Synthesis of a high-affinity Fluorescent PPARγ ligand for high-throughput fluorescence polarization assays

Article abstract of DOI:10.1016/S0968-0896(03)00494-2

Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investigation as molecular targets for the treatment of numerous diseases including Alzheimer's, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARγ subtype complexed with the potent small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein. This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor affinity and maximal effects on fluorescence polarization properties. The recombinant PPARγ ligand binding domain protein bound tightly and specifically to this probe with K _d=61±14 nM as determined by FP measurements. Competition binding assays with known PPARγ ligands provided K_i values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. This fluorescent PPARγ probe enables high-throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARγ ligands, and this rational ligand design approach may be applied to other therapeutically important members of the nuclear hormone receptor superfamily.

Full text of DOI:10.1016/S0968-0896(03)00494-2

Bioorganic & Medicinal Chemistry 11 (2003) 4325–4332
Synthesis of a High-Affinity Fluorescent PPARꢀ Ligand for
High-Throughput Fluorescence Polarization Assays
Michael J. DeGrazia,a Jerry Thompson,b John P. Vanden Heuvelb
and Blake R. Petersona,*
aDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA
bDepartment of Veterinary Sciences andCenter for Molecular Toxicology, The Pennsylvania State University,
University Park, PA 16802, USA
Received 15 July 2003; accepted 23 July 2003
Abstract—Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investiga-
tion as molecular targets for the treatment of numerous diseases including Alzheimer’s, asthma, atherosclerosis, inflammation,
multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARg subtype complexed with the potent
small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous
and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon
atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of
GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein.
This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor
affinity and maximal effects on fluorescence polarization properties. The recombinant PPARg ligand binding domain protein bound
tightly and specifically to this probe with Kd=61ꢀ14 nM as determined by FP measurements. Competition binding assays with
known PPARg ligands provided Ki values that were highly correlated with analogous values obtained by scintillation proximity
(SP) assays. This fluorescentPPAR g probe enables high-throughput and homogenous FP assays for the identification of novel
endogenous and exogenous PPARg ligands, and this rational ligand design approach may be applied to other therapeutically
important members of the nuclear hormone receptor superfamily.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
and the more recently described GI26257028ꢁ30 (1, Fig.
1) exert their therapeutic effects by targeting the PPARg
subtype. Given the importance of PPARg in human
disease, significanteffortis currenlty focused on hte
discovery of improved therapeutics that modulate
activities of this receptor and the identification of nat-
ural endogenous ligands. To facilitate these studies, we
report here the design, synthesis, binding properties, and
utility of compound 2, a derivative of GI262570 as a
fluorescentPPAR g ligand suitable for high-throughput
fluorescence polarization (FP) assays directed at the dis-
covery of novel endogenous and exogenous PPARg
ligands.
Peroxisome proliferator-activated receptors (PPARs)
are members of the nuclear hormone receptor super-
family of ligand-regulated transcription factors.1ꢁ7
These proteins comprise an important class of thera-
peutic targets thought to be involved in Alzheimer’s
disease, asthma, atherosclerosis, inflammation, multiple
sclerosis, cancer, and diabetes.8ꢁ11 The PPARg subtype
is involved in glucose and lipid homeostasis, adipocyte
differentiation, and insulin sensitivity.12 The binding of
endogenous ligand(s) such as Prostaglandin J2 to
PPARg results in the formation of heterodimers with
RXR proteins, interaction with co-activators, and reg-
ulation of the expression of target genes.11,13ꢁ19 Several
antidiabetic drugs including the thiazolidinediones20ꢁ27
Ligand Design
We employed the X-ray crystal structure of the hetero-
dimeric ligand binding domain (LBD) co-complex of
RXRa/9-cis-retinoic acid, PPARg/GI262570, and an
*Corresponding author. Tel.+1-814-865-2969; fax:+1-814-863-8403;
e-mail: brpeters@chem.psu.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00494-2

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tionally restricted upon protein binding, potentially
providing a strong effect on the fluorescence polariza-
tion properties of the probe.
Synthesis
Preparation of 2 commenced with optimization of the
synthesis of 3-iodobenzamide 633,34 as outlined in
Scheme 1. This product was cyclized with bromoketo-
ester 4 prepared from 3 as previously reported35 to
afford the meta-iodophenyloxazole 7. Reduction of 7
with lithium borohydride provided the primary alcohol
8, which was subjected to a Mitsunobu reaction36ꢁ38
with phenol 9, prepared as previously reported.28 The
linked methyl ester product 10 was hydrolyzed to acid
11. This acid (11) was synthesized as an analogue of
GI262570 (1) to provide a high-affinity iodo-modified
ligand of PPARg for subsequentcompeititon binding
experiments. Ester 10 was also modified by Sonagashira
coupling39,40 with Boc-protected propargylamine (12)41
to provide the side-chain coupled product 13. The
methyl ester group of 13 was hydrolyzed under basic
conditions, the Boc-carbamate removed under acidic
conditions, and the resulting amine acylated with fluor-
escein isothiocyanate (FITC) to afford the fluorescein-
modified GI262570 derivative 2 (Scheme 1) in 7.8%
yield over 10 steps. This compound was isolated as the
weakly fluorescentfree acid form shown in Scheme 1.
Dissolution of 2 in buffer atpH 7.4 generates the highly
fluorescent anionic tautomer shown in Figure 1.
Figure 1. Structures of PPARg ligands. The novel fluoresentprobe 2
was designed from the X-ray crystal structure of PPARg-bound 1.
SRC-1 peptide,10,31,32 to design the fluorescent ligand 2
as a PPARg probe. The GI262570 agonistwas chosen
for modification because of its high affinity (Ki=1.2
nM)28 for PPARg and the availability of high-resolution
structural information regarding the protein-bound
complex (Fig. 2). As shown in Figure 2, examination of
the PPARg–GI262570 subunit revealed that the pro-
tein-bound drug is nearly completely encapsulated by
protein except on one face of the phenyl oxazole moiety.
A phenyl carbon atom meta to the oxazole moiety is
exposed to the aqueous environment, and this carbon
lies at the bottom of aꢂ11-A deep cavity with minimal
dimensions of ꢂ4 by ꢂ9 A (Fig. 2). This structural
information indicated that a linear functional group
might be installed at this meta carbon to project a
fluorophore into the adjacent empty protein cavity to
minimize unfavorable interactions between the fluoro-
phore-terminated side chain and the protein. Moreover,
modeling indicated that the relatively flat fluorophore
derived from fluorescein isothiocyanate might be rota-
Fluorescence Polarization Assay Development
To examine the potential effectiveness of compound 2 as
a fluorescent probe, the FP properties of this compound
were initially evaluated as a function of glycerol con-
centration. This highly viscous solvent slows the molec-
ular tumbling rate, and these solutions provided a
method to examine the dynamic range of FP values.42
As shown in Figure 3, the FP of 2 was highly sensitive
to glycerol, indicating that a reduction in tumbling rate
resulting from protein binding might be readily detected
by FP measurements. This suggested that 2 would
comprise a good candidate for FP studies in biochem-
ical systems.
To optimize the conditions required for use of 2 as a
fluorescentPPAR g probe, fluorescence intensity and
fluorescence polarization (mP) values were quantified as a
function of the concentration of 2. As shown in Figure 4
(Panel A), fluorescence intensity increased linearly with
respect to compound concentration whereas FP values
were insensitive to concentrations of 2 ator below 400
nM. Furthermore, a probe concentration of 100 nM
appeared to be optimal for further experiments. To
examine binding of PPARg to this probe, the murine
PPARg LBD was added to buffer containing 2 at100
nM. The affinity (Kd) of 2 for the PPARg LBD was
determined to be 61ꢀ14 nM by non-linear leastsquares
curve fitting of the data shown in Figure 4 (Panel B).
Competition with 50 mM of the non-fluorescent iodo-sub-
stituted precursor (11) substantially reduced FP values,
Figure 2. X-ray crystal structure of the PPARg GI262570 complex
(PDB# 1FM9). The solvent-exposed carbon atom meta to the oxazole
of bound GI262570 is shown in the ribbon model (A) and electrostatic
surface model (B).

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4327
confirming the specificity of the small molecule–protein
interaction. Based on these results, the optimal PPARg
concentration for subsequent competition experiments
was determined to be ꢂ400 nM (ꢂ15 ng/mL).
competition with 2. Competition studies with a series of
known PPARg ligands such as conjugated linoleic acids
(CLA) analyzed in high-throughput format on a Pack-
ard Fusion microtiter plate reader provided the dis-
placementcurves shown in Figure 5 (Panel B). The
structures of these ligands are shown in Figure 6.
To validate the high-affinity probe 2 in a competition
assay format, the Ki of the iodo precursor 11 for the
PPARg LBD was measured. Displacementof the bound
probe 2 by specific ligands such as 11 should diminish
observed mP values. As shown in Figure 5 (Panel A),
titration of 11 provided Ki=50ꢀ10 nM, which con-
firmed that specific PPARg ligands can be identified by
The Ki values determined from competition binding
experiments were compared with previously reported
scintillation proximity (SP) competition assays.43 As
shown in Table 1, an excellentcorrelation was generally
observed between the Ki values determined by FP and
analogous SP measurements. However, analysis of these
compounds by FP assay was found to better predict the
biological effects of compounds under investigation.
For example, the 9Z11Z–CLA isomer is known to
exhibitsignificantacitviyt in a PPAR g-mediated model
of adipogenesis.44 In contrast, the 9E11E–CLA isomer
Figure 3. Fluorescence polarization calibration curve of 2 (100 nM) in
glycerol/phosphate-buffered saline (pH 7.4). The high viscosity of gly-
cerol decreases the tumbling rate of the fluorescent small molecule to
enhance the measured FP value.
Scheme 1. Reagents: (a) Br2/CHCl3, 0–25 ꢃC, 17 h; (b) SOCl2, reflux, 3
h; (c) liquid NH3, ꢁ78 to ꢁ55 ꢃC; (d) 125 ꢃC, 4 h; (e) LiBH4, THF,
50 ꢃC, 1.5 h; (f) DEAD, PPh3, THF, 24 h; (g) LiOH, THF/MeOH
(3:1), 3 h. (h) Pd(PPh3)2Cl2, CuI, TEA, 60 ꢃC, 0.5 h; (i) LiOH, THF/
MeOH, (3:1) 3 h; (j) TFA, CH2Cl2, 0.5 h; (k) FITC, TEA, EtOH/THF
(3:2) 3.5 h.
Figure 4. Optimization of conditions for fluorescence polarization
assays. Panel A: Fluorescence intensity and polarization values as a
function of the concentration of 2 in PBS (pH 7.4). Panel B: Fluores-
cence polarization values from addition of mPPARg LBD to 2 (100
nM) in PBS. In competition experiments, the non-fluorescent com-
pound 11 was added ata final concentration of 50 mM.

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Table 1. Comparison of fluorescence polarization (FP) and pre-
viously reported43 scintillation proximity (SP) assays with PPARg in a
competitive binding format
PPARg ligand
FP assay Ki (mM)
SP assay Ki (mM)
PGJ2
0.63
9.22
9.14
3.63
731
4.54
7.03
14.6
62.8
0.34
N/A
6.45
N/A
4.70
7.10
N/A
5.50
N/A
CLA mixture
9Z11E-CLA
9Z11Z-CLA
9E11E-CLA
10E12Z-CLA
11Z13E-CLA
Furan-CLA
CEA
N/A, data not available.
demonstrate that this probe is sensitive to subtle differ-
ences in molecular interactions in solution that allow
detailed analysis of equilibrium binding events.
Discussion
We report the synthesis of a fluorescent PPARg ligand
(2) suitable for high-throughput fluorescence polariza-
tion (FP) assays. This assay format was investigated
because FP has recently emerged as one of the best
methods for quantifying molecular interactions in solu-
tion.42 The FP method enables rapid, non-radioactive
determination of the affinities of small molecules for
proteins in homogeneous solution-phase assays using
either direct or competitive binding experiments. This
technique exploits the observation that fluorescent
molecules excited with plane-polarized light emit pho-
tons in the same plane provided that the molecule
remains stationary throughout the excited state (ꢂ4
nanoseconds for the fluorescein fluorophore).45 Small
fluorescent molecules tumble rapidly in solution, which
causes photons to be emitted in a different plane from
that of the initial excitation. In contrast, large molecules
such as proteins move little during the exited state
interval, and emitted photons remain highly polarized
with respect to the excitation plane. Hence, small fluor-
escentmolecules are depolarized when free in soluiton
due to rapid tumbling but become highly polarized
upon binding to a slowly tumbling protein receptor.
This change in FP allows highly accurate determination
of the affinities for protein receptors of both fluorescent
small molecules and non-fluorescentsmall molecules
that compete for binding with a fluorescent probe to a
macromolecular target. The homogeneous format of FP
assays facilitates high-throughput screening of small
molecule ligands against cognate protein receptors.
Figure 5. High-throughput competition binding experiments employ-
ing fluorescentprobe 2 (100 nM). Panel A: Determination of Ki for
compound 11. Panel B: Competition assays with known PPARg
ligands. PGJ2, prostaglandin J2. CLA, conjugated linoleic acid. The
CLA mixture includes 9Z11E–CLA (35%), 10E12Z–CLA (35%), and
9Z11Z–CLA + 9E11E–CLA (5%).
Compound 2 was designed based on an X-ray crystal
structure of the PPARg ligand binding domain bound
to the small molecule agonist GI262570. A solvent
exposed atom of this bound compound at the bottom of
an open and narrow protein channel was modified with
a linear alkyne linker that was coupled to the fluorescein
fluorophore. This modification was designed to mini-
mize loss of affinity upon binding of the fluorescent
probe to the protein. Comparison of the Ki value of the
related iodo-substituted precursor 11 (50ꢀ10 nM) with
Figure 6. Structures of prostaglandin J2 (15) and conjugated linoleic
acid (16–22) ligands of PPARg.
is known to exhibit much lower activity in this model.
Although the SP assay does not indicate significant dif-
ferences between these structurally similar isomers, the
FP assay with 2 indicated a 200-fold difference in Ki
(Table 1). These results illustrate the advantages of 2 as
a probe for high-throughput screening by FP, and

M. J. DeGrazia et al. / Bioorg. Med. Chem. 11 (2003) 4325–4332
4329
the Kd of fluorescentprobe 2 (61ꢀ14 nM) revealed that
modification of this compound with the linear side
chain and appended fluorophore resulted in only a
slight reduction in affinity for the receptor. The Ki
values of known PPARg ligands were measured in high-
throughput competition FP assays and compared with
previously reported scintillation proximity (SP) assays.
This comparison revealed that the Ki values measured
by FP more closely paralleled the biological activity of
PPARg ligands in whole-cell assays. The application of
fluorescentprobe 2 in high-throughput assay formats
may significantly facilitate the discovery of endogenous
and exogenous PPARg ligands. Moreover, the rational
ligand design approach described herein holds sig-
nificant potential for the generation of other fluorescent
probes of nuclear hormone receptors.
cannulated over 45 min into freshly distilled, liquefied
ammonia (500 mL) at ꢁ78 ꢃC. This mixture was allowed
to stir for 45 min and then warmed to ꢁ40 ꢃC for 15 h.
Excess ammonia was removed by evaporation at 22 ꢃC
to yield an off-white solid that was suspended in dH2O
(300 mL), filtered, and washed with dH2O (4ꢄ50 mL).
Evaporation in vacuo gave 6 as a white solid (19.2 g
97%). Mp 185–186 ꢃC; 1H NMR (DMSO-d6,
400.13 MHz) d 8.22 (s, 1H), 8.06 (s, 1H), 7.87 (d, 2H,
J=5.8 Hz), 7.47 (s, 1H), 7.26 (t, 1H, J=5.9); 13C NMR
(DMSO-d6, 75.57) d 166.4, 139.7, 136.3, 135.9, 130.4,
126.8, 94.6; IR (KBr): nmax 3337.6, 3158.0, 1665.6,
1561.1, 658.2 cmꢁ1; low-resolution MS (ESI+) m/z 248
(MH+). Anal. calcd for C7H6INO: C, 34.03; H, 2.45; N,
5.67. Found: C, 34.13; H, 2.42; N, 5.55.
Ethyl[2-(3-iodophenyl)-5-methyl-1,3-oxazol-4-yl]acetate
(7). A solution of Br2 (5.82 g, 36.4 mmol) in CHCl3 (10
mL) was added dropwise over 2 h to a stirring solution
of ethyl propionylacetate (3) (5.25 g, 36.4 mmol) in
CHCl3 (30 mL) at0 ꢃC. The reaction mixture was
allowed to stir for an additional 30 min and warmed to
22 ꢃC for 17 h. The reaction vessel was exposed to the
atmosphere, and air was bubbled in for 1 h. The
remaining liquid was dried (Na2SO4) and concentrated
in vacuo to afford 4 as a crude yellow liquid, which was
used immediately without further purification. Com-
pound 6 (5.0 g, 20.2 mmol) was combined with 4 (8.13
g, 36.4 mmol) and the slurry was heated to 125 ꢃC. After
4 h, the resulting dark-red solution was allowed to cool
Materials and Methods
General
All reactions, except those in aqueous media, were per-
formed under an argon atmosphere using standard
techniques for the exclusion of moisture. All commer-
cial reagents for synthesis were purchased from Aldrich
(Milwaukee, WI) or Acros (Morris Plains, NJ) and used
without further purification. The protein ligands Pros-
taglandin J2 (BioMol, Plymouth Meeting, PA), CLA
mixture (Pharmanutrients, Lake Bluff, IL), and con-
jugated linoleic acids (Sigma) were obtained from com-
mercial sources. Tetrahydrofuran and diethyl ether were
distilled from sodium benzophenone ketyl under nitro-
gen. Dichloromethane, triethylamine, and hexanes were
distilled from calcium hydride under nitrogen. Thin-
layer chromatography (TLC) was performed on silica
gel 60 F254 plates (EM Science, Kansas City, MO, 0.25
mm). ICN (Costa Mesa, CA) SiliTech Silica Gel (32–33
ꢃ
t o 22 C and immediately purified by column chroma-
tography (150 g SiO2) eluting with Et2O/hexanes (1:9)
followed by Et2O/hexanes (3:10) to afford 7 as a tan-
nish-white solid (4.2 g, 56%). Mp 68–69 ꢃC; H NMR
1
(CDCl3, 400.13 MHz) d 8.34 (t, 1H, J=1.5 Hz), 7.92 (d,
1H, J=6.7 Hz), 7.71 (d, 1H, J=6.8 Hz), 7.14 (t, 1H,
J=7.9 Hz), 4.18 (q, 2H, J=7.2 Hz), 3.55 (s, 2H), 2.36
(s, 3H), 1.27 (t, 3H, J=7.1 Hz); 13C NMR (CDCl3,
100.61 MHz) d 170.4, 157.0, 146.4, 138.8, 134.9, 130.4,
130.0, 129.5, 125.2, 94.4, 61.3, 32.3, 14.4, 10.5; IR
(KBr): nmax 2983.3, 1730.0, 1648.1, 1543.3, 1413.0,
1372.2, 797.7, 722.9 cmꢁ1; low-resolution MS (ESI+)
m/z 372 (MH+). Anal. calcd for C14H14INO3: C,
45.30; H, 3.80; N, 3.77. Found: C, 45.24; H, 3.79; N,
3.65.
1
mm) was employed for column chromatography. All H
and 13C NMR Spectra were recorded using Bruker
DPX-300, AMX-360, DRX-400, and AMX-2-500 MHz
spectrometers at ambient temperature (22 ꢃC). NMR
signals were referenced to internal CHCl3 (dH 7.27) and
CDCl3 (dC 77.23) or DMSO-d5 (dH 2.50) and DMSO-d6
(dC 39.5) peaks in parts per million (ppm). Mass spectral
data was obtained using a Quattro II (Micromass Inc.,
St. Paul, MN) and Mariner (Perseptive Biosystems,
Foster City, CA) spectrometers from either The Uni-
versity of Texas at Austin Mass Spectrometry Facility
(FAB) or The Pennsylvania State University Mass
Spectrometry Facility (ESI and CI). Infrared spectra
were obtained with a Perkin–Elmer 1600 Series FTIR.
Elemental analyses were performed by Midwest Micro-
lab, LLC (Indianapolis, IN, USA). Melting points are
uncorrected.
2-[2-(3-Iodophenyl)-5-methyl-1,3-oxazol-4-yl]ethanol (8).
A solution of LiBH4 in THF (2 M, 6.7 mL, 12.0 mmol)
was slowly added via a syringe to a stirrꢃing solution of 7
(2.21 g, 6.0 mmol) in THF (20 mL) at0 C. The reaction
mixture was allowed to warm to 22 ꢃC and then heated
ꢃ
t o 50 C for 1.5 h. The solution was cooled, poured into
ice–water (100 mL), acidified to pH ꢂ2 with aqueous
HCl (1 M), and extracted with EtOAc (3ꢄ25 mL). The
organic extract was dried (Na2SO4) and concentrated to
give a yellow oil that was purified by column chroma-
tography (50 g SiO2) with EtOAc/hexanes (7:3) to
afford 8 (1.72 g, 87%) as a white solid. Mp 102–103 ꢃC;
1H NMR (CDCl3, 400.13 MHz) d 8.31 (t, 1H, J=1.6
Hz), 7.92 (d, 1H, J=6.4 Hz), 7.72 (d, 1H, J=6.8 Hz),
7.15 (t, 1H, J=7.9 Hz), 3.92 (t, 1H, J=5.8 Hz), 3.17 (bs,
1H), 2.72 (t, 2H, J=5.73), 2.32 (s, 3H); 13C NMR
(CDCl3, 100.61 MHz) d 157.0, 144.0, 138.9, 134.8,
3-Iodobenzamide (6). 3-Iodobenzoic acid (20 g, 80.6
mmol) was refluxed in excess SOCl2 (150 mL) for 3 h.
The reaction was cooled to room temperature and the
remaining SOCl2 was evaporated in vacuo to afford the
acid chloride as a yellow liquid that was used immedi-
ately without further purification. This acid chloride
(21.5 g, 80.6 mmol) was dissolved in THF (300 mL) and

4330
M. J. DeGrazia et al. / Bioorg. Med. Chem. 11 (2003) 4325–4332
134.4, 130.5, 129.4, 125.2, 94.5, 61.9, 28.4, 10.3; IR
(KBr): nmax 3329.9, 1961.7, 1641.9, 1543.5, 1467.1,
1130.5, 1056.0, 886.1, 732.4 cmꢁ1; low-resolution MS
(ESI+) m/z 330 (MH+). Anal. calcd for C12H12INO2:
C, 43.79; H, 3.67; N, 4.26. Found: C, 43.91; H, 3.57; N
4.15.
1179.1, 701.0 cmꢁ1; low-resolution MS (APCIꢁ) m/z
671 (MHꢁ). Anal. calcd for C34H29INO5: C, 60.72; H,
4.35; N, 4.17. Found: C, 60.73; H, 4.42; N, 4.09.
Methyl (2S)-2-[(2-benzoylphenyl)amino]-3-(4-{2-[2-(3-{3-
[(tert - butoxycarbonyl)amino] - 1- propynyl}phenyl) - 5 -
methyl-1,3-oxazol-4-yl]ethoxy}phenyl)propanoate (13).
Compounds 10 (405 mg, 0.59 mmol) and 12 (114 mg,
0.74 mmol) were added to Pd(PPh3)2Cl2 (20.7 mg, 5
mol%) in triethylamine (30 mL) and this solution was
extensively degassed under Ar. CuI (11.2 mg, 10 mol%)
was added with degassing under Ar, and this mixture
was heated to 60 ꢃC for 0.5 h. The reaction was cooled
Methyl (2S)-2-[(2-benzoylphenyl)amino]-3-(4-{2-[2-(3-io-
dophenyl-5-methyl-1,3-oxazol-4-yl]ethoxy}phenyl) pro-
panoate (10). A solution of diethyl azodicarboxylate
(1.2 g, 7.0 mmol) in THF (70 mL) was added over 0.5 h
to a solution of 9 (1.74 g, 4.6 mmol), 8 (1.68 g, 5.1
mmol), and PPh3 (1.8 g, 7.0 mmol) in THF (170 mL) at
22 ꢃC. The resulting yellow solution was stirred for 24 h
and concentrated in vacuo to give a viscous yellow oil
that was purified by column chromatography (75 g
SiO2) eluting with EtOAc/hexanes (3:17) to afford 10 as
a yellowish foam (1.91 g, 61%). Mp 104–105 ꢃC; 1H
NMR (CDCl3, 400.13 MHz) d 8.90 (d, 1H, J=8.1 Hz),
8.34 (t, 1H, J=1.6 Hz), 7.94 (d, 1H, J=8.7 Hz), 7.73 (d,
1H, J=8.8 Hz), 7.62–7.59 (m, 2H), 7.54–7.43 (m, 4H),
7.36–7.32 (m, 1H), 7.19–7.14 (m, 3H), 6.83 (d, 2H,
J=9.5 Hz), 6.64 (d, 1H, J=9.3 Hz), 6.58 (t, 1H, J=8.3
Hz), 4.09 (q, 1H, J=7.9 Hz), 4.20 (t, 2H, J=7.3 Hz),
3.70 (s, 3H), 3.24–3.09 (m, 2H), 2.95 (t, 2H, J=7.3 Hz),
2.36 (s, 3H); 13C NMR (CDCl3, 100.61 MHz) d 199.4,
173.2, 157.0, 157.9, 150.4, 145.9, 140.5, 138.8, 135.7,
135.1, 134.8, 133.3, 131.1, 130.5, 130.4 (ꢄ2), 129.8,
129.3 (ꢄ2), 128.6, 128.2 (ꢄ2), 125.2, 118.4, 115.1, 114.8
(ꢄ2), 111.9, 94.5, 66.7, 58.2, 52.4, 38.3, 26.5, 10.5; IR
(KBr): nmax 3289.7, 2951.2, 2872.1, 1737.8, 1627.7,
1569.8, 936.8, 750.4 cmꢁ1; low-resolution MS (APCI+)
m/z 687 (MH+). Anal. calcd for C35H31IN2O5: C, 61.23;
H, 4.55; N, 4.08. Found: C, 61.11; H, 4.59; N, 4.00.
ꢃ
t o 22 C, diluted with dH2O and extracted with EtOAc
(3ꢄ20 mL). This organic extract was dried (Na2SO4)
and concentrated in vacuo to afford a faint red oil that
was purified by column chromatography (40 g SiO2)
eluting with EtOAc/hexanes (3:7) to afford the product
1
as a yellow oil (332 mg, 79%). H NMR (DMSO-d6,
400.13 MHz) d 8.65 (d, 1H, J=7.9 Hz), 7.87 (d, 2H,
J=7.9 Hz), 7.59–7.34 (m, 10H), 7.09 (d, 2H, J=8.4 Hz),
6.81 (d, 3H, J=8.4 Hz), 6.62 (t, 1H, J=7.4 Hz), 4.64 (q,
1H, J=6.4 Hz), 4.14 (t, 2H, J=6.4 Hz), 4.01 (d, 2H,
J=5.1 Hz), 3.64 (s, 3H), 3.15–3.03 (m, 2H), 2.88 (t, 2H,
J=6.3 Hz), 2.31 (s, 3H), 1.40 (s, 9H); 13C NMR
(DMSO-d6, 100.61 MHz) d 198.3, 172.3, 157.4, 157.2,
155.3, 149.5, 145.5, 140.0, 135.1, 134.7, 132.9, 132.4,
131.1, 130.3 (ꢄ2), 129.6, 128.7 (ꢄ2), 128.2 (ꢄ2), 128.1,
128.0, 127.5, 125.2, 123.2, 117.4, 114.9, 114.3 (x2),
112.4, 88.6, 80.6, 78.3, 66.0, 56.3, 52.0, 36.7, 30.1, 28.2
(ꢄ3), 25.5, 9.8; IR (film): nmax 3307.1, 2975.9, 1710.9,
1624.8, 1249.3, 735.0, 702.4 cmꢁ1; high-resolution MS
(APCI+), Mass calcd for C43H43N3O7: 713.310. Found
(MH+) 714.315.
(2S)-2-[(2-Benzoylphenyl)amino]-3-(4-{2-[2-(3-iodophe-
nyl)-5-methyl-1,3-oxazol-4-yl]ethoxy}phenyl) propanoic
acid (11). Aqueous LiOH (2 M, 0.33 mL) was added to
a solution of 10 (150 mg, 0.22 mmol) in THF/MeOH
(3:1, 8 mL) and the reaction stirred at 22 ꢃC for 3 h. This
reaction mixture was diluted with dH2O (25 mL), acid-
ified to pHꢂ2 with aqueous HCl (1 M), and extracted
with EtOAc (3ꢄ15 mL). The organic extracts were dried
(Na2SO4) and concentrated to give a yellow oil that was
purified by column chromatography (30 g SiO2) eluting
with MeOH/CH2Cl2 (1:19) to afford 11 as a crude yel-
low oil. Trituration of this oil with Et2O/hexanes (1:1,
4ꢄ25 mL) yielded a yellowish-orange foam that was
washed with Et2O/hexanes (1:1, 3ꢄ15 mL) to afford 11
as a brightyellow solid (123 mg, 83%). Mp 144–145 ꢃC;
1H NMR (CDCl3, 400.13 MHz) d 8.86 (bs, 1H), 8.32 (s,
1H), 7.91 (d, 1H, J=7.9 Hz), 7.72 (d, 1H, J=7.9 Hz)
7.59 (d, 2H, J=7.1 Hz), 7.53–7.42 (m, 4H), 7.35 (t, 1H,
J=7.7 Hz), 7.21 (d, 2H, J=8.5 Hz), 7.13 (t, 1H, J=7.9
Hz), 6.80 (d, 2H, J=8.4 Hz), 6.68 (d, 1H, J=8.5 Hz),
6.61 (t, 1H, J=7.5 Hz), 4.38 (bs, 1H), 4.14 (t, 2H,
J=6.4 Hz), 3.29–3.13 (m, 2H), 2.93 (t, 2H, J=6.4 Hz),
2.33 (s, 3H); 13C NMR (CDCl3, 100.61 MHz) d 199.5,
176.3, 158.03, 158.01, 150.2, 146.0, 140.3, 138.9, 135.7,
135.2, 134.9, 133.2, 131.2, 130.6 (ꢄ2), 130.5, 129.5,
129.4 (ꢄ2), 128.4, 128.3 (ꢄ2), 125.3, 118.7, 115.6, 114.9
(ꢄ2), 112.3, 94.5, 66.7, 58.1, 37.0, 26.3, 10.5; IR (KBr):
nmax 3323.0, 2935.3, 1733.9, 1625.0, 1572.8, 1251.7,
(2S)-2-[(2-Benzoylphenyl)amino]-3-(4-{2-[2-(3-{3-[tert-bu-
toxycarbonyl)amino]-1-propynyl}phenyl)-5-methyl-1,3-
oxazol-4-yl]ethoxy}phenyl)propanoic acid (14). Aqueous
LiOH (2 M, 0.55 mL) was added to a solution of 13 (262
mg, 0.37 mmol) in THF/MeOH (3:1, 16 mL) and the
reaction stirred at 25 ꢃC for 3 h. This mixture was dilu-
ted with dH2O (20 mL), quickly acidified with ice cold
aqueous HCl (1 M), and immediately extracted with
EtOAc (3ꢄ15 mL). This organic extract was dried
(Na2SO4), the solvent removed in vacuo, and the residue
purified by column chromatography (30 g SiO2) eluting
with MeOH/hexanes (1:49) to give 14 as a crude yellow
oil, which was triturated with a mixture of Et2O/hex-
anes (1:1, 4ꢄ15 mL) to afford a yellowish-white solid.
This solid was collected and washed with Et2O/hexanes
(1:1, 3ꢄ10 mL) to provide 14 as a brightyellow solid
1
(205 mg, 79%). H NMR (DMSO-d6, 400.13 MHz) d
8.68 (d, 1H, J=7.6 Hz), 7.87–7.85 (m, 2H), 7.58–7.47
(m, 7H), 7.40 (t, 2H, J=7.3 Hz), 7.34 (d, 1H, J=7.6
Hz), 7.11 (d, 2H, J=8.4 Hz), 6.84–6.79 (m, 3H), 6.58 (t,
1H, J=7.5 Hz), 4.52 (q, 1H, J=6.3 Hz), 4.12 (t, 2H,
J=6.6 Hz), 4.02 (d, 2H, J=5.4 Hz), 3.18–3.01 (m, 2H),
2.87 (t, 2H, J=6.4 Hz), 2.30 (s, 3H), 1.40 (s, 9H); 13C
NMR (DMSO-d6, 100.61 MHz) d 198.2, 173.3, 157.4,
157.1, 155.3, 149.7, 145.5 (ꢄ2), 139.8, 135.0, 134.8,
132.9, 132.4, 131.0, 130.4, 129.6, 128.7 (ꢄ2), 128.6,
128.2 (ꢄ2), 128.1, 127.5, 125.2, 123.1, 117.3, 114.5,
114.2 (ꢄ2), 112.4, 88.6, 80.7, 78.3, 65.0, 56.3, 36.5, 30.2,

M. J. DeGrazia et al. / Bioorg. Med. Chem. 11 (2003) 4325–4332
4331
28.2 (ꢄ3), 25.5, 9.8; IR (film): nmax 3415.4, 2257.0,
1653.6, 1248.5, 1166.4, 1025.6, 827.1, 765.3 cmꢁ1; high-
resolution MS (APCI+), Mass calcd for C42H41N3O7:
699.294. Found (MH+) 700.302.
slurry was then subjected to three cycles of sonication at
4 ꢃC (30 s, micro-tip installed, duty cycle=50%, out-
put=5) with a 30-s rest period between cycles. This
extract was frozen in liquid N2 for 2 min followed by
warming in a 37 ꢃC water bath until thawed. This lysate
was applied to a column of Ni-agarose (1 mL bed
volume per liter of bacterial culture) that had been pre-
equilibrated with HEPES loading buffer until the OD260
was reduced to 0.05. The loaded column was washed
with buffer (20 mM HEPES, pH=7.9) containing imi-
dazole (25 mM) and glycerol (10%) until the OD260 was
reduced to 0.01. The PPARg–LBD was eluted in buffer
(20 mM HEPES, pH=7.9) containing imidazole (200
mM) and glycerol (10%). The protein concentration
was determined by BCA assay (Pierce, bovine serum
albumin standard) and protein purity was verified by
SDS-PAGE (purity visualized by coomassie staining
>90%).
(2S)-2-[(2-Benzoylphenyl)amino]-3-{4-[2-(2-{3-[3-({[30,60-
dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,90-xanthen-5-
yl)amino]carbonothioyl}amino) - 1- propynyl]phenyl} - 5 -
methyl-1,3-oxazol-4-yl)ethoxy]phenyl} propanoic acid
(2). Compound 14 (47 mg, 0.07 mmol) was treated with
trifluoroacetic acid/‘wet’ CH2Cl2 (3:47, 10 mL) at25 ꢃC
for 1 h. The solution was diluted with NaOH/dH2O
(1:9, 10 mL) and extracted with EtOAc (3ꢄ10 mL). The
organic extract was dried (Na2SO4) and concentrated in
vacuo to give the free amine as a crude oil. This amine
(40.3 mg, 0.07 mmol) was added to 5-fluorescein iso-
thiocyanate, isomer I (26 mg, 0.07 mmol) in EtOH/THF
(3:2, 5 mL), cooled to 0 ꢃC, and triethylamine (10 mL,
0.07 mmol) was added. The resulting orange reaction
mixture was warmed to 22 ꢃC and stirred in the dark for
4 h. The reaction was concentrated in vacuo in the dark
to give an orange solid that was purified by column
chromatography (20 g SiO2) eluting with AcOH/
MeOH/CH2Cl2 (2:3:95) to provide 2 as a yellowish-
orange solid (37 mg, 55%). 1H NMR (DMSO-d6,
500.13 MHz) d 8.65 (d, 1H, J=7.0 Hz), 8.47 (s, 1H),
7.90 (s, 1H), 7.76 (t, 2H, J=8.3 Hz), 7.56–7.45 (m, 6H),
7.41–7.36 (m, 2H), 7.32–7.31 (m, 1H), 7.16 (d, 1H,
J=8.4 Hz), 7.10 (d, 2H, J=8.3 Hz), 6.83–6.80 (m, 4H),
6.67–6.66 (m, 2H), 6.60–6.53 (m, 5H), 5,17 (s, 2H), 4.47
(d, 1H, J=5.7 Hz), 4.13 (t, 2H, J=6.6 Hz), 3.34 (bs,
4H), 3.16–2.99 (m, 2H), 2.89 (t, 2H, J=6.4 Hz), 2.33 (s,
3H); 13C NMR (DMSO-d6, 125.76 MHz) d 198.1, 173.3,
172.0, 168.8, 159.5 (ꢄ2), 158.2, 157.1, 152.5, 151.9 (ꢄ2),
149.8, 145.5, 145.3, 142.6, 139.8, 139.7, 137.1, 135.0,
134.7, 132.8, 131.0, 130.4 (ꢄ2), 129.5, 129.2, 129.1,
128.8, 128.6 (ꢄ2), 128.2 (ꢄ2), 127.6, 127.2, 125.3, 124.4,
123.6, 123.4, 117.2, 116.8, 114.4, 114.2 (ꢄ2), 112.6,
112.4, 112.0, 109.8 (ꢄ2), 102.2 (ꢄ2), 83.1, 69.7, 66.1,
56.4, 36.5, 25.6, 21.1, 9.9; IR (KBr): nmax 2923.2, 1611.3,
1452.8, 1249.5, 1179.3, 832.0, 752.8 cmꢁ1; high-resolu-
tion MS (FAB+), Mass calcd for C58H44N4O10S:
988.278. Found (MH+) 989.284.
Fluorescence polarization assays
Assays employed compound 2 (conc=100 nM for Ki
and Kd measurements) in binding buffer (10 mM
HEPES, pH=7.9, 159 mM NaCl, 2 mM MgCl2, 5 mM
DTT). The data shown in Figure 4 and the competition
data with 11 in Figure 5 (Panel A) was obtained with a
Panvera Beacon 2000 instrument. The competition data
with PGJ2 (15), CLA compounds (16–22), and the CLA
mixture (Figure 5, Panel B) was obtained on 96-well
plates with a Packard Fusion Universal Microplate
Analyzer. Values of Kd and Ki were calculated by non-
linear least squares curve fitting using binding models
from GraphPad Prism 3.0 (San Diego, CA, USA).
Acknowledgements
We thank Ms. Kathleen Veety for assistance with che-
mical synthesis. We thank Mr. Scott Martin for assis-
tance with fluorescence polarization measurements. We
thank the NIH (R01-CA83831 to B.R.P. and R01-
DK49009 to J.P.V.H.) and the Penn State College of
Agricultural Sciences Seed Grant Program for financial
support.
Production of bacterially expressed PPARꢀ
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firmed by automated dideoxynucleotide sequencing and
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