Design, Synthesis, and Biological Evaluation of Indole Biphenylcarboxylic Acids as PPARγ Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.5b00218

The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.

Full text of DOI:10.1021/acsmedchemlett.5b00218

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Biological Evaluation of Indole
Biphenylcarboxylic Acids as PPARγ Antagonists
Alice Asteian, Anne-Laure Blayo, Yuanjun He, Marcel Koenig, Youseung Shin, Dana S. Kuruvilla,
Cesar A. Corzo, Michael D. Cameron, Li Lin, Claudia Ruiz, Susan Khan, Naresh Kumar, Scott Busby,
David P. Marciano, Ruben D. Garcia-Ordonez, Patrick R. Griffin, and Theodore M. Kamenecka*
Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #A2A, Jupiter, Florida
33458, United States
S
* Supporting Information
ABSTRACT: The thiazolidinediones (TZD) typified by rosiglitazone are the only
approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM).
Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a
number of severe side effects including congestive heart failure, edema, weight gain,
and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with
high affinity but do not induce transactivation of the receptor, yet they act as insulin
sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of
PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure−activity relationship studies led to the identification
of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-
induced obese mice.
KEYWORDS: PPARγ, nuclear receptor, indole, diabetes
he peroxisome proliferator-activated receptors (PPARs)
are ligand-dependent transcription factors and members
with their ability to block the obesity-linked phosphorylation of
PPARγ at serine 273 (pS273), and counter AEs correlate with
the magnitude of agonism.16 Importantly, blockage of pS273
was shown to be mediated via a conformational change in the
ligand binding domain of PPARγ and not by kinase inhibition.
Subsequently, compounds were developed (e.g., SR1664) that
bind PPARγ with high affinity and block pS273, yet are devoid
of agonism but still afford robust antidiabetic activity in rodent
models of diabetes.1 These findings clearly suggest that it is
possible to separate agonism of the receptor from efficacy, and
such efforts should yield compounds with improved therapeutic
index. In this Letter, we report the discovery and structure−
activity relationship (SAR) of a novel series of indole-based
carboxylic acids related to SR1664.
This project initiated with the prospect of identifying or
synthesizing noncovalent antagonists of PPARγ.17 Covalent
antagonists have been identified but are unlikely to be
developed as therapeutics.18,19 It was not clear if this proposal
was even feasible at the onset, but it entailed a challenge that
would shed light on the hypothesis that one could separate
agonism of PPARγ from its efficacy as a potential diabetic
therapeutic. Without access to a high-throughput screen, we
investigated several alternative approaches to identify antago-
nists of PPARγ. One approach involved starting with known
PPARγ partial agonists from both the primary and patent
literature and designing out the agonism. With a plethora of
T
of the nuclear receptor superfamily that regulate gene
expression patterns of diverse biological processes.2 The
PPARs play a key role in transcriptional regulation of genes
involved in lipid and glucose metabolism and inflammation.
The PPAR family consists of three genes, PPARα, PPARγ, and
PPARβ/δ with each having different tissue distribution,
selectivity, and responsiveness to ligands. PPARγ has been
implicated in several disorders including atherosclerosis,
diabetes, obesity, and cancer. Thus, the search for potent and
selective modulators of PPARγ to be used as therapies to treat
these pathologies is well justified.3 In fact PPARγ agonists have
for many years represented a promising approach to treat
insulin resistance associated with type 2 diabetes.4,5 PPARγ is
the molecular target of the thiazolidinedione (TZD) class of
antidiabetic drugs rosiglitazone and pioglitazone.6 These drugs
were developed prior to their molecular mechanism of action
being determined. Both compounds are high affinity full
agonists of PPARγ. While the TZD class of drugs exhibits
robust effects on glucose metabolism and insulin sensitivity, it is
associated with a myriad of undesirable side effects (adverse
events; AEs) including fluid retention with increased risk of
heart failure, weight gain, loss of bone mineral density, and
cancer.7−9 In efforts to improve the therapeutic index of PPARγ
modulators, partial agonists were developed that proved to be
equally efficacious as insulin sensitizers as full agonists yet with
significantly improved AE profiles.10−15 This disconnect
between agonism and efficacy raised questions about the
mechanism of action of such compounds. Recently it was
reported that efficacy of both full and partial agonists correlated
Received: May 30, 2015
Accepted: August 4, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00218
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potential starting points in the public domain and with little
guidance on where to start, we chose a handful of different
scaffolds and began synthesizing some analogures. Structures
are provided in the Supporting Information (Figure S1).
Partials agonists are defined as weak activators of PPARγ that
elicit the same activation pattern as full agonists, but with a
lower maximal activity. The antihypertensive agent Telmisartan
is an angiotensin II receptor antagonist, but it has also been
reported to function as a weak PPARγ agonist.20,21 Scientists at
GlaxoSmithKline optimized the telmisartan scaffold to afford
active PPARγ antagonists, considerable optimization was
undertaken. We envisioned modifications of all parts of the
molecule as shown in Figure 2. First, we focused our efforts on
GSK 7b, as a potent PPARγ partial agonist (Figure 1A).22
A
Figure 2. SAR optimization of SR1664.
modification of the substituents on the phenyl ring as well as at
the stereogenic center. In the context of expanding the SAR and
improving in vivo properties, we sought to make modifications
to the indole ring as well as to the biphenyl carboxylic acid. The
synthesis of these compounds 15−69 is outlined in Scheme 1
and Schemes S1−S4 in the Supporting Information.
Figure 1. (A) Identification of SR1664 from GSK 7b (SR9034). (B)
Effect of ligands on PPARγ GAL-4 transactivation.
a
Scheme 1. Synthesis of Biphenyl Analogues
group at Amgen developed AMG-131 as a potent and highly
selective PPARγ partial agonist that advanced into Phase 2
clinical trials.23 FK614 from Astellas Pharma is reported as a
selective partial agonist for PPARγ in a GAL4-PPAR trans-
activation assay.11,24 MBX-102 (metaglidasen) is a weak PPARγ
ligand that exhibits both partial agonist and antagonist
activity.25,26 Lastly, Merck identified MRL-24 as a potent
partial agonist of PPARγ, which was ultimately optimized and
advanced into clinical trials as MK-0533.12,27
Roughly two dozen analogues in each series were synthesized
and screened in both a PPARγ binding assay using a
competitive Lanthascreen assay format and a fluorescently
labeled probe as well as in a transactivation assay using PPARγ-
GAL4 and a UAS-Luciferase reporter assay system in an effort
to help identify which series held the most promise.
Compounds that exhibited minimum transactivation in GAL-
4 were profiled using full-length PPARγ and a 5xPPRE-
Luciferase reporter construct. Our definition of an antagonist of
PPARγ is one wherein the transactivation efficiency is ≤10% of
that of rosiglitazone at 10 μM concentration, while still
retaining good affinity for the receptor (i.e., IC50 ≤ 250 nM).
Most analogues synthesized in the different scaffolds in Figure
S1 afforded partial agonists, but several compounds in the GSK
7b series had minimal PPARγ transactivation and looked
promising as PPARγ antagonists. Initial SAR simply focused on
different indole amides and led to the discovery of SR1664
(Figure 1).1 Remarkably, SR1664 has similar affinity for PPARγ
as rosiglitazone, but virtually no activation of the receptor as is
inherent to GSK7b.
a
Reagents and conditions: (a) 2-butanone, HCl, dioxane, reflux; (b)
allyl bromide, K2CO3, DMAC, rt; (c) tert-butyl 4′-(bromomethyl)-
[1,1′-biphenyl]-2-carboxylate, NaH, DMF; (d) Pd(PPh3)4, THF, rt;
(e) R-PhCH(R′)NH2, HATU, DCM, rt; (f) TFA, DCM, rt.
Indole acid (2) was obtained through the condensation of 4-
hydrazine benzoic acid 1 and 2-butanone in high yield followed
by esterification with allyl bromide to give 3. Alkylation of the
indole with tert-butyl 4′-(bromomethyl)-[1,1′-biphenyl]-2-
carboxylate provided 4. Cleavage of the allyl group with
tetrakis(triphenylphosphine)palladium(0) gave acid 5. Lastly,
amide coupling with an amine of interest followed by hydrolysis
of the tert-butyl ester gave final products (Tables 1−4).
For compounds wherein the enantiomerically pure amine
was not commercially available, these were synthesized using
Ellman chemistry28,29 as shown in Scheme S1 in the Supporting
Information. A few modifications to the indole core were also
investigated. The synthesis of these analogues is outlined in
Scheme S2 in the Supporting Information. Finally, we
maintained the biphenylmethyl substitution and explored acid
bioisosteres (Schemes S3 and S4 in the Supporting
Information).
While this was proof-of-principle that one could in fact
identify compounds that bound PPARγ with high affinity with
little to no transactivation of the receptor, very little was known
about the SAR around this scaffold. Additionally, SR1664 had
poor oral in vivo exposure in rodents, and the nitro group
needed to be replaced for potential toxicity concerns. In an
effort to expand the SAR around SR1664 and design orally
We first explored SAR of the left-hand side of the molecule
(Table 1). Initial exploration of the amide group seemed
B
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Table 1. SAR Modification of the Benzamide
a
b
a
b
Cmpd
R
Lantha (IC50, nM) GAL-4 (EC50, nM) Cmpd
R
Lantha (IC50, nM) GAL-4 (EC50, nM)
9034
15
α-ethyl-benzylamine
benzylamine
0.37
1
13(40%)
22
23
24
25
26
27
28
39
4-Me-benzylamine
4-Br-benzylamine
4
14 (30%)
300 (40%)
632 (10%)
2532 (10%)
831 (10%)
363 (30%)
188 (20%)
822 (18%)
14
38
667
1
516 (25%)
1344 (20%)
124 (10%)
196 (35%)
1 (15%)
16
2-Me-benzylamine
2-NH2-benzylamine
2-NO2-benzylamine
3-Me-benzylamine
3-OMe-benzylamine
3-NO2-benzylamine
6
4-NO2-benzylamine
4-t-Bu-benzylamine
thiophen-2-ylmethanamine
diphenylmethanamine
cyclobutanamine
17
32
24
5
18
19
26
32
78
20
4
5665 (40%)
179(40%)
21
20
cyclohexylmethanamine
a
b
Values are means of at least two experiments. All standard deviations are <20%. Transactivation at 10 μM.
simplest by first removing the stereogenic center found in GSK
7b (SR9034). Removing the α-ethyl substituent (15) led to a
slight loss in affinity; however, the compound retained its
partial agonism of the receptor. In general, most types and
positions of substitutions on the phenyl ring were well tolerated
and yielded potent PPARγ partial agonists, which were of little
interest given we were searching for antagonists.
Substitution was tolerated at the ortho-, meta-, and para-
positions with only slight variations in activity. Indeed if we
compare ortho- (18), meta- (21), and para-nitro (24)
analogues, these compounds have similar affinities and
transactivation potentials for PPARγ. The benzyl group was
not required as heterocyclic replacements such as 26 (IC50 = 1
nM, EC50 = 196 nM) were also potent. Surprisingly, the
Table 2. SAR of the Benzyl Amine
Lantha (IC50, GAL-4 (EC50,
a
b
Cmpd
R
R′
stereochemistry
nM)
nM)
9034
30
H
H
H
Et
0.37
3
13 (40%)
53 (20%)
3 (30%)
Et
S
31
Et
R
S
0.13
21
32
4-F
Me
Me
Me
Me
Me
Me
Me
Me
638 (40%)
47 (50%)
2076 (30%)
53 (60%)
4288 (10%)
503 (75%)
516 (20%)
129 (90%)
33
4-F
R
S
0.79
0.90
0.54
80
34
4-OMe
4-OMe
4-NO2
4-NO2
4-Br
sterically encumbering benzhydryl amide (27) with an IC50
=
35
R
S
26 nM and an EC50 = 1 nM is also well tolerated, perhaps
giving an indication of the size of the binding pocket. Lastly,
aryl groups are not required for potency, as simple cycloalkyl
groups (28, 29) afford potent partial agonists as well.
1664
36
R
S
2
37
28
38
4-Br
R
0.73
a
We next examined the effect of the α-substituent in the
benzyl amine. The parent molecule, SR9034, contains a racemic
ethyl group and is a potent PPARγ partial agonist (Table 2).
Separation of the enantiomers led to (S)-30 (IC50 = 3 nM and
EC50 = 53 nM) and (R)-31 (IC50 = 0.13 nM and EC50 = 3
nM), both of which are potent partial agonists; however, the
(R)-enantiomer was an order of magnitude more potent both
in binding and transactivation. We switched from α-ethyl to α-
methyl given the increased access to commercially available
starting materials. This did not seem to affect in vitro activity as
4-substituted α-methylbenzyl amine analogues were equipotent
to the lead SR9034. Introduction of a p-substituent of the
appropriate size led to an unexpected breakthrough. Smaller
groups such as fluorine (S)-32 and (R)-33 or methoxy groups
(S)-34 and (R)-35 did not seem to have much effect on in vitro
activity, with the same general trend observed in that the (R)-
enantiomer was the more potent of the pair. However, the (S)-
4-nitro analogue SR1664, despite good affinity for PPARγ,
exhibited minimal transactivation in GAL-4. The different
effects on transactivation between SR1664 and its enantiomer
36, are quite striking (Figure S2 in the Supporting
Information). SR1664 was also profiled using the full length
PPARγ-reporter assay and exhibited no transactivation at 10
μM. It is possible to observe no transactivation for compounds
that do not get into cells. Therefore, we screened rosiglitazone
Values are means of at least two experiments. All standard deviations
b
are <20%. Transactivation at 10 μM.
in the presence of increasing concentrations of SR1664. This
resulted in a right-fold shift in the rosiglitazone transactivation
curve indicating competitive binding by SR1664 alleviating any
concerns of lack of cell penetrance. Thus, SR1664 was
identified as our first PPARγ antagonist from this series. From
this small subset of analogues, we concluded that not only was
the stereochemistry at the amine alpha-center important but so
was the size of the phenyl substituent. To fully test this,
however, we would need to go beyond what was commercially
available, and synthesize additional amines, and for this, we
turned to Ellman chemistry as highlighted in Scheme S1 (Table
3).
The next set of analogues incorporated the (S)-alpha-methyl
substituent and looked to examine the effect of phenyl
substitution in more detail (Table 3). We introduced
substitution at the 2-, 3-, and 4-positions of the phenyl ring
ranging from small to medium to large with respect to steric
bulk. At the 2-position, substitution was tolerated, but
analogues were all partial agonists of PPARγ. If the substitution
was too large (42), affinity for the receptor is compromised.
The 3-position also tolerated substitution, and as the size of the
substituent increased, we were able to almost completely
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Table 3. SAR for Phenyl Ring Substitution
Table 4. Substitutions at the Stereogenic Center
Lantha (IC50,
GAL-4 (EC50,
a
b
Cmpd
R
stereochemistry
nM)
nM)
53
54
45
55
56
57
58
59
60
H
5
133 (30%)
100(10%)
852 (5%)
100(15%)
133(10%)
60(18%)
CF3
Me
Et
S
S
S
S
S
S
S
S
17
30
9
iPr
80
8
cyclopropyl
nBu
81
1222
97
116(15%)
226(18%)
150 (20%)
iBu
Bn
a
Values are means of at least two experiments. All standard deviations
a
Values are means of at least two experiments. All standard deviations
b
are <20%. Transactivation at 10 μM.
b
are <20%. Transactivation at 10 μM.
led us to maintain the 2,3-dimethylindole substitution in all
future analogues.
remove transactivation (45, 47). The same trend was observed
at the 4-position. Small substitution led to potent partial
agonists, while analogues containing larger substitution led to
antagonists (51, 52).
To put into context the minimal transactivation observed at
10 μM in the GAL-4 assay, the dose response curves for 46 and
51 are included in Figure 3. As can be seen when compared to
We also looked at the effect of modifications to the indole
core itself on PPARγ potency and transactivation (Table S2 in
the Supporting Information). Installation of a benzooxazolone
instead of indole as a central core gave 61, which essentially
wiped out all potency on PPARγ. A benzoimidazolone core was
slightly better tolerated (62) but still led to a 20-fold loss in
affinity. A benzimidazole core was investigated and also led to
potent compounds with minimal PPARγ transactivation;
however, we avoided benzimidazoles of this type in general
given their known activity at the angiotensin II receptor.30 Of
the several heterocyclic cores examined, none provided any
advantages over the indole with regards to potency or activation
of PPARγ and were therefore not pursued.
We next investigated the effect of modifying the acid moiety
in 45 (Table S3 in the Supporting Information). Replacement
with a tetrazole (65) or an oxadiazolone (63, 64) resulted in a
slight decrease in PPARγ activity with minimal PPARγ
transactivation. The hydroxamic acid (66) and amides (67,
68) were also tolerated. Interestingly, nitrile 69 had no activity.
While these acid bioisosteres maintained affinity for PPARγ
with minimal transactivation, several of them proved to be
suboptimal with regards to plasma exposure in rodents and
were hence abandoned as useful substitutions (Table 5). With
Figure 3. Transactivation curves for PPARγ antagonists in (A) GAL4-
PPARγ and (B) full length PPARγ.
rosiglitazone, there is essentially no activation of PPARγ despite
very good affinity for the receptor. Compound 46 was further
profiled for transactivation using full length PPARγ and again
showed no activity (Figure 3).
With this information, we fixed the left-hand side of the
molecule with 3-isopropylphenyl as in 45 and explored the SAR
of the stereogenic center (Table 4). Not surprising, when R =
H (53), the compound is a potent partial agonist, in agreement
with data from the compounds in Table 1. Trifluoromethyl
(54) and ethyl (55) were slightly more potent than α-methyl
analogue 45, with a slight increase in transactivation. Most of
the substitutions here were tolerated with regards to binding
affinity, with the exception of R = iBu (59); however, as the size
of the substitution increased beyond ethyl, there seemed to be a
noticeable increase in transactivation of PPARγ. These results
suggested that the methyl group in the S-configuration was
optimal for PPARγ antagonists in this series. Attempts to
manipulate the methyl groups at the 2,3-position of the indole
core did not offer any advantages with regards to antagonism of
PPARγ (data not shown). Removal of one methyl group led to
a slight loss in affinity and removal of the second methyl group
led to a further drop with no effect on transactivation. Loss in
affinity and potential exposure of the indole ring to metabolism
Table 5. In Vivo Exposures of Selected Biphenyl Analogues
a
Cmpd
45
46
50
51
63
64
65
66
67
[plasma] μM,
2 h
9
19
0.6
5.6
0
0.2
0
0.4
2.5
[plasma] μM,
6 h
3
2.4
0.1
1.9
0
0.01
0
0.03
0.1
a
20 mg/kg p.o.
several promising PPARγ antagonists to choose from, we
investigated oral dosing (p.o.) in mice in an effort to identify
potential candidates for in vivo efficacy studies. When
compounds were dosed orally at 20 mg/kg, we measured
plasma exposure at t = 2 h and t = 6 h. The three carboxylic
acids 45, 46, and 51 had better oral exposure than the
corresponding acid bioisosteres (Table 5). Based on these
results, we chose to evaluate the antidiabetic efficacy of 46 in
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DIO mice, a model of type-2 diabetes and obesity characterized
by hyperglycemia and impaired glucose tolerance. This model
more closely mimics the human condition brought about by
high-fat Western diets than the genetic models using db/db or
ob/ob mice.31
Male C57BL/6J JAX mice were from Charles River and were
maintained on a high fat diet (Research Diets D12451). Mice
were dosed orally once a day (q.d.) for 4 days (20 or 40 mg/
kg) and fasted on Day 4. On the morning of Day 5, animals
were dosed again, and then 180 min later dosed with a glucose
challenge (2 g/kg p.o.).
Baseline blood samples were taken immediately from the tail
vein before dosing (B1; −180 min) and immediately before the
glucose challenge (B2; 0 min). Further blood samples were
taken from the tail vein from all groups at 10, 20, 30, 60, and
120 min. Samples were analyzed for plasma glucose and insulin
content, and a log transformation was used for insulin.
Analysis was by robust regression and included treatment
and assay day as factors and Day 1 body weight, bleeding order,
and Day −4 (baseline) plasma glucose or insulin concentration
as covariates. Data are shown as adjusted means (n = 8), and
standard errors of the mean (SEM) are calculated from the
residuals of the statistical models. Comparisons against vehicle
p.o. q.d. were by Williams’ test for 46. Significances are denoted
by *p < 0.05, **p < 0.01, and ***p < 0.001.
have already reported that antagonists such as SR1664 appear
to be devoid of many of the side effects associated with full
agonism of the receptor, such as weight gain, edema, and bone
mineral loss. It is likely that 46 has a similar profile. We are
currently pursuing X-ray crystallography and HDX (hydrogen−
deuterium exchange) mass spec techniques to further under-
stand the protein dynamics upon ligand binding. We are also
continuing to optimize this scaffold to further improve oral
exposure to support chronic dosing studies in obese rodents.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00218.
Experimental details for key compounds; lanthascreen
and transactivation assays protocols (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-561-228-2207. E-mail: kameneck@scripps.edu.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
After once daily oral administration of 46 for 5 days, a dose-
dependent reduction in fasting glucose was observed (Figure
4). After oral glucose challenge (oGTT), 46 produced a
Funding
National Institutes of Health grant MH084512 (PI: Rosen), the
Abrams Charitable Trust, and the Klorfine Family Fellowship.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AE, adverse events; DCM, dichloromethane; DMAC, dime-
thylacetamide; DMF, dimethylformamide; HATU, 1-[Bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxo-hexafluorophosphate; HCl, hydrochloric acid;
PPAR, peroxisome proliferator-activated receptors; PPTS,
pyridinium p-toluenesulfonate; SAR, structure−activity rela-
tionship; T2DM, treatment of type-2 diabetes; TFA, tifluoro-
acetic acid; THF, tetrahydrofuran; TZD, thiazolidinedione
Figure 4. In vivo efficacy studies with 46 in DIO mice.
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DOI: 10.1021/acsmedchemlett.5b00218
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