2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: Structural design, synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.08.012

The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a novel series of 2-(pyridin-2-yl)-1,3- thiazoles derived from 2-pyridine thiosemicarbazone. The majority of these compounds are potent cruzain inhibitors

Full text of DOI:10.1016/j.ejmech.2014.08.012

European Journal of Medicinal Chemistry 86 (2014) 48e59
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: Structural
design, synthesis and pharmacological evaluation
,
Marcos Veríssimo de Oliveira Cardoso a *, Lucianna Rabelo Pessoa de Siqueira a,
Elany Barbosa da Silva a, Lívia Bandeira Costa a, Marcelo Zaldini Hernandes a,
Marcelo Montenegro Rabello a, Rafaela Salgado Ferreira b, Luana Faria da Cruz b,
c
d
~
ꢀ
^
Diogo Rodrigo Magalhaes Moreira , Valeria Rego Alves Pereira ,
Maria Carolina Accioly Brelaz de Castro d, Paul V. Bernhardt e, Ana Cristina Lima Leite a
a
^
^
^
Departamento de Ciencias Farmaceuticas, Centro de Ciencias da Saúde, Universidade Federal de Pernambuco, 50740-520 Recife, PE, Brazil
b Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, CEP 31270-901 Belo Horizonte, MG, Brazil
c
~
Centro de Pesquisas Gonçalo Moniz, Fundaçao Oswaldo Cruz, CEP, 40296-750 Salvador, BA, Brazil
d
~
~
Centro de Pesquisas Aggeu Magalhaes, Fundaçao Oswaldo Cruz, CEP, 50670-420 Recife, PE, Brazil
e School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a
novel series of 2-(pyridin-2-yl)-1,3-thiazoles derived from 2-pyridine thiosemicarbazone. The majority of
these compounds are potent cruzain inhibitors and showed excellent inhibition on the trypomastigote
form of the parasite, and the resulting structureeactivity relationships are discussed. Together, these data
present a novel series of thiazolyl hydrazones with potential effects against Chagas disease and they
could be important leads in continuing development against Chagas disease.
Received 17 April 2014
Received in revised form
1 August 2014
Accepted 5 August 2014
Available online 6 August 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Chagas disease
Trypanosoma cruzi
Thiazoles
Hydrazones
2-Pyridine thiosemicarbazone
1. Introduction
it, nifurtimox and benznidazole [6,8] (Fig.1). Current chemotherapy
for Chagas disease is unsatisfactory due to its limited efficacy,
Chagas disease, also known as American trypanosomiasis, is a
potentially life-threatening illness caused by the protozoan parasite
Trypanosoma cruzi (T. cruzi) [1]. About 7e8 million people worldwide
are estimated to be infected with T. cruzi, mainly in Latin America.
Over the years, numerous chemical classes against T. cruzi have
become available, but there is still no effective treatment for all
phases of the disease [2]. Recent research has pointed to the
ergosterol biosynthetic pathway as a biochemical target [3,4]. Azole
derivatives such as posaconazole and ravuconazole, have been
tested and presented trypanocidal activity, but they are not yet
available as therapeutics [5e7].
particularly in the chronic phase, with frequent side effects that can
lead to discontinuation of treatment.
Among a number of drug targets being investigated for Chagas
disease, cruzain, the major cysteine protease active in the parasite,
is a prominent candidate [9e12]. Cruzain is a cathepsin-L-like
protease of the papain family thought to be important for intra-
cellular replication and differentiation of the T. cruzi parasite [13].
Among the chemical groups explored for anti-Chagas activity,
thiazolyl hydrazones are noteworthy because of their wide bio-
logical, especially anti-parasitic, activities [14e17].
In 2004, Greenbaum et al. observed that thiosemicarbazones
with a pyridyl moiety inhibit cruzain catalytic activity [18]. Sub-
sequent studies have demonstrated the trypanocidal activity of
thiosemicarbazones and their metal complexes. Recently, Caputo
et al. have demonstrated trypanocidal activity for a series of 4-
arylthiazolylhydrazones [19], with a broad and potent activity for
all forms of the parasite. Recently studies have reported inhibition
Despite the efforts of many investigators in the research of a
new anti-Chagas drugs, only two drugs are currently used to treat
* Corresponding author.
E-mail address: marcosvocardoso@gmail.com (M.V.O. Cardoso).
http://dx.doi.org/10.1016/j.ejmech.2014.08.012
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
49
2. Results and discussion
2.1. Chemistry
2-(pyridin-2-yl)thiosemicarbazones (2aeb) were prepared by
reacting commercially available thiosemicarbazides with the
appropriate aldehyde or ketone (1:1.2 mol ratio) via Schiff base
condensations using ultrasound irradiation in presence of a cata-
lytic amount of AcOH. This reaction condition led to high yields
(80e90%). 2-(pyridin-2-yl)-1,3-thiazoles (3e26) were prepared via
Hantsch cyclization between 2-(pyridin-2-yl)thiosemicarbazones
(2aeb) and substituted 2-bromoacetophenones (Scheme 1). These
reactions proceed well upon refluxing with ethanol (2e4 h), but
here we adapted this to ultrasound conditions at room temperature
[23] using 2-propanol as solvent [24]. This resulted in good yields
(50e85%) and shorter reaction times (60 min in most cases)
compared with the reflux protocol.
Microanalysis and NMR data are compatible with the proposed
compounds. In theory, two geometrical isomers (E and Z) about the
imine (C]N) double bond are possible for the thiosemicarbazones.
However, analysis of the 1H NMR spectra of the target compound
indicated one predominant isomer; the E isomer by comparison with
known analogues [25]. Intramolecular H-bonding involving the
proton attached to N4 (in DMSO) with the imine N-atom leads to a
distinctive singlet around 10.2 ppm [25] and this is also seen here.
Once thiosemicarbazones were characterized, the respective 2-
(pyridin-2-yl)-1,3-thiazoles were characterized by usual spectros-
copy. As exemplified with the 1H NMR analysis of (2-(1-(pyridin-2-
yl)ethylene)hydrazinyl)-4-phenyl-1,3-thiazole (15), the singlet
Fig. 1. Structures of nifurtimox and benznidazole.
of the cysteine protease cruzain by thiosemicarbazones [16]. Our
efforts toward new antichagasic drug since 2010 have led us to a
variety of thiosemicarbazones and thiazolyl hydrazones as trypa-
nocidal agents [16,19e22]. The promising results achieved by
compounds bearing a thiazole ring motivated us to investigate the
trypanocidal activity of novel thiazolyl hydrazones derived from 2-
pyridyl thiosemicarbazone, with changes being made in the phenyl
ring attached in N2. In continuation of our search for bioactive
molecules, we envisaged that the derivatization of the thio-
semicarbazone group into thiazole moiety would generate novel
templates, which are likely to exhibit anti-T. cruzi activity. We also
investigate pyridines as trypanocidal agents because their wide
applicability in organic synthesis, low price and facility in synthesis.
Here, these compounds were tested in vitro against T. cruzi parasite
epimastigote and trypomastigote forms, and cruzain protease. In
this synthetic design of a structureeactivity relationship (SAR) li-
brary, attention was paid to further explore substituents around the
phenyl ring attached in thiazole ring (C9). This study generated
basic SARs about tripomastigote form and cruzain enzyme around
substituents in phenyl ring, and using the scaffold shown in
Scheme 1. Specifically, we report the preparation of twenty-four 2-
(pyridin-2-yl)-1,3-thiazoles (3e27) by ultrasound-assisted synthe-
sis. The synthesized compounds were characterized by IR, NMR and
mass spectral studies. The 2-(pyridin-2-yl)-1,3-thiazoles were
assayed for their in vitro anti-T. cruzi activity against the epi-
mastigote and trypomastigote forms of the parasite. Their cyto-
toxicity in mammalian cell cultures was also investigated. Further
investigations on the possible involvement of 2-(pyridin-2-yl)-1,3-
thiazoles with cruzain activity as potential therapeutic targets were
performed.
peak at
protons occurred as doublets or triplets. For the pyridyl ring, peaks
were observed at 7.76, 8.23, 8.35 and 8.73. For the aromatic ring
coupled to the thiazole ring, doublet and triplet peaks were found
at 7.30, 7.39 and 7.85. For the thiazole ring, a singlet at 7.44 was
found. In addition, the NH proton appeared as broad singlet at
d 2.43 corresponds to the methyl group. The aromatic
d
d
d
d
5.53. The 13C NMR spectrum of (15) the 13C]S resonance from the
parent thiosemicarbazone disappeared while a new 13CeH reso-
nance at ~106 ppm appeared, confirming cyclization in addition to
the resonance at ~169 ppm. Quaternary carbon peaks were
confirmed by DEPT experiments to appear at
d 134.2, 140.1, 150.0
and 168.9. Peaks of the pyridine aromatic carbons were found at
Scheme 1. Synthetic procedures for thiosemicarbazones (2aeb) and 2-(pyridin-2-yl)-1,3-thiazoles (3e26). Reagents and conditions: (a) thiosemicarbazide, 2-propanol, acetic acid
(3 drops), ultrasound irradiation, r.t., 120 min; (b) substituted 2-bromoacetophenones, 2-propanol, CaCO3, ultrasound irradiation, r.t., 60 min; *R: H for all compounds, except to (6)
and (18) where R: Me.

50
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
Table 1
d
123.6, 125.6, 143.9, 147.5, and 150.0. Resonances from the phenyl
Crystal data and structure refinement for compound (4).
ring coupled to the thiazole ring were observed at 126.0, 128.4,
d
129.2 and 134.2. A combination of elemental analysis (C, H, N), UV,
IR and HRMS confirmed the purity and identity of all the
compounds.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C16 H14 N4 S
294.37
296(2)
0.71073
Orthorhombic
Pna21
2.2. X-ray crystallography
9.858(1)
25.602(3)
5.5127(6)
1391.3(3)
The X-ray crystal structure of 2-(2-(pyridin-2-ylmethylene)
hydrazinyl)-4-(4-tolyl)-1,3-thiazole (4) is shown in Fig. 2. Crystal
and refinement data are summarized in Table 1. The dihedral angles
C(11)eC(10)eC(9)eN(4), C(9)eN(4)eC(7)eN(3), C(7)eN(3)eN(2)e
b (Å)
c (Å)
Volume (Å3)
Z
4
rcalc (g cmꢀ3
)
1.405
0.231
616
C(6),
N(2)eC(6)eC(5)eC(4)
and
C(16)eC(13)eC(12)eC(11)
m
(mmꢀ1
F(000)
Crystal size (mm)
range (ꢁ)
)
of ꢀ177.84ꢁ, ꢀ179.48ꢁ, ꢀ176.60ꢁ, ꢀ176.54ꢁ and 178.43ꢁ respectively
indicate that the molecule is close to planar. In the thiazole ring,
two significantly different CeN bonds are found viz. C9eN4
(1.373(4) Å) and C7]N4 (1.265(5) Å) and C8]C9 is clearly a double
bond (1.348(5) Å). It is apparent that compound (4) crystallises in
the Z-isomeric form where intramolecular H-bonding with the
pyridyl ring and the proton attached to N2 is seen (see Fig. 2). In this
case, the E-isomer cannot be stabilised by H-bonding due to the loss
of the proton from N4 of the parent thiosemicarbazone.
0.6 ꢂ 0.1 ꢂ 0.1
q
3.2e25.0
Reflections collected
Independent reflections
Observed reflections [I > 2
Rint
5864
2635
1489
0.0590
s
(I)]
Refinement method
Full-matrix least-squares on F2
Data/restraints/parameters
2635/1/191
0.997
0.0580
Goodness-of-fit on F2
R [I > 2
s
(I)]
wR2 (all data)
0.0871
Absolute structure parameter
ꢀ0.03(11)
0.183 and ꢀ0.144
980106
2.3. Structureeactivity relationships (SAR)
Residual extrema (e Åꢀ3
)
CCDC number
To investigate the importance of substituents on the phenyl ring
for the antiparasitic activity against trypomastigote form, com-
pound (3) (without substituents) was compared with seven para
substituted analogues. It was found that compounds (5), (6), (8) and
(9) showed better anti-T. cruzi activity than (3), while compounds
(4), (7) and (13) were less potent (see Table 2). For the chloro
substituted compounds (9e11), it was observed that the dichloro
compounds (10 and 11) were equally trypanocidal, being 3-fold
more potent than 2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(4-
chlorophenyl)-1,3-thiazole (9). The monobromo compounds (5)
and (6) displayed activity intermediate of the mono and dichloro
compounds. The presence of a methyl group attached to C8 (for
compound 5) did not significantly affect biological activity.
Comparing compound (14) with (3) (naphthyl and aryl,
respectively), it was observed that the influence of a more bulky
substituent increased the trypanocidal activity, probably because of
an increase in lipophilicity. For the nitro compounds (12) and (13),
the position of the substituent affected trypanocidal activity. For
compound (12), (meta substituted), a 2-fold increase in activity was
found relative to (13) (para substituted).
methyl in C6 increases the trypanocidal activity. Compounds
(15)e(26) stand out, show high trypanocidal activity (trypomasti-
gote form); with exception of (25), all compounds are more potent
than reference drug benznidazole.
Table 2
Anti T. cruzi activities of acetylpyridin-2-yl thiosemicarbazone (2a) and 2-(pyridin-
2-yl)-1,3-thiazoles (3e14).
Compd.
Ar
Y strain T. cruzi, IC50
(
m
M)
HepG2, IC50
M)c
Trypomastigotesa
Epimastigotesb
(m
Compounds (15) to (26) (derived from 2-acetyl pyridine)
differed from compounds (3) to (14) (derived from 2-pyridine
carbaldehyde) by a methyl group present in C6 (Scheme 1). Anal-
ysis of the activity of these compounds suggests a trend in which a
2a
3
4
5
6
7
8
9
10
11
12
13
14
Bdz
e
17.3
5.4
16.8
2.2
2.6
72.7
4.8
3.8
1.2
1.2
119.5
8.3
3.8
2.2
18.3
3.5
5.6
3.8
4.8
4
ND
Ph
100
100
100
100
4-CH3Ph
4-BrPh
4-BrPhd
4-FPh
4-CH3OPh
4-ClPh
2,4-diClPh
3,4-diClPh
3-NO2Ph
4-NO2Ph
2-Naphthyl
e
18.20
100
100
100
100
100
100
100
ND
74.2
152.9
2.1
2.5
4.4
8.3
6.6
6.2
a
Determined 24 h after incubation of trypomastigotes with the compounds.
Determined 11 days after incubation of epimastigotes with the compounds. IC50
b
was calculated from at least five concentrations, in triplicate (SD < 10%).
c
IC50 determined for human Hepg2 after 48 h of incubation in the presence of the
Fig. 2. ORTEP view of compound (4) showing intramolecular H-bonding (30% proba-
bility ellipsoids are shown).
compounds.
d
R: methyl, see Scheme 1. Bdz: benznidazole. ND: not determined.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
51
Table 3
Comparing compounds with nitro substituents across the
(3e14) and (15e26) series, the biggest difference was observed for
Anti T. cruzi Activities of acetylpyridin-2-yl thiosemicarbazone (2b) and 2-(pyridin-
2-yl)-1,3-thiazoles (15e26).
compounds (13/25) (meta substituted), with 3.9
mM
against
Compd. Ar
Y strain T. cruzi, IC50
(
m
M)
HepG2, IC50 (m
M)c
74.2
m
M and (12/25), para substituted, with 36.7
mM against
Trypomastigotesa Epimastigotesb
152.9
mM.
Among all of the compounds in this work, the most potent were
(10), (11) and (15). It should be noted that among twenty-four
compounds designed for anti-Chagas activity, twenty compounds
(3, 5, 6, 8e11, 14, 15e24 and 26) displayed better trypanocidal ac-
tivity than benznidazole.
Analysing the antiparasitic activity against epimastigote form,
benznidazole, which is the reference drug, exhibited an IC50 of
6.6 mM. Once again, the majority of derivatives were very active. In
this case, only compounds (6), (20) and (22) were several times less
potent than benznidazole. In contrast, compounds (5), (12),
(15)e(19) and (21) displayed
benznidazole-treated parasites.
a better activity profile than
2b
15
16
17
18
19
20
21
22
23
24
25
26
Bdz
e
2.8
1.1
1.7
2.1
2.3
3.8
2.1
2.1
1.9
3.1
3.9
36.7
2.0
6.2
4.5
2.7
2.5
2.1
2.3
2.8
14.7
2.5
14.9
5.0
2.4
5.2
4.1
6.6
ND
Ph
91.20
100
100
85.11
91.20
100
100
100
100
100
100
100
ND
A recent work describing 2-imino-1,3-thiazoles [16] (derivatives
26e46), that share an aryloxypropylimine instead of a pyridine
ring, displayed lower trypanocidal effects when compared to these
that possess a 2-pyridyl moiety, pointing out the importance of the
2-pyridyl moiety for the trypanocidal activity. Fig. 3 summarizes
the main SAR for anti T. cruzi activity (tripomastigote form).
4-CH3Ph
4-BrPh
4-BrPhd
4-FPh
4-CH3OPh
4-ClPh
2,4-diClPh
3,4-diClPh
3-NO2Ph
4-NO2Ph
2-Naphthyl
e
2.4. Cruzain inhibition activity
Table 4 shows the inhibition of the enzyme cruzain of T. cruzi
(TCC). The experimental protocol described by Ferreira [26] was
utilized in this assay. All compounds were tested at a concentration
a
Determined 24 h after incubation of trypomastigotes with the compounds.
Determined 11 days after incubation of epimastigotes with the compounds. IC50
b
was calculated from at least five concentrations, in triplicate (SD < 10%).
c
of 100
mM, except for cases limited by solubility, in which the
IC50 determined for human Hepg2 after 48 h of incubation in the presence of the
compounds.
screening was performed at either 50 or 75
Table 4). Compounds which inhibited enzyme activity by at least
70% in the screening had their IC50 determined.
m
M (as noted on
d
R: methyl, see Scheme 1. Bdz: benznidazole. Percentage of inhibition is the
average of triplicate runs determined in one experiment. ND: not determined.
Some works describe that 2-imino-1,3-thiazoles are trypanoci-
dal agents by altering the ergosterol biosynthesis instead of
inhibiting the catalytic activity of cruzain [16,19]. In this work we
show that cruzain is potently inhibited by several compounds.
Among all compounds tested, the most actives were (20) and
The para substituted 2-(pyridin-2-yl)-1,3-thiazoles (16e21 and
25) were less active than unsubstituted (2-(1-(pyridin-2-yl)
ethylene)hydrazinyl)-4-phenyl-1,3-thiazole (15), which was the
most potent compound tested in this work. No correlation was
observed between the electron withdrawing properties of the
substituent and trypanocidal activity.
For the isomeric dichloro compounds (22) and (23), we
observed little change in trypanocidal activity. For the nitro com-
pounds (24 and 25), the compound meta substituted (24) was 9-
fold more potent than para substituted (25), (3.9 mM vs 36.7 mM
respectively).
From Table 3, it can be seen that (2-(pyridin-2-yl)ethylene)-1,3-
thiazoles (15e26) (with a methyl attached to C6), had increased the
trypanocidal activity for both trypomastigote and epimastigote in
comparison with (2-(pyridin-2-yl)methylene)-1,3-thiazoles (3e14).
(26), showing the lowest IC50 values (0.04 0.03 mM and 0.01 mM,
respectively). Due to the low potency of some compounds and the
absence of IC50 values, in some cases we made a discussion based in
percentual inhibition of cruzain.
Comparing para substituted compounds; the most active were
(20), (16), (19) and (5), showing percentage of inhibition near 90%.
For the dichloro compounds (10e11), 2-(2-(pyridin-2-ylmethylene)
hydrazinyl)-4-(2,4-dichlorophenyl)-1,3-thiazole (10) did not show
any inhibition of cruzain, in contrast to 2-(2-(pyridin-2-
ylmethylene)hydrazinyl)-4-(3,4-dichlorophenyl)-1,3-thiazole (11),
which displayed weak inhibition. For the nitro compounds (12e13),
Fig. 3. Summary of SAR of trypanocidal activity for compounds (3e26).

52
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
the positional change of the nitro group from para to meta had a
huge impact on potency against cruzain: while the 2-(2-(pyridin-2-
analysis by comparison of compounds (3e14) to compounds
(15e26), which show an analogous difference, we observe that in
seven cases the addition of the methyl considerably improved
cruzain inhibition, while only for two compound pairs (5 vs 17 and
12 vs 24) the opposite effect was observed. Together, these results
indicate the high importance of the methyl group at C6.
ylmethylene)hydrazinyl)-4-(3-nitrophenyl)-1,3-thiazole
shows IC50 ¼ 1.3 0.7 M, 2-(2-(pyridin-2-ylmethylene)hydra-
zinyl)-4-(4-nitrophenyl)-1,3-thiazole (13) does not considerably
inhibit the enzyme at 100 M.
(12)
m
m
To investigate the relative activity of thiosemicarbazone vs
thiazole in cruzain inhibition, compounds (2a) and (3) were
compared. Neither of these compounds inhibits the enzyme at the
concentrations evaluated, but (15), an analogue of (3) differing only
by the addition of a methyl group at C6 was shown to be a
Within the series (15e26), seven compounds had their IC50
values determined, and in five cases the potency was better than
0.5 mM. Among the para substituted compounds, (16) and (19) had
very similar potencies. On the other hand, modifying the methyl
substituent (16) to methoxy (20) increased the potency by an order
of magnitude.
moderately potent inhibitor IC50 ¼ 1.3 0.7
mM. Extrapolating the
For chlorine substituted compounds (21e23), the pattern of
substitution had
a big impact on potency. While the para
Table 4
Comparison of in vitro cruzain inhibition activity of compounds (2ae2b) and (3e26).
substituted compound was inactive, disubstituted compounds
were cruzain inhibitors, and the 3,4-diCl compound (23) was 12
fold more potent than the 2,4-diCl (22). Comparing the isomeric
nitro-compounds (meta (24) and para (25) substituted), no signif-
icant cruzain inhibition was observed.
Compd.
Ar
% Cruzain inhibition at 100
m
Ma
IC50 (m
M)d
2.5. Cytotoxicity activity
To determine the toxicity profile of the compounds, cytotoxicity
assay adapted from Ballell et al. [27] were performed with HepG2
cells, a human liver cell line commonly used for in vitro testing of
toxicity [28]. Cells were incubated with the compounds at con-
centration of 100 mM for 48 h. It is worth mentioning that a com-
2a
2b
1
5
2
5
ND
ND
pound that can kill parasites is necessary but not sufficient to
represent a good drug lead unless it also demonstrates low or non-
toxicity among other criteria. As can be observed in Tables 2 and 3,
only compound (7) (a fluorine derivative) exhibited toxicity for
HepG2 (18.20
mM). The most of compounds were less cytotoxic for
HepG2 at concentrations up to 85.11
mM, demonstrating the low
toxicity profile of the compounds assayed.
2.6. Docking studies
In order to understand a probable mechanism of action, we
investigated the interaction of these compounds with cruzain (PDB
ID: 3IUT) by docking studies. The binding mode for the ligands was
determined as the highest (most positive) score among the possible
solutions for each ligand, generated according to the CHEMPLP
Score Function [29]. Fig. 4 shows the superposition of the best
3
15
4
16
5
17
6
18
7
19
8
20
9
21
10
22
11
23
12
24
13
25
14
26
Ph
Ph
4-CH3Ph
4-CH3Ph
4-BrPh
9
93
23
90
86
ꢀ8
10
27
1b
3
2
4
6
2
4
3
ND
1.3 0.7
ND
0.4 0.2
9.5 7.5
ND
ND
ND
ND
0.3 0.2
ND
0.04 0.03
ND
ND
ND
4.8 3.8
ND
0.4 0.1
1.3 0.7
ND
ND
ND
4-BrPh
4-BrPhe
4-BrPhe
4-FPh
22 3c
89
20 1b
4-FPh
5
4-CH3OPh
4-CH3OPh
4-ClPh
95
2
ꢀ1 4b
4-ClPh
ꢀ3
1
2,4-diClPh
2,4-diClPh
3,4-diClPh
3,4-diClPh
3-NO2Ph
3-NO2Ph
4-NO2Ph
4-NO2Ph
2-Naphthyl
2-Naphthyl
0
3b
7
81
21
89
85
9
4
2
10 5b
12
3
11 5b
3
94
1
3
ND
0.01
a
Values correspond to the average of six measurements, from two independent
experiments, each one in triplicate. Errors are given by the ration between the
standard deviation and the square root of the number of measurements. Com-
pounds evaluated at 100 mM unless otherwise noted.
b
Compounds evaluated at 75
Compounds evaluated at 50
IC50 curves were determined based two independent experiments, each one
m
m
M.
M.
Fig. 4. Superposition of the docking solutions for cruzain-inhibiting compounds (5),
(12), (15), (16), (19), (20), (22), (23) and (26) (blue structures) bound to cruzain (green)
and the crystallographic structure of the “KB2” cocrystallized ligand [30] (red struc-
ture). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
c
d
involving at least seven compound concentrations in triplicates.
e
R: methyl, see Scheme 1. ND: not determined.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
53
is known to accommodate hydrophobic groups. Compared with the
bromophenyl group in (5). The hydrogen bond with the residue
ASP161 is slightly shorter for the molecule (26) (2.5 Å) than for
molecule (5) (2.8 Å). There is also a weak hydrogen bond with the
CYS25 residue (3.4 Å). These finding imbue greater stability to the
complex formed with (26) than (5), with docking scores of 66.09
and 55.29, respectively. These findings corroborate the in vitro
binding assay with the cruzain enzyme.
3. Conclusion
The current investigation has revealed novel structureeactivity
relationships regarding aromatic substitution at the phenyl moiety
of 2-(pyridin-2-yl)-1,3-thiazoles for the design of new drugs for the
treatment of Chagas disease. The novel analogues (3e26) exhibited
improve trypanocidal activity than thiosemicarbazones (2aeb),
while showing a similar potency to standard drug in use for
treatment of Chagas disease, Benznidazole.
Fig. 5. Trend observed between the in vitro (pIC50 for cruzain inhibition) and in silico
(docking score) results, for compounds (5), (12), (15), (16), (19), (20), (22), (23) and
(26).
Compounds (10) and (11) were the most potent thiazoles with
IC50 values of 1.2 mM for each compound. In addition, all of the
docking solutions for compounds that have IC50 values experi-
mentally determined for cruzain (5, 12, 15, 16, 19, 20, 22, 23, 26) and
the crystallographic structure of the “KB2”, a high-efficient cruzain
inhibitor cocrystallized ligand [30]. To compare in silico vs in vitro
cruzain data, IC50 values were first converted into pIC50 values
(equals e log10IC50 for cruzain inhibition, in units of moles per litre).
Fig. 5 shows the trend between the calculated docking scores
and the experimental pIC50 data, which indicates the compounds
with the highest pIC50 being most active. There is a clear correlation
between the docking score and pIC50 supporting the hypothesis
that the most tightly binding thiazoles are also the most potent
cruzain inhibitors (i.e., greater in vitro pIC50 values).
novel thiazoles showed similar or greater trypanocidal efficacy
than Benznidazole, with compounds (10) and (11) exhibiting
highest efficacies overall.
Structureeactivity analysis did not reveal a correlation between
ligands with electron-donating or electron-withdrawing sub-
stituents in terms of trypanocidal activity. However, the in silico
docking studies corroborate with in vitro cruzain inhibition,
showing that the molecules with more stable or positive docking
scores (i.e., greater in silico affinity for cruzain) are also the most
potent cruzain inhibitors (i.e., greater in vitro pIC50 values).
To identify the molecular reasons for the two extremes of po-
tency, the highest (26) (IC50
¼
0.01 nM) and lowest (5)
4. Experimental section
(IC50 ¼ 9.5 nM) in vitro results, we performed a detailed analysis of
the intermolecular interactions with the cruzain target. The prin-
ciple structural differences between these two molecules are as
follows: (i) the presence of a 4-bromophenyl ring linked to the
thiazole ring in molecule (5), instead of a naphthalene group in
molecule (26); (ii) a methyl group on C6 (adjacent to the pyridyl
ring) for molecule (26), rather than hydrogen for molecule (5).
The difference between the binding modes of these two mole-
cules is show in detail in Fig. 6 and Table 5. It appears that the large
hydrophobic naphthalene group in molecule (26) provides a
greater contact surface for interactions with hydrophobic residues
LEU67, MET68, ALA138 and LEU160 in the S2 cruzain subsite, which
4.1. Equipment and reagents
All reagents were used as purchased from commercial sources
(SigmaeAldrich, Acros Organics, Vetec or Fluka). Progress of the
reactions was followed by thin-layer chromatography (TLC) anal-
ysis (Merck, silica gel 60 F254 in aluminium foil). Purity of the target
compounds was confirmed by combustion analysis (for C, H, N, S)
performed by a Carlo-Erba instrument (model EA 1110). Melting
points were determined on a Fisatom 430D electrothermal capil-
lary melting point apparatus and were uncorrected. NMR spectra
were measured on either a Varian UnityPlus 400 MHz (400 MHz for
Fig. 6. Detailed view of the docking solutions for (A) compound (5) and (B) compound (26). Hydrophobic interactions (green) and hydrogen bonds (cyan). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

54
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
cmꢀ1. 1H NMR (300 MHz, DMSO-d6),
d
ppm: 7.35 (t, J ¼ 4.9 Hz, 1H,
Table 5
Docking scores and molecular interactions between cruzain and molecules (5) and
Ar), 7.81 (t, J ¼ 7.7 Hz, 1H, Ar), 8.09 (s, 1H, CH), 8.16 (broad s, 1H,
NH2), 8.26 (d, J ¼ 8.0 Hz, 1H, Ar), 8.35 (broad s, 1H, NH2), 8.55 (d,
J ¼ 4.9 Hz, 1H, Ar), 11.63 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6),
(26).a
Cruzain residues
Molecules
5
d
ppm: 120.2 (C, Ar), 124.0 (C, Ar), 136.5 (C, Ar), 142.5 (C]N), 149.2
26
(C, Ar), 153.3 (C, Ar), 178.3 (C]S, Ar). Anal. Calcd for C7H8N4S: C,
46.65; H, 4.47; N, 31.09; S,17.79; found: C, 46.58; H, 4.50; N, 31.12; S,
17.71. HR-MS (ESI): 181.6730 [MþH]þ.
CYS22
GLY23
CYS25
TRP26
SER64
LEU67
MET68
ALA138
LEU160
ASP161
HIS162
SCORES
e
HC
HC
3.4
HC
e
HC
e
HC
HC
HC
HC
HC
e
4.3.2. 2-[1-(2-Pyridinyl)ethylidene]hydrazinecarbothioamide (2b)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 86%, m.p. (ꢁC): 170. IR (KBr, cmꢀ1): 1553 (C]C), 1612
HC
HC
HC
HC
2.5
HC
66.09
(C]N), 3066 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.37 (s,
2.8
e
3H, CH3), 7.37 (t, J ¼ 6.1 Hz, 1H, Ar), 7.77 (t, J ¼ 7.8 Hz, 1H, Ar), 8.12
(broad s, 1H, NH2), 8.37 (broad s, 1H, NH2), 8.41 (d, J ¼ 8.1 Hz, 1H,
Ar), 8.56 (d, J ¼ 5.5 Hz,1H, Ar),10.30 (s,1H, NH). 13C NMR (75.5 MHz,
55.29
a
HC means “hydrophobic contacts” and the numbers are the hydrogen bond
DMSO-d6),
d ppm: 12.12 (CH3), 120.9 (C, Ar), 123.9 (C, Ar), 136.5 (C,
distances, in Ångstroms.
Ar), 148.1 (C]N), 154.6 (C, Ar), 179.1 (C]S); Anal. Calcd for
C8H10N4S: C, 49.46; H, 5.19; N, 28.84; S, 16.51; found: C, 49.42; H,
5.21; N, 28.79; S, 16.56. HR-MS (ESI): 195.0682 [MþH]þ.
1H and 100 MHz for 13C) or a Bruker AMX-300 MHz (300 MHz for
1H and 75.5 MHz for 13C) instruments. DMSO-d6 and D2O were
purchased from CIL or SigmaeAldrich. Chemical shifts are reported
in ppm and multiplicities are given as s (singlet), d (doublet), t
(triplet), m (multiplet), dd (double doublet), and coupling constants
(J) in hertz. Mass spectrometry experiments were performed on a
LC-IT-TOF (Shimadzu). Unless otherwise specified, ESI was carried
out in the positive ion mode. Typical conditions were: capillary
voltage of 3 kV and cone voltage of 30 V, and peak scan between 50
and 1000 m/z. IR spectra were recorded with a Brucker model IFS66
FT-IR spectrophotometer using KBr pellets.
4.4. General procedure for the synthesis of (3e26). Example for
compound (3)
2-(Pyridin-2-ylmethylene)hydrazinecarbothioamide (2a) (0.5 g,
2.78 mmol) was dissolved in 2-propanol (10 mL) and then the
appropriate 2-bromoacetophenone (see Scheme 1) (0.79 g,
3.05 mmol) and calcium carbonate (0.42 g, 3.05 mmol) were added
to a glass tube. The tube was placed in an ultrasonic bath (40 MHz,
180 V) and irradiated for 60 min, at r.t. Hexane was added and the
mixture was cooled in a freezer overnight. The precipitate was
filtered off and washed with hexane then dried in a desiccator
under vacuum. Additional amount of the desired compound were
obtained from the filtrate after cooling. Pure products were ob-
tained after recrystallization using the solvent system detailed
below for each compound.
4.2. Crystallography
Crystallographic data were collected on an Oxford Diffraction
Gemini S Ultra CCD diffractometer at 296 K using MoKa radiation
(2qmax ¼ 50ꢁ). Data reduction and empirical absorption corrections
were carried out with the CrysAlis Pro program (Oxford Diffraction
vs 171.33.42). The structure was solved by direct methods with
SHELXS86 and refined with SHELXL97 [31]. All non-H-atoms were
refined anisotropically and H-atoms were constrained at their
estimated positions using a riding model. The thermal ellipsoid
diagram was generated with ORTEP3 [32]. All crystallographic
calculations were carried out within the WinGX graphical user
interface [33]. Crystallographic data of compound (4) have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication CCDC 980106. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data
Centre ((Cambridge, UK) via www.ccdc.cam.ac.uk).
4.4.1. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-phenyl-1,3-
thiazole (3)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals. Yield: 86%, m.p. (ꢁC): 170. 1H NMR (300 MHz, DMSO-d6),
d
ppm: 4.47 (broad s, 1H, NH), 7.31 (t, J ¼ 7.2 Hz, 1H, Ar), 7.38e7.52
(m, 4H, 3H of Ar and 1H for CH of thiazole), 7.41 (t, J ¼ 7.3 Hz, 2H,
Ar), 7.49 (d, J ¼ 5.7 Hz, 2H, Ar), 7.86 (d, J ¼ 7.4 Hz, 1H, Ar), 7.97 (t,
J ¼ 9.2 Hz, 1H, Ar), 8.10 (s, 1H, CH), 8.62 (d, J ¼ 4.6 Hz, 1H, Ar). 13
C
NMR (100 MHz, DMSO-d6),
d ppm: 104.5 (SeCH), 120.0 (C, Ar),
124.0 (C, Ar), 125.5 (C, Ar), 127.6 (C, Ar), 128.7 (C, Ar), 139.2 (C, Ar),
147.7 (C, Ar), 150.6 (C, Ar), 151.8 (C]N), 167.5 (SeC]N). IR (KBr,
cmꢀ1): 1553 (C]C), 1612 (C]N), 3066 (NeH) cmꢀ1. Anal. Calcd for
4.3. General modified procedure for the synthesis of known
thiosemicarbazones (2aeb)
C15H12N4S: C, 64.26; H, 4.31; N, 19.98; S, 11.44; Found: C, 64.22; H,
4.38; N, 20.02; S, 11.42. HR-MS (ESI): 281.0842 [MþH]þ.
To a solution of 2-pyridine carbaldehyde (0.75 g, 7 mmol) (or
acetyl-2-pyridine (0.85 g, 7 mmol)) in 2-propanol (10 mL) was
added thiosemicarbazide (0.64 g, 7 mmol) and few drops of acetic
acid. The reaction vessel was placed in an ultrasonic bath (40 MHz,
180 V) and irradiated for 120 min, at r.t. The precipitate was filtered
off, washed with hexane then dried in desiccator under vacuum.
Additional amount of desired compound could be recovered from
the filtrate after cooling.
4.4.2. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-tolyl)-1,3-
thiazole (4)
Crystallization from toluene/hexane 7:3, afforded dark red
crystals, Yield: 79%, m.p. (ꢁC): 197. IR (KBr, cmꢀ1): 1554 (C]N), 1612
(C]N), 3154 (NeH) cmꢀ1 1H NMR (400 MHz, DMSO-d6),
. d ppm:
2.32 (s, 3H, CH3), 7.21 (d, J ¼ 7.9 Hz, 2H, Ar), 7.29 (s, 1H, CH of
thiazole), 7.36 (t, 1H, J ¼ 7.4 Hz, Ar), 7.50 (broad s, 1H, NH), 7.75 (d,
J ¼ 8.1 Hz, 2H, Ar), 7.84 (d, J ¼ 7.6 Hz, 1H, Ar), 7.86 (t, J ¼ 7.6 Hz, 1H,
Ar), 8.06 (s, 1H, CH), 8.57 (d, J ¼ 4.9 Hz, 1H, Ar). 13C NMR (75.5 MHz,
4.3.1. 2-(Pyridin-2-ylmethylene)hydrazinecarbothioamide (2a)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, yield: 91%. m.p. (ꢁC): 167. IR (KBr): (C]C), 1606 (C]N), (NeH)
DMSO-d6), d ppm: 20.8 (CH3), 104.0 (SeCH), 119.1 (C, Ar), 123.6 (C,
Ar), 125.5 (C, Ar), 125.7 (C, Ar), 129.2 (C, Ar), 131.9 (C, Ar), 136.7 (C,
Ar), 141.4 (C, Ar), 149.5 (Ar), 153.2 (C]N), 167.7 (SeC]N); Anal.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
55
Calcd for C16H14N4S: C, 65.28; H, 4.79; N, 19.03; S, 10.89; found: C,
4.4.7. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-
chlorophenyl)-1,3-thiazole (9)
65.26; H, 4.82; N, 19.09; S, 10.84. HR-MS (ESI): 295.0991 [MþH]þ.
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 74%, m.p. (ꢁC): 226. IR (KBr, cmꢀ1): 1552 (C]N), 1612
4.4.3. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-
bromophenyl)-1,3-thiazole (5)
(C]N), 3030 (NeH). 1H NMR (400 MHz, DMSO-d6),
d ppm: 7.48 (d,
J ¼ 8.3 Hz, 2H, Ar), 7.75 (t, J ¼ 6.1 Hz, 1H, Ar), 7.56 (s, 1H, CH of
thiazole), 7.88 (d, J ¼ 8.2 Hz, 2H, Ar), 8.16 (d, J ¼ 8.1 Hz, 1H, Ar), 8.20
(s, 1H, CH), 8.33 (t, J ¼ 7.6 Hz, 1H, Ar), 8.43 (broad s, 1H, NH), 8.75 (d,
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 74%, m.p. (ꢁC): 205. IR (KBr, cmꢀ1): 1569 (C]N), 1574
(C]N), 3151 (NeH). 1H NMR (400 MHz, DMSO-d6),
d
ppm: 3.42 (s,
J ¼ 4.6 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 105.1
1H, NH), 7.29 (s, 1H, CH of thiazole), 7.36 (t, J ¼ 6.6 Hz, 1H, Ar), 7.45
(s,1H, CH), 7.59 (d, J ¼ 8.6 Hz, 2H, Ar), 7.74 (d, J ¼ 8.1 Hz,1H, Ar), 7.80
(d, J ¼ 8.6 Hz, 2H, Ar), 7.86 (t, J ¼ 7.5 Hz, 1H, Ar), 8.06 (d, J ¼ 7.1 Hz,
(SeCH), 119.4 (C, Ar), 123.7 (C, Ar), 127.2 (C, Ar), 128.6 (C, Ar), 132.0
(C, Ar), 133.4 (C, Ar), 137.2 (C, Ar), 141.2 (C, Ar), 149.1 (C, Ar), 149.4 (C,
Ar), 152.8 (C]N), 167.8 (SeC]N). Anal. Calcd for C15H11ClN4S: C,
57.23; H, 3.52; N, 17.80; S, 10.19; found: C, 57.24; H, 3.51; N, 17.82; S,
10.18. HR-MS (ESI): 315.0441 [MþH]þ.
1H), 8.57 (d, J ¼ 4.9 Hz, 1H). 13C NMR (75.5 MHz, DMSO-d6),
d ppm:
105.6 (SeCH), 119.7 (C, Ar), 121.1 (C, Ar), 127.1 (C, Ar), 128.0 (C, Ar),
129.6 (C, Ar), 134.2 (C, Ar), 137.3 (C, Ar), 137.5 (C, Ar), 141.9 (C, Ar),
149.7 (C, Ar), 153.4 (C]N), 168.3 (SeC]N). Anal. Calcd for
4.4.8. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(2,4-
dichlorophenyl)-1,3-thiazole (10)
C
15H11BrN4S: C, 50.15; H, 3.09; N, 15.60; S, 8.93; found: C, 50.11; H,
3.06; N, 15.65; S, 8.97. HRMS (ESI): 358.9608 [MþH]þ.
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 68%, m.p. (ꢁC): 211. IR (KBr, cmꢀ1): 1551 (C]N), 1615
(C]N), 3058 (NeH). 1H NMR (300 MHz, DMSO-d6),
d
ppm:
7.52e7.91 (m, 5H, Ar), 8.15e8.82 (m, 4H, Ar), 12.18 (s, 1H, NH). 13
NMR (75.5 MHz, DMSO-d6), ppm: 111.3 (SeCH),121.9 (C, Ar),125.3
4.4.4. 5-Methyl-2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(4-
bromophenyl)-1,3-thiazole (6)
C
d
Crystallization from toluene/hexane 7:3, afforded yellow crys-
(C, Ar),126.3 (C, Ar),128.1 (C, Ar),130.2 (C, Ar),132.1 (C, Ar),132.7 (C,
Ar), 135.8 (C, Ar), 142.6 (C, Ar), 144.2 (C, Ar), 146.5 (C, Ar), 149.8 (C]
N), 166.9 (SeC]N). Anal. Calcd for C15H10Cl2N4S: C, 51.59; H, 2.89;
N, 16.04; S, 9.18; found: C, 51.57; H, 2.86; N, 16.03; S, 9.15. HR-MS
(ESI): 349.0144 [MþH]þ.
tals, Yield: 58%, m.p. (ꢁC): 202. IR (KBr, cmꢀ1): 1554 (C]N), 1609
(C]N), 3152 (NeH). 1H NMR (300 MHz, DMSO-d6),
d
ppm: 2.43 (s,
3H, CH3), 7.91 (s, 1H, CH), 7.41e7.60 (m, 4H, Ar), 8.03e8.58 (m, 4H,
Ar), 13.05 (broad s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6),
ppm:
d
12.3 (CH3), 118.7 (SeCH), 119.6 (C, Ar), 120.3 (C, Ar), 123.8 (C, Ar),
129.8 (C, Ar), 131.3 (C, Ar), 134.1 (C, Ar), 138.0 (C, Ar), 139.6 (C, Ar),
148.4 (C]N), 167.0 (SeC]N). Anal. Calcd for C16H13BrN4S: C, 51.48;
H, 3.51; N, 15.01; S, 8.59; found: C, 51.53; H, 3.54; N, 15.04; S, 8.62.
HRMS (ESI): 374.9846 [MþH]þ.
4.4.9. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(3,4-
dichlorophenyl)-1,3-thiazole (11)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 59%, m.p. (ꢁC): 217. IR (KBr, cmꢀ1): 1563 (C]N), 1614
(C]N), 3154 (NeH). 1H NMR (400 MHz, DMSO-d6),
d
ppm: 5.22
(broad s, 1H, NH), 7.54e7.88 (m, 5H, Ar), 8.15e8.71 (m, 4H, Ar). 13
NMR (100 MHz, DMSO-d6), ppm: 110.9 (SeCH), 121.6 (C, Ar), 124.9
C
4.4.5. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-
fluorophenyl)-1,3-thiazole (7)
d
(C, Ar), 127.5 (C, Ar), 129.7 (C, Ar), 131.7 (CeCl), 132.2 (C, Ar), 132.7
(CeCl), 134.6 (C, Ar), 142.7 (C, Ar), 143.9 (C, Ar), 146.0 (C, Ar), 149.0
(C]N), 166.4 (SeC]N). Anal. Calcd for C15H10Cl2N4S: C, 51.59; H,
2.89; N, 16.04; S, 9.18; found: C, 51.62; H, 2.85; N, 16.07; S, 9.20.
HRMS (ESI): 349.0068 [MþH]þ.
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 78%, m.p. (ꢁC): 228. IR (KBr, cmꢀ1): 1552 (C]N), 1613
(C]N), 3029 (NeH). 1H NMR (400 MHz, DMSO-d6),
d ppm: 4.92
(broad s, 1H, NH), 7.25 (t, J ¼ 8.8 Hz, 2H, Ar), 7.45 (s, 1H, CH of
thiazole), 7.65 (t, J ¼ 6.3 Hz, 1H, Ar), 7.90 (dd, J ¼ 5.6, 8.8 Hz, 2H, Ar),
8.08 (d, J ¼ 8.0 Hz, 1H, Ar), 8.15 (s, 1H, CH), 8.21 (t, J ¼ 7.5 Hz, 1H, Ar),
4.4.10. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(3-
nitrophenyl)-1,3-thiazole (12)
8.70 (d, J ¼ 5.0 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d
ppm:
105.4 (SeCH), 115.4 (C, Ar), 115.6 (C, Ar), 122.3 (C, Ar), 125.1 (C, Ar),
127.5 (C, Ar), 127.6 (C, Ar), 130.8 (C, Ar), 133.5 (C, Ar), 143.4 (C, Ar),
143.8 (C]N), 148.3 (C, Ar), 149.6 (C, Ar), 160.5 (CeF), 162.9 (CeF),
167.1 (SeC]N). Anal. Calcd for C15H11FN4S: C, 60.39; H, 3.72; N,
18.78; S, 10.75; found: C, 60.37; H, 3.74; N, 18.81; S, 10.77. HRMS
(ESI): 299.0741 [MþH]þ.
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 31%, m.p. (ꢁC): 226. IR (KBr, cmꢀ1): 1355 (NO2),1560 (C]
N), 1596 (C]N), 3413 (NeH). 4.45 (broad s, 1H, NH), 7.67 (t,
J ¼ 6.5 Hz, 1H, Ar), 7.72 (t, J ¼ 8.1 Hz, 1H, Ar), 7.81 (s, 1H, CH of
thiazole), 8.12 (d, J ¼ 8.0 Hz, 1H, Ar), 8.16 (s, 1H, CH), 8.26 (t,
J ¼ 7.9 Hz, 1H, Ar), 8.31 (d, J ¼ 7.9 Hz, 1H, Ar), 8.67 (s, 1H, Ar), 8.72 (d,
J ¼ 4.8 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 108.1
(SeCH), 119.9 (C, Ar), 121.9 (C, Ar), 125.0 (C, Ar), 130.3 (C, Ar), 131.6
(C, Ar), 135.0 (C, Ar), 135.7 (C, Ar), 142.8 (C, Ar), 144.4 (C, Ar), 148.2
(C, Ar), 148.8 (CeNO2), 167.4 (C]N). Anal. Calcd for C15H11N5O2S: C,
55.38; H, 3.41; N, 21.53; S, 9.86; found: C, 55.41; H, 3.39; N, 21.56; S,
9.88. HRMS (ESI): 326.0680 [MþH]þ.
4.4.6. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-
methoxyphenyl)-1,3-thiazole (8)
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 61%, m.p. (ꢁC): 187. IR (KBr, cmꢀ1): 1587 (C]N), 1610
(C]N), 3069 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 3.79 (s,
3H, CH3), 6.97 (d, J ¼ 8.3 Hz, 2H, Ar), 7.29 (s, 1H, CH of thiazole), 7.73
(t, J ¼ 6.2 Hz, 1H, Ar), 7.78 (d, J ¼ 8.3 Hz, 2H, Ar), 8.14 (d, J ¼ 8.3 Hz,
1H, Ar), 8.17 (s, 1H, CH), 8.32 (t, J ¼ 7.8 Hz, 1H, Ar), 8.72 (d, J ¼ 4.8 Hz,
1H, Ar), 12.15 (broad s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6),
4.4.11. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(4-nitrophenyl)-
1,3-thiazole (13)
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 42%, m.p. (ꢁC): 220. IR (KBr, cmꢀ1): 1338 (NO2),1539 (C]
d
ppm: 55.2 (CH3), 103.3 (SeCH), 114.1 (C, Ar), 122.1 (C, Ar), 124.9 (C,
C), 1598 (C]N), 3148 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm:
Ar), 126.9 (C, Ar), 134.3 (C, Ar), 143.2 (C, Ar), 144.1 (C, Ar), 148.8 (C,
Ar), 150.4 (C]N), 167.1 (SeC]N). Anal. Calcd for C16H14N4OS: C,
61.92; H, 4.55; N,18.05; S,10.33; found: C, 61.90; H, 4.57; N,18.08; S,
10.34. HRMS (ESI): 311.0928 [MþH]þ.
4.74 (broad s, 1H, NH), 7.21 (t, J ¼ 6.1 Hz, 1H, Ar), 7.50 (s, 1H, CH of
thiazole), 7.74 (t, J ¼ 7.9 Hz, 1H, Ar), 7.83 (d, J ¼ 8.0 Hz, 2H, Ar), 7.98
(s, 1H, CH), 8.07 (d, J ¼ 9.0 Hz, 2H, Ar), 8.22 (d, J ¼ 9.1 Hz, 2H, Ar),
8.47 (d, J ¼ 4.9 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm:

56
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
110.0 (SeCH), 121.6 (C, Ar), 124.1 (C, Ar), 124.8 (C, Ar), 125.7 (C, Ar),
126.4 (C, Ar), 135.9 (C, Ar), 140.2 (CH2), 142.1 (C, Ar), 145.0 (C, Ar),
146.3 (C, Ar), 148.6 (C, Ar),149.3 (CeNO2), 167.6 (C]N) 168.9 (SeC]
N). Anal. Calcd for C15H11N5O2S: C, 55.38; H, 3.41; N, 21.53; S, 9.86;
found: C, 55.41; H, 3.46; N, 21.56; S, 9.88. HRMS (ESI): 326.0696
[MþH]þ.
N), 170.0 (SeC]N). Anal. Calcd for C16H13BrN4S: C, 51.48; H, 3.51; N,
15.01; S, 8.59; found: C, 51.51; H, 3.54; N, 15.04; S, 8.56. HRMS (ESI):
373.8177 [MþH]þ.
4.4.16. 5-Methyl-(2-(1-(pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
bromophenyl)-1,3-thiazole (18)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
4.4.12. 2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-4-(naphthalen-1-
yl)-1,3-thiazole (14)
tals, Yield: 52%, m.p. (ꢁC): 198. IR (KBr, cmꢀ1): 1565 (C]N), 1604
(C]N), 3164 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.33 (s,
Crystallization from toluene/hexane 7:3, afforded brown crys-
3H, CH3), 2.40 (s, 3H, CH3), 7.28 (t, J ¼ 6.0 Hz, 1H, Ar), 7.60e7.57 (m,
4H, Ar), 7.77 (t, J ¼ 7.7 Hz, 1H, Ar), 7.99 (d, J ¼ 8.0 Hz, 1H, Ar), 8.52 (d,
J ¼ 4.0 Hz, 1H, Ar), 8.81 (broad s, 1H, NH). 13C NMR (75.5 MHz,
tals, Yield: 87%, m.p. (ꢁC): 216. IR (KBr, cmꢀ1): 1540 (C]N), 1602
(C]N), 3112 (NeH). 1H NMR (400 MHz, DMSO-d6),
d ppm: 7.31 (t,
J ¼ 6.0 Hz, 1H, Ar), 7.45 (s, 1H, CH of thiazole), 7.49 (d, J ¼ 7.6 Hz, 2H,
Ar), 7.82 (t, J ¼ 7.4 Hz, 1H, Ar), 7.94e7.85 (m, 5H, Ar), 8.00 (d,
J ¼ 8.6 Hz, 1H, Ar), 8.07 (s, 1H, CH), 8.37 (broad s, 1H, NH), 8.54 (d,
DMSO-d6), d ppm: 12.6 (CH3), 12.7 (CH3), 119.0 (SeCH), 119.9 (C, Ar),
120.6 (C, Ar), 123.7 (C, Ar), 130.3 (C, Ar), 131.7 (C, Ar), 134.8 (C, Ar),
136.9 (C, Ar), 155.4 (C, Ar), 149.0 (C]N), 166.0 (SeC]N). Anal. Calcd
for C17H15BrN4S: C, 52.72; H, 3.90; N, 14.47; S, 8.28; found: C, 52.75;
H, 3.94; N, 14.45; S, 8.26. HRMS (ESI): 387.8449 [MþH]þ.
J ¼ 4.9 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 106.5
(SeCH), 122.5 (C, Ar), 123.8 (C, Ar), 124.2 (C, Ar), 125.2 (C, Ar), 126.1
(C, Ar),126.4 (C, Ar),127.5 (C, Ar), 128.1 (C, Ar), 128.2 (C, Ar), 131.5 (C,
Ar), 132.5 (C, Ar), 132.7 (C, Ar), 133.0 (C, Ar), 142.8 (CH2), 144.4 (C,
Ar), 147.7 (C, Ar), 150.5 (C]N), 167.0 (SeC]N). Anal. Calcd for
4.4.17. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
fluorophenyl)-1,3-thiazole (19)
C
19H14N4S: C, 69.07; H, 4.27; N, 16.96; S, 9.70; found: C, 69.09; H,
Crystallization from toluene/hexane 7:3, afforded yellow crys-
4.24; N, 16.94; S, 9.73. HRMS (ESI): 331.1007 [MþH]þ.
tals, Yield: 73%, m.p. (ꢁC): 209. IR (KBr, cmꢀ1): 1560 (C]N), 3060
(NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.42 (s, 3H, CH3), 6.07
4.4.13. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-phenyl-1,3-
thiazole (15)
(broad s, 1H, NH), 7.25 (d, J ¼ 8.6, 5.6 Hz, 2H, Ar), 7.40 (s, 1H, CH of
thiazole), 7.57 (t, J ¼ 5.0 Hz, 1H, Ar), 7.91 (dd, J ¼ 8.6, 5.6 Hz, 2H, Ar),
8.12 (d, J ¼ 8.0 Hz, 1H, Ar), 8.13 (t, J ¼ 7.2 Hz, 1H, Ar), 8.65 (d,
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 79%; m.p. (ꢁC): 151. IR (KBr, cmꢀ1): 1575 (C]N), 1615
J ¼ 5.0 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 12.4
(C]N), 3058 (NeH). 1H NMR (400 MHz, DMSO-d6),
d
ppm: 2.43 (s,
(CH3), 104.4 (SeCH), 115.4 (C, Ar), 120.2 (C, Ar), 123.7 (C, Ar), 127.5
(C, Ar), 131.2 (C, Ar), 137.8 (C, Ar), 146.0 (C, Ar), 147.7 (C, Ar), 154.0 (C,
Ar), 160.0 (C]N), 169.3 (SeC]N). Anal. Calcd for C16H13FN4S: C,
61.52; H, 4.19; N, 17.94; S, 10.27; found: C, 61.57; H, 4.21; N, 17.95; S,
10.30. HRMS (ESI): [MþH]þ.
3H, CH3), 5.53 (broad s, 1H, NH), 7.30 (t, J ¼ 7.4 Hz, 1H, Ar), 7.39 (t,
J ¼ 7.4 Hz, 2H, Ar), 7.44 (s, 1H, CH of thiazole), 7.76 (t, J ¼ 6.5 Hz, 1H,
Ar), 7.85 (d, J ¼ 7.6 Hz, 2H, Ar), 8.23 (d, J ¼ 8.3 Hz, 1H, Ar), 8.35 (t,
J ¼ 8.0 Hz, 1H, Ar), 8.73 (d, J ¼ 5.5 Hz, 1H, Ar). 13C NMR (75.5 MHz,
DMSO-d6),
d ppm: 13.4 (CH3), 105.9 (SeCH), 123.6 (C, Ar), 125.6 (C,
Ar), 126.0 (C, Ar), 128.4 (C, Ar), 129.2 (C, Ar), 134.2 (C, Ar), 141.0 (C,
Ar), 143.9 (C, Ar), 144.6 (C, Ar), 147.5 (C, Ar), 150.0 (C]N), 168.9
(SeC]N). Anal. Calcd for C16H14N4S: C, 65.28; H, 4.79; N, 19.03; S,
10.89; found: C, 65.31; H, 4.82; N, 19.06; S, 10.87. HRMS (ESI):
295.0985 [MþH]þ.
4.4.18. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
methoxyphenyl)-1,3-thiazole (20)
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 58%, m.p. (ꢁC): 216. IR (KBr, cmꢀ1): 1496 (C]C), 1610
(C]N), 3359 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.42 (s,
3H, CH3), 3.78 (s, 3H, CH3), 7.25 (s, 1H, SeCH), 6.98e7.93 (m, 4H, Ar),
4.4.14. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-tolyl)-1,3-
thiazole (16)
8.19e8.83 (m, 4H, Ar), 10.90 (broad s, 1H, NH). 13C NMR (75.5 MHz,
DMSO-d6), d ppm: 12.7 (CH3), 55.1 (CH3),102.7 (SeCH),114.0 (C, Ar),
Crystallization from toluene/hexane 7:3, afforded yellow crys-
121.7 (C, Ar), 124.4 (C, Ar), 124.5 (C, Ar), 125.9 (C, Ar), 126.9 (C, Ar),
143.6 (C, Ar), 145.4 (C, Ar), 158.9 (C]N), 169.5 (SeC]N). Anal. Calcd
for C17H16N4OS: C, 62.94; H, 4.97; N, 17.27; S, 9.88; found: C, 62.91;
H, 4.95; N, 17.28; S, 9.90. HR-MS (ESI): 325.0975 [MþH]þ.
tals, Yield: 68%, m.p. (ꢁC): 143. IR (KBr, cmꢀ1): 1496 (C]N), 1613
(C]N), 3058 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.31 (s,
3H, CH3), 2.39 (s, 3H, CH3), 3.84 (broad s, 1H, NH), 7.21 (d, J ¼ 8.0 Hz,
2H, Ar), 7.28 (s, 1H, CH of thiazole), 7.35 (t, J ¼ 6.1 Hz,1H, Ar), 7.76 (d,
J ¼ 8.1 Hz, 2H, Ar), 7.83 (t, J ¼ 7.8 Hz, 1H, Ar), 8.02 (d, J ¼ 8.1 Hz, 1H,
Ar), 8.57 (d, J ¼ 4.2 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 12.7
(CH3), 20.7 (CH3), 103.9 (SeCH), 121.7 (C, Ar), 124.4 (C, Ar), 125.8 (C,
Ar), 129.1 (C, Ar), 136.9 (C, Ar), 141.0 (C, Ar), 143.6 (C, Ar), 145.3 (C,
Ar), 148.3 (Ar), 151.7 (C]N), 168.9 (SeC]N). Anal. Calcd for
4.4.19. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
chlorophenyl)-1,3-thiazole (21)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 73%, m.p. (ꢁC): 222. IR (KBr, cmꢀ1): 1574 (C]N), 1603
(C]N), 3359 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.39 (s,
C
17H16N4S: C, 66.21; H, 5.23; N, 18.17; S, 10.40; found: C, 66.26; H,
3H, CH3), 7.35 (t, J ¼ 5.9 Hz, 1H, Ar), 7.42 (s, 1H, CH of thiazole), 7.46
(d, J ¼ 8.4 Hz, 2H, Ar), 7.83 (t, J ¼ 7.8 Hz, 1H, Ar), 7.89 (d, J ¼ 8.4 Hz,
2H, Ar), 8.02 (d, J ¼ 8.0 Hz, 1H, Ar), 8.56 (d, J ¼ 4.2 Hz, 1H, Ar), 11.45
5.26; N, 18.18; S, 10.39. HR-MS (ESI): 309.7527 [MþH]þ.
4.4.15. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
bromophenyl)-1,3-thiazole (17)
(broad s,1H, NH). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 13.4 (CH3),
106.5 (SeCH), 122.6 (C, Ar), 125.3 (C, Ar), 127.7 (C, Ar), 129.2 (C, Ar),
132.6 (C, Ar), 133.6 (C, Ar), 143.0 (C, Ar), 145.5 (C, Ar), 151.7 (C]N),
169.4 (SeC]N). Anal. Calcd for C16H13ClN4S: C, 58.44; H, 3.98; N,
17.04; S, 9.75; found: C, 58.47; H, 3.95; N,17.09; S, 9.71. HR-MS (ESI):
329.8148 [MþH]þ.
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 61%, m.p. (ꢁC): 202. IR (KBr, cmꢀ1): 1567 (C]N), 1615
(C]N), 3029 (NeH). 1H NMR (400 MHz, DMSO-d6),
d ppm: 2.39 (s,
3H, CH3), 7.36 (t, J ¼ 6.3 Hz, 1H, Ar), 7.45 (s, 1H, CH of thiazole), 7.60
(d, J ¼ 8.6 Hz, 2H, Ar), 7.87e7.81 (m, 4H, Ar), 8.02 (d, J ¼ 8.1 Hz, 1H,
Ar), 8.57 (d, J ¼ 4.8 Hz, 1H, Ar), 10.33 (broad s, 1H, NH). 13C NMR
4.4.20. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(2,4-
dichlorophenyl)-1,3-thiazole (22)
(75.5 MHz, DMSO-d6),
d ppm: 12.7 (CH3), 105.8 (SeCH), 120.0 (C,
Ar), 121.0 (C, Ar), 123.9 (C, Ar), 127.4 (C, Ar), 128.0 (C, Ar), 132.0 (C,
Ar), 134.4 (C, Ar), 137.1 (C, Ar), 147.6 (C, Ar), 149.0 (C, Ar), 155.3 (C]
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 62%, m.p. (ꢁC): 227; IR (KBr, cmꢀ1): 1521 (C]N), 1609

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
57
(C]N), 3058 (NeH). 1H NMR (300 MHz, DMSO-d6),
d
ppm: 2.43 (s,
4.5. Cruzain inhibition
3H, CH3), 4.04 (broad s, 1H, NH), 7.43 (s, 1H, CH of thiazole),
7.32e7.88 (m, 3H, Ar), 8.19e8.69 (m, 4H, Ar). 13C NMR (75.5 MHz,
Recombinant cruzain was gently provided by Alison Doak and
Dr. Brian Shoichet, from the University of California San Francisco.
Cruzain activity was measured as previously described [26], by
monitoring the cleavage of the fluorogenic substrate Z-Phe-Arg-
aminomethylcoumarin (Z-FR-AMC) in a Synergy 2 fluorimeter
(Biotek), from the Center of Flow Cytometry and Fluorimetry at the
Biochemistry and Immunology Department (UFMG), using filters of
340 nm for excitation and 440 nm for emission. All assays were
performed in sodium acetate 0.1 M pH 5.5 and in the presence of
5 mM dithiothreitol (DTT) and 0.01% Triton X-100, in a final volume
DMSO-d6),
d ppm: 12.8 (CH3), 105.0 (SeCH), 124.4 (C, Ar), 125.5 (C,
Ar), 127.7 (C, Ar), 128.6 (C, Ar), 134.2 (C, Ar), 141.2 (C, Ar), 143.1 (C,
Ar), 145.4 (C, Ar), 151.7 (C]N), 169.0 (SeC]N). Anal. Calcd for
C
16H12Cl2N4S: C, 52.90; H, 3.33; N, 15.42; S, 8.83; found: C, 52.93; H,
3.35; N, 15.47; S, 8.84. HR-MS (ESI): 362.9432 [MþH]þ.
4.4.21. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(3,4-
dichlorophenyl)-1,3-thiazole (23)
Crystallization from toluene/hexane 7:3, afforded yellow crys-
tals, Yield: 69%, m.p. (ꢁC): 209. IR (KBr, cmꢀ1): 1554 (C]N), 1614
of 200
cruzain was 0.5 nM, and the substrate concentration was 2.5
(Km ¼ 1 M). In all assays, enzyme inhibition was measured after a
10-min pre-incubation of the compounds with enzyme. Com-
pounds were initially screened at 100 M, unless they were insol-
uble at this concentration, in which cases they were tested at 75
or 50 M. If cruzain inhibition higher than 70% was observed at the
mL and on a 96 well plate format. The final concentration of
(C]N), 3061 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 2.43 (s,
mM
m
3H, CH3), 4.12 (broad s, 1H, NH), 7.48 (s, 1H, CH of thiazole),
7.29e7.83 (m, 3H, Ar), 8.12e8.63 (m, 4H, Ar). 13C NMR (75.5 MHz,
m
DMSO-d6),
d ppm: 12.8 (CH3), 110.4 (SeCH), 121.3 (C, Ar), 124.3 (C,
mM
Ar), 127.4 (C, Ar), 128.6 (C, Ar), 145.4 (C, Ar), 145.8 (C, Ar), 146.3 (C,
Ar), 152.2 (C, Ar), 158.1 (C]N), 169.4 (SeC]N). Anal. Calcd for
m
original screening, IC50 was determined based on at least seven
inhibitor concentrations, always after pre-incubation with cruzain
for 10 min. All assays were performed in at least two independent
experiments, each one in triplicates, and were followed for 5 min.
Activity was calculated based on comparison to a DMSO control.
Data was analysed with Prism 5.0 (GraphPad).
C
16H12Cl2N4S: C, 52.90; H, 3.33; N, 15.42; S, 8.83; found: C, 52.89; H,
3.31; N, 15.43; S, 8.80. HR-MS (ESI): 363.0220 [MþH]þ.
4.4.22. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(3-
nitrophenyl)-1,3-thiazole (24)
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 49%, m.p. (ꢁC): 229. IR (KBr, cmꢀ1): 1345 (NO2),1453 (C]
4.6. Parasites
C), 1601 (C]N), 3054 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm:
2.43 (s, 3H, CH3), 4.11(bs, 1H, NH), 7.47 (s, 1H, SeCH), 7.38e7.91 (m,
Epimastigotes of T. cruzi (Y strain) were maintained at 26 ꢁC in
LIT medium (Liver Infusion Tryptose) supplemented with 10% foetal
bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), 1% hemin
(Sigma Co, St. Louis, MO, USA), 1% R9 medium (Sigma Co), and
ꢀ
mg/mL gentamycin (Novafarma, Anapolis, GO, Brazil). Blood-
4H, Ar), 8.15e8.71 (m, 4H, Ar). 13C NMR (100 MHz, DMSO-d6),
d
ppm: 12.8 (CH3), 107.8 (C, Ar), 119.9 (C, Ar), 121.7 (C, Ar), 122.1 (C,
Ar), 124.5 (C, Ar), 130.2 (C, Ar), 131.5 (C, Ar), 135.9 (C, Ar), 138.6 (C,
Ar), 141.1 (C, Ar), 145.5 (C, Ar), 147.5 (C]N), 148.2 (CeNO2), 169.0
(SeC]N). Anal. Calcd for C16H13N5O2S: C, 56.63; H, 3.86; N, 20.64;
S, 9.45; found: C, 56.65; H, 3.89; N, 20.67; S, 9.48. HR-MS (ESI):
340.0851 [MþH]þ.
50
stream trypomastigotes forms of T. cruzi were obtained from su-
pernatants of LLC-MK2 cells previously infected and maintained in
RPMI-1640 medium (Sigma Co) supplemented with 10% FBS, and
50 m
g/mL gentamycin at 37 ꢁC and 5% CO2.
4.4.23. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(4-
nitrophenyl)-1,3-thiazole (25)
4.7. Cytotoxicity for HepG2
Crystallization from toluene/hexane 7:3, afforded orange crys-
tals, Yield: 58%, m.p. (ꢁC): 245. IR (KBr, cmꢀ1): 1577 (C]N), 1596
The HepG2 cytotoxicity assay was adapted from Ballell et al.
[27]. Actively growing HepG2 cells were removed from a T-175 TC
flask using supplemented Eagle's MEM and plated at a density of
3000 cells/well into the 384-well clear-bottom plates using a
Multidrop instrument. Prior to addition of the cell suspension, the
screening compounds (250 nL) were dispensed into the plates with
an Echo 555 instrument. Plates were allowed to incubate at 37 ꢁC at
80% relative humidity for 48 h under 5% CO2. The signal developer,
CellTiter-Glo (Promega) was added to the plates using a Multidrop
(C]N), 3342 (NeH). 1H NMR (400 MHz, DMSO-d6),
d ppm: 2.37 (s,
3H, CH3), 7.30 (t, J ¼ 6.2 Hz, 1H, Ar), 7.63 (s, 1H, CH of thiazole), 7.74
(broad s, 1H, NH), 7.79 (t, J ¼ 7.4 Hz, 1H, Ar), 8.02 (d, J ¼ 8.1 Hz, 1H,
Ar), 8.10 (d, J ¼ 9.0 Hz, 2H, Ar), 8.25 (d, J ¼ 9.0 Hz, 2H, Ar), 8.53 (d,
J ¼ 4.8 Hz, 1H, Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 12.4
(CH3), 108.6 (SeCH), 119.4 (C, Ar), 122.9 (C, Ar), 124.2 (C, Ar), 126.3
(C, Ar), 136.4 (C, Ar), 141.2 (C, Ar), 146.1 (C, Ar), 148.6 (C, Ar), 155.5
(C]N), 172.0 (SeC]N). Anal. Calcd for C16H13N5O2S: C, 56.63; H,
3.86; N, 20.64; S, 9.45; found: C, 56.67; H, 3.87; N, 20.65; S, 9.48.
HR-MS (ESI): 340.0683 [MþH]þ.
and the plates were read using
(PerkinElmer).
a
ViewLux instrument
4.4.24. (2-(1-(Pyridin-2-yl)ethylene)hydrazinyl)-4-(naphthalen-1-
yl)-1,3-thiazole (26)
4.8. Antiproliferative activity for epimastigotes
Crystallization from toluene/hexane 7:3, afforded brown crys-
Epimastigotes were counted in a hemocytometer and then
dispensed into 96-well plates at a cell density of 106 cells/well. Test
inhibitors, dissolved in DMSO, were diluted into five different
tals, Yield: 89%, m.p. (ꢁC): 203. IR (KBr, cmꢀ1): 1556 (C]N), 1614
(C]N), 3035 (NeH). 1H NMR (300 MHz, DMSO-d6),
d ppm: 1.70 (s,
3H, CH3), 3.66 (broad s, 1H, NH), 7.48 (s, 1H, CH of thiazole),
concentrations (1.23, 3.70, 11.11, 33.33, and 100 mg/mL) and added
7.30e7.52 (m, 4H, Ar), 7.81e8.00 (m, 6H, Ar), 8.55 (d, J ¼ 4.9 Hz, 1H,
to the respective wells in triplicate. The plate was incubated for 11
days at 26 ꢁC, and aliquots of each well were collected and the
number of viable parasites were counted in a Neubauer chamber,
and compared to untreated parasite culture. IC50 values were
calculated using non-linear regression on Prism 4.0 GraphPad
software. This experiment was done in duplicate, and Benznidazole
(LAFEPE, Brazil) were used as the reference inhibitors.
Ar). 13C NMR (75.5 MHz, DMSO-d6),
d ppm: 24.0 (CH3), 104.5
(SeCH), 119.0 (C, Ar), 124.0 (C, Ar), 124.1 (C, Ar), 126.4 (C, Ar), 127.6
(C, Ar), 128.0 (C, Ar), 128.1 (C, Ar), 136.7 (C, Ar), 144.3 (C, Ar), 149.4
(C, Ar), 150.3 (C, Ar), 153.7 (C]N), 169.3 (SeC]N). Anal. Calcd for
C
20H16N4S: C, 69.74; H, 4.68; N, 16.27; S, 9.31; found: C, 69.70; H,
4.70; N, 16.31; S, 9.34. HR-MS (ESI): 345.1143 [MþH]þ.

58
M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
[7] P. Veiga-Santos, E.S. Barrias, J.F.C. Santos, T.L. de Barros Moreira, T.M.U. de
4.9. Toxicity for Y strain trypomastigotes
Carvalho, J.A. Urbina, et al., Effects of amiodarone and posaconazole on the
growth and ultrastructure of Trypanosoma cruzi, Int. J. Antimicrob. Agents 40
(2012) 61e71.
Trypomastigotes collected from the supernatant of LLC-MK2
cells were dispensed into 96-well plates at a cell density of
4 ꢂ 105 cells/well. Test inhibitors, dissolved in DMSO, were diluted
into five different concentrations and added into their respective
wells, and the plate was incubated for 24 h at 37 ꢁC and 5% of CO2.
Aliquots of each well were collected and the number of viable
parasites, based on parasite motility, was assessed in a Neubauer
chamber. The percentage of inhibition was calculated in relation to
untreated cultures. IC50 calculation was also carried out using non-
linear regression with Prism 4.0 GraphPad software. Benznidazole
was used as the reference drug.
ꢀ
[8] J.A. Perez-Molina, J. Sojo-Dorado, F. Norman, B. Monge-Maillo, M. Díaz-
ꢀ
~
Menendez, P. Albajar-Vinas, et al., Nifurtimox therapy for Chagas disease
does not cause hypersensitivity reactions in patients with such previous
adverse reactions during benznidazole treatment, Acta Trop. 127 (2013)
101e104.
[9] J. McKerrow, Cysteine protease inhibitors as chemotherapy for parasitic in-
fections, Bioorg. Med. Chem. 7 (1999) 639e644.
[10] A.E. Eakin, M.E. McGrath, J.H. McKerrow, R.J. Fletterick, C.S. Craik, Production
of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi,
J. Biol. Chem. 268 (1993) 6115e6118.
[11] J.H. McKerrow, M.E. McGrath, J.C. Engel, The cysteine protease of Trypanosoma
cruzi as a model for antiparasite drug design, Parasitol. Today 11 (1995)
279e282.
[12] M.E. McGrath, A.E. Eakin, J.C. Engel, J.H. McKerrow, C.S. Craik, R.J. Fletterick,
The crystal structure of cruzain: a therapeutic target for Chagas' disease,
J. Mol. Biol. 247 (1995) 251e259.
4.10. Docking studies
[13] G. Harth, N. Andrews, A.A. Mills, J.C. Engel, R. Smith, J.H. McKerrow, Peptide-
fluoromethyl ketones arrest intracellular replication and intercellular trans-
mission of Trypanosoma cruzi, Mol. Biochem. Parasitol. 58 (1993) 17e24.
[14] S.S. Gawande, S.C. Warangkar, B.P. Bandgar, C.N. Khobragade, Synthesis of
new heterocyclic hybrids based on pyrazole and thiazolidinone scaffolds
as potent inhibitors of tyrosinase, Bioorg. Med. Chem. 21 (2013)
2772e2777.
[15] G.N. Masoud, A.M. Youssef, M.M. Abdel Khalek, A.E. Abdel Wahab,
I.M. Labouta, A.A.B. Hazzaa, Design, synthesis, and biological evaluation
of new 4-thiazolidinone derivatives substituted with benzimidazole ring
as potential chemotherapeutic agents, Med. Chem. Res. 22 (2012)
707e725.
[16] D.R.M. Moreira, S.P.M. Costa, M.Z. Hernandes, M.M. Rabello, G.B. de Oliveira
Filho, C.M.L. de Melo, et al., Structural investigation of anti-Trypanosoma cruzi
2-iminothiazolidin-4-ones allows the identification of agents with efficacy in
infected mice, J. Med. Chem. 55 (2012) 10918e10936.
[17] C. Pizzo, C. Saiz, A. Talevi, L. Gavernet, P. Palestro, C. Bellera, et al., Synthesis of
2-hydrazolyl-4-thiazolidinones based on multicomponent reactions and bio-
logical evaluation against Trypanosoma Cruzi, Chem. Biol. Drug Des. 77 (2011)
166e172.
The structures of all compounds were obtained by application of
the RM1 [34] method, available as part of the SPARTAN 08' program
[35], using internal default settings for convergence criteria.
Docking calculations and analysis was carried using the T. cruzi
cruzain (PDB ID code: 3IUT) as the target [30], in which there was a
cocrystallized complex with an inhibitor (referred to as “KB2”). The
active site was defined as all atoms within a radius of 6.0 Å from
this cocrystallized ligand. The residues GLN19, CYS25, SER61,
LEU67, MET68, ASN70, ASP161, HIS162, TRP184 and GLU208 were
treated as flexible. The GOLD 5.1 program [36] was used for docking
calculations, followed by Binana program [37], which was used to
analyse the molecular interactions present in the best docking so-
lutions, using default setting, except for hydrogen bond distance
(changed to a maximum of 3.5 Å). Figures were generated with
Pymol [38].
[18] D.C. Greenbaum, Z. Mackey, E. Hansell, P. Doyle, J. Gut, C.R. Caffrey, et al.,
Synthesis and structure-activity relationships of parasiticidal thio-
semicarbazone cysteine protease inhibitors against Plasmodium falciparum,
Trypanosoma brucei, and Trypanosoma cruzi, J. Med. Chem. 47 (2004)
3212e3219.
Acknowledgements
We would like to thank the Brazilian National Research Council
(CNPq), Research Foundation of Pernambuco State (FACEPE) and
FIOCRUZ for financial support. M.V.O.C. holds a FACEPE scholarship,
while D.R.M.M. holds a FAPESB scholarship. We also thank the
Department of Fundamental Chemistry-UFPE for recording the 1H
NMR, 13C NMR, LCMS and IR spectra of all compounds. M.V.O.C. is
thankful to P.V.B. in accept a doctoral internship at the University of
Queensland. R.S.F. is thankful to Dr. Anna Tochowicz (University of
California, San Francisco (UCSF), USA) for providing recombinant
cruzain. We also thank members of the Kineto DPU at Glax-
oSmithKline Spain for their support in cytotoxicity studies and
comments on the manuscript. All authors declare no competing
financial interest.
[19] M.E. Caputto, A. Ciccarelli, F. Frank, A.G. Moglioni, G.Y. Moltrasio, D. Vega, et
al., Synthesis and biological evaluation of some novel 1-indanone thiazo-
lylhydrazone derivatives as anti-Trypanosoma cruzi agents, Eur. J. Med. Chem.
55 (2012) 155e163.
[20] M.Z. Hernandes, M.M. Rabello, A.C.L. Leite, M.V.O. Cardoso, D.R.M. Moreira,
D.J. Brondani, et al., Studies toward the structural optimization of novel
thiazolylhydrazone-based potent antitrypanosomal agents, Bioorg. Med.
Chem. 18 (2010) 7826e7835.
[21] A.C.L. Leite, R.S. de Lima, D.R.D.M. Moreira, M.V.D.O. Cardoso, A.C. Gouveia de
Brito, L.M. Farias Dos Santos, et al., Synthesis, docking, and in vitro activity of
thiosemicarbazones, aminoacyl-thiosemicarbazides and acyl-thiazolidones
against Trypanosoma cruzi, Bioorg. Med. Chem. 14 (2006) 3749e3757.
[22] A.C.L. Leite, D.R. de M. Moreira, M.V. de O. Cardoso, M.Z. Hernandes, V.R. Alves
Pereira, R.O. Silva, et al., Synthesis, cruzain docking, and in vitro studies of
aryl-4-oxothiazolylhydrazones against Trypanosoma cruzi, ChemMedChem 2
(2007) 1339e1345.
[23] D.-N. Zhang, J.-T. Li, Y.-L. Song, H.-M. Liu, H.-Y. Li, Efficient one-pot three-
component synthesis of N-(4-arylthiazol-2-yl) hydrazones in water under
ultrasound irradiation, Ultrason. Sonochem. 19 (2012) 475e478.
[24] F. Chimenti, B. Bizzarri, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, et al.,
Appendix A. Supplementary data
A
novel histone acetyltransferase inhibitor modulating Gcn5 network:
cyclopentylidene-[4-(40-chlorophenyl)thiazol-2-yl)hydrazone, J. Med. Chem.
52 (2009) 530e536.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.012.
[25] D.R. Richardson, D.S. Kalinowski, V. Richardson, P.C. Sharpe, D.B. Lovejoy,
M. Islam, et al., 2-Acetylpyridine thiosemicarbazones are potent iron che-
lators and antiproliferative agents: redox activity, iron complexation and
characterization of their antitumor activity, J. Med. Chem. 52 (2009)
1459e1470.
[26] R.S. Ferreira, C. Bryant, K.K.H. Ang, J.H. McKerrow, B.K. Shoichet, A.R. Renslo,
Divergent modes of enzyme inhibition in a homologous structure-activity
series, J. Med. Chem. 52 (2009) 5005e5008.
[27] L. Ballell, R.H. Bates, R.J. Young, D. Alvarez-Gomez, E. Alvarez-Ruiz, V. Barroso,
et al., Fueling open-source drug discovery: 177 small-molecule leads against
tuberculosis, ChemMedChem 8 (2013) 313e321.
[28] V. Mersch-Sundermann, S. Knasmüller, X.-J. Wu, F. Darroudi, F. Kassie, Use of a
human-derived liver cell line for the detection of cytoprotective, anti-
genotoxic and cogenotoxic agents, Toxicology 198 (2004) 329e340.
[29] O. Korb, T. Stützle, T.E. Exner, Empirical scoring functions for advanced
protein-ligand docking with PLANTS, J. Chem. Inf. Model. 49 (2009)
84e96.
References
[1] WHOjChagas disease (American trypanosomiasis).
[2] F. Guhl, J.A. Urbina, Specific chemotherapy of Chagas disease: relevance,
current limitations and new approaches, Acta Trop. 115 (2010) 55e68.
[3] V.G. Duschak, A.S. Couto, An insight on targets and patented drugs for
chemotherapy of Chagas disease, Recent Pat. Antiinfect. Drug Discov. 2 (2007)
19e51.
[4] J.H. McKerrow, P.J. Rosenthal, R. Swenerton, P. Doyle, Development of prote-
ase inhibitors for protozoan infections, Curr. Opin. Infect. Dis. 21 (2008)
668e672.
[5] J.A. Urbina, R. Docampo, Specific chemotherapy of Chagas disease: contro-
versies and advances, Trends Parasitol. 19 (2003) 495e501.
[6] J. Urbina, Chemotherapy of Chagas disease, Curr. Pharm. Des.
8 (2002)
287e295.

M.V.O. Cardoso et al. / European Journal of Medicinal Chemistry 86 (2014) 48e59
59
[30] K. Brak, I.D. Kerr, K.T. Barrett, N. Fuchi, M. Debnath, K. Ang, et al., Nonpeptidic
tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads
for Chagas disease chemotherapy, J. Med. Chem. 53 (2010) 1763e1773.
[34] G.B. Rocha, R.O. Freire, A.M. Simas, J.J.P. Stewart, RM1: a reparameterization of
AM1 for H, C, N, O, P, S, F, Cl, Br, and I, J. Comput. Chem. 27 (2006) 1101e1111.
[35] Spartan “08 Tutorial and User”s Guide: Wavefunction, 2008.
[36] Gold software.
[31] G.M. Sheldrick,
A short history of SHELX, Acta Crystallogr. A 64 (2008)
112e122.
[37] J.D. Durrant, J.A. McCammon, BINANA: a novel algorithm for ligand-binding
characterization, J. Mol. Graph. Model. 29 (2011) 888e893.
[38] The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC, 2002.
[32] L.J. Farrugia, ORTEP -3 for windows e a version of ORTEP-III with a graphical
user interface (GUI), J. Appl. Crystallogr. 30 (1997), 565e565.
[33] L.J. Farrugia, WinGX suite for small-molecule single-crystal crystallography,
J. Appl. Crystallogr. 32 (1999) 837e838.
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