Design, synthesis and molecular modeling studies of novel thiazolidine-2,4-dione derivatives as potential anti-cancer agents

Article abstract of DOI:10.1016/j.molstruc.2017.10.077

A series of novel thiazolidine-2,4-dione derivatives 4a-x have been designed, synthesized and evaluated for potential anti-cancer activity. The anti-cancer activity of synthesized compounds 4a-x were evaluated against selected human cancer cell line of breast (MCF-7) using sulforhodamine B (SRB) method. Among the synthesized compounds, 4x having 2-cyano phenyl group showed significant cytotoxic activity which is comparable to that of adriamycin as standard anti-cancer drug. The SAR study revealed that the substituted phenyl group on oxadiazole ring attached to thiazolidine-2,4-dione moiety showed significant growth inhibitory activity against MCF-7 cell line. The result of molecular modeling studies showed that compounds 4f, 4o and 4x having similar structural alignment as crystal ligand of protein (PDB code: 4DTK) and exhibited hydrogen bond interaction with amino acid residues LYS67, GLU171, ASP128 and ASP186 of PIM-1 kinase. The results of biological activity and docking study revealed that the presence of electron withdrawing group at 2 position of phenyl ring attached to oxadiazole of thiazolidine-2,4-dione scaffold is crucial for better anti-cancer activity.

Full text of DOI:10.1016/j.molstruc.2017.10.077

Accepted Manuscript
Design, synthesis and molecular modeling studies of novel thiazolidine-2,4-dione
derivatives as potential anti-cancer agents
Vivek Asati, Sanjay Kumar Bharti
PII:
S0022-2860(17)31422-9
10.1016/j.molstruc.2017.10.077
MOLSTR 24448
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 August 2017
Revised Date: 21 October 2017
Accepted Date: 22 October 2017
Please cite this article as: V. Asati, S.K. Bharti, Design, synthesis and molecular modeling studies of
novel thiazolidine-2,4-dione derivatives as potential anti-cancer agents, Journal of Molecular Structure
(2017), doi: 10.1016/j.molstruc.2017.10.077.
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Graphical Abstract
“Design, synthesis and molecular modeling studies of novel thiazolidine-2,4-dione
derivatives as potential anti-cancer agents”
Vivek Asati, Sanjay Kumar Bharti *

ACCEPTED MANUSCRIPT
Design, synthesis and molecular modeling studies of novel thiazolidine-2,4-dione
derivatives as potential anti-cancer agents
Vivek Asati, Sanjay Kumar Bharti*
Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur- 495009, Chhattisgarh (India)
E-mail address: skbharti.ggu@gmail.com
Abstract
A series of novel thiazolidine-2,4-dione derivatives 4a-x have been designed, synthesized and
evaluated for potential anti-cancer activity. The anti-cancer activity of synthesized compounds
4a-x were evaluated against selected human cancer cell line of breast (MCF-7) using
sulforhodamine B (SRB) method. Among the synthesized compounds, 4x having 2-cyano phenyl
group showed significant cytotoxic activity which is comparable to that of adriamycin as
standard anti-cancer drug. The SAR study revealed that the substituted phenyl group on
oxadiazole ring attached to thiazolidine-2,4-dione moiety showed significant growth inhibitory
activity against MCF-7 cell line. The result of molecular modeling studies showed that
compounds 4f, 4o and 4x having similar structural alignment as crystal ligand of protein (PDB
code: 4DTK) and exhibited hydrogen bond interaction with amino acid residues LYS67,
GLU171, ASP128 and ASP186 of PIM-1 kinase. The results of biological activity and docking
study revealed that the presence of electron withdrawing group at 2 position of phenyl ring
attached to oxadiazole of thiazolidine-2,4-dione scaffold is crucial for better anti-cancer activity.
Keywords: thiazolidine-2,4-dione; anti-cancer activity; docking; PIM-1 kinase; MCF-7.
* Corresponding author
E-mail address: skbharti.ggu@gmail.com
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1. Introduction
Cancer is a second leading cause of death after cardiovascular diseases [1]. For the treatment of
cancer, several countries spent billions of money, but it is still an exacerbating problem related to
human health [2]. In the past few decades, many chemotherapeutic strategies for cancer
treatment has been proposed, tested and in some cases implemented [3]. Despite the huge efforts
to combat novel chemotherapeutic strategies for the treatment of different types of cancer, this
disease remains one of the major concerns worldwide. Therefore, there is a desperate need to
develop a drug with potential novel mechanisms to combat this disease. Various scientific
studies in literature have shown the targeted therapy on specific kinase as one of the emerging
strategies in cancer chemotherapy [4-6].
Heterocyclic system becomes an important structural motif to develop potential
chemotherapeutic agents [7,8]. Number of drugs based on the heterocyclic system has been
discovered and used as potential chemotherapeutic agents. Exhaustive literature survey revealed
that thiazolidine-2,4-dione (TZD) and rhodanine analogs have been recognized as the privileged
templates in drug design and discovery [9,10]. Several compounds containing the TZD have
been developed as potential anti-cancer agents [11-16], e.g. GSK1059615, (I), a potent,
reversible, ATP-competitive inhibitor of PI3Kα and compound II which showed potent activity
against Raf/MEK/ERK and PI3K/Akt signaling pathways [17,18]. Yang and Huang et al. have
reported troglitazone/ciglitazone analogs ꢀ2CG (III) and ꢀ2TG (IV), as antiproliferative agents
[19,20]. These derivatives produced para substituted benzylidine ring attached to
thizolidinedione showed important role in anti-cancer activity. Similarly, aplysinopsin (V) [21],
indole-derived 2-aminoimidazoline-4-one, AZD1208 (VI) and SMI-4a (VII) [22,23] have been
reported as potential PIM kinase inhibitors (Fig.1).
Recently, PIM kinases have been extensively studied by researcher for their effective role in cell
cycle progression and growth [24]. They generally found in three different isoforms namely
PIM-1, 2 and 3. These kinases are stimulated by exposure of various cytokines, growth factors
and mutagens [25]. Over expression of PIM-1 has been implicated in a wide variety of cancers of
hematological and epithelial origin, such as bladder, gastric, colorectal and prostate carcinomas
[26,27]. In a study, Malinen et al. have reported the over expression of PIM-1 in MCF-7 breast
cancer cell line. They suggested the expression of PIM- 1 is induced by the response of estradiol
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in MCF-7 cells in which estrogens can contribute to breast cancer cell proliferation and
carcinogenesis [28]. Recently, we have also reported 3D QSAR and molecular docking studies of
thiazolidine-2,4-dione analogs on PIM-1 kinase and found promising results [29]. In
continuation with our ongoing work on thiazolidine-2,4-dione scaffold and PIM-1 as molecular
target, present study was aimed to synthesize and evaluate novel thiazolidine-2,4-dione analogs
as potential anti-cancer agents.
2. Experimental
2.1. Materials and methods
All the reagents and solvents were obtained from S.D. Fine chemicals, Sigma–Aldrich, USA,
CDH (central drug house) and E. Merck India Ltd. Melting point apparatus was used for the
measurement of melting points of all derived compounds by using open glass capillaries and are
uncorrected. Progress of the reactions was monitored by using TLC plates (silica gel G), hexane :
ethyl acetate (3 : 2 and 5 : 5, v/v) and benzene: acetone (9:1, v/v) used as solvent systems. The
spots were detected by spraying of iodine vapors or under UV-light. IR spectra were recorded on
FT-IR, Shimadzu Fourier transform spectrophotometer (model no. 8400S) by using KBr pellets
1
in the range of 4000-400 cm-1. The H NMR and 13C NMR spectra of the synthesized
compounds were recorded on Bruker 400 MHz instrument in solvent (DMSO-d6/CHCl3);
chemical shift (δ) values reported in parts per million (ppm) using tetramethylsilane as internal
reference. The splitting pattern of NMR spectra were abbreviated are as follows: s, singlet; bs,
broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet. The mass spectra were
recorded at m/z values by using LC-MS spectrometer, Model Q-ToF Micro, Waters.
2.2. Synthesis of thiazolidine-2,4-dione (1)
A mixture of chloroacetic acid (47.24 g, 0.5 mol) and thiourea (38.06 g, 0.5 mol) in water (50
ml) were placed in a 250 ml round bottom flask. It was stirred for 15 min then cooled, thus a
white precipitate was obtained. To the mixture, 30 ml of conc. HCl was added slowly by a
dropping funnel. The mixture was refluxed for 10 h at 100˚C. The product thus obtained was
filtered and washed with water for three times and dried. Then the product was purified by
recrystallization with ethyl alcohol. Yield: 90%, m.p. 123-125˚C, Rf = 0.64 [hexane: ethyl
acetate (3:2)]. IR (KBr) υ cm-1 3130 (N-H), 3047 (Ar C-H), 1737 (C=O), 619 (C-S-C).
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2.3. Synthesis of 4-formylbenzohydrazide (2)
To a solution of methyl 4-formylbenzoate (4.0 mmol) in ethanol (15 ml), hydrazine hydrate (3
ml) was added slowly with stirring and refluxed for 15 min. After completion of reaction, more
ethanol (15 ml) was added and refluxed for another 12 h. The reaction mixture was cooled and
extracted into ethyl acetate (25 ml), washed with water (15 ml) and dried over Na2SO4. Yield:
85%, m.p. 172-174˚C, Rf = 0.8 [hexane: ethyl acetate (1:1)]. IR (KBr) υ cm-1 3325, 3178 (N-H),
3049 (Ar C-H), 1651 (C=O), 661 (C-S-C); 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H, -
CHO), 8.25 (s, 1H, -N-H), 7.72 (d, J = 9.3 Hz, 2H, ArH), 7.50 (d, J = 8.0 Hz, 2H, ArH), 2.51 (s,
2H, NH2); 13C NMR (DMSO-d6): 165.78 (C=O), 138.92 (C=O), 137.13, 131.60, 127.12, 124.64
(Ar-C); ESI-MS m/z: 165 [(M + 1)+, (13)], 133 (24), 105 (100), 79 (22), 63 (49).
2.4. Synthesis of 4-((2,4-dioxothiazolidin-5-ylidene)methyl)benzohydrazide (3)
In a 250 ml round bottom flask, piperidine (0.03 ml, 0.3 mmol) was added to a solution of
methyl 4-formylbenzohydrazide (0.217 g 1.45 mmol) and thiazolidine-2,4-dione (0.204 g, 1.74
mmol) in EtOH (15 ml) and then the reaction mixture was refluxed for 24 h. The precipitate
formed after completion of the reaction was filtered through Buchner funnel. The precipitate
obtained was washed with water/ethanol (5:1), and then subjected to silica gel column
chromatography using chloroform/methanol (10:1). Yield: 80%, m.p. 230-232ºC, Rf = 0.8
[hexane: ethyl acetate (1:1)]. IR (KBr) υ cm-1 3290,3221 (N-H), 3059 (Ar C-H), 1726, 1680
(C=O), 628 (C-S-C); 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H, -N-H), 8.43 (s, 1H, =C-H),
7.92 (d, J = 9.3 Hz, 2H, ArH), 7.88 (d, J = 8.0 Hz, 2H, ArH), 5.71 (s, 1H, -NH2); 13C NMR
(DMSO-d6): 169.54 (C=O), 164.17 (C=O), 158.03 (C=O), 133.18 (C-CO), 132.58 (=CH-C),
128.14, 127.41, 127.05, 126.78 (Ar-C); ESI-MS m/z: 264 [(M + 1)+, (13)], 204(60), 133 (100),
127 (9).
2.5. General procedure for the synthesis of 5-(4-(5-phenyl-1,3,4-oxadiazol-2-
yl)benzylidene)thiazolidine-2,4-dione (4a - x)
A mixture of 4-((2,4-dioxothiazolidin-5-ylidene)methyl)benzohydrazide (3) (1 mmol),
aliphatic/aromatic acid (1 mmol) and 10 ml POCl3 were placed in a 250 ml flat bottom flask. The
reaction mixture was stirred continuously at 60-70ºC for 14 h. The progress of reaction was
monitored by TLC [benzene: acetone (9:1)]. After completion of reaction the mixture was
concentrated under reduced pressure, poured on crushed ice and neutralized by sodium
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bicarbonate. The precipitate thus obtained was filtered, washed with cold water, dried and
purified by column chromatography using n-hexane : EtOAc as an eluent.
2.5.1. 5-(4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4a)
IR (KBr) υ cm-1 3188 (N-H), 3057 (Ar C-H), 1697 (C=O), 1546 (C=N), 1492 (C=C) and 1273
(C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H, -N-H), 8.26 (s, 1H, =C-H), 8.15 (d, J =
1.76 Hz, 2H, ArH), 7.95 (d, J = 8.40 Hz, 2H, ArH), 7.80 (d, J = 8.36 Hz, 2H, ArH), 7.63 (t, J =
7.92 Hz, 1H, ArH), 7.47 (t, J = 7.32 Hz, 1H, ArH); 13C NMR (DMSO-d6): 169.31 (C=O),
166.17(C=O), 164.37(C=N), 132.61(=CH-C), 130.70, 129.39, 129.18, 128.59, 128.31, 127.01,
126.77(Ar-C); ESI-MS m/z: 350 [(M + 1)+, (10)], 216 (100), 200 (22), 145 (78), 91 (72). (Fig.
S1a-c supporting information)
2.5.2. 5-(4-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4b)
IR (KBr) υ cm-1 IR (KBr) υ cm-1 3182 (N-H), 3062 (Ar C-H), 1658 (C=O), 1546 (C=C), 1278
(C-O-C) and 756 (C-Cl); 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H, -N-H), 8.23 (s, 1H, =C-
H), 8.14 (d, J = 8.36 Hz, 2H, ArH), 7.94 (d, J = 8.40 Hz, 2H, ArH), 7.66 (d, J = 8.28 Hz, 2H,
ArH), 7.54 (d, J = 5.92 Hz, 2H, ArH); 13C NMR (DMSO-d6): 168.17 (C=O), 166.54(C=O),
164.13(O-C=N), 134.61(C-Cl), 132.61(=CH-C), 130.70, 129.18, 128.91, 127.07, 126.97, 123.95,
123.05 (Ar-C); ESI-MS m/z: 384 [(M + 1)+, (10)], 216 (100), 173 (39), 145 (78), 125 (38). (Fig.
S2a-c supporting information)
2.5.3. 5-(4-(5-(2-amino-3,5-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene) thiazolidine-
2,4-dione (4c)
IR (KBr) υ cm-1 3285, 3235, 3163 (N-H), 3057 (Ar C-H), 1678 (C=O), 1535 (C=C), 1278 (C-O-
1
C) and 748 (C-Cl); H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H, -N-H), 8.61 (s, 1H, =C-H),
8.08 (d, J = 6.52 Hz, 2H, ArH), 7.89 (d, J = 6.44 Hz, 2H, ArH), 7.72 (s, 1H, ArH), 5.22 (s, 2H, -
NH2); 13C NMR (DMSO-d6): 168.27 (C=O), 166.58 (C=O), 164.11(O-C=N), 143.61(C-NH2),
132.87 (ClC-CH-CCl), 132.72(CH-CCl-CH), 132.61(=CH-C), 130.70, 129.18, 129.91, 127.07,
126.97, 123.05, 110.05 (Ar-C), 117.59(CNH2-CCl-CH); ESI-MS m/z: 432 [(M + 1)+, (20)], 216
(86), 171 (100), 145 (45), 138 (17). (Fig. S3a-c supporting information)
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2.5.4. 5-(4-(5-(3-chlorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4d)
IR (KBr) υ cm-1 3187 (N-H), 3032 (Ar C-H), 1697 (C=O), 1546 (C=C), 1278 (C-O-C) and 756
1
(C-Cl); H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, -N-H), 8.26 (s, 1H, =C-H), 8.18 (d, J =
8.92 Hz, 2H, ArH), 7.91 (d, J = 7.16 Hz, 2H, ArH), 7.88 (s, 1H, ArH), 7.61 (d, J = 7.16 Hz, 2H,
ArH), 7.58 (t, J = 7.52 Hz, 1H, ArH), 7.54 (d, J = 9.20 Hz, 2H, ArH); 13C NMR (DMSO-d6):
168.57 (C=O), 166.34 (C=O), 164.23(O-C=N), 163.83(O-C=N), 132.62(=CH-C), 130.81(CH-
CCl-CH), 130.70, 129.50, 129.18, 128.91, 127.89, 127.33, 127.07, 126.97, 125.75, 123.85 (Ar-
C); ESI-MS m/z: 384 [(M + 1)+, (10)], 216 (100), 145 (76), 125 (60). (Fig. S4a-c supporting
information)
2.5.5. 5-(4-(5-(2-iodophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4e)
IR (KBr) υ cm-1 3221 (N-H), 3032 (Ar C-H), 1658 (C=O), 1546 (C=C), 1278 (C-O-C) and 545
1
(C-I); H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H, -N-H), 8.30 (s, 1H, =C-H), 8.06 (d, J =
9.96 Hz, 2H, ArH), 7.94 (d, J = 6.36 Hz, 1H, ArH), 7.92 (d, J = 7.8 Hz, 2H, ArH), 7.87 (d, J =
8.0 Hz, 1H, ArH), 7.62 (t, J = 5.92 Hz, 1H, ArH), 7.57 (t, J = 8.88 Hz, 1H, ArH); 13C NMR
(DMSO-d6): 169.32 (C=O), 166.25 (C=O), 164.84(O-C=N), 164.13(O-C=N), 132.52(=CH-C),
139.37, 130.87, 130.43, 129.67, 129.08127.88, 127.62, 127.37, 126.89, 123.95 (Ar-C); ESI-MS
m/z: 475 [(M + 1)+, (9)], 216 (100), 145 (72), 133 (19). (Fig. S5a-c supporting information)
2.5.6. 5-(4-(5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4f)
IR (KBr) υ cm-1 3263, 3199, 3188 (N-H), 3061 (Ar C-H), 1681 (C=O), 1502 (C=C), 1276 (C-O-
C); 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H, -N-H), 8.24 (s, 1H, =C-H), 8.00 (d, J = 9.88
Hz, 2H, ArH), 7.93 (d, J = 6.88 Hz, 2H, ArH), 7.51 (d, J = 6.32 Hz, 2H, ArH), 7.12 (d, J = 8.00
Hz, 2H, ArH), 4.56 (s, 2H, -NH2); 13C NMR (DMSO-d6): 168.57 (C=O), 166.34 (C=O),
164.23(O-C=N), 149.63(C-NH2), 132.52(=CH-C), 131.42, 130.81, 129.08, 127.03, 126.97,
123.85, 122.95, 114.05 (Ar-C); ESI-MS m/z: 365 [(M + 1)+, (8)], 216 (100), 145 (47), 104 (76).
(Fig. S6a-c supporting information)
2.5.7. 5-(4-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4g)
IR (KBr) υ cm-1 3167 (N-H), 3030 (Ar C-H), 1697 (C=O), 1546 (C=C), 1271 (C-O-C), 1070 (C-
1
F). H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, -N-H), 8.84 (s, 1H, =C-H), 8.14 (d, J = 7.2
Hz, 2H, ArH), 7.99 (d, J = 7.76 Hz, 2H, ArH), 7.94 (d, J = 6.2 Hz, 2H, ArH), 7.47 (d, J = 6.36
6

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Hz, 2H, ArH); 13C NMR (DMSO-d6): 168.25 (C=O), 166.03 (C=O), 164.45(O-C=N), 163.33 (C-
F), 132.52 (=CH-C), 133.42, , 130.81, 129.08, 127.03, 126.97, 123.85, 122.95, 116.35 (Ar-C);
ESI-MS m/z: 368 [(M+ + 1), (12)], 253 (13), 216 (100), 145 (68), 109 (46). (Fig. S7a-c
supporting information)
2.5.8. 5-(4-(5-(3-bromophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4h)
IR (KBr) υ cm-1 3209 (N-H), 3032 (Ar C-H), 1658 (C=O), 1546 (C=C), 1280 (C-O-C), 680 (C-
1
Br); H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H, -N-H), 8.89 (s, 1H, =C-H), 8.29 (s, 1H),
8.12 (d, J = 7.28 Hz, 2H, ArH), 7.93 (d, J = 6.76 Hz, 2H, ArH), 7.64 (d, J = 9.44Hz, 1H, ArH),
13
7.55 (t, J = 9.68 Hz, 1H, ArH), 7.47 (d, J = 5.92 Hz, 1H, ArH); C NMR (DMSO-d6): 169.07
(C=O), 166.54 (C=O), 164.33(O-C=N), 163.53 (O-C=N), 132.52 (=CH-C), 132.92, 130.85,
130.71, 129.98, 129.08, 127.37, 127.13, 126.87, 125.55, 123.95 (Ar-C), 118.35 (C-Br); ESI-MS
m/z: 427 [(M + 1)+, 47], 244 (15), 216 (100), 145 (45), 127 (16). (Fig. S8a-c supporting
information)
2.5.9. 5-(4-(5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4i)
IR (KBr) υ cm-1 IR (KBr) υ cm-1 3178 (N-H), 3030 (Ar C-H), 2993 (Ali C-H), 1681 (C=O), 1269
(C-O-C), 1529, 1348 (NO2); 1H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H, -N-H), 8.84 (s, 1H,
=C-H), 8.68 (s, 1H, ArH), 8.45 (d, J = 7.88 Hz, 1H, ArH), 8.36 (d, J = 6.80 Hz, 1H, ArH), 8.28
(d, J = 6.32 Hz, 2H, ArH), 8.20 (s, 1H, ArH), 7.99 (d, J = 7.32 Hz, 2H, ArH), 7.74 (t, J = 8.12
Hz, 1H, ArH); 13C NMR (DMSO-d6): 169.53 (C=O), 166.53 (C=O), 164.63 (O-C=N), 163.73
(O-C=N), 147.72 (C-NO2), 142.29, 135.22, 132.63, 127.38, 127.12, 126.78, 125.53, 123.68 (Ar-
C); ESI-MS m/z: 395 [(M + 1)+, 11], 244 (15), 216 (100), 145 (45), 127 (16). (Fig. S9a-c
supporting information)
2.5.10. 5-(4-(5-(o-tolyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4j)
IR (KBr) υ cm-1 3140 (N-H), 3028 (Ar C-H), 2922 (Ali C-H), 1668 (C=O), 1543 (C=C), 1278
(C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, -N-H), 8.28 (s, 1H, =C-H), 8.05 (d, J =
6.56 Hz, 2H, ArH), 7.98 (d, J = 7.88 Hz, 1H, ArH), 7.86 (d, J = 7.92 Hz, 2H, ArH), 7.50 (t, J =
7.44 Hz, 1H, ArH), 7.47 (t, J = 9.16 Hz, 1H, ArH), 7.30 (d, J = 6.72 Hz, 2H, ArH) 2.53 (s, 3H, -
13
C-H); C NMR (DMSO-d6): 169.32 (C=O), 166.15 (C=O), 165.64 (O-C=N), 164.74 (O-C=N),
135.63 (=CH-C), 132.52, 130.87, 129.67, 129.08, 128.95, 127.88, 127.67, 127.37, 126.87,
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123.95, 123.52 (Ar-C); ESI-MS m/z: 364 [(M + 1)+, (26)], 289 (8), 216 (40), 173 (22), 103 (100).
(Fig. S10a-c supporting information)
2.5.11. 5-(4-(5-(3-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4k)
IR (KBr) υ cm-1 3197 (N-H), 3032 (Ar C-H), 2980 (Ali C-H), 1655 (C=O), 1544 (C=C), 1280
(C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 10.355 (s, 1H, -N-H), 8.28 (s, 1H, =C-H), 8.23 (d, J
= 8.48 Hz, 2H, ArH), 8.05 (d, J = 9.08 Hz, 1H, ArH), 7.96 (s, 1H, ArH), 7.71 (d, J = 7.32 Hz,
1H, ArH), 7.51 (t, J = 7.88 Hz, 1H, ArH), 7.30 (d, J = 8.16 Hz, 1H, ArH), 4.09 (q, 2H, -CH2),
1.335 (t, 3H, -CH3); 13C NMR (DMSO-d6): 169.74 (C=O), 166.03 (C=O), 164.67 (O-C=N),
163.75 (O-C=N), 154.17 (C-OC2H5), 132.22 (=CH-C), 130.91, 130.03, 129.12, 127.38, 127.18,
126.87, 125.56, 123.98, 117.29, 114.29 (Ar-C). (Fig. S11a-c supporting information)
2.5.12. 5-(4-(5-(3,5-dinitrophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
(4l)
IR (KBr) υ cm-1 3150 (N-H), 3080, 3062 (Ar C-H), 1680 (C=O), 1286 (C-O-C), 1535, 1336
(NO2); 1H NMR (400 MHz, DMSO-d6) δ 10.2593 (s, 1H, -N-H), 8.93 (s, 2H, ArH), 8.70 (s, 1H,
13
=C-H), 8.29 (s, 1H, ArH), 8.13 (d, J = 12.88 Hz, 2H ArH,), 8.02 (d, J = 6.08 Hz, 2H, ArH); C
NMR (DMSO-d6): 169.74 (C=O), 166.03 (C=O), 164.67 (O-C=N), 163.75 (O-C=N), 155.63 (C-
NO2), 132.22 (=CH-C), 130.95, 130.61, 129.08, 127.57, 126.87, 125.55, 123.85, 116.05, 103.32
(Ar-C). (Fig. S12a-c supporting information)
2.5.13.
4-(5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)-1,3,4-oxadiazol-2-
yl)benzonitrile (4m)
IR (KBr) υ cm-1 3238, 3197 (N-H), 3086 (Ar C-H), 2993 (Ali C-H), 1670 (C=O), 1546 (C=C),
1282 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H, -N-H), 8.28 (s, 1H, =C-H), 8.24
(d, J = 8.52 Hz, 2H, ArH), 8.09 (d, J = 8.44 Hz, 2H, ArH), 7.99 (d, J = 8.6 Hz, 2H, ArH), 7.91
(d, J = 8.16 Hz, 2H, ArH), 13C NMR (DMSO-d6): 169.07 (C=O), 166.54 (C=O), 164.33 (O-
C=N), 132.52 (=CH-C), 132.32, 130.95, 129.18, 127.47, 126.87, 123.85, 112.33 (Ar-C),
118.54(CN). (Fig. S13a-c supporting information)
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2.5.14.
(4n)
5-(4-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
IR (KBr) υ cm-1 3364 (O-H), 3161 (N-H), 3072 (Ar C-H), 2978 (Ali C-H), 1670 (C=O), 1533
1
(C=C), 1284 (C-O-C); H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H, -N-H), 8.30 (s, 1H, =C-
H), 8.06 (s, 1H, -OH), 7.98 (d, J = 7.88 Hz, 2H, ArH), 7.88 (d, J = 8 Hz, 1H, ArH), 7.62 (t, J =
7.64 Hz, 1H, ArH), 7.48 (t, J = 9.76 Hz, 1H, ArH), 7.30 (d, J = 6.72 Hz, 1H, ArH); 13C NMR
(DMSO-d6): 169.12 (C=O), 166.37 (C=O), 164.27 (O-C=N), 158.62 (C-OH), 132.43 (=CH-C),
131.85, 130.67, 129.05, 127.37, 126.82, 124.55, 120.05, 117.25, 112.32 (Ar-C). (Fig. S14a, b
supporting information)
2.5.15. 5-(4-(5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
(4o)
IR (KBr) υ cm-1 3176 (N-H), 3032 (Ar C-H), 2883 (Ali C-H), 1670 (C=O), 1546 (C=C), 1280
(C-O-C), 758 (C-Cl); 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H, -N-H), 8.63 (s, 1H, ArH),
8.25 (s, 1H, =C-H), 8.05 (d, J = 9.8 Hz, 2H, ArH), 7.88 (d, J = 8.44 Hz, 2H, ArH), 7.78 (d, J =
8.36 Hz, 1H, ArH), 7.67 (d, J = 6.44 Hz, 1H, ArH); 13C NMR (DMSO-d6): 169.02 (C=O),
166.75 (C=O), 164.64 (O-C=N), 135.42 (C-Cl), 133.32 (C-Cl), 132.52 (=CH-C), 130.87, 129.81,
129.08, 128.58, 128.03, 127.87, 127.27, 126.87, 123.95 (Ar-C). (Fig. S15a-c supporting
information)
2.5.16.
(4p)
5-(4-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
IR (KBr) υ cm-1 3380 (O-H), 3113 (N-H), 3080 (Ar C-H), 2968 (Ali C-H), 1691 (C=O),
1
1529(C=C), 1280 (C-O-C); H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H, -N-H), 8.38 (s, 1H,
=C-H), 8.32 (d, J = 4.2 Hz, 2H, ArH), 8.30 (d, J = 4.24 Hz, 1H, ArH), 8.21 (d, J = 10.8 Hz, 2H,
ArH), 8.17 (d, J = 4.28 Hz, 2H, ArH), 5.76 (s, 1H, -OH); 13C NMR (DMSO-d6): 169.35 (C=O),
166.35 (C=O), 165.85 (O-C=N), 157.75 (C-OH), 149.85 (=CH-C), 140.64, 136.63, 131.79,
130.56, 129.46, 123.39, 115.94, 115.06 (Ar-C). (Fig. S16a-c supporting information)
2.5.17. 5-(4-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4q)
IR (KBr) υ cm-1 3168 (N-H), 3084 (Ar C-H), 2941 (Ali C-H), 1680 (C=O), 1535(C=C), 1286 (C-
1
O-C); H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H, -N-H), 9.24 (s, 1H, =C-H), 8.53 (d, J =
9

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8.92 Hz, 1H, ArH), 8.22 (d, J = 8.48 Hz, 1H, ArH), 8.40 (s, 1H), 7.93 (d, J = 6.76 Hz, 2H, ArH),
13
7.65 (d, J = 6.64 Hz, 2H, ArH), 7.56 (t, J = 5.68 Hz, 1H, ArH); C NMR (DMSO-d6): 169.32
(C=O), 166.43 (C=O), 164.84 (O-C=N), 164.13 (O-C=N), 149.89 (N=CH), 148.08 (N=CH),
132.52 (=CH-C), 134.37, 130.43, 129.08, 127.88, 123.97, 123.69, 123.45 (Ar-C). (Fig. S17a-c
supporting information)
2.5.18.
(4r)
5-(4-(5-(trichloromethyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
IR (KBr) υ cm-1 3186 (N-H), 3055 (Ar C-H), 2949 (Ali C-H), 1693 (C=O), 1546(C=C), 1247 (C-
1
O-C), 742, 680(C-Cl); H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, -N-H), 8.81 (s, 1H, =C-
H), 8.10 (d, J = 8.28 Hz, 2H, ArH), 7.95 (d, J = 6.12 Hz, 1H, ArH); 13C NMR (DMSO-d6):
169.18 (C=O), 166.34 (C=O), 163.37 (O-C=N), 162.53 (O-C=N), 132.42 (=CH-C), 130.81,
129.18, 127.33, 126.97, 123.95 (Ar-C). (Fig. S18a-c supporting information)
2.5.19. 5-(4-(5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4s)
IR (KBr) υ cm-1 3142 (N-H), 3045 (Ar C-H), 2995 (Ali C-H), 1680 (C=O), 1546(C=C), 1271 (C-
1
O-C), 1172, 1145 (C-Cl); H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, -N-H), 8.85 (s, 1H,
=C-H), 7.96 (d, J = 6.16 Hz, 2H, ArH), 7.93 (d, J = 6 Hz, 1H, ArH); 13C NMR (DMSO-d6):
169.18 (C=O), 166.34 (C=O), 163.37 (O-C=N), 162.53 (O-C=N), 132.42 (=CH-C), 130.81,
129.18, 127.33, 126.97, 123.95 (Ar-C), 92.35. (Fig. S19a-c supporting information)
2.5.20. 5-(4-(5-(2-hydroxy-3,5-dinitrophenyl)-1,3,4-oxadiazol-2-yl)benzylidene) thiazolidine-
2,4-dione (4t)
IR (KBr) υ cm-1 3343(O-H), 3270 (N-H), 3168, 3108 (Ar C-H), 2916 (Ali C-H), 1693 (C=O),
1546(C=C), 1280 (C-O-C), 1548, 1492 (NO2); 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H, -
N-H), 9.16 (s, 1H, ArH), 8.83 (s, 1H, ArH), 8.80 (s, 1H, =C-H), 8.13 (d, J = 8.56 Hz, 1H, ArH),
7.98 (d, J = 10.08 Hz, 1H, ArH), 4.96 (s, 1H, -OH); 13C NMR (DMSO-d6): 169.57 (C=O),
166.32 (C=O), 164.43 (O-C=N), 163.53 (O-C=N), 148.23, 140.61, 132.32, 131.95, 130.31,
129.08, 127.57, 126.55, 125.87, 123.85, 116.04, 105.32 (Ar-C). (Fig. S20a-c supporting
information)
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2.5.21. 5-(4-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
(4u)
IR (KBr) υ cm-1 3163 (N-H), 3076 (Ar C-H), 2943 (Ali C-H), 1695 (C=O), 1546(C=C), 1255 (C-
O-C); 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H, -N-H), 8.27 (s, 1H, =C-H), 7.96 (d, J = 5.92
Hz, 2H, ArH), 7.90 (d, J = 6.24 Hz, 1H, ArH), 7.86 (d, J = 9.12 Hz, 2H, ArH), 7.62 (t, J = 7.44
Hz, 1H, ArH), 7.57 (t, J = 9.16 Hz, 1H, ArH), 7.29 (d, J = 6.52 Hz, 1H, ArH); 3.95 (s, 3H, -
OCH3); 13C NMR (DMSO-d6): 169.37 (C=O), 166.12 (C=O), 165.64 (O-C=N), 159.43, 132.52
(=CH-C), 131.47, 130.87, 129.08, 127.37, 126.87, 123.67, 119.58, 112.42, 111.92 (Ar-C), 55.48
(-CH3). (Fig. S21a-c supporting information)
2.5.22. 5-(4-(5-(2-nitrophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4v)
IR (KBr) υ cm-1 3166 (N-H), 3086 (Ar C-H), 2916 (Ali C-H), 1670 (C=O), 1500(C=C), 1535,
1346 (-NO2), 1278 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 9.844(s, 1H, -N-H), 8.44 (s, 1H,
=C-H), 8.37 (d, J = 7.52 Hz, 2H, ArH), 8.25 (d, J = 8.0 Hz, 1H, ArH), 8.22 (t, J = 8.8 Hz, 1H,
ArH), 7.99 (d, J = 7.32 Hz, 2H, ArH), 7.95 (d, J = .72 Hz, 1H ArH,), 7.74 (t, J = 8.12 Hz, 1H,
13
ArH); C NMR (DMSO-d6): 169.50 (C=O), 166.68 (C=O), 164.62 (O-C=N), 164.45 (O-C=N),
148.42 (C-NO2), 132.38 (=CH-C), 130.92, 129.48, 129.38, 129.18, 128.38, 127.12, 126.78,
123.68, 116.45, 112.08 (Ar-C). (Fig. S22a-c supporting information)
2.5.23. 5-(4-(5-(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione (4w)
IR (KBr) υ cm-1 3167 (N-H), 3046 (Ar C-H), 2916 (Ali C-H), 1691 (C=O), 1570 (C=C), 1271
(C-O-C), 1016 (C-F). 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H, -N-H), 8.31 (s, 1H, =C-H),
8.07 (d, J = 8.8 Hz, 2H, ArH), 7.95 (d, J = 10.36 Hz, 2H, ArH), 7.92 (d, J = 9.44 Hz, 1H, ArH),
7.85 (t, J = 7.0 Hz, 1H, ArH), 7.62 (t, J = 7.36 Hz, 1H, ArH), 7.54 (d, J = 6.72 Hz, 1H, ArH); 13C
NMR (DMSO-d6): 169.32 (C=O), 166.37 (C=O), 164.72 (O-C=N), 164.62 (O-C=N), 160.47 (C-
F), 132.37 (=CH-C), 130.87, 130.43, 129.67, 129.08, 127.88, 127.62, 127.37, 126.89, 123.95,
114.95 (Ar-C). (Fig. S23a-c supporting information)
2.5.24.
2-(5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)-1,3,4-oxadiazol-2-yl)
benzonitrile (4x)
IR (KBr) υ cm-1 3153 (N-H), 3057 (Ar C-H), 2873 (Ali C-H), 2303 (C≡N), 1697 (C=O), 1543
(C=C), 1272 (C-O-C); 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H, -N-H), 8.41 (s, 1H, C-H),
11

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8.28 (d, J = 7.16 Hz, 2H, ArH), 8.24 (d, J = 8.24 Hz, 1H¸ ArH), 8.17 (t, J = 7.48 Hz, 1H, ArH),
13
7.92 (d, J = 8.16 Hz, 2H, ArH), 7.72 (t, J = 5.36 Hz, 1H, ArH); C NMR (DMSO-d6): 169.25
(C=O), 166.23 (C=O), 165.38 (O-C=N), 164.47(O-C=N), 133.25, 132.57 (=CH-C), 132.34,
130.87, 129.88, 129.04, 127.87, 127.23, 127.04, 126.87, 123.85, 123.35 (Ar-C), 112.33 (CN).
(Fig. S24a-c supporting information)
2.6. In vitro cytotoxicity
The synthesized derivatives of 5-(4-(1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
were evaluated for their anti-cancer activity against selected human cancer cell line of breast
(MCF-7) using sulforhodamine B (SRB) assay. The results of anti-cancer activity were
expressed in terms of growth inhibition (GI50 ꢁM) values and are presented in Table 1. RPMI
1640 medium (10% fetal bovine serum and 2 mM L-glutamine) was used for maintaining the
cell lines. The cells were inoculated into 96 well microtiter plates in 90 µL at 5000 cells per well.
Before addition of testing compounds, the microtiter plates were incubated at 37°C, 95% air, 5%
CO2 and 100 % relative humidity for 24 h. The testing compounds were diluted in DMF for the
preparation of stock solution of 10-2 concentration. During experiment four 10-fold serial
dilutions were prepared using complete medium. Aliquots of 10 µl dilutions of different testing
compounds were added into microtiter plates for preparing final drug concentration. These
microtiter plates were incubated for 48 hours at standard conditions and finally assay was
terminated after addition of cold TCA. The cells were fixed by the addition of cold TCA [50 µl,
30 % (w/v)] and then incubated for further 60 minutes at 4°C. The supernatant was discarded;
the plates were washed at least five times by using tap water and dried in air. The prepared
sulforhodamine B (SRB) solution [(50 µl) at 0.4 % (w/v) in 1 % acetic acid] was added in each
wells and then incubated at room temperature for 20 minutes. After staining the prepared plates,
unbound dye was recovered and residual dye was removed by washing five times using 1 %
acetic acid. After staining, unbound dye was recovered and the residual dye was removed by
washing five times with 1 % acetic acid and dried in air. Bound stain was subsequently eluted
with 10 mM trizma base, and the absorbance was read on an Elisa plate reader at a wavelength
of 540 nm with 690 nm reference wavelength [30,31].
The growth percentage was calculated by average absorbance of the test wells to the average
absorbance of the control wells multiplied by 100. The growth inhibition values (GI50) were
12

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observed by the using of formula [(Ti-Tz)/(C-Tz)] x 100 = 50. Where GI50 represents the 50%
reduction in the net protein increase (as measured by SRB staining) in control cells during the
drug incubation, Tz represents for time zero, C for control growth, and Ti for test growth in the
presence of drug at the four concentration levels.
2.7. Computational studies
2.7.1. In silico prediciton of physicochemical properties
Physicochemical properties of the synthesized compounds 4a-x were calculated using QikProp
module of Schrodinger software. The compounds were first drawn in 2D by ChemDraw then
further processed by using LigPrep module of Schrodinger's software as per standard protocols.
All the prepared structures were introduced into QikProp module for the prediction of
physicochemical properties. QikProp module used for the determination of pharmaceutically
relevant properties such as octanol/water, log S, log BB, overall CNS activity, Caco-2 value,
MDCK cell permeabilities, log Khsa for human serum albumin binding, and log IC50 for HERG
K+ channel blockage [32-36].
2.7.2. Molecular docking
Molecular docking study was performed by GLIDE v3.8 (Schrodinger, LLC, New York) module
for all thiazolidine-2,4-dione derivatives on the binding site of PIM-1 kinase. The crystal
structure of PIM-1 kinase was obtained from protein data bank (PDB code: 4DTK) and
preprocessed by using ‘‘protein preparation wizard’’ in Maestro module v10.3 (Schrodinger,
LLC, New York). Here some other steps like generate states and refinement have also been
processed for addition of hydrogen atoms and disulfide bonds at the missing sites on protein
molecule [37]. The protein structure was changed to a single unit with the removal of water
molecules and other unwanted subunits [38]. After the optimization process, receptor grid
generation was proceeded to the previously attached ligand site. The grid is associated with
different sets of fields for determining shape and properties of the receptor. The ligand binds
with protein residues by using specific force field, generate different poses. The best docked
poses have been ranked on the basis of energy function combining empirical and force-field
terms [39-41].
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3. Results and discussion
3.1. Chemistry
In the present study, twenty-four thiazolidine-2,4-dione derivatives have been synthesized which
are outlined in scheme 1. The starting material thiazolidine-2,4-dione (1) was prepared by the
reaction of chloroacetic acid with thiourea in the presence of hydrochloric acid. The compound
4-formylbenzohydrazide (2) was prepared by the reaction between methyl 4-formylbenzoate and
hydrazine
hydrate
in
ethanol.
The
compound
4-((2,4-dioxothiazolidin-5-
ylidene)methyl)benzohydrazide (3) was obtained by condensation of thiazolidine-2,4-dione with
methyl 4-formylbenzohydrazide using piperidine as a base, refluxing in ethyl alcohol for 24 h.
The general procedure for the synthesis of target compounds 5-(4-(5-phenyl-1,3,4-oxadiazol-2-
yl)benzylidene)thiazolidine-2,4-diones 4a-x were achieved through the versatile and efficient
synthetic route outlined in scheme 1. Briefly, the reaction between 4-((2,4-dioxothiazolidin-5-
ylidene)methyl)benzohydrazide and benzoic acid derivatives was performed by using POCl3 as
cyclizing agent in a refluxing condenser at 60-70ºC for 14 h, followed by the addition of crushed
ice and neutralization by sodium bicarbonate, resulted in precipitate of target compounds. The
1
13
structures assigned to the compounds were supported by the results of IR, H NMR, C NMR
and mass spectral data.
FT-IR spectra of synthesized compound 1, thiazolidine-2,4-dione showed characteristic peaks at
υ cm-1 3130 (-NH) and 1737 (C=O), whereas compound 2 showed peaks at υ cm-1 3325 (NH2),
3178 (-NH) and 1651 (C=O). The FT-IR spectra of compound 3 showed peaks at υ cm-1 3290
(NH2), 3221 (-NH), 1726 (C=O) and 628 (C-S-C). The target compound 4a showed
characteristic IR peaks at υ cm-1 3187 and 1546 of N-H and C=N groups, respectively. Other
derivatives of the series such as 4c, 4d, 4o, 4r showed characteristic FT-IR peaks around υ cm-1
750 due to presence of C-Cl group; 4e at υ cm-1 545 of C-I group; 4g at υ cm-1 1070 of C-F
group; 4h at υ cm-1 680 of C-Br group; 4i at υ cm-1 1529, 1348 of NO2 group. 4n and 4p were
observed absorption band at υ cm-1 3364 and 3380 due to presence of O-H groups, consecutively.
1H NMR spectra of compounds 4 (a-x) showed signals in the range of δ 7.952-8.152 due to the
presence of aromatic protons of central phenyl ring, two singlet at around δ 8.259, 10.255,
indicate the thiazolidine N-H and benzylidene C-H protons, respectively. 1H NMR spectra of 4a
showed two triplet at δ 7.471 and 7.632 due to the presence of unsubstituted phenyl group
14

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protons. Para chloro derivative, 4b showed signals at δ 7.543 and 7.659 due to the presence of
nearby C-H protons whereas compound 4c showed singlet at δ 5.215 due to the presence of
primary amine group. Ortho iodo derivative, 4e showed signals at δ 7.938, 7.616 and 7.565 due
to the presence of ortho, meta and para substituted C-H protons, respectively. Meta nitro
derivative was confirmed by the presence of signals at δ 8.356, 8.446 of nearest C-H protons.
Ortho methyl derivative, 4j showed signals at δ 2.527 due to the presence of methyl C-H proton,
whereas, meta ethoxy derivative, 4k showed δ 1.335 and 4.094 due to the presence of methylene
and methyl C-H protons, consecutively. Compound 4m and 4p with cyanide and hydroxyl
groups at para position of phenyl ring showed peaks in the range of δ 7.914-7.995 and 8.172-
13
8.214 of nearby C-H protons, consecutively. C NMR spectra of compounds 4a-x were shown
peaks at δ 169.31, 166.17 represent carbonyl group, δ 163.83, 164.37 indicative of oxadiazole
carbon, δ 132.61 confirm benzylidene (=C-H) group, δ 130.70, 129.39, 129.18, 128.59, 128.31,
127.01, 126.77 indicative of aromatic carbon (Ar-C) and δ 21.52 accounted for methyl group (-
CH3). Compound 4k showed peak at δ 154.17 of C-OC2H5, compound 4l showed at δ 155.63
indicative of C-NO2, compound 4m showed at δ 118.54 for C=N, compound 4n showed at δ
158.62 for C-OH and compound 4o showed at δ 135.42 for C-Cl. The mass spectrum of
compound 2 displayed a molecular ion peak at m/z 165 and base peak at m/z 105 whereas
compound 3 showed molecular ion peak at m/z 264 and base peak at m/z 133. The compound 4a
showed molecular ion peak at m/z 350 and base peak at m/z 216. The possible fragment of base
peak of compound 4a was (4-((2-hydroxy-4-oxothiazol-5(4H)-ylidene)methylene)cyclohexa-2,5-
dien-1-ylidene)methylium. Compound 4b showed molecular ion peak at m/z 384 with base peak
at 216 and other fragment peaks at m/z 173, 145, 125. Similarly other derivatives of the series
showed their respective m/z value peaks according to molecular mass of the compounds.
3.2. Biological activity
The synthesized compounds 4a-x were evaluated for their anti-cancer activity against selected
human cancer cell line of breast (MCF-7) using sulforhodamine B (SRB) method. The results of
anti-cancer activity are expressed in terms of growth inhibition fifty (GI50 µM) values and are
shown in Table 1. Compound 4x was the most potent derivative of the series against MCF-7
with GI50 value of 0.004 ꢁM. By comparing the activities of 4x with its unsubstituted counterpart
4a, it was revealed that the presence of cyano group at C-4 position of phenyl ring confers the
highest cytotoxic activity against breast cell line, MCF-7. Furthermore, compound 4d showed
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good inhibitory activity against MCF-7 cells with GI50 value of 0.012 ꢁM. Compounds 4c-f, 4h,
4l, 4o, 4t and 4w showed significant improvement in activity compared to unsubstituted
counterpart 4a with GI50 values of 0.028, 0.012, 0.097, 0.055, 0.087, 0.087, 0.031, 0.013 and
0.019 ꢁM, consecutively. Compounds 4n and 4p showed the lowest potency in the series with
GI50 values of 10.0 and 77.0 ꢁM, respectively. Whereas, compounds 4g, 4i-k, 4m, 4q, 4r, 4s, 4u
and 4v showed intermediate inhibitory activities.
The structure activity relationships (SARs) indicate that compounds having oxadiazole ring
attached to p-position (4a-x) of the benzylidene ring were important for anti-cancer activity.
Presence of aromatic ring attached to oxadiazole ring generated potential anti-cancer derivatives.
Compounds with halogen group substitution on oxadiazole attached aromatic ring exhibited
promising activity. Compound 4x with the cyano group at 2 position of the phenyl ring attached
to heterocyclic oxadiazole showed potent activity against MCF-7 cells when compared with
reference drug adriamycin. Substitution at R with electron withdrawing groups such as chloro,
bromo, iodo in compounds 4c, 4d, 4e, 4h, 4o, 4w and 4x showed increase in activity. The
electron donating group (2-methyl) substituted compound 4j showed decrease in activity. The
compound 4d with chloro substitution on the phenyl ring exhibited GI50 value of 0.012 ꢁM better
than unsubstituted compound 4a. Compounds 4h and 4l with 3-Br and 3,5-(NO2)2 groups
showed significant improvement in activity compared to 4a. Compound 4p with 4-OH
substitution on the phenyl ring exhibited lowest potency in the series. Constitutively, 4r and 4s
containing trichloro and trifluoro groups attached to oxadiazole ring showed decrease in activity
compared to other derivatives of the series, attributed to the presence of phenyl ring essential for
the activity. Compounds with 4-substituted phenyl ring showed variation in activities. At this
position, electron withdrawing groups such as fluoro and cyano (4g, 4m, respectively) showed
intermediate inhibitory activities, whereas 4f with electron donating group i.e. amine, exhibited
better inhibitory activity.
3.3. Computational studies
3.3.1. In silico prediction of physicochemical properties
The physicochemical properties of target compounds were summarized in Table 2. Lipophilicity
(Log P) values of all synthesized compounds were in range from 1.83 to 3.34. Compound 4m
was the most lipophilic compound of the series whereas 4P was identified as less lipophilic.
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Molecular weight and polar surface area of compounds were in the range of 339.32 to 475.26
and 106.24 to 225.56, respectively. The compounds were found with optimum number of
hydrogen bonding donors and acceptors. QPPCaco values were found in the range (259) for
compound 4r whereas, compounds 4i and 4p showed poor values. QPlogBB represents the
brain/blood partition coefficient (-4.008 to -0.724) and QPPMDCK represents the apparent
permeability across the MDCK cells were found in the range (>500) for compound 4k. Qikprop
predictions suggested that thiazolidine-2,4-dione derivatives have optimum parameters for anti-
cancer activity and can be considered as a lead molecule for further modifications.
3.3.2. Molecular docking
Molecular modeling studies were performed on Glide v5.8 (Schrodinger, LLC, New York, NY)
to investigate the potential interactions between target compound and PIM-1 kinase (PDB code:
4DTK). All synthesized compounds 4a-x were docked to binding sites of PIM-1 kinase for
studying the essential interactions of compounds with protein to produce anti-cancer activity.
The crystal structure of ligand showed hydrogen bond interaction between oxygen atom of
thiazolidine-2,4-dione with amino acid residues, ASP186 and LYS67, whereas, hydrophobic
groups are positioned between the amino acid residues Leu 44 and Leu 174 of PIM-1 kinase. The
pyrrolidine ring of crystal structure of ligand showed interaction with ASP128 and GLU171 of
PIM-1 kinase. The synthesized compounds 4a-x were allowed similar binding interactions with
the receptor as shown by crystal ligand. The compound 4x showed the best inhibitory activity
against MCF-7 cell line with good docking score (-6.68) against PIM-1 kinase (Fig 2a, b and c).
The substituents of this compound are oriented towards the hydrophobic region comprised of
amino acid residues Leu 44 and Leu 174. The oxygen atom at the second and fourth positions of
the TZD moiety exhibited H-bonding interactions with ASP186 and LYS67 respectively, showed
its essential role for the activity. The docking score with highest affinity also suggested the best
activity of compound against breast cancer cell line. The synthesized compound such as 4f has
shown docking score -6.02 including critical interaction of oxygen with LYS67 and amine with
ASP131 (Fig. 3). This compound showed good alignment with the crystal structure of ligand
with respect to their docking score. 2,4-dichloro group containing compound 4o showed better
interaction with amino acid residues ASP 186, LYS 67 and ASP 131 of PIM-1 kinase than meta
substituted compounds (Fig. 4). All these interactions proved that the synthesized compounds
were shown the best docking results with the receptor cavity of PIM-1 kinase.
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4. Conclusion
In the present study, a series of 5-(4-(5-(1,3,4-oxadiazol-2-yl)benzylidene)thiazolidine-2,4-dione
derivatives have been synthesized, characterized, and screened against MCF-7, breast cancer cell
lines. Among the synthesized compounds, 4x which contains electron withdrawing group
(cyano) at 2 position of phenyl ring attached to oxadiazole of thiazolidine-2,4-dione scaffold
showed potent activity (GI50 = 0.004 ꢁM). The SAR study revealed that phenyl ring is essential
for anti-cancer activity whereas replacement of phenyl ring with trichloro and trifluoro groups
showed decreased activity. Similarly, substitution of electron donating groups on phenyl ring
produced decreased anti-cancer activity. Furthermore docking studies of compounds 4f, 4o and
4x showed the best binding interaction with amino acid residues LYS67, ASP186, ASP131 and
GLU124 of PIM-1 kinase. These derivatives also showed superimposition with the crystal ligand
of protein which validates the docking results of selected compounds. In conclusion, the
structural features of compound 4x may be considered for the development of newer anti-cancer
agents.
Conflict of Interest:
The authors declare no conflict of interest
Acknowledgments:
The authors gratefully acknowledge Indian Council of Medical Research (ICMR), New Delhi for
providing Senior Research Fellowship (SRF) to Mr. Vivek Asati (SRF No.-45/28/2013-
PHA/BMS).
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Fig. captions
Fig. 1. Chemical structures of some representative thiazolidine-2,4-dione based inhibitors
reported in literature.
Fig. 2. a. Docking results showing interaction of compound 4x with amino acid residues ASP
186 and LYS 67 of PIM-1 kinase (PDB ID: 4DTK); b. representation of compound 4x into
receptor cavity PIM-1 kinase; c. 2D interaction diagram of compound 4x with PIM-1 kinase.
Fig. 3. Docking results showing hydrogen bond interaction (yellow dotted lines) of compound 4f
with amino acid residues ASP 186, LYS 67 and ASP 131 of PIM-1 kinase. Here ligand with
green colour represents crystal structure whereas ligand with magenta colour shows synthesized
compound 4f
Fig. 4. Docking results showing hydrogen bond interaction (yellow dotted lines) of compound 4o
with amino acid residues ASP 186, LYS 67 and ASP 131 of PIM-1 kinase. Here ligand with
green colour represents crystal structure whereas ligand with magenta colour shows synthesized
compound 4o
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Table 1 Structure, in vitro antiproliferative activity (GI50) of compounds 4a–x against human
breast cancer cell line (MCF-7)
Comp. No.
R
Yield (%) Melting point (˚C)
GI50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
Ph
4-ClPh
2 NH2, 3,5-Cl2Ph
3-ClPh
2-IPh
4-NH2Ph
4-FPh
3-BrPh
3-NO2Ph
2-CH3Ph
3-OC2H5Ph
78
83
74
69
82
64
71
72
74
77
79
238-240
240-242
256-258
235-237
221-223
234-236
238-240
258-260
212-214
245-247
256-258
2.034
n.d.
0.028
0.012
0.097
0.055
0.466
0.087
0.567
1.572
4j
4k
2.016
0.087
0.651
10.32
0.031
77.13
0.148
2.253
2.29
0.013
1.963
0.671
0.019
0.004
0.00018
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
3,5-(NO2)2 Ph
4-CNPh
2-OHPh
2,4-Cl2Ph
4-OHPh
C5H4N
82
85
82
82
78
68
65
68
68
65
70
75
65
273-275
273-275
216-218
234-236
216-218
226-228
241-244
235-237
255-258
215-217
235-237
216-218
253-255
CCl3
CF3
2-OH, 3,5-(NO2)2Ph
2-OCH3Ph
2-NO2Ph
2-FPh
4x
*ADR
2-CNPh
*ADR = Adriamycin, positive control compound
n.d.: Not determined
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Table 2 Molecular descriptors of thiazolidine-2,4-dione derivatives.
Mol. Wt.
PSA
QPlogPo
/w
QPP
Caco
QPlogB
B
QPP
MDCK
SASA
PercentHumanOral
Absorption
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
590.417
602.978
655.213
552.659
617.362
595.421
628.461
632.116
689.574
523.96
83.712
70.261
87.306
79.041
71.865
85.584
67.594
84.403
20.241
79.435
84.738
73.389
90.767
74.433
67.073
27.192
86.848
87.961
61.951
87.021
85.081
67.874
89.21
349.363
365.363
377.417
339.325
374.373
367.354
394.361
379.389
455.358
341.264
390.628
350.351
418.253
365.363
374.373
439.358
393.416
363.39
110.039
132.642
110.023
120.414
131.709
108.906
153.246
114.598
225.567
111.982
110.812
123.056
107.757
127.78
135.752
207.244
117.727
106.241
158.606
110.037
110.038
136.892
107.632
110.035
2.712
2.006
3.326
2.063
2.067
2.912
2.19
2.831
0.853
2.096
2.843
1.766
3.659
2.126
1.959
1.308
3.248
3.04
192.465
58.063
192.453
172.142
68.169
210.689
35.863
192.336
1.176
176.561
199.024
104.042
233.858
90.743
39.899
2.041
192.441
259.764
19.826
192.474
192.43
48.758
244.068
192.474
-1.214
-1.83
126.48
34.631
126.455
112.113
41.19
213.875
20.572
126.393
0.512
579.865
1298.647
65.058
790.337
56.113
23.086
0.928
126.446
174.898
10.84
-1.322
-1.191
-1.779
-1.094
-2.088
-1.34
-4.008
-0.856
-0.724
-1.472
-0.866
-1.611
-2.075
-3.656
-1.497
-1.096
-2.394
-1.075
-1.115
-1.917
-0.966
-1.08
564.726
583.282
630.328
597.411
629.209
679.161
675.746
610.13
4s
4t
4u
4v
4w
4x
634.849
619.522
599.403
607.357
615.27
394.361
428.259
367.354
364.378
475.26
2.013
3.277
2.946
1.83
3.336
3.201
335.361
228.863
28.673
392.335
311.879
614.488
86.576
383.808
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Scheme 1
Compd.
No.
4a
R
Ph
4b
4c
4d
4e
4-ClPh
2 NH2, 3,5-Cl2Ph
3-ClPh
2-IPh
4f
4-NH2Ph
4-FPh
3-BrPh
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
3-NO2Ph
2-CH3 Ph
3-OC2H5Ph
3,5-(NO2)2 Ph
4-CNPh
2-OHPh
2,4-Cl2Ph
4-OHPh
C5H4N
CCl3
4s
CF3
4t
2-OH,3,5-(NO2)2 Ph
2-OCH3 Ph
2-NO2Ph
2-FPh
4u
4v
4w
4x
2-CNPh
Scheme 1. Reagents: (a) Conc. HCl, water; (b) EtOH; (c) piperidine, EtOH; (d) POCl3.
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I
II
III
NH2
N
O
NH
S
O
IV
V
VI
VII
Fig. 1
s
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