Design, synthesis, and biological evaluation of novel FXR agonists based on auraptene

Article abstract of DOI:10.1016/j.bioorg.2021.105198

Farnesoid X receptor (FXR) has been considered as an attractive target for metabolic disorder and liver injury, while many current FXR agonists suffer from undesirable side effects, such as pruritus. Therefore, it is urgent to develop new structure types different from current FXR agonists. In this study, a series of structural optimizations were introduced to displace the unstable coumarin and geraniol scaffolds of auraptene (AUR), a novel and safe FXR agonist. All of these efforts led to the identification of compound 14, a potent FXR agonist with nearly fourfold higher activity than AUR. Molecular modeling study suggested that compound 14 fitted well with binding pocket, and formed the key ionic bond with His291 and Arg328. In acetaminophen-induced acute liver injury model, compound 14 exerts better therapeutic effect than that of AUR, which highlighting its pharmacological potential in the treatment of drug-induced liver injury.

Full text of DOI:10.1016/j.bioorg.2021.105198

Bioorganic Chemistry 115 (2021) 105198
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and biological evaluation of novel FXR agonists based
on auraptene
,
*
,
,
,*
Qianqian Qiua 1, Yanjuan Wanga 1, Guolong Gua, Fan Yua, Shichao Zhanga, Yining Zhaoa
,
,
*
Bai Lingb
a School of Pharmacy, Yancheng Teachers’ University, Yancheng, PR China
b Department of Pharmacy, Affiliated Hospital 4 of Nantong University, The First People’s Hospital of Yancheng City, Yancheng, PR China
A R T I C L E I N F O
A B S T R A C T
Keyword:
Farnesoid X receptor (FXR) has been considered as an attractive target for metabolic disorder and liver injury,
while many current FXR agonists suffer from undesirable side effects, such as pruritus. Therefore, it is urgent to
develop new structure types different from current FXR agonists. In this study, a series of structural optimizations
were introduced to displace the unstable coumarin and geraniol scaffolds of auraptene (AUR), a novel and safe
FXR agonist. All of these efforts led to the identification of compound 14, a potent FXR agonist with nearly
fourfold higher activity than AUR. Molecular modeling study suggested that compound 14 fitted well with
binding pocket, and formed the key ionic bond with His291 and Arg328. In acetaminophen-induced acute liver
injury model, compound 14 exerts better therapeutic effect than that of AUR, which highlighting its pharma-
cological potential in the treatment of drug-induced liver injury.
N-acetyl-p-aminophenol
FXR
1. Introduction
Nelumal A (3) and Auraptene (AUR, 4) share the same geraniol scaffold
[22,23]. In this study, AUR was selected as lead compound based on its
Nuclear farnesoid X receptor (FXR) [1–3] is a ligand activated nu-
clear receptor transcription factor which involves in several metabolic
[4,5] and inflammatory [6,7] pathways. FXR acts as a sensor for
endogenous bile acids such as chenodeoxycholic acid (CDCA), which
exerts indispensable role in modulating bile acid synthesis, transport
and metabolism [8,9]. In addition, FXR also closely associated with fatty
acid metabolism, glucose homeostasis, liver protection, and intestinal
inflammation [10–13]. The semisynthetic FXR agonist obeticholic acid
has shown encouraging results for the treatment of liver disorders such
as primary biliary cirrhosis and non-alcoholic fatty liver disease
[14–16]. However, consequent side effects including severe pruritus,
increased low-density lipoprotein, and decreased high-density lipopro-
tein tempered these positive findings [17]. Although many non-steroidal
FXR ligands derived from GW4064 (such as Cilofexor, Fig. 1) have been
developed to avoid the side effect of cholic acid derivative, most of these
agonists still suffer from undesirable pruritus [18]. Therefore, it is ur-
gent to develop new structure types different from the series of GW4064.
Currently, there are several FXR agonists with novel scaffolds are
reported, such as compounds 1–4 in Fig. 1 [19–21]. Among them,
promising hepatoprotective effect by activating FXR [23–25]. However,
the coumarin and geraniol scaffolds of AUR are chemically unstable.
Herein, a series of chemically stable scaffolds were used to displace the
coumarin and geraniol groups of AUR (Fig. 2). All of these efforts
resulted in the identification of the optimal compound 14, which
exhibited nearly fourfold higher activity than AUR. Moreover, com-
pound 14 exerts better therapeutic effect on n-acetyl-p-aminophenol
(APAP)-induced acute liver injury.
2. Results and discussion
2.1. Chemistry
Scheme 1 described the synthetic route of compounds 5–8. The key
intermediate geranyl bromide 16 was synthesized from geraniol 15 via
the reaction with PBr3. Cyclization of 17 with ethyl 3-oxobutanoate
provided 18, which was further converted to target compound 5 by
Williamson ether synthesis with 16. The commercially starting material
19 was treated with 16 to afford the target compound 6. The
* Corresponding authors.
E-mail addresses: zhaoyn@yctu.edu.cn (Y. Zhao), 1079014431@qq.com (B. Ling).
1
Qianqian Qiu and Yanjuan Wang contributed equally as first authors.
https://doi.org/10.1016/j.bioorg.2021.105198
Received 29 January 2021; Received in revised form 8 July 2021; Accepted 18 July 2021
Available online 21 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
Fig. 1. Structure of representative FXR agonists.
Fig. 2. Design strategy to extend the chemical space of AUR.
Scheme 1. Reagents and conditions: (a) PBr3, dichloromethane, 0–5 ℃, 1 h; (b) ethyl 3-oxobutanoate, H2SO4, ꢀ 5 ℃ to r.t., 2 h; (c) 2a, K2CO3, acetonitrile, reflux, 6
h; (d) H2SO4, MeOH, reflux, 4 h; (e) LiOH, THF, MeOH, H2O, r.t.
2

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
Scheme 2. Reagents and conditions: (a) chloroacetyl chloride, triethylamine, dichloromethane, r.t.; (b) K2CO3, acetonitrile, reflux, 6 h; (c) LiOH, THF, MeOH, H2O,
r.t.
Table 1
In vitro activities of target compounds on FXR. a
Table 2
Compd.
R
EC50 (μM, Max%)
CDCA
AUR
15.3 (100%)
4.4 (64.7%)
In vitro activities of target compounds on FXR. a
5
6
4.6 (67.2%)
Compd.
R1
R
EC50 (μM, Max%)
CDCA
AUR
15.3 (100%)
4.4 (64.7%)
25.2 (32.8%)
10
11
4-OEt
4-OEt
4.0 (52.8%)
3.5 (61.5%)
7
8
9
6.3 (58.9%)
5.1 (56.3%)
3.9 (62.5%)
12
13
14
4-OEt
2.9 (68.7%)
1.7 (65.9%)
1.2 (73.7%)
4-COOH
3-COOH
a
EC50 value represents the mean of three determinations.
a
EC50 value represents the mean of three determinations.
3

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
Fig. 3. Predicted binding mode of compound 12 (green) in crystal structure of FXR (PDB code: 1OSV). The obeticholic acid and key residues are shown as gray stick,
hydrogen bonds are shown as silvery dashed line.
commercially available material 20 and 23 was converted to 21 and 24
via esterification, and then treated with 16 to give the intermediate 22
and 25 by Williamson ether synthesis, followed by hydrolysis, provided
the target compounds 7 and 8. The compounds 9–14 were synthesized
as shown in Scheme 2. Substituted anilines 26a-c were treated with
chloroacetyl chloride to provide key intermediates 27a-c, which was
converted into compound 10 by Williamson ether synthesis. Conden-
sation of 7-hydroxy-1H-quinolin-2-one with 16 or 27a provided com-
pounds 9 and 11, respectively. Similar procedure afforded compounds
12–14 based on starting material 7-hydroxy-3,4-dihydro-1H-quinolin-2-
one.
increased the strength of interaction with His291 and Arg328. As ex-
pected, the carboxylic acid derivatives 13 and 14 significantly increased
potency. Moreover, the docking studies suggested that both of them
formed hydrogen bond with His444 and Tyr358 (Fig. 4). The carboxylic
acid of compound 13 formed hydrogen bond with Arg328, while the
carboxylic acid of compound 14 formed hydrogen-bonding network
with Arg328 and His291. These results reasonably explained that com-
pound 14 exerted better potency than that of compound 13.
2.3. Effects of compound 14 on APAP-induced acute liver injury
To identify the effects of the optimal compound 14 on drug-induced
liver injury, a high dose of APAP (300 mg/kg) was performed to induce
acute liver injury model. As shown in Fig. 5A-C, a notable liver damage
such as hepatic necrosis, degeneration, and hemorrhage were observed
in APAP-induced model, while AUR and compound 14 treated groups
significantly reduced liver damage. Compound 14 exhibited better he-
patic histopathological improvements than that of AUR. Similar to his-
topathological improvements, the levels of ALT and AST were
significantly decreased in drug-treated groups (Fig. 5D). Moreover,
significantly higher levels of hepatic GSH were observed in AUR and
compound 14 treated groups.
2.2. SAR and molecular modeling study
In order to explore better scaffold to displace the coumarin moiety of
AUR, a series of chemically stable scaffolds were introduced (Table 1).
Compound 5, a direct analog of AUR, revealed a comparative agonistic
activity with AUR. However, the benzofuran-3(2H)-one derivative 6
significantly reduced activity on FXR, suggesting that the 3-position
substituted carbonyl may exert important roles in maintaining the
agonistic activity. Further introducing the phenylacetic acid (7) and
benzoic acid (8) scaffold to simulate the 3-carbonyl group is tolerated in
this area. The displacement of lactone in AUR with amide provided
compound 9, which revealed better activity than AUR.
3. Conclusion
As shown in Table 2, phenoxyacetamide derivative 10 was obtained
by hybrid of compound 2 with AUR, which slightly improved activity
compared to AUR. Similarly, analog 11 exhibited better potency than its
parent compound 9. These results indicated that the phenoxyacetamide
scaffold is superior to geraniol group. Interestingly, compound 12
significantly increased the activity compared to compound 11. The
docking study was performed to better understand the binding mode of
compound 12, which suggested that the carbonyl of compound 12
formed hydrogen-bonding interaction with residues Typ358 and His444
(Fig. 3). Moreover, the additional hydrogen bond was formed between
the oxygen of compound 12 with Arg328. In consideration of the alka-
line amino acids His291 and Arg328 are around the ethyoxyl group, we
wonder whether a carboxylic acid in this position could be further
In this study, AUR was selected as lead compound based on its
promising hepatoprotective effect by activating FXR. A series of chem-
ically stable scaffolds were introduced to displace the coumarin and
geraniol groups of AUR. After SAR and docking studies, the optimal FXR
agonist 14 was identified, and the activity on FXR increased nearly
fourfold compared to AUR. Molecular modeling study suggested that
compound 14 fitted well in binding pocket of FXR, and formed the key
ionic bond with His291 and Arg328. Further study indicated that com-
pound 14 exerts better therapeutic effect on APAP-induced acute liver
injury than that of AUR. All of these results suggested that compound 14
was meaningful for further evaluation as a novel FXR agonist with new
structural type.
4

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
Fig. 4. Predicted binding modes of compound 13 (A) and 14 (B) in crystal structure of FXR (PDB code: 1OSV). The obeticholic acid and key residues are shown as
gray stick, hydrogen bonds are shown as silvery dashed line.
4. Experimental section
4.1.1. (E)-1-bromo-3,7-dimethylocta-2,6-diene, geranyl bromide (16)
Geranyl bromide 16 was synthesized from geraniol 15 via the reac-
tion with PBr3. PBr3 (3.51 g, 12.97 mmol) was added to cooled (0–5 ℃)
solution of geraniol (2 g, 12.97 mmol) in dichloromethane (20 ml) and
the reaction mixture was stirred for 1 h at room temperature. Saturated
aqueous NaHCO3 was added and the product was extracted with ethyl
acetate. The extracts were washed with brine, dried with anhydrous
Na2SO4 and evaporated to give 2.5 g (88.7%) key intermediate 16 as
brown oil.
4.1. General chemistry
All starting materials, reagents and solvents were obtained from
commercial sources and used without further purification unless
otherwise indicated. Purifications by column chromatography were
carried out over silica gel (200–300 mesh) and monitored by thin layer
chromatography performed on GF/UV 254 plates and were visualized
using UV light at 254 and 365 nm. NMR spectra were recorded on a
Bruker ACF-300Q instrument (300 MHz for 1H NMR and 75 MHz for 13
C
4.1.2. (E)-7-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-4-methyl-2H-
chromen-2-one (5)
NMR spectra), chemical shifts are expressed as values relative to tetra-
methylsilane as internal standard, and coupling constants (J values)
were given in hertz (Hz). High resolution mass spectrometry (HRMS)
was performed on AB SCIEX X500R system.
Ethyl 3-oxobutanoate (1.42 g, 10.9 0 mmol) was dissolved in 20 ml
H2SO4 at 0℃, the resulting viscous solution was cooled to about ꢀ 5 ◦C in
an ice bath. Then resorcinol 17 (1 g, 9.08 mmol) was added in batches,
controlling the internal temperature below 0 ◦C. After the addition, the
solution was stirred at room temperature for 2 h. The reaction mixture
5

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
Fig. 5. Effects of compound 14 on APAP-induced acute liver injury. Hematoxylin and eosin (H&E) staining (100 × magnification) of liver in APAP model group (A),
AUR treated group (B), and compound 14 treated group (C). Red arrow indicates degeneration and necrosis, blue asterisk indicates hemorrhage. (D) represents serum
AST, ALT, and liver GSH levels. Data are presented as mean ± SD values (n = 7). Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01,
***p < 0.001 represent significant difference.
was poured into 50 ml ice water, and then the precipitate was collected,
washed with water and dried to obtain 1.3 g (81.3%) the crude product
18 of a white solid. Then, a solution of 18 (0.5 g, 2.84 mmol) and 16
(0.74 g, 3.41 mmol) in acetonitrile (20 ml) was added K2CO3 (1.18 g,
8.51 mmol) at room temperature. The reaction mixture was heated to
reflux with stirring for 6 h. Then the reaction mixture was cooled to
room temperature followed by filtration and the filtrate was concen-
trated under vacuum. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl acetate (1:10,
v/v) as eluent to afford 0.8 g compound 5 as a white solid, yield 92%. 1H
NMR (300 MHz, DMSO‑d6) δ 7.70 – 7.55 (m, 1H), 7.04 – 6.78 (m, 2H),
6.18 (s, 1H), 5.44 (t, J = 6.6 Hz, 1H), 5.04 (t, J = 6.6 Hz, 1H), 4.65 (d, J
= 5.9 Hz, 2H), 2.38 (s, 3H), 2.13 – 1.89 (m, 4H), 1.73 (s, 3H), 1.61 (s,
3H), 1.56 (s, 3H); 13C NMR (75 MHz, DMSO‑d6) δ 161.96, 160.60,
155.15, 153.77, 141.47, 131.50, 126.70, 124.14, 119.49, 113.39,
113.04, 111.49, 101.70, 65.56, 38.82, 26.20, 25.85, 18.55, 17.95,
16.80; HRMS (ESI): calcd for C20H25O3 313.1725; found 313.1712 [M +
H]+.
287.1553 [M + H]+.
4.1.4. (E)-2-(3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)phenyl)acetic acid
(7)
To a solution of 2-(3-hydroxyphenyl)acetic acid (1 g, 6.57 mmol) in
methanol (20 ml) was added 0.5 ml H2SO4 at room temperature and the
mixture was heated to reflux with stirring for 4 h. Then the cooling re-
action mixture was added 50 ml water and the aqueous layer was
extracted with ethyl acetate. The collected organic layer was evaporated
to afford the crude product 21 1 g (91.7%) as a clear oil. The solution of
21 (0.5 g, 3.01 mmol) and 16 (0.78 g, 3.61 mmol) in acetonitrile (20 ml)
was added K2CO3 (1.25 g, 9.03 mmol) at room temperature. The reac-
tion mixture was heated to reflux with stirring for 6 h. Then the reaction
mixture was cooled to room temperature followed by filtration and the
filtrate was concentrated under vacuum. The residue was purified by
silica gel column chromatography using a mixture of petroleum ether/
ethyl acetate (1:10, v/v) as eluent to afford 0.78 g 22 as a clear oil, yield
85.7%. To a solution of the 22 (0.78 g, 2.58 mmol) in 4:4:1 THF/MeOH/
H2O (18 ml) was added LiOH⋅H2O (0.65 g, 15.48 mmol). After stirring at
room temperature for 4 h, the volatiles were removed under reduced
pressure. The residue was acidified with 1 N hydrochloric acid solution,
and then the aqueous layer was extracted with ethyl acetate, and the
collected layer was elaborated in vacuum to afford 0.7 g the targeted
compound 7 as white solid, yield 95%. 1H NMR (300 MHz, DMSO‑d6) δ
7.29 – 7.12 (m, 1H), 6.97 – 6.76 (m, 3H), 5.44 (t, J = 6.6 Hz, 1H), 5.09 (t,
J = 6.9 Hz, 1H), 4.54 (d, J = 6.5 Hz, 2H), 3.54 (s, 2H), 2.14 – 1.93 (m,
4H), 1.72 (s, 3H), 1.66 (s, 3H), 1.60 (s, 3H); 13C NMR (75 MHz,
DMSO‑d6) δ 173.11, 158.81, 140.50, 136.88, 131.51, 130.79, 124.27,
121.99, 120.24, 114.79, 113.05, 64.64, 41.22, 39.42, 26.28, 25.96,
18.03, 16.78; HRMS (ESI): calcd for C18H23O3 287.1725; found
287.1711 [Mꢀ H]-.
4.1.3. (E)-6-((3,7-dimethylocta-2,6-dien-1-yl)oxy)benzofuran-3(2H)-one
(6)
The solution of 19 (0.5 g, 3.33 mmol) and 16 (0.87 g, 4.0 mmol) in
acetonitrile (20 ml) was added K2CO3 (1.38 g, 9.99 mmol) at room
temperature. The reaction mixture was heated to reflux with stirring for
6 h. Then the reaction mixture was cooled to room temperature followed
by filtration and the filtrate was concentrated under vacuum. The res-
idue was purified by silica gel column chromatography using a mixture
of petroleum ether/ethyl acetate (1:10, v/v) as eluent to afford 0.84 g 6
as yellow oil, yield 88%. 1H NMR (300 MHz, DMSO‑d6) δ 7.54 (d, J =
8.7 Hz, 1H), 6.85 (s, 1H), 6.72 (d, J = 8.8 Hz, 1H), 5.47 (t, J = 6.9 Hz,
1H), 4.79 (s, 2H), 4.73 (t, J = 6.6 Hz, 1H), 4.69 (d, J = 7.3 Hz, 2H), 2.18
– 1.92 (m, 4H), 1.74 (s, 3H), 1.65 (s, 3H), 1.59 (s, 3H); 13C NMR (75
MHz, DMSO‑d6) δ 197.21, 175.05, 167.18, 139.54, 132.08, 130.85,
123.55, 121.67, 119.18, 106.54, 101.37, 74.45, 65.28, 39.76, 26.47,
24.68, 18.67, 16.49; HRMS (ESI): calcd for C18H23O3 287.1569; found
4.1.5. (E)-3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)benzoic acid (8)
To a solution of 3-hydroxybenzoic acid (1 g, 7.24 mmol) in methanol
(20 ml) was added 0.5 ml H2SO4 at room temperature and the mixture
6

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
was heated to reflux with stirring for 4 h. Then the cooling reaction
mixture was added 50 ml water and the aqueous layer was extracted
with ethyl acetate. The collected organic layer was evaporated to afford
the crude product 24 0.98 g (89.0%) as a clear oil. The solution of 24
(0.5 g, 3.29 mmol) and 16 (0.86 g, 3.94 mmol) in acetonitrile (20 ml)
was added K2CO3 (1.36 g, 9.86 mmol) at room temperature. The reac-
tion mixture was heated to reflux with stirring for 6 h. Then the reaction
mixture was cooled to room temperature followed by filtration and the
filtrate was concentrated under vacuum. The residue was purified by
silica gel column chromatography using a mixture of petroleum ether/
ethyl acetate (1:10, v/v) as eluent to afford 0.85 g 25 as clear oil, yield
89.5%. To a solution of the 25 (0.85 g, 2.95 mmol) in 4:4:1 THF/MeOH/
H2O (18 ml) was added LiOH⋅H2O (0.74 g, 17.68 mmol). After stirring at
room temperature for 4 h, the volatiles were removed under reduced
pressure. The residue was acidified with 1 N hydrochloric acid solution,
and then The aqueous layer was extracted with ethyl acetate, and the
collected layer was elaborated in vacuum to afford 0.75 g the targeted
compound 8 as a white solid, yield 93%. 1H NMR (300 MHz, DMSO‑d6) δ
7.54 (d, J = 7.6 Hz, 1H), 7.50 – 7.36 (m, 2H), 7.19 (d, J = 8.0 Hz, 1H),
5.41 (t, J = 6.5 Hz, 1H), 5.08 (t, J = 6.5 Hz, 1H), 4.62 (d, J = 6.5 Hz, 2H),
2.13 – 2.00 (m, 4H), 1.74 (s, 3H), 1.63 (s, 3H), 1.57 (s, 3H); 13C NMR (75
MHz, DMSO‑d6) δ 167.66, 158.80, 140.90, 132.62, 131.48, 130.08,
124.20, 121.92, 120.03, 119.96, 115.15, 64.93, 39.35, 26.21, 25.91,
18.00, 16.80. HRMS (ESI): calcd for C17H21O3 273.1569; found
273.1547 [Mꢀ H]-.
4.1.9. N-(4-ethoxyphenyl)-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)
acetamide (11)
Yield 43%, 1H NMR (300 MHz, DMSO‑d6) δ 11.70 (s, 1H), 10.08 (s,
1H), 7.84 (d, J = 9.4 Hz, 1H), 7.77 – 7.43 (m, 3H), 7.10 – 6.79 (m, 4H),
6.35 (d, J = 9.4 Hz, 1H), 4.75 (s, 2H), 4.01 (q, J = 6.8 Hz, 2H), 1.33 (t, J
= 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO‑d6) δ 165.99, 162.72, 160.10,
155.30, 140.96, 140.47, 131.79, 129.75, 121.85, 119.38, 114.83,
114.25, 111.26, 99.68, 63.56, 15.14. HRMS (ESI): calcd for C19H19N2O4
339.1267; found 339.1245 [M + H]+.
4.1.10. N-(4-ethoxyphenyl)-2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)
oxy)acetamide (12)
Yield 51%, 1H NMR (300 MHz, DMSO‑d6) δ 10.08 (s, 1H), 9.94 (s,
1H), 7.59 – 7.47 (m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.94 – 6.82 (m, 2H),
6.60 – 6.50 (m, 2H), 4.59 (s, 2H), 4.05 – 3.91 (m, 2H), 2.80 (dd, J = 8.5,
6.5 Hz, 2H), 2.48 – 2.36 (m, 2H), 1.31 (t, J = 7.0 Hz, 3H). 13C NMR (75
MHz, DMSO‑d6) δ 165.96, 162.75, 160.16, 155.33, 140.92, 131.75,
129.43, 119.32, 114.85, 114.21, 111.25, 102.54, 63.18, 31.12, 24.44,
15.17. HRMS (ESI): calcd for C19H21N2O4 341.1423; found 341.1407
[M + H]+.
4.1.11. General synthetic procedure for target compounds 13–14
A similar process as compounds 10–12 provided the methyl ester of
13 and 14, which was dissolved in 4:4:1 THF/MeOH/H2O (18 ml) and
added LiOH⋅H2O (3 equiv). After stirring at room temperature for 4 h,
the volatiles were removed under reduced pressure. The residue was
acidified with 1 N hydrochloric acid solution, and then the aqueous layer
was extracted with ethyl acetate, and the collected layer was elaborated
in vacuum to afford the targeted compound.
4.1.6. (E)-7-((3,7-dimethylocta-2,6-dien-1-yl)oxy)quinolin-2(1H)-one
(9)
A similar process as compound 6 by using starting material 7-hy-
droxy-1H-quinolin-2-one, which provided compound 9 as yellow solid,
yield 57%. 1H NMR (300 MHz, DMSO‑d6) δ 10.75 (s, 1H), 7.84 (d, J =
9.4 Hz, 1H), 7.64 – 7.57 (m, 1H), 7.09 – 6.87 (m, 2H), 6.35 (d, J = 9.4
Hz, 1H), 5.46 (t, J = 6.6 Hz, 1H), 5.07 (t, J = 6.6 Hz, 1H), 4.68 (d, J =
5.8 Hz, 2H), 2.15 – 1.95 (m, 4H), 1.75 (s, 3H), 1.63 (s, 3H), 1.57 (s, 3H).
13C NMR (75 MHz, DMSO‑d6) δ 164.85, 160.57, 154.35, 151.26, 143.45,
132.51, 125.62, 123.45, 118.51, 114.16, 112.97, 111.24, 101.75, 65.56,
38.82, 26.21, 25.83, 18.51, 16.76. HRMS (ESI): calcd for C19H23NO2
298.1729; found 298.1713 [M + H]+.
4.1.12. 4-(2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)acetamido)
benzoic acid (13)
Yield 35%, 1H NMR (300 MHz, DMSO‑d6) δ 10.56 (s, 1H), 10.11 (s,
1H), 7.93 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.08 (t, J = 7.9
Hz, 1H), 6.56 – 6.48 (m, 2H), 4.71 (s, 2H), 2.80 (dd, J = 8.5, 6.5 Hz, 2H),
2.43 (dd, J = 8.5, 6.5 Hz, 2H). 13C NMR (75 MHz, DMSO‑d6) δ 170.85,
170.62, 167.58, 167.37, 157.50, 142.98, 139.69, 130.83, 128.85,
125.95, 119.34, 116.85, 102.51, 31.12, 24.44. HRMS (ESI): calcd for
C
18H15N2O5 339.1059; found 339.1038 [Mꢀ H]-.
4.1.7. General synthetic procedure for target compounds 10–12
The choroacetylchloride (1.5 equiv) dissolved in dry dichloro-
methane (10 ml) was added dropwise to a solution of substituted aniline
(1 equiv) and triethylamine (3 equiv) in dichloromethane (35 ml),
keeping the temperature at 0 ◦C for 2 h, and then, the mixture was
stirred at room temperature for 16 h. The reaction solution was washed
with 1 N hydrochloric acid, saturated aqueous Na2CO3, and brine in
sequence. The organic layer was dried over anhydrous sodium sulfate,
filtered and evaporated under reduced pressure. The crude product and
starting material were dissolve in acetonitrile (20 ml), and added K2CO3
(5 equiv) at room temperature. The reaction mixture was heated to
reflux with stirring for 6 h. Then the reaction mixture was cooled to
room temperature followed by filtration and the filtrate was concen-
trated under vacuum. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl acetate (1:10,
v/v) as eluent to afford target compounds.
4.1.13. 3-(2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)acetamido)
benzoic acid (14)
Yield 39%, 1H NMR (300 MHz, DMSO‑d6) δ 10.59 (s, 1H), 10.13 (s,
1H), 8.42 – 8.26 (m, 1H), 7.98 – 7.81 (m, 1H), 7.68 (dt, J = 7.7, 1.3 Hz,
1H), 7.53 – 7.37 (m, 1H), 7.10 (d, J = 8.9 Hz, 1H), 6.59 – 6.53 (m, 2H),
4.71 (s, 2H), 2.81 (dd, J = 8.5, 6.5 Hz, 2H), 2.44 (dd, J = 8.5, 6.4 Hz,
2H). 13C NMR (75 MHz, DMSO‑d6) δ 170.85, 167.59, 167.35, 157.54,
139.68, 139.19, 131.73, 129.45, 128.88, 124.90, 124.25, 120.88,
116.80, 108.03, 102.57, 67.49, 31.13, 24.45. HRMS (ESI): calcd for
C
18H15N2O5 339.1059; found 339.1043 [Mꢀ H]-.
4.2. Evaluation for FXR
The human BSEP proximal promoter was amplified from a BAC clone
containing the upstream genomic region of human BSEP and cloned into
the pGL3 Basic vector create a BSEP promoter-reporter plasmid (named
pGL3-hBSEP-luc) [26]. HEK293T cells were transfected with pGL3-
hBSEP-luc and expression vectors encoding human FXR (NR1H4)
using Fugene 6 (Promega) according to manufacturer instructions.
Following transfection, test compounds were added in a 10 point dose
response and cells were incubated at 37 ◦C with 5% CO2 incubator for
24 h. Promoter activity was determined by using Steady-Glo reagent
(Promega). The maximum efficacy of the positive control agonist CDCA
is arbitrarily set at 100%.
4.1.8. N-(4-ethoxyphenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide
(10)
Yield 47%, 1H NMR (400 MHz, DMSO‑d6) δ 10.05 (s, 1H), 8.00 (dd, J
= 9.6, 0.9 Hz, 1H), 7.67 (dd, J = 8.8, 1.2 Hz, 1H), 7.58 – 7.49 (m, 2H),
7.09 – 7.01 (m, 2H), 6.94 – 6.81 (m, 2H), 6.32 (dd, J = 9.5, 1.0 Hz, 1H),
4.57 (s, 2H), 4.05 – 3.92 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, DMSO‑d6) δ 165.76, 161.39, 160.66, 155.62, 155.30, 144.71,
131.71, 129.99, 121.77, 114.83, 113.31, 113.29, 113.20, 102.10, 67.79,
63.54, 15.15. HRMS (ESI): calcd for C19H18NO5 340.1107; found
340.1123 [M + H]+.
7

Q. Qiu et al.
Bioorganic Chemistry 115 (2021) 105198
is a molecular target for the effects of vertical sleeve gastrectomy, Nature 509
(2014) 183.
4.3. Docking procedure
[6] D.A.A. Hollman, A. Milona, K.J. van Erpecum, S.W.C. van Mil, Anti-inflammatory
and metabolic actions of FXR: Insights into molecular mechanisms, BBA-Mol Cell
Biol L 1821 (11) (2012) 1443–1452.
MOE (version 2014.0901, The Chemical Computing Group, Mon-
treal, Canada) was used to perform the docking modeling based on the
reported crystal structure of FXR (PDB ID: 1OSV) [27]. Prior to molec-
ular docking, other crystallized ligands and water were removed, and
the obtained protein was performed by Protonate 3D prior to the
Gaussian Contact surface was draw around the binding pocket. Subse-
quently, the binding pocket with Gaussian Contact surface was isolated.
Then, the selected compound was docked into the binding pocket and
ranked with London dG scoring function. For energy minimization in
binding pocket, MOE Forcefield Refinement was performed and ranked
with London dG scoring function.
[7] R.M. Nijmeijer, R.M. Gadaleta, S.W.C. van Mil, A.A. van Bodegraven, J.B.
A. Crusius, G. Dijkstra, D.W. Hommes, D.J. de Jong, P.C.F. Stokkers, H.
W. Verspaget, R.K. Weersma, C.J. van der Woude, J.M. Stapelbroek, M.E.
I. Schipper, C. Wijmenga, K.J. van Erpecum, B. Oldenburg, S. Bereswill, Dutch
Initiative, Farnesoid X Receptor (FXR) Activation and FXR Genetic Variation in
Inflammatory Bowel Disease, Plos One 6 (8) (2011) e23745.
[8] B. Goodwin, S.A. Jones, R.R. Price, M.A. Watson, D.D. McKee, L.B. Moore,
C. Galardi, J.G. Wilson, M.C. Lewis, M.E. Roth, P.R. Maloney, T.M. Willson, S.
A. Kliewer, A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1
represses bile acid biosynthesis, Mol Cell 6 (3) (2000) 517–526.
[9] A. Gioiello, B. Cerra, S. Mostarda, C. Guercini, R. Pellicciari, A. Macchiarulo, Bile
Acid Derivatives as Ligands of the Farnesoid X Receptor: Molecular Determinants
for Bile Acid Binding and Receptor Modulation, Curr Top Med Chem 14 (2014)
2159–2174.
4.4. Acetaminophen-indcued liver toxicity
[10] Y. Zhang, P.A. Edwards, FXR signaling in metabolic disease, Febs Letters 582
(2008) 10–18.
[11] L. Adorini, M. Pruzanski, D. Shapiro, Farnesoid X receptor targeting to treat
nonalcoholic steatohepatitis, Drug Discov Today 17 (2012) 988–997.
[12] M. Trauner, E. Halilbasic, Nuclear Receptors as New Perspective for the
Management of Liver Diseases, Gastroenterology, 140 (2011) 1120-+.
[13] A. Carotti, M. Marinozzi, C. Custodi, B. Cerra, R. Pellicciari, A. Gioiello,
A. Macchiarulo, Beyond Bile Acids: Targeting Farnesoid X Receptor (FXR) with
Natural and Synthetic Ligands, Curr Top Med Chem 14 (2014) 2129–2142.
[14] F. Nevens, P. Andreone, G. Mazzella, S.I. Strasser, C. Bowlus, P. Invernizzi, J.
P. Drenth, P.J. Pockros, J. Regula, U. Beuers, M. Trauner, D.E. Jones, A. Floreani,
S. Hohenester, V. Luketic, M. Shiffman, K.J. van Erpecum, V. Vargas, C. Vincent, G.
M. Hirschfield, H. Shah, B. Hansen, K.D. Lindor, H.U. Marschall, K.V. Kowdley,
R. Hooshmand-Rad, T. Marmon, S. Sheeron, R. Pencek, L. MacConell,
M. Pruzanski, D. Shapiro, A Placebo-Controlled Trial of Obeticholic Acid in
Primary Biliary Cholangitis, New Engl J Med 375 (2016) 631–643.
[15] S. Fiorucci, C. Di Giorgio, E. Distrutti, Obeticholic Acid: An Update of Its
Pharmacological Activities in Liver Disorders, Handbook of experimental
pharmacology 256 (2019) 283–295.
Eight weeks old male C57BL/6 mice were purchased from Model
Animal Research Center of Nanjing University (Jiangsu, China). All
experimental protocols were approved by the ethical committee at
Yancheng Teachers’ University and conducted according to the Labo-
ratory Animal Management Regulations in China and adhered to the
Guide for the Care and Use of Laboratory Animals published by the
National Institutes of Health (2011). Mice were randomly divided into
groups (n = 7 per group): normal, APAP model, APAP + AUR (20 mg/
kg), APAP + 14 (20 mg/kg). Mice received once daily gavage of drug for
5 days. In the 5th day of dosing drug, overnight-fasted mice received
APAP (300 mg/kg) by intraperitoneal (i.p.) injection. Mice were sacri-
ficed 12 h after APAP injection and blood and livers were collected.
Serum levels of ALT and AST were measured by automatic biochemical
analyzer (AU5811, Japan). Liver GSH was measured by GSH assay kit
(Nanjing Jiancheng, China). Part of the liver sample was fixed in 10%
formalin for histology.
[16] J. Hindson, Obeticholic acid for the treatment of NASH, Nature reviews. Nat Rev
Gastro Hepat 17 (2020) 66.
[17] F. Briand, E. Brousseau, M. Quinsat, R. Burcelin, T. Sulpice, Obeticholic acid raises
LDL-cholesterol and reduces HDL-cholesterol in the Diet-Induced NASH (DIN)
hamster model, Eur J Pharmacol 818 (2018) 449–456.
[18] C. Gege, E. Hambruch, N. Hambruch, O. Kinzel, C. Kremoser, Nonsteroidal FXR
Ligands: Current Status and Clinical Applications, Handbook of experimental
pharmacology 256 (2019) 167–205.
Declaration of Competing Interest
[19] S. Schierle, J. Schmidt, A. Kaiser, D. Merk, Selective Optimization of Pranlukast to
Farnesoid X Receptor Modulators, ChemMedChem 13 (2018) 2530–2545.
[20] L. Hu, Q. Ren, L. Deng, Z. Zhou, Z. Cai, B. Wang, Z. Li, Design, synthesis, and
biological studies of novel 3-benzamidobenzoic acid derivatives as farnesoid X
receptor partial agonist, Eur J Med Chem 211 (2020), 113106.
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.
[21] A. Giancristofaro, A.J.M. Barbosa, A. Ammazzalorso, P. Amoia, B. De Filippis,
M. Fantacuzzi, L. Giampietro, C. Maccallini, R. Amoroso, Discovery of new FXR
agonists based on 6-ECDCA binding properties by virtual screening and molecular
docking, Medchemcomm 9 (10) (2018) 1630–1638.
Acknowledgements
This study was supported by National Natural Science Foundation of
China (No.81803353), Natural Science Foundation of Jiangsu Province
(No. BK20191482 and BK20181478), Lift Project of Jiangsu Association
for Science and Technology for Young Science and Technology Talents
and University Science Research Project of Jiangsu Province
(20KJB350006).
[22] F. Epifano, S. Genovese, E. James Squires, M.A. Gray, Nelumal A, the active
principle from Ligularia nelumbifolia, is a novel farnesoid X receptor agonist,
Bioorg Med Chem Lett 22 (9) (2012) 3130–3135.
[23] J. Wang, T. Fu, R. Dong, C. Wang, K. Liu, H. Sun, X. Huo, X. Ma, X. Yang, Q. Meng,
Hepatoprotection of auraptene from the peels of citrus fruits against 17 alpha-
ethinylestradiol-induced cholestasis in mice by activating farnesoid X receptor,
Food Funct 10 (2019) 3839–3850.
[24] X. Gao, T. Fu, C. Wang, C. Ning, Y. Kong, Z. Liu, H. Sun, X. Ma, K. Liu, Q. Meng,
Computational discovery and experimental verification of farnesoid X receptor
agonist auraptene to protect against cholestatic liver injury, Biochem pharmacol
146 (2017) 127–138.
References
[1] B.M. Forman, E. Goode, J. Chen, A.E. Oro, D.J. Bradley, T. Perlmann, D.J. Noonan,
L.T. Burka, T. McMorris, W.W. Lamph, R.M. Evans, C. Weinberger, Identification of
a nuclear receptor that is activated by farnesol metabolites, Cell 81 (1995)
687–693.
[25] X. Gao, C. Wang, C. Ning, K. Liu, X. Wang, Z. Liu, H. Sun, X. Ma, P. Sun, Q. Meng,
Hepatoprotection of auraptene from peels of citrus fruits against thioacetamide-
induced hepatic fibrosis in mice by activating farnesoid X receptor, Food Funct 9
(5) (2018) 2684–2694.
[2] M. Makishima, A.Y. Okamoto, J.J. Repa, H. Tu, R.M. Learned, A. Luk, M.V. Hull, K.
D. Lustig, D.J. Mangelsdorf, B. Shan, Identification of a nuclear receptor for bile
acids, Science 284 (1999) 1362–1365.
[26] D.C. Tully, P.V. Rucker, D. Chianelli, J. Williams, A. Vidal, P.B. Alper, D. Mutnick,
B. Bursulaya, J. Schmeits, X. Wu, D. Bao, J. Zoll, Y. Kim, T. Groessl, P. McNamara,
H.M. Seidel, V. Molteni, B. Liu, A. Phimister, S.B. Joseph, B. Laffitte, Discovery of
Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment
of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH), J Med
Chem 60 (2017) 9960–9973.
[3] D.J. Parks, S.G. Blanchard, R.K. Bledsoe, G. Chandra, T.G. Consler, S.A. Kliewer, J.
B. Stimmel, T.M. Willson, A.M. Zavacki, D.D. Moore, J.M. Lehmann, Bile acids:
Natural ligands for an orphan nuclear receptor, Science 284 (1999) 1365–1368.
[4] R.M. Gadaleta, M. Cariello, C. Sabba, A. Moschetta, Tissue-specific actions of FXR
in metabolism and cancer, BBA-Mol Cell Biol L 2015 (1851) 30–39.
[5] K.K. Ryan, V. Tremaroli, C. Clemmensen, P. Kovatcheva-Datchary, A. Myronovych,
R. Karns, H.E. Wilson-Perez, D.A. Sandoval, R. Kohli, F. Backhed, R.J. Seeley, FXR
[27] L.Z. Mi, S. Devarakonda, J.M. Harp, C. Han, R. Pellicciari, T.M. Willson,
S. Khorasanizadeh, F. Rastinejad, Structural basis for bile acid binding and
activation of the nuclear receptor FXR, Mol Cell 11 (2003) 1093–1100.
8