A novel thiazolidine compound induces caspase-9 dependent apoptosis in cancer cells

Article abstract of DOI:10.1016/j.bmc.2012.07.016

The forward chemogenomics strategy allowed us to identify a potent cytotoxic thiazolidine compound as an apoptosis-inducing agent. Chemical structures were designed around a thiazolidine ring, a structure already noted for its anticancer properties. Initially, we evaluated these novel compounds on liver, breast, colon and endometrial cancer cell lines. The compound 3 (ALC67) showed the strongest cytotoxic activity (IC₅₀ ～5 μM). Cell cycle analysis with ALC67 on liver cells revealed SubG1/G1 arrest bearing apoptosis. Furthermore we demonstrated that cytotoxicity of this compound was due to the activation of caspase-9 involved apoptotic pathway, which is death receptor independent.

Full text of DOI:10.1016/j.bmc.2012.07.016

Bioorganic & Medicinal Chemistry 20 (2012) 5094–5102
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel thiazolidine compound induces caspase-9 dependent apoptosis
in cancer cells
F. Esra Onen-Bayram b, , Irem Durmaz a, , Daniel Scherman c, Jean Herscovici c, Rengul Cetin-Atalay a,
⇑
a Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Bilkent, 06800 Ankara, Turkey
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Kadıkoy, 34755 Istanbul, Turkey
c UMR 8151 CNRS, U1022 INSERM, Unité de Pharmacologie, Chimique et Génétique et d’Imagerie, Université Paris Descartes, Sorbonne Paris Cité, Chimie-Paris Tech.,
4 Avenue de l’observatoire, 75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The forward chemogenomics strategy allowed us to identify a potent cytotoxic thiazolidine compound as
an apoptosis-inducing agent. Chemical structures were designed around a thiazolidine ring, a structure
already noted for its anticancer properties. Initially, we evaluated these novel compounds on liver, breast,
colon and endometrial cancer cell lines. The compound 3 (ALC67) showed the strongest cytotoxic activity
Received 12 March 2012
Revised 16 May 2012
Accepted 10 July 2012
Available online 20 July 2012
(IC50 ꢀ5
lM). Cell cycle analysis with ALC67 on liver cells revealed SubG1/G1 arrest bearing apoptosis.
Furthermore we demonstrated that cytotoxicity of this compound was due to the activation of
caspase-9 involved apoptotic pathway, which is death receptor independent.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cytotoxic
Cancer
Thiazolidine
Terminal alkyne
Apoptosis
Caspase-9
1. Introduction
resulting in the phagocytosis of membrane-bound apoptotic
bodies. Apoptosis can be triggered by various external or internal
The recent development of proteomics, which is the study of
protein structures and functions in large scale, has led to great
improvements in anticancer drug research.1–3 Through this new
field, novel anticancer targets have been identified, especially by
using chemogenomics, an emerging powerful tool that screens
small-molecule libraries to determine protein functions and drug
candidates.4–8
Biological macromolecules involved in cancer cell growth
mechanisms, metastasis, and tumor angiogenesis constitute the
main targets of current anticancer drug studies. For instance,
proteins involved in mitogenic signal transduction pathways like
HER-29,9,10 and Bcr-Abl tyrosine kinases,11,12 have resulted in
successful treatments of cancer patients. An alternative therapeu-
tic strategy aims at developing apoptosis-inducing agents since
apoptosis is a hallmark of oncogenic cell transformation.13,14
Apoptosis is a highly regulated cell death process that elimi-
nates damaged or malfunctioning cells. It is characterized by
DNA damage-induced chromatin condensation and cell shrinkage
in early stage, followed by nuclear and cytoplasmic fragmentation,
stimuli. Depending on its origin (external or internal) the stimulus
can activate one of two signaling pathways. Both pathways
involve aspartate-specific cysteine proteases or caspases that can
be classified into two groups: initiator caspases and effector
caspases.
The caspases form a cascade that induces the transduction and
signal amplification of apoptotic pathways. Initiator caspases such
as caspase-8 and caspase-9 activate effector caspases upon apopto-
tic signals. These caspases can then activate effector caspases, such
as caspase-3. In turn, the effector caspases cleave key cellular pro-
teins, which lead to the morphological changes observed in cells
undergoing apoptosis.
In this study, we aimed to develop a novel anticancer agent
to activate apoptosis-induced cell death in cancer cell lines.
We synthesized a library of small-molecules around a thiazoli-
dine moiety, as this structure is already noted for its anticancer
properties and thiazolidine derivatives were shown to induce
apoptosis in various cancer cells.15–20 Further, the thiazolidine
heterocycle allows a diverse range of molecular structures in
only a few transformations. We investigated synthesized com-
pounds for their cytotoxicity to several human cancer cell lines
and performed analyses to the cell deaths induced the novel
structures.
⇑
Corresponding author. Tel.: +90 312 290 2503; fax: +90 312 266 5097.
E-mail address: rengul@bilkent.edu.tr (R. Cetin-Atalay).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.016

F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
5095
2. Results
2.1. Preparation of the small-molecule library
The library of tested molecules was developed around a thiazol-
idine core. Three different series of compounds (pyrimidic deriva-
tives, a benzoyl derivatives and N-acetylated triazoles) were
prepared (Chart 1). We have described the synthetic procedures
leading to these structures elsewhere.21
2.2. Identification of cytotoxic activity in cancer cell lines
Preliminary results of the anticancer activity of the synthesized
molecules were obtained using the Giemsa staining, a qualitative
technique in which the dye binds to DNA and allows visualization
of cells attached to culture plates. Cytotoxic effects were moni-
tored on Huh7 hepatocellular carcinoma (HCC) cell lines. The assay
resulted in identifying the lethal effect of the terminal alkyne pre-
cursor of triazoles obtained by acylation of the thiazolidine ring in
the presence of propiolic acid (Scheme 1). None of the remaining
compounds inhibited cell growth (Fig. 1).
Scheme 1. Reagents: (a) benzaldehyde in C2H5OH, H2O (1/1); (b) SOCl2 in absolute
C2H5OH; (c) propiolic acid, DCC in dry CH2Cl2.
From the molecules synthesized and evaluated with Giemsa,
four representative compounds were selected (one thymine deriv-
ative R = C(CH3)3, one benzoyl derivative R1 = (CH2)2-thiophene
R2 = C(CH3)3 and two triazoles R = thiophenethyl and R = amin-
ocyclohexyl) for a sulforhodamine B (SRB) assay, in addition to
compound 3 (ALC67). We confirmed our initial Giemsa assay re-
sults by SRB assays on liver, colon, and breast cancer cell lines.
Except for ALC67, none of the compounds showed significant cyto-
toxicity (Fig. 2).
The quantification of the in vitro antitumor activity of ALC67
was screened on various liver (Huh7, HepG2, Mahlavu, FOCUS),
breast (T47D, MCF7, BT20, CAMA-1), and endometrial (MFE-296)
cancer cell lines using the SRB assay according to the US’ National
Cancer Institute guidelines. The cytotoxic activity of ALC67 was
compared to that of camptothecin (CPT) and 5-fluorouracil (5-
FU), well-known anticancer agents. Promising micromolar IC50
values were obtained for all the tested cell lines (Table 1). The cyto-
toxicity of this compound was further confirmed by real-time cell
analysis (RT-CA), which is based on a time-dependent measure-
ment of the electrical impedance of attached (thus living) cells dur-
ing chemical treatment (Fig. 3). Observed cell death percentages in
the RT-CA with IC100 and IC50 concentrations (determined from the
SRB assays (Table 1)) were highly correlated, except for the Huh7
and CAMA-1 cells. In Huh7, 100% cell death occurred for both
Figure 1. (A) Giemsa staining of Huh7 cell lines with various thiazolidines; cell
death induced by ALC67 is shown in framed wells. (B) The structure of the active
compound 3 (ALC67), the (2RS, 4R)-2-Phenyl-3-propionyl-thiazolidine-4-carboxylic
acid ethyl ester.
concentrations (10 and 5
lM). On the other hand, CAMA-1 showed
a low cell death ratio even in IC100 concentrations (0.02
lM).
CAMA-1 cells grows in multilayers therefore the observed electri-
cal impedance may not reflect the real cell growth.
2.3. Characterization of the cell deaths induced by ALC67
The key feature of apoptotic cell death is DNA fragmentation
due to the activation of endogenous endonucleases. The terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) as-
say is a method that detects fragmented DNA by labeling the ter-
minal ends of nucleic acids. The condensed nuclei observed in
ALC67-treated cancer cells revealed that DNA damage in those
cells is most likely due to apoptosis (Fig. 4). Upon apoptotic stimuli,
the mitochondrial outer membrane permeabilizes and cytochrome
c is released in to the cytoplasm. Because ALC67 induced apoptosis,
we aimed to check the cytochrome c release in the presence of this
compound via immunostaining. And indeed, apoptosis bearing dif-
fuse cytochrome c staining confirmed its induction by ALC67
(Fig. 4).
2.4. Cell cycle arrest induced by ALC67
The apoptotic effect of ALC67 on the cell cycle was further char-
acterized by fluorescence-activated cell sorting (FACS) analysis,
using a propidium iodide stain. This analysis revealed the SubG1/
G1 cell cycle arrest in ALC67-treated Huh7 and Mahlavu cells com-
pared to control cells treated with DMSO (Fig. 5). Untreated HCC
Huh7 and Mahlavu cells showed a normally cycling cells FACS
spectrum, whereas treatment with ALC67 led to cell cycle arrest
at the SubG1/G1 phase (Fig. 5).
Chart 1. Structures of synthesized thiazolidines.

5096
F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
Figure 2. Percent cell death on liver (Huh7), colon (HCT116), and breast (MCF7)
cancer cell lines induced with increasing concentrations (2.5–40 M) of selected
l
compounds E05362 (triazole derivative R = thiophenethyl), E05396 (benzoyl
derivative R1 = (CH2)2-thiophene, R2 = C(CH3)3), E05389 (triazole derivative
R = aminocyclohexyl), E04832 (thymine derivative R = C(CH3)3), and ALC67. Treat-
ment was performed for 72 h in triplicate. NCI-SRB analysis was then applied as
explained in the Methods section. Absorbance values were normalized according to
the DMSO control and Tz. Camptothecin (CPT) was used as a positive control. The
results are representative of three independent experiments; S.D.s are less than
10%.
Figure 3. Real-time percent cell death monitoring for 72 h was performed in the
presence of ALC67 with the IC50 (empty circle) and IC100 (solid triangle) concen-
trations of Table 1. The effect of the compound on cell growth was analyzed using
xCELLigence software. The experiment was done in triplicate; the results were
normalized to the DMSO controls and Tz. The experiments were performed in
triplicate and standard deviations were less 10%.
Table 1
IC50 values of ALC67 in a series of cancer cell lines
a
Tissue
Liver
Cell line
ALC67 IC50
(l
M) CPT IC50
(
l
M) 5FU IC50 (lM)
caspase-9 activity; the caspase-8-specific inhibitor z-IETD-fmk
HepG2
Huh7
MV
FOCUS
HCT116
T47D
MCF7
BT20
10.0 1.5
5.3 0.93
0.41 0.5
5.47 1.5
9.23 0.89
7.62 1.73
4.7 0.81
1.6 0.56
0.01 0.42
0.5 0.3
0.01
0.15
<1
<1
<1
<1
<1
0.07
0.07
<1
5.7
(50
l
M) to quantify caspase-9’s activity when caspase-8 is inacti-
M) to
30.7
9.97
7.69
18.7
8.91
3.5
47.30
1.28
30.68
vated; or the caspase-3-specific inhibitor z-DEVD-fmk (50
l
quantify caspase-9 activity when caspase-3 is inactivated. The
addition of ALC67 led to a significant increase in the activity of cas-
pase-9 (Fig. 6A). Interestingly, such a treatment did not induce any
increase when caspase-3 or caspase-8 were inhibited by their spe-
cific peptide inhibitors (z-DEVD-fmk and z-IETD-fmk, respec-
tively). As depicted in Figure 6A the normalized values were
found to be smaller than 1. These results indicated a central role
of caspase-9 in the cell death process induced by the terminal
alkyne structure.
Colon
Breast
CAMA-1
Endometrial MFE-
296
a
The experiments were performed in triplicate and standard deviations are
shown with
.
Using the same experimental set-up, we also tested the path-
way exhibiting caspase-8 activity but normalized it with the cas-
pase-8 inhibitor. Therefore, initially, HepG2 cells were incubated
for 24 h with one of the caspase inhibitors (the caspase-8-specific
2.5. Investigation of the caspase dependency of apoptosis
To determine the apoptotic pathway that is implied in the de-
tected cell death induced by ALC67, we examined the independent
activities of caspase-8 and caspase-9 by inactivating caspase-3 and
-9, or caspase-8 and -3, respectively, using specific inhibitors.
inhibitor z-IETD-fmk (50
LEHD-fmk (50 M), the caspase-3-specific inhibitor z-DEVD-fmk
(50 M)) or without any of them. Then cells were further treated
lM), the caspase-9-specific inhibitor z-
l
l
Prior to treatment with ALC67 (10
bated for 24 h with one of the following compounds: the caspase-
9-specific inhibitor z-LEHD-fmk (50 M) in order to normalize
l
M), HepG2 cells were incu-
with ALC67, excluding the test tube with the z-IETD-fmk. After
12 h, caspase-8 activity was assessed. Our normalized results
demonstrated that treatment of HepG2 cells with ALC67 does not
l

F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
5097
ALC67
DMSO
Hoechst
Hoechst
TUNEL
TUNEL
A
HepG2
Huh7
FOCUS
Cyt c
Hoechst
Cyt c
Hoechst
B
HepG2
Huh7
FOCUS
Figure 4. Characterization of cell death as apoptosis with 10 lM ALC67 on HepG2, Huh7, and FOCUS liver cancer cells. Cells were incubated 48 h with the cytotoxic
compound. (A) Apoptotic cells displayed condensed nuclei visualized by TUNEL and Hoescht 33258 counterstaining. (B) By immunostaining, diffuse and intense cytochrome c
release from mitochon dria due to apoptosis can be observed in the presence of ALC67.
induce significant activation of caspase-8 even when caspase-9 or
caspase-3 is inhibited (Fig. 6B).
In order to confirm the caspase-9 dependent apoptotic pathway
activation by ALC67, we examined the cleavage patterns of cas-
pase-3, caspase-9 and caspase-8 using specific antibodies. Huh7
activity of thiazolidine rings has emerged only recently.16–20 The
easy, efficient, and rapid introduction of diverse moieties of this
five-membered heterocycle to several sites allows the rapid gener-
ation of small-molecule libraries bearing this core structure. The
recent identification of the anticancer property of 2-arylthiazolidine-
4-carboxylic acid amide compounds led us to design a chemical
library around the 2-phenylthiazolidine-4-carboxylic acid struc-
ture. Substituting the secondary amine of the heterocycle has not
yet been investigated, therefore we synthesized N-acylated struc-
tures to develop novel anticancer agents. Also, given the recent
description of the anticancer activity of some 1-thiazolyl-1,2,3-tri-
azoles27 and our expertise on the generation triazoles with sup-
ported Cu(I) catalysts,28,29 we decided to generate a library with
not only benzoyl and pyrimidine derivatives but also 1,2,3-
triazoles.
cells were incubated for 6, 12 or 24 h with ALC67 (2.5
lM and
5
l
M) or DMSO controls. Treatment with ALC67 caused cleavage
of caspase-9 and therefore activation of its downstream element
caspase-3 (Fig. 7A–C). However, caspase-8 was observed as intact
protein hence the cleaved products of caspase-8 at 43 or 18 kD
was not observed (Fig. 7A, D).
This result is in correlation with the caspase activity assay dem-
onstrated in Figure 6, suggesting that ALC67 treatment induced
caspase-9 dependent apoptotic pathway resulting in caspase-3
cleavage and apoptotic cell death.
When their antitumor activity was evaluated, none of the tria-
zoles gave satisfactory results but, interestingly, their alkyne pre-
cursor presented a considerable impact on hepatocellular, breast,
colon, and endometrial cancer cell lines due to micromolar IC50
values that the molecule exhibited. The detection of apoptotic
3. Discussion
Although the anticancer property of thiazolidinones was
established some time ago,16,22–26 analysis of the antiproliferative

5098
F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
Figure 6. Caspase activity assay with HepG2 cells. Cells were incubated with ALC67
in the absence and 50 M presence of various caspase inhibitor peptides: z-IETD-
l
fmk, z-LEHD-fmk, and z-DEVD-fmk for caspase-8, caspase-9, and caspase-3,
respectively. Then (A) caspase-9 and (B) caspase-8 activities were evaluated after
a
further 12 h of incubation in the presence of ALC67. Two independent
experiments were performed in duplicate. Endogenous caspase-9 (A) and
caspase-8 (B) activities were calculated in the absence of ALC67 with their
associated peptide inhibitors, to which drug-treated caspase activity values were
normalized and represented. The values ⁄p-value <0.05 (t-test) were compared with
cells treated with the caspase-9-specific inhibitor z-LEHD-fmk only; ⁄⁄p-value <0.05
(t-test) were compared with cells treated with the caspase-8-specific inhibitor
z-IETD-fmk only.
morphologies using several staining assays and the confirmation of
the cell death process by additional molecular biology techniques
revealed the apoptotic property of this novel cytotoxic compound.
The diversity of stimuli that can activate and regulate apoptosis
is extensive, and so is the diversity of signaling pathways that lead
to apoptosis. The extrinsic pathway is triggered by the activation of
death receptors, which recruit caspase-8 and either directly acti-
vate caspase-3 to lead to apoptosis (Fig. 8, pathway 1) or activate
a cascade of proteins that stimulates the activity of caspase-9
and caspase-3 (Fig. 8, pathway 2). Another apoptosis route starts
with microtubule or DNA damage, which trigger the activation of
a cascade of proteins, leading to the activation of caspase-9 and -
3 (Fig. 8, pathway 3). In order to determine the caspases involved
in the apoptotic mechanism triggered by the cytotoxic thiazolidine
compound ALC67, we investigated the activities of caspases-8 and
-9, maintaining or repressing the activities of caspase-3 and -9 or
caspase-3 and -8 respectively. Alteration of caspase-8 activity
was not observed in the presence of ALC67; thus the extrinsic
apoptotic pathway activated by death receptors does not seem to
be the one that leads to the observed cell death. To determine
whether the intrinsic apoptotic pathway was involved in ALC67-
induced cell death, the activity of caspase-9 in the presence of this
molecule was evaluated and a significant increase was detected.
Our results indicated that ALC67 stimulated the intrinsic apoptotic
pathway, a finding, consistent with our cytochrome c assay results
(Fig. 4). The release of cytochrome c in the cytosol is widely ac-
cepted to be responsible for the activation of caspase-9; once acti-
vated, caspase-9 activates caspase-3, which eventually results in
the execution of programmed cell death.30,31 Moreover, recent
studies have revealed the existence of a caspase-3-dependent
positive feedback loop that amplifies caspase-9 activity32–34 This
Figure 5. Cell cycle distribution analysis was performed on (A) Huh7 and (B)
Mahlavu liver cancer cells, which were treated with either DMSO or ALC67 (5 lM)
for 12 h and 24 h. Then FACS analysis was performed. The peak at 200 FL2-A
represents 2 N cells (G1-phase) and the peak at 400 represents 4 N cells (G2-phase).
The peak in-between represents S-phase cells. During gating, >4 N cells were
excluded since they showed no variation between control and treated cell groups.

F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
5099
Figure 8. Schematic representation of caspase-9- and -8-dependent apoptotic
pathways and the putative effect of ALC67 on apoptosis-induced cell death. Dashed
lines represent signaling with more than one protein component.
Fluorescent activated cell sorting analysis was performed to as-
sess the effect of ALC67 on the cell cycle. The results indicated
that ALC67 induced SubG1/G1 arrest compared to DMSO-treated
controls, which did not (Fig. 5). Most of the time, SubG1/G1 arrest
is related to apoptosis induction; moreover, this observation corre-
lated with the results of caspase activity assay that showed that
our novel compound induced caspase-9 activity and therefore
the intrinsic apoptotic pathway.
Finally, as no lethal effect was observed either with the 2-phe-
nylthiazolidine-4-carboxylic acid ethyl ester, namely the non-acyl-
ated thiazolidine (2) or with the triazoles resulting from the
bioactive alkyne (ALC67), the toxicity could be attributed to the
three-dimensional organization of the molecule and/or the pres-
ence of the terminal alkyne moiety. The importance of the pres-
ence of a terminal alkyne for cytotoxicity to be acquired has
already been described for several anticancer agents. These similar
compounds are reported to be nucleoside analogues, such as for 3’-
ethynylcytidine (ECyd) and 3’-ethynyluridine (EUrd)37–39, or acety-
lenic antifolates such as CB371740,41 or pralatrexate,42,43 which
were approved in 2009 as the first anticancer agents for the treat-
ment of relapsed or refractory peripheral T-cell lymphoma
(PTCL).44–46 A structure–activity relationship (SAR) study that aims
on the one hand to analyze the impact of this moiety and on the
other hand to improve the bioactivities is currently in progress. It
is also necessary to analyze the impact of the conjugated carbonyl
moiety on the bioactivity since electrophilic centers can trigger
glutathione depletion and hence apoptosis.47 Thus in addition to
the conjugated analogues, propargylamine derivatives will also
be evaluated in the SAR study.
Figure 7. Caspase cleavage in ALC67 treated Huh7 cell lines. Cells were treated
with 2.5 or 5 M ALC67 or DMSO control and incubated for 6, 12 or 24 h. Then (A)
l
activities of caspase-9, caspase-3 and caspase-8 were analyzed by their cleavage
status using specific antibodies against cleaved caspase-9 (c-caspase-9), cleaved
caspase-3 (c-caspase-3) and cleaved and intact caspase-8 by western blot analysis.
Cleaved caspase-8 fragments at 43 and 18 kD were not observed. c-caspase-9 and c-
caspase-3 were analyzed on the same membrane with anti-goat and anti-rabbit
secondary antibodies respectively. Actin is used for equal loading control. Quan-
tification of the gel documents of (B) cleaved-caspase-9, (C) cleaved-caspase-3 and
(D) caspase-8 were calculated by TotalLab Quant software and normalized to actin.
result could explain the inactivation of caspase-9 when cells are
treated by both the cytotoxic molecule and the caspase-3 inhibitor.
Furthermore, our results also displayed a crucial role for caspase-8
in the maintenance of caspase-9 activity, a finding in line with pre-
vious studies showing the amplifying role of caspase-8 in addition
to caspase-3 in Taxol-induced apoptosis.35,36 In addition, the acti-
vation of caspase-9 in the presence of ALC67 suggests a possible
interaction of the molecule with proteins involved in the mito-
chondrial apoptotic signaling pathways. ALC67 induced caspase-9
activation was further confirmed with western blot analysis using
specific antibodies against cleaved forms of caspase-9, caspase-3.
We were unable to observe any bands in cleaved forms of cas-
pase-8, which are expected to be 43 and 18 kD but inactive intact
form of caspase-8 was observed significantly. In order to determine
which proteins are involved microarray analyses could be per-
formed as modifications in gene expression levels can determine
which signaling pathways are disrupted by the presence of the
cytotoxic compound, and consequently reveal its target proteins.
In this study, a library of thiazolidine compounds was synthe-
sized and their cytotoxic activity was evaluated. An alkyne com-
pound, ALC67, was identified as cytotoxic to liver, colon, breast,
and endometrial cancer cells, with comparable IC50 values to that
of CPT and 5-FU. The investigation of the nature of the cytotoxic
effect of ALC67 led determining an apoptotic property that triggers
caspase-9 activity and therefore SubG1/G1 arrest.
4. Experimental section
4.1. Synthesis of the active compound: (2RS, 4R)-2-Phenyl-3-
propionyl-thiazolidine-4-carboxylic acid ethyl ester
4.1.1. (2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid (1)
Sodium hydroxide pellets (2.28 g, 56 .9 mmol) were added
to a solution of L-cystein hydrochlorate monohydrate (10.0 g,

5100
F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
56.9 mmol) in water (37 mL). After complete dissolution, succes-
sively, ethanol (11 mL), benzaldehyde (5.78 ml, 56.9 mmol), and
ethanol (30 mL) were added and the mixture was stirred for 3 h
at room temperature. The resulting precipitate was filtered and
washed with water before being dried under vacuo to lead to the
expected compound quantitatively (11.9 g).
4.4. Sulforhodamine B assay
Cells were plated in 96-well plates (1000–5000 cell/well in
200
lL) and grown for 24 h at 37 °C before being treated with var-
ious concentrations of the tested compounds (from 0.1 to 10
After 72 h of incubation the medium was aspirated, washed once
lM).
1H NMR (DMSO-d6) d 3.07–3.16 (m, 1H), 3.26–3.39 (m, 1H), 3.86
(dd, J = 9.0, 7.8 Hz, 0.4H), 4.22 (dd, J = 7.2, 4.5 Hz, 0.6H), 5.49 (s,
0.4H), 5.66 (s, 0.6H), 7.26–7.52 (m, 5H). 13C NMR (DMSO-d6) d
35.7, 36.2, 65.9, 66.2, 71.8, 72.2, 126.9, 128.1, 128.8, 140.1.
with 1 Â PBS (CaCl2-, MgCl2-free) (Gibco, Invitrogen), and then
50lL of a cold (4 °C) solution of 10% (v/v) trichloroacetic acid
(MERCK) was added. Microplates were left for 1 h at 4 °C. After
aspiration of the solution, plates were washed five times with
deionized water and left to dry. Fifty microliter of a 0.4% (m/v) of
sulforhodamine (Sigma–Aldrich) in 1% acetic acid solution were
added to each well and left at room temperature for 10 min. Then
the sulforhodamine B (SRB) solution was removed and the plates
washed five times with 1% acetic acid before air-drying. Bound sul-
forhodamine B was solubilized in a 200 lL 10 mM Tris-base solu-
tion and the plates were left on a plate shaker for 10 min. The
absorbance was read in a 96-well plate reader at 515 nm.
4.1.2. (2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid ethyl
ester (2)
Thionyl chloride (3.6 ml, 48.9 mmol) diluted in ethanol (30 mL)
was maintained at 0 °C. After the addition of (2RS, 4R)-2-Phenyl-
thiazolidine-4-carboxylic acid (5.00 g, 23.9 mmol), the mixture
was warmed to room temperature and stirred overnight. The sol-
vent was evaporated under reduced pressure and the resulting
crude was dissolved in dichloromethane, washed with a saturated
solution of NaHCO3 (3 Â 20 mL) and water (3 Â 20 mL). The organ-
ic layer was dried (MgSO4) and concentrated under reduced pres-
sure to yield the expected compound as yellow oil (4.14 g, 73%).
1H NMR (CDCl3) d 1.31 (m, 3H) 3.11 (dd, J = 12.0 Hz, J = 9.0 Hz,
0.6H), 3.19 (dd, J = 9.0 Hz, J = 6.0 Hz, 0.4H), 3.39 (dd, J = 9.0 Hz,
J = 6.0 HZ, 0.4H), 3.47 (dd, J = 9.0 Hz, J = 6.0 Hz, 0.6H), 3.98 (dd,
J = 9.0 Hz, J = 6.0 Hz, 0.6H), 4.07 (dd, J = 12.0 Hz, J = 9.1 Hz, 0.4H),
4.25 (m, 2H), 5.57 (s, 0.6H), 5.83 (s, 0.4H), 7.33–7.55 (m, 5H). 13C
NMR (CDCl3) d 14.0, 38.3, 39.7, 61.8, 65.5, 66.1, 71.2, 72.5, 127.0,
127.6, 127.9, 128.2, 130.3, 130.6.
4.5. Cytotoxicity assessment with real-time cell analyzer
For real-time cell analysis (RT-CA, xCELLigence, Roche Applied
Sciences), 50 lL of cell culture media was initially added to each
well of the 96X e-plate (Roche Applied Sciences) to get a steady
impedance value. Then, human cancer cells were seeded in
150 lL of media in varying concentrations of 1000 to 5000 cells/
well. The attachment, spreading, and proliferation of the cells were
monitored every 30 min using the RT-CA in a cell culture incuba-
tor. Approximately 24 h after seeding, when the cells were in the
log growth phase, they were treated with ALC67. For the control,
only DMSO was added to a well. Each experiment was repeated
at least three times. The electronic readout (cell-sensor imped-
ance) was displayed as an arbitrary unit called the cell index (CI).
The CI value was noted every 10 min for the first 24 h and then
every 30 min. The cell inhibition rate (%) = (1 – CI treated cells/
CIDMSO) Â 100.
LC–MS: ELSD 98%, rt = 5.73 min., m/z 238 (M+H)+.
4.1.3. (2RS, 4R)-2-Phenyl-3-propionyl-thiazolidine-4-carboxylic
acid ethyl ester (3)
Propiolic acid (0.246 mL, 4 mmol) was slowly added to a solu-
tion of dicyclohexylcarbodiimide (0.99 g, 4.8 mmol) in anhydrous
CH2Cl2 (10 mL) at 0 °C. After 10 min, a solution of (2RS, 4R)-2-Phe-
nylthiazolidine-4-carboxylate ethyl ester (0.949 g, 4 mmol) in
anhydrous CH2Cl2 (4 mL) was added dropwise, and the mixture
was first stirred at 0 °C for 1 h, and then at room temprature over-
night. The solution was filtered and the extract was evaporated un-
der reduced pressure. The resulting crude was purified on silica gel,
yielding a white solid (0.833 g, 72%).
4.6. Detection of apoptosis
Cells were seeded on coverslips in 6-well plates. After overnight
culture, cells were exposed to ALC67 at a concentration of 5 mM
for 48 h. To determine nuclear condensation by Hoescht 33258
(Sigma–Aldrich) staining, coverslips were washed twice with ice-
cold PBS, fixed in 1 mL of cold methanol for 10 min, and then incu-
1H NMR (CDCl3) d 1.20–1.27 (m, 3H), 2.97 (s, 0.6H), 3.16 (s,
0.4H), 3.19 (dd, J = 7.0 Hz, J = 12.0 Hz, 0.4H), 3.29 (dd, J = 7.0 Hz,
J = 12.0 Hz, 0.4H), 3.35 (d, J = 5.60 Hz, 1.2H), 4.15–4.25 (m, 2H),
4.91 (t, J = 5.60 Hz, 0.6H), 5.18 (t, J = 7.0 Hz, 0.4H), 6.28 (s, 0.4H),
6.40 (s, 0.6H), 7.19–7.62 (m, 5H). 13C NMR (CDCl3) d 14.3, 32.8,
33.9, 62.4, 62.7, 64.1, 65.6, 66.6, 67.9, 75.9, 76.4, 79.6, 81.8,
127.1, 127.3, 128.4, 128.6, 140.5, 152.4, 169.3. LC–MS: ELSD 98%,
rt = 9.82 min., m/z 290 (M+H)+.
bated with 3 lg/mL of Hoescht 33258 for 5 min in darkness. The
coverslips were then rinsed with distilled water, mounted on glass
microscopic slides using 50% glycerol, and examined under fluores-
cent microscopy. A TUNEL assay was performed using the in situ
Cell Death Detection kit (Roche), according to the manufacturer’s
recommendations.
4.2. Cell culture
4.7. Immunofluorescence assay for cytochrome C release
Cancer cells (n = 10) were cultured routinely at 37 °C under 5%
Cytoplasmic cytochrome c was tested by immunofluorescence
staining, as described by Achenbach et al. The cells were grown
on coverslips and fixed with 4% paraformaldehyde for 30 min at
room temperature, then rinsed with PBS and permeabilized in
ice-cold acetone for 10 min. After washing with PBS, the cells were
blocked with 3% BSA in PBS for 1 h at 37 °C and incubated with
anti-cytochrome c monoclonal antibody (BD Biosciences) over-
night at 4 °C. After washing with PBS, the cells were incubated with
FITC-conjugated anti-mouse secondary antibody for 1 h, in dark-
ness, at room temperature. The resulting coverslips were washed
three times with PBS and mounted on glass microscopic slides to
be analyzed by fluorescent microscopy.
CO2 in the standard medium (2 mM
L
-glutamine, 0.1 mM nones-
sential amino acids, 100 units/mL penicillin, 100
lg/mL streptomy-
cin in DMEM, supplemented with 10% FCS (Gibco, Invitrogen)).
4.3. In vitro cell growth assay (Giemsa staining)
Cells were plated into 24-well plates and grown overnight.
Chemicals were dissolved in DMSO and added to the medium at
a concentration of 4 mM and 8 mM. After 48 h of additional incu-
bation, attached cells were stained with Giemsa (Sigma–Aldrich)
and photographed.

F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
5101
4.8. Fluorescence-activated cell sorting analysis
funds, and funds from CNRS and INSERM. F.E. Onen-Bayram was
supported by the French Embassy in Turkey/ARC and the French
Ministry of Education, respectively. The authors thank Bilge Ozturk
for laboratory assistance, Zehra Onen for statistical analysis, and
Ms. R. Nelson for editing the English of the final version of our
manuscript.
Human cancer cell lines of interest were inoculated into 100-
mm culture dishes (100,000–200,000 cells/dish). Twenty-four
hours later, growth medium was replaced by starvation medium
(1% FBS, 1% P/S, 1% NEAA in DMEM) and inoculation was contin-
ued for an additional day. Cells were then treated with the
cytotoxic compound at the desired concentration and incubated
for 24 h before being collected by trypsinization. Pellets were
washed with 1 Â PBS. After centrifugation of the cell suspension,
the supernatant was discarded and the pellets were fixed in ice-
cold 70% ethanol and stored at 4 °C. Before the analysis, the pel-
References
1. Joshi, S.; Tiwari, A. K.; Mondal, B.; Sharma, A. Clin. Chim. Acta 2011, 412,
217.
2. Karagiannis, G. S.; Pavlou, M. P.; Diamandis, E. P. Mol. Oncol. 2010, 4, 496.
3. Liu, R.; Wang, K.; Yuan, K. F.; Wei, Y. Q.; Huang, C. H. Expert Rev. Proteomics
2010, 7, 411.
4. Schreiber, S. L. Bioorg. Med. Chem. 1998, 6, 1127.
5. Lafanechere, L. Comb. Chem. High Throughput Screening 2008, 11, 617.
6. Crews, C. M.; Splittgerber, U. Trends Biochem. Sci. 1999, 24, 317.
7. Scapin, G. Curr. Drug Targets 2006, 7, 1443.
8. Bredel, M.; Jacoby, E. Nat Rev Genet. 2004, 4, 262.
9. Azim, H.; Azim, H. A. Oncology 2008, 74, 150.
10. Browne, B. C.; O’Brien, N.; Duffy, M. J.; Crown, J.; O’Donovan, N. Curr. Cancer
Drug Targets 2009, 9, 419.
11. Lubbert, M.; Muller-Tidow, C.; Hofmann, W. K.; Koeffler, H. P. J. Cell. Biochem.
2008, 104, 2059.
12. Cohen, M. H.; Johnson, J. R.; Pazdur, R. Clin. Cancer Res. 2005, 11, 12.
13. Hunter, A. M.; LaCasse, E. C.; Korneluk, R. G. Apoptosis 2007, 12, 1543.
14. Varfolomeev, E.; Vucic, D. Future Oncol. 2011, 7, 633.
15. Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem. Lett. 2004,
14, 5289.
lets were re-suspended in 500
(25 L PI (Sigma–Aldrich), 5 L 10 mg/mL RNAase A (Fermentas),
0.25 L Triton X-100, and 469.75 L ice-cold PBS) and incubated
lL of propidium iodide solution
l
l
l
l
for 40 min at 37 °C in darkness. After an addition of 3 mL of PBS,
the suspension was centrifuged and the pellets re-suspended in
500 lL of PBS. Cell cycle analysis was conducted with FACSCali-
bur (Becton Dickinson). Data were analyzed and graphs were
prepared using CellQuest software purchased from Becton
Dickinson.
4.9. Caspase activity assay
Cells were plated in 96-well plates in the presence of one of
16. Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. J. Med. Chem. 2005, 48,
2584.
17. Gududuru, V.; Hurh, E.; Sullivan, J.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem.
Lett. 2005, 15, 4010.
18. Li, W.; Wang, Z.; Gududuru, V.; Zbytek, B.; Slominski, A. T.; Dalton, J. T.; Miller,
D. D. Anticancer Res. 2007, 27, 883.
19. Li, W.; Lu, Y.; Wang, Z.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem. Lett. 2007,
17, 4113.
caspase-8 inhibitor peptide z-IETD-fmk (50
tor peptide z-IETD-fmk (50 M), caspase-3 inhibitor peptide
z-DEVD-fmk (50 M), or the DMSO control. After 24 h of incuba-
tion at 37 °C, the medium was removed and replaced with
100 L of a fresh medium containing, in addition to the respective
inhibitors, the tested compound (ALC67) at a concentration of
10 M except for the normalization controls. After 12 h of incuba-
lM), caspase-9 inhibi-
l
l
l
20. Lu, Y.; Wang, Z.; Li, C. M.; Chen, J. J.; Dalton, J. T.; Li, W.; Miller, D. D. Bioorg. Med.
Chem. 2010, 18, 477.
l
tion, caspase activity was measured using the Caspase Glo Assay
Kit (Promega) according to the manufacturer’s recommendations.
21. Onen, F. E.; Boum, Y.; Jacquement, C.; Spanedda, M. V.; Jaber, N.; Scherman, D.;
Myllykallio, H.; Herscovici, J. Bioorg. Med. Chem. Lett. 2008, 18, 3628.
22. Desai, S.; Desai, P. B.; Desai, K. R. Heterocycl. Commun. 1999, 5, 385.
23. Verma, A.; Saraf, S. K. Eur. J. Med. Chem. 2008, 43, 897.
24. Havrylyuk, D.; Zimenkovsky, B.; Lesyk, R. Phosphorus Sulfur and Silicon and
the Related Elements 2009, 184, 638.
4.10. Western blotting
25. Lv, P. C.; Zhou, C. F.; Chen, J.; Liu, P. G.; Wang, K. R.; Mao, W. J.; Li, H. Q.; Yang,
Y.; Xiong, J.; Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 314.
26. Wang, S. B.; Zhao, Y. F.; Zhang, G. G.; Lv, Y. X.; Zhang, N.; Gong, P. Eur. J. Med.
Chem. 2011, 46, 3509.
27. Li, W. T.; Wu, W. H.; Tang, C. H.; Tai, R.; Chen, S. T. Acs Comb. Sci. 2011, 13, 72.
28. Girard, C.; Onen, E.; Aufort, M.; Beauviere, S.; Samson, E.; Herscovici, J. Org. Lett.
2006, 8, 1689.
29. Jlalia, I.; Beauvineau, C.; Beauviere, S.; Onen, E.; Aufort, M.; Beauvineau, A.;
Khaba, E.; Herscovici, J.; Meganem, F.; Girard, C. Molecules 2010, 15, 3087.
30. Hengartner, M. O. Nature 2000, 407, 770.
31. Nicholson, D. W. Cell Death Differ 1999, 6, 1028.
32. Fujita, E.; Egashira, J.; Urase, K.; Kuida, K.; Momoi, T. Cell Death Differ 2001, 8,
335.
33. Denault, J. B.; Eckelman, B. P.; Shin, H.; Pop, C.; Salvesen, G. S. Biochem. J. 2007,
405, 11.
34. Conrad, D. M.; Robichaud, M. R.; Mader, J. S.; Boudreau, R. T.; Richardson, A. M.;
Giacomantonio, C. A.; Hoskin, D. W. Int. J. Oncol. 2008, 32, 1325.
35. von Haefen, C.; Wieder, T.; Essmann, F.; Schulze-Osthoff, K.; Dörken, B.; Daniel,
P. T. Oncogene 2003, 22, 2236.
36. Wieder, T.; Essmann, F.; Prokop, A.; Schmelz, K.; Schulze-Osthoff, K.; Beyaert,
R.; Dörken, B.; Daniel, P. T. Blood 2001, 97, 1378.
37. Hattori, H.; Tanaka, M.; Fukushima, M.; Sasaki, T.; Matsuda, A. J. Med. Chem.
1996, 39, 5005.
Huh7 cells were treated with 2.5 lM and 5 lM ALC67 or DMSO
control for 6, 12 and 24 h. Then equal amounts of cell lysates were
solubilized with 1x loading dye, SRA (or DTT). The protein concen-
tration of the lysates was determined by the Bradford assay. Then
the lysates were denatured for 10 min in 100 °C. 20–50 ng of
proteins were loaded to the gels. NuPAGE NOVEX pre-cast gel sys-
tem was used for throughout the western blot analysis procedures
according to the manufacturer’s protocol. Depending on the
protein length MOPS or MES running buffer was used. After elec-
trophoresis, the proteins were transferred to nitrocellulose mem-
brane (30 V, 90 min) followed by incubation in blocking solution
(5% BSA in 1 Â TBS-T (0.1% tween)) for one hour at room tempera-
ture. Cleaved caspase-9 (Santa Cruz, sc-22182), caspase-3 (Cell sig-
naling, 9662S) and caspase-8 (IC12) (Cell signaling, 9746S) primary
antibodies were used in a ratio of 1:500 in 5% BSA-TBS-T, O/N
+4 °C. Secondary antibodies, anti-goat (Sigma, A8919), anti-rabbit
(Sigma, A6154), anti-mouse (Sigma, A0168), were applied in
1:5000 dilutions in 5%BSA-TBS-T (0.1%) for one hour at room tem-
perature. Actin (Sigma, A5441) primary antibody for equal loading
analysis was used in 1:5000 dilution in 5% milk-powder in TBS-
T(0.1%) for 1 h at room temperature. For visualization of the re-
sults, chemiluminescence was performed with ECL+ kit according
to the manufacturer’s protocol. The chemiluminescence light was
captured on X-ray film.
38. Takatori, S.; Kanda, H.; Takenaka, K.; Wataya, Y.; Matsuda, A.; Fukushima, M.;
Shimamoto, Y.; Tanaka, M.; Sasaki, T. Cancer Chemother. Pharmacol. 1999, 44,
97.
39. Kazuno, H.; Shimamoto, Y.; Tsujimoto, H.; Fukushima, M.; Matsuda, A.;
Sasakio, T. Oncol. Rep. 2007, 17, 1453.
40. Jackson, R. C.; Jackman, A. L.; Calvert, A. H. Biochem. Pharmacol. 1983, 32,
3783.
41. Jackman, A. L.; Taylor, G. A.; Oconnor, B. M.; Bishop, J. A.; Moran, R. G.; Calvert,
A. H. Cancer Res. 1990, 50, 5212.
42. Piper, J. R.; McCaleb, G. S.; Montgomery, J. A.; Kisliuk, R. L.; Gaumont, Y.;
Sirotnak, F. M. J. Med. Chem. 1982, 25, 877.
43. Degraw, J. I.; Colwell, W. T.; Piper, J. R.; Sirotnak, F. M. J. Med. Chem. 1993, 36,
2228.
44. O’Connor, O. A.; Hamlin, P. A.; Portlock, C.; Moskowitz, C. H.; Noy, A.; Straus, D.
J.; MacGregor-Cortelli, B.; Neylon, E.; Sarasohn, D.; Dumetrescu, O.; Mould, D.
Acknowledgments
This work was supported by grants from Turkish State Planning
Organization (DPT) KANILTEK project, Bilkent University local

5102
F. E. Onen-Bayram et al. / Bioorg. Med. Chem. 20 (2012) 5094–5102
R.; Fleischer, M.; Zelenetz, A. D.; Sirotnak, F.; Horwitz, S. Br. J. Haematol. 2007,
139, 425.
45. O’Connor, O. A.; Horwitz, S.; Hamlin, P.; Portlock, C.; Moskowitz, C. H.;
Sarasohn, D.; Neylon, E.; Mastrella, J.; Hamelers, R.; MacGregor-Cortelli, B.;
Patterson, M.; Seshan, V. E.; Sirotnak, F.; Fleisher, M.; Mould, D. R.; Saunders,
M.; Zelenetz, A. D. J. Clin. Oncol. 2009, 27, 4357.
46. O’Connor, O. A.; Pro, B.; Pinter-Brown, L.; Bartlett, N.; Popplewell, L.; Coiffier,
B.; Lechowicz, M. J.; Savage, K. J.; Shustov, A. R.; Gisselbrecht, C.; Jacobsen, E.;
Zinzani, P. L.; Furman, R.; Goy, A.; Haioun, C.; Crump, M.; Zain, J. M.; Hsi, E.;
Boyd, A.; Horwitz, S. J. Clin. Oncol. 2011, 29, 1182.
47. Laborde, E. Cell Death Differ. 2010, 17, 1373.