Synthesis and trypanocidal activity of novel benzimidazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.08.018

The present work reports the synthesis and biological activity of a series of 14 benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the only source of energy for the parasite. In this study, we found four compounds that inhibit TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro studies against T. cruzi epimastigotes showed two compounds that were more active than the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse macrophages (J744 cell line).

Full text of DOI:10.1016/j.bmcl.2015.08.018

Accepted Manuscript
Synthesis and trypanocidal activity of novel benzimidazole derivatives
José Miguel Velázquez-López, Alicia Hernández-Campos, Lilián Yépez-Mulia,
Alfredo Téllez-Valencia, Paulina Flores-Carillo, Rocío Nieto-Meneses, Rafael
Castillo
PII:
S0960-894X(15)00842-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.08.018
BMCL 23012
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 June 2015
30 July 2015
6 August 2015
Please cite this article as: Velázquez-López, J.M., Hernández-Campos, A., Yépez-Mulia, L., Téllez-Valencia, A.,
Flores-Carillo, P., Nieto-Meneses, R., Castillo, R., Synthesis and trypanocidal activity of novel benzimidazole
derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.08.018
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Synthesis and trypanocidal activity of novel benzimidazole
derivatives
José Miguel Velázquez-Lópeza, Alicia Hernández-Camposa, Lilián Yépez-Muliab, Alfredo
Téllez-Valenciac, Paulina Flores-Carilloa, Rocío Nieto-Menesesd, Rafael Castilloa*
aFacultad de Química, Departamento de Farmacia, Universidad Nacional Autónoma de
México, México, DF 04510, México.
bUnidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS,
México, DF 06720, México.
cFacultad de Medicina y Nutrición, Centro de Investigación en Alimentos y Nutrición,
Universidad Juárez del Estado de Durango, Durango 34000, México.
dEscuela Nacional de Ciencias Biológicas, Departamento de Parasitología, IPN, México,
DF 11340, México
*Corresponding author. Tel +52 55 56225287; fax +52 55 56225329.
E-mail address: rafaelc@unam.mx (Rafael Castillo)
Keywords
Benzimidazole derivatives; Chagas disease; Trypanosoma cruzi; Triosephosphate
isomerase

Abstract
The present work reports the synthesis and biological activity of a series of 14
benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of
Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the
only source of energy for the parasite. In this study, we found four compounds that inhibit
TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro
studies against T. cruzi epimastigotes showed two compounds that were more active than
the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse
macrophages (J744 cell line).

Chagas disease (CD), also called American trypanosomiasis, is caused by the protozoan
parasite Trypanosoma cruzi. CD is a life threatening illness considered a Neglected
Tropical Disease (NTD) by the World Health Organization (WHO).1 The parasite is
transmitted to humans through the infected feces of a blood-sucking triatomine bug, which
breaks through skin or mucous membranes during the insect´s bite. CD is also transmitted
by the ingestion of contaminated food, infected blood transfusions, congenital transmission,
or organ transplantation.2 Actually, CD causes more deaths in Latin America than any other
parasitic infection, affecting approximately eight million people, of whom 30-40% have or
will develop cardiomyopathy, digestive megasyndromes, or both.3,4 In spite of this
problem, no immediate prospects for vaccines have been developed. Drug therapy is the
only method currently available for the treatment of Chagas disease.5 In this sense, only
nifurtimox (NFX) and benznidazole (BZ) are widely used as treatment. However, neither of
these drugs are ideal due to their side effects and variable efficacy; depending on the phase
of the disease, they are only effective in the acute and early chronic phases of the infection.6
At the chronic phases, more than 80% of the treated patients with these drugs do not get
parasitological cure. Thus, it is crucial to continue the search for new and better drugs to
treat Chagas disease chemotherapy.7,8
Recently, there has been considerable interest in the inhibition of the parasite’s only energy
supply: the glycolytic pathway, in which triosephosphate isomerase (TIM) plays a crucial
role.9,10 This homodimeric enzyme catalyzes the interconversion of dihydroxyacetone-
phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P).11 Since TIM is present in both T.
cruzi (TcTIM) and human (HsTIM) cells, and it contains a highly conserved catalytic site,
the design of new drugs intended to inhibit the catalytic site must be ruled out. An
alternative, extremely hydrophobic, and non-conserved site at the homodimeric interface of
the enzyme has been proposed to inhibit its activity. In TcTIM, this region is constituted by
aromatic residues, whereas aliphatic and aromatic residues are present in the interface of
HsTIM.12 Several efforts have been made to find a selective inhibitor of TcTIM, in which
heterocyclic systems such as phenazine dioxides, thiadiazines and thiazoles have been
identified as candidates by high-throughput screening.13 A group of amphiphilic
benzothiazole derivatives were also identified by in silico studies.14,15 These works

demonstrate the affinity of the TcTIM interface for molecules with aromatic heterocyclic
systems.
Molecular modifications, such as isosteric replacement, represent a useful strategy to find
new biologically active compounds and help achieve better pharmacokinetic and
pharmacodynamic properties. Ligand-based drug design is one of the most important
molecular modification tools based on the knowledge of other molecules that bind to the
biological target of interest.16 In this context, our research group has synthesized a large
number of benzimidazole derivatives with antiparasitic activities.17,18 Some of these
compounds present activity as TcTIM inhibitors, such as derivatives (I) and (II) shown in
Figure 1. These compounds presented 52% and 48% inhibition at a concentration of 200
µM, respectively.19
Figure 1. Benzimidazole derivatives and the inhibition presented on TcTIM.
Based on the above study, a set of molecules designed by the isosteric replacement of the
thiazole group by pyrazine, pyrimidine, substituted pyridines and substituted anilines
(Table 1) are proposed in this work. Herein, we present the synthesis, characterization and
TcTIM inhibition activities of these compounds. Furthermore, we selected the compounds
which showed a significant inhibition of TcTIM to describe their anti-T cruzi activity and
cytotoxicity assays.
Table 1.
Chemical structure of the benzimidazole derivates 1–14
Compound
R
Compound
R

pyridin-2-yl
pyridin-4-yl
pyrimidin-2-yl
pyrazin-2-yl
phenyl
1
2
3
4
5
6
7
8
6-chloropyridin-2-yl
6-methylpyridin-2-yl
6-carbamoylpyridin-2-yl
5-nitropyridin-2-yl
5-methoxypyridin-2-yl
5-chloropyridin-2-yl
9
10
11
12
13
14
4-chlorophenyl
thiazol-2-yl
5-nitrothiazol-2-yl
Reaction methyl 6-chloro-2-mercapto-1H-benzimidazole-5-carboxylate (15) with the
appropriate 2-chloroacetamides (16–29) by a straightforward substitution reaction afforded
the corresponding final product 1–14 as shown in Scheme 1. Acetone was used as the
solvent and potassium carbonate as a base. The S-methylation reactions were carried out at
low temperatures to avoid N-methylation. The solubility of the compounds obtained and the
electronic effect of aromatic amines in the 2-chloroacetamide were decisive in the yields
obtained.
Scheme 1. Reagents and conditions: (a) NEt3, CHCl3, 0 °C; (b) K2CO3, Acetone, 0 °C.

In turn, 2-chloroacetamides (16–29) were previously synthetized by condensing 2-
chloroacetyl chloride (30) with the appropriate aromatic amines (31–44). Chloroform and
triethylamine were used under a N2 atmosphere.
On the other hand, the synthesis of the common precursor 15 was carried out following the
reactions outlined in Scheme 2. Compound 15 was prepared by methods previously
described by our research group.20 The synthesis started with commercially available 2-
chloro-4-nitrobenzoic acid (45), which was treated with dimethyl sulfate to obtain methyl
2-chloro-4-nitrobenzoate (46). The catalytic reduction of the nitro group with H2 and Raney
nickel at room temperature afforded methyl 4-amino-2-chlorobenzoate (47), which upon
treatment with Ac2O led to methyl 4-(acetylamino)-2-chlorobenzoate (48). Then, 48 was
treated with HNO3/H2SO4 at 0-5°C to give a mixture of two isomers. The required methyl
4-(acetylamino)-2-chloro-5-nitrobenzoate (49) was isolated from the reaction mixture by
crystallization. Subsequently, the acetylamino group was hydrolyzed with KOH in EtOH,
and methyl 4-amino-2-chloro-5-nitrobenzoate (50) was obtained. It is noteworthy that
following this procedure, no hydrolysis of the methyl esters was observed. Compound (50)
was reduced in a hydrogenation apparatus with H2 in the presence of Raney nickel to give
o-phenylenediamine (51), than upon cyclocondensation with ethyl xanthate, generated in
situ, yield methyl 6-chloro-2-mercapto-1H-benzimidazole-5-carboxylate (15). The nitration
and cyclocondensation reactions are the steps that reduce the total yield of this synthesis.
The former gives the 4-(acetylamino)-2-chloro-3-nitrobenzoate isomer as a by-product, and
the latter gives relative low yields of 15 when the xanthate is generated in situ. The best
yields were obtained when the xanthate was previously formed, and then added to the
reaction mixture.

Scheme 2. Reagents and conditions: (a) (Me)2SO4, NaHCO3, DMF; (b) H2, Raney-Ni,
MeOH; (c) Ac2O, AcOH; (d) HNO3, H2SO4, 0 - 5 ºC; (e) MeOH, KOH: (f) H2, Raney-Ni,
MeOH, AcOEt; (g) CS2, KOH, EtOH.|
In vitro inhibition experiments on TIMs
Previously, it has been observed that benzimidazole derivatives I and II exhibit moderate
inhibitory activity on TcTIM.21 In this research, these compounds were used as templates to
make structural changes to determine the influence of the substituents on the inhibitory
activity. Results obtained from the in vitro evaluation on recombinant TcTIM are
summarized in Table 2.
The results indicate some influence of the substituents on the inhibitory activity of TcTIM.
The presence of a chlorine atom at position 6(5) of the benzimidazole nucleus in compound
14 increases the inhibitory activity on the enzyme (69%) when compared with its analog
compound I (52%). None of the substituents at position 2 of the benzimidazole nucleus was
better than that with a 5-nitrothiazol-2-yl (14), although results obtained from the inhibition
with 10 (pirazin-2-yl) and 12 (4-chlorophenyl) are considered.
The compounds with the highest inhibition of the enzyme are those with a 4-chlorophenyl
group (12) and the heterocyclic substituents pyrazine (10) and nitrothiazole (14); this last
one being the most active inhibitor of the enzyme. Compounds having pyridines (1,8) and
aniline (11) show low to moderate activity. Compounds 3–5, with a 6-methylpyridin-2-yl,
6-carbamoylpyridin-2-yl, 6-nitropyridin-2-yl or pyrimidin-2-yl group (9), have zero
inhibitory activity on the enzyme, while 2 and 7, with chlorine attached to the pyridine ring,
have moderate activity. It is worth mentioning that this group of compounds, except 9, had
low solubilities at 200 µM in DMSO. Hence the study was conducted at a lower
concentration of 100 µM.
Table 2.
In vitro inhibitory activity of compounds (1–14) on TcTIM.

Compound
R
Concentration (µM)
% Inhibition TcTIMa
pyridin-2-yl
1
2
200
100
100
100
100
100
100
200
200
200
200
100
200
200
10
21
0
6-chloropyridin-2-yl
6-methylpyridin-2-yl
6-carbamoylpyridin-2-yl
5-nitropyridin-2-yl
5-methoxypyridin-2-yl
5-chloropyridin-2-yl
pyridin-4-yl
3
4
0
5
0
6
4
7
41
33
0
8
9
pyrimidin-2-yl
10
11
12
13
14
pyrazin-2-yl
68
33
65
59
69
phenyl
4-chlorophenyl
thiazol-2-yl
5-nitrothiazol-2-yl
aInhibition of TcTIM activity was measured indirectly quantifying the amount of NADH
consumed by the reduction of DHAP by α-glycerolphosphate dehydrogenase.22
Figure 3 shows the curves of TcTIM inactivation by compounds 10 and 14, while Table 3
shows the TcTIM and HsTIM inhibitory activity for compounds 7, 10, 12, 13 and 14. These
results confirm that compound 14 has a slightly better capacity for TcTIM inhibition than
compound 10. However, compound 10 has a higher selectivity towards TcTIM than
HsTIM; therefore, this compound is proposed as a potential selective TcTIM inhibitor.
Although 7 and 12 have moderate to good TcTIM inhibitory activities and no activity
against HsTIM, it was not possible to calculate the IC50 because of their low solubilities.

Figure 3. Inactivating curves of compounds 10 and 14. The IC50 for each of the compounds
tested in the inactivation of TcTIM was calculated according to reference.23
Table 3.
In vitro inhibitory activity on HsTIM and TcTIM IC50 values.
% Inhibition
Compunds
IC50 TcTIM (µM)
% Inhibition HsTIMa
TcTIM
41 (100 µM)
---
82
0 (100 µM)
6 (200 µM)
0 (100 µM)
0 (200 µM)
15 (200 µM)
7
68 (200 µM)
65 (100 µM)
59 (200 µM)
69 (200 µM)
10
12
13
14
---
223
77
aThese experiments were done exactly as those carried out for the TcTIM inhibition
described in Ref. 22.
In vitro trypanocidal activity
In vitro trypanocidal assays were performed with compounds 7, 10, 12, 13 and 14 since
these presented the highest inhibitory action on TcTIM. The selected group of compounds
was tested against two different strains of T. cruzi: INC-5 and NINOA isolated from
Instituto Nacional de Cardiología and from a patient with Chagas disease in Oaxaca,
México, respectively24 These compounds were tested against the epimastigote forms of T.
cruzi (Table 4). Although the epimastigote is not the infective form of the parasite, the

trypanocidal activity assay against this form is considered valid since several works
demonstrated that the most active compounds against the epimastigote frequently
correspond to the most active compounds against the infective trypomastigote.25,26
Table 4.
In vitro susceptibility of bloodstream epimastigote to compounds 7, 10, 12–14 and NFX.
Compound
R
IC50 INC-5a (µM) IC50 NINOAa (µM)
CC50b (µM)
5-chloropyridin-2-yl
pyrazin-2-yl
4-chloro phenyl
thiazol-2-yl
95.049 ± 1.749
28.672 ± 0.602
186.230 ± 4.103
48.912 ± 0.787
42.516 ± 0.800
50.750 ± 0.839
123.756 ± 2.786
98.799 ± 1.990
56.967 ± 0.961
119.530 ± 2.151
95.363 ± 2.143
89.804 ± 1.138
82.732 ± 1.609
134.580 ± 1.995
90.436 ± 1.426
104.443 ± 1.326
84.895 ± 1.425
131.503 ± 0.490
7
10
12
13
14
Nfx
5-nitrothiazol-2-yl
---
aThe in vitro trypanocidal assays were carried out according to the method previosly
reported, with slight modifications.27
bCC50: concentration that produces the cytotoxicity of 50% J774 cells.28
As shown in Table 4, compound 10 has the best IC50 against the T. cruzi epimastigote INC-
5 strain, better than that of the reference drug, NFX. Compounds 7 and 12 were the least
effective against the INC-5 strain. For this strain, the presence of the pyrazine ring in 10
increases the inhibition of the parasite considerably with respect to compounds with
pyridine (7), aniline (12) and thiazole (13, 14). The results obtained for the inhibition rate
of TcTIM for compounds 10, 13 and 14, and the data obtained in the test with the parasitic
INC-5 strain, it is evident that for all three compounds, there is a significant correlation
between the inhibition of T. cruzi epimastigotes and TcTIM.
On the other hand, the NINOA strain is more resistant to NFX than the INC-5 strain.
Compound 12 (with 4-chloroaniline) has the best IC50 against the NINOA strain, better
than that of the reference drug, NFX. Substituted compounds 7 (with pyridine), 10 (with
pyrazine) and 13 and 14 (with thiazole) were not better than the reference drug.

Another important criterion in the search for molecules with antiprotozoal activity is their
toxicity to mammalian cells. Hence, the in vitro cytotoxicity of 7, 10, and 12-14 was
determined using mouse macrophages from the J744 cell line. As observed in Table 4,
compound 10 presents a better cytotoxicity profile than NFX and the best profile in the
series. The other compounds (7, 12–14) present slightly higher cytotoxicity than the
reference drug.
In summary, the final compounds (1–14) were obtained in moderate to good yields. Only
four compounds (10, 12, 13, 14) inhibited TcTIM significantly with compound 10 being
distinguished from the others since it presented a good inhibition of TcTIM and a very low
activity against HsTIM. Compound 10 also showed better inhibitory activity than NFX
against T. cruzi epimastigotes of the INC-5 strain, and a more favorable cytotoxicity profile
in mammalian cells.
Acknowledgments
We thank CONACyT for financing project 168718 and for the scholarship granted
(295663/225078) to J.M. Velázquez-López. We are also grateful to Rosa Isela del Villar,
Minerva Monroy Barreto, Marisela Gutiérrez Franco and Georgina Duarte for the analytical
support offered. We also thank Anuar Flores Gahona, Rodrigo Aguayo Ortiz from Facultad
de Química, UNAM and Natalie Crespo Velasco from facultad de CIQ-UAEM for the
review of this paper and the helpful suggestions.
Supplementary data
Supplementary data associated with this article can be found in the online version, at:

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Biological assays against T. cruzi: Epimastigotes forms of the NINOA and INC-5
strains (isolated from a patient with Chagas disease in Oaxaca, Mexico and Instituto
Nacional de Cardiología) of T. cruzi were used in this study. T. cruzi epimastigotes
were grown at 28 °C in a liquid medium containing 0.3% yeast extract, 0.9%

tryptose, 0.4% dextrose, 1% disodium phosphate 2-hydrate, 0.36% sodium chloride,
0.04% potassium chloride, 0.15% powdered beef liver, 0.5% brain heart infusion,
and 0.5 to 1.0 mg/100 ml hemin. Stock solutions (10 mg/mL) in DMSO of each test
compound and reference drug were prepared and subsequent dilutions were done
with sterile distilled water. Experiments were carried out using 96-well microplates
containing 1 × 105 epimastigotes/ml. The compounds were dissolved in dimethyl
sulfoxide (DMSO; final solvent concentration not greater than 1%) and were
evaluated at 100, 50, 10, and 5 g/mL. For each experiment there were controls of
parasites growing in the presence of DMSO. The different mixtures and their
corresponding concentrations were added to the wells, and the plates were incubated
at 28 °C for 24 h. All assays were performed in duplicate. The activity was
evaluated using the resarzurin. The plates were read at 535 nm em, 590 nm ex, on a
multiwell plate spectrophotometer. The activity is expressed as IC50 ( g/mL).
28. Citotoxicity test: mouse macrophages from J744 cell line (J744 cells) was used to
assess the cytotoxic effects on mammalian cells. The cells were cultured in standard
culture medium (DMEM with 10 % Fetal Bovine Serum). The cultures were
incubated for 24 h at 37 °C, 5 % CO2 and 95 % relative humidity with different
concentrations of test compounds and reference drug. The final concentration of
DMSO, used as solvent, remained below 1 % in the culture medium. Untreated cells
were included as controls. The cytotoxicity was determined by the resazurin method.
Briefly, resazurin solution were added to each well and the plates were incubated for
3 h. The fluorescence emission was measured as indicated above, (535 nm em, 590
nm ex), on a multiwall plate spectrophotometer. Cytotoxicity was scored as a percent
of metabolic reduction of treated cultures versus untreated control cells.

Graphical abstract