Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity

Article abstract of DOI:10.1021/jm201306q

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the obs

Full text of DOI:10.1021/jm201306q

Article
pubs.acs.org/jmc
Synthesis, Characterization and Biological Evaluation of
Ureidofibrate-Like Derivatives Endowed with Peroxisome
Proliferator-Activated Receptor Activity
L. Porcelli,†,○ F. Gilardi,‡,▽,○ A. Laghezza,§ L. Piemontese,§ N. Mitro,‡ A. Azzariti,† F. Altieri,∥
L. Cervoni,∥ G. Fracchiolla,§ M. Giudici,‡ U. Guerrini,‡ A. Lavecchia,⊥ R. Montanari,# C. Di Giovanni,⊥
A. Paradiso,† G. Pochetti,# G. M. Simone,† P. Tortorella,§ M. Crestani,*,‡,○ and F. Loiodice*,§,○
†Laboratorio di Oncologia Sperimentale Clinica, Istituto Tumori “Giovanni Paolo II”, 70124 Bari, Italy
‡Dipartimento di Scienze Farmacologiche, Universita
̀
degli Studi di Milano, 20133 Milano, Italy
§Dipartimento Farmaco-Chimico, Universita
∥Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Universita
̀
degli Studi di Bari “Aldo Moro”, 70126 Bari, Italy
̀
di Roma “La Sapienza”, 00185 Roma, Italy
⊥Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
#Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00015 Monterotondo Stazione, Roma, Italy
̀
di Napoli “Federico II”, 80131 Napoli, Italy
S
* Supporting Information
ABSTRACT: A series of ureidofibrate-like derivatives was prepared and
assayed for their PPAR functional activity. A calorimetric approach was used to
characterize PPARγ−ligand interactions, and docking experiments and X-ray
studies were performed to explain the observed potency and efficacy. R-1 and
S-1 were selected to evaluate several aspects of their biological activity. In an
adipogenic assay, both enantiomers increased the expression of PPARγ target
genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In
vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a
high fat diet reduced visceral fat content and body weight. Examination of
different metabolic parameters showed that R-1 and S-1 are insulin sensitizers.
Notably, they also enhanced the expression of hepatic PPARα target genes
indicating that their in vivo effects stemmed from an activation of both PPARα
and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in
colon cancer cell lines was also evaluated.
dyslipidemia, atherosclerosis, and cardiovascular diseases.7,8
This receptor subtype is the target of the thiazolidinedione class
INTRODUCTION
PPARs are ligand-dependent transcription factors belonging to
the nuclear receptor superfamily. They control the expression of
genes involved in fatty acid and glucose metabolism and function as
■
of antidiabetic agents (TZDs). TZDs are PPARγ agonists
whose insulin-sensitizing action is well established.9 However,
as full agonists they also stimulate adipocyte differentiation in
cellular lipid sensors that activate transcription in response to the
vitro and weight gain in vivo, which normally aggravates the
binding of a cognate ligand, generally fatty acids and their
eicosanoids metabolites.1−3 As ligand-dependent receptors, PPARs
diabetic state. Additional undesirable side effects associated
with TZD treatment include fluid retention, edema/hemodilu-
form heterodimers with the Retinoid X Receptor (RXR) and adopt
tion, cardiomegaly, anemia, and increased incidence of bone
an active conformation in the presence of a ligand. Additional
fractures.10,11 As a result of the clinical observations mentioned
coregulator proteins are recruited to create a complex that binds to
above, emphasis has shifted to the development of partial
Peroxisome Proliferator Response Elements (PPRE) in target
genes and regulates their expression.4−6
Three main PPAR subtypes have been identified: PPARα
(NR1C1), PPARβ (also known as PPARδ) (NR1C2), and
PPARγ (NR1C3). PPARα is mainly expressed in the liver and
activates a genetic program leading to fatty acid β-oxidation.
PPARβ is more ubiquitously distributed and regulates cellular
functions such as fatty acid catabolism in the skeletal muscle,
agonists or selective PPARγ modulators (SPPARγMs). The
SPPARM approach has recently attracted considerable
attention because it proposes that diverse PPAR ligands,
depending on their chemical structures, would bind in a distinct
manner to PPARs inducing different levels of activation and
distinct conformational changes of the receptor, leading to
differential interactions with coactivators and corepressors.
wound healing, and inflammation. PPARγ plays important roles
in the differentiation and functions of adipocytes and
macrophages with a direct impact on type 2 diabetes,
Received: May 4, 2011
Published: November 14, 2011
© 2011 American Chemical Society
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Structurally diverse modulators or partial agonists, therefore,
are likely to elicit different pharmacological and toxicological
effects depending on the context of tissue, i.e., abundance of
cofactor proteins and target gene. This may enable uncoupling
of the benefits of PPAR activation from the adverse effects
associated with full agonism. In agreement with the SPPARγM
concept, a number of these modulators have already
demonstrated desirable pharmacological profiles in various
rodent models with significantly reduced side effects relative to
those generally observed with existing full agonists.12−24
In a previous work,25 we reported a structural study on two
enantiomeric ureidofibrate-like derivatives (Figure 1) complexed,
activity of these compounds toward PPARγ, as well as PPARα
and PPARβ/δ, was determined by the transactivation assay in
transiently transfected human hepatoblastoma cell line HepG2 or
the monkey kidney cell line COS-1. Moreover, we decided to use
a calorimetric approach (ITC, DSC) to characterize PPARγ−
ligand interactions including the binding affinity. As far as we
know, this is the first case in which calorimetric techniques have
been applied to a series of structurally related PPARγ ligands for
the determination of the thermodynamic parameters associated
with the formation of the receptor−ligand complexes. On the basis
of these results, docking experiments and X-ray studies were
performed to provide a molecular explanation for their different
potency and efficacy. In addition, we evaluated several aspects of
the activity of R-1 and S-1. First of all, we tested the adipogenic
activity of R-1 and S-1 in 3T3-L1 adipocytes and then explored
their in vivo pharmacological properties in a mouse model of
obesity and insulin resistance to study the activity of these
compounds on typical PPARα and PPARγ targets.
Finally, since a large body of evidence shows that PPARγ
ligands exert antitumorigenic effects against a wide variety of
neoplastic cells both in vivo and in vitro,27−31 we evaluated the
capability of the two enantiomers R-1 and S-1 to inhibit cellular
proliferation in colon cancer cell lines. However, we found that
the antiproliferative activity exhibited by these ligands was only
partially related to PPARγ activation.
Chemistry. The synthesis of compounds 2−5, which
followed the procedure previously reported for the preparation
of R-1 and S-1,26 is depicted in Scheme 1 and involved the key
amine intermediates 2a−5a whose condensation with hepta-
noic acid in the presence of hydroxybenzotriazole (HOBT) and
N,N-diisopropylcarbodiimide (DIC) afforded the correspond-
ing amide intermediates 2b−5b. The amide group of these
intermediates was reduced with 1 M borane in THF solution
to give the corresponding amines. The condensation with
2-chloro-benzoxazole, followed by saponification of the ester
function, led to the final acids 2−5. All acids, except 2, were
obtained as colorless oils, which were transformed into sodium
(R-3, S-3, and 4) or cyclohexylamine salts (R-5 and S-5) before
undergoing the biological assay. The synthetic pathways of key
amine intermediates 2a−5a are reported in Scheme 2. The
synthesis of 2a involved the ethyl ester intermediate 2d, which
was prepared by reaction of 4-bromo-phenol with acetone in
the presence of CHBr3 and KOH. The condensation of this
compound with N-vinylphthalimide in the presence of
Pd(AcO)2, tri-o-tolylphosphine and N,N-diisopropylethylamine
in anhydrous CH3CN, followed by hydrogenation at 4 atm in
the presence of Wilkinson catalyst, provided compound 2e.
The hydrazinolysis of the phthalimide moiety led to the desired
amine intermediate. Following the same procedure, both
enantiomers of compound 5a were prepared starting from
R- and S-5d, which were obtained from the condensation of
4-bromo-phenol with S- or R-methylphenyllactate, respectively,
under Mitsunobu conditions. The amines 3a and 4a were
prepared starting from the commercially available tiramine,
which was condensed with carbobenzyloxychloride in the
presence of K2CO3 to afford the N-carbobenzyloxytiramine.
The condensation of this intermediate with R- or S-ethyl-lactate
under Mitsunobu conditions afforded S- or R-3d, respectively,
whereas its condensation with ethyl 2-bromoacetate in the
presence of 95% NaH powder in anhydrous DMF provided the
intermediate 4d. The hydrogenation of S- or R-3d and 4d at 5
atm in the presence of 10% Pd/C in EtOH led to the desired
amines.
Figure 1. Ureidofibrate-like derivatives of the present study.
respectively, with the ligand binding domain (LBD) of PPARγ.
The R-enantiomer, R-1, behaves as a full agonist of PPARγ
whereas the S-enantiomer, S-1, is a less potent partial agonist.
Comparing the X-ray structures of the two complexes we argued
that the partial agonist behavior of S-1 could be ascribed to a
destabilization of the active conformation of helix 12 (H12). In
particular, we showed that the suboptimal conformation of H12,
observed in the PPARγ/S-1 complex, is probably due to a steric
hindrance between the ethyl group, linked to the asymmetric
carbon atom of the ligand, and the crucial residue Q286 of PPARγ,
situated on helix 3 (H3). The importance of the residue Q286
on the transcriptional activity of the receptor was tested by
site-directed mutagenesis which confirmed its key role in the
stabilization of helix 12.26 The functional relevance of this residue
in determining the receptor activity of these enantiomeric
ureidofibrate-like derivatives, prompted us to investigate the
possibility to fine tune the activity of these ligands by modifying
the substituents bound to the stereogenic center at the α position
of the carboxylic group. In this work we report, therefore, the
synthesis and PPARγ functional activity of derivatives 2−5
(Figure 1) in which shorter or longer substituents were introduced
in place of the methyl and/or ethyl of R-1 and S-1. The functional
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a
Scheme 1. Synthesis of PPAR Agonists 2−5
a
(i) Heptanoic acid, HOBT, DIC, CH2Cl2; (ii) 1 M BH3 in THF; (iii) 2-chloro-benzoxazole, (Et)3N, THF; (iv) 1 N NaOH, EtOH, rt; (v) NaHCO3
(for compounds 3 and 4) or cyclohexylamine (for compound 5), EtOH, rt.
a
Scheme 2. Synthesis of Key Amine Intermediates 2a−5a
a
(i) CHBr3, KOH; (ii) MeOH, H2SO4 cat.; (iii) DIAD, Ph3P, anhydrous toluene; (iv) N-vinylphthalimide, Pd(AcO)2, tri-o-tolylphosphine, N,N-
diisopropylethylamine, CH3CN; (v) H2 (4 atm), Wilkinson cat., EtOH, rt; (vi) N2H4·xH2O, EtOH; (vii) Cbz-Cl, K2CO3, EtOH/H2O; (viii) ethyl
2-bromoacetate, 95% NaH, anhydrous DMF; (ix) R- or S-methyllactate, DIAD, Ph3P, anhydrous toluene; (x) H2 (5 atm), 10% Pd/C, EtOH.
Both enantiomers of acids 3 and 5 had enantiomeric excesses
≥95% as determined by HPLC analysis of the chiral stationary
phase (see Supporting Information).
and about five times higher than that of S-1. The introduction
of a hydrogen in place of the same ethyl group gave compound
3 whose enantiomers showed stereoselective activity with R-3
about 15 times more potent than S-3, even though it was 2
times less potent than R-1. A quite good activity was obtained
also with the achiral compound 4 characterized by the presence
of a methylene between the phenolic oxygen and the carboxylic
function; this analogue, in fact, was only 11 times less potent
than R-1. Finally, the two stereoisomers of compound 5 were
tested; this derivative was investigated by analogy with previously
reported phenoxyalkanoic acid PPAR agonists bearing a benzyl
group at α position of the carboxylic group.32−35 Both R-5 and S-5
displayed a fairly good potency although with the lowest efficacy of
the series. For this compound, a small stereoselectivity favorable to
the S-isomer was observed.
RESULTS AND DISCUSSION
■
PPAR Activity. Compounds 2−5 were evaluated first for
their agonist activity on the human PPARγ (hPPARγ) sub-
type in comparison with R-1 and S-1. For this purpose, the
GAL4-PPARγ chimeric receptor was expressed in transiently
transfected HepG2 cells according to a previously reported
procedure.32 The results obtained are reported in Table 1 together
with corresponding data for rosiglitazone used as a reference
compound in the transactivation assay. The maximum induction
obtained with the reference agonist was defined as 100%.
Interestingly, all new ligands were PPARγ activators even
though with an intrinsic activity significantly lower than that of
the full agonist R-1. The substitution of the ethyl group bound
to the stereogenic center of 1 with a methyl afforded the achiral
compound 2, which exhibited potency similar to that of R-1
In order to obtain a more complete pharmacological character-
ization, compounds 2−5 were evaluated also for their agonist
activity on human PPARα and PPARβ/δ subtypes. R-1 and S-1
were assayed only on PPARα given their previously reported
inactivity on the PPARβ/δ subtype.25 The results obtained are
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a
Table 1. Activity of the Tested Compounds in Cell-Based Transactivation Assay
PPARα
PPARγ
PPARδ
compd
R-1
EC50 (μM)
Emax (%)
EC50 (μM)
Emax (%)
EC50 (μM)
ia
Emax (%)
b
b
0.003 0.001
0.056 0.034
0.030 0.016
0.025 0.017
0.620 0.260
0.122 0.058
1.720 0.950
ia
91
75
82
93
78
87
26
ia
3
4
7
6
4
7
7
0.07 0.05
0.59 0.11
0.11 0.06
0.15 0.06
2.30 1.00
0.80 0.40
2.70 1.00
1.70 0.80
ia
116
50
9
ia
b
b
S-1
5
4
2
5
ia
ia
2
50
0.09 0.05
83 24
41 10
R-3
59
0.13 0.09
S-3
32
0.85 0.14
35
7
4
37 11
23
0.57 0.07
40 11
R-5
9
ia
ia
ia
ia
ia
ia
ia
ia
S-5
31 21
ia
Wy-14,643
rosiglitazone
L-165,041
1.6 0.3
100 10
ia
ia
ia
0.02 0.01
ia
100 21
ia
ia
0.021 0.01
100
4
a
b
Efficacy values were calculated as a percentage of the maximum obtained fold induction with the reference compounds. These values correspond
to those previously obtained as reported in ref 25.
provided in Table 1 together with corresponding data for pirinixic
acid (Wy-14,643) and 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-
propoxy]phenoxyacetic acid (L-165,041) used as reference
compounds for PPARα and PPARβ/δ, respectively. Interestingly,
compounds 2−4 turned out to be pan-agonists basically displaying
on both of these receptors the same rank order of activity and
stereoselectivity shown for PPARγ. They acted as potent full
agonists on PPARα, as well as R-1 and S-1, whereas they exhibited
lower potency and efficacy on PPARβ/δ. On the contrary, R-5 was
inactive on PPARβ/δ while displaying a moderate activity on
PPARα; last, S-5 behaved as the only selective PPARγ agonist of
the series. Thus, all of the fibrate analogues (except for S-5) were
more potent on PPARα than PPARγ. It is worth noting that
PPARβ/δ activity was shown only from the compounds with no or
small substituents in the α position of the carboxylic group
confirming what is already reported in the literature for related
fibrate analogues. No PPARβ/δ activity, instead, was observed for
derivatives 1 and 5 endowed with bulkier substituents in the same
position.
Figure 2. DSC thermograms of PPARγ-LBD in the presence and
absence of the ligands of the present study.
The next step was the determination of thermodynamic
parameters relating to the formation of the complexes PPARγ-
LBD/ligand by isothermal titration calorimetry (ITC). This
technique, by measuring the heat absorbed or released by
titrating the protein with a ligand at constant temperature,
yields the stoichiometry of the reaction, the binding enthalpy,
and the affinity constant.
The result of a typical ITC experiment is shown in Figure 3
for binding of R-3. In the upper panel, the heat pulses observed
upon addition of the ligand to PPARγ are plotted as a function
of the injection order. The area of the pulses decreases
progressively until it reaches a constant value due to complete
saturation of the receptor binding sites. The lower panel shows
the integrated heats of reaction plotted against the molar ratio
of total ligand concentration to total oligomeric protein con-
centration, and, superimposed, the simulated curve obtained by
the best fitting of the data according to the “one binding site”
model. The stoichiometry of ligand binding was generally as
expected, with the low accuracy of some results probably due to
errors in the active protein concentration.
Calorimetric Measurements. To investigate the possibil-
ity of using the calorimetric approach for the characterization of
protein−ligand interactions of PPAR ligands, we decided to use
calorimetric techniques to measure the thermodynamic
parameters associated with the formation of the complexes
between ligands 1−4 and PPARγ. Compounds R-5 and S-5
were not tested because of the very low solubility of their
cyclohexylamine salts in the buffer used for the experiments.
Figure 2 shows the differential scanning calorimetry (DSC)
thermograms of PPARγ-LBD in the presence and absence of
the ligands. In these curves, the heat capacity is reported as a
function of temperature with the peaks corresponding to the
protein denaturation. The experimental data showed that
ligand binding, with the only exception of compound 4, shifted
the denaturation peak by about 2−5 °C. As thermal
denaturation was not reversible under these conditions, the
DSC thermograms did not yield useful values of ΔH; however,
they provided useful hints on the degree of global stabil-
ization of the ligand binding domain of PPARγ from the tested
ligands. As known, a global stabilization of PPARγ-LBD also
has a favorable effect on the transactivation activity because of
the indirect stabilization of the activation−function helix 12.
Interestingly, the full agonist R-1 stabilized the receptor better
than the reference compound rosiglitazone, even though this
did not turn out to be beneficial for increasing the potency.
Table 2 shows that both ΔH and TΔS values for tested
compounds 1−4 were favorable to the binding to PPARγ
isoform. The association of the ligands was enthalpy-driven at
25 °C, showing that van der Waals interactions and H-bonds
played an important role in the binding. The only exception
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As far as the binding affinity was concerned, the results (Kd)
for R-1, S-1, and rosiglitazone were in good accordance with
the previously reported data of Ki obtained by the scintillation
proximity assay (SPA), that is, 0.088 μM, 0.971 μM, and
0.074 μM, respectively.25 Importantly, the affinity data for the
enantiomers of compound 3 confirmed a stereoselectivity
favorable to the R-isomer. Thus, the ITC technique could
represent a very useful alternative to SPA given that it avoids
the use of radio-ligands and is less costly.
Molecular Modeling and X-ray Studies. The analysis of
the binding affinity of achiral compounds 2 and 4 showed that
their Kd values did not correlate well with the activity. Ligand 2,
in particular, was one of the most potent agonists of the series
despite its low affinity. To gain more details on the interactions
of PPARγ with the partial agonists S-1, 2, and 4 endowed with
different potency, an approach that combined molecular
docking and X-ray studies was performed.
To this end, the crystal structure of the PPARγ complexed
with the ligand 2 was solved (PDB code: 3R8I) and
successively compared to that of the complexes PPARγ/R-125
(PDB code: 2I4J) and PPARγ/S-125(PDB code: 2I4P).
Interestingly, the analysis of the structures revealed three
different orientations of the carboxylate group that can be
ascribed only to the differences of the substituents on the
carbon atom in α position. As illustrated in Figure 4a, both
oxygens of the carboxylate group of 2 are involved in H-bonds
with Y473, H323, and H449. This arrangement is more similar
to the canonical H-bonding network realized by the potent full
agonist R-1 (Figure 4b), which also has both oxygens engaged in
H-bonds with the triad. On the contrary, only one oxygen of S-1
(Figure 4c) is engaged in H-bonds with the above residues because
of the steric clash between the ethyl group on the asymmetric
carbon atom and the residue Q286 on helix 3 that forced the ligand
to move away from helix 12 provoking a distorted and less
efficacious interaction with this helix.25 Moreover, 2 lacks the
H-bond with S289, whereas its benzoxazole N atom establishes a
H-bond with C285 of helix 3. A water molecule creates a H-bridge
between the benzoxazole O atom and the NH backbone of S342
belonging to the β-sheet. Further hydrophobic contacts with the β-
sheet of the receptor are realized by the long aliphatic chain of 2.
To predict the plausible interactions between compound 4
and PPARγ, molecular docking studies were carried out using
the crystal structure of PPARγ/2 complex. The ligand−receptor
complex was predicted through the automated docking
software GOLD 5.0.1,36 which in several studies was shown
to yield better performances compared to those of other similar
programs.37 The GoldScore-CS docking protocol was adopted
in this study.38 In this protocol, the poses obtained with the
original GoldScore function were rescored and reranked with
the GOLD implementation of the ChemScore function.38,39
Figure 3. Binding of R-3 to PPARγ-LBD. The upper panel shows the
raw data of a representative ITC experiment. The lower panel shows
the corresponding binding isotherm fitted according to the “one
binding site” model.
was S-1 whose enthalpic term was notably lower than that of
R-1. This was in accordance with the crystal structures of the
corresponding complexes of PPARγ with these two enantiomers,
where the less active S-enantiomer realized less productive
interactions with the protein because of a steric clash of its ethyl
group with a residue of the helix 3.25 The more disordered
conformation of S-1, observed in the crystal structure, was also in
accordance with the higher value of the entropic term obtained
by ITC that partly compensated the low enthalpy value.
From Table 2, it is possible to observe, across the series, that the
entropic term decreases by reducing the size of the substituents at
the α position of the carboxylic group. This effect, known as
“hydrophobic effect”, was due to the decreasing buried surface of
contact between the C-alpha substituents and the surface of the
protein resulting in a less favorable entropic contribution.
Table 2. Thermodynamic Parameters Relating to the Formation of the Complexes PPARγ-LBD/Ligand Determined by the ITC
a
Assay
ligand
R-1
n
Kd (μM)
ΔG (kcal/mol)
ΔH (kcal/mol)
TΔS (kcal/mol)
0.92 0.01
0.57 0.07
1.08 0.04
0.22 0.01
0.31 0.01
0.63 0.03
0.43 0.01
0.27
2
−8.9
−7.8
−7.3
−8.0
−7.5
−7.0
−4.3 0.07
−1.1 0.04
−4.1 0.02
−4.5 0.19
−5.8 0.02
−4.6
−6.7
−3.2
−3.5
−1.7
−1.3
−4.4
S-1
2
4.5
1.5
3.3
7.7
0.12
R-3
S-3
4
−5.7 0.04
b
rosiglitazone
−9.4
−5.0
0
a
b
n = molar binding ratio of the ligand−protein interaction (observed stoichiometry). This value was kept fixed during the fitting by Origin.
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Figure 4. H-bond network of compounds 2 (a, green), R-1 (b, white), and S-1 (c, yellow) in the crystal complex with PPARγ represented as a slate
blue ribbon model. (d) Compound 4 (magenta) docked into the PPARγ binding site. Only amino acids involved in the H-bonding network with the
ligand are displayed (white) and labeled. H-bonds discussed in the text are depicted as dashed black lines.
To test the validity of this protocol for the PPAR system, the
crystallized conformation of ligand 2 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: 43 of the poses closely resembled the
crystallized conformation with a heavy atom root-mean-square
deviation (rmsd) ranging from 0.9 to 2.3 Å. ChemScore was
able to rank 25 out of the 43 poses from this cluster as the
highest ranked 25 poses. Figure 1 of Supporting Information
shows the comparison between the predicted docked
conformation of 2 and the one observed in the crystal structure
(rmsd = 1.91 Å). Thus, this docking protocol was considered to
be suitable for the subsequent docking runs for compound 4.
When 4 was docked within the PPARγ binding site, about
Figure 5. Superposition of the crystal structures of PPARγ in complex
with R-1 (white), S-1 (yellow), and 2 (green).
70% of the conformations generated by GOLD adopted only
one highly conserved orientation lying in the same region
occupied by R-1, S-1, and 2. As can be seen in Figure 4d, ligand
4 loses the H-bond with both H323 and S289 side chains as its
carboxylate group assumes a different conformation with
respect to 2, R-1, and S-1. Moreover, the long aliphatic chain
and the benzoxazole ring of the ligand occupy the upper and
lower parts of the distal cavity, respectively, there making
hydrophobic contacts with the surrounding protein residues. In
particular, the benzoxazole moiety of the ligand contacts the
side chain of I341 belonging to the β-sheet.
As reported in Table 1, the potency of 2 is higher than that of
both S-1 and 4 on the same receptor. This behavior can be
interpreted at the molecular level by the more efficient H-
bonding network realized by the carboxylate of 2, with
particular regard to H12. A similar arrangement has been
observed in the crystal complex of PPARγ with the more potent
R-125 (Figure 4b). As shown in Figure 5, R-1 and 2 interact in a
similar way with Y473 of H12, while S-1 forces the side-chain of
the tyrosine to assume a different orientation, probably less
efficacious for a good stabilization of this helix.25 On the
contrary, the less potent S-1 and 4, forming a less effective
H-bonding network, weakly contribute to the H12 stabilization.
As far as the affinity is concerned, a good correlation with the
potency can be observed in the case of the chiral compounds,
where the R-enantiomers show higher affinity and potency with
respect to the S-enantiomers. This correlation is not so evident
for the achiral compounds 2 and 4, where the higher potency,
compared to that of other compounds of the series, could
depend on the better stabilization of H12, as seen in the crystal
structure of 2. As a general rule, the affinity across the series is
also driven by the “hydrophobic effect” which affects the
entropic term. Moreover, our molecular modeling and X-ray
studies show that the partial agonism of the ligands S-1, 2, and
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4 toward PPARγ is in accordance with a better stabilization of
H3 and/or the β-sheet, as discussed extensively in previous
works.25,26
(NEFA), glucose, and insulin were reduced with both R-1
and S-1 (Figure 8). Cholesterol levels were not affected by any
of the tested ligands (data not shown). The changes of the
metabolic profile elicited by the two enantiomers indicated that
these ligands might be insulin sensitizers in vivo. To test this
hypothesis, we carried out the oral glucose tolerance test and
the insulin tolerance test, two standard assays widely used to
assess insulin resistance and basal insulin sensitivity by
following the evolution of glucose levels after a glucose load
or insulin injection, respectively.45 We found that mice on R-1
were able to improve the glucose clearance upon an oral load as
compared to control (HFD) mice (Figure 9A and inset).
Conversely, the glucose clearance following the oral load in
mice on S-1 was slightly improved but did not reach the
statistical significance (Figure 9A and inset). Similarly, the
insulin-induced increase in glucose disposal rate was signifi-
cantly higher in mice treated with R-1, suggesting increased
insulin sensitivity in this group of mice, while the curve with the
S-enantiomer was improved, but the difference with the control
group was not statistically significant (Figure 9B and inset). It
should be mentioned, however, that S-1, although to a lesser
extent than R-1, reduced the circulating levels of glucose and
insulin in fasted mice, reflecting the beneficial outcome of
PPARγ stimulation in vivo with the S-enantiomer. In fact, the
calculation of the homeostatic model assessment of insulin
resistance (HOMA-IR), an index of insulin resistance, shows
that R-1 and S-1 decreased insulin resistance (Figure 9C)
consistent with the reduced plasma levels of glucose and insulin
achieved with these two PPARγ ligands (Figure 8). Notably, by
using a combination of structural studies with techniques to
examine the selective coregulator recruitment, we have recently
shown that S-1 is a partial agonist of PPARγ able to recruit a
different set of coregulators as compared to the R-
enantiomer.26 On the basis of these molecular approaches,
we proposed that this ligand may be a SPPARγM. Yet, since
S-1, as well as R-1, also activates PPARα, it is likely that the
pharmacological profile obtained with these two ligands may
arise from the combined action on these two receptor subtypes.
It should be mentioned that, unlike selective PPARγ agonists,
it is well-known that dual PPARα/γ agonists do not show
weight gain in rodent models of diabetes.46 It will be interesting
in the future to test in cell and animal models whether S-1
is able to uncouple the improved metabolic profile from the
side effects usually observed in patients treated with
thiazolidinediones.47
Adipogenic and in Vivo Activities. We first verified
whether R-1 and S-1 were able to induce adipocyte
differentiation of 3T3-L1 mouse fibroblasts, as PPARγ is a
key determinant in this process. As shown in Figure 6A, both
R-1 and S-1 increased the expression of PPARγ target genes
like fatty acid binding protein 4 (Fabp4), the insulin sensitive
glucose transporter (Glut4), and adiponectin (Acrp30), which
are typically increased in differentiated adipocytes and
represent adipose markers. Furthermore, we observed that
these ligands induced lipid accumulation as a consequence of
differentiation to adipocytes (Figure 6C). In particular, the
quantitation of lipid content confirmed that cells differentiated
in the presence of R-1 or S-1 and accumulated lipids to a level
similar to that observed with the classical differentiation cocktail
containing insulin, dexamethasone and IBMX or with the
PPARγ full agonist rosiglitazone (Figure 6B).
Having ascertained that both R-1 and S-1 induced the
adipogenic program in a PPARγ-dependent manner, we
decided to explore the in vivo pharmacological properties by
administering the two ligands to a mouse model of insulin
resistance induced by the diet. Mice were fed a high fat diet for
16 weeks and then were treated with R-1 (10 mg/kg/day), S-1
(25 mg/kg/day), or the PPARγ agonist rosiglitazone (10 mg/
kg/day) for 2 weeks. At the end of the treatment, the body
weight of mice treated with R-1 was significantly decreased
(12% reduction) as compared to control mice (HFD) (Figure 7A).
The body weight of mice on rosiglitazone was similar to that of
HFD mice, while the decrease of body weight of mice treated
with S-1 (7% reduction) did not reach the statistical signifi-
cance (Figure 7A). The analysis of fat deposits revealed that the
visceral white adipose tissue (WAT) of mice administered with
R-1 or S-1 was dramatically reduced as determined by magnetic
resonance imaging (MRI), whereas rosiglitazone was not
effective on this parameter (Figure 7B and C). In this respect,
the decreased body weight observed with R-1 and the reduced
visceral fat obtained with both the R- and S-enantiomers
represent an advantage versus rosiglitazone, whose admin-
istration is usually associated with increased adipogenesis and
body weight in diabetic patients.40 In a Gal4-based assay, we
had previously shown that both R-1 and S-1 are dual PPARα/γ
ligands;25 therefore, to confirm their behavior as PPARα ligands
we measured the expression of target genes in the liver of these
mice and found that the mRNA levels of the mitochondrial
medium-chain acyl-CoA dehydrogenase (Acadm), long-chain
acyl-CoA dehydrogenase (Acadl), and of the peroxisomal acyl-
Coenzyme A oxidase 1 (Acox1) were significantly increased
by both enantiomers (Figure 2A of Supporting Information).
In parallel, the liver weight of mice treated with R-1 and S-1
rose as compared to that of the control mice (Figure 2B of
Supporting Information). This effect is typically observed in
rodents treated with fibrates and PPARα agonists as confirmed
in the group of mice on fenofibrate (Figure 2B of Supporting
Information).41,42
Effects on Cell Proliferation. Next step in this study was
to evaluate the antitumor effects of R-1 and S-1 in colon cancer
cell lines. We chose these compounds with the aim of
examining if there was difference, with regard to this type of
activity, between full and a partial PPARγ agonists. In fact,
clinical trials on the full agonists TZDs as antineoplastic agents
have shown, so far, conflicting results, justifying the need to
further investigate the anticancer potential of PPARγ agonists.48
The first step was the Western blot analysis of PPARγ
expression in our in vitro panel. The three cell lines utilized in
this study (HT-29, LoVo, and HCT15) showed highly
expressed PPARγ. This expression profile was in support of
the use of these cells for our study and confirmed the existence
of the PPARγ signaling system in such malignant cells
(Figure 10). The capability of R-1 and S-1 to inhibit tumor
cell growth was analyzed using the full PPARγ agonist
troglitazone as a reference compound. All cell lines were
incubated for 1 and 2 days with each compound at various
High body weight and increased content of visceral fat have
been recognized as risk factors for insulin resistance.43,44 The
improvement of morphometric parameters and the decreased
visceral fat observed in mice treated with the two enantiomers
suggest that the metabolic profile of these mice could have
benefited from the administration of R-1 or S-1. In fact, the
levels of circulating triglycerides, nonesterified fatty acids
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Figure 6. Expression of PPARγ target genes and lipid accumulation in murine adipocytes. (Panel A) 3T3-L1 mouse fibroblasts were differentiated in
the presence of the indicated treatments. Total RNA was extracted, and the expression of Fabp4, Glut4 and Acrp30 was measured by real time
qPCR. ctrl, control samples; Ins, samples treated with 5 μg insulin·mL−1; I/D/I, samples treated with 10 μg insulin·mL−1, 1 μM dexamethasone, and
0.5 mM IBMX; Rosi, 1 μM rosiglitazone; R-1, 1 μM R-1; S-1, 1 μM S-1. Data are expressed as the mean SD of triplicate samples. (Panel B)
Spectrophotometric quantification of lipid content after solvent extraction of Oil Red O from mouse adipocytes differentiated in the presence of the
indicated treatments. Results are expressed as the mean SD (n = 4). (**) and (***) indicate statistical significance at p < 0.01 and p < 0.001,
respectively. (Panel C) 3T3-L1 mouse fibroblasts were differentiated to adipocytes in the presence of the indicated treatments. Cells were stained
with Oil Red O, and pictures were taken with an Axiovert 200 microscope at 20× magnification.
concentrations (range 1−100 μM); the cell growth inhibition
was analyzed by the MTT assay, and the IC50 was determined.
A cell proliferation inhibition in dose- and time-dependent
manner was observed. After one day of drug exposure, a cell
growth inhibition was obtained with IC50 higher than 100 μM
(data not shown). The inhibitory effects were significantly
44
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Figure 7. Effect of PPARγ ligands on body weight and visceral fat content in HFD fed mice. Six weeks old C57Bl/6J male mice were fed a high fat
diet for 16 weeks and subsequently were treated with the indicated ligands for two weeks as detailed in the Experimental Section. (Panel A) Body
weight of mice at the end of the treatments (6 mice/treatment group). (Panel B) Quantification of visceral fat by MRI of total body fat; results are
expressed as the percentage of visceral fat area on the total image (the MRI analysis was performed on 3 mice/treatment group). (Panel C)
representative image of MRI performed on mice treated with the indicated ligands. (**) and (***) indicate statistical significance at p < 0.01 and
p < 0.001, respectively.
Figure 8. Plasma levels of glucose, lipids, and hormones in HFD mice treated with PPARγ ligands. The quantification of plasma levels of triglycerides
(panel A), NEFA (panel B), glucose (panel C), and insulin (panel D) was performed on the same mice described in Figure 7 treated with the
indicated ligands. (*), (**), and (***) indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively.
increased when cells were treated for 2 days; in this case, in all cell
lines IC50 ranged between 17 and 65 μM with R-1 and S-1, which
were more potent than troglitazone on LoVo and HCT15 cells.
As shown in Table 3, however, no significantly different anti-
proliferative activity was observed between the two enantiomers.
To investigate if the cell growth inhibition occurred through
PPARγ activation, tumor cell lines were incubated for 48 h with
each of the enantiomers (IC50) and/or the potent PPARγ
antagonist 6 (GW9662)49 (20 μM). Compound 6 is itself an
inhibitor of cell proliferation, but it acts by a PPARγ-
independent mechanism.50 Cell viability was analyzed by the
MTT assay, and in two of the investigated cell lines, 6 neither
inhibited cell proliferation nor reversed growth inhibition
induced by R-1 and S-1 (data not shown); our ligands, in fact,
still induced cell growth suppression, even though PPARγ
was blocked. In such experimental conditions, only HT-29
45
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Figure 9. R-enantiomer is an insulin sensitizer. (Panel A) Four days before sacrifice, HFD mice treated as indicated in the figure were fasted
overnight and underwent an oral glucose tolerance test. Results are expressed as the mean SE of plasma glucose levels at the indicated time points
in at least five animals/group. Area under the curves (AUC) were calculated and plotted (inset). (Panel B) Two days before sacrifice, HFD mice
treated as indicated in the figure were fasted for 6 h and underwent an insulin tolerance test. Results are expressed as the mean SE of plasma
glucose levels at the indicated time points in at least five animals/group. Area under the curves (AUC) were calculated and plotted (inset). (Panel C)
HOMA-IR index was calculated for the indicated treatments. Results are expressed as the mean SD of at least five animals/group. (*) and (***)
indicate statistical significance at p < 0.05 and p < 0.001, respectively.
(Figure 11A). This data suggests the direct involvement of
PPARγ in mediating the antiproliferative activity of R-1 and S-1
in HT-29 cells. In the case of R-1, however, the complete
recovery of proliferation allows one to hypothesize a
simultaneous interference from R-1 and, partially, S-1 in the
pathway responsible for the cytotoxicity of 6, which, as already
mentioned, acts by a PPARγ-independent mechanism. This
could explain why, under cotreatment conditions, 6 did not
exhibit any growth inhibition. Further investigations in this field
are in progress.
To evaluate which phase all the cell lines investigated were
blocked in, we examined the cell cycle by flow cytometry
Figure 10. PPARγ expression. Protein extracts from LoVo, HT-29,
and HCT-15 cells were analyzed by Western blot to analyze the
expression level of PPARγ. β-Actin expression level was used to
normalize the sample value.
analysis. Compared to control cells, an increase of the G0/G1
phase was observed, as shown in Figure 11B for HT-29, after
treatment with R-1 and S-1, at different times, suggesting that
our ligands reduced tumor cell growth via G0/G1 cell arrest.
Next, we verified if the G0/G1 block in HT-29 was mediated
Table 3. Effectiveness of R-1, S-1, and Troglitazone in Colon
Tumour Cell Lines
IC50 (μM)
by PPARγ activation. Flow cytometry of cell cycle revealed that
the activity of R-1 and S-1 on HT-29 was altered when these
cells were simultaneously treated with the PPARγ antagonist 6.
In particular, the G0/G1 block induced by S-1, which appeared
after 2 days of treatment, was resumed by 6. Differently, R-1
blocked the cells already after 1 day, and this block was
persistent after 48 h. In this case, the presence of 6 allowed the
cells to recover the baseline rate of cell cycle progression after
1 day, whereas no response to 6 was observed after 2 days
compd
S-1
HTC-15
HT-29
LoVo
21 1.2
17 1.6
50.9 1.6
50 3.1
41 3.5
50 2.8
17 3.2
19 2.3
65 4.8
R-1
troglitazone
cells showed about 40% loss of viability in response to 6.
Co-treatment of these cells with each of the enantiomers and 6,
instead, resulted in a recovery of proliferation, and this effect
was much more evident when 6 was added to R-1 than to S-1
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Figure 11. (Panel A) PPARγ activation involvement in cell growth inhibition by R-1 and S-1. HT-29 cells were incubated, for two days, with each of
the enantiomers (IC50) and/or 6 (20 μM), and the cell growth modulation was analyzed by MTT assay as described in the Experimental Section.
(Panel B) Cell cycle modulation. HT-29 cells were incubated, for one and two days, with each of the enantiomers R-1 and S-1 (IC50) and/or 6
(20 μM), and the cell cycle was analyzed by flow cytometry. The reported G0/G1 phase percentages are the means of three different experiments SD.
Significance of the differences was p < 0.05 (*).
under the same conditions suggesting that this effect could be
only partially related to PPARγ activation in this cell line
(Figure 11B).
Finally, we evaluated if R-1 and S-1 could effectively induce
apoptosis in the HT-29 cell line. For this purpose, these cancer
cells were treated with both enantiomers and troglitazone
for 24 and 48 h at the corresponding IC50 concentrations.
Fluorescein isothiocyanate-conjugated annexin V was utilized
to detect the externalization of phosphatidylserine that occurs
at an early stage of apoptosis. Propidium iodide was used as a
marker of necrosis due to cell membrane destruction. As shown
in Figure 12, all the compounds effectively induced apoptosis in
a time-dependent manner.
CONCLUSIONS
Figure 12. Apoptotic effect of S-1 and R-1. HT-29 cells were treated
for 2 days with S-1 or R-1 or troglitazone at the corresponding IC50
concentration. Apoptosis detection was performed by Annexin
V-FITC staining assays and propidium iodide (PI), in accordance
■
A short series of ureidofibrate-like derivatives endowed with
PPARγ activity was prepared by modifying the substituents at α
position of the carboxylic group. The use of calorimetric
techniques (DSC, ITC) turned out to be a valuable approach
with the manufacturer’s instructions followed by FACS analysis. The
histogram represents the mean
SEM of apoptosis rates obtained
for the determination of the thermodynamic parameters
associated with the formation of the receptor−ligand
complexes. The behavior of some compounds of the series
was rationalized by performing docking experiments and X-ray
studies which provided a molecular explanation for their
different potency and efficacy. Different aspects of the
biological activity of the full and partial agonists R-1 and S-1,
respectively, were also evaluated. On the basis of the results
obtained in cultured adipocytes and in HFD mice, we conclude
that the two enantiomers activate PPARγ target genes and have
from three independent experiments.
profound effects on the metabolic profile in vivo. These ligands
also activate PPARα in the liver, and this is likely to contribute
to their pharmacological profile. The two enantiomers
improved morphometric and metabolic parameters and
reduced insulin resistance most likely as a consequence of
decreased body weight and visceral fat, which are usually
associated with insulin resistance and type 2 diabetes.43,44
Furthermore, our data obtained in different colon cancer cell
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(2c−5c). The appropriate amine intermediate was dissolved in THF
(15 mL), cooled to 0 °C, and added with N(Et)3 (0.2 mL) and a
solution of 2-chlorobenzoxazole (2.20 mmol) in anhydrous THF
(5 mL). The resulting reaction mixture was stirred for 0.5 h at 0 °C,
0.5 h at room temperature, 2 h at reflux, and 15 h at room
temperature. The organic solvent was evaporated to dryness affording
a solid residue which was chromatographed on a silica gel column
(petroleum ether/ethyl acetate 9:1 as eluent) to give the title
compounds in 42−71% yield.
Methyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}-2-methyl-propanoate (2c). Colorless oil, 71% yield.
S-Methyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}propanoate (S-3c). Colorless oil, 45% yield.
lines indicate that these two stereoisomers not only potently
inhibit cell growth, even though by a mechanism of action only
partially related to PPARγ activation, but also effectively induce
apoptosis in cancer cells. Collectively, the results of this
preliminary integrated study allow one to claim that R-1 and S-
1 represent dual PPARα/γ ligands with promising biological
effects in cellular systems and in an animal model of insulin
resistance and could stand, therefore, as leads for the
development of new molecules for the treatment of type 2
diabetes and/or cancer.
EXPERIMENTAL SECTION
R-Methyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}propanoate (R-3c). Colorless oil, 42% yield.
Ethyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}ethanoate (4c). Yellow oil, 47% yield.
■
Chemical Methods. Column chromatography was performed on
ICN silica gel 60 Å (63−200 μm) as a stationary phase. Melting points
were determined in open capillaries on a Gallenkamp electrothermal
apparatus and are uncorrected. Mass spectra were recorded with a HP
GC/MS 6890-5973 MSD spectrometer, electron impact 70 eV,
equipped with HP chemstation. For GC/MS analysis of acid analytes,
the corresponding methyl esters, obtained by reaction with a solution
S-Methyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}-3-phenyl-propanoate (S-5c). Colorless oil, 66% yield.
R-Methyl 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}-3-phenyl-propanoate (R-5c). Colorless oil, 61% yield.
Preparation of the Final Compounds 2−5. A solution of the
corresponding alkyl ester 2c−5c (0.85 mmol), obtained from the
previous steps, in EtOH (10 mL) and 1 N NaOH (5 mL) was stirred
for 5 h at room temperature. The organic solvent was removed under
reduced pressure and the aqueous phase was acidified with 2 N HCl
and extracted with ethyl acetate. The organic layer was dried over
Na2SO4, filtered, and evaporated to dryness affording the title
compounds in quantitative yields as a white solid (2) or colorless
oils (4, both enantiomers of 3 and 5). The oily acids were transformed
into the corresponding sodium (R-3, S-3, and 4) or cyclohexylamine
salts (R-5 and S-5). For this purpose, to a solution of the appropriate
acid (1.5 mmol) in 95% EtOH (20 mL) was added NaHCO3 (1.5
mmol) or cyclohexylamine (2.0 mmol). The reaction mixture was
stirred overnight at rt. The solvent was evaporated to dryness to give
the desired salts as white solids, which were recrystallized by the
suitable solvent.
2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]phenoxy}-2-
methyl-propanoic Acid (2). White solid, 52% yield (CHCl3/n-hexane).
(−)-S-Sodium 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-
ethyl)]phenoxy}propanoate (S-3). White solid, 45% yield (CHCl3/
n-hexane).
(+)-R-Sodium 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-
ethyl)]phenoxy}propanoate (R-3). White solid, 49% yield (CHCl3/
hexane).
Sodium 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-yl)amino-ethyl)]-
phenoxy}ethanoate (4). White solid, 34% yield (CHCl3/n-hexane).
(−)-S-Cyclohexylammonium 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-
yl)amino-ethyl)]phenoxy}-3-phenyl-propanoate (S-5). White solid,
29% yield (AcOEt/CHCl3).
(+)-R-Cyclohexylammonium 2-{4-[2-(N-Heptyl-N-(benzoxazol-2-
yl)amino-ethyl)]phenoxy}-3-phenyl-propanoate (R-5). White solid,
70% yield (AcOEt/CHCl3).
Preparation of Methyl 2-(4-Bromo-phenoxy)-2-methyl-
propanoate (2d). A solution of KOH (2.92 g, 5.21 mmol) in
H2O (10 mL) was added dropwise to a solution of 4-bromo-phenol
(1.51 g, 8.68 mmol) in acetone (15 mL). After 0.5 h at room
temperature, a first amount of CHBr3 (0.60 mL) was added dropwise,
during 10 min, to the reaction mixture. After 1 h at room temperature,
a second addition of CHBr3 (1.2 mL) was carried out, during 15 min.
The resulting mixture was stirred overnight at room temperature after
which the organic solvent was distilled off. The aqueous phase was
carefully acidified with 6 N HCl and extracted with ethyl acetate. The
collected organic phase was washed with brine, dried over Na2SO4,
filtered, and evaporated to dryness affording a brown oily residue,
which was dissolved in ethyl acetate and extracted five times with
NaHCO3 saturated solution. The aqueous phase was carefully acidified
with 6 N HCl and extracted four times with Et2O. The collected
organic layer was dried over Na2SO4, filtered, and evaporated to
dryness to give a dark red oily residue, which was chromatographed on
a silica gel column (petroleum ether/ethyl acetate/MeOH 7:2:1 as
1
of diazomethane in Et2O, were used. H NMR spectra were recorded
in CDCl3 on a Varian-Mercury 300 (300 MHz) spectrometer at room
temperature (20 °C). Chemical shifts are expressed as parts per
million (δ). The purity of all tested compounds was >95%, as
confirmed by combustion analysis carried out with an Eurovector Euro
EA 3000 model analyzer. Optical rotations were measured with a
Perkin-Elmer 341 polarimeter at room temperature (20 °C):
concentrations are expressed as g(100 mL)−1. The enantiomeric
excesses of the final acids were determined by HPLC analysis 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 an 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 Methyl or Ethyl 2-[4-(2-Heptanoylamino-
ethyl)phenoxy]-α-substituted-ethanoate (2b−5b). Heptanoic
acid (9.00 mmol), HOBT·xH2O (3.00 mmol), and DIC (11.25
mmol) were added to a solution of the appropriate compound 2a−5a
(6.00 mmol) in CH2Cl2 (50 mL). The reaction mixture was stirred for
15 h at room temperature. The organic phase was washed with
NaHCO3 saturated solution, 1 N HCl, and brine, then dried over
Na2SO4, and filtered. The solvent was evaporated to dryness affording
a yellow solid residue which was chromatographed on a silica gel
column (petroleum ether/ethyl acetate from 8:2 to 3:7 as eluents) to
give the desired compounds in 70−89% yields.
Methyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]-2-methyl-prop-
anoate (2b). Yellow oil, 89% yield.
S-Methyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]propanoate (S-
3b). White solid, 71% yield.
R-Methyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]propanoate
(R-3b). White solid, 73% yield.
Ethyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]ethanoate (4b). Yellow
oil, 86% yield.
S-Methyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]-3-phenyl-
propanoate (S-5b). White solid, 70% yield.
R-Methyl 2-[4-(2-Heptanoylamino-ethyl)phenoxy]-3-phenyl-
propanoate (R-5b). White solid, 71% yield.
Preparation of Methyl or Ethyl 2-[4-(2-Heptylamino-ethyl)-
phenoxy]-α-substituted-ethanoate. One molar BH3 in THF
solution (10 mmol) was added, under N2 atmosphere, to a stirred
solution of the appropriate compound 2b−5b (1.22 mmol) in
anhydrous THF (15 mL). The reaction mixture was stirred overnight
at room temperature, then was carefully added with MeOH (15 mL),
and stirred for 0.5 h at reflux. The organic solvent was evaporated to
give a colorless oily residue in quantitative yield, which was used for
the next step without any further purification.
Preparation of Methyl or Ethyl 2-{4-[2-(N-Heptyl-N-(benzox-
azol-2-yl)amino-ethyl)]phenoxy}-α-substituted-ethanoate
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eluent) affording the desired acid as a pale yellow solid in 70% yield. A
solution of this acid (5 mmol) in MeOH (20 mL) and two drops of
concd H2SO4, was stirred for 3 h at reflux, then the solvent was
distilled off, and the residue was dissolved in ethyl acetate. The
resulting solution was washed with NaHCO3 saturated solution and
brine, then the organic phase was dried over Na2SO4, and the solvent
was removed under reduced pressure to give the title compound as a
pale yellow oil in quantitative yield.
Preparation of S- or R-Methyl 2-(4-bromo-phenoxy)-3-
phenyl-propanoate (5d). A solution of diisopropylazodicarboxylate
(DIAD, 2.81 g, 13.90 mmol) in anhydrous toluene (20 mL) was added
dropwise to an ice-bath cooled mixture of R- or S-methyl phenyllactate
(2.51 g, 13.87 mmol), 4-bromophenol (2.45 g, 13.89 mmol), and
triphenylphosphine (3.64 g, 13.88 mmol) in anhydrous toluene (50
mL). The reaction mixture was stirred at room temperature overnight,
under N2 atmosphere. Toluene was evaporated in vacuo, and a mixture
of Et2O and hexane (50 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) affording the desired
compound as a colorless oil in 76% yields.
Preparation of Methyl 2-[4-(2-Phthalimido-2-yl-ethen)-
phenoxy]-2-methyl- or 3-Phenyl-propanoate. A solution of 2d
or S- or R-5d (6.0 mmol), tri-o-tolylphosphine (0.50 mmol), N-vinyl-
phthalimide (6.0 mmol), and N,N-diisopropylethylamine (9.5 mmol) in
anhydrous CH3CN (15 mL) was added, under N2 atmosphere, to a
suspension of Pd(AcO)2 (0.20 mmol) in the same anhydrous solvent
(3 mL). The reaction mixture was stirred for 24 h at reflux, then the
organic solvent was evaporated in vacuo, and CH2Cl2 (20 mL) was added
to the residue. The precipitate was filtered off through a Celite pad, washed
four times with CH2Cl2 (20 mL), and the filtrate was washed with brine
and dried over Na2SO4. The solvent was evaporated to dryness affording a
yellow solid residue, which was chromatographed on a silica gel column
(petroleum ether/ethyl acetate 9:1 as eluent) to give the title compound as
a yellow solid, in 75−89% yield, which was used in the next step without
any further purification.
Methyl 2-[4-(2-Phthalimido-2-yl-ethen)phenoxy]-2-methyl-prop-
anoate. 89% yield.
S-Methyl 2-[4-(2-Phthalimido-2-yl-ethen)phenoxy]-3-phenyl-
propanoate. 87% yield.
R-Methyl 2-[4-(2-Phthalimido-2-yl-ethen)phenoxy]-3-phenyl-
propanoate. 75% yield.
Preparation of Methyl 2-[4-(2-Phthalimido-2-yl-ethyl)-
phenoxy]-2-methyl or 3-Phenyl-propanoate (2e, S- and R-
5e). A solution of the intermediate obtained in the previous step
(6.0 mmol) in THF (35 mL) was added to a stirred suspension of
Wilkinson catalyst (0.40 mmol) in abs EtOH (5 mL). The resulting
mixture was stirred at room temperature under H2 atmosphere
(4 atm) for 5 h. The suspension was filtered through a Celite pad to
remove the catalyst, and the solvent was evaporated to dryness
providing a dark solid residue which was chromatographed on a silica
gel column (petroleum ether/ethyl acetate 7:3 as eluent), affording the
desired compounds as a yellow solids in 70−87% yield.
S- and R-Methyl 2-[4-(2-Amino-ethyl)phenoxy]-3-phenyl-prop-
anoate (5a). 73% and 79% yields, respectively.
Preparation of Benzyl 4-Hydroxy-phenetyl-carbamate. K2CO3
(23.88 g, 172.8 mmol) and benzylchloroformate (8.2 mL, d =
1.195 g · mL−1, 57.6 mmol) were added to a suspension of tiramine
hydrochloride (10.0 g, 57.6 mmol) in H2O (310 mL) and Et2O (250
mL). The resulting mixture was stirred at room temperature for 4 h.
The organic solvent was separated, washed with brine, dried over
Na2SO4, and evaporated to dryness providing the desired compound
as a white solid in 94% yield.
Preparation of Intermediates S- and R-3d. A solution of
diisopropylazodicarboxylate (DIAD, 3.72 g, 18.42 mmol) in anhydrous
THF (30 mL) was added dropwise to an ice-bath cooled mixture of R-
or S-methyl lactate (1.96 g, 18.41 mmol), benzyl 4-hydroxy-phenetyl-
carbamate (4.31 g, 18.40 mmol), and triphenylphosphine (4.84 g, 18.40
mmol) in anhydrous THF (30 mL). The reaction mixture was stirred at
room temperature for 48 h, under N2 atmosphere. THF was evaporated in
vacuo, and a mixture of Et2O and hexane (50 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 (CHCl3/ethyl acetate 98:2 as eluent) affording the desired
compounds as yellow oils in 50 and 57% yields, respectively.
Preparation of 4d. The benzyl 4-hydroxy-phenetyl-carbamate
(4.01 g, 14.74 mmol) was added to an ice-bath cooled suspension of
95% NaH (1.13 g, 45.83 mmol) in anhydrous DMF (80 mL). The
resulting mixture was stirred at 0 °C for 0.5 h, under N2 atmosphere,
and then was added dropwise with a solution of ethyl 2-bromoacetate
(3.69 g, 22.16 mmol) in anhydrous DMF (20 mL). The reaction
mixture was stirred at room temperature for 24 h, under N2
atmosphere. The organic solvent was evaporated in vacuo, and the
residue was dissolved in ethyl acetate. The solution was washed with
ammonium chloride saturated solution, 0.5 N NaOH, and brine, then
was dried over Na2SO4, and the organic solvent was evaporated to
dryness affording the desired compound as a white solid in 95% yield.
Preparation of Intermediates S- and R-3a and 4a. Ten
percent Pd/C (0.1 mmol) was added to a solution of the appropriate
intermediate S-, R-3d, or 4d, (7.50 mmol) in abs. EtOH (100 mL).
The resulting mixture was stirred at room temperature under H2
atmosphere (5 atm) for 1.5 h. The suspension was filtered through a
Celite pad to remove the catalyst, and the solvent was evaporated to
dryness providing the desired compounds as oils in quantitative yields.
PPAR Activity. Reference compounds, medium, and other cell
culture reagents were purchased from Sigma-Aldrich (Milan, Italy).
Plasmids. The expression vectors expressing the chimeric receptor
containing the yeast Gal4-DNA binding domain fused to the human
PPARα, PPARγ, or PPARβ/δ 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.51
Cell Culture and Transfections. Human hepatoblastoma cell line
HepG2 (for PPARα and PPARγ) or monkey kidney cell line COS-1
(for PPARβ/δ) (Interlab Cell Line Collection, Genoa, Italy) were
cultured in Minimum Essential Medium (MEM, HepG2) or Dulbecco’s
Modified Eagle’s Medium (DMEM, COS-1) containing 10% of heat-
inactivated fetal bovine serum, 100 U penicillin G·mL−1, and 100 μg
streptomycin sulfate·mL−1 at 37 °C in a 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-PPARα LBD or Gal4-PPARγ LBD or Gal4-PPARβ/δ
LBD (30 ng), pGal5TKpGL3 (100 ng), and 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 activity was determined using β-D-galactopyranoside
(Sigma, Milan, Italy) as described previously.52 All transfection experiments
were repeated at least twice.
Methyl 2-[4-(2-Phthalimido-2-yl-ethyl)phenoxy]-2-methyl-propa-
noate (2e). 70% yield.
S-Methyl 2-[4-(2-Phthalimido-2-yl-ethyl)phenoxy]-3-phenyl-
propanoate (S-5e). 87% yield.
R-Methyl 2-[4-(2-Phthalimido-2-yl-ethyl)phenoxy]-3-phenyl-
propanoate (R-5e). 85% yield.
Preparation of Intermediates 2a and Both Enantiomers of
5a. N2H4·xH2O (32 mmol) was added to a solution of 2e or S- or R-5e
(5.25 mmol) in absolute EtOH (40 mL). The reaction mixture was stirred
for 1 h at reflux and overnight at rt. The suspension was filtered, and the
organic solvent was evaporated in vacuo to give a yellow solid which was
dissolved in ethyl acetate. The solution was washed with brine, dried over
Na2SO4, and the organic solvent evaporated to dryness affording the
desired compounds in 73−86% yield as yellow oils. The resulting amines
were used in the next step without any further purification.
Methyl 2-[4-(2-Amino-ethyl)phenoxy]-2-methyl-propanoate
(2a). 86% yield.
49
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Journal of Medicinal Chemistry
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Differential Scanning Calorimetry. DSC experiments were
performed with a MicroCal VP-DSC microcalorimeter (MicroCal
Inc., Northampton, MA, USA). The samples were dialyzed against the
Hepes buffer (Hepes 20 mM, pH 8.0, TCEP 1 mM) and gently
degassed before scanning. The LBD of PPARγ was expressed as the
N-terminal His-tagged protein using a pET28 vector and purified as
previously described.25 The protein concentration was 10 μM, and the
ligand concentration was 20 μM. The concentration of PPARγ (30.58
kDa) was determined spectrophotometrically using the extinction
coefficient E0.1% = 0.341 at 280 nm. The reference cell was filled with
the same solvent mixture as that used for the sample, but lacking the
protein. The experiment was performed ranging from 10 to 100 °C,
and the heating rate was 1 °C·min−1. Thermograms were corrected by
subtracting the instrumental baseline, obtained with both cells filled
with the same solvent, and normalized for protein concentration.
When a post-transitional baseline could be determined, a progress
baseline was subtracted; otherwise, a straight line connecting the initial
and the final temperature of the overall transition was used.53 Tm
(temperature of maximum heat capacity) and ΔH (heat reaction) were
calculated using the Origin 7.0 software provided by MicroCal.
Isothermal Titration Calorimetry. ITC experiments were
performed at 25 °C using a MicroCal ITC200 microcalorimeter
(MicroCal Inc., Northampton, MA, USA). PPARγ was extensively
dialyzed against the buffer of choice (Hepes 20 mM, pH 8.0, TCEP 1
mM, or Tris-HCl 20 mM, pH 8.0, and TCEP 1 mM) with Amicon
Ultra filters, and the final exchange buffer was then used to dilute the
ligand stock solutions (20 or 50 mM in DMSO). DMSO was added to
the protein solution at the same percentage of the ligand solution
(below 5%). Samples were centrifuged before the experiments to
eliminate possible aggregates. Protein and ligand solutions were
degassed before use. Titrations were performed at 25 °C. The protein
solution (30−120 μM) was placed in the sample cell, and the ligand
solution (5−15 times more concentrated than the protein) was loaded
into the syringe injector. The titrations involved 19 injections of 2 μL
at 180 s intervals. The syringe stirring speed was set at 1000 rpm.
Reference titrations of ligands into buffer were used to correct for
heats of dilutions. Buffers of different heats of ionization were used
(Hepes and Tris-HCl), but most experiments were performed at pH
8 in Hepes buffer because of its lower enthalpy of ionization (5.0 and
11.3 kcal·mol−1 for Hepes and Tris-HCl at 25 °C, respectively).54
The thermodynamic data were processed with Origin 7.0 software
provided by MicroCal. The values of ΔH were measured for each
titration, and fitting the binding isotherms with a one-site binding
model yielded the values of the association constant (Ka). The
system also gave information of the change in entropy (ΔS). The
binding free energy (ΔG) and dissociation constant (Kd) were
calculated from the experimentally determined values of ΔH and Ka,
using eqs 1 and 2:
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 AMBER99 force field and a constant dielectric of
1, terminating at a gradient of 0.001 kcal mol−1 Å−1.
Docking Simulations. Docking of 2 and 4 to PPARγ was
performed with GOLD, version 5.0.1,36 which uses a genetic algorithm
for determining the docking modes of ligands and proteins. The
binding site was defined as a 13 Å sphere centered on the OH oxygen
of Y473 in the PPARγ structure. The Goldscore-CS docking protocol
was adopted in this study.38 In this protocol, the poses obtained with
the original GoldScore function were rescored and reranked with the
GOLD implementation of the ChemScore function.38,39 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. The top solution obtained
after reranking of the poses with ChemScore was selected to generate
the PPARγ/ligand complexes.
Protein Expression, Purification, and Crystallization. The
LBD of PPARγ was expressed as N-terminal His-tagged protein using
a pET28 vector and purified onto a Ni2+-nitriloacetic acid column (GE
Healthcare) as previously described.25 Crystals of apo-PPARγ were
obtained by vapor diffusion at 18 °C using a sitting drop made by
mixing 2 μL of protein solution (10 mg·mL−1, in 20 mM Tris and
1 mM TCEP, pH 8.0) with 2 μL of reservoir solution (0.8 M sodium
citrate and 0.15 M Tris, pH 8.0). The crystals were soaked for 8 days
in a storage solution (1.2 M sodium citrate and 0.15 M Tris, pH 8.0)
containing the ligand (0.1 mM). The ligand dissolved in DMSO was
diluted in the storage solution so that the final concentration of
DMSO was 0.5%. The storage solution with glycerol 20% (v/v) was
used as the cryoprotectant. Crystals of PPARγ/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 ID14-1
at ESRF, Grenoble). The diffracted intensities were processed using the
programs MOSFLM and SCALA.57 Structure solution was performed
with AMoRe,58 using the coordinates of PPARγ/R-1 (PDB code: 2I4J) as
a starting model. The coordinates were then refined with CNS.59 All data
between 8−2.3 Å were included. The statistics of crystallographic data and
refinement are summarized in Table 1 of the Supporting Information. The
coordinates of PPARγ/2 have been deposited in the Brookhaven Protein
Data Bank (PDB) with the code 3R8I.
Adipogenesis Assay. The adipogenesis assay was performed as
previously described.60 Briefly, 3T3-L1 cells (American Type Culture
Collection, Manassas, VA) were cultured in DMEM supplemented with
10% bovine calf serum. Two days after reaching confluence, differentiation
was induced in DMEM supplemented with 10% fetal calf serum and 1 μM
dexamethasone, 0.5 mM 3-isobuthyl-1-methylxanthine (IBMX), and 10 μg
insulin·mL−1. After 48 h, cells were supplemented with medium containing
10 μg insulin·mL−1 for an additional four days. Alternatively, cells were
differentiated with medium containing 5 μg insulin·mL−1 and 1 μM
rosiglitazone or the other ligands. Medium was replenished with ligands
every other day. Lipid content was determined by the staining with
Oil Red O. Pictures were taken with an Axiovert 200 microscope at
20× magnification. The quantitation of the intracellular dye was performed
after extraction by isopropanol and by reading the absorbance at 550 nm.
Gene Expression in 3T3-L1 Adipocytes. Total RNA was
extracted from 3T3-L1 adipocytes with Trizol (Sigma, Milano, Italy)
followed by purification on RNeasy cartridges (Qiagen, Milano, Italy)
according to the manufacturer’s instructions. One microgram of total
RNA was used to quantitate the mRNA levels of Fabp4, Glut4, and
Acrp30 by real time qPCR using a one-step kit with Taqman probes (Bio-
Rad Laboratories, Milano, Italy) in a CFX 384 thermal cycler (Bio-Rad).
36B4 was used as the house keeping gene for data normalization. The
primer sequences were mouse Fabp4 forward primer 5′-GGC-
GTGGAATTCGATGAA-3′, mouse Fabp4 reverse primer 5′-GCTTGT-
CACCATCTCGTT-3′, and mouse Fabp4 Taqman probe 5′-TGAT-
GCTCTTCACCTTCCTGTCGT-3′; mouse Glut4 forward primer
ΔG = − RT ln(K ) = ΔH − TΔS
(1)
a
K = 1/K
(2)
d
a
where R is the gas constant (1.987 cal·mol−1·K−1), and T is the
working temperature (298 K). The reported parameters are the
average of triplicate measurements. To correct for any discrepancies
in the baseline outlined by the software, a manual adjustment was
performed. In some cases, the parameter Ka was kept fixed during the
refinement to obtain a best fit, expecially at the beginning of the
curve.
Computational Chemistry. Molecular modeling and graphics
manipulations were performed using the molecular operating
environment (MOE)55 and UCSF-CHIMERA software packages,56
running on a 2 CPU (PIV 2.0−3.0 GHZ) Linux workstation.
Ligand and Protein Setup. The core structures of compounds 2
and 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 PPARγ in complex with 2 (PDB code: 3R8I)
were used in the docking experiments. Bound ligand was removed.
50
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Journal of Medicinal Chemistry
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5′-TGTCGCTGGTTTCTCCAACTG-3′, mouse Glut4 reverse primer 5′-
CCATACGATCCGCAACATACTG-3′, and mouse Glut4 Taqman probe
5′-ACCTGTAACTTCATTGTCGGCATGGGTTT-3′; mouse Acrp30
forward primer 5′-AGGCATCCCAGGACATC-3′, mouse Acrp30 reverse
primer 5′-CCTGTCATTCCAACATCTCC-3′, and mouse Acrp30 Taqman
probe 5′-CCTTAGGACCAAGAAGACCTGCATCTC-3′; mouse 36B4
forward primer 5′-AGATGCAGCAGATCCGCAT-3′, mouse 36B4 reverse
primer 5′-GTTCTTGCCCATCAGCACC-3′, and mouse 36B4 Taqman
probe 5′-CGCTCCGAGGGAAGGCCG-3′.
Animal Studies. Six weeks old C57Bl/6J male mice (at least 6
animals/group) (Charles River Laboratories, Calco, Italy) were fed a high
fat diet containing 45% fat as the calorie source for 16 weeks. PPAR ligands
were administered once a day for 2 weeks by oral gavage in 0.5%
hypromellose (Sigma Aldrich). In a preliminary study, different doses for
each compound were tested, and the optimal dosage for compound R-1
and S-1 was 10 mg/kg/day and 25 mg/kg/day, respectively. Fenofibrate
(100 mg/kg/day) and rosiglitazone (10 mg/kg/day) were used as
reference compounds for PPARα and γ activation, respectively. At sacrifice,
blood was taken for the determination of biochemical parameters and
circulating hormones. Body weight was measured, and the livers of mice
were weighed at the end of the experiment and snap frozen in liquid
nitrogen for gene expression analysis. Total plasma cholesterol,
triglycerides, NEFA, and glucose were measured with standard commercial
kits (for cholesterol, Horiba ABX, Roma, Italy; for triglycerides and
glucose, Sentinel Diagnostics, Milano, Italy; for NEFA Wako, Neuss,
Germany). Insulin levels were determined with ELISA kit (Mercodia,
Uppsala Sweden).
of Acadm, Acadl, and Acox1 by real time qPCR using a one-step kit
with Taqman probes (Bio-Rad Laboratories, Milano, Italy) in a CFX
384 thermal cycler (Bio-Rad). 36B4 was used as house keeping gene for
data normalization. The primer sequences were mouse Acadm forward
primer 5′-ACCCAGATCCTAAAGTACCC-3′, mouse Acadm reverse
primer 5′-CGAAAGCAATTCCTCTGGTG-3′, and mouse Acadm Taq-
man probe 5′-TGGCCCATGTTTAGTTCCTTTTTTCCAA-3′; mouse
Acadl forward primer 5′-GAAACCAGGAACTACGTGAAG-3′, mouse
Acadl reverse primer 5′-GCTGTCCACAAAAGCTCT-3′, and mouse
Acadl Taqman probe 5′-CACACATACAGACGGTGCAGCATA-3′;
mouse Acox1 forward primer 5′-TCACGTTTACCCCGGC-3′, mouse
Acox1 reverse primer 5′-CAAGTACGACACCATACCAC-3′, and mouse
Acox1 Taqman probe 5′-CATCAAGAACCTGGCCGTCTGC-3′; and
mouse 36B4 forward primer 5′-AGATGCAGCAGATCCGCAT-3′, mouse
36B4 reverse primer 5′-GTTCTTGCCCATCAGCACC-3′, and mouse
36B4 Taqman probe 5′-CGCTCCGAGGGAAGGCCG-3′.
Statistical Analyses. Statistical analyses were performed via one-
way ANOVA with post-test analysis for multiple group comparisons,
using GraphPad Prism, version 5.0 (GraphPad Software, San Diego,
CA). Differences with p values less than 0.05 were considered
statistically significant.
Effects on Cell Proliferation. Compound 6 and troglitazone
were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock
solutions of these ligands as well as our ureidofibrate-like compounds
were prepared at 20 mM in DMSO and stored in aliquots at −20 °C.
Cell Lines. Our in vitro model was made up of HT-29 colon
adenocarcinoma, LoVo, and HCT15 metastatic colon adenocarcinoma.
Evaluation of Cytotoxicity. Determination of the IC50 was
performed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide (MTT) assay. On day 1, 10,000 cells/well in a volume of
200 μL were seeded in 96-wells plates. In each plate, one column
contained cells not exposed to drugs (control), and 5 columns
contained cells exposed to increasing concentrations of drugs. Each
drug was repeated in 6 identical wells. On day 2, ligands (1, 5, 10, 50,
and 100 μM) were added with different times of drug exposure (1 and
2 days). For each drug, results were expressed as dose−effect curves
with a plot of the fraction of unaffected (surviving) cells versus drug
concentration. The IC50 value was defined as the drug concentration
yielding a fraction of affected (no surviving) cells = 0.5, compared with
that of untreated controls and calculated using Calcusyn software.
Each experiment was done in triplicate.
Western Blot Analysis. Total proteins were extracted from cell
culture by homogenization in a radioimmunoprecipitation assay
(RIPA) buffer (0.5 M NaCl, 1% Triton X-100, 0.5% NP40, 1%
deoxycholic acid, and 3.5 mM sodium dodecyl sulfate (SDS)), with
20% protease inhibitor cocktail (Sigma, Missouri, USA) and measured
by the Bradford method. Total cellular proteins were separated by
electrophoresis on 8% SDS−polyacrylamide gel and electro-trans-
ferred onto PVDF membranes. Membranes were then incubated with
the primary antibody. PPARγ monoclonal antibody was from Cayman
Chemical (Ann Arbor, MI, USA), and β-actin monoclonal antibody
was from Sigma-Aldrich (Missouri, USA). Membranes were then
probed with horseradish peroxidase-labeled secondary antibody, and
the signal was detected by the ECL chemoluminescence assay from
Amersham Pharmacia Biotech (Uppsala, Sweden). Expression level
was evaluated by densitometric analysis using Quantity One software
(Bio-Rad, Hercules, CA, USA), and the β-actin expression level was
used to normalize the sample values.
All animal studies were approved by the local ethical committee and
followed the Italian and European Community legislation.
In Vivo Magnetic Resonance Imaging of Adipose Tissues. At
day 12 of treatment with ligands, mice underwent in vivo magnetic
resonance imaging (MRI) analysis to monitor total body fat
distribution during treatments. Mice were anesthetized with 1%
isoflurane and were analyzed in a 4.7 T Avance II MRI scanner
(Bruker Corporation, Karlsrhue, Germany). After a gradient echo
scout, 16 axial 1 mm thick T1 weighted slices were placed in the
abdominal region spanning from kidneys to bladder included. The
field of view was 30 × 30 mm2 with a matrix of 128 × 128 pixels. Four
averages of a spin echo sequence with TE = 10 ms and TR = 400 ms
were acquired in 3′25″ and provided a very good contrast between fat
and other tissues. In order to maximize consistency between different
animals, the slice immediately frontal with respect to the ilium bone
was chosen for visceral fat estimation and was computed as (fat area)/
(slice area). Areas were semiautomatically delimited and measured
with Adobe Photoshop CS3 (Adobe Systems Inc., San Jose,
California).
Oral Glucose Tolerance Test, Insulin Tolerance Test, and the
Calculation of the HOMA-IR Index. For the oral glucose tolerance
test (OGTT), mice were fasted overnight and were administered 2 g
glucose·kg−1 per os. Blood was taken from the tail at 15 min intervals,
and glucose concentration was determined with a OneTouch Ultra
glucometer (LifeScan, Milano, Italy). For the insulin tolerance test
(ITT), mice were fasted six hours and were administered 0.6 IU·kg−1
of insulin (Eli Lilly, Firenze, Italy) with an intraperitoneal injection.
Blood samples were taken from the tail at 15 min intervals, and
glucose concentration was determined with a OneTouch Ultra
glucometer (LifeScan).
The homeostatic model assessment of insulin resistance (HOMA-
IR) was calculated with the formula
Cell Cycle Analysis. Cells were exposed to the ureidofibrate-like
enantiomers R-1 and S-1, troglitazone (1, 5, 10, 50, and 100 μM), or 6
(20 μM) for 1−2 days. Cells were harvested, washed twice in ice-cold
PBS (pH 7.4), fixed in 4.5 mL of 70% ethanol at −20 °C, then washed
once in ice-cold PBS. The pellet was resuspended in PBS containing 1
mg·mL−1 RNase and 0.01% NP40, and the cellular DNA was stained with
50 μg·mL−1 propidium iodide (Sigma, Missouri, USA). Cells were stored
in ice for 30 min prior to analysis. Cell cycle determinations were carried
out using a FACScan Flow Cytometer (Becton Dickinson), and data were
interpreted using the CellQuest software, provided by the manufacturer.
Apoptosis Determination. Exponentially growing cells were
treated with drug IC50 concentrations for one and two days and then
FPI × FPG
22.5 × 18
where FPI is the fasting plasma insulin concentration (μU·mL−1),
and FPG is the fasting plasma glucose concentration (mg·dL−1).
Gene Expression in the Liver. Total RNA from the livers of
mice treated with the indicated ligands was extracted with Trizol
(Sigma, Milano, Italy) followed by purification on RNeasy cartridges
(Qiagen, Milano, Italy) according to the manufacturer’s instructions.
One microgram of total RNA was used to quantitate the mRNA levels
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Journal of Medicinal Chemistry
Article
harvested and centrifuged at 1,200 rpm for 10 min. The supernatant was
discarded, and the cell pellets were washed in ice-cold PBS and
resuspended in 500 μL of ice-cold 1× binding buffer (0.1 M Hepes/
NaOH, pH 7.4, 1.4 M NaCl, 25 mM CaCl2). Annexin staining was
performed according to the manufacturer’s protocol (annexin V-FITC
apoptosis detection kit I, Becton Dickinson). Cells were stained by the
addition of both 5 μL of annexin V-FITC and 5 μL of propidium iodide
(PI) solution. The annexin V assay allows one to detect apoptosis at a very
early stage. This assay takes advantage of the fact that phosphatidylserine
(PS), which has a strong, specific affinity for annexin V, is translocated
from the inner (cytoplasmic) leaflet of the plasma membrane to the outer
(cell surface) leaflet soon after the induction of apoptosis. PS on the outer
leaflet is, therefore, available to bind labeled annexin V.61 Typically, 10,000
events are collected using excitation/emission wavelengths of 488/525 and
488/675 nm for annexin V and PI, respectively. The samples were
analyzed with FACScan Flow Cytometer and results carried out using
CellQuest software (Becton Dickinson).
for administrative and financial management. This work was
supported by grants from the Cariplo Foundation 2009.2727
(to F.G., F.L., A.L., and G.P.), EU FP6 LSHM-CT2006-037498
(to M.C.), The Giovanni Armenise-Harvard Foundation Career
Development Grant (to N.M.), and Universita degli Studi di
̀
Bari “Aldo Moro”.
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ASSOCIATED CONTENT
* Supporting Information
Physicochemical properties and spectroscopic data for inter-
mediates and final compounds; statistics of crystallographic
data and refinement; superposition of the crystal and docked
structure of 2; activity of R-1 and S-1 enantiomers toward
PPARα. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
Abbreviations Used
PPAR, peroxisome proliferator-activated receptor; NEFA,
nonesterified fatty acids; OGTT, oral glucose tolerance test;
ITT, insulin tolerance test; HOMA-IR, homeostatic model
assessment of insulin resistance; PPRE, peroxisome proliferator
response elements; SPPARM, selective peroxisome proliferator-
activated receptor modulator; NR, nuclear receptor; TZDs,
thiazolidinediones; LBD, ligand binding domain; RXR, retinoid
X receptor; HOBT, hydroxybenzotriazole; DIC, N,N-diisopro-
pylcarbodiimide; DSC, differential scanning calorimetry; ITC,
isothermal titration calorimetry; HFD, high fat diet; WAT,
white adipose tissue; MRI, magnetic resonance imaging; MOE,
molecular operating environment; RIPA, radioimmunoprecipi-
tation assay; RMSF, root-mean-square fluctuation
Accession Codes
PDB ID: 2I4P; 2I4J; 3R8I.
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AUTHOR INFORMATION
Corresponding Author
*(M.C.) Phone: +39 02-50318393. Fax: +39 02-50318391.
E-mail: maurizio.crestani@unimi.it. (F.L.) Phone: +39
080-5442798. Fax: +39 080-5442231. E-mail: floiodice@
farmchim.uniba.it.
■
Present Address
▽University of Lausanne, Center for Integrative Genomics,
Genopode Building, Room 5010, 1015 Lausanne, Switzerland.
Author Contributions
○These authors contributed equally to this work.
ACKNOWLEDGMENTS
We wish to thank Dr. Krister Bamberg (AstraZeneca, Molndal,
Sweden) for kindly providing the Gal4-PPARγ vector and
pET28a-PPARγ-LBD expression vector. We are also grateful to
̀
Miss Elda Desiderio Pinto (Universita degli Studi di Milano)
■
̈
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