Identification of the first potent, selective and bioavailable PPARα antagonist

Article abstract of DOI:10.1016/j.bmcl.2014.03.090

The discovery and SAR of a novel series of potent and selective PPARα antagonists are herein described. Exploration of replacements for the labile acyl sulfonamide linker led to a biaryl sulfonamide series of which compound 33 proved to be suitable for further profiling in vivo. Compound 33 demonstrated excellent potency, selectivity against other nuclear hormone receptors, and good pharmacokinetics in mouse.

Full text of DOI:10.1016/j.bmcl.2014.03.090

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2267–2272
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of the first potent, selective and bioavailable
PPARa
antagonist
⇑
Yalda Bravo , Christopher S. Baccei, Alex Broadhead, Richard Bundey, Austin Chen, Ryan Clark,
Lucia Correa, Jason D. Jacintho, Daniel S. Lorrain, Davorka Messmer, Karin Stebbins, Peppi Prasit,
Nicholas Stock
Inception Sciences, 5871 Oberlin Drive Suite 100, San Diego, CA 92121, 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 SAR of a novel series of potent and selective PPARa antagonists are herein described.
Received 20 January 2014
Revised 25 March 2014
Accepted 26 March 2014
Available online 4 April 2014
Exploration of replacements for the labile acyl sulfonamide linker led to a biaryl sulfonamide series of
which compound 33 proved to be suitable for further profiling in vivo. Compound 33 demonstrated excel-
lent potency, selectivity against other nuclear hormone receptors, and good pharmacokinetics in mouse.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Antagonist
PPAR alpha
Nuclear hormone receptor
Fatty acid oxidation
Cancer
Cancer cells are known to exhibit distinct characteristics from
their ‘normal’ counterparts with regards to their energy require-
ments and metabolism. This divergence in turn, presents research-
ers with a variety of approaches to selectively apply metabolic
stress on cancerous cells without adversely affecting healthy cells.
Warburg observed that most cancer cells are programmed to in-
crease glucose uptake in order to provide the necessary energy
for their proliferative processes. Subsequently, much of the re-
search into aberrant cancer metabolism has, thus far, been focused
on targeting the glycolysis pathway, leaving the other metabolic
pathways largely unexplored.1 Recently, the contribution of fatty
acid oxidation (FAO) to cancer cell function has gained more atten-
tion from the research community.2–4 It has been shown that there
are specific cancer cell types; including prostate, ovarian and renal
cell carcinoma, that are more reliant on fatty acids to satisfy their
metabolic needs.5 It has also been demonstrated that following
detachment of cancer cells from their extracellular matrix (i.e.,
the initial step of cancer metastasis), a metabolic switch towards
increased fatty acid utilization for ATP generation can be seen even
in the most glycolytic cancer cell types.2 Finally, new research sug-
gests that leukemia-initiating cancer (LIC) stem cells may be reli-
ant on FAO for their maintenance and function, hinting at the
possibility of eradicating leukemia through the exhaustion of the
chemo-resistant LIC pool via inhibition of FAO.6
Peroxisome proliferator-activated receptors (PPARs) are a group
of DNA-binding transcription factors within which three distinct
isoforms (i.e., a, b/d and c
) have been identified.7 When activated
by their respective endogenous ligands such as free fatty acids
and eicosanoids, PPARs undergo a series of conformational changes
to accommodate the recruitment of a suitable scaffolding co-activator
protein and to facilitate their hetero-dimerization with 9-cis
retinoic acid receptor (RXR). The resulting ternary complex then
binds to the appropriate peroxisome proliferator response element
(PPRE) on DNA and initiates the transcription and expression of its
target genes. PPARa is implicated primarily in the regulation of
lipid metabolism and as such, its activation leads to an increase
in uptake and catabolism of fatty acids. This phenotype is achieved
via the up-regulation of genes involved in the binding and
transport of fatty acids (i.e., CPT1/2, CACT, and others) for oxidative
processing. Consequently, it follows that antagonism of the PPAR
a
receptor, coupled with the knowledge that certain malignant cells
rely on FAO, represents a novel paradigm to stop the proliferative
and metastatic tendencies of these cancer cells.
Although numerous examples of potent and selective PPAR
a
agonists can be found in the literature; including those that have
been approved clinically for the treatment of hypercholesterol-
emia, hypertriglyceridemia and other related metabolic disor-
ders,8–12 there are only a few isolated reports of confirmed
⇑
Corresponding author. Tel.: +1 858 354 9160.
E-mail address: yaldabravo47@gmail.com (Y. Bravo).
http://dx.doi.org/10.1016/j.bmcl.2014.03.090
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2268
Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2267–2272
Me
Table 1
Me
SAR of acyl sulfonamides
Me
N
HN
Me
Me
Ph
O
CO2H
O
O
CO2H
N
N
N
O
Me Me
N
O
Me
H
O
O
O
4
3
X
S
LY-518674
N
GW-409544
human PPARα agonist: EC50 = 240 nM
N
R
human PPARα agonist: EC50 = 42 nM
H
O
Me Me
N
Me
Me
Me
Me
8 ~ 17
F3C
Me
O
Compound
X
R:
PPAR
IC50
(nM)
a
% Parent compound remainingb
30 min 60 min 24 h
HN
Ph
O
O
O
O
S
O
a
N
H
Et
O
N
N
N
N
H
O
Me Me
O
N
Me
2
8
9
O
O
O
O
O
O
O
O
O
O
C
Ph
2.7 0.9
2.8 1.9
13
6.7 0.6
0.7 0.1
1
4-Me-Ph
4-iPr-Ph
4-CF3-Ph
3-Pyridyl
Cyclohexyl
Cyclopentyl 4.6 1.0
Cyclopropyl 6.6 3.0
3-Furan
4-Pyran
Ph
73.1 1.4
89.5 2.0
63.7 3.4
81.9 3.3
Me
human PPARα antagonist: IC50 = 40 nM
2
GW-6471
0
2.7 0.1
human PPARα antagonist: IC50 = 240 nM
10
11
12
13
14
15
16
17
34 18 102.7 1.9 105.3 3.1
80 18
3
53.7 3.5
0.2 0.1
82.4 2.6
60.5 2.0
21.0 0.8
1.7 0.2
75.4 1.0
86.9 2.9
80.0 2.8
75.2 1.1
43.6 2.0
7.0 0.3
0.1 0.04
66.6 2.0
86.5 3.3
62.3 2.5
Figure 1. Previously reported PPARa
modulators.15
0
78
42 14
60 10
0
PPAR
were reported to be discovered serendipitously, following inde-
pendent SAR campaigns carried out on their respective PPAR ago-
a antagonists. These inhibitors (compounds 1 and 2, Fig. 1)
a
a
Values are the mean of at least three experiments.
For experimental procedure see Ref. 20.
nist precursors (i.e., compounds 3 and 4). The reported initial
program goal was to identify a suitable replacement for their
shared carboxylic acid warhead (highlighted in red). In both cases,
replacement of the carboxylic acid group by either an inverse
amide (i.e., GW6471, compound 1) or an acyl sulfonamide (i.e.,
compound 2) triggered an unanticipated agonist-to-antagonist
b
30 min and 60 min incubation period) and potency against PPAR
a
in our luciferase assay (Table 1). These compounds were readily ac-
cessed through EDC-mediated condensation of acid 5 and sulfon-
amide 7 in the presence of DMAP.16 Sulfonamides that were not
commercially available were themselves synthesized from the cor-
responding sulfonyl chloride and liquid ammonia (Scheme 1).
The incorporation of an aliphatic group such as methyl (8) or
isopropyl (9) at the para position of the terminal phenyl ring deliv-
switch that resulted from the lifting of the PPAR
2 helix, which, in turn, favored the recruitment of a co-repressor
a C-terminal AF-
peptide such as SMRT.
Both antagonists were found to dose-dependently inhibit the
activation of PPAR
known PPAR
agonist) in a cell-based functional assay13 we em-
ployed to drive our SAR. Unfortunately, neither compound proved
suitable for assessing whether the antagonism of PPAR would be
a-driven luciferase expression by GW7647 (a
a
ered PPAR
a antagonists of comparable potency to compound 2.
Both modifications led to a significant improvement in plasma sta-
bility, with the larger isopropyl group being more resistant to-
wards hydrolysis (82% of 9 remained after 60 min of incubation
with murine plasma vs 64% of 8). Unfortunately, the vast majority
a
efficacious in our murine cancer models. Specifically, when mice
were orally administered compound 1, no measurable drug levels
could be detected in the mouse plasma regardless of when the
blood was collected after dose. Although this lack of oral drug
exposure could be mitigated via an alternative mode of adminis-
tration (i.e., intra-peritoneal injection), compound 1’s lack of solu-
bility in conventional dosing vehicles necessitated that the
compound be eventually formulated in neat DMSO. However, this
vehicle choice complicated the interpretation of the resulting
in vivo data. The DMSO vehicle arm was found to exhibit a signif-
icant anti-metastatic effect when compared to conventional vehi-
cles such as 0.5% aqueous methocel or saline (data not shown).14
While compound 2 was found to possess physicochemical proper-
ties amenable for conventional formulation, we quickly discovered
that this compound was very unstable in murine plasma and
underwent an almost instantaneous enzyme-mediated hydrolytic
of acyl sulfonamide 9 (>97%) was still cleaved to PPARa agonist 5
after a 24 h incubation period. Although switching the isopropyl
residue in 9 for its known electron-withdrawing isostere CF3 (10)
further improved the resulting compound’s resistance towards
hydrolytic cleavage at a cost of only a small drop in PPARa potency,
compound 10 could not survive the more rigorous 24 h incubation
protocol unscathed. Replacement of the terminal benzene ring in
compound 2 with a heteroaromatic plate such as 3-pyridyl (11)
proved to be highly detrimental for PPAR
other hand, reducing it to the fully saturated cyclohexane (12)
was tolerated in terms of potency against PPAR . These observa-
tions combine to reveal that the phenyl group in 2 occupies a large,
hydrophobic pocket in the PPAR ligand binding domain but is
a antagonism. On the
a
a
cleavage to the acid; a potent PPARa agonist. As a result, we initi-
ated separate SAR campaigns on both of these scaffolds with the
aim of addressing their respective key liabilities that prohibited
their profiling in vivo. This manuscript will focus on our efforts
at improving the metabolic stability of compound 2 towards
hydrolysis, while preserving its potency and selectivity against
O
S
O
R
Cl
6
Me
N
Me
Me
Me
N
(a)
Me
Me
O
O
R
S
PPARa. Our effort around compound 1 will be disclosed separately
in due course.
H2N
O
O
O
O
7
O
S
O
N
N
N
R
OH
H
Me Me
O
(b)
O
It was initially hypothesized that modification of the steric and/
or the electronic environment adjacent to the cleavage site in com-
pound 2 might afford compounds that are more resistant towards
hydrolysis. In this regard, we first evaluated the impact of the sul-
fonyl substituent on both stability in murine plasma (after a
Me Me
N
N
Me
Me
5
8 ~ 16
Scheme 1. Reagents and conditions: (a) liquid NH3, DCM, À78 °C, 90–95%. (b) EDC,
DMAP, DCM, 40–80%.

Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2267–2272
2269
itself not participating in any stabilizing,
p
-stacking interactions. It
towards enzyme hydrolysis than those without17 and this property
has been exploited for the design of pro-drugs that facilitate the
delivery of non-orally bioavailable sulfonamide drugs. The synthe-
sis of acyl sulfonamide 17 was carried out as detailed in Scheme 2.
Briefly, palladium-catalyzed carbonylation of aryl bromide 18 was
best carried out with triethylsilane as the reducing agent. The
resulting benzaldehyde 19 then readily underwent Reformatsky
reaction with the zincate generated in situ from commercially
is important to stress that while both of these acyl sulfonamides
(11 and 12) were significantly more hydrolytically stable than 2,
these alterations again failed to completely address compound
2’s cleavage liability. Other aliphatic sulfonyl substituents were
subsequently explored, including those cyclic (13 and 14) and het-
erocyclic (i.e., 15 and 16) in nature. As expected, an increase in the
steric bulk around the site of cleavage was accompanied by an
attendant improvement in plasma stability (compare cyclohexyl
12 > cyclopentyl 13 > cyclopropyl 14, and 4-pyran 16 > 3-furan
15). Analogues containing a heteroatom were consistently more
resistant towards enzyme-mediated cleavage, but they were also
found to be less effective at antagonizing PPARa than those with-
out (compare 13 vs 15, and 12 vs 16). Regardless, none of these
alterations were able to deliver a potent, hydrolytically-stable
PPARa antagonist.
It has been previously observed that acyl sulfonamides bearing
an oxygen atom b to the carbonyl group are much more susceptible
available ethyl a-bromoisobutyrate to deliver alcohol 20 in quan-
titative yield. Subsequent ionic de-oxygenation with triethylsilane
and boron trifluoride etherate proceeded without incident and
gave diester 21 in 67% yield. Selective saponification of the more
sterically accessible methyl ester could be achieved with potas-
sium trimethylsilanoate and the resulting carboxylic acid was then
coupled with known hydrazine 22 in the presence of HATU and
Hunig’s base. When heated with an excess of CSA in ethyl acetate,
hydrazine carboxamide 23 underwent cyclization to afford, after
hydrolysis with aqueous lithium hydroxide, triazalone 24. Finally,
OH
Br
CHO
CO2Et
(a)
(b)
MeO2C
MeO2C
Me Me
MeO2C
18
19
20
21
Me
(c)
Me
Me
CO2Et
Me Me
CO2Et
Me Me
(d), (e)
O
MeO2C
EtHN
N
N
H
O
23
(f), (g)
Me
N
Me
Me
Me
Me
Me
Me
Me
Me
MsOH
O
O
O
O
EtHN
N
(h)
NH2
N
S
OH
N
22
N
N
Ph
O
O
Me Me
H
O
Me Me
N
N
Me
24
17
Me
Scheme 2. Reagents and conditions: (a) Pd(PPh3)2Cl2, Na2CO3, Et3SiH, CO, DMF, 80 °C, 53%. (b) Zn, I2, BrC(Me)2CO2Et, sonication, rt, >99%. (c) BF3ÁOEt2, Et3SiH, DCM, 0 °C ? rt,
67%. (d) KOTMS, THF, rt, 92%. (e) compound 22, HATU, Hunig’s base, DMF, rt, 64%. (f) CSA, EtOAc, 80 °C, 75%. (g) 2N aq LiOH, THF, MeOH, rt, 95%. (h) EDC, DMAP, DCM, 88%.
Br
Br
Br
O
O
(a)
(b)
H
N
HO2C
EtHN
H2NHN
N
H
O
N
25
26
27
(c)
Me
Me
Me
Me
Me
Me
O
B
Br
Me
HN
N
(d), (e)
N
O
N
O
O
N
Me
29
28
Me
(f)
Me
N
Me
Me
Me
Me
Me
NH2
X
NHSO2R
X
(g)
N
N
N
O
O
N
N
30
31 ~ 39
Me
Me
Scheme 3. Reagents and conditions: (a) CDI, THF, rt then NH2NH2ÁH2O, rt, 95%. (b) EtN = C@O, DCM, rt, >99%. (c) KOH, MeOH, reflux, 85%. (d) 4-tert-butylbenzyl bromide,
K2CO3, DMF, 45 °C, 93%. (e) Pd(dppf)Cl2, KOAc, bis(pinacolato)diboron, dioxane, 85 °C, 82%. (f) Ar-Br, K2CO3, Pd(PPh3)4, DME, H2O, 85 °C, 65–95%. (g) RSO2Cl, pyridine, rt, 60–
90%.

2270
Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2267–2272
Table 3
using the conditions described earlier in Scheme 1, the requisite
acyl sulfonamide 17 could be cleanly isolated in 88% yield.
Unfortunately, although compound 17 was found to be much more
stable towards hydrolysis than compound 2, the removal of the
oxygen linker did not completely protect 17 from enzyme-mediated
hydrolysis.
Pharmacokinetic profiles of compounds 33 and 37
33
37
CD1 mouse (I.P.)
Dose
Vehicle
Plasma AUC (h
Cmax
30 mpk
Saline
3.4
1.2
1.0
30 mpk
98% saline/2% tween
23
7.2
0.29
lg/mL)
(
l
(
M)
M)
Concurrent with our campaign to stabilize the acyl sulfonamide
handle, we evaluated whether this labile motif in compound 2
Ctrough
l
could be replaced entirely without jeopardizing its PPARa antago-
nism. It was hypothesized that the fibrate core present in 2 could
be effectively mimicked by a six-membered aryl ring.18 The requi-
site biaryl sulfonamide analogues were synthesized as shown in
Scheme 3. Commercially available butanoic acid 25, after its initial
conversion to the corresponding acyl imidazole with CDI, was cou-
pled with hydrazine to give acylhydrazide 26. Subsequent treat-
ment with ethyl isocyanate afforded hydrazine carboxamide 27,
which was then cyclized to triazalone 28 with KOH in refluxing
methanol. This material was first alkylated with 4-tert-butylbenzyl
bromide and then further transformed to pinacol boronate 29 un-
der standard Suzuki–Miyaura borylation conditions. From this ver-
satile intermediate, a variety of amino-aryl and amino-heteroaryl
groups could then be appended via the Suzuki cross-coupling reac-
tion. Finally, the desired biaryl sulfonamides (Table 2, compounds
31–39) were obtained by treatment of aniline 30 with a selection
of commercially available sulfonyl chlorides using pyridine as
solvent.
the para position (32) recovered much of the initial loss in potency.
However, compound 32 was found to be poorly soluble in a variety
of aqueous formulations (0.018 lM in pH = 7.4 aqueous buffer,
Table 2) and could only be formulated in PEG400. As a consequence
of its poor physiochemical properties, no bioavailability was
achieved upon oral administration of compound 32 to rodents. In
order to improve the solubility of this series, installation of hetero-
atoms were explored and 2-pyridyl was found to be optimal in
terms of both PPARa potency and aqueous solubility (compare
32 vs 33 vs 34). Installation of the pyridine led to >80-fold increase
in aqueous solubility. When increasing the nitrogen count at the
terminal aromatic ring further to either a pyrimidine (38) or a
pyrazine (39) potency was lost against PPARa. Sulfonamide 33
was also tested to see whether it recruits SMRT corepressor in a
similar fashion to GW6471. Indeed, using a commercially available
kit where the GST-tagged PPARa ligand binding domain is labeled
with the Tb-anti-GST antibody and the SMRT co-repressor peptide
Although replacement of the fibrate linker in compound 2 with
a simple benzene spacer (31) led to a 200-fold drop in its ability to
antagonize PPARa, moving the sulfonamide group from the meta to
is labeled with fluorescein,19 we witnessed a dose dependent
quenching of the fluorescence signal with an EC50 of 1.4 lM.
Table 2
SAR of biaryl triazalones
Me
Me
Me
NHSO2R
X
N
N
O
N
31 ~ 39
Me
% Parent compound remainingb 24 h
Solubility in pH = 7.4 aqueous bufferc
(lM)
a
Compound
PPAR
a
IC50 (nM)
31
32
33
34
35
36
386 250
21 16
136 8.4
124.7 4.6
140.6 2.4
128.7 2.3
104.8 3.6
133.9 2.3
0.037
0.018
1.5
77 35
122 81
113 63
311 66
0.014
0.423
0.012
37
4800 3492
119.2 2.8
0.082
38
39
114 57
119.4 1.4
131.2 4.2
0.091
4.9
218 153
a
Values are the mean of at least three experiments.
For experimental procedure see Ref. 20.
For experimental procedure see Ref. 21.
b
c

Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2267–2272
2271
M)
Table 4
Nuclear hormone receptor counter screen of compounds 33 and 37
a
a,b
a,c
a,c
a,c
a,c
a,c
PPAR
a
antagonist IC50
(
lM)
PPARa
agonist EC50
(l
M)
PPARd IC50
(l
M)
PPAR
c
IC50
(
lM)
ERb IC50
(lM)
GR IC50
(l
M)
TRb IC50
(l
33
37
0.077 0.035
4.8 3.5
>100
6.0 3.2
>100
15 19
>100
15.2 6.9
34.8 16.1
32.5 19.3
32 19
>100
>69
a
b
c
Values are the mean of at least three separate experiments.
For experimental procedure see Ref. 13.
For experimental procedure see Ref. 25.
compound 33 at 30 mg/kg (IP) to fasted mice,27 we measured a sig-
nificant decrease in plasma FGF21 in the treated animals versus
control (Fig. 2) thus successfully demonstrating functional antago-
4000
3000
2000
nism of the PPAR
In summary, we have designed and synthesized the first series
of potent and selective PPAR antagonists that are suitable for
a
receptor in vivo.28
a
in vivo proof-of-concept experiments. Optimizing the labile acyl
sulfonamide linker in compound 2, we arrived at biaryl sulfon-
amide 33. Understanding of the binding tendencies of this novel
1000
*
series of compounds to PPARa has facilitated the identification of
compound 37 as a structurally similar, negative control. Both of
these compounds will serve as invaluable tools to improve our
understanding of the relationship between FAO and cancer prolif-
eration and survival. Indeed, these data will be disclosed shortly.29
0
Fed
Fasted Cmpd 33
(30mg/kg)
Vehicle
Acknowledgments
Figure 2. Plasma FGF21 concentration after dosing of vehicle control versus 33
given at 30 mg/kg (IP) for 4 days * p < 0.05, t test.
We would like to thank Jill M. Baccei, Brian Stearns and Yen
Truong for their contributions and insightful comments.
With sulfonamide 33 as the reference point, we next examined
the impact of the size of the sulfonyl substituent (R in Table 2) on
References and notes
PPAR
a potency. Changing from phenyl to either methyl (35) or
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demand even further to sulfonamide 37 delivered an extremely
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similar, negative control to compound 33 in our proof-of-concept
experiments. Therefore both compounds 33 and 37 were selected
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served good drug coverage across 24 h (i.e. Ctrough = 1.0
calculated plasma AUC of 3.4 h g/mL. Furthermore with com-
pound 33, the maximum plasma concentration was achieved 1 h
post dose and found to be 1.2 M. Similarly the sodium salt of
compound 37, the proposed negative control to 33, also gave suffi-
cient drug level in mouse using conventional formulation for the
necessary in vivo proof-of-concept experiments. Our initial pro-
gram goal was to design a molecule suitable for assessing whether
lM) with a
l
l
PPARa antagonism would be able to slow down or completely stop
the proliferation of cancer cells both in vitro and in vivo. Therefore,
it was prudent to assess our chosen antagonist and negative con-
trol’s affinity for other nuclear hormone receptors; including estro-
gen receptor beta (ERb) and glucocorticoid receptor (GR), both of
13. The screening of test compounds for activities against human PPAR
were analyzed using a commercial kit, Human PPAR Reporter Assay System
(Indigo Biosciences, Cat. #IB00111). PPAR Reporter cells were dispensed into
96-well assay plates and test compounds were then immediately added to the
wells. For the antagonist assay, PPAR agonist GW7647 (20 nM) was also
a receptors
a
a
a
which have been implicated in the proliferation prostate22
,
added. Following an overnight incubation, the treatment media was discarded
and Luciferase Detection Reagent (LDR) added. The luminescence intensity of
light emission from the ensuing luciferase reaction is directly proportional to
the relative level of PPARa activation in the reporter cells. Luminescence was
read using a Molecular Devices FlexStation 3.
breast23 and renal24 cancer cells (Table 4). As illustrated, com-
pounds 33 and 37 exhibited minimal affinity for this small panel
of nuclear hormone receptors.
14. Lampugnani, M. G.; Pedenovi, M.; Niewiarowski, A.; Casali, B.; Donati, M. B.;
Corbascio, G. C.; Marchisio, P. C. Exp. Cell. Res. 1987, 172, 385.
15. (a) Xu, H. E.; Stanley, T. B.; Montana, V. G.; Lambert, M. H.; Shearer, B. G.; Cobb,
J. E.; McKee, D. D.; Galardi, C. M.; Plunket, K. D.; Nolte, R. T.; Parks, D. J.; Moore,
J. T.; Kliewer, S. A.; Willson, T. M.; Stimmel, J. B. Nature 2002, 415, 813; (b)
Etgen, G. J.; Mantio, N. Curr. Top. Med. Chem. 2003, 3, 1649.
Having identified a selective antagonist of the PPAR
a nuclear
hormone receptor with appreciable exposure after IP dosing we
utilized a mouse model to assess compound 33’s ability to inhibit
PPAR
a known PPAR
tration of a fibrate agonist.26 After 4 consecutive days dosing of
a
target genes in vivo. Fgf21 (Fibroblast growth factor 21) is
a
target gene, induced by either fasting or adminis-
16. Xu, Y.; Mayhugh, D.; Saeed, A.; Wang, X.; Thompson, R. C.; Dominianni, S. J.;
Kauffman, R. F.; Singh, J.; Bean, J. S.; Bensch, W. R.; Barr, R. J.; Osborne, J.;

2272
Y. Bravo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2267–2272
Montrose-Rafizadeh, C.; Zink, R. W.; Yumibe, N. P.; Huang, N.; Luffer-Atlas, D.;
taken at 30 and 60 min time points and precipitated in the same fashion as
Rungta, D.; Maise, D. E.; Mantlo, N. B. J. Med. Chem. 2003, 46, 5121.
17. Larsen, J. D.; Bundgaard, H. Int. J. Pharm. 1987, 37, 87.
time = 0. Precipitated samples are shaken for 5 min and centrifuged at 4000g
for 10 min. 150 lL of supernatant is collected and analyzed for test compound
18. Analysis of the various known PPAR
a
crystal structures in the RCSB protein
using LC-MS/MS. Percent test compound remaining is calculated by dividing
concentration at specific time point by concentration at time equals zero and
then multiplying by 100.
data bank containing a fibrate-derived compound in their respective ligand
binding domain (i.e. PDB: 1KKQ, ligand = GW6471, PDB: 2P54,
ligand = GW735, and PDB: 4BCR, ligand = WY14643) revealed that the
distance between the phenyl ring and the carbon in question ranged from
3.8 to 4.1 angstoms. Similarly, using the known biphenyl crystal structure (i.e.,
PDB: 2XRX), the distance between the phenyl ring on the left and the meta and
para carbons of the phenyl ring on the right are 3.8 and 4.3 angstroms,
respectively.
21. The test compounds and controls (10 mM in DMSO, 10
a pH = 7.4 buffer (490 L/well) on a 96-well plate. The samples were then
briefly vortexed for 2 min before it was shaken on an orbital shaker for 24 h at
room temperature. 200 L of each sample solution was then transferred onto a
MultiScreen filter plate equipped with a polycarbonate membrane and filtered
using Millipore vacuum manifold. The filtrate thus obtained was then
lL/well) were added to
l
l
a
analyzed and quantified by LCMSMS, using the calibration curve constructed
from the corresponding test compound.
22. Signoretti, S.; Loda, M. Am. J. Pathol. 2001, 159, 13. and references cited therein.
23. Pan, D.; Kocherginksy, M.; Conzen, S. D. Cancer Res. 2011, 71, 6360.
24. Mink, K. J.; Jang, J. H.; Lee, J. T.; Choi, K. S.; Kwon, T. K. J. Mol. Med. 2012, 90, 309.
25. Screening of test compounds in Human PPARd, PPARc and the nuclear
hormone receptors, estrogen receptor beta (ERb), glucocorticoid receptor
(GR) and thyroid hormone receptor beta (TRb) were analyzed using reporter
assay system kits from Indigo Biosciences according to the manufacturer’s
instructions.
This would suggest that replacement of a fibrate moiety with an aromatic ring
is feasible.
19. LanthaScreen TR-FRET Coactivator Assays, purchased from Invitrogen
(PV4684), is used to identify antagonists of PPARa following the
26. Badman, M. K.; Pissios, P.; Kennedy, A. R.; Koukos, G.; Flier, J. S.; Maratos-Flier,
E. Cell Metab. 2007, 5, 426.
manufacturer’s instructions except that Fluorescein-SMRT ID2 corepressor
peptide (Invitrogen, PV4423) was substituted for the Fluorescein-PGC1a
peptide provided in the coactivator assay kit. Briefly, increasing
concentrations of compound were added to LBD and corepressor peptide
27. Mice (C57Bl/6) received an IP injection of 33 or vehicle control (saline) daily for
four days. Starting on day 2 of antagonist treatment, mice were fasted for 48 h.
4 h after the final dose, blood was collected into EDTA tubes via terminal
cardiac puncture. Plasma was isolated and analyzed for FGF21 using an ELISA
(R&D Systems, Minneapolis, MN) and 33 concentration using LC/MS methods.
28. Stebbins, K. J.; Broadhead, A. R.; Cabrera, G.; Correa, L. D.; Messmer, D.; Bundey,
R.; Baccei, C.; Bravo, Y.; Chen, A.; Stock, N. S.; Prasit, P.; Lorrain, D. S. J. Pharm.
Exp. Ther. 2014 (manuscript submitted).
29. Messmer, D.; Lorrain, K.; Bravo, Y.; Stock, N.; Bundey, R.A.; Correa, L.; Poon, M.;
Stebbins, K.; Cabrera, G.; Chen, A.; Jacintho, J.D.; Spaner, D.; Prasit, P.; Lorraine,
D. Abstract of Papers, 54th Meeting of the American Society of Hematology,
Atlanta, GA, 2011; Abstract 3879.
solutions with 10 nM of GW7647 (EC80 of the PPAR
assay). After a 2 h incubation at room temperature, the 520/490 TR-FRET ratio
was measured using fluorescence microplate reader (Flex Station 3)
a agonist, measured in this
a
excitation 340 nm, emission 495 nm, and emission 520 nm.
20. A 30 mM DMSO stock solution of test compound is diluted to 100 lM in
acetonitrile. 5
mouse plasma (Bioreclamation Cat# MSEPLEDTA-F) yielding
compound in plasma. Plasma vial is vortexed and 50
immediately precipitated in 200 acetonitrile containing 5 ng/mL
buspirone. This is time zero (t = 0) test compound concentration. Plasma vial
is incubated at 37 °C in water bath and additional 50 L plasma aliquots are
l
L of this 100
l
M solution is spiked into 495
l
1 lM
L of thawed
of test
lL aliquot is
a
l
L
l