In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against cutaneous, mucocutaneous and visceral Leishmania species

Article abstract of DOI:10.1016/j.exppara.2017.11.009

The identification of specific therapeutic targets and the development of new drugs against leishmaniasis are urgently needed, since chemotherapy currently available for its treatment has several problems including many adverse side effects. In an effort to develop new antileishmanial drugs, in the present study a series of 28 N-benzyl-1H-benzimidazol-2-amine derivatives was synthesized and evaluated in vitro against Leishmania mexicana promastigotes. Compounds 7 and 8 with the highest antileishmanial activity (micromolar) and lower cytotoxicity than miltefosine and amphotericin B were selected to evaluate their activity against L. braziliensis 9and L. donovani, species causative of mucocutaneous and visceral leishmaniasis, respectively. Compound 7 showed significantly higher activity against L. braziliensis promastigotes than compound 8 and slightly lower than miltefosine. Compounds 7 and 8 had IC₅₀ values in the micromolar range against the amastigote of L. mexicana and L. braziliensis. However, both compounds did not show better activity against L. donovani than miltefosine. Compound 8 showed the highest SI against both parasite stages of L. mexicana. In addition, compound 8 inhibited 68.27% the activity of recombinant L. mexicana arginase (LmARG), a therapeutic target for the treatment of leishmaniasis. Docking studies were also performed in order to establish the possible mechanism of action by which this compound exerts its inhibitory effect. Compound 8 shows promising potential for the development of more potent antileishmanial benzimidazole derivatives.

Full text of DOI:10.1016/j.exppara.2017.11.009

Experimental Parasitology xxx (2017) 1e8
Contents lists available at ScienceDirect
Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr
In vitro activity of new N-benzyl-1H-benzimidazol-2-amine
derivatives against cutaneous, mucocutaneous and visceral Leishmania
species
,
e
b
b
Rocío Nieto-Meneses a , Rafael Castillo , Alicia Hernandez-Campos ,
ꢀ
b
b
b
ꢀ
Armando Maldonado-Rangel , Jeferson B. Matius-Ruiz , Pedro Josue Trejo-Soto ,
Benjamín Nogueda-Torres , Ma. Auxiliadora Dea-Ayuela , Francisco Bolas-Fernandez ,
a
c
d
ꢀ
ꢀ
b
e, *
ꢀ
ꢀ
ꢀ
Carlos Mendez-Cuesta , Lilian Yepez-Mulia
a Departamento de Parasitología, ENCB-IPN, 11340 Mexico City, Mexico
b Departamento de Farmacia, Facultad de Química, UNAM, 04510 Mexico City, Mexico
c Departamento de Farmacia, Facultad de Ciencias de la Salud, Universidad CEU-Cardenal Herrera, Avda. Seminario s/n, 46113 Moncada, Spain
d Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid Spain
e
ꢀ
ꢀ
Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias-Pediatría, Instituto Mexicano del Seguro Social, 06720 Mexico City, Mexico
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ A series of 28 N-benzyl-1H-benzimi-
dazol-2-amine
synthesized.
derivatives
were
ꢀ Compounds 7 and 8 were very active
against L. mexicana promastigotes
and amastigotes.
ꢀ Compound 8 had high selectivity in-
dex against L. mexicana and
L. braziliensis.
ꢀ Compound
8 inhibited 68.27% the
activity of recombinant Leishmania
mexicana Arginase.
ꢀ Compound 8 is a good scaffold for the
development of new antileishmanial
agents.
a r t i c l e i n f o
a b s t r a c t
The identification of specific therapeutic targets and the development of new drugs against leishmaniasis
are urgently needed, since chemotherapy currently available for its treatment has several problems
including many adverse side effects. In an effort to develop new antileishmanial drugs, in the present
study a series of 28 N-benzyl-1H-benzimidazol-2-amine derivatives was synthesized and evaluated
in vitro against Leishmania mexicana promastigotes. Compounds 7 and 8 with the highest antileishmanial
activity (micromolar) and lower cytotoxicity than miltefosine and amphotericin B were selected to
evaluate their activity against L. braziliensis 9and L. donovani, species causative of mucocutaneous and
Article history:
Received 5 July 2017
Received in revised form
20 October 2017
Accepted 25 November 2017
Available online xxx
visceral leishmaniasis, respectively. Compound
L. braziliensis promastigotes than compound 8 and slightly lower than miltefosine. Compounds 7 and 8
had IC50 values in the micromolar range against the amastigote of L. mexicana and L. braziliensis.
7 showed significantly higher activity against
Keywords:
Benzimidazole derivatives
Antileishmanial activity
ꢀ
* Corresponding author. UIMEIP-Pediatría, Centro Medico Nacional Siglo XXI,
ꢀ
Instituto Mexicano del Seguro Social, Av. Cuauhtemoc 330 Col. Doctores. 06720,
Mexico City, Mexico.
ꢀ
E-mail address: lilianyepez@yahoo.com (L. Yepez-Mulia).
https://doi.org/10.1016/j.exppara.2017.11.009
0014-4894/© 2017 Published by Elsevier Inc.
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

2
R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
Selectivity Index
Arginase
However, both compounds did not show better activity against L. donovani than miltefosine. Compound 8
showed the highest SI against both parasite stages of L. mexicana. In addition, compound 8 inhibited
68.27% the activity of recombinant L. mexicana arginase (LmARG), a therapeutic target for the treatment
of leishmaniasis. Docking studies were also performed in order to establish the possible mechanism of
action by which this compound exerts its inhibitory effect. Compound 8 shows promising potential for
the development of more potent antileishmanial benzimidazole derivatives.
© 2017 Published by Elsevier Inc.
1. Introduction
be good candidates as antileishmanial agents (Keurulainen et al.,
2015); however, this kind of compounds have high carboaromatic
Leishmaniasis is a disease caused by several species of the genus
Leishmania, having a wide range of hosts, including man. It is
considered a neglected tropical disease (NTDs), affecting mainly
developing countries. The World Health Organization (WHO) esti-
mates that approximately 350 million people are living in areas
characterized by active transmission of Leishmania, with 14 million
people directly affected by the disease. There are three main types
of leishmaniasis: cutaneous (CL), the most common form of the
disease, causes ulcers, leading to disfigurement, permanent scars
and in some cases disability; mucocutaneous (MCL), the most
destructive form of the disease, causes partial or total mutilation of
mucous membranes in the nose, mouth and throat; and visceral
kalaeazar (VL), the most severe form of the disease, fatal if left
untreated. It is estimated that there are 300,000 cases of VL and
more than 20,000 to 40,000 annual deaths from this form of the
disease; in the case of CL, it has been reported over one million
cases around the world in the last 5 years (WHO, 2016).
The main drugs available for the treatment of leishmaniasis are
the pentavalent antimonials (SbV), Sodium Stiboglucanate (Pen-
tostam), Meglumine Antimoniate (Glucantime), and Amphotericin
B (Fungizone). Amphotericin B is the first drug of choice for visceral
leishmaniasis in regions with high resistance to treatment with
SbV. Miltefosine is the most recent antileishmanial drug in the
market and the first effective oral treatment against VL, being
recommended as first line drug for childhood VL (Freitas-Junior
et al., 2012). Chemotherapy currently available for leishmaniasis
treatment is far from satisfactory and has several problems
including many adverse side effects, high costs and toxicity (Savoia,
2015; Varela-M et al., 2012). Furthermore, drug resistance to all
known antileishmanial drugs has been reported (Bhattacharya
et al., 2016; Fernandes et al., 2016: Mondelaers et al., 2016; Shaw
et al., 2016; Coelho et al., 2014; Kumar et al., 2014). Therefore, the
identification of specific therapeutic targets and the development
of new drugs are urgently needed.
rings and rigid systems, these properties are associated with low
solubility and pharmacokinetics/pharmacodynamics problems.
Besides, the target of these derivatives on Leishmania was not
characterized.
Among druggable targets of the parasite, important for parasite
survival and proliferation, is the enzyme arginase (ARG) that par-
~
ticipates in the polyamine pathway (Balana-Fouce et al., 2012). L-
ornithine, the amino acid from which polyamines are generated, is
produced from the hydrolysis of L-arginine by ARG. Inhibition of
ARG by N-hydroxyarginine (NOHA) reduces polyamine levels in
Leishmania amastigotes and parasite load (Iniesta et al., 2001).
Leishmania ARG shares 39e43% identity with human ARG (Ilari
et al., 2015); therefore, it is considered a therapeutic target for
the treatment of leishmaniasis.
Previously, we performed a virtual screening study of ZINC
database on the LmARG in order to find inhibitors. From this study,
compounds with N-benzyl-1H-benzimidazole-2-amine scaffold
ꢀ
were identified as potential LmARG inhibitors (Mendez-Cuesta
et al., 2012). Inspired in these results and continuing our search
for benzimidazole derivatives with antileishmanial activity, a new
series of N-benzyl-1H-benzimidazol-2-amine derivatives was syn-
thesized. The main features in these compounds are the substituent
at position 1 of the benzimidazole nucleus, from C4- alkyl groups to
hydrogen; the substitution in the benzenoid ring, with or without
chlorine; and a substituted benzyl group on the 2-amino to increase
the flexibility of the compounds. Nine compounds are not
substituted at positions 5 and/or 6 of the benzimidazole nucleus,
but the others have one or two chlorine atoms at positions 5 and 6.
These compounds will give information about how the activity is
affected with the different substitutions, especially, when positions
5 and 6 of the benzimidazole nucleus are substituted, since it is
known that these positions undergo the first step metabolism,
which could increase the half-life of a possible drug.
The biological activity of the new compounds was initially
evaluated against promastigotes of L. mexicana and those with the
highest antileishmanial activity and lower cytotoxicity than mil-
tefosine and amphotericin B were further tested against the pro-
mastigote and amastigote of L. braziliensis, and L. donovani as well as
L. mexicana amastigotes. In addition, the effect of compounds 7 and
8 on LmARG activity was also evaluated, and in order to know how
these benzimidazole derivatives inhibit the LmARG activity, addi-
tional in silico docking study was performed.
In this regard, benzimidazole derivatives are of wide interest
because of their biological activities and clinical applications
(Alhtar et al., 2016). Their use as antibacterial, antifungal, antima-
larial, antileishmanial as well as anti-inflammatory and anticancer
agents was reviewed by Keri et al. (2015). Our research group has
demonstrated the antiprotozoal activity of benzimidazole de-
rivatives against Giardia intestinalis, Entamoeba histolytica, Tricho-
monas vaginalis. L. mexicana and Trypanosoma cruzi (Melchor-
ꢀ
ꢀ
Doncel de la Torre et al., 2017; Velazquez-Lopez et al., 2016; Díaz-
ꢀ
Chiguer et al., 2012; Hernandez-Luis et al., 2010; Navarrete-
2. Materials and methods
ꢀ
Vazquez et al., 2001; Valdez et al., 2002). Among them, 2-(tri-
fluoromethyl)-1H-benzimidazole derivatives showed promising
in vitro activity against L. mexicana with IC50 values in the range of
2.1. Chemistry
ꢀ
4e24
m
M (Hernandez-Luis et al., 2010). The antileishmanial activity
All N-benzyl-1H-benzimidazol-2-amine derivatives 1e28 were
synthesized by our research group thought a reductive amination
method between 1H-benzimidazol-2-amines 29e39 and alde-
hydes 40e47. The structure of target compounds is shown in
of benzimidazole derivatives has also been demonstrated in other
ꢀ
studies (Mendez-Cuesta et al., 2016; Mota et al., 2014; Oh et al.,
2014). Recently, 2-arylbenzimidazole derivatives have proven to
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
3
Scheme 1. The final compounds were purified by recrystallization
and their structures were confirmed by 1H NMR and 13C NMR
spectroscopy and elemental analyses (see Supplementary
Information).
microplates and allowed to adhere for 24 h at 37 ꢂC in 5% CO2.
The culture medium was then replaced with different concentra-
tions of the compounds (100e0.8 mg/mL), followed by incubation
for another 24 h. Miltefosine and amphotericin B were included as
reference drugs. All assays were carried out in triplicate. Thereafter,
20 mL of a 2.5 mM resazurin solution were added to each well and
2.2. Parasites
the plates were incubated for another 3 h. The fluorescence emis-
sion was measured as described above. All assays were carried out
Leishmania species used: L. mexicana strain MNYC/BZ/62/M379,
L. braziliensis strain MHOM/BR/75/M2903 and L. donovani strain
MHOM/IN/80/DD. Promastigotes were grown in Schneider's Insect
Medium (Sigma-Aldrich) supplemented with 10% heat-inactivated
in triplicate. The half maximal cytotoxicity concentration (CC50
was determined by a Probit analysis.
)
Fetal Bovine Serum (FBS) and 100 U/mL of penicillin plus 100 mg/mL
2.5. Activity evaluation of compounds 7 and 8 against amastigotes
of Leishmania spp
of streptomycin (Sigma-Aldrich), in 25 mL culture flasks. Parasites
grown for 4 days and 7 days were used to evaluate the activity of
the synthesized compounds against promastigotes and intracel-
lular amastigotes, respectively.
The activity of compounds 7 and 8 was evaluated against
amastigotes of L. mexicana, L. braziliensis and L. donovani, species
representative of the three main clinical manifestations of
leishmaniasis.
The activity of compounds 7 and 8 was evaluated using the
resazurin method (Bilbao-Ramos et al., 2012). Briefly, 5 ꢁ 104
macrophages and stationary promastigotes at a 1:10 ratio in 200
2.3. Activity evaluation of benzimidazole derivatives against
promastigotes of Leishmania spp
The activity of all compounds was initially evaluated against
promastigotes of L. mexicana and those with the highest anti-
leishmanial activity and lower cytotoxicity than miltefosine and
amphotericin B were further tested against promastigotes of
L. braziliensis and L. donovani using the resazurin method previ-
ously described by Bilbao-Ramos et al. (2012). For this, 2.5 ꢁ 106
parasites/well were cultured in 96-well microliter plates, com-
pounds were dissolved in DMSO and diluted in the culture medium
m
L/well of culture medium were seeded and the plates were incu-
bated for 24 h at 33 ꢂC, 5% CO2 in humidity chamber. The temper-
ature was then increased to 37 ꢂC for another 24 h. The cells were
then washed several times to remove free non-infective promas-
tigotes, and the final washing medium was replaced with 200
well of culture medium containing 2-fold serial dilutions of the
benzimidazole compound ranging from 100 to 0.8 g/mL. In these
mL/
m
at concentrations ranging from 100 to 0.8
m
g/mL in a final volume of
assays, miltefosine was used as reference drug since it is used for
the treatment of all three forms of leishmaniasis (Vakil et al., 2015).
Amphotericin B was also used as reference drug for L. mexicana
amastigote. After incubation of the plates for 48 h at 37 ꢂC in 5%
CO2, the culture medium was replaced with an equal volume of
lysis solution (Schneider's with 0.048% HEPES and 0.01% SDS) and
maintained at room temperature for 20 min. The lysis solution was
then replaced with Schneider's medium followed by incubation at
26 ꢂC for another 3 days to allow transformation of viable amasti-
gotes into promastigotes and their subsequent proliferation. Ali-
200
m
L. After incubation for 48 h at 26 ꢂC, 20
mL of 2.5 mM resazurin
solution were added to each well, and incubated for 3 h. The
fluorescence intensity (535 nm eexcitation wavelength and
590 nm eemission wavelength) was measured in a fluorometer
€
(Infinite 200, Tecan Group Ltd, Mannedorf, Switzerland). All assays
were carried out in triplicate. The half maximal inhibitory con-
centration (IC50) was determined by a Probit analysis.
2.4. Macrophage cytotoxicity assays
quots of 20 mL of 2.5 mM resazurin were added to each well and the
The cytotoxicity of all compounds was tested using the murine
macrophage cell line J774.2 (ATCC®TIB-67). For this, macrophages
plates were incubated for 3 h. Finally, fluorescence emission was
measured as described above. All assays were carried out in trip-
licate. The half maximal inhibitory concentration (IC50) was
were seeded (5
ꢁ
104 cells/well) in 96-well flat-bottomed
Scheme 1. Synthesis and chemical structure of N-benzyl-1H-benzimidazol-2-amines (1e28). Reagents and conditions: (a) substituted benzaldehyde 40e47, toluene, reflux, (b)
NaBH4, MeOH, 0 ꢂC to room temperature.
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

4
R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
determined by a Probit analysis.
index.html) (Larkin et al., 2007).
2.9. Statistical analysis
All the data was expressed as mean
2.6. Determination of the inhibitory effect of compounds 7 and 8 on
recombinant LmARG activity
standard deviation.
The effect of compounds 7 and 8 on LmARG activity was
determined using the QuantiChom™ Arginase Assay Kit (DARG-
100). Recombinant LmARG was incubated with compounds at
ANOVA and T3 Dunnett's Post hoc test was performed using IBP
SPSS Statistics, in order to determine differences between groups
by analyzing statistical significance. Differences were considered
significant at 0.05 level of confidence.
40
treated enzyme was included as negative control and the ARG
specific inhibitor Nor-NOHA (40 M) was used as positive control.
m
M for 1 h at 25 ꢂC, as recommended by Riley et al. (2011). Non-
m
3. Results and discussion
In order to evaluate the effect of 7 and 8 on human ARG (HsARG),
total extracts were obtained from THP-1 human differentiated
Compounds 1e28 were synthesized by straightforward reduc-
tive amination method with moderate to good yields. The syn-
thesized compounds were purified by crystallization from the
adequate solvent or mixture of solvents. Spectrometric and spec-
troscopic data obtained were consistent with the expected
structures.
The activity of compounds 1e28 was evaluated against
L. mexicana promastigotes. Results are presented in Table 1. It is
seen that compounds 7, 8 and 19 were more active than miltefosine,
while 4, 9 and 11 had similar IC50 values. Compounds 1e28 were
less active than amphotericin B. Compounds 7 and 8 showed the
macrophages treated with 7 and 8 at 40
extracts from non-treated macrophages were included as negative
control and the inhibitor Nor-NOHA (40 M) was used as positive
m
M for 24 h at 37 ꢂC. Total
m
control. Recombinant LmARG and total extracts were incubated
with L-arginine and urea production was quantified as indicative of
ARG activity, following the supplier's instructions. The arginase
activity (units per litter of sample (U/L) was determined by
measuring absorbance at 430 nm in an ELISA reader and calculated
following the supplier's instructions. Experiments were carried out
in duplicate and the percentage of inhibition in relation to control
was calculated.
highest antileishmanial activity with IC50 values of 2.62
mM and
To determine the IC50 of 8 against recombinant LmARG the
QuantiChom™ Arginase Assay Kit was used. For this, recombinant
LmARG was incubated with 8 at different concentrations and the
arginase activity determined as mentioned above. Experiments
were carried out in duplicate. The percentage of inhibition was
calculated and the IC50 value was determined by a Probit analysis.
3.21 M, respectively, being 5.8 and 4.8 times significantly more
m
active than miltefosine (P ꢃ 0.05). It is important to mention that
the IC50 value of miltefosine obtained in this study against the
promastigote of L. mexicana (15.34
reported in other studies (15.7 and 13.45
Enciso et al., 2016). In relation to the IC50 value of amphotericin B
against L. mexicana promastigote reported herein (0.95 M), it is in
the range of the IC50 values reported by Al-Abdely et al. (1998)
against two different L. mexicana isolates (0.5 and 1.0 M). How-
ever, Varela-M et al. (2012) obtained an IC50 of 2.7 M for milte-
fosine and in the case of amphotericin B Escobar et al. (2002)
reported an IC50 in the range of 0.22e0.35 M. Difference in IC50
mM) is in agreement with values
mM) (Wijnant et al., 2017;
m
2.7. Molecular docking
m
Compounds 7 and 8 were built and optimized through an en-
ergy minimization in Spartan’10 (Wavefunction Inc, 2010) using
PM6 semi-empirical method. Polar hydrogens, rotatable bonds and
Gasteiger-Marsilli atomic charges were computed utilizing
MGLTools 1.5.6 (Sanner, 1999). The tridimensional structure of
LmARG was obtained from the Protein Data Bank (PDB ID: 5HJA)
(Hai and Christianson, 2016). During protein preparation, water
molecules and the co-crystal ligand were deleted from the protein,
while the manganese atoms (Mn2þ) were conserved for the cal-
culations. The structure of LmARG was subjected to an energy
minimization employing the AMBER99SB force field by 1000
steepest-descent minimization steps implemented in GROMACS
4.5.5 software. Then, MGLTools 1.5.6 was used to merge all non-
polar hydrogens and to assign Gasteiger charges for each atom of
the macromolecule. Docking calculations were performed using
the AutoDock 4.2 software (Morris et al., 2009; Huey et al., 2007). A
grid box of 50 ꢁ 50 ꢁ 50 points with a grid spacing of 0.375 Å was
calculated for the proper atom types and centered in the catalytic
site. The Lamarckian genetic algorithm was used as a search
method with a total of 20 runs being undertaken with a maximum
number of 5,000,000 energy evaluations and initial populations of
150 conformers. The clusters were ranked by the lowest energy
representative of each cluster. All molecular graphics were pre-
pared with PyMOL 0.99 version.
m
m
values can be attributed to different factors such as the parasite
strain, growth medium and method used for IC50 determination in
the assays.
Compounds 7 and 8, with no substituent in the benzenoid ring,
have a methyl group at position 1 of the benzimidazole nucleus and
an oxygen at position 2 of the benzyl moiety, whether as a hydroxyl
(7) or as a methoxide group (8). Modification of this substitution
pattern leads to a detriment in the activity, as seen in compounds
1e5 with an alkyl group from C2 to C4 and no oxygen at position 2
of the benzyl moiety. In general, all compounds with chlorine
atoms in the benzenoid ring, regardless of having a 1-methyl and an
oxygen at position 2 of the benzyl moiety, were less active than the
reference
drug,
except
19
(5-chloro-N-(5-chloro-2-
hydroxybenzyl)-1-methyl-1H-benzimidazol-2-amine). The same
applies for compounds with 1-H, except 11 (5-chloro-N-(2-
methoxybenzyl)-1H-benzimidazol-2-amine) that is as active as
miltefosine.
The cytotoxicity of all compounds was tested using the murine
macrophage cell line J774.2; results are presented in Table 1. CC50
values for the reference drugs, amphotericin B and miltefosine
were of 6.19 mM and 157.03 mM, respectively. All compounds were
less cytotoxic than amphotericin B. On the other hand, compounds
2, 7e10, 13, 16, 19, 25e28 were less cytotoxic than miltefosine. CC50
value obtained for the reference drug, miltefosine, is agreement
with the value reported by Dea-Ayuela et al. (2016) using murine
2.8. Multiple-sequence alignment of ARG amino acid sequence of
Leishmania spp
Alignments of the amino acid sequence of ARG from L. mexicana
(UniProt: Q6TUJ5), L. braziliensis (UniProt: A4HMH0), and
L. donovani (UniProt: A0A0M4CX5) were performed with the
Clustal-W program server (http://www.ebi.ac.uk/Tools/clustalw2/
macrophage cell line J774.2 (136.4
that other authors using peritoneal macrophages obtained CC50
values for miltefosine in the range of 87.30 Me241.4 M (Stroppa
et al., 2017; Tempone et al., 2017; Bezerra-Souza et al., 2016;
mM). It is important to mention
m
m
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
5
Table 1
Inhibition of proliferation of Leishmania mexicana promastigote, cytotoxicity and selectivity index of compounds 1e28, miltefosine and amphotericin B.
Compound
R1
R2
R3
R4
R5
R6
aIC50
(
mM) SD
bCC50
(m
M) SD
cSI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
CH3CH2
CH3CH2CH2
CH3(CH2)2CH2
(CH3)2CH
CH3CH2
CH3
CH3
CH3
CH3
H
H
H
CH3
CH3
CH3
H
H
H
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
Cl
H
Cl
Cl
Cl
H
Cl
H
Cl
Cl
H
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
H
Cl
Cl
H
H
Cl
H
Cl
Cl
H
H
Cl
H
H
Cl
Cl
H
H
H
H
H
H
OH
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
N(CH3)2
H
H
H
H
H
H
Br
H
Cl
H
107.09 7.75
78.69 3.74
32.19 1.50
19.60 1.87
83.15 2.17
32.06 2.18
2.62 0.79
84.17 0.49
302.55 4.42
61.39 0.49
68.09 0.54
70.67 0.65
100.13 1.33
240.43 2.27
1020 8.5
0.78
3.84
1.91
3.47
0.85
3.12
91.76
317.75
14.05
4.88
4.51
0.18
0.89
0.29
0.19
0.39
0.09
0.22
39.84
0.22
0.55
0.38
0.31
0.30
2.72
1.52
1.83
6.71
10.23
6.5
OCH3
Cl
Br
OCH3
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.21 0.89
13.98 1.83
200.47 6.16
14.53 1.54
406.56 9.55
324.45 6.14
225.14 4.06
438.57 10.62
431.49 24.76
351.03 13.57
187.10 10.52
8.01 1.38
217.87 15.55
62.45 6.60
106.06 10.01
164.78 9.57
89.14 3.45
129.34 6.35
129.95 5.99
145.95 5.47
47.48 1.73
15.34 0.36
0.95 0.0598
196.44 3.34
978.80 5.18
65.59 4.17
75.11 1.36
288.35 4.05
65.29 1.31
87.13 1.92
169.09 2.05
30.18 1.94
40.42 2.81
318.94 3.83
47.90 2.41
34.02 2.38
39.96 2.05
50.61 1.86
26.81 2.08
352.08 9.45
197.43 5.73
267.56 4.64
318.35 5.98
157.03 2.86
6.19 1.614
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
CH3
CH3
CH3
CH3
e
H
miltefosine
amphotericin B
e
SD: Standard deviation of three independent determinations.
a
IC50: Compound concentration that produced a 50% reduction in parasites.
CC50: Compound concentration that produced a 50% reduction in J774.2 Cell.
b
c
SI: Selectivity Index ¼ CC50/IC50
.
Coimbra et al., 2016; Dea-Ayuela et al., 2016). In relation to CC50
value for amphotericin B, Garcia et al. (2017) obtained a CC50 of
of 0.28 and 0.26
respect to miltefosine (IC50 ¼ 0.51
(IC50 ¼ 0.87 M) (P ꢃ 0.05) (Table 2). Other authors have reported
IC50 values for miltefosine and amphotericin B of 0.96 M and
0.51 M (Bilbao-Ramos et al., 2017; Na et al., 2004). However,
Stroppa et al. (2017) reported for miltefosine an IC50 value of 4.2
and Bilbao-Ramos et al. (2017) and Fortin et al. (2012), reported IC50
values for amphotericin of 0.24 M, and 0.07e0.09 M, respectively.
Difference in IC50 values can also be attributed to the parasite strain,
growth medium and method for determination used in the assays.
Compounds 7 and 8 had SI values of 942.8 and 3939.2 against
L. mexicana amastigote (Table 3). Compound 8 was 12.8 and 554
times more selective than miltefosine and amphotericin B,
respectively. In the case of L. braziliensis, 7 and 8 showed slightly
lower antileishmanial activity than the reference drug, miltefosine,
although it was not significantly different (P ꢄ 0.05). In addition,
both compounds were significantly less active against L. donovani
amastigote than miltefosine (P ꢃ 0.05) (Table 2). It is worth noticing
that compound 8 showed the highest SI against L. mexicana pro-
mastigote and amastigote (SI ¼ 317.75 and 3939.2, respectively)
(Table 3).
m
M, respectively, being significantly different with
m
M) and amphotericin B
8.4
et al. (2014) using murine peritoneal macrophages reported a CC50
of 1.08 M. Different CC50 values can be due to the cell type and
mM using murine macrophage cell line J774, meanwhile Ribeiro
m
m
m
m
determination method used in the assays.
mM
Importantly, compounds 7, 8, 9 and 19 showed higher Selectivity
Index (SI) (CC50/IC50) than the reference drugs, miltefosine and
amphotericin B. Compounds 7 and 8 had the highest SI (91.76 and
317.75, respectively) towards L. mexicana promastigotes in com-
m
m
parison to miltefosine and amphotericin
respectively).
B (10.23 and 6.5,
Taking into account their high antileishmanial activity and
lower cytotoxicity in comparison to miltefosine and amphotericin
B, compounds 7 and 8 were selected to search for their wide range
antileishmanial activity against the promastigote and amastigote of
different Leishmania species, responsible for the different clinical
manifestations of the disease. Compound 7 showed significantly
higher activity against L. braziliensis promastigotes than compound
8 (P ꢃ 0.05) and slightly lower than miltefosine (P ꢄ 0.05). In
addition, 7 and 8 did not show better activity against L. donovani
than miltefosine (P ꢃ 0.05) (Table 2).
In order to know more about possible enzyme targets that may
explain the high activity of 7 and 8 against L. mexicana promasti-
gote and amastigote, it was our interest to analyze their ability to
In relation to the in vitro efficacy of compounds 7 and 8 against
the intracellular amastigote of L. mexicana, they showed IC50 values
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

6
R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
Table 2
Inhibition of proliferation of different Leishmania spp by compounds 7 and 8, miltefosine and amphotericin B.
Parasite stage
Promastigotes
IC50(mM) SD
7
8
miltefosine
amphotericin B
L. mexicana
L. braziliensis
L. donovani
2.62 0.79
54.05 10.02
198.47 4.86
3.21 0.89
127.7 21.6
223.8 8.17
15.34 0.36
48.37 11.37
114.68 11.37
0.95 0.06
e
e
Amastigotes
L. mexicana
L. braziliensis
L. donovani
7
8
miltefosine
0.51 0.08
22.67 6.03
66.74 1.05
amphotericin B
0.87 0.03
0.28 0.02
34.22 1.12
222.23 4.8
0.26 0.04
33.78 2.47
218.24 8.46
e
e
IC50: Compound concentration that produced a 50% reduction in parasites.
SD: Standard deviation of three independent determinations.
Table 3
recombinant LmARG, 24.34% and 68.27%, respectively. Compound 8
was more active than Nor-NOHA (32% inhibition) and had an IC50
Selectivity Index (SI) of compounds 7, 8, miltefosine and amphotericin B.
value of 35.9 mM. It is worth noticing that compound 8 did not
inhibit the activity of HsARG, on the contrary, it stimulated the
enzyme activity by 24.21%.
Parasite stage
Promastigotes
SI (Selectivity Index ¼ CC50/IC50
)
7
8
miltefosine
amphotericin B
L. mexicana
L. braziliensis
L. donovani
91.76
4.88
1.33
317.75
9.61
5.48
10.23
3.25
1.37
6.5
e
To elucidate how compounds 7 and 8 inhibited LmARG activity,
an in silico docking study was performed. Compound 8 was the best
ranked with -5.5 kcal/mol followed by 7 with -4.46 kcal/mol. As
seen in Fig. 1, both compounds do not show interactions with res-
idues of the catalytic site and do not show electrostatic interaction
with the Mn2þ atoms. However, these compounds block the cavity
of the catalytic site and are mainly stabilized by hydrogen bond (H-
bond) interactions. Compound 7 shows an H-bond interaction with
residue His139 and hydrophobic interactions with residues Thr257
and Val259. In the case of compound 8, it adopts a conformation
where the benzyl moiety is closer to the catalytic pocket and shows
H-interactions with residues Asn143, Thr148, Ser150 and Asn152.
In addition, the physicochemical descriptors were calculated for
compounds 7 and 8 (Supplementary Material), and they do not
violate the optimal requirements for druggability.
e
Amastigotes
SI (Selectivity Index ¼ CC50/IC50
)
L. mexicana
L. braziliensis
L. donovani
942.8
7.71
1.18
3939.2
30.32
4.69
307.8
6.92
2.35
7.11
e
e
inhibit LmARG activity. LmARG is considered a potential drug target
for the treatment of leishmaniasis since it is essential, not only for
parasite growth and survival, but also in the evasion of the host
immune response (Balana-Fouce et al., 2012; D'Antonio et al., 2013;
Iniesta et al., 2001).
The inhibitory effect of compounds 7 and 8 on recombinant
LmARG activity was experimentally tested using the QuantiChom™
Arginase Assay Kit. Nor-NOHA, considered a specific inhibitor of
LmARG (D'Antonio et al., 2013; Riley et al., 2011) was included as
positive control. Compounds 7 and 8 inhibited the activity of
~
The activity of 8 against L. mexicana can be explained in part by
its inhibitory interactions on LmARG demonstrated both by mo-
lecular docking studies and experimental assays. In an attempt to
Fig. 1. Predicted binding mode and interactions of compounds 7 (a) and 8 (b) with LmARG. 2D interactions were obtained from the PoseView server (http://proteinsplus.zbh.uni-
hamburg.de/#poseview).
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
7
Fig. 2. Alignment among ARG sequences of L. braziliensis, and L. donovani, with the amino acid sequence of L. mexicana ARG. Multiple sequence alignment using ClustalW2-Clustal
Omega. In blue the active site and green the metal binding region. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
explain the lower activity of 8 against visceral species, a sequence
alignment of ARG from the different Leishmania species was per-
formed (Fig. 2). No differences were detected around the active site
(blue) and the metal binding region (green), among cutaneous,
mucocutaneous and visceral species, sharing 75.8% identity among
species. It may be that amino acid differences outside the active site
of L. donovani ARG could influence the affinity binding of compound
8. Further docking studies with the different ARGs will give some
light about their interaction with compound 8.
Another explanation of compound 8 activity against different
Leishmania species could be based on a different L-arginine meta-
bolism in cutaneous and visceral Leishmania species. In fact,
Westrop et al. (2015) performed the metabolomics analysis of the
promastigotes of L. mexicana, L. major and L. donovani, demon-
strating important differences in the amino acid metabolism of
these species, especially tryptophan, aspartate, L-arginine and L-
proline. Regarding L-arginine, authors found that L. mexicana and
L. major internalize this amino acid in greater extent than
L. donovani, detecting more L-ornithine. In addition, it was recently
demonstrated that in L. donovani, although ARG is essential for L-
ornithine and polyamine synthesis, ornithine descarboxylase
appeared to be the rate-limiting enzyme for polyamine production
(Boitz et al., 2016). Therefore, these evidences could explain the
poor activity of compound 8 against visceral species of Leishmania.
Acknowledgements
Nieto-Meneses, as a PhD student from the Programa de Doc-
torado en Ciencias Quimicobiologicas, Escuela Nacional de Ciencias
ꢀ
ꢀ
ꢀ
Biologicas, Instituto Politecnico Nacional was supported by a PhD
ꢀ
fellowship from the IMSS (Grant 062-2013) and CONACyT-Mexico
(Grant 246636). We acknowledge to the UNAM, Posgrado en
Ciencias Químicas and CONACyT for grants to AMR (299651), JBMR
(308208) and PTS (409406/318775). Authors also acknowledge
USAII (Maricela Gutierrez-Franco, Rosa Isela del Villar-Morales,
ꢀ
Nayeli Lopez-Balbiaux, Víctor Lemus-Neri) for the determination
of analyses and spectra and Dr. Antonio Miranda-Duarte for the
statistical analyses. This work was supported by FIS/IMSS/PROT/
G12/1130 and CONACYT (Grant 251726).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.exppara.2017.11.009.
References
Al-Abdely, H.M., Graybill, J.R., Bocanegra, R., Najvar, L., Montalbo, E., Regen, S.L.,
Melby, P.C., 1998. Efficacies of KY62 against Leishmania amazonensis and
Leishmania donovani in experimental murine cutaneous leishmaniasis and
visceral leishmaniasis. Antimicrob. Agents. Chemother. 42, 2542e2548.
Alhtar, W., Khan, M.F., Verma, G., Shaquiquzzaman, M., Rizvi, M.A., Mehdi, S.H.,
Akhter, M., Alam, M.M., 2016. Therapeutic evolution of benzimidazole de-
rivatives in the last quinquennial period. Eur. J. Med. Chem. 126, 705e753.
4. Conclusions
ꢀ
ꢀ
In this study, a series of 28 benzimidazole derivatives were
tested for their activity against different Leishmania species,
responsible for the different clinical manifestations of the disease.
Compound 8 exhibited the highest potency and SI against both
parasite stages of L. mexicana. Compound 8 showed inhibitory ef-
fect on LmARG activity that indeed contributes to its anti-
leishmanial activity, even though its effect on other parasite
molecules cannot be ruled out. Compound 8 is a promising scaffold
for the development of more potent antileishmanial benzimidazole
derivatives.
~
ꢀ
Balana-Fouce, R., Calvo-Alvarez, E., Alvarez-Velilla, R., Prada, C.F., Perez-Pertejo, Y.,
Reguera, R.M., 2012. Role of trypanosomatid's arginase in polyamine biosyn-
thesis and pathogenesis. Mol. Biochem. Parasitol. 181, 85e93.
Bezerra-Souza, A., Yamamoto, E.S., Laurenti, M.D., Ribeiro, S.P., Passero, L.F., 2016.
The antifungal compound butenafine eliminates promastigote and amastigote
forms of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) bra-
ziliensis. Parasitol. Int. 65 (6 Pt A), 702e707.
Bhattacharya, P., Mondal, S., Basak, S., Das, P., Saha, A., Bera, T., 2016. In Vitro sus-
ceptibilities of wild and drug resistant Leishmania donovani amastigotes to
piperolactam A loaded hydroxypropyl-b-cyclodextrin nanoparticles. Acta. Trop.
158, 97e106.
Bilbao-Ramos, P., Sifontes-Rodríguez, S., Dea-Ayuela, M.A., Bolas-Fernandez, F.,
2012. A fluorometric method for evaluation of pharmacological activity against
intracellular Leishmania amastigotes. J. Microbiol. Methods 89, 8e11.
ꢀ
ꢀ
Bilbao-Ramos, P., Dea-Ayuela, M.A., Cardenas-Alegría, O., Salamanca, E., Santalla-
Conflict of interest
ꢀ
ꢀ
Vargas, J.A., Benito, C., Flores, N., Bolas-Fernandez, F., 2017. Leishmaniasis in the
major endemic region of Plurinational State of Bolivia: species identification,
phylogeography and drug susceptibility implications. Acta Trop. 76, 150e161.
The authors declare no conflict of interest.
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009

8
R. Nieto-Meneses et al. / Experimental Parasitology xxx (2017) 1e8
Boitz, J.M., Gilroy, C.A., Olenyik, T.D., Paradis, D., Perdeh, J., Dearman, K., Davis, M.J.,
Yates, P.A., Li, Y., Riscoe, M.K., Ullman, B., Roberts, S.C., 2016. Arginase is essential
for survival of Leishmania donovani promastigotes but not intracellular amas-
tigotes. Infect. Immun. 85 (1) https://doi.org/10.1128/IAI.00554-16 pii:
e00554e16.
Coelho, A.C., Trinconi, C.T., Costa, C.H.N., Uliana, S.R.B., 2014. In vitro and in vivo
miltefosine susceptibility of a Leishmania amazonensis isolate from a patient
with diffuse cutaneous leishmaniasis. PLoS. Negl. Trop. Dis. 8, 1e11.
Coimbra, E.S., Antinarelli, L.M., Silva, N.P., Souza, I.O., Meinel, R.S., Rocha, M.N.,
Soares, R.P., da Silva, A.D., 2016. Quinoline derivatives: synthesis, leishmanicidal
activity and involvement of mitochondrial oxidative stress as mechanism of
action. Chem. Biol. Interact. 260, 50e57.
50e59.
ꢀ
ꢀ
Mendez-Cuesta, C.A., Herrera-Rueda, M.A., Hidalgo-Figueroa, S., Tlahuext, H., Moo-
ꢀ
Puc, R., Chale-Dzul, J.B., Chan-Bacab, M., Ortega-Morales, B.O., Hernandez-
~
ꢀ
Núnez, E., Mendez-Lucio, O., Medina-Franco, J.L., Navarrete-Vazquez, G., 2016.
Synthesis, screening and in silico simulations of anti-parasitic Propamidine/
Benzimidazole derivatives. Med. Chem. 13, 137e148.
~
Mondelaers, A., Sanchez-Canete, M.P., Hendrickx, S., Eberhardt, E., Garcia-
Hernandez, R., Lachaud, L., Cotton, J., Sanders, M., Cuypers, B., Imamura, H.,
Dujardin, J.C., Delputte, P., Cos, P., Caljon, G., Gamarro, F., Castanys, S., Maes, L.,
2016. Genomic and molecular characterization of miltefosine resistance in
Leishmania infantum strains with either natural or acquired resistance through
experimental selection of intracellular amastigotes. PloS. One 11 (4), e0154101.
https://doi.org/10.1371/journal.pone.0154101.
ꢀ
ꢀ
ꢀ
Dea-Ayuela, M.A., Bilbao-Ramos, P., Bolas-Fernandez, F., Gonzalez-Cardenete, M.A.,
2016. Synthesis and antileishmanial activity of C7- and C12-functionalized
dehydroabietylamine derivatives. Eur. J. Med. Chem. 121, 445e450.
Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S.,
Olson, A.J., 2009. AutoDock4 and AutoDockTools4: automated docking with
selective receptor flexibility. J. Comput. Chem. 16, 2785e2791.
Mota, V.Z., de Carvalho, G.S., da Silva, A.D., Costa, L.A., de Almeida-Machado, P.,
Coimbra, E.S., Ferreira, C.V., Shishido, S.M., Cuin, A., 2014. Gold complexes with
benzimidazole derivatives: synthesis, characterization and biological studies.
Biometals 27, 183e194.
Na, Y.M., Lebouvier, N., Le Borgne, M., Pagniez, F., Alvarez, N., Le Pape, P., Le Baut, G.,
2004. Synthesis and antileishmanial activity of 3-imidazolylalkylindoles. Part I.
J. Enzyme. Inhib. Med. Chem. 19 (6), 451e457.
DꢀAntonio, E.L., Ullman, B., Roberts, S.C., Dixit, U.G., Wilson, M.E., Hai, Y.,
Christianson, D.W., 2013. Crystal structure of arginase from Leishmania mex-
icana and implications for the inhibition of polyamine biosynthesis in parasitic
infections. Arch. Biochem. Biophys. 535, 163e176.
ꢀ
ꢀ
Díaz-Chiguer, D.L., Marquez-Navarro, A., Nogueda-Torres, B., de la Luz Leon-
ꢀ
ꢀ
ꢀ
Avila, G., Perez-Villanueva, J., Hernandez-Campos, A., Castillo, R., Ambrosio, J.R.,
ꢀ
ꢀ
Nieto-Meneses, R., Yepez-Mulia, L., Hernandez-Luis, F., 2012. In vitro and in vivo
trypanocidal activity of some benzimidazole derivatives against two strains of
Trypanosoma cruzi. Acta. Trop. 122, 108e112.
ꢀ
ꢀ
ꢀ
ꢀ
Navarrete-Vazquez, G., Cedillo, R., Hernandez-Campos, A., Yepez, L., Hernadez-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Enciso, E., Sarmiento-Sanchez, J.I., Lopez-Moreno, H.S., Ochoa-Teran, A., Osuna-
Luis, F., Valdez, J., Morales, R., Cortes, R., Hernandez, M., Castillo, R., 2001.
Synthesis and antiparasitic activity of 2-(trifluoromethyl)-benzimidazole de-
rivatives. Bioorg. Med. Chem. Lett. 11, 187e190.
ꢀ
ꢀ
Martínez, U., Beltran-Lopez, E., 2016. Synthesis of new quinazolin-2,4-diones as
anti-Leishmania mexicana agents. Mol. Divers 20, 821e828.
Escobar, P., Matu, S., Marques, C., Croft, S., 2002. Sensitivities of Leishmania species
to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and
amphotericin B. Acta Trop. 81, 151e157.
Oh, S., Kim, S., Kong, S., Yang, G., Lee, N., Han, D., Goo, J., Siqueira-Neto, J.L., Freitas-
Junior, L.H., Song, R., 2014. Synthesis and biological evaluation of 2,3-
dihydroimidazo[1,2-a]benzimidazole derivatives against Leishmania donovani
and Trypanosoma cruzi. Eur. J. Med. Chem. 12, 395e403.
Fernandes, A.C., Pedroso, R.B., de Mello, T.F.P., Donatti, L., Venazzi, E.A.S.,
Demarchi, I.G., Aristides, S.M., Lonardoni, M.V., Silveira, T.G., 2016. In vitro
characterization of Leishmania (Viannia) braziliensis isolates from patients with
ꢀ
Ribeiro, T.G., Chavez-Fumagalli, M.A., Valadares, D.G., França, J.R., Rodrigues, L.B.,
ꢀ
Duarte, M.C., Lage, P.S., Andrade, P.H., Lage, D.P., Arruda, L.V., Abanades, D.R.,
different responses to Glucantime® treatment from Northwest Parana, Brazil.
Costa, L.E., Martins, V.T., Tavares, C.A., Castilho, R.O., Coelho, E.A., Faraco, A.A.,
2014. Novel targeting using nanoparticles: an approach to the development of
an effective anti-leishmanial drug-delivery system. Int. J. Nanomedicine 14,
877e890.
ꢀ
Exp. Parasitol. 167, 83e93.
Fortin, A., Hendrickx, S., Yardley, V., Cos, P., Jansen, H., Maes, L., 2012. Efficacy and
tolerability of oleylphosphocholine (OlPC) in a laboratory model of visceral
leishmaniasis. J. Antimicrob. Chemother. 67 (11), 2707e2712.
Freitas-Junior, L.H., Chatelain, E., Kim, H.A., Siqueira-Neto, J.L., 2012. Visceral leish-
maniasis treatment: what do we have, what do we need and how to deliver it?
Int. J. Paristol. Drugs. Drug. Resist 2, 11e19.
Garcia, A.R., Amaral, A.C.F., Azevedo, M.M.B., Corte-Real, S., Lopes, R.C., Alviano, C.S.,
Pinheiro, A.S., Vermelho, A.B., Rodrigues, I.A., 2017. Cytotoxicity and anti-
Leishmania amazonensis activity of Citrus sinensis leaf extracts. Pharm. Biol. 55,
1780e1786.
Riley, E., Roberts, S.C., Ullman, B., 2011. Inhibition profile of Leishmania mexicana
arginase reveals differences with human arginase I. Int. J. Parasitol. 41,
545e552.
Sanner, M.F., 1999. Python: a programming language for software integration and
development. J. Mol. Graph. Model 17, 57e61.
Savoia, D., 2015. Recent updates and perspectives on leishmaniais. J. Infect. Dev.
Ctries. 9, 588, 596.
Shaw, C.D., Lonchamp, J., Downing, T., Imamura, H., Freeman, T.M., Cotton, J.A.,
Sanders, M., Blackburn, G., Dujardin, J.C., Rijal, S., Khanal, B., Illingworth, C.J.,
Coombs, G.H., Carter, K.C., 2016. In vitro selection of miltefosine resistance in
promastigotes of Leishmania donovani from Nepal: genomic and metabolomic
characterization. Mol. Microbiol. 99, 1134e1148.
Hai, Y., Christianson, D.W., 2016. Crystal structures of Leishmania mexicana arginase
complexed with
a, a-disubstituted boronic amino-acid inhibitors. Acta. Crys-
tallogr. F. Struct. Biol. Commun. 72, 300e306.
ꢀ
ꢀ
ꢀ
Hernandez-Luis, F., Hernandez-Campos, A., Castillo, R., Navarrete-Vazquez, G.,
ꢀ
ꢀ
ꢀ
Soria-Arteche, O., Hernandez-Hernandez, M., Yepez-Mulia, L., 2010. Synthesis
and biological activity of 2-(trifluoromethyl)-1H-benzimidazole derivatives
against some protozoa and Trichinella spiralis. Eur. J. Med. Chem. 45,
3135e3141.
Spartan’10, Wavefunction Inc., Irvine, CA 92612 USA.
Stroppa, P.H.,F., Antinarelli, L.M.R., Carmo, A.M.L., Gameiro, J., Coimbra, E.S., da
Silva, A.D., 2017. Effect of 1,2,3-triazole salts, non-classical bioisosteres of mil-
tefosine, on Leishmania amazonensis, 15 Bioorg. Med. Chem. 25 (12),
3034e3045.
Huey, R., Morris, G.M., Olson, A.J., Goodsell, D.S., 2007. A semiempirical free energy
force field with charge-based desolvation. J. Comput. Chem. 28, 1145e1152.
Ilari, A., Fiorillo, A., Baiocco, P., Poser, E., Angiulli, G., Colotti, G., 2015. Targeting
polyamine metabolism for finding new drugs against leishmaniasis: a review.
Mini. Rev. Med. Chem. 15, 243e252.
Tempone, A.G., Ferreira, D.D., Lima, M.L., Costa Silva, T.A., Borborema, S.E.T.,
~
Reimao, J.Q., Galuppo, M.K., Guerra, J.M., Russell, A.J., Wynne, G.M., Lai, R.Y.L.,
Cadelis, M.M., Copp, B.R., 2017. Efficacy of a series of alpha-pyrone derivatives
against Leishmania (L.) infantum and Trypanosoma cruzi. Eur. J. Med. Chem.
139, 947e960.
ꢀ
Iniesta, V., Gomez-Nieto, L.C., Corraliza, I., 2001. The inhibition of arginase by
N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside mac-
rophages. J. Exp. Med. 193, 777e784.
Keri, R.S., Hiremathad, A., Budagumpi, S., Nagaraja, B.M., 2015. Comprehensive re-
view in current developments of benzimidazole-based medicinal chemistry.
Chem. Biol. Drug. Des. 86, 19e65.
Keurulainen, L., Siiskonen, A., Nasereddin, A., Kopelyanskiy, D., Sacerdoti-Sierra, N.,
Leino, T.O., Tammela, P., Yli-Kauhaluoma, J., Jaffe, C.L., Kiuru, P., 2015. Synthesis
and biological evaluation of 2-arylbenzimidazoles targeting Leishmania dono-
vani. Bioorg. Med. Chem. Lett. 25, 1933e1937.
Kumar, A., Das, S., Purkait, B., Sardar, A.H., Ghosh, A.K., Dikhit, M.R., Abhishek, K.,
Das, P., 2014. Ascorbate peroxidase, a key molecule regulating amphotericin B
resistance in clinical isolates of Leishmania donovani. Antimicrob. Agents. Che-
mother. 58, 6172e6184.
Vakil, N.H., Fujinami, N., Shah, P.J., 2015. Pharmacotherapy for leishmaniasis in the
United States: focus on miltefosine. Pharmacotherapy 35, 536e545.
ꢀ ꢀ ꢀ
Valdez, J., Cedillo, R., Hernandez-Campos, A., Yepez, L., Hernandez-Luis, F., Nav-
ꢀ
ꢀ
ꢀ
arrete-Vazquez, G., Tapia, A., Cortes, R., Hernandez, M., Castillo, R., 2002. Syn-
thesis and antiparasitic activity of 1H-benzimidazole derivatives. Bioorg. Med.
Chem. Lett. 12, 2221e2224.
~
Varela-M, R.E., Villa-Pulgarin, J.A., Yepes, E., Müller, I., Modolell, M., Munoz, D.L.,
2012. In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania
spp. and SbV-resistant parasites. PLoS. Negl. Trop. Dis. 6, e1612.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Velazquez-Lopez, J.M., Hernandez-Campos, A., Yepez-Mulia, L., Tellez-Valencia, A.,
Flores-Carrillo, P., Nieto-Meneses, R., Castillo, R., 2016. Synthesis and trypano-
cidal activity of novel benzimidazole derivatives. Bioorg Med. Chem. Lett. 26,
4377e4381.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., Mcgettigan, P.A.,
McWilliam, H., Aristides, S.M., Lonardoni, M.V., Silveira, T.G., 2007. Clustal W
and clustal X version 2.0. Bioinformatics 23, 2947e2948.
Westrop, G.D., Williams, R.A., Wang, L., Zhang, T., Watson, D.G., Silva, A.M.,
Coombs, G.H., 2015. Metabolomic analyses of Leishmania reveal multiple spe-
cies differences and large differences in amino acid metabolism. PLoS. One 10
(9), e0136891. https://doi.org/10.1371/journal.pone.0136891.
World Health Organization (WHO), 2016. Leishmaniasis Home, Leishmaniasis
Control Programme. http://www.who.int/leishmaniasis/en/ (Last time
accessed: December).
Wijnant, G.J., Van Bocxlaer, K., Yardley, V., Murdan, S., Croft, S.L., 2017. Efficacy of
paromomycin-chloroquine combination therapy in experimental cutaneous
leishmaniasis. Antimicrob. Agents. Chemother. 61 (8) https://doi.org/10.1128/
AAC.00358-17 pii: e00358e17.
ꢀ
ꢀ
ꢀ
ꢀ
Melchor-Doncel de la Torre, S., Vazquez, C., Gonzalez-Chavez, Z., Yepez-Mulia, L.,
ꢀ
ꢀ
Nieto-Meneses, R., Jasso-Chavez, R., Saavedra, E., Hernandez-Luis, F., 2017.
Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-
carbohydrazides derivatives as modifiers of redox homeostasis of Trypano-
soma cruzi. Bioorg. Med. Chem. Lett. 27, 3403e3407.
ꢀ
ꢀ
Mendez-Cuesta, C.A., Mendez-Lucio, O., Castillo, R., 2012. Homology modeling,
docking and molecular dynamics of the Leishmania mexicana arginase:
a
description of the catalytic site useful for drug design. J. Mol. Graph. Model 38,
Please cite this article in press as: Nieto-Meneses, R., et al., In vitro activity of new N-benzyl-1H-benzimidazol-2-amine derivatives against
cutaneous, mucocutaneous and visceral Leishmania species, Experimental Parasitology (2017), https://doi.org/10.1016/j.exppara.2017.11.009