New trypanocidal hybrid compounds from the association of hydrazone moieties and benzofuroxan heterocycle

Article abstract of DOI:10.1016/j.bmc.2008.05.038

Hybrid compounds containing hydrazones and benzofuroxan pharmacophores were designed as potential Trypanosoma cruzi-enzyme inhibitors. The majority of the designed compounds was successfully synthesized and biologically evaluated displaying remarkable in

Full text of DOI:10.1016/j.bmc.2008.05.038

Bioorganic & Medicinal Chemistry 16 (2008) 6995–7004
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New trypanocidal hybrid compounds from the association of hydrazone
moieties and benzofuroxan heterocycle
Williams Porcal a, Paola Hernández a, Lucía Boiani a, Mariana Boiani a, Ana Ferreira b, Agustina Chidichimo c,
a,
Juan J. Cazzulo c, Claudio Olea-Azar d, Mercedes González a, , Hugo Cerecetto
*
*
a Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Igua 4225, 11400 Montevideo, Uruguay
b Cátedra de Inmunología, Facultad de Química-Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay
c Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de General San Martín-CONICET,
1650 San Martín, Buenos Aires, Argentina
d Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Hybrid compounds containing hydrazones and benzofuroxan pharmacophores were designed as poten-
tial Trypanosoma cruzi-enzyme inhibitors. The majority of the designed compounds was successfully syn-
thesized and biologically evaluated displaying remarkable in vitro activity against different strains of T.
cruzi. Unspecific cytotoxicity was evaluated using mouse macrophages, displaying isothiosemicarbazone
10 and thiosemicarbazone 12 selectivity indexes (macrophage/parasite) of 21 and 27, respectively. In
addition, the mode of anti-trypanosomal action of the derivatives was investigated. Some of these deriv-
atives were moderate inhibitors of cysteinyl active site enzymes of T. cruzi, cruzipain and trypanothione
reductase. ESR experiments using T. cruzi microsomal fraction suggest that the main mechanism of action
of the trypanocidal effects is the production of oxidative stress into the parasite.
Received 21 April 2008
Revised 16 May 2008
Accepted 16 May 2008
Available online 20 May 2008
Keywords:
Benzofuroxan
Trypanosoma cruzi
ESR
Ó 2008 Elsevier Ltd. All rights reserved.
Cruzipain
Trypanothione reductase
1. Introduction
DNA, lipids, and proteins of the parasite, both by bioreduction of
the nitro group during their metabolization.13,14 Non-selective bio-
Chagas‘ Disease (CD) is the third largest disease burden in Latin
America after malaria and schistosomiasis, all considered as the so-
called neglected diseases. CD is caused by Trypanosoma cruzi
(T. cruzi), a hemoflagellate protozoan (family Trypanosomatidae,
order Kinetoplastida).1,2 This parasitic disease represents a real
health public problem in South America, affecting at least 15 mil-
lion people with more than 25 million at the risk of infection.3
The absence of an efficient chemotherapeutic approach to treat
CD requires urgent attention.4 The only clinically available drugs
for the chemotherapy of CD are two nitroaromatic compounds, Nif-
urtimox (Nfx, LampitÒ, Fig. 1) and Benznidazole (Bnz, RochaganÒ,
Fig. 1).5,6 Both drugs have important disadvantages such as severe
side effects, strain resistance, and variable efficacy.7–11 For these
reasons the development of more safe and efficient drugs against
CD is urgent.12 The mode of action for these drugs is not fully
understood. However, it has been proposed in several studies that
Nfx could act by generation of free radical toxic species through a
redox-cycling process and Bnz by reaction with macromolecules as
reduction of these trypanocidal drugs could be the reason of its
toxic effects in the mammalian host. Other compounds have been
studied as anti-T. cruzi drugs, for example the commercially
available anti-fungal agents KetoconazoleÒ (Ktz, Fig. 1) and Terbi-
nafineÒ (Tbf, Fig. 1), both sterol-membrane biosynthesis inhibi-
tors.15,16 These anti-fungal agents have been shown to have
potent effects against cultures of T. cruzi.
The search for new compounds able to generate oxidative stress
in T. cruzi through selective reduction by oxidoreductases unique
in the parasite is an attractive target in the chemotherapy of
CD.17–21 In this sense, we investigated the capability of the N-oxide
moiety as trypanocidal pharmacophore having the hypothesis that
the N-oxide moiety would act as bioreducible group.22 In previous
works, we found that benzofuroxan (benzo[1,2-c]1,2,5-oxadiazole
N-oxide) derivatives displayed excellent in vitro and in vivo anti-
T. cruzi activities, 5-arylethenylbenzofuroxans and vinyl-
sulfonylbenzofuroxans (I and II, respectively, Scheme 1) being
the best ones.23,24 Studies undertaken by our group suggested
the perturbation of the mitochondrial electron chain, inhibiting
parasite respiration, and that the N-oxide moiety is essential for
the trypanocidal activity.25 A new target recently explored for
the chemotherapy of CD is cruzipain (CP).26 CP is the major
* Corresponding authors. Tel.: +5982 525 8618/216; fax: +5982 525 0749.
E-mail addresses: megonzal@fq.edu.uy (M. González), hcerecet@fq.edu.uy
(H. Cerecetto).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.038

6996
W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
O
N
N
N
O2N
N
N
O
H
SO2
NO2
Nfx
Tbf
Bnz
Cl
N
AcN
N
O
O
O
Cl
N
Ktz
N
Figure 1. Drugs used clinically and experimentally as anti-T. cruzi agents.
O
S
OH
R
N
NH
NH2
O
N
Ar
Me
O
H2N
N
R
N
N
N
H
N
N
Ar
H
H
-R= -Ar,
(I)
(IV)
(III)
(V)
-R= ArSO2-, (II)
-R= variable
Ar= aryl
molecular hybridization
OH
X
O
N
O
O
variable
variable
N
N
N
RHN
N
N
fragment
fragment
H
O
N
N
X= NH, O, S
R= variable
Series 1
Series 2
Scheme 1. Design of hybrid compounds planned from the association of hydrazone moiety and trypanocidal benzofuroxan system.
lysosomal proteinase of T. cruzi, which plays a prominent role in
nutrition of the parasite and in the host/parasite interplay.27,28 In
recent years, several families of compounds such as vinylsulfones,
thioureas, and hydrazone derivatives have been reported as inhib-
itors of CP with in vitro and in vivo trypanocidal activity.29–32 Par-
ticularly, thiosemicarbazone derivatives (III, Scheme 1) were
revealed as a privileged scaffold for the selective inhibition of this
enzyme showing good activity at non-cytotoxic concentrations in
in vitro assays with mammalian cells.33 Some N-acylhydrazones,
like salicylaldehyde N-acylhydrazones IV (Scheme 1), showed
inhibitory activity against the P. falciparum cysteine protease, falci-
pain, and CP.30
Another of the most studied biomolecules as key targets in
T. cruzi is trypanothione reductase (TR).34 All trypanosomatids
have a unique thiol metabolism in which the ubiquitous glutathi-
one reductase (GR: GSSG + NADPH + H+ rightarrow 2 GSH + NADP+)
is replaced by TR. TR is an NADPH-dependent flavoenzyme respon-
sible for maintaining the reducing intracellular milieu and thus
protecting the parasite against oxidative stressors. Trypanothi-
one—N1, N8-bis(glutathionyl)spermidine—is
a low molecular
weight thiol exclusively found in parasitic protozoa of the order
Kinetoplastida. The absence of trypanothione in the mammalian
host together with the sensitivity of trypanosomatids to oxidative
stress renders the enzymes of this parasite-specific thiol metabo-
lism attractive as drug target molecules. A large number of TR
inhibitors have been studied, identifying some that bind to TR
reversibly, some that act as subversive substrates, and also some
that are irreversible inhibitors. The first subversive substrates of
TR described were amidinohydrazone containing nitrofuranes
and naphthoquinones like V (Scheme 1) synthesized by Hender-
son.35 Subversive substrates are reduced by the flavoenzyme in a
single-electron step to the respective radical which then reacts
with molecular oxygen to yield superoxide anion radicals.
In our effort to develop selective and novel trypanocidal com-
pounds, we decided to proceed with a molecular hybridization
strategy. Thus, we combined the hydrazone pharmacophore frag-
ment with the benzofuroxan system aiming to produce novel tem-
plates for the generation of compounds with anti-chagasic activity

W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
6997
S
O
CHO
Cl
H
H
O
H2N
O
N
NHR
N
N
N
N
N
N
NaN3 / DMSO
H
R
N
H
O
O
S
toluene / AcOH / rt
60-100 °C
NO2
1
2, -R = -H
3, -R = -CH2(CH=CH2)
4, -R = -C6H5
Scheme 2. Synthesis of thiosemicarbazone-benzofuroxan hybrid derivatives 2–4.
(Series 1 and 2, Scheme 1). The developed derivatives were exam-
ined for anti-proliferative in vitro activity against two stocks of
T. cruzi, the Tulahuen 2 strain and the CL Brener clone, for the unspe-
cific cytotoxicity against J774 mouse macrophages, and the inhibi-
tion of CP and TR. Studies of the capacity of intraparasite-free
radical production, through ESR spectroscopy, were also performed.
Another designed thiosemicarbazone involved
a
phenoxy-
spacer between the heterocyclic system and the hydrazone moiety
(derivative 12, Scheme 3). Together with derivative 12 the semicar-
bazone and amidinohydrazone analogues, 11 and 13, respectively,
were designed and nucleophilic displacements of bromine, in
derivative 8, by the phenols 5–7 (Scheme 3) were assayed. In these
reaction conditions, only the semicarbazone 11 was obtained ade-
quately, while the desired products 12 and 13 were not generated.
In the reaction with thiosemicarbazone containing phenol 5 the
isothiosemicarbazone 10 was obtained as main product as a result
of the better nucleophilic capability of the sulfur atom. The
chemical structure of this product was established using NMR
(1H-, 13C-), NOE-diff experiments, and two-dimensional experi-
ments such as HMQC and HMBC. To obtain the desired products,
12 and 13, the order of reactions was changed (Scheme 3). Conse-
quently, the thiosemicarbazone 12 and amidinohydrazone 13 were
obtained in a two-step process from condensation of thiosemicar-
bazide and aminoguanidine with aldehyde 9, which was synthe-
sized by reaction of the corresponding bromide derivative 8 with
2. Methods and results
2.1. Chemistry
Thiosemicarbazones belonging to Series 1 (Scheme 1) possess-
ing different lateral chains (hydrogen, allyl, and phenyl), 2–4
(Scheme 2), were synthesized by traditional condensation of com-
mercially available thiosemicarbazides with 5-formylbenzofuro-
xan
1 (Scheme 2). Aldehyde 1 was obtained as previously
reported from 4-chloro-3-nitrobenzaldehyde in a one-pot process,
via SNAr with azide and subsequent cyclization in DMSO as a result
of the pyrolysis of the o-nitrophenylazide intermediate.36
HO
NH
O
N
N
N
X= S
N
S
O
O
H
H
O
N
NH2
N
Br
N
O
H
10
11
N
8
H2N
N
X
X= O
N
HO
5 X=S
6 X=O
O
O
K2CO3 / KI
18-crown-6 / acetone / rt
N
N
O
X= NH
7 X=NH
H2NNH(C=X)NH2
-TsOH / toluene / reflux
p
complex mixture of reaction
or AcONa / EtOH / reflux
H
O
S
HO
O
H2N
N
N
NH2
Br
H
O
N
H
H2N
N
N
p
-TsOH / toluene / rt
K2CO3 / KI
18-crown-6 / acetone / rt
O
S
N
N
O
O
OHC
O
N
O
N
H
NH
12
13
.H2CO3
O
H2N
N
N
H2NHN
NH2
O
NH
N
9
O
AcONa / EtOH / H2O
HCl / reflux
O
N
Scheme 3. Synthesis of semicarbazone-, thiosemicarbazone-, and amidinohydrazone-benzofuroxan hybrid derivatives.

6998
W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
O
1
NOH
NOH
MeO
KCN / MnO2 / MeOH
16
O
O
O
O
O
NO2
NH2
N
N
N
NH2-NH2.H2O
MeO
MeO
FeSO4
H2NHN
O
O
EtOH / rt
N
DMSO / rt
15
14
17
i) NH2-NH2.H2O
EtOH / reflux
Route A
Route B
Salicylaldehyde
HCl / EtOH
ii) Salicylaldehyde
HCl / EtOH / reflux
OH
N
OH
N
O
O
NaClO / NaOH
O
N
EtOH / 0
°C
NO2
N
N
H
and
H
O
N
NH2
NaH / PhI(AcO)2
DMF / 50
18
°
C
19
OH
Route C
NH2
N
O
O
O
O
N
N
N
SOCl2 / CH2Cl2
reflux
Cl
HO
22
O
O
N
Et3N / CH2Cl2 / 0 ºC
21
20
Scheme 4. Attempts to obtain N-acylhydrazone-benzofuroxan hybrid derivative 19.
the phenolate carbanion, p-hydroxybenzaldehyde, generated in
basic medium.
mixture of products, where the desired one was not identified. Cur-
rently, we are working in other synthetic strategies to generate the
desired N-acylhydrazone-benzofuroxan hybrids.
For the preparation of the N-acylhydrazone 19, belonging to
Series 2 (Scheme 1), several synthetic routes were attempted
(Routes A–C, Scheme 4). In the synthetic route A (Scheme 4) it
was planned to prepare the N-acylhydrazone derivative via the
condensation between hydrazide and aldehyde. However, when
methyl ester 14, prepared from aldehyde 1 (Scheme 2), was treated
with hydrazine to obtain the intermediate hydrazide 15 the ring-
opening o-benzoquinone dioxime 16 was isolated as the main
reaction product (Scheme 4). This dioxime could be the result of
the reductive capability of the hydrazine reactant.37 We proposed
as an alternative synthetic process the synthesis of the N-acylhyd-
razone moiety previous to the cyclization step to benzofuroxan
system (Route B). So, the N-acylhydrazone 18 was obtained by sub-
stitution/condensation from 3-amino-4-nitrobenzoic acid methyl
ester, which was synthesized by reduction of the benzofuroxan
14. After that, some attempts to obtain N-acylhydrazone 19 by oxi-
dative cyclization, NaOCl or PhI(Ac2)O, were unsuccessful. Finally,
as the third approach Route C (Scheme 4) was assayed. In this pro-
cedure acyl chloride 21 was reacted with hydrazone 22 yielding a
Unsymmetrical-substitutedaldazines 25–27 (Scheme 5), belong-
ing to Series 2, were synthesized by condensation of aldehyde 1
and hydrazone derivatives 22–24 in moderate to good yields.
All of the proposed structures were established by NMR (1H, 13C,
HMQC, and HMBC experiments), IR, and MS. The purity was estab-
lished by TLC and microanalysis. 1H and 13C NMR spectra of the
benzofuroxans showed broad signals at room temperature due to
the rapid tautomeric equilibrium.23a,25c,38 This phenomenon is ob-
served at room temperature, but is well resolved at low tempera-
ture.23a The tautomerism in benzofuroxan derivatives depends on
solvent, temperature, and both nature and position of substituents.
2.2. Biology
2.2.1. In vitro anti-T. cruzi activity
All hybrid derivatives, and some intermediates, were initially
tested in vitro against the epimastigote form of T. cruzi, Tulahuen
2 strain.22–25 Recently, the existence of the epimastigote form of
R2
R3
R1
O
R2
R3
R1
H
R2
R3
R1
N
1 / AcOH (cat)
N
N
NH2NH2.H2O
EtOH / reflux
N
O
O
N
toluene / rt
NH2
22
23
24
25 -R1 = -OH, -R2 = -H, -R3 = -H
26 -R1 = -H, -R2 = -OH, -R3 = -H
27 -R1 = -H, -R2 = -OH, -R3 = -Br
Scheme 5. Synthesis of hydrazone-benzofuroxan hybrid derivatives 25–27.

W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
6999
Table 1
tial susceptibilities in the two studied strains, the biological behav-
ior of the selected benzofuroxans being similar in both parasite
populations.
In vitro anti-T. cruzi activity of new benzofuroxan derivatives, Nfx, Bnz, Ktz, and Tbf
against Tulahuen 2 strain
Derivative
PGIa
IC50 (l
M)b,c
2.2.2. Unspecific cytotoxicity
Mammal cytotoxicity of the most effective compounds was
studied in vitro using J-774 mouse macrophages as the cellular
2
3
4
5
35
93
98
100
25
70
40
17
52
48
ꢀ25.0
>25.0
8.9
8.7
6.8
>25.0
15.0
>25.0
ꢀ25.0
ꢁ25.0
ꢁ25.0
9
model with doses (50–400
l
M) at least two times higher than
10
11
12
13
25
26
27
the doses used for T. cruzi (25
l
M) (Table 3).23b Ktz and Tbf were
included in the study as trypanocidal references. The thiosemicar-
bazone 4 and amidinohydrazone 3 were the most toxic of the stud-
ied derivatives. The cytotoxicity of these two derivatives against
macrophages is comparable to that observed for the reference drug
Ktz. According to the toxic effects against T. cruzi amidinohydraz-
one 13 and azine 27 were the least selective derivatives with selec-
tivity indexes (SI) lower than 3.4 (Table 3). Aldehyde 9 has a similar
selectivity to that observed for Tbf, while derivatives 10 and 12
were the least toxic against mammal and the most selective among
the studied compounds with SI, for Tulahuen 2 strain, higher than
20.9.
Nfx
Bnz
Ktz
Tbf
100
100
100
100
7.7
7.4
10.0
17.1
a
PGI: percentage of growth inhibition at 25
l
M.
IC50: concentration that produces 50% inhibitory effect.
The results are the means of three independent experiments with a SD less than
b
c
10% in all cases.
2.3. Mechanism of action studies
In order to confirm or discard some possible mechanisms of ac-
tion the following studies were performed with the hybrid deriva-
tives: inhibition of CP, inhibition of TR, and capability of the
developed derivatives to produce intra-parasite free radicals.
T. cruzi as an obligate mammalian intracellular stage has been
revisited and confirmed.39 The compounds were incorporated into
the media at 25 lM and its ability to inhibit parasite growth was
evaluated in comparison to the control (no drug added to the med-
ia) (Table 1). Nfx, Bnz, Ktz, and Tbf were used as the reference try-
panocidal drugs. Besides, the IC50 concentrations (50% inhibitory
dose) were determined for the most active derivatives (Table 1).
Thiosemicarbazones 4 and 12, aldehyde 9 and isothiosemicarbaz-
one 10 were the most active derivatives with IC50 between 6.8
2.3.1. Inhibition of cruzipain
Some of the developed benzofuroxans were tested as possible
inhibitors of T. cruzi CP, following a previously described proce-
dure.24 The assayed derivatives displayed variable capability of
CP inhibition in the assay conditions used (Table 4). They showed
IC50 values between 32 and 100 lM, except for the semicarbazone
11 and the thiosemicarbazones 2 and 3, where almost complete
and 15.0
lM, all of them with IC50 values similar to those of the ref-
erence drugs (IC50 between 7.4 and 17.1
lM). Unsubstituted or al-
lyl-substituted-thiosemicarbazones 2 and 3 were poor parasite
growth inhibitors. However, derivative 4, the phenyl-substituted
thiosemicarbazone, presented excellent activity showing the rele-
vance of the aromatic moiety in the anti-parasite activity. The
semicarbazone 11 and amidinohydrazone 13 exhibited poor activ-
ity compares to thiosemicarbazone 12, indicating in this case the
relevance of the thiocarbonyl moiety. This fact could be explained
in terms of our previous results, where the hydrophilicity and the
presence of a hydrogen-bond-donor moiety in the benzofuroxan
lateral chain decrease the activity.23a On the other hand, aldazines
25–27 were less active against T. cruzi than the reference drugs.
The hybrid designed derivatives phenylthiosemicarbazone 4, thio-
semicarbazone 12, and amidinohydrazone 13, with high, moderate
and poor trypanocidal activity, respectively, against the Tulahuen 2
strain were selected to study against the CL Brener clone (Table 2).
According to these results, it was not possible to observe differen-
lack of inhibition was observed in the studied doses. Thiosemicar-
bazone 12 and amidinohydrazone 13, with IC50 of 43 and 32 lM,
respectively, emerge as CP-inhibitor lead structures for future
chemical optimization through SAR investigation. Clearly, some
structural exigencies for CP-inhibition could be inferred with this
kind of derivatives. On the one hand, as it has been previously de-
scribed, thiosemicarbazone moiety-containing derivatives are bet-
ter inhibitors than semicarbazone analogues (compare the
inhibitions by derivatives 11 and 12).33 In this sense, we identified,
to our knowledge for the first time, the amidinohydrazone moiety
as the best thiosemicarbazone analogue CP-inhibitor. On the other
hand, analyzing azine derivatives 25–27 it was observed, as previ-
ously described,30 that the o-hydroxyl motive is preferential to the
p-hydroxyl one for CP inhibition (compare ortho-derivatives 25 and
27 to para-hydroxyl 26). Comparing these results with the growth
Table 3
Table 2
Cytotoxicity of benzofuroxan derivatives against J-774 mouse macrophages
In vitro anti-T. cruzi activity of selected new benzofuroxan derivatives, Nfx, Bnz, Ktz,
and Tbf against CL Brener
a
Derivative
IC50;macrophage
SIb,c
4
9
<50.0
60.0
142.0
400.0
<50.0
84.0
<5.6
6.9
Derivative
IC50 (l
M)a,b
4
12
13
2.6
19.8
38.2
10
12
13
27
Ktz
Tbf
20.9
26.7
<2.0
<3.4
<5.0
5.1
Nfx
Bnz
Ktz
Tbf
8.5
4.5
5.0
<50.0
88.0
42.0
a
The results are the means of two independent experiments with a SD less than
10% in all cases.
a
IC50: concentration that produces 50% inhibitory effect.
b
b
The results are the means of three independent experiments with a SD less than
10% in all cases.
SI: selectivity index.
SI = IC50,macrophage/IC50,T2,epimastigote
c
.

7000
W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
Table 4
inhibition capability and their lower CP and TR inhibition proper-
ties. For this purpose we used ESR spectroscopy and spin-trapping
tools.41 According to these experiments both thiosemicarbazones
were able to produce free radicals in presence of T. cruzi-micro-
somal fraction. The ESR spectra showed a hyperfine pattern consis-
tent with the N-oxide free radical DMPO-trapped (Fig. 2).25b
Additionally, the most potent anti-T. cruzi hybrid derivative, 4, also
Inhibition of CP and TR activities of some selected hybrid compounds
Derivative
Findings in enzymatic studies
% of TR inhibition (doses, l
M)a
CP, IC50
(
l
M)a
2
3
4
11
12
13
25
26
27
>100b
>100c
78
19 (100) 9 (40)
9 (100) 13 (40)
0 (100) 0 (20)d
0 (100) 0 (20)f
0 (100) 0 (20)f
50 (100) 30 (40)
>100e
43
showed in the ESR spectra the characteristic signals of DMPO-hy-
*
droxyl radical spin adduct (marked with
in Fig. 2,
32
68
100
73
aN = aH = 15.2 G). This could confirm that this compound was able
to produce oxidative stress into the parasite. In the case of the least
potent derivative 12, the presence of hydroxyl radical was not
clearly observed.
g
—
—
—
a
The results are the means of two independent experiments with a SD less than
10% in all cases.
b
7% of inhibition at 100
36% of inhibition at 100
l
l
M.
M.
M the compound starts to precipitate in the assayed buffer.
23% of inhibition at 100 M.
At P20 M the compound starts to precipitate in the assayed buffer.
‘—’, not determined.
3. Conclusions
c
d
e
f
At P40
l
Our results showed that the novel hybrid compounds, like
12, have relevant selective trypanocidal activities. The biological
studies indicated that these derivatives could act mainly by
production of free radical species through the parasite metabo-
lism identifying both N-oxide and hydroxyl free radical as bio-
logical intermediates. On the other hand, the observed
inhibition of cysteinyl active site enzymes of T. cruzi, like CP
and TP, by these compounds was negligible, concluding that
these biochemical pathways are not involved in the mode of ac-
tion of these hybrid compounds. Furthermore, derivative 10,
obtained as a secondary product, showed excellent biological
profile. Deep studies on this non-designed derivative are being
performed. In vivo studies to investigate the ability of these
drugs to decrease the parasitemia of infected mice are currently
underway.
l
l
g
inhibition properties of benzofuroxans, it could be proposed that
the main mode of action of these compounds is not the inhibition
of CP.
2.3.2. Inhibition of T. cruzi trypanothione reductase
Hybrid derivatives 2–4 and 11–12 were studied as reversible
inhibitors of T. cruzi TR, following a previously described proce-
dure.24 The compounds resulted poor TR inhibitors at the doses
tested (Table 4). Some problems with the solubility in the assay
buffer of thiosemicarbazones 4 and 12 and semicarbazone 11 prob-
ably promoted the lack of enzymatic inhibition capability. Amid-
inohydrazone 13 showed the best TR-inhibition capability, with a
IC50 of 100 l
M. As it was previously reported,35,40 the amidinohyd-
4. Experimental
razone moiety could interact positively in the enzymatic site. Com-
paring these results with the growth inhibition properties of
benzofuroxans, it could be proposed that the main mode of action
of these compounds is not the inhibition of TR.
Compounds 1,36 8,25c 2122a, and 22–2442 were prepared accord-
ing to literature procedures. Melting points were determined with
an electrothermal melting point apparatus (Electrothermal 9100)
and were uncorrected. Proton and carbon NMR spectra were re-
corded on a Bruker DPX-400 spectrometer. The chemical shift val-
ues are expressed in ppm relative to tetramethylsilane as internal
standard. In 13C NMR only narrow peaks were reported. ESR spec-
tra were recorded in the X band (9.85 GHz) using a Bruker ECS 106
spectrometer with a rectangular cavity and 50 kHz field modula-
2.4. Free radical generation: ESR studies
In order to study the benzofuroxan’s intraparasite-free-radical
producer ability we selected hybrid compounds, thiosemicarba-
zones 4 and 12. They were selected due to their T. cruzi growth
H
H2N
N
H
H
O
N
DMPO-OH spin adduct
N
N
N
N
O
N
S
OH
N
N
O
O
S
O
N
O
H
DMPO-4 spin adduct
DMPO-12 spin adduct
*
*
*
*
*
*
*
*
3450 3460 3470 3480 3490 3500 3510
3450
3460 3470 3480 3490
3500 3510
3450 3460 3470 3480 3490 3500 3510
[G]
[G]
[G]
Figure 2. ESR spectra. Left and centre: DMPO-benzofuroxan spin adduct obtained with T. cruzi microsomal fraction. Right: pattern of DMPO-hydroxyl radical spin adduct.

W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
7001
tion. Mass spectra were determined either on a MSD 5973 Hew-
lett-Packard or LC/MSD-Serie 100 Hewlett-Packard spectrometers
using electronic impact (EI) or electrospray ionization (ESI), respec-
tively. Infrared spectra were recorded on a Perkin- Elmer 1310
apparatus using potassium bromide tablets for solid and oil
products and the frequencies were expressed in cmꢂ1. Elemental
analyses were carried out on a Fisons EA 1108 CHNS-O instrument
and were within 0.4% of the calculated compositions. Column
chromatography was carried out using Merck silica gel (60–
230 mesh). Most chemicals and solvents were of analytical grade
and used without further purification. All the reactions were
carried out in a nitrogen atmosphere.
washed with toluene and was used without further purification.
Brown solid, 420 mg (88%); 1H NMR (acetone-d6) d: 6.91 (d,
2H,J = 9.4 Hz), 7.32 (br s, 1H), 7.67 (d, 2H,J = 9.4 Hz), 7.80 (br s,
1H), 8.10 (s, 1H), 10.25 (br s, 1H). ESI-MS, m/z: 196 (M+Å+H).
4.6. 4-Hydroxybenzaldehyde semicarbazone (6)44
To a mixture of p-hydroxybenzaldehyde (300 mg, 2.46 mmol) in
EtOH (5.0 mL), a solution of semicarbazide chlorhydrate (274 mg,
2.46 mmol) and NaOAc (200 mg, 2.45 mmol) in water (2 mL) was
added. The mixture was stirred at room temperature for 24 h.
The precipitated solid was collected and washed with toluene,
and was used without further purification. Pale yellow solid,
320 mg (73%); 1H NMR (acetone-d6) d: 6.16 (br s, 2H), 6.89 (d,
2H, J = 8.7 Hz), 7.67 (d, 2H,J = 8.8 Hz), 7.88 (s, 1H), 8.76 (br s, 1H),
9.55 (br s, 1H). ESI-MS, m/z: 180 (M+Å+H).
4.1. General procedure for the synthesis of thiosemicarbazone-
benzofuroxan hybrid derivatives (2–4)
A mixture of 1 (1.0 equiv), the corresponding thiosemicarbazide
(1.0 equiv) and AcOH (catalytic amounts) in dry toluene (12 mL/
mmol) as solvent was stirred at room temperature until the car-
bonyl compound was not present (checked by TLC). The solid prod-
uct was collected and washed with toluene, Et2O and purified as it
is indicated.
4.7. 4-Hydroxybenzaldehyde amidinohydrazone (7)45
A solution of p-hydroxybenzaldehyde (300 mg, 2.46 mmol) in
ethanol (7.0 mL) was treated with an aqueous solution of aminogua-
nidine bicarbonate (30–40%) (430 mg, 3.20 mmol) and with cata-
lytic amounts of HCl (c). The reaction mixture was heated at reflux
for 4 h. The solvent was evaporated in vacuo and the residue was
neutralized with aqueous saturated NaHCO3 and extracted with
EtOAc (3 ꢃ 20 mL). After the work-up the organic layer was evapo-
ratedin vacuoand theresidue corresponding to theproduct, purified
by TLC, was used without further purification. Pale yellow solid,
200 mg (50%); 1H NMR (CD3OD:D2O) d: 6.78 (d, 2H,J = 8.6 Hz), 7.53
(d, 2H,J = 8.6 Hz), 7.96 (s, 1H). ESI-MS, m/z: 179 (M+Å+H).
4.2. Benzofuroxan-5-carboxaldehyde thiosemicarbazone (2)
Purified by crystallization from EtOH; yellow solid (92%); mp
205.0–206.0 °C. 1H NMR (CDCl3) d: 7.86–7.71 (br s, 2H), 8.09 (s,
1H), 8.26 (br s, 1H), 8.30 (s, 1H), 8.37 (s, 1H), 11.72 (s, 1H). 13C
NMR (DMSO-d6) d: 179.4 (C@S), 140.3 (CH@N). EI-MS, m/z (abun-
dance,%): 237 (M+Å, 5), 221 (15), 185 (45), 105 (100). Anal. Calcd for
(C8H7N5O2S): C, 40.50; H, 2.97; N, 29.52. Found: C, 40.37; H, 2.88;
N, 29.35.
4.8. 5-[(4-Formylfenoxy)methyl]benzofuroxan (9)
4.3. N4-Allyl benzofuroxan-5-carboxaldehyde
A mixture of 8 (1.0 g, 4.4 mmol), p-hydroxybenzaldehyde
thiosemicarbazone (3)
(530 mg, 4.4 mmol), K2CO3 (600 mg, 4.4 mmol), KI (30 mg,
0.022 mmol), and 18-crown-6 (10 mg, 0.044 mmol) in acetone
(50.0 mL) was stirred at room temperature for 4 h. The organic sol-
vent was evaporated in vacuo and the residue was treated with
aqueous NaOH 20% (50.0 mL) and extracted with EtOAc
(3 ꢃ 40.0 mL). The organic layer was washed with saturated
sodium chloride solution (40.0 mL) and dried with Na2SO4. Then
the organic solvent was evaporated in vacuo and the residue puri-
fied by column chromatography (SiO2, petroleum ether/EtOAc
(8:2)), yielding derivative 9 as a pale yellow solid (1.1 g, 91%);
mp 136.0–138.0 °C. 1H NMR (CDCl3) d: 5.18 (s, 2H), 7.13 (d, 2H,
J = 8.7 Hz), 7.32 (br s, 1H), 7.57 (br s, 2H), 7.91 (d, 2H, J = 8.7 Hz),
9.93 (s, 1H). 13C NMR (CDCl3) d: 183.7 (C@O), 162.8 (C4), 129.1
(C2), 128.9 (C1), 116.4 (C3), 69.2 (CH2). EI-MS, m/z (abundance,%):
Purified by crystallization from petroleum ether/EtOAc (8:2);
yellow solid (50%); mp 172.0–173.0 °C. 1H NMR (acetone-d6) d:
4.38 (m, 2H), 5.13 (d, 1H, J = 10.3 Hz), 5.25 (d, 1H, J = 17.2 Hz),
6.02 (m, 1H), 7.74–7.62 (br s, 2H), 8.13 (br s, 1H), 8.26 (s, 1H),
8.64 (br s, 1H), 10.76 (br s, 1H). 13C NMR (acetone-d6) d: 179.0
(C@S), 139.5 (CH@N), 134.8 (CH2–CH@CH2), 115.7 (CH2–CH@CH2),
46.7 (CH2–CH@CH2). EI-MS, m/z (abundance, %): 277 (M+Å, 8), 259
(10), 231 (55), 185 (36), 148 (43), 115 (100), 105 (89). Anal. Calcd
for (C11H11N5O2S): C, 47.64; H, 4.00; N, 25.26. Found: C, 47.40; H,
3.89; N, 25.01.
4.4. N4-Phenyl benzofuroxan-5-carboxaldehyde
thiosemicarbazone (4)
270 (M+Å, 5), 254 (13), 149 (34), 133 (100). IR (KBr)
m: 3045, 2870,
1681, 1633, 1600, 1537, 1488, 1373, 1257, 1164, 1112, 1047, 1016,
856, 796, 671, 648, 572. Anal. Calcd for (C14H10N2O4): C, 62.22; H,
3.73; N, 10.37. Found: C, 62.03; H, 3.56; N, 10.12.
Purified by crystallization from petroleum ether/EtOAc (8:2);
yellow solid (63%); mp 166.0–167.0 °C. 1H NMR (acetone-d6) d:
7.25 (t, 1H, J = 7.7 Hz), 7.40 (t, 2H, J = 7.7 Hz), 7.64 (br s, 1H), 7.75
(d, 2H, J = 7.7 Hz), 7.82 (br s, 1H), 8.27 (br s, 1H), 8.35 (s, 1H),
10.03 (br s, 1H), 10.98 (br s, 1H). 13C NMR (acetone-d6) d: 177.4
(C@S), 140.1 (CH@N), 139.9 (C1), 128.4 (C3), 125.9 (C2), 125.4
(C4). EI-MS, m/z (abundance,%): 313 (M+Å, 18), 295 (41), 231 (30),
151 (50), 93 (100). Anal. Calcd for (C14H11N5O2S): C, 53.66; H,
3.54; N, 22.35. Found: C, 53.39; H, 3.30; N, 22.21.
4.9. 1-(4-Hydroxyphenylmethylidene)-S-(benzofuroxan-5-
ylmethyl)isothiosemicarbazone (10)
A mixture of 5 (170 mg, 0.87 mmol), 8 (200 mg, 0.87 mmol),
K2CO3 (120 mg, 0.87 mmol), KI (8 mg, 0.022 mmol), and 18-
crown-6 (2 mg, 0.044 mmol) in acetone (10.0 mL) was stirred at
room temperature for 2 h. The organic solvent was evaporated in
vacuo and the residue purified by column chromatography (SiO2,
petroleum ether/EtOAc (7:3, 1:1)). Derivative 10 was obtained as
a reddish oil that crystallized at 4 °C (80 mg, 27%); 1H NMR (CDCl3)
d: 4.34 (s, 2H), 5.44 (br s, 2H), 6.88 (d, 2H, J = 8.6 Hz), 7.40 (br s, 3H),
7.64 (d, 2H, J = 8.6 Hz), 8.32 (s, 1H). 13C NMR (acetone-d6) d: 159.6
(C4), 159.3 (SAC@NH), 153.7 (NAC@NH), 129.7 (C2), 127.6 (C1),
4.5. 4-Hydroxybenzaldehyde thiosemicarbazone (5)43
A mixture of p-hydroxybenzaldehyde (300 mg, 2.46 mmol), thi-
osemicarbazide (220 mg, 2.46 mmol), and p-TsOH (catalytic
amounts) in dry toluene (5.0 mL) as solvent was stirred at room
temperature for 24 h. The precipitated solid was collected and

7002
W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
115.8 (C3), 32.8 (CH2). EI-MS, m/z (abundance,%): 343 (M+Å, 5), 325
(20), 308 (15), 133 (75), 120 (100). IR (KBr) : 3000–2500, 1701,
1655, 1600, 1520, 1232, 1167, 1105, 1010, 879, 839, 748, 526. Anal.
Calcd for (C15H13N5O3S): C, 52.47; H, 3.82; N, 20.40. Found: C,
52.24; H, 3.60; N, 20.24.
ature. The resulting dispersion was filtered through a short pad of
Celite, the organic phase was concentrated in vacuo, and the resi-
due was used in the next reaction without further purification.
Brown solid (210 mg, 89%). 1H NMR (DMSO-d6) d: 4.00 (s, 3H),
7.59 (br s, 1H), 7.88 (br s, 1H), 8.25 (br s, 1H). EI-MS, m/z (abun-
dance,%): 194 (M+Å, 100), 178 (59),167 (20), 103 (59).
m
4.10. 4-(Benzofuroxan-5-ylmethyloxy)benzaldehyde
semicarbazone (11)
4.14. Methyl 4-amino-3-nitrobenzoate (17)
A mixture of 6 (195 mg, 1.10 mmol), 8 (250 mg, 1.10 mmol),
K2CO3 (151 mg, 1.10 mmol), KI (9 mg, 0.06 mmol), and 18-crown-
6 (3 mg, 0.01 mmol) in acetone (10.0 mL) was stirred at room tem-
perature for 4 h. The precipitated solid was collected and purified
by crystallization from EtOH. Pale yellow solid 50 mg (42%); mp
218.0–219.0 °C. 1H NMR (DMSO-d6) d: 5.23 (s, 2H), 6.40 (br s,
2H), 7.09 (d, 2H, J = 8.8 Hz), 7.49 (br s, 1H), 7.69 (d, 2H,
J = 8.8 Hz), 7.70–7.78 (br s, 2H), 7.79 (s, 1H), 10.08 (s, 1H). 13C
NMR (DMSO-d6) d: 159.3 (C4), 157.6 (C@O), 139.8 (C@NH), 129.0
(C2), 128.9 (C1), 115.8 (C3), 68.9 (CH2). EI-MS, m/z (abundance,%):
A mixture of 14 (370 mg, 1.89 mmol) and FeSO4.7H2O (3.15 g,
12.0 mmol) in DMSO (10.0 mL) as solvent was stirred for 1 h at
room temperature. The mixture was treated with water
(50.0 mL) and extracted with EtOAc (3 ꢃ 10.0 mL). The organic
layer was washed with brine (40.0 mL) and dried over Na2SO4.
Then the organic solvent was evaporated in vacuo and the residue
was used in the next reaction without further purification. Yellow
solid (190 mg, 44%). 1H NMR (DMSO-d6) d: 3.93 (s, 3H), 6.42 (br s,
2H), 6.86 (d, 1H, J = 8.7 Hz), 8.03 (dd, 1H, J = 8.9 Hz, J = 1.9 Hz), 8.87
(d, 1H, J = 2.0 Hz). EI-MS, m/z (abundance,%): 196 (M+Å, 100), 165
(38),135 (34).
327 (M+Å, 1), 311 (6), 278 (8), 238 (11), 133 (100). IR (KBr)
m: 3476,
3273, 2926, 1700, 1655, 1541, 1369, 1259, 1170, 1059, 1014, 946,
854, 789, 553. Anal. Calcd for (C15H13N5O4): C, 55.05; H, 4.00; N,
21.40. Found: C, 54.88; H, 3.89; N, 21.29.
4.15. 4-Amino-20-(2-hydroxybenzylidene)-3-nitrobenzodrazide
(18)
4.11. 4-(Benzofuroxan-5-ylmethyloxy)benzaldehyde
thiosemicarbazone (12)
A solution of the methyl ester 17 (205 mg, 1.05 mmol) and
hydrazine monohydrate (55%) (2.25 mL, 72 mmol) in absolute
EtOH (7.0 mL) was stirred at reflux for 4 h. The solvent was con-
centrated in vacuo, and the reaction mixture was diluted with
water and extracted with EtOAc. The organic layer was dried over
Na2SO4 and evaporated in vacuo. 4-Amino-3-nitrobenzohydrazide
was obtained as an orange solid (190 mg, 93%) and was used in
the next reaction without further purification. 1H NMR (ace-
tone-d6) d: 4.42 (s, 2H), 7.03 (d, 1H, J = 8.9 Hz), 7.72 (s, 2H),
7.84 (dd, 1H, J = 8.8 Hz, J = 1.8 Hz), 8.52 (d, 1H, J = 1.7 Hz), 9.6
A mixture of aldehyde 9 (150 mg, 0.56 mmol), thiosemicarbazide
(50 mg, 0.56 mmol), and p-TsOH (catalytic amounts) in dry toluene
(10 mL/mmol) as solvent was stirred at room temperature until the
carbonyl compound was not present (checked by TLC). The solid
product was collected, washed with EtOH, acetone and purified by
crystallization from EtOH. Yellow pale solid 72 mg (38%); mp
207.0–209.0 °C. 1H NMR (DMSO-d6) d: 5.25 (s, 2H), 7.10 (d, 2H,
J = 8.8 Hz), 7.49 (br s, 1H), 7.77 (br s, 2H), 7.78 (d, 2H, J = 8.8 Hz),
7.90 (br s, 1H), 8.01 (s, 1H), 8.08 (br s, 1H), 11.30 (s, 1H). 13C NMR
(DMSO-d6) d: 178.6 (C@S), 160.0 (C4), 142.8 (C@NH), 129.8 (C2),
128.3 (C1), 115.9 (C3), 68.9 (CH2). EI-MS, m/z (abundance,%): 341
(br s,1H).
A solution of 2-hydroxybenzaldehyde (0.05 mL,
0.51 mmol) in absolute EtOH (2.0 mL) was added to a mixture
of 4-amino-3-nitrobenzohydrazide (100 mg, 0.51 mmol) and cata-
lytic amount of HCl (c) in absolute EtOH (10.0 mL). The final mix-
ture was stirred at room temperature for 1 h. Next, the solvent
was partially concentrated at reduced pressure and the resulting
mixture was poured into cold water. After neutralization with
aqueous NaHCO3 (10%), the precipitate was collected and washed
with petroleum ether. Orange solid (120 mg, 78%). 1H NMR (ace-
tone-d6) d: 6.94 (m, 2H), 7.12 (d, 1H, J = 8.9 Hz), 7.31 (m, 1H), 7.53
(d, 1H, J = 7.4 Hz), 7.87 (s, 2H), 7.97 (dd, 1H, J = 8.9 Hz, J = 1.9 Hz),
8.63 (s, 1H), 8.71 (d, 1H, J = 1.8 Hz), 11.4 (br s, 1H), 11.9 (br s, 1H).
EI-MS, m/z (abundance,%): 300 (M+Å, 19), 181 (36),165 (100), 119
(38).
(M+Åꢂ2, 2), 269 (3), 254 (11),149 (16), 133 (100). IR (KBr)
m: 3280,
2926, 1727, 1645, 1530, 1334, 1267, 1145, 1059, 1015, 956, 8576,
784, 570. Anal. Calcd for (C15H13N5O3S): C, 52.47; H, 3.82; N, 20.40.
Found: C, 52.21; H, 3.72; N, 20.19.
4.12. 4-(Benzofuroxan-5-ylmethyloxy)benzaldehyde
amidinohydrazone (13)
A mixture of 9 (100 mg, 0.37 mmol), aminoguanidine bicarbon-
ate (50 mg, 0.37 mmol), NaOAc (61 mg, 0.74 mmol) and catalytic
amount of HCl (c) in EtOH (3.0 mL) was heated at reflux for 6 h.
Then the mixture was allowed to cool to room temperature, and
the precipitated solid was collected and crystallized from EtOH.
Pale yellow solid 90 mg (75%); mp 215.0–217.0 °C. 1H NMR
(DMSO-d6) d: 5.27 (s, 2H), 7.15 (d, 2H, J = 8.9 Hz), 7.4–7.8 (br s,
6H), 7.84 (d, 2H, J = 8.8 Hz), 8.12 (s, 1H), 11.67 (br s, 1H). 13C
NMR (DMSO-d6) d: 160.4 (C4), 156.3 (NAC@NH), 147.2 (C@NH),
130.1 (C2), 127.7 (C1), 115.9 (C3), 69.0 (CH2). EI-MS, m/z (abun-
4.16. General procedure for the synthesis of unsymmetrical
azines (25–27)
A mixture of 1 (1.0 equiv), the corresponding hydrazone (1.0
equiv), and AcOH (catalytic amounts) in dry toluene (12 mL/mmol)
as solvent was stirred at room temperature until the carbonyl com-
pound was not present (checked by TLC). The solid product was
collected and washed with toluene, Et2O and purified by column
chromatography (SiO2, petroleum ether/EtOAc (8:2)).
dance,%): 310 (M+Åꢂ16, 28), 177 (100), 133 (73). IR (KBr)
m:
3400–2800, 1650, 1610, 1590, 1456, 1385, 1307, 1250, 1047,
1010, 871, 613, 534. Anal. Calcd for (C15H14N6O3): C, 55.21; H,
4.32; N, 25.75. Found: C, 55.03; H, 4.12; N, 25.59.
4.17. 2-[(Benzofuroxan-5-yl)methylene]-1-(2-hydroxybenzyli-
dene)hydrazine (25)
4.13. Methyl benzofuroxan-5-carboxylate (14)
Yellow solid (37%); mp 216.0–217.0 °C. 1H NMR (acetone-d6) d:
7.00 (m, 2H), 7.45 (t, 1H, J = 7.7 Hz), 7.76 (d, 1H, J = 8.8 Hz), 7.88 (br
To a mixture of 1 (200 mg, 1.22 mmol) in MeOH (30.0 mL) was
added KCN (90 mg, 1.34 mmol) and subsequently MnO2 (850 mg,
9.76 mmol). The final mixture was stirred for 6 h at room temper-
s, 1H), 8.05 (br s, 2H), 8.80 (s, 1H), 9.00 (s, 1H), 11.08 (br s, 1H). 13
C
NMR (acetone-d6) d: 164.1 (CH@N), 161.4 (CH@N), 160.3 (C2),

W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
7003
135.3 (C4), 132.0 (C6), 121.2 (C3), 119.2 (C1), 117.6 (C5). EI-MS, m/
compounds (25–1000
l
M) for 48 h. Afterwards, the cells were
z (abundance,%): 282 (M+Å, 14), 266 (8), 256 (55), 213 (39), 129
(56). Anal. Calcd for (C14H10N4O3): C, 59.57; H, 3.57; N, 19.85.
Found: C, 59.40; H, 3.41; N, 19.72.
washed with PBS and incubated (37 °C) with 3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) 0.4 mg/
mL for 3 h. Then, the formazan was dissolved with DMSO
(180 lL) and optical densities were measured. Each concentration
4.18. 2-[(Benzofuroxan-5-yl)methylene]-1-(4-hydroxybenzyli-
dene)hydrazine (26)
was assayed three times and six growth controls were used in each
test. Cytotoxicity percentages (% C) were determined as follows: %
C = [100 ꢂ (ODd ꢂ ODdm)/(ODc ꢂ ODcm)] ꢃ 100, where ODd is the
mean of OD595 of wells with macrophages and different concentra-
tions of the compounds; ODdm is the mean of OD595 of wells with
different compound concentrations in the medium; ODc is the
growth control and ODcm is the mean of OD595 of wells with med-
ium only.
Yellow solid (59%); mp 219.0–220.0 °C. 1H NMR (acetone-d6) d:
6.97 (d, 2H, J = 8.8 Hz), 7.77 (d, 2H, J = 8.8 Hz), 7.86 (br s, 1H), 8.21
(br s, 2H), 8.75 (s, 1H), 8.81 (s, 1H), 8.90 (br s, 1H). 13C NMR (ace-
tone-d6) d: 164.1 (CH@N), 161.4 (CH@N), 161.8 (C4), 132.3 (C2),
125.5 (C1), 119.8 (C3). EI-MS, m/z (abundance,%): 282 (M+Å, 20),
266 (12), 256 (67), 213 (43), 129 (71). Anal. Calcd for
(C14H10N4O3): C, 59.57; H, 3.57; N, 19.85. Found: C, 59.44; H,
3.43; N, 19.78.
4.22. T. cruzi CP inhibition assays
CP was purified to homogeneity from epimastigotes of the
Tulahuen 2 strain by ConA–Sepharose affinity chromatography,
as previously described,47 and its activity was assayed in a reaction
mixture (1 mL) containing (final concentration) 50 mM Tris-ace-
tate buffer, pH 8.0, 0.3 mM Bz–Pro–Phe–Arg–pNA, and 10 mM b-
mercaptoethanol. Absorbance at 410 nm was followed at 30 °C in
a Beckman Model 25 recording spectrophotometer. The derivatives
were added as solutions in DMSO, and the controls contained the
same solvent concentration. E-64 was used as a positive control
of inhibition.
4.19. 2-[(Benzofuroxan-5-yl)methylene]-1-(5-bromo-2-hydro-
xybenzylidene)hydrazine (27)
Yellow solid (75%); mp 201.0–202.0 °C. 1H NMR (acetone-d6)
d: 6.98 (d, 2H, J = 8.7 Hz), 7.57 (dd, 1H, J = 8.8 Hz, J = 2.5 Hz),
7.85 (br s, 1H), 7.95 (d, 1H, J = 2.4 Hz), 8.03 (br s, 2H), 8.86 (s,
1H), 8.93 (br s, 1H), 11.16 (br s, 1H). 13C NMR (acetone-d6) d:
163.8 (CH@N), 162.5 (CH@N), 158.5 (C2), 137.1 (C4), 133.5
(C6), 122.1 (C1), 120.2 (C3), 118.6 (C5). EI-MS, m/z (abun-
dance,%): 360/362 (M+Å, 75), 345 (24), 301 (32), 200 (100). Anal.
Calcd for (C14H9BrN4O3): C, 46.56; H, 2.51; N, 15.51. Found: C,
46.39; H, 2.38; N, 15.41.
4.23. T. cruzi TR inhibition assays
Recombinant T. cruzi TR was prepared according to a published
procedure.48 Trypanothione disulfide was purchased from Bachem,
Heidelberg, Germany. TR activity was measured spectrophotomet-
rically at 25 °C in TR assay buffer (40 mM Hepes, 1 mM EDTA, pH
7.5) as described.40 Stock solutions of the compounds were pre-
pared in DMSO. The assay mixtures (1 mL) contained in TR assay
4.20. In vitro anti-T. cruzi activity
Trypanosoma cruzi epimastigotes (Tulahuen 2 strain or CL Bren-
er clone) were grown at 28 °C in an axenic medium (BHI–ryptose)
complemented with 5% foetal calf serum. Cells were harvested in
the late log phase, resuspended in fresh medium, counted in a Neu-
bauer chamber, and placed in 24-well plates (3 ꢃ 106/mL). Cell
growth was measured as the absorbance of the culture at
610 nm, which was proved to be proportional to the number of
cells present.46 Before inoculation, the media were supplemented
with the indicated amount of the studied compound from a stock
solution in DMSO. The final concentration of DMSO in the culture
media never exceeded 0.8% and the control was run in the presence
of 0.8% DMSO and in the absence of any compound. No effect on
epimastigote growth was observed in the presence of up to 1%
DMSO in the culture medium. The percentage of growth inhibition
was calculated as follows: PGI = {1-[(Ap ꢂ A0p)/(Ac ꢂ A0c)]} ꢃ 100,
where Ap = A600 of the culture containing the compound at day 5;
A0p = A600 of the culture containing the compound right after addi-
tion of the inocula (day 0); Ac = A600 of the culture in the absence of
any compound (control) at day 5; A0c = A600 in the absence of the
compound at day 0. To determine IC50 values, 50% inhibitory con-
centrations, parasite growth was followed in the absence (control)
and presence of increasing concentrations of the corresponding
compound. At day 5, the absorbance of the culture was measured
and related to that of the control. The IC50 value was taken as the
concentration of compound needed to reduce the absorbance ratio
to 50%.
buffer 100 lM NADPH and 105 or 93 lM trypanothione disulfide
(TS2) and varying concentrations of the inhibitor. NADPH, enzyme,
and inhibitor were mixed and the reaction was started by adding
TS2. The absorption decrease at 340 nm due to NADPH consump-
tion was followed. Control assays contained the respective amount
of DMSO instead of inhibitor.
4.24. ESR studies
The free radical production capacity of the new benzofuroxan
derivatives was assessed on T. cruzi-microsomal fraction (4 mg
protein/mL) by Electronic Spin Resonance (ESR) using 5,5-di-
methyl-1-pirroline-N-oxide (DMPO) for spin trapping.25 Benzof-
uroxan derivatives (1 mM, final concentration) were dissolved in
DMF (spectroscopy grade) and the solution was added to a reaction
medium containing 1 mM NADPH, 1 mM EDTA, and 100 mM
DMPO, in 20 mM phosphate buffer, pH 7.4. The final mixture was
transferred to a 50 lL capillary. All of the spectra were registered
in the same scale after 15 scans.
Acknowledgments
Financial support from CSIC (Uruguay) is acknowledged. We
wish to thank Dr. R. Luise Krauth-Siegel, Biochemie-Zentrum-
Heidelberg University, Germany, for performing the helpful TR
inhibition. We thank AMSUD-PASTEUR network for a fellowship
to M.B., CSIC for a fellowship to W.P., Universidad Nacional Gen-
eral San Martín for scholarship to A.C., and PEDECIBA for schol-
arships to P.H., L.B., M.B., and W.P. J.J.C. is a member of the
Research Career of the Argentinean National Research Council
(CONICET).
4.21. Cytotoxicity to mouse macrophages
J-774 mouse macrophages were seeded (100,000 cells/well) in
96-well flat bottomed microplates (Nunclon) with 200 lL of RPMI
1640 medium supplemented with 20% heat-inactivated fetal calf
serum. Cells were allowed to attach for 48 h in a humidified 5%
CO2/95% air atmosphere at 37 °C. Then, cells were exposed to the

7004
W. Porcal et al. / Bioorg. Med. Chem. 16 (2008) 6995–7004
Olea-Azar, C.; Rigol, C.; Opazo, L.; Morello, A.; Maya, J. D.; Repetto, Y.; Aguirre,
References and notes
G.; Cerecetto, H.; Di Maio, R.; González, M.; Porcal, W. J. Chil. Chem. Soc. 2003,
48, 77; (c) Olea-Azar, C.; Rigol; Mendizábal, F.; Cerecetto, H.; Di Maio, R.;
González, M.; Porcal, W.; Morello, A.; Repetto, Y.; Maya, J. D. Lett. Drug Des. Dev.
2005, 2, 294; (d) Aguirre, G.; Boiani, L.; Cerecetto, H.; Di Maio, R.; González, M.;
Porcal, W.; Thomson, L.; Tórtora, V.; Denicola, A.; Möller, M. Bioorg. Med. Chem.
2005, 13, 6324.
1. De Souza, W. Pharm. Des. 2002, 4, 269.
2. Teixeira, A. R. L.; Nitz, N.; Guimaro, M. C.; Gomes, C.; Santos-Buch, C. A.
Postgrad. Med. J. 2006, 82, 788.
3. www.who.int/tdr.
4. Pink, R.; Hudson, A.; Mouriès, M. A.; Bendig, M. Nat. Rev. Drugs Disc. 2005, 4,
727.
26. McKerrow, J. H.; Engel, J. C.; Cafrey, C. R. Bioorg. Med. Chem. 1999, 7, 639.
27. Cazzulo, J. J. Curr. Top. Med. Chem. 2002, 2, 1261.
28. Aparicio, I. M.; Scharfstein, J.; Lima, A. P. Infect. Immun. 2004, 72, 5892.
29. Roush, W. R.; Gwaltney, S. L.; Cheng, J.; Scheidt, K. A.; McKerrow, J. H.; Hansell,
E. J. Am. Chem. Soc. 1998, 120, 10994.
30. Ifa, D. R.; Rodrigues, C. R.; de Alencastro, R. B.; Fraga, C. A. M.; Barreiro, E. J.
J. Mol. Struct. Theochem. 2000, 505, 11.
31. Du, X.; Hansell, E.; Engel, J. C.; Caffrey, C. R.; Cohen, F. E.; McKerrow, J. H. Chem.
Biol. 2000, 7, 733.
32. Doyle, P. S.; Zhou, Y. M.; Engel, J. C.; McKerrow, J. H. Antimicrob. Agents
Chemother. 2007, 51, 3932.
33. Du, X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.; Holler, T. P.; McKerrow, J.
H.; Cohen, F. E. J. Med. Chem. 2002, 45, 2695.
34. Krauth-Siegel, R. L.; Inhoff, O. Parasitol. Res. 2003, 90, S77.
35. Henderson, G. B.; Ulrich, P.; Fairlamb, A. H.; Rosenmerg, I.; Pereira, M.; Sela, M.;
Cerami, A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5374.
36. Porcal, W.; Boiani, M.; Merlino, A.; Gerpe, A.; González, M.; Cerecetto, H. Org.
Proc. Res. Develop. 2008, 12, 156.
5. Rodrigues Coura, J.; Castro, S. L. Mem. Inst. Oswaldo Cruz 2002, 97, 3.
6. Schofield, C. J.; Jannin, J.; Salvatella, R. Trends Parasitol. 2006, 22, 583.
7. Murta, S. M. F.; Gazzinelli, R. T.; Brener, Z.; Romanha, A. J. Mol. Biochem.
Parasitol. 1998, 93, 203.
8. Cerecetto, H.; González, M. Curr. Top. Med. Chem. 2002, 2, 1185.
9. de Mecca, M. M.; Diaz, E. G.; Castro, J. A. Toxicol. Lett. 2002, 136, 1.
10. Kanes Hima, E. N.; Castro-Prado, M. A. Mem. Inst. Oswaldo Cruz 2005, 100, 325.
11. Castro, J. A.; de Meca, M. M.; Bartel, L. C. Hum. Exp. Toxicol. 2006, 25, 471.
12. Nwaka, S.; Ridley, R. G. Nat. Rev. Drug. Disc. 2003, 2, 919.
13. Maya, J. D.; Bollo, S.; Nuñez-Vergara, L. J.; Squella, J. A.; Repetto, Y.; Morello, A.;
Perie, J.; Chauviere, G. Biochem. Pharmacol. 2003, 65, 999.
14. Maya, J. D.; Cassels, B. K.; Iturriaga-Vásquez, P.; Ferreira, J.; Faúndez, M.;
Galanti, N.; Ferreira, A.; Morello, A. Comp. Biochem. Physiol. A Mol. Integr. Physiol.
2007, 146, 601.
15. Lazardi, K.; Urbina, J. A.; de Souza, W. Antimicrob. Agents Chemother. 1990, 34,
2097.
16!. Maldonado, R. A.; Molina, J.; Payares, G.; Urbina, J. A. Antimicrob. Agents
Chemother. 1993, 37, 1353.
17. Steenkamp, D. J. Antioxid. Redox Signal. 2002, 4, 105.
18. Kubata, B. K.; Kabututu, Z.; Nozaki, T.; Munday, C. J.; Fukuzumi, S.; Ohkubo, K.;
Lazarus, M.; Maruyama, T.; Martin, S. K.; Duszenko, M.; Urade, Y. J. Exp. Med.
2002, 196, 1241.
19. Turrens, J. F. Mol. Aspects Med. 2004, 25, 211.
20. Krauth-Siegel, R. L.; Schirmer, R. H.; Bauer, H. Angew. Chem. Int. Ed. 2005, 44, 690.
21. Maya, J. D.; Morello, A. J. Biol. Sci. 2005, 5, 847.
22. (a) Cerecetto, H.; Di Maio, R.; González, M.; Risso, M.; Saenz, P.; Seoane, G.;
Denicola, A.; Peluffo, G.; Quijano, C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1941;
(b) Aguirre, G.; Cerecetto, H.; Di Maio, R.; González, M.; Porcal, W.; Seoane, G.;
Denicola, A.; Ortega, M. A.; Aldana, I.; Monge-Vega, A. Arch. Pharm. 2002, 335,
15.
23. (a) Aguirre, G.; Boiani, L.; Boiani, M.; Cerecetto, H.; Di Maio, R.; González, M.;
Porcal, W.; Denicola, A.; Piro, O. E.; Castellano, E. E.; Sant’Anna, C. M. R.;
Barreiro, E. J. Bioorg. Med. Chem. 2005, 13, 6336; (b) Porcal, W.; Hernández, P.;
Aguirre, G.; Boiani, L.; Boiani, M.; Merlino, A.; Ferreira, A.; Di Maio, R.; Castro,
A.; González, M.; Cerecetto, H. Bioorg. Med. Chem. 2007, 15, 2768; (c) Boiani, L.;
Davies, C.; Arredondo, C.; Porcal, W.; Merlino, A.; Gerpe, A.; Boiani, M.; Pacheco,
J. P.; Basombrío, M. A.; González, M.; Cerecetto, H. Eur. J. Med. Chem. 2008,
doi:10.1016/j.ejmech.2007.12.016.
37. Alexanian, V.; Haddadin, M. J.; Issidorides, C. H.; Nazer, M. Z. Heterocycles 1981,
16, 391.
38. (a) Gasco, A.; Boulton, A. J.. In Advances in Heterocycles Chemistry; Katritzky, A.
R., Boulton, A. J., Eds.; Wiley: New York, 1981; Vol. 29, pp 251–340; (b)
Visentin, S.; Amiel, P.; Fruttero, R.; Boschi, D.; Roussel, C.; Giusta, L.; Carbone,
E.; Gasco, A. J. Med. Chem. 1999, 42, 1422; (c) Ermondi, G.; Visentin, S.; Boschi,
D.; Fruttero, R.; Gasco, A. J. Mol. Struct. 2000, 523, 149.
39. Almeida-de-Faria, M.; Freymuller, E.; Colli, W.; Alves, M. J. Exp. Parasitol. 1999,
92, 263.
40. Jockers-Scherübl, M. C.; Schirmer, R. H.; Krauth-Siegel, R. L. Eur. J. Biochem.
1989, 180, 267.
41. Olea-Azar, C.; Rigol, C.; Mendizábal, F.; Briones, R. Mini-Rev. Med. Chem. 2006, 6,
211.
42. (a) Dabideen, D. R.; Cheng, K. F.; Aljabari, B.; Miller, E. J.; Pavlov, V. A.; Al-Abed,
Y. J. Med. Chem. 2007, 50, 1993; (b) Sousa, C.; Freire, C.; de Castro, B. Molecules
2003, 8, 894; (c) Jain, M. P.; Kumar, S. Talanta 1979, 26, 909.
43. Hakki, E.; Cihat, S.; Mevlut, E.; Nuram, Y. J. Indian Chem. Soc. 1989, 66, 45.
44. Vieites, M.; Buccino, P.; Otero, L.; González, M.; Piro, O. E.; Sánchez Delgado, R.;
Sant’ Anna, C. M. R.; Barreiro, E. J.; Cerecetto, H.; Gambino, D. Inorg. Chim. Acta
2005, 358, 3065.
45. Messeder, J. C.; Tinoco, L. W.; Figueroa-Villar, J. D.; Souza, E. M.; Santa Rita, R.;
de Castro, S. L. Bioorg. Med. Chem. Lett. 1995, 5, 3079.
24. Porcal, W.; Hernández, P.; Boiani, M.; Aguirre, G.; Boiani, L.; Chidichimo, A.;
Cazzulo, J. J.; Campillo, N. E.; Paez, J. A.; Castro, A.; Krauth-Siegel, R. L.; Davies,
C.; Basombrío, M. A.; González, M.; Cerecetto, H. J. Med. Chem. 2007, 50, 6004.
25. (a) Olea-Azar, C.; Rigol, C.; Mendizabal, F.; Briones, R.; Cerecetto, H.; Di Maio,
R.; González, M.; Porcal, W.; Risso, M. Spectrochim. Acta, Part A 2003, 59, 69; (b)
46. Denicola, A.; Rubbo, H.; Rodriguez, D.; Radi, R. Arch. Biochem. Biophys. 1993,
304, 279.
47. Labriola, C.; Sousa, M.; Cazzulo, J. J. Biol. Res. 1993, 26, 101.
48. Sullivan, F. X.; Walsh, C. T. Mol. Biochem. Parasitol. 1991, 44, 145.