Discovery of new PPARγ agonists based on arylopeptoids

Article abstract of DOI:10.1016/j.bmcl.2013.05.034

In this study we present the design, synthesis and biological evaluation of a small, first-generation library of small molecule aromatic amides based on the arylopeptoid skeleton. The compounds were efficiently synthesized using a highly convenient submonomer solid-phase methodology which potentially allows for access to great product diversity. The synthesized compounds were tested for their ability to activate peroxisome proliferator-activated receptors (PPARs) and they all acted as PPARγ agonists in the μM range spanning from 2.5- to 14.7-fold activation of the receptor. This is the first discovery of bioactive molecules based on the arylopeptoid architecture.

Full text of DOI:10.1016/j.bmcl.2013.05.034

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4162–4165
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new PPARc agonists based on arylopeptoids
Kasper Worm-Leonhard a, Thomas Hjelmgaard a, Rasmus K. Petersen b, Karsten Kristiansen b,
John Nielsen c,
⇑
a Department of Chemistry, Section for Chemical Biology and Nanobioscience, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
b Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark
c Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we present the design, synthesis and biological evaluation of a small, first-generation library
of small molecule aromatic amides based on the arylopeptoid skeleton. The compounds were efficiently
synthesized using a highly convenient submonomer solid-phase methodology which potentially allows
for access to great product diversity. The synthesized compounds were tested for their ability to activate
Received 10 April 2013
Revised 7 May 2013
Accepted 9 May 2013
Available online 18 May 2013
peroxisome proliferator-activated receptors (PPARs) and they all acted as PPARc agonists in the lM range
spanning from 2.5- to 14.7-fold activation of the receptor. This is the first discovery of bioactive mole-
cules based on the arylopeptoid architecture.
Keywords:
Lifestyle diseases
Peroxisome proliferator-activated receptors
Aromatic amide
Ó 2013 Elsevier Ltd. All rights reserved.
Solid-phase synthesis
Library synthesis
Metabolic disorders such as obesity, type 2 diabetes mellitus,
insulin resistance and atherosclerosis are escalating globally at
an epidemic rate. Although these so-called ‘lifestyle diseases’ are
related to the balance between energy expenditure and energy
intake, the underlying biochemistry and pharmacology is excep-
tionally complex and include the interaction and orchestration of
numerous receptors such as the peroxisome proliferator-activated
receptors (PPARs).1 PPARs are ligand-activated transcription fac-
tors belonging to the nuclear receptor superfamily. PPARs are
known to play an important role in the general transcriptional con-
trol of numerous cellular processes, including lipid metabolism,
glucose homeostasis, cell cycle progression, cell differentiation,
inflammation and extracellular matrix remodeling. Three subtypes
subpocket of the PPARc ligand binding site while the aromatic tail
can bind to the second and highly hydrophobic subpocket.
Intriguingly, this pharmacophore model can be accommodated
by the structure of N-substituted aminomethyl benzamides, or
arylopeptoids, which is a novel class of aromatic oligoamides.8
We have recently developed highly efficient solution-phase and
solid-phase methodologies for ‘submonomer’ synthesis of arylo-
peptoids in which the arylopeptoid residues are created directly
on the growing chain in an iterative manner by acylation-substitu-
tion cycles (Fig. 2). Only inexpensive and easily accessible acylation
reagents derived from bromo- or chloromethylbenzoic acids are
needed in the acylation steps and in principle, any imaginable pri-
mary amine may be used to install the side chain in the ensuing
substitution steps. This methodology therefore gives facile access
to products of highly diverse nature.
of PPAR have been identified: PPAR
with PPAR being the most extensively investigated isoform as
well as the most highly expressed PPAR subtype in adipocytes
and macrophages.2 A number of small-molecule PPAR
agonists
a, PPARb/PPARd and PPARc,
c
Only few applications of N-substituted aromatic oligoamides
have been demonstrated to date: an example is the formation of
crescent and helical structures in N-substituted benzanilides,9
c
have been identified, comprising the antidiabetic thiazolidinedi-
ones (TZDs) rosiglitazone,3 troglitazone,4 farglitazar,5 and non-
and the use of these as
a-helix mimetics for inhibition of
TZD L-7964496 (Fig. 1). Extensive SAR studies on PPAR
c
ligands
protein–protein p53–hMDM2 interactions.10 Herein, we describe
the discovery of bioactive compounds based on N-substituted
aminomethyl benzamide backbones.
can be summarized in a simplified pharmacophore model where
chemical determinants such as an acidic head group, an aromatic
linker and a hydrophobic (hetero)aromatic tail are key elements.7
The acidic head group can form polar interactions with one
Inspired by the combination of backbone similarities with
PPAR
signed a first generation library of potential PPAR
and 3a–i consisting of an arylopeptoid skeleton (Scheme 2). Since
a number of the existing PPAR agonists contain a phenylpropionic
acid moiety we decided to incorporate para-substituted
c
agonists and our convenient synthetic methods we de-
c
agonists 2a–i
c
⇑
Corresponding author. Tel.: +45 35336706.
E-mail address: john.nielsen@sund.ku.dk (J. Nielsen).
a
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.034

K. Worm-Leonhard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4162–4165
4163
O
O
NH
NH
O
S
N
N
S
O
O
O
O
HO
Rosiglitazone
Troglitazone
OH
O
O
O
N
O
HN
O
S
O
Cl
O
OH
Farglitazar
L-796449
Figure 1. Selection of PPARc agonists.
(O-benzotriazolyl-N,N-tetramethyluronium hexafluorophosphate)
in the substitution steps (Scheme 2).8b Thus, phenylpropionic acid
1 was first reacted with a 2-chlorotrityl chloride polystyrene resin
in CH2Cl2 in the presence of DIPEA. The first side chain was then
installed by reaction with the required primary amine (ethyl-,
isopropyl- or 2,4-dimethoxybenzylamine) in DMSO at 50 °C.
COMU-mediated acylation of the resulting secondary amine with
either 3- or 4-chloromethylbenzoic acid followed by an additional
substitution reaction with one of the three selected primary
amines, and then capping with benzoylchloride completed the
synthesis. Products containing 2,4-dimethoxybenzyl side chains
(2a–e and 3a–e) were cleaved off with TFA–H2O (95:5) which re-
sulted in concomitant removal of the 2,4-dimethoxybenzyl side
chains thereby liberating the secondary amides while the remain-
ing products (2f–i and 3f–i) were cleaved off using HFIP–CH2Cl2
(1:4) as previously described. The desired products 2a–i and 3a–i
were obtained in >97% HPLC purity in 33–42% yield (18–24 mg
product) after preparative HPLC purification.
O
X
R
N
n
O
Iterative "submonomer" synthesis
O
Acylation
Substitution
R
N
H
n
Arylopeptoid
Figure 2. Arylopeptoids: N-substituted aminomethyl benzamides.
The synthesized compounds were analyzed for their ability to
activate PPARc. A mouse embryo fibroblast cell line was tran-
O
O
siently transfected with a Gal4 responsive luciferase reporter and
a plasmid encoding the fusion between the Gal4 DNA binding do-
(a)
OH
OH
Cl
1
main and the human PPAR
very sensitive for identification of PPAR
the luciferase reporter is indicative of ligand dependent stimula-
tion of PPAR . Rosiglitazone was used as a positive control for
PPAR activation. The PPAR activating properties of the com-
c
ligand binding domain. The system is
c
agonists and activation of
Scheme 1. Reagents and conditions: synthesis of 1. Key: (a) formaldehyde (37% aq),
HCl (g), ZnCl2, 80 °C, 12 h.
c
phenylpropionic acid moiety at the C-terminus rather than a ben-
zoic acid moiety. At the second residue the compounds carry either
a para- or a meta-arylopeptoid residue in order to investigate the
possible effects of substitution pattern on the agonist activities.
Furthermore, three types of substituents were installed at the
backbone nitrogens: a simple proton (obtained by acidic removal
of 2,4-dimethoxybenzyl side chains), an ethyl or an isopropyl side
chain. In our previous studies we have established that the amide
group of the arylopeptoid backbone may exist as a cis/trans mix-
ture which may be controlled by proper choice of side chains.8
Thus, an increasing content of cis amide is obtained for simple alkyl
side chains of increasing bulk and therefore, there will be a higher
content of cis amide when installing isopropyl side chains than
ethyl side chains which we speculated may effect the agonist
efficiency.
c
c
pounds were compared to both the DMSO vehicle and the rosiglit-
azone positive control and are depicted in Table 1 as fold activation
over vehicle and relative activation compared to rosiglitazone.
Screening was performed at 10 and 100
synthesized compounds did not display cytotoxicity and were all
PPAR agonists in the M range spanning from 2.5- to 14.7-fold
lM concentration. The
c
l
activation of the receptor. However, no clear trend between struc-
ture and activity could be deduced.
An increased activity in the transactivation assay is only indic-
ative of agonist activity. In order to measure binding to the recep-
tor more directly we furthermore analyzed the ability of selected
compounds to displace a known ligand as determined by an
in vitro competitive binding assay using time-resolved fluores-
cence resonance energy transfer. A terbium labeled anti-GST anti-
Prior to solid-phase synthesis of the designed library we thus
only needed to synthesize the altered starting material 2-[4-(chlo-
romethyl)phenyl]propanoic acid 1 which was obtained in 42%
yield in one step by chloromethylation of 3-phenylpropanoic acid
as described in the literature (Scheme 1).11
body was used to label purified GST-tagged human PPARc ligand
binding domain. Energy transfer from terbium to the tracer, a fluo-
rescent pan PPAR agonist, enabled read-out of each test com-
pound’s ability to displace the tracer. As seen from Table 2 the
three tested compounds (3a, 3e and 3f) were all able to compete
The designed library was then synthesized by direct adaptation
of our previously described solid-phase methodology for
submonomer synthesis of arylopeptoids based on the use of COMU
for binding to the PPAR
PPAR is a rather promiscuous receptor and the relatively large
ligand binding pocket allow for association with a variety of
c-LBD though with relatively low affinity.
c

4164
K. Worm-Leonhard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4162–4165
O
O
Cl
R1
N
O
(a)
(b)
O
Cl
PS
Cl
H
(d)
(c)
O
O
O
O
R1
N
O
O
Cl
R1
N
O
Cl
O
O
(b)
(b)
H
O
R2
N
R1
N
N
R2
R1
N
O
H
O
O
(e), (f)
(e), (f)
a: R1 = H, R2 = H
b: R1 = H, R2 = Et
c: R1 = H, R2 = iPr
d
e
O
O
: R1 = Et, R2 = H
: R1 = iPr, R2 = H
: R1 = Et, R2 = Et
N
R2
R1
N
OH
R2
N
R1
N
OH
f
g
O
2a-i
O
O
: R1 = Et, R2 = iPr
3a-i
h: R1 = iPr, R2 = Et
i: R1 = iPr, R2 = iPr
Scheme 2. Reagents and conditions: solid-phase submonomer synthesis of 2a–i and 3a–i. Key: (a) 1 (1.2 equiv, 0.14 M), DIPEA (6.0 equiv), CH2Cl2, rt, 1 h; (b) R-NH2,
(20 equiv, 2.0 M), DMSO, 50 °C, 1 h; (c) 4-(ClCH2)ArCOOH (3.0 equiv, 1.0 M), COMU (3.5 equiv), DIPEA (7.0 equiv), rt, 20 min; (d) 3-(ClCH2)ArCOOH (3.0 equiv, 1.0 M), COMU
(3.5 equiv), DIPEA (7.0 equiv), rt, 20 min; (e) BzCl (4.0 equiv, 1.0 M), DIPEA (8.0 equiv), CH2Cl2, rt, 15 min; (f) Compounds 2a–e and 3a–e: TFA/water 95:5, rt, 1 h; 2f–i and 3f–i:
HFIP–CH2Cl2 1:4, rt, 30 min.
Table 1
PPAR
activation of all synthesized compoundsa
Table 2
Evaluation of binding affinity of selected compounds
c
Compd
Fold above vehicle
Relative to rosiglitazone (%)
Compd Ligand displacement PPAR
a
PPARd
RXRa
(IC50
,
lM)
activation
activation
activation
Avg.
Std.
Avg.
Std.
3a
3e
3f
62.7
38.7
56.2
À
À
+
À
À
À
À
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
2f
2.5
5.6
6.0
9.3
5.7
7.3
6.2
8.1
12.6
13.4
7.3
11.2
3.6
6.2
6.9
5.8
14.7
5.3
0.6
0.9
1.5
2.1
1.4
1.3
1.6
1.1
1.3
3.1
0.5
2.8
1.5
0.6
0.9
1.5
2.5
1.2
5.4
14.1
13.1
23.5
12.5
18.4
11.8
15.1
21.2
27.2
13.8
20.9
6.9
1.4
2.2
3.2
5.3
3.0
3.3
3.0
2.2
2.2
6.4
0.9
5.3
2.9
1.2
1.4
3.1
4.2
2.4
++
À
Signatures: À, no effect (less than twofold activation); +, activation (more than
twofold, less than fivefold); ++, activation (more than fivefold, less than 10-fold).
identical to the PPAR
of the PPAR -LBD with PPAR
Among the selected compounds only 3a specifically activated
PPAR , whereas 3e and 3f also activated RXR and PPAR , respec-
tively (Table 2).
It has been shown that thiazolidinediones such as rosiglitazone
agonists but also as free fatty acid receptor 1
c
transactivation assay but with substitution
c
a
-, PPARb-/PPARd- or RXR -LBD.
a
3f
c
a
a
2g
3g
2h
3h
2i
11.6
11.6
11.8
24.7
10.7
not only act as PPAR
c
(FFA1) agonists.12 FFA1 responds to free fatty acids and increases
glucose stimulated insulin secretion in pancreatic b-cells, and has
recently also appeared as an interesting target for treatment of
type 2 diabetes.13 This prompted us to screen our ensemble of
compounds towards FFA1 but no agonist or antagonist activity
3i
a
Compounds were screened at 100
l
M concentration.
compounds. Thus, it is not unusual for PPAR
c
to ‘share’ ligands
was observed at a 10 lM level (data not shown). These results indi-
with the family members PPAR
a
and PPARb/PPARd or members
cate that this arylopeptoid-based compound series are selective to-
of the family of obligate heterodimerization partners retinoid X
receptors (RXRs). To evaluate the selected compounds’ receptor
selectivity they were tested in transactivation assays essentially
wards PPAR with no cross-activation of FFA1.
In conclusion, we have used our previously developed solid-
phase methodology for facile synthesis of a small, first-generation

K. Worm-Leonhard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4162–4165
4165
library of small molecule aromatic amides based on an arylopep-
toid backbone. The synthesized compounds did not display cyto-
toxicity and were all PPAR agonists in the M range spanning
from 2.5- to 14.7-fold activation of the receptor. However, no clear
trend between structure and activity could be deduced. Evaluation
of three selected compounds’ (3a, 3e and 3f) binding affinity
showed that they were all able to compete for binding to the
References and notes
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c-LBD although with moderate affinity. With respect to
c, whereas
a
a
the first discovery of bioactive arylopeptoids, further diversifica-
tion of the compounds holds promise that more active and selec-
tive arylopeptoids can be developed.
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The authors are grateful for financial support from the Lund-
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Matthias Kassack at University of Düsseldorf, Germany, are
thanked for testing the compound against FFA1 and Professor
Trond Ulven at University of Southern Denmark, Odense, Denmark
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Supplementary data
Supplementary data (PPAR assays, FFA1 assays, synthetic
procedures, and characterization data for all new compounds)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2013.05.034.
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