Naftifine-analogues as anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1016/j.ejmech.2010.01.052

Chagas disease represents a relevant health problem in Central and South America. The first line of treatment is Nifurtimox and Benznidazole which have a great deal of disadvantages that demands the rapid generation of therapeutic alternatives. Based in our research on aza-thiaheterocycles as anti-Trypanosoma cruzi agents we identified pharmacophores that act through oxidative stress. Here, we describe the synthesis and the activity of new containing bioactive-heterocycles analogues of naftifine as potential T. cruzi membrane sterol biosynthesis inhibitors. Benzimidazole 1,3-dioxides (11 and 13) and quinoxaline 1,4-dioxides (22 and 23) displayed excellent parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated with the compounds showed that any of them are able to accumulate squalene suggesting that in the anti-T. cruzi mechanism of action is not involved the inhibition of sterol biosynthesis. Some derivatives were also tested as antifungal agents. The results obtained in the present work open potential therapeutic possibilities of new compounds for these infectious diseases.

Full text of DOI:10.1016/j.ejmech.2010.01.052

European Journal of Medicinal Chemistry 45 (2010) 2154–2164
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Naftifine-analogues as anti-Trypanosoma cruzi agents
a
a
a
b
b
´
´
Alejandra Gerpe , Lucıa Boiani , Paola Hernandez , Maximiliano Sortino , Susana Zacchino ,
a,
a,
*
*
´
Mercedes Gonzalez , Hugo Cerecetto
a
´
´
´
´
Departamento de Qu´ımica Organica, Facultad de Ciencias-Facultad de Quımica, Universidad de la Republica, Igua 4225, 11400 Montevideo, Uruguay
Catedra de Farmacognosia, Facultad de Ciencias Bioquımicas y Farmaceuticas, Universidad Nacional de Rosario, Rosario, Argentina
b
´
´
´
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas disease represents a relevant health problem in Central and South America. The first line of
treatment is Nifurtimox and Benznidazole which have a great deal of disadvantages that demands the
rapid generation of therapeutic alternatives. Based in our research on aza-thiaheterocycles as anti-Try-
panosoma cruzi agents we identified pharmacophores that act through oxidative stress. Here, we describe
the synthesis and the activity of new containing bioactive-heterocycles analogues of naftifine as potential
T. cruzi membrane sterol biosynthesis inhibitors. Benzimidazole 1,3-dioxides (11 and 13) and quinoxaline
1,4-dioxides (22 and 23) displayed excellent parasite/mammal selectivity indexes. Analysis of the free
sterols from parasite incubated with the compounds showed that any of them are able to accumulate
squalene suggesting that in the anti-T. cruzi mechanism of action is not involved the inhibition of sterol
biosynthesis. Some derivatives were also tested as antifungal agents. The results obtained in the present
work open potential therapeutic possibilities of new compounds for these infectious diseases.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 5 December 2009
Received in revised form
18 January 2010
Accepted 21 January 2010
Available online 28 January 2010
Keywords:
Alkenylamine
Indazole N-oxide
Benzofuroxan
Benzimidazole 1,3-dioxide
Quinoxaline 1,4-dioxide
Anti-T. cruzi agents
1. Introduction
T. cruzi like most pathogenic fungi requires specific 24-alkyl
sterols for cell viability and proliferation in all stages of its life cycle
Chagas disease, or American Trypanosomiasis, remains the
major parasitic disease burden in Central and South America,
despite recent advances in the control of its vectorial and trans-
fusional transmission [1,2]. Current treatments are based on old
and quite unspecific drugs, Nifurtimox (Nfx) and Benznidazole
(Bnz) associated with long-term treatments that may give rise to
severe side effects [3]. In fact, although Nfx and Bnz are able to
eliminate patent parasitemia and to reduce serological titers in
acute and early chronic infections, they are not active against all
Trypanosoma cruzi strains and have significantly low efficacy in
long-term chronic infections. Even though the complete genome of
T. cruzi (T. cruzi) CL Brener clone [4] has been sequenced, not new
chemotherapeutic agents emerge [5]. However, several metabolic
steps essential for parasite survival and for potential use as
chemotherapeutic targets have resulted [6,7]. Regarding to the
validated metabolic steps used to generate new chemotherapeutic
alternatives, membrane sterol biosynthesis is one of the most
promising targets [8].
and cannot use the abundant supply of cholesterol present in its
mammalian hosts [9,10]. The T. cruzi ergosterol biosynthesis
pathway has been chemically validated as a chemotherapeutic
target at several steps [11,12], including squalene epoxidase (SE; EC
1.14.99.7)
a microsomal mono-oxygenase that catalyzes the
conversion of squalene to 2,3-oxidosqualene using molecular
oxygen [9,10]. SE is essential for the synthesis of cholesterol in
mammals and ergosterol in fungi and is potently inhibited by
allylamines, which have been successfully used as antifungal agents
[13,14]. Allylamine derivatives have also shown to be potent in vitro
and in vivo T. cruzi growth inhibitors, acting by a selective reduction
of the parasite’s endogenous membrane sterol levels [15,16].
Specifically, the antifungals terbinafine and naftifine (Tbf and Ntf,
respectively, Fig. 1) [17–20] proved to be promising anti-T. cruzi
agents. Recently our group have described a series of 5-nitrofuranes
and 5-nitrothiophenes able to accumulate squalene, and potentially
inhibit SE, in T. cruzi parasite [21,22]. These compounds (1 and 2,
Fig. 1) have an additional mechanism of action, they are able to
produce oxidative stress and consequently they are dual agents. In
these sense, we have been working in the research and developing
of different oxidative stress producer-heterocycles searching for
new anti-T. cruzi pharmacophores [23]. Interesting molecular hits
were found from its in vitro and in vivo behaviour (3–7, Fig. 1). To
explore new anti-T. cruzi chemical entities we designed hybrid
* Corresponding authors. Tel.: þ598 2 5258618; fax: þ598 2 5250749.
E-mail addresses: megonzal@fq.edu.uy (M. Gonza´lez), hcerecet@fq.edu.uy
(H. Cerecetto).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.052

A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
2155
N
N
O
S
N
N
O2N
N
H
O
O2N
N
H
N
H
O
O
Tbf
Ntf
1
2
O
N
O
O
OCH2Ph
N
CN
N
O2N
Ph
Ph
Cl
Cl
SPh
N
N
N
O
I
N
N
N
N
O
O
O
O
N
3
4
5
6
7
DESIGN
N
linker
heterocycle
S
N
N
N
H
H
8-35
N
n
R
Fig. 1. Anti-T. cruzi agents, squalene accumulator-nitrofuranes and design of the hybrid new generation of compounds.
compounds containing bioactive-heterocycles and alkenylamino
framework. The rationality was that the bioactive-heterocycles
could provide free-radical releaser moities and the alkenylamino
the SE-inhibitor pharmacophores. The synthetic efforts were focus
on five different heterocycles and three different alkenylamino
moieties. Four different N-oxides were used, benzofuroxan, benz-
imidazole 1,3-dioxide, quinoxaline 1,4-dioxide, and indazole
N-oxide and one nitro-containing system, 5-nitroindazole. The
used alkenylamino systems were 3-phenyl-2-propenylamino,
framework present in Ntf, N4-allylthiosemicarbazonyl, moiety
contained in parent compound 2, and homologous of allylamino
moiety. Additionally, the biological effects of all the new
compounds was analyzed using T. cruzi Tulahuen 2 strain as para-
sitic model, and for three compounds the CL Brener clone. The
selectivity towards the parasite was studied by determining the
toxicity of the most active anti-T. cruzi compounds against
mammal-J-774 cells. The sterols level-changes produced by the
hybrid compounds into the parasite were also studied using HPLC
methodology. Furthermore, being some antifungals active against T.
cruzi [15,16], we proposed to evaluate the designed anti-T. cruzi
agents for its in vitro antifungal properties.
or commercially available (E)-4-(3-phenyl-2-propenyl)piperazine
and N4-allylthiosemicarbazide as starting materials. These
compounds were transformed into benzimidazole 1,3-dioxide 11–13
(Scheme 1) by reaction with 2-nitropropane in basic milieu [26,27]
or into quinoxaline 1,4-dioxide 14–23 (Scheme 1) in Beirut condi-
tions [28]. Quinoxaline 24 was obtained from aldehyde IV [29].
Quinoxaline 1,4-dioxides 14–23 were isolated as a mixture of 6- and
7-isomer. For this family 2- and 3-positions were varied with
moieties (–R, –CN, –NH2, –CO2R, –CONHR) with different stereo-
electronic properties. The indazole N1-oxides 25–27 (Scheme 1)
were prepared by reaction of allyl- or homoallylamines V–VII [30]
and o-nitrobenzaldehyde in presence of KCN and basic milieu [31].
The 5-nitroindazoles 28–34 (Scheme 1) were prepared by nucleo-
philic substitution using halides VIII–X [32] and III, 4-(3-phenyl-2-
propenyl)piperazine or (E/Z)-4-pentenylamine (XI, [30]). Being XI
a primary amine, product 35 (Scheme 1) was also obtained in this
reaction as result of a di-substitution process.
All the proposed structures were established by 1H, 13C NMR
(HMQC, HMBC) spectroscopy and MS. The purity was analyzed and
established by TLC and microanalysis, respectively.
2.2. Biological characterization
2. Methods and results
2.2.1. In vitro anti-T. cruzi activity
2.1. Chemistry
The new derivatives, 8–35, were initially tested in vitro against
the epimastigote form of T. cruzi, Tulahuen 2 strain. The existence
of the epimastigote form of T. cruzi as an obligate mammalian
intracellular stage has been revisited and confirmed [33,34]. The
As a first synthetic approach benzofuroxan derivatives 8–10
(Scheme 1) were prepared by simple and efficient procedures, using
bromide I [24] or aldehyde II [25] and N-methylcinnamylamine (III)
compounds were incorporated into the media at 25 mM and their

2156
A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
HN
N
O
O
N
N
N
(III)
N
N
N
N
O
acetone / rt
N
O
O
O
8
11
12
13
NO2
O
N
N
HN
Br
N
N
N
N
O
N
N
O
acetonitrile / rt
S
piperidine
THF / rt
N
S
N
S
(I)
O
O
9
O
O
H
H
N
H
N
H
N
H
N
H2N
N
H
N
H
N
N
N
N
N
N
O
O
O
p-TsOH / toluene / rt
O
(II)
10
O
N
O
O
O
O
H
NC
CN
Ph
R3
N
H
R3
OEt
O
Et3N / DMF / rt
base / rt
base / rt
N
O
(IV)
O
N
O
N
O
O
N
O
S
CN
Ph
N
H
OEt
R3
H2N
R
N
O
R
R
N
N
-R
NH2
N
O
R3
N
O
H
H
p-TsOH / toluene / rt
N
14
15
16
-R3= -CH3, 17
O
N
H
N
H
N
N
N
N
-R3= -CH3, 18
-R3= -Ph, 21
N
S
S
N
O
H
H
N
N
-R3= -CH3, 19
-R3= -Ph, 20
-R3= -CH3, 22
-R3= -Ph, 23
24
H2N
CN
N
n
1)
/ AcOH / KCN
n= 1, E-isomer, -X= -H, 25
n= 2, Z-isomer, -X= -H, 26
n= 2, Z-isomer, -X= -Cl, 27
CHO
NO2
X
(V)-(VII)
N
n
2) Et3N / EtOH
O
X
PhH2CO
NO2
OR
N
OR
N
OCH2Ph
N
N
O2N
O2N
O2N
N
(III) or
HN
N
N
N
N
H2N
or
O
3
3
Y
Y
O
(XI)
-R
X
R
N
K2CO3 / acetonitrile / reflux
28, -R= -CH3,
29, -R= -CH2Ph, -Y-= -O-
30, -R= -CH2Ph, -Y-= -CH2-
-Y-= -O-
35
(VIII), -R= -CH3,
-Y-= -O-, -X= -I
N
(IX)
,
-R= -CH2Ph, -Y-= -O-, -X= -Br
-R= -CH2Ph, -Y-= -CH2-, -X= -Br
(X)
,
31, -R= -CH3,
32, -R= -CH2Ph, -Y-= -O-
33, -R= -CH2Ph, -Y-= -CH2-
-Y-= -O-
N
N
H
N
34, -R= -CH2Ph, -Y-= -O-
Scheme 1. Synthetic procedures used to prepare the Naftifine-analogues.
ability to inhibit the parasite growth was evaluated in comparison
to the control (no drug added to the media) at day 5. The ID50
doses (50% inhibitory dose) were determined for all of them
(Table 1). Nfx, Bnz, Tbf and ketoconazole (Ktz) were used as the
reference trypanosomicidal agents. Some derivatives were also
studied against the high virulent [35,36] CL Brener clone
(Table 1).
5-Nitroindazoles 31 and 34 were the most active anti-T. cruzi
M, near to
2 and 1.5 times more active than the reference drugs Nfx and Bnz.
While the benzimidazole 1,3-dioxide 13, the quinoxaline 1,4-
dioxides 21–23, and the 5-nitroindazoles 29, 30, 32, and 33 dis-
(Tulahuen 2 strain) derivatives, with ID50 ¼ 3.8 and 4.5
m
played ID50 values between 6.5 and 12.0
mM, which also resulted as
active as the references (Nfx, Bnz, Tbf, and Ktz) (Table 1). No

A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
2157
Table 1
and ester containing quinoxaline 1,4-dioxide is preferable to the
benzimidazole 1,3-dioxide system (compare toxicity of 13 and 22
and 23).
Biological Characterization of Naftifine-analogues Against T. cruzi.
Compd.
ID50a(
m
M)
Compd.
ID50a(
m
M)
Compd.
ID50a(
mM)
Activity against Tulahuen 2 strain
8
22.6
(14.0)
(33.0)
22.7
25.0
7.2
14
15
16
17
18
19
20
21
22
23
24
(0.0)b
(0.0)
(6.0)
(34.0)
25.4
(0.0)
(18.0)
9.5
25
26
27
28
29
30
31
32
33
34
35
(28.0)
(30.0)
(20.0)
23.1
10.0
7.8
3.8
6.5
10.6
4.5
(0.0)
2.2.3. In vitro antifungal activity [38,39]
9
Due to some antifungals are actives against T. cruzi [15,16] we
decided to evaluate the designed anti-T. cruzi agents for its in vitro
antifungal properties. To determine the antifungal activity, microor-
ganisms from the American Type Culture Collection (ATCC) or clinical
10
11
12
13
´
isolates provided by the Centro de Referencia en Micologıa (CERE-
Nfx
Bnz
Tbf
Ktz
7.7c
´
´
MIC) from Facultad de Ciencias Bioquımicas y Farmaceuticas (Rosario,
7.4c
12.0
7.5
(21.0)
´
Argentina) or Control Lab from Rıo de Janeiro (Brazil) were used
17.0c
10.0c
(Table 3). Minimal inhibitory concentration (MIC) of each compound
was determined by using broth microdilution techniques for yeasts
[40] and filamentous fungi [41]. For this study we selected some
relevant derivatives with structural modifications at the heterocycle
level. As antifungal reference compounds Tbf, Ktz, and amphotericin
B (AnfB) were used. Although the studied compounds were less
active than the reference compounds the indazole derivatives, i.e. 25,
31, and 33, displayed moderate activities mainly against the derma-
tophytes Microsporum gypseum, Trichophyton rubrum and Tricho-
phyton mentagrophytes that transform them in molecular hits.
Derivative 33 showed the best activity against Cryptococcus neofor-
Activity against CL Brener clone
8
31
33
16.9
7.9
4.5
Nfx
Bnz
8.5c
4.5c
Tbf
Ktz
42.0c
5.0c
a
The results are the means of three independent experiments with an SD less
than 10% in all cases.
b
Values in parenthesis correspond to percentage of T. cruzi growth inhibition at
25.0 mM.
c
From [37].
mans with MIC ¼ 31.2
mg/mL, an interesting result considering that
C. neoformans continues to be an important infection in immuno-
compromised patients, for which nearly 50% is lethal [42].
susceptibility differences between both strains, with the studied
compounds, were observed.
2.2.2. In vitro unspecific cytotoxicity
Mammal cytotoxicity of selected new compounds was studied
in vitro using J-774 mouse macrophages as the cellular model with
2.3. Squalene accumulation analysis
doses (100.0–400.0
used for T. cruzi (25.0
m
M) at least four times higher than the doses
M) (Table 2) [37]. These derivatives were
Since the steroids from the lipid fractions of T. cruzi epi-
mastigotes have been previously described [43], this form was
selected (Tulahuen 2 strain) to study the effects on new derivatives
acting on ergosterol biosynthesis. After a pre-established protocol
(1 ꢀ ID50 per 8 ꢀ 106 cells/mL, 120 h of incubation) [22,44], the
controls (untreated, Tbf- and Nfx-treated) and derivative-treated
parasites were collected and the total lipids were extracted and
analyzed as described previously [12,22,43,45,46]. Quantitative
analyses of squalene (for untreated parasite and Tbf) from sterol
fractions were done by HPLC. None of the studied compounds were
able to accumulate squalene (see Table S1 in Supplementary
Content section). These results demonstrate that the presence, in
the developed derivatives, of the selected alkenylamino moieties is
not enough to guarantee T. cruzi sterol biosynthesis inhibition.
m
selected regarding its in vitro anti-T. cruzi activities and its wide-
spread structural motives at the heterocycle level (Scheme 1). The
selectivity indexes, SI, were expressed as the ratio between ID50 in
macrophages and ID50 in T. cruzi (Tulahuen 2 strain). The benz-
imidazole 1,3-dioxide 11, and the quinoxalines 1,4-dioxides 22 and
23 have similar or better SI values than the biosynthesis-
membrane-sterol inhibitors (Tbf and Ktz), displaying 22 at least
1.3-fold the selectivity of Nfx (Table 2). Clear structural exigencies
could be observed regarding the toxic effects being all the studied
5-nitroindazoles toxics against this mammal system, indepen-
dently of the kind of alkenylamino appendage (see toxicity of 29,
32 and 34). For the same alkenylamino moiety some consider-
ations could be extracted, the benzimidazole 1,3-dioxide is pref-
erable to the benzofuroxan system (compare toxicity of 8 and 11),
3. Discussion
We report the synthesis of twenty-eight new hybrid compounds
and their activity against T. cruzi, macrophages and a wide panel of
fungi. These compounds were designed combining the alkenyla-
mino moieties present in Ntf and compound 2 and a bioactive
heterocycle (benzofuroxan, benzimidazole 1,3-dioxide, quinoxaline
1,4-dioxide, indazole N-oxide, and 5-nitroindazole).
Table 2
Biological Characterization of New Derivatives Against Mammal Macrophages.
a
a
Compd.
ID50
(m
M)
SIb
ID50
(
mM)
SIb
J-774 macrophages
J-774
macrophages
The most interesting derivatives, against T. cruzi (Tulahuen 2
strain and CL Brener clone) and fungi (C. neoformans, M. gypseum,
T. rubrum, and T. mentagrophytes), were the 5-nitroindazole deriv-
atives 31 and 33 (Table 1 and Table 3). However, they were, like Ktz,
toxic against macrophages at the assayed doses (Table 2). According
to the selectivity indexes benzimidazole 1,3-dioxides 11 and 13 and
quinoxaline 1,4-dioxides 22 and 23 could be considered for further
biological studies. Specially, derivative 22 showed better SI than
Nfx that convert it in a lead compound.
8
<100.0
>400.0
109.0
<4.4
>17.6
4.4
28
29
30
31
32
33
34
<100.0
<4.3
11
12
13
21
22
23
<100.0
<100.0
<100.0
<100.0
<100.0
<100.0
<10.0
<12.8
<26.3
<15.4
<9.4
104.0
14.4
<100.0
>400.0
>400.0
<10.5
>53.3
>33.3
<22.2
Nfx
316.0
41.0
Ktz
Tbf
<100.0
339.0
<10.0
19.9
It is clear that the mechanisms of anti-T. cruzi or the modest
antifungal activity are not related to the squalene accumulation. The
structural modifications from the parent compounds, Ntf and 2,
a
The results are the means of three independent experiments with an SD less
than 10% in all cases.
b
SI: selectivity index, ID50,macrophage/ID50,T. cruzi
.

2158
A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
Table 3
Antifungal Activity of Naftifine-analogues Against Filamentous Fungi and Yeasts, Expressed as Minimum Inhibitory Concentration (MIC)/Minimum Fungicidal Concentration
(MFC).
Compd.
MIC/MFC (
m
g/mL)a,b
Ct
Ca
Sc
Cn
Afu
Afl
Ani
Mg
Tr
Tm
10
13
16
18
19
21
24
25
>250
>250
>250
>250
>250
250/>250
>250
>250
125/125
>250
>250
0.78
>250
>250
>250
>250
>250
>250
125/250
>250
>250
62.5/62.5
>250
>250
0.50
>250
>250
>250
>250
250/>250
125/250
>250
>250
62.5/62.5
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
0.50
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
0.50
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
0.50
>250
250/250
>250
>250
250/250
>250
>250
250/250
>250
>250
>250
>250
>250
250/>250
>250
>250
125/125
>250
>250
1.56
125/>250
250/250
250/>250
>250
62.5/125
62.5/125
125/>250
62.5/125
0.125
125/>250
250/250
125/250
>250
62.5/125
62.5/125
125/>250
62.5/125
0.075
125/>250
250/250
125/250
>250
62.5/125
62.5/125
125/>250
62.5/125
0.075
31
32
33
31.2/62.5
0.25
0.39
AnfBc
Tbf
Ktz
1.56
0.5
1.56
0.50
3.12
0.5
0.78
0.125
0.78
0.50
1.56
0.25
0.04
0.05
0.01
0.025
0.025
0.025
0.25
a
MIC ¼ minimum inhibitory concentration, MFC ¼ minimum fungicidal concentration.
b
Ca: Candida albicans ATCC 10231, Ct: Candida tropicalis C 131 2000, Sc: Saccharomyces cerevisiae ATCC 9763, Cn: Cryptococcus neoformans ATCC 32264; An: Aspergillus niger
ATCC 9029, Afu: Aspergillus fumigatus ATCC 26934; Afl: Aspergillus flavus ATCC 9170, Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C113, Tm: Trichophyton
mentagrophytes ATCC 9972.
c
AnfB: amphotericin B.
allowed us to generate compounds with better anti-trypanosomal
activities, but not sharing the mechanism of squalene accumulation.
Structurally talking, the substitution of naphtyl, in Nft, or
5-nitrofuryl moieties, in compound 2, by the different assayed
heterocycles carried out to different anti-T. cruzi profiles (Fig. 2). In
example the benzofuroxan system leads to poorly actives deriva-
tives (see 8 and 10, Fig. 2) while benzimidazole 1,3-dioxide
heterocycle displays compounds with good trypanosomicidal
behaviours (compare compound 2 and 13 activities, Fig. 2). Among
the quinoxaline 1,4-dioxides, the biological behaviour is not
uniform. Quinoxalines 14, 17, 16, 19, 20, and 24 (Fig. 2) were
completely inactive in the assayed conditions. This lack of activity
of 2-ciano-3-amino-derivatives 14 and 16 could be explained for
compounds’ poor solubilities in the biological milieu [47]. While, in
compounds 17,19, 20 and 24, without apparent solubility problems,
the lack of activity could be related to the electronic characteristics
on the 2- and 3-substituents (amide or alkyl moieties). Otherwise,
the 2-ester-substituted derivatives, 22 and 23, display the best
H
N
H
N
O
N
NO2
N
ID50= 8.2
M
Ntf
2
S
O
O
O
O
H
N
H
N
H
H
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
S
S
N
O
O
8
11
10
13
ID50= 22.6
M
ID50= 22.7
M
ID50 25.0
M
ID50= 7.2
M
O
N
O
N
O
O
N
O
N
O
O
N
O
H
N
H
N
H
N
H
N
CN
Ph
CN
Ph
Ph
H
N
H
N
N
N
H
N
N
H
N
N
H
N
N
S
S
N
O
NH2
N
O
CH3
N
O
NH2
N
O
CH3
N
O
S
14
17
16
19
20
ID50 25.0
M
ID50 25.0
M
ID50 25.0
M
ID50 25.0
M
N
ID50 25.0
M
N
O
O
N
O
N
NO2
H
N
H
H
N
H
N
H
N
H
N
N
N
CO2Et
CH3
CO2Et
NC
N
N
O
S
S
S
N
N
N
O
N
O
N
O
N
N
OCH3
O
25
28
22
23
24
ID50 25.0
M
ID50= 23.1
M
O
ID50= 12.0
M
ID50 = 7.5
M
ID50 25.0 M
NO2
NO2
N
N
5
N
N
N
N
OCH2Ph
OCH2Ph
30
29
ID50= 7.8
M
ID50= 10.0
M
Fig. 2. Structural-anti-T. cruzi activity for the Naftifine-analogues. The ID50 refer to Tulahuen 2 strain.

A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
2159
biological profile, being this feature in accordance to our previous
214 (31), 201 (18),177 (24), 158 (24),149 (100),117 (35), 55 (47). IR,
749, 822, 1184, 1246, 1293, 1400, 1563, 1705.
n:
QSAR [47]. The change by an indazole N-oxide heterocycle
promotes less active compounds in contrast to 5-nitroindazolyl
moiety which leads to very active Nft analogues.
5.3. 4-Allyl-1-(N1-oxide-benzo[1,2-c][1,2,5] oxadiazole-5-yl)-
methylidenethiosemicarbazide (10)
4. Conclusions
A mixture of (II) (263 mg, 1.6 mmol), 4-allylthiosemicarbazide
(210 mg, 1.6 mmol) and p-TsOH (catalytic amount) in toluene
(50.0 mL) was stirred at room temperature for 24 h. The precipitate
was filtered and washed with petroleum ether. Yellow solid,
433 mg (98%); mp 172.0–173.0 ꢃC. 1H NMR (acetone-d6) dH: 4.37
(2H, m), 5.24 (1H, d, J ¼ 10.3 Hz), 5.30 (1H, d, J ¼ 16.0 Hz), 5.98 (1H,
s), 7.62 (1H, bs), 7.74 (1H, bs), 8.14 (1H, bs), 8.26 (1H, s), 8.64 (1H,
bs), 10.76 (1H, bs). 13C NMR (acetone-d6) dC: 46.7, 115.8, 134.9, 139.6,
179.1, the benzofuroxan carbons were not detected at room
temperature. EI-MS, m/z (abundance, %): 277 (Mþ, 4), 259 (Mþ ꢁ 18,
4), 231 (62), 185 (30), 163 (28), 148 (41), 115 (100), 105 (78), 81 (28),
We have developed and identified new trypanosomicidal
agents, of which benzimidazole and quinoxaline containing
N-oxide high selectivity indexes. This transforms them in molecular
lead for further structural modifications and further biological
studies, especially in vivo evaluations.
5. Experimental
Some starting materials were commercially available research-
grade chemicals and used without further purification. All solvents
were dried and distilled prior to use. All the reactions were carried
out in a nitrogen atmosphere. Intermediates I, II, and IV–XI and ethyl
benzoylacetate were prepared following synthetic procedures
previously reported [24,25,29,30,32,48]. For the synthetic proce-
dures used to prepare intermediate III, N-phenylbenzoylacetamide
and N-phenylacetoacetamide see Supplementary Content section.
Melting points were determined with an electrothermal melting
point apparatus (Electrothermal 9100) and were 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 on an MSD 5973 Hewlett–Packard spectrometer using
electronic impact at 70 eV (EI). Infrared spectra were recorded on
a Perkin–Elmer 1310 apparatus, using potassium bromide tablets the
frequencies were expressed in cmꢁ1. Microanalyses were performed
on a Fisons EA 1108 CHNS-O instrument and were within ꢂ0.4% of
the calculated compositions. Column chromatography was carried
out using Merck silica gel (60–230 mesh).
56 (85). IR, n: 615, 812, 936, 1107, 1221, 1287, 1358, 1518, 1613, 1701.
5.4. (E)-2,2-Dimethyl-5-[(N-methyl-3-phenyl-2-
propenylamino)methyl]-2H-benzimidazole 1,3-dioxide (11)
A mixture of 8 (148 mg, 0.5 mmol), 2-nitropropane (0.05 mL,
0.5 mmol) and piperidine (0.05 mL, 0.5 mmol) in THF (20.0 mL) was
stirred at room temperature for 4 days. The solvent was evaporated
in vacuo and the residue was purified by column chromatography
(SiO2, ethyl acetate). Red oil, 126 mg (75%). 1H NMR (CDCl3) dH: 1.72
(6H, s), 2.28 (3H, s), 3.23 (2H, d, J ¼ 6.8 Hz), 3.37 (2H, s), 6.27 (1H, dt,
J ¼ 6.8,16.0 Hz), 6.56 (1H, d, J ¼ 16.0 Hz), 7.01 (1H, d, J ¼ 9.6 Hz), 7.16
(1H, s), 7.20 (1H, d, J ¼ 9.6 Hz), 7.25 (1H, d, J ¼ 7.6 Hz), 7.34 (2H, t,
J ¼ 7.6 Hz), 7.40 (2H, d, J ¼ 7.6 Hz). 13C NMR (CDCl3) dC: 24.6, 42.8,
60.5, 61.4, 97.7, 114.1, 115.8, 126.7, 127.3, 128.0, 129.0, 133.4, 133.5,
136.0 (two carbons), 137.2, 144.3. EI-MS, m/z (abundance, %): 337
(Mþ, 7), 321 (Mþ ꢁ 16, 17), 304 (Mþ ꢁ 16-17, 8), 279 (29), 215 (26),
192 (18), 175 (42), 159 (32), 146 (43), 144 (71), 117 (100), 91 (39), 77
(19). IR, n: 695, 747, 970, 1026, 1096, 1181, 1235, 1368, 1404, 1453,
5.1. (E)-5-[(N-Methyl-3-phenyl-2-propenylamino)
1516, 2934.
methyl]benzofuroxan (8)
5.5. (E)-2,2-Dimethyl-5-[4-(3-phenyl-2-propenyl) piperazine-1-yl-
A mixture of (I) (108 mg, 0.5 mmol) and (III) (70 mg, 0.5 mmol)
in acetone (50.0 mL) was stirred at room temperature for 24 h. The
solvent was evaporated in vacuo and the residue was purified by
column chromatography (SiO2, petroleum ether:ethyl acetate, 8:2).
Red oil, 31 mg (23%). 1H NMR (CDCl3) dH: 2.30 (3H, s), 3.26 (2H, d,
J ¼ 6.4 Hz), 3.56 (2H, s), 6.30 (1H, dt, J ¼ 6.4, 15.6 Hz), 6.58 (1H, d,
J ¼ 15.6 Hz), 7.39 (8H, m). 13C NMR (CDCl3) dC: 42.7, 60.5, 61.4, 126.7,
127.0, 128.1, 129.0, 133.6, 137.2, the benzofuroxan carbons were not
detected at room temperature. EI-MS, m/z (abundance, %): 295 (Mþ,
8), 278 (Mþ ꢁ 17, 15), 174 (33), 144 (33), 177 (75), 105 (100), 91 (44),
methyl]-2H-benzimidazole 1,3-dioxide (12)
A mixture of 9 (120 mg, 0.3 mmol), 2-nitropropane (0.04 mL,
0.4 mmol) and piperidine (0.04 mL, 0.4 mmol) in THF (20.0 mL) was
stirred at room temperature for 4 days. The solvent was evaporated
in vacuo and the residue was purified by column chromatography
(SiO2, ethyl acetate). Red solid, 47 mg (35%); mp 154.0–156.5 ꢃC. 1H
NMR (CDCl3) dH: 1.72 (6H, s), 2.56 (8H, bs), 3.23 (2H, d, J ¼ 6.8 Hz),
3.35 (2H, s), 6.29 (1H, dt, J ¼ 6.5, 15.6 Hz), 6.55 (1H, d, J ¼ 15.6 Hz),
6.98 (1H, d, J ¼ 9.8 Hz), 7.16 (1H, bs), 7.19 (1H, d, J ¼ 9.8 Hz), 7.26 (1H,
d, J ¼ 7.2 Hz), 7.32 (2H, t, J ¼ 7.2 Hz), 7.39 (2H, d, J ¼ 7.2 Hz). 13C NMR
(CDCl3) dC: 24.6, 53.3, 53.3, 61.2, 62.5, 97.7, 114.3, 115.8, 126.2, 126.8,
128.0, 129.0, 133.3, 134.0, 136.6, 136.8, 137.2, 143.3. EI-MS, m/z
(abundance, %): 376 (Mþ ꢁ 16, 6), 259 (16), 243 (11), 201 (100), 159
77 (54). IR,
1624, 1725.
n: 714, 1019, 1115, 1267, 1364, 1451, 1489, 1536, 1598,
5.2. (E)-5-[4-(3-Phenyl-2-propenyl)piperazine-1-yl
methyl]benzofuroxan (9)
(20), 144 (18), 117 (61), 91 (18). IR,
n: 695, 745, 970, 1007, 1140, 1260,
1385, 1400, 1650, 2811, 2934, 3450.
A mixture of (I) (103 mg, 0.5 mmol) and (E)-4-(3-phenyl-2-pro-
penyl)piperazine (136 mg, 0.7 mmol) in CH3CN (30.0 mL) was stirred
at room temperature for 12 h. The solvent was evaporated in vacuo
and the residue was purified by column chromatography (SiO2,
petroleum ether:ethyl acetate,1:1). Yellow oil,140 mg (89%).1H NMR
(CDCl3) dH: 2.66 (4H, bs), 2.75 (4H, bs), 3.27 (2H, d, J ¼ 6.8 Hz), 4.49
(2H, s), 6.29 (1H, dt, J ¼ 6.8, 15.6 Hz), 6.57 (1H, d, J ¼ 15.6 Hz), 7.30
(8H, m). 13C NMR (CDCl3) dC: 50.1, 52.9, 59.8, 60.9, 124.0, 126.9, 128.3,
129.0, 135.0, 142.0, the benzofuroxan carbons were not detected at
room temperature. EI-MS, m/z (abundance, %): 350 (Mþ, 1), 272 (17),
5.6. 4-Allyl-1-(2,2-dimethyl-1,3-dioxide-2H-benzimidazole-5-yl)-
methylidenethiosemicarbazide (13)
A mixture of 10 (200 mg, 0.7 mmol), 2-nitropropane (0.08 mL,
0.9 mmol) and piperidine (0.09 mL, 0.9 mmol) in THF (50.0 mL) was
stirred at room temperature for 24 h. The solvent was evaporated in
vacuo and the residue was purified by column chromatography
(SiO2, petroleum ether:ethyl acetate, 1:1). Red solid, 77 mg (34%);
mp >300.0 ꢃC. 1H NMR (CDCl3) dH: 1.75 (6H, s), 4.41 (2H, d,

2160
A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
J ¼ 4.0 Hz), 5.29 (2H, m), 5.98 (1H, m), 7.30 (2H, s), 7.32 (1H, d,
J ¼ 9.0 Hz), 7.38 (1H, d, J ¼ 9.0 Hz), 7.45 (1H, bs), 7.73 (1H, s), 9.87
(1H, bs). 13C NMR (CDCl3) dC: 24.7, 47.4, 98.6, 116.2, 116.8, 118.0,
128.2, 133.4,136.2, 136.6, 136.8, 139.7, 178.0. EI-MS, m/z (abundance,
%): 319 (Mþ, 4), 303 (Mþ ꢁ 16, 3), 286 (Mþ ꢁ 16-17, 6),189 (100), 174
(44), 132 (27), 115 (36), 56 (43).
5.12. (E)-3-Methyl-7(6)-[(N-methyl-3-phenyl-2-
propenylamino)methyl]-N-phenylquinoxaline-2-carboxamide
1,4-dioxide (17)
As equimolecular mixture of 6- and 7-positional isomers. Yellow
solid, 350 mg (77%). 1H NMR (DMSO-d6) dH: 2.22 (3H, s), 2.51 (3H, s),
3.24 (2H, d, J ¼ 6.0 Hz), 3.70/3.82 (2H, s), 6.41 (1H, dt, J ¼ 6.0,
16.0 Hz), 6.60 (1H, d, J ¼ 16.0 Hz), 7.24–8.02 (11H, m), 8.46 (1H, s),
8.50 (1H, d, J ¼ 8.8 Hz), 11.06 (1H, bs). 13C NMR (DMSO-d6) dC: 15.1,
48.9, 60.0, 60.6, 120.4 (two carbons),120.8, 125.6, 127.1, 128.0, 128.3,
129.5 (two carbons), 130.0, 134.4, 138.9 (two carbons), 139.2, 140.0
(two carbons), 142.2, 145.6, 158.8. EI-MS, m/z (abundance, %): 438
(Mþ ꢁ 16, 1), 421 (Mþ ꢁ 16-17, 2), 254 (11), 146 (34), 117 (80), 115
5.7. General procedure for the preparation of derivatives 14–16
A mixture of benzofuroxan (8–10) (0.4 mmol) and malononitrile
(26 mg, 0.4 mmol) was stirred at 0 ꢃC for 10 min. Then, two drops of
Et3N in DMF (0.5 mL) were added and the reaction was stirred at
room temperature for 24–48 h. The desired product was filtered
and washed with petroleum ether.
(100), 91 (78), 77 (65), 65 (48), 51 (30). IR, n: 693, 752, 970, 1223,
1269, 1331, 1447, 1497, 1563, 1599, 1622, 1680, 2789.
5.8. (E)-3-Amino-7-[(N-methyl-3-phenyl-2-
propenylamino)methyl]quinoxaline-2-carbonitrile 1,4-dioxide (14)
5.13. (E)-3-Methyl-N-phenyl-7-[4-(3-phenyl-2-
propenyl)piperazine-1-ylmethyl]quinoxaline-2-carboxamide
1,4-dioxide (18)
Red solid, 114 mg (79%); mp 149.0–151.1 ꢃC. 1H NMR (DMSO-d6)
dH: 2.25 (3H, s), 3.29 (2H, d, J ¼ 6.8 Hz), 3.41 (2H, bs), 3.81 (2H, s),
6.39 (1H, dt, J ¼ 6.8, 16.0 Hz), 6.59 (1H, d, J ¼ 16.0 Hz), 7.25 (1H, d,
J ¼ 7.4 Hz), 7.33 (1H, t, J ¼ 7.4 Hz), 7.45 (1H, d, J ¼ 7.4 Hz), 8.02 (1H,
s), 8.26 (2H, m). 13C NMR (DMSO-d6) dC: 60.0, 62.0, 111.4, 118.9 (two
carbons), 121.2, 127.1, 127.3, 128.4, 129.4, 133.0 (two carbons), 134.0,
137.0, 134.4 (two carbons), 146.6. EI-MS, m/z (abundance, %): 345
(Mþ ꢁ 16,13), 328 (Mþ ꢁ 16-17, 42), 254 (60), 238 (46),199 (70),183
Yellow solid, 199 mg (39%); mp 164.2–165.7 ꢃC. 1H NMR (DMSO-
d6) dH: 2.50 (11H, m), 3.12 (2H, d, J ¼ 5.6 Hz), 3.77 (2H, s), 6.32 (1H,
dt, J ¼ 5.6, 16.4 Hz), 6.54 (1H, d, J ¼ 16.4 Hz), 7.30–7.45 (8H, m), 7.68
(2H, d, J ¼ 8.0 Hz), 7.96 (1H, d, J ¼ 9.2 Hz), 8.43 (1H, s), 8.50 (1H, d,
J ¼ 9.2 Hz), 10.94 (1H, bs). 13C NMR (DMSO-d6) dC: 15.0, 53.5 (two
carbons), 61.0, 61.8, 119.6, 120.0, 120.5, 125.6, 127.1, 127.9, 128.3,
129.4, 130.0, 132.9, 134.2, 137.2, 137.3, 138.6, 138.8 (two carbons),
139.4, 144.2, 158.1. EI-MS, m/z (abundance, %): 493 (Mþ ꢁ 16, 1), 477
(Mþ ꢁ 16-16, 1), 360 (6), 276 (12), 201 (73), 117 (100), 91 (28), 77
(87), 146 (67), 117 (100), 115 (75), 91 (46), 77 (23). IR,
963, 1036, 1117, 1206, 1333, 1617.
n: 691, 745,
(17), 56 (16). IR, n: 691, 749, 967, 1007, 1055, 1154, 1264, 1320, 1445,
1495, 1555, 1599, 1682, 2814.
5.9. (E)-3-Amino-7(6)-[4-(3-phenyl-2-propenyl) piperazine-1-yl-
methyl]quinoxaline-2-carbonitrile 1,4-dioxide (15)
As equimolecular mixture of 6- and 7-positional isomers. Red
solid, 83 mg (50%). 1H NMR (DMSO-d6) dH: 2.51 (8H, bs), 3.10 (2H, d,
J ¼ 6.0 Hz), 3.67/3.71 (2H, s), 6.30 (1H, m), 6.51 (1H, d, J ¼ 15.6 Hz),
7.23 (1H, t, J ¼ 7.6 Hz), 7.32 (2H, t, J ¼ 7.6 Hz), 7.42 (2H, t, J ¼ 7.6 Hz),
8.00 (2H, bs), 8.18 (1H, s), 8.25 (2H, m). 13C NMR (DMSO-d6) dC: 53.5,
53.6, 61.0, 61.1/61.3, 109.0/109.4, 118.9, 119.4, 120.6, 127.1, 128.0,
128.2, 129.4, 132.4, 132.8, 135.8, 136.9, 137.5, 137.6, 146.6/147.0.
EI-MS, m/z (abundance, %): 400 (Mþ ꢁ 16, 2), 384 (Mþ ꢁ 16-16, 10),
267 (19), 201 (100), 183 (51), 117 (63), 91 (17), 77 (9).
5.14. 4-Allyl-1-[1,4-dioxide-3-phenyl-2-(N-phenyl
carbamoyl)quinoxaline-7-yl]methylidene thiosemicarbazide (20)
Yellow solid, 268 mg (54%); mp 241.5–243.1 ꢃC. 1H NMR
(DMSO-d6) dH: 4.27 (2H, bs), 5.15 (2H, dd, J ¼ 10.0, 18.0 Hz), 5.92
(1H, m), 7.10 (1H, dd, J ¼ 7.2, 7.6 Hz), 7.30 (2H, dd, J ¼ 7.6, 8.0 Hz),
7.38 (2H, d, J ¼ 8.0 Hz), 7.49 (3H, m), 7.60 (2H, m), 8.34 (1H, s), 8.52
(1H, d, J ¼ 8.8 Hz), 8.67 (1H, s), 8.74 (1H, d, J ¼ 8.8 Hz), 9.07 (1H, bs),
10.81 (1H, bs), 11.91 (1H, bs). 13C NMR (DMSO-d6) dC: 46.4, 116.1,
119.5, 119.8, 121.0, 125.0, 128.7, 129.0 (three carbons), 129.5, 135.3,
137.9, 138.8 (three carbons), 139.9 (three carbons), 156.9, 178.0. EI-
MS, m/z (abundance, %): 480 (Mþ ꢁ 17, 1), 367 (19), 350 (100), 230
5.10. 4-Allyl-1-(3-amino-2-cyano-1,4-dioxide quinoxaline-7-yl)-
methylidenethiosemicarbazide (16)
(86), 77 (78), 56 (33). IR,
1700.
n: 695, 943, 1094, 1196, 1287, 1329, 1512,
Red solid, 133 mg (97%); mp >400.0 ꢃC. 1H NMR (DMSO-d6) dH
:
4.03 (2H, bs), 4.26 (2H, m), 5.18 (2H, m), 5.93 (1H, m), 8.27 (3H, m),
8.57 (1H, d, J ¼ 5.2 Hz), 8.93/8.99 (1H, bs), 11.73/11.84 (1H, bs). 13C
NMR (DMSO-d6) dC: 46.7, 109.6, 116.5, 118.9, 119.6, 120.7, 132.0,
134.5, 135.8, 137.9 (two carbons), 140.6, 147.1, 178.3. EI-MS, m/z
5.15. Ethyl (E)-3-phenyl-7(6)-[4-(3-phenyl-2-propenyl)piperazine-
1-ylmethyl]quinoxaline-2-carboxylate 1,4-dioxide (21)
(abundance, %): 342 (Mþ þ H, 5), 102 (100). IR,
n: 835, 922, 1105,
As equimolecular mixture of 6- and 7-positional isomers. Yellow
solid, 293 mg (56%). 1H NMR (DMSO-d6) dH: 0.96/0.97 (3H, t,
J ¼ 7.2 Hz), 2.51 (8H, m), 3.12 (2H, d, J ¼ 5.2 Hz), 3.79 (2H, s), 4.14/
4.16 (2H, q, J ¼ 7.2 Hz), 6.31 (1H, dt, J ¼ 5.2, 15.6 Hz), 6.54 (1H, d,
J ¼ 15.6 Hz), 7.24 (1H, d, J ¼ 7.2 Hz), 7.32 (2H, dd, J ¼ 7.2, 7.6 Hz), 7.44
(2H, d, J ¼ 7.6 Hz), 7.56 (5H, m), 7.98 (1H, d, J ¼ 8.8 Hz), 8.41 (1H, s),
8.46 (1H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6) dC: 14.2, 53.5 (two
carbons), 60.9, 61.8, 63.5, 119.5, 121.2, 127.1, 127.8, 128.3, 129.3, 129.5
(two carbons), 130.7, 131.3, 133.0, 134.5, 137.5, 137.7, 138.3, 139.5,
145.0,145.5,160.0. EI-MS, m/z (abundance, %): 508 (Mþ ꢁ 16,1), 491
(Mþ ꢁ 16-16, 1), 291 (14), 233 (11), 219 (21), 201 (100), 117 (82), 91
1188, 1335, 1509, 1624.
5.11. General procedure for the preparation of derivatives 17, 18, 20,
21, and 22
A mixture of the corresponding benzofuroxan (8–10) (1.0 mmol),
the corresponding amide or ester (N-phenylacetoacetamide,
N-phenylbenzoylacetamide, ethyl benzoylacetate, or ethyl acetoa-
cetate) (1.0 mmol), anhydrous K2CO3 (138 mg, 1.0 mmol) in acetone
(50.0 mL) was stirred at room temperature for 48 h. Then, water
(40.0 mL) was added and the precipitate was filtered and washed
with water.
(17), 77 (17), 56 (10). IR,
1331, 1443, 1740, 2803.
n: 695, 743, 849, 1007, 1094, 1152, 1235,

A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
2161
5.16. 4-Allyl-1-(2-ethoxycarbonyl-3-methyl-1,4-
dioxidequinoxaline-7-yl)methylidene thiosemicarbazide (22)
14), 298 (Mþ ꢁ 17-16, 14), 217 (34), 201 (79), 184 (74), 169 (38), 115
(100), 56 (63). IR, n: 677, 938, 1098, 1200, 1318, 1503, 1659, 3195.
Yellow solid, 167 mg (43%); mp 195.1–197.3 ꢃC. 1H NMR (DMSO-
d6) dH: 1.36 (3H, t, J ¼ 7.2 Hz), 2.43 (3H, s), 4.25 (2H, bs), 4.50 (2H, q,
J ¼ 7.2 Hz), 5.14 (2H, dd, J ¼ 10.4, 17.2 Hz), 5.92 (1H, m), 8.06 (1H, d,
J ¼ 9.2 Hz), 8.18 (1H, s), 8.26 (1H, s), 8.45 (1H, d, J ¼ 9.2 Hz), 9.02 (1H,
bs), 11.88 (1H, bs). 13C NMR (DMSO-d6) dC: 14.3, 14.6, 46.4, 63.8,
116.1, 116.6, 131.2, 135.3 (two carbons), 139.9, 140.0, 140.1, 149.6,
149.8, 149.8, 160.1, 178.0. EI-MS, m/z (abundance, %): 389 (Mþ, 1),
372 (Mþ ꢁ 17, 18), 356 (Mþ ꢁ 17-16, 13), 260 (29), 170 (39), 115
5.21. (E)-2-(3-Phenyl-2-propenyl)-2H-indazole-3-carbonitrile
N-oxide (25)
A mixture of 2-nitrobenzaldehyde (166 mg, 1.1 mmol) and (E)-3-
phenyl-2-propenylamine (V) (146 mg, 1.1 mmol) in acetic acid
(20.0 mL) was stirred at room temperature for 24 h. Then, KCN
(143 mg, 2.2 mmol) was added and the reaction was stirred for 48 h.
After that, water (20.0 mL) was added and the intermediate was
extracted with CH2Cl2 (3 ꢀ 20.0 mL), the organic layer was dried
with anhydrous Na2SO4 and the solvent was evaporated in vacuo.
Five drops of Et3N in EtOH (10.0 mL) was added and the reaction
was stirred at room temperature for 48 h. The solvent was evapo-
rated in vacuo, and the residue was fractioned between water
(50.0 mL) and EtOAc (3 ꢀ 20.0 mL). The organic layer was dried
with anhydrous Na2SO4 and the solvent was evaporated in vacuo.
The residue was purified by column chromatography (SiO2, petro-
leum ether:ethyl acetate, 1:1). Yellow solid, 61 mg (20%); mp 120.1–
122.3 ꢃC. 1H NMR (CDCl3) dH: 5.36 (2H, t, J ¼ 6.8 Hz), 6.89 (1H, d,
J ¼ 15.6 Hz), 7.32 (7H, m), 7.70 (1H, d, J ¼ 7.2 Hz), 7.86 (1H, d,
J ¼ 7.2 Hz). 13C NMR (CDCl3) dC: 47.8, 91.1, 110.5, 114.3, 118.8, 118.9,
122.3, 128.8–129.9 (phenyl carbons), 135.2, 137.6, 139.5. EI-MS, m/z
(abundance, %): 275 (Mþ, 2), 259 (Mþ ꢁ 16,1), 117 (100), 115 (37), 91
(100), 56 (66). IR, n: 621, 760, 928, 1007, 1061, 1109,1202,1296, 1329,
1422, 1477, 1534, 1738, 2359.
5.17. General procedure for the preparation of derivatives 19,
and 23
A mixture of 8 (1.0 mmol) and the corresponding amide or ester
(N-phenylacetoacetamide, or ethyl benzoylacetate) (1.0 mmol) was
stirred at 0 ꢃC for 10 min. Then, four drops of Et3N in DMF (1.0 mL)
were added and the reaction was stirred at room temperature for
5–10 days. The desired product was filtered and washed with
petroleum ether.
5.18. 4-Allyl-1-[3-methyl-2-(N-phenylcarbamoyl)-1,4-
dioxidequinoxaline-7(6)-yl]methylidene thiosemicarbazide (19)
(15). IR, n: 586, 750, 962, 1279, 1341, 1439, 1491, 2203.
As equimolecular mixture of 6- and 7-positional isomers.
Orange solid, 144 mg (33%). 1H NMR (DMSO-d6) dH: 2.51 (3H, s),
4.27 (2H, bs), 5.16 (2H, dd, J ¼ 10.4,17.2 Hz), 5.94 (1H, m), 7.21 (1H, t,
J ¼ 7.6 Hz), 7.43 (2H, t, J ¼ 7.6 Hz), 7.68 (2H, t, J ¼ 7.6 Hz), 8.32/8.33
(1H, s), 8.49 (1H, m), 8.66 (2H, m), 9.00 (1H, bs), 10.96 (1H, bs), 11.85
(1H, bs). 13C NMR (DMSO-d6) dC: 15.2, 46.8, 116.5, 119.6/119.9, 120.4,
120.8/121.1, 125.6, 129.8, 130.0, 135.7, 138.0, 137.8, 138.6, 138.7,
139.0, 140.5, 141.0, 157.9, 178.5. EI-MS, m/z (abundance, %): 402
(Mþ ꢁ 17-17, 20), 304 (21), 288 (59), 185 (24), 168 (100), 131 (44),
5.22. (Z)-2-(4-Phenyl-3-butenyl)-2H-indazole-3-carbonitrile
N-oxide (26)
A mixture of 2-nitrobenzaldehyde (166 mg, 1.1 mmol) and (Z)-4-
phenyl-3-butenylamine (VI) (162 mg, 1.1 mmol) in acetic acid
(20.0 mL) was stirred at room temperature for 24 h. Then, KCN
(143 mg, 2.2 mmol) was added and the reaction was stirred for 5
days. After that, water (20.0 mL) was added and the intermediate
was extracted with CH2Cl2 (3 ꢀ 20.0 mL), the organic layer was
dried with anhydrous Na2SO4 and the solvent was evaporated in
vacuo. Five drops of Et3N in EtOH (10.0 mL) was added and the
reaction was stirred at room temperature for 48 h. The solvent was
evaporated in vacuo, and the residue was fractioned between water
(50.0 mL) and EtOAc (3 ꢀ 20.0 mL). The organic layer was dried
with anhydrous Na2SO4 and the solvent was evaporated in vacuo.
The residue was purified by column chromatography (SiO2, petro-
leum ether:ethyl acetate, 7:3). Orange oil, 140 mg (44%). 1H NMR
(CDCl3) dH: 3.04 (2H, m), 4.68 (2H, t, J ¼ 7.0 Hz), 5.73 (2H, m), 6.63
(1H, d, J ¼ 11.2 Hz), 7.09–7.24 (5H, m), 7.39 (2H, m), 7.64 (1H, d,
J ¼ 7.6 Hz), 7.76 (1H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3) dC: 27.1, 46.0,
96.0, 110.6, 114.2, 118.7, 122.0, 125.1, 127.1, 127.2, 128.2–129.0
(phenyl carbons),129.1,133.7,136.0. EI-MS, m/z (abundance, %): 289
(Mþ, 1), 274 (Mþ ꢁ 16, 7), 183 (34), 155 (40), 131 (87), 115 (87), 91
115 (44), 91 (42), 77 (42), 56 (72). IR,
1538, 1659.
n: 741, 920, 1192, 1333, 1507,
5.19. 4-Allyl-1-(2-ethoxycarbonyl-1,4-dioxide-3-
phenylquinoxaline-7-yl)methylidene thiosemicarbazide (23)
Orange solid, 23 mg (5%); mp 171.2–172.6 ꢃC. 1H NMR (CDCl3)
dH: 1.21 (3H, t, J ¼ 3.6 Hz), 4.36 (2H, q, J ¼ 3.6 Hz), 4.45 (2H, m), 5.33
(2H, m), 6.03 (1H, m), 7.54 (3H, m), 7.63 (1H, bs), 7.78 (2H, m), 8.12
(1H, s), 8.21 (2H, m), 8.34 (1H, d, J ¼ 9.4 Hz), 10.05 (1H, bs). 13C NMR
(CDCl3) dC: 14.2, 47.4, 63.0, 117.9, 129.0, 129.0, 129.1, 129.2, 130.3,
130.6, 133.5, 136.0, 137.1, 137.8, 141.1, 142.8, 143.7, 166.8, 178.2. EI-
MS, m/z (abundance, %): 417 (Mþ ꢁ 17-17, 6), 319 (14), 306 (69),
290 (18), 276 (27), 233 (100), 204 (44), 129 (24), 115 (100), 77 (62),
56 (65).
(100), 77 (20). IR,
1701, 2923.
n: 700, 747, 1057, 1206, 1352, 1445, 1493, 1528,
5.20. 4-Allyl-1-(2,3-dimethyl-1,4-dioxide quinoxaline-6-
yl)methylidenethiosemicarbazide (24)
5.23. (Z)-2-[4-(4-Chlorophenyl-3-butenyl)]-2H-indazole-3-
carbonitrile N-oxide (27)
A mixture of (IV) (436 mg, 2.0 mmol), 4-allylthiosemicarbazide
(262 mg, 2.0 mmol) and p-TsOH (catalytic amount) in toluene
(50.0 mL) was stirred at room temperature for 20 h. The precipitate
was filtered and washed with petroleum ether. Yellow solid (from
A mixture of 2-nitrobenzaldehyde (166 mg, 1.1 mmol) and (Z)-4-
(4-chlorophenyl)-3-butenylamine (VII) (200 mg, 1.1 mmol) in
acetic acid (20.0 mL) was stirred at room temperature for 24 h.
Then, KCN (143 mg, 2.2 mmol) was added and the reaction was
stirred for 48 h. After that, water (20.0 mL) was added and the
intermediate was extracted with CH2Cl2 (3 ꢀ 20.0 mL), the organic
layer was dried with anhydrous Na2SO4 and the solvent was
evaporated in vacuo. Five drops of Et3N in EtOH (10.0 mL) was
added and the reaction was stirred at room temperature for 48 h.
EtOH), 543 mg (82%); mp 244.5–247.0 ꢃC. 1H NMR (DMSO-d6) dH
:
2.76 (6H, s), 4.26 (2H, bs), 5.16 (2H, dd, J ¼ 12.0, 16.0 Hz), 5.91 (1H,
m), 8.28 (1H, s), 8.42 (1H, d, J ¼ 8.0 Hz), 8.53 (1H, d, J ¼ 8.0 Hz), 8.62
(1H, s), 9.01 (1H, bs), 11.75 (1H, bs). 13C NMR (DMSO-d6) dC: 15.0,
46.4, 116.1, 119.4, 120.4, 128.6, 135.4, 136.7, 136.8, 137.4, 140.4, 142.3,
142.5, 178.0. EI-MS, m/z (abundance, %): 331 (Mþ, 1), 314 (Mþ ꢁ 17,

2162
A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
The solvent was evaporated in vacuo, and the residue was frac-
tioned between water (50.0 mL) and EtOAc (3 ꢀ 20.0 mL). The
organic layer was dried with anhydrous Na2SO4 and the solvent
was evaporated in vacuo. The residue was purified by column
chromatography (SiO2, petroleum ether:ethyl acetate, 1:1). Yellow
oil, 38 mg (11%). 1H NMR (CDCl3) dH: 3.01 (2H, m), 4.66 (2H, t,
J ¼ 6.8 Hz), 5.78 (1H, m), 6.56 (1H, d, J ¼ 11.6 Hz), 7.02 (2H, d,
J ¼ 8.4 Hz), 7.18 (2H, d, J ¼ 8.4 Hz), 7.40 (2H, m), 7.63 (1H, d,
J ¼ 8.4 Hz), 7.75 (1H, d, J ¼ 8.0 Hz). 13C NMR (CDCl3) dC: 27.2, 46.2,
92.1, 110.9, 114.5, 119.0, 122.4, 126.1, 127.6, 128.7, 128.8, 129.6, 129.9,
133.0, 133.4, 134.8. EI-MS, m/z (abundance, %): 323 (Mþ, 1), 308
5.28. (E)-3-Methoxy-5-nitro-1-{5-[4-(3-phenyl-2-
propenyl)piperazine-1-yl]-3-oxapentyl}-1H-indazole (31)
Orange solid, 182 mg (98%); mp 86.4–87.7 ꢃC. 1H NMR (CDCl3)
dH: 2.47 (10H, m), 3.13 (2H, d, J ¼ 6.8 Hz), 3.50 (2H, t, J ¼ 5.2 Hz), 3.82
(2H, t, J ¼ 5.2 Hz), 4.07 (3H, s), 4.34 (2H, t, J ¼ 5.2 Hz), 6.24 (1H, dt,
J ¼ 6.8, 15.6 Hz), 6.50 (1H, d, J ¼ 15.6 Hz), 7.27 (6H, m), 8.15 (1H, dd,
J ¼ 2.0, 9.4 Hz), 8.54 (1H, d, J ¼ 2.0 Hz). 13C NMR (CDCl3) dC: 49.6,
53.0, 53.5, 56.9, 57.9, 61.1, 69.4, 69.8, 109.9, 112.2, 118.6, 122.6, 125.9,
126.8, 128.0, 128.9, 134.1, 137.1, 141.2, 143.9, 158.7. EI-MS, m/z
(abundance, %): 465 (Mþ, 10), 448 (Mþ ꢁ 17, 22), 293 (13), 229 (13),
(Mþ ꢁ 16, 4), 183 (38), 165 (31), 155 (44), 129 (100), 115 (68). IR,
n
:
215 (100), 172 (18), 117 (72). IR,
1549, 1617, 1713, 2811.
n: 741, 806, 1011, 1142, 1329, 1487,
586, 747, 841, 1013, 1092, 1244, 1293, 1358, 1443, 1493, 2207, 2926.
5.24. General procedure for the preparation of derivatives 28–35
5.29. (E)-3-Benzyloxy-5-nitro-1-{5-[4-(3-phenyl-2-
propenyl)piperazine-1-yl]-3-oxapentyl}-1H-indazole (32)
A mixture of the indazole reactant (VIII, IX or X) (1.0 equiv.),
amines ((III), (E)-4-(3-phenyl-2-propenyl)piperazine or (XI))
(1.0 equiv.), K2CO3 (1.0 equiv.), and KI (catalytic amount) (in the case
of IX or X) in acetonitrile (15.0 mL/0.1 mmol) was heated at reflux
for 10–38 h. The solvent was evaporated in vacuo and the residue
was fractioned by column chromatography (SiO2, ethyl acetate or
CH2Cl2 for 31–33).
Yellow solid (EtOH:H2O, 1:1), 86 mg (62%); mp 98.3–99.6 ꢃC. 1H
NMR (CDCl3) dH: 2.46 (10H, m), 3.13 (2H, d, J ¼ 6.8 Hz), 3.50 (2H, t,
J ¼ 5.6 Hz), 3.86 (2H, t, J ¼ 5.2 Hz), 4.40 (2H, t, J ¼ 5.2 Hz), 5.46 (2H,
s), 6.26 (1H, m), 6.52 (1H, d, J ¼ 15.6 Hz), 7.40 (10H, m), 8.23 (1H, dd,
J ¼ 1.8, 6.8 Hz), 8.67 (1H, d, J ¼ 1.8 Hz). 13C NMR (CDCl3) dC: 49.7,
53.4, 53.9, 58.1, 61.4, 69.7, 69.9, 71.5, 109.9, 112.4, 118.9, 122.8,
126.7–129.0 (phenyl carbons),133.6, 136.6, 137.3, 141.3, 144.0, 158.0.
EI-MS, m/z (abundance, %): 541 (Mþ, 6), 524 (Mþ ꢁ 17, 14), 450 (29),
5.25. (E)-N-[5-(3-Methoxy-5-nitro-1H-indazole-1-yl)-3-
oxapentyl]-N-methyl-3-phenyl-2-propenamine (28)
369 (10), 229 (12), 215 (100), 172 (16), 117 (81), 91 (73). IR,
739, 1009, 1121, 1327, 1485, 1539, 1615, 2807.
n: 617,
Orange oil, 77 mg (94%). 1H NMR (CDCl3) dH: 2.21 (3H, s), 2.53
(2H, t, J ¼ 5.6 Hz), 3.11 (2H, dd, J ¼ 1.2, 6.8 Hz), 3.52 (2H, t,
J ¼ 5.6 Hz), 3.86 (2H, t, J ¼ 5.2 Hz), 4.10 (3H, s), 4.38 (2H, t,
J ¼ 5.2 Hz), 6.18 (1H, dt, J ¼ 6.8, 16.0 Hz), 6.45 (1H, d, J ¼ 16.0 Hz),
7.38 (6H, m), 8.15 (1H, dd, J ¼ 2.0, 9.2 Hz), 8.58 (1H, d, J ¼ 2.0 Hz). 13C
NMR (CDCl3) dC: 31.0, 49.3, 56.2, 56.6, 60.6, 69.5, 69.7, 109.4, 111.9,
118.4, 122.4 (two carbons), 126.3, 127.6, 128.6, 133.2, 136.8, 140.9,
143.6, 158.4. EI-MS, m/z (abundance, %): 410 (Mþ, 3), 393 (Mþ ꢁ 17,
5.30. (E)-3-Benzyloxy-5-nitro-1-{5-[4-(3-phenyl-2-
propenyl)piperazine-1-yl]pentyl}-1H-indazole (33)
Yellow solid, 158 mg (98%); mp 215.2–216.6 ꢃC. 1H NMR
(CDCl3) dH: 1.40 (2H, m), 1.96 (4H, m), 3.04 (2H, t, J ¼ 5.6 Hz), 3.59
(4H, m), 3.87 (2H, d, J ¼ 7.2 Hz), 3.99 (4H, bs), 4.26 (2H, t,
J ¼ 6.4 Hz), 5.46 (2H, s), 6.39 (1H, dt, J ¼ 7.2, 15.6 Hz), 6.84 (1H, d,
J ¼ 15.6 Hz), 7.43 (11H, m), 8.25 (1H, dd, J ¼ 2.0, 9.2 Hz), 8.69 (1H,
d, J ¼ 2.0 Hz). 13C NMR (CDCl3) dC: 23.6, 24.1, 29.1, 48.0, 48.5, 48.6,
57.2, 59.7, 71.5, 109.0, 122.4, 115.0, 119.1, 123.1, 127.6–130.1 (phenyl
carbons), 134.7, 136.7, 141.4, 142.0, 143.1, 158.0. EI-MS, m/z
(abundance, %): 539 (Mþ, 4), 522 (Mþ ꢁ 17, 27), 448 (23), 367 (10),
7), 160 (38), 146 (9), 117 (100), 91 (7). IR,
1487, 1547, 1617.
n: 741, 1142, 1208, 1331,
5.26. (E)-N-[5-(3-Benzyloxy-5-nitro-1H-indazole-1-yl)-3-
oxapentyl]-N-methyl-3-phenyl-2-propenamine (29)
246 (13), 215 (21), 172 (15), 117 (56), 91 (100). IR, n: 696, 739, 808,
Yellow oil, 52 mg (53%). 1H NMR (CDCl3) dH: 2.28 (3H, s), 2.63 (2H,
t, J ¼ 5.6 Hz), 3.20 (2H, d, J ¼ 6.8 Hz), 3.59 (2H, t, J ¼ 5.6 Hz), 3.90 (2H,
t, J ¼ 5.2 Hz), 4.43 (2H, t, J ¼ 5.2 Hz), 5.45 (2H, s), 6.21 (1H, dt, J ¼ 6.8,
16.0 Hz), 6.49 (1H, d, J ¼ 16.0 Hz), 7.40 (11H, m), 8.17 (1H, dd, J ¼ 1.6,
9.2 Hz), 8.64 (1H, d, J ¼ 1.6 Hz). 13C NMR (CDCl3) dC: 42.7, 49.7, 56.4,
60.7, 69.5, 69.9, 71.5, 109.9, 112.4, 118.8, 122.8, 125.2, 126.8–129.0
(phenyl carbons), 134.7, 136.7, 136.9, 141.3, 144.0, 158.1. EI-MS, m/z
(abundance, %): 486 (Mþ, 3), 395 (11), 160 (32), 146 (7), 117 (100), 91
953, 1026, 1140, 1190, 1329, 1375, 1451, 1485, 1541, 1615, 2384,
2942.
5.31. (E/Z)-N-[5-(3-Benzyloxy-5-nitro-1H-indazole-1-yl)-3-
oxapentyl]-5-phenyl-4-pentenamine (34)
Yellow oil, 27 mg (7%). E/Z proportion: 22:78. Isomer E. 1H NMR
(CDCl3) dH: 1.51 (2H, m), 2.16 (2H, m), 2.51 (2H, t, J ¼ 7.4 Hz), 2.63
(2H, t, J ¼ 5.2 Hz), 3.46 (2H, t, J ¼ 5.2 Hz), 3.86 (2H, t, J ¼ 5.2 Hz), 4.39
(2H, t, J ¼ 5.2 Hz), 5.45 (2H, s), 6.20 (1H, m), 6.37 (1H, d, J ¼ 16.0 Hz),
7.41 (11H, m), 8.21 (1H, dd, J ¼ 2.0, 9.4 Hz), 8.67 (1H, d, J ¼ 2.0 Hz).
13C NMR (CDCl3) dC: 30.0, 31.1, 49.5, 49.7, 49.7, 69.9, 71.1, 71.5, 109.7,
112.5, 118.9, 122.8, 123.8–129.1 (phenyl carbons), 130.6, 134.0, 136.6,
138.0, 141.4, 144.0, 158.1. Isomer Z. 1H NMR (CDCl3) dH: 1.51 (2H, m),
2.31 (2H, m), 2.51 (2H, t, J ¼ 7.4 Hz), 2.63 (2H, t, J ¼ 5.2 Hz), 3.46 (2H,
t, J ¼ 5.2 Hz), 3.86 (2H, t, J ¼ 5.2 Hz), 4.39 (2H, t, J ¼ 5.2 Hz), 5.45 (2H,
s), 5.61 (1H, m), 6.43 (1H, d, J ¼ 11.6 Hz), 7.41 (11H, m), 8.21 (1H, dd,
J ¼ 2.0, 9.4 Hz), 8.67 (1H, d, J ¼ 2.0 Hz). 13C NMR (CDCl3) dC: 26.6,
30.6, 49.5, 49.7, 49.7, 69.9, 71.1, 71.5, 109.7, 112.5, 118.9, 122.8,
123.8–129.1 (phenyl carbons), 129.7, 132.6, 136.6, 138.0, 141.4, 144.0,
158.1. EI-MS, m/z (abundance, %): 500 (Mþ, 9), 409 (8), 369 (23), 295
(42). IR, n: 700, 741, 804, 970, 1142, 1329, 1483, 1539, 1615, 2938.
5.27. (E)-N-[5-(3-Benzyloxy-5-nitro-1H-indazole-1-yl)pentyl]-N-
methyl-3-phenyl-2-propenamine (30)
Orange oil, 96 mg (99%). 1H NMR (CDCl3) dH: 1.34 (2H, m), 1.56
(2H, m), 1.92 (2H, m), 2.27 (3H, s), 3.15 (2H, t, J ¼ 6.4 Hz), 3.41 (2H, d,
J ¼ 6.0 Hz), 4.23 (2H, t, J ¼ 6.8 Hz), 5.45 (2H, s), 6.28 (1H, m), 6.50
(1H, d, J ¼ 16.4 Hz), 7.35 (11H, m), 8.21 (1H, dd, J ¼ 2.0, 9.2 Hz), 8.68
(1H, d, J ¼ 2.0 Hz). 13C NMR (CDCl3) dC: 24.7, 27.0, 29.5, 42.2, 48.9,
53.5, 60.3, 71.1, 108.5, 111.9, 118.8, 122.5, 126.3, 126.3, 127.5–128.6
(phenyl carbons), 132.7, 136.3, 137.0, 140.8, 142.6, 157.5. EI-MS, m/z
(abundance, %): 484 (Mþ, 4), 467 (Mþ ꢁ 17, 7), 393 (18), 246 (7), 160
(19), 146 (20), 117 (100), 91 (51). IR,
1453, 1485, 1539, 1615, 2938.
n
: 695, 741, 1140, 1194, 1329,
(6), 279 (11), 205 (7), 174 (72), 144 (15), 117 (14), 91 (100). IR,
739, 1140, 1329, 1485, 1539, 1615, 2936.
n: 700,

A. Gerpe et al. / European Journal of Medicinal Chemistry 45 (2010) 2154–2164
2163
5.32. (E/Z)-N,N-Bis[5-(3-benzyloxy-5-nitro-1H-indazole-1-yl)-3-
oxapentyl]-5-phenyl-4-pentenamine (35)
mL and cells were incubated at 37 ꢃC for 3 h. After removing the
media, formazan crystals were dissolved in DMSO (180 L), and the
m
absorbance at 595 nm was read using a microplate spectrophotom-
eter. Results are expressed as ID50 (compound concentration that
reduce 50% control absorbance at 595 nm). Every ID50 is the average
of three different experiments.
Yellow oil, 242 mg (67%). E/Z proportion: 36:64. Isomer E. 1H
NMR (CDCl3) dH: 1.42 (2H, m), 2.06 (2H, m), 2.33 (2H, t, J ¼ 6.8 Hz),
2.45 (4H, t, J ¼ 5.6 Hz), 3.31 (4H, t, J ¼ 5.6 Hz), 3.77 (4H, t, J ¼ 5.6 Hz),
4.32 (4H, t, J ¼ 5.6 Hz), 5.45 (4H, s), 5.56 (1H, m), 6.34 (1H, d,
J ¼ 15.6 Hz), 7.41 (22H, m), 8.16 (1H, dd, J ¼ 2.0, 9.4 Hz), 8.64 (1H, d,
J ¼ 2.0 Hz). 13C NMR (CDCl3) dC: 27.3, 30.9, 49.7, 54.4, 55.0, 69.9,
70.4, 71.5, 109.9, 112.4, 118.8, 122.7, 126.3–129.1 (phenyl carbons),
130.6, 136.6, 138.0, 138.0, 141.3, 143.9, 158.0. Isomer Z. 1H NMR
(CDCl3) dH: 1.42 (2H, m), 2.22 (2H, m), 2.33 (2H, t, J ¼ 6.8 Hz), 2.45
(4H, t, J ¼ 5.6 Hz), 3.31 (4H, t, J ¼ 5.6 Hz), 3.77 (4H, t, J ¼ 5.6 Hz), 4.32
(4H, t, J ¼ 5.6 Hz), 5.45 (4H, s), 6.14 (1H, m), 6.41 (1H, d, J ¼ 11.6 Hz),
7.41 (22H, m), 8.16 (1H, dd, J ¼ 2.0, 9.4 Hz), 8.64 (1H, d, J ¼ 2.0 Hz).
13C NMR (CDCl3) dC: 26.6, 28.0, 49.7, 54.4, 55.0, 69.9, 70.4, 71.5,
109.9, 112.4, 118.8, 122.7, 126.3–129.1 (phenyl carbons), 129.6, 132.7,
136.6, 138.0, 141.3, 143.9, 158.0. EI-MS, m/z (abundance, %): 841
5.33.3. Microorganisms and media
For the antifungal evaluation, strains from the American Type
Culture Collection (ATCC), Rockville, MD, USA and CEREMIC (C),
´
´
Centro de Referencia en Micologıa, Facultad de Ciencias Bioquımicas
´
y Farmaceuticas, Suipacha 531-(2000)-Rosario (Argentina) were
used: Candida albicans ATCC 10231, Candida tropicalis C 131, Saccha-
romyces cerevisiae ATCC 9763, C. neoformans ATCC 32264, Aspergillus
flavus ATCC 9170, Aspergillus fumigatus ATTC 26934, Aspergillus niger
ATCC 9029, T. rubrum C 110, T. mentagrophytes ATCC 9972, and M.
gypseum C 115.
Strains were grown on Sabouraud-chloramphenicol agar slants
for 48 h at 30 ꢃC, maintained on slopes of Sabouraud-dextrose agar
(SDA, Oxoid) and subcultured every 15 days to prevent pleomor-
phic transformations. Inocula of cells or spore suspensions were
obtained according to reported procedures and adjusted to
103 cells/spores with colony forming units (CFU)/mL [49,50].
(Mþ þ 2H, 50), 840 (Mþ þ H, 100), 501 (19), 345 (27). IR,
n: 700, 754,
806, 967, 1142, 1198, 1329, 1451, 1485, 1539, 1615, 2938.
5.33. Biology
5.33.1. Anti-T. cruzi in vitro test using epimastigotes of Tulahuen 2
strain or CL Brener clone
5.33.4. Antifungal susceptibility testing
T. cruzi epimastigotes (Tulahuen 2 strain or CL Brener clone)
were grown at 28 ꢃC in an axenic medium (BHI-Tryptose) as
previously described [31,32,37], supplemented with 5% fetal bovine
serum (FBS). Cells from a 10-day-old culture (stationary phase)
were inoculated into 50 mL of fresh culture medium to give an
initial concentration of 8 ꢀ 106 cells/mL. Cell growth was followed
by measuring everyday the absorbance of the culture at 600 nm.
Before inoculation, the media were supplemented with the indi-
cated amount of the drug from a stock solution in DMSO.
Compounds 34 and 35 were studied as mixtures of E and Z
geometric isomers (for proportions see synthetic procedures). The
final concentration of DMSO in the culture media never exceeded
0.4% and the control was run in the presence of 0.4% DMSO and in
the absence of any drug. No effect on epimastigote growth was
observed by the presence of up to 1% DMSO in the culture media.
The percentage of growth inhibition (PGI) was calculated as
follows: PGI (%) ¼ {1 ꢁ [(Ap ꢁ A0p)/(Ac ꢁ A0c)]} ꢀ 100, where
Ap ¼ A600 of the culture containing the drug at day 5; A0p ¼ A600 of
the culture containing the drug just after addition of the inocula
(day 0); Ac ¼ A600 of the culture in the absence of any drug (control)
at day 5; A0c ¼ A600 in the absence of the drug at day 0. To
determine ID50 values, 50% inhibitory concentrations, parasite
growth was followed in the absence (control) and presence of
increasing concentrations of the corresponding drug. At day 5, the
absorbance of the culture was measured and related to the control.
The ID50 value was taken as the concentration of drug needed to
reduce the absorbance ratio to 50%.
Minimal Inhibitory Concentrations (MIC) of each compound was
determined by using broth microdilution techniques according to
the guidelines of the Clinical and Laboratory Standards Institute
(CLSI) formely National Committee for Clinical and Laboratory
Standards [49,50] for yeasts (M27-A2) and for filamentous fungi
(M 38 A). MIC values were determined in RPMI 1640 buffered to pH
7.0 with MOPS. Microtiter trays were incubated at 35 ꢃC for yeasts
and Aspergillus spp. and at 28–30 ꢃC for dermatophyte strains in
a moist, dark chamber, and MICs were visually recorded at 48 h for
yeasts, and at a time according to the control fungus growth, for the
rest of fungi. For the assay, stock solution of each compound was
two-fold diluted with RPMI 1640 from 250-0.98
volume ¼ 100 L) and a final DMSO concentration ꢄ1%. A volume of
100 L of inoculum suspension was added to each well, with the
mg/mL (final
m
m
exception of the sterility control where sterile water was added to
the well instead. MIC was defined as the minimum inhibitory
concentration of compound which resulted in total inhibition of the
fungal growth. Ktz, Tbf and AnfB were used as positive controls.
5.33.5. Squalene level analysis
Epimastigote forms of T. cruzi (Tulahuen 2 strain) were main-
tained in an axenic medium (BHI-Tryptose) for analysis of the
effects of the studied compounds on sterol biosynthesis modifica-
tions. The experiments were carried out in cultures at 28 ꢃC and
with strong aeration. The studied compound was added at
a concentration equivalent of ID50 per 8 ꢀ 106 cells/mL as DMSO
solution. Control samples received only the vehicle. Parasites were
exposed to the treatment by 120 h. They were counted by optical
microscopy using a Neubauer chamber. The control and drug-
treated parasites were centrifugated at 3000 rpm during 15 min,
and then the pellets were collected and washed with buffer
phosphate (10.0 mL, 0.05 M, pH 7.4) and centrifugated at 3000 rpm
during 15 min. The pellets were treated with chloroform:methanol
(2:1) during 12 h at 4 ꢃC. Then the organic phases were evaporated
with nitrogen and the residues were treated with acetonitrile
5.33.2. Unspecific mammalian cytotoxicity [22,32]
J-774 murine macrophage-like cells (ATCC, USA) were maintained
by passage in Dulbecco’s modified Eagle’s medium (DMEM) con-
taining 4 mM L-glutamine, and supplemented with 10% heat-
inactivated fetal calf serum. J-774 cells were seeded (1 ꢀ 105 cells/
well) in 96 well microplates with 200 mL 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
(AcCN, HPLC quality) (600
mL) during 1 min and the AcCN solutions
at 37 ꢃC and, then, exposed to compounds (100.0–400.0
mM) for 48 h.
were filtered through a cellulose-RC (0.45 mm, Sartorius) filter. TLC
Afterwards, cell viability was assessed by measuring the
mitochondrial-dependent reduction of MTT (Sigma) to formazan. For
that purpose, MTT was added to cells to a final concentration 0.4 mg/
analyses of neutral lipid fractions were carried out using silica-gel
plates (Merck 5538-7) employing the systems petroleum ether:-
EtOAc or petroleum ether (to see squalene) as eluents. The

2164
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and heating them at 100 ꢃC. Quantitative analyses of squalene from
sterol fractions was done by HPLC using a C-18 Chromosorb column
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(25 cm ꢀ 0.4 cm internal diameter, 10
mm particle size) in a Perkin
Elmer LC-135C/LC-235C Diode Array Detector, Series 410 LC BIO
PUMP, with the UV detector set at 210 nm. The mobile phase
consisted of 100% AcCN and was kept constant at a flow-rate of
0.8 mL/min. The calibration curve of squalene was constructed
(using cholesterol as an internal standard) for quantification of this
sterol in the lipid extracts from T. cruzi and it is the following:
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(
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where A corresponds to the area of the peak at 5.70–5.85 min.
´
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AG and LB thank to PEDECIBA (Uruguay), AG, and PH thank to
ANII (Uruguay), and MS thanks to CONICET for their scholarships.
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