Synthesis and biological evaluation of novel SIPI-7623 derivatives as farnesoid X receptor (FXR) antagonists

Article abstract of DOI:10.1007/s11030-018-9843-2

Most of reported steroidal FXR antagonists are restricted due to low potency. We described the design and synthesis of novel nonsteroidal scaffold SIPI-7623 derivatives as FXR antagonists. The most potent compound A-11 (IC₅₀ = 7.8 ± 1.1 μM) showed better activity compared to SIPI-7623 (IC₅₀ = 40.8 ± 1.7 μM) and guggulsterone (IC₅₀ = 45.9 ± 1.1 μM). Docking of A-11 in FXR’s ligand-binding domain was also studied.

Full text of DOI:10.1007/s11030-018-9843-2

Molecular Diversity
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ORIGINAL ARTICLE
Synthesis and biological evaluation of novel SIPI-7623 derivatives
as farnesoid X receptor (FXR) antagonists
Si-Yun Nian1,2,3 Guo-Ping Wang2,3 Zheng-Li Jiang1 Ying Xiao2 Mo-Han Huang4 Yi-Huan Zhou4
Xiang-Duan Tan4
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•
•
•
•
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Received: 3 November 2017 / Accepted: 13 June 2018
Ó Springer International Publishing AG, part of Springer Nature 2018
Abstract
Most of reported steroidal FXR antagonists are restricted due to low potency. We described the design and synthesis of
novel nonsteroidal scaffold SIPI-7623 derivatives as FXR antagonists. The most potent compound A-11 (IC50-
= 7.8 1.1 lM) showed better activity compared to SIPI-7623 (IC50 = 40.8 1.7 lM) and guggulsterone (IC50-
= 45.9 1.1 lM). Docking of A-11 in FXR’s ligand-binding domain was also studied.
Keywords FXR antagonists Á SIPI-7623 Á Guggulsterone Á Structure activity relationship Á Molecular docking
Abbreviations
Introduction
FXR
LBD
GS
Farnesoid X receptor
Ligand-binding domain
Guggulsterone
Farnesoid X receptor (FXR), a ligand-activated transcrip-
tion factor, is highly expressed in liver, intestine, kidney
and adrenal glands [1]. It contains a ligand-binding domain
(LBD) in the C-terminal, a DNA-binding domain in the
N-terminal and a connection hinge region. After binding to
diverse ligands, the LBD controls the release of corepressor
proteins and the subsequent recruitment of coactivator
proteins [2].
TC
Total cholesterol
Triacylglycerol
TG
LDL-C Low-density lipoprotein cholesterol
Intensive research on finding FXR antagonists has
yielded some low potency steroidal compounds, such as
guggulsterone (GS) [3], sulfated polyhydroxysterols,
16-dehydro-pregnenolone (Fig. 1). The first reported FXR
antagonist GS displayed high affinity to progesterone
receptor, androgen receptor and corticoid receptor [4, 5].
Newly discovered 12u and FXR-99 were identified as
nonsteroidal FXR antagonists with IC50 values of 8.96 and
3.4 lM, respectively, which are useful tools for the study
on FXR antagonists [6–9] (Fig. 1).
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-018-9843-2) contains
supplementary material, which is available to authorized
users.
& Xiang-Duan Tan
tandy@glmc.edu.cn
Our group found in a previous study that nonsteroidal
compound SIPI-7623 was able to decrease total cholesterol
(TC) and triacylglycerol (TG) levels, and increase low-
density lipoprotein cholesterol (LDL-C) level in hyperc-
holesterolemic rats. We found that SIPI-7623 is an FXR
antagonist with an IC50 of 57.7 1.9 lM via TR-FRET
screening. In a dual luciferase assay, SIPI-7623 exhibited
FXR selectivity toward PPARa (IC50 = 44.7 0.8 lM for
FXR, IC50 [ 100 lM for PPARa, EC50 [ 100 lM for
FXR, EC50 [ 100 lM for PPARa. Its derivatives with
1
Department of Clinical Pharmacy, Taizhou Hospital of
Zhejiang Province, Xi Men Street No. 150,
Linhai 317000, Zhejiang Province, China
2
Shanghai Institute of Pharmaceutical Industry, China State
Institute of Pharmaceutical Industry, Shanghai 201203, China
3
Aurisco Pharmaceutical (Yangzhou) Co., Ltd.,
Yangzhou 225100, China
4
College of Pharmacy, Guilin Medical University,
Guilin 541004, China
123

Molecular Diversity
Scheme 1 Reagents and conditions: a CuBr2, EtOAc/CHCl3 (1:1);
b K2CO3, THF
Fig. 1 Example of reported FXR antagonists
Scheme 2 Reagents and conditions: a H2S, CDI, DMF; b compound
2a–2n and 2q, K2CO3, THF
Fig. 2 Design of novel SIPI-7623 derivatives
Scheme 3 Reagents and conditions: a (1) K2CO3, DMF; (2) NaOH,
EtOH; b compound 2j–2o and 2q, K2CO3, THF
ester linker (Y-4, IC50 = 40.1 1.2 lM) and thioester
linker (Y-5, IC50 = 20.4 2.1 lM) slightly increased
FXR antagonistic activity (Fig. 2). In the course of our
search for more potent FXR antagonists, we took SIPI-
7623 as a lead, focused on the substituents on the benzene
ring at region I, the methyl group truncation on region II
and various substituents on region III (Fig. 2). Thus, 61
compounds were designed, synthesized and evaluated for
their activity toward FXR in vitro.
carbonyldiimidazole (CDI) in an H2S atmosphere, com-
pound 3 was converted to compound 4 [13], then reacted
with compounds 2a–2n and 2q using a route identical to
that in Scheme 2 to get compounds A-17–A-30 and Y-5.
The synthetic route of compounds B-1–B-7 (Table 2) is
shown in Scheme 3. 2,5-Dimethylphenol (5) and ethyl
5-bromopentanoate (6), underwent Williamson ether syn-
thesis conditions, and then were hydrolyzed with NaOH to
afford compound 7 [14–16]. After that, compounds 2j–2o
and 2q were reacted with compound 7 in the presence of
K2CO3 to generate B-1–B-7.
Results and discussion
Compounds C-1–C-24 (Table 3) were synthesized as
shown in Scheme 4. Treatment of ethyl isobutyrate (8)
with 1-bromo-3-chloropropane (9) afforded ethyl 5-chloro-
2,2-dimethylpentanoate (10) [17, 18], then compound 10
reacted with appropriate phenol derivatives 11a–11q to
furnish compounds 12a–12q which in turn were hydro-
lyzed with sodium hydroxide in ethanol to afford the desire
compounds 13a–13q. Finally, compounds 13a–13q reacted
with 2n or 2q to obtain compounds C-1–C-24.
Synthesis
The synthetic route of compounds A-1–A-16 (Table 1) and
Y-4 is outlined in Scheme 1. Substituted acetophenones
1a–1q were converted to 2a–2q with CuBr2 [10, 11] and
then reacted with compound 3 in the presence of K2CO3 to
obtain A-1–A-16 and Y-4 [11, 12]. A suitable crystal of A-
4 was obtained by slowly evaporating a mixture of ethyl
acetate and petroleum ether solution at ambient tempera-
ture and characterized by single-crystal X-ray diffraction.
Compounds A-17–A-30 (Table 1) and Y-5 were syn-
thesized as shown in Scheme 2. In the presence of N,N0-
123

Molecular Diversity
a
Cl
O
O
+
Br
Cl
O
O
O
8
9
10
Ar OH
(11a-11q)
c
O
OH
O
O
Ar
Ar
b
O
12a-12q
13a-13q
O
R1
R2
O
O
Fig. 4 SIPI-7623 (green) and C-17 (pink) in FXR-LBD
d
Ar
O
R3
C-1~C-24
Scheme 4 Reagents and conditions: a DMPU, LDA, THF; b K2CO3,
DMF, TBAB; c NaOH, EtOH; d compound 2n or 2q, K2CO3, THF
Biological evaluation
In our initial study, we concluded that SIPI-7623 deriva-
tives with an ester and thioester linker were superior to
derivatives with an amide linker in terms of antagonistic
activity to FXR. We first investigated the effect on FXR
antagonistic activity of different substituted groups on
benzene ring at region I (Table 1). Derivatives without a
hydrophilic hydroxyl group exhibited no activity against
FXR. Interestingly, most of hydrophilic hydroxyl substi-
tuted derivatives, especially the FXR antagonistic activity
of 3,5-dibromo-4-hydroxy derivatives (A-11, A-27), 3,5-
dihydroxy derivatives (A-14, A-30) and 4-hydroxy-3-nitro
derivative (A-29), are three to five fold over that in GS and
SIPI-7623, with IC50 values of 7.8 1.1, 10.8 1.2,
14.8 0.9, 11.1 1.3 and 10.3 1.2 lM, respectively.
From the above results, we concluded that the phenolic
group in region I is a key element for FXR antagonistic
activity in the SIPI-7623 derivatives.
Fig. 5 Molecular solid structure projection of A-4
compounds B1–B7 (Table 2). Compounds B-4 and B-7
were inactive, B-3 and B-5 almost totally lost activity, B-1
(IC50 = 63.5 3.4 lM) and B-6 (IC50 = 27.5 1.3 lM)
displayed weak antagonistic activity compared to GS
(IC50 = 47.8 0.8 lM)
and
SIPI-7623
(IC50-
= 7.8 1.1 lM), respectively. Only compound B-2
(IC50 = 29.0 2.4 lM) exhibited an equivalent antago-
nistic activity compared to A-15 (IC50 = 22.1 2.1 lM).
This indicated that the truncation of methyl group results in
a slightly loss of FXR antagonistic potency.
Furthermore, we investigated the effect of various sub-
stituents in region III, while keeping the hydrophilic
hydroxyl group in region I and methyl group in region II.
All of the designed derivatives demonstrated antagonistic
potency toward FXR, except for C-18 (Table 3). Unfor-
tunately, the antagonistic potency of the designed com-
pounds did not improve.
Docking of compound A-11 into the FXR-LBD revealed
that the phenolic group formed a hydrogen bond with
Arg331 (Fig. 3). This is similar to the binding mode of
SIPI-7623. From the above SAR and docking results, we
propose that the hydrophilic hydroxyl on region I is
essential for FXR antagonistic affinity.
Docking potent analog C-17 into FXR-LBD indicates
that highly lipophilic and sterically substituents in region
III are more favorable than the 2,5-dimethylphenoxy group
in SIPI-7623 (Fig. 4).
Then, we kept the phenolic group in region I and the
methyl groups in region II were removed to generate
Conclusion
SIPI-7623 is an FXR antagonist with weak potency. SAR
studies of its derivatives revealed that the core structure is a
promising scaffold for the development of novel FXR
antagonists. Region I with phenolic group is essential for
Fig. 3 Molecular docking of A-11
123

Molecular Diversity
antagonistic activity, compound with methyl substituted in
region II was more potent than compound with no sub-
stituted in region II, and region III was the key element.
Compound containing 3,5-dibromo-4-hydroxy in region I,
methyl group in region II and 2,5-dimethylphenoxy group
in region III, which is A-11, was the most potent antago-
nist with an IC50 value of 7.8 1.1 lM (Table 1).
temperature for 12 h. After the reaction was completed, the
solution was concentrated. The oil was washed with dilute
HCl and ethyl acetate, dried over MgSO4, filtered, con-
centrated, and the residue was purified by chromatography
to afford compound A-1. Compounds A-2–A-16, Y-4 were
prepared in a similar manner to the synthesis of compound
A-1.
2-Oxo-2-phenylethyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-1) Yield 85.2%; white solid, mp:
58.6–61.6 °C; H NMR (400 MHz, DMSO-d6) d: 7.96 (d,
Experimental
1
J = 8.4 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.56 (t,
J = 7.7 Hz, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.73 (s, 1H),
6.63 (d, J = 7.5 Hz, 1H), 5.48 (s, 2H), 3.93 (t, J = 5.5 Hz,
2H), 2.25 (s, 3H), 2.10 (s, 3H), 1.90–1.63 (m, 4H), 1.24 (s,
6H); ESI–MS m/z: 367 [M - H]-.
Chemistry
All solvents and reagents were commercially available and
used without further purification. Melting points were
determined on WRS-21 melting point apparatus and are
uncorrected. 1H-NMR spectra were recorded on an INOVA
400 (400 MHz) or INOVA 300 (300 MHz) spectrometer
using tetramethylsilane (TMS) as internal standard.
Chemical shifts (d) are reported in parts per million relative
to TMS, and coupling constants (J) are given in Hertz (Hz).
Abbreviations for NMR signals are as follows: s, single; d,
doublet; t, triplet; dd, doublet of doublets; m, multiplet.
Electron-spray ionization mass spectra (ESI–MS) in posi-
tive mode were recorded on a HP5989A mass spectrome-
ter. The purity of all novel compounds was checked by
2-Oxo-2-(p-tolyl)ethyl-5-(2,5-dimethylphenoxy)-2,2-dimethyl-
pentanoate (A-2) Yield 83.7%; white solid, mp:
1
57.7–59.1 °C; H NMR (400 MHz, DMSO-d6) d: 7.86 (d,
J = 8.2 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 6.98 (d,
J = 7.5 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.5 Hz, 1H),
5.43 (s, 2H), 3.93 (t, J = 5.4 Hz, 2H), 2.38 (s, 3H), 2.25 (s,
3H), 2.10 (s, 3H), 1.88 -1.59 (m, 4H), 1.23 (s, 6H); ESI–
MS m/z: 381 [M - H]-.
2-(4-Methoxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanoate (A-3) Yield 84.2%; white solid,
mp: 55.0–57.3 °C; 1H NMR (400 MHz, DMSO-d6) d: 7.94
(d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.98 (d,
J = 7.5 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.5 Hz, 1H),
5.41 (s, 2H), 3.92 (t, J = 7.7 Hz, 2H), 3.85 (s, 3H), 2.25 (s,
3H), 2.10 (s, 3H), 1.87–1.59 (m, 4H), 1.23 (s, 6H); ESI–MS
m/z: 397 [M - H]-, 399 [M ? H]?.
1
TLC and H NMR. All reactions were monitored by TLC
on pre-coated Silica Gel F254 plates (purchased from
Yantai Jiangyou Silicone Development Co., Ltd., China)
with detection by UV.
General procedure for the synthesis
of compounds 2a22q
2-(4-Fluorophenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-4) (Table 4, Fig. 5) Yield 80.8%;
white solid, mp: 50 °C; 1H NMR (400 MHz, DMSO-d6) d:
8.05 (dd, J = 8.8, 5.5 Hz, 2H), 7.39 (t, J = 8.9 Hz, 2H),
6.98 (d, J = 7.5 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.4 Hz,
1H), 5.46 (s, 2H), 3.93 (t, J = 5.3 Hz, 2H), 2.25 (s, 3H),
2.10 (s, 3H), 1.80-1.70 (m, 4H), 1.24 (s, 6H); ESI–MS m/z:
385 [M - H]-.
To a stirred solution of acetophenone 1a (5 mmol) in
chloroform (150 mL) was added ethyl acetate (150 mL),
CuBr2 (10 mmol), and the resulting mixture was stirred at
80 °C for 6 h.
The reaction mixture was filtered, water (150 mL) was
added and the mixture was extracted with dichloromethane
(60 mL 9 3). The combined organic layer was washed
with brine and dried over MgSO4, filtered, evaporated and
purified by flash column chromatography to get compound
2a. Compounds 2b–2q were prepared in a similar manner
to the synthesis of compound 2a (75–90% yield) and were
used without further purification.
2-(4-Chlorophenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-5) Yield 89.2%; white solid, mp:
1
50.4–51.5 °C; H NMR (400 MHz, DMSO-d6) d: 7.97 (d,
J = 8.6 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 6.98 (d,
J = 7.4 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.6 Hz, 1H),
5.46 (s, 2H), 3.93 (t, J = 5.1 Hz, 2H), 2.24 (s, 3H), 2.09 (s,
3H), 1.83–1.55 (m, 4H), 1.23 (s, 6H); ESI–MS m/z: 401
[M - H]-.
General procedure for the synthesis
of compounds A-1–A-16, Y-4
A stirred solution of compound 2a (10.0 mmol) and com-
pound 3 (10.0 mmol) in THF (30 mL) was added K2CO3
(10.0 mmol). The resultant mixture was stirred at room
2-(4-Bromophenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-6) Yield 81.5%; white solid, mp:
52.4–53.0 °C; H NMR (400 MHz, DMSO-d6) d: 7.89 (d,
1
123

Molecular Diversity
Table 1 Structures of A-1–A-30 and screening results
Compd
X
R1
R2
R3
Activation ratio at 10 lMa
Inhibition ration at 50 lMa
ICb50 (lM)
A-1
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
H
H
H
0.2%
0.3%
0.8%
- 0.3%
0.6%
0.8%
8.9%
0.6%
0.0%
1.4%
3.1%
4.1%
16.7%
2.1%
- 0.7%
4.7%
0.3%
0.3%
0.0%
0.6%
0.6%
2.8%
4.7%
0.3%
0.6%
1.1%
- 0.3%
3.0%
3.3%
- 0.7%
ND
11.5%
13.8%
11.8%
10.8%
8.8%
NDd
A-2
H
CH3
OCH3
F
H
ND
A-3
H
H
ND
A-4
H
H
ND
A-5
H
Cl
H
ND
A-6
H
Br
H
11.1%
17.2%
11.5%
11.8%
37.4%
88.1%
66.6%
17.0%
80.1%
78.2%
30.0%
14.8%
19.2%
17.2%
12.1%
9.1%
ND
A-7
H
I
H
ND
A-8
H
CF3
NO2
OH
OH
OH
OH
H
H
ND
A-9
H
H
ND
A-10
A-11
A-12
A-13
A-14
A-15
A-16
A-17
A-18
A-19
A-20
A-21
A-22
A-23
A-24
A-25
A-26
A-27
A-28
A-29
A-30
Y-4
CH3
Br
F
CH3
Br
H
ND
7.8 1.1
33.0 1.2
ND
NO2
OH
H
H
OH
OH
H
14.8 0.9
22.1 2.1
ND
H
H
COOH
H
H
H
ND
S
H
CH3
OCH3
F
H
ND
S
H
H
ND
S
H
H
ND
S
H
Cl
H
ND
S
H
Br
H
10.1%
13.8%
7.1%
ND
S
H
I
H
ND
S
H
CF3
NO2
OH
OH
OH
OH
H
H
ND
S
H
H
14.5%
35.7%
92.2%
68.3%
82.8%
80.9%
ND
ND
S
CH3
Br
F
CH3
Br
H
ND
S
10.8 1.2
30.5 0.9
10.3 1.2
11.1 1.3
42.5 2.3
23.8 1.3
45.9 1.6
40.8 1.7
S
S
NO2
OH
H
H
S
OH
H
O
S
OH
OH
Y-5
GSc
SIPI-7623c
H
H
ND
ND
aResults are given as the mean of two independent experiments
bResults are given as the mean SD of three independent experiments
cUsed as a positive control
dNot determined
123

Molecular Diversity
Table 2 Structures of B-1–B-7 and screening results
Compd
R1
R2
R3
Activation ratio at 10 lMa (%)
Inhibition ration at 50 lMa (%)
ICb50 (lM)
B-1
H
OH
H
H
- 0.3
- 1.1
5.3
50.9
78.6
45.5
10.9
44.6
76.4
12.7
63.5 3.4
29.0 2.4
NDd
B-2
H
OH
OH
H
B-3
OH
F
H
B-4
OH
OH
OH
OH
1.1
ND
B-5
CH3
Br
CH3
Br
0.2
ND
B-6
1.2
27.5 1.3
ND
B-7
GSc
SIPI-7623c
NO2
H
0.8
47.8 0.8
37.7 2.1
aResults are given as the mean of two independent experiments
bResults are given as the mean SD of three independent experiments
cUsed as a positive control
dNot determined
J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 6.98 (d,
J = 7.5 Hz, 1H), 6.72 (s, 1H), 6.63 (d, J = 7.5 Hz, 1H),
5.46 (s, 2H), 3.93 (t, J = 4.9 Hz, 2H), 2.25 (s, 3H), 2.10 (s,
3H), 1.75 (t, J = 10.0 Hz, 4H), 1.23 (s, 6H); ESI–MS m/z:
445 [M - H]-.
2-(4-Hydroxy-3,5-dimethylphenyl)-2-oxoethyl-5-(2,5-dimethy-
lphenoxy)-2,2-dimethylpentanoate (A-10) Yield 61.3%;
white solid, mp: 99.3–102.3 °C; 1H NMR (400 MHz,
DMSO-d6) d: 9.31 (s, 1H), 7.59 (s, 2H), 6.98 (d,
J = 7.5 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.5 Hz, 1H),
5.34 (s, 2H), 3.93 (t, J = 5.6 Hz, 2H), 2.25 (s, 3H), 2.21 (s,
6H), 2.10 (s, 3H), 1.81–1.66 (m, 4H), 1.23 (s, 6H); ESI–MS
m/z: 411 [M - H]-, 413 [M ? H]?,435 [M ? Na]?.
2-(4-Iodophenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-7) Yield 75.1%; white solid, mp:
1
77.2–77.4 °C; H NMR (400 MHz, DMSO-d6) d: 7.96 (d,
J = 8.2 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 6.98 (d,
J = 7.4 Hz, 1H), 6.72 (s, 1H), 6.62 (d, J = 7.3 Hz, 1H),
5.44 (s, 2H), 3.92 (s, 2H), 2.25 (s, 3H), 2.09 (s, 3H), 1.73 (s,
4H), 1.23 (s, 6H); ESI–MS m/z: 493 [M - H]-.
2-(3,5-Dibromo-4-hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethy-
lphenoxy)-2,2-dimethylpentanoate (A-11) Yield 64.1%;
white solid, mp: 83.2–86.2 °C; 1H NMR (400 MHz,
CDCl3) d: 8.04 (s, 2H), 7.02 (d, J = 7.4 Hz, 1H), 6.68 (d,
J = 7.6 Hz, 1H), 6.65 (s, 1H), 5.19 (s, 2H), 3.98 (t,
J = 5.2 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H), 1.98–1.76 (m,
4H), 1.33 (s, 6H); ESI–MS m/z: 541 [M - H]-.
2-oxo-2-(4-(Trifluoromethyl)phenyl)ethyl-5-(2,5-dimethylphe-
noxy)-2,2-dimethylpentanoate (A-8) Yield 80.2%; white
1
solid, mp: 53.5–54.0 °C; H NMR (400 MHz, DMSO-d6)
d: 8.14 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 6.97
(d, J = 7.4 Hz, 1H), 6.71 (s, 1H), 6.62 (d, J = 7.4 Hz, 1H),
5.51 (s, 2H), 3.92 (d, J = 4.8 Hz, 2H), 2.24 (s, 3H), 2.10 (s,
3H), 1.74 (s, 4H), 1.23 (s, 6H); ESI–MS m/z: 435
[M - H]-.
2-(3-Fluoro-4-hydroxyphenyl)-2-oxoethyl-5-(2,5-imethylphe-
noxy)-2,2-dimethylpentanoate (A-12) Yield 65.2%; white
solid, mp: 87.1–88.3 °C; H NMR (400 MHz, CDCl3) d:
7.66 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.04
(dd, J = 15.4, 7.1 Hz, 2H), 6.68 (d, J = 7.6 Hz, 1H), 6.66
(s, 1H), 5.24 (s, 2H), 3.99 (t, J = 5.5 Hz, 2H), 2.33 (s, 3H),
2.21 (s, 3H), 1.91–1.79 (m, 4H), 1.35 (s, 6H); ESI–MS m/z:
401 [M - H]-.
1
2-(4-Nitrophenyl)-2-oxoethyl5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanoate (A-9) Yield 78.1%; oil; 1H NMR
(400 MHz, DMSO-d6) d: 8.37 (d, J = 8.7 Hz, 2H), 8.19 (d,
J = 8.8 Hz, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.72 (s, 1H),
6.62 (d, J = 7.4 Hz, 1H), 5.54 (s, 2H), 3.95–3.90 (m, 2H),
2.25 (s, 3H), 2.10 (s, 3H), 1.74 (s, 4H), 1.24 (s, 6H); ESI–
MS m/z: 412 [M - H]-.
2-(4-Hydroxy-3-nitrophenyl)-2-oxoethyl-5-(2,5-dimethylphe-
noxy)-2,2-dimethylpentanoate (A-13) Yield 57.5%; white
1
solid, mp: 50.2–52.4 °C; H NMR (400 MHz, DMSO-d6)
d: 12.19 (s, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.09 (d,
123

Molecular Diversity
Table 3 Structures of C-1–C-27 and screening results
b
Compd
C-1
R1
H
R2
R3
H
Ar
Activation ratio at 10 µMa
Inhibition ration at 50 µMa
IC50 µM
OH
-0.9%
60.5%
30.2 2.1
C-2
C-3
OH
OH
H
H
OH
OH
-1.5%
-1.5%
79.1%
51.8%
18.5 1.2
55.1 0.6
C-4
C-5
OH
OH
H
H
OH
OH
-0.75
2.7%
74.9%
78.2%
18.7 1.1
16.5 0.8
C-6
C-7
C-8
OH
OH
OH
H
H
H
OH
OH
OH
-0.9%
0.8%
70.85
64.6%
80.2%
27.5 0.6
30.2 2.1
19.5 2.0
-0.9%
C-9
OH
OH
H
H
OH
OH
-0.8%
-0.7%
67.3%
76.4%
25.0 1.8
16.9 1.2
C-10
C-11
OH
H
OH
0.4%
67.3%
29.8 1.4
C-12
C-13
C-14
OH
OH
OH
H
H
H
OH
OH
OH
0.9%
-1.3%
0.3%
80.6%
70.4%
70.9%
16.6 1.6
19.6 2.2
17.0 1.4
C-15
OH
H
OH
-0.2%
69.1%
23.7 2.3
C-16
C-17
OH
OH
H
H
OH
OH
-1.3%
-1.2%
51.8%
78.2%
54.5 2.2
15.9 0.8
C-18
C-19
OH
H
H
OH
H
1.0%
0.2%
21.8%
59.5%
>100
OH
40.6 0.9
C-20
C-21
H
H
OH
OH
H
H
-1.1%
0.4%
64.4%
66.6%
38.3 1.7
38.3 1.2
C-22
C-23
H
H
OH
OH
H
H
0.6%
2.1%
56.3%
74.3%
55.1 3.2
20.3 2.1
C-24
H
OH
H
0.1%
52.1%
59.4 1.2
GSc
43.8 1.6
42.0 2.3
SIPI-7623c
aResults are given as the mean of two independent experiments
bResults are given as the mean SD of three independent experiments
cUsed as a positive control
123

Molecular Diversity
Table 4 Crystal data and structure refinement for A-4
Identification code
A-4
Empirical formula
Formula weight
Temperature
C23 H27 F O4
386.45
296(2) K
Wavelength
1.54178 A
Crystal system, space group
Unit cell dimensions
Volume
Monoclinic, C 2/c
a = 31.1731(4) A alpha = 90°
b = 12.7247(2) A beta = 97.7680(10)°
c = 10.92390(10) A gamma = 90°
4293.40(10) A3
Z, Calculated density
8, 1.196 Mg/m3
Absorption coefficient
F(000)
0.708 mm-1
1648
Crystal size
0.19 9 0.15 9 0.12 mm
Theta range for data collection
Limiting indices
3.76°–67.38°
- 36 B h B 36, - 15 B k B 14, - 12 B l B 2
14602/3731 [R(int) = 0.0202]
96.8%
Reflections collected/unique
Completeness to theta = 67.38
Absorption correction
Max. and min. transmission
Refinement method
Semi-empirical from equivalents
0.7529 and 0.6774
Full-matrix least-squares on F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I [ 2 sigma(I)]
R indices (all data)
3731/0/253
1.057
R1 = 0.0448, wR2 = 0.1263
R1 = 0.0487, wR2 = 0.1308
0.195 and - 0.194 e A-3
Largest diff. peak and hole
J = 8.8 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 6.97 (d,
J = 7.5 Hz, 1H), 6.71 (s, 1H), 6.62 (d, J = 7.5 Hz, 1H),
5.43 (s, 2H), 3.92 (t, J = 5.1 Hz, 2H), 2.24 (s, 3H), 2.09 (s,
3H), 1.81–1.66 (m, 4H), 1.23 (s, 6H); ESI–MS m/z: 428
[M - H]-.
3H), 2.10 (s, 3H), 1.74 (s, 2H), 1.63 (s, 2H), 1.23 (s, 3H),
1.12 (s, 3H); ESI–MS m/z: 383 [M - H]-.
4-(2-((5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanoyl)oxy)
acetyl)benzoic acid (A-16) Yield 32.4%; white solid, mp:
1
104.9–107.9 °C; H NMR (400 MHz, DMSO-d6) d: 8.08
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphe-
noxy)-2,2-dimethylpentanoate (A-14) Yield 65.7%; white
solid, mp: 116.1–117.2 °C; H NMR (400 MHz, DMSO-
d6) d: 9.72 (s, 2H), 6.98 (d, J = 7.4 Hz, 1H), 6.78 (d,
J = 1.8 Hz, 2H), 6.72 (s, 1H), 6.63 (d, J = 7.4 Hz, 1H),
6.52 (s, 1H), 5.33 (s, 2H), 3.93 (t, J = 5.3 Hz, 2H), 2.25 (s,
3H), 2.10 (s, 3H), 1.79–1.65 (m, 4H), 1.23 (s, 6H); ESI–MS
m/z: 399 [M - H]-, 423 [M ? Na]?.
(d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 6.98 (d,
J = 7.4 Hz, 1H), 6.73 (s, 1H), 6.63 (d, J = 7.4 Hz, 1H),
5.50 (s, 2H), 3.93 (d, J = 4.2 Hz, 2H), 2.25 (s, 3H), 2.10 (s,
3H), 1.75 (s, 4H), 1.24 (s, 6H); ESI–MS m/z: 411
[M - H]-.
1
2-(4-Hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanoate (Y-4) Yield 83%; white solid,
1
mp: 105.6–108.2 °C; H NMR (400 MHz, DMSO-d6) d:
2-(3-Hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanoate (A-15) Yield 52.6%; white solid,
mp: \ 50 °C; 1H NMR (300 MHz, DMSO) d: 9.88 (s, 1H),
7.45–7.27 (m, 3H), 7.07 (d, J = 7.7 Hz, 1H), 6.98 (d,
J = 7.3 Hz, 1H), 6.71 (d, J = 6.7 Hz, 1H), 6.62 (d,
J = 7.4 Hz, 1H), 5.39 (s, 2H), 3.95–3.89 (m, 2H), 2.24 (s,
7.80–7.82 (m, 2H), 6.98–7.00 (d, J = 7.6 Hz, 1H),
6.85–6.87 (m, 2H), 6.63–6.64 (m, 2H), 5.6 (s, 1H), 5.23 (s,
2H), 3.94–3.97 (m, 2H), 2.30 (s, 3H), 2.18 (s, 3H),
1.81–1.85 (m, 4H), 1.32 (s, 6H); ESI–MS m/z:
383[M - H]-.
123

Molecular Diversity
S-(2-(4-Chlorophenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanethioate (A-21) Yield 87.1%; oil; 1H
NMR (400 MHz, DMSO-d6) d: 8.02 (d, J = 6.6 Hz, 2H),
7.62 (d, J = 6.6 Hz, 2H), 6.98 (d, J = 6.5 Hz, 1H), 6.71 (s,
1H), 6.62 (d, J = 7.7 Hz, 1H), 4.49 (s, 2H), 3.89 (t,
J = 5.2 Hz, 2H), 2.25 (s, 3H), 2.09 (s, 3H), 1.77–1.61 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 417 [M - H]-.
General procedure for the synthesis
of compound 4
CDI (10.0 mmol) was dissolved in DMF (20 mL), and this
solution was added a solution of the carboxylic acid 3
(10.0 mmol) in DMF (2 mL). Subsequently, the reaction
mixture was stirred at room temperature for 1 h. Then, H2S
gas was bubbled gently through the reaction mixture for
2 h. 0.5 M sulfuric acid (40 mL) was added, and the
mixture was extracted with ethyl acetate (25 mL 9 3). The
organic layer was dried with MgSO4, filtrated, concentrated
and purified by chromatography to yield compound 4.
Yield 75.4%, waxy oil, ESI–MS m/z: 265 [M - H]-.
S-(2-(4-Bromophenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanethioate (A-22) Yield 83.2%; oil; 1H
NMR (400 MHz, DMSO-d6) d: 7.94 (d, J = 8.7 Hz, 2H),
7.76 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 7.4 Hz, 1H), 6.70 (s,
1H), 6.62 (d, J = 7.4 Hz, 1H), 4.48 (s, 2H), 3.89 (t,
J = 5.9 Hz, 2H), 2.25 (s, 3H), 2.09 (s, 3H), 1.76–1.69 (m,
2H), 1.68 -1.62 (m, 2H), 1.22 (s, 6H); ESI–MS m/z: 463
[M - H]-.
General procedure for the synthesis
of compounds A-17–A-30, Y-5
S-(2-(4-Iodophenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanethioate (A-23) Yield 75.9%; oil; 1H NMR
(400 MHz, DMSO-d6) d: 7.94 (d, J = 8.6 Hz, 2H), 7.76 (d,
J = 8.6 Hz, 2H), 6.98 (d, J = 7.6 Hz, 1H), 6.71 (s, 1H),
6.62 (d, J = 7.6 Hz, 1H), 4.46 (s, 2H), 3.89 (t, J = 6.0 Hz,
2H), 2.25 (s, 3H), 2.09 (s, 3H), 1.76–1.69 (m, 2H),
1.68–1.62 (m, 2H), 1.22 (s, 6H); ESI–MS m/z: 509
[M - H]-.
Compounds A-17–A-30, Y-5 were prepared in a similar
manner to that described in the synthesis of compounds A-
1–A-16 from compound 4 in place of compound 3.
S-(2-Oxo-2-phenylethyl)5-(2,5-dimethylphenoxy)-2,2-
dimethylpentanethioate (A-17) Yield 88.4%; oil; 1H NMR
(400 MHz, CDCl3) d: 7.96–7.91 (m, 2H), 7.62 (t,
J = 7.3 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.02 (d,
J = 7.4 Hz, 1H), 6.68 (d, J = 7.7 Hz, 1H), 6.66 (s, 1H),
5.32 (s, 2H), 3.99 (t, J = 5.7 Hz, 2H), 2.33 (s, 3H), 2.21 (s,
3H), 1.94–1.79 (m, 4H), 1.35 (s, 6H); ESI–MS m/z: 383
[M - H]-.
S-(2-Oxo-2-(4-(trifluoromethyl)phenyl)ethyl)-5-(2,5-dimethyl-
phenoxy)-2,2-dimethylpentanethioate (A-24) Yield 76.2%;
oil; 1H NMR (400 MHz, DMSO-d6) d: 8.20 (d, J = 8.1 Hz,
2H), 7.92 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 7.4 Hz, 1H),
6.70 (s, 1H), 6.62 (d, J = 7.5 Hz, 1H), 4.55 (s, 2H), 3.88 (t,
J = 5.9 Hz, 2H), 2.24 (s, 3H), 2.09 (s, 3H), 1.75–1.60 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 451 [M - H]-.
S-(2-Oxo-2-(p-tolyl)ethyl)-5-(2,5-dimethylphenoxy)-2,2-dimethy-
lpentanethioate (A-18) Yield 84.1%; oil; 1H NMR
(400 MHz, DMSO-d6) d: 7.91 (d, J = 8.1 Hz, 2H), 7.35 (d,
J = 8.0 Hz, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.71 (s, 1H),
6.63 (d, J = 11.5 Hz, 1H), 4.46 (s, 2H), 3.89 (t, J = 6.0 Hz,
2H), 2.38 (s, 3H), 2.25 (s, 3H), 2.09 (s, 3H), 1.75–1.63 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 397 [M - H]-.
S-(2-(4-Nitrophenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
1
2,2-dimethylpentanethioate (A-25) Yield 63.7%; oil; H
NMR (400 MHz, DMSO-d6) d: 8.36 (d, J = 8.8 Hz, 2H),
8.23 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 7.4 Hz, 1H), 6.69 (s,
1H), 6.62 (d, J = 7.5 Hz, 1H), 4.56 (s, 2H), 3.88 (t,
J = 5.8 Hz, 2H), 2.24 (s, 3H), 2.08 (s, 3H), 1.75–1.61 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 428 [M - H]-.
S-(2-(4-Methoxyphenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanethioate (A-19) Yield 86.6%; oil; 1H
NMR (400 MHz, DMSO-d6) d: 7.99 (d, J = 9.0 Hz, 2H),
7.06 (d, J = 6.9 Hz, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.70 (s,
1H), 6.62 (d, J = 7.5 Hz, 1H), 4.43 (s, 2H), 3.89 (t,
J = 5.9 Hz, 2H), 3.85 (s, 3H), 2.25 (s, 3H), 2.09 (s, 3H),
1.78–1.62 (m, 4H), 1.22 (s, 6H); ESI–MS m/z: 413
[M - H]-.
S-(2-(4-Hydroxy-3,5-dimethylphenyl)-2-oxoethyl)-5-(2,5-dimet-
hylphenoxy)-2,2-dimethylpentanethioate
(A-26) Yield
61.5%; white solid, mp: 104.1–104.7 °C; 1H NMR
(400 MHz, DMSO-d6) d: 9.28 (s, 1H), 7.64 (s, 2H), 6.98
(d, J = 7.4 Hz, 1H), 6.71 (s, 1H), 6.62 (d, J = 7.3 Hz, 1H),
4.38 (s, 2H), 3.89 (t, J = 5.8 Hz, 2H), 2.23 (d, J = 10.9 Hz,
9H), 2.09 (s, 3H), 1.74–1.63 (m, 4H), 1.22 (s, 6H); ESI–MS
m/z: 427 [M - H]-.
S-(2-(4-Fluorophenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanethioate (A-20) Yield 81.9%; oil; 1H
NMR (400 MHz, CDCl3) d: 7.94 (dd, J = 8.8, 5.3 Hz, 2H),
7.16 (t, J = 8.8 Hz, 2H), 7.00 (d, J = 7.6 Hz, 1H), 6.66 (d,
J = 7.6 Hz, 1H), 6.63 (s, 1H), 5.26 (s, 2H), 3.96 (t,
J = 5.6 Hz, 2H), 2.31 (s, 3H), 2.17 (s, 3H), 1.92–1.73 (m,
4H), 1.32 (s, 6H); ESI–MS m/z: 401 [M - H]-.
S-(2-(3,5-Dibromo-4-hydroxyphenyl)-2-oxoethyl)-5-(2,5-dimeth-
ylphenoxy)-2,2-dimethylpentanethioate (A-27) Yield 59.2%;
white solid, mp: 90.5–92.9 °C; 1H NMR (400 MHz,
DMSO-d6) d: 8.16 (s, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.71
(s, 1H), 6.62 (d, J = 11.2 Hz, 1H), 4.44 (s, 2H), 3.89 (t,
123

Molecular Diversity
J = 5.8 Hz, 2H), 2.25 (s, 3H), 2.09 (s, 3H), 1.76–1.61 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 557 [M - H]-.
purified by chromatography to afford compound 7. Yield
80.5%, waxy oil, ESI–MS m/z: 221[M - H]-.
S-(2-(3-Fluoro-4-hydroxyphenyl)-2-oxoethyl)-5-(2,5-
General procedure for the synthesis
of compounds B-1–B-7
dimethylphenoxy)-2,2-dimethylpentanethioate
(A-
28) Yield 62.5%; white solid, mp: 63.8–66.7 °C; 1H
NMR (400 MHz, DMSO-d6) d: 11.00 (s, 1H), 7.81–7.73
(m, 2H), 7.07 (t, J = 8.8 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H),
6.70 (s, 1H), 6.62 (d, J = 7.6 Hz, 1H), 4.40 (s, 2H), 3.89 (t,
J = 5.8 Hz, 2H), 2.24 (s, 3H), 2.09 (s, 3H), 1.78–1.62 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 417 [M - H]-.
Compounds B-1–B-7 were prepared in a similar manner to
that described in the synthesis of compound A-1.
2-(4-Hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)
pentanoate (B-1) Yield 64.8%; white solid, mp:
80.6–80.9 °C; 1H NMR (400 MHz, DMSO-d6) d: 10.51 (s,
1H), 7.85 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 7.5 Hz, 1H),
6.89 (d, J = 8.8 Hz, 2H), 6.75 (s, 1H), 6.63 (d, J = 7.4 Hz,
1H), 5.38 (s, 2H), 3.97 (t, J = 5.9 Hz, 2H), 2.53–2.50 (m,
2H), 2.26 (s, 3H), 2.10 (s, 3H), 1.85–1.74 (m, 4H); ESI–MS
m/z: 355 [M - H]-.
S-(2-(4-Hydroxy-3-nitrophenyl)-2-oxoethyl)-5-(2,5-
dimethylphenoxy)-2,2-dimethylpentanethioate
(A-
29) Yield 59.4%; waxy oil; 1H NMR (400 MHz, DMSO-
d6) d: 12.14 (s, 1H), 8.50 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H),
7.24 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 7.3 Hz, 1H), 6.70 (s,
1H), 6.62 (d, J = 7.4 Hz, 1H), 4.47 (s, 2H), 3.89 (t,
J = 5.7 Hz, 2H), 2.24 (s, 3H), 2.08 (s, 3H), 1.77–1.62 (m,
4H), 1.22 (s, 6H); ESI–MS m/z: 444 [M - H]-.
2-(3-Hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)
pentanoate (B-2) Yield 59.8%; waxy oil; 1H NMR
(400 MHz, DMSO-d6) d: 9.90 (s, 1H), 7.43–7.33 (m, 2H),
7.33–7.29 (m, 1H), 7.08 (dd, J = 7.9, 2.3 Hz, 1H), 6.99 (d,
J = 7.4 Hz, 1H), 6.75 (s, 1H), 6.63 (d, J = 7.5 Hz, 1H),
5.43 (s, 2H), 3.97 (t, J = 5.9 Hz, 2H), 2.26 (s, 3H), 2.10 (s,
3H), 1.85–1.74 (m, 4H); ESI–MS m/z: 355 [M - H]-, 357
[M ? H]?.
S-(2-(3,5-Dihydroxyphenyl)-2-oxoethyl)-5-(2,5-dimethylphe-
noxy)-2,2-dimethylpentanethioate (A-30) Yield 58.8%;
white solid, mp: 124.5–124.9 °C; 1H NMR (400 MHz,
DMSO-d6) d: 9.68 (s, 2H), 6.98 (d, J = 7.4 Hz, 1H), 6.84
(d, 2H), 6.71 (s, 1H), 6.62 (d, J = 7.4 Hz, 1H), 6.50 (s, 1H),
4.36 (s, 2H), 3.90 (t, J = 5.8 Hz, 2H), 2.25 (s, 3H), 2.10 (s,
3H), 1.76–1.62 (m, 4H), 1.22 (s, 6H); ESI–MS m/z: 415
[M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphenoxy)
pentanoate (B-3) Yield 64.2%; white solid, mp:
1
150.2–151.4 °C; H NMR (400 MHz, DMSO-d6) d: 9.72
S-(2-(4-Hydroxyphenyl)-2-oxoethyl)-5-(2,5-dimethylphenoxy)-
2,2-dimethylpentanethioate (Y-5) Yield 67%; white solid,
mp: 90.3–93.9 °C; 1H NMR (400 MHz, CDCl3) d:
7.82–7.80 (m, 2H), 7.0–6.99 (d,1H, J = 7.2 Hz), 6.88–6.86
(m, 2H), 6.67–6.63 (m, 2H), 5.68 (s, 1H), 5.24 (s, 2H),
3.97–3.94 (m, 2H), 2.30 (s, 3H), 2.18 (s, 3H), 1.86–1.83
(m, 4H), 1.32 (s, 6H); ESI–MS m/z: 423[M ? Na]?.
(s, 2H), 6.99 (d, J = 7.4 Hz, 1H), 6.78 (d, J = 2.0 Hz, 2H),
6.75 (s, 1H), 6.63 (d, J = 7.4 Hz, 1H), 6.51 (s, 1H), 5.35 (s,
2H), 3.97 (t, J = 5.9 Hz, 2H), 2.26 (s, 3H), 2.10 (s, 3H),
1.85–1.74 (m, 4H); ESI–MS m/z: 371 [M - H]-.
2-(3-Fluoro-4-hydroxyphenyl)-2-oxoethyl-5-(2,5-dimethylphe-
noxy)pentanoate (B-4) Yield 55.8%; waxy oil; H NMR
1
(400 MHz, DMSO-d6) d: 7.80–7.66 (m, 2H), 7.09 (t,
J = 9.5 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 6.69 (s, 1H),
6.60 (d, J = 7.4 Hz, 1H), 4.80 (s, 2H), 3.91 (t, J = 5.9 Hz,
2H), 2.28 (t, J = 7.1 Hz, 2H), 2.23 (s, 3H), 2.07 (s, 3H),
1.76–1.65 (m, 4H); ESI–MS m/z: 373 [M - H]-.
General procedure for the synthesis
of compound 7
A solution of compound 5 (10 mmol) and K2CO3
(12 mmol) in DMF (25 mL) was stirred at room temper-
ature for 0.5 h, then compound 6 (12 mmol) and tetrabutyl
ammonium bromide (TBAB, 0.1 mmol) were added into
the above solution. The reaction mixture was stirred
overnight at room temperature; after the reaction was
completed, water was added to the solution and extracted
with DCM (40 mL 9 3). The organic layer was dried with
MgSO4, filtrated and evaporated at reduced pressure; the
residue and NaOH (20 mmol) were dissolved in EtOH
(40 mL), and then the reaction was allowed to 80 °C and
stirred for 2 h. After the reaction was completed, it was
added water (150 mL), extracted with DCM (40 mL 9 3),
evaporated at reduced pressure, and the residue was
2-(4-Hydroxy-3,5-dimethylphenyl)-2-oxoethyl-5-(2,5-
dimethylphenoxy)pentanoate (B-5) Yield 59.5%; white
1
solid, mp: 83.5–85.4 °C; H NMR (400 MHz, DMSO-d6)
d: 7.60 (s, 2H), 6.98 (d, J = 7.1 Hz, 1H), 6.74 (d,
J = 8.4 Hz, 1H), 6.63 (d, J = 7.4 Hz, 1H), 5.37 (s, 2H),
3.97–3.93 (m, 2H), 2.57–2.48 (m, 2H), 2.24 (d,
J = 13.3 Hz, 9H), 2.09 (s, 3H), 1.80-1.70 (m, 4H); ESI–MS
m/z: 383 [M - H]-.
2-(3,5-Dibromo-4-hydroxyphenyl)-2-oxoethyl-5-(2,5-
dimethylphenoxy)pentanoate (B-6) Yield 54.7%; mp: 8
white solid, 88.9–90.2 °C; 1H NMR (400 MHz, DMSO-d6)
d: 8.16 (s, 2H), 6.97 (d, J = 7.4 Hz, 1H), 6.72 (s, 1H), 6.62
123

Molecular Diversity
(d, J = 7.4 Hz, 1H), 4.90 (s, 2H), 3.93 (t, J = 6.0 Hz, 2H),
2.30 (t, J = 7.1 Hz, 2H), 2.25 (s, 3H), 2.09 (s, 3H),
1.76–1.66 (m, 4H); ESI–MS m/z: 513 [M - H]-.
General procedure for the synthesis of C1–C24
Compounds C1–C24 were prepared in a similar manner to
that described in the synthesis of compound A-1.
2-(4-Hydroxy-3-nitrophenyl)-2-oxoethyl-5-(2,5-dimethylphe-
noxy) pentanoate (B-7) Yield 63.2%; white solid, mp:
2-(4-Hydroxyphenyl)-2-oxoethyl-5-(4-(4-chlorobenzoyl)phe-
noxy)-2,2-dimethylpentanoate (C-1) Yield 70.4%; white
1
59.8–61.8 °C; H NMR (400 MHz, DMSO-d6) d: 8.57–
1
8.39 (m, 1H), 8.23–8.03 (m, 1H), 7.28–7.21 (m, 1H), 6.96
(d, J = 7.4 Hz, 1H), 6.71 (s, 1H), 6.61 (d, J = 7.4 Hz, 1H),
4.87 (s, 2H), 3.92 (t, J = 5.9 Hz, 2H), 2.29 (t, J = 7.1 Hz,
2H), 2.24 (s, 3H), 2.08 (s, 3H), 1.77–1.63 (m, 4H); ESI–MS
m/z: 400 [M - H]-.
solid, mp: 114.1–116.2 °C; H NMR (400 MHz, DMSO-
d6) d: 10.50 (s, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.74 (d,
J = 8.8 Hz, 2H), 7.71 (d, J = 8.5 Hz, 2H), 7.61 (d,
J = 8.6 Hz, 2H), 7.09 (d, J = 8.9 Hz, 2H), 6.87 (d,
J = 8.8 Hz, 2H), 5.37 (s, 2H), 4.09 (t, J = 5.8 Hz, 2H),
1.82–1.65 (m, 4H), 1.23 (s, 6H); ESI–MS m/z: 493
[M - H]-, 517 [M ? Na]?.
General procedure for the synthesis
of compound 10
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(4-(4-chloroben-
zoyl)phenoxy)-2,2-dimethylpentanoate (C-2) Yield 64.7%;
white solid, mp: 130.3–131.7 °C; 1H NMR (400 MHz,
DMSO-d6) d: 9.72 (s, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.71
(d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.09 (d,
J = 8.8 Hz, 2H), 6.77 (d, J = 2.1 Hz, 2H), 6.52 (t,
J = 2.1 Hz, 1H), 5.34 (s, 2H), 4.09 (t, J = 6.0 Hz, 2H),
1.85–1.76 (m, 2H), 1.74–1.68 (m, 2H), 1.23 (s, 6H); ESI–
MS m/z: 511 [M ? H]?.
A solution of ethyl isobutyrate (8, 1.13 mol) and 1,3-
dimethylpropyleneurea (5 mL) in THF (160 mL) was
added a solution of lithium diisopropylamide in THF
(790 mL, 2 mol/L) at -50 to -78 °C. The mixture was
stirred for 0.5 h at -78 °C, and 1-bromo-3-chloropropane 9
(1.58 mol) was added, stirred overnight at room tempera-
ture. After the reaction was completed, aqueous HCl (6 N,
250 mL), water (500 mL), ice (500 g) and saturated NH4Cl
solution (400 mL) were added into the reaction mixture.
The mixture was extracted with methyl tert-butyl ether
(250 mL 9 2), washed with saturated NaCl solution
(200 mL), dried with MgSO4, filtered and concentrated,
distillation under vacuum to get compound 10, yield
52.7%, waxy oil, ESI–MS m/z: 192[M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(4-acetylphenoxy)-2,2-
dimethylpentanoate (C-3) Yield 61.5%; white solid, mp:
1
121.5–124.4 °C; H NMR (400 MHz, DMSO-d6) d: 9.72
(s, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H),
6.77 (s, 2H), 6.51 (s, 1H), 5.33 (s, 2H), 4.06 (t, J = 5.7 Hz,
2H), 2.51 (s, 3H), 1.83–1.66 (m, 4H), 1.22 (s, 6H); ESI–MS
m/z: 415 [M ? H]?.
General procedure for the synthesis
of compounds 13a213q
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2,5-difluorophenoxy)-
2,2-dimethylpentanoate (C-4) Yield 58.6%; waxy oil; H
1
NMR (400 MHz, DMSO-d6) d: 9.73 (s, 2H), 7.24 (dd,
J = 9.6, 4.6 Hz, 1H), 7.10 (dd, J = 9.6, 3.7 Hz, 1H), 6.78
(d, J = 2.1 Hz, 2H), 6.72 (dd, J = 8.7, 3.4 Hz, 1H), 6.52 (t,
J = 2.1 Hz, 1H), 5.34 (s, 2H), 4.05–4.01 (m, 2H),
1.81–1.71 (m, 2H), 1.71–1.67 (m, 2H), 1.22 (s, 6H); ESI–
MS m/z: 407 [M - H]-.
A solution of appropriate phenol derivative 11a (10 mmol)
and K2CO3 (12 mmol) in DMF (25 mL) was stirred at
room temperature for 0.5 h, and then compound 10
(12 mmol) and TBAB (0.1 mmol) were added into the
above solution. The reaction mixture was stirred overnight
at 90 °C; after the reaction was completed, water was
added and extracted with DCM (40 mL 9 3). The organic
layer was dried over MgSO4, filtered and evaporated under
reduced pressure to get 12a. Then, compound 12a was
dissolved in EtOH (10 mL), the reaction was stirred at
room temperature and added a solution of NaOH
(20 mmol) in EtOH. The reaction was allowed to 80 °C
and stirred for 2 h. After the reaction was completed, it was
added water (150 mL), extracted with DCM (40 mL 9 3),
concentrated at reduced pressure to afford 13a, which was
used in the next step without further purification. Com-
pounds 13b–13q were prepared in a similar manner to the
synthesis of compound 13a.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2,5-dichlorophenoxy)-
2,2-dimethylpentanoate (C-5) Yield 68.7%; white solid,
1
mp: 151.3–153.9 °C; H NMR (400 MHz, DMSO-d6) d:
9.72 (s, 2H), 7.44 (d, J = 8.5 Hz, 1H), 7.23 (d, J = 2.3 Hz,
1H), 7.01 (dd, J = 8.5, 2.3 Hz, 1H), 6.78 (d, J = 2.2 Hz,
2H), 6.52 (t, J = 2.1 Hz, 1H), 5.34 (s, 2H), 4.09 (t,
J = 5.9 Hz, 2H), 1.83–1.68 (m, 4H), 1.22 (s, 6H); ESI–MS
m/z: 439 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(4-ethylphenoxy)-2,2-
dimethylpentanoate (C-6) Yield 67.1%; white solid, mp:
1
82.5–84.4 °C; H NMR (400 MHz, DMSO-d6) d: 9.72 (s,
2H), 7.10 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H),
123

Molecular Diversity
6.78 (d, J = 2.1 Hz, 2H), 6.52 (t, J = 2.1 Hz, 1H), 5.33 (s,
2H), 3.92 (t, J = 5.7 Hz, 2H), 2.52 (t, J = 5.9 Hz, 2H),
1.79–1.63 (m, 4H), 1.22 (s, 6H), 1.14 (t, J = 7.6 Hz, 3H);
ESI–MS m/z: 399 [M - H]-.
2-(3,5-dihydroxyphenyl)-2-oxoethyl-5-([1,10-biphenyl]-4-yloxy)-
2,2-dimethylpentanoate (C-13) Yield 64.4%; white solid,
mp: 127.4–128.7 °C; H NMR (400 MHz, DMSO-d6) d:
9.73 (s, 2H), 7.60 (t, J = 7.8 Hz, 4H), 7.43 (t, J = 7.8 Hz,
2H), 7.30 (t, J = 7.4 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H),
6.79 (d, J = 2.1 Hz, 2H), 6.52 (t, J = 2.1 Hz, 1H), 5.35 (s,
2H), 4.01 (t, J = 5.9 Hz, 2H), 1.82–1.65 (m, 4H), 1.24 (s,
6H); ESI–MS m/z: 447 [M - H]-, 471 [M ? Na]?.
1
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-2,2-dimethyl-5-((5,6,7,8-
tetrahydronaphthalene-2-yl)oxy)pentanoate
(C-7) Yield
1
61.2%; waxy oil; H NMR (400 MHz, DMSO-d6) d: 9.72
(s, 2H), 6.92 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 2.1 Hz, 2H),
6.64 (dd, J = 8.2, 2.7 Hz, 1H), 6.60–6.58 (m, 1H), 6.52 (t,
J = 2.1 Hz, 1H), 6.40 (d, J = 2.7 Hz, 1H), 5.32 (s, 2H),
3.89 (t, J = 5.7 Hz, 2H), 2.64 (d, 4H), 1.69 (d, 8H), 1.22 (s,
6H); ESI–MS m/z: 425 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-([1,10-biphenyl]-3-yloxy)-
2,2-dimethylpentanoate (C-14) Yield 59.2%; white solid,
mp: 84.6–87.1 °C; 1H NMR (400 MHz, CDCl3) d: 7.59 (d,
J = 7.2 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.40–7.30 (m,
2H), 7.21–7.11 (m, 2H), 6.88 (dd, J = 8.1, 2.1 Hz, 1H),
6.82 (d, J = 2.0 Hz, 2H), 6.51 (t, J = 2.0 Hz, 1H), 5.18 (s,
2H), 4.03 (t, J = 5.9 Hz, 2H), 1.86 (dd, J = 14.2, 6.3 Hz,
4H), 1.33 (s, 6H); ESI–MS m/z: 447 [M - H]-, 471
[M ? Na]?.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-2,2-dimethyl-5-(naphthalen-
1
2-yloxy)pentanoate (C-8) Yield 58.8%; waxy oil; H NMR
(400 MHz, DMSO-d6) d: 9.74 (s, 2H), 7.81 (t, J = 7.6 Hz,
3H), 7.45 (t, J = 7.6 Hz, 1H), 7.36–7.30 (m, 2H), 7.17 (dd,
J = 8.9, 2.5 Hz, 1H), 6.79 (d, J = 2.1 Hz, 2H), 6.53 (t,
J = 2.1 Hz, 1H), 5.35 (s, 2H), 4.08 (t, J = 6.1 Hz, 2H),
1.86–1.72 (m, 4H), 1.24 (s, 6H); ESI–MS m/z: 421
[M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(3-(benzyloxy)phenoxy)-
2,2-dimethylpentanoate (C-15) Yield 53.8%; white solid,
mp: 84.3–86.5 °C; 1H NMR (400 MHz, DMSO-d6) d: 9.74
(s, 2H), 7.44 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H),
7.32 (t, J = 7.1 Hz, 1H), 7.17 (t, J = 8.5 Hz, 1H), 6.79 (d,
J = 2.1 Hz, 2H), 6.58 (d, J = 6.6 Hz, 2H), 6.53 (d,
J = 7.6 Hz, 2H), 5.34 (s, 2H), 5.08 (s, 2H), 3.94 (t,
J = 5.6 Hz, 2H), 1.79–1.66 (m, 4H), 1.22 (s, 6H); ESI–MS
m/z: 477 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-2,2-dimethyl-5-((5-oxo-
5,6,7,8-tetrahydronaphthalen-2-yl)
oxy)pentanoate
(C-
1
9) Yield 67.5%; waxy oil; H NMR (400 MHz, DMSO-
d6) d: 9.72 (s, 2H), 7.81 (d, J = 8.2 Hz, 1H), 6.90 (s, 1H),
6.87 (s, 1H), 6.76 (d, J = 2.1 Hz, 2H), 6.50 (s, 1H), 5.33 (s,
2H), 4.05 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 5.7 Hz, 2H), 2.51
(d, J = 1.9 Hz, 2H), 1.99 (q, 2H), 1.79–1.68 (m, 4H), 1.22
(s, 6H); ESI–MS m/z: 439 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(4-(benzyloxyphenoxy)-
1
2,2-dimethylpentanoate (C-16) Yield 53.5%; waxy oil; H
NMR (400 MHz, DMSO-d6) d: 9.73 (s, 2H), 7.36 (m, 6H),
6.84 (d, J = 2.0 Hz, 2H), 6.81 (s, 1H), 6.68 (d, J = 8.9 Hz,
2H), 6.52 (d, J = 2.1 Hz, 1H), 4.98 (s, 2H), 4.81 (s, 2H),
3.89 (t, J = 5.9 Hz, 2H), 1.76–1.63 (m, 4H), 1.21 (s, 6H);
ESI–MS m/z: 477 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2-acetyl-3-hydrox-
yphenoxy)-2,2-dimethylpentanoate (C-10) Yield 54.9%;
mp: 92.3–94.1 °C; 1H NMR (400 MHz, DMSO-d6) d:
12.20 (s, 1H), 9.73 (s, 2H), 7.34 (s, 1H), 6.78 (d,
J = 2.0 Hz, 2H), 6.56–6.47 (m, 3H), 5.34 (s, 2H), 4.03 (t,
J = 5.3 Hz, 2H), 2.58 (s, 3H), 1.86–1.69 (m, 4H), 1.22 (s,
6H); ESI–MS m/z: 429 [M - H]-, 431 [M ? H]?.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2-(benzyloxy)phe-
noxy)-2,2-dimethylpentanoate (C-17) Yield 57.4%;
1
waxy oil; H NMR (400 MHz, DMSO-d6) d: 9.74 (s, 2H),
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(3-acetyl-5-hydrox-
yphenoxy)-2,2-dimethylpentanoate (C-11) Yield 67.4%;
white solid, mp: 162.6–162.8 °C; 1H NMR (400 MHz,
DMSO-d6) d: 9.80 (s, 1H), 9.71 (s, 2H), 6.93 (s, 2H), 6.77
(d, J = 1.8 Hz, 2H), 6.58 (s, 1H), 6.51 (s, 1H), 5.34 (s, 2H),
3.97 (t, J = 5.7 Hz, 2H), 2.51 (s, 3H), 1.78–1.64 (m, 4H),
1.23 (s, 6H); ESI–MS m/z: 429 [M - H]-.
7.47 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.30 (t,
J = 7.2 Hz, 1H), 7.01 (dd, J = 17.8, 7.6 Hz, 2H), 6.90 (dd,
J = 15.0, 7.3 Hz, 2H), 6.79 (d, J = 2.0 Hz, 2H), 6.54 (s,
1H), 5.32 (s, 2H), 5.09 (s, 2H), 3.98 (s, 2H), 1.75 (s, 4H),
1.21 (s, 6H); ESI–MS m/z: 477 [M - H]-.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-(2-methoxy-5-
methylphenoxy)-2,2-dimethylpentanoate
(C-18) Yield
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-([1,10-biphenyl]-2-yloxy)-
2,2-dimethylpentanoate (C-12) Yield 55.7%; white solid,
mp: 112.9–116.1 °C; 1H NMR (400 MHz, DMSO) d: 9.74
(s, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H),
7.31 (d, J = 7.3 Hz, 3H), 7.09 (d, J = 8.1 Hz, 1H), 7.02 (t,
J = 7.4 Hz, 1H), 6.79 (s, 2H), 6.54 (s, 1H), 5.31 (s, 2H),
3.98 (t, J = 4.8 Hz, 2H), 1.74–1.60 (m, 4H), 1.16 (s, 6H);
ESI–MS m/z: 447 [M - H]-.
1
55.1%; waxy oil; H NMR (400 MHz, DMSO-d6) d: 9.72
(s, 2H), 6.82 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 2.2 Hz, 3H),
6.67 (d, J = 8.2 Hz, 1H), 6.51 (t, J = 2.1 Hz, 1H), 5.33 (s,
2H), 3.92 (t, J = 6.0 Hz, 2H), 3.71 (s, 3H), 2.22 (s, 3H),
1.78–1.65 (m, 4H), 1.22 (s, 6H); ESI–MS m/z: 415
[M - H]-.
123

Molecular Diversity
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-2,2-dimethyl-5-((5-oxo-
5,6,7,8-tetrahydronaphthalen-2-yl)oxy)pentanoate (C-19)
Yield 56.3%; waxy oil; H NMR (400 MHz, CDCl3) d:
7.93 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 6.84 (d,
J = 8.7 Hz, 2H), 6.75 (dd, J = 8.7, 2.0 Hz, 1H), 6.64 (s,
1H), 5.19 (s, 2H), 3.97 (t, J = 5.8 Hz, 2H), 2.84 (t,
J = 5.8 Hz, 2H), 2.55 (t, J = 6.4 Hz, 2H), 2.04–1.98 (m,
2H), 1.90–1.68 (m, 4H), 1.25 (s, 6H); ESI–MS m/z: 423
[M - H]-.
that contained 200 nL of 5 mM testing chemical and 5 nL
of inducer, 520 lM GW4064. The final concentration of
GW4064 was 130 nM in all wells except for DMSO neg-
ative control wells. The final chemical concentration was
50 lM, and the final DMSO concentration was 1% in all
wells. The plates were then spun down after a brief shake
and incubated for 3 h at room temperature. The TR-FRET
signal was then collected for each well with an Envison
plate reader using an excitation wavelength of 340 nm and
emission wavelengths of 665 and 615 nm. The 665/615 nm
ratio from each well was calculated and employed for %
inhibition calculation. The % inhibition for each well was
calculated using the following equation:
1
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-2,2-dimethyl-5-((5,6,7,8-
tetrahydronaphthalen-2-yl)oxy)pentanoate
(C-20) Yield
1
64.2%; waxy oil; H NMR (400 MHz, CDCl3) d: 7.74 (d,
J = 8.8 Hz, 2H), 6.95 (d, J = 8.3 Hz, 1H), 6.84 (dd,
J = 9.2, 2.3 Hz, 2H), 6.68–6.59 (m, 2H), 5.23 (s, 2H), 3.94
(t, J = 5.6 Hz, 2H), 2.77–2.63 (m, 4H), 1.85–1.74 (m, 8H),
1.32 (s, 6H); ESI–MS m/z: 433 [M ? Na]?.
À
Á
130 nM GW4064665nm=615nmÀ Chemical665nm=615nm
À
Á
%Inhibition ¼
 100%
130 nM GW4064665nm=615nm À DMSO665nm=615nm
In the dose response hFXR TR-FRET assay, the general
protocol used for the single point testing was followed with
minor modification. Briefly, 1-to-3 titrated testing chemical
from 20 mM to 60 lM with 100% DMSO and 5 nL of
inducer, 520 lM GW4064 were transferred into a 384-well
black plate, and then 20-lL mixture of 3 nM GST-hFXR,
50 ng Eu-antiGST antibody, 500 nM Biotin-SRC1 and
50 ng streptavidin-APC in assay buffer was added. The
final concentration of GW4064 was 130 nM in all wells
except for the DMSO negative control wells. The final
DMSO concentration was 1% in all wells. The plates were
then spun down after a brief shake and incubated for 3 h at
room temperature. The TR-FRET signal was then collected
for each well using an Envison plate reader using an
excitation wavelength of 340 nm and emission wave-
lengths of 665 nm and 615 nm. The 665/615 nm ratio from
each well was calculated and employed for % inhibition
calculation. The % inhibition for each well was also cal-
culated using the above-mentioned equation. The IC50
value and curve fitting analyses were calculated with
Graphpad Prism 5.
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-([1,10-biphenyl]-4-
yloxy)-2,2-dimethylpentanoate (C-21) Yield 54.1%; waxy
oil; H NMR (400 MHz, CDCl3) d: 7.81 (d, J = 8.8 Hz,
2H), 7.57–7.49 (m, 5H), 7.40 (d, J = 7.7 Hz, 2H), 6.97 (d,
J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 5.23 (s, 2H),
4.03 (t, J = 5.8 Hz, 2H), 1.84 (dd, J = 12.0, 7.2 Hz, 4H),
1.33 (s, 6H); ESI–MS m/z: 433 [M ? H]?.
1
2-(3,5-Dihydroxyphenyl)-2-oxoethyl-5-([1,10-biphenyl]-2-
yloxy)-2,2-dimethylpentanoate (C-22) Yield 55.8%; waxy
1
oil; H NMR (400 MHz, CDCl3) d: 7.73 (d, J = 8.8 Hz,
2H), 7.59–7.52 (m, 3H), 7.43–7.30 (m, 5H), 6.99 (dd,
J = 12.5, 7.9 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.19 (s,
2H), 3.98 (d, J = 4.5 Hz, 2H), 1.72 (d, J = 9.9 Hz, 4H),
1.25 (s, 6H); ESI–MS m/z: 455 [M ? Na]?.
2-(3,5-dihydroxyphenyl)-2-oxoethyl-5-(2,5-dichlorophenoxy)-
2,2-dimethylpentanoate (C-23) Yield 57.6%; waxy oil; 1H
NMR (400 MHz, CDCl3) d: 7.74 (d, J = 8.8 Hz, 2H),
6.91–6.79 (m, 5H), 5.27 (s, 2H), 4.01 (t, J = 6.0 Hz, 2H),
1.95–1.78 (m, 4H), 1.33 (s, 6H); ESI–MS m/z: 425
[M ? H]?.
2-(3,5-dihydroxyphenyl)-2-oxoethyl-5-(4-acetylphenoxy)-2,2-
dimethylpentanoate (C-24) Yield 60.2%; waxy oil; 1H
NMR (400 MHz, CDCl3) d: 7.96–7.89 (m, 2H), 7.77 (d,
J = 8.8 Hz, 2H), 6.90 (dd, J = 10.3, 8.9 Hz, 4H), 5.25 (s,
2H), 4.04 (t, J = 6.0 Hz, 2H), 2.56 (s, 3H), 1.94–1.76 (m,
4H), 1.32 (s, 6H); ESI–MS m/z: 399 [M ? H]?.
Docking study
Receptor preparation
The co-crystallized structure of human FXR with GW4064
˚
analog (PDB code: 3FXV, resolution: 2.26 A) was selected
The characterization data for the synthesized com-
pounds are deposited in Supporting information.
for docking study. The structures were prepared by using
the ‘‘Protein Preparation Wizard’’ module in Maestro [21].
The protein was prepared by adding missing hydrogen
atoms, assigning ligand bond orders, determining ligand
protonation state, removing waters, optimizing hydrogen
bond network and performing a restrained minimization,
etc. Docking grid files were then generated using the
FXR TR-FRET assay [19, 20]
A 20-lL mixture of 3 nM GST-hFXR, 50 ng Eu-anti GST
antibody, 500 nM Biotin-SRC1 and 50 ng streptavidin-
APC in assay buffer was added into a 384-well black plate
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Acknowledgements The work was supported by National Natural
Science Foundation of China (No. 81660571), Guangxi Natural Sci-
ence Foundation of China (No. 2015GXNSFBA139124) and ‘‘Sci-
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