Discovery of INT131: A selective PPARγ modulator that enhances insulin sensitivity

Article abstract of DOI:10.1016/j.bmc.2012.11.058

PPARγ is a member of the nuclear hormone receptor family and plays a key role in the regulation of glucose homeostasis. This Letter describes the discovery of a novel chemical class of diarylsulfonamide partial agonists that act as selective PPARγ modulators (SPPARγMs) and display a unique pharmacological profile compared to the thiazolidinedione (TZD) class of PPARγ full agonists. Herein we report the initial discovery of partial agonist 4 and the structure-activity relationship studies that led to the selection of clinical compound INT131 (3), a potent PPARγ partial agonist that displays robust glucose-lowering activity in rodent models of diabetes while exhibiting a reduced side-effects profile compared to marketed TZDs.

Full text of DOI:10.1016/j.bmc.2012.11.058

Bioorganic & Medicinal Chemistry 21 (2013) 979–992
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of INT131: A selective PPAR
insulin sensitivity
c modulator that enhances
Joshua P. Taygerly a, , Lawrence R. McGee , Steven M. Rubenstein , Jonathan B. Houze ,
⇑
a
a
a
Timothy D. Cushing , Yang Li , Alykhan Motani , Jin-Long Chen , Walter Frankmoelle , Guosen Ye a,à,
a
a
a
a,
a
a,
a,§
a,§
a
a,–
Marc R. Learneda , Juan Jaen , Shichang Miao , Pieter B. Timmermans , Martin Thoolen
,
a,k
a
a
a
Patrick Kearney , John Flygare , Holger Beckmann , Jennifer Weiszmann , Michelle Lindstrom ,
a
a
a
a
b
b
Nigel Walker , Jinsong Liu , Donna Biermann , Zhulun Wang , Atsushi Hagiwara , Tetsuya Iida ,
Hisateru Aramaki , Yuki Kitao , Hisashi Shinkai , Noboru Furukawa , Jun Nishiu , Motonao Nakamura
b
b
b
b
b
b,
a
Amgen, Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States
Japan Tobacco 2-1, Toranomon 2-chome, Minato-ku, Tokyo 105-8422, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
PPAR
cose homeostasis. This Letter describes the discovery of a novel chemical class of diarylsulfonamide par-
tial agonists that act as selective PPAR modulators (SPPAR Ms) and display a unique pharmacological
profile compared to the thiazolidinedione (TZD) class of PPAR full agonists. Herein we report the initial
discovery of partial agonist 4 and the structure–activity relationship studies that led to the selection of
clinical compound INT131 (3), a potent PPAR partial agonist that displays robust glucose-lowering activ-
c is a member of the nuclear hormone receptor family and plays a key role in the regulation of glu-
Received 29 September 2012
Revised 20 November 2012
Accepted 24 November 2012
Available online 10 December 2012
c
c
c
c
Keywords:
INT131
ity in rodent models of diabetes while exhibiting a reduced side-effects profile compared to marketed
TZDs.
PPAR
Type 2 diabetes
Selective PPAR
SPPAR Ms
c
Ó 2012 Elsevier Ltd. All rights reserved.
c
modulator
c
1
. Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disorder, de-
ber of complications including kidney impairment, peripheral neu-
ropathy, blindness, and heart failure. Type 2 diabetes accounts for
over 90% of all cases of diabetes, and it has been estimated that 270
million people will be considered type 2 diabetics by 2025.2 De-
spite extensive research and development of anti-diabetic thera-
pies by the pharmaceutical industry, there remains a significant
need for safe glucose-lowering agents.
,3
fined by the body’s inability to regulate glucose homeostasis as a
result of defective insulin signaling (insulin resistance) and even-
tual impairment of insulin production due to pancreatic b-cell
1
exhaustion. If left untreated, type 2 diabetes can promote a num-
Peroxisome proliferator-activated receptor
c (PPARc) has been
shown to modulate the transcription of genes responsible for adi-
⇑
Corresponding author.
E-mail addresses: josh.taygerly@gmail.com, taygerly@amgen.com (J.P. Taygerly).
Present address: NGM Biopharmaceuticals, Inc., 630 Gateway Boulevard, South
pose differentiation, glucose homeostasis and lipid metabolism1
,3,4
PPAR
c is the target of the thiazolidinedione (TZD) class of anti-
Ò
San Francisco, CA 94080, United States.
diabetic agents that include rosiglitazone (Avandia ; Glaxo-
SmithKline) and pioglitazone (Actos ; Takeda) (Fig. 1). The TZDs
are orally-active small molecule PPAR full agonists that have been
à
Ò
4,5
Present address: GlaxoSmithKline, One Franklin Plaza, Philadelphia, PA 19101,
United States.
c
§
Present address: ChemoCentryx, Inc., 850 Maude Avenue, Mountazin View, CA
clinically validated to increase insulin sensitivity and restore nor-
mal body insulin and glucose levels in hyperglycemic patients.
However, treatment with TZDs has been associated with serious
side effects, including weight gain, peripheral edema, hepatotoxic-
ity, increased risk of congestive heart failure, and bone fracture.
These effects have limited the utility of this class of therapeutic
9
4043, United States.
–
Present address: Edison Pharmaceuticals, Inc., 350 North Bernardo Avenue,
Mountain View, CA 94043, United States.
k
Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080,
United States.
Present address: The University of Tokyo, Department of Biochemistry and
Molecular Biology, Graduate School of Medicine and Faculty of Medicine, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
1
,3,4,6,7
agents.
Other structurally unrelated PPARc full agonists have
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.058

9
80
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
S
Cl
O O
S
CH3
N
O
N
NH
NH
O
O
Cl
Rosiglitazone (1)
Cl
Cl
Et
S
O
N
O
NH
O
O
N
Pioglitazone (2)
INT131 (3)
Figure 1. Marketed TZD PPAR
3).
c
full agonists and selective PPAR
c
modulator INT131
(
displayed similar side-effect profiles, suggesting the possibility
that these undesired effects could be mechanistically associated
with receptor full agonism.8
,9
Recently, we and others have disclosed next-generation ligands
10–12
that selectively modulate the activity of PPAR
c.
These selec-
tive PPAR modulators (SPPAR Ms) have partial agonist activity
c
c
compared to the TZDs, and show increased insulin sensitivity and
robust glucose-lowering activity with a reduction of the side ef-
fects observed with PPARc full agonists, both in animal models
of diabetes and in the clinical setting. The underlying mechanism
for this pharmacological effect is not well understood, although re-
cent studies by Spiegelman and co-workers have shown that insu-
Figure 2. (a) Initial HTS hit 4 and lead 5. (b) Gal4 dose–response curves for HEK293
cells treated with partial agonist 5 (green), full agonist rosiglitazone (1, blue), and
partial agonist 5 after pre-treating cells with 1 lM rosiglitazone (1, red).
2
73
lin sensitivity is related to PPAR
suggesting that PPAR ligands may impart their anti-diabetic ef-
fects through blocking phosphorylation of PPAR by Cdk5 rather
than by agonizing the transcriptional activity of the nuclear recep-
c
phosphorylation state at Ser
,
c
c
activating PPARc by 3.5-fold over basal activity levels as compared
1
3
tor. In any case, these selective PPAR
c
modulators have demon-
with a 20-fold activation in the case of rosiglitazone (1). Diarylsulf-
onamide 5 also antagonizes the activity of rosiglitazone (1) in the
same assay (Fig. 2). Finally, diarylsulfonamide 5 is highly selective
strated that insulin sensitization effects and other side effects
such as edema and weight gain are not directly linked, and thus de-
sired anti-diabetic effects can be decoupled from unwanted side ef-
for PPAR
PPAR , PPARd, FXR, LXR, PXR or RXR or of rodent PPARs was ob-
served below a concentration of 10 M.
c over other nuclear receptors. No activation of human
fects. As a result, selective PPAR
c
modulators may represent
a
improved therapies for the treatment of type 2 diabetes.
l
This Letter describes the discovery of a novel structural class of
Although sulfonamide 5 displayed an attractive in vitro potency
and efficacy profile, it had pharmacokinetic properties that made it
unsuitable for oral administration. When dosed orally in rat, sul-
fonamide 5 showed low oral bioavailability and low overall expo-
sure (oral bioavailability (F ) was 8%, t = 40 min, and the area
small-molecule selective PPARc modulators for the treatment of
type 2 diabetes, culminating in the discovery of INT131 (3,
1
4
Fig. 1), a small molecule therapeutic agent that has clinically
demonstrated robust glucose-lowering ability with a decreased
side effect profile compared to the marketed TZDs.
u
1/2
under the plasma concentration–time curve over 24 h (AUC24 h
)
was 1.6 M h). In vitro, sulfonamide 5 exhibited high clearance
l
2
2
. Results and discussion
in the presence of the S-9 fraction of rat liver microsomes. The pri-
mary metabolites identified after microsomal incubation showed
oxidation at the A-ring methyl and oxidative dealkylation of the
B-ring N-ethylamide (Fig. 3), that likely contributed to the high
clearance and low in vivo exposure. Additionally, sulfonamide 5
.1. Lead identification
A high-throughput screening (HTS) campaign was undertaken
with the goal of identifying lead compounds that demonstrate high
affinity for PPAR but do not fully agonize PPAR function com-
inhibits cytochrome P450-3A4 (CYP3A4) with an IC50 of 0.36
We initially hypothesized that the C-ring chloropyridine might
lM.
c
c
pared to rosiglitazone (1). Two assays were established to guide
this effort. Biochemical affinity was measured in a standard li-
3
gand-displacement assay using [ H]-radiolabeled rosiglitazone
1
5
Cl
O
O
(K
i
= 10 nM). Cell-based potency and efficacy were measured
S
with a chimeric (two hybrid) PPAR -Gal4 transactivation reporter
c
NH
gene assay in HEK293 cells.16 The HTS campaign identified ethyl
ester 4, a moderately potent (IC50 = 80 nM) PPAR ligand with par-
tial agonistic activity in the Gal4 transactivation assay
A
Cl
c
B
H
N
[
O]
CH3
CH3
1
7,18
(
EC50 = 200 nM; eightfold activation over basal activity levels).
Cl
O
O
A brief combinatorial hit expansion effort led to the identification
C
[O]
of N-ethyl amide 5 as a promising lead (Fig. 2). N-ethyl amide 5
N
5
binds PPAR
say (IC50 = 25 nM) and activates PPAR
with an EC50 of 30 nM. Importantly, N-ethyl amide 5 displays re-
c
with increased affinity in the ligand displacement as-
in the cell-based Gal4 assay
CYP3A4
inhibition
c
duced partial agonist character compared to sulfonamide 4 only
Figure 3. Metabolic liabilities associated with diarylsulfonamide 5.

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
981
be responsible for this inhibition given the known propensity of
various pyridines to cause CYP3A4 inhibition by direct heme iron
coordination.
2.3. In vitro SAR studies
1
9
Using the assays described above, a series of analogs with vari-
ous A-ring substituents were profiled for PPAR affinity as well as
c
2
.2. Synthetic chemistry
cell-based potency and efficacy (Table 1). Given the oxidative lia-
bility of the A-ring methyl group, we initially examined removal
of this substituent and were pleased to see that 2,4-dichloroben-
zenesulfonamide 6 had slightly improved biochemical affinity
and cell-based potency compared to parent 2,4-dichloro-5-methyl-
benzenesulfonamide 5 with an efficacy of 25 percent of control. In
fact, a variety of 2,4- and 3,4-disubstitued benzenesulfonamides
exhibited similar in vitro profiles (compounds 6–9). Electron-with-
drawing substituents were preferred on the A-ring. Analogs with a
single substituent at the A-ring 4-position (R , R , R = H, R – H)
were generally less potent (compounds 10–15). Although these li-
gands maintained partial agonist efficacy, they also had larger
shifts between the biochemical IC50 and the cell-based EC50 com-
pared to the 2,4-disubstituted analogs. Electron-donating substitu-
Thus, a medicinal chemistry effort was initiated to develop SAR
around this diarylsulfonamide scaffold with the goal of discovering
PPAR ligands that display reduced oxidative metabolism and
cytochrome inhibition while maintaining the desirable potency
and efficacy profile seen with N-ethyl amide 5. A concise, robust
and scalable synthetic sequence was utilized to prepare a large
c
number of analogs (Scheme 1). S
benzene with an appropriately substituted alcohol or phenol
ArOH) established the B and C rings. Subsequent nitro reduction
with SnCl O generated aniline intermediates that could be
N
Ar displacement of a p-nitrohalo-
1
2
4
3
(
2
ꢀ2H
2
treated with a suitably substituted benzenesulfonyl chloride in
the presence of pyridine to give the target compounds in good
overall yield (50–90% in most cases). This modular sequence facil-
itated rapid access to a large number of analogs with variations of
the aryl A, B, and C-ring substituents.
3
3
ents (R = OCH
3
, 12) and large 4-substituents (R = tert-butyl, 14 or
3
R = SO
2
CH
3
, 15) were particularly deleterious to affinity. Finally,
3
analogs without an A-ring 4-substituent (R = H), such as 2- or
-monosubstituted phenyl analogs, and fully unsubstituted
phenyl A-ring analogs, as well as heterocyclic A-ring analogs and
non-aromatic ring analogs generally lacked biochemical
3
A
2
0
O
O
affinity.
Next, a series of compounds with various substituents on the
aniline B-ring was examined for PPAR affinity and Gal4 potency
S
NO
X
2
NH
R2
c
a, b, c
R1
R1
and efficacy (Table 2). The N-ethylamide could be replaced with
several different substituents. For example, acetophenone 16
5
(R = C(O)CH
3
) displayed nearly identical biochemical and cell-
O
Ar
based potency and efficacy compared to benzamide 6. Small ha-
5
lides and pseudohalides (R = Cl, F, CF
3
, CN; compounds 17–20)
Scheme 1. General synthetic approach to selective PPAR
and conditions: (a) ArOH, Cs CO , DMF, 70 °C; (b) SnCl O, EtOAc, reflux; (c) R -
benzenesulfonylchloride, pyridine, CH
c modulators. Reagents
2
were tolerated, with Cl being the best for binding affinity. Methyl,
S(O)CH , and hydrogen substituents could be accommodated with
3
2
3
2
ꢀ2H
2
2
Cl
2
(50–90% yield over three steps).
Table 1
In vitro activities of A-ring analogs 5–15
R1
A
O
O
R2
R3
S
NH
B
H
N
R4
CH3
Cl
O
O
C
N
PPAR
c
bindinga,b
M)
PPAR
c
transactivationc,d
EC50 (lM)
Efficacye (%)
Compd
R1
R2
R3
R4
IC50
(
l
Rosiglitazone (1)
—
Cl
Cl
Cl
OCF
H
H
H
H
H
—
H
H
H
H
Cl
H
H
H
H
H
H
—
Cl
Cl
CF
Br
Cl
Cl
I
OCH
NO
C(CH
SO CH
—
CH
H
H
H
H
H
H
H
H
H
H
0.20 (±0.003)
0.025 (±0.003)
0.015 (±0.003)
0.050 (±0.014)
0.018 (±0.050)
0.035 (±0.006)
0.16 (±0.05)
0.025 (±0.006)
0.40 (±0.13)
0.049 (±0.015)
>10
0.10
0.03
0.015
0.03
100
18
25
18
15
30
5
6
7
8
9
3
3
3
0.015
0.02
f
f
10
11
12
13
14
15
—
—
0.20
2.0
0.40
>30
>30
40
33
40
0
3
2
H
H
3
)
3
2
3
>10
0
a
b
c
d
e
f
3
Ligand displacement assay using [ H]-radiolabeled rosiglitazone.
Value represents the mean of at least three experiments ± standard deviation.
Value from a single experiment.
Two hybrid PPARc-Gal4 transactivation assay in HEK293 cells.
Efficacy is calculated as the percentage of the maximum activation obtained with rosiglitizone (1), that activates PPARc 20-fold above the basal level.
Not tested.

9
82
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
Table 2
In vitro activities of B-ring analogs 6 and 16–29
Cl
O O
S
NH
A
Cl
B
R5
R6
Cl
O
C
N
Compd
R5
R6
PPAR
IC50
c
bindinga,b
M)
PPAR
c
transactivationc,d
M)
(
l
EC50
(
l
Efficacye (%)
Rosiglitazone (1)
6
—
—
H
H
H
H
H
H
H
H
H
H
H
F
0.20 (±0.003)
0.015 (±0.003)
0.015 (±0.003)
0.10
100
25
18
10
18
10
13
10
15
0
0
0
45
16
80
55
C(O)NHEt
C(O)CH
3
Cl
F
CF
CN
CH
S(O)CH
OCH
H
CO
Cl
0.015
0.010
0.40
1.0
16
17
18
19
20
21
22
23
24
25
26
27
28
29
c
0.021
0.036 (±0.010)
0.066 (±0.027)
0.11 (±0.03)
1.47 (±0.30)
1.44 (±0.18)
0.019 (±0.004)
2.00 (±0.69)
>10
0.018 (±0.001)
0.021 (±0.008)
0.012 (±0.002)
0.031 (±0.007)
3
0.3
0.30
0.60
0.60
>30
>30
>30
0.02
0.006
0.03
0.1
3
3
3
2
H
Cl
C(O)CH
CF
Cl
Cl
CF
3
3
3
a
b
c
3
Ligand displacement assay using [ H]-radiolabeled rosiglitazone.
Value represents the mean of at least three experiments ± standard deviation.
Value from a single experiment.
c-Gal4 transactivation assay in HEK293 cells.
Efficacy is calculated as the percentage of the maximum activation obtained with rosiglitizone (1), that activates PPAR
d
e
Two hybrid PPAR
c
20-fold above the basal level.
increases in both IC50 and EC50 (compounds 21, 22 and 24).
Next, we examined the in vivo PK profile of our improved
PPAR ligands that lack the metabolically labile A-ring methyl
However, a carboxylic acid (CO
ingly, analogs with R = F or OCH (compounds 18 and 23) main-
2
H, 25) was not tolerated. Interest-
c
5
21
3
and B-ring ethylamide found in lead 5. As a class, these com-
pounds could not be properly formulated for intravenous dosing
due to solubility limitations, but nonetheless they were found to
have good oral exposure. Thus we administered compound as a
suspension by oral gavage and evaluated the oral exposure by
comparing the area under the plasma concetration–time curve
measured over 24 h (AUC24 h). We were pleased to see that both
analogs 20 and 27 showed increased exposure in rats compared
to 5 when dosed orally (Table 3). Although these ligands had im-
proved pharmacokinetic profiles, presumably because of reduced
oxidative metabolism, they still inhibited CYP3A4 with IC50 values
around one micromolar. We hypothesized that the substituted
tained comparable biochemical affinity to benzamide 6, but
cellular potency was severely diminished. The origin of this cell
shift is currently not understood. Ligands substituted with small,
electron-withdrawing R and R6 substituents at both the aniline
B-ring 3- and 5-position showed improved affinity and potency
profiles (compounds 26–29). In particular, dichloroaniline 27 binds
5
PPAR
nM and an efficacy of 16% of control. Finally, substitution at the
aniline B-ring C2 or C6 positions generally gave compounds with
diminished PPAR affinity. Similarly, non-aromatic B-rings were
c with an IC50 of 21 nM and activates PPARc with an EC50 of
6
c
2
0
poorly tolerated.
Table 3
Rat PK data and CYP3A4 inhibition data for analogs 5, 20, and 27
Cl
O O
S
NH
Cl
R4
R5
R6
Cl
O
N
Compd
R4
R5
R6
Rat PK PO AUC24 ha
(l
M h)
CYP3A4 inhibition IC50 (lM)
5
CH
H
H
3
H
H
Cl
C(O)NHEt
Cl
Cl
1.6
31
4.8
0.36
0.60
1.20
2
2
0
7
a
Determined for a single 5 mg/kg oral dose in Sprague–Dawley rats.

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
983
Table 4
In vitro activities of C-ring analogs 3, 27 and 30–39
Cl
O O
S
NH
A
R3
B
R5
R6
R7
R3
—
R5
—
R6
—
R7
PPAR
IC50
c
bindinga,b
M)
PPAR
c
transactivation
c,d
CYP3A4 inhibition IC50 (lM)
Compd
1g
e
(l
EC50
(l
M)
Efficacy (%)
—
0.20 (±0.003)
0.10
100
—
Cl
O
O
2
3
3
3
3
3
3
3
3
3
7
0
1
2
3
Cl
Cl
Cl
0.021 (±0.008)
0.006
0.08
0.09
0.20
0.30
0.15
0.10
—
16
0.60
N
N
NC
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CF
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
0.027 (±0.005)
0.019 (±0.002)
0.032 (±0.007)
0.034 (±0.006)
0.017 (±0.001)
0.018 (±0.004)
0.040c
20
25
20
25
40
30
—
0.01
0.001
0.03
0.36
25.0
2.6
H C
O
3
Cl
N
H N(O)C
O
O
2
Cl
N
H C(O)C
3
Cl
N
O
Cl
N
O
O
O
O
O
O
4
5
6
7
Cl
N
N
N
N
H
F
10.0
2.3
0.025c
0.25
0.06
21
42
3
Cl
H
Cl
C(O)CH
3
0.030c
3.0
3
8
9
Cl
Cl
Cl
H
Cl
0.02 (±0.003)
0.48 (±0.05)
0.07
2.0
10
18
1.0
—
N
3
Cl
a
b
c
3
Ligand displacement assay using [ H]-radiolabeled rosiglitazone.
Value represents the mean of at least three experiments ± standard deviation.
Value from a single experiment.
c-Gal4 transactivation assay in HEK293 cells.
Efficacy is calculated as the percentage of the maximum activation obtained with rosiglitizone (1), that activates PPAR
d
e
Two hybrid PPAR
c
20-fold above the basal level.
pyridine C-ring might coordinate iron heme and thus be responsi-
ble for this CYP3A4 inhibition. Concerned about potential drug–
drug interactions, we focused our attention on the SAR of CYP3A4
inhibition with modification of the C-ring chloropyridine.
vey of biaryl heterocyclic C-rings and discovered 3-alkoxyquino-
line analog 3 (INT131), a ligand with an IC50 value of 18 nM and
an EC50 value of 0.1
one (1). This ring system is notable because it imparts a significant
decrease in CYP3A4 inhibition (IC50 = 25 M) to ligand 3. Closely
lM with 30% efficacy compared to rosiglitaz-
A series of C-ring analogs was examined for PPAR
c
affinity, cell-
l
based potency and efficacy, and CYP3A4 inhibition (Table 4). Vari-
ous pyridines with different C3 substituents (compounds 30–33)
had similar biochemical affinity compared to 3-chloropyridine
related analogs 34–37 display similar in vitro profiles and also
show decreased inhibition of CYP3A4. The 6-alkoxyquinoline
C-ring analog 38 maintained PPARc affinity, but without a similar
2
7, but showed reduced potency in the cell-based PPAR
c
-Gal4
decrease in CYP3A4 inhibition. Other alkoxyquinoline isomers lost
significant affinity (data not shown). Additionally, analogs without
aromatic ring nitrogens, such as 3-alkoxynaphthalene 39, exhib-
ited reduced affinity.
transactivation assay and were potent inhibitors of CYP3A4. C-ring
pyridines substituted at the C2 or C6 position generally exhibited
diminished PPARc affinity (data not shown). We undertook a sur-

9
84
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
Table 5
Rat pharmacokinetic properties for compounds 3, and 34–37a
Table 7
Studies in KKAy diabetic mice treated with compounds 1, 3, and 34–37 for 3 days
Compda
Insulind (%)
Blood glucosec (%)
Body weight gaine,f (%)
Compd
PO AUC24 h
(l
M h)
PO Cmax (lM)
1b
3
34
35
36
ꢁ18.9 (±2.5)
ꢁ43.3 (±6.9)
ꢁ37.0 (±5.4)
—
ꢁ18.6 (±5.5)
ꢁ31.3 (±3.0)
ꢁ18.3 (±5.0)
ꢁ5.3
228 (±20)
170 (±28)
195 (±20)
102
143 (±14)
195 (±21)
3
30
105
82
112
3.4
1.1
11
4.8
8.5
0.92
3
3
3
3
4
5
6
7
c
ꢁ44.8 (±12.9)
ꢁ45.5 (±5.6)
ꢁ23.8 (±4.7)
37
ꢁ25.6 (±4.0)
a
Determined for a single 5 mg/kg oral dose in Sprague–Dawley rats.
a
b
c
Dose was 30 mg/kg/day; values reported as mean ± standard deviation (n = 8).
Dose of rosiglitizone (1) was 1.0 mg/kg/day.
Insulin measurements and standard deviations not available.
Change in insulin and blood glucose were calculated as the percentage change
d
Table 6
from levels measured in vehicle control.
a
e
Dose-dependent rat pharmacokinetic properties for compound 3
Percent increase in body weight compared to weight gain in vehicle control
(
weight gain control = 100%).
Food intake was not statistically different between animal groups.
Dose (mg/kg)
PO AUC24 h
(
lM h)
PO Cmax
(
lM)
f
0
0
1
2
0
.1
.3
.0
.0
1.1
2.6
5.1
8.3
71
0.08
0.13
0.39
0.66
4.3
the full agonist rosiglitazone (1). This observation is in agreement
with the reported in vivo efficacy of other SPPARcMs.
1
2
3
1
00
100
6.1
Quinolines 3 and 35–37 were further evaluated in the Zucker
(
fa/fa) fatty rat, an established model for type 2 diabetes that dis-
a
Determined for a single 5 mg/kg oral dose in Sprague–Dawley rats.
2
4
plays hyperglycemia and severe obesity (Table 8). Compound
was administered as a suspension by oral gavage once per day
for 14 days, and then clearance of blood glucose was measured in
an oral glucose tolerance test. We were pleased to see that quino-
lines 3, 35, and 36 displayed blood glucose lowering with a 3 mg/
kg/day dose that was statistically indistinguishable from that pro-
vided by rosiglitazone (1) at a 1 mg/kg dose. In contrast, quinoline
In addition to the attractive in vitro PPARc binding, efficacy, and
CYP3A4 inhibition profiles, quinoline INT131 (3) has a suitable
pharmacokinetic profile for oral dosing. A 5 mg/kg oral dose in
Sprague–Dawley rats resulted in an AUC of 30
lM h over 24 h with
a Cmax of 1.1 M (Table 5). This represents a 19-fold increase in oral
l
3
7 was significantly less efficacious. Even at a daily dose of 30 mg/
exposure compared to initial lead 5. INT131 (3) also exhibits dose-
dependent increases in oral exposure (both AUC and Cmax) at doses
ranging from 0.1–100 mg/kg (Table 6). Compounds 34–37 also dis-
play pharmacokinetic profiles suitable for in vivo oral administra-
kg it caused less glucose-lowering than rosiglitazone at 1 mg/kg/
day. The origin of the disparate efficacies induced by compounds
3
5 and 37 in the KKAy mouse model and the Zucker fatty rat model
is currently unclear but may reflect differences in compound expo-
sure between the two rodent species. In these single-dose studies,
quinolines 3 and 36 caused body weight gain statistically indistin-
guishable from rosiglitazone (1), while quinolines 35 and 37
caused greater weight gain. Additional dose–response efficacy
studies were performed to determine the dose necessary to reduce
blood glucose levels by 30% compared to vehicle control (ED–30%).
In this study, INT131 (3) demonstrated superior in vivo potency
compared to the other quinoline analogs (EDꢁ30% = 0.5 mg/kg).
Additionally, these results revealed that in the single dose study
INT131 (3) was administered at a dose significantly higher than
that required for maximal efficacy. We hypothesized that Zucker
rats administered INT131 (3) at concentrations near the maximum
efficacious dose might display decreased weight gain compared to
rats administered rosiglitazone (1). Thus we undertook more
tion (Table 5). Finally, INT131 (3) is highly selective for PPAR
other nuclear receptors. No activation of human PPAR , PPARd,
FXR, LXR, PXR or RXR or of rodent PPARs was observed below a
concentration of 10 M.
c over
a
l
2
.4. In vivo efficacy studies
Based on suitable in vitro profiles, a lack of CYP3A4 inhibition,
and improved oral PK, several compounds were profiled for
efficacy in validated in vivo models. The well-established KKAy
diabetic mouse model was selected because it displays hyperglyce-
mia, insulin resistance, and severe obesity.22 Mice were fed 30 mg/
kg doses of partial agonists 3, and 34–37 or 1 mg/kg dose of posi-
tive control rosiglitazone (1) for 3 days. On day 4, insulin, blood
glucose, and body weight measurements were taken and compared
to those taken in the vehicle control group (Table 7).
When dosed orally at 30 mg/kg/day, INT131 (3) and quinoline
Table 8
3
6 both showed greater efficacy in lowering blood glucose and
Studies in Zucker (fa/fa) fatty rats treated with compounds 1, 3, and 35–37 for 14 days
23
insulin levels than a 1 mg/kg/day dose of rosiglitazone (1). Addi-
tionally, body weight gain in the animals treated with partial ago-
nists 3 (170% of the weight gain observed in the vehicle control)
was statistically lower than body weight gain in the rosiglitaz-
one-treated group (228% of control), although food intake between
the two rodent groups was statistically identical. Good glucose-
lowering was also observed with quinolines 34 and 37, while poor
efficacy was observed with quinoline 35. Weight gain and efficacy
in glucose lowering did not necessarily trend together. For in-
stance, quinoline 36, a ligand that demonstrated the most robust
glucose-lowering effect, caused less weight gain than less-
efficacious analogs 1, 3, 34, or 37. Finally, these results demon-
strate that in vivo glucose-lowering efficacy is not directly related
Compda Blood glucosed,e (%) Body weight gaind,e,f (%) EDꢁ30% (mg/kg)
g
1b
3
ꢁ31 (±2)
ꢁ30 (±7)
ꢁ34 (±6)
ꢁ21 (±10)
ꢁ19 (±7)
33 (±7)
42 (±6)
55 (±8)
36 (±7)
53 (±13)
—
0.5
3.0
5.0
>30
3
3
5
6
c
37
a
b
c
Dose was 3.0 mg/kg/day.
Dose of rosiglitazone (1) was 1.0 mg/kg/day.
Dose was 30 mg/kg/day.
d
Change in blood glucose and total body weight were calculated as the% change
compared to vehicle control.
e
Mean ± standard deviation (single experiment; n = 6).
f
Food intake was not statistically different between animal groups.
g
Dose necessary to achieve ꢁ30% glucose lowering relative to vehicle control;
to full PPAR
c
receptor activation, since partial agonists 3, 34, 36,
determined from dose–response studies conducted at 0.1, 0.3, 1.0, 3.0, and 80 mg/
kg/day.
and 37 have as good or better efficacy in glucose lowering than

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
985
INT131-treated rats were accompanied by less total body weight
gain, heart weight gain, and lung weight gain than rosiglitazone-
treated rats, suggesting reduced fluid retention with INT131 (3)
treatment compared to rosiglitazone (1) treatment. These and
other in vivo results have been previously described.10 Given its
unique pharmacological and safety profile, INT131 (3) was selected
for further preclinical profiling.
2
.5. Structural biology
To further understand the unique activity of our selective
PPAR
binding domain of PPAR
derived from the SRC-1 co-activator protein, and the X-ray struc-
c
modulators, INT131 (3) was co-crystallized with the ligand
c
(PPAR LBD) in the presence of a peptide
c
2
5
2
6,27
ture was solved with a resolution of 2.4 Å (Fig. 5).
of the PPAR LBD co-crystallized with INT131 (3) was not signifi-
cantly different from other previously reported PPAR LBD struc-
tures, including the orientation of the AF2 helix, which is
The structure
c
Figure 4. Dose-dependent blood glucose levels in Zucker fatty rats treated with
INT131 (3) and rosiglitzaone (1).
c
2
8–30
proposed to be critical for transcriptional activity.
occupies the ligand-binding pocket within the PPAR
lapsed into a U-shaped conformation around Cys
INT131 (3)
LBD, col-
of helix 3
c
thorough dose–response studies. Figure 4 shows the results of a
full dose–response study comparing Zucker rats dosed with either
INT131 (3) or rosiglitazone (1). Both compounds exhibited statisti-
cally similar maximal in vivo efficacy and lowered glucose by >30%
compared to the vehicle control. However as we previously ob-
served, INT131 (3) displayed nearly 10-fold superior potency,
achieving nearly 30% reduction in blood glucose levels with a
2
85
(
(
H3), and partially overlaps the rosiglitazone (1) binding domain
Fig. 5a and b).
Like the central ring of rosiglitazone (1), the phenyl B-ring of
INT131 (3) occupies a narrow hydrophobic channel between helix
3
and Leu
285
and helix 7, sandwiched between Cys
(S–Aryl distance = 3.6 Å)
3
30
(CH
3
–Aryl distance = 3.6 Å) at distances that have been
0
.3 mg/kg dose compared to the 3.0 mg/kg dose required to
31–34
reported for close SHꢀ ꢀ ꢀ
p
and CHꢀ ꢀ ꢀ
p interactions (Fig. 5d).
achieve similar efficacy with rosiglitazone (1). Additionally,
One B-ring Cl substituent is oriented toward helix 3, experiencing
Figure 5. (a and b) X-ray structure of INT131 (3, blue) bound to PPAR
c) X-ray structure of rosiglitazone bound to PPAR LBD showing direct ligand stabilization of the AF2 helix by H-bond between Tyr473 and the thiazolidinedione ring. (d)
LBD showing indirect ligand stabilization of the AF2 helix through water-mediated H-bond network between Tyr473 and
cLBD (grey, AF2 helix in red) with SRC-1 co-activator (yellow) and rosiglitazone (1, pink) superimposed.
(
c
X-ray structure of INT131 bound to PPAR
c
the sulfonamide linker.

9
86
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
hydrophobic interactions with the alkyl chain of Arg288 (Cl–CH
distance = 3.2 Å; not shown in Fig. 5d). The other Cl substituent
is geared into a solvent-exposed area and does not interact with
the protein.
currently being evaluated in advanced clinical trials and shows ini-
tial promise as a novel therapeutic for the treatment of type 2
diabetes.
2
4
0
The quinoline C-ring wraps around helix 3 and occupies the li-
gand entry region between helix 3 and the b-sheet, similar to the
aminopyridine ring of rosiglitazone. Helix 3 Cys2 resides nearly
in the plane formed by the C-ring, 3.6 Å from the quinoline C4, a
known optimal geometry for Sꢀ ꢀ ꢀH–CAryl interactions.35 Addition-
ally, the quinoline face experiences close packing interactions with
4
4
. Experimental section
85
.1. General chemistry methods
1H NMR spectra were obtained on Bruker DRX 400 MHz or Var-
ian Gemini 400 MHz spectrometers. Chemical shifts are reported in
parts per million (ppm). The abbreviations s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet and br = broad are used
throughout. Melting points were determined in open-ended capil-
lary tubes using a FP62 Mettler Toledo melting point apparatus and
are uncorrected. Elemental analysis was performed by Atlantic
Microlab Inc, Norcross, GA and found values are within ±0.4% of
calculated values. Electrospray mass spectra were determined with
a Hewlett Packard Series 1100 MSD ES mass spectrometer. Reac-
tion progress was monitored by thin layer chromatography (TLC)
using Analtech 0.25 mm precoated glass plates, visualized with
3
41
the hydrophobic Ile
The sulfonamide linker and A-ring of INT131 (3) occupy a sig-
nificantly different space in the PPAR LBD compared to the rosig-
side chain of the b-sheet (3.6–4.0 Å).
c
litazone (1) thiazolidinedione ring (Fig. 5c and d). The
diarylsulfonamide binds with the nitrogen lone pair bisecting the
O@S@O angle. Additionally, the sulfone is oriented with one S@O
bond in plane with the aromatic A-ring, geared away from the
ortho-Cl substituent. This is a well-known low-energy conforma-
tion for diarylsulfonamides containing an ortho-substituent on
the sulfone aryl ring. As a consequence, the side chain of Phe363
36
must rotate nearly 180° from its conformation in the rosiglitaz-
UV light, I
raphy was performed using EM Science silica gel 60 (230–
00 mesh). Solvents were of anhydrous quality purchased from
2
, or 7% phosphomolybdic acid in EtOH. Flash chromatog-
3
7
one-bound structure to induce a narrow hydrophobic pocket that
accommodates the A-ring of INT131 (3) and establishes a parallel-
4
3
63
displaced face-to-face aryl–aryl interaction between Phe
and
Aldrich Chemical Co. and were used as received. Reagents were
purchased from commercial vendors and were used as supplied,
unless otherwise stated. Compound 4 was obtained for our screen-
ing library from Maybridge Chemicals as BTB07901.
3
8
the ligand A-ring. The A-ring 4-Cl substituent is tightly fitted into
a region between helix 3 and helix 7 that contains hydrophobic
side chains and does not accommodate large or polar substituents
(Table 3).
The most significant consequence of the INT131 (3) binding
4
.2. General procedure for the synthesis of diarylsulfonamide
mode is that INT131 (3) does not directly interact with the AF2 he-
lix (Fig. 5d). This is in stark contrast to rosiglitazone (1), which par-
ticipates in a direct hydrogen bond between the thiazolidinedione
PPAR ligands 3 and 5–39
c
A 4-halonitrobenzene (1.0 equiv), a hydroxyarene, and Cs
2 3
CO
4
73
nitrogen and Tyr
(Fig. 5c). Instead, stabilization of the AF2 helix
(
1.1 equiv) were combined in DMF and the resulting heteroge-
in the INT131-bound structure occurs through a network of hydro-
gen bonds, utilizing two structurally conserved water molecules
within the TZD pocket (Fig. 5d). One highly stabilized water mole-
neous mixture was stirred vigorously at 25–70 °C until all 4-halo-
nitrobenzene was consumed (2–24 h). The reaction mixture was
diluted with H O and was adjusted to pH 5 with 2 N HCl (aq).
2
The solution was extracted three times with EtOAc, and the organic
layers were combined, washed once with brine solution, dried over
473
cule forms hydrogen bonds to Tyr
of the AF2 helix (2.6 Å) as well
4
49
323
as to His
2.6 Å), that also forms a H-bond to Tyr3 (2.5 Å). The sulfonamide
N–H of INT131 (3) participates with this network through a
(2.9 Å), His
(3.3 Å) and to a second water molecule
27
(
MgSO
diaryl ether that was used directly without further purification.
The nitrodiaryl ether (1.0 equiv) and SnCl O (5.0 equiv) were
ꢀH
4
, filtered and concentrated in vacuo to furnish the desired
3
27
H-bond with Tyr
(2.7 Å), and one sulfonamide oxygen atom
2
2
367
interacts through a weak H-bond with Lys
(3.4 Å) that is also
(3.0 Å). As previously mentioned, it has been
combined in EtOAc (0.5 M), and the heterogeneous mixture was
heated at reflux for 1–5 h. The reaction mixture was allowed to
cool to rt, and equal volumes of 2 N NaOH (aq) solution and EtOAc
were added to precipitate tin salts. The tin salts were removed by
filtration through CeliteÒ and were washed with additional EtOAc.
The filtrate was transferred to a separatory funnel. The layers were
separated and the organic layer was washed once with brine, dried
4
49
H-bonded to His
hypothesized that the agonistic activity of rosiglitazone arises from
ligand stabilization of the flexible AF2 helix into the appropriate
conformation to recruit co-activator proteins and activate the tran-
scriptional machinery.31,39 We propose that the indirect stabiliza-
tion of the AF2 helix by INT131 (3) and other ligands in this series
only partially stabilizes the AF2 helix and results in the observed
partial agonist efficacy profile.
4
over MgSO and concentrated in vacuo to give the desired aniline
that was used directly without further purification.
The aniline (1.0 equiv), a benzenesulfonyl chloride (1.1 equiv),
3
. Conclusions
and pyridine (3.0 equiv) were combined in CH
was maintained at 25–60 °C until the aniline was consumed (1–
24 h). The reaction was diluted with H O, adjusted to pH 5 with
2 N HCl (aq), and extracted three times with EtOAc. The combined
organics were washed with brine, dried over MgSO , and concen-
trated in vacuo to give the desired sulfonamide (50–90% over the
three-step sequence) that was purified by column chromatogra-
phy, trituration, or recrystallization.
2 2
Cl , and the solution
In summary, we have discovered a novel class of selective and
2
potent PPAR
profile compared to the marketed TZD full agonist rosiglitazone
1). Starting from initial hit sulfonamide 4, which had a poor phar-
c modulators that display a unique pharmacological
4
(
macokinetic profile, we undertook structure–activity relationship
studies that culminated in the discovery of INT131 (3), a partial
agonist in the PPARc-Gal4 transactivation assay. In two different
in vivo models for type 2 diabetes, INT131 (3) showed similar glu-
cose-lowering ability and superior in vivo potency with a reduced
side effect profile compared to rosiglitazone (1). X-ray crystallogra-
phy studies revealed a unique binding mode for INT131 (3) that we
propose is responsible for its pharmacological profile. INT131 (3) is
4.2.1. 2,4-Dichloro-N-(3,5-dichloro-4-(quinolin-3-yloxy)
phenyl)benzenesulfonamide (INT131, 3)
3,4,5-Trichloronitrobenzene, quinolin-3-ol (prepared according
4
1
to a known procedure), and 2,4-dichlorobenzene sulfonyl chlo-
ride were combined according to the general procedure to give

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
987
INT131 (3) as a white amorphous solid, mp 192 °C. 1H NMR
1576, 1540, 1472, 1248, 1162 cmꢁ1. MS (ESI): m/z 596.0 (M+H)+.
(
(
7
400 MHz, DMSO-d
d, J = 8.8 Hz, 1H); 8.00 (br d, J = 8.4, 1H); 7.96 (d, J = 2.4 Hz, 1H);
.87 (br d, J = 8.4 Hz, 1H); 7.71 (dd, J = 8.4, 2.0 Hz, 1H); 7.65 (ddd,
J = 8.4, 6.8, 1.6 Hz, 1H); 7.56 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H); 7.44 (d,
J = 2.8 Hz, 1H); 7.30 (s, 2H). 13C NMR (500 MHz, DMSO-d
), 149.8,
44.0, 142.5, 141.5, 139.3, 136.2, 135.0, 132.8, 132.1, 131.8,
29.0, 128.6, 128.3, 127.8, 127.9, 127.5, 127.4, 119.1, 115.5. IR
6
) d 11.40 (s, 1H); 8.85 (d, J = 3.2 Hz, 1H); 8.17
HPLC: 95 Area%.
4.2.6. 2-(5-Chloropyridin-3-yloxy)-5-(3,4-dichlorophenyl-
sulfonamido)-N-ethylbenzamide (9)
2-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
3,4-dichlorophenylbenzenesulfonyl chloride were combined
according to the general procedure to give ligand 9 as an off-white
solid, mp 179 °C. H NMR (400 MHz, DMSO-d
6
1
1
ꢁ1
1
(
film) 3053, 1600, 1576, 1468, 1342, 1161, 982, 748 cm . MS
6
) d 10.58 (s, 1H); 8.37
+
(
ESI): m/z 514.8 (M+H) . Anal. Calcd for C21
H12Cl
4
N
2
O
3
S: C, 49.05;
(d, J = 2 Hz, 1H); 8.27 (br t, 1H); 8.24 (d, J = 2.4 Hz, 1H); 7.91 (d,
J = 2.4 Hz, 1H); 7.87 (d, J = 8.8 Hz, 1H); 7.70 (dd, J = 8.8, 2.4 Hz,
H, 2.35; N, 5.45. Found: C, 49.25; H, 2.44; N, 5.35. HPLC: 99 Area%.
1
H); 7.42 (t, J = 2.4 Hz, 1H); 7.31 (d, J = 2.4 Hz, 1H); 7.19 (dd,
4
.2.2. 2-(5-Chloropyridin-3-yloxy)-5-(2,4-dichloro-5-methyl-
phenylsulfonamido)-N-ethylbenzamide (5)
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
,4-dichloro-5-methylbenzene sulfonyl chloride were combined
according to the general procedure to give ligand 5 as an off-white
J = 8.8, 2.8 Hz, 1H); 7.07 (d, J = 8.8 Hz, 1H); 3.12 (dq, J = 6.4,
7.2 Hz, 2H); 0.92 (t, J = 7.2 Hz, 3H). IR (film) 3403, 3145, 1634,
ꢁ1
+
2
1541, 1338, 1164, 921 cm . MS (ESI): m/z 502.0 (M+H) . Anal.
Calcd for C20 S: C, 47.97; H, 3.22; N, 8.79; Cl, 21.24.
2
2 3 4
H17Cl N O
Found: C, 48.02; H, 3.17; N, 8.33; Cl, 21.23.
1
solid, mp 186–187.5 °C. H NMR (400 MHz, DMSO-d
J = 2.4 Hz, 1H); 8.15 (d, J = 2.4 Hz, 1H); 8.04 (t, J = 5.6 Hz, 1H);
.88 (d, J = 0.8 Hz, 1H); 7.46 (s, 1H); 7.17 (t, J = 2.4 Hz, 1H); 6.91
d, J = 2.4 Hz, 1H); 6.85 (dd, J = 8.8, 2.8 Hz, 1H); 6.73 (d, J = 8.8 Hz,
6
) d 8.23 (d,
4.2.7. 5-(4-Chlorophenylsulfonamido)-2-(5-chloropyridin-3-
yloxy)-N-ethylbenzamide (10)
2-Chloro-5-nitro-N-ethyl-benzamide, 5-chloropyridin-3-ol and
4-chloro-5-methylbenzenesulfonyl chloride were combined
7
(
1
3
5
H); 3.04 (dq, J = 7.6, 6.0 Hz, 2H); 2.28 (s, 3H); 0.86 (t, J = 7.6 Hz,
ꢁ
1
H). IR (film) 3412, 3134, 1636, 1162, 915 cm . MS (ESI): m/z
according to the general procedure to give ligand 10 as an off-
+
1
14.0 (M+H) . Anal. Calcd for C21
H
18
C N
l3 3
O4S: C, 48.99; H, 3.52;
white solid, mp 171 °C. H NMR (400 MHz, DMSO-d
6
) d 10.52 (s,
N, 8.10; Cl, 20.66. Found: C, 48.80; H, 3.51; N, 8.10; Cl, 20.81.
1H); 8.367 (d, J = 2.0 Hz, 1H); 8.25 (br t, 1H); 8.23 (d, J = 2.4 Hz,
H); 7.76 (d, J = 8.4 Hz, 2H); 7.65 (d, J = 8.4 Hz, 2H); 7.40 (t,
1
4
.2.3. 2-(5-Chloropyridin-3-yloxy)-5-(2,4-dichlorophenyl-
sulfonamido)-N-ethylbenzamide (6)
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
,4-dichlorosulfonylbenzenesulfonyl chloride were combined
according to the general procedure to give ligand 6 as an off-white
J = 2.4 Hz, 1H); 7.29 (d, J = 2.4 Hz, 1H); 7.19 (dd, J = 8.8, 2.8 Hz,
1H); 7.04 (d, J = 8.8 Hz, 1H); 3.14 (dq, J = 6.0, 7.2 Hz, 2H); 0.91 (t,
ꢁ1
2
J = 7.2 Hz, 3H). IR (film) 3495, 3407, 1641, 1161, 915, 752 cm .
+
2
2 3 4
MS(EI): m/z 466.0 (M+H) . Anal. Calcd for C20H17Cl N O : C,
51.51; H, 3.67; N, 9.01; Cl, 15.20. Found: C, 51.51; H, 3.68; N,
8.97; Cl, 15.41.
1
solid, mp 179 °C. H NMR (400 MHz, DMSO-d
6
) d 10.90 (s, 1H); 8.37
(
d, J = 1.9 Hz, 1H); 8.25 (t, J = 5.5 Hz, 1H); 8.23 (d, J = 2.4 Hz, 1H);
8
2
.03 (d, J = 8.6 Hz, 1H); 7.90 (d, J = 2.0 Hz, 1H); 7.64 (dd, J = 8.8,
.0 Hz, 1H); 7.40 (t, J = 2.1 Hz, 1H); 7.31 (d, J = 2.7 Hz, 1H); 7.19
4.2.8. 2-(5-Chloropyridin-3-yloxy)-N-ethyl-5-(4-iodophenyl-
sulfonamido)benzamide (11)
(
7
1
dd, J = 8.8, 2.7 Hz, 1H); 7.05 (d, J = 8.8 Hz, 1H); 3.11 (dq, J = 5.8,
2-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
4-iodobenzenesulfonyl chloride were combined according to the
.1 Hz, 2H); 0.91 (t, J = 7.2 Hz, 3H). IR (film) 3407, 1634, 1538,
ꢁ1
+
350, 1223, 1162, 910, 813 cm . MS (ESI): m/z 502.0 (M+H) . Anal.
S: C, 47.93; H, 3.22; N, 8.39; Cl, 21.24.
Found: C, 47.80; H, 3.22; N, 8.36; Cl, 21.51.
general procedure to give ligand 11 as an off-white solid, mp
1
Calcd for C20
H16Cl
3
N
O
3 4
165 °C. H NMR (400 MHz, DMSO-d
6
) d 10.51 (s, 1H); 8.37 (d,
J = 1.9 Hz, 1H); 8.25 (t, J = 5.6 Hz, 1H); 8.24 (d, J = 2.4 Hz, 1H);
7
.97 (d, J = 8.5, 1H); 7.52 (d, J = 8.5, 1H); 7.40 (t, J = 2.2 Hz, 1H);
4
.2.4. 5-(2-Chloro-4-(trifluoromethyl)phenylsulfonamido)-2-(5-
chloropyridin-3-yloxy)-N-ethylbenzamide (7)
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
-chloro-4-trifluoromethylsulfonylbenzenesulfonyl chloride were
combined according to the general procedure to give ligand 7 as
7.30 (d, J = 2.7 Hz, 1H); 7.19 (dd, J = 8.8, 2.7 Hz, 1H); 7.05 (d,
J = 8.8 Hz, 1H); 3.12 (dq, J = 5.8, 7.1 Hz, 2H); 0.92 (t, J = 7.2 Hz,
ꢁ1
2
3H). IR (film) 3412, 3169, 1640, 1266, 1159, 735 cm . MS (ESI):
+
2
m/z 558.1 (M+H) . Anal. Calcd for C20
3 4
H17ClIN O S: C, 43.07; H,
3.07; N, 7.53. Found: C, 42.73; H, 3.14; N, 7.39.
1
an off-white solid, mp 159 °C. H NMR (400 MHz, DMSO-d
6
) d
1
1.06 (s, 1H); 8.36 (d, J = 2 Hz, 1H); 8.25 (br t, 1H); 8.24 (br d,
4.2.9. 2-(5-Chloropyridin-3-yloxy)-N-ethyl-5-(4-methoxy-
phenylsulfonamido)benzamide (12)
2-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
4-methoxybenzenesulfonyl chloride were combined according to
J = 7.6, 1H); 8.22 (d, J = 2.4 Hz, 1H); 8.15 (br s, 1H); 7.94 (d,
J = 8.4 Hz, 1H); 7.40 (t, J = 2.4 Hz, 1H); 7.32 (d, J = 2.8 Hz, 1H); 7.20
(
dd, J = 8.8, 2.8 Hz, 1H); 7.05 (d, J = 8.8 Hz, 1H); 3.10 (dq, J = 6.4,
7
.2 Hz, 2H); 0.90 (t, J = 7.2 Hz, 3H). IR (film) 3416, 3087, 1644,
the general procedure to give ligand 12 as an off-white solid, mp
ꢁ1
+
1
1
320, 1162, 916, 836, 723 cm . MS (ESI): m/z 534.0 (M+H) . Anal.
S: C, 47.20; H, 3.02; N, 7.86; Cl, 13.37; F,
0.67. Found: C, 47.37; H, 3.02; N, 7.85; Cl, 13.37; F, 10.49.
155 °C. H NMR (400 MHz, DMSO-d
6
) d 10.31 (s, 1H); 8.35 (d,
Calcd for C21
1
H16Cl
2
F
3
N
3
O
4
J = 1.6 Hz, 1H); 8.25 (t, J = 5.6 Hz, 1H); 8.21 (d, J = 2.4 Hz, 1H);
7.69 (dd, J = 8.8, 2.0 Hz, 2H); 7.38 (t, J = 2.4 Hz, 1H); 7.29 (d,
J = 2.4 Hz, 1H); 7.19 (dd, J = 8.8, 2.4 Hz, 1H); 7.07 (dd, J = 8.8,
2.0 Hz, 2H); 7.04 (d, J = 8.8 Hz, 1H); 3.10 (dq, J = 6.4, 7.2 Hz, 2H);
0.90 (t, J = 7.2 Hz, 3H). IR (film) 3160, 3144, 2940, 1644, 1223,
4
.2.5. 5-(4-Bromo-2-(trifluoromethoxy)phenylsulfonamido)-2-
(
5-chloropyridin-3-yloxy)-N-ethylamide (8)
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
-bromo-2-trifluoromethoxybenzenesulfonyl chloride were com-
ꢁ
1
ꢁ
2
1156 cm
20ClN
4.36; N, 8.95.
.
MS (ESI): m/z 460.0 (MꢁH) . Anal. Calcd for
4
C
21
H
3 5
O S: C, 54.60; H, 4.36; N, 9.10. Found: C, 54.38; H,
bined according to the general procedure to give ligand 8 as an
1
off-white solid, mp 157 °C. H NMR (400 MHz, DMSO-d
6
) d 8.39
d, J = 1.8 Hz, 1H), 8.30–8.23 (m, 1H), 8.25 (d, J = 2.3 Hz, 1H), 7.92
d, J = 8.5 Hz, 1H), 7.85–7.80 (m, 2H), 7.41–7.39 (m, 1H), 7.31 (d,
(
(
4.2.10. 2-(5-Chloropyridin-3-yloxy)-N-ethyl-5-(4-nitrophenyl-
sulfonamido)benzamide (13)
J = 2.5 Hz, 1H), 7.20 (dd, J = 8.5, 2.3 Hz, 1H), 7.10 (d, J = 9.1 Hz,
H), 3.15–3.08 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). IR (film) 1636,
2-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
4-nitrobenzenesulfonyl chloride were combined according to the
1

9
88
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
general procedure to give ligand 13 as an off-white solid, mp 199–
the general procedure to give ligand 18 as an off-white solid, mp
155 °C. H NMR (400 MHz, DMSO-d ) d 11.05 (s, 1H); 8.39 (d,
6
1
1
2
05 °C. H NMR (400 MHz, DMSO-d
6
) d 10.77 (s, 1H); 8.39 (d,
J = 8.8 Hz, 2H); 8.37 (d, J = 2 Hz, 1H); 8.25 (br t, 1H); 8.23 (d,
J = 2.4 Hz, 1H); 8.01 (d, J = 8.8 Hz, 2H); 7.43 (t, J = 2.4 Hz, 1H);
J = 1.8 Hz, 1H); 8.28 (d, J = 2.4 Hz, 1H); 8.07 (d, J = 8.6 Hz, 1H);
7.90 (d, J = 2.1Hz, 1H); 7.64 (dd, J = 8.6, 2.1 Hz, 1H); 7.52 (t,
J = 2.2 Hz, 1H); 7.24 (t, J = 9.0 Hz, 1H); 7.05 (dd, J = 13.2, 2.5 Hz,
1H); 6.94 (m, 1H). IR (film) 3090, 3074, 2726, 1575, 1508, 1168,
7
.32 (d, J = 2.8 Hz, 1H); 7.21 (dd, J = 8.8, 2.8 Hz, 1H); 7.06 (d,
J = 8.8 Hz, 1H); 3.11 (dq, J = 6.0, 7.2 Hz, 2H); 0.91 (t, J = 7.2 Hz,
ꢁ
1
ꢁ1
+
3
H). IR (film) 1642, 1523, 1163, 914, 895, 738 cm . MS (ESI): m/
1152, 934, 821 cm . MS (ESI): m/z: 449.0 (M+H) . Anal. Calcd for
FN S: C, 45.61; H, 2.25; N, 6.26. Found: C, 45.52; H,
2.22; N, 6.20.
+
z 477.0 (M+H) . Anal. Calcd for C20
4
H17ClN O
6
S: C, 50.37; H, 3.59;
17
C H10Cl
3
2 3
O
N, 11.75; Cl, 7.43. Found: C, 50.44; H, 3.59; N, 11.55; Cl, 7.42.
4
3
.2.11. 5-(4-tert-Butylphenylsulfonamido)-2-(5-chloropyridin-
-yloxy)-N-ethylbenzamide (14)
4.2.16. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-
(trifluoromethyl)phenyl)benzenesulfonamide (19)
2
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
3-Trifluoromethyl-4-chloronitrobenzene, 5-chloropyridin-3-ol
and 2,4-dichlorobenzenesulfonyl chloride were combined accord-
4
-tert-butylbenzenesulfonyl chloride were combined according to
the general procedure to give ligand 14 as an off-white solid, mp
ing to the general procedure to give ligand 19 as an off-white solid,
1
1
1
50 °C. H NMR (400 MHz, DMSO-d
6
) d 10.43 (s, 1H); 8.36 (d,
mp 136 °C. H NMR (400 MHz, DMSO-d
6
) d 11.12 (s, 1H); 8.45 (d,
J = 2.0 Hz, 1H); 8.26 (t, J = 5.7 Hz, 1H); 8.21 (d, J = 2.5 Hz, 1H);
J = 1.8 Hz, 1H); 8.30 (d, J = 2.5 Hz, 1H); 8.05 (d, J = 8.5 Hz, 1H);
7.91 (d, J = 2.1 Hz, 1H); 7.65 (dd, J = 2.6, 2.4 Hz, 1H); 7.65 (dd,
J = 8.6, 2.0 Hz, 1H); 7.47 (d, J = 2.6 Hz, 1H); 7.36 (dd, J = 8.4,
2.6 Hz, 1H); 7.19 (d, J = 8.9 Hz, 1H). IR (film) 3052, 2714, 1575,
7
1
.71 (d, J = 8.6, 2H); 7.60 (d, J = 8.6 Hz, 2H); 7.38 (t, J = 2.2 Hz,
H); 7.30 (d, J = 2.7 Hz, 1H); 7.23 (dd, J = 8.8, 2.8 Hz, 1H); 7.05 (d,
J = 8.8 Hz, 1H); 3.10 (dq, J = 5.8, 7.2 Hz, 2H); 1.27 (s, 9H); 0.91 (t,
ꢁ1
ꢁ1
J = 7.2 Hz, 3H). IR (film) 3408, 2953, 1636, 1164 cm . MS (ESI):
1494, 1321, 1162, 1121, 931, 818 cm . MS (ESI): m/z: 499.0
+
+
m/z 488.0 (M+H) .Anal. Calcd for C24
3
H26ClN O
4
S: C, 59.07; H,
(M+H) . Anal. Calcd for C18
H10Cl
3
3
F N
2
O
3
S: C, 43.40; H, 2.03; N,
5
.37; N, 8.61; Cl, 7.26. Found: C, 58.91; H, 5.35; N, 8.62; Cl, 7.14.
5.54. Found: C, 43.31; H, 2.03; N, 5.54.
4
.2.12. 2-(5-Chloropyridin-3-yloxy)-N-ethyl-5-(4-(methyl-
4.2.17. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-cyano-
phenyl)benzenesulfonamide (20)
3-Cyano-4-chloronitrobenzene, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to
sulfonyl)phenylsulfonamido)benzamide (15)
-Chloro-5-nitro-N-ethylbenzamide, 5-chloropyridin-3-ol and
-methylsulfonylbenzenesulfonyl chloride were combined accord-
ing to the general procedure to give ligand 15 as an off-white solid,
2
4
the general procedure to give ligand 20 as an off-white solid, mp
1
1
mp 156 °C. H NMR (400 MHz, DMSO-d
6
) d 10.77 (s, 1H); 8.37 (d,
162 °C. H NMR (400 MHz, CDCl
3
) d 8.59 (br s, 1H); 8.42 (br s,
J = 2.0 Hz, 1H); 8.27 (t, J = 5.5 Hz, 1H); 8.24 (d, J = 2.4 Hz, 1H);
1H); 8.08 (d, J = 8.5 Hz, 1H); 7.71 (d, J = 1.8 Hz, 1H); 7.60 (d,
J = 2.3 Hz, 1H); 7.53 (dd, J = 8.5, 2.0 Hz, 1H); 7.50–7.45 (m, 2H);
7.22 (br s, 1H); 6.99 (d, J = 8.8 Hz, 1H). IR (film) 3049, 2731,
8
1
.14 (d, J = 8.5, 2H); 8.02 (d, J = 8.4 Hz, 2H); 7.45 (t, J = 2.2 Hz,
H); 7.32 (d, J = 2.7 Hz, 1H); 7.23 (dd, J = 8.8, 2.8 Hz, 1H); 7.06 (d,
ꢁ1
J = 8.8 Hz, 1H); 3.29 (s, 3H); 3.11 (dq, J = 5.8, 7.2 Hz, 2H); 0.91 (t,
2230, 1570, 1489, 1159929, 815 cm . MS (ESI): m/z 455.9
ꢁ1
+
J = 7.2 Hz, 3H). IR (film) 3414, 1639, 1169 cm . MS (ESI): m/z
(M+H) . Anal. Calcd for C18
3 3 3
H10Cl N O S: C, 47.69; H, 2.23; N,
+
5
10.0 (M+H) . Anal. Calcd for C21
3 6 2
H20ClN O S : C, 49.46; H, 3.95;
9.27. Found: C, 47.78; H, 2.34; N, 8.94.
N, 8.24; Cl, 6.95. Found: C, 48.14; H, 3.82; N, 7.93; Cl, 6.85.
4
.2.18. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-methyl-
4
.2.13. N-(3-Acetyl-4-(5-chloropyridin-3-yloxy)phenyl)-2,4-
dichlorobenzenesulfonamide (16)
-Chloro-5-nitroacetophenone, 5-chloropyridin-3-ol and 2,4-dic-
phenyl)benzenesulfonamide (21)
3-Methyl-4-chloronitrobenzene, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to
2
hlorobenzenesulfonyl chloride were combined according to the gen-
eral procedure to give ligand 16 as an off-white solid, mp 149 °C. H
the general procedure to give ligand 21 as an off-white solid, mp
1
1
134 °C. H NMR (400 MHz, CDCl
3
) d 8.28 (br s, 1H); 8.14 (br s,
NMR (400 MHz, CDCl
1
1
3
) d 8.38 (d, J = 1.9 Hz, 1H); 8.23 (d, J = 2.4 Hz,
H); 7.95 (d, J = 8.5 Hz, 1H); 7.55 (d, J = 2 Hz, 1H); 7.53 (d, J = 2.9 Hz,
H); 7.38–7.34 (m, 2H); 7.23 (d, J = 4.4 Hz, 1H); 7.14 (s, 1H); 6.86 (d,
1H); 7.95 (d, J = 8.5 Hz, 1H); 7.55 (d, J = 2.0 Hz, 1H); 7.35 (dd,
J = 8.5, 2.0 Hz, 1H); 7.12 (br s, 1H); 7.09–7.05 (m, 2H); 6.96 (dd,
J = 8.7, 2.6 Hz, 1H); 6.80 (d, J = 8.7 Hz, 1H); 2.15 (s, 3H). IR (film)
ꢁ1
+
J = 8.8 Hz, 1H); 2.54 (s, 3H). IR (film) 3225, 1666, 1481, 1258, 1167,
3259, 1570, 1167, 818 cm . MS (ESI): m/z 445.1 (M+H) . Anal.
Calcd for C18 : C, 48.72; H, 2.95; N, 6.31. Found: C,
8.81; H, 3.03; N, 6.25.
ꢁ1
–
8
19 cm . MS (ESI): m/z: 470.9 (M–H) . HPLC: 95 Area%.
3 2 3
H13Cl N O
4
4
.2.14. 2,4-Dichloro-N-(3-chloro-4-(5-chloropyridin-3-yloxy)
phenyl)benzenesulfonamide (17)
,4-Dichloronitrobenzene, 5-chloropyridin-3-ol and 2,4-dic-
4.2.19. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-
(methylsulfonyl)phenyl)benzenesulfonamide (22)
3
hlorobenzenesulfonyl chloride were combinedaccordingto the gen-
eral procedure to give ligand 17 as an off-white solid, mp 138 °C. H
1-Chloro-2-methanesulfinyl-4-nitrobenzene, 5-chloropyridin-
3-ol and 2,4-dichlorobenzenesulfonyl chloride were combined
1
NMR (400 MHz, DMSO-d
8
1
6
) d 11.03 (s, 1H); 8.40 (d, J = 1.8 Hz, 1H);
.24 (d, J = 2.4 Hz, 1H); 8.06 (d, J = 8.5 Hz, 1H); 7.90 (d, J = 2.0 Hz,
H); 7.65 (dd, J = 8.5, 2.0 Hz, 1H); 7.48 (t, J = 2.2 Hz, 1H); 7.28 (d,
according to the general procedure to give ligand 22 as an off-
1
white solid, mp 90 °C. H NMR (400 MHz, MeOH-d
4
) d 8.37 (d,
J = 2.0 Hz, 1H); 8.25 (d, J = 2.4 Hz, 1H); 8.08 (d, J = 8.6 Hz, 1H);
7.82 (d, J = 2.8 Hz, 1H); 7.69 (d, J = 2.1 Hz, 1H); 7.52–7.46 (m,
3H); 7.10 (d, J = 8.8 Hz, 1H); 3.24 (s, 3H). IR (film) 1571, 1483,
J = 2.5 Hz, 1H); 7.21 (d, J = 8.8 Hz, 1H); 7.10 (dd, J = 8.8, 2.5 Hz, 1H).
ꢁ1
IR (film) 3088, 1574, 1485, 1164, 931 cm . MS (ESI): m/z: 464.9
+
ꢁ1
–
(
M+H) . Anal. Calcd for C17
H
10Cl
4
N
2
O
3
S: C, 43.99; H, 2.17; N, 6.04.
1308, 1167, 1146, 819 cm . MS (ESI): m/z: 505.0 (M–H) . HPLC:
Found: C, 44.06; H, 2.15; N, 6.03.
95 Area%.
4
.2.15. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-fluoro-
phenyl)benzenesulfonamide (18)
-Fluoro-4-chloronitrobenzene, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to
4.2.20. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3-
methoxyphenyl)benzenesulfonamide (23)
3-Methoxy-4-fluoronitrobenzene, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to the
3

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
989
general procedure to give ligand 23 as an off-white solid, mp
1H); 8.07 (d, J = 2.5 Hz, 1H); 8.03 (d, J = 8.6 Hz, 1H); 7.58 (d,
J = 2 Hz, 1H); 7.52 (d, J = 2.5 Hz, 1H); 7.46–7.38 (m, 2H); 7.32 (br
s, 1H); 7.00 (dd, J = 2.5, 2.0 Hz, 1H); 2.46 (s, 3H). IR (film) 3196,
1
1
(
7
(
44 °C. H NMR (400 MHz, CDCl
d, J = 2.4 Hz, 1H); 7.95 (d, J = 8.5 Hz, 1H); 7.55 (d, J = 2.0 Hz, 1H);
.37 (dd, J = 8.5, 2.0 Hz, 1H); 7.15 (s, 1H); 7.04 (m, 1H); 6.91–6.89
m, 2H); 6.26 (dd, J = 8.5, 2.5 Hz, 1H); 3.74 (s, 3H). IR (film) 1716,
3
) d 8.24 (d, J = 2.0 Hz, 1H); 8.11
ꢁ1
1681, 1166, 930, 818, 626, 568 cm . MS (ESI): m/z: 507.1
+
(M+H) . Anal. Calcd for C19
4 2 4
H12Cl N O S: C, 45.08; H, 2.39; N,
ꢁ1
ꢁ
1
574, 1505, 1445, 1341, 1165 cm . MS (ESI): m/z 457.0 (MꢁH) .
5.53. Found: C, 44.71; H, 2.37; N, 5.41.
HPLC: 95 Area%.
4
.2.26. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)-3,5-bis
4
.2.21. 2-(5-Chloropyridin-3-yloxy)-5-(2,4-dichlorophenyl-
(trifluoromethyl)phenyl)benzenesulfonamide (29)
sulfonamido)benzoic acid (24)
2-Bromo-5-nitro-1,3-bis(trifluoromethyl)benzene was synthe-
sized from 2-bromo-1,3-bis(trifluoromethyl)benzene by modifying
Ethyl 2-chloro-5-nitrobenzoate, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to
the general procedure to give methyl-2-(5-chloropyridin-3-
yloxy)-5-(2,4-dichlorophenylsulfonamido)benzoate. This was trea-
ted with LiOH in MeOH at rt overnight to provide ligand 24 as an
off-white solid, mp 232–234 °C. H NMR (400 MHz, DMSO-d
4
2
3
a literature procedure. 1.9 mL of fuming HNO was added to
2.6 mL of concentrated H SO . 2-Bromo-1,3-bis(trifluoromethyl)
2
4
benzene (2.5 g, 8.5 mmol) was added in one portion to the mixture
and heated with stirring to 70 °C for 30 min. The reaction mixture
was cooled to room temperature and poured over 200 mL of ice.
The white crystals were collected by filtration, washed with water,
1
6
) d
1
3.18 (br s, 1H); 10.98 (br s, 1H); 8.31 (d, J = 2 Hz, 1H); 8.13 (d,
J = 2.8 Hz, 1H); 8.02 (d, J = 8.4 Hz, 1H); 7.88 (d, J = 2.4 Hz, 1H);
.63 (dd, J = 8.8, 2.0 Hz, 1H); 7.63 (d, J = 2.8 Hz, 1H); 7.33 (dd,
J = 8.8, 2.8 Hz, 1H); 7.30 (t, J = 2.4 Hz, 1H); 7.15 (d, J = 8.8 Hz, 1H).
and dried under vacuum to give 2.69 g (93%) of product that was
1
7
used without further purification. H NMR (400 MHz, DMSO-d
6
) d
8.7 (s, 2H). This material was combined with 5-chloropyridin-3-ol
ꢁ1
IR (film) 1686, 1560, 1264, 1162, 931, 746 cm . MS (ESI): m/z
and 2,4-dichlorobenzenesulfonyl chloride according to the general
+
1
4
74.9 (M+H) . Anal. Calcd for C18
H
11Cl
3
N
2
O
5
S: C, 45.64; H, 2.34;
procedure to give ligand 29 as an off-white solid, mp 160 °C.
NMR (400 MHz, DMSO-d
.27 (d, J = 2.6 Hz, 1H), 8.17 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 2.0 Hz,
1H), 7.77 (s, 2H), 7.73 (dd, J = 2.1, 8.6 Hz, 1H), 7.64 (t, J = 2.2 Hz,
H
N, 5.91; Cl, 22.45. Found: C, 45.57; H, 2.22; N, 5.92; Cl, 22.63.
6
) d 11.8 (s, 1H), 8.39 (d, J = 1.81 Hz, 1H),
8
4
.2.22. 2,4-Dichloro-N-(4-(5-chloropyridin-3-yloxy)phenyl)
benzenesulfonamide (25)
-Chloronitrobenzene, 5-chloropyridin-3-ol and 2,4-dichloro-ben-
ꢁ1
1H). IR (film) 1474, 1209, 1171, 1126 cm . MS (ESI): m/z 565.0
–
4
(M–H) . Anal. Calcd for C19
9 3 6 2 3
H Cl F N O S: C, 40.34; H, 1.60; N, 4.95.
zenesulfonyl chloride were combined according to the general proce-
dure to give ligand 25 as an off-white solid, mp 139 °C. H NMR
Found: C, 40.42; H, 1.55; N, 5.03.
1
(
400 MHz, DMSO-d
6
) d 10.70 (s, 1H); 8.38 (d, J = 1.6 Hz, 1H); 8.25 (d,
4.2.27. 2,4-Dichloro-N-(3,5-dichloro-4-(5-cyanopyridin-3-
yloxy)phenyl)benzenesulfonamide (30)
J = 2.0 Hz, 1H); 7.98 (d, J = 8.4 Hz, 1H); 7.87 (d, J = 2.0 Hz, 1H); 7.61
(
2
dd, J = 8.4, 2.0 Hz, 1H); 7.48 (t, J = 2.2 Hz, 1H); 7.13 (d, J = 8.4 Hz,
3,4,5-Trichloronitro benzene, 5-hydroxynicotinonitrile (pre-
pared according to a known procedure), and 2,4-dichloro-ben-
zenesulfonyl chloride were combined according to the general
ꢁ1
43
H); 7.03 (d, J = 8.4 Hz, 2H). IR (film) 3280, 1569, 1501, 1168 cm
.
+
MS (ESI): m/z: 431.0 (M+H) . Anal. Calcd for C17
H11Cl
3
N
2 3
O S: C,
47.52; H, 2.58; N, 6.52. Found: C, 47.68; H, 2.62; N, 6.51.
procedure to give ligand 30 as an off-white solid, mp 87–82 °C.
H NMR (400 MHz, CDCl ) d 8.59 (d, J = 1.5 Hz, 1H); 8.45 (d,
3
1
4
.2.23. 2,4-Dichloro-N-(3-chloro-4-(5-chloropyridin-3-yloxy)-5-
J = 2.9 Hz, 1H); 8.05 (d, J = 8.5 Hz, 1H); 7.60 (d, J = 2 Hz, 1H); 7.45
(dd, J = 8.5, 2.0 Hz, 1H); 7.24 (s, 2H); 7.23–7.18 (m, 2H). IR (film)
fluorophenyl)benzenesulfonamide (26)
,4-Dichloro-5-fluoronitrobenzene, 5-chloropyridin-3-ol and 2,4-
dichlorobenzenesulfonyl chloride were combined according to the
ꢁ1
3
2237, 1457, 1166, 818, 621, 569 cm . MS (ESI): m/z 489.9
+
(M+H) . Anal. Calcd for C18
9 4 3 3
H Cl N O S: C, 44.20; H, 1.85; N, 8.59.
general procedure to give ligand 26 as an off-white solid, mp 165–
Found: C, 44.86; H, 2.22; N, 8.28.
1
3
167 °C. H NMR (400 MHz, CDCl ) d 8.31 (d, J = 1.9 Hz, 1H); 8.14 (d,
J = 2.6 Hz, 1H); 8.03 (d, J = 8.5 Hz, 1H); 7.58 (d, J = 2 Hz, 1H); 7.43 (dd,
J = 8.5, 2.0 Hz, 1H); 7.28 (br s, 1H); 7.11 (t, J = 2.2 Hz, 1H); 7.07–7.00
4.2.28. 2,4-Dichloro-N-(3,5-dichloro-4-(5-methylpyridin-3-
yloxy)phenyl)benzenesulfonamide (31)
(
5
FN
m, 2H). IR (film) 3054, 1344, 1166, 1014, 930, 819, 621, 613,
3,4,5-Trichloronitro benzene, 5-methyl-3-hydroxypyridine, and
2,4-dichlorobenzenesulfonyl chloride were combined according to
ꢁ1
ꢁ
62 cm . MS (ESI): m/z: 480.9 (MꢁH) . Anal. Calcd for C17
9 4
H Cl
2 3
O S: C, 42.35; H, 1.88; N, 5.81. Found: C, 42.33; H, 2.03; N, 5.41.
the general procedure to give ligand 31 as an off-white solid, mp
1
2
00–201 °C. H NMR (400 MHz, CDCl
3
) d 8.18 (m, 1H); 8.03 (d,
4
.2.24. 2,4-Dichloro-N-(3,5-dichloro-4-(5-chloropyridin-3-
yloxy)phenyl)benzenesulfonamide (27)
,4,5-Trichloronitro benzene, 5-chloropyridin-3-ol and 2,4-dic-
J = 8.5 Hz, 1H); 7.85 (d, J = 2.7 Hz, 1H); 7.57 (d, J = 2 Hz, 1H); 7.41
(dd, J = 8.5, 2.0 Hz, 1H); 7.20 (s, 2H); 6.99–6.94 (m, 1H); 2.33 (s,
ꢁ1
3
3H). IR (film) 2635(bs), 1463, 1338, 1168, 817, 619, 567 cm .
+
hlorobenzenesulfonyl chloride were combined according to the
MS (ESI): m/z 478.9 (M+H) . Anal. Calcd for C18
4 2 3
H12Cl N O S: C,
general procedure to give ligand 27 as an off-white solid, mp
45.21; H, 2.53; N, 5.86. Found: C, 45.17; H, 2.48; N, 5.89.
1
1
1
1
69 °C. H NMR (400 MHz, DMSO-d
H); 8.23 (d, J = 2.6 Hz, 1H); 8.13 (d, J = 8.5 Hz, 1H); 7.93 (br s,
H); 7.68 (dd, J = 8.6, 2.0 Hz, 1H); 7.50 (m, 1H); 7.25 (s, 2H). IR
6
) d 11.36 (s, 1H); 8.38 (br s,
4.2.29. 5-(2,6-Dichloro-4-(2,4-dichlorophenylsulfonamido)
phenoxy)nicotinamide (32)
ꢁ1
(
film) 3092, 3070, 2786, 1568, 1462, 1165, 819 cm . MS (ESI):
A solution of nitrile 30 in tert-BuOH was treated with powdered
KOH (5 equiv) at rt. The mixture was then maintained at reflux un-
til all starting material was consumed according to TLC analysis.
The mixture was cooled to rt, volatiles were removed in vacuo,
and the resulting residue was dissolved in EtOAc and washed with
1 N HCl (aq) and brine. The organics were dried over MgSO4, fil-
+
m/z: 499.0 (M+H) . Anal. Calcd for C17
H
9
5 2 3
Cl N O : C, 40.95; H,
1
.82; N, 5.62. Found: C, 41.18; H, 1.81; N, 5.55.
4
.2.25. N-(3-Acetyl-5-chloro-4-(5-chloropyridin-3-yloxy)-
phenyl)-2,4-dichlorobenzenesulfonamide (28)
,3-Dichloro-5-nitroacetophenone, 5-chloropyridin-3-ol and
,4-dichlorobenzenesulfonyl chloride were combined according
to the general procedure to give ligand 28 as an off-white solid,
2
tered, and concentrated in vacuo to give ligand 32 as an off-white
1
2
solid (99%), mp 186–188 °C. H NMR (400 MHz, DMSO-d
6
) d 11.37
(br s, 1H); 8.79 (d, J = 1.6 Hz, 1H); 8.43 (d, J = 2.9 Hz, 1H); 8.20 (br s,
1H); 8.14 (d, J = 8.5 Hz, 1H); 7.92 (d, J = 1.9 Hz, 1H); 7.72–7.64 (m,
1
3
mp 174–178 °C. H NMR (400 MHz, CDCl ) d 8.31 (d, J = 2 Hz,

9
90
J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
2
8
C
2
H); 7.49–7.46 (m, 1H); 7.28(s, 2H). IR (film) 3450, 1683, 1171,
163 °C. 1H NMR (400 MHz, DMSO-d
) d 11.04 (s, 1H); 8.79 (d,
6
ꢁ1
+
24, 616, 573 cm . MS (ESI): m/z 507.9 (M+H) . Anal. Calcd for
S: C, 42.63; H, 2.19; N, 8.29. Found: C, 42.78; H,
J = 2.9 Hz, 1H); 8.09 (d, J = 8.5 Hz, 1H); 8.15 (d, J = 8.3 Hz, 1H);
7.98 (d, J = 2.1 Hz, 1H); 7.89 (d, J = 8.2 Hz, 1H); 7.70–7.65 (m,
2H); 7.60–7.58 (m, 1H); 7.57 (d, J = 3.0 Hz, 1H); 7.33 (d,
J = 2.6 Hz, 1H); 7.26 (d, J = 8.8 Hz, 1H); 7.13 (dd, J = 2.6 Hz, 1H). IR
18 4 3 4
H11Cl N O
.20; N, 8.28.
ꢁ1
4
.2.30. N-(4-(5-acetylpyridin-3-yloxy)-3,5-dichlorophenyl)-2,4-
(film) 1488, 1335, 1162, 818, 616 cm . MS (ESI): m/z 481.0
+
dichlorobenzenesulfonamide (33)
,2,3-Trichloro-5-nitrobenzene was coupled to methyl 5-
hydroxynicotinate in the presence of Cs CO at 50 C for 18 h as de-
2 2 3
(M+H) . Anal. Calcd for C21H13Cl FN O S: C, 54.72; H, 2.93; N,
1
6.04; F, 3.96; S, 6.93; Cl, 15.26. Found: C, 54.44; H, 2.83; N, 6.05.
2
3
scribed in the general procedure. The isolated product was used
without further purification to give methyl 5-(2,6-dicholoro-4-
4.2.33. 2-Chloro-N-(3,5-dichloro-4-(quinolin-3-yloxy)phenyl)-4-
(trifluoromethyl)benzenesulfonamide (36)
nitrophenoxy)nicotinate as an off white solid (quantitative yield).
H NMR (400 MHz, DMSO-d ) d 8.98 (d, J = 1.6 Hz, 1H), 8.72 (d,
6
3,4,5-Trichloronitro benzene, 3-hydroxyquinoline, and 2-
chloro-4-trifluoromethylbenzenesulfonyl chloride were combined
1
J = 2.9 Hz, 1H), 8.61 (s, 2H), 7.72 (dd, J = 2.9, 1.7 Hz, 1H), 3.89 (s,
according to the general procedure to give ligand 36 as an off-
+
1
3
H). MS (ESI): m/z 343.0 (M+H) .
white solid, mp 225 °C. H NMR (400 MHz, DMSO-d
6
) d 11.70–
Methyl 5-(2,6-dicholoro-4-nitrophenoxy)nicotinate was then
12.00 (br s, 1H); 8.60–8.67 (m, 1H); 8.35–8.43 (m, 1H); 7.56–8.06
(m, 6H); 7.32–7.38 (m, 2H). MS (ESI): m/z 560.9 (M+H) . Anal.
+
converted to 1-(5-(2,6-dichloro-4-nitrophenoxy)pyridin-3-yl)etha-
none by modifying a literature procedure.4 Specific changes are as
follows: (1) the acid chloride was dissolved in DMF instead of
4
Calcd for C22
3 3 2 3
H12Cl F N O S: C, 46.86; H, 2.15; N, 4.97. Found: C,
47.01; H, 2.26; N, 4.98.
2
PhMe/Et O, (2) the diethylmalonate anion was generated using
NaH in DMF at 0 °C and (3) the coupling reaction was allowed to
stir 4 h as the temperature gradually rose from 0 to 25 °C. The reac-
4.2.34. N-(3-Acetyl-4-(quinolin-3-yloxy)phenyl)-2,4-dichloro-
benzenesulfonamide (37)
tion mixture was then diluted with H
N HCl (aq) and extracted three times with EtOAc. The organic lay-
ers were combined, washed brine, dried over MgSO , and concen-
2
O and adjusted to pH 5 with
2-Chloro-5-nitroacetophenone, 3-hydroxyquinoline, and 2,4-dic-
hlorobenzenesulfonyl chloride were combined according to the gen-
eral procedure to give ligand 37 as an off-white solid, mp 241 °C. H
2
1
4
trated in vacuo to yield a product that was hydrolyzed and purified
6
NMR (400 MHz, DMSO-d ) d 11.58 (s, 1H); 8.86 (d, J = 2.9 Hz, 1H);
as described to give 1-(5-(2,6-dichloro-4-nitrophenoxy)pyridin-3-
8.38 (d, J = 8.4 Hz; 1); 8.23 (s, 1H); 8.01 (d, J = 8.4 Hz, 1H); 7.86 (d,
J = 8.1 Hz, 1H); 7.53–7.68 (m, 3H); 7.46 (d, J = 2.9 Hz, 1H); 7.34 (s,
2H). MS (ESI): m/z 487.0 (M+H) . HPLC: 95 Area%.
1
yl)ethanone (29% yield) as an amorphous solid.
H NMR
+
(
400 MHz, DMSO-d
6
) d 8.99 (d, J = 1.6 Hz, 1H), 8.67 (d, J = 2.9 Hz,
1
H), 8.62 (s, 2H), 7.69 (dd, J = 1.7, 2.9 Hz, 1H), 2.65 (s, 3H). MS
+
(
ESI): m/z 327.0 (M+H) . 1-(5-(2,6-dichloro-4-nitrophenoxy)pyri-
4.2.35. 2,4-Dichloro-N-(3,5-dichloro-4-(quinolin-6-yloxy)
phenyl)benzenesulfonamide (38)
3,4,5-Trichloronitro benzene, 6-hydroxyquinoline, and 2,4-dic-
hlorobenzenesulfonyl chloride were combined according to the
din-3-yl)ethanone was reduced using SnCl O as described in
the general procedure to give 1-(5-(4-amino-2,6-dichlorophen-
2
ꢀH
2
oxy)pyridin-3-yl)ethanone, that was used without further purifica-
1
tion, (quantitative). H NMR (400 MHz, DMSO-d
6
) d 8.92 (d,
general procedure to give ligand 38 as an off-white solid, mp
1
J = 1.6 Hz, 1H), 8.56 (d, J = 2.9 Hz, 1H), 7.44 (dd, J = 3.4, 1.7 Hz,
H), 6.77 (s, 2H), 5.79 (br s, 2H), 2.64 (s, 3H). MS (ESI): m/z 297.0
M+H). The aniline was coupled to 2,4-dichlorobenzenesulfonyl
241 °C. H NMR (400 MHz, DMSO-d
6
) d 11.37 (s, 1H); 8.75 (dd,
1
(
J = 4.4, 1.6 Hz, 1H); 8.23 (dd, J = 8.4, 1.2 Hz, 1H); 8.16 (d,
J = 8.8 Hz, 1H); 8.02 (d, J = 9.2 Hz, 1H); 7.96 (d, J = 2.0 Hz, 1H);
7.71 (dd, J = 8.8, 2.0 Hz, 1H); 7.50 (dd, J = 9.2, 2.8 Hz, 1H); 7.47
(dd, J = 8.4, 4.0 Hz, 1H); 7.29 (s, 2H); 7.05 (d, J = 2.8 Hz, 1H). IR
chloride as described in the general procedure to give ligand 33
1
as an off-white solid (61%), mp 86 °C. H NMR (400 MHz, DMSO-
ꢁ1
d
8
8
6
) d 11.39 (s, 1H), 8.91 (d, J = 1.4 Hz, 1H), 8.53 (d, J = 2.9 Hz, 1H),
.15 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 1.7 Hz, 1H), 7.7 (dd, J = 2.0,
.5 Hz, 1H), 7.45 (dd, J = 1.7, 2.9 Hz, 1H), 7.30 (s, 2H), 2.62 (s, 3H).
(film) 3274, 1573, 1455, 1323, 1147, 960, 862 cm . MS (ESI): m/
+
z 481.0 (M+H) . Anal. Calcd for C21
4 2 3
H12Cl N O S: C, 49.05; H, 2.35;
N, 5.45. Found: C, 49.25; H, 2.44; N, 5.35.
ꢁ1
IR (film) 1694, 1572, 1460, 1277, 1165 cm . MS (ESI): m/z 507.0
+
(
M+H) . Anal. Calcd for C19
H12Cl
4
N
O
2 4
S: C, 45.08; H, 2.39; Cl,
4.2.36. 2,4-Dichloro-N-(3-chloro-4-(naphthalen-2-yloxy)
2
8.02; N, 5.53. Found: C, 46.42; H, 2.92; Cl, 27.47; N, 5.36.
phenyl)benzenesulfonamide (39)
3
,4,5-Trichloronitro benzene, 2-hydroxynaphthalene, and 2,4-
4
.2.31. 2,4-Dichloro-N-(3-chloro-4-(quinolin-3-yloxy)phenyl)
dichlorobenzenesulfonyl chloride were combined according to
benzenesulfonamide (34)
,4-Dichloronitro benzene, 3- hydroxyquinoline, and 2,4-dic-
the general procedure to give ligand 39 as an off-white solid, mp
1
3
152 °C. H NMR (400 MHz, DMSO-d
6
) d 10.95 (s, 1H); 8.06 (d,
hlorobenzenesulfonyl chloride were combined according to the
J = 8.6 Hz, 1H); 7.94 (d, J = 9.0 Hz, 1H); 7.92 (d, J = 2.1 Hz, 1H);
7.90 (d, J = 8.3 Hz, 1H); 7.74 (d, J = 7.9 Hz, 1H); 7.67 (dd, J = 8.6,
2.1 Hz, 1H); 7.48 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H); 7.43 (ddd, J = 8.2,
6.9, 1.4 Hz, 1H); 7.30 (d, J = 2.2 Hz, 1H); 7.23 (dd, J = 8.9, 2.5 Hz,
1H); 7.16–7.12 (m, 2H); 7.10 (dd, J = 8.8, 2.3 Hz, 1H). IR (film)
general procedure to give ligand 34 as an off-white solid, mp
1
1
22 °C. H NMR (400 MHz, DMSO-d
6
) d 11.04 (s, 1H); 8.79 (d,
J = 2.9 Hz, 1H); 8.09 (d, J = 8.5 Hz, 1H); 8.15 (d, J = 8.3 Hz, 1H);
7
2
.98 (d, J = 2.1 Hz, 1H); 7.89 (d, J = 8.2 Hz, 1H); 7.70–7.65 (m,
H); 7.60–7.58 (m, 1H); 7.57 (d, J = 3.0 Hz, 1H); 7.33 (d,
ꢁ1
3281, 1490, 1461, 1162, 819, 624 cm . MS (ESI): m/z 478.0
ꢁ
J = 2.6 Hz, 1H); 7.26 (d, J = 8.8 Hz, 1H); 7.13 (dd, J = 2.6 Hz, 1H). IR
(MꢁH) . Anal. Calcd for C22
3 3
H14Cl NO S: C, 55.19; H, 2.95; N,
ꢁ
1
(
(
film) 1488, 1335, 1162, 818, 616 cm . MS (ESI): m/z 463.0
2.93; Cl, 22.21. Found: C, 54.93; H, 2.84; N, 2.96; 22.44.
+
3 2 3
M+H) . Anal. Calcd for C21H13Cl N O S: C, 52.41; H, 2.73; N,
5
.85. Found: C, 52.41; H, 2.73; N, 5.85.
4.3. General in vitro biology methods
4
.2.32. 2,4-Dichloro-N-(3-fluoro-4-(quinolin-3-yloxy)
4.3.1. Ligand displacement-filtration assay
Displacement of [ H]-rosiglitazone by our PPAR
directly measured in a scintillation proximity assay. A 90
3
phenyl)benzenesulfonamide (35)
,4-Difluoronitro benzene, 3-hydroxyquinoline, and 2,4-dic-
c
ligands was
L reac-
3
l
hlorobenzenesulfonyl chloride were combined according to the
tion slurry containing binding buffer (10 mM Tris-HCl pH 8.0,
general procedure to give ligand 35 as an off-white solid, mp
50 mM KCl, 0.01% NP40, 10 mM dithiothreitol, 0.02% bovine serum

J. P. Taygerly et al. / Bioorg. Med. Chem. 21 (2013) 979–992
991
3
albumin), 350 ng GST-PPAR
glutathione Sepharose 4B beads (Amersham Biosciences) was incu-
bated for 15 minutes at rt. The slurry was then added to 10 L of
PPAR ligand in Me SO that had been allocated into the wells of
c
LBD, 20 nM [ H]-rosiglitazone, and
and Haoda Xu for technical assistance. The Advanced Light Source
is supported by the U.S. Department of Energy under Contract No.
DE-AC03-76SF00098.
l
c
2
a Polyfiltronics Unifilter 350 microassay plate, and the mixture
incubated for 1H with shaking at rt. The filtrate was separated from
bound ligand and protein by vacuum, and the plate containing the
retained material was washed twice with binding buffer before
reading in a Packard TopCount.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.058.
References and notes
4
.3.2. Gal4-PPAR
Co-transfection experiments were carried out using expression
plasmids encoding chimeric GAL4 DNA binding domain-hPPAR li-
c transactivation assay
1. Camp, H. Curr. Opin. Investig. Drugs 2003, 4, 406.
2.
3.
4.
http://www.diabetes.org/home.jsp.
Diamant, M.; Heine, R. J. Drugs 2003, 63, 1373.
(a) Semple, R. K.; Chatterjee, V. K. K.; O’Rahilly, S. J. Clin. Invest. 2006, 116, 581;
c
gand binding domain fusion proteins to drive transcription of UAS-
GAL4-luciferase reporter genes. Transient transfection of HEK293
cells using GenePORTER2 reagent (GTS Inc., San Diego, CA) was car-
ried out according to the manufacturer’s protocol, and luciferase
(
b) Smith, M. Chem. Res. Toxicol. 2003, 16, 679; (c) Oakes, N. D.; Kennedy, C. J.;
Jenkins, A. B.; Laybutt, D. R.; Chisholm, D. J.; Kraegen, E. W. Diabetes 1994, 43,
203.
Stumvoll, M. Expert Opin. Investig. Drugs 2003, 12, 1179.
1
5.
1
5
activity measured 24 h after the addition of compounds.
6. (a) Nesto, R. W.; Bell, D.; Bonow, R. O.; Fonesca, V.; Grundy, S. M.; Horton, E. S.;
Le Winter, M.; Porte, D.; Semenkovich, C. F.; Smith, S.; Young, L. H.; Kahn, R.
Diabetes Care 2004, 27, 256; (b) Kahn, S. E.; Zinman, B.; Lachin, J. M.; Haffner, S.
M.; Herman, W. H.; Holman, R. R.; Kravits, B. G.; Yu, D.; Heise, M. A.; Aftring, R.
P.; Viberti, G. Diabetes Care 2008, 31, 845.
4
4
.4. General in vivo biology methods
7.
(a) Robinson, J. G. Curr. Atheroscler. Rep. 2007, 9, 64; (b) Boden, G.; Zhang, M.
Expert Opin. Investig. Drugs 2006, 15, 243; (c) Patel, C.; Wyne, K. L.; McGuire, D.
K. Diab. Vasc. Dis. Res. 2005, 2, 61; (d) Shearer, B. G.; Billin, A. N. Biochim.
Biophys. Acta 2007, 1771, 1082; (e) Nissen, S. E.; Wolski, K. N. Engl. J. Med. 2007,
.4.1. KKAy mouse studies
Male KKAy obese mice (Harlan, 5–7 weeks old),4 were pre-
5
screened for body weight and a range of clinical parameters (glu-
cose, insulin, leptin, nonesterified free fatty acids (NEFA),
triglycerides, total cholesterol, and HDL cholesterol). Blood sam-
pling was performed from the retro-orbital plexus under ether
anesthesia. The mice were singly housed and randomized into
groups of n = 8/treatment group. Animal holding, room tempera-
ture, and humidity were in conformance with the Guide for the Care
3
56, 2457; (f) Tang, W. H.; Maroo, A. Diabetes Obes. Metab. 2007, 9, 447; (g)
Goldfine, A. B. Curr. Opin. Endocrinol. Diabetes Obes. 2008, 15, 113.
Picard, F.; Auwerx, J. Annu. Rev. Nutr. 2002, 22, 167.
8.
9. A thorough review of small molecule PPAR modulators has recently been
published: Pirat, C.; Farce, A.; Leb e` gue, N.; Renault, N.; Furman, C.; Millet, R.;
Yous, S.; Speca, S.; Berthelot, P.; Desreumaux, P.; Chavatte, P. J. Med. Chem.
2012, 55, 4027.
1
0. (a) Motani, A.; Wang, Z.; Weiszmann, J.; McGee, L. R.; Lee, G.; Liu, Q.; Staunton,
J.; Fang, Z.; Fuentes, H.; Lindstrom, M.; Liu, J.; Biermann, D. H. T.; Jaen, J.;
Walker, N. P. C.; Learned, R. M.; Chen, J.-L.; Li, Y. J. Mol. Biol. 2009, 386, 1301; (b)
Higgins, L. S.; Mantzoros, C. S. PPAR Res. 2008, 1; (c) Li, Y.; Wang, Z.; Motani, A.
Diabetes 2004, 53, A157.
and Use of Laboratory Animals.46 Compounds were dissolved in
EtOH and added to powdered diet (irradiated PicoLabÒ 5053). After
drying overnight to remove EtOH, food was delivered in pre-
weighed food jars on day 1. On day 4, body weight and food intake
were determined, and blood was sampled and assayed for glucose
and insulin. EDTA-treated blood samples were submitted for
hematology and serum samples for clinical chemistry (Quality
Clinical Labs, Mountain View, CA).
11. Li, Y.; Wang, Z.; Furukawa, N.; Escaron, P.; Weiszmann, J.; Lee; Gary; Lindstrom,
M.; Liu, J.; Liu, X.; Xu, H.; Plotnikova, O.; Prasad, V.; Walker, N.; Learned, R. M.;
Chen, J.-L. J. Biol. Chem. 2008, 283, 9168.
12. For reviews of selective PPARc modulators see: (a) Higgins, L. S.; DePaoli, A. M.
Am. J. Clin. Nutr. 2010, 91, 276S; (b) Doshi, L. S.; Brahma, K.; Bahirat, U. A.; Dixit,
A. V.; Nemmani, K. V. S. Expert Opin. Investig. Drugs 2010, 19, 489. and
references within; (c) Zhang, F.; Lavan, B. E.; Gregoire, F. M. PPAR Res. 2007,
Article ID 32696, 7 pages.
4
.4.2. Zucker (fa/fa) fatty rat studies
13. (a) Choi, J. H.; Banks, A. S.; Estall, J. L.; Kajimura, S.; Bostrom, P.; Laznik, D.;
Ruas, J. L.; Chalmers, M. J.; Kamenecka, T. M.; Bluher, M.; Griffin, P. R.;
Spiegelman, B. M. Nature 2010, 466, 451; (b) Choi, J. H.; Banks, A. S.;
Kamenecka, T. M.; Busby, S. A.; Chalmers, M. J.; Kumar, N.; Kuruvilla, D. S.;
Shin, Y.; He, Y.; Bruning, J. B.; Marciano, D. P.; Cameron, M. D.; Laznik, D.;
Jurczak, M. J.; Schürer, S. C.; Vidovi c´ , D.; Shulman, G. I.; Spiegelman, B. M.;
Griffin, P. R. Nature 2011, 477, 477.
Male Zucker fatty (fa/fa) rats (Charles River Laboratories), aged
7
Ò
–8 weeks were allowed free access to irradiated PicoLab 5053 diet
and housed on a 12 h light–dark cycle. Animal holding, room tem-
perature, and humidity were in conformance with the Guide for the
Care and Use of Laboratory Animals.46 Rats were pre-bled via a tail
nick while conscious and samples were analyzed for fed-state glu-
cose, insulin, NEFA, triglycerides and total cholesterol. Area under
the glucose curve (AUC0–120 min) was determined during an oral glu-
cose tolerance test (OGTT; 1 g/10 mL/kg, po) performed in overnight
fasted rats. Blood glucose levels were determined in whole blood ex-
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1
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