Enantio-dependent binding and transactivation of optically active phenylpropanoic acid derivatives at human peroxisome proliferator-activated receptor alpha

Article abstract of DOI:10.1016/S0960-894X(01)00732-6

Optically active phenylpropanoic acid derivatives [(S)-5, and (R)-5] were prepared, and their affinities for peroxisome proliferator-activated receptor (PPAR)α and PPARγ were evaluated. Binding assay and cell-based reporter assay indicated that the activi

Full text of DOI:10.1016/S0960-894X(01)00732-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 333–335
Enantio-Dependent Binding and Transactivation of Optically
Active Phenylpropanoic Acid Derivatives at Human Peroxisome
Proliferator-Activated Receptor Alpha
Hiroyuki Miyachi,* Masahiro Nomura, Takahiro Tanase, Masahiro Suzuki,
Koji Murakami and Katsuya Awano
Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1 Mitarai, Nogi-machi,
Shimotsuga-gun, Tochigi 329-0114, Japan
Received 11 September 2001; accepted 5 November 2001
Abstract—Optically active phenylpropanoic acid derivatives [(S)-5, and (R)-5] were prepared, and their affinities for peroxisome
proliferator-activated receptor (PPAR)a and PPARg were evaluated. Binding assay and cell-based reporter assay indicated that the
activity of these compounds is enantio-dependent, and resides exclusively on the (S)-isomer. # 2002 Elsevier Science Ltd. All rights
reserved.
Introduction
fatty acid flux.7 These results clearly indicate a pivotal
role for PPARa in lipid homeostasis in vivo.
Peroxisome proliferator-activated receptors (PPARs)
are members of the nuclear receptors, which include
steroid receptor, thyroid receptor, retinoid receptor and
others.1 These receptors are ligand-dependent tran-
scription factors. Upon ligand binding, the activated
PPARs heterodimerize with another nuclear receptor,
the retinoid X receptor (RXR),2 and alter the transcrip-
tion of the target genes after binding to specific perox-
isome proliferator response elements (PPREs), which are
a direct repeat of the hexameric AGGTCA recognition
motif separated by one nucleotide (DR1).3 Three sub-
types of PPARs, termed PPARa, PPARd (also known as
PPARb, NUCI, FAAR) and PPARg have been identi-
fied so far in various speices, including humans.4
Fibrate compounds, such as clofibrate and bezafibrate
(Chart 1), have been used for the treatment of hyper-
triglyceridemia for more than 20 years,8 and recently
fenofibrate (Chart 1) was launched in Japan. Although
the molecular target of these drugs remains to be defin-
itively established, recent molecular pharmacological
studies have demonstrated that fibrates activate PPAR’s
at high micromolar concentrations, with poor subtype-
selectivity.9
PPARa, the first isoform to be cloned, in 1990,5 was
found at high density in the liver and regulates the
expression of genes encoding proteins involved in lipid
and lipoprotein metabolism, such as acyl-CoA oxidase,
bifunctional enzyme, liver fatty acid binding protein,
apo A, apo C-III, and so on.6 In addition to the above
in vitro results, PPARa-deficient transgenic mouse
(PPARaÀ/À) exhibited massive hepatic and cardiac
lipid accumulation owing to the inhibition of cellular
*Corresponding author. Tel.: +81-280-56-2201x286; fax: +81-280-
57-1293; e-mail: hiroyuki.miyachi@mb2.kyorin-pharm.co.jp
Chart 1. Chemical structures of the fibrate drugs, rosiglitazone and 5.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00732-6

334
H. Miyachi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 333–335
We considered that more potent and selective activators
of PPARa, especially human PPARa, might have ther-
apeutic utility for the treatment of altered lipid homeo-
stasis in the target organs, especially in the liver.
enantiomeric excesses of both (+)-5 and (À)-5 were
more than 95% ee.14
The absolute configuration of the optically active 5 was
determined by the preparation of the (R)-form of 5
starting from optically active (R)-2-benzylbutanoic
acid15 (Chart 3). (R)-5 exhibited levo rotation, so the
absolute configuration of the (À)-5 was deduced to be
(R) and the antipodal (+)-form of 5 was deduced to be
(S).16
Recently, we have reported the design and the synthesis
of some novel phenylpropanoic acid derivatives as sub-
type-selective PPAR activators,10 and we selected the 2-
ethylphenylpropanoic acid derivative (5; Chart 1) for
further pharmacological study. As a part of our con-
tinuing research directed toward the development of
subtype-selective PPAR activators, we report here the
enantio-dependency of the activity of 5.
In Vitro Studies
There is little information on the differences of binding
and transactivation activity of the enantiomers of syn-
thetic PPARa activators in the case of human PPARa.
Although several optically active peroxisome pro-
liferators that can activate PPARa, such as leucotriene
D4 receptor antagonist (MK-571)11 and clofibric acid
analogues,12 are enantio-dependent, it is not clear whe-
ther these compounds are true PPARa ligands (that can
bind directly to human PPARa).
Transient transactivation assay
Analysis of the transient transactivation activity of the
above compounds on human PPARa and human
PPARg was performed using the reported method.17
The activity was expressed as EC50, which is the con-
centration of the test compound that affords half-max-
imum transactivation activity.
Binding assay
Therefore, we decided to investigate the transactivation
and binding activity of optically active 5, using PPARa
and PPARg (PPARd was not examined, since 5 shows
very weak PPARd transactivation activity). Compound
5 has an asymmetric center at the a-position of the car-
boxylic acid functionality, and it is important to deter-
mine which enantiomer is more potent.
Binding affinity to PPARa and g was determined by
competitive binding assay as previously described.17
Results and Discussions
Under the assay conditions used, no apparent decrease
in optical purity of either enantiomer was observed.18
As can be seen from Table 1, a clear enantio-depen-
dence of the transactivation and binding activity to
human PPAR isoforms was found. In the case of
human PPARa isoform, only the (S)-form exhibited
potent transactivation and binding activity and the cor-
responding (R)-form shows much weaker activities.
Chemistry
Synthetic routes to optically active 5 [(+)-5, and (À)-5]
are outlined in Chart 2. Racemic 5 was prepared as
previously described.10 Optically active 5 was obtained
by the resolution of the corresponding 4-(S)-benzyl-
oxazolidinone imide derivatives by silica-gel column
chromatography and recrystallization, and subsequent
deprotection of the 4-(S)-benzyloxazolidinone.13 The
As for human PPARg isoform, both enantiomers of 5
exhibited weak activities, and no apparent enantio-
dependence was observed. The (S)-form exhibited high
PPARa selectivity in both transactivation and binding
assays, but the degree of subtype-selectivity of the anti-
podal (R)-form was low.
Chart 3. Synthetic routes to the (R)-form of 5. Reagents and condi-
tions: (c) (1) c. H2SO4, MeOH; (2) c.HNO3; (d) (1) 10% Pd/C, EtOH;
(2) NaNO2, dil H2SO4; (3) Na2SO4, dil H2SO4; (e) MeI, K2CO3,
DMF; (f) CHCl2OMe, TiCl4, CH2Cl2; (g) CrO3–H2SO4, acetone;
(h) 4-(trifluoromethyl)benzylamine, 2-chloro-1,3-dimethylimidazolium
chloride, NEt3, CH2Cl2; (i) dil NaOH, MeOH.
Chart 2. Synthetic routes to optically active 5. Reagents and conditions:
(a) (1) Pivaloyl chloride, NEt3, THF; (2) 4-(S)-benzyloxazolidinone,
t-BuOK, THF; (b) LiOH, 30% H2O2, MeOH.

H. Miyachi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 333–335
335
Table 1. Transactivation and binding activities of the enantiomers of
5 with human PPARs
References and Notes
Transactivation (EC50 mM)a Binding (IC50 mM)a
1. Porte, D., Jr.; Schwartz, M. W. Science 1996, 272, 699.
2. Keller, H.; Dreyer, C.; Medin, J.; Mahoudi, A.; Ozato, K.;
Wahli, W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2160.
3. Kliewer, S. A.; Umesono, K.; Noonan, D. J.; Heyman,
R. A.; Evans, R. M. Nature (London) 1992, 358, 771.
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Kroetz, D. L.; Fernandez-Salguero, P. M.; Westphal, H.;
Gonzalez, F. J. Mol. Cell. Biol. 1995, 15, 3012.
Compd Config.
PPARa
PPARg
PPARa
PPARg
(À)-5
(R)
(S)
1.5
0.06
>15
14
24
0.74
>50
44
(+)-5
aTransient transactivation activity and binding activity of the above
compounds on human PPARa and human PPARg were performed
using the previously reported method.17
In this study, we investigated the enantio-dependency of
the human PPARa-selective agonist 5 and found that
the PPARa transactivation and binding activities reside
almost exclusively in the (S)-enantiomer.
8. Fruchart, J.-C.; Duriez, P.; Staels, B. Curr. Opin. Lipidol.
1999, 10, 245.
9. Forman, B. M.; Chen, J.; Evans, R. M. Proc. Natl. Acad.
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10. (a) Nomura, M.; Takahashi, Y.; Tanase, T.; Miyachi, H.;
Ide, T.; Tsunoda, M.; Murakami, K. PCT Int. Appl. WO 00/
75103 (b) Miyachi, H.; Nomura, M.; Tanase, T.; Takahashi,
Y.; Ide, T.; Tsunoda, M.; Murakami, K.; Awano, K. Bioorg.
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ella, V.; Longo, A.; Loiodice, F.; Noonan, D. J.; Feller, D. R.
Chirality 1997, 9, 37.
In the case of rosiglitazone (Chart 1), a potent and
selective PPARg agonist used for the treatment of non-
insulin dependent diabetes mellitus (type II diabetes),
enantio-dependency was also noted, and (S)-rosiglita-
zone exhibited much more potent binding to PPARg
than (R)-rosiglitazone.19 However, the enantio-depen-
dency of (S)-rosiglitazone decreased time-dependently,
probably due to rapid racemization at the C-5 position
of the thiazolidine-2,4-dione ring at physiological pH.20
13. Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem.
Soc. 1982, 104, 1737. ꢀ
A recent X-ray crystallographic analysis of the rosigli-
tazone–human PPARg complex indicated that the sul-
ꢀ
14. (À)-5: [a]D À24 (C 0.40, MeOH), (+)-5: [a]D +24
(C 0.80, MeOH). Analysis of the enantiomeric excess was
performed with a HPLC equipped with a Chirapac OD
column.
15. Meyers, A. I.; Knaus, G.; Kamata, K.; Ford, M. E. J. Am.
Chem. Soc. 1976, 98, 567.
16. The [a]D of (R)-5, prepared from (R)-2-benzylbutanoic
acid, with 92% ee was À21ꢀ (C 0.18, MeOH). The enantio-
meric excess of (R)-5, which was estimated to be about 85%
ee, indicated that a slight epimerization occurred during the
synthesis of (R)-5.
17. Murakami, K.; Tobe, K.; Ide, T.; Mochizuki, T.; Ohashi,
M.; Akanuma, Y.; Yazaki, Y.; Kadowaki, T. Diabetes 1998,
47, 1841.
fur atom of (S)-rosiglitazone is positioned in
a
hydrophobic region of the PPARg ligand-binding
pocket, surrounded by the side chains of the amino
acids F363, Q286, F282, and L469.21 Because the three-
dimensional structures of the ligand-binding domains of
members of the nuclear receptor superfamily are
thought to be well conserved,22 we speculate that the side
chain ethyl group of (S)-5 interacts hydrophobically with
the corresponding hydrophobic region of the PPARa
ligand-binding pocket, although the three-dimensional
structure of human PPARa is not known.23
18. HPLC analysis was performed on Chirapac OD
(0.0046Â0.25 m, flow rate 1.00 mL/min, UV 254 nm, n-hex-
ane/i-PrOH/TFA=95:5:0.2 v/v/v as the eluant).
19. Parks, D. J.; Tomkinson, N. C. O.; Villeneuve, M. S.;
Blanchard, S. G.; Willson, T. M. Bioorg. Med. Chem. Lett.
1998, 8, 3657.
20. Sohda, T.; Mizuno, K.; Kawamatsu, Y. Chem. Pharm.
Bull. 1984, 32, 4460.
21. Nolte, C.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert,
M. H.; Kurokawa, R.; Rosenfeld, M. G.; Willson, T. M.;
Glass, C. K.; Milburn, M. V. Nature (London) 1998, 3958,
137.
22. Uppenberg, J.; Svensson, C.; Jaki, M.; Bertilsson, G.;
Jendeberg, L.; Berkenstam, A. J. Biol. Chem. 1998, 273, 31108.
23. Recently, the X-ray crystal structure of the ligand bind-
ing domain of human PPARa was disclosed. Cronet, P.;
Petersen, J. F. W.; Folmer, R.; Blomberg, N.; Sjoblom, K.;
Karlsson, U.; Lindstedt, E. L.; Bamberg, K. Structure 2001,
9, 699.
In conclusion, 5 shows enantio-dependent transactiva-
tion and binding activities to human PPARa. The
activities were found to reside almost exclusively in the
(S)-enantiomer. Further in vivo pharmacological eva-
luation of the (S)-form of 5 and related compounds, and
a more detailed structure–activity relationship study in
combination with computer-aided molecular modeling
are in progress.
Acknowledgements
The authors wish to thank H. Saito and H. Furuta of
Kyorin Pharmaceutical Co., Ltd., for their analytical
work. Thanks are also due to T. Ide, M. Tsunoda, T.
Nakazawa and W. Hori of Kyorin Pharmaceutical Co.,
Ltd., for their technical assistance.