Synthesis, antimicrobial and cytotoxic activities of some 5-arylidene-4-thioxo-thiazolidine-2-ones

Article abstract of DOI:10.1016/j.ejmech.2008.10.006

Several 5-arylidene-4-thioxo-thiazolidine-2-ones (3a-n) were synthesized and evaluated as antimicrobial agents against representative strains, including multidrug-resistant strains of clinical isolates. Also, the antiproliferative activity was evaluated a

Full text of DOI:10.1016/j.ejmech.2008.10.006

European Journal of Medicinal Chemistry 44 (2009) 2038–2043
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial and cytotoxic activities of some
5-arylidene-4-thioxo-thiazolidine-2-ones
Frederico L. Gouveia a, Renata M.B. de Oliveira a, Tatiane B. de Oliveira b, Ivanildo M. da Silva b,
b
b
b,
*
ˆ
Silene C. do Nascimento , Kesia X.F.R. de Sena , Julianna F.C. de Albuquerque
a Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
b Departamento de Antibio´ticos, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 5-arylidene-4-thioxo-thiazolidine-2-ones (3a–n) were synthesized and evaluated as antimicro-
bial agents against representative strains, including multidrug-resistant strains of clinical isolates. Also,
the antiproliferative activity was evaluated against two human carcinoma cell lines (NCI-H292 and HEp-
2). The compounds containing the 5-arylidene subunit presented greater antimicrobial activities against
Gram positive bacteria, including the multidrug-resistant clinical isolates, than the 4-thioxo-thiazolidine-
2-one. Important SAR information was also gathered, such as the contribution of thiocarbonyl attached at
4-position on the thiazolidine heterocyclic for antimicrobial properties. None of the derivatives exhibited
significant antiproliferative activity against the human carcinoma cell lines.
Received 7 February 2008
Received in revised form 8 September 2008
Accepted 6 October 2008
Available online 17 October 2008
Keywords:
4-Thioxothiazolidinones
Benzylidene derivatives
Antimicrobial activity
Cytotoxic activity
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
antimicrobial activity. For example, the arylidene group at 5-posi-
tion [13,14], the presence of halides in arylidene group [13,15,16]
Infectious diseases are responsible for great number of deaths in
the world population. The reduction of sensibility to antimicrobial
agents in current use has been increasing for a great variety of
pathogens and the resistance to multiple drugs is common for
several microorganisms, especially for Gram positive bacteria.
Infection by methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Enterococci (VRE) presents a difficult problem
for medicine [1–4]. In addition, the treatment of infectious diseases
is more complicated in immuno-suppressed patients, such as those
infected with the HIV, undergoing anticancer therapy or transplants.
Given the evidence for the rapid global spread of resistant clinical
isolates and the appearance of drug-resistant strains among
community acquired infections, the need for discovery or optimi-
zation of antimicrobial agents active against these resistant strains is
of paramount importance. In this context, thiazolidines show
remarkable antimicrobial activity [5,6], in addition to various bio-
logical properties, such as antiproliferative [7,8], antituberculosis [9],
antihyperglycemic [10], antiinflammatory [11], among others [12].
Concurring with previous reports in the literature, illustrating
various thiazolidinone derivatives, 5-arylidene-4-thioxo-thiazoli-
dine-2-one has a number of structural contributors which enable
and non-substitution in nitrogen N-3. In one recent work, the
alkylation at N-3 has not contributed to improve the antimicrobial
properties [17]. Although extensive studies exist regarding the SAR
between thiazolidinone analogues and biological activity, very little
is known about the contribution of thiocarbonyl and the presence
of arylidene subunit attached in 5-position to its antimicrobial
activity.
In view of the facts mentioned above and as part of our
initial efforts to discover potentially active new agents, thirteen
5-arylidene-4-thioxo-thiazolidine-2-ones (3a–n) were synthesized
and evaluated as antimicrobial agents, including against multidrug-
resistant clinical isolates. In addition, the cytotoxicity of these
compounds was tested against two human carcinoma cell lines.
2. Results and discussion
2.1. Chemistry
The title compounds were synthesized as outline in Scheme 1.
Thiazolidine-2,4-dione (1) was prepared using a prior method [18].
Then, 4-thioxo-thiazolidine-2-one (2) was obtained using Law-
esson’s Reagent as an effective thionation reagent [19]. The
compounds 3a–n were prepared by a simple and direct method,
which involves the Knoevenagel condensation with different
* Corresponding author. Av. Professor Moraes Rego, 1235 Cidade Universita´ria,
CEP 50670-901 Recife, Pernambuco, Brazil. Tel./fax: þ55 81 2126 8347.
E-mail address: julianna@ufpe.br (J.F.C. de Albuquerque).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.006

F.L. Gouveia et al. / European Journal of Medicinal Chemistry 44 (2009) 2038–2043
2039
H
O
S
H
O
H
O
R
S
O
N
N
N
R
Lawesson's
Reagent
CHO
CH
S
dioxane
reflux
acetic acid
sodium acetate
reflux
S
S
(1)
(2)
(3a-n)
Yield (15-70%)
3j: R = 2,4-Cl2
3g: R = 2-Cl
3h: R =3-Cl
3i: R = 4-Cl
3a: R = 2-F
3b: R =3-F
3c: R = 4-F
3d: R = 2-Br
3e: R = 3-Br
3f: R = 4-Br
3l: R = 2,6-Cl2
3m: R = 3,4-Cl2
3n: R = 2,3,5-Cl3
Scheme 1. Synthesis of 5-arylidene-4-thioxo-thiazolidine-2-ones (3a–n).
aromatic aldehydes [19]. The structures of the desired compounds
were determined by 1H NMR, 13C NMR, IR and elemental analyses.
In theory, two geometrical isomers (E and Z) around the
exocyclic double bond (CH]C) are possible for the above 5-aryli-
dene-4-thioxo-thiazolidine-2-ones (3a–n). However, all the
synthesized compounds exhibited only the configuration Z, as
expected from the literature and verified by the 1H NMR spectra.
The literature records that the condensation of imidazolidine-2,4-
dione with aromatic aldehydes in acid medium, led only to the Z
isomer [20]. Also, the data of coupled 13C NMR spectra indicated
that 5-benzylidene thiazolidines and imidazolidines exhibit the Z
configuration [19,21,22]. This assignment was also confirmed by X-
ray crystallographic data [23].
when it was compared with compounds 3a–n however, with less
potency than ketoconazole (the reference drug). In contrast, the
biological data indicated that 5-arylidene subunit is essential for
antibacterial activity.
The compounds 2 and 3a–n were also evaluated for antibacterial
activity against a large number of clinical isolates of multidrug-
resistant Gram positive bacteria. The values of the MIC are given in
Table 3 compared with ampicillin and cefalexin, used as stan-
dards. All compounds showed significant inhibitory effects,
especially 5-arylidene derivatives, confirming the importance of
this substitution in increasing activity to inhibit growth of Gram
positive bacteria. The compounds 3a–n were more potent than
cefalexin against most of the microorganisms tested. The most
relevant results were verified for strains S. aureus III (H.C. 21036)
and VI (H.C. 20905), whose inhibitory effect was much greater
than that of ampicillin and cefalexin. It is interesting to note that
the derivative 3n showed a greater inhibitory capacity. This
suggests that the introduction of three atoms of the halogens to
the arylidene group may have exercised an important role to
increase antibacterial properties.
The 1H NMR spectra of the compounds (3a–n) showed one
singlet at
d 7.91–8.29 ppm, correspondent to the methine proton,
which is in agreement with the Z configuration, due to the
deshielding effect of the C]S adjacent. The earlier literature reports
that the methine proton in E isomers appears at lower chemical
shift values, due to the lesser deshielding effect of the sulphur atom
at 1-position [17,24].
2.2. Pharmacology
Table 1
2.2.1. Antimicrobial activity
Inhibitory zone (diameter, mm) of compounds against bacterial and yeast strains
using the disk diffusion method.
The in vitro antimicrobial activity was performed using the
disk diffusion method and the Minimum Inhibitory Concentration
(MIC) with different strains, including multidrug-resistant clinical
isolates. Ampicillin and kanamycin were used as positive controls
for bacteria and ketoconazole for yeast. In a preliminary assay
(Table 1) 5-arylidene-4-thioxo-thiazolidine-2-ones (3a–n) and
the intermediate (2) showed activity against Gram positive and
Candida albicans strains, but did not exhibit activity against Gram
negative strains. Thiazolidine-2,4-dione (1) was inactive for all
microorganisms tested, however, demonstrating that the replac-
ing of the carbonyl by thiocarbonyl increased the antimicrobial
activity.
Compound
Mean zone inhibitiona
Sa
Bs
Ml
Ef
Pa
Ec
Sm
Ms
Ca
1
–
–
–
–
–
–
–
–
–
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3l
19
24
28
29
25
28
23
25
31
27
23
20
25
16
38
NT
NT
21
23
20
26
19
26
18
17
25
22
24
20
27
–
26
25
27
29
18
29
21
18
29
25
24
18
27
21
45
NT
NT
22
20
22
24
19
25
17
16
22
17
18
–
20
19
24
NT
NT
–
–
–
–
–
–
–
–
–
–
–
–
–
–
NT
20
NT
–
–
–
–
–
–
–
–
–
–
–
–
–
–
20
15
NT
–
–
–
–
–
–
–
–
–
–
–
–
–
–
NT
45
NT
18
20
20
25
21
25
17
20
24
20
19
17
20
–
19
16
16
17
13
14
15
12
13
12
15
15
14
–
The values of the MIC against the microorganisms susceptible
in the preliminary test are reported in Table 2. The results showed
significant inhibitory effects, and most compounds exhibit MIC
3m
values in the 2–16 mg/mL range. This class of compounds pre-
3nb
sented high activity against S. aureus, especially the 5-substituted
compounds, when the derivatives 3h and 3j were as active as the
standard drug, ampicillin, but less active than cefalexin, the
second standard used. Besides, the activity against Bacillus subtilis
was found for compounds 3f, 3j and 3n; while last one also was
active against Enterococcus faecalis, in potency comparable with
ampicillin. The derivatives 3f, 3j and 3l exhibited significant
values of MIC for Mycobacterium smegmatis. It is noteworthy that
the intermediate 2 was able to inhibit the proliferation of yeast
Ampicillin
Kanamycin
Ketoconazole
28
NT
NT
NT
40
NT
NT
NT
28
Ampicillin (10
m
g/disc), Kanamycin (30
m
g/disc) and Ketoconazole (25
mg/disc) were
used as positive references; Compounds 1, 2 and 3a–n (300
m
g/disc); –, indicates no
sensitivity or mean zone inhibition lower than 7 mm; NT, not tested; Sa, S. aureus;
Bs, B. subtilis; Ml, M. luteus; Ef, E. faecalis; Pa, P. aeruginosa; Ec, E. coli; Sm, S. mar-
cescens; Ms, M. smegmatis; Ca, C. albicans.
a
Values are mean (n ¼ 3).
b
Solubility problems were observed at higher doses.

2040
F.L. Gouveia et al. / European Journal of Medicinal Chemistry 44 (2009) 2038–2043
Table 2
property. This could be done by putting one or more bulky group(s)
in the arylidene ring [27,28].
Inhibitory activity of compounds 2 and 3a–n expressed as MIC (mg/mL).
Compound
Microorganism
S. aureus B. subtilis M. luteus E. faecalis M. smegmatis C. albicans
3. Conclusion
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3l
3m
3n
8
8
4
4
4
4
4
4
2
4
2
4
4
4
2
ꢂ1
4
32
16
16
8
8
2
16
8
16
2
4
32
16
16
16
8
8
16
8
32
8
16
4
4
2
64
32
32
32
32
16
16
16
8
32
16
16
8
32
32
32
16
32
16
4
16
16
32
4
8
32
64
32
32
64
32
32
32
64
32
32
32
64
This study demonstrated that the bioisosteric replacement of
thiocarbonyl instead of carbonyl in thiazolidine ring, results in an
enhancement of antimicrobial activity. Due to its antibacterial
properties, especially against multidrug-resistant strains of clinical
isolates, the 5-arylidene-4-thioxo-thiazolidine-2-ones identified
here may represent useful starting points for further lead optimi-
zation. Studies involving the mechanism of action are necessary for
a complete understanding of their antimicrobial activity, as well as
structural modifications on the title compounds to improve anti-
proliferative activity.
8
4
2
2
4
32
32
64
>128
NT
2
2
64
NT
Ampicillin
Cefalexin
Ketoconazole NT
NT
NT
ꢂ 1
4. Experimental
ꢂ1
ꢂ1
NT
NT
4.1. Chemistry
NT, not tested.
All melting points were determined using QUIMIS (model
320.23) and are uncorrected. FTIR spectra were recorded on
a BRUKER IFS-66 IR spectrophotometer in KBr pellet. 1H NMR and
13C NMR spectra were measured on VARIAN UNITY PLUS (300 MHz
for 1H and 75.4 MHz for 13C). The chemical shifts are reported in
2.2.2. Cytotoxic activity
The in vitro cytotoxic activity was performed by MTT assay
[25,26] against two human carcinoma cell lines: NCI-H292
(obtained from mucoepidermoid carcinoma of lung) and HEp-2
(obtained from epidermoid carcinoma of the larynx). The inhibitory
effects of compounds 3a–n on the growth of the two cell lines are
shown in Table 4. All compounds showed lower cytotoxicity when
they were compared with vincristine, the standard drug used
(IC50 ¼ 0.04 and 0.003
tively). Moreover, they were not able to inhibit 50% of cell prolif-
eration even at the highest dose used (10 g/mL). The
d
units and the coupling constants (J) are reported in hertz. C, H, N
and S analyses were performed with a Carlo Erba model EA1108
elemental analyzer. Thin layer chromatography was performed on
pre-coated silica plates (Merck Kiesegel 60 F254) and column
chromatography using silica gel (mesh 70–230). The spots could be
visualized easily under ultraviolet light.
mg/mL for NCI-H292 and HEp-2, respec-
m
4.2. General procedure for the preparation of 4-thioxo-thiazolidine-
antiproliferative effect was only achieved at 45.03% for NCI-H292
cells and at 30.10% for HEp-2 cells. Also, we observed that the NCI-
H292 line was more sensitive to synthesized compounds than the
HEp-2 line.
Compounds containing thiazolidinedione ring have already
been reported as apoptosis inducers in cancer cell strains and more
recently have been shown to be active against drug-resistant lung
cancer cells. Thus, although this inhibition is still not sufficient, we
feel that further research would be able to improve the inhibitory
2-one (2)
A mixture of thiazolidine-2,4-dione (1) (0.1 mol) and Lawesson’s
reagent (0.03 mol) in anhydrous dioxane, was heated under reflux
for 24 h. Part of the solvent was evaporated and, after cooling, the
precipitate was filtered, washed with n-hexane and recrystallized
in ethanol. Yield 68%, mp: 140–141 ꢀC. IR (KBr, cmꢁ1): 3115 (NH);
1710 (C]O). 1H NMR (DMSO-d6, 300 MHz, ppm):
NH); 4.60 (s, 2H, –CH2–). Anal. Calcd. for C3H3NOS2 (133.18): C,
d 13.54 (s, 1H,
Table 3
MIC values (in
mg/mL) against clinical isolate of multidrug-resistant Gram positive bacterial strains.
Compound
S. aureus
E. faecalis
Coagulase-
negative
Staphylococcus
I
II
32
III
IV
V
VI
VII
VIII
IX
X
XI
64
32
16
32
2
64
4
32
16
4
XII
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3l
3m
3n
Ampicillin
Cefalexin
32
4
32
2
2
2
4
16
4
2
2
2
64
4
2
2
4
8
4
2
2
2
2
4
2
32
8
2
2
4
8
2
2
2
2
2
4
2
2
2
4
32
8
2
2
4
16
4
2
2
2
32
32
16
32
8
8
8
16
4
16
4
32
32
8
32
16
16
8
32
4
16
4
32
32
8
16
16
8
8
16
4
16
8
16
16
4
32
32
8
16
16
16
8
32
4
16
8
16
16
4
2
64
64
16
16
32
16
64
4
8
2
4
2
8
16
32
16
32
4
8
8
8
4
2
4
4
16
4
4
2
2
2
8
2
2
2
8
8
4
2
2
4
4
2
4
4
4
2
8
2
2
8
8
4
2
8
4
4
4
2
2
2
ꢂ1
32
ꢂ1
64
ꢂ1
2
64
ꢂ1
ꢂ1
8
2
>128
2
>128
64
64
128
128
S. aureus: H.C. 20981 (I), H.C. 21141 (II), H.C. 21036 (III), H.C. 20489 (IV); H.C. 20794 (V) and H.C. 20905 (VI); E. faecalis: H.C. 21752 (VII), H.C. 21944 (VIII), H.C. 24708 (IX) and
H.C. 24962 (X); Coagulase-negative Staphylococcus: H.A.M. 278 (XI) and H.A.M. 338 (XII).

F.L. Gouveia et al. / European Journal of Medicinal Chemistry 44 (2009) 2038–2043
2041
Table 4
Growth inhibitory effects (%) of 3a–n on human carcinoma cell lines.
Compound
10
m
g/mL
5
m
g/mL
2.5
m
g/mL
1.25 mg/mL
NCI-H292
HEp-2
NCI-H292
HEp-2
NCI-H292
HEp-2
NCI-H292
HEp-2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3l
3m
3n
40.41
26.78
38.00
36.87
42.36
36.45
45.03
30.87
41.39
38.37
32.80
43.28
40.50
11.21
20.93
30.10
21.23
23.63
18.23
21.15
18.83
22.25
24.83
8.73
25.18
13.14
31.55
22.94
22.03
16.61
34.19
21.61
35.83
29.28
10.34
35.83
39.68
8.86
19.83
20.56
16.23
ꢁ1.04
15.43
5.97
17.63
16.07
20.03
ꢁ2.39
11.02
22.84
7.78
3.43
17.49
16.19
11.14
1.19
19.88
11.19
28.55
14.69
7.70
1.66
7.03
4.29
2.10
7.32
ꢁ8.34
0.84
ꢁ4.51
10.33
ꢁ1.82
ꢁ0.36
ꢁ11.53
5.44
ꢁ10.82
13.27
4.67
1.10
10.60
3.35
10.01
12.11
4.36
9.18
3.14
ꢁ5.3
ꢁ3.16
ꢁ11.34
ꢁ0.36
0.92
3.91
15.33
ꢁ4.24
5.03
6.83
ꢁ9.16
ꢁ5.45
12.58
16.41
23.54
19.36
33.01
21.75
14.21
27.06; H, 2.27; N,10.52; S, 48.14. Found: C, 27.18; H, 2.47; N, 10.22; S,
48.74.
133.73; 170.54 (C]O); 195.25 (C]S). Anal. Calcd. for C10H6BrNOS2
(300.19): C, 40.01; H, 2.01; N, 4.67; S, 21.36. Found: C, 40.07; H, 2.01;
N, 4.79; S, 21.19.
4.3. General procedure for the preparation of 5-arylidene-4-thioxo-
4.3.5. 5-(3-Bromobenzylidene)-4-thioxo-thiazolidine-2-one (3e)
Yield 40%, mp: 214–215 ꢀC. IR (KBr, cmꢁ1): 3149–3064 (NH);
1685 (C]O); 1587 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
thiazolidine-2-ones (3a–n)
To a solution of compound 2 (0.01 mol) and anhydrous sodium
acetate (0.01 mol) in glacial acetic acid, was added the respective
aromatic aldehyde. The mixture was stirred under reflux for 1–10 h
and then poured into ice-cold water. Then, the precipitate was
filtered, washed with water and n-hexane, dried and purified by
column chromatography.
d
13.96 (s, 1H, NH); 8.02 (s, 1H, –CH]); 7.87 (s, 1H, Ar); 7.71–7.63
(m, 2H, Ar); 7.49 (t, 1H, Ar). 13C NMR (DMSO-d6, 75.4 MHz, ppm,
DEPT): 122.59, 128.53, 131.45, 133.09, 133.29 (CH); 131.32, 133.86,
d
135.82 (Cq); 170.37 (C]O); 195.35 (C]S). Anal. Calcd. for
C10H6BrNOS2 (300.19): C, 40.01; H, 2.01; N, 4.67; S, 21.36. Found: C,
40.21; H, 2.01; N, 4.41; S, 21.56.
4.3.1. 5-(2-Fluorobenzylidene)-4-thioxo-thiazolidine-2-one (3a)
Yield 30%, mp: 159–160 ꢀC. IR (KBr, cmꢁ1): 3163–3080 (NH);
1690 (C]O); 1592 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
4.3.6. 5-(4-Bromobenzylidene)-4-thioxo-thiazolidine-2-one (3f)
Yield 22%, mp: 188–189 ꢀC. IR (KBr, cmꢁ1): 3161–3075 (NH);
1688 (C]O); 1582 (C]C). 1H NMR (Acetone-d6, 300 MHz, ppm):
d
13.97 (s, 1H, NH); 8.14 (s, 1H, –CH]); 7.62–7.54 (m, 2H, Ar); 7.41–
7.35 (m, 2H, Ar). 13C NMR (DMSO-d6, 75.4 MHz, ppm, DEPT):
d
8.09 (s, 1H, –CH]); 7.78–7.73 (m, 2H, Ar); 7.68–7.63 (m, 2H, Ar).
13C NMR (Acetone-d6, 75.4 MHz, ppm):
d
126.15, 130.28, 132.09,
d
116.35 (d, JCF ¼ 21.3), 125.53 (d, JCF ¼ 3.0), 126.39 (d, JCF ¼ 6.97),
133.09, 133.62, 134.08, 134.59, 135.95; 170.83 (C]O); 196.75 (C]S).
Anal. Calcd. for C10H6BrNOS2 (300.19): C, 40.01; H, 2.01; N, 4.67; S,
21.36. Found: C, 40.03; H, 2.31; N, 4.79; S, 21.49.
128.61, 133.23 (d, JCF ¼ 9.0) (CH); 121.50 (d, JCF ¼ 11.0), 131.88, 161.09
(d, JCF ¼ 251.4) (Cq); 170.43 (C]O); 195.41 (C]S). Anal. Calcd. for
C10H6FNOS2 (239.28): C, 50.20; H, 2.53; N, 5.85; S, 26.80. Found: C,
50.42; H, 2.54; N, 5.79; S, 21.09.
4.3.7. 5-(2-Chlorobenzylidene)-4-thioxo-thiazolidine-2-one (3g)
Yield 40%, mp: 166–167 ꢀC. IR (KBr, cmꢁ1): 3166–3080 (NH);
1715 (C]O); 1581 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
4.3.2. 5-(3-Fluorobenzylidene)-4-thioxo-thiazolidine-2-one (3b)
Yield 20%, mp: 103–104 ꢀC. IR (KBr, cmꢁ1): 3080 (NH); 1700
(C]O); 1586 (C]C). 1H NMR (Acetone-d6, 300 MHz, ppm):
d 10.53
d
14.05 (s, 1H, NH); 8.29 (s, 1H, –CH]); 7.65–7.61 (m, 2H, Ar);
7.54–7.49 (m, 2H, Ar). 13C NMR (DMSO-d6, 75.4 MHz, ppm):
128.19,128.93,130.47,130.82,131.59,132.21,132.74,135.13; 170.48
(s, 1H, NH); 8.12 (s, 1H, –CH]); 7.66–7.16 (m, 4H, Ar). Anal. Calcd.
for C10H6FNOS2 (239.28): C, 50.20; H, 2.53; N, 5.85; S, 26.80. Found:
C, 50.60; H, 2.54; N, 5.70; S, 21.19.
d
(C]O); 195.31 (C]S). Anal. Calcd. for C10H6ClNOS2 (255.74): C,
46.97; H, 2.36; N, 5.48; S, 25.07. Found: C, 47.07; H, 2.41; N, 5.48; S,
25.27.
4.3.3. 5-(4-Fluorobenzylidene)-4-thioxo-thiazolidine-2-one (3c)
Yield 18%, mp: 126–127 ꢀC. IR (KBr, cmꢁ1): 3068 (NH); 1727
(C]O); 1573 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
13.89 (s,
1H, NH); 8.06 (s, 1H, –CH]); 7.76–7.71 (m, 2H, Ar); 7.41–7.35 (m,
2H, Ar). 13C NMR (DMSO-d6, 75.4 MHz, ppm, DEPT):
116.70 (d,
4.3.8. 5-(3-Chlorobenzylidene)-4-thioxo-thiazolidine-2-one (3h)
Yield 17%, mp: 112–113 ꢀC. IR (KBr, cmꢁ1): 3081 (NH); 1686
(C]O); 1589 (C]C). 1H NMR (Acetone-d6, 300 MHz, ppm):
d
12.26
(s, 1H, NH); 8.08 (s, 1H, –CH]); 7.70 (s, 1H, Ar); 7.66–7.52 (m, 3H,
Ar). 13C NMR (Acetone-d6, 75.4 MHz, ppm):
129.77, 131.67, 131.88,
d
JCF ¼ 22.05), 133.06 (d, JCF ¼ 9.0), 134.73 (CH); 129.58 (d, JCF ¼ 2.55),
130.12 (d, JCF ¼ 3.0), 163.10 (d, JCF ¼ 250.87) (Cq); 170.54 (C]O);
195.43 (C]S). Anal. Calcd. for C10H6FNOS2 (239.28): C, 50.20; H,
2.53; N, 5.85; S, 26.80. Found: C, 50.40; H, 2.74; N, 5.29; S, 21.11.
d
132.53, 132.79, 135.49, 136.29, 137.45; 189.88 (C]O); 196.56 (C]S).
Anal. Calcd. for C10H6ClNOS2 (255.74): C, 46.97; H, 2.36; N, 5.48; S,
25.07. Found: C, 47.17; H, 2.21; N, 5.48; S, 25.27.
4.3.4. 5-(2-Bromobenzylidene)-4-thioxo-thiazolidine-2-one (3d)
Yield 39%, mp: 214–215 ꢀC. IR (KBr, cmꢁ1): 3161–3080 (NH);
1710 (C]O); 1580 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
4.3.9. 5-(4-Chlorobenzylidene)-4-thioxo-thiazolidine-2-one (3i)
Yield 15%, mp: 183–184 ꢀC. IR (KBr, cmꢁ1): 3161–3080 (NH);
1691 (C]O); 1577 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
14.06 (s, 1H, NH); 8.25 (s, 1H, –CH]); 7.80 (d, 1H, Ar); 7.62–7.52
(m, 2H, Ar); 7.45–7.39 (m, 1H, Ar). 13C NMR (DMSO-d6, 75.4 MHz,
ppm): 126.00, 128.69, 129.10, 132.38, 132.80, 133.73, 133.64,
d
13.93 (s, 1H, NH); 8.04 (s, 1H, –CH]); 7.69–7.66 (m, 2H, Ar); 7.61–
7.58 (m, 2H, Ar). 13C NMR (DMSO-d6, 75.4 MHz, ppm, DEPT):
d

2042
F.L. Gouveia et al. / European Journal of Medicinal Chemistry 44 (2009) 2038–2043
d
129.54, 132.07, 134.33 (CH); 130.43, 132.28, 135.50 (Cq); 170.43
microorganisms tested. The plates were incubated at 35 ꢀC for 24
and 48 h, for bacteria and yeast respectively. Ketoconazole (25 g)
for yeast, ampicillin (10 g) for Gram positive bacteria and kana-
mycin (30 g) for Gram negative bacteria, were used as standard
(C]O); 195.39 (C]S). Anal. Calcd. for C10H6ClNOS2 (255.74): C,
46.97; H, 2.36; N, 5.48; S, 25.07. Found: C, 46.09; H, 2.51; N, 5.48; S,
25.37.
m
m
m
drugs. Paper disks with only DMSO were utilized as negative
controls.
4.3.10. 5-(2,4-Dichlorobenzylidene)-4-thioxo-thiazolidine-2-one
(3j)
A twofold serial dilution technique [30,31] was followed to
determine the minimum inhibitory concentration (MIC) of the
compounds against the susceptible microorganisms in the
preliminary test (yeast and Gram positive bacteria) and against
strains of clinical isolates of multidrug-resistant Gram positive
bacteria. Test compounds, dissolved in DMSO, were added to
culture media (Mu¨ ller–Hinton agar for bacteria and Sabouraud
Liquid Medium for yeast) to obtain final concentrations ranging
Yield 54%, mp: 162–163 ꢀC. IR (KBr, cmꢁ1): 3041 (NH); 1733
(C]O); 1573 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
8.22 (s,
1H, –CH]); 7.85 (d, 1H, Ar); 7.66 (m, 2H, Ar). 13C NMR (Acetone-d6,
75.4 MHz, ppm): 129.73, 131.40, 131.67, 132.68, 133.46, 134.74,
d
137.84, 138.08; 170.87 (C]O); 196.61 (C]S). Anal. Calcd. for
C10H5Cl2NOS2 (290.18): C, 41.39; H, 1.74; N, 4.83; S, 22.10. Found: C,
41.56; H, 1.91; N, 4.78; S, 22.27.
from 128–1 mg/mL. A plate (tube for C. albicans) containing only the
4.3.11. 5-(2,6-Dichlorobenzylidene)-4-thioxo-thiazolidine-2-one
(3l)
culture medium and DMSO was used as negative control. The final
amount applied was of 105 CFU/plate for bacteria and 103 CFU/tube
for yeast. The MIC values were read after incubation at 35 ꢀC for
a period of 20 h (bacteria) and 48 h (yeast). The lowest concen-
tration of the test substance that completely inhibited the growth
Yield 41%, mp: 191–192 ꢀC. IR (KBr, cmꢁ1): 3190–3111 (NH);
1715 (C]O); 1604 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
7.91 (s, 1H, –CH]); 7.60–7.57 (m, 2H, Ar); 7.51–7.45 (m, 1H, Ar).
13C NMR (DMSO-d6, 75.4 MHz, ppm):
d
128.58, 130.15, 131.93,
of the microorganism was recorded as the MIC, expressed in mg/mL.
132.19, 133.04, 138.02, 168.50; 170.43 (C]O); 195.39 (C]S). Anal.
Calcd. for C10H5Cl2NOS2 (290.18): C, 41.39; H, 1.74; N, 4.83; S, 22.10.
Found: C, 41.57; H, 1.96; N, 4.78; S, 22.37.
Ampicillin, cefalexin (bacteria) and ketoconazole (yeast) were used
as standard drugs. All experiments were carried out three times.
4.5. In vitro assay for cytotoxic activity
4.3.12. 5-(3,4-Dichlorobenzylidene)-4-thioxo-thiazolidine-2-one
(3m)
The human lung carcinoma cell line (NCI-H292) and the human
larynx carcinoma cell line (HEp-2) were purchased from the Adolfo
Lutz Institute, Sa˜o Paulo, Brazil. A DMEM (Dulbecco’s Modified
Eagle’s Medium), enriched with 10% of fetal bovine serum, 1% of
Yield 55%, mp: 183–184 ꢀC. IR (KBr, cmꢁ1): 3161–3080 (NH);
1685 (C]O); 1583 (C]C). 1H NMR (DMSO-d6, 300 MHz, ppm):
d
13.99 (s,1H, NH); 8.00 (s,1H, –CH]); 7.94 (d,1H, Jm ¼ 2.1, Ar); 7.78
(d, 1H, Jo ¼ 8.4, Ar); 7.60 (dd, 1H, Jo ¼ 8.3, Jm ¼ 2.1, Ar). 13C NMR
L-glutamine and 1% of antibiotics (penicillin and streptomycin), was
(DMSO-d6, 75.4 MHz, ppm, DEPT):
d 129.27, 131.51, 132.50, 132.77
used for cell cultivation and to perform the tests.
(CH); 131.73, 132.20, 133.14, 134.11 (Cq); 168.49 (C]O); 170.22
(C]S). Anal. Calcd. for C10H5Cl2NOS2 (290.18): C, 41.39; H, 1.74; N,
4.83; S, 22.10. Found: C, 41.46; H, 1.92; N, 4.79; S, 22.27.
The cytotoxic activity was investigated using the MTT assay (3-
(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide). Cell
suspensions were diluted to 105 cells/mL, suitably prepared and
distributed in plates of culture with 96 wells (225
then incubated at 37 ꢀC in a humid atmosphere with 5% of CO2. After
24 h, 25 L of either the synthesized compounds or the reference
drug (vincristine) was added to each well. The plates were incubated
mL in each well),
4.3.13. 5-(2,3,5-Trichlorobenzylidene)-4-thioxo-thiazolidine-2-one
(3n)
m
Yield 70%, mp: 186–187 ꢀC. IR (KBr, cmꢁ1): 3103–3057 (NH);
1753 (C]O); 1600 (C]C). 1H NMR (Acetone-d6, 300 MHz, ppm):
again at 37 ꢀC for 72 h. Then, 25
mL of MTT solution (5 mg/mL) was
added to each well, and the mixture was incubated at 37 ꢀC for 2 h.
At the end of this period, the culture medium with the MTT excess
was aspirated and after that,100 mL of DMSO was added to each well
d
8.24 (s, 1H, –CH]); 7.82 (d, 1H, Jm ¼ 2.4, Ar); 7.62 (d, 1H, Jm ¼ 2.4,
Ar). Anal. Calcd. for C10H4Cl3NOS2 (324.63): C, 37.00; H, 1.24; N,
4.31; S, 19.75. Found: C, 37.02; H, 1.25; N, 4.32; S, 19.85.
to dissolve the formazan crystals. The optical density (OD) of the
wells was measured at 540 nm and compared to the control (cells
with medium only). The assay was conducted in triplicate.
4.4. In vitro assay for antimicrobial activity
The microorganisms used in this study are S. aureus (UFPEDA
01), B. subtilis (UFPEDA 16), M. luteus (UFPEDA 06), E. faecalis
(UFPEDA 138), Pseudomonas aeruginosa (UFPEDA 39), Escherichia
coli (UFPEDA 224), Serratia marcescens (UFPEDA 398), M. smegmatis
(UFPEDA 71) and C. albicans (UFPEDA 1007) obtained from the
cultures collection of the Antibiotics Department of the Federal
University of Pernambuco (UFPE), Brazil. Strains of multidrug-
resistant clinical isolates were also used and consisted of S. aureus
(20981, 21141, 21036, 20489, 20794 and 20905), E. faecalis (21752,
21944, 24708 and 24962) obtained from Bacteriology Department
of University Hospital of the Federal University of Pernambuco and
Coagulase-negative Staphylococcus (278 and 338), obtained from
Bacteriology Department of the Agamenon Magalha˜es Hospital,
Pernambuco, Brazil. The clinical isolates were collected from
various patients hospitalized in several clinics.
Acknowledgments
The authors acknowledge the Research Foundation of Pernam-
buco State (FACEPE), the Brazilian National Research Council
(CNPq) for the financial support and Department of Fundamental
Chemistry of UFPE for the analyses realized. F.L.G. was supported by
FACEPE/CNPq fellowship.
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