New 2-aryloxy-3-phenyl-propanoic acids as peroxisome proliferator-activated receptors α/γ dual agonists with improved potency and reduced adverse effects on skeletal muscle function

Article abstract of DOI:10.1021/jm900941b

The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARα/γ dual agonist 1, allowed the identification of new ligands with improved potency

Full text of DOI:10.1021/jm900941b

6382 J. Med. Chem. 2009, 52, 6382–6393
DOI: 10.1021/jm900941b
New 2-Aryloxy-3-phenyl-propanoic Acids As Peroxisome Proliferator-Activated Receptors r/γ Dual
Agonists with Improved Potency and Reduced Adverse Effects on Skeletal Muscle Function†
Giuseppe Fracchiolla,‡ Antonio Laghezza,‡ Luca Piemontese,‡ Paolo Tortorella,‡ Fernando Mazza,§, Roberta Montanari,
Giorgio Pochetti, Antonio Lavecchia,^ Ettore Novellino,^ Sabata Pierno,# Diana Conte Camerino,# and Fulvio Loiodice*,‡
‡Dipartimento Farmaco-Chimico, Universitaꢀ degli Studi di Bari, via Orabona 4, 70126 Bari, Italia, §Dipartimento di Scienze della Salute,
Universitaꢀ di L’Aquila, 67010 L’Aquila, Italia, Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016
Monterotondo Stazione, Roma, Italia, ^Dipartimento di Chimica Farmaceutica e Tossicologica, Universitaꢀdegli Studi di Napoli “Federico II”, via
Montesano 49, 80131 Napoli, Italia, and #Dipartimento Farmaco-Biologico, Universitaꢀ degli Studi di Bari, via Orabona 4, 70126 Bari, Italia
Received June 26, 2009
The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction
of a linker into the diphenyl system of the previously reported PPARR/γ dual agonist 1, allowed the
identification of new ligands with improved potency on PPARR and unchanged activity on PPARγ. For
the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARγ and docking experiments in
PPARR provided a molecular explanation for their different behavior as full and partial agonists of
PPARR and PPARγ, respectively. Due to the adverse effects provoked by hypolipidemic drugs on
skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle
membrane chloride channel conductance and found that these ligands have a pharmacological profile
more beneficial compared to fibrates currently used in therapy.
Introduction
TZD class of insulin sensitizers has prompted pharmaceutical
companies to concentrate on developing more potent and
dual agonists acting on these two subtypes. Dual-acting
PPARR/γ agonists, in fact, are considered a very attractive
option in the treatment of dyslipidemic type 2 diabetes.11-22
One of the key challenges for the development of a dual
agonist is identifying the optimal receptor subtype selectivity
ratio. PPARγ full agonists, despite their proven benefits,
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.23-25 During the past
decade, therefore, new drugs acting as partial agonists of
PPARγ have been developed with the goal of retaining the
beneficial effects while reducing the adverse effects. Metagli-
dasen (Chart 1), a PPARγ partial agonist, is the most
advanced insulin sensitizer that is currently in phase III
clinical trials. The results of phase II clinical trials showed
that metaglidasen, a prodrug ester that is rapidly and com-
pletely modified in vivo to its mature circulating free acid
form, significantly improved metabolic parameters without
the side effects of fluid retention/edema or weight gain.24
Recently, we reported the synthesis and activity of the new
ligand 1 (Chart 1) whose structure is related to that of the
active metabolite of metaglidasen. This molecule is a dual
PPARR/γ ligand with a partial agonism profile toward the
γ subtype.26 The X-ray structure of the complex with PPARγ
shows that this ligand occupies a branch, named “diphenyl
pocket”, of a new L-shaped region of the PPARγ ligand-
binding domain (LBD) never sampled before by other known
ligands and increases the stabilization of the helix H3, indu-
cing a conformation of the LBD less favorable to the recruit-
ment of coactivators required for full activation of PPARγ.26
As regards PPARR, the ligand assumes a different orientation
Peroxisome proliferator-activated receptors (PPARsa) are
ligand-activated transcription factors belonging to the nuclear
hormone receptor superfamily. They are lipid sensors known
to govern numerous biological processes. There are three
PPAR subtypes, which are the products of distinct genes
and are commonly designated PPARR, PPARγ, and PPARδ.
PPARR is highly expressed in the liver and skeletal muscleand
is the molecular target for the fibrates (e.g., fenofibrate and
gemfibrozil), a class of drugs that lower plasma triglycerides
and increase HDL cholesterol levels in humans.1,2 PPARγ is
expressed most abundantly in adipose tissue and mediates the
antidiabetic activity of the insulin-sensitizing drugs belonging
to the thiazolidindione (TZD) class such as rosiglitazone and
pioglitazone.3 The function of PPARδ is still not fully under-
stood, but recent evidence suggests that this ubiquitously
expressed PPAR isoform has pleiotropic actions that may
govern diverse physiological processes, including the regula-
tion of lipid and lipoprotein metabolism,4,5 insulin sensitiv-
ity,6 cardiac function,7 epidermal biology,8 neuroprotection,9
and gastrointestinal tract function and disease.10 To date,
however, no PPARδ agonist has been fully developed and the
clinical potential of targeting this isotype remains to be clearly
determined. The success of the hypolipidemic fibrates and
†Protein Data Bank identification number (PDB ID) for PPARγ/S-2
and PPARγ/S-4 complexes are 3HOD and 3HO0, respectively.
*To whom correspondence should be addressed. Phone: þ39 080-
5442798. Fax: þ39 080-5442231. E-mail: floiodice@farmchim.uniba.it.
a Abbreviations: PPAR, peroxisome proliferator-activated receptor;
TZD, thiazolidinedione; HDL, high-density lipoprotein; LBD, ligand
binding domain; ClC, chloride channel; TBAF, tetrabutylammonium
fluoride; DBU, 1,8-diazabicycle[5.4.0]undec-7-ene; EDL, extensor digi-
torum longus.
r
pubs.acs.org/jmc
Published on Web 09/23/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6383
Chart 1. Chemical Structures of 1, Metaglidasen, and New
PPAR Agonists 2-15
catalyst and TBAF under Sonogashira conditions,31 followed
by saponification of the ester intermediate, led to the final acid
14. The synthesis of the R- and S-isomers of compounds 2 and 4
was carried out similarly to the corresponding racemates
starting from the readily available (S)- or (R)-ethyl phenyl-
lactates, respectively. Both enantiomers of acids 2 and 4 had
enantiomeric excesses >98% as determined by HPLC ana-
lysis on chiral stationary phase (see Experimental Section).
Synthesis of acids 6, 8, 10, 13, and 15 is represented in
Scheme 2 and involved the key intermediate ester 28, which
was prepared from the condensation of 4-hydroxy-benzalde-
hyde and ethyl phenyllactate according to the procedure
reported in Scheme 1. The reduction of the aldehyde group
of 28 with NaBH4 at 0 ꢀC afforded the corresponding alcohol
whose treatment with PBr3 yielded 29. The subsequent con-
densation of 29 with phenol or thiophenol in the presence of
95% NaH, followed by saponification of the ester intermedi-
ates, led to the target compounds 6 and 8, respectively. For
compound 10, the reductive amination of 28 was carried out
with aniline in the presence of bis(triphenylphosphine)copper-
(I) borohydride and NH2SO3H, following a procedure
reported in the literature.32 The saponification of the corre-
sponding ester intermediate afforded the desired acid. Alter-
natively, the Wittig reaction of 28 with the commercially
available benzyltriphenylphosphonium chloride in the pre-
sence of 1,8-diazabicycle[5.4.0]undec-7-ene (DBU) provided
the cis-ester intermediate of acid 13 as a mixture with the
corresponding geometric isomer (53:47, cis:trans).33 The cis-
isomer was purified by column chromatography and hydro-
lyzed to give 13. Following the same procedure, the synthesis
of compound 15 was achieved using the phenethyltriphenyl-
phosphonium bromide salt; the hydrogenation in the presence
of Wilkinson catalyst of the mixture of propylene geometric
isomers so obtained and alkaline hydrolysis of the ester
intermediate led to the target compound.
in the LBD that stabilizes better the helices H11 and H12,
resultingina full agonismresponse. The identification ofthese
novel ligand-receptor interaction modalities represents a new
hallmark of the partial agonism behavior of certain ligands
and could be exploited for the design of new antidiabetic
agents appropriately targeting the PPARs. Since 1 occupies
only the first branch of the new L-shaped region, we decided
to prepare some analogues characterized by the presence of a
linker, with different length and stereoelectronic properties,
between the aromatic rings of the diphenyl system that could
allow thecompleteoccupation of the entirecavity withthe aim
of evaluating the effects due to a further stabilization of H11,
H12, and the loop 11/12 in terms of potency, efficacy, and
subtype selectivity.
For this purpose, we synthesized the new 2-aryloxy-3-
phenyl-propanoic acids 2-15 reported in Chart 1 and eval-
uated their PPARR/γ activity. Noteworthy, we identified new
ligands with higher potency on PPARR while maintaining
basically unchanged the potency and the partial agonism on
PPARγ. Considering the high degree of stereoselectivity
displayed from these receptors toward the stereoisomer 1,26
the influence of the absolute configuration was also taken into
account for the most interesting derivatives 2 and 4 bearing
one or two methylene groups between the aromatic rings of
the diphenyl system, respectively. X-ray studies with PPARγ
and docking experiments with PPARR have been performed
for the most active isomers S-2 and S-4, providing a molecular
explanation for their different behavior as full and partial
agonists of PPARR and PPARγ, respectively. Finally, since
skeletal muscle is a target of PPAR agonists, the effects of
these two ligands on the function of this tissue were evaluated
by measuring the resting chloride conductance (gCl) sustained
by the voltage-gated chloride channel ClC-1. Previous studies
showed that the reduction of gCl function is one of the
mechanisms of action responsible for myopathies, the most
important of the complications of lipid-lowering treatment
with statins and fibrates.27-30
Results and Discussion
Compounds 2-15 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 accord-
ing to a previously reported procedure.34 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. The
maximum induction obtained with the reference agonist was
defined as 100%.
PPARr Activity. The PPARR activity of racemates 2-15
was examined first in comparison with 1 and Wy-14,643. The
introduction of a methylene between the phenyl rings of
1 afforded the full agonist 2 with a remarkable increase of the
potency. A further lengthening of the spacer to ethylene and
propylene (compounds 4 and 15) provided a progressive
decrease of potency and efficacy. The introduction of isos-
teric substitutions into the spacer gave different results:
a methylene was better than an oxygen atom (2 was about
four times more potent than 3), but when the same replace-
ment was carried out on the ethylenic linker, a more or less
potent agonist was obtained according to the position of the
oxygen atom (5 was three times as potent as 4, but 6 about
twice less potent than 4). A similar behavior was observed,
even though at reversed positions of the heteroatom, with the
introduction of sulfur (compounds 7 and 8), whereas the
Chemistry
Synthesis of compounds 2-5, 7, 9, 11, 12, and 14 is depicted
in Scheme 1 and was carried out starting from ethyl phenyl-
lactate and the suitable 4-substituted phenol, which were
condensed, under Mitsunobu conditions, to obtain the key
intermediates 20-27. The saponification of 20-26 with 1 N
NaOHsolutioninTHF provided thefinalacids 2, 3, 5, 7, 9, 11,
and 12. Compound 4 was obtained by hydrogenation at 4 atm
of 22 in the presence of Wilkinson catalyst followed by
saponification of the ester intermediate. The condensation
of 27 with ethynylbenzene in the presence of palladium

6384 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Fracchiolla et al.
Scheme 1. Synthesis of PPAR Agonists 2-5, 7, 9, 11, 12, and 14
(a) Ph3P, DIAD, anhydrous THF or toluene, rt; (b) 1 N NaOH/THF (1:1), rt; (c) H2 (4 atm), Wilkinson cat., THF/MeOH, rt; (d) ethynylbenzene,
PdCl2(PPh3)2, (n-butyl)4NF xH2O, N2, reflux.
3
Scheme 2. Synthesis of PPAR Agonists 6, 8, 10, 13, and 15
(a) NaBH4, THF/H2O (1:1), 0 ꢀC; (b) PBr3, rt; (c) 95% NaH, phenol or thiophenol, anhydrous THF or DMF, N2, reflux; (d) 1N NaOH/THF (1:1); (e)
aniline, (Ph3P)2CuBH4, NH2SO3H, MeOH, rt; (f) DBU, PhCH2(Ph)3PCl, anhydrous CH3CN, N2, reflux; the isomers mixture was chromatographed on
silica gel; (g) DBU, PhCH2CH2(Ph)3PBr, anhydrous CH3CN, N2, reflux; (h) H2 (8 atm), Wilkinson cat., THF/EtOH, rt.
Table 1. Activity of the Tested Compounds in Cell-Based Transactivation Assay
PPARR
PPARγ
compd
X
EC50 (μM)
efficacy
EC50 (μM)
efficacy
1
2
bond
CH2
CH2
CH2
O
0.19 ( 0.04
0.0159 ( 0.0003
2.4 ( 0.8
0.0115 ( 0.0021
0.060 ( 0.006
0.33 ( 0.18
ia
116 ( 4
115 ( 27
82 ( 2
107 ( 5
126 ( 6
86 ( 16
ia
0.56 ( 0.11
0.58 ( 0.19
5.7 ( 0.4
0.572 ( 0.006
1.21 ( 0.04
0.86 ( 0.04
1.6 ( 0.3
0.40 ( 0.20
2.40 ( 0.23
3.0 ( 0.8
1.4 ( 0.4
0.40 ( 0.07
13 ( 3
9.4 ( 2.3
9.1 ( 0.9
60 ( 8
42 ( 1
21 ( 1
40 ( 4
50 ( 5
39 ( 3
19 ( 4
33 ( 5
43 ( 1
40 ( 4
36 ( 1
33 ( 3
27 ( 6
26 ( 6
40 ( 2
50 ( 1
34 ( 1
39 ( 1
30 ( 6
ia
R-2
S-2
3
4
R-4
CH2CH2
CH2CH2
CH2CH2
CH2O
S-4
5
0.15 ( 0.04
0.107 ( 0.006
0.60 ( 0.13
0.488 ( 0.017
0.31 ( 0.16
3.6 ( 1.5
83 ( 10
112 ( 8
107 ( 13
134 ( 14
92 ( 17
78 ( 4
120 ( 20
106 ( 1
57 ( 4
107 ( 2
36 ( 3
67 ( 15
100 ( 10
ia
6
OCH2
CH2S
SCH2
7
8
9
10
11a
CH2NH
NHCH2
CH2C(O)
CHdCH trans
CHdCH cis
CꢀC
1.4 ( 0.4
1.8 ( 1.0
12
1.75 ( 0.12
0.160 ( 0.022
1.36 ( 0.06
0.72 ( 0.20
1.6 ( 0.3
0.72 ( 0.27
1.5 ( 0.4
0.540 ( 0.017
0.87 ( 0.16
ia
13a
14
15
Wy-14,643
rosiglitazone
CH2CH2CH2
ia
0.039 ( 0.003
100 ( 9
a This compound was tested as sodium salt. ia: inactive at tested concentrations. Efficacy values were calculated as percentage of the maximum
obtained fold induction with the reference compounds (Wy-14,643 for PPARR; rosiglitazone for PPARγ).
presence of the amino group afforded agonists (9, 10) with
potency in the micromolar range, probably due to the
unfavorable capability of this group to function as a hydro-
gen bond donor. Interesting results were also obtained by
substituting the ethylenic spacer of compound 4 with unsatu-
rated two-carbon linkers, which present more constraints
with regard to the bond geometry. Among these ligands, the
cis-derivative 13 was, by far, the most active compound, with
a potency 8 and 10 times higher than the acetylene derivative
14 and the ethanoyl and trans-ethylene derivatives 11 and 12,
respectively. The importance of stereochemistry was con-
firmed by evaluating the activity of two enantiomeric pairs:

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6385
Figure 1. Hydrogen bond network of (A) S-2 (yellow) and (B) S-4 (cyan) in the complex with PPARγ.
Figure 2. Hydrophobic interactions of (A) S-2 and (B) S-4 in the PPARγ internal cavity. Both ligands are colored in yellow.
improved potency compared to the corresponding R-isomer,
whereas S-4 was a little more potent than 1 and four times
more potent than R-4.
With the aim to gain structural biology insight into the role
played by the spacer of the diphenyl system of the most active
ligands in imparting higher potency on PPARR compared to
1 while maintaining basically unchanged the partial agonism
on PPARγ, X-ray crystal studies in PPARγ-LBD and dock-
ing experiments in PPARR have been performed for the most
active isomers S-2 and S-4.
Binding of S-2 and S-4 to PPARγ-LBD. Figure A of the
Supporting Information shows the positioning of S-2 and
S-4, respectively, fitted into the electron density map calcu-
lated in the hydrophobic pocket of PPARγ. Figure 1 sum-
marizes the binding interactions made by the polar head of
S-2 and S-4, respectively, with the surrounding residues.
Figure 3. Superposition of the complexes of PPARγ with 1 (yellow),
S-2 (purple), and S-4 (green).
S-2, in fact, besides displaying 17-fold improved potency
compared to that of 1, was about 200 times more potent than
the corresponding R-isomer, whereas S-4 was equipotent
with 1 and R-4 was completely inactive.
In both cases, one of the carboxylate oxygens forms a
bifurcated H-bond with the Y473 OH and the H449 Nε2
groups; the latter group engages the ligand ether oxygen in a
further H-bond. The other carboxylate oxygen is at H-bonding
distance from H323 Nε2. The benzyl-phenoxy and phenethyl-
phenoxy groups of S-2 and S-4, respectively, are deeply
inserted in the so-called “diphenyl pocket”, forming several
favorable hydrophobic interactions (see Figure 2).
The bottom of the cavity is delineated by the loop 11/12
and is contoured sidewise by H3 and H11. At the bottom of
the cavity, the terminal end of the benzyl-phenoxy and
phenethyl-phenoxy groups faces, on one side, the M463
side-chain of the loop 11/12, realizing strong interactions
between the methyl and the π-cloud. Electrostatic interac-
tions are realized, on the other side, by the edge of the
aromatic ring and the CO group of the Q283 side chain. In
the PPARγ/S-2 complex, the Q286 side chain is also engaged
in such interactions. The short contact between the distal
PPARγ Activity. The PPARγ activity of racemates 2-15
was compared with 1 and rosiglitazone. All compounds
displayed lower potency than that of rosiglitazone and none
of them was able to exhibit full agonist activity. The effects
resulting from the introduction of isosteric substitutions into
the spacer and its lengthening were basically similar to those
on PPARR, whereas a significant change was observed for
analogues containing unsaturated two-carbon linkers. The
more rigid and planar trans-ethylene and acetylene deriva-
tives 12 and 14, in fact, were more potent than cis-derivative
13, whereas the ethanoyl compound 11 still displayed very
low agonist activity. Regarding stereochemistry, the influ-
ence of absolute configuration was confirmed also in this
case; S-2, in fact, displayed equipotency with 1 and 10-fold

6386 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Fracchiolla et al.
Figure 4. (A) S-2 (yellow), as observed in the crystal complex with PPARγ and its possible orientation (cyan) into the additional space of the
diphenyl pocket (white-colored mesh); (B) superposition of the crystal complexes of PPARγ with S-2 (purple) and PPARR with BMS-631707
(white). White-colored mesh represents the additional space of the diphenyl pocket.
Figure 5. Putative orientation of (A) S-2 (white) docked into the hydrophobic pocket of PPARR, superimposed to the structure of the complex
PPARγ/S-2 (purple), and (B) S-4 (white) docked into the hydrophobic pocket of PPARR, superimposed to the structure of the complex
PPARγ/S-4 (purple).
aromatic rings of S-2 and S-4 with M463, Q283, and Q286
are well evidenced by continuous electron density between
the interacting groups (see Figure B in the Supporting
Information).
better stabilize H3 rather than H11 and H12 lose the
character of full agonists.26
In Figure 4B, the CR superposition between the crystal
complex of PPARγ/S-2 and that of the human PPARR
complexed with the R/γ dual agonist BMS-631707 (PDB
code 2REW)38 is shown. It has already been observed26 that
ligands occupying the “diphenyl pocket” in PPARR assume
a different slope into the cavity in order to maintain the
carboxylate H-bond network because of the longer protru-
sion of the Y314 side chain (H323 in PPARγ). It is evident
from Figure 4B that if S-2 could assume in PPARR the same
orientation of BMS-631707, it would occupy the additional
space of the “diphenyl pocket” calculated by VOIDOO,
protruding toward H11 and H12. Moreover, in PPARR, it
would be missing the strong sulfur-arene interactions that could
orient the terminal ring between H3 and the loop 11/12, given
that there is the A454 residue in place of PPARγ M463 and
also H274 in place of Q283.
To test whether a possible occupation of the entire
“diphenyl pocket” could be realized by S-2 and S-4 at the
PPARR-LBD, the binding mode of both compounds was
investigated by docking experiments. To this end, again, the
crystal structure of PPARR/BMS-631707 was employed.
The ligand-receptor complexes were predicted through the
automated docking software GOLD 4.0,39,40 which in sev-
eral studies was shown to yield better performances com-
pared to other similar programs.41-44 The GoldScore-CS
docking protocol45 was adopted in this study. In this proto-
col, the poses obtained with the original GoldScore function
are rescored and reranked with the GOLD implementation
Dual Agonism of S-2 and S-4. The design of these two
compounds as well as the other ligands of the series has been
made with the aim to prepare analogues of 1 that could
occupy the entire “diphenyl pocket” protruding toward
H11 and H12. The superposition of the crystal structures
of the complexes made by the PPARγ-LBD with the three
compounds S-2, S-4, and 1 (Figure 3) shows that, despite the
greater flexibility of the diphenyl-methylene and diphenyl-
ethylene moieties of S-2 and S-4, respectively, these groups
occupy the same portion of the cavity as the diphenyl group
of 1, between H3 and the M463 of the loop 11/12.
Figure 4A shows the possible accommodation of the distal
aromatic ring of S-2, realized by changing the torsional
angles between the two aromatic rings, into the additional
space of the “diphenyl pocket”, calculated by VOIDOO, a
program for computing molecular volumes and for studying
cavities in proteins.35 The possibility of this different posi-
tioning of S-2 has been confirmed by molecular modeling
calculations. The preference of the terminal aromatic rings
for adopting the conformations observed in the crystal
structures might be ascribed to the strong electrostatic
interactions between the π-cloud of the distal aromatic ring
and M463 of the loop 11/12 and Q286 of H3. In particular,
sulfur-arene interactions are known to be strongly attrac-
tive.36,37 The partial agonism of both compounds toward
PPARγ is in accordance with the hypothesis that ligands that

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6387
Figure 6. Effects of in vitro application of 1, S-2, S-4, clofibric acid, and fenofibric acid on gCl of rat EDL muscle. All compounds were tested
at 100 μM concentration. Each bar represents the mean ( SEM of gCl from 10-36 fibers. Student’s t test showed significant differences
between all treated groups and the control group (P < 0.02 or less).
Figure 7. Dose-dependent effects of 1 and S-2 on gCl of rat EDL muscle. Each bar represents the mean ( SEM of gCl from 6-36 fibers.
Student’s t test showed significant differences between all treated groups and the control group (P < 0.05 or less).
of the ChemScore function.45,46 When S-2 and S-4 were
docked within the PPARR binding site, about 60% of the
conformations generated by GOLD adopted only one highly
conserved orientation. Figure 5A shows the putative orien-
tation of S-2 into the hydrophobic pocket of PPARR in
comparison with the structure of the complex PPARγ/S-2.
The ligand assumes a position very similar to that of BMS-
631707, with the distal aromatic ring approaching helix
11 through vdW interactions with V444 and K448; at the
same time, this ring interacts with the residues A454, A455,
and L456 of the loop 11/12. The stabilization of H11, the
loop 11/12, and indirectly, H12, upon ligand binding could
explain the nanomolar activity of S-2 and its character of full
agonist toward PPARR. It is interesting to notice that the
terminal ring of S-2 occupies, as expected, part of the
additional volume of the diphenyl pocket, calculated by
VOIDOO. Additional substitutions on this ring could better
fill this volume with a further stabilization of H11 and H12.
The preparation of S-2 analogues characterized by the
presence on this phenyl of groups with different stereoelec-
tronic properties is under way.
Figure 5B shows S-4 docked into the LBD of PPARR. The
comparison of this structure with that of the complex
PPARγ/S-4 evidence a similar position of the terminal
aromatic ring between H3 and the loop 11/12, with vdW
interactions with A454 and A455 of this loop and with the
residues V270, H274, and Q277 of H3. However, the shifted
position in PPARR toward H11 of the ethylene linker
between the two phenyl groups permits the interaction of
this moiety with the residues V444 and I447 of this helix. This
could explain the almost full agonism of S-4 toward PPARR,
with a small loss of potency and efficacy due to the maintained

6388 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Fracchiolla et al.
contacts with the H3. Also, this case reinforces the hypo-
thesis that a stabilization of H3 could attenuate the full
agonist character of a ligand.
activityon PPARγ. Forthe most interestingstereoisomersS-2
and S-4, X-ray studies in PPARγ and docking studies in
PPARR provided a molecular explanation for their different
behavior as full and partial agonists of PPARR and PPARγ,
respectively. Moreover, the blocking activity of these two
ligands on skeletal muscle chloride conductance, which is
related to myopathies frequently observed in the treatment
with lipid-lowering drugs, resulted in a profile more beneficial
than fibrates currently used in therapy. The high potency on
PPARRaswellasthe partialagonism on PPARγ andthepoor
activity on skeletal muscle function allow, therefore, the claim
that these new molecules may represent the leads of a poten-
tially safer class of drugs with better therapeutic effects in the
treatment of type 2 diabetes.
Effects of 1, S-2, and S-4 on Skeletal Muscle Chloride
Conductance. Lipid-lowering drugs show diffuse myopa-
thies more frequently than other drugs.29 They start with
myalgia, malaise, and muscle tenderness, followed by ele-
vated plasma levels of muscular enzymes.47 Extreme fatigue
may also be a leading symptom, and pain may be present or
absent. Rhabdomyolysis is the most feared side effect in the
clinical use of lipid lowering medications, particularly of
statins. These, either alone or particularly when associated
with fibrates, may give rise to significant muscle necrosis.48
Since the first description of muscular lesions after clofi-
brate therapy,49 myopathies have been attributed to all
lipid-lowering drugs: nicotinic acid,50 fibrates,51-53 and
statins.54 Myopathy as adverse drug reaction of lipid-low-
ering drugs has been characterized biochemically and mor-
phologically, and there exist a few mechanistic hypo-
theses.29 Our previous studies allowed the identification
of the resting chloride conductance (gCl), sustained by the
voltage-gated chloride channel ClC-1, as a cellular target of
statins and fibrates action.30 ClC-1 is a muscle-specific
channel that controls membrane electrical stability and
functional processes such as excitation and contraction. A
reduced ClC-1 function causes disorders related to abnor-
mal action potential firing. Patch clamp studies have
demonstrated a direct inhibition of human ClC-1 channel
expressed in cultured HEK cells by fenofibric acid.55 On
this basis, we decided to test 1, S-2, and S-4 to evaluate the
possibility that these fibrate-like drugs were able, in the
same way, to interfere with the chloride ion flux of skeletal
muscle membrane.
The effects of in vitro application of these compounds on
gCl of rat extensor digitorum longus (EDL) muscle are
shown in Figure 6 in comparison with clofibric acid and
fenofibric acid, active metabolites of clofibrate and fenofi-
brate, respectively. In this experiment, we tested all com-
pounds at 100 μM concentration and found that 1 and S-2
were poorly effective, producing only a 12% and 9% de-
crease of gCl, respectively, similar to fenofibric acid (11%).
S-4 exhibited a little higher potency, producing a 24% block
of gCl similar to 28% block due to clofibric acid. In a second
experiment, we evaluated the effects on gCl produced from
lower doses of 1 and S-2.
As shown in Figure 7, the block activity of the former
vanished at 5 μM (∼2%), whereas the latter was basically
inactive even at 50 μM (∼2%). All compounds were ineffec-
tive on resting potassium conductance (gK). These data
indicate that our compounds are potentially much safer than
fibrates currently used in therapy. In fact, even though
similar in blocking activity of gCl at the tested concentra-
tions, 1, S-2, and S-4 display a potent PPAR agonist activity
at much lower doses than those of clinically used fibrates.
S-2, particularly, is about 3000 and 500 times as potent as
fenofibric acid on PPARR and PPARγ, respectively (EC50 of
fenofibric acid: 30 μM on PPARR and 300 μM on PPARγ).12
Experimental Section
Chemical Methods. Column chromatography was performed
˚
on ICN silica gel 60 A (63-200 μm) as a stationary phase.
Melting points were determined in open capillaries on a Gal-
lenkamp 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 chem-
1
station. H NMR spectra were recorded in CDCl3 (the use of
DMSO-d6 or CD3OD as a solvent is specified) on a Varian-
Mercury 300 (300 MHz) spectrometer at room temperature
(20 ꢀC). Chemical shifts are expressed as parts per million (δ).
For optical isomers, MS and 1H NMR spectra are reported only
for the racemate or one of the two enantiomers. 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. Only for final compounds 4, S-4, and 12
the data related to carbon are þ0.42%, -0.43%, and -0.45%,
respectively. Optical rotations were measured with a Perkin-
Elmer 341 polarimeter at room temperature (20 ꢀC): concentra-
tions are expressed as g (100 mL)-1. The enantiomeric excesses
of acids were determined by HPLC analysis of their methyl
esters, obtained by reaction with a solution of diazomethane in
Et2O, on Chiralcel OD column (4.6 mm i.d. ꢁ 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 detector, a series 200 model pump, and NCI 900 model
interface. Chemicals were obtained from Aldrich (Milan, Italy),
Lancaster (Milan, Italy), or Acros (Milan, Italy) and were used
without any further purification.
Preparation of 4-(Benzylthio)phenol. A solution of benzylbro-
mide (1.35 g, 7.9 mmol) in CHCl3 (20 mL) was added dropwise
to a mixture of 4-mercaptophenol (1.0 g, 7.9 mmol) and Et3N
(1.2 g, 11.85 mmol). The reaction mixture was stirred at reflux
for 1.5 h, then washed with NH4Cl saturated solution. The
organic layer was dried over Na2SO4, filtered, and evaporated to
dryness, affording the title compound as a yellow solid in
quantitative yield. This product was used in the next step with-
out any further purification.
General Procedure for the Preparation of Ethyl 2-(4-Substi-
tuted-phenoxy)-3-phenyl-propanoates (20-27, R- andS-20, R- and
S-22 in Scheme 1 and 28 in Scheme 2). A solution of diisopro-
pylazodicarboxylate (DIAD, 10 mmol) in anhydrous THF or
toluene (20 mL) was added dropwise to an ice-bath cooled
mixture of ethyl phenyllactate (10 mmol), the appropriate
phenol (10 mmol), and triphenylphosphine (10 mmol) dissolved
in the same anhydrous solvent (50 mL). The reaction mixture
was stirred at room temperature overnight, under N2 atmo-
sphere. The organic solvent was evaporated in vacuo, and a
mixture of Et2O and n-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 chromatographed on
a silica gel column (petroleum ether/ethyl acetate 9:1 as eluent),
Conclusion
In conclusion, the introduction of a linker into the diphenyl
system of compound 1, previously reported as a PPARR/γ
dual agonist, allowed the identification of new ligands with
improved potency on PPARR and unchanged partial agonist

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6389
affording the desired compounds as oils or white solids in
32-77% yields.
Both enantiomers of compounds 20 and 22 were prepared
with the same procedure starting from R- or S-ethyl phenyllac-
tate in 35-42% yields.
Ethyl 2-(4-Benzyl-phenoxy)-3-phenyl-propanoate (20). Color-
less oil; 32% yield.
solution of 29 (0.28 mmol) in anhydrous THF (4 mL) was added
dropwise to the reaction mixture, which was refluxed for 5 h. After
distilling off the organic solvent, the residue was treated with ice and
extracted with Et2O. The organic layer was washed with NH4Cl
saturated solution, 0.5 N NaOH and brine, dried over Na2SO4, and
filtered. The solvent was evaporated in vacuo to give a residue, which
was chromatographed on a silica gel column (petroleum ether/ethyl
acetate 95:5 as eluent), affording the desired compounds as pale-
yellow oils.
Ethyl 2-(4-Phenoxy-phenoxy)-3-phenyl-propanoate (21). Yellow
oil; 69% yield.
Ethyl 2-(4-trans-Styryl-phenoxy)-3-phenyl-propanoate (22).
White solid; 38% yield.
Ethyl 2-(4-Phenoxymethyl-phenoxy)-3-phenyl-propanoate. Yield
67%.
Ethyl 2-(4-Benzyloxy-phenoxy)-3-phenyl-propanoate (23). White
solid; 34% yield.
Ethyl 2-(4-Phenylthiomethyl-phenoxy)-3-phenyl-propanoate.
Yield 47%.
Ethyl 2-(4-Benzylthio-phenoxy)-3-phenyl-propanoate (24). Color-
less oil; 45% yield.
Ethyl 2-(4-Benzylamino-phenoxy)-3-phenyl-propanoate (25).
Yellow oil; 33% yield.
Ethyl 2-(4-Phenylacetyl-phenoxy)-3-phenyl-propanoate (26).
Colorless oil; 60% yield.
Ethyl 2-(4-Iodo-phenoxy)-3-phenyl-propanoate (27). White
solid; 77% yield.
Preparation of Ethyl 2-(4-Phenylaminomethyl-phenoxy)-3-
phenyl-propanoate (Scheme 2, Step e). Aniline (0.217 g, 2.3 mmol)
and sulfamic acid (0.224 g, 2.3 mmol) were added to a solution of
28 (0.841 g, 2.80 mmol) in CH3OH (20 mL). After 0.5 h,
(Ph3P)2CuBH4 (1.53 g, 2.5 mmol) was carefully added to the
mixture. After 15 h at room temperature, the solvent was
removed under reduced pressure and the residue dissolved in
ethyl acetate. The organic phase was washed with 1 N HCl, 0.5 N
NaOH, and with brine, then was dried over Na2SO4 and filtered.
The solvent was evaporated to dryness, affording an oily residue
which was chromatographed on a silica gel column (petroleum
ether/ethyl acetate 90:10 as eluent) to give the desired compound
as a yellow oil (1.26 mmol) in 56% yield.
Preparation of Ethyl 2-(4-cis-Styryl-phenoxy)-3-phenyl-pro-
panoate (Scheme 2, Step f). To a stirred solution of benzyl-
triphenylphosphoniumchloride(3.01 g, 7.72 mmol) in anhydrous
CH3CN (15 mL) a solution of 28 (1.54 g, 5.15 mmol) in
anhydrous CH3CN (12 mL) was carefully added, under N2
atmosphere. After 0.5 h, a solution of DBU (1.19 g, 7.72 mmol)
in anhydrous CH3CN (13 mL) was added to the reaction mixture,
which was refluxed for 5 h. The solvent was removed under
reduced pressure and the residue dissolved in ethyl acetate. The
organic phase was washed with NH4Cl saturated solution and
twice with brine, then was dried over Na2SO4 and filtered. The
solvent was evaporated to dryness, affording an oily mixture of
the twostereoisomers whichwerechromatographedona silica gel
column (petroleum ether/ethyl acetate 98:2). The title compound
was obtained as a yellow oil in 19% yield.
Preparation of Ethyl 2-[4-(3-Phenyl-propyl)phenoxy]-3-phen-
yl-propanoate (Scheme 2, Steps g and h). Following the same
procedure reported above, the reaction of 28 with phenethyl-
triphenylphosphonium bromide afforded cis- and trans-ethyl
2-[4-(3-phenyl-propenyl)phenoxy]-3-phenyl-propanoates in 26%
yield. The resultant mixture of stereoisomers (0.505 g, 1.32
mmol) was dissolved in THF (25 mL) and added to a suspension
of Wilkinson catalyst (0.070 g) in abs EtOH (20 mL). The
mixture was stirred at room temperature under H2 atmosphere
(8 atm). After 6 h, the suspension was filtered through a celite
pad to remove the catalyst and the filtrate was concentrated
under reduced pressure to give a brown oil which was chroma-
tographed on a silica gel column (petroleum ether/ethyl acetate
80:20 as eluent), affording the title compound as a colorless oil
(0.503 g, 1.30 mmol) in 98% yield.
Preparation of the Final Acids 2-15. General Procedure
(Schemes 1 and 2). A solution of the corresponding ethyl ester
(5 mmol) in THF (30 mL) and 1 N NaOH (30 mL) was stirred for
2-24 h at room temperature. The organic solvent was removed
under reduced pressure, and the aqueous phase was acidified
with 6 N HCl and extracted with ethyl acetate. The combined
organic layers were dried over Na2SO4 and evaporated to
dryness, affording the final acids in quantitative yields as white
solids or colorless oils.
Ethyl 2-(4-Formyl-phenoxy)-3-phenyl-propanoate (28). Color-
less oil; 68% yield.
Preparation of Ethyl 2-(4-Phenethyl-phenoxy)-3-phenyl-pro-
panoate (Scheme 1, Step c). A solution of 22 (0.280 g, 0.75 mmol)
in THF (20 mL) was added to a stirred suspension of Wilkinson
catalyst (35 mg, 0.0375 mmol) in abs EtOH (2 mL). The resulting
mixture was stirred at room temperature under H2 atmosphere
(4 atm) for 20 h. The suspension was filtered through a celite pad
to remove the catalyst and the solvent was evaporated to
dryness, obtaining a dark solid residue, which was chromato-
graphed on a silica gel column (petroleum ether/ethyl acetate
95:5 as eluent), affording the desired compound as a colorless oil
in 70% yield. Both enantiomers of the title compound were
prepared with the same procedure starting from R- or S-22 in
70-73% yields.
Preparation of Ethyl 2-(4-Phenylethynyl-phenoxy)-3-phenyl-pro-
panoate (Scheme 1, Step d). A mixture of 27 (0.930 g, 2.35 mmol),
phenylacetylene (0.300 g, 2.82 mmol), PdCl2(PPh3)2 (0.045 g,
0.07 mmol), and tetrabutylammonium fluoride hydrate (1.84 g,
7.05 mmol) was refluxed, under N2 atmosphere, for 17 h. Then,
Et2O (10 mL) and H2O (10 mL) were added to the mixture and
the organic phase was washed with brine, dried over Na2SO4,
and filtered. The solvent was evaporated to dryness, affording a
brown oil residue, which was chromatographed on a silica gel
column (petroleum ether/ethyl acetate 95:5 as eluent) to give the
desired compound as a yellow oil in 15% yield.
Preparation of 29 (Scheme 2). NaBH4 (0.145 g, 3.85 mmol)
was added to a stirred and ice-bath cooled solution of 28 (1.044 g,
3.50 mmol) in THF (40 mL) and H2O (40 mL). The reaction
mixture was stirred for 0.5 h at 0 ꢀC and, after distilling off the
organic solvent, the aqueous residue was added with H2O and ice
then extracted with Et2O. The organic phase was washed with
10% Na2CO3 and brine. The organic solvent was dried over
Na2SO4, filtered, and evaporated in vacuo, affording the ethyl
2-(4-hydroxymethyl-phenoxy)-3-phenyl-propanoate as a pale-yel-
low oil in 91% yield. GC/MS, m/z (%): 300 (73) [Mþ], 135 (100)
[C9H11Oþ]. PBr3 (0.810 g, 3.00 mmol) was carefully added to this
intermediate (0.901 g, 3.00 mmol) at 0 ꢀC. The reaction mixture
was stirred for 5 h at room temperature, then poured onto ice and
extracted with Et2O. The organic layer was washed with brine,
dried over Na2SO4, and filtered. The solvent was evaporated to
dryness, affording a solid residue which was chromatographed on
a silica gel column (petroleum ether/ethyl acetate 90:10 as eluent)
to give the title compound in 66% yield.
Preparation of Ethyl 2-(4-Phenoxymethyl-phenoxy)- and 2-(4-
Pheylthiomethyl-phenoxy)-3-phenyl-propanoates (Scheme 2, Step c).
An ice-bath cooled suspension of NaH (95% powder, 1.1 mmol) in
anhydrous THF (6 mL) was carefully added with phenol or
thiophenol (1.1 mmol) under nitrogen atmosphere. After 0.5 h, a
2-(4-Benzyl-phenoxy)-3-phenyl-propanoic Acid (2). Yield 48%.
(S)-2-(4-Benzyl-phenoxy)-3-phenyl-propanoic Acid (S-2). Yield
32%.
(R)-2-(4-Benzyl-phenoxy)-3-phenyl-propanoic Acid (R-2). Yield
56%.

6390 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Fracchiolla et al.
2-(4-Phenoxy-phenoxy)-3-phenyl-propanoic Acid (3). Yield
94%.
2-(4-Phenethyl-phenoxy)-3-phenyl-propanoic Acid (4). Yield
74%.
(S)-2-(4-Phenethyl-phenoxy)-3-phenyl-propanoic Acid (S-4).
Yield 40%.
(R)-2-(4-Phenethyl-phenoxy)-3-phenyl-propanoic Acid (R-4).
Yield 40%.
2-(4-Benzyloxy-phenoxy)-3-phenyl-propanoic Acid (5). Yield
58%.
2-(4-Phenoxymethyl-phenoxy)-3-phenyl-propanoic Acid (6).
Yield 67%.
2-(4-Benzylthio-phenoxy)-3-phenyl-propanoic Acid (7). Yield
67%.
2-(4-Phenylthiomethyl-phenoxy)-3-phenyl-propanoic Acid (8).
Yield 52%.
2-(4-Benzylamino-phenoxy)-3-phenyl-propanoic Acid (9). Yield
24%.
2-(4-Phenylaminomethyl-phenoxy)-3-phenyl-propanoic Acid
(10). Yield 77%.
2-(4-trans-Styryl-phenoxy)-3-phenyl-propanoic Acid (12). Yield
68%.
2-(4-Phenylethynyl-phenoxy)-3-phenyl-propanoic Acid (14).
Yield 70%.
Ligand and Protein Setup. The core structures of compounds
S-2 and S-4 were constructed using standard bond lengths and
bond angles of the MOE fragment library. The carboxylate
group was taken as dissociated. Geometry optimizations were
accomplished with the MMFF94X force field, available within
MOE. The coordinates of PPARR in complex with the R/γ dual
agonist BMS-631707 (PDB code 2REW),38 recovered from
Brookhaven Protein Database,63 were used in the docking
experiments. Bound ligand and water molecules were removed.
A correct atom assignment for Asn, Gln, and His residues was
done, and hydrogen atoms were added using standard MOE
geometries. Partial atomic charges were computed by MOE
using the AMBER99 force field. All heavy atoms were then
fixed, and hydrogen atoms were minimized using the AM-
BER99 force field and a constant dielectric of 1, terminating
-1
at a gradient of 0.001 kcal mol-1
A
.
˚
Docking Simulations. Docking of BMS-631707, S-2 and S-4
to PPARR was performed with GOLD 4.0 version,39,40 which
uses a genetic algorithm for determining the docking modes of
ligands and proteins. An advantage of GOLD over other dock-
ing methods is the program’s ability to account for some
rotational protein flexibility as well as full ligand flexibility.
Specifically, OH groups of Ser, Thr, and Tyr, and amino groups
of Lys are allowed to rotate during docking to optimize
H-bonding to the ligand. GOLD requires a user-defined binding
site. It searches for a cavity within the defined area and considers
all the solvent-accessible atoms in that area as active-site atoms.
The fitness score function that was implemented in GOLD
(GoldScore) is made up of four components that account for
protein-ligand binding energy: protein-ligand hydrogen bond
energy (external H-bond), protein-ligand van der Waals energy
(external vdW), ligand internal vdW energy (internal vdW), and
ligand torsional strain energy (internal torsion). Parameters
used in the fitness function (hydrogen bond energies, atom radii
and polarizabilities, torsion potentials, hydrogen bond direc-
tionalities, and so forth) are taken from the GOLD parameter
file. The fitness score is taken as the negative of the sum of the
energy terms, so larger fitness scores indicated better bindings.
The fitness function has been optimized for the prediction of
ligand binding positions rather than the prediction of binding
affinities, although some correlation with the latter can be also
found.45 The protein input file may be the entire protein
structure or a part of it comprising only the residues that are
in the region of the ligand binding site. In the present study,
GOLD was allowed to calculate interaction energies within a
2-[4-(3-Phenyl-propyl)phenoxy]-3-phenyl-propanoic Acid (15).
Yield 57%.
Compounds 11 and 13 were tested as sodium salts, obtained
after reaction of the corresponding acid (1 mmol) with NaHCO3
(1 mmol) in 95% EtOH (10 mL) and water (1 mL). Then the
solvent was distilled off and the resulting white solid was washed
many times with Et2O.
2-(4-Phenylacetyl-phenoxy)-3-phenyl-propanoic Acid Sodium
Salt (11). Yield 89%.
2-(4-cis-Styryl-phenoxy)-3-phenyl-propanoic Acid Sodium
Salt (13). Yield 67%.
Protein Expression, Purification, and Crystallization. The
LBD of PPARγ was expressed as N-terminal His-tagged protein
using a pET28 vector and purified as previously described.56
Crystals of apo-PPARγ were obtained by vapor diffusion at
20 ꢀC using a sitting drop made by mixing 2 μL of protein
solution (15 mg/mL, in 20 mM Tris, DTT 5 mM, 0.5 mM
EDTA, pH 8.0) with 2 μL of reservoir solution (0.8 M NaCi-
trate, 0.15 mM Tris, 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 dissolved in
DMSO were diluted in the storage solution so that the final
DMSO concentration was 0.5%. The storage solution with
glycerol 20% (v/v) was used as cryoprotectant. Crystals of
PPARγ/S-4 and PPARγ/S-2 belong to the space group C2 with
cell parameters shown in Table 1 of the Supporting Information.
The asymmetric unit is formed by one homodimer.
Structure Determination. X-ray data were collected at 100 K
under a nitrogen stream using synchrotron radiation (beamline
ID14-2 at ESRF, Grenoble). The diffracted intensities were
processed using the programs MOSFLM and SCALA57 for
both complexes. Refinements were performed with CNS58 using
the coordinates of PPARγ/126 (PDB code 3B3K) as a starting
˚
sphere of a 18 A radius centered on the OH atom of Y464 in the
PPARR structure. The Goldscore-CS docking protocol45 was
adopted in this study. To perform a thorough and unbiased
search of the conformation space, each docking run was allowed
to produce 200 poses without the option of early termination,
using standard default settings. With the aim to test the validity
of the Goldscore-CS docking protocol for the human PPARR
system, BMS-631707, the cocrystallized ligand, was first docked
back into its binding site. In this docking run, the 200 poses
produced by GOLD resulted in only one prevailing cluster
on the basis of their conformations: 90 of the poses closely
resembled the cocrystallized conformation with a heavy atom
˚
˚
model. All data between 8-2.6 A were included for PPARγ/S-4
root-mean-square deviation (rmsd) ranging from 0.4 to 1.4 A.
˚
(between 8-2.1 A, for PPARγ/S-2). The statistics of crystal-
ChemScore was able to rank 30 out of the 90 poses from this
cluster as the highest ranked 13 poses. Thus, this docking
protocol was considered to be suitable for the subsequent
docking runs for compounds S-2 and S-4.
The top solution obtained after reranking of the poses with
ChemScore (ChemScore fitness = 45.9 kJ mol-1 for S-2 and
44.4 kJ mol-1 for S-4) was selected to generate the PPARR/
ligand complexes.
Energy Refinement of the Ligand/Receptor Complexes. To
eliminate any residual geometric strain, the obtained complexes
were energy minimized for 5000 steps using combined steepest
descent and conjugate gradient methods until a convergence
lographic data and refinement are summarized in Table 1 of the
Supporting Information. The coordinates of PPARγ/S-2 and
PPARγ/S-4 complexes have been deposited in the Brookhaven
Protein Data Bank (PDB) with the codes 3HOD and 3HO0,
respectively.
Computational Chemistry. Molecular modeling and graphics
manipulations were performed using the molecular operating en-
vironment (MOE)59 and UCSF-CHIMERA software packages,60
running on a 2 CPU (PIV 2.0-3.0 GHZ) Linux workstation.
Energy minimizations were realized by employing the AMBER 9
program,61 selecting the Cornell et al. force field.62

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6391
value of 0.001 kcal mol-1
A
-1. Upon minimization, the protein
parameter measured in chloride free solution was the potassium
conductance (gK). The mean chloride conductance (gCl) was
estimated as the mean gm minus the mean gK. The compounds
1, S-2, S-4, fenofibric acid, and clofibric acid, dissolved in
dimethyl sulfoxide (DMSO), were applied in vitro on muscle
bath, and gm and gK were measured before and 30 min after
addition of increasing concentrations of each compound. The
maximal concentration of DMSO used (0.5%) was without
effect on the parameters studied.
˚
backbone atoms were held fixed. Geometry optimizations were
performed using the SANDER module in the AMBER suite of
programs, employing the Cornell et al. force field to assign
parameters for the standard amino acids. General AMBER
force field (GAFF) parameters were assigned to ligands, while
the partial charges were calculated using the AM1-BCC method
as implemented in the ANTECHAMBER suite of AMBER.
Biological Methods. Medium, other cell culture reagents, and
Wy-14,643 were purchased from Sigma (Milan, Italy). BRL
49653 (rosiglitazone) was obtained by Hefei Scenery Chemical
Co. (Hefei, Anhui, People’s Republic of China). Clofibric acid
and fenofibric acid were obtained by hydrolysis of fenofibrate
and clofibrate (Sigma Aldrich, Italy), respectively.
Plasmids. The expression vectors expressing the chimeric
receptors containing the yeast GAL4-DNA binding domain
fused to the human PPARR or 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.64
Acknowledgment. This work was accomplished thanks to
the financial support of the Ministero dell’Istruzione,
ꢀ
dell’Universita e della Ricerca (MIUR 2005033023) and Uni-
versita degli Studi di Bari (Research Fund 2008).
ꢀ
Supporting Information Available: Spectroscopic data for
intermediates and final compounds; statistics of crystallo-
graphic data and refinement; figures. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
Cell Culture and Transfections. Human hepatoblastoma cell
line HepG2 (Interlab Cell Line Collection, Genoa, Italy) was
cultured in minimum essential medium (MEM) containing
10% of heat-inactivated foetal bovine serum, 100 U penicillin
G mL-1, and 100 μg streptomycin sulfate mL-1 at 37 ꢀC in a
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3
3
humidified atmosphere of 5% CO2. For transactivation assays,
105 cells per well were seeded in a 24-well plate and transfections
were performed after 24 h with CAPHOS (Sigma, Milan, Italy),
a calcium-phosphate method, according to the manufacturer’s
guidelines. Cells were transfected with expression plasmids
encoding the fusion protein GAL4-PPARR LBD or GAL4-
PPARγ LBD (30 ng), pGAL5TKpGL3 (100 ng), pCMVβgal
(250 ng). Four hours after transfection, cells were treated for
20 h with the indicated ligands in triplicate. Luciferase activity in
cell extracts was then determined by a luminometer (VICTOR3
V Multilabel Plate Reader, PerkinElmer). β-Galactosidase acti-
vity was determined using β-D-galactopyranoside (Sigma, Milan,
Italy) as described previously.65 All transfection experiments were
repeated at least twice.
Electrophysiological Recordings of Resting Membrane Chlor-
ide Conductance. The electrophysiological recordings were done
in vitro on the extensor digitorum longus (EDL) muscle dis-
sected from adult Wistar male rats (Charles River Laboratories,
Calco, Italy) under urethane anesthesia (1.2 g kg-1 ip). Soon
after the dissection, rats still anaesthetized were euthanized with
a urethane overdose. All experimental protocols were carried
out in accordance with the Guide for the care and use of
laboratory animals. The EDL muscles were immediately placed
in a 25 mL bath chamber, maintained at 30 ꢀC, and perfused
with normal or chloride-free physiological solution (gassed with
95% O2 and 5% CO2; pH=7.2-7.3).30 The normal (chloride
containing) physiological solution had the following composi-
tion (in mM): 148 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 12 NaHCO3,
0.44 NaH2PO4, 5.5 glucose. The chloride free solution was made
by equimolar substitution of methylsulfate salts for NaCl and
KCl and nitrate salts for CaCl2 and MgCl2. Using the two-
intracellular-microelectrodes technique, in current clamp mode,
the membrane resistance (Rm) and the fiber diameter were
calculated. These parameters were obtained by injecting a hyper-
polarizing constant square-wave current pulse (100 ms duration)
into the muscle fiber through the current electrode and by
recording the resulting voltage deflection with a second micro-
electrode inserted at two distances from the current electrode.
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