3,4,5-Trisubstituted isoxazoles as novel PPARδ agonists: Part 1

Article abstract of DOI:10.1016/j.bmcl.2006.05.055

We report the identification of a novel series of trisubstituted isoxazoles as PPAR activators from a high-throughput screen. A series of structural optimizations led to improved efficacy and excellent functional receptor selectivity for PPARδ. The isoxazoles represent a series of agonists which display a scaffold that lies outside the typical PPAR agonist motif.

Full text of DOI:10.1016/j.bmcl.2006.05.055

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4376–4380
3,4,5-Trisubstituted isoxazoles as novel PPARd agonists: Part 1
Robert Epple,a,* Ross Russo,a Mihai Azimioara,a Christopher Cow,a
Yongping Xie,a Xing Wang,a John Wityak,a Don Karanewsky,a Andrea Gerken,b
Maya Iskandar,b Enrique Saez,b H. Martin Seidelb and Shin-Shay Tianb
aDepartment of Medicinal Chemistry, The Genomics Institute of the Novartis Research Foundation,
10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
bDepartment of Lead Discovery, The Genomics Institute of the Novartis Research Foundation,
10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
Received 27 March 2006; accepted 16 May 2006
Available online 5 June 2006
Abstract—We report the identification of a novel series of trisubstituted isoxazoles as PPAR activators from a high-throughput
screen. A series of structural optimizations led to improved efficacy and excellent functional receptor selectivity for PPARd. The
isoxazoles represent a series of agonists which display a scaffold that lies outside the typical PPAR agonist motif.
Ó 2006 Elsevier Ltd. All rights reserved.
The metabolic syndrome is defined by the presence of
HO2C
O
conditions associated with lipid and/or glucose homeo-
stasis abnormalities. These conditions comprise dyslipi-
demia, obesity, elevated blood pressure, insulin
resistance, and a pro-inflammatory state. All of the
above are recognized risk factors for cardiovascular dis-
ease and type 2 diabetes.1–4
GW501516 (GSK)
α/γ/δ: 1.1/0.9/0.001
S
N
S
CF3
Me
Et
Cl
S
HO2C
L-165461 (Merck)
N
α/γ/δ: 1.3/0.1/0.012
O
O
Pr
The peroxisome proliferator-activated receptors
(PPARs) are ligand-activated transcription factors in-
volved in the regulation of lipid and glucose homeosta-
sis. Dyslipidemia and insulin resistance, two major
components of the metabolic syndrome, are commonly
treated with fibrates and TZDs, drugs that target
PPARa and PPARc, respectively. The pharmacological
role of these receptors has been elucidated by the avail-
ability of these compound classes.5,6 The synthetic li-
gands for the PPARa and PPARc subtypes have been
reviewed extensively.7–11
Figure 1. Early PPARd agonists with reported EC50 values for
transactivation of the three PPAR subtypes in micromolar.
(Fig. 1) have been used for in vivo studies in rodents
and NHPs,12,17–21 and GW501516 is currently in phase
II clinical trials for its antihyperlipidemic properties.
However, the considerable crossactivity of these com-
pounds to other PPAR subtypes makes it difficult to as-
sign their pharmacological effects solely to PPARd.
Herein, we report a compound class with novel structur-
al features and improved selectivity to further investi-
gate the role of PPARd in energy metabolism.
A third subtype, PPARd, is the least well understood,
and is believed to also play a role in energy homeosta-
sis.12 To date only a few reports claim the synthesis of
PPARd agonists.13–16 GW501516 and L-165461
The PPARd agonists reported to date were discovered
using several strategies: GW501516 was optimized from
a library of hydrophobic carboxylates,13 L165461 was
derived from an in silico approach to find novel PPARc
agonists using TZDs as structural templates.14 Recently,
the selective Bayer compound 33 was identified from a
PPARa agonist program,15 and we reported the emer-
Keywords: PPARd agonists; Metabolic syndrome; Energy homeostasis;
Isoxazoles.
*
Corresponding author. Tel.: +1 858 812 1720; fax: +1 858 812
1848; e-mail: repple@gnf.org
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.055

R. Epple et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4376–4380
4377
in vitro PPARd transactivation assays (<20% efficacy
compared to GW501516). To increase activity to a de-
sired full agonist, we first tried to incorporate a carbox-
ylate into the isoxazole scaffold. Based on a comparison
of compound 1 with the above mentioned common ago-
nist motifs and a recent communication of an isoxazole
agonist for the FXR nuclear receptor,22 we hypothesized
that the p-CF3-o-NO2-phenyl group in position 4 of the
isoxazole ring could be replaced by a HG typical for
PPAR activation. The commercially available isoxazole
2 was reduced and chlorinated to intermediate 3, the
chloride was then displaced in neat ethylene glycol
(Scheme 1). Mitsunobu coupling with the HG interme-
diate 4 followed by saponification gave compound 5.
Surprisingly, neither compound 5 nor any other analogs
displaying various L and HG in position 4 showed activ-
ity for the PPARs.
HO2C
HO2C
O
O
Cl
Cl
Cpd 33 (Bayer)
O
α/γ/δ: NA/NA/0.003
N
N
H
CF3
LBX001 (GNF)
O
α/γ/δ: >10/>10//0.01
CF3
Figure 2. Recent PPARd agonists with reported EC50 values for
transactivation of the three PPAR subtypes in micromolar.
gence of LBX001 from computer-aided drug design
(Fig. 2).16
Next, we rationally designed compound 9 from a
superposition of compound 1 with GW501516, which
places the HG in position 5 of the isoxazole ring
(Scheme 2). The oxime 6 was in situ transformed into
the nitrile oxide by NCS chlorination and regioselec-
tively cyclized with propargyl alcohol. The hydroxyl
functionality was mesylated to yield isoxazole 7. Sub-
stitution of intermediate 8 with HG 4 and saponifica-
tion of the corresponding methyl ester gave isoxazole
9. Again, compound 9 was inactive against all three
PPAR subtypes.
In order to discover PPARd selective agonists on novel
structural scaffolds, we performed a high throughput
screen (HTS) on an unbiased library of ꢀ1 million
compounds based on a cell-based transactivation assay
using a Gal4-PPARd LBD chimeric construct. A
number of hits sharing a common isoxazole motif such
as compound 1 were identified (Fig. 3).
A typical PPAR pharmacophore consists of three parts,
namely a head group (HG), a linker (L), and a tail group
(TG).8 The HG usually consists of a phenyl ring bearing
a carboxylate (or surrogate) functionality, which enables
the molecule to form the key hydrogen bonding interac-
tion with the AF2 helix of the receptor and holds the
receptor in an active conformation. A flexible linker L
connects the HG to a hydrophobic TG (usually a het-
eroaromatic system), which fills the rest of the large
and predominantly hydrophobic binding pocket.
Finally, the docking results of compound 1 with the
structure of the PPARd ligand-binding domain co-crys-
tallized with GW243323 revealed best scores when
substituting the aryl ring in position 3 of the isoxazole
with a carboxylate bearing HG. Starting from 3-chlo-
ro-4-hydroxy-phenylacetic acid 10 esterification, trifla-
tion, and palladium-mediated cyanide displacement
gave intermediate 11 (Scheme 3). The cyanide of inter-
mediate 11 was reduced to the aldehyde with Raney-
Nickel alloy, reacted to the oxime with hydroxylamine,
and activated to the chloroxime 12 by NCS chlorina-
tion. The phenol of intermediate 13 was alkylated with
excess 1,2-dibromoethane, then reacted with 2-butyne-
1-ol to give alkyne 14. Cyclization of intermediates 12
and 14 under basic conditions gave a mixture of two reg-
ioisomeric isoxazoles, which could be chromatographi-
cally separated. Saponification of the regioisomer with
the methyl group in position 5 gave the isoxazole 15.
Compound 15 displayed improved potency and efficacy
for activation of PPARd, but was also active on the
PPARa subtype (Table 1). The other regioisomer was
inactive (data not shown).
The isoxazole hits from our HTS did not display a car-
boxylate moiety or a surrogate commonly seen in PPAR
agonists (except for the ortho nitro group in the phenol
substituent), which may explain their low efficacy in the
1
O
2
5
Cl
N
NO2
PPARα PPARγ PPARδ
4
O
3
>10
>10
0.23
(19%)
O
Cl
O
CF3
1
Figure 3. An isoxazole HTS hit from the PPAR screen with EC50 (lM)
and efficacy values for PPAR transactivation.
HO
O
CO2Me
O
O
Cl
N
O
Cl
N
Cl
N
4
a)-b)
c)-e)
O
O
CO2H
Cl
Cl
Cl
Cl
O
CO2H
5
3
2
Scheme 1. Reagents and conditions: (a) LAH, THF, rt, 12 h (92%); (b) SOCl2, DCM, rt, 1 h (91%); (c) ethyleneglycol, NaH, 80 °C, 4 h (65%); (d)
PPh3, DEAD, DCM, rt, 5 h (70%); (e) 1 N LiOH, dioxane, 60 °C, 5 h (95%).

4378
R. Epple et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4376–4380
HS
OH
O
Cl
N
Cl
N
O
CO2Me
Cl
N O
8
a)-c)
S
d)-e)
OMs
Cl
Cl
Cl
O
CO2H
9
7
6
Scheme 2. Reagents and conditions: (a) NCS, DMF, rt, 1 h; (b) propargyl alcohol, NEt3, DCM, rt, 5 h (90%); (c) MsCl, NEt3, DCM, 0 °C, 30 min
(85%); (d) Cs2CO3, CH3CN, rt, 18 h (95%); (e) 1 N LiOH, THF, rt, 12 h (95%).
OH
Cl
Cl
Cl
N
OH
CN
a)-c)
d)-f)
Cl
HO2C
MeO2C
MeO2C
10
11
12
NO2
NO2
OH
g)-h)
O
i)-j)
O
F3C
F3C
13
14
O
Cl
N
NO2
O
O
HO2C
15
CF3
Scheme 3. Reagents and conditions: (a) MeOH, H2SO4, reflux, 8 h, (quant.); (b) Tf2O, NEt3, DCM, 0 °C, 1 h (95%); (c) Zn(CN)2, Pd(PPh3)4, DMF,
80 °C, 30 h (70%); (d) Raney–Nickel alloy, HCO2H, 110 °C, 10 h (55%); (e) H2NOHÆHCl, Na2CO3, H2O, rt, 2 h, (90%); (f) NCS, DMF, HCl (g), rt,
30 min; (g) 1,2-dibromoethane, Cs2CO3, CH3CN, 90 °C, 1 h (75%); (h) 2-butyne-1-ol, 4 M NaOH, lW (150 °C, 3 min) (30%) (i) NEt3, DCE, 90 °C,
6 h (60%); (j) 1 N LiOH, THF, 60 °C, 36 h (95%).
Table 1. In vitro activities for PPARs (subtypes a, c, and d) in cell-based transactivation assays (GAL4-PPAR LBD)
O
Cl
N
R3
R1
X
O
HO2C
R2
Compound
X
R1
R2
R3
a (lM)
c (lM)
d (lM)
%eff
15
CH2O
Me
Me
Me
Me
Me
Me
Et
NO2
NO2
H
CF3
CF3
CF3
H
0.22
0.34
>10
>10
>10
>10
>10
7.11
>10
1.67
>10
>10
>10
>10
>10
1.23
>10
1.25
>10
>10
>10
>10
>10
>10
>10
3.23
>10
>10
>10
>10
>10
>10
0.13
0.004
>10
48
67
—
—
43
63
71
67
26
68
72
66
19
44
80
100
21a
21b
21c
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONMe
CH2O
NO2
CF3
Cl
>10
21d
21e
CF3
Cl
0.26
0.31
0.11
0.09
2.59
0.03
0.01
0.08
3.67
0.19
0.03
0.003
24a
24b
24c
Cl
Cl
Cl
Cl
i-Pr
t-Bu
cyPr
2Fur
Ph
Cl
Cl
Cl
Cl
24d
24e
Cl
Cl
Cl
Cl
24f
25
Ph
Ph
Cl
Cl
Cl
Cl
26
29a
GW501516
COCH2
Ph
Cl
Cl
Efficacies are reported relative to GW501516 (%eff = 100%).
After identification of the HG location in position 3 of
the isoxazole core, we turned our attention to the substi-
tuent in position 4. In order to investigate different sub-
stituents in position 4, we replaced the ether with an
amide functionality. The synthesis shown in Scheme 4
gave not only better access to various analogs, but
also implemented a higher yielding, regioselective cycli-
zation step. t-Butyl acetoacetate 16 was deprotonated

R. Epple et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4376–4380
4379
O
Cl
N
O
a)
b)-d)
OH
R2
OK
O
O
O
e),f)
NH2
R3
R2
OtBu
OtBu
Cl
R3
MeO2C
O
16
17
20a-e
19a: R2 = NO2, R3 = CF3
19b: R2 = NO2, R3 = H
19c: R2 = H, R3 = CF3
19d: R2 = CF3, R3 = CF3
19e: R2 = Cl, R3 = Cl
18
g),h)
O
Cl
N
R2
NH
O
R3
HO2C
O
21a-e
Scheme 4. Reagents and conditions: (a) KN(TMS)2, Et2O, rt, 1 h; (b) 12, CH3CN, 0 °C, 3 h (85%); (c) TFA, rt, 4 h (quant.); (d) SOCl2, toluene,
reflux, 3 h (90%); (e) BrCH2CH2NHBoc, Cs2CO3, CH3CN, 90 °C, 6 h, (50–90%); (f) DCM/TFA 95:5, rt, 1 h (quant.); (g) NEt3, DCM, rt, 30 min
(60%); (h) 1 N LiOH, THF, 60 °C, rt (95%).
to intermediate 17 using potassium bis(trimethylsi-
lyl)amide. Intermediate 12 was in situ transformed into
the nitrile oxide with triethylamine, then cyclized with
enolate 17 at low temperature to give selectively the cor-
rect isoxazole regioisomer. Selective saponification of
the t-butyl protected ester in TFA and activation of
the carboxylate to the acid chloride intermediate 18
was followed by amide formation with the correspond-
ing amines such as 20a–e. Saponification of the head
group methyl ester gave final products 21a–e.
otherwise identical molecules compared to compound
21a led to complete loss of activity. Since a nitro group
presented a potential toxicological liability, we contin-
ued to search for a replacement. A 2,4-bistrifluorometh-
ylphenyl (21d) or a 2,4-dichlorophenyl group (21e) were
the only replacements that maintained some PPARd
activity, although it dropped by a factor of 100.
Last, we examined different substituents in position 5 of
the isoxazole ring. We found that analogs were best
accessible by reacting the appropriate b-ketoester with
amine 20e to give amides 23a–e (Scheme 5), which were
then cyclized and saponified to compounds 24a–e in a
similar fashion to earlier described procedures. The
SAR in this part of the molecule turned out to be rather
broad, with aromatic rings such as a 2-furanyl (com-
pound 24e) or a phenyl (compound 24f) displaying best
activity and selectivity. Other heteroaromatic systems or
introducing substituents into the phenyl ring did not
lead to any further improvement (data not shown).
Switching from the ether of compound 15 to the amide
of compound 21a is not only tolerated, but also im-
proves both potency (4 nM) and selectivity for PPARd
activation (Table 1). We quickly realized that many
amines employed as part of a first round broad survey
in the amide formation gave inactive compounds (data
not shown). The narrowness of the SAR in position 4
was best exemplified by the fact that omitting either
the nitro (21b) or the trifluoromethyl group (21c) in
O
a),b)
Cl
N
c),d)
22a: R1 = Et
R1
O
O
OK
O
Cl
Cl
22b: R1 = iPr
22c: R1 = tBu
22d: R1 = cyPr
22e: R1 = 2-furyl
22f: R1 = Ph
O
R1
OMe
R1
N
H
NH
O
Cl
HO2C
O
Cl
23a-f
24a-f
Scheme 5. Reagents and conditions: (a) 20e, toluene, lW (160 °C, 10 min) (40–60%); (b) KN(TMS)2, Et2O, rt, 1 h; (c) 12, CH3CN, rt, 3 h (60–80%);
(d) 1 N LiOH, THF, rt, 12 h (95%).
O
Cl
Cl
N
HO2C
O
a)-c)
Cl
d),e)
O
OK
Cl
O
O
29a
O
O
Br
O
EtO
Cl
N
Cl
27
28
O
Cl
Cl
HO2C
29b
Scheme 6. Reagents and conditions: (a) 19e, Cs2CO3, CH3CN, 50 °C, 7 h, (90%); (b) acetophenone, NaH, dioxane, reflux, 3 h; (c) KN(TMS)2,
CH3CN, rt, 1 h; (d) 12, CH3CN, rt, 3 h (75%); (e) 1 N LiOH, THF, rt, 12 h (95%).

4380
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(compound 26; synthetic procedure according to
Scheme 3) resulted in a drastic reduction of activity,
whereas a ketomethylene replacement of the amide
(compound 29a) slightly improved both potency and
efficacy. Unfortunately, the depicted synthesis route to
compound 29a (Scheme 6) predominantly gave the
undesired, inactive regioisomer 29b (ratio 29a/
29b ꢀ 1:7).
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In summary, we were able to identify a novel isoxazole
scaffold displaying PPAR activity based on hits derived
from a high throughput screen. In an attempt to im-
prove the activity to a full agonist profile, we designed
and synthesized a number of analogs showing various
degrees of potency, efficacy, and selectivity for the
PPAR subtypes. Compounds 24e and 29a are potent
and highly selective activators of the PPARd receptor.
Notably this series is different from the common PPAR
agonist motifs reported in the literature. The com-
pounds do not seem to require a flexible linker between
functional head and hydrophobic tail, a unique feature
distinct from most reported PPAR modulators. The
isoxazoles presented may be used as tool compounds
for further elucidation of the specific biological roles
of PPARd.
19. Leibowitz, M. D.; Fievet, C.; Hennuyer, N.; Peinado-
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T.; Naito, M.; Auwerx, J.; Yanagisawa, M.; Kodama,
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Acknowledgments
We gratefully acknowledge Drs. T.R. Vedananda, San-
dra Teixeira, and Peter G. Schultz for their helpful dis-
cussions and support.
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