Design, synthesis and cruzain docking of 3-(4-substituted-aryl)-1,2,4-oxadiazole-N-acylhydrazones as anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1016/j.bmc.2009.07.068

Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a valid chemotherapeutic target, since inhibitors of this protease affect the pathology appropriately. By exploring the N-acylhydrazones (NAH) as privi

Full text of DOI:10.1016/j.bmc.2009.07.068

Bioorganic & Medicinal Chemistry 17 (2009) 6682–6691
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and cruzain docking of 3-(4-substituted-aryl)-1,2,4-
oxadiazole-N-acylhydrazones as anti-Trypanosoma cruzi agents
b
b
José Mauricio dos Santos Filho a, , Ana Cristina Lima Leite , Boaz Galdino de Oliveira ,
*
Diogo Rodrigo Magalhães Moreira b, Milena S. Lima c, Milena Botelho Pereira Soares c,d
,
Lucia Fernanda C. C. Leite e
a Departamento de Engenharia Química (DEQ), Centro de Tecnologia e Geociências, Universidade Federal de Pernambuco (UFPE), Rua Prof. Artur Sá S/N, Cidade Universitária,
50740-521 Recife, PE, Brazil
b Departamento de Ciências Farmacêuticas (DCFAR), Centro de Ciências da Saúde, UFPE, Rua Prof. Artur Sá S/N, Cidade Universitária, 50740-520 Recife, PE, Brazil
c Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz (FIOCRUZ), Rua Waldemar Falcão, 121, Candeal 40296-750, Salvador, BA, Brazil
d Hospital São Rafael, Av. São Rafael, 2152. São Marcos 41253-190, Salvador, BA, Brazil
e Universidade Católica de Pernambuco (UNICAP), Rua do Príncipe, 560, Boa Vista, 50050-900 Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a
valid chemotherapeutic target, since inhibitors of this protease affect the pathology appropriately. By
exploring the N-acylhydrazones (NAH) as privileged structures usually present in antiparasitic agents,
we investigated a library of 16 NAH bearing the 3-(4-substituted-aryl)-1,2,4-oxadiazole scaffold (NAH
3a–h, 4a–h). The in vitro bioactivity against epimastigote and trypomastigote forms of T. cruzi was eval-
uated, and some NAH under study exhibited antitrypanosomal activity at concentrations that are not
toxic to mammalian cells. The series of compounds based on the 3-(4-substituted-aryl)-1,2,4-oxadiazole
scaffold revealed the remarkable importance of each substituent at the phenyl’s 4-position for the inhib-
itory activity. Non-nitrated compounds 3a and 4e were found to be as potent as the reference drug, Ben-
znidazole. In addition, the molecular origin of the antitrypanosomal properties for these series was
investigated using docking studies of the TCC structure.
Received 12 May 2009
Revised 22 July 2009
Accepted 26 July 2009
Available online 6 August 2009
Keywords:
Trypanosoma cruzi
Oxadiazole
N-Acylhydrazone
Privileged structures
Molecular docking
Cruzain
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
protease is capable of degrading components of the extracellular
matrix and is involved in the replication and nutrition of the para-
Chagas’ disease or American trypanosomiasis, caused by the
protozoon, Trypanosoma cruzi, and transmitted to humans by in-
fected blood-sucking reduviid bugs of the subfamily Triatominae,
continues to afflict millions of people in Latin American countries.
The treatment of Chagas’ disease is heavily reliant on chemother-
apy using Benznidazole (Bdz), the only World Health Organiza-
tion-recommended drug. There are, however, major concerns
regarding its high toxicity and low effectiveness during the chronic
phase of infection.1,2 Therefore, there has been a considerable
interest in trying to find novel approaches to drug design and bio-
logical targets, as new ways of combating the parasite.3
site.9 In view of these roles, TCC has emerged as a potential target
for drug development in Chagas’ disease treatment.10
The first compounds investigated as potential TCC inhibitors
were peptides and peptidomimetics functionalized using vinyl
sulfones, epoxyketones or diazomethylketones groups. However,
except for the family of dipeptidyl vinyl sulfones, it has generally
been observed that most peptide inhibitors show low selectivity
against related proteases (serine protease chymotrypsin), with a
subsequent toxicity against the mammalian counterparts.11
In order to avoid this undesirable pharmacological profile, the
replacement of a peptide backbone by a more rigid central scaffold
has been considered and, in this scenario, N-acylhydrazones (NAH)
have provided medicinal chemists with a solid strategy for the de-
sign of parasite cysteine protease inhibitors (CPs), including cruz-
ain, falcipain-2, rhodesain and Leishmania cpB ( Fig. 1). In these
initial studies, a set of NAH libraries were investigated, exploring
diverse molecular modifications such as bioisosterism, molecular
simplification and the development of congener series.12–15 More-
over, the use of NAH subunit has enabled the discovery of a
number of lead compounds with potent and selective activity
Molecular targets of the parasite, such as the cruzain,4 trypano-
thione reductase5 and trans-sialidase6 of T. cruzi have proved to be
useful for the medicinal chemistry of new anti-T. cruzi drug candi-
dates.7 The T. cruzi cruzain (TCC) is a member of the papain super-
family of cysteine proteases, with structure and functions similar
to the human enzymes cathepsins B, L, K, S, F, and V.8 This cysteine
* Corresponding author. Tel.: +55 81 21267288; fax: +55 81 21267278.
E-mail address: mauricio_santosfilho@yahoo.com.br (J.M. dos Santos Filho).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.068

J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6683
O
O
aza-vinylogue
relationship
H
R'
N
R'
More conformationally
restricted and chemically
stable than peptides
N
N
H
C
N
H
R1
R
H
+ N=C
peptide scaffold
N-acylhydrazones
(privileged structures)
OH
OH
H
O
OH
O
OH
O
N
N
N
H
N
N
N
N
H
N
N
H
H
Ph
N
OMe
compound ZLIII43A [ref. 12]
active against parasitic CPs
compound LASSBio-334 [ref. 14]
active in cell culture of T. cruzi
compound LASSBio-1016 [ref. 17]
trypanocidal and noncytotoxic in mammalian cells
Figure 1. Design concepts for the NAH subunit as privileged structures.19 Structures of NAH derivatives described as inhibitors of parasitic CPs are listed above.
against the replication of T. cruzi.16,17 For example, the adequate
combination of the NAH subunit attached to a quinoxaline ring re-
sulted the discovery of two new antitrypanosomal agents with IC50
razides (2a–h) by reaction with hydrazine hydrate in ethanol at
0 °C for the duration of 60 min.22
Usually, the condensation reaction between hydrazides and
appropriate 4-substituted phenylaldehydes is achieved by reflux-
ing the reactants under acid catalysis in protic solvent. However,
a mixture of Z/E-isomers is often reported, mainly for reaction pro-
cedures that are only possible at high temperatures or for pro-
longed reaction periods. In a prior study involving the synthesis
of N-acylhydrazone derivatives, a method employing free-catalyst
conditions under reflux developed by our group yielded the E-iso-
mers as major products, although with long reaction times (4 h).23
Theoretical calculations for the configurational and conformational
energies of these Z/E-isomers showed that a planar and more sta-
ble conformation for the E-isomer is achieved, this being the ther-
modynamically preferable product.
Recently, a report described that the NAH derivatives can be
satisfactorily synthesized under acid catalysis for 30 min at rt.24
This mild and rapid procedure was also observed in our laboratory
during the synthesis of NAH derivatives. Interestingly, only a very
short reaction time (10 min) was required to furnish the desired
products, 3a–h and 4a–h, in very good to excellent yields with
no detected by-products. Some physical and chemical properties
of these NAH are summarized in Table 1.
values of 15.9–20 lM against epimastigotes of T. cruzi (Tulahuen 2
strain) at concentrations that do not overtly affect J774 mouse
macrophages, as exemplified by LASSBio-1019 ( Fig. 1). In the
course of this work, in silico studies of molecular docking of these
quinoxaline–NAH were conducted to establish substantial hydro-
gen bond interactions with the TCC structure, which possibly acted
by way of targeting the TCC enzyme.16
From the point of view of medicinal chemistry, NAH are en-
dowed with the interesting capacity of being able to interact
with diverse bioreceptors, owing to their ability to establish H-
bonding sites and the theoretical feasibility of adopting a unique
conformational orientation.18 This profile suggested that the NAH
subunit would be of crucial importance in the process of molec-
ular recognition by the target. Another structural feature of the
NAH moiety is its non-peptidic and achiral nature, which, in
addition to its being synthetically treatable, thereby makes its
suitable for performing structure–activity relationships (SARs).
It stands to reason that the NAH have assumed the position of
authentic privileged structure for the structural design of new
drug candidates, as recently reviewed by Barreiro and co-
workers.19
In this study, we report the synthesis of two new series of 3-(4-
substituted-aryl)-1,2,4-oxadiazole-N-acylhydrazones and their
in vitro activity against epimastigote and trypomastigote forms
of T. cruzi, as well as their interaction with the TCC structure, as
determined by molecular docking studies. In our design, the oxadi-
azole heterocycle was selected as a trypanocide scaffold, for reason
of its previously observed antiparasitic properties,20 and because it
is recognized as a amide bioisoster, in addition to being chemically
more stable than peptide bonds21
2.2. Molecular modeling
Analysis of the structures and conformation of compounds 3a–
h and 4a–h was carried out using the AM1 method,25 available as
part of the BIOMEDCACHE suite of programs,26 using internal default
settings as convergence criteria. Each of the most stable conform-
ers of the E-isomers was subsequently selected for the docking
analysis. The docking analysis was carried out on the TCC binding
site (PDB code: 1U9Q),27,28 where the enzyme’s residues are in
close proximity to the inhibitor named as ‘186’, a fluoromethyl ke-
tone dipeptide that was co-crystallized in complex with the cruz-
ain. This crystal structure is the monomeric catalytic domains
termed X and the active site was narrowed down to all atoms with-
in a sphere of radius 5.0 Å, as determined by the co-crystallized li-
gand ‘186’. The GOLD 4.0 program and HERMES29 interface software
were used for docking calculations, and all solvent molecules and
the co-crystallized inhibitor were removed from the structure.
The theoretical binding profile proposed for 3a–h and 4a–h ligands
with TCC was determined as the highest (most positive) scored of
10 possible solutions for each ligand, ranked according to their fit-
ness scores calculated using the GOLD docking function. We decided
2. Chemistry
2.1. Synthesis
The substituted 3-(4-substituted-aryl)-1,2,4-oxadiazolyl-N-
acylhydrazones 3a–h and 4a–h (Scheme 1) were prepared from
arylamidoximes. After the cyclocondensation reaction between
arylamidoximes and methyloxalyl chloride under reflux in dry
THF, the intermediates bearing an 1,2,4-oxadiazole ring (1a–h)
were obtained and then converted into 1,2,4-oxadiazolylcarbohyd-

6684
J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
O
O
O
OH
O
NH2
O
N
N
Cl
O
N
N
H
NH2NH2.H2O (80%)
EtOH, 0oC, 60min.
O
NH2
N
N
O
THF, reflux, 4.5h
R
R
R
arylamidoximes
(1a-h)
(2a-h)
p-YC6H4CHO,
H2SO4 (cat.)
EtOH, rt, 10min.
Y
O
Y = OH
(4a) R= H
(4b) R= CH3
(4c) R= F
Y = NHCOCH3
(3a) R= H
(3b) R= CH3
O
N
N
N
H
N
H
(3c) R= F
(4d) R= Cl
(4e) R= Br
(4f) R= NO2
(4g) R= OCH3
(4h) R= OH
(3d) R= Cl
(3e) R= Br
(3f) R= NO2
(3g) R= OCH3
(3h) R= OH
R
(3a-h, 4a-h)
Scheme 1. Synthesis of NAH 3a–h and 4a–h.
after 11 days for epimastigotes and 24 h for trypomastigotes of
treatment with each drug. Benznidazole (Bdz) and Gencian Violet
(GV) were used as reference trypanocidal drugs. The SARs were
qualitatively discussed on the basis of the chemical substitutions
performing on the aromatic ring.
Table 1
Physical and chemical properties and parameter of bioavailability for the NAH 3a–h
and 4a–h
Compd
R
Molecular formulaa
Yieldb (%)
Ratioc (E:Z)
C log Pd
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
H
CH3
F
Cl
Br
NO2
OCH3
OH
H
CH3
F
Cl
Br
NO2
OCH3
OH
C18H15N5O3
C19H17N5O3
90
95
93
98
83
90
89
91
98
95
85
95
88
85
90
85
100:0
100:0
100:0
100:0
100:0
100:0
100:0
100:0
88.9:11.1
91.7:8.3
90.9:9.1
90.9:9.1
91.7:8.3
91.7:8.3
88.9:11.1
91.7:8.3
2.19
2.79
2.45
2.91
3.03
2.28
2.30
1.76
2.50
2.89
2.63
3.09
3.20
2.47
2.49
2.03
C
18H14N5O3F
3.2. Toxicity in mice
C18H14N5O3Cl
C18H14N5O3Br
C18H14N6O5
C19H17N5O4
C18H15N5O4
C16H12N4O3
C17H14N4O3
The two most potent NAH derivatives were submitted to an as-
say for general toxicity in mice treated intraperitoneally with com-
pounds 3a and 4e at a single dose of 100 mg/kg. Animals were
monitored for signs of general toxicity, including behavior and
feeding, until 72 h after treatment.
C
16H11N4O3F
C16H11N4O3Cl
C16H11N4O3Br
C16H11N5O5
C17H14N4O4
C16H12N4O4
4. Discussion
4g
4h
The analysis of the 1H NMR spectra of the crude product re-
vealed that most 3a–h compounds essentially adopted the E-con-
figuration in solution and only in some cases were traces of Z-
isomers observed (Table 1). The isomeric ratio of NAH derivatives
was appropriately established by specific signals in the 1H NMR
spectra and confirmed on the basis of data reported in the litera-
ture.23,24 For instance, the NH protons of compounds 3a–h allow
this attribution, albeit with a remarkable tendency to undergo ra-
pid exchange with the solvent. Fortunately, the NHCOCH3 proton
also generates specific signals for each isomer and furnishes a bet-
ter evaluation set because neither superimposition with aromatic
protons nor considerable exchange occurs. On analysis of the split-
ting of the NH of acetamide, the signal at higher chemical shift val-
ues was attributed to the E-isomers, while, in the case of the Z-
isomers the NH proton has a lower deshielding effect. Likewise,
signals associated with the aromatic ring could be of value in
determining this diastereomeric relationship, though in some
cases superimposition of signals led to quantitative limitations.
The E/Z-ratio for the 4a–h series behaved differently, since the
Z-isomers constituted 9–11% of the diastereomeric mixture, de-
spite the fact that no special effort had been made to isolate them.
Tautomerism may be involved during the formation of Z-isomer,
given that it is theoretically feasible that the hydroxyl group may
a
Determined by HRMS.
Calculated from purified products.
Determined by the relative integration of the NHCOCH3 (3a–h) or NH (4a–h)
b
c
signals (1H NMR, 200 MHz) for each isomer.
d
Using the ALOGPS 2.1 database.33
to evaluate the binding patterns of compounds 3a–h and 4a–h at
the TCC active site in theoretical docking terms, and compare these
results with ligand ‘186’ and also with the trypanocidal reference
drug, Bdz.
3. Pharmacology
3.1. Assessment of cytotoxicity and antitrypanosomal
properties
The in vitro cytotoxicity of all compounds was evaluated in cul-
tures of mouse splenocytes, and expressed as the highest non-cyto-
toxic concentration for spleen cells. The compounds were then
tested in vitro against epimastigote (proliferative) and trypomasti-
gote (bloodstream) forms of T. cruzi (Y strain). Anti-T. cruzi proper-
ties were expressed in terms of the IC50 (lM) values, calculated

J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6685
play a part in tautomeric equilibrium in solution via a proton trans-
fer. This kind of tautomerism is consistent with the lability and free
rotation required to form the Z-isomer from the E-isomer, although
additional experimental data are not available at present. The sug-
gested tautomeric conversion is depicted in Scheme 2. In addition
to the pair of signals associated with the NH of the acyl-hydrazone
group and to the split aromatic protons characterizing the E/Z-iso-
mers, the series of compounds, 4a–h, exhibited two different sig-
nals attributed to the hydroxyl group at around d = 10.08 ppm (E-
isomer) and at d = 9.99 ppm (Z-isomer), reinforcing the qualitative
analysis.
Once their structure had been elucidated, all compounds were
tested as antitrypanosomal agents and the results of this are shown
in Table 2. The 3a–h series shares an acetamide group, while the
molecules of the 4a–h series bear a hydroxyl group. Such groups
were selected because of the chemical reactivity of hydroxyl and
the neutral nature of the acetamide. In medicinal chemistry, the
acetamide is considered to be the simplest model for a peptide
fragment. Besides, both chemical groups are endowed with the
capacity to form intermolecular hydrogen bonds with biological
targets. It was observed that such groups generated two distinct
sets of antitrypanosomal agents, and the SARs will first be dis-
cussed separately for each series.
methyl by other substituents (compounds 3c–3h), these inhibitory
properties were lost.
For the 4a–h series of NAH, it was initially observed that,
although of moderate potency against the trypomastigote, the
non-substituted derivative 4a was inactive against the epimasti-
gote form. The replacement of an hydrogen (4a) by methyl (4b)
or methoxy (4g) at the para-position improved potency against
both forms of the parasite, but a concomitant loss of selectivity
was observed in 4b. The potency of methyl (4b) and fluorine (4c)
derivatives was also compared, since methyl and fluorine are of
similar size. The fluorine was twice as potent as the methyl deriv-
ative against epimastigote, although the two were equipotent
against the trypomastigote form.
Analysis of the NAH derivatives bearing halogen substituents
gave rise to the observation of interesting SAR. Compared with
the non-halogenated parent, 4a, each one of the para-halogenated
analogues exhibited an increase in the antitrypanosomal activity,
the order of potency being: 4c (F) > 4e (Br) = 4d (Cl). In fact, these
differences in potency are consistent with the molecular modifica-
tion performed, because the influence of halogen substituents on
biomolecular interactions does not occur because of steric differ-
ences alone, but also as a result of modification of thermodynamic
parameters.32 Apart from the bromine, 4e, which was capable of
inhibiting the parasite at non-cytotoxic concentrations against
mammalian counterparts, the chlorine, 4d, and fluorine, 4c, deriv-
atives were cytotoxic for the mammalian cells.
Analysis of the NAH belonging to the 3a–h series showed that
the non-substituted derivative 3a was very active against epimas-
tigote and trypomastigote forms of T. cruzi at non-cytotoxic con-
centrations. On comparing the inhibitory activity of 3a and the
analogue methyl substituted 3b, the latter proved to be more ac-
tive and to have a potency similar to that of Bdz, although of higher
cytotoxicity among the 3a–h series. By contrast, on replacement of
The nitro derivative (4f) was as potent as the halogenated deriv-
atives, but it proved cytotoxic in mammals. The hydroxyl deriva-
tive (4h) exhibited the weakest antitrypanosomal activity of the
entire series. In fact, the 4h also exhibited the lowest c log P value
OH
O
O
O
O
H
O
N
O
O
N
H
N
N
N
N
N
N
N
H
H
H
N
H
N
N
H
Ar
Ar
Ar
E-isomer
Z-isomer
OH
Scheme 2. Proposal of the tautomeric forms in solution, resulting the formation of Z-isomers.
Table 2
In vitro anti-Trypanosoma cruzi activity of NAH derivatives against Y strain
Compd.
R
Trypomastigotes IC50 in
l
Ma
Epimastigotes IC50 in
l
Ma
Cytotoxicityb
(lg/mL)
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
H
CH3
F
Cl
Br
NO2
OCH3
OH
H
CH3
F
Cl
Br
NO2
OCH3
OH
—
3.6
3.9
Nd.
Nd.
Nd.
Nd.
Nd.
Nd.
35.7
17.9
16.7
21.2
20.5
21.3
32.5
100.3
5.0
14.2
9.8
33 (95)
11 (30)
33
100
33
33
100
33
3.3
<1.1
<1.1
<1.1
33 (97)
<1.1
33 (85)
100
>150
>150
>150
>150
>150
>150
78.3
47.8
21.0
21.6
19.6
25.2
13.8
126.6
6.6
4g
4h
Bdzc
GVc
100
<1.0
—
2.1
—
a
Calculated from seven concentrations using data obtained from at least three independent experiments (SD less than 10% in all cases). Nd., not determined, due to the lack
of activity against epimastigotes.
b
Expressed as the highest concentration tested non-cytotoxic for mouse splenocytes. Values in lM are shown in parentheses.
Bdz is Benznidazole and GV is Gencian Violet.
c

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J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
Table 3
of the 4a–h series (Table 1). One plausible explanation for this
weak activity is the lipophilic effect, because low lipophilicity in of-
ten associated with problems regarding the permeability of the cell
membrane.
Docking results for compounds 3a–h and 4a–h
Compd
R
Docking scorea
Trypomastigotes
b
c
IC50
(l
M)
pIC50
In the light of these results, the structures of NAH 3a–h and 4a–
h (E-isomers) were subjected to docking analysis of the TCC struc-
ture, in an effort to gain information on the molecular origin of the
antitrypanosomal properties of these NHA derivatives. The GOLD
program was used to identify favourable conformations and possi-
ble interaction regions at the active site of the enzyme. All the top-
ranked conformations of 3a–h, 4a–h and Bdz were orientated in
such a way as to insert themselves into the same pocket of the
TCC structure, and their docked locations on the active site were
very close to that occupied by the co-crystallized ligand ‘186’. Fig-
ure 2 shows a superimposition of these docking solutions along-
side the docking of the ligand ‘186’ that was co-crystallized with
TCC.
In the light of this interesting finding, we decided to compare
in vitro and in silico results in order to gain an understanding of
the SARs of this family of NAH. This comparison appears to be rel-
evant, given that the 3a–h and 4a–h NAH represent two congener
series that share structural similarities and whose in vitro data for
IC50 represent a quantitative parameter. Given this, the GOLD score
was plotted against the pIC50 data (determined from IC50 values
against trypomastigotes). The results are shown in Table 3. In addi-
tion, for a better illustration of this comparison, some of the results
in Table 3 were converted into Figure 3, although this illustration
does not constitute a statistical correlation, but a comparison of
in vitro and in silico results.
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
H
CH3
F
Cl
Br
NO2
OCH3
OH
H
CH3
F
Cl
Br
NO2
OCH3
OH
—
45.08
46.62
44.41
46.17
46.68
45.22
45.95
43.45
42.92
44.30
42.09
44.10
46.32
45.91
45.18
43.82
43.83
3.6
3.9
Nd
Nd
Nd
Nd
Nd
Nd
35.7
17.9
16.7
21.2
20.5
21.3
32.5
5.44
5.41
—
—
—
—
—
—
4.45
4.75
4.78
4.67
4.69
4.67
4.49
4.00
5.30
4g
4h
Bdz
100.3
5.0
a
The GOLD docking scores are dimensionless.
See corresponding footnotes in Table 2.
pIC50 = 6 ꢀ (log IC50) of Y strain trypomastigotes, when IC50 values are give in
b
c
lM.
Analysis of this comparison suggests that, with the exception of
a few outliers, the NAH derivatives with more stable GOLD scores on
TCC were also the more potent in vitro antitrypanosomal agents.
For example, Figure 3 shows that the 4e and 4f NAH, which are
the most active of the 4a–h series, also present the most positive
scores, or conversely, possess higher (theoretical) affinity for the
TCC structure.
In an attempt to explain the differences between the docking
scores, an analysis of the polar interactions (hydrogen bonds) for
the docked ligands on the active site of TCC structure are summa-
rized in Table 4, along with the distances between the donor and
acceptor atoms involved in this interaction by way of ligand–en-
zyme complexation. Additionally, details were provided of the
docking positions for a number of NAH derivatives, along with
the position of the crystallographic ligand ‘186’ (Figs. 4–6). Docking
positions were captured using the PYMOL v 0.99X program.30,31
Figure 3. Comparison between the GOLD docking score and the pIC50 values
(trypomastigote, Y strain) for the series 4a–h.
Figure 2. Superimposition of the highest ranked solution of N-acylhydrazones (stick model) in the binding site of TCC (ribbons model), as calculated by GOLD v. 4.0. Left: 3a–h
and Bdz; right: 4a–h and Bdz, alongside the co-crystallized ligand ‘186’ (wireframe model).

J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6687
Table 4
Summary of the hydrophilic (H-phil) intermolecular interactions to the NAH in comparison with the co-crystallized ligand ‘186’
Ligands
Cruzain residues and distance of bondsa
Gln19
Glu205
Gly66
Asp158
Asn69
Cys25
Met68
‘186’
Bdz
3ª
3b
3c
3d
3e
3f
3g
3h
4ª
4b
4c
4d
4e
4f
2.84
2.50–3.47
—
—
—
—
—
—
—
—
—
—
2.80
—
1.66
2.72
1.83
1.73
—
3.05
—
—
—
—
—
—
—
—
3.12
—
—
1.76
—
2.48
1.91
2.86
2.88
2.93
—
—
—
—
—
—
—
—
—
—
—
3.44
—
—
—
—
—
2.99
—
2.97
2.61
2.73
3.11
2.78
—
—
—
—
—
—
—
—
2.58–3.37
3.42
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.56
2.50
3.48
3.42
—
—
2.95
—
—
2.91
2.70
3.08
3.23
3.29
3.39
2.81
2.86
3.56
—
—
4g
4h
1.83
3.36
a
All values are given in angstroms.
Figure 4. Docking pose of compound 3e (stick model) alongside the co-crystallized
ligand ‘186’ (wireframe model) in the TCC binding pocket. White line represents the
hydrogen bonding. Only the key residues are shown for clarity.
Figure 6. Docked pose of compounds 4g (red) and 4h (blue) alongside the co-
crystallized ligand ‘186’ (green, wireframe model) in the TCC binding pocket.
of the TCC, while that of the phenyl adjacent to the 1,2,4-oxadiaz-
ole ring is accommodated on a small hydrophobic pocket, located
close to the Gln19 residue. It is worth pointing out that the mole-
cules of the 4a–h series establish a hydrogen bond (H-bond) with
the Asp158 by way of the carbonyl group of the N-acylhydrazone
subunit. On the other hand, interactions with the Asp158 residue
for the 3a–h series predominantly involve the nitrogen atom of
the N-acylhydrazone subunit. Particularly noticeable are the inter-
actions of the acetamide group (3a–h series) with the Met68 resi-
due, while the hydroxyl group (4a–h series) is generally found to
interact with the Glu205 residue. Furthermore, the pocket that
the 4a–h series occupies is hydrophobic in the vicinity of Gly66
and bounded by an electronegative region marked by Asp158
and Glu205. This provides one initial explanation for the difference
in antitrypanosomal activity observed within the NAH series.
With regard to the SAR within 3e and 4e, the docking analyses
provided a number of explanations. The 3e derivative, which pos-
sesses a bromo substituent, was the NAH of those tested which
presented the most stable GOLD score, but was inactive against
the parasite. By contrast, another bromo analogue (4e) also pre-
sented a stable GOLD score, but in this case, it was much more po-
tent in inhibiting the cell culture of parasites than 3e. In fact,
analysis of the TCC residues predicted by docking studies reveals
Figure 5. Docked pose of compound 4e (stick model) alongside the co-crystallized
ligand ‘186’ (wireframe model) in the TCC binding pocket.
The ‘186’ inhibitor establishes hydrogen bonds with the key res-
idues, Gln19, Gly66 and Asp158, with respective measurements of
2.84, 3.05 and 3.12 Å. Modeling indicated that the N-acylhydrazone
subunit is accommodated on hydrophilic pocket of the active site

6688
J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
that these two compounds have slight differences in terms of dock-
ing interactions with the enzyme. Figure 4 shows the NAH deriva-
tive 3e establishing H-bonds with the Asp158, Asn69 and Met68
residues, with distances of 1.91, 2.56 and 2.34 Å respectively, while
Figure 5 shows that the NAH derivative, 4e, forms H-bonds with
Glu205, Gly66, and Asn69 of 1.83, 3.29, and 3.56 Å in length,
respectively. Likewise, Figure 4 demonstrates that Asp158 is in-
volved in the bidentate H-bond with the oxygen atom on the
1,2,4-oxadiazole ring and with the nitrogen (NH) in the amide
group for derivative 3e. This kind of interaction with the Asp158
residue is a singular observation and was not seen in other NAH
derivatives in the course of this study. Therefore, despite the excel-
lent GOLD score value, this specific interaction predicted for deriva-
tive 3e apparently did not contribute to efficient interaction with
the target.
with chemical shifts d reported in ppm unities relative to the inter-
nal standard TMS, using DMSO-d6 as solvent. Mass spectra were
obtained by using the Finnigan mass spectrometers, model MAT
8200 for low-resolution mass spectrometry (MS) and the MAT 95
for high resolution mass spectrometry (HRMS). Values for HRMS
lie within the permitted limit intervals with resolution of 10000.
The reactions were monitored by thin-layer chromatography
(TLC) performed on silica gel plates prepared with Silica Gel 60
(PF-245 with gypsum, Merck) of a thickness of 0.25 mm. The devel-
oped chromatograms were visualized under ultraviolet light at
254–265 nm. For column chromatography, Merck Silica Gel 60
(230–400 mesh) was used. All common laboratory chemicals were
purchased from commercial sources and used without further
purification. Compounds 1a–h and 2a–h were prepared according
to literature procedures.22
Examination of the TCC complex with the 4g and 4h NAH
showed that neither the hydroxyl (4h) nor the methoxy (4g) group
establishes an H-bond with the enzyme (Figure 6), although 4g re-
sults in a more stable complex (GOLD score) with the TCC than 4h. In
these cases, it is reasonable to suggest that the methoxy group
gives rise to steric change, causing a favourable reorientation of
the TCC pocket, which partly explains why 4g is more potent than
compound 4h.
The final consideration regarding the in silico studies concerns
the manner of inhibition of the TCC enzyme, that is, by covalent
or non-covalent binding. Analysis of the general structures of the
NAH reveals, from a theoretical point of view, that this subunit
possesses sufficient reactivity to establish covalent interactions
with protease, mainly when considering the resonance structures
or tautomers, as previously highlighted.14 Finally, given the dem-
onstrated potency of compounds 3a and 4e, it was decided to per-
form an acute toxicity assay in mice, using a single administration
(ip) of 100 mg/kg. This experiment showed that the tested com-
pounds (3a and 4e) are non-lethal in mice, although the mice pre-
sented few behavioral signs after administration, and suggests that
these are suitable for further in vivo studies of T. cruzi infection.
6.2. General procedure for preparation of compounds 3a–h and
4a–h
To a stirred suspension of 0.01 mol of appropriate hydrazide in
5 mL of ethanol were added 3–4 drops of concentrated sulfuric
acid, when the suspension changes into a clear solution. Then,
the respective aromatic aldehyde (0.015 mol) previously dissolved
in 5 mL of ethanol was added to the mixture at room temperature.
After few seconds, a colored solid fell down and the mixture was
stirred for 10 min prior to addition of 10 mL of water. After vacuum
filtration, washing with cold water/ethanol 1:1 and then with cold
water gave the desired compounds. Recrystallization from diox-
ane/water (1:1, v/v) mixture afforded the powdered final products.
The yields, melting points, spectroscopic and spectrometric data
are listed below for each compound.
6.3. 3-Phenyl-N0-[(4-acetamidophenyl)methylene]-1,2,4-
oxadiazole-5-carbohydrazide (3a)
Mp 273–5 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3413 (N–
H, acetamido), 3326 (N–H, acyl-hydrazone), 3037 (C–H, imine),
1708 (C@O, acetamido), 1671 (C@O, acyl-hydrazone), 1607 (N@C,
imine), 1533 (N@C, heterocyclic); 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.55 (s, 1H, N@CH),
8.10 (dd, J = ꢁ4.0 Hz, 2H, ortho-oxadiazole ArH), 7.69 (s, 4H, ArH),
7.64–7.61 (m, 3H, meta/para-oxadiazole ArH), 2.07 (s, 3H,
NHCOCH3). MS (m/z, %): 349 (M+Å, 100), 204 (8), 147 (39), 118
(92), 103 (36), 43 (34), HRMS, Calcd (Found) for C18H15N5O3:
349.1174 (349.1171).
5. Conclusion
In conclusion, initial investigation of the medicinal chemistry of
anti-T. cruzi agents resulted in the establishment of a set of SARs
and the identification of new bioactive compounds. Among these,
NAH 3a and 4e are especially worthy of note, as they proved to
be potent antitrypanosomal agents and of low toxicity both
in vitro and in vivo, assuming the position of lead-compounds.
Notable chemical features of these two lead-compounds are their
non-nitrated, non-peptidic and achiral natures, since such proper-
ties are considered as important criteria for the development of
antitrypanosomal drug candidates.7 The docking patterns of the
NAH series were similar to those of the ‘186’ co-crystallized inhib-
itor, and display polar interactions with key residues of the active
TCC site, such as Gly66, Asp158 and Asn69. The understanding of
the manner of docking of our N-acylhydrazones on TCC was also
consistent with the SARs observed, thereby providing a useful plat-
form for a future structure-guided design of analogues with opti-
mized potency.
6.4. 3-(4-Methylphenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (3b)
Mp 286–8 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3420 (N–
H, acetamido), 3317 (N–H, acyl-hydrazone), 3029 (C–H, imine),
1706 (C@O, acetamido), 1670 (C@O, acyl-hydrazone), 1609 (N@C,
imine), 1532 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, CONH), 10.1 (s, 1H, NHCOCH3), 8.55 (s, 1H, N@CH),
7.99 (d, J = 7.9 Hz, 2H, ortho-oxadiazole ArH), 7.69 (s, 4H, ArH),
7.42 (d, J = 7.8 Hz, 2H, meta-oxadiazole ArH), 2.40 (s, 3H, CH3),
2.07 (s, 3H, NHCOCH3). MS (m/z, %): 363 (M+Å, 72), 203 (25), 147
(44), 118 (100), 103 (30), 43 (48); HRMS, Calcd (Found) for
C19H17N5O3: 363.1331 (363.1328).
6. Experimental protocols
6.1. Chemistry
6.5. 3-(4-Fluorophenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (3c)
Melting points were determined on a Gallenkamp capillary
apparatus and are uncorrected. Infrared spectra were recorded
using KBr discs on a Perkin–Elmer Paragon 500 FT-IR spectrometer.
1H NMR spectra were recorded on a Bruker DPX-200 spectrometer,
Mp 287–9 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
H, acetamido), 3341 (N–H, acyl-hydrazone), 3029 (C–H, imine),
1710 (CÅO, acetamido), 1672 (CÅO, acyl-hydrazone), 1608 (NÅC,
m = 3424 (N–

J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6689
imine), 1540 (NÅC, heterocyclic). 1H NMR (200Mhz, DMSO-d6) d:
6.10. 3-(4-Hydroxyphenyl)-N0-[(4-acetamidophenyl)meth-
ylene]-1,2,4-oxadiazole-5-carbohydrazide (3h)
12.7 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.54 (s, 1H, NÅCH),
8.15 (t, J = 7.0 Hz, 2H, ortho-oxadiazole ArH), 7.69 (s, 4H, ArH),
7.47 (t, J = ꢁ8.7 Hz, 2H, meta-oxadiazole ArH), 2.06 (s, 3H,
NHCOCH3). MS (m/z, %): 367 (M+Å, 100), 204 (5), 147 (26), 118
(52), 105 (23), 43 (19); HRMS, Calcd (Found) for C18H14N5O3F:
367.1080 (367.1083).
Mp 279–281 °C (from dioxane/H2O); IR (KBr, cmꢀ1
) m = 3550–
3200 (O–H), 3430 (N–H, acetamido), 3337 (N–H, acyl-hydrazone),
1706 (C@O, acetamido), 1667 (C@O, acyl-hydrazone), 1610 (N@C,
imine), 1528 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, CONH), 10.3 (s, 1H, OH), 10.2 (s, 1H, NHCOCH3),
8.55 (s, 1H, N@CH), 7.94 (d, J = 8.8 Hz, 2H, ortho-oxadiazole ArH),
7.70 (s, 4H, ArH), 6.97 (d, J = 8.8 Hz, 2H, meta-oxadiazole ArH),
2.08 (s, 3H, NHCOCH3). MS (m/z, %): 365 (M+Å, 100), 204 (7), 147
(29), 118 (45), 105 (23), 43 (19); HRMS, Calcd (Found) for
C18H15N5O4: 365.1124 (365.1123).
6.6. 3-(4-Chlorophenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (3d)
Mp 292–4 °C (from dioxane/H2O); IR (KBr, cmꢀ1
) m = 3440 (N–H,
acetamido), 3346 (N–H, acyl-hydrazone), 3033 (C–H, imine), 1703
(C@O, acetamido), 1670 (C@O, acyl-hydrazone), 1608 (N@C,
imine), 1534 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.8 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.54 (s, 1H, N@CH),
8.10 (d, J = 8.5 Hz, 2H, ortho-oxadiazole ArH), 7.70 (d, J = 6.7 Hz,
2H, meta-oxadiazole ArH), 7.69 (s, 4H, ArH), 2.06 (s, 3H, NHCOCH3).
MS (m/z, %): 383/385 (M+Å, 100/34), 203 (7), 147 (31), 118 (69), 105
(28), 43 (22); HRMS, Calcd (Found) for C18H14N5O3Cl: 383.0785
(383.0791).
6.11. 3-Phenyl-N0-[(4-hydroxyphenyl)methylene]-1,2,4-
oxadiazole-5-carbohydrazide (4a)
98%; mp 240–1 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3486
(O–H, br), 3248 (N–H), 3035 (C–H, imine), 1682 (C@O), 1607 (N@C,
imine), 1587 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6) d:
12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.51 (s, 1H, N@CH), 8.09 (dd,
J = ꢁ7.0 Hz, 2H, ortho-oxadiazole ArH), 7.64–7.57 (m, 5H, ArH),
6.85 (d, J = 8.5 Hz, 2H, ortho-OH ArH). MS (m/z, %): 308 (M+Å, 53),
189 (22), 145 (15), 119 (100), 106 (58), 77 (41); HRMS, Calcd
(Found) for C16H12N4O3: 308.0909 (308.0913).
6.7. 3-(4-Bromophenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide(3e)
Mp >300 °C (from dioxane/H2O); IR (KBr, cmꢀ1
) m = 3445 (N–H,
6.12. 3-(4-Methylphenyl)-N0-[(4-hydroxyphenyl)methylene]-
acetamido), 3341 (N–H, acyl-hydrazone), 3030 (C–H, imine), 1708
(C@O, acetamido), 1670 (C@O, acyl-hydrazone), 1608 (N@C,
imine), 1539 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.54 (s, 1H, N@CH),
8.02 (d, J = 8.5 Hz, 2H, ortho-oxadiazole ArH), 7.68 (d, J = 8.5 Hz,
2H, meta-oxadiazole ArH), 7.69 (s, 4H, ArH), 2.06 (s, 3H, NHCOCH3).
MS (m/z, %): 427/429 (M+Å, 93/92), 203 (13), 147 (45), 118 (100),
105 (40), 43 (34); HRMS, Calcd (Found) for C18H14N5O3Br:
427.0280 (427.0279).
1,2,4-oxadiazole-5-carbohydrazide (4b)
Mp 235–7 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3441 (O–
H, br), 3320 (N–H), 3030 (C–H, imine), 1673 (C@O), 1604 (N@C,
imine), 1547 (N@C, heterocyclic); 1H NMR (200 MHz, DMSO-d6)
d: 12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH), 7.98 (d,
J = 8.2 Hz, 2H, ortho-oxadiazole ArH), 7.59 (d, J = 8.5 Hz, 2H, meta-
OH ArH), 7,42 (d, J = 8.2 Hz, 2H, meta-oxadiazole ArH), 6.85 (d,
J = 8.7 Hz, 2H, ortho-OH ArH), 2.40 (s, 3H, CH3). MS (m/z, %): 322
(M+Å, 92), 203 (100), 159 (18), 119 (64), 106 (59), 77 (20); HRMS,
Calcd (Found) for C17H14N4O3: 322.1065 (322.1063).
6.8. 3-(4-Nitrophenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (3f)
6.13. 3-(4-Fluorophenyl)-N0-[(4-hydroxyphenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (4c)
Mp >300 °C (from dioxane/H2O); IR (KBr, cmꢀ1
) m = 3420 (N–H,
acetamido), 3350 (N–H, acyl-hydrazone), 3045 (C–H, imine), 1708
(C@O, acetamido), 1667 (C@O, acyl-hydrazone), 1606 (N@C,
imine), 1525 (N@C, heterocyclic); 1H NMR (200 MHz, DMSO-d6)
d: 12.8 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.55 (s, 1H, N@CH),
8.47 (d, J = 9.0 Hz, 2H, ortho-oxadiazole ArH), 8.35 (d, J = 9.0 Hz,
2H, meta-oxadiazole ArH), 7.69 (s, 4H, ArH), 2.06 (s, 3H, NHCOCH3).
MS (m/z, %): 394 (M+Å, 100), 204 (12), 147 (23), 118 (77), 105 (32),
43 (38); HRMS, Calcd (Found) for C18H14N6O5: 394.1025
(394.1032).
Mp 260–2 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3362 (O–
H, br), 3310 (N–H), 3030 (C–H, imine), 1690 (C@O), 1608 (N@C,
imine), 1562 (N@C, heterocyclic); 1H NMR (200 MHz, DMSO-d6)
d: 12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH), 8.14 (dd,
J = ꢁ8.8 Hz, 2H, ortho-oxadiazole ArH), 7.59 (d, J = 8.7 Hz, 2H,
meta-OH ArH), 7,47 (t, J = 8.8 Hz, 2H, meta-oxadiazole ArH), 6.85
(d, J = 8.5 Hz, 2H, ortho-OH ArH). MS (m/z, %): 326 (M+Å, 100), 207
(39), 163 (18), 119 (79), 106 (41), 77 (11); HRMS, Calcd (Found)
for C16H11N4O3F: 326.0815 (326.0804).
6.9. 3-(4-Methoxyphenyl)-N0-[(4-acetamidophenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (3g)
6.14. 3-(4-Chlorophenyl)-N0-[(4-hydroxyphenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (4d)
Mp 294–6 °C (from dioxane/H2O); IR (KBr, cmꢀ1
) m = 3424 (N–H,
Mp 272–4 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3442 (O–
acetamido), 3341 (N–H, acyl-hydrazone), 3044 (C–H, imine), 1703
(C@O, acetamido), 1667 (C@O, acyl-hydrazone), 1610 (N@C,
imine), 1538 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, CONH), 10.2 (s, 1H, NHCOCH3), 8.54 (s, 1H, N@CH),
8.03 (d, J = 8.9 Hz, 2H, ortho-oxadiazole ArH), 7.69 (s, 4H, ArH),
7.16 (d, J = 9.0 Hz, 2H, meta-oxadiazole ArH), 3.85 (s, 3H, OCH3),
2.07 (s, 3H, NHCOCH3). MS (m/z, %): 379 (M+Å, 100), 204 (9), 147
(35), 118 (36), 105 (20), 43 (16); HRMS, Calcd (Found) for
C19H17N5O4: 379.1280 (379.1286).
H, br), 3307 (N–H), 3029 (C–H, imine), 1698 (C@O), 1604 (N@C,
imine), 1583 (N@C, heterocyclic); 1H NMR (200 MHz, DMSO-d6)
d: 12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH), 8.10 (d,
J = 8.7 Hz, 2H, ortho-oxadiazole ArH), 7.70 (d, J = 8.7 Hz, 2H, meta-
OH ArH), 7,59 (d, J = 8.7 Hz, 2H, meta-oxadiazole ArH), 6.85 (d,
J = 8.5 Hz, 2H, ortho-OH ArH). MS (m/z, %): 342/344 (M+Å, 37/15),
223/225 (43/13), 179 (8), 119 (100), 106 (68), 77 (21); HRMS, Calcd
(Found) for C16H11N4O3Cl: 342.0519 (342.0511).

6690
J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6.15. 3-(4-Bromophenyl)-N0-[(4-hydroxyphenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (4e)
ration using a beta-radiation counter (Multilabel Reader, Hidex,
Turku, Finland). The cytotoxicity of the compounds was deter-
mined by comparing the percentage of 3H-thymidine incorporation
(as indicator of viability cell) of drug-treated wells in relation to
untreated wells. Non-cytotoxic concentrations were defined as
those causing a reduction of 3H-thymidine incorporation below
10% in relation to untreated controls.
Mp 278–280 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3447
(O–H, br), 3307 (N–H), 3028 (C–H, imine), 1698 (C@O), 1605
(N@C, imine), 1582 (N@C, heterocyclic); 1H NMR (200 MHz,
DMSO-d6) d: 12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH),
8.02 (d, J = 8.7 Hz, 2H, ortho-oxadiazole ArH), 7.84 (d, J = 8.7 Hz, 2H,
meta-OH ArH), 7,59 (d, J = 8.7 Hz, 2H, meta-oxadiazole ArH), 6.85
(d, J = 8.5 Hz, 2H, ortho-OH ArH). MS (m/z, %): 386/388 (M+Å, 52/
52), 267/269 (44/44), 223/225 (6/7), 119 (100), 106 (51), 77 (14);
HRMS, Calcd (Found) for C16H11N4O3Br: 386.0014 (386.0005).
6.20. In vitro antiproliferative activity
Epimastigotes of T. cruzi (Y strain)34 were cultivated at 26 °C in
liver infusion tryptose medium (LIT) supplemented with 10% fetal
calf serum, 1% hemin, 1% R9 medium and 50 l
g mLꢀ1 gentamycin.
6.16. 3-(4-Nitrophenyl)-N0-[(4-hydroxyphenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (4f)
Parasites (106 cells mLꢀ1) were cultured in a fresh medium in the
absence or in the presence of the compounds being tested (from
stock solution in DMSO). Cell growth was determined after 11 days
of culture by counting viable forms in a hemacytometer, in tripli-
cate. The compounds were used from a stock solution in DMSO.
To determine the IC50, cultures of Y strain epimastigotes in the
presence of different concentrations (0.1, 1.0, 5, 10, 25, 50,
Mp > 300 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3452 (O–H,
br), 3364 (N–H), 3069 (C–H, imine), 1695 (C@O), 1606 (N@C,
imine), 1587 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.7 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH), 8.46 (d,
J = 9.0 Hz, 2H, ortho-oxadiazole ArH), 8.34 (d, J = 9.2 Hz, 2H, meta-
oxadiazole ArH), 7.59 (d, J = 8.7 Hz, 2H, meta-OH ArH), 6.85 (d,
J = 8.5 Hz, 2H, ortho-OH ArH). MS (m/z, %): 353 (M+Å, 74), 234 (2),
190 (4), 119 (100), 106 (36), 77 (7); HRMS, Calcd (Found) for
C16H11N5O5: 353.0760 (353.0767).
100 l
g mLꢀ1) of the compounds were evaluated after 11 days as
described above. IC50 calculation was carried out using non-linear
regression on Prism 4.0 GraphPad software. Y strain T. cruzi try-
pomastigotes were obtained from culture supernatants of Vero cell
line at 37 °C and placed in 96-well plates (4 ꢂ 105 wellꢀ1) in DMEM
medium supplemented with 10% FCS and 50 l
g mLꢀ1 gentamycin.
6.17. 3-(4-Methoxyphenyl)-N0-[(4-hydroxyphenyl)methylene]-
1,2,4-oxadiazole-5-carbohydrazide (4g)
Viable parasites were counted in a hemacytometer 24 h after addi-
tion of compounds by way of trypan blue exclusion. The percent-
age of inhibition was calculated in relation to untreated cultures.
The same procedures were performed for Bdz (reference drug)
and GV (reference for trypomastigote assay) and vehicle alone,
DMSO as blank.
Mp 219–221 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3444
(O–H, br), 3340 (N–H), 3010 (C–H, imine), 1681 (C@O), 1604
(N@C, imine), 1551 (N@C, heterocyclic). 1H NMR (200 MHz,
DMSO-d6) d: 12.6 (s, 1H, NH), 10.1 (s, 1H, OH), 8.50 (s, 1H, N@CH),
8.03 (d, J = 8.9 Hz, 2H, ortho-oxadiazole ArH), 7.59 (d, J = 8.7 Hz, 2H,
meta-OH ArH), 7,15 (d, J = 8.9 Hz, 2H, meta-oxadiazole ArH), 6.82
(d, J = 8.5 Hz, 2H, ortho-OH ArH), 3.85 (s, 3H, OCH3). MS (m/z, %):
338 (M+Å, 93), 219 (99), 175 (14), 148 (100), 133 (65), 119 (50),
106 (53), 77 (21); HRMS, Calcd (Found) for C17H14N4O4: 338.1015
(338.1008).
6.21. Toxicological tests in mice
The selected compounds were resuspended in 100 lL [30%
DMSO:70% H2O, v/v] per dose of 100 mg/kg weight, using 6 ani-
mals per group. The treated animals were monitored for signs of
general toxicity, during 72 h after the treatment. The survival of
drug-treated mice was evaluated and compared to untreated ani-
6.18. 3-(4-Hydroxyphenyl)-N0-[(4-hydroxyphenyl)methylene]-
mals, which received only 100 lL of solution [30% DMSO:70%
1,2,4-oxadiazole-5-carbohydrazide (4h)
H2O, v/v]. This experimental protocol was approved and super-
vised by the Ethics Committee of HEMOPE Hospital (Recife, PE,
Brazil).
Mp 263–5 °C (from dioxane/H2O); IR (KBr, cmꢀ1):
m = 3459 (O–
H, br), 3319 (N–H), 3020 (C–H, imine), 1677 (C@O), 1598 (N@C,
imine), 1556 (N@C, heterocyclic). 1H NMR (200 MHz, DMSO-d6)
d: 12.6 (s, 1H, NH), 10.3 (s, 1H, para-oxadiazole OH), 10.1 (s, 1H,
OH), 8.49 (s, 1H, N@CH), 7.92 (d, J = 8.7 Hz, 2H, ortho-oxadiazole
ArH), 7.59 (d, J = 8.7 Hz, 2H, meta-OH ArH), 6.95 (d, J = 8.8 Hz, 2H,
meta-oxadiazole ArH), 6.85 (d, J = 8.7 Hz, 2H, ortho-OH ArH). MS
(m/z, %): 324 (M+Å, 35), 205 (45), 161 (10), 134 (38), 119 (55), 106
(37), 77 (11), 28 (100); HRMS, Calcd (Found) for C16H12N4O4:
324.0858 (324.0859).
Acknowledgements
This work was supported by grants from the CNPq (# 479982/
2008-2), FACEPE (#APQ-0123-4.03/08), RENORBIO and INCT-CNPq.
D.R.M.M and M.B.P.S are supported by CNPq. We acknowledge the
Laboratory of Medicinal Theoretical Chemistry (UFPE) by the ac-
cess of software and HEMOPE Hospital for supplying mice for tox-
icological tests. Authors are also thankful to Mr. Thomas Dulks and
Mrs. Dorit Kamken (University of Bremen, Germany) for recording
the MS and HRMS of all compounds and José F. O. Costa (FIOCRUZ)
for his technical assistance in the cytotoxicity assay.
6.19. In vitro cytotoxicity
The cytotoxicity of the compounds was determined using BALB/
c mice splenocytes (5 ꢂ 106 cells wellꢀ1) cultured in 96-well plates
in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Chemical
Co., St. Louis, MO) supplemented with 10% of fetal calf serum
References and notes
1. Barrett, M. P.; Burchmore, R. J. S.; Stich, A.; Lazzari, J. O.; Frasch, A. C.; Cazzulo, J.
J.; Krishna, S. Lancet 2003, 362, 1469.
2. Coura, J. R. Mem. Inst. Oswaldo Cruz. 2007, 102, 113.
3. (a) Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19, 495; (b) Cerecetto, H.;
Gonzalez, M. Mini-Rev. Med. Chem. 2008, 8, 1355; (c) Azevedo, W. F., Jr.; Soares,
M. B. P. Curr. Drug Targets 2009, 11, 193; (d) Hunter, W. N. J. Biol. Chem. 2009,
284, 11749; (e) Brenk, R.; Schipani, A.; James, D.; Krasowski, A.; Gilbert, I. H.;
Frearson, J.; Wyatt, P. G. ChemMedChem 2008, 3, 435.
(FCS; Cultilab, Campinas, SP, Brazil) and 50
(Novafarma, Anápolis, GO, Brazil). Each compound was evaluated
in three concentrations (1.1, 3.3, 11, 33, 100
g mLꢀ1), in triplicate.
l
g mLꢀ1 of gentamycin
l
Cultures were incubated in the presence of 3H-thymidine (1
lCi
wellꢀ1) for 24 h at 37 °C and 5% CO2. After this period, the content
of the plate was harvested to determine the 3H-thymidine incorpo-
4. Caffrey, C. R.; Scory, S.; Steverding, D. Curr. Drug Targets 2000, 1, 155.

J. M. dos Santos Filho et al. / Bioorg. Med. Chem. 17 (2009) 6682–6691
6691
5. Siegel, R. L. K.; Comini, M. A. Biochim. Biophys. Acta 2008, 1780, 1236.
6. Neres, J.; Bryce, R. A.; Douglas, K. T. Drug Discovery Today 2008, 13, 110.
7. Moreira, D. R. M.; Leite, A. C. L.; Santos, R. R.; Soares, M. B. P. Curr. Drug Targets
2009, 10, 212.
19. For an excellent definition of privileged structures, see: Duarte, C. D.; Barreiro,
E. J.; Fraga, C. A. M. Mini-Rev. Med. Chem. 2007, 7, 1108.
20. (a) Cerecetto, H.; Maio, R. D.; González, M.; Risso, M.; Saenz, P.; Seoane, G.;
Denicola, A.; Peluffo, G.; Quijano, C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1945;
(b) Havens, C.; Bryant, N.; Asher, L.; Lamoreaux, L.; Perfetto, S.; Brendle, J.;
Werbovetz, K. Mol. Biochem. Parasitol. 2000, 110, 223; (c) Cottrell, D. M.; Capers,
J.; Salem, M. M.; DeLuca-Fradley, K.; Croft, S. L.; Werbovetz, K. A. Bioorg. Med.
Chem. 2004, 13, 2815; (d) Sayed, A. A.; Simeonov, A.; Thomas, C. J.; Inglese, J.;
Austin, C. P.; Williams, D. L. Nat. Med. 2008, 14, 407.
21. (a) Jakopin, Z.; Dolenc, M. S. Curr. Org. Chem. 2008, 12, 850; (b) Borg, S.;
Estenne-Bouthou, G.; Luthman, K.; Csoregh, I.; Hesselink, W.; Hacksell, U. J. Org.
Chem. 1995, 60, 3112; (c) Boeglin, D.; Cantel, S.; Heitz, A.; Martinez, J.;
Fehrentz, J. A. Org. Lett. 2003, 5, 4465.
22. Leite, L. F. C. C.; Ramos, M. N.; da Silva, J. B. P.; Miranda, A. L. P.; Fraga, C. A. M.;
Barreiro, E. J. Farmaco 1999, 54, 747.
23. Santos-Filho, J. M.; de Lima, J. G.; Leite, L. F. C. C.; Ximenes, E. A.; da Silva, J. B. P.;
Lima, P. C.; Pitta, I. R. Heterocycl. Commun. 2005, 11, 29.
24. (a) Bezerra-Netto, H. J. C.; Lacerda, D. I.; Miranda, A. L. P.; Alves, H. M.; Barreiro,
E. J.; Fraga, C. A. M. Bioorg. Med. Chem. 2006, 14, 7924; (b) Carvalho, S. A.; Lopes,
F. A. S.; Wardell, S. M. S. V.; Salomão, K.; Castro, S. L.; Romeiro, N. C.; Silva, E. F.;
Fraga, C. A. M. Bioorg. Med. Chem. 2008, 16, 413.
25. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J. Am. Chem. Soc.
1985, 107, 3902.
8. Cazullo, J. J. Curr. Top. Med. Chem. 2002, 2, 1257.
9. (a) Scharfstein, J.; Schmitz, V.; Morandi, V.; Capella, M. M.; Lima, A. P.; Morrot,
A.; Juliano, L.; Müller-Esterl, W. J. Exp. Med. 2000, 192, 1289; (b) Aparicio, I. M.;
Scharfstein, J.; Lima, A. P. C. A. Infect. Immun. 2004, 72, 5892.
10. Steverding, D.; Caffrey, C. R.; Sajid, M. Mini-Rev. Med. Chem. 2006, 6, 1025.
11. (a) Roush, W. R.; Gonzfilez, F. V.; McKerrow, J. H.; Hansell, E. Bioorg. Med. Chem.
Lett. 1998, 8, 2809; (b) Roush, R. W.; Cheng, J.; Reed, B. K.; Alvarez-Hernandez,
A.; Hansell, E.; McKerrow, J. H.; Engel, C. J. Bioorg. Med. Chem. Lett. 2001, 11,
2759; (c) Jaishankar, P.; Doyle, P. S.; Hansell, E.; McKerrow, J. H.; Zhao, D. M.;
Renslo, A. R. Bioorg. Med. Chem. Lett. 2008, 18, 624.
12. Li, R.; Chen, X.; Gong, B.; Selzer, P. M.; Li, S.; Davidson, E.; Kurzban, G.; Miller, R.
E.; Nuzum, E.; McKerrow, J. H.; Fletterick, R. J.; Gilmor, S. A.; Craik, C. S.; Kuntz,
I. D.; Cohen, F. E.; Kenyon, G. L. Bioorg. Med. Chem. 1996, 4, 1421.
13. (a) Troeberg, L.; Chen, X.; Cheng, M.; Kenyon, G. L.; Flaherty, T. M.; Hua, H.; Cohen,
F. E.; Springer, C.; Lonsdale-Eccles, J. D.; Morty, R. E.; McKerrow, J. H.; Coetzer, T.
H. T. Mol. Med. 2000, 6, 660; (b) Selzer, P. M.; Pingel, S.; Hsieh, I.; Ugele, B.; Chan, V.
J.; Engel, J. C.; Bogyo, M.; Russel, D. G.; Sakanari, J. A.; Mckerrow, J. H. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 11015; (c) Caffrey, C. R.; Schanz, M.; Nkemgu-Njinkeng,
J.; Brush, M.; Hansell, E.; Cohen, F. E.; Flahertly, T. M.; McKerrow, J. H.; Steverding,
D. Int. J. Antimicrob. Agents 2002, 19, 227; (d) Melnyk, P.; Leroux, V.; Sergheraert,
C.; Grellier, P. Bioorg. Med. Chem. Lett. 2006, 16, 31.
26. http://www.CACheSoftware.com.
27. Choe, Y.; Brinen, L. S.; Price, M. S.; Engel, J. C.; Lange, M.; Grisostomi, C.;
Weston, S. C.; Pallai, P. V.; Cheng, H.; Hardy, L. W.; Hartsough, D. S.; Mcmakin,
M.; Tilton, R. F.; Baldino, C. M.; Craik, C. S. Bioorg. Med. Chem. 2005, 13, 2141.
28. http://www.rcsb.org/pdb.
14. Ifa, D. R.; Rangel, C. R.; Alencastro, R. B.; Fraga, C. A. M.; Barreiro, E. J. J. Mol.
Struct. 2000, 505, 11.
15. Rodrigues, C. R.; Flaherty, T. F.; McKerrow, J. H.; Springer, C.; Cohen, F. E. Bioorg.
Med. Chem. Lett. 2002, 12, 1537.
29. http://www.ccdc.cam.ac.uk/products/gold_suite/.
16. Romeiro, N. C.; Aguirre, G.; Hernández, P.; González, M.; Cerecetto, H.; Aldana,
I.; Pérez-Silanes, S.; Monge, A.; Barreiro, E. J.; Lima, L. M. Bioorg. Med. Chem.
2009, 17, 641.
17. (a) Vera-Divaio, M. A. F.; Freitas, A. C.; Castro, H. C.; Albuquerque, S.; Cabral, L.
M.; Rodrigues, C. R.; Albuquerque, M. G.; Martins, R. C. A.; Henriques, M. G. O.;
Dias, L. R. S. Bioorg. Med. Chem. 2009, 17, 295; (b) Zanatta, N.; Amaral, S. S.;
Santos, J. M.; de Mello, D. L.; Fernandes, L. D. S.; Bonacorso, H. G.; Martins, M. A.
P.; Andricopulo, A. D.; Borchhardt, D. M. Bioorg. Med. Chem. 2008, 16, 10236.
18. (a) Fraga, C. A. M.; Barreiro, E. J. Curr. Med. Chem. 2006, 13, 167; (b) Rollas, S.;
Küçükgüzel, S. G. Molecules 2007, 12, 1910.
30. Nogueira, R. C.; Oliveira-Costa, J. F.; de Sá, M. S.; dos Santos, R. R.; Soares, M. B.
P. Curr. Drug Targets 2009, 10, 291.
31. DeLano, W. L. The PYMOL Molecular Graphics System, DeLano Scientific, San
Carlos, CA, USA, 2002, in http://www.pymol.org.
32. Memic, A.; Spaller, M. R. ChemBioChem 2008, 9, 2793.
33. ALOGPS 2.1 Database was Used Accessing the Virtual Computational Chemistry
Laboratory (VCCLABS); http://www.vcclab.org.
34. Leite, A. C. L.; Moreira, D. R. M.; Cardoso, M. V. O.; Hernandes, M. Z.; Pereira, V.
R. A.; Silva, R. O.; Kiperstok, A. C.; Lima, M. S.; Soares, M. B. P. ChemMedChem
2007, 2, 1339.