Design, synthesis, and biological activity of novel PPARγ ligands based on rosiglitazone and 15d-PGJ2

Article abstract of DOI:10.1016/j.bmcl.2005.01.074

To develop novel PPARγ ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ₂. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARγ protein where the alkyl chain of 15d-PGJ₂ is expected to be located.

Full text of DOI:10.1016/j.bmcl.2005.01.074

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1547–1551
Design, synthesis, and biological activity of novel PPARc
ligands based on rosiglitazone and 15d-PGJ2
Shinya Usui, Takayoshi Suzuki, Yoshifumi Hattori, Kazuma Etoh,
Hiroki Fujieda, Makoto Nishizuka, Masayoshi Imagawa, Hidehiko Nakagawa,
Kohfuku Kohda and Naoki Miyata*
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
Received 25 December 2004; revised 30 January 2005; accepted 31 January 2005
Abstract—To develop novel PPARc ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which
are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as
potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group
of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARc protein where the alkyl chain
of 15d-PGJ2 is expected to be located.
Ó 2005 Elsevier Ltd. All rights reserved.
Peroxisome proliferator-activated receptors (PPARa,
PPARc, and PPARd) belong to the nuclear receptor
superfamily and act as ligand-activated transcription
factors.1–3 These receptors play a pivotal roles in regu-
lating the expression of a large number of genes involved
in lipid metabolism and energy balance.4 Many studies
on PPARs have been performed, and these efforts led
to the discovery of the clinically useful thiazolidinedione
(TZD) class of insulin sensitizers such as rosiglitazone5
and pioglitazone6 (Fig. 1), which are potent PPARc
agonists used in the treatment of Type 2 diabetes. How-
ever, the use of TZDs has been limited because of their
poor safety profiles. For example, troglitazone, which
came out to the market first, disappeared from the
market due to its severe hepatic toxicity in 1999,7 and
rosiglitazone is reported to be associated with liver,
cardiovascular, and hematological toxicities.8 We there-
fore initiated a search for non-TZD PPARc ligands with
the goal of finding novel insulin sensitizers. In this letter,
we report the design, synthesis, and biological activity of
non-TZD PPARc ligands based on the structure of ros-
iglitazone and 15-deoxy-D-12,14-prostaglandin J2 (15d-
PGJ2).
The compounds prepared for this study are shown in
Figure 2, and the routes used for their synthesis are illus-
trated in Schemes 1–4. Scheme 1 shows the preparation
of N-(pyridin-2-yl)-N-alkyl derivatives 1–3 bearing a
methyl, ethyl, and propyl group, respectively, on their
nitrogen atom as an alkyl chain.9 The protection of dialk-
ylamine 14a–c by (Boc)2O gave 15a–c.10 The Mitsun-
obu reaction11 was applied to the conversion of 15a–c
into 3-{4-(2-aminoethoxy)phenyl}propanoic acid deriv-
atives 16a–c: treatment of 15a–c with diethylazodicarb-
oxylate, PPh3, and 3-(4-hydroxyphenyl)propanoic acid
methyl ester 21 gave ethers 16a–c. The N-Boc groups
of 16a–c were removed with trifluoroacetic acid to give
amines 17a–c. Treatment of 17a–c with 2-fluoropyri-
dine, or 2-chloropyridine gave N-(pyridin-2-yl)-N-alkyl
compounds 18a–c via nucleophilic aromatic substitu-
tion, and subsequent hydrolysis afforded carboxylic
acids 1–3.
O
O
Me
N
Me
NH
NH
N
S
S
N
O
O
O
Pioglitazone
Rosiglitazone
Figure 1. Structures of rosiglitazone (GSK) and pioglitazone
(Takeda).
Keywords: PPARc ligand; 15d-PGJ2; Insulin sensitizer; Agonist.
*
Corresponding author. Tel./fax: +81 52 836 3407; e-mail: miyata-n@
phar.nagoya-cu.ac.jp
Current address: Faculty of Pharmaceutical Sciences, Musashino
University, 1-1-20 Shin-machi, Nishi-Tokyo City, Tokyo 202-8585,
Japan.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.074

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S. Usui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1547–1551
O
20a : n = 3
20b : n = 4
20c : n = 5
20d : n = 6
20e : n = 7
20f : n = 8
20g : n = 9
H
N
N
Br
N
1 : n = 0
2 : n = 1
3 : n = 2
4 : n = 3
5 : n = 4
6
7
8
9
: n = 5
: n = 6
: n = 7
: n = 8
a
(CH2)nCH3
(CH2)nCH3
OH
N
N
N
O
10 : n = 9
19
20a-g
1-10
O
O
O
OH
OH
b
OMe
OMe
N
N
Br
O
O
HO
22
21
11
23a : n = 3
23b : n = 4
23c : n = 5
23d : n = 6
23e : n = 7
23f : n = 8
23g : n = 9
O
O
X
(CH2)nCH3
OMe
c
12 : X = CH
13 : X = N
N
N
N
O
O
23a-g
12, 13
O
4 : n = 3
5 : n = 4
6 : n = 5
7 : n = 6
8 : n = 7
9 : n = 8
10 : n = 9
Figure 2. Structures of compounds 1–13.
(CH2)nCH3
OH
d
N
N
O
4-10
Boc
N
H
a
b
N
R
OH
R
OH
Scheme 2. Reagents and conditions: (a) CH3(CH2)nNH2, Pd2(DBA)3,
BINAP, t-BuOH, toluene, 80 °C, 7–53%; (b) 1,2-dibromoethane,
K2CO3, THF, 115 °C, 25%; (c) 20a–g, Et3N, KI, THF, 120 °C, 2–
25%; (d) aq NaOH, THF, rt, 81–96%.
15a : R = Me
15b : R = Et
15c : R = Pr
14a : R = Me
14b : R = Et
14c : R = Pr
O
O
OMe
Boc
N
OMe
H
c
N
R
O
R
O
O
17a : R = Me
17b : R = Et
17c : R = Pr
H
16a : R = Me
16b : R = Et
16c : R = Pr
N
a
OMe
N
N
O
O
N
24
25
R
N
OMe
d
O
N
O
b
OH
18a : R = Me
18b : R = Et
18c : R = Pr
N
O
N
11
O
Scheme 3. Reagents and conditions: (a) (i) NaH, DMF, rt; (ii) 22, KI,
90 °C, 72%; (b) aq NaOH, THF, rt, 89%.
R
N
OH
e
N
O
1 : R = Me
2 : R = Et
3 : R = Pr
H
X
Br
X
N
N
Scheme 1. Reagents and conditions: (a) Boc2O, CH2Cl2, 0 °C to rt, 91–
100%; (b) DEAD, PPh3, 3-(4-hydroxyphenyl)propanoic acid methyl
ester 21, THF, 0 °C to rt, 49–63%; (c) TFA, CH2Cl2, 0 °C to rt, 81–
94%; (d) 2-fluoropyridine or 2-chloropyridine, DMF, reflux, 7–38%;
(e) aq NaOH, THF/MeOH, rt, 78–92%.
a
26 : X = CH
19 : X = N
27a : X = CH
27b : X = N
O
N
b
c
OMe
The preparation of the other N-(pyridin-2-yl)-N-alkyl
derivatives 4–10 is outlined in Scheme 2. The prepara-
tion of 2-alkylamino pyridine 20a–g was achieved by
the method of Buchwald:12 treatment of 19 with n-alkyl-
amine, Pd2(DBA)3, BINAP, and t-BuONa in toluene
under reflux. Propanoic acid methyl ester 21 was al-
lowed to react with 1,2-dibromoethane to give ether
22. Coupling between amines 20a–g and ether 22 affor-
ded N-(pyridin-2-yl)-N-alkyl compounds 23a–g, and
subsequent hydrolysis afforded carboxylic acids 4–10.
X
N
O
28a : X = CH
28b : X = N
O
N
OH
X
N
O
12 : X = CH
13 : X = N
Scheme 4. Reagents and conditions: (a) 2-aminopyridine, Pd2(DBA)3,
BINAP, t-BuONa, toluene, 80 °C, 70–88%; (b) (i) NaH, DMF, rt, (ii)
22, KI, 90 °C, 18–30%; (c) aq NaOH, THF, rt, 70–80%.
N-(2-Pyridin-2-yl)-N-aryl derivatives 11–13 were pre-
pared by the procedure outlined in Schemes 3 and 4.

S. Usui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1547–1551
1549
Norharman 24 was reacted with bromide 22 in the pres-
ence of sodium hydride in DMF to give 9H-b-carboline
compound 25, and subsequent hydrolysis gave com-
pound 11 (Scheme 3). Compounds 27a and b were pre-
pared in the same way as 2-alkylamino pyridines 20a–g
(Scheme 4). Compounds 27a and b were allowed to react
with bromide 22 in the presence of sodium hydride in
DMF to give compounds 28a and b. Treatment of 28a
and b with aqueous NaOH gave N-(pyridin-2-yl)-N-phe-
nyl derivative 12 and dipyridinyl derivative 13.
The binding affinity of the compounds for PPARc was
evaluated with a CoA–BAP system (Microsystems).13
In this system, the alkaline phosphatase (AP) activity
is directly proportional to the PPARc-binding affinity
of the ligands.
Figure 3. View of the conformation of 15d-PGJ2 (tube) docked in
PPARc. The hydrophobic and hydrophilic regions are shown in yellow
and blue, respectively.
Since it has been revealed that the TZD ring can be re-
placed by a carboxyl group,14 we initially examined the
binding affinity for PPARc of compound 1, in which the
TZD group of rosiglitazone is replaced by a carboxyl
group. Although compound 1 did not show any activity
at 0.1 and 1 lM, a certain level of activity was observed
at 10 lM (Table 1, line 1). For the further design of
PPARc ligands, we focused on the alkyl chain of 15d-
PGJ2,15,16 an endogenous ligand of PPARc. Since cer-
tain fatty acids with a long alkyl chain are known to
be natural PPARc ligands,17 the hydrophobic moiety
is assumed to be critical for the binding affinity for
PPARc. Our study regarding the binding mode of
15d-PGJ2 in PPARc protein (PDB code 1FM6) by com-
puter calculation (Macromodel 8.1)18 also suggested
that the alkyl chain of 15d-PGJ2 is located in the wide
hydrophobic region of the PPARc ligand-binding cavity
(Fig. 3). However, the crystal structure of a PPARc/ros-
iglitazone complex19 revealed that rosiglitazone does not
have any hydrophobic groups interacting with the
hydrophobic amino acid residues of PPARc. We
hypothesized that the introduction of a hydrophobic
group into compound 1 may increase the affinity for
PPARc (Fig. 4). We therefore designed compounds 2–
10 in which alkyl groups of various lengths were intro-
duced at the 2-aminopyridinyl moiety of compound 1,
and evaluated their ability to bind PPARc. It was found
that the affinity of compounds 1–10 was closely related
to chain length, and the most potent compounds were
heptyl 7, octyl 8, and nonyl 9. In addition, N,N-diaryl
compounds 11–13 exhibited weak activity compared
with compounds 7–9 (Table 1, lines 11–13). We next
compared the binding affinity of compounds 7–9 with
that of rosiglitazone at lower concentrations. As shown
in Figure 5, compound 9 showed the highest activity
among the three, and had only slightly less affinity for
PPARc than did rosiglitazone.
As compound 9 was most active in our study, we used it
for further evaluation. Since it has been reported that
activation of PPARc enhances adipocyte differentia-
tion20 and increases insulin sensitivity, compound 9
was subjected to a rat abdominal preadipocyte differen-
tiation test.21,22 The accumulation of neutral fat in the
cells was observed after the administration of compound
9 at concentrations of 1, 2.5, and 5 lM, and the activity
of compound 9 was found to be comparable to that of
rosiglitazone (Fig. 6).
Table 1. Binding affinity for PPARc of compounds 1–13 at 0.1, 1.0,
and 10 lM.a
Since N-nonyl carboxylic acid 9 had a high level of activ-
ity, we studied its mode of binding to PPARc. A low en-
ergy conformation was calculated when 9 was docked in
a model based on the crystal structure of PPARc using
Macromodel 8.1 software.18 An inspection of the simu-
lated PPARc/9 complex suggested that oxygen atoms of
compound 9 form hydrogen bonds with Ser 289, Tyr
327, and Tyr 473 (Fig. 7). In addition, it was shown that
the nonyl group of 9 is located in the hydrophobic re-
gion formed by Phe 287, Gly 284, Ile 281, Ile 341, and
Met 348 (Fig. 8) where the alkyl chain of 15d-PGJ2 is
calculated to be located (Fig. 3).
0.1 µM
1.0 µM
10 µM
0.6
0.5
0.4
0.3
0.2
0.1
0
In summary, in order to explore novel PPARc ligands,
we prepared several 3-{4-(2-aminoethoxy)phenyl}prop-
anoic acid derivatives designed based on the structures
of rosiglitazone and 15d-PGJ2. Among them, N-(pyri-
din-2-yl)-N-nonyl compound 9 was found to be as
1
2
3
4
5
6
7
8
9 10 11 12 13
-0.1
Compounds
a Values are means of at least three experiments.

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S. Usui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1547–1551
2-aminopyridine moiety
Me
N
COOH
2-aminopyridine moiety
Alkyl chain
N
O
O
R1
N
OH
1
N
O
Me
COOH
Alkyl chain
2-10
O
15d-PGJ2
Figure 4. Structures of compounds 2–10 designed on the basis of the structure of 15d-PGJ2 and rosiglitazone.
7
8
9
rosiglitazone
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0001
0.001
0.01
0.1
1
10
Figure 7. View of the conformation of 9 (tube) docked in PPARc.
Residues around compound 9 and hydrogen bonds are displayed as
wires, and dotted lines, respectively. Figures represent distances in
angstroms.
Concentration (µM)
Figure 5. Binding affinity for PPARc of rosiglitazone and compounds
7–9 at 0.001, 0.01, 0.1, and 1.0 lM. Values are means of at least three
experiments.
Rosiglitazone
9
9
8
7
6
5
4
3
2
1
0
Figure 8. View of the conformation of 9 (tube) docked in PPARc. The
hydrophobic and hydrophilic regions are shown in yellow and blue,
respectively.
0
2
4
6
Concentration of compounds (µM)
Figure 6. Accumulation of fatty acid in rat preadipocytes by rosiglit-
azone and compound 9. Values are means of at least three experiments.
bonds with some hydrophilic amino acid residues, and
the nonyl group appropriately interacts with hydropho-
bic amino acid residues. The findings of this study
will help provide an effective agent for Type 2 diabetes.
potent as rosiglitazone in the binding assay and the prea-
dipocyte differentiation test. Molecular modeling sug-
gested that the carboxylate anion of 9 forms hydrogen

S. Usui et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1547–1551
1551
14. Rybczynski, P. J.; Zeck, R. E.; Dudash, J., Jr.; Combs, D.
W.; Burris, T. P.; Yang, M.; Osborne, M. C.; Chen, X.;
Demarest, K. T. J. Med. Chem. 2004, 47, 196.
Currently, further detailed studies on compound 9 are
under way.
15. Forman, B. M.; Tontonoz, P.; Chen, J.; Brun, R. P.;
Spiegelman, B. M.; Evans, R. M. Cell 1995, 83, 803.
16. Kliewer, S. A.; Lenhard, J. M.; Willson, T. M.; Patel, I.;
Morris, D. C.; Lehmann, J. M. Cell 1995, 83,
813.
Acknowledgements
This work was supported in part by grants from the
Health Sciences Foundation of the Ministry of Health,
Labor, and Welfare of Japan.
17. Kliewer, S. A.; Umesono, K.; Noonan, D. J.; Heyman,
R. A.; Evans, R. M. Nature 1992, 358, 771.
18. Docking and subsequent scoring were performed using
Macromodel 8.1 software. Coordinates of PPARc com-
plexed with rosiglitazone were taken from the Brookhaven
Protein Data Bank (PDB code 1FM6) and hydrogen
atoms were added computationally at appropriate posi-
tions. The structure of a ligand bound to PPARc was
constructed by molecular mechanics (MM) energy mini-
mization. The starting position of a ligand was determined
manually: its carboxyl group was superimposed onto the
TZD ring of crystallographic rosiglitazone. The confor-
mation of the ligand in the active site was minimized by a
MM calculation based upon the OPLS-AA force field with
each parameter set as follows; solvent: water, method:
LBFGS, Max # Iterations: 10,000, converge on: gradient,
convergence threshold: 0.05.
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