Benzoxazinones as PPARγ Agonists. 2. SAR of the Amide Substituent and In Vivo Results in a Type 2 Diabetes Model

Article abstract of DOI:10.1021/jm0301888

A series of benzoxazinones has been synthesized and tested for PPARγ agonist activity. Synthetic approaches were developed to provide either racemic or chiral compounds. In vitro functional potency could be measured through induction of the aP2 gene, a target of PPARγ. These studies revealed that compounds with large aliphatic chains at the nitrogen of the benzoxazinone were the most potent. Substitution of the chain was tolerated and in many cases enhanced the in vitro potency of the compound. Select compounds were further tested for metabolic stability, oral bioavailability in rats, and efficacy in db/db mice after 11 days of dosing. In vivo analysis with 13 and 57 demonstrated that the series has potential for the treatment of type 2 diabetes.

Full text of DOI:10.1021/jm0301888

196
J . Med. Chem. 2004, 47, 196-209
Ben zoxa zin on es a s P P ARγ Agon ists. 2. SAR of th e Am id e Su bstitu en t a n d In
Vivo Resu lts in a Typ e 2 Dia betes Mod el
Philip J . Rybczynski,* Roxanne E. Zeck, J oseph Dudash, J r., Donald W. Combs, Thomas P. Burris,† Maria Yang,
Melville C. Osborne, Xiaoli Chen, and Keith T. Demarest
J ohnson and J ohnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202, Raritan, New J ersey 08869
Received April 21, 2003
A series of benzoxazinones has been synthesized and tested for PPARγ agonist activity.
Synthetic approaches were developed to provide either racemic or chiral compounds. In vitro
functional potency could be measured through induction of the aP2 gene, a target of PPARγ.
These studies revealed that compounds with large aliphatic chains at the nitrogen of the
benzoxazinone were the most potent. Substitution of the chain was tolerated and in many
cases enhanced the in vitro potency of the compound. Select compounds were further tested
for metabolic stability, oral bioavailability in rats, and efficacy in db/db mice after 11 days of
dosing. In vivo analysis with 13 and 57 demonstrated that the series has potential for the
treatment of type 2 diabetes.
In tr od u ction
Natural ligands of PPARγ have been identified. These
endogenous activators include mono- and polyunsatu-
rated fatty acids as well as eicosanoids, with EC50 values
in the micromolar range.9 To date, the most potent
natural agonist identified is 15d-PGJ 2 with a reported
EC50 ) 1-2 µM in cotransfection assays using PPARγ
chimera.10 By comparison, a recent report identified
Saurufuran A from the herb Suarus chinesis as an
agonist with an EC50 ) 16.7 µM in a pFA-GAL4-PPAR
chimera expression construct.11 These and other natural
ligands are considerably less potent than synthetic
ligands (vide infra).
PPARγ is a member of the peroxisome proliferator-
activated receptor family. Its mechanistic role in glucose
and lipid homeostasis has been the subject of extensive
research.1 As a result, PPARγ agonism is a current
treatment for type 2 diabetes. The receptor is widely
distributed in the spleen, colon, adipose tissue, and
macrophages and found to a lesser extent in the liver,
pancreas, and skeletal muscle.2 Activation of PPARγ in
the cell nucleus initiates heterodimerization with an-
other nuclear receptor, the rexinoid receptor (RXR), with
subsequent recruitment of coactivators and induction
of genes that are involved in adipogenesis. Target genes
that are upregulated or downregulated have been
identified from white and brown adipose tissue, skeletal
muscle, and the liver3 (in vitro adipogenesis can be
induced by activation of PPARγ alone or in conjunction
with C/EBPR, although the latter is not sufficient to
promote adipogenesis4). The details of how activation
leads to glucose homeostasis are not fully understood.
Studies suggest that adipogenesis provides increased
lipid metabolism and free fatty acid uptake in adipose
tissue, leading to increased insulin sensitivity and
glucose metabolism in muscle and liver.1a,5,6 In support
of this mechanism, recent evidence shows that a PPARγ
agonist induces glycerol kinase gene expression in
adipocytes, thus promoting triglyceride formation in
that tissue and reducing circulating free fatty acids.7
Alternatively, altered secretion of adipocytokines in
adipose tissue has been proposed as a mechanism of
PPARγ agonist-mediated homeostasis.5 A conflicting
report demonstrates, however, that in heterozygous
PPARγ deficient mice fed a high fat diet, insulin
resistance can be ameliorated with an antagonist of
either PPARγ (bisphenol A diglycidyl ether) or RXR
(HX531).8
Synthetic PPARγ agonists for the treatment of type
2 diabetes12 have proven successful for glucose control
and reduction of HbA1c with the marketed compounds
Rosiglitazone13 and Pioglitazone14,13b (Chart 1). How-
ever, edema and weight gain have been reported in
patients after treatment with PPARγ agonists13b (it
remains to be seen if this is related to individual
compounds or activation of PPARγ, and there continues
to be interest in new compounds for clinical develop-
ment). Additional compounds in clinical and preclinical
development have recently been reviewed.12a,b Com-
pounds reported to be advanced clinical candidates
include Farglitazar (GW262570, Ph III),15 KRP-297 (Ph
II/III),16 Reglitazar (J TT-501, Ph II/III),17 Ragaglitazar
(DRF-2725, Ph II),18 and Tesaglitazar (AZ-242, Ph II).19
We sought a backup for our clinical compound Neto-
glitazone (MCC-555).20 This compound contains a thia-
zolidinedione (TZD) moiety commonly seen in PPARγ
agonists. It has remarkable potency in vivo and shows
promise for the treatment of type 2 diabetes. In an
earlier report, we described our efforts to identify a
backup chemical series and the initial structure-
activity relationship (SAR) work.21 At the time of that
report and during the work described here, there were
no clinical data to dictate the incorporation or avoidance
of any structural features. However, we were excited
to discover a series devoid of the TZD, since this
provided an opportunity to bring a diverse set of ligands
* To whom correspondence should be addressed. Tel: 908.704.4511.
Fax: 908.203.8109. E-mail: prybczyn@prdus.jnj.com.
† Current address: Eli Lilly and Co., Indianapolis, IN 46285.
10.1021/jm0301888 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

Benzoxazinones as PPARγ Agonists
Ch a r t 1. PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 197
Sch em e 1a
of the nitro group and concomitant cyclization to the
benzoxazinone, followed by protection of the primary
alcohol, yielded silyl ether 2. Alkylation of the amide
was achieved by deprotonation of the amide with sodium
hydride and then reaction with the alkylating agent
specified in each experimental (in the synthesis of 55,
alkylation was accomplished with a primary alcohol
under Mitsunobu conditions). A mixture of N- and
O-alkylation products was obtained from 2 under these
deprotonation/alkylation conditions. Therefore, depro-
tection of the tert-butyldimethylsilyl (TBS) ether was
conducted with aqueous methanesulfonic acid in metha-
nol to hydrolyze the O-alkylated products. The N-H and
N-alkyl products were readily separated on silica gel to
provide 2 and Mitsunobu substrate 3. Formation of the
phenyl ether (4) and saponification provided target
compounds 5-12, 15-28, 35, 36, 40, 42, 43, 46, 49, 50,
and 53-56.
The introduction of some of the substituents in the
side chain required additional functional group manipu-
lations. Schemes 2-5 highlight the changes to the
chemistry in Scheme 1. The reagents that could not be
purchased were synthesized, and the methods are
detailed in the Experimental Section. In Scheme 2, 29,
30, 37, and 39 were obtained through manipulation of
differentially protected carboxylic acids. Alkylation of
2 with the benzyl esters of 5-bromopentanoic acid or
6-bromohexanoic acid, followed by deprotection of the
TBS ethers and Mitsunobu etherification, provided
benzyl esters 58 and 59. A portion of each intermediate
was saponified to provide diacids 26 or 27 (see Scheme
1). Alternatively, each benzyl ester was hydrogenated
to a mixed methyl ester/carboxylic acid. Activation of
the acid moiety with 1,1′-carbonyldiimidazole, exposure
to ammonium hydroxide, and saponification of the
a
Reagents and conditions: (a) K2CO3, DMF, 0 °C to room
temperature. (b) (i) H2, Pd/C, EtOH, room temperature; (ii)
TBSOTf, imidazole, DMF, 0 °C to room temperature. (c) (i) NaH,
alkylating agent, DMF, 0 °C to room temperature; (ii) CH3SO3H,
MeOH, water, room temperature. (d) (2-Hydroxyphenyl)acetic acid
methyl ester, Bu3P, 1,1′-(azodicarbonyl)dipiperidine (ADDP), PhH,
10 °C to room temperature. (e) NaOH, MeOH, water, 65 °C.
into preclinical development. In this report, we describe
further efforts to elaborate the SAR of the benzox-
azinone series of compounds.22 The in vitro potency, in
vitro stability toward P450 enzymes in human liver
microsomes, bioavailability in rats, and initial in vivo
efficacy studies are included.
Ch em istr y
Racemic compounds were synthesized as shown in
Scheme 1. 2-Nitrophenol was alkylated with R-bromo-
γ-butyrolactone to provide 1. Catalytic hydrogenation

198 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
Sch em e 2a
a
Reagents and conditions: (a) (i) NaH, Br(CH2)nCO2CH2Ph, DMF, 0 °C to room temperature; (ii) CH3SO3H, MeOH, water, room
temperature; (iii) (2-hydroxyphenyl)acetic acid methyl ester, Bu3P, ADDP, PhH, 10 °C to room temperature. (b) H2, Pd/C, EtOH, room
temperature. (c) (i) 1,1′-Carbonyldiimidazole, CH2Cl2, room temperature, 2 h, and then NH4OH; (ii) NaOH, MeOH, water, 40 °C. (d) (i)
BH3, THF, -50 to 0 °C; (ii) NaOH, MeOH, water, 40 °C.
Sch em e 3a
in Scheme 6 for 13 and 57. In this variation of the
chemistry depicted in Scheme 1, stereochemistry is
introduced from the chiral pool and maintained through-
out the sequence. Mitsunobu reaction of 2-nitrophenol
and (S)-2-hydroxy-γ-butyrolactone provided chiral lac-
tone 65. Reduction of the nitro group, silyl ether
formation, alkylation with 1-iodohexane or 1-bromo-4-
methoxybutane, and deprotection with aqueous HCl
yielded alcohols 66 or 67. Mitsunobu etherification and
saponification with lithium hydroxide provided chiral
products 13 or 57. Chiral high-performance liquid
chromatography (HPLC) analysis of racemic and chiral
intermediates in each case confirmed the presence of a
single enantiomer. In both cases, it was observed that
the more potent of the two enantiomers could be
obtained from the (S)-lactone, with elaboration to final
products. From this, it was inferred that these com-
pounds had (R) absolute configuration, by inversion of
configuration during the Mitsunobu reaction in the first
step.
a
Reagents and conditions: (a) (i) NaH, Br(CH2)mCO2Et, DMF,
0-50 °C; (ii) CH3(CH2)nNH2, MeOH, 45 °C; (iii) CH3SO3H, MeOH,
water, room temperature. (b) (i) (2-Hydroxyphenyl)acetic acid
methyl ester, Bu3P, ADDP, PhH, 10 °C to room temperature; (ii)
NaOH, MeOH, water, 40 °C.
methyl ester yielded compound 29 or 30. Finally,
hydrogenation of each benzyl ester, borane reduction of
the carboxylic acid, and saponification of the methyl
ester provided compound 37 or 39. In Scheme 3,
compounds 31 and 32 were obtained by initial alkylation
of amide 2 with either ethyl bromoacetate or ethyl
4-bromobutyrate. Amidation occurred readily upon ex-
posure to propylamine or methylamine. Deprotection
with methanesulfonic acid provided 60 and 61. Mit-
sunobu etherification and saponification yielded the
target compounds. The chemistry in Scheme 4 provided
compounds 33 and 34. Alkylation of 2 with 6-bromo-
hexyl phthalimide and deprotection of the TBS ether
provided 62. Mitsunobu etherification and deprotection
of the amine yielded 63. Amidation with either acetic
anhydride or methanesulfonyl chloride and saponifica-
tion gave the desired products. In Scheme 5, the keto
group is used to synthesize four additional target
compounds. Grignard addition to 42 with methyl-
magnesium bromide provided 38. The oximes in com-
pounds 44 and 45 were obtained from ketone 42
by condensation with hydroxylamines. Compound 41
was obtained by sodium borohydride reduction of the
ketone in 64 followed by saponification to the target
compound.
Resu lts a n d Discu ssion
In Vitr o SAR Stu d ies. The preliminary results
obtained with this series provided impetus for further
SAR development on the scaffold.21 In those studies, it
was found that the benzyl substituent of the amide
influenced potency (Scheme 1; R ) CH2Ar), the favored
position of the carboxylic acid is in the 2-position of the
phenyl ether, and substitution on the aromatic portion
of the benzoxazinone bicyclic ring is not tolerated. In
the previous and present studies, compounds were
tested in the PPARγ-mediated aP2 gene induction
assay. This target gene of PPARγ has been evaluated
in these laboratories and shown to undergo a large
induction in the presence of agonists.23 As such, it is a
useful marker of in vitro activation of the receptor.
Select compounds were tested for in vitro metabolic
stability and rat oral bioavailability. Racemic and chiral
compounds with acceptable profiles were further tested
for in vivo efficacy.
Chiral compounds arise by virtue of the stereogenic
center at C-2 of the benzoxazinone ring. The stereo-
chemically pure compounds could be obtained by chro-
matographic resolution of racematessas in 13, 14, 47,
48, 51, and 52sor by the stereospecific synthesis shown
Substitution on the amide of the benzoxazinone
started with unsubstituted alkyl side chains, either
branched or linear (Table 1). The effect of homologation

Benzoxazinones as PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 199
Sch em e 4a
a
Reagents and conditions: (a) NaH, 6-bromohexyl phthalimide, DMF, 0 °C to room temperature. (b) (i) (2-Hydroxyphenyl)acetic acid
methyl ester, Bu3P, ADDP, PhH, 10 °C to room temperature; (ii) NH2NH2, EtOH, 60 °C. (c) (i) Ac2O, room temperature; (ii) NaOH,
MeOH, water, 40 °C. (d) (i) CH3SO2Cl, TEA, CH2Cl2, room temperature; (ii) NaOH, MeOH, water, 40 °C.
Sch em e 5a
and octyl linear chains provided compounds with EC50
values of 243, 100, 234, and 300 nM, respectively (11,
12, 15, and 16). The linear nonyl and decyl substituents
reduced the potency to over 1 µM (17 and 18). Optimal
chain length for this scaffold is thus in the range of 5-8
carbons. This was the basis for studies into the role of
chain branching, which provided mixed results. There
was a steady decline in functional potency as the amide
substituent progressed from isoheptyl (19) to ethyl-
cyclohexyl (23) and on to the 5,5-dimethylhexyl, cyclo-
pentylethyl, and cyclopentylpropyl side chains (20-22).
From these data, it appeared that the receptor has
limited space to accommodate the side chains on this
scaffold, so that there was little to be gained from the
additional steric bulk. The effect of electronics on
functional potency will be discussed below.
Compound 12 was separated into enantiomers 13
and 14 by chiral chromatography. Enantiomer 13 was
the more potent of the two, and it was synthesized by
the chemistry in Scheme 6 for in vivo studies (vide
infra).
a
Reagents and conditions: (a) Two equiv of CH3MgBr, THF,
-78 °C to room temperature. (b) H2NOR‚HCl, lutidine, EtOH,
room temperature. (c) (i) NaBH4, EtOH, 0 °C to room temperature;
(ii) NaOH, MeOH, water, 40 °C.
The utility of polar groups in the side chain was
investigated with the compounds shown in Table 2.
After the first two examples, side chains for the com-
pounds in this study were also in the range of 5-8
atoms. Hydroxyethyl and methylene carboxylate deriva-
tives (24 and 25) displayed no improvement over
compound 6. Introducing a carboxylic acid into the side
chain (26-28) likewise provided no advantage. The side
chain was also substituted with a primary (29 and 30)
or secondary amide moiety (31 and 32), as well as a
reverse amide (33) and reverse sulfonamide (34). All of
these target compounds showed poor potency in the
functional assay. The only break in this trend arose from
the unsaturated variation of the hexyl chain (36).25 It
was apparently similar enough to the hexyl chain of 12
to provide a potent agonist. Unfortunately, the double
bond cannot be mimicked by the aforementioned amides.
The nitrile-substituted chain in 35 was superior to the
corresponding carboxylic acid substituent in 28 but was
still not in the potency range obtained from the best
linear compounds in Table 1. The highly polar carboxylic
acids and amides represented by 24-35 indicated that
the receptor favors less polar, linear ligands for activa-
tion of the receptor.
Sch em e 6a
a
Reagents and conditions: (a) Ph3P, DEAD, THF, -20 °C to
room temperature. (b) (i) H2, Pd/C, EtOH, room temperature; (ii)
TBSOTf, imidazole, DMF, 0 °C to room temperature; (iii) NaH,
alkyl halide, DMF, 0-65 °C; (iv) 6 N HCl, MeOH, room temper-
ature. (c) (i) (2-Hydroxyphenyl)acetic acid methyl ester, Bu3P,
ADDP, PhH, 10 °C to room temperature; (ii) LiOH, THF, water,
0 °C to room temperature.
has been well-documented,24 and those principles were
applied to the current scaffold. One to four linear and
branched carbon chains provided low potency com-
pounds (5-10). Extension to the pentyl, hexyl, heptyl,
More interesting results were obtained by substitu-
tion of the alkyl chain with hydroxyl, fluoro, or carbonyl
groups or by replacement of a methylene with oxygen

200 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
Ta ble 1. Benzoxazinones with Linear or Branched Alkyl Substituents
a
b
Each value is the mean of three determinations. Chiral at the C2 position of the benzoxazinone ring.
or sulfur. These substitutions provide linear chains that
are not as polar as those described above. Introducing
a hydroxyl group at the terminus of the pentyl chain
reduced potency (37 vs 11), and branching to the
tertiary alcohol gave a slight improvement (38 vs 37).
However, the linear 6-hydroxyhexyl and 7-hydroxy-
heptyl chains (39 and 40) brought potency back to the
level in the unsubstituted cases (12 and 15) while
reducing lipophilicity. Simply moving the hydroxy from
C6 to C5 of the hexyl substituent reduced potency (41
vs 39). By comparison, potent agonists were obtained
by the incorporation of a ketone into the hexyl or heptyl
side chains (42 and 43). Conversion of the 5-ketohexyl
chain to the corresponding oxime (44, 3.4:1 E/Z mixture)
provided the most potent compound found in this series.
Unfortunately, the oxime was hydrolytically labile and
rapidly reverted to the ketone under acidic conditions
(pH 2). The methyl oxime in 45 (3:1 E/Z mixture)
eliminated all potency, indicating the value of a protic
group in this region. Overall, these results point to a
polarity factor that can be exploited over and above the
sterics explored by the compounds in Table 1.
One or two fluorine atoms could be introduced into
the C5 or C6 position of the hexyl chain (46 and 50)
and the C6 or C7 position of the heptyl chain (49 and
53). The best results were obtained from the hexyl
analogues 46 and 50. Both 46 and 50 were separated
into enantiomers by chiral chromatography (46 was
separated into 47 and 48 while 50 was separated into
51 and 52). Enantiomers 47 and 51 were the better of
each pair, and both are presumed to have (R) absolute
stereochemistry from synthesis beginning with the (S)-
lactone. The most potent enantiomer 51 was taken on
for additional studies (vide infra). Finally, oxygen and
sulfur were introduced into the chain (54-57), with the
best results arising from 56 and its (R)-enantiomer, 57.
Compound 57 was taken on for in vivo studies. It is
interesting to note that 57 was the least potent of the
compounds taken into secondary studies but had one
of the best overall profiles (vide infra).
P h a r m a cok in et ic a n d in Vivo St u d ies. Subse-
quent studies on the series focused on compounds 12,
13, 39, 40, 42, 44, 50, 51, and 57 (Table 3). The in vitro
metabolic stability was determined by incubation with

Benzoxazinones as PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 201
Ta ble 2. Benzoxazinones with Substituted Alkyl Chains
Ta ble 3. Human Liver Microsome Stability and Rat Oral
Bioavailability of Benzoxazinones
rat oral bioavailabilityb
HLMa
AUC
entry
t1/2 (min)
%
(µM h)
t1/2 (h)
12
13
39
40
42
44
50
51
57
53
73
57
76
51
115
95
4
13
27
16
100
39
41
406
327
2.5
2.3
5.8
19.9
84
14.0
14.5
0.6
7.0
9.8
4.2
16.0
7.9
148
76
111
>500
93
88
6.7
a
Incubated at 37 °C with test compound at 5 µM and 1 mg
b
protein/mL microsomal prep. Dosed at 30 mg/kg po as a suspen-
sion in methocel, 3 mg/kg iv. See Experimental Section for details.
human liver microsomes. In this system, values above
30 min were considered acceptable, and all compounds
had high metabolic stability. The analogue with the
hexyl chain (12) and the more potent (R)-enantiomer
(13) had a half-life of 53 and 73 min, respectively. Other
analogues showed little variation by the introduction
of a primary alcohol, ketone, oxime, or fluorine. A
noteworthy difference came from replacing a methylene
(13) with an oxygen (57). It was expected that the
methyl ether would be cleaved, but the compound was
not degraded by the P450s present in the microsomes.
Rat oral bioavailability showed greater variation across
the set of functional agonists. All compounds were dosed
individually at 30 mg/kg po in 0.5% aqueous methocel
for comparison to iv dosing at 3 mg/kg. This test of
exposure after oral dosing showed that racemic 12
provided high exposure at 406 µM h AUC, corresponding
to approximately 100% bioavailability, and the half-life
was 14 h. The high exposure held for the (R)-enantiomer
(13) where the AUC reached 327 µM h, and the half-
life remained at 14.5 h. Continuing through the series,
compounds 39 and 40 showed low exposure after oral
dosing. The analogue 39 had an AUC of 2.5 µM h and
a half-life of 0.6 h. The analogue 40 had an AUC of 2.3
µM h and a half-life of 7 h. The results from the in vitro
microsome test indicated that the compounds were not
susceptible to phase I metabolism. It was not ascer-
tained whether the low exposure was due to phase II
metabolism or poor uptake from the gut, but these
results demonstrated that the hydroxyl group added a
liability to the scaffold. The keto compound 42 provided
similar low exposure after oral dosing, with an AUC of
5.8 µM h and a half-life of 9.8 h. The poor bioavailability
was also presumed to be due to phase II metabolism or
poor uptake. Compound 44 followed this trend with an
AUC of 19.9 µM h and a half-life of 4.2 h. Higher
exposure was obtained from the fluorohexyl compounds
50 and 51 where the exposure seen with the racemic
compound was mirrored by the (R)-enantiomer. Racemic
50 provided an AUC of 84 µM h and a half-life of 16 h,
and chiral 51 had an AUC of 93 µM h and a half-life of
7.9 h. Finally, 57 (the (R)-enantiomer of 55) gave an
AUC of 88 µM h and a half-life of 6.7 h.
Two compounds, 13 and 57, were taken into the 11
day db/db mouse model of type 2 diabetes (Figure 1).
Compound 13 showed a dose-dependent decrease in
plasma glucose over a dose range of 1-30 mg/kg po,
single daily dose. By comparison, 57 showed near
a
b
Each value is the mean of three determinations. Chiral at
the C2 position of the benzoxazinone ring.

202 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
F igu r e 1. Biological profile of two compounds.
maximal effect at doses of 1-30 mg/kg. At this time, it
is not clear why 57 offered such an advantage over 13,
but studies are continuing with these compounds.
In conclusion, the SAR of a PPARγ agonist series has
been developed. Previous work has determined the
optimal location for the carboxylic acid in the phenyl
ether and that substitution of the benzoxazinone aryl
ring was not tolerated.21 This work has demonstrated
that substitution on the amide of the heterocyclic ring
offered enhancement of receptor activation while gener-
ally maintaining bioavailability and resistance to oxida-
tive metabolism. Lipophilic side chains provided the
most potent agonists, and the optimal chain length was
5-8 atoms. These chains could be substituted with
hydroxy, fluorine, carbonyl, or oxime groups. However,
carboxylic acids and amides were not tolerated. Sulfur
and oxygen could be successfully introduced as a
member of the chain. The stereochemistry of the com-
pound was critical to potency. The preferred stereo-
chemistry was inferred to be (R) by virtue of the route
used to obtain enantiomerically pure compounds. Fur-
thermore, two compounds have in vivo efficacy in a db/
db mouse model of type 2 diabetes (Figure 1). Future
communications on this series will describe additional
efficacy testing and preclinical evaluation of the com-
pounds.
Exp er im en ta l Section
Gen er a l Ch em istr y. Purchased reagents and anhydrous
solvents were used as received. Proton NMRs were obtained
with a Bruker 300 MHz in the indicated solvent with chemical
shifts (δ) reported in ppm vs tetramethylsilane and coupling
constants (J ) in Hz. Positive and negative ion loop mass
spectra were obtained with an Agilent 1100 LC/MSD. Elemen-
tal analyses were obtained by Quantitative Technologies, Inc.
(Whitehouse, NJ ) on a Perkin-Elmer 2400 Elemental Analyzer.
The synthesis of the compounds in Scheme 1 is exemplified
by the synthesis of compounds 1-5. The alkylating agent used
for each target compound is noted in the Experimental Section.
If the alkylating agent was synthesized, the experimental
details immediately precede the experimental section for the
target compound where it was used.
3-(2-Nitr op h en oxy)d ih yd r ofu r a n -2-on e (1). A solution
of 2-nitrophenol (50 g, 0.36 mol) in dry dimethyl formamide
(DMF) (200 mL), under N2, was cooled to 0 °C. Potassium
carbonate (74.5 g, 0.54 mol) was added, followed by dropwise
addition of R-bromo-γ-butyrolactone (36 mL, 0.43 mol) in dry
DMF (36 mL). The reaction was stirred at room temperature
for 17 h. Acetic acid (60 mL) was added slowly to control the
CO2 evolution, the mixture was poured into water (4 L)
containing NaCl (200 g), and the solution was washed with
EtOAc. The organic layer was washed with water (5 × 100
mL) and brine (100 mL). The organic layer was then dried
(Na2SO4) and filtered, and the solvent was removed in vacuo.
The phenolic ether (1) was isolated as a pale yellow solid (50
1
g, 0.22 mol, 62%). H (CDCl3): 7.86 (d, J ) 8.1, 1H), 7.58 (t, J

Benzoxazinones as PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 203
) 8.6, 1H), 7.50 (d, J ) 7.8, 1H), 7.16 (t, J ) 7.4, 1H), 5.04 (t,
J ) 7.4, 1H), 4.58 (m, 1H), 4.40 (q, J ) 7.4, 1H), 2.8-2.6 (m,
2H).
ethane. 1H (CDCl3): 7.28-7.18 (m, 2H), 7.05-6.89 (m, 6H),
4.85 (dd, J ) 9.1, 3.6, 1H), 4.23 (m, 2H), 3.99 (m, 2H), 3.63
(dd, J ) 20.9, 15.9, 2H), 2.43 (m, 1H), 2.23 (m, 1H), 1.28 (t, J
) 7.2, 3H). m/z (MH+) 354.2. Anal. (C20H21NO5‚0.25H2O) C,
H, N.
2-(2-t er t -Bu t yld im e t h ylsiloxye t h yl)-4H -b e n zo[1,4]-
oxa zin -3-on e (2). The intermediate 1 (50 g, 0.22 mol) was
suspended in EtOH (550 mL) and then shaken for 3 h with
10% Pd/C and H2 (45 psi) at room temperature. The solution
was filtered through Celite, and the solvent was removed in
vacuo. The amide was obtained as a solid. A solution of the
amide (42.5 g, 0.22 mol) in dry DMF (400 mL), under N2, was
cooled to 0 °C. Imidazole (37.4 g, 0.55 mol) was added in one
portion, followed by addition of TBS chloride (39.8 g, 0.26 mol)
in one portion. The mixture was stirred for 15 h as the ice
bath was thawed to room temperature. The reaction was
poured into water (2 L) containing NaCl (100 g) and washed
with 7:3 Et2O/CH2Cl2 (5 × 120 mL). The organic layer was
washed with water (4 × 100 mL) and brine (100 mL). The
organic layer was then dried (Na2SO4) and filtered, and the
solvent was removed in vacuo. The product 2 was isolated by
silica gel chromatography with hexane/EtOAc and then crys-
tallized from hexane. The silyl ether was obtained as a pale
2-[2-(4-Isopr opyl-3-oxo-3,4-dih ydr o-2H-ben zo[1,4]oxazin -
2-yl)eth oxy]p h en yla cetic Acid (7). Alkylating agent
)
2-iodopropane. 1H (CDCl3): 7.28-7.13 (m, 2H), 7.05-6.89 (m,
6H), 4.74 (m, 2H), 4.21 (m, 2H), 3.63 (dd, J ) 20.2, 16.0, 2H),
2.41 (m, 1H), 2.19 (m, 1H), 1.54 (d, J ) 7.2, 6H). m/z (MH+)
368.1. Anal. (C21H23NO5‚0.25H2O) C, H, N.
2-[2-(3-Oxo-4-p r op yl-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -
2-yl)eth oxy]p h en yla cetic Acid (8). Alkylating agent
)
1
1-iodopropane H (CDCl3): 7.26-7.17 (m, 2H), 7.06-6.89 (m,
6H), 4.82 (dd, J ) 9.2, 3.8, 2H), 4.20 (m, 2H), 3.88 (t, J ) 7.5,
2H), 3.61 (dd, J ) 19.1, 16.2, 2H), 2.45 (m, 1H), 2.23 (m, 1H),
1.68 (hex, J ) 7.6, 2H), 0.97 (t, J ) 7.4, 6H). m/z (M - 1) 368.3.
Anal. (C20H21NO5) C, H, N.
2-[2-(4-Isobu tyl-3-oxo-3,4-dih ydr o-2H-ben zo[1,4]oxazin -
2-yl)eth oxy]p h en yla cetic Acid (9). Alkylating agent
)
1-bromo-2-methylpropane. 1H (CDCl3): 7.28-7.17 (m, 2H),
7.05-6.89 (m, 6H), 4.84 (dd, J ) 9.3, 3.7, 2H), 4.22 (m, 2H),
3.79 (m, 2H), 3.61 (dd, J ) 19.1, 16.1, 2H), 2.44 (m, 1H), 2.24
(m, 1H), 2.08 (hept, J ) 7.0, 1H), 0.94 (m, 6H). m/z (M - 1)
382.3. Anal. (C22H25NO5‚0.15H2O) C, H, N.
1
yellow solid (46.8 g, 0.15 mol, 69% for two steps). H (CDCl3):
6.89 (m, 3H), 6.80 (m, 1H), 4.70 (m, 1H), 3.78 (m, 2H), 2.15
(m, 1H), 1.94 (m, 1H), 0.83 (s, 9H), 0.07 (s, 6H). m/z (MH+)
308.0. Note: The solid is volatile and sublimes when dried
under vacuum for long periods.
2-[2-(4-Bu tyl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -2-
yl)et h oxy]p h en yla cet ic Acid (10). Alkylating agent
)
2-(2-H yd r oxyet h yl)-4-m et h yl-4H -b en zo[1,4]oxa zin -3-
on e (3). A solution of 2 (1.0 g, 3.25 mmol) in dry DMF (35
mL), under N2, was cooled to 0 °C. Sodium hydride (75%
dispersion in oil, 0.105 g, 3.25 mmol) was added, and the
solution was stirred for 30 min at 0 °C. Iodomethane (0.2 mL,
3.25 mmol) was added, the ice bath was removed, and the
solution was stirred overnight. The mixture was poured into
water (180 mL) and washed with Et2O (3 × 60 mL). The
organic layer was washed with water (4 × 40 mL) and brine
(50 mL). The organic layer was then dried (Na2SO4) and
filtered, and solvent was removed in vacuo. The silyl ether
(0.821 g, 2.6 mmol) was dissolved in methanol (15 mL) and
water (0.5 mL) and then treated with CH3SO3H (0.5 mL) and
stirred for 30 min at room temperature. The solvent was
removed, and the product was purified on silica by gel
chromatography with hexane/EtOAc. The product 3 was
obtained as a colorless oil (0.478 g, 2.3 mmol, 70% for two
1-bromobutane. 1H (CDCl3): 7.28-7.17 (m, 2H), 7.04-6.88 (m,
6H), 4.81 (dd, J ) 9.1, 3.8, 2H), 4.20 (m, 2H), 3.92 (t, J ) 7.5,
2H), 3.61 (dd, J ) 19.0, 16.2, 2H), 2.45 (m, 1H), 2.23 (m, 1H),
1.63 (m, 2H), 1.41 (m, 2H), 0.96 (t, J ) 7.3, 3H). m/z (M - 1)
382.3. Anal. (C22H25NO5‚0.1H2O) C, H, N.
2-[2-(3-Oxo-4-p en tyl-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -
2-yl)eth oxy]p h en yla cetic Acid (11). Alkylating agent )
1-bromopentane. 1H (CDCl3): 7.28-7.16 (m, 2H), 7.06-6.88
(m, 6H), 4.81 (dd, J ) 9.2, 3.8, 2H), 4.20 (m, 2H), 3.90 (t, J )
7.6, 2H), 3.61 (dd, J ) 19.2, 16.1, 2H), 2.46 (m, 1H), 2.23 (m,
1H), 1.65 (m, 2H), 1.34 (m, 4H), 0.96 (t, J ) 6.8, 3H). m/z (M
- 1) 396.4. Anal. (C23H27NO5) C, H, N.
2-[2-(4-Hexyl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -2-
yl)et h oxy]p h en yla cet ic Acid (12). Alkylating agent
)
1
1-iodohexane. H (CDCl3): 7.28-7.18 (m, 2H), 7.07-6.89 (m,
6H), 4.83 (dd, J ) 9.1, 3.7, 1H), 4.20 (m, 2H), 3.91 (t, J ) 7.6,
2H), 3.62 (dd, J ) 20.6, 16.0, 2H), 2.44 (m, 1H), 2.23 (m, 1H),
1.65 (m, 2H), 1.33 (m, 6H), 0.88 (t, J ) 6.7, 3H). m/z (MH+)
412.3. Anal. (C24H29NO5) C, H, N.
1
steps). H (CDCl3): 7.01 (m, 4H), 4.72 (dd, J ) 7.2, 5.7, 1H),
3.88 (m, 2H), 3.38 (s, 3H), 2.31-2.10 (m, 2H). m/z (MH+) 208.0.
Meth yl-2-[2-(4-m eth yl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]-
oxa zin -2-yl)eth oxy]p h en yla ceta te (4). A solution of 3 (0.134
g, 0.65 mmol), (2-hydroxyphenyl)acetic acid methyl ester (0.16
g, 0.96 mmol), and tributylphosphine (0.24 mL, 0.96 mmol) in
dry benzene (15 mL), under N2, was cooled to 10 °C. 1,1′-
(Azodicarbonyl)dipiperidine (0.244 g, 0.96 mmol) was added
in one portion, and the solution was stirred at room temper-
ature overnight. The organic layer was washed with 5 N
aqueous NaOH (4 × 5 mL) and brine (5 mL). The organic layer
was then dried (Na2SO4) and filtered, and the solvent was
removed in vacuo. The product was purified by silica gel
chromatography with hexane/EtOAc. The ester 4 was obtained
as a colorless oil (0.148 g, 0.42 mmol, 64%). 1H (CDCl3): 7.28-
7.18 (m, 2H), 7.09-6.89 (m, 6H), 4.79 (dd, J ) 9.5, 3.9, 1H),
4.24 (m, 2H), 3.60 (m, 5H), 3.38 (s, 3H), 2.52 (m, 1H), 2.24 (m,
1H). m/z (MNa+) 378.1.
2-[2-(4-Meth yl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -
2-yl)eth oxy]p h en yla cetic Acid (5). A solution of 4 in MeOH
(15 mL) and 2 N aqueous NaOH (2 mL) was heated to 65 °C
for 2.5 h, cooled to 0 °C, diluted with 10 mL of water, and
acidified with concentrated HCl (0.5 mL). The product was
obtained as a solid by filtration and dried in vacuo at 45 °C
(0.083 g, 0.24 mmol, 78%). 1H (CDCl3): 7.28-7.18 (m, 2H),
7.07-6.89 (m, 6H), 4.87 (dd, J ) 9.0, 3.7, 1H), 4.23 (m, 2H),
3.62 (dd, J ) 21.2, 15.9, 2H), 3.37 (s, 3H), 2.46 (m, 1H), 2.25
(m, 1H). m/z (MH+) 340.1. Anal. (C19H19NO5‚0.15H2O) C, H,
N.
Compound 12 was resolved into enantiomers 13 and 14 by
chiral chromatography with a Chiralcel AD column (2 cm ×
25 cm). The mobile phase was 80:20:0.1 hexane/2-propanol/
TFA, and the flow rate was 6 mL/min. Retention times: (R)
) 23.3 min; (S) ) 29.8 min.
(R)-2-[2-(4-Hexyl-3-oxo-3,4-dih ydr o-2H-ben zo[1,4]oxazin -
2-yl)eth oxy]p h en yla cetic Acid (13). 1H (CDCl3): 7.26-7.19
(m, 2H), 7.03-6.89 (m, 6H), 4.87 (dd, J ) 9.2, 3.5, 1H), 4.22
(m, 2H), 3.91 (t, J ) 7.7, 2H), 3.63 (dd, J ) 21.4, 16.0, 2H),
2.39 (m, 1H), 2.23 (m, 1H), 1.65 (m, 2H), 1.33 (m, 6H), 0.89
(m, 3H). m/z (MH+) 412.3. Anal. (C24H29NO5) C, H, N.
(S)-2-[2-(4-Hexyl-3-oxo-3,4-dih ydr o-2H-ben zo[1,4]oxazin -
2-yl)eth oxy]p h en yla cetic Acid (14). 1H (CDCl3): 7.26-7.19
(m, 2H), 7.03-6.89 (m, 6H), 4.87 (dd, J ) 9.2, 3.5, 1H), 4.22
(m, 2H), 3.91 (t, J ) 7.7, 2H), 3.63 (dd, J ) 21.4, 16.0, 2H),
2.39 (m, 1H), 2.23 (m, 1H), 1.65 (m, 2H), 1.33 (m, 6H), 0.89
(m, 3H). m/z (MH+) 412.3. Anal. (C24H29NO5) C, H, N.
2-[2-(4-Hep tyl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -
2-yl)eth oxy]p h en yla cetic Acid (15). Alkylating agent )
1-iodoheptane. 1H (CDCl3): 7.27-7.19 (m, 2H), 7.06-6.88 (m,
6H), 4.80 (dd, J ) 9.0, 4.0, 1H), 4.20 (m, 2H), 3.90 (t, J ) 7.6,
2H), 3.62 (s, 2H), 2.46 (m, 1H), 2.24 (m, 1H), 1.75-1.28 (m,
10H), 0.92 (m, 3H). m/z (M - 1) 424.1. Anal. (C25H31NO5‚
1.4H2O‚1.0C6H14‚1.0C4H8O2) C, H, N.
2-[2-(4-Octyl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -2-
yl)et h oxy]p h en yla cet ic Acid (16). Alkylating agent
)
2-[2-(4-Eth yl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -2-
yl)eth oxy]p h en yla cetic Acid (6). Alkylating agent ) iodo-
1-bromooctane. 1H (CDCl3): 7.26-7.18 (m, 2H), 7.06-6.88 (m,
6H), 4.80 (dd, J ) 9.1, 3.9, 1H), 4.20 (m, 2H), 3.90 (t, J ) 7.6,

204 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
2H), 3.61 (dd, J ) 18.5, 16.3, 2H), 2.43 (m, 1H), 2.24 (m, 1H),
1.76-1.27 (m, 12H), 0.88 (m, 3H). m/z (M - 1) 438.3. Anal.
(C26H33NO5‚0.2H2O‚0.3C6H14‚0.6C4H8O2) C, H, N.
2-[2-(4-Non yl-3-oxo-3,4-d ih yd r o-2H-b en zo[1,4]oxa zin -
2-yl)eth oxy]p h en yla cetic Acid (17). Alkylating agent )
1-bromononane. 1H (CDCl3): 7.27-7.19 (m, 2H), 7.06-6.88 (m,
6H), 4.80 (dd, J ) 9.2, 4.0, 1H), 4.20 (m, 2H), 3.90 (t, J ) 7.5,
2H), 3.61 (s, 2H), 2.46 (m, 1H), 2.24 (m, 1H), 1.75-1.26 (m,
14H), 0.92 (m, 3H). m/z (M - 1) 452.2. Anal. (C27H35NO5‚
1.4H2O‚1.0C6H14‚0.8C4H8O2) C, H, N.
2H), 7.06-6.88 (m, 6H), 4.82 (dd, J ) 9.0, 3.7, 1H), 4.20 (m,
2H), 3.93 (t, J ) 8.0, 2H), 3.62 (dd, J ) 20.0, 16.0, 2H), 2.43
(m, 1H), 2.23 (m, 1H), 1.80-0.88 (m, 13H). m/z (MH+) 438.3.
Anal. (C24H29NO5) C, H, N.
2-[2-(4-(2-Hyd r oxyeth yl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (24). Alkylating
agent ) 2-bromoethyl acetate. 1H (CDCl3): 7.28-6.89 (m, 8H),
4.86 (dd, J ) 8.8, 4.2, 1H), 4.22 (m, 4H), 3.91 (m, 4H), 2.49
(m, 1H), 2.25 (m, 1H). m/z (M-1) 370.1. Anal. (C20H21NO6‚
0.2H2O) C, H, N.
(2-[2-(2-C a r b o x y m e t h y lp h e n o x y )e t h y l]-3-o x o -2,3-
d ih yd r oben zo[1,4]oxa zin -4-yl)a cetic Acid (25). Alkylating
agent ) ethyl bromoacetate. H (DMSO-d6): 7.24 (d, J ) 7.6,
1H), 7.18 (d, J ) 7.4, 1H), 7.06 (m, 4H), 7.02 (d, J ) 8.1, 1H),
6.89 (t, J ) 7.3, 1H), 4.87 (dd, J ) 9.1, 4.2, 1H), 4.64 (m, 2H),
4.16 (m, 2H), 3.35 (s, 2H), 2.26 (m, 1H), 2.12 (m, 1H). m/z (M-
1) 384.1. Anal. (C20H19NO7‚0.2H2O) C, H, N.
2-[2-(4-Decyl-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]oxa zin -2-
yl)et h oxy]p h en yla cet ic Acid (18). Alkylating agent
)
1
1-bromodecane. 1H (CDCl3): 7.26-7.18 (m, 2H), 7.06-6.88 (m,
6H), 4.80 (dd, J ) 9.1, 3.9, 1H), 4.20 (m, 2H), 3.90 (t, J ) 7.6,
2H), 3.61 (dd, J ) 18.5, 16.2, 2H), 2.45 (m, 1H), 2.24 (m, 1H),
1.75-1.18 (m, 16H), 0.88 (m, 3H). m/z (M - 1) 466.3. Anal.
(C25H31NO5‚0.3C6H14‚0.6C4H8O2) C, H, N.
5-Br om op en ta n oic Ben zyl Ester . Benzyl alcohol (6.3 mL,
60.7 mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide hydrochloride (EDC) (11.6 g, 60.7 mmol) were combined
in CH2Cl2 (110 mL) and cooled to 0 °C. N,N-(Dimethylamino)-
pyridine (0.67 g, 5.5 mmol) and 5-bromovaleric acid (10.0 g,
55.2 mmol) were added, and the reaction was stirred at room
temperature for 7 h. The reaction was poured into water and
extracted with CH2Cl2 (3 × 100 mL). The organic layer was
washed with saturated aqueous NaHCO3 (2 × 50 mL), water
(50 mL), and brine (50 mL). The organic layer was then dried
(Na2SO4) and filtered, and the solvent was removed in vacuo.
The product was obtained as a colorless oil and used in the
synthesis of 26 and 37.
5-(2-[2-(2-Ca r b oxym e t h ylp h e n oxy)e t h yl]-3-oxo-2,3-
d ih yd r oben zo[1,4]oxa zin -4-yl)p en ta n oic Acid (26). 1H
(CD3OD): 7.25-7.15 (m, 2H), 7.09-6.86 (m, 6H), 4.87 (dd, J
) 9.5, 4.2, 1H), 4.18 (m, 2H), 3.98 (m, 2H), 3.57 (dd, J ) 19.1,
16.2, 2H), 2.33 (m, 3H), 2.21 (m, 1H), 1.68 (m, 4H). m/z (M -
1) 426.1. Anal. (C23H25NO7) C, H, N.
2-[2-(4-(5-Met h ylh exyl)-3-oxo-3,4-d ih yd r o-2H -b en zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (19). Alkylating
1
agent ) 1-bromo-5-methylhexane. H (CDCl3): 7.25 (m, 1H),
7.18 (d, J ) 7.3, 1H), 7.06-6.88 (m, 6H), 4.81 (dd, J ) 9.0,
3.8, 1H), 4.20 (m, 2H), 3.90 (t, J ) 7.7, 2H), 3.61 (dd, J ) 20.0,
16.1, 2H), 2.45 (m, 1H), 2.26 (m, 1H), 1.68-1.49 (m, 3H), 1.36
(m, 2H), 1.22 (m, 2H), 0.87 (t, J ) 6.6, 3H). m/z (M - 1) 424.1.
Anal. (C25H31NO5) C, H, N.
1-Br om o-5,5-d im eth ylh exa n e. THF, tert-butylmagnesium
chloride, and CuCN were reacted as described in the literature
to provide (5,5-dimethylhexyloxy)trimethylsilane (Tetrahedron
Lett. 1989, 30, 6393). The silyl ether was cleaved by the
hydrolysis method described in example 3 to provide 5,5-
dimethylhexan-1-ol. The alcohol (0.6 g, 4.6 mmol) was com-
bined with 48% aqueous HBr (10 mL) and heated to reflux
for 3 h. The aqueous layer was washed with 1:1 Et2O/CH2Cl2
(3 × 25 mL). The organic layer was washed with saturated
aqueous NaHCO3 (2 × 25 mL) and brine (25 mL). The organic
layer was then dried (Na2SO4) and filtered, and the solvent
was removed in vacuo. 1-Bromo-5,5-dimethylhexane was
obtained as a colorless oil and used in the synthesis of 20.
2-[2-(4-(5,5-Dim eth ylh exyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (20). Alkylating
agent ) 1-bromo-5,5-dimethylhexane. 1H (CDCl3): 7.28-7.17
(m, 2H), 7.03-6.88 (m, 6H), 4.83 (dd, J ) 9.1, 3.6, 1H), 4.20
(m, 2H), 3.91 (t, J ) 7.5, 2H), 3.63 (dd, J ) 20.0, 16.1, 2H),
2.44 (m, 1H), 2.23 (m, 1H), 1.62 (m, 2H), 1.33 (m, 2H), 1.24
(m, 2H), 0.87 (s, 9H). m/z (M - 1) 438.1. Anal. (C26H33NO5‚
0.4H2O) C, H, N.
6-(2-[2-(2-Ca r b oxym e t h ylp h e n oxy)e t h yl]-3-oxo-2,3-
d ih yd r oben zo[1,4]oxa zin -4-yl)h exa n oic Acid (27). Alkyl-
1
ating agent ) benzyl 6-bromohexanoate. H (CD3OD): 7.25-
6.87 (m, 8H), 4.81 (m, 1H), 4.19 (m, 2H), 3.97 (m, 2H), 3.57
(m, 2H), 2.39-2.17 (m, 4H), 1.64 (m, 4H), 1.40 (m, 2H). m/z
(M - 1) 440.0. Anal. (C24H27NO7) C, H, N.
6-(2-[2-(2-Ca r b oxym e t h ylp h e n oxy)e t h yl]-3-oxo-2,3-
dih ydr oben zo[1,4]oxazin -4-yl)-2,2-dim eth ylh exan oic Acid
(28). Alkylating agent ) 6-bromo-2,2-dimethylhexanitrile.
Exposure to NaOH saponified the ester and the nitrile. 1H
(CDCl3): 7.26 (m, 1H), 7.15 (d, J ) 6.4, 1H), 7.05-6.89 (m,
6H), 4.79 (dd, J ) 9.9, 3.6, 1H), 4.31 (m, 1H), 4.17 (m, 1H),
4.00 (m, 2H), 3.62 (m, 2H), 2.32 (m, 1H), 2.17 (m, 1H), 1.71-
1.19 (m, 6H), 1.16 (s, 3H), 1.14 (s, 3H). m/z (M - 1) 468.1.
Anal. (C26H31NO7) C, H, N.
(2-[2-(4-(4-Cya n o-4,4-d im eth ylbu tyl)-3-oxo-3,4-d ih yd r o-
2H-ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (35).
Alkylating agent ) 6-bromo-2,2-dimethylhexanitrile. The cor-
responding intermediate 4 was saponified with excess LiOH
in THF/water. 1H (CDCl3): 7.26 (m, 1H), 7.18 (d, J ) 7.2, 1H),
7.08-6.89 (m, 6H), 4.82 (dd, J ) 9.1, 3.8, 1H), 4.20 (m, 2H),
3.95 (m, 2H), 3.61 (dd, J ) 19.5, 16.1, 2H), 2.46 (m, 1H), 2.23
(m, 1H), 1.70 (m, 2H), 1.57 (m, 4H), 1.33 (s, 6H). m/z (M - 1)
449.2. Anal. (C26H30N2O5) C, H, N.
2-[2-(4-(2-Cyclopen tyleth yl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (21). Alkylating
agent ) (2-hydroxyethyl)cyclopentane. In the alkylation of the
amide (Scheme 1), a solution of 2 (0.5 g, 1.6 mmol) in THF (5
mL) was cooled to -10 °C. Triphenylphosphine (0.46 g, 1.76
mmol) and diethylazodicarboxylate (DEAD, 0.28 mL, 1.76
mmol) were added, and the solution was stirred overnight at
room temperature. The reaction was diluted with EtOAc (25
mL) and then extracted with 2 N NaOH (2 × 5 mL), water
(10 mL), and brine (10 mL). The organic layer was dried
(Na2SO4) and filtered, and then, the solvent was removed in
vacuo. The reaction product was elaborated to compound 21
with the chemistry in Scheme 1. 1H (CDCl3): 7.28-7.18 (m,
2H), 7.01-6.88 (m, 6H), 4.85 (dd, J ) 9.3, 3.5, 1H), 4.23 (m,
2H), 3.92 (t, J ) 7.8, 2H), 3.62 (dd, J ) 20.0, 16.0, 2H), 2.43
(m, 1H), 2.21 (m, 1H), 1.84 (m, 3H), 1.65 (m, 6H) 1.17 (m, 2H).
m/z (MH+) 424.0. Anal. (C25H29NO5‚0.25H2O) C, H, N.
2-[2-(4-(3-Cyclop en t ylp r op yl)-3-oxo-3,4-d ih yd r o-2H -
ben zo[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (22). Alkyl-
ating agent ) (3-hydroxypropyl)cyclopentane. See the synthe-
2-[2-(4-H e x-5-e n yl-3-oxo-3,4-d ih yd r o-2H -b e n zo[1,4]-
oxazin -2-yl)eth oxy]ph en ylacetic Acid (36). Alkylating agent
) 6-bromo-1-hexene. 1H (CDCl3): 7.25-7.16 (m, 2H), 7.01-
6.88 (m, 6H), 5.79 (m, 1H), 5.03 (m, 1H), 4.96 (m, 1H), 4.81
(dd, J ) 9.1, 3.8, 1H), 4.20 (m, 2H), 3.91 (t, J ) 7.5, 2H), 3.62
(m, 2H), 2.47 (m, 1H), 2.22 (m, 1H) 2.10 (q, J ) 7.1, 2H), 1.67
1
sis of 21 for the synthetic method. H (CDCl3): 7.28-7.18 (m,
(m, 2H), 1.47 (p, J ) 7.2, 2H). m/z (MH+) 432.1. Anal. (C24H27
-
2H), 7.03-6.89 (m, 6H), 4.85 (dd, J ) 9.2, 3.6, 1H), 4.22 (m,
2H), 3.90 (t, J ) 7.7, 2H), 3.63 (dd, J ) 20.3, 15.9, 2H), 2.43
(m, 1H), 2.23 (m, 1H), 1.76-1.52 (m, 10H), 1.39 (m, 3H). m/z
(MH+) 438.1. Anal. (C26H31NO5‚0.12CH2Cl2) C, H, N.
NO5) C, H, N.
7-Br om oh ep tyl Aceta te. Acetic anhydride (1.5 mL, 15.4
mmol) and 7-bromo-1-heptanol (1.6 mL, 10.3 mmol) were
combined in CH2Cl2 (30 mL). DMAP (0.6 g, 10.3 mmol) was
added, and the reaction was stirred at room temperature
overnight. The reaction was diluted with CH2Cl2 (30 mL) and
washed with 1 N HCl (50 mL), 1 N NaOH (50 mL), saturated
2-[2-(4-(2-Cycloh exyleth yl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (23). Alkylating
agent ) 1-bromo-2-cylohexylethane. 1H (CDCl3): 7.28-7.18 (m,

Benzoxazinones as PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 205
NaHCO3 (50 mL), water (50 mL), and brine (50 mL). The
organic layer was dried (Na2SO4) and filtered, and the solvent
was removed in vacuo. The product was obtained as a colorless
oil and used in the synthesis of 40.
7.28-7.17 (m, 2H), 7.05-6.89 (m, 6H), 4.81 (dd, J ) 9./, 3.7,
1H), 4.20 (m, 2H), 3.92 (t, J ) 7.6, 2H), 3.62 (dd, J ) 19.1,
16.2, 2H), 2.44 (m, 1H), 2.23 (m, 1H), 1.90-1.41 (m, 11H). m/z
(MH+). Anal. (C25H29F2NO5) C, H, N.
(2-[2-(4-(7-Hydr oxyh eptyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (40). 1H (CDCl3):
7.27-7.17 (m, 2H), 7.06-6.88 (m, 6H), 4.84 (dd, J ) 9.7, 3.5,
1H), 4.21 (m, 2H), 3.92 (m, 2H), 3.61 (m, 4H), 2.43 (m, 1H),
2.21 (m, 1H), 1.66 (m, 2H), 1.55 (m, 2H), 1.36 (s, 6H). m/z
(MH+) 442.0. Anal. (C25H31NO6) C, H, N.
(2-[2-(3-Oxo-4-(5-oxoh exyl)-3,4-d ih yd r o-2H-ben zo[1,4]-
oxazin -2-yl)eth oxy]ph en ylacetic Acid (42). Alkylating agent
) 1-chloro-5-hexanone. 1H (CDCl3): 7.28-7.18 (m, 2H), 7.05-
6.89 (m, 6H), 4.85 (dd, J ) 9.1, 3.7, 1H), 4.22 (m, 2H), 3.94
(m, 2H), 3.62 (dd, J ) 20.5, 15.9, 2H), 2.50 (m, 2H), 2.41 (m,
1H), 2.23 (m, 1H), 2.13 (s, 3H), 1.64 (m, 4H). m/z (MH+) 426.1.
Anal. (C24H27NO6) C, H, N.
(2-[2-(3-Oxo-4-(6-oxoh ep tyl)-3,4-d ih yd r o-2H-ben zo[1,4]-
oxazin -2-yl)eth oxy]ph en ylacetic Acid (43). Alkylating agent
) 1-bromo-6 heptanone. 1H (CDCl3): 7.28-7.17 (m, 2H), 7.03-
6.89 (m, 6H), 4.81 (dd, J ) 9.3, 3.7, 1H), 4.21 (m, 2H), 3.92
(m, 2H), 3.61 (dd, J ) 19.3, 16.1, 2H), 2.43 (t, J ) 7.3, 3H),
2.23 (m, 1H), 2.13 (s, 3H), 1.64 (m, 4H), 1.38 (m, 2H). m/z (M
- 1) 438.1. Anal. (C25H29NO6) C, H, N.
1-Br om o-6-flu or oh exa n e. Caution: DAST reacts violently
with water. 1-Bromohexan-6-ol (2.0 mL, 15.2 mmol) and DAST
(4.0 mL, 30.5 mmol) were mixed at room temperature and then
stirred at 35 °C for 4 h. The reaction was poured into ice (150
mL) and extracted with CH2Cl2 (3 × 50 mL). The organic layer
was dried (Na2SO4) and filtered, and the solvent was removed
in vacuo. The product was obtained by silica gel chromatog-
raphy with CH2Cl2 and used in the synthesis of 50.
(2-[2-(4-(6-F lu or oh exyl)-3-oxo-3,4-d ih yd r o-2H -b en zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (50). 1H (CDCl3):
7.29-7.19 (m, 2H), 7.03-6.89 (m, 6H), 4.87 (dd, J ) 9.0, 3.5,
1H), 4.52 (t, J ) 6.0, 1H), 4.36 (t, J ) 6.0, 1H), 4.23 (m, 2H),
3.93 (t, J ) 7.6, 2H), 3.63 (dd, J ) 21.8, 15.8, 2H), 2.40 (m,
1H), 2.23 (m, 1H), 1.68 (m, 4H), 1.45 (m, 4H). m/z (MH+) 430.0.
Anal. (C24H28FNO5) C, H, N.
Compound 50 was resolved into enantiomers 51 and 52 by
chiral chromatography with a Chiralpak AD column (2 cm ×
25 cm). The mobile phase was 80:20:0.1 hexane/2-propanol/
TFA, and the flow rate was 9 mL/min. Retention times: (R)
) 19.5 min; (S) ) 24.6 min.
1-Ch lor o-5,5-d iflu or oh exa n e. Caution: (Diethylamino)-
sulfur trifluoride (DAST) reacts violently with water. 1-Chloro-
5-oxohexane (1.37 g, 10.2 mmol) and DAST (2.6 mL, 19.7
mmol) were mixed at room temperature and then stirred at
50 °C overnight. The reaction was poured into ice (150 mL),
adjusted to pH 5, and extracted with Et2O (3 × 50 mL). The
organic layer was washed with water (50 mL) each and brine
(50 mL). The organic layer was then dried (Na2SO4) and
filtered, and the solvent was removed in vacuo. The product
was obtained as a colorless oil, contaminated with <10% of
the starting ketone. The material was used without purifica-
tion in the synthesis of 46.
(2-[2-(4-(5,5-Diflu or oh exyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (46). 1H (CDCl3):
7.30-7.18 (m, 2H), 7.08-6.89 (m, 6H), 4.85 (dd, J ) 9.1, 3.7,
1H), 4.25 (m, 2H), 3.96 (t, J ) 8.0, 2H), 3.66 (dd, J ) 20.5,
15.9, 2H), 2.46 (m, 2H), 2.23 (m, 1H), 1.96-1.52 (m, 9H). m/z
(MH+) 448.1. Anal. (C24H27F2NO5) C, H, N.
(R)-(2-[2-(4-(6-Flu or oh exyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (51). 1H (CDCl3):
7.29-7.19 (m, 2H), 7.03-6.89 (m, 6H), 4.87 (dd, J ) 9.0, 3.5,
1H), 4.52 (t, J ) 6.0, 1H), 4.36 (t, J ) 6.0, 1H), 4.23 (m, 2H),
3.93 (t, J ) 7.6, 2H), 3.63 (dd, J ) 21.8, 15.8, 2H), 2.40 (m,
1H), 2.23 (m, 1H), 1.68 (m, 4H), 1.45 (m, 4H). m/z (MH+) 430.0.
Anal. (C24H28FNO5) C, H, N.
(S)-(2-[2-(4-(6-Flu or oh exyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (52). 1H (CDCl3):
7.25-7.16 (m, 2H), 7.01-6.88 (m, 6H), 4.80 (dd, J ) 9.2, 3.8,
1H), 4.48 (t, J ) 6.0, 1H), 4.36 (t, J ) 6.0, 1H), 4.20 (m, 2H),
3.91 (t, J ) 7.6, 2H), 3.61 (dd, J ) 21.8, 15.8, 2H), 2.44 (m,
1H), 2.23 (m, 1H), 1.67 (m, 4H), 1.45 (m, 4H). m/z (MH+) 430.0.
Anal. (C24H28FNO5‚0.25H2O) C, H, N.
1-Br om o-7-flu or oh ep ta n e. Caution: DAST reacts violently
with water. 1-Bromohexan-7-ol (1.2 mL, 7.7 mmol) and DAST
(1.5 mL, 11.5 mmol) were mixed at room temperature and then
stirred at 50 °C for 4 h. The reaction was poured into ice (150
mL) and extracted with CH2Cl2 (3 × 50 mL). The organic layer
was dried (Na2SO4) and filtered, and the solvent was removed
in vacuo. The product was obtained by silica gel chromatog-
raphy with CH2Cl2 and used in the synthesis of 53.
Compound 46 was resolved into enantiomers 47 and 48 by
chiral chromatography with a Chiralpak AD column (2 cm ×
25 cm). The mobile phase was 80:20:0.1 hexane/2-propanol/
TFA, and the flow rate was 9 mL/min. Retention times: (R)
) 19.0 min; (S) ) 24.4 min.
(2-[2-(4-(7-F lu or oh ep tyl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (53). 1H (CDCl3):
7.28-7.17 (m, 2H), 7.06-6.88 (m, 6H), 4.82 (dd, J ) 9.2, 3.7,
1H), 4.50 (t, J ) 6.1, 1H), 4.35 (t, J ) 6.1, 1H), 4.21 (m, 2H),
3.91 (t, J ) 7.6, 2H), 3.62 (dd, J ) 19.6, 16.0, 2H), 2.44 (m,
1H), 2.23 (m, 1H), 1.66 (m, 4H), 1.39 (m, 6H). m/z (M - 1)
442.1. Anal. (C25H30FNO5) C, H, N.
(R)-(2-[2-(4-(5,5-Diflu or oh exyl)-3-oxo-3,4-d ih yd r o-2H -
ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (47). 1H
(CDCl3): 7.25 (m, 1H), 7.17 (m, 1H), 7.02-6.88 (m, 6H), 4.80
(dd, J ) 9.1, 3.8, 1H), 4.21 (m, 2H), 3.93 (t, J ) 7.5, 2H), 3.60
(dd, J ) 20.4, 16.1, 2H), 2.45 (m, 1H), 2.24 (m, 1H), 1.88 (m,
2H), 1.70 (m, 1H), 1.57 (m, 5H). m/z (MH+) 448.1. Anal.
(C24H27F2NO5) C, H, N.
(2-[2-(3-Oxo-(2-p r op ylsu lfa n ylet h yl)-3,4-d ih yd r o-2H -
b en zo[1,4]oxa zin -2-yl)et h oxy]p h en yla cet ic Acid (54).
Alkylating agent ) 2-chloroethyl-n-propylsulfide. 1H (CDCl3):
7.28-7.17 (m, 2H), 7.05-6.89 (m, 6H), 4.82 (dd, J ) 9.2, 3.7,
1H), 4.21 (m, 2H), 4.10 (q, J ) 9.3, 6.3, 2H), 3.62 (dd, J ) 19.0,
16.1, 2H), 2.74 (dd, J ) 8.7, 6.9, 2H), 2.59 (t, J ) 7.3, 2H),
2.46 (m, 1H), 2.24 (m, 1H), 1.64 (hex, J ) 7.3, 2H), 1.00 (t, J
) 7.3, 3H). m/z (MNa+) 451.9. Anal. (C23H27NO5S) C, H, N.
(2-[2-(4-(3-Eth oxyp r op yl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (55). Alkylating
agent ) 3-ethoxy-1-propanol. See the synthesis of 21 for the
synthetic method. 1H (CDCl3): 7.28-6.89 (m, 8H), 4.82 (dd, J
) 9.1, 3.7, 1H), 4.19 (m, 2H), 4.03 (m, 2H), 3.62 (dd, J ) 18.5,
16.3, 2H), 3.46 (m, 4H), 2.44 (m, 1H), 2.23 (m, 1H), 1.94 (quint,
J ) 6.5, 2H), 1.21 (t, J ) 7.1, 3H). m/z (M - 1) 412.1. Anal.
(C23H27NO6) C, H, N.
(S)-(2-[2-(4-(5,5-Diflu or oh exyl)-3-oxo-3,4-d ih yd r o-2H -
ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (48). 1H
(CDCl3): 7.25 (m, 1H), 7.17 (m, 1H), 7.03-6.88 (m, 6H), 4.80
(dd, J ) 9.1, 3.8, 1H), 4.21 (m, 2H), 3.93 (t, J ) 7.5, 2H), 3.60
(dd, J ) 20.4, 16.1, 2H), 2.45 (m, 1H), 2.22 (m, 1H), 1.87 (m,
2H), 1.70 (m, 1H), 1.57 (m, 5H). m/z (MH+) 448.0. Anal.
(C24H27F2NO5‚0.75H2O) C, H, N.
1-Br om o-6,6-d iflu or oh ep t a n e. Caution: DAST reacts
violently with water. 1-Bromo-6-oxoheptane (1.1 g, 5.7 mmol)
and DAST (1.5 mL, 11.4 mmol) were mixed at room temper-
ature and then stirred at 50 °C overnight. The reaction was
poured into ice (150 mL), adjusted to pH 4, and extracted with
CH2Cl2 (3 × 50 mL). The organic layer was washed with water
(50 mL) and brine (50 mL). The organic layer was then dried
(Na2SO4) and filtered, and the solvent was removed in vacuo.
The product was obtained as a colorless oil, contaminated with
<10% of the starting ketone. The material was used without
purification in the synthesis of 49.
(2-[2-(4-(4-Meth oxybu tyl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (56). Alkylating
agent ) 1-bromo-4-methoxybutane. 1H (CDCl3): 7.26-6.74 (m,
8H), 4.81 (m, 1H), 4.04 (m, 2H), 3.85 (m, 2H), 3.32 (m, 4H),
(2-[2-(4-(6,6-Diflu or oh eptyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (49). 1H (CDCl3):

206 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
3.26 (s, 3H) 2.29 (m, 1H), 2.05 (m, 1H), 1.59 (m, 4H). m/z
(MNa+) 436.0. Anal. (C23H27NO6 ‚ 0.75 H2O) C, H, N.
NaHCO3 (15 mL) and brine (15 mL). The organic layer was
dried (Na2SO4) and filtered, and the solvent was removed in
vacuo. The product was elaborated to compound 31 by the
methods described for compounds 3-5. 1H (CDCl3): 7.32-7.22
(m, 2H), 7.19-6.91 (m, 6H), 6.20 (bs, 1H), 4.94 (dd, J ) 8.5,
4.4, 1H) 4.72 (d, J ) 16.0, 1H), 4.36 (d, J ) 16.0, 1H), 4.32-
4.26 (m, 2H), 3.62 (s, 2H), 3.57-3.18 (m, 2H), 2.61-2.54 (m,
2H), 2.34-2.27 (m, 1H), 1.52-1.43 (m, 2H), 0.86 (t, J ) 7.4,
3H). m/z (MNa+) 449.4. Anal. (C23H26N2O6) C, H, N.
(2-[2-(4-(4-Car bam oylbu tyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (29). Compound
58 (2.0 g, 3.8 mmol, see the synthesis of 26) was dissolved in
EtOH (50 mL) and debenzylated via hydrogenation with 10%
Pd/C at 50 psi for 3.0 h. The reaction was filtered through
Celite, and the solvent was removed in vacuo. A portion of the
product (0.3 g, 0.68 mmol) was dissolved in CH2Cl2 (15 mL).
1,1′-Carbonyldiimidazole (0.22 g, 1.4 mmol) was added, and
the solution was stirred for 2 h. Ammonium hydroxide (0.1
mL, 1.4 mmol) was added, and the reaction was stirred
overnight at room temperature. The reaction was diluted with
water (25 mL), and the pH was adjusted to 7 with 1 N HCl.
The aqueous layer was extracted with EtOAc (3 × 20 mL).
The organic layer was washed with water (25 mL) and brine
(25 mL). The organic layer was then dried (Na2SO4) and
filtered, and the solvent was removed in vacuo. The methyl
ester was saponified by the method for example 5 to yield 29.
1H (CD3OD): 7.26-7.15 (m, 2H), 7.09-6.86 (m, 6H), 4.82 (dd,
J ) 9.4, 4.1, 1H), 4.18 (m, 2H), 3.99 (m, 2H), 3.57 (dd, J )
19.7, 16.2, 2H), 2.39 (m, 1H), 2.25 (m, 3H), 1.67 (m, 4H). m/z
(M-1) 425.0. Anal. (C23H26N2O6) C, H, N.
2-[2-(4-(3-Meth ylca r ba m oylp r op yl)-3-oxo-3,4-d ih yd r o-
2H-ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (32).
Compound 32 was prepared by the methods described for
compound 31 via intermediate 61. 1H (CDCl3): 7.31-6.91 (m,
8H), 5.94 (bs, 1H), 4.88 (dd, J ) 9.1, 3.8, 1H), 4.29-4.19 (m,
2H), 4.08-3.93 (m, 2H), 3.65 (d, J ) 3.4, 2H), 2.81 (d, J ) 5.1,
3H), 2.49-2.41 (m, 1H), 2.29-2.21 (m, 3H), 2.08-1.99 (m, 2H).
m/z (MNa+) 449.4. Anal. (C23H26N2O6) C, H, N.
(2-[2-(4-(6-Acet yla m in oh exyl)-3-oxo-3,4-d ih yd r o-2H -
ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (33). A
solution of the amide 2 (3.0 g, 9.8 mmol) in dry DMF (10 mL)
was added to a suspension of sodium hydride (0.352 g, 11.7
mmol) in dry DMF (15 mL) at 0 °C. After 30 min at 0 °C,
6-bromohexyl phthalimide (4.0 g, 12.7 mmol) was added. The
reaction was stirred for 30 min at 0 °C and then overnight at
50 °C. The reaction was quenched with 1 N aqueous HCl (15
mL), diluted with water (15 mL), and extracted with EtOAc
(3 × 30 mL). The organic layer was washed with water (2 ×
20 mL) and brine (20 mL). The organic layer was then dried
(Na2SO4) and filtered, and the solvent was removed in vacuo.
The crude material (6.2 g, 11.6 mmol) was dissolved in
methanol (150 mL) and water (20 mL) and then treated with
CH3SO3H (4 mL) and stirred for 2 h at room temperature. The
mixture was diluted with EtOAc (150 mL), and then, the
organic layer was washed with saturated aqueous NaHCO3
(50 mL), water (50 mL), and brine (50 mL). The organic layer
was then dried (Na2SO4) and filtered, and the solvent was
removed in vacuo to yield 62. A solution of 62 (5.0 g, 11.8
mmol), (2-hydroxyphenyl)acetic acid methyl ester (3.9 g, 17.8
mmol), and tributylphosphine (4.4 mL, 17.8 mmol) in dry
benzene (200 mL), under N2, was cooled to 10 °C. 1,1′-
(Azodicarbonyl)dipiperidine (4.5 g, 17.8 mmol) was added in
one portion, and the solution was stirred at room temperature
overnight. The organic layer was washed with 5 N aqueous
NaOH (4 × 25 mL) and brine (25 mL). The organic layer was
then dried (Na2SO4) and filtered, and the solvent was removed
in vacuo. The product was obtained as a yellow solid by silica
gel chromatography with hexane/EtOAc (3.1 g, 5.4 mmol).
Hydrazine (0.17 mL, 5.48 mmol) was added to a suspension
of the purified product (2.6 g, 4.57 mmol) in EtOH (13 mL)
and THF (13 mL). The reaction was stirred at 60 °C overnight
and then diluted with MeOH and filtered. The solvent was
evaporated to give 63 (1.46 g, 3.3 mmol) as a white solid.
Compound 63 (0.2 g, 0.45 mmol) was stirred in acetic anhy-
dride (6 mL) overnight. MeOH (10 mL) was added, and the
solvent was removed in vacuo. Purification by reverse phase
HPLC gave the desired acetamide (0.066 g, 0.14 mmol) as a
clear oil. NaOH (1 N, 1 mL, 1 mmol) was added to a solution
of the acetamide (0.066 g, 0.14 mmol) in 5 mL of MeOH. The
reaction was stirred at 45 °C overnight and then acidified to
pH 5 with 1 N HCl and extracted with EtOAc (10 mL). The
organic layer was washed with water (5 mL) and brine (5 mL).
The organic layer was then dried (Na2SO4) and filtered, and
the solvent was removed in vacuo. Compound 33 was obtained
(2-[2-(4-(5-Ca r b a m oylp e n t yl)-3-oxo-3,4-d ih yd r o-2H -
ben zo[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (30). Com-
pound 30 was prepared using the methods described for
1
compound 29. H (DMSO-d6): 7.26-7.17 (m, 2H), 7.11-6.98
(m, 5H), 6.89 (t, J ) 7.4, 1H), 4.80 (dd, J ) 9.2, 4.0, 1H), 4.15
(m, 2H), 3.90 (t, J ) 7.3, 2H), 3.50 (s, 2H), 2.27 (m, 1H), 2.02
(m, 3H), 1.53 (m, 4H), 1.28 (m, 2H). m/z (M-1) 439.1. Anal.
(C24H28N2O6) C, H, N.
(2-[2-(4-(5-Hydr oxypen tyl)-3-oxo-3,4-dih ydr o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (37). Compound
58 (2.0 g, 3.8 mmol, see the synthesis of 26) was dissolved in
EtOH (50 mL) and debenzylated via hydrogenation with 10%
Pd/C at 50 psi for 3.0 h. The reaction was filtered through
Celite, and the solvent was removed in vacuo. A portion of the
product (0.15 g, 0.34 mmol) was dissolved in dry THF (10 mL)
and chilled to -50 °C. Borane-THF (0.7 mL, 0.68 mmol) was
added, and the reaction was stirred while warming to 0 °C
over 8 h. The reaction was quenched with 0.05 N aqueous HCl.
THF was removed in vacuo, and the crude product was
dissolved in EtOAc (150 mL). The organic layer was washed
with water (50 mL) and brine (50 mL). The organic layer was
then dried (Na2SO4) and filtered, and the solvent was removed
in vacuo. The methyl ester was saponified by the method for
1
example 5 to yield 37. H (CD3OD): 7.25-6.87 (m, 8H), 4.82
(dd, J ) 9.4, 4.0, 1H), 4.18 (m, 2H), 3.98 (t, J ) 7.5, 2H), 3.54
(m, 4H), 2.39 (m, 1H), 2.18 (m, 1H), 1.72-1.42 (m, 6H). m/z
(M - 1) 412.1. Anal. (C23H27NO6) C, H, N.
(2-[2-(4-(6-Hyd r oxyh exyl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (39). Compound
39 was prepared using the methods described for compound
37 via intermediate 59. 1H (CDCl3): 7.27-7.16 (m, 2H), 7.02-
6.88 (m, 6H), 4.86 (dd, J ) 9.3, 3.3, 1H), 4.22 (m, 2H), 3.94
(m, 2H), 3.61 (m, 4H), 2.40 (m, 1H), 2.20 (m, 1H), 1.70-1.36
(m, 8H). m/z (M - 1) 426.1. Anal. (C25H31NO6) C, H, N. Anal.
(C24H29NO6) C, H, N.
2-[2-(3-Oxo-4-p r op ylca r ba m oylm eth yl-3,4-d ih yd r o-2H-
ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (31). A
solution of the amide 2 (0.8 g, 2.6 mmol) in dry DMF (5 mL)
was added to a suspension of sodium hydride (0.087 g, 2.86
mmol) in dry DMF (5 mL) at 0 °C. After 30 min at 0 °C, ethyl
bromoacetate (0.35 mL, 3.12 mmol) was added and the reaction
was stirred for 30 min at 0 °C and then overnight at 50 °C.
The reaction was quenched with 1 N HCl (5 mL), diluted with
water (10 mL), and extracted with EtOAc (2 × 15 mL). The
organic layer was washed with water (2 × 10 mL) and brine
(10 mL). The organic layer was then dried (Na2SO4) and
filtered, and the solvent was removed in vacuo. The crude
product (60, 1 g, 2.54 mmol) was dissolved in MeOH (10 mL),
and propylamine (1.46 mL, 17.78 mmol) was added. The
reaction was stirred overnight at 45 °C. The mixture was
diluted with EtOAc (25 mL) and washed with saturated
1
as a clear oil (40 mg, 0.08 mmol). H (CDCl3): 8.16 (bs, 1H),
7.24-7.16 (m, 2H), 7.06-6.87 (m, 6H), 5.29 (bs, 1H), 4.83 (dd,
J ) 9.2, 3.7, 1H), 4.27-4.19 (m, 2H), 3.99-3.85 (m, 2H), 3.61
(s, 2H), 3.19 (dd, J ) 12.8, 6.7, 2H), 2.49-2.39 (m, 1H), 2.27-
2.16 (m, 1H), 1.96 (s, 3H), 1.68-1.63 (m, 2H), 1.49-1.43 (m,
2H), 1.35-1.23 (m, 4H). m/z (MNa+) 491.2. Anal. (C26H32N2O6‚
1.0Na‚0.75H2O) C, H, N.
(2-[2-(4-(6-Met h a n esu lfon yla m in oh exyl)-3-oxo-3,4-d i-
h ydr o-2H-ben zo[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid
(34). Compound 34 was prepared by the methods described
1
for compound 33. H (CDCl3): 7.28-7.16 (m, 2H), 7.07-6.88

Benzoxazinones as PPARγ Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 207
(m, 6H), 4.82 (dd, J ) 9.1, 3.6, 1H), 4.25-4.16 (m, 2H), 3.98-
3.87 (m, 2H), 3.61 (s, 2H), 3.09 (dd, J ) 13.2, 6.6, 2H), 2.92 (s,
3H), 2.46-2.40 (m, 1H), 2.27-2.17 (m, 1H), 1.72-1.62 (m, 2H),
1.59-1.46 (m, 2H), 1.41-1.25 (m, 4H). m/z (MNa+) 527.3. Anal.
(C25H32N2O7S) C, H, N.
g, 0.3 mol) in dry THF (550 mL), under N2, was cooled to -20
°C. A room temperature solution of DEAD (47.5 mL, 0.3 mol)
in THF (20 mL) was added dropwise over 30 min. The reaction
was stirred for 17 h as the cold bath thawed. The mixture was
poured into water (3L) containing NaCl (200 g), and the
solution was washed with a 1:1 ratio of Et2O/EtOAc (6 × 100
mL). The organic layer was washed with water (5 × 100 mL)
and brine (100 mL). The organic layer was then dried (Na2SO4)
and filtered, and the solvent was removed in vacuo. The
product was purified by silica gel chromatography with CH2Cl2/
EtOAc and then crystallized from hexane/EtOAc to yield 65
as a pale yellow solid (21.16 g, 95 mmol, 47%). 1H (CDCl3):
7.86 (d, J ) 8.1, 1H), 7.58 (t, J ) 8.6, 1H), 7.50 (d, J ) 7.8,
1H), 7.16 (t, J ) 7.4, 1H), 5.04 (t, J ) 7.4, 1H), 4.58 (m, 1H),
4.40 (q, J ) 7.4, 1H), 2.8-2.6 (m, 2H). By HPLC, the
enantiomeric purity was >99% for the crystalline sample (8:2
hexane/2-propanol, retention time chiral ) 13.8 min, retention
time racemic ) 11.1 min, 13.7 min).
(R)-4-Hexyl-2-(2-h yd r oxyeth yl)-4H-ben zo[1,4]oxa zin -3-
on e (66). The phenolic ether 65 (21.16 g, 0.095 mol) was
suspended in EtOH (400 mL) and then shaken for 3 h at room
temperature with 10% Pd/C and H2 (45 psi). The solution was
filtered through Celite, and the solvent was removed in vacuo.
The crude benzoxazinone was dissolved in dry DMF (200 mL),
imidazole (16.3 g, 0.24 mol) was added, and the solution was
cooled to 0 °C. TBS chloride (28.6 g, 0.19 mol) was added as a
solid, and the reaction was stirred overnight, under N2, as the
bath thawed. The reaction was poured into water (1.4 L)
containing NaCl (200 g) and washed with a 4:1 ratio of Et2O/
CH2Cl2 (4 × 150 mL). The organic layer was washed with
water (6 × 100 mL) and brine (100 mL). The organic layer
was then dried (Na2SO4) and filtered, and the solvent was
removed in vacuo. The product was isolated by silica gel
chromatography with hexane/EtOAc. Note: The solid is vola-
tile and sublimes when dried under vacuum for long periods.
A solution of the silyl ether (26.75 g, 87 mmol) in dry DMF
(435 mL), under N2, was cooled to 0 °C. Sodium hydride (75%
dispersion in oil, 2.48 g, 83 mmol) was added in four portions
of 0.62 g with 5 min intervals between additions. The solution
was stirred for an additional 40 min at 0 °C. 1-Iodohexane
(12.8 mL, 87 mmol) in dry DMF (25 mL) was added dropwise,
the ice bath was replaced with an oil bath, and the solution
was stirred at 65 °C overnight. The mixture was cooled to room
temperature and poured into water (3 L) containing NaCl (200
g). The aqueous mixture was washed with a ratio of 1:1 Et2O/
EtOAc (4 × 125 mL). The organic layer was washed with water
(6 × 125 mL) and brine (125 mL). The organic layer was then
dried (Na2SO4) and filtered, and the solvent was removed in
vacuo. The product was obtained as a colorless oil by silica
gel chromatography with hexane/EtOAc (26.8 g, 68 mmol,
79%). The silyl ether was dissolved in MeOH (150 mL). HCl
(6 N, 0.5 mL) was added, and the mixture was stirred at room
temperature for 5 h. The solvent was removed in vacuo, and
the product was isolated by silica gel chromatography with
hexane/EtOAc. The primary alcohol 66 was obtained as a
colorless oil (16.7 g, 60 mmol, 88%). 1H (CDCl3): 7.01 (m, 4H),
4.69 (t, J ) 7.0, 1H), 3.88 (m, 4H), 2.44 (t, J ) 5.8, 1H), 2.20
(m, 2H), 1.65 (m, 2H), 1.33 (m, 6H), 0.89 (br t, 3H). m/z (MH+)
278.0. By HPLC, the enantiomeric purity was 97.4:2.1 (9:1
hexane/2-propanol, retention time chiral ) 7.5 min, retention
time racemic ) 7.6 min, 8.5 min).
(R)-2-[2-(4-Hexyl-3-oxo-3,4-dih ydr o-2H-ben zo[1,4]oxazin -
2-yl)eth oxy]p h en yla cetic Acid (13). A solution of 66 (16.7
g, 0.06 mol), (2-hydroxyphenyl)acetic acid methyl ester (15 g,
0.09 mol), and tributylphosphine (22.4 mL, 0.09 mol) in dry
benzene (1 L), under N2, was cooled to 10 °C. 1,1′-(Azodicar-
bonyl)dipiperidine (22.7 g, 0.09 mol) was added in one portion,
and the solution was stirred, with an overhead stirrer, at room
temperature overnight. Water (130 mL) was added, and
stirring was continued for 40 min. The mixture was trans-
ferred to a separatory funnel. The organic layer was washed
with water (4 × 100 mL) and brine (100 mL). The organic
phase was dried (Na2SO4) and filtered, and the solvent was
removed in vacuo. The product was purified by silica gel
(2-[2-(4-(5-Hyd r oxy-5-m eth ylh exyl)-3-oxo-3,4-d ih yd r o-
2H-ben zo[1,4]oxa zin -2-yl)eth oxy]p h en yla cetic Acid (38).
Compound 42 (0.4 g, 1 mmol) was dissolved in dry THF (10
mL), and the solution was cooled to -78 °C. Methylmagnesium
bromide (0.7 mL, 3.0 M in ether) was added, and the reaction
was stirred for 5 h at room temperature. Excess reagent was
quenched with saturated aqueous NH4Cl, and the aqueous
layer was extracted with 1:1 Et2O/CH2Cl2 (3 × 25 mL). The
organic layer was washed with water (25 mL) and brine (25
mL). The organic layer was then dried (Na2SO4) and filtered,
and the solvent was removed in vacuo. The product was
isolated as a colorless oil. 1H (CDCl3): 7.28-7.08 (m, 2H),
6.93-6.81 (m, 6H), 4.78 (m, 1H), 4.21-3.97 (m, 3H), 3.78 (m,
1H), 3.51 (m, 2H), 2.44 (m, 1H), 2.12 (m, 1H), 1.69-1.36 (m,
6H), 1.15 (m, 6H). m/z (M - 1) 440.0. Anal. (C25H31NO6‚
0.75H2O) C, H, N.
(2-[2-(4-(5-Hyd r oxyim in oh exyl)-3-oxo-3,4-d ih yd r o-2H-
ben zo[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (44). Com-
pound 42 (4.72 g, 11 mmol) was stirred at room temperature
as a slurry in EtOH (200 mL). Lutidine (2.6 mL, 22 mmol)
and hydroxylamine (3.8 g, 55 mmol) were added. The mixture
rapidly became clear, and the reaction was complete in 2 h.
Solvent was removed in vacuo, and the residue was dissolved
in EtOAc (100 mL) and water (50 mL). The organic layer was
washed with 0.1 N aqueous HCl (2 × 50 mL), water (50 mL),
and brine (50 mL). The organic layer was then dried (Na2SO4)
and filtered, and the solvent was removed in vacuo. The
product was obtained as a pale yellow solid (4.65 g, 10.5 mmol,
95%) and as a 3.4:1 E/Z mixture of oximes. 1H (CDCl3): 7.30-
7.17 (m, 2H), 7.05-6.90 (m, 6H), 4.87 (dd, J ) 9.4, 3.7) and
4.76 (dd, J ) 10.0, 3.3) 1H, 4.27 (m, 2H), 4.15 (m, 1H), 3.88
(m, 1H), 3.61 (dd, J ) 24.0, 15.4, 2H), 2.56 (m, 1H), 2.38-2.14
(m, 3H), 1.90 (s) and 1.82 (s) 3H, 1.78-1.46 (m, 4H). m/z (MH+)
441.1. Anal. (C24H28N2O6) C, H, N.
(2-[2-(4-(5-Meth oxyim in oh exyl)-3-oxo-3,4-d ih yd r o-2H-
ben zo[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (45). Com-
pound 42 (0.1 g, 0.24 mmol) was stirred at room temperature
as a slurry in EtOH (5 mL). Pyridine (0.1 mL, 1.23 mmol) and
O-methylhydroxylamine (0.098 g, 1.17 mmol) were added. The
mixture rapidly became clear, and the reaction was complete
in 0.5 h. Solvent was removed in vacuo, and the residue was
dissolved in EtOAc (20 mL) and water (10 mL). The organic
layer was washed with 0.1 N aqueous HCl (2 × 50 mL), water
(50 mL), and brine (50 mL). The organic layer was then dried
(Na2SO4) and filtered, and the solvent was removed in vacuo.
The product was obtained as a pale yellow solid (0.087 g, 0.19
1
mmol, 80%) and as a 3:1 E/Z mixture of oximes. H (CDCl3):
7.28-7.15 (m, 2H), 7.05-6.82 (m, 6H), 4.85 (dd, J ) 9.1, 3.6,
1H), 4.21 (m, 2H), 3.82 (s) and 3.80 (s) 3H, 3.62 (dd, J ) 20.4,
16.0, 2H), 2.43 (m, 1H), 2.21 (m, 3H), 1.84 (s) and 1.81 (s) 3H,
1.62 (m, 4H). m/z (MH+) 455.0. Anal. (C25H30N2O6‚0.4H2O) C,
H, N.
(2-[2-(4-(5-Hyd r oxyh exyl)-3-oxo-3,4-d ih yd r o-2H-ben zo-
[1,4]oxazin -2-yl)eth oxy]ph en ylacetic Acid (41). 1H (CDCl3):
7.26-7.17 (m, 2H), 7.00-6.86 (m, 6H), 4.87 (m, 1H), 4.29 (m,
2H), 4.07 (m, 2H), 3.85 (m, 2H), 3.59 (m, 2H), 2.49 (m, 1H),
2.21 (m, 1H), 1.72 (m, 2H), 1.50 (m, 6H), 1.19 (m, 3H). m/z
(MH+) 428.0. Anal. (C24H29NO6‚0.5H2O) C, H, N.
Ster eosp ecific Syn th esis. Analytical chiral HPLC was
performed on a Hewlett-Packard1090 Series II AminoQuant
HPLC fitted with a Daicel Chemical Industries, LTD Chiral-
pak AD column (4.6 mm × 25 cm). The sample concentration
was 1 mg/mL in eluting solvent, the flow rate was 1 mL/min,
and UV detection was at 254 nm. Solvent and retention time
of the chiral and racemic are listed with the individual
experimental.
(R)-3-(2-Nitr op h en oxy)d ih yd r ofu r a n -2-on e (65). A solu-
tion of 2-nitrophenol (27.8 g, 0.2 mol), (S)-(-)-R-hydroxy-γ-
butyrolactone (15.3 mL, 0.2 mol), and triphenylphosphine (78.6

208 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Rybczynski et al.
chromatography with hexane/EtOAc. The ester was obtained
as a colorless oil (24.3 g, 0.057 mol, 95%). H (CDCl3): 7.28-
Sigma Diagnostics Trinder reagent. All of the in vivo data were
analyzed using Prism program (Graphpad, Monrovia, CA), and
statistical analysis was performed using the one way analysis
of variance with a Dunnett’s multiple comparison test.
1
6.89 (m, 8H), 4.76 (dd, J ) 9.4, 4.0, 1H), 4.28-4.21 (m, 2H),
3.92 (t, J ) 7.7, 2H), 3.60 (m, 5H), 2.49 (m, 1H), 2.23 (m, 1H),
1.66 (m, 2H), 1.34 (m, 6H), 0.89 (m, 3H). A solution of the ester
(24.3 g, 0.057 mol) in THF (500 mL) was cooled to 0 °C.
Aqueous LiOH (0.85 N, 200 mL, 0.17 mol LiOH) at 10 °C was
added in one portion. The solution was stirred at room
temperature overnight, open to air. The solution was poured
into water (1 L), and the solution was brought to pH 4 by
portionwise addition of 28.5 mL of 6 N HCl. The aqueous layer
was extracted with CH2Cl2 (4 × 120 mL). The organic layer
was washed with 2:1 water/brine (3 × 150 mL). The organic
layer was then dried (MgSO4) and filtered, and the solvent
was removed in vacuo. The oily residue was diluted with
pentane (1 L) and Et2O (200 mL), heated on a steam bath,
and scratched with a glass rod until the material became a
white solid. The mixture was cooled to 0 °C for 1.5 h and then
filtered and washed with pentane (2 × 100 mL). The amor-
phous white solid was dried under vacuum at 40 °C (17.5 g,
Ack n ow led gm en t. We thank Mary Evangelisto for
the chromatographic resolution of enantiomeric com-
pounds, Richard Adams and Eugene Shmuylovich for
the large scale intermediate of racemic intermediates,
Gary Caldwell and J ohn Masucci for bioavailability
studies, and Mona Patel and Gee-Hong Kuo for careful
reading and helpful discussions on the manuscript.
Refer en ces
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26.
0.043 mol, 75%). mp 80.0-81.5 °C. [R]D ) +31.2 °c ) 1,
25
CHCl3. 1H (CDCl3): 7.28-6.89 (m, 8H), 4.84 (dd, 1H), 4.20 (m,
2H), 3.91 (t, 2H), 3.62 (dd, 2H), 2.43 (m, 1H), 2.23 (m, 1H),
1.65 (m, 2H), 1.33 (m, 6H), 0.88 (m, 3H). By HPLC, the
enantiomeric purity was 99:1 (80:20:0.1 hexane/2-propanol/
trifluoroacetic acid, retention time chiral ) 7.2 min, retention
time racemic ) 7.2 min, 8.7 min).
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(R)-(2-[2-(4-(4-Me t h oxyb u t yl)-3-oxo-3,4-d ih yd r o-2H -
b en zo[1,4]oxa zin -2-yl)et h oxy]p h en yla cet ic Acid (57).
Alkylating agent ) 1-bromo-4-methoxybutane. 1H (CDCl3):
7.26-6.74 (m, 8H), 4.81 (m, 1H), 4.04 (m, 2H), 3.85 (m, 2H),
3.32 (m, 4H), 3.26 (s, 3H) 2.29 (m, 1H), 2.05 (m, 1H), 1.59 (m,
4H). m/z (MH+) 436.0. Anal. (C23H27NO6) C, H, N.
Gen er a l Biology. The PPARγ in vitro aP2 induction assay
was run as described in ref 23. Incubations with human liver
microsomes were run by Absorption Systems (Exton, PA).
Bioa va ila bility. Rats were dosed intravenously (IV) at 3
mg/kg and by oral gavage at 30 mg/kg. The test compound
was formulated for IV dosing as a solution in 10% w/v Solutol
in 5% dextrose in sterile water vehicle (D5W) and formulated
for oral dosing as a uniform suspension in 0.5% methylcellulose
vehicle. Blood samples (0.5 mL) were collected into heparinized
tubes postdose via orbital sinus puncture and then centrifuged
for cell removal. Precisely 200 µL of plasma supernatant was
transferred to a clean vial, frozen with dry ice, and stored at
-70 °C prior to analysis. Four hundred microliters of aceto-
nitrile containing internal standard (Propranolol) was added
to 200 µL of plasma to precipitate proteins. Samples were
centrifuged at 5000g for 3 min, and the supernatant was
removed for analysis by LC-MS-MS. Calibration standards
were prepared by adding appropriate volumes of stock solution
directly into plasma and treatment identically to collected
plasma samples. Calibration standards were typically pre-
pared in the range of 0.1-10 µM for quantitation. LC-MS-MS
analysis was performed using either multiple reaction or
selected ion monitoring for detection of characteristic ions for
each drug candidate and internal standard. Results were
calculated by WinNonlin Pro version 3.1. Oral and intravenous
areas under the concentration vs time curve (AUC) were
compared, to determine the % bioavailability (%F) by the
following formula: dose (IV) × AUC (oral)/dose (oral) × AUC
(IV).
In Vivo Effica cy. Female db/db mice (C57 BLK S/J -m+/+
Leprdb mice (J ackson Labs, Bar Harbor, ME)), about 7 weeks
of age, were maintained on NIH Rat and Mouse/Auto 6F
Reduced Fat Diet #5K52 (PMI Nutrition International Inc.).
Animals were treated with vehicle or compound for 11
consecutive days by oral gavage (n ) 7-8). All mice were
weighed on day 1 prior to dosing and then on day 12. Eighteen
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