Novel quinolone substituted thiazolidin-4-ones as anti-inflammatory, anticancer agents: Design, synthesis and biological screening

Article abstract of DOI:10.1016/j.ejmech.2013.03.011

Nuclear factor-kappaB (NF-κB) has been reported to regulate various genes involved in cancer and inflammation. Accordingly, drugs suppressing or inhibiting NF-κB may possess both anti-inflammatory and anticancer properties. A library of quinolone substituted thiazolidin-4-ones was docked into the active site of NF-κB and the top-ranked 31 compounds were synthesized and evaluated for anti-inflammatory and anticancer activity. The best-ranked compound 6b showed highest anti-inflammatory activity in carrageenan-induced paw edema model. In vitro anticancer studies revealed 1a and 16a as most active compounds against BT-549, HeLa, COLO-205 and ACHN human cancer cell lines. Compounds 1a and 16a exhibited NF-κB dependent anticancer properties and apoptosis mediated cell death. In vivo Ehrlich ascites carcinoma study further confirmed the antitumor activity of 1a and 16a.

Full text of DOI:10.1016/j.ejmech.2013.03.011

Accepted Manuscript
Novel quinolone substituted thiazolidin-4-ones as anti-inflammatory, anticancer
agents: Design, synthesis and biological screening
Sharad Kumar Suthar, Varun Jaiswal, Sandeep Lohan, Sumit Bansal, Anil
Chaudhary, Amit Tiwari, Angel Treasa Alex, Alex Joesph
PII:
S0223-5234(13)00159-1
10.1016/j.ejmech.2013.03.011
EJMECH 6047
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2012
Revised Date: 4 March 2013
Accepted Date: 6 March 2013
Please cite this article as: S.K. Suthar, V. Jaiswal, S. Lohan, S. Bansal, A. Chaudhary, A. Tiwari, A.T.
Alex, A. Joesph, Novel quinolone substituted thiazolidin-4-ones as anti-inflammatory, anticancer agents:
Design, synthesis and biological screening, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.03.011.
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*Highlights (for review)
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Highlights
 Quinolone substituted thiazolidin-4-ones were docked into active site of NF-κB
 Top ranked 31 quinolone substituted thiazolidin-4-ones were synthesized.
 The best-ranked compound 6b showed potent in vivo anti-inflammatory activity.
 Compounds 1a and 16a showed NF-κB dependent anticancer properties.
 In vivo Ehrlich ascites carcinoma model confirmed antitumor activity of 1a and 16a

*Revised Manuscript
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Novel quinolone substituted thiazolidin-4-ones as anti-inflammatory, anticancer agents:
Design, synthesis and biological screening
Sharad Kumar Suthara, Varun Jaiswalb, Sandeep Lohana, Sumit Bansala, Anil Chaudharyc, Amit
Tiwaric, Angel Treasa Alexd, Alex Joespha*
aDepartment of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences,
Manipal-576104, India
bDepartment of Biotechnology and Bioinformatics, Jaypee University of Information
Technology, Waknaghat, Solan-173234, India
cDepartment of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal-576104,
India
dDepartment of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,
Manipal-576104, India
*Corresponding author, Tel.: +91-09448548060; fax: +91-0820-2571998.
E-mail address: alex.joseph@manipal.edu
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ABSTRACT
Nuclear factor-kappaB (NF-κB) has been reported to regulate various genes involved in cancer
and inflammation. Accordingly, drugs suppressing or inhibiting NF-κB may possess both anti-
inflammatory and anticancer properties. A library of quinolone substituted thiazolidin-4-ones
were docked into the active site of NF-κB and the top-ranked 31 compounds were synthesized
and evaluated for anti-inflammatory and anticancer activity. The best-ranked compound 6b
showed highest anti-inflammatory activity in carrageenan-induced paw oedema model. In vitro
anticancer studies revealed 1a and16a as most active compounds against BT-549, HeLa, COLO-
205 and ACHN human cancer cell lines. Compounds 1a and 16a exhibited NF-κB dependent
anticancer properties and apoptosis mediated cell death. In vivo Ehrlich ascites carcinoma study
further confirmed the antitumor activity of 1a and 16a.
Key words: Quinolone, thiazolidin-4-one, NF-κB, Docking, Anti-inflammatory, Anticancer
Abbreviations: NF-κB, Nuclear factor-kappaB; AP-1, activator protein-1; NSAIDs, Nonsteroidal
antiinflammatory drugs; COX, cyclooxygenase; LOX, lipoxygenase; ROS, reactive oxygen
species; DCC, N,N‟-dicyclohexyl carbodiimide; TLC, thin layer chromatography; MTT, (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Bcl-2, B-cell lymphoma 2; Bax, bcl-2
associated X protein; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; RT-PCR, reverse
transcription polymerase chain reaction.
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1. Introduction
Since 1950s rapid progress has been made in the area of cancer cell biology, though most cancer
treatments are still multimodal, involving chemotherapy, radiotherapy, and extensive surgery,
with chemotherapy remains the most significant pharmacological approximation to cancer
treatment [1]. Because of limitations associated with existing agents′ like side effects and
acquired tumor resistance, there is an immediate need of new anticancer agents with better
therapeutic profile [1].
Research over the years has established a strong connection between inflammation and cancer
[2], concluding that cancer is an inflammatory disease [3]. In various pathophysiological
conditions excessive generation of reactive oxygen species (ROS) results in oxidative stress. The
ROS produced by phagocytic leukocytes (e.g., neutrophils, monocytes, macrophages,
eosinophils) during the inflammatory process invade the tissues [4]. The ROS happens to be also
involved in cyclooxygenase-1 (COX-1) and lipoxygenase (LOX) mediated conversion of
arachidonic acid into proinflammatory intermediates and prostaglandins [5, 6]. These ROS could
initiate inflammation by activating redox-sensitive transcription factors such as nuclear factor-
kappaB (NF-κB) and activator protein-1 (AP-1). Conventional nonsteroidal antiinflammatory
drugs (NSAIDs), exhibits anti-inflammatory activity either by selectively inhibiting
cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) or combined suppression of COX-1 and
COX-2 [7]. The upregulation of COX-2 in tumor cells is accompanied by increased expression
of NF-κB [8, 9]. NSAIDs such as, aspirin, ibuprofen, indomethacin and sulindac has been shown
to suppress transcription factor NF-κB, which controls the expression of COX-2, leading to
inhibition of proliferation of tumor cells [3]. Hence suppression of NF-κB which regulates COX-
2 and cyclin D1 (a member of cyclin protein family involved in regulating cell cycle
progression) [3] has been emerged as a key hypothesis for the prevention and treatment of
various cancers.
The known anti-inflammatory drugs such as: Proquazone (1-isopropyl-7-methyl-4-
phenylquinazoline-2(1H)-one) [10], Fluoroquazone (4-(4-fluorophenyl)-7-methyl-1-propan-2-yl-
quinazoline-2-one) [11,12], Tryptanthrin an indolo quinazoline alkaloid [13, 14] bears
quinazolinone moiety and are structurally related to quinolone nucleus. On the other hand,
thiazolidinones [15–17] are important pharmacodynamic heterocyclic nuclei which when
incorporated into different heterocyclic templates, have been reported to possess potent anti-
inflammatory activity. Ruiz et al., reported cytotoxic mechanism of quinolone derivatives as
alkylating agents [1]. The quinolones occupy the catalytic cleft of human DNA-repair O6-
alkylguanine DNA alkyltransferase (MGMT or hAGT), by acting as analogs of the O6-guanine
moiety in the natural substrate, present deep within the binding groove and reaching the catalytic
residue Cys145 [1] while Gududuru et al., found that, thiazolidin-4-one moiety act as a
biomimetic replacement for the phosphate group[18]. Hence, quinolones and thiazolidinones
moieties are biologically proven anticancer and anti-inflammatory pharmacophores and further
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substitution on these scaffolds may further enhance their activity, prompted us to undertake this
problem. The combination of two pharmacophores on the same molecule is a well established
approach for the designing of more potent drugs with dual activity [3,19-27]. Moreover, single
molecule acting on multiple targets is considered as better drug candidate compared to drug
combinations, as administration of single drug will have more predictable pharmacokinetic and
pharmacodynamic properties and improved patient compliance [6,28]. Therefore our current
work highlights design, synthesis, structure-activity relationship (SAR) and biological evaluation
of quinolone substituted thiazolidin-4-ones for their anti-inflammatory and anticancer activity.
2. Results and discussion
2.1 Chemistry
Synthesis of quinolone substituted thiazolidin-4-ones involves 2 steps. In first step, 7-amino-4-
methyl-quinoline-2(1H)-one (3) was synthesized by cyclocondensation of m-phenylenediamine
(1) and ethylacetoacetate (2) (Scheme 1). Step-2 involves a one pot reaction of 7-amino-4-
methyl-quinoline-2(1H)-one with different aldehydes (4) and thioglycolic acid in the presence of
dicyclohexylcarbodiimide (DCC) to yield the quinolone substituted thiazolidin-4-ones (1a-16c)
(Scheme 1). The reactions proceed with initial formation of an imine (the nitrogen of amino
group attacks the carbonyl carbon of aldehyde) and nucleophilc attack of thiol on imino carbon
followed by intramolecular cyclization by loss of water. The later step seems to be critical for
obtaining high yields of thiazolidin-4-ones. N, N‟-dicyclohexyl carbodiimide (DCC) was used as
a dehydrating agent to accelerate the intramolecular cyclization resulted in faster reaction and
improved yields. Progresses of the reactions were monitored by TLC using acetone: chloroform
(7:3) as mobile phase. The synthesized compounds were purified by column chromatography
using silica gel 100-200 mesh and later by recrystallization in acetone to afford white/ yellowish-
white products (1a-16c). Total 31 compounds were synthesized and their yields ranged from
30-74%.
Structures of synthesized title compounds were confirmed by IR, NMR and Mass techniques.
The FT-IR (KBr) spectra displayed characteristic absorption bands corresponding to N-H, C=O,
C=C, cyno, and C-S groups. IR spectra of all quinolone substituted thiazolidin-4-ones showed N-
H stretching in the range of 3319.60–3329.25 cm-1. The vibrational frequency at 1624.12–
1666.55 cm-1 confirmed the presence of carbonyl groups in synthesized compounds. Other
characteristic peaks C=C, C-N, and C-S appeared at 1560.46–1579.75, 1396.91–1415.19, and
640.39–655.53 cm-1 respectively. NMR spectra of all the compounds showed NH proton in the
range of 11.513–11.570 ppm as a broad signal (D2O exchangeable). Formation of thiazolidinone
ring was confirmed by the presence of double doublet peaks between 3.83–4.12 (J= 15.6-16.4
Hz) corresponding to two methylene protons in the 5th position of thiazolidinone nucleus. Peaks
for aromatic, furanyl and thienyl protons in the synthesized compounds appeared between 6.333–
7.738 ppm. The peak for =C-H- proton of quinolone ring was observed beween 6.452–6.881
ppm as singlet while methine –C-H– proton of the cyclized thiazolidinone ring appeared as a
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singlet between 6.298–6.329 ppm. GC-MS spectra showed characteristic molecular ion (M+)
peaks corresponding to the molecular weight of synthesized compounds.
2.2. Biological evaluation
2.2.1. Anti-inflammatory activity
The anti-inflammatory activity of newly synthesized 31 compounds was evaluated by
carrageenan-induced paw oedema model in rats [29] using indomethacin as a reference drug.
Results are expressed as mean ± S.D. (Table 1). Differences between control and treatment
groups were evaluated for statistical significance using one way ANOVA followed by Tukey's
test. The test compounds administered 1 hr prior to carrageenan injection at a dose of 100 mg/kg
body wt. caused significant inhibition of paw oedema volume. Compound 6b, possessing 4-
nitrophenyl substitution on thiazolidinone moiety confers maximum anti-inflammatory activity
followed by 6a bearing 2-nitrophenyl substitution. The presence of fluoro, chloro, and bromo
groups in the meta position of phenyl ring of thiazolidinone moiety exhibited significant
inhibition of paw oedema volume and percentage oedema inhibition values were very close to
most potent compound 6b. Among the test compounds bearing halogen substituted phenyl ring
(2a, 2b, 2c, 3a, 3b, 3c, 4a, 4b, 4c) anti-inflammatory activity increases with electronegativity of
the halogen attached. In case of thiazolidinones with hydroxyl substituted phenyl ring (5a, 5b,
5c), p-hydroxy substituted compound was more active than o- and m-hydroxy substituted
compounds and they were not as active as their corresponding halogen substituted compounds.
The 1-piperidyl ,4-morpholinyl and pyridyl substituted compounds failed to show significant
oedema inhibition, while bromo substituted thienyl (16b) and bromo substituted furanyl
thiazolidinones (15b) showed significant inhibition of paw oedema volume. Incorporation of
electron releasing 5-methyl substitution on thienyl or furanyl ring of thiazolidinones resulted in
loss of activity.
2.2.2. In vitro cytotoxic studies (MTT assay)
, Newly synthesized 31 compounds were screened for their in vitro growth inhibitory activities
against two human cultured cell lines namely, human breast carcinoma cell line (BT-549) and
Human cervical adenocarcinoma cell line (HeLa) at 100 µg/ml by MTT assay method [30].
Results are shown as percentage cytotoxicity after 24 h. (Table 2). The compounds found active
in preliminary screening was further studied for their cytotoxic effect on BT-549 , HeLa,
COLO-205 (human colon adenocarcinoma cell line) and ACHN (human kidney
adenocarcinoma) cell lines and the results are expressed as IC50 ± SD (Table 3). Among the 31
newly synthesized quinolone substituted thiazolidin-4-ones screened for their cytotoxic effect on
BT-549 and HeLa cells, eleven compounds showed percentage cell death greater than 50%
against at least one of the two cell lines used. Among the most active eleven compounds , four
compounds exhibited cell death greater than 50% against both cell lines. The synthesized
compounds, 1a and 16a, showed maximum percentage cytotoxicity at 100 µg/ml. Further
cytotoxicity studies of compound 1a against BT-549, HeLa, COLO-205 and ACHN cell lines
exhibited IC50 values at 39.310, 35.144, 31.382 and 78.861 µg/ml respectively, while compound
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16a against these four cell lines exhibited IC50; 43.047, 27.067, 20.737, and 44.711 µg/ml
respectively. The IC50 of reference drug cisplatin against BT-549, and HeLa cells was found to
be 6.937 and 2.870 µg/ml respectively and IC50 of 5-fluorouracil against COLO-205 and ACHN
was found to be 4.562, and 9.874 µg/ml respectively (Table 3).
The cytotoxicity of all newly synthesized quinolone substituted thiazolidin-4-ones mainly
depends on type of susbstitution on thiazolidinone moiety. The substitution pattern on phenyl,
furanyl and thienyl rings of thiazolidinone moiety showed variation in cytotoxicity. The
compounds with unsubstituted phenyl, thienyl or furanyl rings attached to thiazolidinone moiety
showed the highest percentage of cell death. The electron withdrawing halogen substitution on
phenyl, thienyl and furanyl ring retained the cytotoxicity of parent ring. While the compunds
having electron releasing 4-dimethylamino, 4-methoxy, and 3,4-dimethoxy groups on phenyl
ring resulted in loss of activity. Substitution with six membered heterocyclic structures such as
pyridyl, 1-piperidyl, and 4-morpholinyl rings on thiazolidinone moiety caused drastic loss of
activity.
2.2.3. Acridine orange (AO)–ethidium bromide (EB) cell apoptosis analysis
Based on the results of in vitro cytotoxicity studies of newly synthesized quinolone substituted
thiazolidin-4-ones, two highly active compounds were selected for Acridine orange (AO)–
ethidium bromide (EB) staining to study the compound mediated apoptosis. The phenotypic
study of cells and nuclear changes associated with apoptosis were analyzed by acridine orange
and ethidium bromide (AO/EB) staining [31,32]. Acridine Orange (AO) is a nucleic acid
selective metachromatic stain, which is taken up by both viable and nonviable cells and emits
green fluorescence if intercalated into double stranded nucleic acid. EB intercalates and stains
DNA, providing a red-orange fluorescence. Although it does not stain healthy cells, it can be
used to identify cells that are in the early or final stages of apoptosis which have much more
permeable membranes. COLO-205 cells treated with test drugs (1a and 16a) at a concentration
of their IC50 value showed significant evidence of apoptosis mediated cell death. The treated
cells showed obvious late apoptotic characters after 48 hr treatment of test drugs. Control cells
showed bright green nucleus (Fig. 1) with uniform fluorescence as these cells cannot take up
ethidium bromide, where as test compounds treated cells exhibited orange-red fluorescence due
to staining with ethidium bromide because apoptotic cells with ruptured membrane allow the
entry of ethidium bromide to intercalate into DNA (Fig. 1). The results provide morphological
evidence that compounds 1a and 16a could induce apoptosis and inhibit cell growth.
2.2.4 Hoechst 33258 DNA staining to study nuclear morphological changes
For further characterization of compound mediated apoptosis triggered by compounds 1a and
16a were confirmed by Hoechst 33342 staining [33]. When stained with a nuclear fluorochrome
Hoechst 33342, the normal (untreated control) COLO-205 cells remained uniformly stained with
rounded and unpunctuated nucleus (Fig. 2). The compounds 1a and 16a selected for the study
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showed some characteristic features of apoptosis. Morphological characteristics include nuclear
fragmentation, karyopyknosis and zeiosis (membrane blebbing seen frequently in a dying cell)
(Fig. 2). These observations provide further evidence that an anoikis (form of programmed cell
death which is induced by anchorage-dependent cells detaching from the surrounding
extracellular matrix) occurred when COLO-205 cells were treated with test compounds
selected for the study.
2.2.5. Effects of 1a and 16a on the gene expressions (mRNA) of Bcl-2 and Bax
Bcl-2 is an anti-apoptotic conductor, while Bax is a pro-apoptotic protein. Bcl-2/Bax ratio
regulates release of mitochondrial „cytochrome c‟ to cytosol and mediates apoptosis. In
cancerous cells Bcl-2 is up-regulated while, Bax is down-regulated. Both Bcl-2 and Bax are
target genes of NF-κB [34-37] therefore it is assumed that Inhibition of NF-κB could leads to
down-regulation of Bcl-2 and up-regulation of Bax to inhibit cell proliferation by facilitating
apoptosis. Results of m-RNA expression studies by RT-PCR indicate that test drugs 1a and 16a
significantly down-regulated Bcl-2 and up-regulated Bax in COLO-205 cells (Fig. 3). In
comparison to drug treated cells, increased expression of Bcl-2 and decreased expression of Bax
mRNA was observed in control cells (Fig. 3). The mRNA expression for GAPDH, a
housekeeping gene, was also studied simultaneously with target gene to keep a check on any
background error.
2.2.6 Antitumor activity of compounds against Ehrlich Ascites Carcinoma in Swiss Albino mice
In the Ehrlich ascites carcinoma (EAC) model [38] tumor inoculated control group gained a
maximum weight of 25.96% by day-15 as compared to day-0 weight (Table 4, Fig. 4). Cisplatin
(3.5 mg/kg, i.p. single dose), taken as standard drug, administered on the 10th day of post tumor
inoculation significantly (p<0.05) reduced weight gain as compared to control on day-15 (Table
4). The test compounds 1a and 16a also reduced the weight gain significantly (p<0.05) as
compared to control on day-15. The increase in weight in 1a and 16a treated groups was found to
be 5.93% and 3.86% on day-15 when compared to 25.96% increase in body weight of control
group on corresponding day (Table 4, Fig. 4).
The test compounds 1a and 16a, significantly prolonged life span of animals (Table 5, Fig. 5).
The percentage increase in life span of 1a and 16a treated group was found to be 60.57 and
77.09% respectively as compared to control group. In general 25% increase in life span
compared to control is considered as an effective antitumor response [39]. The mean survival
time for 1a and 16a treated groups was found to be 29.16 and 32.16 days while MST of control
group was recorded as 18.16 days. Cisplatin significantly (p< 0.001) enhanced MST of EAC
induced mice compared to that of control mice and MST for cisplatin treated group was found to
be 38.5 days.
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2.3 Molecular docking study [40]
The 3D structures of NF-κB-DNA complex was taken from Protein Data Bank (PDB code:
1NFK) and co-crystallized DNA was removed. The p50 dimer and p50 monomers (chains A and
B) were used for the virtual screening and structures were prepared by using ChemDraw Ultra
11.0. All the water molecules were removed and hydrogens were added to protein. Library of
seventy molecules was docked into the binding site of receptor using Autodock 4.2. Virtual
screening results are expressed in terms of estimated free energy of binding (docking score)
(Table 6). Estimated free energy of binding for all seventy molecules ranged from -14.87 to -7.75
kcal/mol. Compound 1a showed estimated free energy of binding, -11.64 kcal/mol. Docked
structure of 1a with p50 dimer (Fig. 6) reveals that amine (-NH-) group of quinolone along with
carbonyl group of thiazolidine moiety were involved in hydrogen bonding with Gln 274 and 306
residues. The -NH- group of quinolone formed hydrogen bond with Gln 306 (2.928 Ǻ) while
carbonyl group of 4-thiazolidinone interacted with Gln 306 (2.183 Ǻ) and Gln 274 ( 2.601 Ǻ) by
hydrogen bonding. Phenyl ring of 1a was surrounded by Gln 274, Lys 275, and hydrophobic Phe
307 while Gln 306 was in proximity with –NH- of quinolone moiety. Visual inspection of
docked structure of 6b with receptor (Fig.7) shows carbonyl oxygen of quinolone ,
thiazolidinone and -NH- group of quinolone along with both oxygen atoms of nitro group were
involved in hydrogen bonding with Lys 144, His 141, Lys 144, Lys 144 and Lys 145 amino acids
residues respectively. Hydrogen bond distance between carbonyl oxygen of quinolone and Lys
144 and carbonyl oxygen of 4-thiazolidinone and His 141 was found to be 3.535 and 1.830 Ǻ
respectively. The -NH- of quinolone moiety interacted with Lys 144 with a bond distance of
3.404 Ǻ. Estimated free energy of binding for 6b was found to be -14.87 kcal/mol. In 6b, phenyl
ring was accommodated in the hydrophobic pocket of Val 58 and Cys 59. Similarly carbonyl
oxygen of both quinolone and thiazolidinone and -NH- of quinolone of 16a interacted through
four hydrogen bonds with amino acid residues like Tyr 57 and Lys 144, of the receptor (Fig. 8).
Estimated free energy of binding for 16a was found to be -11.82 kcal/mol. Results of virtual
screening show that carbonyl oxygen and -NH- of the quinolone and carbonyl oxygen of 4-
thiazolidinone were involved in hydrogen bonding with various amino acid residues of NF-κB.
Therefore the combination of two pharmacophores might have played a crucial role in the
pharmacological activity of quinolone substituted thiazolidin-4-ones.
3. Conclusion
Virtual screening of quinolone substituted thiazolidin-4-ones library resulted in the identification
of 31 compounds. Selected compounds were synthesized and evaluated for anti-inflammatory
and anticancer activity. In vivo anti-inflammatory activity of quinolone substituted thiazolidin-4-
ones on carrageenan-induced rat paw oedema model identified compound 6b as a potent anti-
inflammatory agent. Excellent correlation existed between docking score and anti-inflammatory
potency of screened drugs. The cytotoxicity of synthesized compounds were evaluated against
BT-549, HeLa, COLO-205 and ACHN human cancer cell lines. Among the synthesized
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quinolone substituted thiazolidin-4-ones compounds 1a and 16a exhibited the maximum
cytotoxic effect against BT-549, HeLa, COLO-205, and ACHN human cancer cell lines. Both
the compounds 1a and 16a induced distinctly visible morphological changes in COLO-205 cells.
AO/EB apoptosis analysis and Hoechst 33258 nuclear staining confirmed apoptosis mediated
cell death caused by 1a and 16a. The mRNA expression levels of NF-κB target gene Bcl-2 was
down-regulated while of pro-apoptotic gene Bax was up-regulated by 1a and 16a in COLO-205
cells. Antitumor potencies of 1a and 16a were further confirmed by in vivo studies on Ehrlich
ascites carcinoma model. Docking studies revealed that carbonyl oxygen and -NH- of the
quinolone and caybonyl oxygen of 4-thiazolidinone were actively involved in hydrogen bonding
with various amino acids of the receptor. This confirms our hypothesis that conjugation of two
pharmacophores might improve the pharmacological profile of synthesized compounds.
Considering the results of our study compound 1a, 6b and 16a might find an important place in
the new array of molecules targeting NF-κB dependent biological functions. Further research is
needed to determine their potential as anti-inflammatory or anticancer agents.
4. Experimental section
4.1. Chemistry
All chemicals and solvents used were of analytical or reagent grades and were used without
further purification. All the chemicals were purchased from Aldrich, Sigma-aldrich,
Spectrochem, Himedia and SD Fine chemicals limited, India. The purity of all compounds was
established by a single spot on the pre-coated silica gel plates (TLC silica gel F254, Merck,
Germany). The TLC solvent system used was acetone: chloroform (7:3). Iodine vapour was used
as developing reagent. Infrared spectra were recorded on a Shimadzu FTIR-8310 (Shimadzu,
1
Japan) using potassium bromide discs. H NMR spectra were recorded on a Bruker 400 MHz
spectrophotometer (Bruker, USA) in. Chemical shifts are reported in parts per million (δ) units
relative to an internal standard of tetramethylsilane. Coupling constants are given in Hz and the
relative peak areas were in agreement with all assignments. Mass spectra were recorded on a
GC-MS QP 5050, (Shimadzu, Japan). Elemental analyses were performed on Leco CHNS-932
(Leco, St. Joseph, MI, USA). Melting points were determined on a capillary melting point
apparatus (Shital Scientific Industries, India) and are uncorrected.
4.2. Synthesis of 7-amino-4-methyl-quinoline-2(1H)-one
10.8 g (0.1 mol) of m-phenylenediamine (1) and 12.64 ml (0.1 mol) of ethylacetoacetate (2) were
taken in a 250 ml round bottom flask. The reaction mixture was refluxed for 48 h (TLC
controlled). At the end of reaction period, 200 ml of water was added to the flask and the
contents were heated to the boiling temperature of water. The mixture was then filtered; the
filtrate was chilled in refrigerator till the precipitation appeared. The precipitate was collected,
dried in air, recrystallized from methanol and was characterized. (M. P. 270-272 °C, TLC
Solvent system-Acetone: Chloroform- 7: 3) [41].
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4.3. Synthesis of Quinolone substituted thiazolidin-4-ones (1a-16c)
General method: A mixture of appropriate heterocyclic amine (1.0 mmol) (3) and aromatic
aldehyde (1.2 mmol) (4) were stirred in tetrahydrofuran under ice-cold condition for 5 min,
followed by addition of the thioglycolic acid (2.0 mmol). After 5 min, dicyclohexylcarbodiimide
(1.2 mmol) was added to the reaction mixture at 0 °C and the reaction mixture was stirred for an
additional 3-5 h at room temperature to complete the reaction. Progresses of the reactions were
monitored by TLC using acetone: chloroform (7:3) as mobile phase. The precipitated
dicyclohexylurea was filtered off; the filtrate was concentrated to dryness under reduced
pressure. Deionized water was added to the residue and extracted with ethylacetate. The organic
layer was successively washed with 5% aqueous sodium hydrogen carbonate and sodium
chloride. Finally organic layer was dried over anhydrous sodium sulphate. The crude solid
obtained on evaporation of the solvent under reduced pressure was subjected for column
chromatography using silica gel 100-200 mesh using n.hexane: ethyl acetate (7:3) as the solvent
system and further recrystallized in acetone to yield white/ yellowish-white products [42].
4.3.1. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-phenylthiazolidin-4-one (1a). Crystallized
from acetone to give white crystals, yield 44%, mp 170-172 °C. Anal. calcd. for C19H16N2O2S
(336.09): % C, 67.84; H, 4.79; N, 8.33. Found: C, 67.98; H, 4.84; N, 8.24. IR (KBR, cm−1):
3327.32 (N-H), 1629.90 (C=O of thiazolidinone ring), 1577.82 (C=C), 1402.30 (CN), 644.25
(CS). 1H NMR (DMSO-d6, δ ppm): 11.524 (s, 1H, -NH-), 7.118-7.632 (m, 8H, Ar-H), 6.548 (s,
1H, =CH- of quinolone ring), 6.298 (s, 1H, -CH- of thiazolidinone ring), 4.031-4.074 (dd, J =
1.2, 16 Hz, 1H, 5-Ha), 3.889-3.928 (d, J= 15.6 Hz, 1H, 5-Hb), 2.318 (s, 3H, -CH3). GC-MS
(m/z): M+ 336.
4.3.2. 2-(2-fluorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (2a).
Crystallized from acetone to give white crystals, yield 47%, mp 160-161 °C. Anal. calcd. for
C19H15FN2O2S (354.08): % C, 64.39; H, 4.27; N, 7.90. Found: C, 64.54; H, 4.23; N, 7.73. IR
(KBR, cm−1): 3329.25 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1410.01
(CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.558 (s, 1H, -NH-), 7.096-7.623 (m, 7H, Ar-
H), 6.720 (s, 1H, =CH- of quinolone ring), 6.307 (s, 1H, -CH- of thiazolidinone ring), 3.993-
4.035 (d, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.893-3.933 (d, J= 16 Hz, 1H, 5-Hb), 2.319 (s, 3H, -CH3).
GC-MS (m/z): M+ 354.
4.3.3. 2-(3-fluorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (2b).
Crystallized from acetone to give white crystals, yield 40%, mp 152-153 °C. Anal. calcd. for
C19H15FN2O2S (354.08): % C, 64.39; H, 4.27; N, 7.90. Found: C, 64.28; H, 4.25; N, 7.98. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1410.06
(CN), 645.31 (CS). 1H NMR (DMSO-d6, δ ppm): 11.539 (s, 1H, -NH-), 7.109-7.837 (m, 7H, Ar-
H), 6.550 (s, 1H, =CH- of quinolone ring), 6.310 (s, 1H, -CH- of thiazolidinone ring), 4.043-
10

ACCEPTED MANUSCRIPT
4.085 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.889-3.928 (d, J= 15.6 Hz, 1H, 5-Hb), 2.329 (s, 3H, -
CH3). GC-MS (m/z): M+ 354.
4.3.4. 2-(4-fluorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (2c).
Crystallized from acetone to give white crystals, yield 48%, mp 176-177 °C. Anal. calcd. for
C19H15FN2O2S (354.08): % C, 64.39; H, 4.27; N, 7.90. Found: C, 64.48; H, 4.21; N, 7.71. IR
(KBR, cm−1): 3325.39 (N-H), 1666.55 (C=O of thiazolidinone ring), 1560.46 (C=C),
1396.51(CN), 640.39 (CS). 1H NMR (DMSO-d6, δ ppm): 11.533 (s, 1H, -NH-), 7.088-7.598 (m,
7H, Ar-H), 6.557 (s, 1H, =CH- of quinolone ring), 6.307 (s, 1H, -CH- of thiazolidinone ring),
4.041-4.083 (d, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.889-3.928 (d, J= 15.6 Hz, 1H, 5-Hb), 2.326 (s,
3H, -CH3). GC-MS (m/z): M+ 354.
4.3.5. 2-(2-chlorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (3a).
Crystallized from acetone to give white crystals, yield 45%, mp 202-204 °C. Anal. calcd. for
C19H15ClN2O2S (370.05): % C, 61.53; H, 4.08; N, 7.55. Found: C, 61.43; H, 4.10; N, 7.69. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1408.08
(CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.540 (s, 1H, -NH-), 7.121-7.642 (m, 7H, Ar-
H), 6.553 (s, 1H, =CH- of quinolone ring), 6.309 (s, 1H, -CH- of thiazolidinone ring), 4.042-
4.085 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.882-3.921 (d, J= 15.6 Hz, 1H, 5-Hb), 2.315 (s, 3H, -
CH3). GC-MS (m/z): 370 (M+ ), 372 (M+2).
4.3.6. 2-(3-chlorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (3b).
Crystallized from acetone to give white crystals, yield 38%, mp 195-196 °C. Anal. calcd. for
C19H15ClN2O2S (370.05): % C, 61.53; H, 4.08; N, 7.55. Found: C, 61.69; H, 4.11; N, 7.43. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1408.04
(CN), 644.25 (CS). 1H NMR (DMSO-d6, δ ppm): 11.537 (s, 1H, -NH-), 7.129-7.650 (m, 7H, Ar-
H), 6.548 (s, 1H, =CH- of quinolone ring), 6.312 (s, 1H, -CH- of thiazolidinone ring), 4.044-
4.087 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.887-3.927 (d, J= 16 Hz, 1H, 5-Hb), 2.324 (s, 3H, -CH3).
GC-MS (m/z): 370 (M+ ), 372 (M+2).
4.3.7. 2-(4-chlorophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (3c).
Crystallized from acetone to give white crystals, yield 53%, mp 225-227 °C. Anal. calcd. for
C19H15ClN2O2S (370.05): % C, 61.53; H, 4.08; N, 7.55. Found: C, 61.39; H, 4.03; N, 7.69. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1573.97 (C=C), 1406.15
(CN), 644.25 (CS). 1H NMR (DMSO-d6, δ ppm): 11.539 (s, 1H, -NH-), 7.108-7.951 (m, 7H, Ar-
H), 6.561 (s, 1H, =CH- of quinolone ring), 6.310 (s, 1H, -CH- of thiazolidinone ring), 4.045-
4.088 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.890-3.930 (d, J= 16 Hz, 1H, 5-Hb), 2.328 (s, 3H, -CH3).
GC-MS (m/z): 370 (M+ ), 372 (M+2).
4.3.8. 2-(2-bromophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (4a).
Crystallized from acetone to give white crystals, yield 47%, mp 227-228 °C. Anal. calcd. for
11

ACCEPTED MANUSCRIPT
C19H15BrN2O2S (414): % C, 54.95; H, 3.64; N, 6.75. Found: C, 55.10; H, 3.58; N, 6.64. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C),
1411.95(CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.542 (s, 1H, -NH-), 7.108-7.833 (m,
7H, Ar-H), 6.549 (s, 1H, =CH- of quinolone ring), 6.310 (s, 1H, -CH- of thiazolidinone ring),
4.042-4.085 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.889-3.928 (d, J= 15.6 Hz, 1H, 5-Hb), 2.312 (s, 3H,
-CH3). GC-MS (m/z): 414 (M+ ), 416 (M+2).
4.3.9. 2-(3-bromophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (4b).
Crystallized from acetone to give white crystals, yield 44%, mp 205-206 °C. Anal. calcd. for
C19H15BrN2O2S (414): % C, 54.95; H, 3.64; N, 6.75. Found: C, 54.91; H, 3.68; N, 6.53. IR
(KBR, cm−1): 3325.39 (N-H), 1645.33 (C=O of thiazolidinone ring), 1577.82 (C=C),
1400.37(CN), 645.11 (CS). 1H NMR (DMSO-d6, δ ppm): 11.565 (s, 1H, -NH-), 7.136-7.915 (m,
7H, Ar-H), 6.552 (s, 1H, =CH- of quinolone ring), 6.314 (s, 1H, -CH- of thiazolidinone ring),
4.082-4.124 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.881-3.921 (d, J= 16 Hz, 1H, 5-Hb), 2.330 (s, 3H,
-CH3). GC-MS (m/z): 414 (M+ ), 416 (M+2).
4.3.10. 2-(4-bromophenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (4c).
Crystallized from acetone to give white crystals, yield 49%, mp 235-236 °C. Anal. calcd. for
C19H15BrN2O2S (414): % C, 54.95; H, 3.64; N, 6.75. Found: C, 54.88; H, 3.61; N, 6.69. IR
(KBR, cm−1): 3319.60 (N-H), 1660.77 (C=O of thiazolidinone ring), 1576.40 (C=C),
1402.30(CN), 655.53 (CS). 1H NMR (DMSO-d6, δ ppm): 11.541 (s, 1H, -NH-), 7.109-7.833 (m,
7H, Ar-H), 6.550 (s, 1H, =CH- of quinolone ring), 6.311 (s, 1H, -CH- of thiazolidinone ring),
4.042-4.085 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.888-3.928 (d, J= 16 Hz, 1H, 5-Hb), 2.327 (s, 3H, -
CH3). GC-MS (m/z): M+ 414 (M+ ), 416 (M+2).
4.3.11. 2-(2-hydroxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (5a).
Crystallized from acetone to give white crystals, yield 37%, mp 228-230 °C. Anal. calcd. for
C19H16N2O3S (352.09): % C, 64.76; H, 4.58; N, 7.95. Found: C, 64.89; H, 4.53; N, 7.84. IR
(KBR, cm−1): 3327.32 (N-H), 1627.97 (C=O of thiazolidinone ring), 1572.04 (C=C), 1408.08
(CN), 640.39 (CS). 1H NMR (DMSO-d6, δ ppm): 11.551 (s, 1H, -NH-), 7.131-7.623 (m, 7H, Ar-
H), 6.549 (s, 1H, =CH- of quinolone ring), 6.306 (s, 1H, -CH- of thiazolidinone ring), 4.036-
4.078 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.862-3.902 (d, J= 16 Hz, 1H, 5-Hb), 2.316 (s, 3H, -
CH3). GC-MS (m/z): M+ 352.
4.3.12. 2-(3-hydroxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (5b).
Crystallized from acetone to give white crystals, yield 44%, mp 241-242 °C. Anal. calcd. for
C19H16N2O3S (352.09): % C, 64.76; H, 4.58; N, 7.95. Found: C, 64.87; H, 4.61; N, 7.79. IR
(KBR, cm−1): 3327.32 (N-H), 1627.97 (C=O of thiazolidinone ring), 1575.89 (C=C), 1411.23
(CN), 644.25 (CS). 1H NMR (DMSO-d6, δ ppm): 11.528 (s, 1H, -NH-), 7.101-7.589 (m, 7H, Ar-
H), 6.557 (s, 1H, =CH- of quinolone ring), 6.301 (s, 1H, -CH- of thiazolidinone ring), 4.038-
12

ACCEPTED MANUSCRIPT
4.081 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.884-3.923 (d, J= 15.6 Hz, 1H, 5-Hb), 2.317 (s, 3H, -
CH3). GC-MS (m/z): M+ 352.
4.3.13. 2-(4-hydroxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (5c).
Crystallized from acetone to give white crystals, yield 58%, mp 265-266 °C. Anal. calcd. for
C19H16N2O3S (352.09): % C, 64.76; H, 4.58; N, 7.95. Found: C, 64.59; H, 4.55; N, 8.13. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1573.97 (C=C), 1402.30
(CN), 644.25 (CS). 1H NMR (DMSO-d6, δ ppm): 1H NMR (DMSO-d6, δ ppm): 11.513 (s, 1H, -
NH-), 7.068-7.773 (m, 7H, Ar-H), 6.613 (s, 1H, =CH- of quinolone ring), 6.301 (s, 1H, -CH- of
thiazolidinone ring), 4.040-4.083 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.899-3.938 (d, J= 15.6 Hz, 1H,
5-Hb), 2.325 (s, 3H, -CH3). GC-MS (m/z): M+ 352.
4.3.14. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(2-nitrophenyl)thiazolidin-4-one (6a).
Crystallized from acetone to give yellowish-white crystals, yield 34%, mp 234-235 °C. Anal.
calcd. for C19H15N3O4S (381.08): % C, 59.83; H, 3.96; N, 11.02. Found: C, 59.95; H, 4.06; N,
10.89. IR (KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C),
1408.34 (CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.514 (s, 1H, -NH-), 7.182-7.749 (m,
7H, Ar-H), 6.587 (s, 1H, =CH- of quinolone ring), 6.315 (s, 1H, -CH- of thiazolidinone ring),
4.045-4.087 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.892-3.931 (d, J= 15.6 Hz, 1H, 5-Hb), 2.323 (s,
3H, -CH3). GC-MS (m/z): M+ 381.
4.3.15. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(4-nitrophenyl)thiazolidin-4-one (6b).
Crystallized from acetone to give yellowish-white crystals, yield 35%, mp 252-253 °C. Anal.
calcd. for C19H15N3O4S (381.08): % C, 59.83; H, 3.96; N, 11.02. Found: C, 59.77; H, 3.99; N,
11.22. IR (KBR, cm−1): 3327.32 (N-H), 1627.97 (C=O of thiazolidinone ring), 1577.82 (C=C),
1411.19 (CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.548 (s, 1H, -NH-), 7.059-7.676 (m,
7H, Ar-H), 6.575 (s, 1H, =CH- of quinolone ring), 6.324 (s, 1H, -CH- of thiazolidinone ring),
4.042-4.085 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.882-3.922 (d, J= 16 Hz, 1H, 5-Hb), 2.320 (s, 3H, -
CH3). GC-MS (m/z): M+ 381.
4.3.16. 2-(4-(dimethylamino)phenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-
one (7a). Crystallized from acetone to give yellowish-white crystals, yield 57%, mp 255-256 °C.
Anal. calcd. for C21H21N3O2S (379.14): % C, 66.47; H, 5.58; N, 11.07. Found: C, 66.39; H, 5.55;
N, 11.12. IR (KBR, cm−1): 3325.39 (N-H), 1624.12 (C=O of thiazolidinone ring), 1573.97
1
(C=C), 1404.22 (CN), 646.17 (CS). H NMR (DMSO-d6, δ ppm): 11.552 (s, 1H, -NH-), 7.209-
7.587 (m, 7H, Ar-H), 6.563 (s, 1H, =CH- of quinolone ring), 6.319 (s, 1H, -CH- of
thiazolidinone ring), 4.040-4.083 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.896-3.935 (d, J= 15.6 Hz, 1H,
5-Hb), 3.02 (s, 6H, -N(CH3)2), 2.323 (s, 3H, -CH3). GC-MS (m/z): M+ 379.
4.3.17. 2-(4-methoxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one (8a).
Crystallized from acetone to give white crystals, yield 45%, mp 216-217 °C. Anal. calcd. for
13

ACCEPTED MANUSCRIPT
C20H18N2O3S (366.1): % C, 65.55; H, 4.95; N, 7.64. Found: C, 65.68; H, 5.09; N, 7.53. IR (KBR,
cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1407.02 (CN),
1
644.25 (CS). H NMR (DMSO-d6, δ ppm): 11.539 (s, 1H, -NH-), 7.064-7.773 (m, 7H, Ar-H),
6.581(s, 1H, =CH- of quinolone ring), 6.320 (s, 1H, -CH- of thiazolidinone ring), 4.002-4.045
(dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.864-3.904 (d, J= 16 Hz, 1H, 5-Hb), 3.82 (s, 3H, -OCH3), 2.328
(s, 3H, -CH3). GC-MS (m/z): M+ 366.
4.3.18. 2-(3,4-dimethoxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one
(9a). Crystallized from acetone to give white crystals, yield 49%, mp 248-249 °C. Anal. calcd.
for C21H20N2O4S (396.11): % C, 63.62; H, 5.08; N, 7.07. Found: C, 63.51; H, 4.98; N, 6.89. IR
(KBR, cm−1): 3327.32 (N-H), 1624.12 (C=O of thiazolidinone ring), 1575.89 (C=C), 1403.16
(CN), 645.31 (CS). 1H NMR (DMSO-d6, δ ppm): 11.549 (s, 1H, -NH-), 7.123-7.595 (m, 6H, Ar-
H), 6.452 (s, 1H, =CH- of quinolone ring), 6.308 (s, 1H, -CH- of thiazolidinone ring), 4.011-
4.054 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.870-3.910 (d, J= 16 Hz, 1H, 5-Hb), 3.791 (s, 3H, -
OCH3), 3.811 (s, 3H,-OCH3), 2.326 (s, 3H, -CH3). GC-MS (m/z): M+ 396.
4.3.19. 2-(4-hydroxy-3-methoxyphenyl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-
4-one (10a). Crystallized from acetone to give white crystals, yield 66%, mp 269-270 °C. Anal.
calcd. for C20H18N2O4S (382.1): % C, 62.81; H, 4.74; N, 7.33. Found: C, 62.87; H, 4.72; N, 7.21.
IR (KBR, cm−1): 3325.39 (N-H), 1643.41 (C=O of thiazolidinone ring), 1574.17 (C=C), 1402.30
(CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.536 (s, 1H, -NH-), 7.052-7.592 (m, 6H, Ar-
H), 6.598 (s, 1H, =CH- of quinolone ring), 6.305 (s, 1H, -CH- of thiazolidinone ring), 3.990-
4.029 (d, J = 15.6 Hz, 1H, 5-Ha), 3.859-3.898 (d, J= 15.6 Hz, 1H, 5-Hb), 3.81 (s, 3H, -OCH3),
2.329 (s, 3H, -CH3). GC-MS (m/z): M+ 382.
4.3.20. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-styrylthiazolidin-4-one (11a). Crystallized
from acetone to give white crystals, yield 74%, mp 215-216 °C. Anal. calcd. for C21H18N2O2S
(362.11): % C, 69.59; H, 5.01; N, 7.73. Found: C, 69.46; H, 5.13; N, 7.82. IR (KBR, cm−1):
3327.32 (N-H), 1627.97 (C=O of thiazolidinone ring), 1573.97 (C=C), 1406.15 (CN), 1381.08
1
(C=C of cinnamaldehyde), 647.24 (CS). H NMR (DMSO-d6, δ ppm): 11.550 (s, 1H, -NH-),
7.067-7.739 (m, 8H, Ar-H), 6.62 (t, 2H =CH- of styryl group), 6.557 (s, 1H, =CH- of quinolone
ring), 6.321 (s, 1H, -CH- of thiazolidinone ring), 6.11 (m, 2H =CH- of styryl group),4.024-4.066
(dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.874-3.913 (d, J= 15.6 Hz, 1H, 5-Hb), 2.323 (s, 3H, -CH3).
GC-MS (m/z): M+ 362.
4.3.21. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(pyridin-2-yl)thiazolidin-4-one (12a).
Crystallized from acetone to give white crystals, yield 34%, mp 222-224 °C. Anal. calcd. for
C18H15N3O2S (337.09): % C, 64.08; H, 4.48; N, 12.45. Found: C, 64.32; H, 4.50; N, 12.32. IR
(KBR, cm−1): 3327.32 (N-H), 1627.97 (C=O of thiazolidinone ring), 1572.04 (C=C), 1404.22
(CN), 641.10 (CS). 1H NMR (DMSO-d6, δ ppm): 11.557 (s, 1H, -NH-), 7.082-7.689 (m, 7H, Ar-
H), 6.552 (s, 1H, =CH- of quinolone ring), 6.318 (s, 1H, -CH- of thiazolidinone ring), 4.032-
14

ACCEPTED MANUSCRIPT
4.075 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.895-3.935 (d, J= 16 Hz, 1H, 5-Hb), 2.316 (s, 3H, -CH3).
GC-MS (m/z): M+ 337.
4.3.22. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(pyridin-3-yl)thiazolidin-4-one (12b).
Crystallized from acetone to give white crystals, yield 30%, mp 181-182 °C. Anal. calcd. for
C18H15N3O2S, (337.09): % C, 64.08; H, 4.48; N, 12.45. Found: C, 64.18; H, 4.45; N, 12.32. IR
(KBR, cm−1): 3325.39 (N-H), 1633.76 (C=O of thiazolidinone ring), 1575.89 (C=C), 1402.30
(CN), 646.17 (CS). 1H NMR (DMSO-d6, δ ppm): 11.549 (s, 1H, -NH-), 7.077-7.615 (m, 7H, Ar-
H), 6.569 (s, 1H, =CH- of quinolone ring), 6.304 (s, 1H, -CH- of thiazolidinone ring), 4.028-
4.070 (dd, J = 1.2, 15.6 Hz, 1H, 5-Ha), 3.872-3.912 (d, J= 16 Hz, 1H, 5-Hb), 2.324 (s, 3H, -
CH3). GC-MS (m/z): M+ 337.
4.3.23. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(pyridin-4-yl)thiazolidin-4-one (12c).
Crystallized from acetone to give white crystals, yield 37%, mp 241-242 °C. Anal. calcd. for
C18H15N3O2S, (337.09): % C, 64.08; H, 4.48; N, 12.45. Found: C, 64.15; H, 4.50; N, 12.47. IR
(KBR, cm−1): 3326.83 (N-H), 1633.05 (C=O of thiazolidinone ring), 1577.26 (C=C), 1404.48
(CN), 645.74 (CS). 1H NMR (DMSO-d6, δ ppm): 11.561 (s, 1H, -NH-), 7.095-7.692 (m, 7H, Ar-
H), 6.549 (s, 1H, =CH- of quinolone ring), 6.321 (s, 1H, -CH- of thiazolidinone ring), 4.031-
4.074 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.888-3.927 (d, J= 15.6 Hz, 1H, 5-Hb), 2.328 (s, 3H, -
CH3). GC-MS (m/z): M+ 337.
4.3.24. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(piperidin-1-yl)thiazolidin-4-one (13a).
Crystallized from acetone to give white crystals, yield 59%, mp 236-237 °C. Anal. calcd. for
C18H21N3O2S (343.14): % C, 62.95; H, 6.16; N, 12.23. Found: C, 62.85; H, 6.13; N, 12.19. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1408.13
(CN), 644.25 (CS). 1H NMR (DMSO-d6, δ ppm): 11.563 (s, 1H, -NH-), 7.082-7.639 (m, 3H, Ar-
H), 6.530 (s, 1H, =CH- of quinolone ring), 6.318 (s, 1H, -CH- of thiazolidinone ring), 4.033-
4.076 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.896-3.935 (d, J= 15.6 Hz, 1H, 5-Hb), 2.46 (m, 4H, of
piperidinyl group), 1.74 (m, 4H, of piperidinyl group), 1.92 (m, 2H of piperidinyl group ), 2.327
(s, 3H, -CH3). GC-MS (m/z): M+ 343.
4.3.25.
3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-morpholinothiazolidin-4-one
(14a).
Crystallized from acetone to give white crystals, yield 46%, mp 240-241 °C. Anal. calcd. for
C18H19N3O2S (345.11): % C, 59.11; H, 5.54; N, 12.17. Found: C, 58.90; H, 5.51; N, 12.16. IR
(KBR, cm−1): 3327.32 (N-H), 1626.05 (C=O of thiazolidinone ring), 1575.89 (C=C), 1415.19
(CN), 642.32 (CS). 1H NMR (DMSO-d6, δ ppm): 11.554 (s, 1H, -NH-), 7.121-7.646 (m, 3H, Ar-
H), 6.538 (s, 1H, =CH- of quinolone ring), 6.315 (s, 1H, -CH- of thiazolidinone ring), 4.036-
4.079 (dd, J = 1.2, 16 Hz, 1H, 5-Ha), 3.865-3.905 (d, J= 16 Hz, 1H, 5-Hb), 3.55 (m, 4H, of
morpholinyl group), 2.61 (m, 4H, of morpholinyl group), 2.329 (s, 3H, -CH3). GC-MS (m/z): M+
345.
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4.3.26.
2-(furan-2-yl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one
(15a).
Crystallized from acetone to give white crystals, yield 64%, mp 164-165 °C. Anal. calcd. for
C17H14N2O3S (326.07): % C, 62.56; H, 4.32; N, 8.58. Found: C, 62.49; H, 4.33; N, 8.65. IR
(KBR, cm−1): 3327.32 (N-H), 1633.76 (C=O of thiazolidinone ring), 1577.82 (C=C), 1402.30
1
(CN), 646.17 (CS). H NMR (DMSO-d6, δ ppm): 11.570 (s, 1H, -NH-), 6.333-7.657 (m, 6H,
furanyl-H and Ar-H), 6.599 (s, 1H, =CH- of quinolone ring), 6.325 (s, 1H, -CH- of
thiazolidinone ring), 4.036-3.995 (d, J =16.4 Hz, 1H, 5-Ha), 3.834-3.873 (d, J= 15.6 Hz, 1H, 5-
Hb), 2.361 (s, 3H, -CH3). GC-MS (m/z): M+ 326.
4.3.27.
2-(5-bromofuran-2-yl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one
(15b). Crystallized from acetone to give white crystals, yield 65%, mp 152-155 °C. Anal. calcd.
for C17H13BrN2O3S (403.98): % C, 50.38; H, 3.23; N, 6.91. Found: C, 50.42; H, 3.25; N, 6.88.
IR (KBR, cm−1): 3326.72 (N-H), 1632.41 (C=O of thiazolidinone ring), 1574.60 (C=C), 1402.94
1
(CN), 642.34 (CS). H NMR (DMSO-d6, δ ppm): 11.542 (s, 1H, -NH-), 6.676-7.721 (m, 5H,
thianyl-H and Ar-H), 6.518 (s, 1H, =CH- of quinolone ring), 6.322 (s, 1H, -CH- of
thiazolidinone ring), 3.985-4.028 (d, J =16 Hz, 1H, 5-Ha), 3.884-3.923 (d, J= 15.6 Hz, 1H, 5-
Hb), 2.326 (s, 3H, -CH3). GC-MS (m/z): 404 (M+ ), 406 (M+2).
4.3.28.
3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(5-methylfuran-2-yl)thiazolidin-4-one
(15c). Crystallized from acetone to give white crystals, yield 63%, mp 173-174 °C. Anal. calcd.
for C18H16N2O3S (340.09): % C, 63.51; H, 4.74; N, 8.23. Found: C, 63.36; H, 4.69; N, 8.33. IR
(KBR, cm−1): 3327.11 (N-H), 1631.26 (C=O of thiazolidinone ring), 1571.47 (C=C), 1408.32
1
(CN), 645.06 (CS). H NMR (DMSO-d6, δ ppm): 11.562 (s, 1H, -NH-), 6.358-7.680 (m, 5H,
furanyl-H and Ar-H), 6.594 (s, 1H, =CH- of quinolone ring), 6.329 (s, 1H, -CH- of
thiazolidinone ring), 4.038-4.078 (d, J =16Hz, 1H, 5-Ha), 3.846-3.885 (d, J= 15.6 Hz, 1H, 5-
Hb), 2.338 (s, 3H, -CH3 of quinolone ring), 2.211 (s, 3H, -CH3 of thiophen ring). GC-MS (m/z):
M+ 340.
4.3.29. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(thiophen-2-yl)thiazolidin-4-one (16a).
Crystallized from acetone to give white crystals, yield 66%, mp 202-204 °C. Anal. calcd. for
C17H14N2O2S2 (342.05): % C, 56.63; H, 4.12; N, 8.18. Found: C, 56.84; H, 4.16; N, 8.11. IR
(KBR, cm−1): 3325.39 (N-H), 1656.91 (C=O of thiazolidinone ring), 1579.75 (C=C), 1400.37
1
(CN), 646.17 (CS). H NMR (DMSO-d6, δ ppm): 11.547 (s, 1H, -NH-), 6.835-7.636 (m, 6H,
thianyl-H and Ar-H), 6.881 (s, 1H, =CH- of quinolone ring), 6.329 (s, 1H, -CH- of
thiazolidinone ring), 3.983-4.024 (d, J =16.4 Hz, 1H, 5-Ha), 3.903-3.942 (d, J= 15.6 Hz, 1H, 5-
Hb), 2.348 (s, 3H, -CH3). GC-MS (m/z): M+ 342.
4.3.30. 2-(5-bromothiophen-2-yl)-3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)thiazolidin-4-one
(16b). Crystallized from acetone to give white crystals, yield 69%, mp 182-183 °C. Anal. calcd.
for C17H13BrN2O2S2 (419.96): % C, 48.46; H, 3.11; N, 6.65. Found: C, 48.35; H, 3.17; N, 6.84.
IR (KBR, cm−1): 3325.43 (N-H), 1656.80 (C=O of thiazolidinone ring), 1574.79 (C=C), 1405.15
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1
(CN), 644.10 (CS). H NMR (DMSO-d6, δ ppm): 11.545 (s, 1H, -NH-), 6.688-7.738 (m, 5H,
thianyl-H and Ar-H), 6.522 (s, 1H, =CH- of quinolone ring), 6.329 (s, 1H, -CH- of
thiazolidinone ring), 3.984-4.024 (d, J =16 Hz, 1H, 5-Ha), 3.875-3.914 (d, J= 15.6 Hz, 1H, 5-
Hb), 2.321 (s, 3H, -CH3). GC-MS (m/z): 420 (M+ ), 422 (M+2).
4.3.31. 3-(4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)-2-(5-methylthiophen-2-yl)thiazolidin-4-one
(16c). Crystallized from acetone to give white crystals, yield 63%, mp 178-179 °C. Anal. calcd.
for C18H16N2O2S (356.07): % C, 60.65; H, 4.52; N, 7.86. Found: C, 60.74; H, 4.50; N, 7.73. IR
(KBR, cm−1): 3327.17 (N-H), 1650.29 (C=O of thiazolidinone ring), 1569.26 (C=C), 1403.93
1
(CN), 645.84 (CS). H NMR (DMSO-d6, δ ppm): 11.551 (s, 1H, -NH-), 6.824-7.735 (m, 5H,
thianyl-H and Ar-H), 6.881 (s, 1H, =CH- of quinolone ring), 6.329 (s, 1H, -CH- of
thiazolidinone ring), 3.983-4.022 (d, J =15.6 Hz, 1H, 5-Ha), 3.902-3.942 (d, J= 16 Hz, 1H, 5-
Hb), 2.347 (s, 3H, -CH3 of quinolone ring), 2.214 (s, 3H, -CH3 of thiophen ring) . GC-MS (m/z):
M+ 356.
4.4. Biological screening
4.4.1. Animals
Animal care and handling were done as per guidelines of Committee for the Purpose of Control
and Supervision of Experiments on Animals, (CPCSEA), Govt. of India. The experimental
protocol was approved by Institutional Animal Ethics Committee (IAEC), Kasturba Medical
College, Manipal-5761004, India (Registration No. IAEC/KMC/02/2010-2011). Female Swiss
albino mice of 6–8 weeks old (29 ±3 g body weight) were selected for in vivo anticancer study.
Albino rats of charles foster species were used for anti-inflammatory studies. The animals were
acclimatized to the experimental room having temperature 27 ±3 °C, controlled humidity
conditions, and 14:10 h light and dark cycle. The four animals were housed in each
polypropylene cage containing paddy husk as bedding.The mice were fed on autoclaved standard
mice food pellets (Hindustan Lever Ltd., New Delhi) and water ad libitum.
4.4.2. Toxicological study (OECD 425 Guidelines)
An acute toxicity study was done and safe dose was calculated as per OECD test guideline 425
[43].
4.4.3 Anti-inflammatory activity [29]
Carrageenin-induced paw edema test was performed on albino rats of charles foster species by
using the method of Winter et al [44]. Groups of six rats of either sex (body weight 150–200 g),
pregnant females excluded, were given a 100 mg/kg oral dose of test compounds. One hour
later, 0.2 mL of 1% carrageenan suspension in 0.9% NaCl solution was injected subcutaneously,
into the subplantar tissue of the right hind paw of each mouse and the paw volume was measured
with a plethysmometer (UGO Basile 7140, model-7141, Biological research apparatus, Italy) and
then measured again 3 hr later. Indomethacin (10 mg/kg) was used as reference drug. The mean
increase in paw volume was compared with that of control group (six rats received only
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carrageenan, no test compounds) and percent inhibition values were calculated by the formula
given below: % anti-inflammatory activity =1–Dt/Dc× 100. Where Dt represents the percentage
difference in increased paw volume after the administration of test drugs to the rats and Dc
represents the percentage difference of increased volume in the control group.
4.4.4. Anticancer study
4.4.4.1. Cell lines
Human breast carcinoma cell line (BT-549), Human cervical adenocarcinoma cell line (HeLa),
human colon adenocarcinoma cell line (COLO-205) and human kidney adenocarcinoma cell line
(ACHN) were procured from National Centre for Cell Science, Pune, India. The ACHN cells
were maintained in Minimum Essential Medium (MEM), supplemented with 10% FBS, 1% L-
glutamine and 50 μg/ml gentamycin sulphate. The BT-549, HeLa, and COLO-205 cells were
cultured in the RPMI-1640 medium with 2mM L-glutamine adjusted to contain 1.5 g/L sodium
bicarbonate and supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin.
All cell lines were incubated at 37 °C, in 5% CO2 humidified atmosphere. The Ehrlich‟s Ascitic
Carcinoma (EAC) cells to induce cancer in animal model (mice) were obtained from Amala
Cancer Research Center, Amala Nagar, Thrissur, Kerala, India and were maintained as ascites
tumor in Swiss albino mice by intraperitoneal inoculation of 1 ×106 viable cells.
4.4.4.2. In vitro cytotoxic study (MTT assay) [30]
In vitro cytotoxicity of synthesized compounds was evaluated by MTT assay. In brief,
exponentially growing BT-549, HeLa, COLO-205, and ACHN cells were plated in 96-well
plates (104 cells/well, in 100 µl of media) and incubated for 24 h to obtain 60-80% confluency.
The test compounds were solubilized in 50 µl DMSO and were further diluted with media so as
to get DMSO concentration less than 0.25%.The cells were then exposed to different
concentration of test compounds (500, 250, 125, 62.5, 31.25, 15.62, 7.81 µg/ml) in the volume of
100 µl /well. The control wells received only cells containing media and 0.25% DMSO.
Cisplatin was used as positive control. Media was removed after 48 h, and 100 µl MTT reagent
(1 mg/ml) was added to cell cultures, and then kept for incubation at 37 °C for 4 h. The viable
cells developed formazan complex; was solubilized by addition of 100 µl DMSO. The plates
were placed on micro-vibrator for 5 min. The absorbance was recorded on BIOTEK EL X800-
MS microtiter plate reader at 540 nm and percentage cytotoxicity was calculated as (control-
test/control)×100.
4.4.4.3. Acridine orange and ethidium bromide (AO/EB) staining [31,32]
Morphological analysis of apoptosis was done by acridine orange–ethidium bromide dual
staining. COLO-205 cells were seeded at 105 cells/ ml in 12 well plate. After 24 h of incubation
cells were treated with the different concentrations of 1a and 16a for 48 h. After 48 h cell
suspension was collected in a new eppendorf vial and adherent cells were trypsinized and
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transferred to cell suspension. The mixture was centrifuged at 129 g for 5 min. Supernatant was
discarded; the pellet was washed with PBS and centrifuged again. Cells were then stained with
1:1 ratio of AO/EB mixture and viewed and photographed at 400x.
4.4.4.4. Hoechst 33258 DNA staining to study nuclear morphological changes in COLO-205
cells [33]
COLO-205 cells were seeded at density of 105 cells/ml in 12 well plate. When cells reached 70-
80% confluency, old media was removed from wells and cells were treated with test media
having different concentrations of 1a and 16a, incubated for 48 h. After incubation, cells were
fixed with 4% formaldehyde at room temperature for 30 min and stained with Hoechst dye
(5ug/ml) for 15 min at room temperature and washed with PBS three times. Stained cells were
examined under a fluorescence inverted microscope at excitation of 340-380 nm with emission
of 465 nm.
4.4.4.5. RNA preparation [31]
About 4x106 COLO-205 cells/well in 6-well plates were treated with IC50 dose of 1a and 16a and
were incubated for 48 h. The total RNA was extracted from the cells using the total RNA
isolation reagent, TRI Reagent according to the manufacturer's procedure (Sigma-Aldrich), and
then the amount of extracted total RNA was determined by using Shimadzu Biospec-1601
DNA/protein/enzyme analyzer (Shimadzu, Japan).
4.4.4.6. Reverse transcriptase PCR [31]
The extracted total RNA was dissolved in nuclease free water and the RNA was treated with
deoxyribonuclease I (Invitrogen, catalog No. 18068-015). Reverse transcription PCR was
conducted with Super-Script One-Step RT-PCR for Long Templates (Invitrogen, catalog
No.11922-028) with PCR apparatus (Eppendorf, Germany). The primer set used were; Bcl-2: F,
5′-CGACTTCGCCGAGATGTCCAGCCAG-3′, R: 5′-ACTTGTGGCTCAGATAGGCACCCA
G-3′, Bax: F, 5′-CTTCGTCGAGGCCTGTTTAG-3′, R: 5′-ACTGAACCTTGTCCCCACTG-3′,
and for GAPDH: F: 5′-CCATGGAGAAGGCTGGGG-3′, R: 5′-CAAGAGTTGTCATGG
ATGACC-3′, primer pair was used. The parameters for RT-PCR were: 5 min at 95°C;
denaturation 1 min at 95°C; annealing 1 min at 55°C; 40 cycles of: 15 sec at 94°C, 30 sec at
55°C and 1 min at 72°C; final extension of 10 min at 72°C, as described reviously (22). The
amplified products were analyzed on 1.5% agarose gel electrophoresis (Genei, Bangalore, India)
and densitometric analysis of bands was done by ImageJ Software (NIH, USA).
4.4.4.7. Preparation of test solution of compounds for animal activity
The solutions of test compounds were prepared by suspending them in 0.25% carboxymethyl
cellulose (CMC) and administrated i.p. on 10th, 11th, 12th, 13th, and 14th day of tumor
inoculation in the volume of 0.1 ml/10 g mice. All the compounds were tested at the dose of 100
mg/kg body weight. The cisplatin was given as 3.5 mg/kg, i.p. and the dose was selected based
on the past experience with drug and body mass index [45,46].
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4.4.4.8. Induction of Ehrlich Ascites Carcinoma [38]
The Ehrlich Ascites Carcinoma (EAC) tumor model was used for the evaluation of antitumor
activities of test compounds. Female Swiss albino mice were divided into four groups of six
animals each. (Group A. Tumor bearing mice, Group B. Tumor bearing mice treated with one
dose of Cisplatin, Group C. Tumor bearing mice groups treated with compound 1a, and Group
D. Tumor bearing mice groups treated with test compound 16a). The ascites carcinoma-bearing
mice (donor) were taken for the studies after 15 days of tumor transplantation. The ascetic fluid
was drawn using an 18-gauge needle into sterile syringe and small amount was tested for
microbial contamination. The viability of tumor cells was checked by the Tryphan blue exclusion
test and cells were counted using haemocytometer. The ascetic fluid drawn was suitably diluted
with normal saline and viable EAC cells (1×106 cells/mouse) were injected intraperitoneally into
each mouse in an aseptic condition and the day of tumor inoculation was considered as day 0.
The animals were weighed on the day of tumor inoculation and then once in three days thereafter
to assess the tumor development. The test compounds were administered consecutively for 5
days from day 10 to day 14 of tumor inoculation. Cisplatin (single dose, 3.5 mg/kg, i.p.) was
injected on 10th day which served as standard drug. The animals were monitored twice a day for
44 days (till last animal died) to record survival time. Antitumor effects of compounds were
assessed by observation of following parameters.
4.4.4.9. Percentage increase in weight as compared to day 0 weights
The animals are weighed on the day of inoculation and after once in three days in the post
inoculation period, the percentage increase in weight was calculated using the formula: %
Increase in weight= [(animal weight on respective day/animal weigh on day 0) −1]×100 [46].
4.4.4.10. Mean survival time and increase in life span
Total number of days an animal survived from the day of tumor inoculation was counted. The
mean survival time (MST) was calculated by dividing the total number of days animals survived
to the total number of animals in experiment. The percentage increase in lifespan was calculated
using the formula: ILS(%) = [(Mean survival time of treated group/mean survival time of control
group)−1] ×100 [46].
4.4.4.11. Statistical analysis
The statistical analyses were performed by one-way ANOVA, followed by Tukey‟s post hoc test
using GraphPad Prism 5.02. The results are expressed as the mean± S.D. Statistical significance
was set at p<0.05 level.
4.5. Molecular docking into DNA binding site of NF-κB
Library of seventy quinolone substituted thiazolidin-4-one derivatives were prepared by using
chemdraw ultra 11.0. The 3D crystal structure of the NF-κB was obtained from Protein Data
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Bank (PDB codes: 1NFK and 1LE9) [47,48]. The 3D structures of NF-κB, p50-p50 homodimer
(from 1NFK), p50 monomer chain A (p50a from 1NFK), p50 monomer chain B (p50b from
1NFK; p50b from 1LE9) were used for virtual screening [40]. The co-crystallized DNA and
water molecules were removed and AutoDock 4.2 was used for molecular docking studies.
Docking parameters were set to default values on the basis of Lamarckian genetic algorithm
principle [49]. The 31 compounds with highest estimated free energy of binding were selected
synthesis. Synthesized compounds were biologically evaluated for anti-inflammatory and
anticancer study and attempt was made to establish structure-activity relations of compounds to
explore new lead compounds.
Acknowledgements
The authors thank the Indian Institute of Science, Bangalore for NMR spectra and SAIF labs of
Central Drug Research Institute, Lucknow and Punjab University for ESI-MS. We are also
grateful to the Principal, Manipal College of Pharmaceutical Sciences, and Head, Dept. of
Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal University for
providing necessary research facilities.
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Figure Captions
Fig. 1. Apoptosis analysis of COLO-205 cells treated with control and IC50 dose of 1a and 16a
for 48 h. Cells were stained with AO (green) or EB (red) and viewed under fluorescence
microscope at 400x.
Fig. 2. Hoechst 33258 DNA staining for the effects of 1a and 16a on nuclear morphology of
COLO-205 cells. Cells were incubated with IC50 dose of 1a and 16a for 48 h (photographs at
400x).
Fig. 3. The mRNA expression of Bcl-2 and Bax by RT-PCR. COLO-205 cells were treated with
IC50 dose of 1a and 16a for 48 h.
Fig. 4. Effect of test drugs on body weight changes in EAC inoculated mice. All values are the
mean±S.D. of six mice.
Fig. 5. Effect of test drugs on mean survival time in EAC inoculated mice. All the values are
mean±S.D. of six mice. ***P< 0.001 compared to control.
Fig. 6. Stereoview of the complex formed by NF-κB homodimer and the docked compound 1a.
The amino acids Gln 274, and Gln 306 were involved in interaction with compounds.
Fig. 7. Stereoview of the complex formed by NF-κB homodimer and the docked compound 6b.
The amino acids Lys 144, Lys 145, and His141 were involved in interaction with compounds.
Fig. 8. Stereoview of the complex formed by NF-κB homodimer and the docked compound 16a.
The amino acids Lys 144, and Tyr 57 were involved in interaction with compounds.
26

Table(s)
ACCEPTED MANUSCRIPT
Table 1
Effect of test compounds on carrageenan induced paw oedema in rats
Compound
Dose
(mg/kg po)
%Oedema
inhibition ± SDb
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
10
17.80±1.04a
20.35±1.27a
23.58±1.11a
18.96±0.94a
14.5±0.72
1a
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
7a
8a
23.26±1.31a
13.02±0.62
13.96±0.76
22.69±1.18a
12.42±0.84
16.37±0.75a
14.33±0.91
22.90±1.11a
24.14±1.37a
25.16±1.22a
13.29±0.59
21.41±1.13a
20.57±1.41a
12.39±0.82
20.78±1.33a
7.50±0.35
9a
10a
11a
12a
12b
12c
13a
14a
15a
15b
15c
16a
16b
16c
Indomethacin
6.81±0.37
7.62±0.41
2.77±0.23
1.75±0.21
5.16±0.29
23.29±1.42a
10.08±0.93
18.55±1.44a
24.02±1.76a
15.84±1.01a
33.19±1.85a
aOne way ANOVA followed by Tukey's test , a = p<0.05 Vs Control group
b Average of six determinations