Sulindac derivatives that activate the peroxisome proliferator-activated receptor γ but lack cyclooxygenase inhibition

Article abstract of DOI:10.1021/jm700969c

A series of novel derivatives of the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide were synthesized as potential agonists of the peroxisome proliferator-activated receptor gamma (PPARγ). Nonpolar and aromatic substitutions on the benzylidene ring as well as retention of the carboxylic acid side chain were required for optimal activity. Compound 24 was as potent a compound as any other in the series with an EC₅₀ of 0.1 μM for the induction of peroxisome proliferator response element (PPRE)-luciferase activity. Direct binding of compound 24 to PPARγ was demonstrated by the displacement of [³H]troglitazone, a PPARγ agonist, in a scintillation proximity assay. Compound 24 also stimulated the binding of PPARγ to a PPRE-containing oligonucleotide and induced expression of liver fatty-acid binding protein (L-FABP) and adipocyte fatty acid-binding protein (aP2), two established PPARγ target genes. Taken together, these compounds represent potential leads in the development of novel PPARγ agonists.

Full text of DOI:10.1021/jm700969c

J. Med. Chem. 2008, 51, 4911–4919
4911
Sulindac Derivatives That Activate the Peroxisome Proliferator-activated Receptor γ but Lack
Cyclooxygenase Inhibition
Andrew S. Felts,† Brianna S. Siegel,‡ Shiu M. Young,‡ Christopher W. Moth,† Terry P. Lybrand,† Andrew J. Dannenberg,‡
Lawrence J. Marnett,*,† and Kotha Subbaramaiah‡
A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute
of Chemical Biology, Vanderbilt UniVersity Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center,
Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-0146, Department of Medicine, Weill Cornell Medical College,
New York, New York 10065
ReceiVed August 4, 2007
A series of novel derivatives of the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide were
synthesized as potential agonists of the peroxisome proliferator-activated receptor gamma (PPARγ). Nonpolar
and aromatic substitutions on the benzylidene ring as well as retention of the carboxylic acid side chain
were required for optimal activity. Compound 24 was as potent a compound as any other in the series with
an EC50 of 0.1 µM for the induction of peroxisome proliferator response element (PPRE)-luciferase activity.
Direct binding of compound 24 to PPARγ was demonstrated by the displacement of [3H]troglitazone, a
PPARγ agonist, in a scintillation proximity assay. Compound 24 also stimulated the binding of PPARγ to
a PPRE-containing oligonucleotide and induced expression of liver fatty-acid binding protein (L-FABP)
and adipocyte fatty acid-binding protein (aP2), two established PPARγ target genes. Taken together, these
compounds represent potential leads in the development of novel PPARγ agonists.
Introduction
Peroxisome proliferator-activated receptors (PPARs)a are a
group of nuclear hormone receptors that control cellular
metabolism and proliferation through the modulation of gene
expression. They exist as three distinct subtypes: PPARR,
PPARδ, and PPARγ. Upon ligand binding, PPARs, including
PPARγ, form a heterodimer with the retinoid X receptor and
activate gene expression by binding to PPAR response ele-
ments.1 The binding of various endogenous and exogenous
ligands to PPARγ has been shown to regulate cellular dif-
Figure 1. Structures of sulindac sulfide and 2.
ferentiation, apoptosis, glucose homeostasis, and anti-inflam-
matory responses2 (and references within). Thiazolidinediones,
a class of PPARγ agonists that reduce insulin resistance3,4 are
with residues on the AF-2 helix that anchor it close to the
protein, which in turn allows for successful coactivator binding.8
The aromatic center forms a variety of van der Waals interac-
tions with various hydrophobic residues in the binding site, while
the cyclic tail tolerates a more diverse set of substituents that
are free to interact with a fairly large, hydrophobic pocket.6
used to treat type 2 diabetes.3 A wealth of crystallographic
information for agonist bound to PPAR ligand binding domains
(LBDs) as well as extensive SARs have provided structural
insight into the factors controlling receptor binding and activa-
tion as well as subtype selectivity.5–8 These reports, and others,
have described similar binding pockets for all three receptor
subtypes that include a large, concave, mainly hydrophobic
channel. This binding pocket is significantly larger than other
known nuclear receptors, particularly in PPARR and PPARγ
(∼1400 Å)6 and may explain the observed promiscuity of the
PPARs for various types of ligands.5,9,10 Typically, PPAR
agonists consist of an acidic headgroup, traditionally either a
carboxylic acid or a 2,4-thiazolidinedione tethered to an aromatic
center that is linked to cyclic tail region. The acidic headgroup
is crucial for PPAR activation in that it forms key interactions
Previous studies found that a variety of nonsteroidal anti-
inflammatory drugs (NSAIDs) activate PPARs at micromolar
concentrations.11–13 However, the effects of NSAIDs were
heterogeneous. For example, ibuprofen was demonstrated to be
a coactivator of PPARR and γ, while aspirin, acetaminophen,
and piroxicam showed no agonistic activity for any PPAR
subtype.13 Indomethacin and diclofenac were shown to be
selective for PPARγ although certain reports have claimed that
indomethacin can also act as an agonist for PPARR.12 These
compounds are attractive targets for redesign because their
pharmacokinetics have already been extensively studied in
humans. Chemical removal of cyclooxygenase (COX) inhibition
could reduce potential gastrointestinal and cardiovascular side
effects and provide excellent tools for evaluating the contribution
of COX to their overall biological effects. Previous work in
our laboratory has shown that a subtle chemical modification
of sulindac sulfide (i.e., removal of the indenyl methyl group)
abolishes inhibition of cyclooxygenase while retaining activity
at other targets including PPARγ (Figure 1).14
* To whom correspondence should be addressed. Phone: 615-343-7329.
Fax: 615-343-7534. E-mail: larry.marnett@vanderbilt.edu.
†
Vanderbilt University School of Medicine.
Department of Medicine, Weill Cornell Medical College.
‡
a Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; PPAR,
peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator
response element; L-FABP, liver fatty-acid binding protein; aP2, adipocyte
fatty acid-binding protein; COX, cyclooxygenase; DMEM, Dulbecco’s
modified eagle’s medium; FBS, fetal bovine serum; LBD, ligand binding
domain; HRP, horseradish peroxidase.
10.1021/jm700969c CCC: $40.75
2008 American Chemical Society
Published on Web 07/30/2008

4912 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Scheme 1. Synthesis of Benzylidene Derivativesa
Felts et al.
(9) and sulfone (10), gave compounds that were unable to induce
PPRE-luciferase activity. These results would be expected if
the phenyl ring inserted into the large hydrophobic pocket at
the top of the active site analogous to the phenyloxazole tail of
many traditional PPARγ agonists.6 Removal of the benzylidene
ring to give compound 13 also gave an inactive compound.
Anticipating that the benzylidene ring may be making key
hydrophobic interactions in the PPARγ LBD, a series of
nonpolar and aromatic substitutions were examined. Various
substitutions, including hydrogen (14), methyl (15), trifluoro-
methyl (16), and t-butoxy (20), gave derivatives that were less
potent inducers of PPRE-luciferase activity than 2. Other
substitutions, including bromo (12), alkynyl (17), azido (18),
and methoxy (19), yielded derivatives with comparable PPRE-
luciferase activity to 2.
Large aromatic and heteroaromatic systems comprising the
tail end of the pharmacophore are a common feature of other
known PPARγ agonists.7,8,19 A series of naphthyl and biphenyl
substitutions were introduced, resulting in a series of derivatives
that showed a marked increase in potency relative to 2. Although
attachment of a naphthyl ring at the 2-position gave a derivative
(22), which did not result in a significant increase in potency,
attachment at the 1-position yielded highly potent derivatives
(i.e., 24 and 25) with EC50′s of 0.1 µM. It is important to note
that the EC50 as well as the magnitude of the response for 24
was comparable to troglitazone (see below). Interestingly,
incorporation of a nitrogen to give a quinoline derivative
decreased potency relative to 2. Biphenyl substitutions also
resulted in potent derivatives, 27 and 29, with EC50′s of 0.1
µM, although incorporation of a thiomethyl group at the para
postion of the terminal phenyl ring (28) resulted in a significant
drop in potency.
A selection of these derivatives was tested for their ability
to inhibit the two isoforms of cyclooxygenase. As expected,
none of these compounds showed inhibition of either COX-1
or COX-2 (data not shown) up to concentrations of 4 µM. This
further confirmed that the lack of a 2′-methyl group combined
with the isomeric reversal of configuration around the ben-
zylidene double bond eliminates the ability of this class of
compounds to inhibit cyclooxygenase.14
These results point to an interesting SAR for the activation
of PPRE luciferase activity by 2′-des-methyl sulindac sulfide
derivatives. The carboxylic side chain is absolutely required for
optimal activity, consistent with previous observations. The
benzylidene ring shows a bit more flexibility but an overall
preference for nonpolar substituents on the benzylidene ring was
observed. Finally, aromatic systems, particularly naphthyl and
biphenyl, appear to be highly favored at this position.
a Reagents and conditions: (a) R-CHO, 1 N NaOH, MeOH, reflux, 2 h.
Herein we report the design, synthesis, and biological
evaluation of a unique class of PPARγ agonists derived from
sulindac sulfide, an active metabolite of the NSAID sulindac.
Sulindac sulfide has been reported to activate PPARγ15 although
other reports indicate that it does not.16 Furthermore, sulindac
sulfide has been reported to act as an antagonist for PPARδ by
preventing PPARδ/RXR heterodimer from binding to their
recognition sequences.17 The stereochemistry of the benzylidene
double bond is Z in sulindac and sulindac sulfide, whereas it is
E in the 2′-des-methyl derivatives. The altered stereochemistry
presents a unique scaffold from which to evolve novel PPARγ
agonists. Evaluation of a series of 2′-des-methyl sulindac sulfide
analogues identified highly potent compounds that were es-
sentially equipotent to troglitazone, a thiazolidinedione, in the
ability to stimulate PPARγ-dependent transcription and trigger
adipocyte differentiation in cell culture. shRNA knockdown
experiments indicate these compounds are relatively selective
PPARγ agonists, with weak activity at PPARR.
Results
Chemistry. A series of modifications was made to 2′-des-
methyl sulindac sulfide (2) in an effort to explore the chemical
functionalities required for effective activation of PPARγ.
Synthesis of indene 1 was performed according to the procedure
described previously.14 Condensation of the indene with the
appropriate aldehyde was performed using 1 N NaOH in
refluxing methanol (Scheme 1). The aldehydes used were either
commercially available or synthesized using established meth-
ods. Acidic workup followed by filtration or purification using
column chromatography afforded the requisite 2′-des-methyl
sulindac sulfide derivative. Amides of the 2′-des-methyl sulindac
sulfide derivatives were synthesized using standard EDCI
carbodiimide coupling reactions. The final products were
characterized using electrospray ionization mass spectrometry
1
and H NMR and purity was determined using HPLC.
PPRE-Luciferase Reporter Assay. A PPRE-luciferase re-
porter assay was used to test the ability of the 2′-des-methyl
sulindac sulfide derivatives to activate PPARs. HCA-7 colon
cancer cells, which express PPARR, PPARδ, and PPARγ, were
transiently transfected with an expression vector containing a
PPRE-luciferase reporter plasmid.18 Following transfection, cells
were treated with various concentrations of compound for 12 h
and assayed for luciferase activity.
SAR. The series of derivatives of 2 were synthesized, and
the results of the PPRE-luciferase screening assays are sum-
marized in Table 1. A general descriptor of traditional PPARγ
agonists is the requirement of an acidic headgroup that forms
essential hydrogen bonds with key residues on the AF-2 helix
in the LBD. This was verified in the present series with a number
of amide derivatives, none of which activated PPRE-luciferase
(data not shown).
Compound 24 Activates PPARγ and Induces Adipogenesis. On
the basis of the SAR, 24 was as potent an inducer of PPRE
luciferase activity as any other compound in the series. Hence,
this compound was investigated in greater detail. Initially, 24
was compared with troglitazone, a prototypic PPARγ agonist.
Cells were transfected with an expression vector containing
luciferase under the control of a PPRE. Addition of either
compound stimulated comparable concentration-dependent in-
creases in PPRE luciferase activity (Figure 2a). To determine
whether the increase in PPRE luciferase activity reflected direct
binding of 24 to PPARγ, a scintillation proximity assay was
performed. Compound 24 caused a dose-dependent displacement
of [3H]-troglitazone, indicating that 24 binds directly to PPARγ
(Figure 2b). Next, electrophoretic mobility shift assays were
performed to probe for nuclear translocation of PPARγ. Cells
were treated with 24, nuclear lysates were prepared, and the
Having confirmed the requirement of the acidic acid side
chain for PPRE-luciferase activity, a variety of substitutions on
the benzylidene ring were explored in an effort to increase
potency (Table 1). Polar substitutions, including the sulfoxide

Sulindac DeriVatiVes that ActiVate the PPAR γ
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4913
Table 1. Induction of PPRE-Luciferase Activity by Derivatives of 2′-des-Methyl Sulindac Sulfide
a EC50 is the concentration of compound required to achieve 50% of the maximum induction of PPRE-luciferase activity. Means ( SD are shown, n )
3. b A 2-fold induction corresponds to a doubling of luciferase activity relative to vehicle-treated control.
lysates exposed to radiolabeled oligonucleotides containing a
dependent increase in binding of nuclear protein to labeled PPRE
PPRE sequence. Treatment with 24 led to a concentration-
oligonucleotides (Figure 3, lanes 1-5). The specificity of this

4914 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Felts et al.
Figure 3. Compound 24 stimulates PPARγ binding. Nuclear proteins
from HCA7 cells were incubated with a 32P-labeled PPRE containing
oligonucleotide. Cells were treated with vehicle (lane 1) or 24 (0.1
µM, lane 2; 0.25 µM, lane 3; 0.5 µM, lane 4; 1 µM, lane 5) for 30
min. Lanes 6 and 10 represent nuclear extract from cells treated with
1 µM 24 incubated with a 32P-labeled PPRE containing oligonucleotide
and a 100-fold excess of unlabeled oligonucleotide (lane 6) or mutant
PPRE containing oligonucleotide (lane 10). Lanes 7-9 represent nuclear
extract from cells treated with 1 µM 24 incubated with a 32P-labeled
PPRE containing oligonucleotide incubated with 0 µL (lane 7), 1 µL
(lane 8) or 2 µL (lane 9) of antibody to PPARγ. The protein-DNA
complex that formed was separated on a 4% polyacrylamide gel.
Figure 2. Compound 24 is a PPARγ agonist. (a) HCA7 cells were
transfected with 1.8 µg of PPRE luciferase and 0.2 µg of pSVꢀgal.
Cells were treated with 0-2.5 µM 24 or troglitazone for 12 h.
Luciferase activity represents data that have been normalized to
ꢀ-galactosidase. Columns, means; bars, SD; n ) 6. *: p < 0.05,
**: p < 0.01. (b) 24 binds to PPARγ. Competitive binding assays
were performed by scintillation proximity assay for human PPARγ-
ligand binding domain and 50 nM 3H-troglitazone in the presence
of increasing concentrations of nonradioactive 24 as a competitor.
Means and SD are shown, n ) 3.
Figure 4. Compound 24 modulates the expression of known PPARγ
target genes. (a) HCA7 cells were treated with 0-1 µM 24 for 24 h.
Cellular lysate protein (100 µg/lane) was loaded onto a 12.5% SDS-
polyacrylamide gel, electrophoresed, and subsequently transferred onto
nitrocellulose. Immunoblots were probed with antibodies to L-FABP,
aP2, and ꢀ-actin. (b) 30, a PPARγ antagonist, suppresses 24-mediated
induction of L-FABP and aP2. Cells were treated as indicated with
vehicle, 24, 30 alone, or 24 and 30 for 24 h. Cellular lysate protein
(100 µg/lane) was loaded onto a 12.5% SDS-polyacrylamide gel,
electrophoresed, and subsequently transferred onto nitrocellulose. The
immunoblot was probed for L-FABP, aP2, and ꢀ-actin.
binding was also evaluated; binding was abolished when an
excess of unlabeled consensus PPRE oligonucleotide was added
(Figure 3, lane 6). By contrast, binding was unaffected by the
addition of an excess of oligonucleotide in which the PPRE
site was scrambled (Figure 3, lane 10). Supershift analysis
identified PPARγ in the binding complex (Figure 3, lane 9).
To further evaluate the functional significance of 24 as a
PPARγ agonist, we investigated its effects on the levels of
L-FABP and aP2, two established PPARγ target genes.20,21 Cells
were treated with 0-1 µM 24 for 24 h. Following treatment,
cell lysates were prepared and Western blotting was performed.
Compound 24 induced both L-FABP and aP2 (Figure 4a) over
the same concentration range that stimulated PPARγ binding
(Figure 3). Induction of L-FABP and aP2 was suppressed by
pretreatment with 30 (GW9662)22 (Figure 5), a PPARγ antago-
nist (Figure 4b). PPARγ agonists induce adipogenesis;23 hence,
we also investigated whether 24 could stimulate triglyceride
accumulation in 3T3-L1 cells, a murine fibroblast cell line. As
shown in Figure 6, staining with Oil Red O revealed formation
of lipid droplets in cells treated with 24 in a manner similar to
the known PPARγ activator, troglitazone.
Figure 5. Structures of 30 and 31.
shRNA knockdown of PPARr, PPARδ, and PPARγ.
Previous studies have shown that NSAIDs and NSAID deriva-
tives that activate PPARγ also activate PPARR to varying
extents. Western blot analysis indicated that HCA-7 cells contain
PPARR, PPARγ, and PPARδ (Figure 7a). Overexpressing of
shRNAs to the three PPAR isoforms led to selective silencing
of each PPAR (Figure 7a). Knocking down PPARγ abrogated
troglitazone-mediated activation of PPRE-luciferase (Figure 7b).
The inductive effects of 24 were also attenuated when PPARγ
was silenced, although induction was not reduced to the level

Sulindac DeriVatiVes that ActiVate the PPAR γ
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4915
Figure 6. Compound 24 activates adipogenesis in 3T3-L1 cells. Images of Oil Red O stained 3T3-L1 cells were taken after treatment with (a)
vehicle (b) 1 µM troglitazone or (c) 1 µM 24 for 48 h (200×).
Figure 7. Compound 24 is a mixed agonist of PPARγ and PPARR. (a) HCA7 cells were transfected with control shRNA or shRNAs to PPARγ,
PPARR and PPARδ. Cellular protein was isolated (100 µg/lane) and loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently
transferred onto nitrocellulose sheet. Immunoblots were probed with the indicated antibodies. In each case, the shRNA selectively silenced its
receptor. (b-d) shRNA expressing cells were transfected with 1.8 µg PPRE luciferase and 0.2 µg pSVꢀgal. Cells were treated with vehicle, 1 µM
troglitazone, or 1 µM 24 (b); vehicle, 2 µM 31 or 1 µM 24 (c); vehicle, 2.5 µM cPGI or 1 µM 24 (d) for 12 h. Luciferase activity represents data
that have been normalized to ꢀ-galactosidase. Column, means; bars, S.D; n ) 6, *, p < 0.05.
seen in vehicle-treated control (Figure 7b). Compound 31
(GW7647)24 (Figure 5), a PPARR agonist, caused a marked
increase in PPRE-luciferase activity, an effect that was abrogated
when PPARR was silenced (Figure 7c). A small but statistically
significant reduction in the inductive effects of 24 was found
in cells in which PPARR was silenced, indicating that 24 may
act as a weak agonist of PPARR. cPGI, an agonist of PPARδ
was a potent inducer of PPRE-luciferase, an effect that was
abrogated when PPARδ was silenced. In contrast, silencing of
PPARδ did not affect the induction of PPRE-luciferase by 24,
indicating that 24 does not act as an agonist for PPARδ.
Molecular Docking of 24 in the hPPARγ Active Site. To
date, crystal structures of PPARγ in complex with carboxylate-
containing full agonists support a model of receptor activation
in which the bound agonist hydrogen bonds with H323 and the
hydroxyl group of Y473 in the AF-2 helical domain. Stabilized
by this hydrogen-bonding network, the AF-2 domain packs
closely with the protein core, which closes the binding site and
is proposed to activate the receptor.5,7
We generated models for compound 24 bound to PPARγ to
determine whether this molecule might be able to stabilize a
closed, activated receptor structure comparable to those observed

4916 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Felts et al.
Figure 8. PPARγ complex with compound 24. The carboxylate of compound 24 forms hydrogen bonds (shown as dashed yellow lines) with H323
and Y473 of the AF-2 helical domain, displayed in red. The naphthalene moiety of 24 binds in a hydrophobic pocket formed by residues C285,
Y327, L330, M334, V339, L353, M364, and the aliphatic carbons of K367, each colored by atom type. Figure 8 was created with the DINO
program.35
for other full agonists. We used ab initio Hartree-Fock
calculations to compute the global minimum energy structure
for compound 24 and then docked this geometry-optimized
conformation in the receptor using the crystal structure for
the PPARγ complex with the full agonist 2-(5-[3-(7-propyl-
3-trifluoromethylbenzo[d]isoxazol-6-yloxy)propoxy]indol-1-
yl)ethanoic acid (PDB code: 2ATH) as a template. This crystal
structure was chosen because it contained similar structural
elements of the ligand relative to 24 (indole versus indene core)
as well as for sufficient resolution (2.28 Å). Compound 24 was
positioned in the binding site so that its carboxylate group could
form favorable hydrogen bonds with H323 and Y473 in the
AF-2 domain. This ligand orientation placed the large napthalene
substituent neatly into a nearby hydrophobic pocket formed by
residues C285, Y327, L330, M334, V339, L353, M364, and
the aliphatic carbons of K367 (Figure 8). Compound 24 and
protein hydrogen atoms were relaxed with 1000 cycles of
conjugate gradient energy minimization, followed by 2000
additional cycles of minimization for the full receptor-ligand
complex to relieve any residual unfavorable steric interactions.
The rmsd for the minimized PPARγ backbone vs crystal
structure is 0.27 Å.
and aP2. Compound 24 induced the accumulation of lipid in
cells, a finding that is characteristic of PPARγ agonists. shRNA
knockdown of PPARR and PPARγ indicated that 24 is a potent
agonist of PPARγ with only weak activity at PPARR. This
finding contrasts with previous work, demonstrating that certain
NSAIDs are mixed agonists of PPARγ and R with similar
potencies of activation.11 shRNA knockdown of PPARδ indi-
cated that 24 had no effect on PPARδ.25
Our model for the PPARγ-compound 24 complex accom-
modates the ligand well in its global minimum energy confor-
mation and explains the experimental observations. In the model,
the ligand carboxylate forms a hydrogen bond network with
H323 and Y473, comparable to that observed in crystal
structures for other full agonist complexes. This hydrogen-
bonding network recruits the AF-2 helix domain to close the
binding site and activate the receptor. The naphthalene sub-
stituent fits nicely in the large hydrophobic pocket described
above and thus increases favorable protein/ligand contacts
compared to other ligands in the series.
Traditional NSAIDs inhibit COX activity in addition to
having collateral targets such as PPARγ. The derivatives
presented in this report represent structurally distinct deriva-
tions of sulindac sulfide that could not necessarily have been
predicted to retain similar activity. Furthermore, this report
also represents the first published exploration of the structural
requirements necessary for potent PPARγ activation specific
to this class of PPAR agonists. The compounds reported
herein represent a new class of PPARγ activators that lack
the COX inhibitory activity of sulindac sulfide, the parent
NSAID. Derivation of the core structure led to compounds
with potency that was similar to troglitazone, a known
PPARγ agonist. Hence, compounds such as 24 provide
potentially useful tools for understanding the contribution
of the PPARγ response to the overall biological responses
elicited by sulindac sulfide, a compound that can both activate
PPARγ and inhibit COX. Recently, rosiglitazone, a PPARγ
agonist that is used to treat diabetes mellitus, was found to
be efficacious in the treatment of mild to moderately active
ulcerative colitis.26 Future studies will be needed to determine
Discussion
The results from these experiments indicate that sulindac
sulfide as well as its 2′-des-methyl derivatives are potent
inducers of PPARγ. This contradicts an earlier report indicating
that sulindac sulfide does not activate PPARγ.16 The SAR
confirms that the carboxylic side chain is required for activity;
the neutral amide derivatives showed a complete loss of activity.
We also found that nonpolar and aromatic substituents on the
benzylidene ring lead to potent PPARγ agonists. Specifically,
a naphthalene substitution at the 1-position led to compound
24, which had an EC50 of 0.1 µM in a PPRE-luciferase activation
assay. Compound 24 was able to displace [3H]-troglitazone in
a scintillation proximity assay demonstrating that activation of
PPRE-luciferase reflects direct binding to PPARγ. Nuclear
translocation of PPARγ by 24 was confirmed using an elec-
trophoretic mobility shift assay in which a concentration-
dependent increase in binding of nuclear protein to labeled PPRE
oligonucleotides was observed. Moreover, 24 induced known
PPARγ target genes, L-FABP and aP2, and 31, a PPARγ
antagonist, suppressed 24-mediated induction of both L-FABP

Sulindac DeriVatiVes that ActiVate the PPAR γ
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4917
Electrophoretic Mobility Shift Assay. Nuclear extracts were
prepared from cells. For binding studies, oligonucleotides containing
PPRE sites were obtained from Active Motif (Carlsbad, CA). The
complementary oligonucleotides were annealed in 20 mM Tris
(pH7.6), 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol.
The annealed oligonucleotide was phosphorylated at the 5′ end with
[γ-32P]ATP and T4 polynucleotide kinase. The binding reaction
was performed by incubating 5 µg of nuclear protein in 20 mM
HEPES (pH 7.9), 10% glycerol, 300 µg of bovine serum albumin,
and 1 µg of poly (dI·dC) in a final volume of 10 µL for 10 min at
25 °C. The labeled oligonucleotide was added to the reaction
mixture and allowed to incubate for an additional 20 min at 25 °C.
The samples were electrophoresed on a 4% nondenaturing poly-
acrylamide gel. The gel was then dried and subjected to autorad-
iography at -80 °C.
Western Blotting. Cell lysates were prepared by treating cells
with lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween
20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL
trypsin inhibitor, 10 µg/mL aprotinin, and 10 µg/mL leupeptin).
Following sonication, lysates were centrifuged at 4 °C for 10 min
at 10000g to sediment the particulate material. The protein
concentration of the supernatant was measured using a BCA protein
assay kit. SDS-polyacrylamide gel electrophoresis was performed
using 12.5% polyacrylamide gels as described by Laemmli.30 The
resolved proteins (100 mg/lane) were transferred overnight onto a
nitrocellulose membrane and then probed with primary antisera.
Secondary antibody conjugated to HRP was then used. The blots
were then developed with the ECL Western blot detection system
according to the manufacturer’s instructions.
whether the derivatives of sulindac sulfide such as 24 possess
properties that make them attractive leads as drug candidates.
Experimental Section
Materials. BCA protein assay reagent kit and NHC-biotin were
purchased from Pierce Biotechnology, Inc. (Rockford, IL). Nitrocel-
lulose membranes were from Whatman, Inc. (Sanford, ME).
Antibody to ꢀ-actin, secondary antibodies conjugated to horseradish
peroxidase (HRP), and 2-nitrophenyl ꢀ-D-galactopyranoside were
from Sigma-Aldrich Co. (St. Louis, MO). 30 was from EMD
Biosciences, Inc. (San Diego, CA). Polyclonal chicken antiadipocyte
fatty acid-binding protein (aP2) and adipogenesis assay kits were
from Chemicon International Inc. (Temecula, CA). Polyclonal
antisera to liver fatty acid binding protein (L-FABP) was from
Abcam Inc. (Cambridge, MA). Bovine antichicken IgY-HRP
secondary antibody and isoform specific PPAR antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Western blotting detection reagents (ECL) and [32P]-ATP were from
Perkin-Elmer LAS, Inc. (Boston, MA). Plasmid DNA preparation
kits were from Qiagen (Valencia, CA). Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS), OPTI-MEM, and
Lipofectamine 2000 were from Invitrogen Corp. (Carlsbad, CA).
Calf bovine serum (CBS) and 3T3-L1 cells were from the American
Type Culture Collection (Manassas, VA). Luciferase assay sub-
strates A and B and cell lysis buffer were from BD Biosciences &
Co. (Franklin Lakes, NJ). pSVꢀgal was obtained from Promega
Corp. (Madison, WI). The PPRE luciferase and PPAR-γligand
binding domain (PPARγ-LBD) plasmids were gifts from Dr. Ron
Evans (Salk Institute for Biological Sciences, La Jolla, CA).
Streptavidin SPA beads were from Amersham Biosciences (Pis-
cataway, NJ). cPGI and 31 were from Cayman Chemical Co. (Ann
Arbor, MI). Control shRNA and shRNAs to PPARs were obtained
from Superarray Bioscience Corp. (Frederick, MD).
Adipogenesis Assay. The adipogenesis assay was performed
using a kit from Chemicon International. Briefly, 3T3-L1 cells were
grown to 60% confluence. The cells were then placed in growth
medium supplemented with 0.1% 3-isobutyl-1-methylxanthine and
1.0% dexamethasone to initiate adipogenesis for 48 h. The cells
were then treated in growth medium containing 10 µg/mL of
recombinant human insulin supplemented with the test compound
for an additional 2 days. The medium was then replaced with fresh
growth medium for an additional 48 h prior to staining with Oil
Red O. Images were taken using Q-Imaging Retina EX camera
and Olympus IX51 bright field microscope (200x).
Chemistry. HPLC grade solvents obtained from Fischer (Pitts-
burgh, PA) were used for column chromatography. Reagent grade
chemicals were obtained from Aldrich (Milwaukee, WI), Matrix
Scientific (Columbia, SC), Alfa Aesar (Ward Hill, MA), and
Oakwood Products (West Columbia, SC). All other chemicals were
used without further purification. Thin layer chromatography was
performed on silica plates obtained from Analtech (Silica Gel 60
Statistics. Comparisons between groups were made with Stu-
dent’s t test. A difference between groups of p < 0.05 was
considered significant.
F254 precoated). The plates were read by UV fluorescence (254 nm)
or by staining with phosphomolybdic acid (PMA) followed by
heating. Column chromatography was performed using a Biotage
SP1 purification system.
Molecular Modeling. Geometry optimization and conformation
analysis calculations were performed with restricted Hartree-Fock
(RHF) calculations using a 3-21G* basis set with the Gaussian03
package.31 The global minimum energy structure for compound
24 was docking manually in the binding site of the 2ATH crystal
structure7 using the interactive molecular graphics program PSS-
HOW.32 The protein-ligand complex was refined using conjugate
gradient energy minimization and a generalized Born solvent model
with the AMBER9 package.33 A molecular electrostatic potential
for the geometry-optimized conformation of compound 24 was
computed using RHF calculations with a 6-31G* basis set, and the
RESP program34 was then used to fit a set of atom-centered partial
charges that reproduced the electrostatic potential.
Instrumental Analysis. Mass spectra were obtained by elec-
trospray ionization (ESI-MS) on a Finnigan TSQ 7000 triple-
quadrupole spectrometer. 1H NMR spectra were obtained on a
Bruker AC 300 NMR spectrometer using CDCl3 or DMSO-d6 as
the solvent and TMS as an internal standard. All chemical shifts
are reported in ppm downfield from TMS and coupling constants
are reported in hertz.
General Procedure for the Synthesis of Benzylidene Deriva-
tives of Ethyl 2-(5-fluoro-1H-inden-3-yl)ethanoate. To a solution
of the ethyl 2-(5-fluoro-1H-inden-3-yl)ethanoate (1 equiv) and the
appropriate aldehyde (1.2 equiv) was added 1 N NaOH and
methanol. The mixture was stirred at reflux for 2 h. The solution
was cooled, neutralized with 15% HCl, diluted with water, and
extracted with CH2Cl2 (3×). The combined organics were washed
with water, NaHCO3, water, dried (MgSO4), filtered, and concen-
Tissue Culture. The HCA7 human colon cancer cell line was
established from moderately differentiated adenocarcinoma of the
colon.27 The cells were maintained in DMEM supplemented with
100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% FBS.
3T3-L1 cells were grown in DMEM supplemented with 10% CBS.
Creation of Stable Cell Lines. HCA7 cells were transfected
with control shRNA or shRNAs to different PPARs. The Amaxa
nucleofection system (Gaithersburg, MD) was used and the
manufacturer’s instructions employed. Puromycin B resistant cells
were selected.
Transient Transfection Assays. Cells were grown to 40%
confluence in 6-well dishes. For each well, 2 µg of plasmid DNA
were introduced into cells using 6 µg of Lipofectamine 2000 as
per the manufacturer’s instructions. After 6 h of incubation, the
medium was replaced with growth medium for 16 h, followed by
serum-free media containing the compounds for 12 h. The activities
of luciferase and ꢀ-galactosidase were measured.
Scintillation Proximity Assay. The assay was performed as
described previously.28 The PPARγ-LBD was isolated from Es-
cherichia coli as a polyhistidine-tagged fusion protein. Radiolabeled
troglitazone was synthesized as described previously.29 The buffer
for all assays was 50 mM HEPES (pH 7), 50 mM KCl, 5 mM
CHAPS, 0.1 mg/mg BSA. The protein was biotinylated and
immobilized on streptavidin-modified SPA beads. Nonradioactive
24 was used to compete for binding to the PPARγ-LBD using 3H-
troglitazone as the ligand. The assays were performed in the absence
of dithiothreitol.

4918 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Felts et al.
trated in vacuo. All purifications were performed using flash
chromatography.
(E)-2-(5-Fluoro-1-(quinoxalin-6-ylmethylene)-1H-inden-3-yl)-
ethanoic acid (23). 1H NMR (DMF) δ 8.97 (d, J ) 10.2 Hz, 2H),
8.32 (s, 1H), 8.23 (d, J ) 8.7 Hz, 1H), 8.14 (d, J ) 9.0 Hz, 1H),
7.92-7.87 (m, 1H), 7.84 (s, 1H), 7.25 (m, 2H), 7.04 (t, J ) 8.1
Hz, 1H), 3.72, (s, 2H). ESI 331 (M - H+).
(E)-2-(5-Fluoro-1-(4-(methylsulfinyl)benzylidene)-1H-inden-3-
yl)ethanoic acid (9). 1H NMR (DMSO) δ 7.87-7.82 (m, 1H), 7.84
(d, J ) 8.4 Hz, 2H), 7.77 (d, J ) 8.4 Hz, 2H), 7.69 (s, 1H), 7.17
(dd, J ) 2.4, 9.2 Hz, 1H), 7.09 (s, 1H), 7.06 (td, J ) 2.4, 8.7 Hz,
1H), 3.68 (s, 2H), 2.79 (s, 3H). ESI 341 (M - H+).
(E)-2-(5-Fluoro-1-(4-(methylsulfonyl)benzylidene)-1H-inden-3-
(E)-2-(5-Fluoro-1-(naphthalen-1-ylmethylene)-1H-inden-3-yl)-
1
ethanoic acid (24). H NMR (CDCl3) δ 8.12-8.07 (m, 1H), 8.04
(s, 1H), 7.93-7.86 (m, 2H), 7.74 (dd, J ) 4.9, 8.2 Hz, 1H),
7.60-7.50 (m, 4H), 7.04 (dd, J ) 2.3, 8.9 Hz, 1H), 6.99 (td, J )
2.3, 8.3 Hz, 1H), 6.85 (s, 1H), 3.64 (s, 2H). ESI 329 (M - H+).
(E)-2-(5-Fluoro-1-((4-fluoronaphthalen-1-yl)methylene)-1H-in-
1
yl)ethanoic acid (10). H NMR (CDCl3) δ 7.99 (d, J ) 7.8 Hz,
2H), 7.72 (d, J ) 7.8 Hz, 2H), 7.62 (dd, J ) 5.1, 7.8 Hz, 1H), 7.38
(s, 1H), 7.04-6.95 (m, 3H), 3.69 (s, 2H), 3.05 (s, 3H). ESI 357
(M - H+).
1
den-3-yl)ethanoic acid (25). H NMR (DMSO) δ 8.32-8.29 (m,
2H), 8.15-8.06 (m, 2H), 7.75-7.69 (m, 2H), 7.62 (dd, J ) 5.7,
7.9 Hz, 1H), 7.46 (dd, J ) 8.0, 10.6 Hz, 1H), 7.18 (dd, J ) 2.3,
9.3 Hz, 1H), 7.11 (dtd, J ) 2.2, 2.4, 9.7 Hz, 1H), 6.82 (s, 1H),
3.65 (s, 2H). ESI 347 (M - H+).
(E)-4-((3-(Carboxymethyl)-5-fluoro-1H-inden-1-ylidene)methyl)-
benzoic acid (11). 1H NMR (DMSO) δ 8.01 (d, J ) 8.3 Hz, 2H),
7.86 (dd, J ) 5.1, 8.3 Hz, 1H), 7.76 (d, J ) 8.3 Hz, 2H), 7.69 (s,
1H), 7.16 (dd, J ) 2.4, 9.2 Hz, 1H), 7.10-7.04 (m, 2H), 3.68 (s,
2H). ESI 279 (M - CO2H+).
(E)-2-(5-Fluoro-1-(quinolin-8-ylmethylene)-1H-inden-3-yl)etha-
1
noic acid (26). H NMR (DMSO) δ 9.00 (dd, J ) 1.8, 4.2 Hz,
(E)-2-(1-(4-Bromobenzylidene)-5-fluoro-1H-inden-3-yl)ethanoic
acid (12). 1H NMR (CDCl3) δ 7.60 (dd, J ) 4.9, 8.2 Hz, 1H), 7.56
(d, J ) 8.5 Hz, 2H), 7.43 (d, J ) 8.4 Hz, 2H), 7.32 (s, 1H), 7.03
(dd, J ) 2.3, 8.8 Hz, 1H), 6.99 (s, 1H), 6.95 (td, J ) 2.3, 8.4 Hz,
1H), 3.68 (s, 2H). ESI 359 (M - H+).
1H), 8.61 (s, 1H), 8.44 (dd, J ) 1.5, 8.1 Hz, 1H), 8.06-8.00 (m,
2H), 7.93 (dd, J ) 5.1, 8.4 Hz, 1H), 7.73 (t, J ) 7.8 Hz, 1H), 7.63
(dd, J ) 4.2, 8.4 Hz, 1H), 7.19 (dd, J ) 2.4, 9.3 Hz, 1H), 7.11-7.04
(m, 2H), 3.68 (s, 2H). ESI 330 (M - H+).
2-(5-Fluoro-1H-inden-3-yl)ethanoic acid (13). 1H NMR (DMSO)
δ 7.44 (dd, J ) 5.2, 8.2 Hz, 1H), 7.15 (dd, J ) 2.5, 9.5 Hz, 1H),
6.99 (dtd, J ) 1.7, 2.5, 10.0 Hz, 1H), 6.53 (s, 1H), 3.54 (s, 2H),
3.34 (s, 2H). ESI 191 (M - H+).
(E)-2-(1-(Biphenyl-4-ylmethylene)-5-fluoro-1H-inden-3-yl)etha-
1
noic acid (27). H NMR (DMSO) δ 7.86 (dd, J ) 5.1, 8.3 Hz,
1H), 7.78 (m, 4H), 7.74 (d, J ) 7.2 Hz, 2H), 7.67 (s, 1H), 7.49 (td,
J ) 1.7, 7.1 Hz, 2H), 7.39 (tt, J ) 2.1, 7.3 Hz, 1H), 7.19-7.15 (m,
2H), 7.07 (td, J ) 2.4, 8.4 Hz, 1H), 3.69 (s, 2H). ESI 355 (M -
H+).
(E)-2-(1-Benzylidene-5-fluoro-1H-inden-3-yl)ethanoic acid (14).
1H NMR (MeOD) δ 7.70 (dd, J ) 5.0, 8.3 Hz, 1H), 7.62 (d, J )
7.3 Hz, 2H), 7.48 (s, 1), 7.45-7.40 (m, 2H), 7.37-7.31 (m, 1H),
7.07-7.03 (m, 2H), 6.93 (td, J ) 2.4, 8.4 Hz, 1H), 3.65 (s, 2H).
ESI 279 (M - H+).
(E)-2-(5-Fluoro-1-((4′-(methylthio)biphenyl-4-yl)methylene)-1H-
1
inden-3-yl)ethanoic acid (28). H NMR (DMSO) δ 7.86 (dd, J )
5.1, 8.3 Hz, 1H), 7.80-7.55 (m, 7H), 7.36 (d, J ) 7.2 Hz, 2H),
7.18-7.16 (m, 2H), 7.06 (td, J ) 2.5, 8.4 Hz, 1H), 3.67 (s, 2H),
2.52 (s, 3H). ESI 401 (M - H+).
(E)-2-(5-Fluoro-1-(4-methylbenzylidene)-1H-inden-3-yl)ethanoic
acid (15). 1H NMR (CDCl3) δ 7.60 (dd, J ) 4.9, 8.3 Hz, 1H), 7.48
(d, J ) 8.0 Hz, 2H), 7.38 (s, 1H), 7.24 (d, J ) 8.0 Hz, 2H), 7.07
(s, 1H), 7.02 (dd, J ) 2.3, 8.9 Hz, 1H), 6.93 (td, J ) 2.3, 9.2 Hz,
1H), 3.68 (s, 2H), 2.39 (s, 3H). ESI 293 (M - H+).
(E)-2-(5-Fluoro-1-(4-(trifluoromethyl)benzylidene)-1H-inden-3-
yl)ethanoic acid (16). 1H NMR (CDCl3) δ 7.69-7.59 (m, 5H), 7.38
(s, 1H), 7.02 (dd, J ) 2.3, 8.8 Hz, 1H), 6.99-6.92 (m, 2H), 3.68
(s, 2H). ESI 347 (M - H+).
(E)-2-(1-((2′,4′-Difluorobiphenyl-4-yl)methylene)-5-fluoro-1H-in-
1
den-3-yl)ethanoic acid (29). H NMR (DMSO) δ 7.85 (dd, J )
5.1, 8.3 Hz, 1H), 7.77 (d, J ) 8.4 Hz, 2H), 7.68-7.60 (m, 4H),
7.38 (dtd, J ) 2.1, 2.6, 9.3 Hz, 1H), 7.24-7.15 (m, 3H), 7.06 (dtd,
J ) 1.3, 2.4, 8.4 Hz, 1H), 3.69 (s, 2H). ESI 391 (M - H+).
Acknowledgment. This research was supported by research
grants from the National Institutes of Health (CA89450,
CA111469), the New York Crohn’s Foundation, and XL Tech
Group.
(E)-2-(1-(4-Ethynylbenzylidene)-5-fluoro-1H-inden-3-yl)ethanoic
1
acid (17). H NMR (DMSO) δ 7.84 (dd, J ) 5.2, 8.3 Hz, 1H),
7.68 (d, J ) 8.3 Hz, 2H), 7.63 (s, 1H), 7.56 (d, J ) 8.3 Hz, 2H),
7.16 (dd, J ) 2.1, 9.2 Hz, 1H), 7.09 (s, 1H), 7.06 (td, J ) 2.3, 9.1
Hz, 1H), 4.35 (s, 1H), 3.67 (s, 2H). ESI 303 (M - H+).
Supporting Information Available: HPLC traces for all new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(E)-2-(1-(4-(Azidomethyl)benzylidene)-5-fluoro-1H-inden-3-yl)-
1
ethanoic acid (18). H NMR (CDCl3) δ 7.62-7.56 (m, 3H), 7.38
(s, 1H), 7.37 (d, J ) 8.0 Hz, 2H), 7.03-7.00 (m, 2H), 6.94 (td, J
) 2.25, 9.1 Hz, 1H), 4.38 (s, 1H), 3.67 (s, 1H). ESI 334 (M -
H+).
(E)-2-(5-Fluoro-1-(4-methoxybenzylidene)-1H-inden-3-yl)etha-
noic acid (19). 1H NMR (CDCl3) δ 7.60 (dd, J ) 4.9, 8.3 Hz, 1H),
7.55 (d, J ) 8.7 Hz, 2H), 7.35 (s, 1H), 7.08 (s, 1H), 7.02 (dd, J )
2.3, 8.9 Hz, 1H), 6.96 (d, J ) 8.8 Hz, 2H), 6.97-6.90 (m, 1H),
3.86 (s, 3H), 3.69 (s, 2H). ESI 309 (M - H+).
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