Structure-guided modification of isoxazole-type FXR agonists: Identification of a potent and orally bioavailable FXR modulator

Article abstract of DOI:10.1016/j.ejmech.2020.112910

Farnesoid X receptor (FXR) agonists are emerging as potential therapeutics for the treatment of various metabolic diseases, as they display multiple effects on bile acid, lipid, and glucose homeostasis. Although the steroidal obeticholic acid, a full FXR agonist, was recently approved, several side effects probably due to insufficient pharmacological selectivity impede its further clinical application. Activating FXR in a partial manner is therefore crucial in the development of novel FXR modulators. Our efforts focusing on isoxazole-type FXR agonists, common nonsteroidal agonists for FXR, led to the discovery a series of novel FXR agonists bearing aryl urea moieties through structural simplification of LJN452 (phase 2). Encouragingly, compound 11k was discovered as a potent FXR agonist which exhibited similar FXR agonism potency but lower maximum efficacy compared to full agonists GW4064 and LJN452 in cell-based FXR transactivation assay. Extensive in vitro evaluation further confirmed partial efficacy of 11k in cellular FXR-dependent gene modulation, and revealed its lipid-reducing activity. More importantly, orally administration of 11k in mice exhibited desirable pharmacokinetic characters resulting in promising in vivo FXR agonistic activity.

Full text of DOI:10.1016/j.ejmech.2020.112910

European Journal of Medicinal Chemistry 209 (2021) 112910
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Structure-guided modification of isoxazole-type FXR agonists:
Identification of a potent and orally bioavailable FXR modulator
,
b
,
,
b
,
,
Guoshun Luo a 1, Xin Lin a 1, Zhenbang Li c, Maoxu Xiao a b, Xinyu Li d,
Dayong Zhang c **, Hua Xiang a
,
, b, *
a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 210009,
China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
c Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, 210009, PR China
d School of Life and Health Sciences and Warshel Institute for Computational Biology, The Chinese University of Hong Kong, Shenzhen, Guangdong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Farnesoid X receptor (FXR) agonists are emerging as potential therapeutics for the treatment of various
metabolic diseases, as they display multiple effects on bile acid, lipid, and glucose homeostasis. Although
the steroidal obeticholic acid, a full FXR agonist, was recently approved, several side effects probably due
to insufficient pharmacological selectivity impede its further clinical application. Activating FXR in a
partial manner is therefore crucial in the development of novel FXR modulators. Our efforts focusing on
isoxazole-type FXR agonists, common nonsteroidal agonists for FXR, led to the discovery a series of novel
FXR agonists bearing aryl urea moieties through structural simplification of LJN452 (phase 2). Encour-
agingly, compound 11k was discovered as a potent FXR agonist which exhibited similar FXR agonism
potency but lower maximum efficacy compared to full agonists GW4064 and LJN452 in cell-based FXR
transactivation assay. Extensive in vitro evaluation further confirmed partial efficacy of 11k in cellular
FXR-dependent gene modulation, and revealed its lipid-reducing activity. More importantly, orally
administration of 11k in mice exhibited desirable pharmacokinetic characters resulting in promising
in vivo FXR agonistic activity.
Received 6 July 2020
Received in revised form
3 October 2020
Accepted 3 October 2020
Available online 7 October 2020
Keywords:
Farnesoid X receptor (FXR)
Isoxazole
Partial agonists
Oral bioavailability
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
secretion and absorption by directly increases the expression of its
downstream gene bile salt export pump (BSEP) [6]. Additionally,
The farnesoid X receptor (FXR), a nuclear receptor mainly
expressed in the liver, gall bladder and kidney, is physiologically
activated by bile acids and plays a critical role in the maintenance of
bile acid balance [1]. In the liver, activation of FXR prevents bile acid
accumulation through regulation of various genes involved in bile
acid synthesis, transport, excretion, and metabolism [2e4]. Spe-
cifically, FXR activation induces the expression of FXR-small het-
erodimer partner (SHP) that negatively modulates cholesterol 7a-
hydroxylase (CYP7A1) expression, a rate-limiting enzyme involved
in conversion of cholesterol to bile acids [5]. FXR promotes bile acid
FXR is also involved in the regulation of lipid synthesis and meta-
bolism by decreasing mRNA level of sterol regulatory element
binding protein 1c (SREBP-1c) [7]. Therefore, modulation of FXR has
been considered as a valuable therapeutic approach for various
metabolic diseases including primary biliary cirrhosis (PBC), lipid
homeostasis, non-alcoholic steatohepatitis (NASH) and nonalco-
holic fatty liver disease (NAFLD) [8e10].
Since the identification of chenodeoxycholic acid (CDCA) as the
physiological FXR agonist, a variety of synthetic steroidal and
nonsteroidal FXR agonists have been discovered by medicinal
chemistry approaches [11,12] with the obeticholic acid (6a-ethyl-
CDCA, OCA) that was recently approved for the treatment of PBC
leading the pipeline (Fig. 1) [13]. However, higher rates of pruritus
were usually observed in OCA-treated patients [14], which might
be due to the activation of cell surface bile acid receptors by either
OCA or its metabolites [15]. In addition to steroidal ligands [16e19],
emerging efforts have focused on the development of nonsteroidal
* Corresponding author. State Key Laboratory of Natural Medicines, Jiangsu Key
Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
** Corresponding author.
E-mail addresses: cpuzdy@163.com (D. Zhang), xianghua@cpu.edu.cn (H. Xiang).
Same contribution to this paper.
1
https://doi.org/10.1016/j.ejmech.2020.112910
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
Fig. 1. Structure of representative steroidal and isoxazole-type FXR agonists reported. Current work focusing on chemical modifications of LJN452 through structural simplification
and derivatization.
FXR agonists. The most important and widely used nonsteroidal
FXR agonist is the isoxazole-based GW4064 developed by Glaxo-
SmithKline (GSK) [20]. Since then, there have been extensive
optimization efforts to overcome its liabilities such as toxicity,
leading to various agonists possessing an alternative structure to
the stilbene linker while preserving the privileged isoxazole core
[11,12,21]. Except the piperidine-containing LY2562175 [22], typical
isoxazole-type FXR agonists including GW4064, GSK2324 [23] and
LJN452 [24] all demonstrate full FXR agonism with high maximum
efficacy (Fig. 1). From these analogs, tropifexor (LJN452) identified
by Novartis is undergoing clinical phase 2 trials for the treatment of
NASH. However, full activation of FXR might lead to undesirable
cholesterol accumulation including elevated LDL cholesterol, which
has been confirmed in the recent Phase 3 REGENERATE trial of OCA
[14,15,25,26]. Emerging evidence showed that these side effects
probably result from strong FXR-mediated feedback repression of
CYP7A1, a key enzyme for cholesterol metabolism [15,27,28].
Alternatively, partial FXR agonists with low maximum efficacy
capable of activating FXR signaling in moderate or selective man-
ners are emerging as a promising approach to mitigating the
disturbed lipid profile and dealing with metabolic diseases
requiring a long-term treatment [29e32].
Given the fact that no nonsteroidal FXR agonists have been yet
approved for marketing, and many of current available FXR mod-
ulators are biased by poor solubility, poor oral bioavailability, or
insufficient selectivity [8,11,12,33], orally bioavailable agonists
especially that can activate FXR in a partial manner are urgently and
constantly required. Extensive optimization efforts focusing on
modification of ‘link’ moieties and aromatic have successfully
afforded several novel FXR agonists with some entering in clinical
trials [11,12,17]. To identify novel nonsteroidal FXR modulators, we
first focused on the optimization of the widely used isoxazole-
based FXR agonist GW4064 through a ring-closure strategy [34].
In parallel, we also performed structural simplification based on the
clinically used FXR agonist LJN452 of which the central bicyclic
nortropine was replaced with a piperidine drawn from the rare
isoxazole-based partial FXR agonist LY2562175. Furthermore, the
terminal benzothiazole ring was further simplified to aryl urea
structure that are increasingly used in modern drug design in order
to fine-tune crucial drug-like properties [35]. Encouragingly, a
submicromolar FXR agonist with moderate maximum efficacy was
identified in FXR BSEP-luciferase reporter gene assay. Extensive
in vitro and in vivo evaluation further revealed potent and rela-
tively partial modulation of FXR signaling as well as favorable
pharmacokinetics.
2. Results and discussion
2.1. Chemistry
The preparation of designed compounds was according to a
previously reported route shown in Scheme 1 [24]. Firstly, oxime 2
was generated from 2-(trifluoromethoxy)-benzaldehyde 1 through
condensation with hydroxylamine. Chlorination of 2 by NCS pro-
vided chloroxime 3, of which cycloaddition with methyl 3-
cyclopropyl-3-oxopropanoate afforded isoxazole 4. After reduc-
tion of the methyl ester with LiAlH4, the corresponding alcohol 5
was subsequently brominated via Appel reaction to generate
compound 6. Alkylation of 6 with N-Boc-4-hydroxypiperidine un-
der basic conditions resulted in the Boc-protected compound 7,
which was then reacted with TFA to provide intermediate 8. Finally,
target compounds 10a-10i were furnished from intermediate 8
with various substituted anilines 9a-9i under the condition of tri-
phosgene. The benzoic acids 11j, 11k were further prepared from
2

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
Scheme 1. Reagents and conditions: (a) NH2OH$HCl, NaOH, EtOH, 0 ꢀC to rt, 1 h, 93%; (b) NCS, DMF, 25 ꢀC, 1 h, 77%; (c) NaOCH3, methyl 3-cyclopropyl-3-oxopropanoate, THF, ꢁ5 to
35 ꢀC, 12 h, 41%; (d) LiAlH4, THF, 0 ꢀC, 1 h, 92%; (e) CBr4, PPh3, DCM, rt, 1 h, 50%; (f) N-Boc-4-hydroxypiperidine, 18-crown-6, t-BuOK, THF, 12 h, rt, 50%; (g) TFA, DCM, 1 h, rt, 95%; (h)
bis(trichloromethyl)carbonate, Et3N, DCM, 60 ꢀC to rt, 12 h, over 38%; (i) LiOH, THF-H2O, rt, 1 h, 80e86%.
Table 1
corresponding ethyl esters 10, 10 by base-mediated hydrolysis. The
structures of all the final compounds (10a-10i, 11j, 11k) were
confirmed by 1H NMR, 13C NMR and HRMS.
Agonistic effects of synthesized compounds 10a-10j, 11j-11k on FXR.
2.2. Biological evaluation
2.2.1. FXR BSEP-luciferase reporter gene assay
To measure FXR agonistic activity, we performed a cellular
transactivation assay using an FXR BSEP-luciferase reporter gene
assay in HEK293T cells as previously described [36]. The assay that
is used to determine to transcriptional activity of full length FXR on
its response element BSEP promoter is based on a reporter
construct containing FXR expression plasmid and BSEP luciferase
gene reporter. The null-renilla luciferase were co-transfected as
internal control for normalization. The most used full FXR agonist
a
ry
R1
R2
FXR transactivation
EC50
(
m
M)
% efficacy
10a
10b
10c
10d
10e
10f
10g
10h
10i
11j
11k
GW4064
LJN452
H
H
H
8.3
3.2
0.74
7.6
5.2
77 2%
90 1%
91 2%
85 3%
80 2%
CONHCH3
H
COOCH3
H
COOC2H5
H
OCH3
H
COOH
H
CONHCH3
H
COOCH3
H
GW4064 was employed as comparator agonist (EC50 ¼ 0.26
and its transactivation activity at 10 M was defined as 100% acti-
vation (Table 1). The clinical FXR agonist LJN452 was also used as
mM),
m
>10
>10
>10
>10
0.37
0.41
0.26
0.18
8
1%
COOC2H5
36 1%
22 1%
13 1a
86 2%
78 3%
100%
reference agonist which exhibited potent (EC50 ¼ 0.18
mM) and full
H
aCH3
H
FXR activation (90% efficacy relative to GW4064).
All the synthesized compounds were evaluated in vitro by cell-
based FXR transactivation studies. As seen in Table 1, compound
10a without any substitutions in the terminal phenyl demonstrated
moderate FXR activation with EC50 ¼ 8.3 and 77% maximum effi-
cacy relative to GW4064. We further investigated additional amide,
ester and methoxyl groups in meta- and para-position of the
phenylamine partial structure of 10a. The electron-withdrawn
carboxamide and methyl formate substituted in both position
improved FXR activation. Compounds 10b-10e displayed potent
agonistic activities against FXR with EC50 values of 3.2, 0.74, 7.6, and
COOH
e
a
e
e
e
90 3%
a
EC50: the concentration of test compound that gave 50% of the maximum
fluorescence; % efficacy: maximum efficacy of the test compound relative to 10 mM
GW4064 (100%).
derivatives 10h-10i exhibiting week FXR agonistic activity. Further
hydrolysis of 10f-10g provided compounds 11j-11k containing a
typical carboxylic acid pharmacophore that is found in most FXR
5.2
mM, respectively, and over 80% maximum efficacy. However, the
agonists. As expected, compounds 11j (EC50 ¼ 0.37
mM) and 11k
bulkier ethyl derivatives 10f-10g and electron-donating methoxyl
3

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
Fig. 2. Molecular docking and binding modes of compounds 11j (A), 11k (B) and LY2562175 (C). (A). FXR protein is displayed as white cartoon and compounds are displayed as
colored sticks as indicated.
(EC50 ¼ 0.41
m
M) demonstrated potent FXR agonistic activity in
2.2.2. Agonistic effects of compound 11k on FXR nuclear
translocation
comparison to GW4064 (EC50 ¼ 0.26
mM). Surprisingly, compound
11k activated FXR to a less extent (78% efficacy) compared to
LJN452 (92% efficacy).
To further ascertain the agonistic activity of 11k on FXR under
less artificial conditions than in an FXR BSEP-luciferase reporter
gene assay, an immunofluorescence assay was performed to mea-
sure the dynamic of FXR subcellular distribution. The widely used
HepG2 (FXR-rich) cells were selected for the following mechanism
evaluation. Firstly, the cytotoxicity of compound 11k was deter-
mined by MTT assay. As is shown in Fig. 3A, both compound 11k
and GW4064 displayed no significant cytotoxic activity to HepG2
To help clarify the SAR, molecular modeling was performed to
predict the potential molecular interaction mechanisms between
compounds 11j-k and FXR. Due to the structural similarity between
11 and isoxazole-based GW4604, co-crystal structure of the FXR-
GW4064 (PDB: 3DCT) was used for docking analysis. The refer-
ence compound GW4064 was first redocked in FXR to check the
validity of the docking protocol, which revealed a conformation
identical to that in the reported crystal structure (Supporting in-
formation). In agreement with reported X-ray structure of FXR
containing isoxazole-based FXR agonists [24], docking model in
Fig. 2 showed that substituted isoxazole of compound 11j and 11k
forms key H-bond with H447 and fits into hydrophobic pocket,
while the terminal acid is well positioned into the lipophilic pocket
and establishes specific interactions with Arg 331, and Met 265,
which are responsible for their potent FXR transactivation. How-
ever, the H-bond interactions formed by 11k or reported partial
agonist LY2562175 within hydrophilic regions of FXR are slightly
weaker than those observed in 11j. This might in part explain that
11k demonstrated partial agonist-type efficacy, since full agonists
such as CDCA normally establish more and stable hydrophilic in-
teractions to maintain the active conformation [37]. Collectively,
these results indicated that 11k as promising and interesting lead
for further investigation and optimization, and was, therefore,
selected for further in vitro and in vivo evaluation shown in the
following parts.
up to a concentration of 40
with compound 11k at nontoxic concentration of 1
m
M. Further treatment of HepG2 cells
M could
m
markedly promote FXR nuclear translocation as GW4064 did
(Fig. 3B), which further confirmed its agonistic ability against FXR
transcriptional activity.
2.2.3. Activation of compound 11k on FXR and its downstream
effectors
As mentioned above, the resultant FXR agonist 11k demon-
strated potent FXR agonistic activity with EC50 value of 0.41 mM and
78 3% efficacy relative to GW4064, therefore, its agonistic effects
on the expression of FXR downstream effectors were subsequently
quantified in HepG2 cells. For this purpose, the cells were treated
with 11k (1 and 10 mM), the full agonist GW4064 (1 mM) or with
DMSO as control for 16 h, and then FXR target gene mRNA and
protein expression were measured by quantitative RT-PCR and
Western blot respectively. As is shown in Fig. 4A, 11k upregulated
the mRNA expression of FXR and its target genes SHP and BSEP
expression in a dose-dependent manner. It is worth noting that 11k
Fig. 3. (A) Cytotoxicity of compound 11k and GW4064 at various concentrations (0.1, 1, 10, 40 and 80
m
M) on HepG2 cells after incubation for 48 h (values are mean SD; n ¼ 3). (B)
Nuclear translocation of FXR stimulated by compound 11k: HepG2 cells were treated with 11k (1 M) or GW4064 (1
m
m
M) for 16 h. FXR and nuclei was visualized by staining with
Alexa Fluor®488 (green) conjugated secondary antibody and DAPI (blue), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the
Web version of this article.)
4

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
Fig. 4. Dose-dependent partial agonistic activity of 11k on FXR and target effectors. (A) mRNA expression of FXR, SHP and BSEP in HepG2 cells after 16 h incubation with 11k or
GW4064 at indicated concentrations were determined by quantitative RT-PCR. Values are shown as relative fold change of DMSO control group (mean SEM, n ¼ 3; *p < 0.05,
#p < 0.05 vs control). (B) Western blot analysis of the proteins FXR, SHP and BSEP in HepG2 after treatment for 16 h.
at 1
m
M increased the mRNA expression of all three studied genes to
low maximum efficacy is expected to achieve suitable effects on
CYP7A1 repression avoiding disturbing cholesterol homeostasis. To
determine the effects of agonist 11k on CYP7A1 expression, quan-
titative RT-PCR and Western blot were performed in HepG2 cells.
As is shown Fig. 5A and B and supporting information, 11k at 1 mМ
induced less repression of CYP7A1 compared to full agonists
GW4064 and OCA both at mRNA and protein levels, although it
a smaller degree than full agonist GW4064 (1
mM). Furthermore,
consistent with mRNA outcomes, similar trend was seen in protein
expression of FXR, SHP and BSEP (Fig. 4B). All the above results
indicated that 11k activated FXR and its target genes SHP and BSEP
both at mRNA and protein levels in a modest agonist manner
compared to full agonist GW4064 in HepG2 cells.
showed strong inhibition at high concentration of 10
mM. Never-
theless, 11k at 1 М repressed lipogenesis gene SREBP-1c in similar
m
2.2.4. Compound 11k inhibited lipid accumulation by modulation
of FXR target genes
As mentioned above, full FXR agonist obeticholic acid usually
induced robust repression of CYP7A1, which inevitably leads to
undesirable cholesterol accumulation. Partial FXR agonism with
amplitudes as the same concentration of GW4064 did (Fig. 5C). Our
results showed that 11k displayed similar effect on SREBP-1C
downregulation with GW4064 but was apparently less potent on
downregulating CYP7A1 (although not significant), which provide
Fig. 5. Quantitative RT-PCR analysis of CYP7A1 mRNA expression (A), Western blot analysis of the protein CYP7A1 (B) and quantitative RT-PCR analysis of SREBP-1c mRNA
expression (C) in HepG2 cells after treatment with 11k for 16 h (mean SEM, n ¼ 3; *p < 0.05, **p < 0.01 vs control). (D) Graphical representation of morphological and Oil Red O
staining (red) of HepG2 cells. (E) For the quantitative determinations of the accumulated lipids, the Oil Red staining were dissolved in iso-propanol that was measured at 510 nm.
Values are mean SD, n ¼ 3. *P < 0.05, ***P < 0.001 vs. OA-treated group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version
of this article.)
5

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
Table 2
Physicochemical and drug-likeness properties.
Cpd.
MWa
HBAb
HBDc
tPSA (Å2)d
MlogPe
MlogS (mg/L)f
Drug-likeness scoreg
11k
GW4064
LJN452
545.18
541.06
603.15
7
5
8
2
1
1
89.67
58.14
76.83
5.89
8.99
6.81
1.26
0.42
0.69
0.94
0.83
1.18
Table 3
aMW: molecular weight. bHBA: hydrogen-bond acceptors.
Pharmacokinetic properties of compoaad 11k.
cHBD: hydrogen-bond donors. dtPSA: topological polar surface
area. eMlogP: calculated logarithm of the octanol-water partition
coefficient. fMlogS: calculated water solubility. gThe recommended
drug-likeness values are close to 1 calculated by Molsoft/Drug-
likeness prediction tool, shown in supporting information.
Parameter
11k (20 mg/kg p.o.)a
Value
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUC0-t (ng$h/mL)
10.6 1.40
4.80 1.79
2350 57.6
36100 3140
Encouraged by the promising in vitro profile of 11k, we further
conducted in vivo pharmacokinetic studies in male C57BL6/J mice
following oral dose at a single dose of 20 mg/kg body weight. As is
shown in Table 3, compound 11k exhibited favorable oral
bioavailability with high plasma exposure (AUC0-t ¼ 36100 ng h/
mL) and desirable half-life (T1/2 ¼ 10.6 h). Particularly, after a single
oral dose of 20 mg/kg, active concentration above FXR EC50 value
was observed for a tremendous period of around 14 h (Fig. 6A),
resulting in an obvious induction of SHP and BSEP mRNA expres-
sion and repression of CYP7A1 in livers (Fig. 6B). These results
a
Mice dosed at 20 mg/kg p. o., n ¼ 5. Values are expressed as the
mean SD, n ¼ 5.
interesting evidence that 11k would be a selective FXR modulator.
Furthermore, in order to verify the ability of 11k to modulate
lipogenesis and lipid accumulation through FXR pathway, Oil Red O
staining and its subsequent quantification were further performed
using oleic acid (OA)-stimulated HepG2 cells as the lipid loading
cell model (Fig. 5D and E) [38]. Compound 11k dose-dependently
suppressed OA-induced cellular lipid accumulation, reflecting in
the decreased number of oil-stained droplets in 11k-treated groups.
All these data showed that 11k could ameliorate OA-induced lipid
accumulation via FXR activation and the resulting modulation of
target genes.
aa
indicate that 11k was orally active FXR agonist with promising
in vivo efficacy.
3. Conclusion
Based on our ongoing pursuit on isoxazole-type nonsteroidal
FXR modulators, we reported herein the structural simplification
and optimization on a clinical FXR agonist (LJN452), generating a
series of novel FXR agonists screened by FXR BSEP-luciferase re-
porter gene assay. Despite the preliminary structural derivatization,
a potent FXR agonist 11k with similar EC50 potency but lower
maximum efficacy than full FXR agonists was discovered, which
activated FXR-SHP signaling in a selective and moderate manner.
Particularly, moderate suppression of CYP7A1 induced by 11k
indicated a favorable safety profile, which might avoid the draw-
back of undesirable cholesterol accumulation associated with full
agonists such as OCA. However, it is worth noting that the differ-
ence in the modulation of CYP7A1 gene by GW4064 and 11k failed
to be significant, and 11k-mediated gene regulation was still
concentration-dependent from our assay. In contrast, previously
2.2.5. Drug-likeness properties and in vivo studies of 11k
The physicochemical properties of 11k were predicated by
Molsoft (http://molsoft.com/mprop/) to evaluating its drug-
likeness in comparison to GW4064 and LJN452. As shown in
Table 2, all the compounds displayed acceptable drug-likeness
properties (HBA < 10, HBD < 5, TPSA < 90 Å2, and drug-likeness
scores close to 1), while 11k (MlogP ¼ 5.89, MlogS ¼ 1.26)
showed better solubility than GW4064 (MlogP
¼
8.99,
MlogS ¼ 0.42) and LJN452 (MlogP ¼ 6.81, MlogS ¼ 0.89). Collec-
tively, these results together with desirable ADME properties
(supporting information Table S1) indicated that compound 11k is
suitable to be drug-like candidate.
Fig. 6. In vivo evaluation of 11k. (A) Plasma concentration of 11k after oral administration in C57BL6/J mice. The data was the average plasma concentration of five mice at 0.25, 0.5,
1, 2, 4, 8, 12 and 24 h. (B) Quantification RT-PCR analysis of mRNA expression of SHP, BSEP and CYP7A1 in livers 8 h post dose compared to control (100%). Values are expressed as
the mean SEM, n ¼ 3.
6

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
reported potent and partial FXR agonists [32] could reach satura-
tions in gene modulation at a tested range. Although further
investigation is clearly needed to clarify the agonistic character of
11k, our data by directly comparing 11k and GW4064 at the same
dose indicated that 11k was able to selectively modulate FXR target
genes in a distinct manner.
Further in vivo studies showed that 11k displayed preferable
oral bioavailability with desirable half-life (T1/2 ¼ 10.6 h), which
translated into a remarkable modulation of FXR target gene
expression at 8 h post a single oral dose of 20 mg/kg. Collectively,
the urea-containing compound 11k provides a new scaffold for FXR
modulators [11,40,41] and warrants further investigation and
development for the treatment of FXR dependent metabolic or
cardiovascular diseases.
(4.5 g, 18.8 mmol) in CH3OH at ꢁ10 ꢀC. The reaction was warmed to
35 ꢀC and stirred for 12 h. After completion, the reaction mixture
was diluted with water and extracted with ethyl acetate. The
combined organic layer was washed with brine, dried over anhy-
drous sodium sulfate, concentrated under vacuum, and then puri-
fied by silica gel column chromatography using ethyl acetate/
petroleum ether to afford compound 4 as a white solid, 2.5 g in 41%
yield; 1H NMR (DMSO‑d6, 300 MHz)
d 7.65e7.60 (m, 1H, Ph),
7.53(dd, J ¼ 7.6, 2 Hz, 1H, Ph), 7.48e7.43 (m, 2H, Ph), 3.68 (s, 3H,
eCOOCH3), 2.89 (m, J ¼ 6.8 Hz, 1H, eCH(CH2)2), 1.30 (m, J ¼ 6.8 Hz,
4H, eCH(CH2)2); MS (ESI) m/z: 328.1 [MþH]þ.
4.1.4. (5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-
yl)-methanol (5)
LiAlH4 (0.58 g, 15.3 mmol) was added to anhydrous THF at 0 ꢀ
C
4. Experimental protocols
under an inert atmosphere of nitrogen. A solution of compound 4
(2.5 g, 7.6 mmol) in anhydrous THF was added dropwise at 0 ꢀC. The
resulting solution was stirred for 1 h at 0 ꢀC, and the reaction was
quenched by the slow addition of saturated sodium sulfate solution
and subsequent 15% NaOH solution to quench the reaction. The
resulting mixture was filtered through Celite, and the filtrate was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated under vacuum, affording compound 5 as yellow oil,
2.1 g in 92% yield; MS (ESI) m/z: 300.1 [MþH]þ.
4.1. Chemistry
All chemicals and solvents purchased were of analytical grade
and were directly used after without further purification. Reactions
were monitored by thin-layer chromatography (TLC) using pre-
coated silica gel plates (silica gel GF/UV 254), and spots were
visualized under UV light (254 or 365 nm). Column chromatog-
raphy was performed with silica gel (200e300 mesh). High reso-
lution mass spectra (HRMS) were recorded on a Water Q-Tof micro
mass spectrometer. The structure resolution of synthetic com-
pounds was verified by 1H NMR and 13C NMR spectra which were
recorded using a Bruker Avance 400 MHz spectrometer with tet-
ramethylsilane (TMS) as an internal standard. Chemical shifts (d)
are expressed in parts per million; coupling constants (J) are in
hertz (Hz), and the signals are designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. Purity of the compound was
4.1.5. 4-(bromomethyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)
phenyl)isoxazole (6)
Compound 5 (1 g, 3.3 mmol) and PPh3 (1.3 g, 5.0 mmol) were
dissolved in anhydrous DCM at 0 ꢀC. Under an inert atmosphere of
nitrogen, a solution of CBr4 (1.66 g, 5.0 mmol) in anhydrous DCM
was added dropwise at 0 ꢀC. The resulting solution was stirred for
1 h at rt. The reaction mixture was concentrated under vacuum and
directly purified by silica gel column chromatography using ethyl
acetate/petroleum ether to afford compound 6 as a solid, 0.6 g in
analyzed by HPLC (Agilent Eclipse Plus C18 5
and final compounds exhibited the purity greater than 95%.
mM 4.6 ꢂ 250 mm),
50% yield; 1H NMR (CDCl3, 300 MHz)
d 7.55e7.50 (m, 1H, Ph),
7.49e7.45 (m, 1H, Ph), 7.40e7.32 (m, 2H, Ph), 4.27 (s, 2H,
AreCH2eBr), 2.11e1.99 (m, 1H, eCH(CH2)2), 1.24e1.17 (m, 2H,
eCH(CH2)2), 1.17e1.09 (m, 2H, eCH(CH2)2); MS (ESI) m/z: 362.1
[MþH]þ.
4.1.1. (E)-2-(trifluoromethoxy)benzaldehyde oxime (2)
A solution of NH2OH$HCl (2.2 g, 31.7 mmol) in water (20 mL)
was added to a solution of sodium hydroxide (1.2 g, 30 mmol) in
water (20 mL) at 0 ꢀC. The resulting solution was stirred for at 0 ꢀC,
to which was then added a solution of 2-(trifluoromethoxy)-
benzaldehyde 1 (5.0 g, 26.3 mmol) in ethanol (20 mL). The reaction
was stirred at rt for 1 h. After completion, the reaction solution was
diluted with water and extracted with ethyl acetate. The organic
layers were combined, washed with brine, dried over anhydrous
sodium sulfate, and concentrated under vacuum to give 5.0 g of
compound 2 in 93% yield used directly in the next step, MS (ESI) m/
z: 206.1 [MþH]þ.
4.1.6. Tert-butyl 4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxylate (7)
To
a
three-necked bottle was added tert-butyl 4-
hydroxypiperidine-1-carboxylate (0.37 g, 1.8 mmol) and 18-crown
ether-6 (0.48 g, 1.82 mmol) in anhydrous THF at rt. Under an
inert atmosphere of nitrogen, a solution of compound 6 (0.6 g,
1.66 mmol) in anhydrous THF was added dropwise and the
resulting solution was stirred overnight at rt. After completion, the
reaction mixture was diluted with water and extracted with ethyl
acetate. The combined organic layer was washed with brine, dried
over anhydrous sodium sulfate, concentrated under vacuum, and
then purified by silica gel column chromatography using ethyl ac-
etate/petroleum ether to afford compound 7 (0.4 g, 50% yield); 1H
4.1.2. N-hydroxy-2-(trifluoromethoxy)benzimidoyl chloride (3)
NCS (2.5 g, 187 mmol) was slowly added to a stirred solution of
compound 2 (5 g, 24.4 mmol) in DMF (30 mL) under ice bath. The
mixture was stirred for 3 h at room temperature and then diluted
with water and extracted with ethyl acetate. The organic layers
were combined, washed with brine, dried over anhydrous sodium
sulfate, and concentrated under vacuum to give crude compound 3
(4.5 g, 77% yield), MS (ESI) m/z: 240.1 [MþH]þ.
NMR (DMSO‑d6, 300 MHz)
d 7.92e7.61 (m, 4H, Ph), 4.30 (s, 2H,
AreCH2eO-), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2, 2.22 (m, 1H, AreCHe(CH2)2), 1.66 (m, 4H,
-OeCH2eCH2-N-), 1.24 (m, 1H, AreCHe(CH2)2), 1.06 (-O-C-(CH3)3),
1.02, 0.99 (m, 2H, AreCHe(CH2)2); MS (ESI) m/z: 483.1 [MþH]þ.
4.1.3. Methyl 5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazole-4-carboxylate (4)
4.1.7. 5-Cyclopropyl-4-((piperidin-4-yloxy)methyl)-3-(2-
(trifluoromethoxy)phenyl)isoxazole (8)
Compound 7 (0.4 g, 0.83 mmol) was dissolved in anhydrous
DCM (15 mL) at rt. Under an inert atmosphere of nitrogen, TFA
(15 mL) was added dropwise and the resulting solution was stirred
Sodium methoxide (2.6 g, 48.9 mmol) was suspended in CH3OH
(50 mL), and the mixture was cooled to ꢁ10 ꢀC and treated with a
solution of methyl 3-cyclopropyl-3-oxopropanoate (5.3 g,
37.6 mmol) in CH3OH (20 mL) dropwise over 5 min. To above re-
action mixture was then slowly added a solution of compound 3
for
1 h at rt. After completion, the reaction mixture was
7

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
concentrated under vacuum, to afford compound 8 (0.3 g, 95%
yield); MS (ESI) m/z: 383.1 [MþH]þ.
for C28H28F3N3O6 [MþH]þ 559.1915, found 559.1913.
4.1.13. Methyl 3-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoate (10e)
Light yellow oil, yield 38%; 1H NMR (DMSO‑d6, 300 MHz)
4.1.8. General method for the synthesis of final compounds (10a-
10i)
Various substituted anilines 9a-9i (0.3 mmol) was dissolved in
anhydrous DCM at rt. TEA (0.1 mL) and triphosgene (0.47 g,
0.15 mmol) were dissolved in anhydrous DCM at 0 ꢀC, and was
added dropwise to the above aniline solution at 0 ꢀC and the
resultant mixture was stirred at rt for 1 h. To the above mixture was
added a solution of compound 8 in anhydrous DCM and stirred
overnight at rt. The reaction mixture was diluted with water and
extracted with ethyl acetate. The combined organic layer was
washed with brine, dried over anhydrous sodium sulfate, concen-
trated under vacuum, and then purified by silica gel column chro-
matography ether to afford respective target compounds.
d 7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 3.89 (s, 3H,
eCOOCH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2, 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02e0.99 (m, 2H,
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
d 172.1, 159.3, 159.2,
154.8,148.0,139.2,131.7,131.0,128.4,126.8,122.8,120.8,119.8,111.4,
77.7, 77.1, 76.3, 73.6, 59.2, 41.6, 30.5, 8.8, 7.9. HRMS (ESI) m/z: calcd
for C28H28F3N3O6 [MþH]þ 559.1914, found 559.1915.
4.1.14. Ethyl 4-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoate (10f)
Light yellow oil, yield 44%; 1H NMR (DMSO‑d6, 300 MHz)
4.1.9. 4-((5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-
yl)methoxy)-N-phenylpiperidine-1-carboxamide (10a)
d 7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 4.30 (q, 2H,
eCOOCH2CH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2), 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.30 (t, 3H, eCOOCH2CH3), 1.25 (m, 1H,
AreCHe(CH2)2), 1.02e0.99 (m, 2H, AreCHe(CH2)2), 13C NMR
Light yellow oil, yield 41%; 1H NMR (CDCl3, 300 MHz)
d
7.77e7.46 (m, 1H), 7.46e7.38 (m, 1H), 7.37e7.21 (m, 2H),
7.09e6.98 (m, 4H), 6.57 (s, 2H), 3.61 (ddd, J ¼ 11.9, 7.1, 3.9 Hz, 1H),
3.50 (dt, J ¼ 7.6, 3.7 Hz, 4H), 2.22e2.11 (m, 4H), 1.76 (td, J ¼ 7.9,
3.6 Hz, 1H), 1.52 (ddt, J ¼ 12.8, 8.5, 3.9 Hz, 1H), 1.37e1.22 (m, 1H),
(DMSO‑d6, 75 MHz)
d 172.1, 159.3, 154.8, 147.0, 139.2, 131.7, 131.0,
127.5, 126.7, 122.9, 120.4, 119.9, 111.4, 77.8, 77.3, 76.2, 73.6, 59.2,
40.6, 30.5, 8.5, 7.6. HRMS (ESI) m/z: calcd for C29H30F3N3O6 [MþH]þ
574.2185, found 574.2153.
1.19e1.08 (m, 1H). 13C NMR (CDCl3, 75 MHz)
d 171.9, 159.5, 155.0,
146.9, 139.1, 131.8, 131.2, 128.8, 127.0, 123.1, 123.1, 120.8, 120.0, 119.3,
111.6, 77.5, 77.1, 76.7, 73.5, 59.0, 41.4, 30.5, 8.1, 7.6. HRMS (ESI) m/z:
calcd for C26H26F3N3O4 [MþH]þ 502.2883, found 502.2881.
4.1.15. Ethyl 3-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoate (10g)
Light yellow oil, yield 45%; 1H NMR (DMSO‑d6, 300 MHz)
4.1.10. 4-((5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-
4-yl)methoxy)-N-(4-(methylcarbamoyl)phenyl)piperidine-1-
carboxamide (10b)
d
7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 4.30 (q, 2H,
eCOOCH2CH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2), 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.30 (t, 3H, eCOOCH2CH3), 1.25 (m, 1H,
AreCHe(CH2)2), 1.02e0.99 (m, 2H, AreCHe(CH2)2), 13C NMR
Light yellow oil, yield 62%; 1H NMR (DMSO‑d6, 300 MHz)
d 8.86
(s, 1H, eCONHe), 7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, Ar-CH2-O-), 3.49
(m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H, -OeCHe(CH2)2), 2.81 (s, 3H,
eCONHCH3), 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02e0.99 (m, 2H,
(DMSO‑d6, 75 MHz)
d 172.1, 159.3, 154.8, 147.0, 139.2, 131.7, 131.0,
127.5, 126.7, 122.9, 120.4, 119.9, 111.4, 77.8, 77.3, 76.2, 73.6, 59.2,
40.6, 30.5, 8.5, 7.6. HRMS (ESI) m/z: calcd for C29H30F3N3O6 [MþH]þ
574.2085, found 574.2155.
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
d 172.1, 158.2, 155.4,
154.8, 147.0, 139.2, 131.7, 131.0, 128.4, 126.8, 122.8, 120.9, 119.8, 111.5,
77.9, 77.2, 76.3, 74.7, 73.6, 59.2, 41.6, 30.5, 8.3, 7.4. HRMS (ESI) m/z:
calcd for C28H29F3N4O5 [MþH]þ 559.2098, found 559.2095.
4.1.16. 4-((5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-
4-yl)methoxy)-N-(4-methoxyphenyl)piperidine-1-carboxamide
(10h)
4.1.11. 4-((5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-
4-yl)methoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-
carboxamide (10c)
Light yellow oil, yield 39%; 1H NMR (DMSO‑d6, 300 MHz)
d
7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 3.61 (s, 3H,
Light yellow oil, yield 64%; 1H NMR (DMSO‑d6, 300 MHz)
d
8.86
eOCH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2, 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02e0.99 (m, 2H,
(s, 1H, eCONHe), 7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, Ar-CH2-O-), 3.49
(m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H, -OeCHe(CH2)2, 2.81 (s, 3H,
eCONHCH3), 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02e0.99 (m, 2H,
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
d 172.1, 159.3, 154.8,
147.0, 139.2, 131.7, 131.0, 128.4, 126.8, 122.8, 120.8, 119.8, 116.4, 78.0,
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
d
172.1, 159.3, 157.4,
77.4, 76.3, 73.6, 58.2, 41.6, 30.5, 8.6, 7.9. HRMS (ESI) m/z: calcd for
154.8, 147.0, 139.2, 131.7, 131.0, 128.4, 126.8, 122.8, 120.8, 119.8, 111.4,
77.8, 77.5, 76.3, 74.6, 73.6, 59.2, 41.6, 30.5, 8.0, 7.3. HRMS (ESI) m/z:
calcd for C28H29F3N4O5 [MþH]þ 559.2098, found 559.2095.
C
27H28F3N3O5 [MþH]þ 532.2025, found 532.2040.
4.1.17. 3-((5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-
4-yl)methoxy)-N-(4-methoxyphenyl)piperidine-1-carboxamide
(10i)
4.1.12. Methyl 4-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoate (10d)
Light yellow oil, yield 46%; 1H NMR (DMSO‑d6, 300 MHz)
Light yellow oil, yield 38%; 1H NMR (DMSO‑d6, 300 MHz)
d
7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 3.61 (s, 3H,
d
7.48e6.9 (m, 8H, Ar), 4.30 (s, 2H, AreCH2eO-), 3.89 (s, 3H,
eOCH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2, 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02e0.99 (m, 2H,
d 172.1, 159.7, 154.8,
152.0, 149.2, 131.7, 131.0, 128.4, 126.8, 122.8, 120.4, 119.9, 111.9, 77.8,
eCOOCH3), 3.49 (m, 4H, -OeCH2eCH2-N-), 3.37 (m, 1H,
-OeCHe(CH2)2, 2.22 (m, 1H, AreCHe(CH2)2), 1.68 (m, 4H,
-OeCH2eCH2-N-), 1.25 (m, 1H, AreCHe(CH2)2), 1.02, 0.99 (m, 2H,
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
AreCHe(CH2)2), 13C NMR (DMSO‑d6, 75 MHz)
d
172.1, 159.3, 158.6,
154.8, 147.0, 139.2, 131.7, 131.1, 128.4, 126.8, 122.8, 120.8, 119.8, 111.4,
77.8, 77.1, 76.3, 73.6, 59.2, 41.6, 30.5, 7.9, 7.3. HRMS (ESI) m/z: calcd
77.1, 76.3, 73.6, 59.2, 41.6, 30.5, 8.0, 7.3. HRMS (ESI) m/z: calcd for
C
27H28F3N3O5 [MþH]þ 532.2025, found 532.2045.
8

G. Luo, X. Lin, Z. Li et al.
European Journal of Medicinal Chemistry 209 (2021) 112910
4.1.18. General method for the synthesis of final compounds (11j-
11k)
retained, and binding modes presented graphically are represen-
tative of the highest-scored conformations.
LiOH (0.4 mmol) was added to a solution of compound 10f or
10g (0.2 mmol) in THF-H2O (1:1) at rt and the reaction mixture was
stirred for 1 h at rt. The reaction mixture was diluted with water
and adjusted to pH around 4 and extracted with ethyl acetate. The
combined organic layer was washed with brine, dried over anhy-
drous sodium sulfate, concentrated under vacuum, and then puri-
fied by silica gel column chromatography ether to afford respective
target compounds.
4.2.3. MTT assay for cytotoxicity activities
HepG2 Cells were cultured in DMEM medium (containing 10%
(v/v) FBS, 100 U/mL Penicillin and 100 mg/mL Streptomycin) in a 5%
CO2-humidified atmosphere at 37 ꢀC. Cells were trypsinized and
seeded at a density of 1 ꢂ 105/mL into a 96-well plate (100 mL/well)
and incubated at 37 ꢀC, 5% CO2 atmosphere for 24 h. After this time
they were treated with 100 mL/well medium containing test
compounds to provide the final concentration range of 0.1, 1, 10, 40
4.1.19. 4-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoic acid
(11j)
and 80 mmol/L, and re-incubated for a further 48 h 20 mL MTT
(5 mg/mL) was added and cells continued to incubate in darkness at
37 ꢀC for 4 h. The culture medium was then removed carefully and
150 mL DMSO was added. The cells were maintained at room
temperature in darkness for 20 min to ensure thorough color
diffusion before reading the absorbance. The absorbance values
were read at 490 nm for determination of cell viability.
White solid, yield 80%; 1H NMR (DMSO‑d6, 300 MHz)
d 10.81 (s,
1H), 8.72 (s, 1H), 7.90 (m, 2H), 7.79 (m, 2H) 7.58 (m, 4H), 4.43 (s, 2H),
3.59 (m, 4H), 3.12 (m, 2), 2.41 (m,1H),1.67 (m, 2H),1.35 (m, 3H),1.26
(m, 4H). 13C NMR (DMSO‑d6, 75 MHz)
d 172.1, 159.3, 158.6, 154.8,
147.0, 139.2, 131.7, 131.1, 128.4, 126.8, 122.8, 120.8, 119.8, 111.4, 74.8,
60.2, 59.2, 41.6, 30.5, 7.9, 7.3. HRMS (ESI) m/z: calcd for
C
4.2.4. Immunohistochemistry
27H26F3N3O6 [MþH]þ 546.1845, found 546.1841.
HepG2 cells were cultured in DMEM containing 10% FBS and
treated with test compound at indicated concentrations for 16 h.
After HepG2 cells were fixed with 4% PFA in PBS at 37 ꢀC for 30 min,
and washed with PBS for three times, cells were permeabilized
with PBS containing 0.3% Triton X-100 for 10 min. Afterwards the
samples were stained sequentially with primary anti-FXR antibody
(ab187735) and Alexa Fluor 488 (green) conjugated second anti-
body for 1 h at room temperature, and DAPI was used to stain
nuclei. Then cells were imaged on a confocal microscope.
4.1.20. 3-(4-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)
isoxazol-4-yl)methoxy)piperidine-1-carboxamido)benzoic acid
(11k)
White solid, yield 80%; 1H NMR (DMSO‑d6, 300 MHz)
d
10.68 (s,
8.72 (s, 1H), 7.83 (m, 2H), 7.80e7.58 (m, 7H), 3.59 (m, 4H),
4.49 (s, 2H), 3.58 (m, 2H), 3.12 (m, 2), 2.40 (m, 1H), 1.65 (m, 2H), 1.35
(m, 3H), 1.26 (m, 4H). 13C NMR (DMSO‑d6, 75 MHz)
172.0, 159.5,
1H),
d
d
158.7, 154.9, 147.0, 139.4, 131.8, 131.2, 128.5, 126.8, 122.9, 120.8,
119.9,111.5, 74.9, 60.2, 59.2, 41.6, 30.5, 7.9, 7.3. HRMS (ESI) m/z: calcd
for C27H26F3N3O6 [MþH]þ 546.1844, found 546.1840.
4.2.5. Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA was extracted from HepG2 cells pretreated with
test compounds at indicated concentrations for 16 h using RNAiso
Plus (Code. no. 9108) according to the manufacturer’s instructions.
The RT reaction was performed using TaKaRa SYBR Green Master
Mix (Code. no. RR820A) carried out in StepOnePlus™ Real-Time
PCR instrument (ABI). The RNA was reverse transcribed into cDNA
using a PrimeScript™ RT reagent Kit (RR047A). The relative gene
expression quantification was normalized to the GADPH mRNA
expression using the DDCT analysis method. The primers used are
shown in the supplementary data.
4.2. Biological evaluation
4.2.1. FXR activation assay
The expression vector of hFXR and BSEP promoter luciferase
reporter was performed according to previously described [32,39].
HEK293T cells were transfected with human hFXR and BSEP plas-
mids, with pGL4.35 (Promega, USA) used as an internal standard.
HEK293Tcells were grown in DMEM supplemented with 10% FCS at
37 ꢀC and 5% CO2. HEK293Tcells were seeded in 96-well plates with
a density of 10000 cells per well for transfection. 16 h after trans-
fection, the respective test compound or 0.1% DMSO alone as un-
treated control were added in triplicate wells. Following 24 h
incubation with the test compounds, cells were assayed for lucif-
erase activity using GLO-MAX luciferase assay system (Promega)
according to manufacturer’s protocol. Fold activation was obtained
by dividing the mean relative light units of the tested compound at
indicated concentration by the mean relative light units of un-
treated control. Maximum efficacy % was obtained by dividing the
4.2.6. Western blot analysis
HepG2 cells with different treatments for 16 h were washed
twice with PBS, then collected and lysed in lysis buffer for 1 h on the
ice. The lysates were then subjected to centrifugation (13,000 rpm)
at 4 ꢀC for 20 min. Protein concentration in the supernatants was
detected by BCA protein assay (Beyotime, Jiangsu, China). Then
equal amount of protein was separated with 12% SDS-PAGE and
transferred to poly-vinylidene difluoride (PVDF) membranes (Mil-
lipore, Bedford, MA) using a semi-dry transfer system (Bio-rad,
Hercules, CA). Proteins were detected using anti-FXR antibody
(ab187735), anti-SHP1 antibody (ab124942), anti-CYP7A1 antibody
(ab234982) or anti-BSEP antibody (ab155421) overnight at 4 ꢀC
followed by HRP-conjugated secondary antibodies for 1 h at 37 ꢀC.
Enhanced chemiluminescent reagents (Beyotime, Jiangsu, China)
were used to detect the HRP on the immunoblots, and the visual-
ized bands were captured by film.
fold activation of the tested compound at 10 mM by the fold acti-
vation of FXR full agonist GW4604 at 10 mM.
4.2.2. Molecular modeling
The molecular modeling was performed with Discovery Stu-
dio.3.0/CDOCK protocol (Accelrys Software Inc.) based on the
crystal structures of GW4604/FXR (PDB ID: 3DCT) which was
downloaded from Protein Data Bank. Compounds were drown us-
ing ChemBioDraw ultra 14.0, then minimized using Chem3D pro
(Minimize Energy to Minimum RMS Gradient of 0.010) and finally
optimized using Discovery Studio.3.0 (Simulation/energy minimi-
zation) to generate low-energy orientations of the ligand. The
protein and ligand were optimized and charged with CHARMm
force field to perform docking. Up to 10 conformations were
4.2.7. Oil red O staining of HepG2 cells
HepG2 cell cells pretreated with 50 mM oleic acid for 12 h were
incubated with test compounds at indicated concentrations for
16 h. Then, the cells were washed with distilled water twice and
stained with freshly diluted Oil Red O solution for 1 h at rt. Then,
the Oil Red O-stained cells were washed twice with distilled water
9

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the lateral tail vein. After a 10 min centrifugation at 10000 rpm,
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For the analysis of mRNA expression in liver, the 8 h time-point
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PBS and total RNA was extracted using RNA kit following the animal
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with the approval of center for new drug evaluation and research,
China Pharmaceutical University (Nanjing, China).
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
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This work was supported by grants from Postdoctoral Research
Foundation of China (CN) (2019M662007), Natural Science Foun-
dation of China (NSFC, 81874286), and “Double-First-Class” Uni-
versity Project (CPU 2018PZQ02; CPU 2018GY07).
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