Nonacidic Chemotype Possessing N-Acylated Piperidine Moiety as Potent Farnesoid X Receptor (FXR) Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.7b00363

Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure-activity relationship (SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic 10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.

Full text of DOI:10.1021/acsmedchemlett.7b00363

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Nonacidic Chemotype Possessing N‑Acylated Piperidine Moiety as
Potent Farnesoid X Receptor (FXR) Antagonists
Naoki Teno,*,†,‡ Yukiko Yamashita,§ Yusuke Iguchi,§ Ko Fujimori,∥ Mizuho Une,†,§
Tomoko Nishimaki-Mogami,⊥ Takie Hiramoto,† and Keigo Gohda#
‡
§
†Graduate School of Pharmaceutical Sciences, Faculty of Clinical Nutrition, and Faculty of Pharmaceutical Sciences, Hiroshima
International University, 5-1-1 Hirokoshingai, Kure, Hiroshima 737-0112, Japan
∥Faculty of Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094,
Japan
⊥National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan
#Computer-aided Molecular Modeling Research Center, Kansai (CAMM-Kansai), 3-32-302 Tsuto, Otsuka, Nishinomiya 663-8241,
Japan
S
* Supporting Information
ABSTRACT: Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing
transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the
charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure−activity relationship
(SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic
10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with
our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.
KEYWORDS: FXR antagonists, benzimidazole scaffold, N-acylated piperidine, triglyceride accumulation
acidic function tend to be biased against poor selectivity.8,9
arnesoid X receptor (FXR) has been characterized as the
Recently, nonacidic FXR agonists have been reported.10
1−3
Fbile acid nuclear receptor.
It plays a pivotal role in
Nonsteroidal FXR antagonists (1, 2)11,12 are used for the study
regulating bile acids and lipid homeostasis by regulating the
in the treatment of hyperlipidemia (Figure 1). These studies are
based on the following reports: FXR promotes adipocyte
expression of several key genes involved in bile acid (BA)
synthesis, metabolism, and transport in the liver.4 Specific BAs
differentiation and controls adipose cell function.13−15 Addi-
bind and activate FXR, the most potent being chenodeoxycholic
tionally, naturally occurring FXR antagonist, guggulsterone,
acid (CDCA), which is an endogenous ligand of FXR and the
inhibits adipocyte differentiation and induces apoptosis in 3T3-
primary BA found in human bile.1 CDCA transcriptionally
L1 cell.16 Other nonsteroidal FXR antagonists (3, 4) have also
been published (Figure 1).17,18 Some antagonists contain an
represses the expression of cholesterol 7α-hydroxylase
(CYP7A1), the rate-limiting enzyme during bile acid synthesis,
acidic moiety as part of the pharmacophore. The acidic
by inducing that of small heterodimer partner (SHP) in liver.5
components have a tendency for the promiscuous interaction
FXR induces bile acid transporter, bile salt export pump (BSEP),
which transports bile acid from hepatocytes to bile canaliculi and
induces organic solute transporter α(OSTα).6
with off-targets and are susceptible to the conversion to acyl-
Received: September 3, 2017
Accepted: December 27, 2017
GW4064,7 which is used as a reference FXR agonist, had been
published in 2000 (Figure 1). GW4064 and its derivatives with an
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.7b00363
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ACS Medicinal Chemistry Letters
Letter
Table 1. FXR Antagonistic Activity and Cytotoxicity of 6−11
Figure 1. Structures of GW4064 and representative nonsteroidal FXT
antagonists.
glucuronides or acyl-CoA thioesters.19,20 Nonacidic antagonists
would avoid unexpected toxicity and metabolites.
Early in the research process, a novel chemotype 5 with the
acidic moiety as a hit compound was found to show moderate
antagonistic activity against FXR21 (Figure 2). As a hit-to-lead
*
The IC50 values are presented as the means
SD of three
**
independent experiments. Reference 21.
Figure 2. Hit and lead compounds (5 and 6), and regions for
optimization.
Table 2. FXR Antagonistic Activity and Cytotoxicity of 12−17
approach,21 a chemical modification was initiated based on the
structure of 5 to increase its antagonistic potency against FXR.
Eventually, the potency of nonacidic analogue 6 was not
substantially different from that of 5 in FXR time-resolved
fluorescence resonance energy transfer (TR-FRET) binding
assay. It was, however, found that the moderate potency of 6 in
the luciferase reporter gene assay could be obtained in the
absence of the acidic moiety of our new chemotype. The primary
structure−activity relationship (SAR) data enable further
insights: (1) the IC50 values increased with the enhancement
of the hydrophobicity in region A; and (2) replacing the
carboxylic acid of 5 with nonacidic groups in region B had an
impact on FXR antagonism and will be a clue to development of
nonacidic FXR antagonists. Thus, we developed a potent
nonacidic FXR antagonist by focusing on regions A and B in
the structure of 6 as the lead pharmacophore and also evaluated
triglyceride accumulation in 3T3-L1 adipocytes.
The designed compounds listed in Tables 1 and 2 were
synthesized via the procedures shown in Schemes S1−S4
(Supporting Information (SI)). The structure of the prepared
compounds was confirmed by spectroscopic and analytical
techniques. The potent FXR antagonist, T3 (Takeda Pharma-
ceutical Co.),22 used as a reference compound (1), was also
prepared from commercially available starting materials.
1
Characterization of 1 by H NMR, 13C NMR, and HR-MS is
available in the SI.
The first step in tackling derivatization of region A in 6 was
carried out by replacing the cyclohexyl component with N-
substituted piperidine while keeping the methyl group in region
B (R1 = CH3 and R2 = H) (Figure 2). The piperidine derivative,
which can be relatively and easily synthesized from commercial
pyridine derivative, is appropriate for the investigation of
structural diversity of region A. The newly synthesized analogues
were evaluated by a FXR TR-FRET binding assay and a luciferase
reporter assay (Table 1). Additionally, the antagonistic rate of the
*
The IC50 values are presented as the means
independent experiments.
SD of three
analogues is shown in Table S1 (SI). The modification on the
nitrogen atom in piperidine made a profound difference in the
antagonism for FXR.
Positive ionizable functions in the piperidine ring of 7 and 8
revealed an increase in the binding activity to FXR compared to
B
DOI: 10.1021/acsmedchemlett.7b00363
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ACS Medicinal Chemistry Letters
Letter
6. The piperidine of 7 was acylated to evaluate electrically neutral
analogues. This campaign led to the discovery of key analogues
9−11, which exhibited significant improvement in the binding
activity. The N-acylated analogue 9, for instance, appeared to
bind nearly 12-fold greater than 8. In addition, replacing the
acetyl group of 8 with bulky acyl groups (10 and 11) showed the
binding activity approximately 40-times higher than that of 9 in
the binding assay (0.035
0.002 and 0.03
0.004 μM,
respectively), differing from those of 2 and 3 (IC50: 8.96 3.62
μM12 and 7.5 nM,17 respectively). Analogue 10 dose-depend-
ently competed against the binding of GW4064 to FXR (Figure
S1 in SI). However, the potency of 10 in the binding assay is
marginally weaker than that of 1, which is the potent FXR
antagonist.11 In the luciferase assay, the acylated compounds (9−
11) exhibited inhibition against FXR; in particular, 10 and 11
displayed potent FXR antagonism (<0.001 and 0.006 0.0008
nM, respectively). Analogue 10 had substantial changes (more
than a 1000-fold increase) in its antagonist activity compared to
nonacylated piperidines 7 and 8. It turns out that the bulky acyl
moiety (10 and 11) in region A is a key structural element and
contributes substantially to potency.
Figure 3. FXR model complexed with 6 and 10. (A) Complex model of
hFXRα-LBD homodimer (PDB ID: 4OIV, blue wires and green
ribbons) with 10 (yellow) and 6 (pink) was built using AutoDock
Vina1.1.2. (B) Hydrogen bond between the carbonyl group on
piperidine of 10 and His298 (yellow dashed line).
To pursue nonacidic substituents other than CH3 (R1 = CH3,
R2 = H) on benzimidazole, we successively modified the
substituents on benzimidazole (region B) by keeping an acetyl or
isobutyryl moiety and by anticipating a synergistic effect through
a combination of regions A and B (Table 2). In the binding
assays, removal of methyl group (12) enhanced inhibitory
activity against FXR relative to 9, in contrast to analogues bearing
fluoride (13) or chloride (14) substituents on benzimidazole,
which were less active than 9. The same observations were made
with 12−14 in the luciferase assay, showing that both
substitutions (13 and 14) are less favorable in region B. The
two series (9, 12, 13, 14 and 10, 15, 16, 17) shows almost the
same SAR in both assays. For instance, the IC50 values of 13 and
14 in the binding assay are reduced about <10 and 2−3 times as
compared with that of parent compound 9, respectively, and
these trends are shared with 16 and 17 versus 10. However, only
the IC50 value of 12 in the reporter gene assay (0.002 0.003
nM) largely deviates from the SAR. In the combination of
regions A and B reported here, the substituent pattern (R1−R3)
of 9, 10, and 15 facilitated the favorable interaction with FXR.
Notably, robust potency was observed for the substituent pattern
of 15, being nearly equipotent with 10.
To clarify the effects exerted by N-acyl piperidine of 10 toward
FXR, we ran a molecular docking simulation (AutoDock Vina
1.1.2)23 with 10. Our computer-assisted modeling studies
suggested that the binding mode of 10 (yellow) complexed
with LBD of FXR was shown to be similar to that of 6 (pink)
other than around N-acyl piperidine as depicted in Figure 3A.
The obvious difference is that some leeway exists for the
isobutyryl group of 10 to interact. Additionally, modeling studies
predicted that the carbonyl group of isobutyryl in 10 is able to
establish a hydrogen bond with His298 (Figure 3B). The
interaction of an N-acyl group is viewed to affect the antagonism
for FXR. Indeed, comparison of the IC50 values obtained for N-
acylated piperidines 10 or 15 with those for 8 (absence of N-acyl
group) clearly demonstrated that the N-acylated piperidine ring
is a key structural element and contributes to potency. In terms of
the SAR of region B, the results obtained with 13, 14, 16, and 17
reveal that the hydrogen atom and methyl group in region B
cannot be successfully replaced by fluoride or chloride. This
insight into region B was not obtained by modeling studies. The
structures of 10 and 15 are thus combined with the synergism
exerted by the appropriate moieties in two distinct regions.
Eventually, both of the analogues inhibited FXR at low
nanomolar IC50 values in the binding assay and revealed robust
antagonism against FXR.
Large difference in the IC50 values of 10 and 15 reported for
the binding and reporter assays was observed. This would be due
to the proteins used. FXR is used for the luciferase assay; in
contrast, a partial sequence of FXR (ligand binding domain,
LBD) is employed for the binding assay. It is obvious that the
three-dimensional structures of FXR and FXR-LBD would not
be identical; however, a clear SAR on the analogues with N-acyl
group was distinctly observed in each assay and the changes in
the IC50 values in both assays are nearly identical. In contrast,
since 6, 7, and 8 have no N-acyl group, there will be a large
difference in binding to both proteins with the three-dimensional
structures.
Tetrazolium (MTT) colorimetric assays are the most common
employed for the detection of cytotoxicity or cell viability
following exposure to toxic substances,24 and the cytotoxic
activities of 6 and 7−17 were thus evaluated (Tables 1 and 2).
Analogues 10 and 15, which differ from 9 and 12 only by
substituents on the nitrogen atom in piperidine, respectively,
exhibited no cytotoxicity. Relatively strong antagonism in the
luciferase reporter gene assay given by 12 was anticipated to be
overestimated by cytotoxicity. The results obtained in the MTT
assay suggested that cytotoxicity tends to be partially affected by
varying in the substituents on piperidine.
To gain insights into the antagonism of 10, we then
investigated the downstream genes of FXR by real-time reverse
transcription PCR (real-time RT-PCR). The cells cultured in six-
well plates were treated with different concentrations of 10 in the
presence or absence of endogenous agonist, CDCA (Figure
4A,C,E) and synthetic agonist, GW4064 (Figure 4B,D,F). After
24 h, cells were collected. CDCA and GW4064 induced the
expression of BSEP, SHP, and OSTα.5,6 Interestingly, 10
equipotently suppressed the effect of CDCA; namely, by
decreasing the expression levels of three target genes at a low
dose (0.02 μM). Similar to 10, the 1,3,4-trisubstituted-
pyrazolone derivative (2) inhibited the activation of promoters
related to FXR activated by CDCA.12 The effect of GW4064
C
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ACS Medicinal Chemistry Letters
Letter
had neither agonist nor antagonist properties for RXRα, PPAR α,
δ, γ, LXR α, β, VDR, RARα, PXR, and TGR5.
Mouse 3T3-L1 adipocytes have been extensively employed to
examine the cellular and molecular mechanism of adipocyte
differentiation.30 FXR expression was induced during adipocyte
differentiation in them in order to investigate the antiadipogenic
effect of 10 on 3T3-L1 adipocytes. Prior to adipocyte
differentiation, we investigated cell toxicity of 10 in 3T3-L1
cells. Cells were cultured for 6 days in Dulbecco’s modified
Eagle’s medium (DMEM) containing various concentrations of
10 (0−10 μM); no significant toxic effects were observed up to
10 μM in 3T3-L1 cells (Figure 5A).
Figure 4. mRNA expression of CDCA- or GW4064-modulated FXR
target genes was antagonized by 10. Huh-7 cells were treated with
different concentrations of 10 in the presence of 10 μM CDCA (A,C,E)
or 1 μM GW4064 (B,D,F). Data are presented as means SD from n =
3 experiments (*p < 0.01 vs CDCA or GW4064 only). N.C.: Negative
control.
Figure 5. Suppression of lipid accumulation by 10 in adipocytes. (A)
Cell toxicity of 10 on 3T3-L1 adipose cells. The cells were incubated for
6 days in DMEM with various concentrations of 10 (0−10 μM), and cell
toxicity was measured. Data represent means SD (n = 3 independent
experiments). (B) Staining of intracellular lipids by Oil Red O in 3T3-L1
cells. Cells (undifferentiated cells: U) were differentiated into adipocytes
[D] for 6 days in DMEM with 10 (0−5 μM) or guggulsterone (Gu: 10
μM) together without (upper) or with CDCA (5 μM; lower).
Intracellular lipid droplets were stained with Oil Red O. Data are
representative of n = 3 repetitions. (C) Decrease of the intracellular
triglyceride level by 10 in 3T3-L1 cells. The cells (undifferentiated cells:
U; white column) were differentiated [D] into adipocytes for 6 days in
DMEM without (gray column) or with 10 (0.5, 1, or 5 μM; black
columns), or guggulsterone (Gu: 10 μM; purple columns) together
without or with CDCA (5 μM; blue column). Data are presented as
means SD from n = 3 experiments. *p < 0.01 (vs undifferentiated
cells), #p < 0.01 (vs vehicle-treated differentiated cells), and +p < 0.01 (vs
CDCA-treated differentiated cells).
tended to be inhibited dose-dependently by 10. Altogether, these
data indicate the effect of 10 on downstream targeted gene
expressions through FXR inactivation.
CDCA-induced FXR activity was prevented by 10 in the cell-
based luciferase assay (Table 1). Likewise, 10 inhibited the
synthetic agonist GW4064-stimulated FXR activity (Figure
S2A). We next attempted to see whether 10 interacts with
eight other nuclear receptors including retinoid X receptor α
(RXRα) (Figure S2B), peroxisome proliferator-activated
receptor α, γ, and δ (PPAR α, γ, and δ) (Figure S2C−E), liver
X receptor α and β (LXR α and β) (Figure S2F,G), vitamin D
receptor (VDR) (Figure S2H), and retinoic acid receptor α
(RARα) (Figure S2I) according to published methods.21,25,26
Analogue 10 (1 μM) had no agonistic (alone) or antagonistic
(plus the corresponding agonists) effect toward any of these
receptors. Compound 2 reported previously had no effect on six
nuclear receptors other than FXR.12 Furthermore, we showed no
effect of 10 on PXR target gene, CYP3A4 (Figure S3).
TGR5 receptor is the first known G-protein-coupled receptors
specific for deoxycholic acid and lithocholic acid (LCA) but not
for CDCA.27 It has been reported that dual activation of FXR and
TGR5 has a high profile in nonalcoholic fatty liver disease
(NAFLD) and atherosclerosis.28 The combined FXR antago-
nist/TGR5 agonist compounds derived from FXR modulators
have been also developed.29 In this study, no cross-reactivity
between 10 and TGR5 was observed (Figure S2J). The results in
a panel of the receptors revealed that the nonacidic analogue 10
Subsequently, the effect of 10 on adipogenesis in 3T3-L1 cells
was examined. Cells were differentiated into adipocytes for 6
days in DMEM with various concentrations of 10 (0−5 μM) or
guggulsterone (Gu: 10 μM) (top pictures, Figure 5B). The
number of lipid droplets in the cells was increased during
adipogenesis. Results from Oil Red O staining indicated that
exposure to 10 (0−5 μM) dose-dependently reduced the
number of lipid droplets (lower pictures, Figure 5B).
Guggulsterone also decreased the number of cells. In addition,
the cells were differentiated for 6 days in DMEM containing
CDCA with or without 10 (0−5 μM). The number of lipid
droplets in the CDCA-treated differentiated cells increased
greater than in the vehicle-treated differentiated cells. Intra-
D
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cellular lipids were decreased by the cotreatment with 10 in a
dose-dependent manner or with guggulsterone.
REFERENCES
■
(1) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R. M.;
Luk, A.; Hull, M. V.; Lustig, K. D.; Mangelsdorf, D. J.; Shan, B.
Identification of a nuclear receptor for bile acids. Science 1999, 284,
1362−1365.
(2) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.; Consler,
T. G.; Kliewer, S. A.; Stimmel, J. B.; Willson, T. M.; Zavacki, A. M.;
Moore, D. D.; Lehmann, J. M. Bile acids: Natural ligands for an orphan
nuclear receptor. Science 1999, 284, 1365−1368.
To further confirm the decrease of the lipids in adipocytes by
the treatment with 10, we measured the intracellular triglyceride
levels (Figure 5C). The level in the differentiated cells increased
about 5.9-fold in comparison to the undifferentiated cells.
Analogue 10 dose-dependently antagonized the effect of CDCA
on triglyceride accumulation, indicating that it decreased lipid
accumulation in 3T3-L1 cells.
(3) Wang, H.; Chen, J.; Hollister, K.; Sowers, K. C.; Forman, B. M.
Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.
Mol. Cell 1999, 3, 543−553.
Lead optimization efforts for our FXR antagonist series began
with SAR exploration starting from the previously reported
benzimidazole analogue 6. Replacing the cyclohexyl group of 6
with N-acylated piperidine in region A resulted in the discovery
of a key inhibitor, 10, which possessed robust potency as a FXR
antagonist. Concurrent with these investigations, the SAR
exploration of substituents on benzimidazole of 6 was conducted,
which led to the discovery of 10 and 15; both were potent in the
binding and cell assays. The rationale might involve the carbonyl
of the isobutyryl group of piperidine 10 participating in hydrogen
bond interaction with His298 of FXR-LBD (Figure 3), and the
isopropyl moiety of isobutyryl group might affect the putative
hydrophobic interaction with LBD (9 vs 10 and 11, Table 1).
Furthermore, 10 reversed the regulation of CDCA and GW4064
on FXR target gene expressions (BSEP, SHP, and OSTα). The
nonacidic analogue also had greater affinity toward FXR over
TGR5 and other nuclear receptors. Moreover, analogue 10
inhibits differentiation of preadipocytes and reduces intracellular
triglyceride content in 3T3-L1 adipocytes. We envision that the
N-acylated piperidine moiety holds great potential for structural
modifications and on the design of nonacidic FXR antagonists.
Since the robust affinity to FXR is feasible with our nonacidic
analogue, additional studies on hyperlipidemia relevant in vivo
biological activities are in progress, and the results will be
published in due course.
(4) Sinal, C. J.; Tohkin, M.; Miyata, M.; Ward, J. M.; Lambert, G.;
Gonzalez, F. J. Targeted disruption of the nuclear receptor FXR/BAR
impairs bile acid and lipid homeostasis. Cell 2000, 102, 731−744.
(5) Goodwin, B.; Jones, S. A.; Price, R. R.; Watson, M. A.; McKee, D.
D.; Moore, L. B.; Galardi, D.; Wilson, J. G.; Lewis, M. C.; Roth, M. E.;
Maloney, P. R.; Willson, T. M.; Kliewer, S. A. A regulatory cascade of the
nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid
biosynthesis. Mol. Cell 2000, 6, 517−526.
(6) Dawson, P. A.; Lan, T.; Rao, A. Bile acid transporters. J. Lipid Res.
2009, 50, 2340−2357.
(7) Maloney, P. R.; Parks, D. J.; Haffner, C. D.; Fivush, A. M.; Chandra,
G.; Plunket, K. D.; Creech, K. L.; Moore, L. B.; Wilson, J. G.; Lewis, M.
C.; Jones, S. A.; Willson, T. M. Identification of a chemical tool for the
orphan nuclear receptor FXR. J. Med. Chem. 2000, 43, 2971−2974.
(8) Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.;
Chen, L.; Creech, K. L.; Deaton, D. N.; Jones, S. A.; Kaldor, I.; Liu, Y.;
Madauss, K. P.; Marr, H. B.; McFadyen, R. B.; Miller, A. B.; Navas, F., III;
Parks, D. J.; Spearing, P. K.; Todd, D.; Williams, S. P.; Wisely, G. B.
Conformationally constrained farnesoid X receptor (FXR) agonists:
Naphthoic acid-based analogs of GW 4064. Bioorg. Med. Chem. Lett.
2008, 18, 4339−4343.
(9) Singh, N.; Yadav, M.; Singh, A. K.; Kumar, H.; Dwivedi, S. K. D.;
Mishra, J. S.; Gurjar, A.; Manhas, A.; Chandra, S.; Yadav, P. N.; Jagavelu,
K.; Siddiqi, M. I.; Trivedi, A. K.; Chattopadhyay, N.; Sanyal, S. Synthetic
FXR Agonist GW4064 Is a Modulator of Multiple G Protein−Coupled
Receptors. Mol. Endocrinol. 2014, 28, 659−673.
(10) Flesch, D.; Cheung, S.-Y.; Schmidt, J.; Gabler, M.; Heitel, P.;
Kramer, J.; Kaiser, A.; Hartmann, M.; Lindner, M.; Luddens-Damgen,
K.; Heering, J.; Lamers, C.; Luddens, H.; Wurglics, M.; Proschak, E.;
Schubert-Zsilavecz, M.; Merk, D. Nonacidic Farnesoid X Receptor
ASSOCIATED CONTENT
■
̈
̈
S
* Supporting Information
̈
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00363.
Modulators. J. Med. Chem. 2017, 60, 7199−7205.
(11) Amano, Y.; Shimada, M.; Miura, S.; Adachi, R.; Tozawa, R. Effects
of a farnesoid X receptor antagonist on hepatic lipid metabolism in
primates. Eur. J. Pharmacol. 2014, 723, 108−115.
Dose-dependent manner of 10 in TR-FRET and the
affinity between 10 and other nuclear receptors, TGR5;
synthesis and characterization data for 7−17; antagonistic
rate of 6−17; analytical data of 1 and 6, purity
determination (1, 6−17), and biological protocols
(PDF)
(12) Huang, H.; Yu, Y.; Gao, Z.; Zhang, Y.; Li, C.; Xu, X.; Jin, H.; Yan,
W.; Ma, R.; Zhu, J.; Shen, X.; Jiang, H.; Chen, L.; Li, J. Discovery and
optimization of 1,3,4-trisubstituted-pyrazolone derivatives as novel,
potent, and nonsteroidal farnesoid X receptor (FXR) selective
antagonists. J. Med. Chem. 2012, 55, 7037−7053.
(13) Rizzo, G.; Disante, M.; Mencarelli, A.; Renga, B.; Gioiello, A.;
Pellicciari, R.; Fiorucci, S. The farnesoid X receptor promotes adipocyte
differentiation and regulates adipose cell function in vivo. Mol.
Pharmacol. 2006, 70, 1164−1173.
(14) Fiorucci, S.; Rizzo, G.; Antonelli, E.; Renga, B.; Mencarelli, A.;
Riccardi, L.; Morelli, A.; Pruzanski, M.; Pellicciari, R. Cross-Talk
between farnesoid-X-receptor (FXR) and peroxisome proliferator-
activated receptor contributes to the antifibrotic activity of FXR ligands
in rodent models of liver cirrhosis. J. Pharmacol. Exp. Ther. 2005, 315,
58−68.
(15) Feng, S.; Reuss, L.; Wang, Y. Potential of natural products in the
inhibition of adipogenesis through regulation of PPARγ expression and/
or its transcriptional activity. Molecules 2016, 21, 1278.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81 823 73 8942. E-mail: n-teno@ps.hirokoku-u.ac.jp.
ORCID
Naoki Teno: 0000-0002-3940-5473
Ko Fujimori: 0000-0002-2506-0769
Notes
The authors declare no competing financial interest.
(16) Yang, J.-Y.; Della-Fera, M. A.; Baile, C. A. Guggulsterone inhibits
adipocyte differentiation and induces apoptosis in 3T3-L1 cells. Obesity
2008, 16, 16−22.
(17) Xu, Y. Recent progress on bile acid receptor modulators for
treatment of metabolic diseases. J. Med. Chem. 2016, 59, 6553−6579.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Lawrence H. Lazarus for critically reading
the manuscript. We thank Professor Hidetoshi Tahara for data
collection.
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Letter
(18) Yu, D. D.; Lin, W.; Forman, B. M.; Chen, T. Identification of
trisubstituted-pyrazol carboxamide analogs as novel and potent
antagonists of farnesoid X receptor. Bioorg. Med. Chem. 2014, 22,
2919−2938.
(19) Metabolism, Pharmacokinetics and Toxicity of Functional Groups:
Impact of Chemical Building Blocks on ADMET; Smith, D. A., Ed.; RSC
Drug Discovery; Royal Society of Chemistry: Cambridge, U.K., 2010.
(20) Skonberg, C.; Olsen, J.; Grimstrup Madsen, K.; Hansen, S. H.;
Grillo, M. P. Metabolic activation of carboxylic acids. Expert Opin. Drug
Metab. Toxicol. 2008, 4, 425−438.
(21) Teno, N.; Iguchi, Y.; Yamashita, Y.; Mori, N.; Une, M.; Nishimaki-
Mogami, T.; Gohda, K. Discovery and optimization of benzimidazole
derivatives as a novel chemotype of farnesoid X receptor (FXR)
antagonists. Bioorg. Med. Chem. 2017, 25, 1787−1794.
(22) Kitamura, S.; Hosono, H.; Miura, S.; Aoki, K. Preparation of azole
compounds as FXR inhibitors. Jpn. Kokai Tokkyo Koho (2008), JP
2008273847, 13 Nov 2008.
(23) Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient optimization
and multithreading. J. Comput. Chem. 2010, 31, 455−461.
(24) David, T. V.; Philip, S.; Dominic, S.; Anne, M.; Angela, P.;
Michael, R. B. Tetrazolium-based assays for cellular viability: a critical
examination of selected parameters affecting formazan production.
Cancer Res. 1991, 51, 2515−2520.
(25) Tamehiro, N.; Sato, Y.; Suzuki, T.; Hashimoto, T.; Asakawa, Y.;
Yokoyama, S.; Kawanishi, S.; Ohno, Y.; Inoue, K.; Nagao, T.; Nishimaki-
Mogami, T. Riccardin C: A natural product that functions as a liver X
receptor (LXR)α agonist and an LXRβ antagonist. FEBS Lett. 2005, 579,
5299−5304.
(26) Iguchi, Y.; Yamaguchi, M.; Sato, H.; Kihira, K.; Nishimaki-
Mogami, T.; Une, M. Bile alcohols function as the ligands of membrane-
type bile acid-activated G protein-coupled receptor. J. Lipid Res. 2010,
51, 1432−1441.
(27) Thomas, C.; Gioiello, A.; Noriega, L.; Strehle, A.; Oury, J.; Rizzo,
G.; Macchiarulo, A.; Yamamoto, H.; Mataki, C.; Pruzanski, M.;
Pellicciari, R.; Auwerx, J.; Schoonjans, K. TGR5-mediated bile acid
sensing controls glucose homeostasis. Cell Metab. 2009, 10, 167−177.
(28) Jadhav, K.; Xu, Y.; Zhang, Y. Dual activation of bile acid receptors
FXR and TGR5 plays a protective role in non-alcoholic fatty liver disease
and atherosclerosis. FASEB J. 2016, 30, 870−874.
(29) Lamers, C.; Merk, D.; Gabler, M.; Flesch, D.; Kaiser, A.; Schubert-
Zsilavecz, M. SAR studies on FXR modulators led to the discovery of the
first combined FXR antagonistic/TGR5 agonistic compound. Future
Med. Chem. 2016, 8, 133−148.
(30) Cariou, B.; van Harmelen, K.; Duran-Sandoval, D.; van Dijk, T.
H.; Grefhorst, A.; Abdelkarim, M.; Caron, S.; Torpier, G.; Fruchart, J. C.;
Gonzalez, F. J.; Kuipers, F.; Staels, B. The farnesoid X receptor
modulates adiposity and peripheral insulin sensitivity in mice. J. Biol.
Chem. 2006, 281, 11039−11049.
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DOI: 10.1021/acsmedchemlett.7b00363
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