Control of the intracellular levels of prostaglandin E2 through inhibition of the 15-hydroxyprostaglandin dehydrogenase for wound healing

Article abstract of DOI:10.1016/j.bmc.2013.05.049

Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much experimental data suggests that prostaglandin E₂ (PGE₂) plays a role in the prevention of excessive scarring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibitors significantly increased PGE₂ levels in A549 cells. In our continuing attempts to develop highly potent 15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28, 29, and 30 demonstrated IC₅₀ values of 0.048, 0.020, 0.038 and 0.048 μM, respectively. They also increased levels of PGE₂ in A549 cells. Especially, compound 28 significantly increased level of PGE₂ at 260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved wound healing with respect to control.

Full text of DOI:10.1016/j.bmc.2013.05.049

Bioorganic & Medicinal Chemistry 21 (2013) 4477–4484
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Control of the intracellular levels of prostaglandin E2 through
inhibition of the 15-hydroxyprostaglandin dehydrogenase
for wound healing q
Dubok Choi a, , Yu Lan Piao b, , Ying Wu b, Hoon Cho b,
⇑
a Department of Pharmacy, College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea
b Department of Polymer Science & Engineering, Chosun University, Gwangju 501-759, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated
function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much
experimental data suggests that prostaglandin E2 (PGE2) plays a role in the prevention of excessive scar-
ring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed
by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibi-
tors significantly increased PGE2 levels in A549 cells. In our continuing attempts to develop highly potent
15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28,
Received 22 April 2013
Revised 24 May 2013
Accepted 25 May 2013
Available online 4 June 2013
Keywords:
15-PGDH
29, and 30 demonstrated IC50 values of 0.048, 0.020, 0.038 and 0.048 lM, respectively. They also
Prostaglandin E2
Scar
Thiazolidinediones
Transforming growth factor-b
Wound healing
increased levels of PGE2 in A549 cells. Especially, compound 28 significantly increased level of PGE2 at
260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed
in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved
wound healing with respect to control.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
wound healing.3 Many factors influence wound healing, including
growth factors, cytokines, metalloproteinases, and extracellular
The healing of skin wounds is complex and involves the forma-
tion of a clot, an inflammatory response, accumulation of granula-
tion tissue, and the deposition and remodeling of an extracellular
matrix.1 It also requires the interaction of cells in the epidermis
and the dermis and mediators released from inflammatory cells,
fibroblasts, and keratinocytes.2 Re-epithelialization by keratino-
cytes, which is achieved by migration and mitosis of cells in the
epidermis proximal to the wound margin, is a central event in
matrix proteins.2 The exogenous application of some of these fac-
tors has been shown to aid healing.4
Two cytokines, transforming growth factor-b (TGF-b) and
prostaglandin E2 (PGE2), are lipid mediators of inflammation in-
volved in wound healing. Clinical data suggests that the overpro-
duction of TGF-b and suppression of PGE2 are found in cases of
excessive wound scarring compared with normal wound healing.
Hsu et al.5 reported that PGE2 basal levels in hypertrophic burn
scars were significantly lower than those present in normotroph-
ic burn scars. Reno et al.6 showed that the prevention and
control of hypertrophy, especially in burn scars, is achieved
using elastocompression. This compression induced a significant
increase in the release of PGE2, in both the remission and active
stages, suggesting a role for PGE2 in the process of hypertrophy
remission induced by pressure therapy. Kolodsick et al.7 reported
that prostaglandins, mainly PGE2, were important mediators of
dermal wound healing with specific effects on fibroblast behav-
ior and that PGE2 can inhibit the differentiation of fibroblast into
myofibroblasts via the EP2 receptor pathway through the up-
regulation of cyclic adenosine monophosphate (cAMP). Therefore,
PGE2 might play an important role in the prevention of excessive
scarring.
Abbreviations: cAMP, cyclic adenosine monophosphate; DMF, N,N-dimethyl-
formamide; DMSO, dimethyl sulfoxide; DTT, dithiothretitol; EDTA, ethylenedia-
mine-N,N,N0,N0-tetraacetic acid; GST, glutathione S-transferase; NAD+, nicotinamide
adenine dinucleotide (oxidized form); NADH, nicotinamide adenine dinucleotide
(reduced form); PG, prostaglandin; 15-PGDH, 15-hydroxyprostaglandin dehydro-
genase; PPARc, peroxisome proliferators-activated receptor; SDS, sodium dodecyl-
sulfate; TGF-b, transforming growth factor-b; THF, tetrahydrofuran; TLC, thin layer
chromatography.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source
are credited.
⇑
Corresponding author. Tel.: +82 62 230 7635; fax: +82 62 230 2474.
E-mail address: hcho@chosun.ac.kr (H. Cho).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.049

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D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
Prostaglandins are a family of biologically potent fatty acids
agents.25 Previously, we reported that several chemopreventive
agents such as cyclooxygenase inhibitors, flavanoids, phytophenol-
ic compounds, and peroxisome proliferator activated receptor ago-
nists were able to inhibit 15-PGDH at low micromolar range.26 The
results showed that ciglitazone was the most potent inhibitor of 15-
derived from arachidonic acid through the cyclooxygenase
pathway. The wide variety of effects of prostaglandins in many
different cell types is explained with binding to a variety of
mainly G protein-coupled receptors.8 Prostaglandins have a short
lifetime in vivo because they are metabolized rapidly by oxidation
to 15-ketoprostaglandins catalyzed by the cytosolic enzyme
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase
(15-PGDH).9 Therefore, inhibitors of 15-PGDH will be valuable
for the therapeutic management of diseases requiring elevated
prostaglandin levels. Previously, we reported that the antidiabetic
drug ciglitazone and its analogue 5-(4-(2-cyclohexylethoxy)
benzylidene)thiazolidine-2,4-dione were potent inhibitors of
15-PGDH and the inhibition of 15-PGDH may lead to increase
accumulation of PGE2.10,11 Recently, we synthesized different
thiazolidinedione analogues of 15-PGDH inhibitors and evaluated:
the levels of PGE2, the influence of 15-PGDH inhibitor on cochlear
blood flow, and the effects on wounds healing.12 However, the
relationship between thiazolidine-2,4-dione derivatives and
wound healing activity is still unclear.
Several previous studies have evaluated wound healing activity
using various derivatives and natural products for the develop-
ment of an efficient wound healing agent. Jeong13 synthesized var-
ious derivatives of asiatic acid and evaluated the structure–activity
relationship of asiatic acid derivatives for new wound healing
agent. Among them, ethoxymethyl 2-oxo-2,23-isopropylidene-
asiatate showed the strongest and the fastest wounding healing
activity. Furthermore, it left the smallest scar after healing. Haleh-
atty et al.14 characterized a new class of 2-mercapto/2-sele-
nobenzo[h]quinoline-3-carbaldehyde and show significant wound
healing activity and protect oxidative DNA damage from harmful
free radical reactions. Antonov et al.15 studied the natural polysac-
charide chitosan for the wound healing agent in thermal and
chemical burns and possibility of its use. They found that deriva-
tives of chitosan regulate proliferation of fibroblasts and stimulate
normal regeneration of skin. Cristiano et al.16 evaluated the effects
of topical tetracycline on the wound healing process in the skin of
normal and diabetic animals and compared the wound healing
areas in diabetics and their controls after local tetracycline use.
Even though several therapeutic effects from various derivatives
have been documented, there has been no information about the
wound healing effect of thiazolidine-2,4-dione derivatives as 15-
PGDH inhibitors.
PGDH among those cyclooxygenase inhibitors, PPARc agonists, and
chemopreventive agents such as flavanoids and phytophenolic
compounds, which were also shown to be 15-PGDH inhibitors. It
was also interesting to discover that the inhibitory potency of cig-
litazone was higher than those of other thiazolidinediones such as
rosiglitazone and troglitazone. It appears that the nature of the moi-
ety linking through the ether linkage to benzyl-2,4-thiazolidinedi-
one plays an important role in inhibitory potency.10 Ding et al.27
suggested that 15-PGDH decreased the level of proliferative PGE2,
induced apoptosis and functioned like a tumor suppressor. We also
examined the structure–activity relationship of various thiazolidin-
edione derivatives and designed a powerful thiazolidinedione that
inhibited 15-PGDH. Based on the structures of the thiazolidinedi-
one derivatives, we developed a protocol for synthesization of the
various benzylidene thiazolidinedione derivatives using a series
of reactions. The thiazolidinedione derivatives had different sub-
stituents on the phenyl ring and we investigated the inhibition of
15-PGDH on accumulation of PGE2.10
Scheme 1 summarizes the general synthetic routes of thiazoli-
dine-2,4-dione derivatives. p-Hydroxybenzaldehyde, as a starting
material, was reacted with various substituents to afford the
substituted benzaldehyde intermediate in good yield. The interme-
diate obtained was then used for a coupling reaction with thiazol-
idine-2,4-dione to afford the appropriate thiazolidine-2,4-dione
derivatives. Previously, CT-8, a thiazolidinedione derivative, was
found to be a potent inhibitor of 15-PGDH.10 Structure–activity
analysis indicated that the N-methylation of thiazolidine-2,4-
dione, CT-8, abolished the inhibitory activity, whereas the intro-
duction of an ethyl hydroxyl group at amine in CT-8 still had a
good inhibitory effect. Based on the structures of the thiazolidined-
ione derivatives and inhibitory activity, a range of benzylidene
thiazolidinedione derivatives were synthesized with different sub-
stituents on the phenyl ring and their inhibitory activity was eval-
uated. Replacement of the cyclohexylethyl group of CT-8 with the
hetero five-member ring increased the inhibitory potency. How-
ever, replacement of the cyclohexylethyl group with a hetero six-
member ring decreased the inhibitory potency significantly. We
suggested that the hydrogen bond donating groups of thiazoli-
dine-2,4-dione are essential in order to orient the molecule more
favorably toward the binding site in the enzyme.11
In this study, for the development of a potential new drug for
treatment of skin wounds, various thiazolidine-2,4-dione deriva-
tives were newly synthesized and their efficacies and selectivity
were examined by analyzing their structure–activity relationships.
We also evaluated the expression level of PGE2 in A549 cells using
some synthetic derivatives. One of the more potent compounds
was further investigated to elucidate the wound healing effect in
HaCaT confluent monolayers and was compared to the effects of
TGF-b1, the positive control.
Based on our previous results, we synthesized different deriva-
tive of thiazolidine-2,4-dione and investigated the inhibitory activ-
ities of these various synthetic thiazolidine-2,4-diones. All the
synthesized compounds were assayed in vitro against 15-PGDH.
The inhibitory results of synthesized thiazolidine-2,4-dione deriv-
atives are shown Table 1.
The inhibitory activity was strongly affected by the C2 position
of the benzyl ring. Previous studies have shown that the introduc-
tion of a chlorine atom at the C3 of CT-8 showed a great increase in
inhibitory potency. We also introduced a chlorine atom at the C2 of
CT-8 resulting in significantly increased inhibitory potency with an
2. Results and discussion
Currently, heterocyclic compounds have been extensively stud-
ied due to their unique properties and applications in the pharma-
ceutical industry. Among these compounds, thiazolidine
derivatives have become especially noteworthy in recent years.
The thiazolidine-2,4-dione derivatives, especially, have acquired
much importance because of their diverse pharmaceutical applica-
tions. They are used in various treatments such as: in hypoglycemic
and hypolipidemic,17 and as antibactericidal,18 pesticidal,19 anti-
fungal,20 insecticidal,21 anticonvulsant,22 antitubercular,23 antipro-
teinuric and anti-inflammatory,24 antithyroidal and antihistaminic
IC50 values of 0.013 lM, as indicated by compound 1. On the other
hand, replacement of the cyclohexylethyl group with a 2-(propan-
2-yloxy) ethyl (2), 2-(pyridin-2-yl)ethyl (3), 4-nitrobenzyl (4),
2-(propan-2-yloxy)ethyl (9), 2-(pyridin-2-yl)ethyl (11), 2-(4-
methyl-1,3-thiazolidin-5-yl)ethyl (12), or 2-(5-ethylpyridin-2-
yl)ethyl group (20) significantly decreased inhibitory potency.
However, replacement of the cyclohexylethyl with a cyclobutylm-
ethyl (16) or cyclopentylmethyl group (18) increased inhibitory
potency with IC50 values of 0.009 and 0.011 lM, respectively.

D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
R1
4479
R1
O
O
R1
O
R2
R2
R2
b
a
H
H
NH
O
R3-OH
S
HO
R3
O
NH
R3
O
S
O
O
Scheme 1. Synthesis of compounds 1–34. Reagents and conditions: (a) PPh3, DEAD, THF, 25 °C, 18 h; (b) piperidine, AcOH, reflux, 12 h.
Table 1
Table 2
Inhibitory potency of various synthetic thiazolidine-2,4-diones
Inhibitory potency of various synthetic thiazolidine-2,4-diones
R1
R1
O
O
R2
R2
NH
S
NH
S
R3
O
O
n
O
O
Compound R1 R2 R3
IC50 (lM)
Compound
n
R1
R2
IC50 (lM)
CT-8
1
2
3
4
5
6
7
8
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cyclohexylethyl
Cyclohexylethyl
0.051
0.013
1.050
8.915
4.540
0.042
0.211
0.223
0.084
2.738
0.930
4.440
5.165
0.099
0.264
0.020
0.009
0.075
0.011
0.132
2.077
0.100
0.071
27
28
29
30
31
32
33
34
1
2
3
4
1
2
3
4
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
0.048
0.020
0.038
0.048
0.487
0.178
0.052
0.081
2-(Propan-2-yloxy)ethyl
2-(Pyridin-2-yl)ethyl
4-Nitrobenzyl
2-Cyclopentylethyl
2-(Cyclohexyloxy)ethyl
Cl 2-Thiomorpholine 1,1-dioxideethyl
Cl 2-(Thiophen-2-yl)ethyl
Cl 2-(Propan-2-yloxy)ethyl
Cl 4-Nitrobenzyl
Cl 2-(Pyridin-2-yl)ethyl
Cl 2-(4-Methyl-1,3-thiazolidin-5-yl)ethyl
Cl 2-Cyclopentylethyl
Cl Thiophene-2-carbonyl
Cl 2-(Cyclohexyloxy)ethyl
Cl Cyclobutylmethyl
Cl 4-(Prop-1-en-2-yl)cyclohexyl]methyl
Cl Cyclopentylmethyl
Cl 1-Cyclopropylethyl
Cl 2-(5-Ethylpyridin-2-yl)ethyl
Cl 3-Nitrophenyl
9
The enzyme was assayed fluorometrically as described in the text. IC50 value were
determined using NAD+ (250
M) as coenzyme and PGE2 (21 M) as substrate. 15-
PGDH was expressed as a GST fusion enzyme using pGEX-2T vector as described in
10
11
12
13
14
15
16
17
18
19
20
21
22
l
l
the text.
300
*
250
200
150
100
50
Cl 5-Oxo-5,6,7,8-tetrahydronaphthalen-1-
yl
23
24
25
26
H
H
H
H
Cl 2-(5-Nitrofuran-2-yl)ethyl
Cl 3-(-2-Nitroethenyl)phenyl
Cl 1,3-Benzodioxol-5-ylmethyl
Cl Bicyclo[2.2.1]hept-2-ylmethyl
0.123
0.059
0.193
0.165
The enzyme was assayed fluorometrically as described in the text. IC50 values were
determined using NAD+ (250
M) as coenzyme and PGE2 (21 M) as substrate. 15-
PGDH was expressed as a GST fusion enzyme using pGEX-2T vector as described in
the text.
l
l
0
Control
27
28
29
30
For further structure–activity analysis, phenyl was introduced
with different chain lengths to the hydroxyl group of 5-(2 or 3-
chloro-4-hydroxybenzylidene)thiazolidine-2,4-dione and inhibi-
tory activities of these derivatives were tested. The inhibitory re-
sults of the synthesized thiazolidine-2,4-dione derivatives are
shown in Table 2. The inhibitory activity was strongly affected by
the methylene group between phenyl ring and oxygen of 5-(2 or
3-chloro-4-benzyloxybenzylidene) thiazolidine-2,4-dione. Specifi-
cally, addition of one more methylene group between phenyl ring
and the oxygen of 5-(3-chloro-4-(2-cyclohexylethoxy)benzyli-
dene)thiazolidine-2,4-dione increased the inhibitory potency. Fur-
ther addition of methylene groups gradually decreased the
inhibitory potency, as indicated by compounds 29 and 30. It ap-
pears that two methylene groups (28) between the phenyl ring
and oxygen of 5-(3-chloro-4-benzyloxybenzylidene) thiazolidine-
2,4-dione are optimal for inhibitory activity. However, a series of
2-chlorothiazolidine analogues showed that three methylene
groups (33) between the phenyl ring and oxygen are optimal for
inhibitory activity.
Compounds
Figure 1. Inhibition of 15-PGDH in A549 cells. A549 cells were seeded in 6-well
culture plates in RPMI medium containing fetal bovine serum and antibiotic and
incubated overnight in incubator containing 5% CO2 at 37 °C. The A549 cells were
treated with the 15-PGDH inhibitors at 5 lM and the PGE2 levels were assayed by
PGE2 enzyme immunoassay as described in the text. Data were analyzed from
results of 3 independent experiments. ⁄p <0.05, Statistically significant versus the
control.
We evaluated the inhibition of 15-PGDH using various synthetic
thiazolidine-2,4-diones in IL-1b-stimulated A549 cells. The syn-
thetic thiazolidine-2,4-diones were added at various concentra-
tions and incubated for 5 h. Medium was then collected and the
level of PGE2 was analyzed by enzyme immunoassay. The results
show that the level of PGE2 was dose dependently increased (data
not shown). In addition, A549 cells were seeded in 6-well culture
plates in RPMI medium containing fetal bovine serum and antibi-
otic for overnight in incubator containing 5% CO2 at 37 °C. These

4480
D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
250
200
150
100
50
*
*
0
−
−
−
TGF-β1
−
−
+
+
−
−
−
+
−
+
−
+
Compound 28
15-PGDH
Figure 2. Scratch wound healing of HaCaT confluent monolayers. HaCaT cells were plated in a 6-well plate at ca. 5 ꢀ 105 cells per well and maintained in DMEM medium
supplemented with 10% fetal bovine serum and 10 lg/mL mitomycin in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were incubated until ca. 80%
confluence was reached. The cells were mechanically scratched with a sterile 200
l
L pipette tip and incubated under control conditions, in the presence of 1 ng TGF-b1 as
positive control, and in the presence of 5
versus the control.
l
M compound 28, 15-PGDH, and TGF-b1 + 15-PGDH. The cells were observed 48 h after wounding. ⁄p <0.05, Statistically significant
A549 cells were then treated with compound 27, 28, 29, and 30,
each at 5 M and the PGE2 levels were assayed by PGE2 enzyme
on fibroblast behavior. Therefore 15-PGDH inhibitors can be used
to help heal wounds, which require elevated prostaglandin levels.
l
immunoassay. The results show that levels of PGE2 were greatly in-
creased as shown in Figure 1. Especially, compound 28 signifi-
cantly increased the level of PGE2 (260 pg/mL), which was about
fivefold higher than that of control. These results suggest that thia-
zolidinedione derivatives as 15-PGDH inhibitors may have utility
for the therapeutic management of diseases requiring elevated
prostaglandin levels.
To study wound healing, a scratch was made using a sterile
200 lL pipette tip in a HaCaT cell line. Confluent monolayers of Ha-
CaT were scratch wounded as described in the Methods and then
allowed to re-epithelialize for 48 h at 37 °C in the presence or ab-
3. Conclusion
Much experimental data suggests that PGE2 might play a role in
the prevention of excessive scarring. However, it has a very short
half-live in blood, oxidizing to 15-ketoprostaglandins catalyzed
by 15-PGDH. Based on the structures of previously synthesized
thiazolidinedione analogues and their 15-PGDH inhibitory activi-
ties, a series of thiazolidinedione analogues were prepared using
a series of reactions and tested for 15-PGDH inhibitory activity.
Among the various synthetic thiazolidinedione compounds, com-
pound 28 was found to be the most active candidate that signifi-
cantly increased level of PGE2 with 260 pg/mL, which was about
fivefold higher than that of control. These results suggest that thia-
zolidinedione derivatives as 15-PGDH inhibitors may have utility
for the therapeutic management of diseases requiring elevated
prostaglandin levels. 15-PGDH inhibitors can be used to treat dis-
eases such as Raynaud’s and Burerger’s diseases, and diabetic neu-
ropathy, which require elevated prostaglandin levels. In addition,
cell exposed to compound 28 showed significantly improved
wound healing and the wound closure rate was about twofold
higher than that of control. Hence, it could also be useful in wound
healing therapy as it is a directly acting wound healing agent.
sence of compound 28 at 5 lM. Cells exposed to compound 28
showed significantly improved wound healing after 48 h with re-
spect to a control (Fig. 2). However, in the presence of 15-PGDH,
wound closure rate were significantly decreased, as shown in Fig-
ure 2. These findings indicate that PGE2 is an important mediator of
wound healing.
Adult wounds heal with imperfect repair and scar formation,
whereas tissue repair in fetal wounds is regenerative. This result
is thought to be the product of both intrinsic fetal fibroblast prop-
erties and extrinsic soluble mediators such as growth factors, cyto-
kines, and chemokines. Many studies have reported that PGE2 is an
important mediator of dermal wound healing with specific effects

D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
4481
4.4. 15-PGDH assay
4. Experimental procedures
4.1. Chemistry
Assays for the activity of the 15-PGDH inhibitors were per-
formed using a fluorescence spectrophotometer by measuring
the formation of NADH at 468 nm following excitation at
340 nm. 50 mM Tris–HCl (pH 7.5), 0.1 mM DTT, 0.25 mM NAD+,
The thiazolidine-2,4-dione derivatives listed in Tables 1 and 2
were prepared by the method shown in Scheme 1. The connection
of cyclohexylalcohol to the central aryl aldehyde was accom-
plished via Mitsunobu coupling to produce an intermediate alde-
10 lg of the purified enzyme, 21 lM PGE2, and various concentra-
tions of inhibitors (total 2 mL) were added to the cells. Each con-
centration of inhibitor was assayed in triplicate. The fluorescence
of the reaction mixture was read at 340 nm and the activity of
the 15-PGDH inhibitors was determined from a standard curve
prepared from the fluorescence of various concentrations of NADH
at 340 nm.
hyde with
a
yield between 70% and 90%. Knoevenagel
condensation between intermediate aldehyde and thiazolidine-
2,4-dione in refluxing toluene, containing a catalytic amount of
piperidinium acetate, produced the benzylidene-2,4-thiazolidined-
ione, which crystallized from the reaction mixture in high purity.
p-Hydroxybenzaldehyde, as a starting material, was reacted with
various substituents to produce an intermediate substituted benz-
aldehyde with good yield. The intermediate was then used in the
coupling reaction with thiazolidine-2,4-dione to provide the
appropriated thiazolidinedione derivatives. All the compounds
were characterized by 1H NMR.
4.5. Cell culture
A549 cells, derived from a human alveolar type II like adenocar-
cinoma, were cultured in RPMI media supplemented with 10% heat
inactivated fetal bovine serum (Sigma) and 100 lg/mL penicillin.
Cells were maintained and subcultured in 5% CO2 at 37 °C.
4.2. Materials
4.6. PGE2 enzyme immunoassay
PGE2, NAD+, NADH, Glutathione-Sepharose 4B, Dithiothretitol
(DTT), Sodium dodecylsulfate (SDS), EDTA and reduced glutathione
were obtained from Sigma. PGE2 ELISA Kit was purchased from
Thermo Scientific (Rockford, IL, United States). GST gene fusion
pGEX-2T expression vector was obtained from Pharmacia Corp.
The cDNA of human 15-PGDH was cloned from a human placenta
cDNA library as described previously.28 All chemical reagents were
commercially available. UV spectra were obtained using a UV–vis
spectrophotometer (SHIMADZU). TLC plates were prepared using
Kieselgel 60 PF254. Column chromatography was performed using
silica gel (230–400 mesh, Whatman Inc). NMR spectra were re-
corded on a JEOL JNM-LA 300 spectrometer (JOEL, Tokyo, Japan).
Chemical shifts were reported in parts per million (d) and signals
were quoted as s (singlet), d (doublet), t (triplet), q (quartet), and
A549 cells (5 ꢀ 105 cells/well) were seeded in 6-well culture
plates in RPMI medium containing fetal bovine serum and antibi-
otic overnight in CO2 incubator at 5% CO2 and 37 °C. The following
day, 15-PGDH inhibitors (5 lM) were added and media was col-
lected after 12 h of drug treatment. The PGE2 levels were deter-
mined by using PGE2 enzyme immunoassay kits (Thermo
Scientific, Rockford, IL, United States) according to manufacturer’s
protocol.
4.7. Scratch wound test
Scratch wound analysis was performed in confluent monolayers
of HaCaT cells. HaCaT cells were plated in a 6-well plate at ca.
5 ꢀ 105 cells per well and maintained in DMEM medium supple-
mented with 10% fetal bovine serum and 10 lg/mL mitomycin in
m
(multiplet). Various thiazolidinediones were synthesized
according to published procedures.29
a humidified atmosphere containing 5% CO2 at 37 °C. The cells
were incubated until ca. 80% confluence was reached. The cells cul-
tured in the 6-well plate were mechanically scratched with a ster-
4.3. Expression and purification of 15-PGDH
ile 200
presence of 1 ng TGF-b1 as positive control or in the presence of
M compound 28. The width of the wound was measured upon
lL pipette tip and incubated under control conditions in the
The sequence of 15-PGDH cDNA plasmid containing BamHI
and EcoRI sites of the pGEX-2T expression vector was used to
transform Escherichia coli BL-21 LysS. Cells were grown in
5
l
wounding and after treatment using an inverted microscope
equipped with a digital camera.
500 mL LB medium containing 50
lg/mL ampicillin at 37 °C
and 220 rpm until OD600 reached 0.6. Isopropyl b-
D-thiogalacto-
4.8. General procedures for the synthesis of compounds 1–34
side (1 mM) was added and cells were allowed to grow for 12 h
at 25 °C. Cells were then harvested by centrifugin at 4000ꢀg for
30 min at 4 °C. The cell pellets were resuspended in 20 mL of
cold cell lysis buffer [1ꢀPBS buffer (pH 7.4) containing 1 mM
EDTA and 0.1 mM DTT] and sonicated (14 ꢀ 10 s at 4 °C). Dis-
rupted cells were centrifuged at 4000ꢀg for 20 min at 4 °C.
The supernatant was slowly applied to the Glutathione-Sephar-
ose 4B column (about 3 mL) which was equilibrated at 4 °C with
lysis buffer [1ꢀPBS buffer (pH 7.4) containing 1 mM EDTA and
0.1 mM DTT]. After washing with lysis buffer until the OD280
reached less than 0.005. The 15-PGDH was eluted from the Glu-
tathione-Sepharose 4B column by incubation at room tempera-
ture for 5 min with the elution buffer [50 mM Tris–HCL (pH
8.0) containing 10 mM reduced glutathione 1 mM EDTA and
0.1 mM DTT]. The concentration of the purified enzyme was
determined and the purity of the 15-PGDH was assessed by
SDS–PAGE and Coomassie Blue staining method.
Diethyl azodicarboxylate (40% in toluene, 3.74 g, 8.58 mmol)
was slowly added to a stirring solution of cyclohexane ethanol
(1.0 g, 7.80 mmol), 2-chloro-4-hydroxybenzaldehyde (1.22 g,
7.80 mmol) and triphenylphosphine (2.25 g, 8.58 mmol) in THF
(20 mL) over 10 min. at 0 °C. The mixture was then stirred at room
temperature until all of the starting materials dissolved (TLC anal-
ysis). The resulting solution was concentrated under reduced pres-
sure and purified by column chromatography over silica gel
(elution with hexane/ethyl acetate) to produce 1.80 g of intermedi-
ate, 2-chloro-4-(2-cyclohexylethoxy)benzaldehyde (1.64 g, 87%) as
yellow oil. A mixture of 2-chloro-4-(2-cyclohexylethoxy)benzalde-
hyde
(1.0 g,
4.06 mmol),
2,4-thiazolidinedione
(475 mg,
4.06 mmol), piperidine (0.20 mL, 2.03 mmol) and acetic acid
(0.13 mL, 2.03 mmol) in toluene (20 mL) was then added to a
round bottom flask fitted with a Dean–Stark water trap and stirred
under reflux for overnight. After cooling to room temperature, the

4482
D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
precipitate was washed with hexane and dried to produce com-
pound 1.
7.536 (d, J = 11.1 Hz, 1H), 7.315–7.363 (m, 2H), 7.017 (d,
J = 3.3 Hz, 1H), 6.978 (d, J = 8.4 Hz, 1H), 4.354 (t, J = 12.6 Hz, 2H),
3.322 (t, J = 12.6 Hz, 2H).
4.8.1. 5-(2-Chloro-4-(2-cyclohexylethoxy)benzylidene)-
thiazolidine-2,4-dione (1)
4.8.9. 5-(3-Chloro-4-(2-(propan-2-yloxy)ethoxy)benzylidene)-
1,3-thiazolidine-2,4-dione (9)
Compound 1 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.24 g, 85%). 1H NMR (300 MHz,
DMSO-d6) d 12.654 (s, 1H), 7.877 (s, 1H), 7.517 (d, J = 9.0 Hz, 1H),
7.110 (s, 1H), 7.012 (d, J = 9.0 Hz, 1H)), 4.111 (t, J = 13.2 Hz, 2H),
1.434–1.642 (m, 6H), 1.263–1.434 (m, 1H), 1.094–1.221 (m, 4H),
0.847–0.989 (m, 2H).
Compound 9 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.09 g, 77.3%). 1H NMR (300 MHz,
CDCl3) d 7.607 (s, 1H), 7.507 (s, 1H), 7.373 (d, J = 8.4 Hz, 1H),
7.052 (d, J = 8.4 Hz, 1H), 4.255 (t, J = 10.2 Hz, 2H), 3.881 (t,
J = 10.2 Hz, 2H), 1.216 (d, J = 3.9 Hz, 6H).
4.8.2. 5-(2-Chloro-4-(2-(propan-2-yloxy)ethoxy)benzylidene)-
1,3-thiazolidine-2,4-dione (2)
4.8.10. 5-(3-Chloro-4-(4-
nitrobenzyloxy)benzylidene)thiazolidine-2,4-dione (10)
Compound 10 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.09 g, 81.3%). 1H NMR (300 MHz,
DMSO-d6) d 12.680 (s, 1H), 8.407 (s, 1H), 8.302 (d, , J = 8.7 Hz,
2H), 7.760 (d, J = 8.7 Hz, 2H), 7.730 (s, 1H), 7.561 (d, J = 8.4 Hz,
1H), 7.451(d, J = 8.4 Hz, 1H), 5.473 (s, 2H).
Compound 2 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.00 g, 71.4%). 1H NMR (300 MHz,
DMSO-d6) d 12.49 (s, 1H), 7.963 (s, 1H), 7.908 (d, J = 8.4 Hz, 1H),
6.951 (m, 1H), 6.908 (d, J = 8.4 Hz, 1H), 4.195 (t, J = 9.6 Hz, 2H),
2.844 (t, J = 9.6 Hz, 4H), 2.359 (m, 1H) 1.024 (d, J = 5.7 Hz, 6H).
4.8.3. 5-(2-Chloro-3-(2-(pyridin-2-yl)ethoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (3)
4.8.11. 5-(3-Chloro-4-(2-(pyridin-2-yl)ethoxy)benzylidene}-1,3-
thiazolidine-2,4-dione (11)
Compound 3 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.1 g, 79.7%). 1H NMR (300 MHz,
DMSO-d6) d 8.461 (s, 1H), 7.966 (s, 1H), 7.722 (t, J = 13.8 Hz, 1H),
6.836–7.291 (m, 6H), 4.008 (d, J = 2.7 Hz, 2H), 2.982–3.033 (m,
2H), 1.185 (t, J = 13.8 Hz, 2H).
Compound 11 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.03 g, 73.9%). 1H NMR (300 MHz,
DMSO-d6) d 8.971(s, 1H), 8.451 (d, J = 4.8 Hz, 1H), 7.776 (s, 1H),
7.720 (t, J = 17.4 Hz, 1H), 7.649 (s, 1H), 7.416 (d, J = 8.4 Hz, 1H),
7.068 (d, J = 8.4 Hz, 1H), 7.191–7.254 (m, 2H), 4.008 (d,
J = 14.4 Hz, 2H), 3.048 (t, J = 14.4 Hz, 2H).
4.8.4. 5-(2-Chloro-4-(4-
nitrobenzyloxy)benzylidene)thiazolidine-2,4-dione (4)
Compound 4 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (0.88 g, 65.7%). 1H NMR (300 MHz,
DMSO-d6) d 12.640 (s, 1H), 7.887 (s, 1H), 7.704 (d, J = 13.2 Hz,
2H), 7.554 (d, J = 13.2 Hz, 2H), 7.510 (s, 1H) , 7.387 (d, J = 11.4 Hz,
1H), 7.223 (d, J = 11.4 Hz, 1H), 5.419 (s, 2H).
4.8.12. 5-(3-Chloro-4-(2-(4-methyl-1,3-thiazolidin-5-
yl)ethoxy)benzylidene)-1,3-thiazolidine-2,4-dione (12)
Compound 12 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.14 g, 84.4%). 1H NMR (300 MHz,
DMSO-d6) d 12.586 (s, 1H), 8.832 (s, 1H), 7.724 (s, 1H), 7.697 (s,
1H), 7.532 (d, J = 9.0 Hz, 1H), 7.320 (d, J = 9.0 Hz, 1H), 4.318 (t,
J = 11.7 Hz, 2H), 3.274 (t, J = 11.7 Hz, 2H), 2.359 (s, 3H).
4.8.5. 5-(2-Chloro-4-(2-cyclopentylethoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (5)
4.8.13. 5-(3-Chloro-4-(2-cyclopentylethoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (13)
Compound 5 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 83.2%); 1H NMR (300 MHz,
CDCl3) d 8.192 (s, 1H), 7.456 (d, J = 8.7 Hz, 1H), 7.089 (d, J = 3 Hz,
1H), 7.034 (d, J = 2.7 Hz, 1H), 4.047 (t, J = 13.2 Hz, 2H), 3.664–
3.701 (m, 2), 1.811–1.917 (m, 2H), 1.562–1.649 (m, 2H), 1.285 (s,
1H), 1.172–1.261 (m, 3H).
Compound 13 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.18 g, 84.9%). 1H NMR (300 MHz,
DMSO-d6) d 12.992 (s, 1H), 7.720 (s, 1H), 7.689 (s, 1H), 7.545 (d,
J = 11.1 Hz, 1H), 7.317 (d, J = 8.7 Hz, 1H), 4.161 (t, J = 13.2 Hz, 2H),
1.189–1.921 (m, 9H), 1.053–1.212 (m, 2H).
4.8.6. 5-(2-Chloro-4-(2-(cyclohexyloxy)ethoxy)benzylidene)-
1,3-thiazolidine-2,4-dione) (6)
4.8.14. 4-((2,5-Dioxopyrrolidin-3-ylidene)methyl)phenyl
thiophene-2-carboxylate (14)
Compound 6 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 8.176 (s, 1H), 7.211 (d, J = 9, 1H), 7.026 (d, J = 2.4,
1H), 6.920 (d, J = 10.5, 1H), 4.122 (t, J = 6.9, 2H), 3.843 (t, J = 9,
2H), 1.930 (s, 3H), 1.764 (d, J = 5.7, 3H), 1.253–1.311 (m, 4H).
Compound 14 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.695 (s, 1H), 7.730 (s, 1H), 7.636 (d, J = 3.0 Hz, 1H),
7.355 (d, J = 3.6 Hz, 1H), 7.121 (d, J = 8.4 Hz, 2H), 6.968 (d,
J = 7.8 Hz, 1H).
4.8.7. 5-(3-Chloro-4-(2-thiomorpholine 1,1-
4.8.15. 5-(3-Chloro-4-(2-(cyclohexyloxy)ethoxy)benzylidene)-
1,3-thiazolidine-2,4-dione (15)
dioxideethoxy)benzylidene)-2,4-thiazolidinedione (7)
Compound 7 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.26 g, 86%). 1H NMR (300 MHz,
DMSO-d6) d 12.564 (s, 1H), 7.724 (s, 1H), 7.706 (s, 1H), 7.547 (d,
J = 9.0 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 4.263 (t, J = 10.5 Hz, 2H),
3.031–3.119 (m, 8H), 2.990 (t, J = 10.5 Hz, 2H).
Compound 15 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 7.886 (s, 1H), 7.727 (s, 1H), 7.349 (d, J = 8.7 Hz, 1H),
4.259 (t, J = 9.3 Hz, 2H), 3.780 (t, J = 9 Hz, 2H), 1.897–1.975 (m,
2H), 1.628–1.643 (m, 2H), 1.461 (s, 1H), 1.185–1.212 (m, 4H).
4.8.8. 5-(3-Chloro-4-(2-(thiophen-2-
yl)ethoxy)benzylidene)thiazolidine-2,4-dione (8)
4.8.16. 5-(3-Chloro-4-(cyclobutylmethoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (16)
Compound 8 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.21 g, 88.3%). 1H NMR (300 MHz,
DMSO-d6) d 12.585 (s, 1H), 7.725 (s, 1H), 7.708 (d, J = 2.4 Hz, 1H),
Compound 16 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.585 (s, 1H), 7.726 (d, J = 8.7, 1H), 7.523 (d, J = 6.6,

D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
4483
1H), 7.215 (d, J = 7.2, 1H), 4.114 (d, J = 6.3, 2H), 1.952–2.029 (m,
2H), 1.828–1.8947 (m, 4H).
DMSO) d 11.099 (s, 1H), 8.085 (s, 1H), 7.871 (s, 1H), 7.721 (d,
J = 6.3, 1H), 7.394 (s, 1H), 7.229 (d, J = 7.5, 2H), 6.933 (s, 1H).
4.8.17. 5-(3-Chloro-4-((4-(prop-1-en-2-
4.8.25. 5-(4-(1,3-Benzodioxol-5-ylmethoxy)-3-
yl)cyclohexyl)methoxy)benzylidene)-1,3-thiazolidine-2,4-dione
(17)
chlorobenzylidene)-1,3-thiazolidine-2,4-dione (25)
Compound 25 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO) d 11.597 (s, 1H), 7.716 (s, 1H), 7.372 (s, 1H), 7.114 (d,
J = 8.4, 1H), 6.979 (s, 1H), 6.935 (d, J = 8.1, 2H), 6.021 (s, 2H),
5.168 (s, 2H).
Compound 17 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.599 (s, 1H), 7.785 (s, 1H), 7.515 (d, J = 6.6, 1H),
7.299 (d, J = 4.8, 1H), 5.858 (s, 1H), 4.695 (s, 2H), 4.509 (s, 2H),
1.902–2.099 (m, 4H), 1.639 (s, 3H), 1.338–1.505 (m, 2H).
4.8.26. 5-(4-(Bicyclo[2.2.1]hept-2-ylmethoxy)-3-
4.8.18. 5-(3-Chloro-4-(cyclopentylmethoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (18)
chlorobenzylidene)-1,3-thiazolidine-2,4-dione (26)
Compound 26 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.596 (s, 1H), 7.732 (s, 1H), 7.533 (d, J = 2.1, 1H),
7.156 (t, J = 6.3, 1H), 4.084 (d, J = 6.3, 2H), 2.490 (s, 2H), 2.31 (s,
1H), 1.68 (s, 1H), 1.445–1.510 (m, 2H), 1.318–1.388 (m, 3H).
Compound 18 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.601 (s, 1H), 7.726 (d, J = 8.4, 1H), 7.518 (d, J = 6.3,
1H), 7.313 (d, J = 8.7, 1H), 4.022 (d, J = 6.9, 2H), 2.073–2.380 (m,
1H), 1.577–1.754 (m, 2H), 1.381–1.516 (m, 4H), 1.315–1.340 (m,
2H).
4.8.27. 5-(4-(Benzyloxy)-3-chlorobenzylidene)thiazolidine-2,4-
dione (27)
4.8.19. 5-(3-Chloro-4-(2-cyclopropylpropyl)benzylidene)-1,3-
thiazolidine-2,4-dione (19)
Compound 27 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.21 g, 86.4% yield). 1H NMR
(300 MHz, DMSO-d6) d 8.024 (s, 1H), 7.936 (s, 1H), 7.747 (d,
J = 10.2 Hz, 1H), 7.324–7.479 (m, 5H), 7.093 (d, J = 10.2 Hz, 1H),
5.259 (s, 2H).
Compound 19 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO-d6) d 12.598 (s, 1H), 7.732 (d, J = 6.6, 1H), 7.504 (d, J = 6.6,
1H), 7.303 (s, 1H), 4.200 (s, 1H), 1.319 (d, J = 4.2, 3H), 1.101–
1.154 (m, 2H).
4.8.28. 5-(3-Chloro-4-phenylethoxybenzylidene)thiazolidine-
2,4-dione (28)
4.8.20. 5-(3-Chloro-4-(2-(5-ethylpyridin-2-
Compound 28 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.19 g, 86.2% yield). 1H NMR
(300 MHz, DMSO-d6) d 12.547 (s, 1H), 7.894 (s, 1H), 7.694 (s, 1H),
7.533 (d, J = 11.1 Hz, 1H), 7.311–7.348 (m, 5H), 7.248 (d,
J = 11.1 Hz, 1H), 4.367 (t, J = 13.5 Hz, 2H), 3.109 (t, J = 13.5 Hz, 2H).
yl)ethoxy)benzylidene)-1,3-thiazolidine-2,4-dione (20)
Compound 20 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO) d 9.022 (s, 1H), 8.312 (s, 1H), 7.731 (s, 1H), 7.556 (d,
J = 2.4, 1H), 7.492 (s, 1H), 7.296 (s, 1H), 7.005 (d, J = 3.3, 1H),
4.055 (d, J = 7.2, 2H), 3.952 (d, J = 7.5, 2H), 2.496 (s, 3H), 1.183 (s,
2H).
4.8.29. 5-(3-Chloro-4-(3-
phenylpropoxy)benzylidene)thiazolidine-2,4-dione (29)
Compound 29 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.13 g, 83.1% yield). 1H NMR
(300 MHz, DMSO-d6) d 12.538 (s, 1H), 7.714 (s, 1H), 7.706 (s, 1H),
7.533 (d, J = 10.8 Hz, 1H), 7.158–7.311 (m, 6H), 4.143 (t,
J = 12.3 Hz, 2H), 2.802 (t, J = 12.8 Hz, 2H), 2.022–2.093 (m, 2H).
4.8.21. 5-(3-Chloro-4-(3-nitrophenoxy)benzylidene)-1,3-
thiazolidine-2,4-dione (21)
Compound 21 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO) d 11.447 (s, 1H), 7.913 (s, 1H), 7.658 (d, J = 6.9, 2H), 7.399
(d, J = 8.4, 1H), 7.110 (d, J = 8.1, 1H), 6.177 (s, 1H).
4.8.30. 5-(3-Chloro-4-(4-
phenylbutoxy)benzylidene)thiazolidine-2,4-dione (30)
Compound 30 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.04 g, 77.6% yield). 1H NMR
(300 MHz, DMSO-d6) d 12.577 (s, 1H), 7.720 (s, 1H), 7.693 (s, 1H),
7.529 (d, J = 10.5 Hz, 1H), 7.135–7.304 (m, 6H), 4.155 (t,
J = 5.7 Hz, 2H), 2.671 (t, J = 7.2 Hz, 2H), 1.741–1.761 (m, 4H).
4.8.22. 5-(3-Chloro-4-((5-oxo-5,6,7,8-tetrahydronaphthalen-1-
yl)oxy)benzylidene)-1,3-thiazolidine-2,4-dione (22)
Compound 22 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO) d 11.071 (s, 1H), 7.628 (d, J = 2.1, 1H), 7.381 (d, J = 2.4,
1H), 7.113 (d, J = 8.7, 1H), 3.30 (m, 2H), 2.490 (d, J = 5.4, 2H),
1.351 (d, J = 6.9, 1H).
4.8.31. 5-(4-(Benzyloxy)-2-chlorobenzylidene)thiazolidine-2,4-
dione (31)
Compound 31 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (0.98 g, 70.1%). 1H NMR (300 MHz,
DMSO-d6) d 7.850 (s, 1H), 7.561 (d, J = 9.9 Hz, 1H), 7.332–7.499
(m, 6H), 7.221 (d, J = 9.9 Hz, 1H), 5.224 (s, 2H).
4.8.23. 5-(3-Chloro-4-(2-(5-nitrofuran-2-
yl)ethoxy)benzylidene)-1,3-thiazolidine-2,4-dione (23)
Compound 23 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,
DMSO) d 11.099 (s, 1H), 8.978 (s, 1H), 7.634 (d, J = 2.1, 1H), 7.406
(s, 1H), 7.116 (d, J = 8.4, 1H), 4.007 (d, J = 6.9, 4H), 1.096–1.185
(m, 4H).
4.8.32. 5-(2-Chloro-4-phenethoxybenzylidene)thiazolidine-2,4-
dione (32)
Compound 32 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (0.96 g, 69.6%). 1H NMR (300 MHz,
DMSO-d6) d 12.645 (s, 1H), 7.863 (s, 1H), 7.500 (d, J = 7.8 Hz, 1H),
7.188–7.274 (m, 6H), 7.127 (d, J = 7.8 Hz, 1H), 4.323 (t,
J = 13.5 Hz, 2H), 3.064 (t, J = 13.8 Hz, 2H).
4.8.24. 5-(3-Chloro-4-(3-((2-
nitroethenyl)phenoxy)benzylidene)-1,3-thiazolidine-2,4-dione
(24)
Compound 24 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.15 g, 79.2%). 1H NMR (300 MHz,

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D. Choi et al. / Bioorg. Med. Chem. 21 (2013) 4477–4484
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4.8.33. 5-(2-Chloro-4-phenpropoxybenzylidene)thiazolidine-
2,4-dione (33)
Compound 33 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (1.0 g, 72.7%). 1H NMR (300 MHz,
DMSO-d6) d 12.548 (s, 1H), 7.924 (s, 1H), 7.482 (d, J = 7.8 Hz, 1H),
7.200–7.368 (m, 6H), 7.212 (d, J = 7.8 Hz, 1H), 4.139 (t,
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4.8.34. 5-(2-Chloro-4-phenbutoxybenzylidene)thiazolidine-2,4-
dione (34)
Compound 34 was obtained by recrystallization from hexane/
ethyl acetate as a yellow solid (0.98 g, 73.1%). 1H NMR (300 MHz,
DMSO-d6) d 12.64 (s, 1H), 7.81 (s, 1H), 7.511 (d, J = 9.3 Hz, 1H),
7.161–7.296 (m, 6H), 7.105 (d, J = 9.3 Hz, 1H), 4.084 (t,
J = 12.9 Hz, 2H), 2.654 (t, J = 13.8 Hz, 2H), 1.653–1.724 (m, 4H).
19. Hyo, J. G.; Kang, B.; Jeon, R. Arch. Pharm. Res. 2007, 30, 1055.
20. Deohate, P. P.; Deohate, J. P.; Berad, B. N. Asian J. Chem. 2004, 16, 255.
21. Sahu, S. K.; Banerjee, M.; Mishra, S. K.; Mohanta, R. K.; Panda, P. K.; Misro, P. K.
Acta Pol. Pharm. 2007, 64, 121.
22. Altintas, H.; Ates, O.; Uydes-Dogan, B. S.; Alp, F. I.; Kaleli, D.; Ozdemir, O.;
Birteksoz, S.; Otuk, G.; Satana, D.; Uzun, M. Arzneim. Forsch. 2006, 56, 239.
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24. Szeto, C. C.; LiP, K. T. Nephrology 2008, 13, 53.
Acknowledgement
This study was supported by research funds from Chosun Uni-
versity, 2012.
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