Discovery, design and synthesis of Y-shaped peroxisome proliferator- activated receptor δ agonists as potent anti-obesity agents in vivo

Article abstract of DOI:10.1016/j.ejmech.2012.03.055

We have discovered and demonstrated the in vitro and in vivo PPARδ-selective activity of novel Y-shaped agonists. These compounds activated hPPARδ with EC₅₀ values between 1 and 523 nM. Surprisingly, compounds 10a, 11d, 11e and 11f were the mos

Full text of DOI:10.1016/j.ejmech.2012.03.055

European Journal of Medicinal Chemistry 53 (2012) 190e202
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery, design and synthesis of Y-shaped peroxisome proliferator-activated
receptor agonists as potent anti-obesity agents in vivo
d
,
1,
,b,
Jungyeob Ham a 2, Hoosang Hwang a 1, Euno Kim a, Jeong-ah Kim a, Sung Jin Cho a, Jaeyoung Ko a,
Woojin Lee c, Jaehwan Lee a, Harish Holla a, Joydeep Banerjee a, Seokho Kim a, Inho Yang a, Hyun Joo Lee a,
Kyoungjin Shin a, Hyukjae Choi a, Sang-Jip Nam d, Jungae Tak a, Dongyup Hahn a, Taekyung Oh a,
,b,
Dong Hwan Won a, Tae Gu Lee a, Jihye Choi a, Mi Sun Park a, Chaok Seok c, Jungwook Chin a
,
*
,b,
Heonjoong Kang a
*
a Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Republic of Korea
b Research Institute of Oceanography, Seoul National University, NS-80, Seoul 151-747, Republic of Korea
c Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
d College of Pharmacy, Sunchon National University, Suncheon 540-950, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered and demonstrated the in vitro and in vivo PPARd-selective activity of novel Y-shaped
Received 3 November 2011
Received in revised form
28 March 2012
Accepted 29 March 2012
Available online 6 April 2012
agonists. These compounds activated hPPAR
compounds 10a, 11d, 11e and 11f were the most potent and most selective hPPAR
selectivity over the other two subtypes, namely, hPPARa and hPPARg. The PPARd
showed good bioavailability and in vivo efficacy.
d
with EC50 values between 1 and 523 nM. Surprisingly,
agonists with 104-fold
ligands 10a, 11e and 11f
d
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
PPAR
d
Nuclear receptors
Marine natural products
Anti-obesity
GW501516
1. Introduction
found in Xenopus laevis, was previously known as PPAR
humans and has also been known as NUCl [9], PPAR [10] and
FAAR [11].
Previous studies have revealed that PPARd plays an important
role in embryo implantation [12] and that it has physiological
functions in the differentiation of neuronal cells in the central
nervous system (CNS) [13] and in wound healing, exhibiting anti-
inflammatory effects [14,15]. Recent studies have also demon-
b [8] in
d
Peroxisome Proliferator-Activated Receptors (PPARs) comprise
three known subtypes: PPAR
subtypes have differential tissue expression and tissue-specific
functions in vivo. PPAR is mainly expressed in the heart,
kidneys, skeletal muscle and large intestine in humans [2e4] and is
involved in the -oxidation of peroxisomes and mitochondria [5,6].
PPAR is expressed in skeletal muscle at a low level but is mainly
expressed in adipose tissue to induce adipocyte differentiation and
to store energy as fat. PPAR is also involved in the homeostatic
regulation of insulin and glucose [7]. PPAR , which was first
a, PPAR
g
and PPAR
d
[1]. These
a
b
g
strated that PPAR
transformation [15e17]. For example, PPAR
sion of key genes involved in -oxidation in fatty acid catabolism.
PPAR also affects uncoupling proteins (UCPs), which are involved
in energy metabolism, and the effect of PPAR on UCPs has positive
effects on anti-obesity [16,18,19]. The activation of PPAR increases
d
is involved in lipid metabolism and muscle
d
activates the expres-
g
b
d
d
d
d
HDL levels, improves type 2 diabetes without weight change [20],
and favors the treatment of atherosclerosis by inhibiting the genes
associated with atherosclerosis [21]. Therefore, studies on the
regulation of lipid metabolism using PPARd have provided insight
into the development of a treatment strategy for obesity and
related metabolic diseases.
* Corresponding authors. Center for Marine Natural Products and Drug Discovery,
School of Earth and Environmental Sciences, Seoul National University, NS-80,
Seoul 151-747, Republic of Korea. Tel.: þ82 2 880 5730; fax: þ82 2 883 9289.
E-mail addresses: jwchin97@snu.ac.kr (J. Chin), hjkang@snu.ac.kr (H. Kang).
1
These two authors contributed equally to the work.
Korea Institute of Science and Technology, Gangneung Institute, Gangneung
2
210-340, Republic of Korea.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.055

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
191
Merck has reported on a series of synthetic ligands, such as L-
O
631033, that are weak activators of the PPARs [22]. These ligands
resemble fatty acids, with a rigidifying ring located in the middle of
the chain, which is reminiscent of certain eicosanoids. The estab-
lished leukotriene antagonist L-165041 was identified through
O
O
OH
OH
HO
O
O
O
O
O
S
Cl
random screening as an activator of human PPAR
phenylacetic acid derivatives L-796449 and L-783483 have high
affinity (EC50 ¼ 7.9 nM) for human PPAR without subtype speci-
ficity. GW2433, reported by GlaxoSmithKline, is a high affinity
ligand for human PPAR and was demonstrated to be a dual acti-
vator of PPAR and PPAR in a cell-based transfection assay [27].
Recently, GW501516 was discovered through structure-based drug
design and combinatorial chemistry, and this newly identified
compound appeared to be the most effective and selective agonist
d [23e26]. The
L-796449
L-1q5041
d
O
F
Cl
O
O
OH
O
d
Cl
OH
S
N
H
N
S
F3C
d
a
Cl
N
O
GW2433
GW501516
for PPAR
high affinity for hPPAR
selective for PPAR
activated hPPAR and hPPAR
These results suggest that GW501516 might induce undesirable
effects. Previous research has demonstrated that PPAR is impli-
d. According to our results, GW501516 has an extremely
(EC50 ¼ 2.5 nM) and is more than 102-fold
O
d
O
d
over PPARa and PPARg. However, GW501516
a
g above 1 mM concentration (Fig. S1).
Hit , Compound 1
Fig. 1. Chemical structures of PPARd ligands.
a
cated in the development of cardiac hypertrophy caused by
decreased glucose utilization and increased lipid accumulation in
the diabetic heart [28]. In addition, PPAR
causing osteoporosis because of its ability to induce adipogenesis in
progenitor cells derived from bone marrow [29]. In addition, the
g
is a suspected regulator
approximately 12 Å in length. The co-crystal structure of PPAR
with GW2433 revealed that the strong affinity of this ligand to
PPAR was caused by its ability to adopt a Y-shaped conformation
in the hPPAR binding pocket, which may be important for
subtype selectivity. The docking of GW501516 into PPAR with
d
d
well-known agonists of PPAR
g
, rosiglitazone and pioglitazone,
d
being insulin-sensitizers, are associated with a significant increase
d
in the risk of cardiovascular disease [30,31]. Thus, more selective
AutoDock revealed that this ligand occupied only two of three sites
with its L-shaped conformation. An overlay of GW501516 and
PPAR
the present study, we developed highly PPAR
with no activity against the PPAR and PPAR
d
agonists that do not activate PPAR
a
and PPAR
-selective agonists
subtypes at
g are needed. In
d
compound 1 in the PPAR
This overlay suggests that a new Y-shaped compound synthesized
by introducing a phenyl alkyl substituent at the -position to the
d ligand binding pocket is shown in Fig. 2.
a
g
concentrations ranging up to 10
2. Results and discussion
2.1. Chemistry
mM.
a
sulfide could create a ligand that is more potent than GW501516
(Fig. 3).
In our initial design, we synthesized Y-shaped analogs with
various alkyl and arylalkyl substituents at the
a-position to the
sulfide. The synthesis of compounds 10aeg is outlined in Scheme 1.
Compounds 10aeg were prepared from 2-methyl-4-iodophenol in
6 steps. Compound 5 was synthesized using our one-pot method-
ology from the commercially available 2-methyl-iodophenol in 92%
yield [33]. The reaction proceeded through in situ protection of the
hydroxyl group by reaction with isopropyl magnesium bromide,
lithium-halogen exchange with t-BuLi, and the formation of
a lithium thiolate that underwent nucleophilic attack of chlor-
omethyl thiazole 4 in one vessel. Compound 6 was prepared in 90%
yield by reacting the phenolic hydroxyl group with TBDMSCl and
imidazole.
In this paper, we report the development of novel PPARd-
selective ligands, which led to the preparation of Y-shaped agonists.
Using high-throughput screening (HTS) of our marine natural
product libraries, we focused on discovering novel classes of PPAR
d
ligands. In particular, (2R*,4R*)-2,4-dimethyl-4-hydroxy-16-
phenylhexadecanoic acid 1,4-lactone [32] showed an interesting
activity profile (Fig. 1). This finding was not unexpected, as
compound 1 had essential pharmacophores for PPAR
d agonist
activity (an acidic head group and a hydrophobic tail group). The
g-
lactone moiety in compound 1 corresponded to an acidic head
group that allowed hydrogen bonding with the AF-2 helix in the
Compounds 7aeg were synthesized by the alkylation of a-thi-
PPAR
the hydrophobic tail common to PPAR
activity of compound 1 at a molecular level, docking experiments
d
receptor protein. The phenyldecyl group corresponded to
oorganolithiums prepared from the lithiation of compound 6. The
lithiation of compound 6 was achieved by treatment with t-BuLi
d
agonists. To understand the
(2 eq) or LDA (2 eq) in THF at ꢀ78 ꢁC. The corresponding
a-thio-
were performed into the binding pocket of the PPAR
d
receptor
organolithium was alkylated with various electrophiles. Interest-
ingly, the use of 1.2 eq of t-BuLi or LDA produced the corresponding
alkylated compounds 7aeg in low yield (30e45%). However, using
2 equivalents of t-BuLi or LDA afforded compounds 7aeg in high
yield (60e87%).
The deprotection of 7aeg by TBAF provided compounds 8aeg
in quantitative yield. O-Alkylated compounds 9aeg were
obtained by the alkylation of phenol with ethyl bromoacetate
(1.2 eq) in the presence of K2CO3 (2 eq) in 88e91% yield. Finally, o-
alkylated compounds 9aeg were hydrolyzed using 2 N LiOH fol-
lowed by acidification to produce compounds 10aeg in 85e90%
yield. The EC50 values for compounds 10aeg against human PPAR
subtypes listed in Table 1 were determined by an in vitro trans-
fection assay.
using the automated docking program, AutoDock.
The docking of compound 1 into hPPARd revealed that an L-
shaped conformation allowed the lactone group to form hydrogen
bonds with His413, Tyr427, His287 and Thr253. This hydrogen
bonding pattern is expected to be essential for the formation of
a tightly binding ligand complex. The phenyl group of compound 1
was docked into two hydrophobic cavities in two alternate
configurations, similar to the binding mode observed for EPA in
the hPPAR
contains a bundle of thirteen
strands, and its ligand binding pocket is a large cavity within the
protein with a total volume of w1300 Å. The ligand binding
pocket of PPAR assumes a Y-shape, with each of the arms being
d
receptor protein (Fig. 2). The human apo-PPAR
d LBD
a
helices and four small stranded
b
d

192
J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
Fig. 2. Overlay of compound 1 (Lac-2, red and orange) and GW501516 (gray) docked into hPPARd. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
2.2. Biological evaluation
would enhance the affinity for PPAR
other subtypes (PPAR and PPAR ) through filling the Y-shaped
binding pocket. Compound 10a showed excellent potency (1.6 nM)
and 104-fold selectivity for PPAR
over PPAR and PPAR . It was
clear from these results that the compound having a benzyl group
at the -position to the sulfide had enhanced the agonistic activity
for hPPAR with selectivity over the other subtypes (hPPAR and
hPPAR ). In addition, a time-resolved FRET-based binding assay
showed that compound 10a directly binds to hPPAR with an EC50
value of 13 nM (Fig. S2). Based on these results, we synthesized
a series of analogs of 10a to optimize the activity for hPPAR
Compounds 11ae11t were synthesized from intermediate 6 by
d and impart selectivity over
a
g
2.2.1. In vitro evaluation
Remarkably, compounds 10aec (n ¼ 1, 2, 3) displayed excellent
d
a
g
activity for hPPAR
d
(EC50 ¼ 1.6, 2.6 and 6.1 nM, respectively).
However, compounds 10deg (n ¼ 4, 5, 7, and 10), which contain
a
longer phenyl alkyl groups, displayed low activities for hPPAR . It
d
d
a
was evident that the activity linearly decreased with the length of
the alkyl side chain. Interestingly,10e (n ¼ 5) showed a more potent
activity than 10d (n ¼ 4) despite an increased chain length. These
results suggested that the incorporation of a short phenyl alkyl
g
d
d.
group (n ¼ 1 or 2) at the
a-position to the sulfide in GW501516
Fig. 3. Design of a Y-shaped agonist based on molecular modeling studies.

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
193
I
OMgX2
Li
OMgX2
Li S
OMgX2
S
N
Cl
F3C
F3C
i
1) PrMgBr, THF, 0oC
2) t-BuLi, THF, -78oC
3) Sulf ur, -78oC-->rt
OH
S
S
I
OH
S
N
Cl
4)
F3C
N
5
4
a
OTBDMS
OTBDMS
n
S
S
b
S
S
F3C
F3C
N
N
7a-g
6
c
O
O
OH
OEt
n
n
d
S
S
S
S
F3C
F3C
N
N
9a-g
8a-g
e
O
O
OH
n
S
S
F3C
n = 1,2,3,4,5,7,10
N
10a-g
Scheme 1. Reaction conditions: (a) TBDMSCl (1.5 eq), imidazole (2 eq), DMF, r.t.(b) LDA (2 eq), alkyl bromide (1 equiv), THF, ꢀ78 ꢁC(c) TBAF (1.1 equiv), THF, r.t.(d) ethyl bro-
moacetate (1.2 eq), acetone, K2CO3, r.t.(e) 2 N LiOH, 0.5 M NaHSO4.
incorporation of various benzyl substituents or alkyl groups at the
-position to the sulfide. The EC50 values for compounds 11ae11t
The m,p-fluoride-substituted and totally fluoride-substituted
compounds had almost the same activity (entries 10 and 11,
EC50 ¼ 6.7 and 8.1 nM, respectively). Entries 8, 9 and 10 revealed
that the substituents at the o- and p-positions were more important
than the substituent at the m-position. Meanwhile, substitution of
bulky aromatic functional groups on the phenyl group resulted in
a
were derived from an in vitro transfection assay against human
PPAR subtypes (Tables 2and 3). Substitution with an electron-
donating group, such as a methoxy group, at the p-position on
the phenyl group resulted in better activity than substitution with
an electron-withdrawing group (entries 1, 2 and 3, EC50 ¼ 60.0, 9.4
and 4.8 nM, respectively). Compounds with fluoride, chloride, or
trifluoromethyl at the o-position on the phenyl group also dis-
considerable decreases in the PPAR
15, EC50 ¼ 490.1, ia, 523.0 nM). Interestingly, replacing the phenyl
group with an alkyl group resulted in excellent PPAR activity
(entries 16, 17, 18, 19 and 20, EC50 ¼ 1.8, 1.0, 2.3, 1.1, 1.1 nM) but only
moderate activity for hPPAR
d activities (entries 13, 14, and
d
played excellent potency for PPAR
d and were ultra-selective
hPPAR
d
agonists, showing 104-fold selectivity over the other two
a.
subtypes (entries 4, 5, 6, and 7, EC50 ¼ 1.2, 2.1, 1.2, and 2.8 nM,
respectively). The o,p-fluoride-substituted phenyl group showed
2.2.1.1. In vivo evaluation. We also conducted an animal experi-
ment using ten-week-old C57BL/6 mice to investigate the anti-
obesity effect of compound 10a in vivo. Compound 10a was the
better activity for PPAR
d
than did the o,m-fluoride-substituted
group for PPAR
respectively).
d
(entries 8 and 9, EC50 ¼ 4.6 and 16.7 nM,
most potent and selective PPARd agonist and showed good oral

194
J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
agonists with 104-fold selectivity over the other two subtypes,
namely, hPPAR and hPPAR . The most potent and most selective
PPAR ligands, 10a, 11e and 10f, showed good bioavailability and
in vivo efficacy (Figs. 4and 5). These results suggest that our novel
PPAR agonists, especially compounds 10a, 11e and 11f, could be
Table 1
In vitro activities of compounds 10aeg.
a
g
d
d
promising candidates for anti-obesity drugs without causing
adverse effects, such as cardiac hypertrophy and osteoporosis.
4. Experimental
4.1. General
All reactions were performed in oven- and flame-dried glass-
ware under nitrogen atmosphere. Air and moisture sensitive
reagents and solvents were transferred via syringes or cannula, and
they were introduced into the reaction vessel through a rubber
septum. Chemicals obtained from commercial sources were used
without further purification. Flash column chromatography was
carried out on silica gel (230e400 mesh). Analytical thin-layer
chromatography (TLC) was performed with silica gel 60 F254. TLC
plates were visualized with UV light and 5% ammonium dimo-
lybdate or p-anisaldehyde in ethanol with heat. 1H NMR (400 MHz)
in CDCl3 was recorded on a Bruker Avance III 400 MHz NMR
Compound
n
Transactivation EC50(nM)a
HPPAR
a
hPPAR
g
hPPARd
10a
10b
10c
10d
10e
10f
10g
GW501516
1
2
3
4
5
7
ND
ND
ND
ND
ND
ND
ND
229
ND
ND
ND
ND
ND
ND
ND
1.6
2.6
6.1
38.8
11.4
57.2
132.0
2.5
10
>1.2
mM
a
Compounds were screened for agonist activity on PPARs in transiently trans-
fected CV-I cells. EC50 value is the molar concentration of the test compound that
afforded 50% of the maximal reporter activity. ‘ND’ means that its EC50 value is
spectrometer and chemical shifts (d) were expressed in ppm
downfield from the internal tetramethylsilane or with reference to
residual CHCl3. The purity of compounds was assessed by HPLC/MS
spectra.
bigger than 10 mM.
bioavailability (88%) in mice (Table S1). To induce obesity, the mice
were fed a high-fat diet (HFD) containing 35% fat (w/w). Compound
10a was subsequently administered at 10 mg/kg of body weight by
oral intubation daily. During the experiment, no significant differ-
ence in food intake between the vehicle-treated group and the
group treated with compound 10a was observed at any point. After
78 days, the body weight of the vehicle-treated mice had increased
by 102%, whereas the body weight of the compound 10a-treated
mice increased by 60%. Moreover, the compound 10a-treated group
was observed to have decreased serum triglyceride (TG) and free
fatty acid (FFA) levels (29% and 26%, respectively). To test the anti-
obesity effect of compound 10a at the tissue level, we performed
tissue staining using hematoxylin and eosin (Fig. 4). The compound
10a-treated group did not show fatty liver. Furthermore, lipid
accumulation in brown and white adipose tissue was significantly
decreased in the 10a-treated group.
4.2. 2-Methyl-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-
5-yl)methylthio)phenol, compound 5
To
a stirred solution of 2-methyl-4-iodophenol (500 mg,
2.14 mmol) in anhydrous THF (20 ml) at 0 ꢁC was added 2.0 M
isopropyl magnesium chloride solution (1.1 ml, 2.16 mmol). The
reaction mixture was stirred at 0 ꢁC for 10 min under N2 atmo-
sphere and cooled to ꢀ78 ꢁC in dryice-acetone bath. A 1.7 M tert-
butyllithium solution (2.77 ml, 4.70 mmol) was added dropwise to
reaction mixture and stirred at same temperature for 30 min. Sulfur
power (69 mg, 2.14 mmol) was added in one-portion to reaction
mixture and then the reaction mixture was warmed to room
temperature for 1 h. After the reaction mixture was cooled to 0 ꢁC
again, a solution of 4 (624 mg, 2.14 mmol) in THF (3 ml) was added
dropwise to reaction mixture and stirred at room temperature for
1 h. After completion of the reaction, sat. NH4Cl solution (50 ml)
and ethyl acetate (25 ml) were added to reaction mixture. The
organic layer was washed by distilled water, dried by MgSO4, and
concentrated under reduced pressure to give crude compound. The
crude compound was purified by column chromatography on silica
gel (n-hexnane/ethyl acetate ¼ 5/1) to obtain 5 as white crystal
2.2.1.2. In vivo evaluation. To evaluate the in vivo activity of the
newly synthesized and optimized PPARd agonists, eight-week-old
C57BL/6J mice were fed a high-fat diet containing 35% fat to
induce obesity. The control group was orally administered 0.5%
carboxymethyl cellulose in water and fed a high-fat diet, and the
treated groups were orally administered compounds 11e or 11f
(10 mg/kg/day) and fed a high-fat diet. There were six animals in
each group. The results showed that mice treated with the opti-
(728 mg, 86%). 1H NMR (300 MHz, CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz),
7.65 (d, 2H, J ¼ 8.3 Hz), 7.19 (d, 1H, J ¼ 1.5 Hz) 7.01 (dd, 1H, J ¼ 8.2,
2.0 Hz), 6.62 (d, 1H, J ¼ 8.2 Hz), 5.86 (brs, 1H), 4.07 (s, 2H), 2.19 (s,
3H), 2.12 (s, 3H). 13C NMR (75 MHz, CDCl3):
d 163.9, 155.5, 151.7,
mized PPARd agonists had body weight gains that were 35%
137.4, 136.9, 133.5, 131.9 (q, J ¼ 32.6 Hz), 131.7, 126.8, 126.3 (q,
J ¼ 3.9 Hz), 125.8, 123.8, 115.7, 33.2, 16.2, 14.8 HRMS: calcd for
C19H16F3NOS2 [M þ H] þ 395.0625, found 395.0622.
(compound 11e) and 24% (compound 11f) lower than those of the
vehicle group (Fig. 5).
3. Conclusion
4.3. 5-((4-(tert-butyldimethylsilyloxy)-3-methylphenylthio)methyl)-
4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole, compound 6
We synthesized novel Y-shaped PPARd ligands by combining
a known ligand and a marine natural product with automatic
docking experiments. An efficient synthetic route to these analogs
To a stirred solution of 5 (500 mg, 1.26 mmol) in anhydrous DMF
(5 ml) were added imidazole (171 mg, 2.52 mmol) and tert-butyl-
dimethylsilyl chloride (209 mg, 1.38 mmol). The reaction mixture
was stirred at room temperature for 4 h. After completion of the
reaction, the reaction mixture was concentrated under reduced
was also developed. These compounds activated hPPAR
values between 1 and 523 nM. Surprisingly, compounds 10a, 11d,
11e and 11f were the most potent and most selective hPPAR
d with EC50
d

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
195
Table 2
In vitro activities of compounds 11ae11k.
Entry
Compound
R1
R2
Transactivation EC50(nM)a
hPPAR
a
hPPAR
d
hPPAR
l
1
11a
H
NDa
60.0
ND
2
3
11b
11c
H
H
ND
ND
9.4
4.8
ND
ND
4
5
6
11d
11e
11f
H
H
H
ND
ND
ND
1.2
2.1
1.2
ND
ND
ND
7
8
9
11g
11h
11i
H
H
H
ND
ND
ND
2.8
4.6
ND
ND
ND
16.7
(continued on next page)

196
J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
Table 2 (continued )
Entry
Compound
R1
R2
Transactivation EC50(nM)a
hPPAR
ND
a
hPPAR
d
hPPAR
ND
l
10
11j
H
6.7
11
11k
H
ND
8.1
ND
a
’ND’ means that its EC50 value is bigger than 10 mM.
pressure and diluted with ethyl acetate (20 ml) and distilled water
(40 ml). The organic layer was dried with MgSO4, filtered, and
concentrated under reduced pressure to give crude compound. The
crude compound was purified by column chromatography on silica
gel (n-hexnane/ethyl acetate ¼ 10/1) to obtain 6 as white crystal
(5 ml) was added to the reaction mixture. The organic layer was
separated, dried with MgSO4, filtered, and concentrated under
reduced pressure to give the crude compound. This crude
compound was dissolved in acetone (5 ml) at room temperature.
Potassium carbonate (0.76 mmol) and ethyl bromoacetate (55 ml,
(610 mg, 95%). 1H NMR (300 MHz, CDCl3):
d
7.97 (d, 2H, J ¼ 8.1 Hz),
0.50 mmol) were added to the reaction mixture in that order. The
reaction mixture was stirred for further 6 h at room temperature.
After completion of reaction, the reaction mixture was diluted with
ethyl acetate, filtered, dried with MgSO4, and concentrated under
reduced pressure to give the crude compound. The crude
compound was purified by column chromatography on silica gel (n-
hexnane/ethylacetate ¼ 5/1) to obtain 9a as pale yellowish oil
7.65 (d, 2H, J ¼ 8.2 Hz), 7.19 (d, 1H, J ¼ 1.9 Hz) 7.07 (m, 1H), 6.69 (d,
1H, J ¼ 8.3 Hz), 4.11 (s, 2H), 2.21 (s, 3H), 2.11 (s, 3H), 1.01 (s, 9H), 0.21
(s, 6H). 13C NMR (75 MHz, CDCl3):
d 163.2, 154.7, 151.5, 137.1, 136.6,
133.3, 132.4, 131.7, 131.2, 131.0, 130.2, 129.2, 126.6, 126.1, 126.06,
126.01, 125.9, 124.9, 119.4, 32.8, 25.9, 18.5, 16.9, 15.0, ꢀ4.0 HRMS:
calcd. for C19H16F3NOS2 [M þ H] þ 395.0625, found 395.0622.
(177 mg, 94%) 1H NMR (300 MHz, CDCl3):
d
7.98 (d, 2H, J ¼ 8.1 Hz),
7.65 (d, 2H, J ¼ 8.3 Hz), 7.06e7.27 (m, 7H), 6.55 (d, 1H, J ¼ 8.4 Hz),
4.59 (s, 2H), 4.53 (dd,1H, J ¼ 9.7, 5.3 Hz), 4.22 (q, 2H, J ¼ 7.1 Hz), 3.37
(dd,1H, J ¼ 13.7, 5.3 Hz), 3.17 (m,1H) 2.20 (s, 3H),1.83 (s, 3H),1.26 (t,
4.4. 5-(1-(4-(tert-butyldimethylsilyloxy)-3-methylphenylthio)-2-
phenylethyl)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole,
compound 7a
3H, J ¼ 7.2 Hz) 13C NMR (75 MHz, CDCl3):
d 169.1, 163.6, 156.9, 151.8,
138.3, 137.4, 137.3, 136.4, 133.4, 129.3, 128.9, 128.6, 127.2, 126.8,
126.3, 126.2, 126.1, 125.1, 111.8, 65.9, 61.7, 49.1, 44.4, 16.5, 15.1, 14.5.
A stirred solution of 6 (300 mg, 0.59 mmol) in anhydrous THF
(5 ml) was cooled to ꢀ78 ꢁC in dryice-acetone bath under N2
atmosphere. 2.0 M lithiumdiisopropylamide (619
ml, 1.24 mmol)
was added dropwise to reaction mixture at ꢀ78 ꢁC. The reaction
4.6. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-phenylethylthio) phenoxy)acetic acid,
compound 10a
mixture was stirred at ꢀ78 ꢁC for 30 min. A solution of benzyl
bromide (77 ml, 0.65 mmol) in THF (1 ml) was added dropwise
at ꢀ78 ꢁC. After the mixture was stirred at same temperature for
further 30 min, it was quenched by adding sat. NH4Cl solution. The
mixture was diluted by ethyl acetate and the organic layer was
dried with MgSO4, filtered, and concentrated under reduced
pressure to give crude compound. The crude compound was puri-
fied by column chromatography on silica gel (n-hexnane/
ethylacetate ¼ 10/1) to obtain 7a as colorless oil (265 mg, 75%) 1H
To a stirred solution of 9a (150 mg, 0.262 mmol) in ethanol
(5 ml) was added 1 N NaOH (0.27 ml). The reaction mixture was
stirred for 1 h. After completion of the reaction, 1 N HCl was added
to reaction mixture until pH 3 was reached. The acidified mixture
was concentrated under reduced pressure and diluted with ethyl-
acetate and sat. NaCl solution. The organic layer was separated,
dried with MgSO4, filtered, and concentrated under reduced pres-
sure to give 10a as white solid (134 mg, 94%). 1H NMR (600 MHz,
NMR (300 MHz, CDCl3):
d
7.97 (d, 2H, J ¼ 8.1 Hz), 7.65 (d, 2H,
J ¼ 8.2 Hz), 7.03e7.26 (m, 7H) 6.63 (d, 1H, J ¼ 8.3 Hz), 4.51 (dd, 1H,
J ¼ 9.8, 5.3 Hz), 3.37 (dd, 1H, J ¼ 9.8, 5.3 Hz), 3.05 (dd, 1H, J ¼ 13.6,
9.9 Hz), 2.10 (s, 3H), 1.83 (s, 3H), 0.98 (s, 9H), 0.18 (s, 6H) 13C NMR
CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz), 7.66 (d, 2H, J ¼ 8.2 Hz), 7.01e7.26
(m, 7H), 6.55 (d, 1H, J ¼ 8.5 Hz), 4.62 (s, 2H), 4.23 (dd, 1H, J ¼ 9.7,
(75 MHz, CDCl3):
d 163.5, 155.1, 151.8, 138.4, 137.7, 136.5, 133.5,
5.4 Hz), 3.38 (dd,1H, J ¼ 13.8, 5.4 Hz), 3.08 (dd,1H, J ¼ 13.8, 9.7), 2.18
130.3, 129.3, 128.9, 127.2, 126.8, 126.7, 126.2, 126.1, 124.5, 119.4, 49.2,
(s, 3H), 1.77 (s, 3H) 13C NMR (75 MHz, CDCl3):
d 168.9, 163.7, 156.8,
44.4, 26.1, 18.7, 17.1, 15.1, ꢀ3.81, ꢀ3.84.
151.9, 143.2, 137.7, 137.3, 137.2, 133.7, 128.6, 128.4, 126.5, 126.1,
125.8, 111.5, 65.8, 61.6, 47.6, 37.7, 36.2, 31.7, 29.7, 29.5, 29.4, 27.8,
16.3, 15.2, 14.3.
4.5. Ethyl 2-(2-methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)
phenyl)thiazol-5-yl)-2-phenylethylthio)phenoxy)acetate,
compound 9a
4.7. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-3-phenylpropylthio)phenoxy)acetic acid,
compound 10b
To a stirred solution of 7a (200 mg, 0.33 mmol) in THF (5 ml) was
added 1 M tetrabutylammonium fluoride (660 ml, 0.66 mmol) at
room temperature. The reaction mixture was sirred for 1 h. After
completion of the reaction, ethyl acetate (3 ml) and distilled water
1H NMR (300 MHz, CDCl3):
J ¼ 8.3 Hz), 7.26 (m, 3H), 7.12 (m, 4H), 6.55 (d,1H, J ¼ 8.5 Hz), 4.64 (s,
d
7.98 (d, 2H, J ¼ 8.1 Hz), 7.68 (d, 2H,

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
197
Table 3
In vitro activities of compounds 11le11t.
Entry
Compound
R1
R2
Transactiviation EC50(nM)a
hPPAR
ND
a
hPPAR
d
hPPAR
ND
l
12
11l
H
4.4
13
14
11m
H
H
ND
ND
490.1
ND
ND
11n
ia
15
16
17
11o
11p
11q
ND
523.0
1.8
ND
ND
ND
CH3
H
H
135.5
35.0
1.0
18
19
11r
H
432.4
2.3
ND
11s
11t
H
H
591.7
861.3
1.1
1.1
ND
ND
20
a
’ND’ means that its EC50 value is bigger than 10 mM.
2H), 4.20 (dd,1H, J ¼ 8.9, 6.1 Hz), 2.73 (m, 2H), 2.38 (m,1H), 2.20 (m,
NMR (75 MHz, CDCl3): d 172.2, 163.9, 156.6, 151.1, 141.7, 137.9, 137.3,
136.7, 133.9, 128.6, 128.5, 128.3, 126.7, 126.2, 124.9, 111.4, 65.4, 47.5,
37.2, 35.6, 29.6, 16.2, 14.8.
1H), 2.17 (s, 3H), 1.89 (s, 3H).
4.8. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-4-phenylbutylthio)phenoxy)acetic acid, compound 10c
4.9. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-5-phenylpentylthio)phenoxy)acetic acid,
compound 10d
1H NMR (300 MHz, CDCl3):
d
7.94 (d, 2H, J ¼ 8.2 Hz), 7.66 (d, 2H,
J ¼ 8.2 Hz), 7.18 (m, 6H), 6.97 (m, 1H), 6.52 (d, 1H, J ¼ 8.4 Hz), 5.80
(brs, 1H), 4.60 (s, 2H), 4.23 (dd, 1H, J ¼ 8.3, 6.1 Hz), 2.64 (m, 2 Hz),
2.16 (s, 3H), 2.07 (m, 1H), 1.93 (s, 3H), 1.88 (m, 1H), 1.76 (m, 2H) 13C
1H NMR (300 MHz, CDCl3):
J ¼ 8.2 Hz), 7.20 (m, 6H), 6.99 (m, 1H), 6.53 (d, 1H, J ¼ 8.4 Hz), 5.39
d
7.96 (d, 2H, J ¼ 8.2 Hz), 7.66 (d, 2H,

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J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
Fig. 4. Anti-obesity effect of compound 10a. (A) Food intake of vehicle and compound 10a-treated mice, measured over experimental days, did not show any significant difference
between the two groups. (B) The increase in body weight was reduced by administration of compound 10a in high-fat diet-induced obese mice. Compound 10a-treated group
decreased triglyceride (C) and free fatty acid (D) In serum. (n ¼ 6). *P < 0.05; **P < 0.01; ***P < 0.001. (E) Also, H&E staining results showed that compound 10a-treated mice
decreased fat mass in liver, brown adipose tissue (BAT), and white adipose tissue (WAT).
(brs, 1H), 4.61 (s, 2H), 4.23 (dd, 1H, J ¼ 8.8, 6.1 Hz), 2.59 (m, 2H), 2.17
133.8, 128.6, 128.5, 128.3, 126.8, 126.1, 125.9, 124.9, 111.5, 65.3, 47.5,
37.6, 35.9, 31.3, 28.9, 27.7, 16.2, 14.7.
(s, 3H), 1.88 (m, 1H), 1.94 (s, 3H), 1.91 (m, 1H), 1.65 (m, 2H), 1.46 (m,
2H) 13C NMR (75 MHz, CDCl3):
d 172.2, 163.9, 156.6, 151.1, 142.4,
137.8, 137.4, 133.8, 128.5, 128.3, 126.7, 126.1, 126.0, 111.7, 65.4, 47.5,
37.6, 35.8, 31.2, 27.5, 16.2, 14.8.
4.11. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-8-phenyloctylthio)phenoxy)acetic acid, compound 10f
4.10. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-6-phenylhexylthio)phenoxy)acetic acid,
compound 10e
1H NMR (300 MHz, CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz), 7.71 (s, 1H),
7.66 (d, 2H, J ¼ 8.3 Hz), 7.20 (m, 6H), 6.98 (m, 1H), 6.54 (d, 1H,
J ¼ 8.5 Hz), 4.62 (s, 2H), 4.23 (dd, 1H, J ¼ 9.0, 6.0 Hz), 2.58 (m, 2H),
2.17 (s, 3H), 2.03 (m, 1H), 1.95 (s, 3H), 1.89 (m, 1H), 1.61 (m, 2H), 1.28
1H NMR (300 MHz, CDCl3):
J ¼ 8.5 Hz), 7.20 (m, 6H), 6.98 (m,1H), 6.54 (d,1H, J ¼ 8.5 Hz), 4.59 (s,
2H), 4.22 (dd, 1H, J ¼ 9.0, 6.0 Hz), 2.58 (m, 2H), 2.17 (s, 3H), 2.03 (m,
1H), 1.93 (s, 3H), 1.89 (m, 1H), 1.61 (m, 2H), 1.42 (m, 4H) 13C NMR
d
7.96 (d, 2H, J ¼ 8.4 Hz), 7.66 (d, 2H,
(m, 8H) 13C NMR (75 MHz, CDCl3):
d 172.4, 163.9, 156.6, 151.0, 142.9,
137.9, 137.7, 136.7, 133.8, 131.9, 131.5, 128.6, 128.4, 128.2, 126.7, 126.2,
126.1, 125.8, 125.0, 111.5, 65.3, 47.6, 37.7, 36.1, 31.6, 29.4, 29.35, 29.30,
27.9, 16.2, 14.7.
(75 MHz, CDCl3): d 172.4, 163.9, 156.6, 151.1, 142.6, 137.9,137.6, 136.7,

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
199
4.15. 2-(4-(2-(4-Methoxyphenyl)-1-(4-methyl-2-(4-
(trifluoromethyl)phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11c
16
14
12
10
8
Vehicle
Compound 11e
Compound 11f
1H NMR (600 MHz, CDCl3): 8.83 (1H, s, br), 7.91 (2H, d, 7.8 Hz),
7.62 (2H, d, 7.8 Hz), 7.09 (1H, s), 7.01 (1H, d, 7.8 Hz), 6.97 (2H, d,
8.4 Hz), 6.74 (2H, d, 9 Hz), 6.46 (1H, d, 8.4 Hz), 4.48 (1H, q, 5.4 Hz),
4.42 (2H, s), 3.71 (3H, s), 3.30 (1H, q, 5.4 Hz), 3.01 (1H, q, 9.6 Hz),
2.09 (3H, s), 1.78 (3H, s). 13C NMR (150 MHz, CDCl3): 163.85, 158.61,
156.49, 151.36, 137.01, 136.58, 136.50, 133.10, 131.66, 131.44, 130.10,
129.96, 128.98, 126.77, 126.06, 124.96, 123.15, 114.24, 55.31, 48.94,
43.12, 30.47, 16.17, 14.60. HRFABMS (þmode): Calcd. for
C29H26F3NO4S2 [M þ H]þ 573.6462, found 574.1334.
***
***
6
4
2
0
4.16. 2-(4-(2-(2-Chloro-6-fluorophenyl)-1-(4-methyl-2-(4-
(trifluoromethyl) phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11d
0
10
20
30
40
Days
To a stirred solution of 12eeester (156 mg, 0.25 mmol) in THF/
H2O (3:2, 5 ml) was added 2 N LiOH (0.27 ml) at room temperature
and stirred for 2 h. After completion of the reaction, 0.5 M NaHSO4
was added to reaction mixture until pH 2 was reached. The acidified
mixture was concentrated under reduced pressure and diluted
with ethylacetate and sat. NaCl solution. The organic layer was
separated, dried with MgSO4, filtered, and concentrated under
reduced pressure to give 12e as white solid (140 mg, 94%). 1H NMR
(600 MHz, CDCl3): 7.96 (2H, d, 8.4 Hz), 7.66 (2H, d, 7.8 Hz), 7.17 (1H,
s), 7.15 (2H, t, 8.4 Hz), 7.09(1H, dd, 1.2 Hz, 9.6 Hz), 6.92 (1H, m), 6.77
(1H, s, br), 6.55 (1H, d, 8.4 Hz), 4.79 (1H, q, 6.6 Hz), 4.60 (2H, s), 3.45
(1H, q, 7.2 Hz), 3.35 (1H, q, 9 Hz), 2.17 (3H, s), 1.85 (3H, s). 13C NMR
(150 MHz, CDCl3): 172.76, 164.21, 162.60, 160.95, 156.44, 151.17,
136.98, 136.20, 135.55, 132.99, 129.13, 128.39, 126.76, 126.13, 125.57,
125.13, 125.00, 124.15, 114.24, 111.63, 65.29, 45.96, 34.57, 29.92,
16.26, 14.54. HRFABMS (þmode): Calcd. for C28H22ClF4NO3S2
[M þ H]þ 596.0558, found 596.0744(Mþþ1).
Fig. 5. Anti-obesity effect of compounds 11e and 11f in a high-fat diet-induced obese
model. Mice were fed with an HFD containing 35% fat (w/w). Compounds 11e and 11f
were administered to the mice at a dosage of 10 mg/kg with oral intubation daily
(n ¼ 6). ***P < 0.001.
4.12. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-11-phenylundecylthio)phenoxy)acetic acid,
compound 10g
1H NMR (300 MHz, CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz), 7.66 (d, 2H,
J ¼ 8.3 Hz), 7.18 (m, 6H), 6.99 (m, 1H), 6.54 (d, 1H, J ¼ 8.5 Hz), 4.91
(brs, 1H), 4.61 (s, 2H), 4.24 (dd, 1H, J ¼ 9.0, 5.9 Hz), 2.58 (m, 2H), 2.17
(s, 3H), 2.03 (m, 1H), 1.95 (s, 3H), 1.85 (m, 1H), 1.59 (m, 2H), 1.28 (m,
14H).
4.13. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-(4-nitrophenyl)ethylthio)phenoxy)acetic acid,
compound 11a
4.17. 2-(4-(2-(2,6-Difluorophenyl)-1-(4-methyl-2-(4-
(trifluoromethyl)phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11e
1H NMR (600 MHz, CDCl3): 8.83 (1H, s, br), 7.91 (2H, d, 7.8 Hz),
7.62 (2H, d, 7.8 Hz), 7.09 (1H, s), 7.01 (1H, d, 7.8 Hz), 6.97 (2H, d,
8.4 Hz), 6.74 (2H, d, 9 Hz), 6.46 (1H, d, 8.4 Hz), 4.48 (1H, q, 5.4 Hz),
4.42 (2H, s), 3.71 (3H, s), 3.30 (1H, q, 5.4 Hz), 3.01 (1H, q, 9.6 Hz),
2.09 (3H, s), 1.78 (3H, s). 13C NMR (150 MHz, CDCl3): 163.85, 158.61,
156.49, 151.36, 137.01, 136.58, 136.50, 133.10, 131.66, 131.44, 130.10,
129.96, 128.98, 126.77, 126.06, 124.96, 123.15, 114.24, 55.31, 48.94,
43.12, 30.47, 16.17, 14.60. HRFABMS (þmode): Calcd. for
C28H23F3N2O5S2[M þ H]þ 588.6178, found 509.10 79.
1H NMR (300 MHz, CDCl3):
d
7.95 (d, 2H, J ¼ 8.1 Hz), 7.65 (d, 2H,
J ¼ 8.4 Hz), 7.13 (m, 3H), 6.81 (m, 2H), 6.55 (d, 1H, J ¼ 8.4 Hz), 6.01
(brs, 1H), 4.71 (dd, 1H, J ¼ 8.9, 6.8 Hz), 4.63 (s, 2H), 3.35 (dd, 1H,
J ¼ 13.9, 6.7 Hz), 3.23 (dd, 1H, J ¼ 13.9, 9.1 Hz), 2.18 (s, 3H), 1.90 (s,
3H). 13C NMR (75 MHz, CDCl3):
d 172.6, 164.2, 156.4, 151.0, 136.9,
136.8, 136.5, 136.3, 134.3, 132.9, 128.9, 128.7, 128.4, 126.8, 126.1,
125.4, 111.6, 65.3, 46.0, 38.9, 16.3, 14.5. HRFABMS (þmode): Calcd.
for C28H22F5NO3S2 [M þ H]þ 579.6012, found 580.1040.
4.14. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-(4-(trifluoromethyl)phenyl)ethylthio)phenoxy)
acetic acid, compound 11b
4.18. 2-(4-(2-(2-Fluoro-6-(trifluoromethyl)phenyl)-1-(4-methyl-2-
(4-(trifluoromethyl) phenyl)thiazol-5-yl)ethylthio)-2-
methylphenoxy)acetic acid, compound 11f
1H NMR (300 MHz, CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz), 7.67 (d, 2H,
1H NMR (300 MHz, CDCl3):
d
8.44 (br,1H), 7.96 (d, 2H, J ¼ 8.1 Hz),
J ¼ 8.2 Hz), 7.49 (d, 2H, J ¼ 8.0 Hz), 7.21 (d, 2H, J ¼ 8.0 Hz), 7.16 (d,
2H, J ¼ 1.5 Hz), 7.05 (dd, 1H, J ¼ 10.3, 1.9 Hz), 6.56 (d, 1H,
J ¼ 8.5 Hz), 5.34 (s, 1H), 4.63 (s, 2H), 4.52 (dd, 1H, J ¼ 9.4, 5.7 Hz),
3.30 (m, 2H), 2.18 (s, 3H), 1.79 (m, 1H). 13C NMR (150 MHz, CDCl3):
175.00, 163.88, 156.60, 151.53, 141.95, 137.02, 136.60, 135.56, 133.19,
131.59, 129.57, 129.36, 128.34, 126.55, 126.07, 125.64, 124.94,
123.13, 112.51, 48.42, 43.70, 29.92, 16.06, 14.77. HRFABMS
(þmode): Calcd. for C29H23F6NO3S2[M þ H]þ 611.6182, found
612.1102.
7.66 (d, 2H, J ¼ 8.3 Hz), 7.47 (d, 1H, J ¼ 7.8 Hz) 7.34 (m, 1H), 7.19 (t,
1H, J ¼ 9.1 Hz), 7.13 (d, 1H, J ¼ 1.7 Hz), 7.05 (dd, 1H, J ¼ 8.4, 2.2 Hz),
6.54 (d, 1H, J ¼ 8.4 Hz), 4.70 (dd, 1H, J ¼ 8.0, 8.0 Hz), 4.61 (s, 2H),
3.48 (dd, 1H, J ¼ 14.3, 7.2 Hz), 3.37 (dd, 1H, J ¼ 14.2, 8.3 Hz), 2.17 (s,
3H), 1.80 (s, 3H). 13C NMR (75 MHz, CDCl3):
d 172.9, 164.4, 160.5,
156.5, 150.9, 137.0, 136.6, 133.1, 131.9, 131.5, 129.2, 129.0, 128.4,
126.8, 126.2, 126.1, 125.0, 124.2, 122.6, 119.7, 119.3, 111.6, 65.2, 47.4,
33.7, 16.2, 14.4. HRFABMS (þmode): Calcd. for C29H22F7NO3S2
[M þ H]þ 629.6087, found 630.1008.

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J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
4.19. 2-(4-(2-(2,6-Dichlorophenyl)-1-(4-methyl-2-(4-
(trifluoromethyl)phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11g
126.0, 124.5, 111.8, 65.4, 46.3, 30.9, 16.4, 14.9. HRFABMS (þmode):
Calculated for C28H19F8NO3S2 633.5726, found 634.0757(Mþþ1).
4.24. 2-(4-(2-(Biphenyl-4-yl)-1-(4-methyl-2-(4-(trifluoromethyl)
phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)acetic acid,
compound 11l
1H NMR (300 MHz, CDCl3):
d
7.96 (d, 2H, J ¼ 8.1 Hz), 7.66 (d, 2H,
J ¼ 8.3 Hz), 7.08e7.28 (m, 5H), 6.55 (d, 1H, J ¼ 8.5 Hz), 4.85 (dd, 1H,
J ¼ 8.4, 6.8 Hz), 4.61 (s, 2H), 3.59 (dd, 1H, J ¼ 13.7, 6.8 Hz), 3.47 (dd,
1H, J ¼ 13.7, 8.52 Hz), 2.18 (s, 3H), 1.80 (s, 3H). 13C NMR (75 MHz,
1H NMR (600 MHz, CDCl3): 8.55 (1H, s, br), 7.85 (2H, d, 8.4 Hz),
7.54 (2H, d, 7.8 Hz), 7.48 (2H, d, 7.8 Hz), 7.41 (2H, d, 7.8 Hz), 7.37 (2H,
t, 7.8 Hz), 7.29 (1H, t, 7.2 Hz), 7.08 (2H, d, 8.4 Hz), 7.02 (1H, s), 6.96
(1H, d, 8.4 Hz), 6.34 (1H, d, 7.2 Hz), 4.54 (1H, q, 5.4 Hz), 4.16 (2H, s),
3.36 (1H, q, 5.4 Hz), 3.06 (1H, q, 9.6 Hz), 1.94 (3H, s), 1.77 (3H, s). 13C
NMR (150 MHz, CDCl3): 163.59, 156.45, 151.51, 140.68, 139.91,
136.94, 136.81, 136.74, 136.09, 132.99, 131.57, 131.35, 129.55, 128.95,
128.29, 127.50, 127.31, 127.08, 126.78, 126.51, 126.02, 125.36, 124.97,
112.58, 48.55, 43.70, 16.08, 14.87. HRFABMS (þmode): Calculated
for C34H28F3NO3S2 [M þ H]þ 619.7162, found 620.1541.
CDCl3):
d 172.6, 164.2, 156.4, 151.0, 136.9, 136.8, 136.5, 136.3, 134.3,
132.9, 128.9, 128.7, 128.4, 126.8, 126.1, 125.4, 111.6, 65.3, 46.0, 38.9,
16.3, 14.5. HRFABMS (þmode): Calcd. for C28H22Cl2F3NO3S2
[M þ H]þ612.5104, found 612.0449.
4.20. 2-(4-(2-(2,4-Difluorophenyl)-1-(4-methyl-2-(4-
(trifluoromethyl)phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11h
1H NMR (300 MHz, CDCl3):
d
7.95 (d, 2H, J ¼ 8.1 Hz), 7.66 (d, 2H,
J ¼ 8.3 Hz), 7.38 (brs, 1H), 7.16 (m, 1H), 7.03 (m, 2H), 6.74 (m, 2H),
6.56 (d, 1H, J ¼ 8.5 Hz), 4.65 (s, 2H), 4.59 (m, 1H), 3.36 (dd, 1H,
J ¼ 14.0, 6.1 Hz), 0.3.06 (dd, 1H, J ¼ 14.0, 9.3 Hz), 2.18 (s, 3H), 1.87 (s,
4.25. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-(naphthalen-1-yl)ethylthio)phenoxy)acetic acid,
compound 11m
3H). 13C NMR (75 MHz, CDCl3):
d 172.8, 164.1, 156.6, 151.5, 137.2,
136.6, 135.9, 133.2, 132.1, 132.0, 131.6, 128.5, 126.8, 126.2, 126.1,
124.8, 121.0, 120.9, 111.6, 111.3, 104.1, 103.7, 65.2, 47.2, 37.1, 16.3, 14.6.
HRFABMS (þmode): Calcd. for C28H22F5NO3S2 [M þ H]þ 579.6012,
found 580.1040.
1H NMR (300 MHz, CDCl3):
d
7.98 (2H, d, J ¼ 8.0 Hz), 7.76 (1H, m),
7.71 (2H, d, J ¼ 8.1 Hz), 7.67 (2H, d, J ¼ 8.1 Hz), 7.55 (1H, m),
7.43e7.41 (2H, m), 7.22 (1H, dd, J ¼ 8.2, 1.5 Hz), 7.16 (1H, d,
J ¼ 1.5 Hz), 7.1 (1H, d, J ¼ 8.2 Hz), 6.62 (1H, d, J ¼ 8.2 Hz), 4.67e4.64
(1H, m), 4.49 (2H, s), 3.69e3.65 (1H, m), 3.26e3.22 (1H, m), 2.18
4.21. 2-(4-(2-(2,5-Difluorophenyl)-1-(4-methyl-2-(4-
(trifluoromethyl)phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)
acetic acid, compound 11i
(3H, s), 1.79 (3H, s); 13C NMR (75 MHz, CDCl3):
d 173.8, 163.8, 157.3,
151.3, 137.1, 136.6, 136.4, 135.4, 133.5, 133.4, 132.4, 131.5 (q,
J ¼ 32.5 Hz),128.2, 128.1,127.8,127.7,127.6, 127.0, 126.7,126.5, 126.2,
126.0 (q, J ¼ 3.9 Hz), 125.8, 124.9, 123.9, 123.0, 112.0, 67.1, 44.0, 29.7,
16.0, 14.5. HRFABMS (þmode): Calcd. for C32H26F3NO3S2 [M þ H]þ
593.6789, found 594.1384.
1H NMR (600 MHz, CDCl3): 7.81 (2H, d, 7.8 Hz), 7.59 (2H, d,
8.4 Hz), 7.46 (2H, d, 8.4 Hz), 7.16 (2H, d, 8.4 Hz), 7.01(1H, s), 6.93 (1H,
d, 7.8 Hz), 6.35 (1H, d, 7.8 Hz), 4.49 (1H, q, 5.4 Hz), 4.20 (2H, s), 3.36
(1H, q, 6 Hz), 3.11 (1H, q, 9.6 Hz), 2.08 (3H, s), 1.74 (3H, s). 13C NMR
(150 MHz, CDCl3): 163.88, 156.61, 151.53, 141.96, 137.03, 136.60,
135.56, 133.20, 131.81, 131.59, 129.57, 129.46, 128.34, 126.55, 126.07,
125.62, 125.15, 124.94, 123.34, 123.13, 112.48, 48.42, 43.70, 29.92,
16.06, 14.77. HRFABMS (þmode): Calcd. for C28H22F5NO3S2
[M þ H]þ 579.6012, found 580.1040.
4.26. 2-(4-(2-(Anthracen-9-yl)-1-(4-methyl-2-(4-(trifluoromethyl)
phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)acetic acid,
compound 11n
1H NMR (300 MHz, CDCl3):
d 8.37 (1H, S), 8.0e7.93 (6H, m), 7.67
(2H, d, J ¼ 8.0 Hz), 7.42e7.37 (4H, m), 7.17 (1H, d, J ¼ 1.5 Hz), 7.13
(1H, d, J ¼ 8.2 Hz), 6.56 (1H, d, J ¼ 8.2 Hz), 4.75e4.79 (1H, m), 4.62
(2H, s), 4.29e4.27 (1H, m), 4.13e4.11 (1H, m), 2.17 (3H, s), 1.16 (3H,
4.22. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-(3,4,5-trifluorophenyl)ethylthio)phenoxy)acetic
acid, compound 11j
s); 13C NMR (75 MHz, CDCl3):
d 173.8, 164.7, 158.4, 151.6, 137.5, 137.4,
137. 3, 133.8, 132.4, 131.0, 130.7, 129.9, 129.0, 127.8, 127.3, 126.7 (q,
J ¼ 3.9 Hz), 125.7, 124.8, 124.4, 112.7, 64.1, 36.5, 30.4, 16.5, 14.2.
FABMS (þmode): Calcd. for C36H28F3NO3S2 [M þ H]þ 643.50, found
644.0 (Mþ þ 1), 666.0 (Mþ þ Na).
1H NMR (600 MHz, CDCl3): 7.87 (2H, d, 8.4 Hz), 7.58 (2H, d,
8.4 Hz), 7.01(1H, s), 6.95 (1H, d, 8.4 Hz), 6.90 (1H, m), 6.83 (1H, m),
6.73 (1H, m), 6.35 (1H, d, 7.8 Hz), 4.61 (1H, q, 6 Hz), 4.15 (2H, s), 3.32
(1H, q, 5.4 Hz), 3.01 (1H, q, 9.6 Hz), 1.94 (3H, s), 1.86 (3H, s). 13C NMR
(150 MHz, CDCl3): 175.26, 163.79, 159.33, 158.07, 157.71, 156.52,
151.46, 136.68, 135.53, 132.92, 131.71, 128.32, 126.54, 126.03, 125.10,
123.15, 117.86, 116.54, 115.46, 112.72, 46.58, 37.66, 16.03, 14.80.
HRFABMS (þmode): Calcd. for C28H21F6NO3S2 [M þ H]þ 597.5917,
found 580.5914.
4.27. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2,2-diphenylethylthio)phenoxy)acetic acid,
compound 11o
1H NMR (300 MHz, CDCl3):
d
7.73 (2H, d, J ¼ 8.1 Hz), 7.49e7.46
4.23. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)-2-(perfluorophenyl)ethylthio)phenoxy)acetic acid,
compound 11k
(4H, m), 7.34 (2H, t, J ¼ 8.1 Hz), 7.24e7.20 (1H, m), 7.17e7.16 (2H,
m), 7.12e7.09 (2H, t, J ¼ 8.2 Hz), 7.03e7.0 (1H, m), 6.82 (1H, d,
J ¼ 1.6 Hz), 6.76 (1H, dd, J ¼ 8.2, 1.6 Hz), 6.28 (1H, d, J ¼ 8.2 Hz), 5.05
(1H, d, J ¼ 7.5 Hz), 4.32 (1H, d, J ¼ 7.5 Hz), 4.10 (2H, s), 1.85 (3H, s),
1H NMR (300 MHz, CDCl3):
d
8.58 (brs, 1H), 7.96 (d, 2H,
1.70 (3H, s); 13C NMR (75 MHz, CDCl3):
d 175.1, 163.4, 156.6, 151.4,
J ¼ 8.1 Hz), 7.67 (d, 2H, J ¼ 8.3 Hz), 7.14 (d, 1H, J ¼ 1.7 Hz), 7.07 (dd,
1H, J ¼ 8.4, 2.2 Hz), 6.57 (d, 1H, J ¼ 8.5 Hz), 4.66 (m, 1H), 4.63 (s, 2H),
3.35 (dd, 1H, J ¼ 14.2, 7.5 Hz), 3.26 (dd, 1H, J ¼ 14.2, 8.3 Hz), 2.19 (s,
141.7, 141.5, 137.2, 136.7, 136.4, 133.5, 131.2 (q, J ¼ 32.5 Hz), 128.7,
128.6, 128.2, 127.7, 127.2, 126.8, 126.2, 125.7 (q, J ¼ 3.9 Hz), 125.5,
124.8, 124.7, 122.9, 112.5, 67.3, 59.3, 51.8, 15.8, 14.6. HRFABMS
(þmode): Calcd. for C34H28F3NO3S2 [M þ H]þ 619.7162, found
620.1541.
3H), 2.01 (s, 3H). 13C NMR (75 MHz, CDCl3):
d 173.2, 164.7, 156.9,
151.7, 137.4, 136.6, 135.1, 133.5, 132.3, 131.9, 128.9, 127.0, 126.4, 126.3,

J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
201
4.28. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)ethylthio)phenoxy)acetic acid, compound 11p
4.33. Modeling
AutoDock version 3.0.5 was used for the docking calculations.
The protein structure used in the calculation was based on the co-
crystal structure of hPPARd-LBD and GW2433 (PDB code 1GWX).
1H NMR (300 MHz, CDCl3):
d
7.95 (2H, d, J ¼ 8.1 Hz), 7.66 (2H, d,
J ¼ 8.2 Hz), 7.13 (1H, s), 7.06 (1H, d, J ¼ 8.2 Hz), 6.60 (1H, d,
J ¼ 8.3 Hz), 4.47 (2H, s), 4.44 (1H, m), 2.17 (3H, s), 2.10 (3H, s), 1.66
The co-crystallized ligand and crystallographic water molecules
were removed before docking. Missing heavy atoms and hydrogen
atoms were added using the Leap module of the Amber 8 package.
The standard ionization state at neutral pH was considered for all
ionizable residues because there was no apparent salt bridge
between residues with same charge. The affinity grids for docking
were centered on the geometric center of GW2433 with a grid
spacing of 0.375 Å. By visual inspection, we conformed that this
box practically covered the known binding pocket of the receptor.
For consistency of partial charges of the receptor, single-point
calculations were performed to obtain the electrostatic potential
using the HF/6-31G* level of theory. Fitting charges to the elec-
trostatic potential was subsequently performed according to two-
step restrained electrostatic potential (RESP) protocol. Equivalent
atoms were given equal partial charges. Ligand input files for
docking calculations were then prepared using the AutoTors
module in AutoDock 3.0.5, and the atomic solvation parameters
were determined based on the atom types. The Lamarckian
genetic algorithm was selected to search for ligand conformations
that matched the binding site. For each compound, the following
running parameters were used: random initial position, pop-
ulation size of 50, elitism of 1, mutation rate of 0.02, crossover rate
of 0.8, local search rate of 0.06, 4 million energy evaluations and
200 docking trials. The docked conformations from the 200 trials
for each ligand were clustered using a tolerance of a 1.0 Å root-
mean-square deviation and were sorted in terms of docking
energy.
(3H, d, J ¼ 7.0 Hz); 13C NMR (75 MHz, CDCl3):
d 174.2, 163.3, 157.0,
150.1, 138.1, 137.4, 136.7, 133.6, 131.3 (q, J ¼ 32.5 Hz), 128.0, 127.2,
126.4, 125.9 (q, J ¼ 3.9 Hz) 125.0, 124.4, 122.9, 112.0, 66.5, 41.8, 23.5,
15.9, 14.6. HRFABMS (þmode): Calcd. for C22H20F3NO3S2 [M þ H]þ
467.5243, found 468.0915.
4.29. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)propylthio)phenoxy)acetic acid, compound 11q
1H NMR (300 MHz, CDCl3):
d
7.97 (2H, d, J ¼ 8.1 Hz), 7.67 (2H, d,
J ¼ 8.2 Hz), 7.08 (1H, d, J ¼ 1.5 Hz), 6.98 (1H, dd, J ¼ 8.2, 1.5 Hz), 6.63
(1H, d, J ¼ 8.2 Hz), 4.54 (2H, s), 4.20 (1H, m), 2.16 (3H, s), 2.16e2.02
(1H, m), 2.07 (3H, s), 1.92e1.85 (1H, m), 1.03 (3H, t, J ¼ 7.0 Hz); 13C
NMR (75 MHz, CDCl3): d 173.2, 163.6, 157.2, 150.8, 137.2, 137.1, 136.7,
133.4, 131.4 (q, J ¼ 32.5 Hz), 127.9, 126.4, 125.9 (q, J ¼ 3.9 Hz) 125.0,
124.2, 122.8, 120.7, 112.1, 70.0, 31.9, 29.6, 15.8, 14.6, 12.2. HRFABMS
(þmode): Calcd. for C23H22F3NO3S2 [M þ H]þ 481.5509, found
482.1071.
4.30. 2-(2-Methyl-4-(1-(4-methyl-2-(4-(trifluoromethyl)phenyl)
thiazol-5-yl)butylthio)phenoxy)acetic acid, compound 11r
1H NMR (300 MHz, CDCl3):
d
7.95 (2H, d, J ¼ 8.1 Hz), 7.65 (2H, d,
J ¼ 8.2 Hz), 7.12 (1H, d, J ¼ 1.5 Hz), 6.99 (1H, dd, J ¼ 8.2, 1.5 Hz), 6.54
(1H, d, J ¼ 8.2 Hz), 4.62, (2H, s), 4.26 (1H, m), 2.17 (3H, s), 2.05e2.0
(1H, m), 1.96 (3H, s), 1.88e1.83 (1H, m), 1.47e1.42 (2H, m), 0.93 (3H,
t, J ¼ 7.0 Hz); 13C NMR (75 MHz, CDCl3):
d 172.6, 163.9, 156.6, 150.9,
137.8, 137.7, 136.6, 133.8, 131.5 (q, J ¼ 32.5 Hz), 128.2, 126.8, 126.1 (q,
J ¼ 3.9 Hz) 125.1, 124.9, 123.0, 111.5, 65.3, 47.3, 29.9, 21.1 16.2, 14.6,
13.8. HRFABMS (þmode): Calcd. for C24H24F3NO3S2 [M þ H]þ
495.5775, found 496.1228.
4.34. In vitro transfection assay
The assay was performed as described in previous reports
[16]. The monkey kidney cells, CV-1, were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% resin-charcoal-stripped fetal bovine serum, 100 U/ml
penicillin, and 100 g/ml streptomycin in a humidified incubator
(5% CO2 in air) at 37 ꢁC. CV-1 cells were seeded at 6 ꢂ 103 cells
per well in 96-well culture plates and then grown to 70%
confluence before transfection. The cells were washed with
4.31. 2-(2-Methyl-4-(3-methyl-1-(4-methyl-2-(4-(trifluoromethyl)
phenyl)thiazol-5-yl)butylthio)phenoxy)acetic acid, compound 11s
1H NMR (300 MHz, CDCl3):
d
7.88 (2H, d, J ¼ 8.1 Hz), 7.58 (2H, d,
J ¼ 8.2 Hz), 7.03 (1H, d, J ¼ 1.5 Hz), 6.91 (1H, dd, J ¼ 8.2, 1.5 Hz), 6.47
(1H, d, J ¼ 8.2 Hz), 4.54 (2H, s), 4.25 (1H, m), 2.10 (3H, s), 1.86 (3H, s),
1.82e1.61 (3H, m), 0.88 (2H, d, J ¼ 7.0 Hz), 0.83 (2H, d, J ¼ 7.0 Hz);
serum-free medium and then transfected with plasmid
a
13C NMR (75 MHz, CDCl3):
d
175.0, 163.4, 156.4, 150.8, 137.6, 137.3,
mixture containing human PPAR expression vector, b-galactosi-
136.8, 133.5, 131.4 (q, J ¼ 32.5 Hz), 128.2, 127.3, 126.5, 126.0 (q,
J ¼ 3.9 Hz) 125.2, 123.0, 120.8, 112.2, 66.6, 46.7, 29.9, 26.2 23.0, 21.9,
16.1, 14.9. HRFABMS (þmode): Calcd. for C25H26F3NO3S2 [M þ H]þ
509.6040, found 510.1384.
dase, and TK-PPRE-Luc vector by Superfect reagent (QIAGEN).
The 24 h post-transfected cells were washed with serum-free
DMEM and incubated with freshly delipidated 5% FBS DMEM
supplemented with either compounds or DMSO vehicle for 24 h.
After incubation, cell lysates were obtained using cell lysis
buffer, and a luciferase activity was determined upon substrate
addition using a Microlumat Plus Luminometer (Berthold). The
4.32. 2-(4-(2-Cyclopropyl-1-(4-methyl-2-(4-(trifluoromethyl)
phenyl)thiazol-5-yl)ethylthio)-2-methylphenoxy)acetic acid,
compound 11t
luciferase activity was normalized with b-galactosidase activity
using an ONPG buffer. All of the assays were performed in
triplicate.
1H NMR (300 MHz, CDCl3):
d
7.99 (2H, d, J ¼ 8.0 Hz), 7.70 (2H, d,
J ¼ 8.1 Hz), 7.13 (1H, d, J ¼ 1.5 Hz), 7.08 (1H, dd, J ¼ 8.2, 1.5 Hz), 6.62
(1H, d, J ¼ 8.2 Hz), 4.58 (2H, s), 4.42 (1H, m), 2.19 (3H, s), 2.13 (3H,
s), 2.0e1.91 (1H, m), 1.85e1.78 (1H, m), 0.93e0.77 (1H, m),
0.48e0.45 (2H, m), 0.13e0.12 (2H, m); 13C NMR (75 MHz, CDCl3):
Acknowledgments
d
172.9, 163.6, 156.9, 151.6, 137.5, 136.8, 136.6, 133.1, 131.3 (q,
We are grateful to members of our laboratory. This work was
supported by the Marine Biotechnology Program, Ministry of Land,
Transport and Maritime Affairs. H. H., J.H., E. K., J. K., I. Y. and J. C.
were in part supported by the BK21 program, Ministry of Education,
Science and Technology, Korea.
J ¼ 32.5 Hz), 127.9, 126.5, 126.4, 125.8 (q, J ¼ 3.9 Hz), 125.7, 124.9,
124.1, 122.8, 111.5, 54.3, 47.4, 28.8, 15.7, 14.5, 9.2, 4.6, 4.3, HRFABMS
(þmode): Calcd. for C25H24F3NO3S2 [M þ H]þ 507.5882, found
508.1228.

202
J. Ham et al. / European Journal of Medicinal Chemistry 53 (2012) 190e202
[14] N.S. Tan, L. Michalik, N. Noy, et al., Genes Dev. 15 (2001) 3263e3277.
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3444e3449.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.03.055.
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