Synthesis and biological activity of 2-(trifluoromethyl)-1H-benzimidazole derivatives against some protozoa and Trichinella spiralis

Article abstract of DOI:10.1016/j.ejmech.2010.03.050

A series of 2-(trifluoromethyl)-1H-benzimidazole derivatives (1ae1i) were synthesized via Phillips cyclocondensation of a substituted 1,2-phenylenediamine and trifluoroacetic acid. The synthesized compounds were evaluated in vitro against various protozoan parasites: Giardia intestinalis, Entamoeba histolytica, Trichomonas vaginalis and Leishmania mexicana, and they showed nanomolar activities against the first three protozoa tested. The compounds were also tested in vitro and in vivo against the nematode Trichinella spiralis. Compounds 1b, 1c and 1e had the most desirable in vitro antiparasitic profile against all parasites studied. In the in vivo model against T. spiralis, compounds 1b and 1e showed good activity against the adult phase at 75 mg/Kg. However, against the muscle larvae stage, only compound 1f exhibited in vivo antiparasitic efficacy.

Full text of DOI:10.1016/j.ejmech.2010.03.050

European Journal of Medicinal Chemistry 45 (2010) 3135e3141
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of 2-(trifluoromethyl)-1H-benzimidazole
derivatives against some protozoa and Trichinella spiralis
Francisco Hernández-Luis a, Alicia Hernández-Campos a, Rafael Castillo a, Gabriel Navarrete-Vázquez b,
,
Olivia Soria-Arteche c, Manuel Hernández-Hernández d, Lilián Yépez-Mulia e
*
a Facultad de Química, Departamento de Farmacia, UNAM, México, DF 04510, Mexico
b Facultad de Farmacia, UAEM, Cuernavaca, Morelos 62209, Mexico
c Departamento de Sistemas Biológicos, División Ciencias Biológicas y de la Salud, UAM-X, México, DF 04960, Mexico
d Departamento de Biología Celular, CINVESTAV-IPN, México, DF 07000, Mexico
e Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS, México, DF 06720, 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 2-(trifluoromethyl)-1H-benzimidazole derivatives (1ae1i) were synthesized via Phillips
cyclocondensation of a substituted 1,2-phenylenediamine and trifluoroacetic acid. The synthesized
compounds were evaluated in vitro against various protozoan parasites: Giardia intestinalis, Entamoeba
histolytica, Trichomonas vaginalis and Leishmania mexicana, and they showed nanomolar activities against
the first three protozoa tested. The compounds were also tested in vitro and in vivo against the nematode
Trichinella spiralis. Compounds 1b, 1c and 1e had the most desirable in vitro antiparasitic profile against
all parasites studied. In the in vivo model against T. spiralis, compounds 1b and 1e showed good activity
against the adult phase at 75 mg/Kg. However, against the muscle larvae stage, only compound 1f
exhibited in vivo antiparasitic efficacy.
Received 29 January 2010
Received in revised form
29 March 2010
Accepted 31 March 2010
Available online 7 April 2010
Keywords:
2-(Trifluoromethyl)benzimidazole
derivatives
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antiparasitic activity
1. Introduction
The anthelmintic drugs derived from benzimidazole 2-carba-
mates, such as albendazole (ABZ) and mebendazole (MBZ), are
Parasitic diseases are still worldwide problems that have a deep
impact on public health. Infections caused by protozoa such as
Trypanosoma cruzi, Leishmania mexicana, Plasmodium falciparum,
Giardia intestinalis, Entamoeba histolytica or Trichomonas vaginalis,
and helminths such as Trichinella spiralis or Taenia solium are
worldwide spread diseases that affect mainly underdeveloped
countries, where not only tropical or template temperatures prevail,
but also poor sanitary and hygiene conditions are common [1e3].
In spite of the progress made during the last fifty years, in the
understanding of the biochemistry of the parasites and the deve-
lopment of new antiparasitic agents against these diseases, the
availability of adequate chemotherapies remains unsolved. The
increasing resistance of Plasmodium to antimalarics and the lack of
safe drugs against trypanosomiosis and leishmaniosis promote an
ongoing drug research effort to identify mechanistically novel,
nontoxic, cost effective chemotherapies in the treatment of these
sweeping public health problems [4].
used mainly to treat endoparasitic diseases in domestic animals
and humans. These types of compounds are characterized by a high
therapeutic index and low toxicity; however, they find little use in
tissue-dwelling parasites mainly due to poor solubility and
absorption problems [5].
In order to identify the 2-(trifluoromethyl)-1H-benzimidazole
system as a promising scaffold for the development of new anti-
parasitic agents, we have previously synthesized and tested two
series of derivatives of this compound [6,7] and a third one that
included ABZ and MBZ analogues [8]. In the last series some
derivatives showed in vitro activity against G. intestinalis, T. vaginalis
and T. spiralis, although less than ABZ. In view of the previous
results and continuing with our efforts to obtain more information
about the antiparasitic potential of the 2-(trifluoromethyl)-1H-
benzimidazole system we hereby report the synthesis and evalu-
ation of a series of seven new compounds (1ae1g, Table 1) as direct
analogues of two fasciolicide drugs: triclabendazole (TBZ) (1ae1d)
and Alpha (1ee1g) [9]. In addition, two analogues (1h, 1i) of an
antiprotozoal lead, 5,6-dichloro-2-(methylthio)-1H-benzimidazole,
reported previously by our group (Fig. 1) [10] were evaluated.
Derivatives 1ae1i bear a trifluoromethyl group attached at position
2 of the benzimidazole nucleus instead of a methylthio group.
* Corresponding author. Tel.: þ52 55 56276940; fax: þ52 55 56276949.
E-mail address: lilianyepez@yahoo.com (L. Yépez-Mulia).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.050

3136
F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
Table 1
8a, 8b and 11 by 2,3-dichlorophenol or 1-naphtol followed by cata-
Benzimidazole derivatives synthesized.
lytic reduction led to 10a, 10b and 13a, 13b, respectively. The cyclo-
condensation of these intermediates with CF3CO2H, as described
above, gave title compounds 1ce1e, and 1g. Compound 1e was
treated with CH3I and KOH to furnish a mixture of regioisomers from
which 1f was isolated by silica gel column chromatography.
Compounds 1h,1i, 8a, 8b, 9b,11,12b, TBZ and Alphawere prepared in
our laboratory by known procedures [6,9]. The structure of the new
compounds was established from the spectroscopic and spectro-
metric data. ABZ, pentamidine isethionate salt and metronidazole
(MTZ) were purchased from SigmaeAldrich.
1
R
N
N
CF
3
2
R
3
R
Compounds
R1
R2
R3
MW
M.p (ꢁC)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2,3-Cl2C6H3Oa
H
H
H
347.119
361.145
361.145
381.564
362.733
376.759
376.759
255.024
234.605
172e174
137e139
100e101
211e212
110e112
98e99
100e101
236e238
108e109
2,3-Cl2C6H3Oa
CH3
CH3
H
3. Antiprotozoal activity
H
2,3-Cl2C6H3Oa
2,3-Cl2C6H3Oa
Cl
Cl
Cl
C10H7Ob
H
Compounds 1ae1i were evaluated in vitro against G. intestinalis,
E. histolytica, T. vaginalis and L. mexicana.
C10H7Ob
CH3
CH3
H
Cl
Cl
Cl
C10H7Ob
E. histolytica strain HM1-IMSS and T. vaginalis strain GT3 were
cultured in a TYI-S-33 modified medium supplemented with 10%
calf serum. G. intestinalis strain IMSS:0989:1 was maintained in
a TYI-S-33 medium supplemented with 10% calf serum and bovine
bile. In vitro susceptibility assays were performed using a method
previously described [12]. Briefly: 5 ꢀ 104 trophozoites of G. intes-
tinalis or T. vaginalis or 6 ꢀ 103 trophozoites of E. histolytica were
incubated for 48 h at 37 ꢁC with increasing concentrations of test
compounds. ABZ and MTZ were used as standard drugs, and
dimethyl sulfoxide (DMSO) was used as a suitable solvent and
negative control. After incubation, the trophozoites were washed
and subcultured for another 48 h in a fresh medium alone. At the end
of this period, the trophozoites were counted and the 50% inhibitory
concentration (IC50) was calculated by Probit analysis. Experiments
were carried out in triplicate and repeated at least twice.
Cl
H
CH3
a
2,3-dichlorophenoxy.
1-naphthyloxy.
b
The introduction of
a trifluoromethyl group in bioactive
compounds often improves their pharmacodynamic and pharma-
cokinetic properties. In the former, this group has significant effects
on the binding affinity in drugereceptor complexes; meanwhile in
the latter it increases membrane permeability and stability against
metabolic oxidation [11]. In addition to the 2-trifluoromethyl group,
and in order to assess the influence of a hydrogen atom at position 1
of the benzimidazole nucleus on antiparasitic activity, analogues 1b,
1c and 1f, 1g were designed as 1-methyl regioisomers, respectively.
Furthermore, the antiparasitic activity of compounds 1ae1i was
evaluated against protozoa G. intestinalis, E. histolytica, T. vaginalis,
L. mexicana and against the nematode T. spiralis.
The leishmanicidal activity was performed against L. mexicana
promastigotes strain MHOM/MX92/UAY68. Parasites were cultured
at 26 ꢁC in an RPMI 1640 medium supplemented with 10% fetal
bovine serum, 293 mg/L glutamine, 100 U/mL penicillin,100 mg/mL
streptomycin. Promastigotes (2 ꢀ 104 parasites/mL) were sub-
cultured every 72 h. To test the effect of the compounds on Leish-
mania growth, 2 ꢀ 104 promastigotes/mL were cultured in media
2. Chemistry
The synthetic procedures for the preparation of target compounds
(1ae1g) are outlined in Schemes 1 and 2 and were carried out by
well-known methods reported before [6,8,9]. As shown in Scheme 1,
commercial 1-fluoro-4-nitrobenzene (2) reacted with 2,3-dichlor-
ophenol to give 1,2-dichloro-3-(4-nitrophenoxy)benzene (3); this
was reduced with H2 and Raney-Nickel, treated with Ac2O and then
subjected to nitration to give N-[4-(2,3-dichlorophenoxy)-2-nitro-
phenyl]acetamide (4). N-methylation of 4 with dimethyl sulfate and
KOH afforded N-[4-(2,3-dichlorophenoxy)-2-nitrophenyl]-N-meth-
ylacetamide (6). The hydrolysis of 4 and 6, followed by reductionwith
H2 and Raney-Nickel in ethanol, gave the corresponding 1,2-phe-
nylenediamine (5, 7). These intermediates were treated with
CF3CO2H and HCl as a catalyst to furnish 1a and 1b, respectively. For
the synthesis of compounds 1ce1g the reactions shown in Scheme 2
were followed. The nucleophilic substitution of the chlorine atom in
containing final concentrations of 0.1, 0.5, 1.5, 10 and 20 mg/mL of
each compound for 12, 24 and 48 h. DMSO was used as a negative
control and pentamidine was used as a standard drug. The growth
inhibition assays were performed in triplicate for each individual
concentration. Parasites were fixed in 0.14 M NaCl containing 3.7%
formaldehyde and counted under a light microscope in a Neubauer
hematocytometer. At 48 h, the leishmanicidal effect of compounds
1ae1i was expressed as IC50
.
4. Antitrichinellosis activity
Compounds 1ae1i were evaluated in vitro against nematode
T. spiralis muscle larvae (ML) (systemic phase). ABZ was used as
a standard drug and DMSO as a suitable solvent and negative
Cl
Cl
O
O
Cl
Cl
N
N
N
SCH3
SCH
SCH3
3
N
H
N
H
N
H
Cl
Cl
6-chloro-2-(methylthio)-5-(1-naphthyloxy)-
1H-benzimidazole (Alpha)
triclabendazole (TBZ)
5,6-dichloro-2-(methylthio)-
1H-benzimidazole
Fig. 1. Structures of triclabendazole, Alpha and 5,6-dichloro-2-(methylthio)-1H-benzimidazole.

F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
3137
Cl
Cl
Cl
Cl
Cl
Cl
b,c,d
f, b
g
1a
O
NO
O
O
2
NH
2
NH
NHCOCH
2
NO
3
2
3
4
5
e
a
Cl
Cl
F
f, b
g
1b
Cl
Cl
NO
2
O
NO
2
O
NH
2
2
NHCH
NHCOCH
3
3
CH
3
6
7
Scheme 1. Reagents and conditions: (a) K2CO3, 2,3-Cl2C6H3OH, DMF/H20; (b) H2, Raney-Nickel; (c) Ac2O; (d) HNO3/AcO2; (e) KOH, dimethyl sulfate, dimethoxyethane; (f) KOH,
EtOH, heat; (g) CF3CO2H, HCl (cat), heat.
control. T. spiralis ML were obtained according to the procedure of
Dennis et al. [13]. For the assay, 1000 larvae were placed on culture
plates of 24 wells (Nunclon) which contained RPMI 1640 medium
than ABZ against E. histolytica and T. vaginalis. Furthermore, they
were more active than MTZ against E. histolytica; however, only 1b,
1c and 1e were more active than MTZ against T. vaginalis. In
particular, 1b emerged as the most active derivative against both
parasites, being 39 and 13 times more active than MTZ with IC50
values of 9 nM and 16 nM, respectively. Concerning G. intestinalis, all
the tested compounds were more active than MTZ, among which,
compounds 1ee1g showed better activity than ABZ. It seems that
one important factor contributing to the high giardicidal activity
shown, is the 1-naphthyloxy group at position 5 or 6 of the benz-
imidazole nucleus, implying that an increase of the lipophilicity
(Clog P > 6.0) of the 2-(trifluoromethyl)-1H-benzimidazole system
leads to a lower order of IC50. Compounds 1b and 1d were as active
as ABZ with IC50 values of 30 and 31 nM, respectively.
with 0.18, 0.37 and 1.80 mM of the test compounds. The parasites
were then incubated in a humid 5% CO2 atmosphere at 37 ꢁC for 3
days, the medium and the compounds were changed each day.
After incubation, the viability of the parasites was determined by
the MTT colorimetric method described by Comley et al. [14].
Selected compounds were tested in vivo against the intestinal
(adult worm) and systemic stage (ML) of T. spiralis at 75 mg/Kg. At
the intestinal level, some compounds were also tested at 50 mg/Kg
(Table 3). Biological assays were performed as previously described
[15]. ABZ, TBZ and Alpha were used as standard drugs. Animal
experiments were performed according to Norma Oficial Mexicana
(NOM-062-Z00-1999) published on August 22, 2009.
When derivatives 1ae1i were evaluated against L. mexicana
promastigotes, compounds 1b, 1c, 1e and 1h showed IC50 values
<25
mM, while 1g showed moderate activity (Table 2). The
5. Results and discussion
remainder of the compounds (1a, 1d, 1f, 1i) showed no appreciable
activity. Compounds 1c and 1e were the most effective
5.1. Antiprotozoal activity
(IC50 ¼ 4.10
showed lower leishmanicidal activity than pentamidine
M). Data presented in Fig. 2 show the dose-dependent
mM and 13.78 mM, respectively); nevertheless, they
Derivatives 1ae1i showed amebicidal activity in vitro in the
nanomolar range (Table 2). These compounds were more active
(CI50 ¼ 2.42
m
effect of compounds 1b, 1c, 1e and 1h on L. mexicana promastigotes
growth. In general, the compounds significantly affected the
R1
R2
R1
R2
NO2
R3
viability of the parasite at 10 and 20 mg/mL. At these concentrations,
a
c
1c, 1g
the leishmanicidal activity of compounds 1c, 1e and 1h was similar
at 48 h. Among regioisomers 1b and 1c the activity profile was
similar between 12 and 24 h; however, at 48 h they showed
important differences, being 1c the most active compound. This
behavior may derive from the ability of these compounds to
interact with its receptor and not due to their ability to enter the
parasite since their lipophilicity is the same (Clog P ¼ 5.67) (Table
2). The different activity of compounds 1b and 1c is reflected in
NHCH3
NHCH3
b
b
8a: R1 = H, R2 =Cl
8b: R1 = R2 = Cl
9a: R1 = H, R2 = C6H3Cl2O, R3 = NO2
10a: R1 = H, R2 = C6H3Cl2O, R3 = NH2
9b: R1 = Cl, R2 = C10H7O, R3 = NO2
10b: R1 = Cl, R2 = C10H7O, R3 = NH2
the IC50 value (24 mM vs 4.10 mM, respectively). It is worth noticing
R1
Cl
NH2
NH2
R2
c
that the leishmanicidal activity depended on the presence of the
2,3-diclorophenoxy group at position 6 since compound 1c showed
6 times higher leishmanicidal activity than compound 1b. In
addition, the leishmanicidal activity of compound 1a, with no
methyl at position 1, compared with that of compounds 1b and 1c,
both with a methyl group at position 1, indicates that the methyl
group is critical for antiprotozoal activity. This finding is similar to
results previously obtained against G. intestinalis, E. histolytica and
T. vaginalis with other benzimidazole regioisomers derivatives
[6,8]. Nonetheless, in this study a different behavior was shown by
compounds 1ee1g. The leishmanicidal activity of compound 1e
a
1d, 1e
NO2
Cl
Cl
d
11
b
b
12a: R1 = C6H3Cl2O, R2 = NO2
13a: R1 = C6H3Cl2O, R2 = NH2
12b: R1 = C10H7O, R2 = NO2
13b: R1 = C10H7O, R2 = NH2
1f
Scheme 2. Reagents and conditions: (a) K2CO3, 2,3-Cl2C6H3OH, DMF/H2O, or K2CO3,
1-naphtol, DMF; (b) H2, Raney-Nickel; (c) CF3CO2H, HCl (cat), heat; (d) CH3I, KOH,
acetone.

3138
F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
Table 2
In vitro antiprotozoal and anthelmintic activity of benzimidazole derivatives.
Compounds
Clog Pa
G. intestinalis
(IC50 M)
E. histolytica
(IC50 M)
T. vaginalis
(IC50 M)
L. mexicana
T. spiralis (% reduction,
0.18 M)
T. spiralis (% reduction,
0.37 M)
T. spiralis (% reduction,
1.80 M)
,
m
,
m
,
m
(IC50
NEb
,
mM)
m
m
m
1a
1b
1c
1d
1e
1f
1g
1h
5.36
5.67
5.67
5.88
6.14
6.31
6.31
3.75
3.30
3.07
ꢃ0.02
2.47
0.054
0.030
0.063
0.031
0.005
0.023
0.012
0.078
0.042
0.037
1.228
NDc
0.018
0.009
0.019
0.020
0.019
0.038
0.017
0.011
0.046
56.600
0.350
NDc
0.535
0.016
0.110
NDc
0.086
0.970
0.451
0.235
0.238
1.592
0.216
NDc
20 ꢂ 3
54 ꢂ 2
44 ꢂ 2
NEb
24 ꢂ 4
62 ꢂ 2
48 ꢂ 3
NEb
29 ꢂ 5
80 ꢂ 3
67 ꢂ 2
NEb
24.00
4.10
NEb
13.78
65.23
50.12
20.80
NEb
43 ꢂ 3
15 ꢂ 2
6 ꢂ 2
NEb
50 ꢂ 2
29 ꢂ 1
14 ꢂ 3
NEb
65 ꢂ 3
63 ꢂ 3
18 ꢂ 3
19 ꢂ 2
39 ꢂ 3
67 ꢂ 6
NDc
1i
ABZ
MTZ
pentamidine
NEb
NEb
NEb
58.6 ꢂ 2
61.9 ꢂ 3
NDc
NDc
NDc
2.421
NDc
NDc
NDc
a
Calculated from ACD/Labs software v.9.0 (Advanced Chemistry Development, Inc.).
NE: no effect.
ND: not determined.
b
c
was higher than regioisomers 1f and 1g, indicating that when the
substituent is 1-naphthyloxy at 5 or 6, the presence of the methyl
group at position 1 decreases the antiprotozoal activity.
position 1. The opposite effect, however, was observed in the case of
Alpha analogues 1f and 1g versus 1e. Regarding regioisomers,
1be1c or 1fe1g, their antiparasitic profile was related to the rela-
tive position of the substituent and the methyl group: the 2,3-
dichlorophenoxy group or 1-naphthyloxy group at position 5 led to
more active compounds (1b, 1f) than those with the substituent at
position 6 (1c, 1g) respectively.
It is well known that the lipophilicity of a drug, determined as
Clog P, is an important factor involved in parasite penetration [16].
High lipophilicity values are suggestive of good permeability via
a passive transcellular uptake mechanism. Nonetheless, our data
demonstrate that the increase of Clog P of benzimidazole synthe-
sized (Table 2) did not increase antitrichinellosis activity. In fact,
although ABZ and compound 1b have a Clog P of 3.07 and 5.67
respectively, the percentage of metabolic reduction of T. spiralis ML
5.2. Antitrichinellosis activity
The in vitro activity of 1ae1i against T. spiralis ML is presented in
Table 2. Compound 1b was the most effective nematocidal agent,
reducing the parasite metabolic activity by 54% and 62% at the
lowest concentrations used (0.18 mM and 0.37 mM), compared with
58% and 61.9% shown by ABZ at the same concentrations.
Furthermore, 1b reduced the metabolic activity of T. spiralis ML by
80%, when used at 1.80 mM, being more active than ABZ (67%). The
activity of compounds 1c, 1e and 1f increased with the concen-
tration; in particular, the nematocidal activity of compound 1f
was similar at 0.18 mM and 0.37 mM.
increased from 15% at 0.18 mMe63% at 1.80 mM, while 1a, 1d, 1g, 1h
Compounds 1b, 1e and 1f were further tested in vivo against the
intestinal and systemic stage of T. spiralis (Table 3). No dead animals
were registered during the assays. Compound 1e was the most
and 1i exhibited slight or no activity even at the highest concen-
tration. Interestingly, the gain in potency of TBZ analogues 1b and
1c versus 1a is related to the introduction of a methyl group at
700
600
Cl
Cl
600
500
Cl
Cl
CH
O
500
N
O
N
N
400
CF
CF
N
400
300
200
100
0
CH
300
1c
1b
200
100
0
0
12
24
48
0
12
24
48
hours
hours
700
600
500
400
300
200
100
0
600
500
400
300
200
100
0
Cl
Cl
N
CF
N
H
O
N
CF
1h
N
H
Cl
1e
0
12
24
48
0
12
24
48
hours
hours
Fig. 2. Effect of compounds 1b, 1c, 1e and 1h at different concentrations and incubation time on the growth of L. mexicana promastigotes. Control (A), 0.1
1.5 g/mL ( ), 10 g/mL ( ), 20 g/mL ( ).
mg/mL ( ), 0.5 mg/mL ( ),
m
m
m

F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
3139
Table 3
F254 plates (E. Merck) and visualized by irradiation with a UV lamp.
Silica gel 60 (70e230 mesh) was used for column chromatography.
Infrared (IR) spectra were recorded with a FT Perkin Elmer 16 PC
Percentage of adult and muscle larvae load reduction in T. spiralis infected mice
treated with benzimidazole derivatives.
Compounds
Adult phasea
50 mg/Kga
Muscle larvae phaseb
75 mg/Kga
spectrometer on KBr disks, the frequencies (y) are expressed in
cmꢃ1. 1H NMR and 13C NMR spectra were measured with a Varian
EM-390 spectrometer (operating at 300.06 MHz for 1H and
75.45 MHz for 13C). Chemical shifts are given in parts per million
75 mg/Kga
1b
1e
1f
ABZ
TBZ
Alpha
Control
NDc
69
NRd
62
41
28
0
58
80
36
73
7
46
40
64
63
25
24
0
relative to tetramethylsilane (Me4Si,
d
¼ 0); J values are given in Hz.
Splitting patterns have been designated as follows: s, singlet; d,
doublet; q, quartet; dd, doublet of doublet; t, triplet; m, multiplet;
bs, broad singlet. Mass spectra (MS) were recorded on a JEOL JMS-
SX102A spectrometer by electron impact (EI). The high resolution
mass spectra (HRMS) were performed on a Thermo-Electron Trace
GC Ultra spectrometer. Elemental analyses (C, H, N) for new
compounds were performed on Fisons EA 1108 instrument and
agreed with the proposed structures within ꢂ0.4% of the theore-
tical values. Catalytic hydrogenations were carried out in a Parr
shaker hydrogenation apparatus. Starting materials 1-fluoro-4-
nitrobenzene (2), 2,3-dichlorophenol and 1-naphtol were
commercially available (SigmaeAldrich). The Clog P values were
obtained using ACD/labs software v. 9.07.
NDc
0
a
Groups of six mice were infected with 800 T. spiralis ML and treated at day 3
post-infection (p.i.) with a single dose of the drugs. Animals were sacrificed at day 6
p.i., and adults were obtained as previously described [15].
b
Groups of 10 mice were infected with 800 T. spiralis ML and treated with the
drugs at day 28 p.i. for 7 consecutive days. Animals were sacrificed on day 7 after
treatment, and ML were obtained as previously described [15].
c
ND: not determined.
NR: no reduction.
d
active against T. spiralis adult, reducing the parasite burden by 69%
and 80% at 50 mg/Kg and 75 mg/Kg, respectively. It was more active
than ABZ, TBZ and Alpha. Although compound 1e reduced just 40%
the ML load at the systemic level, it was even more active than TBZ
and Alpha, but not more than ABZ. Regarding compound 1b, at
75 mg/Kg, it reduced the adult and ML burden by 58% and 46%,
respectively, being more active than TBZ and Alpha, and less potent
than ABZ.
It is worth noting that compounds 1b and 1e showed a better
antiparasitic activity profile than their respective analogues TBZ
and Alpha. Significantly, the activity of compound 1f against the
adult worm was lower than 1b, 1e and ABZ; however, it was the
most efficient compound against T. spiralis ML, reducing the para-
site burden by 64%, with activity similar to ABZ (63%). The low
solubility and hence the low bioavailability of compounds 1b and
1e could account for its reduced activity at the systemic level.
It is important to mention that although compound 1f showed
low activity against adult T. spiralis, its activity increased against the
systemic phase of the parasite; hence, this compound possibly
behaved as a prodrug, generating compound 1e by N-demethyla-
tion. A similar process could explain the reduction of the in vivo
activity of compound 1b, which could be a prodrug of compound
1a, that showed scarce in vitro activity.
7.1. General procedure for the synthesis of compounds 1ae1e, 1g
The appropriate 1,2-phenylenediamine (5, 7, 11a, 11b, 13a, 13b,
0.0313 mol), 1.6 eq of CF3CO2H and one drop of concentrated HCl
were heated under reflux in a N2 atmosphere for 3e4 h. After
completion of the reaction, the cooled mixture was neutralized
with saturated NaHCO3 solution and the crude product was
extracted with ethyl acetate. The combined organic extracts were
washed with water, dried with Na2SO4 and the solvent was
removed under vacuum. The residue was dissolved in chloroform,
filtered through Al2O3 neutral type and concentrated under
vacuum to obtain the corresponding benzimidazole.
7.1.1. 5(6)-(2,3-Dichlorophenoxy)-2-(trifluoromethyl)-1H-
benzimidazole (1a)
Recrystallized from cyclohexaneeCH2Cl2 to yield a white crys-
talline solid (80% yield). M.p. 172e174 ꢁC; Rf ¼ 0.26 (CHCl3/MeOH
99.5:0.5, UV); IR (y): 2849, 1633, 1577, 1553, 1494, 1448, 1259, 1180,
1141, 962, 906, 767, 714; 1H NMR (DMSO-d6)
d
: 6.95 (dd, 1H, J ¼ 1.5,
8.1, H-60), 7.11 (dd,1H, J ¼ 2.4, 9.0, H-6), 7.26 (d,1H, J ¼ 2.4, H-4), 7.31
(t, 1H, J ¼ 8.1, 8,4, H-50), 7.40 (dd, 1H, J1 ¼ 1.5, J2 ¼ 8.1, H-40), 7.76 (d,
1H, J ¼ 9.0, H-7), 13.90 (br,1H, exchangeable with D2O, NH); 13C
6. Conclusions
NMR (DMSO-d6)
d
: 104.43, 117.22, 118.32, 118.53 (q, J ¼ 269.30, CF3),
118.87, 124.75, 125.56, 127.61, 134.42, 134.66, 137.20, 141.42 (q,
J ¼ 41, C-2), 154.14, 154.36; MS, m/z (abundance,%): 346 (Mþ, 78),
311 (Mþ ꢃ 35, 100), 348 (Mþ þ 2, 51), 350 (Mþ þ 4, 8). Anal. Calcd
for C14H7Cl2F3N2O: C, 48.44; H, 2.03; N, 8.07. Found: C, 48.75; H,
2.09; N, 8.09.
The data obtained indicate that the 2-(trifluoromethyl)-1H-
benzimidazole system is a good scaffold for the development of
new antiparasitic agents. Derivatives 1ae1i showed good anti-
protozoal activity, most of them higher than choice drugs. The
order of parasite susceptibility found was: E. histolytica > G.
intestinalis > T. vaginalis > L. mexicana. Compounds 1b, 1c and 1e
possessed the most desirable in vitro antiparasitic profile against
all parasites studied. In addition, compound 1f had the highest
activity against the T. spiralis systemic stage similar to ABZ. These
outcomes are very promising and our current efforts will focus on
further evaluation of their antiparasitic activity against other
tissue-dwelling parasites and to clarify the possible mechanism of
action.
7.1.2. 5-(2,3-Dichlorophenoxy)-1-methyl-2-(trifluoromethyl)-1H-
benzimidazole (1b)
White solid (80% yield). M.p. 137e139 ꢁC; Rf ¼ 0.71 (CHCl3/
MeOH 99.5:0.5, UV); IR (y): 1871, 1575, 1491, 1448, 1250, 1234, 1176,
1143, 963, 771, 702; 1H NMR (CDCl3)
d: 3.96 (s, 3H, NCH3), 6.82 (dd,
1H, J1 ¼1.5, J2 ¼ 8.1, H-60), 7.12 (t,1H, J1 ¼8.1, J2 ¼ 8.4, H-50), 7.21 (dd,
1H, J1 ¼ 2.4, J2 ¼ 9.9, H-6), 7.25 (dd, 1H, J1 ¼ 1.2, J2 ¼ 8.1, H-40), 7.40
(dd, 1H, J1 ¼ 0.3, J2 ¼ 2.1, H-4), 7.44 (dd, 1H, J1 ¼ 0.3, J2 ¼ 9.0, H-7);
13C NMR (CDCl3)
d: 31. 02, 110.09, 111.20, 117.17, 118.17, 118.77 (q,
7. Experimental
J ¼ 203.92, CF3), 124.47, 125.27, 127.52, 132.67, 134.36, 141.19, 141.59
(q, J ¼ 38, C-2), 153.27, 154.49; MS, m/z (abundance,%): 360 (Mþ,
100), 325 (Mþ ꢃ 35, 98), 362 (Mþ þ 2, 63), 350 (Mþ þ 4, 8). Anal.
Calcd for C15H9Cl2F3N2O: C, 49.89; H, 2.51; N, 7.76. Found: C, 49.75;
H, 2.58; N, 7.79.
Melting points were determined in open capillary tubes with
a Büchi B-540 melting point apparatus and are uncorrected. Reac-
tions were monitored by TLC on 0.2 mm precoated silica gel 60

3140
F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
7.1.3. 6-(2,3-Dichlorophenoxy)-1-methyl-2-(trifluoromethyl)-1H-
benzimidazole (1c)
1H, H-4), 7.62 (d, J ¼ 8.3, H-40), 7.85e7.91 (m, 1H, H-80), 8.26e8.29
(m, 1H, H-50); 13C NMR (DMSO-d6)
d: 31.13, 111.55, 111.70, 118.38 (q,
Recrystallized from ethanol to yield a white crystalline solid (80%
J ¼ 299.14, CF3), 121.86, 123.47, 124.76, 125.56, 126.06, 126.70,
127.76, 134.94, 142.01 (q, J ¼ 39), 149.66, 152.99; MS, m/z (abun-
dance, %): 376 (Mþ, 100). Anal. Calc for C19H12ClF3N2O: C, 60.57; H,
3.21; N, 7.44. Found C, 60.47; H, 3.24; N, 7.18.
yield). M.p.100e101 ꢁC; Rf ¼ 0.73 (CHCl3/MeOH 99.5:0.5, UV); IR (
y):
3062,1889,1622,1579,1522,1483,1451,1413,1259,1185,1131,1095,
966, 905, 1H NMR (CDCl3)
d
: 3.98 (s, 3H, CH3), 6.89 (dd, 1H, J1 ¼ 1.5,
J2 ¼ 8.1, H-60), 6.98 (d, 1H, J ¼ 2.1, H-7), 7.09 (dd, 1H, J1 ¼ 2.4, J2 ¼ 9.0,
H-5), 7.17 (t, 1H, J1 ¼ 2.4, J2 ¼ 9.0, H-50), 7.30 (dd, 1H, J1 ¼1.5, J2 ¼ 8.1,
7.3. General method of synthesis of 1,2-phenylenediamines 5, 7,
10a, 10b, 13a and 13b
H-40), 7.86 (d, 1H, J ¼ 9.0, H-4); 13C NMR (CDCl3)
d: 30. 98, 99.28,
115.99, 118.27, 118.77 (q, J ¼ 271.10, CF3), 122.76, 124.82, 125.63,
127.69, 134.48, 136.61, 137.21, 141.10 (q, J ¼ 38.40, C-2), 154.18,
154.82; MS, m/z (abundance, %): 360 (Mþ, 100), 325 (Mþ ꢃ 35, 97),
362 (Mþ þ 2, 65), 350 (Mþ þ 4, 9). Anal. Calcd for C15H9Cl2F3N2O: C,
49.89; H, 2.51; N, 7.76. Found: C, 49.75; H, 2.93; N, 7.77.
A mixture of adequate substituted 2-nitroaniline (0.0282 mol),
EtOH (100 mL) and 10% Raney-Nickel was hydrogenated at 25 ꢁC
until cessation of H2 uptake. The catalyst was filtered off on
a Whatman paper number 2, washed with EtOH, and the filtrate
concentrated to provide a dark purple-colored liquid, which was
used immediately in a subsequent step without purification.
7.1.4. 5(6)-Chloro-6(5)-(2,3-dichlorophenoxy)-2-(trifluoromethyl)-
1H-benzimidazole (1d)
White solid (50% yield). M.p. 211e212 ꢁC; Rf ¼ 0.37 (CHCl3/MeOH
7.4. Synthesis of precursors 3, 4, and 6
90:10, UV); IR (y
): 3398, 3039, 1576, 1550, 1447, 1274, 1158; 1H NMR
(CDCl3)
d
: 6.74 (dd,1H, J1 ¼1.5, J2 ¼ 8.1, H-60), 7.13 (t, J1 ¼8.4, J2 ¼ 8.1,
7.4.1. 1,2-Dichloro-3-(4-nitrophenoxy)benzene (3)
1H, H-50), 7.27 (dd, J1 ¼1.5, J2 ¼ 8.1,1H, H-40), 7.36 (s,1H, H-4), 7.86 (s,
A mixture of 2 (30 g, 0.2127 mol, 1 eq), K2CO3 (40 g, 0.2894 mol,
1.36 eq), 2,3-dichlorophenol (41.34 g, 0.2551 mol, 1.19 eq), H2O
(20 mL) and DMF (350 mL) was heated at 110 ꢁC for 5 h. The mixture
was allowed to cool to room temperature and poured into ice bath.
The obtained precipitate was filtered and recrystallized from ethyl
acetate to yield a yellow crystalline solid (57 g, 95% yield). M.p.
1H, H-7); 13C NMR (DMSO-d6)
d: 115.97,116.95,117.96 (q, J ¼ 269.30,
CF3), 120.53, 121.43, 121.70, 124.88, 128.72, 132.97, 147.07, 154.14;
MS, m/z (abundance, %): 380 (Mþ, 48), 345 (Mþ ꢃ 35, 100), 382
(Mþ þ 2, 47), 384 (Mþ þ 4, 16). Anal. Calcd for C14H6Cl3F3N2O: C,
44.07; H, 1.58; N, 7.34. Found: C, 45.75; H, 1.63; N, 7.77.
121e122 ꢁC; IR (
y
): 3436, 3077, 1926, 1787, 1612, 1594, 1571, 1510,
7.1.5. 6(5)-Chloro-5(6)-(1-naphthyloxy)-2-(trifluoromethyl)-1H-
benzimidazole (1e)
1484,1445,1339,1254,1216,1166; 1H NMR (299.7, CDCl3)
d: 6.97 (m,
2H, J1 ¼ 2.4, J2 ¼ 9.6, H-20), 7.09 (dd,1H, J1 ¼1.5, J2 ¼ 8.4, H-4), 7.29 (t,
1H, J1 ¼8.1, J2 ¼ 8.1, H-5), 7.42 (dd,1H, J1 ¼1.5, J2 ¼ 8.1, H-6), 8.22 (m,
2H, J1 ¼ 2.4, J2 ¼ 9.6, H-30); MS, m/z (abundance, %): 283 (Mþ, 100).
White solid (64% yield). M.p. 110e112 ꢁC; IR (
1549, 1441, 1392, 1233, 1191; 1H NMR (DMSO-d6)
y
): 3053, 2924,
: 6.75 (d, 1H,
d
J ¼ 7.2, H-20), 7.40 (dd, 1H, J1 ¼ 7.2, J2 ¼ 7.8, H-30), 7.55e7.63 (m, 2H,
H-60, H-70), 7.57 (s, 1H, H-4), 7.70 (d, 1H, J ¼ 7.8, H-40), 7.96e7.99 (m,
1H, H-80), 8.06 (s,1H, H-7), 8.16e8.19 (m, 1H, H-50), 14 (br, 1H,
7.4.2. N-[4-(2,3-Dichlorophenoxy)-2-nitrophenyl]acetamida (4)
A mixture of 3 (11.5 g, 0.040 mol), MeOH (250 mL) and 10%
Raney-Nickel (2 g) was hydrogenated at 25 ꢁC until cessation of H2
uptake. The reaction mixture was filtered off on a Whatman paper
No. 2, washed with EtOH, and the filtrate concentrated to provide
4-(2,3-dichlorophenoxy)aniline (10 g, 97% yield), which was used
immediately in a subsequent step without purification. A stirred
mixture of this compound (10 g, 0.0393 mol), acetic anhydride
(4.46 mL, 0.4724 mol, 1.2 eq) and three drops of H2SO4 was heated
at 80 ꢁC for 3 min. The mixture was cooled, worked up by addition
of cold water, filtered by suction and the crude product was
recrystallized from EtOH to yield a yellow solid (10.5 g, 90% yield).
To a cold mixture of N-[4-(2,3-dichlorophenoxy)phenyl]acetamide
(8 g, 0.0270 mol) and anhydride acetic (44 mL) was added an HNO3
(3 mL, 70.4%) at 0e10 ꢁC. After the addition, the organic layer was
separated, washed with brine and dried with CaCl2. The obtained
precipitate was filtered and recrystallized from EtOH to yield
a yellow crystalline solid (8.32 g, 90% yield). M.p. 122e124 ꢁC;
exchangeable with D2O, NH); 13C NMR (DMSO-d6)
d: 110.40, 110.98,
118.90 (q, J ¼ 320.10, CF3), 121.28, 123.10, 125.01, 125.98, 126.29,
126.88, 127.85,134.54, 141.91 (q, J ¼ 41, C-2), 148.65, 152.76; MS, m/z
(abundance, %): 362 (Mþ, 100). HRMS Calcd for C18H10ClF3N2O
362.733. Found 362.0454.
7.1.6. 5-Chloro-1-methyl-6-(1-naphthyloxy)-2-(trifluoromethyl)-
1H-benzimidazole (1g)
White solid (71% yield). M.p. 98e99 ꢁC; IR (
y): 3053, 1524, 1390,
1239; 1H NMR (CDCl3)
d
: 3.93 (s, 3H, NCH3), 6.84 (dd, 1H, J1 ¼ 7.4,
J2 ¼ 0.8, H-20), 6.97 (s, 1H, H-7), 7.38 (dd, 1H, J1 ¼ 7.8, J2 ¼ 8.0, H-30),
7.50e7.59 (m, 2H, H-60, H-70), 7.67 (d, J ¼ 8.4, H-40), 7.88e7.93 (m,
1H, H-80), 8.02 (s, 1H, H-4), 8.22e8.27 (m, 1H, H-50); 13C NMR
(DMSO-d6) d: 31.11, 100.94, 112.45, 121.84, 123.88, 125.64, 126.30,
126.87, 127.81, 134.99, 137.32, 151.43, 152.75; MS, m/z (abundance,
%): 376 (Mþ, 88), 341 (Mþ ꢃ 35, 100). Anal. Calc for C19H12ClF3N2O:
C, 60.57; H, 3.21; N, 7.44. Found C, 60.34; H, 3.22; N, 7.21.
Rf ¼ 0.5 (CHCl3/MeOH 99.5:0.5, UV); IR (
y
): 3372, 3085, 1693, 1586,
: 2.29 (s, 3H, COCH3),
1520, 1445, 1344, 1256, 970; 1H NMR (CDCl3)
d
7.2. 6-Chloro-1-methyl-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-
benzimidazole (1f)
6.96 (dd, 1H, J1 ¼ 1.5, J2 ¼ 8.1, H-60), 7.23 (t, 1H, J1 ¼ 8.4, J2 ¼ 8.4, H-
50), 7.29 (dd, 1H, J1 ¼ 3.0, J2 ¼ 9.3, H-5), 7.36 (dd, 1H, J1 ¼1.5, J2 ¼ 8.4,
H-40), 7.73 (d, 1H, J ¼ 2.7, H-3), 8.75 (d, J ¼ 9.3, H-6), 10.19 (br, 1H,
NH); MS, (abundance, %): m/z 340 (Mþ, 30), 298 (Mþ ꢃ 42, 100).
Into a stirred mixture of 1e, methyl iodide and acetone was
added a solution of KOH 50% m/v. At the end of the reaction the
mixture was worked up by addition of cold water. The precipitate
was removed by suction and washed several times with water until
neutral pH. The solid residue was a mixture of regioisomers 1f and
1g. Title compound 1f was obtained as a white powder by flash
chromatography. White solid (11% yield). M.p. 100e101 ꢁC; IR
7.4.3. N-[4-(2,3-Dichlorophenoxy)-2-nitrophenyl]-N-
methylacetamide (6)
Into a stirred mixture of 4 (8.30 g, 0.0243 mol) in dimethyl
sulfate (4.60 g, 3.46 mL, 0364 mol, 1.5 eq) and monoglyme (15 mL)
was added a solution of KOH 50% m/v (2.04 g, 0.0364 mol, 1.5 eq) at
32e35 ꢁC. The resulting solution was stirred for 3 h at 32 ꢁC. The
mixture was cooled, worked up by addition of cold water and
extracted with EtOAc. The combined organic extracts were washed
(y d: 3.93 (s, 3H,
):1522, 1483, 1393, 1238, 1186; 1H NMR (CDCl3)
NCH3), 6.76 (dd, 1H, J1 ¼ 7.0, J2 ¼ 0.8, H-20), 7.33 (dd, 1H, J1 ¼ 7.7,
J2 ¼ 8.0, H-30), 7.47(s, 1H, H-7), 7.48e7.57 (m, 1H, H-6, H-70), 7.60 (s,

F. Hernández-Luis et al. / European Journal of Medicinal Chemistry 45 (2010) 3135e3141
3141
with brine, dried with anhydrous Na2SO4 and concentrated in
from the Facultad de Química, USAI, UNAM, for the determination
of all spectra.
vacuo to give a yellow solid (19 g, 95% yield). M.p. 134e135 ꢁC;
Rf ¼ 0.2 (CHCl3/MeOH 99:1, UV); IR (
y
): 3072, 3040, 2930, 1957,
: 1.83 (s, 3H, COCH3),
Role of the funding source: This study was supported by grants
from PAPIIT-UNAM IN210809, CONACYT 80093.
1665, 1541, 1451, 1428, 1267; 1H NMR (CDCl3)
d
3.19 (s, 3H, NeCH3), 7.11 (dd, 1H, J1 ¼1.5, J2 ¼ 8.1, H-60), 7.20 (dd, 1H,
J1 ¼ 2.7, J2 ¼ 8.7, H-5), 7.26 (t, 1H, J1 ¼ 8.1, J2 ¼ 8.1, H-50), 7.33 (d, 1H,
J ¼ 8.7, H-6), 7.44 (dd, 1H, J1 ¼ 1.5, J2 ¼ 8.1, H-40), 7.48 (d, J ¼ 2.3, H-
3); MS, m/z (abundance, %): 354 (Mþ, 1), 308 (Mþ ꢃ 46, 100).
References
[1] A.R. Renslo, J.H. McKerrow, Drug discovery and development for neglected
parasitic diseases. Nat. Chem. Biol. 212 (2006) 701e710.
[2] E. Pozio, World distribution of Trichinella spiralis. Infections in animals and
humans. Vet. Parasitol. 149 (2007) 3e21.
7.5. Synthesis of precursors 9a and 12a
[3] H.H. García, A.E. González, R.H. Gilman, Diagnosis, treatment and control of
Taenia solium cisticercosis. Curr. Opin. Infect. Dis. 16 (2003) 411e419.
[4] M.H. Gelb, W.G. Hol, Parasitology. Drugs to combat tropical protozoan para-
sites. Science 297 (2002) 343e344.
[5] G.C. Cook, Use of benzimidazole chemotherapy in human helminthiases:
indication and efficacy. Parasitol. Today 6 (1990) 133e136.
[6] G. Navarrete-Vázquez, R. Cedillo, A. Hernández-Campos, L. Yepez,
F. Hernández-Luis, J. Valdéz, R. Morales, R. Cortés, M. Hernández, R. Castillo,
Synthesis and antiparasitic activity of 2-(trifluoromethyl)benzimidazole
derivatives. Bioorg. Med. Chem. Lett. 11 (2001) 187e190.
[7] G. Navarrete-Vázquez, M. Rojano-Vilchis, L. Yépez-Mulia, V. Meléndez,
L. Gerena, A. Hernández-Campos, R. Castillo, F. Hernández-Luis, Synthesis and
antiprotozoal activity of some 2-(trifluoromethyl)-1H-benzimidazole bio-
isosteres. Eur. J. Med. Chem. 41 (2006) 135e141.
[8] G. Navarrete-Vázquez, L. Yépez, A. Hernández-Campos, A. Tapia, F. Hernández-
Luis, R. Cedillo, J. González, A. Martínez-Fernández, M. Martínez-Grueiro,
R. Castillo, Synthesis and antiparasitic activity of albendazole and mebendazole
analogues. Bioorg. Med. Chem. 11 (2003) 4615e4622.
[9] A. Hernández-Campos, F. Ibarra-Velarde, Y. Vera-Montenegro, N. Rivera-Fer-
nandez, R. Castillo, Synthesis and fasciolicidal activity of 5-chloro-2-methylthio-
6-(1-naphthyloxy)-1H-benzimidazole. Chem. Pharm. Bull. 50 (2002) 649e652.
[10] J. Valdez, R. Cedillo, A. Hernández-Campos, L. Yepez-Mulia, F. Hernández-Luis,
G. Navarrete, A. Tapia, R. Cortés, M. Hernández, R. Castillo, Synthesis and
antiparasitic activity of 1H-benzimidazole derivatives. Bioorg. Med. Chem.
Lett. 12 (2002) 2221e2224.
[11] K.L. Kirk, Fluorine in medicinal chemistry: recent therapeutic applications of
fluorinated small molecules. J. Fluor. Chem. 127 (2006) 1013e1029.
[12] R. Cedillo-Rivera, B. Chavez, A. Gonzalez-Robles, A. Tapia, L. Yepez-Mulia, In
vitro effect of nitazoxanide against Entamoeba histolytica, Giardia intestinalis and
Trichomonas vaginalis trophozoites. J. Eukaryot. Microbiol. 49 (2002) 201e208.
[13] D.T. Dennis, D.D. Despommier, N. Davis, Infectivity of the newborn larvae of
Trichinella spiralis in the rat. J. Parasitol. 56 (1970) 974e977.
[14] J.C. Comley, S. Townson, M.J. Rees, The further application of MTT-formazan
colorimetry to studies on filarial worm viability. Trop. Med. Parasitol. 40
(1989) 311e316.
A mixture of 8a or 11 (0.0536 mol, 1 eq), K2CO3 (8.14 g,
0.0589 mol, 1.1 eq), 2,3-dichlorophenol (9.61 g, 0.0589 mol, 1.1 eq),
H2O (8 mL) and DMF (60 mL) was heated at 125 ꢁC for 3 h. The
mixture was allowed to cool to room temperature and poured into
ice bath. The obtained precipitate was filtered.
7.5.1. 5-(2,3-Dichlorophenoxy)-N-methyl-2-nitroaniline (9a)
Recrystallized from ethanol to yield a crystalline solid (12.4 g,
81% yield). M.p. 124e126 ꢁC; Rf ¼ 0.27 (hexane/EtOAc 85:15, UV); IR
(y
): 3386, 1631, 1578, 1566, 1508, 1448, 1328, 1258, 1218, 842; 1H
NMR (CDCl3)
d
: 2.94 (s, 3H, NeCH3), 6.13 (dd, 1H, J1 ¼ 2.4, J2 ¼ 9.6,
H-4), 6.27 (d, 1H, J ¼ 2.7, H-6), 7.07 (dd, 1H, J1 ¼ 1.2, J2 ¼ 8.7, H-60),
7.26 (t, 1H, J1 ¼ 8.4, J2 ¼ 8.4, H-50), 7.39 (dd, 1H, J1 ¼ 1.2, J2 ¼ 8.1, H-
40), 8.16 (d, 1H, J ¼ 9.1, H-3), 10.49 (br, 1H, NeH); MS, m/z (abun-
dance, %): 312 (Mþ, 100).
7.5.2. 4-Chloro-5-(2,3-dichlorophenoxy)-2-nitroaniline (12a)
Recrystallized from benzeneeethanol to yield an orange crys-
talline solid (12.5, 70% yield). M.p. 149e150 ꢁC; Rf ¼ 0.59 (hexane/
CHCl3/EtOAc 50:35:15, UV); IR (
y
): 3465, 3344, 3174, 3112, 3039,
1559, 1546, 1224; 1H NMR (CDCl3)
d: 5.93 (s, 1H, H-6), 6.13 (br, 1H,
exchangeable with D2O, NeH), 7.09 (dd, 1H, J1 ¼ 1.80, J2 ¼ 1.2,
J3 ¼ 8.1, H-60), 7.29 (t, 1H, J1 ¼ 8.4, J2 ¼ 8.4, H-50), 7.42 (dd, 1H,
J1 ¼1.8, J2 ¼ 1.2, J3 ¼ 8.1, H-40), 8.26 (s,1H, C-3); MS, m/z (abundance,
%): 332 (Mþ, 94), 262 (Mþ ꢃ 70, 100).
[15] L. Yépez-Mulia, R. Morales-Hurtado, N. Viveros-Guzmán, R. Cedillo,
F. Hernández-Luis, R. Castillo, F. Hernández-Luis, R. Castillo, A. Hernández,
O. Muñoz, Evaluation of albendazole prodrugs in experimental trichinellosis.
Arch. Med. Res. 30 (1999) 368e374.
[16] L.I. Alvarez, M.L. Mottier, C.E. Lanusse, Drug transfer into target helminth
parasites. Trends Parasitol. 23 (2007) 97e104.
Acknowledgments
We are grateful to Rosa Isela del Villar, Georgina Duarte,
Margarita Guzmán, Nayeli López Balbiaux and Marisela Gutiérrez