L. D. Bratton et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3624–3629
3629
hPPARa LBD encoding amino acids 196–468 (GenBank
Accession No. L02932) were used. Volumes of 99 lL of
buffer (50 mM Tris, 10 mM Na–Molybdate, 1 mM EDTA,
and 10% Glycerol, pH 7.6) containing 50 nM of radiola-
beled ligand (3H-2-(4-(3-(4-acetyl-3-hydroxy-2-propylphen-
oxy)propoxy)-phenoxy)acetic acid (34 Ci/mmol) for
PPARd and 3H-2-(4-(2-(3-(2,4-difluorophenyl)-1-heptylure-
ido)ethyl)-phenoxy)-2-methylbutanoic acid (86 Ci/mmol)
for PPARa), 0.2 mg anti-rabbit beads (Amersham,
RPN140), 0.24 lg rabbit anti-GST (Molecular Probes
Inc., A5800), and 0.2 lg purified GST/PPARhLBD were
placed into the wells of Corning 96-well tissue culture
plates. Dimethylsulfoxide (DMSO) (1 lL) or 1 lL of
DMSO containing a test compound at a concentration
sufficient to give a final assay concentration of between
1 nM and 100 lM were added into each well. After
incubation with shaking at room temperature for 30 min,
radioactivity bound to the PPAR LBD-GST fusion protein/
anti-GST/SPA antibody-binding bead complex was
assessed using a Wallac MicroBeta plate reader. The
potency of interaction of a compound with the respective
PPAR LBD was determined as the concentration that
inhibits 50% of the interaction between the respective
PPAR LBD and the radiolabeled ligand.
In the course of our study with these compounds, we
found that there was not a clear correlation between
the PPARd binding affinity and the PPARd functional
activity. In the event, we constructed our structure–
activity relationships with data derived from the recep-
tor binding assay and assessed agonist activity using
the cell based functional assay. Ultimately, compound
28e showed greater than 1000-fold selectivity of PPARd
over PPARa with an EC50 of 59 nM and was thus se-
lected for further study in vivo.
References and notes
1. Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.;
Leitersdorf, E.; Fruchart, J. C. Circulation 1998, 98, 2088.
2. Oliver, W. R., Jr.; Shenk, J. L.; Snaith, M. R. Proc. Natl.
Acad. Sci. U.S.A 2001, 98, 5306.
3. Bordwell, F.; Boutan, J. J. Am. Chem. Soc. 1956, 78, 854.
4. Example for preparing 1,4-benzyloxy-benzylsulfanyl aryl
carboxylic acids from substituted benzyl halides: 1,4-Benzyl-
oxybenzyl alcohol (6a): To a solution of 4-dihydroxymeth-
ylphenol (1.9 g, 15 mmol) in 53 mL of acetonitrile was added
1-bromomethyl-4-trifluoro-methylbenzene (4.0 g, 17 mmol),
followed by cesium carbonate (7.4 g, 23 mmol). The reaction
mixture was stirred at 25 °C for 18 h under nitrogen
atmosphere. The reaction mixture was filtered and the filtrate
was evaporated to afford a crude solid, which was purified by
flash chromatography (silica gel, 30 % ethyl acetate in
hexane) to provide, after drying, 3.27 g (76%) of a white solid.
1,4-Benzyloxybenzyl chloride (7a): To a cold (0 °C) solution
of 6a (3.0 g, 11 mmol) in 40 mL of dichloromethane was
added 3.7 mL of triethylamine (2.7 g, 27 mmol), followed by
1.65 mL of methanesulfonyl chloride (2.4 g, 21 mmol). The
reaction mixture was stirred at 0 °C for 2 h and then at 25 °C
for 18 h. The reaction mixture was evaporated to give a crude
orange oil, which was flash chromatographed (silica gel, 10%
ethyl acetate in hexane) to afford, after drying, 2.3 g (72%) of
an oil.
The human PPARc scintillation proximity assay (SPA) was
used to measure the affinity of ligands for the human PPARc
receptor. The hPPARc LBD encoding amino acids 206–477
(GenBank Accession No. NM_138712.1) was used. Volumes
of 168 lL of buffer (1X PBS, 12 mM b-mercapto ethanol,
0.002% Tween-20, and 9% Glycerol, pH 7.6) containing
40 nM
3H-5-(4-(3-(5-methyl-2-phenyloxazol-4-yl)propa-
noyl)-benzyl)thiazolidine-2,4-dione (9.57 Ci/mmol), 0.3 mg
polylysine-coated yttrium silicate beads (Amersham,
RPNQ0010), and 10 nM purified His-tagged human PPARc
LBD were placed into the wells of a 96-well white assay plate
(Corning 3604). 2 lL of dimethylsulfoxide (DMSO) or 2 lL
of DMSO containing a test compound at a concentration
sufficient to give a final assay concentration binding curve
between 1 nM and 100 lM was added into each well. After
incubation with shaking at room temperature for 2 h,
radioactivity bound to the PPARc LBD-HIS fusion pro-
tein/yttrium bead complex was assessed using a Wallac
MicroBeta plate reader. The potency of interaction of a
compound with the PPARc LBD was determined as the
concentration that inhibits 50% of the interaction between
the PPARc LBD and the radiolabeled ligand.
1,4-Benzyloxybenzylsulfonyl indane ester (12a): To a solu-
tion of 7a (0.45 g, 1.5 mmol) in 9 mL of acetonitrile was
added 4 (0.36 g, 1.5 mmol), followed by cesium carbonate
(0.98 g, 3.0 mmol). The reaction mixture was stirred at 25 °C
for 18 h under nitrogen atmosphere. The reaction mixture
was filtered and the filtrate was evaporated to afford a crude
solid, which was purified by flash chromatography (silica gel,
20% ethyl acetate in hexane) to provide, after drying, 699 mg
(93%) of a white solid.
1,4-Benzyloxybenzylsulfonyl indane carboxylic acid (13a):
To a solution of 12a (0.60 g, 1.2 mmol) in a mixture of 10 mL
of tetrahydrofuran and 2 mL of water was added lithium
hydroxide monohydrate (0.15 g, 3.5 mmol). The reaction
mixture was stirred at 25 °C for 3 h and then evaporated to
give an oily residue. The residue was suspended in 50 mL of
water and then acidified with 1 M hydrochloric acid to pH 2.
The reaction mixture was filtered to collect a white solid,
which was rinsed with water and dried to provide 514 mg
(89%) of the title compound.
8. PPARd chimeric receptor assay (Functional Assay): Tran-
sient transfections assay using the HepG2 hepatoma cell line:
The GAL4 hPPARdLBD, chimeric receptor expression
constructs containing the ligand binding domain for the
human PPARd LBD (encoding amino acids 145–441
GenBank Accession No. NM_006238), was used to
cotransfect cells with GAL4-Luciferase reporter plasmid
(p5Eb-Luc) and b-Gal plasmid. Briefly, HepG2 cells were
seeded in a 100-mm cell culture dish containing 10 mL
DMEM plus 10% serum. Transfection mix was prepared by
combining 15 lg GAL4-Luc plasmid with 15 lg of GAL4-
hPPARdLBD. b-Gal plasmid (1.5 lg) was also added to
each as a control. LipofectAMINE 2000 reagent was used
as suggested by the manufacturer (Invitrogen, Carlsbad,
CA). For each well, 2.4 mL transfection mix was added and
incubated at 37 °C overnight. The next day, transfected
HepG2 cells were reseeded to a 96-well cell culture plate at
the density of 3000 cells per well and compounds were
subsequently added to each well. After 16 h incubation,
cells were then harvested in a lysis buffer (Promega,
Madison, Wisconsin) and luciferase activity was deter-
mined using a luminometer. Luciferase activity was then
normalized with b-Gal activity.
5. Kiong, L.; Tyman, J. J. Chem. Soc. Perkin Trans. 1981, 1,
1942.
6. Pawar, A.; Xu, J.; Jerks, E.; Mangelsdorf, D. J.; Jump, D.
B. J. Biol Chem 2002, 77, 39243.
7. PPAR receptor binding assay: The human PPARd and
PPARa scintillation proximity assay (SPA) was used to
measure the affinity of ligands for the respective human
PPAR receptor. The hPPARd LBD encoding amino acids
145–441 (GenBank Accession No. NM_006238), and