Biological evaluation of novel benzisoxazole derivatives as PPARδ agonists

Article abstract of DOI:10.1016/j.bmc.2011.03.053

We discovered novel peroxisome proliferator-activated receptor δ agonists with a characteristic benzisoxazole ring. Compound 5 exhibited potent human PPARδ transactivation activity. Furthermore, it stimulated the differentiation of oligodendrocyte precurs

Full text of DOI:10.1016/j.bmc.2011.03.053

Bioorganic & Medicinal Chemistry 19 (2011) 3255–3264
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biological evaluation of novel benzisoxazole derivatives as PPARd agonists
⇑
Shogo Sakuma , Tsuyoshi Endo, Takashi Kanda, Hideki Nakamura, Satomi Yamasaki, Tomio Yamakawa
Discovery Research Laboratories, Nippon Chemiphar Co. Ltd, 1-22, Hikokawado, Misato, Saitama 341-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We discovered novel peroxisome proliferator-activated receptor d agonists with a characteristic benzis-
oxazole ring. Compound 5 exhibited potent human PPARd transactivation activity. Furthermore, it stim-
ulated the differentiation of oligodendrocyte precursor cells in vitro. This indicates that this potential
drug may be effective for the treatment of demyelinating disorders such as multiple sclerosis.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 January 2011
Revised 19 March 2011
Accepted 21 March 2011
Available online 31 March 2011
Keywords:
PPARd agonist
Multiple sclerosis
Benzisoxazole ring
Oligodendrocyte precursor cells
1. Introduction
neurons, leading to multiple lesions in various sites. Reflecting this
hypothesis, potential therapeutic drugs targeting disease-related
Multiple sclerosis (MS) is an autoimmune disease that suddenly
manifests itself in persons in their 20s and 30s, accompanying mul-
tiple inflammatory demyelinating disorders in various sites of the
central nervous system (CNS).1–5 Genetic as well as environmental
factors are believed to be implicated in the onset of MS. In Europe,
the incidence of MS is 50 per 100,000 persons and is particularly
high at high latitudes, and the incidence in Japanese individuals
was approximately 5-fold lower than that in Caucasians. However,
due to rapid environmental changes, the incidence in Japan in-
creased 4- to 5-fold in the last 30 years, and currently, approxi-
mately 12,000 Japanese individuals have MS. ‘Conventional MS’
now occurs at similar frequencies in Japanese and Caucasians,
and the peak prevalence rates shifted from persons in their 30s
to younger persons in their 20s.
In addition to three existing interferon (IFN)-b products, a syn-
thetic peptide, ‘Glatiramer,’ comprising major components of mye-
lin basic protein (MBP), which is involved in the onset of MS, was
introduced into medical practice in Europe as a therapeutic anti-
MS drug, and integrin inhibitors were also introduced. Therefore,
annual sales of anti-MS drugs have been rapidly increasing. In
addition, new anti-MS drugs are under development worldwide.
Although causes of the onset of MS are still unknown, a proposed
mechanism is that the immune system destroys myelin sheaths
within neurons, which disturbs signal transmission between
molecules now receive more attention than conventional nonspe-
cific immunotherapy.
Anti-MS drugs can be broadly classified as drugs for symptom-
atic treatment to alleviate acute symptoms for new-onset or recur-
rent MS and disease modifiers to relief physical dysfunction. In
symptomatic treatment, steroids are widely considered highly
effective for MS treatment. However, only IFN-b, glatiramer ace-
tate, and mitoxantrone prevented the advancement of physical
dysfunction in clinical studies, and they were approved as disease
modifiers in Europe. Therefore, recent development efforts concen-
trate on disease modifiers. Major disease modifiers under develop-
ment can be classified into the following three groups: (1) drugs to
inhibit or induce nonspecific immune response; (2) drugs to induce
immunological tolerance specific to MS antigens; and (3) neuro-
protective drugs. As MS is an autoimmune disease targeting the
CNS, various immunological inhibitors have been clinically used,
although most of them had no significant clinical effect. Because
MS is a demyelinating disease, the clinical use of neuroprotective
drugs has been investigated for the treatment of MS. As stated be-
fore, MS is a disease associated with disorders of myelin and its
component, oligodendrocytes. Recent studies clarified that
although normal oligodendrocyte precursor cells (OPCs) can be de-
tected in patient brains, they cannot differentiate and regenerate
myelin because of unknown reasons. Therefore, if differentiation
of precursor cells can be facilitated in patient brains, myelin will
be regenerated, which may lead to the restoration of neural
function.
Abbreviations: MS, Multiple sclerosis; CNS, Central nervous system; OPCs,
Oligodendrocyte precursor cells; PPAR, Peroxisome proliferator-activated receptor.
Peroxisome proliferator-activated receptors (PPARs) are nuclear
receptors that function after heterodimerization with other
⇑
Corresponding author. Tel.: +81 48 952 4311; fax: +81 48 952 0743.
E-mail addresses: sakuma@os.rim.or.jp, s-sakuma@chemiphar.co.jp (S. Sakuma).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.053

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S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
nuclear receptors, such as retinoid X receptors (RXRs). Three PPAR
subtypes, designated as PPAR , PPARd (b), and PPAR , have been
identified. Recent advances in PPAR and PPAR research have re-
transactivation activity and stimulation of oligodendrocyte differ-
entiation by the representative compounds.
a
c
a
c
vealed important roles of these PPARs in lipid and glucose metab-
olism. However, the functions of PPARd are not well understood.
The availability of PPARd transgenic mice and the discovery of
PPARd agonists, especially GW-5015166 and L-1650417 ( Fig. 1),
have stimulated PPARd research, and many reports have shown
that PPARd activation leads to interesting effects such as antiobe-
sity, improved lipid metabolism, and wound healing.8–12
PPARd agonists have been reported to stimulate oligodendro-
cyte differentiation.13 Oligodendrocytes are members of the glial
cell family and form the central myelin sheath. Any disease of
the nervous system in which the myelin sheath of neurons is dam-
aged is considered a demyelinating disorder. The most common
demyelinating disorder is MS, which is characterized by demyelin-
ation of the CNS. In the CNS tissues of MS patients, OPCs are found
to survive the demyelinating damage in MS, but they apparently
fail to proliferate and differentiate.14 Therefore, potent PPARd ago-
nists, which can stimulate oligodendrocyte differentiation, are ex-
2. Chemistry
The synthesis scheme for the benzisoxazole-ring derivatives is
shown below. The outline of the synthesis method for an oxazole
derivative, compound 6, has been reported previously.15 A similar
method was used for synthesizing compounds 3, 4, and 7–17. Thi-
azole derivatives, compounds 5 and 18, by using the method given
below.
In this paper, we have briefly explained the method of synthesis
of compound 5. Key intermediate 22 was synthesized as shown in
Scheme 1. After chlorination of the starting material methyl
4-methyl-3-oxovalerate (19) with sulfuryl chloride, the resulting
intermediate was treated with 4-(trifluoromethyl)thiobenzamide
to obtain a substantial yield of thiazole (20). Compound 20 was
then treated with sodium bis(2-methoxyethoxy)aluminum
hydride to obtain the alcohol (21), which was further treated with
thionyl chloride to produce 5-chloromethyl-4-isopropyl-2-[4-
(trifluoromethyl)phenyl]thiazole (22). Compounds 22 and 23a15
were condensed using LDA to obtain compound 24, which was
then treated with hydrochloric acid to produce compound 25. This
compound was further treated with sodium nitrite and then
hydrolyzed with 75% sulfuric acid to produce compound 26. Com-
pound 26 was condensed with ethyl 2-bromo-2-methylpropionate
in the presence of potassium carbonate in 2-butanone to obtain
compound 27, which was then treated with lithium hydroxide
monohydrate in ethanol to obtain compound 5 (Scheme 2).
pected to be
a possible remedy for dysmyelination and
demyelinating diseases. Based on these findings, the authors have
great interests in compounds with potent PPARd activity, stimulat-
ing us to develop novel compounds. We discovered fibrate deriva-
tive compound 3 by HTS and confirmed that this compound with a
characteristic benzisoxazole ring has PPARd transactivation activ-
ity. Next, we optimized compound 3 whereby we obtained com-
pound 4, which has an isopropyl group at the C5 position of the
oxazole ring, and confirmed that compound 4 has good selectivity
for PPARd.
After the optimization approach to compound 4, we discovered
compound 5, which exhibits the strongest PPARd transactivation
activity in this series of compounds. We also successfully created
compound 6,15 which has a high selectivity for PPARd (Fig. 2). This
paper describes the synthesis method for novel PPARd agonists
with a characteristic benzisoxazole ring and the degree of PPAR
3. Results and discussion
The human PPAR transactivation activity of test compounds
was evaluated by the following method. The mammalian expres-
sion vectors used were pSG5-GAL4-hPPARa, pSG5-GAL4-hPPARc,
Figure 1. Structures of representative PPARd agonists (1: GW-501516 and 2: L-165041).
Figure 2. Lead 3 and optimization process.

S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
3257
Scheme 1. Synthesis of compound 22. Reagents and conditions: (a) (1) SO2Cl2, 0 °C; (2) 4-trifluoromethylthiobenzamide, EtOH, reflux, 71%; (b) NaAlH2(OCH2CH2OCH3)2,
toluene, 0 °C, 69%; (c) SOCl2, Ph-H, 100%.
Scheme 2. Synthesis of target compounds. Reagents and conditions: (a) LDA, THF, À78 °C; (b) 3 M HCl, AcOH, reflux; (c) (1) NaNO2, 25% H2SO4, 0 °C; (2) 75% H2SO4, 120 °C;
(d) ethyl 2-bromo-2-methylpropionate, K2CO3, 2-butanone, room temperature; or ethyl 2-bromoacetate, K2CO3, acetone, room temperature; (e) LiOH monohydrate, EtOH,
H2O, reflux.
and pSG5-GAL4-hPPARd, which express the ligand-binding do-
mains of human PPAR , PPAR , and PPARd, respectively; each of
these vectors was fused to the yeast transcription factor GAL4
DNA binding domain.16 Each receptor expression vector and the
UASx4-TK-LUC17 reporter plasmid were cotransfected into CV-1
cells (kidney fibroblasts isolated from an African green monkey).
with a tert-butyl group decreased the PPARd transactivation activ-
ity. Further, we examined the effect of substitution on the benzene
ring at the C2 position of the oxazole ring. The substitution of a
chloro or hydroxyl group at the C2 position of the benzene ring
led to higher PPARd activation than the substitution of a hydrogen
or trifluoromethyl group. The introduction of an n-butyl or tert-bu-
tyl substituent at the C4 position of the benzene ring increased not
a
c
After the addition of compounds (0.001–100
incubated for 40 h, and luciferase activity was measured. The
PPAR , PPAR , and PPARd transactivation activity of the test com-
pounds was calculated relative to the luciferase activity induced by
M GW-59073518 (PPAR
selective agonist), 10 M rosiglitazone
(PPAR selective agonist), and 0.1 M GW-501516 (PPARd selec-
tive agonist), respectively (Table 1).
lM), CV-1 cells were
only PPARd but also PPARa and c activation. Finally, compound 4,
a
c
with chloro groups at the C2 and the C4 positions of the benzene
ring, was shown to exhibit good PPARd transactivation activity
and good d selectivity. Furthermore, compound 6 having a higher
d selectivity than compound 4 was successfully created by convert-
ing the fibrate structure of compound 4 to the acetic acid structure.
Compound 18, a fibrate derivative that was confirmed to exhibit
strong PPARd transactivation activity, was created by another con-
version of the oxazole ring of compound 4 to a thiazole ring. Com-
pound 5, which was derived by substituting a methyl group at the
C5 position of the benzisoxazole ring of compound 18, exhibited
the most powerful PPARd transactivation activity (EC50 = 0.011
1
l
a
l
c
l
With special attention to the two substituent groups on the
oxazole ring, optimization approach was conducted with com-
pound 3 (PPARd transactivation activity EC50: 14 lM) as a seed
compound. First, the methyl group at the C5 position of the oxazole
ring was replaced with an isopropyl or hexyl group, and it was con-
firmed that the resulting compounds exhibited higher PPARd acti-
vation than the other compounds. Substitution of the methyl group
lM) among the benzisoxazole derivatives.

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S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
Table 1
Peroxisome proliferator-activated receptor (PPAR) transactivation activity of compound 3–18 and representative PPAR agonists as assessed by a cell-based transactivation assay,
using GAL4 DBD-hPPAR LBD
Compound
X
Y
R1
R2
R3
R4
Transactivation activity
EC50 M)
(l
PPAR
a
PPAR
c
PPARd
3
7
8
9
10
11
12
13
14
15
16
4
17
18
5
6
N
N
N
N
N
N
N
N
N
N
N
N
N
S
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
Me
Et
Pr
Pr
iPr
tert-Bu
Hexyl
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
H
H
H
2-Cl
2-Cl
2-Cl
2-Cl
2-OH
2-CF3
4-tert-Bu
4-Bu
2,4-Cl
2,6-Cl
4-CF3
4-CF3
2,4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
12.9
14.7
13.2
13.9
34.1
>10
2.3
10.4
>10
2.9
20.8
25.1
19.1
26.4
31.1
>10
5.9
14.6
>10
1.0
14.9
6.7
9.8
5.4
1.3
>10
2.0
0.9
>10
1.2
0.7
3.3
8.6
2.6
26.3
>10
5.1
0.91
>10
>10
0.10
0.90
4.1
0.1
>10
0.83
0.20
>10
0.010
>10
>10
0.021
0.011
0.025
2.6
>10
>10
0.0017
S
N
GW590735
Rosiglitazone
KRP-297
GW501516
0.40
0.99
This research was conducted on the basis of the forecast that if
the PPARd agonist stimulated the differentiation of adult OPCs in-
side the brains of patients with MS, they could regenerate myelin.
With these adult OPCs as the target cells, the OPCs of the brain of a
rat fetus were used as the model. As the OPCs differentiate into
mature oligodendrocytes in the cell lineage, they specifically devel-
op proteins associated with this process. Among the proteins, O1
and MBP were selected as the differentiation markers. Within the
cell lineage, these proteins exist in the relatively late stage; in other
words, they reach the mature oligodendrocyte stage just before
myelin is formed in the immature oligodendrocyte. For this evalu-
ation, O1 was mainly used. The evaluation methods and an outline
of the results are described below.
area was stained red (O1 stain) such that we could see the pro-
gression in differentiation.20 The T3-treated cells showed a much
greater degree of differentiation than the control. For GW-
501516, too, a clear and more substantial progression of differen-
tiation could be observed as compared to the control group,
although the degree was less than that observed with T3. Figure
In vitro tests were conducted to evaluate the stimulation of oli-
godendrocyte differentiation by new compounds and reference
drugs by using OPCs. OPCs were prepared from primary mixed cell
cultures of the fetal brain cortex of Wistar rats,19 and the OPC-rich
fraction was prepared using the Percoll gradient separation tech-
nique. OPCs were treated with test compounds after 4 days of cul-
ture. The differentiation-stimulating effect was evaluated by an
immunostaining method. Compound was dissolved in dimethyl-
sulfoxide (DMSO), and the final concentration of DMSO in the med-
ium was 0.01%. Each dissolved compound was added to a culture
medium at a concentration of 0.01 or 0.1 lM. For control groups,
0.01% DMSO was added to a culture medium. The cells on the cover
glass were incubated for 3 days at 37 °C with 5% CO2. The cells
were immunostained using anti-oligodendrocyte marker O1 anti-
body and placed on a glass slide. After the slide images of the spec-
imens taken randomly were processed, the areas of stained cells
were measured. Regarding the mean area of the control group as
100%, the mean area of the treated groups was shown as relative
value. The degree of differentiation was determined based on the
increase in the number of stained cells, increased diameter of the
stained cells, and membrane sheet formation.
Figure 3a. Oligodendrocyte differentiation-stimulating effect of T3 and GW-
501516. Seven-day-old primary oligodendrocyte cultures were immunostained
with anti-O1 antibody. T3 and GW were added to the cultures 3 days before fixation.
In the cultures treated with T3 and GW, increase in the number of stained cells,
membrane sheet formation, and increase in cell diameters were observed.
Figure 3a shows the stimulation of oligodendrocyte differentia-
tion by T3 (0.1 lM) and GW-501516 (0.1 lM). For T3, most of the

S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
3259
Figure 3b. Oligodendrocyte differentiation-stimulating effect of GW-501516 and compound 6. Seven-day-old primary oligodendrocyte cultures were immunostained with
anti-O1 antibody. GW-501516 and Compound 6 were added to the cultures 3 days before fixation. In the cultures treated with GW and compound 6, increase in the number
of stained cells, membrane sheet formation, and increase in cell diameters were observed.
3b shows a parallel comparison between compound 6 (0.1
l
M)
potent human PPARd transactivation activity. Moreover, we con-
firmed that compounds 5 and 6 potently stimulate oligodendro-
cyte differentiation. Therefore, we believe that these compounds
may be effective for the treatment of demyelinating diseases such
as MS.
and GW-501516 (0.01 M) for the stimulation of oligodendrocyte
l
differentiation. With both compounds, substantial membrane
sheet formation was observed. Moreover, the number of stained
cells and the proportion of the stained area were also substantial
for both compounds. Figure 4 shows the stimulation of oligoden-
drocyte differentiation by compound 5, which exhibits strong
PPARd transactivation activity. The stained area with compound
5. Experimental
5.1. Chemistry
5 at a concentration of 0.01 lM was double that of the control
group. Membrane sheet formation was also observed. To recon-
firm that each compound stimulated oligodendrocyte differentia-
tion, double staining was conducted by using myelin basic
protein (MBP) in addition to O1. Table 2 shows the stimulation
of oligodendrocyte differentiation by the evaluated compounds,
including the findings of the control group. We confirmed that
compounds 5 and 6 and GW-501516, which exhibit strong PPARd
transactivation activity, stimulated oligodendrocyte differentia-
tion. Moreover, we confirmed that KRP-297, which exhibits weak
PPARd transactivation activity, showed extremely weak stimula-
tion of oligodendrocyte differentiation.
5.1.1. General procedures
Analytical samples were homogeneous as confirmed by TLC,
and the spectroscopic results were consistent with the assigned
strctures. 1H NMR was performed on a JEOL JNM-A400 spectrome-
ter by using deuterated chloroform (CDCl3) and deuterated DMSO
(DMSO-d6) as the solvent. Fast atom bombardment mass spectra
(FAB-MS, HR-MS) were obtained on a JEOL JMS-SX102A spectrom-
eter. IRs were measured using a HORIBA FT-720 spectrometer. Col-
umn chromatography was performed on silica gel [Wako gel C-300
or C-400]. Thin layer chromatography was performed on silica gel
(Merck TLC). The following abbreviations for solvents and reagents
are used: ethanol (EtOH), isopropyl alcohol (IPA), tetrahydrofuran
(THF), diethylether (Et2O), ethyl acetate (EtOAc), N,N-dimethyl-
formamide (DMF), acetic acid (AcOH), sulfuryl chloride (SO2Cl2),
thionyl chloride (SOCl2), and lithium diisopropylamide (LDA).
4. Conclusion
We discovered novel PPARd agonists with a characteristic ben-
zisoxazole ring. Representative compounds 5 and 6 exhibited

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S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
Figure 4. Oligodendrocyte differentiation-stimulating effect of compound 5. Seven-day-old primary oligodendrocyte cultures were immunostained with anti-O1 antibody.
Compound 5 was added to the cultures 3 days before fixation. In the cultures treated with compound 5, increase in the number of stained cells, membrane sheet formation,
and increase in cell diameters were observed.
extracted with EtOAc. The organic layer was washed with water
Table 2
and brine and then dried over Na2SO4. The solvent was evaporated
to give the crude product, which was recrystallized from
EtOAc–hexane to produce the target compound as a pale yellow
solid (19.0 g, yield 69%).
Evaluation of the stimulatory effect of compound 5, 6 and reference compounds on
the differentitation of OPCs
Compound
Concentration of compound
0.03
0.01
lM
lM
0.1 lM
1H NHR (400 MHz, CDCl3) d: 1.35 (d, 6H, J = 7 Hz), 1.82 (t, 1H,
J = 6 Hz), 3.15 (m, 1H), 4.87 (d, 2H, J = 6 Hz), 7.67 (d, 2H, J = 8 Hz),
8.04 (d, 2H, J = 8 Hz). FAB-MS (m/z): 302 (M+1).
5
6
Active
NTa
Active
NT
Active
NT
Active
NT
Active
Active
Active
Inactive
Active
GW-501516
KRP-297
T3
NT
Active
5.1.4. 5-Chloromethyl-4-isopropyl-2-[4-(trifluoromethyl)
phenyl]thiazole (22)
a
Not tested.
To
a stirred solution of 4-isopropyl-2-[4-(trifluoromethyl)
phenyl]thiazole-5-methanol (21) (10.3 g, 34.2 mmol) in benzene
(100 mL) was added SOCl2 (3.84 ml, 52.3 mmol) for 30 min at
0 °C. The mixture was stirred at room temperature for 20 h, and
then the solvent was removed in vacuo. The residue was dried in
vacuo to give the target compound as a brown solid (yield 100%).
1H NHR (400 MHz, CDCl3) d: 1.36 (d, 6H, J = 7 Hz), 3.18 (m, 1H),
4.71 (s, 2H), 7.68 (d, 2H, J = 8 Hz), 8.02 (d, 2H, J = 8 Hz).
5.1.2. Methyl 4-isopropyl-2-[4-(trifluoromethyl) phenyl]thia
zole-5-carboxylate (20)
Methyl 4-methyl-3-oxovalerate (19) (28.1 g, 195 mmol) was
cooled to 5 °C, and to this solution, SO2Cl2 (15.7 mL, 195 mmol)
was added dropwise over 45 min, stirred for 30 min, and allowed
to room temperature. To this solution, 4-(trifluoromethyl)thioben-
zamide (30.8 g, 150 mmol) in EtOH (15 mL) was added and re-
fluxed for 2 h. The reaction mixture was cooled, and to this
solution, IPA was added. The insoluble material was filtered and
washed with IPA. Water was added to the filtrates, which were
stirred at room temperature for 1 h. The precipitated product
was filtered and washed with a solution of IPA/H2O = 1:2 (50 mL)
and dried under a vacuum. The crude product was recrystallized
from IPA/H2O to give the target compound as a pale brown crystal
(34.9 g, yield 71%).
5.1.5. 6-Acetamido-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)
phenyl]-5-thiazolyl]ethyl]-5-methyl-1,2-benzisoxazole (24)
6-Acetamido-3,5-dimethyl-1,2-benzisoxazole (23a)15 (9.18 g,
45.0 mmol) was dissolved in dry THF (315 mL). To the solution,
2 M LDA solution (53.0 mL, 106 mmol) was added dropwise for
40 min at À78 °C under a nitrogen atmosphere, and the mixture
was stirred for 15 min at À78 °C, after which a THF solution
(100 mL) of 5-chloromethyl-4-isopropyl-2-[4-(trifluoromethyl)
phenyl]thiazole (22) (14.39 g, 45.0 mmol) was added dropwise
for 45 min. The mixture was stirred for 1 h under the same condi-
tions and warmed slowly to room temperature. A saturated aque-
ous ammonium chloride solution and EtOAc was added to the
reaction mixture. The organic layer was washed with water and
brine and dried over Na2SO4. After the organic solvent was re-
moved under reduced pressure, the residue was purified by col-
umn chromatography on silica gel with hexane/EtOAc (1:1) to
give the target compound as pale yellow crystals (7.40 g, yield
34%).
1H NHR (400 MHz, CDCl3) d: 1.35 (d, 6H, J = 7 Hz), 3.90 (s, 3H),
3.15 (m, 1H), 7.70 (d, 2H, J = 8 Hz), 8.10 (d, 2H, J = 8 Hz).
5.1.3. 4-Isopropyl-2-[4-(trifluoromethyl)phenyl]thiazole-5-
methanol (21)
To the solution of methyl 4-isopropyl-2-[4-(trifluoromethyl)
phenyl]thiazole-5-carboxylate (20) (30.0 g, 91.1 mmol) in dry tolu-
ene (300 mL) was added dropwise sodium bis(2-methoxyethoxy)
aluminum hydride toluene solution (39.6 g, 137 mmol) at 0 °C.
After 2 h, the reaction mixture was acidified with HCl aq and

S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
3261
1H NMR (CDCl3, 400 MHz) d: 1.25 (d, 6H, J = 7 Hz), 2.26 (br s,
3H), 2.32 (s, 3H), 3.04 (m, 1H), 3.26 (dd, 2H, J = 6 Hz, 8 Hz), 3.37
(dd, 2H, J = 6 Hz, 8 Hz), 7.12 (br s, 1H), 7.65 (d, 2H, J = 8 Hz), 7.99
(d, 2H, J = 8 Hz), 8.40 (br s, 1H).
pionate (27)(8.85 g,15.8 mmol)wassuspendedinanEtOH(180 mL)-
H2O (45 mL). Lithium hydroxide monohydrate (1.33 g, 31.6 mmol)
was added to the solution, and the mixture was refluxed for 2 h. Ice
was added to the reaction mixture. The mixture was acidified with
3 M HClandextractedwithEtOAc. Theorganiclayerwaswashedwith
brine and dried over Na2SO4. After the solvent was removed, the res-
idue was crystallized in EtOAc (9 mL)-hexane (90 mL). Pale yellow
crystal was recrystallized from EtOH–H2O. The crystals were filtered,
washed with water, air-dried overnight, and further dried in vacuo to
give the target compound as a white powder (7.90 g, yield 94%).
1H NMR (CDCl3, 400 MHz) d: 1.23 (d, 6H, J = 7 Hz), 1.72 (s, 6H),
2.28 (s, 3H), 3.02 (m, 1H), 3.2–3.4 (m, 4H), 6.93 (s, 1H), 7.25 (s, 1H),
7.65 (d, 2H, J = 9 Hz), 8.00 (d, 2H, J = 9 Hz). Mp (dec) 166–168 °C.
IR (KBr) cmÀ1: 3000, 1720, 1620, 1520, 1450, 1370, 1320, 1280,
1160, 1120, 1060, 850, 820. FAB-MS (m/z): 533 (M+1). HR-MS calcd
for C27H28F3N2O4S 533.17219, [M+H]+, found 533.17182.
5.1.6. 6-Amino-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)
phenyl]-5-thiazolyl]ethyl]-5-methyl-1,2-benzisoxazole(25)
6-Acetamido-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)phenyl]-
5-thiazolyl]ethyl]-5-methyl-1,2-benzisoxazole (24) (18.0 g, 36.9
mmol) was suspended in 4 M HCl (360 mL) and AcOH (180 mL).
The suspension was heated under reflux for 24 h. Then reaction mix-
ture was cooled to room temperature, poured into ice-cold water,
and neutralized with 10 M NaOH aq. After EtOAc was added to the
mixture, the organic layer was washed with brine and dried over
Na2SO4. After the solvent was removed under reduced pressure,
the crude crystal in the residue was filtered and washed with hexane
to give the target compound as pale brown crystals (16.8 g, yield
100%).
5.1.10. 6-Acetamido-3-[2-[2-(2,4-dichlorophenyl)-4-isopropyl-
4-oxazolyl]ethyl]-5-methyl-1,2-benzisoxazole (29)
1H NMR (CDCl3, 400 MHz) d: 1.25 (d, 6H, J = 7 Hz), 2.21 (s, 3H),
3.05 (m, 1H), 3.21 (dd, 2H, J = 6 Hz, 9 Hz), 3.35 (dd, 2H, J = 6 Hz,
9 Hz), 4.01 (br s, 2H), 6.75 (s, 1H), 7.14 (s, 1H), 7.64 (d, 2H,
J = 8 Hz), 8.02(d, 2H, J = 8 Hz).
The titled compound was synthesized from 6-acetamido-3,5-
dimethyl-1,2-benzisoxazole (23a)15 and 4-chloromethyl-2-(2,
-dichlorophenyl)-5-isopropyloxazole (28)15 in the same manner
as that described for 24: 1H NMR (CDCl3, 400 MHz) d: 1.10 (6H,
d, J = 7 Hz), 2.24 (3H, br s), 2.26(3H, s), 2.92 (1H, m), 3.05 (2H, t,
J = 7 Hz), 3.33 (2H, t, J = 7 Hz), 7.16 (1H, br s), 7.28 (1H, s), 7.32
(1H, dd, 1H, J = 2 Hz, 9 Hz), 7.51 (1H, d, J = 2 Hz), 7.91 (1H, d, 1
J = 9 Hz), 8.34 (1H, br s).
5.1.7. 6-Hydroxy-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)
phenyl]-5-thiazolyl]ethyl]-5-methyl-1,2-benzisoxazole (26)
6-Amino-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)phenyl]-5-thi
azolyl]ethyl]-5-methyl-1,2-benzisoxazole (25) (15.4 g, 34.6 mmol)
was suspended in 25% H2SO4 (170 mL). An aqueous sodium nitrite
(3.10 g, 45.0 mmol) was added to the suspension while it was cooled
with ice. After stirring for 20 min under the same conditions, the
mixture was added dropwise to 75% H2SO4 and then heated to
130 °C. The mixture was refluxed for 3 h under the same conditions,
cooled to room temperature, and poured into ice-cold water. After
EtOAc was added to the mixture, the organic layer was dried over
Na2SO4. After the solvent was removed under reduced pressure,
the crude crystal in the residue was filtered and washed with hexane
to give the target compound as pale brown crystals (8.36 g, yield
54%).
5.1.11. 6-Amino-3-[2-[2-(2,4-dichlorophenyl)-4-isopropyl-4-
oxazolyl]ethyl]-5-methyl-1,2-benzisoxazole (30)
The titled compound was synthesized in the same manner as
that described for 25: 1H NMR (CDCl3, 400 MHz) d: 1.10 (6H, d,
J = 7 Hz), 2.14 (3H, s), 2.92 (1H, m), 3.03 (2H, t, J = 7 Hz), 3.27
(2H, t, J = 7 Hz), 3.97 (2H, br s), 6.72 (1H, s), 7.13 (1H, s), 7.32
(1H, dd, J = 2 Hz, 8 Hz), 7.51 (1H, d, J = 2 Hz), 7.92 (1H, d, 1
J = 8 Hz), 8.34 (1H, br s).
1H NMR (CDCl3, 400 MHz) d: 1.24 (d, 6H, J = 7 Hz), 2.30 (s, 3H),
3.04 (m, 1H), 3.2–3.4 (m, 4H), 5.31 (s, 1H), 6.93 (s, 1H), 7.22 (s, 1H),
7.65 (d, 2H, J = 9 Hz), 8.00 (d, 2H, J = 9 Hz).
5.1.12. 3-[2-[2-(2,4-Dichlorophenyl)-4-isopropyl-4-
oxazolyl]ethyl]-6-hydroxy-5-methyl-1,2-benzisoxazole (31)
The titled compound was synthesized in the same manner as
that described for 26: 1H NMR (CDCl3, 400 MHz) d: 1.11 (6H, d,
J = 7 Hz), 2.22 (3H, s), 2.92 (1H, m), 3.05 (2H, t, J = 7 Hz), 3.29
(2H, t, J = 7 Hz), 6.15 (1H, br s), 6.15(1H, br s), 6.88 (1H, s), 7.19
(1H, s), 7.32 (1H, dd, J = 2 Hz, J = 9 Hz), 7.51 (1H, d, J = 2 Hz), 7.90
(1H, d, J = 9 Hz).
5.1.8. Ethyl 2-[3-[2-[4-isopropyl-2-[4-(trifluoromethyl)phenyl]-
5-thiazolyl]ethyl]-5-methyl-1,2-benzisoxazol-6-yloxy]-2-
methylpropionate (27)
6-Hydroxy-3-[2-[4-isopropyl-2-[4-(trifluoromethyl)phenyl]-5-
thiazolyl]ethyl]-5-methyl-1,2-benzisoxazole (26) (8.36 g, 18.7
mmol), ethyl 2-bromo-2-methylpropionate (14.6 g, 74.8 mmol),
and K2CO3 (10.3 g, 74.8 mmol) were suspended in 2-butanone
(5.0 mL). The suspension was refluxed for 45 h and cooled to room
temperature. The mixture was poured into ice-cold water and
added to EtOAc. The organic layer was washed with brine and
dried over Na2SO4. After the solvent was removed, the crude in
the residue was purified by column chromatography on silica gel
with hexane/EtOAc (10:1–5:1) to give the target compound as a
pale yellow oil (8.85 g, yield 85%)
5.1.13. Ethyl [3-[2-[2-(2,4-dichlorophenyl)-4-isopropyl-4-
oxazolyl]ethyl]-5-methyl-1,2-benzisoxazol-6-yloxy]acetate (32)
The titled compound was synthesized in the same manner as
that described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.11 (6H, d,
J = 7 Hz), 1.30 (3H, t, J = 7 Hz),2.23 (3H, s), 2.91 (1H, m), 3.04 (2H,
t, J = 7 Hz), 3.31 (2H, t, J = 7 Hz), 4.28 (2H, q, J = 7 Hz), 4.69 (2H,
s), 6.80 (1H, s), 7.24 (1H, s), 7.33 (1H, dd, J = 2 Hz, J = 8 Hz), 7.51
(1H, d, J = 2 Hz), 7.91 (1H, d, J = 9 Hz).
1H NMR (CDCl3, 400 MHz) d: 1.23 (d, 6H, J = 7 Hz), 1.24 (t, 3H,
J = 7 Hz),1.67 (s, 6H), 2.26 (s, 3H), 3.02 (m, 1H), 3.2–3.4 (m, 4H),
4.25 (q, 2H, J = 7 Hz), 6.77 (s, 1H), 7.25 (s, 1H), 7.65 (d, 2H,
J = 9 Hz), 8.00 (d, 2H, J = 9 Hz).
5.1.14. [3-[2-[2-(2,4-Dichlorophenyl)-4-isopropyl-4-oxazolyl]
ethyl]-5-methyl-1,2-benzisoxazol-6-yloxy]acetic acid (6)
The titled compound was synthesized in the same manner as
that described for 5: 1H NMR (CDCl3, 400 MHz) d:1.12 (d, 6H,
J = 7 Hz), 2.24 (s, 3H), 2.9–3.0 (m, 1H), 3.04 (dd, 2H, J = 7 Hz,
8 Hz), 3.27 (dd, 2H, J = 7 Hz, 8 Hz), 4.74 (s, 2H), 6.82 (s, 1H), 7.20
(s, 1H), 7.33 (dd, 1H, J = 2 Hz, 9 Hz), 7.52 (d, 1H, J = 2 Hz), 7.88 (d,
1H, J = 9 Hz). Mp (dec) 182–184 °C. HR-MS calcd for
5.1.9. 2-[3-[2-[4-Isopropyl-2-[4-(trifluoromethyl)phenyl]-5-thi
azolyl]ethyl]-5-methyl-1,2-benzisoxazol-6-yloxy]-2-methylpro
pionic acid (5)
Ethyl
2-[3-[2-[4-isopropyl-2-[4-(trifluoromethyl)phenyl]-5-
C
24H22Cl2N2O5: 488.0906 [M]+, found 488.0885.
thiazolyl]ethyl]-5-methyl-1,2-benzisoxazol-6-yloxy]-2-methylpro-

3262
S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
5.1.15. 2-[3-[2-[5-Methyl-2-phenyl-4-oxazolyl]ethyl]-1,2-
benzisoxazol-6-yloxy]-2-methylpropionic acid (3)
1H, J = 2 Hz, 8 Hz), 6.99 (d, 1H, J = 2 Hz), 7.24 (d, 1H, J = 8 Hz),
7.3–7.5 (m, 3H), 7.90 (d, 1H, J = 8 Hz).
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-5-methyl-2-
phenyloxazole in the same manner as that described for 5: 1H
NMR (DMSO-d6, 400 MHz) d: 1.77 (s, 6H), 2.19 (s, 3H), 2.96 (t,
2H, J = 7 Hz), 3.25(t, 2H, J = 7 Hz), 6.86(dd, 1H, J = 2 Hz, J = 9 Hz),
6.96 (d, 1H, J = 2 Hz), 7.45–7.50 (m, 3H), 7.71 (d, 1H, J = 9 Hz),
7.85–7.95 (m, 2H), 13.2 (br s, 1H). FAB-MS (m/z): 407 (M+1).
IR (KBr) cmÀ1: 3000, 2950, 2900, 1720, 1700, 1620, 1610, 1560,
1520, 1500, 1475, 1460, 1440, 1380, 1280, 1180, 1140, 1120, 1040,
1020 980, 840, 780, 740. FAB-MS (m/z): 469 (M+1).
5.1.21. 2-[3-[2-[5-tert-Butyl-2-(2-chlorophenyl)-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (11)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 5-tert-butyl-4-chloromethyl-
2-(2-chlorophenyl)oxazole in the same manner as that described
for 5: Mp (dec) 127–129 °C. 1H NMR (CDCl3, 400 MHz) d: 1.26
(6H, d, J = 7 Hz), 1.65 (s, 6H), 3.17 (t, 2H, J = 7 Hz), 3.31 (t, 2H,
J = 7 Hz), 6.81 (dd, 1H, J = 2 Hz, 8 Hz), 7.00 (d, 1H, J = 2 Hz), 7.28
(d, 1H, J = 8 Hz), 7.3–7.4 (m, 2H), 7.93 (m, 1H).
5.1.16. 2-[3-[2-[2-(2,4-Dichlorophenyl)-5-isopropyl-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (4)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-(2,4-
dichlorophenyl)-5-isopropyloxazole (28) in the same manner as
that described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.13 (6H, d,
J = 7 Hz), 1.66 (s, 6H), 2.95 (m, 1H), 3.04 (t, 2H, J = 7 Hz), 3.27 (t,
2H, J = 7 Hz), 6.82 (dd, 1H, J = 2 Hz, 8 Hz), 6.99 (d, 1H, J = 2 Hz),
7.28 (d, 1H, J = 8 Hz), 7.32 (dd, 1H, J = 2 Hz, 8 Hz), 7.51 (d, 1H,
J = 2 Hz), 7.87 (d, 1H, J = 8 Hz).
IR (KBr) cmÀ1: 3855, 3737, 3433, 2978, 2974, 2872, 2347, 1707,
1703, 1620, 1572, 1549, 1522, 1498, 1421, 1383, 1367, 1346, 1284,
1184, 1146, 1055, 982, 837, 816, 768, 744, 739, 609. FAB-MS (m/z):
483 (M+1).
IR (KBr) cmÀ1: 3635, 2939, 1705, 1618, 1562, 1498, 1466, 1460,
1385, 1381, 1286, 1184, 1147, 1107, 1053, 976, 841, 816, 417. FAB-
MS (m/z): 504 (M+1).
5.1.22. 2-[3-[2-[2-(2-Chlorophenyl)-5-hexyl-4-oxazolyl]ethyl]-
1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (12)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-(2-chloro-
phenyl)-5-hexyloxazole in the same manner as that described for
5: 1H NMR (CDCl3, 400 MHz) d: 0.86 (t, 3H, J = 7 Hz), 1.2–1.3 (m,
6H), 1.4–1.6 (m, 2H), 1.66 (s, 6H), 2.55 (t, 2H, J = 7 Hz), 3.02 (t,
2H, J = 8 Hz), 3.25 (t, 2H, J = 8 Hz), 6.80 (dd, 1H, J = 2 Hz, 10 Hz),
6.98 (d, 1H, J = 2 Hz), 7.2–7.5 (m, 4H), 7.90 (dd, 1H, J = 2 Hz,
10 Hz). FAB-MS (m/z): 512 (M+1).
5.1.17. 2-[3-[2-[5-Ethyl-2-phenyl-4-oxazolyl]ethyl]-1,2-benziso
xazol-6-yloxy]-2-methylpropionic acid (7)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-5-ethyl-2-
phenyloxazole in the same manner as that described for 5: 1H
NMR (DMSO-d6, 400 MHz) d: 1.3 (t, 3H, J = 7 Hz), 1.69 (s, 6H),
2.19 (s, 3H), 2.58 (q, 2H, J = 7 Hz), 3.02 (t, 2H, J = 7 Hz), 3.22(t, 2H,
J = 7 Hz), 6.79(dd, 1H, J = 2 Hz, J = 9 Hz), 7.01 (d, 1H, J = 2 Hz), 7.22
(d, 1H, J = 9 Hz), 7.4–7.5 (m, 3H), 7.9–8.0 (m, 2H). FAB-MS (m/z):
421 (M+1).
5.1.23. 2-[3-[2-[2-(2-Hydroxyphenyl)-5-isopropyl-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (13)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-5-isopropyl-
2-(2-methoxyphenyl)oxazole in the same manner as that
described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.16 (d, 6H,
J = 7 Hz), 1.66 (s, 6H), 2.95 (m, 1H), 3.06 (t, 2H, J = 7 Hz), 3.31 (t,
2H, J = 7 Hz), 6.8–7.1 (m, 4H), 7.32 (m, 1H), 7.40 (d, 1H, J = 9 Hz),
7.76 (dd, 1H, J = 1 Hz, J = 9 Hz). FAB-MS (m/z): 451 (M+1).
5.1.18. 2-[3-[2-[2-Phenyl-5-propyl-4-oxazolyl]ethyl]-1,2-
benzisoxazol-6-yloxy]-2-methylpropionic acid (8)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-phenyl-5-
propyloxazole in the same manner as that described for 5: 1H
NMR (DMSO-d6, 400 MHz) d: 0.85 (t, 3H, J = 7 Hz), 1.5–1.7 (m,
2H), 1.64 (s, 6H), 2.55(t, 2H, J = 7 Hz), 3.03 (t, 2H, J = 8 Hz), 3.19(t,
2H, J = 8 Hz), 6.76 (dd, 1H, J = 2 Hz, J = 9 Hz), 7.00 (d, 1H, J = 2 Hz),
7.20 (d, 1H, J = 9 Hz), 7.4–7.5 (m, 3H), 7.9–8.0 (m, 2H). FAB-MS
(m/z): 435 (M+1).
5.1.24. 2-[3-[2-[5-Isopropyl-2-[2-(trifluoromethyl)phenyl]-4-
oxazolyl]ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic
acid (14)
5.1.19. 2-[3-[2-[2-(2-Chlorophenyl)-5-propyl-4-oxazolyl]ethyl]-
1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (9)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-(2-chloro-
phenyl)-5-propyloxazole in the same manner as that described
for 5.
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-5-isopropyl-
2-[2-(trifluoromethyl)phenyl]oxazole in the same manner as that
described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.11 (d, 6H,
J = 7 Hz), 1.65 (s, 6H), 2.95 (m, 1H), 3.04 (t, 2H, J = 7 Hz), 3.28 (t,
2H, J = 7 Hz), 6.18 (1H, dd, J = 2 Hz, J = 8 Hz), 7.00 (d, 1H, J = 2 Hz),
7.26 (d, 1H, J = 8 Hz), 7.5–7.7 (m, 2H), 7.80 (d, 1H, J = 8 Hz), 8.02
(d, 1H, J = 8 Hz). FAB-MS (m/z): 503 (M+1).
1H NMR (DMSO-d6, 400 MHz) d: 0.85 (t, 3H, J = 7 Hz), 1.45–1.65
(m, 2H), 1.66 (s, 6H), 2.52 (t, 2H, J = 7 Hz), 3.04 (t, 2H, J = 8 Hz), 3.29
(t, 2H, J = 8 Hz), 4.24 (q, 2H, J = 7 Hz), 6.82 (dd, 1H, J = 2 Hz,
J = 9 Hz), 7.00 (d, 1H, J = 2 Hz), 7.3–7.4 (m, 3H), 7.45 (m, 1H), 7.85
(m, 1H). FAB-MS (m/z): 469 (M+1).
5.1.25. 2-[3-[2-[2-(4-tert-Butylphenyl)-5-isopropyl-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (15)
The titled compound was introduced from 6-acetamido-
3-methyl-1,2-benzisoxazole (23b) and 2-(4-tert-butylphenyl)-4-
chloromethyl-5-isopropyloxazole in the same manner as that
described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.16 (d, 6H,
J = 7 Hz), 1.36 (s, 9H), 1.70 (s, 6H), 2.95 (m, 3H), 3.17 (t, 2H,
J = 8 Hz), 6.76 (1H, dd, J = 2 Hz, J = 8 Hz), 7.00 (d, 1H, J = 2 Hz),
7.15 (d, 1H, J = 8 Hz), 7.47 (d, 2H, J = 8 Hz), 7.93 (d, 2H, J = 8 Hz).
FAB-MS (m/z): 491 (M+1).
5.1.20. 2-[3-[2-[2-(2-Chlorophenyl)-5-isopropyl-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (10)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-(2-chloro-
phenyl)-5-isopropyloxazole in the same manner as that described
for 5. Mp (dec) 100–105 °C.
1H NMR (CDCl3, 400 MHz) d: 1.13 (d, 6H, J = 7 Hz), 1.66 (s, 6H),
2.95 (m, 1H), 3.04 (t, 2H, J = 7 Hz), 3.27 (t, 2H, J = 7 Hz), 6.80 (dd,

S. Sakuma et al. / Bioorg. Med. Chem. 19 (2011) 3255–3264
3263
5.1.26. 2-[3-[2-[2-(4-Butylphenyl)-5-isopropyl-4-oxazolyl] eth
yl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (16)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 2-(4-butylphenyl)-4-chloro-
methyl-5-isopropyloxazole in the same manner as that described
for 5: 1H NMR (CDCl3, 400 MHz) d: 0.94 (t, 3H, J = 7 Hz), 1.16 (d,
6H, J = 7 Hz), 1.3–1.5 (m, 2H), 1.6–1.7 (m, 2H), 1.68 (s, 6H), 2.66
(t, 2H), 2.9–3.1 (m, 3H), 3.21 (t, 2H, J = 7 Hz), 6.77 (1H, dd,
J = 2 Hz, J = 8 Hz), 7.00 (d, 1H, J = 2 Hz), 7.20 (d, 1H, J = 8 Hz), 7.25
(d, 2H, J = 8 Hz), 7.90 (d, 2H, J = 8 Hz). FAB-MS (m/z): 491 (M+1).
activity) was measured using a microluminoreader (Type MLR-
100, Corona Electrics Co., Ltd). The luciferase activity of each con-
struct was corrected by the transfection efficiency, which was
calculated from the b-galactosidase activity. The assay method of
b-galactosidase activity was as follows: A portion (50
solubilized sample was transferred into another 96-well plate;
100 L of 2-nitrophenyl-b-galactopyranoside solution was added
lL) of the
l
and incubated for 5 min at room temperature. Fifty microliters of
reaction stopping solution (1 M sodium carbonate solution) was
added. Then, the absorbance at 414 nm was measured.
The PPAR
test compounds was calculated relative to the luciferase activity
induced by 1 M GW-590735 (PPAR selective agonist), 10
rosiglitazone (PPAR selective agonist), and 0.1 M GW-501516
(PPARd selective agonist), respectively.
a, PPARc, and PPARd transactivation activity of the
5.1.27. 2-[3-[2-[2-(2,6-Dichlorophenyl)-5-isopropyl-4-oxazolyl]
ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic acid (17)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 4-chloromethyl-2-(2,6-
dichlorophenyl)-5-isopropyloxazole in the same manner as that
described for 5: MP(dec) 161–163 °C. 1H NMR (CDCl3, 400 MHz)
d: 1.08 (t, 3H, J = 7 Hz), 1.63 (s, 6H), 2.85 (m, 1H), 3.07 (t, 2H,
J = 7 Hz), 3.31 (t, 2H, J = 7 Hz), 6.81 (1H, dd, J = 2 Hz, J = 8 Hz),
6.99 (d, 1H, J = 2 Hz), 7.3–7.4 (m, 4H).
l
a
lM
c
l
5.2.2. Oligodendrocyte differentiation-stimulating effects of
test compounds
Oligodendrocyte progenitors were isolated from glial cells of
the cerebral cortex of fetal Wistar rats (gestational age, 18–
19 days). On the day before cell seeding, cover glasses were coated
IR (KBr) cmÀ1: 3467, 2976, 2873, 2521, 1722, 1624, 1605, 1560,
1518, 1498, 1468, 1433, 1385, 1383, 1367, 1340, 1321, 1271, 1200,
1176, 1130, 1095, 1041, 974, 933, 883, 839, 793, 771, 750, 600, 569,
482. FAB-MS (m/z): 504 (M+1).
with poly-D-lysine for 1 day. The density of cells was adjusted to
1 Â 104 cells/glass and seeded onto the coated cover glasses laid
in the dish. Cells were then incubated at 37 °C with 5% CO2 for
4 days. Drug-containing medium was prepared as follows: drugs
were dissolved in DMSO at 1 Â 10À1 mol/L, diluted with DMSO to
prespecified concentrations, and stored at 4 °C. The stock medium
was further diluted 100-fold with medium and added onto 24-well
5.1.28. 2-[3-[2-[4-Isopropyl-2-[4-(trifluoromethyl)phenyl]-5-
thiazolyl]ethyl]-1,2-benzisoxazol-6-yloxy]-2-methylpropionic
acid (18)
The titled compound was introduced from 6-acetamido-3-
methyl-1,2-benzisoxazole (23b) and 5-chloromethyl-4-isopropyl-
2-[4-(trifluoromethyl)phenyl]thiazole (22) in the same manner as
that described for 5: 1H NMR (CDCl3, 400 MHz) d: 1.23 (d, 6H,
J = 7 Hz), 1.68 (s, 6H), 3.0–3.1 (m, 1H), 3.1–3.4 (m, 4H), 6.92 (dd,
1H, J = 2 Hz, J = 9 Hz), 7.05 (d, 1H, J = 2 Hz), 7.42 (d, 1H, J = 8 Hz),
7.65 (d, 1H, J = 8 Hz), 8.00 (d, 1H, J = 8 Hz). FAB-MS (m/z): 519 (M+1).
culture plates at 500 lL/well. Plates were covered with cell-seeded
cover glasses and incubated at 37 °C with 5% CO2 for 3 days. The
control group was cultured in medium containing 0.01% DMSO
only. Afterward, anti-O1 antibody was added onto slide glasses
for immunostaining. Cell samples were randomly photographed,
and cell-stained area was measured by image processing for each
sample. Relative areas to the mean value of the control group
(set at 100%) were calculated for each group.
5.2. Pharmacology
Acknowledgements
5.2.1. Measurement of PPAR
transactivation activity
The PPARa, PPARc, and PPARd transactivation activity of each
compound was measured in the manner described below.
a, PPARc, and PPARd
The authors would like to thank Dr. Masui and Mr. Hara for
extending support during our research period. We are also deeply
grateful to Mr. Kobayashi and Mr. Mochizuki for their contribution
to the synthesis and analysis of compounds and to Dr. Asou from
the Tokyo Metropolitan Institute of Gerontology for guidance re-
lated to the establishment of a system to assess oligodendrocytes.
No funding sources were involved in this study.
Plasmid: Receptor expression plasmids (GAL4-hPPARa, LBD
GAL4-hPPARc LBD, GAL4-hPPARd LBD), a reporter plasmid
(UASx4-TK-LUC), and a b-galactosidase expression plasmid (PGAL)
similar to that described by Kliewer et al.21 were used.
Transfection: CV-1 cells were seeded in 24-well culture plates at
a density of 2 Â 105 cells per well and cultured for 24 h in OPTI-
Supplementary data
MEM I Reduced Serum Medium (Life Technologies, 500 lL/well)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.053.
containing 4% fetal bovine serum (FBS). After washing with OPTI-
MEM, a transfection mixture (250 L/well) containing 0.03 g of
GAL4-hPPARd LBD, 0.25 g of UASx4-TK-LUC, 0.35 g of b-GAL,
and 2 L of lipofection reagent, DMRIE-C (Life Technologies) was
l
l
l
l
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