Synthesis and?biological activity of?4-thiazolidinones, thiosemicarbazides derived from diflunisal hydrazide

Article abstract of DOI:10.1016/j.ejmech.2005.11.005

Two novel series of 4-thiazolidinone derivatives, namely 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylic acid [2-(5-nitro-2-furyl/substituted phenyl)-4-thiazolidinone-3-yl]amides (5a-g) and 2-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonylhydrazono)-3-a lkyl/a

Full text of DOI:10.1016/j.ejmech.2005.11.005

European Journal of Medicinal Chemistry 41 (2006) 353–359
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and biological activity of 4-thiazolidinones,
thiosemicarbazides derived from diflunisal hydrazide
Güniz Küçükgüzel a,*, Ayla Kocatepe a, Erik De Clercq b, Fikrettin Şahin c,d, Medine Güllüce d
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Istanbul 34668, Turkey
b
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
c
Department of Genetic and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, İstanbul 34755, Turkey
d
Biotechnology Application and Research Center, Ataturk University, Erzurum 25240, Turkey
Received 11 June 2005; received in revised form 28 October 2005; accepted 3 November 2005
Available online 18 January 2006
Abstract
Two novel series of 4-thiazolidinone derivatives, namely 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylic acid [2-(5-nitro-2-furyl/substituted
phenyl)-4-thiazolidinone-3-yl]amides (5a–g) and 2-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonylhydrazono)-3-alkyl/aryl-4-thiazolidinones (6a–
e) together with 5-(2′,4′-difluoro-4-hydroxybiphenyl-5-yl)-2-cyclohexylamino-1,3,4-oxadiazole (7a) have been synthesized as title compounds.
1-(2′,4′-Difluoro-4-hydroxybiphenyl-3-carbonyl)-4-alkyl/arylthiosemicarbazides (4a–g) were also obtained and used as intermediate to give the
title compounds. All synthesized compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv,
antiviral and antimicrobial activities against various virus, bacteria and fungi strains.
© 2006 Elsevier SAS. All rights reserved.
Keywords: 4-Thiazolidinone; Anti-HIV activity; Diflunisal; Thiosemicarbazide
1. Introduction
inhibitors and protease inhibitors have been clinically used
for the treatment of HIV infections. The result of observations
culminated in the discovery of 4-thiazolidinone as a new class
of highly potent non-nucleoside inhibitors [9–12]. Tuberculosis
is a chronic infection disease caused by several species of my-
cobacteria. The incidence of tuberculosis is increasing world
wide, partly due to poverty and inequity and partly to the
HIV/AIDS pandemic, which greatly increase the risk of infec-
tious proceeding to overt disease. During recent years, Myco-
bacterium tuberculosis and microorganisms increased resis-
tance against drugs. Therefore, there is need to develop the
new, potent, fast-acting antimicrobial, antiviral and antimyco-
bacterial drugs with low toxicity.
4-Thiazolidinone derivatives of diflunisal, prepared from di-
flunisal hydrazide-hydrazones can give corresponding 2′,4′-di-
fluoro-4-hydroxybiphenyl-3-carboxylic acid (Diflunisal) as a
metabolite via hydrolytic route. 4-Thiazolidinones of diflunisal
were also designed as possible dual acting antimicrobial/anti-
tuberculosis agents possessing anti-inflammatory properties via
4-Thiazolidinone derivatives are known to possess antibac-
terial [1–5], antifungal [6–8], antiviral [9–13] and antitubercu-
losis [4,14–16] properties (Fig. 1). Peptidoglycan is an essen-
tial component of the cell wall of both Gram-positive and
Gram-negative bacteria. 4-Thiazolidinones have been reported
as novel inhibitors of the bacterial enzyme Mur B which is a
precursor acting during the biosynthesis of peptidoglycan [17].
Human immunodeficiency virus type 1 (HIV-1) was identified
in the development of acquired immune deficiency syndrome
(AIDS). Reverse transcriptase is a key enzyme, packaged with-
in HIV virion capsid, which plays an essential role in the re-
plication of virus. Combinations of reverse transcriptase nu-
cleoside inhibitors, reverse transcriptase non-nucleoside
* Corresponding author. Tel.: +90 216 349 12 16; fax: +90 216 345 29 52.
E-mail address: gkucukguzel@marmara.edu.tr (G. Küçükgüzel).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.11.005

354
G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
its active metabolite, diflunisal against pain and inflammatory
events due to the cell damage arising from tuberculosis and
accompanying infectious diseases. In view of the above obser-
vations, the synthesis of novel and related compounds were
aimed at investigating biological activities of these compounds.
NMR and EI-mass spectral data. Physical and spectral data of
the synthesized compounds are given in Tables 1 and 2.
Thiosemicarbazides (4a–g) were also characterized by UV
absorption bands at 243–258 nm [19] whereas IR spectra dis-
played C=O bands at 1630–1668 cm–1 and C=S bands at
1223–1247 cm–1 [20].
4-Thiazolidinones (6a–e) obtained from thiosemicarbazide
gave absorption bands at 221–222 nm in UV spectra and lac-
2. Chemistry
1
tam C=O stretching bands of the ring at 1716–1736 cm–1. H-
NMR and 13C-NMR data were also in agreement with the for-
mation of 4-thiazolidinone ring. Signals appeared at 3.72–4.02
ppm in the 1H-NMR spectra were attributed to 4-thiazolidinone
methylene group. On the other hand, peaks resonated at 33.72,
156.19 and 171.90 [4] ppm in the 13C-NMR spectrum of com-
pound 2-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonylhydrazo-
no)-3-ethyl-4-thiazolidinones (6c), assigned for S–CH2, C=N
and C=O moieties, confirms the carbon skeleton of 4-thiazoli-
dinone ring. Compound (7a), which was obtained from (4g),
gave absorption bands at 258 nm (instead of 221–222 nm) in
UV and C=N stretching band at 1642 cm–1 (instead of C=O
band at 1716–1736 cm–1) in the IR spectrum. Moreover, mi-
croanalysis revealed that 6a–c had no sulfur atom leading to
the conclusion that the structures of these compounds must
be different from 4-thiazolidinone. Together with microanaly-
2′,4′-Difluoro-4-hydroxybiphenyl-3-carboxylic acid (analge-
sic and anti-inflammatory drug, Diflunisal) and methanol in the
presence of a few drops of concentrated sulfuric acid were
heated and 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylic acid
methyl ester (1) was isolated. By heating this methyl ester and
hydrazine-hydrate in methanol 2′,4′-difluoro-4-hydroxybiphe-
nyl-3-carboxylic acid hydrazide (2) was obtained. After con-
densing hydrazide with aromatic aldehydes in ethanol, 2′,4′-di-
fluoro-4-hydroxybiphenyl-3-carboxylic acid [(5-nitro-2-furyl/
substituted phenyl) methylene] hydrazides (3a–g) were ob-
tained as previously reported by Küçükgüzel et al. [18]. 2′,4′-
Difluoro-4-hydroxybiphenyl-3-carboxylic acid [2-(5-nitro-2-
furyl/substituted phenyl)-4-thiazolidinone-3-yl]amides (5a–g)
were obtained by refluxing the related hydrazide-hydrazones
(3a–g) and thioglycolic acid in dry benzene for 9–16 h using
a Dean–Stark water separator. Compound (2) [18] and alkyl/
aryl isothiocyanates were heated in ethanol to yield original 1-
(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonyl)-4-alkyl/arylthio-
semicarbazides (4a–g). The thiosemicarbazides were then re-
acted with ethyl α-bromoacetate in the presence of anhydrous
sodium acetate in absolute ethanol to yield 2-(2′,4′-difluoro-4-
hydroxybiphenyl-3-carbonylhydrazono)-3-alkyl/aryl-4-thiazoli-
dinones (6a–e) . However, formation of desired 4-thiazolidi-
nones from 1-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonyl)-4-
cyclohexylthiosemicarbazide (4g) failed and instead 5-(2′,4′-di-
fluoro-4-hydroxybiphenyl-5-yl)-2-cyclohexylamino-1,3,4-oxa-
diazole (7a) was obtained (Scheme 1). Purity of the synthe-
sized compounds in this study was confirmed by thin layer
chromatography and microanalysis. Structures of these com-
1
sis, lack of H-NMR resonances observed with S–CH2 func-
1
tion in the H-NMR spectrum of (7a) proved that ring closure
starting from (4g) resulted in formation of 1,3,4-oxadiazole
ring instead of 4-thiazolidinone (Scheme 2). This was not sur-
prising as a similar event was previously reported [4,21]. At-
tempts to synthesize 4-thiazolidinones, by the reaction of 1-ar-
oyl-4-alkyl/aryl thiosemicarbazides with α-halogeno acids (or
esters), have been reported to result in formation of 1,3,4-ox-
adiazole ring system instead of 4-thiazolidinone in some con-
ditions. First step of this reaction is thought to be S-alkylation
of thiosemicarbazide in its thiol form (due to sodium acetate
used) (Scheme 2). Second step involves leaving of ethanol to
give 4-thiazolidinone or ethyl mercaptoacetate leading to 1,3,4-
oxadiazole ring closure (Scheme 2). Electronic and steric prop-
erties of the substituent at the 4-position of thiosemicarbazide
1
pounds were characterized using UV, FT-IR, H-NMR, 13C-
Fig. 1.

G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
355
Scheme 1. Synthetic route to compounds 4a–g, 5a–g 6a–e and 7a. Reagents and conditions: (a) CH3OH/H2SO4, reflux; (b) NH2NH2.H2O, reflux; (c) Ar–CH=O/
EtOH, reflux; (d) R–NCS/EtOH, reflux; (e) HS–CH2–COOH/dry benzene; (f) Br–CH2–COOEt/anhyd. CH3COONa–abs. EtOH (R being aryl or alkyl; see Table 1).
seems to be a determinant for a selection between one of these
two possible pathways. Previous reports on these types of com-
pounds reveal that a bulky substituent like cyclohexyl facili-
tates ethyl mercaptoacetate expulsion resulting in oxadiazole
ring formation whilst small alkyl groups lead to 4-thiazolidi-
none ring via ethanol loss.
lected compound 2′,4′-Difluoro-4-hydroxybiphenyl-3-car-
boxylic acid[2-(5-nitro-2-furyl)-4-thiazolidinone-3-yl)]amide
(5a) displayed molecular ion at m/z 461 which confirmed its
molecular weight. Other important fragmentations were loss of
sulfur and CH2SH from the molecular ion as evidenced by the
fragment ions at m/z 429 and m/z 415, respectively.
Another series of 4-thiazolidinones (5a–g), prepared from
diflunisal hydrazide-hydrazones (3a–g), were characterized by
UV absorption bands at 222–226 nm and IR absorption bands
due to 4-thiazolidinone C=O stretching at 1681–1721 cm–1.
Their 1H-NMR spectra exhibited resonances at 5.86–6.35,
3.61–3.98 and 3.69–4.13 ppm assigned for 4-thiazolidinone,
3. Biological activity
3.1. Antimicrobial activity
1
CH and S–CH2, respectively. In the H-NMR spectra, methy-
The antimicrobial activities of the synthesized compounds
against laboratory strains 56 bacterial species and isolates of
six fungi and a yeast species which described previously [18]
were tested by using disc-diffusion [22] and microdilution as-
say [23]. 2′,4′-Difluoro-4-hydroxybiphenyl-3-carboxylic acid
[(5-nitro-2-furyl) methylene] hydrazide (3a) [18] which is start-
ing compound of (5a) have shown activity against Staphylo-
coccus epidermis HE-5 and Staphylococcus aureus HE-9 at
18.75 and 37.5 μg/ml, respectively. However, compound (5a)
containing 4-thiazolidinone ring, which was obtained by the
reaction of compound (3a) with thioglycolic acid, has not ex-
lene protons of the 4-thiazolidinone ring displayed two signals
appearing as doublets at 3.61–3.98 and 3.69–4.13 ppm due to
the non-equivalent, geminal methylene protons [4] interacting
with the chiral center at position 2. This phenomenon was not
observed with (6a–e) lacking the asymmetric carbon. 13C-
NMR data of selected prototype 2′,4′-difluoro-4-hydroxybiphe-
nyl-3-carboxylic acid[2-(4-fluorophenyl)-4-thiazolidinone-3-
yl)]amide (5f), also supported the structure of 4-thiazolidinone
ring via S–CH2, CH and C=O resonances appeared at 30.14,
61.93 and 169.8 ppm [4], respectively. The EI-MS of the se-

356
G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
Table 1
Physical and spectral data for 4a–g, 5a–g, 6a–e and 7a
a
d
Compound
R
Molecular formula
m.p. (°C)
Rf
UV
IR (KBr) ν (cm–1
)
ethanol
λmax
319
b
4a
CH3
C15H13F2N3O2S
210–2
0.74
3346 (Ar–OH), 3225 (N–H), 1657(amide, C=O), 1269,
1203 (C=S), 1142 (Ar–F).
243
205
b
4b
4c
CH2CH=CH2
C2H5
C17H15F2N3O2S
C16H15F2N3O2S
C20H15F2N3O2S
C21H17F2N3O2S
202–4
0.79
0.78
0.79
0.82
0.72
0.86
0.71
324
246
206
320
245
206
333
258
206
330
251
206
333
247
204
324
245
205
302
275
226
206
336
275
226
206
340
282
224
202
343
268
224
200
348
272
226
201
332
272
226
204
338
222
204
349
275
222
207
342
281
209
200
345
275
221
205
3385 (Ar–OH), 3216 (N–H), 1668 (amide, C=O), 1301,
1195 (C=S), 1143 (Ar–F)
b
205–6
3393 (Ar–OH), 3196 (N–H), 1672 (amide, C=O), 1309,
1200 (C=S), 1142 (Ar–F).
b
4d
4e
C6H5
185–9
3269 (Ar–OH, N–H), 1641 (amide, C=O), 1275, 1215
(C=S), 1140 (Ar–F).
b
C6H4CH3(4)
188–9
3293 (Ar–OH), 3186 (N–H), 1641 (amide, C=O), 1290,
1244 (C=S), 1144 (Ar–F).
b
4f
C6H4 OCH3(4) C21H17 F2 N3O3S
180–4
3310 (Ar–OH), 3225 (N–H), 1630 (amide, C=O), 1299,
1247(C=S), 1176 (Ar–F).
b
4g
5a *
C6H11
C20H21 F2 N3O2S
210–2
3371 (Ar–OH), 3190 (N–H), 1664 (amide, C=O), 1297,
1197 (C=S).
c
5-Nitro-2-furyl
C
20H13 F2 N3O6S
163–5
3331 (Ar–OH, N–H), 1685 (thiazolidinone, C=O), 1654
(amide, C=O).
c
5b
5c
5d
5e
5f
C6H5
C22H16 F2 N2O3S. C2H5OH 164–6
0.85
0.85
0.83
0.85
0.83
3391 (Ar–OH), 3246 (N–H), 1696 (thiazolidinone, C=O),
1647 (amide, C=O).
c
C6H4 Cl(4)
C6H4 F(2)
C6H4 F(3)
C6H4 F(4)
C22H15 ClF2 N2O3S
222–6
3186 (Ar–OH, N–H), 1682 (thiazolidinone, C=O), 1649
(amide, C=O).
c
C22H15 F3N2O3S. 1/2
C2H5OH
178–80
3397 (Ar–OH, N–H), 1697 (thiazolidinone, C=O), 1647
(amide, C=O).
c
C22H15 F3N2O3S. 1/2
C2H5OH
100–2
3262 (Ar–OH, N–H), 1701 (thiazolidinone, C=O), 1653
(amide, C=O).
c
C22H15 F3N2O3S
188–92
3310 (Ar–OH), 3197 (N–H), 1721 (thiazolidinone, C=O),
1651 (amide, C=O).
c
5g
6a
C6H4CH3(4)
CH3
C23H18 F2N2O3S. 1/2 H2O 180–2
C17H13 F2N3O3S. 1/2 H2O 252–4
0.86
0.28
3192 (Ar–OH, N–H), 1681 (thiazolidinone, C=O), 1648
(amide, C=O).
b
3166 (Ar–OH, N–H), 1718 (thiazolidinone, C=O), 1653
(amide, C=O), 1617 (C=N), 1148 (Ar–F).
b
6b
6c
CH2CH=CH2
C2H5
C19H15 F2N3O3S
226–30
(decomposed)
0.69
0.62
1721 (thiazolidinone, C=O), 1653 (amide, C=O), 1620
(C=N), 1145 (Ar–F).
b
C18H15 F2N3O3S. 1/2H2O 208–10
3591 (Ar–OH), 1716 (thiazolidinone, C=O), 1654
(amide, C=O), 1619 (C=N), 1143 (Ar–F).
(decomposed)

G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
357
Compound
a
d
R
Molecular formula
C23H17 F2N3O3S. 2H2O
m.p. (°C)
Rf
UV etha- IR (KBr) ν (cm–1
nol λmax
)
b
6d
C6H4CH3(4)
238–45
0.69
0.52
0.89
333
266
222
345
280
222
326
277
258
204
3445 (Ar–OH, N–H), 1736 (thiazolidinone, C=O), 1655
(amide, C=O), 1603 (C=N), 1145 (Ar–F).
(decomposed)
b
6e
7a
C6H4 OCH3(4) C23H17 F2N3O4S
244–60
1738 (thiazolidinone, C=O), 1655 (amide, C=O), 1605
(C=N), 1146 (Ar–F).
(decomposed)
b
C6H11
C20H17 F2N3O2
204–6
3218 (Ar–OH, N–H), 1642 (amide, C=O), 1593 (C=N),
1139 (Ar–F).
a : Microanalysis for the synthesized compounds Anal. Cal/Found: 4a C, 53.41; H, 3.88; N, 12.46; S, 9.50/C, 53.80; H, 3.67; N, 12.28; S, 9.65. 4b C, 56.19;
H, 4.16; N, 11.56; S, 8.82/C, 56.58; H, 4.14; N, 11.41; S, 9.02. 4c C, 54.69; H, 4.30; N, 11.96; S, 9.12/C, 54.89; H, 4.65; N, 11.64; S, 9.15. 4d C, 60.14; H,
3.79; N, 10.52; S, 8.03/C, 60.43; H, 4.20; N, 10.45; S, 8.16. 4e C, 61.01; H, 4.14; N, 10.16; S, 7.75/C, 61.47; H, 3.77; N, 10.18; S, 7.29. 4f C, 58.73; H, 3.99;
N, 9.79; S, 7.45/C, 58.99; H, 3.84; N, 9.73; S, 7.00. 4g C, 59.25; H, 5.22; N, 10.36; S, 7.91/C, 59.31; H, 5.29; N, 10.52; S, 7.87. 5b C, 61.01; H, 4.69; N,
5.93; S, 6.78/C, 60.84; H, 4.62; N, 6.08; S, 6.95. 5c C, 57.33; H, 3.28; N, 6.08; S, 6.96/C, 57.45; H, 3.06; N, 6.11; S, 6.99. 5d C, 59.09; H, 3.88; N, 5.99; S,
6.86/C, 59.15; H, 3.82; N, 6.77; S, 6.70. 5e C, 59.09; H, 3.88; N, 5.99; S, 6.86/C, 58.64; H, 3.71; N, 5.72; S, 6.20. 5f C, 59.46; H, 3.40; N, 6.30; S, 7.21/C,
59.90; H, 3.30; N, 6.12; S, 6.88. 5g. C, 61.46; H, 4.26; N, 6.23; S, 7.13/C, 60.56; H, 3.83; N, 6.52; S, 7.94. 6a C, 52.85; H, 3.65; N, 10.87; S, 8.30/C, 53.24;
H, 3.23; N, 10.85; S, 8.31. 6b C, 56.57; H, 3.75; N, 10.42; S, 7.95/C, 56.71; H, 3.33; N, 10.41; S, 7.97. 6c C, 53.99; H, 4.035; N, 10.49; S, 8.00/C, 53.53; H,
Table 2
1H-NMR spectral data of 4a–g, 5a–g, 6a–e and 7a
Compound
1H-NMR
4a (CDCl3 + CD3COCD3)
2.93 (s, 3H, CH3), 6.73–7.80 (m, 7H, Ar–H and CHCl3), 7.65 (s, 1/5H, NHCH3), 8.02–8.29 (s, 1/5H, Ar–OH), 9.94 (s, 1/5H,
NHCS), 11.13–12.07 (s, 1/5H, CONH).
4b (CDCl3 + CD3COCD3)
4c (CDCl3 + CD3COCD3)
4d (CDCl3 + CD3COCD3)
4e (CDCl3 + CD3COCD3)
4f (DMSO-d6)
4.16 (s, 2H, N–CH2–CH=CH2), 4.94–4.97 (d, 1H, CH=CH2, cis, J = 10.3 Hz), 5.05–5.09 (d, 1H, CH=CH2, trans, J = 17.2 Hz),
5.73–5.79 (m, 1H, CH=CH2), 6.83–7.89 (m, 7H, Ar–H and CHCl3), 7.57 (s, 1/2H, NHCH2).
1.07 (s, 3H, CH3), 3.55 (q, 3H, CH2), 6.84–7.89 (m, 7H, Ar–H and CHCl3), 7.64–7.85 (b, 1/2H, NHCH2), 8.42 (s, 1/2H, Ar–
OH), 10.06 (s, 1/2H, NHCS), 11.12–12.17 (s, 1/5H, CONH).
6.81–7.91 (m, 12H, Ar–H and CHCl3), 8.26–9.14 (b, 1H, NHC6H5), 9.37 (s, 1H, Ar–OH), 10.33 (s, 1H, NHCS), 11.57 (s, 1H,
CONH).
2.86 (s, 3H, CH3), 6.92–7.96 (m, 10H, Ar–H), 8.61 (b, 1/5H, NH–C6H4–CH3), 9.41 (s, 1/5H, Ar–OH), 10.21–10.47 (b, 1/5H,
NHCS), 11.50–11.87 (s, 1H, CONH).
3.90 (s, 3H, OCH3), 7.05–8.23 (m, 10H, Ar–H), 9.92 (s, 2H, NH–C6H4–CH3 and Ar–OH), 10.56–11.74 (b, 1H, NHCS), 12.20
(s, 1H, CONH).
4g (CDCl3 + DMSO-d6)
5a (CDCl3 + CD3OD)
5b (CDCl3 + CD3OD)
5c (CD3OD)
1.07–1.96 (m, 10H, C6H11), 3.51–3.70 (m, 1H, C6H11), 7.05–8.23 (m, 10H, Ar–H), 9.92 (s, 2H, NH–C6H4–CH3 and Ar–OH),
10.56–11.74 (b, 1H, NHCS), 12.20 (s, 1H, CONH).
3.71, 3.86 (2d and each 1H, S–CH2, J = 15.8 Hz), 5.99 (s, 1H, S–CH–N), 6.75–7.85 (m, 9H, Ar–H and CHCl3), 10.55 (s, 1H,
Ar–OH), 11.19–11.55 (s, 1H, CONH).
1.10 (t, 3H, CH3–CH2–OH), 3.55 (m, 2H, CH3–CH2–OH), 3.67, 3.82 (2d and each 1H, S–CH2, J = 15.9 Hz), 5.92 (s, 1H, S–
CH–N), 6.76–7.77 (m, 12H, Ar–H and CHCl3), 10.18 (s, 1H, Ar–OH), 10.97–11.67 (s, 1H, CONH).
3.67, 3.80 (2d and each 1H, S–CH2, J = 16.0 Hz), 5.91 (s, 1H, S–CH–N), 6.77–7.79 (m, 10H, Ar–H), 10.16 (s, 1H, Ar–OH),
11.29 (s, 1H, CONH).
5d (DMSO-d6)
1.21 (t, 3H, CH3–CH2–OH), 3.63 (m, 2H, CH3–CH2–OH), 4.51 (s, 1H, CH3–CH2–OH), 3.98, 4.13 (2d and each 1H, S–CH2,
J = 15.9 Hz), 6.35 (s, 1H, S–CH–N), 7.18–8.02 (m, 10H, Ar–H), 10.67 (s, 1H, Ar–OH), 11.81 (s, 1H, CONH).
1.40 (t, 3H, CH3–CH2–OH), 3.87 (m, 2H, CH3–CH2–OH), 3.94, 4.10 (2d and each 1H, S–CH2, J = 16.2 Hz), 6.18 (s, 1H, S–
CH–N), 6.97–7.61 (m, 11H, Ar–H and CHCl3), 9.62 (s, 1H, Ar–OH), 11.17 (b, 1H, CONH).
3.61, 3.69(2d and each 1H, S–CH2, J = 15.9 Hz), 5.86 (s, 1H, S–CH–N), 6.69–7.58 (m, 11H, Ar–H and CHCl3), 9.90 (s, 1/3H,
Ar–OH), 11.15–11.40 (b, 1/3H, CONH).
5e (CDCl3)
*5f (CDCl3 + CD3COCD3)
5g (CDCl3 + CD3OD)
2.24 (s, 3H, CH3–C6H4), 3.68, 3.81 (2d and each 1H, S–CH2, J = 15.9 Hz), 5.89 (s, 1H, S–CH–N), 6.77–7.95 (m, 11H, Ar–H
and CHCl3), 10.20 (s, 1/5H, Ar–OH), 11.28 (s, 1/5H, CONH).
6a (CD3OD)
6b (CD3OD)
3.25 (s, 3H, CH3–N), 3.91 (2H, S–CH2), 6.82–7.47 (m, 6H, Ar–H), 8.07 (s, 1H, Ar–OH), 10.63–11.06 (s, 1H, CONH).
3.85 (2H, S–CH2), 4.35–4.39 (m, 2H, N–CH2–CH= CH2), 5.12 (d, 1H, CH=CH2, cis, J = 10.2 Hz), 5.21 (d, 1H, CH=CH2,
trans, J = 17.1 Hz), 5.85 (m, 1H, CH=CH2), 6.76–7.39 (m, 6H, Ar–H), 8.02 (s, 1H, Ar–OH).
1.22 (s, 3H, CH3–N), 3.72–4.02 (m, 4H, S–CH2 and CH3–CH2–N), 6.81–7.46 (m, 6H, Ar–H), 8.06 (s, 1H, Ar–OH).
2.21 (s, 3H, CH3–C6H4-), 4.00–4.79 (m, 2H, S–CH2), 7.20–8.05 (m, 11H, Ar–H and Ar–OH), 11.99 (s, 1H, CONH)
3.97 (s, 2H, S–CH2), 4.43 (s, 3H, O–CH3), 7.18–7.75 (m, 10H, Ar–H), 8.04 (s, 1H, Ar–OH), 11.92 (s, 1H, CONH).
1.26–2.16 (m, 10H, C6H11), 3.69 (m, 1H, C6H11), 5.45 (s, 1H, Ar–OH), 6.93–7.72 (m, 6H, Ar–H), 9.25–10.90 (b, 1H, –NH–
C6H11).
**6c (CD3OD)
6d (DMSO-d6 + CD3OD)
6e (DMSO-d6)
7a (CDCl3)
*Compound 5f: 13C-NMR (DMSO-d6) δ 30.14 (thiazolidinone C5), 61.93 (thiazolidinone C2), 105.33 (biphenyl C9), 112.78 (biphenyl C11), 112.99 (4-fluorophe-
nyl C3, C5), 116.27 (biphenyl C5), 118.43 (biphenyl C3), 124.68 (4-fluorophenyl C2, C6), 126.08 (biphenyl C12), 130.15 (biphenyl C1), 131.02 (biphenyl C2),
132.44 (biphenyl C6), 135.35 (biphenyl C7), 135.42 (4-fluorophenyl C1), 158.65 (biphenyl C4), 161.16 (4-fluorophenyl C4,), 118.43 (biphenyl C8), 164.44 (bi-
phenyl C10), 166.95 (amide, C=O), 169.80 (thiazolidinone, C=O). **Compound 6c: 13C-NMR (DMSO-d6) δ 13.06 (CH3–CH2–N), 33.72 (thiazolidinone CH2),
38.39 (CH3–CH2–N), 105.29 (biphenyl C11), 112.81 (biphenyl C9), 118.42 (biphenyl C5), 125.08 (biphenyl C3), 130.32 (biphenyl C2), 132.57 (biphenyl C6),
133.93 (biphenyl C12), 156.19 (thiazolidinone C2), 158.59 (biphenyl C8), 160.25 (biphenyl C4), 160.95 (biphenyl C10), 164.25 (amide, C=O), 171.90 (thiazolidi-
none, C=O).

358
G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
Scheme 2. Mechanism of 4-thiazolidinone or 1,3,4-oxadiazole ring closure from 1-acyl-4-alkyl/aryl thiosemicarbazides.
hibited antimicrobial activity. According to these results, it was
understood that 4-thiazolidinone and thiosemicarbazide deriva-
tives of diflunisal had no antimicrobial activity against the
tested microorganisms.
Difluoro-4-hydroxybiphenyl-3-carbonyl)-4-methylthiosemicar-
bazide (4a) and 1-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbo-
nyl)-4-allylthiosemicarbazide (4b) exhibited antiviral inhibi-
tions against Vaccinia virus and Herpes simplex virus-1 TK-
KOS at 16 and 9.6 μg ml–1, respectively. 1-(2′,4′-Difluoro-4-
hydroxybiphenyl-3-carbonyl)-4-phenylthiosemicarbazide (4d)
3.2. Antimycobacterial activity
and
1-(2′,4′-difluoro-4-hydroxybiphenyl-3-carbonyl)-4-(4-
methylphenyl)thiosemicarbazide (4e) exhibited antiviral inhibi-
tions against Herpes simplex virus-1 TK-KOS and Punto Toro
virus at 9.6 and 0.64 μg ml–1, respectively. As conclusion, it
was understood that diflunisal thiosemicarbazides were exhib-
ited, more potent antiviral action than corresponding diflunisal
4-thiazolidinones.
The synthesized compounds were tested in vitro for their
antimycobacterial activity. Primary screening was conducted
at 6.25 μg.ml–1 against M. tuberculosis H37Rv in BACTEC
12B medium using a broth microdilution assay, the Microplate
Alamar Blue Assay (MABA) [24]. Rifampin was used as the
standard in the tests. None of the compounds were considered
for further evaluation as they had mycobacterial inhibitions less
than 90% at 6.25 μg ml–1. 1-(2′,4′-Difluoro-4-hydroxybiphe-
nyl-3-carbonyl)-4-ethylthiosemicarbazide (4c) was found to
provide %25 inhibition of mycobacterial growth of M. tuber-
culosis H37Rv in the primary screen at 6.25 μg ml–1.
4. Experimental protocols
All chemical compounds were purchased from Fluka. Diflu-
nisal was provided by Sanovel Pharmaceuticals. Melting points
were taken on Buchi-530 apparatus and were uncorrected. Mi-
croanalysis were performed on a Leco CHNS-932 instrument.
UV Spectra were recorded on a Beckman DU 530 spectrophot-
ometer (1 mg per 100 ml in EtOH). IR spectra were run on
3.3. Antiviral activity
Compounds (4a–g), (5a–g), (6a–c) and (7a) were tested for
antiviral activity and cytotoxicity in various viral test systems,
according to previously published procedures [25–29] . None
of the compounds inhibited vesicular stomatitis virus, Cox-
sackie virus, respiratory syncytical virus, parainfluenza-3 virus,
reovirus, Sindbis virus, Punto Toro virus, Herpes simplex virus
type 1 and 2 and vaccinia virus-induced cytopathicity at sub-
toxic concentrations in HeLa, Vero or Hel cell culture. 1-(2′,4′-
1
BIO-RAD FTS-135 FT-IR spectrophotometer. H-NMR and
13C-NMR spectra were obtained on a Bruker AVANC-DPX
400 instrument. EI-mass spectra were performed using a Micro
MS Zabspec Double Focusing Magnetic sector. The purities of
the synthesized compounds were checked using thin layer
chromatography in ethylacetate/petroleum ether (50:50, v/v)
(λ = UV 254 nm, t = 28 °C)

G. Küçükgüzel et al. / European Journal of Medicinal Chemistry 41 (2006) 353–359
359
4.1. 1-(2′,4′-Difluoro-4-hydroxybiphenyl-3-carbonyl)-4-alkyl/
References
arylthiosemicarbazides (4a–g)
[1] Ö. Ateş, A. Kocabalkanlı, G. Sanış-Ötük, A.C. Ekinci, A. Vidin, Arz-
neim.-Forsch/Drug, Res. 47 (1997) 1134–1138.
A solution of 0.01 mol of (2) [18] and equimolar amount of
appropriate isothiocyanate in 60 ml of EtOH was heated under
reflux for 1–2 h. The precipitate obtained was filtered off,
washed with water and recrystallized from ethanol.
[2] Ö. Ateş, H. Altıntaş, G. Ötük, Arzneim.-Forsch/Drug, Res. 50 (2000)
569–575.
[3] A. Kocabalkanlı, Ö. Ateş, G. Ötük, Arch.Pharm.Med.Chem. 334 (2001)
35–39.
[4] Ş.G. Küçükgüzel, E.E. Oruç, S. Rollas, F. Şahin, A. Özbek, Eur. J. Med.
Chem. 37 (2002) 197–206.
4.2. 2′,4′-Difluoro-4-hydroxybiphenyl-3-carboxylic acid[2-(5-
nitro-2-furyl/substituted phenyl)-4-thiazolidinone-3-yl)]amide
(5a–g)
[5] C.G. Bonde, N.J. Gaikwad, Bioorg. Med. Chem. 12 (2004) 2151–2161.
[6] N. Cesur, Z. Cesur, N. Ergenç, M. Uzun, M. Kiraz, Ö. Kasımoğlu, D.
Kaya, Arch. Pharm. (Weinheim) 327 (1994) 271–272.
[7] N. Karali, E. İlhan, A. Gürsoy, M. Kiraz, Farmaco 53 (1998) 346–349.
[8] H.T.Y. Fahmy, Boll.Chim.Farmaco 140 (2001) 422–427.
A mixture of (3a–g) [18] (0.01 mol) and thioglycolic acid
(0.01 mol) was refluxed in dry benzene (100 ml) using a
Dean–Stark water separator. Excess benzene was evaporated
in vacuo. The resulting residue was triturated with saturated
NaHCO3 solution until CO2 evolution ceased. The solid was
washed with water, dried and recrystallized from ethanol-
water.
[9] M.L. Barreca, A. Chimirri, L.D. Luca, A.M. Monforte, P. Monforte, A.
Rao, M. Zappalà, J. Balzarini, E. De Clercq, C. Pannecouque, M. Witv-
rouw, Bioorg. Med. Chem. Lett. 11 (2001) 1793–1796.
[10] A. Rao, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte, P. Mon-
forte, C. Pannecouque, M. Zappalà, Farmaco 57 (2002) 747–751.
[11] A. Rao, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte, P. Mon-
forte, C. Pannecouque, M. Zappalà, Farmaco 58 (2003) 115–120.
[12] M.L. Barreca, A. Chimirri, E. De Clercq, L.D. Luca, A.M. Monforte, P.
4.3. 2-(2′,4′-Difluoro-4-hydroxybiphenyl-3-
carbonylhydrazono)-3-alkyl/aryl-4-thiazolidinone (6a–e)
Monforte, A. Rao, M. Zappalà, Farmaco 58 (2003) 259–263.
[13] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq, L.D. Luca,
A.M. Monforte, P. Monforte, C. Pannecouque, M. Zappalà, Farmaco 59
(2004) 33–39.
0.01 mol of appropriate thiosemicarbazide (4a–f) and
0.011 mol of ethyl bromoacetate were refluxed in 30 ml of
absolute EtOH in the presence of 0.04 mol of anhydrous
NaOAc for 2 h. The reaction mixture was cooled, diluted with
water and allowed to stand overnight. The solid precipitated
was washed with water, dried and recrystallized from ethanol.
[14] L. Bukowski, M. Janowiec, Z. Zwolska-Kwiek, Z. Andrezejczyk, Phar-
mazie 53 (1998) 373–376.
[15] N. Ulusoy, Arzneim.-Forsch/Drug, Res. 52 (2002) 565–571.
[16] K. Babaoglu, M.A. Page, V.C. Jones, M.R. McNeil, C. Dong, J.
H. Naismith, R.E. Lee, Bioorg. Med. Chem. Lett. 13 (2003) 3227–3230.
[17] C.J. Andres, J.J. Bronson, S.V. D’Andrea, M.S. Deshpande, P.J. Falk, K.
A. Grant-Young, W.E. Harte, H.T. Ho, P.F. Misco, J.G. Robertson, D.
Stock, Y. Sun, A.W. Walsh, Bioorg. Med. Chem. Lett. 10 (2000) 715–
717.
4.4. 5-(2′,4′-difluoro-4-hydroxybiphenyl-5-yl)-2-
cyclohexylamino-1,3,4-oxadiazole (7a)
[18] Ş.G. Küçükgüzel, A. Mazi, F. Şahin, S. Öztürk, J.P. Stables, Eur. J. Med.
Chem. 38 (2003) 1005–1013.
0.01 mol of appropriate thiosemicarbazide (4g) and 0.011
mol of ethyl bromoacetate were refluxed in 30 ml of absolute
EtOH in the presence of 0.04 mol of anhydrous NaOAc for
2 h. The reaction mixture was cooled, diluted with water and
allowed to stand overnight. The solid precipitated was washed
with water, dried and recrystallized from ethanol.
[19] S. Rollas, Doğa 7 (1983) 65–73.
[20] A. Gürsoy, Ş. Demirayak, Z. Cesur, J. Reisch, G. Ötük, Pharmazie 45
(1990) 246–250.
[21] N. Cesur, Z. Cesur, A. Gürsoy, Arch. Pharm. (Weinheim) 325 (1992)
623–624.
[22] R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolke, Manual
Clin. Microbiol. 6 (1995) (Washington).
[23] J.R. Zgoda, J.R. Porter, Pharmaceutical Biol. 39 (2001) 221–225.
Acknowledgements
[24] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004–1009.
[25] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn,
Diflunisal was supplied by Sanovel Pharmaceutical Industry
Inc. We thank Dr. J.A.M. from the Tuberculosis Antimicrobial
Acquisition and Coordinating Facility (TAACF), National In-
stitute of allergy and Infections Diseases Southern Research
Institute, GWL Hansen’s Disease Centre, Colorado State Uni-
versity, Birmingham, AL, USA, for the in vitro evaluation of
antimycobacterial activity using M. tuberculosis H37Rv. This
study was supported by Scientific Research Project Commis-
sion of Marmara University (Project number: HEA-DYD-052/
051201 and SAĞ-YY-010/020103).
J. Desmyter, E. De Clercq, J. Virol. Methods 20 (1988) 309–321.
[26] M. Popovic, M.G. Sarngadharan, E. Read, R.C. Gallo, Science 224
(1984) 497–500.
[27] F. Barré-Sinoussi, J.C. Chermann, M.T. Nugeyre, S. Chamaret, J. Grest,
C. Dauget, C. Axler-Blin, F. Vézinet-Brun, C. Rouzioux, W. Rozen-
baum, L. Montagnier, Science 220 (1983) 868–871.
[28] I. Miyoshi, H. Taguchi, I. Kobonishi, S. Yoshimoto, Y. Ohtsuki, Y. Shir-
aishi, T. Akagi, Gann. Monogr 28 (1982) 219–228.
[29] E. De Clercq, J. Descamps, G. Verhelst, R.T. Walker, A.S. Jones, P.
F. Torrence, D. Shugar, J. Infect. Dis. 141 (5) (1980) 563–574.