Study of 5-nitroindazoles' anti-Trypanosoma cruzi mode of action: Electrochemical behaviour and ESR spectroscopic studies

Article abstract of DOI:10.1016/j.ejmech.2008.07.018

New indazole derivatives have been developed to know about structural requirements for adequate anti-Trypanosoma cruzi activity. In relation to position 1 of indazole ring, we have observed that a butylaminopentyl substituent (14) affords good activity, b

Full text of DOI:10.1016/j.ejmech.2008.07.018

European Journal of Medicinal Chemistry 44 (2009) 1545–1553
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Study of 5-nitroindazoles’ anti-Trypanosoma cruzi mode of action:
Electrochemical behaviour and ESR spectroscopic studies
a
,
Jorge Rodrıguez b, Alejandra Gerpe c, Gabriela Aguirre c, Ulrike Kemmerling d, Oscar E. Piro e, Vicente
´
f
d
a,
c
,
c,
**
*
**
´
´
J. Aran , Juan Diego Maya , Claudio Olea-Azar , Mercedes Gonzalez
, Hugo Cerecetto
a
´
´
´
´
Departamento de Qu´ımica Inorganica y Analıtica, Facultad de Ciencias Quımicas y Farmaceuticas, Universidad de Chile, Santiago, Chile
Departamento de Qu´ımica, Facultad de Ciencias Basicas, Universidad Metropolitana de Ciencias de la Educacion, Santiago, Chile
Departamento de Qu´ımica Organica, Facultad de Ciencias/Facultad de Quımica, Universidad de la Republica, Montevideo, Uruguay
d Departamento de Farmacolog´ıa Molecular y Cl´ınica, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Departamento de Fısica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata and Instituto IFLP (CONICET), C.C. 67, 1900 La Plata, Argentina
Instituto de Quımica Medica (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
b
c
´
´
´
´
e
´
f
´
´
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2008
Received in revised form 11 July 2008
Accepted 15 July 2008
New indazole derivatives have been developed to know about structural requirements for adequate anti-
Trypanosoma cruzi activity. In relation to position 1 of indazole ring, we have observed that a butyla-
minopentyl substituent (14) affords good activity, but N-oxidation of
u-tertiary amino moiety yields
completely inactive compounds (17, 18); the substituent at position 3 of indazole ring affects drastically
the in vitro activity, 3-OH derivative 13 being completely inactive. On the other hand, since compound 22,
denitro-analogue of active compound 4, does not show activity, the 5-nitro substituent of indazole ring
seems to be essential. Intramolecular cyclization of side chain at position 1 also affords inactive
compounds (19, 20). The electrochemical studies showed that the trypanocidal 5-nitroindazole deriva-
tives yielded nitro-anion radical via one-electron process at physiological pH. This electrochemical
behaviour occurs in the parasite according to ESR experiment with the T. cruzi microsomal fraction
showing that 5-nitroindazole derivatives suffer bio-reduction without reactive oxygen species
generation.
Available online 23 July 2008
Keywords:
5-Nitroindazole
Anti-T. cruzi agents
Mode of action
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
that any drugs could replace Nfx and Bnz. Therefore, the search for
new and better drugs against T. cruzi is of paramount importance.
American trypanosomiasis or Chagas’ disease, in which we have
centered the present study, has like etiological agent the Trypano-
soma cruzi, protozoan parasite with complex life cycle, endemic of
Latin America. Latest data from the WHO indicates that over 24
million people are infected or at least serologically positive for T.
cruzi [1]. Currently, this pathology is treated with nitroheterocyclic
agents such as nifurtimox (Nfx) and benznidazole (Bnz) [2]. Both
display effectiveness similar in the acute and undetermined phases
of this disease, but the latter is preferred due to its simpler
administration and that it shows less severe secondary effects; in
fact, Nfx has been retired from the market in many countries. It is
believed that they act through a bio-reduction of the NO2 group,
but their mechanisms of action are different [3,4]. Different ther-
apeutic targets are currently being studied but it does not appear
Despite their toxicity, nitroaromatic compounds are generally
very potent against T. cruzi and have been considered as important
hits for molecular modifications [5,6]. We had previously described
the in vitro anti-T. cruzi activity of some 5-nitroindazole derivatives,
i.e. 1–7 (Scheme 1), some of them having remarkable anti-
epimastigote properties, and displaying also certain antineoplastic
activity that confirms the previously observed parallelism between
R2
N
-X-
-R1
-R2 Compound
O2N
-O-
-O-
-O-
-dimethelylamine -OH
-piperidino
-piperidino
1
2
3
-OH
-OCH3
N
R1
-CH2- -dimethelylamine -OBn
4
5
6
7
X
-O-
-CH2- -piperidino
-O- -piperidino
-dimethelylamine -OBn
-OBn
-OBn
* Corresponding author. Fax: þ56 2 7370567.
** Corresponding authors.
further structural modifications
E-mail address: colea@uchile.cl (C. Olea-Azar).
Scheme 1.
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.018

1546
J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
R2
N
R2
O2N
O2N
amine/K2CO3
N
N
N
acetonitrile reflux
Br
R1
X
X
-R2 Compound
Compound
-R2
-X-
-R2
-X-
-O-
-OH
-OH
-OCH3
-OBn
8
-O- -morpholino
-CH2- -butylamino -OCH3
-OH
13
14
15
16
-CH -
9
-CH22-
-O-
10
-O-
-morpholino
-OBn
-OBn
OBn
N
11
12
-CH2- -butylamino
O2N
-OBn
-CH2-
m-CPBA
-O- -dimethylamine -OBn
-O- -piperidino -OBn
5
N
7
CH2Cl2 reflux
K2CO3
N
O
acetonitrile reflux
O
Compound
N
-X- Compound
O2N
N
-dimethylamine
-piperidino
17
18
-O-
-CH2-
19
20
N
X
Scheme 2.
antichagasic and anticancer activities [7a]. Interestingly, some of
these 5-nitroindazoles did not show significant unspecific cyto-
toxicity against macrophages [7a].
Recently, we have also demonstrated the effectiveness in an in
vivo acute model of Chagas disease of the 5-nitroindazoles 3 and 5
(Scheme 1) [7b]. In order to observe the chemoprophylactic effects
of these derivatives we tested the anti-trypomastigote (CL Brener
clone) activity in blood presenting derivatives 3–5 (Scheme 1)
trifluoroacetic acid, were assayed unsuccessfully obtaining the
de-benzylated derivatives 1 and 2 (Scheme 1), as main reaction
products. For the generation of a backbone in 1-position with
restricted mobility, reactants 8 and 9 were cyclized in the presence
of potassium carbonate in acetonitrile at reflux to afford the cor-
responding fused indazolinones 19 and 20 (Scheme 2) [9]. The
generation of a derivative lacking the 5-nitro substituent, was
assayed unsuccessfully for the reduction of the nitro group, in the
presence of Fe/HCl (Scheme 3)a, to generate intermediate 21.
However, in these conditions the complexation of indazole system
took place without generation of the desired amine 21. Conse-
quently, we assayed the synthetic procedure shown in Scheme 3b
starting from 3-hydroxy-1H-indazole 23 and affording the desired
derivative 22, 5-denitro-analogue of active compound 4, in good
global yield [10–13]. All new compounds were characterized by
NMR (1H, 13C, HMQC, and HMBC), MS and IR analyses and their
purity was established by TLC and microanalysis. Single crystals of
derivative 13 adequate for structural X-ray diffraction studies1 were
remarkable activities at a dose of 250 mg mL [7b]. In this first
approach it was done mainly modifications at the lateral chain in
1- and 3-positions by varying –X–, –R1 and –R2 (see Scheme 1).
Some theoretical and experimental physicochemical studies were
done in order to understand the mode of trypanocidal action of
these compounds, however, some structural modifications and
deep studies on mechanism of action would be performed [7a,8].
Herein, we describe the synthesis of new 5-nitroindazoles
modifying mainly the substitution in R1, including lipophilic and
hydrophilic amino-substituents, the flexibility in backbone at the
1-position and avoiding the presence of the nitro moiety at the
5-position (Scheme 1). We also describe the anti-T. cruzi activities.
The performed structural modifications together with electro-
chemical and ESR studies allow us to get inside the mechanism of
action.
1
(a) In derivative 13 the 5-nitro group is nearly coplanar with the indazole ring.
As expected, intramolecular distances within the phenyl ring correspond to
a delocalized bond structure [C–C bond lengths in the range from 1.373(3) to 1.
404(3) Å]. Within the fused heterocycle ring the double C4–N2 bond distance is 1.
311(2) Å, significantly shorter (13s) than single C5–N3 bond length [1.338(2) Å] and
2. Methods and results
single C8–N3 bond distance [1.452(2) Å]. The 4-thiomorpholine ring is folded back
onto the fused ring group and shows a single bonded chair conformation. C–C
distances are 1.510(3) and 1.517(2) Å; C–N distances are equal to each other [1.
480(2) Å]; S–C bond lengths are also equal to each other within experimental
accuracy [1.800(3) Å]. The molecules are arranged in the lattice as centrosymmetric
dimers where the hydroxyl group of a monomer forms a strong O–H/N bond with
the 4-thiomorpholine N-atom of an inversion related molecule [d(O3/N4) ¼ 2.
592 Å, d(H3/N4) ¼ 1.435 Å, :(O3–H3/N4) ¼ 165.2ꢀ]. Fractional atomic coordi-
nates and equivalent displacement parameters, full bond distances and angles,
anisotropic displacement parameters along with detailed crystal data, data collec-
tion procedure, structure determination method and refinement results are given
as Supporting information. (b) Enraf-Nonius (1997–2000). COLLECT. Nonius BV,
Delft, The Netherlands. (c) K. Harms, S. Wocadlo, XCAD4 – CAD4 Data Reduction,
University of Marburg, Marburg, Germany, 1995. (d) PLATON, A Multipurpose
Crystallographic Tool, Utrecht University, Utrecht, The Netherlands, A.L. Spek, 1998.
(e) G.M. Sheldrick, SHELXS-97. Program for Crystal Structure Resolution. University of
Go¨ttingen: Go¨ttingen, Germany, 1997. (f) G.M. Sheldrick, SHELXL-97. Program for
Crystal Structures Analysis. University of Go¨ttingen: Go¨ttingen, Germany, 1997. (g) C.
K. Johnson, ORTEP-II. A Fortran Thermal-Ellipsoid Plot Program. Report ORNL-5138,
Oak Ridge National Laboratory, Tennessee, USA, 1976.
2.1. Synthesis
Three different modifications at the level of R1 substituent were
done: inclusion of a lipophilic amino substituent (thiomorpholino
moiety), hydrophilic amino substituent (N-oxide of amino moiety),
and donor hydrogen-bond amino substituent (butylamino moiety).
These derivatives were prepared following the pathways shown in
Scheme 2. Using bromides 8–12 as common starting materials, the
reaction with thiomorpholine or butylamine in the presence of
potassium carbonate afforded derivatives 13–16 in good yields [7].
In reference to the N-oxidation of amino-substituents in the lateral
chain, m-chloroperbenzoic acid was used successfully generating
regio-selectively products 17 and 18 (Scheme 2), without oxidation
of indazole nitrogen atoms. On the other hand, the derivatives
treated with urea–hydrogen peroxide complex, in the presence of

J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
1547
a
OBn
N
OBn
N
OBn
N
O2N
H2N
Fe / HCl
reflux
N
N
N
4
21
22
N
N
N
OBn
N
O2N
FeIII
x
N
R
b
OH
N
OH
OBn
N
ClCO2Et
Py reflux
BrBn/Cs2CO3
acetone reflux
N
N
N
N
H
CO2Et
CO2Et
23
24
25
KOH
OBn
OBn
N
OBn
N
Br(CH2)4Br/K2CO3
acetone reflux
dimethylamine
reflux
N
N
N
N
H
(CH2)4Br
22
27
26
N
Scheme 3.
obtained by slow evaporation from an EtOH solution. Fig. 1 shows
the ORTEP molecular drawing of derivative 13.
intensity cross-peaks, 7H-indazole and 40H- and 20H-lateral chain
protons of the lateral chain and 6H-indazole and 50H-lateral chain
protons, and less intense cross-peaks. These results indicate that
the 3-oxapentyl lateral chain could be in a folded conformation
wherein the major cross-peak is between 7H and 50H protons.
Furthermore, cross-peaks does not appear between the protons of
the lateral chain and 4H proton of indazole moiety indicating some
preferential folding in solution that involves only 6H- and
7H-indazole protons. This result is in agreement with the distri-
bution in solid state.
In order to study the preferential conformation of these deriv-
atives in solution, two-dimensional rotating frame nuclear Over-
hauser effect spectroscopy (ROESY) experiments were carried out
in DMSO. An expansion of the ROESY spectrum of derivative 13 is
shown in Fig. 2. The inspection of the ROESY spectra allowed the
establishment of spatial proximities between several aromatic
hydrogens of the indazole moiety and the lateral chain protons. The
two-dimensional spectrum shows several intramolecular cross-
peaks, i.e. between 7H-indazole and 50H-lateral chain protons, high
Fig. 1. Derivative 13-dimer molecular graphic displaying the numbering of molecular
structure. Displacement ellipsoids are drawn at the 30% probability level.
Fig. 2. Partial contour plot of the two-dimensional ROESY spectrum of derivative 13 in
DMSO-d6.

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J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
Table 2
2.2. Biological characterization: anti-trypanosomal activity
Effect of 5-nitroindazole derivatives upon T. cruzi trypomastigote viability after 24 h
treatment
All the compounds were tested in vitro against CL Brener clone
of epimastigote forms of T. cruzi at different concentrations. Table 1
shows the percentage of growth inhibition (% GI) of the indazole
Compound
Viabilitya
4
5
6
14
16
Nfx
0.49 ꢃ 0.007
0.56 ꢃ 0.003
0.29 ꢃ 0.007
40.04 ꢃ 0.100
ꢁ0.37 ꢃ 0.005
50.89 ꢃ 0.058
derivatives at 25 mM. The most active of the new indazole deriva-
tives was 16 with IC50 value in the same order as that of the
reference drug (Nfx), while compound 14 showed medium activity
at the assayed concentration.
The most active 5-nitroindazole derivatives 4–6, 14, and 16
(Table 1) and Nfx were tested upon non-replicative trypomastigote
forms of the parasite. These forms are found in mammalian blood
and are the target for antichagasic drugs. A viability test such as
MTT reduction was carried out using drug concentrations equiva-
lent to IC50 values for epimastigotes. The value of viabilities (Table
2) ranged from 0 to 40% compared with untreated control. The new
derivative 14 displayed similar activity of that of Nfx while parent
compounds 4–6 and the new developed derivative 16 showed
stronger activity against trypomastigote-T. cruzi than Nfx. This
result confirms the trend observed upon epimastigote forms and
previously reported trypomastigote activities and in vivo activities
found for these 5-nitroindazole derivatives.
a
Drug concentrations used were the IC50 obtained from epimastigote form of
T. cruzi (CL Brener clone, Table 1). For details see Section 5.
displayed at more negative potential around ꢁ0.8 V (IIc), which
corresponds to the electroreduction of the protonated hydroxyl-
amine group to form the corresponding amino derivative. The
hydroxylamine group generated in the nitro-reduction could
accept one or two protons and both the mono- and the diproto-
nated forms could undergo reduction at potentials more negative
than the corresponding nitro group [15]. In this media, such
reduction occurs in a two-electron step (see Eq. (2)). At higher pH
values, usually at pH greater than about 4, the height of this wave
decreases with increasing pH and at pH > 6, physiological pH,
virtually no reduction is observed [15]. The decrease reflects the
decrease in rate of protonation with increasing pH. Fig. 3b shows
the results at pH 7 observing that the reductive peak (IIc), corre-
sponding to the formation of amino derivative, disappears.
Hydroxylamine derivative formed is a highly reactive species,
which could undergo numerous chemical and electrochemical
reactions. In our case, it could be electrooxidized to the corre-
sponding nitroso derivative. At pH 7 it is possible to observe in the
voltammogram a reversible couple (IVa/IVc) which appears at
sufficiently low potential, ꢁ0.10 V corresponding to the reversible
reduction of nitroso derivative that was formed by electrooxidation
of the hydroxylamine derivative. In this reversible couple, the
cathodic peak appears only after the accumulation of at least one
cyclic voltammogram indicating that the step is hydroxylamine
oxidation dependent. The nature of the peaks was recognized early
by nitrobenzene (Eq. (3)) [15,16].
2.3. Study of anti-T. cruzi mechanism of action
In order to study if oxidative stress mediates the biological
action of these derivatives we performed some experiments related
to this fact. We studied the indazoles’ electrochemical behaviour in
protic media and the indazoles’ production of free radicals into the
parasite, by ESR experiments.
2.3.1. Electrochemical behaviour
Electrochemical behaviour in protic media for this family of
indazole derivatives was studied using buffer Britton–Robinson/
EtOH (70:30) adjusting to pH 2.0, 7.1 and 9.0. A hanging mercury
drop electrode was used as the working electrode. Fig. 3a shows the
cyclic voltammogram obtained at pH 2.0 when a solution of deri-
vate 14 was electrolyzed. Two irreversible reduction peaks are
observed, a first peak around ꢁ0.4 V could correspond to the
electroreduction of the nitro group (Ic) by four electrons, to form
the hydroxylamine derivative, similar to the results previously
observed for other 5-nitroimidazole derivatives in protic medium
at the same pH (Eq. (1)) [14]. The second irreversible peak was
R—NO2 D4eL D4HD /R—NHOH DH2O
(1)
R—NHOHD2 D2eL D HD /R—NH2 DH2O
R—NHOH 4 R—NO D 2eL
(2)
(3)
Table 1
In vitro antichagasic activity of indazole derivatives
Compound
% GIa,b (IC50
, mM)
1
2
3
4
5
6
7
13
14
15
16
17
18
19
20
22
Nfx
3c
9c
On the other hand, during the reduction at physiological pH
(Fig. 3b) it was possible to note that the peak (Ic) corresponding to
the nitro group four-electron reduction wave is split into two peaks
(Ic and IIIc), which suggests that in neutral and alkaline solutions
the four-electron reduction occurs in two separate steps. The one-
electron reduction from R–NO2 to R–NOꢂ2ꢁ (nitro-anion radical) (Ic)
(Eq. (4)) precedes the three-electron reduction process (wave IIIc)
(Eq. (5)). Both reduction waves are shifted to more negative
potentials, ꢁ0.56 and ꢁ1.41 V, respectively. These results show that
R–NOꢂ2ꢁ radical anion is stabilized in neutral and alkaline media.
Previous reports show similar results for cyclic voltammetry of
nitrobenzene in alkaline medium on Au electrode [15,16].
25c
100 (12.5)c
80 (16.4)c
100 (6.6)c
24c
0
58 (22.0)
20
94 (11.0)
0
6
6
4
18
100 (3.4)
R—NO2 DeL /R—NOꢂ2L
(4)
a
Values expressed as percentage of inhibition of control growth (% GI).
M concentration.
Results are the means of three different experiments with SD less than 10% in all
cases.
Compounds tested at 25
m
b
R—NOꢂ2L D 3eL D 4HD /R—NHOH D H2O
(5)
c
From Ref. [7].

J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
1549
Fig. 3. (a) Derivative 14 (1 mM) cyclic voltammogram in protic media (buffer Britton–Robinson/EtOH 70:30 þ 0.1 M KCl), pH 2.0, speed 2.0 V sꢁ1. (b) Derivative 14 (1 mM) cyclic
voltammogram in protic media (buffer Britton–Robinson/EtOH 70:30 þ 0.1 M KCl), pH 7.1, speed 2.0 V sꢁ1
.
On the other hand, previous reports had indicated that C]N
indazole moiety reduction takes place at high reduction potentials,
around 1.6 V, in protic medium [17,18]. Nevertheless, when a solu-
tion of derivative 22 was used in the electrochemical studies, this
was not electrolyzed under our work conditions. This result indi-
cates that in our conditions the nitro group is the only electro-
active species in 5-nitroindazole derivatives. In addition, the same
behaviour was obtained for 5-nitroindazolone derivatives 19 and
20.
spectrum being in agreement with the trapped nitro-anion radical
species which is generated from the nitro-bio-reduction. The six
lines have the following hyperfines aN ¼ 15.95 G and aH ¼ 23.00 G
consistent with the trapped nitro-anion radical (marked with * in
Fig. 4c). Moreover, these results could be indicating that the mode
of action of these derivatives does not stimulate, unlike Nfx, the
superoxide, hydroxyl radical or hydrogen peroxide generation
[23,24]. However, it makes evident the presence of 5-nitroindazole
reduced metabolites, which could be probably involved in its try-
panocidal activity showing a mechanism of action similar to that of
benznidazole [25–27].
2.3.2. ESR spectroscopic studies
In order to analyse the capacity of these compounds to generate
free radicals into the parasite we incubated our derivatives with
T. cruzi microsomes in the presence of NADPH, and the spin trap-
ping DMPO [19–22]. Fig. 4 shows the ESR spectrum obtained when
DMPO was added to the system 14–T. cruzi microsomes, this
3. Discussion
The newly developed 5-nitroindazole derivatives allow us to
extract some structural requirements for adequate trypanocidal
Fig. 4. ESR spectra of DMPO–14 radical adducts obtained with T. cruzi microsomes. The ESR spectra were observed 25 min after incubation at 28 ꢀC with T. cruzi microsomal fraction
(4 mg protein/mL), NADPH (1 mM), in phosphate buffer (20 mM), pH 7.4 (A) DMSO (10 v/v) and DMPO (100 mM), (B) 14 (1 mM in DMSO 10 v/v) and (C) 14 (2 mM in DMSO 10 v/v)
and DMPO (100 mM). Spectrometer conditions: microwave frequency 9.75 GHz, microwave power 20 mW, modulation amplitude 0.87 G, time constant 0.25 s, number of scans 10.

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J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
activity. The kind of indazole 3-substituent affects drastically the in
vitro activity, producing 3-OH substituent a complete loss of
activity and 3-OBn substituent the most active compounds in each
series of analogues (compare activity of derivatives 13 and 15, or
parent compounds 1 and 5). The change of piperidino substituent
by a thiomorpholino moiety does not improve the activity
(compare activity of derivative 15 and parent compound 7);
however, if the change is by a butylamino substituent the activity
increases (compare activity of derivative 14 and parent compound
3). Neither the N-oxidation of amino substituent in indazole at the
1-position (compare activity of derivative 17 and parent compound
7, or derivative 18 and parent compound 5) nor the cyclization
(derivatives 19 and 20) produces an improvement in the trypano-
cidal activity. The absence of the 5-nitro moiety produces a loss of
activity (compare activity of derivative 22 and parent compound 4).
The electrochemical studies, in protic media and different pH,
showed different reduction behaviour of the trypanocidal
5-nitroindazole derivatives, yielding hydroxylamine via a single
four-electron reduction at pH 2.0 and nitro-anion radical via one-
electron process at physiological pH (7.1). In these conditions, the
final hydroxylamine derivative formed was electrooxidized to the
corresponding nitroso-analogue. This electrochemical behaviour
occurs into the parasite according to ESR experiment with the T.
cruzi microsomal fraction showing that 5-nitroindazole derivatives
suffer bio-reduction without reactive oxygen species generation.
However, in spite of that electrochemical characteristics are similar
for all family, we believe that other factors would affect behaviour in
the biological activities. The reduction mechanism could be different
in biological system. On the other hand, the structural features of the
5-NI derivatives revealed a mimetic similarity with non-peptide
inhibitors represented by the tricyclic ring structures which are
competitive inhibitors of trypanothione reductase (TR). The tricyclic
moiety of these compounds was shown to lodge against the
hydrophobic wall of TR active site [33]. In addition, the presence of
protonatable amino groups in a long alkylamino chain is indis-
pensable for TR recognition. In this sense, the trypanocidal activity
observed could be the result of a mixed mechanism that considers
the bio-reduction and an uncompetitive inhibitor of TR.
were recorded on a Perkin Elmer 1310 spectrometer, using potas-
sium bromide tablets; the frequencies are expressed in cmꢁ1
.
DC-Alufolien silica gel 60 PF254 (Merck, layer thickness 0.2 mm)
and silica gel 60 (Merck, particle size 0.040–0.063 mm) were used
for TLC and flash column chromatography, respectively. Elemental
analyses were obtained from vacuum-dried samples (over phos-
phorous pentoxide at 3–4 mm Hg, 24 h at room temperature) and
performed on a Fisons EA 1108 CHNS-O analyser.
5.2. N-[5-(3-Hydroxy-5-nitro-1H-indazol-1-yl)-3-
oxapentyl]thiomorpholine (13)
A mixture of 8 (1.03 g, 3.1 mmol) and thiomorpholine (1.03 mL,
6.2 mmol) in acetonitrile was heated at reflux for 32 h. The obtained
suspension was allowed to stand at 4 ꢀC overnight and then the
crystallized tertiary amine was collected by filtration, washed with
cold ethanol and air dried, 1.04 g (95%). 1H NMR (400 MHz, DMSO-
d6)
d
ppm: 2.34 (2H, t, J ¼ 5.6 Hz), 2.44 (8H, m), 3.30 (1H, br), 3.41
(2H, t, J ¼ 5.6 Hz), 3.74 (2H, t, J ¼ 5.0 Hz), 4.38 (2H, t, J ¼ 5.0 Hz), 7.64
(1H, d, J ¼ 9.6 Hz), 8.14 (1H, dd, J ¼ 2.0, 9.6 Hz), 8.65 (1H, d,
J ¼ 2.0 Hz); 13C NMR (100 MHz, DMSO-d6)
d ppm: 27.90, 49.09,
55.52, 58.55, 69.25, 69.46, 111.27, 112.34, 119.29, 122.22, 140.64,
143.68, 157.37. MS (EI): m/z (%) 352 (Mþ, 6), 278 (34), 149 (100), 116
(59), 91 (34), 69 (47), 57 (54). Anal. (C15H20N4O4S) C, 51.12; H, 5.72;
N, 15.90; S, 9.10.
5.3. General method for the preparation of 14–16
A mixture of the corresponding bromide (10, 11 or 12, 1 equiv),
the corresponding amine (thiomorpholine or butylamine, 2 equiv)
and potassium carbonate (2 equiv) was heated at reflux for 21 h (for
14) or 14 h (for 15 and 16). After that, the mixture was evaporated to
dryness and treated with aqueous potassium carbonate (5%, 30 mL)
and dichloromethane (3 ꢄ 30 mL). The combined organic layers
were dried (MgSO4) and evaporated to dryness to obtain the
desired tertiary amines as the corresponding free bases.
5.3.1. N-[5-(3-Methoxy-5-nitro-1H-indazol-1-
yl)pentyl]butylamine (14)
4. Conclusion
Yield: 96%. 1H NMR (400 MHz, CDCl3)
d ppm: 0.93 (3H, t,
J ¼ 7.4 Hz), 1.40 (4H, m), 1.94 (6H, m), 2.93 (4H, m), 4.11 (3H, s), 4.21
(2H, t, J ¼ 6.9 Hz), 7.35 (1H, d, J ¼ 9.3 Hz), 8.23 (1H, dd, J ¼ 2.1,
9.3 Hz), 8.62 (1H, d, J ¼ 2.1 Hz), 9.38 (1H, br); 13C NMR (100 MHz,
The results presented above indicate that 5-indazole system
constitutes a start point for further drug development. The mech-
anism of action proves to be related to the production of reduced
species of the nitro moiety similar to that observed with Bnz. The
results provide supporting evidence to stimulate further in vivo
studies of the new compounds in appropriate animal models of
Chagas’ disease.
CDCl3)
d ppm: 13.85, 20.48, 24.42, 25.57, 28.17, 29.12, 47.62, 47.84,
48.61, 56.95,109.07,112.13,119.08,123.04,141.24,143.14,158.73. MS
(EI): m/z (%) 334 (Mþ, 1), 319 (44), 246 (46), 206 (43), 160 (22), 98
(50), 86 (100). Anal. (C17H26N4O3) C, 61.06; H, 7.84; N, 16.75.
5.3.2. N-[5-(3-Benzyloxy-5-nitro-1H-indazol-1-yl)-3-
oxapentyl]thiomorpholine (15)
5. Experimental
Yield: 90%. 1H NMR (400 MHz, CDCl3)
d ppm: 2.49 (2H, t,
5.1. Chemistry
J ¼ 5.6 Hz), 2.60 (5H, m), 3.49 (2H, t, J ¼ 5.6 Hz), 3.87 (2H, t,
J ¼ 5.2 Hz), 4.40 (2H, t, J ¼ 5.2 Hz), 5.46 (2H, s), 7.43 (6H, m), 8.24
(1H, dd, J ¼ 2.0, 9.2 Hz), 8.69 (1H, d, J ¼ 2.0 Hz); 13C NMR (100 MHz,
Compounds 1–12 and 26 were prepared according to literature
procedures [7a,12,13]. All 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. 1H NMR
(300 or 400 MHz) and 13C NMR (75 or 100 MHz) spectra were
recorded on a Varian Unity 300, Varian Inova 400 or Bruker DPX-
400 spectrometers. The chemical shifts are reported in parts per
CDCl3) d ppm: 28.03, 49.68, 55.55, 58.78, 69.41, 69.85, 71.50, 109.78,
112.00, 118.87, 122.76, 128.54, 128.77, 128.98, 136.61, 141.38, 141.40,
144.00, 158.08. MS (EI): m/z (%) 442 (Mþ, 1), 351 (7), 129 (22), 116
(100), 91 (37). Anal. (C22H26N4O4S) C, 59.71; H, 5.92; N, 12.66; S,
7.25.
5.3.3. N-[5-(3-Benzyloxy-5-nitro-1H-indazol-1-
yl)pentyl]butylamine (16)
million from TMS (d scale). J values are given in hertz. The assign-
ments have been performed by means of different standard
homonuclear and heteronuclear correlation experiments (ROESY,
HMQC and HMBC). Electron impact (EI) mass spectra were obtained
at 70 eV on a Shimadzu GC–MS QP 1100 EX instrument. IR spectra
Yield: 95%. 1H NMR (400 MHz, CDCl3)
d ppm: 0.92 (3H, t,
J ¼ 7.4 Hz), 1.39 (4H, m), 1.86 (6H, m), 2.89 (4H, m), 4.22 (2H, t,
J ¼ 6.9 Hz), 5.44 (2H, s), 7.43 (6H, m), 8.22 (1H, d, J ¼ 8.4 Hz), 8.67
(1H, d, J ¼ 2.1 Hz), 9.38 (1H, br); 13C NMR (100 MHz, CDCl3)
d ppm:

J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
1551
13.88, 20.48, 24.22, 25.60, 28.22, 29.16, 47.61, 47.84, 48.64, 71.48,
109.08, 112.32, 119.16, 122.98, 128.51, 128.74, 128.95, 136.63, 141.31,
143.09, 157.97. MS (EI): m/z (%) 410 (Mþ, 1), 367 (12), 246 (52), 140
(21), 91 (100), 86 (39). Anal. (C23H30N4O3) C, 67.29; H, 7.37; N, 13.65.
28.88, 28.94, 43.45, 49.29, 108.83, 115.00, 122.40, 127.16, 142.00,
144.94, 159.00. MS (EI): m/z (%) 247 (Mþ, 80), 219 (10), 177 (10).
Anal. (C12H13N3O3) C, 58.29; H, 5.30; N, 16.99.
5.6. N,N-Dimethyl-5-(3-benzyloxy-1H-indazol-1-yl)pentylamine
(22)
5.4. General method for the preparation of 17 and 18
A mixture of the corresponding amine (5 or 6, 1 equiv) and m-
chloroperbenzoic acid (4 equiv) in dichloromethane (50 mL/mmol
of amine) was heated at reflux for 20 h. The mixture was then
evaporated to dryness and treated with aqueous potassium
carbonate (5%, 30 mL) and dichloromethane (3 ꢄ 30 mL). The
combined organic layers were dried (MgSO4) and evaporated to
dryness. The desired compound was purified by column chroma-
tography (SiO2, CH2Cl2/MeOH (95:5)).
3-Benzyloxy-1-(5-bromopentyl)-1H-indazole (27). A mixture of
26 (1 equiv), 1,5-dibromopentane (5 equiv), potassium carbonate
(2 equiv) in acetone (10 mL/mmol of 26) as solvent was heated at
reflux for 24 h. After that acetone was evaporated in vacuo and the
residue treated with water, brine and chloroform (3 ꢄ 30 mL). The
combined organic layers were evaporated and added to chro-
matographic column (SiO2, CHCl3/MeOH (95:5)) to obtain the
intermediate 27 that was used in the next reaction. N,N-Dimethyl-5-
(3-benzyloxy-1H-indazole)pentylamine (22).
A
mixture of
5.4.1. N-[5-(3-Benzyloxy-5-nitro-1H-indazol-1-yl)-3-
oxapentyl]piperidine N-oxide (17)
compound 27 (1 equiv) and dimethylamine (5.6 M in ethanol)
(2.2 equiv) in ethanol (30 mL/mmol of 27) was stirred at room
temperature for 48 h and then evaporated to dryness. The residue
was treated with aqueous sodium hydroxide (0.25 N) and extracted
with chloroform (4 ꢄ 25 mL). The organic layer was dried with
MgSO4 and evaporated in vacuo. 1H NMR (300 MHz, DMSO-d6)
Yield: 72%. 1H NMR (400 MHz, CDCl3)
d ppm: 1.17 (1H, m), 1.35
(2H, m),1.57 (1H, m), 2.07 (2H, m), 2.77 (2H, t, J ¼ 10.3 Hz), 3.00 (2H,
t, J ¼ 11.2 Hz), 3.18 (2H, m), 3.84 (2H, m), 3.97 (2H, m), 4.38 (2H, m),
5.43 (2H, s), 7.29 (1H, d, J ¼ 8.8 Hz), 7.39 (3H, m), 7.52 (2H, m), 8.19
(1H, d, J ¼ 8.8 Hz), 8.64 (1H, s); 13C NMR (100 MHz, CDCl3)
d
ppm:
d
ppm: 1.18 (2H, q, J ¼ 7.5 Hz), 1.41 (2H, q, J ¼ 7.5 Hz), 1.75 (2H, q,
21.27, 22.27, 49.26, 65.06, 66.64, 69.52, 69.80, 71.75, 109.51, 112.37,
118.88, 122.69, 128.52, 128.72, 128.97, 136.52, 141.27, 143.95, 158.09.
MS (EI): m/z (%) 313 (Mþ ꢁ 127, 4), 269 (15), 98 (64), 91 (100). IR
(KBr, cmꢁ1): 2941, 1614, 1540, 1484, 1329, 1199, 1143, 960. Anal.
(C23H28N4O5) C, 62.71; H, 6.41; N, 12.72.
J ¼ 7.5 Hz), 2.22 (6H, s), 2.31 (2H, t, J ¼ 7.2 Hz), 4.19 (2H, t, J ¼ 6.9 Hz),
5.37 (2H, s), 7.03 (2H, t, J ¼ 7.8 Hz), 7.39 (4H, m), 7.49 (2H, m), 7.59
(1H, d, J ¼ 8.1 Hz); 13C NMR (100 MHz, CDCl3)
d ppm: 24.18, 26.02,
29.29, 44.62, 47.89, 58.71, 70.51, 109.68, 111.74, 119.52, 119.80,
127.69, 127.85, 128.10, 128.52, 137.34, 141.51, 155.03. Anal.
(C21H27N3O) C, 74.74; H, 8.06; N, 12.45.
5.4.2. N,N-Dimethyl-5-(3-benzyloxy-5-nitro-1H-indazol-1-yl)-3-
oxapentylamine N-oxide (18)
5.7. Biology
Yield: 40%. 1H NMR (400 MHz, CDCl3)
d ppm: 2.98 (3H, s), 3.44
(2H, t, J ¼ 5.1 Hz), 3.87 (4H, m), 4.37 (2H, t, J ¼ 5.0 Hz), 5.40 (2H, s),
7.28 (1H, d, J ¼ 8.2 Hz), 7.37 (3H, m), 7.50 (2H, m), 8.17 (1H, dd,
J ¼ 1.6, 8.2 Hz), 8.60 (1H, d, J ¼ 1.6 Hz); 13C NMR (100 MHz, CDCl3)
5.7.1. Epimastigote culture and growth inhibition assays
T. cruzi epimastigotes, CL Brener clone, were grown at 28 ꢀC in
an axenic medium (BHI-tryptose) as previously described, supple-
mented with 5% fetal bovine serum (FBS) [7,19,28–32]. Cells from
a 10-day old culture (stationary phase) were inoculated into 50 mL
of fresh culture medium to give an initial concentration of 1 ꢄ106
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 indicated amount of the drug from
a stock solution in DMSO. The final concentration of DMSO in the
culture media never exceeded 0.4% and the control was run in the
presence of 0.4% DMSO and in the absence of any drug. No effect on
epimastigotes’ growth was observed in the presence of up to 1%
DMSO in the culture media. The percentage of inhibition was
calculated as follows: % ¼ {1 ꢁ [(Ap ꢁ A0p)/(Ac ꢁ A0c)]} ꢄ 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 deter-
mine IC50 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 IC50
value was taken as the concentration of drug needed to reduce the
absorbance ratio to 50%.
d
ppm: 46.01, 49.65, 59.03, 59.49, 65.43, 69.62, 69.81, 71.46, 109.91,
112.41, 118.85, 122.79, 128.52, 128.72, 128.93, 136.53, 141.26, 143.86,
158.00. MS (EI): m/z (%) 313 (Mþ ꢁ 87, 3), 91 (82), 71 (30), 58 (100),
44 (11). IR (KBr, cmꢁ1): 2850, 1617, 1540, 1516, 1377, 1329, 1200,
1140, 952, 706. Anal. (C20H24N4O5) C, 59.99; H, 6.04; N, 13.99.
5.5. General method for the preparation of 19 and 20
A mixture of the corresponding bromide (8 or 9, 1 equiv) and
potassium carbonate (1 equiv) in acetonitrile (10 mL/mmol of
bromide) was heated at reflux for 28 h. Then, the mixture was
evaporated to dryness and treated with aqueous potassium
carbonate (5%, 30 mL) and dichloromethane (3 ꢄ 30 mL). The
combined organic layers were dried (MgSO4) and evaporated to
dryness to obtain the desired compound.
5.5.1. 9-Nitro-1,2,4,5-tetrahydro-[1,4,5]oxadiazepino[4,5-
a]indazole-11-one (19)
Yield: 55%. 1H NMR (400 MHz, CDCl3)
d ppm: 4.00 (4H, t,
J ¼ 4.2 Hz), 4.24 (2H, m), 4.38 (2H, m), 7.12 (1H, d, J ¼ 9.1 Hz), 8.40
(1H, dd, J ¼ 2.1, 9.1 Hz), 8.81 (1H, d, J ¼ 2.1 Hz); 13C NMR (100 MHz,
CDCl3)
d ppm: 46.66, 52.44, 70.27, 70.60, 109.19, 115.67, 122.40,
127.63, 135.93, 146.18, 159.28. MS (EI): m/z (%) 249 (Mþ, 100), 218
(20), 177 (15), 149 (12), 131 (14), 103 (11). Anal. (C11H11N3O4) C,
53.01; H, 4.45; N, 16.86.
5.7.2. Trypomastigotes and viability assay
VERO cells were infected with SN-3 strain of T. cruzi metacyclic
trypomastigotes from 15 days old epimastigote cultures. Subse-
quently, the trypomastigotes harvested from this culture were used
to reinfect further VERO cell cultures at 1 ꢄ106 parasites per 25 cm2
density. VERO cell cultures infected with trypomastigotes were
incubated at 37 ꢀC in humidified air and 5% CO2 for 5–7 days. After
that time, the culture medium was collected, centrifuged at 3000g
5.5.2. 2-Nitro-7,8,9,10-tetrahydro-[1,2]diazepino[1,2-a]indazol-
12(6H)-one (20)
Yield: 60%. 1H NMR (400 MHz, CDCl3)
d ppm: 1.89 (6H, m), 4.13
(2H, m), 4.21 (2H, m), 7.12 (1H, d, J ¼ 9.2 Hz), 8.35 (1H, dd, J ¼ 2.1,
9.2 Hz), 8.81 (1H, d, J ¼ 2.1 Hz); 13C NMR (100 MHz, CDCl3)
d ppm:

1552
J. Rodr´ıguez et al. / European Journal of Medicinal Chemistry 44 (2009) 1545–1553
for 5 min, and the trypomastigote-containing pellet was resus-
pended in RPMI supplemented with fetal bovine serum 5% and
penicillin–streptomycin at a final density of 1 ꢄ107 parasites/mL.
210 ꢄ 106 trypomastigotes are equivalent to 1 mg of protein or
12 mg of wet weight.
Viability assays were performed using the MTT reduction
method as described previously [34]. 1 ꢄ107 trypomastigotes were
incubated in FBS-RPMI culture medium at 37 ꢀC for 24 h with or
without the studied compounds. An aliquot of the parasite
suspension was extracted and incubated in a 96-flat bottomed well
plate and MTT was added at 0.5 mg/mL final concentration, incu-
bated at 28 ꢀC for 4 h and then made soluble with 10% SDS–0.1 mM
HCl and incubated overnight. Formazan formation was measured at
570 nm with reference wavelength at 690 nm in a multiwell reader
(Labsystems MultoskanMS, Finland). Untreated parasites were used
as negative controls (100% of viability).
crystallographic data, are given as Supporting information. Crys-
tallographic data (excluding structure factors) for the structure in
this paper have been deposited at the Cambridge Crystallographic
Data Centre as supplementary publication number CCDC-683743.
Copies of the data can be obtained, free of charge, on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: 144-(0)1223-
336033 or e-mail: deposit@ccdc.cam.ac.uk].
Acknowledgements
Financial support from PEDECIBA (Uruguay), CICYT (Spain,
project SAF 2006-04698) and Collaborative Projects FONDECYT
´
1071068/7070282, Consejo Superior de Investigaciones Cientıficas
(Spain) 2006CL0035 CSIC 16/07-08, MECESUP UMC-0204 and
CONICET (Argentina) is acknowledged. The authors thank RTPD
network and MECESUP UCH-0408 for the scholarship to J.R. and
´
PEDECIBA-QUIMICA for a scholarship to A.G. O.E.P. is a research
5.8. Cyclic voltammetry
fellow of CONICET. Proyecto Anillo ACT 29 CONICYT/PBCT; Proyecto
Bicentenario Red 07, FONDECYT 1061072.
In protic media, the solutions were prepared starting from
a stock solution 0.1 M of sample, 5-NI, in DMSO. The final solution
was prepared through corresponding dilution to obtain a sample
with final concentration, on the voltammetric cell, of 1.0 mM.
Experiments were run in buffer Britton–Robinson/EtOH (70:30)
and 0.1 M of KCl, at different pH, which were used for the dilutions.
The pH was previously adjusted adding small amounts of concen-
trated NaOH or HCl, depending on the desired final pH. CV was
carried out using a Metrohm 693VA instrument with a 694VA
Stand convertor and a 693VA Processor, under a nitrogen atmo-
sphere at room temperature, using a three-electrode cell. A hanging
mercury drop electrode was used as the working electrode, a plat-
inum wire as the auxiliary electrode, and saturated calomel (SC) as
the reference electrode.
Appendix. Supporting information
Crystal data and structure solution methods and refinement
results for derivative 13 (Table S1), listings of atomic fractional
coordinates and equivalent isotropic parameters (Table S2), full
intramolecular bond distances and angles (Table S3), anisotropic
displacement parameters (Table S4) and hydrogen atoms’ positions
and isotropic displacement parameters (Table S5) are provided.
Supplementary data associated with this article can be found in the
online version, at doi:10.1016/j.ejmech.2008.07.018.
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b ¼ 10.037(2),
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a
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