Synthesis of a novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones as promising anti-breast cancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.103569

A novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones 4–16 has been designed and synthesized. Preliminary screening of these compounds for their anti-breast cancer activity revealed that compounds 5, 7, and 9 possess the highest anti-cancer activities. The anti-tumor effects of compounds 5, 7, and 9 were evaluated against human breast cancer cell lines (MCF-7 and MDA-MB-231) and human breast cancer cells. They were also evaluated against normal non-cancerous breast cells, isolated from the same patients, to conclude about their use in a potential targeted therapy. Using MTT uptake method, these three compounds 5, 7, and 9 blunt the proliferation of these cancer cells in a dose-dependent manner with an IC₅₀ of 1.27, 1.50 and 1.31 μM respectively. Interestingly, using flow cytometry analysis these three compounds significantly mediated apoptosis of human breast cancer cells without affecting the survival of normal non-cancerous breast cells that were isolated from the same patients. Mechanistically, these compounds blunt the proliferation of MCF-7 breast cancer cells by robustly decreasing the phosphorylation of AKT, mTOR and the expression of VEGF and HIF-1α. Most importantly, compounds 5, 7, and 9 without affecting the phosphorylation and expression of these crucial cellular factors in normal non-cancerous breast cells that were isolated from the same patients. Additionally, using Western blot analysis the three compounds significantly (P < 0.05) decreased the expression of the anti-apoptotic Bcl-2 members (Bcl-2, Bcl-_XL and Mcl-1) and increased the expression of the pro-apoptotic Bcl-2 members (Bak, Bax and Bim) in MCF-7, MDA-MB-231 and human breast cancer cells making these breast cancer cells susceptible for apoptosis induction. Taken together, these data provide great evidences for the inhibitory activity of these compounds against breast cancer cells without affecting the normal breast cells.

Full text of DOI:10.1016/j.bioorg.2020.103569

Journal Pre-proofs
Synthesis of a novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones as
promising anti-breast cancer agents
Hussein El-Kashef, Gamal Badr, Nagwa Abo El-Maali, Douaa Sayed, Patricia
Melnyk, Nicolas Lebegue, Rofida Abd El-Khalek
PII:
S0045-2068(19)31679-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103569
YBIOO 103569
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
8 October 2019
2 December 2019
2 January 2020
Please cite this article as: H. El-Kashef, G. Badr, N. Abo El-Maali, D. Sayed, P. Melnyk, N. Lebegue, R. Abd El-
Khalek, Synthesis of a novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones as promising anti-breast
cancer agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103569
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Synthesis of a novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones as
promising anti-breast cancer agents
1
*
2*
1
3
Hussein El-Kashef, Gamal Badr , Nagwa Abo El-Maali , Douaa Sayed , Patricia
4
4
3
Melnyk , Nicolas Lebegue and Rofida Abd El-Khalek
1Department of Chemistry, Faculty of Science, Assiut University; Assiut 71516, Egypt.
2Laboratory of Immunology, Zoology Department, Faculty of Science, Assiut University,
Assiut 71516, Egypt.
3Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University; Assiut
7
1516, Egypt.
4Univ. Lille, Inserm, CHU Lille, UMR-S1172, JPArc - Centre de Recherche Jean-Pierre
Aubert Neurosciences et Cancer, F-59000 Lille, France
*
Corresponding authors:
Prof. Dr. Hussein El-Kashef
Email address: elkashef@aun.edu.eg
Prof. Dr. Gamal Badr
Tel: +20 1005075881
Tel: +20 1023370090
Email address: badr73@yahoo.com
1

Abstract
A novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones 4-16 has been designed and
synthesized. Preliminary screening of these compounds for their anti-breast cancer activity
revealed that compounds 5, 7, and 9 possess the highest anti-cancer activities. The anti-
tumor effects of compounds 5, 7, and 9 were evaluated against human breast cancer cell
lines (MCF-7 and MDA-MB-231) and human breast cancer cells. They were also evaluated
against normal non-cancerous breast cells, isolated from the same patients, to conclude
about their use in a potential targeted therapy. Using MTT uptake method, these three
compounds 5, 7, and 9 blunt the proliferation of these cancer cells in a dose-dependent
manner with an IC50 of 1.27, 1.50 and 1.31 µM respectively. Interestingly, using flow
cytometry analysis these three compounds significantly mediated apoptosis of human
breast cancer cells without affecting the survival of normal non-cancerous breast cells that
were isolated from the same patients. Mechanistically, these compounds blunt the
proliferation of MCF-7 breast cancer cells by robustly decreasing the phosphorylation of
AKT, mTOR and the expression of VEGF and HIF-1α. Most importantly, compounds 5,
7
, and 9 without affecting the phosphorylation and expression of these crucial cellular
factors in normal non-cancerous breast cells that were isolated from the same patients.
Additionally, using Western blot analysis the three compounds significantly (P < 0.05)
decreased the expression of the anti-apoptotic Bcl-2 members (Bcl-2, Bcl-XL and Mcl-1)
and increased the expression of the pro-apoptotic Bcl-2 members (Bak, Bax and Bim) in
MCF-7, MDA-MB-231 and human breast cancer cells making these breast cancer cells
susceptible for apoptosis induction. Taken together, these data provide great evidences for
the inhibitory activity of these compounds against breast cancer cells without affecting the
normal breast cells.
Key Words: Thiazolidinediones; thiazolidine-2,4-diones; apoptosis; Bcl-2 family; anti-
breast cancer.
2

1
. Introduction
Cancer, which affected about 18.1 million new cases in 2018, is the second leading cause
of death after the heart diseases across the world [1]. Among the various types of malignant
tumors reported so far, breast cancer is the most frequent cancer in women and represents
the second most prominent reason for deaths among women [2, 3]. Despite the big efforts
devoted to fight against different types of cancer, yet this disease remains one of the major
concerns worldwide. Therefore, the discovery of new and potent anticancer drugs is always
in demand.
Thiazolidine-2,4-diones (TZDs), represent a very important class of heterocyclic
compounds which is highly explored in the literature. TZDs possess a wide spectrum of
biological activities such as antidiabetic [4-9], anti-infectious [10-15], anti-inflammatory
[
16, 17], analgesic [18], antioxidant [19], immunomodulatory [20], overactive bladder
inhibitory [21] and insecticide [22] activities. TZDs also possess inhibitory activity against
tyrosinase [23], hyperlipidimia [24] and acute liver injury [25].
Furthermore, it was reported [26] that TZDs affected cancer development, progression and
metastasis, among which the Raf/MEK/extracellular signal regulated kinase (ERK) [27],
phosphatidylinositol 3-kinase (PI3K)/AKT [28], Wnt signal transduction pathways [29],
PIM-1 and PIM-2 protein kinases overexpression [30], androgen receptor stimulated genes
[
31] and peroxisome proliferator-activated receptors (PPARs) signaling cascades [32]
which are the most commonly up-regulated in human cancers. Most importantly, two
recent reports demonstrated anti-breast cancer activity of TZDs, but the molecular
mechanisms underlying the treatment of breast cancer cells with these compounds were
not clarified [31,33]. Thus, in our present study three synthesized TZDs (5, 7 and 9) were
3

evaluated against human breast cancer cell lines (MCF-7 and MDA-MB-231) and human
breast cancer cells. They were also evaluated against human normal non-cancerous breast
cells which were isolated from the same breast cancer patients. We also monitored the
molecular mechanisms underlying treatment of breast cancer cells with the three
thiazolidinediones 5, 7 and 9.
4

2
. Results and discussion
.1. Chemistry
2
In the present study, some novel thiazolidine-2,4-dione (TZD) derivatives were
synthesized using thiazolidine-2,4-dione (1) [34] as a starting material. This latter
compound has the advantage of possessing two reactive centers: the active methylene
group at position 5 and the nucleophilic nitrogen anion at position 3, of its potassium salt,
which together allow the synthesis of a variety of TZD derivatives. Accordingly, the active
methylene group of compound 1 was reacted with 3,4,5-trimethoxybenzaldehyde via
Knoevenagel
reaction
to
give
the
corresponding
(Z)-5-(3,4,5-
trimethoxybenzylidene)thiazolidine-2,4-dione isomer (2). This reaction was carried out in
boiling anhydrous toluene in the presence of few drops of piperidine and glacial acetic acid
using a Dean-Stark apparatus. The idea behind using the 3,4,5-trimethoxybenzaldehyde, in
the above condensation reaction, was to mimic the 3,4,5-trimethoxybenzene moiety found
in many natural anticancer drugs described in the literature such as combretastatin A-4 [35]
or colchicine [36] (Figure 1).
5

Compound 2 was then converted into its potassium salt 3 by treatment with an ethanolic
KOH solution (Scheme 1).
Reagents and conditions: (a) i. water, 0-5°C, 20 min, ii. HCl, reflux, 12 h, (b) anhydrous toluene, drops of
piperidine and CH
3
COOH, reflux, 15 h. (c) KOH/ethanol, 80 ºC, 20 min.
Scheme 1. General synthesis of compound 3.
Both compounds 2 and 3 were used as synthons in the synthesis of the title compounds.
Thus, when Mannich reaction was applied to compound 2 using piperidine, morpholine or
piperazine as a secondary amine, the corresponding thiazolidinedione derivatives 4-6 were
obtained (Scheme 2).
Reagents and conditions: (a) formaldehyde, piperidine, ethanol, reflux, 6h. (b) formaldehyde, morpholine,
ethanol, reflux, 6h. (c) formaldehyde, piperazine, ethanol, reflux, 6h.
Scheme 2. General synthesis of compound 4-6.
6

Alternatively, other N-substituted TZD derivatives 7-16 could be obtained via the reaction
of the salt 3 with halogeno- and dihalogeno compounds. Thus, when 3 was reacted with
some chloro compounds, such as chloroacetonitrile, 2-chloroacetamide, ethyl
chloroacetate, and chloroacetone in boiling DMF, the corresponding (Z)-N-substituted 5-
(3,4,5-trimethoxybenzylidene)thiazolidinediones 7-10 were obtained (Scheme 3).
Reagents and conditions: (a) chloroacetonitrile, DMF, reflux, 6h. (b) 2-chloroacetamide, DMF, reflux, 6h.
(
c) ethyl chloroacetate, DMF, reflux, 6h. (d) chloroacetone, DMF, reflux, 6h.
Scheme 3. General synthesis of compounds 7-10.
1
The H-NMR spectra of the basic structure of compounds 4-10 showed characteristic
signals of the 9 protons of the three methoxy groups (in addition to those of the other
substituents at the N atom). In deuterated chloroform, these 9 protons appeared as a singlet
at  3.92 ppm as in compounds 4, 6 and 7 (Figure 2A). However, in deuterated DMSO,
they appeared as two singlets at  3.70-3.74 and 3.84-3.85 ppm, integrated for 3 protons
(
of the 4-methoxy substituent) and 6 protons (of the 3,5-dimethoxy substituents) as in
compounds 5, 8-10 respectively (Figure 2B). The exocyclic methylene group of
compounds 4-10 appeared as a singlet at  4.24-4.73 ppm (H , Figures 2A and 2B). The
two aromatic protons appeared as a singlet at  6.75-6.98 ppm (H , Figures 2A and 2B).
3
1
The methylidene proton -CH= appeared downfield as a singlet at 7.80-7.95 ppm (H ,
2
7

Figures 2A and 2B), deshielded by the anisotropic effect exerted by the nearby carbonyl
group, proving a Z-configuration [19, 37]. No signal indicating the formation of the E-
configuration was observed, since the chemical shift of the -CH= proton in this case should
appear around 6.25 ppm [19]. The unambiguous exclusive formation of Z-configuration of
these products was confirmed by X-ray analysis as was shown in our X-ray report [38].
1
3
The C-NMR spectra are consistent with the structure of all the compounds obtained.
On the other hand, a series of (Z)-bis(5-(3,4,5-trimethoxybenzylidene)thiazolidin-3-
yl)alkanes 11-16 with alkane spacers was obtained through the reaction of 3 with
dihalogeno alkanes in DMF under reflux (Scheme 4). All analyses of 11-16 are consistent
with the structures obtained.
Reagents and conditions: dihalogeno alkanes, DMF, reflux, 6 hr.
Scheme 4. General synthesis of compound 11-16.
2
.2. Biology
The treatment of drug-resistant cancer is a clinical challenge, and thus searching for novel
anticancer drugs is critically important [39]. Therefore, an intense research effort is
underway to reveal the underlying mechanisms of apoptosis and cancer progression [40].
Clinically, many chemotherapeutic drugs provide a satisfactory response upon their first
exposure to the tumors, but they cause a variety of side effects to the patients. Therefore,
8

there is an urgent need for potent and selective anticancer drugs [41]. We focused our
attention in the present study to investigate the anti-tumor potential of the three most potent
TZD derivatives and to clarify the underlying molecular mechanisms in the human breast
carcinoma cell lines MDA-MB-231 and MCF-7. We also monitored whether these
analogues exert cytotoxic effects on both human breast carcinoma cell and non-
tumorigenic normal breast cell. There are several mechanisms of anticancer activity of
thiazolidine-2,4-dione derivatives already discussed in the literature. The most known ones
are induction of apoptosis, cell differentiation, and cell cycle arrest [42]. Proliferation is a
crucial process in the maintenance and progression of cancer cells. The ability of cancer
cells to escape from apoptosis and continue to proliferate is one of the fundamental
hallmarks of cancer and is a major target for cancer treatment [39]. In the present study,
we found that compounds 5, 7 and 9 inhibit the proliferation and cell viability of human
breast cancer cells.
2
.2.1. Compounds 5, 7 and 9 blunt the proliferation and cell viability of human
breast cancer cells
Preliminary anti-proliferative screening of all the compounds prepared has been performed
against MCF-7 cell line using MTT uptake method to identify the most potent compounds.
Human breast cancer cells were isolated from breast cancer patients who were admitted to
South Egypt Cancer Institute, Assiut University, Egypt. The isolated breast cancer cells
were then treated with different concentrations of compounds 5, 7 and 9 for 12-h and their
anti-tumor effect was monitored using MTT uptake method. These compounds showed a
dose-dependent anti-proliferative effect with an IC50 of 1.27, 1.50 and 1.31 µM
respectively (Figure 3). These experiments were performed in triplicate and the results
9

were expressed as the mean percentage of viable cells ± SEM. These results were
confirmed by a Trypan blue exclusion test, which was used to determine cell viability and
numerical cell counts simultaneously in all experiments.
2
.2.2. Compounds 5, 7 and 9 robustly mediate apoptosis of human breast cancer
cells.
The inhibition of cancer cell proliferation, the cessation of cell cycle progression and the
induction of apoptosis have all been targeted in chemotherapeutic strategies for the
treatment of various types of cancer. Given the anti-proliferative activity of compounds 5,
7
and 9 against human breast cancer cells, we next evaluated their impact on the induction
of apoptosis in human normal non-cancerous breast cells and in human breast cancer cells,
that were isolated from the same patients, using propidium iodide (PI)/Annexin V double
staining, followed by flow cytometry analysis. Both human breast cancer- and normal cells
were treated with 1.27, 1.50 and 1.31 µM of compounds 5, 7 and 9 respectively for 12 h.
The data from a representative experiment are presented in the dot plot shown, and the
percentages of viable cells (lower left quadrant), early apoptotic cells (lower right quadrant)
and late apoptotic cells (upper right quadrant) were determined (Figure 4A). In the human
normal non-cancerous breast cells, the percentages of apoptotic cells (early and late
apoptotic) were 5% in untreated cells, 5%, 4.5% and 8%, in 5-, 7-, and 9-treated cells,
respectively. However, in human breast cancer cells the percentages of apoptotic cells
(early and late apoptotic) were 12.5% in untreated cells, 65.5%, 77%, and 69%, in 5-, 7-,
and 9-treated cells, respectively. Pooled data from three different experiments are
expressed as the mean percentage of apoptotic cells ± SEM revealed that compounds 5, 7
and 9 significantly (P < 0.05) induced apoptosis of human breast cancer cells without
1
0

affecting the normal non-cancerous breast cells. The effect of compounds 7 (Figure 4B,
closed black bars), 9 (Figure 4C, closed gray bars) and 5 (Figure 4D, hatched bars) on the
induction of apoptosis of human breast cancer cells were significantly obvious.
2
.2.3. Treatment with compounds 5, 7 and 9 induces apoptosis in human breast
cancer cells through direct effect on PI3K/AKT/mTOR, VEGF and HIF-1α
signaling
The mechanism of induced apoptosis upon treatment with compounds 5, 7 and 9 was
investigated by treating MCF-7 cell lines with 1.27, 1.50 and 1.31 µM of 5, 7 and 9
respectively for 12 h, followed by lysing the cells in lysis buffer. Western blot analysis was
then used to analyze the phosphorylation of AKT and mTOR, and the expression of VEGF
and HIF-1α. The results of a representative experiment (out of five) on MCF-7 breast
cancer cells showed that the treatment with compounds 5, 7 and 9 markedly decreased the
phosphorylation of AKT (Figure 5A) and mTOR (Figure 5C), and also decreased the
expression levels of VEGF and HIF-1α (Figure 5E) compared with the medium (0)-treated
cells. The phosphorylation levels of AKT and mTOR were normalized to the total relevant
proteins (T-AKT and T-mTOR) and expression levels of VEGF and HIF-1α were
normalized to total β-actin protein levels. The data from 5 independent experiments
performed on MCF-7 cells showed that the treatment with compounds 5, 7 and 9
significantly (P < 0.05) inhibited the phosphorylation of AKT (Figure 5B) and mTOR
(Figure 5D) and the expression levels of VEGF and HIF-1α (Figure 5F) compared with
medium (0)-treated cells. This experiment was then repeated using both human normal
non-cancerous breast cells and human breast cancer cells which were isolated from the
same breast cancer patients.
1
1

The results of a representative experiment (out of five) showed that the phosphorylation of
AKT (Figure 6A) and mTOR (Figure 6C), and the expression of VEGF and HIF-1α
(Figure 6E) were obviously increased in human breast cancer cells in comparison with
human non-cancerous breast cells. Additionally, treatment of human breast cancer cells
with compounds 5, 7 and 9 markedly decreased the phosphorylation of AKT (Figure 6A)
and mTOR (Figure 6C) and also decreased the expression levels of VEGF and HIF-1α
(Figure 6E) compared with the medium (0)-treated cells. Interestingly, no obvious effects
were observed with the tested compounds on human non-cancerous breast cells. As
previously, after normalization of the data obtained from five independent experiments, the
results clearly indicated that the treatment with compounds 5, 7 and 9 significantly (P <
0
.05) inhibited the phosphorylation of AKT (Figure 6B) and mTOR (Figure 6D) and the
expression levels of VEGF and HIF-1α (Figure 6F) compared with medium (0)-treated
cells.
2
.2.4. Treatment with compounds 5, 7 and 9 induces apoptosis in human breast cancer
cells through direct effects on the Bcl-2 family members.
We have investigated the expression levels of Bcl-2 family members (including the anti-
apoptotic members Bcl-2, Bcl-XL and Mcl-1, and the pro-apoptotic members Bak, Bax and
Bim) after treatment with 1.27, 1.50 and 1.31 µM of these three compounds 5, 7 and 9
respectively. The results of a representative experiment (out of five) on MCF-7 cell line
demonstrated that treatment with compounds 5, 7 and 9 markedly decreased the expression
levels of the anti-apoptotic Bcl-2 members Bcl-2, Bcl-XL and Mcl-1 (Figure 7A) and
clearly increased the expression levels of the pro-apoptotic Bcl-2 members Bak, Bax and
Bim (Figure 7B) compared with the medium (0)-treated cells. The expression levels of the
1
2

analyzed proteins were normalized to total GAPDH protein levels. These results were
significant (P < 0.05), as shown in Figures 7C and 7D, and revealed that the decreased
expression of the anti-apoptotic Bcl-2 members was accompanied by an increase in the
expression of the pro-apoptotic Bcl-2 members, which favors apoptosis induction. Similar
results were obtained using MDA-MB-231 human breast cancer cell line (Figure 8).
This experiment was then repeated using both human normal non-cancerous breast cells
and human breast cancer cells that were isolated from the same patients. The results of a
representative experiment (out of five) clearly illustrated that the expression levels of the
anti-apoptotic Bcl-2 members Bcl-2, Bcl-XL and Mcl-1 (Figure 9A) were clearly up-
regulated, while the expression levels of the pro-apoptotic Bcl-2 members Bak, Bax and
Bim (Figure 9B) were clearly down-regulated in the human breast cancer cells as
compared with the expression levels in the human normal non-cancerous breast cells.
Additionally, treatment of human breast cancer cells with compounds 5, 7 and 9 markedly
decreased the expression levels of the anti-apoptotic Bcl-2 members Bcl-2, Bcl-XL and Mcl-
1
(Figure 9A) and clearly increased the expression levels of the pro-apoptotic Bcl-2
members Bak, Bax and Bim (Figure 9B) compared with the medium (0)-treated cells.
Normalized results allowed considering the impact of compounds 5, 7 and 9 on the
repression of the anti-apoptotic Bcl-2 members and the induction of the pro-apoptotic
proteins (Figures 9C and 9D).
Interestingly, the modulatory effects of compounds 5, 7 and 9 on the expression of Bcl-2
family members were restricted only on the human breast cancer cells without affecting
the human normal non-cancerous breast cells.
1
3

3
. Conclusion
A new series of thiazolidine-2,4-diones incorporating the 5-(3,4,5-trimethoxybenzylidene)
moiety, found in some natural anticancer drugs, has been designed and synthesized. The
synthesized TZDs were tested for their anti-breast cancer activity against human cancer
cell lines (MCF-7 and MDA-MB-231) and human breast cancer cells and also against
normal non-cancerous breast cells that were isolated from the same patients. Compounds
5
, 7 and 9 were shown to be the most active derivatives against breast cancer by evaluating
their activities using MTT assay, flow cytometry and Western blot analysis. Compounds
, 7 and 9 inhibit the proliferation of breast cancer cells in a dose dependent manner with
5
an IC50 of 1.27, 1.50 and 1.31 µM respectively. Interestingly, they induced apoptosis of
human breast cancer cells without affecting the normal non-cancerous breast cells. They
mediated apoptosis in MCF-7, MDA-MB-231 and human breast cancer cells through
decreasing the expression of the anti-apoptotic Bcl-2 members (Bcl-2, Bcl-XL and Mcl-1)
and increasing the expression of the pro-apoptotic Bcl-2 members (Bak, Bax and Bim).
Furthermore, the synthesized three compounds decreased the phosphorylation of AKT and
mTOR and also decreased the expression levels of VEGF and HIF-1α.
1
4

Experimental section
.1. Chemistry
.1. 1. General methods
3
4
All melting points were determined on a Stuart melting point apparatus SMP3 and are
uncorrected. IR spectra were recorded on a Nicolet iS10 FT-IR spectrometer using KBr
1
wafer technique. The H-NMR spectra were recorded on a Bruker Avance 300
1
13
1
13
spectrometer operating at 300 MHz ( H) or 75 MHz ( C). H- and C-NMR chemical
shifts were reported in parts per million (ppm) and were referenced to the residual proton
1
13
peaks in deuterated solvents; CDCl
3
(7.26 ppm for H and 76.90 ppm for C) and DMSO-
1
13
d
6
(2.50 ppm for H and 39.70 ppm for C). Multiplicities are represented by s (singlet), d
(
doublet), t (triplet), q (quartet), and m (multiplet). Coupling constants (J) are reported in
Hertz (Hz). Mass spectra were recorded with an LCMS (Waters Alliance Micromass ZQ
000). LCMS analysis was performed using a Waters XBridge C18 column (5 µm particle
size column, dimensions 50 mm x 4.6 mm). A gradient starting from 98% H O/formate
buffer 5 mM (pH 3.8) and reaching 100% CH CN/ formate buffer 5 mM (pH 3.8) within
min at a flow rate of 2 ml/min was used followed by a return to the starting conditions
2
2
3
4
within 1 min. Elemental analyses (C, H, N) were carried out using a Perkin Elmer 240 C
Micro analyzer and they were found in a good agreement with the theoretical values within
±
0.4%. Thiazolidine-2,4-dione (1) was prepared according a literature procedure [34].
.1.2. (Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dione (2)
A mixture of equimolar amounts (20 mmol) of thiazolidine-2,4-dione (1) (2.34 g) and
,4,5-trimethoxybenzaldehyde (3.92 g) in anhydrous toluene (50 ml) was heated under
reflux in the presence of few drops of acetic acid and piperidine in a flask equipped with a
4
3
1
5

Dean-Stark apparatus. The reaction mixture was heated under reflux for 15 h. After
cooling, the solid product obtained was filtered, washed with little toluene and dried.
Recrystallization from cyclohexane gave light yellow crystals, yield 5.49 g (93%), mp 185-
-
1
1
2
87 ºC. IR (ν cm ): 3176 (NH), 3020 (CH arom.), 2992 (CH aliph.), 2950 (CH aliph.),
1
836 (CH aliph.), 1748 (C=O), 1698 (C=O), 1606, 1576, 1506, and 1451 (C=C arom.). H-
NMR (300 MHz, DMSO-d
Ar-H), 7.25 (s, 1H, -CH=), 12.59 (s, 1H, NH); C NMR (75 MHz, DMSO-d
OCH ), 60.6 (OCH ), 107.9 (2 CH), 122.9 (C), 128.9 (C), 132.5 (C), 139.8 (CH), 153.6
2C), 167.7 (C=O), 168.3 (C=O). MS (ESI ) m/z 294.3 [M-H] . Anal. calcd. for
S (295.31): C, 52.87; H,4.44; N, 4.74. Found: C, 52.90; H, 4.19; N, 4.66 %.
.1.3. (Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dione potassium salt (3)
6
):  3.72 (s, 3H, -OCH
3
), 3.82 (s, 6H, 2 -OCH
3
), 6.91 (s, 2H,
1
3
6
):  56.4 (2 -
3
3
-
-
(
C
13
H13NO
5
4
A solution of potassium hydroxide (0.062 g, 1.1 mmol) in ethanol (3 ml) was added to a
hot solution of compound 2 (0.3 g, 1 mmol) in ethanol (3 ml). The reaction mixture was
then heated with stirring at 80 ºC for 20 min. The white fluffy long needles obtained were
filtered, washed with ethanol and dried. Yield (0.28 g (83%), mp 250-255 ºC (decomp.).
-1
IR (ν cm ): 3026 (CH arom.), 2955 (CH aliph.), 2849 (CH aliph.), 1742 (C=O), 1690
1
(
(
)
C=O), 1607, 1577, 1507, 1577 and 1454 (C=C arom.). H-NMR (300 MHz, DMSO-d
6
) δ
ppm): 3.68 (s, 3H, -OCH
3
), 3.80 (s, 6H, 2 -OCH
. C-NMR (75 MHz, DMSO-d ) δ (ppm): 56.2 (2 OCH
22.8 (C), 132.0 (C), 136.0 (C), 137.7 (CH), 153.3 (2 C), 176.0 (C=O), 183.3 (C=O). MS
3
), 6.86 (s, 2H, ArH), 7.25 (s, 1H, =CH-
1
3
6
3
), 60.5 (OCH ), 106.8 (2 CH),
3
1
-
-
(
ESI ) m/z 294.3 [M-H] . Anal. calcd. for C13
H
12KNO S (333.40): C, 46.83; H, 3.63; N,
5
4
.20. Found: C, 46.58; H, 3.50; N, 4.00 %.
1
6

4
.1.4. General procedure for the synthesis of compounds 4-6.
To a mixture of piperidine (0.1 ml, 1 mmol), morpholine (0.087 ml, 1 mmol) or piperazine
0.08 ml, 1 mmol) and formaldehyde (0.5 ml, 13.5 mmol) in methanol (5 ml), compound
(0.3 g, 1 mmol) was added. The reaction mixture was stirred under reflux for 6 hr in the
(
2
presence of few drops of acetic acid. After cooling the solid product obtained was filtered,
washed with water and dried. Recrystallization from ethanol gave crystals.
4
2
.1.4.1.
(Z)-3-(Piperidin-1-ylmethyl)-5-(3,4,5-trimethoxybenzylidene)thiazolidine-
,4-dione (4)
-1
White crystals, yield 0.26 g (65%), mp 147-149 ºC. IR (ν cm ): 3008 (CH arom.), 2921
(CH aliph.), 2833 (CH aliph.), 1739 (C=O), 1682 (C=O), 1605, 1574, 1501 and 1452 (C=C
1
arom.). H-NMR (300 MHz, CDCl
3
) δ (ppm): 1.40 (m, 2H, CH
), 3.92 (s, 9H, 3 O-CH ), 4.73 (s, 2H, CH
ArH), 7.80 (s, 1H, =CH-). C-NMR (75 MHz, CDCl ) δ (ppm): 23.7 (CH
2
), 1.55 (m, 4H, 2 CH
2
),
2
.60 (t, 4H, J = 5.4 Hz, 2 N-CH
2
3
2
), 6.75 (s, 2H,
1
3
3
2
), 25.9 (2 CH
2
),
5
1
2
1.9 (2 -NCH
2
), 56.2 (2 -OCH
3
), 61.0 (-OCH
3
), 64.5 (-NCH
2
N-), 107.5 (2 CH), 120.7 (C),
+
28.8 (C), 133.7 (C), 140.2 (CH), 153.6 (2 C), 167.6 (C=O), 168.9 (C=O). MS (ESI ) m/z
+
96.0 [M+H-CH
2
-piperidine] . Anal. calcd. for C19
H
24
N
2
5
O S (392.47): C, 58.15; H, 6.16;
N, 7.14. Found: C, 58.00; H, 6.26; N, 6.99 %.
4
2
.1.4.2. (Z)-3-(Morpholin-4-ylmethyl)-5-(3,4,5-trimethoxybenzylidene)thiazolidine-
,4-dione (5)
-1
Yellow crystals, yield 0.36 g (90%), mp 143-145 ºC. IR (ν cm ): 3017 (CH arom.), 2952
(CH aliph.), 2854 (CH aliph.), 1740 (C=O), 1682 (C=O), 1604, 1575, 1501 and 1449 (C=C
1
arom.). H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 2.53 (t, 4H, J = 4.5 Hz, 2 -NCH
2
), 3.55
(
t, 4H, J = 4.5 Hz, 2 -OCH
2
), 3.74 (s, 3H, -OCH ), 3.84 (s, 6H, 2 -OCH ), 4.57 (s, 2H,
3
3
1
7

1
3
CH
0.9 (2 CH
CH), 120.9 (C), 129.0 (C), 133.7 (C), 140.0 (CH), 153.7 (2 C), 167.2 (C=O), 168.9 (C=O).
2
), 6.95 (s, 2H, ArH), 7.87 (s, 1H, =CH-). C-NMR (75 MHz, DMSO-d
6
) δ (ppm):
5
2
), 56.5 (2 -OCH ), 60.7 (-OCH ), 63.7 (-N-CH -N-), 66.5 (2 CH
3
3
2
2
), 108.0 (2
+
+
MS (ESI ) m/z 296.0 [M+H-CH
C, 54.81; H, 5.62; N, 7.10. Found: C, 54.60; H, 5.80; N, 6.84 %.
.1.4.3. 3,3'-(Piperazine-1,4-diylbis(methylene))bis(5-((Z)-3,4,5-trimethoxy
benzylidene)thiazolidine-2,4-dione) (6)
Yellow crystals, yield 0.60 g (84%), mp 205-207 ºC. IR (ν cm ): 3000 (CH arom.), 2992
2
-morpholine] . Anal. calcd. for C18
H N
22 2
6
O S (394.44):
4
-1
(CH aliph.), 2944 (CH aliph.), 2828 (CH aliph.), 1737 (C=O), 1678 (C=O), 1603, 1576,
1
1
504 and 1466 (C=C arom.). H-NMR (300 MHz, CDCl
3
) δ (ppm): 2.70 (s. 8H,
) 4.71 (s, 4H, 2 -NCH N-), 6.74
s, 4H, ArH), 7.81(s, 2H, 2 -CH=). C-NMR (75 MHz, CDCl )δ (ppm): 50.4 (4CH ), 56.2
piperazine), 3.92 (s, 6H, 2 -OCH
3
), 3.93 (s, 12H, 4 -OCH
3
2
1
3
(
(
3
2
4 OCH
3
), 61.0 (2 OCH
3
), 63.1 (2CH
2
), 107.5 (4C), 120.3 (2C), 128.6 (2C), 134.1 (2C),
+
1
40.3 (2CH), 153.6 (4C), 167.3 (C=O), 168.8 (C=O). MS (ESI ) m/z 296.0 [M+H-H
2
-
+
substituted piperazine] . Anal. calcd. for C32
H
36
N
4
O
10
2
S (700.78): C, 54.85; H, 5.18; N,
8
.00. Found: C, 54.66; H, 5.30; N, 7.89 %.
.1.5. General procedure for the synthesis of compounds 7-10.
4
A mixture of the potassium salt 3 (0.33 g, 1 mmol) and chloroacetonitrile (0.082 g, 0.07
ml, 1.1 mmol), chloroacetamide (0.1 g, 1.1 mmol), ethyl chloroacetate (0.135 g, 0.116 ml,
1
.1 mmol), chloroacetone (0.1 g, 0.09 ml, 1.1 mmol) in DMF (5 ml) was heated under
reflux for 6 hr. After cooling the solid product obtained was filtered, washed with water,
dried and recrystallized.
1
8

4
.1.5.1. ((Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dione-3-yl)acetonitrile
7)
Yellow needles (recrystallization from ethanol), yield 0.26 g (87%), mp 173-175 ºC. IR (ν
(
-1
cm ): 3026 (CH arom.), 2998 (CH aliph.), 2836 (CH aliph.), 2222 (CN), 1732 (C=O), 1681
1
(
C=O), 1608, 1573 and 1448 (C=C arom.). H-NMR (300 MHz, CDCl
3
) δ (ppm): 3.93
1
3
(
s, 9H, 3 -OCH
) δ (ppm): 28.2 (CH
CN), 118.5 (C), 127.9 (C), 136.3 (C), 141.0 (CH), 153.7 (2 C), 164.1 (C=O), 166.1 (C=O).
3
), 4.63 (s, 2H, CH
2
), 6.75 (s, 2H, ArH), 7.92 (s, 1H, =CH-). C-NMR (75
MHz, CDCl
3
2
), 56.3 (2 -OCH ), 61.1 (-OCH ), 109.5 (2 CH), 112.8
3
3
(
+
+
MS (ESI ) m/z 335.0 [M+H] . Anal. calcd. for C15
H
14
N
2
5
O S (334.35): C, 53.89; H, 4.22;
N, 8.38. Found: C, 53.72; H, 4.33; N, 8.18 %.
4
.1.5.2. 2-((Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dion-3-yl)acetamide
8)
White crystals (recrystallization from ethanol: dioxane mixture (1:3)), yield 0.26 g (83%),
(
-1
mp 274-275 ºC (decomposition). IR (ν cm ): 3384, 3304 and 3264 (NH
2
) 3026 (CH arom),
2
974 (CH aliph.), 2839 (CH aliph.), 1740 (C=O), 1682 (C=O), 1604, 1579, 1508 and 1448
1
(
C=C arom.). H-NMR (300 MHz, DMSO-d
-OCH ), 4.24 (s, 2H, CH ), 6.97 (s, 2H, ArH), 7.32 (s, 1H, NH), 7.73 (s, 1H, NH), 7.92
s, 1H, =CH-). C-NMR (75 MHz, DMSO-d ) δ (ppm): 43.8 (N-CH ), 56.5 (2 -OCH ),
), 108.1 (2 CH), 120.6 (C), 128.9 (C), 133.9 (C), 140.1 (CH), 153.7 (2 C),
6
), δ (ppm): 3.70 (s, 3H, -OCH ), 3.85 (s, 6H,
3
2
3
2
1
3
(
6
2
3
6
0.7 (-OCH
65.7 (C=O), 167.3 (C=O), 167.5 (C=O). MS (ESI ) m/z 353.0 [M+H] . Anal. calcd. for
S (352.36): C, 51.13; H, 4.58; N, 7.95. Found: C, 50.89; H, 4.60; N, 7.78 %.
.1.5.3. Ethyl ((Z)-5-(3,4,5-trimethoxybenzylidene)thiazolidine-2,4-dion-3-yl)acetate
9)
3
+
+
1
C
15
H
16
N
2
O
6
4
(
1
9

White crystals (recrystallization from ethanol), yield 0.22 g (64%), mp 145-147 ºC. IR (ν
-1
cm ): 3018 (CH arom.), 2999 (CH aliph.), 2942 (CH aliph.), 2842 (CH aliph.), 1760
1
(
C=O), 1739 (C=O), 1689 (C=O), 1602, 1575, 1504 and 1469 (C=C arom.). H-NMR (300
MHz, DMSO-d
s, 6H, 2 –OCH
ArH), 7.95 (s, 1H, =CH-). C-NMR (75 MHz, DMSO-d
6
) δ (ppm): 1.22 (t, 3H, J = 7.1 Hz, OCH
), 4.18 (q, 2H, J = 7.1 Hz, OCH CH ), 4.50 (s, 2H, CH
) δ (ppm): 14.4 (CH
2
CH
3
), 3.74 (s, 3H, -OCH
), 6.98 (s, 2H,
), 42.7 (-
3
), 3.84
(
3
2
3
2
1
3
6
3
NCH
2
), 56.5 (2 -OCH
3
), 60.7 (-OCH
3
), 62.1 (OCH
2
), 108.2 (2 CH), 119.9 (C), 128.7 (C),
1
34.8 (C), 140.3 (CH), 153.7 (2C), 165.3 (C=O), 167.1 (C=O), 167.3 (C=O). MS (ESI+)
+
m/z 382.0 [M+H] . Anal. calcd. for C17
H
19NO S (381.40): C, 53.54; H, 5.02; N, 3.67.
7
Found: 53.38; H, 5.15; N, 3.47 %.
4
.1.5.4. (Z)-3-(2-Oxopropyl)-5-(3,4,5-trimethoxybenzylidene)thiazolidine-2,4-dione
10)
Yellow crystals (recrystallization from ethanol), yield 0.152 g (82%), mp 139-140 ºC. IR
(
-1
(
ν cm ): 3026 (CH arom.), 2939 (CH aliph.), 2839 (CH aliph.), 1748 (C=O), 1726 (C=O),
1
1
691 (C=O), 1604, 1576, 1506 and 1456 (C=C arom.). H-NMR (300 MHz, DMSO-d
6
) δ
), 4.67 (s, 2H, CH ),
.98 (s, 2H, ArH), 7.92 (s, 1H, =CH-). C-NMR (75 MHz, DMSO-d ) δ (ppm): 27.5
), 50.8 (-NCH ), 56.3 (2 -OCH ), 60.7 (-OCH ), 108.2 (2 CH), 120.3 (C), 128.8 (C),
(ppm): 2.25 (s, 3H, CH
3
), 3.74 (s, 3H, -OCH
3
) 3.84 (s, 6H, 2 -OCH
3
2
1
3
6
6
(CH
3
2
3
3
1
34.4 (C), 140.1 (CH), 153.7 (2 C), 165.5 (C=O), 167.3 (C=O), 200.8 (C=O). MS (ESI+)
+
m/z 352.0 [M+H] . Anal. calcd. for C16
H
17NO S (351.37): C, 54.69; H, 4.88; N, 3.99.
6
Found: C, 54.48; H, 4.97; N, 3.71 %.
2
0

4
.1.6. General procedure for the synthesis of compounds 11-16.
A mixture of the potassium salt 3 (0.33 g, 1 mmol) and dichloroethane (0.098 g, 0.07 ml,
1
0
mmol), 1,3-dichloropropane (0.113 g, 0.1 ml, 1 mmol), 1,4-dibromobutane (0.216 g,
.118 ml, 1 mmol), 1,5-dibromopentane (0.23 g, 0.136 ml, 1 mmol), 1,6-dibromohexane
(0.244 g, 0.153 ml, 1 mmol) or 1,8-dibromoctane (0.272 g, 0.184 ml, 1 mmol) in DMF (5
ml) was heated under reflux for 6 hr. After cooling the solid product obtained was filtered,
washed with water, dried and recrystallized.
4
(
.1.6.1. 1,2-Bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)ethane
11)
White crystals (recrystallization from ethanol), yield 0.49 g (88%), mp 237-238 ºC. IR (ν
-1
cm ): 3018 (CH arom.), 2931 (CH aliph.), 2825 (CH aliph.), 1739 (C=O), 1678 (C=O),
1
1
607, 1577, 1507 and 1465 (C=C arom.). H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 3.72
N-), 6.93 (s, 4H, 2 ArH),
) δ (ppm): 48.6 (2 -NCH ), 56.5 (4 -
), 108.2 (4 CH), 120.1 (2 C), 128.7 (2 C), 134.6 (2 C),
(s, 6H, 2 -OCH
3
), 3.82 (s, 12H, 4 -OCH
3
), 3.93 (s, 4H, -N(CH
.87 (s, 2H, 2 =CH-). C-NMR (75 MHz, DMSO-d
), 60.7 (OCH ), 66.8 (OCH
40.2 (2 CH), 153.7 (4 C), 165.3 (2 C=O), 167.2 (2 C=O). MS (ESI ) m/z 617.1 [M+H] .
Anal. calcd for C28 (616.66): C, 54.54; H, 4.58; N, 4.54. Found: C, 54.32; H,
2
)
2
1
3
7
6
2
OCH
3
3
3
+
+
1
H
28
2 10 2
N O S
4
.78; N, 4.50 %.
4
(
.1.6.2. 1,3-Bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)propane
12)
Yellow crystals (recrystallization from ethanol), yield 0.223 g (39%), mp 174-176 ºC. IR
-1
(
ν cm ): 3037 (CH arom.), 2931 (CH aliph.), 2837(CH aliph.), 1735 (C=O), 1678 (C=O),
1
1
607, 1577, 1507 and 1454 (C=C arom.). H-NMR (300MHz, DMSO-d
6
) δ (ppm): 2.01
(m, 2H, J = 6.5 Hz, CH
2
), 3.72 (m, 10H, 2 -OCH
3
and 2 NCH
2
), 3.81 (s, 12H, 4 -OCH ),
3
2
1

1
3
6
.9 (s, 4H, ArH), 7.85 (s, 2H, 2 =CH). C-NMR (75 MHz, DMSO-d ) δ (ppm): 25.2
6
(
CH
2
), 41.8 (2-NCH
2
), 56.4 (4 OCH
3
), 60.6 (2 OCH
3
), 108.1 (4 CH), 120.5 (2 C), 128.8 (2
+
C), 133.6 (2 C), 140.0 (2 CH), 153.6 (4 C), 166.0 (2 C=O), 167.7 (2 C=O). MS (ESI ) m/z
+
6
5
4
31.1 [M+H] . Anal. calcd for C29
H
30
N
2
O
10
S (630.68): C, 55.23; H, 4.79; N, 4.44. Found:
2
5.00; H, 4.98; N, 4.31 %.
.1.6.3. 1,4-bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)butane
13)
(
White crystals (recrystallization from ethanol), yield 0.205 g (71%), mp 207-208 ºC. IR (ν
-1
cm ): 3020 (CH arom.) 2936 (CH aliph.), 2836 (CH aliph.), 2835 (CH aliph.), 1742 (C=O),
1
1
678 (C=O), 1608, 1579, 1506 and 1463 (C=C arom.). H-NMR (300MHz, DMSO-d
6
) δ
), 3.82 (s,
), 6.92 (s, 2H, ArH), 7.86 (s, 2H, 2 =CH). C-NMR (75MHz, DMSO-d ) δ
), 41.5 (2 -NCH ), 56.5 (4 -OCH ), 60.7 (2 -OCH ), 108.1 (4 CH), 120.7
2 C), 128.9 (2 C), 133.6 (2 C), 140.0 (2 CH), 153.7 (4 C), 166.1 (2 C=O), 167.9 (2 C=O).
(ppm): 1.60 (m, 4H, CH
2
-CH
2
), 3.66 (m, 4H, 2 NCH
2
), 3.72 (s, 6H, 2 -OCH
3
1
3
1
2H, 4 -OCH
3
6
(ppm): 24.8 (2 CH
2
2
3
3
(
+
+
MS (ESI ) m/z 645.1 [M+H] . Anal. calcd. for C30
H
32
N
2
O
10
2
S (644.71): C, 55.89; H, 5.00;
N, 4.35. Found: C, 55.66; H, 5.17; N, 4.16 %.
4
.1.6.4. 1,5-Bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)pentane.
14)
Off-white crystals (recrystallization from ethanol:dioxane mixture (1:3)), yield 0.212 g
(
-1
(
75%), mp 147-148 ºC. IR (ν cm ): 3014 (CH arom.), 2943 (CH aliph.), 2825 (CH aliph.),
1
1
742 (C=O), 1678 (C=O), 1608, 1579, 1506 and 1463 (C=C arom.). H-NMR (300MHz,
DMSO-d
NCH ), 3.72 (s, 6 H, 2 -OCH
CH-). C-NMR (75 MHz, DMSO-d
6
) δ (ppm): 1.20 (m, 2H, CH
), 3.80 (s, 12 H, 4 -OCH
) δ (ppm): 23.7 (CH
2
), 1.60 (m, 4H, 2 CH
), 6.90 (s, 4H, ArH), 7.84 (s, 2H, 2
), 27.0 (2 CH ), 41.6 (2 N-
2
), 3.60 (t, 4H, J = 4.6 Hz, 2 -
2
3
3
1
3
=
6
2
2
2
2

CH
39.9 (2 CH), 153.6 (4 C), 166.1 (2 C=O), 167.8 (2 C=O). MS (ESI ) m/z 659.1 [M+H] .
Anal. calcd for C31 (658.74). C, 56.52; H, 5.20; N, 4.25. Found: C, 56.42; H,
2
), 56.4 (4 OCH
3
), 60.6 (2 OCH
3
), 108.0 (4 CH), 120.7 (2 C), 128.9 (2 C), 133.6 (2 C),
+
+
1
H
34
N
2
O S
10 2
5
.35; N, 4.06 %.
.1.6.5. 1,6-Bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)hexane
4
(15)
Yellow needles (recrystallization from ethanol-dioxane mixture (1:3)), yield 0.25 g (80%),
-1
mp 203-205 ºC. IR (ν cm ): 3017 (CH arom.), 2941 (CH aliph.), 2836 (CH aliph.), 1740
1
(
C=O), 1676 (C=O), 1609, 1577, 1507 and 1454 (C=C arom.). H-NMR (300 MHz,
CDCl
NCH
MHz, CDCl
2 -OCH ), 107.5 (4 CH), 120.5 (2 C), 128.7 (2 C), 133.8 (2 C), 140.2 (2 CH), 153.6 (4 C),
66.3 (2 C=O), 167.8 (2 C=O). MS (ESI ) m/z 673.2 [M+H] . Anal. calcd. for C32
(672.76): C, 57.13; H, 5.39; N, 4.16. Found: C, 56.87; H, 5.47; N, 3.99 %.
.1.6.6. 1,8-Bis(5-Z-(3,4,5-trimethoxybenzylidine)thiazolidin-2,4-dione-3-yl)octane
16)
3
) δ (ppm): 1.40 (m, 4H, 2 CH
), 3.92 (s, 18H, 6 -OCH ), 6.75 (s, 4H, ArH), 7.82 (s, 2H, 2 =CH-). C-NMR (75
) δ (ppm): 26.2 (2 CH ), 27.6 (2 CH ), 41.8 (2 -NCH ), 56.2 (4 -OCH ), 61.0
2
), 1.70 (m, 4H, 2 CH
2
), 3.76 (t, 4H, J = 7.2 Hz, 2 -
1
3
2
3
3
2
2
2
3
(
3
+
+
1
H
36
N
2
O S
10 2
4
(
A mixture of the potassium salt 3 (0.66 g, 2 mmol) and 1,8-dibromoctane (0.272 g, 0.184
ml, 1 mmol) in DMF (5 ml) was heated under reflux for 6 hr. After cooling the solid product
obtained was filtered, washed with water and dried. Recrystallization from dioxane gave
-1
yellow needles, yield 0.212 g (71%), mp 168-170 ºC. IR (ν cm ): 3015 (CH arom.), 2939
(CH aliph.), 2854 (CH aliph.), 1742 (C=O), 1679 (C=O), 1607, 1576, 1507 and 1453 (C=C
1
arom.). H-NMR (300 MHz, CDCl
3
) δ (ppm): 1.35 (m, 8H, 4 CH
2
), 1.65 (m, 4H, 2 CH ),
2
2
3

3
2
4
1
7
.75 (t, 4H, J = 7.15 Hz, 2 -NCH
2
), 3.92 (s, 18H, 6 -OCH
=CH-). C-NMR (75 MHz, CDCl ) δ (ppm): 26.6 (2 CH
2.0 (2 -NCH ), 56.2 (4 -OCH ), 61.0 (2 -OCH
3
), 6.75 (s, 4H, ArH), 7.84 (s, 2H,
1
3
3
2
), 27.7 (2 CH ), 28.9 (2 CH ),
2
2
2
3
3
), 107.5 (4 CH), 120.6 (2 C), 128.7 (2 C),
+
33.7 (2 C), 140.2 (2 CH), 153.6 (4 C), 166.3 (2 C=O), 167.8 (2 C=O). MS (ESI ) m/z
+
01.2 [M+H] . Anal. calcd. For C34
H
40
N
2
O
10
2
S (700.82): C, 58.27; H, 5.75; N, 4.00.
Found: C, 58.00; H, 5.83; N, 3.88 %.
4
. 2. Biological assays
. 2. 1. Cell culture and reagents
Human MDA-MB-231 and MCF-7 breast cancer cells were obtained from the Vacsera
Giza, Egypt), and were maintained in a culture medium consisting of MEM supplemented
4
(
with 10% heat-inactivated fetal bovine serum (FBS, EuroClone, Life Science Division,
Milan, Italy) as previously described [43]. The cell cultures were free of Mycoplasma, as
assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Boehringer,
Mannheim, Germany). The anti-proliferative effect of the three thiazolidinediones 5, 7 and
9
was evaluated on these cell lines as well as on the human breast cancer cells that were
isolated from patients with breast cancer was determined using the 3-(4,5-dimethylthiazol-
2
-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as previously described [44].
6
The cells were plated at 1 × 10 cells/ml in 2 ml of culture medium in six-well Costar plates
(Corning, Corning, NY). The cells were treated with different concentrations of 5, 7 and 9
for 12 h, and cytotoxicity was expressed as a relative percentage of the OD values measured
in the control (medium) and chemical compounds-treated cells. Morphological changes
2
4

were observed after exposure to medium and these chemical compounds using a phase-
contrast inverse microscope (Olympus, Japan).
4
.2.2. Isolation of human normal non-cancerous breast cells and human breast
cancer cells.
Human breast cancer cells and human normal non-cancerous breast cells were
obtained from the same three female patients at the time of surgery after informed
written consent was obtained. Patients were treated at South Egypt Cancer Institute,
Assiut University, Egypt.
Patients
Age
Patient History
Patient 1
Patient 2
Patient 3
New case
4
6
5
6
4
3
Subjected to a surgery and received
chemotherapy and radiotherapy.
New case
4
.2.3. Apoptosis detection and flow cytometry analysis
The percentage of breast cancer cells undergoing apoptosis was determined by flow
cytometry after 12 hours of incubation with medium or 5, 7 and 9. Dead cells were
identified using the Trypan blue exclusion test. However, to distinguish between viable,
early apoptotic and late apoptotic cells, the human breast cancer cells and human normal
non-cancerous breast cells were washed and incubated in PBS containing 30% human AB
serum (4°C for 30 minutes) prior to staining with Annexin V-FITC and propidium iodide
(PI) (15 minutes at 25°C) using a commercial kit according to the manufacturer’s
instructions (Abcam, Canada). The cells were analyzed by flow cytometry using a
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FACSCalibur flow cytometer (BD-Pharmingen) within 1 hour of staining, and the
percentage of cells undergoing apoptosis was determined as previously described [45-47].
4
.2.4. Western blot analyses
Western blot analysis was performed as previously described [46, 47]. Briefly, whole-cell
lysates were prepared from human breast cancer cells and human normal non-cancerous
breast cells which were isolated from the same breast cancer patients, as well as from
human breast cancer cell lines (MDA-MB-231 and MCF-7). Cells were firstly treated for
1
7
1
1
2 h with medium (0) or the three TZDs 5, 7 and 9 in RIPA buffer (20 mM Tris-HCl, pH
.5, 120 mM NaCl, 1.0% Triton X100, 0.1% SDS, 1% sodium deoxycholate, 10% glycerol,
mM EDTA and 1% protease inhibitor cocktail, Roche). Following centrifugation at
6,000 × g at 4°C for 15 min, the protein concentrations in the supernatants were
determined using a protein assay kit (Bio-Rad, Hercules, CA) and BCA protein assay
method. Equal amounts of whole-cell protein (50 µg) were mixed with reducing sample
buffer (0.92 M Tris-HCl, pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2-Mercapto-
ethanol) and separated using discontinuous SDS-PAGE. Proteins were transferred onto
nitrocellulose membranes using a Bio-Rad Trans-Blot electrophoretic transfer device, and
the membranes were blocked for 1 h at room temperature with 1% BSA or 5% skim milk
dissolved in TBS (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 0.1%
Tween 20. The membranes were then incubated in the same blocking buffer with anti-
phospho-AKT (p-AKT), anti-active total-AKT (T-AKT), anti-phospho-mTOR (p-mTOR),
anti-total-mTOR (T-mTOR), anti-VEGF, anti-HIF-1α, anti-β-actin, anti-Bcl-2, anti-Bcl-
XL, anti-Mcl-1, anti-Bak, anti-Bax, anti-Bim or anti-GAPDH antibodies (all from Cell
Signaling Technology, Beverly, MA). The blots were thoroughly rinsed and then incubated
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6

with an HRP-labeled species-matched secondary antibody for another 1 h.
Protein bands
were detected using enhanced chemiluminescence reagents (ECL, SuperSignal West Pico
Chemiluminescent Substrate, Perbio, Bezons, France), and the ECL signals were recorded
on Hyperfilm ECL. To quantify band intensities, the films were scanned, saved as TIFF
files and analyzed using NIH ImageJ software.
4
.2.5. Statistical analyses
Data were first tested for normality (using Anderson–Darling test) and for variances
homogeneity prior to any further statistical analysis. Data were normally distributed and
were expressed as the mean ± standard error of the mean (SEM). Significant differences
among groups were analyzed using a one-way analysis of variance using SPSS software,
*
version 17. Differences were considered statistically significant at P < 0.05. P < 0.05, 5-,
7
- and 9-treated cells vs. medium-treated cells.
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7

Conflict of interest:
The authors declare no conflicts of interest. This manuscript has not been published or
submitted elsewhere. This work complies with the Ethical Policies of this Journal and has
been conducted under internationally accepted ethical standards after relevant ethical
review. All authors have approved the final article.
Acknowledgments
We gratefully acknowledge the financial support from IMHOTEP program. Also we are
thankful to the laboratory of Immunology of the Zoology Department, Faculty of Science,
Assiut University and to the Clinical Pathology Department of South Egypt Cancer
Institute. We express our sincere thanks to the NMR facility (Lille 2 University), the
Région Nord-Pas de Calais (France), the Ministère de la Jeunesse, de l'Education Nationale
et de la Recherche (MJENR) and the Fonds Européens de Développement Régional
(FEDER).
Appendix A. Supplementary data
IR, NMR and MS spectra are available as supplementary data.
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