Optimization of 2-(3-(arylalkyl amino carbonyl) phenyl)-3-(2-methoxyphenyl) -4-thiazolidinone derivatives as potent antitumor growth and metastasis agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.068

A series of 2,3-diaryl-4-thiazolidinone derivatives were synthesized and evaluated for their antiproliferative properties against two well-known cancer cell lines (A549 as human lung cancer and MDA-MB-231 as human breast cancer). Structure activity relationship (SAR) analysis resulted in the discovery of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-methoxy-phenyl)-4-thiazolidinone derivatives with high potent inhibitory effects on the proliferation of both cancer cell lines. Furthermore, several compounds with potent antiproliferative activities displayed excellent inhibitory activities on migration with an IC₅₀ of about 0.05 μM on MDA-MB-231 cells in two different migration assays. In particular, compound 39 was indicated to suppress tumor growth and metastasis as well as promote survival rate. Intriguingly, this series of analogs have been indicated to inhibit tumor cell proliferation through inducing cell cycle arrest. These results suggested that the new series of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-methoxyphenyl)-4-thiazolidinone derivatives could be regarded and developed as novel highly potential anticancer agents in the future.

Full text of DOI:10.1016/j.ejmech.2014.04.068

European Journal of Medicinal Chemistry 80 (2014) 340e351
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Optimization of 2-(3-(arylalkyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone derivatives as potent antitumor
growth and metastasis agents
,
,
,
Jing Wu a 1, Linxi Yu a 1, Feifei Yang a 1, Jingjie Li a, Peng Wang a, Wenbo Zhou a, Liwen Qin a,
Yunqi Li a, Jian Luo a, Zhengfang Yi a , Mingyao Liu b, Yihua Chen a
,
a,
,
*
*
a Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical Sciences and School of Life Sciences, East China Normal University,
500 Dongchuan Rd, Shanghai 200241, China
b Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston 77030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,3-diaryl-4-thiazolidinone derivatives were synthesized and evaluated for their anti-
proliferative properties against two well-known cancer cell lines (A549 as human lung cancer and MDA-
MB-231 as human breast cancer). Structure activity relationship (SAR) analysis resulted in the discovery
of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-methoxy-phenyl)-4-thiazolidinone derivatives with high
potent inhibitory effects on the proliferation of both cancer cell lines. Furthermore, several compounds
with potent antiproliferative activities displayed excellent inhibitory activities on migration with an IC50
Received 16 February 2014
Received in revised form
9 April 2014
Accepted 23 April 2014
Available online 24 April 2014
of about 0.05 mM on MDA-MB-231 cells in two different migration assays. In particular, compound 39
Keywords:
was indicated to suppress tumor growth and metastasis as well as promote survival rate. Intriguingly,
this series of analogs have been indicated to inhibit tumor cell proliferation through inducing cell cycle
arrest. These results suggested that the new series of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone derivatives could be regarded and developed as novel highly poten-
tial anticancer agents in the future.
Thiazolidinone derivatives
Antitumor growth and metastasis
Structureeactivity relationships
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
overall poor prognosis [8]. As we know, cancer is characterized by
abnormal cell proliferation as well as migration, which is regarded
Cancer, as a major public health problem, is one of the three
most common causes of death in the world [1]. Among which, lung
cancer is the leading cause of cancer death worldwide for both men
and women with 5-year survival rates between 10% and 16% [2e5].
Breast cancer is the most common malignancy among women and
comprises more than 18% of all female cancers [6,7]. To date, both of
lung cancer and breast cancer are aggressive and correlated with
as a hallmark of the most aggressive and advanced epithelial tu-
mors prior to the metastatic stage [9], and responsible for the death
of above 90% solid cancer patients [10e12]. Thiazolidinone scaffold
has been well-known as a privileged structure in modern medicinal
chemistry [13]. For example, thiazolidinone derivatives have been
mentioned for various biological targets and diverse pharmaco-
logical properties such as anti-inflammatory [14], antiviral [15],
antiproliferative [16,17], anti-HIV [18], hypoglycemic [19], and
antitumor activity [20e22] (Fig. 1). In addition, two small molecule
inhibitors of the actin-related protein 2/3 (Arp2/3) complex with 4-
thiazolidinone structure 1 and 2 (CK-548 and CK-869, Fig. 2) have
been reported [23]. Since the Arp2/3 complex, as a key actin cyto-
skeleton regulator in cell motility, is mainly activated by the up-
stream regulator of N-WASP, which is overexpressed in most of
cancer cells [24], the compounds with Arp2/3 complex inhibition
may have potential anti-metastasis effect against cancer [9].
However, compound 1 or 2 was only shown low to mild inhibition
with micromolar grade against Arp2/3 complex as well as tumor
Abbreviations: A549, human lung adenocarcinome cells; MDA-MB-231, human
breast cancer cell line; Arp2/3, actin-related protein 2/3; SAR, structureeactivity
relationship; DCC, N,N0-dicyclohexyl-carbodiimide; THF, tetrahydrofuran; DMF,
N,N-dimethylformamide; EDC, N-(3-dimethylamino propyl)-N0-ethylcarbodiimide
hydrochloride; HOBt, 1-hydroxy-benzotriazole; SRB, sulforhodamine B; PE, petro-
leum ether; EA, ethyl acetate.
* Corresponding authors.
E-mail addresses: zfyi@bio.ecnu.edu.cn (Z. Yi), yhchen@bio.ecnu.edu.cn
(Y. Chen).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.04.068
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
341
Cl
S
R
Br
COOH
O
S
O
O
N
N
N
S
O
N
H
Cl
N
O
N
H
F
O
antimycobacterial activity
S
anti-HIV activity
anti-inflammatory activity
O
S
N
O
F
F
O
N
HN
R
1
HN
O
NH
S
CF
O
3
R
2
anti-colon cancer activity
anti-lung cancer activity
anti-prostate cancer activity
Fig. 1. Structures of several known 4-thiazolidinones with different biological activities.
cell migration [9,23], which would result in increasing risk because
of off-target effect and non-specificity in cell behavior in vivo, and
therefore excluded its possible use in clinical future [25,26]. Thus,
discovering novel 2,3-diaryl-4-thiazolidinone derivatives as more
potent anti-cancer agents and exploring the mechanisms of this
series of compounds interacting with specific targets are the
highest priority of our research.
importantly, some compounds were further investigated their
dramatic enhancement effect against cancer cell migration. In
particular, the compound 39 with potent anti-proliferative and
anti-migration activity was identified and showed strong anti-
tumor growth and anti-tumor metastasis effects as well as pro-
moted survival rate in vivo, which would be further optimized and
developed as anticancer agents.
In this report, in order to seek more suitable and potent thia-
zolidinone inhibitors as anti-tumor growth and anti-tumor
metastasis agents against lung cancer and breast cancer, we
designed and synthesized a series of 2,3-diaryl-4-thiazolidinone
derivatives based on the scaffold of 1 and 2 as shown in Fig. 2.
Firstly the substitutions of two aromatic rings were focused on
modifying and evaluating their anti-proliferative properties
respectively against non-small cell lung cancer cell line A549 and
breast cancer cell line MDA-MB-231. Secondly the substitutions in
“C” ring were modified for diverse skeletons in detail. Finally a li-
brary of compounds with arylalkyl amino carbonyl substitutions in
the 3-position of 2-aryl group were identified as the highest po-
tency in investigated compounds, some of which exhibited about
hundred-fold increase in inhibition in vitro relative to 1. More
2. Chemistry
Synthesis of the 2,3-diaryl-4-thiazolidinone derivatives fol-
lowed the general procedures illustrated in Schemes 1 and 2. Two
routine protocols were developed for the synthesis of 2,3-diaryl-4-
thiazolidinones [27e31]. One protocol was that several commer-
cially available or synthetic aldehydes and amines were heated
under refluxing in toluene with a water separator connected to the
apparatus. When no further evidence of water elimination was
observed, mercaptoacetic acid was applied to the reaction and
continued to reflux [26]. The other was that two of the three re-
actants, an aldehyde and an amine were treated in THF under ice
bath cooled for 5 min, then mercaptoacetic acid and DCC were
Br
C
S
B
N
Cl
Br
H
R3
O
OH
A
R2
O
N
( )n
C
1 (CK-548)
S
S
C
S
N
N
Br
O
OMe
N
O
A
C
O
OMe
R1
S
B
N
17-31
4-13
35-44
O
A
O
O
2 (CK-869)
Fig. 2. Structural optimization based on 1 (CK-548) and 2 (CK-869) series step by step.

342
J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
O
Ar
S
1
a or b
SH
Ar CHO + Ar NH
2
+
HO
1
2
N
Ar
2
O
3
4-30
Reagents and conditions: (a) CH3Ph, reflux, 140 oC; (b) DCC, THF, 0 oC then to rt.
Scheme 1. Synthesis of thiazolidinones 4e30. Reagents and conditions: (a) CH3Ph, reflux, 140 ꢀC; (b) DCC, THF, 0 ꢀC then to rt.
employed to the reaction mixture and stirred for additional several
hours [10]. Compounds 4e30 were prepared by either of these two
protocols. Further, some of 2,3-diaryl-4-thiazolidinone derivatives
could be further converted to other corresponding analogs based
on the structure diversity, and compounds 32e44 could be pre-
pared via coupling reactions of compound 31 with different amines.
decreased compared with 1. It was worth mentioning that com-
pound 7, substituted with a methoxy group on the ortho-position of
“A” ring, exhibited IC50 values of 1.87 mM for A549 and 4.52 mM for
MDA-MB-231, which displayed that the methoxy group at ortho-
position was more suitable among the investigated groups at “A”
ring for increasing anti-proliferative activity. An additional
methoxy group substitution at para-position on the same phenyl
ring showed just a little decrease (compounds 2 vs 7) for anti-
proliferation activity. Compound 13 without substitution in the
phenyl ring showed very weak activities against both tumor cell
lines, which indicated that 2-position substitution of phenyl with
alkoxy groups might be suitable. Replacement of the substituted
aniline with other aromatic amines (compounds 14e15) or
aliphatic amine (16) caused significant reduction in potency against
both cancer cell lines, which suggested that the substituted 2-
methoxyphenyl structure was more preferred to the anti-
proliferative effect in investigated groups against two tumor cell
lines.
3. Results and discussion
3.1. Structureeactivity relationships against tumor cell proliferation
Tumor cell unlimited proliferation is one of the important fac-
tors in the tumor progression and development, and thus the target
compounds were firstly evaluated for their antiproliferation effi-
cacy. Initially, “A” ring was selected for investigation with retaining
3-bromophenyl and 4-thiazolidinone scaffolds (Fig. 2, Table 1), and
compounds 4e16 were evaluated for their anti-proliferative activ-
ity against A549 (non-small cell lung cancer cell) and MDA-MB-231
Secondly, the SAR on the 3-bromophenyl group (“C” ring) of
compound 7 was next explored (Fig. 2, Table 2). The effect of ortho-,
meta-, and para-substitutions of phenyl group of “C” ring was
compared with the introduction of Bre to different positions (7, 17
and 18). According to the results listed in Table 2, the substitution
with Bre at meta-position (7) exhibited the best inhibitory activity,
while peBr substituted 18 had the lowest activity against both
cancer cell lines. Removal of the substitution of this phenyl ring
(compound 19) or replacement of the 3-bromophenyl of compound
7 with several heterocyclic aromatic groups (compounds 20e22)
dramatically decreased the inhibitory activities. To evaluate
whether phenyl group in “C” ring played an essential role against
cancer cells, we also inserted one methylene group between phenyl
and thiazolidinone scaffold to obtain compound 23, and the results
showed the modification could not increase the activities against
cancer cells either. In summary, these results demonstrated that
(human breast cancer cell) via the sulforhodamine
screening assay. As shown in Table 1, the proliferation inhibition of
hit compound 1 (CK-548) was modest (IC50 is 9.95 M for A549 and
26.98 M for MDA-MB-231). The positions and the electrical
B (SRB)
m
m
properties of substituent were explored as two of major factors
which were responsible for the inhibitory activities of the com-
pounds. For example, the substitutions with electron withdrawing
groups at this position decreased the anti-proliferative activity,
such as compound 10, while the substitution of several compounds
with electron donating groups showed better or comparable
inhibitory activity. In most instances the ortho-position sub-
stitutions were found to be more effective than the meta- or para-
position substitutions (compound 4e6 and 7e9). Therefore, the
compounds with electron donating groups at ortho-position of “A”
ring were further designed and synthesized. Unfortunately, the
results showed the potency of these compounds seemed to be
O
OH
O
OMe
O
R
COOH
a, b
c
S
S
S
d
CHO
N
N
OMe
N
O
O
O
OMe
OMe
32-44
30
31
Reagents and conditions: (a) MeOH, SOCl2, reflux; (b) 2-methoxyaniline, mercaptoacetic acid,
o
o
CH3Ph, reflux, 140 C; (c) LiOH·H2O, MeOH; (d) amine, EDC, HOBt, DMF, 0 C 15 min to rt
3h.
Scheme 2. Synthesis of compounds 31e44. Reagents and conditions: (a) MeOH, SOCl2, reflux; (b) 2-methoxyaniline, mercaptoacetic acid, CH3Ph, reflux, 140 ꢀC; (c) LiOH$H2O,
MeOH; (d) amine, EDC, HOBt, DMF, 0 ꢀC 15 min to rt 3 h.

J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
343
Table 1
Table 3
IC50 values of compounds (1e2, 4e16) against A549 and MDA-MB-231 cells prolif-
IC50 values of compounds (24e31) against A549 and MDA-MB-231 cells prolifera-
eration.
tion.
R
Br
S
N
S
N
O
OMe
R
O
1-2, 4-16
7, 24-31
Compd.
R
IC50 (mM)
Compd.
R
IC50 (mM)
A549
9.95
14.76
13.69
37.09
>50
1.87
>50
8.98
43.71
22.56
37.59
>50
>50
>50
MDA-MB-231
A549
MDA-MB-231
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
2-OH-5-Cl-Ph
2,4-(OMe)2-Ph
2-OH-Ph
3-OH-Ph
4-OH-Ph
2-OMe-Ph
3-OMe-Ph
4-OEt-Ph
3-NO2-Ph
2-NHCH3-Ph
2-N(CH3)2-Ph
Ph
2-thiazolyl
2-pyrimidinyl
PhCH2CH2
26.98
17.11
32.63
17.71
>50
7
Bre
Cle
Fe
NO2e
MeOe
eOH
BrCH2e
COOMe
COOH
1.87
8.18
>50
11.31
>50
4.52
13.92
>50
42.98
>50
>50
>50
23.07
>50
24
25
26
27
28
29
30
31
4.52
>50
47.49
18.03
>50
>50
>50
>50
>50
>50
>50
14.38
0.29
>50
cell lines. Therefore, more stabilized compounds such as amide
with a similar structural performance of compound 30 were quite
naturally designed and prepared for further optimization.
20.67
In order to explore the effect of amide group in meta-position, as
shown in Table 4, many compounds with an arylalkyl amino
carbonyl group on the meta-position of the phenyl ring (“C” ring)
were explored for their anti-proliferative activities. Compounds
which bore di-substitution on the nitrogen of amide (32e34)
showed almost no effects on both cancer cell lines. Excitingly, most
compounds with arylalkyl amino carbonyl substitutions indicated
high anti-proliferation effect against both of tumor cell lines.
Analysis of compounds with mono-substituted nitrogen (35e38)
indicated that carbon chain length between nitrogen and benzene
ring greatly affected the inhibitory potency. In tested compounds
phenylethyl amino group (37) showed better potency against A549
more diverse substitutions at meta-position of phenyl group of “C”
ring should be available for further investigation.
Further SAR analysis of the replacements of Bre was performed
on the meta-position of phenyl of “C” ring with other more sub-
stitutions (Table 3). The results suggested that reduced activities
were observed when the Br (7) was replaced by other halogens
such as Cl (24) or F (25). An obvious decrease in activities was also
observed when compounds substituted with electron-donating
groups (compounds 27e29) or strong electron withdrawing
group like nitro (compound 26). Thus compound 30 with a sub-
stitution of formic ester was selected to synthesize and showed a
more potent effect than compound 7 against A549 (Table 3). Since
formic ester could be easily hydrolyzed to formic acid in vivo,
compound 31, which was a hydrolysis product of compound 30,
was prepared but showed very weak effect against the same tumor
cell line (IC50 ¼ 0.96
moderate inhibitory effect (IC50 ¼ 5.06
better than aniline (35) which obviously decreased the inhibitory
activities (IC50 > 25 M for A549). Further modifications based on
mM) than benzyl amine (36) which had a
mM for A549), and also much
m
compound 37 were explored. Compounds 39, and 41e43 with
substitution on the para-position displayed a much better anti-
Table 2
IC50 values of compounds (17e23) against A549 and MDA-MB-231 cells prolifera-
tion.
proliferation efficacy (IC50 ¼ 0.21e0.13
mM for A549, IC50 ¼ 0.23e
0.050 M for MDA-MB-231) than compound 37.
m
R
S
3.2. Compound 39 inhibited tumor cell proliferation through
N
inducing cell cycle arrest in A549 cells and MDA-MB-231 cells
O
OMe
To further examine the possible mechanism responsible for this
series of compounds-mediated tumor cell proliferation inhibition,
compound 39 was selected for evaluation of its effect on cell cycle
distribution and apoptosis assays. As shown in Fig. 3A and B,
7, 17-23
Compd.
R
IC50 (mM)
compound 39 caused a significant G2/M phase arrest at 1.0 mM in
A549
MDA-MB-231
both tumor cells compared with the control. The percentages of G2/
M phase cells were increased from 11.58% to 48.34% (for MDA-MB-
231) and 3.71%e42.41% (for A549). Results of apoptosis assay
indicated that 48 h treatment of compound 39 displayed little
significant impact on tumor cell apoptosis (Fig. 3C and D). These
results supported that compound 39 inhibited tumor cell prolifer-
ation mainly through inducing cell cycle arrest other than
apoptosis.
7
3-Br-Ph
2-Br-Ph
4-Br-Ph
Ph
4-pyridinyl
2-thiopheneyl
2-quinolinyl
Bn
1.87
4.52
>50
>50
>50
>50
>50
>50
>50
17
18
19
20
21
22
23
31.54
48.49
>50
>50
>50
>50
>50

344
J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
Table 4
be metastasized by breast cancer [32]. Compound 39 was thus
investigated for its potential inhibition against breast cancer
metastasis in mouse tail vein injection tumor metastasis model
in vivo. One week prior to luciferase-labeled MDA-MB-231 cells tail
vein injection, compound 39 with 1.0 mg/kg/day or 5.0 mg/kg/day
or vehicle control was administrated. Continued treatment for the
duration of the experiment (21 days) after cancer cell injection, the
effect of compound 39 was examined by bioluminescence imaging
assay. As shown in Fig. 7A and B, treatment of compound 39
significantly reduced the development of lung metastases in a
dose- and time-dependent manner. Administration of 5.0 mg/kg/
day compound 39 significantly blocked the MDA-MB-231 cell lung
metastases, and photon flux in the lungs of mice was reduced by
96.5 ꢁ 1.5% compared to control mice after 21-day treatment
(Fig. 7B). In addition, survival analysis showed that mice with the
treatment of compound 39 had significantly longer survival rate
than the mice with vehicle control treatment (Fig. 7C), while the
body weight of drug treatment and control group showed little
difference (Fig. 7D), which might indicate that the toxicity of
compound 39 was tolerated under the effective dosage.
IC50 values of compounds (32e44) against A549 and MDA-MB-231 cells prolifera-
tion.
O
S
OMe
N
R
O
32-44
Compd.
R
IC50 (mM)
A549
MDA-MB-231
30
32
33
34
35
36
37
38
39
40
41
42
43
44
eOMe
eNEt2
eN(Allyl)2
piperidine
PhNHe
PhCH2NHe
0.29
>50
>50
>50
>25
5.06
0.96
31.62
0.21
8.52
0.47
0.16
0.13
0.72
23.07
>50
>50
>50
>25
>25
>25
12.32
0.23
13.93
1.36
0.18
0.050
1.77
Ph(CH2)2NHe
Ph(CH2)3NHe
4-Br-Ph(CH2)2NHe
2-Br-Ph(CH2)2NHe
4-F-Ph(CH2)2NHe
4-Cl-Ph(CH2)2NHe
4. Conclusion
4-MeO-Ph(CH2)2NHe
4-OH-Ph(CH2)2NHe
In this study, we reformed and synthesized a series of 2,3-diaryl-
4-thiazolidinone compounds based on the initial hit structure and
evaluated their effects against tumor cell proliferation and migra-
tion. The electrical characters and the positions of substituents in
aromatic rings were discussed. A new series of 2-(3-(arylalkyl
amino carbonyl)phenyl)-3-(2-methoxy- phenyl)-4-thiazolidinone
derivatives were optimized with step by step modification
through SAR analysis. Several of these compounds with arylalkyl
amino carbonyl tail in 3-position of one aromatic ring exhibited
highly potent antiproliferative inhibition with a submicromolar
range against non-small lung cancer cell line A549 and breast cell
line MDA-MB-231. Among these, compounds 39 and 42 showed the
3.3. Compounds suppressed breast cancer cell MDA-MB-231
migration in vitro
As previously mentioned, cell migration is an important charac-
teristic of tumor metastasis, which remains responsible for the 90%
of cancer deaths [10]. Six compounds were thus chosen for further
assessment for their inhibitory activities against cancer cell migra-
tion with highly potent anti-proliferation efficacy in two different
tumor cell lines. The results of these compounds on MDA-MB-231
cell migration in wound healing assay were shown in Fig. 4. Com-
pound 1 exhibited little migration efficacy on MDA-MB-231 cell with
most potent anti-migration activities with an IC50 of 0.01e0.05 mM
the concentration of 0.50 mM. Excitingly, our compounds 37, 39 and
42e44 displayed much better inhibitory potency than 1 (Fig. 4A and
B), especially the compounds 39 and 42, whose IC50 values of cell
in the wound healing and transwell migration models. Further
experiments in vivo indicated that compound 39 inhibited tumor
growth and metastasis in two different animal models. All of above
results demonstrated that these new series of 2-(3-(arylalkyl amino
migration were less than 0.05 mM (Fig. 4C and D). Compounds 39 and
42 were further evaluated for the anti-migration effect on MDA-MB-
231 cell via transwell migration assay (Fig. 5). The results indicated
that both compound 39 and 42 strongly inhibited MDA-MB-231 cell
migration in a dose-dependent manner with IC50 values about 0.01e
carbonyl)phenyl)-3-(2-methoxyphenyl)-4-thiazolidinone
de-
rivatives had highly potent inhibitory effects on both tumor growth
and metastasis. Further work is in progress to optimize this series of
compounds with the aim of increasing their potency as well as
elucidating its detailed mechanism interacting with corresponding
targeted proteins.
0.05 mM in this assay, as shown in Fig. 5.
3.4. Compound 39 inhibited tumor growth in A549 xenograft
mouse model
5. Experimental section
In order to evaluate the efficacy of our compounds against tu-
mor growth in vivo, we selected compound 39 for testing its effi-
cacy in human lung carcinoma cancer cells A549 xenograft mouse
tumor formation model, as presented in Fig. 6. Our results showed
that after a 28-day treatment with 39 (5.0 mg/kg/day and 10.0 mg/
kg/day), both tumor volume (Fig. 6A and C) and tumor weight
(Fig. 6B) in treated groups were significantly less than those in the
control group. In addition, the body weight of three groups of
tumor-bearing mice as well as control had no significant difference
(Fig. 6D), which might suggest that the mice could tolerate the
effective doses of compound 39.
5.1. General methods for chemistry
Reagents were purchased from Adamas-beta Ltd, Sigmae
Aldrich Inc., J&K Inc., or Aladdin-reagents Inc., and used without
further purification unless otherwise specified. All reactions were
carried out with the use of standard techniques under an inert at-
mosphere (Ar or N2). 1H and 13C NMR spectra were generated on a
Varian 300 MHz or Bruker 500 Hz instrument and obtained as
CDCl3 or DMSO-d6 solutions (reported in ppm), using CDCl3 as the
reference standard (7.26 and 77.00 ppm) or DMSO-d6 (2.50 and
39.51 ppm). Coupling constants (J) are reported in Hertz (Hz).
Standard abbreviations indicating spin multiplicities are given as
follow: s (singlet), d (doublet), t (triplet), q (quartet), br (broad) or
m (multiplet). High resolution mass spectra were gathered on
Bruker MicroTOF-Q II LCMS instrument operating in electrospray
ionization (ESI). HPLC (Agilent Technologies 1200 Series) was
3.5. Compound 39 inhibited breast cancer metastasis in vivo
As we know, lungs and bone are the most preferred sites for
breast cancer metastasis although nearly any tissue in the body can

J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
345
Fig. 3. Effect of compound 39 in cell cycle arrest and apoptosis assays.

346
J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
Fig. 4. Effect of several different compounds against breast cancer cell MDA-MB-231 migration in a wound healing migration assay. A and C) Tumor cells were seeded in six-well
plates. A “wound” was created after the cells grew into 80e90% confluence. The compounds with 0.5 or 0.05 mM concentration were added to each well and the cells were incubated
at 37 ꢀC for 24 h, all the groups were fixed and photographed. B or D) Dotted lines indicate the area occupied by the initial scraping and migrated cells were quantified by manual
counting. The means and error bars representing 95% confidence intervals from three independent experiments are presented.
employed for purity determination, using the following method:
Eclipse XDB C18 column, 5
5.2. General procedure for the synthesis of compounds 4, 7, 10e12,
16e17 and 20e30 (method A) [26,30]
m
m, 4.6 mm ꢂ 150 mm, column tem-
perature 40 ꢀC; solvent A: water; solvent B: methanol; gradient of
40e70% B (0e10 min), 70e90% B (10e15 min), 90e40% B (15e
20 min); flow rate of 1.5 mL/min. Compound purity was deter-
mined by high pressure liquid chromatography (HPLC) with a
confirming purity of ꢃ95% for all of the final biologically tested
compounds.
To a solution of commercially available aldehyde (1.0 mmol) and
amine (1.0 mmol) in toluene (10 mL) were heated under reflux
(140 ꢀC) with a water separator connected to the apparatus. When
no further evidence of water elimination was observed, mercap-
toacetic acid (1.5 mmol) was applied to the reaction and continue to

J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
347
Fig. 5. Compounds 39 and 42 inhibit migration of breast cancer MDA-MB-231 in a transwell migration model. A, B) 5 ꢂ 104 MDA-MB-231 cells with the indicated doses of 39 and 42
were seeded in the top-chamber. The bottom-chambers were filled with 10% fetal bovine serum (FBS). Fixed concentrations of compounds were added in both chambers. The cancer
cells were allowed to migrate for 12 h. The stained migrated purple cells in the images were photographed. C, D) Dotted lines indicate the area occupied by the initial scraping and
migrated cells were quantified by manual counting. The means and error bars representing 95% confidence intervals from three independent experiments are presented.
Fig. 6. Compound 39 inhibits lung (A549) tumor growth in the xenograft mouse tumor model. A) Representative photos of A549 tumor on mice at day 28 after different treatment
of intralesional administration (DMSO, 5.0 mg/kg/day, 10.0 mg/kg/day). B) Average weight of the tumors in the xenograft model in control group and 39 treated group. C) Summary
results of the A549 tumor volume in control group and 39-treated group. D) Records of the mice body weight during the treatment of 39 on A549 tumors.

348
J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
Fig. 7. Compound 39 suppress breast cancer metastasis on mouse tail vein injection model with different dose. A) Tumor metastasis over a 21-day period by bioluminescence
analysis. B) Quantitative analysis of metastatic cells in lung by bioluminescence analysis. C) KaplaneMeier analysis of mouse survival after breast tumor mouse tail vein injection. D)
Records of the mice body weight during the treatment of 39 on breast tumor (MDA-MB-231) mouse tail vein injection tumor metastasis model.
reflux to eliminate water. The solvent was concentrated under
reduced pressure. The crude products were purified by column
chromatography to give the terminal target products.
(168 mg, 4.0 mmol) in H2O (2.0 mL) was employed to the reaction
mixture and stirred for 10 min under ice cooling then at room
temperature for overnight. The solvent was concentrated under
reduced pressure. The reaction mixture was treated with 10% HCl to
pH ¼ 1 and extracted with ether, washed with water and dried over
anhydrous Na2SO4. The crude product was purified by column
chromatography (SiO2, petroether/ethyl acetate) to give compound
5.3. General procedure for the synthesis of compounds 5e6, 8e9,
13e15, 18e19 (method B) [28,30]
A solution of commercially available aldehyde (2.0 mmol) and
amine (1.0 mmol) was stirred in THF (10 mL) under ice water bath
for 5 min, and then mercaptoacetic acid (3.0 mmol) and DCC
(1.2 mmol) were employed to the reaction mixture under ice water
bath for another 15 min and stirred for additional several hours at
room temperature. The products were prepared by extraction
filtration and purified by column chromatography to give the ter-
minal target products.
31 as a white solid. Yield: 69%; 1H NMR (300 MHz, CDCl3)
d 8.07 (br
s,1H), 7.97 (d, J ¼ 7.8 Hz,1H), 7.59 (d, J ¼ 7.8 Hz,1H), 7.38 (dd, J ¼ 7.8,
7.8 Hz, 1H), 7.19 (dd, J ¼ 7.2, 8.4 Hz, 1H), 6.93 (d, J ¼ 6.3 Hz, 1H), 6.86
(d, J ¼ 8.4 Hz, 1H), 6.81 (dd, J ¼ 7.5, 7.8 Hz, 1H), 6.17 (s, 1H), 4.03e
3.90 (m, 2H), 3.84 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 171.62,
170.62, 154.74, 139.70, 132.98, 130.67, 130.04, 129.73, 129.57, 128.70,
124.96, 120.83, 111.93, 64.27, 55.62, 33.14. HR MS (ESI) calcd for
[C17H15NO4S þ Na]þ 352.0614, found 352.0612.
To a solution of compound 31 (99 mg, 0.30 mmol) in anhydrous
DMF (5.0 mL) were added EDC (75 mg, 0.39 mmol) and HOBt
(45 mg, 0.33 mmol) and then stirred under ice water bath for 5 min,
then the selected amine (0.36 mmol) were employed to the reac-
tion mixture for another 15 min at 0 ꢀC and stirred at room tem-
perature for 3 h. The mixture was extracted with ethyl acetate,
washed with water and brine, and then dried over anhydrous
Na2SO4. The crude product was purified by column
5.4. General procedure for the preparation of compounds 31 and
32e44
5.4.1. 3-(3-(2-methoxyphenyl)-4-oxo-2-thiazolidinyl)benzoic acid
(31)
To a solution of compound 30 (343 mg, 1.0 mmol) in MeOH
(8 mL) was stirred under ice water bath, and then LiOH$H2O

J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
349
chromatography (SiO2, petroether/ethyl acetate) to afford target
CDCl3): d 171.33, 166.51, 154.75, 139.72, 138.04, 134.56, 130.72,
compounds 32e44.
129.99, 129.64, 128.71, 127.92, 127.60, 127.41, 126.53, 125.19, 120.86,
112.12, 64.25, 55.69, 44.06, 33.07. HR MS (ESI) calcd for
[C24H22N2O3S þ Na]þ 441.1243, found 441.1255.
5.4.2. 2-(3-N,N-diethylamino carbonylphenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (32)
Reaction of 31 and diethylamine and then purification to give
the target compound 32. Yield: 32%; 1H NMR (300 MHz, CDCl3)
d
5.4.7. 2-(3-(phenylethyl amino carbonyl)phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (37)
7.40 (d, J ¼ 8.7 Hz, 1H), 7.32e7.27 (m, 3H), 7.19 (dd, J ¼ 7.8, 7.8 Hz,
The title compound was synthesized according to the procedure
of preparing 32 except using 2-phenyl ethylamine instead of
1H), 6.87 (dd, J ¼ 7.5, 8.1 Hz, 2H), 6.78 (dd, J ¼ 7.2, 7.5 Hz,1H), 6.10 (s,
1H), 3.98e3.87 (m, 2H), 3.83 (s, 3H), 3.50 (br s, 2H), 3.08 (br s, 2H),
diethylamine. Yield: 64%. 1H NMR (300 MHz, CDCl3)
d 7.66 (br s,
1.21 (br s, 3H), 1.00 (br s, 3H). 13C NMR (125 MHz, CDCl3):
d 171.28,
1H), 7.52 (d, J ¼ 7.5 Hz, 1H), 7.46 (dd, J ¼ 0.6, 7.2 Hz, 1H), 7.36e7.28
(m, 4H), 7.24e7.15 (m, 3H), 6.86 (ddd, J ¼ 1.5, 7.8, 8.4 Hz, 2H), 6.78
(dd, J ¼ 7.5, 7.8 Hz,1H), 6.11 (s,1H), 6.05 (t, J ¼ 5.1 Hz,1H), 3.98e3.86
(m, 2H), 3.80 (s, 3H), 3.69 (q, J ¼ 6.3 Hz, 2H), 2.92 (t, J ¼ 6.9 Hz, 2H).
170.46, 154.81, 139.36, 137.36, 130.21, 129.59, 129.62, 128.58, 126.91,
126.02, 125.21, 120.77, 111.96, 64.31, 55.66, 33.11, 30.89, 29.65. HR
MS (ESI) calcd for [C21H24N2O3S þ Na]þ 407.1400, found 407.1403.
13C NMR (125 MHz, CDCl3):
d 171.31, 166.68, 154.73, 139.74, 138.80,
5.4.3. 2-(3-N,N-diallylamino carbonylphenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (33)
134.85, 130.65, 130.07, 129.63, 128.75, 128.67, 127.19, 126.57, 126.48,
125.14, 120.83, 112.04, 64.23, 55.66, 41.11, 35.55, 33.07. HR MS (ESI)
calcd for [C25H24N2O3S þ Na]þ 455.1400, found 455.1402.
The title compound was synthesized according to the procedure
of preparing 32 except using diallylamine instead of diethylamine.
Yield: 66%; 1H NMR (300 MHz, CDCl3)
d 7.42e7.39 (m, 1H), 7.36 (s,
1H), 7.31e7.26 (m, 2H), 7.18 (dd, J ¼ 7.5, 8.1 Hz, 1H), 6.89e6.85 (m,
2H), 6.78 (dd, J ¼ 7.5, 7.5 Hz,1H), 6.09 (s, 1H), 5.85 (br s, 1H), 5.63 (br
s, 1H), 5.24e5.09 (m, 4H), 4.20e4.00 (m, 2H), 3.92 (dd, J ¼ 1.2,
3.6 Hz, 2H), 3.83 (s, 3H), 3.65 (br s, 2H). 13C NMR (125 MHz, CDCl3):
5.4.8. 2-(3-(phenylpropyl amino carbonyl)phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (38)
The title compound was synthesized according to the procedure
of preparing 32 except using 3-phenyl propylamine instead of
d
171.24, 170.93, 154.79, 139.40, 136.34, 132.98, 132.58, 130.21,
diethylamine. Yield: 90%. 1H NMR (300 MHz, CDCl3)
d 7.64 (br s,
129.63, 129.09, 128.61, 127.29, 126.21, 117.64, 111.98, 64.25, 55.67,
33.09, 29.64. HR MS (ESI) calcd for [C23H24N2O3S þ Na]þ 431.1400,
found 431.1408.
1H), 7.49 (dd, J ¼ 1.5, 7.8 Hz, 2H), 7.33e7.27 (m, 3H), 7.21e7.15 (m,
4H), 6.90 (dd, J ¼ 1.5, 7.5 Hz, 1H), 6.86 (d, J ¼ 8.4 Hz, 1H), 6.78 (dd,
J ¼ 7.5, 7.8 Hz, 1H), 6.12 (s, 1H), 6.08 (t, J ¼ 5.4 Hz, 1H), 3.99e3.87 (m,
2H), 3.82 (s, 3H), 3.46 (q, J ¼ 6.6 Hz, 2H), 2.71 (t, J ¼ 7.5 Hz, 2H),
5.4.4. 2-(3-(1-piperidinyl)carbonylphenyl)-3-(2-methoxyphenyl)-
4-thiazolidinone (34)
1.99e1.89 (m, 2H). 13C NMR (125 MHz, CDCl3):
d 171.43, 166.67,
154.78, 141.37, 139.76, 134.82, 128.67, 128.53, 128.34, 127.16, 126.48,
126.06, 120.86, 112.08, 64.29, 55.71, 39.84, 33.46, 33.11, 30.98. HR
MS (ESI) calcd for [C26H26N2O3S þ Na]þ 469.1556, found 469.1554.
The title compound was synthesized according to the procedure
of preparing 32 except using piperidine instead of diethylamine.
Yield: 50%. 1H NMR (300 MHz, CDCl3)
d
7.41 (d, J ¼ 7.2 Hz,1H), 7.33e
7.27 (m, 3H), 7.18 (ddd, J ¼ 1.5, 8.4, 8.7 Hz, 1H), 6.89e6.85 (m, 2H),
6.78 (dd, J ¼ 7.2, 7.8 Hz, 1H), 6.11 (s, 1H), 3.97e3.91 (m, 2H), 3.83 (s,
5.4.9. 2-(3-(4-bromophenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (39)
3H), 3.67 (br s, 2H), 3.12 (br s, 2H), 1.66 (br s, 4H), 1.44 (br s, 2H). 13
C
NMR (125 MHz, CDCl3):
d 171.25, 169.46, 154.79, 139.26, 136.62,
The title compound was synthesized according to the procedure
of preparing 32 except using 4-bromophenyl ethylamine instead of
130.26, 129.56, 128.82, 128.67, 127.46, 126.41, 125.17, 120.71, 111.93,
64.28, 55.65, 33.09, 24.47. HR MS (ESI) calcd for
[C22H24N2O3S þ Na]þ 419.1400, found 419.1401.
diethylamine. Yield: 88%. 1H NMR (300 MHz, CDCl3):
d 7.67 (s, 1H),
7.54e7.47 (m, 2H), 7.45 (d, J ¼ 8.1 Hz, 2H), 7.33 (dd, J ¼ 7.8, 7.8 Hz,
1H), 7.20 (ddd, J ¼ 1.8, 8.1, 9.0 Hz, 1H), 7.10 (d, J ¼ 8.1 Hz, 2H), 6.87
(ddd, J ¼ 1.2, 7.5, 8.1 Hz, 2H), 6.79 (dd, J ¼ 7.5, 7.8 Hz, 1H), 6.12 (s,
1H), 6.05 (t, J ¼ 5.1 Hz, 1H), 3.99e3.88 (m, 2H), 3.81 (s, 3H), 3.66 (q,
J ¼ 6.6 Hz, 2H), 2.88 (t, J ¼ 6.9 Hz, 2H). 13C NMR (125 MHz, CDCl3):
5.4.5. 2-(3-(phenyl amino carbonyl)phenyl)-3-(2-methoxyphenyl)-
4-thiazolidinone (35)
The title compound was synthesized according to the procedure
of preparing 32 except using aniline instead of diethylamine. Yield:
d
171.34, 166.75, 154.80, 139.91, 137.78, 134.72, 131.76, 130.83,
70%. 1H NMR (300 MHz, CDCl3)
d 7.96 (s, 1H), 7.85 (s, 1H), 7.72 (d,
130.52, 130.09, 129.69, 128.79, 127.15, 126.49, 125.21, 120.90, 120.47,
112.12, 64.26, 55.73, 41.00, 35.04, 33.11. HR MS (ESI) calcd for
[C25H23BrN2O3S þ Na]þ 533.0505, found 533.0504.
J ¼ 7.5 Hz, 1H), 7.59 (d, J ¼ 7.5 Hz, 1H), 7.52 (d, J ¼ 7.2 Hz, 1H), 7.39e
7.33 (m, 3H), 7.21e7.12 (m, 2H), 6.91 (dd, J ¼ 1.2, 7.8 Hz, 1H), 6.85 (d,
J ¼ 8.1 Hz, 1H), 6.78 (dd, J ¼ 7.2, 7.2 Hz, 1H), 6.17 (s, 1H), 3.99e3.86
(m, 2H), 3.81 (s, 3H). 13C NMR (125 MHz, CDCl3):
d 171.46, 165.01,
154.75, 139.80, 137.84, 135.20, 131.03, 130.05, 129.74, 128.96, 128.82,
127.59, 126.75, 125.09, 124.54, 120.88, 120.30, 112.11, 64.31, 55.73,
33.13. HR MS (ESI) calcd for [C23H20N2O3S þ Na]þ 427.1087, found
427.14096.
5.4.10. 2-(3-(2-bromophenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (40)
The title compound was synthesized according to the procedure
of preparing 32 except using 2-bromophenyl ethylamine instead of
diethylamine. Yield: 86%. 1H NMR (300 MHz, CDCl3):
d 8.01 (s, 1H),
5.4.6. 2-(3-(benzyl amino carbonyl)phenyl)-3-(2-methoxyphenyl)-
4-thiazolidinone (36)
7.70 (br s, 1H), 7.58 (d, J ¼ 7.8 Hz, 2H), 7.47 (d, J ¼ 8.1 Hz, 1H), 7.32
(dd, J ¼ 7.5, 7.8 Hz, 1H), 7.26 (br s, 1H), 7.21e7.11 (m, 2H), 6.88e6.84
(m, 2H), 6.78 (dd, J ¼ 7.2, 7.5 Hz, 1H), 6.17 (t, J ¼ 5.1 Hz, 1H), 6.12 (s,
1H), 4.00e3.88 (m, 2H), 3.81 (s, 3H), 3.71 (q, J ¼ 6.6 Hz, 2H), 3.09 (t,
The title compound was synthesized according to the procedure
of preparing 32 except using benzyl amine instead of diethylamine.
Yield: 51%. 1H NMR (300 MHz, CDCl3)
d
7.76 (s, 1H), 7.64 (d,
J ¼ 6.9 Hz, 2H). 13C NMR (125 MHz, CDCl3):
d 171.32, 166.83, 154.77,
J ¼ 7.2 Hz, 1H), 7.49 (d, J ¼ 7.5 Hz, 1H), 7.39e7.32 (m, 5H), 7.16 (ddd,
J ¼ 0.6, 7.5, 7.5 Hz, 1H), 6.89 (d, J ¼ 7.5 Hz, 1H), 6.83 (d, J ¼ 8.1 Hz,
1H), 6.77 (dd, J ¼ 7.5, 7.8 Hz, 1H), 6.47 (br s, 1H), 6.13 (s, 1H), 4.59 (d,
J ¼ 5.7 Hz, 2H), 3.96e3.83 (m, 2H), 3.78 (s, 3H). 13C NMR (125 MHz,
139.75, 138.27, 134.77, 132.95, 131.03, 130.70, 130.09, 129.65, 128.69,
128.38, 127.69, 127.30, 126.49, 125.17, 124.54, 120.85, 112.08, 64.27,
55.70, 39.89, 35.55, 33.10. HR MS (ESI) calcd for
[C25H23BrN2O3S þ Na]þ 533.0505, found 533.0504.

350
J. Wu et al. / European Journal of Medicinal Chemistry 80 (2014) 340e351
5.4.11. 2-(3-(4-fluorophenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (41)
purchased from Caliper Life Sciences, Inc (Hopkinton, MA). All cells
were incubated at 37 ꢀC under a humidified 5% CO2 in RPMI 1640
medium (Gibco, Scotland, UK).
The title compound was synthesized according to the procedure
of preparing 32 except using 4-fluorophenyl ethylamine instead of
diethylamine. Yield: 89%. 1H NMR (300 MHz, CDCl3):
d
7.68 (br s,
5.5.2. SRB proliferation assay
1H), 7.53 (d, J ¼ 7.5 Hz, 1H), 7.48 (d, J ¼ 7.5 Hz, 1H), 7.32 (dd, J ¼ 7.8,
7.8 Hz, 1H), 7.21e7.16 (m, 3H), 7.01 (dd, J ¼ 8.7, 8.7 Hz, 2H), 6.87
(ddd, J ¼ 1.8, 8.1, 8.1 Hz, 2H), 6.79 (dd, J ¼ 7.5, 7.5 Hz,1H), 6.12 (s,1H),
6.09 (t, J ¼ 5.1 Hz, 1H), 3.99e3.87 (m, 2H), 3.81 (s, 3H), 3.65 (q,
J ¼ 6.9 Hz, 2H), 2.89 (t, J ¼ 5.1 Hz, 2H). 13C NMR (125 MHz, CDCl3):
The sulforhodamine B (SRB) assay was used to evaluate cell
viability. After seeded onto 96-well plates for 24 h, cells were
treated with the compounds at specified concentrations and the
same amount of DMSO (as control). 48 h later, the cells were fixed
with cold 50% trichloroacetic acid (TCA), then incubated for 1 h at
4 ꢀC. The plates were washed with deionized water five times. Cells
d
171.36, 166.72, 162.61, 160.66, 154.78, 139.85, 134.77, 130.75,
130.19,130.12,129.67, 128.73,127.158, 126.49,120.85,115.53,115.36,
112.07, 64.25, 55.69, 41.23, 34.77, 33.07. HR MS (ESI) calcd for
[C25H23FN2O3S þ Na]þ 473.1306, found 473.1321.
of each well were stained with 50 mL of 0.4% (W/V) SRB solution
dissolved in 1% acetic acid for 10 min then washed with 1% acetic
acid five times to remove the unbound dye. The bound dye was
dissolved with 100 mL of 10 mM unbuffered Tris base for determi-
5.4.12. 2-(3-(4-chlorophenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (42)
nation of optical densities at the wavelength of 515 nm in a
microplate reader SpectraMAX 190 Microplate Reader.
The title compound was synthesized according to the procedure
of preparing 32 except using 4-chlorophenyl ethylamine instead of
5.5.3. Cell cycle analysis
diethylamine. Yield: 88%. 1H NMR (300 MHz, CDCl3):
d
7.67 (br s,
The effect of compound treatment on cell cycle was measured
by PI staining as previously described [33]. Cells were plated in
60 mm2 dishes and exposed to compounds. Treated cells were
detached using trypsin-EDTA and fixed with 70% ethanol. After
1H), 7.53 (d, J ¼ 7.5 Hz, 1H), 7.48 (dd, J ¼ 1.2, 7.8 Hz, 1H), 7.34e7.28
(m, 3H), 7.19e7.13 (m, 3H), 6.87 (ddd, J ¼ 1.5, 7.2, 8.7 Hz, 2H), 6.79
(dd, J ¼ 7.5, 8.7 Hz, 1H), 6.12 (s, 1H), 6.06 (t, J ¼ 5.1 Hz, 1H), 3.99e
3.87 (m, 2H), 3.81 (s, 3H), 3.66 (q, J ¼ 6.9 Hz, 2H), 2.89 (t, J ¼ 6.9 Hz,
centrifugation, the cells were treated with RNase A (10
m
m
g/mL) for
g/mL) for
2H). 13C NMR (125 MHz, CDCl3):
d 171.35, 166.76, 154.81, 139.93,
15 min at 37 ꢀC and stained with propidium iodide (2.0
137.25, 134.73, 132.46, 130.83, 130.13, 129.69, 128.80, 127.14, 126.50,
125.22, 120.90, 112.12, 64.26, 55.73, 41.06, 34.99, 33.11. HR MS (ESI)
calcd for [C25H23ClN2O3S þ Na]þ 489.1010, found 489.1013.
30 min at 37 ꢀC in the dark. The DNA content per cell was evaluated
in a FACS caliber (Becton Dickison, San Diego, CA, USA).
5.5.4. Wound healing migration assay
5.4.13. 2-(3-(4-methoxyphenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (43)
Cells were seeded onto sterile 12-well plates and incubated at
37 ꢀC in complete RPMI 1640 medium to 80e90% confluence for the
experiment, then changed for fresh serum-free media for another
12 h. Cell monolayer was scraped by sterile 0.1 mL pipette tips. The
cells were washed twice with PBS and the media in each well was
replaced with 1.0 mL complete RPMI 1640. The compounds at
specified concentrations were added to each well and the cells
were incubated at 37 ꢀC for 24 h under complete conditions. Cell
migration was observed and photographed by the microscope for
the statistic and image analysis of each treatment. The migrated
cells were manually quantified. The percentage inhibition of
migrated cells was expressed using 100% as the value assigned for
untreated group.
The title compound was synthesized according to the procedure
of preparing 32 except using 4-methoxyphenyl ethylamine instead
of diethylamine. Yield: 89%. 1H NMR (300 MHz, CDCl3):
d 7.67 (br s,
1H), 7.53 (d, J ¼ 7.5 Hz, 1H), 7.47 (d, J ¼ 8.1 Hz, 1H), 7.31 (dd, J ¼ 7.8,
8.1 Hz, 1H), 7.19 (d, J ¼ 6.6 Hz, 1H), 7.14 (d, J ¼ 8.4 Hz, 2H), 6.88 (d,
J ¼ 8.4 Hz, 1H), 6.79 (dd, J ¼ 7.5, 7.8 Hz, 1H), 6.12 (s, 1H), 6.05 (t,
J ¼ 5.1 Hz, 1H), 3.99e3.85 (m, 2H), 3.80 (s, 6H), 3.66 (q, J ¼ 6.9 Hz,
2H), 2.86 (t, J ¼ 6.9 Hz, 2H). 13C NMR (125 MHz, CDCl3):
d 170.35,
165.71, 157.36, 153.80, 138.82, 133.93, 129.69, 128.73, 128.67, 127.72,
126.19, 125.51, 119.88, 113.13, 111.09, 63.28, 54.70, 54.27, 40.32,
33.67, 32.10. HR MS (ESI) calcd for [C26H26N2O4S þ Na]þ 485.1505,
found 485.1500.
5.5.5. Boyden chamber migration assay
5.4.14. 2-(3-(4-hydroxyphenylethyl amino carbonyl) phenyl)-3-(2-
methoxyphenyl)-4-thiazolidinone (44)
5 ꢂ 104 MDA-MB-231 cells in 100
m
L serum-free medium were
added in upper chamber, and 600
mL of medium with 10% fetal
The title compound was synthesized according to the procedure
of preparing 32 except using 4-hydroxyphenyl ethylamine instead
bovine serum (FBS) was added at the bottom. Fixed concentrations
of compounds were added in both chambers. Cells were allowed to
migrate for approximately 10 h. Cells with non-migrated in upper
chamber were removed using cotton swabs. The migrated cells
were fixed in paraformaldehyde for 1 h and stained with 0.1%
crystal violet. Migrated cells in 7e8 randomly selected fields were
counted under an inverted microscope.
of diethylamine. Yield: 90%. 1H NMR (300 MHz, CDCl3):
d 7.64e7.63
(m, 1H), 7.55 (s, 1H), 7.28e7.26 (br s, 3H), 7.20 (ddd, J ¼ 2.1, 6.0,
8.1 Hz, 2H), 7.09 (d, J ¼ 7.5 Hz, 2H), 6.84 (dd, J ¼ 8.4, 8.7 Hz, 3H), 6.77
(d, J ¼ 7.2 Hz, 1H), 6.05 (s, 1H), 6.00 (t, J ¼ 5.1 Hz, 1H), 3.92 (s, 2H),
3.80 (s, 3H), 3.78e3.71 (m, 1H), 3.61e3.52 (m, 1H), 2.87 (q,
J ¼ 6.6 Hz, 2H). 13C NMR (125 MHz, CDCl3):
d 171.97, 166.82, 155.02,
154.75,139.72,135.24,130.85,130.23,130.04,129.97,129.92,128.75,
127.81, 125.86, 124.94, 120.93, 115.96, 112.11, 64.48, 55.72, 41.05,
34.38, 33.14. HR MS (ESI) calcd for [C25H24N2O4S þ Na]þ 471.1349,
found 471.1340.
5.5.6. Apoptosis assay
Apoptotic cells were monitored with Annexin V-FITC Apoptosis
Detection Kit I (BD Biosciences). Cells were washed with cold PBS,
resuspended in binding buffer, and incubated with Annexin V-FITC/
PI staining solution for 15 min following the manufacturer’s in-
structions. Then samples of 10,000 stained cells were analyzed by
flow cytometry (FACS Calibur, BD biosciences).
5.5. Biology
5.5.1. Cell culture
Human lung adenocarcinoma cell line A549 was obtained from
American Type Culture Collection (ATCC). The luciferase-labeled
human mammary adenocarcinoma MDA-MB-231 cell line was
5.5.7. Xenograft tumor growth assay
7 ꢂ 106 A549 cells were injected into nude mice’s lower back. At
the same day, nude mice were randomly divided into 3 groups

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We thank Dr. Songpo Guo for comments and advice. This work
was partially supported by the grants from National Basic Research
Program of China (2012CB910404), National Major Scientific and
Technological Special Project for “Significant New Drugs Develop-
ment” (2013ZX09507001), National Natural Science Foundation
of China (81202407, 81272463), The Science and Technology
Commission of Shanghai Municipality (11DZ2260300 and
12XD1406100), Research Fund for the Doctoral Program of Higher
Education of China (20120076120029), the Fundamental Research
Funds for the Central Universities (78210048), Large Instruments
Open Foundation of East China Normal University (2013-76) and
Innovation Program of Shanghai Municipal Education Commission
(13zz034).
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