In vivo anti-chagas vinylthio-, vinylsulfinyl-, and vinylsulfonylbenzofuroxan derivatives

Article abstract of DOI:10.1021/jm070604e

New benzofuroxans were developed and studied as antiproliferative Trypanosoma cruzi agents. Compounds displayed remarkable in vitro activities against different strains, Tulahuen 2, CL Brener and Y. Its unspecific cytotoxicity was evaluated using human macrophages being not toxic at a concentration at least 8 times, and until 250 times, that of its T. cruzi IC₅₀. Some biochemical pathways were studied, namely parasite respiration, cysteinyl active site enzymes and reaction with glutathione, as target for the mechanism of action. Not only T. cruzi respiration but also Cruzipain or trypanothione reductase were not affected, however the most active derivatives, the vinylsulfinyl- and vinylsulfonyl-containing benzofuroxans, react with glutathione in a redox pathway. Furthermore, the compounds showed good in vivo activities when they were studied in an acute murine model of Chagas' disease. The compounds were able to reduce the parasite loads of animals with fully established T. cruzi infections.

Full text of DOI:10.1021/jm070604e

6004
J. Med. Chem. 2007, 50, 6004-6015
In ViWo Anti-Chagas Vinylthio-, Vinylsulfinyl-, and Vinylsulfonylbenzofuroxan Derivatives‡
Williams Porcal,† Paola Herna´ndez,† Mariana Boiani,† Gabriela Aguirre,† Luc´ıa Boiani,† Agustina Chidichimo,X
Juan J. Cazzulo,X Nuria E. Campillo,§ Juan A. Paez,§ Ana Castro,| R. Luise Krauth-Siegel, Carolina Davies,#
Miguel AÄ ngel Basombr´ıo,# Mercedes Gonza´lez,*,† and Hugo Cerecetto*,†
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias and Facultad de Qu´ımica, UniVersidad de la Repu´blica, MonteVideo, Uruguay,
Instituto de InVestigaciones Biotecnolo´gicas and Instituto Tecnolo´gico de Chascomu´s (IIB-INTECH), UniVersidad Nacional de General San
Mart´ın - CONICET, San Mart´ın, Argentina, Instituto de Qu´ımica Me´dica, CSIC, Madrid, Spain, Neuropharma S.A., Madrid, Spain,
Biochemie-Zentrum Heidelberg, Ruprecht-Karls-UniVersita¨t, Heidelberg, Germany, and Instituto de Patolog´ıa Experimental,
UniVersidad de Salta, Salta, Argentina
ReceiVed May 24, 2007
New benzofuroxans were developed and studied as antiproliferative Trypanosoma cruzi agents. Compounds
displayed remarkable in Vitro activities against different strains, Tulahuen 2, CL Brener and Y. Its unspecific
cytotoxicity was evaluated using human macrophages being not toxic at a concentration at least 8 times,
and until 250 times, that of its T. cruzi IC50. Some biochemical pathways were studied, namely parasite
respiration, cysteinyl active site enzymes and reaction with glutathione, as target for the mechanism of
action. Not only T. cruzi respiration but also Cruzipain or trypanothione reductase were not affected, however
the most active derivatives, the vinylsulfinyl- and vinylsulfonyl-containing benzofuroxans, react with
glutathione in a redox pathway. Furthermore, the compounds showed good in ViVo activities when they
were studied in an acute murine model of Chagas’ disease. The compounds were able to reduce the parasite
loads of animals with fully established T. cruzi infections.
Introduction
decades ago.4 Both drugs are active in the acute phase of the
disease, but its efficacies are very low in the established chronic
phase. What is more, differences in drug susceptibility among
different T. cruzi strains lead to varied parasitological cure rates
according to the geographical area. Extensive work, in the last
two decades, has helped to understand the molecular basis of
the antichagasic activity of both drugs currently used in the
clinic.5 Nfx acts via the reduction of the nitro group to a
nitroanion radical that in term reacts with oxygen to produce
superoxide, a highly toxic metabolite, in a process known as
redox-cycling. The mechanism of action of Bnz also involves
nitro reduction, but reduced intermediates act covalently modi-
fying bio-macromolecules.6 Most frequent side effects of these
drugs include anorexia, vomiting, peripheral polyneuropathy,
and allergic dermopathy that are probably a result of oxidative
or reductive damage to the host’s tissue and are thus inextricably
linked to its antiparasitic activity.1 Other drugs have been
analyzed as anti-T. cruzi agents in the last years, among them
the antifungal Ketoconazole (1-[4-[4-(2R,4S)-2-(2,4-dichloro-
phenyl)-2-(1-imidazol-1-ylmethyl)-1,3-dioxolan-4 -yl]methoxy]-
phenyl-1-piperazinylethanone) (Ktz) and Terbinafine (N-[(2E)-
6,6-dimethyl-2-hepten-4-yn-1-yl]-N-methyl-1-naphthalene-
methanamine) (Tbf) have been demonstrated to act as T. cruzi
membrane sterol inhibitors.7 These antifungal agents were found
as excellent inhibitors of the sterol-membrane biosynthesis.4a
It has been pointed out that drugs that produce oxidative stress
by redox-cycling may be selective, as long as they are selectively
reduced by oxidoreductases that are unique to the parasite.8 The
same could be said for drugs that produce reductive damage
such as Bnz. Following this reasoning, our group has been
looking for less toxic and more selective antichagasic drugs by
using an N-oxide moiety as the bioreductive group.9 Thus,
benzofuroxan derivatives (benzo[1,2-c]1,2,5-oxadizole N-oxide)
were described for the first time as in Vitro anti-T. cruzi agents,10
displaying activities similar or higher than the reference drugs
(Chart 1).11-14 Some of these studies demonstrated that the
N-oxide moiety is essential for activity; additionally it was found
Parasitic diseases affect hundreds of millions people around
the world, mainly in underdeveloped countries. Since parasitic
protozoa are eukaryotic, they share many common features with
its mammalian host, making the development of effective and
selective drugs a hard task. Despite the great effort that has
been made in the discovery of unique targets that afford
selectivity, many of the drugs used today to treat this parasitosis
have serious side effects. Diseases caused by Trypanosomatidae,
which share a similar state regarding drug treatment, include
Chagas disease, the causative agent of which is Trypanosoma
cruzi (T. cruzia).1 This trypanosomatid alone is responsible for
an infected population of nearly 20 millions, and more than
200 million are at risk.2 Although sequencing of the T. cruzi
genome was recently completed,3 no new drugs have been
described yet. Drugs currently used in the treatment of Chagas
disease are two nitroaromatic heterocyles, Nifurtimox (4-(5-
nitrofurfurylindenamino)-3-methylthiomorpholine 1,1-dioxide)
(Nfx) produced only in El Salvador by Bayer, and Benznidazole
(N-benzyl-2-(2-nitro-1H-imidazol-1-yl)acetamide) (Bnz) pro-
duced by Roche. They were introduced empirically over three
‡ Part of this research is presented in the Uruguayan patent of inven-
tion: Cerecetto, H.; Di Maio, R.; Gonza´lez, M.; Porcal, W.; Denicola, A.
UR Patent 28,019, 2003: Derivados de 5-etenilbenzofuroxano, proced-
imiento de preparacio´n y utilizacio´n.
* To whom correspondence should be addressed. Dr. Hugo Cerecetto
and Dr. Mercedes Gonza´lez, Facultad de Ciencias, Igua´ 4225, Montevideo
(11400), Uruguay. E-mail: hcerecet@fq.edu.uy; megonzal@fq.edu.uy;
phone: (5982) 525-8618/216; fax: (5982) 525-0749.
† Universidad de la Repu´blica.
X Universidad Nacional de General San Mart´ın - CONICET.
§ CSIC.
| Neuropharma S.A.
Ruprecht-Karls-Universita¨t.
# Universidad de Salta.
a Abbreviations: T. cruzi, Trypanosoma cruzi; Nfx, Nifurtimox; Bnz,
Benznidazole; Ktz, Ketoconazole; Tbf, Terbinafine; CP, cruzipain; TR,
trypanothione reductase; GR, glutathione reductase; GSH, glutathione;
GSSG, glutathione oxidate; MCPBA, m-chloroperbenzoic acid; pi, postin-
fection; HB, hydrogen bond.
10.1021/jm070604e CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/26/2007

Benzofuroxans as Antitrypanosomatid Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6005
Chart 1. Experimentally Active Compounds against T. cruzi and Strategy to Design Novelty Structures
that some structural motives are important for optimal activity.
Specially, derivatives 1 and 2 (Chart 1) porting a phenylethenyl
moiety were some of the best anti-T. cruzi benzofuroxans
developed by us.15 Furthermore, our studies proved that some
benzofuroxans generate free radicals in biological medium16,17
and some others inhibit parasite respiration.13
identifying those that bind to TR reversibly,26 that act as
subversive substrates (turncoat/sabotage inhibitors),27 or that are
irreversible inhibitors. Only a few irreversible TR inhibitors are
known.28 One is ajoene (7, Chart 1), the spontaneous degradation
product of allicin, the major sulfur component of garlic. Ajeone
is a subversive substrate and a covalent inhibitor of both
reductases, and the modified enzymes show increased oxidase
activity.29 Recently, we described a family of hybrid metallic
complexes which possess as the main mechanism of action the
production of oxidative stress and the irreversible inhibition of
TR.28b
In the field of developing effective agents acting on key
targets in T. cruzi two of the most studied biomolecules are
Cruzipain (CP) and trypanothione reductase (TR). CP is an
endoproteinase able to digest proteins such as casein, bovine
serum albumin, and denatured hemoglobin.18 It is the major
lysosomal proteinase of T. cruzi and can thus be expected to
play a prominent role in nutrition of the parasite. In addition,
other roles have been proposed for the enzyme, i.e., CP may
be involved in the penetration of trypomastigote into the
mammalian cell19,20 and in the differentiation steps of the
parasite’s life cycle.21 Engel and co-workers22 demonstrated that
vinylsulfonyl derivative 3 (Chart 1), among others, arrested
growth of epimastigotes of T. cruzi at concentrations as low as
10 µM and caused cell death after 72 h. Others irreversible
inhibitors have been described (4-6, Chart 1).23 Benzofuroxan
6, identified from a virtual screening using a comprehensive
compound library, was one of the most interesting inhibitors23c
and, according to the authors, presents the additional advantage
that it adheres to Lipinski’s “rule of 5” for becoming a potential
drug.
On the other hand, all trypanosomatids have a unique thiol
metabolism in which the ubiquitous glutathione reductase
(GR: GSSG + NADPH + H+ f 2 GSH + NADP+) is replaced
by TR. TR is an NADPH-dependent flavoenzyme responsible
for maintaining the reducing intracellular milieu and thus
protecting the parasite against oxidative stressors. Trypanothione,
N1,N8-bis(glutathionyl)spermidine, is a low molecular weight
thiol exclusively found in parasitic protozoa of the order
Kinetoplastida.24 Direct precursors for the biosynthesis of
trypanothione are the tripeptide glutathione (GSH) and the
polyamine spermidine (Figure 1). The absence of trypanothione
from the mammalian host together with the sensitivity of
trypanosomatids to oxidative stress renders the enzymes of this
parasite specific thiol metabolism attractive as drug target
molecules.25 A large number of TR inhibitors have been studied,
Using compounds 1-7 (Chart 1) as a template for the design
of new active anti-T. cruzi agents, we have planned the
development of molecules that incorporate these recognized
pharmacophores. In the present work, vinylthio-, vinylsulfinyl-,
and vinylsulfonyl-containing benzofuroxans (Chart 1) were
synthesized as entities that conjugate two different pharma-
cophores, namely the benzofuroxan system and the vinylthio
moieties. The derivatives were examined for antiproliferative
in Vitro activity against three different T. cruzi strains, Tulahuen
2, CL Brener, and Y. Unspecific cytotoxicity of these derivatives
was evaluated in Vitro against human macrophages, THP-1 cells.
For the best derivatives, biochemical studies were performed
to get an insight into its mode of action. Effects on oxygen
uptake, inhibition of two essential cysteinyl active site enzymes,
CP and TR, and chemical reaction with GSH were analyzed.
Docking studies were used to explain the compounds’ lack of
CP inhibition activity. Experimental chemotherapy studies were
also performed using a murine model of Chagas disease with
Tulahuen 2 infection, evaluating the blood trypomastigote and
antibody levels.
Results
Chemistry. Two different approaches were used to obtain
the two-pharmacophore-containing derivatives, the condensa-
tion/oxidation procedure to generate phenylthio-, phenylsulfinyl-,
and phenylsulfonylvinyl derivatives, and substitution/elimination
step to generate the vinylsulfonylmethyl derivative. In the first
approach (Table 1), attempts to obtain sulfone 17 or 18 were
performed through the condensation of sulfone 930 with aldehyde

6006 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Porcal et al.
Figure 1. De noVo trypanothione synthesis from glutathione and spermidine.
Table 1. Attempt to Obtain Derivatives 17/18 from Sulfone 9
teristic of the substituent.35 At 303 K, 1H and 13C NMR spectra
of the benzofuroxans showed broad signals due to the rapid
tautomeric equilibrium. When NMR experiments were carried
out at low temperature, the aromatic region showed narrow
peaks corresponding to both tautomers.14,16,36,37
Biology. In Vitro Antitrypanosomatid Activity. The exist-
ence of the epimastigote form of T. cruzi as an obligate
mammalian intracellular stage has been revisited38,39 and
confirmed recently. Therefore, compounds were tested in Vitro
against the epimastigote form of the parasite.40 As a first
screening, the ability of developed derivatives to inhibit the
growth of the epimastigote form of T. cruzi (Tulahuen 2, CL
Brener ,and Y strains) was evaluated at 25 µM, and the IC50
was determined for the most active compounds (Table 2). This
family of derivatives was evaluated against the in ViVo
susceptible Tulahuen 2 strain and CL Brener clone and the in
ViVo partially Nfx- and Bnz-resistant Y strain.41 Parasites were
grown in the presence of the compound for 5 days, and the
percentage of growth inhibition was determined against control
(no drug added to the medium) as explained in the Experimental
Section.42 Apolar benzofuroxans, 13-18 and 23, showed good
activity at 25 µM against the epimastigote form of the three
different strains, and the IC50 was determined. The lower activity
of derivative 22, the most polar developed benzofuroxan, could
be explained in term of our previous results11,15 being the
consequence of 22 hydrophilicity and the presence of a hydrogen
bond donor moiety in the benzofuroxan lateral chain.
conditions
n-BuLi/THF/0 °C
results
5-hydroxymethylbenzofuroxan,
5-hydroxymethylbenzofurazana
no reaction
t-BuOK/THF/r.t. (several days)
NaOH (50%)/CH2Cl2-H2O/ r.t.
decomposition
a Reduction products.
10;11 however, in the different assayed reaction conditions the
desired products were not observed. When n-butyllithium (n-
BuLi) was used as base, the formyl group was reduced to the
corresponding alcohol, yielding 5-(hydroxymethyl)benzofurox-
an, as well as the deoxygenated benzofuroxan system (benzo-
furazan derivative) as a secondary product. Besides, attempts
to obtain the intermediate â-hydroxysulfone or sulfones 17/18
using t-BuOK or NaOH as base, were fruitless (see Table 1).
Consequently, the Boden-Wittig process31 was assayed using
phosphonium salt 1232 and aldehyde 10 (condition c, Scheme
1), generating the vinylthio derivatives 13 and 14, as chromato-
graphically separated geometric isomers, in good yields. Deoxy-
genated analogues, benzofurazans, were marginally generated
in these olefination reactions even though Boden’s mild
conditions were used. Selective oxidation of sulfides 13 and
14 were performed using classical conditions. Vinylsulfinyl
derivatives 15 and 16 were obtained using MCPBA at low
temperature (-78 °C) and vinylsulfonyl derivatives 17 and 18
using H2O2 in acetic acid at reflux.33 In the MCPBA oxidation,
small amounts of derivatives 17/18 were obtained, less than 5%.
In both conditions (MCPBA and H2O2), no olefin oxidation was
observed. In the second chemical approach, a vinylsulfonylm-
ethyl derivative was prepared (Scheme 2). For this, bromide
2012 was reacted with 2-mercaptoethanol, producing derivative
21 in good yield which was converted successively in sulfones
22 and 23 as it is shown in Scheme 2.34
Unspecific Cytotoxicity. Cytotoxicity of the studied com-
pounds against mammalian cells was evaluated in Vitro at 25-
1000 µM, using THP-1 human macrophages as the cellular
model (Table 3).14,43 In the study, Ktz and Tbf were included
as trypanocidal references. Z stereoisomers, 2, 16, and 18, were
more toxic against human macrophages than the E analogues,
maybe as consequence of better solubilities in the assay’s bio-
logical medium. The new derivatives were less or as toxic as
the parent compounds. Remarkably, phenylsulfonylvinyl deriva-
tive 17 showed the best selectivity indexes, being poorly toxic
against the mammalian system at concentrations that are at least
250, 50,, and 80 times that of its IC50 against T. cruzi epi-
mastigotes, Tulahuen 2, CL Brener, and Y strains, respectively.
In ViWo Anti-T. cruzi Evaluation.44-46 The best anti-T. cruzi
agents in Vitro against Tulahuen 2 strain, compounds 13-18
as equimolecular mixtures of geometric isomers, 13:14 (1:1),
15:16 (1:1) and 17:18 (1:1), were evaluated in ViVo in a murine
model of acute Chagas disease. In this preliminary study, female
Swiss mice were inoculated intraperitoneally with 2000 blood
trypomastigotes, and treatment began 5 days post-infection with
oral administration of 60 mg/kg/day of each compound during
30 days. The administration was done using a saline:Tween 80
(95:5) (vehicle) solution. A group treated in the same manner
with vehicle (control) was included. The level of parasitemia
was determined weekly47 (Figure 2), the mortality was observed
daily, and serological tests were performed 60 and 90 days post-
infection (Table 4). None of the animals treated with benzo-
furoxans died during the treatment while in the control group
the survival fraction was 75%.
All of the proposed structures were established by NMR (1H,
13C, COSY, HMQC, and HMBC experiments), IR, and MS.
The purity was established by TLC and microanalysis. The
stereochemistry around the olefinic carbon-carbon bond was
established using the corresponding 1H NMR coupling constant.
It is well-known that benzofuroxan derivatives exist at room
temperature as a mixture of tautomers. The benzo substituent
could occupy the 5- or 6-position, and the proportion of both
tautomers in the equilibrium depends on the electronic charac-

Benzofuroxans as Antitrypanosomatid Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6007
Scheme 1a
a Reagents and conditions: (a) CH2O/HCl (concd)/toluene/50 °C. (b) PPh3/toluene/reflux. (c) K2CO3/18-crown-6/toluene/reflux. (d) MCPBA/CH2Cl2/-
78 °C to rt. (e) H2O2 (30%)/AcOH/reflux.
Scheme 2a
a Reagents and conditions: (a) NBS/DBPO/CCl4/reflux. (b) 2-mercaptoethanol/t-BuOK/THF/-10 °C. (c) MCPBA/CH2Cl2/ 0 °C to rt. (d) MsCl/Et3N/
CH2Cl2/0°.
Table 2. In Vitro Anti-T. cruzi Activity
IC50 (µM)a,b
compd
T2c
CLBd
Y
1
2
10.8
7.0
2.6
3.8
2.6
0.7
1.6
1.1
>25.0
14.6
7.7
7.4
10.0
17.1
7.5
15.7
4.7
3.5
15.1
5.0
6.2
9.0
7.6
10.0
8.8
1.5
5.0
2.1
-
12.3
6.5
3.8
9.8
44.7
13
14
15
16
17
18
22
23
Nfx
Bnz
Ktz
Tbf
7.6
6.1
e
-
9.1
8.5
4.5
5.0
42.0
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. c T2: Tulahuen 2. d CLB: CL Brener. e Not determined.
Figure 2. Parasitemia in the murine model of acute Chagas disease.
Control (untreated) animals (9) and those receiving 60 mg/kg/d of 13:
14 (1:1) (O), 15:16 (1:1) (0), or 17:18 (1:1) (∆).
Table 3. Cytotoxicity of Benzofuroxan Derivatives to THP-1 Human
Macrophages
SIc
Table 4. Differences in the Level of Anti-T. cruzi Antibodies,
Expressed in Absorbance Units (abs), between days 60th and 90th
Postinfection for the Five Studied Groups
compd
THP-1 IC50 (µM)a,b
T2d
10.2
8.9
47.7
150.0
255.6
99.1
< 12.0
18.8
4.4
19.3
CLBe
Y
1
2
109.9
62.6
14.7
4.0
8.2
17.7
7.0
treatment
∆Aa
15
16
17
18
22
23
Ktz
Tbf
124.0
105.0
409.0
109.0
300.0
274.0
44.0
14.1
70.0
81.8
51.9
-
22.3
4.5
7.4
21.0
53.8
17.9
vehicle
0.028
0.071
Bnz b
13:14 (1:1)
15:16 (1:1)
17:18 (1:1)
0.051
-0.096
-0.052
f
-
30.1
8.8
7.8
a ∆A: Absorbance at 490 nm, day 90 pi - Absorbance at 490 nm, day
60 pi. b Bnz treatment: 100 mg/kg/day, orally administered during 30 days
(see Experimental Section).
329.3
a IC50: concentration that produces 50% inhibitory effect. b The results
are the means of two different experiments with a SD less than 10% in all
cases. c SI: selectivity index ) IC50,macrophage/IC50,epimastigote d T2: Tulahuen
.
benzofuroxan-treated animals, complete and permanent sup-
pression of parasitemia was observed from day 41 pi that
followed near zero parasitemia from day 35 pi. Benzofuroxan
parasitemia levels, during all the studied days, were in agreement
with the in Vitro benzofuroxan behavior, showing in ViVo
activities, in general, in the order 17:18 > 15:16 > 13:14. No
signs of toxicity were observed during the animals’ treatment
with the equimolecular mixtures 13:14, 15:16, or 17:18. All the
2. e CLB: CL Brener strain. f Not determined.
The three compound mixtures were able to diminish the
trypomastigote on the day of the maximum parasitic charge,
day 26 post-infection (pi) (compare parasites’ charge for
untreated animals, 716 tryp/100 fields, to 17:18-treated animals,
76 tryp/100 fields, 15:16-treated animals, 160 tryp/100 fields,
and to 13:14-treated animals, 334 tryp/100 fields). For the

6008 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Table 5. Inhibition of T. cruzi Cruzipain by 1, 2, 13, 15-18
Porcal et al.
pounds, 1 and 2, or as the equimolecular mixture (1:2, 1:1),
caused nearly 50% inhibition at 50 and 100 µM, respectively
(Table 5). Derivative 19 did not produce CP inhibition in the
assayed conditions. Consequently, it could be said that the
benzofuroxan system is not a CP inhibitor. On the other hand,
the studied vinylthio-, vinylsulfinyl-, and vinylsulfonyl-contain-
ing benzofuroxans, 13 and 15-18, were only weak inhibitors
under the experimental conditions. The benzofuroxans’ lack of
CP inhibition was explained by docking studies.49-51 In Table
6 are shown the residues in the binding site involved in ligand-
CP complex formation. Ligands 17 and 18 were compared to
inhibitor 3 as a reference structure. The vinylsulfonyl inhibitor
3 forms a covalent irreversible attachment to the active-site
cysteine residue Cys25 of CP with the vinyl carbon adjacent to
the Phe moiety. The Phe moiety of the inhibitor 3 binds in the
S2 pocket of CP through a hydrogen bond (HB) between the
nitrogen (Na, see Figure 3a) to the CdO of Gly66. In the CP
complex structure, HBs are formed between a SO2 oxygen and
hydrogen-donor side chains of the enzyme. Thus, one of the
SO2 oxygens has HBs with Gln19 (Nꢀ2) and His159 (Nδ1).
The other SO2 oxygen makes HBs with Gln19 (Nꢀ2) and Trp177
(Nꢀ1) of the CP. In addition, compound 3 could establish a HB
with Asp158 as well as aromatic interactions with Trp177. The
interaction data predicted with the LPC program52 reveal that
the benzofuroxans 17 and 18 covalently bound to CP (Cys25)
show one hydrogen bond between the vinylsulfonyl oxygen and
His159 (Nδ1) and aromatic interaction with Trp177. However,
the hydrogen bonds with Asp158, Gln19, and Gly66 do not
exist, these interactions being crucial for inhibition (Table 6).
The ∆Gbind and Kd of each complex calculated with the STC
program53 and the experimental IC50 (µM) values are shown in
Table 6, showing that the predicted values of ∆Gbind are in
agreement with the experimental results.
compd
inhibitor concn (µM)
inhibition (%)a,b
1
10
20
12
0
50
100
10
20
50
43
22c
19
11
20
44
0
2
100
10
1:2 (1:1)
13
25
50
10
23
42
0
25
50
10
15
25
2
15
20
50
100
10
0
24d
12
0
16
20
50
100
10
23
8d
3
17
0
20
50
100
10
0
35d
0
18
2
20
50
100
10
3
24d
19
100
E-64e
a The control assays contained the respective amount of DMSO. b The
values are the mean of at least two independent measurements that differed
by less than 10%. c Solubility problems. d At 50 µM concentration or higher
the compounds were not completely soluble. e For structure see Chart 1.
animals survived until the end of the parasitemia study (day
55). None of the animals treated with the benzofuroxans or Bnz,
used as control in the antibodies studies, showed negative anti-
T. cruzi serology. However, equimolecular mixtures of sulfox-
ides, 15:16, and sulfones, 17:18, decreased antibodies levels
between day 60 and 90 (Table 4), showing higher performance
than Bnz in this assay. Differences in the level of anti-T. cruzi
antibodies are in agreement with the parasitemia findings.
Mechanism of Action Studies. In order to confirm or exclude
some possible mechanisms of action, the following studies were
performed: effect on the parasite respiration, inhibition of CP
and TR, and reaction with glutathione.
Oxygen Uptake Effect. The ability of benzofuroxans to
modify parasite respiration was studied. Derivatives 17 and 18
were studied as described in Experimental Section, investigating
its effect on parasite respiration.48 None of them inhibit parasite
respiration or increase the oxygen consumption as result of
oxygen redox-cycling.
Inhibition of T. cruzi Trypanothione Reductase Studies.
Derivatives 15-18 were first studied as reversible inhibitors
of T. cruzi TR. The assays contained NADPH, TR, and 100
µM of each inhibitor and trypanothione disulfide (TS2) which
corresponds to a substrate concentration of 6 × Km.27b The
parent compound 1 was assayed at 40 µM because at higher
concentrations it precipitated in the buffer. Under these condi-
tions, none of the benzofuroxans proved to be inhibitors of TR.
Only compound 15 showed a slight inhibition of 13%. An
interesting observation was made when compounds 15-18 were
assayed. The color of the assay solutions changed from slight
yellow at the beginning to light-brown at the end of the assay.
This was a first indication that reduced trypanothione [T(SH)2]
generated during catalysis reacted with compounds 15-18 (see
following section). In a second series of kinetics, derivatives
15-18 were studied for its ability to inactivate reduced TR in
a time-dependent fashion. None of the compounds caused more
than 15% inactivation, the vinylsulfinyl derivative 16 and
vinylsulfonyl derivative 18 being the best inactivators at 100
µM (Table 7). These results indicate that any of the studied
benzofuroxan derivatives possess TR irreversible inhibition as
the main anti-T. cruzi mode of action.
Inhibition of T. cruzi Cruzipain Studies. Some of the new
benzofuroxans together with the parent compounds and meth-
ylbenzofuroxan, 19, were studied as inhibitors of T. cruzi CP
at 10-100 µM. None of the assayed derivatives resulted in good
inhibitors of CP at the studied doses. Only the parent com-
Table 6. Cruzipain (CP) Residues in Contact with the Studied Compounds, 3, 17 and 18, and Theoretical ∆GBind and Kd and Experimental IC50
Values of the Studied Complexes
complex
Gly66 (O)
HBb (Nac)
Gln19 (Nꢀ2)
His 159 (Nδ1)
HB (SdO)
Trp177
Asp158 (O)
HB (Nbc)
∆Gbind (kcal/mol)
-8.33
Kd (kcal/mol)
IC50 (µM)
1f29a
HB (OdSdO )
aromatic (Ph)
HB (OdS)
aromatic (Ph)
aromatic (Ph)
7.8 × 10-7
0.008d
e
CP-17
CP-18
-
-
-
HB (SdO)
HB (SdO)
-
-
-4.45
-4.01
0.5 × 10-3
> 50.0
> 50.0
-
1.1 × 10-3
a 1f29: complex CP-3, according to PDB code. b HB: hydrogen bond. c According to Figure 3a. d From reference 54. e No interaction.

Benzofuroxans as Antitrypanosomatid Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6009
Figure 3. (a) Inhibitor 3. (b) Structural superposition of 1f29 (inhibitor 3 in green) and the model complex with compounds 17 (yellow) and 18
(red). (c) Zoom of the structural superposition of 1f29 (inhibitor 3 in standard colors) and the model complex with compounds 17 (green) and 18
(purple).
Table 7. Time-Dependent Inactivation of Reduced T. cruzi
Trypanothione Reductase (TR) by 15-18
derivative 14. The first compounds were able to react with GSH,
at 28 °C, after 2 min of incubation. After 48 h, parent compound
volume activity (∆A/min) at
2 was unable to react with GSH. Taking into account the UV-
signal shifts, the GSH reacted mainly via a redox pathway,
producing GSSG and the corresponding vinylthio- and vinyl-
sulfinyl derivatives from compound 18 and the vinylthio-
containing benzofuroxan from 16 (Figure 4c). These results were
confirmed by isolation and characterization of the redox
products. Kinetic studies were not performed; however, it was
qualitatively observed that compound 16 reacted with GSH
faster than compound 18. Oxadiazole reduction product forma-
tion (the corresponding dioxime; see Figure 4c) was not
observed in the UV spectrum (data not shown).
preincubation time (h)
reaction mixturea
0 h
1 h
3 h
4 h
24 h
-
b
NADPH, TR, buffer, DMSO 0.104 0.105
(control 1)
-
0.104
15, TR, buffer (control 2)
15, NADPH, TR, buffer
0.104 0.086
0.103 0.105
NADPH, TR, buffer, DMSO 0.112 0.108 0.118
(control 1)
-
-
0.091
0.105
-
-
-
0.095
16, TR, buffer (control 2)
16, NADPH, TR, buffer
0.101 0.107
-
-
0.108
0.081
-
0.107 0.107 0.090
-
NADPH, TR, buffer, DMSO 0.104 0.105
(control 1)
-
0.104
17, TR, buffer (control 2)
17, NADPH, TR, buffer
NADPH, TR, buffer, DMSO 0.104 0.105
(control 1)
0.102 0.110
0.101 0.099
-
-
-
0.106
0.098
0.104
-
-
-
Discussion
The new benzofuroxan derivatives, 13-18, 22, and 23, are
efficiently obtained using condensation/oxidation or substitution/
oxidation procedures and chromatographically isolated as pure
stereoisomeric forms. Except derivative 22, the new benzo-
furoxans maintain or improve the parent compounds’ activities
in Vitro against parasite T. cruzi; for example, derivatives 16 or
18 are at least 3 times more active than parent compound 2 in
Vitro. Furthermore, these new benzofuroxans have low mam-
malian cytotoxicity; in our in Vitro model the new compounds
are less or as toxic as the parent compounds, derivatives 16-
18, TR, buffer (control 2)
18, NADPH, TR, buffer
0.104 0.102
0.097 0.084
-
-
-
-
-
0.088
a For conditions see Experimental Section. b Not determined.
Reactivity with Glutathione. In order to confirm the capacity
of the new benzofuroxans to react with thiols in the reductive
metabolism of the parasite, GSH was used as a model
compound. The reactions between benzofuroxans, 2, 14, 16, or
18, and GSH were followed spectroscopically in the UV-visible
region. The Z-isomers were selected because of their high
solubility under the assay conditions and for their optimum
activity compared to the E-isomers. The results are shown in
Figure 4. Clearly, it was confirmed that the vinylsulfinyl and
vinylsulfonyl derivatives, 16 and 18, react with this biological
thiol faster than the parent compound 2 and the vinylthio
18 having the best selectivity index (expressed as IC50,macrophage
/
IC50,epimastigote). The activity does not relate to inhibition of
cellular respiration, CP, or TR. According to docking studies,
the lack of CP inhibition is the result of structural motive
deficiency in the developed compounds (see Table 5 and Figure
3). In the case of TR, molecular details using docking were not
studied, but according to our results, it is possible to summarize

6010 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Porcal et al.
Figure 4. Studies of the reaction between benzofuroxans 14, 16, and 18 and GSH using UV-visible spectroscopy. (a) Spectra changes with time
for compound 16 treated with GSH. Inset: spectrum of derivative 14 (16’s and 18’s reduction end-product) and 16 and 18 after GSH treatment.
(b) Spectra changes with time for compound 18 treated with GSH. Inset: spectrum of GSH. (c) Proposed redox pathway for derivatives 15/16 and
17/18.
that only presence of sulfoxide or vinylthio moieties, as in ajoene
(Chart 1), is not enough for TR inhibition capacity. Any of the
studied compounds were able to inactivate reduced TR in a time-
dependent manner.
the enzyme GR maintaining the GSH level (see upper pathway
in Figure 5). On the contrary, in the parasitic cells compounds
15-18 could decrease GSH levels irreversibly because TR is
unable to reduce GSSG effectively (see lower pathways in
Figure 5), although another possibility is that GSSG could
deplete TSH, tryparedoxin (Tpx), and/or TR as a result of
chemical55,56 or biochemical24,57 pathways (Figure 5, lower
pathways). This proposal is in agreement with the differential
in Vitro cytotoxicity of studied sulfoxides and sulfones (compare
selectivity index values, Table 3, for Z-derivatives 16 and 18
with the Z-parent compound 2).
Furthermore, the oral treatments of Tulahuen 2-infected mice
at 60 mg/kg/day with the most in Vitro active benzofuroxans,
for a total of 30 doses, produce parasitological eradication and
complete animal survival and in some cases decrease the anti-
T. cruzi antibody levels at the end of the experimental protocol.
According to these findings, the equimolecular mixture of
The mammal/parasite selectivity for these compounds could
be explained in terms of the vinylsulfinyl and vinylsulfonyl
derivatives’ capability to react with GSH in a redox process.
First, it is well-known that GSH is one of the essential precursors
of TSH synthesis in the parasite cells (Figure 1). Second, T.
cruzi has much lower GSH levels than those of the mammalian
host. Therefore, in parasites the depletion of GSH levels, and
consequently TSH levels, could be more dangerous than the
GSH diminution in mammals. While in mammals GSH syn-
thesis can be inhibited in up to 80-90% without evidence of
toxicity, in T. cruzi this situation aggravates the precarious
defense against oxidative stress.55 Accordingly, in mammalian
cells the effects of derivatives 15-18 could be precluded by

Benzofuroxans as Antitrypanosomatid Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6011
reactions were carried out in a nitrogen atmosphere. The typical
workup included washing with brine and drying the organic layer
with sodium sulfate before concentration.
Triphenyl(phenylthiomethyl)phosphonium Chloride (12). A
solution of 11 (1.0 g, 6.3 mmol) and Ph3P (1.65 g, 6.3 mmol) in
dry toluene (10.0 mL) was heated at reflux for 12 h. Then the
mixture was allowed to cool to room temperature, and the white
crystalline solid was collected and washed with petroleum ether
and ethyl ether. This procedure was repeated until absence of PPh3
in the organic solvent (checked by TLC). White solid, 1.8 g (68%).
1
mp 225.0-227.0 °C; H NMR (CDCl3) δ 5.51 (d, J ) 9.6 Hz,
2H), 7.22 (m, 3H), 7.34 (m, 2H), 7.65 (m, 2H), 7.76 (m, 7H), 7.88
(m, 6H); ESI-MS, m/z: 385.1 (M+. - Cl); Anal. (C25H22ClPS) C,
H, N, S.
5(E and Z)-[2-(Phenylthio)vinyl]benzo[1,2-c]1,2,5-oxadiazole
N1-Oxide (13 and 14). A mixture of 10 (0.5 g, 3 mmol),
phosphonium salt 12 (1.3 g, 3 mmol), K2CO3 (0.4 g, 3 mmol),
18-crown-6 (10 mg, 0.03 mmol), and dry toluene as solvent was
stirred at reflux during 2.5 h. Then the mixture was allowed to
cool to room temperature, the brown solid was collected and washed
with toluene, and the organic solvent was evaporated in Vacuo. The
residue was purified by column chromatography (SiO2, petroleum
ether:EtOAc (95:5 to 90:10)), yielding derivative 13 as an orange-
yellow solid (0.37 g, 45%) and derivative 14 as an orange solid
(0.26 g, 32%). 13: mp 79.0-80.0 °C; 1H NMR (CDCl3): 6.97 (d,
1H, J ) 15.4 Hz), 7.40 (bs, 1H), 7.50-7.60 (bs, 2H), 7.61 (d, 1H,
J ) 15.2 Hz), 7.62 (m, 2H), 7.68 (d, 1H, J ) 7.8 Hz), 8.00 (d, 2H,
J ) 7.4 Hz); EI-MS, m/z (abundance, %): 270 (M+., 100), 254
(94), 238 (14), 223 (47), 210 (100), 165 (62); Anal. (C14H10N2O2S)
Figure 5. Speculative mechanisms of vinylsulfinyl- or vinylsulfonyl-
benzofuroxan selective mammal cytotoxicities.
vinylsulfinyl derivatives 15 and 16 has the best in ViVo activity,
the Z-isomer 16 being the best in Vitro anti-T. cruzi agent against
Tulahuen 2 and the Nfx, Bnz-partially resistant Y strains.
Furthermore, no signs of toxicity in the studied animals, during
the treatment, were observed.
Conclusions
The new benzofuroxan derivatives developed in this study
were in Vitro active against different strains of the protozoa T.
cruzi. In general, parasite toxic effects were not associated with
mammal cytotoxicity in macrophages. Derivative 17, with a
1
C, H, N, S. 14: mp 89.0-90.0 °C; H NMR (CDCl3): 6.48 (d, J
) 10.8 Hz, 1H), 6.85 (d, J ) 10.8 Hz, 1H), 7.50-7.60 (bs, 3H),
7.41 (m, 3H), 7.51 (d, J ) 7.5 Hz, 1H); EI-MS, m/z (abundance,
%): 270 (M+., 100), 254 (87), 238 (9), 223 (38), 210 (100), 165
(33); Anal. (C14H10N2O2S) C, H, N, S.
phenylsulfonylvinyl substituent, possesses the best IC50,macrophage
/
IC50,epimastigote ratio (more than 200 in the case of Tulahuen 2
strain). The rest of the new derivatives did not possess unspecific
cytotoxicity in human macrophages at a concentration at least
8 times that of its IC50 against T. cruzi (i.e., derivative 15, with
a phenylthiovinyl substituent). The structural motives included
in these compounds, the vinylsulfinyl and vinylsulfonyl moieties,
could act as thiol-depleting entities. The inhibition of cysteinyl
active site enzymes, CP and TR, was excluded as the main
mechanism of action. Additionally, after 30 day treatments, these
vinylthio, vinylsulfinyl, and vinylsulfonyl derivatives were able
to reduce the parasitemia of acute infected animals with
Tulahuen 2 strain without toxicity signs. The effects of the
sulfoxides and sulfones were also demonstrated with the anti-
T. cruzi antibody level modifications. In summary, a new family
of antiparasitic agents was developed that displays promising
in ViVo activity, deserving further study. The synthesis of new
derivatives in order to complete QSAR analysis and preclinical
studies, such as doses, schedule, strains, and toxicological
studies, is currently in progress.
5(E and Z)-[2-(Phenylsulfinyl)vinyl]benzo[1,2-c]1,2,5-oxadia-
zole N1-Oxide (15 and 16). A solution of 13 or 14 (0.25 g, 0.93
mmol) in CH2Cl2 (6.0 mL) was cooled to - 78 °C while a solution
of m-chloroperbenzoic acid (0.16 g, 0.93 mmol) in CH2Cl2 (2.0
mL) 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 × 10.0 mL). After the workup of the
combined organic layers, the residue was purified by column
chromatography (SiO2, petroleum ether:EtOAc (8:2)), yielding
derivative 15 as a yellow solid (0.12 g, 45%) or derivative 16 as a
1
brown solid (0.11 g, 42%). 15: mp 120.0-122.0 °C; H NMR
(CDCl3): 6.99 (d, 1H, J ) 15.4 Hz), 7.38 (d, 1H, J ) 15.5 Hz),
7.40-7.60 (bs + m, 6H), 7.70 (m, 2H); EI-MS, m/z (abundance,
%): 286 (M+., 8), 270 (5), 254 (41), 224 (23), 125 (100); Anal.
1
(C14H10N2O3S) C, H, N, S. 16: mp 106.0-107.0 °C; H NMR
(CDCl3): 6.75 (d, 1H, J ) 10.8 Hz), 7.32 (d, 1H, J ) 10.8 Hz),
7.60 (m, 3H), 7.75 (bs + m, 5H); EI-MS, m/z (abundance, %):
286 (M+., 4), 270 (3), 224 (45), 125 (100); Anal. (C14H10N2O3S)
C, H, N, S.
5(E and Z)-[2-(Phenylsulfonyl)vinyl]benzo[1,2-c]1,2,5-oxa-
diazole N1-Oxide (17 and 18). To a stirred solution of 13 or 14
(0.2 g, 0.74 mmol) in glacial AcOH (3.0 mL) was slowly added
H2O2 (30%) (0.3 mL), and the mixture was heated at reflux for 20
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
derivative 17 as a yellow solid (0.19 g, 86%) or derivative 18
as a yellow solid (0.13 g, 58%) and derivative. 17: mp 168.0-
170.0 °C; 1H NMR (CDCl3): 6.97 (d, 1H, J ) 15.4 Hz), 7.40 (bs,
1H), 7.50-7.60 (bs, 2H), 7.61 (d, 1H, J ) 15.2 Hz), 7.62 (m, 2H),
7.68 (d, 1H, J ) 7.8 Hz), 8.00 (d, 2H, J ) 7.4 Hz); EI-MS, m/z
(abundance, %): 302 (M+., 5), 286 (15), 203 (33), 125 (100); Anal.
Experimental Section
Compounds 1, 2, 10, 11, 19, and 20 were prepared according to
literature procedures.11,12,15,32 Melting points were determined with
an electrothermal melting point apparatus (Electrothermal 9100)
and are uncorrected. Proton and carbon NMR spectra were recorded
on a Bruker DPX-400 spectrometer. The chemical shifts values
are expressed in ppm relative to tetramethylsilane as internal
standard. Mass spectra were determined either on a MSD 5973
Hewlett-Packard or LC/MSD-Serie 100 Hewlett-Packard spectrom-
eters using electronic impact (EI) or electrospray ionization (ESI),
respectively. Microanalyses 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
1
(C14H10N2O4S) C, H, N, S. 18: mp 150.0-152.0 °C; H NMR
(CDCl3): 6.70 (d, 1H, J ) 12.0 Hz), 7.03 (d, 1H, J ) 12.0 Hz),
7.40-7.69 (bs+t, 6H), 7.87 (d, 2H, J ) 7.4 Hz); EI-MS, m/z

6012 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Porcal et al.
(abundance, %): 302 (M+., 2), 286 (8), 203 (18),125 (100); Anal.
(C14H10N2O4S) C, H, N, S.
PBS and incubated (37 °C) with 0.4 mg/mL 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) for 3 h. Then,
formazan was dissolved with DMSO (180 µL), and optical densities
were measured. Each concentration was assayed three times, and
six growth controls were used in each test. Cytotoxicity percentages
(%C) were determined as follows: %C ) [100 - (ODd - ODdm)/
(ODc - ODcm)] × 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 compound
concentrations in the medium, ODc is the growth control, and ODcm
is the mean of OD595 of wells with medium only.
5-(2-Hydroxyethylthiomethyl)benzo[1,2-c]1,2,5-oxadiazole N1-
Oxide (21). A solution of t-BuOK (0.15 g, 1.32 mmol) and
2-mercaptoethanol (0.10 mL, 1.32 mmol) in dry THF (10.0 mL)
was added dropwise to a solution of 20 (0.30 g, 1.32 mmol) in dry
THF (10.0 mL). The temperature was maintained at -10 °C for
30 min and then warmed to room temperature for 2 h. The solvent
was removed in Vacuo and then poured into water (30.0 mL) and
extracted with AcOEt (3 × 10.0 mL). After the workup of the
combined organic layers, the residue was purified by column
chromatography (SiO2, petroleum ether/EtOAc (7:3)), yielding
derivative 21 as a pale yellow oil (0.12 g, 40%). 1H NMR
(CDCl3): 2.67 (t, J ) 5.9 Hz, 2H), 3.76 (s, 2H), 3.80 (t, J ) 5.8
Hz, 2H), 3.94 (m, 1H), 7.33 (bs, 1H), 7.55 (bs, 2H); ESI-MS, m/z:
227 (M+. + H); Anal. (C9H10N2O3S) C, H, N, S.
In ViWo Anti-T. cruzi Activity (acute model). Swiss female mice
(45 days old, 25-30 g) were infected by intraperitoneal injection
of 2000 blood trypomastigotes. One group of four animals was used
as control, treated with vehicle (saline:Tween 80, 95:5), and three
groups of five animals were treated with the three mixtures of
studied benzofuroxans. For serologic studies, one group of four
animals treated with Bnz was included. Treatments were started 5
days after animal infection. Compounds were administered orally
as a vehicle suspension (0.1 mL/mouse), at a dose of 60 mg/kg/
day for benzofuroxan derivatives and 100 mg/kg/day for Bnz, during
30 days (6 day treatment, 1 day rest, until 30 doses completed).
The level of parasitemia was checked weekly by counting in a
Neubauer chamber the number of parasites in 5 µL of blood drawn
from the tail of the mice and diluted 1:10 in ammonium chloride
solution. Quantitative evaluation of circulating anti-T. cruzi antibod-
ies, at days 60 and 90 postinfection, was carried out by the use of
an enzyme-linked immunospot assay. The sera diluted to 1:100 was
reacted with an antigen constituted by a soluble homogenate of T.
cruzi epimastigotes.45 The results are expressed as the ratio of the
absorbance (A) of each serum sample at 490 nm to the cutoff value.
The cutoff for each reaction was the mean of the values determined
for the negative controls plus three times the standard deviation.
Animals. Animals were housed in wire mesh cages at 20 (
2 °C with natural light-dark cycles. The animals were allowed to
feed “ad libitum” to a standard pellet diet and water and were used
after a minimum of 3 days acclimation to the housing conditions.58
The experimental protocols with animals were evaluated and
supervised by the local Ethics Committee, and the research adhered
to the Principles of Laboratory Animal Care.59 Animals were
evaluated by supervision of international protocols, and they were
sacrificed in a humane way in accordance with recognized
guidelines on experimentation. At the end of experiments they were
anesthetized with ethyl ether and sacrificed by cervical dislocation.
Oxygen Uptake.17 T. cruzi epimastigotes (Tulahuen 2 strain)
from 5 days of growth were harvested by centrifugation (800g for
10 min), washed with a solution of NaCl (0.14 M), KCl (2.7 mM),
Na2HPO3 (8 mM), KH2PO4 (15 mM), pH 7.4 (medium A), and
then resuspended in medium A-glucose (5.5 mM glucose). Protein
concentration was determined by the biscinchoninic acid assay.
Oxygen consumption was determined with a water-jacketed Clark
electrode (YSI Model 5300) using a 2 mL reaction mixture in
medium A-glucose at 28 °C. The electrode was calibrated with
oxygen-saturated medium A at 28 °C (220 µM O2). The amount
of parasites used in each assay was always the equivalent to 0.2
mg of protein/mL (40 × 106 parasites/ mL). Drugs were added at
a 200 µM final concentration in DMSO. Control was run in the
absence of drug and with 0.1% DMSO. Cyanide-insensitive
respiration was determined after addition of KCN at a final
concentration of 20 mM.
5-(2-Hydroxyethylsulfonylmethyl)benzo[1,2-c]1,2,5-oxadiaz-
ole N1-Oxide (22). A solution of 21 (0.11 g, 0.49 mmol) in CH2-
Cl2 (4.0 mL) was cooled at 0 °C while a solution of m-chloroper-
benzoic acid (0.21 g, 1.22 mmol) in CH2Cl2 (2.0 mL) was added
dropwise. The mixture was warmed to room temperature until
derivative 21 was not present (SiO2, petroleum ether:EtOAc (8:
2)). After the workup, the residue was purified by column
chromatography (SiO2, petroleum ether:EtOAc (100 to 90:10)),
white solid (0.79 g, 63%). mp 128.0-130.0 °C; 1H NMR
(CDCl3): 3.30 (t, J ) 5.5 Hz, 2H), 4.11 (m, 2H), 4.49 (m, 1H),
4.64 (s, 2H), 7.55 (bs, 1H), 7.71 (bs, 2H); ESI-MS, m/z: 259 (M+.
+ H); Anal. (C9H10N2O5S) C, H, N, S.
5-Vinylsulfonylmethylbenzo[1,2-c]1,2,5-oxadiazole N1-Oxide
(23). A solution of 22 (55 mg, 0.21 mmol) in CH2Cl2 (5.0 mL),
Et3N (75 µL, 0.53 mmol), and methanesulfonyl chloride (0.20 µL,
0.23 mmol) was stirred at 0 °C for 20 min. The mixture was washed
with a saturated solution of NH4Cl (5.0 mL), dried, concentrated
in Vacuo, and purified by column chromatography (SiO2, petroleum
ether/EtOAc (8:2)), yielding 23 (25 mg, 49%) as a pale yellow
1
solid. mp 134.0-136.0 °C; H NMR (CDCl3): 4.62 (s, 2H), 6.25
(d, J ) 9.2 Hz, 1H), 6.28 (d, J ) 2.5 Hz, 1H), 6.97 (dd, J ) 9.9
Hz, J ) 6.7 Hz, 1H), 7.52 (bs, 1H), 7.67 (bs, 2H); ESI-MS, m/z:
241 (M+. + H); Anal. (C9H8N2O4S) C, H, N, S.
In Vitro Anti-T. cruzi Activity. Trypanosoma cruzi epimastig-
otes (Tulahuen 2 strain, Brener strain, Y strain) were grown at
28 °C in an axenic medium (BHI-tryptose) complemented with 5%
fetal calf serum. Cells from 5-day-old culture (stationary phase)
were inoculated to 50 mL of fresh culture medium to give an initial
concentration of 1 × 106 cells/mL. Cell growth was followed by
measuring everyday the absorbance of the culture at 600 nm. Before
inoculation, the medium was supplemented with the indicated
amount of the studied compound from a stock solution in DMSO.
The final concentration of DMSO in the culture medium never
exceeded 0.4%, and the control was run in the presence of 0.4%
DMSO and in the absence of compound. No effect on epimastigote
growth was observed in the presence of up to 1% DMSO in the
culture medium. The percentage of inhibition was calculated as
follows: % ) {1 - [(Ap - A0p)/(Ac - A0c)]}100, where Ap ) A600
of the culture containing the drug at day 5; A0p ) A600 of the culture
containing the compound just after the 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 compound at
day 0. To determine IC50 values, 50% inhibitory 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
the control. The IC50 value was taken as the concentration of
compound needed to reduce the absorbance ratio to 50%.
T. cruzi Cruzipain (CP) Inhibition Assays. Experimental
Studies. Cruzipain was purified to homogeneity from epimastigotes
of the Tulahuen 2 strain by ConA-Sepharose affinity chromatog-
raphy, as previously described,60 and its activity was assayed in a
reaction mixture (1 mL) containing (final concentration) 50 mM
Tris-acetate buffer, pH 8.0, 0.3 mM Bz-Pro-Phe-Arg-pNA, and 10
mM â-mercaptoethanol. Absorbance at 410 nm was followed at
30 °C on a Beckman Model 25 recording spectrophotometer. The
inhibitors were added as solutions in DMSO, and the controls
contained the same solvent concentration. E-64 was used as
reference.
Cytotoxicity to Human Macrophages. THP-1 human mac-
rophages were seeded (100000 cells/well) in 96-well flat bottom
microplates (Nunclon) with 200 µL of RPMI 1640 medium
supplemented with 20% heat-inactivated fetal calf serum. Cells were
allowed to attach for 48 h in a humidified 5% CO2/95% air
atmosphere at 37 °C. Then, cells were exposed to the compounds
(25-1000 µM) for 48 h. Afterward, the cells were washed with

Benzofuroxans as Antitrypanosomatid Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6013
Theoretical Studies. CP-docking studies were first performed
on a set of well-known ligands covalently bound to the active-site
Cys25 of CP54 (for example, compound 3 in Table 6). In this study
the high-resolution crystal structure of CP bound with phenyl-
containing vinylsulfone inhibitor 3 (pdb code 1f2954) was used as
a reference structure, adding protein hydrogen atoms and calculating
partial charges with the MMFF94 procedure51 implemented in the
Sybyl 6.9 program.50 The molecular docking studies with 17 and
18 were carried out using Flexidock,49 implemented in Sybyl 6.9,
minimizing by use of the MMFF94 force field and the MMFF94
charges. The structures of the ligands 17 and 18 were built with
standard bond lengths and angles using Sybyl, and their energies
were minimized using the Powel method with a conjugated gradient
of <0.001 kcal/mol convergent criteria provided by the MMFF94
force field and electrostatic charges based on the same method.
An initial model of the complex CP-inhibitor for compounds 17
and 18 was obtained by transference of system coordinates of
vinylsulfonyl derivatives 17 and 18 to the structure of the CP
complex (pdb code 1f2A) using the option Fit Monomer of Sybyl,
and the covalent bond between Cys 25 and the vinyl carbon at the
â position of SO2 group of 17 and 18 was created. Once the ligand
was situated manually in the active site of the protein, both side
chains and the ligand were minimized using the MMFF94 force
field and the MMFF94 charges while leaving the backbone rigid
using the Powel method with a conjugated gradient of <0.03 kcal/
mol convergent criteria. These complexes represent the entry for
the docking studies. A molecular docking approach was employed
to identify the more favorable interaction of 17 and 18 in the binding
site of the CP receptor. During the flexible docking analysis, the
protein was considered rigid except the residues involved in the
binding site and the ligands were considered flexible. The default
FlexiDock parameters were utilized in all cases, with iterations set
to 30000, obtaining a series of model complexes. Analysis of the
receptor-ligand complex models generated was based on the
hydrogen bond and aromatic and hydrophobic interactions predicted
with the LPC program52 and the free energy and dissociation
constants obtained from the difference accessible surface area
obtained using the STC program.53
T. cruzi Trypanothione Reductase (TR) Inhibition Assays.
Recombinant T. cruzi TR was prepared according to a published
procedure.61 Trypanothione disulfide was purchased from Bachem,
Heidelberg, Germany. TR activity was measured spectrophoto-
metrically at 25 °C in TR assay buffer (40 mM HEPES, 1 mM
EDTA, pH 7.5) as described.27b Stock solutions of the compounds
were prepared in DMSO. The assay mixtures (1 mL) contained
TR assay buffer, 100 µM NADPH, 105 or 93 µM trypanothione
disulfide (TS2), and varying concentrations of the inhibitor.
NADPH, enzyme, and inhibitor were mixed, and the reaction was
started by adding TS2. The absorption decrease at 340 nm due to
NADPH consumption was followed. Control assays contained the
respective amount of DMSO instead of inhibitor.
Test of Time-Dependent Inactivation of Reduced TR. In a
total volume of 50 µL of TR assay buffer (40 mM HEPES, 1 mM
EDTA, pH 7.5), 170 mU TR was incubated at 25 °C with 100 µM
inhibitor in the presence of 160 µM NADPH. At different times
between 0 and 24 h, 5 µL aliquots were removed, and the remaining
activity was measured in a standard TR assay. Because of the 200-
fold dilution, reversible inhibition was not recorded under these
conditions. Two control series contained buffer, TR, and NADPH
or buffer, TR, and inhibitor.
Acknowledgment. Financial support from Collaborative
Project UdelaR (Uruguay) - CSIC (Spain) (#2006UY0009) is
acknowledged. We thank AMSUD-PASTEUR network for a
scholarship to M.B. and PEDECIBA for scholarships to W.P.
and M.B. P.H., G.A., and L.B. are PEDECIBA students.
Supporting Information Available: Elemental analysis re-
sults for benzo[1,2-c]1,2,5-oxadiazole N1-oxide derivatives. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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