Identification of a novel ppar-γ agonist through a scaffold tuning approach

Article abstract of DOI:10.3390/ijms19103032

Peroxisome proliferator-activated receptors (PPARs) are important targets in metabolic diseases including obesity, metabolic syndrome, diabetes, and non-alcoholic fatty liver disease. Recently, they have been highlighted as attractive targets for the trea

Full text of DOI:10.3390/ijms19103032

International Journal of
Molecular Sciences
Article
Identification of a Novel PPAR-γ Agonist
through a Scaffold Tuning Approach
Hyo Jin Gim †, Yong-Sung Choi †, Hua Li, Yoon-Jung Kim, Jae-Ha Ryu and Raok Jeon *
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Sookmyung Women’s University,
Cheongpa-ro 47-gil 100, Yongsan-gu, Seoul 04310, Korea; hjgim83@gmail.com (H.J.G.);
uriys2@gmail.com (Y.-S.C.); cooldog227@hotmail.com (H.L.); yoonjungkim73@gmail.com (Y.-J.K.);
ryuha@sookmyung.ac.kr (J.-H.R)
* Correspondence: rjeon@sookmyung.ac.kr; Tel.: +82-2-710-9571
† These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 August 2018; Accepted: 2 October 2018; Published: 4 October 2018
Abstract: Peroxisome proliferator-activated receptors (PPARs) are important targets in metabolic
diseases including obesity, metabolic syndrome, diabetes, and non-alcoholic fatty liver disease.
Recently, they have been highlighted as attractive targets for the treatment of cardiovascular diseases
and chronic myeloid leukemia. The PPAR agonist structure is consists of a polar head, a hydrophobic
tail, and a linker. Each part interacts with PPARs through hydrogen bonds or hydrophobic interactions
to stabilize target protein conformation, thus increasing its activity. Acidic head is essential for PPAR
agonist activity. The aromatic linker plays an important role in making hydrophobic interactions
with PPAR as well as adjusting the head-to-tail distance and conformation of the whole molecule.
By tuning the scaffold of compound, the whole molecule could fit into the ligand-binding domain to
achieve proper binding mode. We modified indol-3-ylacetic acid scaffold to (indol-1-ylmethyl)benzoic
acid, whereas 2,4-dichloroanilide was fixed as the hydrophobic tail. We designed, synthesized,
and assayed the in vitro activity of novel indole compounds with (indol-1-ylmethyl)benzoic acid
scaffold. Compound 12 was a more potent PPAR-γ agonist than pioglitazone and our previous hit
compound. Molecular docking studies may suggest the binding between compound 12 and PPAR-γ,
rationalizing its high activity.
Keywords: PPAR; agonist; indole; scaffold; tuning
1. Introduction
Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the
nuclear receptor superfamily. There are three subtypes of PPARs, designated as PPAR- , - , and - ),
α
γ
δ(β
which exhibit different tissue expression profiles and modulate specific physiological functions. PPARs
play a critical role in the regulation of multiple genes that regulate glucose and lipid metabolism
and energy homeostasis [
are important molecular targets for the development of new drugs for metabolic diseases, such as
obesity, metabolic syndrome (MetS), diabetes, and non-alcoholic fatty liver disease (NAFLD) [ 11].
1–5]. Because they are involved in multiple metabolic pathways, PPARs
6
–
Recent studies have shown that activation of PPARs not only regulates metabolic pathways but also
mediates various biological effects related to inflammation, apoptosis, oxidative stress, and vascular
function [12–16]. These effects seem to be beneficial in other disease conditions. It has been reported
that the anti–diabetic PPAR agonists are also effective in cardiovascular disease (CVD) [17
thyroid [20,21], colorectal [21], and lung cancer [22] and chronic myeloid leukemia (CML) [23–25].
Currently, the PPAR- agonists, fibrates (e.g., gemfibrozil, Figure 1) are used to treat dyslipidemia,
whereas the PPAR- agonists, thiazolidinediones (TZDs; e.g., rosiglitazone, Figure 1) are used to treat
–19],
α
γ
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type 2 diabetes mellitus (T2DM). However, the use of TZDs is associated with various adverse effects,
particularly weight gain, bone fractures, cardiovascular complications, and edema [26 28]. No drugs
in the market have been identified to target PPAR- ). Continuous efforts are being made by many
–
δ(β
research groups and pharmaceutical companies worldwide to develop potent and safe therapeutic
agents for the treatment of PPAR-associated diseases. In particular, they aim to develop pan, dual,
or selective agonists of the three PPAR subtypes [29–33]. Representative PPAR agonists are depicted
in Figure 1.
Figure 1. Structures of the representative PPAR agonists and newly designed compounds.
All subtypes of PPAR share structural and functional feature similar to those of other nuclear
receptors. Crystal structures of human PPAR-
three-dimensional structure of the ligand binding domains (LBDs). An antiparallel sandwich of
12 -helices (helix 1 to helix 12) and a three-stranded antiparallel -sheet are forming a large ligand
binding cavity in the core of the LBD. The central cavity spans between the AF-2 domain within
C-terminal -helix 12 and the three-stranded antiparallel -sheet [34]. Interestingly, despite a common
general structure of the LBD, ligand binding to PPARs shows both species and isotype specificities.
The ligands for PPAR- or PPAR- should be able to adopt a U-shaped conformation and an L-shaped
conformation for PPAR-δ(β) [35].
α, PPAR-γ, and PPAR-δ(β) have revealed a common
α
β
α
β
α
γ
A typical PPAR agonist consists of three parts: a hydrophobic tail moiety, a polar head group
(usually bearing a carboxylic acid functionality) and a linker which consists of flexible methylene units
and an aromatic ring (Figure 2A). The acidic head group is crucial for PPAR activation. It forms an
H-bonding network with a part of the PPAR that mainly contains the critical polar residues, such as
Gln286, Ser289, His323, Tyr327, Lys367, His449, and Tyr473 in PPAR-
γ (see Figure 2B). The TZD moiety
(acidic head group of rosiglitazone) makes several specific interactions with amino acids. The carbonyl

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groups of the TZD make hydrogen bonds with His323 and His449. His323 forms a secondary hydrogen
bond with Tyr473 in the AF-2 domain. The nitrogen of the TZD head group is within hydrogen-bonding
distance of the hydroxyl group of Tyr473. Lys367 forms another secondary hydrogen bond with the
ligand, at residue His449. The conformation of the TZD head group and the participating amino
acids are fixed by these primary and secondary hydrogen bonds [34]. This H-bonding network
stabilizes the conformation of the AF-2 domain allowing it to bind with the coactivator proteins.
Therefore, the presence of the acidic head in the right position is important in the development of
PPAR agonists. Another part of the PPAR agonists is the hydrophobic moiety that mainly interacts
with the hydrophobic residues in the LBD. The hydrophobic tail occupies the large cavity of the
LBD by interacting with the hydrophobic residues, such as Ile281, Gly284, Ile341, and Leu353 in
PPAR-γ (see Figure 2B). The linker wraps the central helix 3 (H3) and interacts with the surrounding
hydrophobic residues. In addition, it acts as a linker between the acidic head and hydrophobic tail
groups to allow the fitting of the whole compound into the LBD, where proper interactions could
be achieved. Therefore, fine tuning of the linker to adjust the atomic distance or three-dimensional
arrangement is a powerful approach for structural optimization to increase the biological activity
of compounds.
Figure 2.
(
A
) Schematic features of PPAR agonists and the concept of the novel target compounds;
LDB and rosiglitazone (PDB code: 2PRG). Only the critical amino
(
B) Crystal structure of PPAR-γ
acids are displayed and labeled. Polar residues for the key interaction (capped stick) and hydrophobic
residues (gray line) are depicted. Rosiglitazone is displayed in green and oxygen, nitrogen and sulfur
atom is marked in red, blue and yellow respectively.
Previously, we identified a series of (alkoxyindol-3-yl)acetic acids as PPAR agonists [36–38].
Through stepwise structural modification and optimization, benzyloxy-containing indol-3-ylacetic
acid analogs ( and ) were identified as PPAR- dual agonists. Compound lowered blood glucose,
1
2
γ
/
δ
2
insulin, and glycated hemoglobin (HbA1c) levels without causing weight gain; additionally, it reduced
the accumulation of lipids and the size of the adipocytes in db/db mice. These findings indicated the
potential of the indol-3-ylacetic acid as a core scaffold for anti-diabetic and anti-dyslipidemic drugs.
In our previous series of PPAR agonists, the indole group held the critical polar functional group.
In addition, the N-benzyl group of the indole ring significantly affected the PPAR activity. Hence,
we performed further modifications of the indole-based PPAR agonists.
Based on the indol-3-ylacetic acid scaffold, we introduced the dichloroanilide group into the
hydrophobic tail to increase the potency and selectivity. Some reported compounds with the
2,4-dichloroanilide moiety showed a significant PPAR agonist potency, and even small changes
to the 2,4-dichlorophenyl substitution resulted in a marked change in potency. For example, Bayer
compound 33 (Figure 1) showed a potent and selective activity as PPAR-δ agonist with EC50 value
of 3 nM and >1000-fold selectivity. The 2,4-disubstituted anilines were superior to other substitution
patterns [39]. Luckhurst et al. drew inspiration from Bayer compound 33 and combined the anilide
portion with isoindoline, tetrahydroisoquinoline, and benzazepine scaffolds. The structure-activity
relationship study confirmed that the 2,4-dichloroanilide compounds were the most potent of any
other derivatives [40,41]. Although interactions between 2,4-dichloroanilide and LBD of PPAR is

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not specified in those studies, 2,4-dichloroanilide moiety is multifunctional which can make many
kinds of interactions including aromatic pi-interaction, halogen-hydrogen bonding, and hydrogen
bonding through aromatic phenyl, dichloro and amide group, respectively. Furthermore, newly
designed 2,4-dichloroanilide substituted analogues show proper U-shaped binding mode similar to
rosiglitazone in our preliminary molecular modeling study.
For further structural optimization, including re-positioning of the critical acidic head group,
we set the change in the hydrophobic tail group as a starting point for our journey (Figure 2).
2. Result and Discussion
The synthesis of dichloroanilide-linked indole compounds is illustrated in Schemes 1–3.
For the introduction of the dichloroanilide moiety as
a
hydrophobic tail,
2,4-dichloroaniline and 2-chloroacetyl chloride underwent a direct substitution reaction to
yield 2-chloro-N-(2,4-dichlorophenyl)acetamide (3; Scheme 1).
◦
Scheme 1. Reaction conditions: (a) triethylamine, CH2Cl2, 0 C.
Hydroxyindol-3-ylacetic acid methyl ester compounds (4a,4b) were prepared as previously
described [32]. The amino and hydroxyl groups of the indole were benzylated under basic
conditions. The O-benzyl group was selectively deprotected in the following hydrogenation step.
Then, the developed free hydroxyl group was conjugated with compound 3. The methyl esters were
hydrolyzed using LiOH to yield the corresponding carboxylic acid compounds (5–8; Scheme 2).
◦
Scheme 2. Reaction conditions: (a) benzyl bromide, Cs2CO3, DMF, 80 C; (b) H2, Pd/C, EtOAc/EtOH,
RT; (c) compound 3, Cs2CO3, MeCN, RT; (d) LiOH·H2O, THF/MeOH/H2O, RT.
The starting O-benzyl and tert-butyldiphenylsilyl (TBDPS)-protected hydroxyindoles were
synthesized under typical conditions. Compounds with the 3-benzyl group were prepared by
reaction of compound 9a
the 3-position of the O-TBDPS-protected compounds (9e
,
b
with benzyl alcohol, whereas the prenyl group was introduced at
) using prenyl bromide. Compounds
–
f
with methyl carboxylate groups at various positions of the N-linked benzyl group were directly
synthesized by substitution reaction between the amino group of the indole and the 2-, 3-,
or 4-(methoxycarbonyl)methyl-substituted benzyl chloride. The O-benzyl and TBDPS protecting
groups were removed by conventional hydrogenation and tetrabutylammonium fluoride (TBAF),
respectively. O-Alkylation and hydrolysis of the esters yielded the target compounds (12–21;Scheme 3).

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Scheme 3. Reaction conditions: (a) benzyl alcohol, KOH, xylene, reflux; (b) prenyl bromide, Na2CO3,
MeCN/H2O (9:1), RT; (c) methyl 2-, 3- or 4-(chloromethyl)benzoate, NaH, THF, reflux; (d) H2,
Pd/C, EtOAc/EtOH, RT; (e) TBAF, THF, RT; (f) compound
THF/MeOH/H2O, RT.
3
, Cs2CO3, MeCN, RT; (g) LiOH H2O,
·
The structures of all the target compounds (5–8, 12–21) were confirmed by their proton nuclear
magnetic resonance (1H NMR) spectra and high-resolution mass spectrometry (HRMS), and they
were cross-checked against the structures of their methyl ester precursors (P5–8, P12–21) which were
1
characterized by the H and 13C NMR spectra. Analytical data are provided in the Materials and
Methods section.
The PPAR activities of the synthesized dichloroanilide-linked indole analogs were
assessed using a standard cell-based transactivation assay in CV-1 cells. GW7647 [42],
pioglitazone, and GW0742 [43] were used as positive controls for PPAR- , - , and -δ, respectively.
α
γ
These three positive controls presented the maximum activation for each PPAR subtype. The activity
of the compounds was presented as the activation percentage (%) relative to the maximum values of
the positive controls at the indicated concentrations. PPAR-
the tested compounds are summarized in Tables 1 and 2.
α, -γ, and -δ transactivation activities of

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Table 1. In vitro cell-based PPAR transactivation activity a.
% max b
Cpd. No.
Alkoxy position
R1
PPAR-α
PPAR-γ
PPAR-δ
5
6
7
5
6
5
6
H
H
benzyl
benzyl
−38.13 ± 0.06
1.12 ± 0.24
5.60 ± 0.14
8.66 ± 0.23
31.20 ± 0.38
71.87 ± 0.91
11.84 ± 0.35
8.27 ± 0.33
44.35 ± 0.77
65.41 ± 0.70
21.41 ± 0.21
31.68 ± 0.22
8
The agonistic activity of the compounds (10 µ
M) was assayed on PPAR-Luc in CV-1 cells; b Relative activation
a
with respect to the maximum activation obtained with GW7647 (10 µM), pioglitazone (10 µM), and GW0742 (10 µM)
for PPAR-α, -γ, and -δ, respectively.
Table 2. In vitro cell-based PPAR transactivation activity a.
% max b
Cpd. No.
Alkoxy position
R2
R3
PPAR-α
PPAR-γ
PPAR-δ
12
13
14
15
16
17
18
19
20
21
a
5
6
5
6
5
6
5
6
5
6
H
H
H
H
H
2-CO2H
2-CO2H
3-CO2H
3-CO2H
4-CO2H
4-CO2H
2-CO2H
2-CO2H
2-CO2H
2-CO2H
105.08
±
0.41 189.21
±
1.67
94.86 ± 0.20
86.70 ± 0.53
23.85 ± 0.51
86.09 ± 0.31
-1.07 ± 0.14
4.87 ± 0.24
75.88 ± 0.35
19.40 ± 0.54
8.92 ± 0.48
22.16 ± 0.59
51.84 ± 0.25
10.33 ± 0.07
78.15 ± 0.04
-19.98 ± 0.08
-14.44 ± 0.16
23.79 ± 0.11
7.25 ± 0.12
-5.93 ± 0.14
-2.30 ± 0.19
74.54 ± 0.94
42.01 ± 0.52
126.55
±
0.92
0.29 ± 0.06
-0.84 ± 0.16
93.20 ± 0.59
19.03 ± 0.13
5.10 ± 0.20
9.55 ± 0.32
H
benzyl
benzyl
prenyl
prenyl
The agonistic activity of the compounds (10
µ
M) was assayed on PPAR-Luc in CV-1 cells; b Relative activation
with respect to the maximum activation obtained with GW7647 (10 µM), pioglitazone (10 µM), and GW0742 (10 µM)
for PPAR-α, -γ, and -δ, respectively.
Compounds
tail at the 5- and 6- positions, respectively. However, these compounds showed minimum agonistic
activities to PPARs. Their N-benzylated analogs ( ) showed an increase in the activity, which is in
line with the trends observed in a previous study. However, their activities were much lower than
those of the previous hit compounds and (134.4 and 100.5% activity on PPAR- , respectively) [33].
This decline in the activity could be explained by the length and rigidity of the linker between the
hydrophobic tail and indole ring. As shown in Figure 1, the linker in compounds and consists
of four rotatable bonds between the two planar aromatic ring systems. Rosiglitazone also has three
bonds, which are freely rotatable. In contrast, compounds have three rotatable bonds; however,
5 and 6 were the simplest indoly-3-ylacetic acids containing the dichloroanilide
7,8
1
2
γ
1
2
5–8
two of them (same as three atoms) are linked to a planar carbonyl system. This non-flexibility could
result in a difference in the docking poses of the compounds.
To better understand the structural features of the synthesized compounds in the active site of the
PPAR LBD, a docking study of compounds
Sybyl-X (Figure 3). The crystal structure of PPAR-
1
and
5
γ
was carried out using Surflex Dock interfaced with
LBD complexed with the PPAR- selective agonist,
γ

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rosiglitazone (PDB code: 2PRG) was selected to perform the docking of compounds [34]. The polar
head groups of rosiglitazone (green) and compound (magenta) revealed the H-bonding interaction
1
with the polar residues in the hydrophilic pocket. The indole ring acted as an aromatic linker, similar
to the benzene ring of rosiglitazone. Thus, the aromatic linker of each compound was well-overlapped
in the docking position and made the compound U-shaped. The pyridine ring of rosiglitazone was
located in the hydrophobic pocket of PPAR-
The benzene ring of compound was well-aligned with the pyridine ring of rosiglitazone. The docking
pose of compound (red) might explain its low activity. The acidic head group could form hydrogen
bonds, similar to those of compound . The newly introduced 2,4-dichloroanilide moiety was located
γ and interacted with the hydrophobic amino acid residues.
1
5
1
near the hydrophobic pocket; however, it was misaligned with the pyridine ring of rosiglitazone.
Moreover, the benzene ring, a part of the indole moiety, did not superimpose with that of other
compounds. This might due to the non-flexibility of the linker. The rigid carbonyl plane was located at
the corner; thus, the linker could not turn smoothly. Structural modification was needed to compensate
this rigidity. The N-benzyl group of compounds 7 and 8 might make additional interaction with the
side pocket. Nevertheless, we tried to use this benzyl group as an aromatic linker to hold the polar
acidic head.
Figure 3. Binding modes of hit compound
1 (magenta) and compound 5 (red) in the active site of
PPAR- LBD (PDB code: 2PRG; co-crystallized with rosiglitazone (green)). Only the critical amino
γ
acids that interacted with the docked ligands are displayed and labeled. Several residues for the key
interaction (capped stick), hydrophobic residues (gray line), and hydrogen bonds (red-dotted lines) are
depicted. The numbers indicate the atomic distance (Å).
N-Benzylated indole derivatives, which had a carboxylic acid moiety at the ortho-,
meta-, or para-position of the benzyl group, were synthesized.
Among the 5-alkoxy
compounds, the ortho-carboxybenzyl-substituted compound 12 was the most active, whereas the
meta-carboxybenzyl-substituted compound 15 showed the highest activity among the 6-alkoxy series.
We supposed that the activity was also affected by the distance between the alkoxy and carboxyl
groups. 5-Alkoxy was a little far from the benzyl-substituted amine in the indole structure (pseudo-para),
and the ortho-positioned carboxyl group was the best to fit into the hydrophilic pocket. However,
6-alkoxy was at the pseudo-meta position of the indole structure, thus the carboxyl group at the meta
position showed better activity. Molecular modeling guided us to introduce additional substituents at

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the 3-position of the indole moiety, which could be placed in the extra back pocket near the indole
structure. Thus, 3-benzyl- or prenyl-substituted N-(2-carboxybenzyl)indole compounds were also
prepared. Unexpectedly, all analogs (18–21) with a substitution at the 3-position of the indole moiety
exhibited weak PPAR agonistic activity.
A docking study of compound 12 was also carried out using the same system as described
above. Compound 12 showed a U-shape binding mode wrapping around H3, similar to that of
rosiglitazone (Figure 4). The carboxyl group of compound 12 was located in the hydrophilic pocket
and interacted with the key polar residues, Ser289, His323, and Tyr473 by H-bonding. Next to the
acidic head group, the benzene ring was positioned in the hydrophobic region formed by Phe282,
Phe363, and Leu453. The indole ring acted as an aromatic linker, similar to the benzene ring of
rosiglitazone. In addition, the dichloroanilide group was well-fitted in the hydrophobic pocket along
with rosiglitazone. The indole group was located close to H3, and dichloroanilide was positioned
between H3 and
β2. Overall, the dichloroanilide moiety of compound 12 was well-aligned with
the hydrophobic tail of rosiglitazone (Figure 4), in contrast to that of compounds
5
(Figure 3).
The dichlorophenyl group of compound 12 could interact with the Ile281, Gly284, Ile341, and Leu353
residues, which form the hydrophobic pocket. In particular, the ortho-chloro atom was located 3.43 Å
away from the carbonyl oxygen of Gly284 to make a halogen bonding interaction. Therefore, the high
activity of compound 12 might be attributed to the proper binding and suitable interactions between
compound 12 and PPAR-γ.
Figure 4. Binding modes of compound 12 (blue) in the active site of PPAR-γ LBD (PDB code: 2PRG;
co-crystallized with rosiglitazone (green)). Only the critical amino acids that interacted with the docked
ligands are displayed and labeled. Several residues for the key interaction (capped stick), hydrophobic
residues (gray line), and hydrogen bonds (red-dotted lines) are depicted. The numbers indicate the
atomic distance (Å).
Another potent compound, 15 belonged to the 6-alkoxyindole series. The carboxylic acid at
the meta position of the N-benzyl group made 4 pairs of hydrogen bonds with the Ser289 and
Tyr473 residues in the hydrophilic pocket (Figure 5). The carboxylic acid group of compound 15
was aligned similarly to that of compound 12; however, the other part of compound 15 (from the

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benzyl group to the 2,4-dichloroanilide tail) was slightly moved back to locate the acid in the center
of the hydrophilic region. Because of this movement, compound 15 was possibly less coordinated
with the surrounding hydrophobic residues than compound 12. The 6-alkoxyindole analog with
ortho-carboxybenzyl group (13) and 5-alkoxyindole analog with meta-carboxybenzyl group (14) made
less H-bonds with the polar residues because their position in 3D was influenced by the conformation
of the whole molecule (data not shown). The findings of the docking study could explain the
results of the in vitro transactivation assay. The substitution position of carboxylic acid on the
benzyl group highly affected the PPAR activity, implying that the optimal positioning and matched
combinations of the acidic head and lipophilic tail groups were essential. Taken together, we identified
the N-(carboxylbenzyl)-substituted indole as a novel and promising scaffold for development of
therapeutic agents targeting PPARs.
Figure 5. Binding modes of compound 12 (blue) and compound 15 (yellow) in the active site of
PPAR-γ LBD (PDB code: 2PRG; co-crystallized with rosiglitazone). Only the critical amino acids that
interacted with the docked ligands are displayed and labeled. Several residues for the key interaction
(capped stick), hydrophobic residues (gray line), and hydrogen bonds (red-dotted lines) are depicted.
The numbers indicate the atomic distance (Å).
PPAR coactivator recruit assay was performed to determine the potency of the 4 most active
compounds. Binding of agonist to the PPAR LBD causes a conformational change around helix 12
in the LBD, resulting in higher affinity for the coactivator peptide. When the terbium label on the
anti-GST antibody complexed with PPAR is excited at 340 nm, energy is transferred to the fluorescein
label on the coactivator peptide and detected as emission at 520 nm. The time-resolved fluorescence
resonance energy transfer (TR-FRET) ratio of 520:495 is calculated and used to determine the EC50
from a dose response curve of the compound. Based on the biology of the PPAR-coactivator peptide
interaction, this ligand EC50 is a composite value representing the amount of ligand required to bind to
receptor, effect a conformational change, and recruit coactivator peptide. Potency of compounds show
similar order to their activity (Table 3). Notably, compound 12 showed EC50 of 1.96 nM to PPAR-
which was 1,970 fold selective to PPAR- and 16,600 fold to PPAR- . The second potent compound
15 revealed EC50 of 32.3 nM to PPAR- whereas >100 M to other PPAR subtypes. These results
γ
α
δ
γ
µ

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were huge improvement from our previous hit compounds (
1
δ
and
ligand binding pocket is significantly
because of the narrowing of the pocket adjacent to the AF2
domain. Ligands such as TZDs show little or no binding to PPAR- . Compound 11 and 15 have ortho or
meta substituted benzoic acid as an acidic head group. Their acidic head groups might seem to be too
large to fit within the narrow PPAR- pocket. In contrast, the compound and contains an acetic acid
head group that complements the narrow PPAR- ligand binding. And docking study of compound
11 with crystal structure of PPAR- (PDB code: 3G8I; co-crystallized with aleglitazar) and PPAR-
(PDB code: 3TKM; co-crystallized with GW0742) revealed misaligned conformation compared to their
co-crystallized ligands, aleglitazar (PPAR- agonist) and GW0742 (PPAR- agonist) respectively
2), which exhibited PPAR-γ/δ dual
agonistic activity at micromolar concentration. The PPAR-
smaller than those of PPAR- and PPAR-
α
γ
δ
δ
1
2
δ
α
δ
α
/γ
δ
(data not shown). The potency and selectivity of compound 11 could be explained by harmonization of
steric effect of acidic head group, length and rigidity of hydrophobic tail, and properly tuned aromatic
linker system.
Table 3. In vitro PPAR coactivator recruit assay a.
EC50 (µM)
Cpd. No.
Alkoxy position
R2
R3
PPAR-α
PPAR-γ
PPAR-δ
12
13
15
18
5
6
6
5
H
H
H
2-CO2H
2-CO2H
3-CO2H
2-CO2H
3.87
16.9
>100
>100
0.00196
0.571
0.0326
>11.1
32.6
>100
>100
28.1
benzyl
a
The agonistic activity of the compounds was determined using TR-FRET coactivator recruit assay.
3. Materials and Methods
3.1. General Information
Most reagents and solvents were purchased from commercial sources and used without
purification, with the following exceptions. Tetrahydrofuran (THF) was distilled from sodium
benzophenone ketyl. Acetonitrile (MeCN), methylene chloride (CH2Cl2), dimethylformamide
(DMF), and triethylamine were distilled from calcium hydride under nitrogen atmosphere. Column
chromatography was performed using silica gel 60 (230–400 mesh, Merck) with the indicated solvents.
1
Thin-layer chromatography (TLC) was performed using Kieselgel 60 F254 plates (Merck). H and 13
C
NMR spectra were recorded using a Varian Inova 400 or Bruker Avance III HD500 spectrometer using
CDCl3, CD3OD, or (CD3)2SO as solvents. Chemical shifts ( ) were expressed in parts per million (ppm)
δ
downfield from an internal standard, tetramethylsilane. HRMS data were recorded using an Agilent
6530 QTOF mass spectrometer with an electrospray (ESI) interface with Agilent jet stream technology
in the negative ion mode.
3.2. Experimental Procedures and Analytical Data of Compounds
3.2.1. 2-Chloro-N-(2,4-Dichlorophenyl)Acetamide (3)
Triethylamine (1.1 equiv.) was added to a solution of 2,4-chloroaniline in CH2Cl2 (0.5 M) and
stirred at room temperature for 30 min. Then, the reaction mixture was cooled to 0 ◦C. Chloroacetyl
chloride (1.2 equiv.) was added dropwise while the temperature was maintained. After the completion

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of the reaction, water was added to the reaction mixture to quench the reaction. The organic layer
was separated and washed with water thrice. Then, it was dried with brine and MgSO4, filtered,
and concentrated under vacuum. The residue was purified by silica gel column chromatography using
1
n-hexane and EtOAc (5:1) to yield a quantitative amount of the title compound
3
: H NMR (400 MHz,
CDCl3)
δ 8.89 (s, 1H), 8.34 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.27 (dd, J = 8.8, 2.4 Hz, 1H),
4.23 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 164.0, 132.5, 130.2, 129.1, 128.1, 124.1, 122.0, 43.2.
3.2.2. Synthetic Procedure for O-Alkylation of 5- or 6-Hydroxyindole Compounds
The prepared 5- or 6-hydoxyindole compounds were dissolved in anhydrous MeCN (0.5 M), and
Cs2CO3 (1.5 equiv.) was added and stirred at room temperature for 30 min. Compound 3 (1.2 equiv.)
was added to the reaction mixture then stirred until the reaction was complete. Then, the reaction
mixture was diluted with water and extracted with EtOAc. The combined organic extracts were
washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography using an appropriate mixture
of n-hexane and EtOAc as an eluent to yield the corresponding alkoxyindole compounds (the methyl
ester form of the final target compounds; yield = 65–86%).
Methyl 2-(5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetate (P5): 1H NMR
(400 MHz, CDCl3)
δ 9.12 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 8.10 (s, 1H), 7.41 (d, J = 2.4 Hz, 1H),
7.31 (d, J = 8.8 Hz, 1H), 7.28 (dd, J = 8.8, 2.3 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H),
6.96 (dd, J = 8.8, 2.4 Hz, 1H), 4.70 (s, 2H), 3.75 (d, J = 0.5 Hz, 2H), 3.70 (s, 3H); 13C NMR (100 MHz,
CDCl3)
δ 172.4, 167.2, 151.7, 132.9, 132.3, 129.6, 129.0, 128.1, 127.9, 124.6, 123.9, 122.1, 112.7, 112.5, 108.7,
103.1, 68.9, 52.2, 31.3.
Methyl 2-(6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetate (P6): 1H NMR
(400 MHz, CDCl3)
7.38 (d, J = 2.3 Hz, 1H), 7.25 (dd, J = 8.7, 2.5 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 6.88 (dd, J = 8.6, 2.3 Hz,
1H), 6.84 (d, J = 2.0 Hz, 1H), 4.63 (s, 2H), 3.76 (s, 2H), 3.71 (s, 3H); 13C NMR (100 MHz, CDCl3)
172.7,
δ 9.06 (s, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.24 (s, 1H), 7.52 (d, J = 8.6 Hz, 1H),
δ
167.0, 153.8, 136.6, 132.7, 129.6, 129.0, 128.0, 123.8, 123.02, 122.97, 122.0, 120.1, 110.0, 108.5, 96.3, 68.4,
52.2, 31.2.
Methyl 2-(1-benzyl-5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetate (P7):
1H NMR (400 MHz, CDCl3)
δ 9.11 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 2.4 Hz, 1H), 7.32–7.24
(m, 4H), 7.18 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.14 (s, 1H), 7.12–7.09 (m, 2H), 6.92 (dd, J = 8.9,
2.5 Hz, 1H), 5.26 (s, 2H), 4.69 (s, 2H), 3.74 (d, J = 0.6 Hz, 2H), 3.70 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
172.4, 167.2, 151.6, 137.3, 132.9, 132.8, 129.5, 128.98, 128.95, 128.6, 128.5, 128.1, 127.9, 126.9, 123.8,
122.1, 112.4, 111.2, 107.4, 103.4, 68.9, 52.2, 50.5, 31.2.
Methyl 2-(1-benzyl-6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetate (P8):
1H NMR (400 MHz, CDCl3)
δ 9.00 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H),
7.38 (d, J = 2.4 Hz, 1H), 7.31–7.22 (m, 4H), 7.13–7.08 (m, 2H), 7.06 (s, 1H), 6.87 (dd, J = 8.6, 2.3 Hz, 1H),
6.76 (d, J = 2.2 Hz, 1H), 5.22 (s, 2H), 4.61 (s, 2H), 3.75 (s, 2H), 3.70 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 172.5, 166.9, 153.8, 137.0, 132.8, 129.6, 129.0, 128.0, 127.9, 127.2, 126.9, 123.9, 123.8, 122.1, 120.5, 109.6,
107.8, 95.3, 68.5, 52.2, 50.3, 31.2.
Methyl 2-((5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoate (P12):
1H NMR (400 MHz, CDCl3)
δ
9.11 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 8.12–7.98 (m, 1H), 7.39 (d, J = 2.3 Hz
1H), 7.33–7.29 (m, 2H), 7.28–7.25 (m, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.17–7.12 (m, 2H), 6.90 (dd, J = 8.9
2.4 Hz, 1H), 6.54 (d, J = 3.0 Hz, 1H), 6.48–6.44 (m, 1H), 5.76 (s, 2H), 4.68 (s, 2H), 3.94 (s, 3H)
(100 MHz, CDCl3) 167.2, 151.6, 140.1, 133.1, 132.9, 132.7, 131.2, 130.1, 129.1, 129.0, 128.1, 127.8, 127.4,
127.1, 123.8, 122.1, 121.0, 112.2, 111.0, 104.9, 101.8, 95.9, 68.9, 52.4, 49.0.
,
,
;
13C NMR
δ

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Methyl 2-((6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl) benzoate (P13):
1H NMR (400 MHz, CDCl3)
1H), 7.36 (d, J = 2.4 Hz, 1H), 7.30–7.26 (m, 2H), 7.25–7.22 (m, 1H), 7.08 (d, J = 3.2 Hz, 1H), 6.88 (dd, J = 8.6
δ
8.99 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.07–8.02 (m, 1H), 7.59 (d, J = 8.6 Hz
,
,
2.3 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 6.55 (dd, J = 3.2, 0.7 Hz, 1H), 6.49–6.44 (m, 1H), 5.73 (s, 2H),
4.59 (s, 2H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
166.9, 153.6, 139.8, 136.9, 133.0, 132.8, 131.2,
129.5, 128.9, 128.7, 128.0, 127.8, 127.4, 127.0, 124.4, 123.8, 122.2, 122.0, 109.9, 102.0, 95.2, 68.5, 52.4, 48.8.
Methyl 3-((5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl) benzoate (P14):
1H NMR (400 MHz, CDCl3)
δ 9.10 (s, 1H), 8.45 (d, J = 8.9 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.89 (s, 1H),
7.38 (d, J = 2.3 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.25 (dd, J = 8.9, 2.3 Hz, 1H), 7.21–7.13 (m, 4H),
6.91 (dd, J = 8.8, 2.4 Hz, 1H), 6.51 (d, J = 2.6 Hz, 1H), 5.32 (s, 2H), 4.66 (s, 2H), 3.89 (s, 3H); 13C NMR
(100 MHz, CDCl3)
δ 167.0, 151.5, 137.8, 132.7, 132.3, 131.1, 130.7, 129.4, 129.3, 129.2, 128.99 128.95, 128.8,
127.92, 127.88, 123.6, 121.9, 112.0, 110.7, 104.9, 101.8, 68.7, 52.2, 50.1.
Methyl 3-((6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl) benzoate (P15):
1H NMR (400 MHz, CDCl3)
δ 8.98 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.88 (s, 1H),
7.57 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.24 (dd, J = 8.9, 2.4 Hz, 1H),
7.18 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 3.2 Hz, 1H), 6.86 (dd, J = 8.6, 2.3 Hz, 1H), 6.75 (d, J = 2.1 Hz, 1H),
6.52 (dd, J = 3.2, 0.8 Hz, 1H), 5.29 (s, 2H), 4.60 (s, 2H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 166.8,
153.6, 137.6, 136.6, 132.7, 131.2, 130.9, 129.5, 129.10, 129.09, 128.9, 128.2, 128.03, 127.99, 124.5, 123.7,
122.3, 122.0, 109.8, 102.2, 95.1, 68.5, 52.3, 50.0.
Methyl 4-((5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl) benzoate (P16):
1H NMR (400 MHz, CDCl3)
δ 9.10 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H), 7.96 (d, J = 8.2 Hz, 2H),
7.39 (d, J = 2.4 Hz, 1H), 7.27 (dd, J = 8.7, 2.1 Hz, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.16 (d, J = 3.1 Hz
,
,
1H), 7.15–7.10 (m, 3H), 6.91 (dd, J = 8.9, 2.4 Hz, 1H), 6.53 (d, J = 3.1 Hz, 1H), 5.36 (s, 2H), 4.68 (s, 2H)
3.89 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
167.2, 151.7, 142.6, 132.9, 132.5, 130.3, 129.8, 129.7, 129.5,
129.3, 129.0, 128.1, 126.6, 123.8, 122.1, 120.0, 112.3, 110.9, 105.1, 102.0, 68.8, 52.3, 50.3.
Methyl 4-((6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl) benzoate (P17):
1H NMR (400 MHz, CDCl3)
8.97 (s, 1H), 8.38 (d, J = 8.9 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H),
δ
7.58 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 2.3 Hz, 1H), 7.24 (dd, J = 9.1, 2.1 Hz, 1H), 7.11 (d, J = 8.2 Hz
,
,
2H), 7.09 (d, J = 3.2 Hz, 1H), 6.87 (dd, J = 8.6, 2.2 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 6.54 (d, J = 3.1 Hz
1H), 5.32 (s, 2H), 4.60 (s, 2H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
166.8, 153.7, 142.3, 136.7, 132.7,
130.3, 129.8, 129.6, 128.9, 128.3, 128.0, 126.6, 124.5, 123.7, 122.3, 122.0, 109.9, 102.3, 95.0, 68.5, 52.3, 50.1.
Methyl 2-((3-benzyl-5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)
benzoate (P18): 1H NMR (400 MHz, CDCl3)
9.06 (s, 1H), 8.43 (d, J = 8.9 Hz, 1H), 8.07–7.99
δ
(m, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.36–7.22 (m, 7H), 7.20–7.14 (m, 1H), 7.09 (d, J = 8.8 Hz, 1H),
7.02 (d, J = 2.3 Hz, 1H), 6.91–6.84 (m, 2H), 6.52–6.46 (m, 1H), 5.70 (s, 2H), 4.62 (s, 2H), 4.10 (s, 2H),
3.92 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 167.5, 167.2, 151.3, 141.0, 140.3, 133.3, 133.0, 132.9, 131.1,
129.5, 129.0, 128.7, 128.52, 128.48, 128.42, 128.0, 127.8, 127.3, 127.0, 126.1, 123.8, 122.1, 114.9, 112.1, 111.0,
103.6, 68.9, 52.3, 48.8, 31.7.
Methyl
2-((3-benzyl-6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)
8.98 (s, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.06–8.01
benzoate (P19): 1H NMR (400 MHz, CDCl3)
δ
(m, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 2.3 Hz, 1H), 7.32–7.22 (m, 7H), 7.22–7.16 (m, 1H), 6.84–6.79
(m, 2H), 6.70 (d, J = 2.0 Hz, 1H), 6.54–6.49 (m, 1H), 5.67 (s, 2H), 4.58 (s, 2H), 4.11 (s, 2H), 3.94 (s, 3H);
13C NMR (100 MHz, CDCl3)
δ 167.5, 166.9, 153.8, 141.2, 140.0, 137.5, 133.0, 132.8, 131.2, 129.6, 129.0,
128.8, 128.5, 128.0, 127.9, 127.4, 127.04, 126.95, 126.1, 124.0, 123.8, 122.1, 120.7, 115.4, 109.3, 95.3, 68.6,
52.4, 48.7, 31.7.
Methyl
2-((5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-3-(3-methylbut-2-en-1-yl)-1H-indol-
9.01 (s, 1H), 8.40 (d, J = 8.9 Hz, 1H),
1-yl)methyl) benzoate (P20): 1H NMR (500 MHz, CDCl3)
δ

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8.06–8.02 (m, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 2.3 Hz, 1H), 7.31–7.28 (m, 2H), 7.24 (d, J = 2.3 Hz,
1H), 6.86 (dd, J = 8.6, 2.2 Hz, 1H), 6.83 (s, 1H), 6.69 (d, J = 2.2 Hz, 1H), 6.52–6.48 (m, 1H), 5.67 (s, 2H),
5.45–5.40 (m, 1H), 4.60 (s, 2H), 3.96 (s, 3H), 3.45 (d, J = 6.8 Hz, 2H), 1.77 (s, 3H), 1.75 (s, 3H); 13C NMR
(125 MHz, CDCl3)
δ 167.4, 166.9, 153.7, 140.1, 137.4, 132.9, 132.7, 132.1, 131.1, 129.4, 128.9, 127.9, 127.7,
127.2, 126.9, 125.7, 123.9, 123.7, 122.9, 122.0, 120.4, 115.7, 108.9, 95.2, 68.5, 52.2, 48.5, 25.7, 24.1, 17.8.
Methyl 2-((6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-3-(3-methylbut-2-en-1-yl)-1H-indol-
1-yl)methyl) benzoate (P21): 1H NMR (500 MHz, CDCl3)
9.13 (s, 1H), 8.47 (d, J = 8.9 Hz, 1H),
8.07–8.01 (m, 1H), 7.41 (d, J = 2.3 Hz, 1H), 7.31 (dt, J = 6.1, 4.1 Hz, 2H), 7.28 (dd, J = 8.9 2.3 Hz, 1H),
δ
,
7.14 (d, J = 2.4 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 6.91 (s, 1H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H), 6.50–6.46
(m, 1H), 5.70 (s, 2H), 5.43–5.38 (m, 1H), 4.70 (s, 2H), 3.95 (s, 3H), 3.44 (d, J = 7.0 Hz, 2H), 1.78 (s, 3H),
13C NMR (125 MHz, CDCl3)
1.75 (s, 3H); δ 167.5, 167.2, 151.1, 140.4, 133.2, 132.9, 132.8, 132.2, 131.0,
129.4, 128.9, 128.4, 127.9, 127.7, 127.3, 127.2, 127.0, 123.7, 122.8, 122.0, 115.2, 111.9, 110.8, 103.4, 68.9, 52.2,
48.6, 25.7, 24.1, 17.9.
3.2.3. Synthetic Procedure for Hydrolysis of the Methyl Esters to Yield the Target Acid Compounds
LiOH H2O (1.5 equiv.) was added to a solution of the alkoxyindole compounds in
·
THF/MeOH/H2O (1 M, 2:1:1 v/v/v) and stirred at room temperature. After completing the reaction,
the reaction mixture was concentrated under reduced pressure, acidified with 1N HCl solution,
and extracted with EtOAc. The combined organic extracts were washed with water and brine, dried
over anhydrous MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica
gel column chromatography using an appropriate mixture of CHCl3 and MeOH as an eluent to obtain
the target compounds 6–9 and 13–22 with 59–92% yield.
2-(5-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetic acid (5): 1H NMR (400 MHz,
CDCl3/CD3OD)
δ 8.14 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.35 (dd, J = 8.8, 2.3 Hz, 1H),
7.31 (d, J = 8.8 Hz, 1H), 7.19 (s, 2H), 6.94 (dd, J = 8.6, 2.3 Hz, 1H), 4.73 (s, 2H), 3.71 (s, 2H); HRMS(ESI):
m/z 391.0263 [M-H]− (calcd for C18H13Cl2N2O4 = 391.0258).
2-(6-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetic acid (6):1H NMR (400 MHz,
CDCl3/CD3OD)
δ 8.20 (d, J = 8.8 Hz, 1H), 7.75 (s, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 2.3 Hz,
1H), 7.31 (dd, J = 8.8, 2.4 Hz, 1H), 7.08 (s, 1H), 6.99 (d, J = 2.1 Hz, 1H), 6.83 (dd, J = 8.7, 2.3 Hz, 1H),
4.70 (s, 2H), 3.65 (s, 2H); HRMS(ESI): m/z 391.0256 [M-H]− (calcd for C18H13Cl2N2O4 = 391.0258).
2-(1-Benzyl-5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetic acid (7): 1H NMR
(400 MHz, CDCl3/CD3OD)
δ 8.34 (d, J = 8.9 Hz, 1H), 7.50 (s, 1H), 7.45 (d, J = 2.3 Hz, 1H),
7.32 (d, J = 2.3 Hz, 1H), 7.31–7.26 (m, 2H), 7.24–7.18 (m, 3H), 7.13 (d, J = 6.6 Hz, 2H), 6.94 (dd, J = 9.2,
2.2 Hz, 1H), 5.30 (s, 2H), 4.72 (s, 2H), 3.74 (s, 2H); HRMS(ESI): m/z 481.0724 [M-H]− (calcd for
C25H19Cl2N2O4 = 481.0727).
2-(1-Benzyl-6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-3-yl)acetic acid (8): 1H NMR
(400 MHz, CDCl3/CD3OD) δ 8.25 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 2.2 Hz, 1H),
7.30 (dd, J = 8.8, 2.4 Hz, 1H), 7.28–7.21 (m, 3H), 7.16–7.11 (m, 3H), 6.89 (dd, J = 8.7, 2.0 Hz, 1H),
−
6.84 (d, J = 2.0 Hz, 1H), 5.29 (s, 2H), 4.66 (s, 2H), 3.74 (s, 2H); HRMS(ESI): m/z 481.0726 [M-H] (calcd
for C25H19Cl2N2O4 = 481.0727).
2-((5-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(12):
1H NMR (400 MHz, CDCl3/CD3OD)
δ
8.38 (d, J = 8.9 Hz, 1H), 8.03–7.99 (m, 1H), 7.42–7.39
(m, 1H), 7.28 (dd, J = 8.9, 2.4 Hz, 1H), 7.24–7.20 (m, 2H), 7.19 (d, J = 2.4 Hz, 1H), 7.12 (d, J = 3.1 Hz,
1H), 7.10 (d, J = 8.9 Hz, 1H), 6.82 (dd, J = 8.8, 2.4 Hz, 1H), 6.49 (d, J = 2.6 Hz, 1H), 6.42–6.38 (m, 1H),
5.75 (s, 2H), 4.66 (s, 2H); HRMS(ESI): m/z 467.0549 [M-H]− (calcd for C24H17Cl2N2O4 = 467.0571).
2-((6-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(13):
1H NMR (400 MHz, CDCl3/CD3OD)
δ
8.32 (d, J = 8.9 Hz, 1H), 8.09–8.05 (m, 1H), 7.60 (d, J = 8.6 Hz
,

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1H), 7.41 (d, J = 2.4 Hz, 1H), 7.39 (d, J = 0.7 Hz, 1H), 7.30–7.25 (m, 3H), 7.13 (d, J = 3.1 Hz, 1H), 6.89 (dd,
J = 8.6, 2.2 Hz, 1H), 6.81 (d, J = 1.9 Hz, 1H), 6.55 (d, J = 3.2 Hz, 1H), 6.52–6.48 (m, 1H), 5.77 (s, 2H), 4.63
(s, 2H); HRMS(ESI): m/z 467.0550 [M-H]− (calcd for C24H17Cl2N2O4 = 467.0571).
3-((5-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(14):
1H NMR (400 MHz, CDCl3/CD3OD)
δ
8.37 (d, J = 8.8 Hz, 1H), 7.99–7.90 (m, 3H), 7.44 (d, J = 2.3 Hz
,
1H), 7.36 (t, J = 7.6 Hz, 1H), 7.30 (dd, J = 8.8, 2.4 Hz, 1H), 7.24–7.20 (m, 4H), 6.93 (dd, J = 8.9, 2.6 Hz,
1H), 6.51 (d, J = 3.2 Hz, 1H), 5.38 (s, 2H), 4.70 (s, 2H); HRMS(ESI): m/z 467.0558 [M-H]− (calcd for
C24H17Cl2N2O4 = 467.0571).
3-((6-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
1H NMR (400 MHz, CDCl3/CD3OD)
8.25 (d, J = 8.8 Hz, 1H), 7.94–7.87 (m, 2H), 7.55 (d, J = 8.6 Hz
1H), 7.42–7.38 (m, 1H), 7.28 (s, 1H), 7.26 (dd, J = 8.8, 2.4 Hz, 1H), 7.20–7.15 (m, 1H), 7.10 (d, J = 2.7 Hz
acid
(15):
δ
,
,
1H), 6.86 (dd, J = 8.6, 2.2 Hz, 1H), 6.82 (s, 1H), 6.48 (d, J = 3.1 Hz, 1H), 5.28 (s, 2H), 4.63 (s, 2H);
HRMS(ESI): m/z 467.0557 [M-H]− (calcd for C24H17Cl2N2O4 = 467.0571).
4-((5-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(16):
1H NMR (400 MHz, CDCl3/CD3OD)
δ
8.17 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H),
7.44 (d, J = 2.4 Hz, 1H), 7.28 (dd, J = 8.9, 2.3 Hz, 1H), 7.23 (d, J = 3.0 Hz, 1H), 7.20–7.16 (m, 2H),
7.11 (d, J = 8.1 Hz, 2H), 6.89 (dd, J = 8.9, 2.2 Hz, 1H), 6.45 (d, J = 3.2 Hz, 1H), 5.39 (s, 2H), 4.68 (s, 2H);
HRMS(ESI): m/z 467.0545 [M-H]− (calcd for C24H17Cl2N2O4 = 467.0571).
4-((6-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
1H NMR (400 MHz, CDCl3/CD3OD)
9.06 (s, 1H), 8.33 (dd, J = 8.9, 3.1 Hz, 1H), 7.96 (d, J = 8 Hz
2H), 7.59 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 7.28 (dd, J = 8.8, 2.3 Hz, 1H), 7.16–7.11 (m, 3H),
acid
(17):
δ
,
6.89 (dd, J = 8.6, 2.2 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 3.2 Hz, 1H), 5.36 (s, 2H), 4.64 (s, 2H)
;
HRMS(ESI): m/z 467.0549 [M-H]− (calcd for C24H17Cl2N2O4 = 467.0571).
2-((3-Benzyl-5-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(18): 1H NMR (400 MHz, (CD3)2SO)
δ
9.59 (s, 1H), 7.92 (d, J = 7.1 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H),
7.70 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.35 (t, J = 7.6 Hz, 2H), 7.31–7.19 (m, 6H), 7.14 (d, J = 6.7 Hz, 1H),
7.10 (s, 1H), 6.85 (d, J = 9.2 Hz, 1H), 6.44 (d, J = 7.2 Hz, 1H), 5.72 (s, 2H), 4.71 (s, 2H), 4.03 (s, 2H);
HRMS(ESI): m/z 557.1010 [M-H]− (calcd for C31H23Cl2N2O4 = 557.1040).
2-((3-Benzyl-6-(2-((2,4-dichlorophenyl)amino)-2-oxoethoxy)-1H-indol-1-yl)methyl)benzoic
acid
(19): 1H NMR (400 MHz, (CD3)2SO)
δ
10.43 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H),
7.56 (d, J = 6.7 Hz, 1H), 7.38 (dd, J = 8.7, 2.2 Hz, 1H), 7.30–7.20 (m, 6H), 7.17–7.10 (m, 2H), 7.09–7.00
(m, 2H), 6.70 (d, J = 6.5 Hz, 1H), 6.66 (dd, J = 8.5, 1.8 Hz, 1H), 5.66 (s, 2H), 4.74 (s, 2H), 3.99 (s, 2H);
HRMS(ESI): m/z 557.1010 [M-H]− (calcd for C31H23Cl2N2O4 = 557.1040).
2-((5-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-3-(3-methylbut-2-en-1-yl)-1H-indol-1-
yl)methyl)benzoic acid (20): 1H NMR (400 MHz, CDCl3/CD3OD)
δ
8.33 (1H, d, J = 8.8 Hz),
J =5.6, 3.6 Hz), 7.54 (1H, d, J = 8.8 Hz), 7.41 (1H, d, J = 2.0 Hz), 7.31–7.25 (3H, m),
6.88–6.85 (2H, m), 6.75 (1H, d, J = 2.0 Hz), 6.50 (1H, dd, J = 5.6, 3.2 Hz), 5.70 (2H, s), 5.43 (1H, t, J = 7.2
Hz), 4.62 (2H, s)
3.46 (2H, d, J = 6.8 Hz), 1.77 (3H, s), 1.76 (3H, s); HRMS(ESI): m/z 535.1178 [M-H]−
8.09 (1H, dd
,
,
(calcd for C29H25Cl2N2O4 = 535.1197).
2-((6-(2-((2,4-Dichlorophenyl)amino)-2-oxoethoxy)-3-(3-methylbut-2-en-1-yl)-1H-indol-1-
yl)methyl)benzoic acid (21): 1H NMR (400 MHz, CDCl3/CD3OD)
δ 8.31 (1H, d, J = 8.8 Hz),
8.08–8.05 (1H, m), 7.55 (1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 2.4 Hz), 7.31–7.26 (3H, m), 6.89–6.86
(2H, m), 6.78 (1H, d, J = 2.0 Hz), 6.52-6.49 (1H, m), 5.71 (2H, s), 5.44 (1H, t, J = 7.2 Hz), 4.64 (2H,
s), 3.47 (2H, d
C29H25Cl2N2O4 = 535.1197).
,
J = 7.2 Hz), 1.78 (3H, s), 1.77 (3H, s); HRMS(ESI): m/z 535.1177 [M-H]− (calcd for

Int. J. Mol. Sci. 2018, 19, 3032
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3.3. In Vitro PPAR Transactivation Assay
Transient transfection and luciferase assay was carried out. CV-1 cells were seeded in 48-well
plates at a density of 1.5
105 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) with
containing 10% fetal bovine serum (FBS). The cells were transiently transfected with plasmid mixtures
containing PPAR- expression vector and tk-PPRE-luciferase (Luc) vector for 6 h and then
×
α/γ
treated with the samples for 24 h. HEK293t cells were seeded in a 60-mm dish at a density of
1.5 × 106 cells/dish. After transfection with the plasmid mixtures containing PPAR-
δ expression
vector and Luc vector for 6 h, the cells were transferred to a 96-well plate and treated with the
samples. To normalize the transfection efficiency, a
The luciferase activity in the cell lysates was measured using a luciferase assay system (Promega Corp.,
Madison, WI, USA), and the -galactosidase activity was determined by measuring the absorbance at
410 nm using an ELISA plate reader. The data were expressed as the relative luciferase activity divided
by the -galactosidase activity. All constructs were kindly gifted by Dr. Ronald M. Evans at The Salk
β-galactosidase plasmid was cotransfected.
β
β
Institute (La Jolla, CA, USA).
3.4. Molecular Modeling
All molecular modeling calculations were carried out using Surflex Dock interfaced with SYBYL-X
software, version 2.1.1 running on a system with Window 7 Home Premium K 64-bit OS. In this
automated docking program, the flexibility of the ligands, proteins, and biomolecules was considered.
The ligand was built in an incremental fashion, where each new fragment was added in all possible
positions and conformations to a pre-placed base fragment inside the active site. All the molecules
used for docking were sketched in SYBYL, and the energy minimizations were performed using Tripos
Force Field and Gasteiger–Huckel charge with 100,000 iterations of the conjugate gradient method with
a convergence criterion of 0.05 kcal/mol. To prepare the proteins, all hydrogens and MMFF94 charges
were added, and the side-chain amides were fixed. A staged minimization was performed using
Tripos Force Field and MMFF94 charge with 10,000 iterations of the Powell method with a convergence
criterion of 0.5 kcal/mol without the initial optimization. The 3D coordinates of the active sites were
determined from the X-ray crystal structures of PPAR-γ (PDB code: 2PRG), reported as a complex
with rosiglitazone.
3.5. In vitro PPAR Coactivator Recruit Assay
EC50 of compounds were determined by recruitment of transcriptional coactivators using the
TR-FRET based Lanthascreen coactivator assays (Invitrogen, Carlsbad, CA, USA). Which was serviced
by Thermo Fisher Scientific (Medicon, WI, USA) according to the LanthaScreen TR-FRET coregulator
protocol and assay conditions. Coregulator peptide PGC1a, TRAP220/DRIP2 and C33 was used for
the coactivator recruit assay of PPAR-α, -γ, and -δ, respectively.
4. Conclusions
In conclusion, we designed and synthesized several dichloroanilide-linked indole derivatives
and examined their potent and selective PPAR activity. The PPAR transcriptional activities of these
compounds were tested using a cell-based reporter assay. Compound 12 exhibited significant activity
as a PPAR-
PPAR coactivator recruit assay revealed EC50 of compound 12 with 1.96 nM. And it showed 1970-fold
selectivity to PPAR- and 16,600-fold to PPAR- . The docking study provided structural insights
of compound 12 in association with PPAR- and rationalized its good PPAR- agonistic activity.
γ agonist, with approximately 2-fold higher PPAR-γ activity than that of pioglitazone.
α
δ
γ
γ
Therefore, the N-(carboxybenzyl)indole was suggested as an original framework for the next generation
of PPAR ligands. In addition, our scaffold tuning approach could be an expeditious way to optimize
the activity of compounds. This novel PPAR-γ agonist and scaffold tuning approach is expected to

Int. J. Mol. Sci. 2018, 19, 3032
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broaden the bottleneck of new drug discovery for CVD, CML and various types of solid tumors as
well as metabolic diseases.
Author Contributions: R.J. conceived and designed the experiments; H.J.G., Y.-J.K. and H.L. performed the
experiments; H.J.G., Y.-J.K. and Y.-S.C. analyzed the data; J.-H.R. contributed reagents/analysis tools; Y.-S.C. wrote
the paper. Authorship must be limited to those who have contributed substantially to the work reported.
Funding: This research was supported by the National Research Foundation of Korea (NRF) Grant
funded by the Korean government (MSIP) (No. 2011-0030074 and 2014R1A2A1A11052761), the Ministry of
Education (NRF-2016R1A6A3A11932164 and NRF-2017R1A6A3A11034603) and Sookmyung Women’s University
Specialization Program Funding.
Conflicts of Interest: The authors declare no conflict of interest.
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