Synthesis and antiproliferative effect of novel 4-thiazolidinone-, pyridine- and piperazine-based conjugates on human leukemic cells

Article abstract of DOI:10.1016/j.ejmech.2014.05.009

The present work reveals the synthesis and antiproliferative effect of a series of 2, 3 disubstituted 4-thiazolidinone analogues on human leukemic cells. The chemical structures of newly synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Compound methyl 3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-yl)thiazolidin-2-yl) benzoate (5) displayed potent activity (IC₅₀9.71, 15.24 and 19.29 μM) against Nalm6, K562, Jurkat cells. Cell cycle analysis and mitochondrial membrane potential further confirmed that compound 5 is cytotoxic and able to induce cell death.

Full text of DOI:10.1016/j.ejmech.2014.05.009

Accepted Manuscript
Synthesis and antiproliferative effect of novel 4- thiazolidinone-, pyridine- and
piperazine- based conjugates on human leukemic cells
Kothanahally S. Sharathkumar, Ananda Hanumappa, Mahesh Hegde,
Kereyagalahally H. Narasimhamurthy, Sathees C. Raghavan, Kanchugarakoppal S.
Rangappa
PII:
S0223-5234(14)00423-1
10.1016/j.ejmech.2014.05.009
EJMECH 6965
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 February 2014
Revised Date: 14 April 2014
Accepted Date: 2 May 2014
Please cite this article as: K.S. Sharathkumar, A. Hanumappa, M. Hegde, K.H. Narasimhamurthy, S.C.
Raghavan, K.S. Rangappa, Synthesis and antiproliferative effect of novel 4- thiazolidinone-, pyridine-
and piperazine- based conjugates on human leukemic cells, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.05.009.
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ACCEPTED MANUSCRIPT
R = H = IC50 = 9.71 µM
A series of new 4-thiazolidinone-, pyridine- and piperazine- based conjugates were synthesized,
characterized and screened for their anticancer activity against nalm6, k562 and jurkat cell lines.
Few of them showed good potency to induce apoptosis in leukemia cells.

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative effect of novel 4- thiazolidinone-, pyridine- and
piperazine- based conjugates on human leukemic cells
Kothanahally S. Sharathkumar a, Ananda Hanumappa a, Mahesh Hegde a, Kereyagalahally H.
Narasimhamurthy a, Sathees C. Raghavan b, Kanchugarakoppal S. Rangappa a *
a
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore-
570006.
b Department of Biochemistry, Indian Institute of Science, Bangalore-560012.
*CORRESPONDING AUTHOR:
Tel. No.: +91-821-2419661 Fax: +91-821-2500846
*E-mail: rangappaks@gmail.com/rangappaks@chemistry.uni-mysore.ac.in
Abstract: The present work reveals the synthesis and antiproliferative effect of a series of 2,
3 disubstituted 4- thiazolidinone analogues on human leukemic cells. The chemical structures
1
of newly synthesized compounds were confirmed by IR, H NMR, 13C NMR and mass
spectral analysis. Compound methyl 3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-
yl)thiazolidin-2-yl)benzoate (5) displayed potent activity (IC50 9.71, 15.24 and 19.29 µM)
against Nalm6, K562, Jurkat cells. Cell cycle analysis and mitochondrial membrane potential
further confirmed that compound 5 is cytotoxic and able to induce cell death.
Key words: Thiazolidinones, Propylphosphonic anhydride (T3P®), Leukemia, Cytotoxicity,
Chemotherapeutics, Apoptosis.
1. Introduction
Cancer is the uncontrolled proliferation and spread of cells, these proliferating cells
divide rapidly (cell cycle is accelerated) and it alters cell-cell communication and leads to
DNA damage. Leukemia is the most common childhood malignancy, it accounts for 30% of
all cancers diagnosed in children under 15 years of age in developing countries. Therefore,
there is an urgent need for new therapeutics, which could act as anti-leukemic agents with
less or minimal side effects.
Heterocyclic compounds attracted a lot of attention because of its wide spread
biological activities. In specific, five membered heterocycles with two heteroatoms received a
special attention with proven utility in medicinal chemistry [1-3], among them 4-
thiazolidinones represents an important class of five membered heterocycles. Thiazolidinones
has been considered as a magic moiety (wonder nucleus) and they display diverse biological
activities such as anticancer [4-6], anti-inflammatory [7], antimicrobial [8], anticonvulsant
[9], antifungal [10], antitubercular [11], anti HIV [12,13] and as entamoebahistolytica
inhibitors [14]. The 4-thiazolidinone moiety is very versatile and has featured in many drugs
(Fig 1) and several compounds with 4-thiazolidinone core structure were found to kill
selectively drug resistant cancer cells and induce cell death [15]. Tripathi et al. [16] have
given a detailed study on the synthetic approach and biological significance of 4-
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thiazolidinones, the review is an endeavour to highlight the progress of 4-thiazolidinone in
the field of chemical biology. In addition, piperazine- and pyridine conjugated pyrimidine
derivatives are well explored in the literature as selective inhibitors of CDK4/6 [17,18].
In this context, we have recently published a series of thioxothiazolidinone derivatives
endowed with good activity towards the human leukemia cells [19]. Continuing our efforts in
this field and considering the pharmacological activities of thiazolidinones, a new piperazine
substituted pyridine conjugated 4- thiazolidinone derivatives were synthesized and screened
for their antiproliferative activity against different human leukemic cell lines. Interestingly,
few of the synthesized compounds showed significant improvement in the cytotoxicity
profile against different leukemia cell lines in dose- dependent manner, in particular
compound 5 exhibited notable cytotoxicity against Nalm6 cell line.
Herein, we report a mild and simple protocol for the synthesis of a library of highly
functionalised 4- thiazolidinone derivatives 5, 8a-f and 9a-d and their antiproliferative
activities against Jurkat, K562 and Nalm6 human leukemia cell lines.
2. Results and discussion
2.1. Chemistry:
In our current research, a series of novel 4-thiazolidinone-, pyridine- and piperazine
based conjugates 8a-f and 9a-d (Table 1) bearing different level of substituents were
synthesized as shown in scheme-1 & scheme-2. After an exhaustive exploration of the
reaction conditions varying the solvents, equivalents and catalysts, optimal parameters to
prepare title compounds 8a-f and 9a-d were found.
We have synthesized tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (3)
using literature reported method [20]. Displacement of the bromine atom of 5-bromo-2-
nitropyridine with 1-Boc piperazine followed by reduction of nitro group using 10%
palladium on carbon in ethanol under hydrogen atmosphere afforded amino pyridine (3).
N-Bocpiperazine substituted 2-amino pyridine (3) was used as an amine component to
construct 4-thiazolidinone ring. Condensation of methyl 4-formyl-3-methoxy-benzoic acid
methyl ester with 2-amino pyridine (3) followed by cyclization with mercaptoacetic acid gave
the required product tert-butyl 4-(6-(2-(2-methoxy-4-(methoxycarbonyl)phenyl)-4-
oxothiazolidin-3-yl)pyridin-3-yl)piperazine-1-carboxylate (4) in good yield. This particular
reaction is the key step in our whole reaction scheme, so we examined this reaction with
different catalyst under various reaction conditions. Among all, propylphosphonic anhydride
(T3P®) was found to be an efficient cyclodehydrating agent to construct the 4-thiazolidinone
ring. This protocol is having many advantages over the literature reported methods like one
pot operation, short reaction time, mild reaction condition and ease of product purification
with excellent yield. It is noteworthy to mention that, the Boc group of piperazine ring was
unaffected throughout the reaction. Previously we have reported the synthesis of 4-
thiazolidinones using T3P®-DMSO media as an oxidising as well as cyclodehydrating agent
[21].
Removal of Boc protecting group was easily achieved by the treatment of 4 with
aqueous HCl at room temperature. Column purification gave the key intermediate methyl 3-
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methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-yl)thiazolidin-2-yl)benzoate (5) in good
yield of 90%.
The requisite title compounds 8a-f were synthesized by the reaction of 5 with
different benzene sulfonyl chlorides 6a-f in presence of triethyl amine in dichloromethane
solvent. In similar reaction condition the other series 9a-d were synthesized by reacting 5
with different benzyl chlorides 7a-d in good yields.
2.2. Biological studies
2.2.1. Piperazine substituted pyridine conjugated 4- thiazolidinones (5, 8a-f and 9a-d)
induces cytotoxicity on leukemic cell lines
Inhibition of cell proliferation is a key factor for chemotherapeutic agents. In the
present study we used trypan blue and MTT assay to investigate the effect of piperazine
substituted pyridine conjugated 4-thiazolidinones (5, 8a-f and 9a-d) induced cytotoxicity on
three different leukemic cell lines (Jurkat, K562 and Nalm6) (Fig 2), following treatment
with different concentrations of the compound (Table 2). The trypan blue dye exclusion
assay showed significant reduction in cell viability compared to control upon treatment with
5. Besides, MTT assay also showed significant anti proliferation activity, which was
comparable with trypan blue assay. Out of the three cell lines tested, the Nalm6 cell line was
more sensitive to the experimental compound 5, compared to Jurkat and K562 cells, and was
selected for further studies.
2.2.2. Cell cycle analysis following treatment with compound 5 on Nalm6 cells
After preliminary investigations of trypan blue dye exclusion and MTT assays, we
tested the effect of 5 on cell cycle progression by using fluorescence activated cell sorter
(FACS). Nalm6 cells were harvested after 48h of compound 5 treatment (5, 10 and 20ꢀM).
Cells were processed, stained with propidium iodide and subjected to flowcytometric
analysis. The histogram of control (DMSO treated) cells showed standard pattern of cell
cycle progression. Upon addition of 5 to Nalm6 cells, a concentration dependent change was
observed in the cell cycle distribution. However, such change was minimal when the cells
were treated with 5, 10 ꢀM of compound 5. We noted that there was remarkable
accumulation of SubG1 cell population at 20 ꢀM as well as G2/M arrest (Fig 3). These
results suggested that compound 5 arrests the cells at G2/M phase and induces cell death
which was resulted in increased level of SubG1 phase population, thereby affecting its
proliferation.
2.2.3. Piperazine substituted pyridine conjugated 4- thiazolidinone (5) induces cell death
by affecting mitochondrial membrane potential
To determine the mechanism by which compound 5 induces cell death, JC-1 staining
assay was performed. Effect of compound 5 on mitochondrial membrane potential was
determined by JC-1 staining followed by flow cytometry. Changes in the mitochondrial
membrane potential was determined by red verses green fluorescence by JC-1 dye where
healthy mitochondria gives out red fluorescence because of J-aggregates of JC-1 dye and
apoptotic/dead cells show green fluorescence because of lack of mitochondrial membrane
potential. Panels representative of flowcytometric density plots were used to determine the
3

ACCEPTED MANUSCRIPT
results which are depicted in (Fig 4), where cells exposed to the mitochondrial stressor 2, 4-
DNP was used as positive control. DMSO treated cells served as the vehicle control.
Minimum 10,000 events were acquired and analyzed by using Cell Quest Pro software (BD,
USA). Results showed that compound 5 induced a significant depolarization of mitochondrial
membrane potential in a dose-dependent manner on leukemic cells.
3. Conclusion
In summary, the cytotoxicity assays were used for preliminary screening of 4-
thiazolidinone-, pyridine- and piperazine- based conjugates. Among all the screened
derivatives, compound 5 was found to be most potent against leukemia cell lines at lower
concentrations. Further we carried out several methods to elucidate the mechanism by which
the compound to induced the cell death. In Nalm6 cells, compound 5 induced cell cycle arrest
and caused depolarization of mitochondrial membrane potential. Hence the methyl 3-
methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridine-2-yl)thiazolidin-2-yl)benzoate
represent a promising new alternative for an antileukemic agent.
(5)
may
4. Experimental section
4.1. Chemistry
The progress of all reactions was monitored by TLC, which was performed on 2.0-5.0
cm aluminum sheets precoated with silica gel 60 F 254 to a thickness of 0.25 mm (Merck)
1
using UV light for visualization. H NMR and 13C NMR spectra were recorded on an
Agilent-varian NMR spectrometer operating at 400 and 100 MHz, respectively. Chemical
shifts are given in δ values (ppm) using tetramethylsilane as the internal standard. Mass
spectra were recorded using high resolution mass spectrometer (ESI). Infrared spectra were
recorded in nujol/KBr on Shimadzu FT-IR model 8300 spectrophotometer. Chromatographic
separations were carried out on 60:120 silica gel.
4.1.1. Tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (2)
A solution of 1-Boc-piperazine (15.0 mmol) and 5-bromo-2-nitropyridine (5.00 mmol) in N-
methyl pyrrolidone (15 mL) was stirred at 1200C for 3 h. The reaction mixture was diluted
with water and the precipitate was collected by filtration to give 2 (80%) as a colorless
powder. 1H NMR (400 MHz, DMSO-d6): δ 8.10-8.08 (d, J = 9.2 Hz, 1H, ArH), 7.96-7.95 (d,
J = 3.0 Hz, 1H, ArH), 7.33-7.30 (dd, J = 3.4 Hz, 1H, ArH), 3.66–3.63 (m, 4H, piperazine-
CH2), 3.43–3.41 (m, 4H, piperazine-CH2), 1.47 (s, 9H, t-butyl-CH3).
4.1.2. Tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (3)
A suspension of 2 and 10% palladium on carbon in ethanol was hydrogenated for 2 h. The
reaction mixture was filtered through celite and concentrated in vacuo to give 3 as brown oil.
1H NMR (400 MHz, DMSO-d6): δ 7.77-7.76 (d, J = 2.1 Hz, 1H, ArH), 7.27-7.14 (dd, J = 3.0
Hz, 1H, ArH), 6.51 (d, J = 9.0 Hz, 1H, ArH), 3.61–3.59 (m, 4H, piperazine-CH2), 3.39–3.36
(m, 4H, piperazine-CH2), 1.48 (s, 9H. t-butyl-CH3).
4.1.3. Tert-butyl 4-(6-(2-(2-methoxy-4-(methoxycarbonyl)phenyl)-4-oxothiazolidin-3-
yl)pyridin-3-yl)piperazine-1-carboxylate (4)
4

ACCEPTED MANUSCRIPT
4-formyl-3-methoxy-benzoic acid methyl ester (1.0 mmol), amine 3 (1.0 mmol), thioglycolic
acid (1.0 mmol) were stirred in ethyl acetate, propylphosphonic anhydride (T3P®) (1.5 mmol)
was added to the reaction mixture at 00C. The reaction kept for stirring at room temperature
for 3h. After completion of the reaction, the mixture was diluted with water (20 mL) and
neutralized by adding 10% NaHCO3 solution. The product was extracted with ethyl acetate
(10 mL × 2) and the combined organic layers were washed with water followed by brine
solution. The organic phase was dried over anhydrous Na2SO4. The solvent was dried under
reduced pressure to afford a desired product as off white solid which was pure enough to
1
perform the next reaction. H NMR (400 MHz, DMSO-d6): δ 7.90-7.89 (d, J = 2.4 Hz, 1H,
ArH), 7.75-7.72 (m, 2H, ArH), 7.46-7.42 (dd, J = 2.8 Hz, 1H, ArH), 7.34-7.30 (dd, J =
3.2Hz, 1H, ArH), 6.99-6.97 (d, J = 8.2 Hz, 1H, ArH), 6.72 (s, 1H, thiazolidinone-CH), 3.96-
3.90 (m, 2H, thiazolidinone-CH2), 3.71 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.61-3.58 (m, 4H,
piperazine-CH2), 3.27-3.25 (m, 4H, piperazine-CH2), 1.51 (s, 9H, t-butyl-CH3).
4.1.4.
Methyl
3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-yl)thiazolidin-2-
yl)benzoate (5)
Compound 4 was stirred at ambient temperature in dioxane and 4M HCl for 2 h. The solvent
was evaporated in vacuum, the product was precipitated by adding diethyl ether. The
precipitate was collected by filtration and neutralized with 10% NaHCO3 solution and
extracted with ethylacetate (2 times). The combined organic layer was washed with water
followed by brine solution and dried over anhydrous Na2SO4, organic solvent was evaporated
under reduced pressure to afford the crude product 5, which was purified by silica-gel column
chromatography using chloroform and methanol (1:1) as an eluent to give compound 5 as a
pale-yellow solid. IR γmax (KBr) 3280, 3110, 2901, 1820, 1748, 1740, 1681, 1510, 1488,
1
1440, 1246, 1001 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.89-7.88 (d, J = 2.8 Hz, 1H,
ArH), 7.76-7.70 (m, 2H, ArH), 7.44-7.39 (dd, J = 2.4 Hz, 1H, ArH), 7.33-7.30 (dd, J = 3.4
Hz, 1H, ArH), 6.95-6.93 (d, J = 8.8 Hz, 1H, ArH), 6.70 (s, 1H, thiazolidinone-CH), 4.02-3.90
(m, 2H, thiazolidinone-CH2), 3.73 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.65-3.62 (m, 4H,
piperazine-CH2), 3.27-3.25 (m, 4H, piperazine-CH2). 13C NMR (100 MHz, DMSO-d6): δ
170.7, 166.1, 157.5, 142.9, 136.0, 134.4, 132.5, 131.6, 130.1, 125.0, 120.1, 118.5, 112.1,
80.0, 62.5, 56.0, 52.0, 48.6, 34.0 cm-1; HRMS (ESI) m/zCalcd for C21H24N4O4SNa [M+Na]+
451.5047, found 451.5057.
4.1.5. General procedure for the synthesis of compounds 8(a-f)
To
a
solution of methyl 3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-
yl)thiazolidin-2-yl)benzoate 5 (1.00 mmol) and triethylamine (1.5 mmol) in dry
dichloromethane at 00C were added substituted benzene sulfonyl chlorides 6a-f (1.00 mmol).
The reaction mixture was stirred at 00C for about 30 minutes and the stirring was continued at
room temperature for about 3–4 h (completion of the reaction was monitored by TLC). After
completion of the reaction, the reaction mixture was diluted with water and extracted with
dichloromethane (2 times), the combined organic layer was washed with water followed by
brine solution and dried over anhydrous Na2SO4. The solvent was dried under reduced
pressure to afford a desired product.
5

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4.1.5.1. Methyl 3-methoxy-4-(3-(5-(4-(4-methoxyphenylsulfonyl)piperazin-1-yl)pyridin-
2-yl)-4-oxothiazolidin-2-yl)benzoate (8a)
Yield: 88%; Pale yellow liquid; IR γmax (nujol) 3045, 1742, 1651, 1582, 1449, 1381, 1369,
1
1320, 1245, 1165, 1065 cm-1; H NMR (400 MHz, DMSO-d6) : δ 8.00-7.98 (d, J = 9.0 Hz,
1H, ArH), 7.84-7.82 (d, J = 8.2 Hz, 2H, ArH), 7.78-7.77 (d, J = 3.2 Hz, 1H, ArH), 7.58-7.57
(d, J = 2.2 Hz, 1H, ArH), 7.45-7.43 (dd, J = 2.4 Hz, 1H, ArH), 7.37-7.34 (dd, J = 3.2 Hz, 1H,
ArH), 7.12-7.10 (d, J = 9.0 Hz, 1H, ArH), 7.06-7.04 (d, J = 8.2 Hz, 2H, ArH), 6.64 (s, 1H,
thiazolidinone-CH), 4.03-3.99 (dd, J = 2.2 Hz, 1H, thiazolidinone-CH2(HA)), 3.83-3.79 (m,
1H, thiazolidinone-CH2(HB)), 3.89 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
3.29-3.25 (m, 4H, piperazine-CH2), 2.98-2.95 (m, 4H, piperazine-CH2). 13C NMR (100 MHz,
DMSO-d6) : δ 171.1, 166.3, 160.1, 158.2, 144.7, 143.1, 136.2, 134.6, 132.4, 131.8, 131.5,
130.1, 126.3, 125.2, 120.1, 118.3, 114.2, 112.2, 62.4, 56.1, 55.8, 52.1, 48.6, 33.8. HRMS
(ESI) m/z Calcd for C28H30N4O7S2Na [M+Na]+ 621.6904, found 621.6915.
4.1.5.2. Methyl 3-methoxy-4-(3-(5-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)pyridin-2-
yl)-4-oxothiazolidin-2-yl)benzoate (8b)
Yield: 78%; Dark brown liquid; IR γmax (nujol) 3040, 1740, 1668, 1587, 1535, 1449, 1385,
1
1370, 1350, 1242, 1165, 1060. cm-1; H NMR (400 MHz, DMSO-d6) : δ 8.42-8.39 (m, 2H,
ArH), 8.02-7.99 (m, 2H, ArH), 7.919-7.911 (d, J = 3.2 Hz, 1H, ArH), 7.65-7.63 (d, J = 9.2
Hz, 1H, ArH), 7.575-7.570 (d, J = 2.0 Hz, 1H, ArH), 7.46-7.43 (dd, J = 2.4 Hz, 1H, ArH),
7.36-7.33 (dd, J = 3.2 Hz, 1H, ArH), 7.00-6.98 (d, J = 9.2 Hz, 1H, ArH), 6.67 (s, 1H,
thiazolidinone-CH) 4.04-4.00 (dd, J = 1.2 Hz, 1H, thiazolidinone-CH2(HA)), 3.83-3.78(m,
1H, thiazolidinone-CH2(HB)), 3.72 (s, 6H, OCH3), 3.21-3.19 (m, 4H, piperazine-CH2), 3.05-
3.03 (m, 4H, piperazine-CH2).13C NMR (100 MHz, DMSO-d6) : δ 170.9, 166.3, 158.0, 150.6,
144.2, 142.8, 140.9, 135.8, 133.4, 131.5, 129.5, 129.0, 125.2, 120.3, 118.4, 113.1, 61.8, 56.3,
52.4, 47.5, 45.9, 33.3. HRMS (ESI) m/z Calcd for C27H27N5O8S2Na [M+Na]+ 636.6620,
found 636.6637.
4.1.5.3. Methyl 4-(3-(5-(4-(mesitylsulfonyl)piperazin-1-yl)pyridin-2-yl)-4-oxothiazolidin-
2-yl)-3-methoxybenzoate (8c)
Yield: 82%; Pale yellow oil; IR γmax (nujol) 3054, 2987, 1742, 1669, 1582, 1448, 1383,
1
1372, 1251, 1168, 1058 cm-1; H NMR (400 MHz, DMSO-d6) : δ 7.92-7.91 (d, J = 3.0 Hz,
1H, ArH), 7.66-7.64 (d, J = 9.0 Hz, 1H, ArH), 7.58-7.57 (d, J = 2.4 Hz, 1H, ArH), 7.45-7.42
(dd, 2.4 Hz, 1H, ArH), 7.35-7.32 (dd, J = 3.2 Hz, 1H, ArH), 7.21 (s, 2H), 6.94-6.92 (d, J =
9.0 Hz, 1H, ArH), 6.69 (s, 1H, thiazolidinone-CH), 4.03-4.00 (dd, J = 1.4 Hz, 1H,
thiazolidinone-CH2(HA)), 3.83-3.81 (m, 1H, thiazolidinone-CH2(HB)), 3.73 (s, 3H, OCH3),
3.72 (s, 3H, OCH3), 3.18-3.16 (m, 4H, piperazine-CH2), 3.03-3.00 (m, 4H, piperazine-CH2).
2.62 (s, 6H, CH3), 2.58 (s, 3H, CH3).13C NMR (100 MHz, DMSO-d6) : δ 171.0, 166.2, 158.8,
145.2, 144.6, 142.8, 141.2, 137.3, 136.0, 134.5, 132.4, 131.7, 130.3, 130.1, 125.2, 120.0,
118.9, 112.2, 61.9, 56.2, 52.2, 48.9, 34.2, 22.5, 22.0. HRMS (ESI) m/z Calcd for
C30H34N4O6S2Na [M+Na]+ 633.7442, found 633.7451.
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4.1.5.4. Methyl 4-(3-(5-(4-(2,5-dichlorophenylsulfonyl)piperazin-1-yl)pyridin-2-yl)-4-
oxothiazolidin-2-yl)-3-methoxybenzoate (8d)
Yield: 77%; Dark brown gummy solid; IR γmax (nujol) 3050, 1737, 1658, 1576, 1450, 1380,
1
1367, 1240, 1163, 1062, 730 cm-1; H NMR (400 MHz, DMSO-d6) : δ 8.01-7.99 (d, J = 9.2
Hz, 1H, ArH), 7.89-7.88 (d, J = 3.2 Hz, 1H, ArH), 7.84-7.83 (d, J = 2.8 Hz, 1H, ArH), 7.70-
7.68 (d, J = 6.8 Hz, 2H, ArH), 7.58-7.57 (d, J = 2.4 Hz, 1H, ArH), 7.44-7.41 (dd, J = 2.2 Hz,
1H, ArH), 7.35-7.32 (dd, J = 3.4 Hz, 1H, ArH), 7.01-6.99 (d, J = 9.0 Hz, 1H, ArH), 6.72 (s,
1H, thiazolidinone-CH), 4.04-4.00 (dd, J = 1.2 Hz, 1H, thiazolidinone-CH2(HA)), 3.85-3.82
(m, 1H, thiazolidinone-CH2(HB)), 3.74 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.27-3.24 (m, 4H,
piperazine-CH2), 3.07-3.05 (m, 4H, piperazine-CH2). 13C NMR (100 MHz, DMSO-d6) : δ
170.9, 166.0, 159.1, 144.9, 142.7, 141.4, 135.9, 134.5, 133.3, 132.9, 132.5, 131.6, 130.6,
130.1, 129.7, 125.0, 119.9, 118.3, 112.2, 62.4, 56.2, 52.1, 48.7, 34.1. HRMS (ESI) m/z Calcd
for C27H26Cl2N4O6S2Na [M+Na]+ 660.5545, found 660.5553.
4.1.5.5. Methyl 3-methoxy-4-(4-oxo-3-(5-(4-(phenylsulfonyl)piperazin-1-yl)pyridin-2-
yl)thiazolidin-2-yl)benzoate (8e)
Yield: 87%; Yellow gummy liquid; IR γmax (nujol) 3050, 1738, 1650, 1575, 1450, 1375,
1
1365, 1240, 1162, 1066 cm-1; H NMR (400 MHz, DMSO-d6) : δ 7.90-7.89 (d, J = 2.8 Hz,
1H, ArH), 7.74-7.69 (m, 3H, ArH), 7.65-7.61 (m, 3H, ArH), 7.59-7.58 (d, J = 2.4 Hz, 1H,
ArH), 7.46-7.44 (dd, J = 2.4 Hz, 1H, ArH), 7.34-7.31 (dd, J = 3.2 Hz, 1H, ArH), 7.00-6.98
(d, J = 9.2 Hz, 1H, ArH), 6.68 (s, 1H, thiazolidinone-CH), 4.05-4.00 (dd, J = 1.2 Hz, 1H,
thiazolidinone-CH2(HA)), 3.83-3.79 (m, 1H, thiazolidinone-CH2(HB)), 3.724 (s, 3H, OCH3),
3.720 (s, 3H, OCH3), 3.17-3.15 (m, 4H, piperazine-CH2), 2.94-2.91 (m, 4H, piperazine-CH2).
13C NMR (100 MHz, DMSO-d6) : δ 170.9, 166.2, 158.0, 144.2, 142.8, 135.8, 134.8, 133.8,
133.4, 131.5, 129.9, 129.0, 128.0, 125.2, 120.3, 118.4, 113.1, 61.8, 56.2, 52.4, 47.5, 46.0,
33.3. HRMS (ESI) m/z Calcd for C27H28N4O6S2Na [M+Na]+ 591.6644, found 591.6652.
4.1.5.6.
Methyl
4-(3-(5-(4-(4-fluorophenylsulfonyl)piperazin-1-yl)pyridin-2-yl)-4-
oxothiazolidin-2-yl)-3-methoxybenzoate (8f)
Yield: 78%; Pale brown liquid; IR γmax (nujol) 3043, 1739, 1648, 1588, 1448, 1385, 1371,
1
1245, 1163, 1060, 1090 cm-1; H NMR (400 MHz, DMSO-d6) : δ 7.92-7.91 (d, J = 2.6 Hz,
1H, ArH), 7.86-7.84 (d, J = 7.8 Hz, 2H, ArH), 7.82-7.81 (d, J = 3.2 Hz, 1H, ArH), 7.61-7.60
(d, J = 2.4 Hz, 1H, ArH), 7.46-7.43 (dd, J = 2.8 Hz, 1H, ArH), 7.39-7.36 (dd, J = 3.2 Hz, 1H,
ArH), 7.34-7.32 (d, J = 7.8 Hz, 2H, ArH), 7.00-6.98 (d, J = 8.8 Hz, 1H, ArH), 6.71 (s, 1H,
thiazolidinone-CH), 4.00-3.95 (dd, J = 1.2 Hz, 1H, thiazolidinone-CH2(HA)), 3.86-3.83 (m,
1H, thiazolidinone-CH2(HB)), 3.75 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.19-3.16 (m, 4H,
piperazine-CH2), 2.93-2.90 (m, 4H, piperazine-CH2). 13C NMR (100 MHz, DMSO-d6) : δ
170.9, 166.6, 166.1, 158.8, 144.9, 143.1, 136.2, 135.3, 134.4, 132.6, 131.8, 130.7, 130.1,
125.2, 120.4, 118.7, 115.4, 112.2, 62.8, 56.2, 51.8, 48.4, 33.2. HRMS (ESI) m/z Calcd for
C27H27FN4O6S2Na [M+Na]+ 609.6549, found 609.6558.
4.1.6. General procedure for the synthesis of compounds 9(a-d)
7

ACCEPTED MANUSCRIPT
To a solution of methyl 3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl) pyridin-2-
yl)thiazolidin-2-yl)benzoate 5 (1.00 mmol) and triethylamine (1.5 mmol) in dry
dichloromethane at 00C were added substituted benzyl chlorides 7a-d (1.00 mmol). The
reaction mixture was stirred at 00C for about 30 minutes and the stirring was continued at
room temperature for about 4–5 h (completion of the reaction was monitored by TLC). After
completion of the reaction, the reaction was diluted with water and extracted with
dichloromethane (2 times), the combined organic layer was washed with water followed by
brine solution and dried over anhydrous Na2SO4. The solvent was dried under reduced
pressure to afford a desired product.
4.1.6.1. Methyl 3-methoxy-4-(3-(5-(4-(3-methylbenzyl)piperazin-1-yl)pyridin-2-yl)-4-
oxothiazolidin-2-yl)benzoate (9a)
Yield: 74%; Yellow liquid; IR γmax (nujol) 3057, 1735, 1658, 1579, 1445, 1383, 1369, 1240,
1
1167, 1061 cm-1; H NMR (400 MHz, DMSO-d6) : δ 7.90-7.89 (d, J = 2.6 Hz, 1H, ArH),
7.66-7.64 (d, J = 9.2 Hz, 1H, ArH), 7.59-7.58 (d, J = 2.2 Hz, 1H, ArH), 7.46-7.43 (dd, J = 2.4
Hz, 1H, ArH), 7.41-7.38 (m, 1H, ArH), 7.34-7.30 (dd, J = 3.2 Hz, 1H, ArH), 7.19 (s, 1H,
ArH), 7.10-7.08 (d, J = 9.0 Hz, 1H, ArH), 7.06-7.01 (m, 2H, ArH), 6.68 (s, 1H,
thiazolidinone-CH), 4.04-4.01 (dd, J = 1.2 Hz, 1H, thiazolidinone-CH2(HA)), 3.86-3.83 (m,
1H, thiazolidinone-CH2(HB)), 3.74 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.63 (s, 2H, benzyl-
CH2), 3.21-3.19 (m, piperazine-CH2), 3.05-3.03 (m, piperazine-CH2), 2.29 (s, 3H, Ar-CH3).
13C NMR (100 MHz, DMSO-d6) : δ 170.8, 166.1, 158.8, 144.9, 142.7, 138.8, 138.2, 136.2,
134.6, 132.2, 131.4, 130.9, 130.1, 128.2, 127.1, 125.7, 125.1, 120.3, 118.9, 112.4, 64.2, 62.6,
56.2, 52.1, 48.9, 33.4, 21.2. HRMS (ESI) m/z Calcd for C29H32N4O4SNa [M+Na]+ 555.6538,
found 555.6547.
4.1.6.2.
Methyl
3-methoxy-4-(4-oxo-3-(5-(4-(3,4,5-trimethoxybenzyl)piperazin-1-
yl)pyridin-2-yl)thiazolidin-2-yl)benzoate (9b)
Yield: 71%; Brown gummy solid; IR γmax (nujol) 3049, 1737, 1658, 1586, 1451, 1392,
1
1369, 1249, 1176, 1058 cm-1; H NMR (400 MHz, DMSO-d6) : δ 7.88-7.87 (d, J = 2.8 Hz,
1H, ArH), 7.66-7.64 (d, J = 9.2 Hz, 1H, ArH), 7.57-7.56 (d, J = 2.4 Hz, 1H, ArH), 7.44-7.41
(dd, J = 2.2 Hz, 1H, ArH), 7.38-7.35 (dd, J = 3.0 Hz, 1H, ArH), 7.02-7.00 (d, J = 8.8 Hz, 1H,
ArH), 6.76 (s, 2H, ArH), 6.66 (s, 1H, thiazolidinone-CH), 4.06-4.03 (dd, J = 1.2 Hz, 1H,
thiazolidinone-CH2(HA)), 3.88-3.85 (m, 1H, thiazolidinone-CH2(HB)), 3.76 (s, 3H, OCH3),
3.73 (s, 12H, OCH3), 3.64 (s, 2H, benzyl-CH2), 3.26-3.23 (m, 4H, piperazine-CH2), 3.04-3.02
(m, 4H, piperazine-CH2).13C NMR (100 MHz, DMSO-d6) : δ 170.7, 166.2, 158.7, 152.1,
144.6, 142.6, 137.7, 136.0, 134.1, 132.6, 131.8, 130.3, 129.6, 125.1, 120.2, 118.6, 112.3,
103.6, 64.3, 62.5, 60.8, 56.2, 52.0, 48.7, 34.1. HRMS (ESI) m/z Calcd for C31H36N4O7SNa
[M+Na]+ 631.7051, found 631.7061.
4.1.6.3.
Methyl
3-methoxy-4-(3-(5-(4-(3-nitrobenzyl)piperazin-1-yl)pyridin-2-yl)-4-
oxothiazolidin-2-yl)benzoate (9c)
Yield: 70%; Pale yellow gummy solid; IR γmax (nujol) 3037, 1739, 1652, 1593, 1541, 1452,
1389, 1378, 1363, 1245, 1165, 1065 cm-1; 1H NMR (400 MHz, DMSO-d6) : δ 8.01-8.00 (d, J
8

ACCEPTED MANUSCRIPT
= 2.8 Hz, 1H, ArH), 7.98-7.96 (d, J = 9.2 Hz, 1H, ArH), 7.88-7.86 (m, 1H, ArH), 7.84 (s, 1H,
ArH), 7.74-7.71 (m, 1H, ArH), 7.62-7.60 (d, J = 8.2 Hz, 1H, ArH), 7.57-7.56 (d, J = 2.4 Hz,
1H, ArH), 7.45-7.42 (dd, J = 2.4 Hz, 1H, ArH), 7.37-7.34 (dd, J = 3.2 Hz, 1H, ArH), 7.00-
6.98 (d, J = 8.8 Hz, 1H, ArH), 6.72 (s, 1H, thiazolidinone-CH), 4.04-4.01 (dd, J = 1.4 Hz,
1H, thiazolidinone-CH2(HA)), 3.84-3.81 (m, 1H, thiazolidinone-CH2(HB)), 3.74 (s, 3H,
OCH3), 3.72 (s, 3H, OCH3), 3.63 (s, 2H, benzyl-CH2), 3.36-3.33 (m, 4H, piperazine-CH2),
3.01-2.99 (m, 4H, piperazine-CH2). 13C NMR (100 MHz, DMSO-d6) : δ 170.9, 166.2, 158.6,
147.9, 144.6, 142.6, 136.8, 136.0, 135.1, 134.3, 132.5, 131.7, 130.2, 129.3, 125.2, 122.4,
120.1, 118.8, 112.0, 63.9, 62.6, 56.2, 52.1, 48.7, 34.1. HRMS (ESI) m/z Calcd for
C28H29N5O6SNa [M+Na]+ 586.6248, found 586.6259.
4.1.6.4.
Methyl
4-(3-(5-(4-(3,4-dichlorobenzyl)piperazin-1-yl)pyridin-2-yl)-4-
oxothiazolidin-2-yl)-3-methoxybenzoate (9d)
Yield: 73%; Pale brown gummy solid; IR γmax (nujol) 3048, 1740, 1658, 1588, 1451, 1392,
1
1369, 1252, 1178, 1060, 786 cm-1; H NMR (400 MHz, DMSO-d6) : δ 8.00-7.99 (d, J = 2.6
Hz, 1H, ArH), 7.97-7.95 (d, J = 8.8 Hz, 1H, ArH), 7.74-7.72 (d, J = 8.0 Hz, 1H, ArH), 7.58-
7.57 (d, J = 2.2 Hz, 1H, ArH), 7.43-7.41 (dd, J = 2.4 Hz, 1H, ArH), 7.36-7.33 (dd, J = 3.2Hz,
1H, ArH), 7.31 (s, 1H, ArH), 7.17-7.15 (d, J = 8.0 Hz, 1H, ArH), 6.99-6.97 (d, J = 8.8 Hz,
1H, ArH), 6.73 (s, 1H, thiazolidinone-CH), 4.05-4.02 (dd, J = 1.2 Hz, 1H, thiazolidinone-
CH2(HA)), 3.83-3.80 (m, 1H, thiazolidinone-CH2(HB)), 3.75 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 3.64 (s, 2H, benzyl-CH2), 3.38-3.35 (m, 4H, piperazine-CH2), 3.03-3.00 (m, 4H,
piperazine-CH2). 13C NMR (100 MHz, DMSO-d6) : δ 170.9, 166.2, 158.7, 144.9, 142.7,
136.1, 135.2, 134.4, 132.5, 131.8, 131.6, 130.1, 129.8, 129.0, 128.6, 125.2, 120.2, 118.6,
112.3, 63.7, 62.6, 56.1, 51.8, 48.8, 34.3. HRMS (ESI) m/z Calcd for C28H28Cl2N4O4SNa
[M+Na] + 610.5173, found 610.5182.
4.2. Biology
The human chronic myelogenous leukemia (CML) cell line, K562, human T cell
lymphoblast-like cell line Jurkat and B cell leukemia cell line Nalm6 were selected for the
purpose of preliminary anticancer screening of newly synthesized compounds. To assess the
cytotoxicity, trypan blue dye exclusion assays, MTT assay were employed as described
earlier [22]. Further, cell cycle analysis, mitochondrial membrane potential assay were also
performed in order to understand the mode of cell death after treatment with compound 5
[23,24].
4.2.1. Cell lines and culture
Human cell lines, K562, Jurkat (T-cell leukemia) were purchased from National Center for
Cell Science, Pune, India. Cells were grown in RPMI 1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 100 U/mL of Penicillin, and 100 ꢀg of
streptomycin/mL and incubated at 370C in a humidified atmosphere containing 5% CO2.
4.2.2. Trypan blue dye exclusion assay:
9

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The effect of 5, 8a-f and 9a-d on cell viability of Nalm6 cells was determined by Trypan blue
dye exclusion assay [25]. Nalm6 cells were seeded at a density of 1x 105 cells /ml, cultured
for 24 h and synthesized compounds were added at a concentration of 5, 10 and 20 µM.
DMSO (Sigma Aldrich, USA) treated cells were used as vehicle control. Cells were
harvested after 48 h and 72 h time intervals and suspended in 0.4% Trypan blue (Sigma
Aldrich, USA) and the viable cells were counted using haemocytometer. Experiments were
repeated at least 3 times and the values obtained were plotted against different time points.
(Fig. 2 A, C, E)
4.2.3. MTT assay
Cell proliferation was further assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay which is based on the ability of viable cells to metabolize a
yellow tetrazolium salt to violet formazan. Exponentially growing Jurkat, K562 and Nalm6
cells (5 × 104 cells/well) were plated 24 well plate and incubated with 5, 10 and 20 ꢀM of
compounds 5, 8a–f and 9a-d, cells were harvested after 48 h and 72 h of treatment and
incubated with MTT (0.5 mg/mL) at 370C in 96 well plate. The blue MTT formazan
precipitate was then solubilized in detergent (50% final concentration of N, N-
dimethylformamide and 10% of sodium dodecyl sulfate). Absorbance was measured at
570 nm using ELISA plate reader. The mean absorbance of culture medium was used as the
blank and was subtracted. All measurements were performed in triplicate and each
experiment was repeated at least three times.
4.2.4. Cell cycle analysis using Fluorescent activated cell sorter (FACS) analysis
Cellular DNA content of Nalm6 cells were treated with different concentration of 5 treated
with 5, 10 and 20 ꢀM (Approximately 0.75 x 105 cells/ml), cells were harvested after 48 h,
washed with PBS, RNase (50 µg/ml) (Sigma Aldrich, USA) treatment was given and finally
stained with propidium iodide [26] (Sigma Aldrich, USA) and subjected to flow cytometry
(FACS Verse, BD Biosciences, USA) using Cell Quest Pro Software, excitation 488nm laser
and emission at 560/670nm. A minimum of 10,000 cells were recorded per sample and
histograms were analyzed with Flowing Software (Version 2.5).
4.2.5. Mitochondrial membrane potential assay
Changes of mitochondrial trans membrane potential (∆ψm) were analyzed by flow cytometry
using a sensitive dye called JC-1(5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo
carbocyanine iodide (Calbiochem, USA) [23]. Nalm6 cells were treated with compound 5 (5,
10 and 20 µM), cells were harvested after 48 h and incubated with JC-1 (0.5 ꢀM) at 370C for
30 min. Finally, cells were washed with 1X PBS and the cells were subjected to flow
cytometric analysis, using Cell Quest Pro software an excitation at 488nm laser and emission
at 530/630, 580/610 nm. 10,000 cells were acquired per sample and 2, 4-Dinitrophenol (2, 4-
DNP) used as positive control. Results were analyzed in WinMDI 2.9 software and data were
presented.
4.2.6. Statistical analysis:
10

ACCEPTED MANUSCRIPT
Statistical analysis was done by using Graph Pad software prism 5.1, values were expressed
as mean ± SEM for samples and statistical analysis was performed. One-way ANOVA
followed by Dunnett test, in each case experimental samples were compared with control and
significance was determined. The values were considered as statistically significant, if the p-
value was equal to or less than 0.05.
Acknowledgments
We thank Dr. Mrinal Srivastava and Ms. Vidya Gopalakrishnan for help in cell culture
experiments. The authors are grateful to the IFCPAR, CSIR, DST, DST-JSPS, DST
sponsored Indo-South Africa and Indo-Korea Projects, Govt. of India for financial support to
K.S.R. for the project vide NO.IFC/4303-1/2010-11 Dated 22-12-2010. No. 01 (2434) /10/
EMR-II Dated 28.12.2010. DST NO: F.NO.SR/SO/HS-006/2010 (G) Dated 29.08.2011.
DST/INT/JAP/P-79/09 Dated 20-05-2009. DST/INT/SA/P-05/2011-12, Dated 10-01-2012.
No. INT/indo-Korea/122/2011-12, Dated 13.09.2011.
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Table-1. Derivatives of 4-thiazolidinone 8a-f and 9a-d
Entry Compound
R
Yield (%)a
1
2
8a
8b
88
78
82
3
4
8c
8d
77
5
6
8e
8f
87
78
7
8
9a
9b
74
71
13

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9
9c
9d
70
73
O2N
10
a Isolated yield based on intermediate 5
Table- 2. The antiproliferative effects of thiozalidinone-, pyridine- and piperazine- based
conjugates (5, 8a-f and 9a-f) in Jurkat, K562 and Nalm6 cells
Compound
Cell line (IC50 in µM)
Jurkat
19.29
45.24
53.96
>100
>100
52.92
78.26
>100
40.96
37.44
60.99
K562
15.24
54.52
65.21
53.54
>100
65.58
>100
>100
>100
68.69
>100
Nalm6
9.71
5
8a
29.40
>100
>100
27.39
48.44
>100
27.34
>100
>100
>100
0.0041
8b
8c
8d
8e
8f
9a
9b
9c
9d
Paclitaxel
0.0045 0.0055
14

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Figure captions
Fig 1: Structure of lead compounds belongs to 4-thiazolidinone.
Fig 2: Evaluation of cytotoxicity following addition of (5, 8a-f and 9a-9d) on Nalm6 cells. A, C
and E shows trypan blue assay for compounds 5, 8a-f and 9a-9d respectively, after 48 h and 72 h
of treatment (5, 10, 20 µM). B, D and F MTT assay after addition of 5, 8a-f and 9a-9d. In all
panels “C” stands for DMSO treated vehicle control.
Fig 3: Cell cycle analysis of Nalm6 cells following 5 treatment: Nalm6 cells treated with 5 (5, 10
and 20 µM) for 48h, harvested and stained with propidium iodide and subjected to flow
cytometry. (A) Histogram obtained after FACS analysis for Nalm6 cells. (B) Bar graph shows
the percentage of cells in the subG1, G1, S and G2/M phases of the cell cycle. The data
presented is derived from two independent experiments and error bars indicated. For each
samples 10,000 cells were used for cell sorting
.
Fig 4: Detection of loss of mitochondrial membrane potential followed by 5 treatment. Nalm6
cells treated with 5 (5, 10 and 20 µM) for 48h, harvested and stained with JC-1 dye and analyzed
by flow cytometry. 2, 4- DNP treated Nalm6 cells were served as positive control. (A) Density
plot representing JC-1 stained cells at different concentration of 5. (B) Bar diagram showing
ratio of red versus green fluorescent cells.
15

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Figures
O
S
N
N
O
S
O
N
O
NH
O
Cl
Etozoline
Ralitoline
Fig 1: Structure of lead compounds belongs to 4-thiazolidinone.
16

ACCEPTED MANUSCRIPT
Scheme-1
Reagents and conditions : (a) 1-Boc-piperazine, N-methyl pyrrolidone, 1200C, 3h; (b) 10%
palladium on carbon, ethanol, H2, 2h; (c) 4-formyl-3-methoxy-benzoic acid methyl ester,
Mercaptoacetic acid, T3P®, EtOAc, 00C-RT, 3h; (d) Dioxane, 4M HCl, RT, 2h.
Scheme-2
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ACCEPTED MANUSCRIPT
A
B
5
8a
8b
8c
1.0
0.8
0.6
0.4
0.2
0.0
1.0
1.0
1.0
48h
72h
48h
72h
48h
72h
48h
72h
0.8
0.6
0.4
0.2
0.0
0.8
0.8
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
**
**
***
***
***
***
Concentration (µM
)
Concentration (µM)
Concentration (µM)
Concentration (µM)
5
8a
8b
8c
150
100
50
150
100
50
150
48h
72h
150
100
50
48h
72h
48h
72h
48h
72h
100
50
0
*
*
**
***
0
0
0
Concentration (µM)
Concentration (µM)
Concentration (µM
)
Concentration (µM)
C
8d
8e
8f
9a
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
48h
72h
48h
72h
48h
72h
48h
72h
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
D
8d
8e
8f
9a
150
150
100
50
150
100
50
150
100
50
48h
72h
48h
72h
48h
72h
48h
72h
100
50
0
0
0
0
Concentration (µM
)
Concentration (µM
)
Concentration (µM
)
Concentration (µM)
E
F
9b
9c
9d
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
48h
72h
48h
72h
48h
72h
Concentration (µM)
Concentration (µM)
Concentration (µM)
9b
9c
9d
48h
72h
150
100
50
150
100
50
48h
72h
150
100
50
48h
72h
0
0
0
Concentration (µM
)
Concentration (µM)
Concentration (µM
)
Fig 2: Evaluation of cytotoxicity following addition of (5, 8a-f and 9a-9d) on Nalm6 cells. A, C
and E shows trypan blue assay for compounds 5, 8a-f and 9a-9d respectively, after 48 h and 72 h
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ACCEPTED MANUSCRIPT
of treatment (5, 10, 20 µM). B, D and F MTT assay after addition of 5, 8a-f and 9a-9d. In all
panels “C” stands for DMSO treated vehicle control.
Fig 3: Cell cycle analysis of Nalm6 cells following 5 treatment: Nalm6 cells treated with 5 (5, 10
and 20 µM) for 48h, harvested and stained with propidium iodide and subjected to flow
cytometry. (A) Histogram obtained after FACS analysis for Nalm6 cells. (B) Bar graph shows
the percentage of cells in the subG1, G1, S and G2/M phases of the cell cycle. The data
presented is derived from two independent experiments and error bars indicated. For each
samples 10,000 cells were used for cell sorting
.
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A
2.4-DNP
5 μM
Cells + Dye
B
10 μM
20 μM
Red
150
100
50
Green
0
C
5
10
20
2 4-DNP
Concentration (µM
)
Fig 4: Detection of loss of mitochondrial membrane potential followed by 5 treatment. Nalm6
cells treated with 5 (5, 10 and 20 µM) for 48h, harvested and stained with JC-1 dye and analyzed
by flow cytometry. 2, 4- DNP treated Nalm6 cells were served as positive control. (A) Density
plot representing JC-1 stained cells at different concentration of 5. (B) Bar diagram showing
ratio of red versus green fluorescent cells.
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Highlights
ꢀ Synthesis and characterization of novel series of 4-thiazolidinone-, pyridine- and
piperazine- based conjugates.
ꢀ All the synthesized compounds were screened for their anticancer activity against human
leukemic cells.
ꢀ Different assays were performed to investigate their potency to induce apoptosis in
leukemic cells.
ꢀ Compound 5 displayed potent activity against Nalm6 cell line.

ACCEPTED MANUSCRIPT
1H NMR and13C NMR spectra of compounds 8b and 8e
1H NMR spectra of compound 8b
1

ACCEPTED MANUSCRIPT
1H NMR spectra of compound 8b extended (aliphatic region)
2

ACCEPTED MANUSCRIPT
1H NMR spectra of compound 8b extended (aromatic region)
3

ACCEPTED MANUSCRIPT
13C NMR spectra of compound 8b
4

ACCEPTED MANUSCRIPT
13C NMR spectra of compound 8b extended
5

ACCEPTED MANUSCRIPT
13C NMR spectra of compound 8b extended
6

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1H NMR spectra of compound 8e
7