Synthesis, anti-Toxoplasma gondii and antimicrobial activities of benzaldehyde 4-phenyl-3-thiosemicarbazones and 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids

Article abstract of DOI:10.1016/j.bmc.2007.09.025

In the present communication, a new series of 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids (2a-p) have been synthesized. Benzaldehyde 4-phenyl-3-thiosemicarbazones substituted (1a-p) were also obtained and used as intermediate

Full text of DOI:10.1016/j.bmc.2007.09.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 446–456
Synthesis, anti-Toxoplasma gondii and antimicrobial activities
of benzaldehyde 4-phenyl-3-thiosemicarbazones
and 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-
5-thiazolidineacetic acids
a
a
a
a
´
ˆ
Thiago M. de Aquino, Andre P. Liesen, Rosa E. A. da Silva, Vania T. Lima,
b
c
a
c
´
Cristiane S. Carvalho, Antoˆnio R. de Faria, Janete M. de Arau´jo, Jose G. de Lima,
c
b
a,
*
´
´
Antonio J. Alves, Edesio J. T. de Melo and Alexandre J. S. Goes
aDepartamento de Antibio´ticos, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
bLaborato´rio de Biologia Celular, Universidade Estadual do Norte Fluminense, Rio de Janeiro 28015-620, Brazil
c
ˆ ˆ
Departamento de Ciencias Farmaceuticas, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
Received 3 August 2006; revised 29 August 2007; accepted 12 September 2007
Available online 18 September 2007
Abstract—In the present communication, a new series of 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids
(2a–p) have been synthesized. Benzaldehyde 4-phenyl-3-thiosemicarbazones substituted (1a–p) were also obtained and used as inter-
mediate to give the title compounds. All synthesized compounds were characterized by IR, 1H and 13C NMR. The in vitro anti-Tox-
oplasma gondii activity of 1a–p and 2a–p was evaluated. The 4-thiazolidinones (2a–p) were screened for their in vitro antimicrobial
activity. For anti-Toxoplasma gondii activity, in general, all compounds promoted decreases in the percentage of infected cells lead-
ing to parasite elimination. These effects on intracellular parasites also caused a decrease in the mean number of tachyzoites. In addi-
tion, most of the 4-thiazolidinones showed more effective toxicity against intracellular parasites, with IC50 values ranging from 0.05
to 1 mM. According to results of antimicrobial activity, compounds 2f, 2l, and 2p showed best activity against Mycobacterium
luteus, 2c was more active against Mycobacterium tuberculosis, and 2g, 2l, and 2n showed same activity as nistatin (standard drug)
against Candida sp. (4249).
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
water infected with cysts, transplacental transmission
of the parasite to the fetus, or immunosuppression
in adults.4–7
Parasitic diseases, as toxoplasmosis, affect millions of
people, and they are responsible for some of the most
important and prevalent diseases of humans and
domestic animals. These diseases result in considerable
morbidity and mortality worldwide, especially in
developing countries.1–3 The toxoplasmosis is caused
by an intracellular parasite, Toxoplasma gondii, and
is associated with severe pathologies, including pneu-
monia, myocarditis, pulmonary necrosis, and severe
neurological disorders as psychomotor or mental
retardation. In humans, infection with T. gondii com-
monly occurs either following ingestion of food or
In immunocompetent patients, the infection with T. gon-
dii can cause symptoms as fever, headache, or myalgia.
However, serious cases can result in toxoplasmic
encephalitis (characterized by intracerebral mass lesions)
with mortality rates exceeding 30%.6,8,9 The current
therapy for clinical treatment of toxoplasmosis is isola-
tion or combination of chemotherapy using pyrimeth-
amine and sulfadiazine. This utilization is limited,
primarily because of high toxicity and serious side effects
for the host individual and its ineffectiveness in eliminat-
ing intracellular parasites. So, the discovery of less-toxic
and more efficacious parasite-specific drugs becomes
necessary against the infection by T. gondii and treat-
ment of toxoplasmosis.10–17
Keywords: Thiosemicarbazone; 4-Thiazolidinone; Antimicrobial activ-
ity; Anti-Toxoplasma gondii activity.
*
Corresponding author. E-mail: ajsg@ufpe.br
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.025

T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
447
18
´
In recent works, Tenorio et al. have reported the syn-
thesis and evaluation of anti-T. gondii activity of thiose-
micarbazones substituted at arylhydrazone moiety with
nitro substituents at ortho, meta, and para positions,
and 4-thiazolidinones substituted at N-3 position with
phenyl, methyl and hydrogen substituents, and with
the same groups at arylhydrazone moiety (Chart 1). This
paper was pioneer, because of the inexistence of studies
in the literature involving the action of these two classes
of compounds on intracellular T. gondii. It is important
to show a structural similarity between 4-thiazolidinones
synthesized from thiosemicarbazones, as both deriva-
tives possess the Ph–C@N–N–C–(S)–N moiety (Chart
1; A). According to biological results, the 4-thiazolidi-
none derivatives with a phenyl substituent at N-3 posi-
tion showed best values of IC50 for both infected cells
and intracellular parasites. In order to investigate the
anti-T. gondii activity of new structural analogues, we
describe the synthesis, elucidate the structure and
in vitro biological activities of thirty-two new
compounds, by fixing the phenyl group at N-4 and
N-3 positions in the thiosemicarbazones (1a–p) and
4-thiazolidinones (2a–p), respectively (Chart 1; B), and
introducing electron-withdrawing or electron-donating
substituents at arylhydrazone moiety in both derivatives
(Chart 1; C).
potential, due to inefficient parasite elimination and an
increasing number of resistant properties developed by
the pathogens.17
2. Results and discussion
2.1. Chemistry
The syntheses of the thiosemicarbazones (1a–p) and
4-thiazolidinones (2a–p) have been carried out utilizing
´
the same methodology previously reported by Tenorio
et al.18 (Scheme 1). Thiosemicarbazones (1a–p) were
characterized by H NMR, where signals at 9.89–11.95
1
and 9.13–10.25 ppm were attributed to NH and NH–
Ar groups.18 The IR spectra of the 4-thiazolidinones
(2a–p) showed absorption bands at about 1731–
1707 cmÀ1 characteristic for C@O stretching vibration
(acid group), and 1621–1606 cmÀ1 associated with
C@O amide I band. In addition, absorption bands
around 1360–1337 cmÀ1 characteristic for NCS bending
vibration provided confirmatory evidence for ring clo-
1
sure.19,26–28 Further support was obtained from the H
NMR spectra, where it did not display signs of the thi-
osemicarbazone protons (NH), and peaks resonated at
171.5–171.8, 161.6–167.0, and 42.3–42.5 ppm in the
13C NMR spectra assigned for C@O, C@N, and SCH
moieties. On the other hand, 1H NMR spectra exhibited
resonance assigned to the SCH group of the thiazolidine
ring appearing as triplet or double doublet (ABX pat-
tern) at 4.42–4.59 ppm due to the interaction with meth-
ylene protons of the acetyl group.29 The CH@N protons
in these structures were observed in the 8.13–8.43 ppm
region.30 In summary, all the synthesized compounds
exhibited satisfactory spectral data consistent with the
structures.
According to the literature, thiosemicarbazones, as a
class, and compounds which have the 4-thiazolidinone
ring are reported to possess various biological activities,
as antimicrobial, anti-inflammatory, antiviral, antipara-
sitic, and antituberculosis.19–25 As part of our studies to
discover new active compounds, thiosemicarbazones
(1a–p) and 4-thiazolidinones (2a–p) were screened for
in vitro anti-T. gondii activity. As thiosemicarbazones
18
synthesized by Tenorio et al. do not show activity
´
against various bacteria and fungal species, only the
4-thiazolidinones (2a–p) were evaluated for antimicro-
bial activity. The aim of these biological assays is to dis-
cover new drugs with antimicrobial and antiparasitic
2.2. Biological activities
The synthesized and purified thiosemicarbazones (1a–p)
and 4-thiazolidinones (2a–p) were submitted to incuba-
tion for 24 h in cultures of Vero cells infected with
T. gondii. After drug treatment, the cultures showed
decreasing percentage of infection. As a consequence
the mean number of normal tachyzoites decreased
(Tables 1–3). The compounds 1h, 2b, 2e, 2k, and 2l
had an effective action on intracellular parasite multi-
plication. The action of these compounds for a 50%
inhibition of parasite growth was 60.1 mM, which rep-
resents a range of about 12.5–30 lg/mL. However, sul-
fadiazine concentration for 50% inhibition is the same
A
B
R1
N
A
H
N
NH R1
O
N
B
Cyclization
N
S
S
N
R2
R2
COOH
C
C
Chart 1.
O
O
CHO
H
N
NH
S
O
N
R
S
C
O
N
N
H2N NH
NH
acetic acid
EtOH; H2O
reflux
S
N
toluene; DMF
reflux
R
R
COOH
Scheme 1. a; R = hydrogen, b; R = 4-chloro, c; R = 2,4-dichloro, d; R = 3,4-dichloro, e; R = 3-methyl, f; R = 2-fluoro, g; R = 4-fluoro, h;
R = 3-methoxy-4-hydroxy, i; R = 4-dimethylamino, j; R = 3-chloro, k; R = 4-methyl, l; R = 3-methoxy, m; R = 4-methoxy, n; R = 2,4-dimethoxy,
o; R = 3,4,5-trimethoxy, p; R = 3,5-di-tert-butyl-4-hydroxy.

448
T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
Table 1. Effect of 1a–p and 2a–p on cultures of Vero cells infected with T. gondii
Compound
% Infected Vero cellsa
Untreated (control)
Treated (mM)
5
0.1
2
8
20
1a
1b
83.07 6.3
77.64 0.17
61.09 2.12
59 1.4
82.5 4.2
77.82 10.6
34.8 9.19
33.28 13.4
38.02 22.6
39.82 4.9
28.13 0.7
31.83 8.4
38.7 5.6
31.32 4.9
21.96 6.36
26.86 9.89
14.32 0.7
28 10
13.52 1.4
21.05 0.8
12.08 2.82
4.54 1.4
0
0
0
0
0
0
0
0
0
0
0
0
0
1.49
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
70.01 5.6
49.33 14
49.18 7.7
66.23 9.89
91.5 3.5
1c
1d
1e
56.98 6.36
93.16 3.53
22.72 1.41
29.29 0.71
1f
1g
55.76 4
80.4 0.7
46.59 13.5
80.05 11.3
76.14 31
44.52 2.12
51.08 18.3
53.6 16.9
50.43 3.53
73 19
35.2
2
19.83 6
2.6 0.53
1h
1i
19.7 0.17
35.81 0.7
60.69 19
36.78 6.3
34.82 17
29.39 0.7
41.44 7.7
72.04 8.4
64.07 12
48.5 4.2
35.3 2.47
18.08 4.2
0
19.3 0.17
18.14 5.6
52.06 12
27.84 18.4
9.7 2.8
19 7.01
9.2 4.2
24.32 16
30.51 4.9
14.6 6.3
0
74.06 22
69.21 10.4
61.33 13.4
64.09 15
70.32 5.6
83.3 36
13.46 2.12
24.49 0.7
1j
1k
2.96 0.7
5.9 1.41
1l
1m
12.36 0.7
3.6 2.8
1n
1o
80.73 19.09
62.66 0.53
78.8 2.12
82.46 4.9
68.08 10.6
76.37 9.89
63.44 3.53
69.7 4.9
23.17 2.8
6.42 7.12
1p
2a
0
2b
2c
68.15 19
63.23 0.17
0
19.05 0.7
0
0
2d
2e
108
6
101
66 4.2
6
0
10.13 3.53
63.93 5.6
59.88 2.82
62.9 17
15.54 2.8
16.14 4.2
10.2 4.8
41.96 5.65
0
16.19 2.82
0
2f
2g
70.79 2.12
21.7 8.8
0
0
0
2h
2i
51.45 10.9
71.66 10.6
53.73 1.4
83.63 0.7
41.47 15
32.5 0.7
46.02 1.4
50.58 2.12
53.74 9.19
39.2 4.2
3.5 0.01
0
0
0
2j
2k
34.8 3.53
1.23 0.17
28.71 1.41
4.51 0.35
39.5 4.9
14.33 6.36
0
33.5
0
7
0
0
4.16 0.71
2l
2m
34.16 12.7
27.32
7.3 7.7
5.9 0.7
0
7
0
2n
2o
72.51 0.7
64.85 8.4
57.47 3.67
61.18 0.17
80.3 7.7
66.36 2.12
55.57 14
0
15.63 0.7
0
7.2 0.7
0
7.37 0.88
2p
Hydroxyurea
Sulfadiazine
53.55 8.83
58.23 5.65
53.84 0.7
22.78 9.19
48.71 0.7
10.4 5.65
29.55 0.7
6.84
1
a Values are mean SD (n = 3).
for host cell and parasites, about 3 mM (Table 3),
justifying its toxic effects. Moreover, some
thiosemicarbazones and 4-thiazolidinones have been
found to be more effective than hydroxyurea (reference
drug).
It was the difficulty to access intracellular taquizoites
that are harnessed by the chemical structure of the intra-
cellular environment, which led us to the use of these
compounds in the mM (millimolar) range. However,
these results are interesting because they demonstrate
the effective anti-T. gondii action of these new com-
pounds. Future researches can be done from these mol-
ecules’ structures to enable them to access the T. gondii
intravacuolar and make them effective in micro- to
nanomolar range.
In the concentrations of 2, 5, and 8 mM, some com-
pounds were highly toxic because very few or no
infected cells and intracellular parasites could be ob-
served, especially for 2d, 2i, 2k, and 2p (in the concentra-
tion of 2 mM), and for 2b, 2f, 2g, 2h, and 2n (in the
concentration of 5 mM). For higher concentrations,
the parasitophorous vacuole was enlarged and
contained distorted parasites. These cytostatic effects
were dependent on the drug concentrations. In the con-
centration of 20 mM for all drugs, no infected cells or
intracellular parasites were observed. In general, 4-thia-
zolidinones were more effective that thiosemicarbazones
for interrupting the T. gondii growth in lower concentra-
tions. According to Table 3, the toxicity of all com-
pounds was more effective against intracellular
parasites, except for 2g, 2i, 2j, 2m, and 2p. Similar values
were obtained with hydroxyurea but not with
sulfadiazine.
4-Thiazolidinone derivatives (2a–p) were preliminarily
tested for antimicrobial activity by the disc diffusion
method, and the results are reported in Tables 4 and
5. These results indicated that 2p showed best activities
against Staphylococcus aureus, Streptococcus faecalis,
Mycobacterium phlei, M. smegmatis, and M. tuberculo-
sis. 2p and 2o were more effective against Bacillus subtil-
is, 2m against Candida albicans, and various compounds
showed significant inhibition against Micrococcus luteus
and Candida sp. (IMUR 4249). None of the compounds
showed inhibitory effects against Klebsiella pneumoniae,
C. sp. (IMUR 1224), and C. sp. (IMRU 720). Only 2n
showed a little activity against Saccharomyces cerevisiae.

T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
Table 2. Effect of 1a–p and 2a–p on the intracellular multiplication of T. gondii
449
Compound
Mean number of intracellular parasitesa
Untreated (control)
Treated (mM)
0.1
2
5
8
2
20
1a
1b
413 1.4
446 33
431 46
717 57
575 83
461 9.19
541 61
574 66
462 25
663 103
445 92
565 55
545 65
771 62
596 48
504 2.82
467 24
341 58
614 49
890 15
442 20
512 7.7
163 42
458 80
558 144
369 79
417 33
447 153
278 0.7
560 58
391 65
224 22
804 36
487 22
429 45
434 101
359 47
577 43
510 12
406 8.4
332 132
368 42
290 35
526 45
330 31
409 22
439 72
684 29
461 14
517 57
336 71
235 92
530 33
822 85
266 47
504 7.7
367 3.5
109 19
103 11
371 90
232 14
67 13.4
195 4.5
32 11
63 16
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
15 12
0
1c
1d
60 2.12
140 4.2
25 2.82
10 14.14
11 7.5
1
0
0
1
1
0.7
1e
1f
1g
1
0.7
1h
1i
7
10
5
0.7
143 25
367 51
326 40
68 24
20 14
218 52
42 10
26 4.2
31 25
0
1j
1k
1l
1m
7
16
1.4
2
2
7
0
0.17
2.8
112
7
1n
1o
352 7.07
277 5.6
401 101
176 25
32 0.35
100 24
0
15 18
96 26
138 41
61 3.53
1p
2a
2
0
0
0
0
2.8
8
0
5
0
2.19
2b
2c
7.7
2d
2e
44 2.82
29 14
34 7.77
0
16 10
2f
2g
0
0
0
0
0
0
0
0
0
12
9
2
4
0
2h
2i
385 29
146 2.82
273
71 24
0
0
0
2j
2k
181 40
0
145 9.1
0
91 17
2l
2m
144 24
202 26
401 18
249 70
190 9.12
463 125
448 9.19
28 0.7
35 113
50 10
59 24
0
1
28 2.12
2.12
2n
2o
0
13 13
0
1
0
6
0
0.53
1.4
2p
Hydroxyurea
Sulfadiazine
12 3.5
393 30
7 0,7
28 24
a Values are mean SD (n = 3).
The values of minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC) or min-
imum fungicidal concentration (MFC) of the com-
pounds with mean zone inhibition (MZI) above or
equal to 18 mm are reported in Tables 6 and 7. The re-
sults showed that the compounds 2f, 2l, and 2p have less
MIC and MBC values against M. luteus when compared
with the standard drug (chloramphenicol). Similarly, 2c
is more active that rifampicin against M. tuberculosis. In
addition, the compounds 2f, 2g, 2l, and 2n showed equal
values for MIC and MFC against Candida sp. (IMUR
4249) when compared with nistatin.
with hydroxyurea and sulfadiazine. The toxicity of
majority compounds was more effective against intra-
cellular parasites, with IC50 values ranging from 0.05
to 1 mM.
The 4-thiazolidinones (2a–p) were evaluated in vitro
against bacteria and fungal species. The compounds 2f
(MIC = 40 lg/mL; MBC = 30 lg/mL), 2l (MIC =
40 lg/mL; MBC = 30 lg/mL), and 2p (MIC = 35 lg/
mL; MBC = 30 lg/mL) were more active than chloram-
phenicol against M. luteus, 2c (MIC = 70 lg/mL;
MBC = 60 lg/mL) was more active than rifampicin
against M. tuberculosis, and the compounds 2g, 2l, and
2n (MIC = 80 lg/mL; MBC = 40 lg/mL) showed same
activity as nistatin against Candida sp. (IMUR 4249).
3. Conclusion
The thiosemicarbazone (1a–p) and 4-thiazolidinone
(2a–p) derivatives were synthesized and characterized
based on their physical, analytical, and spectral data.
All the prepared compounds were evaluated in vitro
against T. gondii intracellular. All of them significantly
reduced the percentage of infected cells and mean num-
ber of tachyzoites per cell in 2 mM concentration. Var-
ious compounds showed best results when compared
It can be concluded that thiosemicarbazones and 4-thia-
zolidinones provide interesting leads for anti-T. gondii
drug discovery. Modifications to improve the potency
of these derivatives by diversification of the ring at
hydrazone moiety are currently under progress in our
laboratory. Further studies involving mechanistic action
are necessary to fully understand their anti-T. gondii
activity. In addition, the antimicrobial results confirm

450
T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
Table 3. IC50 values of 1a–p and 2a–p for uninfected cells, infected
cells and intracellular parasites in mM
cooled to ambient temp. After, the precipitate was col-
lected with filter under vacuum and washed with water.
White crystals; yield 91%; mp 193–195 ꢁC; IR (KBr):
3297 and 3158 (NH), 1540 (C@N), 1442 (N–CS–N),
Compound
IC50 (mM)a
Uninfected
cells
Infected
cells
Intracellular
parasites
1
1265 and 1198 (C@S); H NMR (300 MHz, CDCl3): d
10.47 (s, 1H, NH), 9.22 (s, 1H, NH–Ar), 8.00 (s, 1H,
CH@N), 7.65–7.68 (m, 4H, Ar–H), 7.40–7.45 (m, 5H,
Ar–H), 7.29–7.30 (m, 1H, Ar–H), 13C NMR
(75.4 MHz, CDCl3): d 175.8 (C@S), 142.8 (CH@N),
137.7 (Cq Ar), 132.9 (Cq Ar), 130.7 (CH Ar), 128.9
(CH Ar), 128.8 (CH Ar), 127.4 (CH Ar), 126.2 (CH
Ar), 124.5 (CH Ar).
1a
1b
5
0.05
1
5
7
2
5
5
0.05
1
4
0.05
1.5 0.05
10
6
1c
1d
1
0.05
0.05
0.05
1
2
2
1
1
0.05
0.05
0.05
0.05
0.05
6
1
1e
5
0.05
0.05
1f
1g
4
1.5 0.05
10 1.5
2
1
2
7
4
2
2
2
3
5
4
0.05
0.05
0.05
0.05
1
1h
1i
6
0.05
0.05
1
0.1 0.05
6
1
3
3
1
1
2
2
4
1
0.05
0.05
0.05
0.05
0.05
0.05
0.05
1
4.1.1. 4-Chlorobenzaldehyde 4-phenyl-3-thiosemicarba-
zone (1b). White crystals; yield 92%; mp 199–201 ꢁC;
IR (KBr): 3305 and 3134 (NH), 1537 (C@N), 1445
(N–CS–N), 1265 and 1194 (C@S); 1H NMR
(300 MHz, CDCl3): d 10.21 (s, 1H, NH), 9.16 (s, 1H,
NH–Ar), 7.92 (s, 1H, CH@N), 7.60–7.66 (m, 4H, Ar–
H), 7.38–7.45 (m, 4H, Ar–H), 7.26–7.30 (m, 1H, Ar–
H), 13C NMR (75.4 MHz, CDCl3): d 175.7 (C@S),
141.6 (CH@N), 137.5 (Cq Ar), 136.6 (Cq Ar), 131.4
(Cq Ar), 129.2 (CH Ar), 128.8 (CH Ar), 128.5 (CH
Ar), 126.4 (CH Ar), 124.7 (CH Ar).
1j
1k
10
8
1
1l
1m
5
0.05
0.06
0.05
1
0.05
0.05
0.06
1
6
1n
1o
5
10
1p
2a
10 1.5
1
0.05
5
2
2
4
9
2
2
0.05
0.06
0.05
0,05
1
0.05
2b
2c
1.5 0.05
1.5 0.05
1.5 0,05
0,5 0.05
1
1
0.05
0,05
2d
2e
1
2
0.05
0.05
0.5 0.05
0.05
0.05 0.02
0.05
0.05 0.03
0.05
2f
2g
0.05
0.05
1
4.1.2. 2,4-Dichlorobenzaldehyde 4-phenyl-3-thiosemicarb-
azone (1c). White crystals; yield 91%; mp 186–187 ꢁC;
IR (KBr): 3251 and 3142 (NH), 1537 (C@N), 1439
(N–CS–N), 1262 and 1199 (C@S); 1H NMR
(300 MHz, CDCl3): d 10.33 (s, 1H, NH), 9.15 (s, 1H,
NH–Ar), 8.32 (s, 1H, CH@N), 7.87 (d, 1H, J = 8.4 Hz,
Ar–H), 7.65 (dd, 1H, J = 8.4, 1.5 Hz, Ar–H), 7.66 (s,
1H, Ar–H), 7.38–7.43 (m, 3H, Ar–H), 7.23–7.30 (m,
2H, Ar–H), 13C NMR (75.4 MHz, CDCl3): d 175.8
(C@S), 138.5 (CH@N), 137.5 (Cq Ar), 136.7 (Cq Ar),
135.1 (Cq Ar), 129.9 (CH Ar), 129.2 (Cq Ar), 128.8
(CH Ar), 127.8 (CH Ar), 127.6 (CH Ar), 126.3 (CH
Ar), 124.4 (CH Ar).
0.05
2h
2i
0.1 0.005
0.05
10 0.05
3
0.05
0.05
1
1
2j
2k
3
0.1 0.05
0.05
2
1
5
6
5
2
1
1
8
0.05
1
0.05 0.02
0.1 0.05
0.5 0.05
0.5 0.05
0.5 0.05
2l
2m
3
0.5 0.05
0.05
0.05
0.06
0.05
0.006
0.05
0.05
2n
2o
2
1
1
0.05
0.05
0.05
2p
Hydroxyurea
Sulfadiazine
1
0.05
0.1 0.05
3 0.05
0.5 0.05
0.05
3
a Values are mean SD (n = 3).
4.1.3. 3,4-Dichlorobenzaldehyde 4-phenyl-3-thiosemicar-
bazone (1d). White crystals; yield 86%; mp 206–208 ꢁC;
IR (KBr): 3310 and 3134 (NH), 1545 (C@N), 1466
(N–CS–N), 1268 and 1191 (C@S); 1H NMR
(300 MHz, CDCl3): d 11.95 (s, 1H, NH), 10.25 (s, 1H,
NH–Ar), 8.34 (d, 1H, J = 1.8 Hz, Ar–H), 8.11 (s, 1H,
CH@N), 7.81 (dd, 1H, J = 8.4, 1.8 Hz, Ar–H), 7.67 (d,
1H, J = 8.4 Hz, Ar–H), 7.20-7.53 (m, 5H, Ar–H), 13C
NMR (75.4 MHz, CDCl3): d 175.6 (C@S), 142.0
(CH@N), 133.3 (Cq Ar), 132.7 (Cq Ar), 131.9 (Cq
Ar), 131.4 (Cq Ar), 130.8 (CH Ar), 129.2 (CH Ar),
129.1 (CH Ar), 127.9 (CH Ar), 125.5 (CH Ar), 125.3
(CH Ar).
the fact that the 4-thiazolidinone ring has great biologi-
cal potential.
4. Experimental
¨
The melting points were determined on BUCHI-535
apparatus and are uncorrected. IR spectra were mea-
sured on BRUKER IFS-66 IR spectrophotometer. H
1
NMR were recorded on UNITY PLUS-300 MHz-VAR-
IAN spectrometer by using tetramethylsilane as internal
standard. The chemical shifts are reported in d units,
and coupling constants (J) are reported in hertz. TLC
development was conducted on 0.25 mm silica gel plates
(60F254, Merck).
4.1.4. 3-Methylbenzaldehyde 4-phenyl-3-thiosemicarba-
zone (1e). White crystals; yield 89.5%; mp 165–166 ꢁC;
IR (KBr): 3297 and 3147 (NH), 1540 (C@N), 1444
(N–CS–N), 1265 and 1202 (C@S); 1H NMR
(300 MHz, CDCl3): d 10.05 (s, 1H, NH), 9.21 (s, 1H,
NH–Ar), 7.91 (s, 1H, CH@N), 7.67 (d, 2H, J = 8.1 Hz,
Ar–H), 7.40-7.49 (m, 4H, Ar–H), 7.23-7.34 (m, 3H,
Ar–H), 2.39 (s, 3H, CH3), 13C NMR (75.4 MHz,
CDCl3): d 175.6 (C@S), 143.4 (CH@N), 138.6 (Cq
Ar), 137.7 (Cq Ar), 132.9 (Cq Ar), 131.5 (CH Ar),
4.1. Representative procedure for (1a–q). Benzaldehyde
4-phenyl-3-thiosemicarbazone (1a)
To a solution of 0.0119 mol of 4-phenylthiosemicarbaz-
ide in 11 mL of EtOH and 22 mL of water were added
0.0125 mol of benzaldehyde and 0.55 mL of acetic acid.
The mixture was stirred under reflux for another 1 h and

T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
451
Table 4. Inhibitory zone diameters (mm) of 2a–p against tested
bacterial strains by disc diffusion method
Ar–H), 7.66 (d, 2H, J = 7.8 Hz, Ar–H), 7.38 (t, 3H,
J = 7.8 Hz, Ar–H), 7.26 (t, 3H, J = 7.2 Hz, Ar–H),
7.19 (t, 3H, J = 7.2 Hz, Ar–H), 7.14 (t, 3H, J = 9.3 Hz,
Ar–H), 13C NMR (75.4 MHz, CDCl3): d 175.6 (C@S),
160.5 (Cq Ar), 136.1 (Cq Ar), 132.3 (CH Ar), 132.1
(CH Ar), 128.8 (CH Ar), 126.8 (Cq Ar), 126.2 (CH
Ar), 124.5 (CH Ar), 124.4 (CH Ar), 124.3 (Cq Ar),
116.2 (CH Ar).
Compound
Mean zone inhibition (MZI) in mma
Sa Bs Ml Ec Kp Sf Mp Ms Mt
2a
2b
—
—
15 20
13 21
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12 07 09 14
15 12 15 17
15 12 16 22
11 12 13 18
2c
2d
10 17 20
11 16 19
2e
—
—
—
—
—
—
—
—
22
11
—
—
—
4.1.6. 4-Fluorobenzaldehyde 4-phenyl-3-thiosemicarba-
zone (1g). White crystals; yield 95%; mp 178 ꢁC; IR
(KBr): 3313 and 3134 (NH), 1548 (C@N), 1447 (N–
CS–N), 1230 and 1198 (C@S); 1H NMR (300 MHz,
CDCl3): d 10.38 (s, 1H, NH), 9.16 (s, 1H, NH–Ar),
7.96 (s, 1H, CH@N), 7.63–7.70 (m, 4H, Ar–H), 7.39–
7.45 (m, 2H, Ar–H), 7.25–7.30 (m, 1H, Ar–H), 7.08–
7.14 (t, 2H, J = 7.8 Hz, Ar–H), 13C NMR (75.4 MHz,
CDCl3): d 175.6 (C@S), 165.8 (Cq Ar), 162.4 (Cq Ar),
142.0 (CH@N), 137.6 (Cq Ar), 129.3 (CH Ar), 129.2
(CH Ar), 128.8 (CH Ar), 126.3 (Cq Ar), 124.7 (CH
Ar), 116.1 (CH Ar).
2f
2g
15 25
14 15
16 07 13 14
16 07 12 14
2h
2i
—
—
—
—
—
10
16
16
—
—
12
12
11
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2j
2k
2l
2m
14 09 22
—
—
—
11 16
15
19 25
2n
2o
—
2p
Rifampicin
26 19 22
18 16 26 23
31 20 49 14 NT 20 25 30 28
Chloramphenicol 25 27 50 27 25 NT NT NT NT
Rifampicin (100 lg/disc) and chloramphenicol (100 lg/disc) were used
as positive reference; compounds 2a–p (300 lg/disc).
—, indicates no sensitivity or MZI lower that 7 mm.
NT, not tested.
4.1.7. 4-Hydroxy-3-methoxybenzaldehyde 4-phenyl-3-thi-
osemicarbazone (1h). White crystals; yield 88.5%; mp
176–177 ꢁC; IR (KBr): 3325 and 3166 (NH), 1551
(C@N), 1447 (N–CS–N), 1268 and 1199 (C@S); 1H
NMR (300 MHz, DMSO-d6): d 11.69 (s, 1H, NH),
9.98 (s, 1H, NH–Ar), 9.54 (s, 1H, OH), 8.05 (s, 1H,
CH@N), 7.55 (t, 3H, J = 8.4 Hz, Ar–H), 7.37 (t, 2H,
J = 7.5 Hz, Ar–H), 7.20 (t, 2H, J = 8.4 Hz, Ar–H),
6.81 (d, 1H, J = 7.5 Hz, Ar–H), 3.84 (s, 3H, OCH3),
13C NMR (75.4 MHz, DMSO-d6): d 175.5 (C@S),
148.3 (Cq Ar), 146.9 (Cq Ar), 143.4 (CH@N), 137.8
(Cq Ar), 128.8 (CH Ar), 126.3 (Cq Ar), 125.3 (CH
Ar), 124.8 (CH Ar), 122.8 (CH Ar), 114.6 (CH Ar),
108.2 (CH Ar), 56.1 (OCH3).
Sa, S. aureus; Bs, B. subtilis; Ml, M. luteus; Ec, E. coli; Kp,
K. pneumoniae; Sf, S. faecalis; Mp, M. phlei; Ms, M. smegmatis; Mt,
M. tuberculosis.
a Values are mean (n = 3).
128.7 (CH Ar), 128.7 (CH Ar), 127.9 (CH Ar), 126.2
(CH Ar), 124.7 (CH Ar), 21.2 (CH3).
4.1.5. 2-Fluorobenzaldehyde 4-phenyl-3-thiosemicarba-
zone (1f). White crystals; yield 93%; mp 181–182 ꢁC;
IR (KBr): 3297 and 3165 (NH), 1535 (C@N), 1442
(N–CS–N), 1263 and 1198 (C@S); 1H NMR
(300 MHz, CDCl3): d 10.40 (s, 1H, NH), 9.22 (s, 1H,
NH–Ar), 8.19 (s, 1H, CH@N), 7.85 (t, 1H, J = 7.2 Hz,
4.1.8. 4-Dimethylaminobenzaldehyde 4-phenyl-3-thiosem-
icarbazone (1i). Light yellow crystals; yield 81.5%; mp
208–210 ꢁC; IR (KBr): 3325 and 3298 (NH), 1594
Table 5. Inhibitory zone diameters (mm) of 2a–p against tested fungal strains by disc diffusion method
Compound
Mean zone inhibition (MZI) in mma
S. cerevisiae
Candida sp. (IMUR 1224)
Candida sp. (IMUR 720)
C. albicans
Candida sp. (IMUR 4249)
2a
2b
—
—
—
—
—
—
—
—
—
—
—
12
—
—
20
—
—
—
—
—
—
—
—
—
—
—
—
—
—
30
—
—
—
—
—
—
—
—
—
—
—
—
—
—
20
08
08
07
07
09
07
08
—
—
—
15
13
10
09
26
—
20
14
21
—
20
22
16
11
18
16
20
15
—
32
2c
2d
2e
2f
2g
2i
2j
2l
2m
2n
2o
2p
Nistatin
Nistatin (100 lg/disc) was used as positive reference; compounds 2a–p (300 lg/disc).
—, indicates no sensitivity or MZI lower that 7 mm.
a Values are mean (n = 3).

452
T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
Table 6. Minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) in lg/mL of selected compounds
Ar), 8.18 (br s, 1H, Ar–H), 8.13 (s, 1H, CH@N), 7.74
(m, 1H, Ar–H), 7.52 (m, 2H, Ar–H), 7.40 (m, 4H, Ar–
H), 7.22 (m, 1H, Ar–H), 13C NMR (75.4 MHz,
DMSO-d6): d 176.3 (C@S), 141.7 (CH@N), 139.0 (Cq
Ar), 136.3 (Cq Ar), 133.7 (Cq Ar), 130.4 (CH Ar),
129.6 (CH Ar), 128.0 (CH Ar), 127.0 (CH Ar), 126.4
(CH Ar), 126.1 (CH Ar), 125.5 (CH Ar).
Organism
Compound
MICa
MBCb
(lg/mL)
(lg/mL)
M. luteus
2a
2b
100
90
90
80
50
50
55
30
30
100
30
40
2c
2d
60
60
2e
2f
60
40
4.1.10. 4-Methylbenzaldehyde 4-phenyl-3-thiosemicarba-
zone (1k). White crystals; yield 77.5%; mp 189–190 ꢁC;
IR (KBr): 3297 and 3142 (NH), 1548 (C@N), 1439
(N–CS–N), 1260 and 1198 (C@S); 1H NMR
(300 MHz, CDCl3): d 10.19 (s, 1H, NH), 9.21 (s, 1H,
NH–Ar), 7.93 (s, 1H, CH@N), 7.66 (dt, 2H, J = 1.2,
8.1 Hz, Ar–H), 7.57 (d, 2H, J = 8.1 Hz, Ar–H), 7.42
(dt, 2H, J = 1.5, 6.6 Hz, Ar–H), 7.27 (dt, 2H, J = 1.2,
8.1 Hz, Ar–H), 7.22 (d, 1H, J = 8.1 Hz, Ar–H), 2.39 (s,
3H, CH3), 13C NMR (75.4 MHz, CDCl3): d 175.6
(C@S), 143.2 (CH@N), 141.2 (Cq Ar), 137.7 (Cq Ar),
130.2 (Cq Ar), 129.6 (CH Ar), 128.7 (CH Ar), 127.4
(CH Ar), 126.2 (CH Ar), 124.6 (CH Ar).
2l
2o
40
110
35
2p
Chloramphenicol
50
S. faecalis
2p
Chloramphenicol
200
70
190
60
S. aureus
2p
Chloramphenicol
50
25
45
20
M. smegmatis
M. tuberculosis
2p
Rifampicin
150
140
140
130
2c
2d
70
100
110
110
60
90
4.1.11. 3-Methoxybenzaldehyde 4-phenyl-3-thiosemicar-
bazone (1l). White crystals; yield 94%; mp 154 ꢁC; IR
(KBr): 3325 and 3155 (NH), 1545 (C@N), 1442 (N–
CS–N), 1276 and 1194 (C@S); 1H NMR (300 MHz,
CDCl3): d 10.75 (s, 1H, NH), 9.21 (s, 1H, NH–Ar),
7.98 (s, 1H, CH@N), 7.65 (d, 2H, J = 8.4 Hz, Ar–H),
7.42 (t, 2H, J = 7.8 Hz, Ar–H), 7.20-7.35 (m, 4H, Ar–
H), 6.97 (dt, 1H, J = 1.2, 8.1 Hz, Ar–H), 3.84 (s, 3H,
OCH3), 13C NMR (75.4 MHz, CDCl3): d 175.7 (C@S),
159.8 (Cq Ar), 143.0 (CH@N), 137.6 (Cq Ar), 134.3
(Cq Ar), 129.9 (CH Ar), 128.8 (CH Ar), 126.3 (CH
Ar), 124.7 (CH Ar), 120.4 (CH Ar), 116.6 (CH Ar),
111.9 (CH Ar), 55.3 (OCH3).
2p
Rifampicin
100
100
a MIC, minimum inhibitory concentration (the lowest concentration
that inhibited the bacterial growth).
b MBC, minimum bactericidal concentration (the lowest concentration
at which no bacterial growth was observed).
Table 7. Minimum inhibitory concentration (MIC) and minimum
fungicidal concentration (MFC) in lg/mL of selected compounds
Compound
MICa(lg/mL)
MFCb (lg/mL)
2b
2d
150
120
80
140
100
70
2f
2g
4.1.12. 4-Methoxybenzaldehyde 4-phenyl-3-thiosemicar-
bazone (1m). White crystals; yield 94%; mp 178–179 ꢁC;
IR (KBr): 3325 and 3145 (NH), 1543 (C@N), 1447 (N–
CS–N), 1249 and 1198 (C@S); 1H NMR (300 MHz,
CDCl3): d 9.89 (s, 1H, NH), 9.18 (s, 1H, NH–Ar),
7.87 (s, 1H, CH@N), 7.64 (dt, 4H, J = 2.1, 9 Hz, Ar–
H), 7.41 (dt, 2H, J = 2.1, 7.2 Hz, Ar–H), 7.25 (dt, 1H,
J = 1.5, 7.2 Hz, Ar–H), 6.93 (dt, 2H, J = 1.8, 7.8 Hz,
Ar–H), 3.85 (s, 3H, OCH3), 13C NMR (75.4 MHz,
CDCl3): d 175.4 (C@S), 161.7 (Cq Ar), 142.9 (CH@N),
137.8 (Cq Ar), 129.1 (CH Ar), 128.7 (CH Ar), 126.1 (CH
Ar), 125.5 (Cq Ar), 124.5 (CH Ar), 114.3 (CH Ar), 55.4
(OCH3).
80
80
70
70
2l
2n
80
80
70
70
Nistatin
Microorganism: Candida sp. (IMUR 4249).
a MIC, minimum inhibitory concentration (the lowest concentration
that inhibited the fungal growth).
b MFC, minimum fungicidal concentration (the lowest concentration
at which no fungal growth was observed).
(C@N), 1442 (N–CS–N), 1257 and 1182 (C@S); 1H
NMR (300 MHz, DMSO-d6): d 11.59 (s, 1H, NH),
9.92 (s, 1H, NH–Ar), 8.03 (s, 1H, CH@N), 7.70 (d,
2H, J = 8.7 Hz, Ar–H), 7.59 (d, 2H, J = 7.5 Hz, Ar–
H), 7.35 (t, 2H, J = 7.8 Hz, Ar–H), 7.18 (t, 1H,
J = 7.5 Hz, Ar–H), 7.72 (d, 2H, J = 8.7 Hz, Ar–H),
2.97 (s, 6H, N(CH3)2), 13C NMR (75.4 MHz, DMSO-
d6): d 174.8 (C@S), 151.5 (Cq Ar), 143.9 (CH@N),
139.2 (Cq Ar), 129.0 (CH Ar), 127.9 (CH Ar), 125.5
(CH Ar), 125.0 (CH Ar), 121.1 (Cq Ar), 111.6 (CH Ar).
4.1.13. 2,4-Dimethoxybenzaldehyde 4-phenyl-3-thiosemi-
carbazone (1n). Light yellow crystals; yield 93.5%; mp
201–202 ꢁC; IR (KBr): 3309 and 3194 (NH), 1543
(C@N), 1453 (N–CS–N), 1284 and 1206 (C@S); 1H
NMR (300 MHz, DMSO-d6): d 11.71 (s, 1H, NH),
10.00 (s, 1H, NH–Ar), 8.43 (s, 1H, CH@N), 8.19 (d,
1H, J = 8.7 Hz, Ar–H), 7.57 (d, 2H, J = 7.2 Hz, Ar–
H), 7.36 (t, 2H, J = 8.1 Hz, Ar–H), 7.19 (t, 1H,
J = 7.2 Hz, Ar–H), 6.62 (d, 1H, J = 1.8 Hz, Ar–H),
6.58 (d, 1H, J = 8.7 Hz, Ar–H), 3.82 (s, 6H, OCH3),
13C NMR (75.4 MHz, DMSO-d6): d 175.3 (C@S),
162.5 (Cq Ar), 159.3 (Cq Ar), 139.1 (CH@N), 138.6
(Cq Ar), 127.9 (CH Ar), 127.7 (CH Ar), 125.7 (CH
4.1.9. 3-Chlorobenzaldehyde 4-phenyl-3-thiosemicarbazone
(1j). White crystals; yield 96%; mp 194–195 ꢁC; IR
(KBr): 3297 and 3139 (NH), 1551 (C@N), 1442 (N–
CS–N), 1265 and 1194 (C@S); 1H NMR (300 MHz,
DMSO-d6): d 11.93 (s, 1H, NH), 10.25 (s, 1H, NH–

T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
453
Ar), 125.1 (CH Ar), 114.7 (Cq Ar), 106.3 (CH Ar), 97.8
(CH Ar), 55.7 (OCH3), 55.4 (OCH3).
1623 (NC@O), 1575 and 1548 (C@N), 1341 (NCS),
1248 (N–N@C), 1034 (CS); 1H NMR (75.4 MHz,
DMSO-d6): d 12.84 (br s, 1H, CO2H), 8.34 (s, 1H,
CH@N), 7.74–7.77 (m, 2H, Ar–H), 7.38-7.56 (m, 7H,
Ar–H), 4.57 (t, 1H, J = 5.7 Hz, CH), 3.13 (d, 2H,
J = 5.7 Hz, CH2), 13C NMR (75.4 MHz, DMSO-d6): d
173.7 (CO2H), 171.8 (C@O), 165.4 (C@N), 154.8
(CH@N), 135.3 (Cq Ar), 135.1 (Cq Ar), 132.9 (Cq
Ar), 129.3 (CH Ar), 129.1 (CH Ar), 128.9 (CH Ar),
128.7 (CH Ar), 128.2 (CH Ar), 42.5 (CH), 36.7 (CH2).
4.1.14. 3,4,5-Trimethoxybenzaldehyde 4-phenyl-3-thiose-
micarbazone (1o). Light yellow crystals; yield 91%; mp
161–162 ꢁC; IR (KBr): 3299 and 3177 (NH), 1556
(C@N), 1417 (N–CS–N), 1262 and 1191 (C@S); 1H
NMR (300 MHz, CDCl3): d 10.31 (s, 1H, NH), 9.13
(s, 1H, NH–Ar), 7.88 (s, 1H, CH@N), 7.63 (d, 2H,
J = 8.1 Hz, Ar–H), 7.43 (t, 2H, J = 8.1 Hz, Ar–H),
7.28 (t, 1H, J = 7.5 Hz, Ar–H), 6.88 (s, 2H, Ar–H),
3.92 (s, 9H, OCH3), 13C NMR (75.4 MHz, CDCl3): d
175.4 (C@S), 153.4 (Cq Ar), 143.6 (CH@N), 140.2 (Cq
Ar), 137.6 (Cq Ar), 128.7 (CH Ar), 128.3 (Cq Ar),
126.4 (CH Ar), 125.1 (CH Ar), 104.5 (CH Ar), 60.8
(OCH3), 56.1 (OCH3).
4.2.2. 2-[[(2,4-Dichlorophenyl)methylene]hydrazono]-4-
oxo-3-phenyl-5-thiazolidineacetic acid (2c). White crys-
tals; yield 63%; mp 236–237 ꢁC; IR (KBr): 1724
(C@O), 1614 (NC@O), 1565 and 1538 (C@N), 1337
(NCS), 1244 (N–N@C), 1037 (CS); 1H NMR
(300 MHz, DMSO-d6): d 12.85 (br s, 1H, CO2H), 8.43
(s, 1H, CH@N), 7.98 (d, 1H, J = 8.7 Hz, Ar–H), 7.72
(d, 1H, J = 2.1 Hz, Ar–H), 7.38–7.57 (m, 6H, Ar–H),
4,59 (t, 1H, J = 5.8 Hz, CH), 3.13 (d, 2H, J = 5.8 Hz,
CH2), 13C NMR (75.4 MHz, DMSO-d6): d 173.8
(CO2H), 171.8 (C@O), 167.0 (C@N), 152.3 (CH@N),
135.9 (Cq Ar), 135.9 (Cq Ar), 134.4 (Cq Ar), 130.1
(Cq Ar), 129.5 (CH Ar), 129.1 (CH Ar), 128.8 (CH
Ar), 128.1 CH Ar), 42.6 (CH), 36.6 (CH2).
4.1.15. 3,5-Bis(1,1-dimethylethyl)4-hydroxybenzaldehyde-
4-phenyl-3-thiosemicarbazone (1p). Yellow crystals; yield
100%; mp 204–205 ꢁC; IR (KBr): 3317 and 3137 (NH),
1535 (C@N), 1439 (N–CS–N), 1268 and 1201 (C@S);
1H NMR (300 MHz, CDCl3): d 9.68 (s, 1H, NH), 9.17
(s, 1H, NH–Ar), 7.84 (s, 1H, CH@N), 7.67 (dd, 2H,
J = 1.5, 8.4 Hz, Ar–H), 7.48 (s, 2H, Ar–H), 7.42 (dt,
2H, J = 1.5, 7.5 Hz, Ar–H), 7.26 (dt, 1H, J = 1.2,
7.8 Hz, Ar–H), 5.56 (s, 1H, OH), 1.45 (s, 18H, CH3),
13C NMR (75.4 MHz, CDCl3): d 175.2 (C@S), 156.5
(Cq Ar), 144.5 (CH@N), 137.9 (Cq Ar), 136.5 (Cq
Ar), 128.8 (CH Ar), 126.1 (CH Ar), 124.7 (CH Ar),
124.5 (CH Ar), 124.2 (Cq Ar), 34.3 (Cq C(CH3)3),
30.1 (CH3).
4.2.3. 2-[[(3,4-Dichlorophenyl)methylene]hydrazono]-4-
oxo-3-phenyl-5-thiazolidineacetic acid (2d). White crys-
tals; yield 45.5%; mp 254–256 ꢁC; IR (KBr): 1730
(C@O), 1611 (NC@O), 1569 and 1536 (C@N), 1340
(NCS), 1241 (N–N@C), 1033 (CS); 1H NMR
(300 MHz, DMSO-d6): d 12.82 (br s, 1H, CO2H), 8.34
(s, 1H, CH@N), 7,92 (s, 1H, Ar–H), 7.72 (s, 2H, Ar–
H), 7.38-7.56 (m, 5H, Ar–H), 4.59 (t, 1H, J = 6.0 Hz,
CH), 3.14 (d, 2H, J = 6.0 Hz, CH2), 13C NMR
(75.4 MHz, DMSO-d6): d 173.8 (CO2H), 171.8 (C@O),
166.2 (C@N), 155.4 (CH@N), 135.1 (Cq Ar), 134.8
(Cq Ar), 133.0 (Cq Ar), 131.7 (Cq Ar), 131.2 (CH Ar),
129.2 (CH Ar), 129.1 (CH Ar), 128.8 (CH Ar), 128.1
(CH Ar), 127.2 (CH Ar), 42.6 (CH), 36.6 (CH2).
4.2. Representative procedure for (2a–q). 2-[(Phenyl-
methylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic
acid (2a)
A solution of 0.0078 mol of benzaldehyde 4-phenyl-3-
thiosemicarbazone and 0.0352 mol of maleic anhydride
in 50 mL of dried toluene was stirred until reflux, and
2 mL of DMF was added until complete solubilization.
The mixture was stirred under the same conditions till
the completion of the reaction (5–9 h). After, the solvent
was evaporated at reduced pressure and the crude prod-
uct was extracted with ethyl acetate twice. The organic
layer was treated with anhydrous sodium sulfate and
evaporated again. Finally the product was purified by
recrystallization from MeOH/water. White crystals;
yield 76%; mp 212–214 ꢁC; IR (KBr): 1707 (C@O),
1621 (NC@O), 1582 and 1555 (C@N), 1344 (NCS),
1252 (N–N@C), 1030 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.83 (br s, 1H, CO2H), 8.33 (s, 1H,
CH@N), 7,74 (dd, 2H, J = 2.1, 6.6 Hz, Ar–H), 7.38–
7.56 (m, 8H, Ar–H), 4.57 (t, 1H, J = 6.0 Hz, CH), 3.13
(d, 2H, J = 6.0 Hz, CH2), 13C NMR (75.4 MHz,
DMSO-d6): d 173.7 (CO2H), 171.8 (C@O), 164.8
(C@N), 157.7 (CH@N), 135.1 (Cq Ar), 134.0 (Cq Ar),
130.8 (CH Ar), 129.0 (CH Ar), 128.8 (CH Ar), 128.7
(CH Ar), 128.2 (CH Ar), 127.7 (CH Ar), 42.5 (CH),
36.7 (CH2).
4.2.4. 2-[[(3-Methylphenyl)methylene]hydrazono]-4-oxo-
3-phenyl-5-thiazolidineacetic acid (2e). White crystals;
yield 67%; mp 221–222 ꢁC; IR (KBr): 1724 (C@O),
1615 (NC@O), 1582 and 1546 (C@N), 1351 (NCS),
1232(N–N@C), 1034 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.83 (br s, 1H, CO2H), 8.28 (s, 1H,
CH@N), 7.25–7.54 (m, 9H, Ar–H), 4.57 (t, 1H,
J = 6 Hz, CH), 3.13 (d, 2H, J = 6 Hz, CH2), 2.33 (s,
3H, CH3), 13C NMR (75.4 MHz, DMSO-d6): d 173.7
(CO2H), 171.8 (C@O), 164.7 (C@N), 157.8 (CH@N),
138.0 (Cq Ar), 135.2 (Cq Ar), 134.0 (Cq Ar), 131.5
(CH Ar), 129.1 (CH Ar), 128.7 (CH Ar), 128.2 (CH
Ar), 127.9 (CH Ar), 125.2 (CH Ar), 42.5 (CH), 36.7
(CH2), 20.9 (CH3).
4.2.5. 2-[[(2-Fluorophenyl)methylene]hydrazono]-4-oxo-3-
phenyl-5-thiazolidineacetic acid (2f). Light yellow crys-
tals; yield 60%; mp 217 ꢁC; IR (KBr): 1721 (C@O),
1621 (NC@O), 1582 and 1548 (C@N), 1344 (NCS),
1232 (N–N@C), 1034 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.85 (br s, 1H, CO2H), 8.37 (s, 1H,
CH@N), 7.90 (dt, 1H, J = 1.5, 8.4 Hz, Ar–H), 7.43-
4.2.1. 2-[[(4-Chlorophenyl)methylene]hydrazono]-4-oxo-3-
phenyl-5-thiazolidineacetic acid (2b). White crystals;
yield 56.5%; mp 245–247 ꢁC; IR (KBr): 1721 (C@O),

454
T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
7.56 (m, 4H, Ar–H), 7.38–7.41 (m, 2H, Ar–H), 7.24-7.31
(m, 2H, Ar–H), 4.58 (t, 1H, J = 6.0 Hz, CH), 3.13 (d,
2H, J = 6.0 Hz, CH2). 13C NMR (75.4 MHz, DMSO-
d6): d 173.8 (CO2H), 171.8 (C@O), 166.1 (C@N), 162.6
(Cq Ar), 159.2 (CH@N), 150.6 (CH Ar), 135.1 (Cq
Ar), 132.9 (CH Ar), 129.1 (CH Ar), 128.8 (CH Ar),
128.1 (CH Ar), 127.4 (CH Ar), 124.9 (CH Ar), 121.4
(Cq Ar), 42.6 (CH), 36.7 (CH2).
(m, 2H, Ar–H), 7.37–7.55 (m, 7H, Ar–H), 4.58 (t, 1H,
J = 5.4 Hz, CH), 3.13 (d, 2H, J = 5.4 Hz, CH2),
13C NMR (75.4 MHz, DMSO-d6): d 173.8 (CO2H),
171.8 (C@O), 168.4 (CH@N), 165.9 (C@N), 136.2 (Cq
Ar), 135.1 (Cq Ar), 133.6 (Cq Ar), 130.8 (CH Ar), 130.4
(CH Ar), 129.1 (CH Ar), 128.8 (CH Ar), 128.2 (CH Ar),
127.0 (CH Ar), 126.2 (CH Ar), 42.5 (CH), 36.7 (CH2).
4.2.10. 2-[[(4-Methylphenyl)methylene]hydrazono]-4-oxo-
3-phenyl-5-thiazolidineacetic acid (2k). White crystals;
yield 54%; mp 224–226 ꢁC; IR (KBr): 1727 (C@O), 1615
(NC@O), 1573 and 1556 (C@N), 1347 (NCS), 1236 (N–
4.2.6. 2-[[(4-Fluorophenyl)methylene]hydrazono]-4-oxo-3-
phenyl-5-thiazolidineacetic acid (2g). White crystals;
yield 56%; mp 227–229 ꢁC; IR (KBr): 1731 (C@O),
1621 (NC@O), 1592 and 1550 (C@N), 1347 (NCS),
1226 (N–N@C), 1040 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.83 (br s, 1H, CO2H), 8.34 (s, 1H,
CH@N), 7.80 (dd, 2H, J = 8.7, 5.7 Hz, Ar–H), 7.38-
7.56 (m, 5H, Ar–H), 7.29 (t, 2H, J = 8.7 Hz, Ar–H),
4.57 (t, 1H, J = 6.0 Hz, CH), 3.13 (d, 2H, J = 6.0 Hz,
CH2), 13C NMR (75.4 MHz, DMSO-d6): d 173.7
(CO2H), 171.8 (C@O), 165.2 (Cq Ar), 164.9 (C@N),
161.9 (Cq Ar), 156.6 (CH@N), 135.2 (Cq Ar), 130.7
(Cq Ar), 130.0 (CH Ar), 129.1 (CH Ar), 128.7 (CH
Ar), 128.2 (CH Ar), 42.5 (CH), 36.7 (CH2).
1
N@C), 1034 (CS); H NMR (300 MHz, DMSO-d6): d
12.84 (br s, 1H, CO2H), 8.27 (s, 1H, CH@N), 7.62 (d,
2H, J = 8.4 Hz, Ar–H), 7.36-7.55 (m, 5H, Ar–H), 7.25
(d, 2H, J = 8.4 Hz, Ar–H), 4.55 (t, 1H, J = 6 Hz, CH),
3.11 (d, 2H, J = 6 Hz, CH2), 2.33 (s, 3H, CH3), 13C
NMR (75.4 MHz, DMSO-d6): d 173.7 (CO2H), 171.8
(C@O), 164.3 (C@N), 157.7 (CH@N), 140.8 (Cq Ar),
135.2 (Cq Ar), 131.3 (Cq Ar), 129.4 (CH Ar), 129.0 (CH
Ar), 128.7 (CH Ar), 128.2 (CH Ar), 127.7 (CH Ar), 42.5
(CH), 36.8 (CH2), 21.1 (CH3).
4.2.11. 2-[[(3-Methoxyphenyl)methylene]hydrazono]-4-oxo-
3-phenyl-5-thiazolidineacetic acid (2l). White crystals;
yield 57%; mp 235–237 ꢁC; IR (KBr): 1727 (C@O),
1615 (NC@O), 1582 and 1559 (C@N), 1341 (NCS),
1241 (N–N@C), 1030 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.76 (br s, 1H, CO2H), 8.28 (s, 1H,
CH@N), 7.28–7.55 (m, 8H, Ar–H), 7.01-7.04 (m, 1H,
Ar–H), 4.56 (t, 1H, J = 5.7 Hz, CH), 3.78 (s, 3H,
OCH3), 3.13 (d, 2H, J = 5.7 Hz, CH2), 13C NMR
(75.4 MHz, DMSO-d6): d 173.6 (CO2H), 171.5 (C@O),
164.6 (C@N), 159.4 (Cq Ar),157.5 (CH@N), 135.3 (Cq
Ar), 135.1 (Cq Ar), 129.8 (CH Ar), 128.9 (CH Ar),
128.6 (CH Ar), 128.0 (CH Ar), 120.2 (CH Ar), 116.4
(CH Ar), 112.6 (CH Ar), 55.0 (OCH3), 42.4 (CH), 36.7
(CH2).
4.2.7.
2-[[(4-Hydroxy-3-methoxyphenyl)methylene]-
hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acid (2h).
Beige crystals; yield 44.5%; mp 219–221 ꢁC; IR (KBr):
1724 (C@O), 1621 (NC@O), 1592 and 1562 (C@N),
1347 (NCS), 1249 (N–N@C), 1030 (CS); 1H NMR
(300 MHz, DMSO-d6): d 12.82 (br s, 1H, CO2H), 9.67
(s, 1H, Ar–OH), 8.18 (s, 1H, CH@N), 7.36–7.55 (m,
5H, Ar–H), 7.31 (d, 1H, J = 1.8 Hz, Ar–H), 7.16 (dd,
1H, J = 1.8, 8.1 Hz, Ar–H), 6.83 (d, 1H, J = 8.1 Hz,
Ar–H), 4.55 (t, 1H, J = 5.7, CH), 3.79 (s, 3H, OCH3),
3.13 (d, 2H, J = 5.7, CH2), 13C NMR (75.4 MHz,
DMSO-d6): d 173.7 (CO2H), 171.8 (C@O), 162.9
(C@N), 157.9 (CH@N), 149.6 (Cq Ar), 147.8 (Cq Ar),
135.3 (Cq Ar), 129.1 (CH Ar), 128.7 (CH Ar), 128.2
(CH Ar), 125.5 (Cq Ar), 122.6 (CH Ar), 115.5 (CH
Ar), 110.2 (CH Ar), 55.5 (OCH3), 42.4 (CH), 36.8
(CH2).
4.2.12. 2-[[(4-Methoxyphenyl)methylene]hydrazono]-4-oxo-
3-phenyl-5-thiazolidineacetic acid (2m). White crystals;
yield 55.5%; mp 211–213 ꢁC; IR (KBr): 1711 (C@O),
1615 (NC@O), 1579 and 1552 (C@N), 1344 (NCS),
1245 (N–N@C), 1024 (CS); 1H NMR (300 MHz,
DMSO-d6): d 12.74 (br s, 1H, CO2H), 8.24 (s, 1H,
CH@N), 7.68 (d, 2H, J = 8.4 Hz, Ar–H), 7.37-7.55 (m,
5H, Ar–H), 7.00 (d, 2H, J = 8.4 Hz, Ar–H), 4.55 (t,
1H, J = 5.4 Hz, CH), 3.80 (s, 3H, OCH3), 3.10 (d, 2H,
J = 5.4 Hz, CH2), 13C NMR (75.4 MHz, DMSO-d6): d
173.5 (CO2H), 171.5 (C@O), 163.2 (C@N), 161.3 (Cq
Ar),157.2 (CH@N), 135.1 (Cq Ar), 129.3 (CH Ar),
128.9 (CH Ar), 128.5 (CH Ar), 128.0 (CH Ar), 126.6
(Cq Ar), 114.2 (CH Ar), 55.2 (OCH3), 42.3 (CH), 36.7
(CH2).
4.2.8. 2-[[(4-Dimethylaminophenyl)methylene]hydrazono]-
4-oxo-3-phenyl-5-thiazolidineacetic acid (2i). Dark red
crystals; yield 36%; mp 230–231 ꢁC; IR (KBr): 1724
(C@O), 1606 (NC@O), 1529 (C@N), 1360 (NCS), 1239
1
(N–N@C), 1030 (CS); H NMR (300 MHz, DMSO-d6):
d 12.82 (br s, 1H, CO2H), 8.13 (s, 1H, CH@N), 7.36–
7.56 (m, 7H, Ar–H), 6.71 (d, 2H, J = 8.7 Hz, Ar–H),
4.53 (t, 1H, J = 5.7 Hz, CH), 3.11 (d, 2H, J = 5.7 Hz,
CH2), 2.95 (s, 6H, N(CH3)2), 13C NMR (75.4 MHz,
DMSO-d6): d 173.6 (CO2H), 171.8 (C@O), 161.6
(C@N), 157.9 (CH@N), 151.9 (Cq Ar), 135.3 (Cq Ar),
129.2 (CH Ar), 129.0 (CH Ar), 128.6 (CH Ar), 128.2
(CH Ar), 121.2 (Cq Ar), 111.6 (CH Ar), 42.4 (CH),
39.7 (N(CH3)2), 36.9 (CH2).
4.2.13. 2-[[(2,4-Dimethoxyphenyl)methylene]hydrazono]-
4-oxo-3-phenyl-5-thiazolidineacetic acid (2n). White crys-
tals; yield 62.5%; mp 202–203 ꢁC; IR (KBr): 1717
(C@O), 1607 (NC@O), 1555 (C@N), 1344 (NCS), 1244
4.2.9. 2-[[(3-Chlorophenyl)methylene]hydrazono]-4-oxo-3-
phenyl-5-thiazolidineacetic acid (2j). Beige crystals; yield
58%; mp 237–239 ꢁC; IR (KBr): 1711 (C@O), 1621
(NC@O), 1573 (C@N), 1341 (NCS), 1258 (N–N@C),
1040 (CS); H NMR (300 MHz, DMSO-d6): d 12.84 (br
s, 1H, CO2H), 8.33 (s, 1H, CH@N), 7.69–7.75
1
(N–N@C), 1023 (CS); H NMR (300 MHz, DMSO-d6):
d 12.73 (br s, 1H, CO2H), 8.37 (s, 1H, CH@N), 7.79 (d,
1H, J = 8.1 Hz, Ar–H), 7.36-7.55 (m, 5H, Ar–H), 6.61
(m, 2H, Ar–H), 4.54 (t, 1H, J = 6 Hz, CH), 3.81 (s,
1

T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
455
3H, OCH3), 3.79 (s, 3H, OCH3), 3.10 (d, 2H, J = 6 Hz,
CH2), 13C NMR (75.4 MHz, DMSO-d6): d 173.4
(CO2H), 171.5 (C@O), 163.1 (C@N), 162.7 (Cq Ar),
159.6 (Cq Ar), 152.5 (CH@N), 135.1 (Cq Ar), 128.8
(CH Ar), 128.4 (CH Ar), 128.0 (CH Ar), 127.3 (CH
Ar), 114.6 (Cq Ar), 106.6 (CH Ar), 98.1 (CH Ar), 55.6
(OCH3), 55.4 (OCH3), 42.3 (CH), 36.8 (CH2).
ried out using the Student’s t test. P values <0.05 were
considered as significant. Data shown are representative
of thirteen in triplicate. Finally, the IC50 values for
infected cells and intracellular parasites for all com-
pounds were obtained after 24 h exposure in the concen-
trations ranging of 0.01–30 mM, in triplicate per assay,
by a non-linear regression using exclusion test with try-
pan blue.18
4.2.14.
2-[[(3,4,5-Trimethoxyphenyl)methylene]hydra-
zono]-4-oxo-3-phenyl-5-thiazolidineacetic acid (2o). Light
brown crystals; yield 45%; mp 119–120 ꢁC; IR (KBr):
1724 (C@O), 1615(NC@O), 1585 and 1559 (C@N), 1354
(NCS), 1239 (N–N@C); 1H NMR (300 MHz, CDCl3): d
12.80 (br s, 1H, CO2H), 8.18 (s, 1H, CH@N), 7.26–7.54
(m, 5H, Ar–H), 6.94 (s, 2H, Ar–H), 4.42 (dd, 1H,
J = 3.6, 8.1 Hz, CH), 3.87 (s, 9H, OCH3), 3.31 (dd, 1H,
J = 3.6, 17.7 Hz, CH2a), 3.18 (dd, 1H, J = 8.1, 17.7 Hz,
CH2b), 13C NMR (75.4 MHz, CDCl3): d 173.7 (CO2H),
171.7 (C@O), 164.1 (C@N), 157.7 (CH@N), 153.1 (Cq
Ar), 135.2 (Cq Ar), 129.5 (Cq Ar), 129.0 (CH Ar), 128.7
(CH Ar), 128.2 (CH Ar), 104.9 (CH Ar), 60.1 (OCH3),
55.8 (OCH3), 42.5 (CH), 36.6 (CH2).
4.4. Assay in vitro for antimicrobial activity
Bacteria and fungal species used in the antimicrobial
evaluation were obtained from Departamento de Anti-
´
bioticos and Instituto de Micologia cultures collections,
Universidade Federal de Pernambuco, Brazil. Namely,
Staphylococcus aureus (ATTC 6538), Bacillus subtilis
(UFPEDA 16), M. luteus (ATTC 2225), Escherichia
coli (ATTC 25922), K. pneumoniae (ATTC 29665),
Streptococcus faecalis (ATTC 6057), Mycobacterium
phlei (UFPEDA 70), Mycobacterium smegmatis
(UFPEDA 71), Mycobacterium tuberculosis (DAUFPE
82), Saccharomyces cerevisiae (UFPEDA 07), Candida
sp. (IMUR 720), Candida sp. (IMUR 1224), Candida
sp. (IMUR 4249), and Candida albicans (UFPEDA
1007) species. The antibacterial and antifungal activi-
ties are reported preliminarily utilizing disc diffusion
method.33 In this method, disks containing known
amounts of an antimicrobial agent were placed on
the surface of an agar plate that has been inoculated
with a standardized suspension of microorganisms
tested. Paper discs with only DMSO were used as neg-
ative controls. The MZI (Mean zone inhibition) for
chloramphenicol and rifampicin (antibacterial), and
nistatin (antifungal) was referred to as a reference va-
lue (mm). All experiments were carried out three times
and repeated if the results differed. All compounds hav-
ing MZI of more or equal to 18 mm were selected for
MIC and MBC or MFC.
4.2.15. 2-[[(3,5-Bis(1,1-dimethylethyl)4-hydroxyphenyl)-
methylene]hydrazono]-4-oxo-3-phenyl-5-thiazolidineace-
tic acid (2p). White crystals; yield 77%; mp 246–247 ꢁC;
IR (KBr): 1730 (C@O), 1615 (NC@O), 1585 (C@N),
1358 (NCS), 1245 (N–N@C), 1030 (CS); 1H NMR
(300 MHz, DMSO-d6): d 12.81 (br s, 1H, CO2H), 8.20
(s, 1H, CH@N), 7.35–7.54 (m, 7H, Ar–H), 4.52 (t, 1H,
J = 5.7 Hz, CH), 3.11 (d, 2H, J = 5.7 Hz, CH2), 1.39
(s, 18H, C(CH3)3), 13C NMR (75.4 MHz, DMSO-d6):
d 173.7 (CO2H), 171.7 (C@O), 162.7 (C@N), 158.5
(CH@N), 156.7 (Cq Ar), 138.9 (Cq Ar), 135.3 (Cq
Ar), 129.0 (CH Ar), 128.6 (CH Ar), 128.2 (CH Ar),
125.4 (Cq Ar), 124.7 (CH Ar), 42.4 (CH), 36.7 (CH2),
34.4 (Cq C(CH3)3), 30.1 (CH3).
4.3. Assay for anti-Toxoplasma gondii activity
For MIC and MBC or MFC assays,34,35 a stock solution
(1 mg/mL) of test compounds was prepared in dimethyl-
sulfoxide solvent. Further, the serial dilution of test
compounds was carried out and the concentrations used
ranged from 10 to 220 lg/mL. Test compounds at vari-
ous concentrations were added to culture medium in a
test tube and different strains were inoculated at 108 bac-
teria/mL concentration. Tryptic Soy Agar and Nutrient
Agar (for antibacterial), and Sabouraud Liquid Medium
(for antifungal) were utilized for culture medium. The
tubes were incubated at 37 ꢁC (antibacterial) or 30 ꢁC
(antifungal) for 24–48 h and then examined for the pres-
ence or absence of growth organisms tested. Chloram-
phenicol, rifampicin, and nistatin were used as
antibacterial and antifungal substances. The MIC values
were obtained from the lowest concentration of the test
compounds where the tubes remained clear, indicating
that the bacterial or fungal growth was completely
inhibited at this concentration. The MBC or MFC val-
ues were measured by inoculating the broths used for
MIC determinations onto drug-free medium. The
MBC or MFC were the first dilution where no growth
is observed. The MIC, MBC, and MFC values were ex-
pressed in lg/mL.
Tachyzoites from the virulent RH strain of T. gondii
were maintained by intraperitoneal passages in Swiss
mice and were collected in Ringer’s solution at pH 7.2,
48 h after infection. Animals were used following Exper-
imental Research Ethical International Committees.
Vero cells (Kidney fibroblasts from African green mon-
keys) were incubated with T. gondii Tachyzoites (para-
site/host cell 5:1 relationship) for 1 h, washed twice
with phosphate-buffered saline solution (PBS) to remove
extracellular parasites, and incubated for 24 h at 37 ꢁC
in the presence of medium 199 supplemented with 5% fe-
tal calf serum (FCS). After, cells infected with T. gondii
were incubated with test compounds in the concentra-
tions of 0.1, 2, 5, 8, and 20 mM for 24 h. Hydroxyurea
and sulfadiazine were utilized as reference substances.
All compounds were added to the infected cells during
intense parasite proliferation. The infected cultures were
washed thrice with PBS, fixed with Bouin’s fixative,
stained with Giemsa, and observed under a light micro-
scope (63· objective Axioplan, Zeiss, Jena, Germany).
The percentage of infected cells and the mean number
of intracellular parasites were determined by examina-
tion of at least 400 cells.31,32 Statistical analysis was car-

456
T. M. de Aquino et al. / Bioorg. Med. Chem. 16 (2008) 446–456
19. C¸ apan, G.; Ulusoy, N.; Ergenc¸, N.; Kiraz, M. Monats-
hefte fu¨r Chemie 1999, 130, 1399.
Acknowledgments
The authors acknowledge Coordenac¸a˜o de Aperfeic¸oa-
`
20. Vigorita, M. G.; Ottana, R.; Monforte, F.; Maccari, R.;
Trivato, A.; Monforte, M. T.; Taviano, M. F. Bioorg.
Med. Chem. Lett. 2001, 11, 2791.
21. Rawal, R. K.; Prabhakar, Y. S.; Katti, S. B.; Clercq, E.
Bioorg. Med. Chem. 2005, 13, 6771.
22. Babaoglu, K.; Page, M. A.; Jones, V. C.; McNeil, M. R.;
Dong, C.; Naismith, J. H.; Lee, R. E. Bioorg. Med. Chem.
Lett. 2003, 13, 3227.
´
mento de Pessoal de Nıvel Superior (CAPES), Conselho
Nacional de Desenvolvimento Cientıfico e Tecnologico
´
´
`
(CNPq) and Fundac¸a˜o de Amparo a Pesquisa do Esta-
do do Rio de Janeiro (FAPERJ).
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