Synthesis and antiparasitic activity of 2-(Trifluoromethyl)benzimidazole derivatives

Article abstract of DOI:10.1016/S0960-894X(00)00619-3

2-(Trifluoromethyl)benzimidazole derivatives substituted at the 1-, 5-, and 6-positions have been synthesized and in vitro tested against the protozoa Giardia lamblia, Entamoeba histolytica, and the helminth Trichinella spiralis. Results indicate that all the compounds tested are more active as antiprotozoal agents than Albendazole and Metronidazole. One compound (20) was as active as Albendazole against T. spiralis. These compounds were also tested for their effect on tubulin polymerization and none inhibited tubulin polymerization.

Full text of DOI:10.1016/S0960-894X(00)00619-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 187±190
Synthesis and Antiparasitic Activity of 2-(Tri¯uoromethyl)-
benzimidazole Derivatives
a
b
a
Gabriel Navarrete-Vazquez, Roberto Cedillo, Alicia Hernandez-Campos,
 Â
b
a
a
b
Lilia
Â
n Ye
Â
Rafael Corte
Â
pez, Francisco Hernandez-Luis, Juan Valdez, RauÂ
c
s, Manuel Hernandez and Rafael Castillo
Â
l Morales,
a,Ã
c
Â
aDepartamento de Farmacia, Facultad de QuõÂmica, UNAM, CU, Mexico D.F. 04510, Mexico
bUnidad de InvestigacioÂn M e dica en Enfermedades Infecciosas y Parasitarias, IMSS, Mexico D.F. Mexico
cDepartamento de BiologõÂa Celular, CINVESTAV, IPN. Mexico D.F. Mexico
Received 23 August 2000; revised 1 November 2000
AbstractÐ2-(Tri¯uoromethyl)benzimidazole derivatives substituted at the 1-, 5-, and 6-positions have been synthesized and in vitro
tested against the protozoa Giardia lamblia, Entamoeba histolytica, and the helminth Trichinella spiralis. Results indicate that all the
compounds tested are more active as antiprotozoal agents than Albendazole and Metronidazole. One compound (20) was as active
as Albendazole against T. spiralis. These compounds were also tested for their e€ect on tubulin polymerization and none inhibited
tubulin polymerization. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Since the sites of action of the di€erent benzimidazole
carbamates are the same, these compounds have addi-
tive action and show crossed resistance, which may
undermine their future therapeutic value.10
Parasitic infections, such as helminthiosis and proto-
zoosis, are still a major problem in developing countries,
a€ecting mainly the infant population. Recent studies
have shown that benzimidazole carbamates, Albenda-
zole and Mebendazole, used mainly as anthelmintic
agents, tested in vitro are e€ective against Giardia lam-
blia and Trichomonas vaginalis, but not against Enta-
As part of our search for basic information about the
structural requirements for antiprotozoal and anthel-
mintic activity, we have synthesized the 2-(tri-
¯uoromethyl)benzimidazole derivatives 16±22 (Table 1).
The in vitro antiparasitic activity of these compounds
on two intestinal protozoa (Giardia lamblia and Enta-
moeba histolytica) and one tissue-dwelling helminth
1�3
moeba histolytica. Clinical assays have demonstrated
that Albendazole is as e€ective as Metronidazole for
4�6
giardiosis.
(Trichinella spiralis) is presented. The activity of 16±22
on rat brain tubulin polymerization is also reported.
The anthelmintic activity of benzimidazole carbamates
has been related to their selective antimitotic activity,
due to the preferential binding to helmintic tubulin over
7
mammalian tubulin. Similarly, the action of Albenda-
zole against G. lamblia also involves the interaction with
tubulin of the Giardia cytoskeleton and other parasitic
Chemistry
The required substituted 1,2-phenylenediamines (9±15)
were prepared by reduction of the corresponding 2-
nitroanilines (1±8) with H , Raney-nickel. The product
2
8
protozoa. It is suggested that one of the requirements
for this action is that the substituted benzimidazole
bears a hydrogen atom at the 1-position and a methoxy-
of reduction, without isolation, was cyclocondensed
with 50% aqueous tri¯uoroacetic acid to give the cor-
responding 2-(tri¯uoromethyl)benzimidazole derivative
9
carbonylamino at the 2-position.
(
by treatment of the symmetrical precursors 16 and 18
with iodomethane, respectively (Scheme 1).
16±22).11 Compounds 19 and 22 could also be prepared
Ã
Corresponding author. Fax: +1-52-5-6-22-53-29; e-mail: rafaelc@
servidor.unam.mx
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00619-3

1
88
G. Navarrete-V a zquez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 187±190
4
3
Solid compounds were puri®ed by recrystallization. The
structure of the puri®ed compounds was established by
spectroscopic and spectrometric data.
Brie¯y: 5Â10 trophozoites of G. lamblia or 6Â10 tro-
phozoites of E. histolytica were incubated for 48 h at
37 C with increasing concentrations of compounds 16±
19�25
ꢀ
2
2, Albendazole and Metronidazole. As the negative
control, trophozoites were incubated with solvent
dimethylsulphoxide) used in the experiments. After the
(
Biological Assays
Culture
incubation, the trophozoites were washed and sub-
cultured for another 48 h in fresh medium alone. At the
end of this period, trophozoites were counted and the
50% inhibitory concentration (IC ) was calculated by
Probit analysis. Experiments were carried out in tripli-
cate and repeated at least twice.
G. lamblia strain IMSS:0989:1 was cultured in TYI-S-33
modi®ed medium, supplemented with 10% calf serum
50
1
and bovine bile. E. histolytica strain HM1-IMSS was
maintained in TYI-S-33 medium supplemented with
1
performed using a method previously described.
0% bovine serum.12 In vitro susceptibility assays were
T. spiralis muscle larvae (parenteral phase) were
13
obtained according to the procedure of Dennis et al.
1
For the assay, 1000 larvae were placed in culture plates
of 24 wells (Nunclon), which contained RPMI 1640
medium, with 0.0001, 0.001, 0.01, 0.1, and 1 mg/mL of
Table 1. Synthesized 2-(tri¯uoromethyl)benzimidazoles (16±22)
the compounds tested. The parasites were then incu-
ꢀ
bated in a humid 5% CO atmosphere at 37 C for 3
2
days, changing the medium and the compounds each
day. Albendazole was used in this test as a positive
control, and the solvent employed as a negative control.
After the incubation, the viability of the parasites was
determined by the colorimetric method described by
Townson et al.,14 with some modi®cations.
R1
R2
R3
Compound
15
16
17
18
19
20
21
22
H
Cl
Cl
H
Cl
H
H
H
Cl
H
H
Cl
Cl
H
H
H
CH
CH
CH
Inhibition of tubulin polymerization assay
3
3
3
Sixty Winstar rats were sacri®ced with an intra-perito-
neal overdose of sodium pentobarbital (200 mg/kg body
weight) and their brains were quickly removed and
homogenized in RB bu€er (MES 0.1 M, pH 6.8, EGTA
Cl
CH
3
1
mM, MgCl 1 mM). Tubulin was puri®ed according to
2
16
the methods reported by Shelanski. Tubulin poly-
merization was carry out by low shear falling ball
viscosimetry, calibrated previously with glycerol solu-
tions of known viscosity in vitrex capillary tubes
(
100 mL, 1.3 mm diameter). In this assay, the poly-
merization of tubulin is indirectly calculated by relative
viscosity.17 The inhibition of polymerization was asses-
sed after the incubation of tubulin with di€erent con-
centrations of compounds 16±22 and Albendazole.
Dimethylformamide (DMF) was used as solvent. Data
1
8
calculation and analysis were performed as described.
Results and Discussion
Compounds 16±22 could be obtained in good yields as
white solids with sharp melting points. The spectro-
scopic and spectrometric data are consistent with the
expected structures.
Biological assay results shown in Table 2, indicate that,
with exception of 17, all compounds were more active
against G. lamblia than Metronidazole (drug of choice
in the treatment of giardiosis and amoebiasis); com-
pound 20 was as active as Albendazole. In relation to the
activity against E. histolytica, all compounds were more
active than Metronidazole and even Albendazole. It is
interesting to mention that compound 21 (6-chloro-1-
methyl-2-(tri¯uoromethyl) benzimidazole) was 43 and
Scheme 1. Synthesis of 2-(tri¯uoromethyl)benzimidazole derivatives
16±22).
(

G. Navarrete-V a zquez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 187±190
189
7
000 times more active than Metronidazole and Alben-
Regarding the anthelmintic activity of 16±22, it is inter-
esting to note that 20 had similar activity than Alben-
dazole against T. spiralis muscle larvae, and that this
compound also showed a very good activity against
both protozoa tested (Table 3).
dazole, respectively (it is known that Albendazole is not
active against E. histolytica). On the other hand, com-
pounds 19±22, with a methyl group at the 1-position,
were as active against E. histolytica as 16±22 with an H
at that position, suggesting that the H at the 1-position
is not necessary for antiprotozoal activity. The fact that
The tubulin polymerization data shown in Table 4 indi-
cate that Albendazole inhibited the polymerization of
tubulin at low concentrations, whereas 16±22 did not
upset polymerization of tubulin, even at high con-
centrations. These results support the idea that one of
the requirements for the action on tubulin of the 5(6)-
substituted benzimidazole is the presence of an hydrogen
atom at the 1-position and a methoxycarbonylamino at
19±22 had very good activity against E. histolytica, an
protozoan in which the major cytoskeleton component
is actin, instead of tubulin, suggests a di€erent mechan-
ism of action from that of 2-benzimidazole carbamate
compounds.
9
Table 2. In vitro susceptibility of Giardia lamblia and Entamoeba
histolytica to compounds 16±22, Metronidazole and Albendazole
the 2-position. Compounds 16±18 have an H in the 1-
position but do not possess a methoxycarbonylamino at
the 2-position, and compounds 19±22 do not bear any
of the radicals mentioned in these positions. These facts
suggest that the mechanism of action of carbamate
benzimidazoles is di€erent of that of compounds 16±22.
It means that the hydrogens in the 1- and 2-positions
are important for the binding of benzimidazoles to
tubulin but not for the antiparasitic e€ect.
Compound
G. lamblia IC50
mM)
E. histolytica IC50
(mM)
(
Metronidazole
Albendazole
1.220
0.037
0.107
1.282
0.078
0.064
0.042
0.127
0.260
0.350
56.330
0.069
0.022
0.011
0.040
0.046
0.008
0.033
1
1
1
1
2
2
2
6
7
8
9
0
1
2
The results obtained with compounds 16±22 as anti-
protozoal agents are very promising, since they broaden
the knowledge about the activity of these versatile deri-
vatives of benzimidazole. It is also important to
emphasize that the results of inhibition of polymeriza-
tion of tubulin give us a good idea about the di€erences in
the mode of action of di€erent benzimidazoles. Further
studies in this area are in progress in our laboratory.
Table 3. Percentage of viability reduction of T. spiralis muscle larvae
after 3 days of incubation with compounds 16±22 and Albendazolea
3Â10�
mM
6
3Â10
� 5
3Â10
� 4
3Â10
� 3
Compound
mM
mM
mM
Albendazole
6
nr
nr
nr
nr
2
13
1
nr
1
1
9
20
3
nr
9
30
9
b
16
17
18
19
20
21
22
Acknowledgements
nr
19
12
39
nr
15
We thank CONACyT for ®nancing project 25920M,
and DGAPA IN204998. We are grateful to Rosa Isela
Â
del Villar, Georgina Duarte, Margarita Guzman, and
9
16
nr
9
nr
2
nr
4
Marisela Gutierrez from School of Chemistry, UNAM,
Â
for the determination of all spectra. Also, we thank
Amparo Tapia for carrying out the biological assays.
a
Values are means of three experiments.
nr, no reduction observed.
b
Table 4. E€ect of compounds 16±22 and Albendazole in tubulin
polymerization
References and Notes
Viscositya
1
. Cedillo-Rivera, R.; Mun
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. Chavez, B.; Cedillo-Rivera, R.; Martinez-Palomo, A. J.
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Ä
oz, O. J. Med. Microbiol. 1992, 37,
Compound
3Â10�
5
3Â10
� 3
0.015
b
0.024
b
b
b
mM
mM
mM
mM
2
Albendazole 7.04 (Æ0.7) 2.07 (Æ0.1)
ndc
nd
3
1
. Fears, S. D.; O'Jare, J. Antimicrob. Agents Chemother.
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1
1
1
1
2
2
2
6
7
8
9
0
1
2
7.55 (Æ0.3) 7.56 (Æ0.1) 7.34 (Æ0.2) 6.92 (Æ0.1)
6.59 (Æ0.6) 6.70 (Æ0.3) 7.14 (Æ0.4) 6.47 (Æ0.1)
7.00 (Æ0.2) 7.03 (Æ0.1) 6.68 (Æ0.7) 7.22 (Æ0.1)
6.68 (Æ0.9) 7.41 (Æ0.3) 7.31 (Æ0.3) 7.16 (Æ0.1)
7.30 (Æ0.2) 7.58 (Æ0.9) 6.94 (Æ0.2) 7.54 (Æ0.3)
6.44 (Æ0.8) 7.39 (Æ0.1) 7.07 (Æ0.7) 7.26 (Æ0.7)
6.93 (Æ0.6) 7.63 (Æ0.9) 6.63 (Æ0.3) 6.94 (Æ0.7)
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. Rodriguez-Garcia, R.; Aburto-Bandala, M.; Sanchez-Mal-
donado, M. Bol. Med. Hosp. Infant. Mex. 1996, 53, 173.
Ä
oz-Garcia, R.;
6
aTubulin viscosity after the polymerization assay, without any com-
pound or Albendazole=7.66 (it represents 100% polymerization). The
incubation of tubulin with the solvent used, DMF 1%=7.42.
Values are means of three experiments, standard deviation is given in
parentheses.
7
1
. Friedman, P. A.; Platzer, E. G. Biochem. Biophys. Acta
990, 630, 271.
b
8. Reynoldson, J. A.; Thompson, R. C.; Horton, R. J. Para-
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c
nd, not determined

1
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1
1
0. Singh, S.; Sharma, S. Med. Res. Rev. 1991, 11, 581.
1. Holan, G.; Samuel, E. L.; Ennis, B. C.; Hinde, R. W. J.
6
NMR (300 MHz, DMSO-d ) d 13.41 (bs, 1H), 7.67 (bs, 2H),
+
7.292±7.38 (dd, 1H, J=8.6 Hz); MS: m/z 220 M .
21. 5,6-Dichloro-2-(tri¯uoromethyl)-1H-benzimidazole (18).
Chem. Soc. 1967, 20.
2. Chavez, B.; Espinoza, M.; Cedillo, R.; Ramirez, A.; Mar-
tinez, A. Arch. Med. Res. 1992, 23, 63.
ꢀ
1
1
Toluene (yield 84.5%); mp 236±238 C; H NMR (300MHz,
+
DMSO-d ) d 7.692 (s, 1H), 8.004 (s, 1H); MS: m/z 220 M .
6
1
1
1
3. Dennis, D. T.; Despommier, D. D.; Davis, N. J. Parasitol.
970, 56, 974.
4. Townson, D. H.; Morris, D. L. Trans. Roy. Soc. Trop.
22. 1-Methyl-2-(tri¯uoromethyl)benzimidazole (19). Ethanol±
ꢀ
1
water (yield 85.2%); mp 98±100 C; H NMR (300 MHz,
DMSO-d ) d 7.8 (d, 1H), 7.76 (d, 1H), 7.465 (t, 1H), 7.365 (t,
1H), 3.91 (s, 3H); MS: m/z 200 M .
6
+
Med. Hyg. 1989, 83, 664.
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1
Ä
oz, O. Arch. Med.
23. 5-Chloro-1-methyl-2-(tri¯uoromethyl)benzimidazole (20).
ꢀ
1
Petroleum ether (yield 80.7%); mp 108±109 C; H NMR
(300 MHz, DMSO-d ) d 7.85 (d, 1H, J=2 Hz), 7.42 (dd, 1H,
J=8 Hz), 7.371 (dd, 1H, J=8 Hz), 3.945 (s, 3H); MS: m/z 234
1
6
+
1
1
1
1
7. MacLean-Fletcher, S. D.; Pollard, T. D. J. Cell. Biol.
980, 85, 414.
M .
24. 6-Chloro-1-methyl-2-(tri¯uoromethyl)benzimidazole (21).
ꢀ
1
8. Pollard, T. D. Meth. Cell. Biol. 1982, 24, 301.
9. 2-(Tri¯uoromethyl)-1H-benzimidazole (16). Ethanol±
Cyclohexane (yield 81.5%); mp 158±160 C; H NMR (300 MHz,
DMSO-d ) d 7.77 (d, 1H, J=8 Hz), 7.44 (d, 1H, J=2 Hz), 7.33
6
ꢀ
1
+
water (yield 80.8%); mp 209±211 C; H NMR (300 MHz,
DMSO-d ) d 8.218 (bs, 1H), 7.754 (m, 2H), 7.412 (m, 2H);
MS: m/z 186 M .
0. (6)-Chloro-2-(tri¯uoromethyl)-1H-benzimidazole
Column chromatography (yield, 68.03%); mp 197±199 C; H
(dd, 1H, J=8 Hz), 3.915 (s, 3H); MS: m/z 234 M .
25. 5,6-Dichloro-1-methyl-2-(tri¯uoromethyl) benzimidazole
(22). Ethanol (yield 82.1%); mp 164±165 C; H NMR
6
+
ꢀ
1
2
(17).
1
6
(300 MHz, DMSO-d ) d 7.94 (s, 1H), 7.56 (s, 1H) 3.94 (s, 3H);
ꢀ
+
MS: m/z 268 M .