Design, synthesis, and evaluation of potent novel peroxisome proliferator-activated receptor γ indole partial agonists

Article abstract of DOI:10.1016/j.bmcl.2019.126664

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve insulin sensitivity by blocking PPARγ phosphorylation at S273; however

Full text of DOI:10.1016/j.bmcl.2019.126664

Journal Pre-proofs
Design, synthesis, and evaluation of potent novel peroxisome proliferator-acti-
vated receptor γ indole partial agonists
Venkateswararao Eeda, Dan Wu, Hui-Ying Lim, Weidong Wang
PII:
S0960-894X(19)30614-6
DOI:
https://doi.org/10.1016/j.bmcl.2019.126664
BMCL 126664
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
26 July 2019
28 August 2019
2 September 2019
Please cite this article as: Eeda, V., Wu, D., Lim, H-Y., Wang, W., Design, synthesis, and evaluation of potent novel
peroxisome proliferator-activated receptor γ indole partial agonists, Bioorganic & Medicinal Chemistry Letters
(2019), doi: https://doi.org/10.1016/j.bmcl.2019.126664
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Design, Synthesis, and Evaluation of Potent Novel Peroxisome Proliferator-Activated
Receptor γ Indole Partial Agonists
Venkateswararao Eedaa1, Dan Wua1, Hui-Ying Limb, & Weidong Wanga*
aDepartment of Medicine, Division of Endocrinology, bDepartment of Physiology,
Harold Hamm Diabetes Center, The University of Oklahoma Health Science Center, 941 Stanton
L. Young Boulevard, Oklahoma City, Oklahoma 73104, United States
*Corresponding author: weidong-wang@ouhsc.edu
1
These authors contribute equally.

Abstract
Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for
glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve
insulin sensitivity by blocking PPARγ phosphorylation at S273; however, their full agonism on
PPARγ also causes significant unwanted side effects. The indole derivative UHC1 displays
insulin-sensitizing effect by acting as a partial agonist through the inhibition of PPARγ S273
phosphorylation, but without full agonist-associated side effects; however, its potency leaves
much to be desired. Herein we report the design and synthesis of potent indole analogs as partial
PPARγ agonists via the structure-activity relationship studies. Our studies revealed that
vanillylamine and piperonyl benzylamine at Site 1 are favored to bind PPARγ with either
biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, compound WO91A with
vanillylamine at Site 1 displays highly potent PPARγ binding affinity (IC50 =16.7 nM), over 30-
fold more potent than the parental compound UHC1, yet with less side effect-associated
transactivation activity.

Type 2 Diabetes (T2D), a current pandemic, usually develops in obese and insulin-resistant
subjects with the onset of insulin-producing β cell dysfunction and death. The thiazolidinedione
(TZD) drugs such as rosiglitazone (Rosi) and pioglitazone are effective in improving insulin
sensitivity and are used for the treatment of insulin resistance and T2D.1 However, the current
TZD drugs are associated with long-term serious side effects such as weight gain, fluid retention,
loss of bone mass and increased risk of congestive heart failure.2, 3 As a result, prescription of
such drugs has recently declined significantly.
TZDs are full agonists of peroxisome proliferator-activated receptor γ (PPARγ).1 PPARγ
is a member of the nuclear receptor family of transcription factors and plays an important role
in the regulation of insulin sensitivity, adipogenesis, and glucose homeostasis by regulating
the transcription of many genes involved in these processes.4 TZDs hence activates the
PPARγ-dependent expression of genes responsible for insulin sensitivity; however, their full
agonism of PPARγ also activates the expression of genes associated with unwanted side
effects.
Recently, it has been reported that the insulin-sensitizing properties of TZDs are attributed
to their ability to block the cyclin-dependent kinase 5 (CDK5) or extracellular signal-regulated
6
kinase (ERK)-dependent PPARγ phosphorylation at S273.5,
Blockage of PPARγ S273
phosphorylation led to the restoration of obesity-induced dysregulation in insulin-sensitivity
relevant genes including adiponectin and adipsin.6 Importantly, this effect is independent of the
full agonism of TZDs that is associated with adverse effects.6 Compounds that block the PPARγ
S273 phosphorylation but without full PPARγ agonism could therefore be promising direction
for T2D drug development.

Several chemical structures have so far been identified to bind to PPARγ and inhibit PPARγ
S273 phosphorylation but without fully stimulating the transcriptional activity of PPARγ; these
compounds, including partial agonists6-9 or non-agonist PPARγ ligands10, 11, improve insulin
sensitivity with diminished adverse effects associated with full agonist TZDs in vivo. The
mechanisms by which these ligands inhibit PPARγ S273 phosphorylation remains incompletely
understood; however, one recent report indicates that ligand binding at the alternate binding site
of PPARγ directly block the S273 from phosphorylation by kinases.12
The indole-based derivatives SR1664 and UHC1 (in Figure 1) were reported as non-agonist
synthetic ligands of PPARγ as they inhibit phosphorylation of S273 and are anti-diabetic in vivo
but do not exhibit full agonist activity.10, 11 These derivatives thus represent a new generation of
improved anti-diabetic scaffold through PPARγ. However, these compounds have less potency
in binding affinity or poor pharmacokinetic properties.10, 11 In this study, we report the synthesis
of a series of novel analogs of compounds based on the UHC1 scaffold that possesses highly
potent PPARγ binding affinity but with weak PPARγ transcription activation. These compounds
therefore represent more effective therapeutics over the current indole-derived PPARγ ligands
and provides a promising approach to treat T2D.

O
O
N
O
H
N
N
S
N
HO2C
O2N
NH
O
Rosiglitazone
(Full agonist)
SR1664
O
O
N
H
N
N
H
N
HO2C
Site 1
N
Manipulated
positions
UHC1
Site 2
Current Lead
Figure 1. Chemical structures of PPAR-g full agonist Rosiglatazone (Rosi), non- or partial
agonists (SR1664 and UHC1), and current lead SAR design.
The syntheses of 2a-j and 3a-e are shown in Scheme 1 and substituents are listed in Table 1.
Key substituted indole acid derivatives were prepared as previous reported.13 Compounds 2a-j
(Scheme 1A) and 3a-e (Scheme 1B) were prepared by coupling commercially available
substituted benzylamines with indole acid derivative by using HATU coupling reagent in DCM
at ambient temperature. Analogs 2a-j were obtained after deprotection of t-butyl group with TFA.
All these synthesized compounds were listed in Table 1 and characterized by physical and
spectral analysis data that confirmed their assigned structures.
Scheme 1.

A
B
Reagents and conditions: (a) HATU, DIPEA, DCM at rt; (b) TFA/DCM
Compound 5 was synthesized using mono-alkylation as shown in Scheme 2. Intermediate 4 was
prepared by alkylating of 4-aminobenzoate in DMF in presence of potassium carbonate followed
by hydrolysis of ester to obtain acid intermediate 4. Compound 5 was obtained by HATU
coupling with corresponding amines in DCM at ambient temperatures.
Scheme 2.
O
O
Br
O
HO
HO
N
O
H
a, b
c, d
NH
NH
NH2
COO
COOH
5
4
Reagents and conditions: (a) K2CO3, DMF; (b) KOH, EtOH; (c) HATU, DIPEA, 3-OH
Benzylamine, DCM; (d) TFA/DCM

To identify more potent analogs of UHC1, we synthesized a series of novel compounds with
structure-activity-relationship (SAR) studies. The newly synthesized compounds were initially
tested for their binding affinity to the ligand binding domain of PPARγ protein using an in vitro
competition binding assay. We first focused our efforts on the modification of the amide group
in the left-hand side of the UHC1 compound. Elimination of the amide group that produces the
key intermediate (1), as seen in Scheme 1, led to a significant loss in PPARγ binding affinity,
indicating that the amide function is compulsory for the binding (Table 1).
We next examined the electron withdrawing substituents on the benzyl group of amides (Scheme
1A) and found that the 4-sulfonamide substituted benzamide (2a) showed comparable
biochemical affinity as the parent UHC1 compound (IC50 0.312 µM for 2a vs. 0.536 µM for
UHC1). Encouraged by this outcome, we then introduced a pyridine ring at the amide side that
contributed to significant improvement in PPARγ binding affinity (2b, IC50 0.034 µM). These
results indicate that the electron-withdrawing substituents are favored on the left-hand side. To
investigate other electronic effects, we synthesized compound (2c) with a piperonylamine
functional group. Compound 2c (IC50 0.015 µM) demonstrated a 20-fold improvement in binding
affinity compared to 2a. Replacement of the phenyl ring by non-aromatic piperidine moiety led
to the resultant compound (2d, IC50 12.120 µM) significantly losing its binding affinity,
indicating that phenyl substituents are essential for the PPARγ binding at that position. On the
other hand, compound (2e, herein we refer it as WO91A), where the piperonyl splits into the OH
and OMe groups as the vanillyl amine derivative, exhibited the most potent binding affinity to

PPAR-γ with IC50 = 16.7 nM (Figure 3A). Such strong affinity may be attributed to the -OH
forming hydrogen bond with the binding pocket of the PPARγ ligand binding domain.
Next, we determined whether shortening of the length of R1 ring may contribute to the improved
binding affinity. We first prepared a shortened analog of 2d with a piperidine-4-amine group (2f),
which turned out to exhibit even poorer binding affinity compared to 2d (IC50 41.280 µM for 2f
vs. 12.120 µM for 2d). Subsequently, we extended the length of the analog by preparing a
tyramine derivative (2g) that maintained significant binding affinity (IC50 0.048 µM). These
results suggest that shortening of the length of benzyl to phenyl ring appears to have no impact
on PPARγ binding affinity. Previous reports have also shown that acidic groups increase ligand
affinity to the PPARγ binding pocket.14, 15 We applied this concept to our analog preparation by
synthesizing compound (2h), which possesses a phenoxy acetic acid in the R1 site (see Scheme
1A). However, compared to 2e (WO91A), 2h exhibited significantly less potent PPARγ binding
affinity (IC50 0.066 µM), although it is comparable to that of compounds with no acidic group
(e.g. 2g).
In the next set of analogs, we explored the importance of biphenyl substituents at the indole site.
We prepared compounds 3a-e by switching the biphenyl substituent with a 3-trifluoromethyl
benzyl group (Table 1, Scheme 1B). First, we chose the most potent compound WO91A
obtained from the above SAR analysis, which possesses the vanillyl amine on the left side. We
replaced the biphenyl group in WO91A with the 3-trifluoromethyl benzyl group and found that
the resultant compound 3a maintained significant PPARγ binding affinity compared to its
biphenyl counterpart WO91A. Moreover, 3b also retained its same binding affinity as its
biphenyl counterpart 2c. Analogs 3c and 3d exhibited reasonable binding affinity too. Together,
these results suggest that the trifluoromethyl benzyl moiety retains potent binding affinity to

PPARγ similar to their biphenyl counterpart. We next introduced an amino acid link on the left
hand-side while keeping the trifluoromethyl benzyl group at the indole site so as to balance the
ClogP for solubility purpose. Surprisingly, the tyrosine analog 3e exhibited a binding affinity for
PPARγ significantly worse than WO91A. Lastly, we examined the importance of indole group
per se. Breaking the indole ring into the non-cyclic aniline derivative (compound 5, seen in
Scheme 2) did not offer any advantage for PPARγ binding affinity; in fact, 5 exhibited poorer
PPARγ binding affinity than most of indole-based 2- and 3-series analogs, indicating the
importance of indole group in PPARγ binding.
Table 1. SAR evaluation of PPARγ binding IC50, inhibition constant, and transactivation activity
values of synthesized compounds.
Comp.
ID
R1
PPARγ
Inhibition Constant Transactivation
Binding
(IC50, µM)a
4.866
(Ki, µM)b
activity (%)c
1
4'-methyl-[1,1'-biphenyl]-2-
carboxylic acid
3.799
16
2a
2b
2c
2d
4-SO2NH2-benzylamine
Pyridin-2-ylmethylamine
Piperonylamine
0.312
0.034
0.015
12.120
0.017
41.280
0.048
0.066
0.244
0.027
0.012
9.461
0.013
32.225
0.038
0.052
0
15
36
11
15
17
31
56
piperidin-4-ylmethanamine
2e (WO91A) Vanillylamine
2f
2g
2h
Piperidine-4-amine
Tyramine
2-(3-(aminomethyl)phenoxy)
acetic acid
3a
3b
3c
3d
3e
5
UHC1
Vanillylamine
0.010
0.016
0.185
0.048
0.346
0.325
0.536
0.076
0.008
0.013
0.144
0.038
0.270
0.254
0.440
0.059
46
32
59
27
44
42
45
100
Piperonyl benzylamine
Pyridin-2-ylmethylamine
2,5-di methoxy benzylamine
Tyrosine
3-Hydroxy Benzylamine
Pyridin-3-ylmethylamine
-
Rosiglitazone

a IC50 (the concentrations that reach half-maximal inhibition) for PPARγ binding was calculated
with GraphPad Prism from the data of ten 3-fold serial titration points. b Inhibition constant (Ki)
c
calculated as detailed in Experimental Section. Transactivation activity is defined as the
maximum activity that a compound achieves and reported as % compared to that of Rosi, which
is designated as 100%. All experiments were performed in triplicate.
Together, these SAR studies indicate that both vanillylamine (WO91A and 3a) and piperonyl
benzylamine (2c and 3b) at Site 1 convey favorable PPARγ binding affinity regardless whether it
is biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, the SAR identified WO91A
as the most potent derivative for PPARγ binding and reveals that WO91A binds to PPARγ with
significantly more favorable chemical properties than UHC1. We next used the in silico docking
modeling method (Autodock) to compare the docking simulations of WO91A and UHC1 with
PPARγ LBD. The docking scores for binding affinity (UHC1 -10.2 kcal/mol vs. WO91A -11.3
kcal/mol) suggests that WO91A fits better than UHC1 in the pocket within the LBD of PPARγ
(Figure 2A, 2B). Notably, in our simulation, both UHC1 and WO91A occupy the alternate
ligand-binding site (Figure 2A, 2B), a site comprised of H2’-H3, -sheet, and  loop regions,
which was previously identified in the PPARγ LBD structure complexed with a partial agonist
MRL20.16 This docking mode is distinct from the canonical PPARγ ligand binding pocket
occupied by full agonists, which usually comprises H3, H3-4 loop, H11 and H12.17 Further,
although SR1664 and its derivative UHC1 were previously reported to bind to the canonical
ligand binding pocket similar to that of a full agonist10, 18, our docking model is consistent with a
recent X-ray crystal structure study that SR1664 binds to the alternate binding site of PPARγ.12

Our docking simulation further showed that WO91A formed hydrogen bonds with multiple
amino acids in the alternate binding site (Figure 2C). The two carboxylate oxygens of WO91A
formed hydrogen bonds with both Arg280 and Gln283 on helix H3 of PPARγ LBD. The amide
oxygen of WO91A also forms hydrogen bond with Ser342 on -sheet. In contrast, UHC1 formed
hydrogen bond with Ser342 only (Figure 2D). These results may explain the more potent binding
affinity of PPARγ with WO91A than UHC1.
Figure 2. WO91A binds to PPARγ LBD in a distinct mode. Docking simulation was performed
with crystal structure of PPARγ LBD (Protein Data Bank PDB ID 5GTO) with Autodock Vina.
A. Docking simulation of the PPARγ LBD:WO91A complex. WO91A is in red. Docking score

for affinity shown. B. Docking simulation of the PPARγ LBD:UHC1 complex. UHC1 is in red.
Docking score for affinity shown. C, D. Schematic representation of atomic interaction between
PPARγ LBD and WO91A (C) or UHC1 (D). hydrogen bonds were shown in brown dashed lines
with donor-acceptance distances in angstroms.
The indole derivatives SR1664 and UHC1 were reported to act as non-agonist PPARγ ligands in
that similar to full agonist Rosi, they bind to PPARγ and increase insulin sensitivity, but in
contrast to Rosi, they do not or weakly activate the transactivation activity of PPARγ, which
probably explain their less or minimal side effects as observed for Rosi.10, 11 We therefore
investigated the transactivation activity of the above newly synthesized compounds. PPARγ
serves as a transcription factor to activate the expression of a number of genes under the
control of promoter carrying PPARγ-responsive element (PRE). To determine the effect of
these UHC analogs on PPARγ transactivation activity, we transfected HEK293 cells with both
PPARγ-expressing plasmid and 3xPRE-Luciferase reporter plasmid and treated the cells with
compounds in a dose-dependent manner (with increasing concentrations from 0.001M to 10
μM). As shown in Table 1 and Figure 3B, compared to the full PPARγ agonist Rosi that
markedly increased the reporter activity, the parental compound UHC1 activated the reporter as
an approximately 45% of strength of Rosi. We noticed that although UHC1 was reported as non-
agonist ligand10, the data obtained here indicates that UHC1 maintains significant activity on
PPARγ reporter activation. The majority of our newly synthesized compounds that exhibited
high PPARγ binding affinity activated the reporter in a manner similar to or less potently than
UHC1, indicating that they are partial agonists of PPARγ. WO91A is in particular of interest in

that it exhibited not only the highest PPARγ binding affinity among all active compounds (its
IC50 is over 30-fold lower than that of the parental compound UHC1) but also the lower
transactivation activity of PPARγ compared to UHC1 (15% for WO91A vs. 45% for UHC1) .
Figure 3. WO91A acts as partial PPARγ agonist. A. The in vitro competition binding assays was
performed as in Experimental sections. Florescence polarization value (mP) was measured at 485
nm (excitation) and at 535 nm (emission). B. Transactivation activity of compounds in HEK293
cells transfected with PPARγ2 and PPRE X3-TK-luc reporter. Transactivation activity was
presented as fold change against DMSO vehicle.

We then chose WO91A for further biological evaluation. PPARγ phosphorylation at S273 is
reported to be responsible for obesity-related insulin resistance.6 Indole derivatives such as
UHC1 inhibits PPARγ phosphorylation at S273 to improve insulin sensitivity. It is reported that
ligand binding to the PPARγ alternate binding site directly blocks the S273 phosphorylation.12
We therefore investigated whether WO91A suppresses the phosphorylation of PPARγ at S273.
As shown in Figure 4A, like Rosi and UHC1, WO91A significantly inhibited the S273
phosphorylation of PPARγ in 3T3-L1 differentiated adipocytes in the presence of TNF which is
known to activate CDK5 to phosphorylate PPARγ at S273, by Western blotting using PPARγ
phospho-S273-specific antibody. Of note, WO91A inhibited the S273 phosphorylation to a
greater degree than UHC1 (Figure 4A). PPARγ S273 phosphorylation is associated with
repression of a set of genes that are believed to be responsible for insulin sensitivity and
dephosphorylation of PPARγ S273 led to the derepression of these genes.6 We investigated the
effect of WO91A on the expression of S273 phosphorylation -associated genes. We selected
Adiponectin, Adipsin, and Car3, three genes that were reported to be associated with insulin
sensitivity for this purpose. We observed that WO91A treatment caused the upregulation of
mRNA levels of Adiponectin, Adipsin, and Car3 in 3T3-L1 differentiated adipocytes (Figure 4B-
D). Together, these results indicate that WO91A inhibits PPARγ phosphorylation.

A
Rosi
+
UHC1
+
91A
+
DMSO DMSO
Treatment
TNF-α
_
+
*
pPPARγ
PPARγ
B
C
D
Adiponectin
Adipsin
Car3
18
16
14
12
10
8
6
4
2
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
6
5
4
3
2
1
0
DMSO Rosi UHC1 WO91A
DMSO Rosi UHC1 WO91A
DMSO Rosi UHC1 WO91A
Figure 4. WO91A inhibits PPARγ S273 phosphorylation and controls the expression of genes
associated with S273 phosphorylation. A. Phosphorylation of PPARγ S273 in 3T3-L1
differentiated preadipocytes treated with TNF 10 ng/ml for 1 h in the presence of compounds at
10 M. pSer273 of PPARγ was detected by Western blotting using PPARγ pSer273-specific
antibody. * undifferentiated parental 3T3-L1 cells. B-D, Relative mRNA expression levels of
pSer273-associated genes Adiponectin (B), Adipsin (C), and Car3 (D) in 3T3-L1 differentiated
with differentiation media in the presence of compounds at 10 M by qRT-PCT.
Activation of PPARγ by full agonists such as Rosi not only inhibits PPARγ phosphorylation to
improve insulin resistance but also regulates additional events including such side effects as fluid
retention and fat and weight gain, events that partial or non-agonists exhibit weak or minimal
impact. One such event is adipogenesis. We therefore investigated whether WO91A affects
adipogenesis in a cell-based assay. As a master regulator of adipose differentiation, the full
agonism of PPARγ by TZDs drives 3T3-L1 mouse embryonic fibroblast cells to differentiate

into adipocyte. When 3T3-L1 cells were differentiated into adipocytes in the presence of
differentiation media, Rosi treatment markedly induced the differentiation of adipocytes, as
indicated by the widespread presence of lipid droplets with positive Oil Red O staining,
whereas there was little or no Oil Red O staining when treated with DMSO (Figure 5A, 5B).
In contrast to Rosi, both WO91A and UHC1 exhibited significantly reduced Oil Red O
staining, but WO91A achieved so with a greater degree (Figure 5C, 5D), indicative of its little
effect on adipogenesis. Next, we investigated the impact of WO91A on the expression of genes
responsible for adipogenesis. We chose genes FABP4, LPL and Glut4 as representatives for this
purpose. Full agonist Rosi is known to induce the expression of these adipogenic genes during
adipogenesis. Indeed, Rosi treatment of differentiated 3T3-L1 pre-adipocytes significantly
upregulated the mRNA levels of FABP4, Glut4, and LPL genes (Figure 5E-G). In contrast,
WO91A only weakly increased the expression these genes at the contration of 10 M (Figure
5E-G).

Rosi
UHC1
WO91A
DMSO
A
D
B
C
Oil Red O
E
F
G
FABP4
LPL
Glut4
6
5
4
3
2
1
0
5
4
3
2
1
0
2.5
2.0
1.5
1.0
0.5
0.0
DMSO Rosi
UHC1 WO91A
DMSO Rosi
UHC1 WO91A
DMSO Rosi UHC1 WO91A
Figure 5. WO91A does not induce adipogenesis. A-D, Oil Red O staining in 3T3-L1 cells
differentiated with differentiation media in the presence of compounds at 10 M. E-G,
Relative mRNA expression levels of adipogenic genes FABP4 (E), LPL (F), and Glut4 (G) in
3T3-L1 differentiated with differentiation media in the presence of compounds at 10 M by
qRT-PCT.
In this study, we have described here the design and synthesis of a series of derivatives of
antidiabetic compound UHC1. These studies combine in vitro competition binding assay and
transcriptional activity assay, allowing the identification of derivatives that exhibit potent
PPARγ binding affinity but yet minimal PPARγ transactivation activity. These SAR studies
indicate that both vanillylamine (WO91A and 3a) and piperonyl benzylamine (2c and 3b) at Site

1 are favored to bind PPARγ in an indole scaffold with either biphenyl or 3-trifluoromethyl
benzyl group at Site 2. In particular, compound WO91A with vanillylamine at Site 1 and a
biphenyl group at Site 2 conveys highly potent PPARγ binding affinity (IC50 =16.7 nM, Ki = 13
nM), over 30-fold more potent than the parental compound UHC1, which may serve as a
foundation for further drug development endeavor. Our further results indicate that WO91A
inhibits PPARγ S273 phosphorylation but plays little role in other full agonist-dependent but
S273 phosphorylation-independent events such as adipogenesis. In summary, these results
present a clearer picture of how future efforts for new therapeutics targeting PPARγ can be
directed for high binding potency but without eliciting transactivation activity.
Acknowledgements:
This work was supported by Oklahoma Center for the Advancement of Science and Technology
and National Institutes of Health (DK108887, DK116017) to W.W.

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