Synthesis and antiprotozoal activity of nitazoxanide-N-methylbenzimidazole hybrids

Article abstract of DOI:10.1016/j.bmcl.2013.10.011

A series of a novel hybrid compounds between nitazoxanide and N-methylbenzimidazole were synthesized starting from the corresponding N-methyl-2-nitroanilines. The new hybrid compounds (1-13) were evaluated in vitro against Giardia intestinalis, Entamoeba

Full text of DOI:10.1016/j.bmcl.2013.10.011

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6838–6841
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiprotozoal activity
of nitazoxanide–N-methylbenzimidazole hybrids
Olivia Soria-Arteche a,b, Alicia Hernández-Campos b, Lilián Yépez-Mulia c, Pedro Josué Trejo-Soto b,
Francisco Hernández-Luis b, Jorge Gres-Molina b, Luis A. Maldonado d, Rafael Castillo b,
⇑
a Departamento de Sistemas Biológicos, División Ciencias Biológicas y de la Salud, UAM-Xochimilco, México, DF 04960, Mexico
b Facultad de Química, Departamento de Farmacia, UNAM, México, DF 04510, Mexico
c Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS, México, DF 06720, Mexico
d Instituto de Química, UNAM, México, DF 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of a novel hybrid compounds between nitazoxanide and N-methylbenzimidazole were
Received 6 August 2013
Revised 1 October 2013
Accepted 4 October 2013
Available online 10 October 2013
synthesized starting from the corresponding N-methyl-2-nitroanilines. The new hybrid compounds
(1–13) were evaluated in vitro against Giardia intestinalis, Entamoeba histolytica, Trichomonas vaginalis.
NTZ, MTZ and ABZ were used as drug standards. Experimental evaluations revealed all of the new
compounds (1–13) were active and showed strong activity against the three protozoa, particularly with
E. histolytica where the IC50 values ranged between 3 and 69 nM.
Keywords:
Benzimidazole
Nitazoxanide
Hybrid compounds
Entamoeba histolytica
Giardia intestinalis
Trichomonas vaginalis
Overall, compounds 2, 5, 7, 8, 9, 11 and 12 stood out with values lower than 87 nM for all three pro-
tozoa, comparatively better than the reference drugs.
Ó 2013 Elsevier Ltd. All rights reserved.
Parasitic diseases are the second most common cause of mortal-
ity worldwide.1,2 Protozoan infections caused by Entamoeba
histolytica and Giardia intestinalis have a high morbidity and
mortality index in developing countries. The World Health Organi-
zation (WHO) estimates that 50 million and 280 million people
worldwide get infected with these two parasites respectively,
affecting mainly the infant population.3–5 Trichomoniasis, caused
by Trichomonas vaginalis, is not less important. Trichomoniasis is
a sexually transmitted disease for which WHO estimates 170
million new infections annually worldwide.4,5
Current drug therapies to control protozoan infections include
nitroheterocycle derivatives. One of these derivatives is metronida-
zole (MTZ), a nitroimidazole and the drug of choice to treat giardi-
asis, amebiasis and trichomoniasis.5 Common side effects of MTZ
include nausea, diarrhea, numbness, and tingling extremities.6 In
addition, animal and bacterial studies suggest that MTZ may be
mutagenic and carcinogenic.7 Parasite resistance to MTZ has also
been reported.4 It is thus imperative to search for alternative ther-
apeutic options. Although albendazole (ABZ) and mebendazole
(MBZ) have been found effective in vitro against T. vaginalis8 and
G. intestinalis,9 their poor intestinal absorption due to low aqueous
solubility has limited their use as antiprotozoal agents.10
Another commonly antiparasitic drug used is nitazoxanide
(NTZ), a nitrothiazole, that was discovered in 1980 by Rossignol
and was first used against helminthes.11 It was later found to be
a more effective treatment than MTZ against G. intestinalis and E.
histolytica. Currently, it is used as an alternative treatment to ABZ
and MBZ for intestinal helminthes and anaerobic bacterias.12 NTZ
is generally well tolerated, with no severe adverse effects reported
in human trials. Adverse effects are mild and transient, mainly re-
lated to the gastrointestinal tract, and include abdominal pain,
diarrhea, and nausea.13,14
We would like to emphasize that present day therapies still rely
on drugs developed decades ago, and in many cases the clinical
usefulness of these drugs is limited by their poor efficacy and/or
high toxicity. Also, drug resistance is spreading rapidly and the
development of new drugs does not keep up with the needs. The
increasing number of reports of refractory cases with these nitro-
heterocyclic drugs has made the search for new therapeutic agents
of utmost importance.
As part of our research program to find new molecules for the
treatment of these protozoan diseases we have synthesized a large
number of benzimidazole derivatives bearing different substitu-
ents at the positions 1, 2, 5, and 6, and tested them in vitro against
⇑
Corresponding author. Tel.: +52 5 56 22 52 87; fax: +52 5 56 22 53 29.
E-mail address: rafaelc@unam.mx (R. Castillo).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.011

O. Soria-Arteche et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6838–6841
6839
G. intestinalis, E. histolytica and T. vaginalis.15–19 In most cases, the
activity of these compounds against E. histolytica and G. intestinalis
proved to be more effective than MTZ.
products of reduction were immediately cyclocondensed with
HCOOH, CH3COOH, CF3COOH in the presence of catalytic amounts
of HCl to give the corresponding benzimidazole derivative32 20–22
and 25–30,33–35 while the crude product of reduction of 14 was
cyclocondensed with ethyl xantate, generated with CS2, EtOH,
H2O, and KOH,36 to afford the corresponding 2-mercaptobenzimi-
dazole (23),37 that was treated with CH3I to give 2-(methylthio)
derivative 24.38
The antiprotozoal activity of MTZ, NTZ, and ABZ involves differ-
ent mechanisms of action. For MTZ the current evidence suggests
that the mode of action is by reduction of the nitro group to a toxic
nitro radical, via a redox mechanism involving the pyruvate ferre-
doxin oxidoreductase (PFOR) system.20–22 As for NTZ, unlike MTZ,
it has been reported that the mode of action is by specific inhibi-
tion of PFOR and is independent of reduced ferredoxin.23 In addi-
tion, recently it has been reported that MTZ and NTZ can also be
reduced via other nitroreductases, such as thioredoxin reductase
and GlNR1.24–28 Finally, the mode of action of ABZ is the inhibition
of microtubule polymerization by selectively binding to the b-
tubulin monomer of the parasite.29
We now report the synthesis and antiparasitic activity against
G. intestinalis, E. histolytica, and T. vaginalis of novel compounds
1–13 (Fig. 1). We designed these compounds following a molecular
hybridization strategy, which combines the pharmacophoric moie-
ties of different bioactive compounds to generate a new hybrid
molecule that can act on multiple targets, looking for synergic ac-
tion with pharmacokinetic advantages over concomitant adminis-
tration of two different free drugs.30 In our approach we linked, via
a carboxamide group, the pharmacophoric portion of NTZ which
contains the nitrothiazole group, responsible for its antiprotozoal
activity, to N-methylbenzimidazole derivatives which our group
has previously reported with high antiprotozoal activity.15–19
Compounds 1–13 were synthesized according to the pathway
described in Scheme 1.
Hydrolysis of benzimidazole esters 20–27 and 28–3039 with
potassium hydroxide afforded the corresponding carboxylic acids
31–37and 38–40. Carboxylic acids 16, 17 and 19 were hydroge-
nated to the corresponding N-methyl-o-phenylendiamines which
in turn were cyclocondensed with 1,3-dicarbomethoxi-S-meth-
ylisothiourea to give carbamates 40–42, respectively.40–42 In the fi-
nal steps of the sequence, acids 31–42 and 38–40, 43 were then
converted to their corresponding imidazolide derivative using
1,10-carbonyldiimidazole (CDI)43,44 and condensed with 2-amino-
5-nitrothiazole to obtain the final hybrid compounds 1–9 and
10–13, respectively.45 The structure of each hybrid compound syn-
thesized (1–13) was confirmed on the basis of its spectroscopic and
spectrometric data.
Compounds 1–13 were then evaluated in vitro against G. intes-
tinalis, E. histolytica, and T. vaginalis.46 NTZ, MTZ and ABZ were used
as drug standards. The solvent DMSO was run simultaneously as a
negative control.47 The results of the biological assays against these
three protozoa are summarized in Table 1.
All of the new compounds (1–13) were found to be active
against the three protozoa, particularly against E. histolytica where
the IC50 values ranged from 3 to 69 nM.
The starting compounds 14–19 were prepared in our laboratory
from the proper benzoic acid derivative through a series of reac-
tions that include esterification, acetylation, nitration, N-methyla-
tion, and hydrolysis. Reduction of N-methylnitroanilines 14 and
16 with H2, Pd/C (5% or 10%), and 15, 17–19 with Ni-Raney affor-
ded the corresponding N-methyl-o-phenylenediamines.31 These
Overall, compounds 2, 5, 7, 8, 9, 11, and 12 stood out with IC50
values lower than 87 nM for all three protozoa, comparatively bet-
ter than the reference drugs. Against G. intestinalis, compounds 2, 8,
11 and 12 had similar activity to ABZ, while they were more active
than MTZ and less active than NTZ. Against E. histolytica, all hybrids
were more active than ABZ and MTZ, most of them were as active
Figure 1. Design and structure of nitazoxanide–N-methylbenzimidazole hybrids.

6840
O. Soria-Arteche et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6838–6841
Scheme 1. Reagents and conditions: (a) Pd/C 5% or 10%, AcOEt or MeOH; (b) Ni-Raney, MeOH; (c) HCO2H or CH3CO2H or CF3CO2H, HCl (cat), heat, or CS2, KOH–EtOH, H2O, 40–
50 °C, then CH3I, (CH3)CO, K2CO3;(d) KOH 30%, MeOH; (e) CH3O2CNHC(SCH3)NCO2CH3, MeOH, 60 °C; (f) CDI, 2-amino-5-nitrothiazole, solvent, 60 °C.
Table 1
were the most effective of these series against E. histolytica. These
compounds also had the same IC50 value and are therefore consid-
ered bioisosterically equivalent. It is worth noting that the substi-
tuent methylthio in compound 4 featured the best amebicidal
activity of all hybrid compounds. Among all compounds, 4 was
the most selective against E. histolytica.
Compounds with a carbamate group at position 2 of the benz-
imidazole nucleus (5, 9, 13) showed good activity against the three
parasites. Also, the activity of the compound with the carboxamide
group at position 6 was more active than when the group was at
position 5. Finally, when there is a hydrogen at position 2, the anti-
parasitic activity is lower than when there is another substituent.
These results show that molecular hybridization is a valuable
tool for the design of new drugs. Based on the information ob-
tained, compounds 2, 4 and 12 are very promising leads for the de-
sign and development of new antiprotozoal agents.
In vitro susceptibility of G. intestinalis, E. histolytica and T. vaginalis to compounds 1–
13, MTZ, ABZ, and NTZ
IC50
intestinalis
(
l
M) G.
IC50
histolytica
(
l
M) E.
IC50 (lM) T.
vaginalis
1
2
3
4
5
6
7
8
0.254 0.008
0.027 0.002
0.19 0.002
0.218 0.017
0.045 0.004
0.096 0.015
0.071 0.004
0.022 0.001
0.041 0.007
0.039 0.004
0.027 0.002
0.021 0.005
0.118 0.009
0.015 0.002
0.046 0.009
0.069 0.007
0.006 0.004
0.032 0.004
0.003 0.000
0.033 0.006
0.037 0.006
0.026 0.004
0.011 0.005
0.021 0.016
0.027 0.004
0.023 0.000
0.006 0.002
0.060 0.005
0.054 0.002
55.274 18.845
0.429 0.029
0.340 0.015
0.082 0.005
0.356 0.007
0.208 0.007
0.070 0.018
0.129 0.011
0.087 0.006
0.023 0.005
0.055 0.005
0.114 0.010
0.070 0.018
0.041 0.005
0.128 0.002
0.107 0.009
1.656 0.104
0.213 0.004
9
10
11
12
13
NTZ
ABZ
Acknowledgments
MTZ 1.224 0.021
This work was supported by project 43269 and 80093 from
CONACyT. We are grateful to Amparo Tapia for carrying out the
biological assays and to Rosa Isela del Villar, Víctor Manuel Arroyo
Sánchez, Georgina Duarte, Margarita Guzmán, Marisela Gutiérrez,
Nayeli López-Balbiaux and Hugo Lemus for the analytical support.
as NTZ. The hybrids 2, 4 and 12 stood out with activities between 3
and 6 nM. Finally, for T. vaginalis, all hybrids were more active than
ABZ and most of them more active than MTZ and NTZ; in this test,
compounds 2, 8, 9, 11 and 12 surpassed the rest.
Based on the biological activity assays, compounds containing
chlorine were not more effective against protozoan than those
without chlorine. Compounds with a methyl group (2, 7, 11) or a
trifluoromethyl group (3, 8, 12), at position 2 of the benzimidazole
nucleus, performed the best against the three parasites with IC50
values of 6–87 and 6–41 nM, respectively. Compounds 2 and 12
Supplementary data
Supplementary data associated with this article can be found,
in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2013.10.011. These data include MOL files and InChiKeys
of the most important compounds described in this article.

O. Soria-Arteche et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6838–6841
37. Methyl 2-mercapto-1-methyl-1H-benzimidazole-6-carboxylate
6841
The
(23).
References and notes
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acidified to pH 4 with 20% HCl solution. The suspension was filtered and
purified as needed to give the corresponding acid.
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46. Biological assays: Trichomonas vaginalis strain GT3, Giardia intestinalis isolate
IMSS:0981:1 and Entamoeba histolytica strain HM1-IMSS were used in all the
experiments. Trophozoites of G. intestinalis were maintained in a TYI-S-33
medium supplemented with 10% calf serum and bovine bile. E. histolytica and
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with 10% bovine serum. Briefly, 5 ꢀ 104 trophozoites of G. intestinalis or T.
vaginalis, or 6 ꢀ 103 trophozoites of E. histolytica were incubated for 48 h at
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as solutions in DMSO. As a negative control, parasite cultures received an equiv
amount of DMSO only, while ABZ, MTZ and NTZ were included as positive
controls. At the end of the treatment period, the cells were washed and
subcultured for another 48 h in a fresh medium to which no drug was added.
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inhibitory concentration (IC50), together with the respective 95% confidence
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immediately used in the next cyclocondensation reactions.
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heated at the reflux point for 12–18 h under N2. After cooling, the reaction
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obtained was collected by suction filtration and purified by recrystallization
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