Identification and characterization of 4-chloro-N-(2- {[5-trifluoromethyl)- 2-pyridyl] sulfonyl} ethyl)benzamide (GSK3787), a selective and irreversible peroxisome proliferator-activated receptor δ (PPARδ) antagonist

Article abstract of DOI:10.1021/jm900464j

4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3 (GSK3787) was identified, as a potent and selective ligand for PPARδ with good pharmacokinetic properties. A detailed binding study using mass spectral, analysis confirmed covalent binding to Cys249 within the PPARδ binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARδ and inhibited basal CPT1 a gene transcription. Compound 3 is a PPARδ antagonist with utility as a tool to elucidate PPARδ cell biology and pharmacology.

Full text of DOI:10.1021/jm900464j

J. Med. Chem. 2010, 53, 1857–1861 1857
DOI: 10.1021/jm900464j
Identification and Characterization of 4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide
(GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist
Barry G. Shearer,*,† Robert W. Wiethe,† Adam Ashe,† Andrew N. Billin,§ James M. Way,§ Thomas B. Stanley,
Craig D. Wagner, Robert X. Xu,^ Lisa M. Leesnitzer,¥ Raymond V. Merrihew,¥ Todd W. Shearer,‡ Michael R. Jeune,‡
John C. Ulrich,‡ and Timothy M. Willson#
†Department of Metabolic Chemistry, ‡Department of Drug Metabolism and Pharmacokinetics, Metabolic Diseases Centre of Excellence for
Drug Discovery, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, North Carolina 27709, §Discovery Technology Group, Department
of Analytical Biochemistry and Biophysics, ^Department of Computational and Structural Sciences, ¥Department of Screening and Compound
Profiling, and #Discovery Medicinal Chemistry, Molecular Discovery Research, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park,
North Carolina 27709
Received April 10, 2009
4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3 (GSK3787) was identified as
a potent and selective ligand for PPARδ with good pharmacokinetic properties. A detailed binding
study using mass spectral analysis confirmed covalent binding to Cys249 within the PPARδ binding
pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of
PPARδ and inhibited basal CPT1a gene transcription. Compound 3 is a PPARδ antagonist with utility
as a tool to elucidate PPARδ cell biology and pharmacology.
Introduction
obese primates. More significantly, PPARδ agonist 1 has
recently been reported to reduce serum triglycerides and
prevent the reduction of HDL-c and apo-A1 levels in seden-
tary human volunteers and increase muscle fatty acid oxida-
tion in patients with insulin resistance.6 These positive results
collectively implicate PPARδ as a promising target for the
novel treatment of metabolic disorders.
A number of PPARδ activating ligands have been dis-
closed, but there remains a need for additional small molecule
regulators of PPARδ with a range of functional activity
profiles to use as tools to further decipher the biological
pathways regulated by PPARδ. Novo Nordisk recently re-
ported a selective PPARδ partial agonist that corrected
plasma lipid parameters and improved insulin sensitivity in
a high fat fed transgenic mouse model highlighting the
potential use of PPARδ partial agonists as novel agents for
the treatment of dyslipidemia.7 We identified a novel class of
anthranilic acids as potent and selective PPARδ partial
agonists with a distinct binding mode.8 Compounds in this
series act as potent partial activators on PPARδ target gene
expression in human skeletal muscle cells.
Recently, we reported the identification of selective PPARδ
antagonist 2 (GSK0660) which has utility as a tool for
elucidating the biological role of PPARδ in vitro.9 However,
a limitation of antagonist 2 is that it lacks oral bioavailablilty.
In this communication, we describe the identification and
characterization of a chemically distinct PPARδ antagonist,
4-chloro-N-(2-{[5-trifluoromethyl)-2-pyri-
dyl]sulfonyl}ethyl)benzamide 3 (GSK3787, Figure 2), that
was identified from our high-throughput screen of the GSK
compound collection.
Nuclear receptors represent an important class of receptor
targets for drug discovery. The peroxisome proliferator-acti-
vated receptors (PPARsa) are ligand activated transcription
factors belonging to the nuclear receptor superfamily and play
key roles in multiple physiological pathways. Three PPAR
receptor subtypes with distinct tissue distributions, designated
as PPARR, PPARγ, and PPARδ, have been identified. The
PPARs are significantly involved in the control of fatty acid
metabolism and represent attractive therapeutic targets.1
PPARR is an important regulator of fatty acid catabolism in
the liver and is the known target receptor for the fibrate class
of lipid lowering drugs.2 PPARγ activators are established
insulin sensitizers and lower plasma glucose.3 Rosiglitazone
and pioglitazone are potent PPARγ agonists currently mar-
keted for the treatment of type 2 diabetes.
There are currently no available drugs targeting PPARδ.
While PPARδ remains the least understood subtype, it is now
recognized as a regulator of genes involved in fatty acid
oxidation, reverse cholesterol transport, and carbon substrate
utilization in skeletal muscle.4,5 Further evidence linking
PPARδ to important roles in lipid homeostasis and glucose
disposal is growing. For example, in rodent models of type 2
diabetes, the PPARδ agonist 1 (GW501516, Figure 1)
improves insulin resistance and lowers plasma glucose. Agonist
1 has also been shown to correct the metabolic syndrome in
*To whom correspondence should be addressed. Phone: 919-483-
9860. Fax: 919-315-6964. E-mail: barry.g.shearer@gsk.com.
aAbbreviations: apo-A1, apolipoprotein A1; CPT1a, carnitine pal-
mitoyltransferase 1a; Cys, cysteine; HCl, hydrogen chloride; HDLc,
high density lipoprotein cholesterol; His, histidine; LCMS, liquid chro-
matography-mass spectrometry; N-Boc, N-tert-butoxycarbonyl;
PDK4, pyruvate dehydrogenase kinase-4; PPAR, peroxisome prolifera-
tor-activated receptor; SAR, structure-activity relationship.
The pyridylsulfone 3 was identified as a potent and selective
hPPARδ ligand (pIC50 = 6.6) with no measurable affinity for
hPPARR or hPPARγ (pIC50 < 5) in our standard in vitro
r
2010 American Chemical Society
Published on Web 02/03/2010
pubs.acs.org/jmc

1858 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Shearer et al.
ligand displacement assay.10 Surprisingly, 3 failed to activate
the receptor in a standard hPPARδ-GAL4 chimera cell-based
reporter assay.11 This result suggested that pyridylsulfone 3
might be a hPPARδ antagonist. To test this hypothesis, we
measured the activity of pyridylsulfone 3 in a standard GAL4
chimera cell-based reporter antagonist assay in which an EC80
dose of the PPARδ agonist 1 was added to the cells.12
Pyridylsulfone 3 completely antagonized the activity of agonist
1 with a pIC50 of 6.9 (n = 2). Compound 3 was inactive against
hPPARR and hPPARγ in similar functional antagonist assays.
Further screening in similar cell-based reported assays using the
mouse PPARδ receptor showed pyridylsulfone 3 failed to
activate the receptor and antagonized the activity of agonist 1
with a pIC50 of 6.9 and 94% maximal inhibition (n = 2). Thus,
pyridylsulfone 3 is a selective PPARδ antagonist with equipo-
tent species activity against the human and mouse receptor.
synthesized to investigate the SAR and identify the key
pharmacophore. We focused on three regions of the molecule:
the pyridyl ring, the aliphatic linker, and the right-hand-side
amide group. The synthesis of these compounds is detailed in
Scheme 1. Sulfur alkylation of 2-mercaptopyridine 4 with
N-Boc protected aminoalkyl bromide 5 in DMF provided
2-thiopyridine 6. Oxidation of the sulfide to the sulfone 7 with
potassium peroxymonosulfate followed by removal of the
N-Boc protecting group with 4 N HCl in dioxane afforded
aminosulfone 8. Acylation of the primary amine with aroyl
chlorides readily provided the final targets3 and 9-20 in good
overall yields.
Results and Discussion
The ability ofthese compoundstobind toeach ofthe PPAR
subtypes was measured in vitro in a ligand displacement
assay.10 Functional PPARδ activity was measured in stan-
dard cell-based GAL4 chimera reporter assays using agonist11
and antagonist formats.12 These results are summarized in
Table 1. Data for PPARδ antagonist 2 are included for
comparison. Substitution of the arylamide ring did not have
a significant effect on binding affinity. Replacement of the
para chloro substitutent with a trifluoromethoxy group gave a
Chemistry
We initiated an effort to evaluate the developability of this
new class of PPARδ antagonist. A series of compounds were
Table 1. Human PPARδ Binding and Functional Potency of Substi-
tuted Pyridylsulfonesa
hPPAR binding
pIC50
hPPARδ
reporter
compd
n
R1
R2
R
γ
δ
pIC50 % inh
2
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
6.8
6.7
7.0
6.7
6.0
6.5
6.8
6.9
6.9
7.3
<5
5.9
<5
<5
6.5
6.9
NT
6.6
NT
6.7
6.7
6.6
6.6
7.1
NT
NT
NT
NT
100
100
3
1
1
1
1
1
1
1
1
1
2
2
1
1
CF3 4-Cl
CF3 4-OCF3
CF3 4-Ph
9
10
11
12
13
14
15
16
17
18
19
20
100
CF3 4-c-C6H12
CF3 4-i-Bu
CF3 2,4-diCl
CF3 3-Br, 4-Cl
100
100
100
100
99
Figure 1. Structures of PPAR ligands.
CF3 3-Me, 4-Cl <5
CF3 2-F, 4-Br
CF3 4-Cl
CF3 4-OCF3
<5
<5
<5
<5
<5
Me
H
4-Cl
4-Cl
a Values had a standard deviation of e10% (n g 2). Reported
functional data are for the antagonist format of the hPPARδ-GAL4
chimera reporter assay. NT = not tested.
Figure 2. Structure of pyridylsulfone 3 (GSK3787).
Scheme 1a
a (a) Et3N, DMF, room temp; (b) potassium peroxymonosulfate, acetone-water, room temp; (c) 4 N HCl, dioxane, room temp; (d) ArCOCl, Et3N,
CH2Cl2, room temp.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1859
Figure 3. Proposed mechanism of covalent binding for 5-trifluoro-
methyl-2-pyridylsulfones.
small increase in binding potency (pIC50 = 7.0). However,
substitution of the 4-chloro group with bulky lipophilic
groups did not improve potency. The binding of the 4-phenyl
analogue 10 was equipotent to pyridylsulfone 3, while the
bulkier lipophilic cyclohexyl analogue 11 was significantly
weaker. The 4-isobutyl substituted analogue 12 exhibited
similar potency and activity as pyridylsulfone 3. Disubstitu-
tion of the arylamide ring did not increase binding or func-
tional potency as evidenced by analogues 13-15. One
compound, however, that did show improved binding po-
tency was the 2-fluoro-4-bromo analogue 16 (pIC50 = 7.3).
All of the compounds tested for functional activity in the cell-
based GAL4 chimera reporter assays profiled as antagonists.
The length of the aliphatic linker is critical for activity.
Extension of the middle two-carbon chain of trifluoro-
methoxy 9 to the three-carbon linker analogue 18 significantly
reduced binding affinity (pIC50 = 5.9). In addition, 17, the
one-carbon extended analogue of pyridylsulfone 3, was not
active. Substitution of the pyridine ring is also critical for
activity. Removal of the trifluoromethyl substituent as in
analogue 20 produced an inactive compound. Replacement
of the trifluoromethyl group with a methyl substituent pro-
vided inactive 19. In addition, the corresponding sulfide
analogue of pyridylsulfone 3 fails to bind to PPARδ, indicat-
ing that the sulfur must be oxidized for activity (data not
shown). These results suggest that a strong electron with-
drawing group para to the sulfone group is required for
activity.
On the basis of this SAR, we raised the question as to
whether or not the para electron withdrawing group was
facilitating a covalent binding interaction between the pyridyl
ring and a nucleophilic residue within the receptor binding
pocket that could lead to displacement of the sulfone group.
Ligands binding irreversibly to the PPARs have been pre-
viously reported. For example, Merck published data show-
ing that 21 (L-764406) was a PPARγ partial agonist that
covalently bound within the ligand binding domain to a
specific cysteine residue.13 Compound 22 (GW9662) is a
PPARγ antagonist structurally distinct from antagonist 21
that has also been shown to bind covalently to cysteine
residues in the ligand binding pocket.14
Figure 4. LC-MS/MS spectrum of peptide fragment generated
from trypsin digestion of hPPARδ LBD-antagonist 3 complex.
To identify the exact site of covalent binding within the
PPARδ binding pocket, a detailed binding study of pyridyl-
sulfone 3 with PPARδ was undertaken. A sample of His-
PPARδ (165-441) was incubated with pyridylsulfone 3 for
90 min. Routine LC/MS analysis of the protein sample
showed a mass increase of 145 Da corresponding exactly to
the additional mass of the 5-trifluoromethyl-2-pyridyl frag-
ment from the ligand. Tryptic digestion was performed, and
mass mapping of the digest identified a single peptide frag-
ment with a mass size 145Da greater thanpredicted. Rigorous
analysis of the resulting m/z and product ion data for this
fragment by LC-MS/MS identified Cys249, detected at 671.2
[M þ 2H]2þ as the site of covalent modification. This site was
confirmed by a strong y ion series (Figure 4).
Given that pyridylsulfone 3 has a reactive moiety, we
sought to ascertain the potential of this compound for un-
desired nonselective reactivity. A semiquantitative experi-
ment was undertaken to determine chemical reactivity where-
in pyridylsulfone 3 (2 mg, 1 equiv) and N-acetylcysteine
(10 mg, 12 equiv) were stirred in DMSO (0.6 mL) for 24 h
at room temperature and then 8 h at 60 ꢀC. LCMS was used to
monitor the disappearance of pyridylsulfone 3 and the ap-
pearance of any new products. Analysis of the mixture after
24 h at room temperature and 8 h at 60 ꢀC showed pyridyl-
sulfone 3 remained without the appearance of any new
products. Thus, pyridylsulfone 3 appears to be a chemically
stable PPARδ antagonist.
To test our hypothesis, pyridylsulfones 3 and 9, two analo-
gues with different molecular weights, were independently
incubated with PPARδ and the binding was monitored
by mass spectra analysis. Both compounds gave the same
exact mass equivalent to PPARδ þ 145 mass units with
complete conversion within 1 h of incubation. This sugges-
ted that both compounds covalently added the same frag-
ment to the receptor. The common structural feature for
these two antagonists that could add 145 mass units is
the trifluoromethylpyridine group. On the basis of these
results, the general mechanism in Figure 3 was proposed for
covalent binding of this series of 5-trifluoromethyl-2-pyridyl-
sulfones.
Pyridylsulfone 3 was further studied for its effects on the
expression of two key PPARδ regulated genes in human
skeletal muscle cells as previously described.9 The target genes
CPT1a and PDK4 play an important role in energy home-
ostasis. CPT1a regulates fatty acid β-oxidation in skeletal
muscle cells,15 while PDK4 plays a key role in skeletal muscle
metabolism by contributing to the regulation of glucose
metabolism.16 Compound 23 (GW0742) is a full PPARδ
agonist that robustly induces target genes CPT1a and
PDK4.9 In our first experiment, 10 nM agonist 23 was added

1860 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Shearer et al.
In an effort to probe the role of PPARδ in the modulation of
colon cancer, we tested the PPARδ antagonist 3 in a panel of
colorectal cancer cell lines (SW480, HCT116, DLD1, RKO)
and noncolorectal cell lines (A549, HEK293).20 Antagonist 3
was incubated in each of these cell lines for 3 days at
concentrations up to 10 μM. Staurosporine and actinomyocin
D were included as positive controls. Antagonist 3 had no
measurable effect on the inhibition of cell proliferation and
was not significantly different from DMSO vehicle in all cell
lines. These results do not support the hypothesis that inhibi-
tion of PPARδ may provide effective antiproliferative activity
against colorectal cancer. However, these preliminary experi-
mental results are based on a single tool compound and more
detailed studies are required to fully understand the potential
role of PPARδ inhibition in the etiology of cancer.
Finally, pharmacokinetic studies were conducted with pyr-
idylsulfone 3 to determine if this antagonist has potential use
as an in vivo tool compound. Pyridylsulfone 3 was adminis-
tered intravenously (0.5 mg/kg) and orally (10 mg/kg) to male
C57BL/6 mice. Mean clearance (CL) and volume of distribu-
tion at steady state (Vss) following iv administration were 39 (
11 (mL/min)/kg and 1.7 ( 0.4 L/kg, respectively. Following
oral administration, good exposure (Cmax = 881 ( 166 ng/mL,
Figure 5. Antagonism of agonist 23 induced expression of target
genes PDK4 and CPT1a by pyridylsulfone 3.
AUCinf = 3343 ( 332 h ng/mL), half-life (2.7 ( 1.1 h), and
3
bioavailability (F = 77 ( 17%) were observed. Thus, pyri-
dylsulfone 3 has pharmacokinetic properties suitable for use
as an in vivo PPARδ antagonist tool compound in mice.
In this disclosure, we have described the identification of
4-chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)-
benzamide 3 as an irreversible antagonist of human and
mouse PPARδ that covalently modifies Cys249 within the
ligand binding pocket. Pyridylsulfone 3 has been shown to
antagonize the induction of PPARδ-regulated target genes in
skeletal muscle cells. In preliminary in vitro studies, we
demonstrated that this PPARδ antagonist is not a regulator
of colon cancer cell proliferation. Antagonist 3 has good oral
pharmacokinetic properties and represents a new tool for use
in the further elucidation of PPARδ biology.
Figure 6. Antagonism of the basal expression of PPARδ target
genes PDK4 and CPT1a by pyridylsulfone 3.
Experimental Section
to human skeletal muscle cells to stimulate the expression of
target genes. Pyridylsulfone 3 was then tested at various doses
for its ability to antagonize the agonist 23 stimulated tran-
scription of CPT1a and PDK4 (Figure 5). Pyridylsulfone 3
effectively antagonized gene expression, suggesting that it can
block the activated PPARδ receptor’s activity.
In a second experiment, the effect of pyridylsulfone 3 on
the expression of the same PPARδ regulated target genes in
the absence of the agonist 23 was examined to determine the
compound’s ability to antagonize basal gene transcription.
Pyridylsulfone 3 was administered to human skeletal muscle
cells at various doses and found to effectively antagonize the
gene expression of CPT1a but not PDK4 (Figure 6). This
suggests that pyridylsulfone 3 may selectively block the basal
activity of the receptor on some PPARδ target genes high-
lighting the potential utility of this compound as a useful tool
for further elucidating the biological role of PPARδ.
PPARδ has been implicated in the cross-talk of signaling
cascades involved in the progression of colorectal cancer.17,18
Although PPARδ expression is elevated in most human
colorectal cancers, the role of PPARδ in colon carcinogenesis
remains controversial and highly debated because of conflict-
ing experimental results reported in the literature.19 It has
been hypothesized that PPARδ antagonists may offer utility
for the prevention and/or treatment of colorectal cancer.17
Solvents and reagents were reagent grade and used without
purification. Reactions involving air or moisture sensitive re-
agents were carried out under a nitrogen atmosphere. All H
1
NMR spectra were recorded on a Varian 400 MHz spectrometer.
Chemicalshifts(δ) arereporteddownfieldfromtetramethylsilane
(Me4Si) in parts per million (ppm) of the applied field. Peak
multiplicities are abbreviated: singlet, s; broad singlet, bs; doub-
let, d;triplet, t;quartet, q;multiplet, m. Couplingconstants(J) are
reported in hertz. LCMS analyses were conducted using a Waters
Acquity UPLC system with UV detection performed from 210 to
350 nm with the MS detection performed on a Waters Acquity
SQD spectrometer. Analytical thin layer chromatography (TLC)
was used to monitor reactions. Plates (2.5 cm ꢀ 7.5 cm) precoated
with silica gel 60 F254 of 250 μm thickness were supplied by EM
Science. Combustion microanalyses were performed by Atlan-
tic Microlab, Inc., Norcross, GA. Purities of key compounds
were >95% as determined by 1H NMR and combustion micro-
analysis.
General Procedure for the Preparation of Substituted 2-Thio-
pyridines 6. The procedure for 1,1-dimethylethyl (2-{[5-
trifluoromethyl)-2-pyridyl]thio}ethylcarbamate (6, R1 = CF3,
n = 1) is representative. To a stirred solution of 5-trifluoro-
methyl-2-mercaptopyridine (4.00 g, 22.3 mmol) in DMF
(80 mL) was added Et3N (5.81 g, 57.4 mmol) followed by a
solution of 2-(N-Boc-amino)ethyl bromide (5.44 g, 24.3 mmol)
in DMF (20 mL). After being stirred for 3 h, the mixture was

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1861
poured into EtOAc, washed with water, brine, and dried over
Na2SO4. Solvent was removed under reduced pressure to give
the desired pyridyl sulfide as a white solid (7.04 g, 98%) which
was used without further purification. 1H NMR (400 MHz,
DMSO-d6) δ 1.36 (9H, s), 3.23 (4H, m), 7.06 (1H, br s), 7.54 (1H,
d, J = 8.6 Hz), 7.99 (1H, dd, J = 8.6, 2.2 Hz), 8.78 (1H, s).
General Procedure for the Preparation of Substituted 2-Sulfo-
nylpyridines 7. The procedure for 1,1-dimethylethyl (2-{[5-
(5) Lopez-Soriano, J.; Chiellini, J.; Maffei, M.; Grimaldi, P. A.;
Argiles, J. M. Roles of Skeletal Muscle and Peroxisome Proliferator-
Activated Receptors in the Development and Treatment of
Obesity. Endocr. Rev. 2006, 27, 318–329.
(6) (a) Sprecher, D. L.; Massien, C.; Pearce, G.; Billin, A. N.; Perlstein,
I.; Willson, T. M.; Hassall, D. G.; Ancellin, N.; Patterson, S. D.;
Lobe, D. C.; Johnson, T. G. Triglyceride: High-Density Lipopro-
tein Cholesterol Effects in Healthy Subjects Administered a Per-
oxisome Proliferator Activated Receptor δ Agonist. Arterioscler.,
trifluoromethyl)-2-pyridyl]sulfonyl}ethylcarbamate (7, R1
=
ꢀ
Thromb., Vasc. Biol. 2007, 27, 359–365. (b) Riserus, U.; Sprecher, D.;
Johnson, T.; Olson, E.; Hirschberg, S.; Liu, A.; Fang, Z.; Hegde, P.;
Richards, D.; Sarov-Blat, L.; Strum, J. C.; Basu, S.; Cheeseman, J.;
Fielding, B. A.; Humphreys, S. M.; Danoff, T.; Moore, N. R.; Murga-
troyd, P.; O'Rahilly, S.; Sutton, P.; Willson, T.; Hassall, D.; Frayn, K. N.;
Karpe, F. Activation of Peroxisome Proliferator-Activated Receptor
(PPAR)δ Promotes Reversal of Multiple Metabolic Abnormalities,
Reduces Oxidative Stress, and Increases Fatty Acid Oxidation in
Moderately Obese Men. Diabetes 2008, 57, 332–339.
CF3, n = 1) is representative. To a stirred solution of pyridyl
sulfide 6 (R1 = CF3, n = 1) (7.03 g, 21.8 mmol) in 4:1 acetone-
water (20 mL) was added Oxone (29.6 g, 48.1 mmol). The
mixture was stirred overnight and then evaporated under redu-
ced pressure to remove the acetone. The remaining aqueous
suspension was partitioned between EtOAc and water. The
organic layer was washed with brine, dried over Na2SO4, and
filtered. Solvent was removed under reduced pressure to give the
desired pyridylsulfone as a white solid (5.75 g, 74%) which was
used without further purification. 1H NMR (400 MHz, DMSO-d6)
δ 1.27 (9H, s), 3.30 (2H, q, J = 6.2 Hz), 3.67 (2H, t, J = 6.4 Hz),
6.81 (1H, t, J = 5.4 Hz), 8.24 (1H, d, J = 8.2 Hz), 8.63 (1H, dd,
J = 8.2, 1.6 Hz), 9.24 (1H, s).
General Procedure for the Preparation of Substituted 2-Sulfo-
nylpyridylamines 8. The procedure for (2-{[5-trifluoromethyl)-2-
pyridyl]sulfonyl}ethyl)amine (8, R1 = CF3, n = 1) is represen-
tative. 4 N HCl in dioxane (100 mL) was added to pyridylsulfone 7
(R1 = CF3, n = 1) (4.50 g, 12.7 mmol), and the mixture was
stirred for 3 h. The resulting white suspension was diluted with
Et2O (100 mL) and stirred for 10 min. The white solid was collected,
washed thoroughly with Et2O, and dried in vacuo to give the
hydrochloride salt of the desired aminosulfone (3.49 g, 95%). This
material was used without further purification. 1H NMR (400 MHz,
DMSO-d6) δ 3.21 (2H, m), 3.89 (2H, m), 8.11 (3H, bs), 8.30 (1H, d,
J = 8.2 Hz), 8.65 (1H, dd, J = 8.2, 1.8 Hz), 9.27 (1H, s).
General Procedure for the Preparation of Substituted 2-Sulfo-
nylpyridylbenzamides 3 and 9-20. The procedure for 4-chloro-
N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3
is representative. To a stirred suspension of aminosulfone
hydrochloride 8 (R1 = CF3, n = 1) (3.49 g, 12.0 mmol) in
CH2Cl2 (100 mL) was added Et3N (5.74 g, 56.7 mmol). The
mixture immediately became homogeneous. 4-Chlorobenzoyl
chloride (2.20 g, 12.6 mmol) was added to the mixture, and
stirring was continued overnight. The mixture was poured into 1
N aqueous HCl and extracted with CH2Cl2. The organic layer
was washed with brine, dried over Na2SO4, filtered, and evapo-
rated under reduced pressure. The remaining solid was recrys-
tallized from EtOAc-hexane to afford 5-trifluoromethyl-2-
pyridylsulfone 3 (4.25 g, 90%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 3.61 (2H, m), 3.87 (2H, m), 7.43 (2H,
d, J = 8.5 Hz), 7.58 (2H, d, J = 8.5 Hz), 8.19 (1H, d, J = 8.2 Hz),
8.45 (1H, d, J = 8.2 Hz), 8.53 (1H, t, J = 5.3 Hz), 9.02 (1H, s).
LRMS (ES) m/z 393 (M þ 1). Anal. Calcd for C15H12N2-
O3SClF3: C, 45.87; H, 3.08; N, 7.13; S, 8.16; Cl, 9.03. Found:
C, 45.89; H, 3.02; N, 7.08; S, 8.05; Cl, 9.06.
(7) Sauerberg, P.; Olsen, G. S.; Jeppesen, L.; Mogensen, J. P.; Petters-
son, I.; Jeppesen, C. B.; Daugaard, J. R.; Galsgaard, E. D.; Ynddal,
L.; Fleckner, J.; Panajotova, V.; Polivka, Z.; Pihera, P.; Havranek,
M.; Wulff, E. M. Identification and Synthesis of a Novel Selective
Partial PPARδ Agonist with Full Efficacy on Lipid Metabolism in
Vitro and in Vivo. J. Med. Chem. 2007, 50, 1495–1503.
(8) Shearer, B. G.; Patel, H. S.; Billin, A. N.; Way, J. M.; Winegar, D.
A.; Lambert, M. H.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.;
Huet, S.; Willson, T. M. Discovery of a Novel Class of PPARδ
Partial Agonists. Bioorg. Med. Chem. Lett. 2008, 18, 5018–5022.
(9) Shearer, B. G.; Steger, D. J.; Way, J. M.; Stanley, T. B.; Lobe, D.
C.; Grillot, D. A.; Iannone, M. A.; Lazar, M. A.; Willson, T. M.;
Billin, A. N. Identification and Characterization of a Selective
Peroxisome-Activated Receptor β/δ (NR1C2) Antagonist. Mol.
Endocrinol. 2008, 22, 523–529.
(10) Brown, P. J.; Smith-Oliver, T. A.; Charifson, P. S.; Tomkinson, N.
C. O.; Fivush, A. M.; Sternbach, D. D.; Wade, L. E.; Orband-
Miller, L.; Parks, D. J.; Blanchard, S. G.; Kliewer, S. A.; Lehmann,
J. M.; Willson, T. M. Identification of Peroxisome Proliferator-
Activated Receptor Ligands from a Biased Chemical Library.
Chem. Biol. 1997, 4, 909–918.
(11) Graham, R. A.; Goodwin, B.; Merrihew, R. V.; Krol, W. L.;
LeCluyse, E. L. Cloning, Tissue Expression, and Regulation of
Beagle Dog CYP4A Genes. Toxicol. Sci. 2006, 92, 356–367.
(12) The antagonist mode assay is run the same as described for the
agonist mode assay11 except cells are incubated with competing
agonist GW501516 (human = 2 nM; mouse = 6 nM) prior to
addition to compound plates.
(13) Elbrech, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.;
Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moeller, D. E. L-
764406 Is a Partial Agonist of Human Peroxisome Proliferator-
Activated Receptor γ. J. Biol. Chem. 1999, 274, 7913–7922.
(14) Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins,
J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J.
M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.;
Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional
Consequences of Cysteine Modification in the Ligand Binding
Sites of Peroxisome Proliferator Activated Receptors by
GW9662. Biochemistry 2002, 41, 6640–6650.
(15) Takahashi, S.; Tanaka, T.; Kodama, T.; Sakai, J. Peroxisome
Proliferator-Activated Receptor δ (PPARδ), a Novel Target Site
for Drug Discovery in Metabolic Syndrome. Pharmacol. Res. 2006,
53, 501–507.
(16) Sugden, M. C.; Holness, M. J. Recent Advances in Mechanisms
Regulating Glucose Oxidation at the Level of the Pyruvate Dehy-
drogenase Complex by PDKs. Am. J. Physiol.: Endocrinol. Metab.
2003, 284, E855–E862.
Acknowledgment. We gratefully acknowledge Dr. Chris-
topher Shelton, Heather L. Fenderson, and Dr. William
Zuercher for providing cell proliferation data of pyridylsul-
fone 3 in colorectal cancer cell lines.
(17) Wang, D.; Wang, H.; Guo, Y.; Ning, W.; Katkuri, S.; Wahli, W.;
Desvergne, B.; Dey, S. K.; DuBois, R. N. Crosstalk between peroxi-
some proliferator-activated receptor δ and VEGF stimulates cancer
progression. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19069–19074.
(18) Wang, D.; DuBois, R. N. Inflammatory Mediators and Nuclear
Receptor Signaling in Colorectal Cancer. Cell Cycle 2007, 6, 682–685.
(19) Peters, J. M.; Hollingshead, H. E.; Gonzalez, F. J. Role of Peroxi-
some-Proliferator-Activated Receptor β/δ (PPARβ/δ) in Gastroin-
testinal Tract Function and Disease. Clin. Sci. 2008, 115, 107–127.
(20) Cell proliferation assay details: Luo, L.; Carson, J. D.; Molnar, K.
S.; Tuske, S. J.; Coales, S. J.; Hamuro, Y.; Sung, C.; Sudakin, V.;
Auger, K. R.; Dhanak, D.; Jackson, J. R.; Huang, P. S.; Tummino,
P. J.; Copeland, R. A. Conformation-Dependent Ligand Regula-
tion of ATP Hydrolysis by Human KSP: Activation of Basal
Hydrolysis and Inhibition of Microtubule-Stimulated Hydrolysis
by a Single, Small Molecule Modulator. J. Am. Chem. Soc. 2008,
130, 7584–7591.
References
(1) Berger, J.; Moller, D. E. The Mechanisms of Action of PPARs.
Annu. Rev. Med. 2002, 53, 409–435.
(2) Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.; Leiters-
dorf, E.; Furchart, J.-C. Mechanism of Action of Fibrates on Lipid
and Lipoprotein Metabolism. Circulation 1998, 98, 2088–2093.
(3) Spiegleman, B. M.; Flier, J. S. Adipogenesis and Obesity: Round-
ing Out the Big Picture. Cell 1996, 87, 377–389.
(4) Burdick, A. D.; Kim, D. J.; Peraza, M. A.; Gonzalez, F. J.; Peters, J.
M. The role of Peroxisome-Activated Receptor-β/δ in Epithelial
Cell Growth and Differentiation. Cell. Signalling 2006, 18, 9–20.