Second generation of 5-ethenylbenzofuroxan derivatives as inhibitors of Trypanosoma cruzi growth: Synthesis, biological evaluation, and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.01.009

In vitro growth inhibitory activity of 21 new 5-ethenylbenzofuroxan derivatives against the protozoan parasite Trypanosoma cruzi, the causative agent of American trypanosomiasis, was studied. The designed compounds possess the previously described exigencies for optimal anti-parasite activity, the 5-ethenylbenzofuroxanyl moiety with different substituents. The synthetic key for preparing the derivatives was the Wittig procedure, that when 5-formylbenzofuroxan was used as the electrophile the corresponding deoxygenated products were marginally generated. Four of the new derivatives displayed remarkable in vitro activities against the epimastigote form of three strains of T. cruzi, Tulahuen 2, CL Brener, and Y. While the three deoxygenated analogues biologically assayed resulted inactives. Unspecific cytotoxicity was evaluated using human macrophages and active derivatives were not toxic at a concentration at least 13 times that of its IC₅₀ against T. cruzi (CL Brener strain). From the preliminary structure-activity relationship studies lipophilicity and electronic requirements were found relevant to anti-T. cruzi activity. Active compounds are more lipophilic than inactive ones and it was also identified that an optimum value of R Swain-Lupton's descriptor is required for optimal activity.

Full text of DOI:10.1016/j.bmc.2007.01.009

Bioorganic & Medicinal Chemistry 15 (2007) 2768–2781
Second generation of 5-ethenylbenzofuroxan derivatives as
inhibitors of Trypanosoma cruzi growth: Synthesis, biological
evaluation, and structure–activity relationshipsq
a
a
a
a
a
´
´
Williams Porcal, Paola Hernandez, Gabriela Aguirre, Lucıa Boiani, Mariana Boiani,
Alicia Merlino,a Ana Ferreira,b Rossanna Di Maio,a Ana Castro,c,
a,
a,
*
*
´
Mercedes Gonzalez and Hugo Cerecetto
aDepartamento de Quı´mica Orga´nica, Facultad de Quı´mica-Facultad de Ciencias,
Universidad de la Repu´blica, 11400 Montevideo, Uruguay
bCa´tedra de Inmunologı´a, Facultad de Quı´mica-Facultad de Ciencias, Universidad de la Repu´blica, 11400 Montevideo, Uruguay
cInstituto de Quı´mica Me´dica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Received 1 August 2006; revised 6 December 2006; accepted 1 January 2007
Available online 12 January 2007
Abstract—In vitro growth inhibitory activity of 21 new 5-ethenylbenzofuroxan derivatives against the protozoan parasite Trypano-
soma cruzi, the causative agent of American trypanosomiasis, was studied. The designed compounds possess the previously described
exigencies for optimal anti-parasite activity, the 5-ethenylbenzofuroxanyl moiety with different substituents. The synthetic key for
preparing the derivatives was the Wittig procedure, that when 5-formylbenzofuroxan was used as the electrophile the corresponding
deoxygenated products were marginally generated. Four of the new derivatives displayed remarkable in vitro activities against the
epimastigote form of three strains of T. cruzi, Tulahuen 2, CL Brener, and Y. While the three deoxygenated analogues biologically
assayed resulted inactives. Unspecific cytotoxicity was evaluated using human macrophages and active derivatives were not toxic at a
concentration at least 13 times that of its IC50 against T. cruzi (CL Brener strain). From the preliminary structure–activity relationship
studies lipophilicity and electronic requirements were found relevant to anti-T. cruzi activity. Active compounds are more lipophilic
than inactive ones and it was also identified that an optimum value of R Swain-Lupton’s descriptor is required for optimal activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Trypanosoma cruzi is transmitted in rural areas to
humans and other mammals by reduviid bugs such as
Rhodnius prolixus and Triatoma infestans.3 The main
route of transmission is the result of blood-sucking
activity of Chagas’ disease vectors on mammals when
feeding in a cyclic process. The parasite presents differ-
ent morphological forms in a complex life cycle: amasti-
gote, the mammalian multiplicative intra-cellular form;
trypomastigote, the non-dividing bloodstream form that
invades tissues; metacyclic trypomastigotes, the non-
dividing highly infective form that invades mammalian
tissues via wounds provoked by vector blood sucking
action; epimastigotes, the replicative form that exists
in the Chagas’ disease vector. Recently, the existence
of the epimastigote form as an obligate mammalian
intra-cellular stage has been revisited4,5 and confirmed.6
Despite the progress achieved in the study of T. cruzi
biochemistry and physiology, in which several crucial
enzymes for parasite survival, absent in the host, have
Chagas’ disease or American trypanosomiasis is an
important health problem that affects around 20 million
people in Central and South America. Around 2–3 mil-
lion individuals develop the typical symptoms of this
disease that results in 50,000 yearly deaths.1,2 The caus-
ative agent of this disease is the hemoflagellate protozo-
an Trypanosoma cruzi (T. cruzi).
Keywords: Ethenylbenzofuroxans; Wittig reaction; T. cruzi.
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-ete-
´
´
y utilizacion.
nilbenzofuroxano, procedimiento de preparacion
*
Corresponding authors. Tel.: +598 2 5258618x216; fax: +598 2 525 07
49 (H.C.); e-mail: hcerecet@fq.edu.uy
Present address: Neuropharma S.A. Avda. de la Industria, 52.
Parque Tecnolo´gico de Madrid, Tres Cantos, 28760 Madrid, Spain.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.009

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2769
been identified as potential targets for the design of new
drugs,7,8 the chemotherapy to control this parasitic
infection remains undeveloped. The pharmacology is
based on old and quite unspecific drugs associated with
long-term treatments that give rise to severe side
effects. In fact, although Nifurtimox (4-([5-nitrofurfury-
family of compounds produce free radicals intra-parasit-
ically.16 Electronic properties of the compounds play an
important role on their effectiveness as anti-T. cruzi
agents.15,17,18 Compounds 1–5 are good examples of
the previously reported 3-D QSAR models using in vitro
anti-T. cruzi activities.15 From these previous biological
results new lead compounds were identified. 5-Ethenyl
derivatives of benzofuroxan were more active than the
other benzofuroxans studied, thus we decided to prepare
new derivatives containing the ethenyl moiety on the
heterocyclic system (Fig. 2).
lidene]-amino)-3-methylthio
morpholine-1,1-dioxide,
Nfx, Fig. 1) and Benznidazole (N-benzyl-2-nitro-1-imi-
dazoleacetamide, Bnz, Fig. 1), the only two drugs cur-
rently in use for clinical treatment of this disease, are
able to wipe out parasitemia and to reduce serological
titers, they are not specific enough to all T. cruzi strains
and they do not guarantee complete cure. Both drugs act
via the reduction of the nitro group. In the case of Nfx,
reduction generates an unstable nitro anion radical
which produces highly toxic reduced oxygen species.
Whereas, Bnz involves covalent modification of macro-
molecules by nitro reduction intermediates.9 The side
effects of these drugs result from the oxidative or reduc-
tive damage in the host’s tissues and are thus inextrica-
bly linked to its anti-parasitic activity. Despite these
limitations, some studies involving nitroimidazole deriv-
atives have been recently described.10,11 Other drugs
have been evaluated as anti-T. cruzi agents in the last
years, among them the antifungals Ketoconazole (Ktz,
Fig. 1) and Terbinafine (Tbf, Fig. 1) have demonstrated
to act as T. cruzi membrane sterol inhibitors.7
In the present work, the development of new 5-ethe-
nylbenzofuroxanyl derivatives (6–22, Figs. 4–8) and
some deoxygenated analogues and their anti-T. cruzi
activities are described. Unspecific mammal cytotoxicity
of some selected derivatives, and the parent compounds
1 and 2, was evaluated in vitro against human macro-
phages THP-1. Theoretical studies were developed in
order to obtain insight into the structural requirements
for optimum activity.
2. Methods and results
2.1. Synthesis
Two different general Wittig procedures were employed
to prepare the 5-ethenyl derivatives (Fig. 3). In the first
case (Fig. 3a), the Wittig reactions were performed
using 5-formylbenzofuroxan as electrophile and the
We have shown that benzofuroxan derivatives (Fig. 2)
possess high in vitro anti-T. cruzi activity against Tulah-
uen 2 strain12–15 and we have demonstrated that this
O
N
N
N
O2N
N
N
H
O
SO2
NO2
Nfx
Bnz
Cl
N
Ac N
N
O
O
O
Cl
N
Ktz
Tbf
N
Figure 1. Drugs used clinically and experimentally as anti-T. cruzi agents.
- inclusion of heterocycle systems
- E/Z isomers
IC50a (μΜ)
10.8
Ref.
-R
Isomeric geometry
E
Z
-Ph
1
7.0
R
O
O
N
N
-p-Cl-Ph
-p-CF3-Ph
-p-NO2-Ph
-H
E:Z (1:1)
E:Z (1:1)
4.3
9.8
7.2
> 25.0
2
3
4
5
R
H
O
O
O
O
N
N
N
N
Z
-
1-4
5
a In vitro activity on T. cruzi (Tulahuen 2 strain).
- inclusion of electron withdrawing moieties
- inclusion of electron donor moieties
- variation of the position
- inclusion of aliphatic moieties
Figure 2. Parent benzofuroxan, anti-T. cruzi activity14,15 and designed structural modifications.

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W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
O
a
b
O
O
N
N
H
R
R
O
O
N
N
N
N
CH2X
CH2PPh3X
R
R
O
O
+
(I)
(II)
NH2
NHCOCH3
O2N
O2N
R
NO2
R
N
N
O
NHCOCH3
CH3
CH2PPh3Br
(III)
(IV)
Figure 3. General procedures used to prepare desired derivatives.
corresponding phosphonium salts, II, prepared from the
halide I. Deoxygenated analogues were marginally gen-
erated in these olefination reactions eventhough Boden’s
mild conditions were used.19 The second approach
(Fig. 3b), designed to avoid the N-oxide reduction in
the olefination conditions, involved the use of the
phosphonium salt III that was reacted with the corre-
sponding aldehydes. Further, hydrolysis and oxidation
in one-pot20 produced the desired products in good
yield.
was converted into 9E by reaction with sodium azide
in one-pot SNAr/cyclization procedure.
Derivatives of the series of 5-(phenylethenyl) benzofuro-
xan with mesomeric electron withdrawing moieties in
the phenyl substituent were prepared as depicted in
Figure 5. Derivatives 10 and 11 were prepared from
the corresponding phosphonium salt (IIe and IIf) and
5-formylbenzofuroxan, both derivatives were obtained
as chromatographically separable E and Z isomers. In
both cases the reduced derivatives were observed but
they were not isolated. The preparation of the o-nitro
derivative 12 was done following the general procedure
shown in Figure 3b. The alkenes (IVbE and IVbZ) were
submitted, independently, to hydrolysis/oxidation pro-
cess to yield both geometric isomers, namely 12E and
12Z.
Derivatives of the series of 5-(phenylethenyl) benzofuro-
xan with mesomeric electron donor moieties in the phen-
yl substituent were prepared using the procedures shown
in Figure 4. Derivatives 6 and 7 were prepared following
the general procedure shown in Figure 3a. For deriva-
tive 6 it was possible to isolate geometric isomers, name-
ly 6E and 6Z, and the corresponding deoxygenated
analogues, 6Ed and 6Zd. For derivative 7 only the E iso-
mer was obtained although the deoxygenated derivative
was chromatographically observed as a fluorescent spot.
But it was not isolated in the chromatographic purifica-
tion process from the reaction mixture.
The two new halo derivatives developed, 13 and 14, are
shown in Figure 6. The m-fluoro derivative 13 was
prepared using the Wittig–Boden process with 5-form-
ylbenzofuroxan as electrophile, observing the formation
of the deoxygenated analogues which were not isolated.
The desired product was obtained as the chromato-
graphically inseparable mixture of geometric isomers,
namely 13E/Z, in a proportion E:Z = 1:1. For the p-bro-
mo derivative 14 was successfully assayed the general
methodology shown in Figure 3b. As in the preparation
of derivative 8 (Fig. 4b), the reaction between the phos-
phonium salt III and p-bromobenzaldehyde in Boden’s
conditions yielded the phosphonium salt’s hydrolysis
product (Fig. 6b). So, stronger conditions were em-
ployed generating the inseparable mixture of E and Z
isomer alkenes, IVc, that were submitted to the hydroly-
sis/oxidation process to yield the desired product as the
E isomer.
In order to avoid the formation of secondary products,
the methodology depicted in Figure 3b was assayed in
the derivative 8’s preparation. The reaction between
the phosphonium salt III and the poor electrophile
p-dimethylaminobenzaldehyde, in the typical Boden’s
conditions, yielded the phosphonium salt’s hydrolysis
product (Fig. 4b).21,22 In order to diminish this undesir-
able reaction, strong base, t-BuOK, and higher temper-
ature, boiling toluene, were employed yielding the
desired alkene IVa as a mixture of geometric isomers.
Attempts to transform IVa into 8 using one-pot hydro-
lysis/oxidation procedure were unsuccessful. Finally,
derivative 8 was obtained as E isomer (Fig. 4b) from
the phosphonium salt IIc and 5-formylbenzofuroxan.23
Marginally deoxygenated derivative, 8Ed, was generated
and it was isolated and spectroscopically characterized.
Three different derivatives of parent compound 5
(Fig. 2), where non-aromatic substituents were included
in the ethenyl moiety, were developed as shown in
Figure 7. While derivative 15 was isolated as the geomet-
ric isomeric mixture, 15E/Z, the stabilized phosphonium
salt methyloxycarbonyl methyl triphenyl phosphonium
bromide IIi generated exclusively the E isomer, namely
In the synthesis of p-hydroxy derivative 9 (Fig. 4c) was
employed the phosphonium salt IId23 reacting with
4-chloro-3-nitrobenzaldehyde. Then the E-alkene V

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2771
R2
a
R1
O
O
N
N
O
N
O
N
CH2P+Ph3 X-
H
O
CH2X
PPh3
-R1,-R2= -OCH2O-, 6E, 42 %
6Z, 23 %
toluene
K2CO3 / 18-c-6
THF / reflux
R2 reflux
R2
-R1= -OCH3, -R2= -H, 7E, 60 %
R2
R1
R1
R1
IIa, -R1,-R2= -OCH2O-, -X= -Cl, 15 %
IIb, -R1= -OCH3, -R2= -H, -X= -Br, 35 %
N
O
N
-R1,-R2= -OCH2O-, 6Ed, 2 %
O
b
6Zd, 3 %
CH3
H
N(CH3)2
K2CO3 / 18-c-6
THF / reflux
NO2
CH2P+Ph3Br-
NHCOCH3
N(CH3)2
O
NO2
H
NHCOCH3
i)HCl (c) / EtOH
N(CH3)2
reflux
decomposition
III
t-BuOK / 18-c-6
ii) NaOH / NaClO
0 ºC
AcNH
toluene / reflux
IVa E/Z, 40 %
NO2
(H3C)2N
O
CH2P+Ph3 AcO-
O
O
CHO
N
N
N
N
H
O
O
i) NaBH4 / EtOH
K2CO3 / 18-c-6
THF / reflux
ii) PPh3 / AcOH
toluene / reflux
40 %
(H3C)2N
8E, 26 %
N(CH3)2
N(CH3)2
N
O
N
IIc
8Ed, 3 %
OH
c
O
CH2P+Ph3 AcO-
HO
NO2
Cl
H
NaN3 / DMSO
60 - 90 ºC
O
N
N
K2CO3 / 18-c-6
O
DMF / reflux Cl
OH
V, 17 %
NO2
IId
9E, 26 %
Figure 4. Series of 5-(phenylethenyl)benzofuroxan with mesomeric electron donor moieties in the phenyl substituent.
16E. Derivative 17 was obtained following the other
methodology where the phosphonium salt III was react-
ed with n-pentanal yielding the chromatographically
inseparable mixture of alkenes IVd that after hydroly-
sis/oxidation generated the desired product as the iso-
meric mixture 17E/Z in a proportion E:Z = 1:1.
Furthermore, heterocyclic analogues of parent compounds
1 and 4 (Fig. 2) were prepared as shown in Figure 8.
Derivatives 18–21 were prepared from the phosphonium
salt III (Fig. 3a and b), yielding the alkenes with different
isomeric distribution. In the case of the thienyl deriva-
tive, intermediate IVe E was generated maintaining this

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W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
O
a
b
O
R1
N
O
N
H
CH2X
CH2P+Ph3 X-
O
PPh3
N
N
O
toluene
reflux
K2CO3 / 18-c-6
THF / reflux
R1
R1
-R1= -CO2H, 10E, 52 %
10Z, 24 %
IIe, -R1= -CO2H, -X= -Br, 80 %
-R1= -CN,
11E, 42 %
11Z, 25 %
IIf, -R1= -CN, -X= -Br, 35 %
O2N
NO2
O
CH2P+Ph3Br-
H
K2CO3 / 18-c-6
THF / reflux
NO2
i) HCl (c) / EtOH
AcNH
reflux
IVb E , 14 %
IVb Z , 46 %
NHCOCH3
NO2
ii) NaOH / NaClO
0 ºC
III
O
N
12E, 14 %
12Z, 88 %
O
NO2
N
Figure 5. Series of 5-(phenylethenyl)benzofuroxan with mesomeric electron withdrawing moieties in the phenyl substituent.
O
a
O
N
CH2P+Ph3 Br-
H
CH2Br
O
N
PPh3
O
N
N
toluene
reflux
K2CO3 / 18-c-6
THF / reflux
F
O
F
F
IIg, 80 %
13E/Z, 66 %
O
b
CH3
H
Br
K2CO3 / 18-c-6
THF / reflux
NO2
CH2P+Ph3Br-
NHCOCH3
Br
O
NO2
H
NHCOCH3
Br
III
i) HCl (c) / EtOH
reflux
t-BuOK / 18-c-6
AcNH
toluene / reflux
IVc E/Z, 20 %
NO2
ii) NaOH / NaClO
0 ºC
Br
O
N
O
14E, 95 %
N
Figure 6. Series of 5-(phenylethenyl)benzofuroxan with halogen in the phenyl substituent.
isomeric distribution after the hydrolysis/oxidation reac-
tion. However, for the 5-nitrothienyl analogue the inter-
mediate was produced as the Z isomer (IVf Z) that after
the hydrolysis/oxidation reaction changed the geometric
distribution to the most stable isomer, product 19E. In
the reaction between 5-formylbenzofuroxan and phos-
phonium salt III the Z isomer was generated, namely
20Z, and after the one-pot process the geometric

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2773
O
a
b
O
N
H
O
O
N
PPh3
R
CH2Br
R
CH2P+Ph3 Br-
N
N
O
R
toluene
reflux
K2CO3 / 18-c-6
THF / reflux
IIh, -R= -cyclohexyl, 7 %
IIi, -R= -CO2Me, 40 %
-R= -cyclohexyl, 15E/Z, 40 %
-R= -CO2Me,
16E, 53 %
+
-
(CH2)3CH3
CH2P Ph3Br
CH3(CH2)3CHO
NO2
i) HCl (c) / EtOH
reflux
t-BuOK / 18-c-6
AcNH
toluene / reflux
IVd E/Z , 31 %
NHCOCH3
NO2
ii) NaOH / NaClO
0 ºC
III
O
N
H3C(H2C)3
O
N
17E/Z, 51 %
Figure 7. Series of 5-ethenylbenzofuroxan with non-aromatic substituent in the ethenyl moiety.
a
CH2P+Ph3Br-
Y
i) HCl (c) / EtOH
reflux
Y
S
O
N
O
N
Y
CHO
S
S
K2CO3 / 18-c-6
THF / reflux
ii) NaOH / NaClO
0 ºC
NO2
AcNH
NHCOCH3
NO2
III
IVe E, -Y= -H, 75 %
IVf Z, -Y= -NO2, 49 %
18E, -Y= -H,
67 %
19E, -Y= -NO2, 26 %
O
b
c
CH2P+Ph3Br-
O
O
N
H
i) HCl (c) / EtOH
reflux
O
N
N
N
N
O
O
K2CO3 / 18-c-6
THF / reflux
ii) NaOH / NaClO
0 ºC
N
NO2
NHCOCH3
NO2
N
III
N
O
NHCOCH3
O
O
20Z, 34 %
21Z, 55 %
N
O
H
Ac2O
reflux
O
O
N
N
N
N
+
O
N
22E, 20 %
Figure 8. Series of 5-ethenylbenzofuroxan with heterocyclic substituent in the ethenyl moiety.
distribution was conserved however the N-oxide reduc-
tion was observed. A detailed study showed that the N-
oxide moiety was lost after hydrolysis conditions (HCl
(concd)/EtOH/reflux). For the preparation of the 4-
pyridinyl derivative, the acidity of methyl protons of 4-
picoline was used. In the conditions depicted in Figure
8c, the E isomer of derivative 22 was generated with a
low proportion of the deoxygenated analogue.
All new compounds were characterized by NMR (1H,
13C, COSY, and HETCOR experiments), IR, and MS.
The purity was established by TLC and microanalysis.
The stereochemistry around olefinic carbon–carbon
bond was established using the corresponding 1H
NMR coupling constant. It is well established that
benzofuroxan derivatives exist at room temperature
as a mixture of tautomers (i.e., Fig. 9a). The benzo

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W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
a
b
O
N
N
N
O
N
N
T = 303 K
O
N
8E
Ha'
Ha
O
c
T = 233 K
T = 288 K
T = 276 K
Ha’
Ha
Figure 9. (a) Tautomeric equilibrium of derivative 8E. (b) 1H NMR spectra of 8E in CDCl3 at 303 K. (c) Series of 1H NMR spectra of 8E in CDCl3
at different temperatures.
substituent could occupy the 5- or 6-position and the
proportion of both tautomers in the equilibrium de-
pends on the electronic characteristic of the substitu-
strain.27,28 The in vitro studies were completed for the
parent compounds 1–414,15 testing them against Brener
and Y strains. As a first screening the compounds were
incorporated into the media at 25 lM and their ability
to inhibit the growth of the epimastigote form of the
parasite, percentage of growth inhibition, was evaluated
in comparison to the control (no compound added to
the media) at day 5. Nfx, Bnz, Ktz, and Tbf were used
as the reference trypanocidal drugs. In order to confirm
the relevance of the N-oxide moiety in the activity, deox-
ygenated analogues were included in the biological stud-
ies. The IC50 concentration (which causes a 50%
reduction in parasite growth) was assessed for the most
active derivatives (Table 1).
1
ent.24 At room temperature (303 K) H and 13C NMR
spectra of the benzofuroxans showed broad signals
due to the rapid tautomeric equilibrium (i.e., Fig. 9b).
When NMR experiments were carried out at low tem-
perature (e.g., 233 K), the aromatic region showed nar-
row peaks corresponding to both tautomers
(Fig. 9c).15,18,25,26 In chloroform solution, (approximate-
ly 10% w/v) at 303 K, compound 8E shows broad reso-
nance signals due to incomplete coalescence (Fig. 9b).
While, on cooling, the broad signals were resolved below
270 K and at 233 K it was possible to record the com-
plete series of spectra (1H, 13C NMR, and HMQC,
HMBC, and COSY experiments). The ratio of 5- and
6-tautomer could be determined at low temperature
using specific chemical shifts. For example at 233 K, in
CDCl3, the chemical shifts of protons Ha and Ha0 (see
Fig. 9a and c) allow us to determine the tautomeric pro-
portion that resulted 5-tautomer:6-tautomer = 42:58.
2.2.2. In vitro mammal cytotoxicity. Unspecific mammal
cytotoxicity of some selected benzofuroxans was evalu-
ated in vitro at 25–400 lM, using THP-1 human macro-
phages as the cellular model (Table 2).29 In the study,
parent compounds 1 and 2, as pure isomers and as geo-
metric isomeric mixtures, and trypanocidal agents Ktz
and Tbf were included.
2.2. Biological characterization
2.3. Structure–activity relationships
2.2.1. In vitro anti-trypanosomatid activity. The new ben-
zofuroxan derivatives were tested in vitro against three
different strains of T. cruzi, Tulahuen 2, CL Brener,
and the Nfx and Bnz partially resistant strain, Y
First, 12 substituted-phenylethenyl benzofuroxans were
used in the structure–activity relationship studies, the
parent compounds 1–4 and the new derivatives 6–11,

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2775
Table 1. In vitro biological activity of parent compounds, new benzofuroxan derivatives, Nfx, Bnz, Ktz, and Tbf
Compound
Ref.
Nfx Bnz
Ktz
Tbf
1
2
3
4
5
6
6d
7
8
8d
Isomer
T2c
E
Z
E:Z E:Z
Z
—
E
Z
E
Z
E
E
E
IC50 (lM)a,b
7.7 7.4
10.0 17.1 10.8 7.0 4.3 9.5 7.0 >25.0 5.3
2.9 >25.0 >25.0 8.0 15.7 >25.0
9.0 >25.0 >25.0 5.0 4.4
8.5 >25.0 >25.0 8.5 22.0
e
CLBd 8.5 4.5
5.0
9.8
42.0 7.5
44.7 6.2
15.7 7.5 4.5 10.5
9.0 9.0 10.0 13.5
—
—
9.0
7.3
>25.0
—
Yf
6.5 3.8
Compound
Ref.
Isomer
9
10
11
12
13
14
15
16
17
18
19
E
20
21
22
E
E
Z
E
Z
E
Z
E:Z
E
E:Z
E
E:Z
E
Z
Z
E
IC50 (lM)a,b
T2
10.2 >25.0 >25.0 >25.0 >25.0 26.4 24.4 2.5 8.7 13.6 >25.0 19.7 0.6
6.0
24.0 >25.0 15.0
CLB
Y
22.5
25.0
—
—
—
—
—
—
—
—
11.1 13.7 10.0 2.5
7.5 5.5
—
—
—
—
12.8 15.0 3.5
12.2 4.5
—
—
—
—
—
24.0
—
—
—
a IC50, concentration that causes a 50% reduction in parasite growth.
b The results are means of three different experiments with a SD less than 10% in all cases.
c T2, Tulahuen 2 strain.
d CLB, CL Brener strain.
e Not determined.
f Y, Y strain.
Table 2. Cytotoxicity of benzofuroxan derivatives, reduced analogues,
and reference compounds to THP-1 human macrophages
descriptors were studied. Eq. 1 shows the best one-vari-
able relationship obtained where activity is correlated, in
a quadratic manner, with the Swain-Lupton’s constant
R (Fig. 10).
Compound
THP-1 IC50 (lM)a,b
IC50,macrophage
/
IC50,epimastigote
T2c
10.2
8.9
CLBd Ye
17.7
7.0
log10ðIC50Þ ¼ 0:95ðꢀ0:09Þ þ 1:34ðꢀ0:49ÞR
þ 1:71ðꢀ0:63ÞR2
1E
1Z
109.9
62.6
14.7
4.0
f
1E:Z(1:1)
2E
2Z
149.0
>200.0g
97.0
—
—
—
—
—
—
—
n ¼ 12; r ¼ 0:680; s ¼ 0:256; F ¼ 3:88;
—
—
p ¼ 0:061:
ð1Þ
2E:Z(1:1)
66.7
15.5
8.9
7.4
6E
6Z
220.0
120.0
81.0
41.5
41.4
24.4
13.3
30.1
14.1
This discrete result could be modestly improved, in the r2
correlation coefficient, including lipophilic descriptor in
the analysis (r = 0.700, s = 0.298, F = 2.58, p = 0.128).
Although the other statistical parameters (s, F, and p)
diminished their quality in this correlation, it shows the
relevance of lipophilicity in the biological activity. Simi-
larly, molar refractivity (MR) could be studied together
with R and R2 however the quality of the statistical cor-
relation resulted worse than the statistical parameters
obtained in Eq. 1 (r = 0.681, s = 0.272, F = 2.30,
p = 0.154). Again, the participation of this descriptor
showed the relevance of substituent volume in the bio-re-
sponse. Besides, the correlation matrix for the used phys-
icochemical descriptors was performed and cross-
correlations between the descriptors were not obtained.
Thus, these parameters are orthogonal, allowing its safe
use in the multilinear regression relationship.32,33
6E:Z(1:1)
6Zd
8E
—
—
—
>400.0
>400.0
<40
>25
<40
>90
<40
>18
Ktz
Tbf
44.0
329.3
4.4
19.3
8.8
7.8
4.5
7.4
a IC50, concentration that produces a 50% reduction in cell viability.
b The results are means of two different experiments with a SD less
than 10% in all cases.
c T2, Tulahuen 2 strain.
d CLB, CL Brener strain.
e Y, Y strain.
f Not determined.
g Solubility problems.
13, and 14. For derivatives 1, 6, 10, and 11 the biological
activities of the E isomeric forms were considered. The
IC50 in Tulahuen 2 strain was used as the biological
activity. log10 (IC50) was used as the dependent variable
in the linearization procedure. In the equations n repre-
sents the number of data points, r2 is the correlation
coefficient, s is the standard deviation of the regression
equation, and F value is related to the F-statistical anal-
ysis (Fischer test). As the independent variables were
employed the previously tabulated substituent constants
related to the electronic, the lipophilic, and the steric
properties (Hammett constants rm, rp, Swain-Lupton’s
constants F and R, lipophilic constant p, and molar
refractivity MR).30,31 One-variable and multivariable
regressions between the activity and the physicochemical
In order to include in the SAR study all the developed
benzofuroxan derivatives, molecular modeling studies
were performed by calculating their stereoelectronic
properties. These properties were determined using
DFT calculations.34,35 A detailed conformational search
for each of the molecules was performed, using MM
methods, to find the minimum energy and highest abun-
dance conformer. The geometry of this conformer was
fully optimized by applying B3LYP/6-31G*//AM1 in
the gas phase that allows us to obtain acute results with
low time of computational calculi. Then, single point
B3LYP/6-31G* calculation was performed.36–38 The

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W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
1,6
1,4
1,2
1,0
0,8
0,6
0,4
a
b
1,3
1,2
1,1
1,0
0,9
0,8
0,7
0,6
-1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4
0,4
0,6
0,8
1,0
1,2
1,4
1,6
log10(IC50) experimental
Figure 10. (a) R versus log10(IC50). (b) Plot of log10(IC50) experimental versus calculated values from Eq. 1. IC50 refers to results on Tulahuen 2
strain.
Table 3. Results of the t-test analysis performed on the two benzofuroxan populations, actives and inactives, and on the corresponding two
populations of studied theoretical descriptors (clogP and gap)
a
Compound
IC50
clogP
t = 2.08, p = 0.05
Gap
t = 2.06, p = 0.05
Most actives
Less actives
Mean = 6.2
Mean = 22.9
t = 6.56, p = 2.2 · 10ꢁ6
Mean = 4.49
Mean = 3.82
Mean = 2.97
Mean = 3.10
a For compounds with IC50 > 25.0 lM it was used IC50 = 30.0 lM in this study.
properties determined and examined in this study were
total energy, magnitude of dipolar moment, HOMO’s
and LUMO’s energies, gap (E_LUMO–E_HOMO), vol-
ume, and the logarithm of the partition coefficient of the
non-ionized molecules (logP). Theoretical logP (clogP)
was calculated using the Villar method, implemented in
PC SPARTAN 04 package39 at the AM1 semiempirical
level. Non-statistical significant correlations were ob-
tained when the different theoretical descriptors were
studied as independent variable, but a clear tendency
was observed between activities and clogP, the most ac-
tive compounds possess the highest clogP, or activities
and gap, the most active compounds possess in general
the lowest gap. These facts were studied dividing the
benzofuroxans into two groups of compounds, the most
actives against Tulahuen 2 strain (IC50 < 11 lM) and the
less actives against the parasite (IC50 P 11 lM). These
two groups of activities resulted significantly different
at 2.2 · 10ꢁ6 level (Table 3). Then, the corresponding
two groups of clogP or gap, for the most and less active
derivatives, were submitted to a t-test analysis. The
clogP values, for both pre-defined populations of com-
pounds, resulted significantly different at a 0.05 level
and the gap values at a level lower than 0.05.
Figure 11 shows, graphically, the distribution of the
HOMO and the LUMO maps in some relevant benzo-
furoxans. In these graphics it is possible to observe the
differences in the LUMO distributions between active
and inactive derivatives against Tulahuen 2 strain
(Fig. 11a). The LUMO is located at the heterocyclic car-
bons for the active derivatives (see arrows in Fig. 11a)
and at the olefinic carbons for the inactive ones. While
the HOMO distributions show less differences between
active and inactive derivatives (Fig. 11b).
a
b
6E
13E
18E
6E
13E
18E
10E
11E
21Z
10E
11E
21Z
Figure 11. (a) Electron density (transparent gray, isovalue 0.02) and LUMO (solid black, isovalue 0.039) isosurfaces for active (up) and inactive
derivatives (down), against Tulahuen 2 strain. (b) Electron density (transparent gray, isovalue 0.02) and HOMO (solid black, isovalue 0.039)
isosurfaces for active (up) and inactive derivatives (down), against Tulahuen 2 strain.

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2777
3. Discussion
in terms of gap (E_LUMO–E_HOMO) the most active
derivatives possessing the lowest gap. Further, the most
reactive derivatives with lowest gaps are the most
active against T. cruzi. Analyzing the gap’s effect in
the activity we studied the localization of frontier molec-
ular orbitals, HOMO and LUMO, in some selected
derivatives. It is possible to observe that nitrogen 1
and carbon 4 of the benzofuroxanyl system contribute
to the LUMO of active derivatives (arrows in
Fig. 11a). This fact could be involved in some differen-
tial nucleophilic or electronation processes. In reference
to the HOMO’s contribution, the clearest difference is
related to the contribution of the olefinic carbons in
the active benzofuroxans (arrows in Fig. 11b). Maybe,
this region interacts with a specific counterpart in a bio-
logical receptor.
In this second generation of 5-ethenylbenzofuroxan
derivatives were found excellent anti-T. cruzi agents. It
was not possible to observe different susceptibilities of
the benzofuroxans against the three studied strains
resulting, in general, equally active in the three strains.
On the other hand, clear differences were not observed
in the activities between the different isomeric forms of
the alkenes. The most active derivatives, 6, 13, 14, and
19, displayed good to excellent IC50 against the three
strains and in the same order as the IC50 of clinical drugs
Nfx and Bnz. The halo derivatives 13 and 14 possess
better activities than the parent compound, the chloro-
derivative 2, being the fluoro-analogue 13 one of the
most active developed benzofuroxans against Tulahuen
2 strain and the bromo-analogue 14 the most active
against CL Brener strain. The derivative 6 with an elec-
tron-donor substituent, in both isomeric forms, possess-
es good capacity to inhibit the three studied strains. The
5-nitrothienyl derivative 19 shows excellent activity
against CL Brener and Y, and good activity against
Tulahuen 2 strain. The thienyl derivative, 18, is the best
compound against Tulahuen 2 strain, with acceptable
IC50 against the two other evaluated strains. As previ-
ously, it was evidenced12–15,40,41 that the deoxygenated
analogues result inactives showing the relevance of the
N-oxide moiety in the anti-T. cruzi activity. In general,
the electron-withdrawing substituted derivatives result
inactives against the epimastigote form of the parasite.
The derivatives substituted in the ethenyl moiety by a
non-aromatic group, like 15 and 17, possess medium
activity showing that aromatic moieties are not funda-
mental for adequate anti-parasite activity. According
to the mammal cytotoxicity studies, the benzofuroxan
parents and new derivatives result selective against the
parasite. Z stereoisomers were more toxic than the E
analogues, maybe as a consequence of their best solubil-
ities in the biological medium. The new derivatives were
less toxic than the parent compounds. Remarkably, the
electron-donor derivatives, compounds 6, in both ste-
reoisomeric forms, and 8E showed low mammal cyto-
toxicity at a concentration that was at least 25 times
that of its IC50 against T. cruzi epimastigotes (Tulahuen
2 strain). The corresponding deoxygenated derivative,
6Zd, resulted non-toxic in the assayed conditions
(IC50,macrophage > 400.0 lM) however its T. cruzi activity
is very low.
4. Conclusions
The results presented above indicate that the in vitro
activity of new 5-ethenylbenzofuroxanyl derivatives
against T. cruzi epimastigotes is superior to that of
the first generation members possessing lower mam-
malian cytotoxicity. These results provide supporting
evidence for further in vivo studies of these com-
pounds in appropriate animal models for Chagas’
disease.
5. Experimental
5.1. Chemistry
All starting materials were commercially available
research-grade chemicals and used without further puri-
fication. Compounds 1E, 1Z, 2E:Z, 3E:Z, 4Z, 5-form-
ylbenzofuroxan, and the phosphonium salts IIc,
IId, and III were prepared following literature
procedures.14,15,23 The purity of the intermediates II,
IV and compound V was established by 1H NMR anal-
ysis. All solvents were dried and distilled prior to use.
All the reactions were carried out in a nitrogen atmo-
sphere. The typical work-up included washing with
brine and drying the organic layer with sodium sulfate
before concentration in vacuo. Melting points were
determined using a Leitz Microscope Heating Stage
Model 350 apparatus or a Mettler FP82 + FP80 appara-
tus and are uncorrected. Elemental analyses were ob-
tained from vacuum-dried samples (over phosphorus
pentoxide at 3–4 mm Hg, 24 h at room temperature)
and performed on a Fisons EA 1108 CHNS-O analyser.
Infrared spectra were recorded on a PerkinElmer 1310
apparatus, using potassium bromide tablets; the fre-
The performed SAR studies showed that in the series of
substituted-phenylethenyl derivatives some electronic
requirements are needed for a good activity. According
to Eq. 1 optimum value of R Swain-Lupton’s descriptor
is required for optimal activity. Substituent volume and
lipophilic characteristics of phenyl’s substituents could
play some role in the activity but not good statistical
results were obtained between these descriptors and
bio-response. The study of all the population, using
molecular modeling, enables to identify some tendency
between the theoretically studied properties and bio-
activity. In one hand, more lipophilic compounds result
more active than less lipophilic benzofuroxans. On the
other hand, some electronic requirement was evidenced
quencies are expressed in cmꢁ1 1H NMR spectra
.
were recorded on a Bruker DPX-400 (at 400 MHz)
instrument, with tetramethylsilane as the internal
reference and in the indicated solvent; the chemical
shifts are reported in ppm. J values are given in Hz.
The 1H NMR signals reported were obtained at
303 K. Mass spectra were recorded on a Shimadzu
GC-MS QP 1100 EX instrument, using electronic
impact at 70 eV.

2778
W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
5.2. General procedure for the synthesis of phosphonium
salts (II)
5.97 (s, 2H), 6.59 (d, J = 12.0, 1H), 6.74 (m, 3H), 6.78
(d, J = 12.0, 1H), 7.28 (d, J = 9.7, 1H), 7.65 (d,
J = 9.5, 1H), 7.69 (s, 1H); MS, m/z (abundance, %):
266 (M+Å, 100), 249 (33), 236 (20), 209 (50), 177 (91);
IR m 2890, 1600, 1500, 1356, 1286, 841.
A mixture of triphenylphosphine (1.1 equiv) and the
corresponding halide (1.0 equiv) in toluene was heated
at reflux for 8–12 h. Then the mixture was allowed to
cool to room temperature and the white crystalline solid
was collected, washed with petroleum ether and ethyl
ether, and dried in vacuum.
5.3.5. 5E-[2-(4-Methoxyphenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (7E). Yellow solid, mp 158.0–
159.0 ꢁC (Found: C, 66.9; H, 4.3; N, 10.1.
C15H12N2O3 requires C, 67.2; H, 4.5; N, 10.4); 1H
NMR (CDCl3) d 3.87 (s, 3H), 6.96 (d, J = 8.7, 2H),
6.99 (d, J = 16.0, 1H), 7.16 (d, J = 16.0, 1H), 7.51 (d,
J = 8.7, 2H), 7.58 (bs, 3H); MS, m/z (abundance, %):
268 (M+Å, 79), 252 (3), 208 (100), 165 (85); IR m 1603,
1537, 960.
5.3. General procedure for the synthesis of 5-ethenylben-
zofuroxan derivatives 6–8, 10, 11, 13, 15, and 16
A mixture of 5-formylbenzofuroxan (1.0 equiv), the cor-
responding phosphonium salt II (1.1 equiv), K2CO3
(1.1 equiv), 18-crown-6 (0.01 equiv), and THF (5.0 mL/
mmol) as solvent was stirred at reflux until the carbonyl
compound was not present (SiO2, petroleum ether/
EtOAc (8:2)). After the work-up, the residue was puri-
fied by column chromatography (SiO2, mixtures of
petroleum ether/EtOAc).
5.3.6. 5E-[2-(4-Dimethylaminophenyl)ethenyl]benzo[1,2-
c]1,2,5-oxadiazole N1-oxide (8E). Orange-brown solid,
mp 150 (d) ꢁC (Found: C, 67.9; H, 5.3; N, 14.6.
C16H15N3O2 requires C, 68.3; H, 5.4; N, 14.9); 1H
NMR (CDCl3) d 3.04 (s, 6H), 6.73 (d, J = 8.8, 2H),
6.90 (d, J = 16.2, 1H), 7.16 (d, J = 16.2, 1H), 7.45 (d,
J = 8.8, 2H), 7.60 (bs, 3H); MS, m/z (abundance, %):
265 (M+Åꢁ16, 6), 235 (14), 209 (2), 148 (28), 135 (57),
63 (100); IR m 3050, 2900, 1599, 1482, 1450, 1188, 814.
5.3.1. 5E-[2-(3,4-Methylendioxyphenyl)ethenyl]benzo[1,2-
c]1,2,5-oxadiazole N1-oxide (6E). Orange-brown needles,
mp 183.7–184.5 ꢁC (from acetonitrile:water) (Found: C,
63.5; H, 3.3; N, 9.7. C15H10N2O4 requires C, 63.8; H,
1
3.6; N, 9.9); H NMR (CDCl3) d 5.98 (s, 2H) 6.91 (d,
5.3.7. 5E-[2-(4-Dimethylaminophenyl)ethenyl]benzo[1,2-
c]1,2,5-oxadiazole (8Ed). From the chromatographic
purification process of 8E was also isolated the deoxy-
genated analogue. Orange solid, mp 172.0–175.0 ꢁC
(Found: C, 72.1; H, 5.4; N, 15.5. C16H15N3O requires
J = 8.0, 1H), 7.16 (dd, J = 1.6, J = 7.9, 1H), 7.27 (d,
J = 1.6, 1H), 7.29 (d, J = 16.3, 1H), 7.48 (d, J = 16.3,
1H), 7.50 (bs, 1H), 7.61 (bs, 1H), 7.86 (bs, 1H); MS
m/z (abundance, %): 282 (M+Å, 65), 266 (11), 247 (9),
222 (100), 163 (70); IR m 1625, 1379, 1273, 1039.
1
C, 72.4; H, 5.7; N, 15.8); H NMR (CDCl3) d 3.05 (s,
6H), 6.75 (d, J = 8.8, 2H), 6.99 (d, J = 16.2, 1H), 7.22
(d, J = 16.2, 1H), 7.48 (d, J = 8.7, 2H), 7.64 (s, 1H),
7.75 (d, J = 9.5, 1H), 7.79 (d, J = 9.5, 1H); MS, m/z
(abundance, %): 250 (M+Åꢁ15, 10), 208 (7), 132 (8),
166 (17), 57 (100); IR m 2910, 1599, 1362, 872, 814.
5.3.2.
5Z-[2-(3,4-Methylendioxyphenyl)ethenyl]benzo-
[1,2-c]1,2,5-oxadiazole N1-oxide (6Z). Yellow-orange
needles, mp 98.9–100.3 ꢁC (from hexane:EtOAc)
(Found: C, 63.7; H, 3.5; N, 9.7. C15H10N2O4 requires
1
C, 63.8; H, 3.6; N, 9.9); H NMR (CDCl3) d 5.98 (s,
2H), 6.50 (d, J = 12.0, 1H), 6.70 (s, 1H), 6.75 (s, 2H),
6.77 (d, J = 12.0, 1H), 7.12 (bs, 1H), 7.28 (bs, 2H);
MS, m/z (abundance, %): 282 (M+Å, 70), 266 (5), 247
(11), 222 (100), 163 (70); IR m 1620, 1514, 873.
5.3.8. 5E-[2-(4-Carboxyphenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (10E). Yellow solid, mp 299.0–302.0
ꢁC (Found: C, 63.9; H, 3.5; N, 9.8. C15H10N2O4 requires
1
C, 63.8; H, 3.6; N, 9.9); H NMR (DMSO-d6) d (ppm)
7.55 (d, J = 16.5, 1H), 7.63 (d, J = 17.0, 1H), 7.76 (d,
J = 8.2, 2H), 7.79 (bs, 1H), 7.96 (bs, 2H), 7.99 (d,
J = 8.2, 2H); MS, m/z (abundance, %): 282 (M+Å, 10),
265 (15), 236 (20), 212 (100), 131 (60); IR m 2548,
1670, 1610, 1292, 847.
5.3.3. 5E-[2-(3,4-Methylendioxyphenyl)ethenyl]benzo[1,2-
c]1,2,5-oxadiazole (6Ed). From the chromatographic
purification process of 6E and 6Z was also isolated the
deoxygenated analogue 6Ed. Orange solid, mp 180.0–
182.0 ꢁC (Found: C, 67.9; H, 3.5; N, 10.1.
C15H10N2O3 requires C, 67.7; H, 3.8; N, 10.5); 1H
NMR (CDCl3) d 6.03 (s, 2H), 6.87 (d, J = 8.0, 1H),
7.05 (dd,J = 1.5, J = 8.0, 1H), 7.05 (d, J = 16.0, 1H),
7.12 (d,J = 1.5 1H), 7.22 (d, J = 16.0, 1H), 7.71 (s,
1H), 7.73 (dd,J = 1.2, J = 9.5, 1H), 7.83 (d, J = 9.5,
1H); MS, m/z (abundance, %): 266 (M+Å, 90), 249 (52),
236 (37), 209 (30), 177 (100); IR m 2895, 1610, 1570,
1539, 1492, 1257, 1035, 954, 927.
5.3.9. 5Z-[2-(4-Carboxyphenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (10Z). Yellow solid, mp 155.0–
158.0 ꢁC (Found: C, 63.8; H, 3.3; N, 9.6. C15H10N2O4
1
requires C, 63.8; H, 3.6; N, 9.9); H NMR (DMSO-d6)
d 6.84 (d, J = 12.3, 1H), 6.97 (d, J = 12.4, 1H), 7.07
(bs, 1H), 7.38 (d, J = 8.1, 2H), 7.54 (bs, 2H), 7.87 (d,
J = 8.2, 2H); MS, m/z (abundance, %): 282 (M+Å, 10),
265 (15), 236 (20), 212 (100), 131 (60); IR m 2544,
1680, 1609, 1287, 887.
5.3.4.
5Z-[2-(3,4-Methylendioxyphenyl)ethenyl]benzo-
[1,2-c]1,2,5-oxadiazole (6Zd). From the chromatograph-
ic purification process of 6E and 6Z was also isolated the
deoxygenated analogue 6Zd. Yellow solid, mp 81.0–
83.0 ꢁC (Found: C, 67.5; H, 3.4; N, 10.3. C15H10N2O3
5.3.10. 5E-[2-(4-Cyanophenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (11E). Orange-brown solid, mp
193.0–196.0 ꢁC (Found: C, 68.2; H, 3.4; N, 15.9.
C15H9N3O2 requires C, 68.4; H, 3.5; N, 16.0); 1H
NMR (CDCl3) d 7.50 (bs, 1H), 7.61 (bs, 1H), 7.62
1
requires C, 67.7; H, 3.8; N, 10.5); H NMR (CDCl3) d

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2779
(s, 2H), 7.68 (bs, 1H), 7.83 (d, J = 8.3, 1H), 7.86 (bs,
1H), 7.90 (d, J = 8.4, 2H); MS, m/z (abundance, %):
263 (M+Å, 84), 247 (55), 230 (64), 215 (30), 203 (82),
190 (100), 177 (34); IR m 2222, 1615, 1525, 820.
10.8. C14H10N2O3 requires C, 66.1; H, 4.0; N, 11.0);
1H NMR (CDCl3) d 6.92 (d, J = 8.6, 2H), 7.24 (d,
J = 16.4, 1H), 7.47 (d, J = 16.4, 1H), 7.56 (d, J = 8.6,
2H), 7.60 (bs, 2H), 7.87 (bs, 1H), 8.68 (bs, 1H); MS,
m/z (abundance, %) 254 (M+, 48), 238 (7), 194
(72), 165 (100), 139 (20), 115 (59); IR m (cmꢁ1) 3400,
1715, 1655, 1540, 1458, 1396, 1482, 1450, 1213, 1113,
1080.
5.3.11. 5Z-[2-(4-Cyanophenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (11Z). Yellow solid, mp 145.0–
147.0 ꢁC (Found: C, 68.1; H, 3.6; N, 15.8. C15H9N3O2
1
requires C, 68.4; H, 3.5; N, 16.0); H NMR (DMSO-
d6) d 6.94 (d, J = 12.3, 1H), 7.01 (d, J = 12.4, 1H), 7.18
(bs, 1H), 7.47 (bs, 2H), 7.54 (d, J = 8.3, 2H), 7.73 (d,
J = 8.3, 2H); MS, m/z (abundance, %): 263 (M+Å, 91),
247 (18), 230 (39), 215 (25), 203 (100), 190 (59), 177
(41); IR m 2228, 1607, 1520, 826.
5.5. General procedure for the synthesis of 5-ethe-
nylbenzofuroxan derivatives 12, 14, and 17–21
Synthesis of IVb–f and 20. A mixture of III (1.0 equiv),
the corresponding aldehyde (1.0 equiv), K2CO3
(1.0 equiv), 18-crown-6 (0.01 equiv), and THF (5 mL/
mmol) as solvent was stirred at reflux until the carbonyl
compounds were not present (SiO2, petroleum ether/
EtOAc (8:2)). After the work-up, the residue was puri-
fied by column chromatography (SiO2, petroleum
ether/EtOAc (9:1)).
5.3.12. 5Z/E-[2-(3-Fluorophenyl)ethenyl]benzo[1,2-c]1,2,
5-oxadiazole N1-oxide (13E/Z). Yellow solid (Found:
C, 65.3; H, 3.2; N, 10.5. C14H9FN2O2 requires C, 65.6;
1
H, 3.5; N, 10.9); H NMR (CDCl3) d 6.61 (d, J = 12.2,
1H), 6.84 (d, J = 12.2, 1H), 6.98 (m, 4H), 7.12 (d,
J = 16.5, 1H), 7.21 (d, J = 16.2, 1H), 7.23–7.41 (m,
7H), 7.59 (bs, 3H); MS, m/z (abundance, %): 256 (M+Å,
31), 240 (30), 196 (84), 183 (100), 170 (56), 146 (39);
IR m 1611, 1582, 1524, 781, 681.
Synthesis of 5-ethenylbenzofuroxan derivatives 12, 14,
17–19, and 21. The corresponding alkene (IVb–f or 20)
(1.0 equiv), HCl (c) (10.0 equiv), and ethanol (12 mL/
mmol) as solvent were heated at reflux during 1–2 h.
The reaction mixture was cooled at room temperature
and ethanolic NaOH (0.16% w/v, 1.25 equiv) was added
and an intense red solution was obtained. The mixture
was cooled at 0 ꢁC and aqueous solution of NaClO
(2.8 M, 4.0 equiv) was added dropwise, and the reaction
mixture was stirred during 30 min. After the work-up,
the residue was purified by column chromatography
(SiO2, petroleum ether/EtOAc (9:1)).
5.3.13. 5Z/E-[2-Cyclohexylethenyl]benzo[1,2-c]1,2,5-oxa-
diazole N1-oxide (15E/Z). Brown oil (Found: C, 68.4; H,
6.2; N, 11.1. C14H16N2O2 requires C, 68.8; H, 6.6; N,
11.5); 1H NMR (CDCl3) d 1.25 (bs, 6H), 1.75 (bs,
5H), 5.70 (m, 1H), 6.25 (m, 1H), 7.20 (bs, 2H), 7.40
(bs, 1H); MS, m/z (abundance, %) 244 (M+Å, 39), 228
(52), 166 (100); IR m 1655, 1437, 953, 708.
5.3.14. 5E-[2-(Methoxycarbonyl)ethenyl]benzo[1,2-c]1,2,
5-oxadiazole N1-oxide (16E). Yellow-white solid, mp
140.0–143.0 ꢁC (Found: C, 54.3; H, 3.6; N, 12.8.
C10H8N2O4 requires C, 54.6; H, 3.7; N, 12.7); 1H
NMR (CDCl3) d 3.86 (s, 3H), 6.54 (d, J = 16, 1H),
7.50 (bs, 3H), 7.68 (d, J = 16.0, 1H); MS, m/z (abun-
dance, %) 220 (M+Å, 100), 204 (27), 189 (27), 173 (36),
145 (15); IR m 1721, 1613, 1578, 1198, 812.
5.5.1. 5E-[2-(2-Nitrophenyl)ethenyl]benzo[1,2-c]1,2,5-oxa-
diazole N1-oxide (12E). Yellow solid, mp 190 (d) ꢁC
(Found: C, 59.6; H, 3.0; N, 14.5. C14H9N3O4 requires
1
C, 59.4; H, 3.2; N, 14.8); H NMR (CDCl3) d 7.45 (d,
J = 16.3, 1 H), 7.60–7.75 (bs, 3H), 7.82 (t, J = 7.6, 1H),
7.83 (d, J = 16.2, 1H), 7.91 (bs, 1H), 8.02 (d, J = 7.7,
1H), 8.07 (dd, J = 1.2, J = 8.2, 1H); MS, m/z (abun-
dance, %): 283 (M+Å, 25), 267 (5), 223 (6), 181 (10),
120 (19), 92 (95), 91 (100).
5.4. 5E-[2-(4-Hydroxyphenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (9E)
5.5.2. 5Z-[2-(2-Nitrophenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (12Z). Yellow solid, mp 135 (d) ꢁC
(Found: C, 59.2; H, 2.9; N, 14.6. C14H9N3O4 requires C,
59.4; H, 3.2; N, 14.8); 1H NMR (CDCl3) d 6.77 (d,
J = 12.0, 1H), 6.83–6.98 (bs, 1H), 7.16 (d, J = 12.0,
1H), 7.26 (m, 3H), 7.52 (m, 2H), 8.19 (m, 1H); MS,
m/z (abundance, %) 283 (M+Å, 79), 267 (1), 223 (4),
120 (13), 119 (100), 92 (86), 91 (25); IR m 3100, 1620,
1522, 1341, 1462, 793.
5.4.1. 4Z/E-[2-(4-Chloro-3-nitrophenyl)ethenyl]phenol (V).
A mixture of 4-chloro-3-nitrobenzaldeyde (300 mg,
1.60 mmol), the corresponding phosphonium salt IId
(800 mg, 1.90 mmol), K2CO3 (520 mg, 3.80 mmol),
18-crown-6 (5 mg, 0.02 mmol), and DMF (10 mL) as
solvent was stirred at 100–110 ꢁC for 1.5 h. After the
work-up, the residue was purified by column chroma-
tography (SiO2, petroleum ether/EtOAc (0–10%)),
yellow solid (76 mg).
5.5.3. 5E-[2-(4-Bromophenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (14E). Orange solid, mp 170 (d)
ꢁC (Found: C, 52.8; H, 2.7; N, 8.6. C14H9BrN2O2 re-
quires C, 53.0; H, 2.9; N, 8.8); 1H NMR (CDCl3) d
7.09 (d, J = 16.4, 1H), 7.17 (d, J = 16.3, 1H), 7.42 (d,
J = 8.4, 2H), 7.56 (d, J = 8.4, 2H), 7.30–7.65 (bs, 3H);
MS, m/z (abundance, %): 302 (M+Å + 2–16, 2), 300
(M+Åꢁ16, 1), 192 (13), 178 (6), 63 (100); IR m 3100,
2926, 1603, 1568, 1530, 1007, 961, 839.
5.4.2. 5E-[2-(4-Hydroxyphenyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (9E). A mixture of V (20.5 mg,
0.08 mmol), NaN3 (4.4 mg, 68 mmol), and DMSO
(1 mL) as solvent was heated at 60–65 ꢁC for 4 h and
then at 80–90 ꢁC for 3 h. After the work-up, the residue
was purified by column chromatography (SiO2, petro-
leum ether/EtOAc (0–20%)). Yellow solid (5 mg), mp
174.3–175.1 ꢁC (Found: C, 65.9; H, 3.8; N,

2780
W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
5.5.4. 5Z/E-(1-Hexenyl)benzo[1,2-c]1,2,5-oxadiazole N1-
oxide (17E/Z). Red-brown oil (Found: C, 65.9; H, 6.3;
N, 12.5. C12H14N2O2 requires C, 66.0; H, 6.5; N,
(abundance, %): 239 (M+Å, 32), 223 (97), 192 (100), 178
(71); IR m 1612, 1593, 1412, 964, 844, 750.
1
12.8); H NMR (CDCl3) d 0.92 (t, 3H), 0.96 (t, 3H),
5.7. SAR
1.28–1.60 (m, 16H), 5.92 (td, 2H), 6.34 (m, 6H), 7.00–
7.60 (bs, 12H); MS, m/z (abundance, %): 218 (M+Å,
25), 202 (3), 158 (5), 149 (100); IR m 2929, 1650, 1525,
1450, 1337, 1223.
Structural manipulations were performed using
Spartan’04 package. Structures discussed within this
study were modeled using standard bond lengths and
angles, and energy-minimized as implemented in the
Spartan’04 software package, 1.0.1 version. On the more
flexible molecules we performed the corresponding
conformational analysis using the MM methods
(Conformational Distribution module implemented in
Spartan’04 package). All the compounds were built as
the 6-substituted tautomers and as Z- or E-isomers.
When the isomers’ mixture was biologically evaluated
the E-form was used in the SAR study. The conformer
of minimum energy was fully optimized using B3LYP/
6-31G*//AM1.
5.5.5. 5E-[2-(2-Thienyl)ethenyl]benzo[1,2-c]1,2,5-oxadia-
zole N1-oxide (18E). Brown solid, mp 122.0–125.0 ꢁC
(Found: C, 58.9; H, 3.1; N, 11.3; S, 12.7. C121H8N2O2S
requires C, 59.0; H, 3.3; N, 11.5; S, 13.1); H NMR
(CDCl3) d (ppm) 6.89 (d, J = 16.0, 1H), 7.08 (dd,
J = 1.5, J = 3.6 1H), 7.20 (d, J = 3.5, 1H), 7.33 (s,
1H), 7.37 (d, J = 16.0, 1H), 7.40 (bs, 1H), 7.54 (bs,
2H); MS, m/z (abundance, %): 244 (M+Å, 52), 228
(26), 184 (100), 171 (34), 139 (42); IR m 1622, 1570,
945, 702.
5.5.6. 5E-[2-(5-Nitro-2-thienyl)ethenyl]benzo[1,2-c]1,2,5-
oxadiazole N1-oxide (19E). Orange solid, mp 160 (d)
ꢁC (Found: C, 49.6; H, 2.4; N, 14.4; S, 10.8.
C12H7N3O4S requires C, 49.8; H, 2.4; N, 14.5; S,
5.8. Biology
5.8.1. In vitro anti-trypanosomal activity. Trypanosoma
cruzi epimastigotes (Tulahuen 2, CL Brener, and Y
strains) were grown at 28 ꢁC in an axenic medium (BHI-
Tryptose) as previously described,12–15 complemented
with 5% fetal calf serum. Cells were harvested in the late
log phase, resuspended in fresh medium, counted in Neu-
bauer’s chamber, and placed in 24-well plates (3 · 106/
mL). Cell growth was measured as the absorbance of
the culture at 610 nm, which was proved to be propor-
tional to the number of cells present.42 Before inocula-
tion, 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 cul-
ture 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 epimastigotes’ growth was
observed by the presence of up to 1% DMSO in the cul-
ture media. The percentage of growth inhibition was cal-
culated as follows: PGI = {1 ꢁ [(Ap ꢁ A0p)/(Ac ꢁ A0c)]}
· 100, where Ap = A600 of the culture containing the com-
pound at day 5; A0p = A600 of the culture containing the
compound right after addition of the inocula (day 0);
Ac = A600 of the culture in the absence of any compound
(control) at day 5; A0c = A600 in the absence of the com-
pound at day 0. To determine IC50 values, parasite
growth was followed in the absence (control) and pres-
ence of increasing concentrations of the corresponding
compound. The IC50 values were determined as the
compound concentration required to reduce by half the
absorbance of the control (without compound).
1
11.1); H NMR (CDCl3) d 7.42 (d, J = 4.3, 1H), 7.54
(d, J = 16.2, 1H), 7.63–7.80 (bs, 1H), 7.76 (d, J = 16.2,
1H), 7.80–7.95 (bs, 2H), 8.03 (d, J = 4.3, 1H); MS, m/z
(abundance, %): 289 (M+Å, 2), 148 (3), 140 (4), 128
(100), 102 (13); IR m 1611, 1572, 1524, 1329, 812, 731.
5.5.7. 5Z-[2-(4-Acetylamino-3-nitrophenyl)ethenyl]benzo-
[1,2-c]1,2,5-oxadiazole N1-oxide (20Z). Yellow solid, mp
134.0–135.0 ꢁC (Found: C, 56.6; H, 3.5; N, 16.2.
C16H12N4O5 requires C, 56.5; H, 3.6; N, 16.5); 1H
NMR (CDCl3) d 2.31 (s, 3H), 6.71 (d, J = 12.1, 1H),
6.79 (d, J = 12.1, 1H), 7.05 (bs, 1H), 7.34 (bs, 2H),
7.51 (dd, J = 1.9, J = 8.8, 1H), 8.11 (d, J = 1.9, 1H),
8.75 (d, J = 8.8, 1H), 10.34 (bs, 1H); MS, m/z (abun-
dance, %) 340 (M+Å, 3), 324 (44), 282 (100), 252 (18),
205 (35); IR m 3343, 1698, 1620, 1570, 1337.
5.5.8. 5Z-[2-Benzofurazanyl]benzo[1,2-c]1,2,5-oxadiazole
N1-oxide (21Z). Orange-brown solid, mp 125.0–128.0 ꢁC
(Found: C, 59.7; H, 2.9; N, 19.7. C14H8N4O3 requires C,
60.0; H, 2.9; N, 20.0); 1H NMR (CDCl3) d 6.84 (d,
J = 12.3, 1H), 6.92 (d, J = 12.3, 1H), 7.08 (bs, 1H),
7.26 (d, J = 9.3, 1H), 7.37 (bs, 2H), 7.74 (s, 1H), 7.77
(d, J = 9.4, 1H); MS, m/z (abundance, %): 280 (M+Å,
14), 264 (23), 217 (46), 204 (37), 190 (100), 177 (46);
IR m 1609, 1526, 1009, 885.
5.6. 5E-[2-(4-Pyridinyl)ethenyl]benzo[1,2-c]1,2,5-oxadia-
zole N1-oxide (22E)
5.8.2. In vitro cytotoxicity to macrophages. THP-1 hu-
man monocyte-like cells (ATCC, USA) were seeded
(100.000 cells/well) in 96-well flat-bottomed microplates
(Nunclon) and differentiated to macrophages by culture
(5% CO2, 37 ꢁC, 48 h) in the presence of 50 ng/mL phor-
bol-12-myristate-13 acetate (Sigma) in RPMI 1640
medium supplemented with 10% heat-inactivated fetal
calf serum. Afterward, cells were exposed for 48 h to
the compounds (25.0–400.0 lM) or vehicle for control.
Cell viability was then assessed by measuring the
A mixture of 5-formylbenzofuroxan (0.2 g, 1.22 mmol),
4-picoline (0.12 mL, 1.22 mmol), and Ac2O (10 mL)
was heated at reflux during 4 h. After the work-up, the
residue was purified by column chromatography (SiO2,
petroleum ether/EtOAc (8:2)). Brown solid, mp 180.0–
181.5 ꢁC (Found: C, 65.1; H, 3.6; N, 17.4. C13H9N3O2
1
requires C, 65.3; H, 3.8; N, 17.6); H NMR (CDCl3) d
7.40 (bs, 3H), 7.30 (m, 2H), 7.80 (d, J = 15.0, 1H),
7.91 (d, J = 15.0, 1H), 8.71 (d, J = 9.0, 2H); MS, m/z

W. Porcal et al. / Bioorg. Med. Chem. 15 (2007) 2768–2781
2781
O. E.; Castellano, E. E.; Sant’Anna, C. M. R.; Barreiro, E.
J. Bioorg. Med. Chem. 2005, 13, 6336.
16. Olea-Azar, C.; Rigol, C.; Opazo, L.; Morello, A.; Maya, J.
D.; Repetto, Y.; Aguirre, G.; Cerecetto, H.; DiMaio, R.;
´
Gonzalez, M.; Porcal, W. J. Child. Chem. Soc. 2003, 48,
mitochondrial-dependent reduction of 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sig-
ma) to formazan. For that purpose, cells were washed
with PBS and incubated (37 ꢁC) with MTT 0.4 mg/mL
for 3 h. Formazan was dissolved with DMSO (180 lL)
and optical densities were measured at 560 nm. Each
concentration was assayed three times and six additional
controls (cells in medium) were used in each test. Cyto-
toxicity percentages (% C) were determined as follows:
77.
17. Olea-Azar, C.; Rigol, C.; Mendizabal, F.; Briones,
´
R.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.;
´
Porcal, W.; Risso, M. Spectrochim. Acta
59, 69.
A 2003,
%
C = [100 ꢁ (ODc ꢁ ODcm)/(ODc ꢁ ODvm)] · 100,
´
18. Olea-Azar, C.; Rigol, C.; Mendizabal, F.; Cerecetto, H.;
Di Maio, R.; Gonzalez, M.; Porcal, W.; Morello, A.;
´
where ODc is the mean of OD560 obtained for cells incu-
bated with medium (controls), ODcm is the mean of
OD560 cells incubated with different compound concen-
trations, and ODvm is the mean of OD560 of cells incu-
bated with vehicle. The IC50, macrophage values were
determined as the drug concentration required to reduce
by half the absorbance of the control.
Repetto, Y.; Maya, J. D. Lett. Drugs Des. Dev. 2005, 2,
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This work received financial support from Fondo
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