Antiproliferative and tumor inhibitory studies of 2,3 disubstituted 4-thiazolidinone derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.06.069

Abstract 4-Thiazolidinone derivatives were synthesized using T3P-DMSO media as a cyclodehydrating agent. All the molecules were tested for their cytotoxicity against leukemic cell lines. The compound 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)thiazolidin-4-one (4e) with electron donating substituent at para position of phenyl ring displayed considerable cytotoxicity against Reh and Nalm6 cells with an IC₅₀ value of 11.9 and 13.5 μM, respectively. Furthermore, the compound 4e tested for tumor regression studies induced by EAC in Swiss albino mouse. Both in vitro and in vivo results suggested significant antiproliferative activity of compound 4e in Reh cells and mouse tumor tissue treated with compound 4e showed multifocal areas of necrosis and numerous number of apoptotic cells.

Full text of DOI:10.1016/j.bmcl.2015.06.069

Accepted Manuscript
Antiproliferative and tumor inhibitory studies of 2,3 disubstituted 4-thiazolidi-
none derivatives
Kothanahally S. Sharath Kumar, Ananda Hanumappa, Maruthai Vetrivel,
Mahesh Hegde, Yarabhally R. Girish, Thinnali R. Byregowda, Suguna rao,
Sathees C. Raghavan, Kanchugarakoppal S. Rangappa
PII:
S0960-894X(15)00662-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.069
BMCL 22859
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 March 2015
5 June 2015
17 June 2015
Please cite this article as: Sharath Kumar, K.S., Hanumappa, A., Vetrivel, M., Hegde, M., Girish, Y.R., Byregowda,
T.R., rao, S., Raghavan, S.C., Rangappa, K.S., Antiproliferative and tumor inhibitory studies of 2,3 disubstituted
4-thiazolidinone derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/
j.bmcl.2015.06.069
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Antiproliferative and tumor inhibitory studies of 2,3 disubstituted 4-thiazolidinone
derivatives
Kothanahally S. Sharath Kumar,a Ananda Hanumappa,a Maruthai Vetrivel,b Mahesh Hegde,a
Yarabhally R. Girish,a Thinnali R. Byregowda,b Suguna rao,b Sathees C. Raghavan,c
Kanchugarakoppal S. Rangappa a *
aDepartment of Studies in Chemistry, Manasagangotri, University of Mysore, Mysuru-570006.
bDepartment of Veterinary Pathology, Veterinary College, Hebbal, Bengaluru-560024.
cDepartment of Biochemistry, Indian Institute of Science, Bengaluru-560012.
*Corresponding Author:
Tel. No.: +91-821-2419661 Fax: +91-821-2500846
*E-mail: rangappaks@gmail.com/rangappaks@chemistry.uni-mysore.ac.in
Abstract
4-thiazolidinone derivatives were synthesized using T3P®-DMSO media as a cyclodehydrating
agent. All the molecules were tested for their cytotoxicity against leukemic cell lines. The
compound 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)thiazolidin-4-one (4e) with electron
donating substituent at para position of phenyl ring displayed considerable cytotoxicity against
Reh and Nalm6 cells with an IC50 value of 11.9 and 13.5 µM respectively. Furthermore, the
compound 4e tested for tumor regression studies induced by EAC in Swiss albino mouse. Both
in vitro and in vivo results suggested significant antiproliferative activity of compound 4e in Reh
cells and mouse tumor tissue treated with compound 4e showed multifocal areas of necrosis and
numerous number of apoptotic cells.
Keywords
4-thiazolidinone, T3P, Reh, EAC, Tumor regression.
Small molecule libraries have become an important source for the discovery of new drug
molecules. In particular heterocycles with azolidinone ring play an important role in bio-organic
and medicinal chemistry, especially reports concerning 4-thiazolidinone-containing heterocyclic
compounds have gradually increased because of their pharmaceutical applications.1,2
4-thiazolidinone has been considered as a magic moiety (wonder nucleus) and represents an
important class of heterocyclic compounds with a wide spread biological applications.
Thiazolidinone derivatives have been investigated for a range of pharmacologic indications such

as
anti-viral,3
anti-convulsant,4
cardiovascular,5
anti-inflammatory,6
antidiabetic,7
antihyperlipidemic,8 antimicrobial,9 antituberculosis,10 antiparasitis,11 FSH agonist,12
antiarthritic13 and antidiarrhoeal14 activity. In addition, 4-thiazolidinones are known to possess
good activity against different forms of cancer like, breast cancer (MCF-7),15 JNK stimulating
phosphatase-1 (JSP-1),16 tumor necrosis factor (TNFα),17 antiapoptotic biocomplex (Bcl-XL-
BH3),18 integrin αvβ3 receptor,19 colon cancer (HT29)20 and CDK1/Cyclin B inhibiton.21 But
their anti-leukemic effects have been less widely documented.22
From the past few years we have been engaged in the development of new synthetic routes23 for
the synthesis of various bioactive heterocyclic compounds and recently we have reported a novel
propylphosphonic anhydride - DMSO mediated one pot synthesis of 4-thiazolidinone derivatives
from variety of primary and secondary alcohols (Scheme 1).24 That method provides an excellent
protocol for the synthesis of 2,3 disubstituted 4-thiazolidinone derivatives in one pot operation
with excellent yields.25 In continuation of our research work towards the identification of new
synthetic compounds as chemotherapeutic agents,26 we have screened all ten 4-thiazolidinone
derivatives (4a-4j) (Table 1) which were synthesized from different aryl/heteroaryl/acyclic
alcohols and aryl/heteroaryl amines bearing electron donating or electron withdrawing group
with excellent yields. All the synthesized compounds (4a-4j) were characterized by elemental
analysis, mass, IR and NMR spectroscopy. The IR spectra show a characteristic peak at 1720-
1740 cm-1 corresponds to carbonyl group of thiazolidinone. In further, the 1H NMR spectra of all
the compounds showed a multiplet at δ 3.83-4.05 due to the diastereotopic C5H2 protons of 4-
thiazolidinone ring and in the 13C NMR the carbonyl carbon C4 of the thiazolidinone ring
appeared at δ 170.0-171.9.
Scheme: 1 Synthesis of 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)thiazolidin-4-one(4e)

Table 1. Chemical structures of compounds 4a-4j

Proliferation rate of Reh and Nalm6 cells in presence of different concentration of
thiozolidinone derivatives (4a-4j) were tested at 48 h time point using MTT assay.27 Results
showed significant antiproliferative effect of thiozolidinone derivatives on Reh cells compared to
Nalm6 cells. Importantly compound 4e showed potent antiproliferative activity on Reh cells
followed by 4d, 4j and 4f. 4e exhibited IC50 value of 11.9 μM on Reh cells and 13.5 μM on
Nalm6 cells (Table 2). Since 4e showed significant antiproliferative activity on Reh cells, it was
chosen for further mechanistic studies. Significant antiproliferative activity of compound 4e
indicated aberrant cell cycle distribution of compound 4e treated Reh cells. Results indicated that
compound 4e was able to accumulate cells in SubG1 phase of cell cycle in concentration
dependent manner (Fig. 1), suggested that, compound 4e can induce cell death in Reh cells.
Decreased mitochondrial membrane potential is one of the important mechanisms for the
mitochondrial mediated apoptotic cell death, therefore mitochondrial membrane potential of
compound 4e treated Reh cells was investigated.28 Interestingly, increased green fluorescence of
JC-1 dye in a dose dependent manner of compound 4e treatment was observed which indicated
the lowering of the mitochondrial membrane potential. Our results suggest that compound 4e
could decrease mitochondrial membrane potential and induce cell death in leukemic cell line
through apoptotic process (Fig. 2). The compound 4e treatment on mice bearing EAC resulted in
considerable reduction in tumor volume without affecting the function of other organs (Fig. 3).
Besides, the histological evaluation were showed that the morphology and cellular construction
of the organs was unaltered when treated with the compound 4e. Despite existence of
proliferative tumor cells, the tumor tissue microscopically revealed multifocal areas of necrosis
and innumerable number of apoptotic cells (Fig. 4) following treatment with compound 4e.
In conclusion, we have found 2,3 disubstituted 4-thiazolidinone based small molecules are
biologically active against human cancer cells ex vivo. In vivo data suggested tumor regression
property of compound 4e in EAC tumor model. Further studies on synthesized derivatives would
help to identify novel thiazolidinone compounds as potential anticancer therapeutics.

Table 2. Antiproliferative activity of 4-thiazolidinones
Name of the cell line
(IC50 in µM)
Nalm6
>100
49.8
Compound
Reh
>100
>100
>100
29.6
HEK 293T
N.T
4a
4b
4c
4d
4e
4f
N.T
93.4
N.T
42.6
N.T
11.9
69.4
13.2
>100
82.9
>80
N.T
4g
4h
4i
>100
>100
>100
41.4
N.T
>100
>100
>100
N.T
N.T
4j
N.T
*N.T = Not tested
A
Control
5 µM
10 µM
20 µM
B
100
80
60
40
20
0
G1
S
G2/M
SubG1
C
5
10
20
Concentration (M)
Figure 1. Cell cycle analysis of compound 4e treated Reh cells. Compound 4e treated (5, 10 and
20 μM, for 48 h) Reh cells were subjected to cell cycle analysis, DMSO treated cells were used
as vehicle control. A) Histograms representing cell cycle distribution of control and compound
4e treated cells. B) Bar diagram representing percentage of cell population in different phase of
the cell cycle in control and compound 4e treated Reh cells.

A
Cells+JC1
5 µM
10 µM
B
100
80
60
40
20
0
R1
R2
C
5
10
20
2-4.DNP
20 µM
2,4.DNP
Concentration (M)
Figure 2. Analysis of mitochondrial membrane potential of compound 4e treated Reh cells.
Compound 4e treated (5, 10 and 20 μM, for 48 h) Reh cells were subjected to JC-1 staining to
analyze mitochondrial membrane potential. DMSO treated cells were used as control and 2, 4-
DNP treated cells were used as positive control. A) Dot plots representing distribution of red and
green fluorescing Reh cells in control and compound 4e treated cases. B) Bar diagram
representing percentage positive cells for red (R1) and green fluorescent (R2).

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
25mg/kg
0
2
4
6
8
10
12
14
Days
Figure 3. Tumor volume following compound 4e treatment in mice.
Control
Compound 4e
A
B
4X
4X
C
D
10X
10X
Miitotiic cellll
Apoptotiic cellll
Miitotiic cellll
Apoptotiic cellll
Figure 4. A) Section of tumor mass showing solid arrangement of neoplastic cells (4X). C)
Section of tumor mass showing solid arrangement of neoplastic cells (10X). B) Section of tumor
mass treated with compound 4e showing multifocal areas of necrosis and apoptotic cells (4X) D)
Section of tumor mass treated with compound 4e showing multifocal areas of necrosis and
apoptotic cells (10X).
Acknowledgments:
The authors are grateful to the DST-IFCPAR & CSIR 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. A.H. thanks ICMR for SRF (SRF No 45/54/2013-
PHA/BMS, Dated: 08.12.2014) for research fellowship.

References
1.
2.
Kaminskyy, D.; Zimenkovsky, B.; Lesyk, R. Eur. J. Med. Chem. 2009, 44 (9), 3627-36.
Sharath Kumar, K. S.; Mohan, C. D.; Jagadish, S.; Rakesh, K. S.; Ananda, H.; Basappa.;
Rangappa, K. S. Asian J. Pharm. Clin. Res. 2015, 8 (1), 142-148.
3. Barreca, M. L.; Chimirri, A.; De Luca, L.; Monforte, A. M.; Monforte, P.; Rao, A.;
Zappala, M.; Balzarini, J.; De Clercq, E.; Pannecouque, C.; Witvrouw, M. Bioorg. Med. Chem.
lett. 2001, 11 (13), 1793-6.
4.
Amin, K. M.; Rahman, D. E.; Al-Eryani, Y. A. Bioorg. Med. Chem. 2008, 16 (10), 5377-
88.
5.
Bhandari, S. V.; Bothara, K. G.; Patil, A. A.; Chitre, T. S.; Sarkate, A. P.; Gore, S. T.;
Dangre, S. C.; Khachane, C. V. Bioorg. Med. Chem. 2009, 17 (1), 390-400.
6. Ottana, R.; Mazzon, E.; Dugo, L.; Monforte, F.; Maccari, R.; Sautebin, L.; De Luca, G.;
Vigorita, M. G.; Alcaro, S.; Ortuso, F.; Caputi, A. P.; Cuzzocrea, S. Eur. J. Pharmacology.
2002, 448 (1), 71-80.
7.
Ottana, R.; Maccari, R.; Giglio, M.; Del Corso, A.; Cappiello, M.; Mura, U.; Cosconati,
S.; Marinelli, L.; Novellino, E.; Sartini, S.; La Motta, C.; Da Settimo, F. Eur. J. Med. Chem.
2011, 46 (7), 2797-806.
8.
9.
Joy, M. J.; Jacob, N.; Kutty, N. G. Indian Drugs. 2005, (42), 47.
Felise, H. B.; Nguyen, H. V.; Pfuetzner, R. A.; Barry, K. C.; Jackson, S. R.; Blanc, M. P.;
Bronstein, P. A.; Kline, T.; Miller, S. I. Cell host & microbe. 2008, 4 (4), 325-36.
10. Aridoss, G.; Amirthaganesan, S.; Kim, M. S.; Kim, J. T.; Jeong, Y. T., Eur. J. Med.
Chem. 2009, 44 (10), 4199-210.
11.
12.
Carlson, E. E.; May, J. F.; Kiessling, L. L. Chemistry & biology 2006, 13 (8), 825-37.
Wrobel, J.; Jetter, J.; Kao, W.; Rogers, J.; Di, L.; Chi, J.; Perez, M. C.; Chen, G. C.; Shen,
E. S. Bioorg. Med. Chem. 2006, 14 (16), 5729-41.
13. Panico, A. M.; Vicini, P.; Geronikaki, A.; Incerti, M.; Cardile, V.; Crasci, L.; Messina,
R.; Ronsisvalle, S. Bioorg. Chemistry. 2011, 39 (1), 48-52.
14. Mazzoni, O.; di Bosco, A. M.; Grieco, P.; Novellino, E.; Bertamino, A.; Borrelli, F.;
Capasso, R.; Diurno, M. V. Chem. Biol. Drug. Des. 2006, 67 (6), 432-6.
15. Huang, J. W.; Shiau, C. W.; Yang, J.; Wang, D. S.; Chiu, H. C.; Chen, C. Y.; Chen, C. S.
J. Med. Chem. 2006, 49 (15), 4684-9.
16.
17.
Cutshall, N. S.; O'Day, C.; Prezhdo, M. Bioorg. Med. Chem. lett. 2005, 15 (14), 3374-9.
Carter, P. H.; Scherle, P. A.; Muckelbauer, J. A.; Voss, M. E.; Liu, R.-Q.; Thompson, L.
A.; Tebben, A. J.; Solomon, K. A.; Lo, Y. C.; Li, Z.; Strzemienski, P.; Yang, G.; Falahatpisheh,
N.; Xu, M.; Wu, Z.; Farrow, N. A.; Ramnarayan, K.; Wang, J.; Rideout, D.; Yalamoori, V.;
Domaille, P.; Underwood, D. J.; Trzaskos, J. M.; Friedman, S. M.; Newton, R. C.; Decicco, C. P.
Proc. Natl. Acad. Sci. USA. 2001, 98 (21), 11879-11884.
18.
Yuan, J. Nat. Cell Biol. 2001, 3 (2), 173-82.
19. Dayam, R.; Aiello, F.; Deng, J.; Wu, Y.; Garofalo, A.; Chen, X.; Neamati, N. J. Med.
Chem. 2006, 49 (15), 4526-34.
20. Ottana, R.; Carotti, S.; Maccari, R.; Landini, I.; Chiricosta, G.; Caciagli, B.; Vigorita, M.
G.; Mini, E. Bioorg. Med. Chem. lett. 2005, 15 (17), 3930-3.
21. Chen, S.; Chen, L.; Le, N. T.; Zhao, C.; Sidduri, A.; Lou, J. P.; Michoud, C.; Portland, L.;
Degterev, A.; Lugovskoy, A.; Cardone, M.; Mulley, B.; Wagner, G.; Mitchison, T.;
Jackson, N.; Liu, J. J.; Konzelmann, F.; Chi, F.; Tovar, C.; Xiang, Q.; Chen, Y.; Wen, Y.;
Vassilev, L. T. Bioorg. Med. Chem. lett. 2007, 17 (8), 2134-8.

22.
Med. Chem. lett. 2013, 23 (23), 6292-5.
23. (a) Narasimhamurthy, K. H.; Chandrappa, S.; Sharath Kumar, K. S.; Swaroop, T. R.;
Suresh, N.; Nagesh, H. N.; Sekhar, K. V.; Kumar, A.; Shirazi, A. N.; Parang, K. Bioorg.
Rangappa, K. S. Chem. Lett. 2013, 42, 1073-1075. (b) Narasimhamurthy, K. H.; Chandrappa, S.;
Sharath Kumar, K. S.; Harsha, K. B.; Ananda, H.; Rangappa, K. S. RSC Adv. 2014, 4, 34479. (c)
Girish, Y. R.; Sharath Kumar, K. S.; Umashankar, M.; Lokanath, N. K.; Rangappa, K. S.;
Shashikanth, S. RSC Adv. 2014, 4, 55800. (d) Lingaraju, G. S.; Swaroop, T. R.; Vinayaka, A. C.;
Sharath Kumar, K. S.; Sadashiva, M. P.; Rangappa, K. S. Synthesis, 2012, 44, 1373-1379.
24.
Rangappa, K. S. Tetrahedron Lett. 2012, 53 (42), 5619-5623.
25. Synthesis of 3-(3,4-dimethoxyphenyl)-2-(pyridin-3-yl)thiazolidin-4-one (4a): To a
Sharath Kumar, K. S.; Swaroop, T. R.; Harsha, K. B.; Narasimhamurthy, K. H.;
solution of 3-Pyridinemethanol 1a (1.1 mmol) in a mixture of solvents EtOAc: DMSO (4ml:
2ml), was added T3P® (2.5 mmol, 50% solution in ethyl acetate) at 00C, and the resulting
mixture was stirred at room temperature for 1-2h under nitrogen atmosphere. 3,4-
dimethoxyaniline 2a (1.0 mmol) and thioglycolic acid (1.0 mmol) were added once and stirred
further for 1-3 h at room temperature. 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
removed under reduced pressure to afford a crude product which was purified by column
chromatography using hexane: ethyl acetate mixture (8:2) as an eluent.
26.
Rangappa, K. S. Eur. J. Med. Chem. 2014, 81, 341-9.
27. Kavitha, C. V.; Nambiar, M.; Ananda Kumar, C. S.; Choudhary, B.; Muniyappa, K.;
Rangappa, K. S.; Raghavan, S. C. Biochem. Pharmacol. 2009, 77, 348.
28. Hegde, M.; Karki, S. S.; Thomas E. Kumar, S.; Panjamurthy, K.; Ranganatha, S. R.;
Rangappa, K. S.; Choudhary, B.; Raghavan, S. C. PloS One. 2012, 7, 43632.
Sharath Kumar, K. S.; Ananda H.; Hegde, M.; Narasimhamurthy, K. H.; Raghavan, S. C.;