Thiosemicarbazones derived from 1-indanones as new anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1016/j.bmc.2011.09.037

In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12 and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular docking.

Full text of DOI:10.1016/j.bmc.2011.09.037

Bioorganic & Medicinal Chemistry 19 (2011) 6818–6826
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiosemicarbazones derived from 1-indanones as new
anti-Trypanosoma cruzi agents
María E. Caputto a, Lucas E. Fabian a, Diego Benítez c, Alicia Merlino c,d, Natalia Ríos c, Hugo Cerecetto c,
a,
Graciela Y. Moltrasio b, Albertina G. Moglioni a, Mercedes González c, , Liliana M. Finkielsztein
⇑
⇑
a Química Medicinal, Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Ciudad Autónoma de Buenos Aires, Argentina
b Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Ciudad Autónoma de Buenos Aires, Argentina
c Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
d Laboratorio de Química Teórica y Computacional, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2011
Revised 14 September 2011
Accepted 20 September 2011
Available online 24 September 2011
In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good
anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the
compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds
to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12
and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular
docking.
Keywords:
Thiosemicarbazones
1-Indanones
Ó 2011 Elsevier Ltd. All rights reserved.
Chagas’ disease
Anti-Trypanosoma cruzi agents
Microwave-assisted synthesis
Cruzipain
1. Introduction
region, and require long term treatment, besides they frequently
have toxic side effects.3 Therefore, it is necessary to find a more
Chagas’ disease or American Trypanosomiasis is a parasitic dis-
ease endemic to Latin America, where it affects about 20 million
people. Mortality rates range from 8% to 12%, depending on the
age and physiological state of the patient.1 The causative agent of
this disease is the flagellate protozoan Trypanosoma cruzi (T. cruzi),
which is transmitted to humans either by transfusion of infected
blood, from an infected mother to her child, by the feces of several
species of triatomine bugs that are strictly hematophages, or orally
by contaminated food.
The disease is controlled at present through the elimination of
the vectors with insecticides and serological screening of blood.
Better housing and educational campaigns are also fruitful ap-
proaches. Like other parasitic diseases, Chagas’ disease is associ-
ated with poverty and low educational levels. The development
of vaccines has thus far been unsuccessful.2 Chemotherapy to con-
trol this parasite infection is very limited and is based on the
nitroderivatives Nifurtimox (Nfx) and Benznidazole; these drugs
have variable therapeutic effects according to the geographical
effective and safer therapy against Chagas’ disease.
In this regard, thiosemicarbazones (TSCs) have been described
to present parasitocidal action against T. cruzi.4 TSCs are a class
of small molecules that have been evaluated over the last 50 years
as antiviral,5 antibacterial6 and anticancer compounds.7 Du and
co-workers first introduced the thiosemicarbazone functionality
into compounds designed to inhibit cruzipain,8 the major cysteine
protease expressed in all the life cycle stages of the parasite. This
endoproteinase is the most abundant member among the
cysteine-, serine-, threonine-, and metallo-proteinases, and it is
expressed as a complex mixture of isoforms. The bulk of the
enzyme is lysosomal and is also present in an epimastigote-specific
pre-lysosomal organelle called ‘reservosome’; in addition, some
plasma membrane-bound isoforms and cruzipain forms released
into the medium have been reported. Cruzipain is essential for
infection host cells, replication and metabolism of the parasite
and plays multiple roles in disease pathogenesis, therefore has
been emerged as one of the best targets for the development of
vaccines and drugs with anti-T. Cruzi activity.8
In the search for a pharmacological control of Chagas’ disease,
new 5-nitrofurane derivatives containing thiosemicarbazone moi-
ety and their metallic complexes have been designed and synthe-
sized, showing significant anti-T. cruzi activity.9
⇑
Corresponding authors. Tel.: +598 2 5250749 (M.G.); Tel.: +54 11 49648233;
fax: +54 11 49648252 (L.M.F.).
E-mail addresses: megonzal@fq.edu.uy (M. González), lfinkiel@ffyb.uba.ar (L.M.
Finkielsztein).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.037

M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
6819
Previously, we have reported the evaluation of several TSCs
derived from 1-indanones with different patterns of substitution
in the aromatic ring against bovine viral diarrhea virus (BVDV) as
a surrogate model of hepatitis C virus (HCV).10 The TSC derived
from 5,6-dimethoxy-1-indanone showed the most potent anti-
BVDV activity, with a selectivity index higher than that of the
reference drug Ribavirin.
Taking into account the anti-T. cruzi activity presented by com-
pounds possessing a thiosemicarbazone moiety, we decide to
investigate the potential antichagasic action of TSCs derived from
1-indanones previously synthesized (1–14). The promising antich-
agasic activity showed by these compounds led us to prepare new
TSCs with other substituents on the aromatic ring of the indanic
nucleus and different substitution patterns, as well as N4-substi-
tuted TSCs with phenyl groups (N4-TSCs). In this work, we present
the in vitro anti-proliferative activity of twenty-four TSCs (1–24)
(Fig. 1), including the synthesis of four new TSCs and six new
N4-TSCs, all derived from 1-indanones, on the epimastigote form
of T. cruzi. We developed a microwave-assisted synthesis that com-
prises a three-component coupling reaction for N4-TSCs prepara-
tion in good yields. A structure–activity relationship is also
discussed to highlight the structural requirements for optimal
activity. Docking analysis with cruzipain was performed in order
to explore the capacity of these TSCs to inhibit this enzyme. Also,
experimental cruzipain inhibition capacity was experimentally
investigated for some selected features.
H
N
H
N
H
N
R4
R1
N
N
NH2
R3
R2
R3
R2
S
S
R5
1-18
19-24
Figure 1. General chemical structures of the TSCs and N4-TSCs synthesized.
H
R4
R1
O
R4
R1
N
N
R3
R2
NH2
NH2NHCSNH2
R3
R2
S
EtOH
reflux
1-18
Scheme 1. Synthetic procedure for obtaining TSCs 1–18.
2. Results and discussion
2.1. Chemistry
O
O
O
O2N
TSCs 1–18 were synthesized in high yield from the correspond-
ing 1-indanones by treatment with thiosemicarbazide, according
to the general procedure previously described10 (Scheme 1).
TSCs 15–18 are new compounds. 1-Indanone precursors were
available from different sources: 5-fluoro-1-indanone is a commer-
cial reagent; 4-nitro-1-indanone (25) and 6-nitro-1-indanone (26)
were prepared by nitration of 1-indanone, as shown in Scheme 2;
HNO3/H2SO4
+
CH3NO2
-15ºC
NO2
25
26
Scheme 2. Synthesis of 4-nitro- and 6-nitro-1-indanone.
O
O
ClCH2CH2COCl
AlCl3
H2SO4
Cl
CS2
Br
Br
Br
27
Scheme 3. Synthesis of 5-bromo-4,7-dimethyl-1-indanone.
28
Method A
NH2
O
N
R3
R2
R3
R2
i
ii
H
N
H
N
N
R3
R2
S
R5
Method B
iii
O
N
R3
R2
C
S
+
NH2NH2.H2O
+
R5
Scheme 4. Synthetic routes for obtaining N4-TSCs. Reaction conditions: (i) hydrazine hydrate 85%/isopropanol, reflux; (ii) isothiocyanate/benzene, room temperature; (iii)
acetic acid, isopropanol, MW.

6820
M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
and, finally, 5-bromo-4,7-dimethyl-1-indanone (28) was obtained
following the protocol presented in Scheme 3.
The second methodology (Method B, Scheme 4) is a one-pot
synthesis via a multicomponent coupling reaction, under micro-
wave irradiation (MW). For this environmentally friendly synthesis
toward N4-TSCs, in the same pot we mixed one equivalent of the
corresponding 1-indanone and an excess of 15% of isothiocyanate
and hydrazine hydrate in isopropanol and a catalytic amount of
acetic acid. This route afforded N4-TSCs in good yields and short
reaction times. Comparative results obtained in both synthetic pro-
cesses are shown in Table 1.
The N4-TSCs were obtained by two different synthetic method-
ologies. The first approach (Method A, Scheme 4) involves a proce-
dure that comprises two steps: (1) synthesis of hydrazone from the
corresponding 1-indanone and hydrazine hydrate, (2) preparation
of the desired N4-TSCs by reaction of hydrazone with the suitable
isothiocyanate. This method includes the isolation of each pre-
pared intermediate, which are time, solvent and energy consuming
procedure.
2.2. Anti-T. cruzi evaluation
Table 1
Comparative results obtained in the synthesis of N4-TSCs by Methods A and B
TSCs 1–18 and N4-TSCs 19–24 were evaluated against the epi-
mastigote form of T. cruzi, Tulahuen 2 strain (Table 2). The occur-
rence of the epimastigote form of T. cruzi as an obligate
mammalian intracellular stage has been reevaluated and con-
firmed.11 Furthermore, it should be noted that a good correlation be-
tween the anti-proliferative epimastigote activity and the in vivo
anti-T. cruzi activity was observed with compounds from our chem-
ical library.12 Firstly, the mentioned compounds were assayed at
H
H
N
N
N
R3
S
R2
R5
Compound R2
R3
R5
H
CH3 48
Cl
H
Method A
Method B
Time (h) Yield (%) Time (min) Yield (%)
25
lM concentration; all the TSCs were incorporated into the
growth medium at 25
l
M and its ability to inhibit the growth of
19
20
21
22
23
24
H
H
H
H
H
H
25
89
72
79
77
53
79
20
10
30
15
20
20
89
78
73
74
69
84
the parasite was evaluated by comparison with untreated controls
on day 5 and using Nfx as reference trypanosomicidal drug.12 The
50% inhibitory concentration (IC50) was determined. TSCs derived
from 1-indanones 1–18 exhibited, in some cases, relevant anti-T.
cruzi activities, the most active being the 4,5-dimethoxy-subtituted
4
25
OCH3 OCH3
OCH3 OCH3 CH3 72
OCH3 OCH3 Cl 48
Table 2
In vitro activity of synthesized TSCs
H
H
H
N
R4
N
N
N
N
NH2
R3
R2
R3
R2
S
S
R5
R1
1-18
19-24
PGIb
% Hemolysisc (25
lM)
SId
a
Compound
R1
R2
R3
R4
R5
IC50
(
lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
H
CH3
H
Br
H
CH3
OCH3
H
H
H
OCH3
H
H
NO2
H
H
CH3
H
H
H
H
H
CH3
H
CH3
CH3
CH3
Cl
H
OCH3
H
OCH3
OCH3
Cl
Br
H
H
F
Br
H
H
H
H
H
H
H
H
Cl
CH3
H
H
OCH3
OCH3
H
H
H
H
NO2
H
H
H
H
H
OCH3
OCH3
OCH3
H
H
H
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
18.6
3.6
4.0
4.3
5.7
>50
7.8
6.2
7.5
7.5
>50
1.8
>50
2.3
>50
>50
8.3
25.0
7.7
5.9
25
80.8
100.0
98.5
66.5
100.0
14.4
83.7
84.2
92.1
100.0
19.0
98.9
1.7
0.2
0.5
0.1
0.1
7.7
2.8
0.1
1.0
0.8
7.5
0.1
0.1
0.1
2.4
4.5
0.1
0.2
2.4
2.7
1.6
0.1
0.4
4.2
0.1
—
404.0
200.0
985.0
665.0
12.9
5.1
837
84.2
115.1
13.3
190.0
989.0
170.0
41.2
4.9
310.0
421.0
21.2
25.8
47.5
508.0
168.8
18.5
770
98.8
22.0
3.1
84.2
51.0
69.6
76.0
50.8
67.5
77.7
77.0
100.0
100.0e
H
CH3
Cl
H
CH3
Cl
H
H
H
H
OCH3
OCH3
OCH3
1.0
3.0
1.0
7.7
H
H
Nfx
—
Amphotericin B
0.152e
100.0e
1.0e
a
b
c
IC50: concentration that produces 50% reduction in parasite growth. All the values are the mean of three different experiments.
PGI: percentage of parasite growth inhibition at 25
% Hemolysis: percentage of erythrocytes lysis at 25
l
l
M. All the values are the mean of three different experiments.
M.
d
e
SI: selectivity index, ratio between PGI and % hemolysis.
Taken from Ref. 9h.

M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
6821
4.0x10-2
2.0x10-2
TSC 12 (Table 2). Despite the interesting biological properties of
some of these molecules, it is difficult to highlight in detail their
structure–activity relationship. Nevertheless, some general aspects
merit to be commented: chloro and nitro TSCs were not active
against T. cruzi in culture (6, 13, 15 and 16), except compound 7 with
moderate activity. TSCs with a methyl substituent in the aromatic
ring showed an increased anti-T. cruzi activity (2, 3 and 4). Also, it
should be noted that the relative position of the methoxyl group is
not significant when the TSC is monosubstituted (8, 9 and 10), how-
ever, when the TSC is disubstituted, there is a remarkable difference
in activity (11 and 12). All N4-TSCs (19–24) displayed very good
activities, being more active than the reference drug Nfx, with the
only exception of derivative 21. The increase in the anti-T. cruzi
activity when N4-substitution was performed is clearly noted. More-
over, the 5,6-dimethoxy substitution gives rise to some of the best
anti-T. cruzi agents (compare compounds 22, 23 and 24, with an
4.0x10-3
3.0x10-3
2.0x10-3
1.0x10-3
0.0
1
2 3 4 5 6 7 8 9
10 C11o1m2 p13. I1D4 15 16 17 18 19 20 21 22 23 24
IC50 of 1.0, 3.0 and 1.0
an IC50 >50 M).
lM, respectively, with compound 11, with
l
Figure 2. Dissociation constants of the TSCs studied.
2.3. Unspecific mammalian cell cytotoxicity
TSCs were evaluated in terms of the non-specific cytotoxicity
using human erythrocytes as a mammalian cell model.9h In these
experiments, Amphotericin B was used as a reference drug due
to its recognized hemolytic effects. All the TSCs were evaluated
Table 3
Cruzipain inhibition activities of selected TSC compared to IC50 against T. cruzi
epimastigotes
%Inh CPb (100
lM)
a
Compound
IC50
(
lM)
at 25 lM and compared with the parasite growth inhibition at
the same concentration (Table 2). None of the TSCs studied showed
remarkable unspecific mammalian cytotoxicity, with percentages
of erythrocyte lysis between 0.1 and 7.7. Therefore, the selectivity
indexes (SI), defined as the ratio between the percentages of para-
8
10
11
12
16
22
24
6.2
7.5
>50
1.8
>50
1.0
1.0
—
9.8
0.0
1.7
61.0
24.0
0.0
67.0
100.0
site growth inhibition at 25 lM (PGI) and of hemolysis, showed
values much higher than for Amphotericin B. These results demon-
strate that these TSCs could be considered excellent lead com-
pounds as anti-T. cruzi agents.
30-Bromopropiophenone
thiosemicarbazonec
a
IC50: concentration that produces 50% reduction in parasite growth. All the
values are the mean of three different experiments.
2.4. Molecular docking studies
b
% Inh CP: percentage of cruzipain inhibition at 100 lM.
c
Reference compound.8
Molecular docking models have been shown to adequately pre-
dict the binding mode of different anti-T. cruzi thiosemicarbazone
derivatives to cruzipain.9g,13 Therefore, in order to gain more in-
sights into the nature of the trypanosomicidal activity displayed
by the novel TSCs reported in this work, molecular docking studies
with cruzipain were performed. The theoretical dissociation con-
stants for the compounds studied as potential cruzipain inhibitors
(Kd CP) range from 10ꢀ4 to 10ꢀ2 M, indicating that these molecules
could be moderate inhibitors of the enzyme (Figure 2). It is worth
noticing that TSCs 12, 22 and 24, the most active compounds, are
those with best cruzipain inhibition capacity, with Kd values in
the order of 10ꢀ4 M. On the other hand, TSCs 2, 3, 4 and 23 showed
Kd values one order of magnitude higher, in agreement with their
good anti-T. cruzi activities. In the same sense, TSC 15, an inactive
agent, showed the highest Kd, demonstrating that, in general terms,
this molecular model is able to adequately predict the experimen-
tal data regarding the trypanosomicidal profile of these TSCs. An
exception to this model would be compounds 11 and 16 (inactive
compounds) with Kd values in the order of 10ꢀ4 M.
gated for the first time the mechanism governing the self aggrega-
tion of TSCs in water (in particular compound 11). Overall
information indicated the fast initial formation of negatively-
charged nano-aggregates that gradually grew in size to generate
larger clusters, these structures serving as nuclei for the later crys-
tallization and precipitation of the compound in water.14 We ex-
tended the study of aggregation to the compounds that were
selected to be evaluated as inhibitors of cruzipain, and we ob-
served that these derivatives have a similar tendency to form such
aggregates, therefore the differences in activity should not be
attributed to this phenomenon. The most active anti-proliferative
compounds were those with the highest inhibitory action against
cruzipain (see derivatives 12 and 24). An exception was compound
22, which is an excellent anti-T. cruzi agent, without capacity to in-
hibit cruzipain, this result suggests that this compound could be
acting by another mechanism of action. The lack of activity against
cruzipain of derivative 22 emphasizes the importance of the chlo-
rine-substitution in position 4 of the N4-phenyl ring, being this the
only structural difference between compounds 22 and 24. By com-
paring the cruzipain inhibitory profile of compounds 11 and 12
(1.7% vs 61%), it is clear that 4,5-dimethoxy substitution signifi-
cantly improves the inhibitory activity with respect to the 5,6-
dimethoxy substitution against cruzipain. TSCs 8 and 10, with
moderate activity, are not capable to inhibit cruzipain. The best
inhibitor evaluated against cruzipain was N4-TSC 24, with 67%
inhibition. These results indicate that compounds 12 and 24
2.5. Inhibition of cruzipain
Some TSCs showing variable activity against T. cruzi were se-
lected to evaluate their capacity to inhibit T. cruzi cruzipain. The
selection was based on the values of the dissociation constants of
the TSCs studied. The compounds selected were 8, 10, 11, 12, 16,
22 and 24 and the results are shown in Table 3. (Note: the refer-
ence compound in the experimental assay against cruzipain has a
theoretical Kd of 6.2 ꢁ 10ꢀ4 M.)9h In a previous work, we investi-

6822
M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
Figure 3. Best conformations of TSCs 24 (a), 12 (b) and 22 (c) into cruzipain active-site cleft.
emerge as cruzipain inhibitor lead structures for future chemical
optimization based on rational drug design.
3. Conclusions
The mode of interaction of the best experimental inhibitors 12
and 24 with cruzipain was studied using theoretical models. As
illustrated in Figure 3, both compounds occupy the substrate bind-
ing site by establishing interactions with different residues lining
Here, we developed a microwave-assisted one-pot three-com-
ponent synthesis for a rapid preparation of N4-TSCs with good
yields. The main advantages of this method are its simple purifica-
tion process and its short experimental time for the reaction to be
completed. Among all the twenty-four TSCs prepared in this work,
derivatives 12, 22 and 24 were excellent anti-T. cruzi agents with
excellent selectivity indexes higher than 150, thus indicating that
these TSCs can be lead trypanosomicidals. Also, TSC 12 and 24 dis-
played inhibitory activity against cruzipain, the major cysteine
protease of T. cruzi, pointing this as the target of action. The mode
of action was explained using molecular docking studies.
the cleft. The N
the thiosemicarbazone N4 of inhibitor 24 and N1 of inhibitor 12
(Fig. 3a and b, respectively). N 2 of Gln19 forms a hydrogen bond
e1 hydrogen of Trp177 makes hydrogen bonds to
e
to the thiosemicarbazone N2 hydrogen of 24 and interacts with the
thiosemicarbazone N1 of 12. The Nd1 hydrogen of His159 makes
hydrogen bonds to the thiosemicarbazone N4 of 24. The thiosemi-
carbazone N4 in compound 12 forms a hydrogen bond with the CO
oxygen of Ser176. Moreover, the 4-chlorophenyl moiety of 24
makes a large hydrophobic contact with the aromatic side chain
of Trp177. This aromatic interaction and the hydrogen bond to
His159 are lost in compound 12, which is less stabilized than 24
in the binding cleft. This behavior is in accordance with our exper-
imental results. It is worth noting that the methoxy groups in the
4,5-position of compound 12 are oriented toward the solvent
(Fig. 3b). According to the docking results, compounds 24 and 22
(Fig. 3a and c, respectively) bind to the enzyme in a similar fashion.
Therefore, the lack of activity against cruzipain displayed by the
latter could be related to solubility problems in the conditions of
the assay.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were determined on a Thomas
Hoover apparatus. Thin layer chromatography (TLC) was used to
monitor reactions. Flash chromatography was performed with sil-
ica gel (Merck silica gel 60, 230–400 mesh). IR spectra were re-
corded as KBr pellets using a Perkin Elmer Spectrum One FT-IR
spectrophotometer. 1H and 13C NMR spectra were recorded on a
Bruker 500 MHz spectrometer (see the NMR spectra in the

M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
6823
Supplementary data). High resolution mass spectra were obtained
on Bruker micrOTOF-Q II spectrometer. Micro-analyses were car-
ried out on a Carlo Erba elemental analyzer (Model 1106) and were
within 0.4% of the theoretical values. Microwave-assisted reactions
were carried out in a Microwave Synthesis Reactor Monowave 300
Anton Paar. Absorbance readings were made in a Flex Station 3
Multi-mode Microplate Reader.
and the solid was suspended in water (20 mL), filtered and washed
with water (2 ꢁ 5 mL), EtOH (5 mL), CH2Cl2 (5 mL) and finally hex-
ane (2 ꢁ 5 mL). TSCs were recrystallized from ethanol.
4.1.4.1. 4-Nitro-indan-1-one thiosemicarbazone (15). Yield:
89%, mp: decompose before melting. IR m
/cmꢀ1 (KBr): 3410 (N–
H), 3225 (N–H), 3137 (N–H), 1586 (C@N), 1098 (C@S). 1H NMR
(DMSO-d6) d ppm: 2.95 (m, 2H, CH2), 3.49 (m, 2H, CH2), 7.60 (m,
1H, H-Ar),), 8.14 (s, 1H, NH), 8.20 (d, J = 8.1 Hz, 1H, H-Ar), 8.31 (s,
1H, NH), 8.36 (d, J = 7.8 Hz, 1H, H-Ar), 10.49 (s, 1H, NH). 13C NMR
(DMSO-d6) d ppm: 27.2, 29.7, 125.5, 128.0, 128.7, 141.9, 143.8,
145.5, 154.1, 178.9. HRMS (ESI) m/z (M+H)+ calcd for C10H11N4O2S
251.05972, found 251.05961. Anal. Calcd for C10H10N4O2S: C,
47.99; H, 4.03; N, 22.39; S, 12.81. Found: C, 48.06; H, 4.04; N,
22.34; S, 12.78.
4.1.1. 4-Nitro-indan-1-one (25) and 6-nitro-indan-1-one (26)
To a well-stirred mixture of H2SO4 (13.9 mL of a 98% solution)
and HNO3 (2.6 mL of a 65% solution) was added at ꢀ10 °C a solu-
tion of 1-indanone (1.0 g, 7.6 mmol) in nitromethane (1.1 mL).
The addition rate was carefully adjusted to raise the temperature
from ꢀ10 to ꢀ5 °C during 30 min. The reaction mixture was stirred
further for 15 min at this temperature (attention has to be paid to
temperature and reaction time!). After ice-water hydrolysis
(200 mL), the yellow precipitate was collected and extracted with
CH2Cl2 (2 ꢁ 20 mL). The organic phase was washed with a KHCO3
solution 5% (2 ꢁ 15 mL) washed with saturated brine (10 mL),
dried over anhydrous Na2SO4 and evaporated in vacuo. The residue
was separated by flash chromatography using hexane–ethyl ace-
tate (1:0.2) as eluent. Compound 25, yield: 0.94 g, 80%, mp: 98–
100 °C (mp lit.15 100–101 °C) 1H NMR (CDCl3) d ppm: 2.80 (m,
2H, CH2), 3.53 (m, 2H, CH2), 7.61 (m, 1H, H-Ar), 8.08 (d,
J = 8.5 Hz, 1H, H-Ar), 8.47 (d, J = 8.3 Hz, 1H, H-Ar). Compound 26,
yield: 0.23 g, 20%, mp: 73–74 °C (mp lit.16 72–73 °C). 1H NMR
(CDCl3) d ppm: 2.83 (m, 2H, CH2), 3.28 (m, 2H, CH2), 7.61 (d,
J = 8.5 Hz, 1H, H-Ar), 8.44 (dd, J = 2.2 Hz, J = 8.5 Hz, 1H, H-Ar),
8.56 (d, J = 2.2 Hz, 1H, H-Ar).
4.1.4.2. 6-Nitro-indan-1-one thiosemicarbazone (16). Yield:
78%, mp: decompose before melting. IR m
/cmꢀ1 (KBr): 3414 (N–
H), 3306 (N–H), 3160 (N–H), 1582 (C@N), 1082 (C@S). 1H NMR
(DMSO-d6) d ppm: 2.96 (m, 2H, CH2), 3.17 (m, 2H, CH2), 7.60 (d,
J = 8.5 Hz, 1H, H-Ar), 8.18 (d, J = 8.5 Hz, 1H, H-Ar), 8.27 (s, 1H,
NH), 8.38 (s, 1H, NH), 8.79 (s, 1H, H-Ar), 10.42 (s, 1H, NH). 13C
NMR (DMSO-d6) d ppm: 27.8, 28.8, 117.3, 125.0, 126.8, 140.0,
147.7, 154.1, 155.7, 179.0. HRMS (ESI) m/z (M+H)+ calcd for
C
C
10H11N4O2S 251.05972, found 251.05928. Anal. Calcd for
10H10N4O2S: C, 47.99; H, 4.03; N, 22.39; S, 12.81. Found: C,
48.04; H, 4.04; N, 22.41; S, 12.83.
4.1.4.3. 5-Fluor-indan-1-one thiosemicarbazone (17). Yield:
90%, mp: 229–230 °C. IR m
/cmꢀ1 (KBr): 3415 (N–H), 3226 (N–H),
4.1.2. 1-(4-Bromo-2,5-dimethyl-phenyl)-3-chloro-propan-1-one
(27)
3140 (N–H), 1594 (C@N), 1085 (C@S). 1H NMR (DMSO-d6) d
ppm: 2.90 (m, 2H, CH2), 3.06 (m, 2H, CH2), 7.14 (m, 1H, H-Ar),
7.20 (dd, J = 1.6 Hz, J = 9.2 Hz, 1H, H-Ar), 7.93 (dd, J = 5.7 Hz,
J = 7.5 Hz, 1H, H-Ar), 7.98 (s, 1H, NH), 8.19 (s, 1H, NH), 10.25 (s,
1H, NH). 13C NMR (DMSO-d6) d ppm: 27.7, 28.4, 112.4 (d,
J = 18.0 Hz), 114.8 (d, J = 19.2 Hz), 123.9, 134.5, 151.3 (d,
J = 7.3 Hz), 155.6, 164.0 (d, J = 197.6 Hz), 178.6. HRMS (ESI) m/z
(M+H)+ calcd for C10H10FN3NaS 246.04717, found 246.04706. Anal.
Calcd for C10H10FN3S: C, 53.79; H, 4.51; F, 8.51; N, 18.82; S, 14.36.
Found: C, 53.87; H, 4.54; F, 8.48; N, 18.80; S, 14.40.
AlCl3 (2.60 g, 19.5 mmol) was added to a magnetically stirred
solution of 2-bromo-1,4-dimethylbenzene (1 mL, 7.2 mmol) and
b-chloro-propionyl chloride (0.92 mL, 9.6 mmol) in CS2 (7 mL) at
0 °C over a period of 30 min. The reaction mixture was heated un-
der reflux for a further 30 min. The resulting dark-brown solution
was cooled at room temperature and carefully poured onto ice
and was extracted with CH2Cl2 (20 mL), washed with water
(2 ꢁ 10 mL), dried over anhydrous Na2SO4, and evaporated in va-
cuo. Yield: 1.22 g, 61%, mp: 82–83 °C (mp lit.17 84–85 °C). 1H
NMR (CDCl3) d ppm: 2.40 (s, 3H, CH3), 2.44 (s, 3H, CH3), 3.34 (t,
J = 6.5 Hz, 2H, CH2), 3.87 (t, J = 6.5 Hz, 2H, CH2), 7.44 (s, 1H, H-
Ar), 7.49 (s, 1H, H-Ar).
4.1.4.4. 5-Bromo-4,7-dimethyl-indan-1-one thiosemicarbazone
(18). Yield: 92%, mp: 274–275 °C. IR m
/cmꢀ1 (KBr): 3426 (N–H),
3247 (N–H), 3199 (N–H), 1588 (C@N), 1083 (C@S). 1H NMR
(DMSO-d6) d ppm: 2.25 (s, 3H, CH3), 2.49 (s, 3H, CH3), 2.93 (m,
2H, CH2), 2.98 (m, 2H, CH2), 7.22 (s, 1H, NH), 7.35 (s, 1H, H-Ar),
8.31 (s, 1H, NH), 10.21 (s, 1H, NH). 13C NMR (DMSO-d6) d ppm:
21.9, 22.3, 27.9, 28.6, 122.2, 126.6, 128.6, 135.4, 141.4, 149.7,
158.3, 178.6. HRMS (ESI) m/z (M+H)+ calcd for C12H15BrN3S
312.01646, found 246.01530. Anal. Calcd for C12H14BrN3S: C,
46.16; H, 4.52; Br, 25.29; N, 13.46; S, 10.27. Found: C, 46.22; H,
4.53; Br, 25.56; N, 13.44; S, 10.30.
4.1.3. 5-Bromo-4,7-dimethyl-indan-1-one (28)
Chloroketone 27 (1.00 g, 3.6 mmol) was added in small portions
with swirling to concentrated H2SO4 (14 mL). The resulting solu-
tion was heated on a silicon bath at 90 °C. After 1 h, the reaction
mixture was cautiously poured onto ice and then extracted with
ethyl acetate (50 mL). The organic layer was washed with a solu-
tion of 10% NaOH (2ꢂ ꢁ 20 mL), water (20 mL), dried over anhy-
drous Na2SO4, and evaporated in vacuo. The residue was purified
by flash chromatography using CH2Cl2–hexane (1:1) as eluent.
Yield: 0.75 g, 75%, mp: 102–104 °C (mp lit.18 105–106). 1H NMR
(CDCl3) d ppm: 2.35 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.66 (t,
J = 6.0 Hz, 2H, CH2), 2.99 (t, J = 6.0 Hz, 2H, CH2), 7.32 (s, 1H, H-Ar).
4.1.5. General procedure for the synthesis of N4-TSCs (19–24)
Method A: In the first step, a mixture of 1-indanone or 5,6-dime-
thoxy-1-indanone (3.8 mmol) and hydrazine hydrate 85% (0.9 mL)
in isopropanol (5 mL) was heated under reflux for 1 h. After cool-
ing, the solvent was evaporated and the residue was dissolved in
CH2Cl2 (20 mL), dried over anhydrous Na2SO4 and evaporated
again to render the crude derivative. Hydrazones were recrystal-
lized from methanol, the physical data were in according with
those reported in the literature.19 In the second step, to a solution
of the hydrazone (2.5 mmol) in benzene (10 mL) was added drop-
wise a solution of the corresponding isothiocyanate (3.0 mmol) in
benzene (5 mL). The resulting mixture was left stirring at room
temperature monitored by TLC until the hydrazone was consumed.
4.1.4. General procedure for the synthesis of TSCs 15–1810
A suspension of corresponding 1-indanone (1.2 mmol) and thi-
osemicarbazide (2.7 mmol) in absolute ethanol (20 mL) was heated
under reflux for 30 min, then concentrated H2SO4 (0.10 mL) was
added and the heating was continued until the 1-indanone was
consumed. The conversion of 1-indanone in the corresponding
TSC was monitored by TLC on silica gel 60 F254 using chloro-
form–ethanol (1:0.1) as eluent. The solvent was removed in vacuo

6824
M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
The solid obtained was filtered, washed with benzene (5 mL) and
with ethanol (2 mL).
H-Ar), 7.20 (d, J = 7.9 Hz, 2H, H-Ar), 7.49 (d, J = 7.9 Hz, 2H, H-Ar),
8.53 (s, 1H, NH), 9.18 (s, 1H, NH). 13C NMR (DMSO-d6) d ppm:
21.1, 28.3, 28.5, 56.0, 56.2, 104.8, 108.2, 126.4, 129.0, 129.9,
134.9, 137.2, 142.9, 149.3, 152.5, 158.8, 176.6. HRMS (ESI) m/z
(M+H)+ calcd for C19H22N3O2S 356.14272, found 356.14177. Anal.
Calcd for C19H21N3O2S: C, 64.20; H, 5.95; N, 11.82; S, 9.02. Found:
C, 64.12; H, 5.97; N, 11.79; S, 9.04.
Method B: A mixture of 1-indanone or 5,6-dimethoxy-1-inda-
none (0.38 mmol), hydrazine hydrate 85% (21
lL), the correspond-
ing isothiocyanate (0.43 mmol), acetic acid (20
lL) and ethanol
(0.5 mL) in a glass tube equipped with a screw cap and magnetic
agitation, was placed in a microwave synthesizer at 90 °C (30 W,
2.5 bar). After completion of the reaction, monitored by TLC, the
obtained mixture was suspended in water (5 mL), filtered and
washed with ethanol (2 mL). N4-TSCs were recystallized from
ethanol.
4.1.5.6. 5,6-Dimethoxyindan-1-one N-(4-chlorophenyl)thiosem-
icarbazone (24). Mp: 190–192 °C. IR m
/cmꢀ1 (KBr): 3313 (N–H),
3194 (N–H), 1587 (C@N), 1079 (C@S). 1H NMR (CDCl3) d ppm:
2.82 (m, 2H, CH2), 3.12 (m, 2H, CH2), 3.93 (s, 3H, OCH3), 3.94 (s,
3H, OCH3), 6.83 (s, 1H, H-Ar), 7.15 (s, 1H, H-Ar), 7.35 (d,
J = 8.7 Hz, 2H, H-Ar), 7.63 (d, J = 8.7 Hz, 2H, H-Ar), 8.48 (s, 1H,
NH), 9.22 (s, 1H, NH). 13C NMR (DMSO-d6) d ppm: 28.3, 28.5,
56.1, 56.2, 104.9, 108.2, 128.0, 128.4, 129.7, 129.8, 138.7, 143.1,
149.2, 152.6, 159.3, 176.5. HRMS (ESI) m/z (M+Na)+ calcd for
4.1.5.1. Indan-1-one N-phenylthiosemicarbazone (19)20
202–204 °C. IR
/cmꢀ1 (KBr): 3267 (N–H), 3193 (N–H), 1595
. Mp:
m
(C@N), 1097 (C@S). 1H NMR (CDCl3) d ppm: 2.84 (m, 2H, CH2),
3.21 (m, 2H, CH2), 7.29–7.43 (m, 6H, H-Ar), 7.69 (d, J = 7.7 Hz, 2H,
H-Ar), 7.78 (d, J = 7.8 Hz, 1H, H-Ar), 8.53 (s, 1H, NH), 9.67 (s, 1H,
NH). 13C NMR (DMSO-d6) d ppm: 27.9, 28.7, 1229, 125.6, 126.0,
127.3, 128.5, 129.2, 131.2, 138.1, 139.6, 149.4, 158.3, 176.8. HRMS
(ESI) m/z (M+H)+ calcd for C16H16N3S 282.10594, found 282.10569.
Anal. Calcd for C16H15N3S: C, 68.30; H, 5.37; N, 14.93; S, 11.40.
Found: C, 68.39; H, 5.35; N, 14.90; S, 11.43.
C18H18ClN3NaO2S 398.07005, found 398.06860. Anal. Calcd for
C18H18ClN3O2S: C, 57.52; H, 4.83; Cl, 9.43; N, 11.18; S, 8.53. Found:
C, 57.58; H, 4.82; N, 11.14; S, 8.51.
4.2. Biological evaluation
4.1.5.2. Indan-1-one N-(4-methylphenyl)thiosemicarbazone
4.2.1. In vitro anti-trypanosomal activity
(20)20
.
Mp: 173-175 °C. IR
m
/cmꢀ1 (KBr): 3301 (N–H), 3197 (N–
T. cruzi epimastigotes (Tulahuen 2 strain) were grown at 28 °C
in an axenic medium (BHI-Tryptose) as previously described,9,12
complemented with 5% fetal calf serum. Cells were harvested in
the late log phase, re-suspended in fresh medium, counted in
Neubauer’s chamber and placed in 24-well plates (2 ꢁ 106/mL).
Cell growth was measured as the absorbance of the culture at
590 nm, which was proved to be proportional to the number of
cells. Before inoculation, the media were supplemented with the
indicated amount of the studied compound from a stock solution
in DMSO. The final concentration of DMSO in the culture media
never exceeded 1% and the control was run in the presence of
1% DMSO and in the absence of any compound. No effect on
epimastigotes growth was observed by the presence of up to 1%
DMSO in the culture media. Nfx and Amphotericin B were used
as the reference trypanosomicidal drugs. The percentage of
growth inhibition was calculated as follows {1 ꢀ [(Ap ꢀ A0p)/
(Ac ꢀ A0c)]} ꢁ 100, where Ap = A590 of the culture containing
the studied compound at day 5; A0p = A590 of the culture contain-
ing the studied compound right after addition of the inocula (day
0); Ac = A590 of the culture in the absence of any compound (con-
trol) at day 5; A0c = A590 in the absence of the compound at day
0. To determine IC50 values, parasite growth was followed in the
absence (control) and presence of increasing concentrations of the
corresponding compound. The IC50 values were determined as the
drug concentrations required to reduce by half the absorbance of
that of the control (without compound).
H), 1589 (C@N), 1102 (C@S). 1H NMR (CDCl3) d ppm: 2.36 (s, 3H,
CH3), 2.84 (m, 2H, CH2), 3.20 (m, 2H, CH2), 7.20 (d, J = 8.2 Hz, 2H,
H-Ar), 7.31 (m, 1H, H-Ar), 7.37–7.42 (m, 2H, H-Ar), 7.52 (d,
J = 8.2 Hz, 2H, H-Ar), 7.76 (d, J = 7.9 Hz, 1H, H-Ar), 8.56 (s, 1H,
NH), 9.27 (s, 1H, NH). 13C NMR (DMSO-d6) d ppm: 21.0, 27.9,
28.7, 122.9, 126.0, 127.3, 129.0, 129.1, 131.1, 134.8, 137.0, 138, 1,
149.3, 158.1, 176.9. HRMS (ESI) m/z (M+Na)+ calcd for C17H17N3NaS
318.10354, found 318.10263. Anal. Calcd for C17H17N3S: C, 69.12;
H, 5.80; N, 14.22; S, 10.85. Found: C, 69.04; H, 5.79; N, 14.18; S,
10.88.
4.1.5.3. Indan-1-one N-(4-chlorophenyl)thiosemicarbazone
(21). Mp: 192–194 °C. IR
m
/cmꢀ1 (KBr): 3261 (N–H), 3224 (N–
H), 1589 (C@N), 1083 (C@S). 1H NMR (CDCl3) d ppm: 2.84 (m,
2H, CH2), 3.20 (m, 2H, CH2), 7.23 (m, 1H, H-Ar), 7.25–7.37 (m,
4H, H-Ar), 7.65 (d, J = 7.8 Hz, 2H, H-Ar), 7.76 (d, J = 7.8 Hz, 1H, H-
Ar), 8.56 (s, 1H, NH), 9.32 (s, 1H, NH). 13C NMR (DMSO-d6) d
ppm: 28.0, 28.7, 122.9, 126.0, 127.2, 127.7, 128.4, 129.6, 131.2,
138.1, 138.6, 149.4, 158.6, 176.8. HRMS (ESI) m/z (M+Na)+ calcd
for C16H14ClN3NaS 338.04892, found 338.04925. Anal. Calcd for
C16H14ClN3S: C, 60.85; H, 4.47; Cl, 11.23; N, 13.31; S, 10.15. Found:
C, 68.39; H, 5.35; N, 14.90; S, 11.43.
4.1.5.4. 5,6-Dimethoxyindan-1-one N-phenylthiosemicarbazone
(22). Mp: 204–205 °C. IR m
/cmꢀ1 (KBr): 3248 (N–H), 3201 (N–H),
1596 (C@N), 1078 (C@S). 1H NMR (CDCl3) d ppm: 2.83 (m, 2H,
CH2), 3.12 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 3.94 (s, 3H, OCH3),
6.83 (s, 1H, H-Ar), 7.16 (s, 1H, H-Ar), 7.40 (m, 3H, H-Ar), 7.66 (d,
J = 7.6 Hz, 2H, H-Ar), 8.48 (s, 1H, NH), 9.27 (s, 1H, NH). 13C NMR
(DMSO-d6) d ppm: 28.4, 28.5, 56.0, 56.2, 104.8, 108.2, 125.7,
108.2, 125.7, 126.5, 128.6, 129.8, 139.7, 143.0, 149.3, 152.6,
158.9, 176.6. HRMS (ESI) m/z (M+H)+ calcd for C18H20N3O2S
342.12707, found 342.12578. Anal. Calcd for C18H19N3O2S: C,
63.32; H, 5.61; N, 12.31; S, 9.39. Found: C, 63.37; H, 5.59; N,
12.34; S, 9.41.
4.2.2. Unspecific mammalian cytotoxicity
Red blood cell lysis assay.9h Human blood collected in sodium
citrate solution (3.8%) was centrifuged at 1500 rpm for 10 min at
4 °C. The plasma supernatant was removed and the erythrocytes
were suspended in ice cold PBS. The cells were again centrifuged
at 1500 rpm for 10 min at 4 °C. This procedure was repeated two
more times to ensure the removal of any released hemoglobin.
Once the supernatant was removed after the last wash, the cells
were suspended in PBS to get a 2% w/v red blood cell solution. A
volume of 400
tion 50, 100 and 200
Amphotericin (final concentration 1.5
400 L of the 2% w/v red blood cell solution in ten microcentrifuge
tubes for each concentration and incubated for 24 h at 37 °C.
Complete hemolysis was attained using neat water yielding the
l
L of studied compounds, in PBS (final concentra-
M), negative control (solution of PBS), or
M) were added to
4.1.5.5. 5,6-Dimethoxyindan-1-one N-(4-methylphenyl)thio-
l
semicarbazone (23). Mp: 179–181 °C. IR
m
/cmꢀ1 (KBr): 3262
B
l
(N–H), 3192 (N–H), 1593 (C@N), 1084 (C@S). 1H NMR (CDCl3) d
ppm: 2.35 (s, 3H, CH3), 2.83 (m, 2H, CH2), 3.11 (m, 2H, CH2), 3.92
(s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.82 (s, 1H, H-Ar), 7.15 (s, 1H,
l

M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
6825
100% control value (positive control). After incubation, the tubes
were centrifuged and the supernatants were transferred to new
tubes. The release of hemoglobin was determined by spectrophoto-
metric analysis of the supernatant at 405 nm. Results were ex-
pressed as percentage of the total amount of hemoglobin
released by action of the compounds. This percentage is calculated
using the equation: Hemolysis percentage (%) = [(A1 ꢀ A0)/A1
water] ꢁ 100, where A1 is the absorbance at 405 nm of the test
sample at t = 24 h, A0 is the absorbance at 405 nm of the test sam-
ple at t = 0 h, and A1 water is the absorbance at 405 nm of the po-
sitive control (water) at t = 24 h. The experiments were done by
quintuplicate.
were centered on S atom of the catalytic cysteine 25 residue. A grid
spacing of 0.375 Å (approximately one fourth of the length of car-
bon–carbon covalent bond) and a distance-dependent function of
the dielectric constant27 were used for the calculation of the ener-
getic map. Fifty runs were generated by using Lamarckian Genetic
Algorithm searches. Default settings were used with an initial pop-
ulation of 150 randomly placed individuals, a maximum number of
7.0 ꢁ 105 energy evaluations, and a maximum number of 2.7 ꢁ 104
generations. A mutation rate of 0.02 and a crossover rate of 0.8
were chosen. In all cases the pose with best score from multiple
docking of the same ligand were chosen.
Acknowledgments
4.2.3. Cruzipain inhibitory activity9h
Cruzipain (6
ing 50 mM PBS pH 7.3 or acetate buffer pH 5.3, 5 mM DTT and
100 M compound for 5 min at room temperature. Fluorogenic
substrate Z-Phe-Arg-AMC (KM = 1.8 M) was added to a concentra-
tion of 10 M, and increase in fluorescence (excitation 380 nm and
emission 460 nm) was monitored for 10 min at room temperature
in a 96 well-microplate Varioskan spectrofluorometer and spectro-
photometer. Compounds were added as solutions in DMSO and po-
sitive controls contained only buffered solvent. The final assay
lM) was incubated in a reaction mixture contain-
We thank CONICET and PEDECIBA-ANII for scholarship to M.E.C.
and D.B. respectively. Financial supports from UBA, Prosul-CNPq,
RIDIMEDCHAG-CYTED, CSIC.
l
l
l
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.037.
volume was 100 lL and the final DMSO concentration never ex-
References and notes
ceeded 10%. The cruzipain inhibitor reference compound 3’-bro-
mopropiophenone thiosemicarbazone,8 was included in the
1. World Health Organization (WHO). Control of Chagas Disease. Technical
Reports Series, 2002; Vol. 905, pp. 1–109.
2. Maya, J. D.; Cassels, B. K.; Iturriaga-Vásquez, P.; Ferreira, J.; Faúndez, M.;
Galanti, N.; Ferreira, A.; Morello, A. Comp. Biochem. Physiol. 2007, 146, 601.
3. (a) Coura, J. R.; De Castro, S. L. Mem. Inst. Oswaldo Cruz 2002, 97, 3; (b)
Cerecetto, H.; González, M. Pharmaceuticals 2010, 3, 810.
4. Wilson, H. R.; Revankar, G. R.; Tolman, R. L. J. Med. Chem. 1994, 17, 760.
5. (a) Mishra, V.; Pandeya, S. N.; Pannecouque, C.; Witvrouw, M.; De Clercq, E.
Arch. Pharm. 2002, 335, 183; (b) Condit, R. C.; Easterly, R.; Pacha, R. F.; Fathi, Z.;
Meis, R. J. Virology 1991, 185, 857; (c) Teitz, Y.; Ronen, D.; Vasover, A.;
Stematsky, T.; Riggs, J. L. Antiviral Res. 1994, 24, 305.
6. Kasuga, N. C.; Sekino, K.; Ishikawa, M.; Honda, A.; Yokoyama, M.; Nakano, S.;
Shimada, N.; Koumo, C.; Nomiya, K. J. Inorg. Biochem. 2003, 96, 298.
7. Feun, L.; Modiano, M.; Mao, L. J.; Marini, A.; Savaraj, Plezia. P.; Almassian, B.;
Colacino, E.; Fischer, J.; MacDonal, S. Cancer Chemother. Pharmacol. 2002, 50,
223.
8. (a) Du, X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.; Holler, T. P.; McKerrow,
J. H.; Cohen, F. E. J. Med. Chem. 2002, 45, 2695; (b) Cazzulo, J. J. Curr. Top. Med.
Chem. 2002, 2, 1261; (c) Duschak, V. G.; Couto, A. S. Curr. Med. Chem. 2009, 16,
3174; (d) Cazorla, S. I.; Frank, F. M.; Becker, P. D.; Arnaiz, M.; Mirkin, G. A.;
Corral, R. S.; Guzman, C. A.; Malchiodi, E. L. J. Infect. Dis. 2010, 202, 136.
9. (a) Aguirre, G.; Cerecetto, H.; González, M.; Gambino, D.; Otero, L.; Olea-Azar,
C.; Rigol, C.; Denicola, A. Bioorg. Med. Chem. 2004, 12, 4885; (b) Rigol, C.; Olea-
Azar, C.; Mendizábal, F.; Otero, L.; Gambino, D.; González, M.; Cerecetto, H.
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2005, 61, 2933; (c) Otero, L.; Vieites,
M.; Boiani, L.; Denicola, A.; Rigol, C.; Opazo, L.; Olea-Azar, C.; Maya, J. D.;
Morello, A.; Krauth-Siegel, R. L.; Piro, O. E.; Castellano, E.; González, M.;
Gambino, D.; Cerecetto, H. J. Med. Chem. 2006, 49, 3322; (d) Otero, L.; Maya, J.
D.; Morello, A.; Rigol, C.; Barriga, G.; Rodríguez, J.; Folch, C.; Norambuena, E.;
González, M.; Olea-Azar, C.; Cerecetto, H.; Gambino, D. Med. Chem. 2008, 4, 11;
(e) Pérez-Rebolledo, A.; Teixeira, L. R.; Batista, A. A.; Mangrich, A. S.; Aguirre, G.;
Cerecetto, H.; González, M.; Hernández, P.; Ferreira, A. M.; Speziali, N. L.;
Beraldo, H. Eur. J. Med. Chem. 2008, 43, 939; (f) Pagano, M.; Demoro, B.; Toloza,
J.; Boiani, L.; González, M.; Cerecetto, H.; Olea-Azar, C.; Norambuena, E.;
Gambino, D.; Otero, L. Eur. J. Med. Chem. 2009, 44, 4937; (g) Rodrigues, C.;
Batista, A. A.; Ellena, J.; Castellano, E. E.; Benítez, D.; Cerecetto, H.; González, M.;
Teixeira, L. R.; Beraldo, H. Eur. J. Med. Chem. 2010, 45, 2847; (h) Merlino, A.;
Benitez, D.; Chavez, S.; Da Cunha, J.; Hernández, P.; Tinoco, L. W.; Campillo, N.
E.; Páez, J. A.; Cerecetto, H.; González, M. Med. Chem. Commun. 2010, 1, 216.
10. Finkielsztein, L. M.; Castro, E. F.; Fabian, L. E.; Moltrasio, G. Y.; Campos, R. H.;
Cavallaro, L. V.; Moglioni, A. G. Eur. J. Med. Chem. 2008, 43, 1767.
analysis as a control, 100% at 100
of at least three experiments.
lM. The values represent means
4.3. Docking studies
TSCs 1–24 were virtually screened for their capability of acting
as cruzipain inhibitors using SurflexDock21 method implemented
in the molecular modeling package Sybyl 8.1.22 SurflexDock is a
new docking methodology that combines Hammerhead’s empirical
scoring function23 with a molecular similarity method to generate
putative alignment of ligands. SurflexDock employs an idealized
active site ligand (called a protomol) as a target to generate puta-
tive alignments of molecules or molecules fragments.24 These
putative poses are achieved using the Hammerhead scoring func-
tion. Before performing docking, all compounds were minimized
using the Conjugate Gradient algorithm with a conjugated gradient
of <0.001 kcal/mol convergent criteria provided by the MMFF94
force field25 and MMFF94 electrostatic charges. TSCs 1–24 were
docked into the binding site of cruzipain (PDB ID 1F29). Using
1F29 as reference complex, we generated a protomol, a computa-
tional representation of the intended binding site to which puta-
tive ligands are aligned. SurflexDock’s protomol use CH4, C@O
and NH fragments and its purpose is to direct the initial placement
of the ligands during the docking process. Protomol construction
was based on protein residues present in the active site of the en-
zyme. Each docking of putative ligands returned up to 50 scored
poses, with the score consisting of an affinity score expressed as
ꢀlog10 (Kd).
Besides, molecular docking of compounds 12, 22 and 24 into the
three-dimensional X-ray structure of T. cruzi cruzipain (PDB code
1EWL) was carried out using Autodock 4.2 software, as imple-
mented through the graphical user interface (GUI) AutoDockTools
(ADT 1.5.4).26 ADT was employed to setup the enzymes: all hydro-
gen atoms were added, Gasteiger charges were calculated, and
nonpolar hydrogens were merged to carbon atoms. The 3D struc-
tures of ligand molecules were obtained from molecular mechanics
calculation using the MMFF94 force filed25 as implemented in the
molecular modeling package Sybyl 8.1.22 ADT was used to generate
docking input files. In all docking experiments a grid box size of
60 ꢁ 60 ꢁ 66 points in x, y, and z directions was used, and the maps
11. (a) Faucher, J. F.; Baltz, T.; Petry, K. G. Parasitol. Res. 1995, 81, 441; (b) Almeida-
de Faría, M.; Freymuller, E.; Colli, W.; Alves, M. J. Exp. Parasitol. 1999, 92, 263;
(c) Tyler, K. M.; Engman, D. M. Int. J. Parasitol. 2001, 31, 472.
12. (a) Porcal, W.; Hernández, P.; Boiani, M.; Aguirre, G.; Boiani, L.; Chidichimo, A.;
Cazzulo, J. J.; Campillo, N. E.; Paez, J. A.; Castro, A.; Krauth-Siegel, R. L.; Davies,
C.; Basombrío, M. A.; González, M.; Cerecetto, H. J. Med. Chem. 2007, 50, 6004;
(b) Boiani, M.; Boiani, L.; Denicola, A.; Torres de Ortiz, S.; Serna, E.; Vera de
Bilbao, N.; Sanabria, L.; Yaluff, G.; Nakayama, H.; Rojas de Arias, A.; Vega, C.;
Rolón, M.; Gómez-Barrio, A.; Cerecetto, H.; Gonzalez, M. J. Med. Chem. 2006, 49,
3215; (c) Boiani, L.; Davies, C.; Arredondo, C.; Porcal, W.; Merlino, A.; Gerpe, A.;
Boiani, M.; Pacheco, J. P.; Basombrío, M. A.; Cerecetto, H.; González, M. Eur. J.
Med. Chem. 2008, 43, 2229; (d) Boiani, L.; Gerpe, A.; Arán, V. J.; Torres de Ortiz,

6826
M. E. Caputto et al. / Bioorg. Med. Chem. 19 (2011) 6818–6826
S.; Serna, E.; Vera de Bilbao, N.; Sanabria, L.; Yaluff, G.; Nakayama, H.; Rojas de
Arias, A.; Maya, J. D.; Morello, A.; Cerecetto, H.; González, M. Eur. J. Med. Chem.
2009, 44, 1034.
18. Miller, R. B.; Stowell, J. G.; Dugar, S.; Moock, T. E.; Jenks, C. W.; Farmer, S. C.;
Phan, B.; Wujcik, C. E.; Olmstead, M. M. Tetrahedron 2002, 58, 6061.
19. Lansbury, P. T.; Mancuso, N. R. J. Am. Chem. Soc. 1966, 88, 1205.
20. Misra, V. S.; Varma, R. S.; Agarwal, S. J. Indian Chem. Soc. 1975, 52, 981.
21. Jain, A. N. J. Med. Chem. 2003, 46, 499.
13. Merlino, A. M.; Otero, L.; Gambino, D.; Coitiño, E. L. Eur. J. Med. Chem 2011, 46,
2639.
14. Glisoni, R. J.; Chiappetta, D. A.; Finkielsztein, L. M.; Moglioni, A. G.; Sosnik, A.
New. J. Chem. 2010, 34, 2047.
15. Hasbun, J. A.; Barker, K. K.; Merthes, M. P. J. Med. Chem. 1973, 16, 847.
16. Musso, D. L.; Cochran, F. R.; Kelley, J. L.; McLean, E. W.; Selph, J. L.; Rigdon, G. C.;
Faye Orr, G.; Davis, R. G.; Cooper, B. R.; Styles, V. L.; Thompson, J. B.; Hall, W. R.
J. Med. Chem. 2003, 46, 399.
22. SYBYL 8.1, Tripos Inc., 1699 South Hanley Rd., St. Louis, MO 63144, USA, 2008.
23. Welch, W.; Ruppert, J.; Jain, A. N. Chem. Biol. 1996, 3, 449.
24. Ruppert, J.; Welch, W.; Jain, A. N. Protein Sci. 1997, 6, 524.
25. Clark, M.; Cramer, R. D., III; Van Opdenbosch, N. J. Comput. Chem. 1989, 10,
9822.
26. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
27. Goodford, P. J. J. Med. Chem. 1985, 28, 849.
17. Raposo, M. M. M.; Pereira, A. M. B.; Oliveira-Campos, A. M. F.; Shannon, P. V. R.
J. Chem. Res. (S) 1999, 8, 466.