Identification of a PPARδ agonist with partial agonistic activity on PPARγ

Article abstract of DOI:10.1016/j.bmcl.2009.04.151

The discovery and optimization of a series of potent PPARδ full agonists with partial agonistic activity against PPARγ is described.

Full text of DOI:10.1016/j.bmcl.2009.04.151

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3550–3554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a PPARd agonist with partial agonistic activity on PPAR
c
b
c
a
a
Richard V. Connors a, , Zhulun Wang , Martin Harrison , Alex Zhang , Malgorzata Wanska ,
*
Steve Hiscock d, Brian Fox a, Michael Dore e, Marc Labelle f, Athena Sudom b, Sheree Johnstone b,
Jinsong Liu b, Nigel P. C. Walker b, Anne Chai g, Karen Siegler g, Yang Li g, Peter Coward g,
*
a Department of Chemistry, Amgen, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Department of Molecular Structure, Amgen, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
c AstraZeneca Inc., Alderley Park, Cheshire SK10 4TF, UK
d Astex Therapeutics, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, UK
e Novartis Inc., 250 Massachusetts Ave., Cambridge, MA 02139, USA
f Lundbeck Research USA Inc., 215 College Road Paramus, NJ 07652-1431, USA
g Department of Biology, Amgen, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and optimization of a series of potent PPARd full agonists with partial agonistic activity
against PPAR is described.
Received 26 February 2009
Revised 28 April 2009
Accepted 30 April 2009
Available online 9 May 2009
c
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
PPARd
PPARg
Agonist
Modulator
(HDL). Rosiglitazone (Avandia ), a selective agonist of PPARc,
11 en-
TM
The peroxisome proliferator-activated receptors (PPARs) are
members of the nuclear receptor family of ligand-regulated tran-
hances insulin sensitivity, reduces plasma glucose an average of
60–80 mg/dL and exerts a moderate corrective effect on TGs, FFA
scription factors.1 There are three subtypes: PPAR
a
(NR1C1), PPARd
(NR1C2, also called PPARb), and PPAR
c
(NR1C3). The PPARs are
and HDL in type
2
diabetes mellitus (T2DM) patients.
essentially dietary fat sensors that maintain lipid and glucose
homeostasis through control of a network of genes involved in
metabolism. Their endogenous ligands include a diverse array of
GW501516, a selective agonist of PPARd, decreases low-density
lipoprotein (LDL), TGs and insulin by 29%, 56% and 48%, respec-
tively, and increases HDL by 79% in obese rhesus monkeys.12 Stud-
ies in rodents suggest that activation of PPARd also has beneficial
effects on obesity and insulin resistance.13 Considerable research
effort has focused on the development of selective PPAR agonists
for the treatment of T2DM and metabolic syndrome.1 However,
the importance of controlling both lipid and glucose levels in
fatty acids, eicosanoids and oxysterols.2 PPAR
a is expressed mainly
in the liver and, to a lesser degree, in muscle and heart tissue. Its
principal function is control of fatty acid metabolism,3 especially
in response to a prolonged fast.4 PPAR
c is expressed primarily in
adipose tissue, where it acts as a master regulator of adipocyte for-
mation.5 PPARd is expressed in many tissues, albeit at low levels in
the liver. Its primary functions are control of fatty acid catabolism
and energy homeostasis.6,7
T2DM has stimulated interest in PPAR
agonists, as well as PPAR /d/
pan agonists.16 As T2DM patients
receiving rosiglitazone often encounter significant weight gain,11
there has also been a drive to reduce this adverse effect. PPAR
a/
c14 and PPARd/c15 dual
a
c
Synthetic ligands have helped elucidate the biology of PPAR
a/c
receptors and revealed their utility as drug targets. Fenofibrate
dual agonists held promise in achieving this goal, owing to reports
TM
(Tricor ), a PPARa agonist that was developed as a hypolipidemic
that fibrates reduce weight gain in rodents without impacting food
before the PPARs were discovered,8–10 reduces triglycerides (TG)
and free fatty acids (FFA) and raises high-density lipoprotein
intake.17 Other workers have pursued PPARd/ dual agonists,18 rea-
c
soning that the increased fatty acid oxidation attending PPARd acti-
vation19 might diminish weight gain. Prompted by evidence that
PPAR
have opted to combine PPARd full agonism and PPAR
c
partial agonists show a reduced weight gain profile,20 we
* Corresponding authors. Tel.: +1 650 244 2890 (R.V.C.).
E-mail address: rconnors@amgen.com (R.V. Connors).
c
partial
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.151

R. V. Connors et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3550–3554
3551
agonism into a single ligand. Thus, here we describe our effort to
develop a PPARd/ agonist/modulator with acceptable pharmaco-
kinetic properties for in vivo studies.
c
In a recent patent,21 we disclosed a class of compounds possess-
ing potent PPARd agonist activity that increases HDL levels in high-
fat-fed Sprague–Dawley rats. Compound 1 (Fig. 1) was selected
from this series as our lead because of its favorable properties as
a PPARd/
for PPARd (Ki = 0.007
vation assay (Gal4 EC50 = 0.018
GW50151612). Its affinity for PPAR
it acts as partial agonist in the cell based assay (Gal4
EC50 = 0.46, 20% relative to rosiglitazone11). It is selective for PPARd
and PPAR versus PPAR (PPAR Ki = 1.1 M, Gal4 EC50 >10 M).
c
agonist/modulator. Most notably, 1 exhibits high affinity
M) and full agonism in the PPARd transacti-
M, 93% efficacy relative to
is weaker (Ki = 0.42 M) and
l
l
c
l
a
c
a
a
l
l
Finally, 1 exhibits acceptable PK properties, with a clearance in
rat of 0.06 L/h/kg and a Vdss of 0.7 L/kg following 0.5 mg/kg iv dos-
ing, and an oral bioavailability of 99% after 5.0 mg/kg po dosing.
Our strategy for optimizing 1 as a PPARd/c agonist/modulator
was to probe the effect of substitutes for the Cl atom on the central
ring, and when improved central ring analogs were identified, to
prepare libraries in which the CF3 aryl is diversified.
The affinity of compounds towards PPARd was evaluated using
a scintillation proximity assay, while their affinity for PPAR
assessed using a filtration assay.22 The selectivity of compounds
was evaluated using a PPAR scintillation proximity assay as a
counter-screen. Compounds exhibiting Ki values less than 0.1
in the ligand binding assays were assessed for agonist activity in
a transactivation assay in CV-1 cells, using a PPAR ligand binding
domain/GAL4 DNA binding domain expression construct and lucif-
erase reporter gene.23
The general synthetic route to the core structure of 1 is de-
scribed in Scheme 1.24 Ethyl aryloxyacetate 3 was obtained on
treatment of 2,3-dimethylphenol with ethylbromoacetate in
DMF/Cs2CO3. Chlorosulfonation of 3 followed by Sn reduction
yielded mercaptan 5, which was then converted to aldehyde 6 on
treatment with 4-fluoro-3-methoxymethyleneoxybenzaldehyde25
and Cs2CO3 in DMF. Compound 7 was then obtained via NaBH4
reduction of aldehyde 6, followed by formation of the benzyl chlo-
ride with MsCl/TEA. SN2 displacement of benzyl chloride 7 with 4-
hydroxybenzotrifluoride in DMF/Cs2CO3 furnished compound 8. In
the final steps, the MOM protecting group in 8 was removed with
4 N HCl in dioxane, the nascent phenol was alkylated with an
c was
a
lM
Scheme 1. Reagents and conditions: (a) ethylbromoacetate, Cs2C03, DMF, 50 °C,
4 h, 95%; (b) chlorosulfonic acid, 0 °C–rt, 4 h, 85%; (c) Sn, EtOH, HCl/dioxane,
0–80 °C, 4 h, 80%; (d) 4-fluoro-3-methoxymethyleneoxybenzaldehyde, Cs2C03,
DMF, 50 °C; 16 h, 60% (e) (1) NaBH4/THF 0 °C–rt, 4 h, 95%; (2) MsCl/TEA, 0 °C–rt,
16 h, 95%; (f) 4-hydroxybenzotn-fluonde, Cs2C03, DMF, 60 °C, 16 h, 60%; (g) (1)
4NHC1/dioxane, 0 °C, l h, 95%; (2) Br(CH2)3OMe, Cs2C03, DMF, 60 °C, 4 h, 80%; (3)
NaOH/EtOH, 0 °C–rt, 2 h, 95%.
appropriate alkyl halide in DMF/Cs2CO3 and the ethyl ester was
saponified in NaOH/EtOH.
The first series of compounds examined the effect of replacing
the Cl atom of 1 with ethers (Table 1). As compound 1 is already
appreciably lipophilic, preference was given to low molecular
weight and polar central ring substitutents. In a further effort to re-
duce cLog P, the 2,3-dimethyl phenyl mercaptan was used in the
synthesis instead of the tetrahydronaphthyl headpiece. Previous
experience with 1 suggested that the 2,3-dimethyl headpiece
would be tolerated but would likely modulate PPARa/c selectiv-
ity.21 As the data in Table 1 show, ligands with heteroatom-con-
taining substituents on the central ring generally have lower
activity in the dGal4 functional assay. While compounds 9, 11, 12
and 16 all showed strong binding to PPARd, only weak activity
was observed in the dGal4 cell assay. The data in Table 1 also show
a correlation between the basicity of nitrogens present in central
ring substituents, PPARd affinity and dGal4 activity. Pyridines 14
and 15, for instance, both showed low-nM affinity for PPARd, while
the morpholine analog 17 was 5-fold less potent and the tertiary
amine 18 was inactive. All four compounds showed lower activity
in the dGal4 assay, while the most basic ligands 17 and 18 were
inactive. Encouragingly, compound 10 stood out for its high affinity
for PPARd, its activity in the PPARd cell-based assay, and its binding
affinity for PPARc
. The cocrystal of 10 in PPARd26 revealed a full
agonist binding mode,27 in which the three contiguous aryl rings
wrap around helix-3 (H3), the carboxylate interacts with Y473,
H323 and H449, and the CF3-aryl is positioned in close proximity
to C285 (Fig. 2). The propargyl substituent projects into the central
ligand binding pocket (LBP), aligned along the H7 backbone to-
wards H5. The acetylene bond is parallel to the F368 amide car-
bonyl and within pi-stacking distance of the side chain phenyl.
Figure 1. Published PPAR ligants and lead compound 1.

3552
R. V. Connors et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3550–3554
Table 1
In vitro binding and transactivation data for the central ring side-chain ether library
Compound
SPA K; (
dSPA K; (
Filt Kj (
Gal4EC50
%Max activationb
dGal4EC50
M)a
%Max activationc
9
10
11
12
13
14
15
16
17
18
a
l
l
l
M)
M)
M)
1.2
0.008
2.7
>10
—
1.9
88%
>10
—
0.21
0.004
0.30
6.1
48%
0.054
86%
3.0
1.3
0.011
5.9
>10
—
1.7
95%
>10
—
1.2
0.016
2.2
>10
—
1.1
89%
>10
—
0.94
0.035
0.66
>10
—
1.3
0.036
4.1
>10
—
2.9
78%
>10
—
1.3
0.047
5.0
>10
—
4.1
76%
>10
—
1.1
0.070
7.6
>10
—
7.1
84%
>10
—
1.2
0.27
4.0
>10
—
>10
—
>10
—
>10
>10
>10
>10
—
>10
—
>10
—
c
a
{
l
M)a
{
l
2.2
95%
>10
—
c
Gal4EC50
(l
M)a
%Max Activationd
40%
a
EC50 values are the molar concentrations of test compounds that afford 50% of the maximal reporter activity.
Relative to GW2433.
Relative to GW501516.
Relative to rosiglitazone.
b
c
d
PPAR
PPARd or PPAR
as well with very similar dGal4 activities for 19–21, but with
potencies trending from 4.9 M (43%) for compound 19 to 0.16 lM
c
affinity increases nearly 40-fold with minimal effects on
. This trend is observed in the functional assays
Gal4
a
c
l
(34%) for 21. Examination of hydroxyl-substituted analogs of 20
and 21 revealed that compound 24 posesses a nearly 10-fold in-
crease in PPARd affinity. The PPARd cocrystal structures of 21 and
24 are essentially identical to that shown for 10 (Fig. 2), except
for the proximity of the pentynyl hydroxyl in 24 and the F327
amide carbonyl on H5.28 Despite having significant affinity for
PPARd/
in the cell-based assays. Compound 21 is the best exemplar of
the series, maintaining sub- M activity in the Gal4 assay and high
c, the hydroxyl-substituted analogs are less active than 21
l
c
activity in the dGal4 assay. The cocrystal of compound 21 in
PPARc29 (Fig. 3) revealed a binding geometry in which its three
rings are linearly aligned along H3, with the carboxylate-AF2 inter-
action, known to be required for full agonism,27 replaced by a close
association with the R288 side chain. The pentynyl group, crucial
Figure 2. X-ray crystal structure of compound 10 in PPARd.26
for PPARc affinity, occupies a hydrophobic pocket circumscribed
by H3 and H7. The difference in binding mode between the two
PPAR isoforms is highlighted in Figure 4.
Armed with a side chain substituent conferring full agonistic
activity on PPARd and partial agonistic activity on PPARc, attention
was next directed to optimizing the CF3-aryl tailpiece. The com-
pounds listed in Table 3 summarize the results obtained for a fo-
cused library of tailpiece variants. Shifting the tailpiece
trifluoromethyl function to the meta and ortho positions progres-
This suggests that ligands with larger central ring substituents
have lower binding affinities because H7 and F368 form a rela-
tively small pocket which is well-filled by the acetylene, but too
small for larger substituents.
Insights derived from the compound 10 PPARd cocrystal struc-
ture were figured into the design of the next set of compounds.
As in vitro data for compound 121 suggested that the selectivity be-
sively diminishes PPARd affinity and functional activity. PPAR
c
tween PPARa and PPARc might be favorably impacted by shifting
affinity is maintained in the meta-substituted ligand 29 but is re-
duced in 30, which is consistent with the cell-based data. Replacing
the trifluoromethyl function with Cl diminishes PPARd affinity and
to the tetrahydronaphthyl headpiece, it was used as the scaffold
in this compound set. In selecting side chain substituents, empha-
sis was placed on appending heteroatoms to the end of the propar-
gyl substituent that could potentially form productive interactions
functional activity, as well as PPAR
analog, 32 shows a similar profile, with concomitant loss of PPAR
affinity. Pyridyl analog 33 is less active in the PPARd/ cellular as-
c cellular activity. The 2-methyl
c
with H5 in PPARd, while exploring new binding territory in PPAR
As the data for compound 19 in Table 2 show, shifting to the tetra-
hydronaphthyl headpiece does impact PPAR affinity, increasing
c.
c
says, while changing the CF3-aryl oxygen to nitrogen (compound
34) diminishes binding to all PPAR isoforms. Saturated aliphatic
substituents (35–37) diminish PPARd binding, but shift
cacy into the full agonist range. While compound 21 emerged with
a/c
the Ki for each roughly fourfold. More encouragingly, as methyl or
ethyl groups are added to the propargyl terminus of 19 (20–21),
cGal4 effi-

R. V. Connors et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3550–3554
3553
Table 2
In vitro binding and transactivation data for the central ring alkynyl ether library
Compound
SPA Ki ( M)
dSPA Ki ( M)
Filt Ki ( M)
Gal4EC50
M)a
%Max
activationb
19
20
1.1
0.006
0.077
>10
21
22
1.9
0.022
0.084
>10
23
1.7
0.002
0.21
>10
24
1.7
0.0006
0.043
>10
25
2.9
0.006
0.079
>10
26
2.0
0.009
0.020
>10
27
1.7
0.004
0.044
>10
28
1.0
0.016
0.025
>10
a
l
l
l
0.96
0.007
1.2
0.88
0.005
0.033
>10
c
a
3.9
(l
9%
—
—
—
—
—
—
—
—
—
8Gal4EC50
%Max
(
l
M)a 0.052
87%
0.043
83%
0.044
94%
1.4
89%
0.22
91%
2.0
99%
1.2
99%
0.34
86%
0.47
96%
0.68
99%
activationc
c
Gal4EC50
%Max
activationd
(
l
M)a 4.9
43%
0.70
23%
0.16
34%
2.2
81%
5.9
79%
>10
—
3.3
99%
0.61
21%
1.4
38%
1.6
66%
a
b
c
EC50 values are the molar concentrations of test compounds that afford 50% of the maximal reporter activity.
Relative to GW2433.
Relative to GW501516.
Relative to rosiglitazone.
d
Figure 4. X-ray crystal structure of compound 10 in PPARd with compound 21,
from an aligned PPAR
, superimposed.29
c
Figure 3. X-ray crystal structure of compound 21 in PPARc 29
.
the best balance of PPARd and PPAR
set, compounds such as 29, 33 and 36 show that the tailpiece sub-
stituent can impart a range of efficacies on PPAR
c
activity of the ligands in this
and a Vdss of 1.4 L/kg following 0.5 mg/kg iv dosing, and an oral
bioavailability of 26% after 2.0 mg/kg po dosing. Compound 21 is
shown to adopt a full agonist geometry in PPARd,28 and a predom-
c.
During the course of these studies, central ring and tailpiece
inantly antagonist or partial agonist binding geometry in PPARc 29
.
modifications of compound 1 have yielded compound 21, a full
Modification of the tailpiece aryl yields ligands with subtype selec-
agonist of PPARd (Ki = 0.005
partial-agonist activity on PPAR
[34%] M) and selectivity against PPAR
>10 M). Compound 21 displays a clearance in rat of 0.5 L/h/kg
l
M, Gal4 EC50 = 0.044 [94%]
(Ki = 0.033 M, Gal4 EC50 = 0.16
(Ki = 0.88 M, Gal4 EC50
l
M), with
tivities ranging from PPARd full agonists with partial agonistic
c
l
activity on PPAR
compound 21 a useful tool for evaluating the in vivo effects of
PPARd full agonism combined with partial PPAR agonistic activity.
c to PPARc full agonists. These properties render
l
a
l
l
c

3554
R. V. Connors et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3550–3554
Table 3
In vitro binding and transactivation data for selected tailpiece aryl ether analogs
Compound
SPA Ki ( M)
dSPA Ki ( M)
Filt Ki ( M)
Gal4EC50
%Max activationb
8Gal4EC50
M)a
%Max activationc
21
29
1.3
30
31
2.0
32
0.8
33
7.9
0.011
0.084
>10
—
34
35
36
2.6
37
4.1
0.12
0.11
>10
—
a
l
l
l
0.88
0.005
0.033
>10
0.93
0.12
0.17
>10
—
1.1
88%
4.5
5.9
0.66
>10
>10
—
3.1
75%
—
5.5
1.2
0.20
>10
—
>10
—
1.5
96%
0.035
0.030
>10
—
0.42
83%
0.30
26%
0.036
0.039
>10
—
0.35
87%
0.56
37%
0.028
0.15
>10
—
0.37
70%
0.61
38%
0.15
0.076
>10
—
0.90
95%
0.50
92%
c
a
(
l
M)a
—
(l
0.044
94%
0.16
34%
1.2
1.5
99%
0.49
65%
81%
0.39
73%
c
Gal4EC50
(l
M)a
%Max activationd
64%
—
a
EC50 values are the molar concentrations of test compounds that afford 50% of the maximal reporter activity.
Relative to GW2433.
Relative to GW501516.
Relative to rosiglitazone.
b
c
d
Auwerx, J.; Yanagisawa, M.; Kodama, T.; Sakai, J. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 15924.
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22. hPPARd ligand binding was directly measured using a scintillation proximity
assay. 0.1 ng/
ml) and 0.1 ug/
l
l protein, 10 nM radioligand ([3H]-GW2433, 50 Ci/mmol, 1 mCi/
ll polylysine SPA beads were incubated for 1 h at room
temperature in binding buffer (10 mM K2HPO4, 10 mM KH2PO4, 2 mM EDTA,
50 mM NaCl, 10% (v/v) glycerol, 2 mM CHAPS, and 1 mM DTT, pH 7.1). Plates
were read on a Packard TopCount. Conditions for hPPAR
hPPARd except that 2 ng/ l protein and 2 ug/ l beads were used. For hPPAR
ligand binding was directly measured using a filtration assay. 2.4 ng/ l protein,
a were the same as
l
l
c,
l
20 nM 3H-rosiglitazone (20 Ci/mmol, 1 mCi/ml), and 10% glutathione bead
slurry were incubated for 1 h at room temperature in binding buffer (10 mM
Tris–HCl, 50 mM KCl, 1 mM DTT, 0.01% NP-40, and 0.02% BSA, pH 8.0) in a Uni-
Filter 350 96-well assay plate (Polyfiltronics). The reactions were filtered under
vacuum and washed twice with binding buffer. Scintillant was added and
plates were read on a Packard TopCount.
23. CV1 cells were plated at 15,000 cells/well in 96-well plates in DMEM/F12 + 10%
charcoal/dextran-treated FBS. Cells were transfected with plasmid DNA
encoding the DNA-binding domain of the yeast transcription factor GAL4
fused to the ligand binding domain of hPPARa, hPPARd, or hPPARc, and a
luciferase reporter gene driven by four copies of the Gal4 promoter. Effectene
was used as the transfection reagent. Four hours post-transfection, test
compounds were added, and luciferase activity was measured 20 h later.
24. All compounds were characterized by 1H NMR and LC/MS and their purity
determined to be greater than 95% by reverse phase HPLC.
25. 4-Fluoro-3-methoxymethyleneoxybenzaldehyde was prepared from 4-fluoro-
3-methoxybenzaldehyde via BBr3 demethylation followed by protection of the
resulting phenol with MOM-Cl/TEA.
26. PPARd X-ray structure of compound 10 (3GZ9).
27. Nagy, L.; Schwabe, W. R. Trends Biochem. Sci. 2004, 29, 317.
28. Unpublished results.
29. PPARc X-ray structure of compound 21 (3H0A).