Synthesis and in vitro trichomonicidal, giardicidal and amebicidal activity of N-acetamide(sulfonamide)-2-methyl-4-nitro-1H-imidazoles

Article abstract of DOI:10.1016/j.ejmech.2009.01.005

Two new series of imidazole derivatives (acetamides: 1-8 and sulfonamides: 9-15) were synthesized using a short synthetic route. Compound 1 as well as the intermediate 16g were characterized by X-ray crystallography. Imidazole derivatives 1-15 were tested in vitro against three unicellular parasites (Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica) in comparison with benznidazole (Bzn) and metronidazole. Compound 1 [N-benzyl-2-(2-methyl-4-nitro-1H-imidazol-1-yl)acetamide] was 2 times more active than Bzn against T. vaginalis and G. intestinalis and it was as active as Bzn against E. histolytica. Sulfonamides showed selective toxicity against E. histolytica over the other parasites. Toxicity assay showed that all compounds are non-cytotoxic against MDCK cell line. The results revealed that compounds 1-15 have antiparasitic bioactivity in the micromolar range against the parasites tested, and could be considered as benznidazole bioisosteres.

Full text of DOI:10.1016/j.ejmech.2009.01.005

European Journal of Medicinal Chemistry 44 (2009) 2975–2984
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro trichomonicidal, giardicidal and amebicidal activity
of N-acetamide(sulfonamide)-2-methyl-4-nitro-1H-imidazolesq
a
b
c
a
´
´ ˜
´
´
Emanuel Hernandez-Nunez , Hugo Tlahuext , Rosa Moo-Puc , Hector Torres-Gomez , Reyna Reyes-
b
c
a
a,
*
´
Martınez , Roberto Cedillo-Rivera , Carlos Nava-Zuazo , Gabriel Navarrete-Vazquez
a Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
b Centro de Investigaciones Qu´ımicas, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
c
´
Unidad Interinstitucional de Investigacio´n Me´dica, IMSS-Facultad de Medicina, UADY, Me´rida, Yucatan 97000, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new series of imidazole derivatives (acetamides: 1–8 and sulfonamides: 9–15) were synthesized
using a short synthetic route. Compound 1 as well as the intermediate 16g were characterized by X-ray
crystallography. Imidazole derivatives 1–15 were tested in vitro against three unicellular parasites
(Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica) in comparison with benznidazole
(Bzn) and metronidazole. Compound 1 [N-benzyl-2-(2-methyl-4-nitro-1H-imidazol-1-yl)acetamide] was
2 times more active than Bzn against T. vaginalis and G. intestinalis and it was as active as Bzn against E.
histolytica. Sulfonamides showed selective toxicity against E. histolytica over the other parasites. Toxicity
assay showed that all compounds are non-cytotoxic against MDCK cell line. The results revealed that
compounds 1–15 have antiparasitic bioactivity in the micromolar range against the parasites tested, and
could be considered as benznidazole bioisosteres.
Received 12 July 2008
Received in revised form
12 December 2008
Accepted 8 January 2009
Available online 19 January 2009
Keywords:
4-Nitroimidazoles
Trichomonas vaginalis
Giardia intestinalis
Entamoeba histolytica
Benznidazole
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Benznidazole [Bnz, RadanilÒ], a 2-nitroimidazole derivative, is
an important drug for Chaga’s Disease, and has been used in other
Parasitic diseases are still major problem in developing coun-
tries, affecting hundreds of millions people around the world. Since
parasites are eukaryotic, they share many common features with
their mammalian host, making the development of effective and
selective drugs a hard task. Despite the great effort that has been
done in the discovery of unique targets that afford selectivity, many
of the drugs used today have serious side effects [1].
Some heterocyclic nucleus used mainly as antiparasitic drugs
are: benzimidazoles-2,5(6)-substituted and 2- or 5-nitroimidazoles
[2,3]. Nitroimidazoles are attractive and important reagents in
organic synthesis [4–6]. The 5-nitroimidazole core, found particu-
larly in metronidazole, the most commonly antiparasitic drug, is
accepted as drug of choice for anti-infectious chemotherapy against
anaerobic bacteria and parasites and also for the radiosensitization
of hypoxic tumors [7].
parasitic diseases [8].
The Bnz mode of action is related to reductive metabolism, it
functions as prodrug and must be activated by an NADH-depen-
dent, mitochondrially localized, bacterial-like, type
reductase [9].
I nitro-
It is well-known that the imidazole pharmacophore is an
important structural core in medicinal chemistry that shows
a broad spectrum of pharmacological activities. Several compounds
containing the nitroimidazole scaffold have been used as antipar-
asitic [10,11], antimycobacterial [12], antibacterial [13,14], antitu-
moral [15], antioxidant, antifungal, [16] and calcium channel
antagonist agents [17].
As a part of our search for basic information about the structural
requirements for new antiparasitic activities of an old drug
(Benznidazole), we have synthesized a series of novel N-acetamide
(sulfonamide)-2-methyl-4-nitro-1H-imidazoles as analogues of
this drug. The in vitro antiparasitic activity of these compounds on
intestinal unicellular parasites (Giardia intestinalis and Entamoeba
histolytica), and a urogenital tract parasite (Trichomonas vaginalis) is
also reported in this paper.
q
´
´ ˜
Taken in part from the PhD Thesis of Emanuel Hernandez-Nunez.
* Corresponding author. Tel./fax: þ52 777 3297089.
E-mail address: gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.005

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´
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Table 1
NO2
N
Predictive values of antiparasitic effect calculated with PASS for compounds 1–15.
N
N
N
Ar(Bn)
N
H
N
H
O2N
Ar
O
O
Compound
Antiparasitic effect
(formerly
antiprotozoal)
Benznidazole (Bnz)
Pa
Pi
1
2
3
4
5
6
7
8
Benzyl
Phenyl
4-Fluorophenyl
4-Chlorophenyl
4-Cyanophenyl
4-Nitrophenyl
0.651
0.679
0.645
0.661
0.629
0.685
0.657
0.639
0.006
0.006
0.007
0.006
0.007
0.006
0.006
0.006
R
N
N
n
N
H
O2N
2,6-Dichlorophenyl
3-(Trifluoromethyl)phenyl
n = 0, 1
O
Imidazole acetamide analogues 1-8
O
N
N
S
R
O
O2N
O
O
N
Compound
Ar
Antiparasitic effect
(formerly antiprotozoal)
N
S
Pa
Pi
O2N
9
NHCOCH3
H
CH3
Cl
F
0.563
0.617
0.591
0.599
0.571
0.616
0.574
0.012
0.006
0.008
0.008
0.011
0.007
0.010
R
10
11
12
13
14
15
Imidazole sulfonamide analogues 9-15
Fig. 1. Drug design of title compounds.
NO2
OCH3
Pa ¼ Probability of activity, Pi ¼ Probability of inactivity.
2. Results and discussion
2.1. Drug design of derivatives 1–15
In our ongoing research on imidazole derivatives we have
designed the compounds 1–8, on the basis of the structure of
antiparasitic drug benznidazole (Bzn, Fig. 1). In order to find new
antiparasitic bioactivities of this old drug (besides antichagasic
activity), we decided to exchange the nitro group from position 2 to
position 4, and add an extra-methyl group at position 2, as in
metronidazole core. The resulting 2-methyl-4-nitroimidazole
scaffold was kept and it was hybridized with sulfonamide phar-
macophore as shown in compounds 9–15, in order to improve the
antiparasitic activity. The design was also based on the biological
activity predictions made by the computer software PASSÒ
(Prediction of activity spectra for substances). This software illus-
trates the predicted activity spectrum of a compound as probable
activity (Pa) and probable inactivity (Pi) with the accuracy of
prediction reported to be as high as 85% [18–21].
H
N
Cl
i or ii
Cl
Ar(Bn)
Cl
+ (Bn)Ar-NH2
O
O
18
a-h
16a-h
O2N
N
N
O2N
H
iii
N
N
Cl
Ar(Bn)
+
H
N
N
H
Ar(Bn)
O
1-8
16a-h
O
17
O2N
O
N
2.1.1. In silico PASSÒ screening
N
O2N
N
S
O
iv
An approach to computer-aided prediction of the general bio-
logical activity spectra on the basis of chemical structure of
a compound has been developed and marketed as computer
program PASSÒ [18,19,21]. This software is based on a robust
analysis of structure–activity relationships in a heterogeneous
training set [20] including many thousands of compounds from
different chemical series. Using PASS predictions the number of
N
+ Cl
S
R
H
17
O
O
9-15
19-25
R
Scheme 1. Reagents: (i) Et3N, CH2Cl2; (ii) NaHCO3, Acetone; (iii) K2CO3, CH3CN; (iv)
KOH, DMAP (cat.), triethylamine, CH2Cl2.

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2977
Table 2
2.2. Chemistry
Comparison of yields of synthesized 2-chloro-N-(aryl)acetamides 16a–h obtained
through two methodologies.
The
a
-chloroacetamides adequately substituted 16a and 16b–h,
Compd
Ar
Conditions
Reaction time (h)
were synthesized by condensation of 2-chloroacetyl chloride (18)
with benzylamine (a) and 4-substituted anilines (b–h) respectively,
through two methodologies (Scheme 1). In the first one, we used
methylene chloride as solvent and triethylamine as base. In the
second one, we change the solvent and the base for acetone and
sodium bicarbonate, respectively, affording the desired compounds
with yields ranging from 70 to 90%. The reactions conditions,
duration of reaction and yields of the products prepared are listed
in Table 2.
Bimolecular nucleophilic substitution in 16a–h with 4(5)-nitro-
2-methyl-1H-imidazole anion (17) afforded compounds 1–8
(Scheme 1). Solid compounds were purified by recrystallization and
the structure of the pure compounds was established by spectro-
scopic data. Crystals suitable for X-ray crystallography were
obtained for compounds 1 and 16g.
Et3N/CH2Cl2
% yield
NaHCO3/acetone
% yield
16a
16b
16c
16d
16e
16f
Bn
Ph
96.3
92.3
71.1
93.8
94.1
96.5
88.4
87.5
–
24
24
48
24
24
24
24
48
89.3
89.4
86.1
81.8
84.8
89.5
90.2
4-F-Ph
4-Cl-Ph
4-CN-Ph
4-NO2-Ph
2,6 diCl-Ph
3-CF3-Ph
16g
16h
actives in the selected compounds can be increased by up to
17-fold [18]. Thus, PASS-based computer pre-screening of large
databases of diverse compounds can increase the probability of
finding bioactive new chemical agents, and reduce the number of
compounds that have to be synthesized and tested
experimentally.
Compounds 9–15 were prepared in a single step (Scheme 1),
starting from 4(5)-nitro-2-methyl-1H-imidazole anion (17), via
a coupling reaction with arylsulfonyl chlorides 19–25, in the
presence of a catalytic amount of 4-dimethylaminopyridine and
triethylamine. Title compounds were recovered with 11–89%
yields. Compounds were purified by recrystallization or by
column chromatography. The chemical structures of the synthe-
sized compounds were confirmed on the basis of their spectral
data.
Before the establishment of an in vitro antiparasitic assay, we
obtained predictive values concerning biological activities by
comparing the chemical structures of the designed compounds
1–15, with structures or substructures of more than 46,000 well-
known biologically active drugs included in the database of PASSÒ
[20]. Results of prediction are presented as estimates of the
probability Pa that the compounds are active. For Pa values ꢀ0.7,
the corresponding compound is very likely to reveal this activity
in experiments, but in that case, the chance of the compound
being the analogue of a known pharmaceutical agent is also high.
For Pa values between 0.5 and 0.7, the compound is likely to
reveal this activity in experiments and the compound exhibits less
similarity to the known pharmaceutical agents. For Pa values
lower than 0.5, the compound is unlikely to reveal this activity in
experiments, but if the presence of this activity is confirmed in
experiments, the compound might be a new biologically active
chemical entity. Predictive antiparasitic values for compounds
1–15 are summarized in Table 1. Pa values estimated for anti-
parasitic activity for compounds 1–8 (acetamides) were higher
than 0.6 for all structures. These results indicated that compounds
1–8 could be likely to reveal this activity in experiments and the
compounds exhibit less similarity to the known antiparasitic
drugs. For sulfonamides series 9–15, Pa values were also in the
range of 0.5–0.6; hence, the structures are likely to reveal anti-
parasitic activity in experiments. For Bzn, the Pa value estimated
for antiparasitic activity was 0.497, due to the lack of information
about the other parasiticidal bioactivities rather than
trypanocidal.
In the nuclear magnetic resonance spectra (1H NMR;
d ppm), the
signals of the respective protons of the compounds were verified on
the basis of their chemical shifts, multiplicities and coupling
constants. In compounds 3–6, 9, and 11–15, the aromatic region of
the 1H NMR spectrum contained an A2B2 pattern signals ranging
from d
H 7.04 to 8.24 ppm, attributable to H-20, H-30, H-50 and H-60,
of the 4-substituted benzene ring. Also, we observed in all
compounds a characteristic pattern for 2-methyl-4-nitroimidazole:
a singlet methyl signal ranging from 2.26 to 2.33, and a singlet
signal ranging 8.31–8.46 ppm, attributable to H-5 of heterocyclic
ring. A singlet peak was observed for compounds 1–8, assigned to
a methylene group found in 2-substituted acetamide.
2.3. Crystallography
Single crystals of compounds 1 and 16g were grown by slow
evaporation at room temperature of DMSO and acetone, respec-
tively. Perspective views of the molecular structures of compounds
1 and 16g are shown in Fig. 2. The most relevant crystallographic
data for 1 and 16g have been summarized in Table 3. Selected bond
lengths, bond angles, and torsion angles are outlined in Table 4. In
both cases, the packing is stabilized by hydrogen bonds [22].
Fig. 2. The molecular structures of 1 and 16g showing the atom labeling scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small
spheres of arbitrary radii.

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2978
Table 3
van der Waals radii of the chlorine atoms and exhibits an obtuse
angle in C(2)–Cl(1)/Cl(3) [(C)Cl/Cl–(C) 3.437 Å, C–Cl/Cl
152.99ꢂ] (Fig. 4b).
Crystallographic data for compounds 1 and 16g.
Crystal data
1
16g
In conclusion, the crystal packing of compounds 1 and 16g is
determined by N–H/N and N–H/O hydrogen bonds, respec-
tively, with consequent formation of polymeric chains. On the
other hand, the Cl/Cl donor–acceptor interactions detected in the
intermediate 16g are important because halogen atoms that are
covalently bond to carbon atoms are known to form cooperative
short contacts to hydrogen, nitrogen, oxygen, sulfur and other
halogens, this short contacts are implicated in all the fields where
design and manipulation of aggregation phenomena play a key
role [23–28].
Formula
C13H14N4O3
0.23 ꢃ 0.27 ꢃ 0.31
274.28
Orthorhombic
Pna21
C8 H6Cl3NO
0.15 ꢃ 0.17 ꢃ 0.23
238.49
Orthorhombic
P212121
Crystal size (mm)
MW (g molꢁ1
)
Crystal System
Space group
Cell parameters
a (Å)
13.0874(13)
8.4150(8)
11.9285(12)
1313.7(2)
4
4.7166(15)
10.917(3)
18.405(5)
947.7(7)
4
b (Å)
c (Å)
V (Å3)
Z
m
r
(mmꢁ1
calcd (g cmꢁ3
)
0.102
1.387
0.921
1.671
)
Data collection
limits (Å)
hkl limits
No. collected refl.
q
2.88 <
q
< 27.5ꢂ
2.17 < q< 25.0
ꢁ17,16; ꢁ10,10; ꢁ15,
ꢁ5,5; ꢁ12,8; ꢁ21,11
2.4. In vitro antiparasitic effect
1,514,245
3387
No. ind refl. (Rint
Refinement
)
1586 (0.038)
1000 (0.077)
In this study fifteen new nitroimidazole derivatives (1–15), were
synthesized and tested in vitro as antiparasitic agents against G.
intestinalis, T. vaginalis and E. histolytica. The main features of these
compounds are:
R [I > 2sigma(I)]
Rw (all data)
No. of variables
GOF
0.074
0.170
186
1.273
ꢁ0.17
0.27
0.053
0.116
110
1.123
ꢁ0.52
0.50
Drmin (e Åꢁ3
Drmax (e Åꢁ3
)
)
- The 2-nitroimidazole found in Bzn was replaced by 2-methyl-
4-nitroimidazole moiety in compounds 1–15.
- The substitution of the hydrogen atom at position 4 of the
benzene ring by cyano, nitro, halo (–F, –Cl), or 3-tri-
fluoromethyl and 2,6-(Cl)2 groups in order to determine bio-
isosteric equivalence, enhancement of solubility and potential
antiparasitic activity in compounds 1–8.
In the crystal structure of 1 an intermolecular N4–H4/N2i
hydrogen bond [symmetry code: (i) ½ ꢁ x, ½ þ y, ½ þ z] links the
molecules into chains running in zigzag along the diagonal of the bc
plane (Fig. 3), with H4/N2i ¼ 2.21(5) Å, N4/N2i ¼ 3.024(6) Å and
N4–H4/N2i ¼ 167(4)ꢂ.
- The replacement of sulfonamide group instead of acetamide
group found in Bzn in compounds 9–15.
In the crystal lattice of 16g the molecules are linked through
intermolecular N1–H1/O1ii hydrogen bonds [symmetry code: (ii)
ꢁ1 þ x, y, z] into chains running along the a axis (Fig. 4a), with
H1/O1ii ¼ 2.05(7) Å,
N1/O1ii ¼ 2.827(8) Å
and
N1–H1/
Biological assays results against the three unicellular parasites
tested are summarized in Table 5. Comparison was made among
new compounds and the antiparasitic drug benznidazole. In order
to compare bioactivities, 2-methyl-4(5)-nitroimidazole (Mni) and
metronidazole (Met), were also tested.
O1ii ¼ 167(7)ꢂ. The packing is further stabilized by intermolecular
C(2)–Cl(1)/Cl(3)iii interhalogen contacts [symmetry code: (iii)
½ þ x, ½ ꢁ y, 2 þ z] [23–28]. The C(2)–Cl(1)/Cl(3)–C(8) interaction
has a Cl/Cl separation that is slightly shorter than the sum of the
In the first series (acetamide derivatives 1–8), compounds 1,
3, 4 and 6 showed high bioactivity (<10
mM) against T. vaginalis.
Compound 1, with benzylacetamide substituent (Bzn true
analogue), was two-fold more active than Bzn and five-fold
more potent than Mni. The same pattern was observed in
a
Table 4
Selected bond lengths, bond angles and torsion angles for compound 1 and 16g.
1
16g
compound 4, which bears
a 4-chloroacetanilide substituent.
Bond lengths (Å)
C(1)–C(3)
C(1)–N(2)
C(1)–N(3)
C(2)–N(2)
C(2)–N(1)
N(1)–C(3)
N(3)–O(1)
N(3)–O(2)
Compounds 3 and 6 (4-fluoro and 4-nitroacetanilide substituent,
respectively) were three times more potent than Bzn and six-
fold more actives than Mni. Compound 5 (4-CN) was as active as
Bzn and three-fold more potent than Mni, whereas compounds
1.340(7)
1.344(6)
1.438(5)
1.308(6)
1.361(5)
1.348(6)
1.224(7)
1.210(7)
1.206(6)
1.328(6)
C(2)–Cl(1)
C(6)–Cl(2)
C(1)–N(1)
N(1)–C(7)
C(7)–O(1)
C(8)–Cl(3)
1.735(9)
1.759(8)
1.416(10)
1.346(10)
1.216(9)
1.771(8)
2
(4-H),
7 (3-CF3) and 8 [2,6-(Cl)2] were less potent than
reference drugs.
For G. intestinalis, compounds 1, 4–6 were more active than
Bzn and Mni. The most potent compound was 5, which was two
times more active than Bzn. Compounds 3 and 7 were as active
C(5)–O(3)
C(5)–N(4)
Bond angles (deg)
N(2)C(2)N(1)
N(2)C(1)N(3)
C(1)N(3)O(1)
C(1)N(3)O(2)
O(3)C(5)N(4)
N(4)C(5)C(4)
Torsion angles (deg)
O(1)N(3)C(1)C(3)
O(2)N(3)C(1)N(2)
N(1)C(4)C(5)O(3)
O(3)C(5)N(4)C(6)
N(4)C(6)C(7)C(8)
as Bzn. Nitroimidazoles
compounds.
2 and 8 were the least active
111.3(4)
Cl(1)C(2)C(1)
Cl(2)C(6)C(1)
C(1)N(1)C(7)
N(1)C(7)O(1)
C(7)C(8)Cl(3)
119.6(6)
120.7(5)
116.3(5)
119.1(5)
125.0(4)
112.8(4)
119.9(6)
123.7(7)
123.7(8)
110.9(6)
Against E. histolytica, only compound 1 was as active as Bzn
and two-fold more potent than Mni. Compounds 2–8 showed
low bioactivity against this parasite. It is important to note that
none of the compounds assayed were more active that
metronidazole.
With these results, we could conclude that compounds 1–8
displayed a selective toxicity against T. vaginalis over the other
unicellular parasite tested. However, compound
ꢁ2.8(7)
ꢁ2.5(7)
23.3(7)
0.1(8)
Cl(1)C(2)C(1)N(1)
Cl(2)C(6)C(1)N(1)
C(1)N(1)C(7)O(1)
O(1)C(7)C(8)Cl(3)
2.9(11)
0.6(11)
ꢁ1.5(15)
13.5(12)
1 (which is
115.2(6)
a benznidazole true congener), was more active than this drug

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2979
Fig. 3. A view of the crystal packing compound 1 showing the formation of zigzag chains. Hydrogen bonds are represented by dotted lines and H atoms not involved in hydrogen-
bonding interactions have been omitted for clarity.
against the three parasites tested. The re-positioning of the nitro
group and the addition of methyl substituent in 1, make the
molecule more active than the parent drug (Bzn).
2.5. In vitro cytotoxic effect
Compounds 1–15 were evaluated for their toxicity against
In the last series (sulfonamide derivatives 9–15), biological
assay results against T. vaginalis showed that all compounds were
more active than Bzn and Mni. In particular, compounds 9, 11, 12,
14 and 15, substituted at position 4 of the benzenesulfonamide
scaffold with acetamido, methyl, chloro, nitro and methoxy
MDCK cell line. Compounds 2–15 were non-cytotoxic, with IC50
s
ranging from 321.30 to 1270.30 M. The selectivity index (SI) of the
m
compounds defined as the ratio of cytotoxicity to biological activity
(SI ¼ CC50 MDCK cells/IC50 parasites) was calculated. It is generally
considered that biological efficacy is not due to in vitro cytotoxicity
when SI ꢀ 10. Most of the compounds possessing good in vitro
antiparasitic activity have shown decent selectivity index (Table 5).
groups respectively, showed high bioactivity (<10 mM).
Compound 14 was the most active showing an IC50 ¼ 2.93
mM
(six-fold more potent than Bzn and fourteen-fold more potent
than Mni). Against G. intestinalis, compounds 10–15 were more
potent than Bzn and Mni, whereas compound 9 was as active as
the two reference drugs. Compounds 14 and 15 were the most
actives against this parasite; showing two times more potency
than reference drugs.
Compound 1 showed a CC50 of 77.55
mM, whereas metronidazole
displayed a CC50 ¼ 68
mM. All compounds were less cytotoxic than
metronidazole.
Since nitroimidazole mode of action is related to reductive
metabolism, they must be activated by an NADH-dependent,
mitochondrially localized, bacterial-like, type I nitroreductase.
According to the position of the nitro substituent in the imidazole
ring (2-, 4- and 5-), the reduction potentials are different. In
relation to their biological activity, 2-nitroimidazole derivatives
are preferably used as radiosensitizers, while 5-nitroimidazole
derivatives are mainly used as antiparasitic or antibacterial drugs,
and 4-nitroimidazoles are relatively more inert [29]. However, the
descriptions of the mutagenic and carcinogenic properties of
some 2- and 5-nitroimidazole derivatives have also increased the
In vitro antiamoebic activity exhibited by compounds 9–15
was acceptable. All of them showed high bioactivity in the range
of 3.55–8.56 mM. Compounds 9 and 11 were as active as Bzn and
two times more active than Mni. With these biological results,
we could conclude that the introduction of benzenesulfonamide
core instead of acetamide substituent enhances the antiparasitic
activity of 2-methyl-4-nitroimidazole derivatives. However, none
of the compounds assayed were more active that metronidazole.
Fig. 4. Packing diagrams of compound 16g showing the formation of chains along a axis. Hydrogen bonds and interhalogen contacts are represented by dotted lines and H atoms
not involved in hydrogen bonding have been omitted for clarity.

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2980
Table 5
Physicochemical properties and in vitro antiparasitic bioactivity of imidazoleacetamides 1–8 and imidazolesulfonamides 9–15.
R
N
N
n
N
O2N
H
O
Cmpd
n
R
Mp (ꢂC)
Yield (%)
MS FAB (þ)
IC50
(m
M)
Selectivity index
CC50/IC50
T. vaginalis
G. intestinalis
E. histolytica
CC50
(
m
M) MDCK cell
T.v
10
46
148
70
47
120
11
G. i
E. h
1
2
3
4
5
6
7
8
1
0
0
0
0
0
0
0
H
H
4-F
4-Cl
4-CN
4-NO2
2,6-diCl
3-CF3
198.0–200.9
292.0–293.8
287.0–289.9
297.0–300.2
280.7–282.0
317.0–319.1
220.1–223.9
250.0–254.0
90.1
80.1
72.0
92.1
81.5
79.8
61.4
60.7
275
261
279
295
286
306
329
329
7.98
24.64
5.61
9.18
14.20
6.63
17.70
49.11
21.04
16.02
11.25
15.44
22.02
37.59
3.96
26.60
11.19
18.11
41.23
15.34
25.28
29.08
77.55
1136.60
828.06
642.35
672.37
798.03
396.49
435.03
4
23
39
40
60
52
18
12
20
43
74
35
16
52
16
15
37.02
67.43
6
O
O
N
N
S
O2N
R
Cmpd
R
Mp (ꢂC)
EIMS
IC50
(
m
M)
Selectivity index
CC50/IC50
T. vaginalis
G. intestinalis
E. histolytica
CC50
(
m
M) MDCK cell
T.v
G. i
E. h
9
–NHCOCH3
–H
–CH3
–Cl
–F
–NO2
–OCH3
152.0–154.0
129.0–131.3
147.1–150.2
174.3–175.8
152.5–155.1
160.1–161.1
160.0–161.5
324
267
281
301
285
312
297
8.73
12.52
9.96
7.62
11.48
2.93
8.41
20.18
11.99
13.97
15.34
10.62
7.50
3.89
6.74
3.55
4.03
4.44
7.28
8.56
1031.39
1270.30
987.59
578.37
813.32
611.35
321.30
118
101
99
76
71
51
106
71
38
77
265
188
278
144
183
84
10
11
12
13
14
15
209
38
82
49
6.55
38
N
NO2
N
H
Mni
Bnz
Met
40.74
18.62
0.29
20.40
22.58
5.36
8.38
4.27
0.77
N
O2N
N
H
N
O
N
N
NO2
OH
68.0
interest in minor mutagenic 4-nitroimidazoles [14,30]. Although
the nitro reduction is crucial for any biological activity, there are
still no conclusive results about the incidence of the nitro position
in their biological activity [31].
promising since many of the compounds showed activity compa-
rable with benznidazole, and even better potency. Bzn is an anti-
chagasic drug, but this study also shows other antiparasitic
bioactivities. The bioactivity observed against these three unicel-
lular parasites suggests that compounds 1–8 (acetamide deriva-
tives) showed mainly trichomonicidal and giardicidal properties,
whereas compounds 9–15 (sulfonamide derivatives) shown anti-
amoebic activity. None of the compounds assayed were more active
than metronidazole. Toxicity assay showed that all compounds are
non-cytotoxic against MDCK cell line.
3. Conclusion
We have synthesized new N-acetamide(sulfonamide)-
2-methyl-4-nitro-1H-imidazoles 1–15, and screened them for their
in vitro antiparasitic activity. The obtained results are very

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2981
This study demonstrated that the bioisosteric replacement of
2-nitroimidazole ring by 2-methyl-4-nitroimidazole scaffold,
results in retention and an enhancement of antiparasitic bioactivity.
Further optimization and pharmacokinetic characterization of this
series are in progress in our laboratory.
261.1085. Anal. Calcd. for C12H12N4O3: C, 55.38; H, 4.65; N, 21.53.
Found: C, 55.14; H, 4.61; N, 21.34.
4.2.3. N-(4-Fluorophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
acetamide (3)
Yield 0.80 g (91.3%) of white solid. Mp 287.0–289.9 ꢂC. 1H NMR
4. Experimental
(400 MHz, DMSO-d6)
d
2.31 (s, 3H, CH3), 5.03 (s, 2H, CH2CO), 7.39
(dd, 2H, H-20, J ¼ 8.8 Hz), 7.63 (dd, 2H, H-30, J ¼ 5.0 Hz, JH3 –
0
¼ 9.0 Hz), 8.35 (s, 1H, H-5), 10.79 (s, 1H, N–H). 13C NMR (100 MHz,
F0
4.1. Instruments
12.7 (CH3), 49.3 (CH2CO), 115.4 (d, 2C, C-30, C-50, JC3 –
0
DMSO-d6)
d
¼ 21.2 Hz), 120.9 (d, 2C, C-20, C-60, JC2 –F ¼ 21.2 Hz), 123.4 (C-5),
0
0
Melting points were determined on an EZ-Melt MPA120 auto-
mated melting point apparatus from Stanford Research Systems
and are uncorrected. Reactions were monitored by TLC on 0.2 mm
precoated silica gel 60 F254 plates (E. Merck). 1H and 13C NMR were
recorded on a Varian INOVA 400 and Varian Mercury 200 instru-
ments, respectively. Chemical shifts are given in ppm relative to
F
134.8 (C-10), 145.0 (C-4), 146.0 (C-2), 158.1 (d, C-40, JC4 –
0
¼ 238.3 Hz), 164.2 (C]O). MS (FABþ): m/z 279 (M þ H)þ. HRMS
F
(FABþ) Calcd. for C12H12N4O3F [M þ H]þ 279.2465, found: 279.1194.
4.2.4. N-(4-Chlorophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
acetamide (4)
tetramethylsilane (Me4Si,
d
¼ 0) in DMSO-d6; J values are given in
Hz. The following abbreviations are used: s, singlet; d, doublet; q,
quartet; dd, doublet of doublet; t, triplet; m, multiplet; bs, broad
signal. MS were recorded on a JEOL JMS-700 spectrometer by
Electron Impact or Fast Atom Bombarded [(FABD)]. Predictive
values of antiparasitic activities were also investigated using the
chemistry software server PASS (http://195.178.207.233/PASS/)
[19]. Starting materials benzylamine (a), aniline (b), 4-fluoroaniline
(c), 4-chloroaniline (d), 4-aminobenzonitrile (e), 4-nitroaniline (f),
2,6-dichloroaniline (g), 3-(trifluoromethyl)aniline (h), 2-methyl-
4(5)-nitro-1H-imidazole (17), 2-chloroacetyl chloride (18), and 4-
substituted benzenesulfonyl chlorides (19–25) were commercially
available from Aldrich and used without purification.
Yield 0.52 g (74.1%) of white solid. Mp 297.0–300.2 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d 2.31 (s, 3H, CH3), 5.01 (s, 2H, CH2CO), 7.39 (d,
2H, H-30, J ¼ 8.8 Hz), 7.62 (d, 2H, H-20, J ¼ 8.8 Hz), 8.33 (s, 1H, H-5),
10.60 (s, 1H, N–H). 13C NMR (100 MHz, DMSO-d6)
d 12.6 (CH3), 49.3
(CH2CO), 120.7 (2C, C-20, C-60), 123.4 (C-5), 127.3 (C-40), 128.7 (2C,
C-30, C-50), 137.2 (C-10), 145.0 (C-4), 146.0 (C-2), 164.4 (C]O). MS
(FABþ): m/z 295 (M þ H)þ. HRMS (FABþ) Calcd. for C12H12N4O3Cl
[M þ H]þ 295.7011, found: 295.0586. Anal. Calcd. for C12H11N4O3Cl:
C, 48.91; H, 3.76; N, 19.01. Found: C, 48.60 H, 3.55; N, 18.90.
4.2.5. N-(4-Cyanophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
acetamide (5)
Yield 0.85 g (94.6%) of white solid. Mp 280.7–282.0 ꢂC. 1H NMR
4.2. General method of synthesis of N-aryl-2-(2-methyl-4-nitro-
1H-imidazol-1-yl)acetamides (1–8)
(400 MHz, DMSO-d6)
4H, H-20, H-30), 8.35 (s,1H, H-5),11.23 (s,1H, N–H).13C NMR (100 MHz,
DMSO-d6)
12.6 (CH3), 49.5 (CH2CO), 105.4 (C-40), 118.7 (CN), 119.2
d 2.31 (s, 3H, CH3), 5.10 (s, 2H, CH2CO), 7.87 (m,
d
A solution of 2-methyl-4(5)-nitro-1H-imidazole (3.93 mmol,
1.0 equiv) and potassium hydroxide (4.72 mmol, 1.2 equiv) in
acetonitrile (10 mL) was stirred for 30 min until the anion was
formed (yellow solution). This mixture was added to a solution of
(2C, C-2, C-60),123.4 (C-5),133.3 (2C, C-30, C-50),142.6 (C-10), 145.0 (C-
4), 146.0 (C-2), 165.2 (C]O). MS (FABþ): m/z 286 (M þ H)þ. HRMS
(FABþ) Calcd. for C13H12N5O3 [M þ H]þ 286.2655, found: 286.1410.
substituted
a
-chloroacetamides 16a–h (4.33 mmol, 1.1 equiv) in
4.2.6. 2-(2-Methyl-4-nitro-1H-imidazol-1-yl)-N-(4-
nitrophenyl)acetamide (6)
acetonitrile (5 mL) and stirred under reflux for 8 h. Solvent was
removed under vacuum, and the residue obtained was washed
with water. The crude solid product was then recrystallized from
appropriate solvent.
Yield 0.85 g (88.2%) of white solid. Mp 317.0–319.1 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d 2.27 (s, 3H, CH3), 5.13 (s, 2H, CH2CO), 7.86 (d,
2H, H-30, J ¼ 8.8 Hz), 8.17 (d, 2H, H-20, J ¼ 9.6 Hz), 8.33 (s, 1H, H-5),
11.73 (s, 1H, N–H). 13C NMR (100 MHz, DMSO-d6)
d 13.2 (CH3), 50.2
4.2.1. N-Benzyl-2-(2-methyl-4-nitro-1H-imidazol-1-yl)
acetamide (1)
(CH2CO),119.7 (2C, C-2, C-60),124.1 (C-5),125.7 (2C, C-30, C-50),143.1
(C-4),145.4 (C-40),145.8 (C-10), 146.8 (C-2),166.3 (C]O). MS (FABþ):
m/z 306 (M þ H)þ.
Yield 0.91 g (83.9%) of white solid. Mp 198.0–200.9 ꢂC. 1H NMR
(400 MHz, DMSO-d6)
d
2.26 (s, 3H, CH3), 4.33 (d, 2H, CH2Ph, J ¼ 6.0 Hz),
4.85 (s, 2H, CH2CO), 7.24–7.36 (m, 5H, Ph), 8.31(s,1H, H-5), 8.82 (dd,1H,
4.2.7. N-(2,6-Dichlorophenyl)-2-(2-methyl-4-nitro-1H-imidazol-1-
yl)acetamide (7)
N–H, J ¼ 6 Hz). 13C NMR (100 MHz, DMSO-d6)
d 12.6 (CH3), 42.5
(CH2Ph), 48.8 (CH2CO),123.3 (C-5),126.9 (C-40),127.3 (C-20, C-60),128.3
(C-30, C-50), 138.6 (C-10), 145.0 (C-4), 145.7 (C-2), 165.4 (C]O). MS
(FABþ): m/z 275 (M þ H)þ. HRMS (FABþ) Calcd. for C13H15N4O3
[M þ H]þ 275.2826, found: 275.1259. Anal. Calcd. for C13H14N4O3: C,
56.93; H, 5.14; N, 20.43. Found: C, 57.05; H, 5.23; N, 19.77.
Yield 0.30 g (30.0%) of white solid. Mp 220.1–223.9 ꢂC. 1H NMR
(400 MHz, DMSO-d6) d 2.33 (s, 3H, CH3), 5.11 (s, 2H, CH2CO), 7.38 (dd,
1H, H-40, J ¼ 8.0 Hz), 7.57 (d, 2H, H-30, J ¼ 8.0 Hz), 8.38 (s, 1H, H-5),
10.43 (b, 1H, N–H). 13C NMR (100 MHz, DMSO-d6)
d 12.6 (CH3), 48.7
(CH2CO), 123.5 (C-5), 128.6 (2C, C-30, C-50), 129.6 (C-40), 132.0 (C-10),
133.3 (2C, C-20, C-60), 145.0 (C-4), 145.8 (C-2), 164.7 (C]O). MS
(FABþ): m/z 329 (M þ H)þ. HRMS (FABþ) Calcd. for C12H10N4O3Cl2
[M þ H]þ 329.1388, found: 329.0239. Anal. Calcd. for C12H10N4O3Cl2:
C, 43.79; H, 3.06; N, 17.02. Found: C, 42.58; H, 3.26; N, 16.98.
4.2.2. 2-(2-Methyl-4-nitro-1H-imidazol-1-yl)-N-
phenylacetamide (2)
Yield 0.46 g (75.8%) of white solid. Mp 292.0–293.8 ꢂC. 1H NMR
(400 MHz, DMSO-d6)
d 2.31 (s, 3H, CH3), 5.00 (s, 2H, CH2CO), 7.08
(dd, 1H, H-40, J ¼ 7.6 Hz,), 7.33 (dd, 2H, H-30, J ¼ 7.8 Hz, J ¼ 7.8 Hz),
4.2.8. 2-(2-Methyl-4-nitro-1H-imidazol-1-yl)-N-[3-
(trifluoromethyl)phenyl]acetamide (8)
7.58 (d, 2H, H-20, J ¼ 7.6 Hz), 8.46 (s, 1H, H-5), 8.82 (s, 1H, N–H). 13C
NMR (100 MHz, DMSO-d6)
d
12.7 (CH3), 49.3 (CH2CO), 119.1 (2C,
Yield 0.92 g (71.5%) of white solid. Mp 250.0–254.0 ꢂC. 1H NMR
C-20, C-60),123.5 (C-5),123.7 (C-40),128.9 (2C, C-30, C50),138.3 (C-10),
145.0 (C-4), 146.0 (C-2), 164.2 (C]O). MS (FABþ): m/z 261 (M þ H)þ.
HRMS (FABþ) Calcd. for C12H15N4O3 [M þ H]þ 261.2560 found:
(400 MHz, DMSO-d6) d 2.32 (s, 3H, CH3), 5.08 (s, 2H, CH2CO), 7.44
(d, 1H, H-40, J ¼ 7.6 Hz), 7.58 (d, 1H, H-50, J ¼ 8.0 Hz), 7.81 (d, 1H,
H-60, J ¼ 8.0 Hz), 8.12 (s, 1H, H-20), 8.36 (s, 1H, H-5), 11.17 (s, 1H,

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2982
N–H). 13C NMR (100 MHz, DMSO-d6)
d
12.7 (CH3), 49.4 (CH2CO),
(C-400), 145.4 (C-2). MS (EI) m/z (% int. rel.) 301 (10), 175 (93), 111
(100), 75 (27). Anal. Calcd. for C10H8ClN3O4S: C, 39.81; H, 2.67; N,
13.93. Found: C, 39.56; H, 2.54; N, 13.85.
115.2 (C-20), 120.0 (C-40), 122.7 (q, CF3, JC3 –F ¼ 258.0 Hz), 129.6
(C-30), 130.1 (C-60), 139.2 (C-50), 146.0 (C-2), 145.0 (C-4), 165.0
(C]O). MS (FABþ): m/z 329 (M þ H)þ. HRMS (FABþ) Calcd. for
C13H12N4O3F3 [M þ H]þ 329.2540, found: 329.0950.
0
4.3.5. 1-[(4-Fluorophenyl)sulfonyl]-2-methyl-4-nitro-1H-imidazole
(13)
Yield 0.15 g (14.3%) of white solid. Mp 152.5–155.1 ꢂC. 1H NMR
4.3. General method of synthesis of 1-(arylsulfonyl)-2-methyl-4-
nitro-1H-imidazoles (9–15)
(200 MHz, CDCl3)
d
2.59 (s, 3H, H-20), 7.36 (dd, 2H, H-300, H-500,
J ¼ 9.0, J ¼ 3.3 Hz), 8.05 (dd, 2H, H-200, H-600, J ¼ 8.0, J ¼ 3.0 Hz), 8.21
(s, 1H H-4). 13C NMR (50 MHz CDCl3) 15.4 (C-20), 117.9–118.2 (C-300,
d
To a solution of 2-methyl-4(5)-nitro-1H-imidazole (3.00 mmol) in
dichloromethane (10 mL) were added triethylamine (3.30 mmol,
1.1 equiv) and a catalytic amount of 4-dimethylaminopyridine (DMAP).
C-500, J ¼ 23.3 Hz), 118.7 (C-4), 131.2–131.3 (C-200, C-600, J ¼ 9.8 Hz),
132.1 (C-5, C-100), 145.3 (C-2), 167.0 (C-400, JC–F ¼ 259.5 Hz). MS (EI)
m/z (% int. rel.) 285 (19), 159 (100), 95 (96), 75 (16). Anal. Calcd. for
C10H8FN3O4S: C, 42.11; H, 2.83; N, 14.73. Found: C, 41.70; H, 2.86; N,
15.17.
After stirring at room temperature for 15 min,
a solution of
4-substituted benzenesulfonyl chloride (3.30 mmol, 1.1 equiv) in 5 mL
of dichloromethane was added droop by droop. The reaction mixture
was stirred at 40 ꢂC under nitrogen atmosphere for 6–10 h. After
complete conversion as indicated by TLC, the solvent was removed in
vacuo, the residue was neutralized with saturated NaHCO3 solution,
and the aqueous layer was extracted with ethyl acetate (3 ꢃ 15 mL),
washed with water (3 ꢃ 20 mL), and dried over anhydrous Na2SO4. The
solvent was evaporated in vacuo and the precipitated solids were
recrystallized from a mixture of ethanol–water.
4.3.6. 2-Methyl-4-nitro-1-[(4-nitrophenyl)sulfonyl]-1H-imidazole
(14)
Yield 0.13 g (10.8%) of white solid. Mp 160.1–161.1 ꢂC. 1H NMR
(200 MHz, DMSO-d6)
d
2.18 (s, 3H, 200-CH3), 7.87 (d, 2H, H-300, H-500,
13
J ¼ 8.0 Hz), 8.24 (d, 3H, H-200, H-600, J ¼ 8,0 Hz). C NMR (50 MHz
DMSO-d6)
d
14.2 (C-20), 120 (C-4), 123.8 (C-500, C-300), 127.5 (C-200,
C-400, C-600), 146.9 (C-100), 148.0 (C-2), 155.0 (C-5). MS (FABþ) m/z 312
(Mþ). Anal. Calcd. for C10H8N4O6S: C, 38.46; H, 2.58; N,17.94. Found:
C, 37.84; H, 2. 63; N, 17.50.
4.3.1. N-{4-[(2-Methyl-4-nitro-1H-imidazol-1-yl)
sulfonyl]phenyl}acetamide (9)
Yield 0.55 g (43.4%) of white solid. Mp 152.0–154.0 ꢂC. 1H NMR
4.3.7. 1-[(4-Methoxyphenyl)sulfonyl]-2-methyl-4-nitro-1H-
imidazole (15)
(200 MHz, DMSO-d6)
d
2.07 (s, 3H, 400-COCH3), 2.36 (s, 3H, 20-CH3),
7.57 (s, 4H, H-200, H-300, H-500, H-600), 8.27 (s, 1H, H-4) 10.08 (s, 1H,
´
Yield 1.03 g (89%) of white solid. Mp 160–161.5 ꢂC. 1H NMR
N–H). 13C NMR (50 MHz, DMSO-d6)
d 3.8 (C-2), 24.0 (COCH3), 118.0
(200 MHz, CDCl3)
d
2.52 (s, 3H, H-20), 3.87 (s, 3H, OCH3), 7.04 (dd,
(C-300, C-500), 119.5 (C-4), 126.7 (C200, C600), 139.8 (C-5), 142.0 (C-100),
145.1 (C-2), 146.2 (C-400), 168.4 (CO). MS (EI): m/z 324. Anal. Calcd.
for C12H12N4O5S: C, 44.44; H, 3.73; N, 17.28. Found: C, 44.05; H,
3.28; N, 17.80.
2H, H-300, H-500, J ¼ 8.8 Hz, J ¼ 1.6 Hz), 7.68 (dd, 2H, H-200, H-600,
J ¼ 8.8 Hz, J ¼ 1.6 Hz), 8.14 (s, 1H, H-4). 13C NMR (50 MHz CDCl3)
d
17.5 (C-2), 56.6 (O–CH3),11.8 (C-4),115.6 (C-500, C-300),128. 7 (C-100),
131.2 (C-5), 131.2 (C-5, C-200, C-600), 142.5 (C-2), 166.3 (C-400). MS (EI)
m/z (% int. rel.) 297 (7), 171 (100), 107 (48), 77 (34).
4.3.2. 2-Methyl-4-nitro-1-(phenylsulfonyl)-1H-imidazole (10)
Yield 0.15 g (14%) of white solid. Mp 129.0–131.3 ꢂC. 1H NMR
4.4. General method of synthesis of 2-chloro-N-arylacetamides
(16a–h)
(200 MHz, DMSO-d6) d
2.47 (s, 3H, 20-CH3), 7.37 (m, 3H, H-400, H-500,
H-600, J ¼ 8.0, J ¼ 2.0, Hz), 7.65 (m, 2H, H-200 H-600, J ¼ 8.0, J ¼ 2.0 Hz),
8.25 (s, 1H, H-4). 13C NMR (50 MHz, DMSO-d6)
d
14.8 (C-20), 119.0
4.4.1. Method A
(C-4), 125.7 (C-200, C-600), 127.9 (C-300, C-500), 128.5 (C-400), 144.0
(C-100), 128.5 (C-5), 144.5 (C-100), 149.9 (C-2). MS (EI) m/z (% int. rel.)
267 (20),141 (70), 77 (100). Anal. Calcd. for C10H9N3O4S: C, 44.94; H,
3.39; N, 15.72. Found: C, 44.02; H, 4.07; N, 15.52.
A solution of substituted amine a–h (6.20 mmol, 1 equiv) and
triethylamine (6.83 mmol, 1.1 equiv) in dichloromethane (10 mL)
was added over a period of 30 min to a solution of 2-chloroacetyl
chloride (6.83 mmol, 1.1 equiv) in dichloromethane (3 mL). The
reaction mixture was kept under stirring until the product was
formed as a solid (24 h). Dichloromethane was removed under
vacuum, and the residue obtained washer with water.
4.3.3. 2-Methyl-1-[(4-methylphenyl)sulfonyl]-4-nitro-1H-
imidazole (11)
Yield 0.53 g (48.4%) of white solid. Mp 147.1–150.2 ꢂC. 1H
4.4.2. Method B
NMR (200 MHz, CDCl3)
d
2.5 (s, 3H, 400-CH3), 2.57 (s, 3H, 20-CH3),
A solution of substituted amine a–h (17.99 mmol, 1 equiv) and
sodium bicarbonate (21.60 mmol, 1.2 equiv) in acetone (15 mL) was
added over a period of 30 min to a solution of 2-chloroacetyl
chloride (19.80 mmol, 1.1 equiv) in acetone (5 mL) The reaction
mixture was kept under stirring until the product was formed as
a solid (24 h). Acetone was removed under vacuum, and the residue
obtained was washed with water.
7.46 (s, 2H, H-200, H-600, J ¼ 8.4, J ¼ 2.4 Hz), 7.67 (s, 2H, H-300, H-500
J ¼ 8.4, J ¼ 2.4 Hz), 8.21 (s, 1H, H-4). 13C NMR (50 MHz CDCl3)
d
15.3 (CH3-C-20), 22.2 (CH3-C-400), 118.7 (C-4), 128.1 (C-200, C-600),
131.0 (C-300, C-500) 133.1 (C-5) 145.1 (C-100) 148.0 (C-2). MS (EI) m/
z (% int. rel.) 281 (10), 155 (70), 91 (100), 65 (23). Anal. Calcd. for
C11H11N3O4S: C, 46.97; H, 3.94; N, 14.94. Found: C, 46.63; H, 3.91;
N, 14.74.
4.4.3. N-Benzyl-2-chloroacetamide (16a)
4.3.4. 1-[(4-Chlorophenyl)sulfonyl]-2-methyl-4-nitro-1H-imidazole
(12)
White solid, Mp 93.4–94.8 ꢂC (Lit. 92–93 ꢂC [32]).
Yield 0.51 g (45.1%) of white solid. Mp 174.3–175.8 ꢂC. 1H NMR
4.4.4. 2-Chloro-N-phenylacetamide (16b)
(200 MHz, CDCl3)
d
2.59 (s, 3H, 20-CH3), 7.65 (m, 2H, H-300, H-500,
White solid, Mp 136.0–139.0 ꢂC (Lit 134–135 ꢂC [33]).
J ¼ 8.7, J ¼ 2.7 Hz), 7.94 (m, 2H, H-200, H-600, J ¼ 8.7, J ¼ 2.7 Hz), 8.21
(s, 1H, H-4). 13C NMR (50 MHz CDCl3)
d
: 15.4 (C-20), 118.7 (C-4),
4.4.5. 2-Chloro-N-(4-fluorophenyl)acetamide (16c)
129.0 (C-200, C-600), 129.5 (C-5), 130.7 (C-300, C-500), 134.5 (C-100), 143.4
Grey solid, Mp 112.0–115.0 ꢂC (Lit. 109–112 ꢂC [34]).

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E. Hernandez-Nun˜ez et al. / European Journal of Medicinal Chemistry 44 (2009) 2975–2984
2983
4.4.6. 2-Chloro-N-(4-chlorophenyl)acetamide (16d)
of G. lamblia or T. vaginalis or E. histolytica were incubated for 48 h
at 37 ꢂC with increasing concentrations of synthesized compounds,
Benznidazole, metronidazole and 2-methyl-4(5)-nitro-1H-imid-
azole. As the negative control, trophozoites were incubated with
DMSO used in the experiments. After the incubation, the tropho-
zoites were washed and subcultured for another 48 h in fresh
medium alone. At the end of this period, 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.
White solid, Mp 169.8–172.0 ꢂC (Lit 168–170 ꢂC [33]). 1H NMR
(200 MHz, CDCl3)
J ¼ 8.8 Hz); 7.33 (d, 2H, H-2, H-6, J ¼ 8.8 Hz); 9.56 (bs, 1H, N–H)
ppm; 13C NMR (50 MHz, CDCl3)
42.9 (CH2Cl); 120.8 (2C, C-2, C-6);
d
3.88 (s, 2H, COCH2Cl); 7.00 (d, 2H, H-3. H-50,
d
128.3 (2C, C-3, C-5); 128.5 (C-4); 136.3 (C-1); 164.4 (C]O) ppm.
4.4.7. 2-Chloro-N-(4-cyanophenyl)acetamide (16e)
Beige solid, Mp 184.0–186.6.0 ꢂC. 1H NMR (200 MHz, CDCl3)
d
3.90 (d, 2H, COCH2Cl, J ¼ 2.0 Hz); 7.33 (dd, 2H, H-2, H-6, J ¼ 2.0,
8.8 Hz); 7.53 (dd, 2H, H-3, H-5, J ¼ 2.0, 8.8 Hz); 9.92 (s, 1H, N–H)
ppm, 13C NMR (50 MHz, CDCl3)
43.0 (CH2Cl), 106.4 (C-4), 118.5
d
4.6.2. Cytotoxicity test
(CN), 119.5 (2C, C-2, C-6), 132.6 (2C, C-3, C-5), 142.0 (C-1), 165.0
(C]O) ppm.
Cytotoxicity on host cells is a very important criterion for
assessing the selectivity of the observed antiparasitic activity. The
cytotoxicity assay was performed according to Rahman et al. [42],
where, 1.5 ꢃ104 viable cells from the cell line were seeded in
a 96-well plate and incubated for 24–48 h. Dog kidney cells (MDCK)
were grown in DMEM media supplemented with 10% (v/v) Fetal
Bovine Serum with 100 U/mL penicillin and 100 mg/mL strepto-
mycin and maintained at 37 ꢂC in a 5% CO2 atmosphere with 95%
humidity. When cells reached >80% confluence, the medium was
replaced and the cells were treated with the compounds at 6.25,
4.4.8. 2-Chloro-N-(4-nitrophenyl)acetamide (16f)
Green solid, Mp 184.0–185.6 ꢂC (Lit 181–183 ꢂC [33]).
4.4.9. 2-Chloro-N-(2,6-dichlorophenyl)acetamide (16g)
White solid, Mp 178.0 ꢂC (Lit. light Brown 172 ꢂC [35]). 1H NMR
(200 MHz, DMSO-d6)
d 4.34 (s, 2H, COCH2Cl), 7.56 (d, 2H, H-3, H-5,
J ¼ 8.0 Hz), 7.37 (d, 1H, H-4, J ¼ 7.4, Hz), 10.26 (s, 1H, N–H) ppm, 13C
NMR (50 MHz, DMSO-d6)
d
42.3 (CH2Cl), 128.5 (2C, C-3, C-5), 129.5
12.5, 25, 50 and 100
mg/mL dissolved in DMSO at a maximum
(C-4), 132.1 (C-1), 133.5 (2C, C-2, C-6), 164.8 (C]O) ppm.
concentration of 0.05%. After 72 h of incubation, 10
mL of a 0.005%
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) solution (5 mg/mL) was added to each well and incubated at
37 ꢂC for 4 h. The medium was removed and the formazan,
a product generated by the activity of dehydrogenases in cells, was
dissolved in acidified isopropanol (0.4 N HCl). The amount of
MTT–formazan is directly proportional to the number of living cells
and was determined by measuring the optical density (OD) at
540 nm using a Bio-assay reader. Metronidazole was used as
a positive control, whereas untreated cells were used as negative
controls. The concentration of the crude extract that killed 50% of
the cells (CC50) was calculated by GraphPad Prim 4 software. All
determinations were performed in triplicate [42].
4.4.10. 2-Chloro-N-[3-(trifluoromethyl)phenyl] acetamide (16h)
White solid, Mp 73.3–75.5 ꢂC. 1H NMR (200 MHz, CDCl3)
d ppm:
4.22 (s, 2H, COCH2Cl); 7.47 (d, 1H, H-4, J ¼ 8.0 Hz); 7.47 (dd, 1H, H-5,
J ¼ 8.0 Hz); 7.77 (d, 1H, H-6, J ¼ 8.0 Hz); 7.85 (s, 1H, H-2); 8.40 (bs,
1H, N–H) ppm; 13C NMR (50 MHz, CDCl3)
d 43.0 (CH2Cl); 117.0 (q,
C-2, JC–F ¼ 3.8 Hz); 122.0 (q, C-40, JC–F ¼ 6.0 Hz); 122.7 (q, C-3,
JC–F ¼ 65.15 Hz); 123.3 (C-6); 129.0 (C-5); 127.2 (q, CF3,
JC–F ¼ 320.0 Hz); 137.3 (C-1); 164.2 (C]O).
4.5. X-ray crystallography
X-ray diffraction studies were performed on a Bruker-APEX
diffractometer with
monochromator: graphite). Frames were collected at 100 K for
compound 1 and 293 K for compound 16g via -rotation at 10 s
a
CCD area detector
(
lMOK ¼ 0.71073 Å,
a
Acknowledgments
u/4
This work was supported in part by grant from PROMEP-SEP,
UAEMOR-PTC-131 (GNV). We are grateful to Ismael Leon-Rivera,
Victoria Labastida and Maria Medina from the CIQ, UAEM for the
determination of all mass spectra. We are indebted to Luis Domi-
nguez-May, Juan Chale Dzul and Nina Mendez-Domınguez from
the UADY, for their technical assistance during antiparasitic
susceptibility assays.
per frame (SMART) [36]. The measured intensities were reduced to
F2 and corrected for absorption with SADABS (SAINT-NT) [37].
Corrections were made for Lorentz and polarization effects. Struc-
ture solution, refinement and data output were carried out with the
SHELXTL-NT program package [38,39]. Non-hydrogen atoms were
refined anisotropically. All hydrogen atoms (except for H4 in
compound 1 and H1 in compound 16g) were placed in geometri-
cally calculated positions using a riding model. Atoms H4 and H1
were located in a difference Fourier map and were freely isotropi-
cally refined. Crystallographic data for the structures reported in
this paper have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publications nos. CCDC-
692184, 692185. Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: (þ44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk, www:
http://www.ccdc.cam.ac.uk).
´
´
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