Design, synthesis and biological evaluation of 2-(2-amino-5(6)-nitro-1H-benzimidazol-1-yl)-narylacetamides as antiprotozoal agents

Article abstract of DOI:10.3390/molecules22040579

Parasitic diseases are a public health problem affecting millions of people worldwide. One of the scaffolds used in several drugs for the treatment of parasitic diseases is the benzimidazole moiety, a heterocyclic aromatic compound. This compound is a crucial pharmacophore group and is considered a privileged structure in medicinal chemistry. In this study, the benzimidazole core served as a model for the synthesis of a series of 2-(2-amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-arylacetamides 1-8 as benznidazole analogues. The in silico pharmacological results calculated with PASS platform exhibited chemical structures highly similar to known antiprotozoal drugs. Compounds 1-8 when evaluated in silico for acute toxicity by oral dosing, were less toxic than benznidazole. The synthesis of compounds 1-8 were carried out through reaction of 5(6)-nitro- 1H-benzimidazol-2-amine (12) with 2-chlroactemides 10a-h, in the presence of K₂CO₃ and acetonitrile as solvent, showing an inseparable mixture of two regioisomers with the -NO₂ group in position 5 or 6 with chemical yields of 60 to 94%. The prediction of the NMR spectra of molecule 1 coincided with the experimental chemical displacements of the regioisomers. Comparisons between the NMR prediction and the experimental data revealed that the regioisomer endo-1,6-NO₂ predominated in the reaction. The in vitro antiparasitic activity of these compounds on intestinal unicellular parasites (Giardia intestinalis and Entamoeba histolytica) and a urogenital tract parasite (Trichomonas vaginalis) were tested. Compound 7 showed an IC₅₀ of 3.95 μM and was 7 time more active against G. intestinalis than benznidazole. Compounds 7 and 8 showed 4 times more activity against T. vaginalis compared with benznidazole.

Full text of DOI:10.3390/molecules22040579

molecules
Article
Design, Synthesis and Biological Evaluation of
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-
arylacetamides as Antiprotozoal Agents
2
1
,
2
3
4
Emanuel Hernández-Núñez *, Hugo Tlahuext , Rosa Moo-Puc , Diego Moreno ,
5
6
María Ortencia González-Díaz and Gabriel Navarrete Vázquez
1
CONACYT, Departamento de Recursos del Mar, Centro de Investigación y de Estudios Avanzados del IPN,
Unidad Mérida, Mérida 97310, Yucatán, Mexico
2
3
4
5
6
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,
Cuernavaca 62210, Morelos, Mexico; tlahuext@uaem.mx
Unidad Interinstitucional de Investigación Médica, Instituto Mexicano del Seguro Social/Universidad
Autónoma de Yucatán, Mérida 97150, Yucatán, Mexico; moopuc@gmail.com
Facultad de Ingeniería, Universidad Autónoma de Yucatán, Mérida 97310, Yucatán, Mexico;
diegomorenor@gmail.com
CONACYT, Centro de Investigación Científica de Yucatán, Mérida 97200, Yucatán, Mexico;
maria.gonzalez@cicy.mx
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca 62209, Morelos, Mexico;
gabriel_navarrete@uaem.mx
*
Correspondence: emanuel.hernandez@cinvestav.com or ehernandeznu@conacyt.mx; Tel.: +52-999-942-9400
Academic Editor: Diego Muñoz-Torrero
Received: 24 January 2017; Accepted: 31 March 2017; Published: 4 April 2017
Abstract: Parasitic diseases are a public health problem affecting millions of people worldwide. One
of the scaffolds used in several drugs for the treatment of parasitic diseases is the benzimidazole
moiety, a heterocyclic aromatic compound. This compound is a crucial pharmacophore group
and is considered a privileged structure in medicinal chemistry. In this study, the benzimidazole
core served as a model for the synthesis of a series of 2-(2-amino-5(6)-nitro-1H-benzimidazol-1
-
yl)-N-arylacetamides
with PASS platform exhibited chemical structures highly similar to known antiprotozoal drugs.
Compounds when evaluated in silico for acute toxicity by oral dosing, were less toxic than
benznidazole. The synthesis of compounds were carried out through reaction of 5(6)-nitro-
H-benzimidazol-2-amine (12) with 2-chlroactemides 10a , in the presence of K CO and acetonitrile
as solvent, showing an inseparable mixture of two regioisomers with the -NO group in position 5
1–8 as benznidazole analogues. The in silico pharmacological results calculated
1–8
1–8
1
–h
2
3
2
or 6 with chemical yields of 60 to 94%. The prediction of the NMR spectra of molecule 1 coincided
with the experimental chemical displacements of the regioisomers. Comparisons between the NMR
prediction and the experimental data revealed that the regioisomer endo-1,6-NO predominated in
2
the reaction. The in vitro antiparasitic activity of these compounds on intestinal unicellular parasites
(
Giardia intestinalis and Entamoeba histolytica) and a urogenital tract parasite (Trichomonas vaginalis)
were tested. Compound showed an IC50 of 3.95 M and was 7 time more active against G. intestinalis
showed 4 times more activity against T. vaginalis compared
7
µ
than benznidazole. Compounds
with benznidazole.
7 and 8
Keywords: benzimidazole; antiprotozoal; NMR prediction
Molecules 2017, 22, 579; doi:10.3390/molecules22040579
www.mdpi.com/journal/molecules

Molecules 2017, 22, 579
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1
. Introduction
Parasitic diseases are a public health problem affecting millions of people worldwide [
1]. Parasites
are eukaryotic and share common features with their mammalian host. This has motivated the search
for effective and selective drugs, although the task remains a difficult one. While significant efforts
have been made in the discovery of new therapeutic targets that are more selective and more potent,
these can also come with side-effects that can be severe [
research that can be classified in drug repositioning (old drugs for pharmacological therapies to current
diseases) [ ], the search of new drugs obtained of the nature (plants, organism’s marine, terrestrial,
bacteria, to mention some) [ ], the rational design of drugs (medicinal chemistry) [ ] and the evaluation
of new chemical entities in in silico evaluations [ ]. Benzimidazole, a heterocyclic aromatic chemical
2]. The current status of anti-parasitic drug
3
4
5
6
compound, is a crucial pharmacophore group and a privileged structure in medicinal chemistry. Many
heterocyclic nuclei are present in antiparasitic drugs, such as 5(6)-substituted benzimidazoles and
2
-aminonitrobenzimidazoles [
common antiparasitic drug and the drug of choice for anti-infectious chemotherapy against anaerobic
protozoa and other parasites [ ]. Benznidazole [Bnz, Rosanil], a 2-nitroimidazole derivative, is an
7,8]. The 2-aminobenzimidazole core is found in albendazole, the most
9
important drug for Chagas disease, and has been used in other parasitic diseases [10]; For example:
Nava-Zuazo et al. (2014) proposed the use of Bnz as an alternative treatment against Giardia intestinalis,
Entamoeba histolytica and Trichomonas vaginalis in in vitro experiments [11] and Bernandino et al.
used the drug against four Leishmania species [12]. Currently, the pharmaceutical industry uses
old drugs with known pharmacokinetic parameters for new diseases in order to save time in the drug
development, a process called drug repositioning [13]. Bnz’s mode of action is related to reductive
metabolism. It functions as a prodrug and must be activated by an NADH-dependent, mitochondrially
localized, bacterial-like, type I nitroreductase [14]. Several compounds containing the 5(6)-nitro
or 2-amino-benzimidazole scaffold have been used as antiparasitic [15], antimycobacterial [16],
antimicrobial [17] and antifungal [18], anthelmintic [19], antitumoral [20], antioxidant [21], analgesic
and anti-inflammatory[22], antihypertensive and vasorelaxant [23] agents. As a part of our search
for basic information about the structural requirements for new antiparasitic activities of an old drug
(
Bnz), we synthesized a series of novel 2-(2-amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-arylacetamide
benzoanalogues 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) are reported in this paper.
2
. Results and Discussion
2
.1. Drug Design of Benznidazole Derivatives
The compounds shown in Table 1 were designed based on the structures of benznidazole, a drug
used to treat trypanosomiasis, and widely recommended as an antiprotozoal agent [24,25].
Table 1. Calculated activity and toxicity in silico profiles for compounds 1–8 and antiprotozoal choice drugs.
Antiparasitic Effect
LD50 (mg/Kg)
(Trichomonas vaginalis)
Compound
Mouse
Rat
Pa
Pi
ip
p.o.
ip
po
1
2
3
4
5
6
7
8
0.313
0.319
0.250
0.304
0.201
0.324
0.292
0.201
0.333
0.705
0.023
0.020
0.065
0.027
0.122
0.018
0.033
0.122
0.015
0.002
300
460
490
490
380
430
290
280
150
1100
1300
1600
830
840
960
1200
920
1100
1200
280
430
330
530
460
800
1200
280
440
480
1300
1200
830
670
970
320
670
900
750
620
1100
1
0
Bnz
Pa = Probability of activity Pi = Probability of inactivity ip = Intraperitoneal po = Oral.

Molecules 2017, 22, 579
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Common constituents of these compounds are the imidazole ring and the presence of the nitro
group, which is located in 2-position. The first design consideration was to join the benznidazole to
generate a series of imidazole derivatives that were reported in previous studies [26]. The second
consideration in this work was the introduction of an aromatic ring between the nitro group and the
imidazole following the principle of vinylogy [27] to generate 5(6)-nitrobenzimidazole derivatives.
Finally, the third consideration was to change the nitro group present in 2-position for the amine
group, favoring the formation of hydrogen bonds and increasing solubility in aqueous systems [28
Scheme 1).
]
(
Scheme 1. Drug design of benznidazole analogues.
2
.2. Evaluation in Silico (PASS and ACDTox/Suite)
Predictive values concerning antiparasitic activities were obtained by comparing the chemical
structures of the compounds designed with the structures or substructures of more than 30,000
well-known biologically active drugs. Prediction results are presented as estimates of the probability,
Pa, that the compounds are active and Pi, the probability that compounds are not active. For Pa values
=
0.7, the corresponding compound is very likely to reveal this activity in experiments, however 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
values for 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-arylacetamide 1–8 are summarized in Table 1.
These show biological activities against trichomonas. Pa values estimated for trichomonas activity
were lower than 0.5 for all compounds. For benznidazole the Pa values estimated for trichomonas
activity were 0.497 and Pa values estimated for antiprotozoal (Trichomonas vaginalis) activity were 0.705.
These results indicated that 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-arylacetamide 1–8 exhibited
chemical structures highly similar to known antiprotozoal drugs.
Computational prediction of toxicity [29] has been performed in drug development in order to
anticipate potentially harmful substances. The toxicity parameters of all the benznidazole compounds
were calculated with ACD/Tox Suite software (v. 2.95) [30].
The acute toxicity of a chemical is defined as a dose that is lethal to 50% of treated animals (LD ).
50
The acute toxicity can be viewed as a ‘cumulative potential’ to cause various acute effects and death of

Molecules 2017, 22, 579
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animals [31]. In these predictions, all compounds evaluated in silico in mice by oral dosing were less
toxic than benznidazole. However, intraperitoneal (i.p.) route of administration the compounds are
shown to be more toxic than benznidazole.
2
.3. Chemical Synthesis and Characterization
The appropriately substituted -chloroacetamides 12a
of arylanilines with 2-chloroacetyl chloride (11) (Scheme 2). In the methodology, we used
α
–h, were synthesized by condensation
a–h
methylene chloride as the solvent and triethylamine as the base, producing the desired compounds
with yields ranging from 70%–90%. The reaction conditions and product yields are listed in a previous
article [26]. Compound 10 was prepared as shown in Scheme 1 [32]. Treatment of 4-nitro-1,2-
◦
phenylenediamine (
(6)-nitro-1H-benzimidazol-2-amine (10). The synthesis of compounds
the reaction of 5(6)-nitro-1H-benzimidazol-2-amine (10) with 2-chloroacetamides 12a
of K CO and acetonitrile as solvents, as shown in Scheme 2. Solid compounds were purified
9
) with cyanogen bromide in a mixture of diglyme/water (4:1) at 90 C gave
5
1–8
were carried out through
, in the presence
–
h
2
3
by recrystallization or by column chromatography and the structure of the pure compounds was
established by spectroscopic data. The compounds 1–8 were obtained in good yields (Table 2).
Scheme 2. Reagents: (i) BrCN, Diglime/water (4:1); (ii) NaHCO , acetone.
3
The chemical structures of the synthesized compounds were confirmed based on their spectral
data (NMR and mass spectra), and their purity ascertained by microanalysis. The elemental analysis
was 5 ppm less than the theoretical values obtained for FAB-MS. Physical constants of the title
compounds are shown in Table 2.
1
In the nuclear magnetic resonance spectra, two possible products were observed by H- and
C-NMR. Figure 1a shows the H-NMR spectrum of compound 1, where the signals corresponding
13
1
to the benzylic protons found at approximately 4.4 ppm and the methylene hydrogens alpha to the
carbonyl group at 4.8 ppm are observed in a ratio of 75:25.

Molecules 2017, 22, 579
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Table 2. Physicochemical properties, and in vitro antiprotozoal bioactivity benznidazole analogues 1–8.
IC50 (µM)
Reaction Yield
MS FAB
(+)
◦
Cmpd. m.p. ( C)
Reigoisomers
Mixture Ratio
Time (h)
(%)
T. vaginalis
G. lamblia
E. histolytica
1
2
3
4
5
6
7
8
279.0–282.0
285.0–287.8
313.0–315.0
294.0–296.0
286.0–289.0
309.0–312.0
303.0–306.0
282.0–284.0
192.0–194.0
5.0
5.0
7.0
12.0
8.0
8.0
18.0
18.0
5.0
90.1
80.1
72.0
92.1
81.5
79.8
61.4
60.7
94.1
326
297
330
345
337
357
381
380
179
75:25
60:40
70:30
69:31
57:43
59:41
55:45
61:39
50:50 *
11.34
26.76
15.61
14.66
11.16
7.09
5.92
5.62
22.12
11.26
10.44
16.47
15.26
14.45
16.52
9.20
18.43
46.16
26.14
28.28
21.07
13.23
34.01
45.60
46.80
3.84
28.39
1
0
Bnz
- - -
- - -
- - -
- - -
- - -
18.62
22.58
4.27
ND = not determinate; * Tautomeric equilibrium.
7
5:25
Figure 1.
(
a
) 1H-NMR spectrum for compound
1
in the region between 4 and 5 ppm and (
b
) 1H-NMR
◦
spectra acquired at different temperature from 20 to 100 C (400 MHz, DMSO-d ).
6
Based on the NMR data, to confirm the formation of regioisomers a variable temperature NMR
experiment was performed (Figure 1b), in which the existence of a tautomer mixture would be

Molecules 2017, 22, 579
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confirmed by rapid interconversion between the two components of the mixture. The coalescence
temperature would be where a single signal is observed for each of the protons of the structure. In this
experiment, coalescence with increasing temperature was not observed, leading to the conclusion that
the mixture analyzed did not correspond to the formation of tautomers, but rather to the generation of
regioisomers (Scheme 1).
Thus, the existence of a mixture of regioisomers was proposed and derivative allocation was
performed. The formation of the regioisomer mixture could be explained by the possible formation
of hydrogen bonds between the carbonyl oxygen group and the hydrogen of the amino group. This
favors the formation of both regioisomers and the hydrogen bond, which gives them a conformation
in three dimensions composed of a 6-membered ring, anchoring the conformations observed for
these compounds and reducing the energy to a global minimum to stabilize the molecules (Figure 2).
To strengthen the hypothesis of the formation of a mixture of regioisomers, two-dimensional NMR
experiments were performed, in which a correlation of protons was observed in the NOESY spectrum
at 4.8 ppm (–CH ) with the corresponding aromatic proton of the benzimidazole, in this case with the
2
1
H-7, indicating that the possible structure is binding to the N , which would generate a mixture of
regioisomers (Figure 2).
H-7, 7.19,7.20
H-11, 4.83, 4.87
◦
Figure 2. NOESY NMR experiment (mixing time 100 ms, temperature 25 C, Varian Oxford 400 MHz).

Molecules 2017, 22, 579
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1
In the structural elucidation of the benzimidazole derivatives, the signals ( H-NMR;
δ
ppm) of the
respective protons of the compounds were verified based on their chemical shifts, multiplicities and
1
coupling constants. In compounds
ranging from
1–
8
, the aromatic region of the H-NMR spectrum contained signals
0 0 0 0
7.04–8.24 ppm, attributable to H-2 , H-3 , H-5 and H-6 , of the 4-substituted benzene
δ
ring. In all compounds, observations included a characteristic pattern for NHCH CO (H-11), a singlet
2
methylene signal ranging from 4.83 to 5.11, and a broad signal ranging from 7.03 to 7.63, attributable to
the NH of the heterocyclic ring. One peak (J
m
= 2.0 Hz) was observed for compounds
1–
8, assigned
2
to hydrogen H-4 or H-7 found in aromatic benzimidazole (Table 3).
Table 3. NMR predictions for reigoisomers and tautomer compounds.
Experimental (δ, DMSO-d6)
1H-NMR 13C-NMR
In Silico (δ, DMSO-d6)
1H-NMR
13C-NMR
Label
a
b
a
b
a
b
c
d
a
b
c
d
1
2
3
4
5
6
7
8
9
- - -
- - -
- - -
7.93
7.87
- - -
7.19
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
- - -
7.52
8.57
- - -
8.10
- - -
6.89
- - -
- - -
7.54
8.60
- - -
8.29
- - -
7.13
- - -
- - -
7.18
- - -
8.44
8.71
- - -
- - -
- - -
- - -
- - -
158.05 159.58
- - - - - -
114.67 118.04 6.75
140.14 138.70 - - -
109.32 113.38 8.38
107.06 103.48 8.74
141.88 141.88
142.81 149.73
- - -
44.87
165.93 166.08
- - -
163.58 166.01 161.77 158.55
- - - - - - - - - - - -
- - -
7.97
- - -
7.95
7.20
- - -
120.50 121.30 121.08 119.48
152.59 148.66 149.48 152.78
121.72 125.74 126.13 121.83
107.17 106.15 108.73 109.66
145.53 140.09 138.02 144.21
151.13 157.75 183.91 151.70
- - -
- - -
5.54
4.30
- - -
5.15
4.61
- - -
- - -
- - -
- - -
- - -
- - -
1
1
1
1
1
1
2
3
4
5
6
0
1
2
3
7.03
4.83
- - -
8.73
4.32
- - -
7.03
4.87
- - -
8.78
4.33
- - -
- - -
44.72
5.82
4.31
- - -
5.32
4.62
- - -
7.50
7.63
7.61
7.62
7.46
3.81
4.25
- - -
4.98
4.60
- - -
7.44
7.75
7.70
7.63
7.72
5.77
3.74
- - -
4.61
4.77
- - -
7.63
7.69
7.68
7.74
7.47
- - -
50.92
- - -
50.93
- - -
49.35
- - -
48.10
173.12 173.57 172.21 169.12
- - -
48.44
145.57 144.75 147.96 147.03
135.56 135.36 135.06 134.90
134.15 134.59 134.90 134.12
134.88 134.92 133.23 134.04
134.30 134.11 134.47 134.53
135.25 135.38 136.46 135.65
- - -
42.39
- - -
48.56
- - -
47.53
- - -
48.10
4
42.41
138.77 134.31
0
0
0
0
0
0
7.23–7.36
127.20 127.15 7.48
127.20 128.19 7.64
126.79 127.13 7.62
127.20 128.19 7.63
127.20 127.15 7.50
7.23–7.36
7.23–7.36
7.23–7.36
7.23–7.36
Scheme 3 describes the proposed reaction mechanism for the formation of the regioisomers.
Initially, the electron pair of the nitrogen exo located in C-2 is introduced and is directed towards
the N-3 endo (tautomeric electron transfer to Petrova et al.) [33]. The anion formed nucleophilic ally
1
displaces the chlorine from the amide, where the N enters its electron pair system to regenerate
aromaticity, leading to the formation of the reigoisomer mixture. These were inseparable from one
another using conventional methods, such as crystallization and open column chromatography.

Molecules 2017, 22, 579
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Some examples of mixtures of regioisomers have been previously described in the literature,
for instance Chen and Willis (2015) reported that a combination of arynes, generated using fluoride
from the corresponding 2-(trimethylsilyl)aryl triflates and 3-hydroxy-4-aminothiadiazoles, leads
to the selective formation of 3-amino-substituted benzo[d]isothiazoles to obtain an inseparable
mixture of regioisomers [34]. The diprop-2-ynylamines 4a–c were treated with methyl prop-2-ynyl
ether under standard conditions. The obtained product, isoindolinone, was isolated by column
chromatography as an inseparable 1:1 mixture of regioisomers [35]. On the other hand, in a
study of anticancer compounds, an inseparable mixtures of 2 regioisomers were obtained from
6-[N-(2-t-butyldimethylsilyloxyethyl)-N-methylamino]-2-bromo-3-methoxy-1,4,5,8-triptycenetetraone;
and 6-[N-(4-hydroxybutyl)-N-methylamino]-2-bromo-3-methoxy-1,4,5,8-triptycenetetraone by silica
gel column chromatography [36]. In addition, Meng et al. reported the synthesis of a regioisomer
mixture (1:1 ratio) of 5(6)-methyl-1-(1-phenylethyl)-1H-benzo[d]imidazole [37].
Scheme 3. Mechanism of the formation of reigoisomers mixture 1,5 and 1,6.
2
.4. NMR Prediction
The H and 13C theoretical and experimental chemical shifts (
1
δ
), isotropic shielding values (
σ
)
1
13
and the assignments of fourth compounds are included in Table 3. The observed H and C-NMR
spectra of the compounds 1a and 1b in DMSO-d solvent are shown in the Table 3, respectively. All
6
calculations of the NMR shielding constants have been performed using gauge-including atomic
orbitals (GIAO) approximation provided by GAUSSIAN 09,[38] in DMSO solvent. Nwchem program
package (version 6.5) was employed [39]. Geometry optimizations were carried out at the DFT
level with the B3LYP exchange-correlation functional [40] and Def2-TZVP [41] basis set. Harmonic
vibrational frequencies were also analyzed at the same level to characterize the nature of the stationary
molecules. The isotropic shielding values were used to calculate the isotropic chemical shifts with
respect to tetramethylsilane (TMS).
1
13
The H and C chemical shifts are referenced from the DMSO-d solvent resonances, which are
6
assigned as 2.50 and 39.5 ppm, respectively. The experimental chemical shifts of proton attached to
nitrogen for the compounds 1a and 1b were shifted towards downfield due to the hydrogen bonding
interaction and the signals were observed at 7.03 and 8.73 ppm, respectively. The computed NMR
chemical shifts of NH protons of compounds 1a and 1b were found at 5.54, 5.82, 5.15 and 5.32 ppm,
respectively. The calculated value of N-H proton for compound 1 remains unacceptable apart from
the experimental values, since the chemical shift associated with this proton is not correctly described
by the continuum model. For the compounds 1a and 1b, the difference between the theoretical and
experimental N-H chemical shifts is probably caused by intermolecular hydrogen bonding. This
discrepancy can be explained by the DFT calculations belonging to an isolated molecule in gas phase
while the experimental data belong to the same molecule in solid state phase. The experimental

Molecules 2017, 22, 579
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chemical shift of methylene protons (H-11) was shifted towards downfield due to the electronegativity
of nitrogen atom and the signals were observed at 4.83 and 4.87 ppm for the compounds 1a and
1b, respectively. Corresponding signals for the phenyl substituted methylene protons (H-14) were
also observed at the 4.32 and 4.33 4 ppm, respectively. The computed NMR chemical shifts of
methylene protons (H-11) of compounds 1a and 1b were found at 4.30 and 4.31, 4.61 and 4.62 ppm,
respectively. The difference between the chemical shifts was 0.21 with an estimated error of 4.5% for
the method used in this calculation for these protons. The protons of the benzimidazole group were
experimentally found at 7.93, 7.87 and 7.19 ppm in the DMSO solution for compound 1a (H-4, H-6 and
H-7, respectively); at 7.20, 7.95 and 7.97 ppm for compound 1b (H-4, H-5 and H-7, respectively). For
comparison, our theoretical calculations yielded in 7.52, 8.57 and 8.10 ppm for compounds 1b, and
1
6
.75, 8.38 and 8.74 ppm for 1a. Calculated H-NMR peaks are in agreement with both experimental
(
Table 3).
For the chemical displacements of 13C, the experimental and theoretical displacements were
compared, revealing only small differences (Table 3). It was concluded from the data collected in
13
Table 3 that experimental and theoretical values of C-NMR were more compatible than those values
acquired for H-NMR.
1
With regard to the protons of the benzimidazole group for the tautomers 1c and 1d, the estimated
error between the experimental values and theoretical calculations is about 11.1% (see Table 3 for
chemical shift values) suggesting that these isomers were not formed. The 13C theoretical data do not
correspond to those obtained experimentally (Table 3).
2
.5. In Vitro Antiprotozoal Assays
In this study eight new 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-Arylacetamides 1–8 were
tested in vitro as antiprotozoal agents against G. intestinalis, T. vaginalis and E. histolytica. The main
features of these compounds are the substitution of the hydrogen atom at position 3, 4 or 6, 7 by
different fluorine, nitro, chloro, trifluoromethyl and cyanide substituent groups which were used to
determine bioisosteric equivalence, enhancement of solubility, and potential antiprotozoal activity.
The results of the biological assays are shown in Table 2, All synthesized compounds were more
active against G. intestinalis than benznidazol. Compound
against this parasite than Bnz due to the presence of the trifluoromethyl group in the meta position.
For T. vaginalis, benznidazole presents an IC50 of 18.62 M. Conversely, compounds and are
three times more potent than benznidazole. We can see that the activity increases when we have
an –NO group (compound 4) or 2,6-dichloro substituent (compound ). These groups are needed
to maintain trichomonacidal activity in subsequent molecular changes. For Entamoeba histolytica,
benznidazole was the most active, presenting an IC50 of 4.27 M, and all synthesized compounds
, with a second –NO group as a
8 is observed to be 7 times more active
µ
4
7
7
2
µ
were less active than benznidazole. It is observed that compound
substituent, obtained the best amoebicidal activity of the series.
4
2
The compounds shown in Table 2 are fully compatible with Lipinski’s rule [42
,43], which should
allow for the development of additional antiprotozoal analogues. Their advantages include:
•
Physical properties known to be compatible with desirable pharmacokinetics (low molecular
weight, favorable Clog P, favorable hydrogen bond donating and accepting capabilities);
Potency and efficacy, with IC50 values at the low micromolar level;
Simple synthetic access and thus low production costs;
Bioisosteric groups improving the likelihood of reasonable solubility.
•
•
•
When performing the in silico evaluation with the PASS@ program where the Activity Probability
(
Pa) is predicted and the data are sorted from highest to lowest, they maintain the following
sequence: (4-NO -Ph) > (Ph) > (Bn) > (4-Cl-Ph) > (2,6-diCl-Ph) > (4-F-Ph) > (4-CN-Ph) >
(3-CF -Ph); In contrast to the experimental data for Trichomonas vaginalis, the following scheme was
6
2
1
4
7
3
5
2
8
3
found: 8(3-CF -Ph) > 7(2,6-diCl-Ph) > 6(4-NO -Ph) > 5(4-CN-Ph) > 1(Bn) > 4(4-Cl-Ph) > 3(4-F-Ph) >
3 2

Molecules 2017, 22, 579
10 of 16
2
(Ph), correlation of in silico-in vitro activity is low, indicated by a ratio
7 (2,6-dichlorophenyl) and 5
(
4-CN-Ph). Biological activity prediction programs (PASS@) provide information on the relationship of
structure and biological activity before developing the synthesis, but it is necessary to perform the
experiments to test them.
In summary, we synthesized a series of benzimidazole derivatives obtaining an inseparable
regioisomers mixture, with the 1,6-substituted compound as predominant isomer. All compounds
show good antiprotozoal profile against G. intestinalis and T. vaginalis in comparison with benznidazole.
3
. Materials and Methods
3
.1. Chemistry
Melting points were determined on an EZ-Melt MPA120 automated melting point apparatus from
Stanford Research Systems (Sunnyvale, CA, USA) and are uncorrected. Reactions were monitored by
1
TLC on 0.2 mm precoated silica gel 60 F254 plates (E. Merck KGaA, Darmstadt, Germany). H-NMR
(
400 MHz) and 13C-NMR (100 MHz) spectra were recorded on Varian Oxford (Varian, Palo Alto,
CA, USA). Chemical shifts are given in ppm relative to tetramethylsilane (Me Si, δ = 0) in DMSO-d6;
4
values are given in Hz. The following abbreviations are used: s, singlet; d, doublet; q, quartet;
dd, doublet of doublet; t, triplet; m, multiplet; br, broad signal. MS were recorded on a JEOL
JMS-700 spectrometer (JEOL, Tokyo, Japan) by Fast atom bombardment [FAB (+)]. Clog p values
were obtained using Molinspiration (www.molinspiration.com; Slovensky Grob, Slovak Republic).
Predictive values of antiprotozoal activities were also investigated using the PASS chemistry software
server (http://www.ibmh.msk.su/PASS/), per the mathematical model and database developed by
Lagunin et al. [44]. Starting materials were commercially available from Sigma Aldrich (Sigma-Aldrich,
St. Louis, MI, USA) and used without purification.
3
.2. General Method of Synthesis of 2-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-Arylacetamides 1–8
The appropriate N-arylacetamide (1.2 mol) was added dropwise to a solution of 5-nitro-1H-
◦
benzoimidazol-2-ylamine (1.0 mol) in acetonitrile (30 mL) and potassium carbonate (2.2 mol) at 90 C.
The mixture was stirred at reflux for between 5 and 18 h. The solvent was removed under vacuum and
the residue was suspended in water. The precipitates were filtered and dried. Crude compounds were
recrystallized from DMSO:H O mixture (50:50) or purified by column chromatography.
2
◦
2
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-benzylacetamide (
1
). Yield: 90.1%; m.p. 268–271 C; Yellow
1
13
solid, H-NMR and C-NMR refer to a 75:25 mixture of regioisomers.
1
7
5% Regioisomer of (2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-benzylacetamide (1a). H-NMR. (400 MHz,
DMSO-d6)
δ 4.32 (2H, d, JNH-H = 5.6 Hz, NHCH2Ph); 4.83 (2H, s, NHCH2CO); 7.03 (2H, br, NH ); 7.19
2
(
J
(
1
1H, d, Jorto = 8.4 Hz, H-7); 7.23–7.36 (5H, m, Ph); 7.87 (1H, dd, Jmeta = 2.4 Hz, Jorto = 8.4 Hz, H-6); 7.93 (d,
meta = 2.0 Hz, 1H, H-4); 8.73 (t, JNH-H = 6.0 Hz, 1H, CONH) ppm. 13C-NMR (100 MHz, DMSO-d )
δ
42.4
6
0
0
0
NHC
H Ph); 44.9 (NH
C
H CO); 107.1 (C-7); 109.3 (C-6); 114.7 (C-4); 126.8 (C-4 ); 127.2 (2C, C-2 , C-6 );
28.2 (2C, C-3 , C-5 ); 138.8 (C-1 ); 140.1 (C-5); 141.9 (C-7a); 142.8 (C-3a); 158.1(C-2); 165.9 (C=O) ppm.
2
2
0
0
0
1
2
5% Regioisomer of (2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-benzylacetamide (1b). H-NMR (400 MHz,
DMSO-d6)
δ 4.33 (2H, d, JNH-H =5.6 Hz, NHCH2Ph); 4.87 (2H, s, NHCH2CO); 7.03 (2H, br, NH ); 7.20
2
(
1H, d, Jorto = 8.4 Hz, H-4); 7.23–7.36 (m, 5H, Ph); 7.95 (1H, dd, Jmeta = 2.6 Hz, Jorto = 8.6 Hz, H-5); 7.97
13
(1H, d, Jmeta = 2.0 Hz, H-7); 8.78 (1H, t, JNH-H = 5.6 Hz, CONH) ppm. C-NMR (100 MHz, DMSO-d ) δ
6
0
0
4
2.4 (NH
C
H Ph); 44.7 (NH
C
H CO); 103.5 (C-7); 113.5 (C-6); 118.5 (C-4); 127.15 (2C, C-2 , C6 ); 127.11
2
2
0
0
0
0
(
C-4 ), 128.2 (2C, C-3 , C-5 ); 134.3 (C-1 ); 138.7 (C-5); 141.9 (C-7a); 149.7 (C-3a); 159.6 (C-2); 166.1 (C=O)
+
+1
ppm. MS (FAB): m/z 326 (M H ).
◦
2
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-phenylacetamide (
2
). Yield: 80.1%; m.p. 285–288 C; Yellow
1
13
solid, H-NMR and C-NMR indicate a 60:40 mixture of regioisomers.

Molecules 2017, 22, 579
11 of 16
1
6
0% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-phenylacetamide (2a). H-NMR (400 MHz,
0
δ 5.05 (2H, s, NHCH2CO); 7.06 (1H, dd, Jorto = 7.2 Hz, Jorto = 7.2 Hz, H-4 ); 7.22 (2H,
d, Jorto = 9.2 Hz, H-2 , H-6 ); 7.32 (2H, d, Jorto = 8.0 Hz, H-3 , H-5 ); 7.61 (2H, br, NH); 7.86 (1H, d,
DMSO-d6)
0
0
0
0
J
orto = 8.0 Hz, H-6); 7.96 (1H, d, Jorto = 8.0 Hz, H-7); 8.11 (1H, s, H-4); 10.52 (1H, br, CONH) ppm.
1
3
0
C-NMR (100 MHz, DMSO-d )
δ
45.2 (NH
C
H CO); 103.9 (C-7); 113.5 (C-6); 118.2 (C-4); 123.5 (C-4 );
6
2
0
0
0
0
0
1
27.2 (2C, C-2 , C-6 ); 128.2 (2C, C-3 , C-5 ); 138.8 (C-1 ); 140.1 (C-5); 142.1 (C-7a); 142.8 (C-3a); 158.1
(C-2); 165.9 (C=O) ppm.
1
4
0% Regioisomer of 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-phenylacetamide (2b). H-NMR (400 MHz,
0
δ 5.01 (2H, s, NHCH CO); 7.06 (1H, dd, Jorto = 7.2 Hz, Jorto = 7.2 Hz, H-4 ); 7.22 (2H, d,
2
DMSO-d6)
J
1
MHz, DMSO-d )
(
ppm. MS (FAB): m/z 312 (M H ).
0 0 0 0
orto = 9.2 Hz, H-2 , H-6 ); 7.32 (2H, d, Jorto = 8.0 Hz, H-3 , H-5 ); 7.63 (br, 2H, NH); 7.86 (d, Jorto = 8.8 Hz,
13
H, H-4); 7.96 (1H, d, Jorto = 8.0 Hz, H-5); 8.32 (s, 1H, H-7); 10.52 (1H, br, CONH) ppm. C-NMR (100
H CO); 107.4 (C-7); 109.4 (C-6); 114.9 (C-4); 119.1 (2C, C-2 , C-6 ); 123.5
C-4 ). 128.9 (2C, C-3 , C-5 ); 138.8 (C-1 ); 140.5 (C-5); 142.1 (C-7a); 142.9 (C-3a); 158.4 (C-2); 164.9 (C=O)
0
0
δ
45.6 (NH
C
6
2
0
0
0
0
+
+1
◦
2
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-(4-fluorophenyl) acetamide (
3
). Yield: 72.0%; m.p. 313.0–315.0 C;
1
13
Yellow solid, H-NMR and C-NMR indicate a 70:30 mixture of regioisomers.
1
7
0% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-(4-fluorophenyl) acetamide (3a). H-NMR
(
J
400 MHz, DMSO-d )
δ
4.98 (2H, s, NHCH CO); 7.08 (2H, br, NH); 7.17 (2H, dd, Jorto = 8.8 Hz,
6
2
0
0
0
orto = 8.8 Hz, H-3 , H5 ); 7.30 (1H, d, Jorto = 8.8 Hz, H-7); 7.62 (2H, dd, Jorto = 5.2 Hz, Jorto = 8.8 Hz, H-2 ,
0
H6 ); 7.87 (1H, dd, Jmeta = 2.4 Hz, Jorto = 8.8 Hz, H-6); 7.95 (1H, d, Jmeta = 2.8 Hz, H-4); 10.47 (1H, s,
CONH) ppm. 13C-NMR (100 MHz, DMSO-d )
δ 45.3 (NHCH CO); 107.2 (C-7); 109.3 (C-6); 114.7 (C-4);
2
6
0
0
0
0
0
1
1
15.3 (d, JC-F = 56.2 Hz, 2C, C-3 , C-5 ); 120.7 (d, J = 7.6 Hz, 2C, C-2 , C-6 ); 135.0 (C-1 ); 140.3 (C-5);
41.9 (C-7a); 142.8 (C-3a); 158.1 (C-2); 159.3 (d, JC-F = 56.2 Hz, C-4 ); 164.6 (C=O) ppm.
C-F
0
1
3
0% Regioisomer of 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-(4-fluorophenyl) acetamide (3b). H-NMR
(
J
400 MHz, DMSO-d ) δ 5.02 (2H, s, NHCH CO); 7.33 (2H, br, NH); 7.17 (2H, dd, Jorto = 8.8 Hz,
6 2
0 0
orto = 8.8 Hz, H-3 , H5 ); 7.21 (1H, d, Jorto = 8.8 Hz, H-4); 7.62 (2H, dd, Jmeta = 5.2 Hz, Jorto = 8.8 Hz,
0
0
H-2 , H6 ); 7.87 (1H, dd, Jmeta = 2.4 Hz, Jorto = 8.8 Hz, H-5); 8.12 (1H, d, Jmeta = 2.4 Hz, H-7); 10.47 (1H, s,
CONH) ppm. 13C-NMR (100 MHz, DMSO-d )
45.1 (NH H CO); 103.7 (C-7); 113.3 (C-6); 118.0 (C-4);
20.7 (d, J = 7.6 Hz, 2C, C-2 , C-6 ); 159.3 (d, J = 56.2 Hz, C-4 ); 115.3 (d, J = 56.2 Hz, 2C, C-3 ,
δ
C
2
6
0
0
0
0
1
C-F
C-F
C-F
0
0
C-5 ); 134.6 (C-1 ); 138.8 (C-5); 141.9 (C-7a); 149.6 (C-3a); 156.7 (C-2); 164.7 (C=O) ppm. MS (FAB): m/z
3
+
+1
30 (M H ).
◦
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-(4-chlorophenyl) acetamide (4). Yield: 92.1%; m.p. 294.0–296.0 C;
2
1
13
Yellow solid, H-NMR and C-NMR indicate 69:31 mixture of regioisomers.
1
6
(
(
9% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-(4-chlorophenyl) acetamide (4a). H-NMR
400 MHz, DMSO-d6) 5.04 (2H, s, NHCH CO); 7.32 (2H, br, NH); 7.37 (1H, d, Jorto = 8.8 Hz, H-7); 7.63
2H, d, Jmeta = 2.4 Hz, Jorto = 5.2 Hz, H-3 , H-5 ); 7.65 (2H, d, Jmeta = 2.0 Hz, Jorto = 5.2 Hz, H-2 , H-6 );
.96 (1H, d, Jmeta = 2.8 Hz, Jorto = 8.8 Hz, H-6); 8.12 (1H, d, Jmeta = 2.0 Hz, H-4); 10.56 (s, 1H, CONH)
δ
2
0
0
0
0
7
13
ppm. C-NMR (100 MHz, DMSO-d )
(
δ
45.2 (NH
C
H CO); 103.9 (C-7); 113.4 (C-6); 118.1 (C-4); 120.5
2C, C-2 ,C-6 ); 126.9 (C-4 ); 128.7 (2C, C-3 ,C-5 ); 137.6 (C-1 ); 138.8 (C-5); 141.9 (C-7a); 149.6 (C-3a);
6
2
0
0
0
0
0
0
1
59.7 (C-2); 165.1 (C=O) ppm.
1% Regiisomer of 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-(4-chlorophenyl) acetamide (4b). H-NMR
400 MHz, DMSO-d6) 4.99 (2H, s, NHCH CO); 7.06 (2H, br, NH); 7.21 (1H, d, Jorto = 8.8 Hz, H-4); 7.63
2H, d, Jmeta = 2.4 Hz, Jorto = 5.2 Hz, H-3 , H-5 ); 7.65 (2H, d, Jmeta = 2.0 Hz, Jorto = 5.2 Hz, H-2 , H-6 );
.86 (1H, d, Jmeta = 2.0 Hz, Jorto = 8.8 Hz, H-5); 7.96 (1H, d, Jmeta = 2.0 Hz, H-7); 10.59 (s, 1H, CONH)
1
3
(
(
δ
2
0
0
0
0
7
13
ppm. C-NMR (100 MHz, DMSO-d )
δ
45.3 (NH
C
H CO); 107.3 (C-7); 109.3 (C-6); 114.8 (C-4); 120.5
6
2
0
0
0
0
0
0
(
2C, C-2 , C-6 ); 126.9 (C-4 ); 128.7 (2C, C-3 , C-5 ); 134.6 (C-1 ); 140.3 (C-5); 141.9 (C-7a); 142.7 (C-3a);
+
+1
1
58.6 (C-2); 164.9 (C=O) ppm. MS (FAB): m/z 346 (M H ).

Molecules 2017, 22, 579
12 of 16
◦
2
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-(4-cyanophenyl) acetamide (
5
). Yield: 81.5%; m.p. 254.0–256.7 C;
1
13
Yellow solid, H-NMR and C-NMR indicate 57:43 mixture of regioisomers.
1
5
7% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-(4-cyanophenyl) acetamide (5a). H-NMR
(
(
400 MHz, DMSO-d )
δ
0
5.08 (2H, s, NHCH CO); 7.07 (2H, br, NH); 7.22 (1H, d, Jorto = 8.8 Hz, H-7); 7.79
6
2
0
0
0
4H, s, H-2 , H-3 , H-5 , H-6 ); 7.85 (1H, d, Jorto = 8.8 Hz, H-6); 7.94 (1H, d, Jmeta = 2.0 Hz, H-4); 10.84 (1H,
s, CONH) ppm. 13C-NMR (100 MHz, DMSO-d )
δ
0
45.3 (NHCH CO); 103.9 (C-7); 105.2 (C-4 ); 113.4
2
6
0
0
0
0
0
(
(
C-6); 118.1 (C-4); 118.9 (CN); 119.2 (2C, C-2 ,C-6 ); 133.4 (2C, C-3 , C-5 ); 134.6 (C-1 ); 138.9 (C-5); 142.8
C-7a); 149.6 (C-3a); 159.6 (C-2); 165.8 (C=O) ppm.
1
4
3% Regioisomer of 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-(4-cyanophenyl) acetamide (5b). H-NMR
(
(
400 MHz, DMSO-d )
δ
0
5.03 (2H, s, NHCH CO); 7.32 (2H, br, NH); 7.33 (1H, d, Jorto = 8.8 Hz, H-4); 7.79
6
2
0
0
0
4H, s, H-2 , H-3 , H-5 , H-6 ); 7.85 (1H, d, Jorto = 8.8 Hz, H-5); 7.97 (1H, d, Jmeta = 2.0 Hz, H-7); 10.87 (1H,
s, CONH) ppm. 13C-NMR (100 MHz, DMSO-d )
δ
0
45.5 (NHCH CO); 103.3 (C-7); 105.2 (C-4 ); 109.4
2
6
0
0
0
0
0
(
C-6); 114.8 (C-4); 118.9 (CN); 119.2 (2C, C-2 , C-6 ); 133.4 (2C, C-3 , C-5 ); 134.6 (C-1 ); 140.3 (C-5); 142.7
+
+1
(
C-7a); 142.0 (C-3a); 158.1 (C-2); 165.7 (C=O) ppm. MS (FAB): m/z 337 (M H ).
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-(4-nitrophenyl) acetamide ( ). Yield: 79.8%; m.p. 309.0–312.0 C;
Yellow solid, H-NMR and C-NMR indicates a 59:41mixture of reigoisomers.
9% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-(4-nitrophenyl) acetamide (6a). H-NMR
400 MHz, DMSO-d6) 5.11 (2H, s, NHCH CO); 7.34 (2H, br, NH); 7.36 (1H, d, Jorto = 8.8 Hz, H-7); 7.85
2H, dd, Jmeta = 2.8 Hz, Jorto = 5.2 Hz, H-2 , H-6 ); 7.86 (2H, dd, Jmeta = 2.4 Hz, Jorto = 8.0 Hz, H-3 , H-5 );
◦
2
6
1
13
1
5
(
(
δ
2
0
0
0
0
13
8
(
.19 (1H, d, Jmeta = 2.0 Hz, H-4); 8.25 (1H, d, Jorto = 8.4 Hz, H-6); 11.04 (1H, s, CONH) ppm. C-NMR
0
100 MHz, DMSO-d )
δ
45.4 (NH
C
H CO); 104.0 (C-7); 144.9 (C-4 ); 113.4 (C-6); 118.1 (C-4); 118.7 (2C,
6
2
0
0
0
0
0
C-2 , C-6 ); 125.1 (2C, C-3 , C-5 ); 134.6 (C-1 ); 138.9 (C-5); 142.2 (C-7a); 149.6 (C-3a); 159.6 (C-2); 166.0
(C=O) ppm.
4
1% Regioisomer compound 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-(4-nitrophenyl) acetamide (6b).
1
H-NMR (400 MHz, DMSO-d6)
H-4); 7.83 (1H, d, Jmeta = 2.4 Hz, H-7); 7.95 (2H, dd, Jmeta = 2.8 Hz, Jorto = 5.2 Hz, H-2 , H-6 ); 7.96 (2H,
δ 5.06 (2H, s, NHCH CO); 7.09 (2H, br, NH); 7.22 (d, Jorto = 8.8 Hz, 1H,
2
0
0
0
0
dd, Jmeta = 2.4 Hz, Jorto = 8.0 Hz, H-3 , H-5 ); 8.25 (1H, d, Jorto = 8.4 Hz, H-5); 11.01 (1H, s, CONH) ppm.
13
0
C-NMR (100 MHz, DMSO-d )
δ
45.6 (NH
C
H CO); 107.4 (C-7); 144.7 (C-4 ); 109.4 (C-6); 114.8 (C-4);
6
2
0
0
0
0
0
118.7 (2C, C-2 , C-6 ); 125.1 (2C, C-3 , C-5 ); 134.6 (C-1 ); 140.3 (C-5); 142.0 (C-7a); 142.7 (C-3a); 158.1
+
+1
(C-2); 165.8 (C=O) ppm. MS (FAB): m/z 357 (M H ).
2
3
-(2-Amino-5(6)-nitro-1H-benzimidazol-1-yl)-N-(2,6-dichlorophenyl) acetamide (7). Yield: 61.4%; m.p.
03.0–306.0 C; Yellow solid, H-NMR and C-NMR indicate a 55:45 mixture of reigoisomers.
◦
1
13
5
5% Regioisomer of 2-(2-amino-6-nitro-1H-benzimidazol-1-yl)-N-(2,6-dichlorophenyl) acetamide (7a).
1
H-NMR (400 MHz, DMSO-d6)
δ 5.11 (2H, s, NHCH CO); 7.21 (1H, d, Jorto = 8.4 Hz, H-7); 7.36
2
0
0
0
(2H, dd, Jmeta = 3.8 Hz, Jorto = 8.4 Hz, H-3 , H-5 ); 7.41 (2H, br, NH); 7.54 (1H, d, Jorto = 8.0 Hz, H-4 );
7
.97 (1H, d, Jmeta = 2.6 Hz, Jorto = 8.6 Hz, H-6); 8.02 (1H, d, Jmeta = 2.4 Hz, H-4); 10.45 (1H, s, CONH)
13
ppm. C-NMR (100 MHz, DMSO-d )
δ
44.6 (NH
C
H CO); 103.7 (C-7); 109.4 (C-6); 114.8 (C-4); 128.5
6
2
0
0
0
0
0
0
(
2C, C-3 , C-5 ); 129.5 (2C, C-2 , C-6 ); 132.2 (C-1 ); 133.4 (C-4 ); 138.7 (C-5); 142.0 (C-7a); 142.9 (C-3a);
1
58.0 (C-2); 165.2 (C=O) ppm.
4
5% Regioisomer of 2-(2-amino-5-nitro-1H-benzimidazol-1-yl)-N-(2,6-dichlorophenyl)acetamide (7b).
1
H-NMR (400 MHz, DMSO-d6)
δ 5.08 (2H, s, NHCH CO); 7.12 (2H, br, NH); 7.21 (1H, d, Jorto = 8.8 Hz,
2
0
0
0
H-4); 7.34 (2H, dd, Jmeta = 3.6 Hz, Jorto = 8.4 Hz, H-3 , H-5 ); 7.55 (1H, d, Jorto = 8.0 Hz, H-4 ); 7.91 (1H, d,
J
meta = 2.0 Hz, Jorto = 8.8 Hz, H-5); 7.94 (1H, d, Jmeta = 2.4 Hz, H-7); 10.45 (1H, s, CONH) ppm. 13C-NMR
0
0
(
100 MHz, DMSO-d )
δ
44.8 (NH
C
H CO); 107.1 (C-7); 113.4 (C-6); 118.24 (C-4); 128.5 (2C, C-3 , C-5 );
6
2
0
0
0
0
129.5 (2C, C-2 , C-6 ); 134.1 (C-1 ); 133.4 (C-4 ); 140.0 (C-5); 142.0 (C-7a); 149.8 (C-3a); 159.6 (C-2); 165.3
+
+1
(C=O) ppm. MS (FAB): m/z 381 (M H ).

Molecules 2017, 22, 579
13 of 16
2
6
-(2-Amino-5(6)-nitro-2,3-dihydro-1H-benzimidazol-1-yl)-N-[3-(trifluoromethyl)phenyl]acetamide (
0.7%; m.p. 282.0–284.0 C; Yellow solid, H-NMR and C-NMR indicate 61:39 mixture of regioisomers.
8
). Yield:
◦
1
13
6
1% Regioisomer of 2-(2-amino-6-nitro-2,3-dihydro-1H-benzimidazol-1-yl)-N-[3-(trifluoromethyl)phenyl]
1
acetamide (8a). H-NMR (400 MHz, DMSO-d6)
δ
5.07 (2H, s, NHCH CO); 7.32 (2H, br, NH); 7.22
2
0
(1H, d, Jorto = 8.4 Hz, H-7); 8.10 (1H, d, Jmeta = 2.8 Hz, H-2 ); 7.85 (2H, dd, Jmeta = 2.8 Hz, Jorto = 5.2 Hz,
0
0
0
H-6 ); 7.86 (2H, dd, Jmeta = 2.4 Hz, Jorto = 8.0 Hz, H-4 , H-5 ); 8.15 (1H, d, Jmeta = 2.4 Hz, H-4); 7.96 (1H,
dd, Jmeta = 2.2 Hz, Jorto = 8.6 Hz, H-6); 10.73 (1H, s, CONH) ppm. C-NMR (100 MHz, DMSO-d )
13
δ
6
0 0 0
CH CO); 103.9 (C-7); 109.4 (C-6); 115.0 (C-2 ); 119.8 (C-4 ); 118.1 (C-4); 118.1 (C-6 ); 122.5 (q,
2
4
5.2 (NH
0
0
0
JC-F = 277.8 Hz, CF ); 129.5 (q, J = 33.4 Hz, C-3 ); 130.1 (C-5 ); 134.6 (C-1 ); 138.9 (C-5); 141.9 (C-7a);
3
C-F
1
49.7 (C-3a); 159.7 (C-2); 165.6 (C=O) ppm.
3
9% Regiosiomer of 2-(2-amino-5-nitro-2,3-dihydro-1H-benzimidazol-1-yl)-N-[3-(trifluoromethyl)phenyl]
1
acetamide (8b). H-NMR (400 MHz, DMSO-d6)
δ 5.06 (2H, s, NHCH CO); 7.09 (2H, br, NH); 7.22
2
(
1H, d, Jorto = 8.8 Hz, H-4); 7.83 (1H, d, Jmeta = 2.4 Hz, H-7); 7.95 (2H, dd, Jmeta = 2.8 Hz, Jorto = 5.2 Hz,
0
0
0
0
H-2 , H-6 ); 7.96 (2H, dd, Jmeta = 2.4 Hz, Jorto = 8.0 Hz, H-4 , H-5 ); 8.25 (1H, d, Jorto = 8.4 Hz, H-5); 11.01
13
(
1H, s, CONH) ppm. C-NMR (100 MHz, DMSO-d )
δ
45.3 (NH
= 277.8 Hz, CF ); 129.5 (q, J
C
H CO); 107.3 (C-7); 113.4 (C-6); 115.0
6
2
0
0
0
0
(
C-2 ); 119.8 (C-4 ); 114.8 (C-4); 118.1 (C-6 ); 122.5 (q, J
= 33.4 Hz, C-3 );
C-F
3
C-F
0 0
1
40.3 (C-5 ); 134.6 (C-1 ); 140.3 (C-5); 141.9 (C-7a); 142.8 (C-3a); 158.1 (C-2); 165.4 (C=O) ppm. MS (FAB):
+
+1
m/z 381 (M H ).
NMR spectra of compounds 1–8 can be found in Supplementary Materials.
3
.3. Biological Assays
G. intestinalis strain IMSS:0696:1 was cultured in a TYI-S-33 modified medium, supplemented with
0% calf serum and bovine bile. The T. vaginalis strain GT3 and E. histolytica HM1-IMSS were cultured
1
in TYI-S-33 medium, supplemented with 10% bovine serum. In vitro susceptibility assays were
4
performed using a method described previously [26
,
45]. Briefly, 4
×
10 trophozoites of G. intestinalis
◦
or T. vaginalis or E. histolytica were incubated for 48 h at 37 C with increasing concentrations of the
synthesized benznidazole and metronidazole compounds. Trophozoites incubated in a culture medium
with DMSO were used as the negative control in the experiments. After incubation, trophozoites
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 (IC ) was calculated by Probit
50
analysis. Experiments were carried out in triplicate and repeated at least twice.
3
.4. Computational Chemistry
All calculations of the NMR shielding constants have been performed using gauge-including
atomic orbitals (GIAO) [38] in DMSO solvent. We employed the nwchem program package
version 6.5) [39]. Geometry optimizations were carried out at the DFT level with the B3LYP
(
exchange-correlation functional [40], with Def2-TZVP [41] basis set. Harmonic vibrational frequencies
are also analyzed at the same level to characterize the nature of the stationary molecules.
4
. Conclusions
We synthesized a series of benzimidazole derivatives obtaining inseparables mixtures of
regioisomers, with the 1,6-substituted compound as the predominant one. Products were characterized
1
13
by H- and C-NMR, where we corroborated that the predominant regioisomer was substituted at
the 6-position. In addition, computational calculations of the regioisomer mixtures conformed the
match between the predicted chemical displacements with the experimental findings, confirming
the substitution of the nitro group in position 6 as the predominant regioisomer. The mixture of
regioisomers demonstrated interesting activity against two intestinal parasites: Giardia and Trichomonas.
Compound
8 obtained an IC50 of 3.84 µM against G. intestinalis, and an IC50 of 5.62 µM against
T. vaginalis. This compound bears a –CF substituted in the meta position of the aryl acetamide.
3

Molecules 2017, 22, 579
14 of 16
All compounds show good antiprotozoal profiles against G. intestinalis and T. vaginalis in comparison
with benznidazole.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: We thank María Medina Pintor and Victoria Labastida Galván from Centro de Investigaciones
Químicas, UAEM, for the determination of all mass spectra. Supported in part by CONACyT (CB-2015-01)
2
54321. The authors acknowledge to the General Coordination of Information and Communications Technologies
(
CGSTIC) at CINVESTAV for providing HPC resources on the Hybrid Cluster Supercomputer “Xiuhcoatl” of
the LANCAD CINVESTAV node and ABACUS Laboratory of Applied Mathematics and High Performance
Computation of CINVESTAV-IPN, Project CONACT-EDOMEX-2011-C01-165873, that have contributed to the
research results reported within this paper.
Author Contributions: Emanuel Hernández-Núñez and Gabriel Navarrete Vazquez designed and synthesized
all compounds, and analyzed all data of paper; Rosa Moo-Puc performed the in vitro evaluation of the parasites;
Hugo Tlahuext provided information on nuclear magnetic resonance and mass spectrometry plus support in
data analysis; María Ortencia Gonzalez-Diaz and Diego Moreno carried out the computational calculations of
reigoisomers and tautomeric mixtures.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).