Anti-trypanosomatid benzofuroxans and deoxygenated analogues: Synthesis using polymer-supported triphenylphosphine, biological evaluation and mechanism of action studies

Article abstract of DOI:10.1016/j.ejmech.2009.09.009

Hybrid vinylthio-, vinylsulfinyl-, vinylsulfonyl- and vinylketo-benzofuroxans developed as anti-trypanosomatid agents, against Trypanosoma cruzi and Leishmania spp., have showed low micromolar IC₅₀ values. The synthetic route to access to these derivatives was an efficient Wittig reaction performed in mild conditions with polymer-supported triphenylphosphine (PS-TPP). Additionally, the benzofurozan analogues, deoxygenated benzofuroxans, were prepared using PS-TPP as reductive reagent in excellent yields. The trypanosomicidal and leishmanocidal activities of the benzofuroxan derivatives were measured and also some aspects of their mechanism of action studied. In this sense, inhibition of mitochondrial dehydrogenases activities, production of intra-parasite free radicals and cruzipain inhibition were studied as biological target for the anti-trypanosomatid identified compounds. The trypanosomicidal activity could be the result of both the parasite-mitochondrion function interference and production of oxidative stress into the parasite.

Full text of DOI:10.1016/j.ejmech.2009.09.009

European Journal of Medicinal Chemistry 44 (2009) 5055–5065
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-trypanosomatid benzofuroxans and deoxygenated analogues: Synthesis
using polymer-supported triphenylphosphine, biological evaluation and
mechanism of action studies
a
a
a
a
a
b
´
Diego Castro , Lucia Boiani , Diego Benitez , Paola Hernandez , Alicia Merlino , Carmen Gil ,
c
a,
a,
a,
*
*
*
´
Claudio Olea-Azar , Mercedes Gonzalez , Hugo Cerecetto , Williams Porcal
a
´
´
´
´
Departamento de Qu´ımica Organica, Facultad de Ciencias-Facultad de Quımica, Universidad de la Republica, Igua 4225, 11400 Montevideo, Uruguay
b Instituto de Qu´ımica Me´dica-CSIC, Madrid, Spain
c
´
´
´
´
Departamento de Qu´ımica Inorganica y Analıtica, Facultad de Ciencias Quımicas y Farmaceuticas, Universidad de Chile, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Hybrid vinylthio-, vinylsulfinyl-, vinylsulfonyl- and vinylketo-benzofuroxans developed as anti-trypa-
nosomatid agents, against Trypanosoma cruzi and Leishmania spp., have showed low micromolar IC50
values. The synthetic route to access to these derivatives was an efficient Wittig reaction performed in
mild conditions with polymer-supported triphenylphosphine (PS-TPP). Additionally, the benzofurozan
analogues, deoxygenated benzofuroxans, were prepared using PS-TPP as reductive reagent in excellent
yields. The trypanosomicidal and leishmanocidal activities of the benzofuroxan derivatives were
measured and also some aspects of their mechanism of action studied. In this sense, inhibition of
mitochondrial dehydrogenases activities, production of intra-parasite free radicals and cruzipain inhi-
bition were studied as biological target for the anti-trypanosomatid identified compounds. The trypa-
nosomicidal activity could be the result of both the parasite-mitochondrion function interference and
production of oxidative stress into the parasite.
Received 29 July 2009
Received in revised form
3 September 2009
Accepted 7 September 2009
Available online 18 September 2009
Keywords:
Hybrid benzofuroxans
Trypanosoma cruzi
Leishmania spp.
Mitochondrial dehydrogenases
Free radicals
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
sense, new structural hits reported in the literature and patents
have been developed showing preclinical efficacy in different
Diseases caused by Trypanosomatidae, which share a similar
state regarding drug treatment, include Chagas’ disease (CD) (Try-
panosoma cruzi) and Leishmaniasis (Leishmania spp.). These try-
panosomatids alone are responsible for an infected population of
nearly 30 millions and more than 400 millions are at risk. CD is
a systemic chronic parasitic infection caused by a hemoflagellate
protozoan, Trypanosoma cruzi (T. cruzi), which is transmitted
primarily by species of a blood-feeding triatomine insect known as
Vinchuca or Chinche [1,2].
Recently, the World Health Organization (WHO) estimates that
8 million people are infected with CD, and approximately 11,000
deaths are reported annually [3]. Despite this situation, signifi-
cant scientific progress on the search and pharmacological
actions of effective agents to treat CD has been made. In this
models [4–9]. While various classes of trypanosomicidal agents
have been described in the last years, Nifurtimox (Nfx, LampitÒ,
Fig. 1) and Benznidazole (Bnz, RochaganÒ, Fig. 1), two nitro-
aromatic compounds, are even today the only clinically available
drugs for the chemotherapy of CD [10]. Both nitro-derivatives have
exhibited significant activity in the acute phase, with up to 80% of
parasitological cures in treated patients, while in the chronic phase,
less than 20% of treated patients are parasitologically cured [3]. The
bioreduction of the nitro group to unstable nitroanion radicals,
which react to produce highly toxic reactive oxygen species
(superoxide anion, hydrogen peroxide and hydroxyl radical) and
covalent modification of macromolecules by nitroreduction inter-
mediates are the mechanism of action proposed to Nfx and Bnz,
respectively [11,12].
Unfortunately, the emergence of T. cruzi parasite strains
resistant to Nfx and Bzn and severe side effects of these drugs
have eroded its efficacies [13–16]. Non selective bioreduction of
these trypanosomicidal drugs could be the reason for their side
effects in the mammalian host. This fact increases the urgency of
* Corresponding authors. Tel.: þ598 2 525 86 18x216; fax: þ598 2 525 07 49.
E-mail addresses: megonzal@fq.edu.uy (M. Gonza´lez), hcerecet@fq.edu.
uy (H. Cerecetto), wporcal@fq.edu.uy (W. Porcal).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.009

5056
D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
O
N
N
N
O2N
N
N
H
O
SO2
NO2
Nfx
Bnz
Ph
Ph
O
O
O
O
O
H
N
H
O
N
S
Ph
O
N
H
N
N
H
Ph
O
O
O
O
Vinylsulfone derivative (I)
Vinyl-α-ketoester derivative (II)
Ph
O-
O
P
N+
Mtf
O
O
15
Fig. 1. Drugs used clinically and experimentally as anti-T. cruzi and anti-Leishmania agents.
the search and development for novel, effective and safer agents
to treat this neglected disease. To develop new drugs to combat
efficiently this parasitic infection research should be directed
toward key differences between the metabolism of the mammal
and the parasite. In this sense, several biochemical pathways
have been identified as potential differential target such as the
suggested the perturbation of the mitochondrial respiratory
chain, inhibiting parasite respiration, and the N-oxide moiety
were essential for the trypanosomicidal activity [27,28]. After
a
cluster methodology study, Hansch’s series design, 5-
phenylethenylbenzofuroxan (1, Scheme 1) was revealed as
excellent hit for the design of new trypanosomicidal compounds
[27]. After that, further studies were performed over this skel-
eton in order to study the influence of structural modifications
on the biological activity [29–31] finding vinylthio-, vinylsulfinyl-
and vinylsulfonyl-benzofuroxan derivatives (2–5, Scheme 1) with
remarkable in vitro activities against different T. cruzi strains and
good in vivo activities in an acute murine model of Chagas’
disease.
The drugs of choice for the treatment of leishmaniasis are
sodium stibogluconate (PentostamÒ), meglumine antimoniate
(GlucantimeÒ), pentamidine and liposomal amphotericin B, but
these sometimes meet with failure [32]. Besides, WHO/TDR
develops a research program with Miltefosine (Mtf, Fig. 1), a very
promising leishmanocidal drug [33,34]. This illness, associated with
poverty, does not attract the investment from pharmaceutical
companies as a result of the lack of commercial reasons; conse-
quently, efforts to develop new and safer drugs have been carried
out mainly by academic institutions. In this sense, we have also
enzyme cruzipain,
a
cysteine protease, characterized by
a cysteine-thiol active-site responsible to attack the carbonyl of
the amide bond [17–19]. Cruzipain is essential to many biological
processes evidences have indicated it is critically required for
intracellular parasite survival. Several covalent inhibitors, struc-
turally related to Michael-like systems such as vinylsulfone I and
vinylketone II (Fig. 1), have been reported to inhibit cruzipain
preventing parasite intracellular growth and differentiation
[20,21]. Also generation of oxidative stress in T. cruzi, through
selective reduction of drugs by oxidoreductases unique in the
parasite, is an attractive target [22,23]. In 1999, the benzo[1,2-
c]1,2,5-oxadiazole N-oxide heterocycle (benzofuroxan, i.e. deriv-
atives 1–5 Scheme 1) was reported by our group as T. cruzi
growth inhibitor [24]. Our initial studies indicated that the N-
oxide group of this heterocycle system could act as bioreducible
group into the parasite generating free radical species [24–26].
On the other hand some studies developed by our group
O
S
n
O
N
O
N
O
N
O
O
N
O
S
O
O
O
N
N
N
N
O
1, E-isomer IC50=10.7μM
Z-isomer IC50=7.0 μM
2, n=0 IC50=2.6μM (2E),IC50=3.8μM (2Z)
3, n=1 IC50=2.6μM (3E),IC50=0.7μM (3Z)
4, n=2 IC50=1.6μM (4E),IC50=1.1μM (4Z)
5 IC50=14.6μM
6
O
S
O
n
O
N
O
O
N
III, n=0
IV, n=1
V, n=2
R
VI
R
O
N
N
Scheme 1. Previous and new designed anti-trypanosomatid benzofuroxans.

D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
5057
studied new series of structural hits against different leishmania
strains [35–38] finding N-oxide derivatives as potential anti-leish-
mania agents (i.e. compound 6, Scheme 1).
the bases NaH or sodium bis(trimethylsilyl)amide (NaHMDS)
followed by the condensation with formylbenzofuroxan 7 with
the concomitant cleavage of the resin through Wittig reaction
resulting in the free chalcone-like and the polymer-supported
triphenylphosphine oxide (PS-TPPO, Scheme 3). Filtration of the
reaction through a pad of silica yielded benzofuroxanyl chalcone-
like compounds 9–12 with good overall yields. In these conditions,
1H-NMR showed that chalcone-like compounds 9–12 were isolated
as the E geometric isomers around the olefinic group, the deoxy-
genated analogues were removed, by chromatography, as
secondary products in very low yields.
To obtain vinylsulfone, i.e. 21, the solid-phase synthesis
strategy shown in Scheme 4 was first attempted. However, the
desired vinylsulfone was not isolated or generated in very low
yield. Different oxidation conditions were attempted to generate
the resin-attached sulfone. When m-CPBA [44] was used the
vinylthio-derivative 15E/Z (Scheme 5) was obtained as the main
product after the Wittig process. This fact could indicate both
phosphonium phenyls’ steric impediment and bromine-anion’s
stereo-electronic effects in the proximity of the sulfur atom
diminish its susceptibility to oxidant attack. Attempts with oxone
or urea-hydrogen peroxide complex (Scheme 4) render little
amount, less than 5%, of the desired product, 21, after the Wittig
process.
On the basis of these results, the vinylthio-derivatives, 2, 14–16,
were firstly obtained, as chromatographically separated geometric
isomers, through Wittig solid-phase synthesis (Scheme 5). Subse-
quently, vinylsulfinyl-derivatives, 3, 17–19, and vinylsulfonyl-
derivatives, 4, 20–22, were selectively obtained in solution using
m-CPBA at low temperature or H2O2 (30%) in acetic acid at reflux,
respectively.
To corroborate the effect of the N-oxide moiety in the trypano-
somicidal activity of the new hybrid benzofuroxans, some were
converted into the corresponding deoxygenated analogues (ben-
zofurazans). Previously, we reported the synthesis of benzofurazans
from the corresponding benzofuroxans with triphenylphosphine in
boiling ethanol [27]. The byproduct triphenylphosphine oxide
generated in this reaction is often hard to separate from the desired
product. The use of PS-TPP as reductant agent in mild conditions
rendered the benzofurazans 23–29 in excellent yields allowing to
separate the byproduct, PS-TPPO bya single filtration (Table 1). All of
the proposed structures were established by 1H-, 13C-NMR (HMQC,
HMBC) spectroscopy and MS. The purity was established by TLC and
microanalysis.
Herein, structural modifications on benzofuroxans 2–4 were
planned in order to recognize the effects on the anti-T. cruzi activity
when different substituted-phenyls and Michael-acceptors were
used. Therefore, we designed new structures based on the associ-
ation of benzofuroxan heterocycle with Michael-like systems as for
example vinylsulfonyl and the bioisoster vinylketo moiety present
in cruzipain inhibitors (Scheme 1, structures III–VI). We have
developed a small library of benzofuroxan derivatives employing
a polymer-supported triphenylphosphine (PS-TPP) as reagent.
Triphenylphosphine is one of the most widely used phosphorus-
containing reagents in organic synthesis for many types of trans-
formations such as Mitsunobu and Wittig reactions. However, the
byproduct triphenylphosphine oxide, generated by these reactions,
is often difficult to separate from the end-product. The use of
commercially available PS-TPP leads to much simpler workups and
products isolation [39,40]. So, following the use of a PS-TPP, the
triarylphosphine oxide remains attached to the resin (PS-TPPO) and
it could be separated only by filtration. The PS-TPP could be
regenerated after reduction of the triarylphosphine oxide with
trichlorosilane. The designed benzofuroxan derivatives were effi-
ciently synthesized via Wittig reaction using a PS-TPP. Besides, the
deoxygenated derivatives, benzofurazans, were prepared by reac-
tion of the corresponding N-oxides with PS-TPP. The developed
derivatives were examined for antiproliferative in vitro activity
against T. cruzi Tulahuen 2 strain and CL Brener clone, against
promastigote form of Leishmania braziliensis (MHOM/BR/00/
LTB300) and Leishmania pifanoi (MHOM/VE/57/LV135) strains and
for unspecific cytotoxicity against J774 mouse macrophages. Initial
studies of mitochondrial dehydrogenases inhibition, cruzipain
inhibition and capacity to produce intra-parasite free radical,
through electronic spin resonance (ESR) spectroscopy, were also
performed to investigate the mechanisms of action.
2. Chemistry
As occurs with benzofuroxanyl vinylsulfone 4 [30], attempts to
obtain benzofuroxanyl chalcone-like derivative 9 (Scheme 2) in
solution through classical aldolic conditions were fruitless, causing
complete decomposition of the starting heterocycle or reduction
products (benzofurazan). This could be explained as the result of the
well-known ring opening susceptibility of this heterocycle by
nucleophilic attack. Benzofuroxan system reacts very easily with
a large number of electron-rich species, as for example enolates,
yielding various classes of products such as ring-opening derivatives,
other heterocycle derivatives and deoxygenated derivatives [41–43].
Consequently, a solid-phase synthetic strategy was chosen for
the synthesis of benzofuroxanyl chalcone-like compounds 9–12
using commercially available 2-bromoacetophenones (8a–d) and
PS-TPP, via Wittig conditions (Scheme 3). Treatment of PS-TPP with
the 2-bromoacetophenones 8a–d yielded the corresponding
phosphonium salt (evaluated by FT-IR for the appearance of new
bands near to 1670 cmꢀ1 corresponding to the stretching vibration
of the C]O bond). Then this phosphonium salts were reacted with
Table 1
Synthesis of benzofurazan derivatives using PS-TPP.
Benzofurazan
X
R
Yield (%)a
Purity (%)b
23
24
25
26
27
28
29
S
H
H
H
H
Cl
OCH3
NO2
93
96
95
90
94
92
88
98
96
97
98
96
97
96
O
O
O
O
O
SO
SO2
CO
CO
CO
CO
Aldolic condensation
N
N
H
Ph
Ph
O
O
N
N
NaH / THF / r.t
or
NaOH / EtOH-H2O / r.t
Acetophenone
7
9
a
After filtration and washing (see Experimental Section).
From 1H-NMR analysis.
b
Scheme 2. Attempt to obtain the benzofuroxanyl chalcone-like 9 in solution.

5058
D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
Br
Ph
Ph
P
Ph
P
O
O
Ph
Br
DMF / 70-80 ºC
12 h
+
R
R
R= H
Cl
8a
8b
PS -TPP
NaHMDS or NaH
THF / 1h / r.t
OCH3 8c
NO2
8d
O
O
N
O
N
H
O
P
Ph
P
O
O
Ph
Ph
O
Ph
N
7
+
O
N
R
R
THF / 8-12 h / 50 ºC
R= H
Cl
9
10
PS -TPPO
OCH3 11
NO2 12
Scheme 3. Solid-phase synthesis of benzofuroxanyl chalcone-like compounds 9–12.
3. Biology
observed. The deoxygenated derivatives 23–29 possess less trypa-
nosomicidal activity than the corresponding N-oxide analogues and
reference drugs, showing that N-oxide moiety is imperative for
adequate anti-T. cruzi activity. The vinylsulfonyl-derivative 25, with
3.1. In vitro trypanosomicidal activity
All hybrid benzofuroxans, and some intermediates, were tested
in vitro against T. cruzi, using epimastigote form of Tulahuen 2 strain
and CL Brener clone. The existence of the epimastigote form of
T. cruzi as an obligate mammalian intracellular stage has been
revisited and confirmed [45]. The IC50 concentration (50% inhibitory
growth concentration) for each compound was evaluated in
comparison tothe control (no drug added to the media). Nfx and Bnz
were used as the trypanosomicidal reference drugs. Table 2 shows
the effect of developed benzofuroxan derivatives on the growth of T.
cruzi. Chalcone-like compounds 9, 11 and 12, vinylthio 15E and 16E,
vinylsulfinyl 17Z and 18Z and vinylsulfonyl 20Z and 21Z were the
IC50 25.0 and 18.0 mM for Tulahuen 2 and CL Brener, respectively,
possesses higher anti-T. cruzi activity than the corresponding
vinylthio- and vinylsulfinyl-derivatives, 23 and 24, respectively,
showing that the electrophilic center in this vinylsulfonyl moiety
could be important for adequate trypanosomicidal activity. It was
not possible to evidence clear different benzofuroxans-activity
patterns from Tulahuen 2 strain and CL Brener clone biological data.
In general, similar activity-profiles were observed in both parasitic
systems, i.e. one of the most active on Tulahuen 2 strain, derivative
15E, was the most active against CL Brener, or one of the least active
onTulahuen 2, derivative 19Z, was also one of the least active against
CL Brener (Table 2).
most active compound with IC50 between 1.0 and 5.0 mM, all with
potencies similar to the reference drugs. The most active derivatives
are the vinylthio-benzofuroxan 15E and 17Z. Substitution on the
phenyl group of the chalcone-like 10, with chloro group results with
moderate trypanosomicidal activity against Tulahuen 2 strain. No
clear relationship between geometric isomerism and activity was
3.2. In vitro leishmanocidal activity
Initially, the parent compounds 2–5,
a phenylvinylthio-,
a
phenylvinylsulfinyl-,
a
phenylvinylsulfonyl-, and an
Assayed oxidation conditions
Br
Ph
Br
Ph
Ph
P
Ph
P
O
O
m-CPBA (3, 5 and 10 equiv)
CH2Cl2 / 0ºC or r.t / 1-24 h
S
S
Cl
or
Cl
Oxone (2,2 equiv)
DMF-H2O / 24 h
or
H2N(CO)NH2.H2O2 (3 equiv)
CH2Cl2 / rt / 12 h
i) NaH / THF / r.t / 1h
O O
S
O
ii) 7
N
O
N
Cl
21
Scheme 4. Attempt to obtain the vinylsulfone 21 through solid-phase synthesis.

D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
5059
Br
Ph
Ph
i) CH2O / HBr
O
P
S
S
N
SH
Toluene / 50-60 ºC / 3 h
i) NaH / THF / r.t / 1h
O
N
R
R
R
O
ii)
Ph
P
o
ii)
R= H, 2
N
R= H, 13a
F, 13b
Ph
H
O
F, 14
Cl, 15
CH3, 16
N
Cl, 13c
CH3, 13d
THF / 8-12 h / 50 ºC
Toluene / 50 ºC / 48 h
PS-TPP
m-CPBA / CH2Cl2
-78 at 0 ºC / 2 h
H2O2 / AcOH
reflux / 4 h
O
S
O O
S
O
N
O
N
O
O
N
N
R
R
R= H, 4
F, 20
R= H, 3
F, 17
Cl, 21
Cl, 18
CH3, 19
CH3, 22
Scheme 5. Solid-phase synthesis of vinylthio 2, 14–16 and traditional synthesis of vinylsulfinyl 3, 17–19 and vinylsulfonyl 4, 20–22.
alkylvinylsulfonyl-derivative were selected to be studied as
leishmanocidal agents. According to the results on the leishma-
nocidal test two of the new phenylvinylthio-derivatives, 14 and
15, were also studied biologically. They were tested in vitro
against promastigote form of L. braziliensis (MHOM/BR/00/
LTB300) and L. pifanoi (MHOM/VE/57/LV135) strains. Viability of
parasite was assessed colorimetrically using the MTT assay
[46,47]. For each derivative, the percentage of cytotoxicity was
and 50.0 mM, the IC50 was calculated and reported in Table 3. Mtf
was used as the reference leishmanocidal drug. The most active
benzofuroxans are the vinylthio 2 (Z and E isomers), 14 (Z and E
isomers) and 15 (E isomer) and the vinylsulfinyl 3 (Z isomer)
finding the same profile of activity than in T. cruzi parasite. The
vinylthio-benzofuroxan 2Z and 15E are at least 10 times more
active than reference leishmanocidal drug in vitro against pro-
mastigote form of L. braziliensis. Clearly, in leishmanocidal
activity, the Z-geometric isomers were more active than the
E-ones. Benzofuroxans 2E, 2Z, 14E, 14Z, and 15E were assayed in
vitro against both Leishmania strains, and all of them were found
to exhibit slightly higher leishmanocidal activity against L. bra-
ziliensis than against L. pifanoi.
initially determined at 25 mM as it is indicated in Experimental
Section and then in a concentration-response assay, between 0.5
Table 2
In vitro anti-T. cruzi activity of new benzofuroxan derivatives, Nfx and Bnz.
M)a, b
Compd.
IC50
T2c
(m
M)a, b
3.3. Unspecific cytotoxicity
Compd.
IC50
(
m
T2c
5.0
CLBd
nde
CLBd
Unspecific mammalian cytotoxicity of the developed benzo-
furoxans was evaluated in vitro in the range of 1.0–400.0 mM,
9
20
21
22
7.5 (E)
2.7 (Z)
4.8 (E)
1.3 (Z)
4.3 (E)
3.6 (Z)
42.0
6.3 (E)
2.9 (Z)
4.8 (E)
2.3 (Z)
9.6 (E)
5.1 (Z)
25.0
10
11
25.0
4.1
nd
nd
Table 3
IC50 Values of selected benzofuroxans against different Leishmania spp strains.
12
14
2.4
5.0 (E)
nd
23
24
Compd.
IC50 (m
M)a
5.5 (E)
5.9 (Z)
1.0 (E)
2.8 (Z)
4.7 (E)
4.1 (Z)
6.2 (E)
3.2 (Z)
6.7 (E)
4.7 (Z)
6.0 (E)
8.4 (Z)
8.5
41.0
32.0
12.0 (Z)
1.9 (E)
4.2 (Z)
1.9 (E)
4.2 (Z)
4.5 (E)
1.6 (Z)
5.5 (E)
2.7 (Z)
4.6 (E)
6.7 (Z)
7.7
L. braziliensis
L. pifanoi
15
16
17
18
19
25
26
27
28
29
Bnz
25.0
97.0
18.0
nd
LTB300
LV135
2E
2Z
3E
3Z
4E
4Z
5
14E
14Z
15E
Mtf
1.5
0.9
31.5
4.6
21.5
10.8
16.8
4.3
4.3
4.5
ndb
nd
nd
nd
>100.0
>100.0
>100.0
7.4
nd
nd
nd
nd
4.3
4.5
4.6
(85.5c)
Nfx
4.5
1.7
0.9
9.0
a
IC50: concentration that produces 50% inhibitory effect.
b
The results are the means of three different experiments with a SD less than 10%
in all cases.
a
The results are the means of three independent experiments with a SD less than
10% in all cases.
c
T2: Tulahuen 2 strain.
CLB: CL Brener clone.
nd: not determined.
d
e
b
nd: not determined.
Compound tested at 5.0 mM, % of growth inhibition is reported.
c

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D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
Table 4
Table 5
Cytotoxicity of benzofuroxan derivatives against J-774 mouse macrophages.
Inhibition of T. cruzi cruzipain by 2, 9, 11, 20, 24 and 25.
Compd.
IC50
(m
M)a
SIb, c
Compd.
IC50
(
m
M)a
SIb, c
Compd.
CP percentage of inhibition (%)a, b
25 50
2E/Z
3E
3Z
4E
4Z
9
10
11
12
14E
30.0
30.0
14.0
16.0
11.0
47.0
28.0
31.0
32.0
35.0
10.0d
11.5
20.0
10.0
10.0
9.4
1.1
7.6
13.3
7.0
14Z
18E
18Z
22E
24
25
26
27
28
43.0
18.0
10.0
3.6
4.0
6.3
1.1
>10.0
3.1
m
M
m
M
100 mM
2
9
11
20
24
25
11.0
0.0
nd
22.0
0.0
nd
30.0
0.0
13.0
0.0
0.0
0.0
8.0
>400.0
78.0
102.0
251.0
111.0
131.0
0.0
nd
0.0
nd
1.1
0.0
0.0
<2.5
<1.1
<1.3
a
The control assays contained the respective amount of DMSO.
The values are the mean of at least two independent measurements that
b
29
differed by less than 10%.
a
The results are the means of two independent experiments with a SD less than
10% in all cases.
b
SI: selectivity index.
c
braziliensis. The percentage of mitochondrial dehydrogenase
activities compared to untreated control was assessed using the
colorimetric MTT assay performed at very short times, no more
than 240 min of incubation, following previously described
procedures [37,38,50]. Table 6 shows the results obtained for the
studied benzofuroxans as inhibitors of T. cruzi mitochondrial
dehydrogenases and the comparation with the reference drugs Nfx
and Bnz, some relevant features could be observed. The parent
compound, vinylsulfone 5 showed the highest inhibition of mito-
chondrial dehydrogenases with a value of 58.5%. The parent
compound, vinylthio 2, vinylsulfoxide 3Z and vinylsulfones 4E and
20 and chalcone-like derivative 9 show some degree of mito-
chondrial dehydrogenases inhibition, with values between 14.5 and
25.0%. The result for chalcone-like derivative 9 is in agreement with
some previous observations describing chalcones ability to inhibit
mitochondrial parasite proteins [49–51]. On the other hand, the
rest of the new developed benzofuroxans studied as well as the
reference trypanosomicidal drugs, Nfx and Bzn, were not inhibitors
of mitochondrial dehydrogenases at the studies time. In order to
study the inhibitory effect of benzofuroxan’s in Leishmania mito-
chondrial dehydrogenases we selected the most effective inhibitors
of T. cruzi mitochondrial dehydrogenases, i.e. vinylsulfoxide 3Z and
vinylsulfones 4E and 5, and compare with the leishmanocidal drug
Mtf. All the studied compounds exhibited a clear time-dependent
inhibitory effect after 30 min of incubation (Fig. 2).
SI ¼ IC50, macrophage/IC50, T2, epimastigote
.
d
Considering the IC50 ¼ 3.0
mM.
using J774 mouse macrophages as the cellular model (Table 4).
The cytotoxicity of the chalcone-like compounds 9–12 against
macrophages is comparable to that observed for the vinylthio-
benzofuroxan 2, vinylsulfinyl-benzofuroxan 3 and vinylsulfonyl-
benzofuroxan 4. In particular, chalcone-like derivative 9 resulted
less toxic against the macrophages than the corresponding
vinylsulfone-benzofuroxan bioisoster 4E with similar order of
selectivity index (SI). Chalcone-like derivatives 9 and 12 were the
most selective among the new studied benzofuroxans (Table 4)
being the SI, for Tulahuen 2 strain, near to 10. These results are in
agreement with previous reports of selective anti-T. cruzi
synthetic chalcones [48]. These chalcone-like benzofuroxans
could be considered promising agents for further anti-
trypanosomal
therapy.
The
deoxygenated
chalcone-like
compounds 26–29 show lower levels of macrophage toxicities
than the corresponding N-oxide analogues, 9–12, however their SI
are worse as result of their lower trypanosomicidal activity. The
substitution on the phenyl moiety, by –F, –Cl, or –CH3, of the new
vinylthio-, vinylsulfinyl-, and vinylsulfonyl-benzofuroxans do not
contribute in the improvement of SI values (compare SI value of
unsubstitutedphenyl vinylthio-derivative 2E/Z to that of 4-fluo-
rophenyl-vinylthio-ones 14E and 14Z, or SI value of unsub-
stitutedphenyl vinylsulfinyl-derivatives 3E and 3Z to that of
4-chlorophenylvinylsulfinyl-ones 18E and 18Z, and SI value of
unsubstitutedphenylvinylsulfonyl 4E to that of 4-methyl-
phenylvinylsulfonyl-one 22E). No clear relationship between
geometric isomerism and activity was observed.
3.4.3. Free radical generation: ESR studies
Three of the most T. cruzi-active hybrid benzofuroxans, 9, 14 and
20 were studied as intra-parasite free radical producers using ESR
spectroscopy and spin-trapping tools. In the spin trapping tech-
nique, the cyclic nitrone 5,5-dimethylpyrroline-N-oxide (DMPO) is
the most used spin trapping compound due to the unique ESR
spectrum for trapping hydroxyl and superoxide radical [52].
3.4. Mechanism of action studies
In order to determine the mechanism of action the following
studies were performed: inhibition of cruzipain (CP), inhibition of
the mitochondrial dehydrogenases activity and capability to
produce intra-parasite free radicals.
Table 6
Inhibition of T. cruzi mitochondrial dehydrogenases.
Compd.
Percentage of inhibition (%)a, b
2E/Z (1:1)
3Z
4E
5
14.5
25.0c
25.0c
58.5
21.8
0.0
3.4.1. Inhibition of T. cruzi cruzipain
Selected developed benzofuroxans and benzofurazans were
studied as inhibitors of T. cruzi CP, following a previously described
procedure [30]. None of the assayed compounds resulted to be good
9
14E/Z (1:1)
20E/Z (1:1)
21E/Z (1:1)
Nfx
inhibitors of this enzyme at the studied doses (25.0–100.0
mM)
17.6
4.0
(Table 5) showing these Michael acceptor-system, linked to both
heterocycles, were not enable to inhibit this biomolecule.
4.0c, d
7.0c, e
Bnz
a
120 min of incubation.
Tulahuen 2 strain.
Y strain.
Taken from reference [37].
3.4.2. Inhibition of T. cruzi and Leishmania
mitochondrial dehydrogenases
Some of the developed benzofuroxans were tested as possible
inhibitors of mitochondrial dehydrogenases in T. cruzi and L.
b
c
d
e
Taken from reference [38].

D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
5061
120
100
80
60
40
20
0
access to the desired benzofuroxans by using Wittig reaction, by
a mild method, is achieved involving adequate aldehyde and
PS-TPP. Studies developed to determine the mechanism of action
showed that trypanosomicidal and leishmanocidal activities of
these hybrid benzofuroxans might be result of the interference
on the parasite-mitochondrion function and the production of
oxygen oxidative species into the parasite. Further structural
optimization, QSAR, and in vivo activities in an acute murine
model of Chagas’ disease and Leishmaniosis are currently
underway.
3Z
4E
5
Mtf
30
60
90
120
240
5. Experimental
-20
time (min)
5.1. Chemistry
Fig. 2. Inhibition mitochondrial dehydrogenases (%) compared to untreated L. brazil-
iensis promastigotes. Mtf activity was taken from reference [38].
Compounds 2–5 and 7 were prepared according to procedures
previously described [24,30]. Melting points were determined with
an electrothermal melting point apparatus (Electrothermal 9100)
and are uncorrected. Proton and carbon NMR spectra were recor-
ded on a Bruker DPX-400 spectrometer. The chemical shifts values
are expressed in ppm relative to tetramethylsilane as internal
standard. In 13C-NMR data only narrow peaks were reported. ESR
spectra were recorded in the X band (9.85 GHz) using a Bruker ECS
106 spectrometer with a rectangular cavity and 50 kHz field
modulation. Mass spectra were determined either on a MSD 5973
Hewlett–Packard or LC/MSD-Serie 100 Hewlett–Packard spec-
trometers using electronic impact (EI) or electrospray ionization
(ESI), respectively. Infrared spectra were recorded on a Perkin–
Elmer 1310 apparatus, using potassium bromide tablets for solid
and oil products (the frequencies are expressed in cmꢀ1). Micro-
analyses were performed 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 analytical grade and used
without further purification. All the reactions were carried out in
a nitrogen atmosphere.
Recently, we have developed a group of heteroarylnitrones, espe-
cially a furoxanylnitrone derivative (a(Z)-(3-methylfuroxan-4-yl)-
N-tert-butylnitrone, FXN), with the ability to directly trap and
stabilize oxygen, carbon, and sulfur-centered free radicals [53].
Consequently, the free radical production capacity of these ben-
zofuroxans was assessed on T. cruzi-microsomal fraction by ESR
using DMPO and FXN for spin trapping. The ESR spectra of the free
radical generated by chalcone-like derivative 9 in the T. cruzi-
microsomal fraction, and trapped by DMPO, showed a pattern
consistent with the trapped N-oxide free radical together with the
well known DMPO-OH$ adduct (Fig. 3) [31]. Though the ESR spectra
of the free radical generated by the vinylthio-benzofuroxan 14 and
vinylsulfonyl-benzofuroxan 20 in the T. cruzi-microsomal fraction,
and trapped with FXN, showed a pattern consistent with trapped
hydroxyl free radical (Fig. 3) [53]. These experiments clearly
demonstrated that this family of compounds is able to produce
oxygen oxidative species into the parasite which could be one of the
modes of action of these hybrid compounds.
4. Conclusions
5.1.1. General procedure for the synthesis of the
5E-[2-(arylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide
derivatives (9–12)
To a suspension of a polymer-bound triphenylphosphine (0.5 g,
3 mmol/g phosphine) in dry DMF (3.5 mL/mmol) was added 2
equivalents of the corresponding 2-bromoacetophenone. The
We have identified new benzofuroxan derivatives as prom-
ising anti-trypanosomatid agents. To our knowledge this is the
first time that benzofuroxan containing structures are reported
also as leishmanocidal compounds. An efficient synthetic route
FXN-14(E/Z) spin adduct
DMPO-9 spin adduct
FXN-20E spin adduct
450
3460 3470 3480 3490 3500 3510 35
[G]
3460 3470 3480 3490 3500 35
[G]
3460 3470 3480 3490 3500 35
[G]
Fig. 3. ESR spectra. DMPO and FXN-benzofuroxan spin adduct obtained with T. cruzi-microsomal fraction.

5062
D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
mixture was stirred for 12 h at 70–80 ꢁC. The support was cooled,
carefully filtered and extensively washed with dry toluene
(2 ꢂ10 mL), dry dichloromethane (2 ꢂ10 mL) and dry diethyl ether
(2 ꢂ10 mL). The brown powders were dried at reduced pressure.
According to the weight increase the loading was estimated to be in
2.9 mmol phosphonium salt per gram support [54]. To a suspension
of polymer-bound phosphonium salt (1 equiv.) in THF was added to
1 M solution of NaHMDS in THF (1.5 equiv.) at room temperature
yielding the ylide within 60 min. Excess base was carefully removed
by extensive washing of the support with dry THF (30 mL). The
support was resuspended in dry THF (3 mL) and the for-
mylbenzofuroxan 7 (0.80 equiv.) was added at room temperature.
The mixture was stirred 8–12 h at 50 ꢁC, filtered through a pad of
silica and washed with THF (2 ꢂ 10 mL), and diethyl ether
(2 ꢂ10 mL). The combined organic layers were concentrated at
reduced pressure to give the corresponding vinylketobenzofuroxan.
(0.80 equiv.) was added at room temperature. The mixture was
stirred 8–12 h at 50 ꢁC, filtered through a pad of silica and washed
with THF (2 ꢂ 10 mL), and diethyl ether (2 ꢂ 10 mL). The combined
organic layers were concentrated at reduced pressure and the
residue purified by column chromatography (SiO2, petroleum
ether/EtOAc (95:5)) to afford spectroscopically pure compounds.
5.1.2.1. 5E-[2-(4-fluorophenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N-oxide (14E). Yellow solid (50%); mp 106.0–108.5 ꢁC. 1H-NMR
(CDCl3)
d
: 6.44 (d, 1H, J ¼ 15.5 Hz), 7.07 (d, 1H, J ¼ 15.5 Hz), 7.15 (t,
2H), 7.36 (bs, 2H), 7.50 (m, 2H), 7.53–7.48 (bs, 1H). EI-MS, m/z
(abundance, %): 288 (Mþ$, 100), 272 (40), 256 (8), 228 (74), 183 (50),
127 (47). Anal. (C14H9FN2O2S) C, H, N, S.
5.1.2.2. 5Z-[2-(4-fluorophenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N-oxide (14Z). Yellow solid (25%); mp 95.0–96.5 ꢁC. 1H-NMR
(CDCl3)
d
: 6.47 (d, J ¼ 10.8 Hz, 1H), 6.75 (d, J ¼ 10.8 Hz, 1H), 7.13 (m,
5.1.1.1. 5E-[2-(phenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole
2H), 7.40 (bs, 1H), 7.48 (m, 2H), 7.54–7.47 (bs, 2H). EI-MS, m/z
(abundance, %): 288 (Mþ$, 100), 272 (39), 256 (6), 228 (76), 183 (53),
127 (49). Anal. (C14H9FN2O2S) C, H, N, S.
N-oxide (9). Yellow solid (53%); mp 170.0–171.0 ꢁC. 1H-NMR
(CDCl3)
d: 7.54–7.70 (bs, 3H), 7.58 (d, 1H, J ¼ 15.3 Hz), 7.59 (d, 1H,
J ¼ 10.8 Hz), 7.65 (m, 2H), 7.79 (d, 1H, J ¼ 15.6 Hz), 8.06 (d, 2H,
J ¼ 7.7 Hz). EI-MS, m/z (abundance, %): 266 (Mþ$, 85), 250 (58), 233
(11), 220 (9), 206 (41), 178 (34), 77 (100). Anal. (C15H10N2O3) C, H, N.
5.1.2.3. 5E-[2-(4-chlorophenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N-oxide (15E). Yellow solid (39%); mp 110.0–113.0 ꢁC. 1H-NMR
(CDCl3)
d
: 6.54 (d, 1H, J ¼ 15.5 Hz), 7.04 (d, 1H, J ¼ 15.6 Hz), 7.41 (m,
4H), 7.45–7.34 (bs, 3H). ESI-MS, m/z: 305.0 (Mþ$ þ H), 327.0
5.1.1.2. 5E-[2-(4-chlorophenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (10). Yellow solid (50%); mp 193.5–195.0 ꢁC. 1H-NMR
(Mþ$ þ Na). Anal. (C14H10N2O2S) C, H, N, S.
(CDCl3)
d
: 7.50–7.70 (bs, 3H), 7.54 (d, 2H, J ¼ 8.5 Hz), 7.57 (d, 1H,
J ¼ 15.7 Hz), 7.80 (d, 1H, J ¼ 15.7 Hz), 8.00 (d, 1H, J ¼ 8.4 Hz). ESI-MS,
5.1.2.4. 5Z-[2-(4-chlorophenyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole N-oxi-
m/z: 301 (Mþ$ þ H). Anal. (C15H9ClN2O3) C, H, N.
de (15Z). Yellow solid (32%), mp 128–130 ꢁC. 1H-NMR (CDCl3)
d:
6.62 (d, J ¼ 10.8 Hz, 1H), 6.77 (d, J ¼ 10.8 Hz, 1H), 7.41 (d, J ¼ 8.1 Hz,
2H), 7.43 (d, J ¼ 8.1 Hz, 2H), 7.54–7.37 (bs, 3H). ESI-MS, m/z: 305.0
(Mþ$ þ H). Anal. (C14H9ClN2O2S) C, H, N, S.
5.1.1.3. 5E-[2-(4-methoxyphenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
diazole N-oxide (11). Yellow solid (47%); mp 165.5–167.0 ꢁC.
1H-NMR (CDCl3)
d
: 3.61 (s, 3H), 7.01 (d, 2H, J ¼ 8.6 Hz), 7.44–7.65
(bs, 3H), 7.48 (d, 1H, J ¼ 15.8 Hz), 7.40 (d, 2H, J ¼ 8.9 Hz), 7.67 (d, 1H,
5.1.2.5. 5E-[2-(4-methylphenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
J ¼ 15.7 Hz). ESI-MS, m/z: 297 (Mþ$ þ H). Anal. (C16H12N2O4) C, H, N.
N-oxide (16E). Yellow solid (53%); mp 129.0–131.0 ꢁC. 1H-NMR
(CDCl3)
d
: 2.41 (s, 3H), 7.03 (d, 1H, J ¼ 16.0 Hz), 7.12 (d, 1H,
J ¼ 15.3 Hz), 7.25 (d, 2H, J ¼ 7.9 Hz), 7.46 (d, 2H, J ¼ 8.0 Hz), 7.30–
7.40(bs, 2H), 7.61 (bs,1H). EI-MS, m/z (abundance, %): 284 (Mþ$, 20),
268 (25), 252 (67), 238 (52), 217 (39), 191 (100). Anal.
(C15H12N2O2S) C, H, N, S
5.1.1.4. 5E-[2-(4-nitrophenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (12). Yellow solid (35%); mp 171.5–173.0 ꢁC. 1H-NMR
(CDCl3)
d
: 7.44–7.65 (bs, 3H), 7.50 (d, 1H, J ¼ 15.8 Hz), 7.56 (d, 2H,
J ¼ 8.9 Hz), 7.78 (d, 1H, J ¼ 15.7 Hz), 8.24 (d, 1H, J ¼ 8.8 Hz). ESI-MS,
m/z: 312 (Mþ$ þ H). Anal. (C15H9N3O5) C, H, N.
5.1.2.6. 5Z-[2-(4-methylphenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N-oxide (16Z). Brown oil (30%); 1H-NMR (CDCl3)
d: 2.40 (s, 3H),
5.1.2. General procedure for the synthesis of the
6.55 (d, 1H, J ¼ 10.8 Hz), 6.83 (d, 1H, J ¼ 10.8 Hz), 7.20 (d, 1H,
J ¼ 8.1 Hz), 7.51 (d, 1H, J ¼ 8.0 Hz), 7.30–7.45(bs, 2H), 7.63 (bs, 1H).
EI-MS, m/z (abundance, %): 284 (Mþ$, 100), 268 (59), 252 (34), 238
(15), 217 (56), 191 (58). Anal. (C15H12N2O2S) C, H, N, S.
5(E/Z)-[2-(arylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide (2,14–16)
A mixture of paraformaldehyde (1.89 g, 0.063 mmol), toluene
(12 mL) and HBr (48%, 35 mL) was heated at 50 ꢁC and treated
dropwise within 30 minutes with a solution of corresponding tio-
phenol (0.04 mmol) in toluene (12 mL). Finally, the mixture was
stirred for 60 minutes at 50 ꢁC. The organic layer was washed with
cold water, dried with Na2SO4 and used in the next reaction
without further purification. A suspension of a polymer-bound
triphenylphosphine (3.0 g, 3 mmol/g phosphine) in the crude of the
bromomethylation process was stirred for 48 h at 50 ꢁC. The
support was filtered and washed with dry toluene (2 ꢂ 20 mL), dry
dichloromethane (2 ꢂ 20 mL) and dry diethyl ether (2 ꢂ 20 mL).
The brown powder was dried at reduced pressure. According to the
weight increase the loading was estimated to be in 2.85 mmol
phosphonium salt per gram support. To a suspension of polymer-
bound phosphonium salt (1 equiv.) in THF was added NaH
(1.5 equiv.) at room temperature generating the ylide within
60 min. The excess of the base was carefully removed by extensive
washing of the support with dry THF. The support was resuspended
5.1.3. General procedure for the synthesis of the 5(E/Z)-[2-
(arylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide (17–19)
A solution of the vinylthio-derivative E or Z (1 equiv.) in CH2Cl2
(6.0 mL/mmol) was cooled to ꢀ78 ꢁC while a solution of m-chlor-
operbenzoic acid (1 equiv.) in CH2Cl2 (2.0 mL/mmol) was added
dropwise during 5 min. The mixture was stirred and warmed to
room temperature for 2 h and then poured into saturated sodium
bicarbonate solution (10.0 mL) and the mixture extracted with
CH2Cl2 (3 ꢂ10.0 mL). After the workup of the combined organic
layers, the residue was purified by column chromatography (SiO2,
petroleum ether: EtOAc (8:2)), yielding spectroscopically pure
compounds.
5.1.3.1. 5E-[2-(4-fluorophenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
in dry THF (6 mL/g resin) and the formylbenzofuroxan
7
zole N-oxide (17E). Yellow solid (51%); mp 117.0–119.0 ꢁC. 1H-NMR

D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
5063
(CDCl3): 6.98 (d, 1H, J ¼ 15.4 Hz), 7.30 (m, 2H), 7.41 (d, 1H,
J ¼ 15.3 Hz), 7.55–7.34 (bs, 3H), 7.73 (m, 2H). EI-MS, m/z (abun-
dance, %): 304 (Mþ$, 2), 288 (1), 275 (10), 256 (100), 240 (24), 196
(41). Anal. (C14H9FN2O3S) C, H, N, S.
5.1.4.3. 5E-[2-(4-chlorophenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (21E). Pale yellow solid (42%); mp 178.0–180.0 ꢁC. 1H-
NMR (CDCl3)
d
: 6.96 (d, 1H, J ¼ 15.4 Hz), 7.37 (bs, 1H), 7.45–7.65 (bs,
2H), 7.59 (d, 2H, J ¼ 8.6 Hz), 7.67 (d, 1H, J ¼ 15.4 Hz), 7.92 (d, 2H,
J ¼ 8.6 Hz). ESI-MS, m/z: 337 (Mþ$ þ H), 359 (Mþ$ þ H). Anal.
(C14H9ClN2O4S) C, H, N, S.
5.1.3.2. 5Z-[2-(4-fluorophenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (17Z). Yellow solid (54%); mp 114.0–116.0 ꢁC. 1H-NMR
(CDCl3)
d
: 6.69 (d, 1H, J ¼ 10.7 Hz), 7.07 (d, 1H, J ¼ 10.7 Hz), 7.30 (m,
5.1.4.4. 5Z-[2-(4-chlorophenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
2H), 7.48–7.62 (bs, 3H), 7.70 (m, 2H). EI-MS, m/z (abundance, %):
304 (Mþ$, 21), 288 (6), 275 (31), 256 (100), 240 (13), 215 (27), 196
(59). Anal. (C14H9FN2O3S) C, H, N, S.
zole (21Z). Pale yellow solid (49%); mp 117.0–118.0 ꢁC. 1H-NMR
(CDCl3)
d
: 6.67 (d, 1H, J ¼ 12.0 Hz), 7.06 (d, 1H, J ¼ 12.0 Hz),
7.50–7.55 (bs,1H), 7.54 (d, 2H, J ¼ 8.7 Hz), 7.62–7.58 (bs, 2H), 7.83 (d,
2H, J ¼ 8.7 Hz). ESI-MS, m/z: 337 (Mþ$ þ H), 359 (Mþ$ þ H). Anal.
(C14H9ClN2O4S) C, H, N, S.
5.1.3.3. 5E-[2-(4-chlorophenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (18E). Yellow solid (44%); mp 185.0–187.0 ꢁC. 1H-NMR
(CDCl3)
d
: 6.98 (d,1H, J ¼ 15.4 Hz), 7.38 (d,1H, J ¼ 15.4 Hz), 7.55–7.34
5.1.4.5. 5E-[2-(4-methylphenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
(bs, 3H), 7.55 (d, 2H, J ¼ 8.5 Hz), 7.65 (d, 2H, J ¼ 8.6 Hz). ESI-MS, m/z:
diazole N-oxide (22E). Yellow solid (27%); mp 157.0–158.0 ꢁC.
321.0 (Mþ$ þ H), 343.0 (Mþ$ þ Na). Anal. (C14H9ClN2O3S) C, H, N, S.
1H-NMR (CDCl3)
d
: 2.43 (s, 3H), 6.98 (d, 1H, J ¼ 15.4 Hz), 7.30–7.45
(bs, 1H), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.50–7.65 (bs, 2H), 7.64 (d, 1H,
J ¼ 15.4 Hz), 7.86 (d, 2H, J ¼ 8.3 Hz). ESI-MS, m/z: 317 (Mþ$ þ H).
Anal. (C15H12N2O4S) C, H, N, S.
5.1.3.4. 5Z-[2-(4-chlorophenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (18Z). Pale yellow solid (52%); mp 127.0–129.0 ꢁC. 1H-
NMR (CDCl3)
d: 6.67 (d, 1H, J ¼ 10.7 Hz), 7.08 (d, 1H, J ¼ 10.8 Hz),
7.63–7.53 (bs, 3H), 7.57 (d, 2H, J ¼ 8.5 Hz), 7.62 (d, 2H, J ¼ 8.6 Hz).
ESI-MS, m/z: 321.0 (Mþ. þ H), 343.0 (Mþ. þ Na). Anal.
(C14H9ClN2O3S) C, H, N, S.
5.1.4.6. 5Z-[2-(4-methylphenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
diazole N-oxide (22Z). Pale yellow solid (14%); mp 116.0–118.0 ꢁC.
1H-NMR (CDCl3)
1H, J ¼ 10.8 Hz), 7.16 (d, 2H, J ¼ 8.4 Hz), 7.40–7.55 (bs, 3H), 7.45 (d,
2H, J ¼ 8.3 Hz). ESI-MS, m/z: 317 (Mþ$ þ H). Anal. (C15H12N2O4S) C,
H, N, S.
d
: 2.42 (s, 3H), 6.71 (d, 1H, J ¼ 10.9 Hz), 7.21 (d,
5.1.3.5. 5E-[2-(4-methylphenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (19E). Orange solid (21%); mp 160.0–162.0 ꢁC. 1H-NMR
(CDCl3): 2.44 (s, 3H), 6.99 (d, 1H, J ¼ 15.4 Hz), 7.35 (d, 1H,
J ¼ 15.4 Hz), 7.38 (d, 2H, J ¼ 8.3 Hz), 7.54–7.42 (bs, 3H), 7.60 (d, 2H,
J ¼ 8.1 Hz). ESI-MS, m/z: 301.0 (Mþ$ þ H). Anal. (C15H12N2O3S) C,
H, N, S
5.1.5. General procedure for the synthesis of the deoxygenated
analogues (23–29)
To
a suspension of a polymer-bound triphenylphosphine
(2 equiv.) in THF (50 mL/mmol) was added 1 equivalent of the
corresponding benzofuroxan derivative. The mixture was stirred
for 12 h at room temperature. Then, the support was carefully
filtered through a pad of silica and washed with THF and diethyl
ether. The combined organic layers were concentrated under
reduced pressure to give the corresponding benzofurazan
derivatives.
5.1.3.6. 5Z-[2-(4-methylphenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (19Z). Pale yellow solid (16%); mp 68.0–70.0 ꢁC. 1H-
NMR (CDCl3)
d
: 6.72 (d, 1H, J ¼ 10.9 Hz), 7.12 (d, 1H, J ¼ 10.8 Hz),
7.60–7.50 (bs, 3H), 7.45 (d, 2H, J ¼ 8.3 Hz), 7.58 (d, 2H, J ¼ 8.4 Hz).
ESI-MS, m/z: 301.0 (Mþ. þ H). Anal. (C15H12N2O3S) C, H, N, S.
5.1.4. General procedure for the synthesis of the 5(E/Z)-[2-(arylsul
fonyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole derivatives (20–22)
To a solution of the vinylthio-derivative E or Z (1 equiv.) in
glacial AcOH (4.0 mL/mmol) was slowly added H2O2 (30%) (0.4 mL/
mmol), and the mixture was heated under reflux for 30 min. The
reaction mixture was cooled, neutralized with aqueous NaHCO3,
and extracted with EtOAc. After the workup of the combined
organic layers, the residue was purified by column chromatography
(SiO2, petroleum ether/EtOAc (8:2)), yielding spectroscopically
pure compounds.
5.1.5.1. 5E-[2-(phenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole N-oxide
(23). Yellow–orange oil (93%); 1H-NMR (CDCl3)
d: 7.04 (d, 1H,
J ¼ 15.6 Hz), 7.21 (d, 1H, J ¼ 15.5 Hz), 7.40 (dd, 1H), 7.45–7.55 (m,
3H), 7.58 (d, 2H), 7.87 (d, 1H), 8.01 (s, 1H). ESI-MS, m/z: 255
(Mþ$ þ H). Anal. (C14H10N2OS) C, H, N, S.
5.1.5.2. 5E-[2-(phenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N-oxide (24). Brown solid (96%); mp 120.5–122.0 ꢁC. 1H-NMR
(CDCl3)
d
: 7.04 (d, 1H, J ¼ 15.4 Hz), 7.51 (d, 1H, J ¼ 15.4 Hz), 7.55 (dd,
1H, J ¼ 9.4 Hz, J ¼ 1.3 Hz), 7.57 (m, 3H), 7.74 (m, 2H), 7.85 (d, 1H,
J ¼ 9.4 Hz), 7.89 (s, 1H). ESI-MS, m/z: 271 (Mþ$ þ H). Anal.
(C14H10N2O2S) C, H, N, S.
5.1.4.1. 5E-[2-(4-fluorophenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (20E). Pale yellow solid (56%); mp 134.0–135.0 ꢁC. 1H-
NMR (CDCl3)
d
: 6.99 (d, 1H, J ¼ 15.4 Hz), 7.28 (t, J ¼ 8.3 Hz), 7.35–
7.45 (bs, 1H), 7.50–7.70 (bs, 2H), 7.67 (d, 1H, J ¼ 15.4 Hz), 8.01 (m,
2H). EI-MS, m/z (abundance, %): 320 (Mþ., 64), 304 (13), 255 (6), 221
(11), 160 (71), 143 (100). Anal.(C14H9FN2O4S) C, H, N, S.
5.1.5.3. 5E-[2-(phenylsulfonyl)vinyl]benzo[c]1,2,5-oxadiazole N-oxide
(25). Brown oil (95%); 1H -NMR (CDCl3)
d
: 7.06 (d, 1H, J ¼ 15.4 Hz),
7.53 (dd, 1H, J ¼ 9.4 Hz, J ¼ 1.1 Hz), 7.58–7.69 (m, 3H), 7.78 (d, 1H,
J ¼ 15.4 Hz), 7.90 (d, 1H, J ¼ 9.4 Hz), 8.00 (m, 2H). ESI-MS, m/z: 287
(Mþ$ þ H). Anal. (C14H10N2O3S) C, H, N, S.
5.1.4.2. 5Z-[2-(4-fluorophenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N-oxide (20Z). Pale yellow solid (46%); mp 132.5–134.0 ꢁC. 1H-
NMR (CDCl3)
d
: 6.72 (d, 1H, J ¼ 10.9 Hz), 7.05 (d, 1H, J ¼ 10.9 Hz),
5.1.5.4. 5E-[2-(phenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxadiazole (26).
7.25 (t, J ¼ 8.1 Hz), 7.40–7.65 (bs, 3H), 7.86 (m, 2H). EI-MS, m/z
(abundance, %): 320 (Mþ., 51), 304 (4), 255 (11), 221 (18), 160 (45),
143 (68), 95 (100). Anal. (C14H9FN2O4S) C, H, N, S.
Pale yellow solid (90%); mp 180.5–182.0 ꢁC. 1H-NMR (CDCl3)
d: 7.56
(t, 2H), 7.66 (m, 1H), 7.69 (d, 1H, J ¼ 15.8 Hz), 7.79 (dd, 1H, J ¼ 1.1 Hz,

5064
D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
J ¼ 9.4 Hz), 7.89 (d, 1H, J ¼ 15.7 Hz), 7.95 (d, 2H, J ¼ 9.4 Hz), 8.05 (s,
(FBS). Different concentration of studied compounds dissolved in
DMSO were added and maintained for 5 days. Afterwards, the cells
were washed with PBS and incubated (37 ꢁC) with 0.4 mg/mL MTT
1H), 8.07 (2H, J ¼ 7.2 Hz). EI-MS, m/z (abundance, %): 250 (Mþ$
,
100), 233 (18), 205 (19), 165 (25), 105 (54), 77 (75). Anal.
(C15H10N2O2) C, H, N.
(Sigma) for 3 h. Then, formazan was dissolved with DMSO (180 mL),
and optical densities were measured. Each concentration was
assayed three times, and six growth controls were used in each test.
Cytotoxicity percentages (PCyt (%)) were determined as follows:
PCyt ¼ [100 ꢀ (ODdꢀ ODdm)/(ODc ꢀ ODcm)] ꢂ 100, where ODd is
the mean of OD595 of wells with parasites and different concen-
trations 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
medium only. The IC50 value was taken as the concentration of drug
needed to reduce the absorbance ratio to 50%.
5.1.5.5. 5E-[2-(4-chlorophenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
diazole (27). Yellow solid (94%); mp 210.0–212.0 ꢁC. 1H-NMR
(CDCl3)
d
: 7.69 (d, 2H, J ¼ 8.0 Hz), 7.92 (d, 1H, J ¼ 15.6 Hz), 8.18 (d,
1H, J ¼ 10.0 Hz), 8.22 (d, 1H, J ¼ 15.6 Hz), 8.26 (2H, J ¼ 8.4 Hz), 8.31
(1H, J ¼ 9.6 Hz), 8.54 (s, 1H). ESI-MS, m/z: 285 (Mþ$ þ H). Anal.
(C15H9ClN2O2) C, H, N.
5.1.5.6. 5E-[2-(4-methoxyphenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
diazole N-oxide (28). Yellow solid (92%); mp 183.0–185.0 ꢁC.
1H-NMR (CDCl3)
d
: 3.60 (s, 3H), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.90 (s, 1H,
J ¼ 15.6 Hz), 8.18 (d, 1H, J ¼ 10.4 Hz), 8.22 (d, 1H, J ¼ 15.6 Hz), 8.26
(d, 2H, J ¼ 8.4 Hz), 8.32 (d, 1H, J ¼ 9.6 Hz), 8.55 (s, 1H). ESI-MS, m/z:
281 (Mþ$ þ H). Anal. (C16H12N2O3) C, H, N.
5.2.3. Cytotoxicity to macrophages
J-774 murine macrophage-like cells (ATCC, USA) were main-
tained by passage in Dulbecco’s modified Eagle’s medium (DMEM)
containing 4 mM L-glutamine, and supplemented with 10% heat-
5.1.5.7. 5E-[2-(4-nitrophenylcarbonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
inactivated foetal calf serum. J-774 cells were seeded
(100.000 cells/well) in 96 well flat bottom microplates (Nunclon)
diazole (29). Yellow solid (88%); mp 205.0–207.0 ꢁC. 1H-NMR
(CDCl3)
d
: 7.98 (d, 1H, J ¼ 15.6 Hz), 8.20 (d, 1H, J ¼ 9.6 Hz), 8.25 (d,
with 200 mL of RPMI 1640 medium supplemented with 20% heat-
inactivated foetal 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 compounds (1.0–400.0 mM) for 48 h. After-
wards, the cells were washed with PBS and incubated (37 ꢁC) with
MTT (0.4 mg/mL) for 3 h. Then, the formazan was dissolved with
1H, J ¼ 15.6 Hz), 8.33 (d, 1H, J ¼ 10.0 Hz), 8.42 (d, 2H, J ¼ 9.0 Hz),
8.45 (d, 2H, J ¼ 8.8 Hz), 8.58 (s, 1H). ESI-MS, m/z: 296 (Mþ$ þ H).
Anal. (C15H9N3O4) C, H, N.
5.2. Biology
DMSO (180 mL) and optical densities were measured. Each
5.2.1. In vitro anti-T. cruzi activity using Tulahuen 2 strain
concentration was assayed three times and six growth controls
were used in each test. Cytotoxicity percentages (% C) were deter-
mined as follows: % C ¼ [100 ꢀ (ODdꢀ ODdm)/(ODcꢀ ODcm)] ꢂ
100, where ODd is the mean of OD595 of wells with macrophages
and different concentrations of the compounds; ODdm is the mean
of OD595 of wells with different compounds concentration in the
medium; ODc is the growth control and ODcm is the mean of OD595
of wells with medium only.
Trypanosoma cruzi epimastigotes (Tulahuen 2) were grown at
28 ꢁC in an axenic medium (BHI-tryptose) complemented with 5%
foetal calf serum. Cells were harvested in the late log phase,
resuspended in fresh medium, counted in a Neubauer chamber, and
placed in 24-well plates (3 ꢂ106/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 [35,36]. Before inocu-
lation, 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 ¼ {1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢂ 100, where Ap ¼ A610 of the
culture containing the compound at day 5; A0p ¼ A610 of the culture
containing the compound right after addition of the inocula (day
0); Ac ¼ A610 of the culture in the absence of any compound
(control) at day 5; A0c ¼ A610 in the absence of the compound at day
0. To determine IC50 values, 50% inhibitory growth concentrations,
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%.
5.2.4. Mitochondrial dehydrogenase activities [37,38]
Mitochondrial dehydrogenase activities were measured in 24-
well plates. One million of T. cruzi epimastigotes (Tulahuen 2 and Y
strains) or one million of L. braziliensis promastigotes (LTB300) in
500
20
m
L of the corresponding medium were seeded in each well, and
m
M of studied compounds was added. Two wells with untreated
parasites were maintained as controls corresponding to the given
time of treatment. The cultures were incubated at 28 ꢁC. At the
different time incubations, the parasites were counted, and the
colorimetric MTT dye-reduction assay was performed, the tetra-
zolium salt being converted into purple formazan by mitochondria.
Fifty
to each well, and plates were incubated for an additional 2 h. The
reaction was stopped by addition of 500 L of acidic isopropanol
mL of a solution containing 5 mg/mL of MTT in PBS were added
m
(0.4 mL HCl of 10 N in 100 mL isopropanol). The absorbance was
measured at 570 nm. Under our conditions, compounds did not
interfere with the reaction mixture. Percentage of mitochondrial
dehydrogenase activities (%) was determined using untreated
parasites-activities as 100%.
5.2.2. Viability of CL Brener clone, LTB300 strain of L. braziliensis
and LV135 strain of L. pifanoi
Trypanosoma cruzi epimastigotes (CL Brener clones) were grown
as it is indicated above. L. braziliensis (MHOM/BR/00/LTB300 strain)
and L. pifanoi (MHOM/VE/57/LV135) promastigotes were grown at
28 ꢁC in an axenic-RPMI medium supplemented with 5% FBS as
previously described [35–38]. Cell-culture plates consisting of 24
wells were filled at 1 mL/well with the corresponding parasite strain
culture during its exponential growth in the corresponding medium.
BHI-Tryptose medium supplemented with 5% foetal bovine serum
5.2.5. ESR studies
The free radical production capacity of the new benzofuroxan
derivatives was assessed on T. cruzi-microsomal fraction (4 mg
protein/mL) by ESR using DMPO and FXN for spin trapping
[25,26,31]. Benzofuroxan 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 or FXN, in 20 mM phosphate buffer, pH ¼ 7.4.

D. Castro et al. / European Journal of Medicinal Chemistry 44 (2009) 5055–5065
5065
[25] C. Olea-Azar, C. Rigol, F. Mendiza´bal, R. Briones, H. Cerecetto, R. Di Maio,
The final mixture was transferred to a 50 mL capillary. All of the
spectra were registered in the same scale after 15 scans.
´
M. Gonzalez, W. Porcal, M. Risso, Spectrochim. Acta Part A 59 (2003) 69–74.
[26] C. Olea-Azar, C. Rigol, M. Morillo, J.D. Maya, Y. Repetto, G. Aguirre, H. Cerecetto,
´
R. Di Maio, M. Gonzalez, W. Porcal, J. Chil. Chem. Soc. 48 (2003) 3–5.
[27] G. Aguirre, L. Boiani, H. Cerecetto, R. Di Maio, M. Gonza´lez, W. Porcal,
L. Thomson, V. To´ rtora, A. Denicola, M. Mo¨ller, Bioorg. Med. Chem. 13 (2005)
6324–6335.
Acknowledgements
Financial support from PDT (Uruguay) is acknowledged. We
thank to Dr. J.J. Cazzulo, Instituto de Investigaciones Biotecnologicas,
Universidad Nacional de General San Martın-CONICET, Argentina,
for performing helpful in the cruzipain isolation and purification.
We thank PEDECIBA for scholarship to LB, PEDECIBA-ANII for
scholarship to AM and PH, and UdelaR for scholarship to DB.
[28] G. Aguirre, L. Boiani, M. Boiani, H. Cerecetto, R. Di Maio, M. Gonza´lez,
´
´
W. Porcal, A. Denicola, O.E. Piro, E.E. Castellano, M.R. SantAnna, E.J. Barreiro,
Bioorg. Med. Chem. 13 (2005) 6336–6346.
´
[29] W. Porcal, P. Herna´ndez, G. Aguirre, L. Boiani, M. Boiani, A. Merlino, A. Ferreira,
R. Di Maio, A. Castro, M. Gonza´lez, H. Cerecetto, Bioorg. Med. Chem. 15 (2007)
2768–2781.
´
[30] W. Porcal, P. Hernandez, M. Boiani, G. Aguirre, L. Boiani, A. Chidichimo,
J.J. Cazzulo, N.E. Campillo, J.A. Paez, A. Castro, R.L. Krauth-Siegel, C. Davies,
´
´
M.A. Basombrıo, M. Gonzalez, H. Cerecetto, J. Med. Chem. 50 (2007) 6004–6015.
[31] W. Porcal, P. Herna´ndez, L. Boiani, M. Boiani, A. Ferreira, A. Chidichimo,
J.J. Cazzulo, C. Olea-Azar, M. Gonza´lez, H. Cerecetto, Bioorg. Med. Chem. 16
(2008) 6995–7004.
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