In vitro evaluation of arylsubstituted imidazoles derivatives as antiprotozoal agents and docking studies on sterol 14α-demethylase (CYP51) from Trypanosoma cruzi, Leishmania infantum, and Trypanosoma brucei

Article abstract of DOI:10.1007/s00436-019-06206-z

There is an urgent need to discover and develop new drugs to combat parasitic diseases as Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei), and leishmaniasis (Leishmania ssp.). These diseases are considered among the 13 most unattended diseases worldwide according to the WHO. In the present work, the synthesis of 14 arylsubstituted imidazoles and its molecular docking onto sterol 14α-demethylase (CYP51) was executed. In addition, the compounds, antiprotozoal activity against T. brucei, T. cruzi, Trypanosoma brucei rhodesiense, and Leishmania infantum was evaluated. In vitro antiparasitic results of the arylsubstituted imidazoles against T. brucei, T. cruzi, T.b. rhodesiense, and L. infantum indicated that all samples from arylsubstituted imidazole compounds presented interesting antiparasitic activity to various extent. The ligands 5a, 5c, 5e, 5f, 5g, 5i, and 5j exhibited strong activity against T. brucei, T. cruzi, T.b. rhodesiense, and L. infantum with IC₅₀ values ranging from 0.86 to 10.23?μM. Most samples were cytotoxic against MRC-5 cell lines (1.12 < CC₅₀ < 51.09?μM) and only ligand 5c showed a good selectivity against all tested parasites. According to the results of the molecular docking, the aromatic substituents in positions 1, 4, and 5 have mainly stabilizing hydrophobic interactions with the enzyme matrix, while the oxygen from NO₂, SO₃H, and OH groups interacts with the Fe²⁺ ion of the Heme group.

Full text of DOI:10.1007/s00436-019-06206-z

Parasitology Research
https://doi.org/10.1007/s00436-019-06206-z
IMMUNOLOGY AND HOST-PARASITE INTERACTIONS - ORIGINAL PAPER
In vitro evaluation of arylsubstituted imidazoles derivatives
as antiprotozoal agents and docking studies on sterol 14α-demethylase
(
CYP51) from Trypanosoma cruzi, Leishmania infantum,
and Trypanosoma brucei
1
1
1
2
3
Julio Alberto Rojas Vargas & América García López & Yulier Pérez & Paul Cos & Matheus Froeyen
Received: 25 April 2018 /Accepted: 4 January 2019
#
Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
There is an urgent need to discover and develop new drugs to combat parasitic diseases as Chagas disease (Trypanosoma cruzi),
sleeping sickness (Trypanosoma brucei), and leishmaniasis (Leishmania ssp.). These diseases are considered among the 13 most
unattended diseases worldwide according to the WHO. In the present work, the synthesis of 14 arylsubstituted imidazoles and its
molecular docking onto sterol 14α-demethylase (CYP51) was executed. In addition, the compounds, antiprotozoal activity
against T. brucei, T. cruzi, Trypanosoma brucei rhodesiense, and Leishmania infantum was evaluated. In vitro antiparasitic
results of the arylsubstituted imidazoles against T. brucei, T. cruzi, T.b. rhodesiense, and L. infantum indicated that all samples
from arylsubstituted imidazole compounds presented interesting antiparasitic activity to various extent. The ligands 5a, 5c, 5e, 5f,
5
g, 5i, and 5j exhibited strong activity against T. brucei, T. cruzi, T.b. rhodesiense, and L. infantum with IC values ranging from
50
0
.86 to 10.23 μM. Most samples were cytotoxic against MRC-5 cell lines (1.12 < CC < 51.09 μM) and only ligand 5c showed a
50
good selectivity against all tested parasites. According to the results of the molecular docking, the aromatic substituents in
positions 1, 4, and 5 have mainly stabilizing hydrophobic interactions with the enzyme matrix, while the oxygen from NO ,
2
2
+
SO H, and OH groups interacts with the Fe ion of the Heme group.
3
.
.
.
.
Keywords Arylsusbtituted imidazoles Trypanosoma cruzi Trypanosoma brucei Trypanosoma b. rhodesiense Leishmania
.
.
infantum In vitro evaluation Molecular docking
Section Editor: Sarah Hendrickx
Introduction
*
Julio Alberto Rojas Vargas
jarojas@uo.edu.cu
Trypanosomatidae form a family of unicellular eukaryotic
parasites from the order Kinetoplastida, phylum Euglenozoa,
supergroup Excavates. Many human pathogens possess a
number of species of kinds Leishmania and Trypanosoma.
The life cycles of these organisms turn out to be very complex,
constantly moving between insects and mammalian hosts
América García López
america@uo.edu.cu
Paul Cos
paul.cos@uantwerpen.be
(Guedes da Silva et al. 2017; Lepesheva et al. 2015).
Matheus Froeyen
mathy.froeyen@rega.kuleuven.be
Leishmaniasis is a common disease in many continents,
each year 20,000 to 30,000 deaths and almost 1 million of
new cases are reported, arriving to report oneself around 12
million infected people (Lepesheva et al. 2015). It is caused by
unicellular eukaryotic organisms from genus Leishmania
1
2
Chemistry Department, Exact and Natural Science Faculty, Oriente
University, Santiago de Cuba, Cuba
Laboratory of Microbiology, Parasitology and Hygiene (LMPH), S7,
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
University of Antwerp, Wilrijk, Belgium
(
Trypanosomatidae family). Analogous to other protozoan
parasites, Leishmania has a complex life cycle, transmitted
via the bite of female phlebotomine sandflies. The parasite
exist in two forms namely promastigote in sandfly and
3
Medicinal Chemistry, Department of Pharmacy, Rega Institute, KU
Leuven, Leuven, Belgium

Parasitol Res
amastigote in human macrophage (Bates 2007; Rodríguez
et al. 2006). It is exhibited in three clinical form: visceral
leishmaniasis (Leishmania donovani, Leishmania infantum/
Leishmania chagasi), also known as kala-azar; cutaneous
leishmaniasis caused by Leishmania major, L. donovani,
Leishmania tropica, and Leishmania aethiopica; and muco-
cutaneous leishmaniasis caused by Leishmania braziliensis.
American trypanosomiasis, more known as Chagas dis-
ease, is usually transmitted by the bites of triatomine insects,
also known as Bkissing bugs.^ These insects or their feces
carry the flagellate parasite Trypanosoma cruzi (T. cruzi) and
can also contaminate foods such as fruits juices, resulting in
foodborne. It is an endemic disease in Central and South
America, a few cases occur occasionally in the southern
USA. This disease has become a growing problem in non-
endemic areas, where infections may not be recognized by
many people, and where the few effective drugs may not be
generally available (Lepesheva et al. 2015).
cytokinesis, stopping cell growth to cause death of the cell
membrane (Roberts et al. 2003). Extensive work is performed
already considering CYP51 as target. Silva et al. (Guedes da
Silva et al. 2017) have analyzed the effectiveness of two clas-
ses of drugs using CYP51: VNI and VFV. Schel et al. (2016)
have demonstrated the anti-fungal activity of VT-1161 and
VT-1129, among many other application reports with the
use of CYP51 target (Singh et al. 2015; Anusha et al. 2015).
Here, we report the synthesis, in vitro antiprotozoal activ-
ity, and cytotoxicity of 14 arylsubstituted imidazoles, as well
as its molecular docking onto sterol 14α-demethylase
(CYP51). We have chosen the CYP51 target because all ana-
lyzed compounds possess the principal pharmacophore fea-
tures of CYP51 inhibitors reported by Vita et al., where they
state that an active compound must contain a heterocycle with
a nitrogen able to interact with the Fe atom present in the
Heme group and having two hydrophobic groups near to it
(De Vita et al. 2016).
Human African trypanosomiasis, known commonly as
sleeping sickness, is caused by infection with protozoans of
the genus Trypanosoma. It is transmitted by their only vector
tsetse fly (Glossina genus) bites, which have acquired their
infection from human or other animals that contain in her
organism other human parasites (WHO 2017). This disease
has two clinical manifestations, Trypanosoma brucei
gambiense (T.b. gambiense) and Trypanosoma brucei
rhodesiense (T.b. rhodesiense). The T.b. gambiense causes a
more chronic infection that is responsible for over 90% of
cases, whereas T.b. rhodesiense causes an acute infection that
is responsible for a smaller proportion of the overall human
African trypanosomiasis disease burden. The disease develops
rapidly and invades the central nervous system (WHO 2017).
The patient may exhibit some of the following symptoms:
fevers, skin eruptions, irregular febrile episodes, headaches,
malaise, exhaustion, anorexia, extreme thirst, muscle and joint
pains, anemia, rash, coma, and ultimately death. Sleeping
sickness is endemic in 36 Sub-Saharan African countries, with
an 85% case reduction since year 2000 and less than 3000
cases in year 2015, a record low with around > 60 million
people are at risk (WHO 2017).
Materials and methods
General procedure for the synthesis of substituted
imidazoles
The arylsubstituted imidazoles were obtained by reaction of
aromatic aldehydes, primary amines, benzils, and ammonium
acetate (Fig. 1) catalyzed by boric acid (Shelke et al. 2009).
All reagents used had commercial grade. Melting points were
determined in open capillaries using a Digital Melting Point
1
13
apparatus WRS-2A. H-NMR and C-NMR spectra were
recorded with a Bruker DRX-600 spectrometer obtained in
DMSO-d and MeOD solutions. The FT-IR spectra were re-
6
corded on a Shimadzu-FTIR Infrared spectrometer in KBr
−1
(
νmax in cm ). The reaction occurred at the maximum energy
area in the reaction vessel, where was slightly lower than the
level of the water and the temperature was controlled at 60 °C.
Reaction conditions are as follows: aldehyde 1 (1 mmol),
amine 2 (1 mmol), ammonium acetate 3 (1.5 mmol), benzil 4
(
1 mmol), H BO (5 mol%), and propylene glycol (15 mL)
3 3
Sterol 14α-demethylase (CYP51, EC.1.14.13.70) has been
found as a vital target for the development of anti-fungal, anti-
leishmania, anti-trypanosoma drugs and also for the design of
cholesterol-lowering drugs. Crystal structures of protozoan
sterol 14α-demethylases provide an opportunity for
structure-directed development of such inhibitors. The en-
zyme is responsible for catalyzing the elimination of the
alpha-methyl group of the sterol, which leads to the formation
of cholesterol in vertebrates, the well-known ergosterol in
fungi, and several ergosterol alkylated derivatives
heating under reflux for 6 h.
The products (5a-n) in Table 1 were confirmed by the
1
13
analysis of spectra H-NMR, C-NMR, FT-IR, mass spectra,
and the melting points obtained for each compounds.
Antiparasitic activity
All compounds were in vitro tested against T. cruzi Tulahuen
CL2, β-galactosidase strain (nifurtimox-sensitive),
L. infantum (MHOM/MABE/67 amastigotes), and T. brucei
(Squib-427 strain, suramin-sensitive) and T.b. rhodesiense
(strain STIB-900).
(
phytosterol) for plants and protozoa (Buckner and Urbina
012). The production of ergosterol in fungus and protozoa,
unlike mammals, is lethal when it accumulates, affecting
2

Parasitol Res
Fig. 1 Synthesis of arylsubstituted imidazoles
Compound stock solutions were prepared in DMSO at
0 mM. The compounds were serially pre-diluted (2-fold or
-fold) in DMSO followed by a further (intermediate) dilution
in demineralized water to assure a final in-test DMSO concen-
tration of < 1%. Test plates are identical for all screens and
produced as a single batch. The results were expressed as %
reduction in parasite burdens compared to control wells and an
IC50 (50% inhibitory concentration) was calculated. The
in vitro screening was carried out according to the previously
described procedure (Cos et al. 2006).
Nonidet in 100 ml PBS. The change in color was measured at
540 nm after 4 h incubation at 37 °C. Benznidazole was
included as reference drug. The test compound is classified
as inactive when the IC50 is higher than 30 μM. When IC50
lies between 30 and 5 μM, the compound is regarded as being
moderate active. When the IC50 is lower than 5 μM, the com-
pound is classified as highly active on the condition that it also
demonstrates selective action (absence of cytotoxicity).
Trypanosoma brucei brucei Squib 427 strain (suramin-
sensitive) and T.b. rhodesiense (strain STIB-900) were used
for screening. The strains are maintained in Hirumi (HMI-9)
medium, supplemented with 10% inactivated fetal calf serum.
All cultures and assays were conducted at 37 °C under an
2
4
Trypanosoma cruzi, Tulahuen CL2, β-galactosidase strain
nifurtimox-sensitive) was used. The strain was maintained on
(
MRC-5SV2 (human lung fibroblast) cells in MEM medium,
supplemented with 200 mM L-glutamine, 16.5 mM NaHCO3,
and 5% inactivated fetal calf serum. All cultures and assays
were conducted at 37 °C under an atmosphere of 5% CO2.
Assays were performed in sterile 96-well microtiter plates,
each well containing 10 μl of the watery compound dilutions
atmosphere of 5% CO . Assays were performed in sterile
2
96-well microtiter plates, each well containing 10 μl of the
compound dilutions together with 190 μl of the parasite sus-
4
3
pension (1.5 × 10 parasites/well—T.b. brucei, and 4 × 10
parasites/well—T.b. rhodesiense). Parasite growth was com-
pared to untreated-infected (100% parasite growth) and unin-
fected controls (0% growth). After 3 days incubation, parasite
growth was assessed fluorimetrically after addition of 50 ml
resazurin per well. After 6 h (T.b. rhodesiense) or 24 h (T.b.
brucei) at 37 °C, fluorescence was measured (λ 550 nm, λ
4
together with 190 μl of MRC-5 cell/parasite inoculum (2.10
5
cells/ml + 2.10 parasites/ml). Parasite growth was compared
to untreated-infected controls (100% growth) and non-
infected controls (0% growth) after 7 days incubation.
Parasite burdens were assessed after adding the substrate
CPRG (chlorophenol red β-D-galactopyranoside): 50 μl/
well of a stock solution containing 15.2 mg CPRG + 250 μl
ex
em
590 nm). Suramin (T.b. brucei and T.b. rhodesiense) was
included as the reference drugs. The compound is classified
as inactive when the IC50 is higher than 5 μM. When IC50 lies
between 5 and 1 μM, the compound is regarded as being
moderate active. When the IC50 is lower than 1 μM, the com-
pound is classified as highly active on the condition that it also
demonstrates selective action (absence of cytotoxicity).
Leishmania infantum MHOM/MA(BE)/67 was maintained
in the golden hamster (Mesocricetus auratus) and spleen
amastigotes were collected for infection. Primary peritoneal
mouse macrophages (PMM) were used as host cells and were
collected 2 days after peritoneal stimulation with a 2% potato
starch suspension. Assays were performed in 96-well micro-
titer plates, each well containing 10 μl of the compound dilu-
tions together with 190 μl of macrophage-parasite inoculum
Table 1 Chemical structure of arylsubstituted imidazole
Entry
Compound
R
R
R
1
R
2
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Fu
Fu
Fu
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Fu
Fu
Fu
Ph-CH
Ph-CH
Ph-CH
Ph-CH
2
2
2
2
p-N(CH
p-OH
H
3
)
2
p-OCH
3
m-CH
o-CH
m-CH
p-CH
3
-C
-C
-C
-C
H-C
6
H
5
-
m-NO
2
3
6
H
5
-
p-NO
p-NO
p-NO
2
5g
5h
5i
3
H
6 5
-
2
2
3
6
H
5
-
p-SO
3
6
H
5
p-N(CH
3
)
2
2
5
6
(
3.10 cells + 3.10 parasites/well // RPMI-1640 + 5% FCSi).
1
0
5j
–
p-N(CH
3
)
After 5 days incubation, total amastigote burdens were micro-
scopically assessed after Giemsa staining. Miltefosine was
included as reference drug. A test compound is classified as
inactive when the IC50 is higher than 30 μM. When IC50 lies
between 30 and 10 μM, the compound is regarded as moder-
ately active. If the IC50 is lower than 10 μM, the compound is
1
1
5k
5l
Fu-CH
2
4-OH, 3-CH
3
O
1
1
1
2
3
4
–
4-OH, 3-CH
3
O
5m
5n
–
6 5
C H
Fu-CH
2
6 5
p-OH-C H
Ph phenyl, Fu furyl

Parasitol Res
classified as highly active on the condition that it also demon-
strates selective action (absence of cytotoxicity against prima-
ry peritoneal macrophages).
structures 3L4D and 4BJK contained identical domains; we
selected chain A for the docking experiments. Structure 3KSW
contained only one chain. Finally, the resulting prepared 3D
structure of the proteins was saved as PDB file, using
AutodockTools 1.5.6 (Sanner 1999).
In vitro cytotoxicity evaluation on human fibroblasts
(
MRC-5 cell line)
Kollman united atom charges, solvation parameters, and
polar hydrogens were added to the receptor. Since the ligands
are not peptides, Gasteiger charges were assigned and non-
polar hydrogens were merged. ChemBioDraw (Evans 2014)
was used to draw and design the structures of arylsubstituted
imidazoles (Fig. 1) taking into account reference Planche et al.
(2009). ChemBio3D Ultra 12.0 (Evans 2014) was used to
optimize the geometry, running a MMFF94 energy minimiza-
tion of the 3D structures. The Gasteiger charge calculation
method (Gasteiger and Marsili 1980) was used and partial
charges were added to the ligand atoms prior to docking, via
AutodockTools.
MRC-5SV2 cells were cultured in MEM + Earl’s salts-medi-
um, supplemented with L-glutamine, NaHCO , and 5%
inactivated fetal calf serum. All cultures and assays were con-
ducted at 37 °C under an atmosphere of 5% CO . Assays were
performed in 96-well microtiter plates, each well containing
1
of MRC-5SV2 inoculum (1.5 × 10 cells/ml). Cell growth was
compared to untreated-control wells (100% cell growth) and
medium-control wells (0% cell growth). After 3 days incuba-
tion, cell viability was assessed fluorimetrically after addition
of resazurin (λex 550 nm, λem 590 nm). Tamoxifen was used
as positive control. The compound is classified non-toxic
when the IC50 is higher than 30 μM. Between 30 and
3
2
0 μl of the watery compound dilutions together with 190 μl
5
Identification of binding site residues
1
0 μM, the compound is regarded as moderately toxic.
The binding site residues for CYP51—T. cruzi, CYP51—T.
brucei, and CYP51—L. infantum were identified from the
analysis of 3KSW, 4BJK, and 3L4D available in the RCSB
Protein Data Bank. CYP51—L. infantum in the active site
involves the amino acids Leu355, Met357, Leu358, Met459,
Val212, Phe104, Met105, Tyr115, Ala114, Phe109, Gly282,
Met283, Phe289, Leu129, Ala290, and Val460 (Hargrove
et al. 2011), CYP51—T. cruzi involves the amino acids
Tyr103, Met106, Phe110, Tyr116, Leu127, Ala297, Phe290,
Ala291, Thr295, Leu356, Leu357, Met358, Met360, Val461,
Met460, and Ala287 (Lepesheva et al. 2010), and CYP51—T.
brucei involves the amino acids Tyr116, Tyr103, Met106,
Phe105, Phe290, Ala287, and Phe110 (Choi et al. 2013) ac-
cording to the references.
When the IC50 is lower than 10 μM, the compound is classi-
fied as highly toxic.
Molecular docking
Ligands and proteins preparation
Molecular modeling was performed using the high-performance
computing capabilities of the Cluster of Chemistry Department
of University of Oriente running the Linux operating system
Debian 8.0 distribution. The pdb file was prepared using the
software UCSF Chimera molecular graphic system, version
1.10.227 (Pettersen et al. 2004). Cytochrome P450 14α-sterol
demethylase, CYP51, of the species T. cruzi, T. brucei, and
L. infantum was selected: CYP51 of T. cruzi in complex with
an inhibitor VNF ((4-(4-chlorophenyl)-N-[2-(1H-imidazol-1-yl)-
Grid box preparation and docking
1-phenylethyl]benzamide)—PDB ID: 3KSW (CYP51—T. cruzi)
(
Lepesheva et al. 2010) with a resolution of 3.05 Å, CYP51 of
Docking experiment were performed between arylsubstituted
imidazoles and CYP51—T. cruzi and CYP51—L. infantum
proteins. AutoDock requires pre-calculated grid maps, one
for each atom type, present in the ligand being docked, as it
stores the potential energy arising from the interaction with the
macromolecule. This grid must surround the region of interest
(active site) in the macromolecule.
T. brucei bound to (S)-N-(3-(1H-indol-3-yl)-1-oxo-1-(pyridin-4-
ylamino)propan-2-yl)-3,3′-difluoro-(1,1′-biphenyl)-4-
carboxamide—PDB ID: 4BJK (CYP51—T. brucei) (Choi et al.
2
013) with a resolution of 2.67 Å and CYP51 of L. infantum in
complex with fluconazole—PDB ID: 3L4D (CYP51—L.
infantum) (Hargrove et al. 2011) with resolution of 2.75 Å. The
Table 2 Grid box parameters
selected for the target enzymes
Enzyme
x, y, z
Size
Spacing (Å)
coordinates of left of box
(points)
CYP51—T. cruzi
2.48
26.996
− 28.96
58.216
22.191
− 1.658
26.493
50 × 50 × 50
50 × 50 × 50
50 × 50 × 50
0.375
0.375
0.375
CYP51—L. infantum
CYP51—T. brucei
31.917
26.642

Parasitol Res
Grid box parameters (Table 2) were set by using
synthetic approaches is still desirable and much in demand.
The synthesized imidazole derivatives were characterized af-
ter recrystallization from an appropriate solvent by recording
their H-NMR, C-NMR, FT-IR, and MS spectra. The results
of the synthesis are summarized in Table 3.
AutoDockTools (ADT), a free graphic user interface of
MGL software packages (version 1.5.6rc3) (Sanner 1999).
The molecular docking program AutoDock version 4.2
1
13
(
Sanner 1999) was employed to perform the docking experiment.
The Lamarckian Genetic Algorithm was used during the process
to explore the best conformational space for the ligand with a
population size of 50 individuals. The maximum number of
generations and evaluations were set at 27,000 and 2,500,000,
respectively. Other parameters were set as default. After complete
execution of Autodock, 50 conformations of the ligands in com-
plex with the receptor were obtained, which were ranked based
on binding energy and inhibition constant (Ki).
To check the docking procedure and given the similar
structure (imidazole and triazole rings) between the com-
pounds here reported, we choose known CYP51 inhibitors,
VNF ((R)-N-(2-(1H-imidazol-1-yl)-1-phenylethyl)-4′-
chloro-[1,1′-biphenyl]-4-carboxamide) to CYP51—T. cruzi
According to the results shown in Table 3, all synthesized
compounds have a similar melting point and good yield com-
pared with the reference.
Spectral data results
(
5a) 4-(1-benzyl-4,5-diphenyl-1H-imidazol-2-yl)-N,N-
dimethylaniline. MW: 429.57 mp: 151–153 °C (Reported:
1
1
50–152 °C (Kantevari et al. 2006)). H-NMR (600 MHz,
DMSO-d ): δ ppm 8.10–7.18 (m, 19H, Ar-H), 5,12 (s, 2H,
6
1
3
CH ), 3.34 (s, 6H, CH × 2). C-NMR (150 MHz, DMSO-
2
3
d6): δ ppm 155.2, 150.42, 141.3, 137.78, 131.51, 129.38,
1
1
28.94, 128.75, 128.60, 128.49, 128.35, 127.48, 126.38,
25.61, 118.42, 111.99(Carom), 47.68 (CH ), 41.02 (CH x2).
(
Friggeri et al. 2014), ketoconazole (KET) for CYP51—T.
brucei (Hargrove et al. 2012), and VFV ((R)-N-(1-(3,4′-
difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phe-
nyl-1,3,4-oxadiazol-yl)benzamide) for CYP51—L. infantum
(
(
2
+
3
Mass spectrum (ES+). 430.23 [M + H] , 387.18, 340.18,
97.13.
5b) 4-(1-benzyl-4,5-diphenyl-1H-imidazol-2-yl) phenol.
MW: 402.49 mp: 136–139 °C (Reported: 135–138
2
(
Lepesheva et al. 2015), which were rebuilt and redocked
Fig. 2).
1
(Khalafi-Nezhad et al. 2016)). H-NMR (600 MHz, DMSO-
d ): δ ppm 9.76 (s, 1H, OH), 6.74–7.46 (m, 19H), 5.11 (s, 2H,
6
1
3
CH2), C-NMR (150 MHz, DMSO-d ): δ ppm 158.04,
6
Results and discussion
153.84, 141.40, 137.57, 136.52, 134.75, 130.88, 130.07,
1
29.63, 128.80, 128.55, 128.10, 127.18, 126.10, 125.70,
Synthesis and characterization of arylsubstituted
imidazoles
121.57, 115.46 (C ), 47.63(CH ). Mass spectrum (ES+).
arom
2
+
403.18 [M + H] , 313.13, 297.14.
5c) 1-benzyl-2,4,5-triphenyl-1H-imidazole. MW: 386.50
(
Since arylsubstituted imidazoles have become increasingly
useful and important in the pharmaceutical field, the develop-
ment of clean, high-yielding, and environmentally friendly
mp: 158–160 °C (Reported: 155–158 (Khalafi-Nezhad et al.
1
2016)). H-NMR (600 MHz, DMSO-d ): δ ppm 6.75–7.69
6
1
3
(m, 20H, Ar-H), 5,13 (s, 2H, CH2). C-NMR (150 MHz,
Fig. 2 Structure of control
compounds VNF (a), KET (b),
and VFV (c)
a
b
c

Parasitol Res
Table 3 Physical data and yields
of arylsubstituted imidazoles
a
Product
R
R
R
1
R
2
Yield
%)
M.P. (°C)
(observed)
M.P. (°C)
(literature)
(
b
c
5
a
Ph Ph Ph-CH
Ph Ph Ph-CH
Ph Ph Ph-CH
Ph Ph Ph-CH
Ph Ph m-CH
Ph Ph o-CH
Ph Ph m-CH
Ph Ph p-CH
Ph Ph p-SO
Ph Ph
Ph Ph Fu-CH
2
p-N(CH
p-OH
H
3
)
2
89
88
90
91
89
86
89
88
88
84
87
151–153
136–139
158–160
165–168
165–166
235–236
221–223
219–222
109–112
255–257
244–245
150–152
5
b
2
135–138
c
5
c
2
155–158
c
5
d
2
p-OCH
3
167–169
5
e
3
-C
-C
-C
-C
H-C
6
H
5
-
m-NO
2
–
–
–
5
f
3
6
H
5
-
p-NO
p-NO
p-NO
2
5
g
3
H
6 5
-
2
2
b
5
h
3
6
H
5
-
219–220
5
i
3
6
H
5
p-N(CH
3
)
2
2
–
256–259
–
d
5
j
–
p-N(CH
3
)
5
k
2
4-OH,
3
3
-CH O
5
l
Fu Fu
Fu Fu
–
–
4-OH,
3
83
189–190
–
-CH
3
O
e
5
m
C H
6 5
82
89
196–198
155–158
197–198
5
n
Fu Fu Fu-CH
2
p-OH-C
6
H
5
–
a
b
c
d
e
Indicated yields refer to isolated products
Kantevari et al. (2006)
Khalafi-Nezhad et al. (2016)
Eidi et al. (2016)
Kang et al. (2011)
1
DMSO-d ) δ ppm 153.04, 141.33, 137.20, 137.36, 133,58,
found). H-NMR (600 MHz, DMSO-d ): δ ppm 7.12–8.69
6
6
3
H
1
1
1
31.12, 130.43, 129.7, 129.10, 127.49, 126.94, 126.18,
(m, 18H, Ar-H), 2.33 (s, 3H, CH3). C-NMR (150 MHz,
DMSO-d ): 149.63, 148.89, 144.32, 141.74, 137,36, 135.60,
132.30, 131.72, 130.36, 129.65, 129.53, 127.68, 126.93,
126.65, 124.10, 122.36, 117.76(Carom), 17.57(CH ). IR
(KBr): υmax3080(C=Carom), 1685(C=N), 1580(N-O),
1500(C=C ), 1345(C-NO ). δ
Ho-sust) cm . Mass spectrum (ES+). 432.17 [M + H]
387.18, 342.12, 297.14.
(5g) 2-(4-nitrophenyl)-4,5-diphenyl-1-(m-tolyl)-1H-imid-
azole. MW: 431.50 mp: 221–223 °C (Reported: none report-
25.67, 125.26 (C ), 47.73 (CH ). Mass spectrum (ES+
arom
2
6
+
)
. 387.18 [M + H] 297.13.
5d) 1-benzyl-2-(4-methoxyphenyl)-4,5-diphenyl-1H-im-
idazole. MW: 416.52 mp: 165–168 °C (Reported: 167–169
(
3
1
(
Khalafi-Nezhad et al. 2016)). H-NMR (600 MHz, DMSO-
860(C-NAr-NO2), 740(C-
arom
−
2
max
1
+
d ): δ ppm 6,75–8.01 (m, 17H, Ar-H), 5.65 (s, 2H, CH ),
6
H
2
1
3
3.81 (s, 3H, OCH3). C-NMR (150 MHz, DMSO-d6):
159.66, 153.70, 147.20, 141.30, 138.82, 137.49, 134.30,
130.70, 129.80, 129.60, 128.59, 127.66, 125.63, 123.20,
114.15(Carom), 55.26(CH ), 47.66(CH ). Mass spectrum
1
ed found). H-NMR (600 MHz, MeOH-d ): δ ppm 7.33–
8.31 (m, 12H, Ar-H), 2.21 (s, 3H, CH ). C-NMR
(150 MHz, DMSO-d ): 149.63, 148.89, 144.32, 141.74,
3
2
4
H
+
13
(
ES+). 417.19 [M + H] 327.14, 221.14.
5e) 2-(3-nitrophenyl)-4,5-diphenyl-1-(m-tolyl)-1H-imid-
3
(
6
azole. MW: 431.50 mp: 165–166 °C (Reported: none report-
137,36, 135.60, 132.30, 131.72, 130.36, 129.65, 129.53,
127.68, 126.93, 126.65, 124.10, 122.36, 117.76(Carom),
1
ed found). H-NMR (600 MHz, DMSO-d ): δ ppm 7.10–
6
H
1
3
8
.80 (m, 18H, Ar-H), 2,35 (s, 3H, CH ). C-NMR
17.57(CH ). IR (KBr): υ
3058 (C=Carom), 1650 (C=N),
3
3
max
(
150 MHz, DMSO-d ): 149.80, 148.73, 146.04, 145.00,
43.97, 139.15, 138.12, 136.04, 134.86, 132.72, 131.00,
30.08, 129.99, 129.57, 127.83, 126.07, 123.16, 122.19,
1599 (C=C ), 1335 (C-NO ), 853 (C-N
) cm . Mass spectrum (ES+). 432.17 [M + H] 342.12,
sust
179.09, 149.02.
), 767 (C-H
6
arom
2
Ar-NO2 m-
+
−1
1
1
1
18.82, 115.25(Ca r o m), 21.4 (CH ). IR (KBr):
(5h) 2-(4-nitrophenyl)-4,5-diphenyl-1-(p-tolyl)-1H-imid-
azole. mp: 219–222 °C (Reported: 219–220 °C (Kantevari
3
υmax3058(C=Carom), 169(C=N), 1575(N-O), 1460 y
1
8
H] 418.19, 342.12, 297.13, 221.13, 179.08.
5f) 2-(4-nitrophenyl)-4,5-diphenyl-1-(o-tolyl)-1H-imidaz-
ole. MW: 431.50 mp: 235–236 °C (Reported: none reported
1
502(C=Carom), 1335 (C-NO ). δ
853(C-NAr-NO2), 710 y
et al. 2006)). MW: 431.50. H-NMR (600 MHz, MeOH-
2
max
−1
10(C-H ) cm . Mass spectrum (ES+). 432.17 [M +
m-sust
d ): δ ppm 7.32–8.21 (m, 18H, Ar-H), 2.14 (s, 3H, CH ).
4
13
H
3
+
C-NMR (150 MHz, MeOH-d ): δ ppm, 148.81145.56,
4 H
(
139.16, 137.06, 133.29, 130.39, 129.72, 129.61, 129.52,
129.43, 128.92, 128.89, 127.28, 126.82, 125.21, 124.35,

Parasitol Res
Table 4 Antitrypanosomal, antileishmanial (IC50, μM ± SD), and cytotoxic (MRC-5) (CC50, μM ± SD) activities of arylsubstituted imidazoles.
Biological experiments were performed in duplicate
Products
MRC-5
T.b. brucei
T.b. rhod.
T. cruzi
L. infantum
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
1.56 ± 0.2
3.59 ± 0.41
39.85 ± 5.13
3.36 ± 0.34
35.21 ± 7.21
2.11 ± 0.11
4.21 ± 0.29
2.29 ± 0.21
72.22 ± 9.15
3.75 ± 0.33
4.37 ± 0.43
12.33 ± 2.34
32.00 ± 4.12
35.11 ± 5.89
16.23 ± 1.78
0.86 ± 0.06
37.11 ± 3.88
3.77 ± 0.29
44.11 ± 4.99
2.24 ± 0.31
5.17 ± 0.56
3.19 ± 0.31
14.56 ± 2.67
3.29 ± 0.33
4.37 ± 0.44
12.09 ± 1.33
11.99 ± 1.87
15.53 ± 2.19
24.89 ± 4.12
1.09 ± 0.01
35.12 ± 5.88
4.07 ± 0.51
34.13 ± 4.87
2.07 ± 0.24
1.93 ± 0.23
1.80 ± 0.43
17.66 ± 3.2
4.31 ± 0.51
0.96 ± 0.03
3.78 ± 0.02
18.19 ± 0.20
16.99 ± 1.77
3.81 ± 0.45
1.02 ± 0.02
> 64.0
> 64.0
38.62 ± 5.33
> 64.0
10.23 ± 1.16
> 64.0
2.29 ± 0.31
2.03 ± 0.14
1.12 ± 0.06
51.09 ± 4.76
4.66 ± 0.32
4.11 ± 0.42
3.67 ± 0.12
13.22 ± 1.22
14.67 ± 1.11
3.23 ± 0.33
10.2 ± 1.44
2.07 ± 0.21
8.20 ± 0.81
1.99 ± 0.19
40.89 ± 6.21
4.11 ± 0.51
4.07 ± 0.42
16.44 ± 1.77
64.99 ± 8.81
48.23 ± 5.51
25.47 ± 2.45
g
h
i
j
k
l
m
n
Tamoxifen
Suramin
0.03 ± 0.01
0.04 ± 0.01
Benznidazole
Miltefosine
2.89 ± 0.56
10.07 ± 4.23
T. cruzi, Trypanosoma cruzi; L. infantum, Leishmania infantum; T.b. brucei, Trypanosoma b. brucei; T.b. rhod., Trypanosoma b. rhodesiense; MRC-5,
human fetal lung fibroblast cell line
(
Carom). 22.15(CH ). IR (KBr): υmax 3050 (C=Carom), 1615
(150 MHz, DMSO-d ): 151.35, 145.87, 143.10, 139.60,
3
6
(
C=N), 1500 (C=Carom), 1338 (C-NO ), δ
860 (C-NAr-
max
136.40, 130.69, 130.09, 128.72, 128.53, 128.31, 127.40,
2
−1
NO2), 840 (C-H
) cm . Mass spectrum (ES+). 432.17
p-sust
126.88, 126.3, 126.21, 113.93, 112.49 (Carom), 40.01
+
−
[
M + H] 342.12, 301.14, 179.09, 149.02, 137.08.
(CH x2) ppm. Mass spectrum (ES−). 495.14 [M-H] ,
3
(
5i) 4-(2-(4-(dimethylamino)phenyl)-4,5-diphenyl-1H-
338.17, 214.01, 172, 155.
imidazol-1-yl)benzenesulfonic acid. MW: 495.60 mp: 109–
(5j) 4-(4,5-diphenyl-1H-imidazol-2-yl)-N,N-
dimethylaniline. MW: 339.44 mp: 255–257 °C (Reported:
256–259 °C (Eidi et al. 2016)). H-NMR (600 MHz,
1
1
12 °C (Reported: none reported found). H-NMR
1
(
7
600 MHz, DMSO-d ): δH ppm 9.66 (s, 1H, OH), 6.47–
6
1
3
.97(m, 18H, Ar-H), 2.99 (s, 6H, CH × 2). C-NMR
DMSO-d ): δ ppm 12.36 (s, 1H, NH), 6.80–7.97 (m, 14H,
3
6
H
Table 5 Selectivity index (SI) of
the arylsubstituted imidazole
from T. cruzi, L. infantum, T.b.
brucei, and T.b. rhodesiense
Products
MRC-5/T.b. brucei
MRC-5/T.b. rhod.
MRC-5/T. cruzi
MRC-5/L. infantum
5a
0.43
> 1.61
11.5
1.81
> 1.72
10.2
1.43
> 1.82
9.49
> 1.87
1.11
1.53
> 1
5
b
5
c
3.78
> 1
5
d
> 1.81
1.09
0.48
0.49
0.71
1.24
0.94
0.29
0.41
0.42
0.20
> 1.45
1.02
0.39
0.35
3.50
1.42
0.94
0.30
1.10
0.94
0.13
5
e
1.11
0.24
0.56
1.25
1.13
1
5
f
1.05
0.62
2.89
1.08
4.28
0.97
0.73
0.86
0.85
5
g
5
h
5
i
5
j
5
k
0.22
0.20
0.30
0.13
5
l
5
m
5
n

Parasitol Res
Table 6 Binding energy and
inhibition constant of the
arylsubstituted imidazoles and
control CYP51 inhibitors VNF,
KET, and VFV
Test comp.
CYP51—T. cruzi
ΔG (kcal/mol)
CYP51—L. infantum
ΔG (kcal/mol)
CYP51—T. brucei
Ki
Ki
ΔG
Ki
(nM)
(nM)
(kcal/mol)
(nM)
5
a
− 9.91
− 9.98
− 9.61
− 10.41
− 13.32
− 13.63
− 13.80
− 13.49
− 14.54
− 9.48
− 9.25
− 7.24
− 7.31
− 9.59
− 9.65
–
54.32
48.54
90.61
23.31
0.17
− 11.13
− 10.37
− 10.74
− 10.95
− 12.88
− 9.22
− 9.40
− 8.44
− 6.39
− 9.84
− 10.61
− 7.75
− 8.19
− 10.26
–
7.00
25.10
13.31
9.46
− 10.58
− 9.84
− 9.76
− 9.97
− 14.47
− 14.75
− 14.01
− 12.56
− 10.58
− 8.68
− 10.15
− 8.43
− 9.07
− 9.83
–
17.68
61.38
69.82
49.08
0.02
5
b
5
c
5
d
5
e
0.36
5
f
0.10
175.52
128.58
649.09
20,800
61.34
16.18
2080
1000
29.97
–
0.02
5
g
0.08
0.05
5
h
0.13
0.62
5
i
0.02
17.55
437.39
36.02
665.25
226.01
62.73
–
5
j
112.65
167.21
4950
4370
93.98
85.03
–
5
k
5
l
5
m
5
n
VNF
VFV
KET
− 10.29
–
28.60
–
–
–
–
–
− 11.37
4.60
1
3
1
Ar-H), 2.99 (s, 6H, CH x2). C-NMR (150 MHz, DMSO-
(Reported: none reported found). H-NMR (600 MHz,
DMSO-d ): δ ppm 9.28 (s, 1H, OH), 6.85–7.72 (m, 16H,
3
d ): δ ppm 154.21, 150.36, 146.80, 128.44, 127.84, 126.56,
6
H
6
H
1
3
1
18.60, 112.03, 111.11(Carom), 43.45 (CH x2). Mass spec-
Ar-H, Furanyl), 4.46 (s, 2H, CH ), 3.83 (s, 3H, CH ). C-
2 3
NMR (150 MHz, DMSO-d ): δH ppm 148.72, 147.77,
147.22, 146.08, 142.06, 138.62, 128.50, 128.74, 127.82,
127.52, 127.15, 121.77, 118.52, 115.70, 112.08, 110.92,
3
+
trum (ES+). 340.18 [M + H] , 297.14.
5k) 4-(1-(furan-2-ylmethyl)-4,5-diphenyl-1H-imidazol-2-
yl)-2-methoxyphenol. MW: 422.48 mp: 244–245 °C
6
(
Fig. 3 Superimposition of the best poses of the ligand + control drugs for CYP51—T. cruzi in a 5i (tan) + VNF (sky blue) and b 5f (tan) + KET (sky blue)
for CYP51—T. brucei included in the binding pocket

Parasitol Res
5e
5f
5g
5h
Fig. 4 Similar interaction between ligands 5e, 5f, 5g, and 5h with the enzyme CYP51—T. brucei
1
05.42, 109.46(Carom), 55.79 (CH ), 40.01 (CH ). Mass spec-
(5n) 4-(4,5-di(furan-2-yl)-1-(furan-2-ylmethyl)-1H-imidazol-
2-yl)phenol. MW: 392.46 mp: 155–158 (Reported: none report-
3
2
+
trum (ES+). 423.17 [M + H] , 343.14, 297.14.
5l) 4-(4,5-di(furan-2-yl)-1H-imidazol-2-yl)-3-
1
(
ed found). H-NMR (600 MHz, DMSO-d ): δ ppm 9.79 (s,
6
H
methoxyphenol. MW: 276.30 mp: 189–190 °C (Reported: none
1H, Ar-OH), 3.42 (s, 2H, CH ), 6.87–7.92 (m, 13H, Ar-H,
2
1
13
reported found). H-NMR (600 MHz, DMSO-d ): δ ppm 3.87
Furanyl). C-NMR (150 MHz, DMSO-d ): δ ppm 158.50,
6
H
6
H
(
s, 3H, CH3), 9.33 (s, 1H, OH), 6.57–7.81 (m, 9H, Ar-H,
157.91, 153.67, 151.30, 146.19, 142.9, 142.1, 130.75, 128.48,
127.80, 126.98, 121.73, 115.96, 107.70(Carom), 37.80 (CH2).
Mass spectrum (ES+). 373.12 [M + H] 293.09, 277.10.
13
Furanyl), 12.62 (s, 1H, NH). C-NMR (150 MHz, DMSO-
d ): δ ppm 144.71, 142.46, 141.80, 129.33, 128.97, 121.29,
+
6
H
1
20.91, 118.91, 115.67, 112.81, 111.45, 108.18, 106.79(Carom),
+
55.84(CH ). Mass spectrum (ES+). 323.10 [M + H] , 293.09,
3
201.08.
Antiprotozoal and cytotoxic effects
(
5 m) 4,5-di(furan-2-yl)-2-phenyl-1H-imidazole. MW:
2
2
76.30 mp: 196–198 (Reported: 197–198 °C (Kang et al.
The antiparasitic activities and the cytotoxicity of the test and
reference compounds are listed in Table 4. All compounds were
tested in vitro against the tripomastigotes stage of T.b. brucei and
T.b. rhodesiense and amastigote stage of T. cruzi and L. infantum.
The cytotoxicity of all arylsubstituted imidazoles against MRC-5
cell lines (human lung fibroblasts) was evaluated and the
1
011)). H-NMR (600 MHz, DMSO-d ): δ ppm 6.58–8.10
6
H
1
3
(
(
m, 11H, Ar-H, Furanyl), 12.87 (s, 1H, NH). C-NMR
150 MHz, DMSO-d ): δH ppm 149.34, 146.32, 144.48,
6
142.69, 141.94, 129.89, 128.78, 119.42, 111.96, 106.98(Carom).
+
Mass spectrum (ES+). 277.09 [M + H] , 201.07.

Parasitol Res
Fig. 5 Superimposition of the
best pose of the 5e (tan) + VFV
(sky blue) included in the binding
pocket of CYP51—L. infantum
a
b
Fig. 6 Docked structures of compound 5d in CYP51—T. cruzi; a two-dimensional representations of interaction by the Ligplot software (Wallace et al.
996); b three-dimensional representation of the interaction the ligand (tan) and Heme group (cyan)
1

Parasitol Res
selectivity indices (SI) (SI = CC50 MRC-5 cells/IC50 protozoa)
were calculated (Table 5) (Ehata et al. 2012).
Ligands 5k and 5l have a similar structure in position two of
the imidazoles with a phenyl group joined to a hydroxyl (4-
The imidazoles 5a, 5c, 5e, 5f, 5g, 5i, 5j, 5k, and 5n exhib-
ited IC50 values lower than 5 μM against T. cruzi. The ligands
OH) substituent and a methoxyl (3-CH O) group.
3
None of studied arylsubstituted imidazoles showed a
higher activity than the reference drug suramin towards T.b.
rhodesiense, demonstrating the lower sensitivity for this class
of compounds (De Vita et al. 2016). Only the ligand 5a
(IC = 0.86 μM) possessed an IC value comparable with
5
l, 5m, and 5h had a moderate activity with IC50 values of
1
8.19, 16.99, and 17.66 μM, respectively. Only the ligands 5d
and 5b, both with similar structure (Table 1) substituted in
position two of the arylsubstituted imidazole by a hydroxyl
5
0
50
(
OH) and a methoxy (OCH ), showed a low activity. The most
the control drug suramin.
3
active compounds 5a, 5f, 5g, and 5j showed lower IC values
Compounds 5a, 5c, 5e, 5f, 5g, 5i, and 5j presented similar
antileishmanial activity compared with the control drug
miltefosine, but they showed a pronounced cytotoxicity on
MRC-5 cells (Table 4), indicating the lack of selectivity to-
wards L. infantum (Table 5).
5
0
than the reference compound benznidazole (Table 4).
However, only compound 5c with an IC50 of 4.07 μM pre-
sented a good selectivity towards T. cruzi with a SI of 9.49
(
Table 5).
On the other hand, against T.b. brucei and T.b. rhodesiense,
Compound 5c had the highest selectivity to the
Trypanosoma family with a SI of 9.49, 11.5, and 10.2 for
T. cruzi, T.b. brucei, and T.b. rhodesiense, respectively
(Table 5).
the active compounds were 5a, 5c, 5e, 5f, 5g, 5i, and 5j. The
most active compound against T.b. brucei was 5e (IC50
.11 μM), while for T.b. rhodesiense the most active com-
pound was 5a with an IC = 0.86 μM. Both compounds were
=
2
5
0
less active than the reference compounds suramin for T.b.
brucei (0.03 μM) and T.b. rhodesiense (0.04 μM). For T.b.
rhodesiense, the ligands with a good activity were 5h, 5k, 5l,
and 5m, with IC50 values between 11.99 and 15.53 μM.
Molecular docking
The procedure adopted for the molecular docking was used
for all designed arylsubstituted imidazoles (Table 1) with each
a
b
Fig. 7 Docked structures of compound 5g in the CYP51—T. cruzi; a 2D interaction by the Ligplot software; b 3D representation of the interaction the
ligand (tan) and the heme group (cyan)

Parasitol Res
target selected. The docked poses for each of the compounds
have been evaluated and the pose with the lowest binding free
energy and inhibition constant was thereby chosen (Table 6).
The lowest binding free energy (i.e., best docking score)
and inhibition constant indicates the highest ligand/protein
affinity. The docking studies were done in comparison with
control drugs (i.e., known inhibitors of the target enzymes).
The control drugs were redocked with each protein target cho-
sen, VNF for CYP51—T. cruzi, VFV for CYP51—L.
infantum, and KET for CYP51—T. brucei.
12.56 kcal/mol, respectively, and Ki values very low com-
pared with the KET control. The best ligand 5f showed sim-
ilar conformation respect to KET control (Fig. 3b) and the
ligands 5e, 5g, and 5h presented similar values (ΔG and Ki)
to ligand 5f, due to the same interaction with the Heme group
through the oxygen presented in the NO group (Fig. 4).
2
For enzyme CYP51—L. infantum with the control drug
VFV, the binding energy was found to be − 10.29 kcal/mol
with a Ki = 28.60 nM. The values of VFV are only exceeded
by ligands 5a, 5b, 5c, 5d, 5e, 5k were found to have − 11.13,
− 10.37, − 10.74, − 10.95, − 12.88, and − 10.61, respectively,
for the binding energy and Ki values very low respect to the
VFV control (Table 6). The best ligand was 5e and we can
observe in Fig. 5 the superimposition between VFV and 5e,
where both ligands interacted directly with the Heme group.
It should be noted that ligand 5e appeared to bind in
the three receptor enzymes (CYP51—T. cruzi, CYP51—
L. infantum, CYP51—T. brucei) and in all cases this
ligand had lower ΔG values when compared to the
control drugs. Some similar behavior occurs with the
ligands 5a and 5k binding to CYP51—L. infantum and
CYP51—T. brucei and 5f, 5g, 5h, and 5i binding with
CYP51—T. cruzi and CYP51—T. brucei. All of them
show better binding values than the control drugs. Our
molecular docking studies revealed that all compounds
(5a–n) in one way or another interacted with the 14 α-
As shown in Table 6, the binding energies (ΔG) and inhi-
bition constant (Ki) of the control drug VNF in the docking
with CYP51—T. cruzi were found to be − 9.65 kcal/mol and
8
5.03 nM, respectively. However, the value for almost de-
signed imidazoles was found to be higher than the VNF con-
trol, with ΔG value between − 9.25 and − 14.54 kcal/mol and
low values of Ki. The ligand 5i shows the better values (ΔG =
−
14.54 kcal/mol and Ki = 0.02 nM) and a similar conforma-
tion compared with the VNF control (Fig. 3a), other ligands
showed better values and only the ligands 5l and 5m showed
not accepted values of binding energies and inhibition con-
stant compared with the control.
In the case of CYP51—T. brucei, the binding energy (ΔG)
of the control drug ketoconazole was − 11.37 kcal/mol (Ki =
4
.60 nM), exceeded only by the ligands 5e, 5f, 5g, and 5h with
a binding energy of − 14.47, − 14.75, − 14.01, and −
a
b
Fig. 8 Docked structures of compounds 5i (a) and 5l (b) in the CYP51—T. cruzi pocket represented using the Ligplot software

Parasitol Res
demethylase enzyme for each species (CYP51—T. cruzi,
CYP51—L. infantum, CYP51—T. brucei) by hydropho-
bic interactions or azole-Heme coordination and π-π
and π-cation interactions.
hydrophobic interaction with Ala291, Ala287, Phe290,
Phe110, Met106, Tyr116, Tyr103, Pro99, His420,
Leu356, and Thr295 similar as seen with the ligands
5a, 5b, 5c, 5d, 5j, 5k, 5m, and 5n where they share
the same residues involved in the active site.
This study with CYP51—T. cruzi demonstrated that in
some ligands (5a, 5b, 5c, 5d, 5j, 5k, 5m, and 5n) there are
hydrophobic interactions with the Heme group (Fig. 6).
We see also hydrogen bond interactions with amino acid
residue Arg361 (5b, 5d, and 5n). All ligands showed hydro-
phobic interaction with Ala291, Ala287, Phe290, Phe110,
Met106, Tyr116, Tyr103, Pro99, His420, Leu356, and
Thr295. They shared some residues Ala291, Ala287,
Phe290, Phe110, Met106, Tyr116, Tyr103, Leu356, and
Thr295 involved in the active site according to Lepesheva
et al. (2010). The ligands 5e, 5f, 5g, and 5h showed an elec-
In the case of CYP51—L. infantum, only the ligands 5e and
5h showed interaction between one of the NO group’s oxy-
2
gens (O32) with the heme group (Fig. 9a) and in the ligand 5i
complex the interaction was with the SO H group’s oxygen
3
atoms (Fig. 9b).
On the other hand, the ligands 5a, 5b, 5c, 5d, 5j, 5k, 5l, 5m,
and 5n were found to have hydrophobic interactions with
Met359, Val212, Pro209, Me459, Val356, Met357, Val460,
Thr294, Ala290, Leu355, Phe109, Met105, Phe289, Tyr115,
Tyr102, Phe104, and Hem481 (Fig. 10). These ligands share
some residues Leu355, Met357, Val212, Phe104, Met105,
Tyr115, Phe109, and Ala290 involved in the active site ac-
cording to Hargrove et al. (2011).
Compounds 5a, 5b, 5c, 5d, 5i, 5j, 5k, 5l, and 5m were found
to establish hydrophobic interactions with CYP51—T. brucei
(Fig. 11) that involved the residues Val461, Ala291, Phe290,
Thr295, His294, Phe105, Tyr103, Met106, Tyr116, Leu127,
tronic interaction between the NO group (electron pair of
2
2
+
oxygen O33) and the Fe of Hem488 group (Fig. 7); however
for ligand 5i and 5l, the interaction with the Hem488 is with
SO H (electron pair of O20) for 5i and OCH (electron pair of
3
3
O25) for 5l (Fig. 8).
The ligands 5e, 5f, 5g, and 5h also showed the in-
teraction with the heme group. We could observe the
a
b
Fig. 9 Docked structures of compounds 5e (a) and 5i (b) in the CYP51—L. infantum pocket represented using the Ligplot software

Parasitol Res
a
b
Fig. 10 Docked structures of compound 5b in the CYP51—L. infantum; a 2D interaction by Ligplot software, b 3D representation of the interaction the
ligand (tan) and Heme group (cyan)
Ala115, Ala287, Phe110, Hem1450, and His294. All those com-
pounds (5a, 5b, 5c, 5d, 5i, 5j, 5k, 5l, and 5m) share some
residues: Tyr116, Tyr103, Met106, Phe105, Phe290, Ala287,
and Phe110 involved in the active site (Choi et al. 2013).
a
b
Fig. 11 Docked structures of compound 5a in the CYP51—T. brucei; a 2D interaction by Ligplot software, b 3D representation of the interaction the
ligand (tan) and Heme group (cyan)

Parasitol Res
CBC
O
CMC
C
CAC
SG
C3CC2C
CA
Cys422
C1C
CBB
CAB
N
C4C
CB
CMD
CHD
CHC
2
.28
C1D
NC
C4B
Ala291
ND
FE
NB
C3B
C2D
C3D
C2B
CAD
2.46
CMB
C4D
NA
C1B
CHA
CH2B.44
O1D
Phe290
CBD
His294
C1A
C4A
C2 AC 3A
O01
CGD
Hem1450
O2D
CAA
CMA
C03
C17
C08
O16
Val461
C18
CBA
O2A
C04
C07
C15
C19
O1 CA GA
C05
C06
C14
C09
Thr295
N13
N10
Leu356
C11C271
C24
C12
Met358
C25
C29
C20
O21
C23
Met460
O26
C28
C22
C27
Leu208
Phe105
Met106
a
b
Fig. 12 Docked structures of compounds 5f (a) and 5n (b) in the CYP51—T. brucei pocket represented using the Ligplot software
On the other hand, the ligands 5e, 5f, 5g, 5h, and 5n were
observed to interact with the Heme group through the NO2
function for 5e, 5f, 5g, 5h and with OH for 5n (Fig. 12).
These results reveal that the position of the substituent in
the phenyl joined to the imidazole ring in the four positions
affected strongly the orientation and the subsequently interac-
tion of the imidazoles with the Heme iron atom in the binding
site of the enzyme.
identified between oxygen fundamentally present in NO ,
2
2
+
SO H, and OH groups and the Fe ion of the Heme group.
3
The reported results can partly justify and support the possible
use of some of these imidazoles for the treatment of parasitic
diseases such as sleeping sickness, leishmaniasis, and in some
extent American trypanosomiasis named Chagas disease, with
no apparent toxic effects in patients.
Acknowledgements This work has been supported by the Belgian
Development Cooperation through VLIR-UOS (Flemish Interuniversity
Council-University Cooperation for Development) in the context of the
Institutional University Cooperation programme with Universidad de
Oriente, Santiago de Cuba, Cuba started in 2013.
Conclusions
In conclusion, the present investigation has described the
chemical synthesis and antiprotozoal, cytotoxic activities of
several arylsubstituted imidazoles as well as their molecular
docking in the active site of CYP51 of those protozoa. The
reported results showed that all samples possessed interesting
in vitro antiprotozoal activity at different extents. The ligand
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