Indanylacetic acid derivatives carrying aryl-pyridyl and aryl-pyrimidinyl tail groups-new classes of PPAR γ/δ and PPAR α/γ/δ agonists

Article abstract of DOI:10.1016/j.bmcl.2006.11.025

Modulation of PPAR activities represents an attractive approach for the treatment of diabetes with associated cardiovascular complications. The indanylacetic acid structural motif has proven useful in the generation of potent and tunable PPAR ligands. Mod

Full text of DOI:10.1016/j.bmcl.2006.11.025

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1056–1061
Indanylacetic acid derivatives carrying aryl-pyridyl and
aryl-pyrimidinyl tail groups—new classes of PPAR c/d
and PPAR a/c/d agonists
Louis-David Cantin,a,* Sidney Liang,a Herbert Ogutu,a Christiana I. Iwuagwu,a
Ken Boakye,c William H. Bullock,a Michael Burns,c Roger Clark,a Thomas Claus,c
Fernando E. delaCruz,c Michelle Daly,c Frederick J. Ehrgott,a Jeffrey S. Johnson,a
Christine Keiper,c James N. Livingston,c Robert W. Schoenleber,a Jeffrey Shapiro,c
Christopher Town,b Ling Yang,c Manami Tsutsumic and Xin Maa
aDepartment of Chemistry Research, Bayer Pharmaceuticals Corporation, West Haven, CT 06516, USA
bDepartment of Research Technologies, Bayer Pharmaceuticals Corporation, West Haven, CT 06516, USA
cDepartment of Metabolic Disorders Research, Bayer Pharmaceuticals Corporation, West Haven, CT 06516, USA
Received 20 October 2006; revised 7 November 2006; accepted 8 November 2006
Available online 15 November 2006
Abstract—Modulation of PPAR activities represents an attractive approach for the treatment of diabetes with associated cardiovas-
cular complications. The indanylacetic acid structural motif has proven useful in the generation of potent and tunable PPAR
ligands. Modification of the substituents on the linker and the heterocycle tail group allowed for the modulation of the selectivity
at the different receptor subtypes. Compound 33 was evaluated in vivo, where it displayed the desired reduction of glucose levels and
increase in HDL levels in various animal models.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Diabetes, together with its complications, was responsi-
ble for 1 in 5 deaths in the US in 2002.1 The World
Health Organization is now forecasting that by the
year 2030, 366 million people will suffer from diabetes
worldwide,2 with 90% of these cases due to non-insu-
lin-dependent (or Type II) diabetes.1 As the disease
progresses, numerous complications gradually erode
the quality of life of patients. The greater prevalence
of cardiovascular complications2 in diabetics under-
scores the need to develop antidiabetic agents capable
of lowering HbA1c levels while improving the lipid
profile of patients. Such multi-action agents may hold
promise for delaying the progression of the disease,
as suggested by the increasing evidence identifying
obesity as a cause factor of Type II diabetes and con-
comitant cardiovascular complications.
Correction of the elevated glucose levels in patients with
Type II diabetes is typically achieved through modula-
tion of insulin production, of glucose neogenesis, or by
improving glucose uptake by muscle tissues in response
to insulin.3 The gradual increase in skeletal muscle tissue
resistance to insulin has also been linked to a cascade of
events leading to Type II diabetes and eventually to b-
cell failure. Physicians have attempted to slow the pro-
gression of insulin resistance by the use of insulin sensi-
tizers such as PPAR (peroxizome proliferator-activated
receptor) gamma agonists. This class of agents, typified
by the glitazones, has received significant attention from
the pharmaceutical industry leading to the successful
development of two agents, rosiglitazone and pioglitaz-
one, currently used for the management of elevated glu-
cose levels.4 The sustained research interest in these
agents is due to the various roles that PPARs can play
in controlling glucose homeostasis, as well as triglycer-
ide (TG) and cholesterol levels.5 Indeed, PPARa ago-
nists, such as fibrates, have played a long-standing role
in modulation of triglyceride levels,6 and there is now
increasing evidence that PPARd agonists can improve
Keywords: PPAR agonist; Indanyl acetic acid; Diabetes; Cardiovascu-
lar; Triglycerides; HDL; Cholesterol; SAR.
*
Corresponding author. Tel.: +1 203 812 3968; e-mail: david.cantin.
b@bayer.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.025

L.-D. Cantin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1056–1061
1057
high-density lipoprotein (HDL)/low-density lipoprotein
(LDL) profiles in animals.7
O
COOH
d
a, b, c
MeO
MeO
A previous report from our laboratories disclosed
PPARa/c agonists incorporating a novel indanylacetic
acid head-group.8 This work led to the identification
of compounds capable of correcting glucose levels, as
well as modulating TG levels. Herein, we report on fur-
ther modifications to indanylacetic acid, which lead to
PPAR agonists that incorporate PPARd activity.
MeO
3
4
COOEt
COOH
e, f
HO
5
6
99% e.e.
Starting from our first generation agonists (1, Fig. 1), we
hypothesized that linking an unsubstituted indanylacetic
acid with six-membered heteroaryl group using a flexible
linker could provide us with a dual or triple agonist (2,
Fig. 1).
Scheme 1. Reagents and conditions: (a) Zn, ethyl bromoacetate, THF,
40 ꢁC; (b) H2 (40 psi), 5% Pd/C, EtOH, 99% yield (two steps); (c)
NaOH, H2O, EtOH, reflux, 80% yield; (d) (S)-(À)-a-methylbenzyl-
amine, acetone; recrystallization; 1 N HCl, EtOAc, 35% yield, 99% ee;
(e) TMSCl, EtOH, 98% yield; (f) AlCl3, EtSH, CH2Cl2, 5–10 ꢁC, 96%
yield.
The synthesis of the indane fragment (Scheme 1) started
from indanone 3 which was converted to the corre-
sponding enoate under Reformatsky-type conditions.9
Subsequent reduction and hydrolysis provided acid 4,
which underwent chemical resolution using (S)-(À)-a-
methylbenzylamine to provide 5 in good yield and high
enantiomeric excess. With 5 in hand, esterification of 5
followed by demethylation provided the right-hand
fragment 6 of the target compounds.
CO2Et
Me
Me
a, b, c
Cl
N
N
4'
N
Me
Cl
Cl
N
O
N
7
8
CO2H
The synthesis of the left-hand moiety of the target com-
pounds was achieved starting from an appropriately
substituted 2,4-dichloropyridine (7) onto which the ami-
no alcohol linker was introduced chemoselectively at the
C4 position using sodium carbonate in ethanol (Scheme
2). Subsequent etherification using a modified Mitsun-
obu protocol allowed for the introduction of the indane
6 in good yield, followed by treatment with NaH and
methyl iodide to provide the key intermediate 8. Finally,
Suzuki cross-coupling, followed by saponification, pro-
vided compound 9.
Me
N
d, e
4'
N
N
O
Me
R3
9
Scheme 2. Reagents and conditions: (a) 1,3-propanolamine, NaHCO3,
EtOH, 95% yield; (b) 6, ADDP, PPh3, THF, 95% yield; (c) NaH, MeI,
DMF, 60% yield; (d) Ar–B(OH)2, PdCl2(dppf)ÆCH2Cl2, Na2CO3, H2O,
1,4-dioxane, toluene; (e) LiOH, H2O, THF, EtOH, 40–60% yield (two
steps).
We began our optimization work by exploring the SAR
at the C2 position of the pyrimidine. The task of opti-
mizing for all three activities was simplified since most
variations at C2 led to potent PPARd ligands (Table
1). With our attention focused on improving PPARa
and PPARc potencies, we began by introducing lipo-
philic groups at the para-position of the C2 phenyl ring
(11–14, Table 2), which provided increased potency at
PPARc and PPARa. Introduction of both electron-do-
Table 1.
CO2H
Me
N
2
N
N
O
Me
R3
Compound R3
IRBA10
(EC50, nM) FRET
PPARa11
PPARd11
FRET
(EC50, nM) (EC50, nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
H
678
780
145
29
6610
1150
200
24
9
4-Me
4-Et
4-iPr
10
34
28
58
12
83
7
990
641
4-t-Bu
4-EtO
156
161
3090
1390
6550
2120
1130
4390
3540
2370
4-MeO 7010
4-Ac
4-F
237
860
4-Cl
2800
670
7
3-Me
3-EtO
3-Cl
41
7
6930
582
23
Figure 1.

1058
L.-D. Cantin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1056–1061
Table 2.
CO2H
(R1/R2)
6
5
N
N
N
O
Me
Compound R1/R2 IRBA10
PPARa11
PPARd11
FRET
(EC50, nM) (EC50, nM)
(EC50, nM) FRET
23
24
12
25
26
H
5-F
800
360
145
950
600
565
425
200
120
920
25
16
10
2
Figure 2.
5-Me
5-Et
6-Me
11
often difficult, we sought nonetheless to identify the ma-
jor metabolites to help guide our optimization work. We
suspected that the N-methyl group might be the origin
of this instability (Fig. 2), which was confirmed by
in vitro metabolite identification studies. Although we
observed other less prevalent metabolites, the N-de-
methylated products were consistently the major metab-
olites observed.
nating (15 and 16) and withdrawing groups (17–19)
tended to decrease the PPARa potency relative to lipo-
philic substituents. Migration of the substituent to the
meta-position of the phenyl ring caused a loss in potency
at PPARa and PPARd as exemplified by compounds 20
and 22. In contrast, a 3-EtO group (21) caused a signif-
icant increase in potency at PPARd over the corre-
sponding 4-EtO (15), while the PPARa potency
remained unaffected. These group migrations caused dif-
ferent effects on PPARc potency where the 3-EtO (21)
showed decreased potency and the 3-Cl (22) provided
a moderate (4- to 5-fold) increase in potency vs its
4-Cl analog (19). Further substitution at the ortho-posi-
tion, or introduction of two substituents (2,4 or 3,4),
led to compounds with decreased potency at one or
more receptor subtypes.
Compound 27 was found to be a weak ligand for all
receptors except PPARd (56 nM). Modifications of the
linker in an attempt to block metabolism revealed that
this area of the molecule was critical for maintaining
the potency at all three receptor subtypes. Indeed, intro-
duction of sterically hindered groups or cyclization led
to a decrease in potency (data not shown). However,
exchanging the N–Me group for an oxygen atom to re-
move the site of metabolism was better tolerated (IRBA:
1600 nM, PPARa FRET: 900 nM, PPARd FRET:
5 nM). The activity profile of the ether-linked agonists
was optimized by incorporating our findings with those
of a related project,14 where a phenyl ring bearing a thi-
azole in the para-position to the linker was beneficial.
Turning our attention to the effects of the substituents
on the pyrimidine ring, it rapidly became apparent that
the size and location of the substituent was critical for
retaining the activity (Table 2). Indeed, while the 5-
methyl group (12) was optimal, the 5-ethyl and 6-methyl
groups (25 and 26) decreased potency at either PPARa
or PPARc. In addition, introduction of a fluorine atom
at position 5 (24) was tolerated by all three receptor
subtypes.
Using the 2,4-substituted pyrimidines did not offer the
opportunity to introduce a substituent in the para-posi-
tion to the linker. Additionally, we had previously deter-
mined that the other pyrimidine isomers were less
favored. We therefore opted to change the heterocycle
to a pyridine, which allowed for the desired substituent
orientation and was expected to provide similar physico-
chemical properties to the pyrimidines.
Having completed the initial optimization, several com-
pounds were selected for further pharmacological profil-
ing. Cell assays12 performed on compound 12 confirmed
its potent agonist activity at PPARa and d13 with a 10-
fold decrease in potency from the human receptor to the
mouse receptor. Surprisingly, compound 13 proved to
be more potent than expected as a PPARa agonist in
CV-1 cells, while maintaining the same species prefer-
ence for the human receptor.13
CO2Et
NC
NC
a, b
N
Cl
N
N
O
O
O
O
R3
28
29
30
CO2H
S
R4
Having identified compounds with the targeted activity
profile, we evaluated the pharmacokinetic profile of sev-
eral analogs. An initial screen using rodent and human
microsomes revealed that the compounds were rapidly
metabolized. Subsequent in vivo experiments confirmed
an overall trend toward low plasma exposure when
either compound 12 or 13 was dosed orally in rodents
(mice and rats). Although establishing a direct correla-
tion between microsomal stability and oral exposure is
c, d, e
N
Scheme 3. Reagents and coinditions: (a) 1,3-propanediol, NaH, DMF,
83% yield; (b) 6, ADDP, PPh3, THF, 67% yield; (c) H2S, Et2NH,
DMF, 86% yield; (d) 2-haloketones, EtOH, 70 ꢁC; (e) LiOH, H2O,
THF, EtOH, 60–80% yield (two steps).

L.-D. Cantin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1056–1061
1059
Table 3.
CO2H
R4
N
O
O
Compound
R4
IRBA10 (EC50, nM)
PPARa11 FRET (EC50, nM)
PPARd11 FRET (EC50, nM)
S
31
32
264
421
3
5
N
S
511
6780
N
O
33
34
35
1390
807
9
3
S
N
N
S
EtO
411
A series of O-linked pyridines bearing a thiazole substi-
tuent were synthesized as described in Scheme 3. Addi-
tion of 1,3-propane diol to 5-chloronicotinonitrile (28),
followed by Mitsunobu etherification with indane 6,
provided intermediate 29. Conversion of the nitrile
group to the corresponding thioamide was achieved by
exposure to H2S in DMF at 40 ꢁC. Subsequent Han-
tzsch-type condensation of diversely substituted haloke-
tones, followed by basic hydrolysis, provided the target
compound 30.
25
0
Compound 33
-25
ED50~6.7mpk
***
-50
-75
***
A series of thiazoles were prepared, from which com-
pound 33 emerged as a compound of potential interest
(Table 3). Although the potency at PPARa was weaker
than anticipated, the potent PPARc and d activities
prompted us to further profile this compound. The
activity profile translated well to human cell assays,15
however compound 33 showed a significant decrease in
potency (>20-fold) in mouse PPARd cell assay (human
PPARd in CV-1 cells (IC50) 56 nM; mouse PPARd in
CV-1 cells (IC50) 1390 nM).
0
5
10
15
20
25
30
mpk Compound
Figure 3. Effects of 33 on blood glucose levels in db/db mice after 7
days of treatment.
day 0
day 11
16000
12000
8000
4000
0
Pleasingly, compound 33 provided the desired improve-
ment in pharmacokinetic profile in rodents [e.g., rats
(PO and IV, 3 mpk) PO AUC 26 lM h, Cmax 2.8 lM; F
48%, t1/2 = 5 h]. This improved exposure profile, which
was found to be similar in mice, allowed us to evaluate
compound 33 in diabetic animal models. Compound 33
reduced plasma glucose levels in db/db mice in a dose-de-
pendent manner with an estimated ED50 value of 6.7 mpk
after 7 days of dosing (Fig. 3).16 At a dose of 10 mpk, com-
pound 33 proved to be as efficacious in lowering glucose as
the maximally effective dose of rosiglitazone in this model
(not shown). Similarly, when administered for 10 days at a
dose of 10 mpk, compound 33 normalized glucose toler-
ance in Zucker fa/fa rats (Fig. 4).16
***
Vehicle
Compound 33
Figure 4. Effects of 33 on glucose tolerance in Zucker fa/fa rats after 10
days of treatment.
assessed using hApoA1 mice. Oral administration of
33 to hApoA1 mice at 30 mpk (to account for the weaker
cell potency at murine PPARd) for 10 days16 reduced
The ability of compound 33 to modulate TG and HDL-
cholesterol levels through its effects on PPARd was

1060
L.-D. Cantin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1056–1061
medium containing 0.5 lM human IGF-1 and test com-
pound. After treatment, the medium was replaced with
medium free of IGF-1 and compound, and incubated for 4
days. After washing the cells with buffer, they were
incubated with 0.1 nM 125I–insulin and ( ) 100 nM
unlabeled insulin, and incubated at rt for 1 h. The cells
were then washed 3· with buffer, dissolved with 1 N
NaOH, and the amount of radioligand bound was
measured using a gamma counter. Values reported are
means of at least two experiments.
plasma triglycerides [À32 7% (p < 0.05)] and raised
HDL-cholesterol [22 7% (p < 0.05)].
In summary, we have shown that the indanylacetic acid
group is a versatile head-group, which can be combined
to diverse tail groups to generate PPAR agonists with
different receptor subtype selectivity. The optimization
of the tail group allowed for the identification of both
PPARc/d dual and PPARa/c/d triple agonists. The anti-
diabetic properties of this scaffold were demonstrated,
together with added benefits of correcting TG and
HDL levels associated with agonism of PPARd.17
11. PPARa and PPARd activity was measured using a
fluorescence resonance energy transfer (FRET) assay
using the human PPARa and PPARd ligand-binding
domains. Test compounds were incubated in 96-well plates
with europium-labeled anti-GST antibody, GST-tagged
PPAR ligand-binding domain, biotinylated REB-binding
protein, and streptavidin-labeled APC (Wallac, AD0065).
The plate was read in a fluorimeter with an excitation
wavelength of 340 nm and emission wavelengths of 615
and 640 nm. Values reported are means of at least two
experiments.
12. PPARa and PPARd activity was measured using a cell-
based GAL4 transactivation assay for human and mouse
ligand-binding domain in CV-1 cells. CV-1 cells were
seeded in 96-well plates at 2.8 · 104 cells per well, grown
overnight in standard media containing 10% fetal bovine
serum, and then transiently transfected using the Lipo-
fectamine/Plus procedure. The cells in each well were
transfected with plasmids containing the Gal4/PPAR-
LBD fusion, UAS/firefly luciferase, and Renilla luciferase.
After an overnight incubation with media containing 10%
FBS treated with charcoal/dextran, test compounds were
added and the cells were incubated for an additional 24 h.
The plates were processed using the Promega Dual
Luciferase kit and read on a Packard Topcount. EC50
values were determined based on a dose–response curve
and the percent maximum stimulation was assessed by
comparison to reference compounds. Values reported are
means of at least two experiments.
13. Compound 12: human PPARd CV-1 cells IC50 30 nM;
human PPARa CV-1 cells IC50 175 nM; mouse PPARd
CV-1 cells IC50 370 nM; mouse PPARa CV-1 cells IC50
670 nM.Compound 13: human PPARd CV-1 cells IC50
49 nM; human PPARa CV-1 cells IC50 237 nM; mouse
PPARd CV-1 cells IC50 610 nM; mouse PPARa CV-1 cells
IC50 2530 nM.
14. Rudolph, J.; Chen, L.; Majumdar, D.; Bullock, W.H.;
Burns, M.; Choi, S.; Claus, T.; Dela Cruz, F.E.; Daly, M.;
Ehrgott, F.J.; Johnson, J.S.; Livingston, J.N.; Nophsker,
M.; Schoenleber, R.W.; Shapiro, J.; Tomlinson, S.; Town,
C.; Yang, L.; Tsutsumi, M.; Ma, X. Abstracts of Papers,
232nd ACS National Meeting, Sept 10–14, 2006; MEDI-
377.
Acknowledgments
´
´
We thank Jefferson Chin, Laszlo Musza, and Anthony
Paiva for NMR and LC-MS support. We are also grate-
ful to Drs. Roger A. Smith and Derek Lowe for helpful
discussions.
References and notes
1. Stumvoll, M.; Goldstain, B. J.; van Haeften, T. W. The
Lancet 2005, 365, 1333.
2. Zimmet, P.; Alberti, K. G.; Shaw, J. Nature 2001, 414,
782.
3. Krentz, A. J.; Bailey, C. J. Drugs 2005, 66, 385.
4. Campbell, I. W. Curr. Mol. Med. 2005, 5, 349.
5. For leading references, see: (a) Staels, B.; Fruchart, J.-C.
Diabetes 2005, 54, 2460; (b) Cheng, P. T. W.; Mukherjee,
R. PPARs as targets for metabolic and cardiovascular
diseases 2005, 5, 741.
6. Robillard, R.; Fontaine, C.; Chinetti, G.; Fruchart, J.-C.;
Fibrates Staels, B. Handb. Exp. Pharmacol. 2005, 170, 389.
7. Oliver, W. R.; Shenk, J. L.; Snaith, M. R.; Russell, C. S.;
Plunket, K. D.; Bodkin, N. L.; Lewis, M. C.; Winegar, D.
A.; Sznaidman, M. L.; Lambert, M. H.; Xu, H. E.;
Sternbach, D. D.; Kliewer, S. A.; Hansen, B. C.; Willson,
T. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5306.
8. Lowe, D. B.; Bifulco, N.; Bullock, W. H.; Claus, T.;
Coish, P.; Dai, M.; Dela Cruz, F. E.; Dickson, D.; Fan,
D.; Hoover-Litty, H.; Li, T.; Ma, X.; Mannelly, G.;
Monahan, M.-K.; Muegge, I.; O’Connor, S.; Rodriguez,
M.; Shelekhin, T.; Stolle, A.; Sweet, L.; Wang, M.; Wang,
Y.; Zhang, C.; Zhang, H.-J.; Zhang, M.; Zhao, K.; Zhao,
Q.; Zhu, J.; Zhu, L.; Tsutsumi, M. Bioorg. Med. Chem.
Lett. 2006, 16, 297.
9. All compounds described gave consistent 1H NMR and
LC/MS data. For more details on their synthetic prepa-
rations, see: Cantin, Louis-David; Choi, Soongyu; Clark,
Roger B.; Hentemann, Martin F.; Ma, Xin; Rudolph,
Joachim; Liang, Sidney X.; Akuche, Christiana; Lavoie,
Rico C.; Chen, Libing; Majumdar, Dyuti; Wickens, Philip
L. Preparation of indaneacetic acid derivatives and their
use as pharmaceutical agents, WO 2004058174.
10. The PPARc activity was assessed by IRBA, a cell-based
assay performed in mouse 3T3-L1 pre-adipocytes. This
assay measures the ability of a test compound to cause an
increase in the number of insulin receptors and hence is an
index of the insulin sensitizing activity. 3T3-L1 cells were
seeded in 96-well tissue culture plates using DMEM
containing 10% fetal bovine serum, 1% pen/strep, and
2 mM L-glutamine, and were grown until they were 2 days
post-confluent. Cells were then treated for 2 days with
15. Compound 33: human PPARd CV-1 cells IC50 56 nM;
human PPARa CV-1 cells IC50 > 6000 nM; mouse PPARd
CV-1 cells IC50, 1390 nM.
16. In vivo studies (mice and rats). All animals were purchased
at 6 weeks of age and were maintained on standard
laboratory rodent chow ad libitum. db/db and hApoA1
Mice experiments. Female db/db mice and male hApoA1
mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). The hApoA1 mice were used within 2 weeks
of their arrival, whereas the db/db mice were maintained
on diet for 3–4 weeks and 8–10 weeks, respectively, prior
to starting the study. The average body weight was 25 g
for the db/db mice and 50 g for the hApoA1 mice. The
animals were weighed and tail-bled prior to the start of
study. Plasma from db/db mice was analyzed for glucose
levels using either
a Beckman Glucose Analyzer 2

L.-D. Cantin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1056–1061
1061
(Beckman Instruments, Fullerton, CA) or a Technicon Axon
autoanalyzer (Bayer HealthCare LLC, Tarrytown, NY).
Plasma from hApoA1 mice was analyzed for triglyceride
and HDL-cholesterol levels using the Axon autoanalyzer.
The animals were arranged into the appropriate number
of groups with each group having the same mean plasma
glucose levels (db/db mice) or plasma triglyceride levels
(hApoA1 mice) prior to dosing. All animals then were
orally dosed once daily with vehicle (0.5% methylcellulose
in water), a positive control compound, or compound 33.
The db/db and hApoA1 mice were dosed for 7 days. All
animals were fed ad libitum throughout the study.
Approximately 24 h after the last dose, the animals were
weighed and bled again and the plasma analyzed for
glucose, triglycerides or HDL-cholesterol. Calculation of
percent change due to drug treatment. In order to evaluate
the effects of compound 33 treatment on glucose levels and
on triglyceride and HDLc levels, it is useful to calculate
the percent change in glucose or triglyceride and HDLc
that is due to drug treatment. This takes into account any
changes that may have occurred in the vehicle-treated
animals during the study. The percent change due to drug
treatment is calculated as follows: (((Tf/Ti)/(Vf/
tolerance experiments. Female Zucker Fatty (fa/fa) rats
were purchased from Charles River (Wilmington, MA).
Animals were maintained on diet for 8–10 weeks, prior to
starting the study. The average body weight was 471 g.
Female fa/fa rats were fasted overnight and given a 2 g/kg
IPGTT. Blood glucose was measured from tail to tip
blood using a Glucometer (Bayer HealthCare LLC, Mish-
awaka, IN) just prior to the glucose load and after 15, 30, 60,
90, and 120 min. The glucose area-under-the-curve (AUC)
was calculated over the 0-to-120 min using the trapezoidal
method, and the rats were grouped with equivalent mean
glucose AUC values. Starting a week later, the rats were
dosed daily with vehicle (0.5% methylcellulose) or 10 mg/kg
compound 33 for 10 days. The rats were fasted overnight
and a 2 g/kg IPGTT performed again on day 11.Statistical
analysis. All results are expressed as the means SEM for
the number of animals indicated in the figure legends.
ANOVA was used to evaluate the effects of positive control
drugs and compound 33 using InStat (GraphPad Software
Inc., San Diego, CA). The Tukey–Kramer multiple com-
parisons test was used for the parametric ANOVA.
Whenever a non-parametric ANOVA was required, the
Kruskal–Wallis test was used. Results were considered
significant at p < 0.05.
Vi)) À 1) 100 Tf and Ti are the final and initial values
*
of either plasma glucose or serum triglyceride levels in the
drug-treated animals, respectively, and Vf and Vi are the
same for the vehicle-treated animals. A positive number
denotes a drug-induced increase, whereas a negative
number denotes a drug-induced decrease. fa/fa rat glucose
17. Xu, Y.; Etgen, G. J.; Broderick, C. L.; Canada, E.;
Gonzalez, I.; Lamar, J.; Montrose-Rafizadeh, C.; Oldham,
B. A.; Osborne, J. J.; Xie, C.; Shi, Q.; Winneroski, L. L.;
York, J.; Yumibe, N.; Zink, R.; Mantlo, N. J. Med. Chem.
2006, 49, 5649.