Design, synthesis, and evaluation of a novel series of α-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) α/δ dual agonists for the treatment of metabolic syndrome

Article abstract of DOI:10.1016/j.bmc.2006.09.001

A series of α-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARα/δ). Structure-activity relationship studies indicated that the shape of the linking group and the sha

Full text of DOI:10.1016/j.bmc.2006.09.001

Bioorganic & Medicinal Chemistry 14 (2006) 8405–8414
Design, synthesis, and evaluation of a novel series of a-substituted
phenylpropanoic acid derivatives as human peroxisome
proliferator-activated receptor (PPAR) a/d dual agonists
for the treatment of metabolic syndrome
Jun-ichi Kasuga,a Daisuke Yamasaki,b Yoko Araya,a Aya Nakagawa,a
Makoto Makishima,c Takefumi Doi,b Yuichi Hashimotoa and Hiroyuki Miyachia,*
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita city, Osaka 565-0871, Japan
cSchool of Medicine, Nihon University, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
Received 16 August 2006; revised 31 August 2006; accepted 2 September 2006
Available online 25 September 2006
Abstract—A series of a-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated
receptors alpha and delta (PPARa/d). Structure–activity relationship studies indicated that the shape of the linking group and the
shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype
transactivation. Structure–activity relationships among the amide series (10) and the reversed amide series (13) are similar, but
not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating
that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket
of PPAR. a-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARa/d dual agonists.
Representative compounds exhibited marked nuclear receptor selectivity for PPARa and PPARd. Subtype-selective PPAR activa-
tion was also examined by analysis of the mRNA expression of PPAR-regulated genes.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
paired glucose tolerance, elevated blood pressure, ele-
vated 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 at-
tack, stroke, heart failure, and sudden cardiac death.2
Differences in genetic background, diet, physical activi-
ty, age, gender, and nutrition all affect the prevalence
of the metabolic syndrome.3 Metabolic syndrome is an
extremely important diagnostic indication for the identi-
fication of high-risk patients for multiple risk factor
modification to prevent or delay adverse cardiovascular
events. Affected individuals have visceral obesity, im-
According to the current unifying definition, key ele-
ments of the metabolic syndrome include insulin resis-
tance, abnormal glucose metabolism, hypertension,
atherogenic dyslipidemia (low HDL cholesterol or high
triglycerides), and obesity. For clinical diagnosis of the
metabolic syndrome, there are currently two similar sets
of diagnostic criteria. The World Health Organization
definition requires at least one of the three major fea-
tures, i.e., type 2 diabetes, impaired glucose tolerance,
and insulin resistance, plus at least two of the minor fea-
tures, which include hypertension, obesity, hypertrigly-
ceridemia, and microalbuminuria.1 The National
Cholesterol Education Program Adult Treatment Panel
III Guidelines require three of five clinical criteria,
including abdominal adiposity, hypertriglyceridemia,
low HDL, hypertension, and fasting hyperglycemia, to
make a diagnosis.4 From both sets of criteria, it is clear
that losing weight and restoration of serum glucose and
Keywords: PPAR; PPARa; PPARd; PPARa/d dual agonist; Metabolic
syndrome.
*
Corresponding author. E-mail: miyachi@iam.u-tokyo.ac.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.001

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J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
lipid parameters to normal levels are of primary impor-
tance for treatment.
ligands of PPARc and PPARa, respectively, much
research has been focused on these metabolic nuclear
receptor subtypes as therapeutic targets for the treat-
ment of metabolic syndrome. In contrast, although
PPARd was discovered more than 15 years ago, research
interest in it has been limited. However, after 2001, the
availability of PPARd-knockout animals and the selec-
tive ligands, such as GW-501516 (3), prompted us to
examine the role of PPARd in fatty acid
metabolism.10,11
Therefore, identification of the molecular targets of the
transducers critically involved in the control of glucose
and lipid homeostasis is crucial for developing new ther-
apeutic agents for the treatment of metabolic syndrome.
Metabolic nuclear receptors (NR), are particularly
attractive target molecules, since they have been found
to play a central role in maintaining cellular and
whole-body glucose and lipid homeostasis. These NRs
are activated by a wide variety of physiological ligands,
including dietary fatty acids, cholesterol metabolites,
and xenobiotic compounds, thereby modulating the
transcriptional networks of the target response genes.
Among these receptors, special attention has been paid
for more than a decade to the members of the peroxi-
some proliferator-activated receptor (PPAR) family.
Studies using GW-501516 (3) as a chemical tool point
to key roles for PPARd in lipid metabolism, insulin
resistance, and foam cell and macrophage activation
in atherosclerosis. GW501516 (3) treatment significant-
ly increased HDL cholesterol levels, possibly in associ-
ation with decreased lipoprotein lipase activity, in
insulin-resistant middle-aged obese rhesus monkeys.12
The results showed that GW501516 (3) causes a dra-
matic dose-dependent increase in serum HDL choles-
PPARs are members of the nuclear receptor superfamily,
and are activated by endogenous saturated and unsatu-
rated fatty acids and their metabolites, as well as synthetic
ligands.5 PPARs are heterogeneous, and three subtypes
have been identified to date: PPARa [officially NR1C1],
PPARd/b [NR1C2], and PPARc [NR1C3]. Each PPAR
subtype appears to be differentially expressed in a tissue-
specific manner, and to play a pivotal role in lipid,
lipoprotein, and glucose homeostasis.6 PPARa is mostly
expressed in the tissues involved in lipid oxidation, such
as liver, kidney, skeletal, cardiac muscle, and adrenal
glands. PPARc is expressed in adipose tissue, macro-
phages, and vascular smooth muscles. PPARd is mainly
expressed in skeletal muscle and adipose tissues, but is
ubiquitously expressed in other tissues in extremely small
amounts. PPARs heterodimerize with another nuclear
receptor partner, retinoid X receptor (RXR), and the het-
erodimers regulate gene expression by binding to a specif-
ic consensus DNA sequence, termed PPRE (peroxisome
proliferator responsive element), which is a direct repeat
of the hexameric AGGTCA recognition motif separated
by single nucleotide (DR1), present in the promoter
region of target genes.7
terol and
a reduction in LDL cholesterol and
triglycerides. In a primate model of the metabolic
syndrome, GW501516 (3) dose-dependently lowered
plasma insulin levels, without any adverse effect on
glycemic control.12 Similarly, in ob/ob mice,
GW501516 (3) markedly improved glucose tolerance
and insulin resistance,12 although the underlying
mechanism remains unclear.13
In vitro studies suggested that PPARd activation by
GW501516 (3) in cultured macrophages results in
increased expression of the reverse cholesterol transport-
er ABC A1 and enhances the efflux of cholesterol.12 All
these observations suggest that PPARd may also be an
effective target for the treatment of metabolic syndrome.
Considering the above-mentioned findings, the metabol-
ic function(s) of PPARd seem to be mainly targeted to
adipose tissue and smooth muscle, via fatty acid oxida-
tion and energy uncoupling. If this is so, a compound
that can effectively activate both PPARa and PPARd
might have additive and/or synergistic positive effect(s)
in the treatment of metabolic syndrome, modulating
both hepatic fatty acid oxidation through PPARa, and
fatty acid oxidation and energy uncoupling in muscle
and adipose tissue through PPARd. There are a few
examples of PPARa/d dual agonists in the literature,
including compounds 5,14 6,15 and 716 (Fig. 1), but their
activities are not so high and the structural variety is
poor. Therefore, there is considerable interest in creating
novel PPARa/d dual agonists from both basic scientific
and clinical points of view.
PPARc was first identified as a master regulator of adi-
pocyte differentiation, but more recent molecular-bio-
logical studies have indicated that PPARc activation is
also linked to the expression of many important genes
that affect energy metabolism, such as the TNF-a, lep-
tin, and adiponectin genes.8
PPARa regulates the expression of genes encoding for
proteins involved in lipid and lipoprotein homeostasis.9
For example, it regulates genes involved in fatty acid
uptake (such as fatty acid binding protein, FABP), b-ox-
idation (acyl-CoA oxidase), and x-oxidation (cyto-
chrome P450). It down-regulates apolipoprotein C-III,
a protein that inhibits triglyceride hydrolysis by lipopro-
tein lipase, and it also regulates genes involved in reverse
cholesterol transport, such as apolipoprotein A-I and
apolipoprotein A-II.9
Previously, we designed and synthesized a series of
substituted phenylpropanoic acid derivatives as human
PPARa-selective agonists, using KRP-297,17 a PPARc/
a dual agonist with the 2-methoxybenzamide structural
motif, as a lead compound. We found that KCL (2),
an a-ethylphenylpropanoic acid derivative, exhibited
potent, human PPARa-selective agonistic activity.18–21
As a part of our continuing research directed toward
the structural development of subtype-selective PPAR
agonists, we tried to construct structurally new
Based on the findings that antidiabetic thiazolidine-2,
4-diones (glitazones) and antidyslipidemic fibrates are

J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
8407
O
O
O
S
F
OH
OH
O
N
H
Me
Me
Me
F
F
N
H
N
MeO
F
F
1
2
O
Me
Me
O
O
O
OH
OH
F
O
OH
Me
S
N
F
S
F
O
O
Me
3
4
O
O
Cl
F
O
O
O
O
O
N
OH
Cl
OH
N
H
F
N
N
Cl
N
N
S
O
F
O
O
6
O
F
F
OH
5
7
Figure 1. Structures of representative PPARa (1 (GW-9645), 2 (KCL)), PPARd (3 (GW-501516), 4), and dual PPARa/d (5 (GW-2433), 6, 7) agonists.
Compounds (10a–h) were prepared from substituted
benzoic acid derivatives (8a–d) in 2 steps. Compounds
8a–d21 were condensed with substituted benzylamine,
followed by alkaline hydrolysis to afford the desired
products (Scheme 1).22 The reversed amide derivatives
(13b–h, 13l–m) and the sulfonamide derivative 13a were
prepared from substituted benzylamine derivatives
(11a–d) by means of procedures similar to those used
for the preparation of compounds 10 (Scheme 2).
R1
R2
O
O
O
X
OH
OH
N
H
F
F
R3
O
Me
O
Me
F
KCL
Figure 2. Structural development of KCL.
PPARa/d dual agonists, using KCL with the 2-meth-
oxybenzamide structure as a lead compound (Fig. 2).
It was anticipated that small manipulations of the struc-
ture of KCL would open up characteristic new PPAR
subtype selectivities.
Optically active a-ethyl-substituted phenylpropanoic
acid derivatives with the reversed amide linkage (13i–k,
13n) were prepared as per a previously described
method.23
2. Chemistry
3. Results and discussion
The synthetic routes to the present series of a-substituted
phenylpropanoic acids are outlined in Schemes 1 and 2.
The transactivation activity of the present series of
compounds towards each of the PPARs is summarized
O
O
O
O
O
O
X
OEt
OH
N
H
N
OEt
a
b
HO
z
z
H
R
R
R
X
X
Y
Y
8a (X = OMe, R = H)
8b (X = OMe, R = Me)
8c (X = OMe, R = Et)
8d (X = F, R = Et)
9a-h
10a-h
Scheme 1. Synthesis of 10a–h. Reagents: (a) substituted benzylamine, ClCO2Et, TEA, CH2Cl2; (b) aq-NaOH, EtOH.
O
O
O
A
A
OEt
OH
N
H
N
H
H2N
MeO
11a (R = H)
OEt
c)
d)
z
z
R
R
R
MeO
MeO
Y
Y
12a, 12b-h, 12l-m
A = SO2 (12a)
A = CO (12b-h, 12l-m)
13a, 13b-h, 13l-m
A = SO2 (13a)
A = CO (13b-h, 13l-m)
11b (R = Me)
11c (R = Et)
11d (R =n-Pr)
Scheme 2. Synthesis of 13a–h, 13l–m. Reagents: (c) Substituted benzoyl chloride (or substituted benzenesulfonyl chloride), TEA, CH2Cl2; (d) aq-
NaOH, EtOH.

8408
J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
Table 1. In vitro functional PPAR transactivation activity of substituted phenylpropanoic acids
O
X
O
R
N
H
OH
z
A
Y
a
EC50 (nM)
Compound
X
Y
Z
A
Stereo
PPARa
PPARd
PPARc
2
MeO
F
Et
Et
Et
Et
H
H
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
Rac
Rac
Rac
Rac
Rac
Rac
70
700
iab
3000
iab
10a
10b
10c
10d
10e
H
iab
73
MeO
MeO
MeO
MeO
2-F
3-F
3-F
3-F
730
590
iab
870
1700
iab
32
500
19
Me
1900
8200
Compound
X
Y
R
Stereo
PPARa
PPARd
PPARc
10f
10g
10h
MeO
MeO
MeO
Et
Et
Et
4-phenoxybenzyl
fluoren-2-yl-methyl
pyrene-1-yl-methyl
Rac
Rac
Rac
15
1200
3000
iab
2000
7000
iab
500
iab
a Compounds were screened for agonist activity on PPAR-GAL4 chimeric receptors in transiently transfected HEK-293 cells as described. EC50 value
is the molar concentration of the test compound that affords 50% of the maximal reporter activity.
b ‘‘ia’’ means inactive (no apparent activity) at the concentration of 10 lM.
Table 2. In vitro functional PPAR transactivation activity of substi-
tuted phenylpropanoic acids
those of the non-fluorinated compound 10a (although
10b shows somewhat increased activity toward PPARc,
O
F
F
its activity is still low). Interestingly, some steric features
of the hydrophobic tail are critically important for
PPARa activity and the selectivity in the present series.
The 4-phenoxybenzyl derivative (10f) exhibited potent
PPARa transactivation activity and about 100-fold
PPARa selectivity (vs. PPARd and PPARc), while the
conformationally restricted fluoren-2-ylmethyl deriva-
tive (10g) and bulky pyren-1-ylmethyl derivative (10h)
exhibited only weak activity or did not exhibit PPARa
transactivation activity at the concentration of 10 lM.
F
B
OH
O
Me
a
EC50 (nM)
Compound
B
PPARa
PPARd
PPARc
2
CH2NHCO
SO2NHCH2
CONHCH2
70
iab
19
700
iab
3000
iab
13a
13b
200
2600
a See footnote (a) in Table 1.
b See footnote (b) in Table 1.
As already mentioned, the substituent at the a-position
of the carboxyl group is important for the potency for
PPARa,21 i.e., 10d, which does not have an a-substitu-
ent, exhibited only weak PPARa transactivation activity
(though it showed more than 100-fold PPARa selectivi-
ty), while introduction of a methyl group (10e) increased
the PPARa activity by more than 10-fold, though the
PPARd and PPARc transactivation activities are still
weak, so that this compound retains PPARa selectivity.
As a whole, the amide-type derivatives (10) tended to
exhibit a PPARa-selective transactivation profile, and
we did not find PPARa/d dual agonists in this series of
compounds.
in Tables 1–3, together with the results for the lead com-
pound, racemic KCL. First, we investigated the effect of
the introduction of a fluorine atom in racemic KCL, be-
cause we expected that a fluorinated compound would
bind more tightly to PPAR by effectively excluding the
water molecule located in the hydrophobic binding pock-
et of PPAR. Compound 10a, which has a fluorine atom
on the central benzene ring, instead of a methoxyl group,
however, showed considerably decreased PPARs trans-
activation activity as compared with racemic KCL; it
did not exhibit apparent PPAR transactivation activity
at the concentration of 10 lM. This result might indicate
that the methoxyl group located at the ortho position of
the amide carbonyl group is involved in a hydrogen-
bonding interaction that restricts the conformation of
the central benzene part of the molecule. As for substit-
uents at the hydrophobic tail part, introduction of fluo-
rine apparently did not affect PPAR transactivation
activity or selectivity. Compounds 10b and 10c, which
have a fluorine atom at the 2 or 3 position of the distal
benzene ring, respectively, exhibited PPARa and PPARd
transactivation activities that were comparable with
Previously, we found that the 3-atom unit linker –CH2–
NH–CO– imparted potent PPARa transactivation activ-
ity, but shortening or lengthening of the linking group
considerably decreased the activity.23 We also noted that
a flexible linker, such as –CH2–CH2–CH2– or –CH2–
CH2–O–, decreased PPARa transactivation activity,
but caused PPARd transactivation activity to appear.
Therefore, we focused our attention on a hybrid type
linker, i.e., –CO–NH–CH2– (13b), and found that this
linker increased both PPARa and PPARd transactiva-
tion activity to some extent, as compared with the amide
type (–CH2–NH–CO–) linker (2). We speculated that

J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
8409
Table 3. In vitro functional PPAR transactivation activity of substituted phenylpropanoic acids
O
O
R
N
H
OH
z
A
Y
X
a
EC50 (nM)
Compound
X
Y
Z
A
Stereo
PPARa
PPARd
PPARc
13b
13c
13d
13e
13f
13g
13h
13i
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
Et
Et
H
4-CF3
4-CF3
4-CF3
4-F
rac
rac
rac
rac
rac
rac
rac
S
19
10
200
24
2600
2200
6000
ia(b)
iab
2-F
3-F
3-CF3
H
Et
Et
11
51
250
200
19
2000
iab
H
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
Me
n-Pr
Et
H
210
120
12
iab
H
63
10
3600
1900
4900
iab
2-F
3-F
3-F
13j
13k
Et
Et
S
12
150
23
840
R
Compound
X
Y
R
Stereo
PPARa
PPARd
PPARc
13l
MeO
MeO
MeO
Et
Et
Et
4-phenoxyphenyl
pyrene-1-yl
pyrene-1-yl
rac
rac
S
8.5
24
10
120
66
800
iab
iab
13m
13n
40
a See footnote (a) in Table 1.
b See footnote (b) in Table 1.
the appearance of a conformationally flexible group and/
or atom, such as –CH2– or –O–, adjacent to the
central benzene ring might favor both PPARa and
PPARd activity, while introduction of a more conform-
ationally restricted group (–CO–) next to the central
benzene ring might be unfavorable for PPARd activity.
These results might reflect differences in the shape and
environment of the hydrophobic cavity hosting the dis-
tal benzene ring between PPARa and PPARd.
activity on both PPARa and PPARd, while the antipo-
dal (R) isomer 13k exhibited far less potency. Therefore,
we concluded that both PPARa and PPARd transacti-
vation activities reside exclusively in the (S)-enantiomer,
and both 13i and 13j show dual-agonist activity toward
PPARa and PPARd.
Interestingly, steric tolerance of the hydrophobic tail
part is somewhat different between the amide series of
compounds and the reversed amide series of com-
pounds. In the case of R = 4-phenoxybenzyl (10f, 13l),
10f and 13l exhibited almost equipotent PPARa transac-
tivation activity. As for PPARd, 13l still exhibited a sub-
micromolar order EC50, while the activity of 10f was
about 10 times less potent than that of 13l. As a result,
the PPARd/a selectivity ratio of 10f is superior to that of
13l (80-fold selective vs. 10-fold selective). The difference
in the sterical factor is extremely evident in the case of
R = pyren-1-yl group (10h, 13m–13n). Although 10h
did not exhibit apparent PPARs transactivation activity
We then prepared the sulfonamidemethyl linker deriv-
ative 13a, but this compound did not exhibit apparent
PPAR transactivation activity. The hydrophilic sulfon-
amide moiety might interact unfavorably with the
hydrophobic pocket of each PPAR ligand binding do-
main. Based on the above results, we further prepared
the ‘reversed amide’ series of compounds (13c–n). As
can be seen in Table 3, some noteworthy results were
obtained. In contrast to the amide series, the introduc-
tion of fluorine at the 2 or 3 position of the distal
benzene ring increased the PPARd transactivation
activity. The position of the distal benzene substitu-
ents is crucial, since compound 13e, which has a fluo-
rine atom at the 4 position and a trifluoromethyl
group at the 3 position, showed considerably de-
creased PPAR transactivation activity. This is consis-
tent with the previously obtained structure–activity
relationship result that steric bulkiness at the 4-posi-
tion is an important factor for potent PPARa transac-
tivation activity.21
80
0.1
M
1 M
60
40
20
0
Considering the results obtained above, we then pre-
pared optically active derivatives, 13i, 13j, and 13k. As
can be seen from Table 3, a clear enantio-dependency
of the transactivation activity toward the PPARa and
PPARd isoforms was found. Compound 13j, which
has (S) configuration, exhibited potent transactivation
a
a
a
h
l
a
t
ha
R
l
m
ha
p
h
o
p
l
r
XR
F
e
D
V
p
l
a
p
t
m
l
l
a
d
a
n
a
g
a
R
R
o
R
R
R
A
A
C
R
X
A
X
P
A
L
R
R
PP
P
PP
Figure 3. Cross-nuclear receptor reactivity of 0.1 lM, and 1 lM 13i.

8410
J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
Figure 4. Regulation of PPAR targeted gene expression by various PPAR ligands.
at the concentration of 10 lM, 13m still exhibited potent
dual PPARa/d transactivation activity, comparable with
those of 13c and 13d. These differences might be a result
of interaction of the distal hydrophobic tail part of 10h
and 13m with different amino acids in the large hydro-
phobic ligand binding pocket, probably due to the dif-
ference in the conformational flexibility of these linker
groups. In the case of the pyren-1-yl derivative, PPARa
and PPARd transactivation activities also reside in the
(S)-enantiomer, 13n.
At first, we investigated the effects of the representative
PPAR-subtype selective agonists, fenofibrate (Feno; a
PPARa-selective agonist), GW-501516 (GW; a PPARd-
selective agonist), and troglitazone (Tro; a PPARc-selec-
tive agonist), to transactivate these genes in Huh-7. As
indicated in Figure 4, when Huh-7 cells were treated
with 50 lM Feno, 0.1 lM GW, and 10 lM Tro (the
indicated concentrations of each compound were en-
ough to exhibit subtype-selective transactivation activi-
ty), these three genes mRNA expressions were
augmented subtype selectively. These results indicated
that each PPAR-subtype in Huh-7 is functionally active.
ADRP gene expression was significantly augmented
with these three PPAR agonists. CPT1A gene expression
was greatly augmented by Feno and GW, indicating
that expression of this gene is mediated through the
PPARa and PPARd pathways. ANGPTL4 gene expres-
sion was greatly augmented only by Tro, which indicates
that expression of this gene is mediated through the
PPARc pathway. Treatment with 0.1 lM 13i augmented
CPT1A gene expression to an extent comparable with
that obtained with 0.1 lM GW, and had little effect on
expression of ANGPTL4 gene, probably due to the
weak activity of 13i toward PPARc.
In order to investigate the nuclear receptor selectivity
(cross-reactivity) of the representative compound 13i,
we determined the transactivation activity of 13i on rep-
resentative nuclear receptors (PPARs, VDR, FXR,
LXRa, RARa, and RXRa). As can be seen from
Figure 3, 13i is specific for PPARa and PPARd because
it did not significantly activate VDR, PPARc, LXRa,
RARa or RXRa at concentrations up to 1 lM 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 to both PPARa
and PPARd.
To demonstrate the ability of compound 13i to activate
the genes which have peroxisome proliferator responsive
element (PPRE) in the promoter region in the cell level,
we examined changes in expression of representative
PPAR-mediated genes in human hepatocellular carci-
noma Huh-7. We chose carnitine palmitoyl acyl-CoA
transferase 1A (CPT1A), angiopoietin-like protein 4
(ANGPTL4), and adipocyte differentiation-related pro-
tein (ADRP) as the representative PPAR target genes.
For each gene of human were reported to possess PPRE
in the promoter region.24–26 CPT1A is the key enzyme in
the carnitine-dependent transport across the mitochon-
drial inner membrane and its deficiency results in a de-
creased rate of fatty acid b-oxidation. ANGPTL4 is a
member of the angiopoietin/angiopoietin-like gene fam-
ily and encodes a glycosylated, secreted protein with a
fibrinogen C-terminal domain. This gene is the target
of peroxisome proliferation activators, such as fibrate
class antihyperlipidemic agents. ADRP is associated
with the globule surface membrane material. This pro-
tein is a major constituent of the globule surface. In-
crease in the level of ADRP mRNA is one of the
earliest indications of adipocyte differentiation.
These results indicate that the representive compound
13i is an effective PPARa/d dual agonist at the cellular
level.
In summary, we have developed a series of potent hu-
man PPARa/d dual agonists, which possess potent
PPARa/d transactivation activity with high selectivity
over PPARc. In vivo pharmacological evaluation of
representative compounds is awaited.
4. Experimental
4.1. General
Melting points were determined by using a Yanagimoto
hot-stage melting point apparatus and are uncorrected.
Elemental analyses were carried out in the Microanalyt-
ical Laboratory, Faculty of Pharmaceutical Sciences,
University of Tokyo, and were within plus or minus
0.3% of the theoretical values. NMR spectra were
recorded on a JEOL JNM-GX500 (500 MHz) spectrom-
eter. Chemical shifts are expressed in ppm relative to tet-

J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
8411
ramethylsilane. Mass spectra were recorded on a JEOL
JMS-DX303 spectrometer.
cedure similar to that used for 10b. Mp 120–121 °C;
1H NMR (500 MHz, CDCl3) d 9.18 (s, 1H), 7.81 (d,
1H, J = 7.9 Hz), 7.72 (d, 1H, J = 5.5 Hz), 7.55 (d, 1H,
J = 7.9 Hz), 7.41 (s, 1H), 7.26 (m, 5H), 6.46 (d, 1H,
J = 5.5 Hz), 5.00 (m, 1H), 4.76 (s, 2H), 4.22 (s, 2H),
3.95 (m, 1H), 3.61 (m, 1H), 1.80 (m, 3H), 1.56 (m,
3H); HRMS (FAB+) calcd for C21H22F4NO4 (M+H)+;
470.1779. Found 470.1762. Anal. (C21H21F4NO4) C,
H, N.
4.1.1. 2-Ethyl-3-[4-methoxy-3-[N-[[2-fluoro-4-(trifluoro-
methyl)phenyl]methyl]carbamoyl]phenyl] propanoic acid
(10b). Ethyl 3-(3-carboxy-4-methoxyphenyl)-2-ethyl-
propionate (8c)21 (400 mg, 1.43 mmol) was dissolved
in 10 mL of dry dichloromethane, and the solution
was cooled to À10 °C to À15 °C. Triethylamine
(0.5 mL, 3.58 mmol) was added under stirring, fol-
lowed by the addition of ethyl chlorocarbonate
(185 mg, 1.71 mmol) dissolved in 5 mL of dry dichlo-
romethane. The mixture was stirred for 10 min at
À10 °C, then 2-fluoro-4-(trifluoromethyl)benzylamine
(392 mg, 1.71 mmol) dissolved in 5 mL of dry dichlo-
romethane was added dropwise. Stirring was contin-
ued for 30 min at À10 °C, then for 7 h at room
temperature and finally the mixture was left to stand
overnight. It was washed with aqueous citric acid,
aqueous sodium hydrogen carbonate, and brine, then
dried over anhydrous sodium sulfate and concentrat-
ed. The residue was purified by silica gel column chro-
matography (eluant n-hexane: AcOEt = 1:1 v/v) to
afford 270 mg of ethyl 2-ethyl-3-[4-methoxy-3-[N-[[4-
(trifluoromethyl)phenyl]methyl]carbamoyl]phenyl] pro-
pionate (9b) as a yellow oil.
4.1.4.
3-[4-Methoxy-3-[N-[[4-(trifluoromethyl)phenyl]-
methyl]carbamoyl]phenyl]propanoic acid (10d). This
compound was prepared from 8a21 and 4-(trifluoro-
methyl)benzylamine by means of a procedure similar
to that used for 10b. Mp 141–143 °C; 1H NMR
(500 MHz, CDCl3) d 9.18 (s, 1H), 7.81 (d, 1H,
J = 7.9 Hz), 7.72 (d, 1H, J = 5.5 Hz), 7.55 (d, 1H,
J = 7.9 Hz), 7.41 (s, 1H), 7.26 (m, 5H), 6.46 (d, 1H,
J = 5.5 Hz), 5.00 (m, 1H), 4.76 (s, 2H), 4.22 (s, 2H),
3.95 (m, 1H), 3.61 (m, 1H), 1.80 (m, 3H), 1.56 (m,
3H); HRMS (FAB+) calcd for C19H18F4NO4 (M+H)+;
400.1169. Found 400.1172.
4.1.5.
3-[4-Methoxy-3-[N-[[4-(trifluoromethyl)phenyl]-
methyl]carbamoyl]phenyl]-2-methylpropanoic acid (10e).
This compound was prepared from 8b21 and 4-(trifluo-
romethyl)benzylamine by means of a procedure similar
to that used for 10b. Mp 125–127 °C; 1H NMR
(500 MHz, CDCl3) d 8.32 (s, 1H), 8.04 (d, 1H,
J = 2.6 Hz), 7.54 (t, 1H, J = 7.7 Hz), 7.29 (dd, 1H,
J = 7.7, 2.6 Hz), 7.20 (d, 1H, J = 7.2 Hz), 7.17 (d, 1H,
J = 7.2 Hz), 6.90 (d, 1H, J = 7.0 Hz), 4.69 (d, 2H,
J = 6.0 Hz), 3.93 (s, 3H), 3.00–3.04 (m, 1H), 2.73–2.78
(m, 1H), 2.66–2.71 (m, 1H), 1.16 (d, 3H, J = 6.8 Hz);
HRMS (FAB+) calcd for C20H20F4NO4 (M+H)+;
414.1339. Found 414.1328. Anal. (C20H20F4NO4) C,
H, N.
A
mixture of 9b (270 mg), 15 mL ethanol, and
15 mL of a 1 mol/L aqueous solution of sodium
hydroxide was stirred for 4 h at 50 °C, then concen-
trated under reduced pressure. The residue was dis-
solved in water and acidified with dil. HCl. The
precipitate formed was collected by filtration, dried,
and recrystallized from ethyl acetate to afford
165 mg (27%) of the title compound as colorless
1
prisms: mp 102–103 °C; H NMR (500 MHz, CDCl3)
d 0.96 (t, 3H, J = 7.3 Hz), 1.53–1.72 (m, 2H), 2.59–
2.66 (m, 1H), 2.77 (dd, 1H, J = 13.7, 6.8 Hz), 2.96
(dd, 1H, J = 13.7, 8.3 Hz), 3.92 (s, 3H), 4.73 (d,
2 H, J = 5.9 Hz), 6.90 (d, 1H, J = 8.3 Hz), 7.29 (dd,
1H, J = 8.3, 2.4 Hz), 7.47 (d, 1H, J = 8.3 Hz), 7.59
(d, 2H, J = 7.8 Hz), 8.08 (d, 1H, J = 2.4 Hz), 8.32
(t, 1H, J = 5.9 Hz); HRMS (FAB+) calcd for
C21H22F4NO4 (M+H)+; 428.1485. Found 428.1453.
Anal. (C21H21F4NO4) C, H, N.
4.1.6. 2-Ethyl-3-[4-methoxy-3-[N-[[4-(phenoxy)phenyl]-
methyl]carbamoyl]phenyl] propanoic acid (10f). This
compound was prepared from 8c21 and 4-(phen-
oxy)benzylamine by means of a procedure similar to
that used for 10b. Mp 123–124 °C; 1H NMR
(500 MHz, CDCl3) d 9.18 (s, 1H), 7.81 (d, 1H,
J = 7.9 Hz), 7.72 (d, 1H, J = 5.5 Hz), 7.55 (d, 1H,
J = 7.9 Hz), 7.41 (s, 1H), 7.26 (m, 5H), 6.46 (d, 1H,
J = 5.5 Hz), 5.00 (m, 1H), 4.76 (s, 2H), 4.22 (s, 2H),
3.95 (m, 1H), 3.61 (m, 1H), 1.80 (m, 3H), 1.56 (m,
3H); HRMS (FAB+) calcd for C26H28NO5 (M+H)+;
434.1970. Found 434.1967. Anal. (C26H27NO5) C,
H, N.
4.1.2. 2-Ethyl-3-[4-fluoro-3-[N-[[4-(trifluoromethyl)phen-
yl]methyl]carbamoyl]phenyl]propanoic acid (10a). This
compound was prepared from 8b21 and 4-(trifluoro-
methyl)benzylamine by means of a procedure similar
to that used for 10b. Mp 99–100 °C; 1H NMR
(500 MHz, CDCl3) d 9.18 (s, 1H), 7.81 (d, 1H,
J = 7.9 Hz), 7.72 (d, 1H, J = 5.5 Hz), 7.55 (d, 1H,
J = 7.9 Hz), 7.41 (s, 1H), 7.26 (m, 5H), 6.46 (d, 1H,
J = 5.5 Hz), 5.00 (m, 1H), 4.76 (s, 2H), 4.22 (s, 2H),
3.95 (m, 1H), 3.61 (m, 1H), 1.80 (m, 3H), 1.56 (m,
3H); FAB-MS m/z 398 (M+H)+; Anal. (C20H19F4NO4)
C, H, N.
4.1.7. 2-Ethyl-3-[4-methoxy-3-[N-[[fluoren-1-yl]methyl]-
carbamoyl]phenyl] propanoic acid (10g). This compound
was prepared from 8c21 and fluoren-1-yl-methylamine
by means of a procedure similar to that used for 10b.
1
Mp 151–153 °C; H NMR (500 MHz, CDCl3) d 8.25
(s, 1H), 8.10 (s, 1H), 7.75 (m, 2H), 7.53 (m, 2H), 7.36
(m, 2H), 7.26 (m, 2H), 6.88 (d, J = 7.7 Hz, 1H), 4.74
(d, J = 5.6 Hz, 2H), 3.88 (s, 5H), 2.94 (m, 1H), 2.80
(m, 1H), 2.63 (m, 1H), 1.62 (m, 2H), 0.96 (t,
J = 6.4 Hz, 3H); MS m/z 430 (M+H)+; Anal.
(C27H24NO4) C, H, N.
4.1.3. 2-Ethyl-3-[4-methoxy-3-[N-[[3-fluoro-4-(trifluoro-
methyl)phenyl]methyl]carbamoyl]phenyl] propanoic acid
(10c). This compound was prepared from 8c21 and 3-flu-
oro-4-(trifluoromethyl)benzylamine by means of a pro-

8412
J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
4.1.8. 2-Ethyl-3-[4-methoxy-3-[N-[[pyren-1-yl]methyl]car-
bamoyl]phenyl] propanoic acid (10h). This compound
was prepared from 8c21 and pyren-1-yl-methylamine
by means of a procedure similar to that used for 10b.
4.1.11.
2-Ethyl-3-[4-methoxy-3-[N-[2-fluoro-4-(trifluoro-
methyl)benzoylamino]methyl]phenyl]propanoic acid (13c).
This compound was prepared from 11c21 and 2-fluo-
ro-4-trifluoromethylbenzoyl chloride by means of a
procedure similar to that used for 13a. Mp 108–
110 °C; 1H 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.
(C21H21F4NO4) C, H, N.
1
Mp 132–134 °C; H NMR (500 MHz, CDCl3) d 8.34
(d, 2H, J = 8.8 Hz), 8.30 (s, 1H), 8.13–8.19 (m, 5H),
7.99–8.05 (m, 3H), 7.23 (d, 1H, J = 8.1 Hz), 6.78 (d,
1H, J = 8.8 Hz), 5.36 (d, 2H, J = 5.1 Hz), 3.65 (s, 3H),
2.94–2.98 (m, 1H), 2.76—2.80 (m, 1H), 2.64–2.65 (m,
1H), 1.64–1.69 (m, 1H), 1.57–1.61 (m, 1H), 0.96 (t,
3H, J = 7.5 Hz); FAB-MS m/z 393 (M+H)+; Anal.
(C30H27NO4) C, H, N.
4.1.9. 2-Ethyl-3-[4-methoxy-3-[N-[4-(trifluoromethyl)ben-
zenesulfonylamino]methyl]phenyl]propanoic acid (13a).
Ethyl 3-(3-aminomethyl-4-methoxyphenyl)-2-ethylpro-
pionate HCl (11c)21 (212 mg, 0.70 mmol) was dis-
solved in 20 mL of dry dichloromethane and 1.2
equiv of triethylamine was added under stirring, fol-
lowed by the addition of 4-(trifluoromethyl)benze-
nesulfonyl chloride (177 mg, 0.70 mmol) dissolved in
5 mL of dry dichloromethane. Stirring was continued
overnight at room temperature. The mixture was
washed with aqueous citric acid, aqueous sodium
hydrogen carbonate, and brine, then dried over anhy-
drous sodium sulfate and concentrated. The residue
was purified by silica gel column chromatography
(eluant n-hexane: AcOEt = 1:1 v/v) to afford 297 mg
of ethyl 2-ethyl-3-[4-methoxy-3-[N-[4-(trifluorometh-
yl)benzenesulfonylamino]methyl]phenyl]propanoic acid
(12a) as an oil. A mixture of 12a (295 mg), 15 mL eth-
anol and 15 mL of 1 mol/L aqueous solution of sodi-
um hydroxide was stirred for 4 h at 50 °C, then
concentrated under reduced pressure. The residue
was dissolved in water, and acidified with dil. HCl.
The precipitate that formed was collected by filtration,
dried, and recrystallized from ethyl acetate to afford
235 mg (85%) of the title compound: mp 130–131 °C;
1H NMR (500 MHz, CDCl3) d 7.80 (d, J = 8.5 Hz,
2H), 7.60 (d, J = 8.1 Hz, 2H), 6.97 (dd, J = 8.1,
2.1 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.54 (d,
J = 8.1 Hz, 1H), 5.45 (br, 1H), 4.16 (d, J = 6.0 Hz,
2H), 3.65 (s, 3H), 2.79 (dd, J = 13.6, 8.5 Hz, 1H),
2.60 (dd, J = 13.7, 6.4 Hz, 1H), 2.50 (m, 1H), 1.62
(m, 1H), 1.56 (m, 1H), 0.95 (t, J = 7.2 Hz, 3H);
HRMS (FAB+) calcd for C20H22F4NO5S (M+H)+;
445.1171. Found 445.1171.
4.1.12.
2-Ethyl-3-[4-methoxy-3-[N-[3-fluoro-4-(trifluoro-
methyl)benzoylamino]methyl]phenyl]propanoic acid (13d).
This compound was prepared from 11c21 and 4-fluoro-
3-trifluoromethylbenzoyl chloride by means of a proce-
dure similar to that used for 13a. Mp 120–121 °C; H
NMR (500 MHz, CDCl3) d 7.60 (m, 3H), 7.14 (s, 1H),
7.10 (dd, J = 8.5, 2.1 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H),
6.77 (br, 1H), 4.57 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H),
2.88 (dd, J = 13.7, 8.5 Hz, 1H), 2.72 (dd, J = 13.7,
6.0 Hz, 1H), 2.56 (m, 1H), 1.66 (m, 1H), 1.58 (m, 1H),
0.96 (t, J = 7.3 Hz, 3H); MS (FAB); 428 (M+H)+; Anal.
(C21H21F4NO4) C, H, N.
1
4.1.13.
2-Ethyl-3-[4-methoxy-3-[N-[4-fluoro-3-(trifluoro-
methyl)benzoylamino]methyl]phenyl]propanoic acid (13e).
This compound was prepared from 11c21 and 3-fluoro-
4-trifluoromethylbenzoyl chloride by means of a proce-
dure similar to that used for 13a. Oil; 1H NMR
(500 MHz, CDCl3) d 8.07 (m, 1H), 8.00 (m, 1H), 7.26
(t, J = 9.4 Hz, 1H), 7.18 (m, 1H), 7.14 (dd, J = 8.1,
6.0 Hz, 1H), 6.85 (m, 2H), 5.70 (br, 1H), 4.61 (d,
J = 5.6 Hz, 2H), 3.89 (s, 3H), 2.92 (dd, J = 13.7,
8.5 Hz, 1H), 2.75 (dd, J = 13.7, 7.7 Hz, 1H), 2.59 (m,
1H), 1.68 (m, 1H), 1.61 (m, 1H), 0.99 (t, J = 7.7 Hz,
3H); HRMS; (M+H)+; calcd for C21H22F4NO4,
428.1485. Found 428.1488.
4.1.14. 3-[4-Methoxy-3-[N-[[4-(trifluoromethyl)benzoyl-
amino]methyl]phenyl]propanoic
acid
(13f).
This
compound was prepared from 11a21 and 4-(trifluoro-
methyl)benzoyl chloride by means of a procedure similar
to that used for 13a. Mp 166–167 °C; 1H NMR
(500 MHz, CDCl3) d 7.86 (d, 2H, J = 8.3 Hz), 7.68 (d,
2H, J = 8.3 Hz), 7.20 (d, 1H, J = 2.1 Hz), 7.14 (dd, 1H,
J = 8.1, 2.1 Hz), 6.84 (d, 1H, J = 8.1 Hz), 6.72 (s, 1H),
4.62 (d, 2H, J = 6.0 Hz), 3.87 (s, 3H), 2.91 (t, 2H,
J = 7.7 Hz), 2.65 (d, 2H, J = 7.7 Hz); HRMS; (M+H)+;
calcd for C21H22F4NO4, 382.1236. Found 382.1266.
4.1.10. 2-Ethyl-3-[4-methoxy-3-[N-[4-(trifluoromethyl)-
benzoylamino]methyl]phenyl]propanoic acid (13b). This
compound was prepared from 11c21 and 4-trif-
luoromethylbenzoyl chloride by means of a proce-
dure similar to that used for 13a. Mp 133–135 °C;
1H NMR (500 MHz, CDCl3)
d
7.79 (d, 2H,
4.1.15. 3-[4-Methoxy-3-[N-[[4-(trifluoromethyl)benzoyl-
amino]methyl]phenyl]-2-methylpropanoic acid (13g). This
compound was prepared from 11b21 and 4-(trifluoro-
methyl)benzoyl chloride by means of a procedure simi-
lar to that used for 13a. Mp 129–130 °C; 1H NMR
(500 MHz, CDCl3) d 7.85 (d, 2H, J = 8.1 Hz), 7.67 (d,
2H, J = 8.1 Hz), 7.18 (d, 1H, J = 2.1 Hz), 7.10 (dd, 1H,
J = 8.1, 2.1 Hz), 6.82 (d, 1H, J = 8.1 Hz), 6.74 (s, 1H),
4.61 (d, 2H, J = 5.5 Hz), 3.87 (s, 3H), 2.96 (dd, 1H,
J = 13.7, 6.8 Hz), 2.76–2.64 (m, 2H), 1.18 (d, 3H,
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 C21H22F3NO44/
5H2O C, 59.51; H, 5.57; N, 3.30. Found: C,
59.27; H, 5.18; N, 3.21.

J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
8413
J = 6.8 Hz); HRMS; (M+H)+; calcd for C20H21F3NO4,
396.1422. Found 396.1423.
2H), 7.07 (dd, 1H, J = 8.6, 2.1 Hz), 7.02 (d, 2H,
J = 8.1 Hz), 6.97 (d, 2H, J = 8.5 Hz), 6.79 (d, 1H,
J = 8.1 Hz), 6.66 (t, 1H, J = 4.6 Hz), 4.57 (d, 2H,
J = 6.0 Hz), 3.84 (s, 3H), 3.50 (br, 1H), 2.87 (dd, 1H,
J = 13.7, 8.6 Hz), 2.71 (dd, 1H, J = 13.7, 6.4 Hz), 2.55
(m, 1H), 1.65 (m, 1H), 1.58 (m, 1H), 0.95 (t, 3H,
J = 7.3 Hz, 3H); MS (FAB); 434(M+H)+; Anal.
(C26H27NO5) C, H, N.
4.1.16. 3-[4-Methoxy-3-[N-[[4-(trifluoromethyl)benzoyl-
amino]methyl]phenyl]-2-n-propylpropanoic acid (13h).
This compound was prepared from 11d21 and 4-(trifluo-
romethyl)benzoyl chloride by means of a procedure sim-
1
ilar to that used for 13a. Mp 129—130 °C; H NMR
(500 MHz, CDCl3) d 7.85 (d, 2H, J = 8.3 Hz), 7.66 (d,
2H, J = 8.3 Hz), 7.16 (d, 1H, J = 2.1 Hz), 7.09 (dd, 1H,
J = 8.5, 2.1 Hz), 6.81 (d, 1H, J = 8.5 Hz), 6.77 (s, 1H),
4.58 (d, 2H, J = 6.0 Hz), 3.85 (s, 3H), 2.87 (dd, 1H,
J = 13.7, 9.0 Hz), 2.71 (dd, 1H, J = 13.7, 6.0 Hz), 2.63
(m, 2H), 1.63 (m, 1H), 1.48 (m, 1H), 1.36 (m, 2H),
0.90 (d, 3H, J = 7.3 Hz); HRMS; (M+H)+; calcd for
C20H21F3NO4, 424.1769. Found 424.1736.
4.1.21. 2-Ethyl-3-[4-methoxy-3-[N-[[pyren-1-yl]carbony-
laminomethyl]phenyl]propanoic
acid
(13m).
This
compound was prepared from 11c21 and pyren-1-ylcar-
bonyl chloride by means of a procedure similar to that
1
used for 13a H NMR (500 MHz, DMSO-d6) d 12.08
(s, 1H), 9.04 (s, 1H), 8.50 (d, 1H, J = 9.4 Hz), 8.33–
8.36 (m, 3H), 8.22–8.27 (m, 3H), 8.18 (d, 1H,
J = 7.7 Hz), 8.12 (t, 1H, J = 7.7 Hz), 7.23 (s, 1H), 7.09
(d, 1H, J = 8.3 Hz), 6.94 (d, 1H, J = 8.3 Hz), 4.54 (d,
2H, J = 5.6 Hz), 3.84 (s, 3H), 2.80 (dd, 1H, J = 13.7,
7.3 Hz), 2.62 (dd, 1H, J = 13.7, 7.3 Hz), 2.44–2.49 (m,
1H), 1.45–1.55 (m, 2H), 0.85 (t, 3H, J = 7.3 Hz); HRMS;
(M+H)+; calcd for C30H28NO4, 466.2018. Found
466.1984. Anal. (C30H27NO4) C, H, N.
4.1.17. (S)-2-Ethyl-3-[4-methoxy-3-[N-[3-fluoro-4-(trifluoro-
methyl)benzoylamino]methyl]phenyl]-propanoic acid (13i).
This compound was prepared according to the reported
method.23 Mp 120–121 °C; 1H NMR (500 MHz,
CDCl3) d 7.60 (m, 3H), 7.14 (s, 1H), 7.10 (dd, J = 8.5,
2.1 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.77 (br, 1H),
4.57 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 2.88 (dd,
J = 13.7, 8.5 Hz, 1H), 2.72 (dd, J = 13.7, 6.0 Hz, 1H),
2.56 (m, 1H), 1.66 (m, 1H), 1.58 (m, 1H), 0.96 (t,
J = 7.3 Hz, 3H); HRMS; (M+H)+; calcd for
C21H21F4NO4, 428.1485. Found 428.1453. Anal.
(C21H21F4NO4) C, H, N.
4.1.22. (S)-2-Ethyl-3-[4-methoxy-3-[N-[[pyren-1-yl]car-
bonylaminomethyl]phenyl]propanoic acid (13n). This
compound was prepared according to the reported
method.23 1H-NMR (500 MHz, CDCl3) d 8.46 (d,
1H, J = 9.4 Hz), 8.17 (m, 2H), 7.98 (m, 5H), 7.32
(s, 1H), 7.10 (d, 1H, J = 5.6 Hz), 6.80 (d, 1H,
J = 5.6 Hz), 6.63 (m, 1H), 4.70 (m, 2H), 3.82 (s,
3H), 2.87 (m, 1H), 2.74 (m, 1H), 2.56 (m, 1H),
1.50 (m, 2H), 0.94 (t, 3H, J = 7.5 Hz); HRMS;
(M+H)+; calcd for C30H28NO4, 466.2018. Found
466.2029. Anal. (C30H27NO4) C, H, N.
4.1.18. (S)-2-Ethyl-3-[4-methoxy-3-[N-[2-fluoro-4-(trifluoro-
methyl)benzoylamino]methyl]phenyl]-propanoic acid (13j).
This compound was prepared according to the reported
method.23 Mp 102–103 °C; 1H 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); HRMS; (M+H)+; calcd
for C21H21F4NO4, 428.1485. Found 428.1453. Anal.
(C21H21F4NO4) C, H, N.
4.2. Cell culture and cotransfection assay
Human embryonic kidney HEK293 cells were cul-
tured in DMEM containing 5% fetal bovine serum
and antibiotic-antimycotic (Nacalai) at 37 °C in a
humidified atmosphere of 5% CO2 in air. Transfec-
tions were performed by calcium phosphate coprecip-
itation. Eight hours after transfection, ligands were
added. Cells were harvested approximately 16–20 h
after the treatment, and luciferase and ß-galactosi-
dase activities were assayed using a luminometer
and a microplate reader. DNA cotransfection exper-
iments included 50 ng of reporter plasmid, 20 ng
pCMX-ß-galactosidase, 15 ng each receptor expres-
sion 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 ß-ga-
lactosidase control and reported values are means of
triplicate assays.
4.1.19. (R)-2-Ethyl-3-[4-methoxy-3-[N-[2-fluoro-4-(trifluoro-
methyl)benzoylamino]methyl]phenyl]-propanoic acid (13k).
This compound was prepared according to the reported
method.23 Mp 102–103 °C; 1H 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); HRMS; (M+H)+; calcd
for C21H21F4NO4, 428.1485. Found 428.1453. Anal.
(C21H21F4NO4) C, H, N.
4.3. Quantitative real-time PCR study
4.1.20. 2-Ethyl-3-[4-methoxy-3-[N-[[4-phenoxyphenyl]car-
bonylaminomethyl]phenyl]propanoic acid (13l). This com-
pound was prepared from 11c21 and 4-phenoxybenzoyl
chloride by means of a procedure similar to that used
for 13a. Oil; H NMR (500 MHz, CDCl3) d 7.72 (d,
2H, J = 9.0 Hz,), 7.36 (t, 2H, J = 8.1 Hz), 7.16 (m,
Total RNA of human hepatocellular carcinoma Huh-7
cells was isolated using an RNA preparation kit (Isogen;
Nippon Gene Corp.). Total RNA (6 lg) was treated with
DNase I, then the DNase I was inactivated using DNase I
inactivating reagent (Ambion; TURBO DNA-freeTM).
1

8414
J. Kasuga et al. / Bioorg. Med. Chem. 14 (2006) 8405–8414
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First-Strand Synthesis System for RT-PCR (Invitrogen)
with oligo(dT) 12–18 primer.
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(Bio-Rad Laboratories). Amplification specificity was
verified by visualizing PCR products on an ethidium
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Acknowledgments
The work described in this paper was partially support-
ed by a Grant-in-aid for Scientific Research from The
Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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