Synthesis of Naphthyl-, Quinolin- and Anthracenyl Analogues of Clofibric Acid as PPARα Agonists

Article abstract of DOI:10.1111/cbdd.12677

PPARα is a ligand activated transcription factor belonging to the nuclear receptor subfamily, involved in fatty acid metabolism in tissues with high oxidative rates such as muscle, heart and liver. PPARα activation is important in steatosis, inflammation and fibrosis in preclinical models of non-alcoholic fatty liver disease identifying a new potential therapeutic area. In this work, three series of clofibric acid analogues conjugated with naphthyl, quinolin, chloroquinolin and anthracenyl scaffolds were synthesized. In an effort to obtain new compounds active as PPARα agonists, these molecules were evaluated for PPARα transactivation activity. Naphthyl and quinolin derivatives showed a good activation of PPARα; noteworthy, optically active naphthyl derivatives activated PPARα better than corresponding parent compound.

Full text of DOI:10.1111/cbdd.12677

Chem Biol Drug Des 2016; 87: 467–471
Research Letter
Synthesis of Naphthyl-, Quinolin- and Anthracenyl
Analogues of Clofibric Acid as PPARa Agonists
Letizia Giampietro*, Alessandra Ammazzalorso,
Isabella Bruno, Simone Carradori, Barbara
De Filippis, Marialuigia Fantacuzzi, Antonella
Giancristofaro, Cristina Maccallini and
Rosa Amoroso*
tissue; it plays a pivotal role on lipid metabolism by
decreasing both serum triglycerides and free fatty acid
levels, and increasing high-density lipoprotein level (HDL)
(
(
3). PPARa is activated by the fibrate antilipidemic drugs
4). The PPARc controls fatty acid storage and glucose
metabolism by coordinating the expression of genes
involved in lipid metabolism, adipogenesis and inflamma-
tion (5). This receptor is activated by the thiazolidinedione
(TZD) antidiabetic drugs (6). PPARb/d regulates fatty acid
catabolism, insulin sensitivity and energy homeostasis in
muscle and adipose tissue (7). Some phenoxyacetic acid
derivatives, L-165041, GW501516, GW0742 and MBX-
Dipartimento di Farmacia, Universit aꢀ “G. d’Annunzio” di
Chieti, via dei Vestini, 66100, Chieti, Italy
*
Corresponding authors: Letizia Giampietro,
l.giampietro@unich.it; Rosa Amoroso,
ramoroso@unich.it
PPARa is
a ligand activated transcription factor
8
025 are very potent and selective activators of PPARb
belonging to the nuclear receptor subfamily, involved
in fatty acid metabolism in tissues with high oxidative
rates such as muscle, heart and liver. PPARa activation
is important in steatosis, inflammation and fibrosis in
preclinical models of non-alcoholic fatty liver disease
identifying a new potential therapeutic area. In this
work, three series of clofibric acid analogues conju-
gated with naphthyl, quinolin, chloroquinolin and
anthracenyl scaffolds were synthesized. In an effort to
obtain new compounds active as PPARa agonists,
these molecules were evaluated for PPARa transacti-
vation activity. Naphthyl and quinolin derivatives
showed a good activation of PPARa; noteworthy, opti-
cally active naphthyl derivatives activated PPARa bet-
ter than corresponding parent compound.
both in vitro and in vivo. Although PPARb agonists are not
yet in clinical use, human studies have been performed
providing very encouraging findings for the treatment of
metabolic disorders in dyslipidemic obese individuals
(8–10).
The structures of all PPARs subtypes are similar: an amino
terminal activation domain (AF-1), a DNA binding domain
(DBD), a ligand binding domain (LBD) and a second car-
boxy terminal activation domain (AF-2). When a ligand
interacts with PPAR, there is a binding to PPAR respon-
sive elements (PPREs) in the DNA after dimerization with
another nuclear receptor, the retinoid-X receptor (RXR).
The release of corepressors and recruitment of coactiva-
tors regulate the transcription of specific genes (1).
Key words: anthracenyl, clofibric acid, naphthyl, peroxisome
proliferator-activated receptor agonist, quinolin, transactivation
assay
In the last years, the development of new compounds that
activate PPARa has been an important target to better
understand structure–activity relationships (SARs) for
obtaining new hypolipidemic drugs with lower adverse
events. A lot of PPARa-selective agonists have been
reported previously (11,12). The common structure is com-
posed of an aromatic ring and carboxylic acid with or with-
out various spacers. The aromatic ring of the ligands
interacts with the hydrophobic binding pocket and the
acidic moiety forms hydrogen bonds with amino acids on
the AF-2 region in a roughly Y-shaped ligand binding site
Received 16 July 2015, revised 22 August 2015 and
accepted for publication 14 September 2015
Peroxisome proliferator-activated receptors (PPARs) are a
subfamily of ligand-activated nuclear hormone receptors
that are highly expressed in metabolically active tissues,
where regulate genes encoding lipid and glucose metabo-
lism, and overall energy homeostasis. Three PPAR sub-
types, commonly designated as PPARa, PPARc and
PPARd(b), have been identified; these receptors differ in
their tissue distributions, selectivity and responsiveness to
ligands, thus leading to the regulation of different sets of
genes involved in lipid metabolism and energy balance
(13,14).
In the past years, we have synthesized different series of
fibrate derivatives with the aim of obtaining new hypolipi-
demic compounds active as PPARa agonists (15–17). In
this field, stilbenes and chalcones and some of their syn-
thetic derivatives have shown to activate PPARa or to
(1,2). PPARa is highly expressed in metabolically active tis-
sues including liver, muscle, intestine and brown adipose
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12677
467

Giampietro et al.
N
rac-17–20 were obtained by S 2 reaction of phenols 9–12
with ethyl 2-bromo-2-methylpropanoate or ethyl 2-bromo-
propanoate, in the presence of sodium metal in absolute
EtOH, under nitrogen atmosphere at reflux. The basic
hydrolysis of 13–16 and rac-17–20 with 1% alcoholic solu-
tion of KOH gave the acids 1, 3, 5, 7 and rac-2, rac-4,
rac-6, rac-8 (Scheme 1). The phenols 1-naphthol (9),
quinolin-2-ol (10) and 6-chloroquinolin-2-ol (11) are com-
mercially available; conversely, 1-anthrol (12) was synthe-
sized by the reaction of 1-anthrylamine in the presence of
NaHSO and EtOH at reflux (Scheme 2).
3
Figure 1: Chemical modifications of clofibric acid.
The clofibric acid chiral analogues (R)-2 and (S)-2 were
synthesized by using (S)-4-isopropyl-2-oxazolidinone as
chiral auxiliary because of its well-established capability of
optical resolution of the a-aryloxyacetic acids (22). The
lower plasma lipid levels (18,19). Therefore, our research
group has previously synthesized chiral analogues of clofi-
brate with different biological activity related to stereo-
chemistry (20,21).
2
acid rac-2 was chlorinated with SOCl to obtain the acyl
chloride rac-21 that was used to N-acylate the chiral auxil-
iary. The equimolar diastereomeric mixture of chiral imides
The present work describes the synthesis of methylated
and demethylated derivatives of clofibric acid (the active
metabolite of clofibrate), where the aromatic centre is sub-
stituted by naphthalene, quinoline, chloroquinoline or
anthracene scaffolds (Figure 1). The new compounds were
in vitro evaluated for their PPAR agonistic activity using the
reporter gene assay.
2
2 were obtained in good yield and separated on silica
gel. The cleavage with lithium hydroperoxyde led to both
dextrorotatory and levorotatory enantiomers of optical
active acid 2 (Scheme 3). The enantiomeric purity of (R)-2
and (S)-2 was evaluated by a capillary electrophoretic run,
by using b-cyclodextrin as a chiral selector. Analyses
showed enantiomeric excesses up to 98% for the two
tested compounds. As reported elsewhere, the com-
pounds (R)-2 and (S)-2 are levorotatory and dextrorotatory
All compounds (Figure 2) were easily obtained in good
yields by standard synthetic procedures. Esters 13–16 and
Figure 2: New compounds.
2
Scheme 1: Reagents and conditions: (a) ethyl 2-bromo-2-methylpropanoate or ethyl 2-bromopropanoate, Na, EtOH, N , reflux; (b) KOH,
EtOH, reflux.
3 2
Scheme 2: Reagents and conditions: (a) NaHSO , H O, EtOH, reflux.
4
68
Chem Biol Drug Des 2016; 87: 467–471

Naphthalene, Quinoline and Anthracene in PPARa Agonists
Scheme 3: Reagents and conditions: (a) SOCl
O, rt.
, CH Cl
2 2 2
, 40 °C; (b) BuLi, (S)-4-isopropyl-2-oxazolidinone, THF, À78 °C, (c) LiOOH, THF/
H
2
Table 1: In vitro transactivation activity of new clofibric acid analogues
O
COOH
Ar
R1 R2
a
b
b
b
Compound
Ar
R
1
R
2
clogP
PPARa (E%)
PPARc (E%)
PPARd (E%)
1
CH
H
H
3
CH
CH
CH
CH
3
3
3
3
3.269 Æ 0.262
2.929 Æ 0.255
2.929 Æ 0.255
2.929 Æ 0.255
37
50
106
56
23
36
35
12
31
29
12
33
rac-2
(
(
R)-2
S)-2
H
3
CH
H
3
CH
CH
3
2.458 Æ 0.305
2.108 Æ 0.293
43
31
32
23
32
23
rac-4
3
N
5
Cl
CH
H
3
CH
CH
3
3.104 Æ 0.334
2.754 Æ 0.324
37
50
22
35
11
22
rac-6
3
N
7
CH
H
3
CH
CH
3
4.499 Æ 0.263
4.150 Æ 0.256
12
12
14
24
34
15
rac-8
3
Clofibric acid
Cl
CH
3
CH
3
2.724 Æ 0.272
100
–
–
Rosiglitazone
L-165 041
–
–
–
–
100
–
–
100
a
clogP were calculated by using ACD/Labs Extension for CS ChemDraw (version 5.0).
b
Compounds were tested in at least three separate experiments at 150 lM. Efficacy values were calculated as percentage of the maxi-
mum obtained fold induction with the clofibric acid for PPARa, rosiglitazone for PPARc and L-165 041 for PPARd.
Chem Biol Drug Des 2016; 87: 467–471
469

Giampietro et al.
respectively (23). All compounds were purified by column
chromatography on silica gel or by crystallization and fully
characterized by IR, H and C NMR spectroscopy.
full agonist activity on PPARc and PPARd, showing lower
potency than rosiglitazone and L-165 041.
1
13
In conclusion, three series of clofibric acid analogues were
synthesized and in vitro evaluated for human PPARs trans-
activation assay. Compounds with naphthyl, quinolin and
6-chloroquinolin scaffold showed a better increase in the
transcriptional activity of receptor than the anthracenyl
derivatives. The best results were obtained with com-
pounds (R)-2 and (S)-2 that showed EC50 values of
6.9 Æ 0.6 lM and 11.6 Æ 0.9 lM respectively.
To investigate PPARa agonistic potency and subtype
selectivity, all compounds were evaluated for human
PPARa, PPARc and PPARd functional activity by a cell-
based transactivation assay in eukaryotic cells (24). In this
method, we utilized firefly luciferase reporter gene that pro-
vides a good assay sensitivity, dynamic range when quan-
tifying nuclear receptor activity and optimal correlation with
in vivo activity. In this study, we used clofibric acid, rosigli-
tazone and L-165 041, as reference compounds for
PPARa, PPARc and PPARd respectively; results are
expressed as efficacy (E%) relative to positive control.
These results and the clogP calculated with ACD/Labs
Extension for CS ChemDraw (version 5.0) were reported in
Table 1.
Acknowledgments
This study was supported by University “G. d’Annunzio” of
Chieti local grants.
All naphthyl derivatives (1, rac-2, (R)-2 and (S)-2) showed an
activation of PPARa higher than anthracenyl analogues (7 and
rac-8) but similar to quinolin and 6-chloroquinolin derivatives
Conflict of interest
The authors declare no conflict of interest.
(3, rac-4, 5 and rac-6). Probably, the activity is influenced by
the lipophilicity; indeed, these compounds have clogP values
similar to clofibric acid (1 clogP = 3.269 Æ 0.262; rac-2, (R)-2
and (S)-2 clogP = 2.929 Æ 0.255; 3 clogP = 2.458 Æ 0.305;
rac-4 clogP = 2.108 Æ 0.293; 5 clogP = 3.104 Æ 0.334;
rac-6 clogP = 2.754 Æ 0.324; clofibric acid clogP = 2.724 Æ
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Materials and methods.
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