Anticancer, antimicrobial and antiparasitical activities of copper(I) complexes based on N-heterocyclic carbene (NHC) ligands bearing aryl substituents

Article abstract of DOI:10.1080/00958972.2020.1836359

New benzimidazolium salts were synthesized as N-heterocyclic carbene precursors. These NHC precursors were metallated with Cu₂O and CuI in acetone and water under reflux to give novel copper(I) complexes. The structures of these benzimidazolium

Full text of DOI:10.1080/00958972.2020.1836359

Journal of Coordination Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Anticancer, antimicrobial and antiparasitical
activities of copper(I) complexes based on N-
heterocyclic carbene (NHC) ligands bearing aryl
substituents
Nedra Touj , Ibrahim S. Al Nasr , Waleed S. Koko , Tariq A. Khan , Ismail
Özdemir , Sedat Yasar , Lamjed Mansour , Faisal Alresheedi & Naceur Hamdi
To cite this article: Nedra Touj , Ibrahim S. Al Nasr , Waleed S. Koko , Tariq A. Khan , Ismail
Özdemir , Sedat Yasar , Lamjed Mansour , Faisal Alresheedi & Naceur Hamdi (2020): Anticancer,
antimicrobial and antiparasitical activities of copper(I) complexes based on N-heterocyclic
carbene (NHC) ligands bearing aryl substituents, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2020.1836359
To link to this article: https://doi.org/10.1080/00958972.2020.1836359
Published online: 08 Nov 2020.
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2020.1836359
Anticancer, antimicrobial and antiparasitical activities
of copper(I) complexes based on N-heterocyclic carbene
(NHC) ligands bearing aryl substituents
Nedra Touja, Ibrahim S. Al Nasrb,c, Waleed S. Kokoc, Tariq A. Khand, Ismail
e,f
e,f
g
h
€
Ozdemir , Sedat Yasar , Lamjed Mansour , Faisal Alresheedi and
Naceur Hamdic,i
aResearch Laboratory of Environmental Sciences and Technologies (LR16ES09), Higher Institute of
b
Environmental Sciences and Technology, University of Carthage, Hammam-Lif, Tunisia; Department
c
of Biology, College of Science and Arts, Qassim University, Unaizah, Saudi Arabia; Department of
Science Laboratories, College of Science and Arts, Qassim University, Ar Rass, Saudi Arabia;
dDepartment of Clinical Nutrition, College of Applied Health Sciences, Qassim University, Ar Rass,
e
_
€ €
Saudi Arabia; Faculty of Science and Art, Department of Chemistry, Inonu University, Malatya,
f
g
_
€ €
Turkey; Catalysis Research and Application Center, Inonu University, Malatya, Turkey; Zoology
Department, College of Science, King Saud University, Saudi Arabia; Department of Physics, College
of Science, Qassim University, Buraidah, Saudi Arabia; Department of Chemistry, College of Science
h
i
and Arts, Qassim University, Ar Rass, Saudi Arabia
ABSTRACT
ARTICLE HISTORY
Received 6 April 2020
Accepted 18 September 2020
New benzimidazolium salts were synthesized as N-heterocyclic
carbene precursors. These NHC precursors were metallated with
Cu2O and CuI in acetone and water under reflux to give novel
copper(I) complexes. The structures of these benzimidazolium
salts and copper(I) complexes were characterized on the basis of
KEYWORDS
(NHC)Cu(I) Complexes;
benzimidazolium salts;
biological activity
1
elemental analysis, H NMR, 13C NMR, IR and LC–MS spectroscopic
techniques. The (NHC)Cu(I) complexes 3–4 were tested against
MCF7 and MDA-MB-231 cancer cells, Escherichia coli, methicillin-
resistant Staphylococcus aureus (MRSA) and Candida albicans
microorganisms, Leishmania major promastigotes and amasti-
gotes, Toxoplasma gondii parasites and against Vero cell line
in vitro. The synthesized copper NHC carbene complex 4b (1,3-
bis(2,3,4,5,6-pentamdthylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-
2-yl)copper(I) chloride) was the most active against MCF7 cancer
cells (half growth Inhibition Concentrations (IC50) ¼ 0.3 mg mLꢀ1),
as well as the most potent antimicrobial against E. coli (inhibition
zone (IZ) ¼ 23.3 mm), MRSA (IZ ¼ 25.5 mm) and C. albicans (IZ ¼
28.5 mm) besides its antileishmanial activities against L. major pro-
mastigotes and amastigotes (IC50 < 0.04 mg mLꢀ1). Compound 4c
(1,3-bis(4-(tert-butyl)benzyl)-2,3-dihydro-1H-benzo[d]imida-zol-2-
yl)copper(II) bromide) is the most potent anticancer against MDA-
MB-231 cancer cells IC50 ¼ 0.4 mg mLꢀ1). Compound 4e (5,6-
dimethyl-1,3-bis(2,4,6-trimethylbenzyl)-2,3-dihydro-1H-benzo[d]imi-
dazol-2-yl)copper(I) chloride) is the best suitable antitoxoplasmal
CONTACT Naceur Hamdi
Naceur.hamdi@Isste.rnu.tn
Hamdi_Naceur@Yahoo.fr
Research Laboratory of
Environmental Sciences and Technologies (LR16ES09), Higher Institute of Environmental Sciences and Technology,
University of Carthage, Hammam-Lif, Tunisia.
Supplemental data for this article is available online at https://doi.org/10.1080/00958972.2020.1836359.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
N. TOUJ ET AL.
drug candidate due to its SI of 16.5. These candidates need fur-
ther study to identify mode of action and drug standardization.
1. Introduction
The biological properties revealed for benzimidazolium salts include anticancer, anti-
microbial, antiviral, anti-HIV, anti-inflammatory, antioxidant, antihypertensive and anti-
diabetic effects [1–15]. The substitution at carbon between two nitrogen atoms in this
system usually increases the biological activities [5–7]. The activities are also enhanced
upon coordination to the metal ions [16, 17].
The NHC complexes based on isoelectronic less expensive and more abundant transi-
tion metals like silver(I) and copper(I) have been also investigated because they might
behave similarly from an organometallic point of view [18–23]. Indeed, gold(I) and
copper(I) complexes present similar affinities toward selenium and could be considered
to have similar biological mechanism [2–25]. Lazreg et al. [26] assumed that NHC copper
complexes could generate reactive oxygen species (ROS) leading to a DNA strand break,
responsible for the observed cytotoxicity. In the literature, several copper(I) complexes
have been reported to exhibit half-growth inhibition concentrations (IC50) in the micro-
molar (mM) range against different cancer cell lines. These copper(I) complexes are gen-
erally coordinated to phosphane and/or nitrogen ligands [27–33].
Gautier et al. firstly selected the [CuX(NHC)] complexes and compared their activities
on different cancer cell lines such as MCF-7, MCF-7R, LNCaP, HL60, and KB. They demon-
strated that the copper complexes stopped the cell cycle progression at the mitotic G1
phase [34]. Then, the group of Tacke synthesized copper complexes bearing benzyl-sub-
stituted NHC ligands and studied their cytotoxicity against CAKI-1 [35–40].
However, parasitical diseases are still a worldwide medical problem with particular
emphasis on the neglected tropical diseases (NTDs). These diseases are perilously

JOURNAL OF COORDINATION CHEMISTRY
3
dangerous for both civilians and migrants in tropical and subtropical countries. Also,
the changing of the weather in many areas of the world probably leads to new para-
sitic infection. One of the important factors is to develop new drugs against NTDs; few
pharmaceutical companies work for these drugs because they think developing coun-
tries have no ability to afford them. So, there is few numbers of drugs developed
against NTDs [41–44].
The new trends are to discover drug candidates for various NTDs from cheap and
renewable products. Also there are desires to direct the assistance for immunecompro-
mised humans to some infections such as Toxoplasma gondii [41–44]. In the present
work the synthesis and characterization of the new benzimidazolium salts (2a–e) and
their [CuX(NHC)] complexes 3–4 were studied, then investigated for their activities
against cancer cells MCF7 and MDA-MB-231, antimicrobial (Escherichia coli, MRSA
(methicillin-resistant Staphylococcus aureus), Candida albicans) and antiparasitical
(Leishmania major and T. gondii). Vero cell line was tested for cytotoxicity.
2. Experimental
2.1. I-Synthesis of benzimidazolium salts
2.1.1. I-1-Synthesis of benzimidazolium salts 1
A mixture of 5,6-dimethylbenzimidazole (30 mmol, 4.38 g) or benzimidazole (30 mmol,
3.54 g) and potassium hydroxide (30 mmol, 1.68 g) was dissolved in 25 mL of ethanol.
The mixture was stirred for 15 min at room temperature. Then, 30 mmol of benzyl hal-
ide was added gradually. After stirring for one hour at room temperature, the mixture
is refluxed for 16 h. The reaction is followed by TLC. After cooling to room tempera-
ture, the solvent was evaporated under reduced pressure. The residue obtained was
dissolved in 40 mL of dichloromethane. After filtration, the solution is concentrated
under reduced pressure. The yellow solid obtained is dried under vacuum [45].
2.1.1.1. 5,6-Dimethyl-1-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazole (1a). Yield:
1
97%, C21H26N2, MW ¼ 306.5, M.p. ¼ 173 ꢁC. m(CN) ¼ 1448.65 cmꢀ1. H NMR (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,
0
3H, CH3(a)), 2.37 (s, 3H, CH3(b)), 5.16 (s, 2H, H1 ), 7.20 (s, 1H, H7), 7.24 (s, 1H, H4), 7.50 (s,
1H, H2). 13C NMR (CDCl3, 100 MHz), d (ppm): 16.51 (Cc,g), 16.87 (Cd,f), 17.16 (Ce), 20.33
0
0
0
0
(Ca), 20.66 (Cb), 44.27 (C1 ), 109.77 (C7), 120.29 (C4), 127.37 (C5,8), 131.21 (C4 ,5 ,6 ), 131.97
0
0
0
(C6), 133.38 (C3 ,7 ), 133.56 (C9), 136.13 (C2 ), 141.10 (C2).
2.1.1.2. 1-(2,3,4,5,6-Pentamethylbenzyl)-1H-benzo[d]imidazole (1b). Yield: 95%,
C19H22N2, MW ¼ 278.4, M.p. ¼ 121 ꢁC. m(CN) ¼ 1454.84 cmꢀ1
.
1H NMR (CDCl3,
400 MHz), d (ppm): 2.21 (s, 6H, 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). 13C NMR
0
0
(CDCl3, 100 MHz), d (ppm): 16.55 (Ca,e), 16.89 (Cb,d), 17.19 (Cc), 44.42 (C1 ), 109.64 (C7),
0
0
0
0
0
120.33 (C4), 122.37 (C5), 122.87 (C6), 127.12 (C8,9), 133.47 (C4 ,5 ,6 ), 133.55 (C3 ,7 ), 136.28
0
(C2 ), 141.96 (C2).

4
N. TOUJ ET AL.
2.1.1.3. 1-(4-(Tert-Butyl)benzyl)-1H-benzo[d]imidazole (1c). Yield: 92%, C18H20N2, MW ¼
1
264.4, M.p. ¼ 123.6 ꢁC. m(CN) ¼ 1457.59 cmꢀ1. H NMR (CDCl3, 400 MHz), d (ppm): 1.22
0
0
0
(s, 9H, 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). 13C NMR (CDCl3, 100 MHz) d (ppm) 31.28
0
0
0
0
(Ca,b,c), 34.60 (C8 ), 48.53 (C1 ), 110.08 (C7), 120.39 (C4), 122.25 (C5), 123.06 (C6), 125.97
0
0
0
0
0
0
(C4 ,6 ), 126.92 (C3 ,7 ), 132.46 (C2 ,8), 143.20 (C2,9), 151.40 (C5 ).
2.1.1.4. 5,6-Dimethyl-1-(2,4,6-trimethylbenzyl)-1H-benzo[d]imidazole (1d). Yield: 94%,
C19H22N2, MW ¼ 278.4, M.p. ¼ 292.3 ꢁC. m(CN) ¼ 1497.89 cmꢀ1
.
1H NMR (CDCl3,
400 MHz), d (ppm): 2.16 (s, 6H, CH3(c,e)), 2.25 (s, 3H, CH3(d)), 2.31 (s, 3H, CH3(a)), 2.35 (s,
0
0
0
3H, CH3(b)), 5.11 (s, 2H, H1 ), 6.88 (s, 2H, H4 ,6 ), 7.17 (s, 1H, H7), 7.22 (s, 1H, H4), 7.49 (s,
1H, H2). 13C NMR (CDCl3, 100 MHz) d (ppm) 18.52 (Cc,e), 19.26 (Cd), 19.62 (Ca), 20.01
0
0
0
(Cb), 41.99 (C1 ), 108.74 (C7), 119.23 (C4), 126.29 (C5), 128.59 (C4 ,6 ), 130.19 (C8), 131.02
0
0
0
0
(C6), 136.83 (C5 ,9), 137.70 (C3 ,7 ), 139.86 (C2 ), 141.47 (C2).
2.1.1.5. 1-(3,5-Dimethylbenzyl)-5,6-dimethyl-1H-benzo[d]imidazole (1e). Yield: 93%,
C18H20N2, MW ¼ 264.4, M.p. ¼ 124.3 ꢁC. m(CN) ¼ 1446.05 cmꢀ1
.
1H NMR (CDCl3,
300 MHz), d (ppm): 2.18 (s, 6H, CH3(c,d)), 2.27 (s, 3H, CH3(a)), 2.29 (s, 3H, CH3(b)), 5.13 (s,
0
0
0
0
2H, H1 ), 6.70 (s, 2H, H3 ,7 ), 6.85 (s, 1H, H5 ), 7.01 (s, 1H, H7), 7.51 (s, 1H, H4), 7.75 (s, 1H,
H2). 13C NMR (CDCl3, 75 MHz), d (ppm): 20.28 (Ca), 20.61 (Cb), 21.26 (Cc,d), 48.62 (C1 ),
0
0
0
0
110.14 (C7), 120.26 (C4), 124.80 (C3 ,5 ,7 ), 129.78 (C5), 131.11 (C8), 132.22 (C6), 135.70
0
0
0
(C9,2 ), 138.65 (C4 ,6 ), 142.46 (C2).
2.1.2. I-2-Synthesis of benzimidazolium salts 2
In a dried Schlenk tube, (1 g) of 1 and (1 eq) of benzyl halide are dissolved in 2 mL of
N,N-dimethylformamide. The mixture was stirred at 70 ꢁC for 24–48 h and the reaction
was monitored by TLC. At the end of the reaction, the reaction mixture was diluted
with 30 mL of diethyl ether to precipitate the product. After filtration, the white solid
obtained was dried under vacuum and then recrystallized from a mixture of DCM-
ether solvents (1:3) for further purification [46].
2.1.2.1. 5,6-Dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazol-3-ium
chloride (2a). Yield: 96%, C33H43N2Cl, MW ¼ 503.2, M.p. ¼ 307.7 ꢁC. m(CN) ¼
1440.99 cmꢀ1 1H NMR (CDCl3, 400 MHz), d (ppm): 2.22 (s, 30H, CH3), 2.28 (s, 6H,
.
CH3(a,b)), 5.80 (s, 4H, H1 ,1 ), 7.11 (s, 2H, H4,7), 9.85 (s, 1H, H2). 13C NMR (CDCl3,
0
00
0
0
0
0
0
100 MHz), d (ppm): 16.92 (Cc,g,c ,g ), 17.01 (Cd,f,d ,f ), 17.28 (Ce,e ), 20.77 (Ca,b), 48.10
0
00
0
0
0
00 00 00
0
0
00 00
(C1 ;1 ), 113.41 (C4;7), 125.58 (C8;9), 130.42 (C4 ;5 ;6 ;4 ;5 ;6 ), 133.50 (C5;6), 133.74 (C3 ;7 ;3 ;7 ),
0
00
136.88 (C2 ;2 ), 141.35 (C2).
2.1.2.2. 1,3-Bis(2,3,4,5,6-pentamethylbenzyl)-1H-benzo[d]imidazol-3-ium chloride (2b).
1
Yield: 92%, C31H39N2Cl, MW ¼ 475.1, M.p. ¼ 210.6 ꢁC. m(CN)¼ 1553.91 cmꢀ1. H NMR
0
0
0
0
(CDCl3, 400 MHz), d (ppm): 2.15 (s, 12H, CH3(a,e,a ,e )), 2.18 (s, 12H, CH3(b,d,b ,d )), 2.20 (s,
0
0
00
6H, CH3(c,c )), 5.84 (s, 4H, H1 ,1 ), 7.20 (d, 2H, H5,6), 7.34 (d, 2H, H4,7), 10.34 (s, 1H, H2).
13C NMR (CDCl3, 100 MHz), d (ppm): 16.95 (Ca,e,a ,e ), 17.06 (Cb,d,b ,d ), 17.30 (Cc,c ), 48.58
0
0
0
0
0

JOURNAL OF COORDINATION CHEMISTRY
5
0
00
0
00
0
0
00 00
(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
0
0
00 00
0
00
(C3 ;7 ;3 ;7 ), 137.07 (C2 ;2 ), 143.0 (C2).
2.1.2.3. 1,3-Bis(4-(tert-butyl)benzyl)-1H-benzo[d]imidazol-3-ium bromide (2c). Yield:
1
89%, C29H35N2Br, MW ¼ 491.5, M.p. ¼ 296.5 ꢁC. m(CN) ¼ 1562.86 cmꢀ1. H NMR (CDCl3,
0
00
0
0
00 00
400 MHz), d (ppm): 1.20 (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). 13C NMR (CDCl3,
0
0 00 00
0
00
0
00
100 MHz), d (ppm): 31.18 (CH3), 34.67 (C8 ;8 ), 51.27 (C1 ;1 ), 113.78 (C4;7), 126.34
0
0
00 00
0
0
00 00
0
00
(C4 ;6 ;4 ;6 ), 127.09 (C5,6), 128.17 (C3 ;7 ;3 ;7 ), 129.56 (C8,9), 131.41 (C2 ;2 ), 143,0 (C2),
0
00
152.46 (C5 ;5 ).
2.1.2.4. 5,6-Dimethyl-1-(2,3,4,5,6-pentamethylbenzyl)-3-(2,4,6-trimethylbenzyl)-1H-ben-
zo[d]imidazol-3-ium chloride (2d). Yield: 94%, C31H39N2Cl, MW ¼ 475.1, M.p. ¼
272.2 ꢁC. m(CN) ¼ 1567.83 cmꢀ1
.
1H NMR (CDCl3, 400 MHz), d (ppm): 2.17 (s, 6H,
0
00
CH3(a,b)), 2.21 (s, 24H, CH3(c,d,e,f,g,h,i,j)), 5.68 (s, 2H, H1 ), 5.80 (s, 2H, H1 ), 6.83 (s, 2H,
H4 ;6 ), 7.04 (s, 1H, H4), 7.20 (s, 1H, H7), 10.41 (s, 1H, H2). 13C NMR (CDCl3, 100 MHz), d
00 00
0
(ppm): 16.96 (Cd,f), 17.09 (Cc,g), 17.30 (Ce), 20.17 (Ca,b), 20.75 (Ch,j), 21.03 (Ci), 47.46 (C1 ),
00
00 00
0
47.82 (C1 ), 113.09 (C7), 113.54 (C4), 125.19 (C8), 125.75 (C9), 129.96 (C4 ;6 ), 130.3 (C5 ),
0
0
0
0
00
00
00 00
133.52 (C4 ;6 ), 133.92 (C3 ;7 ), 136.83 (C5 ), 136.90 (C2 ), 137.15 (C3 ,7 ), 137.88 (C5;6),
0
139.32 (C2 ), 142.17 (C2).
2.1.2.5. 5,6-Dimethyl-1,3-bis(2,4,6-trimethylbenzyl)-1H-benzo[d]imidazol-3-ium chloride
1
(2e). Yield: 96%, C29H35N2Cl, MW ¼ 447.1, M.p. ¼ 245.6 ꢁC. m(CN) ¼ 1567.86 cmꢀ1. H
0
0
0
NMR (CDCl3, 400 MHz), d (ppm): 2.23 (s, 6H, CH3(a,b)), 2.29 (s, 18H, CH3(c,c ,d,d ,e,e )), 5.79
(s, 4H, H1 ,1 ), 6.91 (s, 6H, Harom), 10.92 (s, 1H, H2). 13C NMR (CDCl3, 100 MHz), d (ppm):
0
00
0
0
0
0
00
20.21 (Cc,e,c ,e ), 20.74 (Ca,b), 21.06 (Cd,d ), 47.23 (C1 ;1 ), 113.29 (C4,7), 125.40 (C8,9), 130.07
0
0
00 00
0
00
0
00
0
0
00 00
(C4 ,6 ;4 ,6 ), 130.25 (C5 ,5 ), 136.88 (C2 ,2 ), 137.91 (C3 ,7 ;3 ;7 ), 139.56 (C5,6), 142.70 (C2).
2.2. II-Synthesis of copper complexes
2.2.1. II-1-Synthesis of copper complexes CuI 3
A standard Schlenk was loaded with benzimidazolium salts 2 (1 mmol), copper iodide
(1 mmol, 0.19 g) and K2CO3 (2 mmol, 0.276 g). The resulting mixture was heated under
reflux in acetone at 60 ꢁC under argon for 24 h. After this time, the mixture was fil-
tered on a silica column, eluting with dichloromethane. The product obtained was
concentrated with a rotary evaporator and dried under vacuum affording white com-
plexes 3a–3e [47].
2.2.1.1. (5,6-Dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihydro-1H-benzo[d]imi-
dazol-2-yl)copper(I) iodide (3a). Yield: 68%, C33H42N2ICu, MW ¼ 657.2, M.p. ¼ 265.6 ꢁC.
1
m(CN) ¼ 1448.51 cmꢀ1. H NMR (CDCl3, 400 MHz), d (ppm): 2.12 (s, 12H, CH3(c,g,c,g )),
00
0
00
0
0
00
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). 13C NMR (CDCl3, 100 MHz), d (ppm): 16.10 (Cc,g,c ,g ), 16.25 (Cd,f,d ,f ),
0
0
0 0
0
0
00
0
0
00 00
16.36 (Ce,e ), 19.46 (Ca,b), 47.80 (C1 ;1 ), 110.85 (C4;7), 126.35 (C8;9), 131.79 (C4 ;6 ;4 ;6 ),
0
00
0
0
00 00
0
00
131.9 (C5 ;5 ), 132.09 (C5;6), 132.91 (C3 ;7 ;3 ;7 ), 135.67 (C2 ;2 ), 184.99 (C2). Anal. Calc. for

6
N. TOUJ ET AL.
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).
2.2.1.2. (1,3-Bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)copper(
I) iodide (3b). Yield: 72%, C31H38N2ICu, MW ¼ 629.1, M.p. ¼ 245.3 ꢁC. m(CN) ¼
1419.02 cmꢀ1 1H NMR (CDCl3, 400 MHz), d (ppm): 2.13 (s, 12H, CH3(a,e,a ,e )), 2.17 (s,
.
0
0
0
0
0
0
00
12H, CH3(b,d,b ,d )), 2.22 (s, 6H, CH3(c,c )), 5.47 (s, 4H, H1 ,1 ), 7.08 (d, 2H, H5;6), 7.15 (d, 2H,
H4,7). 13C NMR (CDCl3, 100 MHz), d (ppm): 16.11 (Ca,e,a ,e ), 16.26 (Cb,d,b ,d ), 16.38 (Cc,c ),
0
0
0
0
0
0
00
0
00
0
0
00 00
48.13 (C1 ;1 ), 110.61 (C4;7), 122.76 (C5;6), 126.13 (C5 ;5 ), 132.08 (C4 ;6 ;4 ;6 ), 132.98
0
0
00 00
0
00
(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): 501.2398
[M-I]þ (Calc. for C31H38N2Cu: 501.2331).
2.2.1.3.
(1,3-Bis(4-(tert-butyl)benzyl)-2,3-dihydro-1H-benzo[d]imida-zol-2-yl)copper(I)
iodide (3c). Yield: 66%, C29H34N2ICu, MW ¼ 601.1, M.p. ¼ 208.1 ꢁC. m(CN) ¼
1476.56 cmꢀ1. H NMR (DMSO-d6, 400 MHz), d (ppm): 1.20 (s, 18H, CH3), 5.71 (s, 4H,
1
H1 ,1 ), 7.28 (s, 6H, H4 ,6 ,4 ,6 ;5,6), 7.39 (d, 4H, H3 ,7 ,3 ,7 ), 7.59 (d, 2H, H4,7). 13C NMR (DMSO-
0
00
0
0
00 00
0
0
00 00
0
0
0
0
00
00 00
d6, 100 MHz), d (ppm): 36.23 (Ca,b,c,a ,b ,c ), 39.43 (C8 ,8 ), 55.85 (C1 ,1 ), 117.18 (C4,7), 128.74
0
0
00 00
0
0
00 00
0
00
0
00
(C5,6), 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): 473.2044 [M-I]þ (Calc. for C29H34N2Cu: 473.2018).
2.2.1.4.
(1,3-Bis(3,5-dimethylbenzyl)-5,6-dimethyl-2,3-dihydro-1H-benzo[d]imidazol-2-
yl)copper(I) iodide (3d). Yield: 63%, C27H30N2ICu, MW ¼ 573, M.p. ¼ 232.5 ꢁC. m(CN) ¼
1440.29 cmꢀ1. H NMR (CDCl3, 300 MHz), d (ppm): 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). 13C NMR (CDCl3,
1
0
0
0
00
0
0
0
00 00 00
0
0
0
00
75 MHz), d (ppm): 19.40 (Ca,b), 20.28 (Cc,d,c ,d ), 51.42 (C1 ,1 ), 110.96 (C4,7), 124.29
0
0
00 00
0
00
0
00
0
0
00 00
(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 C27H30N2ICu (%): 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
C27H30N2Cu: 383.2443).
2.2.1.5. (1,3-Bis(4-(tert-butyl)benzyl)-5,6-dimethyl-2,3-dihydro-1H-benzo[d]imidazol-2-
yl)copper(I) iodide (3e). Yield: 65%, C31H38N2ICu, MW ¼ 629.1, M.p. ¼ 225.4 ꢁC. m(CN)
1
¼ 1365.35 cmꢀ1. H NMR (CDCl3, 300 MHz), d (ppm): 1.21 (s, 18H, CH3(c,d,e,c ,d ,e )), 2.13
0
0
0
0
00
0
0
00 00
(s, 6H, CH3(a;b)), 4.97 (s, 4H, H1 ,1 ), 6.62 (s, 2H, H4,7), 7.20 (d, 4H, H3 ,7 ,3 ,7 ), 7.24 (d, 4H,
H4 ,6 ,4 ,6 ). 13C NMR (CDCl3, 75 MHz), d (ppm): 18.92 (Ca;b), 30.29 (Cc,d,e,c ,d ,e ), 33.47
0
0
00 00
0
0
0
0
00
0
00
0
0
00 00
0
0
00 00
(C8 ,8 ), 43.49 (C1 ,1 ), 108.40 (C4,7), 124.61 (C4 ,6 ,4 ,6 ), 126.10 (C3 ,7 ,3 ,7 ), 126.51 (C8,9),
0
00
0
00
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).
2.2.2. II-2-Synthesis of copper complexes CuX 4
A round bottom flask was charged with benzimidazolium salt 2 (2 mmol), copper
oxide (1.3 mmol, 0.186 g) and deionized water (6 mL). The mixture was heated under

JOURNAL OF COORDINATION CHEMISTRY
7
reflux for 24 h. After this time, the reaction mixture was allowed to cool to room tem-
perature, then diluted with DCM and the organic fraction was extracted. The organic
extract was dried with MgSO4 and concentrated under reduced pressure. The product
obtained was purified by chromatography on a silica column, eluting with dichlorome-
thane to give the desired product in the form of an off-white solid [48].
2.2.2.1. (5,6-Dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihy-dro-1H-benzo[d]imi-
dazol-2-yl)copper(I) chloride (4a). Yield: 70%, C33H42N2ClCu, MW ¼ 565.7, M.p. ¼
265.6 ꢁC. m(CN) ¼ 1447.07 cmꢀ1
.
1H NMR (CDCl3, 300 MHz), d (ppm): 2.12 (s, 12H,
0
0
0
0
0
0
00
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, 4H, H1 ,1 ), 6.82 (s,
2H, H4,7).13C NMR(CDCl3, 75 MHz), d (ppm): 15.99 (Cc,g,c ,g ), 16.21 (Cd,e,f,d ,e ,f ), 19.45
0
0
0
0 0
0
00
0
0
00 00
0
00
(Ca,b), 48,0 (C1 ;1 ), 110.77 (C4,7), 126.49 (C8,9), 131.78 (C4 ,6 ;4 ;6 ), 131.83 (C5 ;5 ), 132.13
0
0
00 00
0
00
(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): 529.2619
[M-Cl]þ (Calc. for C33H42N2Cu: 529.2644).
2.2.2.2.
(1,3-Bis(2,3,4,5,6-pentamdthylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-2-yl)
copper(I) chloride (4b). Yield: 74%, C31H38N2ClCu, MW ¼ 537.7, M.p. ¼ 238.8 ꢁC. m(CN)
¼ 1435.31 cmꢀ1. H NMR (CDCl3, 300 MHz), d (ppm): 2.22 (s, 12H, CH3(a,e,a ,e )), 2.25 (s,
1
0
0
0
0
0
0
00
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). 13C NMR (CDCl3, 75 MHz), d (ppm): 17.05 (Ca,e,a ,e ), 17.26 (Cb,c,d,b ,c ,d ), 49.37
0
0
0
0
0
0
00
0
00
0
0
00 00
(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
0
0
00 00
0
00
(C3 ,7 ,3 ,7 ), 136.58 (C2 ;2 ), 186.03 (C2). Anal. Calc. for C31H38N2ClCu (%): C, 69.25; H,
7.12; N, 5.21. Found (%): C, 69.37; H, 7.19; N, 5.34. HR-MS(ESI), (m/z): 501.2391 [M-Cl]þ
(Calc. for C31H38N2Cu: 501.2331).
2.2.2.3.
(1,3-Bis(4-(tert-butyl)benzyl)-2,3-dihydro-1H-benzo[d]imida-zol-2-yl)copper(I)
bromide (4c). Yield: 63%, C29H34N2BrCu, MW ¼ 554.1, M.p. ¼ 208.1 ꢁC. m(CN) ¼
1
1
0
00
1479.23 cm . H NMR (CDCl3, 300 MHz), d (ppm): 1.21 (s, 18H, CH3), 5.55 (s, 4H, H1 ,1 ),
7.19-7.34 (m, 12H, Harom). 13C NMR (CDCl3, 75 MHz), d (ppm): 30.23 (Ca,b,c,a ,b ,c ), 33.56
0
0
0
0
00
0
00
0
0
00 00
0
0
00 00
(C8 ,8 ), 51.58 (C1 ,1 ), 110.96 (C4,7), 123.0 (C5,6), 124.96 (C4 ,6 ,4 ,6 ), 126.27 (C3 ,7 ,3 ,7
)
,
0
00
0
00
130.99 (C8,9), 132.79 (C2 ,2 ), 150.49 (C5 ,5 ), 184.60 (C2). 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):
473.2043 [M-Br]þ (calc. for C29H34N2Cu: 473.2018).
2.2.2.4.
(5,6-Dimethyl-1-(2,3,4,5,6-pentamethylbenzyl)-3-(2,4,6-trimethylbenzyl)-2,3-
dihydro-1H-benzo[d]imidazol-2-yl)copper(I) chloride (4d). Yield: 68%, C31H38N2ClCu,
MW ¼ 537.7, M.p. ¼ 231.3 ꢁC. m(CN) ¼ 1450.56 cmꢀ1
.
1H NMR (CDCl3, 400 MHz), d
(ppm): 2.22 (s, 15H, CH3(c,d,e,f,g)), 2.27 (s, 6H, CH3(h,j)), 2.30 (s, 6H, CH3(a,b)), 2.32 (s, 3H,
0
00
00 00
CH3(i)), 5.38 (s, 2H, H1 ), 5.54 (s, 2H, H1 ), 6.67 (s, 1H, H7), 6.89 (s, 2H, H4 ;6 ), 7.10 (s, 1H,
H4). 13C NMR (CDCl3, 100 MHz), d (ppm): 17.10 (Cc,g), 17.15 (Cd,f), 17.10 (Ce), 20.42 (Ca),
0
00
20.51 (Cb), 20.66 (Ch,j), 21.04 (Ci), 47.69 (C1 ), 49.58 (C1 ), 111.35 (C7), 112.18 (C4), 127.13
00 00
0
0
0
0
0
00
(C4 ,6 ), 127.62 (C8,9), 129.91 (C4 ,5 ,6 ), 132.98 (C5,6), 134.14 (C3 ,7 ), 136.86 (C5 ), 137.55
00 00
0
00
(C3 ,7 ), 138.57 (C2 ,2 ), 183.86 (C2)). Anal. Calc. for C31H38N2ClCu (%): C, 69.32; H, 7.17;
N, 5.30. Found (%): C, 69.2; H, 7.12; N, 5.21. HRMS (ESI), (m/z): [M þ Cl], found 571.4046.

8
N. TOUJ ET AL.
C31H38N2CuCl2 requires 571.1708; [M-Cl]þ, found 501.2463. C31H38N2Cu requires
501.2331; found 439.3121. C31H39N2 requires 439.3113.
2.2.2.5. (5,6-Dimethyl-1,3-bis(2,4,6-trimethylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-2-
yl)copper(I) chloride (4e). Yield: 72%, C29H34N2ClCu, MW ¼ 509.6, M.p. ¼ 229.3 ꢁC.
1
m(CN) ¼ 1465.85 cmꢀ1. H NMR (CDCl3, 400 MHz), d (ppm): 2.25 (s, 18H, CH3(c,c ,d,d ,e,e ),
0
0
0
2.33 (s, 6H, CH3(a;b)), 5.45 (s, 4H, H1 ,1 ), 6.85 (s, 2H, H4,7), 6.94 (s, 4H, H4 ,6 ,4 ,6 ). 13C
0
00
0
0
00 00
0
0
0
0
00
NMR (CDCl3, 100 MHz), d (ppm): 20.50 (Cc,d,e,c ,d ,e ), 21.11 (Ca,b), 48.28 (C1 ;1 ), 111.80
0
0
00 00
0
00
0
0
00 00
00
(C4;7), 127.27 (C4 ;6 ;4 ;6 ), 130.11 (C5,6,8,9), 133.12 (C5 ;5 ), 137.52 (C3 ,7 ;3 ,7 ), 139.04 (C2;2 ),
183.78 (C2). Anal. Calc. for C29H34N2ClCu (%): C, 68.41; H, 6.77; N, 5.61. Found (%): C,
68.35; H, 6.72; N, 5.50. HRMS (ESI), (m/z): [M-Cl]þ, found 473.1990. C29H34N2Cu requires
473.2018; found 411.2756. C29H35N2 requires 411.2800.
2.3. Biological activities
2.3.1. Anticancer cytotoxicity activities
3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was used to evalu-
ate the efficacy of the compounds against MDA-MB-231 and MCF7 cells as carried out
previously [23]. The cells were cultured into 96-well plates (Corning, USA) at a density
of 5 ꢂ 105 cells per well in 200 lL medium. After 24 h, cells were treated with different
concentrations (10, 5, 2.5, 1 mg mLꢀ1) of compounds. After 48 h incubation period,
20 lL MTT solution was added to each well and cells were further incubated for 2 h at
37 ꢁC. At the end, the media were removed by aspiration and then 200 lL 0.1% HCl-
MeOH was added to dissolve formazan crystals. The OD value was read at 490 nm on
a microplate reader (Thermo MULTISCAN FC, China). MeOH-treated cells were used as
controls (negative). The number of replications for the anticancer cytotoxic studies
was three. The compounds were screened colorimetrically using MTT cell assay.
The relative cell viability was calculated using the following formula:
Relative cell viability ð%Þ ¼ ðOD treated=OD controlÞ ꢂ 100 ð%Þ
2.4. Disk diffusion method
R
Nutrient agar plates were used for culturing E. coli (ATCCV 10418) and MRSA
R
(ATCCV3359) while potato dextrose agar of (HiMedia, India) was used for culturing and
R
C. albicans (ATCCV 90028) for 24 h at 35 ꢁC. The cultures were obtained from the
American Type Culture Collection (ATCC). The disc diffusion technique was used to
assess the antimicrobial activities [22]. Sterile saline solution (0.9%) was used for
microbes and the turbidity was adjusted to 0.5 OD value using a spectrophotometer
(Labomed Inc., USA). Sterilized cotton swab was used to put the inoculum in to the
surface of agar plates. Sterile blank disks (6-mm) were loaded with 10 mL of com-
pounds stock solution (10 mg/mL) giving a concentration of 50 mg/disc. Commercial
tetracycline discs (30 mg per disc) were used as positive controls and methanol as a
negative control for comparison. The petri-plates were incubated at 35 ꢁC for 24 h. The
inhibition zones (IZ) were assessed as diameters (mm) produced by the compounds
on the test microoganisms.

JOURNAL OF COORDINATION CHEMISTRY
9
2.5. Leishmania major cell isolation, culture conditions, and assays
Leishmania major promastigotes were collected from indoor male patient and main-
tained at 26 ꢁC in Schneider’s Drosophila medium (Invitrogen, USA) supplied by 10%
inactivated fetal bovine serum (FBS, Invitrogen, USA) with antibiotics using tissue cul-
ture flask followed by weekly transfers, and finally cryopreserved in liquid nitrogen
with amount of 3 ꢂ 106 parasite mLꢀ1 [49].
For assessing the compounds activity. Promastigotes from logarithmic-phase cul-
tured in phenol red-free RPMI 1640 medium (Invitrogen, USA) with 10% FBS were sus-
pended on 96-well plates to yield 106 cells mLꢀ1 (200 mL/well). Compounds and
reference drug AmB were added using different concentrations (100, 33, 11, 3.7, 1.2,
0.4, 0.14, and 0.04 mg mLꢀ1). DMSO (1%) was used as negative control. Plates were
incubated at 26 ꢁC for 72 h. MTT colorimetric assay was used for assessing the number
of viable promastigotes. The samples were analyzed using an ELISA reader at 570 nm.
Obtained IC50 values resulted from three repeated experiments [49].
For evaluating compounds against L. major amastigotes, peritoneal macrophages
from female BALB/c mice (6–8 weeks of age) were collected [50], then 5 ꢂ 104 cells/
well were seeded on 96-well plates in phenol red-free RPMI 1640 medium with 10%
FBS for 4 h at 37 ꢁC in 5% CO2 to enhance the adhesion of the cells. PBS was used
for washing after the media discarded. About 200 mL containing L. major promasti-
gotes solution was added per well. For allowing infection with amastigotes the
plates were incubated for 24 h at 37 ꢁC in humidified 5% CO2. Then, PBS was used
for washing the infected macrophages and covered with fresh phenol red-free RPMI
1640 medium containing compounds and AmB at different concentrations (100, 33,
11, 3.7, 1.2, 0.4, 0.14, and 0.04 mg mLꢀ1) and incubated at 37 ꢁC in humidified 5%
CO2 for 72 h. DMSO (1%) was used as negative control. The percentage of infected
macrophages were evaluated microscopically after removing medium, washing, fix-
ation, Giemsa staining. Obtained IC50 values resulted from three repeated experi-
ments [49].
2.6. Toxoplasma gondii cell line, culture conditions, and assay
R
Vero cells (ATCCV CCL81TM, USA) were used for the cultivation of T. gondii tachy-
zoites of the RH strain obtained from State Key Laboratory for Agrobiotechnology,
China Agricultural University, Bejing, China. Complete RPMI 1640 medium with 10%
FBS inactivated was used for culturing the cells in a humidified 5% CO2 at 37 ꢁC. 96-
Well plates (5 ꢂ 103 cells/well in 200 mL RPMI 1640 medium) were used for the culti-
vation of the Vero cells and then incubated at 37 ꢁC and 5% CO2 for 24 h, followed
by removal of medium and washing the cells with PBS. Then, RPMI 1640 medium
with 2% FBS containing tachyzoites (RH strain) of T. gondii at a parasites:Vero cells
ratio of 5:1 were added. After incubation at 37 ꢁC and 5% CO2 for 5 h, cells were
washed with PBS and then treated with compounds and ATO reference drug with
different concentrations (100, 33, 11, 3.7, 1.2, 0.4, 0.14, and 0.04 mg mLꢀ1) while
DMSO (1%) was used as negative control. 1% toluidine blue was used for staining
the cells after 72 h incubation at 37 ꢁC and 5% CO2 followed by washing with PBS
and fixation in 10% formalin. Inverted photomicroscope was for the determination

10
N. TOUJ ET AL.
of the infection index (number of cells infected from 200 cells tested) of T. gondii.
Then % inhibition was calculated as:
ð
Þ
Inhibition ð%Þ ¼ ðI Control ꢀ I ExperimentalÞ= I Control ꢂ 100
Then effects of test compounds on parasite growth were expressed as IC50 values,
from three repeated experiments [44, 51].
2.7. In vitro cytotoxicity assay
For assessing compounds cytotoxicity, MTT assay was used against Vero cells as previ-
ously described against cancer cells according to OECD guidelines [52]. Complete
RPMI 1640 medium with 10% FBS was used for culturing Vero cells in 96-well plates (5
103 cells/well/200 mL) then incubated for 24 h at 5% CO2 and 37 ꢁC. Cells were washed
with PBS and treated with test compounds for 72 h at varying concentrations (100, 33,
11, 3.7, 1.2, 0.4, 0.14, and 0.04 mg mLꢀ1) in medium with 10% FBS. Some cells were
treated with medium in 2% FBS to be kept as negative control. Then the media was
removed by aspiration and 50 mL of RPMI 1640 medium containing 14 mL MTT (5 mg
mLꢀ1) was added followed by 4 h incubation. The supernatant was removed again
and 200 mL DMSO was added to each well with continuous shaking for 15 min. A
FLUOstar OPTIMA spectrophotometer was applied for colorimetric analysis
(k ¼ 540 nm). Cytotoxic effects were expressed by CC50 values (concentration that
caused a 50% reduction in viable cells). Three different experiments were repeated for
CC50 calculation [44].
3. Results and discussion
3.1. Synthesis and characterization
N-heterocyclic carbene ligands have been proven very popular in the last 20 years.
Conjugate reduction of a,b-unsaturated ketones and esters, the hydrosilylation of
ketones, the cyclopropanation of terminal alkenes, as well as olefinations, carbene
transfer reactions, aziridination of olefins and methylenation of aldehydes are among
some examples of the uses of Cu-NHC complexes (specifically (IPr) CuCl) in modern
catalysis. Finally, these catalysts are air- and moisture-stable and they can be used as
precursors to synthesize more air-sensitive complexes [53].
All the benzimidazolium salts as NHC precursors were already prepared similarly to
the published procedures [54, 55]. As shown in Scheme 1, benzimidazolium salts 2a–e
were synthesized in good yields by quaternization of 1 in DMF at 70 ꢁC for 03 days
with the corresponding arylchlorides. The benzimidazolium salts 2a–e are stable to air
1
and moisture, both in the solid state and in solution. They were characterized by H-
NMR, 13Cf Hg NMR, IR and elemental analysis techniques.
1
1
The structures of the benzimidazolium salts 2 can be easily confirmed by the H
NMR spectroscopic data. The characteristic carbonic protons (NCHN) are located at
9.85, 10.34, 11.83, 10.41, 10.92, 11.51, 11.42, and 11.63 ppm, respectively. The corre-
sponding methylene protons appear as singlets at 5.8, 5.84, 5.75, 5.68, 5.79, 5.68, 5.65,
and 5.77 ppm, respectively, which are comparable to the reported values [56–58]. As

JOURNAL OF COORDINATION CHEMISTRY
11
Scheme 1. Protocol synthesis benzimidazolium salts 2a–e.
expected, the absence of pro-carbenic protons can be observed upon coordination of
the benzimidazolium salts with the copper(I), confirming formation of the NHC–Cu(I)
complexes 3–4. In the 13C NMR spectra, the signals for the carbene carbon atoms of
salts 2a–e appear at 141.3, 143.0, 143.0, 143, 142.1, 142.07, 141.6, 141.6, 141.8, and
141.83 ppm, respectively, which are consistent with signals for other NHC–Cu(I) com-
plexes [59]. The [CuCl(NHC)] complexes 4 were already synthesized following the
Nolan–Cazin procedure using Cu2O as metal precursor for 24 h in water under reflux
and the appropriate benzimidazole. These metal(II) complexes were obtained as white
solids in 63–74% yield.
Complexes 3a-e were obtained by reacting benzimidazolium salts 2a–c with K2CO3
in acetone under reflux for 24 h in acetone in the presence of CuI in good yields
(Scheme 2).
In recent years, copper N-heterocyclic carbene complexes (Cu-NHCs) have found
applications as catalysts [60] and as NHC transfer reagents [61]. In the clear majority of
these cases, the copper is present in oxidation state Cu(I), where the soft–soft acid–-
base interaction, Cu(I)–C, results in a highly stable complex. Also, when the NMR of
the solution in the NMR tube was taken again days later, same NMR spectrum had
been seen.
The elemental analysis data of [CuCl(NHC)] complexes 3–4 are in agreement with
the theoretical values for the synthesized complexes. The benzylic –CH2– proton sig-
0
00
nals H1 ,1 for complex [CuCl(NHC)] 2e as representative were observed at 5.45 ppm,
while the aromatic protons appeared at d between 6.85 and 7.26 ppm. The carbene
carbon signal of 2e was observed at 183.7 ppm in the 13C NMR spectrum, while the
0
00
C1 ,1 carbon signals were at d 48.2 ppm. The mass spectrum of 2e gave the most
prominent peaks at m/z ¼ 279 and 411, respectively.
The 1H NMR spectra of the Cu-NHC complexes (I) 3–4 showed less intense and
downfield shifted benzimidazole signals as compared to the free ligands. In 13C NMR
spectra of the complexes, a downfield shift in C ¼ N resonance of the ligands upon
complexation indicates the binding of benzimidazolium salts to copper through the
imine nitrogen atom. The aromatic carbons of benzene ring resonate between 112
and 141 ppm. In case of the ligands the parallel ring carbons behave as equivalent but

12
N. TOUJ ET AL.
Scheme 2. Protocol synthesis of [CuCl(NHC)] complexes 3–4.
Table 1. Anticancer activity of Cu-NHC complexes 3–4.
Anticancer activity, IC50 (in mM)
Cu–NHC complexes (II) 3–4
MCF7 MDA-MB-231
3a
3b
0.943
NA
0.806
NA
3c
3d
1.181
NA
1.364
NA
3e
4a
4b
4c
NA
NA
0.795
0.595
1.155
NA
1.095
1.153
0.794
NA
4d
4e
NA
0.697
NA
Tetracycline
NA, not active.
in the complexes they showed separate signals. The methyl peak in the Cu-NHC com-
plexes (I) 3–4 is observed around 16 ppm. These results are in agreement with the
data of other such complexes [58, 62–67].
3.2. Biological evaluation
3.2.1. Anticancer evaluation
Table 1 indicates that the five compounds were highly efficient and active against the
two types of cancer cells investigated in this study. For MCF7, the five compounds
(4b, 4a, 3a, 4c, and 3c) had IC50 (IC50 is the lethal concentration required to kill 50%
of the population) values less than 1 mg mLꢀ1 with an IC50 ¼ 0.3, 0.4, 0.6, 0.6, and
0.7 mg mLꢀ1, respectively; for MDA-MB-231, the same compounds had IC50 values of

JOURNAL OF COORDINATION CHEMISTRY
13
Table 2. Antimicrobial profile of synthesized derivatives Cu–NHC complexes
(I) 3–4a.
Antimicrobial activity (50 mg/disc)
Cu–NHC complexes (I) 3–4
E. coli
MRSA
C. albicans
3a
3b
3c
3d
18
16
2
1
15 0.5
15 00
18.0 00
10. 1.0
13 00
16.0 00
28.0 00
10.0 00
0.00 00
14 00
16 0.6
0.6
12 00
8
3e
4a
4b
21.0
23
1
1
22
25
2
1
25.0 00
28 1
4c
4d
4e
2a
21 0.6
18
13 00
12
23.0 001
0.5
12 00
11
28.0 00
1
8
0.0 00
14 00
10
2b
2c
13
11
11
10
12
12
2d
2e
12
13
11
12
12
13
Tetracycline
Fluconazole
20
–
16
–
–
24
Values are mean value standard deviation of three different replicates.
aThe concentration was 50 mg.
0.6, 0.6, 0.5, 0.4, and 0.8 mg mLꢀ1, respectively. The other compounds showed no activ-
ity against the cancer cells. These results agree with the previous results of Kizrak
et al. [68], who studied different compounds of silver and gold N-heterocyclic carbene
complexes against a number of human cancer cells and finally stated that these types
of research provide exciting opportunities for discovering new types of metallodrugs.
We can conclude our results enhance the discovery of new anticancer drugs based on
metal complex compounds (Table 1).
3.2.2. Antimicrobial activities
A simple inspection of Table 2 indicates that the highest antibacterial activity against
E. coli was reported by 4b with inhibition zone (IZ) 23.3 mm followed by 4a and 4c
with IZ ¼ 21 mm for all of them. Also, 4b is the most active against MRSA with IZ ¼
25.5 mm followed by 4c (IZ ¼ 23 mm) and 4a (IZ ¼ 22.5 mm). 4b also proved the best
antifungal activity against C. albicans (IZ ¼ 28.5 mm) followed by 3b and 4c (IZ ¼
28 mm) and then 4a (IZ ¼ 25 mm). NHC metals particularly silver synthesized com-
pounds as well as copper derivatives were found to have potent antibacterial activities
[69, 70]. Our results here agree with this finding. These results have shown that benzi-
midazolium salts 2a–e precursors have lower antibacterial activity when compared
with the Cu–NHC complexes (I) 3–4, which have promising results, with good activity
against bacteria and fungi, although the mechanism of antimicrobial activity is
not known.
3.3.3. Antileishmanial activities
Table 3 shows all the compounds except 4e revealed antileishmanial activity against
both L. major amastigotes and promastigotes in vitro with IC50 lower than 9 mg mLꢀ1
.
Seven compounds, 3c, 3e, 4a, 4b, 4c, and 4d, possess IC50 lower than 0.4 mg mLꢀ1
against L. major promastigotes while four of them (3c, 4b, 4c, and 4d) showed IC50

14
N. TOUJ ET AL.
Table 3. Antileishmanial activity of Cu–NHC complexes against L. major promastigotoes and
amastigotes.
Cu–NHC
complexes
CC50 of Vero
Amastigote IC50
Promastigotes
(II) 3–4
cells at mM
at mM
IC50 at mM
Amastigote SI
Promastigote SI
3a
3b
3c
3d
3e
4a
4 b
4c
4d
4e
1.035
6.358
<0.665
1.431
<0.636
<0.707
1.692
<0.722
0.948
125.588
7.4 2.64
1.978
6.358
<0.665
2.967
6.199
4.949
<0.744
<0.722
<0.744
56.907
0.46 0.07
12.173
12.875
<0.665
4.712
<0.636
<0.707
<0.744
<0.722
<0.744
147.763
0.78 0.09
0.52
0.83
–
0.48
–
–
>2
–
0.08
0.42
–
0.30
–
–
>2
–
>1
2.15
16
>1
0.86
9.49
AmB
Amphotericin B is an antileishmania reference drug.
Table 4. Antitoxopolasmal activity of Cu-NHC complexes (I) 3–4 against T. gondii.
Cu–NHC complexes (I) 3–4
CC50 of Vero cells at mM
Antitoxoplasma IC50 at mM
SI
3a
3b
3c
3d
3e
4a
4 b
4c
1.035
5.404
<0.665
1.431
<0.636
<0.707
1.692
<0.722
0.948
<0.608
11.444
<0.665
4.188
<0.636
<0.707
4.835
<0.722
1.562
7.653
>1
0.47
–
0.34
–
–
0.35
–
4d
4e
ATO
0.62
16.51
103.33
126.374
9.3 2.08
0.09 0.02
ATO, Atovaquone is an antitoxoplasma reference drug.
lower than 0.4 mg mLꢀ1 against L. major amastigotes. Compounds 4b and 4d are less
toxic and their SI over 1 for both L. major promastigotes and amastigotes. In recent
studies a group of metal-based complexes were found of strong antileishmanial activ-
ities against L. major promastigotes and Trypanosoma cruzi due to their interference
with DNA formation [70], also some gold complexes against L. infantum promastigotes
and amastigotes [71]. These results support our results here for Cu–NHC complexes as
antileishmanial against L. major promastigotes and amastigotes in vitro.
3.3.4. Antitoxoplasmal activities
Table 4 indicates that all the compounds possess good antitoxoplasmal activity against
T. gondii in vitro with IC50 lower than 7.5 mg mLꢀ1. Five of them (3a, 3c, 3e, 4a, and
4d) have IC50 lower than 0.4 mg mLꢀ1, followed by 4d, 3d, 4b, 4e, and 3 b with IC50
¼
0.84, 2.4, 2.6, 3.9, and 7.2 mg mLꢀ1, respectively. The table also shows 4e possesses
very good SI with 16.51, which indicates the safety of this compound as an antitoxo-
plasmal drug candidate. However, NHC–carbene metal complexes had not previously
been investigated against T. gondii, but some of their derivatives mainly of silver and
gold were found of good antiparasitical activities against some of apicomplexes proto-
zoa such as Plasmodium spp. [72]. This finding agrees with our results for NHC-copper
complexes against T. gondii.

JOURNAL OF COORDINATION CHEMISTRY
15
4. Conclusions
In the present investigation, the synthesized copper NHC–carbene complex 4b was
found to be the most active against MCF7 cancer cells, as well as the most potent
antimicrobial against E. coli, MRSA and C. albicans besides its antileishmanial activities
against L. major promastigotes and amastigotes. Compound 4c is the most potent
anticancer against MDA-MB-231 cancer cells. Compound 4e is the most suitable anti-
toxoplasmal drug candidate from all investigated compounds due to its SI of 16.5.
Further pharmacological investigations based on the mode of action and drug stand-
ardization are highly recommended for these drug candidates.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was funded by the Research Supporting Project (RSP2020/75), King Saud University,
Riyadh Saudi Arabia.
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