5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells

Article abstract of DOI:10.1016/j.ejmech.2016.03.089

The article presents the synthesis of 5-ene-4-thiazolidinone derivatives with pyrazole core linked by enamine group. The structure and purity of compounds were confirmed by analytical and spectral data including X-ray analysis. Target compounds were scree

Full text of DOI:10.1016/j.ejmech.2016.03.089

Accepted Manuscript
5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells
Julia Senkiv, Nataliya Finiuk, Danylo Kaminskyy, Dmytro Havrylyuk, Magdalena
Wojtyra, Iryna Kril, Andrzej Gzella, Rostyslav Stoika, Roman Lesyk
PII:
S0223-5234(16)30272-0
10.1016/j.ejmech.2016.03.089
EJMECH 8514
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 March 2016
Revised Date: 28 March 2016
Accepted Date: 31 March 2016
Please cite this article as: J. Senkiv, N. Finiuk, D. Kaminskyy, D. Havrylyuk, M. Wojtyra, I. Kril, A. Gzella,
R. Stoika, R. Lesyk, 5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.089.
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.

ACCEPTED MANUSCRIPT
5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells
Julia Senkiva, Nataliya Finiuka, Danylo Kaminskyyb, Dmytro Havrylyukb, Magdalena
Wojtyrab, Iryna Krilb, Andrzej Gzellac, Rostyslav Stoikaa, Roman Lesykb,*
a
Institute of Cell Biology of National Academy of Sciences of Ukraine, 14/16 Drahomanov
Str., Lviv 79005, Ukraine
b Danylo Halytsky Lviv National Medical University, 69 Pekarska Str., Lviv 79010, Ukraine
c Poznan University of Medical Sciences, 6 Grunwaldzka Str., Poznan 60780, Poland
X
R
N
Y
S
X=O, Y=S
X=S, Y=O
N
N
R
N
R

5-Ene-4-thiazolidinones induce apoptosis in mammalian leukemia cells
ACCEPTED MANUSCRIPT
Julia Senkiva, Nataliya Finiuka, Danylo Kaminskyyb, Dmytro Havrylyukb, Magdalena Wojtyrab, Iryna
Krilb, Andrzej Gzellac, Rostyslav Stoikaa, Roman Lesykb,*
a
Institute of Cell Biology of National Academy of Sciences of Ukraine, 14/16 Drahomanov Str., Lviv
79005, Ukraine
b Danylo Halytsky Lviv National Medical University, 69 Pekarska Str., Lviv 79010, Ukraine
c Poznan University of Medical Sciences, 6 Grunwaldzka Str., Poznan 60780, Poland
Keywords: 5-ene-4-thiazolidinones, anticancer activity, leukemia, apoptosis, ROS.
Corresponding author
Tel.: +380322755966; Fax: +380322757734;
E-mail: dr_r_lesyk@org.lviv.net, dr_lesyk@meduniv.lviv.ua (R. Lesyk)
1

Abstract
ACCEPTED MANUSCRIPT
The article presented the synthesis of 5-ene-4-thiazolidinone derivatives with pyrazole core linked
by enamine group. The structure and purity of compounds were confirmed by analytical and spectral
data including X-ray analysis. Target compounds were screened for their anticancer activity and
selective antileukemic action was confirmed. 5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one (compound 1) was selected as most active
agent against HL-60 and HL-60/ADR cell lines; IC50=118 nM / HL-60 with low toxicity towards
pseudonormal cells. The mitochondria-depended apoptosis was identified as the main mode of 1 action.
Moreover compound’s effect induces G0/G1 arrest of the treated cells and causes inhibition of cell
division and is related with activation of ROS production.
2

1. Introduction
ACCEPTED MANUSCRIPT
4-Thiazolidinones and related heterocycles are intensively explored for design of new drug-like
molecules [1-3]. Despite the variety of 4-thiazolidinone bearing compounds, the search for new
antibacterial, antiviral, anti-inflammatory, and antidiabetic agents [4,5] is the main direction associated
with the thiazolidinone framework. Nowadays research area is mainly focused on a design of new
anticancer agents [6]. A majority of the biologically active 4-thiazolidinones belong to the 5-ylidene (5-
ene) derivatives containing the exocyclic double bond. This is reflected in thesis about a crucial role of
the C5 substituent (namely 5-ene) in the pharmacological effect [7-11]. Conjugation of the 5-ene
fragment to the carbonyl group at C4 position of the thiazolidine core makes the compounds to be
electrophilic and potentially reactive due to a possible Michael addition of the nucleophilic protein
residues to the exocyclic double bond [12]. This property can characterize 5-ene-4-thiazolidinones as
frequent hitters or pan assay interference compounds that are treated as useless in the drug discovery
process because of their possible insufficient selectivity [3,13,14]. This view is disproved in many
studies and a large number of lead-compounds belong to the mentioned 5-ene-4-thiazolidinones. From
another hand, such Michael acceptors are among the most effective activators of the Nrf2 through the
Keap1 modification that opens new perspectives in treatment of inflammation and cancer [15,16].
Positive perspective of above mentioned thiazolidinone sub-type is linked to a polypharmacological
approach in drug discovery, where the affinity toward various targets is regarded as anadvantage [16].
Moreover, 5-ene-4-thiazolidinones are the examples of privileged scaffolds that can be treated as drug-
like molecular structures providing baseline affinity for the whole protein family [17]. Besides, better
results in drug design can be expected when responses are evoked by the multipoint interventions in
more than one mechanism and in different targets, following a concept of multi-target drugs. The
exploitation of 5-ylidene diversity allows achieving such desired combinations, for instance within the
hybrid pharmacophore approach [17]. A significant biological potency of compounds bearing 5-ene-4-
thiazolidinone fragment in combination with the heterocyclic moieties (such as thiazole, pyrazole,
flavone, chromone, furan, etc.) or other molecular fragments was discovered [7,9,19-22]. Such approach
can be treated as a perspective tool for improving selectivity.
3

Numerous papers and patents are devoted to design and development of new anticancer agents
ACCEPTED MANUSCRIPT
based on 5-ene-4-thiazolidinones [1,6]. Research in this field can be outlined in the following directions:
i) screening of the libraries of compounds for further hits optimization; ii) design of high affinity ligands
to "validated" anticancer targets; iii) the creation of the hybrid molecules combining several attractive
scaffolds [23]; iv) search for anticancer agents among compounds/drugs with other biological activities
(anti-inflammatory, antidiabetic, antimicrobial, etc.) [4,5,24].
Despite a long list of potential and validated bio-targets for new anticancer agents, there is no
systematization of the 4-thiazolidinones as anticancer agents. It was discovered that 5-ene-4-
thiazolidinones can act as: 1) inhibitors of the antiapoptic protein-protein interactions – between Bcl-2
and Bax family, as well as inhibitors of their interaction with the receptors’ domains [25,26]; 2)
inhibitors of JSP-1 – “atypical” dual-specific phosphatases family member (JNK-stimulating
phosphatase-1) [26]; 3) COX inhibitors [28] because of their potential anticancer activity; 4) inhibitors
of insulin-like growth factor-1 receptor [29]; 5) selective inhibitors of extracellular signal-regulated
kinases-1 and 2 (ERK1/2) [30]; 6) modulators of estrogen-related receptor-α (ERR-α) [31]; 7) Pim-1
and Pim-2 inhibitors [32]; 8) inhibitors of CDK1 [33,34] and CDK1/cyclin B [35]; 9) inhibitors of Polo-
like kinase 1 (Plk1) [36]; 10) agents with SHP-2 inhibitory action (SHP-2, a non-receptor protein
tyrosine phosphatase that mediates cell signaling via growth factors and cytokines acting via the
RAS/MAP kinase pathway [37]; 11) tumor necrosis factor (TNFα) inhibitors [38]; 12) integrin αvβ3
receptor inhibitors [39] etc. Besides, the 5-ylidene rhodanines can act as “Myc-Max compounds” that
inhibit or reverse association between c-Myc and Max [40], etc.
Many chemotherapeutics exert the anticancer activity by triggering apoptotic cell death and
exhibit excellent antiproliferative properties [41]. Despite high affinity to different molecular targets,
apoptosis induced by 4-thiazolidinones has been demonstrated in various cancer cells [42,43]. The
apoptosis-depended mode of action is mainly related here with the inhibition of Bcl-2/Bcl-XL function,
although other effects of 5-ene-4-thiazolidinones in the apoptotic signaling pathway involving
mitochondria-mediated apoptosis have been described [44]. 4-Thiazolidinones also decrease
mitochondrial membrane potential in leukemic cells that is one of the most important mechanisms for
the mitochondrial mediated apoptotic cell death [45]. As an example, MKT 077 (1-ethyl-2-[[3-ethyl-5-
4

(3-methyl-2(3H)-benzothiazolylidene)-4-oxo-2-thiazolidinylidene]methyl]pyridinium chloride) has been
ACCEPTED MANUSCRIPT
reported as an antitumor agent with apoptosis related mechanism of action which is related to its
differential interaction with the Hsp70 (Heat shock protein 70 kDa) allosteric states and with the
reactivation of p53 function [46].
Evolution of the adaptive mechanisms provides cancer cells with the ability to evade apoptotic
execution and bestows a survival advantage upon them. Most tumors develop a multi-drug resistance
that is often associated with the over-expression of the membrane permeability-glycoprotein (P-gp). It is
firmly established that P-gp-mediated multi-drugresistance is a major obstacle to a successful cancer
chemotherapy [47]. Data were obtained suggesting that 4-thiazolidinones (e.g. MMPT (5-[(4-
methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone)
and
DBPT
(5-(2,4-
dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidine) inhibit growth of drug-resistant cancer cells
(human non-small-cell lung and colon cancer) independent on P-gp and p53 status and induce apoptosis
[48].
Cancer cells are characterized by a loss of differentiation control and changes in cell cycling. The
cell cycle is important in cancer treatment because many therapies work the best when cells are actively
or rapidly dividing. For example, some chemotherapy drugs work by targeting cells in a particular phase
of the cell cycle, such as G1, S or G2 [49]. Anticancer effect of 5-ene-4-thiazolidinones can be achieved
by a reversible blocking of cell cycle progression at the G2/M phase border that leads to induction of
apoptosis [33]. FACS analysis of the action of 5-arylfurfurylidene-4-thiazolidinone-3-carboxylic acids
possessing strong antiproliferative activity towards human leukemia cells showed a remarkable
accumulation of sub-ploid cells, the sub-G1 phase (G0/G1) followed by a decline of both G1 and G2/M
phases, and the observed growth inhibition could be due to apoptosis induction [34]. The related 5-ene-
4-thiazolidinones with heterocyclic fragments induced cell cycle arrest of leukemic cells at the G2/M
phase and increased number of cells in the sub-G1 phase leading to cell death, as a result of
depolarization of the mitochondrial membrane potential. Similar data were obtained at the action of 2-
heterylamino-4-thiazolidinones that induced dissipation of the mitochondrial membrane potential, as
well as redox changes in treated HT29 cells accumulated in the G2/M and sub-G0/G1 phases of cell cycle
[11]. Treatment of cancer cells with MMPT and DBPT also led to a time-dependent arrest of cells in the
5

G2/M phase. 5-Ene-4-thiazolidinones may act as an ATP-competitive inhibitors of Plk1 (a key regulator
ACCEPTED MANUSCRIPT
of mitotic progression and cell division in the eukaryotes) in a concert with cyclin-dependent kinase 1–
cyclin B1 and Aurora kinases to orchestrate a wide range of critical cell cycle events leading to a
prometaphase-like mitotic (G2/M) arrest [36,50].
The antileukemic effect of variety 5-ene-4-thiazolidinones is one of the most prominent directions
that was demonstrated in our studies [2,7,9,21], as well as in investigations of other scientists [34]. The
crucial role of the C5-ene fragment of 4-thiazolidinones in the antileukemic effect was also confirmed,
and its complication was shown as a potent direction for compounds optimization.
Present work is an extension of our ongoing efforts towards a search for new 4-thiazolidinone-
based anticancer agents. Here we addressed the synthesis of new 5-ene-4-thiazolidinones, evaluation of
their anticancer effects in the leukemia and carcinoma cells in vitro, and study of signaling pathways
involved in apoptosis induction in tumor cells treated with the synthesized 4-thiazolidinones.
2. Results
2.1. Chemistry
Design of molecular structure of target compounds was based on our former findings [9,10,51], as
well as on following items: i) belonging of target compounds to the 5-ene-4-thiazolidinone sub-type; ii)
combination of pyrazoline and thiazolidinone cores within hybride pharmacophore approach; iii)
introduction of substituents in N3 position of main core – as a phase of compounds modification aimed
at a decrease of its general toxicity [52]; iv) geometry of molecular structures (C5 ring, thiazolidinone
core and N3 residue) meets the suggesting that the three-ring system might be required for the
anticancer activity [48,53]; v) introduction of OH group in ortho position of the phenyl moiety (C5
fragment) is desirable for anticancer effect realization [54].
Synthesis of the 4-thiazolidinone derivatives was performed according to earlier described 2-
stages protocol based on the modification of methylene group of the thiazolidinone core [55,56]. The
procedure included condensation with triethyl orthoformate, and the obtained 5-ethoxy-4-
thiazolidinones were converted into the appropriate enamines under the reaction with appropriate 3,5-
6

diaryl-2-pyrazolines [51] (Fig. 1). Rhodanine аnd isorhodanine were used as basic cores in order to
ACCEPTED MANUSCRIPT
establish structure-activity relationships.
Fig. 1.
Structure of the synthesized compounds was confirmed by the elemental analysis and
1
spectroscopic data (1H NMR, 13C NMR and LCMS). H NMR spectra of synthesized compounds
showed characteristic patterns of an AMX system for protons at positions 4 and 5 of pyrazoline ring.
The proton of the methylene group (=CH) showed a singlet at 7.19-7.95 ppm (Z-isomer), and broad
singlet of the NH proton of the thiazolidinone ring appeared at 12.32-12.97 ppm.
The X-ray analysis (Fig. 2) revealed that the crystal of compound 1 is a channel-forming solvate
crystal with strongly disordered ethanol molecules. There are four symmetry-independent molecules A –
D and three and a half solvent molecules in the asymmetric part of the unit cell. The solute molecules
differ strongly in terms of conformation and the differences are related to the arrangement of two cyclic
systems, i.e. 2-hydroxyphenyl and 3-acetoxyphenyl (Fig. 3). The other systems in 5-(3-phenyl-4,5-
dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one moiety, i.e. 2-thioxothiazolidin-4-one, 5-
dihydropyrazole and phenyl group, in molecules A – D are approximately coplanar. 2-Hydroxyphenyl
and 3-acetoxyphenyl rings form with the mean plane of 5-(3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-2-thioxothiazolidin-4-one the dihedral angles of 88.65(6), 86.11(7), 71.98(5) i 70.20(6)°
and 86.80(6), 52.35(6), 103.37(7), 113.45(7)°, respectively.
Fig. 2.
The arrangement of the mentioned systems are also described with torsion angles C2−N3−C7−C8
and N19−C23−C30−C31 which reveal values of -95.1(3), 123.7(3), 76.3(3), -114.4(3)° and -84.4(3), -
98.3(3), -87.3(3), -90.7(2)°, respectively.
In molecules A and C the carbonyl oxygen atom O15 of acetoxyl group is disordered and takes
two alternative positions a and b (Fig. 3) with site occupancy factors in molecule A of 0.910(7) and
0.090(7), in molecule C of 0.514(9) and 0.486(9), respectively. In molecule D the whole acetoxyl group
is disordered where the atoms O13D, C14D, O15D, C16D (Fig. 2 and 3) take two alternative positions
with site occupancy factors of 0.911(3) and 0.089(3), respectively.
Fig. 3.
7

ACCEPTED MANUSCRIPT
2.2. Screening of anticancer activity
In vitro screening of anticancer activity of the synthesized compounds towards human
promyelocytic leukemia cells of HL-60 line and human ovarian carcinoma cells of Skov3 line was
performed by the MTT assay. Doxorubicin was used as a reference drug in positive control. Tested
compounds were added to cultured cells in different concentrations (0 – 10.0 ꢀM) and the cells were
treated for 72 h. The screening results were expessed as IC50 and outlined in the Table 1. The tested
compounds possessed different antiproliferative action that was generally weaker comparing with the
doxorubicin, although some of them possess micromolar meaning of the ІС50.
Table 1
N-substituted derivatives were characterized by higher level of activity (comparison of
compounds 8 and 9), presence of the aromatic moiety is better in comparison with the aliphatic one
(compounds 7, 4, 3 and 1, 2). It should be stressed that position of the substituent in the benzene
fragment (N3-possition of thiazolidinone core) is of great significance here (e.g. different level of
activity of compounds 1 and 2). These findings need further investigation since there were no such
peculiarities when the activities of the compounds 18 and 12, 13 were compared. It was found that OH
group in the ortho position (R2, Fig. 1) was optimal (comparison of 12, 13 аnd 14), while a complication
of arylidene fragments of the pyrazoline ring could be considered as a direction to increase of the
anticancer activity (compounds 2, 11 аnd 12). Compounds based on isorhodanine core (19, 20)
possessed higher antiproliferative activity that argues a relevance of this direction.
In general, tested compounds were less active towards Skov3 cells growing in a substrate-
dependent manner comparing with such activity towards HL-60 cells growing in suspension: compound
18: IC50 = 0.163 ꢀM versus 0.400±0.052 ꢀM, compound 2: IC50 = 0.244 ꢀM versus 9.400±2.04 ꢀM, and
compound 3: IC50 = 0.341 ꢀM versus 2.880±0.192 ꢀM, correspondingly.
Among tested compounds, 1 and 8 possessed the highest antiproliferative activity (IC50 0.12 and
0.1 ꢀM, correspondingly) towards human leukemia HL-60 cells, however, they were not affecting
substrate-dependent human ovarian carcinoma cells of Scov3 line. The compound 1 was selected for
further in depth study, and it was screened in different concentrations (0.01-10.0 ꢀM) towards a set of
8

the mammalian tumor cell lines of different origin. A 72 h continuous drug exposure protocol was used
ACCEPTED MANUSCRIPT
with the MTT assay.
In vitro screening was held towards the pseudonormal cell lines (HEK 293T, KS 483) and L929
transformed cell line, all growing in a substrate-dependent manner. There was no inhibition of cell
viability observed in the case of testing HEK 293T and KS 483 cells (Fig. 4). Compound 1 also did not
possess toxic action towards L929 cells. Thus, proliferation of the pseudonormal cells was not inhibited
under the effect of compound 1.
Fig. 4.
The anticancer effect in vitro of the compound 1 was also studied using human leukemia Jurkat T-
cells, murine leukemia L1210 cells, and human breast carcinoma MCF-7 cells. It was found that (Z)-5-
[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one possessed a cytotoxic action towards L1210 cells (IC50 = 0.7 ꢀM) and Jurkat
cells (IC50 = 1.1 ꢀM), however, its toxicity towards MCF-7 cells was expressed only in high doses (IC50
= 8.3 ꢀM), suggesting leukemia cell specificity of its action.
Following prominent effect of compound 1 on growth of human leukemia HL-60 cells, the HL-
60/ADR (adriamycin-resistant cells with over-expression of P-glycoprotein) sub-line of these cells was
involved in the study. Compound 1 was less effective toxicant for the HL-60/ADR cells (IC50 = 1.00
ꢀМ) than for their wild type (IC50 = 0.12 ꢀM) (Fig. 4), such effect was much more prominent than the
effect of doxorubicin – “golden standart” in the chemotherapy (IC50 = 8.8 ꢀМ). The mechanisms
responsible for leukemia cell specificity of cytotoxic action of the 1 should be studied additionally.
The cytotoxic effect of compound 1 towards HL-60 and HL-60/ADR human leukemia cells was
dose- and time-dependent (3, 6, 24, 48 and 72 h). It demonstrated the antiproliferative action towards
HL-60 as soon as in 6 h after its addition to cultured cells (IC50 = 0.5 ꢀМ), while its toxicity towards
HL-60/ADR was prominent only after 24 h (IC50 = 1.0 ꢀМ) (Fig. 5).
Fig. 5.
2.3. Mode of action
The anticancer effect of cisplatin and doxorubicin (“classical” anticancer drugs) is accompanied
by an increase in the intracellular level of the reactive oxygen species (ROS) that may contribute to their
9

therapeutic effect [57]. To investigate whether the 4-thiazolidinone derivatives kill tumor cells by free
ACCEPTED MANUSCRIPT
radicals/ROS-depended manner, we have monitored the 1 action using well known antioxidants – N-
acetylcysteine (NAC) and ascorbic acid (AA). The NAC is an analogue and precursor of the glutathione
and it displays cancer preventive properties not only at early stages of carcinogenesis process but also
during its advanced stages. NAC is a source of sulfhydryl groups in cells and scavenger for free radicals.
It interacts with ROS such as OH· and H2O2, thus, preventing apoptosis and promoting cell survival
[41]. NAC (1.0 mM) had a protective effect on the cytotoxic action of compound 1 towards leukemia
cells over a period of 72 h exposure (Fig. 6).
Fig. 6.
Ascorbic acid deficiency is accompanied by an increased oxidative stress and tissue injury
including oxidant-induced necrotic cell death. It prevents oxidative modification of both cytosolic and
membrane components of cells [58]. We found that AA (25.0 ꢀМ) under compound 1 treatment
significantly enhanced survival of HL-60 and HL-60/ADR cells compared to the level of compound 1
action (Fig. 6). Thus, cytotoxic effect of compound 1 in tumor cells was blocked by the NAC and AA
which inhibited the action of ROS. The ROS are an effective weapon used by the anticancer
chemotherapeutics against tumor cells [11]. Probably, the compound 1 induced the accumulation of
ROS in human leukemia HL-60 and HL-60/ADR cells that causes its antitumor effect under most
probably mitochondria-mediated apoptosis.
In the next experiments, the apoptosis-inducing potential of compound 1 was studied in more
detail. Supravital double staining with FITC-conjugated Annexin V and propidium iodide of the HL-60
cells treated for 48 h with 1 (0.1 and 1.0 ꢀM) showed that tested compound induced apoptosis in these
leukemia cells (Fig. 7B).
Western-blot analysis of proteins of treated cells was used to explore the molecular mechanisms
underlying the pro-apoptotic activity of the compound 1. The lysates were obtained from human
promyelocytic leukemia HL-60 cells treated for 48 h with (Z)-5-[5-(2-hydroxyphenyl)-3-phenyl-4,5-
dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one (1) (0.1; 0.5; 1.0 ꢀM) or
doxorubicin (0.1 ꢀM), used as a positive control. It was suggested that antitumor action of the 4-
thiazolidinones is associated with their affinity to pro- and anti-apoptotic members of the family of Bcl-
10

2 proteins [25]. Here we have found that under the action of 1 the level of the pro-apoptotic protein Bax
ACCEPTED MANUSCRIPT
was increased, while the level of the anti-apoptotic protein Bcl-XL was decreased (Fig. 7A).
Thus, we have studied activation of the MAPKs such as JNK, ERK and p38 in HL-60 cells treated
with the compound 1 (Fig. 7A). It was found that compound 1 markedly stimulated phosphorylation of
the MAPK p38 at 48 h after the start of treatment. We have found that compound 1 inhibited c-Jun N-
terminal kinase (JNK) phosphorylation that can mediate inhibition of c-Jun phosphorylation [48].
Inhibition of JNK is accompanied by the activation of STAT 3 [52], we have found the increasing of
pSTAT3 expression (Fig. 7A). Besides, we have found the activation of ERK1/2 and Endo Gunder the
action (48 h) of compound 1.
Fig. 7.
A loss of proper control of the cell cycle in the mammalian cells is one of the main engines
leading to cellular transformation [59]. We studied the effect of compound 1 on cell cycling in HL-60
leukemia cells treated with that compound for 8 and 48 h. The obtained flow cytometry data (Fig. 8A)
demonstrate that compound 1 affects HL-60 cell cycle and increases a percentage of cells in the G0/G1
phase, while the doxorubicin increased the number of the G2/M phase cells [60]. Thus, compound 1
cause the G0/G1arrest of cells that differs it from the pharmacological profile of the related 4-
thiazolidinones.
As shown in Fig. 8B, the treatment of HL-60 leukemia cells with compound 1 caused an increase
in the level of p21 and Cdk1 proreins. p21 directly inactivates Cdk2 by blocking cell transition from the
G1 to S phase of cell cycle. That was proved by the flow cytometry results (Fig. 8A).
Fig. 8.
We found that (Z)-5-[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-
acetoxyphenyl)-2-thioxothiazolidin-4-one (1) inhibited phosphorylation of the Rb (Retinoblastoma)
protein and decreased the amount of the Cyclin D3 in HL-60 leakemia cells (Fig. 8B). The activity of
this protein is required for the G1/S transition in the cell cycle. This correlates with the obtained results
of the Western-blot analysis of expression of pRb (Fig. 8B).
Summarizing, compound 1 induces apoptosis with activation of Bax, MAPKs, Endo G by
mitochondria-mediated pathway and causes G0/G1 arrest in cell cycle of the treated cells.
11

ACCEPTED MANUSCRIPT
3. Discussion
In vitro screening of the anticancer activity of synthesized 4-thiazolidinone derivatives towards
human promyelocytic leukemia cells of HL-60 line showed that among tested compounds (Z)-5-[5-(2-
hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-
4-one 1 and (Z)-5-[5-(4-dimethylaminophenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-
acetoxyphenyl)-2-thioxothiazolidin-4-one 8 possessed the highest antiproliferative activity (IC50 0.12
and 0.10 ꢀM, correspondingly), however, they were not affecting human ovarian carcinoma cells of
Scov3 line growing in a substrate-dependent manner. Tested compounds based on the isorhodanine core
possessed higher antiproliferative activity that argues the relevance of this direction. Moreover,
modification of pyrazoline core could be considered as a perspective direction for increase of the
anticancer activity of new N-substituted 5-ene-thiazolidinones. Proliferation of the pseudonormal cells
was not inhibited under the effect of compound 1. Similar results were obtained when the rat myoblast-
derived cells of L-6 line were treated [51]. Recently, we described a marked anti-trypanosomal activity
of such compounds [50]. Taken together, these data may suggest an existence of common mechanisms
of their cytotoxic activity towards tumor and protozoa cells [5,24]. Thus, the synthesized substances can
be considered as so-called multi-target compounds (see above).
A resistance to the anthracycline antibiotics and other chemotherapeutics due to P-glycoprotein
(P-gp)-mediated export of xenobiotics is a frequent problem in cancer treatment. Compound 1 was more
effective toxicant for the HL-60/ADR cells (IC50 = 1.00 ꢀМ) than the effect of doxorubicin (IC50 = 8.8
ꢀМ). It was shown that structurally related compounds were not inhibiting P-glycoproteins and acted
independently on the P-glycoprotein mechanisms [47,48,53]. Such characteristics could be inherent to
the compounds under this study, since their structure does not correspond to structural requirements of
inhibitors of P-glycoproteins [53]. Presented findings confirm our statement about leukemia-specific
targeting of the 5-ene-4-thiazolidinone derivatives [7,9].
The mechanisms responsible for cytotoxic action of the compound 1 were also studied. The
anticancer effect of cisplatin and doxorubicin is known to be accompanied by an increase in the
intracellular level of the reactive oxygen species that contribute to their therapeutic effect [57]. High
12

levels of ROS in mitochondria (as a primary source of ROS) can result in free radical attack of
ACCEPTED MANUSCRIPT
membrane phospholipids and cause mitochondrial membrane depolarization [61]. Moreover, ROS play
a key role in the mitochondria-mediated apoptosis. Thus, compound 1 induced the accumulation of ROS
in human leukemia HL-60 and HL-60/ADR cells that causes its antitumor effect under most probably
mitochondria-mediated apoptosis. This is an irreversible step associated with the attack of membrane
phospholipids, release of mitochondrial factors, and triggering caspase cascades [62]. It was reported
that ROS level increased under the action of 5-ene-4-thiazolidinones in HT29 and CEM cells, and it was
inhibited by the AA [11,63]. Besides, it was shown that 5-(4-hydroxy-3-methoxybenzylidene)-2-thioxo-
4-thiazolidinone significantly elevated the Parkin and Miro2 expression levels in the acute MPTP-
treated mice, and stabilized mitochondrial membrane potential and ATP synthesis in MPP+-treated
Neuro-2a cells [64]. It is also worth to note about a search for potent antioxidants among the 4-
thiazolidinones including compounds with the anticancer potential [65]. 2-Benzo[d]isothiazolylimino-5-
benzylidene-4-thiazolidinones were shown to possess high antioxidant effect, NO lowering and GAGs
restoring capability, and also reduced the production of the metalloproteinases and NF-κB expression
[66]. It is desired that new anticancer agents do not only induce the apoptosis of tumor cells leading to
quick phagocytosis of the remnants of tumor cells by the macrophages, but also possess a capability to
diminish the negative side effects of applied chemotherapy. Thus, novel 5-ylidene-4-thiazolidinone
derivatives could be of great use in that regard.
It was reported that the anticancer effects of related 4-thiazolidinones could be realized by
apoptosis induction in different ways – through PARP-, MAPK-, JNK-, Bcl-2-, CDK1/cyclin B
inhibition or be dependent on caspase cascades [6,48].
It was suggested that antitumor action of 4-thiazolidinones is associated with their affinity to
pro- and anti-apoptotic members of the family of Bcl-2 proteins [25]. These proteins can stimulate the
mitochondrial outer membrane permeabilization and a release of proteins of the inter-membrane space
into the cytosol. After induction of the apoptotic stimuli by the anticancer drugs, the pro-apoptotic
proteins of that family, including Bax, Bak and Bad, are induced in accordance with their sensitivity to
the anticancer drugs [67]. The homodimerized Bax acts on the VDAC (Voltage-dependent anion
channel) localized in the outer membrane of mitochondria, thus, resulting in a release of cytochrome c
13

which activates the caspase cascade [68,69]. It was also reported that a release of cytochrome c is
ACCEPTED MANUSCRIPT
inhibited by the antiapoptotic proteins, such as Bcl-2 and Bcl-XL [68]. Here we have found that under
the action of compound 1 the level of the pro-apoptotic Bax protein was increased, while the level of the
antiapoptotic Bcl-XL protein was decreased.
It is known that cellular stresses and stimuli can induce apoptosis via modulating the mitogen-
activated protein kinases (MAPK) signaling pathway [48,70] (inhibition of JNK, and activation of p38,
ERK under the action of 1). The activated p38 was shown to phosphorylate p53 [46]. Inhibition of JNK
is accompanied by the activation of STAT 3 (pSTAT3 expression increasing). The activation/inhibition
of JNK modulates the phosphorylation of mitochondrial proteins during apoptosis. ERK can regulate
cell growth at the transcription level of an early response gene, such as c-fos, mediated by STAT3 (Ser
727) activation. Activation of ERK also causes phosphorylation of the nuclear substrate STAT3 that
correlates well with our results. Activation of the ERK1/2 and EndoG leads to a release of cytochrome c
and subsequent formation of the apoptosomes through the mitochondria-mediated apoptosis pathway
[70]. It was also reported that 4-thiazolidinone derivatives (e.g. MMPT) induced apoptosis in lung
cancer cells by caspase-3, -8, -9, and poly(ADP-ribose) polymerase cleavage and cytochrome c release
from the mitochondria [48]. Besides, a loss of proper control of cell cycle in the mammalian cells is one
of the main engines leading to cellular transformation [59]. Compound 1 causes the G0/G1arrest of cells
that differs it from the pharmacological profile of the related 4-thiazolidinones.
Treatment of the HL-60 leukemia cells with compound 1 caused an increase in the level of p21
and Cdk1 proteins. As a proliferation inhibitor, p21 can play an important role in preventing tumor
development. The mechanisms by which p21 may promote apoptosis could be related to its ability to
affect the components of the DNA repair machinery [59]. P21 directly inactivates Cdk2 by blocking the
transition from the G1 to S phase of cell cycle. Our findings demonstrate a correlation between the p21
expression and induction of the pro-apoptotic Bax protein. P21 induces the expression of the pro-
apoptotic Bax protein to the levels sufficient to inhibit the apoptotic Bcl-2 inhibitor that represents its
counterpart [71]. Elevation in the cyclin-dependent kinase 1 (Cdk1) plays a key role in control of the
eukaryotic cell cycle by modulating centrosome cycle, as well as a mitotic onset. It promotes the G2/M
14

transition and regulates G1 progress and the G1-S transition via association with the multiple interphase
ACCEPTED MANUSCRIPT
cyclins [71,72].
We have also found inhibition of phosphorylation of Rb and decrease in the amount of Cyclin D3
in the HL-60 leukemia cells under the action of compound 1. During cell cycle, phosphorylation of the
Rb protein along the G1 phase is exerted by the CDK2/CyclinE complex and leads to a release of the
E2F transcription factor that activates expression of genes needed for the S phase. Rb is a tumor
suppressor protein that in the dephosphorylated form does not release E2F, thus, avoiding cell cycle
progression by inhibiting the E2F activity [72]. The inhibition of Cyclin D3 correlates with the G0/G1
arrest in these cells. Cyclin D3 was shown to interact with the cyclin dependent kinases 4 and 6
(CDK4/6) and was involved in phosphorylation of the tumor suppressor Rb protein [73]. In conclusion,
the compound 1 induces apoptosis by the mitochondria-mediated pathway and causes G0/G1arrest in cell
cycle of the leukemia cells.
4. Conclusion
The panel of 5-ene-4-thiazolidinones was synthesized and screened for their anticancer activity in
vitro. (Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one (1) was selected as most active agent with a selective action against the
leukemic cells. It inhibits growth of human leukemia cells (HL-60 line) being applied even in the
nanomolar concentrations (IC50 = 118 nM). In addition, it possesses low toxicity towards the
pseudonormal cells. The obtained data suggest the mitochondria-depended apoptosis as the main mode
of 1’s action; compound 1 also induces the G0/G1arrest of the treated cells and causes inhibition of cell
division.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and methods
15

2-Thioxo-4-thiazolidinone (rhodanine) and N3-substituted-rhodanines, 4-thioxo-2-thiazolidinone
ACCEPTED MANUSCRIPT
(isorhodanine) [74], 3,5-diaryl-4,5-dihydro-1H-pyrazole [75] were used as starting reagents and
prepared according to the method described previously.
Melting points of newly synthesized compounds were measured in open capillary tubes on a
BUCHI B-545 melting point apparatus and are uncorrected. The elemental analysis (C, H, N) were
performed using a Perkin Elmer 2400 CHN analyzer. Analyses indicated by symbols of the elements or
1
functions were within ± 0.4% of the theoretical values. The H NMR spectra were recorded on Varian
Gemini 400 MHz and 13C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d6 using
tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units with use of δ scale.
Mass spectra were obtained using electrospray ionization techniques on an Agilent 1100 Series LCMS.
Analytical HPLC were performed on an Agilent 1100 HPLC with Diode Array Detection. Purity of all
compounds was determined to be ≥ 95% by the HPLC. The peak purity was checked with the UV
spectra.
4.1.2. General procedure of synthesis of 5-(3,5-diaryl-4,5-dihydropyrazol-1-ylmethylene)-2-
thioxothiazolidin-4-ones and 5-(3,5-diaryl-4,5-dihydropyrazol-1-ylmethylene)-4-thioxothiazolidin-2-
ones.
A
mixture of 5-ethoxymethylene-2-thioxothiazolidin-4-one or 5-ethoxymethylene-4-
thioxothiazolidin-2-one (5 mmol) [51] and appropriate 3,5-diaryl-4,5-dihydro-1H-pyrazole (5 mmol)
was refluxed in 10 mL of ethanol during 1 h. After cooling, the crystalline product was separated by
filtration, washed with ethanol, and dried. Re-crystallization from DMF:EtOH (1:1) rendered the target
product in a pure form.
4.1.2.1. (Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-
2-thioxothiazolidin-4-one (1). Spectral and analytical data are described [51].
4.1.2.2. (Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(4-acetoxyphenyl)-
1
2-thioxothiazolidin-4-one (2). Yield 74%, mp 277-279 ºC. H NMR (400 MHz, DMSO-d6): δ 10.03 (s,
16

1H, OH), 7.90 (d, 2H, J = 7.2 Hz, arom), 7.56-7.62 (m, 3H, arom), 7.46 (s, 1H, =CH), 7.32 (d, 2H, J =
ACCEPTED MANUSCRIPT
8.5 Hz, arom), 7.21-7.28 (m, 4H, arom), 6.84-6.92 (m, 2H, arom), 5.76 (dd, 1H, J = 11.5, 6.3 Hz,
CH2CH), 3.91 (dd, 1H, J = 18.0, 11.7 Hz, CH2CH), 3.51 (dd, 1H, J = 18.2, 6.4 Hz, CH2CH), 2.29 (s,
13
3H, CH3). C NMR (100 MHz, DMSO-d6): δ 193.9, 169.0, 165.9, 159.9, 155.2, 150.3, 133.3, 133.2,
131.1, 130.1, 130.0, 129.9, 129.2, 129.1, 127.1, 124.9, 122.4, 119.2, 115.9, 93.1, 62.0, 40.8, 20.9.
LCMS (ESI+) m/z 516 (M+H)+. Calcd. for C27H21N3O4S2: C, 62.90; H, 4.11; N, 8.15; Found: C, 62.70;
H, 4.40; N, 8.00%.
4.1.2.3.
(Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-methyl-2-
1
thioxothiazolidin-4-one (3). Yield 72%, mp 313-315 ºC. H NMR (400 MHz, DMSO-d6): δ 10.02 (s,
1H, OH), 7.84-7.90 (m, 2H, arom), 7.54-7.60 (m, 3H, arom), 7.43 (s, 1H, =CH), 7.20-7.25 (m, 2H,
arom), 6.88 (d, 1H, J = 7.7 Hz, arom), 6.86 (t, 1H, J = 7.5 Hz, arom), 5.73 (dd, 1H, J = 11.6, 6.5 Hz,
CH2CH), 3.88 (dd, 1H, J = 18.1, 11.8 Hz, CH2CH), 3.51 (dd, 1H, J = 18.1, 6.5 Hz, CH2CH), 3.28 (s,
13
3H, CH3). C NMR (100 MHz, DMSO-d6): δ 193.2, 165.9, 159.7, 155.2, 133.0, 131.0, 130.0, 130.0,
129.1, 129.0, 127.0, 124.9, 119.2, 115.9, 92.9, 62.0, 40.6, 30.6. LCMS (ESI+) m/z 396 (M+H)+. Calcd.
for C20H17N3O2S2: C, 60.74; H, 4.33; N, 10.62; Found: C, 60.90; H, 4.50; N, 10.50%.
4.1.2.4.
(Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-ethyl-2-
thioxothiazolidin-4-one (4). Yield 78%, mp 234-236 ºC. 1H NMR (400 MHz, DMSO-d6): δ 9.99 (s, 1H,
OH), 7.86 (m, 2H, Hz, arom), 7.53-7.60 (m, 3H, arom), 7.40 (s, 1H, =CH), 7.23 (d, 2H, J = 6.4 Hz,
arom), 6.88 (d, 1H, J = 6.5 Hz, arom), 6,85 (t, 1H, J = 7.1 Hz, arom), 5.72 (dd, 1H, CH2CH, J = 11.1,
6.2 Hz), 3.96 (m, 2H, CH2CH3), 3.90 (dd, 1H, CH2CH, J = 18.1, 11.5 Hz), 3.50 (dd, 1H, CH2CH, J =
13
18.2, 6.2 Hz), 1.09 (t, 3H, J = 6.7, Hz, CH3). C NMR (100 MHz, DMSO-d6): δ 193.5, 166.3, 160.4,
155.8, 133.7, 131.7, 130.6, 129.7, 129.6, 127.7, 125.5, 119.9, 116.5, 93.3, 62.5, 41.1, 31.0, 12.6. LCMS
(ESI+) m/z 410 (M+H)+. Calcd. for C21H19N3O2S2: C, 61.59; H, 4.68; N, 10.26; Found: C, 61.70; H,
4.80; N, 10.10%
17

4.1.2.5.
(Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(pyridin-3-yl)-2-
ACCEPTED MANUSCRIPT
thioxothiazolidin-4-one (5).Yield 72%, mp 292-294 ºC. 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H,
OH), 8.63 (d, 1H, J = 4.7 Hz, arom), 8.50 (brs, 1H, arom), 7.90 (d, 2H, J = 6.5 Hz, arom), 7.80 (d, 1H, J
= 8.2 Hz, arom), 7.55-7.61 (m, 4H, arom), 7.45 (s, 1H, =CH), 7.22-7.26 (m, 2H, arom), 6.89 (d, 1H, J =
7.8 Hz, arom), 6.87 (t, 1H, J = 7.5 Hz, arom), 5.77 (dd, 1H, J = 11.6, 6.7 Hz, CH2CH), 3.92 (dd, 1H, J =
13
17.9, 11.6 Hz, CH2CH), 3.55 (dd, 1H, J = 17.9, 6.2 Hz, CH2CH). C NMR (100 MHz, DMSO-d6): δ
194.0, 165.7, 160.2, 155.2, 149.5, 149.4, 136.7, 133.4, 132.9, 131.2, 130.1, 130.0, 129.1, 129.0, 127.1,
124.9, 123.9, 119.3, 115.9, 93.0, 62.1, 41.0. LCMS (ESI+) m/z 459 (M+H)+. Calcd. for C24H18N4O2S2:
C, 62.86; H, 3.96; N, 12.22; Found: C, 63.00; H, 4.10; N, 12.30%.
4.1.2.6. (Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-trifluoromethyl-
1
phenyl)-2-thioxothiazolidin-4-one (6). Yield 81%, mp 276-278 ºC. H NMR (400 MHz, DMSO-d6):
δ10.03 (s, 1H, OH), 7.90 (d, 2H, J = 5.9 Hz, arom), 7.83 (d, 1H, J = 8.0 Hz, arom), 7.72-7.77 (m, 2H,
arom), 7.64 (d, 2H, J = 8.2 Hz, arom), 7.59 (d, 2H, J = 6.7 Hz, arom), 7.46 (s, 1H, =CH), 7.22-7.26 (m,
2H, arom), 6.89 (d, 1H, J = 8.3 Hz, arom), 6.86 (t, 1H, J = 6.9 Hz, arom), 5.77 (dd, 1H, J = 11.4, 6.2 Hz,
CH2CH), 3.55 (dd, 1H, J = 18.1, 11.7 Hz, CH2CH), 3.51 (dd, 1H, J = 18.0, 5.9 Hz, CH2CH). 13C NMR
(100 MHz, DMSO-d6): δ 193.8, 165.7, 160.1, 155.2, 136.8, 133.9, 131.2, 130.2, 130.1, 130.0, 129.7 (q,
J = 32 Hz), 129.1, 129.1, 127.1, 126.0, 125.7, 125.0 (q, J = 272 Hz), 124.9, 119.2, 115.9, 93.1, 62.0,
41.1. LCMS (ESI+) m/z 526 (M+H)+. Calcd. for C26H18F3N3O2S2: C, 59.42; H, 3.45; N, 8.00; Found: C,
59.60; H, 3.60; N, 7.80%.
4.1.2.7.
(Z)-3-(5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-2-
1
thioxothiazolidin-4-one-3-yl)-propionic acid ethyl ester (7). Yield 75%, mp 258-260 ºC. H NMR (400
MHz, DMSO-d6): δ 10.02 (s, 1H, OH), 7.82-7.90 (m, 2H, arom), 7.53-76 (m, 3H, arom), 7.41 (brs, 1H,
=CH), 7.23 (m, 2H, arom), 6.82-6.90 (m, 2H, arom), 5.72 (dd, J = 11.6, 5.6 Hz 1H, CH2CH), 4.17 (t,
2H, J = 7.3 Hz, CH2), 4.01 (q, 2H, J = 7.1 Hz, CH2), 3.88 (dd, J = 18.3, 11.9 Hz, 1H, CH2CH), 3.52 (dd,
13
J = 17.9, 6.1 Hz, 1H, CH2CH,), 2.60 (t, 2H, J = 7.1Hz, CH2), 1.14 (t, 3H, J = 7.1Hz, CH3). C NMR
(100 MHz, DMSO-d6): δ 193.7, 170.8, 166.2, 160.6, 155.8, 133.8, 131.709, 130.6, 130.6, 129.7, 129.6,
18

127.7, 125.5, 119.8, 116.5, 93.0, 62.6, 60.8, 50.0, 40.7, 31.6, 14.4. LCMS (ESI+) m/z 482 (M+H)+.
ACCEPTED MANUSCRIPT
Calcd. for C24H23N3O4S2: C, 59.86; H, 4.81; N, 8.73; Found: C, 60.00; H, 5.00; N, 8.60%.
4.1.2.8.
(Z)-5-[5-(4-Dimethylaminophenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-
acetoxyphenyl)-2-thioxothiazolidin-4-one (8). Spectral and analytical data are described [51].
4.1.2.9.
(Z)-5-[5-(4-Dimethylaminophenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-2-
thioxothiazolidin-4-one (9). Spectral and analytical data are described [51].
4.1.2.10. (Z)-5-[3,5-Di-(4-chlorophenyl)-4,5-dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(10). Yield 72%, mp 289-291 ºC. 1H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H, NH), 7.83 (d, 2H, J =
8.4 Hz, arom), 7.65 (d, 2H, J = 8.6 Hz, arom), 7.51 (d, 2H, J = 8.4 Hz, arom), 7.41 (d, 2H, J = 8.4 Hz,
arom), 7.32 (s, 1H, =CH), 5.65 (dd, 1H, J = 11.1, 6.2 Hz, CH2CH), 4.00 (dd, 1H, J= 18.2, 11.9 Hz,
13
CH2CH), 3.46 (dd, 1H, J = 18.3, 5.8 Hz, CH2CH). C NMR (100 MHz, DMSO-d6): δ 195.9, 168.2,
157.5, 138.9, 135.6, 133.4, 132.3, 129.2, 129.1, 128.9, 128.8, 97.1, 64.2, 41.4. LCMS (ESI+) m/z
434/436/438 (M+H)+. Calcd. for C19H13Cl2N3OS2: C, 52.54; H, 3.02; N, 9.67; Found: C, 52.70; H, 3.20;
N, 9.80%.
4.1.2.11.
(Z)-5-[3,5-Di-(4-chlorophenyl)-4,5-dihydropyrazol-1-ylmethylene]-3-(4-acetoxyphenyl)-2-
1
thioxothiazolidin-4-one (11). Yield 70%, mp 284-286 ºC. H NMR (400 MHz, DMSO-d6): δ 7.85 (d,
2H, J = 8.1 Hz, arom), 7.67 (d, 2H, J = 7.9 Hz, arom), 7.55 (s, 1H, =CH), 7.52 (d, 2H, J = 7.9 Hz,
arom), 7.42 (d, 2H, J = 8.0 Hz, arom), 7.31 (d, 2H, J = 8.0 Hz, arom), 7.25 (d, 2H, J = 8.2 Hz, arom),
5.74 (dd, 1H, J = 10.6, 5.6 Hz, CH2CH), 4.05 (dd, 1H, J = 17.4, 10.8 Hz, CH2CH), 3.51 (dd, 1H, J =
13
16.8, 5.3 Hz, CH2CH), 2.29 (s, 3H, CH3). C NMR (100 MHz, DMSO-d6): δ 194.9, 169.6, 166.7,
158.9, 151.0, 136.4, 134.0, 133.9, 133.825, 130.5, 129.8, 129.5, 129.4, 129.3, 129.2, 123.0, 108.4, 95.0,
64.8, 41.9, 21.4. LCMS (ESI+) m/z 568/570/572 (M+H)+. Calcd. for C27H19Cl2N3O3S2: C, 57.04; H,
3.37; N, 7.39; Found: C, 57.20; H, 3.30; N, 7.20%.
19

4.1.2.12.
(Z)-5-[5-(2-Hydroxyphenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-ylmethylene]-3-(4-
ACCEPTED MANUSCRIPT
1
acetoxyphenyl)-2-thioxothiazolidin-4-one (12). Yield 79%, mp 241-242 ºC. H NMR (400 MHz,
DMSO-d6): δ 10.04 (s, 1H, OH), 8.32 (s, 1H, arom), 8.12 (brs, 2H, arom), 8.00-8.08 (m, 3H, arom), 7.98
(d, 2H, J = 8.8 Hz, arom), 7.52-7.66 (m, 3H, arom), 7.50 (s, 1H, =CH), 7.32 (d, 2H, J = 8.7 Hz, arom),
7.21-7.30 (m, 2H, arom), 6.90 (d, 1H, J = 8.3 Hz, arom), 6.87 (t, 1H, J = 7.0 Hz, arom), 5.81 (dd, 1H, J
= 11.5, 6.0 Hz, CH2CH), 4.02 (dd, 1H, J = 17.3, 11.6 Hz, CH2CH), 3.66 (dd, 1H, J = 18.2, 5.5 Hz,
13
CH2CH), 2.30 (s, 3H, CH3). C NMR (100 MHz, DMSO-d6): δ 193.9, 168.9, 165.9, 159.9, 155.2,
150.4, 133.8, 133.4, 133.12, 132.6, 129.9, 128.9, 127.8, 127.4, 127.0, 126.7, 126.4, 125.0, 123.64,
122.9, 122.4, 119.3, 115.9, 93.3, 62.1, 41.0, 20.9. LCMS (ESI+) m/z 566 (M+H)+. Calcd. for
C31H23N3O4S2: C, 65.82; H, 4.10; N, 7.43; Found: C, 66.00; H, 4.30; N, 7.20%.
4.1.2.13.
(Z)-5-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-ylmethylene]-3-(4-
1
acetoxyphenyl)-2-thioxothiazolidin-4-one (13). Yield 80%, mp 283-285 ºC. H NMR (400 MHz,
DMSO-d6): δ 8.30 (s, 1H, arom), 8.11 (brs, 2H, arom), 7.98-8.03 (m, 3H, arom), 7.58-7.62 (m, 2H,
arom), 7.52 (d, 2H, J = 7.5 Hz, arom), 7.44 (d, 2H, J = 8.0 Hz, arom), 7.33 (d, 2H, J = 8.5 Hz, arom),
7.26 (d, 2H, J = 8.0 Hz, arom), 5.79 (dd, 1H, J = 10.6, 5.5 Hz, CH2CH), 4.17 (dd, 1H, J = 17.3, 10.9 Hz,
CH2CH), 3.63 (dd, 1H, J = 17.3, 5.5 Hz, CH2CH), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
δ 194.8, 169.6, 166.7, 159.9, 151.0, 145.1, 139.8, 134.5, 134.1, 133.9, 133.8, 133.2, 130.5, 129.8, 129.4,
129.3, 129.2, 128.5, 128.4, 127.9, 127.7, 123.0, 94.9, 64.7, 42.0, 21.4. LCMS (ESI+) m/z 584/586
(M+H)+. Calcd. for C31H22ClN3O3S2: C, 63.74; H, 3.80; N, 7.19; Found: C, 63.90; H, 4.00; N, 7.10%.
4.1.2.14.
(Z)-5-[5-(4-Methoxyphenyl)-3-(naphthalen-2-yl)-4,5-dihydropyrazol-1-ylmethylene]-3-(4-
1
acetoxyphenyl)-2-thioxothiazolidin-4-one (14). Yield 80%, mp 256-258 ºC. H NMR (400 MHz,
DMSO-d6): δ 8.32 (s, 1H, arom.), 8.13 (brs, 2H, arom.), 7.99-8.05 (m, 2H, arom.), 7.60-76.3 (m, 2H,
arom.), 7.50 (s, 1H. =CH), 7.38 (d, J = 7.8 Hz, 2H, arom.), 7.32 (d, J = 7.8 Hz, 2H, arom.), 7.26 (d, J =
8.5 Hz, 2H, arom.), 7.01 (d, 2H, J = 8.5 Hz, arom.), 5.70 (m, 1H, CH2CH), 4.14 (dd, 1H, J = 18.5, 11.0
Hz, CH2CH), 3.77 (s, 3H, OCH3), 3.64 (dd,1H, J = 19.0, 6.0 Hz, CH2CH), 2.30 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6): δ 194.3, 169.2, 166.2, 159.8, 159.7, 150.6, 144.3, 134.1, 133.5, 132.8, 132.0,
20

130.1, 129.0, 128.90, 128.8, 128.6, 128.0, 128.0, 127.6, 127.2, 123.1, 122.6, 114.8, 94.0, 64.7, 55.3,
ACCEPTED MANUSCRIPT
41.5, 21.0. LCMS (ESI+) m/z 580 (M+H)+. Calcd. for C32H25N3O4S2: C, 66.30; H, 4.35; N, 7.25; Found:
C, 66.50; H, 4.50; N, 7.10%.
4.1.2.15. (Z)-5-[5-(2-Hydroxyphenyl)-3-(naphthalen-2-yl)-4,5-dihydropyrazol-1-ylmethylene]-3-methyl-
1
2-thioxothiazolidin-4-one (15). Yield 75%, mp 280-282 ºC. H NMR (400 MHz, DMSO-d6): δ 9.95 (s,
1H, OH), 8.28 (brs, 1H, arom.), 8.21-8.24 (m, 1H, arom), 8.09 (brs, 1H, arom), 7.93-8.06 (m. 2H,
arom), 7.56-7.59 (m, 2H, arom), 7.46 (s, 1H, =CH), 7.20-7.25 (m, 2H, arom), 6.91 (d, 1H, J = 7.6 Hz,
arom), 6.86 (t, 1H, J = 6.4 Hz, arom), 5.78 (dd, 1H, J = 11.2, 6.7 Hz, CH2CH), 4.03 (dd, 1H, J = 18.0,
13
11.0 Hz, CH2CH), 3.64 (dd,1H, J = 18.2, 6.4 Hz, CH2CH), 2.45 (s, 3H, CH3). C NMR (100 MHz,
DMSO-d6): δ 193.9, 166.6, 158.0, 155.8, 135.8, 134.1, 133.3, 130.6, 129.2, 128.3, 128.0, 127.6, 127.3,
127.0, 125.6, 124.2, 123.5, 119.9, 116.5, 93.7, 62.6, 15.2,. LCMS (ESI+) m/z 446 (M+H)+. Calcd. for
C24H19N3O2S2: C, 64.70; H, 4.30; N, 9.43; Found: C, 64.90; H, 4.10; N, 9.30%.
4.1.2.16.
(Z)-5-[5-(2-Hydroxyphenyl)-3-(naphthalen-2-yl)-4,5-dihydropyrazol-1-ylmethylene]-3-
1
(piridin-3-yl)-2-thioxothiazolidin-4-one (16). Yield 81%, mp 260-262 ºC. H NMR (400 MHz, DMSO-
d6): δ 10.05 (s, 1H, OH), 8.64 (m, 1H, arom), 8.52 (s, 1H, arom), 8.42 (s, 1H, arom), 8.33 (s, 1H, arom),
8.23 (d, 1H, J = 8.7 Hz, arom), 8.13 (brs, 2H, arom), 7.91-8.08 (m, 4H, arom), 7.82 (d, 2H, J = 7.5 Hz,
arom), 7.58-7.65 (m, 4H, arom), 7.53 (s, 1H, =CH), 7.23-7.29 (m, 2H, arom), 6.90 (d, 1H, J = 7.8 Hz,
arom), 6.87 (t, 1H, J = 7.3 Hz, arom), 5.75 (dd, 1H, J = 11.0, 6.5 Hz, CH2CH), 4.03 (dd, 1H, J = 17.5,
11.5 Hz, CH2CH), 3.68 (dd, 1H, J = 17.6, 6.4 Hz, CH2CH). 13C NMR (100 MHz, DMSO-d6): δ 193.9,
165.7, 160.2, 155.2, 149.5, 149.4, 136.8, 133.9, 133.4, 132.9, 132.6, 130.1, 129.0, 128.7, 128.6, 127.8,
127.5, 127.0, 124.9, 123.9, 122.9, 119.3, 115.9, 93.2, 62.2, 41.1 LCMS (ESI+) m/z 509 (M+H)+. Calcd.
for C28H20N4O2S2: C, 66.12; H, 3.96; N, 11.02; Found: C, 66.30; H, 4.20; N, 10.90%.
4.1.2.17.
(Z)-5-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-ylmethylene]-3-(3-
acetoxyphenyl)-2-thioxothiazolidin-4-one (17). Spectral and analytical data are described [51].
21

4.1.2.18.
(Z)-5-[5-(4-Chlorophenyl)-3-(naphthalen-2-yl)-4,5-dihydropyrazol-1-ylmethylene]-2-
ACCEPTED MANUSCRIPT
1
thioxothiazolidin-4-one (18). Yield 81%, mp 283-285 ºC. H NMR (400 MHz, DMSO-d6): δ 12.96 (s,
1H, NH), 8.26 (s, 1H, arom), 8.09 (d, 1H, J = 8.7 Hz, arom), 8.06 (d, 1H, J = 8.9 Hz, arom), 8.00 (t, 2H,
J = 8.9 Hz, arom), 7.59-7.64 (m, 2H, arom), 7.52 (d, 2H, J = 8.3 Hz, arom), 7.44 (d, 2H, J = 8.3 Hz,
arom), 7.37 (s, 1H, arom), 5.73 (dd, 1H, J = 11.5, 6.3 Hz, CH2CH), 4.13 (dd, 1H, J = 18.2, 11.4 Hz,
13
CH2CH), 3.59 (dd, 1H, J = 18.1, 6.1 Hz, CH2CH). C NMR (100 MHz, DMSO-d6): δ 195.8, 168.2,
162.2, 158.6, 139.1, 133.8, 133.3, 132.5, 132.4, 129.2, 128.9, 128.6, 128.6, 127.8, 127.4, 127.0, 122.9,
96.9, 64.1, 41.5. LCMS (ESI+) m/z 450/452 (M+H)+. Calcd. for C23H16ClN3OS2: C, 61.39; H, 3.58; N,
9.34; Found: C, 61.50; H, 3.70; N, 9.20%.
4.1.2.19. (Z)-5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-ylmethylene]-4-thioxothiazolidin-
2-one (19). Spectral and analytical characteristics are described [51].
4.1.2.20.
(Z)-5-[5-(4-Chlorophenyl)-3-(naphtalen-2-yl)-4,5-dihydropyrazol-1-ylmethylene]-4-
1
thioxothiazolidin-2-one (20). Yield 82%, mp 293-285 ºC. H NMR (400 MHz, DMSO-d6): δ 12.52 (s,
1H, NH), 8.18 (s, 1H, =CH), 8.05 (d, 1H, J = 8.5 Hz, arom), 7.91-7.97 (m, 4H, arom.), 7.52-7.58 (m,
2H, arom), 7.40-7.48 (m, 4H, arom), 5.82 (dd, 1H, J = 10.9, 5.9 Hz, CH2CH), 4.12 (dd, 1H, J = 17.9,
11.5 Hz, CH2CH), 3.53 (dd, 1H, J = 18.0, 5.9 Hz, CH2CH). 13C NMR (100 MHz, DMSO-d6): δ 187.7,
172.9, 160.6, 139.4, 137.5, 134.6, 134.1, 133.1, 129.9, 129.7, 129.6, 129.3, 129.2, 128.5, 128.4, 127.8,
127.7, 123.8, 105.7, 65.3, 41.5. LCMS (ESI+) m/z 450/452 (M+H)+. Calcd. for C23H16ClN3OS2: C,
61.39; H, 3.58; N, 9.34; Found: C, 61.50; H, 3.60; N, 9.10%.
4.1.3. Crystal structure determination of 5-[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one (1).
Crystal Data: C27H21N3O4S2, 0.75(C2H6O), Mr = 550.14, triclinic, space group P-1, a =
11.41562(10) Å, b = 15.6249(2) Å, c = 29.8642(4) Å, α = 88.0515(11)°, β = 86.1259(9)°, γ =
78.3790(9)°, V = 5204.56(11) Å3, Z = 8, Dcalc = 1.404 /cm3, ꢀ = 2.226 mm−1, T = 130(2) K.
22

Data Collection: A yellow plate crystal (CH3CH OH) of 0.16 mm × 0.08 mm× 0.05 mm was used
ACCEPTED MA2NUSCRIPT
to record 52573 (Cu Kα radiation, θmax = 76.5°) intensities on an Agilent SuperNova four-circle
diffractometer equipped with an Atlas CCD detector (CrysAlis PRO, Version 1.171.37.35; Agilent
Technologies Ltd. (Oxford Diffraction): Yarnton, UK, 2014) using mirror monochromatized Cu Kα
radiation from a high-flux microfocus source (λ = 1.54178 Å). Accurate unit cell parameters were
determined by least-squares techniques from the θ values of 25621 reflections, θ range 2.9−76.0°. The
data were corrected for Lorentz polarization and for absorption effects (CrysAlis PRO, Version
1.171.37.35; Agilent Technologies Ltd. (Oxford Diffraction): Yarnton, UK, 2014). The 20510 total
unique reflections (Rint = 0.0304) were used for structure determination.
Structure Solution and Refinement. The structure was solved by direct methods (SHELXS-97),
and refined against F2 for all data (SHELXL-97) [76]. The positions of the H atoms bonded to O36A,
O36B, O36C and O36D atoms were obtained from the difference Fourier maps and were refined freely.
The remaining H atoms were placed geometrically in calculated positions and were refined with a riding
model, with C−H = 0.98 Å (CH3), 0.99 Å (CH2), 1.00 Å (Csp3H), 0.95 Å (CarH) and Uiso(H) = 1.2Ueq(C)
or 1.5Ueq(C) for methyl H atoms. The methyl groups were refined as rigid groups, which were allowed
to rotate. Final refinement converged with R = 0.0520 (for 17462 data with F2 > 4σ(F2)), wR = 0.1382
(on F2 for all data), and S = 1.068 (on F2 for all data). The largest difference peak and hole was 0.649
and -0.480 e Å−3. The molecular illustrations were drawn using ORTEP-3 for Windows [77].
Crystallographic
data
for
(Z)-5-[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one have been deposited at the Cambridge
Crystallographic Data Centre (CCDC), 12 Union ROAD, Cambridge CB2 1EZ (U.K.) (phone, (+44)
1223/336-408; fax, (+44) 1223/336-033; e-mail, deposit@ccdc.cam.ac.uk; World Wide Web,
http://www.ccdc.cam.ac.uk (deposition no. CCDC 1432661)).
4.2. Biological experiments
4.2.1. Materials
Stock solutions of 4-thiazolidones (10 mM) were prepared in the dimethyl sulfoxide (Sigma-
Aldrich, USA), and additionally dissolved in culture medium prior to addition to the cell culture. 3-(4,5-
23

Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (99.5% pure), DMEM, N-acetyl-
ACCEPTED MANUSCRIPT
cysteine (NAC), ascorbic acid (AA), propidium iodide (PI), Hoechst-33258 and FITC-conjugated
Annexin Vwere obtained from Sigma-Aldrich, USA; RPMI and fetal bovine serum were obtained from
APP, Austria. Doxorubicin was purchased from Pharmachemie B.V., the Netherlands.
4.2.2. Cell cultures
Human promyelocytic leukemia cells of HL-60 line, adriamycin-resistant cells of HL-60/ADR
line with over-expression of P-glycoprotein, and human ovarian carcinoma cells of Skov3 line were
obtained from the American type culture collection (ATCC, Minnesota, USA). Human acute T-cell
leukemia cells of Jurkat line, mouse lymphocytic leukemia cells of L1210 line, human breast
adenocarcinoma cells of MCF-7 line, transformed mouse fibroblasts of L929 line, human embryonic
kidney cells of HEK293T line, and murine mesenchymal preosteoblastic cells of KS 483 line were
obtained from Collection at R.E. Kavetsky Institute of Experimental Pathology, Oncology and
Radiology (Kyiv, Ukraine). Cells were grown in the RPMI or DMEM culture medium supplemented
with 10% fetal bovine serum. Cells were cultivated in the CO2-thermostate at 37oC in atmosphere of
95% air and 5% CO2.
4.2.3. Cytotoxicity assays
In vitro screening of anticancer activity of the synthesized compounds and doxorubicin used as a
reference drug control towards cancer cell lines was measured by the MTT test [78]. Tumor cells were
seeded for 24 hours in 96-well microtiter plates at a concentration of 2,000 substrate-dependent
cells/well or 10,000 suspension cells/well (100 ꢀL/well), after that cells were incubated for 72 hours
with various additions of the synthesized compounds (0; 0.01; 0.1; 0.5; 1.0; 10.0 ꢀM). MTT which is
converted to dark blue, water insoluble MTT formazan by the mitochondrial dehydrogenases, was used
to determine viable cells according to the manufacturer’s protocol (Sigma-Aldrich, USA). The IC50 of
tested compounds was calculated as a lethal concentration of drug killing 50 % cells in comparison with
an untreated culture.
24

For estimating the impact of the antioxidants (AA and NAC) on the cytotoxic activity of 4-
ACCEPTED MANUSCRIPT
thiazolidinones, different doses of drugs were used in combination with cell pretreatment for 30 min
with the non-toxic doses of the antioxidants (NAC at 1.0; 2.0 mM and AA at 25.0; 100.0 ꢀM).
4.2.4. Fluorescent microscopy
Duplicate slides were prepared for each cell type/treatment group and >500 cells were counted in
each sample. FITC-conjugated Annexin V and PI double staining were performed in order to detect
early apoptotic events in HL-60 cells treated with 4-thiazolidinones. In 48 h after the addition of the
tested compound, the HL-60 cells were pelleted by centrifugation at 2,000 rpm, washed twice with 1x
PBS, and incubated for 15 min in the Annexin V binding buffer containing 1/20 volume of FITC-
conjugated Annexin V solution and PI (20.0 ꢀg/mL). 10 ꢀL of cell suspension were added on slides and
cover glasses were placed over them. Сells were examined under Zeiss AxioImager A1 fluorescent
microscope (Carl Zeiss, Germany).
4.2.5. Western blot analysis
After 48 h exposure of the tested compound, cell proteins were isolated, resolved by the
SDS/PAGE, and transferred onto a polyvinylidenedifluoride (PVDF) membrane for Western blotting, as
described [60]. The following antibodies were used in this study at a 1:1.000 dilution: anti-p38, anti-
phospho-ERK ½ (Thr 202/Tyr 204), anti-EndoG, anti-phospho-STAT3 (Tyr 705), anti-p21
(Waf1/Cip1), anti-phospho-cdc2 (Tyr 15) (Cdk1), anti-phospho-Rb (Ser 807/811) (Cell Signaling
Technology, USA), anti-Bax (sc-6236), anti-Bcl-XL/S (sc-634), anti-JNK (sc-571), anti-CyclinD3 (C-
16, sc-182) (Santa Cruz Biotec, USA). Equal loading of each lane was evaluated by the immunoblotting
of the same membrane with the anti-beta-actin monoclonal mouse AC-15 (Sigma-Aldrich, USA). All
secondary peroxidase-labelled antibodies (Cell Signaling, USA) were used at working dilution of
1:5,000.
4.2.6. Cell cycle analysis
25

HL-60 cells were seeded into 6-well plates and then treated for 8 and 48 h with the tested
ACCEPTED MANUSCRIPT
compounds at different concentrations. To analyze cell cycle distribution, cells were collected, washed
with the PBS, fixed with 70% ethanol, and stored at –20°C. For analysis, cells were transferred in the
PBS, incubated with RNAse (10 ꢀg/ml) for 30 min at 37°C, treated for 30 min with propidium iodide (5
ꢀg/ml), and analyzed by flow cytometry (Becton Dickinson, Palo Alto, CA).
4.2.7. Statistical analysis
All data are presented as the mean ± standart deviation (SD). Results were analyzed and illustrated
with GraphPad Prism (version 5; GraphPad Software, San Diego, CA). Statistical analyses were
performed using two-way ANOVA with Bonferroni post-tests (apoptosis induction, tumor growth). P-
value of <0.05 was considered as statistically significant.
Acknowledgement
The authors support all people of good will currently struggling for the liberty and justice in
Ukraine. The authors thank the Association for Regional Cooperation in the Fields of Health, Science
and Technology (RECOOP HST Association) and Dr. Sandor Vari (president of the RECOOP HST
Association) for their support. This work was partially supported by the West-Ukrainian Biomedical
Research Center (for Julia Senkiv and Natalia Finiuk in 2015/2016).
Supplementary data
The representative 1H and 13C NMR spectra of synthesized compound are presented.
References
[1] A.C. Tripathi, S.J. Gupta, G.N. Fatima, P.K. Sonar, A. Verma, S.K. Saraf, 4-Thiazolidinones: The
advances continue, Eur. J. Med. Chem. 72 (2014) 52–77.
[2] R.B. Lesyk, B.S. Zimenkovsky, 4-Thiazolidones: centenarian history, current status and perspectives
for modern organic and medicinal chemistry, Cur. Org. Chem. 8(16) (2004) 1547–1577.
26

[3] T. Tomašic, L.P. Mašic. Rhodanine as a scaffold in drug discovery: a critical review of its
ACCEPTED MANUSCRIPT
biological activities and mechanisms of target modulation, Expert Opin. Drug Dis. 7(7) (2012) 549-560.
[4] V.S. Jain, D.K. Vora, C.S. Ramaa, Thiazolidine-2,4-diones: progress towards multifarious
applications, Bioorg. Med. Chem. 21(7) (2013) 1599–1620.
[5] A. Kryshchyshyn, D. Kaminskyy, P. Grellier, R. Lesyk, Trends in research of antitrypanosomal
agents among synthetic heterocycles, Eur. J. Med. Chem. 85 (2014) 51–64.
[6] V. Asati, D.K. Mahapatra, S.K. Bharti, Thiazolidine-2,4-diones as multi-targeted scaffold in
medicinal chemistry: potential anticancer agents, Eur. J. Med. Chem. 87 (2014) 814–833.
[7] D. Kaminskyy, D. Khyluk, O. Vasylenko, L. Zaprutko, R. Lesyk, A facile synthesis and anticancer
activity evaluation of spiro[thiazolidinone-isatin] conjugates, Sci. Pharm. 79(4) (2011) 763–777.
[8] D. Kaminskyy, B. Zimenkovsky, R. Lesyk, Synthesis and in vitro anticancer activity of 2,4-
azolidinedione-acetic acids derivatives, Eur. J. Med. Chem. 44 (2009) 3627–3636.
[9] D. Havrylyuk, B, Zimenkovsky, O, Vasylenko, A, Gzella, R. Lesyk, Synthesis of new 4-
thiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity, J. Med.
Chem. 55(20) (2012) 8630–8641.
[10]D. Kaminskyy, I. Subtel'na, B. Zimenkovsky, O. Karpenko, A. Gzella, R. Lesyk, Synthesis and
evaluation of anticancer activity of 5-ylidene-4-aminothiazol-2(5H)-one derivatives, Med. Chem.
(Shariqah UAE), 11(6) (2015) 517–530.
[11]G. Revelant, S. Huber-Villaume, S. Dunand, G. Kirsch, H. Schohn, S. Hesse, Synthesis and
biological evaluation of novel 2-heteroarylimino-1,3-thiazolidin-4-ones as potential anti-tumor agents,
Eur. J. Med. Chem. 94 (2015) 102–112.
[12]B.Z. Ahn, D.E. Sok, Michael acceptors as a tool for anticancer drug design, Curr. Pharm. Design,
2(3) (1996) 247–262.
[13]J.B. Baell, G.A. Holloway, New substructure filters for removal of pan assay interference
compounds (PAINS) from screening libraries and for their exclusion in bioassays, J. Med. Chem. 53
(2010) 2719−2740.
27

[14]T. Mendgen, C. Steuer, C.D. Klein, Privileged scaffolds or promiscuous binders: a comparative
ACCEPTED MANUSCRIPT
study on rhodanines and related heterocycles in medicinal chemistry, J. Med. Chem. 55(2) (2012) 743–
753.
[15]H.J. Forman, K.J.A. Davies, F. Ursini, How do nutritional antioxidants really work: nucleophilic
tone and para-hormesis versus free radical scavenging in vivo, Free Rad. Biol. Med. 66 (2014) 24–35.
[16]J.C.W. Hildyard, C. Ämmälä, I.D. Dukes, S.A. Thomson, A.P. Halestrap, Identification and
characterization of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate
carrier, BBA Bioenergetics, 1707(2-3) (2005) 221–230.
[17]J.B. Baell, Observations on screening-based research and some concerning trends in the literature,
Future Med. Chem. 2(10) (2010) 1529–1546.
[18]B. Meunier, Hybrid molecules with a dual mode of action: dream or reality?, Acc. Chem. Res.
41(1) (2008) 69–77.
[19]Q. Zhang, H. Zhou, S. Zhai, B. Yan, Natural product-inspired synthesis of thiazolidine and
thiazolidinone compounds and their anticancer activities, Curr. Pharm. Design, 16(16) (2010) 1826–
1842.
[20]D. Havrylyuk, B. Zimenkovsky, R. Lesyk, Synthesis, biological activity of thiazolidinones bearing
indoline moiety and isatin based hybrids, Mini Rev. Org. Chem. 12(1) (2015) 66–87.
[21]D. Kaminskyy, B. Bednarczyk-Cwynar, O. Vasylenko, O. Kazakova, B. Zimenkovsky, L.
Zaprutko, R. Lesyk, Synthesis of new potential anticancer agents based on 4-thiazolidinone and
oleanane scaffolds, Med. Chem. Res. 21(11) (2012) 3568–3580.
[22]S. Avdieiev, L. Gera, D. Havrylyuk, R.S. Hodges, R. Lesyk, V. Ribrag, Y. Vassetzky, V. Kavsan,
Bradykinin antagonists and thiazolidinone derivatives as new potential anti-cancer compounds, Bioorg.
Med. Chem. 22(15) (2014) 3815–3823.
[23]K.Nepali, S.Sharma, M.Sharma, P.M.S. Bedi, K.L. Dhar, Rational approaches, design strategies,
structure activity relationship and mechanistic insights for anticancer hybrids, Eur. J. Med. Chem. 77
(2014) 422–487.
28