Synthesis and in vivo anticancer and antiangiogenic effects of novel thioxothiazolidin-4-one derivatives against transplantable mouse tumor

Article abstract of DOI:10.1007/s00044-009-9187-7

A series of novel thioxothiazolidin-4-one derivatives 5(a-g) were synthesized by the coupling of different amines containing aliphatic, substituted aromatic, and heterocyclic moieties, such as oxadiazol, pyrazole, isoxazole, and piperazine with 2-(5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo- 2-thioxothiazolidin-3-yl)acetic acid. All compounds were characterized by ¹H NMR, LCMS, FTIR and elemental analysis. In this study, we investigated the possibility that these novel thioxothiazolidin-4-one derivatives 5(a-g) inhibits tumor growth and tumor induced angiogenesis using mouse Ehrlich Ascites Tumor (EAT) as a model system. Our results demonstrated that the compounds significantly reduced ascites tumor volume, cell number, and increased the life span of EAT-bearing mice. In addition, the compounds manifested strong antiangiogenic effects and suppressed tumor induced endothelial proliferation in the mice peritoneum. From our findings, it is noted that the derivatives 5(a-e) may be possible candidates for anticancer therapy with the ability to inhibit tumor angiogenesis and tumor cell proliferation.

Full text of DOI:10.1007/s00044-009-9187-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:236–249
DOI 10.1007/s00044-009-9187-7
ORIGINAL RESEARCH
Synthesis and in vivo anticancer and antiangiogenic
effects of novel thioxothiazolidin-4-one derivatives
against transplantable mouse tumor
S. Chandrappa Æ H. Chandru Æ A. C. Sharada Æ K. Vinaya Æ
C. S. Ananda Kumar Æ N. R. Thimmegowda Æ
P. Nagegowda Æ M. Karuna Kumar Æ K. S. Rangappa
Received: 7 May 2008 / Accepted: 17 March 2009 / Published online: 23 April 2009
¨
Ó Birkhauser Boston 2009
Abstract A series of novel thioxothiazolidin-4-one derivatives 5(a–g) were
synthesized by the coupling of different amines containing aliphatic, substituted
aromatic, and heterocyclic moieties, such as oxadiazol, pyrazole, isoxazole,
and piperazine with 2-(5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thiox-
othiazolidin-3-yl)acetic acid. All compounds were characterized by 1H NMR,
LCMS, FTIR and elemental analysis. In this study, we investigated the possibility
that these novel thioxothiazolidin-4-one derivatives 5(a–g) inhibits tumor growth
and tumor induced angiogenesis using mouse Ehrlich Ascites Tumor (EAT) as a
model system. Our results demonstrated that the compounds significantly reduced
ascites tumor volume, cell number, and increased the life span of EAT-bearing
mice. In addition, the compounds manifested strong antiangiogenic effects and
suppressed tumor induced endothelial proliferation in the mice peritoneum. From
our findings, it is noted that the derivatives 5(a–e) may be possible candidates for
anticancer therapy with the ability to inhibit tumor angiogenesis and tumor cell
proliferation.
Keywords Thioxothiazolidin-4-one ꢀ EAT cells ꢀ Lifespan ꢀ Anticancer ꢀ
Antiangiogenesis
S. Chandrappa ꢀ K. Vinaya ꢀ C. S. Ananda Kumar ꢀ N. R. Thimmegowda ꢀ
P. Nagegowda ꢀ K. S. Rangappa (&)
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
e-mail: rangappaks@gmail.com; rangappaks@chemistry.uni-mysore.ac.in
H. Chandru ꢀ A. C. Sharada
Department of Biochemistry, Yuvaraja’s College, University of Mysore, Mysore 570005, India
M. Karuna Kumar
Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570006,
India

Med Chem Res (2010) 19:236–249
237
Introduction
Cancer is a disease of worldwide importance. Its incidence in the developed
countries is increasing, and its mortality occupies the second rank in the order
of death causes. Similar tendency can be observed in the developing world: the
gradual improvement in the life expectancy is associated with an elevated cancer
incidence and mortality. Accordingly, we might assume that malignancy will be
soon a global problem with its consecutive burden. Therefore, it is easy to
understand that cancer therapy is in the focus of common interest. For now,
the treatment of any malignancy is based on surgery, radiotherapy, and drug
therapy.
The growth and metastasis of cancer cells are dependent on angiogenesis.
Angiogenesis has been functionally defined as the sprouting of new vessels from
preexisting blood vessels and is an essential process in wound healing.
Furthermore, angiogenesis is a critical process in tumor cells invasion (Zetter,
1998). It is deemed that only those cells that have completed neovascularization
are capable of growing violently in size and volume, especially when the tumor
size is beyond 2–3 mm. The process of angiogenesis induced by tumor cells
consists of several distinct stages, including slow growth of tumor cells without
blood vessels and then releasing specific angiogenic growth factors (Fontanini
et al., 1999).
In the rapid developing field of small molecule combinatorial chemistry, it
particularly appeals easy way for the synthesis of small heterocyclic compound
libraries (Gordon et al., 1994; Gordon and Steele, 1995; Green, 1995; Murphy et al.,
1995; Ruhland et al., 1996). An important application of small molecule libraries is
the preparation of a directed or focused combinatorial library for assay against a
specific biological target. As part of our search for biologically active compounds
with sulphur- and nitrogen-containing heterocycles, 4-thiazolidinones substituted in
the 2 position were proven to be biologically very potent and selective (McLamore
et al., 1952; Sobin, 1952; Tanabe et al., 2006). A wide spectrum of pharmacological
activities has been reported for these compounds. Some of these therapeutic areas
include antimicrobial (Franzen, 2000), anticancer (Lakhan and Rai, 1987; Hour
et al., 2000), antiviral (Hamel et al., 1996), anticonvulsant (Corbett et al., 2000;
Archana et al., 2003), antifungal (Samir et al., 2007; Bartroli et al., 1998), anti-
inflammatory (Goel et al., 1999), analgesic (Smith and Dewitt, 1996), and
antiproliferative activities (Herschman, 1996; Veeresa et al., 2004). Thiazolidinones
derivatives possess broad pharmacological action on the central nervous system,
especially anti-HIV agents (Barreca et al., 2001) and cyclooxygenase (COX)
inhibitors (Chen et al., 2004).
In this context, the synthesis of libraries centered on a known lead compound are
valuable for the evaluation of the tumor growth inhibitory activities of novel
synthetic thiazolidinone analogs on Ehrlich ascites tumor cells in vivo. The
observations suggest that the synthetic thiazolidinone analogs most likely target
tumor proliferation and tumor progression.

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Chemistry
Thiazolidinone derivatives 5(a–g) were prepared by the method summarized in
Scheme 1. Initially the compound 3 was synthesized by the condensation reaction
with rhodhanine-3-acetic acid 1 with phenyl furfural 2 by using sodium acetate and
acetic acid. The obtained yield was found to be 80%. Finally, the obtained
intermediate 3 was treated with substituted aliphatic, aromatic and heterocyclic
amines in presence of N-methyl morpholine, HATU, and N,N-dimethyl formamide
as a solvent at room temperature to give the target molecules with a good yield
ranging from 76–86% with high purity. All the synthesized molecules 5(a–g) were
1
characterized by H NMR, LCMS, IR, and elemental analysis.
Results and discussion
Chemistry
The coupling reaction of thiazolidinone moiety with different substituted amines
1
was confirmed by the H NMR and IR data. The disappearance of –NH2 peak and
Cl
Cl
O
O
Gla AcOH/CH3COONa
1200 C, 10 h
O
OHC
O
+
S
HOOC
N
HOOC
N
S
S
S
2
3
1
Cl
O
O
NMM, HATU, DMF
O
N
S
r.t, 5 h, R-NH2 4(a-g)
R
N
H
S
5(a-g)
Where R-NH2=
N
O
N
NH
O
H2N
N
N
H3C
N
CH3
F
H2N
4a
4c
4b
NH2
N
O
NH2
OH
NH2
H3COOC
O
NH2
4d
4e
4f
4g
Scheme 1 Synthesis of thiazolidinone derivatives 5(a-g)

Med Chem Res (2010) 19:236–249
239
–COOH peaks confirms the formation of our target molecules. From IR data,
appearance of HN-C=O symmetric stretching frequency ranging from 3478–
3385 cm-1 and appearance of –C=O in thiazolidinone at 1637–1603 cm-1 confirms
the formation of products. All the synthesized molecules 5(a–g) were evaluated for
their anticancer activity.
Biology
The peritoneum of the mice were cut open and the inner lining of the peritoneal
cavities were examined for angiogenesis in both control and compound 5(a–g)
treated tumor bearing mice and photographed. All data were analyzed by using one-
way ANOVA and observed for significance at P \ 0.05 level.
In continuation of our research on anticancer and angioinhibitory activities, we
report the growth inhibitory and anti-angiogenic effects of the novel synthetic
thiazolidinone analogs 5(a–g) on the Ehrlich’s ascites tumor cells grown in the
peritoneal cavity of Swiss albino mice (Chandru and Sharada, 2007a, b; Chandru
et al., 2007a, b; Ananda Kumar et al., 2008). The compounds 5(a–e) inhibited the
tumor growth resulting in increase in the Medial Survival Time (MST) and %
Increase in Life Span (ILS) of tumor bearing mice. The tumor growth delay by
the compounds was further supported with the decrease in cell number and ascites
volume. These findings suggest that the effect of compounds is on the
proliferating tumor cells. Our reports agree with the earlier studies on in vitro
antiproliferative activity against human colon cancer cell lines of thiazolidinone
(Roman et al., 2006). Few thiazolidinone possess in vitro antiproliferative activity
by acting as inhibitors of translation initiation process. Various thiazolidinone
(Veeresa et al., 2004; Rosaria et al., 2005) have been reported for antitumor
activities (Chimirri et al., 1986). Over expression of COX has been shown to be
associated with a wide variety of cancers and the role of COX in tumor cell
proliferation has been demonstrated (Williams et al., 1999). Thiazolidinones
derivatives are found to be cyclooxygenase (COX) inhibitors (Chen et al., 2004).
The results on anti-angiogenic studies of 5(a–g) compounds demonstrated that, in
addition to being good anti-tumor agents, the compounds can also act as effective
angiogenic inhibitors. These compounds prevented the proliferation of endothelial
cells in tumor bearing mouse peritoneum. Tumor angiogenesis is a complex process
of proliferation and migration of endothelial cells towards the cancer mass that
secretes a variety of growth factors (Kato et al., 1994). Thiazolidinone have been
demonstrated as potential antitumor agents on human colorectal carcinoma
(Vigorita et al., 1997; Vigorita et al., 2001; Ottana et al., 2002), and in this study,
the angioinhibitory effects may be due to inhibition of growth factors which requires
further investigations.
The coupling of different amines containing aliphatic, substituted aromatic and
heterocyclic moieties such as oxadiazol, pyrazole, isoxazole, piperazine with 2-(5-
(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
leads to the production of novel compounds 5(a–g). Among the derivatives,
compound 5b exhibited maximum tumor growth inhibition compared to 5a due to
the presence of pyrazole with cyclopropyl ring, where as 5a substituted with a

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Med Chem Res (2010) 19:236–249
oxadiazol moiety in the phenyl ring exhibited lower tumor growth inhibitory effects.
Compound 5e contains a phenyl ring with good electron donating methoxy group
and compound 5c contains phenyl ring with electron withdrawing fluorine group
and 5d has phenolic group and all these compounds exhibited comparable antitumor
response. However, the compounds 5f with isoxazole moiety and 5g with butyl
chain did not show effective antitumor response. The additional modification and
diversification of functional groups in order to improve the anti-cancer activity is
currently in progress.
Conclusion
Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazo-
lidin-3-yl)acetic acid derivatives gave good yield with purity. The results of the
present findings are encouraging, and the compounds 5(a–e) have shown good anti-
tumor and anti-angiogenic effects against transplantable mouse Ehrlich ascites
tumor. Further studies on the thiazolidinone derivatives are of great importance
because the compounds may lead to potential therapeutic agents for treatment of
cancer.
Experimental
Melting points were determined using SELACO-650 hot stage melting point
apparatus and were uncorrected. Infrared (IR) spectra were recorded using a Jasco
FTIR-4100 series. Nuclear magnetic resonance (1H NMR) spectra were recorded on
Shimadzu AMX 400-Bruker, 400 MHz spectrometer using DMSO-d6 as a solvent
and TMS as internal standard (chemical shift in d ppm). Spin multiplets are given as
s (singlet), d (doublet), t (triplet), and m (multiplet). Mass and purity were recorded
on a LC-MSD-Trap-XCT. Elemental (CHNS) analyses were obtained on Vario EL
III Elementar. Silica gel column chromatography was performed using Merck 7734
silica gel (60–120 mesh) and Merck made TLC plates.
Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid (3)
A mixture of 3-rhodanine-3-acetic acid 1 (1.0 g, 0.01 mmol), 5-(3-chlorophenyl)-
furfural 2 (1.08 g, 0.01 mmol) and anhydrous sodium acetate (1.3 g, 0.03 mmol)
were taken in 10 ml glacial acetic acid. The reaction mixture was heated to 120 °C
in an oil bath for 10 h. Then the reaction mixture was cooled, filtered, and washed
with ether. A reddish solid compound was obtained (1.75 g, 75%). The schematic
representation of the synthesized compound is shown in Scheme 1. 1H NMR
(DMSO-d6, 400 MHz) d: 10.5 (s, 1H, –COOH), 7.9 (d, 1H, Ar–H), 7.6 (t, 1H, Ar–
H), 7.5 (d, 1H, Ar–H), 7.4 (d, 2H, Ar–H), 7.1 (d, 1H, Ar–H), 4.5 (s, 2H, –CH2). MS:
379.97.

Med Chem Res (2010) 19:236–249
241
Procedure for the synthesis of 4-(5-methyl-1,2,4-oxadiazol-
3-yl)benzeneamine (4a)
A mixture of 4-amino benzonitrile (1.0 g, 8.4 mmol), hydroxyl amine hydrochlo-
ride (0.64 g, 9.2 mmol), and KOH (1.4 g, 25 mmol) were taken in methanol
(10 ml). The reaction mixture was refluxed for 2 h. After completion of the
reaction, the solvent was removed under reduced pressure. The resulting residue
was dissolved in water and extracted with ethyl acetate. The organic layer was dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained
oxime was cyclized using sodium ethoxide (1.09 g, 16.0 mmol) in presence of ethyl
acetate (3 volume) to get 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzeneamine as off
white solid. The schematic representation is shown in Scheme 2. 1H NMR (DMSO-
d6, 400 MHz) d: 7.6 (dd, 2H, Ar–H), 6.6 (dd, 2H, Ar–H), 5.7 (bs, 2H, –NH2), 2.5
(s, 3H, –CH3).
Procedure for the synthesis of 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-
5-amine (4b)
1-(4-methoxybenzyl)-2-methylhydrazine salt (1.0 g, 5.36 mmol) and 3-cyclopro-
pyl-3-oxopropanenitrile (0.85 g, 5.36 mmol) were taken in ethanol, and then
sodium ethoxide (1.09 g, 16.0 mmol) was added. The reaction mixture was refluxed
for 2 h. After completion of the reaction, the solvent was removed under reduced
pressure. The workup procedure was same as discussed earlier. The obtained
1
product was white solid. The schematic representation is shown in Scheme 3. H
NMR (CDCl3, 400 MHz) d: 7.13 (d, 2H, Ar–H), 6.9 (d, 2H, Ar–H), 5.2 (s, 1H,
Ar–H), 5.1 (s, 2H, –CH2), 3.8 (s, 3H, –OCH3), 3.3 (br s, 2H, –NH2), 1.9 (m, 1H,
–CH), 0.9 (m, 2H, –CH2), 0.7 (m, 2H, –CH2).
OH
N
NH2OH.HCl, KOH
MeOH, Reflux, 2 h
N
N
EtOH/EtCOONa
O
H2N
H2N
CN
H2N
C NH2
CH3COOEt, reflux,
1.5 h
CH3
4a
Scheme 2 Synthesis of 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzeneamine (4a)
CH3
OCH3
O
H2N
O
CH2CN
EtOH/EtCOONa
reflux, 2 h
N
N
+
NH
NH2 HCl
4b
Scheme 3 Synthesis of 1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-amine (4b)

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Med Chem Res (2010) 19:236–249
General procedure for the synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a–g)
A solution of 2-(5-((5-(4-chlorophenyl) furan-2-yl)methylene)-4-oxo-2-thioxothia-
zolidin-3-yl)acetic acid 3 (1.0 eq) and appropriate amines (1.0 eq) in dry DMF was
taken. N-methyl morpholine (NMM) (3.0 eq) and 10% of the HATU catalyst were
added to the reaction mixture. The reaction mixture was stirred for 5 h at room
temperature, and progress of the reaction was monitored by TLC. After completion
of the reaction, water was added and the reaction mixture was filtered and finally
washed with ether and dried under vacuum.
Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)-N-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)acetamide
(5a)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), 4-(5-methyl-1,2,4-oxa-
diazol-3-yl)benzeneamine 4a (0.05 g, 0.01 mmol), N-methyl morpholine (0.04 g,
0.03 mmol), and 0.01 g HATU. The product obtained was reddish solid (0.12 g,
1
80%). M.P (°C): 251–253. H NMR (DMSO-d6, 400 MHz) d: 10.51 (s, 1H, –NH),
7.94 (d, 2H, Ar–H), 7.80 (d, 2H, Ar–H), 7.72 (d, 1H, =CH), 7.46 (d, 2H, Ar–H), 7.3
(d, 1H, Ar–H), 7.01 (d, 1H, Ar–H), 6.68 (d, 1H, Ar–H), 4.53 (s, 2H, –CH2), 3.1 (s,
3H, –CH3). MS (-1): 535.70. IR (KBr, cm-1): 3441, 1603, 1364, 1190, 1057. Anal.
calcd. for C25H17ClN4O4S2 (%): C-55.91, H-3.19, N-10.43, S-11.94. Found C-
55.88, H-3.16, N-10.40, S-11.89.
Synthesis of N-(1-(4-methoxybenzyl)-3-cyclopropyl-1H-pyrazol-5-yl)-2-(5-((5-
(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetamide (5b)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), 1-(4-methoxybenzyl)-3-
cyclopropyl-1H-pyrazol-5-amine 4b (0.07 g, 0.01 mmol), N-methyl morpholine
(0.04 g, 0.03 mmol), and 0.01 g HATU. The product obtained was reddish solid
(0.14 g, 85%). M.P (°C): 254-256. 1H NMR (DMSO-d6, 400 MHz) d: 11.01 (s, 1H,
–NH), 7.93 (d, 1H, =CH), 7.61 (m, 2H, Ar–H), 7.52 (d, 2H, Ar–H), 7.44 (d, 1H, Ar–
H), 7.2 (d, 2H, Ar–H), 7.23 (d, 2H, Ar–H), 5.24 (s, 1H, Ar–H), 5.13 (s, 2H, –CH2),
4.9 (s, 2H, –CH2), 3.83 (s, 3H, –OCH3), 1.84 (m, 1H, –CH), 0.9 (2H, m, –CH2), 0.76
(m, 2H, –CH2). MS (-1): 602.10. IR (KBr, cm-1): 3478, 1609, 1331, 1190, 1057.
Anal. calcd. for C30H25ClN4O4S2 (%): C-59.54, H-4.16, N-9.26, S-10.60. Found C-
50.50, H-4.12, N-9.22, S-10.56.

Med Chem Res (2010) 19:236–249
243
Synthesis of 5-((5-(4-chlorophenyl)furan-2-yl)methylene)-3-(2-(4-(2-
fluorophenyl)piperazin-1-yl)-2-oxoethyl)-2-thioxothiazolidin-4-one (5c)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)aceticacid 3 (0.1 g, 0.01 mmol), 1-(2-fluorophenyl)pipera-
zine 4c (0.05 g, 0.01 mmol), N-methyl morpholine (0.04 g, 0.03 mmol), and 0.01 g
HATU. The product obtained was reddish solid (0.12 g, 77%). M.P (°C): 234–236.
1H NMR (DMSO-d6, 400 MHz) d: 7.94 (d, 1H, =CH), 7.82 (d, 2H, Ar–H), 7.62 (m,
1H, Ar–H), 7.5 (m, 2H, Ar–H), 7.42 (d, 1H, Ar–H), 7.1 (m, 4H, Ar–H), 5.04 (s, 2H,
–CH2), 3.6-3.7 (s, 4H, N–CH2), 2.9-3.0 (s, 4H, N–CH2). MS (?1): 542.04. IR (KBr,
cm-1): 1613, 2925, 1502, 1329, 1208. Anal. calcd. for C26H21ClFN3O3S2 (%): C-
57.61, H-3.90, N-7.75, S-11.83. Found C-57.57, H-3.86, N-7.71, S-11.79.
Synthesis of methyl 2-(2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetamido)-3-(4-hydroxyphenyl)propanoate (5d)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), methyl 2-amino-3-(4-
hydroxyphenyl)propanoate 4d (0.06 g, 0.01 mmol), N-methyl morpholine (0.04 g,
0.03 mmol), and 0.01 g HATU. The product obtained was reddish solid (0.126 g,
1
84%). M.P (°C): 228–230. H NMR (DMSO-d6, 400 MHz) d: 9.43 (s, 1H, –NH),
8.7 (bs, 1H, –OH), 7.9 (d, 2H, Ar–H), 7.8 (d, 3H, Ar–H), 7.73 (d, 1H, =CH), 7.4 (d,
2H, Ar–H), 7.3 (d, 1H, Ar–H), 7.02 (d, 1H, Ar–H), 6.68 (d, 1H, Ar–H), 4.6 (d, 2H,
–CH2), 4.5 (s, 2H, –CH2), 3.6 (d, 3H, –COOCH3), 4.4 (t, 1H, –CH). MS (?1):
555.10. IR (KBr, cm-1): 3434, 1637, 1316, 1200. Anal. calcd. for C26H21ClN2O6S2
(%): C-56.06, H-3.80, N-5.03, S-11.51. Found C-56.01, H-3.76, N-4.98, S-11.47.
Synthesis of N-(4-methoxyphenethyl)-2-(5-((5-(4-chlorophenyl)furan-2-
yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetamide (5e)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), 2-(4-methoxyphenyl)eth-
anamine 4e (0.04 g, 0.01 mmol), N-methyl morpholine (0.04 g, 0.03 mol), and
0.01 g HATU. The product obtained was reddish solid (0.11 g, 80%). M.P (°C):
219–221. 1H NMR (DMSO-d6, 400 MHz) d: 10.12 (s, 1H, –NH), 7.9 (d, 1H, =CH),
7.8 (m, 3H, Ar–H), 7.6 (m, 3H, Ar–H), 7.5 (m, 3H, Ar–H), 7.3 (d, 1H, Ar–H), 5.2 (t,
2H, –CH2), 4.9 (t, 2H, –CH2), 4.5 (s, 2H, –CH2), 3.8 (s, 3H, –OCH3), 4.5 (s, 2H,
–CH2), 3.6 (d, 3H, –COOCH3), 4.4 (t, 1H, –CH). MS (?1): 513. IR (KBr, cm-1):
3461, 1634, 1405, 1325, 1191. Anal. calcd. for C25H21ClN2O4S2 (%): C-58.53,
H-4.13, N-5.46, S-12.50. Found C-58.49, H-4.09, N-5.42, S-12.45.
Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)-N-(isoxazol-3-yl)acetamide (5f)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), isoxazol-3-amine 4f

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Med Chem Res (2010) 19:236–249
(0.02 g, 0.01 mmol), N-methyl morpholine (0.04 g, 0.03 mmol), and 0.01 g HATU.
1
The product obtained was reddish solid (0.126 g, 86%). M.P (°C): 249–251. H
NMR (DMSO-d6, 400 MHz) d: 10.6 (s, 1H, –NH), 7.9 (d, 1H, =CH), 7.7 (m, 2H,
Ar–H), 7.6 (m, 2H, Ar–H), 7.4 (m, 2H, Ar–H), 7.3 (d, 1H, Ar–H), 4.5 (s, 2H, –CH2).
MS (-1): 445. IR (KBr, cm-1): 3423, 1632, 1330, 1190, 954. Anal. calcd. for
C19H12ClN3O4S2 (%): C-51.18, H-2.71, N-9.42, S-14.38. Found C-51.15, H-2.68,
N-9.38, S-14.34.
Synthesis of N-butyl-5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidine-3-carboxamide (5g)
It was obtained from 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid 3 (0.1 g, 0.01 mmol), butan-1-amine 4g (0.02 g
0.01 mmol), N-methyl morpholine (0.04 g 0.03 mol), and 0.01 g HATU. The
1
product obtained was reddish solid (0.11 g, 80%). M.P (°C): 210-212. H NMR
(DMSO-d6, 400 MHz) d: 11.0 (s, 1H, -NH), 7.86 (d, 1H, =CH), 7.7 (d, 1H, Ar–H),
7.68 (d, 1H, Ar–H), 7.53 (m, 1H, Ar–H), 7.45 (m, 2H, Ar–H), 7.36 (d, 1H, Ar–H),
4.5 (s, 2H, –CH2), 3.6 (t, 2H, –CH2), 3.55 (m, 2H, –CH2), 3.05 (m, 2H, –CH2), 2.13
(t, 3H, –CH3). MS (?1): 435.2. IR (KBr, cm-1): 3385, 1637, 1405, 1116. Anal.
calcd. for C19H17ClN2O3S2 (%): C-54.21, H-4.07, N-6.66, S-15.24. Found C-54.18,
H-4.02, N-6.62, S-15.21.
Biology
In vivo anti-cancer activities of synthetic novel thiazolidinones analogs
Animals and tumor
Inbred Swiss albino mice of 6–8 weeks old weighing 25 5 g of either sex were
used for the experiments. Ehrlich ascites tumor was grown in adult Swiss albino
mice intraperitoneally. Experimental animals were prepared by injecting 5 9 106
viable tumor cells into intraperitoneal cavity of Swiss mice. Tumor growth was
followed by recording the animal weights. EAT cells started exponential growth
phase from the seventh day after tumor cell injection and the animal succumb to the
ascites tumor burden on 14–18 days after injection.
Animal survival
Seven days after tumor cell injection, the animals were divided into groups of five
each and were treated as follows: 1) control: 0.2 ml of 0.1% DMSO was given on
days 7, 9, and 11 after tumor transplantation; 2) compound-treated groups: the
compounds 5(a–g) were given to seven different groups of tumor bearing mice.
Three doses of the compounds 5(a–g) (100 mg/kg body. wt) were injected ip into
the mice on days 7, 9, and 11 after tumor transplantation. All the mice were weighed
on the day of tumor inoculation and at weekly intervals. Animal survival was

Med Chem Res (2010) 19:236–249
245
recorded up to 28 days. The tumor response was assessed on the basis of median
survival time (MST) and increase in life span (% ILS) (Sharada et al., 1996). MST
and % ILS were calculated from the mortality data with in the observation period.
Enhancements of life span by 25% are more over that of the control was considered
as effective anti-tumor response.
In vivo tumor growth inhibition
After 7 days of tumor cell injection the animals were divided into groups of ten
each; the control group received 0.2 ml of 0.1% DMSO on days 7, 9, and 11 after
tumor transplantation. The compounds 5(a–g) were given to seven different groups
of tumor bearing mice as scheduled above. The tumor inhibitory effect of the
compounds on EAT cell growth was assessed by counting cell number and ascites
volume. On day 12, the control and compounds 5(a–g)-treated tumor-bearing mice
were killed and an incision was made in the abdominal region. EAT cells along with
the ascites fluid were harvested in a beaker containing 2 ml of saline and
centrifuged at 3000 rpm for 10 minutes at 4 °C. The volume of ascites fluid was
obtained by subtracting the volume of saline added from the volume of the
supernatant. The harvested EAT cells were resuspended in 0.9% saline and counted
using a hemocytometer.
The vehicle-treated control animals developed tumor and died within 14–
18 days; median survival time (MST) was 16 days. Three doses of 100 mg/kg body
weight of thiazolidinone analogs 5(a–g) treatments on days 7, 9, and 11 after tumor
transplantation increased the MST and % ILS. The compound 5a showed 23 days of
MST with 43.75% ILS, but the compound 5g was ineffective and produced 12.5%
Table 1 Effect of synthetic novel thiazolidinone analogs 5(a–g) on survival of mice bearing Ehrlich
ascites tumor
Treatment
groups
No. of
animals
Schedule
days
MST
(days)a
ILS (%)
–
Av. Wt changeb
8.52 0.41
Control
5
7, 9, 11
16
(0.1%DMSO)
5a
5b
5c
5d
5e
5f
5
5
5
5
5
5
5
7, 9, 11
7, 9, 11
7, 9, 11
7, 9, 11
7, 9, 11
7, 9, 11
7, 9, 11
23*
25*
21*
20*
22*
19
43.75
56.25
31.25
25
-3.4 0.5
-3.71 0.48
-2.6 0.34
-2.22 0.28
-2.63 0.31
-2.41 0.52
-2.3 0.61
37.5
18.25
12.5
5g
18
5(a–g) dose in 100 mg/kg body wt
a
Median survival time (MST) and ILS % was calculated from the mortality data within the observation
period
b
Determined on 12th day of treatment
* Significant difference from control (P \ 0.05)

246
Med Chem Res (2010) 19:236–249
ILS, which is less than the effective anti-tumor response (25% ILS). The compound
5e increased the % ILS, which was doubled to that produced by the compound 5f.
The compound 5c have shown considerable increase in lifespan of animals. The %
ILS reached 50% in the 5d-treated group. The highest tumor response 56.25% ILS
was observed in 5b-treated group. The decrease in animal body weights in
compound-treated groups compared with the control is an indication of inhibition of
tumor cell proliferation (Table 1).
10
9
8
*
*
*
7
6
5
4
3
2
1
0
*
*
*
*
Control 5a
5b
5c
5d
5e
5f
5g
Treatments
Fig. 1 Effect of the compounds 5(a–g) on ascites volume of EAT bearing mice. The bar graph represents
the effect of the compounds on ascites volume. All treatments showed a significant decrease in ascites
volume from that of control (P \ 0.05). The error bars represent standard deviation of the mean
2500
2000
*
*
*
1500
*
*
*
*
1000
500
0
5a
5b
5c
5d
5e
5f
5g
Control
Treatments
Fig. 2 Effect of the synthetic compounds 5(a–g) on cell number of EAT-bearing mice. The bar graph
represents the effect of the compounds on cell number. All treatments showed a significant decrease in
cell number compared with control (P \ 0.05). The error bars represent standard deviation of the mean

Med Chem Res (2010) 19:236–249
247
The inhibitory effect of thiazolidinone analogs 5(a–g) on EAT cells in vivo was
evaluated in terms of volume of ascites and total number of cells in mice treated
with vehicle or compounds. The mean value of ascites volume and cell number in
control animals was found to be 7.6 0.9 ml (Fig. 1) and 1,840 1.62 9 106
cells/mouse, respectively (Fig 2). Among the compounds 5(a–g) treatment, the best
response was obtained with the compound 5b. This compound decreased the mean
ascites volume to 4.8 0.49 ml with corresponding reduction of mean cell number
to 1,080 0.79 9 106 cells/mouse. The compounds 5a and 5e showed mean
ascites volume of 5.2 0.68 ml and 5.7 1.26 ml, respectively. The compound
5c and 5d were equally effective in reducing ascites volume and cell number.
Although the compounds 5f and 5g were ineffective in prolonging the life span of
Fig. 3 Suppression of in vivo angiogenesis by synthetic compounds 5(a–g). Peritoneal lining of tumor
bearing mice treated with vehicle (0.1% DMSO) and thiazolidinone analogs were inspected for anti-
angiogenesis effects. Inhibition of angiogenesis was more prominent in 5(a–g)-treated mice compared
with control

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Med Chem Res (2010) 19:236–249
EAT-bearing mice, 5f showed significant decrease in mean value of ascites volume
and cell number (Figs. 1 and 2).
Examination of tumor induced peritoneal endothelial proliferation of EAT-
bearing mice is a reliable model of in vivo angiogenesis. It is evident from the
results that more number of blood vessels were seen in the peritoneum of control
EAT-bearing mice compared with 5(a–g)-treated mice, which demonstrated
reduced vasculature (Fig. 3).
Acknowledgments The authors are grateful to UGC, Govt. of India for financial support to Prof. K. S.
Rangappa under the projects vide No. F. 31-143/2005(SR) and UGC-SAP (Phase I) vide No. F. 540/10/
2004-05 (SAP I). One of the authors (K. Vinaya) is grateful to CSIR, New Delhi, for financial support
under CSIR SRF order No. 09/119(0172)2K8 EMR-I. The CHNS data from the instrument granted by
DST-FIST programme order No. SR/FST/CSI-051/2002 are greatly acknowledged.
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