Benzo[1,2-c]1,2,5-oxadiazole N-oxide derivatives as potential antitrypanosomal drugs. Part 3: Substituents-clustering methodology in the search for new active compounds

Article abstract of DOI:10.1016/j.bmc.2005.05.020

The results of a study on the use of Hansch's series design, cluster methodology, for the generation of new benzo[1,2-c]1,2,5-oxadiazole N-oxide derivatives as antitrypanosomal compounds are described. In vitro activity of these compounds was tested against Tulahuen 2 strain of Trypanosoma cruzi. Clearly, the Hansch methodology allowed identifying two cluster-substituents suitable for further structural modifications. The most effective drugs, derivatives 11, 18, and 21, with 50% inhibitory concentration (IC₅₀) of the same order as that of the reference drug, represent an excellent structural point of chemical modifications for the design of future drugs. Preliminary results from the study of the mechanism of action of these benzofuroxans point to perturbation of the mitochondrial electron chain, inhibiting parasite respiration.

Full text of DOI:10.1016/j.bmc.2005.05.020

Bioorganic & Medicinal Chemistry 13 (2005) 6324–6335
Benzo[1,2-c]1,2,5-oxadiazole N-oxide derivatives as potential
antitrypanosomal drugs. Part 3: Substituents-clustering
methodology in the search for new active compoundsq
a
Gabriela Aguirre, Lucıa Boiani, Hugo Cerecetto,
a
a, ,
*
Rossanna Di Maio,a,*
´
a
Mercedes Gonzalez, Williams Porcal, Ana Denicola, Matıas Mo¨ller,
a
b
b
´
Leonor Thomson and Veronica Tortora
´
b
b
´
´
a
´
´
´
´
Departamento de Quımica Organica, Facultad de Quımica-Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay
b
´ ´ ´ ´
Laboratorio de Fisicoquımica Biologica y Enzimologıa, Facultad de Ciencias, Universidad de la Republica,
11400 Montevideo, Uruguay
Received 19 April 2005; revised 9 May 2005; accepted 9 May 2005
Available online 22 August 2005
Abstract—The results of a study on the use of HanschÕs series design, cluster methodology, for the generation of new benzo[1,2-c]1,2,
5-oxadiazole N-oxide derivatives as antitrypanosomal compounds are described. In vitro activity of these compounds was tested
against Tulahuen 2 strain of Trypanosoma cruzi. Clearly, the Hansch methodology allowed identifying two cluster-substituents suitable
for further structural modifications. The most effective drugs, derivatives 11, 18, and 21, with 50% inhibitory concentration (IC50) of the
same order as that of the reference drug, represent an excellent structural point of chemical modifications for the design of future
drugs. Preliminary results from the study of the mechanism of action of these benzofuroxans point to perturbation of the mitochondrial
electron chain, inhibiting parasite respiration.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
acute phase followed by a life-long chronic phase. The
acute infections are commonly asymptomatic stages.
ChagasÕ disease (CD) or American trypanosomiasis is
produced by several strains of the protozoan parasite
of the order Kinetoplastida Trypanosoma cruzi.1 This
disease represents a serious public health problem in
the countries and zones where it is endemic (21 countries
in Central and South America) because there are no
effective methods of immunoprophylaxis or chemother-
apy. Currently, 16–18 million persons are infected and
100 million are at risk.2 Cases have also been reported
in the United States.3 In humans, T. cruzi induces an
In some cases, especially in children, fever, swelling of the
lymph glands, enlargement of the liver and spleen, or lo-
cal inflammation at the site of infection have been evi-
denced. Patients with chronic infections are affected by
heart lesions or pathological dilations of the digestive
tract (megacolon and megaesophagus) and disorders of
nerve conduction of these organs. Patients with severe
chronic disease become progressively more ill and ulti-
mately die of heart failure.4 CD, and their consequences,
in immunocompromised patients has been reported as an
opportunistic infection that potentially produces mor-
bidity and loss of the patientsÕ quality of life.5 The forms
of T. cruzi present in the human host are the bloodstream
trypomastigote and the intracellular replicative amasti-
gote form. The existence of the epimastigote form as
an obligate mammalian intracellular stage has been
revisited6 and confirmed recently.7 The wide tissue distri-
bution of intracellular T. cruzi amastigotes during both
acute and chronic phases makes CD a more difficult sub-
ject for drug targeting in comparison to leishmaniasis
where amastigotes are restricted to macrophages and
Keywords: Benzofuroxan; T. cruzi; HanschÕs series design; Mitochon-
drial parasite respiration.
q Part of this research is presented in the Uruguayan patent of
´
invention: Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Porcal, W.;
Denicola, A. UR Patent No. 28.019, 2003: Derivados de 5-
´
´
etenilbenzofuroxano, procedimiento de preparacion y utilizacion.
Corresponding authors. Tel.: +598 2 5258618 x 216; fax: +598 2 525
07 49 (H.C.); e-mail: hcerecet@fq.edu.uy
*
´ ´
Present address: Igua 4225, Laboratorio de Quımica Organica,
´
´ ´
Instituto de Quımica Biologica, Facultad de Ciencias, Universidad
de la Repu´blica, 11400 Montevideo, Uruguay.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.020

G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
6325
human African trypanosomiasis is characterized in the
second stage of the disease by the presence of trypano-
somes in the central nervous system and cerebrospinal
fluid. Since vaccinations against trypanosomatic infec-
tions are still under development,8 the need for improv-
ing chemoprophylactic/chemotherapeutic approaches in
the control of these pathogens is indisputable. The
current chemotherapy against CD is still inadequate.9
The main drugs in use are Nifurtimoxꢁ (Nfx) and Ben-
znidazoleꢁ; however, they have undesirable side effects
and are yet inefficient to treat chronic CD, which can
be regarded as a disease with no cure. Furthermore,
the production and commercialization of Nfx have been
discontinued by the pharmaceutical company.
acceptor and donor.13b The use of five substituent-clus-
ters in the design of the new benzofuroxans enabled us
to identify lead compounds, so it was unnecessary to
study the superior number of Hansch clusters (i.e., 10,
20, or 60). To corroborate the biological relevance of
the N-oxide group some derivatives were converted into
the corresponding deoxygenated analogues. In addition,
the heme-reduction products of some derivatives (i.e., o-
nitroanilines) were prepared to investigate their activity
and to compare with the parent compounds.
The biological activity was evaluated in vitro against
Tulahuen 2 strain of T. cruzi. Finally, in order to gain
insight into the mechanism of action, the benzofuroxansÕ
capacity to produce reactive nitrogen and oxygen species
was studied. Thus, their NO production capacity, their
parasiteÕs respiration inhibition capacity, and their po-
tential production of superoxide were analyzed.
We have previously reported a series of benzo[1,2-c]-
1,2,5-oxadiazole N-oxide derivatives (benzofuroxans)
with anti-T. cruzi activity.10 Derivatives 1–3 (Fig. 1) pro-
duced the best in vitro trypanosome inhibitors. These
compounds were designed as new antitrypanosomal
agents with the hypothesis that the N-oxide moiety
would be responsible for generating toxic radical spe-
cies. For some of these benzofuroxans, free radical gen-
eration was proved and the radical species characterized
using ESR spectroscopy.11 Lipophilic–hydrophilic bal-
ance of the compounds also played an important role
in their effectiveness as antichagasic drugs. In addition,
the cytotoxicity of these compounds against mammalian
fibroblasts was comparable to that of Nfx.10a
2. Methods and results
2.1. Synthesis
Derivatives 4, 5, and 6 were prepared, using the appro-
priate nitrophenyl azides reactants, by cyclocondensa-
tion in boiling toluene (Scheme 1).14 Derivative 5 was
converted into nitrile 8 via cluster II-derivative 7.15
Alcohol 9 was obtained as previously reported from
aldehyde 5.10a Cluster I-derivatives 10 and 11 were also
obtained using derivative 5. Thus, derivative 10 was ob-
tained via a free solvent-Knoevenagel process between
aldehyde 5 and malononitrile in the presence of basic-
Al2O3.16 The Beirut reaction between benzofuroxan
and malononitrile to obtain quinoxaline dioxide was
minimized in these conditions.
Since these benzofuroxans were developed as analogues
of previously described nitrofuran derivatives,12 system-
atic structural modifications were not used in the design
process.
Herein, we report the design of new benzofuroxans
using substituents-clustering tools previously described
by Hansch and co-workers.13 The new structures were
designed initially based on the five substituent-clusters
shown in Chart 1. Hansch and co-workers established
these clusters using the substituent variables p, F, R,
MR, and the capacity to establish hydrogen bonds as
When derivative 5 was reacted at low temperature with
nitromethane and potassium t-butoxide followed by
treatment with methanesulfonyl chloride and triethyl-
amine,17 derivative 11 was obtained with a moderate
global yield (11% in two steps). The E-stereochemistry
of derivative 11 was established using the corresponding
1H NMR coupling constant between olefinic protons
(J = 13.7 Hz). Under the conditions used for the first
step (i.e., nitroaldolic process), formation of the corre-
sponding 1-hydroxybenzimidazole 3-oxide from benzo-
furoxan and nitromethane was not detected.18 The
cluster III-benzofuroxan derivatives 14–18 were pre-
pared as shown in Scheme 2.10a,14c,19 Derivative 14
was prepared from o-nitroaniline by reaction with so-
dium hypochlorite in basic medium while derivatives
15–17 were prepared, using the appropriate nitrophenyl
azide reactants by cyclocondensation in boiling toluene.
Chloromethyl derivative 18 was prepared as previously
described from alcohol 9.10a In this process deoxygen-
ated derivative 19 was obtained as a secondary product.
From the reaction between chloride 18 and excess of
potassium iodide, in the presence of crown ether 18-
crown-6, was obtained cluster IV-derivative 20 (Scheme
2). The reaction was difficult to follow by TLC (SiO2,
diethyl ether/ethyl acetate (10%)), because of the very
close Rf values of reactant 18 and product 20.
Figure 1. Benzofuroxan derivatives with anti-T. cruzi activity.
Chart 1. Clusters and benzofuroxan substituents selected from
HanschÕs series design methodology.

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G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
Scheme 1. Synthesis of cluster I- and cluster II-benzofuroxan derivatives.
Scheme 2. Synthesis of cluster III- and cluster IV-benzofuroxan derivatives.
Bodens–Wittig methodology, depicted in Scheme 3, was
used to prepare the olefin 21.20 In this process both
geometric isomers, Z-21 and E-21, were generated and
isolated independently using column chromatography
(SiO2, petroleum ether/ethyl acetate (10%)). Attempts
to obtain phenyl derivative 22 from bromo derivative
17, using Suzuki methodology, were fruitless. Bro-
mobenzofuroxan, 17, did not react under any of the as-
sayed conditions.21 Derivative 22 was obtained from
o-nitroaniline following the sequence shown in Scheme
3 with a good global yield (42% from o-nitroaniline).
To prepare cluster V-derivatives (compounds 25 and
26, Scheme 4) nucleophilic substitution processes were
assayed using bromo derivative 17 as reactant.22

G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
6327
Scheme 3. Synthesis of derivatives 21 and 22 from cluster IV.
Scheme 4. Synthesis of cluster V-benzofuroxan derivatives.
However, it was not possible to obtain the desired prod-
ucts. In the case of reaction with sodium methoxide the
deoxygenated derivative 24 was obtained as the main
product, whereas when bromo-derivative 17 was treated,
under different conditions, with secondary amines (i.e.,
morpholine), o-nitrophenylhydrazine derivatives were
obtained.23 Finally, derivatives 25 and 26 were obtained
from the corresponding o-nitroaniline as shown in
Scheme 4.14c,24
diate 23 (Scheme 3), the 21-deriving o-nitroaniline was
developed. Compound 28 (an inseparable mixture of
Z- and E-isomers) was prepared following the sequence
shown in Scheme 5.
Benzofuroxan derivatives exist as a mixture of isomers
at room temperature (A and B, Fig. 2); equilibrium con-
centrations are dependent on a number of factors (i.e.,
the nature and the position of the substituents at the
ring).26 The room temperature (303 K) 1H and 13C
NMR spectra of the benzofuroxans show benzo-protons
and -carbons as broad peaks, indicating extensive
benzofuroxan tautomerism. The spectra (1H, 13C NMR,
and HMQC, HMBC, and COSY experiments) were sim-
plified at low temperature, from 263 to 230 K, where it
was observed that one of these isomers predominated.27
In order to corroborate the effect of the N-oxide moiety
in the displayed activity, besides the sub-products 19
(Scheme 2) and 24 (Scheme 3), we developed the 21-
deoxygenated analogue 27 (Scheme 5). The reaction
between E/Z-21 with triphenylphosphine (Ph3P), in boil-
ing ethanol, yielded derivative E/Z-27.25 Otherwise, to
study the effect of the possible ferrous reduction-
metabolic product in the biological response, besides
commercial products 12 and 13 (Scheme 2) and interme-
All new compounds were identified by IR, MS, 1H
NMR, 13C NMR, COSY, and HETCOR experiments,

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G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
Scheme 5. Synthesis of deoxy derivative 27 and o-nitroaniline 28.
Table 1. In vitro activity of benzofuroxan derivatives and Nfx on
T. cruzi (Tulahuen strain) at 25 lM
Compound
Percentage of growth inhibition (%)a,b
Nfx
2
100.0
90.0
Cluster I
4
Figure 2. Benzofuroxan tautomeric equilibria at room temperature.
89.0
67.0
9.0
5
and their purity was established by TLC and
microanalysis.
8
10
11
66.0
92.0
2.2. Biological characterization
Cluster II
6
7
9
1.0
38.0
21.0
2.2.1. Antitrypanosomal activities. All compounds were
tested in vitro against T. cruzi. Epimastigote forms of
T. cruzi, Tulahuen 2 strain, were grown in axenic media
as described in Section 5. The compounds were incorpo-
rated into the media at 25 lM and their ability to inhibit
the parasite growth was evaluated in comparison to the
control (deprived of drug). Nfx was used as the reference
trypanocidal drug. Growth of the parasite was followed
by measuring the increase in absorbance at 600 nm,
which was proved to be proportional to the number of
cells present.10,12 The percentage of inhibition, summa-
rized in Table 1, was calculated as follows: % =
{1 À [(Ap À A0p)/(Ac À A0c)]} · 100, where Ap = A600 of
the culture containing the drug at day 5; A0p = A600 of
the culture containing the drug right after addition of
the inocula (day 0); Ac = A600 of the culture in the ab-
sence of any drug (control) at day 5; A0c = A600 in the
absence of the drug at day 0. The IC50 concentration
(50% inhibitory concentration) was assessed for com-
pounds presenting higher trypanocidal activity. Read-
ings were done on day 5 of growth, and IC50 values
determined as the drug concentration required to reduce
by half, the absorbance of that of the control (without
drug) (Fig. 3).
Cluster III
14
15
16
17
18
35.0
22.0
2.0
5.0
97.0
Cluster IV
20
E-21
93.0
93.0
Z-21
E-21:Z-21 (50:50)
22
100.0
97.0
31.0
Cluster V
25
26
10.0
26.0
a Inhibition of epimastigotes growth, concentration = 25 lM.
b The results are the means of three different experiments with SD less
than 10% in all cases.
ity, derivative 5, and two benzofuroxans with low activ-
ity, derivatives 8 and 25. The capacity to act as nitric
oxide (NO)-releasing agents, effects on mitochondria-
dependent superoxide production and parasite respira-
tion28 were studied for selected benzofuroxans.
2.3. Approaches to the study of mechanism of action
In order to gain insight into the molecular mechanism of
antitrypanosomal activity of benzofuroxans, we devel-
oped some preliminary studies on a selected series of
derivatives. We selected for the studies three benzofu-
roxans with high antitrypanosomal activity, derivatives
11, 18, and E-21, one benzofuroxan with medium activ-
2.3.1. Evaluation of benzofuroxans as NO donors.
According to recent descriptions, some benzofuroxan
derivatives are capable of acting as NO-releasing com-
pounds.15 NO is an important messenger implicated in
the regulation of numerous biological processes.29 In

G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
6329
derivative 11 was able to yield nitrite both in the absence
(10% nitrite yield after 1 h at 37 ꢂC) and in the presence
of thiol (15% nitrite yield after 1 h at 37 ꢂC) (Fig. 4).
2.3.2. Oxygen uptake and cyanide effect. The six selected
benzofuroxans and Nfx were studied as described in
Section 5, investigating their effect on parasite respira-
tion.30 Figure 5A shows time-dependent inhibition of
T. cruzi respiration by selected benzofuroxan deriva-
tives. Clearly, it was seen that the most active benzofu-
roxans did not increase oxygen uptake but rather
significantly inhibited cellular respiration. Figure 5B
shows the effect of cyanide on parasite respiration on
incubation with the benzofuroxans. Fifty micromolar
of NaCN was able to inhibit respiration up to 95% in
the control run, thus, cyanide-insensitive respiration
only 5%; whereas incubation with Nfx significantly in-
creased it to 50%. On the other hand, the effect of the
benzofuroxan derivatives on cyanide-insensitive oxygen
uptake was minor, except for derivatives 5, 11, and E-
21 (Fig. 5B). However, the observed result with deriva-
tive 5 could be an artifact due to the special reactivity
of this compound. Thus, in an independent experiment
it was demonstrated, using TLC, that derivative 5
(25 lM) rapidly reacted with NaCN (50 lM) dramati-
cally reducing cyanide concentration in the medium.
Figure 3. Dose–response curves as anti-T. cruzi for selected benzo-
furoxan derivatives, 11 (s), E-21 (n), E-21:Z-21 (50:50) (,), and Nfx
(j).
particular, it plays a crucial role in the immune response
by its cytotoxic action on macrophages and leukocytes.
Thus, benzofuroxan-trypanocidal activities could be
related to their potential NO release capacity. Griess
methodology was used as a primary screening of
NO-donor capacities, measuring nitrite yields generated
by benzofuroxans incubated either with or without
20 mM cysteine (1 h at 25 or 37 ꢂC). Most derivatives
ꢀ
tested did not release NO, even at high concentrations
(400 lM) and in the presence of excess of cysteine. Only
3. Discussion
Some of the benzofuroxan derivatives investigated show
excellent antitrypanosomal activity, similar to that of
Nfx, at 25 lM. In particular, derivatives 11 and 18 re-
sulted in the most active benzofuroxans described until
now. The Hansch methodology proved, in this case, to
be an excellent tool in the search for new anti-T. cruzi
agents. Clearly, it was observed that substituents con-
tained in clusters I and IV yielded the most active com-
pounds. The mesomeric electron-withdrawing properties
of cluster I-substituents could be used to explain the
activity and in the case of cluster IV-substituents the vol-
ume should be taken into account to explain this fact.27
Figure 4. Nitrite yield by benzofuroxan derivatives after 1 h at pH 7.4
and 37 ꢂC, in the absence (white columns) or in the presence (black
columns) of 50-fold excess of cysteine.
Figure 5. (A) Time-dependent inhibition of T. cruzi respiration in the presence of 25 lM of benzofuroxan derivatives 11 (.), 18 (d), and E-21 (m).
Control was run with no drug and 0.5% DMSO (j). (B) Effect of cyanide (NaCN, 50 lM) on respiration. Left: Oxygen consumption by T. cruzi
(1 mg protein) incubated for 5 days in the absence (A) or presence (B) of 25 lM of derivative E-21. The numbers represent rates of oxygen
consumption (in lmol minÀ1 mg proteinÀ1). Right: NaCN-insensitive oxygen uptake. For each benzofuroxans (25 lM) and control (no drug), the
ratio of rates of oxygen consumption before and after addition of NaCN was calculated as percentage, as mean SD of two independent
experiments (*statistical difference with control for StudentÕs t test, p < 0.05).

6330
G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
On the other hand, it is possible to observe that substit-
uents contained in clusters II, III (except for derivative
18), and V yielded the least active compounds. The sub-
stituents acting as hydrogen bond donors belong to clus-
ter II and mesomeric electron-releasing substituents to
cluster V. These properties could be playing some role
in the observed deficient trypanocidal activity.27 Four
out of five derivatives in cluster III were inactive. How-
ever, derivative 18 of cluster III is one of the most active
developed products. In this case the substituent chloro-
methyl could be relocated in another cluster, or the
Hansch methodology used with a superior number of
clusters (10, 20 or 60). Nevertheless, in our opinion,
derivative 18 was especially active because it shares par-
ticular structural characteristics that enhance its activity:
the benzylic-like electrophilic center (chloromethyl sub-
stituent). This good electrophile center, such as in deriv-
ative 20 or in parent compound 3, could react with
biological nucleophiles (amines and phosphates from
DNA, thiols, alcohols, and amines from proteins)
enhancing its cytotoxicity. Derivatives 20 and 21 (as
Z-isomer or E:Z-mixture) showed IC50 values similar
to the parent compound 2 and of the same order as
Nfx. However, derivative 21 resulted in a more attrac-
tive lead compound because its structure allows further
synthetic modifications. For the different geometric iso-
mers of derivative 21 we could observe that the spatial
distribution did not significantly affect the activity. The
50:50 mixture of Z- and E-isomers showed similar
IC50 to that of the Z-isomer.
culture medium in the in vitro tests, we evaluated some
benzofuroxan metabolic products in order to investigate
whether the benzofuroxan bio-activities were the result
of the hemine-reduction products (o-nitroanilines).
Clearly, it was demonstrated that the trypanocidal activ-
ity is not due to the corresponding ferrous-reduced
derivatives (Table 2). When the ferrous-reduced metab-
olite (28) of the most active derivative 21 was evaluated
in vitro, a complete lack of activity was observed. The
same behavior was observed for the ferrous-reduced
metabolite of derivative 15, compound 13. However,
o-nitroanilines 12 and 23 presented similar activities as
the corresponding benzofuroxans, 14 and 22, respec-
tively, but this result could be due to the toxicity of
anilines.
In order to gain insight into the metabolism of benzofu-
roxans, the generation of metabolite 28 (as Z and E mix-
ture) in the T. cruzi culture treated with compound 21
(as Z and E mixture) was investigated. Using TLC anal-
ysis, it was possible to observe the formation of a new
metabolic product after 4 h of treatment. This com-
pound did not correspond to the o-nitroaniline 28. The
structure of this metabolite was not further character-
ized but could be like the mammalian microsomal-
metabolite recently described for benzofuroxan.33
The benzofuroxan-trypanocidal activity is not the result
of their NO-release capacity, since only the active nitro-
alkene 11 yielded nitrite but at a concentration 200-fold
superior to the corresponding IC50. This capacity could
be related to the substituent (2-nitroalkenyl) rather than
the benzofuroxan system. Also, it was observed that the
benzofuroxansÕ trypanocidal activity is related to their
capacity to inhibit cellular respiration. In the absence
of drugs, cyanide-insensitive respiration is minor (close
to 5%), suggesting that cytochrome aa3 is the main T.
cruzi terminal oxidase as observed before.30 Treatment
with Nfx increased the cyanide-insensitive respiration
to more than 50%, suggestive of superoxide/H2O2 pro-
duction. This is in agreement with previous ESR results
consistent with the trapping of hydroxyl radical
(DMPO–OH adduct) when incubating an Nfx analogue
with T. cruzi homogenates and the spin trap.11b How-
ever, treatment with the most active benzofuroxans did
not increase the cyanide-insensitive respiration to such
high levels (up to 22% for derivative 11), suggesting a
different mechanism of action than nitrofuranes like
Nfx.
As observed previously,10a,31 the absence of the N-oxide
moiety produced a total loss of activity, confirming that
this group plays a role in the mechanism of the benzofu-
roxansÕ trypanosome toxicity (compare activity of deriv-
ative 21 with activity of deoxy-derivative 27, Table 2). In
the same manner, it was possible to observe the dra-
matic decay in the activity of derivative 18 when it was
deoxygenated (19). In the case of derivative 17 and the
deoxy-analogue 24 the difference in activity was not
too clear because derivative 17 itself was poorly active.
Recently, the capacity of ferrous salts and oxyhemoglo-
bin to reduce benzofuroxan derivatives to the corre-
sponding o-nitroanilines was described,32 suggesting
that blood is a possible site for metabolizing benzofu-
roxans. Because hemine is incorporated into the T. cruzi
Table 2. In vitro activity on T. cruzi of deoxygenated and ferrous-
reduced derivatives at 25 lM
On the other hand, if we consider the potential use of
benzofuroxan derivatives as drugs, these should present
adequate behavior in vivo according to LipinskiÕs rules
and their topological polar surface area (TPSA).34
Lipinski described the desired ranges for certain proper-
ties thought to be important for pharmacokinetics and
drug development. They are: C log P < 5, number of
hydrogen bond donors 65, number of hydrogen bond
acceptors 610, and molecular weight <500. A com-
pound that fulfills at least three of these criteria adheres
to LipinskiÕs rule. Another very helpful parameter for
the prediction of absorption is the polar surface area
(PSA), defined as the sum of surfaces of polar atoms
Percentage of growth inhibition (%)a,b
Parent compound
Deoxygenated
analogue
Ferrous-reduced
derivative
14, 35.0
15, 22.0
—
—
12, 26.0
13, 0.0
—
17, 5.0
18, 97.0
24, 0.0
19, 28.0
27, 10.0
—
—
28, 0.0
23, 41.0
E-21:Z-21 (50:50), 97.0
22, 31.0
a Inhibition of epimastigotes growth, concentration = 25 lM.
b The results are the means of three different experiments with SD less
than 10% in all cases.

G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
Table 3. Nfx and the properties of benzofuroxans to fulfill Lipinski rule
6331
Ref
Lipinski rule
TPSAb,c
ClogPa
No. of H-bond donorsb
No. of H-bond acceptorsb
Mol wt
No. of criteria met
Rule
4
<5
65
0
610
7
<500
181.0
164.0
207.0
184.5
276.0
238.0
287.0
At least 3
3.92
2.07
4.36
2.88
2.95d
4.76
8.51
All
All
All
All
All
All
3
97.3
68.6
97.3
51.5
51.5
51.5
108.7
5
0
5
11
18
20
21
Nfx
0
7
0
4
0
4
0
4
0
8
a Theoretical LogP (ClogP) was calculated using Villar method, at AMI semiempirical method, implemented in SpartanÕ04, 1.0.1 version, suite of
programs.36
b http://www.molinspiration.com/cgi-bin/properties.
c TPSA, topological polar surface area.
d This value was obtained from http://www.molinspiration.com/cgi-bin/properties because parameters for iodine atom are not available into Villar
method.
in a molecule, which provides good correlation with
experimental transport data (intestinal absorption,
Caco-2 monolayers penetration, and blood–brain bar-
rier crossing).35 Table 3 lists such physicochemical prop-
erties for the trypanocidal benzofuroxan derivatives. All
of the potent antiparasitic benzofuroxans presented
herein are fully compatible with LipinskiÕs rule and pos-
sess better topological polar surface area (TPSA) than
Nifurtimox.
Stage Model 350 apparatus and are uncorrected. Ele-
mental analyses were obtained from vacuum-dried sam-
ples (over phosphorous pentoxide at 3–4 mmHg, 24 h at
room temperature) and performed on a Fisons EA 1108
CHNS-O analyzer, and were within 0.4% of theoretical
values. Infrared spectra were recorded on a Perkin-
Elmer 1310 apparatus, using potassium bromide tablets
for solid and oil products (the frequencies are expressed
in cmÀ1). 1H NMR and 13C NMR spectra were recorded
on a Bruker DPX-400 (at 400 and 100 MHz) instru-
ment, with tetramethylsilane as the internal reference
and in the indicated solvent; the chemical shifts are re-
4. Conclusions
1
ported in ppm. The H and 13C NMR signals reported
A new series of benzo[1,2-c]1,2,5-oxadiazole N-oxide
was synthesized using a facile methodology. The preli-
minary antitrypanosomal studies of these derivatives
showed that some of them exhibit significant in vitro
activity. The N-oxide moiety of these compounds partic-
ipates in the pharmacophore, whereas the ferrous-
reduced analogues (potential metabolites using heme
from the culture) are not responsible for the antitry-
panosomal activity observed. The excellent in vitro
activities suggest that compounds like 11, 18, 20, and
21 (as a mixture of geometric isomers), and to a lesser
extent derivatives 4 and 5, represent a new class of
anti-T. cruzi agents. Further structural optimization
and in vivo studies to investigate the ability of these
drugs to decrease the parasitemia of infected mice and
the toxicity are currently under way.
were obtained at room temperature. In 13C NMR data
only narrow peaks were reported. Bruker software was
used to perform NOE, COSY, HMQC, and HMBC
experiments. Mass spectra were recorded on a Shimadzu
GC-MS QP 1100 EX instrument, for electronic impact
at 70 and 20 eV, or chemically ionized with methane.
5.1.1. 5-(2,2-Dicyanoethenyl)-N1-oxidebenzo[1,2-c]1,2,5-
oxadiazole (10). A mixture of aldehyde 5 (300 mg,
1.8 mmol), Al2O3 (typ T, 550 mg), malononitrile
(120 mg, 1.8 mmol), and dried CH2Cl2 (3 mL) was stir-
red at room temperature for 24 h. Then, the Al2O3
was filtered and washed with excess of CH2Cl2. The or-
ganic solvent was evaporated in vacuo and the residue
was purified by crystallization from CHCl3, brown
needles (230 mg, 40%); mp 135.0–136.5 ꢂC; IR mmax
1
3073, 2228, 1605, 1570, 1485 cmÀ1; H NMR (acetone-
d6, 400 MHz) d: 7.93 (br s, 1H), 8.03 (br s, 1H), 8.24
(br s, 1H), 8.44 (s, 1H); 13C NMR (acetone-d6,
100 MHz) d: 86.51, 112.72, 113.75, 134.50; MS, m/z
(abundance): 212 ([M]+ꢀ, 100%), 196 (27%), 166 (17%),
152 (27%). Anal. (C10H4N4O2) C, H, N.
5. Experimental
5.1. Chemistry
All starting materials were commercially available re-
search-grade chemicals and were used without further
purification. Compounds 4–9, 14–18, 25, and 26 were
prepared according to the literature.10,15,19–21 All sol-
vents were dried and distilled prior to use. All the reac-
tions were carried out in a nitrogen atmosphere. The
typical work-up included washing with brine and drying
the organic layer with sodium sulfate. Melting points
were determined using a Leitz Microscope Heating
5.1.2.
oxadiazole (11)
5-(2-Nitroethenyl)-N1-oxidebenzo[1,2-c]1,2,5-
5.1.2.1. 5-(1-Hydroxy-2-nitroethyl)-N1-oxidebenzo[1,
2-c]1,2,5-oxadiazole. A mixture of CH3NO2 (160 mg,
2.6 mmol), t-BuOK (32 mg, 0.3 mmol), and THF:t-BuOH
(1:1, 26 mL) as solvent was cooled at 0 ꢂC. Then, a solution
of aldehyde 5 (400 mg, 2.4 mmol) in THF (12 mL) was
added slowly. The final mixture was stirred during 24 h

6332
G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
at 0 ꢂC. The solvent was evaporated in vacuo and the resi-
due was treated with aqueous HCl (5%) (20 mL) and ex-
tracted with AcOEt (3 · 10 mL). After the work-up the
organic solvent was evaporated in vacuo. The product
was obtained as an oil (300 mg, 55%) and was used in
12%), 149 (100%), 133 (9%), 89 (54%). Anal.
(C7H5IN2O2) C, H, N.
5.1.5. 5-(2E/Z-Phenylethenyl)-N1-oxidebenzo[1,2-c]1,2,5-
oxadiazole (21). A mixture of aldehyde 5 (100 mg,
1
the next reaction without further purification; H NMR
0.6 mmol),
benzyltriphenylphosphonium
chloride
(acetone-d6, 400 MHz) d: 2.95 (br s, 1H), 4.73 (dd,
J1 = 9.3 Hz, J2 = 13.0 Hz, 1H), 4.95 (dd, J1 = 3.2 Hz,
J2 = 13.0 Hz, 1H), 5.69 (dd, J1 = 2.8 Hz, J2 = 9.4 Hz,
1H), 7.61 (br s, 2H), 7.70 (br s, 1H).
(280 mg, 0.7 mmol), K2CO3 (100 mg, 0.7 mmol), 18-
crown-6 (1.8 mg), and THF (3 mL) as solvent was stir-
red at reflux during 15 min. The solvent was evaporated
in vacuo, the residue was treated with brine (5 mL) and
extracted with EtOAc (3 · 10 mL). After the work-up
the organic layer was evaporated in vacuo. The residue
was purified by column chromatography (SiO2, petro-
leum ether/EtOAc (9:1)), yielding derivative E-21 as a
yellow solid (73 mg, 50%) (Rf = 0.75, SiO2, petroleum
ether/EtOAc (8:2)) and derivative Z-21 as a yellow solid
(19 mg, 13%) (Rf = 0.65, SiO2, petroleum ether/EtOAc
(8:2)); E-21: mp 143.8–145.5 ꢂC; IR mmax 1614, 1572,
5.1.2.2. 5-(2-Nitroethenyl)-N1-oxidebenzo[1,2-c]1,2,5-
oxadiazole (11). A mixture of 5-(1-hydroxy-2-nitro-
ethyl)-N1-oxidebenzo[1,2-c]1,2,5-oxadiazole
(300 mg,
1.3 mmol), MeSO2Cl (152 mg, 1.3 mmol) and CH2Cl2
as solvent (1.4 mL) was stirred at 0 ꢂC for 2 h. Et3N
(0.25 mL) was added and the mixture was stirred for
30 min. Then EtOAc (30 mL) was added and the organic
layer was washed with H2O (10 mL), aqueous HCl
(10%) (10 mL), and brine (10 mL). After the work-up
the organic solvent was evaporated in vacuo. The resi-
due was purified by column chromatography (SiO2,
petroleum ether/EtOAc (9:1)) and the product was crys-
tallized from EtOH, orange solid (100 mg, 20%); mp
1525, 1485, 962, 758, 735 cmÀ1 1H NMR (CDCl3,
;
400 MHz) d: 7.09 (d, J = 16.3 Hz, 1H), 7.21 (d,
J = 16.3 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 7.40 (t,
J = 7.6 Hz, 2H), 7.54 (d, J = 7.3 Hz, 2H), 7.60 (br s,
3H); 13C NMR (acetone-d6, 100 MHz) d: 109.00 (br s),
114.00 (br s), 119.00 (br s), 126.52, 127.46, 129.36,
129.43, 133.61, 136.32; MS, m/z (abundance): 238
([M]+ꢀ, 90%), 222 (16%), 192 (16%), 178 (100%), 149
(3%). Anal. (C14H10N2O2) C, H, N. Z-21: mp 61.5–
141.5–143.0 ꢂC; IR mmax 3100, 1635, 1608, 1533 cmÀ1
;
1H NMR (acetone-d6, 400 MHz) d: 7.76 (br s, 1H),
7.86 (br s, 1H), 8.17 (br s + d, J = 13.7 Hz, 2H), 8.18
(d, J = 13.7 Hz, 1H); 13C NMR (acetone-d6, 100 MHz)
d: 118.29 (br s), 136.29, 140.87; MS, m/z (abundance):
207 ([M]+ꢀ, 100%), 191 (12%), 166 (17%), 160 (55%),
144 (21%). Anal. (C8H5N3O4) C, H, N.
62.9 ꢂC; IR mmax 1610, 1570, 1520, 1480 cmÀ1
;
1H
NMR (CDCl3, 400 MHz) d: 6.74 (d, J = 12.2 Hz, 1H),
6.94 (d, J = 12.2 Hz, 1H), 7.18 (br s, 1H), 7.33 (br s,
5H), 7.42 (br s, 2H); MS, m/z (abundance): 238 ([M]+ꢀ,
3%), 222 (30%), 192 (25%), 178 (26%), 149 (40%). Anal.
(C14H10N2O2) C, H, N.
5.1.3. 5-Chloromethylbenzo[1,2-c]1,2,5-oxadiazole (19).
A mixture of alcohol 9 (1.0 g, 6.0 mmol), SOCl2
(1.3 mL, 18.0 mmol), and DMF (0.15 mL) was stirred
at reflux during 3 h. The mixture was poured onto
aqueous NaHCO3–ice and the aqueous mixture was
extracted with EtOAc (3 · 30 mL). The combined
extracts were dried and evaporated in vacuo. The resi-
due was purified by column chromatography (SiO2,
petroleum ether/EtOAc (9:1)), yielding derivative 18 as
a colorless oil (440 mg, 40%) (Rf = 0.40, SiO2, petroleum
ether/EtOAc (8:2)) and derivative 19 as a colorless oil
(110 mg, 10%) (Rf = 0.45, SiO2, petroleum ether/EtOAc
5.1.6. 5-Phenyl-N1-oxidebenzo[1,2-c]1,2,5-oxadiazole (22)
5.1.6.1. 4-Iodo-2-nitroaniline. A mixture of o-nitroan-
iline (1.0 g, 7.3 mmol), I2 (910 mg, 3.6 mmol), HIO3
(640 mg, 3.6 mmol), and acetic acid (10 mL) was stirred
at 50 ꢂC during 3 h. The solvent was evaporated in va-
cuo, the residue was treated with H2O (20 mL) and
neutralized with aqueous NaOH (6 N). The orange pre-
cipitate was filtered and dried (1.80 g, 94%) and was
used in the next reaction without further purification;
1H NMR (CDCl3, 400 MHz) d: 6.11 (br s, 2H), 6.62
(d, J = 8.7 Hz, 1H), 7.58 (dd, J1 = 2.0 Hz, J2 = 8.7 Hz,
1H), 8.44 (d, J = 2.0 Hz, 1H); MS, m/z (abundance):
264 ([M]+ꢀ, 100%), 218 (26%), 91 (17%).
(8:2)); 19: IR mmax 1600, 1540, 1450 cmÀ1 1H NMR
;
(CDCl3, 400 MHz) d: 4.66 (s, 2H), 7.46 (dd,
J1 = 1.4 Hz, J2 = 9.3 Hz, 1H), 7.84 (d, J = 1.4 Hz, 1H),
7.88 (d, J = 9.5 Hz, 1H); MS, m/z (abundance): 168
([M]+ꢀ, 60%), 133 (70%), 117 (20%). Anal. (C7H5ClN2O)
C, H, N.
5.1.6.2. 4-Phenyl-2-nitroaniline (23). A mixture of
4-iodo-2-nitroaniline (132 mg, 0.5 mmol), NaOH
(80 mg, 2 mmol), phenylboronic acid (67 mg,
0.55 mmol), PdCl2 (2 mg), and H2O (1 mL) and MeOH
(2 mL) as solvent was stirred at room temperature dur-
ing 24 h. Then, the reaction was heated at 100 ꢂC during
2 h. The solvent was evaporated in vacuo and the resi-
due was treated with aqueous HCl (5%), pH neuter,
and extracted with EtOAc (3 · 10 mL). After the
work-up the organic layer was evaporated in vacuo.
The residue, brown solid, was the product (80 mg,
75%) and was used in the next reaction without further
purification; 1H NMR (acetone-d6, 400 MHz) d: 7.07 (br
s, 2H), 7.20 (d, J = 8.8 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H),
7.46 (t, J = 8.0 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.78
5.1.4. 5-Iodomethyl-N1-oxidebenzo [1,2-c]1,2,5-oxadiaz-
ole (20). A mixture of chloride 18 (100 mg, 0.54 mmol),
KI (450 mg, 2.7 mmol), 18-crown-6 (680 mg, 2.7 mmol),
and DMF as solvent (2 mL) was stirred at room temper-
ature for 4 h. Then H2O (20 mL) was added and ex-
tracted with EtOAc (3 · 20 mL). After the work-up the
organic solvent was evaporated in vacuo. The residue
was purified by column chromatography (SiO2, petro-
leum ether/EtOAc (95:5)), colorless oil that crystallized
at 4 ꢂC (45 mg, 30%); IR mmax 2957, 1620, 1593, 1537,
1480 cmÀ1 1H NMR (CDCl3, 400 MHz) d: 4.42 (s,
;
2H), 7.5 (br s, 3H); MS, m/z (abundance): 276 ([M]+ꢀ,

G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
6333
(dd, J1 = 2.2 Hz, J2 = 8.7 Hz, 1H), 8.33 (d, J = 2.2 Hz,
1H); 13C NMR (acetone-d6, 100 MHz) d: 120.27,
123.32, 126.38, 127.23, 127.44, 129.19, 129.33, 134.56,
139.37, 145.62; MS, m/z (abundance): 214 ([M]+ꢀ,
100%), 184 (4%), 168 (32%).
at reflux for 8 h. The precipitate, corresponding with
the desired product, was filtered and washed with dry
toluene (white needles (11.4 g, 62%)). The salt was used
without further purification.
5.1.8.3. 4-(2E/Z-Phenylethenyl)-2-nitroaniline (28). A
mixture of benzaldehyde (150 mg, 1.4 mmol) (4-acetyl-
5.1.6.3. 5-Phenyl-N1-oxidebenzo[1,2-c]1,2,5-oxadiaz-
ole (22). NaOH (8 mg) was dissolved in EtOH 95%
(5 mL) with heating. Then, 23 (34 mg, 0.16 mmol) was
added to the hot ethanolic solution and an intense red
solution was obtained. The mixture was cooled at
0 ꢂC, aqueous NaClO (2.8 M) (0.2 mL) was added drop-
wise, and the reaction was stirred during 20 min. The
solvent was evaporated in vacuo and the residue was
neutralized with aqueous HCl (10%) and extracted with
EtOAc (3 · 10 mL). After the work-up the organic layer
was evaporated in vacuo. The residue was purified by
column chromatography (SiO2, petroleum ether/EtOAc
(9:1)), red-brown solid (20 mg, 59%); mp 122.0–
amino-3-nitrophenyl)methyltriphenyl
phosphonium
bromide (760 mg, 1.4 mmol), K2CO3 (200 mg,
1.4 mmol), 18-crown-6 (20.0 mg), and THF (10 mL) as
solvent was stirred at reflux during 8 h. The solvent
was evaporated in vacuo, the residue was treated with
brine (5 mL) and extracted with EtOAc (3 · 10 mL).
After the work-up the organic layer was evaporated
in vacuo. The residue was purified by column chromat-
ography (SiO2, petroleum ether/EtOAc (9:1)), yielding
4-(2E/Z-phenylethenyl)-2-nitroacetanilide as an orange
oil (332 mg, 84%). A mixture of this product (236 mg,
0.8 mmol), concentrated hydrochloride acid (0.3 mL),
and ethanol (10 mL) as solvent was heated at reflux dur-
ing 1 h. The solvent was evaporated in vacuo and the
residue was neutralized with aqueous saturated NaH-
CO3 and extracted with EtOAc (3 · 10 mL). After the
work-up the organic layer was evaporated in vacuo
and the residue corresponding to the product, pure by
TLC, was used without further purification (oil,
1
123.5 ꢂC; IR mmax 1610, 1566, 1525, 756, 715 cmÀ1; H
NMR (CDCl3, 400 MHz) d: 7.4 (m, 5H), 7.5 (br s,
3H); MS, m/z (abundance): 212 ([M]+ꢀ, 81%), 196
(2%), 166 (2%), 152 (58%). Anal. (C12H8N2O2) C, H, N.
5.1.7. 5-(2E/Z-Phenylethenyl)benzo[1,2-c]1,2,5-oxadiaz-
ole (27). A mixture of E/Z-21 (100 mg, 0.4 mmol),
Ph3P (110 mg, 0.4 mmol) and EtOH (30 mL) as solvent
was heated at reflux for 2 h. The EtOH was evaporated
in vacuo. The residue was washed with petroleum ether
to eliminate the unreacted Ph3P and then purified by col-
umn chromatography (SiO2, petroleum ether/EtOAc
(0–10%)), yellow oil (70 mg, 75%); IR mmax 3050, 1624,
1
188 mg, 98%). H NMR (CDCl3, 400 MHz) d: 6.14 (br
s, 2H), 6.83 (dd, J1 = 3.0 Hz, J2 = 8.7 Hz, 1H), 7.01 (s,
2H), 7.27 (br t, J = 7.7 Hz, 1H), 7.37 (br t, J = 7.3 Hz,
2H), 7.50 (br d, J = 6.6 Hz, 2H), 7.61 (d, J = 8.7 Hz,
1H), 8.24 (s, 1H); MS, m/z (abundance): 240 ([M]+ꢀ,
75%), 224 (9%).
1
1541, 962, 694 cmÀ1. H NMR (CDCl3, 400 MHz) d:
6.68 (d, J = 12.0 Hz, 0.5H), 6.89 (d, J = 12.0 Hz,
0.5H), 7.20 (d, J = 16.0 Hz, 0.5H), 7.28 (m, 3.5H), 7.36
(t, J = 7.3 Hz, 0.5H), 7.43 (t, J = 7.2 Hz, 1H), 7.59
(d + d + s, J = 7.9 Hz, J = 16.0 Hz, 1.5H), 7.69 (s,
0.5H), 7.76 (d, J = 8.8 Hz, 0.5H), 7.77 (d, J = 8.1 Hz,
0.5H), 7.84 (d, J = 9.7 Hz, 0.5H); MS, m/z (abundance):
222 ([M]+ꢀ, 32%), 205 (82%), 191 (45%), 165 (100%).
Anal. (C14H10N2O) C, H, N.
5.1.8.4. 5-(2E/Z-Phenylethenyl)-N1-oxidebenzo[1,2-c]-
1,2,5-oxadiazole (21). The crude of the hydrolysis pro-
cess (above reaction) was neutralized with NaOH
(150 mg) and then NaOH (150 mg) was added. The mix-
ture was cooled at 0 ꢂC, aqueous NaClO (2.8 M)
(1.5 mL) was added dropwise and the reaction was stir-
red during 30 min. Aqueous NaClO (2.8 M) (1.5 mL)
was added again and the mixture was stirred during
30 min. The solvent was evaporated in vacuo and the
residue was neutralized with aqueous HCl (10%) and ex-
tracted with EtOAc (3 · 10 mL). After the work-up the
organic layer was evaporated in vacuo. The residue,
brown solid (75%), was chromatographically (TLC)
identical with E/Z-21 obtained by the other procedure.
5.1.8. 4-(2E/Z-Phenylethenyl)-2-nitroaniline (28)
5.1.8.1. 4-Bromomethyl-2-nitroacetanilide. A mixture
of 4-methyl-2-nitroacetanilide (10.0 g, 52.0 mmol),
NBS (7.3 g, 41.0 mmol), PDBO (0.5 g, 2.1 mmol), and
CCl4 (100 mL) as solvent was heated under reflux during
5 h. Then, the precipitate, succinimide, was filtered and
washed with CCl4. The organic phase was evaporated
in vacuo and the residue corresponding to the product,
pure by TLC, was used without further purification
5.2. Biology
Trypanosoma cruzi epimastigotes (Tulahuen 2 strain)
were grown at 28 ꢂC in an axenic medium (BHI-Tryp-
tose) as previously described,10,12,37 complemented with
5% fetal calf serum. Cells from a 10-day-old culture (sta-
tionary phase) were inoculated into 50 mL of fresh cul-
ture medium to give an initial concentration of 1 · 106
cells/mL. Cell growth was followed by measuring every-
day the absorbance of the culture at 600 nm. Before
inoculation, the media were supplemented with the indi-
cated amount of the drug from a stock solution in
DMSO. The final concentration of DMSO in the culture
media never exceeded 0.4% and the control was run in
the presence of 0.4% DMSO and in the absence of any
1
(13.2 g, 95%). H NMR (CDCl3, 400 MHz) d: 2.31 (s
3H), 4.49 (s, 2H), 7.68 (dd, J1 = 2.1 Hz, J2 = 8.8 Hz,
1H), 8.25 (d, J = 2.1 Hz, 1H), 8.79 (d, J = 8.8 Hz, 1H),
10.34 (br s, 1H); 13C NMR (CDCl3, 100 MHz) d:
25.99, 31.34, 123.05, 126.32, 133.50, 135.14, 136.36,
136.82, 169.38; MS, m/z (abundance): 272/274 ([M]+ꢀ,
3/3%), 193 (45%), 151 (100%).
5.1.8.2. (4-Acetylamino-3-nitrophenyl)methyl triphenyl
phosphonium bromide. A mixture of 4-bromomethyl-2-
nitroacetanilide (13.2 g, 48 mmol), Ph3P (12.6 g,
48 mmol) in anhydrous toluene (100 mL) was heated

6334
G. Aguirre et al. / Bioorg. Med. Chem. 13 (2005) 6324–6335
A. J.; Stumf, R. J.; Kirchhoff, L. V.; Dodd, R. Y. J. Infect.
Dis. 1997, 176, 1047; (c) Cohen, J. E.; Tsai, E. C.;
Ginsberg, H. J.; Godes, J. Surg. Neurol. 1998, 49, 324.
4. Umezawa, E. S.; Stolf, A. M.; Corbett, C. E.; Shikanai-
Yasuda, M. A. Lancet 2001, 357, 797.
5. (a) Dutra, W. O.; Martins-Filho, O. A.; Cancado, J. R.;
Pinto-Dias, J. C.; Brener, Z.; Gazzinelli, G.; Carvalho, J.
F.; Colley, D. G. Scand. J. Immunol. 1996, 43, 88; (b)
drug. No effect on epimastigotes growth was observed
by the presence of up to 1% DMSO in the culture media.
To determine IC50 values, 50% inhibitory concentra-
tions, parasite growth was followed in the absence (con-
trol) and presence of increasing concentrations of the
corresponding drug. At day 5, the absorbance of the cul-
ture was measured and related to the control. The IC50
value was taken as the concentration of drug needed to
reduce the absorbance ratio to 50%.
´
´
Concetti, H.; Retequi, M.; Perez, G.; Perez, H. Hum.
Pathol. 2000, 31, 120; (c) Corti, M. AIDS Patient Care
STDS 2000, 14, 581.
6. (a) Almeida-de-Faria, M.; Freymuller, E.; Colli, W.;
Alves, M. J. Exp. Parasitol. 1999, 92, 263; (b) Faucher,
J. F.; Baltz, T.; Petry, K. G. Parasitol. Res. 1995, 81, 441.
7. Tyler, K. M.; Engman, D. M. Int. J. Parasitol. 2001, 31,
472.
8. (a) Wrightsman, R. A.; Manning, J. E. Vaccine 2000, 18,
1419; (b) Sepulveda, P.; Hontebeyrie, M.; Liegeard, P.;
Mascilli, A.; Norris, K. A. Infect. Immun. 2000, 68, 4986.
5.3. Studies of mechanism of action
ꢀ
5.3.1. Screening of benzofuroxans as NO donors. Nitric
oxide (ꢀNO) release was assessed by determining the
ꢀ
main aerobic decomposition product of NO, nitrite.38a
The benzofuroxans were assayed at a final 400 lM con-
centration in 0.1 M phosphate buffer, pH 7.4, either with
or without 20 mM cysteine for 1 h at 25 or 37 ꢂC. All as-
says contained 8% DMSO. Nitrite content was then
determined by Griess reaction.38b Derivative E-21 was
not assayed due to poor solubility.
´
9. Cerecetto, H.; Gonzalez, M. Curr. Topics Med. Chem.
2002, 2, 1185.
10. (a) Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.;
´
Saenz, P.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano,
C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1941; (b)
´
Porcal, W.; Seoane, G.; Denicola, A.; Ortega, M. A.;
Aldana, I.; Monge, A. Arch. Pharm. 2002, 335, 15.
11. (a) Olea-Azar, C.; Rigol, C.; Mendizabal, F.; Briones, R.;
5.3.2. Oxygen uptake.37 Trypanosoma cruzi epimastig-
otes (Tulahen 2 strain) from 5 days of growth were har-
vested by centrifugation (815 g for 15 min), washed and
resuspended in 0.14 M NaCl, 2.7 mM KCl, 8 mM Na2H-
PO3, 15 mM KH2PO4, pH 7.4 (medium A). Protein con-
centration was determined by Biuret assay in the
presence of 0.2% deoxycolate. T. cruzi cells (1 mg pro-
tein/mL) were incubated at 28 ꢂC with or without
25 lM of benzofuroxan derivatives, and aliquots were
taken with time to measure respiration (oxygen uptake).
Oxygen consumption was determined with a water-jack-
eted Clark electrode (YSI Model 5300) using 1.5 mL
reaction mixtures in medium A at 28 ꢂC. The electrode
was calibrated with oxygen-saturated medium A at
28 ꢂC (220 lM O2). The amount of parasites used in each
assay was always the equivalent of 1 mg of protein/mL.
Aguirre, G.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.;
´
Cerecetto, H.; Di Maio, R.; Risso, M.; Gonzalez, M.;
Porcal, W. Spectrochim. Acta, Part A 2003, 59, 69; (b)
Olea-Azar, C.; Rigol, C.; Opazo, L.; Morello, A.; Maya, J.
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´
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Financial support from CSIC-UdelaR and PEDECIBA
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