Synthesis, biological evaluation, and molecular modeling investigation of new chiral fibrates with PPARα and PPARγ agonist activity

Article abstract of DOI:10.1021/jm0502844

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular diseases, obesity, and diabetes. Medications targeted to PPARs have been esta

Full text of DOI:10.1021/jm0502844

J. Med. Chem. 2005, 48, 5509-5519
5509
Synthesis, Biological Evaluation, and Molecular Modeling Investigation of New
Chiral Fibrates with PPARr and PPARγ Agonist Activity
Alessandra Pinelli,† Cristina Godio,† Antonio Laghezza,‡ Nico Mitro,† Giuseppe Fracchiolla,‡
Vincenzo Tortorella,‡ Antonio Lavecchia,§ Ettore Novellino,§ Jean-Charles Fruchart,| Bart Staels,|
Maurizio Crestani,† and Fulvio Loiodice*,‡
Dipartimento di Scienze Farmacologiche, Universita` degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italia,
Dipartimento Farmaco-Chimico, Universita` degli Studi di Bari, via Orabona 4, 70126 Bari, Italia, Dipartimento di Chimica
Farmaceutica, Universita` degli Studi di Napoli, via Montesano 49, 80131 Napoli, Italia, and U545 Inserm, Departement
d'Atherosclerose, Institut Pasteur de Lille and Faculte´ de Pharmacie, Universite´ de Lille 2, 1 rue du Prof Calmette,
Lille Cedex 59019, France
Received March 30, 2005
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors
that govern lipid and glucose homeostasis playing a central role in cardiovascular diseases,
obesity, and diabetes. Medications targeted to PPARs have been established to treat hyper-
lipidemia (fibrates) and insulin resistance (glitazones). Thus, there is significant interest in
developing new and specific ligands for these receptors. Here, we present the results of the
screening of new ligands of PPARR and PPARγ. Optical isomers of new chiral fibrates were
synthesized and tested in cell-based assays. Compound (S)-7 showed a dual PPARR/γ activity,
and its stereochemistry was crucial in receptor activation. Protease protection experiments
suggested that this compound binds directly to PPAR. Moreover, computational studies showed
that it properly docks to PPARR and γ ligand binding pockets. Interestingly, (S)-7 exhibited
only a modest capacity to induce the differentiation of murine fibroblasts 3T3-L1 into adipocytes
compared to rosiglitazone, a well-known PPARγ agonist.
Introduction
another nuclear receptor, the 9-cis-retinoic acid receptor
(RXR),13,14 forming a complex that interacts with specific
DNA response elements within promoter regions of
target genes. When activated by agonist ligand binding,
this heterodimer complex recruits transcription coacti-
vators and regulates the transcription of genes involved
in the control of lipid and carbohydrate metabolism.
Fibrates are a class of drugs that reduce serum
triglycerides and increase HDL cholesterol through
activation of PPARR which is expressed predominantly
in the liver.15 This receptor activation has also been
shown to produce antiinflammatory effects in vascular
cells with possible beneficial effects in the prevention
of atherosclerosis.16 Thiazolidine-2,4-diones (TZDs or
glitazones), on the other hand, are antidiabetic agents
that improve the blood glucose level in type 2 diabetes
by an insulin-sensitizing mechanism related to a selec-
tive activation of PPARγ subtype.17 Compounds with
dual PPARR/PPARγ activity, thus, appear well-suited
for the treatment of diabetic patients with the additional
risk factor of dyslipidemia. So far, therefore, a relatively
high number of dual PPARR and PPARγ agonists have
been described.18-29
Recently,30 we reported the effects on PPARR of some
chiral analogues 2 of clofibric acid 1 (Figure 1), the
active metabolite of the hypolipidemic drug clofibrate.
The potency of these compounds in activating the
receptor was increased by the presence of a methylene
group between the aromatic ring and the phenolic
oxygen and by the enlargement of the aromatic moiety
to a biphenyl group. On the other hand, a more
extensive aromatic portion is present, also, in the series
of new aryloxyacetic acids31,32 3 with dual PPARR/
Type 2 diabetes, previously referred to as non-insulin-
dependent diabetes mellitus (NIDDM), accounts for over
90% of the diabetic cases reported in the western world.
This metabolic disease, which is characterized by pro-
gressive insulin secretory dysfunction and insulin re-
sistance at major target tissues such as skeletal muscle,
liver, and adipose tissue,1-8 is also linked to a wide
spectrum of other pathophysiologic conditions including
dyslipidemia (hypertriglyceridemia, decreased serum
HDL cholesterol, increased small dense LDL particles),
hypertension, hyperuricemia, increased plasminogen
activator inhibitor-1 (PAI-1), abnormal fibrinolytic sys-
tem, and abdominal obesity.9,10 Several drugs are cur-
rently available for the treatment of type 2 diabetes
including various insulin formulations, sulfonylureas,
biguanides, and R-glucosidase inhibitors. Among the
many approaches being evaluated for the discovery of
new agents,10,11 one of the most promising is certainly
represented by the exploitation of peroxisome prolifera-
tor-activated receptor (PPAR) ligands. The PPARs were
cloned a decade ago as orphan members of the super-
family of nuclear transcription factors that includes the
receptors for steroid, retinoid, and thyroid hormones.12
There are three PPAR subtypes which are the products
of distinct genes and are commonly designated PPARR,
PPARγ, and PPARâ/δ. The PPARs heterodimerize with
* To whom correspondence should be addressed. Phone: +39 080-
5442798. Fax: +39 080-5442231. E-mail: floiodice@farmchim.uniba.it.
† Universita` degli Studi di Milano.
‡ Universita` degli Studi di Bari.
§ Universita` degli Studi di Napoli.
| Universite´ de Lille 2.
10.1021/jm0502844 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/04/2005

5510 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Pinelli et al.
Figure 1. Clofibric acid and chiral analogues.
Figure 2. Dual PPARR/PPARγ agonists.
Figure 3. New chiral clofibric acid analogues.
Scheme 1a
PPARγ activity as well as in netoglitazone 4, a thiazo-
lidinedione PPARR/PPARγ agonist established as an
antidiabetic drug in animal models of Type 2 diabetes
(Figure 2).33,34 Moreover, these analogues 2 displayed
a high degree of stereoselectivity when either a phenyl
or an n-propyl group was located in R to the carboxylic
function; in fact, the (S)-isomers were much more active
than the (R)-isomers. Afterward, another example of
stereoselectivity was reported in a patent for the chiral
clofibric acid analogue halofenate, whose levo-isomer
was shown to significantly reduce plasma glucose dif-
ferently from the dextro-isomer.35 For this enantiomer,
described only very recently as a selective PPAR modu-
lator, a Phase 2 trial was started with the name of
metaglidasen.36
The combination of these structural elements led us
to the formulation of the new chiral clofibric acid
analogues 5 and 6 (Figure 3) in which the aromatic
moiety is represented by a naphthyl and a 4-(4-chlo-
rophenyl)benzyl group, respectively, and the substituent
on the stereogenic center by an n-propyl alkyl chain.
Additionally, compound 7 (Figure 3) was investigated
as representative of the structural simplification of
â-aryl-R-oxysubstituted propanoic acids for which recent
reports have suggested very potent in vitro and in vivo
PPARR and PPARγ activities.20,21,28,29,37 The develop-
ment of molecules with reduced molecular weight, in
fact, does not necessarily provide weak PPAR ago-
nists32,38,39 and, on the other hand, improves pharma-
cokinetic and pharmaceutic properties.39
Compounds 5-7 were synthesized in optically active
form and both (R)- and (S)-isomers were evaluated for
their PPARR/PPARγ activity by the transactivation
assay, a powerful and widely used assay whose good
correlation with in vivo activity is generally accepted.
Only the enantiomer (S)-7 showed an interesting dual
agonist activity and this prompted us to investigate
other analogues of this series (8-15, Figure 3) in which
the influence of different substituents on the phenolic
ring was examined. According to the results obtained
with compound 7, all of these analogues were synthe-
sized only as the (S)-isomers.
a (a) CH2N2, Et2O; (b) 2-naphthol, PPh3, DEAD, THF, rt; (c) NaH
95%, 4-BrC6H4CH2Br, DMF, rt; (d) Pd(PPh3)4, K2CO3, 4-ClC6H4-
B(OH)2, toluene, 80 °C; (e) THF, 1 N NaOH (1:1).
of compound (S)-5 is depicted in Scheme 1 and involved
the esterification of (R)-2-hydroxypentanoic acid with
diazomethane and the condensation with 2-naphthol
under Mitsunobu conditions followed by saponification
to the desired acid. On the other hand, the reaction of
(R)-2-hydroxypentanoic acid with 4-bromobenzyl bro-
mide and subsequent esterification with diazomethane
led to the intermediate bromobenzyl ether 17 whose
Suzuki cross-coupling reaction with 4-chlorophenylbo-
ronic acid, followed by saponification with NaOH in
THF/H2O, provided compound (R)-6 (Scheme 1).
The synthesis of the isomers (R)-5 and (S)-6 was
carried out in identical fashion starting from (S)-2-
hydroxypentanoic acid. Analogues (S)-7, 8-12, and
15 were prepared via a Mitsunobu reaction of the
suitable 4-substituted phenol or 2-naphthol with (R)-
methyl (or ethyl) phenyllactate, followed by saponifica-
tion of the so obtained products 19b-h to the desired
acids (Scheme 2). In the same way the isomer (R)-7 was
obtained starting from (S)-methyl phenyllactate. For
compounds 13 and 14, the Mitsunobu reaction of 4-bro-
mophenol or 4-hydroxybenzaldehyde with (R)-methyl (or
ethyl) phenyllactate yielded 19i and 19a, respectively;
the former was reduced with NaBH4, the latter was
condensed with 2-thiopheneboronic acid under Suzuki
conditions. The subsequent saponification of the inter-
Chemistry. Both enantiomers of compounds 5 and
6 were obtained starting from the optical isomers of
2-hydroxypentanoic acid readily prepared from com-
mercially available (L)- and (D)-norvaline.40 Synthesis

Chiral Fibrates with PPARR and -γ Agonist Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5511
Scheme 2a
introduced in place of the chlorine in 7 affected both
PPARR and PPARγ agonist activity almost with the
same rank order (Table 1). The introduction of the
strong electron-withdrawing trifluoromethyl group pro-
vided compound 8 with increased PPARR and γ potency,
but slightly lower efficacy toward both receptor isotypes.
However, the introduction of the acetyl group with
similar electronic characteristics, but poor lipophilicity,
afforded compound 10 with less agonist activity on both
PPARR and PPARγ. Similar results were obtained with
9 in which the presence of the electron-donating meth-
oxy group reduced the PPARγ activity, particularly.
A new significant improvement in both potency and
efficacy was obtained with the more lipophilic aromatic
rings as substituents. These compounds (11, 14, 15)
were full PPARR agonists, at least as potent as Wy-14,-
643, and displayed also a remarkable activity on PPARγ
(Table 1). In particular, compound 11 showed the most
interesting dual activity profile being also a full PPARγ
agonist only approximately 10 times less potent than
rosiglitazone. Similar results were obtained, also, with
the introduction of a short alkyl group on the phenolic
ring (compound 12), although with a reduced potency
on PPARγ. On the contrary, the substitution of the ethyl
chain of 12 with a polar and hydrogen bond forming
hydroxymethyl group (compound 13) dropped the PPARR
activity and produced a very weak activation of PPARγ.
To ascertain the presence of the known possible species-
specific selectivity of PPARR ligands,29 the more inter-
esting compounds of the series ((S)-7, 8, 11, 12, 14),
together with clofibric acid, were also tested in the
murine PPARR (mPPARR) transactivation assay. All the
compounds, except clofibric acid, exhibited an increased
potency particularly notable in the thiophene derivative
(14) which was 6 times more potent on mPPARR than
on hPPARR, although less efficacious (Table 1).
The same compounds were also tested with the
PPARâ/δ subtype but in this case no activation was
observed (data not shown), suggesting that these mol-
ecules are PPARR/γ selective ligands.
At this point, we selected compound (S)-7 and tested
its ability to promote differentiation of murine fibroblast
3T3-L1 cells to adipocytes, a process in which PPARγ
receptors play a major role.42,43 At the concentration of
25 µM, (S)-7 was capable of inducing lipid accumulation
although with less efficacy than rosiglitazone as judged
by the different morphology of cells exposed to rosigli-
tazone and (S)-7, respectively (Figure 4). As expected,
treatment of 3T3-L1 cells with (R)-7 and Wy-14,643 did
not result in any morphological modification (Figure 4).
a (a) PPh3, DIAD, toluene, rt; (b) Pd(PPh3)4, K2CO3, 2-thiophene-
boronic acid, toluene, 80 °C; (c) THF, 1 N NaOH (1:1); (d) NaBH4,
THF/H2O (1:1), 0 °C.
mediate esters so obtained led to the target compounds
(Scheme 2).
All of the final optically active acids 5-15 had
enantiomeric excesses >98% as determined by HPLC
analysis on chiral stationary phase (see Experimental
Section).
Results and Discussion
Compounds 5-15, as well as clofibric acid, were
evaluated for their agonist activity on the human
PPARR (hPPARR) and PPARγ (hPPARγ) subtypes. For
this purpose, GAL4-PPAR chimeric receptors were
expressed in transiently transfected HepG2 cells ac-
cording to a previously reported procedure.41 The results
obtained (Table 1) were compared with corresponding
data for Wy-14,643 and rosiglitazone used as reference
compounds in the PPARR and PPARγ transactivation
assays, respectively. Maximum obtained fold induction
with the reference agonist was defined as 100%.
The enantiomers of compounds 5-7 were examined
first (Table 1). (R)-6 and (S)-6 displayed a weak activity
on PPARR and a negligible activation of PPARγ, but the
substitution of the chloro-biphenylmethylene system in
6 with the much more planar naphthalene ring gave 5
whose enantiomers behaved differently. (S)-5, in fact,
was a more potent and selective PPARR agonist than
clofibric acid and almost as efficacious as Wy-14,643,
although less potent, whereas (R)-5 was virtually inef-
fective on both receptor subtypes. However, the removal
of the phenylmethylene moiety in 6 as well as the
replacement of the n-propyl group on the stereogenic
center with benzyl afforded 7 which exhibited the most
interesting profile. Its (S)-enantiomer displayed a re-
markable dual PPARR/PPARγ activity; its efficacy on
PPARR was higher than that of clofibric acid and
comparable to that of Wy-14,643 with the potency about
half of the reference compound. In addition, (S)-7 was
much more active than clofibric acid in activating
PPARγ, although not as potent and effective as rosigli-
tazone. The stereochemistry still played a crucial role
being the (R)-enantiomer virtually ineffective on both
PPAR isoforms.
To further investigate the characteristics of (S)-7, we
performed the protease protection assay, a practical
technique used to assess the capacity of a ligand to bind
to a receptor and consequently to alter its conformation.
This conformational change is reflected by the increased
resistance of the receptor LBD to partial digestion by
proteases.44,45
As shown in Figure 5, a typical protease protection
pattern was obtained when hPPARγ was incubated with
(S)-7. In fact, incubation of the receptor with increasing
concentrations of protease (trypsin or proteinase K) in
the absence of ligand leads to the complete digestion of
the receptor. In contrast, when PPARγ was preincu-
bated with (S)-7 or with the thiazolidinedione ligand
According to the results obtained with compound 7,
its analogues 8-15, in which the influence of different
substituents on the phenolic ring was examined, were
prepared only as the (S)-isomers. The substituents

5512 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Pinelli et al.
Table 1. Activity of the Tested Compounds in Cell-Based Transactivation Assaya
hPPARR
hPPARγ
mPPARR
compound
EC50 (µM)
efficacy
EC50 (µM)
efficacy
EC50 (µM)
efficacy
R-5
3.9 ( 0.87
8.4 ( 0.5
10.5 ( 1.62
95.6 ( 8.75
36.6 ( 5.09
29.8 ( 3.12
14.3 ( 1.85
99.4 ( 12.4
80.7 ( 11.3
80.7 ( 9.77
68.3 ( 7.17
124.2 ( 10.9
113.8 ( 1.45
38.2 ( 7.51
93.2 ( 8.5
130.4 ( 13
89.7 ( 12.2
100 ( 9.71
i.a.
12.5 ( 2.49
7.2 ( 1.5
4.5 ( 1.04
7.8 ( 1.3
10.6 ( 2.77
11.5 ( 1.85
8 ( 1.68
-
-
S-5
-
-
-
-
-
R-6
4.0 ( 0.12
3.9 ( 0.69
10.9 ( 1.85
3.9 ( 0.01
0.5 ( 0.12
2.5 ( 0.08
13.5 ( 2.19
0.5 ( 0.04
0.7 ( 0.06
28.7 ( 2.91
0.9 ( 0.14
1.4 ( 0.05
39.6 ( 4.1
1.6 ( 0.3
5.4 ( 1.04
8.9 ( 1.85
10.5 ( 2.43
2.7 ( 0.18
0.9 ( 0.16
20.1 ( 1.76
12.4 ( 0.54
0.5 ( 0.08
3.6 ( 0.52
25.7 ( 3.64
1.6 ( 0.29
1.5 ( 0.09
309 ( 87.3
i.a.
-
S-6
-
R-7
-
S-7
58.8 ( 6.01
47.1 ( 7.11
47.1 ( 4.28
18.8 ( 3.12
91.8 ( 9.02
116 ( 14.6
9.7 ( 1.34
61.2 ( 11.96
42.4 ( 8.6
31 ( 1.73
i.a.
1.3 ( 0.23
0.27 ( 0.05
49.1 ( 7.8
8
9
106.9 ( 12.3
-
-
-
-
10
11
0.2 ( 0.07
0.35 ( 0.03
99.3 ( 10.23
127.4 ( 3.29
-
12
13
-
14
0.15 ( 0.03
70.7 ( 10.1
-
67.2 ( 13.53
100 ( 5.61
-
15
-
clofibric acid
Wy-14,643
rosiglitazone
42 ( 6.4
0.04 ( 0.01
-
i.a.
0.039 ( 0.003
100 ( 9.06
a i.a. ) inactive at the tested concentrations. Values are mean ( SEM. Efficacy values were calculated as percentage of the reference
compounds (Wy-14,643 for PPARR; rosiglitazone for PPARγ).
Figure 4. Effect of rosiglitazone and (S)-7 on differentiation of 3T3-L1 cells. After reaching confluence, cells were stimulated
with 1 µM dexamethasone, 1 µM IBMX, and 2 µM insulin for 2 days. Then the DEX/MIX medium was removed and cells were
treated for 10 days with the following ligands: a. vehicle; b. 0.1 µM rosiglitazone; c. 25 µM (S)-7; d. 25 µM (R)-7; e. 25 µM Wy-
14,643.
rosiglitazone, a major protease-resistant fragment of
27kDa was observed. This presumably is the result of
a conformational change leading to the activation of the
receptor. These results confirm that (S)-7 binds directly
to the PPARγ LBD and induces a conformational change
in PPARγ.
Docking of (S)-7 and 11 into the hPPARR revealed a
consistent set of recurring binding modes. For the two
investigated ligands, well-clustered docking results
could be obtained. As shown in Table 2, the 50 inde-
pendent docking runs carried out for each ligand gener-
ally converged to a small number of different positions
(“clusters” of results differing by less than 1.5 Å rmsd).
Generally, the top clusters (i.e. those with the most
favorable ∆Gbind) were also associated with the highest
frequency of occurrence, which suggests a good conver-
gence behavior of the search algorithm. The best results
in terms of free energy of binding were all located in a
similar position at the active site. The most important
interactions are summarized in Table 2, and a graphical
representation of the binding modes is given in Figure
6.
To better understand the activity of the most inter-
esting compounds (S)-7 and 11 at a molecular level,
docking experiments were performed into the binding
pockets of the hPPARR and γ receptors21,46 making use
of the automated docking program AutoDock.47 As a
preliminary test of the docking method, GW409544, a
potent full agonist on both PPARR and PPARγ,46 was
docked into the PPARR crystal structure. The docking
test indicated that the cluster of similar conformations
with the lowest energy docked structure reproduced
very closely the crystallographic binding mode of
GW409544 to PPARR.46 The hydrogen bond network
predicted by the AutoDock program was virtually
identical to that found in the crystal structure. This
docking test provided validation for using this program
to perform docking studies of our ligands to PPARs.
In the most frequently occurring and most favorable
result (-10.6 kcal/mol, found 28 times out of 50), the
(S)-enantiomer of 7 is found in the center of the active
site (Figure 6A), in the same orientation of the experi-
mentally determined PPARR-bound conformation of
GW409544. A binding mode very similar was also found

Chiral Fibrates with PPARR and -γ Agonist Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5513
Figure 5. (S)-7 induces an agonist-like conformational change in PPARγ. 35S-GAL4-PPARγ LBD was synthesized in vitro and
was subsequently preincubated with ethanol (control) or 25 µM (S)-7 or 1 µM rosiglitazone, then incubated with distilled water
or increasing concentration of trypsin (panel A) or proteinase K (panel B). Digestion products were analyzed by SDS-PAGE
followed by autoradiography. An asterisk indicates the 27 kDa protease-resistant fragment of hPPARγ.
Table 2. Results of 50 Independent Docking Runs for Ligands (S)-7 and 11a
ligand
Ntot
focc
∆Gbind
surrounding residues
PPARR
(S)-7
10
12
28
17
-10.6
-11.0
Phe273, Cys276, Thr279, Ser280, Tyr314, Ile317, Phe318, Leu321, Met325, Met330, Ile354, Phe351,
Met355, Tyr464
11
Ile272, Phe273, Cys276, Thr279, Ser280, Tyr314, Phe318, Leu321, Met330, Met335, Leu344, Leu347,
Phe351, Ile354, Tyr464
PPARγ
(S)-7
11
12
27
36
10
-8.5
-10.3
Phe282, Cys285, Ser289, Arg288, His323, Tyr327, Leu330, Met334, Phe363, Met364, Tyr473, His449
Ile281, Phe282, Cys285, Ser289, Arg288, His323, Tyr327, Leu330, Met334, Leu353, Leu356, Phe360,
Phe363, Met364, Tyr473, His449
a Ntot is the total number of clusters; the number of results in the top cluster is given by the frequency of occurrence, focc; ∆Gbind is the
estimated free energy of binding for the top cluster results and is given in kcal/mol. The last column shows the contacting residues for the
binding mode of the top cluster. Only residues with at least five van der Waals contacts to the ligand are shown. Residues that form
hydrogen bonds with the ligand are highlighted in bold.
for compound 11 (Figure 6B) with a ∆G of -11.0 kcal/
phenyl of both ligands. It is worth noting that the
Cys276 side chain is in close contact with both aromatic
rings of the ligand, making additional hydrophobic
interactions.
mol (found 17 times out of 50).
The ligands adopt a U-shaped conformation that
allows the carboxylate group to form hydrogen bonds
ꢀ
with Tyr314, Tyr464, and Ser280. The His440 N 2 is
The 4-chlorophenoxy ring of (S)-7 points to a large
hydrophobic cleft lined by residues Ile354, Met355,
Phe351, and Phe273. Particularly, the electron-rich
benzene ring of Phe273 appears to be optimally oriented
for a favorable π-stacking interaction with the electron-
deficient 4-chlorobenzene ring of the ligand: the planes
of the two aromatic rings are fairly parallel and sepa-
rated by a distance of 5.3 Å. As shown in Figure 6B,
this hydrophobic cleft appears sufficiently large to
accommodate the biphenyl group of 11, making exten-
sive hydrophobic contacts with the residues Ile272,
Phe273, Leu344, Leu347, Phe351, Ile354, and Met355.
hydrogen bonded to Tyr464 Oη, contributing to stabilize
the hydrogen bonding network. This hydrogen bonding
pattern is expected to be essential for the formation of
a tight binding ligand complex which stabilizes a charge
clamp48 between the C-terminal activation function 2
(AF-2) helix and a conserved lysine residue on the
surface of the receptor. The benzyl group of both (S)-7
and 11 is bound into a hydrophobic cavity formed by
Thr279, Ile317, Phe318, Leu321, Met325, and Met330
side chains. The Phe318 aromatic ring appears in a
suitable orientation for a T-shaped interaction with the

5514 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Pinelli et al.
Figure 6. (S)-7 (A) and 11 (B) enantiomers docked into the PPARR binding site. Only amino acids located within 5 Å distance
from the bound ligand are displayed and labeled. The ligand atoms are shown in red. The hydrogen bonds discussed in the text
are depicted as yellow dashed lines.
Figure 7. (S)-7 (C) and 11 (D) enantiomers docked into the PPARγ binding site. Only amino acids located within 5 Å distance
from the bound ligand are displayed and labeled. The ligand atoms are shown in yellow. The hydrogen bonds discussed in the
text are depicted as yellow dashed lines.
To understand the hPPARγ activity and investigate
possible binding modes, compounds (S)-7 and 11 were
also docked into the hPPARγ. AutoDock showed that a
very clear preference for a single position in the active
site could be obtained for (S)-7: the result with top
binding energy (-8.5 kcal/mol) was found 36 times out
of 50 (Table 2). The structure of the ligand revealed a
binding mode very similar to that previously described
for (S)-7 into PPARR (Figure 7C). The carboxylic group
of (S)-7 is involved in hydrogen bonds with Tyr473 and
Ser289. The side chains of His323 (Tyr314 in PPARR)
and His449 (His440 in PPARR), which are involved in
hydrogen bond formation in other PPARγ receptor/
ligand complexes,48-50 appear somewhat too far away
for significant hydrogen-bonding contributions so that
the activation of the receptor by the ligand could be only
partial, explaining thus the partial agonist properties
of (S)-7 at PPARγ.
pothesis of a covalent interaction with the receptor at
the level of the chlorine atom as previously described
for an irreversible PPARγ ligand,51 for which a specific
cysteine residue was identified as the attachment site.
On the other hand, differently from that compound, in
our series the replacement of the chlorine in (S)-7 with
other substituents still retains the PPARγ activity.
Figure 8 shows a superimposition of (S)-7 on rosiglita-
zone bound to the PPARγ with only the residues
involved in hydrogen bonding. The benzyl and 4-chlo-
rophenoxy rings of (S)-7 occupy the two previously
described hydrophobic pockets.
Phe363 and Phe282 are involved in hydrophobic
contacts with the 4-chlorophenoxy ring of the ligand.
Particularly, the benzene ring of Phe363 makes a
favorable π-stacking interaction with the aromatic ring
of (S)-7. The Phe282 side chain also interacts with the
aromatic ring of the ligand via a T-shaped interaction.
Such interactions would be consistent with the activity
trend of these compounds showing that lipophilic and
This different binding mode of the ligand within the
PPARγ receptor seems, therefore, to exclude the hy-

Chiral Fibrates with PPARR and -γ Agonist Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5515
rosiglitazone but similar or greater insulin-sensitizing
activity.36,38,50,52-56
In conclusion, we prepared a new series of chiral
clofibric acid analogues some of which were potent
PPARR agonists as well as PPARγ agonists. For the
partial PPARγ agonist (S)-7, the lower potency and
efficacy on PPARγ was associated with a lower adipo-
genic effect distinguishing it from conventional PPARγ
agonists and suggesting it is a PPARγ modulator. In
particular, due to the reduced efficiency in the stimula-
tion of adipocyte differentiation and the ensuing fat
accretion and weight gain, (S)-7 could represent a very
promising candidate as the lead for the design of new
dual PPARR/PPARγ drugs to be used in the therapy of
human metabolic disorders such as type 2 diabetes,
atherosclerosis, and obesity in patients with high car-
diovascular risk.
Experimental Section
Chemical Methods. Column chromatography was per-
formed on ICN silica gel 60 Å (63-200 µm) as the stationary
phase. Melting points were determined in open capillaries on
a Gallenkamp electrothermal apparatus and are uncorrected.
Mass spectra were recorded with a HP GC/MS 6890-5973 MSD
spectrometer, electron impact 70 eV, equipped with HP
1
chemstation. H NMR spectra were recorded in CDCl3 either
on a Varian EM-390 (when 90 MHz is indicated), using
tetramethylsilane as the internal standard, or on a Varian-
Mercury 300 (300 MHz) spectrometer. Chemical shifts are
expressed as parts per million (δ). Microanalyses of solid
compounds were carried out with an Eurovector Euro EA 3000
model analyzer; the analytical results are within (0.4% of
theoretical values. Optical rotations were measured with a
Perkin-Elmer 341 polarimeter at room temperature (20 °C):
concentrations are expressed as g/100 mL. The enantiomeric
excesses of acids were determined by HPLC analysis of their
methyl esters, obtained by reaction with an ethereal solution
of diazomethane, on Chiralcel OD, AD or OD-R columns (4.6
mm i.d. × 250 mm, Daicel Chemical Industries, Ltd., Tokio,
Japan). For this purpose, small amounts of the (R)-enanti-
omers of 8-15 were also prepared starting from (S)-methyl
phenyllactate. Analytical liquid chromatography was per-
formed on a PE chromatograph equipped with a Rheodyne
7725i model injector, a 785A model UV/Vis detector, a series
200 model pump and NCI 900 model interface. Chemicals were
from Aldrich and were used without any further purification.
Complete 1H NMR, MS, and elemental analysis data are given
in the Supporting Information.
(S)-Methyl 2-(2-Naphthyloxy)pentanoate (16). A solu-
tion of diethylazodicarboxylate (DEAD, 8 mmol) in anhydrous
THF (30 mL) was added dropwise to an ice-bath cooled mixture
of equimolar amounts of (R)-methyl 2-hydroxypentanoate
(prepared from (R)-2-hydroxypentanoic acid40 with a solution
of diazomethane in diethyl ether), 2-naphthol and triphen-
ylphosphine in anhydrous THF (15 mL). The reaction mixture
was stirred at room-temperature overnight, under N2 atmo-
sphere. THF was evaporated in vacuo and a mixture of Et2O
and hexane (30 mL, 1:1) was added to the residue. The
resulting precipitate was filtered off and the filtrate was
evaporated to dryness. The residue was chromatographed on
silica gel column (petroleum ether/ethyl acetate 8:2 as eluent)
affording the desired compound as a pale yellow oil in 82%
yield.
Figure 8. Superimposition of (S)-enantiomer of 7 (by-atom)
on the PPARγ-bound conformation of rosiglitazone48 (green).
Indicated are also the residues involved in hydrogen bonding
to (S)-7, with their residue types and sequence numbers
written in white, and the numbers of the helices in the active
site region written in cyan.
electron-withdrawing substituents in the para position
of the phenoxy ring increase the potency. In fact, the
electron-withdrawing trifluoromethyl group of com-
pound 8 allows to favorably realize a π-stacking charge-
transfer interaction with the electron-rich ring of Phe363.
In case of 11, AutoDock furnished two top-ranked
clusters. Besides the top result with a ∆G of -10.3 kcal/
mol (found 10 times out of 50), a second result is
obtained with a ∆G of -10.7 kcal/mol (found 7 times).
Interestingly, both results are located in the active site
and share the binding mode of (S)-7, respectively. In
contrast to the results observed for (S)-7, the carboxylic
group of 11 forms hydrogen bonds with Ser289, Tyr327,
and Tyr473. His449 is involved in indirect recognition
of the ligand, which is mediated through Tyr327. In fact,
ꢀ
the Oη of this tyrosine is hydrogen bonded with the N 2
of His449 (Figure 7D). These results support the crucial
role of the hydrogen bonding pattern involving the four
residues mentioned above for ligand binding and agonist
activity of 11 in PPARγ.
The lower potency and efficacy of (S)-7 on PPARγ
compared to rosiglitazone, however, could be a favorable
peculiarity of this molecule as well as its dual PPARR/
PPARγ activity. Differently, in fact, from conventional
PPARγ agonists or antagonists, (S)-7 could behave as
a PPARγ modulator, that is a ligand able to suitably
act as a full agonist, partial agonist or antagonist based
on the tissue involved. Compounds with this pharma-
cological profile have been previously described and, in
some cases, were found to display a partial PPARγ
agonism together with lower adipogenic effect than
The (R)-enantiomer of 16 was prepared with the same
procedure starting from (S)-methyl 2-hydroxypentanoate;
yield: 85%.
(R)-Methyl 2-(4-Bromobenzyloxy)pentanoate (17). A
solution of (R)-2-hydroxypentanoic acid40 (17 mmol) in anhy-
drous DMF (25 mL) was added dropwise, under N2 atmo-
sphere, to a stirred and ice-bath cooled suspension of NaH 95%
powder (53 mmol) in anhydrous DMF (50 mL). After the gas

5516 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Pinelli et al.
evolution had ceased, a solution of 4-bromobenzyl bromide (14
mmol) in anhydrous DMF (25 mL) was added dropwise to the
mixture which was stirred overnight at room-temperature.
Ethyl acetate (25 mL) was added to quench the reaction
mixture which was evaporated to dryness; the residue was
suspended in ethyl acetate and poured into cooled 6 N HCl.
The organic layer was separated, and the aqueous phase was
extracted with ethyl acetate. The collected organic phases were
washed with a saturated solution of NH4Cl and extracted with
a saturated solution of NaHCO3. The aqueous layer was
acidified with 6 N HCl and extracted with ethyl acetate. The
organic layer was dried over Na2SO4 and evaporated to dryness
affording the crude acid as a yellow oil. After dissolving in Et2O
(10 mL), a freshly prepared diazomethane ethereal solution
was added up to persistent yellow coloring. The solvent was
removed under vacuum and the desired ester was obtained,
as a yellow oil, after purification by column chromatography
on silica gel (petroleum ether/CHCl3 7:3 and then 4:6 as
eluents). Yield: 32%.
(S)-Methyl 2-(2-naphthyloxy)-3-phenyl-propanoate
(19h): pale yellow oil; 72% yield.
(S)-Ethyl 2-(4-formylphenoxy)-3-phenyl-propanoate
(19i): pale yellow oil; 71% yield.
(S)-Methyl 2-[4-(2-Thienyl)phenoxy]-3-phenyl-propan-
oate. This compound was prepared by the same procedure
reported for 18 starting from 19a and 2-thiopheneboronic acid.
White solid; 52% yield.
(S)-Ethyl 2-[4-(Hydroxymethyl)phenoxy]-3-phenyl-
propanoate. To an ice-bath cooled solution of 19i (0.76 g, 2.55
mmol) in (1:1) THF/H2O (20 mL), NaBH4 (0.096 g, 2.55 mmol)
was added. The reaction mixture was stirred at 0 °C for 15
min and then was poured into brine and extracted with Et2O.
The organic layer was dried over Na2SO4 and evaporated in
vacuo to give a yellow pale oil (0.88 g) which was chromato-
graphed on silica gel column (petroleum ether/ethyl acetate
8:2 as eluent) affording the desired compound as a colorless
oil in 72% yield.
General Procedure for the Preparation of the Final
Acids. A solution of the corresponding methyl or ethyl esters
(5 mmol) in THF (30 mL) and 1 N NaOH (30 mL) was stirred
at room temperature for 1-6 h. The organic layer was removed
under reduced pressure, and the residue was acidified with 6
N HCl and extracted with Et2O. The organic layer was dried
over Na2SO4 and evaporated to dryness affording the final
acids as white solids which were recrystallized from hexane
or CHCl3/hexane (34-95% yields).
(+)-(R)-2-(2-Naphthyloxy)pentanoic acid [(R)-5]: 34%
yield; mp: 139-40 °C (hexane); [R]D ) +59 (c 1.0, MeOH); e.e.
) 99% (methyl ester on Chiralcel OD-R column, CH3CN/H2O
60:40 as the mobile phase, flow rate: 0.4 mL/min, detection:
254 nm).
(-)-(S)-2-(2-Naphthyloxy)pentanoic acid [(S)-5]: 43%
yield; mp: 140-2 °C (hexane); [R]D ) -59 (c 1.0, MeOH); e.e.
) 99% (for experimental conditions, see (R)-5).
(+)-(R)-2-[4-(4-Chlorophenyl)benzyloxy]pentanoic acid
[(R)-6]: 47% yield; mp: 97-8 °C (hexane); [R]D ) +56 (c 1.0,
MeOH); e.e. ) 98% (methyl ester on Chiralcel AD column,
hexane/i-PrOH 98:2 as the mobile phase, flow rate: 0.5 mL/
min, detection: 254 nm).
(-)-(S)-2-[4-(4-Chlorophenyl)benzyloxy]pentanoic acid
[(S)-6]: 50% yield; mp: 99-100 °C (hexane); [R]D ) -58 (c
1.0, MeOH); e.e. ) 99% (for experimental conditions, see (R)-
6).
(+)-(R)-2-(4-Chlorophenoxy)-3-phenyl-propanoic acid
[(R)-7]: 42% yield; mp: 117-8 °C (CHCl3/hexane); [R]D ) +12
(c 1.0, MeOH); e.e. ) 98% (methyl ester on Chiralcel OD
column, hexane/i-PrOH 98:2 as the mobile phase, flow rate:
0.5 mL/min, detection: 230 nm).
(-)-(S)-2-(4-Chlorophenoxy)-3-phenyl-propanoic acid
[(S)-7]: 60% yield; mp: 116-7 °C (CHCl3/hexane); [R]D ) -13
(c 1.0, MeOH); e.e. ) 99% (for experimental conditions, see
(R)-7).
(-)-(S)-2-(4-Trifluoromethylphenoxy)-3-phenyl-pro-
panoic acid (8): 95% yield; mp: 117-8 °C (CHCl3/hexane);
[R]D ) -17 (c 1.0, MeOH); e.e. ) 99% (methyl ester on Chiralcel
OD column, hexane/i-PrOH 98:2 as the mobile phase, flow
rate: 0.5 mL/min, detection: 230 nm).
(-)-(S)-2-(4-Methoxyphenoxy)-3-phenyl-propanoic acid
(9): 34% yield; mp: 94-5 °C (CHCl3/hexane); [R]D ) -11 (c
1.0, MeOH); e.e. ) 98% (methyl ester on Chiralcel OD column,
hexane/i-PrOH 98:2 as the mobile phase, flow rate: 0.5 mL/
min, detection: 230 nm).
(-)-(S)-2-(4-Acetylphenoxy)-3-phenyl-propanoic acid
(10): 74% yield; mp: 90-1 °C (CHCl3/hexane); [R]D ) -15 (c
1.0, MeOH); e.e. ) 98% (methyl ester on Chiralcel AD column,
hexane/i-PrOH 90:10 as the mobile phase, flow rate: 0.5 mL/
min, detection: 280 nm).
The (S)-enantiomer of 17 was prepared with the same
procedure starting from (S)-2-hydroxypentanoic acid;40 yield:
44%.
(R)-Methyl 2-[4-(4-Chlorophenyl)benzyloxy]pentanoate
(18). A solution of 17 (2 mmol) in anhydrous toluene (3 mL)
was added dropwise, in N2 atmosphere, to a warm (80 °C) and
stirred suspension of 4-chlorophenylboronic acid (4 mmol),
anhydrous K2CO3 (3 mmol), and tetrakis(triphenylphosphine)-
palladium (0.07 mmol) in anhydrous toluene (3 mL). The
resulting mixture was stirred at 80 °C for 7 h and filtered on
Celite which was washed with ethyl acetate. The organic
solution was washed with 0.5 N NaOH, 15% citric acid, and
brine, dried over Na2SO4, and evaporated to dryness affording
a dark solid. The desired product was obtained as a yellow oil
after purification by column chromatography on silica gel
(petroleum ether/ethyl acetate 9.5:0.5 and then 8:2 as eluents)
in 55% yield.
The (S)-enantiomer of 18 was prepared with the same
procedure starting from the (S)-enantiomer of 17; yield: 70%.
General Procedure for the Preparation of Methyl or
Ethyl 2-(4-Substituted-aryloxy)-3-phenyl-propanoates
(19a-i). A solution of diisopropylazodicarboxylate (DIAD, 10
mmol) in anhydrous toluene (20 mL) was added dropwise to
an ice-bath cooled mixture of (R)-methyl or ethyl phenyllactate
(10 mmol), the appropriate phenol (5 mmol), and triphen-
ylphosphine (10 mmol) in anhydrous toluene (55 mL). The
reaction mixture was stirred at room-temperature overnight,
under N2 atmosphere. Toluene was evaporated in vacuo, and
a mixture of Et2O and hexane (40 mL, 1:1) was added to the
residue. The resulting precipitate was filtered off, and the
filtrate was evaporated to dryness. The residue was chromato-
graphed on silica gel column (petroleum ether/ethyl acetate
9:1 as eluent) affording the desired compounds as oils or white
solids in 40-92% yields.
(S)-Methyl 2-(4-bromophenoxy)-3-phenyl-propanoate
(19a): white solid; 76% yield.
(S)-Methyl 2-(4-chlorophenoxy)-3-phenyl-propanoate
(19b): pale yellow oil; 72% yield.
The (R)-enantiomer of 19b was prepared with the same
procedure starting from (S)-methyl phenyllactate; yield: 68%.
(S)-Methyl 2-(4-trifluoromethylphenoxy)-3-phenyl-
propanoate (19c): pale yellow oil; 92% yield.
(S)-Methyl 2-(4-Methoxyphenoxy)-3-phenyl-propanoate
(19d). After column chromatography, a GC analysis of the oil
obtained showed the presence of an impurity in the ratio of
about 1:1. However, 19d (45% yield, approximately, on the
base of GC analysis) was used for the next step without any
further purification.
(S)-Methyl 2-(4-acetylphenoxy)-3-phenyl-propanoate
(19e): pale yellow oil; 73% yield.
(S)-Methyl 2-(4-phenylphenoxy)-3-phenyl-propanoate
(19f): white solid; 52% yield.
(-)-(S)-2-(4-Phenylphenoxy)-3-phenyl-propanoic acid
(11): 76% yield; mp: 149-50 °C (CHCl3/hexane); [R]D ) -1 (c
1.0, MeOH); e.e. ) 99% (methyl ester on Chiralcel OD column,
hexane/i-PrOH 98:2 as the mobile phase, flow rate: 0.5 mL/
min, detection: 230 nm).
(S)-Methyl 2-(4-ethylphenoxy)-3-phenyl-propanoate
(19g): colorless oil; 40% yield.

Chiral Fibrates with PPARR and -γ Agonist Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5517
(-)-(S)-2-(4-Ethylphenoxy)-3-phenyl-propanoic acid
(12): 72% yield; mp: 84-5 °C (hexane); [R]D ) -10 (c 1.0,
MeOH); e.e. ) 97% (methyl ester on Chiralcel AD column,
hexane/i-PrOH 98:2 as the mobile phase, flow rate: 1 mL/min,
detection: 225 nm).
(-)-(S)-2-[4-(Hydroxymethyl)phenoxy]-3-phenyl-pro-
panoic acid (13): 80% yield; mp: 128-9 °C (CHCl3/hexane);
[R]D ) -12 (c 1.0, MeOH); e.e. ) 99% (methyl ester on Chiralcel
AD column, hexane/i-PrOH 95:5 as the mobile phase, flow
rate: 1 mL/min, detection: 280 nm).
turer (Promega). The transcription/translation reaction was
subsequently aliquoted and ethanol or rosiglitazone or com-
pound (S)-7 was added. These mixtures were incubated for 20
min at 25 °C. The protease was added and digestions were
allowed to proceed for 10 min at 25 °C. The reactions were
terminated by the addition of gel loading buffer and boiling
for 5 min. The products of the digestion were separated by
electrophoresis through a 12% polyacrylamide-SDS gel. After
electrophoresis, the gel was dried and the radiolabeled diges-
tion products were visualized by autoradiography.
Computational Chemistry. Molecular modeling and graph-
ics manipulations were performed using the SYBYL software
package (Sybyl Molecular Modeling System, version 6.9, Tripos
Inc., St. Louis, MO), running it on a Silicon Graphics R12000
workstation. Model building of (S)-7 and 11 was accomplished
with the TRIPOS force field61 available within SYBYL. Point
charges were calculated as Kollman-all-atom62 for proteins and
Gasteiger-Marsili63 for ligands. Energy minimizations were
realized by employing the Insight II/Discover program (Insight
II Molecular Modeling Package and Discover 2.2000 Simula-
tion Package, MSI Inc., San Diego, CA), selecting the CVFF
force field.64
Docking Simulations. Docking was performed with ver-
sion 3.05 of the program AutoDock.47 It combines a rapid
energy evaluation through precalculated grids of affinity
potentials with a variety of search algorithms to find suitable
binding positions for a ligand on a given protein. While the
protein is required to be rigid, the program allows torsional
flexibility in the ligand.
Docking to PPARR and PPARγ was carried out using the
empirical free energy function and the Lamarckian genetic
algorithm, applying a standard protocol, with an initial
population of 50 randomly placed individuals, a maximum
number of 1.5 × 106 energy evaluations, a mutation rate of
0.02, a crossover rate of 0.80, and an elitism value of 1.
Proportional selection was used, where the average of the
worst energy was calculated over a window of the previous 10
generations. For the local search, the so-called pseudo-Solis
and Wets algorithm was applied using a maximum of 300
iterations per local search. The probability of performing local
search on an individual in the population was 0.06, and the
maximum number of consecutive successes or failures before
doubling or halving the local search step size was 4. Fifty
independent docking runs were carried out for each ligand.
Results differing by less than 1.5 Å in positional root mean-
square deviation (rmsd) were clustered together and repre-
sented by the result with the most favorable free energy of
binding.
(1) Ligand Setup. Molecular models of compounds (S)-7
and 11 were constructed using standard bond lengths and
bond angles of the SYBYL fragment library. The carboxylate
group was taken as dissociated. Geometry optimizations were
realized with the SYBYL/MAXIMIN2 minimizer by applying
the BFGS (Broyden, Fletcher, Goldfarb, and Shannon) algo-
rithm and setting an rms gradient of the forces acting on each
atom of 0.05 kcal/mol Å as the convergence criterion. Atomic
charges were assigned using the Gasteiger-Marsili formal-
ism,63 which is the type of atomic charges used in calibrating
the AutoDock empirical free energy function. Finally, the
compounds were setup for docking with the help of AutoTors,
the main purpose of which is to define the torsional degrees
of freedom to be considered during the docking process. The
number of flexible torsions defined for each ligand is as
follow: five in (S)-7 and six in 11.
(+)-(S)-2-[4-(2-Thienyl)phenoxy]-3-phenyl-propanoic
acid (14): 44% yield; mp: 154-5 °C (CHCl3/hexane); [R]D
)
+8 (c 1.0, MeOH); e.e. ) 99% (methyl ester on Chiralcel OD
column, hexane/i-PrOH 90:10 as the mobile phase, flow rate:
0.5 mL/min, detection: 230 nm).
(+)-(S)-2-(2-Naphthyloxy)-3-phenyl-propanoic acid (15):
76% yield; mp: 133-4 °C (CHCl3/hexane); [R]D ) +19 (c 1.0,
MeOH); e.e. ) 99% (methyl ester on Chiralcel OD column,
hexane/i-PrOH 98:2 as the mobile phase, flow rate: 0.5 mL/
min, detection: 230 nm).
Biological Methods. Medium and other cell culture re-
agents, Wy-14,643, clofibric acid, insulin, dexamethasone and
isobutylmethylxanthin (IBMX) were purchased from Sigma
(St. Louis, MO). BRL 49653 (rosiglitazone) was obtained by
Hefei Scenery Chemical Co. (Hefei, Anhui, Popular Republic
of China).
Plasmids. The expression vectors expressing the chimeric
receptors containing the yeast GAL4-DNA binding domain
fused to either the human PPARR/PPARγ ligand binding
domain (LBD) and the reporter plasmid for these GAL4
chimeric receptors (pGAL5TKpGL3) containing five repeats
of the GAL4 response elements upstream of a minimal
thymidine kinase promoter that is adjacent to the luciferase
gene were described previously.57 The murine PPARR LBD and
the human PPARδ LBD expression plasmids in the pSG5
vector were kind gifts from Dr. Krister Bamberg (AstraZeneca,
Mo¨lndal, Sweden).
Cell Culture and Transfections. Human hepatoblastoma
cell line HepG2 (American Type Culture Collection, Manassas,
VA) was cultured in Dulbecco’s Modified Eagle Media/F-12
Nutrient (1:1) Mixture (D-MEM/F-12) containing 10% of heat
inactivated Foetal Calf Serum, 100 U penicillin G/mL and 100
µg streptomycin sulfate/mL at 37 °C in a humidified atmo-
sphere of 10% CO2. For transactivation assay 105 cells/well
were seeded in a 24 well plate in triplicate and transfections
were performed with a modification of the calcium-phosphate
method.58 Cells were transfected with expression plasmids
encoding the fusion protein GAL4-PPARR LBD or GAL4-
PPARγ LBD (30 ng), pGAL5TKpGL3 (100 ng), pCMVâgal (300
ng). After transfection, cells were treated for 20 h with the
indicated ligands. Luciferase activity in cell extracts was then
determined by a luminometer (LUMAT LB 9501 Berthold).
â-Galactosidase activity was determined using â-D-galactopy-
ranoside (Calbiochem) as described previously.59 All transfec-
tion experiments were repeated at least twice.
3T3-L1 Differentiation. Murine fibroblast 3T3-L1 cells
from American Type Culture Collection were cultured in
D-MEM with 10% heat inactivated bovine calf serum, 100 U
penicillin G/mL, and 100 µg streptomycin sulfate/mL at 37 °C
in a humidified atmosphere of 10% CO2. For differentiation
experiments, 6 × 104 cells/well were seeded in six-well plates
in duplicate. After reaching confluence, usually after 5-6 days,
cells were stimulated to differentiate as described.60 Briefly,
2 days postconfluent preadipocytes were treated with complete
medium containing 1 µM dexamethasone, 1 µM IBMX, and 2
µM insulin (DEX/MIX medium). After 2 days, the DEX/MIX
medium was removed, cells were washed three times with
medium and then treated with ligands for 10 days. Medium
was replenished with appropriate ligands every 2 days.
Limited Proteolysis Digestion Analysis. Protease diges-
tion was carried out as described previously44 with minor
modifications. The plasmid pGAL4-hPPARγ LBD was used to
synthesize 35S-radiolabeled PPARγ in a coupled transcription/
translation system according to the protocol of the manufac-
(2) Protein Setup. Crystal structures of PPARR in complex
with GW409544 (entry code: 1K7L)46 and PPARγ in complex
with rosiglitazone (entry code: 1KNU),21 recovered from
Brookhaven Protein Database, were used. The structures were
setup for docking as follows: polar hydrogens were added using
the PROTONATE utility.47 To optimize the hydrogen positions,
the structures were subjected to a short energy minimization
using the Discover module of InsightII, in accordance with the
type of force field and protein charges of the AutoDock
empirical free energy function. Solvation parameters were

5518 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Pinelli et al.
(17) Willson, T. M.; Cobb, J. E.; Cowan, D. J.; Wiethe, R. W.; Correa,
I. D.; Prakash, S. R.; Beck, K. D.; Moore, L. B.; Kliewer, S. A.;
Lehmann, J. M. The Structure-Activity Relationship between
Peroxisome Proliferator-Activated Receptor γ Agonism and the
Antihyperglycemic Activity of Thiazolidinediones. J. Med. Chem.
1996, 39, 665-668.
(18) Cronet, P.; Petersen, J. F. W.; Folmer, R.; Blomberg, N.; Sjo¨blom,
K.; Karlsson, U.; Lindstedt, E.-L.; Bamberg, K. Structure of the
PPARR and -γ ligand binding domain in complex with AZ 242:
Ligand selectivity and Agonist Activation in the PPAR family.
Structure 2001, 9, 699-706.
(19) Brooks, D. A.; Etgen, G. J.; Rito, C. J.; Shuker, A.; Dominianni,
S. J.; Warshawsky, A. M.; Ardecky, R.; Paterniti, J. R.; Tyhonas,
J.; Karanewsky, D. S.; Kauffman, R. F.; Broderick, C. L.;
Oldham, B. A.; Montrose-Rafizadeh, C.; Winneroski, L. L.; Faul,
M. M.; McCarthy, J. R. Design and Synthesis of 2-Methyl-2-
{4-[2-(5-methyl-2-aryloxazol-4-yl)ethoxy]phenoxy}propionic
Acids: A New Class of Dual PPARR/γ Agonists. J. Med. Chem.
2001, 44, 2061-2064.
(20) Lohray, B. B.; Lohray, V. B.; Bajji, A. C.; Kalchar, S.; Poondra,
R. R.; Padakanti, S.; Chakrabarti, R.; Vikramadithyan, R. K.;
Misra, P.; Juluri, S.; Mamidi, N. V. S. R.; Rajagopalan, R. (-)3-
[4-[2-(Phenoxazin-10-yl)ethoxy]phenyl]-2-ethoxypropanoic Acid
[(-)DRF2725]: A Dual PPAR Agonist with Potent Antihyper-
glycemic and Lipid Modulating Activity. J. Med. Chem. 2001,
44, 2675-2678.
(21) Sauerberg, P.; Pettersson, I.; Jeppesen, L.; Bury, P. S.; Mogensen,
J. P.; Wassermann, K.; Brand, C. L.; Sturis, J.; Wo¨ldike, H. F.;
Fleckner, J.; Andersen, A.-S. T.; Mortensen, S. B.; Svensson, L.
A.; Rasmussen, H. B.; Lehmann, S. V.; Polivka, Z.; Sindelar, K.;
Panajotova, V.; Ynddal, L.; Wulff, E. M. Novel Tricyclic-R-
alkyloxyphenylpropionic Acids: Dual PPAR R/γ Agonists with
Hypolipidemic and Antidiabetic Activity. J. Med. Chem. 2002,
45, 789-804.
(22) Etgen, G. J.; Oldham, B. A.; Johnson, W. T.; Broderick, C. L.;
Montrose, C. R.; Brozinick, J. T.; Misener, E. A.; Bean, J. S.;
Bensch, W. R.; Brooks, D. A.; Shuker, A. J.; Rito, C. A.;
McCarthy, J. R.; Ardecky, R. A.; Tyhonas, J. S.; Dana, S. L.;
Bilakovics, J. M.; Paterniti, J. R., Jr.; Ogilvie, K. M.; Liu, S.;
Kauffman, R. F. A Tailored Therapy for the Metabolic Syndrome,
The Dual Peroxisome Proliferator-Activated Receptor-R/γ Ago-
nist LY465608 Ameliorates Insulin Resistance and Diabetes
Hyperglycemia While Improving Cardiovascular Risk Factors
in Preclinical Models. Diabetes 2002, 51, 1083-1087.
(23) Ebdrup, S.; Pettersson, I.; Rasmussen, H. B.; Deussen, H.-J.;
Jensen, A. F.; Mortensen, S. B.; Fleckner, J.; Pridal, L.; Nygaard,
L.; Sauerberg, P. Synthesis and Biological and Structural
Characterization of the Dual-Acting Peroxisome Proliferator-
Activated Receptor R/γ Agonist Ragaglitazar. J. Med. Chem.
2003, 46, 1306-1317.
(24) Xu, Y.; Rito, C. J.; Etgen G. J.; Ardecky, R. J.; Bean, J. S.; Bensch,
W. R.; Bosley, J. R.; Broderick, C. L.; Brooks, D. A.; Dominianni,
S. J.; Hahn, P. J.; Liu, S.; Mais, D. E.; Montrose-Rafizadeh, C.;
Ogilvie, K. M.; Oldham, B. A.; Peters, M.; Rungta, D. K.; Shuker,
A. J.; Stephenson, G. A.; Tripp, A. E.; Wilson, S. B.; Winneroski,
L. L.; Zink, R.; Kauffman, R. F.; McCarthy, J. R. Design and
Synthesis of R-Aryloxy-R-methylhydrocinnamic Acids: A Novel
Class of Dual Peroxisome Proliferator-Activated Receptor R/γ
Agonists. J. Med. Chem. 2004, 47, 2422-2425.
(25) Koyama, H.; Miller, D. J.; Boueres, J. K.; Desai, R. C.; Jones,
B.; Berger, J. P.; MacNaul, K. L.; Kelly, L. J.; Doebber, T. W.;
Wu, M. S.; Zhou, G.; Wang, P.; Ippolito, M. C.; Chao, Y.-S.;
Agrawal, A. K.; Franklin, R.; Heck, J. V.; Wright, S. D.; Moller,
D. E.; Sahoo, S. P. (2R)-2-Ethylchromane-2-carboxylic acids:
Discovery of Novel PPARR/γ Dual Agonists as Antihyperglycemic
and Hypolipidemic Agents. J. Med. Chem. 2004, 47, 3255-3263.
(26) Takamura, M.; Sakurai, M.; Yamada, E.; Fujita, S.; Yachi, M.;
Takagi, T.; Isobe, A.; Hagisawa, Y.; Fujiwara, T.; Yanagisawa,
H. Synthesis and biological activity of novel R-substituted
â-phenylpropionic acids having pyridin-2-ylphenyl moiety as
antihyperglycemic agents. Bioorg. Med. Chem. 2004, 12, 2419-
2439.
added to the final protein file using the ADDSOL utility of
AutoDock 3.05. The grid maps representing the proteins in
the actual docking process were calculated with AutoGrid. The
grids (one for each atom type in the ligand, plus one for
electrostatic interactions) were chosen to be sufficiently large
to include not only the active site but also significant portions
of the surrounding surface. The dimensions of the grids were
thus 30 Å × 30 Å × 30 Å, with a spacing of 0.375 Å between
the grid points. Refinement of the receptor/ligand complexes
was achieved by energy minimization using the steepest
descent and conjugate gradient methods, permitting only the
ligand and the side chain atoms of the protein to relax.
Acknowledgment. This work was accomplished
thanks to the financial support of the Ministero
dell′Istruzione, dell′Universita` e della Ricerca (MIUR
2003033405), and of the University of Milano
(FIRST2003/2004 to M.C.).
Supporting Information Available: Spectroscopic data
for intermediates and final compounds and microanalysis data
for acids 5-15. This material is available free of charge via
the Internet at http:// pubs.acs.org.
References
(1) De Franzo, R. A.; Bonadonna, R. C.; Ferrannini, E. Pathogenesis
of NIDDM. Diabetes Care 1992, 15, 318-368.
(2) Eriksson, J.; Franssila-Kallunki, A.; Ekstrand, A.; Saloranta, C.;
Widen, E.; Schalin, C.; Groop, L. Early metabolic defects in
persons at increased risk for non-insulin-dependent diabetes
mellitus. N. Engl. J. Med. 1989, 321, 337-343.
(3) Lillioja, S.; Mott, D. M.; Spraul, M.; Ferraro, R.; Foley, J. E.;
Ravussin, E.; Knowler, W. C.; Bennett, P. H.; Bogardus, C.
Insulin resistance and insulin secretory dysfunction as precur-
sors of non-insulin-dependent diabetes mellitus. Prospective
studies of Pima Indians. N. Engl. J. Med. 1993, 329, 1988-1992.
(4) Pimenta, W.; Korytkowski, M.; Mitrakou, A.; Jenssen, T.; Yki-
Jarvinen, H.; Evron, W.; Dailey, G.; Gerich, J. Pancreatic beta-
cell dysfunction as the primary genetic lesion in NIDDM.
Evidence from studies in normal glucose-tolerant individuals
with a first-degree NIDDM relative. JAMA 1995, 273, 1855-
1861.
(5) Fernandez-Castaner, M.; Biarnes, J.; Camps, I.; Ripolles, J.;
Gomez, N.; Soler, J. Beta-cell dysfunction in first-degree relatives
of patients with non-insulin-dependent diabetes mellitus. Diabet.
Med. 1996, 13, 953-959.
(6) Volk, A.; Renn, W.; Overkamp, D.; Mehnert, B.; Maerker, E.;
Jacob, S.; Balletshofer, B.; Haring, H. U.; Rett, K. Insulin action
and secretion in healthy, glucose tolerant first degree relatives
of patients with type 2 diabetes mellitus. Influence of body
weight. Exp. Clin. Endocrinol. Diabetes 1999, 107, 140-147.
(7) Weyer, C.; Bogardus, C.; Mott, D. M.; Pratley, R. E. The natural
history of insulin secretory dysfunction and insulin resistance
in the pathogenesis of type 2 diabetes mellitus. J. Clin. Invest.
1999, 104, 787-794.
(8) Weyer, C.; Tataranni, P. A.; Bogardus, C.; Pratley, R. E. Insulin
resistance and insulin secretory dysfunction are independent
predictors of worsening of glucose tolerance during each stage
of type 2 diabetes development. Diabetes Care 2001, 24, 89-94.
(9) Reaven, G. M. Role of insulin resistance in human desease.
Diabetes 1988, 37, 1597-1607.
(10) Moneva, M. H.; Dagogo-Jack, S. Multiple drug targets in the
management of type 2 diabetes. Curr. Drug Targets 2002, 3,
203-221.
(11) Hulin, B. New hypoglycaemic agents. Prog. Med. Chem. 1994,
31, 1-58.
(12) Issemann, I.; Green, S. Activation of a member of the steroid
hormone receptor superfamily by peroxisome proliferators.
Nature 1990, 347, 645-650.
(27) Murakami, K.; Tobe, K.; Ide, T.; Mochizuki, T.; Ohashi, M.;
Akanuma, Y.; Yazaki, Y.; Kadowaki, T. A novel insulin sensitizer
acts as a coligand for peroxisome proliferator-activated recep-
tor-R (PPAR-R) and PPAR-γ: effect of PPAR-R activation on
abnormal lipid metabolism in liver of Zucker fatty rats. Diabetes
1998, 47, 1841-1847.
(28) Liu, K. G.; Smith, J. S.; Ayscue, A. H.; Henke, B. R.; Lambert,
M. H.; Leesnitzer, L. M.; Plunket, K. D.; Willson, T. M.;
Sternbach, D. D. Identification of a Series of Oxadiazole-
Substituted R-Isopropoxy Phenylpropanoic Acids with Activity
on PPARR, PPARγ, and PPARδ. Bioorg. Med. Chem. Lett. 2001,
11, 2385-2388.
(13) Kliewer, S. A.; Umesono, K.; Noonan, D. J.; Heyman, R. A.;
Evans, R. M. Convergence of 9-cis-retinoic acid and peroxisome
proliferator signaling pathways through heterodimer formation
of their receptors. Nature 1992, 358, 771-774.
(14) Mangelsdorf, D. J.; Evans, R. M. The RXR heterodimers and
orphan receptors. Cell 1995, 83, 841-850.
(15) Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.; Leit-
ersdorf, E.; Fruchart, J.-C. Mechanism of action of fibrates on
lipid and lipoprotein metabolism. Circulation 1998, 98, 2088-
2093.
(16) Fruchart, J.-C.; Duriez, P.; Staels, B. Peroxisome proliferator-
activated receptor-alpha activators regulate genes governing
lipoprotein metabolism, vascular inflammation and atheroscle-
rosis. Curr. Opin. Lipidol. 1999, 10, 245-257.
(29) Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B. R. The
PPARs: From Orphan Receptors to Drug Discovery. J. Med.
Chem. 2000, 43, 527-550.

Chiral Fibrates with PPARR and -γ Agonist Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5519
(30) Rangwala, S. M.; O′Brien, M. L.; Tortorella, V.; Loiodice, F.;
Longo, A.; Noonan, D. J.; Feller, D. R. Stereoselective effects of
chiral clofibric acid analogues on rat peroxisome proliferator-
activated receptor R (rPPARR) activation and peroxisomal fatty
acid â-oxidation. Chirality 1997, 9, 37-47.
(31) Liu, K.; Xu, L.; Jones, A. B. Preparation of 1,2-benzoxazolyloxy-
acetic acids and analogues as PPAR agonists for treatment of
diabetes and lipid disorders. Merck & Co. Inc., USA, PCT Int.
Appl. 2001, WO 0160807; Chem. Abstr. 2001, 135, 180757.
(32) Adams, A. D.; Hu, Z.; von Langen, D.; Dadiz, A.; Elbrecht, A.;
MacNaul, K. L.; Berger, J. P.; Zhou, G.; Doebber, T. W.; Meurer,
R.; Forrest, M. J.; Moller, D. E.; Jones, A. B. O-arylmandelic
acids as highly selective human PPAR alpha/gamma agonists.
Bioorg. Med. Chem. Lett. 2003, 13, 3185-3190.
(33) Wulff, E. M.; Jeppesen, L.; Bury, P. S.; Mogensen, J. P.; Fleckner,
J.; Andersen, A.-S. T.; Wassermann, K.; Sauerberg, P. Charac-
terization of a future generation insulin sensitizers in vitro and
in vivo. Diabetes 2001, 50 (Suppl. 2), Abst. 2210-PO.
(34) Sorbera, L. A.; Castan˜er, J.; del Fresno, M.; Silvestre, J.
Netoglitazone. Drugs Future 2002, 27, 132-139.
(35) Luskey, K. L.; Luo, J. Preparation and use of (-)-(3-trihalo-
methylphenoxy)(4-halophenyl)acetates for treatment of insulin
resistance, type 2 diabetes, hyperlipidemia and hyperuricemia.
Metabolex, Inc., USA.; Diatex, Inc., U.S. Pat. Appl. 20030220399,
2003; Chem. Abstr. 140, 4856.
(36) Karpf, D. B.; Flores-Lozano, F.; Rosenstock, J. MBX-102, a novel
insulin sensitizer for type 2 diabetes that does not cause weight
gain or edema. Presented at the 3rd International Symposium
on PPARs: Efficacy and Safety, Monte Carlo (Monaco), March
19-23, 2005. See also http://www.metabolex.com/pipeline.html.
(37) Buckle, D. R.; Cantello, B. C. C.; Cawthorne, M. A.; Coyle, P. J.;
Dean, D. K.; Faller, A.; Haigh, D.; Hindley, R. M.; Jefcott, L. J.;
Lister, C. A.; Pinto, I. L.; Rami, H. K.; Smith, D. G.; Smith, S.
A. Non thiazolidinedione antihyperglycemic agents. 1: R-Het-
eroatom substituted â-phenylpropanoic acids. Bioorg. Med.
Chem. Lett. 1996, 6, 2121-2126.
(38) Rocchi, S.; Picard, F.; Vamecq, J.; Gelman, L.; Potier, N.; Zeyer,
D.; Dubuquoy, L.; Bac, P.; Champy, M.-F.; Plunket, K. D.;
Leesnitzer, L. M.; Blanchard, S. G.; Desreumaux, P.; Moras, D.;
Renaud, J.-P.; Auwerx, J. A unique PPARγ ligand with potent
insulin-sensitizing yet weak adipogenic activity. Mol. Cell 2001,
8, 737-747.
(39) Cobb, J. E.; Blanchard, S. G.; Boswell, E. G.; Brown, K. K.;
Charifson, P. S.; Cooper, J. P.; Collins, J. L.; Dezube, M.; Henke,
B. R.; Hull-Ryde, E. A.; Lake, D. H.; Lenhard, J. M.; Oliver, W.,
Jr.; Oplinger, J.; Pentti, M.; Parks, D. J.; Plunket, K. D.; Tong,
W.-Q. N-(2-Benzoylphenyl)-L-tyrosine PPAR Agonists. 3. Struc-
ture-Activity Relationship and Optimization of the N-Aryl
Substituent. J. Med. Chem. 1998, 41, 5055-5069.
(48) Nolte, R. T.; 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. Ligand binding and co-activator assembly of
the peroxisome proliferator-activated receptor-gamma. Nature
1998, 395, 137-143.
(49) Xu, H. E.; Lambert, M. H.; Montana, V. G.; Parks, D. J.;
Blanchard, S. G.; Brown, P. J.; Sternbach, D. D.; Lehmann, J.
M.; Wisely, G. B.; Willson, T. M.; Kliewer, S. A.; Milburn, M. V.
Molecular recognition of fatty acids by peroxisome proliferator-
activated receptors. Mol. Cell 1999, 3, 397-403.
(50) Oberfield, J. L.; Collins, J. L.; Holmes, C. P.; Goreham, D. M.;
Cooper, J. P.; Cobb, J. E.; Lenhard, J. M.; Hull-Ryde, E. A.; Mohr,
C. P.; Blanchard, S. G.; Parks, D. J.; Moore, L. B.; Lehmann, J.
M.; Plunket, K.; Miller, A. B.; Milburn, M. V.; Kliewer, S. A.;
Willson, T. M. A peroxisome proliferator-activated receptor γ
ligand inhibits adipocyte differentiation. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 6102-6106.
(51) Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt,
T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E.
L-764406 is a partial agonist of human peroxisome proliferator-
activated receptor γ. The role of Cys313 in ligand binding. J. Biol.
Chem. 1999, 274, 7913-7922.
(52) Berger, J. P.; Petro, A. E.; Macnaul, K. L.; Kelly, L. J.; Zhang,
B. B.; Richards, K.; Elbrecht, A.; Johnson, B. A.; Zhou, G.;
Doebber, T. W.; Biswas, C.; Parikh, M.; Sharma, N.; Tanen, M.
R.; Thompson, G. M.; Ventre, J.; Adams, A. D.; Mosley, R.;
Surwit, R. S.; Moller, D. E. Distinct properties and advantages
of a novel peroxisome proliferator-activated protein gamma
selective modulator. Mol. Endocrinol. 2003, 17, 662-676.
(53) Misra, P.; Chakrabarti, R.; Vikramadithyan, R. K.; Bolusu, G.;
Juluri, S.; Hiriyan, J.; Gershome, C.; Rajjak, A.; Kashireddy, P.;
Yu, S.; Surapureddi, S.; Qi, C.; Zhu, Y. J.; Rao, M. S.; Reddy, J.
K.; Ramanujam, R. PAT5A: a partial agonist of peroxisome
proliferator-activated receptor gamma is a potent antidiabetic
thiazolidinedione yet weakly adipogenic. J. Pharmacol. Exp.
Ther. 2003, 306, 763-771.
(54) Reginato, M. J.; Bailey, S. T.; Krakow, S. L.; Minami, C.; Ishii,
C.; Tanaka, H.; Lazar, M. A. A potent antidiabetic thiazo-
lidinedione with unique peroxisome proliferator-activated recep-
tor γ-activating properties. J. Biol. Chem. 1998, 273, 32679-
32684.
(55) Kurosaki, E.; Nakano, R.; Shimaya, A.; Yoshida, S.; Ida, M.;
Suzuki, T.; Shibasaki, M.; Shikama, H. Differential effects of
YM440 a hypoglycemic agent on binding to a peroxisome
proliferator-activated receptor γ and its transactivation. Bio-
chem. Pharmacol. 2003, 65, 795-805.
(40) Winitz, M.; Bloch-Frankenthal, L.; Izumiya, N.; Birnbaum, S.
M.; Baker, C. G.; Greenstein, J. P. Studies on diastereoisomeric
R-amino acids and corresponding R-hydroxy acids. VII. Influence
of â-configuration on enzymic susceptibility. J. Am. Chem. Soc.
1956, 78, 2423-2430.
(56) Acton, J. J., III; Black, R. M.; Jones, B.; Moller, D. E.; Colwell,
L.; Doebber, T. W.; MacNaul, K. L.; Berger, J.; Wood, H. B.
Benzoyl 2-methyl indoles as selective PPARγ modulators. Bioorg.
Med. Chem. Lett. 2005, 15, 357-362.
(57) Raspe, E.; Madsen, L.; Lefebvre, A.-M.; Leitersdorf, I.; Gelman,
L.; Peinado-Onsurbe, J.; Dallongeville, J.; Fruchart, J.-C.; Berge,
R.; Staels, B. Modulation of rat liver apolipoprotein gene
expression and serum lipid levels by tetradecylthioacetic acid
(TTA) via PPARR activation. J. Lipid Res. 1999, 40, 2099-2110.
(58) Crestani, M.; Stroup, D.; Chiang, J. Y. L. Hormonal regulation
of the cholesterol 7R-hydroxylase gene (CYP7). J. Lipid Res.
1995, 36, 2419-2432.
(59) Hollons, T.; Yoshimura, F. K. Variation in enzymatic transient
gene expression assays. Anal. Biochem. 1989, 182, 411-418.
(60) Rubin, C. S.; Hirsch, A.; Fung, C.; Rosen, O. M. Development of
hormone receptors and hormonal responsiveness in vitro. Insulin
receptors and insulin sensitivity in the preadipocyte and adipo-
cyte forms of 3T3-L1 cells. J. Biol. Chem. 1978, 253, 7570-7578.
(61) Vinter, J. G.; Davis, A.; Saunders: M. R. Strategic approaches
to drug design. 1. An integrated software framework for molec-
ular modeling. J. Comput.-Aided Mol. Des. 1987, 1, 31-55.
(62) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio,
C.; Alagona, G.; Profeta, S., Jr.; Weiner, P. A new force field for
molecular mechanical simulation of nucleic acids and proteins.
J. Am. Chem. Soc. 1984, 106, 765-784. Details of the imple-
mentation are given in Sybyl 6.5 Theory Manual, Tripos, St.
Louis, MO, 1998, p 441.
(41) Lehmann, J. M.; Moore, L. B.; Smith-Oliver, T. A.; Wilkinson,
W. O.; Willson, T. M.; Kliewer, S. A. An antidiabetic thiazo-
lidinedione is a high affinity ligand for peroxisome proliferator-
activated receptor γ (PPARγ). J. Biol. Chem. 1995, 270, 12953-
12956.
(42) Brun, R. P.; Kim, J. B.; Hu, E.; Spiegelman, B. M. Peroxisome
proliferator-activated receptor γ and the control of adipogenesis.
Curr. Opin. Lipidol. 1997, 8, 212-218.
(43) Camp, H. S.; Chaudhry, A.; Leff, T. A novel potent antagonist
of peroxisome proliferator-activated receptor γ blocks adipocyte
differentiation but does not revert the phenotype of terminally
differentiated adipocytes. Endocrinology 2001, 142, 3207-3213.
(44) Allan, G. F.; Xiaohua, L.; Tsai, S. Y.; Weigel, N. L.; Edwards, D.
P.; Tsai, M.-J.; O′Malley, B. W. Hormone and antihormone
induce distinct conformational changes which are central to
steroid receptor activation. J. Biol. Chem. 1992, 267, 19513-
19520.
(45) Berger, J.; Bailey, P.; Biswas, C.; Cullinan, C. A.; Doebber, T.
W.; Hayes, N. S.; Saperstein, R.; Smith, R. G.; Leibowitz, M. D.
Thiazolidinediones produce a conformational change in peroxi-
somal proliferator-activated receptor-γ: binding and activation
correlate with antidiabetic actions in db/db mice. Endocrinology
1996, 137, 4189-4195.
(46) Xu, H. E.; Lambert, M. H.; Montana, V. G.; Plunket, K. D.;
Moore, L. B.; Collins, J. L.; Oplinger, J. A.; Kliewer, S. A.;
Gampe, R. T., Jr.; McKee, D. D.; Moore, J. T.; Willson, T. M.
Structural determinants of ligand binding selectivity between
the peroxisome proliferator-activated receptors. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 13919-13924.
(47) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.; Olson, A. J. Automated docking using a
Lamarckian genetic algorithm and an empirical binding free
energy function. J. Comput. Chem. 1998, 19, 1639-1662.
(63) Gasteiger, J.; Marsili, M. Iterative partial equilization of orbital
electronegativity - A rapid access to atomic charges. Tetrahe-
dron 1980, 36, 3219-3228.
(64) Hagler, A. F.; Lifson, S.; Dauber, P. Consistent force field studies
of intermolecular forces in hydrogen-bonded crystals. J. Am.
Chem. Soc. 1979, 101, 5122-5130.
JM0502844