Synthesis and antiprotozoal activity of novel 2-{[2-(1H-imidazol-1-yl) ethyl]sulfanyl}-1H-benzimidazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.05.012

A series of 19 new 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives was synthesized starting from the properly substituted 1,2-phenylendiamine. These compounds have hydrogen or methyl at position 1; while hydrogen, chlorine, ethoxy or methoxycarbonyl group is at position 5 and/or 6. The novel compounds were tested against protozoa Trichomonas vaginalis, Giardia intestinalis and Entamoeba histolytica. Experimental evaluations revealed strong activity for all tested compounds, having IC ₅₀ values in the nanomolar range, which were even better than metronidazole, the drug of choice for these parasites.

Full text of DOI:10.1016/j.bmcl.2013.05.012

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4221–4224
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiprotozoal activity of novel 2-{[2-(1H-imidazol-
1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives
Jaime Pérez-Villanueva a, Alicia Hernández-Campos a, Lilián Yépez-Mulia b, Carlos Méndez-Cuesta a,
Oscar Méndez-Lucio a, Francisco Hernández-Luis a, Rafael Castillo a,
⇑
a Facultad de Química, Departamento de Farmacia, UNAM, DF 04510, Mexico
b Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS, DF 06720, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 February 2013
Revised 1 May 2013
Accepted 7 May 2013
Available online 15 May 2013
A series of 19 new 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives was synthe-
sized starting from the properly substituted 1,2-phenylendiamine. These compounds have hydrogen or
methyl at position 1; while hydrogen, chlorine, ethoxy or methoxycarbonyl group is at position 5 and/
or 6. The novel compounds were tested against protozoa Trichomonas vaginalis, Giardia intestinalis and
Entamoeba histolytica. Experimental evaluations revealed strong activity for all tested compounds, having
IC50 values in the nanomolar range, which were even better than metronidazole, the drug of choice for
these parasites.
Keywords:
Benzimidazole
Entamoeba histolytica
Giardia intestinalis
Trichomonas vaginalis
Ó 2013 Elsevier Ltd. All rights reserved.
Parasitic infections caused by protozoa still represent a major
public health problem in developing countries. Some important
intestinal protozoa include Giardia intestinalis and Entamoeba his-
tolytica, causal agents of giardiosis and amebiosis, respectively.
According to the World Health Organization (WHO), there are esti-
mated 280 million giardiosis cases each year. Interestingly, G. intes-
tinalis has been classified as the most common diagnosed flagellate
in the intestinal tract.1,2 Infection by this protozoan usually pro-
duces diarrhea and associated symptoms. Furthermore, it is worth
mentioning that child incidence is very high and is associated with
malnutrition, linear growth retardation and poor cognitive func-
tion.3,4 Regarding E. histolytica, WHO estimated that this protozoan
causes severe disease in 50 million people each year and has been
classified as one of the most common causes of death from para-
sitic disease.2,5 In addition to the common symptoms such as diar-
rhea and dysentery, this protozoan can penetrate the intestinal
mucosa and migrate to other organs causing severe damage.1,2,5
In addition to intestinal infections, the genitourinary infection
caused by T. vaginalis (trichomonosis) is estimated to be more than
180 million new cases annually.1 Although trichomonosis in men is
generally asymptomatic or mild, in women it causes severe symp-
toms and consequences. The infection produces deep inflammation
of the genital tract and has been associated with preterm labor,
low-birth weight, sterility, cervical cancer and a predisposition to
HIV infection.6,7 For these three diseases, metronidazole (MTZ)
has been successfully used as the drug of choice for more than
40 years; however, its side effects and the development of resistant
strains limit its use.1 Although some additional chemotherapeutic
agents are available (e.g. tinidazole and nitazoxanide used in the
treatment of giardiosis), it is still important to have more options
of treatment, because of different individual response to drugs.
During the last years, an important number of benzimidazole
derivatives have been synthesized and evaluated as antiprotozoals
by our group and other external research groups.8–15 Moreover,
studies based on the emerging concept of the activity landscape
were undertaken to find out the structure–activity relationships
(SAR) of benzimidazole derivatives as trichomonicidal and giardi-
cidal agents.16,17 Besides, quantitative structure–activity relation-
ships (QSAR) studies based on comparative molecular field
analysis (CoMFA) and comparative molecular similarity indices
analysis (CoMSIA) were carried out for amebicidal and trichomon-
icidal benzimidazole derivatives.18,19 Although some important
conclusions have been highlighted with our previous synthetic
and SAR studies, the information accumulated is still limited.
Therefore, increasing the database of benzimidazole derivatives re-
mains of paramount importance to have access to new SAR fea-
tures that can lead to the optimization of benzimidazole
derivatives as antiprotozoals. Expanding our previous work, herein
we report the synthesis of 19 new 2-{[2-(1H-imidazol-1-
yl)ethyl]sulfanyl}-1H-benzimidazole derivatives (35–53), Fig. 1.
The design of the new benzimidazole derivatives was based on
previous SAR conclusions derived from our former studies
(Fig. 1).16–19 It was previously observed that substituted alkylthio
⇑
Corresponding author. Tel.: +52 556 22 52 87; fax: +52 556 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.2013.05.012

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J. Pérez-Villanueva et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4221–4224
Figure 1. Some SAR conclusions for benzimidazole derivatives tested against G. intestinalis (Gi), E. histolytica (Eh), and T. vaginalis (Tv) which lead to the structure of the 19
new derivatives (35–53) synthesized in this work.
groups at position 2 of the benzimidazole nucleus lead to potent
compounds against T. vaginalis and G. intestinalis.12,17,19 Some of
the most active compounds were those with hydrogen acceptors
or an aromatic ring attached to the alkyl chain. The new derivatives
synthesized in this work contain an alkylthio imidazole group at
position 2, a substituent which combines both features. In addition,
hydrogen or methyl substituents were present at position 1 of the
new derivatives. It is worth noting that both, hydrogen and methyl
derivatives, have been reported as strong trichomonicidal and
giardicidal agents.9,14 Also, hydrogen, chlorine, carbomethoxy and
ethoxy substituents were considered at position 5 and/or 6 for
the new derivatives. As previous QSAR studies of benzimidazole
derivatives suggest, the substituents at position 6 are favorable
for trichomonicidal and giardicidal activity,16,17,19 whereas substit-
uents at position 5 play an important role for amebicidal activity.18
It is important to emphasize that SAR conclusions previously re-
ported are highly dependent on the database analyzed.
with carbon disulfide in the presence of potassium hydroxide
and ethanol to give the respective 2-mercaptobenzimidazole
(18–34).20,21 These were then alkylated with 1-(2-chloroethyl)-
1H-imidazole to afford title compounds 35–37, 39, 40, and 42–
53.22 Finally, compounds 38 and 41 were obtained by N-methyla-
tion of compounds 35 and 37, respectively.23 The intermediate 1-
(2-chloroethyl)-1H-imidazole was prepared under solid–liquid
phase transfer-catalysis conditions as described previously by
Bogdal et al.,24 in which commercial imidazole was alkylated with
1,2-dichloroetane in the presence of sodium hydroxide, potassium
carbonate and tetrabutylammonium chloride. Compounds 35–53
were obtained as solids with fair global yields and purity. The
compounds were characterized using 1H and 13C nuclear magnetic
resonance,25 mass spectrometry and high resolution mass spec-
trometry (See supplementary data for structure characterization,
yields and melting points).
The in vitro antiprotozoal activity of compounds 35–53 was
tested following the method previously described for T. vaginalis,
G. intestinalis and E. histolytica.8–15,26 The results obtained from
the biological assays are shown in Table 1. All tested compounds
Compounds 35–53 were prepared according to the sequence of
reactions shown in Scheme 1. The first step was the cycloconden-
sation of the properly substituted 1,2-phenylenediamine (1–17)
Scheme 1. Synthesis of compounds 35–53: (a) CS2, KOH, EtOH/H2O, 40–50 °C, 8 h; (b) ClCH2CH2Cl, Bu4NCl, NaOH, K2CO3, 50 °C, 3 h; (c) CH3CN, KOH or DBU, 60 °C, 8 h; (d)
CH3I, (CH3)2CO, K2CO3.

J. Pérez-Villanueva et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4221–4224
4223
Table 1
Chemical structures and biological activity of benzimidazole derivatives 35–53
R1
R2
R3
IC50
(l
M) T. vaginalis
IC50
(lM) G. Intestinalis
IC50 (lM) E. histolytica
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
H
H
H
CH3
CH3
CH3
CH3
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
H
Cl
H
Cl
H
0.1780 0.0087
0.1704 0.0076
0.1405 0.0045
0.1490 0.0027
0.1281 0.0072
0.1349 0.0024
0.0963 0.0022
0.1108 0.0023
0.0727 0.0105
0.1138 0.0055
0.0869 0.0101
0.0774 0.0067
0.0926 0.0020
0.0717 0.0053
0.0991 0.0044
0.0717 0.0038
0.0698 0.0063
0.0761 0.0094
0.0980 0.0042
0.2360 0.0160
1.5905 0.0113
0.1167 0.0029
0.0807 0.0025
0.0766 0.0090
0.0871 0.0082
0.0626 0.0039
0.0666 0.0024
0.0688 0.0065
0.0777 0.0023
0.0549 0.0021
0.0569 0.0045
0.0556 0.0020
0.0616 0.0067
0.0442 0.0020
0.0566 0.0020
0.0465 0.0044
0.0452 0.0083
0.0356 0.0084
0.0083 0.0023
0.0208 0.0042
1.2260 0.1250
0.0370 0.0030
0.1474 0.0058
0.0735 0.0076
0.0591 0.0023
0.0774 0.0055
0.0672 0.0110
0.0660 0.0129
0.0642 0.0043
0.0612 0.0023
0.0579 0.0021
0.0522 0.0067
0.0428 0.0040
0.0427 0.0112
0.0513 0.0040
0.0428 0.0044
0.0620 0.0044
0.0408 0.0019
0.0460 0.0063
0.0298 0.0047
0.0148 0.0042
0.3798 0.1461
56.5334 18.8445
COOCH3
COOCH3
H
H
Cl
CH3
CH3
CH3
CH3
H
COOCH3
COOCH3
H
Cl
H
Cl
OCH2CH3
OCH2CH3
H
Cl
Cl
COOCH3
COOCH3
OCH2CH3
OCH2CH3
H
H
CH3
CH3
CH3
CH3
51
52
53
Cl
OCH2CH3
OCH2CH3
Metronidazole
Albendazole
showed good activity in the nanomolar range. It is worth noting
that all compounds were more potent than metronidazole, the
drug of choice for these three parasites. The SAR nature of the data-
base presented in this work is essentially continuous. That is, the
new set of compounds is in agreement with the similarity principle
which establishes that structurally similar compounds have similar
biological activities. This conclusion is supported since all mole-
cules in the dataset have less than 15-fold variation in potency.
However, these low variations are enough to support some SAR
conclusions and are in agreement with most of the observations
previously reviewed (Fig. 1).
Results in Table 1, for compounds 35–41, show that dichloro
substitution at positions 5 and 6 in compounds 37 and 41 is
slightly favorable for antiprotozoal activity, as compared with
non-substituted compounds 35 and 38, respectively. Furthermore,
compounds 35 and 38 show a favorable effect of the methyl group
at position 1 against the three protozoa tested. This same tendency
was observed for compounds 37 and 41 against T. vaginalis, but no
important changes were observed for G. intestinalis and E. histolyti-
ca. It is interesting to note that all the 1-methyl compounds show
comparable or better activity than their respective 1H analogue.
The only exceptions are compounds 44 and 47, which have a
slightly lower activity against T. vaginalis as compared with their
1H analogues 42 and 43, respectively. These results agree with
the previous SAR models that suggest that hydrogen at position 1
is favorable for trichomonicidal activity. In general, compounds
43–53, which have a carbomethoxy or an ethoxy substituent at
positions 5, 6 present the best activities. In particular, compounds
50–53, with an ethoxy substituent, have similar or higher giardi-
cidal potency than albendazole. It is important to emphasize that
compound 52, which showed a very strong giardicidal activity,
was the only one whose activity increased 14-fold against G. intes-
tinalis, as compared with unsubstituted analogue 35, which was
the less active compound against all the protozoa tested. Addition-
ally, compounds 50–53 were particularly active against T. vaginalis
and E. histolytica as compared with albendazole, which is poorly
active. Compounds 50–53 can be considered among the most po-
tent benzimidazole derivatives reported against the three proto-
zoa. Noteworthy, antiprotozoal activities are in agreement with
the previous SAR studies that indicate the importance of the sub-
stituent size at position 5 and 6 of the benzimidazole nucleus for
the antiprotozoal activity.16–19 Also, the strong activity of com-
pounds 35–53 agrees with the previous SAR observation that alkyl-
thio groups at position 2, having a hydrogen acceptor or an
aromatic ring, lead to potent compounds.
The mechanism of action for the antiprotozoal activity of com-
pounds 35–53 is still unknown. Interestingly, we found in previous
studies that benzimidazole derivatives that are not benzimidazole
carbamates (BC) do not inhibit b-tubulin polymerization as anti-
parasitic BC do.8,9 Therefore, a different mechanism of action is im-
plied in the antiprotozoal activity of the current derivatives.
In summary, compounds 35–53 resulted in new structures
which were able to be synthesized with acceptable yields starting
from the properly substituted 1,2-phenylendiamine. The novel
compounds were characterized by using spectrometric and spec-
troscopic methods. Biological assays revealed that the novel com-
pounds have strong activity against T. vaginalis, G. intestinalis and
E. histolytica, which is better than metronidazole. Particularly, com-
pounds 50–53 exhibited the best activities; among these, com-
pound 52 was the most active against G. intestinalis. The new
compounds expand our database of benzimidazole derivatives
and increase our knowledge concerning the structural require-
ments for antiprotozoal activity. These results are useful for the de-
sign of new stronger antiprotozoal compounds.
Acknowledgements
This work was supported by project 80093 from CONACyT.
J.P.-V. acknowledges the CONACyT scholarship 173896/173896
and Carso Health Institute for the PhD student scholarship
S0710443. We are grateful to Amparo Tapia for carrying out the
biological assays and to Rosa Isela del Villar, Nuria Estearau,

4224
J. Pérez-Villanueva et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4221–4224
19. Pérez-Villanueva, J.; Medina-Franco, J. L.; Caulfield, T. R.; Hernández-Campos,
A.; Hernández-Luis, F.; Yépez-Mulia, L.; Castillo, R. Eur. J. Med. Chem. 2011, 46,
3499.
Georgina Duarte, Margarita Guzmán and Marisela Gutiérrez for the
analytical support.
20. VanAllan, J. A.; Deacon, B. D. Org. Synth. Coll. Vol. 1963, 4, 569.
21. General procedure for the synthesis of 2-mercaptobenzimidazole derivatives (18–
34): the proper 1,2-phenylenediamine 1–17 (16 mmol) was added over a
stirred solution of ethanol (50 mL), water (5 mL), potassium hydroxide
(19.2 mmol) and carbon disulfide (19.2 mmol). The mixture was heated
overnight under a N2 atmosphere at 50 °C. The cold reaction mixture was
then neutralized with acetic acid and poured into 250 mL of cold water for
complete precipitation. The solid was separated and dried using vacuum
filtration. The crude product was purified by recrystallization, adding norite to
the solvent.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.05.
012.
References and notes
22. General procedure for the synthesis of 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-
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solution) or DBU (6.6 mmol, for ester derivatives 23–28). Then, 1-(2-
chloroethyl)-1H-imidazole (10.2 mmol) was added and the mixture was
heated at 60 °C for 6–8 h. Next, the cold reaction mixture was neutralized
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residue was extracted with ethyl acetate (30–50 mL), the organic phase was
washed thrice with 20 mL of water, then dried with anhydrous sodium sulfate,
and evaporated under vacuum. The residue was purified by column
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25. Some signals in 13C NMR for 1H substituted benzimidazole derivatives are
duplicated due to tautomeric effect. This same effect decreases the intensity of
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26. 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
T. vaginalis trophozoites were maintained in TYI-S-33 medium supplemented
with 10% bovine serum. Briefly, 5 Â 104 trophozoites of G. intestinalis or T.
vaginalis, or 6 Â 103 trophozoites of E. histolytica were incubated for 48 h at
37 °C with different concentrations of the compound to be tested, each added
as solutions in DMSO. As a negative control, parasite cultures received an
equivalent amount of DMSO only, while ABZ and MTZ 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.
The trophozoites were then counted with a haemocytometer and the 50%
inhibitory concentration (IC50), together with the respective 95% confidence
limit was calculated by Probit analysis. Experiments were carried out in
triplicate and repeated at least twice.