Synthesis, biological evaluation and molecular investigation of fluorinated peroxisome proliferator-activated receptors α/γ dual agonists

Article abstract of DOI:10.1016/j.bmc.2012.01.025

PPARs are transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new agonists for these receptors. Given that the introduc

Full text of DOI:10.1016/j.bmc.2012.01.025

Bioorganic & Medicinal Chemistry 20 (2012) 2141–2151
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular investigation of fluorinated
peroxisome proliferator-activated receptors
a/c dual agonists
Giuseppe Fracchiolla a, Antonio Laghezza a, Luca Piemontese a, Mariagiovanna Parente a,
c
c
b
Antonio Lavecchia b, , Giorgio Pochetti , Roberta Montanari , Carmen Di Giovanni ,
⇑
Giuseppe Carbonara a, Paolo Tortorella a, Ettore Novellino b, Fulvio Loiodice a,
⇑
a Dipartimento Farmaco-Chimico, Università degli Studi di Bari ‘Aldo Moro’, Via Orabona 4, 70126 Bari, Italy
b Dipartimento di Chimica Farmaceutica e Tossicologica, ‘Drug Discovery’ Laboratory, Università di Napoli ‘Federico II’, Via Domenico Montesano 49, 80131 Napoli, Italy
c Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016 Monterotondo Stazione, Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
PPARs are transcription factors that govern lipid and glucose homeostasis and play a central role in car-
diovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new agonists
for these receptors. Given that the introduction of fluorine generally has a profound effect on the physical
and/or biological properties of the target molecule, we synthesized a series of fluorinated analogs of the
Received 21 October 2011
Revised 13 January 2012
Accepted 16 January 2012
Available online 28 January 2012
previously reported compound 2, some of which turned out to be remarkable PPARa and PPARc dual ago-
nists. Docking experiments were also carried out to gain insight into the interactions of the most active
derivatives with both receptors.
Keywords:
Structure–activity relationships
Carboxylic acids
Ó 2012 Elsevier Ltd. All rights reserved.
Nuclear receptors
Crystallographic studies
Docking experiments
1. Introduction
tion of lipid and lipoprotein metabolism,8 insulin sensitivity,9
cardiac function,10 epidermal biology,11 neuroprotection,12 and
Peroxisome proliferator-activated receptors (PPARs) are
members of the nuclear receptor superfamily, and three subtypes
(PPARa, PPARc, and PPARd) have been identified to date. They con-
trol the expression of genes involved in fatty acid metabolism and
function as cellular lipid sensors that activate transcription in
response to the binding of a cognate ligand, generally fatty acids
and their eicosanoids metabolites.1,2
gastrointestinal tract function and disease.13 To date, however,
no PPARd agonist has been fully developed and the clinical poten-
tial of targeting this isotype remains to be clearly determined.
Given the importance of simultaneously controlling both glu-
cose and lipid levels, dual-acting PPARa/c agonists, are considered
a very attractive option in the treatment of dyslipidemic type 2
diabetes.14–19 One of the key challenges for the development of a
dual agonist is identifying the optimal receptor subtype selectivity
PPARa is highly expressed in the liver and skeletal muscle and is
the molecular target for the fibrates (e.g., fenofibrate and gemfibro-
ratio. In particular, PPARc full agonists, despite their proven bene-
zil), a class of drugs that lower plasma triglycerides and increase
fits, possess a number of deleterious side effects such as weight
gain, peripheral edema, increased risks of congestive heart failure
and higher rate of bone fracture.20,21 During the last decade, there-
HDL cholesterol levels in humans.3,4 PPAR
c is the most extensively
studied among the PPAR subtypes and plays important roles in the
functions of adipocytes, muscles, and macrophages with a direct
impact on type 2 diabetes, dyslipidemia, atherosclerosis, and car-
diovascular diseases.5,6 This receptor is expressed most abundantly
in adipose tissue and mediates the antidiabetic activity of the
insulin-sensitizing drugs belonging to the thiazolidinedione (TZD)
class such as rosiglitazone and pioglitazone.7 PPARd is more ubiq-
uitously distributed and recent evidence suggests that this isoform
may govern diverse physiological processes, including the regula-
fore, new drugs acting as partial agonists or modulators of PPAR
c
have been developed with the goal of retaining the beneficial ef-
fects while reducing the adverse effects. A number of these drugs
has already demonstrated desirable pharmacological profiles in
various rodent models with significantly reduced side effects rela-
tive to those generally observed with existing full agonists.22–29
We recently reported a series of (S)-
noic acids 1 (Fig. 1) some of which were potent PPAR
as well as PPAR agonists.
For the partial PPAR agonist 2, the lower potency and efficacy
on PPAR was associated with a lower adipogenic effect distin-
guishing it from conventional PPAR agonists and suggesting it is
a-aryloxy-b-phenylpropa-
a
agonists
c
c
⇑
Corresponding authors. Fax: +39 081678613 (A.L.); +39 0805442231 (F.L.).
E-mail addresses: lavecchi@unina.it (A. Lavecchia), floiodice@farmchim.uniba.it
c
(F. Loiodice).
c
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.025

2142
G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
in different positions of the aryloxy group; in the set of compounds
9–17, the chlorine atom was maintained in the para position of the
phenoxy moiety, except for 17, while introducing the same substit-
uents either on one of the aromatic rings or both of them. The
preparation of the analog 10 was carried out because, as previously
shown,33 the introduction of an additional chlorine on the benzylic
O
COOH
O
COOH
R
Cl
1
2
R = CF3, CH3O, COCH3, Ph, C2H5, CH2OH, 2-Th, Cl
moiety of 2 increased the potency on both PPARa and PPARc iso-
types. Finally, we prepared the fluoro analogs 18 and 19 in which
the benzylic methylene was removed and the phenyl was directly
linked to the stereogenic carbon by analogy with the active metab-
Figure 1. (S)-a-aryloxy-b-phenylpropanoic acids endowed with PPARa/c dual
activity.
a PPARc
modulator.30 In particular, due to the reduced efficiency in
the stimulation of adipocyte differentiation and the ensuing fat
accretion and weight gain, 2 could represent a very promising can-
didate as the lead for the design of new dual PPARa/PPARc drugs to
olite 20 of the known PPARc selective modulator metaglidasen
which is currently in Phase II clinical development as an oral urico-
suric agent for the treatment of hyperuricemia in patients with
type 2 diabetes.34
be used in the therapy of human metabolic disorders such as type 2
diabetes, atherosclerosis, and obesity in patients with high cardio-
vascular risk.
Due to the peculiarity of compound 2, we decided to better
understand its mechanism of action at the molecular level and,
for this reason, solved the crystal structure of its complex with
Considering the high degree of stereoselectivity generally dis-
played by PPARs towards S isomers, some derivatives were pre-
pared, when synthetically feasible, only in this configuration (3,
6, 7 and 9); S-10, instead, was obtained by fractional crystallization
of the diastereomeric esters achieved from racemate with (R)-
pantolactone.
PPARc-LBD. In addition, with the aim to investigate the possibility
The PPARa and PPARc activity of all derivatives was evaluated
to fine-tune the activity of this ligand, we synthesized a new series
of its analogs in which fluorine atom or trifluoromethyl group were
introduced on the aromatic rings in place of chlorine or as addi-
tional substituents. Fluoro or trifluoromethyl substituents gener-
ally have a profound effect on the physical and/or biological
properties of the target molecule. Their introduction, in fact, be-
yond improving metabolic stability by blocking metabolically
labile sites, can modulate physicochemical properties, such as lipo-
philicity or acidity, change molecular conformation, and increase
binding affinity by exploiting specific interactions of fluorine with
the target protein.31,32
by the transactivation assay, a powerful and widely used assay that
is generally accepted to correlate well with in vivo activity. For the
most potent agonists 6 and S-10 docking experiments were per-
formed on both PPAR isoforms to gain more details on the interac-
tions at a molecular level and propose a binding mode explaining
the SAR data.
2. Results and discussion
2.1. Chemistry
Herein we report, therefore, the crystal structure of the complex
PPARc-LBD/2 as well as the synthesis and biological activity of the
analogs shown in Figure 2.
In a first set of compounds (3–8), the chlorine atom was re-
moved and substituted by either fluorine or trifluoromethyl group
Synthesis of compounds 3–5, 7–9, 11 and 12 was carried out as
previously reported for analog 6,30 and is depicted in Scheme 1.
The procedure started from ethyl phenyllactate and the suitable
phenol, which were condensed, under slightly modified Mitsunobu
conditions,35 to give the corresponding ethyl ester intermediates.
The saponification of these intermediates with 1 N NaOH solution
in THF provided the desired final acids. For the S enantiomeric
acids 3, 6, 7 and 9, (R)-ethyl phenyllactate was used as starting
compound.
Racemates 10 and 13–17 were prepared by condensation of the
suitable diethyl ariloxymalonate, obtained as reported in a previ-
ous work,36 with the appropriate substituted benzyl bromide in
the presence of 95% NaH followed by alkaline hydrolysis and ther-
mal decarboxylation at 160 °C (Scheme 2). The S isomer of com-
pound 10 was obtained by fractional crystallization followed by
alkaline hydrolysis of the diastereomeric esters mixture achieved
through condensation of the racemate with R-pantolactone
(Scheme 2).
O
COOH
n
X
Y
Cpd
2
Abs. Config.
X
Y
H
n
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
S
S
-
4-Cl
4-F
3-F
2-F
3
H
4
H
5
-
H
6
S
S
-
4-CF3
3-CF3
H
7
H
8
2-CF3
H
The absolute configuration of this enantiomer was determined
on the basis of circular dichroism analysis; the S configuration
was assigned to the levorotatory isomer whose CD curve shows a
negative Cotton effect in the 235–270 nm region and a positive
Cotton effect around 225 nm. The same effects are also present in
the CD curve of the isomer 9 which was used as a reference
9
S
-
4-Cl, 2-F
4-Cl, 2-F
4-Cl, 2-F
4-Cl, 3-F
4-Cl, 3-CF3
4-Cl
H
10
S-10
11
12
13
14
15
16
17
18
19
20
4-Cl
4-Cl
H
S
-
-
H
-
2-F
3-F
4-F
3-F
3-F
H
-
4-Cl
-
4-Cl
-
4-Cl, 2-F
2-CF3
a, b
ArOH
+
HO
COOEt
O
COOH
Ar
-
-
4-Cl, 2-F
4-Cl, 2-F
3-CF3
-
4-Cl
4-Cl
(a)
2-9, 11, 12
Figure 2. Fluorinated
a-aryloxy-b-phenylpropanoic acids reported in the present
study. (a) The active metabolite (20) of metaglidasen is the levo-isomer of uncertain
Scheme 1. Reagents and conditions: (a) DVB–Ph3P, DIAD, anhydrous toluene, rt; (b)
absolute configuration.
1 N NaOH, THF, rt.

G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
2143
F
O
COOH
O
COOEt
COOEt
O
COOH
a, b, c
d, e
Br
Ar
Ar
+
from 10
X
Cl
Cl
X = 4-Cl, 2-F, 3-F, 4-F
X
10, 13-17
S-10
Scheme 2. Reagents and conditions: (a) 95% NaH powder, anhydrous DMF, 55 °C; (b) 1 N NaOH, 95% EtOH, reflux; (c) decarboxylation at 160 °C; (d) (R)-pantolactone, DCC,
DMAP, anhydrous THF; fractional cristallization from n-hexane/CHCl3; (e) 1 N NaOH, THF.
compound for its high structural similarity. The S configuration of
9, in fact, was assigned on the basis of the known stereochemical
course of its synthetic pathway.
Compounds 18 and 19 were obtained as described in Scheme 3
by condensation of sodium 4-chloro-2-fluorophenate with the cor-
responding ethyl 2-bromo-2-aryl acetate in absolute EtOH, fol-
lowed by saponification of the ester intermediates.
All the optically active acids had enantiomeric excesses >98% as
determined by HPLC analysis on chiral stationary phase (see
Section 4).
2.2. Binding of 2 to PPARc-LBD
Figure 1 of the Supplementary data shows the positioning of 2
fitted into the electron density map calculated in the hydrophobic
pocket of PPARc. The orientation of this ligand was similar to those
previously reported for other
a-aryloxy-b-phenylpropanoic
acids.35 Figure 3 summarizes the binding interactions made by
the polar head of 2 with the residues that are generally involved
in the canonical intermolecular H-bonding network in the pres-
ence of carboxylate containing ligands.
Figure 3. Hydrogen bond network of 2 in complex with PPARc.
One of the carboxylate oxygens formed a bifurcated H-bond
with the Y473 OH and the H323 N
oxygen was at H-bonding distance from H449 N
e
2 groups; the other carboxylate
2 which, in turn,
e
engaged the ligand ether oxygen in a further H-bond. Interestingly,
also the chlorine atom, as previously reported for other substitu-
ents, was able to force the side chain of Phe282 to assume a new
conformation allowing the access to the new region called ‘diphe-
nyl pocket’.35 Moreover, the Cl atom made electrostatic interac-
tions with the side-chain of Q286 and the sulphur atom of M463,
contributing in this way to stabilize the conformation of the loop
11/12 (Fig. 4).
2.3. Pharmacology
Compounds 3–19 were evaluated for their agonist activity to-
ward the human PPARa (hPPARa) and PPARc (hPPARc) subtypes
in comparison with 2 and the active metabolite 20 of metaglidasen.
For this purpose, GAL4-PPAR chimeric receptors were expressed in
transiently transfected HepG2 cells according to a previously re-
ported procedure.30 The results obtained were also compared with
corresponding data for Wy-14,643 and pioglitazone used as refer-
Figure 4. Chlorine atom interactions in the complex PPARc/2.
ence compounds in the PPAR
a
and PPAR
c
transactivation assays,
respectively (Table 1). Maximum obtained fold induction with
the reference agonist was defined as 100%.
The results obtained from compounds 3–8 showed that PPAR
activity depends on the type and the position of the substituent.
The para-fluoro derivative 3, in fact, displayed a marked reduction
of the activity on both PPAR isoforms compared to 2 and similar ef-
fects were obtained with the ortho-fluoro analogs 4 and 5 even
though these compounds were tested as racemates.
F
ONa
F
Br
COOEt
O
COOH
a, b
+
Cl
Cl
On the contrary, trifluoromethyl analogs provided different re-
sults. In this case, in fact, the most potent derivative turned out
to be the para-substituted analog 6. This compound, in fact,
showed sub-micromolar potency acting basically as a full agonist
X
X
X = H or Cl
18, 19
Scheme 3. Reagents and conditions: (a) abs EtOH, reflux; (b) 2 N NaOH, THF, rt.
on PPARa and partial agonist on PPARc. A remarkable decrease

2144
G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
Table 1
and 19 turned out to be markedly lower than 9 and 10 on both
PPAR isoforms and very similar to that of metaglidasen on PPAR
however, both analogs showed, differently from metaglidasen,19
comparable potency and better efficacy on PPAR isotype. The dual
PPAR activity of these two newly metaglidasen analogs sug-
gests that they are promising ligands for which a further evalua-
tion of the pharmacological properties in vitro and in vivo could
be beneficial.
Activity of compounds tested in the cell-based transactivation assay
c;
Compd
PPAR
M)
a
PPAR
M)
c
a
EC50
(
l
Emax (%)
EC50
(
l
Emax (%)
a/c
2
3
4
5
6
7
8
9
10
S-10
11
12
13
14
15
16
4.9 1.0
24.8 4.5
90
55
57
36
3
3
6
6
9.6 1.5
35.2 2.5
77
58
33
26
59
8
9
3
2
9
21
7
41
19
4
4
22.3 2.5
0.5 0.1
9.8 0.5
i.a.
81 11
0.9 0.2
15.9 0.5
30 20
71
4
44 11
13
84 10
2.4. Docking studies
i.a.
101
6
2.2 0.6
1.4 0.2
1.0 0.2
6.5 1.6
2.5 0.1
6.6 0.1
5.1 0.1
5.4 0.1
4.5 1.3
i.a.
5
2.5 0.1
0.9 0.3
0.5 0.2
14.1 2.9
5.8 3.9
4.9 1.5
3.5 0.3
5.2 1.2
2.4 0.5
11.6 3.3
32 19
To gain more details on the interactions of the most active ago-
nists 6 and S-10 with both PPARc and PPARa at a molecular level
and to propose a binding mode that explains the SAR data, docking
experiments were performed into the X-ray crystal structures of
76 12
76 22
69
70
40
6
9
9
51
59 11
66
65 18
6
21 11
2
59
52
57
83
20
11
24
13
i.a.
100
4
3
1
6
5
3
5
3
both PPARc complexed with 2 (PDB code: 3CDP) and PPARa com-
66
4
plexed with the
a/c dual agonist BMS-631707 (PDB code:
2REW).37 Ligand docking calculations were performed with the
aid of Glide 5.7 in standard precision (SP) mode.38
101
i.a.
1
17
18
19
20
19
8
88 21
In order to generate a validated docking protocol for the PPAR
system, it was necessary to conduct a docking pose validation first.
The crystallized conformation of 2 from the receptor/ligand com-
plex structure was utilized for conducting this validation. Ten
poses of 2 were generated using the Glide SP mode and re-ranked
by GlideScore, Emodel and Glide energy score. The top ranked solu-
tion (Fig. 5a and b) for all the three scoring functions was the same
and resulted practically identical to the corresponding X-ray pose,
as evident from a root-mean-square deviation (rmsd) value of less
than 1 Å.
10.3 0.8
i.a.
1.6 0.3
i.a.
38
6
16.4 0.8
10 1.0
i.a.
i.a.
100 10
i.a.
Wy-14,643
Pioglitazone
0.57 0.07
6
i.a.: Inactive at tested concentrations (up to 5 or 10
culated as the percentage of the maximum obtained fold induction with the ref-
erence compounds (Wy-14,643 for PPAR ; pioglitazone for PPAR ).
lM). Efficacy values were cal-
a
c
of PPAR activity was observed in the meta-CF3 analog 7, whereas
the racemate 8, resulting from the introduction of CF3 in the ortho
position of the phenoxy moiety, displayed very low potency and
Furthermore, the H-bonds predicted by Glide SP for 2 were vir-
efficacy on PPAR
c
and no activity on PPAR
a
.
tually identical to those found into the crystallographic PPAR
complex. Thus, in this study the two ligands 6 and S-10 were
docked in both PPAR and PPAR using the same docking protocol
as used in pose validation and the top ranked poses of each ligand
based on GlideScore, Emodel and Glide energy score were
extracted.
c/2
Next, we examined the activity of derivatives 9–17 which were
characterized by the presence of F and/or CF3 either on one of the
aromatic rings or both of them while maintaining, with the excep-
tion of 17, the chlorine atom on the para position of the phenoxy
a
c
moiety. Interestingly, both PPARa and PPARc activity of most com-
pounds turned out not to be particularly affected, compared to 2,
by the introduction of the substituents. In other words, the electro-
static interaction formed by the chlorine in the receptor pocket
seems to be one of the forces driving the binding mode so that
any other interaction formed from additional substituents is not
able to produce remarkable modifications. In fact, the analog 17,
which was the only compound lacking the chlorine, showed no
When the ligands were docked into PPARc, the best scoring
trend was observed for Glide energy followed by Emodel and Glide
score (Table 2).
Glide energy ranked 2, 6 and S-10 in the same order as their bio-
logical activity with the S-10 pose getting the highest score. The
Emodel score ranked S-10 in the first place but 2 was scored higher
than 6. The rank order for GlideScore was the worst wherein the
weakest ligand 2 was ranked highest and the most potent S-10
was ranked second. A break up of the Glide energy term showed
that the coulombic term (ecoul) had a better correlation with the
biological activity as compared to the van der Waals term (evdw).
Comparison of the ligand structures shows that the activity of
these molecules correlates well with the presence of carboxylate
and fluorine groups and corroborates with the docking results,
which point towards the importance of the electrostatic interac-
activity on PPARa and very low potency and efficacy on PPARc.
Moreover, the presence of an additional chlorine on the para posi-
tion of the benzylic moiety significantly increased the activity; the
analog 10, in fact, was about 3 and 10 times more potent than 2 on
PPARa and PPARc, respectively, while maintaining the same effi-
cacy. Given the relevant activity of this last compound, we decided
to prepare and test S-10 which confirmed to be the eutomer dis-
playing approximately a two-fold increase in potency on both
PPAR isoforms compared to racemate.
tions. As regards PPARa, the three scoring functions reported in Ta-
Finally, we decided to test the two analogs 18 and 19 with the
aim to evaluate the importance of the benzylic methylene of this
series of ligands on PPAR activity. These two compounds, in fact,
derived from 9 and 10, respectively, by removing the methylene
unit between the stereogenic center and the phenyl ring. The pref-
erence went to these two derivatives due to their high structural
similarity with the active metabolite 20 of the selective modulator
ble 2 showed a different trend. GlideScore ranked 6 and S-10 in the
same order as their biological activity with the 6 pose having the
highest score. On the contrary, Emodel and Glide energy scores
ranked S-10 higher than 6.
The top ranked poses generated by Glide for 6 and (S)-10 within
the PPARc crystal structure were found to occupy the same spatial
position as the crystallized compound 2. As can be seen in Figure 6,
the carboxylate group of both ligands engaged H-bonds with H323,
Y473, and H449 side chains involved in the receptor activation,
while the substituted-phenoxy and -benzyl moieties were lodged
in two hydrophobic pockets, respectively. In particular, the
p-CF3-phenoxy ring of 6 and the o-F-p-Cl-phenoxy ring of S-10
occupied the first cavity of the ligand-binding pocket lined by
hydrophobic residues such as F363 of H7, L453, I456 of H11,
of PPARc metaglidasen, which is currently in Phase II clinical
development as an oral uricosuric agent for the treatment of
hyperuricemia in patients with type 2 diabetes.34 Even though pro-
vided with low potency and efficacy, in vitro and in vivo preclinical
studies revealed that metaglidasen has antidiabetic and hypolipi-
demic activity similar to full agonists without causing some of
the typical PPARc side effects of glitazones. The activity of 18

G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
2145
Figure 5. (a) Overlay of the crystallized conformation of 2 (cyan) and the top ranked pose (magenta) based on GlideScore, Emodel and Glide energy score. (b) Plot showing the
heavy atom rmsd of the ten poses with the the crystallized conformation of 2 ranked on the basis of GlideScore (red), Emodel (magenta) and Glide energy score (green).
Table 2
Docking scores for the best ranked poses of compounds 2, 6 and S-10 into PPAR
c
and 6 and S-10 into PPARa
Compd
EC50
(lM)
GlideScore
(kcal/mol)
Emodel
(kcal/mol)
Glide energy
(kcal/mol)
evdw
ecoul
PPAR
c
2
6
S-10
9.6 1.5
0.9 0.2
0.5 0.2
À8.42
À7.30
À7.87
À47.33
À43.07
À52.22
À34.26
À34.51
À36.15
À33.66
À30.38
À31.93
À0.60
À4.13
À4.22
PPARa
6
S-10
0.5 0.1
1.0 0.2
À10.28
À9.59
À73.22
À73.50
À43.00
À46.91
À34.47
À37.73
À8.53
À9.18
M463, L465 of the loop 11/12 and F282, C285, Q286 of H3. On the
other side, the benzyl ring of 6 and the p-Cl-benzyl ring of S-10
were oriented towards the second hydrophobic cavity, interacting
with the surrounding amino-acid residues C285 of H3, L330, I326
of H5 and M364 of H7.
As depicted in Figure 6, the top ranked poses obtained by Glide
for 6 and S-10 were found to dock well also into the PPAR binding
a
site through four H-bonds and many van der Waals contacts. The
carboxylate group of the ligands formed a network of H-bond
interactions with the residues S280 on H3, Y314 on H5, H440 on
H11 and Y464 on H12. H440 was found to engage a further H-bond
with the ether oxygen of the two ligands. In addition, the p-CF3-
phenoxy ring of 6 and the o-F-p-Cl-phenoxy ring of S-10 were lo-
cated in the first binding cavity and made hydrophobic contacts
with F351, I354 of H7, V444, I447 of H11, A454 of the loop 11/12
and F273, C276, Q277 of H3. The benzyl ring of 6 and the p-Cl-ben-
zyl ring of S-10 fitted into the second hydrophobic cavity formed
by residues T279, C276 of H3, I317, F318 L321 of H4, M330 of b-
sheet, and M355 of H7.
Visual inspection of both PPARc/6 and PPARc/S-10 complexes
revealed that one p-CF3 fluorine atom of 6 formed a H-bond with
the NH2 group of Q286, while the o-F atom of S-10 established a
H-bond with the SH group of C285. The FÁ Á ÁH–N and FÁ Á ÁH–S dis-
tance is 2.2 Å for both complexes and the corresponding FÁ Á ÁH–N
and FÁ Á ÁH–S angles are 147.6° and 117.0°, respectively. All these
values fall in the range considered acceptable for FÁ Á ÁH–X (X = O,
N or S) H-bonds.39 It has been argued that fluorine rarely engages
in H-bonds in small molecule X-ray crystal structures.39 However,
in protein pockets where ligands are immobilized by a variety of
forces, they appear to be more common. FÁÁÁX distances beyond
3.0 Å can be regarded as dipole–dipole interactions that most likely
provide small stabilizing contributions (61 kcal molÀ1) for the ob-
served binding poses. Our results are consistent with the SAR data
3. Conclusions
In conclusion, we prepared and tested a new series of fluori-
nated analogs of the previously reported PPAR
a/c dual agonist
showing that 6 and S-10 are the most potent PPAR
series with EC50 values of 0.9 and 0.5 M, respectively.
From analysis of docking models we also argued that 6 and S-
10, which have a weak transactivation profile towards PPAR , pref-
c ligands of the
2. The displacement of the chlorine atom of this ligand generally
resulted in compounds with lower potency and efficacy on both
isoforms, whereas the introduction of additional fluorine or CF3
into the molecular structure of 2 did not markedly affect the activ-
ity. This allows to hypothesize that the electrostatic interactions
made from chlorine with PPAR residues, as seen in the X-ray struc-
l
c
erentially stabilize H3 through closer hydrophobic contacts or H-
bonds made with residues of this helix (Q286 and C285). This rela-
tionship is in agreement with our previous findings regarding two
enantiomeric ureidofibrate-like derivatives complexed with
ture of the complex PPARc/2, are determinant for agonist activity.
Only the substitution of chlorine with CF3 or the introduction of an
additional fluorine in the ortho position of aryloxy ring of 2 affor-
ded significantly more potent and effective agonists and docking
experiments were performed to rationalize this peculiar behavior.
The presence of the methylene unit between the stereogenic center
and the phenyl ring of 2 also turned out to be important for
PPARc, showing partial and full agonism, respectively, towards this
nuclear receptor.40 Even in that case, while the full agonism of one
enantiomer could be related to stronger interactions with H11,
H12, and the loop 11/12, the partial agonism of the other enantio-
mer could be ascribed to closer contacts with the residue Q286 of
H3.

2146
G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
Figure 6. Compounds 6 (a, yellow) and S-10 (b, orange) docked into the PPAR
c binding site are represented as a slate hot pink ribbon model. Compounds 6 (c) and S-10 (d)
docked into the PPAR binding site are represented as a green ribbon model. Only amino acids directly implicated in the ligand binding are displayed (white) and labeled. H-
a
bonds discussed in the text are depicted as dashed black lines.
activity. Its removal, in fact, decreased potency and efficacy as
shown by derivatives 18 and 19 having high structural similarity
with the active metabolite of metaglidasen. Differently from
0.5%. The storage solution with 20% glycerol (v/v) was used as cryo-
protectant. Crystals of PPAR /2 belong to the space group C2 with
cell parameters shown in Table 1 of the Supplementary data. The
c
metaglidasen, however, both these analogs showed a PPAR
a/c
asymmetric unit is formed by one homodimer.
dual activity which makes them promising ligands for which a fur-
ther evaluation of the pharmacological properties in vitro and
in vivo could be beneficial.
4.2. Structure determination
X-ray data were collected at 100 K under a nitrogen stream
using synchrotron radiation (beamline ID14-1 at ESRF, Grenoble).
4. Experimental section
The diffracted intensities were processed using the programs MOS-
41
FLM and SCALA
.
Refinements were performed with CNS42 using
4.1. Protein expression, purification and crystallization
the coordinates of apo-PPARc (PDB code 1PRG) as a starting mod-
el.43 All data between 8 and 2.8 Å were included in the refinement.
The statistics of crystallographic data and refinement are summa-
rized in Table 1 of the Supplementary data. The coordinates of
The LBD of PPAR
tein using a pET28 vector and purified as previously described.40
Crystals of apo-PPAR were obtained by vapor diffusion at 20 °C
using sitting drop made by mixing 2 L of protein solution
(15 mg mLÀ1, in 20 mM Tris, 5 mM DTT, 0.5 mM EDTA, pH 8.0)
with 2 L of reservoir solution (0.8 M NaCitrate, 0.15 mM Tris,
c was expressed as N-terminal His-tagged pro-
c
PPARc/2 complex have been deposited in the Brookhaven Protein
Data Bank (PDB) with the code 3CDP.
l
l
4.3. Computational resources
pH 8.0). The crystals were soaked for 7 days in a storage solution
(1.2 M NaCitrate, 0.15 M Tris, pH 8.0) containing the ligands
(0.1 mM). The ligands were dissolved in ethanol and diluted in
the storage solution so that the final ethanol concentration was
Molecular modeling and graphics manipulations were per-
formed using Maestro 9.244 running on a Linux workstation with
an Intel Core i7 920 CPU and 12 GB of RAM. Glide 5.738 was used

G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
2147
for all docking calculations. Figures were generated using Pymol
1.0.45
After transfection, cells were treated for 20 h with the indicated li-
gands. Luciferase activity in cell extracts was then determined by a
luminometer (VICTOR3 V Multilabel Reader, PerkinElmer). b-Galac-
4.4. Ligand and receptor preparation
tosidase activity was determined using b-D-galactopyranoside (Sig-
ma, Milan, Italy) as described previously.50 All transfection
experiments were repeated at least twice.
Molecular models of compounds 2, 6 and S-10 were built and
minimized with the Maestro module of the Schrödinger molecular
modeling package. The Polak–Ribiere conjugate gradient (PRCG)
method with a 0.001 kcal molÀ1 ÅÀ1 convergence threshold was
used for the geometry optimization calculations. These structures
were then incorporated into the LigPrep 2.5 module of Schröding-
er.46 The protonation state for all ionizable groups was set at neu-
tral pH 7.
4.9. Chemical methods
Column chromatography was performed on ICN Silica Gel 60 Å
(63–200 lm) as a stationary phase. Melting points were deter-
mined in open capillaries on a Gallenkamp electrothermal appara-
tus and are uncorrected. Mass spectra were recorded on an HP GC/
MS 6890-5973 MSD spectrometer, electron impact 70 eV,
equipped with HP chemstation or an Agilent LC/MS 1100 Series
LC/MSD Trap System VL spectrometer, electrospray ionization
(ESI). 1H NMR spectra were recorded in CDCl3 (the use of other sol-
vents is specified) on a Varian-Mercury 300 (300 MHz) spectrome-
ter. Chemical shifts are expressed as parts per million (ppm, d).
Microanalyses of solid compounds were carried out with an Euro-
vector Euro EA 3000 model analyzer; the analytical results are
within 0.4% of theoretical values. Only for final compounds 3, 4,
9, 18 and 19 the data related to carbon are 0.64%, À0.85%, 0.74%,
0.67% and 0.72%, respectively.
Optical rotations were measured with a Perkin-Elmer 341
polarimeter at room temperature (20 °C): concentrations are ex-
pressed as g 100 mLÀ1. The CD curves were registered on a J-810
model JASCO spectropolarimeter. The enantiomeric excesses of
acids were determined by HPLC analysis of acids or their methyl
esters, obtained by reaction with an ethereal solution of diazo-
methane, on Chiralcel OD or AD columns (4.6 mm i.d. Â 250 mm,
Daicel Chemical Industries, Ltd, Tokyo, Japan). Analytical liquid
chromatography was performed on a PE chromatograph equipped
with a Rheodyne 7725i model injector, a 785A model UV/vis detec-
tor, a series 200 model pump and NCI 900 model interface. Chem-
icals were from Aldrich (Milan, Italy), Lancaster (Milan, Italy) or
Across (Milan, Italy) and were used without any further
purification.
The crystal structures of PPAR
c
in complex with 2 (PDB code:
dual agonist BMS-
3CDP) and of PPAR in complex with the
a
a/c
631707 (PDB code: 2REW)37 were prepared using the ‘Protein
Preparation Wizard’ panel of Schrödinger molecular modeling
package. In particular, using the ‘preprocess and analyze structure’
tool, the bond orders were assigned, all the hydrogen atoms were
added, the bound ligand and all the water molecules were deleted.
Using Epik 2.2,47 a prediction of the heterogroups ionization and
tautomeric states was performed. An optimization of the H-bond-
ing network was performed using the ‘H-bond assignment’ tool. Fi-
nally, using the ‘impref utility’, the positions of the hydrogen atoms
were optimized by keeping all the heavy atoms in place.
4.5. Docking simulations
2, 6 and S-10 were docked into both PPARc and PPARa binding
sites using the Glide 5.7 program in SP mode.38,48 The binding re-
gion was defined by a 10 Â 10 Â 10 Å box centered on the crystal-
lized conformation of ligand 2 for PPAR
c and BMS-631707 for
PPARa. A scaling factor of 0.8 was applied to the van der Waals ra-
dii. Default settings were used for all the remaining parameters.
Ten poses were generated for each ligand and re-ranked using
the scoring terms available in Glide.
4.6. Biological methods
Medium, other cell culture reagents, pioglitazone and Wy-
14,643 were purchased from Sigma (Milan, Italy). The active
metabolite of metaglidasen was synthesized in-house.
4.10. Preparation of ethyl 2-aryloxy-3-phenylpropanoates
4.10.1. General procedure
To an ice-bath cooled suspension of Ph3P–PS (2% DVB,
4.7. Plasmids
3 mmol gÀ1, 13.8 mmol) in anhydrous toluene (35 mL) was added
dropwise
a
solution of diisopropylazodicarboxylate (DIAD,
The expression vectors expressing the chimeric receptors con-
taining the yeast GAL4-DNA binding domain fused to either the hu-
man PPARa/PPARc ligand binding domain (LBD) and the reporter
plasmid for these GAL4 chimeric receptors (pGAL5TKpGL3) con-
taining five repeats of the GAL4 response elements upstream of a
minimal thymidine kinase promoter that is adjacent to the lucifer-
ase gene were described previously.49
13.5 mmol) in anhydrous toluene (35 mL). The resulting mixture
was stirred for 0.5 h. A solution of racemic or (R)-ethyl phenyllac-
tate (11 mmol) in anhydrous toluene (50 mL) and corresponding
phenol (10 mmol) was added at room temperature. The reaction
mixture was stirred overnight at rt, under N2 atmosphere. The solid
was filtered off and the filtrate was evaporated to dryness. The res-
idue was chromatographed on silica gel column (petroleum ether/
ethyl acetate 9:1 or 8:2 as eluent), affording the desired com-
pounds as pale yellow oils in 58–92% yields.
4.8. Cell culture and transfections
Human hepatoblastoma cell line HepG2 (Interlab Cell Line Col-
lection, Genoa, Italy) was cultured in Minimum Essential Medium
(MEM) containing 10% of heat inactivated Foetal Bovine Serum,
4.10.2. (S)-Ethyl 2-(4-fluoro-phenoxy)-3-phenylpropanoate
78% yield; 1H NMR: d = 1.18 (t, 3H, CH3CH2O), 3.21–3.23 (m, 2H,
PhCH2), 4.17 (q, 2H, CH3CH2O), 4.68–4.72 (m, 1H, CH), 6.74–6.79,
6.88–6.94 and 7.22–7.32 (m, 9H, aromatics); GC/MS, m/z (%): 288
(83) [M]+, 215 (30), 177 (100).
100 U penicillin G mLÀ1 and 100 g streptomycin sulfate mLÀ1 at
l
37 °C in a humidified atmosphere of 5% CO2. For transactivation as-
say 105 cells/well were seeded in a 24-well plate in triplicate and
transfections were performed after 24 h, with the CAPHOSÒ (Sig-
ma, Milan, Italy), a calcium–phosphate method, according to the
manufacturer guidelines. Cells were transfected with expression
4.10.3. Ethyl 2-(3-fluoro-phenoxy)-3-phenylpropanoate
76% yield; 1H NMR: d = 1.19 (t, 3H, CH3CH2O), 3.22–3.25 (m, 2H,
PhCH2), 4.17 (q, 2H, CH3CH2O), 4.74–4.78 (m, 1H, CH), 6.54–6.78
and 7.13–7.31 (m, 9H, aromatics); GC/MS, m/z (%): 288 (35) [M]+,
215 (38), 177 (100).
plasmids encoding the fusion protein GAL4-PPAR
a LBD or GAL4-
PPAR LBD (30 ng), pGAL5TKpGL3 (100 ng), pCMVbgal (250 ng).
c

2148
G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
4.10.4. Ethyl 2-(2-fluoro-phenoxy)-3-phenylpropanoate
92% yield; 1H NMR: d = 1.18 (t, 3H, CH3CH2O), 3.23–3.30 (m, 2H,
PhCH2), 4.17 (q, 2H, CH3CH2O), 4.77–4.82 (m, 1H, CH), 6.78–7.08
and 7.24–7.33 (m, 9H, aromatics); GC/MS, m/z (%): 288 (33) [M]+,
215 (22), 177 (100).
4.11.4. Diethyl 2-(2-trifluoromethyl-phenoxy)malonate
14% yield; 1H NMR: d = 1.30 (t, 6H, 2CH3), 4.32 (q, 4H, 2CH2),
5.22 (s, 1H, CH), 6.88–6.91, 7.08–7.13, 7.44–7.50 and 7.61–7.64
(m, 4H, aromatics); GC/MS, m/z (%): 320 (44) [M]+, 200 (36), 171
(100), 143 (37).
4.10.5. (S)-Ethyl 2-(3-trifluoromethyl-phenoxy)-3-
phenylpropanoate
4.12. Preparation of diethyl 2-aryloxy-2-benzyl-malonates
70% yield; 1H NMR: d = 1.19 (t, 3H, CH3CH2O), 3.22–3.32 (m, 2H,
PhCH2), 4.11–4.23 (m, 2H, CH3CH2O), 4.79–4.84 (m, 1H, CH), 6.97–
7.06 and 7.19–7.37 (m, 9H, aromatics); GC/MS, m/z (%): 338 (11)
[M]+, 177 (100), 135 (47), 105 (32).
4.12.1. General procedure
A
solution of the suitable diethyl aryloxy-malonate,36
(10 mmol) in anhydrous DMF (25 mL) was added dropwise to a
suspension of NaH (95% powder, 18 mmol) in anhydrous DMF
(20 mL) at 0 °C. After stirring for 20 min at rt, a solution of the suit-
able substituted benzylbromide (12 mmol) in anhydrous DMF
(15 mL) was added dropwise and the resulting reaction mixture
stirred at 60 °C for 15–20 h. The solvent was removed under re-
duced pressure and the residue poured into water and extracted
with diethyl ether. The organic layer was washed with saturated
ammonium chloride solution, dried over Na2SO4 and the solvent
evaporated in vacuo to give an oily residue which was chromato-
graphed on silica gel column (petroleum ether/ethyl acetate 95:5
as eluent). The title compounds were obtained as pale yellow oils
in 30–45% yields.
4.10.6. Ethyl 2-(2-trifluoromethyl-phenoxy)-3-
phenylpropanoate
58% yield; 1H NMR: d = 1.14 (t, 3H, CH3CH2O), 3.25–3.34 (m, 2H,
PhCH2), 4.14 (q, 2H, CH3CH2O), 4.87–4.91 (m, 1H, CH), 6.74–7.02,
7.21–7.41 and 7.55–7.58 (m, 9H, aromatics); GC/MS, m/z (%): 338
(6) [M]+, 177 (100), 135 (56).
4.10.7. (S)-Ethyl 2-(4-chloro-2-fluoro-phenoxy)-3-
phenylpropanoate
59% yield; 1H NMR: d = 1.19 (t, 3H, CH3CH2O), 3.21–3.34 (m, 2H,
PhCH2), 4.17 (q, 2H, CH3CH2O), 4.73–4.78 (m, 1H, CH), 6.70–6.75,
6.91–6.97, 7.04–7.09 and 7.21–7.32 (m, 8H, aromatics); GC/MS,
m/z (%): 324 (8) [M+2]+, 322 (25) [M]+, 177 (100), 135 (87).
4.12.2. Diethyl 2-(2-fluoro-4-chloro-phenoxy)-2-(4-chloro-benzyl)-
malonate
35% yield; 1H NMR: d = 1.15 (t, 6H, 2CH3CH2O), 3.53 (s, 2H,
ArCH2), 4.15 (q, 4H, 2CH3CH2O), 6.93–7.26 (m, 7H, aromatics);
GC/MS, m/z (%): 432 (2) [M+4]+, 430 (11) [M+2]+, 428 (17) [M]+,
283 (29), 237 (100).
4.10.8. Ethyl 2-(4-chloro-3-fluoro-phenoxy)-3-
phenylpropanoate
92% yield; 1H NMR: d = 1.19 (t, 3H, CH3CH2O), 3.23 (d, 2H,
PhCH2), 4.19 (q, 2H, CH3CH2O), 4.72 (t, 1H, CH), 6.54–6.67 and
7.19–7.32 (m, 8H, aromatics); GC/MS, m/z (%): 324 (15) [M+2]+,
322 (47) [M]+, 177 (100), 105 (68).
4.12.3. Diethyl 2-(4-chloro-phenoxy)-2-(2-fluoro-benzyl)-
malonate
35% yield; 1H NMR: d = 1.15 (t, 6H, 2CH3CH2O), 3.67 (s, 2H,
ArCH2), 4.18 (q, 4H, 2CH3CH2O), 6.83–7.27 (m, 8H, aromatics);
GC/MS, m/z (%): 396 (29) [M+2]+, 394 (83) [M]+, 221 (100).
4.10.9. Ethyl 2-(4-chloro-3-trifluoromethyl-phenoxy)-3-
phenylpropanoate
61% yield; 1H NMR: d = 1.20 (t, 3H, CH3CH2O), 3.25 (d, 2H,
PhCH2), 4.25 (m, 2H, CH3CH2O), 4.77 (d, 1H, CH), 6.87–7.01,
7.14–7.15 and 7.23–7.35 (m, 8H, aromatics); GC/MS, m/z (%): 374
(8) [M+2]+, 372 (24) [M]+, 177 (100), 135 (69), 105 (46).
4.12.4. Diethyl 2-(4-chloro-phenoxy)-2-(3-fluoro-benzyl)-
malonate
30% yield; 1H NMR: d = 1.16 (t, 6H, 2CH3CH2O), 3.67 (s, 2H,
ArCH2), 4.17 (q, 4H, 2CH3CH2O), 6.71–6.89 and 7.16–7.19 (m, 8H,
aromatics); GC/MS, m/z (%): 396 (32) [M+2]+, 394 (90) [M]+, 321
(49), 221 (100).
4.11. Preparation of diethyl 2-aryloxy-malonates
4.11.1. General procedure
A mixture of diethyl chloromalonate (19.1 mmol) and suitable
sodium phenate (20.1 mmol), prepared from an equivalent amount
of the corresponding phenol and sodium in abs EtOH, was stirred
and heated under reflux in acetone (100 mL) for 4 h. The solvent
was removed under reduced pressure, the residue was taken up with
water and extracted with ethyl acetate. The combined organic ex-
tracts were washed with brine, dried over Na2SO4 and concentrated
to give an oily residue which was chromatographed on silica gel col-
umn (petroleum ether/ethyl acetate 9:1 as eluent). The desired com-
pounds were obtained as pale yellow oils in 14–77% yield.
4.12.5. Diethyl 2-(4-chloro-phenoxy)-2-(4-fluoro-benzyl)-
malonate
45% yield; 1H NMR: d = 1.14 (t, 6H, 2CH3CH2O), 3.55 (s, 2H,
ArCH2), 4.16 (q, 4H, 2CH3CH2O), 6.81–6.99 and 7.12–7.26 (m, 8H,
aromatics); GC/MS, m/z (%): 396 (19) [M+2]+, 394 (51) [M]+, 221
(100).
4.12.6. Diethyl 2-(2-fluoro-4-chloro-phenoxy)-2-(3-fluoro-
benzyl)-
malonate
37% yield; 1H NMR: d = 1.15 (t, 6H, 2CH3CH2O), 3.56 (s, 2H,
ArCH2), 4.16 (q, 4H, 2CH3CH2O), 6.93–7.13 and 7.20–7.26 (m, 7H,
aromatics); GC/MS, m/z (%): 414 (18) [M+2]+, 412 (51) [M]+, 221
(100).
4.11.2. Diethyl 2-(4-chloro-phenoxy)malonate
77% yield; 1H NMR: d = 1.29 (t, 6H, 2CH3), 4.30 (q, 4H, 2CH2),
5.14 (s, 1H, CH), 6.80–7.26 (m, 4H, aromatics); GC/MS, m/z (%):
288 (34) [M+2]+, 286 (100) [M]+, 141 (89).
4.12.7. Diethyl 2-(2-trifluoromethyl-phenoxy)-2-(3-fluoro-benzyl)-
malonate
4.11.3. Diethyl 2-(2-fluoro-4-chloro-phenoxy)malonate
58% yield; 1H NMR: d = 1.30 (t, 6H, 2CH3), 4.31 (q, 4H, 2CH2),
5.15 (s, 1H, CH), 6.99–7.16 (m, 3H, aromatics); GC/MS, m/z (%):
306 (21) [M+2]+, 304 (62) [M]+, 231 (17), 159 (100), 147 (32).
36% yield; 1H NMR: d = 1.08 (t, 6H, 2CH3CH2O), 3.59 (s, 2H,
ArCH2), 4.13 (q, 4H, 2CH3CH2O), 6.91–7.39 and 7.59–7.62 (m, 8H,
aromatics); GC/MS, m/z (%): 428 (7) [M]+, 221 (100).

G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
2149
4.12.8. Preparation of ethyl 2-(4-chloro-2-fluoro-phenoxy)-2-
arylacetates
(100); Anal. Calcd for C15H13FO3: C, 69.22; H, 5.03. Found: C,
69.57; H, 5.02.
Sodium 4-chloro-2-fluoro-phenate (12 mmol) was dissolved in
abs. EtOH (30 mL) and added dropwise to a solution of ethyl 2-bro-
mo-2-arylethanoate (10 mmol) in abs. EtOH (90 mL). The resulting
mixture was refluxed for 24 h and the solvent was evaporated to
dryness. The resulting residue was dissolved in ethyl acetate,
washed twice with 0.5 N NaOH and brine, dried over Na2SO4 and
the solvent was evaporated to dryness affording a yellow oil resi-
due. The desired compounds were obtained as colorless oily by col-
umn chromatography on silica gel using petroleum ether/ethyl
acetate 98:2 as eluent.
4.13.5. (S)-2-(3-Trifluoromethyl-phenoxy)-3-phenylpropanoic
acid (7)
88% yield (n-hexane); mp: 62–64 °C; [
a
]
D À22 (c 1.0 in MeOH);
1H NMR: d = 3.24–3.37 (m, 2H, CH2), 4.87–4.91 (m, 1H, CH), 6.91–
7.07 and 7.17–7.39 (m, 10H, 9 aromatics + COOH, D2O exchanged);
ESI-MS m/z (%) negative: 309 (100) [MÀH]À1, 161 (55), 147 (8); ee
>99% (HPLC: Chiralcel OD column; hexane/i-propanol/TFA
95:5:0.05, flow rate 1 mL minÀ1, detection 254 nm); Anal. Calcd
for C16H13F3O3: C, 61.94; H, 4.22. Found: C, 62.34; H, 4.28.
4.12.9. Ethyl 2-(4-chloro-2-fluoro-phenoxy)-2-phenylacetate
43% yield; 1H NMR: d = 1.20 (t, 3H, CH3CH2O), 4.20 (q, 2H,
CH3CH2O), 5.60 (s, 1H, CH), 6.85–7.01, 7.10–7.14, 7.34–7.43 and
7.53–7.57 (m, 8H, aromatics); GC/MS, m/z (%): 310 (3) [M+2]+,
308 (9) [M]+, 163 (100).
4.13.6. 2-(2-Trifluoromethyl-phenoxy)-3-phenylpropanoic acid
(8)
84% yield (CHCl3/n-hexane); mp: 210–212 °C; 1H NMR (DMSO-
d6): d = 2.93–3.24 (m, 2H, CH2), 4.43–4.47 (m, 1H, CH), 6.87–6.92,
7.09–7.28 and 7.38–7.47 (m, 10H, 9 aromatics + COOH, D2O ex-
changed); ESI-MS m/z (%) negative: 309 (94) [MÀH]À1, 161 (100),
147 (5); Anal. Calcd for C16H13F3O3: C, 61.94; H, 4.22. Found: C,
61.88; H, 4.22.
4.12.10. Ethyl 2-(4-chloro-2-fluoro-phenoxy)-2-(4-chloro-
phenyl)acetate
46% yield; 1H NMR: d = 1.20 (t, 3H, CH3CH2O), 4.21 (m, 2H,
CH3CH2O), 5.57 (s, 1H, CH), 6.84–7.14 and 7.33–7.52 (m, 7H, aro-
matics); GC/MS, m/z (%): 346 (1) [M+4]+, 344 (3) [M+2]+, 342 (4)
[M]+, 269 (26), 197 (100).
4.13.7. (S)-2-(4-Chloro-2-fluoro-phenoxy)-3-phenylpropanoic
acid (9)
95% yield (CHCl3/n-hexane); mp: 88–89 °C; [
a
]
À20 (c 1.0 in
D
MeOH); 1H NMR: d = 3.23–3.36 (m, 2H, CH2), 4.79–4.83 (m, 1H,
CH), 5.10 (bb, 1H, COOH, D2O exchanged), 6.68–6.74, 6.91–6.97,
7.05–7.10 and 7.23–7.35 (m, 8H, aromatics); GC/MS, m/z (%)
(methyl ester): 310 (9) [M+2]+, 308 (27) [M]+, 163 (70), 121
(100); ee >99% (HPLC: Chiralcel AD column, hexane/i-propanol/
TFA 95:5:0.02, flow rate 1 mL minÀ1, detection 254 nm); Anal.
Calcd for C15H12ClFO3: C, 61.13; H, 4.10. Found: C, 61.87; H, 4.07.
4.13. Preparation of the final acids 3–5, 7–9, 11, 12, 18 and 19
4.13.1. General procedure
To a stirred solution of the corresponding ethyl ester (1 mmol)
in THF (10 mL) was added 2 N NaOH (10 mL). The reaction mixture
was stirred for 4–8 h. THF was evaporated in vacuo and the aque-
ous phase was acidified with 2 N HCl and extracted with Et2O. The
combined organic layers were washed twice with brine, dried over
Na2SO4 and evaporated to dryness affording the title acids, except
for 4, as white solids, which were recrystallized from n-hexane or
CHCl3/n-hexane.
The acid 4, obtained as a colorless oil, was dissolved in 95%
EtOH (10 mL) and added with a solution of NaHCO3 (1 equiv) in
H2O (1 mL). The reaction mixture was stirred for 2 h and then
the solvent was evaporated to dryness affording the corresponding
sodium salt as a white solid which was recrystallized from MeOH/
CHCl3.
4.13.8. 2-(4-Chloro-3-fluoro-phenoxy)-3-phenylpropanoic acid
(11)
96% yield (CHCl3/n-hexane); mp: 116–117 °C; 1H NMR:
d = 3.21–3.34 (m, 2H, CH2), 4.76–4.80 (m, 1H, CH), 5.51 (bb, 1H,
COOH, D2O exchanged), 6.55–6.67 and 7.20–7.34 (m, 8H, aromat-
ics); GC/MS, m/z (%) (methyl ester): 310 (6) [M+2]+, 308 (18)
[M]+, 163 (31), 121 (100); Anal. Calcd for C15H12ClFO3: C, 61.13;
H, 4.10. Found: C, 60.87; H, 4.03.
4.13.9. 2-(4-Chloro-3-trifluoromethyl-phenoxy)-3-
phenylpropanoic acid (12)
4.13.2. (S)-2-(4-Fluoro-phenoxy)-3-phenylpropanoic acid (3)
85% yield (CHCl3/n-hexane); mp: 78–79 °C; 1H NMR: d = 3.23–
3.37 (m, 2H, CH2), 4.82–4.87 (m, 1H, CH), 6.87–6.91, 7.14–7.15
and 7.20–7.36 (m, 9H, 8 aromatics + COOH, D2O exchanged); ESI-
MS m/z (%) negative: 345 (3) [M+2ÀH]À1, 343 (10) [MÀH]À1, 195
(100); Anal. Calcd for C16H12ClF3O3: C, 55.75; H, 3.51. Found: C,
55.87; H, 3.57.
98% yield (n-hexane); mp: 85–86 °C; [a] +19 (c 1.0 in MeOH);
D
1H NMR: d = 3.20–3.33 (m, 2H, CH2), 4.76–4.80 (m, 1H, CH), 6.75–
6.81, 6.89–6.96 and 7.24–7.34 (m, 10H, 9 aromatics + COOH, D2O
exchanged); GC/MS, m/z (%) (methyl ester): 274 (41) [M]+, 163
(52), 121 (100); ee >99% (HPLC, methyl ester: Chiralcel OD column,
n-hexane/i-propanol 95:5, flow rate 1 mL minÀ1
,
detection
280 nm); Anal. Calcd for C15H13FO3: C, 69.22; H, 5.03. Found: C,
69.86; H, 5.09.
4.13.10. 2-(4-Chloro-2-fluoro-phenoxy)phenylacetic acid (18)
98% yield (CHCl3/n-hexane); mp: 110–111 °C; 1H NMR: d = 5.63
(s, 1H, CH), 6.84–6.90, 6.96–7.00, 7.10–7.14, 7.34–7.44 and 7.52–
7.56 (m, 9H, 8 aromatics + COOH, D2O exchanged); ESI-MS m/z
(%) negative: 281 (13) [M+2ÀH]À1, 279 (40) [MÀH]À1, 235 (3),
129 (100); Anal. Calcd for C14H9FClO3: C, 59.91; H, 3.59. Found:
C, 60.58; H, 3.69.
4.13.3. Sodium 2-(3-Fluoro-phenoxy)-3-phenylpropanoate (4)
99% yield; mp: 250 °C dec.; 1H NMR: d = 3.10–3.23 (m, 2H, CH2),
4.49–4.53 (m, 1H, CH), 6.53–6.65 and 7.10–7.35 (m, 9H, aromat-
ics); Anal. Calcd for C15H12FO3Na: C, 63.83; H, 4.29. Found: C,
62.98; H, 4.21.
4.13.11. 2-(4-Chloro-2-fluoro-phenoxy)-2-(4-chloro-
phenyl)acetic acid (19)
4.13.4. 2-(2-Fluoro-phenoxy)-3-phenylpropanoic acid (5)
58% yield (CHCl3/n-hexane); mp: 90–91 °C; 1H NMR: d = 3.24–
3.37 (m, 2H, CH2), 4.83–4.88 (m, 1H, CH), 6.75–6.83, 6.90–7.10
and 7.22–7.36 (m, 10H, 9 aromatics + COOH, D2O exchanged);
GC/MS, m/z (%) (methyl ester): 274 (14) [M]+, 163 (65), 121
99% yield (n-hexane); mp: 110–12 °C; 1H NMR: d = 4.57 (bb, 1H,
COOH, D2O exchanged), 5.61 (s, 1H, CH), 6.84–6.89, 6.98–7.02,
7.11–7.16, 7.36–7.42 and 7.47–7.54 (m, 7H, aromatics); ESI-MS
m/z (%) negative: 317 (11) [M+4ÀH]À1, 315 (64) [M+2ÀH]À1, 313

2150
G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
(100) [MÀH]À1, 269 (31), 129 (57); Anal. Calcd for C14H8FCl2O3: C,
6.74–6.79, 6.91–7.08 and 7.18–7.30 (m, 8H, aromatics); ESI-MS m/z
(%) negative: 293 (46) [MÀH]À1, 165 (100), 127 (38), 121 (19); Anal.
Calcd for C15H12ClFO3: C, 61.13; H, 4.10. Found: C, 61.51; H, 4.16.
53.36; H, 2.88. Found: C, 54.08; H, 3.00.
4.13.12. Preparation of pantolactone esters of 10
(R)-Pantolactone (12 mmol), dimethylaminopyridine (DMAP,
0.1 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 10 mmol) were
added, under N2 atmosphere, to a stirred solution of the racemic acid
10 (12 mmol) in anhydrous THF (50 mL). The reaction mixture was
stirred at room temperature for 48 h, afterwards the precipitate
was filtered off and the organic phase was evaporated to dryness.
The mixture of diastereomeric esters was obtained, as a white solid,
by column chromatography on silica gel using petroleum ether/
ethyl acetate 95:5 as eluent. The recrystallization of the mixture
from CHCl3/n-hexane 1:7 afforded the desired (S,R)-diastereomer.
4.14.6. 2-(4-Chloro-phenoxy)-3-(4-fluoro-phenyl)propanoic
acid (15)
57% yield; mp: 108–109 °C; 1H NMR: d = 3.17–3.30 (m, 2H,
CH2), 4.42 (bb, 1H, COOH, D2O exchanged), 4.74–4.78 (m, 1H,
CH), 6.73–6.78, 6.94–7.12 and 7.19–7.34 (m, 8H, aromatics); GC/
MS, m/z (%) (methyl ester): 310 (12) [M+2]+, 308 (34) [M]+, 181
(57), 139 (100); Anal. Calcd for C15H12ClFO3: C, 61.13; H, 4.10.
Found: C, 61.27; H, 4.05.
4.14.7. 2-(4-Chloro-2-fluoro-phenoxy)-3-(3-fluoro-
phenyl)propanoic acid (16)
4.13.13. (R)-Pantolactone ester of (S)-2-(4-chloro-2-fluoro-
phenoxy)-3-(4-chloro-phenyl)propanoic acid
80% yield; mp: 107–108 °C; 1H NMR: d = 3.23–3.36 (m, 2H,
CH2), 4.79–4.84 (m, 1H, CH), 6.73–6.78, 6.94–7.12 and 7.19–7.34
(m, 8H, 7 aromatics + COOH, D2O exchanged); GC/MS, m/z (%)
(methyl ester): 328 (11) [M+2]+, 326 (32) [M]+, 181 (36), 139
(100); Anal. Calcd for C15H11ClF2O3: C, 57.62; H, 3.55. Found: C,
57.76; H, 3.58.
13% yield; 1H NMR: d = 0.93 and 1.03, (s, 6H, CHC(CH3)2, major
diastereomer), 3.26–3.40 (m, 2H, CHCH2), 3.97–4.05 (m, 2H, CH2O-
CO), 4.87–4.91 (m, 1H, ArOCHCOO), 5.35 (s, 1H, CHC(CH3)2), 6.82–
6.88, 6.99–7.12 and 7.27–7.37 (m, 7H, aromatics); GC/MS, m/z (%):
444 (1) [M+4]+, 442 (7) [M+2]+, 440 (10) [M]+, 295 (49), 165 (100);
de = 99% as determined by comparing the signals integrations of
the methyls of the pantolactone moiety (d = 0.96 and 1.10 for the
minor diastereomer).
4.14.8. 2-(2-Trifluoromethyl-phenoxy)-3-(3-fluoro-
phenyl)propanoic acid (17)
70% yield; mp: 166–168 °C; 1H NMR: d = 3.28–3.38 (m, 2H,
CH2), 4.96–5.00 (m, 1H, CH), 6.72–7.61 (m, 9H,
8 aromat-
4.14. Preparation of the final acids 10, S-10 and 13–17
ics + COOH, D2O exchanged); ESI-MS m/z (%) negative: 327 (80)
, 165 (20), 161 (100), 121 (5); Anal. Calcd for
C16H12F4O3: C, 58.54; H, 3.68. Found: C, 58.83; H, 3.74.
[MÀH]À1
4.14.1. General procedure
The suitable diethyl malonate (3 mmol), obtained from the reac-
tion described above, was refluxed under stirring with 1 N NaOH
(3 mL) in 95% EtOH (12 mL) for 4–6 h. The organic solvent was dis-
tilled off under reduced pressure and the resulting aqueous phase
washed with Et2O, acidified to pH 2 with 6 N HCl and extracted with
Et2O. The combined organic extracts were dried over Na2SO4 and the
solvent removed under reduced pressure. The resulting mixture was
heated at 160 °C for 2 h affording the desired acids as white solids
which were purified by recrystallization from n-hexane/CHCl3.
Acknowledgments
This work was accomplished thanks to the financial support of
the Università degli Studi di Bari ‘Aldo Moro’ (Research Fund 2009)
and of the CARIPLO Foundation (file 2009-2727).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.025.
4.14.2. 2-(4-Chloro-2-fluoro-phenoxy)-3-(4-chloro-
phenyl)propanoic acid (10)
82% yield; mp: 132–134 °C; 1H NMR: d = 3.20–3.33 (m, 2H,
CH2), 4.77–4.81 (m, 1H, CH), 6.72–6.78, 6.96–7.00, 7.07–7.11 and
7.23–7.30 (m, 8H, 7 aromatics + COOH, D2O exchanged); ESI-MS
m/z (%) negative: 327 (53) [MÀH]À1, 181 (54), 145 (100); Anal.
Calcd for C15H11Cl2FO3: C, 54.74; H, 3.37. Found: C, 54.98; H, 3.42.
References and notes
1. Berger, J. P.; Akiyama, T. E.; Meinke, P. T. Trends Pharmacol. Sci. 2005, 26, 244.
2. Kliewer, S. A.; Sundseth, S. S.; Jones, S. A.; Brown, P. J.; Wisely, G. B.; Koble, C. S.;
Devchand, P.; Wahli, W.; Willson, T. M.; Lenhard, J. M.; Lehmann, J. M. Proc. Natl
Acad. Sci. USA 1997, 94, 4318.
3. Elisaf, M. Curr. Med. Res. Opin. 2002, 18, 269.
4. Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.; Leitersdorf, E.; Fruchart, J.
C. Circulation 1998, 98, 2088.
5. Evans, R. M.; Barish, G. D.; Wang, Y. X. Nat. Med. 2004, 10, 355.
6. Semple, R. K.; Chatterjee, V. K.; O’Rahilly, S. J. Clin. Invest. 2006, 116, 581.
7. Campbell, I. W. Curr. Mol. Med. 2005, 5, 349.
8. Sprecher, D. L.; Massien, C.; Pearce, G.; Billin, A. N.; Perlstein, I.; Willson, T. M.;
Hassall, D. G.; Ancellin, N.; Patterson, S. D.; Lobe, D. C.; Johnson, T. G.
Arterioscler. Thromb. Vasc. Biol. 2007, 27, 359.
4.14.3. (S)-2-(4-Chloro-2-fluoro-phenoxy)-3-(4-chloro-
phenyl)propanoic acid [S-10]
99% yield; mp: 86–88 °C; [
(HPLC: Chiralcel AD column; hexane/i-propanol/TFA 99:1:0.04;
flow rate 1 mL minÀ1
detection 254 nm); Anal. Calcd for
15H11Cl2FO3: C, 54.74; H, 3.37. Found: C, 54.41; H, 3.37.
a]
À6 (c 1.0 in MeOH); ee >99%
D
;
C
9. Reilly, S. M.; Lee, C. H. FEBS Lett. 2008, 582, 26.
10. Madrazo, J. A.; Kelly, D. P. J. Mol. Cell. Cardiol. 2008, 44, 968.
11. Schmuth, M.; Jiang, Y. J.; Dubrac, S.; Elias, P. M.; Feingold, K. R. J. Lipid Res. 2008,
49, 499.
12. Iwashita, A.; Muramatsu, Y.; Yamazaki, T.; Muramoto, M.; Kita, Y.; Yamazaki,
S.; Mihara, K.; Moriguchi, A.; Matsuoka, N. J. Pharmacol. Exp. Ther. 2007, 320,
1087.
13. Peters, J. M.; Hollingshead, H. E.; Gonzalez, F. J. Clin. Sci. (Lond.) 2008, 115, 107.
14. Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B. R. J. Med. Chem. 2000, 43,
527.
4.14.4. 2-(4-Chloro-phenoxy)-3-(2-fluoro-phenyl)propanoic
acid (13)
55% yield; mp: 113–114 °C; 1H NMR: d = 3.23–3.42 (m, 2H,
CH2), 4.82–4.87 (m, 1H, CH), 6.73–6.78, 6.91–7.10 and 7.17–7.32
(m, 9H, 8 aromatics + COOH, D2O exchanged); GC/MS, m/z (%)
(methyl ester): 310 (13) [M+2]+, 308 (38) [M]+, 139 (100); Anal.
Calcd for C15H12ClFO3: C, 61.13; H, 4.10. Found: C, 61.27; H, 4.08.
15. Henke, B. R. J. Med. Chem. 2004, 47, 4118.
16. Ebdrup, S.; Pettersson, I.; Rasmussen, H. B.; Deussen, H. J.; Frost Jensen, A.;
Mortensen, S. B.; Fleckner, J.; Pridal, L.; Nygaard, L.; Sauerberg, P. J. Med. Chem.
2003, 46, 1306.
17. Devasthale, P. V.; Chen, S.; Jeon, Y.; Qu, F.; Shao, C.; Wang, W.; Zhang, H.; Cap,
M.; Farrelly, D.; Golla, R.; Grover, G.; Harrity, T.; Ma, Z.; Moore, L.; Ren, J.;
Seethala, R.; Cheng, L.; Sleph, P.; Sun, W.; Tieman, A.; Wetterau, J. R.; Doweyko,
4.14.5. 2-(4-Chloro-phenoxy)-3-(3-fluoro-phenyl)propanoic
acid (14)
50% yield; mp: 125–127 °C; 1H NMR: d = 3.20–3.32 (m, 2H,
CH2), 4.77–4.81 (m, 1H, CH), 5.97 (bb, 1H, COOH, D2O exchanged),

G. Fracchiolla et al. / Bioorg. Med. Chem. 20 (2012) 2141–2151
2151
A.; Chandrasena, G.; Chang, S. Y.; Humphreys, W. G.; Sasseville, V. G.; Biller, S.
A.; Ryono, D. E.; Selan, F.; Hariharan, N.; Cheng, P. T. J. Med. Chem. 2005, 48,
2248.
32. Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
33. Fracchiolla, G.; Laghezza, A.; Piemontese, L.; Carbonara, G.; Lavecchia, A.;
Tortorella, P.; Crestani, M.; Novellino, E.; Loiodice, F. ChemMedChem 2007, 2,
641.
34. Gregoire, F. M.; Zhang, F.; Clarke, H. J.; Gustafson, T. A.; Sears, D. D.; Favelyukis,
S.; Lenhard, J.; Rentzeperis, D.; Clemens, L. E.; Mu, Y.; Lavan, B. E. Mol.
Endocrinol. 2009, 23, 975.
18. Koyama, H.; Miller, D. J.; Boueres, J. K.; Desai, R. C.; Jones, A. B.; Berger, J. P.;
MacNaul, K. L.; Kelly, L. J.; Doebber, T. W.; Wu, M. S.; Zhou, G.; Wang, P. R.;
Ippolito, M. C.; Chao, Y. S.; Agrawal, A. K.; Franklin, R.; Heck, J. V.; Wright, S. D.;
Moller, D. E.; Sahoo, S. P. J. Med. Chem. 2004, 47, 3255.
19. Shi, G. Q.; Dropinski, J. F.; McKeever, B. M.; Xu, S.; Becker, J. W.; Berger, J. P.;
MacNaul, K. L.; Elbrecht, A.; Zhou, G.; Doebber, T. W.; Wang, P.; Chao, Y. S.;
Forrest, M.; Heck, J. V.; Moller, D. E.; Jones, A. B. J. Med. Chem. 2005, 48, 4457.
20. Grey, A. Osteoporos. Int. 2008, 19, 129.
35. Montanari, R.; Saccoccia, F.; Scotti, E.; Crestani, M.; Godio, C.; Gilardi, F.;
Loiodice, F.; Fracchiolla, G.; Laghezza, A.; Tortorella, P.; Lavecchia, A.; Novellino,
E.; Mazza, F.; Aschi, M.; Pochetti, G. J. Med. Chem. 2008, 51, 7768.
36. Carbonara, G.; Fracchiolla, G.; Loiodice, F.; Tortorella, P.; Conte-Camerino, D.;
De Luca, A.; Liantonio, A. Il Farmaco. 2001, 56, 749.
37. Wang, W.; Devasthale, P.; Farrelly, D.; Gu, L.; Harrity, T.; Cap, M.; Chu, C.;
Kunselman, L.; Morgan, N.; Ponticiello, R.; Zebo, R.; Zhang, L.; Locke, K.; Lippy,
J.; O’Malley, K.; Hosagrahara, V.; Kadiyala, P.; Chang, C.; Muckelbauer, J.;
Doweyko, A. M.; Zahler, R.; Ryono, D.; Hariharan, N.; Cheng, P. T. Bioorg. Med.
Chem. Lett. 2008, 18, 1939.
38. Glide, version 5.7; Schrodinger, LLC: New York, 2011.
39. (a) Dunitz, J. D.; Taylor, R. Chem. Eur. J. 1997, 3, 89; (b) Dunitz, J. D.
ChemBioChem 2004, 5, 614.
40. Pochetti, G.; Godio, C.; Mitro, N.; Caruso, D.; Galmozzi, A.; Scurati, S.; Loiodice,
F.; Fracchiolla, G.; Tortorella, P.; Laghezza, A.; Lavecchia, A.; Novellino, E.;
Mazza, F.; Crestani, M. J. Biol. Chem. 2007, 282, 17314.
21. Shearer, B. G.; Billin, A. N. Biochim. Biophys. Acta 2007, 1771, 1082.
22. Dropinski, J. F.; Akiyama, T.; Einstein, M.; Habulihaz, B.; Doebber, T.; Berger, J.
P.; Meinke, P. T.; Shi, G. Q. Bioorg. Med. Chem. Lett. 2005, 15, 5035.
23. Reifel-Miller, A.; Otto, K.; Hawkins, E.; Barr, R.; Bensch, W. R.; Bull, C.; Dana, S.;
Klausing, K.; Martin, J. A.; Rafaeloff-Phail, R.; Rafizadeh-Montrose, C.; Rhodes,
G.; Robey, R.; Rojo, I.; Rungta, D.; Snyder, D.; Wilbur, K.; Zhang, T.; Zink, R.;
Warshawsky, A.; Brozinick, J. T. Mol. Endocrinol. 2005, 19, 1593.
24. Martin, J. A.; Brooks, D. A.; Prieto, L.; Gonzalez, R.; Torrado, A.; Rojo, I.; Lopez de
Uralde, B.; Lamas, C.; Ferritto, R.; Dolores Martin-Ortega, M.; Agejas, J.; Parra,
F.; Rizzo, J. R.; Rhodes, G. A.; Robey, R. L.; Alt, C. A.; Wendel, S. R.; Zhang, T. Y.;
Reifel-Miller, A.; Montrose-Rafizadeh, C.; Brozinick, J. T.; Hawkins, E.; Misener,
E. A.; Briere, D. A.; Ardecky, R.; Fraser, J. D.; Warshawsky, A. M. Bioorg. Med.
Chem. Lett. 2005, 15, 51.
41. Leslie, A.; Janisch, S. Health Serv. J. 1992, 102, 26.
25. Burgermeister, E.; Schnoebelen, A.; Flament, A.; Benz, J.; Stihle, M.; Gsell, B.;
Rufer, A.; Ruf, A.; Kuhn, B.; Marki, H. P.; Mizrahi, J.; Sebokova, E.; Niesor, E.;
Meyer, M. Mol. Endocrinol. 2006, 20, 809.
26. Allen, T.; Zhang, F.; Moodie, S. A.; Clemens, L. E.; Smith, A.; Gregoire, F.; Bell, A.;
Muscat, G. E.; Gustafson, T. A. Diabetes 2006, 55, 2523.
27. Carmona, M. C.; Louche, K.; Lefebvre, B.; Pilon, A.; Hennuyer, N.; Audinot-
Bouchez, V.; Fievet, C.; Torpier, G.; Formstecher, P.; Renard, P.; Lefebvre, P.;
Dacquet, C.; Staels, B.; Casteilla, L.; Penicaud, L. Diabetes 2007, 56, 2797.
28. Kim, M. K.; Chae, Y. N.; Kim, H. S.; Choi, S. H.; Son, M. H.; Kim, S. H.; Kim, J. K.;
Moon, H. S.; Park, S. K.; Shin, Y. A.; Kim, J. G.; Lee, C. H.; Lim, J. I.; Shin, C. Y. Arch.
Pharm. Res. 2009, 32, 721.
42. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-
Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr., Sect. D 1998, 54, 905.
43. 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. Nature 1998, 395,
137.
44. Maestro, version 9.2; Schrödinger, LLC, New York, NY, 2011.
45. DeLano, Warren L. The PyMOL Molecular Graphics System. DeLano Scientific
LLC, San Carlos, CA, USA. http://www.pymol.org/.
46. LigPrep, version 2.5; Schrödinger, LLC, New York, NY, 2011.
47. Epik, version 2.2; Schrödinger, LLC: New York, 2009.
29. Motani, A.; Wang, Z.; Weiszmann, J.; McGee, L. R.; Lee, G.; Liu, Q.; Staunton, J.;
Fang, Z.; Fuentes, H.; Lindstrom, M.; Liu, J.; Biermann, D. H.; Jaen, J.; Walker, N.
P.; Learned, R. M.; Chen, J. L.; Li, Y. J. Mol. Biol. 2009, 386, 1301.
30. Pinelli, A.; Godio, C.; Laghezza, A.; Mitro, N.; Fracchiolla, G.; Tortorella, V.;
Lavecchia, A.; Novellino, E.; Fruchart, J. C.; Staels, B.; Crestani, M.; Loiodice, F. J.
Med. Chem. 2005, 48, 5509.
48. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
49. Raspe, E.; Madsen, L.; Lefebvre, A.-M.; Leitersdorf, I.; Gelman, L.; Peinado-
Onsurbe, J.; Dallongeville, J.; Fruchart, J.-C.; Berge, R.; Staels, B. J. Lipid. Res.
1999, 40, 2099.
31. Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
50. Hollons, T.; Yoshimura, F. K. Anal. Biochem. 1989, 182, 411.