Synthesis and biological evaluation of some novel 1-indanone thiazolylhydrazone derivatives as anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.013

A series of novel 4-arylthiazolylhydrazones (TZHs) derived from 1-indanones were synthesized in good yields (66-92%) in a simple procedure using microwave irradiation and then characterized by spectroscopy studies. The compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against the epimastigote, trypomastigote and amastigote forms of the parasite. Most TZHs displayed excellent activity, and were more potent and selective than the reference drug Benznidazole, used in the current chemotherapy. Analysis of the free sterols from parasite incubated with the compounds showed that inhibition of ergosterol biosynthesis is a possible target for the action of these new TZHs. In particular, TZH 9 emerged as a promising antichagasic compound to be evaluated in animal models.

Full text of DOI:10.1016/j.ejmech.2012.07.013

European Journal of Medicinal Chemistry 55 (2012) 155e163
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of some novel 1-indanone thiazolylhydrazone
derivatives as anti-Trypanosoma cruzi agents
,
María E. Caputto a, Alejandra Ciccarelli b, Fernanda Frank c d, Albertina G. Moglioni a,
,
Graciela Y. Moltrasio e, Daniel Vega f, Elisa Lombardo b, Liliana M. Finkielsztein a
*
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 Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP, UBA-CONICET), Hospital de Clínicas José de San Martín, Avenida Córdoba 2351, 1120, Ciudad Autónoma de Buenos Aires,
Argentina
c Cátedra de Inmunología e Instituto de Estudios de la Inmunidad Humoral (IDEHU), CONICET-UBA, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113,
Ciudad Autónoma de Buenos Aires, Argentina
d Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121, Ciudad Autónoma de Buenos Aires,
Argentina
e Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Ciudad Autónoma de Buenos Aires, Argentina
f Departamento Física de la Materia Condensada, Gerencia de Investigación y Aplicaciones, Comisión Nacional de Energía Atómica y ECyT, Universidad Nacional de General San
Martín, Av. Gral. Paz 1499, 1650, San Martín, Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4-arylthiazolylhydrazones (TZHs) derived from 1-indanones were synthesized in good
yields (66e92%) in a simple procedure using microwave irradiation and then characterized by spec-
troscopy studies. The compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against
the epimastigote, trypomastigote and amastigote forms of the parasite. Most TZHs displayed excellent
activity, and were more potent and selective than the reference drug Benznidazole, used in the current
chemotherapy. Analysis of the free sterols from parasite incubated with the compounds showed that
inhibition of ergosterol biosynthesis is a possible target for the action of these new TZHs. In particular,
TZH 9 emerged as a promising antichagasic compound to be evaluated in animal models.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 17 February 2012
Received in revised form
21 May 2012
Accepted 7 July 2012
Available online 20 July 2012
Keywords:
Thiazolylhydrazones
Chagas’s disease
Anti-Trypanosoma cruzi compounds
Microwave assisted synthesis
1. Introduction
Current specific chemotherapy relies on two compounds:
Nifurtimox (Nfx) and Benznidazole (Bnz), associated with long-
Chagas’s disease, also called American Trypanosomiasis, is
a parasitic disease caused by the kinetoplastid protozoan Trypa-
nosoma cruzi (T. cruzi). Despite recent advances in the control of its
vectorial and transfusional transmission [1,2], it is endemic from
Southern California to Argentina, and afflicts 24 million people [3].
The parasite has a complex life cycle, which includes obligatory
stages in the mammalian host and in the vector. In the host, T. cruzi
has two forms: an intracellular dividing form (amastigote) and
a non-dividing highly infective bloodstream form (trypomastigote)
that can invade host cells. Two forms also occur in the vector: one
replicative (epimastigote) and one non-replicative (metacyclic
trypomastigote) [4,5].
term treatments that may give rise to severe side effects [6]. In
fact, although Nfx and Bnz are able to eliminate patent parasitemia
and to reduce serological titers in acute and early chronic infections
[7,8], they have significantly low efficacy in long-term chronic
infections [9]. The side effects of these drugs result from the
oxidative damage in the host tissues and are thus inextricably
linked to their anti-parasitic activity [10].
Recently, we have reported the synthesis of new thio-
semicarbazones derived from 1-indanones. These compounds
possess an important activity against T. cruzi with selectivity
indexes higher than that of the reference drug Nfx. Some of them
are also able to inhibit cruzipain, the major cysteine protease of the
parasite [11]. In continuation of our search for bioactive molecules,
we envisaged that the transformation of the thiosemicarbazone
group into thiazolylhydrazone moiety would generate novel
templates which are likely to exhibit anti-T. cruzi activity.
* Corresponding author. Tel.: þ54 11 49648233; fax: þ54 11 49648252.
E-mail address: lfinkiel@ffyb.uba.ar (L.M. Finkielsztein).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.013

156
M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
Thiazolylhydrazones (TZHs) have been of interest to medicinal
and hydrobromide forms previously described by Chimenti and
coworkers [14,15].
chemists because of their biological activities. They have been
described as antibacterial [12,13], antifungal [14] and anti-
inflammatory agents [13]. Chimenti and coworkers developed
a series of selective inhibitors of the A and B isoforms of mono-
aminooxidase (MAO) with the thyazolylhydrazone function in their
structure [15].
Several studies have shown that commercially available ergos-
terol biosynthesis inhibitors having a thiazole or triazole nucleus,
which are highly successful for the treatment of fungal diseases
(such us ketoconazole, itraconazole, posaconazole or ravuconazole)
have suppressive effects against T. cruzi infections in humans or
experimental animals [7,16].
Due to the good results obtained when the synthesis was per-
formed in DMF under MW irradiation (method c), we decided to
explore the preparation in DMF, but with conventional heating.
Surprisingly, the products obtained with this methodology were
not the same as those obtained with method c. The new products
had different melting points and showed different spectroscopic
data; hence, we decided to investigate the structure of the resulting
compounds in detail.
All the compounds synthesized were characterized by spectral
studies. None of the compounds obtained gave crystals adequate to
carry out an X-ray analysis. For structural elucidation and
In this work, we present the preparation of seventeen TZHs
derived from 1-indanones in a simple manner, by microwave-
assisted synthesis. The synthesized compounds were fully charac-
terized by IR, NMR and mass spectral studies. TZHs were assayed
for their in vitro anti-T. cruzi activity against the epimastigote,
trypomastigote and amastigote forms of the parasite. Their cyto-
toxicity in mammalian cell cultures was also investigated, and the
structural requirements for their optimal activity are discussed.
Further, investigations on the possible involvement of TZHs with
cruzipain activity and ergosterol biosynthesis, as possible thera-
peutic targets, were performed.
comparison, we chose TZHs 1 and 4 and the counterpart
compounds obtained by conventional heating in DMF (18 and 19
respectively). To facilitate the structural assignment of the selected
compounds, we carried out the cyclohexanone thiosemicarbazone
condensation with phenacyl chloride and 4-methyl-phenacyl
chloride in both reaction conditions (20, 21 by microwave irradia-
tion and 22, 23 by conventional heating) (Fig. 1). Spectroscopic data
of TZHs 20 and 21 were coincident with those of the hydrochloride
and hydrobromide forms previously described [15c,18]. In addition,
the patterns observed in the IR and 1H and 13C NMR spectra were
similar for TZHs 1, 4, 20 and 21.
TZH 21 was obtained in a crystalline form suitable to perform an
X-ray crystallographic study. Analysis of the ORTEP diagram
confirmed that the compound was forming a hydrochloride, due to
the release of HCl (g) in the condensation reaction. The detailed
study of the crystal structure demonstrated that C2-N3 and C2-N13
distances are 1.321 Å and 1.343 Å respectively, indicating that
a resonance phenomenon occurs between the atoms involved
(Fig. 2) [19].
Bearing in mind the similarity observed in the spectroscopic
data of TZHs 1, 4, 20 and 21, which correspond to hydrochloride
forms, we treated these compounds with NaHCO310% overnight.
The melting points and IR spectra of the resulting products were
coincident with those of compounds 18, 19, 22 and 23. This led us to
consider the possibility that the compounds from the reaction
performed in DMF with conventional heating were the corre-
sponding free bases.
Hydrochloride TZHs and free base TZHs (compounds 1, 4, 20, 21
and 18, 19, 22 and 23 respectively) can be discriminated through
the analysis of 1H NMR, 13C NMR and IR spectroscopic data. The
NMR signals of all compounds considered were similar when the
spectra were recorded in DMSO-d6, but different when they were
recorded in CDCl3. Clearly, there is solvent effect acting on the
hydrochloride/free base equilibrium. Table 3 reports these data.
In the 1H NMR spectra, there is a broad and low intense signal
with one proton integral at 12.92, 12.78, 12.76 and 12.26 ppm for
TZHs 1, 4, 20 and 21, respectively. Since the same signal was shifted
downfield by about 4 ppm for free base TZHs (18 and 22, not
observed for compounds 19 and 23), it was assigned to the
hydrazone NH proton (D2O exchangeable). In addition, an extra
signal between 13.20 and 14.25 ppm appeared in the spectra of the
hydrochloride TZHs, indicating that 1, 4, 20 and 21 are in the salt
2. Results and discussion
2.1. Chemistry
Hantzsch reaction of
a-halocarbonyl compounds with thio-
amide derivatives provides a useful method for the preparation of
the thiazole nucleus [17]. In general, this process requires high
temperatures and long reaction times and often produces low
yields. Here, we report the successful use of microwave (MW)
irradiation to enhance this classical method, by the condensation of
a
-chloroketones with thiosemicarbazones for the preparation of
TZHs (Scheme 1).
With the aim to find the optimal reaction conditions to
synthesize these derivatives using MW irradiation, we investigated
the preparation of TZH derived from thiosemicarbazone of 5,6-
dimethoxy-1-indanone and phenacyl chloride using three
different solvents at the same temperature: a) EtOH, b) acetonitrile,
and c) DMF. Results were compared with those obtained when the
reaction takes place using the conventional method (reflux in
EtOH) (Table 1).
When the selected TZH was synthesized using method c, the
desired product was achieved in 30 s at 80 ꢀC with very good yields
(92%). Taking this result into account, we prepared a series of TZHs
by reacting thiosemicarbazones derived from 1-indanones, with
different substituents in the aromatic ring, as starting materials,
and several phenacyl chlorides in the presence of a few drops of
DMF. The TZHs obtained under optimized reaction conditions are
summarized in Table 2. Spectroscopic data of the synthesized
compounds were in accordance with those of TZHs resulting from
cyclic ketone thiosemicarbazone derivatives as the hydrochloride
Scheme 1. Synthesis of TZHs by condensation of 1-indanone thiosemicarbazone derivatives and phenacyl chlorides.

M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
157
Table 1
Synthesis of TZH derived from thiosemicarbazone of 5,6-dimethoxy-1-indanone and phenacyl chloride in different reaction conditions.
.
Time (min)
Yieldb (%)
Reflux in EtOH
240
8
12
45
53
51
92
MW irradiation in EtOHa (method a)
MW irradiation in acetonitrilea (method b)
MW irradiation in DMFa (method c)
0.5
a
Temperature: 80 ꢀC.
Yields refer to pure products.
b
Table 2
TZHs synthesized under MW irradiation in DMF.
H
N
N
N
R1
S
R4
R2
R3
.
TZHs
R1
R2
R3
R4
Yielda (%)
1
2
3
4
5
6
7
8
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
CH3
H
H
CH3
CH3
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
OCH3
NO2
H
H
H
H
H
H
CH3
Cl
H
CH3
Cl
H
CH3
Cl
Cl
Cl
Cl
Cl
H
CH3
H
71
76
74
92
77
86
85
76
83
68
91
81
67
66
73
84
79
9
10
11
12
13
14
15
16
17
H
H
Fig. 2. ORTEP diagram for compound 21.
CH3
H
CH3
CH3
H
(1615e1630 cmꢁ1). These observations also confirm that the
hydrochloride and the free base are the chemical structures that
correspond to both series of compounds. The analysis of the allows
establishing significant differences in C10 chemical shifts of both
forms, which are deshielded and shielded by over 10 ppm in the
hydrochloride and free base TZHs respectively. The marked
downfield shift of C10 (164.5, 165.9, 165.4 and 163.2 ppm) in
compounds 1, 4, 20 and 21 compared to 18, 19, 22 and 23 is due to
increased positive charge density on that carbon. Further, the
chemical shifts observed for C4 in the 13C NMR spectra of the free
base TZHs are in agree with previously assignments [21].
H
H
CH3
a
Time reaction: 30 s. Temperature reaction: 80 ꢀC.
form. Further, IR spectra of 18, 19, 22 and 23 exhibited a sharp
intense band between 3305 and 3395 cmꢁ1, characteristic for NH
stretching, whereas spectra of the compounds in the hydrochloride
form showed a weak broad band between 2550 and 2780 cmꢁ1
,
corresponding to NHþ vibration of the salt form, in agreement with
the literature [20]. The vibration band of the C]N moiety of the
thiazole nucleus appeared between 1555 and 1565 cmꢁ1, whereas
the band in the salt form was shifted to a higher frequency
2.2. In vitro anti-T. cruzi activity
The compounds were biologically evaluated in aqueous solu-
tions obtained from stock solutions of the hydrochloride forms of
TZHs in DMSO, whose final concentration did not exceed 1% DMSO.
As noted previously, under these conditions, both forms (hydro-
chloride and free base) showed identical spectroscopic
characteristics.
The new compounds, 1e17, were initially tested in vitro against
the epimastigote form of T. cruzi, Tulahuen 2 strain. TZHs were
incorporated into the media at 15, 10, 7.5, 5 and 2.5 mM concen-
trations. Table 4 shows the IC50 epi (50% inhibitory concentration)
value determined for each of them. Bnz was used as the reference
Fig. 1. TZHs derived from the reaction of cyclohexanone under MW irradiation in DMF
thiosemicarbazone with phenacyl chloride (20) and 4-methyl-phenacyl chloride (21).

158
M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
Table 3
the bloodstream form of the parasite. The results observed in this
assay demonstrate that, in general, compounds with a methyl
substituent in position R1 and R3 are the most active of the series.
TZHs 5, 7, 8, 9,10,15,16, and 17 were analyzed to determine their
activity against the intracellular replicative amastigotes. The
compounds selected were the most active against the epimastigote
and trypomastigote forms of the parasite, except for compound 15.
The results obtained can be seen in Table 5. The compounds 7, 8, 9,
1H NMR and 13C NMR spectroscopy data of the hydrochloride TZHs and free base
TZHs selected.
10, 15 and 17 showed excellent activity, with IC50
values
amas
.
ranging from 0.09 to 1.35
mM, being TZH 9 the most active
Compound
Solvent
C10
C2
C5
C4
NH
H5
compound of this series (IC50 amas ¼ 0.09
m
M). In particular, TZH 9
showed very good correlation of activity against the three forms
evaluated. These results make compound 9 an excellent candidate
for further biological studies.
1
18
1/18
4
19
4/19
20
22
20/22
21
23
CDCl3
CDCl3
DMSO-d6
CDCl3
CDCl3
DMSO-d6
CDCl3
CDCl3
DMSO-d6
CDCl3
CDCl3
164.5
155.9
157.5
165.9
156.7
158.7
165.4
158.6
156.5
163.2
156.7
155.3
169.9
169.3
169.9
168.6
169.3
170.2
170.4
170.7
170.4
170.4
170.9
170.0
100.6
103.5
104.3
100.1
103.3
103.3
101.2
103.0
103.8
100.8
102.6
102.2
142.3
151.3
148.4
140.5
151.2
152.3
140.9
148.8
149.8
140.4
150.9
150.1
12.92
8.75
6.74
6.90
7.31
6.71
6.87
7.26
6.67
6.70
7.24
6.60
6.75
7.27
11.16
12.78
a
-
2.3. In vitro unspecific cytotoxicity
10.90
12.76
8.15
The mammal cytotoxicity of the new compounds was studied
in vitro using Vero cells as the cellular model. None of the seven-
10.85
12.26
teen TZHs caused cell death up to a concentration of 100
(Bnz CC50 ¼ 82.79 M).
mM
a
-
m
21/23
DMSO-d6
10.91
a
Not observed.
2.4. Mechanism of action studies
As mentioned above, compounds with antichagasic activity
structurally related to TZHs, such as thiosemicarbazones and thia-
zoles, act by inhibition of cruzipain and sterol biosynthesis
respectively. In order to determine a probable mechanism of action,
we decided to investigate the effect on these two probable thera-
peutic targets of the synthesized compounds. The following studies
were performed: inhibition of cruzipain and sterol biosynthesis
analysis, both studies were done on the epimastigote stage.
trypanosomicidal agent. Compounds 8, 9, 14, 16, 17 were the most
active anti-T. cruzi derivatives, with IC50 epi values between 4.08 and
4.69
while TZHs 3, 6, 7, 12, 13, 15 displayed moderate activities, with IC50
epi values between 5.24 and 8.62 M. Derivatives 1, 2, 4, 5, 10 and 11
mM, which also resulted more active than the reference drug,
m
were less potent than Bnz. These data suggest that better trypa-
nocidal activity may be attained when R1, R2 or R3 is a methyl
group; on the other hand, the nature of the substituent in the R4
position is not decisive in the anti-T. cruzi activity observed.
The lytic effect on mouse blood trypomastigotes was also eval-
uated for compounds 1e17 (Table 4). When analyzing the relative
potency of the compounds tested in comparison to Bnz, we found
better activities for TZHs 8, 9, 10 and 16. Compounds 5, 7, 13, 15 and
17 showed moderate activities (around 2 times lower than that of
the reference drug). The other derivatives were poorly active against
2.4.1. Inhibition of T. cruzi cruzipain
TZHs 2, 5, 7, 8, 9, 10, 15, 16, and 17 were studied as inhibitors of
T. cruzi cruzipain, following a previously described procedure
[22,23]. E-64 [L-trans-epoxy-succinyl-leucyl-amido(4-guanidino)
butane] was used as reference drug [23]. None of the assayed
compounds resulted to be inhibitors of this enzyme at the studied
doses (10, 20 and 50 mM) (Table 6).
Table 4
2.4.2. Sterol biosynthesis analysis
Biological characterization of TZHs against the epimastigote and trypomastigote
After a pre-established protocol (2.5 ꢂ 107 cells/mL, 24 h of
incubation) [24], the controls (untreated, terbinafine-treated) and
TZHs-treated parasites were collected and the total lipids were
extracted and analyzed as described previously [25,26]. Qualitative
analyses ofneutrallipid fractions werecarriedout usingTLC. None of
the studied compounds were able to accumulate lanosterol (Fig. 3).
forms of T. cruzi.
a,c
b,c
TZHs
IC50 epi
(
m
M)
IC50 trypo
(mM)
1
2
3
4
5
6
7
8
18.99 ꢃ 2.59
>15
8.62 ꢃ 0.25
10.14 ꢃ 0.43
>15
>>500
>>500
308.41 ꢃ 8.66
195.22 ꢃ 8.36
69.57 ꢃ 7.02
403.69 ꢃ 11.46
48.91 ꢃ 6.86
17.24 ꢃ 3.44
20.01 ꢃ 1.99
19.72 ꢃ 2.63
596.95 ꢃ 11.33
372.16 ꢃ 7.43
61.10 ꢃ 4.79
172.42 ꢃ 10.32
87.01 ꢃ 5.16
22.74 ꢃ 2.11
49.53 ꢃ 2.87
30.26 ꢃ 2.85
7.17 ꢃ 0.56
7.08 ꢃ 0.68
4.36 ꢃ 0.49
4.53 ꢃ 0.66
11.78 ꢃ 0.55
17.05 ꢃ 0.78
7.15 ꢃ 0.53
8.59 ꢃ 0.65
4.69 ꢃ 0.43
5.24 ꢃ 0.27
4.27 ꢃ 0.24
4.08 ꢃ 0.20
5.39 ꢃ 0.25
Table 5
In vitro activity of the TZHs selected against the
amastigote form of T. cruzi.
9
10
11
12
13
14
15
16
17
Bnz
a,b
TZHs
IC50 amas
(mM)
5
7
8
9
10
15
16
17
7.72 ꢃ 0.68
0.68 ꢃ 0.09
0.72 ꢃ 0.11
0.09 ꢃ 0.02
0.84 ꢃ 0.14
1.35 ꢃ 0.18
3.96 ꢃ 0.32
0.27 ꢃ 0.06
a
IC50 epi : concentration (in
m
M) that inhibits 50% of epimastigote form of T. cruzi
a
growth.
IC50 amas : concentration (in mM) that inhibits 50%
b
IC50 trypo: concentration (in
T. cruzi lysis.
m
M) that inhibits 50% of trypomastigote form of
of amastigote form of T. cruzi growth.
b
All data are expressed as meansꢃstandard devi-
c
All data are expressed as meansꢃstandard deviations of three separate exper-
ations of three separate experiments, running in
duplicates or triplicates.
iments, running in duplicates or triplicates.

M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
159
Table 6
microwave-assisted synthesis. Most of these new derivatives
exhibited promising activity against epimastigotes and against
trypomastigote and amastigote forms, which are the mammal
stages of T. cruzi. We identified compound 9 as an excellent
candidate for further molecular structural modifications and bio-
logical studies, especially in vivo evaluations. Preliminary results
from our laboratory showed that some TZHs could be able to
produce the inhibition of the enzymatic system involved in
the sterols biosynthetic route. Further studies to confirm the
probable mechanism of action of these novel compounds will be
performed.
Effect on T. cruzi cruzipain activity of selected TZHs.
Compound
Nonea
2
Concentration (
m
M)
Inhibition (%)
0
8
5
5
0
0
0
3
0
0
4
2
0
3
0
3
0
0
0
0
0
0
3
0
0
3
0
0
62
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
20
50
10
5
7
8
4. Experimental
9
4.1. Chemistry
Melting points (uncorrected) were determined on a Thomas
Hoover apparatus. Thin layer chromatography (TLC) was used to
monitor reactions. Reactions were carried out in a Microwave
Synthesis Reactor Microwave$300 Anton Paar. IR spectra were
recorded 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. High resolution mass spectra
were obtained on Bruker micrOTOF-Q II spectrometer. Single
crystal X-Ray diffraction data were collected at room tempera-
ture, using a Gemini A diffractometer, Oxford Diffraction. Data-
collection strategy and data reduction followed standard
procedures implemented in the CrystAlisPro software. The
structures were solved using program SHELXS-97 and refined
using the full-matrix LS procedure with SHELXL-97. Anisotropic
displacement parameters were employed for non-hydrogen
atoms and H atoms were treated. All H atoms were located at
the expected positions and they were refined using a riding
model. In the final cycle of refinement, LS weights of the form
10
15
16
17
E-64
a
None: assay that corresponds to the enzyme incubated in the absence of drug
but in presence of the respective amount of DMSO.
Among TZHs studied, compounds 8 and 9 seem to accumulate
squalene in different degrees with the concomitant decrease of
ergosterol. These results suggest that inhibition of the biosynthesis
of ergosterol is a possible target for the action of these TZHs.
w ¼ 1/[
s
2(Fo2) þ (a*P)2 þ b*P], where P ¼ [(Fo2) þ (2*F2c)]/3, were
employed. All the 1-indanones were commercially purchased,
except 5-methyl-1-indanone [27] and 4-nitro-1-indanone which
was prepared according to the protocol previously described
[11]. Phenacyl chlorides were synthesized as reported in our
previous communication [28].
3. Conclusions
Seventeen 4-arylthiazolylhydrazones derived from 1-indanones
were prepared with good yields in a simple manner by using
Fig. 3. a) TLC analysis of lipid extracts from epimastigote T. cruzi treated with terbinafine (Tbf), 2, 8, 9 and with 17. Erg: ergosterol, Lan: lanosterol, Sq: squalene, C: extract without
treatment. b) Relative intensities of bands were quantified by densitometry using Scion image software (Scion). Results are expressed in arbitrary units.

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M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
4.1.1. Reaction of 1-indanone thiosemicarbazone derivatives with
phenacyl chlorides under microwave irradiation. General procedure
(ESI) m/z (M þ H)þ calcd for C21H22N3O2S 380.14272, found
380.14330.
In
a typical procedure a mixture of thiosemicarbazone
(0.010 mmol), phenacyl chloride (0.013 mmol) and the appropriate
solvent (0.15 mL) (method a: EtOH, method b: acetonitrile, method
c: DMF) in a borosilicate boiling tube, was placed in a microwave
synthesizer at 80 ꢀC. After completion of the reaction (monitored by
TLC), the mixture was suspended in water, filtered, and washed
with EtOH and hexane. Compounds 1e17, 20 and 21 (hydrochloride
forms) were prepared according to method c. All synthesized TZHs
were crystallized from EtOH.
4.1.1.6. 4-(4-Chlorophenyl)-2-(2-(5,6-dimethoxy-2,3-dihydro-1H-inden-
1-ylidene)hydrazinyl)thiazolium chloride (6). Mp: 226e228 ꢀC. IR
n/
cmꢁ1 (KBr): 2710 (NHþ), 1618 and 1588 (C]N). 1H NMR (500 MHz;
CDCl3):
d
: 3.12 (s, 4H, 2ꢂ CH2), 3.92 (s, 3H, OCH3), 3.95 (s, 3H, OCH3),
6.71 (s, 1H, H-thiazole), 6.82 (s, 1H), 7.15 (s, 1H), 7.45 (d, 2H,
J ¼ 8.2 Hz, HeAr), 7.64 (d, 2H, J ¼ 8.2 Hz, HeAr), 12.68 (s, 1H, NH).
13C NMR (DMSO-d6)
d: 28.0, 28.1, 55.5, 55.6, 102.6, 104.2, 108.1,
127.2,128.6,129.4,131.9,133.3,141.4,148.5,148.8,151.6,157.6,169.6.
HRMS (ESI) m/z (M þ H)þ calcd for C20H19ClN3O2S 400.08810,
found 400.08868.
4.1.1.1. 2(2-(2,3-Dihydro-1H-inden-1-ylidene)hydrazinyl)-4-phenylt-
hiazolium chloride (1). Mp: 209e210 ꢀC. IR
n
/cmꢁ1 (KBr): 2714
(NHþ), 1618 and 1586 (C]N). 1H NMR (500 MHz; CDCl3):
d
: 3.13 (m,
4.1.1.7. 2-(2-(4-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
2H, CH2), 3.18 (m, 2H, CH2), 6.74 (s, 1H, H-thiazole) 7.32 (t, 1H,
J ¼ 7.6 Hz, HeAr), 7.37 (d, 1H, J ¼ 7.6 Hz, HeAr), 7.41e7.50 (m, 4H,
HeAr), 7.71 (d, 2H, J ¼ 7.1 Hz, HeAr), 7.79 (d, 1H, J ¼ 7.6 Hz, HeAr),
4-phenylthiazolium chloride (7). Mp: 225e227 ꢀC. IR
n
/cmꢁ1 (KBr):
2712 (NHþ), 1618 (C]N). 1H NMR (500 MHz; CDCl3):
d: 2.31 (s, 3H,
CH3), 3.08 (m, 2H, CH2), 3.14 (m, 2H, CH2), 6.72 (s, 1H, H-thiazole),
7.24 (m, 2H, HeAr), 7.43e7.49 (m, 3H, HeAr), 7.62 (t, 1H, J ¼ 4.4 Hz,
HeAr), 7.70 (d, 2H, J ¼ 7.3 Hz, HeAr), 12.89 (s, 1H, NH), 13.74 (s, 1H,
12.91 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 27.9, 28.6, 104.3, 121.2,
126.1, 126.3, 127.6, 128.3, 129.2, 130.7, 134.6, 137.9, 148.6, 150.1, 157.7,
169.9. HRMS (ESI) m/z (M þ H)þ calcd for C18H16N3S 306.10594,
found 306.10677.
NH). 13C NMR (DMSO-d6)
d: 18.0, 27.0, 27.5,103.8,118.2, 125.6, 127.3,
127.7, 128.7, 130.5, 134.3, 134.7, 137.4, 147.1, 149.3, 157.3, 169.5.
HRMS (ESI) m/z (M þ H)þ calcd for C19H18N3S 320.12159, found
320.12238.
4.1.1.2. 2(2-(2,3-Dihydro-1H-inden-1-ylidene)hydrazinyl)-4-p-tolylth-
iazolium chloride (2). Mp: 209e211 ꢀC. IR /cmꢁ1 (KBr): 2654 (NHþ),
n
1618 and 1580 (C]N). 1H NMR (500 MHz; CDCl3):
d: 2.38 (s, 3H,
4.1.1.8. 2-(2-(4-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
CH3), 3.14 (m, 2H, CH2), 3.20 (m, 2H, CH2), 6.66 (s, 1H, H-thiazole),
7.27 (d, 2H, J ¼ 8.0 Hz, HeAr), 7.32 (m, 1H, HeAr), 7.37 (d, 1H,
J ¼ 7.5 Hz, HeAr), 7.43 (m, 1H, HeAr), 7.58 (d, 2H, J ¼ 8.0 Hz, HeAr),
7.78 (d, 1H, J ¼ 7.8 Hz, HeAr), 12.88 (s, 1H, NH), 13.52 (s, 1H, NH). 13C
4-p-tolylthiazolium chloride (8). Mp: 231e233 ꢀC. IR /cmꢁ1 (KBr):
n
2719 (NHþ), 1619 and 1591 (C]N). 1H NMR (500 MHz; CDCl3):
d:
2.31 (s, 3H, CH3), 2.39 (s, 3H, CH3), 3.07 (m, 2H, CH2), 3.13 (m, 2H,
CH2), 6.65 (s, 1H, H-thiazole), 7.24 (m, 2H, HeAr), 7.27 (d, 2H,
J ¼ 7.3 Hz, HeAr) 7.58 (d, 2H, J ¼ 7.3 Hz, HeAr), 7.62 (t,1H, J ¼ 4.4 Hz,
NMR (DMSO-d6) d: 28.0, 28.7, 104.3, 121.3, 126.0, 127.5, 128.2, 129.1,
130.6, 134.6, 138.1, 141.6, 144.6, 148.5, 157.6, 169.6. HRMS (ESI) m/z
HeAr),12.84 (s,1H, NH). 13C NMR (DMSO-d6)
d: 18.0, 20.8, 26.7, 27.5,
(M þ H)þ calcd for C19H18N3S 320.12159, found 320.12229.
102.9, 118.2, 125.5, 127.3, 129.2, 130.6, 131.7, 134.7, 137.0, 137.4, 147.1,
148.1, 157.5, 169.3. HRMS (ESI) m/z (M þ H)þ calcd for C20H20N3S
334.13724, found 334.13779.
4.1.1.3. 4-(4-Chlorophenyl) 2-(2-(2,3-dihydro-1H-inden-1-ylidene)
hydrazinyl)thiazolium chloride (3). Mp: 213e215 ꢀC. IR
n
/cmꢁ1
(KBr): 2627 (NHþ), 1615 and 1583 (C]N). 1H NMR (500 MHz;
CDCl3): : 3.10 ( m, 2H, CH2), 3.16 (m, 2H, CH2), 6.74 (s, 1H, H-
4.1.1.9. 4-(4-Chlorophenyl)-2-(2-(4-methyl-2,3-dihydro-1H-inden-1-
d
ylidene)hydrazinyl)thiazolium chloride (9). Mp: 236e238 ꢀC. IR
thiazole), 7.32 (t,1H, J ¼ 7.7 Hz, HeAr), 7.37 (d, 1H, J ¼ 7.7 Hz, HeAr),
n
/cmꢁ1 (KBr): 2694 (NHþ), 1623 and 1590 (C]N). 1H NMR
7.45 (m, 3H, HeAr), 7.66 (d, 2H, J ¼ 8.5 Hz, HeAr), 7.78 (d, 1H,
(500 MHz; CDCl3): d: 2.31 (s, 3H, CH3), 3.08 (m, 2H, CH2), 3.13 (m,
J ¼ 7.7 Hz, HeAr), 12.68 (s, 1H, NH). 13C NMR (DMSO-d6)
d
: 28.1,
2H, CH2), 6.73 (s, 1H, H-thiazole), 7.24 (m, 2H, HeAr), 7.45 (d, 2H,
J ¼ 7.3 Hz, HeAr), 7.61 (t, 1H, J ¼ 4.8 Hz, HeAr), 7.64 (d, 2H,
28.9, 105.2, 121.4, 126.6, 127.8, 128.0, 129.4, 130.9, 132.7, 134.3, 138.2,
148.7, 149.6, 157.7, 170.4. HRMS (ESI) m/z (M þ H)þ calcd for
C18H15ClN3S 340.06752, found 340.06817.
J ¼ 7.3 Hz, HeAr),12.74 (s,1H, NH). 13C NMR (DMSO-d6)
d: 18.0, 27.0,
27.5, 104.6, 118.2, 127.3, 127.4, 128.7, 130.5, 132.0, 133.5, 134.7, 137.4,
147.1, 148.7, 157.2, 169.6. HRMS (ESI) m/z (M þ Na)þ calcd for
C19H17ClN3S 354.08262, found 354.08300.
4.1.1.4. 2-(2-(5,6-Dimethoxy-2,3-dihydro-1H-inden-1-ylidene)hydra-
zinyl)-4-phenylthiazolium chloride (4). Mp: 219e221 ꢀC. IR /cmꢁ1
n
(KBr): 2689 (NHþ), 1615 and 1592 (C]N). 1H NMR (500 MHz;
CDCl3):
4.1.1.10. 4-(4-Chlorophenyl)-2-(2-(4-methoxy-2,3-dihydro-1H-inden-
d
: 3.12 (s, 4H, 2ꢂ CH2), 3.92 (s, 3H, OCH3), 3.95 (s, 3H,
1-ylidene)hydrazinyl)thiazolium chloride (10). Mp: 233e235 ꢀC. IR
n
/
OCH3), 6.71 (s, 1H, H-thiazole), 6.80 (s, 1H, HeAr), 7.16 (s, 1H,
HeAr), 7.45 (m, 3H, HeAr), 7.69 (d, 2H, J ¼ 7.3, HeAr), 12.84 (s, 1H,
cmꢁ1 (KBr): 2702 (NHþ), 1619 (C]N). 1H NMR (500 MHz; CDCl3):
d:
2.87 (m, 2H, CH2), 3.09 (m, 2H, CH2), 3.88 (s, 3H, OCH3), 6.82 (s, 1H,
H-thiazole), 6.85 (d, 1H, J ¼ 7.6 Hz, HeAr), 7.31 (d, 1H, J ¼ 7.6 Hz,
HeAr), 7.39 (m, 3H, HeAr), 7.71 (d, 2H, J ¼ 8.5 Hz, HeAr), 12.77 (s,
NH). 13C NMR (DMSO-d6)
d: 28.7, 28.8, 56.2, 56.3,103.3, 104.2,108.8,
126.3, 128.5, 129.4, 130.0, 134.8, 142.5, 149.4, 150.2, 152.3, 158.7,
170.2. HRMS (ESI) m/z (M þ H)þ calcd for C20H20N3O2S 366.12707,
found 366.12786.
1H, NH), 13.20 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 25.7, 28.0, 55.6,
105.0, 111.7, 113.3, 127.7, 129.1, 129.3, 132.3, 134.1, 136.0, 139.8,
149.6, 156.7, 156.9, 170.0. HRMS (ESI) m/z (M þ Na)þ calcd for
C19H16ClN3NaOS 392.05948, found 392.05835.
4.1.1.5. 2-(2-(5,6-Dimethoxy-2,3-dihydro-1H-inden-1-ylidene)hydra-
zinyl)-4-p-tolylthiazolium chloride (5). Mp: 221e223 ꢀC. IR /cmꢁ1
n
(KBr): 2714 (NHþ), 1627 and 1591 (C]N). 1H NMR (500 MHz;
CDCl3):
4.1.1.11. 4-(4-Chlorophenyl)-2-(2-(4-nitro-2,3-dihydro-1H-inden-1-
d
: 2.39 (s, 3H, CH3), 3.12 (s, 4H, 2ꢂ CH2), 3.92 (s, 3H,
ylidene)hydrazinyl)thiazolium chloride (11). Mp: 230e232 ꢀC. IR
OCH3), 3.95 (s, 3H, OCH3), 6.63 (s, 1H, H-thiazole), 6.82 (s, 1H),
7.16 (s, 1H), 7.27 (d, 2H, J ¼ 8.1 Hz, HeAr), 7.58 (d, 2H, J ¼ 8.1 Hz,
HeAr), 12.82 (s, 1H, NH), 13.99 (s, 1H, NH). 13C NMR (DMSO-d6)
n
/cmꢁ1 (KBr): 2698 (NHþ), 1621 and 1590 (C]N). 1H NMR
(500 MHz; CDCl3): d: 2.97 (m, 2H, CH2), 3.66 (m, 2H, CH2), 6.87 (s,
1H, H-thiazole), 7.38 (d, 2H, J ¼ 8.5 Hz, HeAr), 7.53 (t, 1H,
J ¼ 7.6 Hz, HeAr), 7.70 (d, 2H, J ¼ 8.5 Hz, HeAr), 8.10 (d, 1H,
J ¼ 7.6 Hz, HeAr), 8.23 (d, 1H, J ¼ 7.6 Hz, HeAr), 12.81 (s, 1H, NH).
d
: 20.1, 28.0, 28.2, 55.5, 55.6, 102.6, 104.1, 108.1, 125.5, 129.2,
129.6, 131.2, 137.1, 141.6, 148.4, 148.8, 151.7, 158.1, 169.2. HRMS

M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
161
13C NMR (DMSO-d6)
129.5, 132.4, 134.0, 142.0, 143.7, 146.1, 149.8, 154.2, 169.7. HRMS
(ESI) m/z (M þ H)þ calcd for C18H14ClN4O2S 385.05205, found
385.05300.
d
: 28.0, 30.0, 105.4, 125.5, 126.9, 127.7, 129.1,
125.9, 126.0, 129.7, 131.7, 136.7, 137.5, 138.2, 145.8, 149.6, 157.1, 169.8.
HRMS (ESI) m/z (M þ H)þ calcd for C20H20N3S 334.13724, found
334.13811.
4.1.1.18. 2-(2-Cyclohexylidenehydrazinyl)-4-phenylthiazolium chlo-
4.1.1.12. 4-(4-Chlorophenyl)-2-(2-(5-methyl-2,3-dihydro-1H-inden-
ride (20) [18]. Yield: 68%, mp: 170e172 ꢀC. IR /cmꢁ1 (KBr): 2586
n
1-ylidene)hydrazinyl)thiazolium chloride (12). Mp: 245e246 ꢀC. IR
(NHþ), 1614 and 1567 (C]N). 1H NMR (500 MHz; CDCl3):
d:
n
/cmꢁ1 (KBr): 2681 (NHþ), 1619 and 1592 (C]N). 1H NMR
1.62e1.79 (m, 6H, CH2), 2.38 (m, 2H, CH2), 2.60 (m, 2H, J ¼ 6.4 Hz,
(500 MHz; CDCl3):
d
: 2.42 (s, 3H, CH3), 3.14 (m, 4H, 2ꢂ CH2), 6.71
CH2), 6.67 (s, 1H, H-thiazole), 7.40e7.48 (m, 3H, HeAr), 7.67 (d, 2H,
(s, 1H, H-thiazole), 7.13 (d, 1H, J ¼ 7.8 Hz, HeAr), 7.19 (s, 1H, HeAr),
J ¼ 6.9 Hz, HeAr),12.78 (s,1H, NH).13C NMR (DMSO-d6)
d: 25.6, 26.0,
7.46 (d, 2H, J ¼ 8.5 Hz, HeAr), 7.66 (d, 2H, J ¼ 8.5 Hz, HeAr), 7.69 (d,
27.3, 27.8, 35.2, 103.7, 126.0, 128.0, 129.1, 134.8, 149.8, 156.5, 170.5.
1H, J ¼ 7.8 Hz, HeAr), 12.76 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 21.7,
28.2, 28.4, 104.9, 121.0, 126.6, 127.7, 128.5, 129.1, 134.0, 140.4, 148.7,
149.3, 157.1, 170.1. HRMS (ESI) m/z (M þ H)þ calcd for C19H17ClN3S
354.08262, found 354.08282.
4.1.1.19. 2-(2-Cyclohexylidenehydrazinyl)-4-p-tolylthiazolium chlo-
ride (21) [15c]. Yield: 70%, mp: 165e167 ꢀC. IR /cmꢁ1 (KBr): 2692
n
(NHþ), 1615 and 1589 (C]N). 1H NMR (500 MHz; CDCl3):
d:
1.59e1.79 (m, 6H, CH2), 2.36 (m, 2H, CH2), 2.38 (s, 3H, CH3), 2.60 (m,
2H, CH2), 6.60 (s, 1H, H-thiazole), 7.27 (d, 2H, J ¼ 8.1 Hz, HeAr), 7.57
(d, 2H, J ¼ 8.1 Hz, HeAr),12.26 (s,1H, NH),14.18 (s,1H, NH). 13C NMR
4.1.1.13. 4-(4-Chlorophenyl)-2-(2-(6-methyl-2,3-dihydro-1H-inden-
1-ylidene)hydrazinyl)thiazolium chloride (13). Mp: 225e227 ꢀC. IR
n
/cmꢁ1 (KBr): 2687 (NHþ), 1618 and 1591 (C]N). 1H NMR
(DMSO-d6) d: 20.8, 25.1, 25.5, 26.8, 27.1, 34.7, 102.2, 125.4, 129.1,
(500 MHz; CDCl3):
(s, 1H, H-thiazole), 7.46 (m, 3H, HeAr), 7.58 (s,1H, HeAr), 7.66 (m,
3H, HeAr), 12.79 (s, 1H, NH). 13C NMR (DMSO-d6)
: 21.2, 28.1,
28.2, 104.9, 121.1, 125.8, 127.6, 129.0, 131.4, 132.2, 136.6, 138.1,
145.5, 149.2, 157.0, 169.9. HRMS (ESI) m/z (M þ H)þ calcd for
C19H17ClN3S 354.08262, found 354.08306.
d
: 2.41 (s, 3H, CH3), 3.13 (m, 4H, 2ꢂ CH2), 6.74
132.1, 136.6, 150.1, 155.3, 170.0.
d
4.1.2. Reaction of 1-indanone thiosemicarbazone derivatives with
phenacyl chlorides under conventional heating. general procedure
A suspension of thiosemicarbazone (0.5 mmol) and phenacyl
chloride (0.5 mmol) in DMF (30 mL) was heated under reflux until
completion of the reaction (monitored by TLC). The mixture was
poured onto ice and the resulting solid was filtered, washed with
EtOH and hexane. Compounds 18, 19, 22 and 23 were prepared
according to this methodology. All synthesized TZHs were crystal-
lized from EtOH.
4.1.1.14. 2-(2-(5-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
4-phenylthiazolium chloride (14). Mp: 227e229 ꢀC. IR /cmꢁ1 (KBr):
n
2678 (NHþ), 1615 and 1591 (C]N). 1H NMR (500 MHz; CDCl3):
d:
2.40 (s, 3H, CH3), 3.12 (m, 4H, 2ꢂ CH2), 6.72 (s, 1H, H-thiazole), 7.13
(d, 1H, J ¼ 8.0 Hz, HeAr), 7.17 (s, 1H, HeAr), 7.46 (m, 3H, HeAr), 7.66
(d, 1H, J ¼ 8.0 Hz, HeAr), 7.70 (d, 2H, J ¼ 8.5 Hz, HeAr), 12.68 (s, 1H,
4.1.2.1. 2(2-(2,3-Dihydro-1H-inden-1-ylidene)hydrazinyl)-4-phenyl-
NH), 13.46 (s, 1H, NH). 13C NMR (DMSO-d6)
d: 21.7, 28.2, 28.4, 104.1,
thiazole (18). Yield: 67%, mp: 158e160 ꢀC. IR
n
d
/cmꢁ1 (KBr): 3331
: 2.76 (m, 2H, CH2),
121.0, 126.0, 126.6, 128.1, 128.5, 129.1, 134.7, 135.5, 140.4, 148.8, 149.7,
157.5, 169.9. HRMS (ESI) m/z (M þ H)þ calcd for C19H18N3S 320.12159,
found 320.12204.
(NeH),1559 (C]N).1H NMR (500 MHz; CDCl3):
3.16 (m, 2H, CH2), 6.90 (1H, s, H-thiazole) 7.32e7.42 (m, 7H, HeAr),
7.80 (d, 2H, J ¼ 7.2 Hz, HeAr), 8.49 (s, 1H, NH). 13C NMR (CDCl3)
d:
26.3, 28.4, 103.7, 121.6, 125.4, 125.9, 127.2, 127.7, 128.6, 130.2, 134.9,
137.7,147.5,151.3,156.0,169.3. The 13C NMR spectrum was similar to
that observed for compound 1 when it was recorded in DMSO-d6.
4.1.1.15. 2-(2-(5-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
4-p-tolylthiazolium chloride (15). Mp: decompose before melting. IR
n
/cmꢁ1 (KBr): 2732 (NHþ),1618 and 1591 (C]N). 1H NMR (500 MHz;
CDCl3):
d
: 2.38 (s, 3H, CH3), 2.40 (s, 3H, CH3), 3.13 (m, 4H, 2ꢂ CH2),
4.1.2.2. 2-(2-(5,6-Dimethoxy-2,3-dihydro-1H-inden-1-ylidene)hydra-
6.64 (s, 1H, H-thiazole), 7.13 (d, 1H, J ¼ 8.0 Hz, HeAr), 7.17 (s, 1H,
HeAr), 7.27 (d, 2H, J ¼ 8.2 Hz, HeAr) 7.58 (d, 2H, J ¼ 8.2 Hz, HeAr),
7.66 (d, 1H, J ¼ 8.0 Hz, HeAr), 12.81 (s, 1H, NH). 13C NMR (DMSO-d6)
zinyl)-4-phenylthiazole (19). Yield: 62%, mp: 204e205 ꢀC. IR
n
d
/cmꢁ1
: 2.74
(KBr): 3304(NeH), 1558 (C]N). 1H NMR (500 MHz; CDCl3):
(m, 2H, CH2), 3.02 (m, 2H, CH2), 3.90 (s, 3H, OCH3), 3.96 s, 3H,
OCH3), (6.78 (s, 1H, HeAr), 6.87 (s, 1H, H-thiazole), 7.21 (s, 1H,
HeAr), 7.30 (m, 1H, HeAr), 7.38 (m, 2H, HeAr) 7.80 (d, 2H,
d
: 21.3, 21.7, 28.2, 28.5, 103.2, 121.0, 126.0, 126.6, 128.5, 129.7, 135.6,
137.3, 140.3, 148.7, 157.5, 169.9. HRMS (ESI) m/z (M þ H)þ calcd for
C20H20N3S 334.13724, found 334.13787.
J ¼ 7.2 Hz, HeAr), 8.49 (s, 1H, NH). 13C NMR (CDCl3)
d: 27.0, 28.4,
56.0, 56.1, 103.0, 103.3, 107.3, 125.9, 127.7, 128.6, 129.8, 134.9, 141.0,
149.2, 151.2, 151.8, 156.7, 169.3. The 13C NMR spectrum was similar
to that observed for compound 4 when it was recorded in DMSO-d6
4.1.1.16. 2-(2-(6-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
4-phenylthiazolium chloride (16). Mp: 222e224 ꢀC. IR /cmꢁ1 (KBr):
n
2647 (NHþ), 1619 and 1592 (C]N). 1H NMR (500 MHz; CDCl3):
d:
2.40 (s, 3H, CH3), 3.08 (m, 2H, CH2), 3.14 (m, 2H, CH2), 6.74 (s, 1H, H-
thiazole), 7.24 (m, 2H, HeAr), 7.43 (m, 1H, HeAr), 7.47(m, 1H, HeAr),
7.58 (s, 1H, HeAr), 7.71 (d, 2H, J ¼ 7.1 Hz, HeAr), 12.72 (s, 1H, NH). 13C
4.1.2.3. 2-(2-Cyclohexylidenehydrazinyl)-4-phenylthiazole (22). Yield:
55%, mp:128e130 ꢀC. IR /cmꢁ1 (KBr): 3394 (NeH), 1561 (C]N). 1H
n
NMR (500 MHz; CDCl3): d: 1.57e1.70 (m, 6H, CH2), 2.25e2.35 (m,
NMR (DMSO-d6)
d: 21.3, 28.2, 28.4, 104.2, 121.4, 125.9, 126.1, 128.2,
4H, CH2), 6.69 (s, 1H, H-thiazole), 7.27e7.36 (m, 3H, HeAr), 7.66 (d,
129.1, 131.7, 136.7, 138.1, 145.8, 149.3, 157.6, 169.9. HRMS (ESI) m/z
2H, J ¼ 7.4 Hz, HeAr). 13C NMR (CDCl3)
d: 26.1, 26.4, 27.6, 28.0, 35.8,
(M þ H)þ calcd for C19H18N3S 320.12159, found 320.12187.
103.0, 126.5, 129.0, 129.5, 133.6, 148.8, 158.6, 170.7. The 13C NMR
spectrum was similar to that observed for compound 20 when it
was recorded in DMSO-d6
4.1.1.17. 2-(2-(6-Methyl-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)-
4-p-tolylthiazolium chloride (17). Mp: 224e226 ꢀC. IR /cmꢁ1 (KBr):
n
2729 (NHþ), 1619 and 1592 (C]N). 1H NMR (500 MHz; CDCl3):
d:
4.1.2.4. 2-(2-Cyclohexylidenehydrazinyl)-4-p-tolylthiazole (23). Yield:
2.41 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.16 (m, 4H, 2ꢂ CH2), 6.70 (s, 1H,
H-thiazole), 7.28 (m, 2H, HeAr), 7.30 (d, 2H, J ¼ 8.5 Hz, HeAr), 7.61 (s,
1H, HeAr), 7.62 (d, 2H, J ¼ 8.5 Hz, HeAr), 12.83 (s, 1H, NH), 14.25 (s,
63%, mp: 85e87 ꢀC. IR /cmꢁ1 (KBr): 3389 (NeH), 1556 (C]N). 1H
n
NMR (500 MHz; CDCl3): d: 1.62e1.72 (m, 6H, CH2), 2.26e2.43 (m, 4H,
CH2), 2.36 (s. 3H, CH3), 6.75 (s,1H, H-thiazole), 7.18 (d, 2H, J ¼ 8.0 Hz,
1H, NH). 13C NMR (DMSO-d6)
d: 21.3, 21.4, 28.2, 28.4, 103.3, 121.4,
HeAr), 7.64 (d, 2H, J ¼ 8.0 Hz, HeAr), 8.15 (s, 1H, NH). 13C NMR

162
M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
(CDCl3)
d
: 21.9, 26.1, 26.2, 27.5, 35.7, 102.6, 126.5, 126.8, 130.0, 131.6,
parasites. The IC50
mastigote form) was estimated by lineal regression analysis from
the %I values and the decimal logarithm (log) of drug concentration.
(50% inhibitory concentration on trypo-
trypo
132.3,138.3,150.9,156.7,170.9. The 13C NMR spectrumwas similar to
that observed for compound 21 when it was recorded in DMSO-d6
4.2. Biology
4.2.4. Amastigote growth inhibition assay
For the analysis of intracellular parasites (amastigotes form), 96-
well microplate were seeded with J774 murine macrophage cells
4.2.1. Parasites and mammalian cells
T. cruzi epimastigotes (Tulahuen strain, Tul 2 stock) were grown
at 28 ꢀC in a liquid medium containing 0.3% yeast extract, 0.9%
tryptose, 0.4% dextrose, 1% disodium phosphate 2-hydrate, 0.36%
sodium chloride, 0.04% potassium chloride, 0.15% powered beef
liver, 0.5% brain heart infusion and 0.5e1.0 mg/100 mL hemin.
T. cruzi bloodstream trypomastigotes from Tulahuen strain, Tul 2
stock were obtained from infected CF1 mice by cardiac puncture, at
the peak of parasitemia on day 15 postinfection. Trypomastigotes
were routinely maintained by infecting 21-days-old CF1 mice
(weighing 23.8 ꢃ 2.6 g).
line, at 5 ꢂ 103 cells/well in 100
ml volumes and incubated 2 h at
37 ꢀC and 5% CO2. Cells were infected with transfected blood-
trypomastigotes expressing the b-galactosidase gene (clone C4) at
a parasite:cell ratio 10:1. After 24 h of contact, cell cultures were
washed twice with PBS to remove free parasites and the
compounds (2e100 mM) were added in 200 ml fresh complete RPMI
medium without phenol red (to avoid interference with the assay
absorbance readings at 570 nm). Each concentration was tested in
duplicate. After 7 days, the assay were developed by addition of the
detergent Nonidet P-40 (1% final concentration) and CPRG (100 mM
Vero cells line was cultivated in MEM or RPMI, respectively,
supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/mL
streptomycin and 0.292 mg/mL glutamine.
final concentration). Plates were incubated at 37 ꢀC for 4 h. Wells
with galactosidase activity turned the media from yellow to red,
and this was quantified by absorbance at 570 nm in a Microplate
Reader (Bio-Rad Laboratories). Because that assayed compounds
are colored and present significant absorbance at 570 nm, controls
including uninfected J774 cells with RPMI alone (0% infection
control for untreated infected well) and uninfected J774 cells with
RPMI and different doses of assayed compounds (0% infection
control for treated infected well) were done. The percentage of
inhibition was calculated as: %I ¼ [1ꢁ(AitꢁAnit/(AicꢁAnic] ꢂ 100,
where Ait represents the mean A570 value recorded for treated
infected wells, Anit represents the mean A570 value recorded for
treated non-infected wells, Aic represents the mean A570 value
recorded for untreated infected wells and Anic represents the mean
A570 value recorded for untreated non-infected wells. The IC50 amas
(50% inhibitory concentration on amastigote form) was estimated
by lineal regression analysis from the %I values and the decimal
logarithm (log) of drug concentration.
4.2.2. Animals
Outbred CF1 male and inbred C3H/HeN female mice were
nursed at the Departamento de Microbiología, Facultad de Medic-
ina, Universidad de Buenos Aires. Animals received human care and
were treated in accordance with guidelines established by the
Animal Care and Use Committee of the Argentine Association of
Specialists in Laboratory Animals (AADEALC).
4.2.3. In vitro trypanocidal activity assay
To evaluate the growth inhibition of T. cruzi epimastigote,
parasites from a 5 days-old culture were inoculated into fresh
culture medium to reach an initial concentration of 1.5e2.5 ꢂ 106
cells/mL. Cells were cultured with different concentrations of
compounds (usually of 1.50e15
mM or 0.5e2.5 mM) for 4 days. Bnz
(2.50e15 M) was used as the reference trypanocidal drug (positive
m
control). The compounds ability to inhibit growth of the parasite
(antiproliferative activity) was evaluated, in triplicate, in compar-
ison to the control without drug. Cells growth was followed by
counting the number of cells per mL of culture using a Neubauer
chamber and was expressed as cellular density (CD). For the count
only the cells showing motility (parasites without motility were
dead as demonstrated by positive staining with trypan blue) were
taking into account. The percentage of inhibition (%I) was calcu-
lated as: %I ¼ {1ꢁ[(CD5teCD0t)/(CD5ceCD0c)]} ꢂ 100, where CD5t is
cellular density of treated parasites at day 5; CD0t is cellular density
of treated parasites just immediately after adding the drug (day 0);
CD5c is cellular density of untreated parasites (control) at day 5; and
CD0c is cellular density of untreated parasites at day 0. The IC50 epi
(50% inhibitory concentration on epimastigote forms) was esti-
mated by lineal regression analysis from the %I values and the
decimal logarithm (log) of drug concentration.
4.2.5. Cytotoxicity assay
Cytotoxic activity was evaluated in vitro, using Vero cells. The
cells were cultured under the standard conditions described above.
Cells 9 ꢂ 105 cell/mL were seeded in a 24-well plate and after 48 h
different concentrations of compounds (12.5e200.0
mM) or Bnz
(3.0e3000 mM) were added. After 24 h of incubation, cells were
washed twice with PBS and 3-(4,5-dimethylthiazol-2yl)-2,5-
diphenyltetrazolium bromide (MTT) from a stock solution (5 mg/
mL) was added at a final concentration of 0.5 mg/mL. Plates were
incubated for 1 h at 37 ꢀC. Finally, blue precipitates were dissolved
in 0.5 ml of dimethyl sulfoxide (DMSO) and were read on a plate
reader (Spectra CountÔ BS 10001) at a wavelength of 570 nm.
Values from blank plates containing only medium and reagents
were subtracted from the values of the samples. The values of
absorbance showed a good correlation with viable cells counts
using trypan blue. All MTT assays were repeated at least three times
by using four samples per assay.
The trypanocidal effects of different compounds and Bnz were
also tested on bloodstream trypomastigotes according to a stan-
dard WHO protocol with minor modifications [29]. Briefly, mouse
blood containing trypomastigotes was diluted in culture medium to
adjust the parasite concentration to 1.5 ꢂ 106 cells/ml. Parasites
4.2.6. Cruzipain inhibition assay
Cruzipain was isolated from a cell-free extract of T. cruzi epi-
mastigotes by ConA-Sepharose affinity chromatography, as previ-
ously described [22]. The enzymatic activity was assayed in an
incubation system contained Tris-acetate buffer pH 8.2 (50
2-mercaptoethanol (10 mol) and the synthetic chromogenic
substrate Bz-Pro-Phe-Arg-pNA (0.3 mol) in a total volume of 1 ml.
The reaction was followed spectrophotometrically at 410 nm on
a Hewlett Packard 8452AeDiode Array spectrophotometer. The
drugs were added as solutions in DMSO and the controls were
prepared with the correspondent amount of the same solvent. E-64
were seeded (150
different concentrations of compounds (0.30e350
m
l/well) in duplicate in a 96-well microplate, and
M) were added.
m
mmol),
Plates were incubated for 24 h, and the remaining alive parasites
were counted in a Neubauer chamber as previously described [30].
The results were expressed as the percentage of lysed parasites (%I)
relative to the number of parasites in the control (without adding
the drug): %I ¼ [1ꢁ(CDt/(CDc)] ꢂ 100, where CDt is cellular density
of treated parasites and CDc is cellular density of untreated
m
m

M.E. Caputto et al. / European Journal of Medicinal Chemistry 55 (2012) 155e163
163
2,4-disubstituted thiazoles as possible antimicrobial agents, Eur. J. Med. Chem.
43 (2008) 261e267.
[L-trans-epoxy-succinyl-leucyl-amido(4-guanidino)butane]
used as reference drug [23].
was
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4.2.7. Ergosterol biosynthesis analysis
T. cruzi epimastigotes (2.5 ꢂ 107 ells/mL) were cultured for 24 h at
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