Synthesis, molecular modeling studies and biological evaluation of fluorine substituted analogs of GW 501516

Article abstract of DOI:10.1016/j.bmc.2011.10.020

(±)-2-Fluoro-2-(2-methyl-4-(((4-methyl-2-(4-(trifluoromethyl)phenyl) thiazol-5-yl)methyl)thio)phenoxy)acetic acid (2a) has been prepared and subjected to biological testing against all three subtypes of the PPARs. This compound exhibited agonist effects w

Full text of DOI:10.1016/j.bmc.2011.10.020

Bioorganic & Medicinal Chemistry 19 (2011) 6982–6988
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling studies and biological evaluation of fluorine
substituted analogs of GW 501516
Calin C. Ciocoiu a, Aina W. Ravna b, Ingebrigt Sylte b, Arild C. Rustan c, Trond Vidar Hansen a,
⇑
a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO BOX 1068, Blindern, N-0316 Oslo, Norway
b Research group of Medical Pharmacology and Toxicology, Department of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
c School of Pharmacy, Department of Pharmaceutical Biosciences, University of Oslo, PO BOX 1068, Blindern, N-0316 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
( )-2-Fluoro-2-(2-methyl-4-(((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methyl)thio)phen-
Received 30 June 2011
Revised 16 September 2011
Accepted 7 October 2011
Available online 17 October 2011
oxy)acetic acid (2a) has been prepared and subjected to biological testing against all three subtypes of the
PPARs. This compound exhibited agonist effects with EC50 values of 560 and 55 nM against PPAR
a and
PPARd, respectively, in a luciferase assay. Moreover, compound ( )-2a also exhibited potent ability to
induce oleic acid oxidation in a human myotube cell assay with EC50 = 3.7 nM. Compound ( )-2a can
be classified as a dual PPAR
against the PPAR receptor. Molecular modeling studies revealed that both enantiomers of 2a bind to
the PPARd receptor with similar binding energies.
a/d agonist with a 10-fold higher potency against the PPARd receptor than
Keywords:
GW 501516
Agonist
a
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorine
PPARa
PPARd
1. Introduction
substitution of a hydrogen atom by a fluorine atom is a well known
strategy for modification of lead compounds in drug discovery.13
We recently published triazole- and thiazole-based analogs of
Peroxisome proliferator-activated receptors (PPARa, PPARd, and
PPAR
c
) are ligand-activated transcription factors that belong to the
GW 501516 (1) which exhibited dual PPARa
/d effects.14,15 Herein,
nuclear hormone receptor superfamily.1 Literature data underline
the role of the three subtypes in the expression of genes responsi-
ble for the lipid and carbohydrate metabolism by interacting with
specific DNA peroxisome proliferator response elements (PPRE).2
we report the synthesis and biological evaluation of analogs of 1
where one hydrogen atom at the alpha-carbon atom to the carbox-
ylic acid moiety has been replaced with a fluorine atom. We rea-
soned that such substitutions would afford new agonists with a
stronger carboxylic acid with potential stronger binding affinity
to the PPARs. These efforts led to the identification of three dual
Molecules activating PPAR
a have beneficial effects on lipid
metabolism by decreasing both serum triglycerides and free fatty
acid levels, and increasing high-density lipoprotein level (HDL).3
PPARa/d agonists (Fig. 1). As of today, only a few dual PPARa/d ago-
PPAR
c
agonists improve glucose tolerance in type 2 diabetic pa-
nists have been reported.16
tients.4 Several studies have suggested that PPARd plays an impor-
tant role in regulating lipid metabolism and energy homeostasis in
muscle and adipose tissues.5–9 Recently, dual agonists became of
interest since it has been envisioned they could combine the
beneficial effects of two different subtype receptors avoiding their
negative actions. Dual activation of PPARs could be a useful tool
against several diseases such as metabolic disorders, type 2
diabetes and cardiovascular diseases.10
The fluorine atom has found a wide range of applications in
medicinal chemistry.11 For example, the fluorine atom can partici-
pate in hydrogen bonding interactions and alter the physical and
chemical properties of organic compounds.12 Therefore, bioisosteric
2. Results and discussion
2.1. Chemistry
Phenols 3a–3c, synthesized as described before,15,17 were alkyl-
ated with ( )-ethyl 2-bromo-2-fluoroacetate to yield esters ( )-
4a–4c. Basic aqueous hydrolysis of ( )-4a–4c afforded the racemic
acids ( )-2a–( )-2c in 54–68% yield over the two steps (Scheme 1).
2.2. Biological evaluation
GW 501516 (1) and compounds ( )-2a–( )-2c, at five different
concentrations, were exposed for 96 h to fully differentiated
human skeletal muscle cells cultured in 96 well plates. After this
⇑
Corresponding author. Tel.: +47 22857450; fax: +47 22855947.
E-mail address: t.v.hansen@farmasi.uio.no (T.V. Hansen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.020

C. C. Ciocoiu et al. / Bioorg. Med. Chem. 19 (2011) 6982–6988
6983
Figure 1. Structures of GW501516 and analogues ( )-2a-( )-2c.
Scheme 1. Synthesis of compounds ( )-2a-( )-2c. (i) ( )-Ethyl 2-bromo-2-fluoroacetate, Cs2CO3, CH3CN; (ii) LiOH, H2O, THF.
period of time, the oleic acid oxidation level was measured by
detection of the accumulation of 14C-labeled oxidized oleic acid.18
The EC50-values for compounds ( )-2a–( )-2c obtained using this
assay have been compiled in Table 1.
Introduction of a fluorine atom on the alpha-carbon to the car-
boxylic group of GW 501516 (1) led to compound ( )-2a that
exhibited decreased potency compared to GW 501516 (1). The
EC50-values were determined to be 0.03 nM for 1 and EC50 = 3.7 nM
for ( )-2a, respectively, in the human skeletal muscle cells assay
(Table 1).
According to Sternbach and co-authors, the substituent in the
ortho-position in the A-ring of GW 501516 (1) is important for
the selectivity of the activation of each of the three PPARs.19 Hence,
we wanted to investigate how the activity and selectivity of the
new agonists will be effected by introducing small alkyl groups
in this position. Substitution of the methyl group of ring A in com-
pound ( )-2a with an ethyl group led to compound ( )-2b that was
almost as potent as ( )-2a with EC50 = 5.0 nM. When an iso-propyl
group was introduced instead of the methyl group, the potency de-
creased significantly. For compound ( )-2c the EC50-value was
measured as 31.4 nM. Hence, potent agonist effects were observed
in this cell-based assay for all three compounds.
The activation towards each subtype of the PPARs were then
investigated in a luciferase cell assay.20 Compounds ( )-2b and
( )-2c exhibited similar activities towards the PPAR
EC50 = 710 and 735 nM, respectively, while ( )-2a exhibited
slightly higher activity against PPAR (EC50 = 560nM, Table 1).
a subtype with
a
Regarding PPARd activation, compounds ( )-2b (EC50 = 290)
and ( )-2c (EC50 = 580) were both less potent than ( )-2a
(EC50 = 55 nM). However, compound ( )-2a was less potent than
Table 1
Substitution pattern (see Fig. 1) and EC50-values of prepared compounds
a
b
b
b
Compound
R1
EC50 (nM)
PPAR
a
EC50 (nM)
PPAR
c
EC50 (nM)
PPARd EC50 (nM)
( )-2a
( )-2b
( )-2c
Methyl
Ethyl
Iso-propyl
—
—
—
3.7 0.8
5.0 1.1
31.4 5.1
0.03 0.01
nd
560 25
710 25
735 20
>1000
>1000
>1000
>1000
912 25
>1000
55
4
290 12
580 28
2.9 0.4
>1000
GW 501516 (1)
EHAc
32
5
BRLd
>1000
>1000
48
8
>1000
a
EC50-value measured from the oleic acid oxidation human myotube cell assay. Results are of three independent experiments with quadruplicate replicates shown. Values
are expressed as means SD.
b
EC50-value measured at different concentration of agonist against each of the three PPARS using the luciferase-based transient transfection system assay. Values are
expressed as means SD.
c
(2E,4E,8Z,11Z,14Z,17Z)-Eicosa-2,4,8,11,14,17-hexaenoic acid.
Rosiglitazone.
d

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GW 501516 (1) against PPARd. No activation towards PPAR
c
was
The docking poses were compared with the binding mode of the
reported agonist 2,3-dimethyl-4-((2-(prop-2-yn-1-yloxy)-4-((4-
(trifluoromethyl)phenoxy)methyl)phenyl)sulfanylphenoxy)acetic
acid in the PPARd X-ray crystal structure.21 The comparison re-
vealed that docking pose A was more in agreement with the bind-
ing mode of the agonist in the PPARd X-ray crystal structure than
pose B. The interaction energies are given in Table 2.
The docking of (S)-2a and (R)-2a revealed key interactions with
amino acids Arg284, Cys285, His323, His449 and Tyr473 (Fig. 2a–
d). In pose A, the fluorine atom of both (S)-2a and (R)-2a was ori-
ented towards Met453 (Fig. 2a and c). The carboxylic group of
(S)-2a was rotated towards Thr289 (Fig. 2a), while the carboxylic
group of (R)-2a was oriented towards His323, His449 and Tyr473
(Fig. 2c). The orientation of the carboxylic group of (R)-2a was al-
observed for any of the compounds ( )-2a–( )-2c (Table 1). Appar-
ently, introducing a larger substituent in the ortho-position in the
A-ring of 1 afforded a reduction of the potency against the PPARd
receptor for the new fluoro-analogs of lead compound GW
501516 (1). These observations are in accord with the structural–
activity studies performed on 1 for activation of PPARd.19 Introduc-
ing a fluoro-atom at the alpha-carbon atom to the carboxylic acid
moiety had a positive effect on PPARa activation. Hence, the com-
pounds ( )-2a–( )-2c can be classified as dual PPARa/d agonists.
2.3. Molecular modeling
Molecular modeling studies were performed in order to investi-
gate the binding ability of the two enantiomers of compound ( )-
2a exhibited towards the PPARd receptor. The docking indicated
that both (S)-2a and (R)-2a could be oriented in two putative posi-
tions at the binding site. In the following these positions are
termed pose A and pose B. The main differences between the
two poses were the orientation of the carboxyl acid end of the mol-
ecules, and hence also of the fluorine atom. The two docking poses
of (S)-2a and (R)-2a in PPARd are shown in Figure 2a–d.
Table 2
Interaction energies of (S)-2a and (R)-2a oriented in pose A and pose B
Pose
(S)-2a (kcal/mol)
(R)-2a (kcal/mol)
A
B
À15.82
À14.78
À14.03
À15.23
Figure 2 (a–d). The two docking poses of (S)-2a and (R)-2a in PPARd.

C. C. Ciocoiu et al. / Bioorg. Med. Chem. 19 (2011) 6982–6988
6985
Figure 3. The superposition of (S)-2a and (R)-2a in docking pose A together with a known agonist in the PPARd X-ray crystal structure.
Figure 4 (a–d). (S)-2a and (R)-2a docked into PPARa (4a-4b) and PPARc (4c-4d), respectively.

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C. C. Ciocoiu et al. / Bioorg. Med. Chem. 19 (2011) 6982–6988
most identical to carboxylic group of the agonist in the PPARd X-
ray crystal structure. In pose B, the fluorine atom of both (S)-2a
(Fig. 2b) and (R)-2a (Fig. 2d) was oriented towards Pro359. The
carboxylic group of (R)-2a was oriented towards Phe282, while
the carboxylic group of (S)-2a was oriented towards Cys285.
Series (Eclipse XDB-C18, 5
Agilent 6310 ion trap. According to LC/MS spectra, all final com-
pounds submitted to the biological testing had a purity >99%.
l
m 4.6 Â 150 mm), coupled with an
4.2. Ethyl 2-fluoro-2-(2-methyl-4-((4-methyl-2-(4-trifluoro
The superposition of (S)-2a and (R)-2a in docking pose
A
methylphenyl)thiazol-5-yl)methylthio)phenoxy)-acetate (( )-
4a)
with the known agonist 2,3-dimethyl-4-((2-(prop-2-yn-1-yloxy)-
4-((4-(trifluoromethyl)-phenoxy)methyl)phenyl)sulfanylphenoxy)
acetic acid in the PPARd X-ray crystal structure is depicted in
Figure 3.
Cs2CO3 (143 mg, 0.44 mmol) was added to
a solution of
2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)thiazol-5-
yl)methylthio)phenol (3a) (160 mg, 0.4 mmol)in dry CH3CN
(10 mL). To this mixture was added dropwise a solution of ethyl
According to calculated binding energies, the affinity of both
(S)-2a and (R)-2a for the PPARd receptor is similar, with the affinity
of (S)-2a slightly higher than that of (R)-2a. When comparing (S)-
2a and (R)-2a in pose A with the agonist binding mode in the
PPARd X-ray crystal structure, the binding mode of (R)-2a was
more in agreement with the agonist binding model in the PPARd
X-ray crystal structure (Fig. 3). Since our molecular modeling stud-
ies revealed that (S)-2a and (R)-2a have similar binding affinity, we
did not investigate the asymmetric synthesis or racemate resolu-
tion. In order to investigate differences in intermolecular interac-
2-bromo-2-fluoroacetate (60 lL, 0.48 mmol) in dry CH3CN (3 mL).
The mixture was stirred over night at rt under argon, then diluted
with water and extracted with ethyl acetate (3 Â 100 mL). The or-
ganic layers were combined, dried over MgSO4, and concentrated.
The residue was purified by column chromatography on silica gel
with hexane/ethyl acetate (3:1) as eluent to give ( )-4a as a yellow
oil in 88% yield (177 mg, 0.35 mmol). 1H NMR (300 MHz, CDCl3):
d = 7.93 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 2.1,
1H), 7.16 (dd, J = 8.4, 2.1 Hz, 1H), 6.99 (dd, J = 8.3, 0.7 Hz, 1H),
5.84 (d, J = 59.4 Hz, 1H), 4.32 (qd, J = 7.1, 0.7 Hz, 2H), 4.13 (s, 2H),
2.22 (s, 3H), 2.21 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR
(75 MHz, CDCl3): d = 163.77 (d, J = 31.7 Hz), 163.11, 154.11 (d,
J = 2.8 Hz), 151.32, 136.64 (distorted q, J = 1.3 Hz), 135.22, 131.22,
131.20 (q, J = 32.6 Hz), 130.26, 129.73 (d, J = 1.6 Hz), 129.38,
126.29, 125.77 (q, J = 3.8 Hz), 123.86 (q, J = 272.2 Hz), 116.68 (d,
J = 1.0 Hz), 102.76 (d, J = 232.6 Hz), 62.49, 31.70, 15.88, 14.81,
13.88; MS (EI) m/z500.1 [M+H]+; HRMS calcd for C23H21F4NO3S2
[M]+: 499.0899; found: 499.0900.
tions between (S)-2a and (R)-2a with PPAR
both enantiomers were also docked into PPAR
a
, PPAR
c
and PPARd,
. These
a
and PPARc
studies are shown in Figure 4a–d.
When (S)-2a and (R)-2a were docked into PPAR
was observed that the sulfur atoms of both of the ligands inter-
acted with Cys285 (PPARd) and Cys276 (PPAR ), respectively.
The corresponding amino acid in PPAR is Ser285. These observa-
tions may, at least in part, explain the dual agonist effects towards
PPAR , and PPARd.
The molecular modeling studies reported herein will be used for
a and PPARc, it
a
c
a
the future development of more potent dual PPARa/d agonists.
4.3. Ethyl 2-fluoro-2-(2-ethyl-4-((4-methyl-2-(4-trifluorometh
ylphenyl)thiazol-5-yl)methylthio)phenoxy)-acetate (( )-4b)
3. Conclusions
The title compound was prepared in 80% yield (122 mg,
0.24 mmol) as a yellow oil from 2-ethyl-4-((4-methyl-2-(4-trifluo-
Bioisosteric substitution of one of the two hydrogen atoms on
the alpha-carbon atom to the carboxylic group with a fluorine atom
romethylphenyl)thiazol-5-yl)methylthio)phenol
(3b)(123 mg,
led to three new dual PPAR
(1), these substitutions had a positive effect on PPAR
while the activation of PPARd was reduced. No significant activity
a
/d agonists. Compared to GW 501516
0.3 mmol) according to the general procedure described for ( )-4a.
1H NMR (300 MHz, CDCl3): d = 7.94 (d, J = 8.1 Hz, 2H), 7.62 (d,
J = 8.2 Hz, 2H), 7.22–7.14 (m, 2H), 7.00 (dd, J = 8.3, 1.1 Hz, 1H),
5.86 (d, J = 59.4 Hz, 1H), 4.33 (qd, J = 7.1, 1.5 Hz, 2H), 4.13 (s, 2H),
2.60 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H), 1.12 (t,
J = 7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3): d = 163.78 (d,
J = 31.8 Hz), 163.15, 153.91 (d, J = 2.9 Hz), 151.40, 136.66 (distorted
q, J = 1.3 Hz), 135.49 (d, J = 1.6 Hz), 134.18, 131.57, 131.24 (q,
J = 32.6 Hz), 130.31, 129.30, 126.31, 125.80 (q, J = 3.8 Hz), 123.87
(q, J = 272.2 Hz), 116.53 (d, J = 1.0 Hz), 102.70 (d, J = 232.4 Hz),
62.49, 31.85, 22.98, 14.77, 14.04, 13.89; MS (EI) m/z 514.1 [M+H]+;
HRMS calcd for C24H23F4NO3S2 [M]+: 513.1055; found: 513.1062.
a
activation,
was observed against PPARc. The binding pocket in the PPARd
receptor has been characterized as large.22 Based on the results re-
ported herein, the introduction of a small substituent, such as a
fluorine atom, at the alpha-carbon atom to the carboxylic acid moi-
ety, affords potent dual PPARa/d agonists. Molecular modeling
studies and calculated binding energies showed that the affinity
of both (S)-2a and (R)-2a for the PPARd receptor is similar. Based
on these results, it is likely that the enantiomers of the agonists re-
ported herein will exhibit similar potency as agonists against
PPARd and PPARa, respectively.
4.4. Ethyl 2-fluoro-2-(2-isopropyl-4-((4-methyl-2-(4-trifluoro
methylphenyl)thiazol-5-yl)methylthio)phenoxy)-acetate (( )-
4c)
4. Experimental
4.1. General methods
The title compound was prepared in 89% yield (155 mg,
0.29 mmol) as a yellow oil from 2-isopropyl-4-((4-methyl-2-(4-tri-
fluoromethylphenyl)thiazol-5-yl)methylthio)phenol (3c) (140 mg,
0.33 mmol) according to the general procedure described for ( )-
4a. 1H NMR (300 MHz, CDCl3): d = 7.93 (d, J = 8.1 Hz, 2H), 7.62 (d,
J = 8.3 Hz, 2H), 7.23–7.16 (m, 2H), 7.00 (dd, J = 9.1, 1.0 Hz, 1H),
5.86 (d, J = 59.3 Hz, 1H), 4.33 (qd, J = 7.1, 1.6 Hz, 2H), 3.26 (hept,
J = 6.9 Hz, 1H), 2.15 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H), 1.11 (d,
J = 6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3): d = 163.75 (d,
J = 31.9 Hz), 163.14, 151.44, 153.42 (d, J = 3.0 Hz), 139.73 (d,
J = 1.6 Hz), 136.64 (distorted q, J = 1.2 Hz), 131.94, 131.70, 131.22
(q, J = 32.6 Hz), 130.35, 129.23, 126.28, 125.78 (q, J = 3.8 Hz),
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 per
million (ppm, 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). Melt-
ing points were measured using a Barnstead Electrothermal appa-
ratus and are uncorrected. Flash chromatography was performed
on Silica Gel 60 (40–63 lm, Fluka). Mass spectra were recorded
at 70 eV with Fission’s VG Pro spectrometer. High resolution mass
spectra were performed with a VG Prospec mass spectrometer. LC/
MS analyses were performed on an Agilent Technologies 1200

C. C. Ciocoiu et al. / Bioorg. Med. Chem. 19 (2011) 6982–6988
6987
123.86 (q, J = 272.1 Hz), 116.52 (d, J = 1.1 Hz), 102.73 (d,
4.8. Measurement of oleic acid oxidation18
J = 232.4 Hz), 62.48, 31.97, 26.81, 22.47, 14.70, 13.88; MS (EI) m/z
528.1 [M+H]+; HRMS calcd for C25H25F4NO3S2 [M]+: 527.1212;
found: 527.1219.
Satellite cells were isolated from the Musculus obliquusinternus
abdominis of healthy donors. The biopsies were obtained with in-
formed consent and approval by the Regional Committee for Re-
search Ethics, Oslo, Norway. The cells were cultured at 37 °C in a
humidified atmosphere of 5% CO2 and 95% air in DMEM (5.5 mM
glucose) with 2% FCS, 2% Ultroser G, penicillin/streptomycin (P/S,
4.5. 2-Fluoro-2-(2-methyl-4-((4-methyl-2-(4-trifluoro
methylphenyl)thiazol-5-yl)methylthio)phenoxy)acetic acid
(( )-2a)
100 U/mL/ 100 lg/mL) and amphotericin B (1.25 lg/mL) 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 per-
formed after 7 days of differentiation, and preincubation with ago-
nists was started after 3 days. The agonists were added at the
following five concentrations: 100, 10, 1, 0.1, 0.01 nM. The sub-
To a stirred solution of ( )-4a (177 mg, 0.35 mmol) in THF
(10 mL) and H2O (5 mL), at 0 °C, was added slowly 215 lL of
2.0 M LiOH. The reaction mixture was stirred until TLC indicated
complete hydrolysis. The mixture was diluted with 50 mL H2O,
acidified with 0.1 M HCl, extracted with diethyl ether,
(3 Â 50 mL), dried over MgSO4, and concentrated. The residue
was recrystallized from ethyl acetate/hexane to give ( )-2a as a
colorless solid in 65% yield (108 mg, 0.23 mmol). Mp 161–162 °C;
1H NMR (300 MHz, DMSO-d6): d = 8.05 (d, J = 8.1 Hz, 2H), 7.81 (d,
J = 8.3 Hz, 2H), 7.32 (d, J = 1.9 Hz, 1H), 7.25 (dd, J = 8.5, 1.9 Hz,
1H), 7.09 (d, J = 8.4 Hz, 1H), 6.27 (d, J = 58.9 Hz, 1H), 4.43 (s, 2H),
2.26 (s, 3H), 2.16 (s, 3H); 13C NMR (75 MHz, DMSO-d6):
d = 165.37 (d, J = 30.2 Hz), 161.85, 153.12 (d, J = 2.4 Hz), 151.17,
136.48 (distorted q, J = 1.3 Hz), 133.66, 131.27, 129.90, 129.69 (q,
J = 32.1 Hz), 128.79, 128.72 (d, J = 1.2 Hz), 126.34, 126.08 (q,
J = 3.7 Hz), 124.00 (q, J = 272.1 Hz), 116.25, 102.31 (d,
J = 228.8 Hz), 29.79, 15.57, 14.70; MS (EI) m/z 470.10 [MÀH]À;
HRMS calcd for C21H17F4NO3S2 [M]+: 471.0586; found: 471.0570.
strate, (1-14C)oleic acid (1
with 10 mM HEPES and 1 mM
l
Ci/mL, 100
lM), was given in DPBS
L
-carnitine. A 96-well UNIFILTERÒ
micro plate was mounted on top of the CellBINDÒ plate as de-
scribed before,18 and the cells were incubated at 37 °C for 4 h.
The CO2 trapped in the filter was counted by liquid scintillation
(MicroBetaÒ, PerkinElmer) and normalized against protein con-
tent.EC50-values were calculated with GraphPad Prism, version
4.Student’s t-test was employed for determination of statistical
significance.
4.9. Luciferase-based transient transfection system20
COS-1 cells (ATCC no. CRL 1650) were cultured in DMEM sup-
plemented with
L
-glutamine (2 mM), penicillin (50 U/mL), strepto-
g/mL), and 10% inactivated FBS.
mycin (50 g/mL), fungizone (2.5
l
l
4.6. 2-Fluoro-2-(2-ethyl-4-((4-methyl-2-(4-trifluoromethyl
phenyl)thiazol-5-yl)methylthio)phenoxy)acetic acid (( )-2b)
The cells were incubated at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air and used for transient transfections. Cells were
plated in six-well plates 1 day before transfection. Transient trans-
fection by lipofectamin 2000 (Invitrogen, Carlsbad, CA) was per-
formed as described. Each well received 990 ng plasmid: 320 ng
reporter ((UAS)5-tk-LUC) (UAS = upstream activating sequence
and LUC = luciferase), 640 ng pGL3 basic (empty vector) and
The title compound was prepared in 67% yield (78 mg,
0.16 mmol) as a colorless solid from ( )-4b (122 mg, 0.24 mmol)
according to the general procedure described for ( )-2a. Mp 159–
160 °C; 1H NMR (300 MHz, DMSO-d6): d = 8.01 (d, J = 7.8 Hz, 2H),
7.78 (d, J = 7.9 Hz, 2H), 7.32–7.20 (m, 2H), 7.11 (d, J = 8.1 Hz, 1H),
6.28 (d, J = 58.9 Hz, 1H), 4.39 (s, 2H), 2.54 (q, J = 7.4 Hz, 2H), 2.20
(s, 3H), 1.06 (t, J = 7.3 Hz, 3H);13C NMR (75 MHz, DMSO-d6):
d = 165.39 (d, J = 30.2 Hz), 161.86, 152.90 (d, J = 2.6 Hz), 151.24,
136.50 (distorted q, J = 1.0 Hz), 134.40, 132.75, 131.30 (d,
J = 0.9 Hz), 130.43, 129.68 (q, J = 32.0 Hz), 128.61, 126.32, 126.10
(q, J = 3.8 Hz), 123.99 (q, J = 272.4 Hz), 116.07, 102.25 (d,
J = 228.9 Hz), 29.97, 22.51, 14.62, 14.09. MS (EI) m/z 484.21
[MÀH]À, HRMS calcd for C22H19F4NO3S2 [M]+: 485.0742; found:
485.0725.
30 ng expression plasmid of either pSG5-GAL4-hPPAR
a, pSG5-
GAL4-hPPARd and pSG5-GAL4-hPPAR . The agonists were added
c
at the following five concentrations: 1000, 100, 10, 1 and 0.1 nM
to the media 5 h after transfection. The positive controls and DMSO
(negative control) were 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, Sun-
nyvale, CA) and normalized against protein content. The following
compounds were used as positive controls: (2E,4E,8Z,11Z,14Z,17Z)-
eicosa-2,4,8,11,14,17-hexaenoic acid (EHA), GW 501516 (1) and
4.7. 2-Fluoro-2-(2-isopropyl-4-((4-methyl-2-(4-trifluoromethyl
phenyl)thiazol-5-yl)methylthio)phenoxy)acetic acid (( )-2c)
rosiglitazone (BRL) for PPARa, PPARd, and PPARc, respectively. Stu-
dent’s t-test was employed for determination of statistical
significance.
The title compound was prepared in 76% yield (109 mg,
0.22 mmol) as a colorless solid from ( )-4c (155 mg, 0.29 mmol)
according to the general procedure described for ( )-2a. Mp
120–121 °C; 1H NMR (300 MHz, DMSO-d6): d = 8.02 (d,
J = 8.2 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.28 (dd, J = 8.5, 2.3 Hz,
1H), 7.19 (d, J = 2.2 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.28 (d,
J = 58.9 Hz, 1H), 4.39 (s, 2H), 3.19 (hept, J = 6.9 Hz, 1H), 2.15 (s,
3H), 1.08 (d, J = 6.9 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d = 165.37 (d, J = 30.2 Hz), 161.88, 152.41 (d, J = 2.5 Hz), 151.29,
138.56 (d, J = 1.4 Hz), 136.51 (distorted q, J = 1.3 Hz), 131.38,
130.76, 130.45, 129.66 (q, J = 32.0 Hz), 128.50, 126.31, 126.11
(q, J = 3.8 Hz), 123.99 (q, J = 271.8 Hz), 116.13, 102.28 (d,
J = 228.9 Hz), 30.17, 26.38, 22.23 (d, J = 4.4 Hz), 14.56.MS (ESI)
m/z 498.34 [MÀH]À, HRMS calcd for C23H21F4NO3S2 [M]+:
499.0899; found: 499.0881.
4.10. Molecular modeling
The ICM (‘Internal Coordinate Mechanics’) program23 (version
3.6-1h) was used for docking and calculation of receptor–ligand
interaction energies. The X-ray crystal structure of PPARd in com-
plex with the agonist (2,3-dimethyl-4-((2-(prop-2-yn-1-yloxy)-4-
((4-(trifluoromethyl)phenoxy)methyl)phenyl)sulfanyl)-phenoxy)
acetic acid have been solved (PDB code: 3GZ9).21 The structure of
PPARd from this complex was converted to an ICM object and
the receptor maps were calculated based on the agonist position
in the X-ray crystal structure complex. (S)-2a and (R)-2a were
modeled using the ICM molecule editor and docked into PPARd

6988
C. C. Ciocoiu et al. / Bioorg. Med. Chem. 19 (2011) 6982–6988
10. Balakumar, P.; Rose, M.; Ganti, S. S.; Krishan, P.; Singh, M. Pharmacol. Res. 2007,
56, 91.
using interactive docking, and the interaction energy was calcu-
lated using the calcBindingEnergy macro of ICM.
11. (a) Hagmann, W. K. J. Med. Chem. 2008, 51, 4359; (b) Yamazaki, T.; Taguchi, T.;
Ojima, I. Fluorine Med. Chem. Chem. Biol. 2009, 3; (c) Hodgetts, K. J.; Combs, K. J.;
Elder, A. M.; Harriman, G. C. Annu. Rep. Med. Chem. 2010, 45, 429; (d) Ojima, I.
Fluorine in Medicinal Chemistry Chemical Biology; Wiley, 2009.
12. Strunecka, A.; Patocka, J.; Connett, P. J. Appl. Biomed. 2004, 2, 141.
13. Ismail, F. M. D. J. Fluorine Chem. 2002, 118, 27.
Acknowledgments
Professor G. Hege Thoresen is gratefully acknowledged for fruit-
ful discussions and assistance with biological testing. Inven2A/S is
gratefully acknowledged for financial support.
´
14. Ciocoiu, C. C.; Nikolic, N.; Nguyen, H. H.; Thoresen, G. H.; Aasen, A. J.; Hansen, T.
V. Eur. J. Med. Chem. 2010, 45, 3047.
15. Ciocoiu, C. C.; Ravna, A. W.; Sylte, I.; Hansen, T. V. Arch. Pharm. Chem. Life Sci.
2010, 343, 612.
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