New perspectives on the synthesis and antichagasic activity of 3-alkoxy-1-alkyl-5-nitroindazoles

Article abstract of DOI:10.1016/j.ejmech.2013.12.025

The synthesis and antiprotozoal activity of some 3-alkoxy-1-alkyl- (1, 4) and 3-alkoxy-1-(ω-aminoalkyl)-5-nitroindazoles (2, 3, 5-8) against different morphological forms of Trypanosoma cruzi are reported. These compounds were prepared using simple alkylation reactions and, usually, taking advantage of the reactivity of some indazole-derived betaines previously studied by us. Most indazole derivatives showed in vitro activities similar or higher than those of the reference drug benznidazole; this fact, along with low unspecific cytotoxicities against Vero cells shown by some of them, led to very good selectivity indexes (SI). The high efficiency of 5-nitroindazoles 1 and 2 against T. cruzi was confirmed by further in vitro studies on infection rates and by an additional in vivo study in a murine model of acute and chronic Chagas disease. Complementary analyses of the changes in the metabolites excreted by the parasite and on the ultrastructural alterations induced after treatment with indazole derivatives 1 and 2 were also conducted.

Full text of DOI:10.1016/j.ejmech.2013.12.025

European Journal of Medicinal Chemistry 74 (2014) 124e134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New perspectives on the synthesis and antichagasic activity
of 3-alkoxy-1-alkyl-5-nitroindazoles
Beatriz Muro a, Felipe Reviriego a, Pilar Navarro a, Clotilde Marín b,
,
Inmaculada Ramírez-Macías b, María José Rosales b, Manuel Sánchez-Moreno b
,
**
,
Vicente J. Arán a
*
a Instituto de Química Médica (IQM), CSIC, c/ Juan de la Cierva 3, E-28006 Madrid, Spain
b Departamento de Parasitología, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antiprotozoal activity of some 3-alkoxy-1-alkyl- (1, 4) and 3-alkoxy-1-(u-aminoalkyl)-
Received 13 September 2013
Received in revised form
12 December 2013
Accepted 22 December 2013
Available online 3 January 2014
5-nitroindazoles (2, 3, 5e8) against different morphological forms of Trypanosoma cruzi are reported.
These compounds were prepared using simple alkylation reactions and, usually, taking advantage of the
reactivity of some indazole-derived betaines previously studied by us. Most indazole derivatives showed
in vitro activities similar or higher than those of the reference drug benznidazole; this fact, along with
low unspecific cytotoxicities against Vero cells shown by some of them, led to very good selectivity
indexes (SI). The high efficiency of 5-nitroindazoles 1 and 2 against T. cruzi was confirmed by further
in vitro studies on infection rates and by an additional in vivo study in a murine model of acute and
chronic Chagas disease. Complementary analyses of the changes in the metabolites excreted by the
parasite and on the ultrastructural alterations induced after treatment with indazole derivatives 1 and 2
were also conducted.
Keywords:
Nitroindazoles
In vitro assays
In vivo assays
Chagas disease
Cytotoxicity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
In this context, we have reported in the last years the synthesis
and antichagasic properties of some 5-nitroindazole derivatives,
Chagas disease or American trypanosomiasis is a major para-
sitosis originally endemic to poor rural areas of Latin America,
mainly transmitted by hematophagous triatomine insects and
caused by the trypanosomatid (Kinetoplastid) protozoan parasite
Trypanosoma cruzi. At present because of intense international
migrations it can also be found in other areas such as Western
Europe, USA, Australia, etc, countries in which the infection is
transmitted by other routes. Only two drugs, nifurtimox and
benznidazole are currently available for the treatment of Chagas
disease. This chemotherapy, however, is unsatisfactory because
these drugs exhibit limited efficacy in the chronic phase of the
disease and also severe toxic side effects. Thus, it is clear that the
development of new drugs for the treatment of this parasitosis is
urgently needed. Several excellent articles covering different as-
pects of Chagas disease chemotherapy [1] have been published
recently.
mainly 1-substituted 3-alkoxy-1H-indazoles [2e4], 2-substituted
3-alkoxy-2H-indazoles [4] and 1,2-disubstituted indazolin-3-ones
[4], as well as those of some 4-substituted and 1,4-disubstituted
7-nitroquinoxalin-2-ones [5,6]. It has been proposed [2e4,7] that
the mentioned nitro-group bearing heterocycles act upon intra-
cellular nitro reduction followed by redox cycling leading to reac-
tive oxygen species (ROS) (like nifurtimox) [8] or producing
electrophilic metabolites (like benznidazole) [8] able to damage
essential biomolecules of parasites. Nevertheless, the classical
mechanisms of action proposed for nifurtimox and benznidazole
are currently under revision [9] and consequently, that of our
nitroheterocycles need to be investigated more thoroughly.
Inhibition of trypanothione reductase has also been suggested ac-
cording to molecular modelling studies carried out with 7-
nitroquinoxalin-2-one derivatives [6].
Considering the interest of 3-alkoxy-1-alkyl-5-nitroindazole
scaffold [2e4] in the field of antichagasic agents, and that
u-(dia-
lkylamino)alkyl chains are found in some of the most active re-
ported indazoles [2,3] and quinoxalines [6], in this article we
describe the synthesis and antichagasic properties of a new family
of 5-nitroindazole-derived primary, secondary and tertiary amines
* Corresponding author. Tel.: þ34 915622900; fax: þ34 915644853.
** Corresponding author. Tel.: þ34 958242369; fax: þ34 958243174.
E-mail addresses: msanchem@ugr.es (M. Sánchez-Moreno), vjaran@iqm.csic.es
(V.J. Arán).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.025

B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
125
(2, 3, 5e8); the reported low solubility of some indazole derivatives
containing lipophilic substituents [4] led us to introduce in the
present compounds an oxaalkyl chain directed to improve their
solubility in aqueous media. Compounds 1 and 4 were also pre-
pared and tested as simple models containing the 3-alkoxy-1-alkyl-
5-nitroindazole scaffold (Fig. 1).
Alkoxyindazolyl)alkyl halides 16 and 17, chromatographically
separated from the corresponding 1,2-disubstituted indazolinones
18 and 19, were then treated with an excess of methylamine in
ethanol to afford secondary amines 2 and 5, respectively. The latter,
treated with an additional equivalent of the required halides 16 or
17, yielded tertiary amines 3 and 6.
Antichagasic properties were initially evaluated in vitro against
epimastigote, amastigote and trypomastigote forms of T. cruzi; Vero
cells were used in order to determine unspecific cytotoxicity of our
compounds. This study was complemented by infectivity assays on
Vero cells carried out with products showing highest activity and
selectivity index (SI) values, i.e., compounds 1 and 2. In vivo try-
panocidal activities of these compounds in a murine model of acute
and chronic phases of Chagas disease were also determined.
Furthermore, a 1H NMR study has been conducted in order to
observe changes in the nature and percentage of metabolites
excretion directed to obtain information about the effect of our
compounds on the glycolytic pathway of T. cruzi epimastigotes;
finally, we have also studied the ultrastructural alterations of
parasite epimastigotes treated with our compounds using trans-
mission electron microscopy (TEM).
Finally, treatment of iodide 17 with an excess of ammonia in
ethanol gave primary amine 7 as the main reaction product, along
with a small amount of secondary amine 8; both compounds were
readily separated by chromatography.
Since the control of the reaction of alkyl halides with ammonia
or primary amines is difficult, this method is not usually recom-
mended [14] for the preparation of primary or secondary amines,
respectively; in our case, however, these processes were very clean,
affording primary amine 7 and secondary amines 2 and 5 with
excellent yields.
The structure of all compounds has been established on the
basis of analytical and spectral data. Hydrazides such as 13 appear
in solution (NMR) as mixtures of Z and E rotamers owing to
restricted rotation around the NeCO bond [12]. In this case, the Z
rotamer/E rotamer ratios, determined by 1H NMR as previously
reported for related compounds [12], are ca. 58:42 and 65:35 in
CDCl3 and (CD3)2SO, respectively.
2. Results and discussion
As previously observed for related betaines [12], the presence on
the quaternary nitrogen atom of compound 14 hinders the
conformational equilibrium of morpholine ring. Thus the aniso-
chronic NCH2 protons can be easily distinguished in the 1H NMR
spectrum as Hax and Heq by comparison with the spectra of previ-
ously described piperidine analogues [12]. A similar effect has been
observed for OCH2 protons but in this case the assignment of the
signals is not easy, and they have been distinguished in the spectral
description as HA and HB.
On the other hand, 3-alkoxy-1-alkylindazoles (1, 4, 16, 17) can be
easily distinguished by NMR from the isomeric 1,2-disubstituted
indazolinones (10, 11, 18, 19, respectively) also arising from the
alkylation of 1-substituted indazol-3-ols [11].
2.1. Chemistry
Compounds 1 and 4 were prepared by alkylation of 1-methyl-5-
nitroindazol-3-ol 9 (Scheme 1). As expected [10,11], mixtures of
N1,O- and N1,N2-dialkyl derivatives were obtained, but chromato-
graphical separation of 3-alkoxy-1-alkylindazoles (1, 4) from the
isomeric 1,2-dialkylindazolinones (10, 11) was very simple.
Compounds 2, 3, 5e8 were prepared according to the pathway
shown in the Scheme 1. The key intermediate is indazolium-3-olate
14, belonging to a class of betaines which synthesis [12] and reac-
tivity [13] have previously been studied by some of us. These betaines
are easilyavailable bycyclization of2-halogenobenzohydrazides such
as13 which, inturn, wasobtainedbyacylation of 4-aminomorpholine
with the corresponding acid chloride 12 [12].
2.2. In vitro anti-T. cruzi evaluation
Treatment of betaine 14 with hydrobromic acid [13] yielded
u-
(3-hydroxyindazolyl)alkyl bromide 15. Alkylation of this compound
with benzyl bromide or methyl iodide afforded, as expected for 1-
substituted indazol-3-ols [10,11], mixtures of O- (16, 17) and N2-
alkyl (18, 19) derivatives; partial halogen exchange (Finkelstein
reaction), completed by treatment with sodium iodide in acetone,
In vitro activity of compounds 1e8 on epimastigotes, amasti-
gotes and trypomastigotes [15,16] of T. cruzi, the unspecific cyto-
toxicity against Vero cells and the corresponding selectivity indexes
(SI) are gathered in Table 1. First we prepared an epimastigotes
(extracellular insect vector stage) culture and in a further step we
infected Vero cells with metacyclic forms of parasite, which were
takes also place during the methylation reaction (NMR).
u-(3-
Fig. 1. Chemical structure of indazole derivatives 1e8 studied in the present work.

126
B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
All compounds show high activities against the three parasite
forms, of the same order or slightly lower (up to ca. one half) or
higher than those of the reference drug. 3-Benzyloxy derivatives
are in general more active than the corresponding 3-methoxy an-
alogues (compare 1 vs 4, 2 vs 5 and 3 vs 6). This effect has previ-
ously been observed for other 3-alkoxy-1-alkylindazoles [2,3];
since for related 3-BnO and 3-MeO derivatives the reduction po-
tentials corresponding to formation of nitro anion radical in aprotic
solvents are similar (ca. ꢀ1.2 V) [3,17], we assume, as suggested for
1,2-disubstituted indazolinones [4], that more lipophilic 3-
benzyloxy derivatives are better substrates for parasite nitro-
reductases than 3-methoxy compounds. On the other hand, dif-
ferences between activities of
a primary amine and the
corresponding NeMe derivative (7 vs 5), or between indazole and
bisindazole derivatives (2 vs 3, 5 vs 6 and 7 vs 8) are scarce. Great
differences are found, however, in the unspecific cytotoxicity
against Vero cells and, of course, in the selectivity indexes (SI)
resulting from these data; we are not able to find, however, a clear
relationship between toxicity and structure. Taking into account all
data included in Table 1, compounds 1 and 2, with SI values
exceeding 14e23 times those of benznidazole depending on the
parasite stage are the best compounds; on the other hand, com-
pound 8 shows rather good trypanocidal activity but it is also
highly toxic for Vero cells, thus resulting SI values (SI < 1) so
regrettable as those of benznidazole, currently used for the treat-
ment of Chagas disease. Trypanocidal activities of the same order
have been published for related 1-[u-(dialkylamino)alkyl]indazole
derivatives; nevertheless, data provided for the latter are hetero-
geneous (changes in the T. cruzi strain and in the model of host cells,
differences in the way of expressing trypanocidal activity and un-
specific cytotoxicity data, SI values not determined, etc.) [2,3] and a
direct comparison with those obtained in this article is difficult.
Unfortunately, this situation is rather frequent as has been stressed
by some authors [16].
On the other hand, high activities found for the simple indazole
derivatives 1 and 4 are very interesting and merit further research;
in fact, low activity values against T. cruzi epimastigotes have been
reported for 1-methyl-3-phenethoxy- and 1-methyl-3-(2-
naphthylmethoxy)-5-nitroindazole, closely related analogues of
compound 1 [4].
With the aim of obtaining more accurate information about the
products showing highest activity and SI values in the previously
described assays, compounds 1 and 2, the spreading of the parasite
in Vero cells was studied by measuring the rates of infection and
the average number of amastigotes and trypomastigotes present
during a 10-days treatment (Fig. 2). Vero cells were infected with
metacyclic forms of T. cruzi and the gradual conversion of the latter
into amastigotes was observed in the invaded cells. During the
mentioned period the rate of infection of the host cells gradually
increased reaching at the end of experiment 99% of invasion
(Fig. 2A).
The test was repeated in the presence of the reference drug and
of compounds 1 and 2 at their IC25 concentrations. It was found that
the rate of infection decreased in all cases in relation to the control,
showing compounds 1 and 2, with reductions in the infection rate
of 82% and 78% respectively, a much higher efficiency than benz-
nidazole (19% of decrease).
Scheme 1. Synthesis of final indazole derivatives 1e8, intermediates 13e17 and sec-
ondary reaction products 10, 11, 18 and 19.
Concerning the average number of amastigotes per Vero cell
(Fig. 2B), the results were consistent with those mentioned above
for infection rates. After treatment, compounds 1 and 2 signifi-
cantly reduced the number of amastigotes per cell in 76% and 63%,
respectively, while benznidazole showed only a decrease of 37%.
In relation to the number of trypomastigotes/mL found in the
culture medium (Fig. 2C), a maximum was reached in the control on
day 10 (ca. 9.5 ꢁ 103), but this value was substantially reduced by
converted into amastigotes (intracellular mammalian host forms).
Activity against trypomastigotes (extracellular peripheral blood
mammalian host stage) was determined using infected mice blood
collected after infection with the parasite. Compounds 1e8 as well
as the standard drug benznidazole were assayed at concentrations
of 1e100
mM and from the obtained data IC50 values shown in
Table 1 were calculated.

B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
127
Table 1
In vitro activity, unspecific cytotoxicity and selectivity index (SI) found for 5-nitroindazole derivatives 1e8 on extra- and intracellular forms of T. cruzi.
Compounds
T. cruzi IC50
(
m
M)a
Vero cells IC50
(
m
M)a
SIb
Epimastigote
forms
Intracellular
amastigote forms
Trypomastigote
forms
Epimastigote
forms
Intracellular
amastigote forms
Trypomastigote
forms
Benznidazole
15.9 ꢂ 1.1
17.6 ꢂ 0.7
19.1 ꢂ 0.9
26.6 ꢂ 1.4
24.7 ꢂ 1.8
26.7 ꢂ 2.0
24.6 ꢂ 0.8
27.3 ꢂ 2.4
31.2 ꢂ 2.6
23.3 ꢂ 4.6
14.1 ꢂ 1.4
16.6 ꢂ 0.9
16.5 ꢂ 0.6
32.6 ꢂ 2.2
23.4 ꢂ 4.0
27.9 ꢂ 2.1
26.5 ꢂ 1.7
25.7 ꢂ 2.0
16.4 ꢂ 3.2
12.0 ꢂ 1.3
14.5 ꢂ 0.5
16.5 ꢂ 0.8
24.8 ꢂ 1.8
20.5 ꢂ 1.3
20.4 ꢂ 0.7
20.7 ꢂ 1.7
26.4 ꢂ 2.3
13.6 ꢂ 0.9
193.7 ꢂ 14.9
213.6 ꢂ 21.5
128.4 ꢂ 8.8
183.1 ꢂ 11.4
87.6 ꢂ 7.4
0.8
0.6
0.8
1
2
3
4
5
6
7
8
11.0 (14)
11.2 (14)
4.8 (6)
7.4 (9)
3.3 (4)
4.1 (5)
3.2 (4)
0.5 (1)
13.7 (23)
12.9 (21)
7.8 (13)
5.6 (9)
3.7 (6)
4.6 (8)
3.3 (5)
0.6 (1)
16.1 (20)
14.7 (18)
7.8 (10)
7.4 (9)
4.3 (5)
6.3 (8)
4.2 (5)
0.6 (1)
129.6 ꢂ 9.5
87.2 ꢂ 5.5
16.9 ꢂ 1.1
a
IC50 ¼ the concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100
mM).
b
Selectivity index ¼ IC50 Vero cells/IC50 extracellular and intracellular forms of parasite. In brackets: number of times that compound SI exceeds the reference drug SI on the
different morphological forms of parasite.
the reference drug (41%) and, especially, by compounds 1 and 2
(80% and 75%, respectively). In summary, the results of spreading of
parasite in Vero cells are in agreement with those of trypanocidal
activity reported in Table 1 for intracellular and extracellular forms
of T. cruzi.
Consequently, they excrete into the medium a considerable part of
the hexose skeleton as partially oxidised fragments in the form of
fermented metabolites. The nature and percentage of catabolism
compounds depend on the pathway used for glucose metabolism
by each trypanosomatid species and the life-cycle stage considered,
but CO2, succinate, acetate,
L-lactate, pyruvate, L-alanine and
ethanol are usually produced [19,20]. Succinate is important
because its main role is to maintain the glycosomal redox balance,
which allows the reoxidation of NADH produced in the glycolytic
pathway. Succinic fermentation has the advantage of requiring only
half of the phosphoenolpyruvate produced to maintain the NADþ/
NADH balance, while the remaining pyruvate is converted inside
the mitochondrion and the cytosol into acetate, L-lactate, L-alanine
or ethanol, according to the degradation pathway followed by each
species [20].
To gain information concerning the effect of our indazole de-
rivatives on glucose metabolism in the parasites, the final excretion
products were identified. These data were obtained by recording
the 1H NMR spectra of epimastigotes from T. cruzi after treatment
with the studied compounds at their IC25 concentrations. The re-
sults were compared with a control of epimastigotes maintained in
a cell-free medium for four days after inoculation with the parasite,
2.3. In vivo anti-T. cruzi evaluation
Considering the good results obtained in the previously
mentioned in vitro assays, in vivo activity of compounds 1 and 2 was
studied on infected female BALB/c mice. Drugs currently used
against Chagas disease are effective in the acute phase of the dis-
ease, but its efficacy in the chronic phase is very controversial. It
was therefore decided to evaluate the activity of our compounds 1
and 2 on murine models of both phases, using benznidazole as the
reference drug. For experiments in the acute phase, the first 30 days
after infection were considered, while the effect on the chronic
phase was studied between 30 and 120 days after infection. Mice
were inoculated with metacyclic forms of T. cruzi and treatment
with test compounds (1 mg/kg/day doses) was initiated by intra-
peritoneal route 5 days after infection and continued for 5 addi-
tional days. The study included different control groups as
described in the Experimental section. During the study of the
activity in the acute phase, the level of parasitemia was determined
every 3e4 days. Fig. 3 shows the number of circulating trypomas-
tigotes/mL of blood vs days elapsed since infection. On the day of
maximum parasite load (ca. 14 days after infection) all tested
compounds significantly decreased the number of trypomastigotes.
On day 30, at the end of the acute phase observation period,
compounds 1 and 2 reduced the level of parasitemia by 33% and
39%, respectively, values significantly higher than that found for the
reference drug (23%). No mouse died in any of our experiments
performed either with the control or with compounds 1 and 2 at
the doses used; however, the survival percentage for the mice
treated with benznidazole was only about 80%.
showing the characteristic signals of CH3 of ethanol, acetate,
alanine and -lactate, and CH2 signal of succinate.
L-
L
When T. cruzi epimastigotes were treated with compounds 1
and 2, the succinate excretion appeared clearly decreased (35% and
13%, respectively), probably showing the disruption of glycosomal
or mitochondrial enzymes involved in its biosynthesis [20] (Table 3,
Fig. S1).
On the other hand,
from pyruvate; compound 1 seems to inhibit cytosolic enzymes
responsible for the transformation of pyruvate to -lactate (un-
known enzyme) (23% of reduction) and -alanine (alanine amino-
L-lactate, L-alanine and ethanol originate
L
L
transferase) (19% of reduction) thereby forcing its metabolism to
ethanol (pyruvate decarboxylase and NAD-linked alcohol dehy-
drogenase) (11% of increase) [20]. Among the pyruvate-derived
metabolites, compound 2 only affects the lactate levels (17% of
decrease), those of alanine and ethanol remaining unchanged.
Moreover, acetate excretion does not change after treatment with
compounds 1 and 2, showing the absence of disturbances of en-
zymes responsible for the production of this metabolite at mito-
chondrial level [20].
In connection with the activity in the chronic phase of the dis-
ease, Table 2 shows the differences in the level of anti-T. cruzi an-
tibodies between 30 and 120 days after infection followed by the
mentioned treatment. As can be seen, compound 1 and especially 2,
were much more effective than benznidazole, reducing signifi-
cantly the antibodies levels with respect to the control.
2.4. Metabolites excretion study
2.5. Ultrastructural alterations
It is well established that trypanosomatids are unable to
completely degrade glucose to CO2 under aerobic conditions. Their
characteristic catabolism is mediated by glycolytic enzymes most of
them placed in the organelle called the glycosome [18].
Ultrastructural alterations induced on T. cruzi epimastigotes by
indazole derivatives 1 and 2 at their IC25 concentrations have been
studied by transmission electron microscopy (TEM) (Fig. 4). One of

128
B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
Fig. 3. Parasitemia in the murine model of acute Chagas disease: effect of benznidazole
(BZN) and 5-nitroindazole derivatives 1 and 2 (1 mg/kg/day doses during 5 days).
Table 2
Differences in the level of anti-T. cruzi antibodies between
days 30 and 120 post-infection for benznidazole and for
derivatives 1 and 2.
Compoundsa
D
Ab
Control (untreated)
Benznidazole
0.146
0.110
1
2
ꢀ0.025
ꢀ0.141
a
Dose of 1 mg/kg/day for five consecutive days, admin-
istered by the intraperitoneal route.
b
D
A ¼ absorbance at 490 nm on day 120 post-infection
e
absorbance at 490 nm on day 30 post-infection
(expressed in absorbance units).
appearance, with undulations and discontinuities, perhaps as a
result of the mentioned altered cytoskeleton.
3. Conclusions and future outlooks
The biological evaluation has shown that all compounds tested
exhibit adequate antichagasic activity against the three studied
morphological stages of T. cruzi; although the establishment of a
clear structureeactivity relationship is difficult, the more lipophilic
3-OBn derivatives show, in general, higher activities than the cor-
responding 3-OMe analogues. Moreover, the large differences
observed in the cytotoxicity of the compounds against Vero cells
leads to dramatic differences in the values of selectivity indexes in
relation to those of the antichagasic standard drug benznidazole;
thus compounds 1 and 2 stand out for their high effectiveness,
further confirmed through additional infectivity assays and an
in vivo murine model of Chagas disease. Unfortunately, trypanoci-
dal activity data provided previously for other indazole derivatives
are very heterogeneous and a direct comparison with those ob-
tained in the present article cannot be carried out. Nevertheless,
Fig. 2. Effect of benznidazole (BZN) and 5-nitroindazole derivatives 1 and 2 (IC25
concentrations) on the infection rate and growth of T. cruzi: (A) rate of infection; (B)
mean number of amastigotes per infected Vero cell; (C) number of trypomastigotes/mL
in the growth medium.
evident morphological changes was the presence of very little
electron-dense cytoplasm observed in the majority of treated
protozoa; another very significant alteration was a large vacuoli-
zation (V), with some lipidic type vacuoles occupying in some cases
most of the cytoplasm (figure not shown). Mitochondria (M) and
glycosomes (G) were damaged or decreased, in agreement with the
metabolic changes described in the previous section. They are also
very frequent parasites showing appearance of being dead (DP) and
the death of a large number of parasites. These phenomena are also
observed when T. cruzi is treated with benznidazole, causing
disruption of the parasite cytoskeleton with disorganization of the
microtubule structure and plasma membrane with scalloped
Table 3
Variation in the 1H NMR peaks corresponding to catabolites excreted by T. cruzi
epimastigotes in the presence of 5-nitroindazole derivatives 1 and 2 with respect to
the control test.a
Compounds
Lac
Ala
A
S
Eth
1
2
ꢀ23%
ꢀ17%
ꢀ19%
¼
¼
ꢀ35%
ꢀ13%
þ11%
¼
¼
(e) peak decrease; (þ) peak increase; (¼) no difference detected.
a
Lac, L-lactate; Ala, L-alanine; A, acetate; S, succinate; Eth, ethanol.

B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
129
Fig. 4. Ultrastructural alterations observed by TEM in epimastigotes of T. cruzi either untreated (control) or treated with 5-nitroindazole derivatives 1 and 2, showing organelles
with their characteristics: kinetoplast (K); vacuoles (V); mitochondrion (M); glycosomes (G); cytoskeleton microtubules (MT); altered cytoplasmic membrane (MC); dead parasites
(DP). Control: untreated parasites, bar: 1
compound 2, bar: 1 m.
mm; BZN: parasites treated with benznidazole, bar: 1.59 mm; 1: parasites treated with compound 1, bar: 1 mm. 2: parasites treated with
m
although much work remains to be done in order to increase our
knowledge in the field of structureeactivity relationships, the po-
tential of indazole scaffold in the development of new antichagasic
drugs is confirmed by our present results.
(Merck, particle size 0.040e0.063 mm) for flash column chroma-
tography. Solvents and reagents were obtained from different
commercial sources and used without further purification. Micro-
analyses were performed on a Heraeus CHN-O-RAPID analyzer and
were within the ꢂ0.3% of the theoretical values.
We suggest here, mainly on the basis of metabolic changes
caused by indazoles 1 and 2, that these compounds may interfere
with some glycosomal or mitochondrial enzymes involved in the
catabolism of T. cruzi. For the future, in order to continue our
studies, we have planned the synthesis of other 5-nitroindazoles,
including fluorescent derivatives that could label the organelles
constituting their primary target, as well as the identification of the
products arising from the bioreduction of 5-nitroindazoles medi-
ated by trypanosomal nitroreductases. In fact, related studies
recently carried out for nifurtimox and benznidazole [9] have
greatly advanced the understanding of their mode of action and
allowed banishing old assumptions.
4.1.2. 3-Benzyloxy-1-methyl-5-nitro-1H-indazole (1)
A mixture of 1-methyl-5-nitro-1H-indazol-3-ol (9) [4,13] (1.93 g,
10 mmol), benzyl bromide (1.88 g, 11 mmol) and potassium car-
bonate (4.00 g, excess) in acetone (100 mL) was refluxed for 5 h.
Inorganic salts were removed by filtration and, after evaporation of
acetone and addition of water (100 mL), the crude mixture of
benzyl derivatives was extracted with chloroform (3 ꢁ 50 mL). This
extract was concentrated and applied to the top of a column which
was eluted first with chloroform to afford the desired 3-benzyloxy
derivative 1 (2.24 g, 79%); mp 130e132 ꢃC (2-propanol) [21]; Rf 0.25
(CHCl3), 0.89 (CHCl3eMeOH 10:1). 1H NMR (CDCl3):
d 8.66 (d,
J ¼ 2.1 Hz,1H, 4-H), 8.21 (dd, J ¼ 9.3, 2.1 Hz,1H, 6-H), 7.50 (m, 2H, Ph
4. Experimental section
2-, 6-H), 7.39 (m, 3H, Ph 3-, 4-, 5-H), 7.21 (d, J ¼ 9.3 Hz,1H, 7-H), 5.41
(s, 2H, OCH2), 3.92 (s, 3H, CH3); 13C NMR (CDCl3):
d 157.33 (C-3),
4.1. Chemistry
142.80 (C-7a), 140.66 (C-5), 136.07 (Ph C-1), 128.51 (Ph C-3, -5),
128.30 (Ph C-4), 128.03 (Ph C-2, -6), 122.41 (C-6), 118.53 (C-4),
111.80 (C-3a), 108.43 (C-7), 70.97 (OCH2), 35.47 (CH3); MS (EI): m/z
(%) 283 (30) [M]þ, 206 (1), 192 (3), 150 (2), 146 (3), 104 (3), 103 (3),
91 (100).
4.1.1. General methods
Melting points (mp) were determined in a Stuart Scientific
melting point apparatus SMP3. 1H (300 MHz) and 13C (75 MHz)
NMR spectra were recorded on a Bruker Avance 300 spectrometer.
Further elution of the column with a chloroformemethanol
(30:1) mixture afforded the known [11] 2-benzyl-1-methyl-5-
nitro-1,2-dihydro-3H-indazol-3-one (10) (0.34 g, 12%); Rf 0.00
(CHCl3), 0.78 (CHCl3eMeOH 10:1).
The chemical shifts are reported in ppm from TMS (d scale) but
were measured against the solvent signal [(CD3)2SO: dH 2.49, dC
39.50; CDCl3: dH 7.24, dC 77.00]. The assignments have been per-
formed by means of different standard homonuclear and hetero-
nuclear correlation experiments (NOE, gHSQC and gHMBC). To
simplify the description of the NMR spectra, bis(indazolylalkyl)
amines have been numbered as simple compounds. Electron
impact (EI) and electrospray (ESþ) mass spectra were obtained on a
Hewlett Packard 5973 MSD (70 eV) and on a Hewlett Packard 1100
MSD spectrometer, respectively. DC-Alufolien silica gel 60 PF254
(Merck, layer thickness 0.2 mm) was used for TLC, and silica gel 60
4.1.3. 3-Methoxy-1-methyl-5-nitro-1H-indazole (4)
A mixture of 1-methyl-5-nitro-1H-indazol-3-ol (9) [4,13] (1.16 g,
6 mmol), methyl iodide (2 mL, excess) and potassium carbonate
(1.93 g, 14 mmol) in acetone (50 mL) was stirred at room temper-
ature for 24 h and then refluxed for 1 h. The reaction mixture was
treated as described for compound 1, affording the known [13]

130
B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
3-methoxy derivative 4 (0.66 g, 53%) [Rf 0.30 (CHCl3), 0.86 (CHCl3e
MeOH 10:1)] and 1,2-dimethyl-5-nitro-1,2-dihydro-3H-indazol-3-
one (11) (0.56 g, 45%) [Rf 0.00 (CHCl3), 0.44 (CHCl3eMeOH 10:1)].
Alternatively, direct alkylation of 5-nitro-1H-indazol-3-ol [22]
(1.07 g, 6 mmol) with an excess of methyl iodide following the same
procedure afforded 0.80 g (64%) of 3-methoxy-1-methylindazole 4
and 0.41 g (33%) of the mentioned 1,2-dimethylindazolinone 11.
4.1.7. 5-(3-Benzyloxy-5-nitro-1H-indazol-1-yl)-3-oxapentyl
bromide (16)
A mixture of bromide 15 (3.30 g, 10 mmol), benzyl bromide
(1.88 g, 11 mmol) and potassium carbonate (4.00 g, excess) in
acetone (100 mL) was stirred at room temperature for 24 h and
then refluxed for 2 h. Inorganic salts were removed by filtration
and, after evaporation of acetone and addition of water (100 mL),
the crude mixture of benzyl derivatives was extracted with chlo-
roform (3 ꢁ 50 mL). This extract was dried (MgSO4), absorbed on
silica gel and applied to the top of a column which was eluted first
with hexane in order to remove excess of benzyl bromide and then
with chloroform to afford the title compound 16 (2.82 g, 67%); mp
91e93 ꢃC (2-propanol); Rf 0.25 (CHCl3), 0.76 (CHCl3eMeOH 50:1).
4.1.4. 2-Chloro-N-morpholino-5-nitrobenzamide (13)
A solution of 2-chloro-5-nitrobenzoyl chloride (12) [12] (14.30 g,
65 mmol) in dry diethyl ether (200 mL) was added at room tem-
perature during 30 min to
a well stirred solution of 4-
aminomorpholine (6.64 g, 65 mmol) in 0.6 M aq sodium hydro-
gencarbonate (150 mL). After 2 h the ether was evaporated, and the
solid in suspension collected by filtration, washed with water
(150 mL) and air-dried (14.86 g, 80%); mp 198e200 ꢃC (decomp.)
1H NMR [(CD3)2SO]:
d
8.51 (d, J ¼ 2.2 Hz, 1H, 40-H), 8.17 (dd, J ¼ 9.4,
2.2 Hz, 1H, 60-H), 7.72 (d, J ¼ 9.4 Hz, 1H, 70-H), 7.56 (m, 2H, Ph 2-, 6-
H), 7.39 (m, 3H, Ph 3-, 4-, 5-H), 5.43 (s, 2H, 30-OCH2), 4.47 (t,
J ¼ 5.0 Hz, 2H, 5-H), 3.83 (t, J ¼ 5.0 Hz, 2H, 4-H), 3.62 (t, J ¼ 5.6 Hz,
(ethanol); Rf 0.49 (CHCl3eMeOH 10:1). 1H NMR (CDCl3):
d 8.40 (d,
2H, 2-H), 3.41 (t, J ¼ 5.6 Hz, 2H, 1-H); 13C NMR [(CD3)2SO]:
d 156.74
J ¼ 2.7 Hz, 1H, 6-H, Z and E rot.), 8.21 (Z rot.) and 8.15 (E rot.) (both
dd, J ¼ 8.8, 2.7 Hz) (1H, 4-H), 7.59 (Z rot.) and 7.58 (E rot.) (both d,
J ¼ 8.8 Hz) (1H, 3-H), 6.78 (Z rot.) and 6.67 (E rot.) (both br s) (1H,
NH), 3.86 (m, Z rot.) and 3.51 (br m, E rot.) (4H, 20-, 60-H), 3.00 (m, Z
rot.) and 2.79 (br m, E rot.) (4H, 30-, 50-H) (Z rot./E rot. ratio: 58/42);
(C-30), 143.22 (C-70a), 140.32 (C-50), 136.28 (Ph C-1), 128.42 (Ph C-3,
-5), 128.16 (Ph C-4), 128.12 (Ph C-2, -6), 121.92 (C-60), 117.39 (C-40),
110.85 (C-30a), 110.73 (C-70), 70.54 (30-OCH2), 70.09 (C-2), 68.46 (C-
4), 48.43 (C-5), 31.98 (C-1); MS (ESþ): m/z 444 [Mþ2 þ Na]þ, 442
[M þ Na]þ, 422 [Mþ2 þ H]þ, 420 [M þ H]þ. Anal. calcd. for
13C NMR [(CD3)2SO, signals of Z rot. (65% of the mixture)]:
d 161.57
C18H18BrN3O4 (420.26): C 51.44; H 4.32; N 10.00. Found: C 51.38; H
(CO),146.03 (C-5),137.40 (C-2),136.72 (C-1),131.24 (C-3),125.59 (C-
4), 123.80 (C-6), 65.85 (C-20, -60), 54.60 (C-30, -50); MS (EI): m/z (%)
285 (2) [M]þ, 254 (4), 201 (12),184 (29),138 (21),110 (18),101 (100),
85 (53). Anal. calcd. for C11H12ClN3O4 (285.68): C 46.25; H 4.23; N
14.71. Found: C 46.38; H 4.10; N 14.48.
4.33; N 10.05.
Further elution of the column with a chloroformemethanol
(50:1) mixture yielded several byproducts among which we were
able to identify 5-(2-benzyl-5-nitro-3-oxo-1,2-dihydro-3H-inda-
zol-1-yl)-3-oxapentyl bromide (18) (0.42 g, 10%); mp 106e108 ꢃC
(ethanol); Rf 0.00 (CHCl3), 0.26 (CHCl3eMeOH 50:1). 1H NMR
4.1.5. 5-Nitro-1H-indazol-1-spiro-40-morpholinium-3-olate (14)
A mixture of hydrazide 13 (4.28 g, 15 mmol) and sodium
hydrogencarbonate (3.36 g, 40 mmol) in ethanol (200 mL) was
refluxed for 24 h. After cooling, the reaction mixture was absorbed
on silica gel and applied to the top of a column of the same material
which was eluted with chloroformemethanol mixtures (10:1 to
5:1), affording the title betaine (3.55 g, 95%); mp 234e238 ꢃC
(CDCl3):
d
8.81 (d, J ¼ 2.2 Hz, 1H, 40-H), 8.34 (dd, J ¼ 9.0, 2.2 Hz, 1H,
60-H), 7.34e7.16 (m, 6H, 70-H, PhH), 5.19 (s, 2H, 20-CH2), 4.02 (t,
J ¼ 4.9 Hz, 2H, 5-H), 3.47 (t, J ¼ 5.7 Hz, 2H, 2-H), 3.44 (t, J ¼ 4.9 Hz,
2H, 4-H), 3.15 (t, J ¼ 5.7 Hz, 2H, 1-H); 13C NMR [(CD3)2SO]:
d 161.09
(C-30), 149.90 (C-70a), 141.04 (C-50), 136.16 (Ph C-1), 128.70 (Ph C-3,
-5), 127.78 (Ph C-4), 127.26 (Ph C-2, -6), 126.58 (C-60), 120.21 (C-40),
115.17 (C-30a), 112.67 (C-70), 69.99 (C-2), 67.03 (C-4), 46.90 þ(C-5),
44.89 (20-CH2), 31.93 (C-1); MS (EI): m/z (%) 421 (21) [Mþ2] , 419
(21) [M]þ, 375 (2), 282 (7), 177 (4), 131 (3), 109 (4), 107 (4), 91 (100).
Anal. calcd. for C18H18BrN3O4 (420.26): C 51.44; H 4.32; N 10.00.
Found: C 51.24; H 4.57; N 10.12.
(ethanol); Rf 0.12 (CHCl3eMeOH 10:1). 1H NMR [(CD3)2SO]:
d 8.58
(dd, J ¼ 8.7, 2.1 Hz, 1H, 6-H), 8.51 (dd, J ¼ 8.7, 0.6 Hz, 1H, 7-H), 8.23
(dd, J ¼ 2.1, 0.6 Hz, 1H, 4-H), 4.31 (m, 4H, 20-, 60-HA and 30-, 50-Hax),
4.05 (m, 2H, 20-, 60-HB), 3.09 (m, 2H, 30-, 50-Heq); 13C NMR
[(CD3)2SO]: d 169.41 (C-3),157.39 (C-7a),149.96 (C-5),132.70 (C-3a),
126.17 (C-6), 119.11 (C-7), 118.06 (C-4), 63.51 (C-30, -50), 62.53 (C-20,
-60); MS (EI): m/z (%) 249 (47) [M]þ, 219 (36), 218 (37),191 (100),161
(52), 146 (33), 145 (45), 133 (21), 117 (32), 103 (23), 90 (55). Anal.
calcd. for C11H11N3O4 (249.22): C 53.01; H 4.45; N 16.86. Found: C
53.08; H 4.49; N 16.79.
4.1.8. 5-(3-Methoxy-5-nitro-1H-indazol-1-yl)-3-oxapentyl iodide
(17)
A mixture of bromide 15 (1.98 g, 6 mmol), methyl iodide (2 mL,
excess) and potassium carbonate (1.93 g, 14 mmol) in acetone
(50 mL) was stirred at room temperature for 24 h and then refluxed
for 2 h. Inorganic salts were removed by filtration and, after
evaporation of acetone and addition of water (100 mL), the mixture
of O- and N2-methyl derivatives was extracted with chloroform
(3 ꢁ 50 mL). This extract was dried (MgSO4), concentrated and
applied to the top of a column which was eluted first with chlo-
roform to afford a 22/78 mixture (1H NMR, molar ratio) of the ex-
pected oxapentyl bromide and the corresponding iodide 17. In
order to complete the halogen exchange, this mixture was dis-
solved in a saturated solution of sodium iodide in acetone (15 mL)
and refluxed for 3 h. The solvent was evaporated to dryness, water
(50 mL) was added and the solid in suspension was extracted with
chloroform (3 ꢁ 50 mL). Evaporation of dried (MgSO4) organic layer
afforded pure iodide 17 (1.17 g, 50%); mp 86e88 ꢃC (methanol); Rf
4.1.6. 5-(3-Hydroxy-5-nitro-1H-indazol-1-yl)-3-oxapentyl bromide
(15)
A mixture of betaine 14 (7.73 g, 31 mmol) and 48% aq hydro-
bromic acid (50 mL) was refluxed for 3 h. After cooling and addition
of water (100 mL), the precipitated solid was collected by filtration,
washed with plenty water and air-dried (8.39 g, 82%); mp 185e
189 ꢃC (ethanol); Rf 0.46 (CHCl3eMeOH 10:1). 1H NMR [(CD3)2SO]:
d
11.45 (br s, 1H, OH), 8.63 (dd, J ¼ 2.2, 0.5 Hz, 1H, 40-H), 8.13 (dd,
J ¼ 9.3, 2.2 Hz, 1H, 60-H), 7.65 (dd, J ¼ 9.3, 0.5 Hz, 1H, 70-H), 4.39 (t,
J ¼ 5.3 Hz, 2H, 5-H), 3.81 (t, J ¼ 5.3 Hz, 2H, 4-H), 3.64 (t, J ¼ 5.5 Hz,
2H, 2-H), 3.43 (t, J ¼ 5.5 Hz, 2H, 1-H); 13C NMR [(CD3)2SO]:
d 156.44
(C-30), 142.74 (C-70a), 139.81 (C-50), 121.44 (C-60), 118.41 (C-40),
111.45 (C-30a), 110.40 (C-70), 70.12 (C-2), 68.51 (C-4), 48.13 (C-5),
31.99 (C-1); MS (EI): m/z (%) 331 (20) [Mþ2]þ, 329 (20) [M]þ, 285
(1), 249 (16), 218 (3), 206 (22), 192 (100), 176 (14), 160 (6), 146 (50),
107 (7), 91 (6). Anal. calcd. for C11H12BrN3O4 (330.13): C 40.02; H
3.66; N 12.73. Found: C 39.97; H 3.68; N 12.81.
0.12 (CHCl3), 0.86 (CHCl3eMeOH 10:1). 1H NMR (CDCl3):
d 8.59 (d,
J ¼ 2.0 Hz, 1H, 40-H), 8.19 (dd, J ¼ 9.3, 2.0 Hz, 1H, 60-H), 7.35 (d,
J ¼ 9.3 Hz, 1H, 70-H), 4.37 (t, J ¼ 5.1 Hz, 2H, 5-H), 4.08 (s, 3H, OCH3),
3.86 (t, J ¼ 5.1 Hz, 2H, 4-H), 3.59 (t, J ¼ 6.5 Hz, 2H, 2-H), 3.06 (t,
J ¼ 6.5 Hz, 2H, 1-H); 13C NMR [(CD3)2SO]:
d
157.52 (C-30), 143.32 (C-

B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
131
70a), 140.27 (C-50), 121.94 (C-60), 117.36 (C-40), 110.81 (C-70), 110.68
(C-30a), 70.57 (C-2), 68.18 (C-4), 56.52 (OCH3), 48.44 (C-5), 5.10 (C-
1); MS (EI): m/z (%) 391 (38) [M]þ, 264 (1), 220 (17), 206 (100), 190
(7), 174 (6), 160 (42), 155 (9), 131 (3), 103 (6), 90 (4), 89 (4). Anal.
calcd. for C12H14IN3O4 (391.16): C 36.85; H 3.61; N 10.74. Found: C
36.93; H 3.40; N 10.81.
193 (49), 174 (18), 160 (20), 149 (7), 131 (4), 117 (5), 103 (14), 89 (8).
Anal. calcd. for C13H18N4O4 (294.31): C 53.05; H 6.16; N 19.04.
Found: C 53.19; H 6.01; N 19.15.
4.1.11. N-Methylbis[5-(3-benzyloxy-5-nitro-1H-indazol-1-yl)-3-
oxapentyl]amine (3)
Further elution of the column with a chloroformemethanol
(50:1) mixture yielded N2-methyl derivatives [22/78 mixture (1H
NMR, molar ratio) of oxapentyl bromide and the corresponding
iodide], which after treatment with sodium iodide in acetone as
described afforded 5-(2-methyl-5-nitro-3-oxo-1,2-dihydro-3H-
indazol-1-yl)-3-oxapentyl iodide (19) (0.98 g, 42%); mp 127e129 ꢃC
(2-propanol); Rf 0.00 (CHCl3), 0.55 (CHCl3eMeOH 10:1). 1H NMR
A mixture of amine 2 (0.89 g, 2.4 mmol), bromide 16 (1.01 g,
2.4 mmol) and potassium carbonate (0.35 g, 2.5 mmol) in aceto-
nitrile (100 mL) was refluxed for 48 h. After filtration of salts and
evaporation of solvent, the desired product was isolated by column
chromatography; elution with chloroformemethanol (100:1 to
50:1) mixtures afforded the title tertiary amine 3 (1.45 g, 85%); Rf
0.00 (CHCl3), 0.36 (CHCl3eMeOH 10:1). Mp (96e98 ꢃC) as well as
analytical and some spectral data [1H NMR and MS (ESþ)] have
(CDCl3):
d
8.72 (d, J ¼ 2.2 Hz, 1H, 40-H), 8.33 (dd, J ¼ 9.2, 2.2 Hz, 1H,
60-H), 7.26 (d, J ¼ 9.2 Hz, 1H, 70-H), 4.15 (t, J ¼ 4.9 Hz, 2H, 5-H), 3.58
previously been reported [23]. 13C NMR [(CD3)2SO]: 156.70 (C-30),
d
(t, J ¼ 4.9 Hz, 2H, 4-H), 3.50 (s, 3H, CH3), 3.48 (t, J ¼ 6.2 Hz, 2H, 2-H),
143.18 (C-70a), 140.24 (C-50), 136.25 (Ph C-1), 128.42 (Ph C-3, -5),
128.17 (Ph C-4), 128.13 (Ph C-2, -6), 121.80 (C-60), 117.35 (C-40),
110.75 (C-30a), 110.72 (C-70), 70.52 (30-OCH2), 68.66 (C-2), 68.53 (C-
4), 56.45 (C-1), 48.59 (C-5), 42.40 (CH3).
2.96 (t, J ¼ 6.2 Hz, 2H, 1-H); 13C NMR [(CD3)2SO]:
d
160.07 (C-30),
148.36 (C-70a), 140.72 (C-50), 126.29 (C-60), 120.05 (C-40), 115.10 (C-
30a), 112.01 (C-70), 70.55 (C-2), 66.97 (C-4), 46.62 (C-5), 29.06 (CH3),
4.91 (C-1); MS (EI): m/z (%) 391 (87) [M]þ, 375 (1), 273 (1), 220 (1),
206 (100), 192 (9), 190 (11), 177 (4), 160 (51), 155 (21), 146 (9), 131
(10), 119 (2), 104 (5), 103 (5), 91 (4). Anal. calcd. for C12H14IN3O4
(391.16): C 36.85; H 3.61; N 10.74. Found: C 36.83; H 3.52; N 10.63.
Alternatively, halogen exchange can be completed before chro-
matographic separation of O- and N2-methyl derivatives.
4.1.12. N-Methylbis[5-(3-methoxy-5-nitro-1H-indazol-1-yl)-3-
oxapentyl]amine (6)
This compound was prepared as described for 3-benzyloxy
analogue 3, starting from amine 5 (0.53 g, 1.8 mmol), iodide 17
(0.70 g, 1.8 mmol) and potassium carbonate (0.28 g, 2 mmol) in
acetonitrile (50 mL); column chromatography, eluted with
dichloromethane-methanol mixtures (50:1 to 20:1), afforded the
desired compound 6 (0.83 g, 83%); Rf 0.31 (CHCl3eMeOH 10:1). Mp
(122e124 ꢃC) as well as analytical and some spectral data [1H NMR
and MS (EI)] have previously been reported [23]. 13C NMR (CDCl3)
4.1.9. N-Methyl-5-(3-benzyloxy-5-nitro-1H-indazol-1-yl)-3-
oxapentylamine (2)
A mixture of bromide 16 (1.68 g, 4 mmol) and a 33% (ca. 8 M)
solution of methylamine in ethanol (50 mL), was stirred at room
temperature for 24 h. The mixture was then evaporated to dryness
and, after addition of 2 N aq hydrochloric acid (50 mL), extracted
with diethyl ether (3 ꢁ 50 mL). Solid potassium carbonate was
added to the acidic layer until pH 10 and the resulting suspension
extracted with chloroform (3 ꢁ 50 mL). The organic layer was
separated, dried (MgSO4) and evaporated to afford practically pure
amine 2 (1.36 g, 92%); mp 84e86 ꢃC (2-propanol); Rf 0.00 (CHCl3),
d
158.31 (C-30), 143.57 (C-70a), 140.74 (C-50), 122.17 (C-60), 118.28 (C-
40), 111.77 (C-30a), 109.46 (C-70), 69.57 (C-2), 69.41 (C-4), 57.12 (C-1),
56.49 (OCH3), 49.20 (C-5), 42.88 (NCH3).
4.1.13. 5-(3-Methoxy-5-nitro-1H-indazol-1-yl)-3-oxapentylamine
(7) and bis[5-(3-methoxy-5-nitro-1H-H-indazol-1-yl)-3-oxapentyl]
amine (8)
0.08 (CHCl3eMeOH 10:1). 1H NMR [(CD3)2SO]:
d
8.52 (d, J ¼ 2.1 Hz,
1H, 40-H), 8.18 (dd, J ¼ 9.2, 2.1 Hz, 1H, 60-H), 7.72 (d, J ¼ 9.2 Hz, 1H,
70-H), 7.55 (m, 2H, Ph 2-, 6-H), 7.40 (m, 3H, Ph 3-, 4-, 5-H), 5.43 (s,
2H, 30-OCH2), 4.46 (t, J ¼ 4.9 Hz, 2H, 5-H), 3.75 (t, J ¼ 4.9 Hz, 2H, 4-
H), 3.34 (t, J ¼ 5.6 Hz, 2H, 2-H), 2.41 (t, J ¼ 5.6 Hz, 2H, 1-H), 2.10 (s,
A stirred suspension of iodide 17 (1.14 g, 2.91 mmol) in a satu-
rated solution of ammonia in ethanol (30 mL) was heated in an
autoclave at 70 ꢃC for 72 h. The mixture was then evaporated to
dryness and, after addition of 10% aq potassium carbonate (50 mL),
extracted with chloroform (3 ꢁ 50 mL). The organic layer was dried
(MgSO4), concentrated and applied to the top of a flash chroma-
tography column which was eluted with chloroformemethanol
mixtures (20:1 to 5:1) to afford, in this elution order, secondary
amine 8 (94 mg, 12%) and primary amine 7 (700 mg, 86%).
Compound 7: mp 70e72 ꢃC (previous sintering) (hexane); Rf
3H, CH3); 13C NMR [(CD3)2SO]: 156.76 (C-30),143.27 (C-70a),140.32
d
(C-50), 136.27 (Ph C-1), 128.44 (Ph C-3, -5), 128.20 (Ph C-4), 128.18
(Ph C-2, -6), 121.89 (C-60), 117.45 (C-40), 110.79 (C-30a), 110.75 (C-70),
70.56 (30-OCH2), 69.67 (C-2), 68.64 (C-4), 50.61 (C-1), 48.59 (C-5),
35.94 (CH3); MS (ESþ): m/z 393 [M þ Na]þ, 371 [M þ H]þ. Anal.
calcd. for C19H22N4O4 (370.40): C 61.61; H 5.99; N 15.13. Found: C
61.35; H 6.10; N 15.07.
0.06 (CHCl3eMeOH 10:1). 1H NMR [(CD3)2SO]:
d
8.46 (d, J ¼ 2.0 Hz,
1H, 40-H), 8.17 (dd, J ¼ 9.5, 2.0 Hz, 1H, 60-H), 7.70 (d, J ¼ 9.5 Hz, 1H,
70-H), 4.45 (t, J ¼ 4.9 Hz, 2H, 5-H), 4.04 (s, 3H, OCH3), 3.76 (t,
J ¼ 4.9 Hz, 2H, 4-H), 3.28 (t, J ¼ 5.5 Hz, 2H, 2-H), 2.48 (t, J ¼ 5.5 Hz,
4.1.10. N-Methyl-5-(3-methoxy-5-nitro-1H-indazol-1-yl)-3-
oxapentylamine (5)
Starting from iodide 17 (0.59 g, 1.5 mmol) and a 33% (ca. 8 M)
solution of methylamine in ethanol (30 mL), following the proce-
dure described for compound 2, pure amine 5 was obtained (0.41 g,
93%); mp 71e73 ꢃC (hexane) [hydrochloride (5ꢁ HCl), mp 202e
204 ꢃC (ethanol)]; Rf 0.04 (CHCl3eMeOH 10:1). 1H NMR (CDCl3)
2H, 1-H); 13C NMR [(CD3)2SO]: 157.47 (C-30), 143.27 (C-70a), 140.22
d
(C-50), 121.88 (C-60), 117.36 (C-40), 110.67 (C-70), 110.55 (C-30a), 72.83
(C-2), 68.55 (C-4), 56.48 (OCH3), 48.53 (C-5), 41.17 (C-1); MS (ESþ):
m/z 303 [M þ Na]þ, 281 [M þ H]þ. Anal. calcd. for C12H16N4O4
(280.28): C 51.42; H 5.75; N 19.99. Found: C 51.57; H 5.89; N 19.72.
Compound 8: mp 105e107 ꢃC (2-propanol); Rf 0.37 (CHCl3e
d
8.59 (d, J ¼ 2.1 Hz, 1H, 40-H), 8.18 (dd, J ¼ 9.3, 2.1 Hz, 1H, 60-H), 7.30
(d, J ¼ 9.3 Hz, 1H, 70-H), 4.35 (t, J ¼ 5.1 Hz, 2H, 5-H), 4.07 (s, 3H,
MeOH 10:1). 1H NMR [(CD3)2SO]:
d
8.40 (d, J ¼ 2.1 Hz, 2H, 40-H),
OCH3), 3.81 (t, J ¼ 5.1 Hz, 2H, 4-H), 3.44 (t, J ¼ 5.1 Hz, 2H, 2-H), 2.56
8.08 (dd, J ¼ 9.3, 2.1 Hz, 2H, 60-H), 7.59 (d, J ¼ 9.3 Hz, 2H, 70-H), 4.38
(t, J ¼ 4.9 Hz, 4H, 5-H), 4.01 (s, 6H, OCH3), 3.69 (t, J ¼ 4.9 Hz, 4H, 4-
H), 3.24 (t, J ¼ 5.4 Hz, 4H, 2-H), 2.35 (t, J ¼ 5.4 Hz, 4H, 1-H); 13C NMR
(t, J ¼ 5.1 Hz, 2H,1-H), 2.26 (s, 3H, NCH3); 13C NMR (CDCl3):
d 158.35
(C-30), 143.60 (C-70a), 140.78 (C-50), 122.20 (C-60), 118.37 (C-40),
111.83 (C-30a), 109.23 (C-70), 70.47 (C-2), 69.44 (C-4), 56.45 (OCH3),
51.09 (C-1), 49.16 (C-5), 36.17 (NCH3); MS (EI): m/z (%) 295 (5)
[M þ H]þ, 294 (1) [M]þ, 263 (8), 251 (7), 237 (7), 219 (100), 206 (29),
[(CD3)2SO]:
d
157.43 (C-30), 143.17 (C-70a), 140.12 (C-50), 121.74 (C-
60), 117.25 (C-40), 110.57 (C-70), 110.53 (C-30a), 70.00 (C-2), 68.59 (C-
4), 56.43 (OCH3), 48.49 (C-5), 48.39 (C-1); MS (ESþ): m/z 566

132
B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
[M þ Na]þ, 544 [M þ H]þ. Anal. calcd. for C24H29N7O8 (543.53): C
4.2.5. In vitro activity: trypomastigotes assay (extracellular forms)
Metacyclogenesis was induced by culturing a 5-day-old culture
of epimastigotes that was harvested by centrifugation at 7000 g for
10 min at 10 ꢃC. The parasites were then incubated for 2 h at 28 ꢃC at
a density of 5 ꢁ 108 cells/mL in TAU medium (190 mM NaCl, 17 mM
KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM phosphate buffer, pH 6.0).
Thereafter, the parasites were incubated at a 1:100 dilution (final
epimastigotes concentration: 5 ꢁ 106 cells/mL) for 96 h at 28 ꢃC in
53.03; H 5.38; N 18.04. Found: C 52.96; H 5.34; N 17.91.
4.2. Biological evaluation
4.2.1. Parasite strain culture
Epimastigotes of T. cruzi SN3 strain (IRHOD/CO/2008/SN3) iso-
lated from domestic Rhodnius prolixus from Guajira (Colombia) [24]
were cultivated in vitro in medium trypanosomes liquid (MTL) with
10% inactivated foetal bovine serum and were kept in an air at-
mosphere at 28 ꢃC in Roux flasks (Corning, USA) with a surface area
of 75 cm2, according to a previously described methodology [25].
TAU3AAG medium (TAU supplemented with 10 mM
50 mM sodium -glutamate, 2 mM sodium -aspartate and 10 mM
-glucose) in 25 mL culture flasks with a layer of culture medium
that was not more than 1 cm in depth [27].
L-proline,
L
L
D
The activity (% of parasites reduction) was compared with the
control following a reported methodology [28] with some minor
modifications performed in our laboratory. The assay of drug ac-
tivity against T. cruzi was carried out using blood from Balb/c albino
mice collected during the parasitemia peak (7th day) after infection
with the SN3 strain of T. cruzi. The infected blood was diluted with
non-infected murine blood to the concentration of
4 ꢁ 106 trypomastigotes/mL and then diluted to 1:2 in RPMI 1640
mediumeGIBCO (2 ꢁ 106 trypomastigotes/mL). Stock solutions of
the compounds were prepared in dimethyl sulfoxide. A sample of
4.2.2. Cell culture and cytotoxicity tests
Vero cells (EACC number 84113001) originally obtained from
monkey kidney were grown in RPMI (Gibco), supplemented with
10% inactivated foetal bovine serum in a humidified 95% air, 5% CO2
atmosphere at 37 ꢃC for two days. The cytotoxicity test for Vero cells
was performed according to a previously described methodology
[25]. After 72 h of treatment, cell viability was determined by flow
cytometry. Thus, 100
mL) was added and incubated for 10 min at 28 ꢃC in darkness. Af-
terwards, 100 L/well of fluorescein diacetate (100 ng/mL) was
mL/well of propidium iodide solution (100 mg/
infected blood and drugs (100, 50, 25, 10 and 1
mM) were added to
m
the well of a 96-microwell plate providing a final volume of 200 mL
added and incubated under the same conditions. Finally, the cells
were recovered by centrifugation at 400 g for 10 min and the
precipitate washed with phosphate buffered saline (PBS). Flow
cytometric analysis was performed with a FACSVantage flow cy-
tometer (Becton Dickinson). The percentage viability was calcu-
lated in comparison with the control culture. The IC50 was
calculated using linear regression analysis from the Kc values of the
in order to calculate IC50 values. To reproduce the blood bank
conditions, plates were incubated at 4e8 ꢃC for 24 h. The experi-
ments were repeated three times. Each solution was examined
microscopically for parasite counting using the Neubauer chamber.
4.2.6. In vitro activity: infection assays
Vero cells were grown under the same conditions expressed in
the amastigotes assay during two days. Afterwards, the cells were
infected in vitro with metacyclic forms of T. cruzi at a ratio of 10:1.
The drugs (IC25 concentrations) were added immediately after
infection and were incubated for 12 h at 37 ꢃC in 5% CO2. The non-
phagocytosed parasites and the drugs were removed by washing,
and then the infected cultures were grown for 10 days in fresh
medium. Fresh culture medium was added every 48 h. The drug
activity was determined from the percentage of infected cells and
the number of amastigotes per infected cell and that of trypo-
mastigotes in the medium was also determined [29], in treated and
untreated cultures, in methanol-fixed and Giemsa-stained prepa-
rations. The percentage of infected cells and the mean number of
amastigotes per infected cell were determined by analysing 200
host cells distributed in randomly chosen microscopic fields. Values
represented in Fig. 2 are the means of three separate experiments;
in all cases experimental values fall within two standard deviations
from the mean.
concentrations employed (1e100 mM).
4.2.3. In vitro activity: epimastigotes assay (extracellular forms)
According to a reported procedure [25], the obtained com-
pounds and the reference drug (benznidazole) were dissolved in
dimethyl sulfoxide, which at a final concentration of 0.01% was
shown to be nontoxic and without inhibitory effects on parasite
growth. The compounds were added to the culture medium at
dosages of 100, 50, 25, 10 and 1 mM. The effects of each compound
against T. cruzi epimastigotes were tested at 72 h using a Neubauer
haemocytometric chamber. The antichagasic effect is expressed as
the IC50, i.e. the concentration required to give 50% of growth in-
hibition, calculated by linear regression analysis from the Kc values
of the concentrations employed. Results gathered in Table 1 are
averages of four separate experiments.
4.2.4. In vitro activity: amastigotes assay (intracellular forms)
Vero cells were grown in RPMI (Gibco) supplemented with 10%
inactivated foetal bovine serum and were kept in a humidified at-
mosphere of 95% air and 5% CO2 at 37 ꢃC. Cells were seeded at a
density of 1 ꢁ 104 cells/well in 24-well microplates (Nunc) with
rounded coverslips on the bottom and cultured for 2 days. After-
wards the cells were infected in vitro with metacyclic forms [26] of
T. cruzi at a ratio of 10:1 during 24 h. The non-phagocytosed par-
asites were removed by washing, and then the drugs (at 100, 50, 25,
4.2.7. In vivo trypanocidal activity assay
Groups of three BALB/c female mice (6e8 weeks old, 20e25 g)
maintained under standard conditions were infected with 1 ꢁ 105
T. cruzi metacyclic forms by the intraperitoneal route. The animals
were divided into the following groups: (I) group 1: uninfected (not
infected and not treated); (II) group 2: untreated (infected with
T. cruzi but not treated); (III) group 3: uninfected [not infected and
treated with 1 mg/kg body weight/day, for five consecutive days
(5e10 days postinfection) by the intraperitoneal route] [30] and
(IV) group 4: treated [infected and treated for five consecutive days
(5e10 days postinfection) with the tested compounds and benz-
nidazole]. This animal experiment was performed with the
approval of the ethical committee of the University of Granada.
10 and 1 mM) were added. Vero cells with the drugs were incubated
for 72 h at 37 ꢃC in 5% CO2. Drug activity was determined on the
basis of number of amastigotes in treated and untreated cultures in
methanol-fixed and Giemsa-stained preparations. The number of
amastigotes was determined by analysing 200 host cells distributed
in randomly chosen microscopic fields. The antichagasic effect is
expressed as the IC50. Results given in Table 1 are averages of four
separate experiments.
A blood sample (5
treated mouse was taken and diluted 1:15 (50
buffer:0.1 M citric acid, 0.1 M sodium citrate and 20
m
L) drawn from the mandibular vein of each
L of citrate
L of lysis
m
m

B. Muro et al. / European Journal of Medicinal Chemistry 74 (2014) 124e134
nitroindazole derivatives, Bioorg. Med. Chem. 13 (2005) 3197e3207;
133
buffer at pH 7.2:2 M TriseCl, MgCl2). The parasites were counted by
the Neubauer chamber. The number of bloodstream T. cruzi forms
was recorded every 3e4 days from 5 to 30 days postinfection. The
number of trypomastigote forms was expressed as trypomasti-
gotes/mL. The obtained mean values are gathered in Fig. 3; exper-
imental values lie in each case within two standard deviations from
the mean.
Circulating anti-T. cruzi antibodies, at days 30 and 120 post-
infection, were evaluated quantitatively by an enzyme-linked
immunoassay. The blood, diluted to 1:50 in PBS, was reacted with
an antigen composed of an excreted FeeSOD of T. cruzi epi-
mastigotes [31]. The results were expressed as the ratio of the
absorbance of each sample at 490 nm to the cutoff value. The cutoff
for each reaction was the mean of the values determined for the
negative controls plus three times the standard deviation.
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Cultures of T. cruzi epimastigotes (initial concentration
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chemical shifts were expressed in parts per million (ppm,
d scale),
using sodium 2,2-dimethyl-2-silapentane-5-sulphonate as the
reference signal. The chemical shifts used to identify the respective
metabolites were consistent with those previously described [32].
4.2.9. Ultrastructural alterations
Epimastigotes of T. cruzi were cultured at
a density of
5 ꢁ 105 cells/mL in the corresponding medium containing the
compounds tested at their IC25 concentrations. After 96 h, these
cultures were centrifuged at 400 g for 10 min, and the pellets
produced were washed in PBS and then mixed with 2% (v/v)
paraformaldehyde/glutaraldehyde in 0.05 M cacodylate buffer (pH
7.4) for 4 h at 4 ꢃC. Following this, the pellets were prepared for
transmission electron microscopy (TEM) study using a previously
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The authors thank the Spanish MICINN for financial support
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F.R. acknowledges a CSIC JAE-Doc contract co-funded by EU (ESF,
European Social Fund).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.12.
025. These data include MOL files and InChiKeys of the most
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