Synthesis and biological evaluation of novel 2-heteroarylimino-1,3-thiazolidin-4-ones as potential anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2015.02.053

A series of 35 heteroarylimino-1,3-thiazolidinones with three sites of functionalization were synthesized and their antiproliferative properties were studied. The in vitro screening by MTT assay was performed against five cancer cell lines (human colon cancer cell lines HT29, HCT116 and SW620 and breast cancer cell lines MCF7 and MDA-MB-231). It was observed that N3-substituted thiazolidinones had moderate activities whereas 5-benzylidene thiazolidinones showed promising activities. To investigate the mechanism of action, detailed biological studies of six selected compounds (those presenting the lower mitotic index) were carried out on the human colon cancer HT29 cell line. Cell cycle assay revealed that those compounds induced cell accumulation in G2/M and in subG0/G1 phases of cell cycle. Moreover, dissipation of mitochondria membrane potential was observed as well as redox changes in treated cells.

Full text of DOI:10.1016/j.ejmech.2015.02.053

Accepted Manuscript
Synthesis and biological evaluation of novel 2-heteroarylimino-1,3-thiazolidin-4-ones
as potential anti-tumor agents
Germain Revelant, Sophie Huber-Villaume, Sandrine Dunand, Gilbert Kirsch, Hervé
Schohn, Stéphanie Hesse
PII:
S0223-5234(15)00149-X
10.1016/j.ejmech.2015.02.053
EJMECH 7735
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 September 2014
Revised Date: 26 February 2015
Accepted Date: 27 February 2015
Please cite this article as: 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, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.053.
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
Graphical abstract
H
O
N
N
S
S
R
Cl
4(a-l)
4b
4c
4e
R = p-methoxyphenyl
R = p-methylphenyl,
R = p-dimethylaminophenyl
4k
R = 3,4-dimethoxyphenyl

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel 2-heteroarylimino-1,3-thiazolidin-4-ones as
potential anti-tumor agents
Germain Revelant1,a, Sophie Huber-Villaume2,b, Sandrine Dunand1, Gilbert Kirsch1,
Hervé Schohn2, ** and Stéphanie Hesse1, *
1.
Université de Lorraine - UMR CNRS 7565 - Structure et Réactivité des
Systèmes Moléculaires Complexes – Equipe 3 (HECRIN) - 1 Boulevard Arago, 57070
Metz Technopôle, France
2.
Université de Lorraine - UMR CNRS 7565 - Structure et Réactivité des
Systèmes Moléculaires Complexes – Equipe 5 (2MIC) – Campus Bridoux – rue du
Général Delestraint – 57070 Metz Cedex, France
*Corresponding author: Tel.: +33 3 87 54 74 97. Fax: + 33 3 87 31 58 01.
** Corresponding author: Tel.: +33 3 87 37 84 09. Fax:+ 33 3 87 31 58 01
E-mail addresses: herve.schohn@univ-lorraine.fr (H. Schohn), stephanie.hesse@univ-lorraine.fr (S.
Hesse)
a and b, Authors contributed equally to this article
Keywords : thiazolidinone, thiophene, thiazole, antiproliferative activity, colon cancer
Abstract
A series of 35 heteroarylimino-1,3-thiazolidinones with three sites of functionalization were
synthesized and their antiproliferative properties were studied. The in vitro screening by MTT
assay was performed against five cancer cell lines (human colon cancer cell lines HT29,
HCT116 and SW620 and breast cancer cell lines MCF7 and MDA-MB-231). It was observed
that N3-substituted thiazolidinones had moderate activities whereas 5-benzylidene
thiazolidinones showed promising activities. To investigate the mechanism of action, detailed
biological studies of six selected compounds (those presenting the lower mitotic index) were
carried out on the human colon cancer HT29 cell line. Cell cycle assay revealed that those
compounds induced cell accumulation in G2/M and in subG0/G1 phases of cell cycle.
Moreover, dissipation of mitochondria membrane potential was observed as well as redox
changes in treated cells.

ACCEPTED MANUSCRIPT
1. Introduction
Colon cancer is one of the most frequent cancer diseases in the world. Most popular anti-
cancer drugs belong to molecule groups which interfere with the progression of tumour cell
through cell cycle progression by limiting DNA synthesis or DNA damage reparation. In
addition, another group of drugs consists in the inhibition either of the formation of mitotic
spindle or in protein function, especially protein activity, involved in these mechanisms [1].
Other strategies consist in the development of chemotherapeutic drugs which inhibit G2/M
transition concomitantly with apoptosis cell death, such as thiazolidinone derivatives [2].
Thiazolidinone moiety belongs to an important family of heterocyclic scaffold presenting
diverse biological activities such as antimicrobial [3], antidiabetic [4] or anticonvulsant [5].
This scaffold has attracted continuing interest over the years [6-7]. Especially, 2-imino-4-
thiazolidinones have also been investigated for their pharmacological properties. For example,
darbufelone has showed anti-inflammatory activity [8], whereas 2-heteroarylimino-1,3-
thiazolidin-4-ones, especially thiazolylimino- and benzothiazolylimino- derivatives, were
described as antifungal or antibacterial compounds [9, 10]. 5-Benzylidene-2-phenylimino-1,3-
thiazolidin-4-ones I were reported for their anticancer activity [11] and a complete SAR study
was realized on their anti-tumour activity against non-small cell lung cancer cell line H460
and its paclitaxel-resistant variant H460taxR (Fig. 1) [12]. 2-Phenylimino-5-(3-
methoxybenzylidene)-3-propyl-1,3-thiazolidin-4-one II was proved to induce cell growth
arrest after human colon adenocarcinoma-derived HT29 cells exposure for 72h (IC50
=
64.1µM) [13]. Recently thiazolidin-4-ones III substituted with indolin-2-one fragment were
evaluated against four human cancer cells as HT29, human gastric H460, mammary
adenocarcinoma-derived MDA-MB-231 and sarcoma-derived SMMC-7721 cells, and the
compounds showed promising antiproliferative activity [14].
Novel 2-(heteroarylimino)-1,3-thiazolidin-4-ones IV with three different sites of
functionalization were synthesized and tested for their antiproliferative effect against human
colon and breast cancer–derived cells by MTT assay. Based on this first screening, several
molecules were selected to investigate their mechanism of action. Especially, cell cycle
distribution, induction of apoptosis and redox changes were studied.
2. Results and discussion
2.1. Chemistry
A first series of six 2-[5-aryl-3-thienyl]imino-1,3-thiazolidin-4-ones 1-6 were synthesized
according to the synthetic pathway described in Scheme 1. 3-Aminothiophenes were reacted
with chloroacetyl chloride and then with ammonium thiocyanate [15]. A second series of

ACCEPTED MANUSCRIPT
compounds 4(a-l) were synthesized starting from 4, the most active compound of the first
series. For this purpose, derivative 4 was condensed with several aldehydes in presence of
pyrrolidine in methanol (Scheme 1 and Supporting Information S1.1).
To test effect of nitrogen N3 substitution on antiproliferative activity, the synthesis of a third
series of compounds was carried out using two different pathways highlighted in Scheme 2.
Firstly, 3-aminothiophenes were converted into the corresponding isothiocyanates. Those
compounds were then either condensed with p-anisidine to give the corresponding thioureas
which were reacted with ethyl chloroacetate and cyclized to led compounds 7-11 (steps b and
c in Scheme 2) [16] or isothiocyanates were condensed with chloroacetamides in basic media
to allow formation of compounds 12-17 (step d in Scheme 2 and Supporting Information
S1.2).
Finally, variation was made on heteroarylimino moiety. Derivatives 18-19 were synthesized
starting from 3-aminoselenophene [17] and aminothiazole. Those thiazolidinones were then
modified by introducing substituents on position 5 to give derivatives 20-24 (Scheme 3 and
Supporting Information S1.3).
2.2. Biological results
2.2.1. In-vitro cytotoxicity
Using the MTT method, antiproliferative activity of synthesized compounds was evaluated
against human colon cancer cell lines (HT29, HCT116 and SW620) and breast cancer cells
(MCF7 and MDA-MB-231). Among compounds 1-6 (Table 1), compound 4 inhibited HT29
cell growth with an IC50 estimated at 17.1 ± 7.2 µM but had however no effect on the other
cell lines used (IC50> 50µM). It should also be noted that compound 18, selenophene
analogue of compound 4, displayed no cytotoxicity whatever the cell line tested (Table 1)
whereas compound 19, thiazole analogue of compound 4, showed moderate anticancer
activity against HT29 and HCT116 cells (IC50 values estimated at 8.9 ± 2.1 µM and 23.8 ±
6.8 µM, respectively).
Derivatives 4 (a-l), obtained starting from 4, presented diverse subtitutions at 5-position of the
thiazolidine ring (Scheme 2 and Table 1). With exception of derivatives 4g and 4i (bearing
respectively 4-hydroxybenzylidene and heptylidene), all those compounds were more potent
than 4 (IC50< 12 µM on HT29 cells). 4-Methoxybenzylidene (4b), 4-methylbenzylidene (4c),
4-chlorobenzylidene (4d), 4-dimethylaminobenzylidene (4e), 4-bromobenzylidene (4f), 2-
naphtylidene (4h) and 4-fluorobenzylidene (4l) substitutions gave the higher cell growth
inhibitory effect. Similar results were obtained when the experiments were performed with the
other cell lines with some exceptions: SW620 cell exposure to compounds 4b and 4h led to
IC50 estimated at 28.1 ± 12.4 and > 50 µM, respectively. A similar IC50 was calculated when

ACCEPTED MANUSCRIPT
HT29 (11.5 ± 7.0 µM) or MDA-MB-231cells (10.9 ± 0.5 µM) were treated with compound
4j, but higher IC50values were obtained when the other cell lines were tested.
Compounds 7-17 presenting diverse substitutions on N3 of the thiazolidinone ring were also
evaluated (Scheme 2 and Table 1). With the exception of compound 9, the synthesized
derivatives were less efficient to alter colorectal or mammary cell growth than compounds
4(a-l).
Finally, derivatives 20-24, synthesized starting from 19, were evaluated for their respective
cytotoxicity (Scheme 3 and Table 1). IC50 values were at the micromolar range after cell
exposure to compounds 20-22. Similar results were obtained when cells were exposed to
compound 23, with the exception of SW620 cells (IC50 = 20.3 ± 2.8 µM). In addition,
treatment with compound 24 was efficient to inhibit HT29 and HCT116 cell growth, but it
had no effect when the other cell lines were used. IC50 values were however greater than those
obtained for compounds 20-22 (Table 1).
Collectively, compounds 4(a-l) and 20–23 reduced colorectal cell growth. Some of those
compounds were selected to further investigate mechanisms of HT29 cell death.
2.2.2. Cell cycle assay
To confirm results obtained by MTT procedure, HT29 cells were treated with each compound
at concentration corresponding to IC50 value over three days with daily medium changes.
Cells were counted by the Trypan blue method. In these conditions, cell growth inhibition
reached 90 % when cells were exposed to compounds 4b, 4c, 4e or 4k (Fig. 2A). Lower
inhibitions were obtained with compound 4d (25%), 4f (25%), 4h (50%), 4j (40%), 4l (25%)
or 9 (50%). However, when HT29 cells were treated with compounds 20-23 at concentration
corresponding to IC50 value, cell growth inhibition was less than 25% (Fig. 2B). HT29 cells
were therefore treated with 1-10 µM of each compound (Fig. 2B). In these experimental cell
culture conditions, cell growth inhibition reached 90 %, when HT29 cells were exposed to 2.5
µM of compound 20 or 22. Growth inhibition was estimated at 40% after cell exposure to
10µM of compound 21 (Fig. 2B). When cells were treated with compound 23, less than 5 %
cell growth inhibition was observed whatever the concentration tested (not shown).
Cell growth kinetic curves were further established and HT29 cell viability was tested by flow
cytometry after treatment with compound 4b, 4c, 4e or 4k over three days (Fig. 3A). Each
compound altered dramatically cell growth and levels of living cells decreased concomitantly
(Fig. 3B). Similar results were obtained after treatment with 2.5 µM of compound 20 or 22
(Fig. 3C and 3D) but the effect on cell viability was lower as compared to the results obtained
with compound 4 derivatives suggesting that those compounds had rather a cytostatic impact
on HT29 growth.

ACCEPTED MANUSCRIPT
Cell distribution in cell cycle phases after cell exposure to each compound was analyzed. Cell
accumulation in G2/M phase was found after cell exposure to compound 4b, 4c, 4e or 4k
(Table 2). When HT29 cells were treated with compound 20 or 22, cells accumulated slightly
in G2/M phase. Interestedly, cell exposure to compound 4b, 4c, 4e, 4k, 20 or 22 involved cell
accumulations in sub-G1 fraction, supporting that each treatment triggered cells to apoptosis
cell death (Table 2). Moreover, it was observed by the use of DAPI probe loaded on treated
cell layers that chromatin condensation and nuclear fragmentation occurred in treated cells
(Supporting Information S2). Cell death was associated also with the loss of mitochondrial
membrane potential (ψm) as demonstrated by the absence of J-aggregates when HT29 cells
were exposed to each compound together with JC-1 fluorescent probe (Supporting
Information S2).
Since IC50 was near or less than 10 µM (Table 1), impact of compounds 4a, 4f, 4j and 9 was
also studied (Fig. 2A). In brief, whatever the molecule tested, cell exposure to each compound
was not associated to cell accumulation in sub-G1 fraction. Cell treatment with compound 4a,
4f or 4j increased cell distribution in S-phase of cell cycle whereas cells accumulated slightly
in G0/G1 after exposure to compound 9. Collectively, those results suggested that molecule
treatment was associated rather to cytostatic cell growth arrest.
2.2.3. Changes in redox statut
Oxidative stress is related to the induction of autophagy, the cell defence against cell injury.
Ultimately, activation of autophagy process is linked to cell death in association or not with
apoptosis [18, 19]. As oxidative stress induces apoptosis [20], ROS and RNS levels were
quantified in cells exposed to thiazolidin-4-one derivatives 4b, 4c, 4e, 4k, 20 or 22.
Fluorescent probe DCF-2DA was used to quantify any change in ROS levels after cell
exposure to thiazolidin-4-one derivatives over 6 hours (Fig. 4). ROS levels reached a peak
one hour after compound 4b treatment (Fig. 4A). ROS levels increased with the maximum
reached at 4 hours and they decreased over 6 hours to ROS content found in DMSO-treated
cells. A similar situation was obtained when cells were exposed to compound 4k; but ROS
levels reached a peak 30 min after drug treatment. Compound 4e treatment increased
gradually ROS production and reached maximum at 4 hours; ROS levels decreased slightly
thereafter. It was similar to that obtained when HT29 cells were treated with compound 4c.
Similar ROS production curve was observed when cells were exposed to compound 20. ROS
levels reached a peak at 3 hours whereas compound 22 did not alter ROS content within the
cells over 6 hours-treatment duration (Fig. 4B).
DAF-2DA detects reactive oxygen- and nitrogen-derived species and notably nitric oxide.
DHR123 probe is used to detect peroxynitryl radical. As shown in table 3 and with the
exception of compound 22, each compound treatment involved the production of RNS species
detected by DAF-2DA probe. In addition slight but significant decreases of peroxynitryl

ACCEPTED MANUSCRIPT
radical levels were obtained when cells were treated with compound 4b, 4c, 4e, 4k or 22,
whereas peroxynitryl content increased after cell exposure to compound 20 (Table 3).
Since thiazolidin-4-one derivative treatment induced the production of reactive species with
the exception of compound 22, the cytoplasmic GSH content was quantified as it is often
depleted in the presence of an excess of reactive species (Table 3). GSH content was
decreased up to 30 % when cells were exposed to compound 4c whereas 10-15%-GSH
decreases were obtained after cell exposure to compounds 4b and 4e. In contrast, there was no
significant change in GSH levels in cell homogenates treated with compound 4k or 20.
Finally, pre-treatment with ascorbic acid, an antioxidant, protected cells against stress injury
but it did not rescue HT29 cell growth (Table 4) supporting that the oxidative stress observed
was not solely involved in cell death observed.
Moreover, generation of reactive species could activate the NF-2E related factor 2/Kelch like-
ECH-associated protein 1 (Nrf2/Keap1) pathway, involved to protect cells against oxidative
stress [21]. Activation of the Nrf2/Keap1 pathway leads to the stabilization of Nrf2 which
translocates in nuclei and binds to Maf protein. The complex, after binding to specific DNA
sequence, defined as antioxidant response element localised in the promoter of Nfr2/Maf
target genes, increases the transcription of genes involved in reactive species-mediated
response such as Heme Oxygen-1 (HO-1), NADH quinone oxidase-1 (NQO1) or glutamyl-
cysteyl ligase (GCL). As shown in figures 4C and 4D, HT 29 treatment with derivatives 4c
and 4d did not induced HO-1 expression. In contrast, NQO-1 and GCL expression were
increased supporting that other pathways could regulate the expression of the enzymes as
described previously [22, 23]. However, the increases of GCL expression, which is the rate-
limiting enzyme in the synthesis of GSH, correlated well with the decreases of GSH levels
observed when cells were treated with thiazolidin-4-one derivatives 4c and 4e (Table 3 and
Fig. 4C). Similarly, cell exposure to derivative 4k did not affect GSH content and GCL
expression stayed constant (Table 3 and Fig. 4D). Finally, the distribution of Nrf2 protein by
cell fractionation was also studied. However, Nfr2 protein levels were not increased in nuclei
homogenates prepared from treated cells over 6 hours. Similar results id est absence of Nrf2
translocation and no increase of HO-1, NQO-1 or GCL respective expression, were obtained
when HT29 cells were treated with compounds 4b, 4e, 20 and 22 (Supporting Information S3
and S4).
2.2.4. Autophagy detection
As cell growth arrest and redox changes in treated cells have been observed, autophagy
detection was realized. As shown in figure 5A, acidic vesicles contents were increased within
HT29 treated cells over two days, as demonstrated by the use of acridine orange dye.
Moreover, beclin-1 expression, a marker of autophagosome formation, was induced after cell
exposure to each molecule (Fig. 5B). Thus, our results supported that HT29 exposure to

ACCEPTED MANUSCRIPT
thiazolidin-4-one derivatives was associated with autophagy process. However, it could not be
concluded whether autophagy process results in HT29 cell apoptosis cell death or whether
compound treatment is associated to autophagy cell death as previously discussed [24].
3. Conclusions
35 Thiazolidinone derivatives were tested for their antiproliferative activity against a panel of
five cancer cell lines. It was shown that substitution with an aryl group on N3 of
thiazolidinone ring reduced dramatically the anti-cancer activity whereas substitution by a
benzylidene group on position 5 is really important. Especially, it was found that compounds
with a p-methoxybenzylidene (4b), p-methylbenzylidene (4c), p-dimethylaminobenzylidene
(4e) and 3,4-dimethoxybenzylidene (4k) enhanced the impact on HT29 cell growth arrest. For
these compounds, cell underwent apoptosis cell death after cell accumulation in G2/M phase
of the cell cycle, as described previously with another thiazolidinone derivative. On the other
hand, it was demonstrated that cell exposure to the selected compound was associated with
the production of reactive species, and the induction of autophagy which could lead to cell
death [24]. Of note, increasing the production of reactive species represents also an alternative
among the chemotherapeutic strategies against cancer cells (for review, [25]). Therefore,
further studies are needed to decipher cell death mechanism involved in HT29 cell exposed to
the selected thiazolidin-4-one derivatives.
4.
Experimental
4.1. Chemistry
All solvents were of reagent grade and, when necessary, were purified and dried by standard
methods. All chemicals were purchased from Acros or Sigma-Aldrich. All reactions were
routinely checked by TLC analysis on an Alugram SIL G/UV254 (Macherey-Nagel) with spots
visualized by UV light. The concentration of solutions after reactions and extractions involved
the use of a rotary evaporator operating at reduced pressure. Organic solutions were dried
over anhydrous magnesium sulfate, and column chromatography was performed using silica
gel 60 (50-200 µm diameter). Melting points were determined in open capillaries on a Stuart
SMP3 apparatus and are uncorrected. The 1H and 13C NMR spectra were measured on an AC
Bruker 250 MHz spectrometer in CDCl3 or DMSO-d6; chemical shifts are reported in parts
per million (ppm). All coupling constants (J) are given in Hz. MS spectra were recorded on an
Agilent Technologies GC-MS instrument equipped with a 7683 injector, 6890N gas
chromatograph and a 5973 mass selective detector. The mass spectrometer was operated in EI
mode at 70 eV, and MS spectra were recorded from m/z 50 to 650. Elemental analyses (C, H,
N, S) were used to confirm the purity of all compounds (>95%) and were performed on a
CHN ThermoScientific Flash 2000 apparatus.
Thienyliminothiazolidinones 1-6 and their N-methoxyphenyl derivatives 7-11 were obtained
via already reported procedures respectively [15] and [16].
4.1.1. General method for the functionalization of 5-position of the thiazolidinone.
Access to compounds 4(a-l) and 20-24

ACCEPTED MANUSCRIPT
Thiazolidinone 4 or 19 (0.5 mmol) was dissolved in methanol (20 mL) and pyrrolidine (0.5
mmol) was added. The corresponding aldehyde (0.5 mmol) was added under stirring and the
mixture was refluxed overnight. After cooling to room temperature, the precipitating product
was filtered, washed with methanol (10 mL) and diethyl ether (10 mL), and dried.
It must be noted that 13C NMR of some following products can not be performed due to their
low solubility.
4.1.2. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-benzylidene-1,3-thiazolidin-4-one (4a)
1
Yellow solid ; yield : 56% ; mp 269°C ; H NMR (DMSO-d6) δH7.18 (s, 1 H, CH), 7.40 (s, 1
13
H, CH), 7.45-7.78 (m, 10 H, 10 × CH), 12.11 (s, 1 H, NH); C NMR (DMSO-d6) δC 112.8,
118.3, 121.1, 126.8, 129.1, 129.2, 129.6, 129.8, 130.6, 131.9, 132.2, 132.4, 133.3, 133.8,
141.1, 141.8; HRMS (ESI) : m/z calcd for [C20H13ClN2OS2 + H]+ : 397.0231 ; found :
397.0256.
4.1.3. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-methoxybenzylidene)-1,3-
thiazolidin-4-one (4b)
1
Yellow solid ; yield : 75% ; mp 285°C ; H NMR (DMSO-d6) δH 3.77 (s, 3 H, OCH3), 7.03-
13
7.16 (m, 3 H, 3 × CH), 7.39-7.77 (m, 8 H, 8 × CH) , 12.17 (s, 1 H, NH); C NMR (DMSO-
d6) δC 55.3, 114.8, 120.9, 126.8, 126.9, 129.1, 129.2, 129.7, 130.9, 131.5, 131.7, 131.9, 132.2,
132.4, 132.6, 141.8, 160.6; HRMS (ESI) : m/z calcd for [C21H15ClN2O2S2 + H]+ : 427.0336 ;
found : 427.0354.
4.1.4. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-methylbenzylidene)-1,3-thiazolidin-
4-one (4c)
Yellow solid ; yield : 69% ; mp 284°C ; 1H NMR (DMSO-d6) δH2.31 (s, 3 H, CH3), 7.17 (s, 1
H, CH), 7.27-7.52 (m, 7 H, 7 × CH), 7.58-7.77 (m, 3 H, 3 × CH) , 12.26 (s, 1 H, NH); HRMS
(ESI) : m/z calcd for [C21H15ClN2OS2 + H]+ : 411.0387 ; found : 411.0402.
4.1.5. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-chlorobenzylidene)-1,3-thiazolidin-
4-one (4d)
1
Pale Brown solid ; yield : 71% ; mp 282°C ; H NMR (DMSO-d6) δH7.16 (s, 1 H, CH), 7.39-
7.77 (m, 10 H, 10 × CH) , 12.27 (s, 1 H, NH); HRMS (ESI) : m/z calcd for [C20H12Cl2N2OS2
+ H]+ : 430.9841 ; found : 430.9844.
4.1.6. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-dimethylaminobenzylidene)-1,3-
thiazolidin-4-one (4e)
Orange solid ; yield : 79% ; mp 288°C ; 1H NMR (DMSO-d6) δH 2.96 (s, 6 H, 2 × CH3), 6.75-
6.84 (m, 2 H, 2 × CH), 7.15 (s, 1 H, CH), 7.36-7.71 (m, 8 H, 8 × CH) , 12.02 (s, 1 H, NH);
HRMS (ESI) : m/z calcd for [C22H18ClN3OS2 + H]+ : 440.0653 ; found : 440.0655.
4.1.7. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-bromobenzylidene)-1,3-thiazolidin-
4-one (4f)
Green solid ; yield : 68% ; mp 298°C ; 1H NMR (DMSO-d6) δH7.16 (s, 1 H, CH), 7.39 (s, 1 H,
CH), 7.45-7.77 (m, 9 H, 9 × CH), 12.33 (s, 1 H, NH); HRMS (ESI) : m/z calcd for
[C20H12BrClN2OS2 + H]+ : 476.9315 ; found : 476.9322.

ACCEPTED MANUSCRIPT
4.1.8. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-hydroxybenzylidene)-1,3-
thiazolidin-4-one (4g)
1
Green solid ; yield : 76% ; mp 253°C ; H NMR (DMSO-d6) δH6.84-6.92 (m, 2 H, 2 × CH),
7.13 (s, 1 H, CH), 7.38-7.70 (m, 8 H, 8 × CH) , 12.33 (s, 1 H, NH); 13C NMR (DMSO-d6) δC
112.4, 116.2, 118.8, 121.0, 124.2, 124.6, 126.8, 129.1, 130.1, 131.8, 132.0, 132.2, 132.4,
141.0, 141.7, 159.3; HRMS (ESI) : m/z calcd for [C20H13ClN2O2S2 + H]+ : 413.0180 ; found :
413.0218.
4.1.9. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(2-naphtylidene)-1,3-thiazolidin-4-one
(4h)
1
Yellow solid ; yield : 89% ; mp 288°C ; H NMR (DMSO-d6) δH7.21 (s, 1 H, CH), 7.41-8.17
(m, 13 H, 13 × CH) , 12.29 (s, 1 H, NH), 13C NMR (DMSO-d6) δC 112.2, 112.8, 118.1, 120.8,
124.1, 126.7, 126.8, 128.1, 128.3, 129.1, 129.2, 129.3, 131.1, 131.3, 131.8, 132.1, 132.2,
132.4, 132.7, 134.4, 141.1, 141.9; HRMS (ESI) : m/z cald for [C24H15ClN2OS2 + H]+ :
447.0387 ; found : 447.0385.
4.1.10. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-heptylidene-1,3-thiazolidin-4-one (4i)
1
Brown solid ; yield : 60% ; mp 191°C ; H NMR (DMSO-d6) δH0.78-0.84 (m, 3 H, CH3),
1.22-1.39 (m, 8 H, 4 × CH2), 2.10-2.14 (m, 2 H, CH2), 6.70-6.73 (m, 1 H, CH), 7.07 (s, 1 H,
CH), 7.34 (s, 1 H, CH), 7.44-7.52 (m, 2 H, 2 × CH), 7.63-7.71 (m, 2 H, 2 × CH), 12.05 (s, 1H,
NH); 13C NMR (DMSO-d6) δC 13.9, 21.9, 27.3, 28.3, 30.9, 32.5, 112.4, 118.2, 120.9, 126.7,
129.1, 131.9, 132.2, 132.3, 132.5, 134.6, 141.0, 141.6; HRMS (ESI) : m/z calcd for
[C20H21ClN2OS2 + H]+ : 405.0857 ; found : 405.0859.
4.1.11. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(2-pyridylmethylene)-1,3-thiazolidin-
4-one (4j)
1
Orange solid ; yield : 76% ; mp 291°C ; H NMR (DMSO-d6) δH7.11 (s, 1 H, CH), 7.31-7.50
(m, 3 H, 3 × CH), 7.59-7.92 (m, 5 H, 5 × CH), 8.67-8.75 (m, 2 H, 2 × CH), 12.19 (s, 1 H,
13
NH); C NMR (DMSO-d6) δC 112.7, 118.3, 121.0, 123.4, 123.6, 126.7, 126.9, 127.3, 129.1,
132.3, 137.3, 137.4, 141.0, 141.7, 149.2, 151.5, 151.6; HRMS (ESI) : m/z cald for
[C19H12ClN3OS2 + H]+ : 398.0183 ; found : 398.0179.
4.1.12. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(3,4-dimethoxybenzylidene)-1,3-
thiazolidin-4-one (4k)
Yellow solid ; yield : 72% ; mp 242°C ; 1H NMR (DMSO-d6) δH3.77 (s, 3 H, OCH3), 3.82 (s,
3 H, OCH3), 7.10-7.21 (m, 4 H, 4 × CH), 7.41-7.48 (m, 2 H, 2 × CH), 7.57-7.68 (m, 4 H, 4 ×
CH), 12.18 (s, 1 H, NH); HRMS (ESI) : m/z calcd for [C22H17ClN2O3S2 + H]+ : 457.0442 ;
found : 457.0442.
4.1.13. 2-{[5-(4-Chlorophenyl)-3-thienyl]imino}-5-(4-fluorobenzylidene)-1,3-thiazolidin-
4-one (4l)
1
Orange solid ; yield : 54% ; mp 223°C ; H NMR (DMSO-d6) δH7.18 (s, 1 H, CH), 7.30-7.51
(m, 5 H, 5 × CH), 7.60-7.77 (m, 5 H, 5 × CH) , 12.21 (s, 1 H, NH); 13C NMR (DMSO-d6) δC
116.2, 116.6, 121.0, 126.8, 126.9, 129.1, 129.2, 130.1, 130.5, 132.0, 132.1, 132.2, 132.4,
132.6, 141.1, 141.8; HRMS (ESI) : m/z cald for [C20H12ClFN2OS2 + H]+ : 415.0136 ; found :
415.0160.

ACCEPTED MANUSCRIPT
4.1.14. 2-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]imino}-5-(4-dimethylaminobenzylidene)-
1,3-thiazolidin-4-one (20)
1
Orange solid ; yield : 86% ; mp> 300°C ; H NMR (DMSO-d6) δH3.05 (s, 6 H, 2 × CH3),
6.89 (d, 2 H, 2 × CH, J= 8.75Hz), 7.51-7.62 (m, 5 H, 5 × CH), 8.01 (d, 2 H, 2 × CH,
J=8.75Hz), 7.93 (s, 1 H, CH), 12.51 (s, 1 H, NH); HRMS (ESI) : m/z calcd for
[C21H9ClN4OS2 + H]+ : 441.0605 ; found : 441.0615.
4.1.15. 2-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]imino}-5-(4-methylbenzylidene)-1,3-
thiazolidin-4-one (21)
Yellow solid ; yield : 68% ; mp> 300°C ; 1H NMR (DMSO-d6) δH2.38 (s, 3 H, CH3), 7.40 (d,
2 H, 2 × CH, J= 8.25Hz), 7.53-7.58 (m, 4 H, 4 × CH), 7.66 (s, 1 H, CH), 7.93 (s, 1 H, CH),
7.97 (d, 2 H, 2 × CH, J=8.25Hz), 12.68 (s, 1 H, NH); HRMS (ESI) : m/z calcd for
[C20H14ClN3OS2 + H]+ : 412.0340 ; found : 412.0348.
4.1.16. 2-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]imino}-5-(4-methoxybenzylidene)-1,3-
thiazolidin-4-one (22)
1
Yellow solid ; yield : 79% ; mp> 300°C ; H NMR (DMSO-d6) δH3.86 (s, 3 H, OCH3), 7.20
(d, 2 H, 2 × CH, J= 8.5Hz), 7.59-7.71 (m, 5 H, 5 × CH), 7.97 (s, 1 H, CH), 8.01 (d, 2 H, 2 ×
CH, J=8.5Hz) ,12.74 (s, 1 H, NH);HRMS (ESI) : m/z calcd for [C20H14ClN3O2S2 + H]+ :
428.0289 ; found : 428.0290.
4.1.17. 2-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]imino}-5-(2-naphtylidene)-1,3-thiazolidin-4-one
(23)
Yellow solid ; yield : 85% ; mp> 300°C ; 1H NMR (DMSO-d6) δH7.53-7.65 (m, 4 H, 4 × CH),
7.79 (d, 1 H, CH, J=9Hz), 7.88 (s, 1 H, CH), 7.98-8.05 (m, 5 H, 5 × CH), 8.14 (d, 1 H, CH,
J=9Hz), 8.29 (s, 1 H, CH), 12.79 (s, 1 H, NH); HRMS (ESI) : m/z calcd for [C23H14ClN3OS2
+ H]+ : 440.0340 ; found : 440.0346
4.1.18. 2-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]imino}-5-(4-fluorobenzylidene-1,3-
thiazolidin-4-one (24)
Yellow solid ; yield : 82% ; mp> 300°C ; 1H NMR (DMSO-d6) δH7.46-7.53 (m, 2 H, 2 × CH),
7.60 (d, 2 H, 2 × CH, J=8.5Hz), 7.73-7.79 (m, 3 H, 3 × CH), 7.96 (s, 1 H, CH), 7.98 (d, 2 H, 2
× CH, J=8.5Hz), 12.79 (s, 1 H, NH); HRMS (ESI) : m/z calcd for [C19H11ClFN3OS2 + H]+ :
416.0089 ; found : 416.0096.
4.1.19. General procedure for the synthesis of N-substituted thiazolidinones 12-17
To a mixture of the corresponding chloroacetamide (0.5 mmol) and potassium carbonate (0.75
mmol) in acetonitrile (5 mL) was added portionwise the corresponding isothiocyanate (0.5
mmol). The mixture was then heated at 40°C overnight. After cooling to room temperature,
the mixture was poured into cold water (25 mL) and extracted with ethyl acetate. The organic
layer was dried with magnesium sulfate, filtered and evaporated under reduced pressure. The
expected product was purified using silica gel chromatography (Eluent : Cyclohexane/Ethyl
acetate 3/1).

ACCEPTED MANUSCRIPT
4.1.20. 3-(4-Chlorophenyl)-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-thiazolidin-4-one
(12)
Pale brown solid ; yield : 71% ; mp 80°C ; 1H NMR (CDCl3) δH 4.03 (s, 2 H, CH2), 6.75 (d, 1
H, CH, J=1.5Hz), 7.01 (d, 1 H, CH, J=1.5Hz), 7.29-7.34 (m, 4 H, 4 × CH), 7.45-7.50 (m, 4 H,
13
4 × CH); C NMR (CDCl3) δC 32.9, 110.9, 120.1, 126.7, 129.1, 129.4, 129.6, 132.6, 133.0,
133.6, 134.9, 142.7, 146.6, 155.2, 171.0; HRMS (ESI) : m/z calcd for [C19H12Cl2N2OS2 + H]+
: 418.9841 ; found : 418.9835.
4.1.21. 3-Phenyl-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-thiazolidin-4-one (13)
1
Pink solid ; yield : 83% ; mp 152°C ; H NMR (CDCl3) δH4.05 (s, 2 H, CH2), 6.75 (d, 1 H,
CH, J=1.5Hz), 7.01 (d, 1 H, CH, J=1.5Hz), 7.31-7.38 (m, 4 H, 4 × CH), 7.46-7.52 (m, 5 H, 5
× CH) ; 13C NMR (CDCl3) δC 41.3, 110.8, 120.3, 119.8, 126.7, 127.9, 129.0, 129.4, 132.7,
133.5, 134.7, 142.5, 146.4, 155.1, 171.3; HRMS (ESI) : m/z calcd for [C19H13ClN2OS2 + H]+ :
385.0231 ; found : 385.0234.
4.1.22. 3-(4-Methylphenyl)-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-thiazolidin-4-one
(14)
1
Brown solid ; yield : 90% ; mp 107°C ; H NMR (CDCl3) δH2.33 (s, 3 H, CH3), 3.97 (s, 2 H,
CH2), 6.67 (d, 1 H, CH, J=1.5Hz), 6.91 (d, 1 H, CH, J=1.5Hz), 7.16 (d, 2 H, 2 × CH, J=9Hz),
7.18-7.21 (m, 4 H, 4 × CH), 7.39 (d, 2 H, 2 × CH, J=9Hz) ; 13C NMR (CDCl3) δC 21.3, 32.9,
110.8, 120.3, 126.7, 127.7, 129.2, 130.1, 132.0, 132.7, 133.5, 139.2, 142.4, 147.0, 155.8,
171.4; HRMS (ESI) : m/z calcd for [C20H15ClN2OS2 + H]+ : 399.0387 ; found : 399.0383.
4.1.23. 3-(4-Dimethylaminophenyl)-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-
thiazolidin-4-one (15)
Brown solid ; yield : 84% ; mp 123°C ; 1H NMR (CDCl3) δH3.01 (s, 6 H, 2 × CH3), 4.01 (s, 2
H, CH2), 6.73 (d, 1 H, CH, J=1.5Hz), 6.79 (d, 2 H, 2 × CH, J=9Hz), 7.03 (d, 1 H, CH,
J=1.5Hz), 7.18 (d, 2 H, 2 × CH, J=9Hz), 7.33 (d, 2 H, 2 × CH, J=8.5Hz), 7.48 (d, 2 H, 2 ×
CH, J=8.5Hz) ; 13C NMR (CDCl3) δC 32.8, 40.5, 110.7, 112.6, 120.4, 126.6, 128.4, 129.0,
132.8, 133.4, 142.3, 147.3, 149.5, 150.5, 156.4, 171.8; HRMS (ESI) : m/z calcd for
[C21H18ClN3OS2 + H]+ : 428.0653 ; found : 428.0655.
4.1.24. 3-(3,4-Dimethoxyphenyl)-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-thiazolidin-
4-one (16)
Pale brown solid ; yield : 59% ; mp 181°C ; 1H NMR (CDCl3) δH3.90 (s, 3 H, OCH3), 3.91 (s,
3 H, OCH3), 4.02 (s, 2 H, CH2), 6.75 (d, 1 H, CH, J=1.5Hz), 6.83 (d, 1 H, CH, J=2Hz), 6.92
(Dd, 1 H, CH, J=8.5Hz, J=2Hz), 6.95 (d, 1 H, CH, J=8.5Hz), 7.01 (d, 1 H, CH, J=1.5Hz),
13
7.32 (d, 2 H, 2 × CH, J=8Hz), 7.47 (d, 2 H, 2 × CH, J=8Hz) ; C NMR (CDCl3) δC 32.9,
56.0, 110.9, 111.2, 111.3, 120.3, 120.4, 126.7, 127.3, 129.0, 132.7, 133.5, 142.5, 147.0, 150.0,
155.9, 171.5; HRMS (ESI) : m/z calcd for [C21H17ClN2O3S2 + H]+ : 445.0442 ; found :
445.0427.
4.1.25. 3-(1-Naphtyl)-2-{[5-(4-chlorophenyl)-3-thienyl]imino}-1,3-thiazolidin-4-one (17)
1
Yellow solid ; yield : 92% ; mp 98°C ; H NMR (CDCl3) δH 4.10 (s, 2 H, CH2), 6.65 (d, 1 H,
CH, J=2Hz), 7.22 (d, 1 H, CH, J=2Hz), 7.22 (d, 2 H, 2 × CH, J=8.5Hz), 7.36 (d, 2 H, 2 × CH,
13
J=8.5Hz), 7.40–7.58 (m, 4 H, 4 × CH), 7.85–7.93 (m, 3 H, 3 × CH) ; C NMR (CDCl3) δC

ACCEPTED MANUSCRIPT
33.2, 110.8, 120.3, 121.9, 125.6, 126.6, 126.7, 126.9, 127.3, 128.8, 129.0, 129.5, 130.2, 131.5,
132.6, 133.4, 134.6, 142.4, 146.9, 155.3, 171.4; HRMS (ESI) : m/z calcd for [C23H15ClN2OS2
+ H]+ : 435.0387 ; found : 435.0376
4.1.26. General procedure for the synthesis of heteroaryliminothiazolidinones 18-19
starting from aminoheterocycle
To a solution of the corresponding heteroarylamine (10 mmol) in dry DMF (10 mL) was
added dropwise chloroacetyl chloride (11 mmol) and the mixture was stirred at room
temperature for 2h. The mixture was then poured into cold water (50 mL) and the formed
precipitate was filtered, washed with water (2 × 10 mL) and dried.
The obtained chloroacetamide (5 mmol) was then dissolved in ethanol (20 mL) and
ammonium thiocyanate was added (10 mmol). The mixture was refluxed for 3h and stirred at
room temperature overnight. The precipitating product was filtered, washed with water (10
mL) and petroleum ether (10 mL), and dried.
4.1.27. 2-{[5-(4-Chlorophenyl)-3-selenophenyl]imino}-1,3-thiazolidin-4-one (18)
Brown solid ; yield : 78% ; mp 264°C ; 1H NMR (DMSO-d6) δH4.00 (d, 2 H, CH2, J=8.6Hz),
7.48 (m, 2 H, 2 × CH), 7.61 (m, 2 H, 2 × CH) , 7.69 (s, 1 H, CH) , 8.19 (s, 1 H, CH) 11.43 (s,
1 H, NH); 13C NMR (DMSO-d6) δC35.4, 116.9, 123.8, 127.1, 129.1, 132.5, 134.2, 138.1,
146.7, 177.5, 187.9; HRMS (ESI) : m/z calcd for [C13H9ClN2OSSe + H]+ : 356.9359 ; found :
356.9354.
4.1.28. 2-{[4-(4-chlorophenyl)-1,3-thiazol-2-yl]imino}-1,3-thiazolidin-4-one (19)
1
Pink solid ; yield : 68% ; mp> 300°C ; H NMR (DMSO-d6) δH4.03 (s, 2 H, CH2), 7.50 (d, 2
H, 2 × CH, J=8Hz), 7.89 (s, 1 H, CH), 7.96 (d, 2 H, 2 × CH, J=8Hz), 12.14 (s, 1 H, NH);
13C NMR (DMSO-d6) δC 34.9, 111.2, 127.3, 128.8, 132.4, 132.8, 149.6, 163.6, 169.2, 174.1;
HRMS (ESI) : m/z cald for [C12H8ClN3OS2 + H]+ : 309.9870 ; found : 309.9876
4.2.Biological methods
4.2.1. Cell culture
HT29 (APCmut, P53mut, K-RASwt) and HCT116 (APCwt, P53wt, K-RASmut) cell lines have been
established from colon adenocarcinoma tissue and represented colon cancer cell model
behaving chromosomic and microsatellite instability, respectively. SW620 cells (APCwt,
P53mut, K-RASmut) were originated from a lymphoid nodule and have been characterised as
metastatic cells. MCF7 cells derived from mammary adenocarcinoma tissue. MDA-MB-231
cells were isolated from pleural effusion and characterised as metastatic cells. Colon cancer
cells were cultivated in Dulbecco minimum essential medium DMEM (Eurobio, Courtaboeuf,
France) supplemented with 10% (v/v) heat-inactivated (30 min at 56°C) fetal calf serum
(Eurobio), 50 ꢀg/mL gentamycin (Invitrogen, Paisley, UK) and 2 mM L-Glutamine
(Eurobio). Mammary cancer cell were cultivated in RPMI medium containing 10% heat-
inactivated fetal calf serum, 2mM L-Glutamine and 1% Penicillin/streptomycin (Eurobio).
Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.
4.2.2. Determination of IC50
IC50 was calculated from results obtained by the thiazolyl blue tetrazolium bromide (MTT)
procedure. In brief, cancer cells were seeded in 96 well-plates at 104 cells/well (Dutscher,
Molsheim, France). 24 hours after seeding, increasing concentrations of each compound

ACCEPTED MANUSCRIPT
(range from 0.01 to 100µM) were added to each well for 72 hours at 37°C. After incubation,
the medium was discarded and 100µl/well of MTT solution (0.5 mg/ml diluted in medium)
were added and incubated for 2 hours. Water-insoluble MTT formazan blue crystals were
finally dissolved in DMSO. Each plate was read at 570 nm. IC50 was calculated using
GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Data are expressed as IC50
S.E.M. obtained from at least two independent experiments (n = 16).
±
4.2.3. Cell growth kinetics
HT29 cells were seeded in 6 well-plates. 48 hours after seeding, cells were treated daily with
each molecule over three days. Dilutions of each drug were performed in DMSO prior to
addition in the cell medium. Control cells were treated with 0.1% (v/v) of DMSO. At the
indicating time, cells were harvested by trypsination (Trypsin /EDTA solution, Eurobio).
100µl of cell suspension were mixed to an equal volume of 0.04% (v/v) Trypan blue dye
solution and living cells were counted using a Malassez hematimeter under microscopy. Data
are means ± S.E.M of triplicate determination from two independent experiments. Results are
expressed either as mitotic index (number of compound-treated cells related to control cells
treated by 0.1% (v/v) DMSO used as compound vehicle) or cells counts (x105 cells/ml, in
Figure 3).
4.2.4. Cell viability assay
Cell viability assay was performed with s imultaneous double-staining procedure using 2’,7’ –
dichlorofluorescein diacetate (DCF-DA, viable cells) and propidium iodide (PI, dead cells).
Two days after seeding, HT29 cells were treated with each molecule corresponding to the
concentration of IC50 value for various times up to 24 hours with the exception of compound
20 and 22, tested at 2.5 µM. Cells were loaded with 50µM DCF-DA and 25 µg/mL PI during
15 min at 37°C. After incubation, cells were harvested by trypsination, washed with PBS and
finally suspended in 1 ml of the same buffer. Fluorescence was measured in 30 000
cells/sample by flow cytometry (FL-1 and FL-3; FACSCalibur, BD Sciences, Le Pont de
Claix, France). Data are means ± S.E.M. of triplicate determinations from two independent
experiments. Results are expressed in percentage of viable cells (negative to IP, and positive
to DCF-2DA) with respect to total cells/events quantified.
4.2.5. Cell cycle analysis
HT29 cells were seeded in 6 well-plates. 48 hours after seeding, cells were treated with each
molecule. Cells layers were washed twice with phosphate buffer saline (PBS), harvested by
trypsination and fixed in 70% (v/v) ethanol solution for 2 hours and stored at -20°C at least 1
hour. Cells were then centrifuged at 1000g for 5 min at 4°C, washed with PBS and
centrifuged again. Pellets were suspended in 1 ml PBS containing 50 µg/ml propidium iodide,
20 µg/ml of RNAse A (Sigma-Aldrich France, St Quentin Fallavier, France) and 0.1 % (v/v)
Triton X100 for 20 min and they were analyzed by flow cytometry (FL2-A; Calibur). Results
were quantified using the CellQuest software (BD Sciences) and cell cycle distribution was
analysed using Modfit sofware (Verity Software House, Topsham, Maine, USA). Results are
expressed as percentage of treated HT29 cells in each cell cycle phase. Cells in sub-G1
fraction are expressed as the percentage of total cells analysed in each experiment. Data are
means ± S.E.M. of triplicate determinations from three independent experiments.
4.2.6.Cell-living imaging

ACCEPTED MANUSCRIPT
HT29 cells were seeded in 35mm petri dishes and treated with each compound over two days.
After incubation, cell layers were fixed in 70% (v/v) ethanol solution for 5 min, rinced twice
with PBS. 1 µM 4',6'-diamidino-2-phenylindol (DAPI) in PBS was added for 15 min. In
parallel, similar experimental conditions were carried on with supplementation of 1 µM
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) probe
diluted in DMSO. JC-1 was used to demonstrate alteration of mitochondrial membrane
potential (Ψm). After compound treatment, JC-1 probe was added to the medium for 15 min
at 37°C. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) at 50 µM for
15 min was used in order to dissipate Ψm which can be visualized by the absence of J-
aggregates under red fluorescent filter. After incubation, cell layers were washed rapidly with
warmed PBS (37°C) twice. Pictures were captured under UV fluorescent microscopy (Nikon
Eclipse 80i, Nikon) equipped with a digital camera (DXM1200F, Nikon). They were further
processed using NIS-Element AR 2.3 software (Nikon).
4.2.7. Quantification of oxygen and nitric oxide-derived species
Two days after seeding, HT29 cells were treated with each molecule corresponding to the
concentration of IC50 value for various times up to 24 hours with the exception of compound
20 and 22, tested at 2.5 µM. Cells were loaded with 50µM DCF-DA during 15 min at 37°C
for ROS detection, 1 µM DAF-2DA during 30 min at 37°C for RNS detection, or 25µM DHR
during 20 min at 37°C for peroxynitryl radical detection. Cell exposure to 1mM tert-butyl for
one hour was used as a positive ROS-induced control. After incubation, cells were harvested
by trypsination, washed with PBS and finally suspended in 1 ml of the same buffer.
Fluorescence was measured in 30 000 cells/sample by flow cytometry (FL-1). When used,
1mM ascorbic acid, as an antioxidant, was added one hour before drug treatment and cells
were prepared as described. Results were expressed as percentage of results obtained with
treated cells related to cells treated with DMSO used as compound diluent. Data are means ±
S.E.M of triplicate determination from three independent experiments.
4.2.8. GSH content determination
HT29 cells were grown in 6 well-plates. 48 hours after seeding, cells were exposed to each
molecule corresponding to the concentration of IC50 value for 24 hours. Cell layers were
washed three times with ice-cold PBS. They were then scrapped with 10% (v/v) perchloric
acid ice-cold solution (pH 8.0) containing 2 mM EDTA. The homogenates were centrifuged
at 15 000 g for 15 min at 4°C. The supernatant were stored at -80°C until analysis. GSH
content was determined by a fluorescence-based microtiter plate method [26]. Perchloric acid-
precipitated proteins (pellets) were solubilized in daily made 1 N NaOH prior to protein
content determination using a modification of the procedure of Lowry et al. [27]. GSH
contents were calculated as nanomole of GSH/mg of protein. Results are expressed in
percentage as content of intracellular GSH related to that in DMSO-treated cells (relative
GSH content). Data are means ± S.E.M of triplicate determination from three independent
experiments.
4.2.9. Western blotting
HT29 cells were seeded in 25 cm2-flasks. Total protein homogenates were prepared from
drug- and DMSO-treated cells as follows; cell layers were washed twice with ice cold PBS
and they were scrapped with 25 mMHepes/KOH (pH 7.5) buffer containing 10 mM EDTA,
40 mMKCl, 0.5 %(v/v) Igepal (Nonidet P40), 1 mM DTT and 0.1% (v/v) protease inhibitor
mixture (Sigma). Homogenates were collected and left on ice for 30 min before centrifugation
at 15,000g for 30 min at 4°C. Supernatants were stored at -80°C until use. 50 µg protein from

ACCEPTED MANUSCRIPT
whole cell homogenates were resolved in 10-15% SDS PAGE and they were transferred onto
nitrocellulose membranes. The saturation step was performed in Tris saline buffer (50 mM
Tris/HCl (pH 7.4) containing 0.15 M NaCl and 0.01 % (v/v) Tween 20), supplemented with
5% (w/v) non-fat milk or BSA. The primary antibody prepared in the same buffer was then
added. Membranes were incubated overnight at 4°C. The following antibodies were used:
antibody against GCL (5529-1; Epitomics), HO-1 (1922-1; Epitomics), NQO-1 (2618-1;
Epitomics) and GAPDH (MAB374; Millipore). Washing steps (3 times for 5 min) were
performed with Tris saline buffer. The blots were then incubated 45 min in this buffer
containing the horseradish peroxidase-conjugated species-specific antibody, and finally
washed 3 times for 5 min with the same buffer. Blots were developed by chemo-luminescence
detection according to the manufacturer’s protocol (Santa Cruz Biotech). Each blot was
stripped and used to detect human GAPDH (1: 10 000) using the same protocol. Bands were
quantified and normalized to GAPDH content. The ratio, level of protein of interest versus
GAPDH content, in DMSO-treated cells was considered as 1.00. Results shown are
representative of one out of three independent experiments.
4.2.10. Detection of autophagy process
HT29 cells were seeded in 6 well-plates and they were treated as described before for two
days with daily medium changes. Cells were incubated with 1µg/ml acridine orange for 15
min, then harvested by trypsination and suspended in PBS before flow cytometry analysis.
Fluorescence (FL-3) was measured in 30 000 cells/sample. Results were analysed using
CellQuest and Cyflogic software and they are expressed in arbitrary unit related to those
obtained with DMSO-treated cells. Results are means ± S.E.M. of triplicate determinations
from three independent experiments. In parallel, cells were cultivated in 25 cm2-flasks and
treated. Total protein homogenates were prepared and western blots were performed, using
Beclin-1 (1:500, BD Biosciences) and GAPDH antibodies, respectively, as a marker of
autophagosome formation and an internal marker for protein loading.
4.2.11. Statistics
Differences between results from control and drug-treated cells were analysed by Student’s t
test and any difference was considered significant at p < 0.05.
Acknowledgments
We thank the ‘Ministère de l’Enseignement Supérieur et de la Recherche’ for Ph.D. grants
to G.R. and S. H.-V.
References
1. E. Pasquier, S. Honore, D. Braguer, Microtubule-targeting agents in angiogenesis: where
do we stand?, Drug Resist. Updat. 9 (2006) 74-86.
2. L.T. Vassilev, C. Tovar, S. Chen, D. Knezevic, X. Zhao, H. Sun, D.C. Heimbrook, L.
Chen, Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1,
Proc. Natl Acad. Sci. USA 103 (2006) 10660-10665.
3.
A. Deep, S. Jain, P.C. Sharma, S.K. Mittal, P. Phogat, M. Malhotra, Synthesis,
characterization and antimicrobial evaluation of 2,5-disubstituted-4-thiazolidinone
derivatives, Arabian J. Chem. 7 (2014) 287-291.

ACCEPTED MANUSCRIPT
4.
R. Maccari, R.M. Vitale, R. Ottana, M. Rocchiccioli, A. Marrazzo, V. Cardile, A.C.E.
Graziano, P. Amodeo, U. Mura, A. DelCorso, Structure-activity reliationships and molecular
modelling of new 5-arylidene-4-thiazolidinone derivatives as aldose reductase inhibitors and
potential anti-inflammatory agents, Eur. J. Med. Chem. 81 (2014) 1-14.
5.
A.P.G. Nikalje, A.N. Shaikh, S.I. Shaikh, F.A.K. Khan, J.N. Sangshetti, D.B. Shinde,
Microwave assisted synthesis and docking study of N-(2-oxo-2(4-oxo-2-substituted
thiazolidin-3-ylamino)ethyl)benzamide derivatives as anticonvulsant agents, Bioorg. Med.
Chem. Lett. 24 (2014) 5558-5562.
6.
A.K. Jain, A. Vaidya, V. Ravichandran, S.K. Kashaw, R.K. Agrawai, Recent
developments and biological activities of thiazolidinone derivatives: a review, Bioorg. Med.
Chem. 20 (2012) 3378-3395
7.
A.C. Tripathi, S.J. Gupta, G.N. Fatima, P.K. Sonar, A. Vermab, S.K. Saraf, 4-
thiazolidinones: the advances continue..., Eur. J. Med. Chem 72 (2014), 52-77 and references
therein.
8.
P.C. Unangst, D.T. Connor, W.A. Cetenko, R.J. Sorenson, C.R. Kostlan, J.C. Sircar,
C.D. Wright, D.J. Schrier, R.D. Dyer, Synthesis and biological evaluation of 5-[[3,5-bis(1,1-
dimethylethyl)-4-hydroxyphenyl)methylene]oxazoles, -thiazoles, and –imidazoles: novel dual
5-lipoxygenase and cyclooxygenase inhibitors with anti-inflammatory activity, J. Med. Chem.
(1994) 322-328.
9.
K. Omar, A. Geronikaki, P. Zoumpoulakis, C. Camoutsis, M. Sokovic, C. Ciric, J.
Glamoclija, Novel 4-thiazolidinone derivatives as potential antifungal and antibacterial drugs,
Bioorg. Med. Chem. 18 (2010) 426-432.
10.
P. Vicini, A. Geronikaki, M. Incerti, F. Zani, J. Dearden, M. Hewitt, 2-
analogues of 2-thiazolylimino-5-
Heteroarylimino-5-benzylidene-4-thiazolidinones
benzylidene-4-thiazolidinones with antimicrobial activity: synthesis and structure-activity
relationship, Bioorg. Med. Chem. 16 (2008) 3714-3724.
11.
S. Xu, W. Guo, F. Teraishi, J. Pang, K. Kaluarachchi, L. Zhang, J. Davis, F. Dong, B.
Yan, B. Fang, Anticancer activity of 5-benzylidene-2-phenylimino-1,3-thiazolidin-4-one
(BPT) analogs, Med. Chem. (2006) 597-605.
12.
H.Y. Zhou, S.H. Wu, S.M. Zhai, A.F. Liu, Y. Sun, R.S. Li, Y. Zhang, S. Ekins, P.W.
Swaan, B.L. Fang, B. Zhang, B. Yan, Design, synthesis, cytoselective toxicity, structutre-
activity relationships, and pharmacophore of thiazolidinone derivatives targeting drug-
resistant lung cancer cells, J. Med. Chem. 51 (2008) 1242-1251.
13.
R. Ottana, S. Carotti, R. Maccari, I. Landini, G. Chiricosta, B. Caciagli, M.G. Vigorita,
E. Mini, In vitro antiproliferative activity against human colon cancer cell lines of
representative 4-thiazolidinones, Bioorg. Med. Chem. Lett. 15 (2005) 3930-3933.

ACCEPTED MANUSCRIPT
14.
S. Wang, Y. Zhao, G. Zhang, Y. Lv, N. Zhang, P. Gong, Design, synthesis and
biological evaluation of novel 4-thiazolidinones containing indolin-2-one moiety as potential
antitumor agent, Eur. J. Med. Chem. 46 (2011) 3509-3518.
15.
thienylimino-1,3-thiazolidin-4-ones, Synthesis (2010) 2543-2546.
16. G. Revelant, S. Hesse, G. Kirsch, Synthesis of novel N-subtituted 2-(hetero)arylimino-
1,3-thiazolidin-4-ones, Synthesis (2010) 3319-3324.
17. G. Revelant, S. Hesse, G. Kirsch, Synthesis of novel 2-aminothieno[3,2-d]thiazoles
I. Abdillahi, G. Revelant, Y. Datoussaid, G. Kirsch, Synthesis of novel 2-
and selenolo[3,2-d]thiazoles, Tetrahedron (2011) 9352-9357.
18. M. Dodson, V. Darley-Usmar, J. Zhang, Cellular metabolic and autophagic pathways:
traffic control by redox signaling, Free Radic. Biol. Med. 63 (2013) 207-221.
19. Z. Liu, M.J. Lenardo, Reactive oxygen species regulate autophagy through redox-
sensitive proteases, Dev. Cell 12 (2007) 484–485.
20. F. Teraishi, S. Wu, J. Sasaki, L. Zhang , W. Guo, J.J. Davis, F. Dong, B. Fang,
Identification of a novel synthetic thiazolidin compound capable of inducing c-Jun NH2-
terminal kinase-dependent apoptosis in human colon cancer cells, Cancer Res 65 (2005)
6380-6387.
21. S.K. Niture, J.W. Kaspar, J. Shen, A.K. Jaiswal, Nrf2 signaling and cell survival,
Toxicol. Appl. Pharmacol. 244 (2010) 37-42.
22. T. Xie, A.K. Jaiswal, AP-2-mediated regulation of human NAD(P)H: quinone
oxidoreductase 1 (NQO1) gene expression, Biochem Pharmacol 51 (1996) 771-778.
23. J. Wu, H. Xu, P.F. Wong, S. Xia, J. Xu, J. Dong, Icaritin attenuates cigarette smoke-
mediated oxidative stress in human lung epithelial cells via activation of PI3K-AKT and Nrf2
signaling, Food Chem. Toxicol. 64 (2014) 307-313.
24.
mechanisms: a radical therapeutic approach?, Nat. Rev. Drug Discov. 8 (2009) 579-591.
25. M.M. Hippert, P.S. O'Toole, A. Thorburn, Autophagy in cancer: good, bad, or both?
Cancer Res. 66 (2006) 9349-9351.
D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated
26.
K. Lewicki, S. Marchand S, L. Matoub, J. Lulek, J. Coulon J, P. Leroy,
Development of a fluorescence-based microtiter plate method for the measurement of
glutathione in yeast. Talanta 70 (2006) 876-882.
27.
O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with
the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265-275.

ACCEPTED MANUSCRIPT
H
N
H
N
O
O
R2
NH2
N
R2
N
a
b
S
S
for 4
R1
S
R1
S
S
R
Cl
4(a-l)
1-6
1
R1 = p-methylphenyl, R2 = H
R1 = p-methoxyphenyl, R2 = H
1 = phenyl, R2 = H
R1 = p-chlorophenyl, R2 = H
R1, R2 = naphtyl
4a
4b
4c
4d
4e
4f
R = phenyl
4g
4h
4i
4j
4k
4l
R = p-hydroxyphenyl
R = 2-naphtyl
R = hexyl,
R = 2-pyridinyl
R = 3,4-dimethoxyphenyl
R = p-fluorophenyl
2
3
4
5
6
R = p-methoxyphenyl
R = p-methylphenyl,
R = p-chlorophenyl
R = p-dimethylaminophenyl
R = p-bromophenyl
R
R1 = tert-butyl, R2 = H
Scheme 1. Reagents and conditions: (a) i) ClCH2COCl, dry DMF, rt, 2h ii) NH4SCN, EtOH, reflux, 3h (46-
90%); (b) RCHO, pyrrolidine, MeOH, reflux, 20h (56-89%).
H
N
R2
R1
NH2
H
N
R2
R1
NCS
R2
b
a
R1
7
R1 = Phenyl ;R2, R', R'' = H ; R = OMe
S
S
OMe
S
HN
R
S
8
R1 = p-methylphenyl ;R2, R', R'' = H ; R = OMe
9
R
R
1 = p-methoxyphenyl ;R2, R', R'' = H ; R = OMe
1 = p-chlorophenyl ;R2, R', R'' = H ; R = OMe
O
d
10
11
12
13
14
15
16
17
Cl
R1, R2 = dihydronaphtyl ; R', R'' = H ; R = OMe
1 = p-chlorophenyl ;R2, R', R'' = H ; R = Cl
R1 = p-chlorophenyl ;R2, R, R', R'' = H
1 = p-chlorophenyl ;R2, R', R'' = H ; R = Me
c
R''
R'
R
R'
N
R
R
R''
R2
S
R1 = p-chlorophenyl ;R2, R', R'' = H ; R = NMe2
R1 = p-chlorophenyl ;R2, R'' = H ; R, R' = OMe
R1 = p-chlorophenyl ;R2, R = H ; R', R" = C4H4
N
R1
S
O
7-17
Scheme 2. Reagents and conditions: (a) CSCl2, NaHCO3, CHCl3, r t, 2h; (b) p-anisidine, dry CH2Cl2, r t, 18h;
(c) ClCH2COOEt, NaOAc, dry DMF, reflux, 24h; (d) K2CO3, CH3CN, 40°C.
H
O
20
21
22
23
24
R = p-dimethylaminophenyl
R = p-methylphenyl
R = p-methoxyphenyl
R = 2-naphtyl
N
NH2
N
a
S
Se
R = p-fluorophenyl
18
Se
Cl
Cl
O
O
HN
HN
NH2
b
N
N
N
S
S
R
N
a
S
N
S
19
S
Cl
20-24
Cl
Cl
Scheme 3. Reagents and conditions: (a) i) ClCH2COCl, dry DMF, rt, 2h ii) NH4SCN, EtOH, reflux, 3h; (b)
RCHO, pyrrolidine, MeOH, reflux, 20h.

ACCEPTED MANUSCRIPT
Table 1: IC50 determination for thiazolidin-4-one derivatives
IC50 (µM)
Compound
MDA-
MB-231
>50
HT29
HCT116
SW620
MCF7
>50
>50
>50
>50
>50
>50
>50
>50
1
2
>50
>50
>50
>50
>50
>50
3
17.1 ± 7.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
4
5
>50
>50
>50
>50
>50
6
10.5 ± 5.8
3.8 ± 0.9
6.4 ± 1.5
2.8 ± 2.0
1.5 ± 1.2
2.0 ± 1.5
>50
3.7 ± 3.1
33.5 ± 4.4
11.5 ± 7.0
10.9 ± 3.1
4.1 ± 0.6
38.3 ± 7.7
> 50
11.7 ± 1.1
24.6 ± 7.8
> 50
36.6 ± 3.9
41.4 ± 7.4
28.9 ± 8.6
29.2 ± 4.3
14.1 ± 2.7
> 50
11.2 ± 2.2
1.3 ± 0.1
1.0 ± 0.1
11.6 ± 3.4
0.5 ± 0.1
3.3 ± 0.1
ND
3.0 ± 0.6
ND
21.6 ± 1.1
1.7 ± 0.2
7.4 ± 0.7
ND
>50
> 50
18.0 ± 0
1.2 ± 0.2
0.9 ± 0.04
6.4 ± 0.3
0.8 ± 0.01
3.5 ± 0.2
ND
1.8 ± 0.1
ND
10.9 ± 0.5
1.9 ± 0.1
ND
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
7
8
28.1 ± 12.4
1.5 ± 0.1
4.6 ± 0.7
0.8 ± 0.1
0.7 ± 0.6
ND
1.0 ± 0.1
0.9 ± 0.1
6.7 ± 0.3
0.4 ± 0.05
2.5 ± 0.3
> 50
1.2 ± 0.1
> 50
30.2 ± 6.5
7.6 ± 0.7
> 50
>50
ND
>50
>50
8.7 ± 1.6
ND
ND
>50
1.9 ± 0.4
> 50
ND
>50
2.7 ± 0.2
> 50
41.0 ± 9.0
2.7 ± 0.05
> 50
> 50
3.5 ± 0.1
33.0 ± 6.6
ND
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ND
ND
ND
ND
> 50
>50
> 50
> 50
ND
ND
ND
ND
> 50
ND
>50
ND
ND
> 50
ND
49.8 ± 11.9
ND
> 50
19.5 ± 1.7
>50
18.7 ± 2.6
> 50
46.9 ± 4.8
ND
> 50
ND
> 50
> 50
ND
> 50
8.9 ± 2.1
0.5 ± 0.1
0.5 ± 0.2
0.4 ± 0.1
1.8 ± 0.3
2.9 ± 0.7
23.8 ± 6.8
0.2 ± 0.02
0.5 ± 0.1
0.4 ± 0.03
1.2 ± 0.03
7.5 ± 4.3
>50
0.20 ± 0.1
0.89 ± 0.1
0.05 ± 0.03
20.3 ± 2.8
> 50
0.1 ± 0.02
0.4 ± 0.05
0.1 ± 0.02
0.3 ± 0.1
>50
0.3 ± 0.01
0.8 ± 0.1
0.3 ± 0.3
0.9 ± 0.05
> 50
Viability was assessed by the MTT procedure as described in the biological methods section. IC50Values are
means ± S.E.M. ND ; not determined

ACCEPTED MANUSCRIPT
Table 2: Cell cycle distribution after cell exposure to thiazolidin-4-one derivatives
Sub G1
G0/G1
phasec
G2/M
Treatmenta
S phasec
fractionb
phasec
DMSO (0.1%)
6.9 ± 0.5
21.6 ± 2.4*
21.5 ± 1.8*
29.0 ± 1.9*
20.6 ± 2.2*
2.3 ± 0.1
46.1 ± 0.4
1.2 ± 0.4#
1.2 ± 0.2#
1.5 ± 0.2#
2.3 ± 0.9#
67.7 ± 0.5
23.9 ± 0.3
18.2 ± 1.6#
14.8 ± 2.0#
14.8 ± 0.7#
11.3 ± 0.9#
21.1 ± 1.5
20.7 ± 1.0
17.2 ± 1.3#
30.0 ± 2.0
80.6 ± 1.2#
84.0 ± 1.8#
83.7 ± 0.7#
86.4 ± 0.6#
11.3 ± 1.0
18.7 ± 0.7#
24.1 ± 1.4#
4b
4c
4e
4k
DMSO (0.1%)
16.2 ± 0.7* 60.6 ± 0.1#
20.9 ± 1.4* 58.7 ± 0.4#
20
22
a, HT29 cells were treated with each compound as described in the biological methods section. b, results are
expressed as percentage of cells in the sub-G1 fraction related to total cells analysed. c, results are expressed as
#
percentage of cells in each phase of cell cycle. * and , significant statistical difference (P < 0.05) at Student’s t
test; comparison between HT29-treated cells with respect to cells exposed to DMSO.
Table 3: Redox changes within HT29 cells exposed to thiazolidin-4-one derivatives
Treatment
RNS content
GSH content
DAF-2DA
DHR 123
0.1% (v/v) DMSO
100.0 ± 1.0
169.7 ± 3.6*
155.0 ± 0.3*
294.4 ± 8.6*
116.3 ± 1.1*
100.0 ± 4.2
100.0 ± 2.0
88.8 ± 4.1*
73.9 ± 8.3*
84.6 ± 3.7*
108.1 ± 7.9*
85.9 ± 3.2*
95.0 ± 3.2*
80.1 ± 2.3*
83.6 ± 4.9*
4b
4c
4e
4k
0.1% (v/v) DMSO
100.0 ± 1.0
100.0 ± 0.5
100.0 ± 10.5
109.1 ± 5.2
144.4 ± 10.9*
157.6 ± 4.6*
20
103.3 ± 1.7
80.9 ± 0.7*
117.0 ± 4.8
22
RNS and GSH levels were quantified as described in the biological methods section. *, significant statistical
result (P < 0.05) at Student’s t test; comparison between HT29-treated cells with respect to cells exposed to
DMSO.

ACCEPTED MANUSCRIPT
Table 4: Impact of thiazolidin-4-one derivatives on HT29 cells pre-treated with
antioxidant.
ROS content
Mitotic index
Compound
+ 1mM
ascorbic acid*
65.7 ± 4.7
Compound
+ 1mM
alone
alone
ascorbic acid
1.12 ± 0.03
0.03 ± 0.02
0.02 ± 0.01
0.02 ± 0.01
0.04 ± 0.01
1.08 ± 0.04
0.09 ± 0.02
0.1% (v/v) DMSO
100.0 ± 3.5
123.1 ± 0.3
172.0 ± 5.5
177.8 ± 1.6
141.6 ± 2.6
100.0 ± 5.6
252.5 ± 14.5
1.00 ± 0.08
0.03 ± 0.01
0.02 ± 0.01
0.02 ± 0.01
0.04 ± 0.01
1.00 ± 0.07
0.13 ± 0.02
97.0 ± 3.1
4b
102.5 ± 4.8
133.6 ± 4.8
112.1 ± 3.9
64.7 ± 2.6
4c
4e
4k
0.1% (v/v) DMSO
158.8 ± 8.5
20
ROS levels and mitotic index were calculated as described in the biological methods section. *, significant
statistical result (P < 0.05) at Student’s t test; comparison between HT29 cells pre-treated with ascorbic acid
before addition of each compound with respect to cells exposed to each compound alone.

ACCEPTED MANUSCRIPT
O
O
S
O
C
N
RN
N
HN
A
O
Y
N
S
S
N
N
X
S
HN
R'
R
O
B
H3CO
III
I
II
IV
Fig. 1. Thiazolidinones described for their pharmacological activities (I-III) and target compounds IV.
Fig. 2. HT29 cell growth inhibition of compound 4 and its derivatives.
HT29 cells were grown with compounds 4, 4(a-l), and 9 (A) at the concentration corresponding to IC50 value,
and with 0.5-10 µM with compounds 20 and 22 (B) for 72 hours. Results are expressed as mitotic index (number
of compound-treated cells related to control cells treated by 0.1% (v/v) DMSO used as compound vehicle)
*, significant difference (p<0.05) at Student’s t test between HT29-treated cells with respect to control cells,
exposed to DMSO used as compound vehicle.

ACCEPTED MANUSCRIPT
Fig. 3. HT29 cell growth kinetic and cell viability after cell exposure to compounds 4b, 4c, 4e, 4k, 20 and 22.
Kinetic growth (A and C) and cell viability assays (B and D) were performed as described in the biological
methods section. HT29 cells treated with compound 4b, 4c, 4e, 4k at the concentration corresponding to IC50
value (A and B) and with 2.5 µM of compound 20 and 22 (C and D). Statistical analysis demonstrated that
significant difference (P < 0.05) at Student’s t test was obtained in A-D (comparison between HT29-treated cells
with respect to cells exposed to DMSO).

ACCEPTED MANUSCRIPT
Fig. 4. Thiazolidin-4-one derivative treatment is associated with ROS production and modulation of antioxidant
enzyme expression.
A and B: ROS production was calculated in HT29 treated cells with compound 4a, 4c, 4e or 4k, and compound
20 or 22 as described in the biological methods section. C and D: Western blotting was performed as described
in the Biological methods section. Respective mouse or rabbit diluted antibodies against HO-1 (1: 1 000), NQO1
(1:1 000), GCL (1: 500) were used. GAPDH antibodies (1: 10 000) were used as an internal loading control
Typical results obtained when HT29 cells were treated with compound 4c and 4k.

ACCEPTED MANUSCRIPT
Fig. 5. Autophagy processes in HT29 exposure to thiazolidin-4-one derivatives.
A. autophagy process was quantified inHT29 cells exposed to each compound as described in the biological
methods section.*, significant difference at Student t test (P<0.05) with respect to DMSO-treated cells.
B. Detection of beclin-1 expression during autophagosome formation by western blotting. The experiments were
performed with rabbit antibody against human beclin-1 as described in the biological methods section. Typical
results out of three independent experiments obtained with protein homogenates prepared from HT29 cells
treated with each compound over two days. Mouse antibody against human GAPDH was used as sample
loading.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT