Synthesis, molecular modeling and preliminary biological evaluation of a set of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole as potential antibacterial, anti-Trypanosoma cruzi and antifungal agents

Article abstract of DOI:10.1016/j.bmc.2011.09.009

A series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives was synthesized and their activity screened in vitro against Staphylococcus aureus, Trypanosoma cruzi, and Candida albicans. The bioactivity was expressed as minimum inhibitory concentration (MIC) for S. aureus strains, and as fifty-percent inhibitory concentration (IC₅₀) of parasite population growth for T. cruzi. A molecular modeling approach was performed to establish qualitative relationships regarding the biological data and the compounds' physicochemical properties. The 5-(4-OC4H9Ph, 5μ), and 5-(4-CO₂CH₃Ph, 5o) derivatives were the most active compounds for S. aureus ATCC 25923 (MIC = 1.95 -1.25 μg/mL) and T. cruzi (IC₅₀ = 7.91 μM), respectively. Also, a preliminary evaluation against C. albicans involving some compounds was performed and the 5-(4-CH ₃Ph, 5e) derivative was the most active compound (MIC = 3.28-2.95 μg/mL). In this preliminary study, all synthesized 3-acetyl-2,5- disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives were active against all microorganisms tested.

Full text of DOI:10.1016/j.bmc.2011.09.009

Bioorganic & Medicinal Chemistry 19 (2011) 6292–6301
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and preliminary biological evaluation of a set
of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole as potential
antibacterial, anti-Trypanosoma cruzi and antifungal agents
⇑
Marina Ishii , Salomão Dória Jorge, Alex Alfredo de Oliveira, Fanny Palace-Berl, Ieda Yuriko Sonehara,
Kerly Fernanda Mesquita Pasqualoto, Leoberto Costa Tavares
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 580, Bloco 16, Butantã,
São Paulo, SP 05508-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2011
Revised 29 August 2011
Accepted 6 September 2011
Available online 10 September 2011
A series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives was synthesized and their
activity screened in vitro against Staphylococcus aureus, Trypanosoma cruzi, and Candida albicans. The bioac-
tivity was expressed as minimum inhibitory concentration (MIC) for S. aureus strains, and as fifty-percent
inhibitory concentration (IC50) of parasite population growth for T. cruzi. A molecular modeling approach
was performed to establish qualitative relationships regarding the biological data and the compounds’
physicochemical properties. The 5-(4-OC4H9Ph, 5l), and 5-(4-CO2CH3Ph, 5o) derivatives were the most
Keywords:
active compounds for S. aureus ATCC 25923 (MIC = 1.95 –1.25
tively. Also, a preliminary evaluation against C. albicans involving some compounds was performed and the
5-(4-CH3Ph, 5e) derivative was the most active compound (MIC = 3.28–2.95 g/mL). In this preliminary
lg/mL) and T. cruzi (IC50 = 7.91 lM), respec-
Oxadiazolines
Bacterial resistance
MRSA
l
study, all synthesized 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives were active
VISA
against all microorganisms tested.
Trypanosoma cruzi
Candida albicans
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
antibacterial5 and antiprotozoal6 agents and they constitute an
important class of heterocyclic compounds presenting high bio-
Although many efforts have been made, bacterial and protozoal
infections are still responsible for high rates of morbidity and mor-
tality throughout the world, being an important threat to the public
health system, especially in the developing countries. It is esti-
mated that about 15.7% of infections are caused by Staphylococcus
aureus,1 while Trypanosoma cruzi infections affect around 20,000
people per year,2,3 and 21,000 deaths are attributed to Chagas’ dis-
ease every year.2
The incidence of bacterial resistance to antibiotic therapy,
including here the emergence of multidrug resistant pathogens,
as well as the often poor safety profile and lack of efficacy of the cur-
rently available drugs, reinforces the need for the development of
new and potent chemical entities or an improvement in the activity
of well-known chemical compounds. Considering this statement,
the synthesis of analogues can be seen as an efficient approach to
optimize an active chemical structure and design new drugs, since
simple structural changes can lead to better biological activities
through modifications of physicochemical properties.
logical activity levels. Other studies, in which the
2,3-dihydro-1,3,4-oxadiazoline ring was into the molecular struc-
ture, have indicated antimicrobial,7 anticonvulsivant,8 antifungal,7
and anti-inflammatory9 activity related to that structural moiety.
The main advantage of the cyclization step, in synthesis, and the
introduction of an acetyl group is the presence of an oxadiazole
ring that may improve pharmacokinetic efficacy due to its in-
creased ability to establish hydrogen bonding interactions and
confer enhanced metabolic stability. Because of that, it could be
considered as an important structural motif for designing new
drugs.10
Previous studies11,12 showed that molecular modification tech-
niques applied to nitrocompounds is an advantageous strategy for
the definition of potential new candidates regarding anti-infective
chemotherapy. Thus, in this study, the purpose was the design,
synthesis, and evaluation of the biological activity of a novel set
of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole deriva-
tives as an alternative to overcome the currently anti-infective
drugs’ resistance and, consequently, to establish more effective
therapies.
Since the 1940s, nitroheterocyclic compounds, including deriv-
atives with a five-membered heterocyclic ring,4 have been used as
Additionally, a molecular modeling approach was carried out to
identify possible qualitative property-activity relationships. For
that, the lipophilic (LP) and electrostatic potential (EP) maps were
⇑
Corresponding author. Tel.: +55 11 3091 3726; fax: +55 11 3815 6386.
E-mail address: marishii@usp.br (M. Ishii).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.009

M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
6293
the properties primarily considered. As it is well known, the lipo-
philicity or hydrophobicity property is quite important to the diffu-
sion of any compound through the bacterial biological system. This
property can generally be expressed by the calculated n-octanol/
water partition coefficient (C log P). The map of EP is related to the
electronic density distribution and considers, for example, the con-
tribution of substituent groups to the whole molecular electronic
density.
silane). It was observed that the absence of a singlet signal
around d 12 ppm, indicating the proton of amidic nitrogen and
a singlet at d 7.74 ppm was the evidence of the 2,3-dihydro-
1,3,4-oxadiazoline ring group formation. The presence of a singlet
signal near to d 2.29 ppm confirmed the three protons related to
the acetyl group. Analyzing the 13C NMR spectra, signals can be
observed near to 168 and 21 ppm, which indicate the presence
of carbonyl and alkyl group, respectively. The 1H NMR, 13C
NMR, and elemental analysis data were in agreement with the
proposed structures.
Moreover, the steric atomic hindrance was also calculated to
verify the contribution of substituent volume and its relationship
with the biological data.
2.2. Biological activities and molecular modeling findings
2. Results and discussion
2.1. Chemistry
The biological activities of 3-acetyl-2,5-disubstituted-2,3-dihy-
dro-1,3,4-oxadiazole derivatives against S. aureus (ATCC 25923,
SP3/R33, and VISA3 strains) were determined using the minimum
inhibitory concentration (MIC) method. The percentage of growth
inhibition for the T. cruzi epimastigote form (Y strain) was ex-
pressed as IC50 values, which represent the compound concentra-
tion responsible for inhibiting 50% of the parasite population
growth. Results are shown in Table 1.
For the S. aureus ATCC strain, ampicillin was used as control
drug. The MIC values indicated as the most and least active com-
pounds the 4-OC4H9 (5l) and the 4-C3H7 (5h) substituted deriva-
tives, respectively. The MIC values found for 5l ranged from 1.95
In this study, the synthesis of fifteen 3-acetyl-2,5-disubsti-
tuted-2,3-dihydro-1,3,4-oxadiazole derivatives was performed in
four steps: the synthesis of 4-substituted methyl esters (2a–o)
from commercially available substituted benzoic acids (1a–o),
synthesis of 4-substituted benzhydrazides (3a–o) from those
methyl esters, synthesis of Schiff’s bases (4a–o) from reaction
with 5-nitro-2-thiophenecarboxaldehyde, and synthesis of 3-acet-
yl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives
(5a–o) from the Schiff’s bases (Fig. 1).
to 1.25 lg/mL, and for 5h varied from 4.61 to 3.28 lg/mL. The
The synthesis of 4-substituted benzoic acid 4-substituted
N0-((5-nitrothiophen-2-yl)methylene)benzohydrazide
(4a–o)
two compounds have a hydrophobic character as represented
through the LP texture maps as well as expressed by the C log P
values (see Fig. 2A and Table 2). The 4-OC4H9 and 4-C3H7 substitu-
ent groups do provide the same electronic features to the 5l and 5h
compounds as can be visualized through the EP opaque maps
(Fig. 2B). The 4-OC4H9 and 4-alkyl substituent have an electron
donating effect on the aromatic ring (red to green color more
delimited on the aromatic ring).
Indeed, as the alkyl chain length was increased the activity of
3-acetyl-2,5-disubstituted-1,3,4-oxadiazoline derivatives progres-
sively decreased for the S. aureus ATCC strain [4-CH3 (5f) ꢀ 4-
C2H5 (5g) ꢀ 4-C3H7 (5h)] (Table 1). This trend might be explained
by the molecular hydrophobicity and/or steric hindrance features.
A longer alkyl chain could impair the approximation of enzymes,
for example, which would be acting as potential targets. The
shown to be simple and practical, achieving yield in the range from
80% to 90%, as described in previous studies.14 On the other hand,
for the novel series of 3-acetyl-2,5-disubstituted-1,3,4-oxadiazo-
line derivatives (5a–o), the parameters of reaction time and tem-
perature were adjusted for each compound. The minimal
temperature for the product formation was 120 °C, considering that
in less severe conditions the reaction time did not translate into a
marked improvement for the reaction yield.
During the synthesis procedure, thin layer chromatography
(TLC) analysis was performed to verify the formation of 3-acet-
yl-2,5-disubstituted-1,3,4-oxadiazolines. The structural elucida-
tion of these derivatives was confirmed through 1H NMR and
13C NMR spectra analysis, considering chemical shifts (d) related
to the residual solvent signal or internal standard (tetramethyl-
Figure 1. Synthesis of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles derivatives. Reaction conditions: (I): CH3OH, H2SO4/reflux/4 h; (II): N2H4 64% aq/75 °C/
10 min; (III) 5-nitro-2-thiophenecarboxaldehyde, H2O, H2SO4, CH3COOH, C2H5OH/reflux/1 h; (IV) acetic anhydride/120 °C/8 h.

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M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
Table 1
In vitro antibacterial and antiprotozoal activity of the series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives
Compound
R
Staphylococcus aureus
Trypanosoma cruzic
ATCC 25923 MICd
(l
g/mL)
SP3/R33a MICd
(
lg/mL)
VISA3a,b MICd
(l
g/mL)
IC50
(
lM)
e
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
–H
–Cl
–Br
–I
3.07–2.46
2.46–1.97
2.95–2.66
3.58–2.87
3.28–2.62
3.28–2.62
3.36–2.69
4.61–3.28
2.79–2.23
2.95–2.36
4.10–3.28
1.95–1.25
2.66–2.39
2.95–2.66
3.38–2.95
6.59–5.90
0.10–0.20
—
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
2.50–1.25
5.00–2.50
2.50–1.25
5.00–2.50
2.50–1.25
2.50–1.25
—
1.25–0.63
5.00–2.50
5.00–2.50
5.00–2.50
5.00–2.50
5.00–2.50
5.00–2.50
2.50–1.25
1.25–0.63
5.00–2.50
5.00–2.50
10.00–5.00
2.50–1.25
1.25–0.63
1.25–0.63
10.00–5.00
32.00–16.00
1.00–0.50
—
10.24
10.85
10.16
12.10
14.25
11.47
10.74
9.90
10.67
26.64
12.20
11.47
9.85
–CF3
–CH3
–C2H5
–C3H7
–OCH3
–OC2H5
–OC3H7
–OC4H9
–NO2
–CN
8.19
7.91
120.46
—
—
–CO2CH3
Nifuroxazide
Ampicilin
Vancomycin
Benznidazole
32.00–16.00
1.00–0.50
—
—
20.84
a
Resistant to amoxicilin/clavulanic acid, ampicilin, cephazoline, cephotaxime, cephalotine, ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem,
nitrofurantoin, norfloxacin, oxacillin, penicillin, rifampicin, trimethoprim/sulfametoxazole.
b
Vancomycin-intermediate Staphylococcus aureus strain.
Epimastigote form of Y strain.
Values corresponding to the average of three repetitions.
Concentration of compound which causes 50% parasite growth inhibition.
c
d
e
calculated steric hindrance15 values found for the alkyl substitu-
ent 4-CH3, 4-C2H5, and 4-C3H7 were 3.46, 6.26, and 9.15, respec-
tively. The van der Waals volumes of these substituent groups can
be visualized in Figure 3.
Regarding the alkoxy derivatives, the same behavior was ob-
served for the S. aureus ATCC strain when the alkyl chain length
was increased up to three carbon atoms [4-OCH3 (5i) ꢀ 4-OC2H5
(5j) ꢀ 4-OC3H7 (5k)] (see Table 1). The calculated steric hindrance
values found for the alkoxy substituent 4-OCH3, 4-OC2H5, and 4-
OC3H7 were 4.47, 7.20, and 10.07, respectively, corroborating for
that assumption (see Fig. 3). For biological activity evaluation, as
the bioactivity was, measured using MIC method, none specific
target was actually addressed.
Otherwise, the calculated partition coefficient (C log P), is the
major factor in the permeability of the tested compound through
the cell membrane and can be directly related to the produced bio-
logical effect.20 According to Table 2, the C log P values found for the
compounds 5f, 5g, and 5h were 1.47, 1.93, and 2.38, respectively.
The compounds 5i, 5j, and 5k presented C log P values of 0.97,
1.32, and 1.84, correspondingly. As expected, the presence of a polar
heteroatom at the 4-alkyl substituent group decreased the global
hydrophobicity of the 3-acetyl-2,5-disubstituted-1,3,4-oxadiazo-
line derivatives. Visually, regarding the LP texture maps (see
Fig. 4), which are intimately related to the C log P values, the dark
brown color on the region of the 4-alkyl substituent group was in
general replaced by a green to light brown color in comparison to
the 4-alkoxy moiety.
the non-substituted compound (5a), indicating that molecular
structural modifications, specially as in the synthesis of analogs,
correspond to an advantageous strategy for obtaining more effec-
tive therapeutic alternatives. A quantitative structure–activity
relationship study is currently in progress to provide more infor-
mation and understanding of the mechanism of action of these
derivatives.
Ampicillin and vancomycin were used as control drugs for the
S. aureus SP3/R33 and VISA3 strains, respectively. The MIC values
were the same for all synthesized compounds when they were
tested against the S. aureus SP3/R33 strain (2.50–1.25 lg/mL), ex-
cept for the 4-OC3H7 (5k) and 4-NO2 (5m) derivatives (Table 1).
The compound 5m presented the lowest C log P value (ꢁ1.32,
see Table 2), which is probably due to the hydrophilic character
attributed to its substituent group (4-NO2) by the calculation
method. None conclusive explanation regarding the MIC values
found for the S. aureus SP3/R33 strain can be made based upon
the substituent groups or related physicochemical properties
pointed out here.
Again, for the S. aureus VISA3 strain, the MIC values did not
change significantly, and ranged mostly from 5.00 to 2.50
lg/mL, ex-
cept for the 4-OC4H9 (5l) derivative (10.00–5.00 g/mL) (Table 1).
l
This finding might suggest that when the alkoxy substituent group
presents more than three carbon atoms in its chain the activity
against the VISA3 strain could be reduced. The 4-OC4H9 substituent
group has a steric atomic hindrance value of 12.99, which is bigger
than those found for the 4-OC2H5 and 4-OC3H7 derivatives (7.20
and 10.07, respectively). The compounds 4-OCH3 (5i), 4-CN (5n),
4-CO2CH3 (5o), and the non-substituted compound (5a) showed
The compounds 4-Cl (5b), 4-OCH3 (5i), 4-OC2H5 (5j), 4-OC4H9
(5l), 4-NO2 (5m), and 4-CN (5n) presented higher bioactivity than

M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
6295
Figure 2. Comparison of the physicochemical properties calculated for the 4-C3H7 (5h) and 4-OC4H9 (5l) substituted derivatives. (A) LP texture maps and C log P values (Sybyl
8.0 package).18 Brown color (0.081) indicates hydrophobic regions and blue color (ꢁ0.067) denotes hydrophilic areas. (B) EP opaque maps using GaussView 3.0 (Gaussian Inc.
1993–2003). Red color indicates negative values of EP (ꢁ0.024) or higher electronic density regions while blue color denotes positive values (0.024) or lower electronic
density distribution areas.
Table 2
compound has a more hydrophilic character in comparison to 5j
(see Table 2, C log P values). Considering the data found for the
T. cruzi epimastigote form, when the length of alkoxy chain was
increased the biological activity did not progressively decrease.
The 4-OC3H7 (5k) and 4-OC4H9 (5l) derivatives presented IC50 val-
C log P values found for the series of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-
oxadiazole derivatives using Ghose et al. method (1998)16 and SLN17 (Sybyl 8.018
)
Compound
–R
C log P values
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
–H
–Cl
–Br
–I
0.98
1.65
1.73
1.56
1.93
1.47
1.93
2.38
0.97
1.32
1.84
2.29
ꢁ1.32
0.82
0.71
ues of 12.20 and 11.47 lM, respectively.
Some of the tested compounds showed antiprotozoal biological
activity equivalent to the non-substituted compound (5a), follow-
ing the trend observed for the multidrug-resistant S. aureus strains.
The 4-OCH3 (5i) derivative, for example, presented a C log P value
quite similar to the non-substituted compound (5a), which proba-
bly reflected in its anti-T. cruzi activity. The LP texture maps of the
compounds 5a and 5i are showed in Figure 5. All compounds were
more active than benznidazole at the same assay conditions.
An initial screening for the antifungal activity of these com-
–CF3
–CH3
–C2H5
–C3H7
–OCH3
–OC2H5
–OC3H7
–OC4H9
–NO2
–CN
pounds established MIC ranges of values from 8.00 to 7.20
for the 4-CF3 (5e) derivative; from 7.20 to 6.78 g/mL for 4-
OC4H9 (5l); from 6.48 to 5.83 g/mL for 4-OC2H5 (5j); from 4.72
to 4.25 g/mL for 4-Cl (5b); and from 3.28 to 2.95 g/mL for 4-
lg/mL
–CHCO2
l
l
the same activity and the lowest MIC values (see Table 1). Addition-
ally, they presented C log P values between zero and one [0.97 (5i),
0.82 (5n), 0.71(5o), and 0.98 (5a)] (Table 2), which could indicate a
preferential range for the hydrophobicity feature regarding the bio-
activity against the VISA3 strain.
l
l
CH3 (5f), which was the most active antifungal agent tested. The
concentration ranges used for the determination of antifungal
activity varied depending upon the solubility of the compound in
the medium used.
Observing the biological data, it could be hypothesized that
the molecular recognition process between the set of 3-acetyl-
2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives (li-
gands) and the multidrug-resistant strains, SP3/R33 and VISA3
(targets), would be distinct since different ranges of MIC values
were found.
Considering the calculated properties, the most (5f) and the
least (5e) active compounds have a hydrophobic character, ex-
pressed by the C log P values and visually represented by the LP
texture maps (see Fig. 6B). The steric atomic hindrance contribu-
tions found for the 4-CF3 (5e) and 4-CH3 (5f) substituent groups
were 2.82 and 3.41, respectively, indicating that there is not a sig-
nificant difference for this geometrical or structural feature
(Fig. 6C). Otherwise, the electronic property is indeed distinct
regarding the two substituent groups. The 4-CH3 (5f) substituent
group has an inductive electron donating effect on the aromatic
system (red to yellow color on the aromatic ring—Fig.6A) whereas
the 4-CF3 (5e) substituent group presents an inductive electron
The most active compound against the epimastigote form of T.
cruzi was the 4-CO2CH3 (5o) derivative (IC50 value of 7.91
The 5o compound was threefold more active than 5j (4-OC2H5),
the less active compound (IC50 value of 26.64 M) (Table 1).
lM).
l
The 4-CO2CH3 (5o) and 4-OC2H5 (5j) derivatives present steric
hindrance values of 5.86 and 7.20, respectively. Also, the 5o

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M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
Figure 3. Visualization of the substituent groups’ volumes through van der Waals surfaces (ViewerLite 5.0, Accelrys Inc. 2002) for the compounds 5f–k. The molecules are
displayed in ball-stick scaled model (carbon atoms are in gray color, oxygen in red, nitrogen on blue, sulfur in yellow, and hydrogen atoms in white).
Figure 4. LP texture maps calculated for the compounds 5f–k using the Sybyl 8.0 package.18 Brown color (0.081) indicates hydrophobic regions and blue color (ꢁ0.067)
denotes hydrophilic areas.
withdrawing effect on that molecule moiety (green color on the
aromatic ring—Fig. 6A).
wide spectrum of activity, which will be more efficiently explored
through the addition of more compounds to this series.
Although the MIC and IC50 values of the new oxadiazoline com-
pounds presented in this study showed lower activity than the
antibiotic drugs used as reference, they present a remarkable and
Thus, in order to elucidate the bioactivity profile of this new
class of compounds, the extension of the series of analogues is
ongoing, and it is expected to provide a better understanding of

M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
6297
Figure 5. LP texture maps and C log P values found for the compound 5a and 5i using the Sybyl 8.0 package.18 Brown color (0.081) indicates hydrophobic regions and blue
color (ꢁ0.067) denotes hydrophilic areas. The molecules are displayed in ball-stick model (carbon atoms are in gray color, oxygen in red, nitrogen on blue, sulfur in yellow,
and hydrogen atoms in white) (GaussView 3.0, Gaussian Inc. 1993–2003).38
the relationships between the 4-substitutent groups, their physico-
chemical properties, and the overall microbial activity.
and 1.0 mL (2.0 mmol) of sulfuric acid. The solvent was concen-
trated and the product obtained washed with cold water. In some
cases, as for non-substituted compounds, the ester showed an oily
aspect and, to promote its precipitation, the compound was cooled
by immersion in dry ice–ethanol bath.
3. Conclusion
Biological screening results showed that some compounds have a
promising profile to be considered as attractive chemical entities for
the development of new antiprotozoal and antibacterial agents. The
4-OC4H9 (5l) derivative was the most active compound against the S.
aureus ATCC 25923 strain, while the 4-OCH3 (5i), 4-CN (5n), and 4-
CO2CH3 (5o) derivatives exhibited good activity against the VISA3
(SP3/R33) strain, similarly to the non-substituted compound and
close to vancomycin. Regarding the initial assays for the C. albicans
strain, the 4-CH3 (5f) and the 4-Cl (5b) derivatives were showed as
more active compounds. Their MIC ranges varied from 3.28 to
4.1.2. General procedure for the synthesis of benzhydrazides
(3a–o)20
Hydrazine hydrate 64% (v/v) (30.0 mL, 0.33 mol) was heated up
to 50–60 °C. The methyl ester 3 (0.01 mol) was added and the mix-
ture was heated at reflux for 10 min. The cooling was performed
sequentially in water bath, followed by ice bath and dry ice–etha-
nol bath. The precipitate was filtered and washed with cold water.
4.1.3. General procedure for the synthesis of Schiff’s bases (4a–o)
Schiff’s bases were synthesized by heating at reflux 5-nitro-
thiophen-2-carboxaldehyde (0.01 mol) and benzhydrazides (3)
(0.01 mol) in water, sulfuric acid, acetic acid, and methanol
(8:7:8:20 v/v) for 1 h. After cooling, the mixture was poured into
cold water to precipitate the Schiff’s bases.
2.95 lg/mL and from 4.72 to 4.25 lg/mL, respectively, two-fold
more active than 4-CF3 (5e), 4-OC4H9 (5l), and 4-OC2H5 (5j). Further
studies are needed to confirm these in vitro results and investigate
the mode of action of these derivatives. The calculated physico-
chemical and structural properties, using molecular modeling tech-
niques, provided suitable information to explain the resulting
biological data allowing the establishment of qualitative property-
activity relationships.
4.1.4. General procedure for the synthesis of 2,3-dihydro-1,3,4-
oxadiazolines derivatives (5a–o)
0.5 g of the corresponding Schiff’s base was dissolved in 100 mL
acetic anhydride at 85 °C and the mixture heated at 120 °C for 8 h.
The reaction medium was evaporated to 10% volume then cooled,
allowing the product to precipitate.
4. Experimental
4.1. Chemistry
4.1.4.1.
dro-1,3,4-oxadiazole (5a).
3-Acetyl-5-phenyl-2-(5-nitrothiophen-2-yl)-2,3-dihy-
White solid (72%); mp 103.0–
NMR spectra were recorded on a BRUKER ADPX Advanced
(300 MHz) spectrometer employing DMSO-d6 solutions with tetra-
methylsilane as internal standard. Melting points were determined
using Micro-Química MQAPF-301 apparatus and elemental analysis
was performed on a Perkin-Elmer 24013 CHN Elemental Analyzer.
104.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d, 1H, H8,
J = 4.3 Hz), 7.87 (d, 2H, H15,19, J = 8.6 Hz), 7.63 (s, 1H, H2), 7.56 (m,
3H, H16/17/18), 7.47 (d, 1H, H7, J = 4.3 Hz), 2.29 (s, 3H, H22); 13C
NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.3 (C20), 154.4 (C5),
151.6 (C6), 146.6 (C9), 132.2 (C14), 129.5 (C17), 129.1 (C16, C18),
127.1 (C8), 126.6 (C15, C19), 123.2 (C7), 86.7 (C2), 20.9 (C22); Anal.
Calcd for (C14H11N3O4S): C, 52.99; H, 3.49; N, 13.24. Found: C,
53.06; H, 3.59; N, 13.24.
4.1.1. General procedure for the synthesis of methyl esters (2a–
o)20
Each substituted benzoic acid (1) (0.04 mol) was heated at re-
flux for 4 hours in 50.0 mL (1.23 mol) of anhydrous methanol

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M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
Figure 6. Comparison of the physicochemical and structural properties calculated for the 4-CH3 (5f) and 4-CF3 (5e) substituted derivatives. (A) EP opaque maps using
GaussView 3.0 (Gaussian Inc. 1993–2003).38 Red color indicates higher electronic density regions (ꢁ0.024) while blue color denotes lower electronic density areas (0.024). (B)
LP texture maps and C log P values (Sybyl 8.0 package).18 Brown color (0.081) indicates hydrophobic regions and blue color (ꢁ0.067) denotes hydrophilic areas. (C)
Visualization of the substituent groups’ volumes through van der Waals surfaces (ViewerLite 5.0, Accelrys Inc. 2002) and respective steric atomic hindrance values.19 The
molecules are displayed in ball-stick scaled model (carbon atoms are in gray color, oxygen in red, nitrogen on blue, sulfur in yellow, fluorine in cyan, and hydrogen atoms in
white).
4.1.4.2. 3-Acetyl-5-(4-chlorophenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5b). Yellow solid (70%); mp
130.0–131.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.08 (d,
1H, H8, J = 4.3 Hz), 7.87 (d, 2H, H15/19, J = 8.1 Hz), 7.63 (d, 2H, H16/
18, J = 8.2 Hz), 7.58 (s, 1H, H2), 7.48 (d, 1H, H7, J = 4.3 Hz), 2.29 (s,
3H, H22); 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.4 (C20),
153.6 (C5), 151.6 (C6), 146.4 (C9), 136.9 (C17), 129.5 (C14), 129.3
(C15, C19), 128.4 (C16, C18), 127.1 (C8), 122.1 (C7), 87.0 (C2), 20.9
(C22); Anal. Calcd for (C14H10ClN3O4S): C, 47.80; H, 2.87; N, 11.95.
Found: C, 47.88; H, 2.67; N, 11.95.
4.1.4.4. 3-Acetyl-5-(4-iodophenyl)-2-(5-nitrothiophen-2-yl)-2,3-
dihydro-1,3,4-oxadiazole (5d). Yellow solid (55%); mp 156.0–
157.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d, 1H, H8,
J = 4.1 Hz), 7.94 (d, 2H, H16/18, J = 8.6 Hz), 7.62 (d, 2H, H15/19,
J = 8.6 Hz), 7.57 (s, 1H, H2), 7.47 (d, 1H, H7, J = 4.3 Hz), 2.29 (s, 3H,
H22); 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.4 (C20),
154.0 (C5), 151.7 (C6), 146.5 (C9), 138.0 (C16, C18), 129.5 (C14),
128.3 (C15, C19), 127.2 (C8), 127.2 (C8), 122.7 (C7), 99.9 (C2), 21.0
(C22); Anal. Calcd for (C14H10IN3O4S): C, 37.94; H, 2.27; N, 9.48.
Found: C, 37.89; H, 2.29; N, 9.37.
4.1.4.3. 3-Acetyl-5-(4-bromophenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5c). Yellow solid (42%); mp
142.0–143.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.01 (d,
1H, H8, J = 4.3 Hz), 7.74 (d, 2H, H15/19, J = 9.0 Hz), 7.70 (d, 2H, H16/
18, J = 9.0 Hz), 7.51 (s, 1H, H2), 7.42 (d, 1H, H7, J = 4.3 Hz), 2.23 (s,
3H, H22); 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.9 (C20),
154.3 (C5), 152.2 (C6), 147.0 (C9), 132.7 (C16, C18), 129.9 (C14),
129.0(C15, C19), 127.6 (C8), 126.3 (C7), 123.0 (C17), 87.6 (C2), 21.5
(C22); Anal. Calcd for (C14H10BrN3O3S): C, 42.44; H, 2.54; N, 10.61.
Found: C, 42.38; H, 2.57; N, 10.63.
4.1.4.5.
3-Acetyl-5-(4-trifluoromethylphenyl)-2-(5-nitrothio-
Yellow solid
phen-2-yl)-2,3-dihydro-1,3,4-oxadiazole (5e).
(59%); mp 135.0–136.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm):
8.08 (d, 1H, H8, J = 4.3 Hz), 8.07 (d, 2H, H15/19, J = 8.4 Hz), 7.93 (d,
2H, H16/18, J = 8.4 Hz), 7.62 (s, 1H, H2), 7.51 (d, 1H, H7, J = 4.3 Hz),
2.31 (s, 3H, H22); 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 168.1
(C20), 153.8 (C5), 152.2 (C6), 146.8 (C9), 132.4 (C14), 131.9 (C17),
130.0 (C15, C19), 128.0 (C16, C18), 127.8 (C8), 126.7 (C7), 126.6
(CF3), 87.9 (C2), 21.5 (C22); Anal. Calcd for (C15H10F3N3O4S): C,
46.76; H, 2.62; N, 10.91. Found: C, 46.90; H, 2.92; N, 11.02.

M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
6299
4.1.4.6. 3-Acetyl-5-(4-methylphenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5f). Yellow solid (48%); mp
127.0–128.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d,
1H, H8, J = 4.3 Hz), 7.76 (d, 2H, H15/19, J = 7.9 Hz), 7.55 (s, 1H, H2),
7.46 (d, 1H, H7, J = 4.3 Hz), 7.37 (d, 2H, H16/18, J = 7.9 Hz), 2.39 (s,
3H, CH3), 2.29 (s, 3H, H22); 13C NMR {H} (DMSO-d6, 75 MHz): d
(ppm): 167.2 (C20), 154.6 (C5), 151.6 (C6), 146.7 (C9), 142.5 (C17),
129.7 (C16, C18), 129.5 (C14), 127.0 (C8), 126.6 (C15, C19), 120.4
(C7), 86.6 (C2), 21.1 (C22), 21,0 (CH3); Anal. Calcd for
(C15H13N3O4S): C, 54.37; H, 3.95; N, 12.68. Found: C, 54.21; H,
4.03; N, 12.58.
2H, CH2, J = 6.3 Hz), 2.27 (s, 3H, H22), 1.75 (m, 2H, CH2, J = 7.2 Hz),
0.98 (t, 3H, CH3, J = 7.2 Hz); 13C NMR {H} (DMSO-d6, 75 MHz): d
ppm): 167.6 (C20), 162.2 (C17), 155.1 (C5), 152.0 (C6), 147.3 (C9),
130.0 (C8), 115.5 (C7), 129.1 (C15, C19), 127.5 (C14), 129.0 (C16,
C18), 86.9 (C2), 69.8 (CH2), 22.3 (CH2), 21.5 (C22), 10.7 (CH3); Anal.
Calcd for (C17H17N3O5S): C, 54.39; H, 4.56; N, 11.19. Found: C,
54.50; H, 4.56; N, 11.14.
4.1.4.12. 3-Acetyl-5-(4-n-butoxyphenyl)-2-(5-nitrothiophen-2-
yl)-2,3-dihydro-1,3,4-oxadiazole (5l). Yellow solid (32%); mp
116.0–117.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d,
1H, H8, J = 4.3 Hz), 7.78 (d, 2H, H15/19, J = 9.0 Hz), 7.53 (s, 1H, H2),
7.45 (d, 1H, H7, J = 4.2 Hz), 7.08 (d, 2H, H16/18, J = 9.0 Hz), 4.05 (t,
2H, CH2, J = 6.4 Hz), 2.27 (s, 3H, H22), 1.72 (m, 2H, CH2, J = 6.6 Hz),
1.40 (m, CH2, J = 7.4 Hz), 0.94 (t, 3H, CH3, J = 7.4 Hz; 13C NMR {H}
(DMSO-d6, 75 MHz): d (ppm): 167.6 (C20), 162.9 (C17), 155.1 (C5),
152.1 (C6), 147.4 (C9), 130.0 (C8), 129.1 (C15, C19), 127.5 (C7),
115.7 (C16, C18), 115.5 (C14), 86.8 (C2), 68.1 (CH2), 31.0 (CH2), 21.4
(C22), 19.1 (CH2), 14.9 (CH3); Anal. Calcd for (C18H19N3O5S): C,
55.52; H, 4.96; N, 10.79. Found: C, 56.01; H, 3.51; N, 11.15.
4.1.4.7.
3-Acetyl-5-(4-ethylphenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5g). Yellow solid (62%); mp
108.0–109.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.06 (d,
1H, H8, J = 4.1 Hz), 7.79 (d, 2H, H16/18, J = 8.1 Hz), 7.56 (s, 1H, H2),
7.46 (d, 1H, H7, J = 4.3 Hz), 7.39 (d, 2H, H15,19, J = 8.1 Hz), 2.68 (m,
2H, CH2, 7.1 Hz), 2.29 (s, 3H, H22), 1.17 (t, 3H, CH3, J = 7.1 Hz); 13C
NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.7 (C20), 155.1 (C5),
152.1 (C6), 149.0 (C9), 147.3 (C17), 130.02 (C14), 129.0 (C15, C19),
127.5 (C8), 127.3 (C16, C18), 121.2 (C7), 87.1 (C2), 28.6 (CH2), 21.49
(C22), 15.5 (CH3); Anal. Calcd for (C16H15N3O4S): C, 55.64; H, 4.38;
N, 12.17. Found: C, 55.58; H, 4.54; N, 11.98.
4.1.4.13. 3-Acetyl-5-(4-nitrophenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5m). Yellow solid (57%); mp
186.0–187.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.40 (d,
2H, H16/18, J = 9.0 Hz); 8.11 (d, 2H, H15/19, J = 9.0 Hz); 8.07 (d, 1H,
H8, J = 4.6 Hz) 7.63 (s,1H, H2); 7.52 (d, H7, J = 4.3 Hz); 2.33 (3H,
H22). 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.7 (C20),
153.0 (C5), 152.2 (C6), 149.4 (C9), 146.2 (C17), 129.3 (C14), 128.0
(C8), 127.2 (C7, C15, C19), 124.1 (C16, C18), 87.8 (C2), 21.0 (C22);
Anal. Calcd for (C14H10N4O6S): C, 46.41; H, 2.78; N, 15.46. Found: C,
46.50; H, 2.76; N, 15.30.
4.1.4.8.
3-Acetyl-5-(4-n-propylphenyl)-2-(5-nitrothiophen-2-
yl)-2,3-dihydro-1,3,4-oxadiazole (5h). White solid (65%); mp
73.0–74.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.06 (d, 1H,
H8, J = 4.0 Hz), 7.78 (d, 2H, H15/19, J = 8.1 Hz), 7.56 (s, 1H, H2), 7.46
(d, 1H, H7, J = 4.0 Hz), 7.37 (d, 2H, H18,16, J = 8.1 Hz), 2.62 (t, 2H,
CH2, J = 7.6 Hz), 2.28 (s, 3H, H22), 1.62 (m, 2H, CH2, J = 7.3 Hz),
0.89 (t, 3H, CH3, J = 7.2 Hz); 13C NMR {H} (DMSO-d6, 75 MHz): d
(ppm): 167.2 (C20), 154.6 (C5), 151.6 (C6), 146.9 (C9), 146.7 (C17),
129.5 (C14), 129.1 (C15, C19), 127.0 (C8), 126.6 (C16, C18), 120.71
(C7), 86.6 (C2), 37.1 (CH2), 23.67 (CH2), 21.0 (C22), 13.5 (CH3);
Anal. Calcd for (C17H17N3O4S): C, 56.81; H, 4.77; N, 11.69. Found: C,
57.13; H, 5.11; N, 11.72.
4.1.4.14. 3-Acetyl-5-(4-cyanophenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5n). Yellow solid (53%); mp
136.0–137.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.08 (d,
2H, H15/19, J = 4.2 Hz); 8.02 (s, 3H, H18/16 e H8); 7.62 (s, 1H, H2),
7.51 (d, H7, J = 4.2 Hz); 2.32 (3H, H22). 13C NMR {H} (DMSO-d6,
75 MHz): d (ppm): 167.7 (C20), 157.3 (C5), 153.2 (C6), 152.0 (C9),
146.3 (C14), 133.0 (C16, C18), 129.5 (C8), 127.5 (C7), 127.3 (C15,
C19), 118.0 (CN), 114.3 (C17), 87.6 (C2), 21.1 (C22). Anal. Calcd for
(C15H10N4O4S): C, 52.63; H, 2.94; N, 16.37. Found: C, 49.76; H,3.78;
N, 15.83.
4.1.4.9.
3-Acetyl-5-(4-methoxyphenyl)-2-(5-nitrothiophen-2-
yl)-2,3-dihydro-1,3,4-oxadiazole (5i). White solid (51%); mp
109.0–110.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.08 (d,
1H, H8, J = 4.3 Hz), 7.82 (d, 2H, H15/19, J = 8.8 Hz), 7.56 (s, 1H, H2),
7.47 (d, 1H, H7, J = 4.1 Hz), 7.12 (d, 2H, H16/18, J = 8.6 Hz), 3.9 (s,
3H, OCH3), 2.29 (s, 3H, H22); 13C NMR {H} (DMSO-d6, 75 MHz): d
(ppm): 167.2 (C20), 162.3 (C17), 154.5 (C5), 151.6 (C6), 146.9 (C9),
129.5 (C8), 128.6 (C15, C19), 127.0 (C7), 115.4 (C14), 114.6 (C16,
C18), 86.5 (C2), 55.5 (OCH3), 21.0 (C22); Anal. Calcd for
(C15H13N3O5S): C, 51.87; H, 3.77; N, 12.10. Found: C, 51.75; H,
3.69; N, 11.87.
4.1.4.15.
3-Acetyl-5-(4-acetoxyphenyl)-2-(5-nitrothiophen-2-
yl)-2,3-dihydro-1,3,4-oxadiazole (5o). Yellow solid (75%); mp
120.0–121.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.06(d, 1H,
H8, J = 4.3 Hz); 7.93 (d, 2H, H16/18, J = 8.6 Hz); 7.57 (s, 1H, H2); 7.48
(d, H7, J = 4.3 Hz); 7.35 (d, 2H, H15/19 J = 8.6 Hz); 2.32 (6H, H22 e
OCOCH3); 13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 168.8 (C20),
167.4 (C@O), 154.0 (C5), 153.3 (C6), 151.7 (C17), 146.6 (C9), 129.4
(C14), 128.2 (C15, C19), 127.1 (C8), 122.8 (C16, C18), 120.8 (C7),
87.0 (C2), 21.0 (C22), 20.8 (CH3). Anal. Calcd for (C16H13N3O6S): C,
51.20; H, 3.49; N, 11.19. Found: C, 51.10; H, 3.55; N, 11.06.
4.1.4.10. 3-Acetyl-5-(4-ethoxyphenyl)-2-(5-nitrothiophen-2-yl)-
2,3-dihydro-1,3,4-oxadiazole (5j).
Yellow solid (56%); mp
108.0–109.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d,
1H, H8, J = 4.3 Hz), 7.79 (d, 2H, H15/19, J = 8.6 Hz), 7.54 (s, 1H, H2),
7.45 (d, 1H, H7, J = 4.3 Hz), 7.08 (d, 2H, H16/18, J = 8.6 Hz), 4.11 (q,
2H, CH2, J = 6.9 Hz), 2.27 (s, 3H, H22), 1.35 (t, 3H, CH3, J = 6.9 Hz);
13C NMR {H} (DMSO-d6, 75 MHz): d (ppm): 167.6 (C20), 162.0
(C17), 155.0 (C5), 152.0 (C6), 147.4 (C9), 130.0 (C8), 129.1 (C15,
C19), 127.7 (C7), 127.5 (C14), 115.5 (C16, C18), 86.9 (C2), 64.0
(CH2), 21.5 (C22), 14.9 (CH3); Anal. Calcd for (C16H15N3O5S): C,
53.18; H, 4.18; N, 11.63. Found: C, 52.73; H, 3.91; N, 11.20.
4.2. Biology
The biological activity of the nitrocompound derivatives was
evaluated against the epimastigote form of T. cruzi Y strain, S aureus
ATCC 25923, multidrug-resistant S. aureus 3SP/R33 and VISA3
strains, and Candida albicans ATCC537Y.
4.1.4.11. 3-Acetyl-5-(4-n-propoxyphenyl)-2-(5-nitrothiophen-2-
yl)-2,3-dihydro-1,3,4-oxadiazole (5k). White solid (50%); mp
102.0–103.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 8.07 (d,
1H, H8, J = 4.3 Hz), 7.79 (d, 2H, H15/19, J = 9.0 Hz), 7.54 (s, 1H, H2),
7.45 (d, 1H, H7, J = 4.3 Hz), 7.10 (d, 2H, H16/18, J = 9.0 Hz), 4.01 (t,
4.2.1. In vitro anti-T. cruzi activity
The parasite epimastigote form, at the stationary phase and from
a culture incubated for 10 days at 28 °C, was diluted into liver infu-
sion tryptose (LIT) medium to obtain a final concentration of
2.0 ꢂ 106 parasites per mL. The LIT medium was prepared as

6300
M. Ishii et al. / Bioorg. Med. Chem. 19 (2011) 6292–6301
described in a previous study.13 A preliminary assay using the inoc-
ula at logarithmic phase was performed, indicating the derivative
compounds that presented a good potential profile. The nitroderiv-
atives and benznidazole (BZN), the standard drug, were separately
dissolved in dimethylsulphoxide, DMSO, (Labsynth, São Paulo,
Brazil) and diluted into LIT culture medium to obtain a concentra-
in their neutral forms using as starting geometry a derivative of
1,3,4-oxadiazole whose crystallographic information was retrieved
from Ref. 30. Each model was energy-minimized in the MM+ force
field31 without any constrains (HyperChem 7.5132). The partial
atomic charges were calculated employing the AM1 semiempirical
method.33 Molecular dynamics (MD) simulations [1 ns; size step
1 fs; 310 K] were carried out using the MOLSIM 3.2 program.34 The
lowest-energy conformer selected from MD simulation of each
compound was the input structure for calculating the physico-
chemical and structural properties explored in this study. Electro-
static partial atomic charges (CHELPG)35 were computed using the
B3LYP/3-21G⁄ method36,37 implemented in the GAUSSIAN 03 W pro-
gram.38 The EP property was calculated in a cube surface using
an isovalue of 0.0004 (GAUSSIAN 03 W).38 Negative values of EP (high-
er electronic density) are denoted as intense red while positive val-
ues are mapped in blue (lower electronic density) on the molecular
surfaces. The LP property was computed onto a Connolly molecular
surface39,40 employing a sphere probe of 1.4 Å radii (Sybyl 8.0
package18). This property and the C log P values were calculated
applying the Ghose and co-workers method16 and Sybyl Line Nota-
tion (SLN)17 (Sybyl 8.0 package18). The resulting LP maps were
evaluated regarding the color ramps, which range from dark brown
(hydrophobic or lipophilic region on molecular surface) to blue
(hydrophilic area on molecular surface). It is noteworthy that the
color scheme is easy to interpret if the blue is associated with
water and the brown as oil/fat. Furthermore, the atomic steric hin-
drance of each model was calculated from the covalent radii values
and geometrical distances, using the Calculator Plugins at the
Marvin 5.3.8 software.19 This structural feature is additive, mean-
ing that the total steric hindrance of a substituent group corre-
sponds to the sum of each atomic steric hindrance calculated.19
tion from 1.25 to 40.0 lM, depending on the solubility of the com-
pounds. An aliquot of the inocula containing 2.0 ꢂ 106 viable
parasites per mL21,22 was added to the compounds being evaluated
and incubated at 28 °C for 5 days. After this period, the amount of
viable parasites was determined using a spectrophotometer (T80
plus, PG Instruments, United Kingdom) at 580 nm (10 mm light
path length). The assays were performed in triplicate and the
remaining parasites were counted in a Neubauer chamber. The
anti-T. cruzi activity was determined by the relation.23–28
PGI ¼ 1 ꢁ ½ðAP ꢁ A0PÞ=ðA
Þꢃ ꢂ 100;
CꢁA0C
where: PGI = percentage of growth inhibition; Ap = A580 of the cul-
ture containing the drug at day 5; A0p = A580 of the culture contain-
ing the compound after the addition of the inoculums at day 0;
Ac = A580 of the culture in the absence of any compound at day 5;
Aoc = A580 of the culture in the absence of any compound at day 0.
4.2.1.1. IC50 determination. The inhibitory concentration
50%, IC50, was calculated from the percentage of inhibition growth
using a dose–response curve for each case, using at least four con-
centration values.
4.2.2. Antibacterial activity20
Minimal Inhibitory Concentration (MIC) of the compounds was
determined with 96-well microtiter plates containing twofold se-
rial dilutions of the compounds in Tryptic Soy Broth (TSB, Sigma-
AldrichÒ) medium. Stock solutions of the compounds were pre-
pared in DMSO/TSB 1:10 v/v. Concentrations ranged from 0.1 to
Acknowledgments
The authors thank Professor Elza Masae Mamizuka, from the
Department of Clinical and Toxicological Analysis, USP, for pro-
viding the 3SP/R33 and VISA3 S. aureus strains and Professor
Maria Júlia Manso Alves, from Chemistry Institute for providing
the epimastigote form of T. cruzi strain. The authors thank Brazil-
ian scientific committees, CNPq and FAPESP for the financial
support.
80 lg/mL, using nifuroxazide, ampicilin, and vancomycin as drug
controls. Bacterial suspensions were prepared by turbidity adjust-
ment to a density of 0.5 on the McFarland scale and further dilu-
tion in sterile physiologic saline solution and TSB. The plates
were incubated at 35 °C for 18 h, and the lowest concentration of
compound at which there was no visible growth was considered
as being the MIC value. Readings at 24 and 48 h were carried out
for sterility control. All assays were performed in triplicate.
References and notes
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