Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2

Article abstract of DOI:10.1016/j.bmc.2012.03.016

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by hereditary mutations of bile salt export pump (BSEP), such as E297G BSEP, which is a folding-defective mutant that is unable to traffic beyond the endoplasmic reticulum (ER). 4-Phen

Full text of DOI:10.1016/j.bmc.2012.03.016

Bioorganic & Medicinal Chemistry 20 (2012) 2940–2949
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structural development of small molecules that enhance
transport activity of bile salt export pump mutant associated
with progressive familial intrahepatic cholestasis type 2
Takashi Misawa a, Hisamitsu Hayashi b, Yuichi Sugiyama b, , Yuichi Hashimoto a,
⇑
⇑
a Institute of Molecular and Cellular Biosciencies, The University of Tokyo, 1-1-1 Bunkyo-ku, Tokyo 113-0032, Japan
b Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by hereditary mutations of bile salt
export pump (BSEP), such as E297G BSEP, which is a folding-defective mutant that is unable to traffic
beyond the endoplasmic reticulum (ER). 4-Phenylbutyric acid (4-PBA) enhances the cell surface expres-
sion and transport capacity of E297G BSEP, but has a relatively high dose (1 mM or more) is required to
show the effect. Here, we show that bile acids possibly act as pharmacological chaperones, promoting the
proper folding and trafficking of E297G BSEP. We also describe the discovery and structural development
of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 February 2012
Revised 5 March 2012
Accepted 5 March 2012
Available online 14 March 2012
Keywords:
Bile salt export pump
Bile acid
Farnesoid X receptor
Mutant
1. Introduction
be folding-defective (misfolded). As a result, it is retained within
the cell on endoplasmic reticulum (ER), and does not acquire the
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a
recessive hereditary liver disease, characterized by cholestasis
and jaundice in the first year of life and leading to cirrhosis and
death before adulthood, unless liver transplantation is carried
out.1 Although ursodeoxycholic acid has been widely used in treat-
ment of the disease, it is considered to be a symptomatic treatment
which is effective only in a small proportion of patients (approx
10%).2 Therefore, alternative agents for the treatment of PFIC2
are urgently needed. Many studies have been performed in PFIC2
patients, and the cause of the disease was shown to be a hereditary
defect in the expression of bile salt export pump (BSEP/ABCB11).
BSEP is a member of the ATP-binding cassette transmembrane
transporter family and mediates the excretion of monovalent bile
acids (such as taurocholate) from liver cells. Genomic analysis of
PFIC2 patients has revealed many kinds of missense, premature
termination, frame-shift and splicing-junction mutations in the
BSEP gene.3 Among them, two missense mutations, E297G
(Glu297Gly) and D482G (Asp482Gly), are frequently observed in
PFIC2 patients.4,5 Generally, wild-type (WT) BSEP is synthesized
and folded at the endoplasmic reticulum (ER). Then, correctly
folded BSEP acquires Golgi-related glycosylation and is trafficked
to the plasma membrane. But, the E297G mutant is considered to
Golgi-related glycosylation. Hayashi et al. reported that the
E297G and D482G mutations cause a reduction of BSEP on plasma
membrane, but importantly, they found that the transport function
of both mutated BSEPs is almost normal if they localized correctly.5
Thus, the folding-defective nature, linked to abnormal traffick-
ing (retention at ER), of E297G mutant lies at the core of the etiol-
ogy of PFIC2, at least in part. If E297G BSEP could be induced to fold
properly, the mutant protein could be transported to the correct
localization, where it should be functional, providing a promising
strategy for treatment of E297G BSEP-related PFIC2. Hayashi
et al. reported that sodium 4-phenylbutylic acid (4-PBA) enhanced
the cell-surface expression and transport capacity of E297G BSEP
by prolonging the half-life of cell surface-resident BSEP, through
modulating its ubiquitination status and AP2-mediated internali-
zation.5–7 Although 4-PBA can be beneficial for the treatment for
PFIC2, a relatively high dose (1 mM or more) is required to rescue
the function of the mutant BSEP in vivo. Therefore, the develop-
ment of more potent compounds is a pivotal work to improve clin-
ical application of the medical therapy for PFIC2. Based on this
background, we searched for novel compounds able to enhance
the transport capacity of E297G BSEP at lower doses.
Previously, we and others have investigated the usefulness of
pharmacological chaperones for treatment of diseases caused by
folding-defective membrane proteins.8–12 A pharmacological chap-
erone is defined as a small molecule that specifically binds to its
⇑
Corresponding authors. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495 (Y.H.).
E-mail addresses: yu-one.sugiyama@nifty.com (Y. Sugiyama: biological part),
hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto: chemical part).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.016

T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
2941
2.50
2.00
1.50
1.00
0.50
0.00
much lower dose levels than 4-PBA.13 In the cases of some fold-
ing-defective mutant proteins, including mutated rhodopsin, it
has been shown that their ligands (substrates, inhibitors, modula-
tors, etc.) act as pharmacological chaperones.8 Therefore, we hoped
that bile acids, which are substrates of BSEP, might act as pharma-
cological chaperones of E297G BSEP, consequently enhancing the
cell-surface expression and transport capacity of E297G BSEP.
Here, we show that bile acids do act as pharmacological chaper-
ones of E297G BSEP. We also describe the discovery and structural
development of non-steroidal compounds with potent pharmaco-
logical chaperone activity for E297G BSEP.
CA
0µM
0uM
10µM
10uM
100µM
10 uM
WT
E297G
2. Results and discussion
Figure 1. Concentration-dependence of the effect of cholic acid (CA) on [3H]TC
accumulation in MDCK II cells expressing wild type (WT) BSEP or E297G BSEP.
Vertical scale: The amounts of [3H]TC accumulated in the MDCK II cells transfected
with WT BSEP, E297G BSEP or GFP were measured by means of radioactivity (dpm).
The ratio between the [3H]TC accumulated in the BSEP-transfected MDCK II cells
and that in the GFP-transfected cells are presented.
2.1. Effects of bile acids on [3H]TC accumulation
To screen compounds that enhance the transport capacity of
E297G BSEP, we established a functional assay system, using Ma-
din-Darby canine kidney II (MDCKII) cells expressing Na+-tauro-
cholate cotransporting polypeptide (NTCP) and WT or E297G
BSEP. The amount of [3H]taurocholate ([3H]TC) accumulation in
cells expressing E297G BSEP was higher than that in cells
target protein and induces or promotes proper folding and traffick-
ing of the protein.13 The advantages of pharmacological chaper-
ones are considered to include high specificity and efficacy at
Table 1
Decreasing effect of bile acids on [3H]TC accumulation
O
R2
H
R3
H
H
HO
H
R1
Compd
R1
R2
R3
Accumulation of [3H] TC (%)
100
lM
10
73
lM
Cholic acid (CA)
Glycocholic acid (GC)
Taurocholic acid (TC)
Deoxycholic acid (DCA)
Chenodeoxycholic acid (CDCA)
Ursodeoxycholic acid (UDCA)
OH
OH
OH
H
OH
OH
OH
OH
H
OH
40
Glycine
Taurine
OH
OH
OH
108
105
65
b-OH
46
43
50
76
a-OH
H
Table 2
Decreasing effect of GW4064 and derivatives on [3H]TC accumulation
O
N
O
R4
R1
R1
N
R3
O
R2
Compd
R1
R2
R3
R4
Accumulation of [3H]TC (%) at 10
lM
CDCA
GW4064
6g
6a
6b
6c
6d
6e
6f
15a
15b
15c
15d
15e
5c
50
33
111
66
49
31
53
59
70
64
85
93
80
35
93
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
3-COOH
2-COOH
4-COOH
3-COOMe
H
Me
n-Bu
Bn
Phenethyl
1-Naphthyl
2-Naphthyl
Bn
Bn
Bn
Bn
Bn
Bn

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T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
HOOC
N
O
N
HOOC
O
O
Cl
HOOC
Cl
N
Cl
O
GW4064
Fexeramine
AGN-34
Figure 2. Structures of representative FXR ligands.
(a)
(b)
4
3.5
3
2.5
2
1.5
1
3.5
3
2.5
2
1.5
1
0.5
0
0.5
0
1
2
3
4
1
2
3
4
0
1
10
0
1
10
3
3
GW4064
Concentration (µM)
6c
Concentration (µM)
Figure 3. Concentration-dependence of the effects of GW4064 (a), and 6c (b) on [3H]TC accumulation in MDCK II cells expressing E297G BSEP. Vertical scale: same as the
legend of Figure 1.
Scheme 1. Synthetic routes to the present series of amide compounds. Reagents and conditions: (a) 2-chloro-4-hydroxybenzaldehyde, K2CO3, DMF, rt; (b) 2-methyl-2-
butene, NaClO2, NaH2PO4, t-BuOH, H2O, rt; (c) (i) methyl 3-aminobenzoate, MsCl, Et3N, THF, rt; (d) NaH, RBr, DMF, rt; (e) LiOH, H2O, THF, rt.
expressing WT BSEP because of the cellular efflux function of
[3H]TC by BSEP and the lower cell-surface expression of E297G
BSEP compared with WT BSEP (Fig. 1).14 We first examined the ef-
fect of several BSEP substrates and other related bile acids, that is,
cholic acid (CA), glycocholic acid (GC), taurocholic acid (TC), cheno-
deoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxy-
cholic acid (UDCA), on the function of E297G BSEP. As shown in
Table 1, the bile acids examined, with the exceptions of GC and

T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
2943
Scheme 2. Synthetic routes to the present series of compounds. Reagents and conditions: (a) (i) NH2OH–HCl, NaOH, EtOH, 90 °C; (ii) NCS, DMF, rt; (iii) 4-methyl-3-
oxopentanoic acid methyl ester, Et3N, NaOMe, MeOH, rt; (b) DIBAL, THF, 0 °C; (c) CBr4, PPh3, DCM, rt; (d) 2-chloro-4-hydroxybenzaldehyde or 4-hydroxybenzaldehyde, K2CO3,
DMF, rt; (e) 2-methyl-2-butene, NaClO2, NaH2PO4, t-BuOH, H2O, rt; (f) (i) methyl aminobenzoates (for 13a, b, d, e) or 16 (for 14c), MsCl, Et3N, THF, rt; (g) NaH, BnBr, DMF, rt;
(h) LiOH, H2O, THF, rt; (i) benzaldehyde, AcOH, NaBH(OAc)3, DCM, rt.
0.08
2
0.07
0.06
1.5
1
0.05
0.04
0.03
0.02
0.01
0
0.5
0
control
control
6c (10 µM)
6c (10 uM)
control
control
4-PBA (1000uM)
4-PBA (1 mM)
Figure 4. Effect of 4-PBA and 6c on [3H]TC transport across the apical membrane in monolayers of MDCK II cells expressing E297G BSEP. The clearance (PSapical) was
determined as described in the Experimental section. Each bar represents the mean SE of triplicate determinations.
TC, possessed moderate to potent BSEP-function promoting activ-
ity. These compounds dose-dependently decreased the amount of
[3H]TC accumulation in the concentration range of 1–100
(Fig. 1). CDCA showed the most potent activity among the tested
lM

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T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
bile acids, decreasing[3H]TC accumulation by 50% at 10
l
M (Ta-
compounds are outlined in Scheme 2. Substituted isoxazoles 10a
and 10b, which were prepared as described in the literature,20
were reacted with 4-hydroxybenzaldehyde (11a) or 2-chloro-4-
hydroxybenzaldehydes (11b–c). The resulting aldehydes were oxi-
dized to the carboxylic acids 12, which were condensed with
methyl aminobenzoate to afford the amide linker derivatives
13a–b, 13d–e, and 14c. N-Alkylation (14a–b and 14d–e) and
saponification of the ester yielded carboxylic acid derivatives
15a–e.
ble 2). We speculated that bile acids increased the cell-surface
expression of E297G BSEP by acting as pharmacological chaper-
ones, thereby decreasing [3H]TC accumulation in cells expressing
E297G BSEP.
2.2. Molecular design for novel non-steroidal compounds
Though CDCA showed potent activity, it has steroid skeleton, and
so might influence other proteins, such as nuclear receptors. There-
fore, we searched for active compounds with a non-steroidal skele-
ton. For this purpose, we focused on farnesoid X receptor (FXR)
ligands. FXR is a well-characterized member of the so-called meta-
bolic subfamily of nuclear receptors, and is a transcriptional sensor
for bile acids. CDCA is an endogenous FXR ligand,15,16 and acts as a
signaling molecule that governs lipid, steroid, and cholesterol
homeostasis.17 FXR is involved in possesses such as glucose utiliza-
tion, inflammation, and carcinogenesis.18,19 After deorphanization
of FXR in 1999, extensive studies were directed to the creation of
FXRligandsthatcouldmodulateFXR-mediatedspecificgeneexpres-
sion, and several steroidal ornon-steroidalFXR ligandshave beenre-
ported (Fig. 2).20–22 We hypothesized that non-steroidal FXR ligands
might act as a structural equivalent of CDCA and enhance the trans-
port capacity of E297G BSEP. First, we investigated whether FXR li-
gands enhance the transport capacity of E297G BSEP. We found
that GW4064, which is a potent non-steroidal agonist for FXR,
dose-dependently decreased [3H]TC accumulation (Fig. 3a).
GW4064hastheadvantagethatitsbasicarchitecture isdistinctfrom
that of CDCA, and it shows no activity towards other nuclear recep-
tors, including the retinoic acid receptor.20 GW4064 seemed to be
superior to CDCA in its efficacy to decrease [3H]TC accumulation in
cells expressing E297G BSEP (Table 2), and was selected as a lead
compound for further structural development.
In the case of GW4064, a brief SAR report from GSK researchers
disclosed that a carboxyl group is essential and preferably located
at the 3-position (meta-position) for FXR-agonistic activity.20 We
evaluated the BSEP-function-promoting activity of regioisomers,
that is, the carboxyl group was shifted to the ortho-(15d) or para-
position (15e) from the meta-position, as well as the effect of ester-
ification of the para-carboxyl group (5c). As shown in Table 2, ester
derivative 5c had no effect on [3H]TC accumulation in cells
expressing E297G BSEP at 10 lM. On the other hand, ortho-substi-
tution (15d) decreased the BSEP function-promoting activity, while
para-substitution (15e) or meta-substitution (6c) did not affect the
BSEP function-promoting activity. These results indicate that a car-
boxyl substituent at the meta- or para- position is important for
interaction with E297G BSEP.
Next, we turned our attention to SAR of the Cl substituent. We
prepared derivatives in which one of the Cl groups was substituted
with a hydrogen (15a and 15b) or a methyl group (15c), but these
derivatives showed only weak BSEP function-promoting activity.
2.4. Transcellular transport assay
Finally, we evaluated the effectiveness of 6c in a transcellular
transport assay with MDCK II cells expressing E297G BSEP.
[3H]TC accumulation is determined by several parameters, includ-
ing cellular efflux of [3H]TC mediated by BSEP, but in the transcel-
lular transport assay with MDCK II monolayers, the function of
BSEP can be directly evaluated by measuring the efflux of [3H]TC
across the apical membrane after addition of [3H]TC on the basal
side, because BSEP is localized on apical membrane of MDCK II
monolayers.5 To directly confirm the effect of compounds on the
function of BSEP, PSapical, a kinetic parameter representing [3H]TC
transport across the apical membrane, was calculated. Treatment
2.3. Structure–activity relationship of GW4064 derivatives
Kainuma and co-workers reported that the double bond of
GW4064 can be replaced with a carbamoyl moiety without loss
of FXR activity.23 Similarly, we designed and prepared secondary
amide 6g, N-methyl amide 6a, N-butyl amide 6b, N-benzyl amide
6c, N-phenethyl amide 6d, and N-naphthyl amide 6e and 6f deriv-
atives, and examined whether these derivatives decrease [3H]TC
accumulation in cells expressing E297G BSEP. Synthetic routes to
the present series of compounds are outlined in Scheme 1. Substi-
tuted isoxazoles (1), which were prepared as described in the liter-
ature,20 were reacted with 2-chloro-4-hydroxybenzaldehyde (2).
The resulting aldehydes were oxidized to the carboxylic acids 3,
which were condensed with ethyl aminobenzoate to afford the
amide linker derivatives 4. N-Alkylation (5a–f) and saponification
of the ester yielded carboxylic acid derivatives 6a–g.
with 1 mM 4-PBA and 10 lM 6c increased PSapical by 3- and 7-fold,
respectively (Fig. 4). Thus, 6c enhanced the transport capacity at
much lower concentration than 4-PBA.
3. Conclusion
We focused on the idea that the transport capacity of E297G
BSEP might be increased by pharmacological chaperones that pro-
mote proper trafficking of the mutant BSEP. First, we established
that bile acids decrease [3H]TC accumulation in cells expressing
E297G BSEP. Moreover, we discovered that non-steroidal
GW4064 and its analogs also show this activity. Compound 6c in-
We evaluated the effect of the prepared compounds on [3H]TC
accumulation. As shown in Table 2, secondary amide derivative
6g was ineffective at 10 lM, but introduction of an alkyl chain or
a benzyl/phenethyl/naphthyl group at the amide linkage resulted
in BSEP-function-promoting activity. N-Benzyl derivative 6c
showed the most potent activity among the amide derivatives,
and dose-dependently decreased [3H]TC accumulation (Fig. 3b).
The pharmacological chaperone-like efficiency of the N-alkyl/N-
benzyl amide derivatives 6a, b, c, g decreased in the order of
6c > 6b > 6a >> 6g, and that of N-benzyl/phenethyl/naphthylamide
derivatives 6c–f decreased in the order of 6c > 6d > 6e > 6f. These
results suggest that the size of the binding pocket hosting the cen-
tral region of GW4064 derivatives might be limited to accommo-
date a benzyl or smaller group.
creased PSapical by 7-fold at 10 lM. Further studies to examine in
detail the molecular mechanism(s) of the enhancement of E297G
BSEP trafficking and the effects of these compounds on other BSEP
mutants are in progress.
4. Experimental
4.1. General
Proton nuclear magnetic resonance spectra (1H NMR) were re-
corded on a JEOL JNMGX500 (500 MHz) spectrometer in the indi-
cated solvent. Chemical shifts (d) are reported in parts per
Next, we turned our attention to SAR of the carboxyl group and
substituents such as Cl. Synthetic routes to the present series of the

T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
2945
million relative to the internal standard tetramethylsilane. High-
resolution mass spectra (HRMS) and fast atom bombardment
(FAB) mass spectra were recorded on a JEOL JMA-HX110 mass
spectrometer. Bile acids were purchased from Aldrich, Tokyo Kasei
Kogyo, Wako Pure Chemical Industry, and Kanto Kagaku, and used
without purification. Flash column chromatography was per-
formed using silica gel 60 (particle size 0.060–0.210 mm) supplied
by Kanto Kagaku.
5.0 mM glucose, and 1.53 mM CaCl2, adjust to pH 7.4). After incu-
bation with [3H]TC solution for 2 h, cells were washed three times
with ice-cold Krebs–Henseleit buffer. Cells were dissolved in 2 N
HCl and neutralized with 4 N NaOH, and [3H]TC was measured
by liquid scintillation counting.
4.3. Chemistry
4.3.1. 2-Chloro-4-{[5-isopropyl-3-(2,6-dichlorophenyl)isoxazol-
4-yl]methoxy}benzaldehyde (2)
4.2. Biology
To a solution of 1 (261 mg, 0.748 mmol) in DMF (5.0 mL) were
added 2-chloro-4-hydroxybenzaldehyde (120 mg, 0.766 mmol)
and potassium carbonate (210 mg, 1.53 mmol), and the mixture
was stirred for 3.0 h at room temperature. Then H2O and AcOEt
were added to it. The organic layer was washed with brine, dried
over MgSO4, and concentrated. The residue was purified by silica
4.2.1. Materials and cell culture
[3H]Taurocholate (TC) (2 Ci/mmol) was purchased from NEN Life
Sciences (Boston MA). The BD Adeno-X-Adenoviral Expression Sys-
tem (BD Biosciences, Palo Alto, CA) was used to establish human
WT and E297G BSEP recombinant adenoviruses as previously de-
scribed.14 As a control, recombinant adenovirus containing green
fluorescence protein (GFP) was used. Madin-Darby canine kidney
II (MDCK II) cells were cultured in Dulbecco’s modified Eagle’s med-
ium (D-MEM) containing 10% FBS, penicillin and streptomycin mix-
ture at 37 °C in a humidified atmosphere of 5% CO2 in air.
gel chromatography (n-hexane/AcOEt = 4/1 v/v) to afford
2
(245 mg, 77%) as a colorless oil; 1H NMR (500 MHz, CDCl3) d
10.30 (s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.33
(t, J = 7.6 Hz, 1H), 6.79 (s, 1H), 6.75 (d, J = 8.6 Hz), 4.80 (s, 2H),
3.32 (septet, J = 7.3 Hz, 1H), 1.44 (d, J = 7.3 Hz, 6H); MS (FAB,
[M+H]+) m/z 424.
4.2.2. Generation of recombinant adenovirus
The recombinant adenoviruses containing wild-type BSEP,
E297G BSEP, or GFP were constructed as described previously.14
The BSEP cDNA was amplified via polymerase chain reaction(PCR)
with Ex-Taq (Takara Bio, Shiga, Japan) from the commercially avail-
able cDNA library (Clontech, PaloAlto, CA). Cryptic bacterial pro-
moter was inactivated as previously described24 before being
cloned into the adenovirus shuttle vector pShuttle (Clontech). Con-
struction of the PFIC2 mutants was performed using a Qui-
kChangeXL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
The BSEP cDNA was sequenced in an ABI 377DNA Sequencer (Ap-
plied Biosystems, Foster City, CA). The sequence of the wild-type
BSEP cDNA was identical to that published (accession number,
AF091582).
The cloned wild-type and two mutated cDNAs were introduced
into the adenovirus vector (Clontech). There combinant adenovi-
ruses were produced using the adenovirus expression system,
and the titer was checked with an Adeno-X Rapid Titer Kit (Clon-
tech). As a control, recombinant adenoviruses containing green
fluorescence protein (GFP) were used.
4.3.2. 2-Chloro-4-{[5-isopropyl-3-(2,6-dichlorophenyl)isoxazol-
4-yl]methoxy}benzoic acid (3)
To a solution of 2 (795 mg, 1.82 mmol) in t-BuOH (15. 0 mL)
were added NaH2PO4 (267 mg, 2.25 mmol), 2-methyl-2-butene
(830 lL, 8.0 mmol), NaClO2 (674 mg, 7.45 mmol) and H2O
(5.0 mL), and the mixture was stirred overnight at room tempera-
ture. Then H2O and AcOEt were added to it. The organic layer was
washed with brine, dried over MgSO4, and concentrated. The
residue was purified by silica gel chromatography (n-hexane/
AcOEt = 2/1 v/v) to afford 3 (797 mg, 97%) as a colorless solid; 1H
NMR (500 MHz, CDCl3) d 7.96 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 7.6 Hz,
2H), 7.33 (t, J = 7.6 Hz, 1H), 6.85 (s, 1H), 6.71 (d, J = 8.6 Hz), 4.78
(s, 2H), 3.32 (septet, J = 7.3 Hz, 1H), 1.44 (d, J = 7.3 Hz, 6H); MS
(FAB, [M+H]+) m/z 440.
4.3.3. Methyl3-{2-chloro-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-benzoylamino}benzoate (4)
To a solution of 3 (230 mg, 0.523 mg) in dry THF (20 mL) were
added Et3N (150 lL, 0.725 mmol), MsCl (50.0 lL, 0.628 mmol)
and methyl 3-aminobenzoate (100 mg, 0.654 mmol) at 0 °C, and
the mixture was stirred for 2.5 h at room temperature. Then H2O
and AcOEt were added to it. The organic layer was washed with
brine, dried over MgSO4, and concentrated. The residue was puri-
fied by silica gel chromatography (n-hexane/AcOEt = 2/1 v/v) to af-
ford 4 (255 mg, 85%) as a colorless oil; 1H NMR (500 MHz, CDCl3) d
8.12 (d, J = 15.2 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz,
1H), 7.77 (d, J = 8.6 Hz, 2H), 7.47–7.40 (m, 2H), 7.34 (t, J = 8.0 Hz,
1H), 6.82 (s, 1H), 6.77 (d, J = 8.6 Hz, 1H), 4.78 (s, 2H), 3.92 (s, 3H),
3.33 (septet, J = 7.3 Hz, 1H), 1.44 (d, J = 7.3 Hz, 6H); MS (FAB,
[M+H]+) m/z 573.
4.2.3. Transcellular transport assay
MDCK II cells were seeded on Transwell membrane inserts
(pore size 3 lM; Falcon, Bedford, MA) in 24-well plates at a density
of 1.5 Â 105 cells per insert. After 2 days’ culture, confluent cells
were infected with recombinant adenovirus containing cDNA for
WT, E297G, BSEP or GFP at 50 MOI. Cells were cultured for 24
hours after infection and subsequently treated with compounds.
Then, 24 hours after treatment, transcellular transport assays were
performed as described.5 The apparent efflux clearance across the
apical membrane (PSapical) was calculated by dividing the steady-
state rate for the transcellular transport of [3H]TC determined over
2 h by the cellular concentration of [3H]TC determined at the end
of the experiment (2 h).
4.3.4. Methyl3-{N-methyl-2-chloro-4-{[3-(2,6-dichlorophenyl)-
5-isopropylisoxazol-4-yl]methoxy}-benzamido}benzoate (5a)
Sixty percent Sodium hydride (2.2 mg, 0.055 mmol) was rinsed
twice with n-hexane under an argon atmosphere to remove min-
eral oil, and DMF (0.50 mL) was added to it. To the suspension
was added 4 (29 mg, 0.050 mmol) in DMF (0.50 mL), and the whole
4.2.4. [3H]TC accumulation assay
MDCK II cells were seeded on 24-well plates at a density of
1.5 Â 105 cells. After 1 day of culture, confluent cells were infected
with recombinant adenovirus containing cDNA for WT, E297G
BSEP or GFP at 50 MOI. Cells were cultured for 24 h after infection
and subsequently treated with compounds. Then, 24 h after treat-
ment, the medium was removed and cells were washed with
Krebs–Henseleit buffer (118 mM NaCl, 23.8 mM NaHCO3,
4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES,
was stirred for 30 min at 0 °C. Methyl iodide (6.3 lL, 0.10 mmol)
was added to the mixture and stirring was continued for 12 h at
ambient temperature. Then 10 mL of water was added and the
whole was extracted with AcOEt. The extract was washed with
brine, dried over MgSO4, and evaporated. The residue was purified
by silica gel column chromatography (n-hexane/AcOEt = 3:1 to 2:1

2946
T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
v/v) to afford 5a (29 mg, 98%) as a colorless oil; 1H NMR (500 MHz,
DMSO-d6, 100 °C) d 7.77–7.41 (m, 5H), 7.39 (t, J = 7.4 Hz, 1H), 7.18
(d, J = 8.5 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 6.7 Hz, 1H), 4.81 (s, 2H),
3.84 (s, 3H), 3.42 (m, 1H), 3.31 (s, 3H), 1.31 (d, J = 6.7 Hz, 6H); MS
(FAB, [M+H]+) m/z 587.
whole was extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO4, and concentrated. The residue was
purified by silica gel chromatography (n-hexane/AcOEt = 1/1 v/v)
to afford 6a (104 mg, 86%) as pale yellow solid; mp 89–92 °C, 1H
NMR (500 MHz, CDCl3)
d 8.17 (d, J = 14.6 Hz, 2H), 8.09 (d,
J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H),
7.50 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.35 (t, J = 7.9 Hz,
1H), 6.83 (s, 1H), 6.78 (d, J = 8.5 Hz, 1H), 4.79 (s, 2H), 3.35–3.32
(m, 1H), 1.44 (d, J = 6.7 Hz, 6H); HRMS (FAB, [M+H]+) calcd for
4.3.5. Methyl 3-{N-butyl-2-chloro-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-benzamido}benzoate (5b)
Thiscompound wasprepared from4 (50 mg, 87.1 lmol)bymeans
ofaproceduresimilartothatusedfor5a. Compound5b(164 mg, 78%)
as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d 7.78 (s, 2H), 7.37 (d,
J = 7.9 Hz, 2H), 7.31 (t, J = 7.9 Hz, 1H), 7.22 (s, 2H), 6.94 (d, J = 8.6 Hz,
2H), 6.55 (s, 1H), 6.42 (d, J = 8.6 Hz, 1H), 4.61 (s, 2H), 3.90 (s, 2H),
4.78 (s, 2H), 3.24 (septet, J = 7.3 Hz, 1H), 1.36 (d, J = 7.3 Hz, 9H), 0.92
(t, J = 7.3 Hz, 3H); MS (FAB, M+H+) m/z 629.
C27H22Cl3N2O5 559.0594, found 559.0579.
4.3.11. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-butylbenzamido}benzoic
acid (6b)
This compound was prepared from 5b (6.30 mg, 10.0 lmol) by
means of a procedure similar to that used for 6a. Compound 6b
(4.50 mg, 73%) as pale yellow oil; 1H NMR (500 MHz, CDCl3) d
7.83 (s, 2H), 7.36 (d, J = 7.9 Hz, 2H), 7.30 (t, J = 7.9 Hz, 3H), 6.96
(d, J = 8.6 Hz, 1H), 6.55 (s, 1H), 6.44 (d, J = 8.6 Hz, 1H), 4.62 (s,
2H), 3.94 (s, 2H), 3.24 (septet, J = 7.3 Hz, 1H), 1.36 (d, J = 7.3 Hz,
9H), 0.92 (t, J = 7.3 Hz, 3H); MS (FAB, [M+H]+) m/z 615. HRMS
(FAB, [M+H]+) calcd C31H30Cl3N2O5 615.1220, found 615.1171.
4.3.6. Methyl 3-{N-phenylmethyl-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]-methoxy}-
benzamido}benzoate (5c)
This compound was prepared from 4 (255 mg, 0.444 mmol) by
means of a procedure similar to that used for 5a. Compound 5c
(217 mg, 74%) as a colorless solid; 1H NMR (500 MHz, CDCl3) d
7.74–6.97 (m, 13H), 6.56 (s, 1H), 6.42 (d, J = 8.5 Hz, 1H), 5.12 (s,
2H), 4.61 (s, 2H), 3.87 (s, 3H), 3.27–3.21 (m, 1H), 1.36 (d,
J = 6.7 Hz, 6H); FAB:MS m/z 663 (M+H)+.
4.3.12. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-benzylbenzamido}benzoic
acid (6c)
4.3.7. Methyl 3-{N-(2-phenylethyl)-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]-
methoxy}benzamido}benzoate (5d)
This compound was prepared from 5c (6.3 mg, 10.0 lmol) by
means of a procedure similar to that used for 6a. Compound 6c
(4.5 mg, 73%) as pale yellow solid; mp 73–76 °C, 1H NMR
This compound was prepared from 4 (78.0 mg, 0.136 mmol) by
means of a procedure similar to that used for 5a. Compound 5d
(67.0 mg, 73%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d
7.76–6.40 (m, 15H), 4.61 (s, 2H), 4.16 (s, 2H), 3.94 (s, 3H), 3.24 (br,
1H), 3.00 (s, 2H), 1.36 (d, J = 6.7 Hz, 6H); MS (FAB, [M+H]+) m/z 677.
(500 MHz, DMSO-d6, 100 °C) d 7.65–7.22 (m, 12H), 7.16 (d,
J = 8.5 Hz, 1H), 6.69 (s, 1H), 6.56 (d, J = 8.5 Hz, 1H), 5.03 (s, 2H),
4.79 (s, 2H), 3.40–3.35 (m, 1H), 1.31 (d, J = 6.5 Hz, 6H); HRMS
(FAB, [M+H]+) calcd for C34H28Cl3N2O5 649.1064, found 649.1072.
4.3.13. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-[2-
phenylethyl]benzamido}benzoic acid (6d)
4.3.8. Methyl 3-{N-(1-naphtylmethyl)-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]-
methoxy}benzamido}benzoate (5e)
This compound was prepared from 5d (30.5 mg, 44.2 lmol) by
This compound was prepared from 4 (52.0 mg, 90.6
l
mol) by
means of a procedure similar to that used for 6a. Compound 6d
(20.2 mg, 68%) as a pale yellow solid; mp 52–55 °C, 1H NMR
(500 MHz, DMSO-d6, 100 °C) d 7.84–6.42 (m, 15H), 4.62 (s, 2H),
4.18 (s, 2H), 3.23 (br, 1H), 3.02 (s, 2H), 1.36 (d, J = 6.7 Hz, 6H); HRMS
(FAB, [M+H]+) calcd for C35H23Cl3N2O5 663.1220, found 663.1225.
means of a procedure similar to that used for 5a. 5e (27.0 mg,
37%) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) d 8.28 (d,
J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H),
7.64–7.50 (m, 4H), 7.35 (d, J = 7.3 Hz, 2H), 7.30 (d, J = 7.3 Hz, 1H),
7.23 (t, J = 7.3 Hz, 1H), 7.14 (s, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.72
(d, J = 7.3 Hz, 1H), 6.54 (s, 1H), 6.39 (d, J = 8.6 Hz, 1H), 5.61 (s,
2H), 3.83 (s, 3H), 3.22 (septet, J = 7.0 Hz, 1H), 1.34 (d, J = 7.0 Hz,
6H); MS (FAB, [M+H]+) m/z 713.
4.3.14. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-[1-
naphtylmethyl]benzamido}benzoic acid (6e)
This compound was prepared from 5e (27.2 mg, 37.8 lmol) by
4.3.9. Methyl 3-{N-(2-naphtylmethyl)-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]-
methoxy}benzamido}benzoate (5f)
means of a procedure similar to that used for 6a. Compound 6e
(14.7 mg, 53%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d
8.29 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz,
1H), 7.70–7.67 (m, 3H), 7.57 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 7.3 Hz,
1H), 7.34 (d, J = 7.3 Hz, 2H), 7.28 (d, J = 7.3 Hz, 1H), 7.24 (t,
J = 7.3 Hz, 1H), 7.15 (s, 1H), 6.96 (d, J = 7.3 Hz, 2H), 6.78 (d,
J = 7.3 Hz, 1H), 6.54 (s, 1H), 6.40 (d, J = 8.0 Hz, 1H), 5.63 (s, 2H),
4.60 (s, 2H), 3.22 (septet, J = 7.0 Hz, 1H), 1.34 (d, J = 7.0 Hz, 6H);
FAB:MS m/z 698 (M)+. HRMS (FAB, [M+H]+) calcd for
This compound was prepared from 4 (50.4 mg, 87.1 lmol) by
means of a procedure similar to that used for 5a. Compound 5f
(36.1 mg, 58%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d
7.84–7.67 (m, 4H), 7.52–7.50 (m, 2H), 7.47–7.43 (m, 2H), 7.36 (d,
J = 8.0 Hz, 2H), 7.33–7.28 (m, 2H), 7.05 (t, J = 8.0 Hz, 1H), 7.00 (s,
2H), 6.56 (s, 1H), 6.44 (d, J = 8.0 Hz, 1H), 5.28 (s, 2H), 4.62 (s, 2H),
3.84 (s, 3H), 3.24 (septet, J = 7.0 Hz, 1H), 1.36 (d, J = 7.0 Hz, 6H);
MS (FAB, [M+H]+) m/z 713.
C38HC30ClN2O5 699.1220, found 699.1213.
4.3.15. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-[2-
naphtylmethyl]benzamido}benzoic acid (6f)
This compound was prepared from 8f (36.0 mg, 50.4
4.3.10. 3-{2-Chloro-{4-[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}-N-methylbenzamido}benzoic
acid (6a)
l
mol) by
To a solution of 5a (124 mg, 0.216 mmol) in THF (2.20 mL) was
added 1 M LiOH (1.30 mL). The reaction mixture was stirred over-
night at room temperature, then acidified with 2 M HCl and the
means of a procedure similar to that used for 6a. Compound 6f
(20.8 mg, 57%) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) d
7.81–7.73 (m, 4H), 7.67 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.47–7.43

T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
2947
(m, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.12–7.01
(m, 3H), 6.56 (s, 2H), 6.44 (d, J = 8.0 Hz, 1H), 5.30 (s, 2H), 4.62 (s,
2H), 3.23 (septet, J = 7.0 Hz, 1H), 1.35 (d, J = 7.0 Hz, 6H); HRMS
(FAB, [M+H]+) calcd for C38HC30ClN2O5 699.1220, found 699.1196.
12c (90.3 mg, 91%) as a colorless oil; 1H NMR (500 MHz, CDCl3) d
7.95 (d, J = 8.6 Hz, 1H), 7.22 (t, J = 7.9 Hz, 1H), 7.09 (d, J = 7.9 Hz,
1H), 6.85 (d, J = 2.4 Hz, 1H), 6.70 (dd, J = 8.6, 2.4 Hz, 1H), 4.62 (s,
2H), 3.30 (septet, J = 7.4 Hz, 1H), 2.11 (s, 6H), 1.43 (d, J = 7.4 Hz,
6H).
4.3.16. 3-{2-chloro-4-[(3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl)methoxy]benzamido}benzoic acid (6g)
This compound was prepared from 4 (60.7 mg, 0.105 mmol) by
means of a procedure similar to that used for 6a. Compound 6g
(36.1 mg, 61%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d
8.26 (s, 1H), 8.15–8.08 (m, 2H), 7.88 (d, J = 8.0 Hz, 1H), 7.74 (d,
J = 8.6 Hz, 2H), 7.48 (t, J = 8.0 Hz, 1H), 7.41–7.31 (m, 3H),, 6.81 (s,
1H), 6.75 (d, J = 8.6 Hz, 1H), 4.77 (s, 2H), 3.33 (septet, J = 7.3 Hz,
1H), 1.44 (d, J = 7.3 Hz, 6H); HRMS (FAB, [M+H]+) calcd for
4.3.23. Methyl 3-{2-chloro-4-[(5-isopropyl-3-phenylisoxazol-4-
yl)methoxy]benzamido}benzoate (13a)
This compound was prepared from 12a (187 mg, 0.503 mmol)
by means of a procedure similar to that used for 4. Compound
13a (186 mg, 94%) as a colorless powder; 1H NMR (500 MHz,
CDCl3) 8.17 (s, 1H), 8.13 (s, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.88–7.83
(m, 2H), 7.65–7.63 (m, 2H), 7.47–7.45 (m, 3H, 7.01 (d, J = 2.4 Hz,
1H), 6.96 (dd, J = 9.1, 2.4 Hz, 1H), 4.90 (s, 2H), 3.93 (s, 3H), 3.29
(septet, J = 6.9 Hz, 1H), 1.41 (d, J = 6.9 Hz, 6H).
C28H24Cl3N2O5 573.0751, found 573.0720.
4.3.17. 2-Chloro-4-[(5-isopropyl-3-phenyl-isoxazol-4-
yl)methoxy]benzaldehyde (11a)
4.3.24. Methyl 3-{4-[(5-isopropyl-3-phenylisoxazol-4-
yl)methoxy]benzamido}benzoate (13b)
This compound was prepared from 10a (92.7 mg, 0.650 mmol)
by means of a procedure similar to that used for 2. Compound
11a (218 mg, 94%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3)
d 10.35 (s, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.61 (dd, J = 7.9, 2.4 Hz, 2H),
7.47–7.42 (m, 3H), 6.97 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 8.5, 2.4 Hz,
1H), 4.92 (s, 2H), 3.27 (septet, J = 7.4 Hz, 1H), 1.41 (d, J = 7.4 Hz,
6H); MS (FAB, [M+H]+) m/z 356.
This compound was prepared from 12b (300 mg, 0.738 mmol)
by means of a procedure similar to that used for 4. Compound
13b (230 mg, 68%) as a colorless powder; 1H NMR (500 MHz,
CDCl3) 8.10 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.6 Hz, 3H),
7.45 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 8.0 Hz,
1H), 6.85 (d, J = 8.6 Hz, 2H), 4.80 (s, 2H), 3.93 (s, 3H), 3.34 (septet,
J = 6.8 Hz, 1H), 1.43 (d, J = 6.8 Hz, 6H).
4.3.18. 4-{[3-(2,6-Dichlorophenyl)-5-isopropylisoxazol-4-
yl]methoxy}benzaldehyde (11b)
4.3.25. Methyl 2-{2-chloro-4-[(3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl)methoxy]- benzamido}benzoate (13d)
This compound was prepared from 3 (147 mg, 0.256 mmol) by
means of a procedure similar to that used for 4. Compound 13d
(108 mg, 56%) as a pale yellow solid; 1H NMR (500 MHz, CDCl3) d
8.85 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.61–7.55 (m, 2H),
7.41 (d, J = 8.0 Hz, 2H), 7.33 (t, J = 8.6 Hz, 1H), 7.12 (t, J = 8.0 Hz,
1H), 6.84 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 4.76 (s, 2H),
3.89 (s, 3H), 3.33 (septet, J = 7.9 Hz, 1H), 1.44 (d, J = 7.9 Hz, 6H);
MS (FAB, [M+H]+) m/z 574.
This compound was prepared from 10b (349 mg, 1.00 mmol) by
means of a procedure similar to that used for 2. Compound 11b
(406 mg, quant.) as a pale yellow oil; 1H NMR (500 MHz, CDCl3) d
9.85 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.32
(t, J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 4.81 (s, 2H), 3.33 (septet,
J = 7.4 Hz, 1H), 1.43 (d, J = 7.4 Hz, 6H); MS (FAB, [M+H]+) m/z 390.
4.3.19. 2-Chloro-4-{[3-(2,6-dimethylphenyl)-5-
isopropylisoxazol-4-yl]methoxy}benzaldehyde (11c)
This compound was prepared from 10c (100 mg, 0.324 mmol)
by means of a procedure similar to that used for 2. Compound
11c (96.9 mg, 77%) as a pale yellow oil; 1H NMR (500 MHz, CDCl3)
d 10.30 (s, 1H), 7.82 (d, J = 9.1 Hz, 1H), 7.23 (t, J = 7.9 Hz, 1H), 7.09
(d, J = 7.9 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 9.1, 2.4 Hz,
1H), 4.64 (s, 2H), 3.31 (septet, J = 7.4 Hz, 1H), 2.11 (s, 6H), 1.41
(d, J = 7.4 Hz, 6H); MS (FAB, [M+H]+) m/z 384.
4.3.26. Methyl 4-{2-chloro-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}- benzamido}benzoate (13e)
This compound was prepared from 6 (147 mg, 0.256 mmol) by
means of a procedure similar to that used for 4. Compound 13e
(107 mg, 56%) as a pale yellow solid; 1H NMR (500 MHz, CDCl3) d
8.21 (s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.70
(d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.35 (t, J = 8.6 Hz, 1H),
6.82 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.78 (s, 2H), 3.91
(s, 3H), 3.33 (septet, J = 7.9 Hz, 1H), 1.44 (d, J = 7.9 Hz, 6H); MS
(FAB, [M+H]+) m/z 574.
4.3.20. 2-Chloro-4-[(5-isopropyl-3-phenylisoxazol-4-
yl)methoxy]benzoic acid (12a)
This compound was prepared from 11a (218 mg, 0.613 mmol)
by means of a procedure similar to that used for 3. Compound
12a (187 mg, 82%) as a pale yellow powder; 1H NMR (500 MHz,
CDCl3) d 8.05 (d, J = 8.6 Hz, 1H), 7.64–7.62 (m, 2H), 7.47–7.43 (m,
3H), 7.04 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 8.6, 2.5 Hz, 1H), 4.90 (s,
1H), 3.28 (septet, J = 6.7 Hz, 1H), 1.41 (d, J = 7.4 Hz, 6H).
4.3.27. Methyl 3-{N-benzyl-2-chloro-4-[(5-isopropyl-3-
phenylisoxazol-4-yl)methoxy]benzamido}- benzoate (14a)
This compound was prepared from 13a (53.7 mg, 0.105 mmol)
by means of a procedure similar to that used for 5a. Compound
14a (12.0 mg, 20%) as a colorless oil; 1H NMR (500 MHz, CDCl3) d
7.78 (d, J = 8.6 Hz, 1H), 7.74 (s, 1H), 7.56 (d, J = 7.3 Hz, 2H), 7.44
(t, J = 7.3 Hz, 1H), 7.38 (t, J = 8.6 Hz, 1H), 7.31–7.27 (m, 5H), 7.16
(t, J = 8.6 Hz, 1H), 7.07 (t, J = 8.6 Hz, 2H), 6.75 (s, 2H), 6.61 (d,
J = 8.6 Hz, 1H), 5.15 (s, 2H), 4.72 (s, 2H), 3.87 (s, 3H), 3.20 (septet,
J = 6.9 Hz, 1H), 1.34 (t, J = 6.9 Hz, 1H).
4.3.21. 4-{[3-(2,6-Dichlorophenyl)-5-isopropylisoxazol-4-
yl]methoxy}benzoic acid (12b)
This compound was prepared from 11b (390 mg, 1.00 mmol) by
means of a procedure similar to that used for 3. Compound 12b
(330 mg, 81%) as a colorless powder; 1H NMR (500 MHz, CDCl3) d
7.97 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.32 (t, J = 8.5 Hz,
1H), 6.81 (d, J = 8.5 Hz, 2H), 4.80 (s, 2H), 3.33 (septet, J = 7.4 Hz,
1H), 1.43 (d, J = 7.4 Hz, 6H).
4.3.28. Methyl 3-{N-benzyl-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}- benzamido}benzoate (14b)
This compound was prepared from 13b (53.0 mg, 98.3 lmol) by
4.3.22. 2-Chloro-4-{[3-(2,6-dimethylphenyl)-5-
means of a procedure similar to that used for 5a. Compound 14b
(35.9 mg, 57%) as a colorless oil; 1H NMR (500 MHz, CDCl3) d
7.76 (d, J = 7.9 Hz, 1H), 7.71 (s, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.30–
7.27 (m, 8H), 7.22 (d, J = 8.6 Hz, 3H), 7.16 (t, J = 7.9 Hz, 1H), 6.94
isopropylisoxazol-4-yl]methoxy}benzoic acid (12c)
This compound was prepared from 11c (95.9 mg, 0.247 mmol)
by means of a procedure similar to that used for 3. Compound

2948
T. Misawa et al. / Bioorg. Med. Chem. 20 (2012) 2940–2949
(d, J = 7.3 Hz, 1H), 6.51 (d, J = 8.6 Hz, 2H), 5.12 (s, 2H), 4.66 (s, 2H),
3.87 (s, 3H), 3.26 (septet, J = 7.3 Hz, 1H), 1.36 (d, J = 7.3 Hz, 6H); MS
(FAB, [M+H]+) m/z.629.
(26 mg, 74%) as pale yellow oil. 1H NMR (500 MHz, CDCl3) d 7.83
(d, J = 7.9 Hz, 1H), 7.75 (s, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.28–7.27
(m, 5H), 7.24–7.19 (m, 4H), 7.02 (d, J = 7.9 Hz, 1H), 6.53 (d,
J = 7.9 Hz, 2H), 5.14 (s, 2H), 4.67 (s, 2H), 3.26 (septet, J = 7.3 Hz,
1H), 1.35 (d, J = 7.3 Hz, 6H); HRMS (FAB, [M+H]+) calcd
4.3.29. Methyl 3-(benzylamino)benzoate (16)
To a solution of methyl 3-aminobenzoate (150 mg, 1.00 mmol) in
dry DCM (5.0 mL) was added benzaldehyde (200 mg), acetic acid
C34H29Cl2N2O5 615.1454, found 615.1434.
(200
l
L) and NaBH (OAC)3 (600 mg, 2.83 mmol) at 0 °C. The reaction
4.3.35. 3-(N-Benzyl-2-chloro-4-((3-(2,6-dimethylphenyl)-5-
isopropylisoxazol-4-yl)methoxy)- benzamido)benzoic acid
(15c)
mixture was stirred at rt overnight, and partitioned between AcOEt
and water. The organic layer was washed with brine, dried over
MgSO4, andconcentrated. Theresiduewaspurifiedbysilicagelchro-
matography (AcOEt/hexane = 1/10) to afford 16 (138 mg, 57%) as a
colorless powder; 1H NMR (500 MHz, CDCl3) 7.39–7.32 (m, 6H),
7.29 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 8.0 Hz,
1H), 4.37 (s, 2H), 4.16 (s, 1H), 3.88 (s, 3H); MS (FAB, [M]+) m/z 241.
This compound was prepared from 14c (45.6 mg, 72.3 lmol) by
means of a procedure similar to that used for 6a. Compound 15c
(33.1 mg, 75%) as a pale yellow foam; 1H NMR (500 MHz, CDCl3)
d 7.81 (d, J = 6.1 Hz, 1H), 7.76 (s, 1H), 7.31–7.28 (m, 5H), 7.21–
7.15 (m, 2H), 7.08–6.99 (m, 4H), 6.54 (s, 1H), 6.44 (d, J = 7.3 Hz,
1H), 5.14 (s, 2H), 4.46 (s, 2H), 3.23 (septet, J = 6.9 Hz, 1H), 2.04 (s,
6H), 1.36 (d, J = 6.9 Hz, 6H); HRMS (FAB, [M]+) calcd C36H33ClN2O5
608.2078, found 608.2086.
4.3.30. Methyl 3-{N-benzyl-2-chloro-4-{[3-(2,6-
dimethylphenyl)-5-isopropylisoxazol-4-yl]methoxy}-
benzamido}benzoate (14c)
This compound was prepared from 12c (25.3 mg, 0.104 mmol) by
means of a procedure similar to that used for 4. Compound 14c
(45.5 mg, 78%) as a pale yellow foam; 1H NMR (500 MHz, CDCl3) d
7.74–7.71 (m, 2H), 7.31–7.27 (m, 6H), 7.20 (t, J = 7.9 Hz, 2H), 7.11 (t,
J = 7.9 Hz, 2H), 7.05 (d, J = 7.3 Hz, 2H), 6.99 (t, J = 8.6 Hz, 2H), 6.54 (s,
1H), 6.42 (d, J = 7.9 Hz, 1H), 5.12 (s, 2H), 4.45 (s, 2H), 3.86 (s, 3H),
3.23 (septet, J = 6.9 Hz, 1H), 2.04 (s, 6H), 1.36 (d, J = 6.9 Hz, 6H).
4.3.36. 2-{N-Benzyl-2-chloro-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}- benzamido}benzoic acid
(15d)
This compound was prepared from 14d (35.9 mg, 52.7 lmol) by
means of a procedure similar to that used for 6a. Compound 15d
(19.1 mg, 55%) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) d
7.93 (d, J = 8.0 Hz, 1H), 7.37–7.31 (m, 4H), 7.30–7.27 (m, 3H),
7.19 (t, J = 8.0 Hz, 2H), 7.10 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 8.6 Hz,
1H), 6.56 (d, J = 2.5 Hz, 1H), 6.38 (dd, J = 8.0, 2.5 Hz, 1H), 5.99 (d,
J = 14.6 Hz, 1H), 4.59 (s, 2H), 4.15 (d, J = 14.6 Hz, 1H), 3.21 (septet,
J = 7.9 Hz, 1H), 1.34 (dd, J = 16.4, 7.9 Hz, 6H);HRMS (FAB, [M+H]+)
calcd for C34H27Cl3N2O5 649.1064, found 649.1074.
4.3.31. Methyl 2-{N-benzyl-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]methoxy}-
benzamido}benzoate (14d)
This compound was prepared from 12c (50 mg, 87.1 lmol) by
means of a procedure similar to that used for 4. Compound 14d
(35 mg, 61%) as pale yellow foam;1H NMR (500 MHz, CDCl3) d
7.79 (d, J = 8.0 Hz, 1H), 7.38–7.34 (m, 2H), 7.30 (t, J = 8.6 Hz, 2H),
7.29 (d, J = 8.0 Hz, 1H), 7.20–7.13 (m, 4H), 7.00 (d, J = 8.0 Hz, 1H),
6.84 (d, J = 8.6 Hz, 1H), 6.55 (d, J = 2.5 Hz, 1H), 6.36 (dd, J = 8.0,
2.5 Hz, 1H), 5.89 (d, J = 14.6 Hz, 1H), 4.59 (s, 2H), 4.13 (d,
J = 14.6 Hz, 1H), 3.95 (s, 3H), 3.22 (septet, J = 7.9 Hz, 1H), 1.34
(dd, J = 16.4, 7.9 Hz, 6H); MS (FAB, [M+H]+) m/z 663.
4.3.37. 4-{N-Benzyl-2-chloro-4-{[3-(2,6-dichlorophenyl)-5-
isopropylisoxazol-4-yl]methoxy}- benzamido}benzoic acid
(15e)
This compound was prepared from 6e (40.1 mg, 60.2 lmol) by
means of a procedure similar to that used for 6a. Compound 15e
(25.4 mg, 55%) as a pale yellow oil. 1H NMR (500 MHz, CDCl3) d
7.81 (d, J = 7.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.29–7.21 (m, 6H),
7.00 (d, J = 7.3 Hz, 3H), 6.59 (s, 1H), 6.45 (d, J = 7.3 Hz, 1H), 5.10
(s, 2H), 4.62 (s, 2H), 3.24 (septet, J = 7.9 Hz, 1H), 1.35 (d,
J = 7.9 Hz, 6H);HRMS (FAB, [M+H]+) calcd C34H27Cl3N2O5
649.1064, found 649.1020.
4.3.32. Methyl 4-{N-benzyl-2-chloro-4-{[3-(2,6-
dichlorophenyl)-5-isopropylisoxazol-4-yl]methoxy}-
benzamido}benzoate (14e)
This compound was prepared from 13e (43.1 mg, 74.9 lmol)
according to 4. Compound 14e (41.8 mg, 82%) as a pale yellow so-
lid; 1H NMR (500 MHz, CDCl3) d 7.76 (d, J = 8.0 Hz, 2H), 7.37–7.27
(m, 8H), 6.99 (d, J = 7.3 Hz, 3H), 6.59 (s, 1H), 6.45 (d, J = 7.3 Hz,
1H), 5.10 (s, 2H), 4.62 (s, 2H), 3.84 (s, 3H), 3.25 (septet, J = 7.9 Hz,
1H), 1.37 (d, J = 7.9 Hz, 6H); MS (FAB, [M+H]+) m/z 663.
Acknowledgments
The work described in this paper was partially supported by
Grants-in Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science. We thank Ms. Asami Hattori
for excellent technical support on the biological experiments.
4.3.33. 3-{N-Benzyl-2-chloro-4-{[5-isopropyl-3-phenylisoxazol-
4-yl]methoxy}benzamido}benzoic acid (15a)
This compound was prepared from 14a (12.8 mg, 20.5 lmol) by
References and notes
means of a procedure similar to that used for 6a. Compound 15a
(3.90 mg, 33%) as a pale yellow foam; 1H NMR (500 MHz, CDCl3)
d 7.83 (d, J = 7.3 Hz, 1H), 7.77 (s, 1H) 7.55 (d, J = 6.7 Hz, 2H), 7.42
(d, J = 7.3 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.31–7.27 (m, 5H), 7.20
(t, J = 8.6 Hz, 1H), 7.13–7.08 (m, 2H), 6.74 (s, 1H), 6.62 (d,
J = 7.9 Hz, 1H), 5.16 (s, 2H), 4.72 (s, 2H), 3.20 (septet, J = 6.9 Hz,
1H), 1.33 (d, J = 6.9 Hz, 6H); HRMS (FAB, [M+H]+) calcd
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