Fibrate-derived N-(methylsulfonyl)amides with antagonistic properties on PPARα

Article abstract of DOI:10.1016/j.ejmech.2012.10.019

The identification of novel PPAR ligands represents an attractive research to fully understand the complex biological pathways regulated by these receptors. Selective PPAR modulators, inverse agonists and antagonists of three PPAR isoforms could help to clarify biological effects on lipid and glucose homeostasis. Here we describe the identification of a group of N-(methylsulfonyl)amides, derived from PPARα agonist carboxylic acids. Transactivation and FRET assay confirmed an antagonist behaviour on PPARα for some of these compounds, with submicromolar IC₅₀. A preliminary analysis on selectivity α/γ revealed different profiles of inhibition or activation.

Full text of DOI:10.1016/j.ejmech.2012.10.019

European Journal of Medicinal Chemistry 58 (2012) 317e322
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fibrate-derived N-(methylsulfonyl)amides with antagonistic properties on PPAR
a
Alessandra Ammazzalorso, Alessandra D’Angelo, Antonella Giancristofaro, Barbara De Filippis,
Mauro Di Matteo, Marialuigia Fantacuzzi, Letizia Giampietro, Pasquale Linciano, Cristina Maccallini,
*
Rosa Amoroso
Dipartimento di Farmacia, Università “G. d’Annunzio”, via dei Vestini, Chieti 66100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification of novel PPAR ligands represents an attractive research to fully understand the
complex biological pathways regulated by these receptors. Selective PPAR modulators, inverse agonists
and antagonists of three PPAR isoforms could help to clarify biological effects on lipid and glucose
homeostasis. Here we describe the identification of a group of N-(methylsulfonyl)amides, derived from
Received 14 September 2012
Received in revised form
8 October 2012
Accepted 11 October 2012
Available online 23 October 2012
PPAR
a
a
agonist carboxylic acids. Transactivation and FRET assay confirmed an antagonist behaviour on
for some of these compounds, with submicromolar IC50. A preliminary analysis on selectivity a/g
PPAR
revealed different profiles of inhibition or activation.
Keywords:
Ó 2012 Published by Elsevier Masson SAS.
PPARs
PPAR antagonist
Transactivation assay
FRET
1. Introduction
(SPPARMs) [7], inverse agonists [8], and antagonists [9] of the three
PPAR isoforms have attracted much interest as investigative tools,
to fully understand the biology and pharmacology of PPARs. To date
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated transcription factors that belong to the nuclear receptor
only few selective PPAR
GW6471 [10], and triazolone-based compounds from Lilly [11], able
to revert the activity of full agonists. The strategy of blocking PPAR
activation has found several applications in elucidating the bio-
logical pathways controlled by PPAR [12]. In addition, a new study
by Laurenti et al. suggested an interesting role of PPAR antagonists
a antagonists have been described:
superfamily [1]. The three PPAR isoforms, PPAR
their tissue distributions, selectivity and responsiveness to ligands.
PPAR plays a role in the clearance of circulating or cellular lipids
via the regulation of gene expression involved in lipid metabolism
in liver and skeletal muscle [2]. PPAR promotes adipocyte differ-
entiation to enhance blood glucose uptake [3], whereas PPAR is
involved in lipid oxidation and cell proliferation [4]. PPAR subtypes
share similar structure: an N-terminal ligand-independent
a, g, and b/d, vary in
a
a
a
g
a
b
/d
in glioblastoma models in reducing lipid droplets, related to
tumour malignancy grade [13].
a
In the search for novel compounds acting on PPARa, we recently
transactivation domain (AF1), followed by a highly conserved
DNA binding domain (DBD) and a C-terminal ligand binding
reported the synthesis and biological evaluation of a family of N-
(phenylsulfonyl)amides containing a benzothiazole scaffold, found
domain (LBD) containing
a
ligand-dependent transactivation
to be PPAR
compound, showing a dose-dependent inhibition profile on PPAR
activation promoted by GW7647, with a micromolar potency
a inhibitors [14]; compound 1 was found the best
function (AF2). PPAR ligands induce binding to PPAR responsive
elements (PPREs) in the DNA after dimerization with another
nuclear receptor, the retinoid-X receptor (RXR) [5].
A number of potent and selective PPAR agonists have been
disclosed [6], but to date it remains the need for additional small
regulators of PPARs, in order to further decipher the biological
pathways regulated by these receptors. Selective PPAR modulators
a
(IC50 ¼ 6.5
mM); it was able to decrease CPT1A gene expression
(Fig. 1).
By comparing results obtained from our studies and Lilly’s sul-
fonimidic triazolones from literature, we suppose the crucial role of
N-phenylsulfonyl moiety in determining an antagonistic behaviour
on PPARa, suggesting the steric hindrance of sulfonimide could be
the key feature determining activation or block of the receptor. We
thought to replace the phenyl ring with a less hindered substituent,
such a methyl, to verify that hypothesis.
* Corresponding author. Tel.: þ39 0871 3554686; fax: þ39 0871 3554911.
E-mail address: ramoroso@unich.it (R. Amoroso).
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.10.019

318
A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 58 (2012) 317e322
We performed the biological evaluation of derivatives 2aeg and
O
S
N
O
O
3e6 by a standard transactivation assay [17], combined with
a competitive time-resolved fluorescence resonance energy trans-
fer (TR-FRET) assay [18]. In the transactivation assay, GAL4-PPAR
chimeric receptors were expressed in eukaryotic cells HEK293;
we utilized firefly luciferase reporter gene technology to provide
optimal assay sensitivity and dynamic range when quantifying
nuclear receptor activity. The assays were performed using agonist
and antagonist formats. In the antagonist mode the experimental
procedure is the same except that cells are incubated with an
agonist before the addition of tested compounds. New compounds
S
S
N
Cl
H
1
Fig. 1. Benzothiazole-based N-(phenylsulfonyl)amide, PPARa antagonist.
In this work we present the synthesis of new N-(methylsulfonyl)
amides, derived fromPPAR agonistcarboxylicacids. The bioisosteric
a
were tested at 150
mM, and reference compounds 7 and 8 at 150 mM
replacement of the carboxylic group with a sulfonimidic one is
a useful strategy in drug discovery. The sulfonimidic moiety shows
a very similar profile to carboxylic group in terms of acidity and H-
bond properties. It might be interesting to evaluate the influence of
this bioisosteric modification in PPARa ligands, where the carboxylic
head has been described as a structural key involved in a H-bond
network within the ligand binding domain [15].
and 1 M, respectively. The results, expressed as fold activation, are
shown in Table 1. Among benzothiazole-based compounds, all
derivatives show an antagonistic profile, except for 2a, that acti-
m
vates PPAR
a good inhibition of the receptor, with submicromolar IC50 values,
as for 2c, 2e, and 2g (IC50 ¼ 0.6, 0.8 and 0.9 M, respectively).
Methanesulfonimidic derivatives of fibrates 3e6 gave a different
profile in PPAR activation: fenofibrate analogue 4 does not
a
(EC50 ¼ 1.59
mM). Some of these compounds exhibit
m
a
2. Results and discussion
produce a notable activation or repression of basal activity, clofi-
brate and gemfibrozil derivatives 3 and 6 are agonists (EC50 ¼ 10.7
In this paper we describe the synthesis and biological evaluation
of a first group of benzothiazole-based compounds (2aeg), and
a second group derived from classical fibrates clofibrate, fenofi-
brate, bezafibrate and gemfibrozil (3e6). We selected clofibric acid
(7) and GW7647 (8) as reference compounds (Fig. 2).
and 7.1 mM, respectively), whereas bezafibrate analogue shows an
antagonistic profile, also if with a poor potency (IC50 ¼ 68.5
The dose-dependent antagonistic profile of 2e and 5 is illus-
trated in Fig. 3: both decrease the activation promoted by 8 (2 M)
at concentrations 1e150 M, without showing PPAR activation
mM).
m
m
a
The N-(methylsulfonyl)amides 2aeg were easily obtained by the
direct coupling of corresponding carboxylic acids with meth-
anesulfonamide, in the presence of 1-ethyl-3-[3-dimethylaminop
ropyl]carbodiimide hydrochloride (EDC) and 4-dimethylamino
pyridine (DMAP) (Scheme 1). The synthesis of starting acids has
been reported elsewhere [16]. Commercial fibrates (clofibric acid,
bezafibrate and gemfibrozil) were reacted as described, whereas
fenofibrate was previously hydrolyzed byaqueous NaOH 2 N, inTHFat
60 ꢀC.
when used alone. Similar trends were found for all compounds
showing an antagonistic behaviour.
The different effect produced by tested compounds on PPAR
a
does not follow a clear trend of structureeactivity relationship. The
nature of substituent R3, among benzothiazole derivatives 2aeg,
could be a structural feature involved in the activation or block of
the receptor, with only methyl derivative 2a acting as agonist. It is
also evident that steric hindrance on sulfonimidic moiety does not
represent the key factor modulating the receptor activity: both
R2
R1
O
O
S
N
O
O
O
O
O
O
O
S
S
Cl
O
S
O
S
N
H
N
H
N
H
R3
Cl
O
2a-g
3
4
O
O
O
O
S
O
O
N
H
O
O
S
O
N
H
N
H
Cl
6
5
O
O
S
OH
O
O
OH
N
N
Cl
H
7
8
Fig. 2. N-(Methylsulfonyl)amides benzothiazole-based or derived from classical fibrates and reference compounds.

A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 58 (2012) 317e322
319
A
preliminary overview on subtype selectivity was also
addressed by a transactivation experiment on PPAR isoform. Each
compound was tested on PPAR at the concentration correspond-
R2
R1
O
R2
R1
O
S
N
S
N
O
O
g
S
S
S
OH
N
H
g
R3
R3
ing to IC50 (or EC50) on alpha isoform. The comparison of FA values
outlined different behaviours for tested compounds (Fig. 5).
2a-g
Compounds 1 and 2e were found mainly inhibitors of PPARa,
without a notable influence on gamma isoform; 2f and 2g showed
inhibition of both PPAR subtypes, whereas four derivatives (2b, 2c,
Scheme 1. Reagents and conditions: methanesulfonamide, EDC, DMAP, dry CH2Cl2,
ꢀCer.t., 24 h.
0
2d and 5) showed mixed properties, producing inhibition of PPAR
and activation of PPAR . At last, a group of compounds (2a, 3 and 6)
gave a different, interesting profile, producing strong activation of
PPAR with a moderate PPAR activity, so promising a dual
agonistic activity.
These results look of some interest, with benzothiazole deriva-
tives 2aeg showing a correlation between chemical structure and
selectivity. We observed a decreasing ability to activate PPARa by
increasing the steric hindrance of substituent R3, in alpha to
a
methane- and benzenesulfonimidic derivatives can act as antago-
nists, or, in same cases, as agonists.
g
We decided to further analyze the binding of our compounds to
g
a
PPAR
method based on the fluorescence resonance energy transfer. The
competitive ligand binding to PPAR results in a loss of FRET signal,
observed at proper wavelengths. In a first experiment we per-
formed FRET assay testing our compounds at 100 M, with 7 and 8
(1 M) as positive controls. Also compound 1 was included as
a positive control: we demonstrated elsewhere its ability to
produce an antagonistic effect on PPAR activation at micromolar
a by means of a TR-FRET assay, a robust and well established
a
m
m
carbonylic group. The presence of a n-propylic group or more
hindered alkyls, or a phenyl ring, produces antagonism on PPAR
The nature of this substituent, indeed, has a different impact on
PPAR : if R3 is less hindered, the compound is able to activate the
a.
a
concentration [14]. Results are expressed as the ratio of fluores-
cence intensity at 520 nm (fluorescein emission excitated by
terbium emission) and 495 nm (terbium emission). Relative FRET
values were normalized for 7e100%. All tested compounds show
a significant decrease in FRET ratio, with affinity profiles similar
or better than positive controls. Among benzothiazole derivatives,
higher binding affinities were found for 2b and 2e (137.4 and
116.7%, respectively); all fibrate derivatives, except for 5, showed
positive binding affinities compared to clofibric acid (128.5, 121.2
and 118.0% for 4, 3 and 6, respectively) (Fig. 4).
g
receptor, whereas n-hexyl or phenyl derivatives are antagonists.
Reported data confirm the possibility to obtain PPAR ligands
lacking in carboxylic head, so disrupting the classical H-bond
network present in carboxylic agonists. The different steric
hindrance on sulfonimidic moiety is probably important, but it
does not represent the key factor modulating the receptor activity.
Whereas benzenesulfonimidic derivatives were found mainly
antagonists, the methylsulfonyl analogues here presented show
activation or inhibition on PPARa.
Starting from these data, we selected compounds showing
a high FRET ratio in order to derive the IC50 in the FRET-based
competitive ligand binding assay. Selected compounds (2b, 2c, 2e
and 2g) were tested at five concentrations, in three independent
experiments. Results are summarized in Table 2, where combined
data from transactivation and FRET are reported. The FRET assay
fully confirmed the transactivation results, as it is evident from the
nice agreement among the calculated IC50 values. Only a slight
difference, in terms of IC50 value, was observed for compound 2b,
that appears the more potent derivative in the FRET assay, but not
3. Conclusions
A group of novel N-(methylsulfonyl)amides were designed and
synthesized as bioisosteric derivatives of fibrates. The combined
biological approach, including transactivation and FRET assay,
revealed an interesting profile for some of these compounds. A
preliminary screening on PPAR
behaviour; we identified PPAR
dependent inhibition profile and submicromolar IC50, but also
potential dual antagonists and agonists.
g
a
isoform revealed different
antagonists, with dose-
a
in the transactivation experiment (IC50 ¼ 0.48 and 8.87
respectively). The comparison of these data allowed us to point out
the good binding affinities to PPAR of the newly synthesized N-
mM,
a/g
a
4. Experimental section
(methylsulfonyl)amides, with a quite good potency profile.
4.1. Chemistry
Table 1
In vitro PPAR
a transactivation of synthesized compounds.
Melting points were determined on a Büchi B-540 apparatus and
are uncorrected. Infrared spectra were recorded on a FT-IR 1600
PerkineElmer spectrometer. NMR spectra were run at 300 MHz on
Compd
R1
R2
R3
FAa
IC50
(
m
M)b
EC50 (m
M)c
2a
2b
2c
2d
2e
2f
2g
3
4
5
6
7
H
H
OEt
OEt
H
H
H
Me
1.84 ꢁ 0.07
0.36 ꢁ 0.07
0.28 ꢁ 0.06
0.47 ꢁ 0.12
0.08 ꢁ 0.02
0.04 ꢁ 0.01
0.07 ꢁ 0.03
1.70 ꢁ 0.10
1.10 ꢁ 0.10
0.44 ꢁ 0.04
1.55 ꢁ 0.05
1.6 ꢁ 0.22
e
1.59 ꢁ 0.04
n-Pr
n-Pr
i-Pr
n-But
n-Hex
Ph
8.87 ꢁ 0.08
e
a Varian instrument; chemical shifts (d) are reported in ppm. Micro-
Cl
Cl
Cl
Cl
Cl
0.6 ꢁ 0.01
e
analyses were carried out with an Eurovector Euro EA 3000 model
analyser and the analytical results were within 0.4% of the theoretical
values. Commercial reagents were used as received from Aldrich or
Fluka.
46.5 ꢁ 1.31
e
0.8 ꢁ 0.01
e
H
H
5.1 ꢁ 0.51
e
0.9 ꢁ 0.02
e
e
10.7 ꢁ 0.61
e
e
4.2. General procedure for the synthesis of N-(methylsulfonyl)
amides
68.5 ꢁ 3.21
e
e
e
e
7.1 ꢁ 0.32
55.0 ꢁ 3.9
0.2 ꢁ 0.02
To a cooled mixture (0e5 ꢀC) of proper acids (1.0 mmol) in dry
dichloromethane (15 mL), 1-ethyl-3-[3-dimethylaminopropyl]car-
8
2.8 ꢁ 0.23
a
FA: Fold activation. Compounds were tested in at least three separate experi-
M. Only 8 was tested at 1
M. The results are expressed ꢁ SEM.
Compounds were tested in at least three separate experiments at five concen-
trations, from to 150 M, in the presence of (2 M). The results are
expressed ꢁ SEM.
ments at 150
m
m
bodiimide hydrochloride (EDC, 1.0 mmol, 177.0
mL) and 4-
b
dimethylaminopyridine (DMAP, 1.0 mmol, 122.2 mg) were added,
under stirring in a nitrogen atmosphere. After 15 min, meth-
anesulfonamide (1.1 mmol) was added, and the mixture was
allowed to warm to room temperature. After stirring overnight, the
1
m
8
m
c
Compounds were tested in at least three separate experiments at five concen-
trations, from 1 to 150
M. The results are expressed ꢁ SEM.
m

320
A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 58 (2012) 317e322
Fig. 3. Dose-dependent antagonistic profile of 2e and 5.
reaction was diluted with dichloromethane (15 mL), washed with
2 N HCl (3 ꢂ 30 mL), dried on Na2SO4. After evaporation of solvent
under reduced pressure, crude products were purified on silica gel
(eluent dichloromethane/methanol 9:1).
4.2.3. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)
pentanamide (2c)
White needles, 53% yield, m.p. 150e151 ꢀC. IR (KBr) 1716, 1549,
1345 and 1171 cmꢃ1
;
1H NMR (CDCl3)
d
0.97 (t, 3H, J ¼ 7.2 Hz,
CH3CH2), 1.43e1.62 (m, 2H, CH2CH3), 1.80e2.17 (m, 2H, CH2CH),
3.27 (s, 3H, CH3SO2), 4.22 (t, 1H, J ¼ 7.5 Hz, CH), 7.36 (dd, 1H,
J ¼ 8.7 Hz, J ¼ 2.1 Hz, CH Ar), 7.69 (d, 1H, J ¼ 8.7 Hz, CH Ar), 7.93 (d,
4.2.1. 2-[(6-Ethoxy-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)
propanamide (2a)
Yellowish needles, 62%yield, m.p.136e138 ꢀC. IR (KBr) 3210,1726,
1H, J ¼ 2.1 Hz, CH Ar), 11.69 (bs, 1H, NH); 13C NMR (CDCl3)
d 13.8
1685, 1558 cmꢃ1
;
1H NMR (CDCl3)
d
1.44 (t, 3H, J ¼ 6.9 Hz,
(CH3CH2), 20.5 (CH2CH3), 31.2 (CH2CH), 41.4 (CH3SO2), 50.2 (CH),
121.6, 122.1 and 126.3 (CH Ar), 133.3, 133.5, 152.4 and 169.1 (C Ar),
169.9 (C]O). Anal. (C13H15ClN2O3S3) C, H, N.
CH3CH2OAr),1.61 (d, 3H, J ¼ 7.5 Hz, CH3CH), 3.26 (s, 3H, CH3SO2), 4.07
(q, 2H, J¼ 6.9Hz,ArOCH2CH3), 4.32(q,1H, J¼ 7.5 Hz, CHCH3), 7.06(dd,
1H, J ¼ 9.0 Hz, J ¼ 2.4 Hz, CH Ar), 7.22 (d,1H, J ¼ 2.4 Hz, CH Ar), 7.81 (d,
1H, J ¼ 9.0 Hz, CH Ar), 12.49 (bs, 1H, NH); 13C NMR (CDCl3)
d
15.0
4.2.4. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-3-methyl-N-
(methylsulfonyl)butanamide (2d)
(CH3CH2OAr), 15.3 (CH3CH), 41.3 (CH3SO2), 45.1 (CHCH3), 64.4
(ArOCH2CH3), 105.0, 116.5 and 122.2 (CH Ar), 136.4, 145.9, 157.5 and
163.4 (C Ar), 170.6 (C]O). Anal. (C13H16N2O4S3) C, H, N.
White solid, 67% yield, m.p. 129e130 ꢀC. IR (KBr) 3191, 1686,
1461, 1348, 1136 cmꢃ1
;
1H NMR (CDCl3)
d
1.15 (dd, 6H, J ¼ 6.9 Hz,
CH3CHCH3), 2.50 (m, 1H, CH3CHCH3), 3.27 (s, 3H, CH3SO2), 3.98 (t,
1H, J ¼ 7.2 Hz, CHS), 7.37 (dd, 1H, J ¼ 8.7 Hz, J ¼ 2.1 Hz, CH Ar), 7.70
(d, 1H, J ¼ 8.7 Hz, CH Ar), 7.92 (d, 1H, J ¼ 2.1 Hz, CH Ar), 11.56 (bs, 1H,
4.2.2. 2-[(6-Ethoxy-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)
pentanamide (2b)
White needles, 57% yield, m.p. 136e138 ꢀC. IR (KBr) 3163,
NH); 13C NMR (CDCl3)
d 20.0 and 21.0 (CH3CHCH3), 28.6
1715, 1692, 1335 and 1136 cmꢃ1
;
1H NMR (CDCl3)
d
0.95 (t, 3H,
(CH3CHCH3), 41.4 (CH3SO2), 58.2 (CHS), 121.6, 122.1 and 126.3 (CH
Ar), 133.3, 133.5, 152.5 and 168.7 (C Ar), 169.8 (C]O). Anal.
(C13H15ClN2O3S3) C, H, N.
J ¼ 7.2 Hz, CH3CH2CH2), 1.44 (t, 3H, J ¼ 6.9 Hz, CH3CH2OAr), 1.42e
1.63 (m, 2H, CH3CH2CH2), 1.78e2.17 (m, 2H, CH3CH2CH2), 3.26 (s,
3H, CH3SO2), 4.07 (q, 2H, J ¼ 6.9 Hz, ArOCH2CH3), 4.13 (t, 1H,
J ¼ 7.2 Hz, CHCH2), 7.06 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.4 Hz, CH Ar),
7.22 (d, 1H, J ¼ 2.4 Hz, CH Ar), 7.82 (d, 1H, J ¼ 9.0 Hz, CH Ar),
4.2.5. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)
hexanamide (2e)
12.29 (bs, 1H, NH); 13C NMR (CDCl3)
d
13.8 (CH3CH2CH2), 14.9
White solid, 63% yield, m.p. 120e122 ꢀC. IR (KBr) 3194, 1694,
(CH3CH2OAr), 20.5 (CH2CH2CH3), 31.3 (CH2CH2CH3), 41.3
(CH3SO2), 50.4 (CHCH2), 64.4 (ArOCH2CH3), 105.0, 116.5 and
122.3 (CH Ar), 136.4, 146.0, 157.5 and 163.2 (C Ar), 170.4 (C]O).
Anal. (C15H20N2O4S3) C, H, N.
1460, 1345, 1134 cmꢃ1
;
1H NMR (CDCl3)
d
0.91 (t, 3H, J ¼ 7.2 Hz,
CH3CH2), 1.24e1.59 (m, 4H, CH2), 1.78e2.25 (m, 2H, CH2), 3.27 (s,
3H, CH3SO2), 4.21 (t, 1H, J ¼ 7.2 Hz, CHS), 7.37 (dd, 1H, J ¼ 8.7 Hz,
J ¼ 2.1 Hz, CH Ar), 7.69 (d, 1H, J ¼ 8.7 Hz, CH Ar), 7.93 (d, 1H,
J ¼ 2.1 Hz, CH Ar), 11.69 (bs, 1H, NH); 13C NMR (CDCl3)
d 14.0
(CH3CH2), 22.4, 29.0 and 29.3 (CH2), 41.4 (CH3SO2), 50.4 (CH), 121.6,
122.1 and 126.3 (CH Ar), 133.3, 133.5, 152.4 and 169.1 (C Ar), 169.9
(C]O). Anal. (C14H17ClN2O3S3) C, H, N.
4.2.6. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)
octanamide (2f)
White solid, 70% yield, m.p.102e104 ꢀC. IR (KBr) 3213,1706,1430,
1346, 1146 cmꢃ1; 1H NMR (CDCl3)
d
0.86 (t, 3H, J ¼ 7.2 Hz, CH3CH2),
Table 2
Combined data obtained for selected compounds by transactivation and FRET assay.
Compd
Transactivation assay
IC50 M)
FRET assay
(m
Normalized FRET
IC50 (mM)
ratio % (520/495 nm)
2b
2c
2e
2g
8.87 ꢁ 0.08
0.6 ꢁ 0.01
0.8 ꢁ 0.01
0.9 ꢁ 0.02
137.4
80.9
116.7
95.3
0.48 ꢁ 0.01
0.89 ꢁ 0.03
0.60 ꢁ 0.01
0.95 ꢁ 0.07
Fig. 4. Normalized FRET ratios (520/495 nm) obtained for tested compounds.

A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 58 (2012) 317e322
321
4.2.11. 5-(2,5-Dimethylphenoxy)-2,2-dimethyl-N-(methylsulfonyl)
pentanamide (6)
White solid, 66% yield, m.p. 97e98 ꢀC. IR (KBr) 3456, 1714, 1331,
1158 cmꢃ1; 1H NMR (CDCl3)
1.26 (s, 6H, C(CH3)2), 1.74e1.76 (m, 4H
d
CH2CH2), 2.17 (s, 3H, CH3Ph), 2.29 (s, 3H, CH3Ph), 3.26 (s, 3H,
CH3SO2), 3.93 (t, 2H, CH2O), 6.60 (s, 1H, CH Ar), 6.66 (d, 1H,
J ¼ 7.5 Hz, CH Ar), 6.99 (d,1H, J ¼ 7.5 Hz, CH Ar), 8.25 (bs,1H, CONH);
13C NMR (CDCl3)
d 16.0 and 21.6 (CH3Ph), 25.1 (C(CH3)2), 37.2
(OCH2CH2), 41.6 (CH3SO2), 43.6 (OCH2CH2CH2), 67.5 (OCH2), 82.4
(C(CH3)2), 112.2 and 121.1 (CH Ar), 123.7 (C Ar), 130.6 (CH Ar), 136.8
and 156.9 (C Ar), 176.8 (CONH). Anal. (C16H25NO4S) C, H, N.
4.3. In vitro PPAR transactivation assay
Fig. 5. Activation or inhibition profile on PPARa and PPARg isoforms.
HEK293A cells were maintained in growth medium composed of
DMEM (Sigma) supplemented with 10% FBS (Gibco), 1% penicillin/
streptomycin (Sigma),1%MEMnon-essentialaminoacid(Sigma)and
1.24e2.13 (m, 10H, CH2), 3.27 (s, 3H, CH3SO2), 4.21 (t, 1H, J ¼ 7.2 Hz,
CHS), 7.37 (dd,1H, J ¼ 8.7 Hz, J ¼ 2.1 Hz, CH Ar), 7.69 (d,1H, J ¼ 8.7 Hz,
CH Ar), 7.93 (d, 1H, J ¼ 2.1 Hz, CH Ar), 11.70 (bs, 1H, NH); 13C NMR
1% sodium pyruvate MEM 100 mM (Sigma). The PPARa or PPARg
ligand-binding activity of the test compounds was determined using
transient transfection assay. The HEK293A cells were plated in white
96-well plates and cultured until 70e80% confluency for 16 h. Before
transfection, the culture medium was replaced by fresh serum-free
medium. The cells were transiently transfected with 50 ng of
reporter plasmid, 20 ng of renilla, 40 ng of pGEM, and 30 ng of each
receptor expression plasmid per well by the calcium phosphate
coprecipitation method. Test compounds were added after 6 h. After
16e18 h treatment, the cellular luciferase activity was determined
using commercial firefly luciferase assay according to the supplier’s
instructions (Promega). The results were normalized to the renilla
activity to correct the transfection efficiencies.
(CDCl3)
d 14.2 (CH3CH2), 22.7, 27.2, 29.0, 29.3 and 31.6 (CH2), 41.4
(CH3SO2), 50.5 (CH),121.6,122.1 and 126.3 (CH Ar),133.3,133.5,152.4
and 169.1 (C Ar), 169.9 (C]O). Anal. (C16H21ClN2O3S3) C, H, N.
4.2.7. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-N-(methylsulfonyl)-
2-phenylacetamide (2g)
White solid, 58% yield, m.p. 176e178 ꢀC. IR (KBr) 1697, 1548,
1351, 1177, 1128 cmꢃ1; 1H NMR (CDCl3)
d 3.29 (s, 3H, CH3SO2), 5.48
(s, 1H, CHS), 7.34e7.49 (m, 6H, CH Ar), 7.69 (d, 1H, J ¼ 8.4 Hz, CH Ar),
7.94 (d, 1H, J ¼ 1.8 Hz, CH Ar), 11.21 (bs, 1H, NH); 13C NMR (CDCl3)
d
41.5 (CH3SO2), 54.4 (CHS), 121.6, 122.2, 126.2, 129.1, 129.4 and
129.6 (CH Ar), 132.2, 133.4, 133.5, 152.6 and 168.2 (C Ar), 168.3 (C]
4.4. Time-resolved fluorescence resonance energy transfer (TR-
O). Anal. (C16H13ClN2O3S3) C, H, N.
FRET) analysis
4.2.8. 2-(4-Chlorophenoxy)-2-methyl-N-(methylsulfonyl)
propanamide (3)
TR-FRET assay was performed by using LanthaScreenÔ TR-FRET
PPAR
a competitive binding assay kit (Invitrogen, PV4892). This
White solid, 65% yield, m.p. 153e155 ꢀC. IR (KBr) 3221, 1703,
ligand-binding assay is based on the competitive displacement of
a labelled pan-agonist, as described by the manufacturer (Invi-
1334, 1165 cmꢃ1; 1H NMR (CDCl3)
d
1.51 (s, 6H, C(CH3)2), 3.34 (s, 3H,
CH3SO2), 6.87 (d, 2H, J ¼ 8.7 Hz, CH Ar), 7.28 (d, 2H, J ¼ 8.7 Hz, CH
Ar), 9.00 (bs, 1H, NH); 13C NMR (CDCl3)
24.5 (C(CH3)2), 41.6
trogen). Briefly, for the PPAR
glutathione S-transferase (GST) antibody is used to indirectly
label the PPAR LBD. When a fluorescent ligand (tracer; the PPAR
a assay a terbium-labelled, anti-
d
(CH3SO2), 82.4 (C(CH3)2), 123.5 and 129.8 (CH Ar), 130.1 and 151.6 (C
Ar), 173.7 (CONH). Anal. (C11H14ClNO4S) C, H, N.
a
pan-agonist) binds to the receptor, energy transfer from the anti-
body to the tracer occurs, and a high TR-FRET ratio is observed.
4.2.9. 2-[4-(4-Chlorobenzoyl)phenoxy]-2-methyl-N-
(methylsulfonyl)propanamide (4)
Competitive ligand binding to the PPAR
compound’s ability to displace the tracer from PPAR
a loss of FRET signal between the antibody and the tracer.
Compounds, except for 8 (1 M), were screened at 100 M and the
a
LBD is detected by a test
a
, resulting in
White solid, 73% yield, m.p. 159e161 ꢀC. IR (KBr) 3077, 1708,
1642, 1288, 1121 cmꢃ1; 1H NMR (CDCl3)
d
1.62 (s, 6H, C(CH3)2), 3.33
m
m
(s, 3H, CH3SO2), 6.99 (d, 2H, J ¼ 8.4 Hz, CH Ar), 7.47 (d, 2H, J ¼ 8.4 Hz,
assay performed following the manufacturer’s instructions. After
a 2 h incubation, fluorescence was measured using a fluorescence
counter (Spectramax Gemini-XS, Molecular Devices) at emission
wavelengths of 495 or 520 nm, with excitation at 340 nm. TR-FRET
signal was measured. The data were calculated as TR-FRET ratio by
dividing the emission signal of the acceptor (fluorescein, 520 nm)
by the emission signal of the donor (terbium, 495 nm). Binding
activity is reported as Inhibitory Concentration (IC50) calculated in
CH Ar), 7.72 (d, 2H, J ¼ 8.4 Hz, CH Ar), 7.78 (d, 2H, J ¼ 8.4 Hz, CH Ar),
8.88 (bs, 1H, NHCO); 13C NMR (CDCl3)
d 24.7 (C(CH3)2), 41.6
(CH3SO2), 82.2 (C(CH3)2), 120.4, 128.9, 131.4 and 132.2 (CH Ar),
133.0, 136.0, 139.1 and 157.3 (C Ar), 173.5 (CONH), 194.3 (COPh).
Anal. (C18H18ClNO5S) C, H, N.
4.2.10. 4-Chloro-N-[2-(4-{1,1-dimethyl-2-[(methylsulfonyl)amino]-
2 oxoethoxy}phenyl)ethyl]benzamide (5)
a concentration range of 1e100 mM. This parameter represents test
ligand concentration at which 50% of the labelled pan-agonist is
displaced by the test ligand.
White solid, 77% yield, m.p. 184e185 ꢀC. IR (KBr) 3400, 1710,
1629, 1334, 1126 cmꢃ1; 1H NMR (DMSO)
d 1.43 (s, 6H, C(CH3)2), 2.76
(t, 2H, J ¼ 7.2 Hz, CH2Ph), 3.23 (s, 3H, CH3SO2), 3.41e345 (m, 2H,
CH2NH), 6.78 (d, 2H, J ¼ 8.4 Hz, CH Ar), 7.14 (d, 2H, J ¼ 8.4 Hz, CH Ar),
7.51 (d, 2H, J ¼ 8.4 Hz, CH Ar), 7.80 (d, 2H, J ¼ 8.4 Hz, CH Ar), 8.62 (t,
Acknowledgements
1H, NHCO), 11.93 (bs, 1H, NHSO2); 13C NMR (DMSO)
d
24.7 (C(CH3)2),
The authors gratefully acknowledge the Italian MIUR for finan-
cial support and Dr. David J. Mangelsdorf (Dallas, TX) for the PPAR
plasmids and Dr. Antonio Moschetta for helpful comments and
suggestions.
34.8 (CH2Ph), 41.5 (CH2NH), 41.6 (CH3SO2), 80.7 (C(CH3)2), 120.2,
129.0, 129.7 and 130.1 (CH Ar), 133.9, 134.2, 136.5 and 153.4 (C Ar),
165.7 (CONH), 174.9 (CONHSO2). Anal. (C20H23ClN2O5S) C, H, N.

322
A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 58 (2012) 317e322
{[5-trifluoromethyl)- 2-pyridyl] sulfonyl} ethyl)benzamide (GSK3787),
References
a
d
selective and irreversible peroxisome proliferator-activated receptor
(PPAR ) antagonist, J. Med. Chem. 53 (2010) 1857e1861;
d
[1] (a) L. Michalik, W. Wahli, Peroxisome proliferator-activated receptors: three
isotypes for a multitude of functions, Curr. Opin. Biotech. 10 (1999) 564e570;
(b) A.K. Hihi, L. Michalik, W. Wahli, PPARs: transcriptional effectors of fatty acids
and their derivatives, Cell. Mol. Life Sci. 59 (2002) 790e798;
(b) J.i. Kasuga, S. Ishida, D. Yamasaki, M. Makishima, T. Doi, Y. Hashimoto,
H. Miyachi, Novel biphenylcarboxylic acid peroxisome proliferator-activated
receptor (PPAR)
d selective antagonists, Bioorg. Med. Chem. Lett. 19 (2009)
6595e6599;
(c)M. Moreno, A. Lombardi, E. Silvestri, R. Senese, F. Cioffi, F. Goglia, A. Lanni, P. De
Lange, PPARs: nuclearreceptors controlled by, andcontrolling, nutrient handling
through nuclear and cytosolic signaling, PPAR Res. (2010). art. no. 435689.
(c) G. Deng, Z. Liu, F. Ye, X. Luo, W. Zhu, X. Shen, H. Liu, H. Jiang, Tryptophan-
containing dipeptide derivatives as potent PPAR antagonists: design,
synthesis, biological evaluation, and molecular modeling, Eur. J. Med. Chem.
43 (2008) 2699e2716;
(d) P.M. Toth, S. Naruhn, V.F.S. Pape, S.M.A. Dörr, G. Klebe, R. Müller,
W.E. Diederich, Development of improved PPARb/d inhibitors, Chem. Med.
g
[2] (a) J.C. Fruchart, Peroxisome proliferator-activated receptor-alpha (PPARa): at
the crossroads of obesity, diabetes and cardiovascular disease, Atherosclerosis
205 (2009) 1e8;
(b) D.H. Van Raalte, M. Li, P.H. Pritchard, K.M. Wasan, Peroxisome proliferator-
Chem. 7 (2012) 159e170.
activated receptor (PPAR)-
a
:
a
pharmacological target with
a
promising
- bigger than fat,
, the
[10] H.E. Xu, T.B. Stanley, V.G. Montana, M.H. Lambert, B.G. Shearer, J.E. Cobb,
D.D. McKee, C.M. Galardi, K.D. Plunket, R.T. Nolte, D.J. Parks, J.T. Moore,
S.A. Kliewer, T.M. Willson, J.B. Stimmel, Structural basis for antagonist-
future, Pharm. Res. 21 (2004) 1531e1538.
[3] (a) S.A. Kliewer, T.M. Willson, The nuclear receptor PPAR
Curr. Opin. Genet. Dev. 8 (1998) 576e581;
g
mediated recruitment of nuclear co-repressors by PPARa, Nature 415 (2002)
813e817.
(b) S. Rocchi, J. Auwerx, Peroxisome proliferator-activated receptor
g
ultimate liaison between fat and transcription, Brit. J. Nutr. 84 (Suppl. 2)
[11] G.J. Etgen, N. Mantlo, PPAR ligands for metabolic disorders, Curr. Top. Med.
Chem. 3 (2003) 1649e1661.
[12] (a) M.R. Romano, M.D. Lograno, Involvement of the peroxisome proliferator-
activated receptor (PPAR) alpha in vascular response of endocannabinoids
in the bovine ophthalmic artery, Eur. J. Pharmacol. 683 (2012) 197e203;
(b) L. Ding, X. Liang, D. Zhu, Y. Lou, Peroxisome proliferator-activated receptor
(2000) S223eS227;
(c) A. Koppen, E. Kalkhoven, Brown vs white adipocytes: the PPAR
g cor-
egulator story, FEBS Lett. 584 (2010) 3250e3259.
[4] K.-D. Wagner, N. Wagner, Peroxisome proliferator-activated receptor beta/
delta (PPAR ) acts as regulator of metabolism linked to multiple cellular
b/d
functions, Pharmacol. Ther. 125 (2010) 423e435.
a
is involved in cardiomyocyte differentiation of murine embryonic stem cells
[5] (a) K. Motojima, Peroxisome proliferator-activated receptor (PPAR): structure,
mechanisms of activation and diverse functions, Cell. Struct. Funct. 18 (1993)
267e277;
in vitro, Cell. Biol. Int. 31 (2007) 1002e1009;
(c) D.R. Sagar, D.A. Kendall, V. Chapman, Inhibition of fatty acid amide
hydrolase produces PPAR-a-mediated analgesia in a rat model of inflamma-
tory pain, Br. J. Pharmacol. 155 (2008) 1297e1306.
[13] G. Laurenti, E. Benedetti, B. D’Angelo, L. Cristiano, B. Cinque, S. Raysi, M. Alecci,
M.P. Cerù, M.G. Cifone, R. Galzio, A. Giordano, A. Cimini, Hypoxia induces
(b) Y. Brélivet, N. Rochel, D. Moras, Structural analysis of nuclear receptors:
from isolated domains to integral proteins, Mol. Cell. Endocrinol. 348 (2012)
466e473.
[6] (a) B.G. Shearer, W.J. Hoekstra, Recent advances in peroxisome proliferator-
activated receptor science, Curr. Med. Chem. 10 (2003) 267e280; (b)
F. Chang, L.A. Jaber, H.D. Berlie, M.B. O’Connell, Evolution of peroxisome
proliferator-activated receptor agonists, Ann. Pharmacother. 41 (2007) 973e
983;
peroxisome proliferator-activated receptor
a (PPARa) and lipid metabolism
peroxisomal enzymes in human glioblastoma cells, J. Cell. Biochem. 112
(2011) 3891e3901.
[14] A. Ammazzalorso, A. Giancristofaro, A. D’Angelo, B. De Filippis, M. Fantacuzzi,
L. Giampietro, C. Maccallini, R. Amoroso, Benzothiazole-based N-(phenyl-
(c) A. Ciudin, C. Hernández, R. Simó, Update on cardiovascular safety of PPAR-
gamma agonists and relevance to medicinal chemistry and clinical pharma-
cology, Curr. Top. Med. Chem. 12 (2012) 585e604.
sulfonyl)amides as a novel family of PPARa antagonists, Bioorg. Med. Chem.
Lett. 21 (2011) 4869e4872.
[15] P. Cronet, J.F.W. Petersen, R. Folmer, N. Blomberg, K. Sjöblom, U. Karlsson, E.-
L. Lindstedt, K. Bamberg, Structure of the PPAR and - ligand binding domain
[7] (a) P.L. Feldman, M.H. Lambert, B.R. Henke, PPAR modulators and PPAR pan
agonists for metabolic diseases: the next generation of drugs targeting
peroxisome proliferator-activated receptors? Curr. Top. Med. Chem. 8 (2008)
728e749;
a
g
in complex with AZ 242; ligand selectivity and agonist activation in the PPAR
family, Structure 9 (2001) 699e706.
[16] L. Giampietro, A. Ammazzalorso, A. Giancristofaro, F. Lannutti, G. Bettoni, B. De
Filippis, M. Fantacuzzi, C. Maccallini, M. Petruzzelli, A. Morgano, A. Moschetta,
R. Amoroso, Synthesis and biological evaluation of 2-heteroarylthioalkanoic
acid analogues of clofibric acid as peroxisome proliferator-activated receptor
(b) C. Pirat, A. Farce, N. Lebègue, N. Renault, C. Furman, R. Millet, S. Yous,
S. Speca, P. Berthelot, P. Desreumaux, P. Chavatte, Targeting peroxisome
proliferator-activated receptors (PPARs): development of modulators, J. Med.
Chem. 55 (2012) 4027e4061.
a
agonists, J. Med. Chem. 52 (2009) 6224e6232.
[8] (a) S. Lieber, F. Scheer, W. Meissner, S. Naruhn, T. Adhikary, S. Müller-Brüs-
selbach, W.E. Diederich, R. Müller, (Z)-2-(2-Bromophenyl)-3-{[4-(1-methyl-
[17] (a) I.G. Schulman, R.A. Heyman, The flip side: identifying small molecule
regulators of nuclear receptors, Chem. Biol. 11 (2009) 639e646;
(b) L.H. Naylor, Reporter gene technology: the future looks bright, Biochem.
Pharmacol. 58 (1999) 749e757.
[18] (a) G. Zhou, R. Cummings, Y. Li, S. Mitra, H.A. Wilkinson, A. Elbrecht,
J.D. Hermes, J.M. Schaeffer, R.G. Smith, D.E. Moller, Nuclear receptors have
distinct affinities for coactivators: characterization by fluorescence resonance
energy transfer, Mol. Endocrinol. 12 (1998) 1594e1604;
piperazine)amino]phenyl}acrylonitrile (DG172): an orally bioavailable PPAR
-selective ligand with inverse agonistic properties, J. Med. Chem. 55 (2012)
2858e2868;
(b) R.P. Trump, J.E. Cobb, B.G. Shearer, M.H. Lambert, R.T. Nolte, T.M. Willson,
R.G. Buckholz, S.M. Zhao, L.M. Leesnitzer, M.A. Iannone, K.H. Pearce, A.N. Billin,
b/
d
W. Hoekstra, Co-crystal structure guided array synthesis of PPAR
g inverse
agonists, Bioorg. Med. Chem. Lett. 17 (2007) 3916e3920.
(b) G. Zhou, R. Cummings, J. Hermes, D.E. Moller, Use of homogeneous time-
resolved fluorescence energy transfer in the measurement of nuclear receptor
activation, Methods 25 (2001) 54e61.
[9] (a) B.G. Shearer, R.W. Wiethe, A. Ashe, A.N. Billin, J.M. Way, T.B. Stanley,
C.D. Wagner, R.X. Xu, L.M. Leesnitzer, R.V. Merrihew, T.W. Shearer, M.R. Jeune,
J.C. Ulrich, T.M. Willson, Identification and characterization of 4-chloro-N-(2-