Synthesis, docking, and in vitro activity of thiosemicarbazones, aminoacyl-thiosemicarbazides and acyl-thiazolidones against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2006.01.034

A novel series of thiosemicarbazone and aminoacyl-thiazolidones derivatives were synthesized. Their structure suggests that these compounds could have anti-Trypanosoma cruzi activity. Biological evaluation indicates that some of these compounds are able t

Full text of DOI:10.1016/j.bmc.2006.01.034

Bioorganic & Medicinal Chemistry 14 (2006) 3749–3757
Synthesis, docking, and in vitro activity of
thiosemicarbazones, aminoacyl-thiosemicarbazides
and acyl-thiazolidones against Trypanosoma cruzi
Ana Cristina Lima Leite,a,* Renata Souza de Lima,a Diogo Rodrigo de M. Moreira,a
a
a
´
Marcos Verıssimo de O. Cardoso, Ana Carolina Gouveia de Brito,
Luciene Maria Farias dos Santos,a Marcelo Zaldini Hernandes,b Alice Costa Kiperstok,c
Ricardo Santana de Limac and Milena B. P. Soaresc
a
ˆ
ˆ
Laborato´rio de Planejamento, Avaliac¸a˜o e Sı´ntese de Fa´rmacos—LABSINFA, Departamento de Ciencias Farmaceuticas,
Universidade Federal de Pernambuco, Rua Prof. Artur Sa´ S/N, Cidade Universita´ria, 50740-520 Recife, PE, Brazil
b
ˆ
ˆ
Laborato´rio de Quı´mica Teo´rica Medicinal—LQTM, Departamento de Ciencias Farmaceuticas, Universidade Federal de
Pernambuco, Rua Prof. Artur Sa´ S/N, Cidade Universita´ria, 50740-520 Recife, PE, Brazil
cCentro de Pesquisas Gonc¸alo Moniz/FIOCRUZ—Rua Waldemar Falca˜o, 121, Candeal, 40296-750 Salvador, BA, Brazil
Received 29 September 2005; revised 12 January 2006; accepted 13 January 2006
Available online 3 February 2006
Abstract—A novel series of thiosemicarbazone and aminoacyl-thiazolidones derivatives were synthesized. Their structure suggests
that these compounds could have anti-Trypanosoma cruzi activity. Biological evaluation indicates that some of these compounds are
able to inhibit the growth of T. cruzi in concentrations non-cytotoxic to mammalian cells. Docking studies were carried out in order
to investigate the binding pattern of these compounds for the T. cruzi cruzain (TCC) protein, and these showed a significant
correlation with experimental data.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
this reason, cruzain (aka cruzipain) and trypanothione
reductase (TR) are specific targets in the search for novel
and selective inhibitors and subversive substrates.1,5
Chagas’ disease is a serious health problem that affects
around 20 million people in Central and South Ameri-
cas. The protozoan Trypanosoma cruzi is the causative
agent of this disease. Current therapy is based on nifur-
timox and benznidazole, drugs capable of eliminating
parasitaemia and reducing serological titers in the acute
phase of infection but not effective for all T. cruzi
strains, especially in the chronic phase of infection. In
addition, these drugs may cause serious adverse side
effects due to their high toxicity.1–4
Cruzain is the major cysteine protease of T. cruzi, and is
released at all life cycle stages of the parasite, but deliv-
ered to different cellular compartments at each stage.
This enzyme is essential for replication of the intracellu-
lar parasite and appears to have potential for new anti-
trypanosomal chemotherapy.5
The forms of T. cruzi present in the human host are the
bloodstream trypomastigote and the intracellular repli-
cative amastigote. The epimastigote form, an obligate
mammalian intracellular stage, has been confirmed
recently.6 Since vaccinations against trypanosomatic
infections are still under development, the need for
new drugs is indisputable.
To develop new drugs to combat parasitic infections, re-
search is often directed toward key differences between
the metabolism of the mammal and the parasite. For
Keywords: Thiosemicarbazones; Acyl-hydrazones; Aminoacyl-thiazoli-
dones; Anti-Trypanosoma cruzi compounds; Docking studies.
The trypanocidal activity of several aromatic and
heterocyclic hydrazones and acyl-hydrazine-hydrazone
has been reported.2–4 In addition, some derivatives with
*
Corresponding
+0558121268510; e-mail: acllb2003@yahoo.com.br
author.
Tel.:
+0558133418511;
fax:
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.034

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A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
thiosemicarbazone and semicarbazone scaffolds were
synthesized and screened to evaluate their ability for
interaction with cruzain, and it was revealed that thiose-
micarbazones are active against the cysteine protease,
indicating that electronic and steric differences between
a sulfur and an oxygen atom make a substantial differ-
ence to biological activity.5
order to obtain the aminoacyl-2-methyl-3-thiosemicab-
azides, a condensation reaction between 2-methyl-3-
thiosemicabazide and amino acids (tert-butoxycarbonyl-
glycine, valine, and phenylalanine), BOP Castro’s
reagent (benzotriazolyloxy-tris-(dimethylamino) phos-
phonium hexafluorophosphate) was the most efficient
carbonyl activant used. Compounds 1a–c were obtained
with a yield of 32–46% (Scheme 1).11
A novel class of anti-infective molecules currently not
used clinically is the thiazole peptides. Some 4-thiazoli-
done derivatives have antibacterial, antituberculosis,
antifungal,7 and antiparasitic8,9 activities, among others.
Some thiazole peptides, a family of natural products, are
known for their potent antibacterial activity, predomi-
nantly resulting from their inhibitory activity on protein
synthesis.10
To obtain compounds 3 and 5a–c (Scheme 2) we used
the symmetrical anhydride (Boc)2O (di-tert-butylpyro-
carbonate) to protect the amine function of thiosemicar-
bazide. The intermediate compound 2 was obtained with
a yield of 55%. In the second step, the cyclization was
accomplished through the reaction of the Boc-thiosemi-
carbazide and chloroacetic acid in the presence of sodi-
um acetate, using ethanol as a solvent under reflux to
obtain 3 with a yield of 78%. For the removal of the
Boc-protecting group of 4-thiazolidone, classical cleav-
age conditions using TFA/CH2Cl2 (1:1) give rise to
unprotected 4-thiazolidone 4 (98% yield). The last stage
involves condensation of the a-amino acids (tert-butyl-
oxycarbonyl-L-phenylalanine, or a tert-butyloxycarbon-
yl-L-glycine, or tert-butyloxycarbonyl-L-proline) with
the amine moiety of the 4-thiazolidone using dic-
During previous studies aiming to discover structures
endowed with trypanocidal activity, we synthesized
derivatives that possess a thiosemicarbazone or a hydra-
zine-thiazolidone scaffold. A series of arylthiosemicar-
bazone or aminoacyl-thiazolidone derivatives were
synthesized. Here, we report the synthesis of acyl-hydra-
zine, thiosemicarbazone, and acyl-thiazolidone and the
in vitro evaluation of their ability to inhibit the growth
of epimastigote and trypomastigote forms of T. cruzi,
as well as a docking analysis under T. cruzi cruzain
(TCC). The compounds designed were thus analyzed
as potential ligands for cruzain.
yclohexylcarbodiimide
(DCC)
and
N-hydroxy-
succinimide (OH-Su) as carbonyl activating agents.
This method resulted in average yields of 68–80%.12,13
Compounds 10a,c, and b, were prepared according to
two strategies. Different thiophenols were reacted with
bromoacetaldehyde diethyl acetal (10a and c) or chloro-
propanone (10b) in the presence of KOH/Cu to produce
the corresponding ketone (6) and diethyl acetals (7).
After treatment in acid medium, compound 7 produced
the aldehyde 8. Aldehydes and ketones were condensed
with thiosemicarbazide to obtain thiosemicarbazones 9.
Cyclization of precursor 9 with chloroacetic acid or a-
chloropropionic acid generated thiazolidone-hydrazone
derivatives (Scheme 3).9
2. Chemistry
2.1. Synthesis
Initially we developed three series of peptidyl-thiazoli-
done and peptidyl-2-methyl-3-thiosemicarbazides. In
S
S
BOP/TEA
NH2N
CH3
C
NH2
Boc-NH-CH(R)-CO NH-N-C-NH2
Boc-Glycine
2.2. Molecular modeling
CH3
Boc-Phenylalanine
Boc-Valine
1a: R= H
1b: R= CH2C6H5
Gly
Phe
Val
The structures and conformational analysis of com-
pounds 1, 3, 5, and 10 were obtained through the appli-
cation of the AM114 method available as part of the
1c: R= CH(CH3)2
Scheme 1. Synthesis of aminoacyl-thiosemicarbazides.
S
S
S
(Boc)2O/NaOH
CH2ClCOOH
Boc
NH
NH
C
NH2
Boc
NH2NH
C
NH2
O
NHNH
N
3
2
CF3COOH
S
S
DCC/OH-Su
-
+ NH NH
O
Boc-NH-CH(R)-CO NH NH
TFA
O
3
Boc-Phenylalanine
Boc-glycine
N
N
4
Boc-Proline
5a: R= H
5b: R=CH2-C6H5 Phe
5c: Pro
Gly
Boc
N
Scheme 2. Synthesis of the aminoacyl-4-thiazolidone derivatives.

A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
3751
SCH2COCH3
S
ClCH2COCH3
KOH/Cu
SCH2C=N-NH-C-NH2
R1
SH
R
R
S
6
NH2NHCNH2
R2-CHCOOH
9
Cl
R
SCH2CH(OEt)2
H3O+
SCH2CHO
BrCH2CH(OEt)2
KOH/Cu
R2
S
N
S
N
H
N
O
8
7
R
R1
R
R= Cl R1= H R2= CH3 10a
R=H R1= CH3 R2=CH3 10b
R
R=CH3 R1= H R2= H
10c
Scheme 3. Synthesis of the 4-thiazolidone-2-arylthiosemicarbazone derivatives.
BioMedCache software package,15 using internal de-
fault settings for convergence criteria.
3. Pharmacology
3.1. Cytotoxicity
Molecules 10a and b were synthesized and tested as
racemic mixtures, so the molecular modeling treats
the two enantiomers (R and S) independently and
the docking procedure uses both isomers for each
compound.
The compounds were tested in different concentrations
in mouse spleen cell cultures as described in Section 6.
The highest non-toxic concentration of each compound
was then used in subsequent assays to evaluate their
anti-T. cruzi activity.
The docking analysis was first carried out on the T. cruzi
cysteine protease cruzain (TCC) binding site (structure
1U9Q,16 taken from the RCSB Protein Data Bank,
<http://www.rcsb.org/pdb/>), where the residues are
bonded more closely to the ligand known as ‘186’, co-
crystallized in complex with cruzain. This crystal struc-
ture is present in a monomeric form, with a chain named
X, and there is a ligand binding domain. The ‘186’ li-
gand was extracted from the complex, and during the
calculations the active site was defined to lie within a
3.2. Anti-Trypanosoma cruzi activity
The compounds were tested in vitro against epimasti-
gote (Y and Colombian strains) and trypomastigote
(Colombian strain) forms of T. cruzi at non-cytotoxic
concentrations as outlined in Section 6. Table 1 shows
the percentage of inhibition for the derivatives under
evaluation and the IC50 for Y strain epimastigotes.
˚
cube of about 18 A in each direction, centered on the
coordinates (X = 3.357; Y = 11.064; and Z = 6.235) of
the co-crystallized ligand, certainly covering the active
site region. Inside this pre-defined region, the grids of
probe atom interaction energies were computed at a res-
4. Results and discussion
In the course of our search for new structures, including
an aminoacyl or a hydrazine-hydrazone moiety, a num-
ber of synthesis strategies were employed. First, we
developed three series of peptidyl-thiazolidone, pepti-
dyl-2-methyl-3-thiosemicarbazide, and thiazolidone-thi-
osemicarbazone. Ten compounds were identified as
active against T. cruzi. We observed a thiosemicarba-
zone and/or hydrazine-4-thiazolidone (Figs. 1a and b)
scaffold common to several analogues. The thiosemicar-
bazone (Fig. 1a) scaffold has been described as possess-
ing useful properties, such as low molecular weight,
reasonable ClogP, good hydrogen bonding properties,
and easy and economical synthetic routes.5
˚
olution of 0.20 A and the ligand probes were then
docked using the Genetic Algorithm followed by a Local
Search procedure (GA_LS), also known as a Lamarck-
ian Genetic Algorithm (LGA),17 and the 50 lowest ener-
gy structures were stored for further analysis.
The docking analysis of compounds 1, 3, 5, and 10 was
carried out using the AutoDockTools (ADT) v1.118 and
Autodock v3.0.519 programs, using the default parame-
ters for all the variables, except for the number of dock-
ing runs (50), the maximum number of energy
evaluations on Genetic Algorithm-GA (25,000,000),
and the maximum number of generations in GA
(10,000). Preliminary calculations indicate that these
three specific parameter modifications provide signifi-
cant improvement in the results obtained using the
docking procedure, thereby producing more stable bind-
ing of the ligands. The active site was treated as a rigid
molecule, whereas the ligands were treated as flexible,
which means that all non-ring torsions were maintained
(active).
In the present work, the compounds were tested at non-
cytotoxic concentrations on the epimastigote form of Y
and Colombian strains and the trypomastigote form of
Colombian strain. As shown in Table 1, in the case of
compounds 1a–c, 1b (phenylalanine) was the most active
against the epimastigote form of Y and Colombian
strains. Compound 1c, in a high concentration, was
inactive after the 11th day of treatment. In the case of
compounds 3 (intermediate) and 5a–c, no decrease in

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A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
Table 1. Biological properties of compounds
Compound
Percentage of growth inhibitionc (%)
Y Epimastigote Colombian
Concentration
(lmol/L)a
Colombian
trypomastigote
at 24 h
epimastigote
at 11 days
b
11ꢁ day
IC50
1a
1b
38.1
283.7
328.5
4.3
50.0
92.3
7.0
114.4
85.1
495.7
142.6
73.4
83.3
30.5
31.9
128.4
45.0
1.8
50.0
95.0
50.0
55.6
11.1
55.6
0.0
5.4
8.1
1c
5.4
3
5a
52.2
0.0
44.0
48.0
36.0
12.0
27.0
75.7
21.6
100.0
3.4
2.6
5b
5c
8.7
30.4
31.8
340.8
35.8
38.2
65.2
89.0
100.0
92.3
99.7
10a
10b
10c
Bzd
20.0
60.0
50.0
77.8
a Determined by cytotoxicity assay using mouse splenocytes.
b lmol/L.
c Percentage of growth inhibition calculated as described in Section 3.1.
d Bz, Benznidazole.
Table 2. Docking results for compounds 1, 3, 5, and 10
a
b
S
S
Compound
pIC50 = ꢀlog IC50
Free energy of binding
(kcal/mol)
O
Ar
N
(with IC50 in mol/L)
H2N
N
H
NH2
N
NH
1a
1b
1c
3.94
4.07
3.30
3.85
4.13
4.08
4.52
4.50
3.89
4.35
ꢀ5.48
ꢀ6.27
ꢀ5.67
Figure 1. Thiosemicarbazone and thiazolidone scaffold.
3
ꢀ5.36
5a
5b
5c
ꢀ5.27
cytotoxicity was observed, indicating that the use of the
aminoacyl moiety in the thiazolidone scaffold has no ef-
fect on cytotoxicity. Only compound 5c (a proline deriv-
ative) was five times more active than intermediate 3,
and about twice as active as 5a and b. However, this
compound was inactive against the Colombian strain.
With regard to the aryl-thiazolin-hydrazone series (com-
pounds 10a–c), biological analyses revealed that 10b (in
racemic form) was the least cytotoxic and the most ac-
tive against the Colombian strain in a high concentra-
tion. Compound 10a (in racemic form) was the most
active at the concentrations tested. This compound pos-
sesses a chlorine substituent in the phenyl ring system
and has shown itself to be active against epimastigote,
in comparison with benznidazole. In general, this series
showed good trypanocidal activity. In accordance with
previous works, our results confirm the importance of
the thiosemicarbazone scaffold for trypanocidal
activity.5
ꢀ6.88
ꢀ7.01
10a
10b
10c
ꢀ6.92 (R) and ꢀ7.10 (S)
ꢀ7.02 (R) and ꢀ7.01 (S)
ꢀ6.67
The most stable docking solutions for the complexes be-
tween compounds 1, 3, 5, and 10 and TCC are presented
in Table 2, along with the free energy of binding (DG)
values. In order to investigate possible correlations be-
tween experimental and theoretical data, the free energy
of binding (kcal/mol) was plotted against the pIC50
(equals ꢀlog IC50 of the Y strain epimastigotes) values.
The pIC50 and free energy of binding values are present-
ed in Table 2 and plotted in Figure 2.
Figure 2. Correlation between the free energy of binding (kcal/mol)
and the pIC50 (equals ꢀlog IC50) values for molecules 1, 3, 5, and 10.
For molecules 10a and b were used the both enantiomers, R and S. The
stippled line at the diagonal is only for visualization purposes, in order
to make easier the identification of the correlation between the
variables.
er values for pIC50, are those with most negative free
energy of binding, showing that the molecules with more
stable or negative binding energies are also the most
Figure 2 provides evidence of a correlation between the-
oretical binding (docking) and the IC50 data, that is, the
most active molecules or the compounds that have high-

A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
3753
active (greater affinity for TCC), at least when the Y
strain is considered.
in TCC reveals important and specific interactions,
which are important for describing the affinity of such
molecules to the cruzain. These results confirm that
the thiosemicarbazone scaffold is a potential anti-T. cru-
zi agent. Additional structural optimization, in vivo
activity studies and the characterization of the pharma-
cokinetics of these compounds are currently underway.
In order to compare the binding pattern of these mole-
cules, the compound that produced the best result (i.e.,
the most stable complex) in docking analysis—the S
enantiomer of molecule 10a was analyzed, along with
the crystallographic structure of TCC, and the results
of this comparison can be seen in Figure 3.
6. Experimental
6.1. Chemistry
Figure 3 shows that compound 10a (S enantiomer) (ball
and stick model) establishes hydrogen bonds with the
GLY66, MET68, and ASN69 residues of the TCC bind-
ing site (colored line model with three letter codes in yel-
All melting points were determined using a Thomas
Hoover apparatus and are uncorrected. IR spectra were
˚
low), with 2.258, 1.894 and 2.078 A measured distances,
1
respectively. It should be pointed out that the ligand
‘186’, co-crystallized with TCC in the structure 1U9Q,
also shows an important hydrogen bond with the resi-
due GLY66, in the same ligand binding domain.
obtained using KBr pellets. H and 13C NMR spectra
were measured using a Varian UNITYplus-300 MHz
NMR spectrophotometer using DMSO-d6 as solvent
and tetramethylsilane as an internal standard. Thin-
layer chromatography (TLC) was carried out on silica
Figure 4 illustrates the important interactions between
the 10a (S enantiomer) compound and the TCC, after
docking studies, in similar a fashion to Figure 3. These
interactions include three important hydrogen bonds
(as in Fig. 3) and also hydrophobic interactions between
ligand and target residues. The LIGPLOT program20
was used to produce Figure 4.
gel plates with a fluorescence indicator of F254
(0.2 mm, E. Merck); the spots were visualized in UV
light and by spraying with a 2% ethanol solution of nin-
hydrin or charing reagent. Column chromatography
was performed on silica using Kieselgel 60 (230–
400 mesh, E. Merck). All reagents used in the present
study were of analytical grade.
For comparison reasons, one can see in Figure 5 the
crystallographic structure of the ligand named ‘186’ at
the binding site of TCC, in order to understand the
selection of this ligand-interaction site.
6.1.1. General procedure for compounds 1a–c. The N-
protected amino acid (1 mmol) was dissolved in DMF
(10 mL) containing 1 mmol of 2-methyl-3-thiosemicar-
bazide and BOP (1 mmol). The solution was cooled in
an ice bath, and TEA (0.7 mL) was added to it. The
preparation was left to stand for 12 h at room tempera-
ture. Then, dichloromethane (50 mL) was added under
stirring. The organic layer was washed several times
with sodium bicarbonate (50 mL), water (50 mL), 1 M
citric acid (50 mL), water (50 mL), dried over sodium
sulfate, and concentrated in vacuo. The residue was
purified using silica gel column chromatography, with
ethyl acetate–hexane (1:1) as a solvent, and produced
a white powder when crystallized with diethyl ether–
hexane.
5. Conclusion
New thiosemicarbazones, aminoacyl-thiosemicarbaz-
ides, and aminoacyl-thiazolidones were synthesized
using accessible methodologies. Some derivatives exhibit
significant in vitro activity against epimastigote T. cruzi,
particularly compounds 5c and 10a. The docking results
corroborate the experimental IC50 data, and a detailed
analysis of the binding characteristics of these ligands
Figure 3. Important hydrogen bonds between the S enantiomer of compound 10a (ball and stick model) and residues (colored line model with three
letter codes in yellow) of the TCC structure, obtained by the docking procedure. The distances for hydrogen bonding are given in angstrom and are
labeled in blue. The rest of the protein structure was suppressed for clarification purposes.

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A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
Figure 4. Schematic figure of the important interactions between the 10a (S enantiomer) compound and the TCC, including hydrogen bonds and
hydrophobic interactions with specific residues. The legend for this figure is presented on the bottom.
6.1.1.1.
1-[2-(tert-Butoxycarbonyl)aminoacetyl]-2-
128.32, 128.71, 137.23, 156.08, 169.63, 170.63;
C16H24N4O3S.
methylthiosemicarbazide (1a). Yield: 42%; mp 92–4 ꢁC;
Rf = 0.62 (ethyl acetate); IR (KBr/cmꢀ1): 1087, 1243,
1311, 1625, 2928, 3035, 3326; 1H NMR (DMSO-d6/
ppm) d: 1.36 (s, 9H, (CH3)3), 1.54 (t, J = 7.4, 3H,
CHb), 3.35 (s, 3H, N2–CH3), 4.30 (s, 2H, NH2), 5.03
(d, J = 5.9, 2H, CHa), 7.38 (t, J = 5.9, 1H, NHa), 8.24
(d, J = 8.3, 1H, N2–H), 9.40 (s, 1H, N1–H); 13C NMR
(DMSO-d6/ppm) d: 28.36, 35.65, 46.01, 78.96, 158.08,
162.52, 169.88; C11H14N4O3S.
6.1.1.3. 1-[2-(tert-Butoxycarbonyl)-2-isopropyl-amino-
acetyl]-2-methylthiosemicarbazide (1c). Yield: 74%; mp
134–6 ꢁC; Rf = 0.57 (ethyl acetate); IR (KBr/cmꢀ1):
1044, 1203, 1511, 1668, 2973, 3204, 3304; 1H NMR
(DMSO-d6/ppm) d: 1.59 (s, 9H, (CH3)3), 2.42 (s, 3H,
N2–CH3), 2.50 (t, J = 8.7, 2HCHb), 4.30 (s, 2H,
NH2), 4.48–4.56 (1H, CHa), 7.95 (s, 1H, NHa), 9.85
(s, 1H, N1–H); 13C NMR (DMSO-d6/ppm) d: 18.96,
28.21, 28.79, 36.98, 78.81, 156.54, 170.32, 182.15;
C12H24N4O3S.
6.1.1.2.
1-[2-(tert-Butoxycarbonyl)-2-benzyl-amino-
acetyl]-2-methylthiosemicarbazide (1b). Yield: 79%; mp
102–3 ꢁC; Rf = 0.68 (ethyl acetate); IR (KBr/cmꢀ1):
1114, 1245, 1416, 1668, 2941, 2995, 3332; 1H NMR
(DMSO-d6/ppm) d: 1.23–1.32 (s, 9H, (CH3)3), 2.50
(t, J = 8.7, 2H, CHb), 3.35 (s, 3H, N2–CH3), 4.30 (s,
2H, NH2), 4.48–7.43–7.48 (m, 5H, Ar), 4.56 (1H,
CHa), 6.92 (d, J = 7.1, 1H, NHa), 8.80 (d, J = 8.3,
1H, N2–H), 9.35 (s, 1H, N1–H); 13C NMR (DMSO-
d6/ppm) d: 28.48, 38.48, 46.08, 56.75, 79.25, 124.36,
6.1.2. General procedure for compounds 2 and 5a–c.
6.1.2.1.
1-[tert-Butyloxycarbonyl]thiosemicarbazide
(2). A solution of thiosemicarbazide (1 mmol), dioxane
(20 mL), water (10 mL), and 1 N NaOH (11 mL) was
stirred in an ice-water bath. Di-tert-butyl pyrocarbonate
(2.4 g, 11 mmol) was added and stirring was continued
at room temperature for 30 min. The solution was con-

A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
3755
Figure 5. Schematic figure of the crystallographic structure of ligand named ‘186’ into TCC, including hydrogen bonds and hydrophobic interactions
with specific residues. The legend for this figure is presented in Figure 4.
centrated in vacuo to about 10–15 mL, cooled in an ice-
water bath, covered with a layer of ethyl acetate
(30 mL), and acidified with a dilute solution of KHSO4
to pH 2–3 (Congo paper). The aqueous phase was
extracted using ethyl acetate (15 mL) and the extraction
repeated. The ethyl acetate extracts were pooled, washed
with water (twice, 30 mL each time) dried over anhy-
drous Na2SO4, and evaporated it in vacuo.
trifluoroacetic acid and dichloromethane (10 mL) to ob-
tain the deprotected hydrazino-4-thiazolidone (4). The
N-protected amino acid in dimethylformamide under
0 ꢁC, 6.8 mmol of the dicyclohexylcarbodiimide,
5.1 mmol of N-hydroxysuccinimide, and 1.7 mmol of
intermediate 4 was added and was allowed to warm to
room temperature. Subsequently, the reaction mixture
was filtered, and the filtrate was treated with ethyl ace-
tate. The organic phase was washed with NaHCO3,
water and aqueous NaCl, dried over Na2SO4 solution,
and concentrated. The residue was treated with hexane
and filtered.
Yield: 37%; mp 170–2, Rf = 0.72 (ethyl acetate), IR
(KBr/cmꢀ1): 1052, 1285, 1720, 3189, 3375; 1H NMR
(DMSO-d6/ppm) d: 1.45 (m, 9H, (CH3)3), 7.32 (s, 2H,
NH2), 7.75 (s, 1H, N–H), 8.78 (s, 1H, N–H).
Yield: 76%; mp 165–7, Rf = 0.68 (7:3, ethyl acetate/hex-
1
6.1.2.2. 1-(tert-Butyloxycarbonylamino)-2-(4-oxo-4,5-
dihydrothiazol-2-yl)hydrazide (3). The mixture of pro-
tected thiosemicarbazide (2 mmol), a-chloroacetic acid,
and sodium acetate (2 mmol) in ethanol (10 mL) was
stirred for 6 h at reflux temperature. When the reaction
had been completed, the mixture was left at ꢀ4 ꢁC for
72 h. Then, hexane was added and the solid was filtered.
Ethyl acetate was added and the organic phase was
washed with water, dried (over anhydrous sodium sul-
fate), and evaporated. The Boc-hydrazino-4-thiazoli-
done intermediate (3) was treated with a mixture of
ane), IR (KBr/cmꢀ1): 1250, 1456, 1717, 3135; H NMR
(DMSO-d6/ppm) d: 1.47 (m, 9H, (CH3)3), 3.93 (2H,
CH2), 9.39 (s, 1H, N–H), 11.30 (s, 1H, N–H); 13C
NMR (DMSO-d6/ppm) d: 27.76, 31.85, 76.85, 155.23,
167.92, 173.98; C8H13N3O3S.
6.1.2.3. 2-(4-Oxo-4,5-dihydrothiazol-2-yl)-1-(2-[tert-
Yield:
68%; mp 152–4 ꢁC, Rf = 0.6 (7:3, ethyl acetate/hexane),
IR (KBr/cmꢀ1): 1244, 1536, 1710, 3328; 1H NMR
(DMSO-d6/ppm): 1.24 (s, 9H, (CH3)3), 2.41 (s, 2H,
butoxycarbonylamino]ethane)hydrazide
(5a).

3756
A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
CH2), 3.66 (dd, J = 6.2 Hz, 2H CHa), 5.55 (d,
J = 7.7 Hz, 1H, NH), 7.48 (t, J = 6.2 Hz, 1H, NHa);
13C NMR (DMSO-d6/ppm) d: 27.99, 31.45, 45.25,
55.79, 80.20, 125.39, 128.56, 144.31, 156.14, 170.02,
170.92, 175.92; C17H22 N4O4S.
6.1.3.3.
N-(4-Oxo-2-thiazolin-2yl)-N0-p-methylthio-
ethylidenehydrazone (10c). Yield: 30%; mp 155–6 ꢁC,
recrystallizing solvent: ethanol (95%)/H2O. IR (KBr/
cmꢀ1): 1595, 1635, 1730, 2990; 1H NMR (DMSO-d6/
ppm): 2.21 (s, 3H, CH3), 3.70 (s, 2H, CH2), 3.72 (d,
J = 12Hz, 2H, CH2), 7.20 (m, 4H, aromatic), 7.61 (t,
J = 12 Hz, 1H, CH); 13C NMR (DMSO-d6/ppm) d:
20.57, 34.25, 39.50, 128.87, 129.16, 130.97, 135.81,
155.87, 174.20; C13H16ON3S2O.
6.1.2.4. 2-(4-Oxo-4,5-dihydrothiazol-2-yl)-1-(2(S)N-
[tert-butoxycarbonylamino]-3-(phenyl)propane)hydrazide
(5b). Yield: 98%; mp 159–60 ꢁC, Rf = 0.76 (7:3, ethyl
acetate/hexane), IR (KBr/cmꢀ1): 1245, 1574, 1736,
1
3329; H NMR (DMSO-d6/ppm) d: 1.24–1.32 (s, 9H,
6.2. Biology
(CH3)3), 1.73 (d, J = 10.49 Hz, 2H, CHb), 2.83 (s, 2H,
CH2), 5.61 (d, J = 7.4 Hz, 1H, NH), 4.60–4.50 (m, 1H
CHa), 7.33–7.26 (m, 5H, Ar); 13C NMR (DMSO-d6/
ppm) d: 28.39, 31.95, 41.20, 77.52, 149.53, 158.56,
170.64, 170.86, 178.32; C13H20O4N4S.
6.2.1. Cytotoxicity assay. The cytotoxicity of the com-
pounds was determined using BALB/c mice splenocytes
(5· 106 cells/well) cultured in 96-well plates in Dul-
becco’s Modified Eagle’s Medium (DMEM, Sigma
Chemical Co., St. Louis, MO) supplemented with 10%
of fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil)
6.1.2.5. 2-(4-Oxo-4,5-dihydrothiazol-2-yl)-1-(2(S)N-
[tert-butoxycarbonylamino]-2-pyrrolidine)hydrazide (5c).
Yield: 74%; mp 105–4 ꢁC, Rf = 0.64 (7:3, ethyl acetate/
hexane), IR (KBr/cmꢀ1): 1132, 1574, 1699, 3328; 1H
NMR (DMSO-d6/ppm): 1.37–1.40 (s, 9H, (CH3)3),
1.83–1.91 (m, 2H, CHb), 2.02–2.07 (m, 2H, CHc), 2.50
(t, J = 3.2 Hz, 2H, CHd), 2.81 (s, 2H, CH2), 4.57 (dd,
J = 3.5 Hz, 3.89, 1H, CHa), 5.58 (d, J = 7.1 Hz, 1H,
NH); 13C NMR (DMSO-d6/ppm) d: 24.49, 28.33,
30.25, 33.37, 45.32, 61.72, 80.01, 154.51, 168.49,
171.01, 175.79; C13H20O4N4S.
´
and 50 lg/mL of gentamycin (Novafarma, Anapolis,
GO, Brazil). Each compound was evaluated in three
concentrations (1, 10, and 100 lg/mL), in triplicate. Cul-
tures were incubated in the presence of H-thymidine
3
(1 lCi/well) for 24 h at 37 ꢁC and 5% CO2. After this
period, the plate content was harvested to determine
3
the H-thymidine incorporation using a beta-radiation
counter (b-matrix 9600, Packard). The toxicity of the
compounds was determined comparing the percentage
3
of H-thymidine incorporation (as indicator of cell via-
bility) of drug-treated wells in relation to untreated
wells. Non-toxic concentrations were defined as those
causing a reduction of 3H-thymidine incorporation
below 10% in relation to untreated controls.
6.1.3. General procedure for compounds 10a–c. Thiophe-
nols were reacted with bromoacetaldehyde diethyl acetal
(20 mmol) or choro-2-propanone (20 mmol) in the pres-
ence of KOH (20 mmol) and Cu (20 mmol) to produce
the corresponding diethyl acetals and ketones. The
diethyl acetals were then refluxed with 2 N H2SO4,
thereby producing aldehydes. The aldehydes and ke-
tones (15 mmol) were condensed to 2-methyl-3-thiosem-
icarbazide (15 mmol) in glacial acetic medium (20 mL)
to produce the thiosemicarbazone intermediate. Cycliza-
tion of these intermediates with a-chloropropionic
(5 mmol) acid in the presence of sodium acetate
(5 mmol) generated cyclized 4-thiazolidone derivatives
with a yield of 30% and 67%.
6.2.2. Anti-T. cruzi assay. Epimastigotes of T. cruzi (Y
and Colombian strains) were cultivated at 26 ꢁC in liver
infusion tryptose medium (LIT) supplemented with 10%
fetal calf serum, 1% hemin, 1% R9 medium, and 50 lg/
mL gentamycin. Parasites (106 cells/mL) were cultivated
in a fresh medium in the absence or in the presence of
the compounds being tested or 0.01 mg/mL benznidazol
(Rochagan, Roche). Cell growth was determined after
11 days of culture by counting viable forms in a hema-
cytometer. The compounds were prepared from a stock
solution in DMSO. To determine the IC50, cultures of Y
strain epimastigotes in the presence of different concen-
trations of the compounds were evaluated after 11 days
as described above. IC50 calculation was carried out
using non-linear regression on Prism 4.0 GraphPad
software. Colombian strain T. cruzi trypomastigotes
were obtained from culture supernatants of LCC-
MK2 cell line and placed in 96-well plates (4· 105/well)
in DMEM supplemented with 10% FCS and 50 lg/mL
gentamycin. Compounds were added at non-toxic con-
centrations, in triplicate. Viable parasites were counted
in a hemacytometer 24 h after addition of compounds
by way of trypan blue exclusion. The percentage of inhi-
bition was calculated in relation to untreated cultures.
6.1.3.1. N-(4-Oxo-5-methyl-2-thiazolin-2yl)-N0-p-chlo-
rophenylthioethylidenehydrazone (10a). Yield: 67%; mp
146–7 ꢁC, recrystallizing solvent: ethanol (95%)/H2O.
IR (KBr/cmꢀ1): 1600, 1640, 1720, 2990; 1H NMR
(DMSO-d6/ppm): 1.50 (d, J = 12 Hz, 3H, CH3), 3.90
(d, J = 10.8 Hz 2H, CH2), 4.20 (m, 1H, CH), 7.44 (m,
4H, Ar), 7.70 (t, J = 12Hz, 1H, CH); 13C NMR
(DMSO-d6/ppm) d: 20.85, 35.29, 40.52, 129.12, 129.54,
144.26, 159.02, 175.20; C12H12ON3S2Cl.
6.1.3.2. N-(4-Oxo-5-methyl-2-thiazolin-2yl)-N0-phe-
nylthio-2-propylidenehydrazone (10b). Yield: 44%; mp
138–9 ꢁC, recrystallizing solvent: absolute ethanol. IR
(KBr/cmꢀ1): 1590, 1645, 1725, 3090; 1H NMR
(DMSO-d6/ppm): 1.23 (d, J = 7.2 Hz 3H, CH3), 1.70
(d, 3H, CH3), 3.63 (d, J = 7.8 Hz 2H, CH2), 3.93 (m,
1H, CH), 7.20 (m, 5H, aromatic); 13C NMR (DMSO-
d6/ppm) d: 20.57, 34.25, 39.50, 128.87, 129.16, 130.97,
135.81, 155.87, 174.20; C13H16ON3S2O.
Acknowledgments
We thank the Brazilian National Research Council
(CNPq), the Research Foundation of Pernambuco State

A. C. L. Leite et al. / Bioorg. Med. Chem. 14 (2006) 3749–3757
3757
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