Design, synthesis, and evaluation of potent, structurally novel peroxisome proliferator-activated receptor (PPAR) δ-selective agonists

Article abstract of DOI:10.1016/j.bmc.2007.05.023

A series of 3-(4-alkoxyphenyl)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) δ-selective agonists, based on our previously discovered potent human PPARα/δ dual agonist TIPP-401 as a lead compound. Structure-activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the 4-position, and the n-butoxy compound exhibited the most potent PPARδ transactivation activity and highest PPARδ selectivity. The (S)-enantiomer of a representative compound exhibited extremely potent PPARδ transactivation activity, comparable with or somewhat superior to that of the known PPARδ-selective agonist, GW-501516. The representative compound regulated the expression of genes involved in lipid and glucose homeostasis, and should be useful not only as a chemical tool to study PPARδ function, but also as a candidate drug for the treatment of metabolic syndrome.

Full text of DOI:10.1016/j.bmc.2007.05.023

Bioorganic & Medicinal Chemistry 15 (2007) 5177–5190
Design, synthesis, and evaluation of potent, structurally
novel peroxisome proliferator-activated receptor (PPAR)
d-selective agonists
Jun-ichi Kasuga,a Izumi Nakagome,b Atsushi Aoyama,a Kumiko Sako,a
Michiyasu Ishizawa,c Michitaka Ogura,c Makoto Makishima,c Shuichi Hirono,b
Yuichi Hashimotoa and Hiroyuki Miyachia,*
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bSchool of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
cSchool of Medicine, Nihon University, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
Received 17 April 2007; revised 6 May 2007; accepted 8 May 2007
Available online 13 May 2007
Abstract—A series of 3-(4-alkoxyphenyl)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated
receptor (PPAR) d-selective agonists, based on our previously discovered potent human PPARa/d dual agonist TIPP-401 as a lead
compound. Structure–activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the
4-position, and the n-butoxy compound exhibited the most potent PPARd transactivation activity and highest PPARd selectivity.
The (S)-enantiomer of a representative compound exhibited extremely potent PPARd transactivation activity, comparable with or
somewhat superior to that of the known PPARd-selective agonist, GW-501516. The representative compound regulated the expres-
sion of genes involved in lipid and glucose homeostasis, and should be useful not only as a chemical tool to study PPARd function,
but also as a candidate drug for the treatment of metabolic syndrome.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
elevated blood pressure, elevated triglycerides, and low
HDL-cholesterol.
The increasing incidences of obesity, type 2 diabetes,
and dyslipidemia, and their consequences in terms of
cardiovascular morbidity and mortality, represent a
considerable public health problem.1 The metabolic syn-
drome is composed of an accumulation of metabolic and
cardiovascular risk factors that predispose to heart
attack, stroke, heart failure, and sudden cardiac death,2
and differences in genetic background, diet, physical
activity, age, gender, nutrition, and so on, all affect its
prevalence.3 This syndrome is an extremely important
diagnostic indication for the identification of high-risk
patients for multiple risk factor modification to prevent
or delay adverse cardiovascular events. Affected individ-
uals have visceral obesity, impaired glucose tolerance,
According to the current unifying definition, key ele-
ments of the metabolic syndrome include insulin resis-
tance, abnormal glucose metabolism, hypertension,
atherogenic dyslipidemia, and obesity.1,4 Based on these
criteria, it is obvious that losing weight and restoration
of serum glucose and serum lipid parameters to normal
levels are of primary importance. From this point of
view, the identification of molecular targets critically
involved in the regulation of energy balance and glucose
and lipid homeostasis is important for the creation of
the new therapeutic agents for the treatment of meta-
bolic syndrome. Nuclear receptors (NRs) are particu-
larly attractive molecular targets to study, since they
play a central role in maintaining energy balance and
in cellular and whole-body glucose and lipid homeosta-
sis. The NRs are activated by a wide variety of small
physiological ligands, such as dietary unsaturated fatty
acids, their metabolites, and various xenobiotics, there-
by modulating the transcriptional networks of the target
response genes. Among these receptors, special attention
Keywords: PPAR; PPARd; PPARd-selective agonist; Metabolic
syndrome.
*
Corresponding author. Tel.: + 81 03 5841 7848; fax: +81 03 5841
8495; e-mail: miyachi@iam.u-tokyo.ac.jp
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.023

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
has been paid to the members of the peroxisome prolif-
erator-activated receptor (PPAR) family (NR1Cs) for
more than a decade.
in association with decreased lipoprotein lipase activity,
in insulin-resistant middle-aged obese rhesus monkeys.12
In a primate model of the metabolic syndrome, PPARd
activation lowered plasma insulin levels, without any
adverse effect on glycemic control.12 Similarly, in the
case of ob/ob mice, PPARd activation markedly
improved glucose tolerance and insulin resistance,12
although the underlying mechanism remains unclear.13
PPARs are activated by endogenous saturated and
unsaturated fatty acids and their metabolites, as well
as synthetic ligands.5 Three subtypes of PPARs have
been identified to date, that is, PPARa, PPARc, and
PPARd.6 Each PPAR subtype is differentially expressed
in a tissue-specific manner, and they have pivotal roles
in lipid, lipoprotein, and glucose homeostasis.7 PPARa
is mostly expressed in the tissues involved in lipid oxida-
tion, such as liver, kidney, skeletal, cardiac muscle, and
adrenal glands.8 PPARc is expressed in adipose tissue,
macrophages, and vascular smooth muscles.9 In con-
trast, PPARd is ubiquitously expressed, though it is
mainly found in skeletal muscle and adipose tissues.10
PPARs function after heterodimerization with another
nuclear receptor partner, retinoid X receptor (RXR),
and regulate gene expression by binding to a specific
consensus DNA sequence, termed PPRE (peroxisome
proliferator responsive element), located in the pro-
moter region of target genes.7
In addition to these animal-model studies, recent preli-
minary phase I/II clinical studies of GW-501516 demon-
strated that a PPARd agonist elevated HDL-cholesterol
levels (phase I) and decreased triglyceride (phase I,
phase I/II), total cholesterol (phase I/II), and apoB100
levels (phase I/II).14 All these observations suggest that
PPARd may be an effective target for the treatment of
metabolic syndrome.
Several PPARd-selective agonists have been reported in
the literature, though most are derivatives of GW-
501516 and L-165041, that is, (2-methyl)phenoxyacetic
acid derivatives.15–20 We designed and synthesized a
series of substituted phenylpropanoic acid derivatives
as human PPARa/d dual agonists, using KCL, a
PPARa selective agonist with the 2-methoxybenzamide
structural motif, as a lead.21–25 As a part of our contin-
uing research directed toward the structural develop-
ment of characteristic subtype-selective PPAR
agonists, we tried to construct structurally new
PPAR/d selective agonists.
Based on the findings that the glitazone-class antidia-
betic agents and fibrate-class antidyslipidemic agents
are ligands of PPARc, and PPARa, respectively, much
research interest has been focused on these two meta-
bolic NR subtypes as therapeutic targets for the treat-
ment of type II diabetes and dyslipidemia. In contrast,
research interest in PPARd has been limited, perhaps
because of its ubiquitous distribution. However, the
availability of PPARd-knockout animals and selective
ligands, especially GW-501516, developed by Glaxo-
SmithKline, prompted us to examine the involvement
of PPARd in fatty acid metabolism, insulin resistance,
reverse cholesterol transport, inflammation, and so
on.11 For example, ligand-mediated PPARd activation
significantly increased HDL-cholesterol levels, possibly
Previously, we have designed and synthesized a potent
human PPARa/d dual agonist, TIPP401, based on
KCL, a PPARa-selective agonist, as a lead compound
(Fig. 1).27–29 Two structural modifications of KCL, that
is, a change of the linking group from –CH2NHCO– to
–CONHCH2–, and the introduction of a fluorine atom
at the 2-position of the distal benzene ring, affording
TIPP401, had increased the PPARd transactivation
O
O
O
OH
O
OH
O
F
OH
S
F
S
F
O
O
N
GW-501516
L-165041
O
OH
O
O
O
OH
Cl
N
H
N
Cl
Cl
F
GW-2433
O
EPA
O
O
F
O
OH
OH
N
H
N
H
F
F
F
F
O
CH3
O
CH3
F
F
KCL; PPARα selective
TIPP-401; PPAR α/δ dual
Figure 1. Structures of the representative ligands possessing PPARd activity, GW-501516, L-165041, eicosapentaenoic acid (EPA), GW-2433, and
our PPAR ligands KCL and TIPP-401.

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5179
activity with retention of the PPARa activity. We antic-
ipated that if we could further increase the PPARd activ-
ity of TIPP-401, while weakening its PPARa activity, we
might obtain a structurally new type of PPARd-selective
agonist, distinct from the well-known phenoxyacetic
acid derivative GW-501516.
Based on these insights, our working hypothesis was
that, if we could connect one more sterically bulky
hydrophobic side chain to the backbone of TIPP-401,
directed toward the upper cavity of PPARd, it should
have the effect of strengthening the PPARd activity,
while weakening the PPARa activity.
In order to create PPARd-selective agonists based on
TIPP-401, we took into account the results of X-ray
crystallographic analyses of PPARd complexed with a
natural unsaturated fatty acid, eicosapentaenoic acid
(EPA), and the synthetic ligand GW-2433.30 The PPAR
ligand-binding pocket is reported to form a large Y-
shaped cavity that extends from the C-terminal helix
to the b-sheet lying between helices H3 and H6. EPA
binds to the cavity in two distinct conformations, that
is, ‘tail-up’ and ‘tail-down’ conformations. The carboxyl
group and the first eight carbon units take almost the
same configuration in both conformations. However,
the distal hydrophobic tail part of the tail-up conformer
of EPA was bent upwards into the upper cavity of the
Y-shaped pocket, while in the tail-down conformer,
the hydrophobic tail part was bent downwards into
the bottom cavity of the Y-shaped pocket. In the case
of the phenoxyacetic acid derivative GW-2433, which
exhibited moderate PPARa/d dual activity, there are
two hydrophobic tail parts, and the two substituents
project into the two cavities occupied by the hydropho-
bic tail part of EPA in the two different binding con-
formers. Contrary to the case of PPARd, none of the
PPARa and PPARc agonists whose binding modes have
been solved by X-ray crystallography takes the tail-up
conformation, although the reason for this is not yet
known. However, we speculated that the amino acid(s)
forming the entrance to the upper cavity might be bulk-
ier in PPARa and PPARc than in PPARd. We previ-
ously suggested that our PPARa-selective agonist
KCL might take a tail-down conformation, based on
molecular modeling studies of the KCL–PPARa com-
plex and the results of site-directed mutagenesis studies
of PPARa (Fig. 2).31,32 TIPP-401 was also considered
to dock into the downward cavity of PPARa, because
Ile272, which is located on the lower half of helix 3, is
critical for potent PPARa transactivation by TIPP-401.
Based on our previously reported binding model of
KCL,32 a methoxy group at the 4-position was expected
to be directed toward the upper cavity, so we prepared
various 3-(4-alkoxyphenyl)propanoic acids (Table 1).
2. Chemistry
The synthetic routes to the present series of compounds
are outlined in Schemes 1 and 2. Compounds (5a–e, 5g–
i) were prepared from 4-alkoxybenzaldehydes (1a–f) in
six steps. 4-Alkoxybenzaldehydes were treated with tri-
ethyl 2-phosphonobutyrate in the presence of t-BuOK
as a base, followed by hydrogenolysis, to afford ethyl
phenylpropionate derivatives (2a–f). In the case of the
preparation of 2f, rebenzylation of phenolic hydroxyl
group was needed. The formylation of 2a–f to afford
3a–f, which was amidealkylated with 4-(trifluorometh-
yl)benzamide
derivatives.
Subsequent
alkaline
hydrolysis afforded the desired products 5a–e, 5g–i.
The 4-benzyloxy derivative (5f) was prepared by hydrog-
enolysis of 4f, rebenzylation with benzyl bromide, and
then alkaline hydrolysis.
Compounds (10a–g), with various alkyl substituents at
the a-position of the carboxylic acid, were prepared
from 4-alkoxybenzaldehydes (1a and 1d) in five steps,
using a similar reaction sequence to that employed for
the synthesis of compounds 5a–e, 5g–i.
An optically active phenylpropanoic acid derivative,
(S)-5i, was prepared, based on Evans’s asymmetric
alkylation method as the key step, as depicted in
Scheme 2. 5-Formylsalicylic acid (11) was benzyl-
esterified (12) and n-butoxylated to give compound
13. The formyl group of 13 was reduced with NaBH4
(14) and bromination of the hydroxyl group afforded
the bromomethyl derivative (15). (R)-N-Butyryl-4-ben-
zyloxazolidinone was treated with 15 under Evans’s
asymmetric alkylation protocol,26 followed by hydrog-
enolysis to afford the key synthetic intermediate 17.
This was reduced with BH3–THF (18), and then oxi-
dation with activated MnO2 afforded the formyl
derivative 19. This was amidealkylated with 2-fluoro-
4-(trifluoromethyl)benzamide,
followed
by
the
removal of the chiral auxiliary to afford the desired
(S)-configuration product, (S)-5i (Scheme 2). The
antipodal (R) enantiomer was prepared via similar
procedures, using (S)-N-butyryl-4-benzyloxazolidinone
as the reagent.
3. Results and discussion
As regards PPARa, introduction of a short-chain
alkoxy group was found to be preferable for the
Figure 2. Molecular modeling of KCL docked to human PPARa
ligand-binding domain.

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
Table 1. PPARs transactivation activity of the present series of compounds
R1
O
O
OH
N
H
F
O
F
R3
R2
F
Compound
R1
R2
R3
EC50 (nM)
PPARd
PPARa
PPARc
5a
5b
5c
5d
5e
5f
H
H
H
H
H
H
Me
Et
Et
Et
Et
Et
Et
Et
18
12
8
170 40
15
2600 400
1800 900
1300 100
1300 100
2300 200
6200 1500
3
4
n-Pr
n-Bu
n-Hexyl
Bn
77 11
520 120
1200 300
>10,000
6.8 0.6
4.8 0.6
45 5
110 20
5g
5h
5i
F
F
F
Me
Et
Et
Et
8.2 1.4
41 8
280 40
21
1
2200 500
650 10
1400 200
n-Pr
n-Bu
1.7 0.1
1.9 0.2
10a
10b
10c
l0d
H
H
H
H
Me
Me
Me
Me
H
210 40
3000 300
210 30
120 10
>10,000
>10,000
Me
n-Pr
n-Bu
19 3
70 21
230 30
3600 600
2400 100
700 150
l0e
l0f
F
F
F
Me
Me
n-Pr
n-Bu
n-Pr
29
5
40
6
1500 100
1000
140 20
820 60
1000 460
220 20
5.1 0.5
1.8 0.3
l0g
GW-501516
n-Bu
1000 170
8600 1200
transactivation activity, that is, the ethoxy (5b) and
methoxy (5a) derivatives exhibited the most potent
activity. In contrast, a longer alkoxy group was prefera-
ble in the case of PPARd transactivation activity, and
the n-butoxy (5d) and n-propoxy (5c) derivatives were
the most potent. These results are consistent with the
idea that the shape and the environment of the hydro-
phobic cavity hosting the alkoxy group at the 4-position
differ somewhat among these PPAR subtypes. These
compounds were basically weak agonists for the PPARc
subtype, with each compound exhibiting an EC50 value
of micromolar order or less.
the environment of the cavity hosting the alkyl group lo-
cated at the a-position of the carboxyl group are similar
in PPARa and PPARd (Table 2).
Considering the results obtained above, we then pre-
pared the optically active derivatives (S)-5i and (R)-5i.
Clear enantio-dependency of the transactivation activity
toward the PPAR subtypes was found, and (S)-5i, which
has S configuration, exhibited more potent transactiva-
tion activity than the antipodal R isomer, (R)-5i. There-
fore, we concluded that the activity resides primarily in
the (S)-enantiomer, but the enantio-selectivity is less
apparent than in the case of the PPARa/d dual agonist,
TIPP-401.29 (S)-5i exhibited extremely potent PPARd
transactivation activity, comparable with or somewhat
superior to that of the known PPARd-selective agonist
GW-501516, and its PPAR subtype selectivity was also
high (Fig. 3).
As the introduction of a fluorine atom on the distal ben-
zene ring, especially at the 2-position, was reported to
enhance the PPAR transactivation activity,27,29 we pre-
pared compounds 5g–i. As expected, 5h and 5i exhibited
enhanced PPARa and PPARd transactivation activities
as compared with the non-fluorinated compounds 5c
and 5d. The PPARd transactivation activity of 5i is com-
parable with that of GW-501516 in our assay system,
and the selectivity for PPARd over both PPARa and
PPARc is more than 100-fold.
In order to investigate the nuclear receptor selectivity
(cross-reactivity) of (S)-5i, we determined the transacti-
vation activity of (S)-5i on representative nuclear recep-
tors (PPARs, VDR, FXR, LXRa, RARa, and RXRa).
As can be seen from Figure 4, (S)-5i seems to be specific
for PPARd (and to a lesser extent to PPARa) because it
did not significantly activate VDR, PPARc, LXRa,
RARa or RXRa at concentrations up to 300 nM (more
than 300-fold higher concentration as compared to that
of EC50 of (S)-5i) under the experimental conditions
used. These results indicate that, although the ligand-
binding domains of nuclear receptors are similar, there
are distinct structural requirements for preferential
binding of (S)-5i to PPARd.
As already mentioned, the substituent at the a-position
of the carboxyl group is also important for the potency
in case of PPARa,22,25 and therefore we investigated the
effect of substitution at this position in the present series
of compounds (Table 1). As regards PPARa, introduc-
tion of an ethyl group (5a) or a methyl group (10b)
was favorable for the transactivation activity, and fur-
ther elongation of the substituent decreased the activity.
Similarly, an ethyl group (5a) or a n-propyl group (10c)
was favorable for PPARd transactivation activity, and
further elongation of the substituent decreased the
activity. These results may mean that the shape and
Moreover, to assess the ability of (S)-5i to activate genes
a
which have
peroxisome proliferator-responsive

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5181
R2
O
O
R4
O
O
O
O
N
H
O
R4
R4
H
O
H
c
F
F
a
b
R3
O
O
O
R3
O
R1
F
R3
R1
O
R1
R1
1a: R1 = Me
1b: R1 = Et
1c: R1 = nPr
1d: R1 = nBu
2a: R1 = Me,
2b: R1 = Et,
2c: R1 = nPr,
2d: R1 = nBu,
R3 = Et, R4 = Me
R3 = Et, R4 = Et
R3 = Et, R4 = Et
R3 = Et, R4 = Et
3a: R1 = Me
3b: R1 = Et
3c: R1 = nPr
3d: R1 = nBu
R3 = Et, R4 = Me
R3 = Et, R4 = Et
R3 = Et, R4 = Et
R3 = Et, R4 = Et
4a: R1 = Me,
4b: R1 = Et,
4c: R1 = nPr,
4d: R1 = nBu,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
R4 = Me
R4 = Et
R4 = Et
R4 = Et
R4 = Et
R4 = Et
R4 = Et
R4 = Et
R4 = Et
1e: R1 = nHexyl
1f: R1 = 4NO2Bn
2e: R1 = nHexyl, R3 = Et, R4 = Et
2f: R1 = 4NO2Bn,R3 = Et, R4 = Et
3e: R1 = nHexyl R3 = Et, R4 = Et
3f: R1 = 4NO2Bn R3 = Et, R4 = Et
4e: R1 = nHex,
4f: R1 = 4NO2Bn, R2 = H, R3 = Et,
R2 = F, R3 = Et,
R2 = F, R3 = Et,
R2 = F, R3 = Et,
4g: R1 = Me,
4h: R1 = nPr,
4i: R1 = nBu,
7a: R1 = Me,
7b: R1 = Me,
7c: R1 = Me,
7d: R1 = Me,
7e: R1 = nBu,
R3 = H, R4 = Me
R3 = Me, R4 = Et
R3 = nPr, R4 = Et
R3 = nBu, R4 = Et
R3 = nPr, R4 = Et
8a: R1 = Me,
8b: R1 = Me,
8c: R1 = Me,
8d: R1 = Me,
8e: R1 = nBu,
R3 = H,
R4 = Me
R3 = Me, R4 = Et
R3 = nPr, R4 = Et
R3 = nBu, R4 = Et
R3 = nPr, R4 = Et
9a: R1 = Me,
9b: R1 = Me,
9c: R1 = Me,
9d: R1 = Me,
9e: R1 = Me,
9f: R1 = Me,
9g: R1 = nBu,
R2 = H, R3 = H,
R4 = Me
R2 = H, R3 = Me, R4 = Et
R2 = H, R3 = nPr, R4 = Et
R2 = H, R3 = nBu, R4 = Et
R2 = F, R3 = nPr, R4 = Et
R2 = F, R3 = nBu, R4 = Et
R2 = F, R3 = nPr, R4 = Et
R2
O
OH
R3
O
d
N
H
F
F
O
R1
F
5a: R1 = Me,
5b: R1 = Et,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
R2 = H, R3 = Et,
5c: R1 = nPr,
O
N
O
5d: R1 = nBu, R2 = H, R3 = Et,
5e: R1 = nHex, R2 = H, R3 = Et,
O
O
OH
5g: R1 = Me,
5h: R1 = nPr,
R2 = F, R3 = Et,
R2 = F, R3 = Et,
H
OEt
N
H
HO
f
e
F
4f
O
F
F
F
5i: R1 = nBu, R2 = F, R3 = Et,
F
F
10a: R1 = Me,
10b: R1 = Me, R2 = H, R3 = Me
10c: R1 = Me, R2 = H, R3 = nPr
10d: R1 = Me, R2 = H, R3 = nBu
R2 = H, R3 = H
6
5f
10e: R1 = Me,
R2 = F, R3 = nPr
10f: R1 = Me, R2 = F, R3 = nBu
10g: R1 = nBu R2 = F, R3 = nPr
Scheme 1. Reagents and conditions: (a) (i) (EtO)2POCH(R3)CO2Et, t-BuOK, THF, rt; (ii) H2, 10% Pd–C, AcOEt, rt; (b) TiCl4, MeOCHCl2,
CH2Cl2, À30 °C; (c) benzamide derivative, (Et)3SiH, TFA, toluene, reflux; (d) aq LiOH, EtOH, rt; (e) H2, 10% Pd–C, AcOEt, rt; (f) (i) BnBr, K2CO3,
DMF, rt; (ii) same as in (d).
O
O
O
O
O
O
O
O
O
O
Br
O
H
O
OH
O
g
h
i
j
HO
HO
H
O
H
O
15
HO
12
11
13
O
14
O
O
O
O
O
O
O
O
O
O
O
O
N
N
HO
N
H
N
O
HO
O
O
O
O
k
l
m
n
O
O
18
19
16
17
F
F
O
O
O
O
O
N
H
N
O
o
p
N
H
OH
F
F
O
F
F
O
F
20
F
(S)-5i
Scheme 2. Reagents and conditions: (g) BnBr, KHCO3, DMF, rt; (h) n-BuI, K2CO3, DMF, rt; (i) NaBH4, EtOH, rt; (j) PBr3, ether, 0 °C; (k)
LiHMDS, (S)-3-n-butyryl-4-benzyloxazolidine-2-one, THF, À30 to 0 °C; (l) H2, 10% Pd–C, AcOEt, rt; (m) BH3–THF, THF, 0 °C, rt; (n) MnO2,
CH2Cl2, rt; (o) 2-fluoro-4-trifluoromethylbenzamide, (Et)3SiH, TFA, toluene, reflux; (p) LiOHÆH2O, 30% H2O2, THF–H2O.
element (PPRE) in the promoter region at the in vivo
level, we examined changes in the PPAR-mediated
expression of representative genes in mice. We chose
peroxisome proliferator-activated receptor gamma coac-
tivator 1a (PGC-1a) and uncoupling protein 3 (UCP3)
as representative PPAR target genes, since the human
genes were reported to possess PPRE in the promoter
region. PGC-1a is a potent transcriptional coactivator
for nuclear receptors.33,34 PGC-1a, which is expressed
in skeletal muscle, is able to induce mitochondrial
myogenesis and to induce preferential synthesis of slow
fibers.35 UCP3 belongs to a family of mitochondrial
uncoupling proteins which uncouple oxidative phos-
phorylation, thereby increasing thermogenesis and
decreasing the formation of ATP.36 Recent evidence
also points to a role of UCP3 in the export of
ionized fatty acids and lipid peroxides from the
mitochondria.37

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
Table 2. Comparison of the enantio-selectivity of 5i for transactivation
PGC-1α
UCP3
of PPARs
4.0
3.0
2.0
1.0
0.0
25.0
20.0
15.0
10.0
5.0
O
F
O
OH
N
H
F
F
O
F
Compound
EC50 (nM)
0.0
Cont.
(S)-5i
Beza
Cont.
(S)-5i
Beza
PPARa
PPARd
PPARc
Figure 5. Assay of gene induction by (S)-5i. Tissues were harvested
from C57BL/6 mice dosed for 3 days with vehicle, (S)-5i (3 mg/kg), or
bezafibrate (30 mg/kg) by sc. The expression of PGC-1a and UCP3
genes was analyzed by real-time PCR analysis. Induction (fold) is
reported relative to the vehicle control.
(S)-5i
(R)-5i
GW-501516
250 40
620 60
1000 460
0.91 0.2
8.3 1.5
1.8 0.3
1100 160
7000 1900
8600 1200
10000.0
7500.0
5000.0
2500.0
0.0
PPARδ
PPARα
PPARγ
study (Figs. 6 and 7) (details will be reported elsewhere).
Comparison of the CoMFA counterplots with the crys-
tal structure of the PPARd ligand-binding domain pro-
vided information about how the structural changes of
the agonists affect their activities. As can be seen in
Figure 6, hydrogen bonding interaction was observed
between carbonyl oxygen of the reversed-amide type lin-
ker and threonine 288 (T288) of PPARd (Fig. 6, left pa-
nel), while such a hydrogen-bonding interaction was not
found between carbonyl oxygen of the amide-type linker
and T288 (Fig. 6, right panel). This might be one of the
reasons why the change of the linker from amide type to
reversed-amide type enhanced the PPARd transactiva-
tion activity by 10-fold.
-10.0
-9.5
-9.0
-8.5
log (M)
-8.0
-7.5
-7.0
-6.5
Figure 3. Dose-dependency of (S)-5i for transactivation of PPARs.
50
In this CoMFA model (Fig. 7), the introduction of a ste-
rically bulky group near the methoxy group at the 4-po-
sition in the present series favors the activity, and this
was deduced to be related to the presence of the upper
pocket in the Y-shaped ligand-binding domain of
PPARd. We speculate that the side chain n-butoxy
group of (S)-5i fits into the upper pocket formed by
the hydrophobic amino acids L330, K367, and F368.
Further molecular modeling and X-ray crystallographic
studies are on-going.
3 nM
30 nM
300 nM
40
30
20
10
0
cont.
PPARα PPARδ PPAR
VDR
FXR
LXRα
RARα
RXRα
In summary, we have developed a series of potent hu-
man PPARd-selective agonists, which possess potent
PPARd transactivation activity with high selectivity
over the other PPAR subtypes. In vivo pharmacological
evaluation of a representative compound is planned.
Figure 4. Nuclear receptor selectivity (cross-reactivity) of (S)-5i.
We first investigated the effects of bezafibrate (Beza;
30 mg, sc), which has PPARd transactivation activity.
As indicated in Figure 5, 30 mg Beza augmented expres-
sion of the PGC-1a and UCP3 genes by 2.5- and 10-
fold, respectively. Treatment with 3 mg (S)-5i, about
one-tenth of the amount of Beza, also augmented
expression of the PGC-1a and UCP3 genes by 2.5-
and 15-fold, respectively. These results indicate that
the representative compound (S)-5i is an effective
PPARd-selective agonist at the in vivo level.
4. Experimental
4.1. General
Melting points were determined by using a Yanagimoto
hot-stage melting point apparatus and are uncorrected.
1H NMR spectra were recorded on a JEOL JNM-
GX500 (500 MHz) spectrometer. Chemical shifts are ex-
pressed in parts per million relative to tetramethylsilane.
Mass spectra were recorded on a JEOL JMS-DX303
spectrometer.
We have successfully obtained a potent and selective,
structurally novel PPARd agonist, (S)-5i. In order to
investigate the structure–activity relationship, and the
reason for the PPARd selectivity, we analyzed the
three-dimensional structure–activity relationship by
means of the comparative molecular field analysis
(CoMFA)38,39 method, and the molecular modeling
4.1.1. Ethyl 2-[1-(4-methoxyphenyl)methyl]butyrate (2a).
Potassium t-butoxide (5.6 g, 49.9 mmol) was suspended

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5183
O
O
F
O
F
O
OH
OH
N
N
H
H
F
F
F
F
MeO
MeO
F
F
T289
H323
Y473
W264
T288
H449
I333
O
F
O
L255
I249
OH
N
I363
H
F
F
V348
MeO
L330
K367
F
F352
F368
Figure 6. Predicted mode of binding of the amide derivative and the reversed-amide derivative to PPARd. The steric map is shown in green and
yellow, and the electrostatic map in blue and red. Orange, hydrophobic amino acids; green, hydrophilic amino acids; magenta, amide derivative and
reversed-amide derivative. Hydrogen bonds are shown as yellow dotted lines.
Figure 7. Comparison of the CoMFA counterplot of the steric field based on TIPP-401 (right) and the superimposition of TIPP-401 on the ligand-
binding domain of PPARd (left). The CoMFA steric counter map is shown in green and yellow. Green, areas in which bulky atomic groups are
sterically favorable for the activity; yellow, areas in which bulky atomic groups are unfavorable for the activity.
in 15 mL of dehydrated tetrahydrofuran under argon
and cooled with ice. Triethyl 2-phosphonobutyrate
(12.0 mL, 50.4 mmol) dissolved in 15 mL of dehydrated
tetrahydrofuran was added dropwise. When the addi-
tion was completed, the mixture was stirred for 1 h, then
4-methoxybenzaldehyde (5.45 g, 40.0 mmol) was added
dropwise. The mixture was stirred for 6 h at room tem-
perature. The reaction mixture was concentrated and
mixed with ice-water. The whole was extracted with
ethyl acetate, washed with water and brine, dried over
anhydrous magnesium sulfate, and concentrated. The
residue was purified by silica gel column chromatogra-
phy (eluant; n-hexane/ethyl acetate = 3:1, v/v) to afford
9.31 g (99%) of the title compound as colorless crystals
(mixture of the geometric isomers). 1H NMR
(500 MHz, CDCl3) d 7.59 (s, 1H), 7.36 (d, J = 8.5 Hz,
2H), 6.92 (d, J = 8.5 Hz, 2H), 4.26 (q, J = 7.3 Hz, 2H),
3.83 (s, 3H), 2.57 (q, J = 7.3 Hz, 2H), 1.34 (t,
J = 7.3 Hz, 3H), 1.18 (t, J = 7.3 Hz, 3H); MS (FAB)
235 (M+H)+.
J = 8.5 Hz, 2H), 4.07 (m, 2H), 3.78 (s, 3H), 2.86 (dd,
J = 13.7, 8.5 Hz, 1H), 2.69 (dd, J = 13.7, 6.4 Hz, 1H),
2.53 (m, 1H), 1.63 (m, 1H), 1.55 (m, 1H), 1.16 (t,
J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); MS (FAB)
237 (M+H)+.
4.1.2. Ethyl 2-[1-(4-ethoxyphenyl)methyl]butyrate (2b).
This compound was prepared from 4-ethoxybenzalde-
hyde and triethyl 2-phosphonobutyrate by means of a
procedure similar to that used for 2a. 1H NMR
(500 MHz, CDCl3) d 7.06 (d, J = 8.8 Hz, 2H), 6.79 (d,
J = 8.8 Hz, 2H), 4.09–4.04 (m, 2H), 4.00 (q,
J = 7.1 Hz, 2H), 2.86 (dd, J = 13.9, 8.6 Hz, 1H), 2.68
(dd, J = 13.9, 6.8 Hz, 1H), 2.55–2.49 (m, 1H), 1.66–
1.52 (m, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.16 (t,
J = 7.3 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H); MS (FAB)
250 (M+).
4.1.3. Ethyl 2-[1-(4-n-propoxyphenyl)methyl]butyrate
(2c). This compound was prepared from 4-n-propoxy-
benzaldehyde and triethyl 2-phosphonobutyrate by
1
This compound (9.31 g, 39.7 mmol) was hydrogenated
with 10% Pd–C (1.00 g) in 100 mL of ethanol affording
9.38 g (y; quant.) of the title compound. 1H NMR
(500 MHz, CDCl3) d 7.07 (d, J = 8.5 Hz, 2H), 6.80 (d,
means of a procedure similar to that used for 2a. H
NMR (500 MHz, CDCl3) d 7.06 (d, J = 8.5 Hz, 2H),
6.80 (d, J = 8.5 Hz, 2H), 4.07 (m, 2H), 3.88 (t,
J = 6.8 Hz, 2H), 2.86 (dd, J = 13.7, 8.5 Hz, 1H), 2.68

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
(dd, J = 13.7, 6.8 Hz, 1H), 2.52 (m, 1H), 1.79 (m, 2H),
1.63 (m, 1H), 1.55 (m, 1H), 1.17 (t, J = 7.3 Hz, 3H),
1.02 (t, J = 7.7 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); MS
(FAB) 264 (M+).
4.1.8. Ethyl 2-[1-(4-ethoxy-3-formylphenyl)methyl]buty-
rate (3b). This compound was prepared from 2b by
1
means of a procedure similar to that used for 3a. H
NMR (500 MHz, CDCl3) d 10.5 (s, 1H), 7.62 (d,
J = 2.6 Hz, 1H), 7.33 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 6.88
(d, J = 8.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 4.09–4.03
(m, 2H), 2.88 (dd, J = 14.1, 8.6 Hz, 1H), 2.72 (dd,
J = 14.1, 6.4 Hz, 1H), 2.57–2.51 (m, 1H), 1.67–1.51 (m,
2H), 1.46 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 7.3 Hz, 3H),
0.92 (t, J = 7.7 Hz, 3H); MS (FAB) 279 (M+H)+.
4.1.4. Ethyl 2-[1-(4-n-butoxyphenyl)methyl]butyrate (2d).
This compound was prepared from 4-n-butoxybenzalde-
hyde and triethyl 2-phosphonobutyrate by means of a
procedure similar to that used for 2a. 1H NMR
(500 MHz, CDCl3) d 7.05 (d, J = 8.5 Hz, 2H), 6.79 (d,
J = 8.5 Hz, 2H), 4.07 (m, 2H), 3.93 (t, J = 6.4 Hz, 2H),
2.76 (m, 2H), 2.52 (m, 1H), 1.75 (m, 2H), 1.62 (m,
2H), 1.48 (m, 2H), 1.16 (t, J = 7.3 Hz, 3H), 0.97 (t,
J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); MS (FAB)
278 (M+).
4.1.9. Ethyl 2-[1-(3-formyl-4-n-propoxyphenyl)methyl]-
butyrate (3c). This compound was prepared from 2c
by means of a procedure similar to that used for 3a.
1H NMR (500 MHz, CDCl3) d 10.49 (s, 1H), 7.63 (d,
J = 2.3 Hz, 1H), 7.33 (dd, J = 8.5, 2.3 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 4.06 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H),
2.88 (dd, J = 13.9, 8.5 Hz, 1H), 2.71 (dd, J = 13.9,
6.4 Hz, 1H), 2.54 (m, 1H), 1.86 (m, 2H), 1.63 (m, 1H),
1.54 (m, 1H), 1.17 (t, J = 7.3 Hz, 3H), 1.06 (t,
J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); MS (FAB)
293 (M+H)+.
4.1.5. Ethyl 2-[1-(4-n-hexyloxyphenyl)methyl]butyrate
(2e). This compound was prepared from 4-n-hexyloxy-
benzaldehyde and triethyl 2-phosphonobutyrate by
1
means of a procedure similar to that used for 2a. H
NMR (500 MHz, CDCl3) d 7.06 (d, J = 8.5 Hz, 2H),
6.79 (d, J = 8.5 Hz, 2H), 4.07 (m, 2H), 3.92 (t,
J = 6.8 Hz, 2H), 2.86 (dd, J = 13.9, 8.5 Hz, 1H), 2.67
(dd, J = 13.9, 6.4 Hz, 1H), 2.52 (m, 1H), 1.76 (m, 2H),
1.63 (m, 1H), 1.55 (m, 1H), 1.45 (m, 2H), 1.33 (m,
4H), 1.17 (t, J = 7.3 Hz, 3H), 0.91 (m, 6H); MS (FAB)
306 (M+).
4.1.10. Ethyl 2-[1-(4-n-butoxy-3-formylphenyl)methyl]-
butyrate (3d). This compound was prepared from 2d
by means of a procedure similar to that used for 2a.
1H NMR (500 MHz, CDCl3) d 10.5 (s, 1H), 7.62 (d,
J = 2.6 Hz, 1H), 7.33 (dd, J = 8.6, 2.6 Hz, 1H), 6.88 (d,
J = 8.6 Hz, 1H), 4.06 (m, 4H), 2.79 (m, 2H), 2.54 (m,
1H), 1.82 (m, 2H), 1.64 (m, 2H), 1.52 (m, 2H), 1.17 (t,
J = 7.3 Hz, 3H), 0.99 (t, J = 7.3 Hz, 3H), 0.92 (t,
J = 7.3 Hz, 3H); MS (FAB) 307 (M+H)+.
4.1.6. Ethyl 2-{1-[4-(4-nitrophenyl)methoxyphenyl]methyl}
butyrate (2f). This compound was prepared from 4-benzyl-
oxybenzaldehyde and triethyl 2-phosphonobutyrate by
means of a procedure similar to that used for 2a, and
subsequent 4-nitrobenzylation. 1H NMR (500 MHz,
CDCl3) d 8.24 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.6 Hz,
2H), 7.09 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H),
5.14 (s, 2H), 4.04–4.09 (m, 2H), 3.92 (t, J = 6.8 Hz,
2H), 2.86 (dd, J = 13.9, 8.5 Hz, 1H), 2.67 (dd, J = 13.9,
6.4 Hz, 1H), 2.87 (dd, J = 14,1, 9.0 Hz, 1H), 2.70 (dd,
J = 14.1, 6.8 Hz, 1H), 2.50–2.56 (m, 1H), 1.51–1.67 (m,
2H), 1.16 (t, J = 7.3 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H);
MS (FAB) 357 (M+).
4.1.11. Ethyl 2-[1-(3-formyl-4-n-hexyloxyphenyl) methyl]-
butyrate (3e). This compound was prepared from 2e by
means of a procedure similar to that used for 3a. H
1
NMR (500 MHz, CDCl3) d 10.48 (s, 1H), 7.62 (d,
J = 2.1 Hz, 1H), 7.33 (dd, J = 8.5, 2.1 Hz, 1H), 6.87 (d,
J = 8.5 Hz, 1H), 4.01–4.03 (m, 4H), 2.88 (dd, J = 13.9,
8.5 Hz, 1H), 2.71 (dd, J = 13.9, 6.4 Hz, 1H), 2.54 (m,
1H), 1.82 (m, 2H), 1.64 (m, 1H), 1.55 (m, 1H), 1.47
(m, 2H), 1.34 (m, 4H), 1.17 (t, J = 7.3 Hz, 3H), 0.93–
0.89 (m, 6H); MS (FAB) 355 (M+H)+.
4.1.7. Ethyl 2-[1-(3-formyl-4-methoxyphenyl)methyl]-
butyrate (3a). To a solution of 2a (9.38 g, 39.7 mmol)
and 500 mL of anhydrous dichloromethane was added
titanium tetrachloride (30.0 mL), followed by dichlo-
romethylmethylether (12.0 mL) at À20 °C, under
argon atmosphere. The mixture was stirred overnight
at room temperature. The mixture was poured into
30 mL of 1.5 mol/L HCl and separated the dichloro-
methane layer. The organic layer was washed with satd
NaHCO3 solution and brine, dried over anhydrous
sodium sulfate, and concentrated. The residue was
purified by silica gel column chromatography (eluant;
n-hexane/ethyl acetate = 4:1, v/v) to afford 10.5 g
(y; quant.) of the title compound as a pale brown oil.
1H NMR (500 MHz, CDCl3) d 10.43 (s, 1H), 7.62
(d, J = 2.6 Hz, 1H), 7.35 (dd, J = 8.5, 2.6 Hz, 1H),
6.90 (d, J = 8.5 Hz, 1H), 4.06 (m, 2H), 3.90 (s, 3H),
2.88 (dd, J = 13.7, 9.0 Hz, 1H), 2.72 (dd, J = 13.7,
6.4 Hz, 1H), 2.54 (m, 1H), 1.63 (m, 1H), 1.55 (m,
1H), 1.16 (t, J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H);
MS (FAB) 265 (M+H)+.
4.1.12. Ethyl 2-{1-[3-formyl-4-(4-nitrophenyl)methoxy-
phenyl]methyl}butyrate (3f). This compound was pre-
pared from 2f by means of a procedure similar to that
used for 3a. 1H NMR (500 MHz, CDCl3) d 10.5 (s,
1H), 8.24 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 2.1 Hz, 1H),
7.60 (d, J = 9.0 Hz, 2H), 7.33 (dd, J = 8.6, 2.6 Hz, 1H),
6.88 (d, J = 8.6 Hz, 1H), 5.25 (s, 1H), 4.01–4.10 (m,
2H), 2.88 (dd, J = 14,1, 9.0 Hz, 1H), 2.72 (dd, J = 14,1,
6.0 Hz, 1H), 2.50–2.56 (m, 1H), 1.50–1.66 (m, 2H),
1.14 (t, J = 7.3 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H); MS
(FAB) 357 (M+).
4.1.13. 2-{4-Methoxy-3-[(4-trifluoromethylbenzoylamino)-
methyl]phenylmethyl}butyric acid (5a). A mixture of 3a
(750 mg,
2.83 mmol),
4-trifluoromethylbenzamide
(1.75 g, 8.48 mmol), triethylsilane (1.36 mL, 8.48 mmol),
trifluoroacetic acid (0.65 mL, 8.48 mmol), and 50 mL of
dehydrated toluene was refluxed for 24 h. The mixture
was evaporated, and the residue was purified by silica

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5185
gel column chromatography (eluant; n-hexane/ethyl ace-
tate = 2:1, v/v) to obtain 900 mg (73%) of 4a as a colorless
oil. A mixture of 4a (900 mg, 2.06 mmol), 15 mL of etha-
nol, and 15 mL of a 1 mol/L aqueous solution of lithium
hydroxide was stirred overnight at 50 °C, then concen-
trated under reduced pressure. The residue was sus-
pended in water and acidified with dil HCl. The
precipitate formed was collected by filtration, dried, and
recrystallized from n-hexane–ethyl acetate to afford
620 mg (74%) of the title compound as colorless prisms.
Mp 133–135 °C. 1H NMR (500 MHz, CDCl3) d 7.79 (d,
2H, J = 8.7 Hz), 7.24 (d, 2H, J = 8.1 Hz), 7.17 (d, 1H,
J = 2.1 Hz), 7.10 (d, 1H, J = 8.1 Hz), 6.80 (d, 1H, J =
8.7 Hz), 6.69 (s, 1H), 4.58 (d, 2H, J = 6.0 Hz), 3.85 (s,
3H), 2.86–2.90 (m, 1H), 2.70–2.74 (m, 1H), 2.55–2.59
(m, 1H), 1.63–1.70 (m, 1H), 1.55–1.60 (m, 1H), 0.95 (t,
3H, J = 7.3 Hz). FAB MS m/z 423 (M+H)+. Anal. Calcd
for C21H22F3NO4 4/5H2O C, 59.51; H, 5.57; N, 3.30.
Found: C, 59.27; H, 5.18; N, 3.21.
(500 MHz, CDCl3) d 7.85 (d, J = 8.3 Hz, 2H), 7.66 (d,
J = 8.3 Hz, 2H), 7.15 (d, J = 2.1 Hz, 1H), 7.08 (dd,
J = 8.5, 2.1 Hz, 1H), 6.79 (m, 2H), 4.60 (d, J = 5.6 Hz,
2H), 3.99 (t, J = 6.4 Hz, 2H), 2.88 (dd, J = 13.7,
8.5 Hz, 1H), 2.71 (dd, J = 13.7, 6.4 Hz, 1H), 2.56 (m,
1H), 1.79 (m, 2H), 1.65 (m, 1H), 1.59 (m, 1H), 1.43
(m, 2H), 1.29 (m, 4H), 0.95 (t, J = 7.3 Hz, 3H), 0.86 (t,
J = 6.8 Hz, 3H). HRMS (FAB) calcd for C26H33F3NO4
480.2362; found: 480.2378 (M+H)+.
4.1.18. 2-{3-[(2-Fluoro-4-trifluoromethylbenzoylamino)-
methyl]-4-methoxyphenylmethyl}butyric acid (5g). This
compound was prepared from 3a by means of a proce-
1
dure similar to that used for 5a. Mp 108–110 °C. H
NMR (500 MHz, CDCl3) d 8.22 (t, 1H, J = 7.7 Hz),
7.4 (m, 3H), 7.17 (s, 1H), 7.10 (d, 1H, J = 8.1 Hz),
6.81 (d, 1H, J = 8.1 Hz), 4.63 (d, 2H, J = 6.0 Hz), 3.87
(s, 3H), 2.89 (dd, 1H, J = 14.1, 8.5 Hz), 2.73 (dd, 1H,
J = 14.1, 8.5 Hz, 1H), 2.57 (m, 1H), 1.80 (br, 1H), 1.66
(m, 1H), 1.59 (m, 1H), 0.96 (t, 3H, J = 7.5 Hz);
MS (FAB) 428(M+H)+; Anal. Calcd for C21H21F4NO4,
C 59.02, H 4.95, N 3.28. Found: C, 59.05; H, 5.21; N,
3.14.
4.1.14. 2-{4-Ethoxy-3-[(4-trifluoromethylbenzoylamino)-
methyl]phenylmethyl}butyric acid (5b). This compound
was prepared from 3b by means of a procedure similar
to that used for 5a. Mp 94–95 °C. 1H NMR
(500 MHz, CDCl3) d 7.85 (d, J = 8.1 Hz, 2H), 7.66 (d,
J = 8.1 Hz, 2H), 7.15 (d, J = 2.1, 1H), 7.08 (dd,
J = 8.3, 2.1 Hz, 1H), 6.84 (s, 1H), 6.79 (d, J = 8.3 Hz,
1H), 4.61 (d, J = 6.0 Hz, 2H), 4.07 (q, J = 6.8 Hz, 2H),
2.89 (dd, J = 14.1, 2.6 Hz, 1H), 2.71 (dd, J = 14.1,
6.4 Hz, 1H), 2.59–2.53 (m, 1H), 1.69–1.55 (m, 2H),
1.43 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 7.3 Hz, 3H).
HRMS (FAB) calcd for C22H25F3NO4 424.1736; found:
424.1736 (M+H)+.
4.1.19. 2-{3-[(2-Fluoro-4-trifluoromethylbenzoylamino)-
methyl]-4-n-propoxyphenylmethyl}butyric acid (5h). This
compound was prepared from 3c by means of a proce-
1
dure similar to that used for 5a. Mp 124–125 °C. H
NMR (500 MHz, CDCl3) d 8.24 (t, J = 8.1 Hz, 1H),
7.51 (d, J = 8.1 Hz, 1H), 7.39 (m, 2H), 7.16 (d,
J = 1.9 Hz, 1H), 7.07 (dd, J = 8.5, 1.9 Hz, 1H), 6.79 (d,
J = 8.5 Hz, 1H), 4.64 (d, J = 5.6 Hz, 2H), 3.97 (t,
J = 6.4 Hz, 2H), 2.89 (dd, J = 13.9, 8.1 Hz, 1H), 2.71
(dd, J = 13.9, 6.4 Hz, 1H), 2.57 (m, 1H), 1.85 (m, 2H),
1.65 (m, 1H), 1.59 (m, 1H), 1.06 (t, J = 7.7 Hz, 3H),
0.95 (t, J = 7.3 Hz, 3H). HRMS (FAB) calcd for
C23H26F4NO4 456.1798; found: 456.1792 (M+H)+.
4.1.15. 2-{4-n-Propoxy-3-[(4-trifluoromethylbenzoylami-
no)methyl]phenylmethyl}butyric acid (5c). This compound
was prepared from 3c by means of a procedure similar
1
to that used for 5a. Mp 124 °C. H NMR (500 MHz,
CDCl3) d 7.84 (d, J = 8.1 Hz, 2H), 7.66 (d, J = 8.1 Hz,
2H), 7.15 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.5, 1.9 Hz,
1H), 6.79 (m, 2H), 4.61 (d, J = 5.6 Hz, 2H), 3.96 (t,
J = 6.4 Hz, 2H), 2.88 (dd, J = 13.7, 8.5 Hz, 1H), 2.71
(dd, J = 13.7, 6.4 Hz, 1H), 2.56 (m, 1H), 1.82 (m, 2H),
1.66 (m, 1H), 1.58 (m, 1H), 1.04 (t, J = 7.3 Hz, 3H),
0.95 (t, J = 7.3 Hz, 3H). HRMS (FAB) calcd for
C23H27F3NO4 438.1892; found: 438.1903 (M+H)+.
4.1.20. 2-{3-[4-n-Butoxy-(2-fluoro-4-trifluoromethylbenzoyl-
amino)methyl]phenylmethyl}butyric acid (5i). This com-
pound was prepared from 3d by means of a procedure
1
similar to that used for 5a. Mp 109–110 °C. H NMR
(500 MHz, CDCl3) d 8.23 (t, J = 7.9 Hz, 1H), 7.51 (d,
J = 7.9 Hz, 1H), 7.39 (m, 2H), 7.16 (d, J = 2.1 Hz, 1H),
7.07 (dd, J = 8.3, 2.1 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H),
4.63 (d, J = 6.0 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H), 2.89
(dd, J = 13.9, 8.5 Hz, 1H), 2.70 (dd, J = 13.9, 6.4 Hz,
1H), 2.56 (m, 1H), 1.81 (m, 2H), 1.69–1.48 (m, 4H),
0.99–0.94 (m, 6H). HRMS (FAB) calcd for C24H28F4NO4
470.1954; found: 470.1940 (M+H)+.
4.1.16. 2-{4-n-Butoxy-3-[(4-trifluoromethylbenzoylami-
no)methyl]phenylmethyl}butyric acid (5d). This com-
pound was prepared from 3d by means of a procedure
similar to that used for 5a. Mp 91–94 °C. 1H NMR
(500 MHz, CDCl3) d 7.84 (d, J = 8.4 Hz, 2H), 7.66 (d,
J = 8.4, 2H), 7.14 (m, 1H), 7.07 (m, 1H), 6.83 (m, 1H),
6.79 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 3.99
(t, J = 6.4 Hz, 2H), 2.88–2.70 (m, 2H), 2.55 (m, 1H),
1.78 (m, 2H), 1.64–1.57 (m, 2H), 1.48 (m, 2H), 0.95
(m, 6H); HRMS (FAB) calcd for C24H29F3NO4
452.2049; found: 452.2046 (M+H)+.
4.1.21. Ethyl 2-{4-hydroxy-3-[(4-trifluoromethylbenzoyl-
amino)methyl}butyrate (6). This compound was pre-
pared from 3f by means of a procedure similar to that
1
used for 5a, and subsequent hydrogenolysis. H NMR
(500 MHz, CDCl3) d 8.97 (s, 1H), 7.87 (d, J = 8.5 Hz,
2H), 7.67 (d, J = 8.1 Hz, 2H), 7.01 (dd, J = 8.1, 2.1 Hz,
1H), 6.93 (d, J = 2.1 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H),
4.52 (d, J = 6.4 Hz, 1H), 4.00–4.06 (m, 2H), 2.80 (dd,
J = 13.7, 8.5 Hz, 1H), 2.64 (dd, J = 13.7, 6.4 Hz, 1H),
2.49–2.55 (m, 1H), 1.53–1.66 (m, 2H), 1.12 (t,
J = 6.4 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H); MS (FAB)
424 (M+H)+.
4.1.17. 2-{4-n-Hexyloxy-3-[(4-trifluoromethylbenzoylami-
no)methyl]phenylmethyl}butyric acid (5e). This com-
pound was prepared from 3e by means of a procedure
similar to that used for 5a. Mp 92–94 °C. 1H NMR

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
4.1.22. Ethyl 2-{4-benzyloxy-3-[(4-trifluoromethylbenzoyl-
amino)methyl]benzyl}butyrate. A mixture of 6 (400 mg,
0.944 mmol), anhydrous cesium carbonate (301 mg,
0.924 mmol), benzyl bromide (0.110 mL, 0.926 mmol),
and 10 mL of dehydrated N,N-dimethylformamide was
stirred for 16 h at room temperature. The reaction
mixture was diluted with AcOEt, washed with 2 mol/L
of HCl, satd NaHCO3, water, brine, and evaporated.
The residue was purified by silica gel column
chromatography (eluant; n-hexane/ethyl acetate =
4:1–1:1, v/v) to obtain 298 mg (62%) of the title com-
4.1.27. Ethyl 2-[(4-n-butoxyphenyl)methyl]pentanoate (7e).
This compound was prepared from 4-n-butoxybenzalde-
hyde and triethyl 2-phosphonopentanoate by means of a
procedure similar to that used for 2a. 1H NMR
(500 MHz, CDCl3) d 7.04 (d, J = 8.5 Hz, 2H), 6.80 (d,
J = 8.5 Hz, 2H), 3.93 (t, J = 6.4 Hz, 2H), 3.59 (s, 3H),
2.86 (dd, J = 13.7, 8.1 Hz, 1H), 2.67 (dd, J = 13.7, 6.8 Hz,
1H), 2.62 (m, 1H), 1.75 (m, 2H), 1.60 (m, 1H), 1.52–1.42
(m, 3H), 1.30 (m, 2H), 0.97 (t, J = 7.7 Hz, 3H), 0.88 (t,
J = 7.3 Hz, 3H); MS (FAB) 278 (M)+.
1
pound as a colorless oil. H NMR (500 MHz, CDCl3) d
4.1.28. Ethyl (3-formyl-4-methoxyphenyl)propanoate
(8a). This compound was prepared from 7a by means
7.67 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.36–
7.43 (m, 5H), 7.15 (d, J = 2.1 Hz, 1H), 7.08 (dd, J = 8.1,
2.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 5.09
(s, 1H), 4.65 (d, J = 5.5 Hz, 2H), 4.04–4.10 (m, 2H),
2.87 (dd, J = 12.1, 8.5 Hz, 1H), 2.69 (dd, J = 12.1,
6.4 Hz, 1H), 2.51–2.57 (m, 1H), 1.52–1.67 (m, 2H), 1.16
(t, J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H); MS (FAB)
514 (M+H)+.
1
of a procedure similar to that used for 3a. H NMR
(500 MHz, CDCl3) d 10.4 (s, 1H), 7.63 (d, J = 2.6 Hz,
1H), 7.38 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 6.90 (d,
J = 8.6 Hz, 1H), 3.86 (s, 2H), 3.64 (s, 2H), 2.89 (t,
J = 7.7 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H).
4.1.29. Ethyl (3-formyl-4-methoxyphenyl)-2-methylpro-
panoate (8b). This compound was prepared from 7b by
1
means of a procedure similar to that used for 3a. H
4.1.23. 2-{4-benzyloxy-3-[(4-trifluoromethylbenzoylami-
no)methyl]benzyl}butyric acid (5f). This compound was
prepared from ethyl 2-{4-benzyloxy-3-[(4-trifluoro-
methylbenzoylamino)methyl]benzyl}butyrate by means
NMR (500 MHz, CDCl3) d 10.42 (s, 1H), 7.61 (s, 1H),
7.35 (s, 1H), 6.89 (d, J = 8.5 Hz, 1H), 4.07 (m, 2H),
3.89 (s, 3H), 2.96 (m, 1H), 2.66 (m, 2H), 1.15 (m, 6H);
MS (FAB) 251 (M+H)+.
1
of a procedure similar to that used for 5a. H NMR
(500 MHz, CDCl3) d 7.66 (d, J = 8.1 Hz, 2H), 7.56 (d,
J = 8.1 Hz, 2H), 7.37–7.40 (m, 5H), 7.09 (d,
J = 8.1 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 5.07 (s, 2H),
4.60 (d, J = 5.5 Hz, 2H), 2.88 (dd, J = 14.1, 9.0 Hz,
1H), 2.71 (dd, J = 14.1, 6.4 Hz, 1H), 2.51–2.57 (m,
1H), 1.54–1.69 (m, 2H), 0.94 (t, J = 7.7 Hz, 3H); MS
(FAB) 486 (M+H)+.
4.1.30. Ethyl [(3-formyl-4-methoxyphenyl)methyl]pent-
anoate (8c). This compound was prepared from 7c by
1
means of a procedure similar to that used for 3a. H
NMR (500 MHz, CDCl3) d 10.44 (s, 1H), 7.62 (d,
J = 2.1 Hz, 1H), 7.34 (dd, J = 8.5, 2.1 Hz, 1H), 6.90 (d,
J = 8.5 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 2.88 (dd,
J = 13.7, 9.0 Hz, 1H), 2.72 (dd, J = 13.7, 6.4 Hz, 1H),
2.64 (m, 1H), 1.62 (m, 1H), 1.45 (m, 1H), 1.31 (m,
2H), 0.89 (t, J = 7.3 Hz, 3H); MS (FAB) 265 (M+H)+.
4.1.24. Ethyl 3-(4-methoxyphenyl)-2-methylpropionate
(7b). This compound was prepared from 4-methoxy-
benzaldehyde and triethyl 2-phosphonopropionate by
1
means of a procedure similar to that used for 2a. H
4.1.31. Ethyl [(3-formyl-4-methoxyphenyl)methyl]hexa-
noate (8d). This compound was prepared from 7d by
NMR (500 MHz, CDCl3) d 7.08 (d, J = 8.5 Hz, 2H),
6.81 (d, J = 8.5 Hz, 2H), 4.08 (q, J = 7.3 Hz, 2H), 3.78
(s, 3H), 2.95 (dd, J = 13.3, 6.4 Hz, 1H), 2.69–2.59 (m,
2H), 1.19 (t, J = 7.3 Hz, 3H), 1.13 (t, J = 6.8 Hz, 3H);
MS (FAB) 222 (M+).
1
means of a procedure similar to that used for 3a. H
NMR (500 MHz, CDCl3) d 10.44 (s, 1H), 7.62 (d,
J = 2.3 Hz, 1H), 7.34 (dd, J = 8.3, 2.3 Hz, 1H), 6.90 (d,
J = 8.3 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 2.88 (dd,
J = 13.7, 9.0 Hz, 1H), 2.72 (dd, J = 13.7, 6.0 Hz, 1H),
2.60 (m, 1H), 1.63 (m, 1H), 1.49 (m, 1H), 1.28 (m,
4H), 0.87 (t, J = 6.8 Hz, 3H); MS (FAB) 279 (M+H)+.
4.1.25. Ethyl 2-[(4-methoxyphenyl)methyl]pentanoate
(7c). This compound was prepared from 4-methoxybenz-
aldehyde and triethyl 2-phosphonopentanoate by means
1
of a procedure similar to that used for 2a. H NMR
4.1.32. Ethyl [(4-n-butoxy-3-formylphenyl)methyl]pent-
anoate (8e). This compound was prepared from 7e by
means of a procedure similar to that used for 3a. H
(500 MHz, CDCl3) d 7.06 (d, J = 8.5 Hz, 2H), 6.81 (d,
J = 8.5 Hz, 2H), 3.78 (s, 3H), 3.59 (s, 3H), 2.86 (dd,
J = 13.7, 8.1 Hz, 1H), 2.68 (dd, J = 13.7, 6.4 Hz, 1H),
2.62 (m, 1H), 1.60 (m, 1H), 1.46 (m, 1H), 1.30 (m, 2H),
0.88 (t, J = 7.3 Hz, 3H); MS (FAB) 236 (M+).
1
NMR (500 MHz, CDCl3) d 10.47 (s, 1H), 7.61 (d,
J = 2.3 Hz, 1H), 7.31 (dd, J = 8.5, 2.3 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 4.06 (t, J = 6.4 Hz, 2H), 3.60 (s, 3H),
2.88 (dd, J = 13.7, 8.5 Hz, 1H), 2.71 (dd, J = 13.7,
6.0 Hz, 1H), 2.64 (m, 1H), 1.82 (m, 2H), 1.62 (m, 1H),
1.52 (m, 2H), 1.45 (m, 1H), 1.31 (m, 2H), 0.99 (t,
J = 7.7 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H); MS (FAB)
307 (M+H)+.
4.1.26. Ethyl 2-[(4-methoxyphenyl)methyl]hexanoate (7d).
This compound was prepared from 4-methoxybenzalde-
hyde and triethyl 2-phosphonohexanoate by means of a
procedure similar to that used for 2a. 1H NMR
(500 MHz, CDCl3) d 7.06 (d, J = 8.5 Hz, 2H), 6.81 (d,
J = 8.5 Hz, 2H), 3.78 (s, 3H), 3.60 (s, 3H), 2.86 (dd,
J = 13.7, 8.5 Hz, 1H), 2.68 (dd, J = 13.7, 6.4 Hz, 1H),
2.60 (m, 1H), 1.62 (m, 1H), 1.49 (m, 1H), 1.27 (m, 4H),
0.87 (t, J = 6.4 Hz, 3H); MS (FAB) 250 (M)+.
4.1.33. 3-{4-Methoxy-3-[(4-trifluoromethylbenzoylami-
no)methyl]phenyl}propanoic acid (10a). This compound
was prepared from 8a by means of a procedure similar
to that used for 5a. Mp 166–167 °C. 1H NMR

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5187
(500 MHz, CDCl3) d 7.86 (d, J = 8.3 Hz, 2H), 7.68 (d,
J = 8.3 Hz, 2H), 7.20 (d, J = 2.1 Hz, 1H), 7.14 (dd,
J = 8.1, 2.1 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.72 (s,
1H), 4.62 (d, J = 6.0 Hz, 2H), 3.87 (s, 3H), 2.91 (t,
J = 7.7 Hz, 2H), 2.65 (d, J = 7.7 Hz, 2H); HRMS
(FAB) calcd for C19H19F3NO4 382.1266; found:
382.1236 (M+H)+.
similar to that used for 5a. Mp 135 °C. 1H NMR
(500 MHz, CDCl3) d 8.22 (t, J = 8.1 Hz, 1H), 7.50 (d,
J = 8.1 Hz, 1H), 7.39 (m, 2H), 7.15 (d, J = 2.1 Hz, 1H),
7.07 (dd, J = 8.3, 2.1 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H),
4.63 (d, J = 5.6 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H), 2.89
(dd, J = 13.7, 7.7 Hz, 1H), 2.68 (dd, J = 13.7, 6.4 Hz,
1H), 2.63 (m, 1H), 1.80 (m, 2H), 1.61 (m, 1H), 1.53–1.31
(m, 5H), 0.98 (t, J = 7.7 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H).
HRMS (FAB) calcd for C23H26F4NO4 456.1798; found:
456.1779 (M+H)+.
4.1.34. 3-{4-Methoxy-3-[(4-trifluoromethylbenzoylamino)-
methyl]phenyl}-2-methylpropanoic acid (10b). This com-
pound was prepared from 8b by means of a procedure
similar to that used for 5a. Mp 129 °C. 1H NMR
(500 MHz, CDCl3) d 7.85 (d, J = 8.1 Hz, 2H), 7.67 (d,
J = 8.1 Hz, 2H), 7.18 (d, J = 2.1 Hz, 1H), 7.10 (dd,
J = 8.1, 2.1 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 6.74 (s,
1H), 4.61 (d, J = 5.5 Hz, 2H), 3.87 (s, 3H), 2.96 (dd,
J = 13.7, 6.8 Hz, 1H), 2.76–2.64 (m, 2H), 1.18 (d, J =
6.8 Hz, 3H); MS (FAB) 396 (M+H)+. HRMS (FAB) calcd
for C20H20F3NO4 396.1423 (M+H)+; found: 396.1422.
4.1.39. Benzyl 2-n-butoxy-5-formylbenzoate (13). A mix-
ture of 11 (5.00 g, 30.0 mmol), benzyl bromide (3.56 mL,
30.0 mmol), potassium hydrogencarbonate (3.00 g,
30.0 mmol), and 40 mL of N,N-dimethylformamide
was stirrered for 24 h. The mixture was poured into
ice-water and stirred for 2 h. The precipitate was redis-
solved in 100 mL of AcOEt, dried over anhyd MgSO4,
filtered, and concentrated to obtain 13. A mixture of
12, iodobutane (5.52 g, 30.0 mmol), potassium carbon-
ate (5.53 g, 30.0 mmol), and 40 mL of N,N-dimethyl-
formamide was stirred for 24 h. The reaction mixture
was poured into water, and the whole was extracted with
ethyl acetate, washed with water and brine, dried over
anhydrous magnesium sulfate, and concentrated. The
residue was purified by silica gel column chromatogra-
phy (eluant; n-hexane/ethyl acetate = 4:1, v/v) to afford
4.00 g (43%) of the title compound as a colorless oil.
1H NMR (500 MHz, CDCl3) d 9.87 (s, 1H), 8.30 (d,
J = 2.6 Hz, 1H), 7.97 (dd, J = 8.6, 2.6 Hz, 1H), 7.43–
7.44 (m, 2H), 7.32–7.38 (m, 3H), 7.05 (d, J = 8.6 Hz,
1H), 5.35 (s, 2H), 4.10 (t, J = 6.4 Hz, 2H), 1.74–1.80
(m, 2H), 1.42–1.46 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H).
4.1.35. 2-{4-Methoxy-3-[(4-trifluoromethylbenzoylamino)-
methyl]phenylmethyl}pentanoic acid (10c). This com-
pound was prepared from 8c by means of a procedure
1
similar to that used for 5a. Mp 129–130 °C. H NMR
(500 MHz, CDCl3) d 7.85 (d, J = 8.3 Hz, 2H), 7.66 (d,
J = 8.3 Hz, 2H), 7.16 (d, J = 2.1 Hz, 1H), 7.09 (dd,
J = 8.5, 2.1 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.77 (s,
1H), 4.58 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 2.87 (dd,
J = 13.7, 9.0 Hz, 1H), 2.71 (dd, J = 13.7, 6.0 Hz, 1H),
2.63 (m, 2H), 1.63 (m, 1H), 1.48 (m, 1H), 1.36 (m,
2H), 0.90 (d, J = 7.3 Hz, 3H). HRMS (FAB) calcd for
C22H25F3NO4 424.1736; found: 424.1769 (M+H)+.
4.1.36. 2-{4-Methoxy-3-[(4-trifluoromethylbenzoylamino)-
methyl]phenylmethyl}hexanoic acid (10d). This com-
pound was prepared from 8d by means of a procedure
similar to that used for 5a. Mp 151 °C. 1H NMR
(500 MHz, CDCl3) d 7.85 (d, J = 8.1 Hz, 2H), 7.66 (d,
J = 8.1 Hz, 2H), 7.16 (s, 1H), 7.09 (dd, J = 8.3, 2.1 Hz,
1H), 6.81 (d, J = 8.3 Hz, 1H), 6.76 (s, 1H), 4.59 (d,
J = 6.0 Hz, 2H), 3.85 (s, 3H), 2.87 (dd, J = 13.9,
9.0 Hz, 1H), 2.72 (dd, J = 13.9, 5.6 Hz, 1H), 2.61 (m,
1H), 1.65 (m, 1H), 1.51 (m, 1H), 1.31 (m, 4H), 0.87 (t,
J = 6.8 Hz, 3H). HRMS (FAB) calcd for C23H27F3NO4
438.1892; found: 438.1909 (M+H)+.
4.1.40. Benzyl 5-bromomethyl-2-n-butoxybenzoate (15).
To a solution of 13 (3.55 g, 11.4 mmol) and 70 mL of
ethanol was added NaBH4 (0.50 g, 13.3 mmol) portion-
wise at 0 °C, and stirred for 2 h at room temperature.
The excess ethanol was evaporated, and the residue
was poured into water and the whole was extracted with
CH2Cl2. The extract was washed with water and brine,
dried over anhydrous magnesium sulfate, and concen-
trated. The residue was purified by silica gel column
chromatography (eluant; n-hexane/ethyl acetate = 4:1,
v/v) to afford 3.26 g (91%) of the intermediate alcohol
14 as a colorless oil. A mixture of 14 (3.26 g, 10.4 mmol),
phosphorus tribromide (0.50 mL, 5.32 mmol), and
70 mL of dehydrated ether was stirred for 1 h at 0 °C.
The reaction mixture was poured into saturated ammo-
nium chloride solution, and the whole was extracted
with ether. The extract was washed with water and
brine, dried over anhydrous magnesium sulfate, and
concentrated. The residue was purified by silica gel col-
umn chromatography (eluant; n-hexane/ethyl ace-
tate = 10:1, v/v) to afford 2.74 g (70%) of the title
compound as a colorless oil. 1H NMR (500 MHz,
CDCl3) d 7.83 (d, J = 2.1 Hz, 1H), 7.48–7.32 (m, 6H),
6.92 (d, J = 9.0 Hz, 1H), 5.34 (s, 2H), 4.47 (s, 2H),
4.02 (t, J = 6.4 Hz, 2H), 1.75 (m, 2H), 1.44 (m, 2H),
0.92 (t, J = 7.3 Hz, 3H); MS (FAB) 377, 379 (M+H)+.
4.1.37. 2-{4-Methoxy-3-[(2-fluoro-4-trifluoromethylbenzoyl-
amino)methyl]phenylmethyl}pentanoic acid (10e). This com-
pound was prepared from 8c by means of a procedure
similar to that used for 5a. Mp 95–97 °C. 1H NMR
(500 MHz, CDCl3) d 8.21 (t, J = 8.1 Hz, 1H), 7.50 (d,
J = 8.1 Hz, 1H), 7.42 (t, J = 5.8 Hz, 1H), 7.37 (d,
J = 11.5 Hz, 1H), 7.15 (d, J = 2.1 Hz, 1H), 7.09 (dd,
J = 8.1, 2.1 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 4.62 (d,
J = 5.6 Hz, 2H), 3.86 (s, 3H), 2.89 (dd, J = 13.7, 8.1 Hz,
1H), 2.69 (dd, J = 13.7, 6.8 Hz, 1H), 2.63 (m, 1H), 1.61
(m, 1H), 1.47 (m, 1H), 1.41–1.30 (m, 2H), 0.89 (t,
J = 7.3 Hz, 3H). HRMS (FAB) calcd for C22H24F4NO4
442.1641; found: 442.1643 (M+H)+.
4.1.38. 2-{4-n-Butoxy-3-[(2-fluoro-4-trifluoromethylbenzoyl-
amino)methyl]phenylmethyl}pentanoic acid (10f). This com-
pound was prepared from 8d by means of a procedure
4.1.41. Benzyl 5-[(S)-2-((R)-4-benzyl-2-oxo-oxazolidine-
3-carbonyl)butyl]2-n-butoxybenzoate (16). (R)-3-(1-Butyr-

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J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
yl)-4-benzyloxazolidin-2-one (1.30 g, 5.25 mmol) and
20 mL of dehydrated tetrahydrofuran were mixed under
an argon atmosphere, and cooled to À60 °C. Under stir-
ring, a 1 mol/L solution of sodium bis(trimethylsi-
lyl)amide in dehydrated tetrahydrofuran (5.80 mL,
5.80 mmol) was added dropwise. After completion of
the addition, the mixture was stirred for 1 h at À15 °C,
recooled to À60 °C, and then a solution of 15 (1.81 g,
4.80 mmol) in dehydrated tetrahydrofuran (20 mL) was
added dropwise. After completion of the addition, the
mixture was further stirred for 1 h while gradually heated
to room temperature. A saturated aqueous solution of
ammonium chloride was added to the reaction mixture,
and the whole was extracted with ethyl acetate. The ex-
tract was washed with water and brine, dried over anhy-
drous sodium sulfate, and concentrated. The residue was
purified by silica gel chromatography (eluant; n-hexane/
ethyl acetate = 5:1, v/v) to afford 1.75 g (67%) of the de-
(dd, J = 13.2, 3.4 Hz, 1H), 3.08 (dd, J = 13.7, 7.7 Hz,
1H), 2.77 (dd, J = 13.7, 6.8 Hz, 1H), 2.58 (dd, J = 13.2,
9.8 Hz, 1H), 1.87 (m, 2H), 1.77 (m, 1H), 1.51 (m, 3H),
0.99 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H); MS
(FAB) 454 (M+H)+.
4.1.43. N-{5-[(S)-2-((R)-4-Benzyl-2-oxo-oxazolidine-3-car-
bonyl)butyl]-2-n-butoxybenzyl}-2-fluoro-4-trifluoromethyl-
benzamide (20). A mixture of 19 (393 mg, 0.898 mmol),
2-fluoro-4-trifluoromethylbenzamide
(0.41 g,
1.98 mmol), triethylsilane (0.80 mL, 4.98 mmol), trifluo-
roacetic acid (0.24 mL, 3.12 mmol), and 15 mL of dehy-
drated toluene was stirred for 2 days at 80 °C. The
mixture was evaporated, and the residue was purified
by silica gel column chromatography (eluant; n-hex-
ane/ethyl acetate = 4:1, v/v) to obtain 376 mg (67%) of
1
the title as a colorless oil. H NMR (500 MHz, CDCl3)
d 8.15 (t, J = 7.9 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.31–
7.16 (m, 7H), 6.92 (m, 2H), 6.82 (d, J = 8.5 Hz, 1H),
4.69–4.57 (m, 3H), 4.12–3.97 (m, 5H), 2.99 (dd,
J = 13.7, 8.5 Hz, 1H), 2.95 (dd, J = 13.5, 3.8 Hz, 1H),
2.75 (dd, J = 13.7, 6.4 Hz, 1H), 2.44 (dd, J = 13.5,
9.0 Hz, 1H), 1.77 (m, 3H), 1.57 (m, 1H), 1.49 (m, 2H),
0.98–0.92 (m, 6H); MS (FAB) 629 (M+H)+.
1
sired compound as a colorless oil: H NMR (500 MHz,
CDCl3) d 7.68 (d, J = 2.1 Hz, 1H), 7.42–7.22 (m, 9H),
7.03 (d, J = 6.8 Hz, 2H), 6.87 (d, J = 8.5 Hz, 1H), 5.28
(s, 2H), 4.64 (m, 1H), 4.14–3.94 (m, 5H), 3.06 (dd,
J = 13.2, 3.4 Hz, 1H), 3.01 (dd, J = 13.7, 8.1 Hz, 1H),
2.75 (dd, J = 13.7, 6.4 Hz, 1H), 2.44 (dd, J = 13.2,
9.8 Hz, 1H), 1.75 (m, 3H), 1.53 (m, 1H), 1.41 (m, 2H),
1.01–0.88 (m, 6H); MS (FAB) 544 (M+H)+.
4.1.44. (S)-2-{3-[4-n-Butoxy-(2-fluoro-4-trifluoromethyl-
benzoylamino)methyl]phenylmethyl}butyric acid ((S)-5i).
Compound 20 (376 mg, 0.598 mol) was dissolved in
8 mL tetrahydrofuran and 2 mL of water under an argon
atmosphere with ice-cooling. To this solution was added
30% aqueous hydrogen peroxide (2.4 mL, 23.7 mmol).
Then 1.2 mL of a 1 mol/L solution of lithium hydroxide
monohydrate was added, and the mixture was stirred
further for 1 h under ice-cooling. 1.2 mL of a solution
of sodium hydrogen sulfite was added dropwise to the
mixture and the whole was stirred for 30 min. The reac-
tion mixture was concentrated, poured into ice-water,
acidified with dil HCl, and then extracted with methylene
chloride. The extract was washed with brine, dried over
anhydrous magnesium sulfate, and concentrated. The
residue was dissolved in ethyl acetate and n-hexane,
and allowed to stand. The precipitated crystals were col-
lected by filtration and dried. A second crop of crystals
was obtained from the filtrate. The first and second crops
were combined, washed with the mixed solvent of n-hex-
ane and ethyl acetate (4:1–3:1, v/v), and dried to afford
155 mg (55%) of the title compound as a colorless crys-
talline powder. Mp 70–71 °C. 1H NMR (500 MHz,
CDCl3) d 8.22 (t, J = 8.1 Hz, 1H), 7.51 (d, J = 8.1 Hz,
1H), 7.40 (m, 2H), 7.16 (d, J = 2.1 Hz, 1H), 7.07 (dd,
J = 8.3, 2.1 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 4.63 (d,
J = 6.0 Hz, 2H), 4.00 (t, J = 6.8 Hz, 2H), 2.90 (dd,
J = 13.9, 8.1 Hz, 1H), 2.69 (dd, J = 13.9, 6.4 Hz, 1H),
2.56 (m, 1H), 1.80 (m, 2H), 1.67–1.46 (m, 4H), 0.98 (t,
J = 7.3 Hz, 3H), 0.95 (t, J = 7.3 Hz, 3H); MS (FAB)
470 (M+H)+. [a]D +20° (c 0.60, CHCl3, 23 °C). Anal.
Calcd for C24H27F4NO4 C, 61.40; H, 5.80; N, 2.98.
Found: C, 61.12; H, 5.73; N, 2.98.
4.1.42. 5-[(S)-2-((R)-4-Benzyl-2-oxo-oxazolidine-3-carbon
yl)butyl]2-n-butoxybenzaldehyde (19). Compound 16
(1.75 g, 3.22 mmol), 100 mg of 10% palladium on car-
bon, and 30 mL of ethyl acetate were mixed and cata-
lytic hydrogenation was carried out at an initial
hydrogen pressure of 98 kPa. After completion of the
reaction, the catalyst was removed by filtration and
the filtrate was washed with ethyl acetate. The reaction
mixture and the washings were combined and concen-
trated. The residue was purified by silica gel chromatog-
raphy (eluant; n-hexane/ethyl acetate = 3:1, v/v) to
afford 1.37 g (94%) of the carboxylic acid derivative 17
as a colorless oil. To a solution of 17 (1.37 g, 3.02 mmol)
and 25 mL of dehydrated tetrahydrofuran was added a
1 mol/L solution of borane–tetrahydrofuran complex
(5.00 mL, 5.00 mmol) dropwise. After completion of
the addition, the mixture was stirred overnight at room
temperature. A saturated aqueous solution of ammo-
nium chloride was added to the reaction mixture, and
the whole was extracted with ethyl acetate. The extract
was washed with water and brine, dried over anhydrous
sodium sulfate, and concentrated. The residue was puri-
fied by silica gel chromatography (eluant; n-hexane/ethyl
acetate = 4:1, v/v) to afford 1.18 g (89%) of the hydroxy-
methyl derivative 18 as a colorless oil. A solution of 18
(1.18 g,
2.68 mmol),
activated-MnO2
(0.50 g,
5.75 mmol), and 30 mL of dehydrated dichloromethane
was stirred overnight at room temperature. The catalyst
was filtered, washed with dichloromethane, and concen-
trated. The residue was purified by silica gel chromatog-
raphy (eluant; n-hexane/ethyl acetate = 4:1, v/v) to
afford 855 g (73%) of the formyl derivative 20 as a color-
less oil. 1H NMR (500 MHz, CDCl3) d 8.05 (d,
J = 2.3 Hz, 1H), 7.52 (dd, J = 8.5, 2.3 Hz, 1H), 7.29–
7.24 (m, 3H), 7.08 (d, J = 6.8 Hz, 2H), 6.97 (d,
J = 8.5 Hz, 1H), 4.68 (m, 1H), 4.24–4.01 (m, 5H), 3.14
4.1.45. (R)-2-{3-[4-n-Butoxy-(2-fluoro-4-trifluoromethyl-
benzoylamino)methyl]phenylmethyl}butyric acid ((R)-5i).
This compound was prepared from (S)-3-(1-Butyryl)-4-
benzyloxazolidin-2-one by means of a procedure similar

J. Kasuga et al. / Bioorg. Med. Chem. 15 (2007) 5177–5190
5189
to that used for (S)-5j. Mp 70–71 °C. 1H NMR
(500 MHz, CDCl3) d 7.65–7.57 (m, 3H), 7.14 (s, 1H),
7.10 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.77
(s, 1H), 4.57 (m, 2H), 3.85 (s, 3H), 2.88 (dd, J = 13.9,
8.5 Hz, 1H), 2.72 (dd, J = 13.9, 6.0 Hz, 1H), 2.56 (m,
1H), 1.67 (m, 1H), 1.58 (m, 1H), 0.96 (t, J = 7.6 Hz,
3H); MS (FAB) 470 (M+H)+. [a]D À25° (c 0.39, CHCl3,
24 °C). Anal. Calcd for C24H27F4NO4 C, 61.40; H, 5.80;
N, 2.98. Found: C, 61.04; H, 5.88; N, 2.85.
regression analyses. The optimal number of components
in the PLS model was determined using the cross-valida-
tion coefficient r2 (called q2) values obtained from the
leave-one-out cross-validation technique. The PLS mod-
el with the highest q2 values was then selected to derive
3D-QSAR models and the poses of ligands to the
PPARd LBD.
Acknowledgments
4.2. Cell culture and cotransfection assay
We thank Katsuyoshi Kawana (Nihon University), for
helpful discussions and technical assistance. The work
described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry
of Education, Culture, Sports, Science and Technology,
Japan, and the Japan Society for the Promotion of
Science.
Human embryonic kidney HEK293 cells were cultured
in DMEM containing 5% fetal bovine serum and antibi-
otic–antimycotic mixture (Nacalai) at 37 °C in a humid-
ified atmosphere of 5% CO2 in air. Transfections were
performed by calcium phosphate coprecipitation. Eight
hours after transfection, ligands were added. Cells were
harvested approximately 16–20 h after the treatment,
and luciferase and b-galactosidase activities were as-
sayed using a luminometer and a microplate reader.
DNA cotransfection experiments included 50 ng of
reporter plasmid, 20 ng of pCMX-b-galactosidase,
15 ng of each receptor expression plasmid, and pGEM
carrier DNA to make a total of 150 ng of DNA per well
in a 96-well plate. Luciferase data were normalized to an
internal b-galactosidase control and reported values are
the means of triplicate assays.
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