E297G mutated bile salt export pump (BSEP) function enhancers derived from GW4064: Structural development study and separation from farnesoid X receptor-agonistic activity

Article abstract of DOI:10.1016/j.bmcl.2012.04.099

Bile salt export pump (BSEP) is a member of the ATP-binding cassette transmembrane transporter family and mediates biliary excretion of bile acids from hepatocytes. Several BSEP mutants, including Glu297Gly (E297G) and Asp482Gly (D482G), cause progressive familial intrahepatic cholestasis type 2. We previously found that compounds based on GW4064, a representative farnesoid X receptor (FXR) agonist, enhanced E297G BSEP transport activity. Here, we conducted a structure-activity relationship analysis of GW4064 derivatives aimed at separating E297G BSEP-function-promoting activity and FXR-agonistic activity. Among newly synthesized reversed-amide derivatives of previously reported GW4064 analogs 2a-2f, we identified 7c as a selective BSEP function enhancer.

Full text of DOI:10.1016/j.bmcl.2012.04.099

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3962–3966
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Bioorganic & Medicinal Chemistry Letters
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E297G mutated bile salt export pump (BSEP) function enhancers derived
from GW4064: Structural development study and separation from farnesoid X
receptor-agonistic activity
Takashi Misawa a, Hisamitsu Hayashi b, Makoto Makishima c, Yuichi Sugiyama b, , Yuichi Hashimoto a,
⇑
⇑
a Institute of Molecular and Cellular Biosciences, 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
c School of Medicine, Nihon University, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Bile salt export pump (BSEP) is a member of the ATP-binding cassette transmembrane transporter family
and mediates biliary excretion of bile acids from hepatocytes. Several BSEP mutants, including Glu297Gly
(E297G) and Asp482Gly (D482G), cause progressive familial intrahepatic cholestasis type 2. We previ-
ously found that compounds based on GW4064, a representative farnesoid X receptor (FXR) agonist,
enhanced E297G BSEP transport activity. Here, we conducted a structure–activity relationship analysis
of GW4064 derivatives aimed at separating E297G BSEP-function-promoting activity and FXR-agonistic
activity. Among newly synthesized reversed-amide derivatives of previously reported GW4064 analogs
2a–2f, we identified 7c as a selective BSEP function enhancer.
Received 9 April 2012
Revised 21 April 2012
Accepted 21 April 2012
Available online 30 April 2012
Keywords:
Bile salt export pump
Bile acid
Farnesoid X receptor
Mutant
Ó 2012 Elsevier Ltd. All rights reserved.
Bile salt export pump (BSEP) is a member of the ATP-binding
cassette transmembrane transporter family and mediates the bili-
ary excretion of monovalent bile acids (such as taurocholate) from
hepatocytes.1–3 It is well known that several BSEP mutants cause
progressive familial intrahepatic cholestasis type 2 (PFIC2) which
is a recessive hereditary liver disease characterized by cholestasis
and jaundice in the first year of life, leading to cirrhosis and death
before adulthood unless liver transplantation is carried out.4
Among the PFIC2-causing mutations, two missense mutations, that
is, E297G (Glu297Gly) and D482G (Asp482Gly), are frequently ob-
served in PFIC2 patients.5,6 The E297G mutant is considered to be
folding-defective (misfolded), and as a result, this mutant BSEP is
retained within the cell on endoplasmic reticulum (ER), and does
not acquire the usual golgi-related glycosylation. Hayashi et al. re-
ported 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
are correctly localized.7 Furthermore, Hayashi et al. reported that
sodium 4-phenylbutyric 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 internalization.6–8
In our previous work, we found that bile acids showed moderate
E297G BSEP-function-promoting activity, and chenodeoxycholic
acid (CDCA) was the most potent function-promoter among the
tested bile acids.9 Furthermore, compounds based on GW4064
[a representative farnesoid X receptor (FXR) agonist10] ( Fig. 1)
enhanced the BSEP transport activity. Among the tested com-
pounds, compound 2d possessed the most potent E297G BSEP-
function-promoting activity at 10 lM (Table 2). We speculated
that these compounds enhanced the transport capacity by
increasing the cell-surface expression of the mutated BSEP.
On the other hand, it was reported that GW4064 derivatives
(2a, 2b, 2d, 2e) possess moderate to potent FXR-agonistic activity.
FXR is a well-characterized member of the so-called metabolic
subfamily of nuclear receptors, and is a transcriptional sensor for
bile acids.12,13 It is well known that FXR-agonists such as CDCA
and GW4064 increase endogenous BSEP mRNA level and lower
the bile acid pool.14 We considered that the contribution of
FXR-agonistic activity to the improvement of BSEP function in
our assay system was negligible, because we had calculated the
ratio of the amount of [3H]taurocholate ([3H]TC) accumulated in
MDCK II cells expressing WT or E297G BSEP versus that in the
MDCK II cells expressing GFP to confirm the effect of GW4064
derivatives on the function of BSEP.
A reduction in the bile acids pool, which may be caused by
FXR-agonists (vide supra), is likely to cause inactivation of TGR5,
which is a member of the G protein-coupled receptors (GPCR),
and hence facilitates the development of obesity and diabetes.15
⇑
Corresponding authors. Address: Institute of Molecular and Cellular Biosci-
ences, The University of Tokyo, 1-1-1 Bunkyo-ku, Tokyo 113-0032, Japan (Y. H.).
Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.099

T. Misawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3962–3966
3963
Figure 1. Structure of GW4064 and E297G BSEP function enhancers.
Table 1
E297G BSEP-function-promoting activity and FXR-agonistic activity of bile acids
R1
R2
Accumulation of [3H]TCa (%) at 100
lM
FXR-agonistic activity EC50
(lM)
c
Compd
CA (3)
OH
H
b-OH
OH
OH
H
40b
65b
46b
43b
NAd
50
DCA (4)
CDCA (5)
UDCA (6)
27
a-OH
H
NAd
The amount of [3H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%.
Date taken from our previous report.9
The EC50 value is the molar concentration of the test compound that affords 50% of the maximal reporter activity.
a
b
c
d
NA = No activity at 10 lM.
Table 2
FXR-agonistic activity of E297G BSEP function enhancers
R1
R2
Accumulation of [3H]TCa (%) at 10
lM
FXR-agonistic activity EC50
(
l
M) and efficacyd (%)
c
Compd
CDCA (5)
GW4064 (1)
50b
33b
111b
66b
49b
31b
53b
59b
80b
35b
93b
27 (41)
0.07 (100)
3.0 (61)
3.1 (45)
1.1 (43)
2.5 (31)
0.84 (30)
3.0 (28)
NAe
2a
2b
2c
2d
2e
2f
2g
2h
2i
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
Bn
Bn
Bn
2.6 (41)
NAe
a
b
c
The amount of [3H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%.
Data taken from our previous report.9
The EC50 value is the molar concentration of the test compound that affords 50% of the maximal reporter activity.
The effect of GW4064 on FXR activation efficacy was defined as 100%.
d
e
NA = No activity at 10 lM.
Therefore, in order to understand the biology and/or pharmacology
of BSEP in more detail, and to examine whether or not FXR-
agonistic activity would be problematic for PFIC2 treatment, we
require selective BSEP function enhancers lacking FXR-agonistic
activity as investigative tools. Here, we investigated the effect of
the previously reported compounds on FXR activation and analyzed

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T. Misawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3962–3966
Figure 2. Maximal FXR transcriptional activation activity (efficacy) of GW4064 and 2d.
Scheme 1. Synthetic route to the present series of reverse amide compounds. Reagents and conditions: (a) TBSCl, imidazole, DMF, rt, quant; (b) monomethylisophthalate,
MsCl, TEA, THF, 0 °C–rt, 78%; (c) TBAF, THF, 0 °C–rt, quant; (d) isoxazole derivative (14), K2CO3, DMF, rt, 87%; (e) R-Br or R-I, NaH, DMF, 0 °C–rt, 28–95%; (f) LiOH, THF, H2O, rt,
29–81%.
their structure–activity relationship (SAR) for FXR-agonistic activ-
ity. We also describe the application of these results to the design
and synthesis of selective BSEP function enhancer candidates.
To evaluate the activities of the compounds, we selected two
assay systems. One was an accumulation assay to evaluate the
E297G BSEP-function-promoting activity, using Madin-Darby
canine kidney II (MDCKII) cells expressing Na+-taurocholate
cotransporting polypeptide (NTCP) and E297G BSEP, as described
previously.9 With this method, we evaluated the amount of
[3H]TC accumulated in MDCK II cells expressing E297G BSEP;
this accumulation is determined by several parameters, including
the cellular efflux of [3H]TC via BSEP. The other was a reporter
gene assay to evaluate the FXR-agonistic activity, including the
EC50 value, and efficacy compared with those of the full agonist
GW4064, using CMX-GAL4N-hFXR as the recombinant receptor
gene, TK-MH100x4-LUC as the reporter gene, and the CMX
b-galactosidase gene for normalization, as previously reported.11
We first tested the FXR-agonistic activity of various bile acids,
that is, cholic acid (CA; 3), deoxycholic acid (DCA; 4), chenodeoxy-
cholic acid (CDCA; 5), and ursodeoxycholic acid (UDCA; 6). We pre-
viously reported that these bile acids possess moderate E297G
BSEP-function-promoting activity.9 Compounds 4 and 5 showed
weak FXR-agonistic activity, whereas compounds 3 and 6 showed
no FXR-agonistic activity (Table 1). These results suggest that
E297G BSEP-function-promoting activity can be separated from
FXR-agonistic activity.

T. Misawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3962–3966
3965
Table 3
E297G BSEP-function-promoting activity and FXR-agonistic activity of 7a–7e
Compd
R
Accumulation of [3H]TCa (%) at 10
lM
FXR-agonistic activity EC50 (l
M) and efficacyb (%)
CDCA
GW4064
2d
7a
7b
7c
7d
7e
50
33
31
75
60
42
50
59
27 (41)
0.07 (100)
2.5 (31)
NAc
H
Me
n-Bu
Bn
NAc
NAc
>10
0.41 (35)
1-Naphthyl
a
b
c
The amount of [3H]TC accumulated in MDCK II cells expressing E297G BSEP was defined as 100%.
The effect of GW4064 on FXR activation efficacy was defined as 100%.
NA = no activity at 10 lM.
Figure 3. Concentration dependency of the effect of 2d, 7a, and 7c–7d on [3H]TC accumulation in MDCK II cells expressing E297G BSEP. The amount of [3H]TC accumulated in
MDCK II cells expressing E297G BSEP was defined as 100%.
Next, we investigated the FXR-agonistic activity of our previ-
ously reported E297G BSEP function enhancers derived from
GW4064 (2a–2i, Table 2). E297G BSEP function enhancers 2a–f
and 2h showed weak FXR-agonistic activity, while 2g and 2i were
inactive. Broadly speaking, the EC50 values for FXR-agonistic activ-
ity of 2a–2f and 2h were about one order of magnitude higher
derivative 2d showed the most potent BSEP function enhancing
activity, and decreased [3H]TC accumulation with the potency of
31% at 10 lM (Table 2). The FXR-agonistic activities of 2a–2f are
summarized in Table 2. The secondary amide derivatives 2a and
N-alkyl/benzyl/phenethyl/naphthyl amide derivatives 2b–2f acti-
vated FXR in the order of 2e > 2c > 2d > 2a = 2b = 2f. These results
indicated that the size of the binding pocket of FXR hosting the
central portion of GW4064 derivatives might be sufficient to
accommodate a phenethyl or smaller group, being similar in this
respect to the corresponding site of BSEP.
Then, we turned our attention to the SAR of the carboxyl group
of the benzoic acid moiety. In the case of GW4064, a brief SAR re-
port from GSK researchers disclosed that a carboxyl group is essen-
tial and is preferably located at the 3-position (meta-position) for
(0.84–3.1 lM) than that of GW4064 (1, 0.07 lM). We previously
reported the SAR of GW4064 derivatives for BSEP function
enhancing activity, focusing on the stilbene moiety of GW4064,
particularly the two-atom moiety connecting the two aromatic
rings of the stilbene skeleton, which corresponds to the secondary
amide structure of 2a and tertiary amide structures of 2b–2f
[–N(R1)–C(O)– of 2a–f in Figure 1; designated as the central
portion of the molecule in this paper]. Among them, N-benzyl

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T. Misawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3962–3966
FXR-agonistic activity.10 We evaluated the FXR-agonistic activity of
regio-isomers, that is, the carboxyl group was shifted to the ortho-
(2g) or para-position (2h) from the meta-position, as well as the ef-
fect of esterification of the meta-carboxyl group (2i) (Table 2).
ortho-Isomer 2g and ester derivative 2i showed neither E297G
BSEP-function-promoting nor FXR-agonistic activity, whereas the
para-isomer (2h) showed these activities with similar potency to
the meta-isomer 2d. These results might indicate that the carboxyl
group is similarly recognized by the binding pockets of BSEP and FXR.
As expected, E297G BSEP function enhancers (2a–2f, 2h) also
showed FXR-agonistic activity (Table 2). As shown in Table 2, sub-
stitution of the double bond at the central portion of GW4064 (1)
to an amide bond, that is, compound 2d, had no effect on E297G
BSEP-function-promoting activity, but decreased the potency of
FXR-agonistic activity, that is, the EC50 values for GW4064 (1)
clear FXR-agonistic activity (EC50 values for FXR transcriptional
activation activity of 7d and 7e are >10 and 0.41 lM, respectively,
Table 3).
In summary, we confirmed that bile acids and 2a–2f exhibit
both FXR-agonistic activity and E297G BSEP-function-promoting
activity. Based on SAR considerations, we designed and synthe-
sized reversed-amide derivatives of previously reported GW4064
analogs 2a–2f and identified one of the compounds, 7c, as a selec-
tive BSEP function enhancer. Further SAR study and pharmacolog-
ical evaluation of representative compounds are in progress.
Comparison of the pharmacological effects elicited by a BSEP selec-
tive enhancer such as 7c and BSEP/FXR dual activators such as 2d
should lead to a better understanding of the effect of FXR activa-
tion on PFIC2 treatment.
and 2d are 0.07
lM and 2.5
l
M, respectively. The above-men-
Acknowledgements
tioned SAR analysis of FXR-agonistic activity was based on the
measured EC50 values. However, the efficacy of FXR activation
should also be considered. It is well known that full agonists such
as GW4064 fully activate FXR (100% efficacy), whereas partial ago-
nists show lower levels of maximal transcriptional activation than
the full agonist. As mentioned above, the EC50 value of compound
2d is higher than that of GW4064 (1), and in addition, the efficacy
of FXR transcriptional activation activity of 2d is only 21% com-
pared to that of the full agonist GW4064 (Fig. 2). So, we speculated
that the N-substituted amide-type central portion of the molecule
might be the key to separating E297G BSEP-function-promoting
activity from FXR-agonistic activity. To further investigate the
SAR of the N-substituted amide-type central portion, we designed
and synthesized reversed-amide derivatives, 7a–e. The synthetic
route for the preparation of the reversed-amide derivatives is out-
lined in Scheme 1. Substituted isoxazole 14 was prepared by the
method reported previously. 4-Amino-2-chlorophenol (8) was pro-
tected with a TBS group and condensed with monomethylisophth-
alate to afford amide 10. After deprotection of the hydroxyl group,
the resulting compound 11 was reacted with isoxazole derivative
14 to afford reversed-amide derivative 12, whose saponification
gave 7a. Finally, N-alkylation of 12 by saponification of the esters
13b–e yielded carboxylic acid derivatives 7b–7e.
The work described in this paper was partially supported by
Grant-in-aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Sciences and Technology, Japan, and the Japan
Society for the Promotion of Science. We thank Ms. Asami Hattori
for excellent technical support during the biological experiments.
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We evaluated the E297G BSEP-function-promoting activity and
FXR-agonistic activity of the prepared reversed-amide derivatives
7a–7e (Table 3 and Fig. 3). These compounds possessed moderate
to potent E297G BSEP-function-promoting activity. Among the re-
versed-amide derivatives, N-butyl derivative 7c possessed the
most potent activity (accumulation of [3H]TC was reduced to 42%
and 59% at 10 lM and 1 lM, respectively, Table 3 and Fig. 3).
Concerning FXR-agonistic activity, secondary reversed-amide
derivative 7a and N-alkyl reversed-amide derivatives 7b–7c
showed no activity over the concentration range examined (up to
10 lM), whereas N-benzyl/naphthyl derivatives (7d, 7e) showed