Discovery of (2R)-2-(3-{3-[(4-methoxyphenyl)carbonyl]-2-methyl-6- (trifluoromethoxy)-1H-indol-1-yl}phenoxy)butanoic acid (MK-0533): A novel selective peroxisome proliferator-activated receptor γ modulator for the treatment of type 2 diabetes mellitus with

Article abstract of DOI:10.1021/jm900097m

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are used to treat type 2 diabetes mellitus (T2DM). Widespread use of PPARγ agonists has been prevented due to adverse effects including weight gain, edema, and increased risk of congestive

Full text of DOI:10.1021/jm900097m

3846
J. Med. Chem. 2009, 52, 3846–3854
Articles
Discovery of (2R)-2-(3-{3-[(4-Methoxyphenyl)carbonyl]-2-methyl-6-(trifluoromethoxy)-1H-indol-
1-yl}phenoxy)butanoic Acid (MK-0533): A Novel Selective Peroxisome Proliferator-Activated
Receptor γ Modulator for the Treatment of Type 2 Diabetes Mellitus with a Reduced Potential
to Increase Plasma and Extracellular Fluid Volume
John J. Acton, III, Taro E. Akiyama, Ching H. Chang, Lawrence Colwell, Sheryl Debenham, Thomas Doebber,
Monica Einstein, Kun Liu, Margaret E. McCann, David E. Moller,† Eric S. Muise, Yugen Tan, John R. Thompson,
Kenny K. Wong, Margaret Wu, Libo Xu, Peter T. Meinke, Joel P. Berger, and Harold B. Wood*
Merck Research Laboratories, Merck & Co., Inc., RY800-C114, P.O. Box 2000, Rahway, New Jersey 07065
ReceiVed January 23, 2009
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are used to treat type 2 diabetes mellitus
(T2DM). Widespread use of PPARγ agonists has been prevented due to adverse effects including weight
gain, edema, and increased risk of congestive heart failure. Selective PPARγ modulators (SPPARγMs)
have been identified that have antidiabetic efficacy and reduced toxicity in preclinical species. In comparison
with PPARγ full agonists, SPPARγM 6 (MK0533) displayed diminished maximal activity (partial agonism)
in cell-based transcription activation assays and attenuated gene signatures in adipose tissue. Compound 6
exhibited comparable efficacy to rosiglitazone and pioglitazone in vivo. However, with regard to the induction
of untoward events, 6 displayed no cardiac hypertrophy, attenuated increases in brown adipose tissue, minimal
increases in plasma volume, and no increases in extracellular fluid volume in vivo. Further investigation of
6 is warranted to determine if the improvement in mechanism-based side effects observed in preclinical
species will be recapitulated in humans.
Introduction
realized due to undesirable weight gain, peripheral edema, and
anemia (resulting from increased plasma volume) following
prolonged usage.1,2 These adverse effects have precluded dosing
TZDs to achieve maximal clinical efficacy and ruled out their
wider usages. Indeed, TZDs are contra-indicated for T2DM
patients who are at risk for congestive heart failure because the
fluid retention they induce can exacerbate this disease.3 Similar
side effects have been observed in rodents as well,4 and more
recently additional concerns have been raised over generally
increased cardiovascular risk5,6 and possible loss of bone mass
following TZD treatment.7,8
Type 2 diabetes mellitus (T2DMa) is a metabolic disorder
characterized by insulin resistance and hyperglycemia. Histori-
cally, the disease has been associated with obesity resulting from
the sedentary western lifestyle. However, the incidence of T2DM
is quickly emerging as a global medical concern. If the incidence
of T2DM continues unchecked at its current rate, the World
Health Organization predicts there will be 300 million cases
worldwide by the year 2010. While lifestyle changes would
significantly ameliorate the risk or severity of this disease state,
compliance with existing therapies remains a long-standing
problem owing to generally poor tolerability of most T2DM
targeted drugs. One class of effective chemotherapeutic agents
for T2DM is the thiazolidinediones (TZDs, e.g., rosiglitazone
and pioglitazone). These compounds function as sensitizers of
endogenous insulin via activation of the peroxisome proliferator
activated receptor gamma (PPARγ). PPARγ is a member of
the nuclear hormone receptor super family. There are two
PPARγ subtypes (PPARγ1/PPARg2). PPARγ1 is expressed at
low levels in many tissues, while PPARγ2 is highly expressed
in adipocytes. The full potential of these drugs has not been
Considering the beneficial insulin sensitizing effects of TZD
therapy, a significant effort has been devoted to the development
of selective PPARγ modulators (SPPARγMs) that retain the
desirable efficacy for type 2 diabetes but have diminished
potential for, or ideally lack all together, the undesirable PPARγ
mechanism-based side effects.3,9-18 Herein, we describe our
efforts to identify an optimized indole-derived SPPARγM (6,
MK-0533) that demonstrates a reduced potential to cause
increases in heart weight, plasma volume, and extracellular fluid
volume and is equally efficacious to rosiglitazone and piogli-
tazone in its ability to improve diabetes in animal models.
SPPARγM 6 has an ancillary profile suitable to allow its
evaluation of in man.
* To whom correspondence should be addressed. Phone: 732 594 7592.
Fax: 732 594 9556. E-mail: blair_wood@merck.com.
†
Current address: Lilly Research Laboratories, Lilly Corporate Center,
In Vitro SAR and Synthesis. We recently have disclosed a
preliminary summary describing a structurally novel series of
SPPARγMs.19,20 A thorough investigation of the SAR revealed
that the key features exemplified in Figure 1 were critical for
SPPARγM activity. These SPPARγMs are potent partial
agonists in cell-based transcriptional activity assays that, in
DC 0545, Indianapolis, Indiana 46285.
a Abbreviations: PPARγ, peroxisome proliferator-activated receptor γ;
T2DM, type 2 diabetes mellitus; SPPARγM, selective peroxisome prolif-
erator-activated receptor γ modulator; TZD, thiazolidinedione; CYP,
cytochrome P450; SPA, scintillation proximity assay; PV, plasma volume;
ECF, extracellular fluid; TG, triglyceride; FFA, free fatty acid; HW, heart
weight.
10.1021/jm900097m CCC: $40.75  2009 American Chemical Society
Published on Web 06/09/2009

MK-0533: A NoVel SelectiVe PPARγ Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3847
primarily inhibition of simvastatin acid glucuronidation. The
comparatively high systemic plasma exposures required for
useful in vivo efficacy raised concerns for potential drug-drug
interactions with simvastatin, therefore more scaffold optimiza-
tion ensued.
The reversed indole platform offered greater opportunity to
probe modifications to the carbonyl/benzisoxazoyl region.
Introducing an aryl ether in place of the benzoyl or benzisox-
azoyl group afforded potent partial agonists in vitro and in vivo.
However, 3-phenoxy indole 5 was poorly tolerated in vivo with
unacceptable increases in liver enzymes (e.g., ALT and AST)
detected after 2 weeks dosing in a rat tolerability assay.
Ultimately, a desirable ancillary profile was realized after
revisiting the reversed indole scaffold containing a 3-benzoyl
substituent. An improved pharmacokinetic profile could be
obtained by excising the methylene moiety and directly attaching
the phenyl ring to the nitrogen of the indole ring system to yield
compound 6. Compound 6 was a potent PPARγ selective partial
agonist in vitro, was not a potent inhibitor of the P-450 enzymes
(>10 uM against all CYPs), was not a time-dependent inhibitor
of CYP3A4, and demonstrated low irreversible protein binding
in vitro (<10 pmol/mg protein in rat and human liver mi-
crosomes) and in vivo in rat (<10 pmol/mg protein in plasma
and liver).22
Figure 1. Summary of SAR of the indolyl SPPARγMs.
comparison with full agonists, produce a diminished gene
signature in 3T3-L1 adipocytes and display a decreased ability
to induce adipogenic differentiation of cultured rodent and
human preadipocytes.21 The observation that full and partial
agonists interact differently with PPARγ suggests that ligand-
receptor complexes with SPPARγMs may show quantitatively
different interactions with coactivators and corepressors, thereby
providing a physical basis for their differing pharmacological
profiles. However, the ancillary profiles of the compounds
disclosed to date were ill suited to merit clinical investigation
of these compounds in type 2 diabetic subjects. Thus, refining
the search for optimized SPPARγMs with more suitable drug-
like ancillary profiles was a necessary prerequisite prior to
considering evaluation of the potential benefits of these com-
pounds in type 2 diabetic subjects.
SPPARγM 6 was evaluated in vitro in PPAR assays from
multiple species and was found to be a potent ligand for
hPPARγ in SPA-based assays and a partial agonist of PPARγ
in cell-based transcriptional assays with high selectivity for
PPARγ over PPARR (Table 2). Using the human isoforms, >600
times more selectivity was observed between PPARγ versus
PPARR. In preclinical species, 6 also demonstrated good
selectivity between PPARγ and PPARR (>48 times) in vitro.
All the compounds described here demonstrated a high degree
of selectivity over PPARδ subtype. No activity was observed
for this receptor at the highest concentration tested (10 µM) in
the binding assay.
With the structural elements for potent in vitro SPPARγM
activity identified, we turned our attention to the optimization
of the ancillary profile of these molecules. A major liability of
the N-benzoyl indoles was significant loss of the labile anisoyl
group after oral dosing in rats. This liability was exacerbated,
as the resultant indole metabolites were potent CYP inhibitors
and found to accumulate after multiple dosing, raising the risk
for significant drug-drug interaction liabilities (Figure 2).
As the carbonyl oxygen of 1 was integral for binding potency,
fused heterocycles were evaluated as possible isosteric replace-
ments for the benzoyl group. Extensive modification of each
series to maximize intrinsic activity and introduce additional
desirable, drug-like properties was undertaken; the following
compounds (2-6) exemplify optimal derivatives from a given
cohort. For instance, a benzisoxazole proved to be a suitable
replacement for the anisoyl moiety. Although the optimized
N-benxisoxazole SPPARγM 2 was equipotent to the benzoyl
indoles in vitro and in vivo, low levels of the parent indoles
lacking N-substitution (similar to 1a) were still detected
systemically in plasma after orally dosing compounds such as
2. Therefore, further scaffold refinements were investigated to
avoid this troubling P-450 liability. 1,3-Transposition of the
indole nucleus such that the benzoyl group was connected via
a carbon-carbon bond at the 3-position of the indole provided
analogue 3, which is now incapable of debenzoylation in vivo.
This reversed indole maintained potent partial agonist activity
in vitro. However, 3 was found to have poor bioavailability,
low systemic plasma exposure, high clearance, and a short half-
life in preclinical species. In an effort to improve its pharma-
cokinetic profile, additional modification of the reversed indole
platform was initiated. Hybrid analogues were synthesized in
which the benzisoxazole functionality was incorporated onto
the 3-position of the transposed indole core. Such analogues,
typified by 4, while demonstrating good in vitro potency with
improved pharmacokinetic characteristics overall had compara-
tively modest efficacy in rodent models of diabetes. While in
general terms, this modification was viable, hybrid analogue 4
subsequently was found to have undesirable off-target activities,
We have also evaluated SPPARγM 6 in gene expression
studies performed in fully differentiated 3T3-L1 adipocytes
using Agilent 25K spotted microarrays. At PPARγ saturating
concentrations, SPPARγM 6 showed an overall similar but
attenuated gene expression profile compared with the PPARγ
full agonists rosiglitazone and non-TZD carboxylic acid PPARγ
agonist (COOH).23 No robust SPPARγM 6 specific signature
genes were observed (Figure 3). A PPARγ activity score was
developed using a ꢀ-square fitting approach and a set of 4619
reporter genes in order to provide a single relative measure of
the magnitude of their expression and therefore PPARγ activa-
tion.23 These 4619 reporter genes represent the intersection of
the two structurally distinct PPARγ full agonists rosiglitazone
and COOH at p < 0.01, which we believe represents a “true”
PPARγ target gene signature (both directly and indirectly
regulated genes) in the adipocytes. While the activity scores of
full agonists were indistinguishable (both rosiglitazone and
COOH have scores of 1.0), the activity score of SPPARγM 6
was 0.62 (Table 2, last entry) due to the attenuated PPARγ gene
expression profile relative to that of rosiglitazone.
SPPARγM 6 was prepared in good overall yields and high
enantiopurity utilizing the route outlined in Scheme 1. The core
indole was prepared via a Gassman indole synthesis24 to provide
8 in 40-60% isolated yields, subsequent to removal of
the undesired regioisomer. N-arylation of 8 with 3-bromoanisole
was achieved using a catalytic Pd/ligand cocktail to afford 10
in high yields after demethylation with boron tribromide.
Acylation at the 3-position with anisoyl chloride and diethyl

3848 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Acton et al.
Figure 2. N-Acyl indoles are metabolically labile in vivo.
Table 1. Human PPARγ Binding IC50 and Transactivation EC50 Values
compd
IC50 (nM)a
EC50 (nM)b
% max activation
features
full agonist
labile benzoyl
labile benzisoxazole
poor PK profile
off-target activity
poor rodent tolerabilty
optimized scaffold
rosiglitazone
∼200
20-27
100
36
31
25
24
20
25-39%
1
2
3
4
5
6
1
6
1
2
1
9
2
3
1
3
3
2
a IC50 is measured by the complete displacement of a radio-labeled full agonist indicating competitive binding to the receptor in a SPA assay format.
b EC50 is defined as the compound concentration at which 50% of a given compound’s intrinsic maximal response has been reached.
Table 2. Comparison of SPPARγM 6 and Rosiglitazone in Vitro
Potencies across Species and PPARγ Activity Score Based upon Gene
Expression in 3T3-L1 Adipocytes
resistant rodent model of T2DM characterized by hyperglycemia
and hypertriglyceridemia.26 Its in vivo efficacy was compared
to rosiglitazone and pioglitazone as positive controls; the dosage
of each agent was selected such that comparable efficacy was
obtained in all treatment groups. After once daily oral dosing
for 11 days, compound 6 produced a significant reduction in
blood glucose levels (-83%). Rosiglitazone and pioglitazone
performed similarly, affording glucose decrements of 75% and
50%, respectively. On the final day of dosing, the systemic
plasma exposures of each compound were measured. It is
noteworthy that despite the in vitro partial agonist activity in
transactivation assays and attenuated gene signature in adipo-
cytes relative to rosiglitazone, based upon the systemic plasma
drug exposure, SPPARγM 6 was roughly 3-5 times more
potent than pioglitazone or rosiglitazone in this diabetic animal
model (Table 4). Plasma levels of adiponectin, a bona fide
PPARγ target engagement biomarker, also were measured in
the rosiglitzone and SPPARγM treatment groups. Significant
increases in adiponectin were observed in both treatment groups.
in vitro potencies (nM)
SPPARgM 6
rosiglitazone
hPPARγ (IC50)a b
9-14
∼200
,
hPPARR (IC50)b
>50000
>50000
hPPARγ (EC50)c
2-5 (42-68%)
33-68% @ 3000
240-340 (53-64%)
330-440 (52-80%)
370 (35-62%)
0.6
20-27
1-4% @ 3000
na
na
na
1.0
hPPARR (EC50)c
murine PPARR (EC50)c
hamster PPARR (EC50)c
dog PPARR (EC50)c
PPARγ activity score
a PPARγ binding is similar across all species evaluated. Only human
binding data is presented for clarity. b IC50 is measured by the complete
displacement of a radio-labeled full agonist indicating competitive binding
to the receptor in a SPA assay format. c EC50 is defined as the compound
concentration at which 50% of a given compound’s intrinsic maximal
response has been reached.
aluminum chloride followed by a basic workup of the reaction
provided intermediate 11. Coupling the phenol with tert-butyl
(S)-2-hydroxy butyrate afforded the desired acid 6 in high yields
and high enantiomeric excess after removal of the tert-butyl
ester with trifluoroacetic acid (Scheme 1).25
Tolerability in Sprague-Dawley Rats. Recently, reports
have emerged that rosiglitazone causes increased risk of
cardiovascular complications after prolonged usage in humans.5,6
Also, thiazolidinediones have been associated with cardiac
hypertrophy in a number of preclinical species.27 To evaluate
if the partial agonist activity would translate into a different in
vivo profile, SPPARγM 6 was compared head-to-head with
rosiglitazone in Sprague-Dawley rats. Both agents were dosed
daily for 2 weeks at dosages to achieve exposures that were
Pharmacokinetics. Overall, 6 was found to have desirable
pharmacokinetic characteristics in preclinical species with low
to moderate plasma clearance, moderate to good oral bioavail-
ability, and acceptable terminal half-life (Table 3).
Efficacy in db/db Mice. To evaluate its antidiabetic efficacy
in vivo, SPPARγM 6 was dosed in db/db mice, an obese insulin

MK-0533: A NoVel SelectiVe PPARγ Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3849
Figure 3. Comparison of rosiglitazone, COOH, and SPPARγM 6 gene expression heat map of 4619 reporter genes.
Scheme 1. Synthesis of SPPARγM 6
Table 3. Pharmacokinetic Profile of SPPARγM 6 in Preclinical Species
Table 4. Evaluation of SPPARγM 6 in db/db Mice and
Sprague-Dawley Ratsa
PO nAUC
Clp
T1/2
Cmax
Vdss
species
% F
(µM·h)
(mL/min/kg)
(h)
(µM) (L/kg)
db/db mouse
rat
dog
monkey
81
59
25
1.6
2.9
0.8
18.3
7.5
10.1
1.6
3.6
3.2
1.0
2.4
0.5
1.4
1.4
0.7
compd
vehicle
rosiglitazone
6
pioglitazone
dose (mpk)
AUC (µM·h)
gluc. correction
(%)
adiponectin
(µg/mL)
10
320
-79 ( 10
30
60
75
205
-50 ( 11
-83 ( 4
about 10 times greater than the dosages administered to db/db
mice in efficacy studies. In contrast to rosiglitazone, which
produced the expected increases in heart weight (25%) and
brown adipose tissue (BAT, 260%), there was no significant
increase in heart weight observed in animals dosed with
SPPARγM 6. Another distinguishing characteristic was the
diminished augmentation in BAT weight by 6 in comparison
to rosiglitazone. Overall, SPPARγM 6 was well tolerated and
there were no significant findings other than a nominal increase
in liver weight (120%) comparable to that observed with the
PPARR agonist fenofibrate, which was also included as a
control. The increases in liver weight after treatment with 6
reflect the minor murine PPARR activity present with this
compound (Table 4).
14.5 ( 1.7 57.3 ( 3.1b 48.1 ( 6.8b
n.d.d
Sprague-Dawley rat
compound
vehicle
rosiglitazone
6
fenofibrate
dose (mpk)
AUC (µM·h)
heart (g)
150
581
300
954
300
5582
1.12 ( 0.03 1.39 ( 0.07b 1.10 ( 0.03 1.16 ( 0.04
9.0 ( 0.4
263 ( 13
liver (g)
10.5 ( 0.8
19.7 ( 0.9b 18.9 ( 0.3b
537 ( 35b
276 ( 41
BATc (mg)
944 ( 145b
a Values are the means of six animals/group ( the standard error of the
mean. b P < 0.05 compared to vehicle controls (Dunnett’s t test). c BAT )
brown adipose tissue. d nd ) not determined.
used as surrogate markers for induction of edema by PPARγ
agonists.23 Briefly, changes in plasma volume are determined
by Evan’s blue dye dilution, while ECF volume changes are
determined by bioelectrical impedance. Although not frankly
diabetic, insulin resistant fa/fa Zucker rats are hyperinsulinemic
and dyslipidemic. Therefore, the use of this model makes it
possible to evaluate both the insulin sensitizing and mechanism-
based side effects of PPARγ ligands in the same model.
Evaluation of Plasma Volume and Extracellular Fluid
Volume Increases in fa/fa Zucker Rats. On the basis of its
robust efficacy in db/db mice and the unique tolerability profile
in Sprague-Dawley rats, further in vivo characterization of
SPPARγM 6 was undertaken. To evaluate its potential for
mechanism-based side effects, we examined two parameters in
obese fa/fa Zucker rats, plasma volume (PV) and extracellular
fluid volume (ECF) augmentation, that have previously been

3850 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Acton et al.
Figure 4. Comparison of TG, insulin, and FFA lowering between rosiglitazone and SPPARγM 6 in Zucker fa/fa rats. * P < 0.05 compared to
vehicle controls (Dunnett’s t test).
Figure 5. Comparison of PV, ECF, and HW increases between rosiglitazone and SPPARγM 6 in Zucker fa/fa rats. * P < 0.05, ** P < 0.01
compared to vehicle controls (Dunnett’s t test).
Following 7 days of treatment with either rosiglitazone or 6
in Zucker fa/fa rats, the final body weights of treatment groups
were not statistically different from those of fa/fa vehicle
controls. In general, body-weight gain correlated well with food
consumption over the study period regardless of treatment group.
Plasma glucose levels were not elevated in the vehicle control
fa/fa Zucker rats and were unaffected by rosiglitazone or
SPPARγM 6. Plasma trigylcerides (TG), insulin, and free fatty
acids (FFA) were significantly reduced by rosiglitazone, and
SPPARγM 6 indicative of diminished insulin resistance. As
shown in the graphs, rosiglitizone affords maximal reductions
in plasma TG, insulin, and FFA at 10 mpk with no further
decreases upon increasing the dosage to 100 mpk. Similar
reductions in TG, insulin, and FFA are observed at the 30 mpk
dosage of SPPARγM 6. At the end of the study period, systemic
plasma drug levels were measured at each dosage. The
exposures for rosiglitazone were 9.0, 61.1, 583.7, and 6458
µM·h at 0.1, 1, 10, and 100 mg/kg, respectively. The plasma
exposures for SPPARγM 6 were 6, 115.2, 1475, and 4522 µM·h
at 3, 30, 100, and 150 mg/kg, respectively. Compound dose
ranges were selected based upon differences in mouse and rat
PK as well as the dosage needed for efficacy in db/db mouse.
On an exposure basis, SPPARγM 6 is 5 times more potent that
rosiglitazone when dosed to achieve maximal reductions in TG,
insulin, and FFA. This is consistent with the findings in db/db
mice (Figure 4).
Summary
Herein we describe the identification of a novel indole-derived
SPPARγM. In contrast to full agonists, SPPARγM 6 was a
partial agonist in cell-based transcriptional activity assays and
produced an attenuated gene signature in 3T3-L1 adipocytes.
In a widely accepted rodent model of diabetes, db/db mice,
SPPARγM 6 exhibited comparable efficacy to rosiglitazone and
pioglitazone. It also showed similar insulin sensitizing activity
to PPARγ full agonists in insulin resistant fa/fa Zucker rats.
However, with regard to the induction of adverse events,
SPPARγM 6 displayed a number of important benefits vs
rosiglitazone. No cardiac hypertrophy was seen, and only
attenuated increases in brown adipose tissue were noted after
treating Sprague-Dawley rats chronically with high dosages
of 6. In Zucker fa/fa rats, SPPARγM 6 treatment produced only
a trend toward increased plasma volume that plateaued with
increasing doses, while concomitant increases in extracellular
fluid or heart weight were not observed. The robust preclinical
antidiabetic activity and superior ancillary pharmacology of
SPPARγM 6 is sufficient to merit further investigation of its
properties and to determine if the improvement in mechanism-
based side effects observed in preclinical species will be
recapitulated in humans.
Experimental Section
General. All commercial chemicals and solvents were reagent
grade and were used without further purification unless otherwise
specified. All reported yields were isolated yields after chroma-
tography or crystallization. NMR spectra were obtained on a Varian
Inova 500 MHz NMR spectrometer equipped with either a Nalorac
3 mm probe or a Varian indirect detection 3 mm probe. All spectra
run in CDCl3 were referenced to residual CHCl3 at δ 7.26 ppm.
Standard resolution mass spectra were obtained on HP1100 LC/
MS systems; all mass spectra were obtained using electrospray
ionization (EI) in positive ion mode. An LCT time-of-flight mass
spectrometer (Waters, Beverly MA) with LockSpray electrospray
ionization source was used for exact mass analysis. Chemical purity
and enantiopurity was confirmed by HLPC to be >95%. Purity was
determined by HPLC analysis via gradient elution (10%-100%
acetonitrile/water modified with 0.1% TFA over 3 min with a 30 s
Significant increases in PV, ECF volume, and heart weight
(HW) were observed at the 10 mpk dosage of rosiglitazone,
and further increases in these parameters occur at the 100 mpk
dosage. After treatment with SPPARγM 6, no significant
increase in PV was observed at the 30 mpk dosage. However,
there was a modest increase in PV with SPPARγM 6 at 100
mpk, but in contrast to rosiglitazone, this augmentation reached
a plateau upon increasing the dosage up to 150 mpk. Further-
more, SPPARγM 6 did not produce any significant increases
in ECF or HW at any dosage evaluated. On the basis of these
findings, we conclude that SPPARγM 6 afforded efficacy
comparable to rosiglitazone with reduced potential for mech-
anism-based side effects (Figure 5).

MK-0533: A NoVel SelectiVe PPARγ Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3851
hold at 100% acetonitrile followed by a return to 10% acetonitrile/
water in 30 s at 5 mL/min) on a YMC C8 column (5 µm) at λ )
220 nm. Chiral purity determined by treating (6) with TMS-
diazomethane and isolating the methyl ester. Chiral HPLC analysis
of the methyl ester was performed using 85% heptane/15% EtOH
as eluent on a Chiralpak AD column (4.6 mm × 250 mm) at a
flow rate of 0.75 mL/min using a photodiode array detector, plus
single wavelength analysis at 254 nm. The methyl ester of racemic
(6) was also prepared for comparison.
2-Methyl-6-(trifluoromethoxy)-1H-indole (8) step 1. To a
vigorously stirred solution of 3-trifluoromethoxyaniline (7, 5.74 g,
0.032 mol) in methylene chloride (100 mL) at -78 °C (acetone-dry
ice bath) was added dropwise a solution of tert-butyl hypochlorite
(3.5 g, 0.032 mol) in 15 mL of the same solvent. After 10 min, a
solution of methylthio-2-propanone (3.375 g, 0.032 mol) in
methylene chloride (15 mL) was added slowly over 10 min. A slight
exotherm was noted during the addition, resulting in a clear yellow
solution. After stirring for another hour at -78 °C, during which
time the intermediate azasulfonium salt precipitated to form a
suspension, a solution of triethylamine (4.5 mL, 0.032 mol) in
methylene chloride (15 mL) was added slowly over 5 min to give
a clear solution. After an additional 10 min at -78 °C, the cooling
bath was removed and the reaction was allowed to warm to room
temperature. A 50 mL portion of water was added under vigorous
stirring, and the organic layer was separated, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to give 8.75 g of a 2:1
mixture of the desired 6-triluoromethoxy-2-methyl-3-methylthio-
indole and the corresponding 4-trifluoromethoxy isomer as a yellow
oil. The crude product was used in the next step without further
purification. LC-MS (M + 1) 262.
the drying agent, the filtrate was evaporated and the residue purified
by silica gel chromatography to give 397 mg (90%) of the title
compound. H NMR (500 MHz, CDCl3): δ 7.50 (d, Ph, J ) 8.5
Hz, 1H), 7.41 (t, Ph, 1H), 6.99 (d, Ph, J ) 8.5 Hz, 1H), 6.97 (s,
Ph, 1H), 6.94 (dd, ph, J ) 8.2, 2.5 Hz, 1H), 6.91 (dd, Ph, J ) 7.5,
1.4 Hz, 1H), 6.81 (t, Ph, 1H), 6.38 (s, Ph, 1H), 5.02 (s, OH, 1H),
2.30 (s, 2-CH3, 3H).
1
[1-(3-Hydroxyphenyl)-2-methyl-6-(trifluoromethoxy)-1H-in-
dol-3-yl](4-methoxyphenyl)methanone (11). First, 242 mg (0.788
mmol) of (10) was dissolved in methylene chloride (4 mL) and
cooled to -20 °C. Then a solution of diethylaluminum chloride in
toluene (1.8M, 1.23 mL) was added slowly (over 1-2 min) and
stirred for 5-15 min. Then added a solution of 4-methoxybenzoyl
chloride (377 mg, 2.21 mmol) in methylene chloride (1 mL) and
allowed to stir overnight while slowly reaching room temperature.
pH 7.0 buffer was added dropwise until gas evolution ceased and
then partitioned. The aqueous layer was extracted twice more with
methylene chloride, and then the combined organic layers were
washed twice with saturated NaCl solution, dried over sodium
sulfate, filtered, and evaporated. The crude isolate was then
dissolved in methanol (5 mL), and sodium hydroxide solution (1.0
M, 1.6 mL) was added. The solution was monitored by TLC for
disappearance of diacyl indole and then neutralized with HCl (1.0
M, 1.6 mL). The reaction mixture was then diluted with water and
extracted with ethyl acetate. The ethyl acetate layer was dried over
sodium sulfate, filtered, evaporated, and the residue purified by silica
gel chromatography to give 222 mg (64% yield) of the title
1
compound. H NMR (500 MHz, CDCl3): δ 7.83 (d, Ph, J ) 8.8
Hz, 2H), 7.45 (t, Ph, 1H), 7.41 (d, Ph, J ) 8.6 Hz, 1H), 7.05 (dd,
Ph, J ) 8.8, 2.4 Hz, 1H), 6.97 (m, Ph, 3H), 6.94 (s, Ph, 1H), 6.91
(dd, Ph, J ) 8.5, 1.4 Hz, 1H), 6.86 (t, Ph, 1H), 6.38 (s, OH, 1H),
3.91 (s, OCH3, 3H), 2.35 (s, 2-CH3, 3H). HRMS: m/e 442.1267
(M + 1) calcd (M + 1) 442.1266.
2-Methyl-6-(trifluoromethoxy)-1H-indole (8) Step 2. A solu-
tion of the crude product from the previous step (8.8 g, 0.032 mol)
in 250 mL of absolute ethanol was placed in a 500 mL, three-
necked, round-bottomed flask fitted with a mechanical stirrer. An
excess of freshly washed W-2 Raney nickel (80 g, 50% slurry in
water, washed twice with water and three times with ethanol) was
added. The mixture was stirred for 4 h (the reaction was monitored
by LC-MS). The Raney nickel was removed by filtration through
celite and washing with ethanol. (CAUTION: W-2 Raney nickel
may ignite spontaneously if allowed to become dry. During the
filtration, a small amount of solvent must be left to cover the
catalyst.) The combined ethanolic solutions were concentrated in
vacuo. Purification by flash chromatography (Biotage 75 Flash, 9:1
hex/EtOAc) gave 4.05 g of the desired product as white solid
(58.1%) and 2.0 g of the 4-trifluoromethoxy indole isomer as an
tert-Butyl(2R)-2-(3-{3-[(4-methoxyphenyl)carbonyl]-2-methyl-
6-(trifluoromethoxy)-1H-indol-1-yl}phenoxy)butanoate (12).
First, 220 mg (0.500 mmol) of (11) was dissolved in tetrahydrofuran
(2.5 mL) and cooled to 0 °C. Then triphenylphosphine (170 mg,
0.650 mmol) and tert-butyl (S)-2-hydroxybutyrate (104 mg, 0.650
mmol) were then added, followed by diethylazodicarboxylate (102
µL, 0.650 mmol). The reaction was stirred overnight and then
directly purified by silica gel chromatography to give 275 mg of
the title compound. 1H NMR (500 MHz, CDCl3): δ 7.84 (d, Ph, J
) 8.9 Hz, 2H), 7.49 (t, Ph, 1H), 7.45 (d, Ph, J ) 8.7 Hz, 1H), 7.05
(br, Ph, 1H), 6.97 (m, Ph, 4H), 6.89 (br, Ph, 1H), 6.87 (br t, Ph,
1H), 4.50 (t, OCH(CH2CH3)CO2tBu, 1H), 3.90 (s, OCH3, 3H), 2.37
(s, 2-CH3, 3H), 2.01 (m, OCH(CH2CH3)CO2tBu, 2H), 1.47 (br s,
OCH(CH2CH3)CO2tBu, 9H), 1.10 (t, OCH(CH2CH3)CO2tBu, 3H).
LC/MS: m/e 584.12 (M + 1).
(2R)-2-(3-{3-[(4-Methoxyphenyl)carbonyl]-2-methyl-6-(trif-
luoromethoxy)-1H-indol-1-yl}phenoxy)butanoic Acid (6). First,
274 mg (0.470 mol) of (12) was dissolved in 4.7 mL of dichlo-
romethane. Then trifluoroacetic acid (2.4 mL, 31.1 mol) was added
and the reaction was stirred until TLC monitoring showed that the
reaction was complete. The volatiles were removed by rotary
evaporation. The residue was dissolved twice in dichloromethane/
hexanes and evaporated. The residue was pumped on high vacuum
for 12 h. It was purified on a silica gel column (70/30/0.33 hexanes/
ethyl acetate/acetic acid) to give clean title compound (236 mg,
95% yield) after isolation. 1H NMR (500 MHz, CDCl3): δ 7.84 (d,
Ph, J ) 8.7 Hz, 2H), 7.50 (t, Ph, 1H), 7.42 (br s, Ph, 1H), 7.08 (br
s, Ph, 1H), 6.970 (m, Ph, 4H), 6.92 (br m, Ph, 2H), 4.67 (br m,
OCH(CH2CH3)CO2H, 1H), 3.90 (s, OCH3, 3H), 2.38 (s, 2-CH3,
3H), 2.07 (m, OCH(CH2CH3)CO2H, 2H), 1.13 (t, OCH(CH2-
CH3)CO2H, 3H). LC/MS: m/e 528 (M + 1). HRMS: m/e 528.1614
(M + 1) calcd (M + 1) 528.1644. HPLC purity 99.7% (tR ) 2.316
min). Chiral purity 99.6% tR13.19 min, 0.1% tR 19.24 min (racemate
50/50 at tR 13.22 and 19.25 min, respectively).
1
oil (28.7%). H NMR (CDCl3, 500 MHz): δ 7.93 (br s, 1H), 7.45
(d, J ) 8.6 Hz, 1H), 7.16 (s, 1H), 6.95 (d, J ) 8.6 Hz, 1H), 6.23
(s, 1H), 2.45 (s, 3H). LC/MS: m/e 215.89 (M + 1). HRMS: m/e
216.0628 (M + 1) calcd (M + 1) 216.0636.
1-(3-Methoxyphenyl)-2-methyl-6-(trifluoromethoxy)-1H-in-
dole (9). 2-Methyl-6-(trifluoromethoxy)-1H-indole (8), 645 mg, 3.0
mmol), 3-bromoanisole (0.456 mL, 3.6 mmol), sodium t-butoxide
(404 mg, 4.2 mmol), trisdibenzylidine dipalladium (206 mg, 0.225
mmol), and 2-di-t-butylphosphinobiphenyl (201 mg, 0.675 mmol)
were stirred in toluene at 80 °C and monitored by TLC (3/1 hexanes/
methylene chloride) or reversed phase HPLC until complete. The
reaction mixture was then cooled, filtered over celite, and the filtrate
evaporated to give a crude isolate, which was purified by silica gel
chromatography to give the title compound (460 mg, 48% yield).
1H NMR (500 MHz, CDCl3): δ 7.51 (d, Ph, J ) 8.5 Hz, 1H), 7.45
(t, Ph, 1H), 7.02 (dd, Ph, J ) 8.4, 2.5 Hz, 1H), 6.99 (m, Ph, 2H),
6.92 (dd, Ph, J ) 8.3, 1.6 Hz, 1H), 6.86 (t, Ph, 1H), 6.39 (s, Ph,
1H), 3.85 (s, OCH3, 3H), 2.30 (s, 2-CH3, 3H). LC/MS: m/e 322.05
(M + 1).
3-[2-Methyl-6-(trifluoromethoxy)-1H-indol-1-yl]phenol (10).
First, 460 mg (1.43 mmol) of (9) was dissolved in 7 mL of
dichloromethane at 0 °C. Then boron tribromide (1.0 N, 2.86 mL)
in dichloromethane was added, the cooling bath was removed, and
the reaction was stirred at room temperature overnight. The reaction
was then quenched with ice for 30 min and partitioned. The organic
was washed with water and dried over sodium sulfate. After filtering
Preparation of Recombinant PPARs and Binding Assay.
Recombinant PPARs were prepared and the receptor binding assays
were performed as previously described.15 Briefly, the full length
human cDNAs for each was subcloned into pGEX-KT expression

3852 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Acton et al.
vectors (Pharmacia, Piscataway, NJ), followed by production of
purified recombinant proteins in Escherichia coli. Using the purified
GST-hPPAR receptors, scintillation proximity assay-based receptor
competitive binding assays were performed in Packard OptiPlate-
96 well polystyrene microplates (Packard BioScience, Meriden, CT)
using [3H2]nTZD3 for PPARγ and PPARR and [3H2]nTZD4 for
PPARδ. Kis were calculated by the equation of Cheng and Prusoff.28
Four-parameter logistic equation used is shown below:
1:1000 or 1:5000, 1, or 5 µL into 5 mL of 1× assay buffer. Each
sample was assayed in duplicate.
Assay Protocol. The assay utilized a Mouse Adiponectin RIA
kit (cat. no. MADP-60HK) purchased from LINCO Research, Inc.
St. Charles, MO. The assay was performed over 2 days and utilizes
125I-labeled murine adiponectin and a multispecies adiponectin rabbit
antiserum to determine the level of adiponectin in serum, plasma,
or tissue culture media by the double antibody/PEG technique. The
adiponectin standards were prepared using recombinant mouse
adiponectin and were used to determine the µg/mL circulating levels
of adiponectin in plasma samples.
(
)
Y ) Bottom + Top - Bottom /1 +
LogEC - X HillSlope (1)
(
)
50
Adiponectin Concentration Determination. The concentrations
of adiponectin in the plasma samples were determined from the
standard curve, which was linear from 1 to 100 ng/mL. Standards
are fit to a one site competition equation using GraphPad Prism
Software purchased from GraphPad Software, Inc., San Diego, CA.
Statistical Analysis. The unknown concentrations of adiponectin
determined by GraphPad Prism Software were exported to the CMG
(Comparing Multiple Groups) Guidance System statistical package,
provided by the Merck Biometrics Research Department. This
provided all statistical data, including point and box plot graphs,
along with pairwise comparison results.
Obese Zucker fa/fa Rat Studies. Nine-ten week old male
Zucker fa/fa rats (Charles River Laboratories, Wilmington, MA)
were housed two animals per cage and were provided food (Harlan
Teklad Diet no. 7012, Madison, WI) and water ad libitum. Animals
were housed in a temperature-, humidity-, and light-controlled room
(21-23 °C, 47-65%, 12-12 h light-dark cycle, n ) 8/group).
Following 7 days of acclimation, rats were treated once daily for
7 days by oral gavage with vehicle (0.25% methylcellulose, 10 mL/
kg), rosiglitazone (0.1, 1, 10, and 100 mg/kg), or SPPARγM 6 (3,
30, 100, and 150 mg/kg).
Bottom is the Y value at the bottom plateau, while Top is the Y
value at the top plateau. Log EC50 is the X value when the response
is halfway between bottom and top. HillSlope describes the
steepness of the curve (GraphPad Prism version 4.00 for Windows,
GraphPad Software, Inc., San Diego, CA).
Cell-Based Transcriptional Activity Assays. COS-1 cells were
cultured and transactivation assays were performed as previously
described.26 Briefly, cells were transfected with a pcDNA3-hPPAR/
GAL4 expression vector, pUAS(5X)-tk-luc reporter vector, and
pCMV-lacz as an internal control for transactivation efficiency using
Lipofectamine (Invitrogen, Carlsburg, CA). After a 48 h exposure
to compounds, cell lysates were produced, and luciferase and
ꢁ-galactosidase activity in cell extracts were determined. Inflection
points (EC50) of normalized luciferase activity were calculated by
the four-parameter logistic equation.
Gene Signature Profiling in 3T3-L1 Cells. 3T3-L1 cells were
grown to confluence in medium A (Dulbecco’s modified Eagle’s
medium with 10% fetal calf serum, 100 units/mL penicillin, and
100 µg/mL streptomycin) at 37 °C in 5% CO2 and induced to
differentiate as previously described.29 Briefly, differentiation was
induced by incubating the cells with medium A supplemented with
methylisobutylxanthine (IBMX), dexamethasone, and insulin for
2 days (days 1 to 2), followed by another 2-day incubation with
medium A supplemented with insulin (days 3 to 4). The cells were
further incubated in medium A for an additional 4 days to complete
the adipocyte conversion (days 5 to 8). At day 8 following the
initiation of differentiation, cells were incubated in medium A (
compounds for 24 h at saturating concentrations: vehicle, rosigli-
tazone (600nM), COOH (3870 nM), or SPPARγM6 (1075 nM).
The doses were chosen to be 1.5 log the inflection point in the
fatty acid binding protein adipogenesis assay performed in 3T3-
L1 cells. Following treatment, total RNA was prepared from the
adipocytes using Trizol reagent (Invitrogen, Carlsbad, CA) and
RNeasy kits (Qiagen, Valencia, CA) according to the manufacturers’
instructions and RNA concentration was estimated from absorbance
at 260 nm. Microarray processing was performed as previously
described.30,31 Briefly, labeled cRNA was hybridized for 48 h onto
Agilent 60mer 2-color spotted microarrays. Individual treatment
samples (including individual vehicle treatment samples) were
hybridized against a vehicle treatment pool. LogRatio and P values
were generated by averaging replicates (3 replicates per treatment)
and the Rosetta Resolver v7.2 system (Rosetta Inpharmatics LLC,
a wholly owned subsidiary of Merck and Co., Inc., Seattle, WA).
LogRatio values represent the difference in regulation of the
compound treated samples versus the vehicle treated sample where
a positive value signifies up-regulation following compound treat-
ment and vice versa. A PPARγ activity score indicative of overall
adipoctye gene expression was generated using a subset of 4619
reporter genes and the ꢀ-square fitting approach, as previously
described.23 This subset of reporter genes is composed of the
intersection of the two PPARγ full agonists, rosiglitazone and
COOH signature reporter genes with P < 0.01.
Evaluation of Insulin Sensitization. Tail nick blood samples
were collected one day before the start of dosing and one day after
seventh dose and plasma glucose (Sigma glucose Trinder assay kit,
St. Louis, MO), insulin (rat insulin RIA from American Laboratory
Products Company, Windham, NH), free fatty acids (FFA) and
triglyceride (both from Roche Diagnostics, Basel, Switzerland)
concentrations were determined.
Bioelectrical Impedance Analysis for Determination of Ex-
tracellular Fluid Volume. Twenty-four hours following the final
dose, rats were anesthetized with ketamine (85 mg/kg, im) and
xylazine (10 mg/kg, im). Animals were positioned on a noncon-
ductive surface in dorsolateral recumbency and extracellular fluid
volume was determined by bioelectrical impedance analysis,
following procedures described by the manufacturer (Hydra ECF/
ICF impedance analyzer model 4, Xitron, San Diego, CA) and by
B. H. Cornish et al.32 and Rutter et al.33 A tetrapolar impedance
monitor was used to measure impedance, and hence the total body
water, over a frequency range of 5 kHz to 1 MHz. Source electrodes
(1 cm × 26G stainless steel needles) were inserted 5 mm
subcutaneously. Detector electrodes were inserted along the midline
at the anterior point of the sternum and the anterior point of the
penis. The distance between electrodes was measured and included
for data modeling.
Plasma Volume Measurement. After determination of extra-
cellular fluid volume, plasma volume was measured in anesthetized
animals using a dye dilution technique following methods described
by Belcher and Harriss34 with minor modifications. Evans blue dye
solution (25 mg/mL in physiological saline) was filtered through a
0.22 µm filter prior to injection into a femoral vein. Twenty minutes
after injection, a heparinized blood sample (2 mL) was withdrawn
from the descending aorta. Plasma was separated by centrifugation
of the blood at 1100g for 15 min.; samples were kept at -80 °C
until assayed. Absorbance of the thawed plasma was read at 620
nm, and plasma Evans blue dye concentrations were calculated
according to a standard curve generated by a serial dilution of the
25 mg/mL Evans blue dye-saline solution. Plasma volume was
calculated by using the dilution factors of Evans blue as shown
below.
Glucose Measurements. db/db mice were used as described
previously.28
Adiponectin Measurements. Serum/Plasma Sample Han-
dling. Serum 5-15 µL per sample of plasma were received. Vehicle
was diluted 1:1000 µL into 5 mL of 1× assay buffer (10 mM
phosphate buffer, pH 7.6 containing 0.08% sodium azide, 0.1%
RIA grade BSA), and all full and partial agonists were diluted either

MK-0533: A NoVel SelectiVe PPARγ Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3853
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[dye]in plasma (2)
Heart Weight Measurements. Animals were then euthanized
by pneumothorax and exsanguination, and the heart was excised,
blotted, and weighed.
Plasma Drug Level Measurements. All plasma samples col-
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SPPARγM 6 measurements.
Animal Care and Handling. All animal experiments and
euthanasia protocols were conducted in strict accordance with the
National Research Council’s Guide for the Care and Use of
Laboratory Animals. Animal experiment protocols were reviewed
and approved by the Institutional Animal Care and Use Committee
of Merck Research Laboratories. The laboratory animal facilities
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for Assessment and Accreditation of Laboratory Animal Care
International.
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