Design and synthesis of benzoxazole containing indole analogs as peroxisome proliferator-activated receptor-γ/δ dual agonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.027

A series of benzoxazole or benzothiazole containing indole analogs, 6-alkoxyindole-2-carboxylic acids and 5-alkoxy-3-indolylacetic acids, were synthesized as novel candidates of PPARγ/δ dual agonists and their ligand activities for PPAR subtypes (α, γ, an

Full text of DOI:10.1016/j.bmcl.2011.03.027

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3057–3061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of benzoxazole containing indole analogs as peroxisome
proliferator-activated receptor-
c/d dual agonists
⇑
Hyo Jin Gim, Ye-Jin Cheon, Jae-Ha Ryu, Raok Jeon
College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzoxazole or benzothiazole containing indole analogs, 6-alkoxyindole-2-carboxylic acids
Received 2 February 2011
Revised 24 February 2011
Accepted 9 March 2011
Available online 24 March 2011
and 5-alkoxy-3-indolylacetic acids, were synthesized as novel candidates of PPAR
their ligand activities for PPAR subtypes ( , and d) were investigated. In transient transactivation assay,
several compounds activated PPAR and d with little activity of PPAR . Putative binding mode of the
compounds 1a and 2a in the active site of PPAR was similar with that of rosiglitazone and the molecular
modeling provides molecular insight to the observed activity.
c/d dual agonists and
a, c
c
a
c
Keywords:
Indole
Ó 2011 Elsevier Ltd. All rights reserved.
Benzoxazole
PPAR
Diabetes
Peroxisome proliferator-activated receptors (PPARs) belong to a
subfamily of ligand-activated nuclear hormone receptors and func-
tion as key regulators of glucose, lipid, and cholesterol metabo-
lism.1 PPARs, upon ligand activation, heterodimerize with
retinoid X receptor (RXR) and the PPAR–RXR complex binds to per-
oxisome proliferator response elements (PPREs) and initiates the
transcription process of the target genes. Three PPAR subtypes
agonist are attractive strategy for the treatment of metabolic
syndrome.5
We made our endeavor for investigation of structural feature
aimed at the discovery of novel PPARc/d or PPAR-pan agonists. De-
spite structural diversity of the PPAR agonists, they share a com-
mon structural feature. Most of PPAR agonists compose of five
common elements; a polar head group (A) connected to an aro-
matic ring (C) through a short linker (B), a linker (D), and a hydro-
phobic tail group (E) (Fig. 1).
have been identified to date as PPAR
PPAR plays a critical role in the regulation of cellular uptake,
activation, and b-oxidation of fatty acid, and its agonists, the fi-
brates, are used to treat dyslipidemia. PPAR which is mainly asso-
a, PPARc
, and PPARd.2
a
Polar head group is a critical binding motif in the active site of
PPAR ligand binding domain (LBD). Various groups such as thiazo-
c
ciated with adipose-related functions regulates lipid metabolism,
lidinedione ring and a-alkoxy-substituted propionic acids have
lipid uptake into adipocytes, glucose homeostasis, and insulin sen-
been investigated as potent polar head groups. However, these po-
lar head groups are prone to racemization under physiological con-
ditions.6 Therefore, it is important to explore new polar head
groups with high affinity and structural stability. Our study was fo-
cused on the novel scaffolds of polar head and short linker to aro-
matic center. To start with, our chemical library was screened to
explore new polar head groups for PPAR ligand and molecular
docking as a computational tool was also employed for this pur-
pose. In vitro functional assay and docking analysis led us to a
number of compounds which showed weak PPAR agonistic activ-
ity. One of these was indole based structure, indole-2-carboxylic
acid, which was expected to be a good replacement of known
acidic head groups. In fact, several indole-based compounds have
been reported as PPAR agonists providing novel scaffold of polar
head group with planar bicyclic system.7–9 In the previous indole
analog, hydrophobic tail was mostly connected to nitrogen of in-
dole through poly methylene liker.
sitivity. PPAR
used as efficient insulin sensitizers in type 2 diabetes. Recently,
PPAR dual agonists have been studied intensively to combine
the insulin sensitizing potential of PPAR
cial activity of lipid regulation of PPAR
c agonists, specifically the thiazolidinediones, are
a/c
c
a
agonist with the benefi-
agonist.3 Most of them,
however, have been discontinued in clinical trial due to the adverse
toxicity profiles such as kidney and cardiovascular toxicity.4 Recent
research has revealed PPARd also play a role in the regulation of re-
verse cholesterol transport and high-density lipoprotein metabo-
lism, and its agonists such as GW501516 may be of use in the
treatment of dyslipidemia, obesity, and insulin resistance.
Regarding the beneficial pharmacological effects of each PPAR
subtypes, the development of PPARc/d dual agonist or PPAR-pan
⇑
Corresponding author. Tel.: +82 2 710 9571; fax: +82 2 715 9571.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.027

3058
H. J. Gim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3057–3061
(A)
(B)
(C)
(D)
(E)
a
N
N
X
Cl
N
Polar
Head
OH
n
Aromatic
Center
Hydrophobic
Tail
X
Linker
Linker
3, X= O, n=1
4, X= S, n=1
5, X= O, n=2
X= O, S
Figure 1. The general topology of synthetic PPAR agonists.
Scheme 1. Synthesis of hydrophobic tail, benzoxazolyl- or benzothiazolylmethyla-
minoalcohol. Reagents and conditions: (a) 2-methylaminoethanol or 3-methyl-
amino-1-propanol, TEA, THF.
In our indole-based analogs, the acidic head group was attached
to the C2- or C3-position of indole ring with/without a short linker
and hydrophobic tail was connected to the C5- or C6-position of in-
dole through poly methylene liker. It allowed the introduction of
extra substituent at the nitrogen of indole leading to the unique
feature in comparison with the reported indole-based PPAR ago-
nists. Herein, we report design, synthesis and biological evaluation
of the novel indole carboxylic acid and indolylacetic acid analogs
with hydrophobic tail of benzoxazole or benzothiazole (Fig. 2).
reaction of hydroxyindole 12 with the alcohol 4 in the presence of
tributylphosphine and 1,10-(azodicarbonyl)dipiperidine (ADDP)
gave compounds 13a–c which were hydrolyzed to final acids 1e–f.
Scheme 4 depicts the synthesis of benzoxazole-linked indolyl-
3-acetic acids. Esterification of 4-hydroxyindolyl-3-acetic acid gave
methyl ester 14, which was converted to 15a and 2d by Mitsunobu
reaction with respective 3 and 5. Introduction of various substitu-
ents to the nitrogen of indoles and the following hydrolysis of the
resulted N-alkylated compounds gave the target products 2b, 2c,
2g, and 2i.
PPARs agonist activities of the synthesized compounds were
evaluated by in vitro transient transactivation assays.11 To com-
pare the efficacy of compounds, GW0746, rosiglitazone and
GW0742 were used as reference compounds in the PPARa, c, and
d transactivation assays, respectively.
We first investigated PPARa, c, d transactivation activity of in-
dole carboxylic acid analogs as shown in Table 1. Introduction of
the substituents at nitrogen of the indole core led to increased
activity for PPAR, especially PPARc. Replacement of hydrophobic
benzoxazole with benzothiazole increased activity as compared
1b and 1c with 1f and 1g, respectively. Among the indole carbox-
ylic acid analogs, N-ethylated benzothiazole 1g showed the highest
To understand binding mode of the indole analogs into the PPAR
c,
docking experiments were also performed.
Synthetic routes for the preparation of compounds 1a–d are
outlined in Schemes 1 and 2. First, hydrophobic tails, benzoxazolyl-
and benzothiazolylmethylaminoalcohol 3–5, were prepared by
N-alkylation of 2-methylaminoethanol or 3-methylamino-1-pro-
panol with 2-chlorobenzoxazole or 2-chlorobenzothiazole
(Scheme 1).
Preparation of benzoxazole-linked indole-2-carboxylic acids
was started from compound 3. Mesylation of alcohol 3 and
O-alkylation of 4-hydroxybenzaldehyde with the mesylate 6 gave
aldehyde 7. Condensation of aldehyde 7 with methyl azidoacetate
in the presence of NaOMe and following thermolysis of the re-
sulted 2-azido-phenylacrylate 8 afforded indole 9a. The N-alkyl-
ation of indole 9a with corresponding alkyl halide in the
presence of base gave compounds 9b–d, which were hydrolyzed
to final acids 1b–d (Scheme 2).
Synthesis of benzothiazole-linked indole-2-carboxylic acids
was carried out in the similar way with that of benzoxazole ana-
logs (Scheme 3). 2-Azido-3-phenylacrylate 10 was prepared by
condensation of 4-methoxybenzaldehyde with methyl azidoace-
tate in the presence of NaOMe and converted to 6-methoxy-1H-in-
dole-2-carboxylate 11a via thermolysis. The N-alkylation of indole
11a and successive demethylation of arylmethyl ether 11a–c affor-
ded 6-hydroxy-1-alkyl-1H-indole-2-carboxylate 12a–c. Mitsunobu
PPAR
c
/d transactivation activity (66% efficacy for PPAR
c and 69%
for PPARd) with low PPAR
a
activity (17%).
We then explored indolylacetic acids which have more flexibil-
ity at the polar head region by adding a methylene linker between
indole core and acidic head, and their PPAR transactivation
activities were described in Table 2. In these series, both acidic
head group and linker were connected to respective C3- and
C5-positions of indole ring instead of C2- and C6-positions in the
O
O
Cl
O
O
NH
N
S
O
O
N
O
Fenofibrate
Rosiglitazone
PPARα agonist
PPARγ agonist
O
O
CO2H
F3C
O
S
OH
O
O
O
S
O
S
N
Tesaglitazar
GW501516
PPARα/γ dual agonist
PPARδ agonist
O
O
N
N
CO H
n
2
O
n
N
R
X
N
O
X = O, S
HO2C
PPAR pan agonist
Figure 2. Chemical structures of some known PPAR agonists and the representative structure of target compounds.

H. J. Gim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3057–3061
3059
N
O
a
b
N
O
N
O
N
N
N
O
CHO
OH
OMs
3
6
7
CO2CH3
N3
c
d
CO2R
N
N
N
N
N
N
O
O
O
H
O
O
8
9a, R = Me
1a, R = H
f
e
CO2R
N
N
R1
O
9b , R = Me, R1=Me
9c , R = Me, R1=Et
9d , R = Me, R1=Benzyl
1b , R = H, R1=Me
1c , R = H, R1=Et
1d , R = H, R1=Benzyl
f
Scheme 2. Synthesis of benzoxazole-linked indole-2-carboxylic acids. Reagents and conditions: (a) MsCl, TEA, CH2Cl2, rt, 2 h; (b) 4-hydroxybenzaldehyde, NaH, DMF; (c)
N3CH2COOMe, NaOMe, MeOH; (d) xylene, heat, 6 h; (e) R1X, NaH (or Cs2CO3), DMF; (f) NaOH, THF (or DMF), MeOH, H2O.
CHO
CO2CH3
N3
a
b
CO2R
N
H3CO
CH3O
CH3O
R1
10
11a, R1= H, R = Me
11b, R1, R = Me
11c, R1= Et, R = Me
c
e
d
CO2R
N
CO2R
N
N
N
O
HO
R1
R1
S
12a, R1= H, R = Me
12b, R1, R = Me
12c, R1= Et, R = Me
13a, R1= H, R = Me
13b, R1, R = Me
13c, R1= Et, R = Me
1e, R1 = H, R = H
1f, R1 = Me, R = H
1g, R1= Et, R = H
f
Scheme 3. Synthesis of benzothiazole-linked indole-2-carboxylic acids. Reagents and conditions: (a) N3CH2COOMe, NaOMe, MeOH; (b) xylene, heat, 6 h; (c) R1X, NaH (or
Cs2CO3), DMF; (d) BBr3; (e) 4, ADDP, PBu3, THF/toluene; (f) NaOH, THF (or DMF), MeOH, H2O.
CO2Me
CO2H
HO
HO
N
CO2R
b
a
O
O
N
n
N
H
N
H
N
H
14
15a, R = Me, n = 1
2a, R = H, n = 1
2d, R = Me, n = 2
2e, R = H, n = 2
d
N
c
CO2R
d
O
O
N
n
N
R1
15b, R1 = Me, R = Me, n = 1
2b, R1 = Me, R = H, n = 1
15c, R1 = Et, R = Me, n = 1
2c, R1 = Et, R = H, n = 1
2f, R1 = CO2Me, R = Me, n = 2
2g, R1 = CO2H, R = H, n = 2
d
d
d
2h, R1 = 4-fluorobenzyl, R = Me, n = 2
2i, R1 = 4-fluorobenzyl, R = H, n = 2
2j, R1 = 4-chlorobenzoyl, R = Me, n = 2
d
Scheme 4. Synthesis of benzoxazole-linked indolyl-3-acetic acids. Reagents and conditions: (a) SOCl2, MeOH; (b) 3 or 5, ADDP, PBu3, THF/toluene; (c) R1X, NaH (or Cs2CO3),
DMF; (d) NaOH, THF (or DMF), MeOH, H2O.

3060
H. J. Gim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3057–3061
Table 1
In vitro functional transactivation activity of 6-alkoxyindole-2-carboxylic acids on
murine PPARa
CO2H
N
N
N
R1
O
X
1
Compd
X
R1
% Maxb (20
lM)
mPPAR
a
mPPAR
c
mPPARd
1a
1b
1c
1d
1e
1f
O
O
O
O
S
H
Me
Et
Bn
H
Me
Et
NA
NA
NA
10.0
NA
NA
NA
NA
NA
NA
33.4
14.6
27.9
68.8
23.3
43.9
NA
29.6
66.4
S
S
13.6
16.7
1g
NA means not active, which is for compounds producing transactivation activity
lower than 10% at 20 M.
l
a
Compounds were tested in at least three separate experiments.
Fold activation relative to maximum activation obtained with GW0746 (1
b
Figure 3. Superposition of the structure of 1a (red), 2a (blue) and crystallized
rosiglitazone (green) in the binding pocket of PPAR
l
M)
c
.
for PPARa, with rosiglitazone (1 lM) for PPARc, and with GW0742 (0.1 lM) for
PPARd.
acids, docking experiments into the hPPAR
main were carried out using Surflex Dock interfaced with SYBYL-
X version 1.2 on Linux. The crystal structure of the human PPAR
c receptor binding do-
Table 2
In vitro functional transactivation activity of 5-alkoxy-3-indolylacetic acids on
murine PPAR
c
a
in complex with rosiglitazone (PDB code: 2PRG)10 was employed
for the docking study. In this automated docking program, the flex-
ibility of the ligands is considered while the protein is considered
as a rigid structure. The molecules for docking were sketched in
the SYBYL and energy minimizations were performed using Tripos
Force Field and Gasteiger–Huckel charge with 20,000 iterations of
conjugate gradient method with convergence criterion of
0.05 kcal/mol. The docking analyses of compounds 1a and 2a were
performed using the internal default parameters for all the
variables.
N
CO2R2
O
O
N
n
N
R1
2
Compd
n
R1
R2
% Maxb (20
lM)
mPPAR
a
mPPAR
c
mPPARd
2a
2b
2c
2d
2e
2f
2g
2h
2i
1
1
1
2
2
2
2
2
2
2
H
H
H
H
Me
H
Me
H
Me
H
NA
28.0
NA
NA
NA
NA
NA
NA
13.3
NA
28.1
58.6
66.7
42.1
18.3
37.1
NA
NA
15.8
28.9
NA
NA
32.9
NA
NA
NA
NA
NA
NA
18.3
According to the crystal structure of rosiglitazone in the LBD of
PPARc, thiazolidinedione ring of rosiglitazone interacts with resi-
dues His323, His449 and Tyr473. Many reports have suggested
that the specific polar interactions are important for the activities
Me
Et
H
H
of PPAR
AF-2 helix in a conformation favoring the binding of co-activators
to PPARc 10
In the predicted binding orientation, both 1a and 2a
c agonists, as this H bonding network could stabilize the
CH2CO2Me
CH2CO2H
CH2(4-F–C6H4)
CH2(4-F–C6H4)
CO(4-Cl–C6H4)
.
were occupied the same spatial position and had specific interac-
tions with the critical amino acids in the binding pocket as the
2j
Me
NA means not active, which is for compounds producing transactivation activity
lower than 10% at 20 M.
l
a
Compounds were tested in at least three separate experiments.
Fold activation relative to maximum activation obtained with GW0746 (1
b
lM)
for PPARa, with rosiglitazone (1 lM) for PPARc, and with GW0742 (0.1 lM) for
PPARd.
indole carboxylic acids. Subsequently, the distance between car-
boxylic acid and oxygen of the linker was kept in both seriese of
indole carboxylic acids and indolylacetic acids. As expected, indol-
ylacetic acids 2a–c revealed higher activity than corresponding in-
dole carboxylic acids 1a–c. Similarly with indole carboxylic acids (
1a–c), N-methyl (2b) or ethyl (2c) substituted analogs of indole
ring revealed higher PPARc transactivation activity than un-substi-
tuted indole (2a) did. However, introduction of para-substituted
benzyl (2h–j), carboxy ester (2f), or carboxyl group (2g) was not
favorable for the activity. Effect of lengthening (2e) of the linker
between indole core and benzoxazole on the activity was not clear.
Most of the active compounds showed higher activity for PPAR
over PPARd. PPAR transactivation activity was ignorable.
c
a
To help interpretation of SAR data and to rationalize the differ-
Figure 4. View of important polar interactions between 1a (red) and 2a (blue) and
ent activities between indole carboxylic acids and indolylacetic
the critical residues of the PPARc.

H. J. Gim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3057–3061
3061
crystallized rosiglitazone did (Fig. 3). Compared the predicted
References and notes
binding mode of 1a with 2a, the carboxy group of 1a made H-bond
only with Tyr473, whereas the carboxy group of 2a made two H-
bonds with Tyr473 and His449 (Fig. 4). This docking result might
1. Oliver, W. R., Jr.; Shenk, J. L.; Snaith, M. R.; Russell, C. S.; Plunket, K. D.; Bodkin,
N. L.; Lewis, M. C.; Winegar, D. A.; Sznaidman, M. L.; Lambert, M. H.; Xu, H. E.;
Sternbach, D. D.; Kliewer, S. A.; Hansen, B. C.; Willson, T. M. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 5306.
2. Braissant, O.; Foufelle, F.; Scotto, C.; Dauca, M.; Wahli, W. Endocrinology 1996,
137, 354.
3. Chaput, E.; Saladin, R.; Silvestre, M.; Edgar, A. D. Biochem. Biophys. Res. Commun.
2000, 271, 445.
4. Balakumar, P.; Rose, M.; Singh, M. Pharmacology 2007, 80, 1.
5. Wilkinson, A. S.; Monteith, G. R.; Shaw, P. N.; Lin, C. N.; Gidley, M. J.; Roberts-
Thomson, S. J. J. Agric. Food. Chem. 2008, 56, 3037.
6. Sohda, T.; Mizuno, K.; Kawamatsu, Y. Chem. Pharm. Bull. 1984, 32, 4460.
7. Dropinski, J. F.; Akiyama, T.; Einstein, M.; Habulihaz, B.; Doebber, T.; Berger, J.
P.; Meinke, P. T.; Shi, G. Q. Bioorg. Med. Chem. Lett. 2005, 15, 5035.
8. Henke, B. R.; Adkison, K. K.; Blanchard, S. G.; Leesnitzer, L. M.; Mook, R. A., Jr.;
Plunket, K. D.; Ray, J. A.; Roberson, C.; Unwalla, R.; Willson, T. M. Bioorg. Med.
Chem. Lett. 1999, 9, 3329.
9. Mahindroo, N.; Peng, Y. H.; Lin, C. H.; Tan, U. K.; Prakash, E.; Lien, T. W.; Lu, I. L.;
Lee, H. J.; Hsu, J. T.; Chen, X.; Liao, C. C.; Lyu, P. C.; Chao, Y. S.; Wu, S. Y.; Hsieh, H.
P. J. Med. Chem. 2006, 49, 6421.
provide the structural insight for the higher PPARc transactivation
activity of indolylacetic acid analogs 2a–c than those of indole car-
boxylic acid analogs 1a–c. Interestingly, an additional polar inter-
action was observed between the oxygen of benzoxazole ring
and Arg288 in the region positioned by hydrophobic tail.
In summary, series of novel indole carboxylic acids and indolyl-
acetic acids were prepared as PPARc/d agonists and carefully inves-
tigated their structural feature. In general, indolylacetic acids were
more active than the corresponding indole carboxylic acids. Dock-
ing analysis imply that indolylacetic acids likely form more favor-
able H bond network in the LBD of PPARc. We suggest that
indolylacetic acid structure might act as a versatile template for
the creation of novel PPAR agonists and further SAR exploration
is underway.
10. Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.;
Rosenfeld, M. G.; Willson, T. M.; Glass, C. K.; Milburn, M. V. Nature 1998, 395,
137.
Acknowledgment
11. Transient transfection and luciferase assay: CV-1 cells were seeded in 48-well
plate at a density of 1.5 ꢀ 105 cells/well in DMEM with 10% FBS. The cells were
transiently transfected with plasmid mixtures containing PPARs expression
vector and tk-PPRE-luciferase (Luc) vector for 6 h, and then treated with
samples for 24 h. To normalize transfection efficiency, b-galactosidase plasmid
was cotransfected. The luciferase activities in cell lysates were measured using
luciferase assay system (Promega Corp., Madison, WI) and the b-gal activities
were measured as the absorbance at 410 nm by using an ELISA plate reader.
Data are reported as relative luciferase activity divided by the b-galactosidase
activity. All the constructs were kindly gifted by Dr. Ronald M. Evans at The
Salk Institute (La Jolla, CA).
This research was supported by the Sookmyung Women’s Uni-
versity Research Grants (2009).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.027.