Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

Article abstract of DOI:10.1016/j.bmc.2009.01.016

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-l) were synthesized. Variation in the functional grou

Full text of DOI:10.1016/j.bmc.2009.01.016

Bioorganic & Medicinal Chemistry 17 (2009) 2576–2584
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxo-
thiazolidin-3-yl)acetic acid derivatives and evaluation of their cytotoxicity
and induction of apoptosis in human leukemia cells
S. Chandrappa a, C. V. Kavitha b, M. S. Shahabuddin b, K. Vinaya a, C. S. Ananda Kumar a, S. R. Ranganatha a,
a,
Sathees C. Raghavan b, , K. S. Rangappa
*
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
b Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-
(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a–l)
were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of com-
pounds bearing amide moiety. Their chemical structures were confirmed by 1H NMR, IR and Mass Spectra
analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antipro-
liferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leu-
kemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed
by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring
plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have
shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating
group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different elec-
tron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation
suggest that 5e is more cytotoxic and able to induce the apoptosis.
Received 5 December 2008
Revised 8 January 2009
Accepted 9 January 2009
Available online 15 January 2009
Keywords:
4-Oxo-2-thioxothiazolidinone
Leukemia
Antiproliferation
Anticancer agents
DNA damage
Apoptosis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Leukemia is one of the major types of cancer affecting a signifi-
cant segment of the population, especially children. Although
Among diseases cancer is considered as the deadliest and a sub-
stantial number of new antineoplastic agents have been discov-
ered. Considerable insight has been gained into the mechanisms
by which many of these compounds affect cellular growth and this
knowledge has been used in the designing of new chemotherapeu-
tic drugs.1 Chemotherapy combined with surgery and/or radiother-
apy still has an important role to play in the therapeutic approach
of cancer.
Cancer cells differ from their normal counterparts in a number
of biochemical processes, particularly during the control of cell
growth and division. One characteristic of cancer cells, that distin-
guishes them from most normal cells, is their high proliferative in-
dex. As a result, targeting of proliferative pathways resulting in cell
death via apoptosis or prevention of cell division via cell cycle ar-
rest, are considered effective strategies for fighting this disease.
development of better chemotherapy regimens has improved
remission induction and overall survival in leukemia, relapse re-
mains a common problem.2,3 Development of innovative therapies
and identification of more effective drugs, therefore, remain high
priorities for leukemia research. Currently, combined anticancer
therapies or multi-acting drugs are clinically preferred to tradi-
tional cytotoxic treatment, with the aim of overcoming resistance
and toxicity drawbacks. These events often prevent successful
treatment and are responsible for reduced survival times.4,5 Hence
in the field of chemotherapeutic drugs, the search for new, more ac-
tive, more selective and less toxic compounds is still very intense,
and new promising anticancer approaches are being tested.6,7
In discovering small anticancer molecules, a notable role is
played by heterocyclic structures, and among these, a growing
attention focuses on the synthesis and study of the biological prop-
erties of compounds containing various combinations of thiazolid-
inone or furfural moieties.8 An important application of small
molecule libraries is the preparation of a directed or focused com-
binatorial library for assay against a specific biological target. 4-
thiazolidinones substituted in the 2 position were proven to be
biologically very potent and selective.9–11 A wide spectrum of
* Corresponding authors. Tel.: +91 80 2293 2674; fax: +91 080 2360 0814 (S.C.R.),
tel.: +91 821 2419661; fax: +91 821 2412191 (K.S.R.).
E-mail addresses: sathees@biochem.iisc.ernet.in (S.C. Raghavan), rangappaks@
chemistry.uni-mysore.ac.in, rangappaks@gmail.com (K.S. Rangappa).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.016

S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
2577
Cl
O
Cl
O
(i)
O
+
OHC
O
HOOC
N
S
HOOC
N
S
S
S
1
2
3
Cl
O
O
O
(ii)
N
S
R
N
H
S
5(a
-l)
Scheme 1. Reagents and conditions: (i) CH3COONa, CH3COOH, 120 °C, 12 h; (ii) isobutyl chloroformate, triethyl amine, THF, R-NH2 4(a–l), rt, 4–5 h. Where 4a = 4-trifluoro
methyl benzyl amine; 4b = 4-cyanophenyl amine; 4c = 4-fluorobenzyl amine; 4d = 5-amino-2-(trifluoromethyl)benzonitrile; 4e = 4-methoxy benzyl amine; 4f = 4-methyl
benzyl amine; 4g = 4-bromo benzyl amine; 4h = 2-chloro benzyl amine; 4i = 1H-tetrazol-5-amine; 4j = hexan-1-amine; 4k = 3- methyl benzyl amine; 4l = butan-1-amine.
pharmacological activities has been reported for these compounds.
These include anticancer,12–14 peptic ulcer,15,16 antimicrobial,17
Table 1
antiviral,18 anticonvulsant,19 antitubercular,20 antifungal,21 anti-
Chemical structure, yield and melting point of the synthesized compounds 5(a–l)
inflammatory,22 analgesic23 and antiproliferative24 activities. Thia-
Compound
R
Yield (%)
Mp (°C)
zolidinone derivatives also possess wide range of pharmacological
action, especially on anti-HIV agents25,26 and ability to inhibit the
bacterial enzyme MurB.27 It is also reported that, groups like furan
and aryl furans contribute to the biological activities28 and these
would result in highly potent and selective anticancer agents.29
The design, synthesis and biological study of new anticancer com-
pounds to obtain compounds with enhanced activity are an ongo-
ing research project in our group. We have recently reported the
synthesis of novel derivatives of diazaspiro bicyclo hydantoin
and induction of apoptosis in leukemia cells.30,31 We also devel-
oped a series of thiazolidinone derivatives and investigated their
anticancer properties against Ehrlich ascites tumor cells.32–34 In
continuation of our research in this field we designed and synthe-
sized novel thiazolidonones having furan moiety.
5a
76
182–184
CF3
5b
5c
5d
5e
5f
82
79
85
88
82
80
79
185–187
192–194
190–192
179–181
188–190
176–178
186–188
CN
F
CN
CF3
In this study, we investigated the antiproliferative activity and
apoptosis induced by the newly synthesized thiazolidinone ana-
logs against human leukemia cells. Our data indicate that com-
pounds with more electron donating groups induce significant
apoptosis rather than electron withdrawing groups.
OCH3
CH3
Br
2. Results and discussion
2.1. Chemical synthesis
5g
5h
Synthesis of the key intermediate 2-(5-((5-(4-chlorophenyl)
furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid
3 is outlined in Scheme 1. Thiazolidine-2,4-dione 1 was treated
with 5-(3-chlorophenyl)- furfural 2, this undergoes aldol conden-
sation to produce adduct 3. This reaction requires use of sodium
acetate as a base and acetic acid as solvent. The reaction of 2-(5-
((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiaz-
olidin-3-yl)acetic acid 3 with different aromatic amines (R-NH2)
were carried out in the presence of base triethyl amine, isobutyl
chloroformate and tetrahydrofuran (THF) as solvent with a good
yield ranging from 76% to 90%. Synthesized molecules 5(a–l) were
structurally characterized by 1H NMR, IR analysis and Mass Spectra
(MS). The coupling of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methy-
lene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid 3 with different
aromatic amines was confirmed by IR and 1H NMR data. Com-
pounds 5(a–l) were confirmed by IR data, which showed disap-
pearance of stretching frequencies of COOH at 2995 cmÀ1. From
1H NMR spectra, this showed appearance of –CONH at
12.03 ppm. The chemical structures, physical data and yield of all
the synthesized compounds are given in Table 1.
Cl
N
N
5i
5j
5k
5l
N
85
90
84
88
195–197
187–189
185–187
182–184
N
H
CH3

2578
S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
2.2. Thiazolidinones 5(a–l) induces cytotoxicity on leukemic
cells
the compounds identified, 5e displayed the most prominent cyto-
toxicity which could be attributed to its electron donating methoxy
group.
Induction of cell death or inhibition of cell proliferation is an
important property for chemotherapeutic agents. In the present
study, we have used trypan blue and MTT assay to investigate
the effect of thiazolidinones 5(a–l) on cell viability of different leu-
kemic cell lines (K562 and Reh). The cells were counted at intervals
of 24 h till the control cells attained stationary phase. Results
showed that addition of compounds 5(a–l) affected the viability
of the cells in most cases in both the leukemic cell lines tested
(Fig. 1 and data not shown). The cytotoxicity induced by the thia-
zolidinones 5(a–l) was in a dose- and time-dependent manner. In
The cytotoxicity induced by thiazolidinones 5(a–l) was further
verified using MTT assay. In order to perform MTT assay, we incu-
bated K562 or Reh cells with 10, 100 and 250 lM of the com-
pounds and cells were harvested at 48 and 72 h. Results showed
that 5e, 5f, 5g, 5h, 5i, 5j, 5k, and 5l were more cytotoxic and ef-
fected in dramatic reduction in cell proliferation at 100 lM or
higher concentration after 48–72 h of treatment (Fig. 2 and data
not shown). Based on these studies the IC50 value for thiazolidinon-
es 5(a–l) were calculated for 72 h (Table 2). Thus our results show
that compounds having electron donating groups have more po-
tent activity. Hence, we decided to compare the results within
the different electron donating groups present at different posi-
tions. For that we have chosen compounds, 5e and 5k bearing
methoxy (para position) and methyl group (meta position),
respectively.
LDH release assay was performed to test the cell damage in-
duced by the compounds 5e and 5k. For this, K562 cells were cul-
tured with 10, 50 and 100 lM of 5e or 5k for 24 and 48 h. The
release of LDH (an indicator of membrane integrity) was measured
as mentioned in ‘Materials and methods’. Results showed a dose-
both the cell lines tested, the lowest concentration (10
zolidinones 5(a–l) was least effective (Fig. 1). However, an increase
in the concentration of thiazolidinones 5(a–l) to 100 or 250 M af-
lM) of thia-
l
fected the cell viability within 48–72 h (Fig. 1B, C and E–L) except
in 5(a, d) (Fig. 1A and D). It was found that the effect was main-
tained even after prolonged incubation periods. Interestingly, the
DMSO control, corresponding to the highest concentrations of
compounds tested did not show any significant toxic effect. Based
on these results we identify that compounds 5e, 5f, 5g, 5h, 5i, 5j,
5k, and 5l induce cytotoxicity in the tested leukemic cells. Among
35
30
25
20
15
10
5
35
30
25
20
15
10
5
control
control
B
E
H
control
C
F
I
35
30
25
20
15
10
5
A
DMSO
DMSO
DMSO
5c-10µM
5c-100µM
5c-250µM
5b-10µM
5b-100µM
5b-250µM
5a-10µM
5a-100µM
5a-250µM
0
0
0
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
Days
60
control
60
Control
D 35
Control
DMSO
5d-10µM
5d-100µM
5d-250µM
DMSO
DMSO
50
40
30
20
10
0
50
40
30
20
10
0
30
25
20
15
10
5
5f-10µM
5f-100µM
5f-250µM
5e-10µM
5e-100µM
5e-250µM
0
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
Days
60
50
40
30
20
10
0
60
control
60
G
control
control
DMSO
DMSO
DMSO
50
40
30
20
10
0
50
40
30
20
10
0
5i-10µM
5i-100µM
5i-250µM
5g-10µM
5g-100µM
5g-250µM
5h-10µM
5h-100µM
5h-250µM
0
1
3
4
5
2Days
0
1
2
3
4
5
0
1
3
4
5
2Days
Days
60
50
40
30
20
10
0
L
control
control
J
control
K 35
60
50
40
30
20
10
0
DMSO
DMSO
DMSO
30
25
20
15
10
5
5l-10µM
5l-100µM
5l-250µM
5k-10µM
5k-100µM
5k-250µM
5j-10µM
5j-100µM
5j-250µM
0
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
Days
Figure 1. Dose-and time-dependent effect of thiazolidinones 5(a–l) on K562 cell survival. Approximately 0.75 Â 105 cells/ml were cultured followed by the treatment of
compound 5(a–l) after 24 h at a concentration of 10, 100 and 250 lM. Besides untreated cells, DMSO treated cells were used as vehicle control. After every 24 h from the time
of addition of compounds, cell viability was determined by trypan blue exclusion assay and data was represented as a graph. Graphs A–L represents the treatment of
compound 5(a–l), respectively against K562 cells.

S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
2579
Figure 2. Determination of the effect of thiazolidinones 5(a–l) on cell proliferation by MTT assay. After 48 and 72 h of exposure, K562 cells with 5(a–l) (10, 100 and 250 lM)
were incubated with MTT (5 mg/ml) in duplicates and resulting blue formazan precipitate was dissolved in detergent and absorbance was measured at 570 nm. Results are
presented as percentage of cell proliferation (the cell viability of vehicle cells were considered as 100%). Error bars are represented in the figure. Graphs A–L represents the
treatment of compound 5(a–l), respectively against K562 cells.
100
80
60
40
20
0
24 hrs
48 hrs
100
80
60
40
20
0
24 hrs
48 hrs
5e
5k
DMSO 10µM
50µM 100µM
Concentration
DMSO 10µM 50µM 100µM
Concentration
Figure 3. Time-and dose dependent LDH release in K562 cells treated with 5e or 5k. K562 cells were incubated for 24 and 48 h with different concentrations of 5e or 5k.
Release of LDH in the medium was measured at 490 nm. Results are presented as percentage of LDH release.
and time-dependent increase in LDH release (Fig. 3), further con-
firming the above results. We observed that 5e, which has a strong
electron donating methoxy group at para position is more potent
than 5k with a less electron donating methyl group at meta
position.
Table 2
2.3. Assessment of cell cycle profile upon treatment with 5e or
5k on K562 cells
IC50 values of thiazolidinones 5(a–l) as determined based on MTT assay
Compound
IC50 (lM)
Data from trypan blue exclusion assay and MTT assay showed
that 5e and 5k induced reduction in the number of live cells. Based
on this, we were interested in studying cell cycle distribution by
fluorescence activated cell sorting (FACS). K562 cells were har-
K562
155
Reh
5a
5b
5c
5d
5e
5f
5g
5h
5i
7
6
170
90
165
9
7
85
4
5
150
>250 15
>250 14
vested after 48 h of compound treatment (10, 50 and 100 lM), PI
40
45
68
64
52
50
48
50
2
2
4
3
3
3
3
3
44
45
75
70
50
55
53
55
2
3
4
3
3
5
4
4
stained and subjected to flow cytometry. The histogram of vehicle
control (DMSO) treated cells showed a standard cell cycle pattern,
which include G1 and G2 peaks separated by S phase peak (Fig. 4).
SubG1 peak (mostly dead cells) was either absent or not promi-
nent. Interestingly upon addition of 5e or 5k to K562 cells, a con-
centration dependent change was observed in the cell cycle
pattern (Fig. 4A–D). However, such a change was less prominent
5j
5k
5l

2580
S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
A
B
5e-100µM
5e-50µM
DMSO
G1
5e-10µM
G1
G1
G2/M
G2/M
G2/M
G0/G1
G0/G1
S
G1
S
G2/M
S
S
G0/G1
G2/M
G0/G1
5k-50µM
DMSO
G1
5k-100µM
5k-10µM
G1
G1
G2/M
G2/M
G1
G0/G1
G2/M
S
S
G0/G1
S
S
G0/G1
G0/G1
G0/G1
100%
80%
60%
40%
20%
0%
C
G0/G1
G2/M
S
D
100%
80%
60%
40%
20%
0%
G2/M
S
G1
G1
DMSO
10µM
50µM
100µM
DMSO
10µM
50µM
100µM
Concentration
Concentration
5e
5k
Figure 4. Cell cycle analysis of K562 cells treated with thiazolidinones 5e or 5k. K562 cells (0.75 Â 105 cells/ml) were incubated at 37 °C with 5e or 5k (10, 50 and 100
lM).
Following 48 h of incubation, cells were fixed and stained with propidium iodide and subjected to FACS analysis. Panel A and B show histograms comparing the effect of 5e
and 5k at specific cell cycle stages. In both the Panel A and B, the first histogram represents DMSO treated cells. Panel C and D show the quantification of cells in different
stages of cell cycle followed by treatment with 5e and 5k, respectively.
when the cells were treated with 5k at 50
l
M when compared
Thiazolidine is an important scaffold known to be associated
with several biological activities.35 Some of the derivatives of 2-
aryl-4-oxo-thiazolidin-3-yl amides have the ability to inhibit the
with 5e at 50 M. Although we were not able to visualize promi-
l
nent arrest at any stage of the cell cycle, we noted that there was
a remarkable accumulation of subploid cells, the subG1 phase
(G0/G1), and decline of both G1 and G2/M (Fig. 4C and D of 5e
and 5k, respectively) phases when compared with untreated cells.
Therefore, our studies further confirm that growth inhibition could
be due to apoptosis.
1
2
3 4
M
1
2
3
4
M
A
B
10.0Kb
5.0
10.0Kb
5.0
2.4. DNA fragmentation induced by 5e or 5k
3.0
2.0
3.0
2.0
The parameter which was considered to assess the DNA damage
upon treatment with compounds was chromosomal DNA fragmen-
tation. The chromosomal DNA extracted from the K562 cells trea-
ted with increasing concentrations (10, 50 and 100 lM) of 5e or 5k
1.0
1.0
after 72 h, was used for agarose gel electrophoresis as described in
‘Materials and methods’. The results showed fragmentation of the
DNA leading to a smear in the lanes in which cells were treated
with 5e or 5k (Fig. 5). The observed smear is the result of DNA
breakage at multiple positions across the chromosomal DNA. The
0.5
0.3
0.5
0.3
intensity of smear increased with the dose. In case of 5e, 50
showed moderate smearing and 100 showed maximum
(Fig. 5A), however, it was limited to a lesser extent even in
100 M concentration in case of 5k. These results further suggest
that 5e having methoxy group induces fragmentation of chromo-
somal DNA leading to apoptosis more efficiently than 5k having
methyl group.
lM
l
M
Figure 5. Detection of 5e or 5k induced DNA damage in K562 cells. The
chromosomal DNA was extracted from K562 cells following treatment with
different concentrations of 5e (A) and 5k (B). The purified DNA was then resolved
on a 1% agarose gel at 30 V for 6 h. In both panels, Lane 1: DMSO; Lane 2–4: K562
l
cells treated with 10, 50 and 100 lM, respectively. ‘M’ is Marker.

S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
2581
5e and 5k showed a remarkable accumulation of subploid cells,
the subG1 phase (G0/G1) followed by the decline of both G1 and
G2/M phases. Therefore, our studies further suggest that the ob-
served growth inhibition could be due to apoptosis. In addition
to that we have also observed the inter nucleosomal DNA fragmen-
tation induced by the compound 5e, which is a characteristic of
apoptosis mediated cytotoxicity.
OCH3
HN
HN
CH3
O
O
O
O
N
N
S
S
S
S
4. Experimental
4.1. Chemistry
O
O
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 Shima-
dzu 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. Silica gel column chromatography was per-
formed using Merck 7734 silica gel (60–120 mesh) and Merck
made TLC plates.
Cl
Cl
5e
5k
Figure 6. Structures of the potent molecules.
growth of different prostate cancer cells with improved selectivity
compared to serine amide phosphates.36 Some representative of 2-
phenylimino-4-thiazolidinones have been investigated as potent
inhibitors of the growth of human colon carcinoma cell lines with
a different COX-2 expression.37–41 Holla et al., reported the 2-
(5-arylfurfurylidene/5-nitrofurfurylidene)-5-aryl-7-(2,4-dichloro-5-
fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-2(1H)-ones exhibited
in vitro antitumour activity with moderate to excellent growth
inhibition against a panel of 60 different cancer cell lines.42 Thia-
zolidine-2,4-dione and 2,4-thione derivatives have been reported
for inhibitors of translation initiation.12 In view of the above find-
ings, the anticancer activity was checked by carrying out the reac-
tions of 2-(5-((5-(4-chlorophenyl) furan-2-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetic acid with different amines containing
substituted aromatic rings.
From the SAR studies, it reveals that the substitution at C-termi-
nal of the phenyl ring play a key role in its biological activity. 5e
and 5k having electron donating groups enhances the activity,
whereas 5(a–d) having electron withdrawing groups (CN, F, CF3)
decreases the activity. Interestingly, compounds 5g, 5h and 5i hav-
ing electron withdrawing bromo (para), chloro (ortho) and tetra-
zole groups, respectively showed good activity. This could be due
to the less electron withdrawing effect of chloro, bromo and tetra-
zole group when compared to fluoro group 5c. On the other hand,
as the electron donating efficiency increases, the activity also in-
creases. Introducing electron donating methoxy and methyl groups
(5e, 5f) on the C-terminal of the phenyl ring at 4th position re-
sulted in increase in the activity, but the position of methyl group
from 4th to 3rd position lead in the loss of activity and also in 5j
and 5l, shows good activity due to the presence of electron donat-
ing alkyl chain. However, further studies are required to under-
stand the exact mechanism of its action. The structures of the
potent molecules are shown in Figure 6.
4.2. 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-chloro phenyl)-furfural 2 (1.08 g, 0.01 mmol) and anhydrous so-
dium acetate (1.3 g, 0.03 mmol) were taken in glacial acetic acid
10 ml. The reaction mixture was heated to 120 °C in an oil bath
for 10 h. Then the reaction mixture was cooled, filtered, was with
ether. Finally get a reddish solid compound (1.75 g, 75%). 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.
4.3. General procedure for the synthesis of 2-(5-((5-(4-chloro-
phenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid derivatives 5(a-l)
A solution of 2-(5-((5-(4-chlorophenyl) furan-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)acetic acid 3 (1.0 equiv) in dry
THF was taken cool to 0–5 °C. Then isobutyl chloroformate
(1.5 equiv) and triethyl amine were added to the cold reaction mix-
ture. The reaction mixture was stirred for 15 min at same temper-
ature, and then adds appropriate amines (1.0 equiv) to the reaction
mixture. Allow the reaction mixture to room temperature and stir-
red 4–5 h. The progress of the reaction was monitored by TLC. After
completion of the reaction, water was added and the reaction mix-
ture was filtered. Finally washed with ether and dried under
vacuum.
4.3.1. Synthesis of N-(4-(trifluoromethyl)benzyl)-5-((5-(4-chloro-
phenyl)furan-2-yl) methylene)-4-oxo-2-thioxothiazolidine-3-
carboxamide (5a)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 4-trifluoro methyl benzyl amine 4a
(0.046 g, 0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol)
and triethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6,
400 MHz) d: 10.52 (s, 1H, –NH), 7.94 (s, 1H, @CH), 7.81 (d, 2H,
Ar-H), 7.72 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.3 (d, 2H, Ar-H),
7.01 (d, 1H, Ar-H), 6.68 (d, 1H, Ar-H), 5.1 (s, 2H, –CH2), 4.53 (s,
2H, –CH2). MS: 536.5. IR (KBr, cmÀ1): 3430, 1641, 1603, 1366,
1190, 1057, 812.
3. Conclusion
In the present study, we have shown that novel thiazolidinone
derivatives 5(a–l) showed moderate to strong antiproliferative
activity against human leukemia cells. Interestingly from the cyto-
toxic assays we noted that the compounds with electron donating
groups at C-terminal of the phenyl ring resulted in an increase in
the activity by inducing cell death. FACS analysis of compounds

2582
S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
4.3.2. Synthesis of N-(4-(cyano)phenyl)-5-((5-(4-chlorophenyl)
furan-2-yl) methylene)-4-oxo-2-thioxothiazolidine-3-
carboxamide (5b)
2.91 (s, 3H, –CH3). MS: 482.9. IR (KBr, cmÀ1): 3430, 1640, 1607,
1367, 1192, 1056, 805.
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 4-cyanophenyl amine 4b (0.031 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.51 (s, 1H, –NH), 7.93 (s, 1H, @CH), 7.79 (d, 2H, Ar-H), 7.7
(d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.31 (d, 2H, Ar-H), 7.1 (d, 1H,
Ar-H), 6.67 (d, 1H, Ar-H), 4.54 (s, 2H, –CH2). MS: 479.8. IR (KBr,
cmÀ1): 3433, 2224, 1638, 1609, 1376, 1196, 1050, 803.
4.3.7. Synthesis of N-(4-bromobenzyl)-2-(5-((5-(4-chlorophenyl)-
furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetamide
(5g)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid
3 (0.1 g, 0.26 mmol), 4-bromo benzyl amine 4 g
(0.048 g, 0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol)
and triethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6,
400 MHz) d: 10.5 (s, 1H, –NH), 7.91 (s, 1H, @CH), 7.76 (d, 2H, Ar-
H), 7.66 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.31 (d, 2H, Ar-H), 7.05
(d, 1H, Ar-H), 6.67 (d, 1H, Ar-H), 5.1 (s, 2H, –CH2), 4.57 (s, 2H, –
CH2). MS: 547.6. IR (KBr, cmÀ1): 3435, 1645, 1607, 1367, 1193,
1056, 804.
4.3.3. Synthesis of N-(4-(fluoro)benzyl)-5-((5-(4-chlorophenyl)
furan-2-yl) methylene)-4-oxo-2-thioxothiazolidine-3-carbox-
amide (5c)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 4-fluorobenzyl amine 4c (0.032 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.51 (s, 1H, –NH), 7.92 (s, 1H, @CH), 7.77 (d, 2H, Ar-H), 7.68
(d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.31 (d, 2H, Ar-H), 7.05 (d, 1H,
Ar-H), 6.68 (d, 1H, Ar-H), 5.12 (s, 2H, –CH2), 4.54 (s, 2H, –CH2).
MS: 486.9. IR (KBr, cmÀ1): 3430, 1635, 1606, 1368, 1192, 1055,
816.
4.3.8. Synthesis of N-(2-chlorobenzyl)-2-(5-((5-(4-chloro- phenyl)-
furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetamide
(5h)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid 3 (0.1 g, 0.26 mmol), 2-chloro benzyl amine 4h
(0.037 g, 0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol)
and triethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6,
400 MHz) d: 10.51 (s, 1H, –NH), 7.93 (s, 1H, @CH), 7.82 (d, 2H,
Ar-H), 7.7 (d, 2H, Ar-H), 7.65 (d, 2H, Ar-H), 7.35 (d, 2H, Ar-H),
7.13 (d, 1H, Ar-H), 6.7 (d, 1H, Ar-H), 5.13 (s, 2H, –CH2), 4.56 (s,
2H, –CH2). MS: 503.5. IR (KBr, cmÀ1): 3436, 1644, 1607, 1369,
1193, 1056, 727.
4.3.4. Synthesis of N-(3-cyano-4-(trifluoromethyl)benzyl)-5-((5-
(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothi-
azolidine-3-carboxamide (5d)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 5-amino-2-(trifluoromethyl)ben-
zonitrile 4d (0.048 g, 0.26 mmol), isobutyl chloroformate
(0.053 g, 0.39 mmol) and triethyl amine (0.078 g, 0.78 mmol). 1H
NMR (DMSO-d6, 400 MHz) d: 10.53 (s, 1H, –NH), 7.96 (s, 1H,
@CH), 7.85 (d, 1H, Ar-H), 7.79 (d, 2H, Ar-H), 7.49 (d, 2H, Ar-H),
7.35 (d, 2H, Ar-H), 7.1 (d, 1H, Ar-H), 6.7 (d, 1H, Ar-H), 4.56 (s, 2H,
-CH2). MS: 547.8. IR (KBr, cmÀ1): 3444, 2225, 1642, 1616, 1367,
1193, 1056, 806, 725.
4.3.9. Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-
4-oxo-2-thioxothiazolidin-3-yl)-N-(1H-tetrazol-5-yl)acetamide
(5i)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 1H-tetrazol-5-amine 4i (0.026 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.45 (s, 1H, –NH), 7.91 (s, 1H, @CH), 7.82 (s, 1H, N–H), 7.7 (d,
2H, Ar-H), 7.65 (d, 2H, Ar-H), 7.35 (d, 1H, Ar-H), 6.7 (d, 1H, Ar-H),
4.5 (s, 2H, –CH2). MS: 464.8. IR (KBr, cmÀ1): 3433, 3306, 1645,
1609, 1365, 1194, 1056.
4.3.5. Synthesis of N-(4-methoxybenzyl)-2-(5-((5-(4-chloro
phenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl) acetamide (5e)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 4-methoxy benzyl amine 4e
(0.035 g, 0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol)
and triethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6,
400 MHz) d: 10.5 (s, 1H, –NH), 7.9 (s, 1H, @CH), 7.76 (d, 2H, Ar-
H), 7.65 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.3 (d, 2H, Ar-H), 7.02
(d, 1H, Ar-H), 6.67 (d, 1H, Ar-H), 5.1 (s, 2H, –CH2), 4.54 (s, 2H,
–CH2), 3.91 (s, 3H, –OCH3). MS: 498.8. IR (KBr, cmÀ1): 3435,
1638, 1606, 1367, 1193, 1056, 805.
4.3.10. Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)-
methylene)-4-oxo-2-thioxo thiazolidin-3-yl)-N-hexylacetamide
(5j)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid
3 (0.1 g, 0.26 mmol), hexan-1-amine 4j (0.026 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.1 (s, 1H, –NH), 7.87 (s, 1H, @CH), 7.7 (d, 2H, Ar-H), 7.65 (d,
2H, Ar-H), 7.35 (d, 1H, Ar-H), 6.7 (d, 1H, Ar-H), 4.45 (s, 2H, –CH2),
3.1 (t, 2H, –NCH2), 1.3–1.02 (m, 8H, –(CH2)4), 0.78 (t, 3H, –CH3).
MS: 462.9. IR (KBr, cmÀ1): 3402, 2852, 1645, 1608, 1364, 1193,
1055.
4.3.6. Synthesis of N-(4-methylbenzyl)-2-(5-((5-(4-chlorophenyl)
furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetamide
(5f)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 4-methyl benzyl amine 4f (0.031 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.51 (s, 1H, –NH), 7.9 (s, 1H, @CH), 7.75 (d, 2H, Ar-H), 7.65
(d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.3 (d, 2H, Ar-H), 7.04 (d, 1H,
Ar-H), 6.67 (d, 1H, Ar-H), 5.1 (s, 2H, –CH2), 4.53 (s, 2H, –CH2),
4.3.11. Synthesis of N-(3-methylbenzyl)-2-(5-((5-(4-chloro-
phenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
yl)acetamide (5k)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
acetic acid 3 (0.1 g, 0.26 mmol), 3-methyl benzyl amine 4 k
(0.031 g, 0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol)
and triethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6,

S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
2583
400 MHz) d: 10.49 (s, 1H, –NH), 7.92 (s, 1H, @CH), 7.73 (d, 2H, Ar-
H), 7.63 (d, 2H, Ar-H), 7.41 (d, 2H, Ar-H), 7.32 (d, 2H, Ar-H), 7.08 (d,
1H, Ar-H), 6.7 (d, 1H, Ar-H), 5.12 (s, 2H, –CH2), 4.56 (s, 2H, –CH2),
2.95 (s, 3H, –CH3). MS: 483. IR (KBr, cmÀ1): 3430, 1640, 1607,
1367, 1192, 1056, 805.
ture medium was used as the blank and was subtracted. IC50 values
(concentration of compound causing 50% inhibition of cell growth)
were estimated after 72 h of compound treatment. The absorbance
of vehicle cells was taken as 100% viability and the values of trea-
ted cells were calculated as a percentage of control and presented
as histograms (Fig. 2).
4.3.12. Synthesis of 2-(5-((5-(4-chlorophenyl)furan-2-yl)-
methylene)-4-oxo-2-thioxo thiazolidin-3-yl)-N-butylacetamide
(5l)
The product obtained was reddish solid from 2-(5-((5-(4-chlo-
rophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-
4.4.4. LDH release assay
Release of lactate dehydrogenase (LDH) is an indicator of mem-
brane integrity and hence cell injury. LDH assay was performed as
per standard protocols to assess the LDH release in the culture
media following the treatment with 5e or 5k (10, 50 and
acetic acid
3 (0.1 g, 0.26 mmol), butan-1-amine 4l (0.019 g,
0.26 mmol), isobutyl chloroformate (0.053 g, 0.39 mmol) and tri-
ethyl amine (0.078 g, 0.78 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 10.13 (s, 1H, –NH), 7.85 (s, 1H, @CH), 7.72 (d, 2H, Ar-H), 7.63
(d, 2H, Ar-H), 7.30 (d, 1H, Ar-H), 6.71 (d, 1H, Ar-H), 4.51 (s, 2H,
–CH2), 3.15 (t, 2H, –NCH2), 1.22–1.02 (m, 4H, –(CH2)2), 0.81 (t,
3H, –CH3). MS: 434.8. IR (KBr, cmÀ1): 3402, 2852, 1645, 1608,
1364, 1193, 1055.
100 l
M) on K562 cells for 24 and 48 h.44 The intracellular LDH
was determined after lysing the cells by freezing at À70 °C and ra-
pid thawing. The LDH release was measured at an absorbance of
490 nm. The percentage of LDH release was calculated as: (LDH
activity in media)/(LDH activity in media + LDH activity in total
cells) Â 100%.
4.4.5. Cell cycle analysis
4.4. Biology
Cellular DNA content was measured by flow cytometry. Approx-
imately 0.75 Â 105 cells/ml were cultured and treated with 10, 50
To investigate the antiproliferative activity of thiazolidinones
which is an important potency indicator for chemotherapeutic
drugs, leukemic cells growing in log phase were treated with 10,
and 100 lM concentrations of 5e or 5k. Cells were harvested after
48 h of treatment, washed, fixed in 70% ethanol and incubated with
RNase A (Sigma–Aldrich, USA). Propidium iodide (PI, 50 g/ml, Sig-
l
100 and 250
l
M. Since the compounds were dissolved in DMSO,
ma–Aldrich, USA) was added half an hour before acquiring the flow
cytometric reading (FACScan, BD Biosciences, USA). A minimum of
10,000 cells were acquired per sample and histograms were ana-
lyzed by using WinMDI 2.8 software.
it was used as vehicle control. The amount of DMSO used was cor-
responding to the DMSO in highest concentration of compound
tested, which did not show any significant effect on the cell lines
tested. To assess the cytotoxicity of newly synthesized thiazolidi-
nones, we employed trypan blue dye exclusion assay, MTT assay,
LDH assay and FACS analysis. In addition to this we also performed
DNA fragmentation assay which is an indicator of apoptosis. Each
experiment was repeated a minimum of two times.
4.4.6. DNA fragmentation assay
DNA fragmentation was performed for elucidating the mode of
action of the investigated compounds, especially with respect to
induction of oligonucleosomal DNA fragmentation (DNA ladder),
which is a characteristic feature of the programmed cell death or
apoptosis.45,46 During the apoptotic process, activated nucleases
degrade the higher order chromatin structure of DNA into mono-
and oligonucleosomal DNA-fragments. Apoptotic degradation of
DNA was analyzed by agarose gel electrophoresis. Briefly, K562
4.4.1. Cell lines and culture
Human cell line, K562 (chronic myelogenous leukemia) was
purchased from National Center for Cell Science, Pune, India and
Reh (B-cell leukemia) cell line was a kind gift from Prof. Michael
Lieber, University of Southern California, USA. Cells were grown
in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine
cells were cultured in presence 5e or 5k at 10, 50 and 100
for 72 h. Cells were harvested and genomic DNA was extracted
using standard protocol. DNA was resuspended in 250 L of TE buf-
lM
serum (FBS), 100 U/mL of Penicillin, and 100
lg of streptomycin/ml
l
and incubated at 37 °C in a humidified atmosphere containing 5%
fer. The DNA samples were run on 1% agarose gel and visualized by
CO2.
ethidium bromide staining and photographed.
4.4.2. Trypan blue exclusion assay
Acknowledgments
To determine the effect of thiazolidinone analogs 5(a–l) on via-
bility of K562 or Reh cells, approximately 0.75 Â 105 cells/ml were
seeded in a 6-well tissue culture plate for 24 h and compounds
This work was supported by UGC, Govt. of India under the Pro-
jects vide no. F. 31-143/2005 (SR), UGC-SAP (Phase I) vide No. F.
540/10/2004-05 (SAP I) and DST-FIST Programmes for KSR. SCR
acknowledges support from Lady Tata Memorial Trust interna-
tional award for leukemia research (London). We thank Dr. Omana
Joy for help related to FACS analysis. One of the authors (K. Vinaya)
is grateful to Council of Scientific and Industrial Research (CSIR),
New Delhi for financial support under CSIR-SRF order No. 09/
119(0172)2K8 EMR-I.
5(a–l) were added at a concentration of 10, 100 and 250 lM. Cells
were collected at intervals of 24 h and resuspended in 0.4% Trypan
blue (viable-unstained and non viable-blue). The number of viable
cells were counted using haemocytometer.
4.4.3. MTT assay
Cell survival was further assessed by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay,43 which is
based on the ability of viable cells to metabolize a yellow tetrazo-
lium salt to violet formazan. Exponentially growing K562 or Reh
cells (1 Â 104 cells/well) were plated in triplicates and incubated
References and notes
1. (a) Verweij, J.; Jonge, M. J. A. Eur. J. Cancer 2000, 36, 1479; (b) Verdecchia, A.;
Mariotto, A.; Capocaccia, R.; Gatta, G.; Micheli, A.; Sant, M.; Berrino, F. Eur. J.
Cancer 2001, 37, 1149; (c) Garret, M. D.; Workman, P. Eur. J. Cancer 1999, 35,
2010.
2. Lowenberg, B.; Downing, J. R.; Burnett, A. N. Engl. J. Med. 1999, 341, 1051.
3. Weltermann, A.; Fonatsch, C.; Haas, O. A.; Greinix, H. T.; Kahls, P.; Mitterbauer,
G.; Jager, U.; Kainz, B.; Geissler, K.; Valent, P.; Sperr, W. R.; Knobl, P.;
Schwarzinger, I.; Gleiss, A.; Lechner, K. Leukemia 2004, 18, 293.
with 10, 100 and 250 lM of 5(a–l). Cells were harvested after 48
and 72 h of treatment and incubated with MTT (5 mg/ml) at
37 °C. The blue MTT formazan precipitate was then solubilized in
detergent (50% final concentration of N,N-dimethylformamide
and 10% of sodium dodecyl sulphate). Absorbance was measured
at 570 nm using ELISA plate reader. The mean absorbance of cul-

2584
S. Chandrappa et al. / Bioorg. Med. Chem. 17 (2009) 2576–2584
4. Mencher, S. K.; Wang, L. G. BMC Clin. Pharmacol. 2005, 5, 3.
5. Jimeno, A.; Hidalgo, M. Crit. Rev. Oncol. Hematol. 2006, 59, 150.
6. Sawyers, C. Nature 2004, 432, 294.
7. Li, Q.; Xu, W. Curr. Med. Chem. Anticancer Agents 2005, 5, 53.
8. Holla, B. S.; Rao, B. S.; Sarojini, B. K.; Akberali, P. M. Eur. J. Med. Chem. 2004, 39, 777.
9. McLamore, W. M.; Celmer, W. D.; Bogert, V. V.; Pennington, F. C.; Solomons, I. A.
J. Am. Chem. Soc. 1952, 74, 2946.
28. Holla, B. S.; Sarojini, B. K.; Rao, B. S.; Akberali, P. M.; Kumari, N. S.; Shetty, V. Il
Farmaco 2001, 56, 565.
29. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2008, doi:10.1016/j.ejmech.2008.09.032.
30. Ananda Kumar, C. S.; Kavitha, C. V.; Vinaya, K.; Benaka Prasad, S. B.;
Thimmegowda, N. R.; Chandrappa, S.; Raghavan, S. C.; Rangappa, K. S. Invest.
New Drugs 2008, doi:10.1007/s10637-008-9179-3.
10. Sobin, B. A. J. Am. Chem. Soc. 1952, 74, 2947.
31. Kavitha, C. V.; Nambiar, M.; Ananda Kumar, C. S.; Choudhary, B.;
Muniyappa, K.; Rangappa, K. S.; Raghavan, S. C. Biochem. Pharmacol. 2008,
77, 348.
32. Anil Kumar, C.; Nanjunda Swamy, S.; Gaonkar, S. L.; Basappa, R. K. S.; Bharathi,
P. S.; Rangappa, K. S. Med. Chem. 2007, 3, 269.
11. Tanabe, Y.; Yamamoto, H.; Murakami, M.; Yanagi, K.; Kubota, Y.; Okumura, H.;
Sanemitsu, Y.; Suzukamo, G. J. Chem. Soc., Perkin Trans I 1995, 7, 935.
12. Chen, H.; Fan, Y. H.; Natarajan, A.; Guo, Y.; Iyasere, J.; Harbinski, F.; Luus, L.;
Christ, W.; Aktas, H.; Halperin, J. A. Bioorg. Med. Chem. 2004, 14, 5401.
13. Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. J. Med. Chem. 2005, 48, 2584.
14. Zhou, H.; Wu, S.; Zhai, S.; Liu, A.; Sun, Y.; Li, R.; Zhang, Y.; Ekins, S.; Swaan, P.
W.; Fang, B.; Zhang, B.; Yan, B. J. Med. Chem. 2008, 1242.
15. Kawasaki, T.; Immaru, D.; Osaka, Y.; Tsuchiya, T.; Ono, S. Eur. Patent 50003 (Cl.
C07D279/06), 1982; Chem. Abstr. 1982, 97, 92300b.
16. Szabadka; Istvan; Harsanyi; Kalman et. al., US Patent-4937252, 1990.
17. Samir, B.; Wesam, K.; Ahmed, A. F. Eur. J. Med. Chem. 2007, 42, 948.
18. Elbarbary, A. A.; Khodair, A. I.; Pedersen, E. B.; Nielsen, C. Monatsh. Chem. 1994,
125, 593.
19. Archana; Srivastava, V. K.; Kumar, A. Ind. J. Pharm. Sci. 2003, 63, 358.
20. Kucukguzel, S. G.; Kocatepe, A.; De Clercq, E.; Sahin, F.; Gulluce, M. Eur. J. Med.
Chem. 2006, 41, 353.
21. Liu, H. L.; Li, Z. C.; Anthonsen, T. Molecules 2000, 5, 1055.
22. Goel, B.; Ram, T.; Tyagi, R.; Bansal, E.; Kumar, A.; Mukherjee, D.; Sinha, J. N. Eur.
J. Med. Chem. 1999, 34, 265.
33. Anil Kumar, C.; Jayarama, S.; Basappa; Bharathi, P. S.; Rangappa, K. S. Invest.
New Drugs 2007, 25, 343.
34. Priya, B. S.; Anil Kumar, C.; Nanjunda Swamy, S.; Basappa; Naveen, S.;
Rangappa, K. S. Bioorg. Med. Chem. Lett. 2007, 17, 2775.
35. (a) Barreca, M. L. Bioorg. Med. Chem. Lett. 2001, 11, 1793; (b) Barreca, M. L.;
De Clercq, E. J. Med. Chem. 2002, 45, 5410; (c) Goel, B.; Kumar, A. Eur. J.
Med. Chem. 1999, 34, 265; (d) Allen, S. Bioorg. Med. Chem. Lett. 2004, 14,
1619.
36. Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem. Lett. 2004,
14, 5289.
37. Dexter, D. L.; Barbosa, J. A.; Calabresi, P. Cancer Res. 1979, 39, 1020.
38. Brattain, M. G.; Fine, W. D.; Khaled, F. M.; Thompson, J.; Brattain, D. E. Cancer
Res. 1981, 41, 1751.
39. Fogh, J.; Trempe, G. In Human Tumor Cells in Vitro; Fogh, J., Ed.; Plenum Press:
New York, 1975; p 115.
23. Manju, T.; Piyush, K.; Kalpana, P.; Triloki, N. B.; Jayanti, P. B. Pharmacology
1985, 31, 260.
40. Tompkins, W. A.; Watrach, A. M.; Schmale, J. D.; Schultz, R. M.; Harris, J. A. J.
Natl. Cancer Inst. 1974, 52, 1101.
24. Ottana, R.; Carotti, S.; Maccari, R.; Landini, I.; Chiricosta, G.; Caciagli, B.;
Vigorita, M. G.; Mini, E. Bioorg. Med. Chem. Lett. 2005, 15, 3930.
25. Barreca, M. L.; Chimirri, A.; Luca, L. D.; Monforte, A. M.; Monforte, P.; Rao, A.;
Zappala, M.; Balzarini, J.; Clercq, E. D.; Pannecouque, C.; Witvrouw, M. Bioorg.
Med. Chem. Lett. 2001, 11, 1793.
26. Balzarini,J.;Orzeszko,B.;Maurin,J.K.;Orzeszko,A.Eur.J.Med.Chem.2007,42,993.
27. Andres, C. J.; Bronson, J. J.; D’Andrea, S. V.; Deshpande, M. S.; Falk, P. J.; Young,
K. A.; Harte, W. E.; Ho, H. T.; Misco, P. F.; Robertson, J. G.; Stock, D.; Sun, Y.;
Walsh, A. W. Bioorg. Med. Chem. Lett. 2000, 10, 715.
41. NCI-Navy Medical Oncology Branch cell line supplement, J. Cell Biochem. Suppl.
1996, 24.
42. Holla, B. S.; Rao, B. S.; Sarojini, B. K.; Akberali, P. M. Eur. J. Med. Chem. 2004, 39,
777.
43. Freimoser, F. M.; Jakob, C. A.; Aebi, M.; Tuor, U. Appl. Environ. Microbiol. 1999,
65, 3727.
44. Korzeniewski, C.; Callewaert, D. M. J. Immunol. Methods 1983, 64, 313.
45. Konstantinov, S. M.; Berger, M. R. Cancer Lett. 1999, 144, 153.
46. Henkels, P. M.; Turchi, J. J. Cancer Res. 1999, 59, 3077.