Synthesis of 2-Hydrazolyl-4-Thiazolidinones Based on Multicomponent Reactions and Biological Evaluation Against Trypanosoma Cruzi

Article abstract of DOI:10.1111/j.1747-0285.2010.01071.x

A series of 18 novel 2-hydrazolyl-4-thiazolidinones-5-carboxylic acids, amides and 5,6-α,β-unsaturated esters were synthesized, and their in vitro activity on cruzipain and T. cruzi epimastigotes was determined. Some agents show activity at 37μm concentration in the enzyme assay. Computational tools and docking were used to correlate the biological response with the physicochemical parameters of the compounds and their cruzipain inhibitory effects.

Full text of DOI:10.1111/j.1747-0285.2010.01071.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01071.x
Chem Biol Drug Des 2011; 77: 166–172
Research Article
Synthesis of 2-Hydrazolyl-4-Thiazolidinones Based
on Multicomponent Reactions and Biological
Evaluation Against Trypanosoma Cruzi
Chiara Pizzo1, Cecilia Saiz1, Alan Talevi2,
The growing knowledge of the basic biology of Trypanosoma cruzi,
the ethiological agent causative of Chagas disease, empowers the
development of new, rationally developed approaches to specific
chemotherapy. The cathepsin L-like cysteine protease termed cruzi-
pain or cruzain is responsible for the major proteolytic activity of all
stages of the parasite life cycle and represents an interesting target
for the development of potential therapeutics for the treatment of
Luciana Gavernet2, Pablo Palestro2,
Carolina Bellera2, Luis Bruno Blanch2,
Diego Benı´tez3, Juan J. Cazzulo4, Agustina
Chidichimo4, Peter Wipf5 and S. Graciela
Mahler1,*
the disease (2). Cruzipain is differentially expressed in the four main
stages of the parasite's biological cycle; it is located in different
cellular compartments and is essential for the survival of T. cruzi
within host cells (3). While its exact function is unknown, it is likely
involved in the degradation of proteins scavenged from the blood
meal of the insect vector (1).
1Departamento de Quꢀmica Orgꢁnica, Cꢁtedra de Quꢀmica
Farmacꢂutica, Universidad de la Repfflblica (UdelaR), Avda. General
Flores 2124, CC1157 Montevideo, Uruguay
2Cꢁtedra de Quꢀmica Medicinal, Departamento de Ciencias
Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de
La Plata (UNLP), La Plata, Argentina
3Laboratorio de Quꢀmica Orgꢁnica, Facultad de Ciencias-Facultad de
Quꢀmica, UdelaR, Montevideo, Uruguay
The computer-assisted, rational search for new drugs has emerged
as a popular strategy for the development of new chemotherapeutic
agents. It involves both target- and ligand-based approaches. In par-
ticular, virtual screening (VS) has proven to be very useful in the
discovery of new chemical entities for common as well as
neglected diseases (4). A VS campaign led to the 2-hydrazolyl-4-
thiazolidinone core as a potential cruzipain inhibitor. Based on this
result, we designed, synthesized, and biologically evaluated a series
of analogs against cruzipain and epimastigotes of T. cruzi. Computa-
tional tools were used to correlate the biological responses with
physicochemical parameters of the compounds and to simulate the
cruzipain–inhibitor interactions by docking calculations.
4IIB-INTECH Universidad Nacional de General San Martꢀn –
CONICET, San Martꢀn, Argentina
5Department of Chemistry, University of Pittsburgh, Pittsburgh, PA
15260, USA
*Corresponding author: S. Graciela Mahler, gmahler@fq.edu.uy
A series of 18 novel 2-hydrazolyl-4-thiazolidinon-
es-5-carboxylic acids, amides and 5,6-a,b-unsatu-
rated esters were synthesized, and their in vitro
activity on cruzipain and T. cruzi epimastigotes
was determined. Some agents show activity at
37 lM concentration in the enzyme assay. Compu-
tational tools and docking were used to correlate
the biological response with the physicochemical
parameters of the compounds and their cruzipain
inhibitory effects.
Methods and Materials
Chemistry
Reactions were monitored by analytical thin-layer chromatography
(TLC) on 0.25-mm silica gel-coated plastic sheets (Polygramꢀ SIL
G ⁄ UV 254; Macherey-Nagel Dꢀren, Germany). Flash chromatography
on Silica gel 60 (J. T. Baker, 40 lm average particle diameter) was
Key words: 2-hydrazolyl-4-thiazolidinone, Chagas disease, cruzipain,
inhibitors, multicomponent connection reaction, Trypanosoma cruzi
Received 19 August 2010, revised 8 December 2010 and accepted for
publication 11 December 2010
1
used to purify the crude reaction mixtures. H and 13C NMR spectra
were recorded at 400 and 100 MHz, respectively, on a Bruker
AVANCE. Chemical shifts (d) are in ppm relative to the residual sol-
vent signal, and coupling constants (J) are reported in Hz. Decou-
pled HMBC spectra were obtained using the Bruker hmbcgplpndqf
pulse sequence using standard parameters. IR spectra were
obtained on a Perkin Elmer 1310 (Wellesley, MA, USA) and Shima-
dzu 8101A FTIR spectrophotometers (Salem, OR, USA). Low-
resolution mass spectra were measured on a GCMS Shimadzu QP
1100-EX spectrometer. High-resolution mass spectra were measured
on VG AutoSpect spectrometer (EI mode). Microwave-assisted reac-
A century after its discovery, Chagas disease still represents a
major public health challenge in Latin America where currently an
estimated 11 million people are currently infected. Because of
growing population movements, an increasing number of Chagas
disease cases have now been detected in non-endemic areas, such
as North America and some European countries. Unfortunately, only
benznidazole and nifurtimox are clinically available for treatment of
the disease, and both demonstrate only limited efficacy during the
acute phase of the disease and show severe side effects (1).
166

Synthesis of 2-hydrazolyl-4-thiazolidinones
tions were carried out on a CEM Discover microwave reactor
equipped with 10-mL vials. Melting points were determined using a
Laboratory Devices Gallenkamp (Loughborough, Leicestershire, UK).
All reactions were carried out in dry, freshly distilled solvents under
anhydrous conditions unless otherwise stated. Yields are reported
for chromatographic and spectroscopic (1H and 13C-NMR) pure com-
pounds unless otherwise stated.
J = 4.6, 1H), 8.31 (d, J = 3.0, 1H), 11.86 (bs, 1H); 13C NMR (DMSO-
d6) 25.6, 37.9, 44.0, 55.4, 78.7–79.3, 114.4, 126.9, 129.3, 155.7,
161.3, 169.2; HRMS calculated for C14H16N4O3S [M)H]+: 321.1021,
found: 321.1022.
(1i) (RS)-2-(2-(4-Chlorobenzylidene)hydrazono)-3-
methyl-4-oxo-5-thiazolidine-N-methylacetamide
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using p-Cl benzaldehyde and methylthiosemicarbazide to give
thiazolidinone 1i (88% yield) as a white solid: mp 225–226 ꢁC; IR
(1f–n) General procedure for the preparation of
thiazolidineacetamides
The preparation of compound 1f is representative.
1
(KBr) 3305, 1717, 1646, 1621, 1578 cm)1; H NMR (CDCl3) 2.68 (dd,
J = 16.1, J = 10.2, 1H), 2.87 (d, J = 4.8, 3H), 3.17 (dd, J = 16.1,
J = 3.8, 1H), 3.33 (s, 3H), 4.38 (dd, J = 10.2, J = 3.8, 1H), 5.58 (s,
1H), 7.39 (d, J = 8.5, 2H), 7.71 (d, J = 8.5, 2H), 8.38 (s, 1H); 13C
NMR (DMSO-d6) 26.4, 30.2, 38.5, 44.0, 129.9, 130.2, 133.9, 136.1,
157.0, 166.0, 170.0, 175.1; HRMS calculated for C14H15ClN4O2S
[M+Na]+: 361.0496, found 361.0498.
(1f) (RS)-2-((2-(Benzylidene)hydrazono)-4-oxo-5-
thiazolidine-N-methylacetamide
To a stirred solution of benzaldehyde (200 mg, 1.9 mmol) in PhMe
(1 mL) and DMF (1 mL) were added thiosemicarbazide 3 (136 mg,
1.5 mmol) and p-TsOH acid (2 mg, 0.01 mmol). The reaction mixture
was heated in stirred microwave vial for 3 min at 90 ꢁC. After for-
mation of thiosemicarbazone derivate, (followed by TLC) maleimide
(138 mg, 1.26 mmol) was added, and the reaction mixture was
heated 10 min at 110 ꢁC in the microwave. The residue was finally
recrystallized from MeOH ⁄ H2O (1:1) to give 1f (237 mg, 65% yield)
as a white solid: mp 253–254 ꢁC dec.; IR (KBr) 3321, 1712, 1644,
(1j) (RS)-2-(2-(2-Bromobenzylidene)hydrazono)-3-
methyl-4-oxo-5-thiazolidine-N-methylacetamide
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using o-Br-benzaldehyde and methylthiosemicarbazide to give
thiazolidinone 1j (55% yield) as a white solid: mp 208–209 ꢁC; IR
1
1569 cm)1
;
1H NMR (DMSO-d6) 2.60 (d, J = 4.6, 3H), 2.64 (dd,
(KBr) 3318, 1716, 1644, 1623, 1609 cm)1; H NMR (DMSO-d6) 2.58
J = 16.2, J = 9.9, 1H), 2.94 (dd, J = 16.2, J = 3.7, 1H), 4.32 (dd,
J = 9.9, J = 3.7, 1H), 7.47 (m, 3H), 7.76 (m, 2H), 8.00 (d, J = 4.6,
1H), 8.40 (s, 1H), 11.94 (bs, 1H); 13C NMR (DMSO-d6) 25.9, 37.8,
44.1, 127.4, 127.7, 128.7, 128.9, 130.7, 134.3, 156.1, 164.7, 169.3,
175.9; HRMS calculated for C13H14N4O2S [M)H]+: 291.0916, found:
291.0918.
(d, J = 4.2, 3H), 2.71 (dd, J = 16.4, J = 9.6, 1H), 2.98 (dd, J = 16.4,
J = 3.6, 1H), 3.19 (s, 3H), 4.39 (dd, J = 9.6, J = 3.6, 1H), 7.43 (m,
1H), 7.49 (t, J = 16.0, J = 8.0, 1H), 7.73 (d, J = 8.0, 1H), 7.98 (d,
J = 8.0, 1H), 8.04 (d, J = 4.2, 1H), 8.67 (s, 1H); 13C NMR (DMSO-d6)
26.0, 29.9, 37.9, 43.6, 124.5, 128.4, 128.7, 132.9, 133.0, 133.8,
155.8, 166.9, 169.6, 174.7; HRMS calculated for C14H15BrN4O2S
[M+Na]+: 404.9997, found: 404.9986.
(1g) (RS)-2-(2-(Thiophen-2-ylmethylene)
hydrazono)-4-oxo-5-thiazolidine-N-
methylacetamide
(1k) (RS)-2-(2-(4-Fluorobenzylidene)hydrazono)-
3-methyl-4-oxo-5-thiazolidin-N-methylacetamide
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using p-F-benzaldehyde and methylthiosemicarbazide to give
thiazolidinone 1k (65% yield) as a white solid: mp 226–227 ꢁC; IR
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using 2-thiophenecarboxaldehyde to give thiazolidinone 1g
(75% yield) as a yellowish solid: mp 246–247 ꢁC dec.; IR (KBr)
1
3324, 1712, 1640, 1571 cm)1; H NMR (DMSO-d6) 2.59 (d, J = 4.7,
(KBr) 2957, 1721, 1698, 1630 cm)1 1H NMR (DMSO-d6) 2.59 (d,
;
3H), 2.64 (m, 1H), 2.92 (dd, J = 16.1, J = 3.5, 1H), 4.29 (dd, J = 9.7,
J = 3.5, 1H), 7.16 (m, 1H), 7.49 (d, J = 4.2 1H), 7.68 (dd, J = 4.2,
J = 0.8, 1H), 7.97 (d, J = 4.7, 1H), 8.54 (s, H), 11.82 (bs, 1H); 13C
NMR (DMSO-d6) 25.5, 37.8, 44.0, 128.0, 129.7, 132.7, 138.9, 150.5,
164.8, 169.7, 176.2; HRMS calculated for C11H12N4O2S2 [M)H]+:
297.0480, found: 291.0487.
J = 4.6, 3H), 2.70 (dd, J = 16.3, J = 9.7, 1H), 2.97 (dd, J = 16.3,
J = 3.7, 1H), 3.17 (s, 3H), 4.37 (dd, J = 9.7, J = 3.7, 1H), 7.32 (t,
J = 12.4, J = 5.4, 2H), 7.85 (m, 2H), 8.03 (d, J = 4.6, 1H), 8.51 (s,
1H); 13C NMR (DMSO-d6) 26.0, 29.8, 38.1, 43.6, 116.4 (d, JCF = 22),
130.4 (d, JCF = 9), 131.3 (d, JCF = 3), 156.6, 164.5 (d, JCF = 245),
165.2, 169.6, 174.6; HRMS calculated for C14H15FN4O2S [M+H]+:
323.0978, found: 323.0973.
(1h) (RS)-2-(2-(4-
Methoxybenzylidene)hydrazono)-4-oxo-5-
thiazolidine-N-methylacetamide
(1l) (RS)-2-(2-(3-Bromobenzylidene)hydrazono)-3-
methyl-4-oxo-5-thiazolidine-N-methylacetamide
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using m-Br-benzaldehyde and methylthiosemicarbazide to give
thiazolidinone 1l (75% yield) as a white solid: mp 214–215 ꢁC; IR
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using p-OMe-benzaldehyde to give thiazolidinone 1h (81%
yield) as a white solid: mp 250–251 ꢁC; IR (KBr) 3320, 1714, 1641,
1
1
1613, 1513 cm)1; H NMR (DMSO-d6) 2.59 (d, J = 4.6, 3H), 2.64 (m,
(KBr) 3309, 1717, 1644, 1621, 1571 cm)1; H NMR (CDCl3) 2.71 (dd,
1H), 2.92 (dd, J = 16.1, J = 3.7, 1H), 3.81 (s, 3H), 4.29 (dd, J = 9.9,
J = 3.7, 1H), 7.01 (d, J = 8.8, 2H), 7.70 (d, J = 8.8, 2H), 7.99 (d,
J = 16.0, J = 10.1, 1H), 2.88 (d, J = 4.8, 3H), 3.19 (dd, J = 16.0,
J = 3.7, 1H), 3.35 (s, 3H), 4.39 (dd, J = 10.1, J = 3.7, 1H), 5.62 (s,
Chem Biol Drug Des 2011; 77: 166–172
167

Pizzo et al.
1H), 7.30 (m, 2H), 7.56 (ddd, J = 7.9, J = 1.8, J = 1.0, 1H), 7.68 (d,
J = 7.9, 1H), 7.94 (d, J = 1.8, 1H), 8.36 (s, 1H); 13C NMR (CDCl3)
26.6, 29.8, 39.4, 43.5, 122.9, 126.9, 130.2, 130.6, 133.6, 136.3,
156.8, 165.0, 169.3, 174.3; HRMS calculated for C14H15BrN4O2S
[M+H]+: 383.0177, found 383.0171.
160.6, 164.7 (d, JCF = 252), 165.1, 166.5; HRMS calculated for
C14H13FN3O3S [M+H]+: 322.0662, found: 322.0650.
(6a) (Z)-2-(4-Methoxybenzylidene)hydrazono-5-
methoxycarbonylmethylene)thiazolidin-4-one
The typical procedure for thiazolidinone 6c preparation was fol-
lowed using p-OMe-benzaldehyde and thiosemicarbazide to give
thiazolidinone 6a (71% yield) as a yellow solid: mp 260–261 ꢁC
(1m) (RS)-2-(2-(1-(3-Bromophenyl)
ethylidene)hydrazono)-3-methyl-4-oxo-5-
thiazolidine-5-N-methylacetamide
1
dec; IR (KBr) 2961, 1725, 1700 1643, 1606 cm)1; H NMR (DMSO-
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using m-Br phenylacetophenone and methylthiosemicarbazide
to give thiazolidinone 1m (88% yield) as a yellowish solid: mp
d6) 3.79 (s, 3H), 3.82 (s, 3H), 6.67 (s, 1H), 7.06 (d, J = 8.8, 2H), 7.78
(d, J = 8.8, 2H), 8.46 (s, 1H); 13C NMR (DMSO-d6) 52.9, 55.9, 114.5,
114.9, 126.7, 130.3, 143.6, 158.8, 159.8, 162.2, 166.4.
1
181–182 ꢁC; IR (KBr) 3313, 1706, 1644, 1609, 1571 cm)1; H NMR
(DMSO-d6) 2.43 (s, 3H), 2.58 (d, J = 4.6, 3H), 2.71 (dd, J = 16.3,
J = 9.5, 1H), 2.97 (dd, J = 16.3, J = 3.6, 1H), 3.21 (s, 3H), 4.36 (dd,
J = 9.5, J = 3.6, 1H), 7.43 (t, J = 16.0, J = 8.0, 1H), 7.66 (d,
J = 8.0, 1H), 7.86 (d, J = 8.0, 1H), 8.01 (t, J = 1.8, 1H); 13C (DMSO-
d6) 14.4, 25.5, 29.4, 37.6, 43.1, 121.9, 125.5, 128.8, 130.7, 132.5,
140.0, 160.0, 164.2, 169.1, 174.2; HRMS calculated for
C15H18BrN4O2S EIMS [M+H]+: 397.0334, found: 397.0338.
(6b) (Z)-2-[4-Chlorobenzylidene)hydrazono]-3-
methyl-5-methoxycarbonylmethylene)
thiazolidin-4-one
The typical procedure for thiazolidinone 6c preparation was fol-
lowed using p-Cl-benzaldehyde to give thiazolidinone 6b (85%
yield) as a yellow solid: mp 204–205 ꢁC; IR (KBr) 2949, 1711, 1703,
1
1621, 1581 cm)1; H NMR (CDCl3) 3.45 (s, 3H), 3.88 (s, 3H), 6.92 (s,
1H), 7.41 (d, J = 8.5, 2H), 7.76 (d, J = 8.5, 2H), 8.43 (s, 1H); 13C
NMR (CDCl3) 29.9, 52.7, 116.1, 129.2, 129.7, 132.4, 137.4, 141.8,
158.7, 161.0, 165.2, 166.6; HRMS calculated for C14H12ClN3O3S
[M+Na]+: 360.0180, found: 360.0179.
(1n) (RS)-2-(2-(4-Trifluoromethyl)benzy
lidene)hydrazono)-3-methyl-4-oxo-5-thiazolidin-
N-methylacetamide
The typical procedure for thiazolidinone 1f preparation was fol-
lowed using p-CF3-benzaldehyde and methylthiosemicarbazide to
give thiazolidinone 1n (90% yield) as a white solid: mp 220–
(6d) (Z)-2-[(4-Trifluoromethyl)
benzyliden)hydrazono]-3-methyl-5-
1
221 ꢁC; IR (KBr) 3294, 1719, 1644, 1623, 1586, 1559 cm)1; H NMR
(CDCl3) 2.69 (dd, J = 23.1, J = 12.0, 1H), 2.87 (d, J = 4.5, 3H), 3.18
(dd, J = 23.1, J = 3.7, 1H), 3.34 (s, 3H), 4.38 (dd, J = 12.0, J = 3.7,
1H), 5.66 (d, J = 4.5, 1H), 7.66 (d, J = 8.2, 2H), 7.88 (d, J = 8.2,
2H), 8.45 (s, 1H); 13C NMR (CDCl3) 26.6, 29.8, 39.3, 43.5, 125.7 (q,
JCF3 = 4 Hz), 126.5 (q, JCF3 = 262 Hz), 128.3, 131.9 (q, JCF3 = 32 Hz),
137.5, 156.7, 165.4, 169.3, 174.3; HRMS calculated for
C15H15F3N4O2S [M+H]+: 373.0946 373.0947.
methoxycarbonylmethylene) thiazolidin-4-one
The typical procedure for thiazolidinone 6c preparation was followed
using p-CF3-benzaldehyde to give thiazolidinone 6d (90% yield) as a
yellow solid: mp 187–188 ꢁC; IR (KBr) 2956, 1711, 1646, 1630 cm)1
;
1H NMR (CDCl3) 3.49 (s, 3H), 3.90 (s, 3H), 6.94 (s, 1H), 7.69 (d,
J = 8.2, 2H), 7.94 (d, J = 8.2, 2H), 8.52 (s, 1H); 13C NMR (CDCl3)
29.8, 52.6, 116.3, 123.8 (q, JCF3 = 273), 125.7 (q, JCF3 = 4), 128.6,
132.6 (q, JCF3 = 33), 137.1, 141.5, 158.3, 161.9, 165.1, 166.5; HRMS
calculated for C15H12F3N3O3S [M+Na]+: 394.0444, found: 394.0443.
(6a–e) General procedure for a, b-unsaturated
thiazolidine esters
The preparation of compound 6c is representative.
(6e) (Z)-2-(4-Methoxybenzylidene)hydrazono)-3-
methyl-5-methoxycarbonylmethylene)
thiazolidin-4-one
(6c) (Z)-2-(4-Fluorobenzylidene)hydrazono)-3-
methyl-5-methoxycarbonylmethylene)
thiazolidin-4-one
The typical procedure for thiazolidinone 6c preparation was fol-
lowed using p-OMe-benzaldehyde to give thiazolidinone 6e (87%
yield) as a yellow solid: mp 192–193 ꢁC; IR (KBr) 2958, 1703, 1631,
1
To a stirred solution of p-F-benzaldehyde (150 mg, 1.2 mmol) in dry
EtOH (5 mL) were added methylthiosemicarbazide (140 mg, 1.3 mmol)
and p-TsOH acid (15 mg, 0.1 mmol). After formation of methylthiose-
micarbazone derivate (followed by TLC) was added dimethyl acetylen-
edicarboxylate (189 mg, 1.3 mmol). The reaction mixture was stirred
at RT for 2 h, and a yellow precipitate was collected. The solid was
recrystallized from ethyl acetate to give 6c (304 mg, 81% yield) as
1604 cm)1; H NMR (CDCl3) 3.45 (s, 3H), 3.88 (d, J = 1.1, 6H), 6.91
(s, 1H), 6.96 (m, 2H), 7.78 (m, 2H), 8.43 (s, 1H); 13C NMR (CDCl3)
30.0, 52.8, 55.8, 114.6, 115.9, 126.9, 130.6, 142.4, 159.8, 162.6,
165.5, 166.9; HRMS calculated for C15H15N3O4S [M+H]+: 334.0856,
found: 334.0856.
yellow solid: mp 214–215 ꢁC; IR (KBr) 3077, 1716, 1633, 1595 cm)1
;
Cruzipain inhibitory activity
1H NMR (CDCl3) 3.45 (s, 3H), 3.88 (s, 3H), 6.92 (s, 1H), 7.15–7.11 (m,
2H), 7.84–7.81 (m, 2H), 8.45 (s, 1H); 13C NMR (CDCl3) 29.7, 52.6,
115.9, 116.1, 130.1 (d, JCF = 3), 130.4 (d, JCF = 9), 141.8, 158.6,
The activity was assayed in a reaction mixture (100 lL) containing
in final concentration: Tris–HCl buffer, pH 7.6 (50 mM), Bz-Pro-Phe-
Arg-pNA (0.150 mM), b-mercaptoethanol (10 mM) and cruzipain
168
Chem Biol Drug Des 2011; 77: 166–172

Synthesis of 2-hydrazolyl-4-thiazolidinones
(0.140 lM). Absorbance at 410 nm was monitored at 30 ꢁC on a
Beckman Model 25 spectrophotometer. The potential inhibitors were
added as solutions in DMSO, and the control inhibitor was E64
(100% inhibition at 10 lM) added at same solvent concentration.
All inhibitors were assayed by duplicate. The percentage of cruzi-
pain inhibition (PCI) was calculated as follows: PCI
(%) = (Ai ⁄ Ao) · 100, where Ai and A0 are the absorbance with and
without inhibitor respectively.
2D autocorrelations and others. Detailed information on the models
used is given in the Data S1.
Conformational analysis and docking
The conformational analysis of the tested compounds was carried
out using the Conformational Search tool from the HyperChem 7.01
software package (Hypercube Inc, 2002). Geometry optimization was
performed through molecular mechanics (MM + force field) with a
gradient convergence threshold of 0.1 kcal ⁄ (ꢁ mol). The five low-
est-energy conformers were then refined at a semiempirical level
with the PM3 base and a gradient convergence threshold of
0.05 kcal ⁄ (ꢁ mol). The selected conformers were refined using
Gaussian03 software (B3LYP ⁄ 6-31G**). Charges were calculated at
the same level of theory. The binding of compound 6b to cruzain
(Figure 3) was analyzed using AutoDockTools 1.5.2 and AutoDock
4.0 docking programs (5).
Trypanocidal activity
Epimastigotes of the Tulhahuen 2 strain were grown at 28 ꢁC in an
axenic medium (BHT-Tryptose) complemented with 10% heat-inacti-
vated fetal calf serum. Cells from 4-day-old culture (exponential
phase) were inoculated to fresh medium to give an initial concen-
tration of 13 · 106 ⁄ mL. After the inoculation, each culture was
supplemented with the inhibitors to a final concentration of 50 lM.
Epimastigotes growth was monitored by counting the parasites in a
Neubauer chamber.
Results and Discussion
The values given are means of parasite densities after 8 days. The
final concentration of DMSO in the culture media was between
0.7% and 1.5%, the control was run in the same amount of DMSO
and in the absence of any compound. The presence of up to 1.5%
of DMSO in the culture medium does not have substantial effect
on the epimastigote growth. The percentage of growth inhibition
Synthesis
A VS strategy was applied to 537 503 chemical structures from the
ZINC 5 database (6) to select new cruzipain inhibitors. 2-Hydrazolyl-
4-thiazolidinone (1) cores were selected for synthesis, from the pos-
sible candidates. These are hybrid molecules that combine thiose-
micarbazones with 4-thiazolidinones, two building blocks with
interesting biological activities. The combination of both pharmaco-
phores has been used to exhibit anti-Toxoplasma gondii (7), and
also anti-Trypanosoma cruzi activity (8).
(PGI)
was
calculated
as
follows:
inhibition
(%) = {1)[(Np)N0p) ⁄ (Nc)N0c)]}% 100, where Np and N0p is the
number of cells of the culture containing the drug at 8 and 0 day
respectively; Nc and N0c is the number of cells of the culture in the
absence of any drug at day 8 and at day 0 respectively.
The selected scaffold 1 presents four regions to explore the chemi-
cal space: the substituents R1 and R2 at the hydrazolyl group, the
substituent R3 at the nitrogen of the thiazolidinone, the presence or
absence of saturation at D5,6, and the substituent R4 in the side
chain (Figure 1).
Effects of inhibitors on the growth of Vero cells
Vero cells were seeded (10 000 cells ⁄ well) in 24-well cell culture
cluster flat bottom (Corning) containing glass coverslips with 500 lL
of MEM medium supplemented with 10% fetal calf serum. Cells
were allowed to attach for 24 h in a humidified 5% CO2 ⁄ 95% air
atmosphere at 37 ꢁC.
We previously developed a Multicomponent Connection Reaction
(MCR) for the synthesis of 2-hydrazolyl-4-thiazolidinones-5-carboxylic
acids. Compounds 1a–e were prepared using a tandem sequence
assisted by microwave irradiation of a reaction mixture containing
aldehydes (2, R2 = H), thiosemicarbazide (3, R3 = H) and maleic
anhydride (4, X = O), see Table 1, entries 1–5 (9). The former pro-
cess, compared to a stepwise reaction, allows the minimization of
waste, amount of solvent, reagents, adsorbents, and energy.
Then, the cells were exposed to the compounds (50 and 100 lM)
for 24 h. Afterwards, the cell culture media was removed.
Fresh MEM medium with 4% fetal calf serum and without inhibi-
tors was added, and the coverslips were incubated at 37 ꢁC for
48 h. The cells were then fixed and stained with May Grunwald–
Giemsa. The effects of inhibitors in Vero cells at 100 lM inhibitor
concentration were determined by contrast phase microscopy com-
parisons to Vero cells controls without inhibitor and classified as
toxic and non-toxic.
A similar tandem sequence was applied for the synthesis of new 2-
hydrazolyl-4-thiazolidinones amides 1f–n, combining aldehydes or
O
6
1
S
5
Virtual screening
R4
R2
R1
The methodology included a 2D QSAR approach based on applica-
tion of linear discriminant analysis (LDA) and multiple linear regres-
sion (MLR) on 0D–2D molecular constitutional and topological
descriptors from Dragon (Milano Chemometrics, 2003), including
constitutional and topological descriptors, counts of functional
groups, counts of fragments, information index, connectivity index,
4
N
O
N 2
N
R3
1
Figure 1: Hydrazolyl-4-thiazolidinones scaffold.
Chem Biol Drug Des 2011; 77: 166–172
169

Pizzo et al.
Table 1: Synthesis of thiazolidinones 1a–n.
OMe
OMe
O
S
O
S
O
R2
N
6
R1
H
H
O
5
6
S
3J = 5–6 Hz
XH
2J = 1–2 Hz
O
4
O
S
R2
R1
H2N
+
X
+
N
Solvent, 100oC
N
N
O
R2
N
H
NH
R3
R1
O
N
N
N
4
N
N
R3
R1
µW
R3
R3
O
R2
2
3
4
1
6
7
X = O, NMe
Figure 2: Thiazolidine 6 and 1,3-thiazine 7 isomers.
Compounds
X
R1
R2
R3
Yield (%)
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
O
O
O
O
p-OMePh
2-thiophenyl
o-FPh
p-NO2Ph
p-ClPh
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
61a
33a
57a
61a
70
1
thiazines 7, see Figure 2. The discrimination by H-NMR and 13C-
NMR for compound 6 or 7 is not trivial, because they have almost
identical signals in the spectra. A useful technique for the identifi-
cation of the two possible isomers was the decoupled HMBC exper-
iment to measure the coupling constant at 3 bonds (10). The 3J
values between H6 and C4 for all products 6a–e were 6.3 to
5.2 Hz, and according to the literature these values correspond to
an exo double bond in thiazolidines 6 (11) (see Table 2). Depending
on the solvent used, a mixture of compounds 6 and 7, in PhMe, or
only the thiazolidinone 6 (in EtOH) was obtained.
O
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
Ph
65b
75b
81b
88b
55b
65b
75b
88b
90
1g
1h
1i
2-thiophenyl
p-OMePh
p-ClPh
o-BrPh
p-FPh
m-BrPh
m-BrPh
p-CF3Ph
10
1j
11
12
13
14
1k
1l
1m
1n
All the compounds were prepared using a one-pot methodology and
provided the desired structures in good yields from readily accessi-
ble starting materials.
aReagents and conditions: PhMe ⁄ DMF, p-TsOH (cat.), 100 ꢁC, lW.
bReagents and conditions: PhMe, p-TsOH (cat.), 100 ꢁC, lW.
ketones (2), hydrazides (3, R3 = H or Me) and N-methylmaleimide
(4, X = NMe), to give the desired amides in good yields (75–90%
Table 1, see entries 6–14).
Cruzipain inhibition
Different thiazolidinone derivatives including carboxylic acids 1a–e,
acetamides 1f–n and a,b-unsaturated esters 6a–e were evaluated
against the cruzipain enzyme (12). Only thiazolidines 6b–e, bearing
an a,b-unsaturated ester, showed interaction with the enzyme at
lM concentration (entries 16–18, Table 3); the remaining thiazolidi-
nones were less active.
Finally, a series of new 2-hydrazolyl-4-thiazolidinone-5,6-a,b-unsatu-
rated esters (6) was synthesized using an MCR between aldehydes
2, thiosemicarbazides 3, and dimethyl acetylenedicarboxylate 5, to
yield compounds 6a–e, in EtOH at r.t. in excellent yields (71–90%),
independent of the presence of electron-withdrawing or electron-
donating groups, Table 2.
The reaction occurs first by thiosemicarbazone formation, followed
by Michael addition of the sulfur atom to the triple bond and then
cyclization to give in theory two possible products thiazolidines 6 or
Table 2: Synthesis of thiazolidinones 6a–e
OMe
O
O
OMe
S
O
H
R2
R1
EtOH
H N
2
+
S
+
N
H
NH
R3
N
R2 R1
N
O
N
R3
2
3
R2 = H
O
OMe
5
6
Compound
R3
R1
3J(C,H) Hz
Yield (%)
Figure 3: Surface representation of the active site of cruzain.
Ligand and important residues were highlighted as stick representa-
tions. Hydrogen atoms are not shown. Colored atoms are as fol-
lows: Carbon atoms in green, nitrogen atom in blue, oxygen atoms
in red, sulfur atoms in yellow, and chlorine atoms in light blue.
Hydrogen bond interaction d = 1.93ꢁ.
6a
6b
6c
6d
6e
H
p-OMePh
p-ClPh
p-FPh
p-CF3Ph
p-OMePh
5.2
5.2
5.2
6.3
5.2
71
85
81
90
87
Me
Me
Me
Me
170
Chem Biol Drug Des 2011; 77: 166–172

Synthesis of 2-hydrazolyl-4-thiazolidinones
Table 3: Biological activities of synthetic thiazolidinones 1a–n,
and 6a–e against cruzipain.
Benznidazole, but only compound 6d showed a moderate activity
(PGI-Y = 37% at 50 lM) (14).
PCI
PCI
PCI
The unspecific cytotoxicity for the most active compounds 1i–n and
6b–e was evaluated in vitro against mammalian cells at 100 and
50 lM, using Vero cells as the cellular model. The results showed
that compounds 1l, 1m, and 1n were non-toxic at the highest
doses assayed, and compound 1m was found to have the best
selectivity as anti-T. cruzi Tul 2 agent, see Table 4.
Entry Compound (%)a Entry Compound (%)a Entry Compound (%)a
1
2
3
4
5
6
7
1a
1b
1c
1d
1e
1f
36
34
6
4
11
3
8
9
1h
1i
22
7
0
15
16
17
18
19
20
–
6a
6b
6d
6e
6c
7
75
65
71
0
10
11
12
13
14
1j
1k
1l
3
17
22
27
1m
1n
E-64
–
100
–
1g
27
Molecular modeling and QSAR studies
aPCI = % of cruzipain inhibition at (Inhibitor) = 100 lM, (Cruzi-
pain) = 0.140 lM, values are means of two values.
E64: trans-Epoxysucciny-L-leucyl-amido(4-guanidino) butane; (L-3-trans-Carb-
oxyoxiran-2-Carbonyl)-L-Leucyl-Admat was used as reference compound.
The mechanistic proposal for cruzipain inhibition involves a nucleo-
philic attack of the cysteine sulfur atom to an electrophilic moiety
able to accept electrons. According to this idea, we decided to gain
insight into the atomic distribution of charges as well as the contribu-
tion of each atom to the frontier orbital. A systematic conformational
analysis and ab initio geometry optimization was carried out as
described in the experimental section. The electronic parameters com-
puted were HOMO and LUMO energies, LUMO and HOMO coeffi-
cients, normalized LUMO and HOMO coefficients for each atom (15).
Common physicochemical properties are frequently related to biologi-
cal activity and the drug-likeness concept, such as theoretical logP
(Moriguchi¢s LogP)mlogP), (mlogP)2, molecular refractivity (MR) and
molecular weight (MW), which also calculated using DRAGON software.
Anti-Trypanosoma cruzi activity and toxicity
According to these assays, a subset of selected compounds was
evaluated in their capability to inhibit the epimastigote form of
T. cruzi (Tulahuen 2, strain) growth, (Table 4). A good correlation
between in vitro anti-T. cruzi epimastigote and in vivo anti-T. cruzi
activities has been reported (13); therefore, we used this biological
test to validate our possible hits.
It is interesting to note that related thiazolidinones were predicted
by Lima et al. (8) as potential cruzipain inhibitors based on docking
calculations, and their activity was supported by anti-T. cruzi (epim-
astigotes) assays. Our results pointed toward a different scenario;
the antiparasitic activity of this core is independent of the cruzipain
inhibition. The most active compounds against T-cruzi Tul-2 1i, j, k,
m are inactive at the enzyme, so this activity is indeed independent
on the cystein protease activity. Thiazolidinone 6c is the only com-
pound active in both assays. We can conclude that these compounds
involve a different mechanism of action. The best results for anti-
T. cruzi activity were obtained with the compounds 1 or 6, having as
substituents: R3 = Me; R2 = H or Me; R1 = Ph bearing an halogen,
independent of the presence or absence of the D5,6 alkene.
The QSAR analysis showed a very poor correlation between cruzi-
pain inhibition (PCI) and the calculated descriptors.
However, better results were obtained between PGI-Tul2 activity
and the descriptors, revealing significant correlations with MR (cor-
relation coefficient r = 0.83), Moriguchi's logP (r = 0.79), and
(mlogP)2 (r = 0.74). The MR explained around 70% of the variance
of the 14 assayed compounds.
Docking
Molecular docking was used to analyze the binding mode of 6b,
the most active compound of the set. The most stable docking con-
formation obtained for 6b is shown in Figure 3. A hydrogen-bond-
ing interaction was predicted between the ester group of the
inhibitor and the TRP184 residue, with a 1.93 ꢁ measured distance.
Additionally, the aromatic ring of 6b makes positive lipophilic inter-
actions with the hydrophobic S2 pocket of the enzyme, character-
ized by LEU60, ALA136, and MET68 residues. The chlorine atom
points toward the end of the cavity, delimited by the GLU105 resi-
due, which is capable of extending its carboxylic group out to avoid
negative interactions with lipophilic ligands. Despite these non-
bonded interactions that stabilized the complex, no interactions
were found between 6b and the CYS25 and ⁄ or HIS162 catalytic
residues and no nucleophilic attack can be predicted. This observa-
tion may explain the observed weak inhibition of cruzipain.
Furthermore, compounds 1a–n and 6a–e were also evaluated
using epimastigotes Y, a resistant strain against Nifurtimox and
Table 4: Anti-Trypanosoma cruzi Tulahuen 2 and cytotoxic activi-
ties
Inhibitiona Cytotoxic
Compound (%) Tul2
Inhibitiona Cytotoxic
categoryb Compound (%) Tul2
categoryb
1a
1b
1c
1d
1i
0
0
0
nd
nd
nd
nd
Toxic
Toxic
Toxic
Non-toxic
1m
1n
6b
6c
60
42
39
50
46
38
100
–
Non-toxic
Non-toxic
Toxic
Toxic
Toxic
Non-toxic
nd
–
0
50
52
50
28
6d
6e
Nfxc
–
1j
1k
1l
Conclusions
aInhibition % was tested at 50 lM.
bDetermined at 100 lM inhibitor concentration by contrast phase microscopy
comparisons to Vero controls; nd = not determined.
New 2-hydrazoyl-4-thiazolidinone scaffolds could be obtained from
commercially available starting materials by using MCR and well-
cNfx: Nifurtimox was used as reference drug.
Chem Biol Drug Des 2011; 77: 166–172
171

Pizzo et al.
established methodologies. Compounds 1i, 1j, 1k, 1m, and 6c
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erately active on the parasite. The mode of action proposed for these
compounds is based on a different mechanism rather than cruzipain
inhibition. Toxicity screening was also performed pinpointing the
amide 1m as the most selective compound. Further structure optimi-
zation is needed to increase the antichagasic activity.
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Acknowledgments
This work was supported by the National Institutes of Health-FIRCA
(R03TW007772). We greatly appreciate Prof. Mercedes Gonzꢂlez for
providing valuable suggestions and corrections to this paper. G.M.
thanks RIDIMEDCHAG-CYTED. A.T., L.G., and J.J.C. are members of
the Research Career of CONICET (Argentina). L.B.-B. and L.G. thank
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Data S1. General procedure for the preparation and characteriza-
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