Structure-based design, synthesis, and nonalcoholic steatohepatitis (NASH)-preventive effect of phenylpropanoic acid peroxisome proliferator- activated receptor (PPAR) α-selective agonists

Article abstract of DOI:10.1016/j.bmc.2011.03.064

A series of α-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) α-selective agonists, based on our PPARα/δ dual agonist 3 as a lead compound. Structure-activity relationship studies clearly indicated that the steric bulkiness and position of the distal hydrophobic tail part are critical for PPARα agonistic activity and PPARα selectivity, as had been predicted from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic steatohepatitis (NASH) in an animal model.

Full text of DOI:10.1016/j.bmc.2011.03.064

Bioorganic & Medicinal Chemistry 19 (2011) 3183–3191
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design, synthesis, and nonalcoholic steatohepatitis
(NASH)-preventive effect of phenylpropanoic acid peroxisome
proliferator-activated receptor (PPAR) a-selective agonists
Shintaro Ban a, Jun-ichi Kasuga b, Izumi Nakagome c, Hiromi Nobusada a, Fusako Takayama a,
Shuichi Hirono c, Hiromu Kawasaki a, Yuichi Hashimoto b, Hiroyuki Miyachi a,
⇑
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
b Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
c School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of
activated receptor (PPAR)
Structure–activity relationship studies clearly indicated that the steric bulkiness and position of the distal
hydrophobic tail part are critical for PPAR agonistic activity and PPAR selectivity, as had been predicted
a
-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-
Received 1 February 2011
Revised 26 March 2011
Accepted 30 March 2011
Available online 8 April 2011
a
-selective agonists, based on our PPAR /d dual agonist 3 as a lead compound.
a
a
a
from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic
steatohepatitis (NASH) in an animal model.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
liver disorder that occurs in patients without significant alcohol
consumption.6,7 NASH is the most prevalent cause of chronic liver
The peroxisome proliferator-activated receptors (PPARs) are
members of the human nuclear receptor family, functioning as li-
gand-dependent transcription factors. These receptors are acti-
vated by the binding of various endogenous fatty acids and their
disease in westernized countries, and patients with NASH and cir-
rhosis are at risk for hepatocellular carcinoma.8
Recent animal studies have demonstrated that acyl CoA oxidase
(AOX)-null mice have defective peroxisomal b-oxidation and exhi-
metabolites, and by synthetic ligands.1 Three subtypes, PPAR
a,
bit steatohepatitis.9 When PPAR
a-null mice were fed with MCD
PPARd, and PPAR
c
have been isolated to date.2 These PPAR sub-
diet, which is deficient in both methionine and choline and high
in sucrose (40%) and fat (10%), they developed severe steatohepa-
titis compared with wild-type mice on the same diet. These data
indicated the critical role of fatty acid disposal pathways in the
progression of hepatic steatosis and steatohepatitis.10 Therefore,
types share a high level of structural homology, but each has dis-
tinct physiological functions. Although PPARd is ubiquitously
expressed, the other two PPAR subtypes show unique tissue distri-
bution patterns, that is, PPAR
cally active tissues, such as liver, kidney, skeletal and cardiac
muscle, and adrenal glands, while PPAR is expressed mainly in
adipose tissue, macrophages, and vascular smooth muscles.3
Among PPARs, PPAR regulates genes involved in uptake and
a is expressed mostly in metaboli-
PPARa agonists have therapeutic potential for the treatment of
c
NASH.
In the continuous study directed toward the development of
subtype-selective PPAR ligand,11–15 we would like to report the de-
a
oxidation of free fatty acids, triglyceride hydrolysis and up-regula-
tion of reverse cholesterol transport, such as apolipoprotein A-I
and apolipoprotein A-II.4 Fibrate-class antihyperlipidemic agents,
such as fenofibrate (1) and bezafibrate (2), which effectively lower
elevated serum triglycerides and moderately increase high-density
sign and synthesis of phenylpropanoic acid-type PPARa agonists.
We also describe the preventive effect of a representative com-
pound against progression of NASH in an animal model.
2. Inhibitor design
lipoproteins (HDL), can bind and activate PPARa. Dut, their affinity
is weak in the high micromolar range), and the subtype selectivity
In order to create PPAR
noic acid structure as a basic framework, we focused on our
PPAR /d dual
/d-dual agonist 3.11 Compound 3 is a potent PPAR
agonist with EC50 values of 10, 12, and 1900 nM for PPAR , PPARd,
and PPAR , respectively.
a-selective agonists with phenylpropa-
is not high (Fig. 1).5
Nonalcoholic steatohepatitis (NASH), consisting of hepatic stea-
tosis accompanied with inflammation and fibrosis, is a progressive
a
a
a
c
Recently, we have succeeded in solving the X-ray crystallo-
graphic structure of 3 complexed with the hPPARd ligand-binding
⇑
Corresponding author. Tel.: +81 086 251 7930.
E-mail address: miyachi@pharm.okayama-u.ac.jp (H. Miyachi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.064

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S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
O
O
O
F
O
Cl
O
COOH
O
OH
O
N
H
F
F
N
H
MeO
O
F
Cl
1
2
3
Figure 1. Representative fibrate class PPARa agonists (1, 2) and our PPARa/d dual agonist 3.
domain (LBD).16 The structure of this complex throws light on the
S-enantiomer preference, the importance of the acidic functional-
ity, and the effect of introduction of fluorine at the 20-position for
PPARd activity. But, unfortunately, this complex lacks the six amino
acid residues from the end of the H20 helix to the beginning of the
H3 helix (residues 265–270). Therefore, we computationally con-
structed a binding model of 3 complexed with full-length hPPARd
LBD structure,17 based on the reported full structure deposited in
PDB. We also constructed a binding model of 3 complexed with
benzaldehyde (4) in five steps. 4-Methoxybenzaldehyde was trea-
ted with triethyl 2-phosphonobutyrate in the presence of tBuOK as
a base, followed by hydrogenolysis with palladium on carbon to af-
ford the ethyl phenylpropionate derivative (6). The formylation of
6 with dichloromethylmethylether in the presence of TiCl4 as a Le-
wis acid to afford 7, which was treated with hydroxylamine hydro-
chloride and subsequent reduction afforded 9. The benzoylation of
9 with substituted benzoyl chlorides in the presence of triethyl-
amine as a base afforded benzamide derivatives (10a–k). Subse-
quent alkaline hydrolysis afforded the desired racemic products
11a–k. Optically active phenylpropanoic acid derivatives, 14a–c,
were prepared as described previously (Scheme 1).
full-length hPPARa LBD, by superimposing 3 on the reported full-
length hPPAR LBD. The results are depicted in Figure 2A and B.
a
Comparing these two binding models, we noticed that the shapes
of the hydrophobic pockets hosting the hydrophobic tail part of 3
(4-trifluoromethyl group) are somewhat different, that is, the
4. Results and discussion
hydrophobic pocket of hPPARa is wider than that of hPPARd.
Therefore, we anticipated that if we could introduce a sufficiently
bulky substituent instead of the 4-trifluoromethyl group, we
The SAR results are summarized in Figure 3. The introduction of
a bulky substituent at the 4-position of the distal benzene ring was
effective to improve PPARa agonistic activity, as expected, that is,
the activity increased in the order of 11a < 11b < 11c < 11d. While
the introduction of a substituent at the 4-position is important to
elicit PPARd agonistic activity (11a vs 11b), the activity did not
seem to correlate well with the bulkiness of the substituent. As re-
would be able to create a hPPAR
introduced substituent would enhance the interaction with the
hydrophobic pocket in the case of the hPPAR LBD, whereas it
a-selective agonist, because the
a
would be too large to enter the pocket in the case of hPPARd
LBD. Based on this working hypothesis, we planned to synthesize
derivatives of 3 bearing a bulky substituent at the hydrophobic tail
part.
gards activity towards PPAR
tive (11a) or weak agonistic (11b–11d). The position of the bulky
substituent is also important for PPAR agonistic activity. The
PPAR agonistic activity increased in the order of 2-position (11f,
c, the compounds were basically inac-
a
3. Chemistry
a
11i) < 3-position (11e, 11h) < 4-position (11d, 11g). In the case of
the ether linker connecting the distal benzene rings (11d, 11j,
and 11k), one atom length (11d) might be optimal, because the
The synthetic routes to the racemic series of compounds are
outlined in Scheme 1. Compound 9 was prepared from 4-methoxy-
Figure 2. (A–E) Molecular modeling structures of PPAR LBD–3 complexes. (A) PPARd LBD–3 complex model (amino acid residues other than 265–270 are taken from our
previous X-ray data (PDB 2znp)); (B) binding mode of 3 to PPARd LBD; (C) PPAR LBD–3 complex model; (D) binding mode of 3 to PPAR LBD; (E) zoomed view of the ligand-
binding domain near the trifluoromethyl group of 3. Cyan represents Ile of PPARd, magenta represents Ile of PPAR
a
a
a
.

S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
3185
O
CHO
COOEt
COOEt
COOEt
COOEt
b
a
8
c
H
MeO
d
MeO
MeO
MeO
6
5
7
4
HO
O
N
COOEt
H2N
MeO
f
COOEt
e
N
H
H
R
MeO
MeO
9
10a-k
O
COOH
g
N
H
R
MeO
11a-k
O
O
O
O
Bn
O
Bn
O
OHC
MeO
X
X
COOH
h
i
N
N
H
N
N
H
O
MeO
O
MeO
O
O
12
13a-c
14a-c
Scheme 1. Synthetic route to the present series of compounds. Reagents and conditions: (a) (EtO)2POCH(Et)CO2Et, 60% NaH, THF, 0 °C to rt, 5 h, 85%; (b) H2, 10% Pd–C, EtOH,
rt, 2 h, quant.; (c) MeOCHCl2, TiCl4, CH2Cl2, ꢀ30 °C to 0 °C, 5 h, 70%; (d) NH2OH HCl, pyridine, EtOH, reflux, overnight, 80%; (e) H2, 10% Pd–C, EtOH, concd HCl, 50 °C, overnight,
90%; (f) substituted benzoyl chloride, triethylamine, CH2Cl2, rt, overnight, 60–80%; (g) aq NaOH, EtOH, rt to 60 °C, overnight, 55–90%; (h) substituted benzamide,
triethylsilane, TFA, toluene, reflux, 48 h, 85–97%; (i) LiOH–H2O, 30% H2O2, THF/H2O = 4:1 (V/V), 0 °C, 2.5 h then rt, 3 h, 60–86%.
Figure 3. Potency of the compounds prepared in this study. Compounds were screened for agonist activity towards PPAR-GAL4 chimeric receptors in transiently transfected
HEK-293 cells as described. The EC50 value is the molar concentration of the test compound that affords 50% of the maximal reporter activity. ‘‘rac’’ means racemic compound.
benzyl derivative (11j) and phenethyl derivative (11k) exhibited
decreased PPAR agonistic activity.
Anticipating an improvement of PPAR
the 4-position trifluoromethyl group of 3 was changed to the phen-
oxy derivative, we next prepared the S enantiomer series of 11d,
for we have succeeded in solving the X-ray crystallographic struc-
tures of PPARa/d-dual agonist 3, complexed with both PPARa and
a
PPARd ligand binding domains as described above. The ethyl
groups of 3 are placed at the deepest binding sites with the head
carboxyl groups and contact the surrounding hydrophobic resi-
dues. When their R isomers approach the ligand-binding pocket,
the ethyl groups may cause a steric clash with the surrounding
a
agonistic activity when

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S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
10 weeks
6 weeks
16 weeks
residues, particularly those on the central parts of the H3 helix,
even though some structural rearrangements could occur in both
the proteins and ligands.
Further addition of a halogen atom at the 4-position of the
phenoxy moiety of 11d was not effective in improving PPARa ago-
control
CDHF
NC
NC
CDHF
CDHF
CDHF
CDHF
NASH
CDHF + OS
nistic activity, especially in the case of the Cl (14b) and Br (14c)
derivatives, but the 4-fluoro derivative (14a) exhibited comparable
Compd.
CDHF + OS + compd. (1 mg/kg/day)
PPAR
Thus, we have successfully obtained a potent, selective, struc-
turally novel PPAR agonist, 14a. As compared to the PPAR /d dual
agonist lead 3, 14a showed twofold greater PPAR agonistic activ-
ity, while the PPARd agonistic activity was considerably decreased
to 1/25. Although the PPAR agonistic activity was also increased
by twofold as compared to 3, this activity still weak compared to
the PPAR agonistic activity. Therefore, we selected 14a for further
in vivo study. The docking modes are depicted in Figure 4A–D.
In order to investigate the therapeutic potential of 14a, we eval-
uated the ability of 14a to block progression of NASH in an animal
model.18,19 Although the pathogenesis of NASH has not yet been
fully elucidated, the ‘two-hit’ theory is widely accepted.20 The first
hit is the accumulation of fatty acids in the liver to cause steatosis.
Steatosis is relatively mild pathological process and is easily
reversible. However, with secondary cellular stress, steatosis can
progress to steatohepatitis.21,22 The rat model used in this study
mimics two hits, by continuous feeding of CDHF diets for 4 weeks
as first hit. Then rats received CDHF diets for another 4 weeks and
treated during this period with intraperitoneal injection of sodium
nitrite every day to induce methemoglobinemia, as a second hit.
The protocols are summarized in Figure 5, the results are summa-
rized in Figures 6 and 7.
a agonistic activity to 11d.
NC: normal chow, CDHF: choline deficient high-fat diet, OS: oxidative stress (sodium nitrite)
a
a
Figure 5. In vivo experimental design.
a
with hypoxemia), and 14a-treated NASH groups are shown in Fig-
ure 6. The livers from CDHF diet-fed rats were significantly greater
in size than those of normal chow-fed rats (Fig. 6b). On the other
hand, extensive liver atrophy was observed in NASH livers
(Fig. 6c), and surface nodules were present. Administration of
14a prevented atrophy and nodule formation, but had no apparent
effect on the features of fatty liver (Fig. 6d). Histopathological
examination of liver tissues stained with hematoxylin exhibited
hepatic macrovesicular steatosis in all CDHF-fed rats (Fig. 6f and
g). No fibrosis was seen in the livers of rats fed normal chow com-
pared with those fed CDHF alone. In the case of the NASH rats, ad-
vanced liver fibrosis and necrosis were observed (Fig. 6k), but such
liver fibrosis was clearly attenuated by 14a (1 mg/kg/day) treat-
ment (Fig. 6l).
Figure 7 shows the changes of aspartate amino transferase
(AST), alanine transaminase (ALT), and alkaline phosphatase
(ALP), which are well-known markers of liver function.23 In this
experiment, levels were significantly higher in the NASH rats as
compared to rats of the CDHF-fed group. In the 1 mg/kg/day 14a-
treated NASH group, liver function was improved, as compared
c
a
Representative histological sections of livers from the normal,
choline deficient high-fat (CDHF) diet-fed, NASH (CDHF diet-fed
Figure 4. (A–E) Molecular modeling structures of PPAR
are taken from our previous X-ray data (PDB 2znp)); (B) PPAR
(D) zoomed view of the ligand-binding domain near the 4-(4-fluorophenoxy) group of 14a.
a
LBD–3, and PPARa LBD–14a complexes. (A) PPARa LBD–3 complex model (amino acid residues other than 265–270
a
LBD–14a complex model; (C) zoomed view of the ligand-binding domain near the trifluoromethyl group of 3;

S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
3187
control
CDHF
NASH
Compd. (1 mg/kg/day)
A
B
C
D
nodule
control
CDHF
NASH
Compd. (1 mg/kg/day)
E
F
G
H
500 µm
500 µm
500 µm
500 µm
control
CDHF
NASH
Compd. (1 mg/kg/day)
I
J
K
L
500 µm
500 µm
500 µm
500 µm
fibrosis
Figure 6. Histological evaluation of liver samples from rats of various groups. Histological evaluation of liver samples of rats from the normal (A), CDHF (B), NASH (C), and
NASH + 1 mg/kg/day 14a (D) groups. The effects of 14a on liver structure were examined by hematoxylin and eosin staining in rats from the normal (e), CDHF (f), NASH (g),
and NASH + 1 mg/kg/day 14a (h) groups. The effects of 14a on liver fibrosis were examined by Masson Trichrome staining in rats from the normal (I), CDHF (J) NASH (K), and
NASH + 1 mg/kg 14a (L) groups. Data show typical results. Scale bar = 500 lm.
200
6
4
2
0
200
AST
ALT
ALP
**
**
150
100
50
**
150
100
50
##
**
**
##
**
##
0
Control
CDHF
NASH
Compd.
Control CDHF
NASH Compd.
(1mg/kg/day)
Control
CDHF
NASH
Compd.
(1mg/kg/day)
(1mg/kg/day)
Data are presented as mean
SEM for 4-12 mice. **p<0.01, compared with control. ##p<0.01, compared with NASH. Assessed by Tukey’s test.
Figure 7. Biochemical markers of hepatobiliary damage (AST, ALT, and ALP levels in serum): AST, ALT, and ALP levels in the four experimental groups (see legend to Fig. 6)
were measured at the end of the study period. Each value represents the mean SEM of five rats. ##p <0.01 versus normal control group, ⁄⁄p <0.01 versus CDHF group (by
ANOVA followed by with Dunnett’s multiple comparison test).
to the NASH group, although the enzyme levels were still increased
compared to the normal chow-fed group. These in vivo findings
6. Experimental
indicate that our PPARa-selective agonist, 14a, can block, or at
6.1. General methods
least delay, the progression of NASH.
Melting points were determined with a Yanagimoto hot-stage
melting point apparatus and are uncorrected. NMR spectra were
recorded on a Varian VXR-300 (1H 300 MHz) spectrometer. Pro-
ton chemical shifts were referenced to the TMS internal stan-
dard. Elemental analysis was carried out with a Yanagimoto
MT-5 CHN recorder elemental analyzer and results were within
0.4% of the theoretical values. FAB-MS was carried out with a
VG70-SE.
5. Conclusion
We have succeeded in creating a potent and selective PPAR
agonist, 14a. We also showed that 14a can block progression of
NASH in an animal model. Our results indicate that PPAR -selec-
tive activation can prevent or ameliorate liver dysfunction and de-
a
a
lay the progression of fatty liver to fibrosis in this model of NASH.

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S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
6.1.1. Ethyl 2-(4-methoxybenzylidene)butanoate (5)
6.1.5. Ethyl 2-(3-(aminomethyl)-4-methoxybenzyl)butanoate
HCl (9)
Sodium hydride (214 mg, 5.35 mmol; 60% oil dispersion) was
suspended in 10 mL of dehydrated tetrahydrofuran under argon
and cooled with ice. Triethyl 2-phosphonobutyrate (1.34 g,
5.31 mmol) dissolved in 20 mL of dehydrated tetrahydrofuran
was added dropwise. When the addition was completed, the mix-
ture was stirred for 1 h, then 4-methoxybenzaldehyde (1.44 g,
5.33 mmol) dissolved in 25 mL of dehydrated tetrahydrofuran
was added dropwise, and the mixture was stirred for a further
4 h at room temperature. The reaction mixture was poured into
ice/water. The whole was extracted with ethyl acetate, and the or-
ganic phase was washed with water and brine, dried over anhy-
drous sodium sulfate, and concentrated. The residue was purified
by silica gel column chromatography (eluant, n-hexane/ethyl ace-
tate = 5:1 v/v) to afford 1.45 g (74%) of the title compound as a yel-
low oil. 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)+.
Compound 5 (6.80 g, 24.3 mmol) was dissolved in 150 mL of
ethanol, 2.5 mL of concd HCl, 10% palladium on carbon (0.70 g)
was added, and hydrogenation was carried out overnight at an ini-
tial pressure of 0.3 MPa. After completion of the reaction, 100 mL of
water was added, the catalyst was removed by filtration, and the
filtrate was concentrated. Ether (300 mL) was added to the residue
and triturated to afford 6.10 g (83%) of the title compound as a col-
orless amorphous solid: 1H NMR(500 MHz, CDCl3) d 8.03 (s, 3H),
7.17 (m, 2H), 6.97 (d, J = 8.2 Hz, 1H), 4.02 (m, 2H), 3.92 (s, 2H),
3.79 (s, 3H), 2.76 (dd, J = 13.6, 8.2 Hz, 1H), 2.64 (dd, J = 13.6,
6.7 Hz, 1H), 2.52 (m, 1H), 1.49 (m, 2H), 1.11 (t, J = 7.0 Hz, 3H),
0.84 (t, J = 7.6 Hz, 3H); MS (FAB) 266 (M+H)+
.
6.1.6. 2-(3-(Benzamidomethyl)-4-methoxybenzyl)butanoic acid
(11a)
To a solution of 9 (302 mg, 1.0 mmol), triethylamine (0.3 mL)
and dichloromethane (10 mL) was added benzoyl chloride
(0.12 mL, 1.0 mmol) at 0 °C, and the mixture was stirred for
30 min at 0 °C, then for 5 h at room temperature. The reaction mix-
ture was washed with dil. HCL and saturated NaHCO3 solution,
dried over anhydrous magnesium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (eluant,
6.1.2. Ethyl 2-(4-methoxybenzyl)butanoate (6)
5 (9.31 g, 39.7 mmol) was dissolved in 200 mL of ethanol, 10%
palladium on carbon (1.10 g) was added, and hydrogenation was
carried out for 3 h at an initial pressure of 353 kPa. After comple-
tion of the reaction, the catalyst was removed by filtration and
the filtrate was concentrated to afford 9.38 g (quant.) of the title
compound as a yellow oil: 1H NMR(500 MHz, CDCl3) d 7.07 (d,
J = 8.5 Hz, 2H), 6.80 (d, 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)+.
n-hexane/ethyl acetate = 2:1 v/v) to afford 297 mg (90%) of the es-
ter intermediate 10a as a yellow oil: 1H NMR(500 MHz, CDCl3) d
7.75 (d, J = 5.3 Hz, 2H), 7.4 (m, 3H), 7.14 (s, 1H), 7.06 (d,
J = 8.1 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.65 (s, 1H), 4.60 (d,
J = 6.0 Hz, 2H), 4.05 (m, 2H), 3.85 (s, 3H), 2.86 (dd, J = 13.7,
8.5 Hz, 1H), 2.68 (dd, J = 13.7, 6.8 Hz, 1H), 2.53 (m, 1H), 1.62 (m,
1H), 1.54 (m, 1H), 1.16 (t, J = 7.3 Hz, 3H), 0.90 (t, J = 7.7 Hz, 3H);
MS (FAB) 370 (M+H)+.
A mixture of 10a (290 mg, 0.79 mmol), 1 mol/L LiOH (2 mL) and
ethanol (5 mL) was stirred at 80 °C overnight. The reaction mixture
was neutralized with HCl and extracted with CHCl3. The extract
was washed with water, brine, dil HCL, and saturated NaHCO3
solution, then dried over anhydrous magnesium sulfate, and con-
centrated. The residue was purified by silica gel column chroma-
tography (eluant, n-hexane/ethyl acetate = 2:1 v/v) to afford
235 mg (85%) of the title compound as a colorless crystalline pow-
der: 1H NMR(500 MHz, CDCl3) d 7.75 (d, J = 7.7 Hz, 2H), 7.47 (m,
1H), 7.40 (t, J = 7.7 Hz, 2H), 7.16 (d, J = 1.7 Hz, 1H), 7.08 (d,
J = 8.1 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.72 (s, 1H), 5 (br, 1H),
4.58 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 2.88 (dd, J = 13.7, 8.5 Hz, 1H),
2.70 (dd, J = 13.7, 6.4 Hz, 1H), 2.55 (m, 1H), 1.64 (m, 1H), 1.56 (m,
1H), 0.94 (t, J = 7.7 Hz, 3H); MS (FAB) 342 (M+H)+; HRMS (M+H)
calcd for 342.1705, found 342.1707 (M+H)+.
6.1.3. Ethyl 2-(3-formyl-4-methoxybenzyl)butanoate (7)
To a solution of 6 (9.38 g, 39.7 mmol) and 500 mL of anhydrous
dichloromethane was added titanium tetrachloride (35.0 mL,
318 mmol), followed by dichloromethyl methyl ether (13.4 mL,
148 mmol) at ꢀ20 °C, under an argon atmosphere. The mixture
was stirred for 6 h at ꢀ20 °C, then poured into dilute HCl solution
and the layers were separated. The organic layer was washed with
water and brine, dried over anhydrous sodium sulfate, and concen-
trated. The residue was purified by silica gel column chromatogra-
phy (eluant, n-hexane/ethyl acetate = 4:1 v/v) to afford 10.5 g
(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)+.
6.1.7. 2-(3-(4-Methylbenzamidomethyl)-4-methoxybenzyl)
butanoic acid (11b)
6.1.4. Ethyl 2-(3-((hydroxyimino)methyl)-4-methoxybenzyl)
butanoate (8)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR(500 MHz, CDCl3) d 7.65 (d, J = 8.1 Hz, 2H),
7.2 (br, 1H), 7.19 (d, J = 7.7 Hz, 2H), 7.15 (d, J = 2.1 Hz, 1H), 7.07
(dd, J = 8.1, 2.1 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.71 (s, 1H), 4.57
(d, J = 5.6 Hz, 2H), 3.83 (s, 3H), 2.88 (dd, J = 13.7, 8.1 Hz, 1H), 2.68
(dd, J = 13.7, 6.4 Hz, 1H), 2.55 (m, 1H), 2.37 (s, 3H), 1.63 (m, 1H),
1.56 (m, 1H), 0.94 (t, J = 7.3 Hz, 3H); MS (FAB) 356 (M+H)+; HRMS
(M+H) calcd for 356.1862, found 356.1849 (M+H)+.
To a solution of 7 (6.8 g, 25.7 mmol), pyridine (3 mL) and etha-
nol (100 mL) was added hydroxylamine hydrochloride (2.1 g
30.2 mmol). The mixture was refluxed for 5 h, then concentrated
and poured into water. The whole was extracted with ethyl ace-
tate, and the organic phase was washed with water, brine, dil
HCL, and saturated NaHCO3 solution, then dried over anhydrous
magnesium sulfate, and concentrated. The residue was purified
by silica gel column chromatography (eluant, n-hexane/ethyl ace-
tate = 5:1 v/v) to afford 6.80 g (95%) of the title compound as a yel-
low oil: 1H NMR(500 MHz, CDCl3) d 8.46 (s, 1H), 7.51 (m, 2H), 7.14
(dd, J = 8.3, 2.6 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 4.07 (m, 2H), 3.83 (s,
3H), 2.87 (dd, J = 13.9, 8.5 Hz, 1H), 2.69 (dd, J = 13.9, 6.4 Hz, 1H),
2.54 (m, 1H), 1.63 (m, 1H), 1.55 (m, 1H), 1.17 (t, J = 7.3 Hz, 3H),
0.91 (t, J = 7.3 Hz, 3H); MS (FAB) 280 (M+H)+.
6.1.8. 2-(4-Methoxy-3-((4-(trifluoromethoxy)benzamido)
methyl)benzyl)butanoic acid (11c)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR(500 MHz, CDCl3) d 7.78 (d, J = 8.6 Hz, 2H),
7.21 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 2.1 Hz, 1H), 7.08 (dd, J = 8.1,

S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
3189
2.1 Hz, 1H), 6.79 (m, 2H), 4.56 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 2.88
(dd, J = 13.7, 8.5 Hz, 1H), 2.69 (dd, J = 13.7, 6.4 Hz, 1H), 2.54 (m,
1H), 1.65 (m, 1H), 1.57 (m, 1H), 0.94 (t, J = 7.3 Hz, 3H); MS (FAB)
426 (M+H)+; HRMS (M+H) calcd for 426.1528, found 426.1539
(M+H)+. Anal. Calcd for C21H21NO4: C, 59.29; H, 5.21; N, 3.29.
Found: C, 59.30; H, 5.45; N, 3.10 (M+H)+.
7.17 (d, J = 2.1 Hz, 1H), 7.08 (dd, J = 8.5, 2.1 Hz, 1H), 6.79 (br, 1H),
6.78 (d, J = 8.6 Hz, 1H), 4.60 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 2.89
(dd, J = 13.7, 7.9 Hz, 1H), 2.69 (dd, J = 13.7, 6.9 Hz, 1H), 2.55 (m,
1H), 1.63 (m, 1H), 1.56 (m, 1H), 0.93 (t, J = 7.3 Hz, 3H); MS (FAB)
418 (M+H)+; HRMS (M+H) calcd for 418.2018, found 418.2007.
Anal. Calcd for C26H27NO4: C, 74.80; H, 6.52; N, 3.35. Found: C,
74.52; H, 6.38; N, 3.15 (M+H)+.
6.1.9. 2-(4-Methoxy-3-((4-phenoxybenzamido)
methyl)benzyl)butanoic acid (11d)
6.1.14. 2-(4-Methoxy-3-((2-phenylbenzamido)methyl)benzyl)
butanoic acid (11i)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR (500 MHz, CDCl3) d 7.72 (d, J = 9.0 Hz, 2H),
7.36 (t, J = 8.1 Hz, 2H), 7.16 (m, 2H), 7.07 (dd, J = 8.6, 2.1 Hz, 1H),
7.02 (d, J = 8.1 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.1 Hz,
1H), 6.66 (t, J = 4.6 Hz, 1H), 4.57 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H),
3.5 (br, 1H), 2.87 (dd, J = 13.7, 8.6 Hz, 1H), 2.71 (dd, J = 13.7,
6.4 Hz, 1H), 2.55 (m, 1H), 1.65 (m, 1H), 1.58 (m, 1H), 0.95 (t,
J = 7.3 Hz, 3H); MS (FAB) 434 (M+H)+; HRMS (M+H) calcd for
434.19675, found 434.1960 (M+H)+. Anal. Calcd for C26H27NO5: C,
72.04; H, 6.28; N, 3.23. Found: C, 71.79; H, 6.38; N, 3.00.
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR(500 MHz, CDCl3) d 7.71 (d, J = 6.8 Hz, 1H),
7.45 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.33 (d, J = 7.3 Hz,
1H), 7.3–7.2 (m, 5H), 7.01 (dd, J = 8.5, 2.1 Hz, 1H), 6.93 (d,
J = 2.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 5.82 (br, 1H), 4.30 (m, 2H),
3.56 (s, 3H), 2.86 (dd, J = 13.7, 8.1 Hz, 1H), 2.65 (dd, J = 13.7,
6.8 Hz, 1H), 2.54 (m, 1H), 1.64 (m, 1H), 1.57 (m, 1H), 0.95 (t,
J = 7.7 Hz, 3H); MS (FAB) 418 (M+H); HRMS (M+H) calcd for
418.2018, found 418.1998 (M+H)+.
6.1.10. 2-(4-Methoxy-3-((3-phenoxybenzamido)methyl)
benzyl)butanoic acid (11e)
6.1.15. 2-(3-((4-(Benzyloxy)benzamido)methyl)-4-
methoxybenzyl)butanoic acid (11j)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR (500 MHz, CDCl3) d 7.46 (d, J = 7.7 Hz, 1H),
3.39 (s, 1H), 7.3 (m, 3H), 7.1 (m, 4H), 7.00 (d, J = 8.5 Hz, 2H), 6.78
(d, J = 8.5 Hz, 1H), 6.72 (t, J = 5.1 Hz, 1H), 4.55 (d, J = 5.6 Hz, 2H),
3.81 (s, 3H), 2.89 (dd, J = 13.7, 8.1 Hz, 1H), 2.68 (dd, J = 13.7,
6.8 Hz, 1H), 2.55 (m, 1H), 1.63 (m, 1H), 1.57 (m, 1H), 0.94 (t,
J = 7.3 Hz, 3H). HRMS (M+H) calcd for 434.19675, found 434.1960
(M+H)+.
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR(500 MHz, CDCl3) d 7.71 (d, J = 9.0 Hz, 2H),
7.4 (m, 5H), 7.16 (d, J = 2.1 Hz, 2H), 7.07 (dd, J = 8.5, 2.1 Hz, 1H),
6.96 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.1 Hz, 1H), 6.64 (s, 1H), 5.09
(s, 2H), 4.55 (m, 2H), 3.84 (s, 3H), 2.87 (dd, J = 13.7, 8.5 Hz, 1H),
2.70 (dd, J = 13.7, 6.0 Hz, 1H), 2.54 (m, 1H), 1.64 (m, 1H), 1.56 (m,
1H), 0.94 (t, J = 7.7 Hz, 3H); MS (FAB) 448 (M+H)+; HRMS (M+H)
calcd for 448.2124, found 448.2111 (M+H)+. Anal. Calcd for
C27H29NO5: C, 72.46; H, 6.53; N, 3.13. Found: C, 72.29; H, 6.76;
6.1.11. 2-(4-Methoxy-3-((2-phenoxybenzamido)methyl)
benzyl)butanoic acid (11f)
N, 2.99.
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR (500 MHz, CDCl3) d 8.36(t, J = 5.6 Hz, 1H),
8.25 (dd, J = 8.1, 1.3 Hz, 1H), 7.9 (br, 1H), 7.3 (m, 3H), 7.17 (m,
2H), 7.13 (d, J = 1.7 Hz, 1H), 7.02 (d, J = 8.1 Hz, 3H), 6.79 (d,
J = 8.1 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 6.0 Hz, 2H),
3.54 (s, 3H), 2.86 (dd, J = 13.7, 7.7 Hz, 1H), 2.63 (dd, J = 13.9,
6.8 Hz, 1H), 2.52 (m, 1H), 1.60 (m, 1H), 1.53 (m, 1H), 0.92 (t,
J = 7.3 Hz, 3H). HRMS (M+H) calcd for 434.1967, found 434.1968
(M+H)+.
6.1.16. 2-(4-Methoxy-3-((4-phenethoxybenzamido)methyl)
benzyl)butanoic acid (11k)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR (500 MHz, CDCl3) d 7.67 (d, J = 8.5 Hz, 2H),
7.25 (m, 5H), 7.13 (s, 2H), 7.05 (d, J = 8.3 Hz, 1H), 6.85 (d,
J = 8.5 Hz, 2H), 6.76 (d, J = 8.3 Hz, 1H), 6.62 (s, 1H), 4.53 (d,
J = 6.0 Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.81 (s, 3H), 3.08 (t,
J = 6.0 Hz, 2H), 2.85 (dd, J = 13.7, 8.5 Hz, 1H), 2.67 (dd, J = 13.7,
6.4 Hz, 1H), 2.52 (m, 1H), 1.61 (m, 1H), 1.54 (m, 1H), 0.91 (t,
J = 7.3 Hz, 3H); MS (FAB) 462 (M+H)+; HRMS (M+H) calcd for
462.2280, found 462.2280 (M+H)+.
6.1.12. 2-(4-Methoxy-3-((4-phenylbenzamido)methyl)benzyl)
butanoic acid (11g)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR (500 MHz, CDCl3) d 7.82 (d, J = 8.1 Hz, 2H),
7.62 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 7.7 Hz, 2H), 7.45 (t, J = 7.3 Hz,
2H), 7.37 (t, J = 7.2 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 7.09 (dd,
J = 8.6, 2.1 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.77 (m, 1H), 4.60 (d,
J = 5.6 Hz, 2H), 3.85 (s, 3H), 2.89 (dd, J = 13.7, 8.5 Hz, 1H), 2.71
(dd, J = 13.6, 6.4 Hz, 1H), 2.56 (m, 1H), 1.65 (m, 1H), 1.58 (m, 1H),
0.95 (t, J = 7.3 Hz, 3H); MS (FAB) 418 (M+H)+; HRMS (M+H) calcd
for 418.2018, found 418.1998. Anal. Calcd for C26H27NO4: C,
74.80; H, 6.52; N, 3.35. Found: C, 74.62; H, 6.57; N, 3.21 (M+H)+.
6.1.17. N-(5-((S)-2-((R)-4-Benzyl-2-oxo-oxazolidine-3-
carbonyl)butyl)-2-methoxybenzyl)-4-(4-fluorophenoxy)
benzamide (13a)
A
mixture of 12 (235 mg, 0.59 mmol), 4-(4-fluorophen-
oxy)benzamide (560 mg, 2.42 mmol), triethylsilane (0.29 mL,
1.8 mmol), trifluoroacetic acid (0.14 mL, 1.9 mmol), and 15 ml
of dehydrated toluene was refluxed for 2 days. The mixture
was evaporated, and the residue was purified by silica gel col-
umn chromatography (eluant; n-hexane/ethyl acetate = 3:1 v/v)
to afford 274 mg (76%) of the title compound as a yellow oil.
1H NMR (500 MHz CDCl3) d 7.71 (d, J = 8.8 Hz, 2H), 7.33–7.18
(m, 6H), 6.98 (d, J = 8.8 Hz, 2H), 6.97–6.94 (m, 2H), 6.87–6.79
(m, 3H), 6.73 (ddd, J = 10.0 Hz, 2.0 Hz, 2.0 Hz, 1H), 6.52 (t,
J = 5.6 Hz, 1H), 4.68–4.55 (m, 3H), 4.15–4.08 (m, 2H), 4.05 (dd,
J = 9.2 Hz, 2.4 Hz, 1H), 3.84 (s, 3H), 3.02–2.96 (m, 2H), 2.77 (dd,
J = 13.6 Hz, 6.4 Hz, 1H), 2.48 (dd, J = 13.6 Hz, 9.2 Hz, 1H), 1.82–
1.67 (m, 1H), 1.66–1.53 (m, 1H), 0.93 (t, J = 7.6 Hz, 3H); MS
(FAB) 611.
6.1.13. 2-(4-Methoxy-3-((3-
phenylbenzamido)methyl)benzyl)butanoic acid (11h)
This compound was prepared from 9 and 4-methylbenzoyl
chloride, using the same procedures as described for the prepara-
tion of 11a: 1H NMR(500 MHz, CDCl3) d 7.99 (s, 1H), 7.69 (m,
2H), 7.59 (d, J = 7.7 Hz, 2H), 7.44 (m, 3H), 7.36 (t, J = 7.7 Hz, 1H),

3190
S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
6.1.18. N-(5-((S)-2-((R)-4-Benzyl-2-oxo-oxazolidine-3-carbonyl)
butyl)-2-methoxybenzyl)-4-(4-chlorophenoxy)benzamide
(13b)
6.1.22. (S)-2-(3-((4-(4-Brophenoxy)benzamido)methyl)-4-
methoxybenzyl)butanoic acid (14c)
This compound was prepared from 13c, using the same proce-
This compound was prepared from 12 and 4-(4-chlorophen-
oxy)benzamide, using the same procedures as described for the
preparation of 13a. 1H NMR (400 MHz CDCl3) d 7.69 (d, J = 8.8 Hz,
2H), 7.32 (d, J = 8.8 Hz, 2H), 7.27–7.18 (m, 5H), 6.99–6.92 (m,
6H), 6.82 (d, J = 8.4 Hz, 1H), 6.50 (t, J = 5.6 Hz, 1H), 4.68–4.55 (m,
3H), 4.15–4.08 (m, 2H), 4.05 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 3.84 (s,
3H), 3.02–2.96 (m, 2H), 2.76 (dd, J = 13.6 Hz, 6.4 Hz, 1H), 2.48
(dd, J = 13.6 Hz, 9.2 Hz, 1H), 1.82–1.68 (m, 1H), 1.66-1.53 (m, 1H),
0.93 (t, J = 7.6 Hz, 3H); MS (FAB) 627, 629 (M+H)+.
dures as described for the preparation of 14a. Mp 106–107 °C; 1H
NMR (500 MHz CDCl3)
d 7.73 (d, J = 9.0 Hz, 2H), 7.46 (d,
J = 9.0 Hz, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.5 Hz, 2.4 Hz,
1H), 6.97 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 6.79 (d,
J = 8.5 Hz, 1H), 6.66 (t, J = 5.6 Hz, 1H), 4.61–4.53 (m, 2H), 3.85 (s,
3H), 2.88 (dd, J = 13.5 Hz, 9.0 Hz, 1H), 2.71 (dd, J = 14.0 Hz, 6.0 Hz,
1H), 2.58–2.54 (m, 1H), 1.70–1.53 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H);
½
a 2D0
ꢁ
¼ þ19; MS (FAB) 512, 514 (M+H)+; HRMS (FAB) calcd for
512.1073; found: 512.1055 (M+H)+.
6.1.19. N-(5-((S)-2-((R)-4-Benzyl-2-oxo-oxazolidine-3-carbonyl)
butyl)-2-methoxybenzyl)-4-(4-bromophenoxy)benzamide
(13c)
6.2. Animals
Six-week-old male Wistar rats were purchased from Shimizu
Animal Co. (Kyoto, Japan) and maintained at the Okayama Univer-
sity animal facility in accordance with the Okayama University
Guidelines on the Care and the Use of Laboratory Animals. The rats
were housed in individual cages in air-conditioned rooms with a
controlled 12-h light-dark cycle. The rats were allowed to accli-
mate to the animal facility and allowed free access to diet and
water ad libitum.
This compound was prepared from 12 and 4-(4-bromophen-
oxy)benzamide, using the same procedures as described for the
preparation of 13a. 1H NMR (500 MHz CDCl3) d 7.70 (d, J = 8.5 Hz,
2H), 7.46 (d, J = 9.0 Hz, 2H), 7.29-7.17 (m, 5H), 7.00-6.89 (m, 6H),
6.82 (d, J = 8.0 Hz, 1H), 6.51 (t, J = 4.0 Hz, 1H), 4.67-4.61 (m, 2H),
4.58 (dd, J = 14.5 Hz, 7.0 Hz, 1H), 4.14–4.09 (m, 2H), 4.05 (dd,
J = 9.0 Hz, 3.5 Hz, 1H), 3.84 (s, 3H), 3.02–2.96 (m, 2H), 2.77 (dd,
J = 13.5 Hz, 6.5 Hz, 1H), 2.48 (dd, J = 13.5 Hz, 9.0 Hz, 1H), 1.81–
1.70 (m, 1H), 1.64-1.54 (m, 1H), 0.93 (t, J = 7.5 Hz, 3H); MS (FAB)
671, 673 (M+H)+.
6.3. Experimental design
At 6 weeks of age, the rats were divided into groups and fed
either control normal diet or choline deficient high-fat (CDHF) diet
(Oriental Yeast Co., Tokyo, Japan) for 4 weeks. At the end of this
period, the 6-week experimental period began. The rats of the nor-
mal control group were continued on normal chow (MF laboratory
chow, Oriental Yeast Co., Tokyo, Japan). The rats of the CDHF group
were continued on CDHF diet. On the other hand, the rats of the
CDHF + hypoxemia (NASH) group continued on CDHF diet and
were also treated with intraperitoneal injection of 40 mg/kg of so-
dium nitrite (NaNO2) every day to induce methemoglobinemia.
The rats of the 14a-treated NASH group were treated in the same
way as the NASH group, but also received 14a 1 mg/kg/day. At
the end of the 6-week experimental period, the rats were sacrificed
under light ether anesthesia, blood was collected from the inferior
vena cava for serum analysis, and livers were resected for histopa-
thological and biochemical analyses. The experimental design is
summarized in Figure 5.
6.1.20. (S)-2-(3-((4-(4-Fluorophenoxy)benzamido)methyl)-4-
methoxybenzyl)butanoic acid (14a)
Compound 13a (267 mg, 0.437 mmol) was dissolved in 24 mL
of tetrahydrofuran and 6 mL of water under an argon atmosphere
with ice-cooling. To this solution was added 30% aqueous hydro-
gen peroxide (0.50 mL, 4.4 mmol). Then a solution of lithium
hydroxide monohydrate (80 mg, 1.8 mmol) in water (1 mL) was
added, and the mixture was stirred further for 2.5 h at 0 °C, and
for 3 h at room temperature. An aqueous solution of sodium
hydrogen sulfite (1.00 g/6 mL) was added to the mixture and
the whole was stirred for 30 min, then acidified with dil HCl,
and extracted with ethyl acetate. The extract was washed with
brine, dried over anhydrous magnesium sulfate and concentrated.
The residue was purified by silica gel column chromatography
(eluant; n-hexane/ethyl acetate = 2:1 v/v) to afford 97 mg (36%)
of the title compound as a colorless amorphous solid. 1H NMR
(500 MHz CDCl3) d 7.75 (d, J = 8.8 Hz, 2H), 7.30 (ddd, J = 8.4 Hz,
8.4 Hz, 6.8 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz,
2.4 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.85 (ddd, J = 8.4 Hz, 2.4 Hz,
0.8 Hz, 1H), 6.81–6.77 (m, 2H), 6.73 (ddd, J = 10.0 Hz, 2.4 Hz,
2.4 Hz, 1H), 6.70 (t, J = 5.6 Hz, 1H), 4.62–4.53 (m, 2H), 3.85 (s,
3H), 2.88 (dd, J = 14.0 Hz, 8.8 Hz, 1H), 2.70 (dd, J = 13.6 Hz,
6.4 Hz, 1H), 2.59–2.51 (m, 1H), 1.71–1.51 (m, 2H), 0.94 (t,
6.4. Biochemical analysis
Serum biochemical markers, AST, ALT, and ALP were deter-
mined spectrophotometrically by using standard kits (Wako Pure
Chemical Industries, Osaka, Japan).
J = 7.6 Hz, 3H); ½a D20
¼ þ19; HRMS (FAB) calcd for 512.1073;
ꢁ
6.5. Histological examination
found: 512.1055 (M+H)+.
Liver tissues were collected, fixed overnight in 10% formalde-
hyde in phosphate-buffered saline, then dehydrated and embed-
ded in paraffin. Liver tissue sections (4 lm thick) were stained
6.1.21. (S)-2-(3-((4-(4-Chlorophenoxy)benzamido)methyl)-4-
methoxybenzyl)butanoic acid (14b)
This compound was prepared from 13b, using the same proce-
with hematoxylin and with Masson trichrome (MT) staining for
histological examination.
dures as described for the preparation of 14a. Mp 108–109 °C; 1H
NMR (400 MHz CDCl3)
d 7.73 (d, J = 9.2 Hz, 2H), 7.32 (d,
J = 9.2 Hz, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 2.4 Hz,
1H), 6.97 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.79 (d,
J = 8.4 Hz, 1H), 6.68 (t, J = 5.6 Hz, 1H), 4.62–4.52 (m, 2H), 3.85 (s,
3H), 2.88 (dd, J = 14.0 Hz, 8.4 Hz, 1H), 2.71 (dd, J = 13.6 Hz, 6.4 Hz,
1H), 2.59–2.52 (m, 1H), 1.71–1.51 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H);
6.6. Statistical analysis
Data are expressed as mean SEM of five rats. Differences
among groups were examined by one-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test. A p va-
lue less than 0.05 was considered statistically significant.
½
a 2D0
ꢁ
¼ þ20; MS (FAB) 468, 470 (M+H)+; HRMS (FAB) calcd for
468.1578; found: 468.1555 (M+H)+.

S. Ban et al. / Bioorg. Med. Chem. 19 (2011) 3183–3191
10. Roden, M. Nat. Clin. Pract. Endocrinol. Metab. 2006, 2, 335.
3191
Acknowledgments
11. Kasuga, J.; Yamasaki, D.; Araya, Y.; Nakagawa, A.; Makishima, M.; Doi, T.;
Hashimoto, Y.; Miyachi, H. Bioorg. Med. Chem. 2006, 14, 8405.
This work was supported in part by the Targeted Proteins
Research Program of the Japan Science and Technology Corpora-
tion (JST) and by the Uehara Memorial Foundation, and the Terumo
Life Science Foundation.
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