New potent 5-nitroindazole derivatives as inhibitors of Trypanosoma cruzi growth: Synthesis, biological evaluation, and mechanism of action studies

Article abstract of DOI:10.1016/j.bmc.2009.10.030

New 5-nitroindazole derivatives were developed and their antichagasic properties studied. Eight compounds (14-18, 20, 26 and 28) displayed remarkable in vitro activities against Trypanosoma cruzi (T. cruzi). Its unspecific cytotoxicity against macrophages

Full text of DOI:10.1016/j.bmc.2009.10.030

Bioorganic & Medicinal Chemistry 17 (2009) 8186–8196
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New potent 5-nitroindazole derivatives as inhibitors of Trypanosoma cruzi
growth: Synthesis, biological evaluation, and mechanism of action studies
c
a,
c
Jorge Rodríguez a,b, Vicente J. Arán f, , Lucía Boiani , Claudio Olea-Azar , María Laura Lavaggi ,
*
*
Mercedes González c, Hugo Cerecetto c, Juan Diego Maya d, Catalina Carrasco-Pozo e, Hernán Speisky Cosoy e
a Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile
b Departamento de Química, Facultad de Ciencias Básicas, Universidad Metropolitana de Ciencias de la Educación, Chile
c Laboratorio de Química Orgánica, Facultad de Ciencias/Facultad de Química, Universidad de la Republica, Uruguay
d Departamento de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Chile
e Unidad de Micronutrientes, Instituto de la Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile
f Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
New 5-nitroindazole derivatives were developed and their antichagasic properties studied. Eight com-
pounds (14–18, 20, 26 and 28) displayed remarkable in vitro activities against Trypanosoma cruzi
(T. cruzi). Its unspecific cytotoxicity against macrophages was evaluated being not toxic at a concentra-
tion at least twice that of T. cruzi IC50, for some derivatives. The electrochemical studies, parasite respi-
ration studies and ESR experiment showed that 5-nitroindazole derivatives not be able to yield a
redox cycling with molecular oxygen such as occurs with nifurtimox (Nfx). The study on the mechanism
of action proves to be related to the production of reduced species of the nitro moiety similar to that
observed with benznidazole.
Received 28 July 2009
Revised 13 October 2009
Accepted 15 October 2009
Available online 20 October 2009
Keywords:
5-Nitroindazole
Trypanosoma cruzi
Mode of action
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ferent.2,3 In fact, Nfx acts via the reduction of the nitro group to
unstable nitroanion radical, which react to produce highly toxic, re-
Chagas disease (American trypanosomiasis) is an insidious,
potentially fatal parasitic disease that is widespread in Latin Amer-
ica affecting 10–14 million or more. This disease is caused by the fla-
gellate Trypanosoma cruzi (T. cruzi). Chagas disease is cause for
epidemiologic concern not only in Latin America but also potentially
for the United States, Canada, Spain, Italy, Israel, and Australia, due
the high rate immigration of them.1 In fact, the development of
new compounds is a clear need for a safe and effective drug for the
treatment of chronic Chagas disease. While generally successful
when used in acute cases, the current standard therapy benznidaz-
ole (RochaganÒ, RadanilÒ, Roche; http://www.roche.com/) or nifur-
timox (Nfx, LampitÒ, Bayer; http://www.bayer.com/) is, at best, only
minimally effective in chronic Chagas disease and is associated with
significant side effects.1 Both compounds have exhibited significant
activity in the acute phase, with up to 80% parasitological cures in
treated patients. However, in the chronic phase, less than 20% of
treated patients are parasitologically cured. These agents function
as prodrugs and must undergo enzyme-mediated activation within
the pathogen to have cytotoxic effects, reactionswhich are catalyzed
by nitroreductases (NTRs). It is believed that they act through a bio-
reduction of the nitro group, but their mechanisms of action are dif-
duced oxygen metabolites (superoxide anion, hydrogen peroxide
and hydroxyl radical).4 Benznidazole, acts via reductive stress and
involves covalent modification of macromolecules by nitroreduc-
tion intermediates.4
Despite their toxicity, nitroaromatic compounds are generally
very potent against T. cruzi and have been considered as important
hits for molecular modifications.5,6 Recently, we have described
the synthesis of a series of 5-nitroindazole derivatives (5-NI) and
also their antiparasitic activity against T. cruzi, showing the
remarkable anti-epimastigote and anti-trypomastigotes properties
of some of them.7–9 However, some of these 5-NI did not show sig-
nificant unspecific cytotoxicity against macrophages.7 Addition-
ally, we have also demonstrated the effectiveness of some 5-
nitroindazoles in an in vivo acute model of Chagas disease.9
On the other hand, previous results showed that N-oxidation of
x
-tertiary amino moiety and the intramolecular cyclization of side
chain, at position 1 of indazole ring, yield completely inactive com-
pounds; affecting drastically the in vitro activity of the substitu-
ents at positions 3 and 5 of indazolic ring wherein the 3-hydroxy
derivatives were completely inactive.8 Besides, the 5-nitro moiety
of indazole ring seems to be essential for the in vitro trypanocidal
activity. Furthermore, ESR experiments employing T. cruzi micro-
somal fraction showed that 5-NI suffer bio-reduction without reac-
tive oxygen species generation.8
* Corresponding authors.
E-mail address: colea@uchile.cl (C. Olea-Azar).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.030

J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
8187
OCH3
series of derivatives was prepared using 3-alkoxy-5-nitroindazoles
(7 and 8, Scheme 2) as common starting materials. These com-
pounds were prepared following the pathways previously reported
by our research group (see Scheme 2).10
Treatment of 3-methoxy-5-nitroindazole 7 with 1,3-dibromo-
propane and treatment of 3-benzyloxy-5-nitroindazole 8 with
1,5-dibromopentane or 1,6-dibromohexane afforded the respective
OBn
Et
Me
Me
R2
N
N
N
O2N
Et
i-Pr
N
3-alkoxy-1-(x-bromoalkyl)-5-nitroindazoles 9–11 (Scheme 3), in
N
O
moderate to good yield; some 1,10-polymethylenebis(3-alkoxy-5-
nitroindazole) derivatives also produced in the alkylation reactions
were easily separated by chromatography. Using bromides 9–11 as
starting materials, the reaction with dimethylamine, pyrrolidine,
piperidine, homopiperidine and tetrahydroisoquinoline afforded
in each case the tertiary amine, 12–18, in good yields (see Scheme
3). On the other hand, direct treatment of 3-alkoxyindazoles 7 and
8 with the required 2-(dialkylamino)ethyl chlorides, afforded ter-
tiary amines 19–28 in good yield (see Scheme 3).
N
i-Pr
R1
[CH2]x
N
N
N
N
x : 2; 3; 5; 6
Scheme 1.
2.2. In vitro trypanocidal activity
In accordance with those findings and considering both the bio-
logical significance of the nitro group and the substituent at posi-
tion 3 of indazole ring, we have devised now a new series of 5-NI
(Scheme 1). They have been functionalized with different length
of side chain at the position 1, two different 3-alkoxy substituent
and differences in the volume of the hydrophilic amino-substitu-
ents at the lateral side chain. The performed structural modifica-
tions together with their effect on anti-T. cruzi activities,
respiration inhibition, redox potential, redox cycling properties
and ESR studies allow us to extract some structural requirements
for adequate trypanocidal of future designed agents.
In order to study the in vitro trypanocidal activity we used the
noninfective epimastigote forms, as in previous studies with other
nitroheterocyclics which have also proved to be efficient against
the trypomastigote and amastigote forms.6 We reported recently
that the 5-NI derivatives showed to be more effective in trypomas-
tigote than in epimastigote forms.8 Consequently, epimastigote
form is a suitable model for studying the mode of action of the
compounds employed in the present study.
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 the IC50 of the 5-NI. The most actives of
the new 5-NI were 14–18, 20, 26 and 28 with IC50 value in the
same order as that of the reference drug (Nfx) being the best deriv-
atives compounds 15, 17 and 20. Besides, these results confirm
that substituent at positions 3 of indazole ring affects drastically
the in vitro activity, such as we reported previously.8 3-OBn com-
pounds being always more active than the corresponding 3-OMe
derivatives. Moreover, the effect on the in vitro activity of the
tested 5-NI derivatives seems to be also related with other addi-
tional factors. In this sense, length of the side chain is a factor to
be considered; trypanocidal activity increases with the increase
2. Results and discussion
2.1. Synthesis
The modifications that were done on the 5-nitroindazole struc-
ture are shown in Scheme 1: variations in the length of side chain,
inclusion of a hydrophilic amino substituent (morpholino moiety)
and lipophilic amino-substituents with different volumes (dimeth-
ylamino, diethylamino, diisopropylamino, pyrrolidino, piperidino,
homopiperidino and tetrahydroisoquinolinyl moieties). The whole
OH
O2N
COOH
O2N
O2N
COOH
HCl
NH2NH2
N
+
Water
reflux
Ethanol
reflux
NHNH2
Cl
N
H
3
2
1
Pyridine
ClCO2Et
OH
OR1
OR1
N
O2N
O2N
BnBr o
O2N
MeI/Cs2CO3
N
N
N
N
Acetone
reflux
KOH/Ethanol
N
H
COOEt
COOEt
4
R1
Compound
R1 Compound
7
8
Me
Bn
5
6
Me
Bn
Scheme 2.

8188
J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
R1
Compound
R1
x
Compound
7
8
3
5
6
9
Me
Bn
Me
Bn
Bn
10
11
OR1
OR1
O2N
O2N
/K CO
Br[CH2]xBr
2
3
N
N
N
Acetone reflux
N
H
[CH2]xBr
2-butanone
reflux
Amine/EtOH
2
Cl[CH ]
2 2R /K2CO3
reflux
for 19-28
for 12-18
OR1
O2N
N
N
[CH2]x R2
R1
Me
Me
Bn
x
R2
Compound
3
3
5
dimethylamino
piperidino
1,2,3,4-
12
13
14
tetrahydroisoquinolyl
Bn
Bn
Bn
Bn
Me
Bn
Me
Bn
Me
Bn
Me
Bn
Me
Bn
5
5
6
6
2
2
2
2
2
2
2
2
2
2
pyrrolidino
15
16
17
18
19
20
21
22
23
24
25
26
27
28
homopiperidino
piperidino
dimethylamino
piperidino
piperidino
dimethylamino
dimethylamino
morpholino
morpholino
diethylamino
diethylamino
diisopropylamino
diisopropylamino
Scheme 3.
of the length chains (compare activity of the pairs 10 and 11, 12
and 21, and 18 and 22). In addition, the amino moiety presence
at the end of the side chain tends to improve the activity (compare
10 with 14, 15 and 16; 11 with 17 and 18). However, hydrophilic
thiomorpholino8 and morpholino moieties do not improve the
activity in relation to that of piperidino derivatives (compare activ-
ities of compounds 20 and 24). Besides the hydrophilic differences
another explanation could be depicted, the electron-withdrawing
effect of the oxygen on the morpholine system tends to decrease
the electron density at the nitrogen making it less basic. If we com-
pare the activity and pKa (calculated using the ChemAxon’s11 pre-
diction plugins) of amino moieties of derivatives 20 (piperidine
pKa = 8.63) and compound 24 (morpholine pKa = 6.46), it is ob-
served that piperidine remains protonated up to physiologic pH
and its activity is at least threefold-higher than compound 24. Fur-
thermore, bulky and branch amino groups tend to improve the
activity (compare activities of derivatives 22, 26 and 28).
In order to check the specific antichagasic activity, unspecific
cytotoxic activity against macrophages was also studied. A viability
test such as MTT reduction was carried out using 25 lM as com-
pounds concentrations. Results are exhibit in Table 1, being unspe-
cific activity expressed as cytotoxicity percentage (% C) compounds

J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
8189
Table 1
Table 2
5-Nitroindazoles in vitro anti-T. cruzi activity (epimastigote form) and unspecific
5-Nitroindazole derivatives electrochemical potential and dimerization ability
cytotoxic activity (% C) against macrophages
Compound
ꢀEPc/mV
k2/(Mꢀ1 sꢀ1
)
E17/mV (NHE)
Compound
aIC50
b%C
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1273
1266
1212
1238
1258
1232
1274
1162
1155
1200
1182
1216
1214
1275
1233
1205
1227
930
178
581
575
528
551
568
546
582
485
479
518
503
532
530
582
546
522
31.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Nfx
67.8
20.7
19.3
>25.0
>>25.0
12.9
7.5
10.5
7.4
9.4
>>25.0
8.4
>>25.0
ꢁ25.0
>>25.0
>>25.0
>>25.0
11.3
>>25.0
9.2
3.4
—
—
—
29
0
15
25
25
33
40
10
27
15
12
0
167
25.1
281
40.3
248
11.1
226
13.1
225
22.0
209
24.1
447
44.1
444
28.7
419
26.4
174
11.4
444
28.7
322
41.3
229
28.3
383
39.5
218
56.5
0
0
30
15
0
28
250
541
45.2
aNfx
486
286(286)
362(380)
484(486)
36
aBnz
1019
1160
—
—
aMetronidazole
a
Refs. 12,13.
40
a
IC50 = concentration (
% C = cytotoxicity percentages, using 25
l
M) that inhibits 50% of T. cruzi growth (CL-Brener clone).
b
l
M as compounds concentrations.
tory with the results obtained in the trypanocidal activity (see
Table 1), in the sense that the substituent at positions 3 of indazole
ring affects drastically the in vitro activity. However, if we consider
the energy needed to reduce the nitro group to the nitro anion rad-
ical and the stabilities of the them, the electrochemical results
indicate that these properties are not affected with different sub-
stituent in position 3-alcoxy.
13, 23–25, and 28 are the least toxic studied 5-NI. Interestedly,
derivatives 14–17, 20, 26 and 28 showed cytotoxicity against mac-
rophages lower than the 40%, the values for the anti-T. cruzi refer-
ence compound (Nfx), being this concentration around twice their
epimastigote IC50 values.
2.3. Electrochemical studies
2.4. Effect on the parasitic respiration
The reduction potential in aprotic media for this family of 5-NI
was studied using DMF as aprotic solvent. The data in aprotic mili-
eu not only describe the situation in that medium but also permit
to obtain biological significance parameters as the E17 It is consid-
ered as indicative of nitro radical anion formation in vivo.12 In this
regards, a good correlation between cathodic peak potential, in
aprotic medium, with E17 (value obtained by pulse radiolysis) has
been previously described. Consequently, we employed the exper-
imental values, in aprotic medium, to calculate the E17 value by
interpolation. Table 2 sorts these values, where we can observe
that the 5-NI show reduction potentials higher than nifurtimox
and benznidazole. This result indicates that reduction could be less
favorable in the biological milieu. However, this kind of com-
pounds could be biotransformated via enzymatic reduction, be-
cause the reduction potential (E17) is comparable with those
obtained for the well-known enzymatic reducible drug metronida-
zole (Table 2). Additionally, it has previously showed that nitro
compounds with reduction potentials lower than E17 <ꢀ400 mV
or Epc <ꢀ1000 mV (in DMF), as it is the case for metronidazole,
the rate of superoxide formation by microsomal enzymes was
not significant.13 Interestingly, this indicates that 5-NI and benzni-
dazole could not be able to yield a redox cycling with molecular
oxygen such as occurs with Nfx.
In order to evaluate if the 5-NI could induce redox cycling, with
the subsequent production of superoxide radical anion and hydro-
xyl radical, oxygen uptake experiments were performed. Table 3
shows Tulahuen epimastigote oxygen uptake in the presence of
some selected 5-NI. Some of the assayed derivatives, 10 and 11,
did not show inhibition of oxygen uptake with respect to untreated
control cells (Table 3). While that the three derivatives with amino
moieties on the side chain, 18, 20 and 22, inhibited respiration be-
tween 39% and 49% at IC50 equivalent concentrations. This result
indicates that the respiratory chain could be an important target
1280
1260
1240
1220
1200
1180
1160
1140
On the other hand, we calculated the k2 dimerization values
(see Table 2), which indicate that in general the 5-NI radical anions
are more stable than the Nfx radical in aprotic milieu which im-
plies a lower deactivation. In this sense, these compounds are a
promising alternative as an antichagasic from the point of view
of the electrochemical parameters. Figure 1 shows a linear rela-
tionship between Epc and k2 values. This means < that whereas
the 5-NI is more difficult to be reduced, the yielded radical is more
stable. Our electrochemical results in aprotic media are contradic-
150
200
250
300
350
400
450
k2 / M-1s-1
Figure 1. Cathodic peak potential values versus dimerization rate constant values
from the one-electron reduction of some selected 5-NI derivatives (12–20, 24 and
28) in non-aqueous medium.

8190
J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
Table 3
All the compounds with IC50 values around o lower than 25 lM
5-Nitroindazole derivatives effect upon oxygen uptake
were biotransformed via enzymatic reduction in a similar way,
independent of the tripanocide activity of them. These results indi-
cate that the bioreduction mechanism is not the only choice to ex-
plain the tripanocide activity of these compounds. In this regard,
the parasitic respiration showed by the compounds 18, 20 and
22 confirm this. In addition, these results show that 5-NI T. cruzi
growth inhibition induction occurs at concentrations that do not
stimulate hydroxyl radical generation indicating that the trypano-
cidal effect does not depend on the effect of oxygen radicals. In this
regard, the 5-NI could display a mechanism of action similar to that
of benznidazole.18–20
a,b,c
Compound Concentration in the assay (mM) (times of
IC50 concentration)
Oxygen
uptake (%)
10
11
18
20
22
2 (32)
38
100
100
100
100
100
52
59
100
62
0.83 (13)
0.42 (7)
2 (34)
0.83 (14)
0.42 (7)
0.42 (18)
0.208 (9)
0.104 (4)
0.83 (20)
0.42 (10)
0.208 (5)
0.42 (8)
0.208 (4)
61
100
51
2.5.2. Reactivity against dpph and galvinoxyl free radicals
Reactivity of tertiary amines towards free radicals species is a
process of high biological interest,21 the associated mechanism
corresponds to an oxidative N-dealkylation of tertiary amines in-
duced by free radicals species.22 On the other hand, the oxidative
N-dealkylation of amines has been reported as the metabolic
mechanism of some xenobiotics and endogenous compounds.21,23
Consequently, information of the reactivity of 5-NI against 2,2-di-
phenyl-1-picrylhydrazyl (dpph) and galvinoxyl free radicals, two
stable lipophilic chromogenic radicals, could be an important point
in order to consider the N-dealkylation like a possible metabolic
mechanism of 5-NI.
In general, all the studied compounds were able to scavenge
both radicals in a percent lower than the 30% (Fig. 3). However,
derivatives 16 and 18 show a scavenger percent higher than 40%.
Reactivity of these derivatives toward dpph and galvinoxyl free
radicals could involve an N-dealkylation mechanism. Scheme 4
shows that N-dealkylation processes maybe proceed directly via
64
a
Percent rate of oxygen consumption compared with that of control (C) cells in
Tulahuen strain.
b
Values correspond to means of three independent experiments with a SD less
than 5% in all cases.
c
Control respiration was 31.5 nanoatoms of oxygen per min and per mg of
protein.
for this type of compounds. As well they point out that these com-
pounds did not have any effects on redox cycling as previously we
reported for other 5-NI.8,9 This result is in agreement with the high
reduction potentials above described. In addition, the effect on the
parasitic respiration of the tested 5-NI seems to be related with the
type of group at the end of side chain, the two derivatives with a
bromine moiety on the side chain have no effect on the parasite
respiration. However, similar derivatives with an amino group in-
stead a bromine inhibit the parasitic respiration. Furthermore, the
presence of nitro group and length of side chain seems do not have
a significant effect on the parasitic respiration.
a-carbon hydroxylation. The reactivity of the N-methylene C–H
bond of tertiary amines has been previously reported such as a
concerted proton–electron transfer mechanism.22 The results seem
to show that rise in the reactivity is related with large side chain
(derivative 18) and the homopiperidine group (derivative 16). Fur-
thermore, the low reactivity exhibit by the great majority of 5-NI
derivatives indicate that the N-dealkylation induces by free radi-
cals is low. However, these results constitutes a point to be consid-
ers for further drug development.
2.5. ESR spectroscopic studies
2.5.1. ESR evidence for enzymatic reduction
Figure 2 shows that the incubation of 5-NI derivative, 28, with
mammalian cytosolic fraction in presence of NADPH and POBN,
as spin-trapping under aerobic conditions, produces a characteris-
tic spectrum that could be identified as the POBN-CH3 adduct. The
six lines have the following hyperfines aN = 15.8 G and aH = 2.6 G
3. Conclusion
The trypanocidal activity of new 5-nitroindazole derivatives has
been reported, where some derivatives (14–18, 20, 26 and 28)
showed interesting properties with activities in the same magni-
tude order that Nfx. Fortunately, some of more active derivatives
(14–17, 20, 26 and 28) do not show significant unspecific cytotox-
icity against macrophages. Besides, taking into account the role of
amine group volume and length of side chain on tripanocidal activ-
ity, new derivatives bearing a quaternized amino moiety and with
a length of side chain between 5- and 6-CH2 could present better
activities and they will be synthesized and tested to confirm it.
The study on the mechanism of action proves to be related to the
production of reduced species of the nitro moiety similar to that
observed with benznidazole. The results provide supporting evi-
dence to stimulate further in vivo studies of the new compounds
in appropriate animal models of Chagas’ disease.
*
consistent with the trapped methyl radical (marked with in
Fig. 2C). The observation here described indicates that 5-NI could
be reduced by cytosolic fraction to a nitro anion radical and the
reoxidation of which is accompanied by hydroxyl radical forma-
tion.14,15 POBN–CH3 adduct was observed in presence of dimethyl-
sulfoxide (DMSO) as a results of hydroxyl radical reaction with the
solvent.16 In spite of POBN–CH3 being a secondary adduct, we have
chosen to use DMSO in order to estimate radical yield due to the
higher stability of this adduct when compared to those of the
POBN–OH or DMPO oxygen radical adduct.16 When we used DMPO
in similar conditions and when we used acetonitrile instead DMSO
it was not observed these signals (Fig. 2B).
Attempts to demonstrate 5-NI capacity to generate superoxide
or hydroxyl radicals into T. cruzi, under similar conditions to those
used with cytosolic fraction, were unsuccessful. However, the incu-
bation of 5-NI (in acetonitrile) with T. cruzi, in the presence of
NADPH and DMPO, generated an ESR spectrum consistent with
the trapping of the nitroheterocycle radical (see Fig. 2D). In agree-
ment to these results 5-NI did not stimulate oxygen consumption
in T. cruzi epimastigote form (see Table 3). Previous reports have
shown formation of both hydroxyl and superoxide radicals con-
comitantly with a increase of the oxygen consumption.17
4. Experimental
4.1. Chemistry
Compounds 2–8 were prepared according to literature proce-
dures.10,24 All starting materials were commercially available re-

J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
8191
Figure 2. ESR spectra of radical adducts obtained with mammalian cytosolic fraction and T. cruzi microsomes. The ESR spectra were observed 5 min after incubation at 37 °C
with mammalian cytosolic fraction (1 mg protein/mL), NADPH (1 mM), in phosphate buffer (20 mM), pH 7.4 (A) 28 (1 mM in acetonitrile 10 v/v), (C) 28 (2 mM in acetonitrile
10 v/v) and POBN (100 mM). The ESR spectra were observed 15 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 (B) 28 (1 mM in acetonitrile 10 v/v), (D) 14, 18, 22 and 28, (2 mM in acetonitrile 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 scans: 10.
search-grade chemicals and used without further purification. All
solvents were dried and distilled prior to use. 1H NMR (300 MHz)
and 13C NMR (75 MHz) experiments have been performed on a
Bruker AVANCE DRX300 spectrometer equipped with a pulse gra-
dient unit capable of producing magnetic field pulse gradients in
the z-direction of 53.5 G cmꢀ1. The spectra have been acquired in

8192
J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
100
90
80
70
60
50
40
30
20
10
0
dpph
CT 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 23 24 25 26 27 28 Nfx
Compound
Figure 3. 5-Nitroindazole derivatives reactivity against to dpph free radical. (a) The reaction mixture contained 1.0 mM DPPH in the presence or absence of 1.0 mM
concentrations of the compounds under study. ESR signals were recorded 1 min following the start of the reaction. Spectrometer conditions were: microwave frequency,
9.72 GHz; microwave power, 20 mW; modulation amplitude, 0.98 G; receiver gain, 59 db; and sweep time, 20.972 s.
O
R2N
CR'
HO
CHR'
R2N
R2N
CH2R'
O
a
R2N H
R'
HC
+
R2N
CH2R'
OH
CH
CH2
b
O
N H
N
R
R
N
R
CH
Scheme 4. Proposed mechanism of tertiary amine oxidation by dpph or galvinoxyl free radicals.
an inversed probe-head at 298 K in 5 mm tubes. The chemical
shifts are reported in parts per million from TMS (d scale). J values
are given in hertz. The assignments have been performed by means
of different standard homonuclear and heteronuclear correlation
experiments (ROESY, HMQC and HMBC). 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. Melting points are uncorrected and
were determined with a Reichert Galen III hot plate microscope
with a DUAL JTEK Dig—Sense thermocouple thermometer. Elemen-
tal analyses were obtained from vacuum-dried samples (over
phosphorous pentoxide at 3–4 mm Hg, 24 h at room temperature)
and performed on a Fisons EA 1108 CHNS-O analyser.
intermediates 9–11 which were used in the next step without fur-
ther purification.
4.3. General method for the preparation of 12–13, 15–18
These compounds were prepared starting from 3-alkoxy-1-bro-
moalkyl-5-nitroindazole derivatives (9–11, 1 equiv) and the corre-
sponding amine (dimethylamine, piperidine, pyrrolidine and
homopiperidine, 2 equiv) in ethanol. The mixture was heated at re-
flux for 24 h and then evaporated to dryness. The residue was trea-
ted with aqueous potassium carbonate (5%, 30 mL) and extracted
with chloroform (3 ꢂ 30 mL). The combined organic layers were
dried (MgSO4) and evaporated to dryness to yield the desired ter-
tiary amines as the corresponding free bases.
4.2. General method for the preparation of 9–11
4.4. General method for the preparation of 19, 21–23, 25
These compound were prepared starting from 3-alkoxy-5-nitro-
indazole derivatives
7
or
8
(1 equiv), potassium carbonate
These compound were prepared starting from 3-alkoxy-5-nitro-
(2 equiv) and the corresponding dibromoalcane (1,3-dibromopro-
pane, 1,5-dibromopentane or 1,6-dibromohexane, 5 equiv) in ace-
tone. The mixture was heated at reflux for 6 h and then
evaporated to dryness. The residue was treated with water and ex-
tracted with chloroform (3 ꢂ 30 mL). The combined organic layers
were dried (MgSO4) and evaporated, and the residue applied to a
chromatography column (SiO2, CHCl3/MeOH (95:5)) to afford the
indazole derivatives 7 and 8 (1 equiv), potassium carbonate
(5 equiv) and the corresponding 2-(dialkylamino)ethyl chlorides
[2-(dimethylamino)ethyl chloride hydrochloride, 2-(diethyl-
amino)ethyl chloride hydrochloride, 1-(2-chloroethyl)piperidine
hydrochloride and 4-(2-chloroethyl)morpholine hydrochloride,
2 equiv)] in 2-butanone. The mixture was heated at reflux for 24 h
and evaporated to dryness. The residue was then treated with

J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
8193
aqueous potassium carbonate (5%, 30 mL) and extracted with chlo-
roform (3 ꢂ 30 mL). The combined organic layers were dried
(MgSO4) and evaporated to dryness to afford the desired tertiary
amines as the corresponding free bases.
127.08 (tetrahydroisoquinolyl C-2), 127.13 (tetrahydroisoquinolyl
C-3), 128.20 (tetrahydroisoquinolyl, C-4), 128.59 (Ph, C-3 and -5),
128.68 (Ph, C-4), 128.92 (Ph, C-2 and -6), 129.04 (tetrahydroiso-
quinolyl, C-1), 131.83 (tetrahydroisoquinolyl, C-5), 136.76 (Ph, C-
1, tetrahydroisoquinolyl, C-6), 142.94 (C-5), 143.25 (C-7a), 157.17
(C-3). Anal. Calcd for C28H31ClN4O3 (471.6): C, 71.31; H, 6.63; N,
11.88. Found: C, 71.44; H, 6.55; N, 10.99.
4.5. General method for the preparation of 14, 20, 24, 26–28
For compound 14, a mixture of 3-alkoxy-1-bromoalkyl-5-nitro-
indazole derivative (10, 1 equiv) and tetrahydroisoquinoline
(2 equiv) in ethanol. The mixture was heated at reflux for 24 h.
For compounds 20, 24, 26–28, a mixture of 3-alkoxy-5-nitroindaz-
ole derivatives 7 and 8 (1 equiv), potassium carbonate (5 equiv)
and the corresponding 2-(dialkylamino)ethyl chlorides [2-(diethyl-
amino)ethyl chloride hydrochloride, 2-(diisopropylamino)ethyl
chloride hydrochloride, 1-(2-chloroethyl)piperidine hydrochloride
and 4-(2-chloroethyl)morpholine hydrochloride, 2 equiv)] in 2-
butanone. The mixture was heated at reflux for 24 h. In each case,
the mixture was then evaporated to dryness and, after addition of
5% aqueous potassium carbonate (30 mL), extracted with chloro-
form (3 ꢂ 30 mL). The combined organic layers were dried (MgSO4)
and evaporated to dryness. The residue was treated with aqueous
0.5 N hydrochloric acid (50 mL) and the insoluble material was re-
moved by extraction with diethyl ether (3 ꢂ 50 mL). Evaporation of
the acidic layer afforded the desired tertiary amines as the corre-
sponding hydrochlorides.
4.5.4. 3-Benzyloxy-5-nitro-1-(5-pyrrolidinopentyl)-1H-indazole
(15)
Yield: 99%; mp 135–139 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.23 (m, 2H, 30-H), 1.59 (m, 2H, 40-H), 1.85 (m, 6H, 20-H, pyrr-
olidino 3 and 4-H), 2.87 (t, J = 7.8 Hz, 2H, 50-H), 3.02 (broad signal,
4H, pyrrolidino 2- and 5-H), 4.33 (t, J = 6.7 Hz, 2H, 10-H), 5.45 (s, 2H,
Bn–CH2), 7.42 (m, 3H, Ph 3-, 4- and 5-H), 7.56 (m, 2H, Ph 2- and 6-
H), 7.77 (d, J = 9.4 Hz, 1H, 7-H), 8.21 (dd, J = 2.0, 9.4 Hz, 1H, 6-H),
8.55 (d, J = 2.0 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6) d
ppm: 23.01 (pyrrolidino C-3 and -4), 23.67 (C-30), 25.78 (C-40),
29.02 (C-20), 48.32 (C-10), 53.53 (pyrrolidino C-2 and -5), 54.43
(C-50), 70.95 (Bn–CH2), 110.64 (C-7), 111.08 (C-3a), 118.02 (C-4),
122.48 (C-6), 128.48 (Ph, C-3 and -5), 128.56 (Ph, C-4), 128.80
(Ph, C-2 and -6), 136.65 (Ph, C-1), 140.75 (C-5), 142.79 (C-7a),
157.03 (C-3). Anal. Calcd for C23H28N4O3 (408.5): C, 67.63; H,
6.91; N, 13.72. Found: C, 67.72; H, 6.83; N, 13.64.
4.5.5. 1-(5-Azepanylpentyl)-3-benzyloxy-5-nitro-1H-indazole
(16)
4.5.1. 1-(3-Dimethylaminopropyl)-3-methoxy-5-nitro-1H-
indazole (12)
Yield: 98%; mp 79–81 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.25 (m, 2H, 30-H), 1.45 (broad signal, 10H, 40-H and azepanyl 3-,
4-, 5- and 6-H), 1.87 (m, 2H, 20-H), 2.85 (broad signal, 6H, 10-H
and azepanyl, 2- and 7-H), 4.34 (t, J = 6.8 Hz, 2H, 50-H), 5.45 (s,
2H, Bn–CH2), 7.41 (m, 3H, Ph 3-, 4- and 5-H), 7.57 (m, 2H, Ph 2-
and 6-H), 7.75 (d, J = 9.3 Hz, 1H, 7-H), 8.22 (dd, J = 2.1, 9.3 Hz, 1H,
6-H), 8.54 (d, J = 2.1 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6)
d ppm: 23.12 (azepanyl, C-4 and -5), 23.67 (C-30), 24.50 (azepanyl,
C-3 and -6), 25.80 (C-20), 29.21 (C-40), 48.30 (C-50), 53.25 (azepanyl,
C-2 and -7), 54.43 (C-10), 70.95 (Bn–CH2), 110.62 (C-7), 111.09 (C-
3a), 118.01 (C-4), 122.50 (C-6), 128.48 (Ph, C-3 and -5), 128.56 (Ph,
C-4), 128.81 (Ph, C-2 and -6), 136.63 (Ph, C-1), 140.73 (C-5), 142.77
(C-7a), 157.01 (C-3). Anal. Calcd for C25H32N4O3 (436.5): C, 68.78;
H, 7.39; N, 12.83. Found: C, 68.88; H, 7.30; N, 12.96.
Yield: 75%; mp 57–60 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.93 (m, 2H, 20-H), 2.15 (s, 6H, N(CH3)2), 2.21 (t, J = 6.9 Hz, 2H, 30-
H), 4.05 (s, 3H, OCH3), 4.31 (t, J = 6.7 Hz, 2H, 10-H), 7.68 (d,
J = 9.3 Hz, 1H, 7-H), 8.19 (dd, J = 2.0, 9.3 Hz, 1H, 6-H), 8.48 (d,
J = 2.0 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6) d ppm: 27.09
(C-20), 45.15 (NCH3), 46.38 (C-10), 55.89 (C-30), 56.86 (OCH3),
110.55 (C-7), 110.79 (C-3a), 117.86 (C-4), 122.35 (C-6), 140.59 (C-
5), 142.94 (C-7a), 157.86 (C-3). Anal. Calcd for C13H18N4O3 (278.3):
C, 56.10; H, 6.52; N, 20.13. Found: C, 56.28; H, 6.42; N, 20.04.
4.5.2. 3-Methoxy-5-nitro-1-(3-piperidinopropyl)-1H-indazole
(13)
Yield: 99%; mp 119–122 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.44 (m, 6H, piperidino 3-, 4- and 5-H), 1.94 (m, 2H, 20-H), 2.18 (m,
2H, 30-H), 4.06 (s, 3H, OCH3), 4.32 (t, J = 6.5 Hz, 2H, 10-H), 7.70 (d,
J = 9.4 Hz, 1H, 7-H), 8.20 (dd, J = 2.1, 9.4 Hz, 1H, 6-H), 8.51 (d,
J = 1.6 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6) d ppm: 24.45
(piperidino C-4), 25.84 (piperidino C-3 and -5), 26.42 (C-20), 46.55
(C-10), 54.25 (C-30), 55.41 (piperidino C-2 and -6), 56.87 (OCH3),
110.75 (C-7, C-3a), 117.87 (C-4), 122.19 (C-6), 140.55 (C-5), 143.10
(C-7a), 157.89 (C-3). Anal. Calcd for C16H22N4O3 (318.4): C, 60.36;
H, 6.97; N, 17.60. Found: C, 60.28; H, 6.85; N, 17.73.
4.5.6. 3-Benzyloxy-5-nitro-1-(6-piperidinohexyl)-1H-indazole
(17)
Yield: 99%; mp 127–130 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.23 (m, 4H, 40-H, 30-H), 1.45 (m, 4H, piperidino 3- and 5-
H), 1.59 (m, 4H, 50-H, piperidino 4-H), 1.79 (m, 2H, 20-H), 2.58
(broad signal, 2H, 60-H), 2.70 (broad signal, 4H, piperidino 2- and
6-H), 4.31 (t, J = 6.7 Hz, 2H, 10-H), 5.44 (s, 2H, Bn–CH2), 7.41 (m,
3H, Ph 3-, 4- and 5-H), 7.56 (m, 2H, Ph 2- and 6-H), 7.75 (d,
J = 9.5 Hz, 1H, 7-H), 8.20 (dd, J = 1.9, 9.5 Hz, 1H, 6-H), 8.54 (d,
J = 1.9 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6) d ppm: 22.93
(piperidino C-4), 24.20 (piperidino C-3 and -5), 24.78 (C-40),
26.07 (C-30), 26.40 (C-50), 29.28 (C-20), 48.46 (C-10), 53.23 (piperidi-
no C-2 and -6), 57.34 (C-60), 70.93 (Bn–CH2), 110.61 (C-7), 111.04
(C-3a), 117.99 (C-4), 122.42 (C-6), 128.49 (Ph C-3 and -5), 128.52
(Ph C-4), 128.77 (Ph C-2 and -6), 136.67 (Ph C-1), 140.69 (C-5),
142.77 (C-7a), 156.99 (C-3). Anal. Calcd for C25H32N4O3 (436.5):
C, 68.78; H, 7.39; N, 12.83. Found: C, 68.89; H, 7.52; N, 12.70.
4.5.3. 3-Benzyloxy-5-nitro-1-[5-(1,2,3,4-tetrahydroisoquinol-2-
yl)pentyl]-1H-indazol hydrochloride (14ꢃHCl)
Yield: 67%; mp 190–195 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.28 (m, 2H, 30-H), 1.77 (m, 2H, 40-H), 1.87 (m, 2H, 20-H),
3.10 (broad signal, 4H, 50-H, tetrahydroisoquinolyl, 7-H), 3.68
(broad signal, 2H, tetrahydroisoquinolyl, 8-H) 4.36 (t, J = 6.5 Hz,
4H, 10-H, tetrahydroisoquinolyl, 10-H), 5.46 (s, 2H, Bn–CH2), 7.26
(m, 4H, tetrahydroisoquinolyl 1-, 2-, 3- and 4-H), 7.40 (m, 3H, Ph
3-, 4- and 5-H), 7.57 (m, 2H, Ph 2- and 6-H), 7.79 (d, J = 9.4 Hz,
1H, 7-H), 8.23 (dd, J = 2.0, 9.4 Hz, 1H, 6-H), 8.57 (d, J = 2.0 Hz, 1H,
4-H), 9.78 (tetrahydroisoquinolyl NH); 13C NMR (100 MHz,
DMSO-d6) d ppm: 23.61 (C-30, tetrahydroisoquinolyl C-7), 25.44
(C-40), 29.10 (C-20), 48.39 (C-10), 49.41 (tetrahydroisoquinolyl, C-
3), 52.51 (tetrahydroisoquinolyl, C-10), 55.47 (C-50), 71.09 (Bn–
CH2), 110.76 (C-7), 111.22 (C-3a), 118.15 (C-4), 122.62 (C-6),
4.5.7. 3-Benzyloxy-1-(6-dimethylaminohexyl)-5-nitro-1H-
indazole (18)
Yield: 85%: mp 45–48 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.21 (m, 4H, 40-H, 30-H), 1.29 (broad signal, 2H, 50-H), 1.78 (m, 2H,
20-H), 2.07 (s, 6H, N(CH3)2), 2.12 (broad signal, 2H, 60-H), 4.29 (t,
J = 6.7 Hz, 2H, 10-H), 5.44 (s, 2H, Bn–CH2), 7.39 (m, 3H, Ph 3-, 4-

8194
J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
and 5-H), 7.56 (m, 2H, Ph 2- and 6-H), 7.74 (d, J = 9.5 Hz, 1H, 7-H),
8.19 (dd, J = 1.9, 9.5 Hz, 1H, 6-H), 8.53 (d, J = 1.9 Hz, 1H, 4-H); 13C
NMR (100 MHz, DMSO-d6) d ppm: 26.57 (C-30, -40 and -50), 29.23
(C-20), 45.14 (N(CH3)2), 48.43 (C-10), 59.23 (C-60), 70.93 (Bn–CH2),
110.53 (C-7 and -3a), 117.97 (C-4), 122.34 (C-6), 128.37 (Ph C-3
and -5), 128.60 (Ph C-4), 128.84 (Ph C-2 and -6), 136.77 (Ph C-1),
140.73 (C-5), 142.82 (C-7a), 156.99 (C-3). Anal. Calcd for
C22H28N4O3 (396.5): C, 66.64; H, 7.12; N, 14.13. Found: C, 66.50;
H, 7.27; N, 14.25.
4.5.12. 3-Methoxy-1-(2-morpholinoethyl)-5-nitro-1H-indazole
(23)
Yield: 79%; mp 112–114 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
2.41 (m, 4H, morpholino 3- and 5-H), 2.71 (t, J = 6.3 Hz, 2H, 20-H),
3.46 (m, 4H, morpholino 2- and 6-H), 4.05 (s, 3H, OCH3), 4.41 (t,
J = 6.3 Hz, 2H, 10-H), 7.75 (d, J = 9.5 Hz, 1H, 7-H), 8.20 (dd, J = 2.1,
9.5 Hz, 1H, 6-H), 8.51 (d, J = 2.1 Hz, 1H, 4-H); 13C NMR (100 MHz,
DMSO-d6) d ppm: 46.44 (C-10), 53.69 (morpholino C-3 and -5),
56.98 (C-20), 57.49 (OCH3), 66.64 (morpholino C-2 and -6), 110.99
(C-7), 111.04 (C-3a), 117.95 (C-4), 122.38 (C-6), 140.72 (C-5),
143.49 (C-7a), 157.94 (C-3). Anal. Calcd for C14H18N4O4 (306.3): C,
54.89; H, 5.92; N, 18.29. Found: C, 54.77; H, 5.79; N, 18.41.
4.5.8. 3-Methoxy-5-nitro-1-(2-piperidinoethyl)-1H-indazole
(19)
Yield: 79%; mp 78–79 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.46 (m, 6H, piperidino 3-, 4- and 5-H), 2.46 (m, 4H, piperidino 2-
and 6-H), 2.76 (t, J = 6.3 Hz, 2H, 20-H), 4.15 (s, 3H, OCH3), 4.48 (t,
J = 6.3 Hz, 2H, 10-H), 7.83 (d, J = 9.5 Hz, 1H, 7-H), 8.28 (dd, J = 1.0,
9.5 Hz, 1H, 6-H), 8.58 (d, J = 1.0 Hz, 1H, 4-H); 13C NMR (100 MHz,
DMSO-d6) d ppm: 24.31 (piperidino C-4), 26.04 (piperidino C-3
and -5), 46.87 (C-10), 54.52 (piperidino C-2 and -6), 56.95 (C-20),
57.85 (OCH3), 110.93 (C-7), 111.02 (C-3a), 117.89 (C-4), 122.25
(C-6), 140.63 (C-5), 143.46 (C-7a), 157.88 (C-3). Anal. Calcd for
C15H20N4O3 (304.3): C, 59.20; H, 6.62; N, 18.41. Found: C, 59.12;
H, 6.72; N, 18.50.
4.5.13. 3-Benzyloxy-1-(2-morpholinoethyl)-5-nitro-1H-indazol
hydrochloride (24ꢃHCl)
Yield: 95%; mp 71–74 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
3.15 (d, J = 7.8 Hz, 2H, morpholino 3- and 5-Heq), 3.47 (d,
J = 12.0 Hz, 2H, morpholino 3- and 5-Hax), 3.62 (d, J = 7.8 Hz, 2H,
morpholino 2- and 6-Heq), 3.76 (t, J = 11.50 2H, 20-H), 3.95 (d,
J = 12.0 Hz, 2H, morpholino 2- and 6-Heq), 4.84 (t, J = 6.5 Hz, 2H,
10-H), 5.48 (s, 2H, Bn–CH2), 7.43 (m, 3H, Ph 3-, 4- and 5-H), 7.58
(m, 2H, Ph 2- and 6-H), 7.88 (d, J = 9.3 Hz, 1H, 7-H), 8.31 (dd,
J = 2.0, 9.3 Hz, 1H, 6-H), 8.61 (d, J = 2.0 Hz, 1H, 4-H), 10.49 (sa,
13
1H, NH); C NMR (100 MHz, DMSO-d6) d ppm: 43.09 (C-10),
4.5.9. 3-Benzyloxy-5-nitro-1-(2-piperidinoethyl)-1H-indazol
hydrochloride (20ꢃHCl)
51.81 (morpholino C-3 and -5), 54.45 (C-20), 63.67 (morpholino
C-2 and -6), 71.22 (Bn–CH2), 111.04 (C-7), 112.01 (C-3a), 118.11
(C-4), 123.07 (C-6), 128.58 (Ph C-3 and -5), 128.74 (Ph C-4),
128.97 (Ph C-2 and -6), 136.61 (Ph C-1), 141.40 (C-5), 143.27 (C-
7a), 157.64 (C-3). Anal. Calcd for C20H23ClN4O4 (418.9): C, 57.35;
H, 5.53; N, 13.38. Found: C, 57.47; H, 5.46; N, 13.50.
Yield: 86%; mp 196–203 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.77 (m, 6H, piperidino 3-, 4- and 5-H), 2.94 (m, 2H, piperi-
dino 1- and 6-Heq), 3.51 (m, 2H, 20-H, 2H, piperidino 1- and 6-
Hax), 4.83 (t, J = 6.5 Hz, 2H, 10-H), 5.47 (s, 2H, Bn–CH2), 7.40 (m,
4H, 7-H and Ph 3-, 4- and 5-H), 7.57 (m, 2H, Ph 2- and 6-H), 7.89
(d, J = 9.3 Hz, 1H, 7-H), 8.30 (dd, J = 2.1, 9.3 Hz, 1H, 6-H), 8.60 (d,
J = 2.1 Hz, 1H, 4-H), 10.49 (sa, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d ppm: 21.70 (piperidino C-4), 22.81 (piperidino C-3
and -5), 43.29 (C-10), 52.80 (piperidino C-2 and -6), 54.32 (C-20),
71.21 (Bn–CH2), 111.03 (C-7), 111.90 (C-3a), 118.09 (C-4), 123.05
(C-6), 128.57 (Ph C-3 and -5), 128.73 (Ph C-4), 128.97 (Ph C-2
and -6), 136.60 (Ph C-1), 141.38 (C-5), 143.25 (C-7a), 157.62 (C-
3). Anal. Calcd for C21H25ClN4O3 (416.9): C, 60.50; H, 6.04; N,
13.44. Found: C, 60.44; H, 6.14; N, 13.36.
4.5.14. 1-(2-Diethylaminoethyl)-3-methoxy-5-nitro-1H-
indazole (25)
Yield: 81%; mp 35–37 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.46 (t, J = 7.1, 6H, N(CH2CH3)2), 2.41 (q, J = 7.1, 4H, N(CH2CH3)2),
2.77 (t, J = 6.0 Hz, 2H, 20-H), 4.05 (s, 3H, OCH3), 4.32 (t, J = 6.0 Hz,
2H, 10-H), 7.71 (d, J = 9.5 Hz, 1H, 7-H), 8.18 (dd, J = 2.1, 9.5 Hz, 1H,
6-H), 8.49 (d, J = 2.1 Hz, 1H, 4-H); 13C NMR (100 MHz, DMSO-d6)
d
ppm: 12.42 (N(CH2CH3)2), 47.06 (N(CH2CH3)2), 47.73(C-10),
52.07 (C-20), 56.95 (OCH3), 110.83 (C-7), 111.10 (C-3a), 117.91
(C-4), 122.23 (C-6), 140.58 (C-5), 143.70 (C-7a), 157.91 (C-3). Anal.
Calcd for C14H20N4O3 (292.3): C, 57.52; H, 6.90; N, 19.17. Found: C,
57.70; H, 7.05; N, 19.05.
4.5.10. 1-(2-Dimethylaminoethyl)-3-methoxy-5-nitro-1H-
indazole (21)
Yield: 83%; mp 73–76 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
2.26 (s, 6H, N(CH3)2), 2.78 (t, J = 6.4 Hz, 2H, 20-H), 4.16 (s, 3H,
OCH3), 4.49 (t, J = 6.4 Hz, 2H, 10-H), 7.86 (d, J = 9.3 Hz, 1H, 7-H),
8.30 (dd, J = 2.1, 9.3 Hz, 1H, 6-H), 8.61 (d, J = 2.1 Hz, 1H, 4-H); 13C
NMR (100 MHz, DMSO-d6) d ppm: 45.69 (N(CH3)2), 47.06 (C-10),
56.98 (C-20), 58.30 (OCH3), 110.85 (C-7), 110.96 (C-3a), 117.97
(C-4), 122.45 (C-6), 140.68 (C-5), 143.30 (C-7a), 157.88 (C-3). Anal.
Calcd for C12H16N4O3 (264.3): C, 54.54; H, 6.10; N, 21.20. Found: C,
54.61; H, 6.19; N, 21.33.
4.5.15. 3-Benzyloxy-1(2-diethylaminoethyl)-5-nitro-1H-indazol
hydrochloride (26ꢃHCl)
Yield: 91%; mp 176–178 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.20 (t, J = 7.2, 6H, N(CH2CH3)2), 3.16 (t, J = 7.2, 4H,
N(CH2CH3)2), 3.55 (t, J = 5.2 Hz, 2H, 20-H), 4.83 (t, J = 6.5 Hz, 2H,
10-H), 5.48 (s, 2H, Bn–CH2), 7.43 (m, 3H, Ph 3-, 4- and 5-H), 7.58
(m, 2H, Ph 2- and 6-H), 7.93 (d, J = 9.5 Hz, 1H, 7-H), 8.31 (dd,
J = 2.1, 9.3 Hz, 1H, 6-H), 8.60 (d, J = 2.1 Hz, 1H, 4-H), 10.77 (sa,
1H, NH); 13C NMR (100 MHz, DMSO-d6) d ppm: 8.82 (N(CH2CH3)2),
43.40 (C-20), 46.98 (N(CH2CH3)2), 49.36 (C-10), 71.21 (Bn–CH2),
111.08 (C-7), 111.91 (C-3a), 118.08 (C-4), 123.07 (C-6), 128.58
(Ph C-3 and -5), 128.72 (Ph C-4), 128.96 (Ph C-2 and -6), 136.63
(Ph, C-1), 141.38 (C-5), 143.40 (C-7a), 157.64 (C-3). Anal. Calcd
for C20H25ClN4O3 (404.9): C, 59.33; H, 6.22; N, 13.84. Found: C,
59.49; H, 6.07; N, 13.67.
4.5.11. 3-Benzyloxy-1-(2-dimethylaminoethyl)-5-nitro-1H-
indazole (22)
Yield: 93%; mp 93–96 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
2.24 (s, 6H, N(CH3)2), 2.78 (t, J = 6.2 Hz, 2H, 20-H), 4.50 (t, J = 6.2 Hz,
2H, 10-H), 5.56 (s, 2H, Bn–CH2), 7.51 (m, 3H, Ph 3-, 4- and 5-H), 7.67
(m, 2H, Ph 2- and 6-H), 7.87 (d, J = 9.3 Hz, 1H, 7-H), 8.30 (dd, J = 2.1,
9.3 Hz, 1H, 6-H), 8.64 (d, J = 2.1 Hz, 1H, 4-H); 13C NMR (100 MHz,
DMSO-d6) d ppm: 45.66 (N(CH3)2), 47.06 (C-10), 56.31 (C-20),
71.01 (Bn–CH2), 110.88 (C-7), 111.18 (C-3a), 118.02 (C-4), 122.46
(C-6), 128.62 (Ph C-3 and -5), 128.67 (Ph C-4), 128.86 (Ph C-2
and -6), 136.79 (Ph C-1), 140.76 (C-5), 143.28 (C-7a), 157.06 (C-
3). Anal. Calcd for C18H20N4O3 (340.4): C, 63.52; H, 5.92; N,
16.46. Found: C, 63.42; H, 5.85; N, 16.53.
4.5.16. 1-(2-Diisopropylaminoethyl)-3-methoxy-5-nitro-1H-
indazol hydrochloride (27ꢃHCl)
Yield: 74%; mp 171–173 °C. 1H NMR (400 MHz, DMSO-d6) d
ppm: 1.33 (d, J = 6.65, 6H, N(CHCH3CH3)2), 1.36 (d, J = 6.65, 6H,
N(CHCH3CH3)2), 3.56 (m, 2H, N(CHCH3CH3)2), 3.72 (t, J = 7.0 Hz,
2H, 20-H), 4.09 (s, 3H, OCH3), 4.81 (t, J = 7.0 Hz, 2H, 10-H), 7.89 (d,

J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
8195
J = 9.3 Hz, 1H, 7-H), 8.29 (dd, J = 2.1, 9.3 Hz, 1H, 6-H), 8.55 (d,
J = 2.2 Hz, 1H, 4-H), 10.02 (sa, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d ppm: 17.19 (N(CHCH3CH3)2), 18.47 (N(CHCH3CH3)2),
45.14 (C-10 and -20), 54.98 (N(CHCH3CH3)2), 57.17 (OCH3), 111.27
(C-7), 111.61 (C-3a), 117.95 (C-4), 122.99 (C-6), 141.33 (C-5),
143.54 (C-7a), 158.47 (C-3). Anal. Calcd for C16H25ClN4O3 (356.9):
C, 53.85; H, 7.06; N, 15.70. Found: C, 53.69; H, 7.19; N, 15.80.
The supernatant was discarded and DMSO was added to dissolve
the formazan. After 10 min. incubation at 37 °C, the absorbance
at 540 nm was measured.
4.7. Oxygen uptake
Tulahuen strain T. cruzi epimastigotes were harvested by 500g
centrifugation, followed by washing and re-suspension in 0.05 M
sodium phosphate buffer, pH 7.4, and containing 0.107 M sodium
chloride. Respiration measurements were carried out polarograph-
ically with a Clark no. 5331 electrode (Yellow Springs Instruments)
in a 53 YSI model (Simpson Electric Co). The chamber volume was
2 mL and the temperature was 28 °C. The amount of parasite used
was equivalent to 2 mg of protein. The IC50 equivalent concentra-
tion corresponds to the final concentration used in the oxygen up-
take experiments. This concentration was calculated considering
that the IC50 (culture growth experiments) was determined using
3 ꢂ 106 parasites/mL, equivalent to 0.0375 mg protein/mL as the
initial parasite mass; 80 ꢂ 106 parasites/mL, equivalent to 1 mg
protein/mL, was used in the oxygen uptake experiments. In order
to maintain the parasite masse drug ratio constant in these two
types of experiments, the original IC50 was corrected by this 26-
fold parasite mass increase in the oxygen uptake experiment. Val-
ues are expressed as mean SD for three independent experi-
ments. No effect of DMSO alone was observed.
4.5.17. 3-Benzyloxy-1-(2-diisopropylaminoethyl)-5-nitro-1H-
indazol hydrochloride (28ꢃHCl)
Yield: 94%; mp 205–207 °C. 1H NMR (400 MHz, DMSO-d6) d ppm:
1.32 (d, J = 6.65, 6H, N(CHCH3CH3)2), 1.34 (d, J = 6.65, 6H, N(CHCH3-
CH3)2), 3.56 (m, 2H, N(CHCH3CH3)2), 3.72 (t, J = 6.9 Hz, 2H, 20-H),
4.81 (t, J = 6.9 Hz, 2H, 10-H), 5.49 (s, 2H, Bn–CH2), 7.42 (m, 3H, Ph
3-, 4- and 5-H), 7.57 (m, 2H, Ph 2- and 6-H), 7.90 (d, J = 9.3 Hz, 1H,
7-H), 8.30 (dd, J = 2.1, 9.3 Hz, 1H, 6-H), 8.59 (d, J = 2.1 Hz, 1H, 4-H),
10.01 (sa, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d ppm: 17.19
(N(CHCH3CH3)2), 18.46 (N(CHCH3CH3)2), 44.09 (C-10), 45.13 (C-20),
54.95 (N(CHCH3CH3)2), 71.22 (Bn–CH2), 111.35 (C-7), 111.68 (C-
3a), 118.02 (C-4), 123.01 (C-6), 128.60 (Ph, C-3 and -5), 128.73 (Ph,
C-4), 128.95 (Ph, C-2 and -6), 136.66 (Ph, C-1), 141.39 (C-5),
143.48 (C-7a), 157.67 (C-3). Anal. Calcd for C22H29ClN4O3 (332.9):
C, 61.03; H, 6.75; N, 12.94. Found: C, 61.21; H, 6.59; N, 12.81.
4.6. Biology
4.6.1. 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, com-
plemented with 5% fetal bovine serum (FBS)7,25–30 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 ꢂ 106 cells/mL. Cell growth was followed by measuring everyday
the absorbance of the culture at 600 nm. Before inoculation, the
media was 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 by the presence of
up to 1% DMSO in the culture media. The percentage of inhibition
was calculated as follows: % = {1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢂ 100,
where Ap = A600 of the culture containing the drug at day 5;
A0p = A600 of the culture containing the drug just after addition of
the inocula (day 0); Ac = A600 of the culture in the absence of any
drug (control) at day 5; A0c = A600 in the absence of the drug at
day 0. To determine IC50 values, 50% inhibitory concentrations, par-
asite 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 re-
duce the absorbance ratio to 50%.
4.8. Preparation of the rat liver cytosolic fractions
Livers were obtained from male Sprague-Dawley rats (198–
202 g) from ‘Centro de Investigaciones Nucleares’ (UdelaR, Montevi-
deo, Uruguay). The animals were allowed food and water ad libitum.
The experimental protocols with animals were adhered to the Prin-
ciples of Laboratory Animal Care. The animals were sacrificed by cer-
vical dislocation and the livers, maintained in an ice bath, were
perfused in situ with an ice-cold KCl (0.9%) solution and washed
with 3 volumes of Tris–HCl (0.05 M)–sucrose (0.25 M) pH 7.4 in a
Potter–Elvehjem glass–Teflon homogeniser. The homogenates were
centrifuged for 30 min at 900g at 4 °C and the supernatant fraction
was centrifuged at 10,000g for 1 h at 4 °C. The pellet was discarded
and the supernatant fraction was further centrifuged at 100,000g
for 1 h at 4 °C. Metabolic assays were carried out with cytosol either
fresh or frozen in Tris–HCl buffer and stored at ꢀ80 °C.
4.9. Cyclic voltammetry
DMSO (spectroscopy grade) was obtained from Aldrich. Tetra-
butylammonium perchlorate (TBAP), used as supporting electro-
lyte, was obtained from Fluka. CV was carried out using a
Metrohm 693VA instrument with a 694VA Stand convertor and a
693VA Processor, in DMSO (ca. 1.0 ꢂ 10ꢀ3 mol Lꢀ1), under a nitro-
gen atmosphere at room temperature, with TBAP (ca. 0.1 mol Lꢀ1),
using a three-electrode cell. A hanging mercury drop electrode was
used as the working electrode, a platinum wire as the auxiliary
electrode and saturated calomel electrode (SCE) as the reference.
Using the Nicholson’s procedure31 the Ipa/Ipc values (RNO2=
RNO2ꢀÅ) was measured from each cyclic voltammogram with scan
rate between 0.1 and 5 V/s. Employing the theoretical approaches
of Olmstead et al. for dimerization,32 the Ipa/Ipc values were in-
4.6.2. Cell culture
Human intestinal epithelial cells (Caco-2 ATCC accession
HTB-37) were maintained in DMEM-F12, supplemented with 10%
fetal bovine serum, penicillin (100 unit/mL), and streptomycin
(0.1 mg/mL), at 37 °C, in a humidified incubator with 5% CO2/95%
air atmosphere. 5 ꢂ 104 cells/mL of culture medium were plated
in 96-well plates.
serted into a working curve to determine the parameter
is described by the equation too
x, which
4.6.3. MTT reduction assay
x
¼ k2C0
s
Cell viability was measured by MTT assay, based on the ability
of live cells to convert thiazolyl blue into a dark blue formazan.
Cells were treated with PBS containing the 5-NI derivatives, 12–
where k2 is the second-order rate constant for the dimerization of
RNO2
ꢀÅ
, C0 is the nitrocompound concentration and s ¼ ðEkꢀ
E1=2Þ=
m
, where Ek is the switching potential, E1/2 is the half-wave po-
is the scan rate.
28 (25
lM) for 2 h. After washing, 0.5 mg/mL MTT were added to
tential and
m
each well and cells were further incubated for 120 min at 37 °C.

8196
J. Rodríguez et al. / Bioorg. Med. Chem. 17 (2009) 8186–8196
4.10. ESR experiments
Acknowledgments
ESR spectra were recorded in the X-band (9.7 GHz) using a Bru-
ker ECS 106 spectrometer with a rectangular cavity and 50 kHz
field modulation. The hyperfine splitting constants were estimated
to be accurate within 0.05 G. The ESR spectra were simulated using
the program WINEPR Simphonia 1.25 version.
Financial support from PEDECIBA (Uruguay), CICYT (Spain, the
grant SAF 2006-04698) and Collaborative Projects FONDECYT
1071068/7070282, Consejo Superior de Investigaciones Científicas
(Spain) 2006CL0035 CSIC 16/07-08, MECESUP UMC-0204. MLL
thanks CSIC-UdelaR (Uruguay) for a scholarship.
4.10.1. Parasites for ESR studies
References and notes
T. cruzi epimastigotes (Tulahuen strain), from our collection,
were grown at 28 °C in Diamond’s monophasic medium as re-
ported earlier, with blood replaced by 4 mM hemin.33 Fetal calf ser-
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a final concentration of 4%. Parasites:
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8 ꢂ 107 cells correspond to 1 mg protein or 12 mg of fresh weight.
ESR spectra were produced using a microsomal fraction (4 mg pro-
tein/mL) obtained from T. cruzi, in a reaction medium containing
1 mM NADPH, and 100 mM DMPO, in 20 mM phosphate buffer,
pH 7.4. All experiments were done after of 15 min of incubation,
to 28 °C, of 5-NI with T. cruzi microsomal fraction, NADPH and
DMPO in an aerobic environment.
9. Boiani, L.; Gerpe, A.; Arán, V. J.; Torres de Ortiz, S.; Serna, E.; Vera de Bilbao, N.;
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4.10.2. Rat cytosolic fractions for ESR studies
The standard incubation mixture, in aerobic conditions, was:
1 mM NADPH in a 0.1 M potassium phosphate buffer (pH 7.4) con-
taining EDTA (1.5 mM) and the corresponding 5-NI (40 lM) dis-
11. www.chemaxon.com.
solved in DMSO or acetonitrile and 100 mM of DMPO or POBN as
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sion was added to give a final protein concentration of 1 mg/mL.
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4.10.3. Assay for Dpph radical
The dpph radical-scavenging capacity of individual selected 5-
NI derivatives was determined with an ESR spectrometry meth-
od.34 Each 5-NI solution was mixed with dpph stock solution to ini-
tiate the 5-NI–radical reaction. All reaction mixtures contained
1.0 mM dpph and 1.0 mM 5-NI, and the control solution contained
no 5-NI. Both dpph and 5-NI solutions were prepared in acetoni-
trile. ESR signals were recorded 1 min following the start of the
reaction. Spectrometer conditions were: microwave frequency,
9.72 GHz; microwave power, 20 mW; modulation amplitude,
0.98 G; receiver gain, 59 db; and sweep time, 20.972 s.
The scavenging activity of each compound was estimated by
comparing the dpph signals in the 5-NI–radical reaction mixture
and the control reaction at the same reaction time, and expressed
as percentage dpp remaining. The dpph radical-scavenging rate of
test compounds was calculated using the formula scavenging
rate = [(A0 ꢀ Ax)/A0] ꢂ 100%, where Ax and A0 are the double-inte-
gral ESR for the first line of samples in the presence and absence
of test compounds, respectively.
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4.10.4. Assay for galvinoxyl radical
The galvinoxyl radical-scavenging capacity of individual selected
5-NI was determined with an ESR spectrometry method. The 5-NI
derivatives (1 mM) were mixed with 1 mM galvinoxyl in all reaction
mixtures, and the control solution contained no 5-NI. Both galvin-
oxyl and 5-NI solutions were prepared in acetonitrile. ESR signals
were recorded 1 min following the start of the reaction. Spectrome-
ter conditions were the same as described above.
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