Antichagasic and trichomonacidal activity of 1-substituted 2-benzyl-5-nitroindazolin-3-ones and 3-alkoxy-2-benzyl-5-nitro-2H-indazoles

Article abstract of DOI:10.1016/j.ejmech.2016.03.036

Two series of new 5-nitroindazole derivatives, 1-substituted 2-benzylindazolin-3-ones (6-29, series A) and 3-alkoxy-2-benzyl-2H-indazoles (30-37, series B), containing differently functionalized chains at position 1 and 3, respectively, have been synthesi

Full text of DOI:10.1016/j.ejmech.2016.03.036

European Journal of Medicinal Chemistry 115 (2016) 295e310
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Antichagasic and trichomonacidal activity of 1-substituted 2-benzyl-
5-nitroindazolin-3-ones and 3-alkoxy-2-benzyl-5-nitro-2H-
indazoles*
,
b
a
,
, c
Cristina Fonseca-Berzal a , Alexandra Ibanez-Escribano b, Felipe Reviriego a
,
ꢀ~
c
c
c
a, b
ꢀ
ꢀ
Jose Cumella , Paula Morales , Nadine Jagerovic , Juan Jose Nogal-Ruiz
,
a
,
b
d
d
ꢀ
ꢀ
Jose Antonio Escario , Patricia Bernardino da Silva , Maria de Nazare C. Soeiro ,
a,
b,
**
a, c, *
ꢀ
ꢀ
Alicia Gomez-Barrio
, Vicente J. Aran
a Moncloa Campus of International Excellence (UCM-UPM & CSIC), Spain
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramon y Cajal s/n, 28040, Madrid, Spain
Instituto de Química Medica (IQM), Consejo Superior de Investigaciones Científicas (CSIC), c/Juan de la Cierva 3, 28006, Madrid, Spain
Laboratorio de Biologia Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21040-900, Rio de Janeiro, Brazil
b
ꢀ
c
ꢀ
d
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of new 5-nitroindazole derivatives, 1-substituted 2-benzylindazolin-3-ones (6e29, series A)
and 3-alkoxy-2-benzyl-2H-indazoles (30e37, series B), containing differently functionalized chains at
position 1 and 3, respectively, have been synthesized starting from 2-benzyl-5-nitroindazolin-3-one 5,
and evaluated against the protozoan parasites Trypanosoma cruzi and Trichomonas vaginalis, etiological
agents of Chagas disease and trichomonosis, respectively. Many indazolinones of series A were efficient
against different morphological forms of T. cruzi CL Brener strain (compounds 6, 7, 9, 10 and 19e21:
Received 23 November 2015
Received in revised form
26 February 2016
Accepted 14 March 2016
Available online 17 March 2016
IC50 ¼ 1.58e4.19
m
M for epimastigotes; compounds 6, 19e21 and 24: IC50 ¼ 0.22e0.54
mM for amasti-
Keywords:
gotes) being as potent as the reference drug benznidazole. SAR analysis suggests that electron-donating
groups at position 1 of indazolinone ring are associated with an improved antichagasic activity. More-
over, compounds of series A displayed low unspecific toxicities against an in vitro model of mammalian
cells (fibroblasts), which were reflected in high values of the selectivity indexes (SI). Compound 20 was
Antiprotozoal drug
Nitroheterocycle
Indazole
Trichomonas vaginalis
Trypanosoma cruzi
also very efficient against amastigotes from Tulahuen and Y strains of T. cruzi (IC50 ¼ 0.81 and 0.60
respectively), showing low toxicity towards cardiac cells (LC50 > 100 M). In what concerns compounds
of series B, some of them displayed moderate activity against trophozoites of a metronidazole-sensitive
isolate of T. vaginalis (35 and 36: IC50 ¼ 9.82 and 7.25 M, respectively), with low unspecific toxicity
towards Vero cells. Compound 36 was also active against metronidazole-resistant isolate
(IC50 ¼ 9.11 M) and can thus be considered a good prototype for the development of drugs directed to
T. vaginalis resistant to 5-nitroimidazoles.
mM,
m
m
a
m
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
Our research group has been pursuing during the last decade
the identification of alternative therapeutic options for parasitic
protozoan infections through the design, synthesis and biological
activity analysis of novel nitroheterocycles. In these previous
studies we reported the activity against Trypanosoma cruzi [1e8],
Trypanosoma brucei [9], Leishmania spp. [2,10] and Trichomonas
vaginalis [1,11e13] of 5-nitroindazole derivatives, mainly 1-
substituted indazol-3-ols [11,12], 2-substituted indazolin-3-ones
[11], 1-substituted 3-alkoxyindazoles [1e5,7,9], 1,2-disubstituted
*
ꢀ
Dedicated to our dear colleague Dr. María Teresa García Lopez, former director
ꢀ
of Instituto de Química Medica (IQM, CSIC, Madrid) for her outstanding contribu-
tion to Medicinal Chemistry, on the occasion of her retirement.
* Corresponding author. Instituto de Química Medica (IQM), Consejo Superior de
Investigaciones Científicas (CSIC), c/Juan de la Cierva 3, 28006, Madrid, Spain.
** Corresponding author. Departamento de Parasitología, Facultad de Farmacia,
Universidad Complutense de Madrid, Plaza Ramon y Cajal s/n, 28040, Madrid,
Spain.
ꢀ
ꢀ
ꢀ
E-mail addresses: agbarrio@ucm.es (A. Gomez-Barrio), vjaran@iqm.csic.es
ꢀ
(V.J. Aran).
http://dx.doi.org/10.1016/j.ejmech.2016.03.036
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

296
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
indazolin-3-ones [5,6,8,13] and 2-substituted 3-alkoxyindazoles
[5,6,13].
to recent data from the World Health Organization, the number of
new cases in adults in 2008 was estimated to be 276.4 million [30].
This protozoan is transmitted only through sexual contact. It is
characterized by a wide range of clinical manifestations that can
cause severe inflammation of genitourinary passages accompanied
by a characteristic vaginal discharge, erythema, pruritus, dysuria,
infertility or the formation of small lesions in the cervix. In men, the
infection can result in non-gonococcal urethritis (NGU) and
impaired sperm viability and motility [31,32]. Human trichomo-
nosis has been associated with various complications such as
problems during pregnancy, “low birth weight” (LBW), preterm
births, etc. [33]. This infection also increases the risk of develop-
ment of cervical [34] and prostate [35] neoplasia, and it is also
related to a greater predisposition to co-infection with other STIs of
bacterial or viral origin [36e38]. However, epidemiological studies
have shown that at least half of the infected women and 80% of men
do not show symptoms, becoming asymptomatic carriers and po-
tential transmitters of this infection [39].
Trichomonosis is preferably treated with metronidazole, intro-
duced to the market by 1960. Currently, metronidazole and tini-
dazole, both from the same family of 5-nitroimidazoles [40], are the
only two drugs approved by the Food and Drug Administration
(FDA) to treat this STI [41]. However, there are no effective alter-
natives for patients who develop side effects or hypersensitivity, or
when its use is contraindicated. Moreover, it is estimated that
approximately 5e10% of diagnosed cases of trichomonosis are
caused by nitroimidazoles-resistant isolates [42,43].
In the present study our efforts have been centered on new
indazole derivatives focusing on two parasitic infections, Chagas
disease and trichomonosis. The reference drugs for both diseases,
i.e., nifurtimox and benznidazole for the former, and metronidazole
for the latter, are nitroheterocyclic compounds [14], and further-
more our 5-nitroindazole derivatives have previously shown, as
mentioned above, activity against both protozoan parasites.
Chagas disease is a silent chronic infection caused by the try-
panosomatid (Kinetoplastid) protozoan parasite T. cruzi. This
parasitosis, originally endemic to poor rural areas of Latin America,
from Mexico to Argentina, is an anthropozoonosis, mainly trans-
mitted in this area by hematophagous triatomine insects [15,16].
The disease is currently endemic to 21 countries, where causes
more than 7000 deaths per year and maintains over 25 million
people at risk for the infection [17]. Now, owing to intense inter-
national migrations and secondary routes of transmission (i.e.,
transfusion of infected blood, contaminated organ transplantation
or congenitally), it can also be found in non-endemic areas such as
Western Europe, USA, or Australia [18], currently affecting about 7
million people worldwide [17].
The initial acute phase of Chagas disease, which appears after 1
week of T. cruzi infection, has low (<10%) mortality usually related
to heart failure and/or meningoencephalitis, but it is often
asymptomatic [15,19]. After 1e2 months, immune response
partially controls the infection but cannot eradicate it completely.
Although the infection persists for life, about 60e70% of untreated
infected people never develop clinical manifestations, entering in
an indeterminate phase of the disease lacking organ affectation.
However, the remaining 30e40%,10e30 years after initial infection,
evolve the symptomatic chronic phase of the disease, characterized
by a life-threatening cardiomyopathy and/or severe digestive
problems (e.g. megacolon and megaesophagus). In fact, Chagas
infection significantly contributes to the global burden of cardio-
vascular disease [15,16]. The differences noticed in clinical pre-
sentations occur as a consequence of six discrete typing units (DTU)
of T. cruzi (TcIeTcVI), which along the endemic area show differ-
ences in their geographical distribution, ecotopes and mammalian
host [20].
The drugs currently used to treat Chagas disease, nifurtimox and
benznidazole, are decades old and have many limitations, including
severe side-effects, low efficacy specially at the later chronic stage
of the disease [21], and other important inconveniences such as the
different response to the treatment registered among divergent
T. cruzi strains [20]. Although benznidazole is the first-line treat-
ment in most countries [17], the drawbacks of the current therapy
make the search for new drugs urgently needed; it is especially
necessary to find effective and safe compounds for the treatment of
prevalent late chronic stage of the disease, where the effectiveness
of benznidazole, despite some controversial reports [19,21], seems
to be very low, especially to prevent heart damage progression [22].
In recent years many compounds exhibiting anti-T. cruzi activity,
as well as different potential molecular targets of parasite, have
been reported [21,23e26].
Antifungal azoles acting as sterols biosynthesis inhibitors such
as posaconazole or ravuconazole seemed to be very promising
candidates but recent clinical studies have been rather disap-
pointing, showing that azoles are less effective than benznidazole,
at least when used as monotherapy [15,27]. In fact, it has recently
been pointed out that nitroheterocyclic derivatives still represent
the only real alternative in the antichagasic fight [28].
Therefore, the search for alternative drugs for the treatment of
both parasitic pathologies is urgently needed.
In the present study we have synthesized and phenotypically
evaluated in vitro against T. cruzi and T. vaginalis two main series of
new indazole derivatives: 1-substituted 2-benzyl-5-nitroindazolin-
3-ones 6e29 (Scheme 1, series A) and 3-alkoxy-2-benzyl-2H-
indazoles 30e37 (Scheme 1, series B), as well as some other
products, i.e., bisindazoles 38 and 39, and quinazolinones 40e43,
arising from the synthetic procedures.
The preparation and study of both series of compounds were
planned considering the previously reported activity against T. cruzi
[5,6,8] and T. vaginalis [13] of related compounds containing
different moieties at position 2 (Me, Ph, Bn, phenethyl and 2-
naphthylmethyl) and alkyl or aryl substituents (Me, Pr, iPr, Bu, Pe,
Bn and Ph) at position 1 of indazolin-3-one ring (related to series A)
or at 3-O of 2H-indazol-3-ol system (related to series B). The most
active compounds against T. cruzi epimastigotes (1e3;
IC50
IC50 ¼ 18.51
¼
0.93e2.39
mM) [5] and T. vaginalis trophozoites (4;
mM) [13] from previous studies are gathered in Fig. 1.
The current new compounds 6e37 have been designed to
explore the antiprotozoal activity of indazoles containing at the
mentioned 1 or 3-O positions unsaturated moieties,
u-substituted
alkyl chains supporting different functional groups or acyl and
sulfonyl residues. Taking in mind that in previous reports [5,6] 2-
benzyl group led to the best antichagasic activities, this substitu-
ent was maintained in the current series A and B. In relation to
physicochemical properties, some of these chemical modifications
have also been planned in order to improve the low water solubility
of the previously studied indazole derivatives [5].
2. Results and discussion
2.1. Chemistry
Many compounds gathered in Scheme 1 were obtained by
alkylation of the previously reported [44] 2-benzyl-5-
nitroindazolin-3-one 5 with the required functionalized halides.
These alkylation reactions afforded, as previously described [5],
Regarding trichomonosis, T. vaginalis (fam. Trichomonadidae) is
the causative agent of this sexually transmitted infection (STI) ac-
counting for over 50% of all curable STIs worldwide [29]. According

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
297
Scheme 1. Synthesis of 1-substituted 2-benzyl-5-nitroindazolin-3-ones 6e29 and 3-alkoxy-2-benzyl-5-nitro-2H-indazoles 30e37.

298
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
Fig. 1. Structure of previously studied antichagasic (1e3) and trichomonacidal (4) 5-
nitroindazole derivatives.
mixtures of the corresponding 1,2-disubstituted indazolin-3-ones
(7e13, 15 and 20e22) and 3-alkoxy-2-alkyl-2H-indazoles
(30e37); the latter were usually minor reaction products which in
some cases were not isolated. Most alkylation reactions were car-
ried out in potassium carbonate/DMF at 100 ꢀC. Under these con-
ditions, however, alkylation with methyl bromoacetate or
bromoacetamide afforded quinazolinones 40 and 41, respectively
(Scheme 2). This rearrangement, due to the acidity of CH2 protons
of methyl acetate or acetamide moieties, has been previously
observed for other 1,2-disubstituted indazolinones [45,46] and for
indazole-derived betaines [47]. Since the alkylation reactions were
carried out with an excess of the corresponding bromoacetic acid
derivative (methyl ester or amide), further alkylation of compounds
40 and 41 at position 1 to yield derivatives 42 and 43 took also
place. Nevertheless, alkylation of compound 5 with methyl bro-
moacetate or bromoacetonitrile in sodium hydrogen carbonate/
acetone at 35 ꢀC afforded the desired compounds 11/32 and 12,
respectively.
Fig. 2. Structure of bisindazole derivatives 38 and 39.
chloroformates or with the corresponding acyl or sulfonyl chlorides
in pyridine afforded 1-alkoxycarbonyl- (25, 26), 1-benzoyl- (28)
and 1-tosyl- (29) indazolinones. 1-Acetyl derivative 27 was pre-
pared from compound 5 and acetic anhydride. In agreement with
previous reports, only the corresponding 1,2-disubstituted de-
rivatives could be isolated from these processes [45].
Compounds 6, 14, 16e19, 23 and 24 (Scheme 1) were obtained
following different procedures from 1,2-disubstituted indazoli-
nones arising from the above mentioned alkylation reactions. Thus,
1-vinyl derivative 6 was prepared from 2-bromoethyl derivative 9
through a dehydrohalogenation reaction. Butyric acid derivative 16
was obtained by hydrolysis (LiOH) of ester 15. Butyramide 17 and
N-methylbutyramide 18 were obtained by treatment of ethyl ester
15 with ammonia or methylamine, respectively; N,N-dimethylbu-
tyramide 19 was prepared from the chloride (SOCl2) of acid 16 and
dimethylamine. 3-Ethoxypropyl derivative 23 was isolated from an
attempted preparation of 3-hydroxypropyl derivative 21 from the
corresponding bromide 10 and sodium hydroxide in ethanol/water.
Finally, 2-acetoxyethyl derivative 24 was prepared by acetylation
with acetic anhydride of the corresponding 2-hydroxyethyl com-
pound 20.
Propargylation reaction leading to compound 8 gave best results
working in potassium carbonate/refluxing acetone; during alkyl-
ation of compound
5 to derivatives 13/33 with methyl 3-
bromopropionate in DMF at 100 ꢀC, dehydrogenation of the latter
to methyl acrylate was observed; best results were obtained in
potassium carbonate/toluene/water under phase transfer catalysis;
nevertheless, large amounts of the corresponding acid 14, arising
from hydrolysis of ester moiety of compound 13, were also
produced.
From the reaction of 5 with 1,2-dibromoethane, O,O’- (38) and
O,N1’-bisindazole (39) derivatives (Fig. 2) could also be isolated.
On the other hand, treatment of compound 5 with the required
Scheme 2. Synthesis of 6-nitroquinazolin-4-ones 40e43.

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
299
The corresponding mixtures of isomeric 1-substituted 2-
benzylindazolin-3-ones (series A) and 3-alkoxy-2-benzyl-2H-
indazoles (series B) obtained in some case (pairs 9/30, 10/31, 11/32,
13/33, 15/34, 20/35, 21/36 and 22/37), as well as some byproducts
(e.g., 38 and 39), were easily separated by column chromatography;
the same procedure was used to purify crude compounds (8, 12, 17
and 23) containing minor impurities. In most cases, products
included in this work are crystalline solids which were easily pu-
rified by recrystallization from an appropriate solvent.
The structure of all compounds has been established on the
basis of analytical and spectral data. The patterns of 1H and 13C NMR
signals of indazole ring found for compounds of series A and for the
isomeric derivatives of series B are rather different, in agreement
with previously reported data [5]. Furthermore, signals of 1-CH2
and Bn CH2 protons of compounds A always appear, respectively, at
higher field than those of OCH2 and Bn CH2 protons of the corre-
sponding isomers B. On the other hand, NMR signals of quinazolin-
4-one 40 are also rather different from those of its indazole-derived
isomers 11 (series A) and 32 (series B); a remarkable feature of
compounds 40e43 is that their Bn CH2 protons, as well as 1-CH2
protons of 42 and 43, are anisochronic and have been distinguished
in the description of 1H NMR spectra as HA and HB.
amastigotes are the intracellular forms present in the mammalian
host cells, thus the high efficiency of these indazolinones is espe-
cially relevant from the point of view of human disease.
Since 1-(2-hydroxyethyl) derivative 20 achieved the best try-
panocidal profile on both epimastigotes (IC50 ¼ 1.58
mM) and
intracellular amastigotes (IC50 ¼ 0.22 M) of CL-B5, further in vitro
m
studies were carried out to explore its potential activity on other
T. cruzi strains. The Tulahuen strain was selected to confirm the
activity of 20 on parasites belonging to DTU TcVI (drug-sensitive
T. cruzi), as well as the Y strain (DTU TcII) to evaluate it over
moderately drug-resistant parasites [52]. The obtained results
(Table 2) show that this compound was rather more efficient than
benznidazole against epimastigotes of Y strain; however, its activity
against the clinically significant forms, i.e., amastigotes of both
strains of T. cruzi, was only slightly higher than that of the reference
drug. Nevertheless, according to recently proposed criteria [53],
compounds displaying IC50 values lower than 10 mM on intracel-
lular amastigotes of DTUs TcII and TcVI, may be considered at an
initial stage of the drug pipeline as promising hits for Chagas dis-
ease chemotherapy. Moreover, the low unspecific toxicity
(LC50 > 100 mM, Table 2) of compound 20 against cardiac cells was
also determined. The absence of cardiotoxicity characteristics
supports the specific trypanocidal activity of compound 20, ac-
cording to the importance this target cell has in the pathology of
Chagas disease [8].
2.2. Biology
2.2.1. In vitro evaluation of anti-T. cruzi activity
In previous works on related 1,2-disubstituted indazolinones we
found that these compounds do not support much variation at
position 2, benzyl group being the best substituent [5,6]; however,
in relation to position 1, our current findings demonstrate that
simple alkyl groups (Me, Pr, iPr, Bu and Pe) present in the previously
reported antichagasic indazolinones are not strictly necessary, and
that a large number of differently substituted groups can be accept
at this position without considerable loss of activity. This fact may
allow in the future improving the physicochemical and pharma-
cokinetic properties of 5-nitroindazolinones, especially their low
solubility in water.
In vitro assays against T. cruzi CL Brener strain (DTU TcVI) were
performed according to a sequential protocol of drug screening in
which activity against epimastigotes (extracellular insect vector
stage of parasite), unspecific cytotoxicity against L929 murine fi-
broblasts (amastigotes host cells) and selectivity indexes (SI) are
simultaneously evaluated. Compounds with selectivity indexes (SI)
on epimastigotes similar or higher than that of benznidazole, were
selected for further testing of activity on amastigotes (intracellular
mammalian host forms) [48].
In vitro activity of compounds 6e43 against epimastigotes and
amastigotes of T. cruzi [49,50], unspecific cytotoxicity against mu-
rine fibroblasts and their corresponding selectivity indexes (SI) are
collected in Table 1. These compounds as well as the standard drug
2.2.2. In vitro evaluation of anti-T. vaginalis activity
Initial studies of activity against T. vaginalis were carried out on
trophozoites of the metronidazole-sensitive JH31A#4 isolate. The
studied compounds were assayed at concentrations of
benznidazole were assayed at concentrations of 0.125e256 mM and
from the obtained data IC50 values shown in Table 1 were calcu-
lated. It can be seen that most 3-alkoxy-2-benzylindazoles (30e37)
exhibited poor activity towards epimastigotes, while many 1,2-
disubstituted indazolinones were much more efficient that the
9.37e300
Table 3. Activity of all compounds was lower than that of the
reference drug metronidazole (IC50 ¼ 1.43 M), but some of them
showed a moderate to good trichomonacidal activity. Indazoles
showing IC50 < 50 M included 1,2-disubstituted indazolinones 19
mM, and the activity results (IC50 values) are collected in
m
reference drug benznidazole (IC50 ¼ 22.73
deserve indazolinones containing at position 1 unsaturated groups
such as vinyl (6) or allyl (7), or -substituted alkyl groups such as 2-
mM). Special mention
m
u
and 20 (series A) and 3-alkoxy-2-alquilindazoles 31 and 33e36
(series B). Compounds 35 and 36, 3-(2-hydroxyethoxy)- and 3-(3-
bromoethyl (9), 3-bromopropyl (10), 3-(dimethylcarbamoyl)propyl
(19), 2-hydroxyethyl (20) and 3-hydroxypropyl (21), with
hydroxypropoxy)indazole derivatives, had
against the parasite with IC50 values of 9.82 and 7.25 m
a
relevant activity
M, respec-
IC50 ¼ 1.58e4.19
mM. On the other hand, indazolinones with a
carboxylic acid side-chain (14, 16), those acylated at position 1 (27,
28) and quinazolinones (40e43), do not exhibit significant activity.
The low antichagasic efficiency of the latter against epimastigotes
of T. cruzi Dm28c clone (DTU TcI) has previously been observed [51].
Most compounds showing high activity against epimastigotes
(6, 7, 9, 19, 20 and 21) display low unspecific cytotoxicity against
murine fibroblasts, giving SI (46 to > 162) much higher than that of
the reference drug benznidazole (SI > 11.26).
tively. These results are in agreement with the previously reported
[13] potential of 3-alkoxy-2-alkyl-2H-indazole scaffold in the field
of trichomonacidal drugs. Other compounds, including quinazoli-
nones 40e43, had low activity or even showed no effect on the
parasite growth and, in this case, their IC50 values could not be
calculated (Table 3).
Further cytotoxicity studies against Vero cells as well as the
determination of the corresponding selectivity indexes (SI) were
In relation to activities and selectivity indexes against amasti-
only conducted for most indazoles displaying IC50 < 50 mM. The
gotes, 2-benzyl-1-(
as acetoxy derivative 24 (IC50 ¼ 0.22e0.25
more effective than benznidazole (IC50 ¼ 0.68
(Table 1). Taking in mind the metabolic instability of ester moiety,
2-acetoxyethyl derivative 24 may act as prodrug of 2-
hydroxyethyl compound 20. It is important to consider that
u
-hydroxyalkyl)indazolinones 20 and 21 as well
M; SI > 1024) were also
M; SI > 376.47)
data, also included in Table 3, show that compounds 19, 20, 31, 33,
35 and 36 display low unspecific cytotoxicity, with percentages of
inhibition of Vero cells growth <20% at 300 mM and SI values be-
tween >6.1 and > 41.4. Once again, 3-alkoxy-1-alkyl-2H-indazoles
35 and 36 (series B), which showed very low percentages of cyto-
m
m
a
toxicity (%C) at the highest concentration tested (300
mM) (%

300
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
Table 1
In vitro activities against T.cruzi CL-B5 epimastigotes and amastigotes (IC50), unspecific cytotoxicities against murine fibroblasts (LC50) and selectivity indexes (SI) found for
compounds 6e43.
Compound
IC50 epimastigotes (
m
M)a
LC50 fibroblasts (
m
M) a
SIb epimastigotes
IC50 amastigotes (
m
M) a
SIc amastigotes
6
7
8
9
10
11
12
13
2.75 0.09
4.19 0.95
10.79 4.80
2.13 0.72
2.90 0.37
26.17 11.31
11.66 2.13
9.19 1.20
>256
160.46 14.89
192.36 21.85
195.27 9.70
>256
25.19 3.16
128.89 10.32
>256
58.35
45.91
18.10
>120.19
8.69
0.47 0.10
1.58 0.60
3.02 0.55
1.14 0.14
341.40
121.75
64.66
>224.56
e
e
4.92
e
e
>21.95
>27.86
ND
3.50 0.83
2.93 1.68
>73.14
>87.37
>256
>256
14
e
e
15
16
11.14 0.59
>256
78.07 8.02
>256
7.01
ND
e
e
e
e
17
18
19
20
21
22
23
24
30.98 3.94
13.29 0.92
3.86 0.37
1.58 0.06
1.68 0.36
6.56 0.87
6.57 0.27
5.43 1.89
49.01 14.58
43.59 5.41
>256
>256
>256
>256
>256
>256
>256
170.41 9.94
>256
122.03 15.98
86.06 13.66
>256
>8.26
>19.26
>66.32
>162.02
>152.38
>39.02
25.94
>47.14
2.49
e
e
1.50 0.23
0.54 0.04
0.22 0.06
0.25 0.12
2.29 0.79
3.31 0.52
0.25 0.14
e
>170.67
>474.07
>1163.64
>1024
>111.79
51.48
>1024
e
25
26
27
1.97
ND
e
e
e
e
28
29
30
>256
28.76 2.67
>256
145.52 6.10
>256
>256
<0.57
>8.90
ND
e
e
e
e
e
e
31
32
33
39.04 13.42
144.71 9.30
>256
59.04 4.61
>256
>256
1.51
>1.77
ND
e
e
e
e
e
e
34
35
25.05 1.75
>256
>256
>256
>10.22
ND
25.36 4.50
e
>10.09
e
36
37
64.91 8.34
>256
>256
>256
>3.94
ND
e
e
e
e
38
>256
>256
ND
e
e
39
>256
>256
ND
e
e
40
41
216.34 14.99
>256
>256
>256
>1.18
ND
e
e
e
e
42
43
>256
>256
205.14 17.32
>256
<0.8
ND
e
e
e
e
Benznidazole
22.73 3.33
>256
>11.26
0.68 0.08
>376.47
e: Not evaluated against amastigotes due to a SI on epimastigotes lower than that of benznidazole or a SI which could not be calculated (ND) owing to the very low activity on
epimastigotes.
ND: Not determined owing to the low activity values (IC50 epimastigotes > 256
m
M).
a
Results of IC50 and LC50 are expressed as the mean SD.
Selectivity indexes for epimastigotes (SI ¼ LC50 L929/IC50 epimastigotes).
b
c
Selectivity indexes for amastigotes (SI ¼ LC50 L929/IC50 amastigotes).
Table 2
In vitro activities (IC50) of compound 20 on intracellular amastigotes of Tulahuen (DTU TcVI), epimastigotes and intracellular amastigotes of Y strain (DTU TcII) and toxicities
(LC50) on primary cultures of cardiac cells.
Compound
IC50 Tulahuen amastigotes (
m
M)a
IC50 Y strain epimastigotes (
m
M)a
IC50 Y strain amastigotes (
m
M)a
LC50 cardiac cells (mM)
20
0.81 0.29
1.66 0.10
3.18 0.37
28.11 1.77
0.60 0.03
1.77 1.11
>100
>100
Benznidazole
a
Results of IC50 are expressed as the mean SD.
C35 ¼ 14.7
4.3; %C36 ¼ 14.5
3.2), were the most interesting
2.3. Physicochemical and pharmacokinetic parameters,
electrostatic potential and structureeactivity relationships
compounds.
Indazoles with the highest SI values against T. vaginalis JH31A#4,
31, 35 and 36, were then assayed against the metronidazole-
resistant isolate IR78 following similar protocols and the obtained
results are gathered in Table 4. Values found for compound 36
In order to get further insights onto the new indazole derivatives
of both series A and B, some physicochemical and pharmacokinetic
parameters (QikProp software), as well as the electrostatic poten-
tials maps (Spartan software) of selected molecules, were calcu-
lated (Supplementary material, Table S1 and Fig. S1).
Neither indazolin-3-one derivatives (series A) nor 3-alkoxy-5-
nitroindazoles (series B) violate Lipinski's and Jorgensen's rules.
The calculated predictions suggest a good human oral bioavail-
ability [71e100%, except for acids 14 (57%) and 16 (64%), and for
(IC50 ¼ 9.11
m
M; SI > 32.9) are very similar to those obtained for the
metronidazole-sensitive isolate (IC50 ¼ 7.25
m
M; SI 41.4),
>
evidencing the lack of cross-resistance between this compound and
the reference drug. Thus, compound 36 can be considered a good
prototype for the future development of drugs against T. vaginalis
isolates resistant to 5-nitroimidazoles.

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
301
Table 3
In vitro activities against T. vaginalis JH31A#4 trophozoites (IC50), unspecific cytotoxicities against Vero cells (CC50) and selectivity indexes (SI) found for compounds 6e31,
33e37 and 40e43.
Compound
IC50
(
m
M)a
CC50
(
m
M)
SIb
Compound
IC50
(m
M)a
CC50
(m
M)
SIb
6
7
8
9
258.43 [200.15e363.67]
358.86 [197.50e498.70]
260.97 [171.51e500.54]
347.74 [242.80e587.54]
80.21 [61.22e112.81]
534.57 [406.21e843.18]
ND
375.54 [304.84e507.72]
ND
192.42 [120.05e429.96]
ND
104.13 [91.47e119.55]
182.01 [151.62e227.51]
48.94 [38.82e61.41]
17.94 [14.30e21.68]
128.44 [110.59e152.07]
284.62 [162.44e402.26]
362.18 [237.01e515.79]
e
e
e
e
e
e
e
e
e
e
e
e
e
e
24
25
26
27
28
29
30
31
33
34
35
36
37
40
41
42
265.63 [196.68e402.26]
418.86 [324.24e615.79]
588.88 [386.67e733.84]
212.91 [174.24e271.41]
197.42 [130.21e326.63]
151.47 [101.14e289.79]
64.75 [45.00e96.83]
18.57 [12.98e24.49]
43.02 [25.11e71.78]
20.63 [17.08e24.54]
9.82 [8.58e11.11]
7.25 [6.49e8.04]
107.45 [97.90e117.89]
ND
ND
58.20 [40.78e84.25]
ND
1.43 [1.13e1.79]
e
e
e
e
e
e
e
e
e
e
e
e
e
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
e
e
e
e
e
e
e
538.85c
>300
e
29.0
>7.0
e
e
e
e
>300
>300
e
>30.5
>41.4
e
e
e
>300
>300
e
>6.1
>16.7
e
e
e
e
e
e
e
e
e
43
e
e
e
e
Metronidazole
>600
>100
e: Not evaluated against Vero cells owing to the low IC50 values.
ND: IC50 values could not be determined owing to the very low activity.
Values of growth inhibition used to calculate IC50 and CC50 displayed a standard deviation of less than 10%.
a
In brackets, 95% confidence intervals.
b
Selectivity indexes (SI ¼ CC50 Vero cells/IC50 trophozoites).
c
95% confidence interval: 399.77e942.38.
Table 4
water solubility; in this sense, values of calculated log S show that
some of the more efficient antichagasic 1,2-disubstituted de-
rivatives (e.g., 6, 7, 19, 20, 22 and 23; log S ¼ ꢁ2.53 to ꢁ3.26) are
more water soluble than the previously reported [5] 2-
benzylindazolinones carrying propyl- (1), isopropyl- (2) or butyl
(3) substituents at position 1 (log S ¼ ꢁ3.51 to ꢁ4.00) (Supple-
mentary material, Table S1).
Among compounds of series A, we have not found a clear rela-
tionship between lipophilicity and trypanocidal properties; in fact
anti-epimastigote activity of 3-bromopropyl derivative 10, one of
In vitro activities against T. vaginalis IR78 trophozoites (IC50), unspecific cytotoxic-
ities against Vero cells (CC50) and selectivity indexes (SI) found for compounds 31, 35
and 36.
Compound
IC50
(
m
M)a
CC50
(
m
M)
SIb
31
35
36
39.12 [34.24e44.55]
49.82 [24.21e71.01]
9.11 [7.16e11.48]
5.78 [4.38e9.43]
538.85c
>300
>300
>600
13.8
>6.0
>32.9
>100
Metronidazole
Values of growth inhibition used to calculate IC50 and CC50 displayed a standard
deviation of less than 10%.
the more lipophilic compounds (IC50 ¼ 2.90
m
M; log P ¼ 3.39), is
a
In brackets, 95% confidence intervals.
b
only slightly lower than that of 2-hydroxyethyl derivative 20, one of
Selectivity indexes (SI ¼ CC50 Vero cells/IC50 trophozoites).
c
95% confidence interval: 399.77e942.38.
the less lipophilic derivatives (IC50 ¼ 1.58
m
M; log P ¼ 1.52).
On the contrary, electron-donating or -withdrawing properties
of substituents at position 1 of series A derivatives seem to play a
crucial role with regard to activity. In fact, indazolinones containing
at this position strong electron-withdrawing substituents, with the
amide 17 (55%)] and they do not detect side effects associated with
CNS (supplementary material, Table S1).
We have previously published that many 1,2-disubstituted
indazolinones (series A analogues) are efficient against T. cruzi,
while most isomeric 2-substituted 3-alkoxy-2H-indazoles (series B
analogues) show no antichagasic activity [5]. As commented in
section 2.4, antiparasitic nitroheterocycles are in fact prodrugs
activated by the parasites metabolism after bioreduction. Taking in
mind that the reduction potentials corresponding to the formation
of nitro anion radicals in aprotic solvents are similar for the pre-
viously described analogues of series A (ꢁ1.04 to ꢁ1.10 V) and B
(ꢁ1.13 to ꢁ1.22 V) [54], it is likely that the former are better sub-
strates for trypanosome nitroreductases than the latter. In fact, the
structures and properties of both kinds of compounds, derived
from two distinct indazole tautomers, are rather different; e.g.,
derivatives of series B (log P ¼ 3.09e5.09) are always significantly
more lipophilic than the corresponding isomers of series A (log
P ¼ 1.38e3.39) according to the calculated log P values (e.g., 1.52 for
20 vs 3.09 for 35) (supplementary material, Table S1). Compounds
of both series show also clearly distinct electrostatic potential
maps, e.g., 11, 20 and 27 (series A) vs 35 (series B) (supplementary
material, Fig. S1).
higher values of Taft's polar substituent constants (
(25; * ¼ 1.81) or Bz (28; * ¼ 2.20) moieties,
* ¼ 2.26), Ac (27;
show little or no activity (Supplementary material, Table S1).
Compounds containing groups with intermediate * values, e.g.,
CH2COOMe (11; * ¼ 1.30) display mod-
* ¼ 1.06) or CH2CN (12;
erate activity and, finally, the most active compounds support
substituents with low * values, e.g., CH]CH2 (6;
* ¼ 0.56),
CH2CH]CH2 (7; * ¼ 0.41), [CH2]2OH (20;
* ¼ 0.00), [CH2]2Br (9;
* ¼ 0.09) or [CH2]3OH (21; * ¼ ꢁ0.04). The low antichagasic ac-
tivities observed for acids 14 and 16 and for amide 17, despite the
appropriate values of *, are probably due to pharmacokinetic is-
sues; compound 17 has the lowest calculated value of log P, and 14
and 16 must be ionized in the culture medium (for epimastigotes,
LIT medium; pH ¼ 7.4), this hindering the penetration across the
parasite membranes.
The molecular electrostatic potential maps of selected com-
pounds from series A (11, 20 and 27) show an overall similarity for
all derivatives. In a more specific analysis, subtle changes in the
electronic density of the carbonyl oxygen can be observed
depending on substituents at position 1 (Supplementary material,
Fig. S1). Thus, the carbonyl oxygen of the inactive 1-acetyl
s*), e.g., COOEt
s
s
s
s
s
s
s
s
s
s
s
s
s
In relation to trypanocidal properties, one of the aims of the
current work was to obtain indazolinone derivatives with improved

302
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
derivative 27 presents lower maximum values of electronegativity
potential (ꢁ161.4 kJ/mol) than the oxygen of the moderately and
highly active compounds 11 (ꢁ201.2 kJ/mol) and 20 (ꢁ191.0 kJ/
mol), respectively. These differences suggest that 3-CO group of
these compounds participates in some important interaction in the
active site of nitroreductases, which is affected by the electroneg-
ativity of the carbonyl oxygen.
accepted for nifurtimox. On the other hand, it has also been pro-
posed that the inhibition of trypanothione reductase [63,64] or the
interference with glycosomal or mitochondrial enzymes involved
in the catabolism of T. cruzi [7] could contribute to the activity of
other 5-nitroindazole derivatives with different substitution pat-
terns. According to these discrepant proposals and taking in
consideration the mentioned recent studies carried out with the
antichagasic and trichomonacidal reference drugs, it is evident that
the mode of action of our indazole derivatives needs to be inves-
tigated more thoroughly.
Regarding trichomonacidal activity, the best compounds, 3-(u-
hydroxyalkoxy)indazoles 35 and 36 (series B), are more active
against JH31A#4 isolate than the previously described and more
hydrophilic analogue 4 [13] (supplementary material, Table S1).
Unfortunately, only a few products have shown activity among
derivatives of series B, and these limited data do not allow us to
establish a structureeactivity relationship.
3. Conclusions and future outlooks
The previously proposed antichagasic potential of 1,2-
disubstituted 5-nitroindazolin-3-one scaffold [5,6,8] is confirmed
in the current study. Compounds 20, 21 and 24 (series A) are very
2.4. Considerations on the mode of action of antiparasitic
nitroheterocycles
efficient against amastigotes of T. cruzi CL Brener strain (IC50 < 1 mM,
SI > 1000) and, additionally, the former displays activities of the
same order against amastigotes of Tulahuen and Y strains. Although
a 2-benzyl group seems convenient for optimized activity [5], many
Classical antichagasic drugs have been accepted to act, after
initial metabolic reduction by trypanosome nitroreductases,
through the final production of reactive oxygen species (ROS)
(nifurtimox) or electrophilic metabolites (benznidazole), able to
damage essential biomolecules of parasites such as thiols, lipids,
proteins, DNA, etc. [55,56]. However, a report showed that the in-
duction of oxidative stress in T. cruzi by nifurtimox was not able to
explain its activity [57]. Effectively, it has recently been proposed
that the trypanocidal effects of nifurtimox can be attributed to the
substituents, e.g. unsaturated chains or
u-substituted alkyl moi-
eties, are accepted at position 1 without severe loss of activity.
Inactive compounds are the result of strong electron-withdrawing
substituents at position 1, e.g., acyl groups, or the presence of a
carboxylic acid moiety in the side chain, i.e., compounds 14 and 16,
which leads to unsuitable pharmacokinetic issues.
In what refers to trichomonacidal activity, some of the new 3-
alkoxy-1-alkyl-2H-indazoles (series B) have shown moderate effi-
ciency. Compound 36 displays similar activity for a metronidazole-
sensitive (JH31A#4) and for a metronidazole-resistant (IR78)
reactivity of an open chain
a,b-unsaturated nitrile arising from
reductive and hydrolytic metabolism, probably acting as a Michael
acceptor [58]. On the other hand, it has been suggested that the
antichagasic effects of benznidazole probably rely on the combined
effects of several of its metabolites, especially those of the final
product, the highly reactive dialdehyde glyoxal, able to produce
adducts, e.g., with guanosine [59]; furthermore, the detection of
covalent conjugates of reduced metabolites of benznidazole with
low molecular weight thiols (e.g., cysteine, glutathione, trypano-
thione, etc.) and other parasite biomolecules has recently been
published; in this study, low molecular weight glyoxal-derived
adducts could not be however detected [60].
Regarding trichomonacidal agents, it has been assumed that
metronidazole and other nitroimidazole drugs act against
T. vaginalis after reduction of NO2 group at hydrogenosomal level;
this process has been suggested to be carried out by reduced
ferredoxin, which is in turn produced by pyruvate:ferredoxin
oxidoreductase or by NAD:ferredoxin oxidoreductase [61].
Furthermore, the involvement of a flavin-dependent thioredoxin
reductase displaying nitroreductase activity towards nitro-
midazoles has recently been proposed [62]. It has been reported
that the reduction of metronidazole yields nitro radicals and,
especially, cytotoxic intermediates leading to the formation of ad-
ducts with parasite proteins and to DNA disruption. A recent study
has shown that metronidazole and tinidazole metabolites deplete
intracellular thiol levels, and that metabolite-protein adducts are
not formed indiscriminately; in fact, only ten non-hydrogenosomal
proteins, most of them associated with thioredoxin-mediated
redox regulation, seem to be mainly affected [62].
isolate of T. vaginalis (IC50 ¼ 7.25 and 9.11
mM, respectively). Thus, 3-
alkoxy-1-alkyl-2H-indazole can be considered a good scaffold for
further chemical modifications directed to treat infections caused
by 5-nitroimidazoles-resistant parasites. Unfortunately, in this case,
the structureeactivity relationship is not clear, and much work
remains to be done to understand the effect of the different sub-
stituents on trichomonacidal activity.
In order to better understand the structureeactivity relation-
ships and the mode of action of nitroindazoles, further studies are
underway aimed to study the biological effect of some analogues
carrying the nitro group at different positions, or supporting other
substituents including
u-aminoalkyl chains directed to improve
their activity and physicochemical and pharmacokinetic properties,
or even containing fluorescent moieties potentially able to label
storage sites and/or the organelles constituting the primary cellular
target of the studied compounds. On the other hand, taking into
account the recently published outstanding articles on the mode of
action of nifurtimox [58], benznidazole [59,60] and metronidazole
[62], studies will be directed to the identification of products
arising from the bioreduction of 5-nitroindazoles mediated by re-
ductases and also to the detection of potential adducts of nitro-
indazole metabolites with parasites biomolecules.
4. Experimental section
4.1. Chemistry
The mode of action of the present 5-nitroindazole derivatives
has not been studied in detail, but for several antiprotozoal 1,2-
disubstituted indazolin-3-ones and 1-substituted indazol-3-ols
the involvement of 5-nitro group seems evident according to pre-
vious reports [5,12,13]. A study of electrochemical and enzymatic
reduction of related 1,2-disubstituted indazolinones (analogues of
series A) and 2-substituted 3-alkoxyindazoles (analogues of series
B) [54] suggested that these compounds could induce oxidative
stress in the parasites, i.e., a mode of action similar to that initially
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 at room temperature (~20 ꢀC) on a
Bruker Avance 300 spectrometer. The chemical shifts are reported
in ppm from TMS (d scale) but were measured against the solvent
signal. The assignments have been performed by means of different
standard homonuclear and heteronuclear correlation experiments

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
303
(NOE, gHSQC and gHMBC). Numbering used in the description of
NMR spectra is indicated in Schemes 1 and 2 and in Fig. 2. 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
(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 0.3% of the theoretical values.
236 (6), 192 (11), 177 (9), 162 (5), 146 (8), 131 (11), 103 (7). Anal.
C16H15N3O4 (C, H, N).
4.1.2.1.4. 2-Benzyl-1-(3-hydroxypropyl)-5-nitro-1,2-dihydro-3H-
indazol-3-one (21). Yield: 0.58 g (48%). Mp 141e143 ꢀC (2-PrOH).
4.1.2.1.5. 2-Benzyl-1-(2-methoxyethyl)-5-nitro-1,2-dihydro-3H-
indazol-3-one (22). Yield: 0.78 g (64%). Mp 116e118 ꢀC (2-PrOH).
4.1.2.1.6. 2-Benzyl-3-[3-(ethoxycarbonyl)propoxy]-5-nitro-2H-
indazole (34). Yield: 0.40 g (28%). Mp 87e89 ꢀC (2-PrOH).
4.1.2.1.7. 2-Benzyl-3-(2-hydroxyethoxy)-5-nitro-2H-indazole
(35). Yield: 0.21 g (18%). Mp 167e169 ꢀC (2-PrOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.90 (d, J ¼ 2.1 Hz, 1H, 4-H), 7.91 (dd, J ¼ 9.6,
2.1 Hz, 1H, 6-H), 7.53 (d, J ¼ 9.6 Hz, 1H, 7-H), 7.31 (m, 5H, Bn aro-
matic H), 5.50 (s, 2H, Bn CH2), 5.16 (t, J ¼ 5.5 Hz, 1H, OH), 4.72 (t,
J ¼ 4.5 Hz, 2H, 10-H), 3.81 (m, 2H, 20-H); 13C NMR [75 MHz,
4.1.2. Alkylation of 2-benzyl-5-nitroindazolin-3-one 5
4.1.2.1. Preparation of allyl (7), 3-(ethoxycarbonyl)propyl (15/34), 2-
hydroxyethyl (20/35), 3-hydroxypropyl (21/36) and 2-methoxyethyl
(22/37) derivatives. A stirred mixture of the starting 2-
benzylindazolinone 5 [44] (1.00 g, 3.71 mmol), the required bro-
mide (4.00 mmol) and potassium carbonate (0.55 g, 4.00 mmol) in
DMF (20 mL) was heated at 100 ꢀC until completion of reaction
[TLC; 30 min (for 7), 12 h (for 22/37) and 12 h followed by 1e3
additions of further amounts of bromide (0.3 mmol) and, eventu-
ally, if needed, base (0.3 mmol) each 6 h (for 15/34, 20/35 and 21/
36)]. The mixture was then evaporated to dryness and, after addi-
tion of water (200 mL), extracted with chloroform (3 ꢂ 50 mL). For
compound 7, the organic phase was dried (MgSO4) and concen-
trated to dryness; the residue was triturated with 2-PrOH (10 mL)
and the insoluble material recovered by filtration and air-dried. For
the remaining compounds, the concentrated chloroform phase was
applied to the top of a chromatography column which was eluted
with chloroform/acetone mixtures (50:1 to 25:1) (for 15/34 and 22/
37) or with the same mixtures (10:1 to 5:1) followed by chloro-
form/methanol (50:1 to 25:1) (for 20/35 and 21/36). In all cases, 3-
alkoxy-2-benzyl-2H-indazoles (34e37) eluted first, followed by the
corresponding 1-substituted 2-benzylindazolinones (15, 20e22).
Spectral and analytical data of compounds 15, 21, 22, 34, 36 and
37 are included as supplementary material.
(CD3)2SO]:
d 150.0 (C-3), 146.9 (C-7a), 140.2 (C-5), 135.9 (Bn C-1),
128.6 (Bn C-3, -5), 128.0 (Bn C-2, -6), 127.9 (Bn C-4), 120.9 (C-4),
119.9 (C-6), 118.0 (C-7), 105.3 (C-3a), 76.4 (C-10), 59.8 (C-20), 51.5 (Bn
CH2); MS (EI): m/z (%) 313 (100) (Mþ), 269 (58), 252 (14), 222 (7),
191 (41), 164 (7), 149 (4), 103 (14). Anal. C16H15N3O4 (C, H, N).
4.1.2.1.8. 2-Benzyl-3-(3-hydroxypropoxy)-5-nitro-2H-indazole
(36). Yield: 0.12 g (10%). Mp 122e124 ꢀC (2-PrOH).
4.1.2.1.9. 2-Benzyl-3-(2-methoxyethoxy)-5-nitro-2H-indazole
(37). Yield: 0.39 g (32%). Mp 109e111 ꢀC (2-PrOH).
4.1.2.2. Preparation of propargyl derivative 8. A stirred mixture of 2-
benzylindazolinone 5 [44] (1.00 g, 3.71 mmol), propargyl bromide
(80% w/w in toluene) (4.00 mmol) and potassium carbonate (0.55 g,
4.00 mmol) in acetone (50 mL) was refluxed for 12 h. The mixture
was then evaporated to dryness and, after addition of water
(200 mL), extracted with chloroform (3 ꢂ 50 mL). The concentrated
chloroform phase was applied to the top of a chromatography
column which was eluted with chloroform/acetone mixtures (50:1
to 30:1) to afford compound 8.
4.1.2.2.1. 2-Benzyl-5-nitro-1-propargyl-1,2-dihydro-3H-indazol-
3-one (8). Yield: 0.62 g (54%). Mp 163e165 ꢀC (EtOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.52 (d, J ¼ 2.1 Hz, 1H, 4-H), 8.47 (dd, J ¼ 9.0,
4.1.2.1.1. 1-Allyl-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one
(7). Yield: 1.04 g (91%). Mp 137e139 ꢀC (2-PrOH). The preparation
of this compound from the corresponding 1-allylindazol-3-ol and
benzyl bromide has been claimed in a patent [65]; however, mp is
not given and the reported 1H NMR spectrum is not in agreement
with that recorded by us in the same solvent. 1H NMR [300 MHz,
2.1 Hz, 1H, 6-H), 7.80 (d, J ¼ 9.0 Hz, 1H, 7-H), 7.27 (m, 5H, Bn aro-
matic H), 5.12 (s, 2H, Bn CH2), 4.96 (d, J ¼ 2.1 Hz, 2H, 10-H), 3.16 (t,
J ¼ 2.1 Hz, 1H, 30-H); 13C NMR [75 MHz, (CD3)2SO]:
d 161.5 (C-3),
151.6 (C-7a), 143.1 (C-5), 136.1 (Bn C-1), 128.6 (Bn C-3, -5), 127.7 (Bn
C-4), 127.6 (C-6, Bn C-2, -6), 119.9 (C-4), 118.8 (C-3a), 114.0 (C-7),
76.6 (C-20), 75.9 (C-30), 44.7 (Bn CH2), 38.5 (C-10); MS (EI): m/z (%)
307 (100) (Mþ), 268 (13), 249 (5), 230 (3), 203 (4), 128 (5), 115 (4),
103 (11). Anal. C17H13N3O3 (C, H, N).
(CD3)2SO]:
d
8.51 (d, J ¼ 2.1 Hz, 1H, 4-H), 8.38 (dd, J ¼ 9.0, 2.1 Hz, 1H,
6-H), 7.70 (d, J ¼ 9.0 Hz, 1H, 7-H), 7.26 (m, 5H, Bn aromatic H), 5.48
(m, 1H, 20-H), 5.21 (dd, J ¼ 17.1, 1.1 Hz, 1H, 30-Htrans), 5.15 (s, 2H, Bn
CH2), 5.10 (dd, J ¼ 10.5, 1.1 Hz, 1H, 30-Hcis), 4.67 (d, J ¼ 6.0 Hz, 2H, 10-
4.1.2.3. Preparation of 2-bromoethyl (9/30) and 3-bromopropyl (10/
31) derivatives and related compounds (38, 39). A stirred mixture of
the starting 2-benzylindazolinone 5 [44] (2.00 g, 7.43 mmol), the
H); 13C NMR [75 MHz, (CD3)2SO]:
d 161.0 (C-3), 149.3 (C-7a), 141.7
(C-5), 136.1 (Bn C-1), 130.3 (C-20), 128.6 (Bn C-3, -5), 127.8 (Bn C-4),
127.4 (Bn C-2, -6), 127.2 (C-6), 120.3 (C-4, -30), 116.4 (C-3a), 112.6 (C-
7), 49.4 (C-10), 44.8 (Bn CH2); MS (EI): m/z (%) 309 (100) (Mþ), 268
(19), 232 (3), 218 (2),164 (3),131 (4), 103 (7). Anal. C17H15N3O3 (C, H,
N).
4.1.2.1.2. 2-Benzyl-1-[3-(ethoxycarbonyl)propyl]-5-nitro-1,2-
dihydro-3H-indazol-3-one (15). Yield: 1.00 g (70%). Oil which so-
lidifies on standing; mp 51e53 ꢀC.
required
a,u-dibromoalkane (40.00 mmol) and potassium car-
bonate (1.10 g, 8.00 mmol) in DMF (50 mL) was heated at 100 ꢀC for
3 h. The mixture was then evaporated to dryness and, after addition
of water (200 mL), extracted with chloroform (3 ꢂ 50 mL). The
organic phase was dried (MgSO4), concentrated and applied to the
top of a chromatography column which was eluted with chloro-
form/acetone mixtures (50:1 to 25:1).
4.1.2.1.3. 2-Benzyl-1-(2-hydroxyethyl)-5-nitro-1,2-dihydro-3H-
Starting from 1,2-dibromoethane, the following compounds
were obtained in this elution order: 2,3-disubstituted indazole 30,
1-vinylindazolinone 6 [70 mg (3%); mp and spectral and analytical
data are given below, section 4.1.4.1.1], 1,2-disubstituted indazoli-
none 9, and the corresponding O,O’- (38) and O,N1’- (39) ethylene
derivatives.
Analogously, starting from 1,3-dibromopropane, the corre-
sponding 2,3-disubstituted indazole 31 and 1,2-disubstituted
indazolinone 10 could be obtained.
indazol-3-one (20). Yield: 0.91 g (78%). Mp 137e139 ꢀC (2-PrOH).
1H NMR [300 MHz, (CD3)2SO]:
d
8.49 (d, J ¼ 2.1 Hz, 1H, 4-H), 8.31
(dd, J ¼ 9.3, 2.1 Hz, 1H, 6-H), 7.60 (d, J ¼ 9.3 Hz, 1H, 7-H), 7.29 (m,
3H) and 7.20 (m, 2H) (Bn aromatic H), 5.20 (s, 2H, Bn CH2), 4.73 (t,
J ¼ 5.1 Hz, 1H, OH), 4.12 (t, J ¼ 4.9 Hz, 2H, 10-H), 3.45 (m, 2H, 20-H);
13C NMR [75 MHz, (CD3)2SO]:
d
160.9 (C-3), 149.2 (C-7a), 140.7 (C-
5), 136.1 (Bn C-1), 128.7 (Bn C-3, -5), 127.8 (Bn C-4), 127.2 (Bn C-2,
-6), 126.4 (C-6), 120.3 (C-4), 114.6 (C-3a), 112.4 (C-7), þ58.4 (C-20),
49.2 (C-10), 44.8 (Bn CH2); MS (EI): m/z (%) 313 (100) (M ), 282 (11),
Spectral and analytical data of compounds 10, 30, 38 and 39 are

304
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
included as supplementary material.
NMR [75 MHz, (CD3)2SO]: d
167.3 (C-20), 160.8 (C-3), 150.1 (C-7a),
4.1.2.3.1. 2-Benzyl-1-(2-bromoethyl)-5-nitro-1,2-dihydro-3H-
141.8 (C-5), 135.9 (Bn C-1), 128.5 (Bn C-3, -5), 127.7 (Bn C-4), 127.4
(Bn C-2, -6), 127.3 (C-6), 120.1 (C-4), 116.3 (C-3a), 112.0 (C-7), 52.0
(CH3), 47.8 (C-10), 44.9 (Bn CH2); MS (ESþ): m/z (%) 705 (23)
([2M þ Na]þ), 683 (25) ([2M þ H]þ), 364 (34) ([MþNa]þ), 342 (100)
([MþH]þ). Anal. C17H15N3O5 (C, H, N).
indazol-3-one (9). Yield: 1.20 g (43%). Mp 172e174 ꢀC (2-PrOH). 1H
NMR [300 MHz, (CD3)2SO]:
d
8.52 (d, J ¼ 2.4 Hz, 1H, 4-H), 8.37 (dd,
J ¼ 9.0, 2.4 Hz, 1H, 6-H), 7.75 (d, J ¼ 9.0 Hz, 1H, 7-H), 7.26 (m, 5H, Bn
aromatic H), 5.19 (s, 2H, Bn CH2), 4.53 (t, J ¼ 6.3 Hz, 2H,10-H), 3.51 (t,
J ¼ 6.3 Hz, 2H, 20-H); 13C NMR [75 MHz, (CD3)2SO]:
d
161.4 (C-3),
4.1.2.4.2. 2-Benzyl-1-cyanomethyl-5-nitro-1,2-dihydro-3H-inda-
zol-3-one (12). Yield: 0.84 g (73%). Mp 146e148 ꢀC (2-PrOH).
4.1.2.4.3. 2-Benzyl-3-(methoxycarbonyl)methoxy-5-nitro-2H-
indazole (32). Yield: 40 mg (3%). Mp 94e96 ꢀC (2-PrOH). 1H NMR
149.3 (C-7a), 141.5 (C-5), 135.9 (Bn C-1), 128.7 (Bn C-3, -5), 127.9 (Bn
C-4), 127.3 (Bn C-2, -6), 127.0 (C-6), 120.4 (C-4), 115.4 (C-3a), 112.6
(C-7), 47.7 (C-10), 45.2 (Bn CH2), 29.2 (C-20); MS (EI): m/z (%) 377
(99) ([Mþ2]þ), 375 (100) (Mþ), 361 (5), 359 (5), 347 (7), 345 (7), 256
(9), 254 (9), 192 (8), 177 (9), 164 (9), 145 (8), 131 (17), 103 (36). Anal.
[300 MHz, (CD3)2SO]:
d
8.83 (d, J ¼ 2.2 Hz, 1H, 4-H), 7.91 (dd, J ¼ 9.6,
2.2 Hz, 1H, 6-H), 7.57 (d, J ¼ 9.6 Hz, 1H, 7-H), 7.33 (m, 5H, Bn aro-
C
16H14BrN3O3 (C, H, N).
matic H), 5.54 (s, 2H, Bn CH2), 5.49 (s, 2H,10-H), 3.74 (s, 3H, CH3); 13C
4.1.2.3.2. 2-Benzyl-1-(3-bromopropyl)-5-nitro-1,2-dihydro-3H-
NMR [75 MHz, (CD3)2SO]: d
168.4 (C-20), 148.7 (C-3), 146.8 (C-7a),
indazol-3-one (10). Yield: 0.90 g (31%). Mp 111e113 ꢀC (2-PrOH).
4.1.2.3.3. 2-Benzyl-3-(2-bromoethoxy)-5-nitro-2H-indazole (30).
Yield: 0.84 g (30%). Mp 140e142 ꢀC (2-PrOH).
140.6 (C-5), 135.6 (Bn C-1), 128.6 (Bn C-3, -5), 127.9 (Bn C-2, -4, -6),
120.1 (C-4), 119.9 (C-6), 118.2 (C-7), 105.1 (C-3a), 69.5 (C-10), 52.2
(CH3), 51.8 (Bn CH2); MS (EI): m/z (%) 341 (100) (Mþ), 325 (1), 282
(2), 268 (16), 252 (2), 236 (2), 222 (2), 191 (2), 164 (2), 103 (3). Anal.
4.1.2.3.4. 2-Benzyl-3-(3-bromopropoxy)-5-nitro-2H-indazole
(31). Yield: 0.84 g (29%). Mp 124e126 ꢀC (2-PrOH). 1H NMR
C17H15N3O5 (C, H, N).
[300 MHz, (CD3)2SO]:
d
8.88 (d, J ¼ 2.4 Hz,1H, 4-H), 7.90 (dd, J ¼ 9.6,
2.4 Hz, 1H, 6-H), 7.54 (d, J ¼ 9.6 Hz, 1H, 7-H), 7.31 (m, 5H, Bn aro-
matic H), 5.47 (s, 2H, Bn CH2), 4.82 (t, J ¼ 5.8 Hz, 2H, 10-H), 3.66 (t,
J ¼ 6.6 Hz, 2H, 30-H), 2.35 (m, 2H, 20-H); 13C NMR [75 MHz,
4.1.2.5. Preparation of 2-(methoxycarbonyl)ethyl derivatives 13/33
and 2-carboxyethyl derivative 14. A stirred solution of the starting
2-benzylindazolinone 5 [44] (1.00 g, 3.71 mmol), potassium car-
bonate (1.38 g,10.00 mmol) and benzyltributylammonium bromide
(0.10 g) in a mixture of water (20 mL) and toluene (20 mL) was
heated at 100 ꢀC for 30 min, and then, methyl 3-bromopropionate
(0.84 g, 5.03 mmol) was added. Further amounts of methyl 3-
bromopropionate (ca. 10 ꢂ 0.25 g) and, eventually, if needed, po-
tassium carbonate to have a basic pH, were added during 2 days.
The reaction mixture was allowed to reach room temperature and
the toluene phase was then separated, dried (MgSO4) and evapo-
rated to dryness. The obtained residue was chromatographed on a
column using chloroform/acetone mixtures (50:1 to 10:1) to afford,
following this elution order, compound 33 and then compound 13.
The remaining water phase was acidified with conc. HCl (pH 1)
and the precipitated solid collected by filtration, dried and applied
to the top of a column which was eluted first with chloroform/
methanol (50:1 to 10:1) mixtures and then with a chloroform/
methanol (10:1) mixture containing 0.5% of acetic acid to afford, in
this elution order, recovered starting indazolinone 5 [70 mg (7%)]
and then acid 14.
(CD3)2SO]:
d 149.4 (C-3), 146.8 (C-7a), 140.3 (C-5), 135.8 (Bn C-1),
128.6 (Bn C-3, -5), 127.9 (Bn C-4), 127.7 (Bn C-2, -6), 120.8 (C-4),
119.9 (C-6), 118.1 (C-7), 104.9 (C-3a), 71.7 (C-10), 51.8 (Bn CH2), 32.0
(C-20), 30.5 (C-30); MS (EI): m/z (%) 391 (99) ([Mþ2]þ), 389 (100)
(Mþ), 280 (7), 269 (98), 252 (43), 222 (12), 191 (38), 164 (14), 123
(48), 121 (50), 103 (27). Anal. C17H16BrN3O3 (C, H, N).
4.1.2.3.5. 1,2-Bis(2-benzyl-5-nitro-2H-indazol-3-yloxy)ethane
(38). Yield: 51 mg (3%). Mp 205e207 ꢀC (1-PrOH).
4.1.2.3.6. 2-Benzyl-1-[2-(2-benzyl-5-nitro-2H-indazol-3-yloxy)
ethyl]-5-nitro-1,2-dihydro-3H-indazol-3-one (39). Yield: 0.24
g
(14%). Mp 207e209 ꢀC (MeNO2).
4.1.2.4. Preparation of (methoxycarbonyl)methyl derivatives 11/32
and cianomethyl derivative 12. A stirred mixture of 2-
benzylindazolinone 5 [44] (1.00 g, 3.71 mmol), methyl bromoace-
tate or bromoacetonitrile (4.5 mmol) and sodium hydrogen car-
bonate (0.42 g, 5.00 mmol) in acetone (30 mL) was heated at 35 ꢀC
for 5 days. The mixture was then evaporated to dryness and, after
addition of water (50 mL), the precipitated solid was collected by
filtration. For 11/32, the obtained solid was washed with acetone
(2 ꢂ 5 mL) affording pure compound 11 (1.03 g). The filtrate was
concentrated to dryness, dissolved in chloroform and applied to the
top of a chromatography column which was eluted with chloroform
and a chloroform/acetone mixture (50:1) to afford, following this
elution order, compound 32 and then an additional amount of
compound 11.
Spectral and analytical data of compounds 13 and 33 are
included as supplementary material.
4.1.2.5.1. 2-Benzyl-1-[2-(methoxycarbonyl)ethyl]-5-nitro-1,2-
dihydro-3H-indazol-3-one (13). Yield: 0.56 g (42%). Mp 127e129 ꢀC
(2-PrOH).
4.1.2.5.2. 2-Benzyl-1-(2-carboxyethyl)-5-nitro-1,2-dihydro-3H-
indazol-3-one (14). Yield: 0.33 g (26%). Mp 224e226 ꢀC (EtOH/
H2O). 1H NMR [300 MHz, (CD3)2SO]:
d 12.39 (br s, 1H, COOH), 8.50
For compound 12, the solid obtained after filtration was directly
chromatographed with chloroform/acetone mixtures (50:1 to 30:1)
to afford pure 1-cyanomethyl derivative.
Alkylation of compound 5 with methyl bromoacetate or bro-
moacetamide in potassium carbonate/DMF at 100 ꢀC afforded the
rearranged quinazolinones 40e43 (see below, section 4.1.5).
Spectral and analytical data of compound 12 are included as
supplementary material.
4.1.2.4.1. 2-Benzyl-1-(methoxycarbonyl)methyl-5-nitro-1,2-
dihydro-3H-indazol-3-one (11). Yield: 1.18 g (93%). Crystals (2-
PrOH) of this compound soften at 150e170 ꢀC, then the com-
pound resolidifies showing a further mp at 195e197 ꢀC, corre-
sponding (TLC) to that of the rearranged quinazolinone 40. 1H NMR
(d, J ¼ 2.1 Hz, 1H, 4-H), 8.37 (dd, J ¼ 9.3, 2.1 Hz, 1H, 6-H), 7.69 (d,
J ¼ 9.3 Hz,1H, 7-H), 7.26 (m, 5H, Bn aromatic H), 5.17 (s, 2H, Bn CH2),
4.27 (t, J ¼ 6.9 Hz, 2H, 10-H), 2.26 (t, J ¼ 6.9 Hz, 2H, 20-H); 13C NMR
[75 MHz, (CD3)2SO]: d
171.9 (C-30), 161.3 (C-3), 149.0 (C-7a), 141.6
(C-5),136.0 (Bn C-1),128.7 (Bn C-3, -5),127.8 (Bn C-4),127.3 (Bn C-2,
-6), 127.1 (C-6), 120.3 (C-4), 116.2 (C-3a), 112.7 (C-7), 45.0 (Bn CH2),
43.1 (C-10), 30.5 (C-20); MS (ESþ): m/z (%) 705 (51) ([2MþNa]þ), 683
(35) ([2MþH]þ), 364 (65) ([MþNa]þ), 342 (100) ([MþH]þ). Anal.
C17H15N3O5 (C, H, N).
4.1.2.5.3. 2-Benzyl-3-[2-(methoxycarbonyl)ethoxy]-5-nitro-2H-
indazole (33). Yield: 0.15 g (11%). Mp 130e132 ꢀC (2-PrOH).
4.1.3. Alkoxycarbonylation, acylation and sulfonylation of 2-benzyl-
5-nitroindazolin-3-one 5: preparation of alkoxycarbonyl (25, 26),
acyl (27, 28) and tosyl (29) derivatives
[300 MHz, (CD3)2SO]:
d
8.54 (d, J ¼ 2.1 Hz, 1H, 4-H), 8.40 (dd, J ¼ 9.3,
2.1 Hz, 1H, 6-H), 7.67 (d, J ¼ 9.3 Hz, 1H, 7-H), 7.27 (m, 5H, Bn aro-
matic H), 5.15 (s, 2H, Bn CH2), 5.09 (s, 2H,10-H), 3.41 (s, 3H, CH3); 13C
To a stirred solution of the starting 2-benzylindazolin-3-one 5

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
305
[44] (1.00 g; 3.71 mmol) in pyridine (15 mL), the required alkyl
chloroformate (4.00 mmol) (for 25, 26), acetic anhydride (2.0 mL,
excess) (for 27) or the corresponding acid chloride (3.90 mmol) (for
28, 29) were slowly added. The reaction mixture was heated at
100 ꢀC during 3 h (for 27), or stirred at room temperature during 1 h
(for 25, 26, 28, 29), and then poured into water (100 mL). The
precipitated solid was collected by filtration, washed with 2% aq.
HCl (50 mL) and with plenty water, and air-dried.
3, -5), 128.1 (C-6), 127.9 (Bn C-4), 127.4 (Bn C-2, -6), 120.4 (C-4),
115.5 (C-3a), 112.1 (C-7), 104.7 (C-20), 47.0 (Bn CH2); MS (EI): m/z (%)
295 (100) (Mþ), 280 (2), 265 (2), 218 (2), 158 (3), 145 (3), 116 (7), 104
(9). Anal. C16H13N3O3 (C, H, N).
4.1.4.2. Preparation of 4-(indazol-1-yl)butiric acid 16. A mixture of
ethyl ester 15 (0.50 g, 1.30 mmol) and lithium hydroxide (0.16 g,
6.68 mmol) in THF/H2O (1:1, 20 mL) was stirred at room temper-
ature for 12 h. THF was then evaporated and the aqueous solution
acidified with conc. aq. HCl (pH 1). The precipitated solid was
collected by filtration, washed with 1% aq. HCl (3 ꢂ 3 mL) and air-
dried.
4.1.4.2.1. 2-Benzyl-1-(3-carboxypropyl)-5-nitro-1,2-dihydro-3H-
indazol-3-one (16). Yield: 0.42 g (91%). Mp 202e204 ꢀC (EtOH/
H2O). Spectral and analytical data of this compound are included as
supplementary material.
Spectral and analytical data of compounds 26, 28 and 29 are
included as supplementary material.
4.1.3.1. 2-Benzyl-1-ethoxycarbonyl-5-nitro-1,2-dihydro-3H-indazol-
3-one (25). Yield: 1.20 g (95%). Mp 135e137 ꢀC (EtOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.56 (d, J ¼ 2.2 Hz,1H, 4-H), 8.52 (dd, J ¼ 9.0,
2.2 Hz, 1H, 6-H), 8.00 (d, J ¼ 9.0 Hz, 1H, 7-H), 7.26 (m, 3H) and 7.11
(m, 2H) (Bn aromatic H), 5.33 (s, 2H, Bn CH2), 4.39 (q, J ¼ 7.0 Hz, 2H,
Et CH2), 1.30 (t, J ¼ 7.0 Hz, 3H, CH3); 13C NMR [75 MHz, (CD3)2SO]:
d
162.6 (C-3), 149.5 (COO), 145.6 (C-7a), 144.2 (C-5), 135.3 (Bn C-1),
4.1.4.3. Preparation of amide 17 and N-methylamide 18. For com-
pound 17, a mixture of ethyl ester 15 (0.50 g, 1.30 mmol) and a
saturated solution of ammonia in methanol (15 mL) was allowed to
stand at room temperature for 10 days. After evaporation of the
solvent and ammonia, in order to remove some minor byproducts,
the residue was directly applied to the top of a column which was
eluted with a chloroform/methanol (25:1) mixture to afford the
required product.
For compound 18, a mixture of ethyl ester 15 (0.50 g, 1.30 mmol)
and 8 M methylamine in ethanol (20 mL) was allowed to stand at
room temperature for 24 h. The reaction mixture was evaporated to
dryness to afford the desired amide.
128.9 (C-6), 128.7 (Bn C-3, -5), 128.0 (Bn C-4), 127.4 (Bn C-2, -6),
119.6 (C-4), 118.2 (C-3a), 116.3 (C-7), 64.7 (Et CH2), 50.0 (Bn CH2),
13.8 (CH3); MS (ESþ): m/z (%) 705 (39) ([2MþNa]þ), 683 (75)
([2MþH]þ), 364 (15) ([MþNa]þ), 342 (100) (MþH]þ). Anal.
C
17H15N3O5 (C, H, N).
4.1.3.2. 2-Benzyl-1-benzyloxycarbonyl-5-nitro-1,2-dihydro-3H-inda-
zol-3-one (26). Yield: 1.45 g (97%). Mp 143e145 ꢀC (2-PrOH).
4.1.3.3. 1-Acetyl-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one
(27). Yield: 1.03 g (89%). Mp 165e167 ꢀC (2-PrOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.56 (d, J ¼ 2.1 Hz, 1H, 4-H), 8.50 (dd, J ¼ 9.3,
4.1.4.3.1. 2-Benzyl-1-(3-carbamoylpropyl)-5-nitro-1,2-dihydro-
3H-indazol-3-one (17). Yield: 0.45 g (98%). Oil which solidifies after
trituration with 2-PrOH. Mp 106e108 ꢀC. Spectral and analytical
data of this compound are included as supplementary material.
4.1.4.3.2. 2-Benzyl-1-[3-(methylcarbamoyl)propyl]-5-nitro-1,2-
dihydro-3H-indazol-3-one (18). Yield: 0.45 g (94%). Mp 159e161 ꢀC
2.1 Hz, 1H, 6-H), 8.30 (d, J ¼ 9.3 Hz, 1H, 7-H), 7.25 (m, 3H) and 7.11
(m, 2H) (Bn aromatic H), 5.33 (s, 2H, CH2), 2.57 (s, 3H, CH3); 13C
NMR [75 MHz, (CD3)2SO]: d 168.4 (Ac CO), 164.0 (C-3), 145.9 (C-7a),
144.1 (C-5), 135.1 (Bn C-1), 128.9 (C-6), 128.7 (Bn C-3, -5), 128.0 (Bn
C-4), 127.6 (Bn C-2, -6), 119.7 (C-4), 118.5 (C-3a), 116.2 (C-7), 51.1
(CH2), 25.0 (CH3); MS (ESþ): m/z (%) 645 (44) ([2MþNa]þ), 623 (3)
([2MþH]þ), 334 (62) ([MþNa]þ), 312 (100) ([MþH]þ). Anal.
(2-PrOH). 1H NMR [300 MHz, (CD3)2SO]:
d
8.52 (d, J ¼ 2.1 Hz, 1H, 4-
H), 8.36 (dd, J ¼ 9.3, 2.1 Hz, 1H, 6-H), 7.67 (br q, J ¼ 4.5 Hz, 1H, NH),
7.61 (d, J ¼ 9.3 Hz, 1H, 7-H), 7.25 (m, 5H, Bn aromatic H), 5.20 (s, 2H,
Bn CH2), 4.03 (t, J ¼ 7.4 Hz, 2H, 10-H), 2.52 (d, J ¼ 4.5 Hz, 3H, CH3),
1.97 (t, J ¼ 7.2 Hz, 2H, 30-H), 1.53 (m, 2H, 20-H); 13C NMR [75 MHz,
C
16H13N3O4 (C, H, N).
4.1.3.4. 1-Benzoyl-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one
(28). Yield: 1.36 g (98%). Mp 164e166 ꢀC (2-PrOH).
(CD3)2SO]: d
171.3 (C-40), 160.8 (C-3), 148.4 (C-7a), 141.2 (C-5), 136.1
(Bn C-1), 128.7 (Bn C-3, -5), 127.8 (Bn C-4), 127.2 (Bn C-2, -6), 127.1
(C-6),120.5 (C-4),115.5 (C-3a),111.9 (C-7), 46.2 (C-10), 44.6 (Bn CH2),
31.3 (C-30), 25.4 (CH3), 22.0 (C-20); MS (ESþ): m/z (%) 759 (17)
([2MþNa]þ), 737 (53) ([2MþH]þ), 391 (22) ([MþNa]þ), 369 (100)
([MþH]þ). Anal. C19H20N4O4 (C, H, N).
4.1.3.5. 2-Benzyl-5-nitro-1-tosyl-1,2-dihydro-3H-indazol-3-one (29).
Yield: 1.54 g (98%). Mp 162e164 ꢀC (1-PrOH).
4.1.4. Transformation of alkylation products
4.1.4.1. Preparation of vinyl derivative 6. A solution of 1-(2-
bromoethyl)indazolinone 9 (0.56 g, 1.49 mmol) and piperidine
(0.35 g, 4.11 mmol) in ethanol (20 mL) was refluxed for 48 h. The
solution was evaporated to dryness and, after addition of 2% aq. HCl
(50 mL), extracted with chloroform (3 ꢂ 50 mL). The organic phase
was dried (MgSO4) and evaporated to dryness to afford the title
compound; similar results were obtained using methylamine (33%
w/w solution in ethanol).
4.1.4.4. Preparation of N,N-dimethylamide 19. A suspension of acid
16 (0.53 g,1.49 mmol) in a mixture of thionyl chloride (1.00 mL) and
chloroform (30 mL) was refluxed for 1 h. After evaporation of the
solvent and excess of thionyl chloride, a solution of dimethylamine
hydrochloride (1.63 g, 19.99 mmol) and potassium carbonate
(2.80 g, 20.26 mmol) in water (20 mL) was added to the oily residue.
The mixture was vigorously stirred for 12 h and then the precipi-
tated product 19 was collected by filtration, washed with water
(3 ꢂ 5 mL) and air dried.
Compound 6 was also obtained as a byproduct arising from
alkylation of 2-benzylindazolinone 5 with 1,2-dibromoethane (see
above, section 4.1.2.3).
4.1.4.4.1. 2-Benzyl-1-[3-(dimethylcarbamoyl)propyl]-5-nitro-1,2-
dihydro-3H-indazol-3-one (19). Yield: 0.51 g (90%). Mp 144e146 ꢀC
(2-PrOH). Spectral and analytical data of this compound are
included as supplementary material.
4.1.4.1.1. 2-Benzyl-5-nitro-1-vinyl-1,2-dihydro-3H-indazol-3-one
(6). Yield: 0.41 g (93%). Mp 116e118 ꢀC (2-PrOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.53 (d, J ¼ 3.0 Hz,1H, 4-H), 8.40 (dd, J ¼ 9.3,
3.0 Hz, 1H, 6-H), 7.71 (d, J ¼ 9.3 Hz, 1H, 7-H), 7.26 (m, 3H) and 7.15
(m, 2H) (Bn aromatic H), 7.08 (dd, J ¼ 14.7, 9.0 Hz, 1H, 10-H), 5.25
(dd, J ¼ 14.7, 1.4 Hz, 1H, 20-Htrans), 5.20 (s, 2H, Bn CH2), 5.10 (dd,
4.1.4.5. Preparation of 3-ethoxypropyl derivative 23. To a solution of
1-(3-bromopropyl) derivative 10 (0.59 g, 1.51 mmol) in ethanol
(100 mL), sodium hydroxide (0.40 g) in water (20 mL) was added,
and the mixture was stirred at room temperature for 3 days.
J ¼ 9.0, 1.4 Hz, 1H, 20-Hcis); 13C NMR [75 MHz, (CD3)2SO]:
d 162.0 (C-
3), 146.7 (C-7a), 142.1 (C-5), 135.5 (Bn C-1), 129.9 (C-10), 128.7 (Bn C-

306
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
Ethanol was then evaporated and the basic solution extracted with
chloroform (3 ꢂ 50 mL). The organic phase was dried (MgSO4),
concentrated and applied to the top of a chromatography column;
elution with chloroform/acetone mixtures (50:1 to 25:1) afforded
first 1-allyl derivative 7 [28 mg (6%)] and then ethyl ether 23.
4.1.4.5.1. 2-Benzyl-1-(3-ethoxypropyl)-5-nitro-1,2-dihydro-3H-
indazol-3-one (23). Yield: 0.38 g (71%). Mp 93e95 ꢀC (2-PrOH).
Spectral and analytical data of this compound are included as
supplementary material.
Spectral and analytical data of compounds 42 and 43 are
included as supplementary material.
4.1. 5.1. 3-Benzyl-2-methoxycarbonyl-6-nitro-1, 2, 3, 4-
tetrahydroquinazolin-4-one (40). Mp 194e196 ꢀC (1-PrOH). 1H NMR
[300 MHz, (CD3)2SO]:
d
8.84 (br s, 1H, 1-H), 8.46 (d, J ¼ 2.7 Hz, 1H, 5-
H), 8.14 (dd, J ¼ 9.0, 2.7 Hz, 1H, 7-H), 7.31 (m, 5H, Bn aromatic H),
6.91 (d, J ¼ 9.0 Hz, 1H, 8-H), 5.49 (s, 1H, 2-H), 5.12 (d, J ¼ ꢁ15.3 Hz,
1H, Bn CHA), 4.33 (d, J ¼ ꢁ15.3 Hz, 1H, Bn CHB), 3.56 (s, 3H, CH3); 13
C
NMR [75 MHz, (CD3)2SO]:
d 169.4 (COOMe), 160.6 (C-4), 151.2 (C-
4.1.4.6. Preparation of 2-acetoxyethyl derivative 24. A suspension of
1-(2-hydroxyethyl) derivative 20 (0.47 g, 1.50 mmol) in acetic an-
hydride (5 mL) was heated at 100 ꢀC for 2 h. The reaction was
evaporated to dryness and the residue triturated with 2-PrOH
(2 mL); the insoluble title compound was collected by filtration,
washed with 2-PrOH (2 ꢂ 2 mL) and air-dried.
8a), 138.3 (C-6), 136.5 (Bn C-1), 128.9 (C-7), 128.5 (Bn C-3, -5), 127.8
(Bn C-2, -6),127.5 (Bn C-4),124.3 (C-5),114.8 (C-8),113.4 (C-4a), 67.0
(C-2), 52.8 (CH3), 47.9 (CH2); MS (ESþ): m/z (%) 705 (41)
([2MþNa]þ), 683 (9) ([2MþH]þ), 364 (43) ([MþNa]þ), 342 (100)
(MþH]þ), 102 (87). Anal. C17H15N3O5 (C, H, N).
4.1.4.6.1. 1-(2-Acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-
indazol-3-one (24). Yield: 0.47 g (88%). Mp 168e170 ꢀC (2-PrOH).
4.1.5.2. 3-Benzyl-2-carbamoyl-6-nitro-1,2,3,4-tetrahydroquinazolin-
4-one (41). Mp 219e221 ꢀC (EtOH). 1H NMR [300 MHz, (CD3)2SO]:
1H NMR [300 MHz, (CD3)2SO]:
d
8.51 (d, J ¼ 2.4 Hz, 1H, 4-H), 8.38
d
8.46 (br s, 1H, 1-H), 8.43 (d, J ¼ 2.7 Hz, 1H, 5-H), 8.10 (dd, J ¼ 9.0,
(dd, J ¼ 9.0, 2.4 Hz,1H, 6-H), 7.66 (d, J ¼ 9.0 Hz,1H, 7-H), 7.25 (m, 5H,
Bn aromatic H), 5.19 (s, 2H, Bn CH2), 4.36 (t, J ¼ 4.8 Hz, 2H, 10-H),
4.07 (t, J ¼ 4.8 Hz, 2H, 20-H), 1.57 (s, 3H, CH3); 13C NMR [75 MHz,
2.7 Hz, 1H, 7-H), 7.66 (br s, 1H, CONHA), 7.46 (br s, 1H, CONHB), 7.33
(m, 5H, Bn aromatic H), 6.82 (d, J ¼ 9.0 Hz, 1H, 8-H), 5.31 (d,
J ¼ ꢁ15.6 Hz, 1H, Bn CHA), 5.17 (s, 1H, 2-H), 3.87 (d, J ¼ ꢁ15.6 Hz, 1H,
(CD3)2SO]:
d
169.5 (Ac CO), 161.2 (C-3), 149.9 (C-7a), 141.3 (C-5),
Bn CHB); 13C NMR [75 MHz, (CD3)2SO]:
d 169.7 (CONH2), 161.0 (C-4),
136.0 (Bn C-1), 128.7 (Bn C-3, -5), 127.9 (Bn C-4), 127.3 (Bn C-2, -6),
126.8 (C-6), 120.3 (C-4), 115.5 (C-3a), 112.5 (C-7), 60.2 (C-20), 45.9
(C-10), 44.8 (Bn CH2), 20.1 (CH3); MS (EI): m/z (%) 355 (100) (Mþ),
325 (2), 295 (5), 282 (6), 204 (6), 177 (9), 131 (10), 103 (7). Anal.
151.7 (C-8a), 137.5 (C-6), 136.6 (Bn C-1), 128.7 (C-7), 128.6 (Bn C-3,
-5), 127.6 (Bn C-2, -6), 127.4 (Bn C-4), 124.0 (C-5), 114.2 (C-8), 113.8
(C-4a), 67.6 (C-2), 47.2 (CH2); MS (ESþ): m/z (%) 675 (13)
([2MþNa]þ), 653 (24) ([2MþH]þ), 349 (5) ([MþNa]þ), 327 (82)
([MþH]þ), 102 (100). Anal. C16H14N4O4 (C, H, N).
C
18H17N3O5 (C, H, N).
4.1.5. Preparation of quinazolinones 40e43
4.1.5.3. 3-Benzyl-2-methoxycarbonyl-1-(methoxycarbonyl)methyl-6-
nitro-1,2,3,4-tetrahydroquinazolin-4-one (42). Mp 158e160 ꢀC
(EtOH).
a) Compounds 40e43 from starting indazolinone 5. A stirred
mixture of the starting 2-benzylindazolinone 5 [44] (1.00 g,
3.71 mmol), the required bromoacetic acid derivative (methyl ester
or amide) (5.00 mmol) and potassium carbonate (0.83 g,
6.00 mmol) in DMF (20 mL) was heated at 100 ꢀC for 45 min. The
reaction was evaporated to dryness and, after addition of water
(150 mL), the precipitated solid was collected by filtration, washed
with water and air-dried. For compounds 40/42, the obtained solid
was triturated with chloroform (15 mL); the insoluble material was
recovered by filtration, washed with chloroform (2 ꢂ 4 mL) and air-
dried to afford 0.63 g of pure compound 40. The filtrate was
concentrated and applied to the top of a column which was eluted
with a chloroform/methanol mixture (50:1) to afford, following
this elution order, compound 42 [0.38 g, 25%] and an additional
amount of compound 40 [overall yield: 0.77 g (61%)].
4.1.5.4. 3-Benzyl-2-carbamoyl-1-carbamoylmethyl-6-nitro-1,2,3,4-
tetrahydroquinazolin-4-one (43). Mp 244e246 ꢀC (MeNO2).
4.2. Biology
4.2.1. Study of antichagasic activity
4.2.1.1. T. cruzi strains culture. CL-B5 and Tulahuen C4 strains, both
stably transfected with Escherichia coli b-galactosidase gene (lacZ)
[66] and the Y strain of T. cruzi, originally isolated from an acute
human case [67] were used throughout the experiments.
Axenic cultures of T. cruzi CL-B5 lacZ epimastigotes were grown
at 28 ꢀC in liver infusion tryptose (LIT) medium, supplemented with
10% heat-inactivated FBS and antibiotics (penicillin 100 U/mL and
For compounds 41/43, the solid obtained after addition of water
was directly chromatographed using chloroform/methanol mix-
tures (30:1 to 10:1) to afford, following this elution order, com-
pound 41 [0.79 g, 65%] and compound 43 [0.06 g (4%)].
streptomycin 100
mg/mL) and continuously maintained in loga-
rithmic growth by weekly passages. Tissue culture-derived trypo-
mastigotes (TCT) of this strain were obtained by infecting L929 cells
with epimastigotes at the stationary phase (14-day-old cultures)
and incubated 24 h at 33 ꢀC in a humidified 5% CO2 atmosphere.
Non-penetrated parasites were removed by washing infected cul-
tures with phosphate-buffered saline (PBS). After the addition of
fresh medium, infected cultures were incubated in similar condi-
tions for 7 days and then TCT obtained in the supernatant.
Similarly, TCT of T. cruzi Tulahuen C4 lacZ were harvested in the
supernatant of L929 cultures previously infected with invasive
forms of the parasite and maintained in RPMIS at 37 ꢀC in a hu-
midified 5% CO2 atmosphere [68].
Regarding the Y strain, epimastigotes were maintained in the
conditions described above. Bloodstream trypomastigotes (BT)
were obtained by heart puncture from infected Swiss mice at the
parasitemia peak day and after their purification, resuspended in
RPMI medium supplemented with 5% heat-inactivated FBS. All
procedures involving mice were carried out in accordance with the
b) Rearrangement of indazolinone 11 to quinazolinone 40. A
mixture of indazolinone 11 (0.51 g, 1.49 mmol) and potassium
carbonate (0.22 g, 1.59 mmol) in acetone (40 mL) was stirred at
room temperature for 12 h. After evaporation of acetone and
addition of water (20 mL), the precipitated solid was collected by
filtration, dried and chromatographed using chloroform/acetone
mixtures (30:1 to 20:1) to afford compound 40 [0.44 g, 87%].
c) Compounds 42 and 43 from 40 and 41, respectively. A mixture
of the corresponding quinazolinone 40 or 41 (1.50 mmol), the
required bromoacetic acid derivative (methyl ester or amide)
(1.70 mmol) and potassium carbonate (0.27 g, 1.95 mmol) in DMF
(20 mL) was heated at 100 ꢀC for 1 h. The reaction was evaporated
to dryness and, after addition of water (100 mL), the precipitated
solid was collected by filtration, washed with water and air-dried to
afford the corresponding 1-substituted quinazolinones 42 [0.50 g
(81%)] and 43 [0.55 g (96%)].

C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
307
guidelines established by the Fiocruz Committee for the Use of
Animals (CEUA LW16/14).
(cell:parasite) and incubated overnight at 33 ꢀC with 5% CO2.
Following the infection, the medium was discarded and cultures
washed with PBS to remove non-penetrated TCT. Infected cultures
were incubated within compounds diluted in fresh MEM for 7 days
in similar conditions of temperature and humidity. Each concen-
tration was assayed in triplicate and controls of compounds, me-
dium, cell growth and infection included in all the plates. By this
4.2.1.2. Mammalian cell cultures. Murine L929 fibroblasts were
grown in plastic culture flasks (75 cm2) and sustained either in
MEM or RPMIS without phenol-red, supplemented as reported [8].
Cell cultures were maintained in a humidified 5% CO2 atmosphere
at 37 ꢀC and sub-passaged once a week using a solution 0.03% EDTA
and 0.05% trypsin in PBS for cell-detachment.
Primary cultures of embryonic cardiac cells were obtained from
Swiss mice as previously described [69]. After their purification,
cultures were sustained in Dulbecco's Modified Eagle's Medium
time, 50 mL of CPRG in 3% Triton X-100 (final concentration 400 mM,
pH 7.4) was added per well and the plates incubated 3 h at 37 ꢀC.
Absorbance was read at 595 nm in an Infinite 200 multifunctional
microplate reader (Tecan) and percentages of amastigote growth
inhibition (%AGI) calculated as reported [71].
(DMEM) supplemented with 2.5 mM CaCl2, 1 mM
L
-glutamine, 5%
The assay on Tulahuen amastigotes was conducted in 96-well
tissue culture plates by distributing 100 mL/well of RPMIS con-
heat-inactivated foetal bovine serum (FBS) (30 min, 56 ꢀC) and 2%
chicken embryo extract. Finally, cardiac cells cultures were main-
tained at 37 ꢀC in an atmosphere of 5% CO2.
taining 4000 L929 cells. The plates were maintained for 24 h at
37 ꢀC with 5% CO2 and afterwards, cells were incubated within TCT
(ratio 1:10) for another 2 h. Next, non-penetrated parasites were
discarded replacing culture medium by fresh one. In order to
establish the infection, the plates were maintained during 48 h at
37 ꢀC with 5% CO2. Then, medium was replaced by solutions of each
compound in fresh RPMIS and the plates incubated for 96 h at the
same conditions of temperature and humidity. Each concentration
was evaluated by triplicate and controls of infection and cell growth
4.2.1.3. Epimastigote susceptibility assays. The activity over T. cruzi
CL-B5 lacZ was evaluated in presence of the substrate CPRG [66,68],
according to the colorimetric method optimized by our research
group [70], with some modifications [71]. The assay was performed
in 96-well microtitre plates by seeding 2.5 ꢂ 105 log-phase epi-
mastigotes/mL (200
mL/well), which were incubated within the
compounds for 72 h at 28 ꢀC. Stock solutions of the studied com-
pounds and the reference drug benznidazole [kindly provided by
included in all the plates. Afterwards, 50 mL of 500 mM CPRG in 0.5%
Nonidet P40 was added to each and the plates returned to the
incubator for 18 h. Finally, absorbance was read at 570 nm in a
SpectraMax 190 microplate reader (Molecular Devices) and the
results expressed as %AGI [68].
ꢀ
^
LAFEPE (Laboratorio Farmaceutico de Pernambuco), Brazil] were
prepared in (CH3)2SO (DMSO) and added to the cultures to give
0.125e256 mM final concentrations. Concentration of DMSO in the
cultures was always <0.2% v/v. Each concentration was tested in
triplicate and growth, medium and drug controls included in all the
Regarding the assay over Y strain amastigotes, 100,000 cardiac
cells/well were seeded in 24-well tissue culture plates provided
with round coverslips previously coated with gelatin and then,
maintained overnight in DMEM at 37 ꢀC with 5% CO2. After 24 h of
celleparasite interaction (ratio 1:10), infected cultures were
washed to remove non-internalized BT and incubated 48 h within
compounds diluted in fresh DMEM. Next, cultures were fixed with
Bouin's fixative and stained with Giemsa. The mean number of
infected cells and the mean number of parasites per infected cell
were scored in 400 host cells by duplicate and thus the endocytic
index (EI) calculated by multiplying these two parameters. Only
parasites with characteristic nuclei and kinetoplast were counted,
since irregular ones were considered as parasites undergoing
death. Activity results were estimated by calculating the percentage
of inhibition of the EI (% EI) [73].
plates. Afterwards, 50
mL of CPRG in 0.9% Triton X-100 was added
per well (final concentration 200
m
M, pH 7.4) and the plates incu-
bated 3 h at 37 ꢀC. Finally, absorbance was read at 595 nm in an
ELx808 ELISA reader (Biotek Instruments Inc.) and percentages of
epimastigote growth inhibition (%EGI) estimated as previously re-
ported [71].
The activity on Y strain epimastigotes was evaluated by applying
the resazurin assay previously standardized by our group [72]. Log-
phase cultures were seeded at a density of 3 ꢂ 106 epimastigotes/
mL in culture tubes and maintained at 28 ꢀC overnight to allow
homogeneous growth. Afterwards, 200 mL per well of cultures were
distributed in 96-well microplates and incubated within the com-
pounds for 48 h at 28 ꢀC. Each concentration was tested in triplicate
and growth, medium and drug controls included in all the plates.
For each strain, the assays were run at the same conditions three
times separately (n ¼ 3). IC50 values were estimated from the
doseeresponse curves obtained by plotting drug concentrations vs
%AGI or %EI. Results are expressed as the mean value of IC50 SD
(standard deviation) (SPSS, v20, IBM).
Then, 20 mL/well of resazurin in PBS (3 mM, pH 7) was added and
the plates maintained at 28 ꢀC for another 5 h. Finally, fluorescence
intensity was measured (AlamarBlue® Assay, U.S. Patent No.
5,501,959; lexc 535 nm, lem 590 nm) in an Infinite 200 multifunc-
tional microplate reader (Tecan) and %EGI estimated.
For both assays, the experiments were run in the same condi-
tions three times separately (n ¼ 3). IC50 values, i.e., the concen-
trations causing 50% of epimastigote growth inhibition (EGI), were
estimated from the doseeresponse curves obtained by plotting
drug concentrations vs %EGI. Results are expressed as the mean
value of IC50 SD (standard deviation) (SPSS, v20, IBM).
4.2.1.5. Unspecific cytotoxicity assays. The cytotoxic profile on
L929 cells was studied in 96-well flat bottom microplates con-
taining 100
m
L of MEM with 15 ꢂ 103 cells/well. After cell attach-
ment (3 h at 37 ꢀC, 5% CO2), fibroblasts were incubated for 48 h at
37 ꢀC and 5% CO2 within compounds diluted in MEM. Each con-
centration was tested in triplicate and controls of the compounds,
medium and cultures included in all the plates. Afterwards, 20 mL of
4.2.1.4. Amastigote susceptibility assays. The activity on intracel-
lular amastigotes was tested by infecting either L929 fibroblasts
with TCT (CL-B5 and Tulahuen strains) or cardiac cells with BT (Y
strain).
resazurin in PBS solution (2 mM, pH 7) was added to each well and
the plates incubated similarly for 3 h. Fluorescence intensity was
measured as aforementioned (lexc 535 nm, lem 590 nm) and per-
centages of unspecific cytotoxicity (%C) calculated [71].
The assay on CL-B5 lacZ amastigotes was performed in 48-well
The toxic effect on cardiac cell cultures was evaluated by seeding
tissue culture plates by using CPRG [72]. According to this, 120
mL
per well 6 ꢂ 104 cardiac cells/100
mL DMEM in 96-well microplates
of MEM containing 10,000 L929 cells were seeded per well and the
plates incubated 2 h at 37 ꢀC in a humidified 5% CO2 atmosphere.
After the attachment, cells were infected with TCT at a 1:6 ratio
previously coated with gelatin and then, incubated 24 h at 37 ꢀC in a
humidified 5% CO2 atmosphere. Afterwards, medium was replaced
by solutions of each compound in fresh DMEM and plates

308
C. Fonseca-Berzal et al. / European Journal of Medicinal Chemistry 115 (2016) 295e310
incubated 48 h in similar conditions of temperature and humidity.
Each concentration was tested in triplicate and controls included in
all the plates. Finally, cell morphology and contractibility were
examined by light microscopy and viability evaluated in presence of
the redox indicator PrestoBlue® according to manufacturer's in-
structions. After 5 h of incubation at 37 ꢀC and 5% CO2, absorbance
was measured at 570 nm and 600 nm in a SpectraMax 190
microplate reader (Molecular Devices) and %C estimated as
described previously [68].
For both assays, all the experiments were developed similarly
for three (n ¼ 3). LC50 values, i.e., the concentrations causing 50% of
cellular lethality, were estimated from de doseeresponse curves
obtained by plotting drug concentrations vs %C. Results are
4.3. Physicochemical and pharmacokinetic parameters
4.3.1. In silico calculation of parameters
A set of 34 physicochemical descriptors was computed using
€
QikProp version 3.5 integrated in Maestro (Schrodinger, LLC, New
York, USA). Relevant QikProp descriptors are presented as supple-
mentary material (Table S1). The 3D conformations used in the
calculation of QikProp descriptors were generated using the pro-
gram Spartan '08 (Wave function, Inc., Irvine CA) as follows: the
structure of each molecule was built from the fragment library
available in the program. Then, ab initio energy minimizations of
each structure at the Hartree-Fock 6-31G* level were performed. A
conformational search was next implemented using Molecular
Mechanics (Monte Carlo method) followed by a minimization of
the energy of each conformer calculated at the Hartree-Fock 6-31G*
level. The global minimum energy conformer of each compound
was used as input for ADME studies with QikProp.
expressed as the mean value of LC50
(SPSS, v20, IBM).
SD (standard deviation)
4.2.2. Study of trichomonacidal activity
4.2.2.1. T. vaginalis culture. The in vitro assays were conducted with
two different isolates from the American Type Culture Collection
(ATCC, Maryland, USA): JH31A#4 (metronidazole-sensitive) and
IR78 (metronidazole-resistant). Trophozoites were cultivated in
TYM (Tripticase-Yeast-Maltose) medium supplemented with 10%
fetal bovine serum (FBS) and 5% of antibiotic solution (100 IU
4.3.2. Taft's
Most values of
material, Table S1) have been obtained from the literature [76]; in
some cases, * values have been calculated from pKas found in the
literature for the corresponding carboxylic acids [77] using the
equation pKa ¼ 4.66e1.62 * [76].
s
* constants
s
* constants for substituents (supplementary
s
penicillin and 100
mg/mL streptomycin), in a humified chamber at
s
37 ꢀC and 5% CO2.
4.3.3. Conformational analysis and electrostatic potential maps
calculation
4.2.2.2. Mammalian cell cultures. The cellular line employed for the
unspecific cytotoxic assays were Vero CCL-81 (ATCC, Maryland,
USA). The cells were cultured in RPMI medium (SigmaeAldrich),
supplemented with 10% FBS and antibiotics solution (100 IU peni-
Global minimum energy analysis of the compounds was per-
formed using ab initio Hartree-Fock (HF) calculations at the 6-31G*
level, within the Spartan ’08 (Wave function, Inc., Irvine, CA). A
conformational search was next implemented using Molecular
Mechanics (Monte Carlo method). Local energy minima were
identified by rotation of a subject torsion angle through 360ꢀ in 60ꢀ
increments (6-fold search), followed by HF 6-31G* energy mini-
mization of each rotamer generated. The electrostatic potential of
the global minimum energy conformer was calculated using the
Hatree-Fock method at the 6-31G* level of theory and was mapped
on the 0.002 isodensity surface of each molecule. The surface was
color-coded according to the potential, with electron-rich regions
colored red and electron-poor regions colored blue (supplementary
material, Fig. S1).
cillin and 100 mg/mL streptomycin) in a humidified 95% air/5% CO2
atmosphere at 37 ꢀC.
4.2.2.3. Trophozoites susceptibility assays. Initially, the studied
compounds were evaluated against the sensitive isolate JH31A#4.
Stock solutions of the studied compounds were prepared in DMSO
and added to glass tubes containing log-phase growth cultures
after 5 h of seeding 105 trophozoites/mL, to achieve 9.37e300
mM
final concentrations of the products. Final concentration of DMSO
was always <0.2% v/v. The biological activity was determined after
24 h of incubation at 37 ꢀC by fluorimetric determination using
resazurin (SigmaeAldrich) as redox dye as previously described
[74]. Metronidazole (SigmaeAldrich) at 25 mM concentration was
used as the reference drug.
Acknowledgments
Trichomonacidal activity against the resistant isolate IR78 was
evaluated only for compounds showing a relevant activity against
JH31A#4 isolate and absence of unspecific cytotoxic effect against
Vero cells [75].
The experiments were performed at least two times in triplicate.
IC50 values and the 95% confidence limits were calculated by probit
analysis (SPSS v20, IBM).
This work was partially supported by the Spanish Ministry of
Economy and Competitiveness (MINECO; ref. SAF2015-66690-R),
by the 911120 UCM-CEI Moncloa research group (E-Health Cluster),
by a Development Cooperation project from the UCM (ref. 9/12) and
by CNPq and FAPERJ. C. F.-B. and A. I.-E. acknowledge Moncloa
Campus of International Excellence (UCM-UPM & CSIC) for their
PICATA predoctoral fellowships. F. R. acknowledges a CSIC JAE-Doc
contract co-funded by EU (ESF, European Social Fund). M. N. C. S. is
research fellow of CNPq and CNE. The authors thank the Program
for Technological Development in Tools for Health-PDTIS-FIOCRUZ
for use of its facilities.
4.2.2.4. Unspecific cytotoxicity assays. Vero cells were seeded
(50,000 cells/well) in 96-well flat-bottom microplates (Nunc) with
100 m mL of
L of medium. After cell attachment for 6 h at 37 ꢀC, 100
medium containing the studied products were added. The unspe-
cific toxicity was determined after 24 h of incubation with the
compounds. Resazurin (1 mM stock solution) was used as redox
dye. After 3 h of incubation, fluorescence was measure in a fluo-
rimeter (Infinite 200, Tecan) (lexc 535 nm, lem 590 nm) following a
method previously published by our group [75].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.036.
Each concentration was assayed per triplicate and in two inde-
pendent assays. The concentration causing 50% of Vero cells growth
inhibition (CC50) and the 95% confidence limits was determined by
probit analysis (SPPS v20, IBM).
References
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