Synthesis, biological evaluation and molecular modeling of GW 501516 analogues

Article abstract of DOI:10.1002/ardp.201000189

Eleven analogues of GW 501516 (1) were prepared and subjected to biological testing in a semi-high throughput human skeletal muscle cell assay. The assay testing indicated that all analogues elicited oxidation of oleic acid. Among the most potent agonists, 2e (2-{2-ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl) thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoic acid), was also subjected to a luciferase-based transfection assay, which showed that this compound is a potent agonist against PPARδ and a moderate agonist against PPARα. Docking of compound 2e into PPARδ revealed that it occupied the agonist binding site and exhibited key hydrogen bonding interactions with His323, His449, and Tyr473. Eleven GW 501516 analogues were prepared and subjected to human skeletal muscle cells. The assay testing indicated that all analogues elicited oxidation of oleic acid. One of the analogues displayed dual agonist effects against both PPARα and PPARδ. Copyright

Full text of DOI:10.1002/ardp.201000189

612
Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
Full Paper
Synthesis, Biological Evaluation and Molecular Modeling of
GW 501516 Analogues
Calin C. Ciocoiu1, Aina W. Ravna2, Ingebrigt Sylte2, and Trond Vidar Hansen1
1 School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, Oslo, Norway
2 Medical Pharmacology and Toxicology, Department of Medical Biology, Faculty of Health Sciences,
University of Tromsø, Tromsø, Norway
Eleven analogues of GW 501516 (1) were prepared and subjected to biological testing in a semi-
high throughput human skeletal muscle cell assay. The assay testing indicated that all analogues
elicited oxidation of oleic acid. Among the most potent agonists, 2e (2-{2-ethyl-4-[(4-methyl-2-(4-
trifluoromethylphenyl)thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoic acid), was also subjected
to a luciferase-based transfection assay, which showed that this compound is a potent agonist against
PPARd and a moderate agonist against PPARa. Docking of compound 2e into PPARd revealed that it
occupied the agonist binding site and exhibited key hydrogen bonding interactions with His323,
His449, and Tyr473.
Keywords: GW 501516 / Agonists / Multi well assay / Molecular modeling / PPARa / PPARd
Received: June 25, 2010; Revised: August 16, 2010; Accepted: August 20, 2010
DOI 10.1002/ardp.201000189
Introduction
and adipose tissues [7–11]. Furthermore, the activation of
PPARd increases HDL levels, attenuates weight gain, and
improves insulin sensitivity [7, 9]. As of today, no drugs that
target the PPARd receptor have been approved, and only a
few selective and potent ligands that target this receptor have
been identified [12–15]. In 2003, scientists from
GlaxoSmithKline reported the compound GW 501516 (1)
(Fig. 1) to be both highly potent and selective against the
PPARd receptor [15]. When obese Rhesus monkeys were
treated with this agonist, an increase in the plasma HDL
level, as well as a decrease in the plasma triglyceride level,
was observed [16]. Based on these observations, compound 1
is an interesting lead compound for the development of
remedies against type-II diabetes and metabolic syndrome.
Moreover, recently dual agonists have received attention as
potential remedies against several diseases such as metabolic
disorders, type-2 diabetes and cardiovascular diseases [17, 18].
Among such possible dual agonists it may be an advantage to
activate simultaneously both PPARa and PPARd by a single
dual compound to effectively reduce the risk of cardiovas-
cular disease [17, 18]. So far only a few dual agonists of this
type have been reported [19–22]. Herein we report the syn-
thesis, biological evaluation, and molecular modeling stud-
ies of analogues of GW 501516 (1) in which structural
modifications at the alpha-carbon atom next to the carboxylic
acid moiety and at the ortho-position of the benzene ring A
Peroxisome proliferator-activated receptors are ligand-acti-
vated transcription factors that belong to the nuclear hor-
mone receptor superfamily [1]. So far, three isotypes have
been identified and characterized: PPARa, PPARd, and
PPARg. PPARs are involved in expression of genes responsible
of the lipid and carbohydrate metabolism by interacting with
specific DNA peroxisome proliferator response elements
(PPRE) [2]. Agonists acting on the PPARa have been shown
to have beneficial effects on lipid metabolism by decreasing
both serum triglycerides and free fatty acid metabolism
levels, but also increasing high-density lipoprotein level
(HDL) [3]. PPARg agonists have the ability to improve glucose
tolerance in type 2 diabetic patients [4]. Dyslipidemia and
insulin resistance, two major components of the metabolic
syndrome and diabetes, are usually treated with either the
fibrate or the thiazolidinedione (TZD) classes of drugs that
target PPARa and PPARg, respectively [4–6]. Several studies
have suggested that PPARd plays an important role in regu-
lating lipid metabolism and energy homeostasis in muscle
Correspondence: Trond Vidar Hansen, School of Pharmacy, Department
of Pharmaceutical Chemistry, University of Oslo, PO BOX 1068, Blindern,
N-0316 Oslo, Norway
E-mail: t.v.hansen@farmasi.uio.no
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
613
Figure 1. Structure of GW 501516 (1) and derivatives 2a–2k.
have been made (Fig. 1). These efforts led to the identification
of a moderate agonist against PPARa while retaining potent
agonist effects against PPARd. Additionally, six other
analogues displayed dual agonist effects at 10 mM against
both PPARa and PPARd.
Reaction between commercially available 4-mercapto-2-
methylphenol, 4a and 4b, respectively, with commercially
available 5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)
thiazole in the presence of Cs2CO3 at ambient temperature
afforded 5a–5c in 69–84% yield. Sulfur-substituted para-mer-
captophenols 5a–5c were then reacted with the correspond-
ing ethyl 2-bromoesters in the presence of Cs2CO3 to yield
esters 8a–8h which after basic aqueous hydrolysis afforded
acids 2a–2h in 51–80% yield (Scheme 1).
Chemistry
Compounds 3a–3e were synthesized according to a literature
procedure [23]. Thiocyanates 3a and 3b were reduced with
LiAlH4 to mercaptophenols 4a and 4b in 71–88% yield.
Treatment of thiocyanates 3c–3e with LiAlH4 afforded
large quantities of the corresponding disulfide dimers.
Scheme 1. Reagents and conditions: (a) LiAlH4, THF; (b) 5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thiazole, Cs2CO3,
CH3CN; (c) i) ethyl 2-bromoacetate, Cs2CO3, CH3CN or ii) ethyl 2-bromopropionate, Cs2CO3, CH3CN; (d) LiOH, THF, H2O, 08C (8a,
8c, 8d, 8f, 8g); (CH3)3COK, THF, H2O, reflux (8b, 8e, 8h).
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Scheme 2. Reagents and conditions: (a)
ethyl 2-bromoacetate, Cs2CO3, CH3CN; (b)
NaBH4, 1,4-dithioerythritol, EtOH; (c) 5-chloro-
methyl-4-methyl-2-(4-trifluoromethylphenyl)thi-
azole, Cs2CO3, CH3CN; (d) LiOH, THF, H2O,
08C.
Hence, compounds 3c–3e were first reacted with ethyl
2-bromoacetate and then reduced with NaBH4 and 1,4-
dithioerythritol to afford 7a–7c in 61–77% yield [24].
Oxygen-substituted para-mercaptophenols 7a–7c were
reacted with 5-chloromethyl-4-methyl-2-(4-trifluoromethyl-
phenyl)thiazole to produce the esters 8i–8k which after basic
aqueous hydrolysis afforded acids 2i–2k in 49–69% yield over
the two steps (Scheme 2).
highly potent with EC ¼ 0.10 nM in the human skeletal
muscle cell assay (Table 1). Substituting one hydrogen atom
from the alpha-carbon atom next to the carboxylic acid
moiety of GW 501516 (1) with a methyl group led to a
decrease in potency (2a: EC50 ¼ 0.65 nM) compared to 1.
Introduction of two methyl groups afforded agonist 2b that
was slightly more potent than 2a (2b: EC50 ¼ 0.24 nM), but
exhibited slightly lower potency than the lead compound 1.
When the methyl group from the R1-position of GW 501516
Biological evaluation
Table 1. Substitution pattern (see Fig. 1) and EC50-values of tested
compounds in the oleic acid oxidation assay.
GW 501516 (1) and compounds 2a–2k at five different con-
centrations were exposed for 96 h to fully differentiated
human skeletal muscle cells cultured in 96-well plates.
After this period of time, the level of oxidation of oleic acid
was measured by detection of accumulated 14C-labeled oxi-
dized oleic acid [25]. The EC50-values for compounds 2a–2k
are presented in Table 1. Compounds 2a–2k were also tested
against all three peroxisome proliferator-activated receptors
(PPARa, PPARd, and PPARg) in a luciferase-based transient
transfection assay (Figs. 2–4).
Compound
R1
R2
R3
EC50 (nM)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
CH3
CH3
Ethyl
Ethyl
Ethyl
H
CH3
H
H
CH3
H
H
CH3
H
H
H
CH3
CH3
H
CH3
CH3
H
CH3
CH3
H
0.65
0.24
0.31
0.36
0.54
4.15
5.79
9.11
5.51
16.60
17.30
0.10
iso-Propyl
iso-Propyl
iso-Propyl
tert-Butyl
Cyclopentyl
Cyclohexyl
CH3
H
H
H
Results and discussion
GW 501516
H
The results from the oxidation of the oleic acid assay are
a
compiled in Table 1. The lead compound GW 501516 (1) was
Results of three experiments
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
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Figure 2. Activation of the ligand-binding domain of PPARa by compounds 2a–2k. Positive control: EHA ((2E,4E,8Z,11Z,14Z,17Z)-eicosa-
2,4,8,11,14,17-hexaenoic acid).
(1) was substituted to an ethyl group, a decrease of the
potency was noticed (2c: EC50 ¼ 0.31 nM). Substituting one
hydrogen atom in 2c with a methyl group alpha-carbon atom
next to the carboxylic acid moiety retained the potency, as
observed for 2d (EC50 ¼ 0.36 nM). Increasing the size of the
ortho-substituent in the benzene ring A (R1), by substitution
with an iso-propyl, tert-butyl, cyclopentyl or cyclohexyl group,
led to a reduction in potency (2f: EC50 ¼ 4.15 nM, 2i:
EC50 ¼ 5.51 nM, 2j: EC50 ¼ 16.60 nM, 2k: EC50 ¼ 17.30 nM).
These substituents may be too bulky for the ligand-binding
domain of PPARd. Crystallographic studies have shown that a
lipophilic pocket in the PPARd ligand-binding domain can
accommodate small substituents at the ortho-position of the
aromatic ring [15, 26]. Changing the methyl group in 2b (R1)
to an ethyl group led to agonist 2e that exhibited an
EC50-value of 0.54 nM. Replacing the ethyl group in 2e with
Figure 3. Activation of the ligand-binding domain of PPARd by compounds 2a–2k. Positive control: GW 501516 (1).
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Figure 4. Activation of the ligand-binding domain of PPARg by compounds 2a–2k. Positive control: BRL (rosiglitazone).
an iso-propyl group afforded agonist 2h (EC50 ¼ 9.11 nM) that
was even less potent than 2e. In the series of compounds 2b,
2e, and 2h, the potency decreased with increasing size of R1, a
trend that was also observed with 2i, 2j, and 2k. Replacement
of the hydrogen atoms from the alpha-carbon atom to the
carboxylic moiety of 2f with one and two methyl groups
diminished the potency compared to 2e, as noticed for 2g
(EC50 ¼ 5.79 nM) and 2h (EC50 ¼ 9.11 nM).
of PPARs has been extensively studied [27] and X-ray crystallo-
graphic structures have been reported for both active and
inactive receptor conformations. In the active receptor con-
formation, the most C-terminal a-helix (helix 12) acts as a lid
closing the binding cavity, while in the inactive state the
binding site is more accessible from the outside. In the
activated receptor conformation of PPARd, the amino acids
His323, His449, and Tyr473 are essential for agonist inter-
actions [28]. In the present study, 2e was docked into
an activated receptor conformation of PPARd, and the
docking showed that 2e was well accommodated to the
activated receptor conformation, with a binding mode very
similar to that of the full PPARd agonist 2-{2,3-dimethyl-4-
[2-prop-2-ynyloxy-4-((4-trifluoromethylphenoxy)methyl)phenyl-
thio]phenoxy}acetic acid (PDB entry: 3GZ9) (Fig. 5). The dock-
ing of 2e revealed key interactions with amino acids Arg284,
Cys285, His323, His449, and Tyr473 (Fig. 5). As for the full
agonist, the acidic group of 2e interacted with His323,
His449, and Tyr473. The trifluoromethyl group had contact
with Arg284. The calculated interaction energy of the 2e—
PPARd complex was –14.9 kcal/mol. The docking mode sup-
ports the observation that compound 2e is a PPARd agonist.
In the series of tested compounds, the potency decreased
with increasing size of the substituent in the R1-position. In
the docked complex of 2e the ethyl group in R1 points
in the direction of Thr289, Ile326, and Phe327. A larger
substituent R1 will produce severe steric interactions with
these residues and this may explain the decrease in potency
when the size of the substituent is increased to iso-propyl, tert-
butyl, cyclopentyl or cyclohexyl groups.
Next, we investigated the effects compounds 2a–2k exhib-
ited on all of the peroxisome proliferator-activated receptors
(PPARa, PPARd, and PPARg) in a luciferase-based transient
transfection system. Compounds 2a–2e, 2g, and 2h showed a
higher activation of both PPARa as well as PPARd at 10 mM
concentrations than the positive controls (Figs. 2 and 3).
Compounds 2i and 2j activated only the PPARd receptor with
the efficacy comparable to the lead compound 1 at 10 mM
(Fig. 3). No notable activation of PPARg was observed (Fig. 4) at
the same concentration of all prepared analogs of 1. To
further investigate the agonist effects of compound 2e, the
EC50-values were determined against all three PPARs using
the aforementioned transfection assay. The EC50-value for 2e
against PPARd was determined to be 5.0 nM, which is slightly
lower than the EC50-value of 1.0 nM reported for GW 501516
(1) [15]. Interestingly, the EC50-value against the PPARa recep-
tor was determined to be 750 nM, which is a moderate
agonist effect. Compound 2e was found to be inactive against
PPARg (EC50 > 5000 nM).
In order to gain information on the binding of 2e with the
ligand-binding domain of the PPARd receptor, molecular
modeling studies were performed. The activation process
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
617
Figure 5. A. 2e docked into PPARd. Color cod-
ing: red O, blue N, grey H, yellow C in 2e, grey C
in PPARd. Coloring of the Ca traces of PPARd
is blue via white to red from N-terminal to C-
terminal. B. The docked complex of 2e (purple)
superimposed onto the X-ray structure complex
of the agonist 2-{2,3-dimethyl-4-[2-prop-2-ynyl-
oxy-4-((4-trifluoromethylphenoxy)methyl)phe-
nylthio]phenoxyacetic acid (green) (PDB entry:
3GZ9).
Conclusions
per million (d) relative to CDCl3 (7.24 ppm for 1H and 77.00 ppm
for 13C) or DMSO-d6 (2.50 ppm for 1H and 39.51 ppm for 13C).
Melting points were measured using a Barnstead Electrothermal
apparatus. Melting points are uncorrected. Flash column chroma-
tography was performed on silica gel 60 (40–63 mm, Fluka). LC/MS
analyses were performed on an Agilent Technologies 1200 Series
(Eclipse XDB-C18, 5 mm 4.6 ꢀ 150mm), coupled with an Agilent
6310 ion trap. According to LC/MS spectra, all final compounds
submitted to the biological testing had a purity >99%.
To the best of our knowledge, very few analogues of GW
501516 (1) have been reported in which modifications at
the alpha-carbon atom next to the carboxylic acid moiety
have been made [21, 22, 29–32]. This moiety is a common
feature for the chemical structures of most PPARd agonists
reported [12–15]. Herein we report that compounds 2a–2d,
2g, and 2h displayed dual agonist effects at 10 mM against
both PPARa and PPARd. Docking of compound 2e into the
ligand binding domain of PPARd supported that compound
2e is a potent PPARd agonist with EC50 ¼ 5 nM. Moderate
potency was observed against PPARa for compound 2e
(EC50 ¼ 750 nM). Since very few dual PPARa/d agonists have
been reported in the literature [19–22], further studies are
underway focusing on the preparation of potent and dual
PPARa/d agonists based on the results reported herein. These
efforts will be reported in due course.
2-Ethyl-4-thiocyanatophenol (3a)
The title compound was prepared as following: To a stirred
solution of 2-ethylphenol (2 mmol, 240 mL), sodium thiocyanate
(520 mg, 6.4 mmol) and methanol (40 mL) at 08C was added a
solution of sodium bromide (206 mg, 2 mmol) and bromine
(206 mL, 2 mmol) in methanol (60 mL). The mixture was stirred
for 3 h under argon at 08C and then diluted with saturated
aqueous solution of NaHCO3. The mixture was extracted
(CH2Cl2, 3 ꢀ 100 mL), the organic phases were combined,
washed with brine, dried (MgSO4), and concentrated. The residue
was purified by column chromatography using hexane/ethyl
acetate (3:1) as eluent to give 3a as a white solid in 70% yield
(252 mg, 1.4 mmol). Mp ¼ 61–628C. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.35 (d, J ¼ 2.4 Hz, 1H), 7.26 (dd, J ¼ 8.4, 2.5 Hz, 1H), 6.83 (d,
J ¼ 8.4 Hz, 1H), 6.71 (br s, 1H), 2.64 (q, J ¼ 7.5 Hz, 2H), 1.23 (t,
J ¼ 7.5 Hz, 3H). 13C-NMR (75 MHz, CDCl3): d ¼ 156.11, 133.40,
133.06, 131.36, 116.79, 112.84, 111.94, 22.75, 13.35.
Experimental
General methods
All dry solvents were commercially available. NMR spectra were
recorded on a Bruker DPX300 spectrometer. Coupling constants
(J) are reported in hertz, and chemical shifts are reported in parts
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J ¼ 2.3 Hz, 1H), 7.03 (dd, J ¼ 8.2, 2.1 Hz, 1H), 6.62 (d, J ¼ 8.2 Hz,
1H), 4.86 (br s, 1H), 3.36 (s, 1H), 3.15 (hept, J ¼ 6.9 Hz, 1H), 1.22 (d,
J ¼ 6.9 Hz, 6H). 13C-NMR (75 MHz, CDCl3): d ¼ 151.60, 135.55,
129.62, 129.58, 119.83, 116.07, 26.97, 22.38.
2-Iso-propyl-4-thiocyanatophenol (3b)
The title compound was prepared in 81% yield (785 mg,
4.07 mmol) as orange oil from 2-iso-propylphenol (5 mmol,
685 mL) following the general procedure. 1H-NMR (300 MHz,
CDCl3): d ¼ 7.34 (d, J ¼ 2.4 Hz, 1H), 7.24 (dd, J ¼ 8.4, 24 Hz, 1H),
6.76 (d, J ¼ 8.4 Hz, 1H), 5.72 (br s, 1H), 3.19 (hept, J ¼ 6.8 Hz, 1H),
1.21 (d, J ¼ 6.9 Hz, 6H). 13C-NMR (75 MHz, CDCl3): d ¼ 155.18,
137.34, 131.17, 131.01, 117.07, 113.27, 112.37, 27.17, 22.19.
2-Methyl-4-{[4-methyl-2-(4-trifluoromethylphenyl)thiazol-
5-yl]methylthio}phenol (5a)
The title compound was prepared as following: To a solution of
4-mercapto-2-methyl-phenol (280 mg, 2 mmol) in dry CH3CN
(40 mL) was added Cs2CO3 (706 mg, 2 mmol). To this mixture
was added dropwise a solution of 5-chloromethyl-4-methyl-2-(4-
trifluoromethylphenyl)thiazole (518 mg, 1.78 mmol) in dry
CH3CN (10 mL). The mixture was stirred for 4 h at ambient
temperature under argon, then diluted with water and extracted
(ethyl acetate, 3 ꢀ 100 mL). The organic layers were combined,
dried (MgSO4) and concentrated. The residue was purified by
column chromatography on silica gel with hexane/ethyl acetate
(3:1) to give 5a as a light yellow solid in 96% yield (676 mg,
1.71 mmol). Mp ¼ 126–1278C. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.93 (d, J ¼ 8.1 Hz, 2H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.19 (d,
J ¼ 1.6 Hz, 1H), 6.91 (dd, J ¼ 8.2, 2.1 Hz, 1H), 6.54 (d, J ¼ 8.2 Hz,
1H), 4.03 (s, 2H), 2.17 (s, 3H), 2.03 (s, 3H). 13C-NMR (75 MHz,
CDCl3): d ¼ 163.78, 155.25, 151.11, 137.10, 136.22 (distorted q,
J ¼ 1.2 Hz), 133.21, 131.49 (q, J ¼ 32.7 Hz), 131.48, 126.45,
125.96 (q, J ¼ 3.8 Hz), 125.54, 123.81 (q, J ¼ 272.3 Hz), 123.16,
115.13, 32.79, 15.76, 14.21.
2-Tert-butyl-4-thiocyanatophenol (3c)
The title compound was prepared in 68% yield (280 mg,
1.35 mmol) as a light yellow solid from 2-tert-butylphenol
(301 mg, 2 mmol) following the general procedure. Mp ¼ 77–
788C. 1H-NMR (300 MHz, CDCl3): d ¼ 7.42 (d, J ¼ 2.4 Hz, 1H), 7.26
(dd, J ¼ 8.3, 2.4 Hz, 1H), 6.73 (d, J ¼ 8.3 Hz, 1H), 6.00 (br s, 1H),
1.38 (s, 9H). 13C-NMR (75 MHz, CDCl3): d ¼ 156.89, 138.86,
131.74, 131.55, 118.25, 112.69, 112.40, 34.90, 29.10.
2-Cyclopentyl-4-thiocyanatophenol (3d)
The title compound was prepared in 83% yield (230 mg,
1.05 mmol) as a yellow oil from 2-cyclopentylphenol (353 mg,
2 mmol) following the general procedure. 1H-NMR (300 MHz,
CDCl3): d ¼ 7.40 (d, J ¼ 2.4 Hz, 1H), 7.26 (dd, J ¼ 8.4, 2.4 Hz,
1H), 6.82 (d, J ¼ 8.4 Hz, 1H), 6.44 (br s, 1H), 3.32–3.18 (m, 1H),
2.19–1.96 (m, 2H), 1.92–1.46 (m, 6H). 13C-NMR (75 MHz, CDCl3):
d ¼ 156.11, 135.13, 131.50, 131.13, 116.93, 112.76, 112.24, 38.99,
32.51, 25.11.
2-Ethyl-4-{[4-methyl-2-(4-trifluoromethylphenyl)thiazol-5-
yl]methylthio}phenol (5b)
2-Cyclohexyl-4-thiocyanatophenol (3e)
The title compound was prepared in 66% yield (290 mg,
0.71 mmol) as a yellow solid from 4a (185 mg, 1.2 mmol)
and 5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thiazole
(311 mg, 1.07 mmol) following the general procedure. Mp ¼
132–1338C. 1H-NMR (300 MHz, CDCl3): d ¼ 7.93 (d, J ¼ 8.1 Hz,
2H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.18 (d, J ¼ 2.2 Hz, 1H), 6.93 (dd,
J ¼ 8.2, 2.3 Hz, 1H), 6.54 (d, J ¼ 8.2 Hz, 1H), 4.03 (s, 2H), 2.57 (q,
J ¼ 7.5 Hz, 2H), 2.01 (s, 3H), 1.15 (t, J ¼ 7.5 Hz, 3H). 13C-NMR
(75 MHz, CDCl3): d ¼ 163.75, 154.82, 151.16, 136.24 (distorted
q, J ¼ 0.9 Hz), 135.72, 133.28, 131.61, 131.48 (q, J ¼ 32.7 Hz),
131.46, 126.44, 125.95 (q, J ¼ 3.8 Hz), 123.81 (q, J ¼ 272.2 Hz),
123.24, 115.38, 32.86, 22.91, 14.16, 13.85.
The title compound was prepared in 61% yield (284 mg,
1.22 mmol) as a yellow oil from 2-cyclohexylphenol (358 mg,
2 mmol) following the general procedure. 1H-NMR (300 MHz,
CDCl3): d ¼ 7.44 (d, J ¼ 2.4 Hz, 1H), 7.31 (dd, J ¼ 8.4, 2.4 Hz,
1H), 6.88 (d, J ¼ 8.4 Hz, 1H), 6.40 (br s, 1H), 3.05–2.87 (m, 1H),
2.10–1.74 (m, 5H), 1.68–1.08 (m, 5H). 13C-NMR (75 MHz, CDCl3):
d ¼ 155.41, 136.66, 131.48, 131.06, 117.05, 112.73, 112.59, 37.04,
32.69, 26.68, 26.03.
2-Ethyl-4-mercaptophenol (4a)
The title compound was prepared as following: A solution of
2-ethyl-4-thiocyanatophenol (3a) (963 mg, 5.38 mmol) in anhy-
drous THF (100 mL) was added cautiously to a mixture of LiAlH4
(215 mg, 5.5 mmol) and anhydrous THF (50 mL) at 08C. The
reaction mixture was stirred at ambient temperature for 4 h
under argon. Adding moist THF, water, and 1.0 M HCl destroyed
the unreacted LiAlH4. The mixture was extracted (ethyl acetate,
3 ꢀ 100 mL). The organic layers were combined, washed with
brine, dried (MgSO4), and concentrated. The residue was purified
by column chromatography using hexane/ethyl acetate (4:1) as
eluent to give 4a as a light colorless oil in 71% yield (585 mg,
3.8 mmol). 1H-NMR (300 MHz, CDCl3): d ¼ 7.28 (d, J ¼ 2.1 Hz,
1H), 7.22 (dd, J ¼ 8.2, 2.3 Hz, 1H), 6.70 (d, J ¼ 8.2 Hz, 1H), 4.86
(s, 1H), 2.61 (q, J ¼ 7.5 Hz, 2H), 1.22 (t, J ¼ 7.5 Hz, 3H). 13C-NMR
(75 MHz, CDCl3): d ¼ 153.78, 132.46, 130.79, 130.24, 128.62,
115.77, 22.77, 13.64.
2-Iso-propyl-4-{[4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl]methylthio}phenol (5c)
The title compound was prepared in 80% yield (576 mg,
1.36 mmol) as a light yellow solid from 4b (321 mg, 1.9 mmol)
and 5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thiazole
(495 mg, 1.7 mmol) following the general procedure. Mp ¼ 135–
1368C. 1H-NMR (300 MHz, CDCl3): d ¼ 7.93 (d, J ¼ 8.1 Hz, 2H),
7.61 (d, J ¼ 8.3 Hz, 2H), 7.19 (d, J ¼ 2.1 Hz, 1H), 6.93 (dd, J ¼ 8.2,
2.2 Hz, 1H), 6.53 (d, J ¼ 8.2 Hz, 1H), 4.03 (s, 2H), 3.17 (hept,
J ¼ 6.9 Hz, 1H), 1.98 (s, 3H), 1.15 (d, J ¼ 6.9 Hz, 6H). 13C-NMR
(75 MHz, CDCl3): d ¼ 163.75, 154.28, 151.24, 136.28 (distorted q,
J ¼ 1.2 Hz), 136.04, 133.28, 133.16, 131.48 (q, J ¼ 32.7 Hz),
131.45, 126.44, 125.95 (q, J ¼ 3.8 Hz), 123.83 (q, J ¼ 272.3 Hz),
123.23, 115.55, 32.91, 26.90, 22.35, 14.10.
2-Iso-propyl-4-mercaptophenol (4b)
Ethyl 2-(2-tert-butyl-4-thiocyanatophenoxy)acetate (6a)
The title compound was prepared as following: to a solution of
2-tert-butyl-4-thiocyanatophenol (3c) (280 mg, 1.35 mmol) in dry
Title compound was prepared in 88% yield (578 mg, 3.44 mmol)
as a light yellow oil from 3b (753 mg, 3.9 mmol) following the
general procedure. 1H-NMR (300 MHz, CDCl3): d ¼ 7.17 (d,
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
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CH3CN (30 mL) was added Cs2CO3 (483 mg, 1.48 mmol). To this
mixture was added dropwise a solution of ethyl 2-bromoacetate
(165 mL, 1.48 mmol) in dry CH3CN (10 mL). The mixture was
stirred for 3 h at ambient temperature, then diluted with water
and extracted (ethyl acetate, 3 ꢀ 100 mL), dried (MgSO4), and
concentrated. The residue was purified by column chromatog-
raphy on silica gel with hexane/ethyl acetate (4:1) to give 6a as a
colorless oil in 79% yield (311 mg, 1.06 mmol). 1H-NMR
(300 MHz, CDCl3): d ¼ 7.44 (d, J ¼ 2.5 Hz, 1H), 7.35 (dd,
J ¼ 8.5, 2.5 Hz, 1H), 6.72 (d, J ¼ 8.6 Hz, 1H), 4.63 (s, 2H), 4.23
(q, J ¼ 7.1 Hz, 2H), 1.38 (s, 9H), 1.26 (t, J ¼ 7.1 Hz, 3H). 13C-NMR
(75 MHz, CDCl3): d ¼ 167.91, 157.98, 141.04, 131.03, 130.93,
114.50, 113.03, 111.51, 65.04, 61.36, 35.18, 29.26, 14.00.
(m, 2H), 2.13–1.95 (m, 2H), 1.88–1.34 (m, 6H), 1.26 (t, J ¼ 7.1 Hz,
3H). 13C-NMR (75 MHz, CDCl3): d ¼ 168.71, 154.54, 136.21, 129.86,
128.84, 120.99, 112.03, 65.65, 61.10, 39.04, 32.71, 25.31, 14.01.
Ethyl 2-(2-cyclohexyl-4-mercaptophenoxy)acetate (7c)
The title compound was prepared in 85% yield (200 mg,
0.68 mmol) as a light yellow oil from 6c (257 mg, 0.80 mmol)
following the general procedure. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.16 (d, J ¼ 2.3 Hz, 1H), 7.05 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.57
(d, J ¼ 8.5 Hz, 1H), 4.57 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.34 (s, 1H),
3.05–2.89 (m, 1H), 1.91–1.66 (m, 5H), 1.50–1.18 (m, 8H). 13C-NMR
(75 MHz, CDCl3): d ¼ 168.73, 153.87, 137.68, 129.75, 128.77,
121.10, 112.01, 65.64, 61.09, 36.89, 32.84, 26.86, 26.21, 14.02.
Ethyl 2-(2-cyclopentyl-4-thiocyanatophenoxy)acetate (6b)
The title compound was prepared in 69% yield (220 mg,
0.72 mmol) as a white solid from 3d (230 mg, 1.05 mmol) and
ethyl-2-bromoacetate (129 mL, 1.16 mmol) following the general
procedure. Mp ¼ 45–468C. 1H-NMR (300 MHz, CDCl3): d ¼ 7.38
(d, J ¼ 2.4 Hz, 1H), 7.31 (dd, J ¼ 8.6, 2.5 Hz, 1H), 6.70 (d,
J ¼ 8.6 Hz, 1H), 4.62 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.43–3.25
(m, 1H), 2.11–1.98 (m, 2H), 1.86–1.45 (m, 6H), 1.26 (t, J ¼ 7.1 Hz,
3H). 13C-NMR (75 MHz, CDCl3): d ¼ 168.11, 157.18, 137.76,
130.72, 130.42, 114.68, 112.42, 111.44, 65.30, 61.31, 39.26,
32.48, 25.20, 13.99.
Ethyl 2-{2-methyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}propanoate (8a)
The title compound was prepared as following: To a solution of 2-
methyl-4-{[4-methyl-2-(4-trifluoromethylphenyl)thiazol-5-yl]me-
thylthio}phenol (5a) (120 mg, 0.3 mmol) in dry CH3CN (10 mL)
was added Cs2CO3 (147 mg, 0.45 mmol). To this mixture was
added dropwise a solution of ethyl 2-bromopropanoate (55 mL,
0.39 mmol) in dry CH3CN (3 mL). The mixture was stirred over
night at ambient temperature under argon, then diluted with
water and extracted (ethyl acetate, 3 ꢀ 100 mL). The organic
layers were combined, dried (MgSO4), and concentrated. The
residue was purified by column chromatography on silica gel
with hexane/ethyl acetate (4:1) to give 8a as a colorless oil in 97%
yield (143 mg, 0.29 mmol). 1H-NMR (300 MHz, CDCl3): d ¼ 7.95
(d, J ¼ 8.1 Hz, 2H), 7.63 (d, J ¼ 8.2 Hz, 2H), 7.18 (d, J ¼ 1.7 Hz,
1H), 7.07 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.55 (d, J ¼ 8.5 Hz, 1H), 4.68 (q,
J ¼ 6.8 Hz, 1H), 4.15 (q, J ¼ 7.1 Hz, 2H), 4.08 (s, 2H), 2.20 (s, 3H),
2.18 (s, 3H), 1.59 (d, J ¼ 6.8 Hz, 3H), 1.20 (t, J ¼ 7.1 Hz, 3H). 13C-
NMR (75 MHz, CDCl3): d ¼ 171.90, 163.04, 156.22, 151.24, 136.72
(distorted q, J ¼ 1.2 Hz), 136.08, 132.06, 131.24 (q, J ¼ 32.6 Hz),
130.74, 128.57, 126.37, 125.82 (q, J ¼ 3.8 Hz), 124.94, 123.91 (q,
J ¼ 272.2 Hz), 112.21, 72.82, 61.22, 32.44, 18.51, 16.15, 14.76,
14.05.
Ethyl 2-(2-cyclohexyl-4-thiocyanatophenoxy)acetate (6c)
The title compound was prepared in 66% yield (257 mg,
0.8 mmol) as a light yellow oil from 3e (285 mg, 1.22 mmol)
and ethyl 2-bromoacetate (149 mL, 1.34 mmol) following the
general procedure. 1H-NMR (300 MHz, CDCl3): d ¼ 7.35 (d,
J ¼ 2.4 Hz, 1H), 7.30 (dd, J ¼ 8.5, 2.5 Hz, 1H), 6.70 (d,
J ¼ 8.6 Hz, 1H), 4.62 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.07–2.91
(m, 1H), 1.93–1.66 (m, 5H), 1.52–1.15 (m, 8H). 13C-NMR (75 MHz,
CDCl3): d ¼ 168.13, 156.53, 139.16, 130.58, 130.30, 114.83,
112.45, 111.42, 65.28, 61.30, 37.06, 32.61, 26.71, 26.06, 13.98.
Ethyl 2-(2-tert-butyl-4-mercaptophenoxy)acetate (7a)
The title compound was prepared as following: To a stirred
solution of ethyl 2-(2-tert-butyl-4-thiocyanatophenoxy)acetate
(6a) (223 mg, 0.76 mmol) in ethanol (20 mL) at 08C 1,4-dithioery-
thritol (154 mg, 1 mmol) and NaBH4 (38 mg, 1 mmol) were
added in portions. The reaction was stirred for 20 min.
Adding 1 M HCl destroyed the unreacted NaBH4. The mixture
was diluted with water and extracted (diethyl ether,
3 ꢀ 100 mL), dried (MgSO4), and concentrated. The residue
was purified by column chromatography on silica gel with
hexane/ethyl acetate (4:1) to give 7a as a yellow solid in 43%
yield (88 mg, 0.33 mmol). Mp ¼ 52–538C. 1H-NMR (300 MHz,
CDCl3): d ¼ 7.25 (d, J ¼ 2.3 Hz, 1H), 7.11 (dd, J ¼ 8.4, 2.3 Hz,
1H), 6.59 (d, J ¼ 8.4 Hz, 1H), 4.58 (s, 2H), 4.24 (q, J ¼ 7.1 Hz,
2H), 3.35 (s, 1H), 1.38 (s, 9H), 1.28 (t, J ¼ 7.1 Hz, 3H). 13C-NMR
(75 MHz, CDCl3): d ¼ 168.60, 155.38, 139.56, 129.99, 129.40,
120.61, 112.46, 65.26, 61.21, 34.89, 29.54, 14.08.
Ethyl 2-methyl-2-{2-methyl-4-[(4-methyl-2-
(4-trifluoromethylphenyl)-thiazol-5-yl)
methylthio]phenoxy}propanoate (8b)
The title compound was prepared in 83% yield (166 mg,
0.33 mmol) as a yellow oil 5a (160 mg, 0.4 mmol) and ethyl 2-
bromo-2-methylpropanoate (73 mL, 0.52 mmol) following the
procedure described for 8a. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.94 (d, J ¼ 8.1 Hz, 2H), 7.63 (d, J ¼ 8.2 Hz, 2H), 7.16 (d,
J ¼ 1.8 Hz, 1H), 7.03 (dd, J ¼ 8.3, 2.2 Hz, 1H), 6.53 (d,
J ¼ 8.5 Hz, 1H), 4.19 (q, J ¼ 7.1 Hz, 2H), 4.08 (s, 2H), 2.18 (s,
3H), 2.15 (s, 3H), 1.56 (s, 6H), 1.20 (t, J ¼ 7.1 Hz, 3H). 13C-NMR
(75 MHz, CDCl3): d ¼ 174.18, 163.01, 154.18, 151.34, 136.80 (dis-
torted q, J ¼ 1.3 Hz), 136.02, 131.45, 130.79 (q, J ¼ 32.7 Hz),
130.66, 130.45, 126.34, 125.82 (q, J ¼ 3.9 Hz), 125.44, 123.92
(q, J ¼ 272.2 Hz), 116.68, 79.22, 61.46, 32.38, 25.36, 16.58,
14.80, 14.03.
Ethyl 2-(2-cyclopentyl-4-mercaptophenoxy)acetate (7b)
The title compound was prepared in 85% yield (166 mg,
0.59 mmol) as a colorless liquid from 6b (215 mg, 0.70 mmol)
following the general procedure. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.18 (d, J ¼ 2.3 Hz, 1H), 7.06 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.58
(d, J ¼ 8.5 Hz, 1H), 4.57 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.40–3.26
Ethyl 2-{2-ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetate (8c)
The title compound was prepared in 93% yield (406 mg,
0.82 mmol) as a white solid from 5b (360 mg, 0.88 mmol) and
ethyl 2-bromoacetate (128 mL, 1.15 mmol) following the
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
procedure described for 8a. Mp ¼ 80–81 C. 1H-NMR (300 MHz,
CDCl3): d ¼ 7.94 (d, J ¼ 8.1 Hz, 2H), 7.63 (d, J ¼ 8.2 Hz, 2H), 7.16
(d, J ¼ 2.1 Hz, 1H), 7.13 (dd, J ¼ 8.3, 2.3 Hz, 1H), 6.58 (d,
J ¼ 8.3 Hz, 1H), 4.59 (s, 2H), 4.21 (q, J ¼ 7.1 Hz, 2H), 4.08 (s,
2H), 2.62 (q, J ¼ 7.5 Hz, 2H), 2.14 (s, 3H), 1.25 (t, J ¼ 7.1 Hz,
3H), 1.14 (t, J ¼ 7.5 Hz, 3H). 13C-NMR (75 MHz, CDCl3):
d ¼ 168.62, 163.01, 156.02, 151.36, 136.76 (distorted q,
J ¼ 1.2 Hz), 134.78, 134.20, 132.26, 131.18 (q, J ¼ 32.6 Hz),
130.68, 126.31, 125.79 (q, J ¼ 3.8 Hz), 125.20, 123.90 (q,
J ¼ 272.8 Hz), 111.52, 65.40, 61.26, 32.47, 23.07, 14.70, 14.06,
13.85.
Ethyl 2-{2-iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}propanoate (8g)
The title compound was prepared in 94% yield (170 mg,
0.32 mmol) as a yellow oil from 5c (144 mg, 0.34 mmol) and
ethyl 2-bromopropanoate (57 mL, 0.44 mmol) following the pro-
1
cedure described for 8a. H-NMR (300 MHz, CDCl3): d ¼ 7.94 (d,
J ¼ 8.1 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.15 (d, J ¼ 2.3 Hz, 1H),
7.10 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.56 (d, J ¼ 8.4 Hz, 1H), 4.71 (q,
J ¼ 6.8 Hz, 1H), 4.13 (q, J ¼ 7.1 Hz, 2H), 4.05 (s, 2H), 3.31 (hept,
J ¼ 6.9 Hz, 1H), 2.09 (s, 3H), 1.59 (d, J ¼ 6.8 Hz, 3H), 1.25–1.07 (m,
9H). 13C-NMR (75 MHz, CDCl3): d ¼ 171.83, 162.97, 155.20,
151.40, 138.43, 136.75 (distorted q, J ¼ 1.3 Hz), 132.45, 132.27,
131.13 (q, J ¼ 32.6 Hz), 130.75, 126.27, 125.75 (q, J ¼ 3.8 Hz),
124.76, 123.88 (q, J ¼ 272.1 Hz), 111.98, 72.44, 61.14, 32.55,
26.78, 22.33, 18.43, 14.59, 14.00.
Ethyl 2-{2-ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}propanoate (8d)
The title compound was prepared in 93% yield (142 mg,
0.28 mmol) as a yellow oil from 5b (125 mg, 0.3 mmol) and
ethyl 2-bromopropanoate (51 mL, 0.39 mmol) following the pro-
Ethyl 2-{2-iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
1
cedure described for 8a. H-NMR (300 MHz, CDCl3): d ¼ 7.94 (d,
thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoate (8h)
The title compound was prepared in 53% yield (96 mg,
0.18 mmol) as a yellow oil from 5c (144 mg, 0.34 mmol) and
ethyl 2-bromo-2-methylpropanoate (66 mL, 0.44 mmol) following
the procedure described for 8a. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.93 (d, J ¼ 8.1 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.12 (d,
J ¼ 2.3 Hz, 1H), 7.06 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.51 (d,
J ¼ 8.4 Hz, 1H), 4.17 (q, J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 3.25 (hept,
J ¼ 6.9 Hz, 1H), 2.09 (s, 3H), 1.57 (s, 6H), 1.17 (t, J ¼ 7.1 Hz, 3H),
1.09 (d, J ¼ 6.9 Hz, 6H). 13C-NMR (75 MHz, CDCl3): d ¼ 174.19,
163.01, 153.26, 151.41, 140.18, 136.75 (distorted q, J ¼ 1.3 Hz),
132.49, 131.70, 131.20 (q, J ¼ 32.6 Hz), 130.76, 126.31, 125.80 (q,
J ¼ 3.8 Hz), 125.06, 123.91 (q, J ¼ 272.0 Hz), 116.24, 78.95, 61.40,
32.52, 26.81, 25.33, 22.44, 14.59, 13.95.
J ¼ 8.1 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.14 (d, J ¼ 2.3 Hz, 1H),
7.09 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.55 (d, J ¼ 8.4 Hz, 1H), 4.70 (q,
J ¼ 6.8 Hz, 1H), 4.14 (q, J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 2.61 (q,
J ¼ 7.5 Hz, 2H), 2.13 (s, 3H), 1.59 (d, J ¼ 6.8 Hz, 3H), 1.18 (t,
J ¼ 7.2 Hz, 3H), 1.13 (t, J ¼ 7.6 Hz, 3H). 13C-NMR (75 MHz,
CDCl3): d ¼ 171.82, 162.92, 155.76, 151.34, 136.76 (distorted q,
J ¼ 1.3 Hz), 134.81, 134.25, 132.24, 131.12 (q, J ¼ 32.6 Hz),
130.70, 126.28, 125.75 (q, J ¼ 3.7 Hz), 123.89 (q, J ¼ 272.2 Hz),
124.82, 111.97, 72.48, 61.13, 32.46, 23.17, 18.42, 14.66, 13.99,
13.83.
Ethyl 2-{2-ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoate (8e)
The title compound was prepared in 83% yield (131 mg,
0.25 mmol) as a yellow oil from 5b (125 mg, 0.3 mmol) and
ethyl 2-bromo-2-methylpropanoate (58 mL, 0.39 mmol) following
the procedure described for 8a. 1H-NMR (300 MHz, CDCl3):
d ¼ 7.93 (d, J ¼ 8.1 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.12 (d,
J ¼ 2.3 Hz, 1H), 7.05 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.52 (d,
J ¼ 8.4 Hz, 1H), 4.17 (q, J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 2.55 (q,
J ¼ 7.5 Hz, 2H), 2.12 (s, 3H), 1.57 (s, 6H), 1.17 (t, J ¼ 7.1 Hz,
3H), 1.10 (t, J ¼ 7.5 Hz, 3H). 13C-NMR (75 MHz, CDCl3): d ¼
174.12, 162.90, 153.79, 151.35, 136.76 (distorted q, J ¼ 1.3 Hz),
135.96, 134.81, 131.65, 131.12 (q, J ¼ 32.6 Hz), 130.68, 126.26,
125.74 (q, J ¼ 3.8 Hz), 125.11, 123.88 (q, J ¼ 272.3 Hz), 116.20,
78.91, 61.36, 32.39, 25.27, 23.40, 14.64, 13.92.
Ethyl 2-{2-tert-butyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetate (8i)
The title compound was prepared as following: To a solution of
ethyl 2-(2-tert-butyl-4-mercaptophenoxy)acetate (7a) (88 mg,
0.33 mmol) in dry CH3CN (10 mL) was added Cs2CO3 (117 mg,
0.33 mmol). To this mixture was added dropwise a solution of 5-
chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thiazole
(98 mg, 0.34 mmol) in dry CH3CN (5 mL). The mixture was
stirred for 4 h at ambient temperature under argon, then diluted
with water and extracted (ethyl acetate, 3 ꢀ 100 mL), dried
(MgSO4), and concentrated. The residue was purified by column
chromatography on silica gel with hexane/ethyl acetate (4:1) to
give 8i as a colorless oil in 94% yield (163 mg, 0.31 mmol). 1H-
NMR (300 MHz, CDCl3): d ¼ 7.94 (d, J ¼ 8.1 Hz, 2H), 7.63 (d,
J ¼ 8.2 Hz, 2H), 7.21 (d, J ¼ 2.2 Hz, 1H), 7.18 (dd, J ¼ 8.2,
2.3 Hz, 1H), 6.60 (d, J ¼ 8.3 Hz, 1H), 4.60 (s, 2H), 4.23 (q,
J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 2.07 (s, 3H), 1.31 (s, 9H), 1.26 (t,
J ¼ 7.1 Hz, 3H). 13C-NMR (75 MHz, CDCl3): d ¼ 168.41, 163.08,
156.98, 151.53, 139.44, 136.77 (distorted q, J ¼ 1.3 Hz), 133.27,
132.80, 130.74, 131.20 (q, J ¼ 32.6 Hz), 126.32, 125.81 (q,
J ¼ 3.8 Hz), 124.66, 123.91 (q, J ¼ 272.1 Hz), 112.16, 65.14,
61.28, 34.86, 32.60, 29.42, 14.60, 14.07.
Ethyl 2-{2-iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetate (8f)
The title compound was prepared in 75% yield (130 mg,
0.26 mmol) as a yellow oil from 5c (144 mg, 0.34 mmol) and
ethyl 2-bromoacetate (49 mL, 0.44 mmol) following the pro-
cedure described for 8a. 1H-NMR (300 MHz, CDCl3): d ¼ 7.93
(d, J ¼ 8.1 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 2.2 Hz,
1H), 7.13 (d, J ¼ 8.3, 2.3 Hz, 1H), 6.58 (d, J ¼ 8.3 Hz, 1H), 4.59 (s,
2H), 4.21 (q, J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 3.31 (hept, J ¼ 6.9 Hz,
1H), 2.09 (s, 3H), 1.24 (t, J ¼ 7.1 Hz, 3H), 1.13 (d, J ¼ 6.9 Hz, 6H).
13C-NMR (75 MHz, CDCl3): d ¼ 168.56, 162.97, 155.45, 151.39,
138.43, 136.72 (distorted q, J ¼ 1.2 Hz), 132.42, 132.26, 131.12
(q, J ¼ 32.6 Hz), 130.70, 126.26, 125.74 (q, J ¼ 3.8 Hz), 125.14,
123.87 (q, J ¼ 271.9 Hz), 111.61, 65.38, 61.20, 32.49, 26.73, 22.31,
14.57, 14.01.
Ethyl 2-{2-cyclopentyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetate (8j)
The title compound was prepared in 58% yield (114 mg,
0.21 mmol) as a yellow oil from 7b (100 mg, 0.36 mmol)
and 5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thiazole
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Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
621
(107 mg, 0.37 mmol) following the procedure described for 8i.
1H-NMR (300 MHz, CDCl3): d ¼ 7.94 (d, J ¼ 8.1 Hz, 2H), 7.63 (d,
J ¼ 8.2 Hz, 2H), 7.18–7.11 (m, 2H), 6.59 (d, J ¼ 8.2 Hz, 1H), 4.59 (s,
2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 4.06 (s, 2H), 3.40–3.22 (m, 1H), 2.11 (s,
3H), 2.06–1.87 (m, 2H), 1.78–1.37 (m, 6H), 1.25 (t, J ¼ 7.1 Hz, 3H).
13C-NMR (75 MHz, CDCl3): d ¼ 168.62, 163.02, 156.15, 151.45,
136.77 (distorted q, J ¼ 1.3 Hz), 136.23, 133.03, 132.25, 131.18
(q, J ¼ 32.6 Hz), 130.74, 126.30, 125.78 (q, J ¼ 3.6 Hz), 125.04,
123.90 (q, J ¼ 272.3 Hz), 111.67, 65.53, 61.25, 38.91, 32.73, 32.55
25.31, 14.66, 14.07.
with 50 mL H2O, acidified with 0.1 M HCl, extracted (diethyl
ether, 3 ꢀ 50 mL), dried (MgSO4), and concentrated. The residue
was recrystallized from ethyl acetate/hexane to give 2b as a white
solid in 45% yield (72 mg, 0.15 mmol). Mp ¼ 125–1268C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 8.02 (d, J ¼ 8.2 Hz, 2H), 7.80 (d,
J ¼ 8.3 Hz, 2H), 7.16 (d, J ¼ 1.8 Hz, 1H), 7.02 (dd, J ¼ 8.7,
2.0 Hz, 1H), 6.79 (d, J ¼ 8.6 Hz, 1H), 4.29 (s, 2H), 2.17 (s, 3H),
2.07 (s, 3H), 1.40 (s, 6H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 173.84,
161.64, 156.36, 150.98, 136.53 (distorted q, J ¼ 1.3 Hz), 134.21,
131.61, 130.81, 129.58 (q, J ¼ 32.0 Hz), 126.84, 126.33, 126.07 (q,
J ¼ 3.8 Hz), 124.00 (q, J ¼ 272.2 Hz), 123.18, 112.45, 73.66, 30.77,
18.75, 15.96, 14.62. MS (ESI) m/z 480.0 [M – H]–, HRMS calcd.
for C23H22F3NO3S2 [M]þ: 481.0993; found: 481.0988.
Ethyl 2-{2-cyclohexyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetate (8k)
The title compound was prepared in 79% yield (151 mg,
0.27 mmol) as a light yellow oil from 7c (100 mg, 0.34 mmol)
2-{2-Ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)thiazol-
5-yl)methylthio]phenoxy}acetic acid (2c)
and
5-chloromethyl-4-methyl-2-(4-trifluoromethylphenyl)thia-
zole (101 mg, 0.35 mmol) following the procedure described
for 8i. 1H-NMR (300 MHz, CDCl3): d ¼ 7.94 (d, J ¼ 8.1 Hz, 2H),
7.62 (d, J ¼ 8.2 Hz, 2H), 7.14 (dd, J ¼ 8.3, 2.3 Hz, 1H), 7.11 (d,
J ¼ 2.2 Hz, 1H), 6.58 (d, J ¼ 8.3 Hz, 1H), 4.58 (s, 2H), 4.21 (q,
J ¼ 7.1 Hz, 2H), 4.05 (s, 2H), 3.01–2.85 (m, 1H), 2.08 (s, 3H),
1.87–1.58 (m, 5H), 1.45–0.99 (m, 8H). 13C-NMR (75 MHz, CDCl3):
d ¼ 168.62, 162.97, 155.53, 151.50, 137.58, 136.76 (d, J ¼ 1.2 Hz),
133.15, 132.34, 131.14 (q, J ¼ 32.6 Hz), 130.67, 126.28, 125.74 (q,
J ¼ 3.7 Hz), 124.99, 123.88 (q, J ¼ 272.3 Hz), 111.65, 65.48, 61.22,
36.79, 32.84, 32.53, 26.83, 26.14, 14.59, 14.05.
The title compound was prepared in 43% yield (86 mg,
0.18 mmol) as a white solid from 8c (206 mg, 0.42 mmol) follow-
ing the procedure described for 2a. Mp ¼ 141–1428C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 8.00 (d, J ¼ 8.1 Hz, 2H), 7.78 (d,
J ¼ 8.4 Hz, 2H), 7.15–7.04 (m, 2H), 6.69 (d, J ¼ 9.0 Hz, 1H), 4.28
(s, 2H), 4.26 (s, 2H), 2.51 (q, J ¼ 7.4 Hz, 2H), 2.13 (s, 3H), 1.05 (t,
J ¼ 7.5 Hz, 3H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 172.85, 161.65,
156.65, 151.03, 136.53 (distorted q, J ¼ 1.2 Hz), 132.95, 132.54,
131.74, 131.16, 129.58 (q, J ¼ 32.0 Hz), 126.28, 122.86, 126.09 (q,
J ¼ 3.8 Hz), 123.99 (q, J ¼ 272.1 Hz), 112.24, 67.33, 30.95, 22.63,
14.55, 13.90. MS (ESI) m/z 466.1 [M
–
H]ꢁ, HRMS calcd.
for C22H20F3NO3S2 [M]þ: 467.0837; found: 467.0858.
2-{2-Methyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}propanoic acid (2a)
The title compound was prepared as following: to a stirred
solution of ethyl 2-{2-methyl-4-[(4-methyl-2-(4-trifluoromethyl-
2-{2-Ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)thiazol-
5-yl)methylthio]phenoxy}propanoic acid (2d)
phenyl)thiazol-5-yl)methylthio]phenoxy}propanoate
(8a)
The title compound was prepared in 50% yield (69 mg, 0.14 mmol)
as a white solid from 8d (142 mg, 0.28 mmol) following the pro-
cedure described for 2a. Mp ¼ 127–1288C. 1H-NMR (300 MHz,
DMSO-d6): d ¼ 12.95 (br s, 1H), 8.02 (d, J ¼ 8.1 Hz, 2H), 7.79 (d,
J ¼ 8.3 Hz, 2H), 7.17 (dd, J ¼ 8.4, 2.3 Hz, 1H), 7.12 (d, J ¼ 2.3 Hz,
1H), 6.73 (d, J ¼ 8.5 Hz, 1H), 4.81 (q, J ¼ 6.7 Hz, 1H), 4.30 (s, 2H),
2.52 (q, J ¼ 7.3 Hz, 2H), 2.13 (s, 3H), 1.49 (d, J ¼ 6.7 Hz, 3H), 1.06 (t,
J ¼ 7.5 Hz, 3H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 172.97, 161.73,
155.23, 151.18, 136.55 (distorted, J ¼ 1.2 Hz), 133.37, 133.05,
131.52, 131.22, 129.62 (q, J ¼ 32.0 Hz), 126.30, 126.08 (q, J ¼
3.7 Hz), 124.22, 124.00 (q, J ¼ 272.0 Hz), 112.42, 71.67, 30.77,
22.70, 18.26, 14.50, 13.85. MS (ESI) m/z 480.1 [M – H]ꢁ, HRMS calcd.
for C23H22F3NO3S2 [M]þ: 481.0993; found: 481.0980.
(170 mg, 0.34 mmol) in THF (10 mL) and H2O (5 mL) at 08C
was added slowly 215 mL of 2.0 M LiOH. The reaction mixture
was stirred until TLC indicated completion of the reaction. The
mixture was diluted with 50 mL H2O, acidified with 0.1 M HCl,
extracted (diethyl ether, 3 ꢀ 50 mL), dried (MgSO4), and concen-
trated. The residue was recrystallized from ethyl acetate/hexane
to give 2a as a white solid in 50% yield (80 mg, 0.17 mmol).
Mp ¼ 78–798C. 1H-NMR (300 MHz, DMSO-d6): d ¼ 12.97 (br s,
1H), 8.03 (d, J ¼ 8.1 Hz, 2H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.22 (d,
J ¼ 1.6 Hz, 1H), 7.15 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.72 (d,
J ¼ 8.5 Hz, 1H), 4.80 (q, J ¼ 6.7 Hz, 1H), 4.33 (s, 2H), 2.19 (s,
3H), 2.13 (s, 3H), 1.50 (d, J ¼ 6.7 Hz, 3H). 13C-NMR (75 MHz,
DMSO-d6): d ¼ 172.92, 161.70, 155.51, 151.08, 136.53 (distorted
q, J ¼ 1.0 Hz), 134.36, 131.55, 130.74, 129.61 (q, J ¼ 31.9 Hz),
127.29, 126.34, 126.11 (q, J ¼ 3.8 Hz), 124.38, 124.00 (q,
J ¼ 272.3 Hz), 112.46, 71.81, 30.54, 18.28, 15.85, 14.60. MS
(ESI) m/z 466.10 [M – H]–, HRMS calcd. for C22H20F3NO3S2 [M]þ:
467.0837; found: 467.0830.
2-{2-Ethyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)thiazol-
5-yl)methylthio]phenoxy}-2-methylpropanoic acid (2e)
The title compound was prepared in 60% yield (74 mg,
0.15 mmol) as a white solid from 8e (131 mg, 0.25 mmol) follow-
ing the procedure described for 2b. Mp ¼ 132–1338C. 1H-NMR
(300 MHz, DMSO-d6) d ¼ 13.07 (br s, 1H), 8.02 (d, J ¼ 8.1 Hz, 2H),
7.80 (d, J ¼ 8.3 Hz, 2H), 7.15 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.13 (d,
J ¼ 2.2 Hz, 1H), 6.62 (d, J ¼ 8.3 Hz, 1H), 4.31 (s, 2H), 2.49 (q,
7.5 Hz, 2H), 2.13 (s, 3H), 1.50 (s, 6H), 1.04 (t, J ¼ 7.5 Hz, 3H).
13C-NMR (75 MHz, DMSO-d6): d ¼ 175.04, 161.73, 153.12,
151.21, 136.54 (distorted q, J ¼ 1.2 Hz), 134.96, 133.54, 131.39,
130.71, 129.62 (q, J ¼ 32.0 Hz), 126.29, 126.10 (q, J ¼ 3.7 Hz),
124.76, 124.00 (q, J ¼ 272.1 Hz), 116.22, 78.32, 30.65, 24.98,
22.92, 14.49, 13.97. MS (ESI) m/z 494.0 [M – H]ꢁ, HRMS calcd.
for C24H24F3NO3S2 [M]þ: 495.1150; found: 495.1173.
2-Methyl-2-{2-methyl-4-[(4-methyl-2-
(4-trifluoromethylphenyl)- thiazol-5-yl)
methylthio]phenoxy}propanoic acid (2b)
The title compound was prepared as following: To a stirred
solution of ethyl 2-methyl-2-{2-methyl-4-[(4-methyl-2-(4-trifluoro-
methylphenyl)thiazol-5-yl)methylthio]phenoxy}propanoate (8b)
(166 mg, 0.33 mmol) in THF (10 mL) and H2O (5 mL) was added
1 mL aqueous solution of 2.0 M t-BuOK. The reaction mixture was
refluxed for 24 h. After the completion, the mixture was diluted
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

622
C. C. Ciocoiu et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
132.34, 131.98, 131.55, 129.62 (q, J ¼ 31.9 Hz), 126.30, 126.13 (q,
J ¼ 3.8 Hz), 124.01 (q, J ¼ 272.1 Hz), 123.64, 113.01, 64.70, 34.38,
31.12, 29.18, 14.32. MS (ESI) m/z 494.0 [M – H]–, HRMS calcd.
for C24H24F3NO3S2 [M]þ: 495.1150; found: 495.1138.
2-{2-Iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetic acid (2f)
The title compound was prepared in 35% yield (43 mg,
0.09 mmol) as a white solid from 8f (130 mg, 0.26 mmol) follow-
ing the procedure described for 2a. Mp ¼ 146–1478C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 12.95 (br s, 1H), 8.02 (d, J ¼ 8.0 Hz, 2H),
7.80 (d, J ¼ 8.2 Hz, 2H), 7.20 (dd, J ¼ 8.4, 1.7 Hz, 1H), 7.07 (d,
J ¼ 1.5 Hz, 1H), 6.80 (d, J ¼ 8.5 Hz, 1H), 4.69 (s, 2H), 4.29 (s, 2H),
3.22 (hept, J ¼ 6.9 Hz, 1H), 2.07 (s, 3H), 1.06 (d, J ¼ 6.9 Hz, 6H).
13C-NMR (75 MHz, DMSO-d6): d ¼ 170.01, 161.77, 154.98, 151.26,
137.09, 136.57 (distorted q, J ¼ 1.2 Hz), 131.59, 131.48, 130.92,
129.61 (q, J ¼ 32.1 Hz), 126.29, 126.10 (q, J ¼ 3.8 Hz), 124.26,
124.00 (q, J ¼ 272.1 Hz), 112.20, 64.73, 30.91, 26.15, 22.16,
2-{2-Cyclopentyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetic acid (2j)
The title compound was prepared in 39% yield (37 mg,
0.07 mmol) as a white solid from 8j (94 mg, 0.18 mmol) follow-
ing the procedure described for 2a. Mp ¼ 88–898C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 8.02 (d, J ¼ 8.1 Hz, 2H), 7.80 (d,
J ¼ 8.3 Hz, 2H), 7.14 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.01 (d,
J ¼ 2.1 Hz, 1H), 6.70 (d, J ¼ 8.5 Hz, 1H), 4.31 (s, 2H), 4.25 (s,
2H), 3.30–3.14 (m, 1H), 2.07 (s, 3H), 1.93–1.77 (m, 2H), 1.63–
1.28 (m, 6H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 173.09, 161.70,
156.70, 151.18, 136.55 (distorted q, J ¼ 1.2 Hz), 134.44, 131.84,
131.46, 131.34, 129.58 (q, J ¼ 31.7 Hz), 126.26, 126.09 (q,
J ¼ 3.9 Hz), 124.00 (q, J ¼ 272.2 Hz), 122.72, 112.27, 67.27,
38.25, 32.34, 31.07, 24.85, 14.50. MS (ESI) m/z 506.2 [M – H]ꢁ,
HRMS calcd. for C25H24F3NO3S2 [M]þ: 507.1150; found 507.1161.
14.43. MS (ESI) m/z 480.1 [M
–
H]ꢁ, HRMS calcd. for
C23H22F3NO3S2 [M]þ: 481.0993; found: 481.0973.
2-{2-Iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}propanoic acid (2g)
The title compound was prepared in 66% yield (113 mg,
0.23 mmol) as a white solid from 8g (183 mg, 0.35 mmol) follow-
ing the procedure described for 2a. Mp ¼ 170–1718C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 12.97 (br s, 1H), 8.03 (d, J ¼ 8.1 Hz, 2H),
7.81 (d, J ¼ 8.2 Hz, 2H), 7.19 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.06 (d,
J ¼ 2.2 Hz, 1H), 6.74 (d, J ¼ 8.6 Hz, 1H), 4.83 (q, J ¼ 6.7 Hz, 1H),
4.29 (s, 2H), 3.21 (hept, J ¼ 6.9 Hz, 1H), 2.06 (s, 3H), 1.50 (d,
J ¼ 6.7 Hz, 3H), 1.06 (d, J ¼ 6.9 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6): d ¼ 172.90, 161.77, 154.67, 151.29, 137.21, 136.58
(distorted q, J ¼ 1.4 Hz), 131.58, 131.48, 131.04, 129.61 (q,
J ¼ 32.0 Hz), 126.30, 126.11 (q, J ¼ 3.7 Hz), 124.13, 124.00 (q,
J ¼ 272.2 Hz), 112.51, 71.59, 30.91, 26.23, 22.09, 18.22, 14.40.
MS (ESI) m/z 494.1 [M – H]ꢁ, HRMS calcd. for C24H24F3NO3S2
[M]þ: 495.1150; found: 495.1147.
2-{2-Cyclohexyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetic acid (2k)
The title compound was prepared in 48% yield (64 mg,
0.12 mmol) as a white solid from 8k (135 mg, 0.25 mmol) follow-
ing the procedure described for 2a. Mp ¼ 144–1458C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 12.94 (br s, 1H), 8.04 (d, J ¼ 8.1 Hz, 2H),
7.81 (d, J ¼ 8.3 Hz, 2H), 7.21 (dd, J ¼ 8.5, 2.2 Hz, 1H), 6.97 (d,
J ¼ 2.2 Hz, 1H), 6.78 (d, J ¼ 8.6 Hz, 1H), 4.68 (s, 2H), 4.27 (s, 2H),
2.91–2.76 (m, 1H), 2.04 (s, 3H), 1.70–1.51 (m, 5H), 1.36–1.04 (m,
5H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 170.06, 161.80, 155.09,
151.42, 136.60 (distorted q, J ¼ 1.0 Hz), 136.13, 131.82, 131.77,
131.51, 129.61 (q, J ¼ 32.1 Hz), 126.30, 126.08 (q, J ¼ 3.6 Hz),
124.02 (d, J ¼ 272.1 Hz), 123.94, 112.14, 64.73, 36.07, 32.26,
30.98, 26.42, 25.58, 14.40. MS (ESI) m/z 520.0 [M – H]–, HRMS
calcd. for C26H26F3NO3S2 [M]þ: 521.1306; found: 521.1310.
2-{2-Iso-propyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}-2-methylpropanoic
acid (2h)
The title compound was prepared in 48% yield (44 mg,
0.09 mmol) as a white solid from 8h (96 mg, 0.18 mmol) follow-
ing the procedure described for 2b. Mp ¼ 122–1238C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 8.03 (d, J ¼ 8.1 Hz, 2H), 7.82 (d,
J ¼ 8.3 Hz, 2H), 7.17 (dd, J ¼ 8.5, 2.2 Hz, 1H), 7.06 (d,
J ¼ 2.2 Hz, 1H), 6.62 (d, J ¼ 8.5 Hz, 1H), 4.30 (s, 2H), 3.16 (hept,
J ¼ 6.9 Hz, 1H), 2.07 (s, 3H), 1.49 (s, 6H), 1.04 (d, J ¼ 6.9 Hz, 6H).
13C-NMR (75 MHz, DMSO-d6): d ¼ 175.15, 161.79, 152.64, 151.31,
139.06, 136.58 (distorted q, J ¼ 1.3 Hz), 131.49, 131.23, 130.98,
129.61 (q, J ¼ 32.0 Hz), 126.30, 126.15 (q, J ¼ 3.6 Hz), 124.57,
124.02 (q, J ¼ 272.2 Hz), 116.36, 78.41, 30.83, 26.34, 25.02,
Measurement of oleic acid oxidation
Satellite cells were isolated from the Musculus obliquus internus
abdominis of healthy donors. The biopsies were obtained with
informed consent and approval by the Regional Committee for
Research Ethics, Oslo, Norway. The cells were cultured in DMEM
(5.5 mM glucose) with 2% FCS, 2% Ultroser G, penicillin/strepto-
mycin (P/S) and amphotericin B until 70–80% confluent. Myoblast
differentiation to myotubes was then induced by changing
medium to DMEM (5.5 mM glucose) with 2% FCS, 25 pM insulin,
P/S and amphotericin B. Experiments were performed after 7 days
of differentiation, and preincubation with agonists was started
after 3 days. The substrate, [1-14C]oleic acid (1 mCi/mL, 100 mM),
was given in DPBS with 10 mM HEPES and 1 mM L-carnitine. A 96-
well UNIFILTER1 microplate was mounted on top of the
CellBIND1 plate as described before [25], and the cells were incu-
bated at 378C for 4 h. The CO2 trapped in the filter was counted by
liquid scintillation (MicroBeta1, PerkinElmer) and normalized
against protein content. EC50-values were calculated with
GraphPad Prism, version 4.
22.21, 14.40. MS (ESI) m/z 508.0 [M
–
H]ꢁ, HRMS calcd.
for C25H26F3NO3S2 [M]þ: 509.1306; found: 509.1295.
2-{2-Tert-butyl-4-[(4-methyl-2-(4-trifluoromethylphenyl)-
thiazol-5-yl)methylthio]phenoxy}acetic acid (2i)
The title compound was prepared in 44% yield (42 mg,
0.08 mmol) as a white solid from 8i (94 mg, 0.18 mmol) follow-
ing the procedure described for 2a. Mp ¼ 105–1068C. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 12.97 (br s, 1H), 8.04 (d, J ¼ 8.1 Hz, 2H),
7.81 (d, J ¼ 8.3 Hz, 2H), 7.26 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.00 (d,
J ¼ 2.3 Hz, 1H), 6.83 (d, J ¼ 8.5 Hz, 1H), 4.69 (s, 2H), 4.26 (s, 2H),
2.01 (s, 3H), 1.23 (s, 9H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 169.82,
161.86, 156.67, 151.45, 138.02, 136.58 (distorted q, J ¼ 1.2 Hz),
Luciferase-based transient transfection system
COS-1 cells (ATCC no. CRL 1650) were cultured in DMEM supple-
mented with L-glutamine (2 mM), penicillin (50 U/mL),
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 10, 612–624
GW 501516 Analogues
623
streptomycin (50 mG/mL), fungizone (2.5 mg/mL), and 10% inac-
tivated FBS. The cells were incubated at 378C in a humidified
atmosphere of 5%CO2 and 95% air and used for transient trans-
fections. Cells were plated in six-well plates 1 day before trans-
fection. Transient transfection by lipofectamin 2000 (Invitrogen,
Carlsbad, CA) was performed as described. Each well received
990 ng plasmid: 320 ng reporter ((UAS)5-tk-LUC) (UAS ¼
upstream activating sequence and LUC ¼ luciferase), 640 ng
pGL3 basic (empty vector) and 30 ng expression plasmid of
either pSG5-GAL4-hPPARa, pSG5-GAL4-hPPARd, and pSG5-GAL4-
hPPARg. 10 mM of the compounds and controls and DMSO
(negative control) was added to the media 5 h after transfection.
Transfected cells were maintained for 24 h before lysis by
reporter lysis buffer. Binding of the ligands to the LBD of
PPARs activates GAL4 binding to UAS, which in turn stimulates
the tk promoter to drive luciferase expression. Luciferase activity
was measured using a luminometer (TD-20/20 luminometer
Turner Designs, Sunnyvale, CA) and normalized against protein
content. The following compounds were used as positive con-
trols: (2E,4E,8Z,11Z,14Z,17Z)-eicosa-2,4,8,11,14,17-hexaenoic acid
(EHA), GW 501516 (1) and rosiglitazone (BRL) for PPARa, PPARd,
and PPARg, respectively. EC50 is the concentration of test com-
pounds needed to induce 50% of the maximum luciferase
activity. The EC50-value is the average of three separate tests.
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energy. The X-ray crystal structure PPARd (PDB entry: 3GZ9) [28],
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