Design, synthesis and metabolic regulation effect of farnesoid X receptor (FXR) antagonistic benzoxepin-5-ones

Article abstract of DOI:10.1016/j.cclet.2017.04.034

A series of benzoxepin-5-ones were designed and synthesized by the cyclization of chalcones which were previously found as FXR antagonists. The cellular FXR antagonism of benzoxepines was investigated, among which the most potent compound 10l was able to reduce the plasma and hepatic triglyceride and plasma ALT levels in mice.

Full text of DOI:10.1016/j.cclet.2017.04.034

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Chinese Chemical Letters
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Original article
Design, synthesis and metabolic regulation effect of farnesoid X
receptor (FXR) antagonistic benzoxepin-5-ones
Guo-Ning Zhanga,b,1, Yi Huana,1, Xing Wanga, Su-Juan Suna, Zhu-Fang Shena,
Wei-Shuo Fanga,
*
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China
b
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Peking Union Medical College, Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of benzoxepin-5-ones were designed and synthesized by the cyclization of chalcones which were
previously found as FXR antagonists. The cellular FXR antagonism of benzoxepines was investigated,
among which the most potent compound 10l was able to reduce the plasma and hepatic triglyceride and
plasma ALT levels in mice.
Received 6 March 2017
Received in revised form 10 April 2017
Accepted 28 April 2017
Available online xxx
© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Farnesoid X receptor
Antagonist
Benzoxepin-5-one
Triglyceride
Plasma ALT
1. Introduction
Extensive studies on the discovery of FXR ligands in the past 1–
2 decades, since the deorphanization of FXR, has resulted in the
Farnesoid X receptor (FXR) [1,2], belonging to the nuclear
receptor superfamily, has emerged as a key player in the control of
multiple metabolic pathways. It is rich in liver and some extra-
hepatic tissues such as the kidney and intestine, which were
constantly exposed to high concentration of bile acids, the
physiological ligands of FXR. It is also highly expressed other
cholesterol-rich tissues such as adrenal glands [2,3].
report of some steroidal- (natural ligand like) and non-steroidal
FXR ligands [9], among which three compounds (obeticholic acid,
Px-102 and LJN-452) entered clinic trials. In May 2016, obeticholic
acid was approved by FDA for the treatment of primary biliary
cholangitis (PBC) in combination with ursodeoxycholic acid
(UDCA) in adults with an inadequate response to UDCA, or as a
single therapy in adults unable to tolerate UDCA, validating the
utility of FXR interacting agents in human [10].
FXR binds to cis-acting elements in the promoters of series
target genes, such as cholesterol 7
a
-hydroxylase (Cyp7A1), bile
Serious side effects were encountered during the application of
FXR full agonists to animals and patients with diabetes and liver
steatosis, such as, inhibition of bile acid synthesis and increased
levels of low-density lipoprotein (LDL) [11,12]. So FXR partial
agonists and antagonists have been actively pursued as alter-
natives to full agonists, although their number and structural
diversity are still limited.
Most of reported FXR antagonists to date are still steroidal
molecules, including the first known antagonist guggulsterone
(Fig. 1) [13,14]. There are also a few synthetic non-steroidal
scaffolds reported to date (Fig. 2), i.e., substituted-isoxazole
derivatives (2a–2b) [15], 1, 3, 4-trisubstituted-pyrazolone (3)
[16], T3 (4) [17], NDB (5) [18], hydroxyacetophenone derivatives
(6a–6c) [19] and 1,3-disubstituted-pyrazole-3-carboxamide (7a–
7c) [20]. In addition to guggulsterone, a family of sesterterpene
salt export pump (BSEP) and Na+-dependent taurocholate
cotransporting polypeptide (NTCP), and modulate their expres-
sions in response to bile acid metabolites [4]. Apart from its crucial
role in maintaining bile acids and cholesterol homeostasis, FXR
also regulates the lipid and glucose metabolism [5]. Previous
studies demonstrated the regulation of FXR benefits to various
diseases such as metabolic disorders, hepatitis, arteriosclerosis and
cancer [6–8]. Thus, FXR has become an attractive therapeutic
target.
* Corresponding author.
E-mail addresses: wfang@imm.ac.cn, weishuo_fang@yahoo.com (W.-S. Fang).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.cclet.2017.04.034
1001-8417/© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: G.-N. Zhang, et al., Design, synthesis and metabolic regulation effect of farnesoid X receptor (FXR)
antagonistic benzoxepin-5-ones, Chin. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.04.034

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Fig. 1. Structures of the first steroidal FXR antagonist gugglesterone (1) and representative non-steroidal FXR antagonists reported in literatures.
Fig. 2. Generation of 1-benzoxepin-5-one from chalcone based FXR antagonists.
suvanine, were also the rare examples for natural products derived
antagonists [21].
the presence of chloromethoxymethane and Hünig’ base [23].
Subsequently, the other phenol group in the mono-protected
compounds 12a–12d was treated with 1,2-dibromoethane and
then cyclized to afford the corresponding benzoxepines 14a–14d.
In the ether formation step, 12a–12d were only partially converted
to 13a–13d in the presence of potassium carbonate as the base,
even at high temperature (110 ꢁC) and prolonged time (20 h). Then
a more soluble and stronger base cesium carbonate was used
instead, and 13a–13d obtained in good to excellent yield (>95%) at
100 mg scale. However, when the reaction was scaled up to 1 g,
only about 30% convertion was observed and byproducts (phenyl-
ethynyl ether and others unidentified) began to appear with
prolonged reaction time, when a magnetic stirring bar was used.
When this reaction was conducted with mechanical stirring
(possibly better mixing), 13a–13d was afforded in good yield at
gram-scale (1g–10 g). Finally, the condensation of benzoxepines
14a–14d with different benzaldehydes in the presence of catalytic
amount of piperidine in solvent-free conditions and subsequent
deprotection under acidic conditions in one-pot afforded corre-
sponding benzoxepin-5-ones 10a–10l in good yields. The spectral
data of most potent compound 10l was shown below.
In the pursuit of new chemotype of FXR interacting agents, we
have discovered chalcones 8a and 8b as moderate FXR antagonists
(IC50 at 10ꢀ5 mol/L), by the screening of a nature product like
library containing 9a–9l. However, those cellular active chalcones
were not active in animal assays, possibly due to their instability in
vivo. Then we tried to optimize their PD and PK properties by the
conformational restriction of chalcones and found benzopyrenes
as FXR antagonists both in vitro and in vivo whereas flavones not
[22]. This suggested the right conformation for chalcones to
interact with FXR is the more extended, low energy comformation
B (Fig. 2). Here we reported an attempt to retain or increase the FXR
interaction by cyclizing the different ring from that in benzopyrene
formation, but still retain the right conformation of chalcones,
leading to the discovery of the benzoxepin-5-one based FXR
antagonists.
2. Results and discussion
2.1. Chemistry
6-Hydroxy-4-(4-methoxybenzylidene)-3,4-dihydrobenzo[b]
As shown in Scheme 1, one phenol in the different substituted
oxepin-5(2H)-one (10l): 1H NMR (CDCl3, 500 MHz):
d
12.13 (s, 1H,
dihydroxy-acetophenones 11a–11d were selectively protected in
OH), 7.55 (s, 1H, COꢀꢀC¼CH), 7.43 (d, 2H, J = 8.5 Hz, H-2', H-6'), 7.36
Please cite this article in press as: G.-N. Zhang, et al., Design, synthesis and metabolic regulation effect of farnesoid X receptor (FXR)
antagonistic benzoxepin-5-ones, Chin. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.04.034

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3
Scheme 1. Synthesis of compounds 10a–10l. Reagents and conditions: a) MOMCl, DIEA, DCM, RT. b) K2CO3 or Cs2CO3, DMF, 80 ꢁC. c).NaH, THF, 80 ꢁC. d) benzaldehyde,
piperidine (cat.). e) HCl (3 mol/L)/MeOH = 5:2 (v/v), reflux.
(CH3, O-CH3), 29.2 (CH2, CH2-CH2-C); ESI-MS: [M+H]+: 297.1; HR-
MS: C18H17O4+: m/z 297.1129 (observed), 297.1121 (calculated).
Experimental procedure for the synthesis of all 1-benzoxepin-
5-one and their spectral data, were shown in Supplementary
material.
2.2. Biological activity
The cellular activities of compounds 10a–10l were screened by
using a mammalian one-hybrid FXR coactivator association assay.
Encouragingly, three compounds (10j–10l) showed moderate to
high antagonistic activities at 10ꢀ5 mol/L against the FXR activation
induced by the same concentration of CDCA (Fig. 3). It was found
that the 6-hydroxyl group in benzoxepin-5-one (10j–10l) is
essential for its FXR antagonistic activity and a 4'-methoxyl group
favourable for higher activity (10j–10l vs. 10g–10i).
Fig. 3. The FXR antagonistic activity of compounds 10a–10l. The luciferase activity
of HEK293T cells treated by 10ꢀ5 mol/L of CDCA was set as 100%. The antagonistic
activity of all compounds was tested at a concertation of 10ꢀ5 mol/L.
The benzoxepin-5-one 10l, the most potent FXR antagonists
among this series of compounds, was chosen to evaluate its
metabolic regulation activity in vivo. It was administrated orally
(200 mg/kg, qd) in diabetic KKay mice for 28 days. The plasma
triglyceride level in 10l treatment group decreased by 33.3% and
27.7% (P < 0.05, P < 0.05, Fig. 4A) after 16 and 28 days of treatment,
respectively. Hepatic triglyceride level was also decreased by 31.6%
(P < 0.05, Fig. 4B) after 28 days’ treatment. The increased level of
ALT (also named as GPT, glutamic pyruvic transaminase) and AST
(also named as GOT, glutamic oxaloacetic transaminase) in plasma
is usually associated with damaged liver. Compound 10l was found
(m,1H, H-8), 6.96 (d, 2H, J = 8.5 Hz, H-3', H-5'), 6.77 (d,1H, J = 8.5 Hz,
H-9), 6.60 (d, 1H, J = 8.0 Hz, H-7), 4.39 (t, 2H, J = 6.0 Hz, O-CH2-CH2),
3.85 (s, 3H, O-CH3), 3.02 (t, 2H, J = 6.0 Hz, C-CH2-CH2); 13C NMR
(CDCl3, 125 MHz): 199.4 (C, CO), 162.9 (C, C-9a), 160.2 (C, C-4'),
159.7 (C, C-6), 138.2 (CH, CO-C-CH), 136.6 (C, CO-C-CH),135.9 (C, C-
8), 131.5 (2C, C-2', C-60), 127.7 (C, C-1'), 115.3 (C, C-5a), 114.2 (2C, C-
3', C-5'), 113.1 (C, C-7), 112.2 (C, C-9), 71.5 (CH2, O-CH2-CH2), 55.3
Fig. 4. Effects of 10l (200 mg/kg) on triglyceride metabolism and plasma ALT and AST level in diabetic KKay mice after 28 days treatment. (A) The plasma triglyceride levels at
16 and 28 days treatment. (B) The hepatic triglyceride level and plasma ALT and AST level after 28 day of treatment. n = 10. In the control group (Con) water (0.05 ml/10 g) was
given by oral gavage every day for 28 days. *P < 0.05 compared with control.
Please cite this article in press as: G.-N. Zhang, et al., Design, synthesis and metabolic regulation effect of farnesoid X receptor (FXR)
antagonistic benzoxepin-5-ones, Chin. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.04.034

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to reduce the plasma ALT level by 23.1% (P < 0.05, Fig. 4B) and had
no effect on the plasma AST level after 28 days of treatment,
indicating this antagonist does not have hepatitis damage and even
could play a hepatic protection role. Besides, it also did not affect
the food and water intake and the body weight of mice.
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chalcones. The most potent antagonist 10l in cells significantly
reduce the triglyceride in plasma and hepatic and plasma ALT level
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Experimental procedure for the synthesis of 1-benzoxepin-5-
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Please cite this article in press as: G.-N. Zhang, et al., Design, synthesis and metabolic regulation effect of farnesoid X receptor (FXR)
antagonistic benzoxepin-5-ones, Chin. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.04.034