Identification and synthesis of a novel selective partial PPARδ agonist with full efficacy on lipid metabolism in vitro and in vivo

Article abstract of DOI:10.1021/jm061202u

The aim was to identify a novel selective PPARδ agonist with full efficacy on free fatty acid (FFA) oxidation in vitro and plasma lipid correction in vivo. Using the triple PPARα,γ,δ agonist 1 as the structural starting point, we wanted to investigate the

Full text of DOI:10.1021/jm061202u

J. Med. Chem. 2007, 50, 1495-1503
1495
Identification and Synthesis of a Novel Selective Partial PPARδ Agonist with Full Efficacy on
Lipid Metabolism In Vitro and In Vivo
Per Sauerberg,* Grith S. Olsen, Lone Jeppesen, John P. Mogensen, Ingrid Pettersson, Claus B. Jeppesen, Jens R. Daugaard,
Elisabeth D. Galsgaard, Lars Ynddal, Jan Fleckner, Vladimira Panajotova,† Zdenek Polivka,† Pavel Pihera,†
Miroslav Havranek,† and Erik M. Wulff
NoVo Nordisk A/S, Diabetes Research Unit, NoVo Nordisk Park, 2760 MåløV, Denmark, and RE&D VUFB, Podebradska 56/186, 180 66 Praha
9, Czech Republic
ReceiVed October 16, 2006
The aim was to identify a novel selective PPARδ agonist with full efficacy on free fatty acid (FFA) oxidation
in vitro and plasma lipid correction in vivo. Using the triple PPARR,γ,δ agonist 1 as the structural starting
point, we wanted to investigate the possibility of obtaining selective PPARδ agonists by modifying only
the acidic part of 1, while holding the lipophilic half of the molecule constant. The structure-activity
relationship was guided by in vitro transactivation data using the human PPAR receptors, FFA oxidation
efficacy performed in the rat muscle L6 cell line, and in vivo rat pharmacokinetic properties. Compound 7
([4-[3,3-bis-(4-bromo-phenyl)-allylthio]-2-chloro-phenoxy]-acetic acid) was identified as a selective, partial
agonist with good oral pharmacokinetic properties in rat. Chronic treatment of high fat fed ApoB100/CETP-
Tgn mice with 7 corrected the plasma lipid parameters and improved insulin sensitivity. These data suggest
that selective PPARδ agonists have the potential to become a novel treatment of dyslipidemia.
Introduction
L-165041 ((4-(3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-pro-
poxy)-acetic acid).8 Later, an increase in plasma HDL-
cholesterol (79%) and a decrease in plasma LDL-c (29%), TG
(56%), and insulin (48%) in obese, normo-glycemic rhesus
monkeys after treatment with the PPARδ selective agonist
GW501516 ({2-methyl-4-[4-methyl-2-(4-trifluoromethyl-phen-
yl)-thiazol-5-ylmethylsulfanyl]-phenoxy}-acetic acid, Table 1)
has increased the therapeutic interest in this third PPAR
receptor.9 An up-regulation of the expression of the reverse
cholesterol transporter ATP-binding cassette A1 (ABC-A1) in
macrophages, fibroblasts, and intestinal cells and an increase
of apolipoprotein A1 (Apo-A1) specific cholesterol efflux was
observed as part of the mechanism responsible for the effects
The peroxisome proliferator-activated receptors (PPARsa) are
transcription factors known to play a central role in regulating
the storage and catabolism of dietary fat. The PPARs were
cloned a little more than a decade ago as orphan members of
the nuclear receptor gene family that includes the steroid,
retinoid, and thyroid hormones receptors.1,2 There are three
PPAR subtypes: PPARR, PPARγ, and PPARδ.3 The PPAR
receptors heterodimerize with the RXR receptor, and the PPAR/
RXR heterodimers then bind to DNA sequences known as DR-1
response elements.4 As with other members of the nuclear
receptor gene family, the PPARs are ligand-activated transcrip-
tion factors. Agonist activation of the receptor results in changes
in the expression level of mRNAs encoded by PPAR target
genes.
The use of PPARγ activators, for example, rosiglitazone
(Table 1) and pioglitazone (glitazones), in the treatment of type
2 diabetes have been established due to their abilities to lower
blood glucose and insulin levels and improve insulin sensitiv-
ity.5,6 Similarly, PPARR activators, for example, fenofibrate
(Table 1) and clofibrate (fibrates), have been used clinically
for more than three decades for their ability to lower plasma
triglycerides (TG) and moderately raise high-density lipoprotein
(HDL)-cholesterol.7 There is no drug available targeting the third
PPARδ receptor, but there is evidence that PPARδ is involved
in lipid homeostasis.
9
of GW501516 on plasma cholesterol parameters. In addition
to the effects on cholesterol homeostasis, GW501516 treatment
was observed to lower plasma glucose and insulin and improve
insulin sensitivity in diabetic ob/ob mice and high fat diet
induced insulin resistant mice.10 Increased oxygen consumption
in vivo suggesting a fuel switch from glucose to free fatty acids
(FFA), as well as increased FFA oxidation in skeletal muscle,
was demonstrated both in vivo and in vitro.10
Two different transgenic mouse models overexpressing
PPARδ in either adipose tissue11 or in muscle tissue12 have
shown up-regulation of genes (LPL, FABP, FAT, CD36, CPT1b,
and ACS) and proteins (UCP-2) responsible for lipid uptake
and metabolism and energy uncoupling. Both types of mice had
reduced adipose tissue and were protected against high fat diet
induced body weight gain. Supportive for the hypothesis of
skeletal muscle being a major target organ were in vitro
treatment of C2C12 muscle cells with GW501516 showing up-
regulated genes involved with TG hydrolysis and FFA oxidation
(LPL, ACS4, CPT1), preferential lipid utilization (PDK4),
energy expenditure (UCP1,-2, -3), and lipid efflux (ABCA1/
G1).13,14 Further, GW501516 was found to increase glucose
uptake independently of insulin and enhance subsequent insulin
stimulation in cultured primary human skeletal muscle cells.15
As a possible mechanism, the phosphorylation and expression
of AMP-activated protein kinase were likewise increased.
The first proposed pharmacological role for PPARδ was in
the regulation of cholesterol homeostasis. Total plasma choles-
terol was raised in db/db mice treated with the PPARδ ligand
* To whom correspondence should be addressed. Telephone:
+4544434858. Fax: +4544663939. E-mail: psa@novonordisk.com.
† RE&D VUFB.
a Abbreviations: FFA, free fatty acid; PPAR, peroxisome proliferator-
activated receptor; TG, triglycerides; HDL, high density lipoprotein; ABC-
A1, ATP-binding cassette A1; LDL, low density lipoprotein; Apo-A1,
apolipoprotein A1; ApoB100/CETP, apo-lipoprotein B/cholesterol ester
transfer protein; ADDP, azodicarboxylic dipiperidide; PK, pharmacokinetic;
EDL, musculus extensor digitorum longus.
10.1021/jm061202u CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/08/2007

1496 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Sauerberg et al.
Table 1. In Vitro hPPARR, rPPARR, hPPARγ, and hPPARδ Transactivation of Test and Reference Compoundsa
a Compounds were tested in at least three separate experiments in at least five concentrations ranging from 0.001 to 30 µM. EC50 is the concentration
giving 50% of the maximal activity observed for a given compound. b For each compound, the efficacy (% max) is given as a relative compared to the
maximal activity of NNC 61-465532 for PPARR. c Rosiglitazone for PPARγ. d Carbacyclin for PPARδ. The results are expressed as means ( SEM. If a
plateau was not reached at the highest concentration of compound tested (30µM), the effect at this concentration was calculated and the maximal effect was
indicated to be greater than this value. In such cases, the EC50 was assigned as >10 µM. If the maximal effect of a compound was less than 10%, no EC50
could be calculated. e Percent relative to GW501516 (272%).

PPARδ Agonist with Full Efficacy on Lipid Metabolism
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1497
Scheme 121 a
a Reagents and conditions: (a) (i) Na, EtOH; (ii) (EtO)2POCH2COOEt, 70 °C. (b) (i) 1 M DIBAL-H in toluene, toluene, room temperature; (ii) HCl
(conc), H2O, room temperature.
Scheme 2a
a Reagents and conditions: (a) P(Bu)3, ADDP, THF, 0 °C to room temperature. (b) 1 N NaOH, EtOH.
Table 2. The Effect of PPAR Agonists on Fatty Acid Oxidation in Rat
In addition to the effects on cholesterol homeostasis and
glycemic control, PPARδ activation has been suggested to
attenuate inflammation and slow the progression of atheroscle-
rosis by direct and indirect mechanisms.16 Nevertheless, these
effects could not be detected in low-density lipoprotein (LDL)-
receptor knock-out mice after treatment with the PPARδ agonist
GW0742 ({2-methyl-4-[4-methyl-2-(4-fluoro-phenyl)-thiazol-
5-ylmethylsulfanyl]-phenoxy}-acetic acid), a close analogue to
GW501516.17 Recently, the same agonist was used to show that
PPARδ treatment increased HDL-cholesterol by 50% and
lowered cholesterol absorption in wild-type mice by 43%.18
L6 Muscle Cellsa
EC50 ( SEM
% max increase
compound
(nM)
of GW ( SEMb
2
3
4
5
6
7
8
212 ( 92
615 ( 265
204 ( 40
224 ( 42
379 ( 113
30 ( 3
69 ( 5
59 ( 5
62 ( 5
78 ( 11
85 ( 8
109 ( 6
75 ( 14
58 ( 6
70 ( 1
100
3 ( 1
Rosiglitazone
fenofibric acid
GW501516
∼5000
∼3000
6 ( 1
Human polymorphism studies further suggested that PPARδ
is involved in cholesterol metabolism.19 Recently, phase 1
clinical trial data from healthy normolipidemic male volunteers
treated for 2 weeks with GW501516 showed that HDL-
cholesterol increased by 19% and TG decreased by 20%,
compared to placebo.20 GW501516 is reported to be in phase 2
clinical trials.
a Average of 3-4 experiments made in triplicate. b Maximal increase is
given as % of maximal increase by GW501516. Maximal increase for
GW501516 was 120 ( 21% over basal.
Results and Discussion
We have previously published on our design of the triple
PPARR,γ,δ activator 1 (Table 1).21 The aim of the present work
was to investigate if it was possible to obtain selective PPARδ
agonists by only modifying the acidic part of 1, while holding
the lipophilic half of the molecule constant.24 At the same time,
we wanted to identify selective PPARδ agonists using trans-
activation assays to investigate the possibility of getting a full
pharmacological response on the desired FFA oxidation/lipid
lowering effects with such agonists.
Removal of the ethoxy group R to the carboxylic acid in 1
gave immediately a selective partial PPARδ agonist 2 (Table
1). The PPARδ transactivation potency and efficacy of 2 was
retained compared to 1, but unfortunately, 2 did not give full
efficacy in the in vitro FFA oxidation assay (Table 2) compared
to GW501516 (which was considered a full PPARδ agonist).
Furthermore, the pharmacokinetic (PK) evaluation in rats
revealed that 2 had very poor oral bioavailability and a very
short half-life (Table 3). This finding is in agreement with a
previous observation on a related compound, where extensive
beta-oxidation was suggested to be the reason for the poor PK
properties.23 The meta-substituted analogue 3 was a ten times
less potent PPARδ agonist than 2. Compound 3 had slightly
better PK properties, but the exposure was still very low. The
one carbon shorter analogues of 2 and 3, 4 and 5, respectively,
were also partial PPARδ agonists. Even though 4 and 5 had
The aim of the present work was to identify novel selective
PPARδ agonists with full efficacy on FFA oxidation in vitro
and plasma lipid correction in vivo. The structural starting point
was the previously published triple PPARR,γ,δ agonist 1.21 The
structure-activity relationship (SAR) was guided by in vitro
transactivation data using the human PPAR receptors,22 FFA
oxidation efficacy performed in the rat muscle L6 cell line, and
in vivo rat pharmacokinetic properties. Compound 7 was
identified as a selective, partial agonist with good oral rat
pharmacokinetic properties in vivo and full efficacy on plasma
lipids in the lipid-metabolism humanized double transgenic apo-
lipoprotein B/cholesterol ester transfer protein (ApoB100/CETP
Tgn) mouse model.
Chemistry
We have earlier described the synthesis of 3,3-bis-(4-
bromophenyl)-prop-2-en-1-ol, 10, from 4,4′-dibromo-benzophen-
one (Scheme 1).21 Coupling of 10 with the hydroxyl/mercapto-
phenylalkylesters 11-16 was done under nonoptimized Mitsunobu
conditions using ADDP (azodicarboxylic dipiperidide) and
tributylphosphine to give the esters 17-23 in good to high yields
(Scheme 2). Basic hydrolysis of the ester intermediates 17-23
gave the desired target molecules 2-8.

1498 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Table 3. Single Dose Rat Pharmacokinetica
Sauerberg et al.
single dose rat pharmacokinetic
b
g
dose
i.v./p.o.
(mg/kg)
Cmax
p.o.
AUCc
p.o.
Fd
p.o.
(%)
T1/2
f
CLe
(mL/min/kg)
Vss
p.o.
(min)
cmpd
(ng/mL)
(ng × min)/mL
(L/kg)
1
2
3
4
5
6
7
8
1.08/2.29
0.20/0.43
0.20/0.45
0.20/0.43
0.20/0.45
0.20/0.45
1.06/2.06
1.18/2.35
1.09/2.13
554
bdh
51
252
419
241
508
887
454
83 534
78
21.4
bdh
27
6.0
bdh
7
11.9
10
7
2.3
bdh
5.3
1.4
bdh
1.4
0.3
0.9
1.0
85
bdh
bdh
407
210
bdh
130
46
bdh
953
447
153
181
59
67 560
156 709
119 351
84 108
79 294
179 505
127 478
95
136
151
612
GW501516
a Rats were given either a single dose i.v. (n ) 8) or a single dose p.o. (n ) 8) of each of the test compounds. At each of the time points (5, 15, 30, 60,
90, 120, 240, and 360 min), one animal was sacrificed and blood samples were analyzed for compound plasma concentration. b Maximum plasma concentration
after oral dosing. c Estimated area under the plasma-concentration time curve after oral dosing. d Oral bioavailability. e Clearance. f Volume of distribution
during steady state. g Oral half-life. h Below detection limit.
Figure 1. Concentration dependent activation of HEK293 cells
transfected with the hPPARδ(LBD)-GAL4 receptor. Comparison of
the hPPARδ potency and efficacy between carbacyclin, 7, and
GW501516.
Figure 3. Compound 7 (magenta) docked into the active site of the
PPARδ receptor crystallized with GW2433. The Connolly surface was
generated around the amino acids within 6 Å from the crystallized
ligand (GW2433). The chlorine (green) in compound 7 occupies a
pocket not used by compounds 2-5 (or GW2433).
Figure 2. Concentration dependent inhibition of the hPPARδ(LBD)-
GAL4 transactivation activity of the full agonist GW501516 by the
partial agonist 7.
better PK properties in the rat (Table 3), none of the compounds
induced full FFA oxidation in the muscle cell line in vitro.
Examination of the published crystal structure of the PPARδ
receptor protein suggested room for a substituent on the central
phenyl group (Figure 3).25 The 3-chlorine-substituted analogue
Figure 4. Dose response curves for 7 and GW501516 on fatty acid
of 4, compound 6, had higher PPARδ efficacy but lower potency
oxidation in rat L6 muscle cells. Effect of increasing concentration of
in the transactivation assay. To improve the potency, the chlorine
compound for 48 h on fatty acid oxidation in L6 muscle cells expressed
group was next introduced in the 2-position and oxygen was
as % increase as compared to untreated cells. The curves represent the
average of experiments in triplicate ( SEM.
replaced by sulfur, as in GW501516.26 In an attempt to block
the metabolism, oxygen was also inserted giving compound 7,
which had 73% PPARδ transactivation efficacy (Table 1 and
Figure 1) but 113% efficacy in the FFA oxidation assay (Table
2 and Figure 4) compared to GW501516. Further, compound 7
and GW501516 had no effect on glucose oxidation in the rat
muscle L6 cell line (data not shown). To verify that 7 really
was a partial PPARδ agonist, increasing concentrations of 7
was added to a fully efficacious concentration (100 nM) of the
full agonist GW501516 (Figure 2). The transactivation efficacy
of GW501516 was reduced to the max efficacy of 7, demon-
strating the partial agonist/antagonist properties of 7. Compound
7 was approximately seven times less potent than GW501516,
whereas the methyl analogue 8 was nearly equipotent to the

PPARδ Agonist with Full Efficacy on Lipid Metabolism
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1499
In support of the published literature,10-15 FFA oxidation in
skeletal muscles was increased (Figure 7), indicating that at least
part of plasma lipid correcting and insulin resistance improving
effects were through this mechanism. The fact that plasma levels
of total ketone bodies (Table 4) were decreased rather than
increased, as seen after fenofibrate treatment (data not shown),
indicated that the beta-oxidation in liver was not part of the
lipid lowering mechanism. Interestingly, the partial agonist 7
corrected the plasma lipid and glucose profile as good as or
even better than the full agonist GW501516. Compound 7
therefore fulfils the criteria of being a selective partial PPARδ
agonist with full efficacy on the desired effects in vivo.
In conclusion, through systematic structural modifications of
the PPARpan agonist 1, we have identified 7, which is a partial
PPARδ agonist in transactivation assay but a full agonist on
FFA oxidation in muscle cells both in vitro and in vivo. Further,
chronic treatment of high fat fed ApoB100/CETP-Tgn mice with
7 corrected the plasma lipid parameters and improved insulin
sensitivity. Although the mechanism behind PPARδ activation
is not fully understood and the side effect profile is not explored,
the data in this report support the notion that selective PPARδ
agonists have the potential to become a novel treatment of
dyslipidemia.
Figure 5. Plasma drug concentration versus time curves obtained from
rats dosed with 7 orally by gavage and intravenously. At each time
point, one rat was sacrificed and drug plasma concentration was
measured.
Experimental Section
structurally similar GW501516. The partial agonist 7 further
had the desired PK properties with high exposure, good oral
bioavailability, low clearance, and sufficiently long half-life
(Table 3 and Figure 5). The methyl analogue 8 had slightly
higher exposure and a longer half-life, but 8 was not a full
agonist in the FFA oxidation assay.
Chemistry. The compounds 17-23 and 2-8 (Scheme 2) were
synthesized without optimization of the reaction conditions. H
NMR data were recorded on a 300 or 400 MHz spectrometer, with
solvent peak as internal reference value (DMSO, 2.50; TMS peak
was used in CDCl3).
1
Ethyl 3-(4-Hydroxy-phenyl)-propionate, 11. To an ice-cooled
solution of 3-(4-hydroxy-phenyl)-propionic acid (8.3 g, 50.0 mmol)
in ethanol (100 mL) was added dropwise thionyl chloride (3.7 mL,
50.7 mmol). The mixture was stirred at room temperature overnight,
concentrated in vacuo, and the residue was purified by kugelrohr
distillation to give 9.6 g (99%) of 11 as a colorless oil.
Ethyl 3-{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-pro-
pionate, 17. Under an atmosphere of nitrogen, azodicarboxylic
dipiperidide (504 mg, 2.0 mmol) was added at 0-5 °C to a stirred
solution of tributylphosphine (404 mg, 2.0 mmol), 11 (388 mg,
2.0 mmol), and 1021(736 mg, 2.0 mmol) in dry THF (50 mL). The
reaction mixture was warmed to room temperature and stirred for
48 h. The reaction mixture was concentrated in vacuo, and water
and ethyl acetate (75 mL each) were added. The aqueous layer
was collected and further extracted with ethyl acetate (2 × 75 mL).
The organic layers were combined, washed with water, dried
(MgSO4), and evaporated. The crude product was then purified by
column chromatography on silica (25% ethyl acetate in heptane as
eluent) to give 1.0 g (92%) of the title compound.
3-{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-propionic
Acid, 2. To a solution of 17 (1.0 g, 2.0 mmol) in toluene (20 mL)
and ethanol (50 mL) was added 1 N NaOH (10.0 mL), and the
reaction mixture was stirred for 16 h at room temperature. The
reaction mixture was concentrated in vacuo, and 1 N HCl (10 mL)
was added. The product was extracted with ethyl acetate (3 × 50
mL). The organic layers were combined, washed with water, dried
(MgSO4), filtered, and concentrated in vacuo. The residue was
recrystallized from warm ethanol (100 mL), which was concentrated
to 60 mL and cooled, to give 600 mg (56%) of the title compound.
Anal. (C24H20Br2O3) C, H.
Ethyl 3-(3-Hydroxy-phenyl)-propionate, 12. To an ice-cooled
solution of 3-(3-hydroxy-phenyl)-propionic acid (20.0 g, 120 mmol)
in ethanol was added dropwise thionyl chloride (8.8 mL, 120 mmol).
The mixture was stirred at room temperature overnight, concentrated
in vacuo, and submitted to flash chromatography (10% ethyl acetate
in toluene eluent) to give 23.3 g (100%) of 12.
The similar rank order of potency and the actual potencies
(EC50 values) of the tested compounds in PPARδ transactivation
(Table 1) and in FFA oxidation in vitro (Table 2), and the
relative lack of FFA oxidation by rosiglitazone and fenofibrate,
support previous findings of FFA oxidation in muscles primarily
being mediated by PPARδ.13,14
Based on the above data, 7 was selected for further in vivo
evaluation in mice. Before testing in mice, 7 was evaluated for
PPAR activity at the mouse receptors. Transactivation data
showed similar mPPARδ activity (7: EC50 ) 0.30 ( 0.11 µM,
167 ( 15%; GW501516: EC50 ) 0.054 ( 0.022 µM, 269 (
39%), as seen in the human assay, although potencies were
slightly lower. Likewise, 7 had no mPPARR or mPPARγ
activity up to 30 µM. The effects on plasma lipids of 7 and
GW501516 were evaluated in the double transgenic mouse
model (ApoB100/CETP-Tgn). The ApoB100/CETP-Tgn mouse
had received the human genes for ApoB100 and CETP. This
resulted in a changed plasma lipid profile from a typical rodent
profile, with high levels of HDL and low levels of LDL, to a
human-like lipid profile, with lower HDL levels and higher LDL
levels. In addition to the genetic modifications, the mice were
fed a high fat diet to induce dyslipidemia and insulin resistance.
At the end of the study, skeletal muscles were removed and
tested for FFA oxidation activity ex vivo.
GW501516 was tested at 6 mg/kg/day p.o., a dose shown to
give full efficacy in mice without liver weight increase.10 The
less-potent compound 7 was tested in three doses: 5, 10, and
20 mg/kg/day p.o. Unlike in previously published studies in
high fat fed C57BL/6J mice,10 PPARδ treatment did not change
food intake or body weight in ApoB100/CETP-Tgn mice (data
not shown). However, treatment with 7 changed plasma lipid
parameters in a dose-dependent manner to a less atherogenic
profile, with increased HDL and decreased LDL and TG (Figure
6A,B and Table 4). At the same time, HbA1c levels were
lowered, suggesting improved insulin sensitivity (Table 4).
3-{3-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-propionic
Acid Ethyl Ester, 18. The compound was synthesized from 12
and 10 as described for compound 17 in a 756 mg (93%) yield.

1500 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Sauerberg et al.
Figure 6. Plasma HDL cholesterol (panel A) and LDL cholesterol (panel B) after 6 weeks treatment of ApoB100/CETP-Tgn mice with GW501516
(9) and 7 (1).
Figure 7. FFA oxidation in skeletal muscle of ApoB100/CETP-Tgn mice after 6 weeks treatment with 7 and GW501516. The mainly oxidative/
slow, musculus soleus (type I fibers), is shown in panel A. The mainly glycolytic/fast, musculus extensor digitorum longus (EDL; type II fibers),
is shown in panel B. There was a significant correlation between increasing doses of 7 and FFA-oxidation in both soleus and EDL.
Table 4. Treatment Effects on Plasma Parameters in ApoB100/CETP-Tgn Mice
% change from vehicle treated controls (6 weeks treatment)
total
total
cholesterol
ketone
bodies
cmpd
HDL
4
LDL
triglycerides
glucose
insulin
HbA1c
GW501516
(6 mg/kg)
7
(5 mg/kg)
7
(10 mg/kg)
7
(20 mg/kg)
-15
-19
-14
2
-19
-14
-49
5
-4
-41
-13
24
14
33
-7
-12
-35
-15
-7
-3
-2
-5
-11
-8
3
-4
-9
-10
-29
-5
-12
3-{3-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-propionic
Acid, 3. The ester 18 (755 mg, 1.4 mmol) was hydrolyzed in 1 N
NaOH (5.6 mL) and ethanol (15 mL) for 16 h at room temperature.
Water (5 mL) was added, and the ethanol was removed by
concentration in vacuo. The mixture was neutralized with 6 N HCl.
The crude product was extracted with ethyl acetate (3 × 30 mL).
The organic layers were combined, dried (MgSO4), and evaporated.
The residue was dissolved in toluene, and the product was
precipitated with petroleum ether to give 430 mg (60%) of the title
compound. Anal. (C24H20Br2O3) C, H.
Methyl {4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-ac-
etate, 19. The compound was synthesized from methyl 4-hydroxy-
phenyl-acetate, 13,27and 10 as described for compound 17 in a 480
mg (62%) yield
{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-acetic Acid, 4.
To a solution of 19 (473 mg, 0.9 mmol) in THF (5 mL) and ethanol
(3 mL) was added 1 N NaOH (3 mL), and the reaction mixture
was stirred at 60 °C for 1 h and at room temperature overnight.
The title compound was isolated as the sodium salt, by filtration,
and washed with ethanol to give 375 mg (81%). Anal. (C23H18-
Br2O3, C6H14N4O2) C, H, N, arginine salt.
Ethyl 3-(3-Hydroxy-phenyl)-acetate, 14. To an ice-cooled
solution of 3-(3-hydroxyphenyl)-acetic acid (21.0 g, 138 mmol) in
ethanol was added dropwise thionyl chloride (10.1 mL. 138 mmol).
The mixture was stirred at room temperature overnight, concentrated
in vacuo, and submitted to flash chromatography (10% ethyl acetate
in toluene eluent) to give 23.8 g (77%) of 14.
Ethyl 3-{3-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-ac-
etate, 20. The compound was synthesized from 14 and 10 as
described for compound 17 in a 701 mg (88%) yield.
3-{3-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-phenyl}-acetic Acid,
5. Compound 20 was hydrolyzed as described for compound 3 to
give 5 in a 701 mg (76%) yield. Anal. (C23H18Br2O3) C, H.
Ethyl 3-Chloro-4-hydroxyphenylacetate, 15. To an ice-cooled
solution of 3-chloro-4-hydroxyphenylacetic acid (10.0 g, 53.0
mmol) in ethanol was added dropwise thionyl chloride (3.9 mL,
53.5 mmol). The mixture was stirred at room temperature for 48 h,
concentrated in vacuo, and submitted to flash chromatography

PPARδ Agonist with Full Efficacy on Lipid Metabolism
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1501
(graduated from toluene to 5% ethyl acetate in toluene eluent) to
give 11.0 g (97%) of 15.
Ethyl {4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-3-chloro-phen-
yl}-acetate, 21. The compound was synthesized from 15 and 10
as described for compound 17 in a 1.0 g (89%) yield.
{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-3-chloro-phenyl}-ace-
tic Acid, 6. Compound 21 was hydrolyzed as described for
compound 4 to give 6 in a 817 mg (82%) yield, isolated by filtration
as the sodium salt. Anal. (C23H19Br2ClO3, C6H14N4O2) C, H, N,
arginine salt.
(6 × 106 cells/flask) the day before transfection to give a confluence
of ∼50% at transfection. A total of 23 µg of DNA containing 17.5
µg pM1(x)LBD(R, γ, or δ), 2.7 µg pRLCMV, 2.2 µg pGL2(Gal4)5,
and 0.55 µg pADVANTAGE was transfected per flask using
FuGene transfection reagent according to the manufacturers instruc-
tions (Roche). The cells were harvested from the flasks 20-24 h
after transfection and plated in 96-well plates (110.000 cells/well
in media w/o phenol red). The relevant dilutions of test compound
were added immediately thereafter. The plates were further
incubated for 20 h before measurement of luciferase activity. The
luciferase assay was performed using the LucLite kit according to
the manufacturers’ instructions (Perkin-Elmer). Briefly, media
including test compound was aspirated and replaced with PBS.
LucLite substrate was added and light emission was quantified on
a Packard TopCount.
Human PPAR R, γ, or δ were obtained by PCR amplification
using cDNA synthesized by reverse transcription of mRNA from
human liver, adipose tissue, and placenta, respectively. Amplified
cDNAs were cloned into pCR2.1 and sequenced. The ligand binding
domain (LBD) of each PPAR isoform was generated by PCR
(PPAR R: aa 167, C-terminus; PPAR γ: aa 165, C-terminus,
PPARδ: aa 128, C-terminus) and fused to the DNA binding domain
of the yeast transcription factor GAL4 by subcloning fragments in
frame into the vector pM1,28 generating the plasmids pM1RLBD,
pM1γLBD, and pM1δLBD. Ensuing fusions were verified by
sequencing. The reporter gene was constructed by inserting an
oligonucleotide encoding five repeats of the GAL4 recognition
sequence (5 × CGGAGTACTGTCCTCCG(AG))29 into the vector
pGL2 promotor plasmid (Promega), generating the plasmid pGL2-
(GAL4)5. The plasmid pRLCMV (Promega) encodes a Renilla
luciferase used in the validation of the assay. The presence of the
pADVANTAGE plasmid (Promega) leads to an enhancement of
the luciferase signal.
All compounds were dissolved in DMSO and diluted at least
1:1000 upon addition to the cells.
Raw data is expressed in relative light units. All curve fittings
were done by nonlinear regression using GraphPad PRISM (Graph-
Pad Software, San Diego, CA).
In the experiment designed to show 7 was a partial agonist that
competes with GW501516 for binding to PPARδ, a dose response
curve of 7 was generated in the presence of 100 nM GW501516.
Fatty Acid Oxidation in L6 Line. The rat skeletal muscle cell
line L6 was cultured at 37 °C in a humidified atmosphere containing
5% CO2 in MEM Alpha (Gibco; 32571-028) supplemented with
10% foetal bovine serum, 100 U/mL penicillin, and 100 µg/mL
streptomycin. When confluent, the cells were differentiated by
changing the medium to MEM Alpha (Gibco; 32571-028)
supplemented with 2% foetal calf serum, 100 U/mL penicillin, and
100 µg/mL streptomycin. Experiments were performed after five
days of differentiation.
Methyl (2-Chloro-4-mercapto-phenoxy)-acetate, 16. A solution
of 2-chlorophenol (25.7 g, 200 mmol), potassium carbonate (41.4
g, 300 mmol), and ethyl bromoacetate (35.1 g, 210 mmol) in
2-butanone (240 mL) was stirred at 100 °C for 24 h. The reaction
mixture was filtered and evaporated. The residue was dissolved in
toluene (100 mL), washed with water (3 × 25 mL), dried, and
evaporated. The residue (37 g, 172 mmol) was dissolved in
dichloromethane (50 mL) and chlorosulfonic acid (93 g, 800 mmol)
was added slowly at -10 °C. The reaction mixture was stirred at
room temperature for 1 h. Ice water (25 mL) was added carefully,
and the mixture was extracted with dichloromethane (3 × 100 mL).
The combined organic phases were washed with water, dried, and
evaporated to give crude ethyl (4-chlorosulfonyl-2-chloro-phenoxy)-
acetate as an oil in a 47.5 g (76%) yield.
To a refluxing solution of red phosphorus (17 g, 566 mmol) and
I2 (2.4 g, 9.4 mmol) in glacial acetic acid (100 mL) was added
slowly a solution of ethyl (4-chlorosulfonyl-2-chloro-phenoxy)-
acetate (47.5 g, 151 mmol) in glacial acetic acid (1000 mL). The
reaction mixture was refluxed for 24 h. Water (20 mL) was added
carefully, and the mixture was refluxed for an additional 1 h. After
cooling, the reaction mixture was filtered and the filtrate was diluted
with water (500 mL). The mixture was extracted with dichlo-
romethane (3 × 100 mL), and the organic phases were washed
with water. After drying, the dichloromethane phase was evaporated
to give crude (2-chloro-4-mercapto-phenoxy)-acetic acid in a 17 g
(52%) yield. The crude acid was dissolved in methanol (50 mL),
and a solution of acetyl chloride (24 g, 310 mmol) in methanol
(200 mL) was added slowly at 10 °C. The reaction mixture was
stirred for 2 h. The reaction mixture was evaporated and the residue
was purified by column chromatography (eluent: ethyl acetate/
heptane (4:1)) to give pure 16 in a 17 g (93%) yield.
Methyl {4-[3,3-Bis-(4-bromo-phenyl)-allylsulfanyl]-2-ethyl-
phenoxy}-acetate, 22. Intermediate 10 (2.1 g, 5.5 mmol) and
tributylphosphine (1.7 g, 8.6 mmol) were dissolved in dry THF
(50 mL) and cooled to 0 °C under an atmosphere of nitrogen.
Azodicarbonyl dipiperidide (2.1 g, 8.6 mmol) was added, and the
reaction mixture was stirred for 5 min. Compound 16 (1 g, 4.2
mmol) was slowly added (5 min) and the stirring was continued
for 12 h at 5 °C. Water (10 mL) was added, and the mixture was
extracted with ethyl acetate (2 × 50 mL). The combined organic
extracts were dried and evaporated. The residue was purified by
HPLC to give the title compound in a 2.4 g (100%) yield.
{4-[3,3-Bis-(4-bromo-phenyl)-allylsulfanyl]-2-chloro-phenoxy}-
acetic Acid, 7. The ester 22 (350 mg, 0.6 mmol) was dissolved in
ethanol (10 mL). NaOH (1 N, 3 mL, 3 mmol) was added at room
temperature, and the reaction mixture was stirred for 18 h at 5 °C,
after which it was treated with 1 N HCl (15 mL) and extracted
with dichloromethane (2 × 20 mL). The combined organic phases
were dried and evaporated to give the title compound in a 230 mg
(68%) yield. Anal. (C23H17Br2ClO3S, C6H14N4O2, H2O) C, H, N,
arginine salt.
Differentiated L6 muscle cells were stimulated with PPARδ
agonist for 42 h (fresh medium and compound were given after
24 h) in differentiation medium, followed by 6 h of stimulation in
differentiation medium supplemented with 0.4 mM palmitate
containing 10 µCi/mL [9,10-3H]-palmitic acid (Perkin-Elmer; NET-
043) conjugated to 2% fatty acid free BSA (Sigma; A7030) by
sonication. The assay was terminated by the addition of 10% TCA.
Upon oxidation of the tritiated palmitate, 3H-H2O is generated and
3
appears in the medium. H-H2O was separated from the medium
by equilibration with water in a separate container at 50 °C o/n.
3
The quantity of H-H2O transferred to the water was measured
Methyl {4-[3,3-Bis-(4-bromo-phenyl)-allylsulfanyl]-2-methyl-
phenoxy}-acetate, 23. The compound was synthesized from methyl
(4-mercapto-2-methyl-phenoxy)-acetate23 (2.06 g, 9.7 mmol) and
10, as described for compound 22, in a 4.0 g (88%) yield.
{4-[3,3-Bis-(4-bromo-phenyl)-allylsulfanyl]-2-methyl-phenoxy}-
acetic Acid, 8. Compound 23 was hydrolyzed as described for
compound 7 to give 8 in a 482 mg (93%) yield. Anal. (C24H20-
Br2O3S) C, H.
by liquid scintillation counting. Compounds tested were diluted in
DMSO as 1000× stocks, giving a final concentration of 0.1%
DMSO in the medium upon stimulation. Concentrations ranging
from 0 to 30× EC50 for the individual compounds (determined in
the hPPARδ transactivation assay) were tested.
Glucose oxidation was measured in the same way as fatty acid
oxidation, with the exception that the tritiated palmitate was replaced
by 10 µCi/mL [5-3H]-D-glucose (Perkin-Elmer; NET-530). As a
positive control for the assay, FCCP (mesoxalonitrile 4-trifluo-
romethoxyphenylhydrazone (Fluka; 21857), a known uncoupler of
PPAR In Vitro Transactivation Assay. HEK293 cells were
grown in DMEM + 10% FCS. Cells were seeded in T-175 flasks

1502 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Sauerberg et al.
oxidative phosphorylation) was included in the glucose oxidation
measurements, and in addition, fatty acid oxidation measurement
for GW501516 was performed simultaneous with the glucose
oxidation assay.
10-3H]-palmitic acid (Perkin-Elmer; NET-043) conjugated to 2%
fatty acid free BSA (Sigma; A7030, as described in Thompson et
al.).31 Incubations were performed at 30 °C, with gentle agitation
(110/min), under continuous gassing with 95% O2-5% CO2. The
assay was terminated by addition of 10% TCA. Upon oxidation of
Data were expressed as fold over unstimulated cells, and curve
fitting was done by nonlinear regression using GraphPad Prism
(GraphPad software, SanDiego, CA). EC50 is the concentration
giving 50% of maximal response observed for a given compound.
To compare the efficacy of each compound, GW501516 was
included in every experiment, and maximal response is given as
% of maximal response of GW501516. Results are given as mean
( SEM from three to four experiments performed in triplicates.
Pharmacokinetics. The procedure has previously been described
in detail.30 Briefly, the compounds were dosed p.o. and i.v. to male
SD rats. The compounds were dissolved in 5% ethanol, 10% HPCD,
and phosphate buffer; pH 7.5-8.0. Blood samples were collected
in EDTA tubes. Each data point represents one animal.
3
the tritiated palmitate, H-H2O is generated and appears in the
medium. 3H-H2O was separated from the medium by equilibration
with water in a separate container at 50 °C o/n. The quantity of
3H-H2O transferred to the water was measured by liquid scintil-
lation counting.
Data are presented as mean ( SEM. Statistical analyses were
done using one-way ANOVA, followed by Dunnett’s multiple
comparison test or unpaired t-test with Welch correction. P values
less than 0.05 were considered significant, *p < 0.05 and **p <
0.01.
Glide2.5 Docking. The structure manipulation and surface
generation was performed in Maestro version 7.0.33 Glide calcula-
tions were performed with Impact version 3.5.34,35 Compound 7
was built in Maestro version 7.0, a formal charge of -1 was
assigned, and the molecule was minimized with the MMFFs force
field. The crystal structure of the PPARδ receptor crystallized with
GW2433 was used (1GWX) for docking.25 Default neutralization
zone (10-20 Å) and minimization (0.30 Å) was used in the
preparation of the protein. In the generation of the grid, default
vdW scaling was used (1.00) and the enclosing box was set 20 Å.
Compound 7 was docked with Glide version 2.5 using the SP mode
and the van der Waals radii of the ligand atoms were scaled by
0.8. Ten poses were generated in the docking procedure, and the
first pose was used.
Plasma samples were analyzed by high turbulence liquid chro-
matography combined with tandem mass spectrometry.
ApoB100/CETPtgn Mice. ApoB100/CETPtgn double transgenic
female mice (B6;SJL-Tgn(CETP)-Tgn(APOB100), Taconic Europe,
8680 Ry, Denmark), 9-11 weeks old, with a mean body weight
of 19.2 g ( 1.4, n ) 8/group, were fed a high fat diet (D01111201,
Research Diets Inc., USA) containing 17 kcal % protein, 43 kcal
% carbohydrate, and 40 kcal % fat to induce dyslipidemia and
insulin resistance. Fresh diet from the freezer was supplied every
day (5 g/mouse), which corresponds to ad libitum feeding. The
high fat feeding was started 3 weeks prior to administration of
compounds and was continued throughout the 6-weeks dosing
period. The animals were allowed to adapt to the laboratory
conditions during the 3-weeks prefeeding period. Tap water was
freely available in the home cages throughout the studies. A normal
12 h/12 h light/dark regime was operative (lights on at 0600 h) in
the stables and room temperature was held at 20-21 °C. All animal
procedures were conducted according to Novo Nordisk A/S Animal
Care approved protocols, and the experiments were done in
compliance with internal animal welfare and national guidelines.
Suspensions of compounds were made in the standard vehicle
0.2% CMC (Merch Eurolab 279294T, Lot 1009855) + 0.4%
Tween-80 (Merch Eurolab 8.22187.0500, Lot S31764043) in saline.
Fresh suspensions were made for seven days of dosing and kept at
+4 °C.
Acknowledgment. The technical assistance of Bjørn Bentz-
en, Alice Jørgensen, Otto Larsson, Per S. Klifforth, Nicolai
Steffensen, Lene Priskorn, Helle Bach, Annette Zeneca, Dorthe
D. Andersen, Trine N. Eriksson, and Helle Fjordvang is highly
appreciated.
Supporting Information Available: 1H NMR data on inter-
mediates and final products. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
The animals were dosed orally by gavage twice daily with either
vehicle or test compound. The dose volume was 10 mL/kg.
Body weight and food intake were measured once weekly. Blood
was drawn after 23 and 46 days treatment from the orbital plexus
(250 µL) in overnight fasted animals under isoflurane anesthesia.
The blood samples were collected in EDTA coated Eppendorf
tubes, centrifuged at 4000 × g for 10 min at 4 °C, and plasma was
transferred to Hitachi cups for analyses on a Hitachi 912 auto-
analyzer (Roche).
The measured parameters were as follows: total cholesterol,
HDL, LDL, triglycerides, glucose, HbA1c (all Roche), total ketone
bodies, and CETP (Wako) and insulin (Screening and Assay Dept.
244). CETP and insulin were measured by ELISA. The CETP level
in plasma was tested after three weeks of treatment with the PPARδ
agonists included in this study. The results showed that the CETP
level was not reduced. In contrast, CETP levels were found to be
up-regulated (data not shown). Why the beneficial effects found
(e.g., high HDL) after the treatment cannot be due to lack of CETP.
At the final blood collection on day 46, musculus soleus (mainly
red muscle, type I oxidative fibers) and musculus extensor digitorum
longus (EDL, mainly white muscle, type IIb glycolytic fibers) were
gently dissected and immediately transferred to pregassed (95%
O2-5% CO2) Krebs-Henseleit (KRH) buffer (in mM: 118.5,
NaCl; 4.7, KCl; 1.2, KH2PO4; 25, NaHCO3; 2.5, CaCl2; 1.2,
MgSO4; and 10, HEPES) supplemented with 10 mM glucose and
2% BSA. Fatty acid oxidation capacity was determined by
incubating the muscles for 90 min in pregassed KRH buffer
supplemented with 10 mM glucose, 60 nM insulin (human from
Novo Nordisk) and 0.2 mM palmitate containing 10 µCi/mL [9,-
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