Synthesis of artemisinin-piperazine-furan ether hybrids and evaluation of in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2021.113295

For the first time, eight novel artemisinin-piperazine-furane ether hybrids (5a?h) were efficiently synthesized and investigated for their in vitro cytotoxic activity against some human cancer and benign cells. The absolute configuration of hybrid 5c was

Full text of DOI:10.1016/j.ejmech.2021.113295

European Journal of Medicinal Chemistry 215 (2021) 113295
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis of artemisinin-piperazine-furan ether hybrids and
evaluation of in vitro cytotoxic activity
*
Meng-Xue Wei , Jia-Ying Yu, Xin-Xin Liu, Xue-Qiang Li, Meng-Wei Zhang, Pei-Wen Yang,
Jin-Hui Yang
State Key Laboratory of High-efficiency Utilization of Coal and Green Chemical Engineering, Ningxia Engineering Research Center for Natural Medicine,
National Demonstration Center for Experimental Chemistry Education, College of Chemistry and Chemical Engineering, Ningxia University, 489 Helanshan
West Road, Yinchuan, 750021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 December 2020
Received in revised form
For the first time, eight novel artemisinin-piperazine-furane ether hybrids (5a¡h) were efficiently
synthesized and investigated for their in vitro cytotoxic activity against some human cancer and benign
cells. The absolute configuration of hybrid 5c was determined by X-ray crystallographic analysis. Hybrids
25 January 2021
5
a¡h exhibited more pronounced growth-inhibiting action on hepatocarcinoma cell lines than their
parent dihydroartemisinin (DHA) and the reference cytosine arabinoside (ARA). The hybrid 5a showed
M)
Accepted 7 February 2021
Available online 16 February 2021
the best cytotoxic activity against human hepatocarcinoma cells SMMC-7721 (IC50 ¼ 0.26 ± 0.03
m
after 24 h. Furthermore, hybrid 5a also showed good cytotoxic activity against human breast cancer cells
MCF-7 and low cytotoxicity against human breast benign cells MCF-10A in vitro. We found the cyto-
Keywords:
Furan ether
Artemisinin
X-ray crystallography
Cytotoxic activity
Cancer cells
4
toxicity of hybrid 5a did not change when tumour cells absorb iron sulfate (FeSO ); thus, we conclude the
anti-tumour mechanism induced by iron ions (Fe2 ) is unclear.
þ
© 2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
the pharmacological properties of the parent compound by
increasing biological efficacy, reducing adverse side effects, modi-
fying the selectivity spectrum (low toxicity), providing better
bioavailability, and introducing new biological properties not found
in the parent compound. The combination of artemisinin-type
drugs with anticancer drugs can enhance the activity of the anti-
cancer drugs in vitro or in vivo [21e24].
Furanone moiety (1a, Fig. 1) attracted a lot of attention because
of its significant biological activities [1e7] and occurrence in many
natural products [8e10]. Natural borneol and menthol are Chinese
herbal medicines with anti-inflammatory effects. In previous work,
our group found that a single epimer of the chiral furan ethers
provides good cytotoxic activity [11,12]. Artemisinin (ARS, 1b)
extracted from Artemisia annua is used in Chinese medicine to treat
chills and fever [13,14]. Since 1993, a large volume of studies have
highlighted the potential of ARS as a novel therapeutic agent for
cancer [15e18]. Dihydroartemisinin (DHA, 1c) exhibited high anti-
cancer activity in Lewis lung carcinoma (LLC) cell line in vitro [19].
Artesunate (ART, 1d) has been reported to kill cancer cells by
inducing oxidative stress and DNA damage; it can also down-
regulate human recombinant protein in ovarian cancer cells [20].
Over the past decade, hybridization of natural products has proven
to be a very effective strategy for medicinal chemistry and drug
design. This process not only saves time and cost, but also improves
Given the therapeutic properties of furan derivatives and the
lack of artemisinin-piperazine-furan ether hybrids reported in the
literature, we designed and synthesized a new series of hybrid
furan ether derivatives possessing an artemisinin-piperazine moi-
ety (5aeh, Scheme 3) to evaluate their in vitro cytotoxicity and
compare with VCR and ARA. Then we also research the effects of
2þ
Fe on cytotoxic activity [25e29].
2. Results and discussion
2.1. Synthesis
Compound 2 was synthesized through a substitution reaction
[
22], using dry dimethyl sulfoxide (DMSO) as a catalyst (Scheme 1).
First, DHA and oxalyl chloride formed artemisinin chloroformyl
formic ester. Then, the amidogen of piperazine replaced the
*
Corresponding author.
E-mail address: weimengxue@nxu.edu.cn (M.-X. Wei).
https://doi.org/10.1016/j.ejmech.2021.113295
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
Fig. 1. Structures of furanone (1a), artemisinin (ARS, 1b), dihydroartemisinin (DHA, 1c), and artesunate (ART, 1d).
obtained by recrystallization in petroleum ether (PE), ethanol
EtOH), and water (H O). Chiral menthyl ethers 4c and 4g were
synthesized by performing one recrystallization in PE/EtOH/H O,
while chiral borneol ether 4h was obtained through multiple
recrystallization in EtOH/H O. An enantiomeric mixture of 4d was
obtained in a ratio of 3:1 ( z 3:1) in the same manner. However,
(
2
2
2
a:b
only the racemates of 4e and 4f were obtained by recrystallization.
Compound 2 was not as stable as expected and needed to be
kept in the refrigerator and avoid thermal reaction. Despite this, the
reaction of 2 with furan ethers 4 is nice, and the yield is almost
quantitative (>95% yield) at room temperature (Scheme 3). Hybrids
5
2
a¡h was obtained in a simple way. Briefly, a mixture of compound
, furan ethers 4, and N,N-diisopropylethylamine (DIPEA) in
dichloromethane (DCM) was stirred for 2e4 h at room tempera-
ture. We obtained the single crystal of 5c by dissolving it in dilute
hydrochloric acid and ethanol, and its structure was corroborated
by X-ray diffraction, as shown in Fig. 2.
Scheme 1. Synthesis of Compound 2.
chloroformyl formic ester to produce the artemisinin-piperazine 2
in 68% yield.
The halogenated furan ethers 4a¡h were synthesized using
alcohol and mucochloric acid or mucobromic acid 3 as raw mate-
rials and concentrated sulphuric acid as a catalyst (Scheme 2). The
reaction requires a water separator to separate the water to move
the balance to the right. Because methanol (MeOH) and ethanol
2.2. Biological activity of the hybrids
Considering that artemisinin can target the liver, all hybrids
including 5a¡h, DHA, VCR and ARA were screened for cytotoxic
activity against human hepatocarcinoma cells SMMC-7721 and
human normal liver cells LO2 using the standard MTT assay in vitro.
The results are shown in Table 1. Comparison of the results showed
(
EtOH) are denser than cyclohexane, we chose 1,2-dichloroethane
as solvent and added methanol and ethanol at regular intervals to
shift the balance to the right. Menthol and borneol would be
carbonized when heated under reflux, so the reaction mixture
required stirring at room temperature (rt) before heating slowly;
however, there was still some carbonization. Intermediate furan
ethers were all solid except for 4a and 4b. The crystalloid was
the inhibitory activity of 5a (IC50 ¼ 0.26 ± 0.03
better than ARA (IC50 0.63 0.04
(IC50 ¼ 0.27 ± 0.03 M) in vitro against human hepatocarcinoma
cells SMMC-7721. Hybrid 5a (IC50 ¼ 0.32 ± 0.03 M after 24 h) also
had lower cytotoxicity against benign cells LO2 than VCR
0.30 0.02 after 24 h). Hybrids 5b
mM after 24 h) was
¼
±
mM) and VCR
m
m
(IC50
¼
±
mM
Scheme 2. Synthesis of furan ethers 4a¡h. Reagents and conditions: (a) MeOH or EtOH, 1,2-dichloroethane, H
then reflux.
2 4 2 4
SO , rt then reflux; (b) menthol or borneol, cyclohexane, H SO , rt
2

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
Scheme 3. Synthesis of novel hybrids 5a¡h.
Fig. 2. X-ray structure of hybrid 5c. Crystallographic data for hybrid 5c is available from the Cambridge Crystallographic Data Centre under reference number CCDC 2012573. The
thermal ellipsoids of all non-hydrogen atoms are shown at 30% probability.
(
IC50 ¼ 0.70 ± 0.04
m
M after 24 h) and 5c (IC50 ¼ 0.64 ± 0.03
m
M
toxicity could be used as an anti-tumour drug by controlling the
dosage.
after 24 h) had less cytotoxic activity against benign cells LO2 than
VCR, ARA, and DHA, although their inhibitory activity against hu-
man hepatocarcinoma cells SMMC-7721 was not the best. Consid-
ering this, a drug with moderate anti-tumour properties and low
SMMC-7721 ¼ human hepatocarcinoma cells; LO2 ¼ human
normal liver cells; VCR ¼ vincristine; ARA ¼ cytosine arabinoside;
mM ¼
mmol/mL. IC50 values represent the compound concentration
3

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
Table 1
36.98% when the concentration of hybrid 5a was 100 mg/mL. It was
Cytotoxic activities against SMMC-7721 and LO2 cells for 5a¡h, VCR, ARA and DHA.
found that hybrid 5a could induce the apoptosis of SMMC-
7721 cells in a concentration-dependent manner. The results indi-
cated that the antiproliferative effect of 5a might be related to the
induction of apoptosis.
We chose hybrid 5a to test anti-tumour activity against other
cancer cells (Table 2). Hybrid 5a had good activity against human
Compound
IC50 (mM)/24 h
SMMC-7721
LO2
5
5
5
5
5
5
5
5
a
b
c
d
e
f
0.26 ± 0.03
0.56 ± 0.03
0.52 ± 0.05
0.57 ± 0.03
0.29 ± 0.04
0.43 ± 0.04
0.39 ± 0.04
0.48 ± 0.03
0.27 ± 0.03
0.63 ± 0.04
>0.7
0.32 ± 0.03
0.70 ± 0.04
0.64 ± 0.03
0.46 ± 0.04
0.26 ± 0.03
0.50 ± 0.03
0.36 ± 0.04
0.48 ± 0.01
0.30 ± 0.02
0.43 ± 0.04
0.22 ± 0.04
breast cancer cells MCF-7 (IC50 ¼ 0.08 ± 0.03
mM) after 48 h in vitro.
The cytotoxic activity of hybrid 5a against hepatocarcinoma cells
SMMC-7721 significantly improved as time progressed, IC50 from
g
h
0.26 ± 0.03
mM (Table 1) to 0.11 ± 0.04 mM (Table 2) after 24 h and
VCR
ARA
DHA
48 h, respectively. Studies have shown that some cancer cells can
2þ
import Fe through abnormal iron transporters in their mem-
branes, while normal cells seldom have abnormal iron transporters
[
26,27]. We tested the cytotoxic activity of hybrid 5a against human
hepatocarcinoma cells SMMC-7721, MHCC97H, and HCCLM3 in the
(
m
M) required to inhibit tumour cell proliferation by 50% and were
presence of FeSO4 for 48 h in vitro. Instead of directly using the
2
þ
2þ
calculated using concentrations from triplicate measurements.
As can be seen in Fig. 3, Hybrid 5a showed the best inhibitory
activity against human hepatocarcinoma cells SMMC-7721, and the
inhibitory rate increased with increasing concentration. As shown
in Fig. 4, the annexin V-FITC (AV) and propidium iodide (PI) staining
were used and performed on flow cytometry to explore the
apoptotic cells mediated by 5a. SMMC-7721 cells were treated with
medium containing Fe and 5a, Fe was added when culturing
cells, which is a good way to simulate the process of cancer cells
2
þ
absorbing Fe . Hybrid 5a showed similar activity against human
2
þ
hepatocarcinoma cells with or without Fe . In our opinion, the
2þ
anti-tumour mechanism induced by Fe
remains unclear. The
morphological changes of cells induced by hybrid 5a are shown in
Fig. 5. Hybrid 5a can effectively inhibit the proliferation of tumour
cells in vitro.
5
a at different concentrations of 0.1, 10, and 100 mg/mL, respec-
tively. As can be seen from Fig. 4, the percentage of early and late
apoptotic cells increased (lower right quadrant and upper right
quadrant, respectively) as the concentration of hybrid 5a increased.
The average cell apoptosis including the percentage of early and
late apoptotic cells, across three parallel experiments, reached to
SMMC-7721, MHCC97H, HCCLM3, HUH-7
¼
human hep-
atocarcinoma cells. HCT116 human colon cancer cells.
¼
CCD18CO ¼ human normal colon cells. MCF-7 ¼ human breast
cancer cells. MCF-10A ¼ human normal breast cells.
m
M ¼
mmol/mL.
IC values represent the compound concentration (
5
0
mM) required to
Fig. 3. Inhibitory effect of 5a¡h, VCR, ARA and DHA with various concentrations on SMMC-7721 and LO2 cells for 24 h.
4

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
Fig. 4. Flow cytometric analysis of SMMC-7721 cells using different concentrations of hybrid 5a (0.1, 10, and 100
mg/mL); each concentration was detected three times in parallel.
The percentage of cells positive for AV and/or PI was reported in the quadrants including cells in the lower left quadrant (live cells), lower right quadrant (early apoptotic cells),
upper right quadrant (late apoptotic cells), and upper left quadrant (necrotic cells).
Table 2
hepatocarcinoma cells SMMC-7721 than the parent DHA, and two
references (VCR and ARA). Hybrid 5a also had good cytotoxicity
against human breast cancer cells MCF-7, and low cytotoxicity
against human breast benign cells MCF-10A in vitro. Hybrid 5a also
has good activity against other hepatocarcinoma cells (MHCC97H,
HCCLM3, HUH-7) in vitro. We find that the mechanism of anti-
tumour induced by iron ion was not-quite explanation. Iron ion
can’t increase the cytotoxic activity of hybrid 5a. The results could
provide a useful reference for research involving anti-tumour
drugs.
Cytotoxic activities of 5a against cancer and benign cells.
Cells
IC50 (mM)/48 h
SMMC-7721
SMMC-7721
MHCC97H
MHCC97H
HCCLM3
HCCLM3
HUH-7
HCT116
CCD18CO
MCF-7
0.11 ± 0.04
0.10 ± 0.04
0.10 ± 0.04
0.09 ± 0.05
0.10 ± 0.03
0.08 ± 0.03
0.16 ± 0.03
0.10 ± 0.03
0.10 ± 0.03
0.08 ± 0.03
0.22 ± 0.04
a
a
a
MCF-10A
4
4
4
. Experimental
a
4
Cancer cells were cultured with 100 mmol/L FeSO .
.1. Synthesis and characterization
inhibit tumour cell proliferation by 50% and were calculated using
concentrations from triplicate measurements.
.1.1. General
All starting materials and reagents were purchased from
commercially available sources and used without further purifica-
1
3
. Conclusion
tion, unless otherwise indicated. H NMR spectra were recorded on
a Bruker Avance III 400 MHz spectrometer. Chemical shifts (in ppm)
13
For the first time, a series of artemisinin-piperazine-furan ether
were calibrated with CDCl
3
.
C NMR spectra were obtained by
hybrids were synthesized efficiently, and the absolute configura-
tion of hybrid 5c was determined by X-ray crystallographic analysis.
The cytotoxic activities of the hybrids are presented in this article.
Hybrid 5a showed higher inhibitory activity against human
using the same NMR spectrometer and were calibrated with CDCl .
3
Infrared (IR) spectra of compounds dispersed in potassium bromide
were recorded using a Spectrum Two spectrometer, and values are
ꢀ
1
reported as
n in cm . High-resolution mass spectrum (HRMS) of
5

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
g/mL after 48 h in (A) SMMC-7721 cells, (B) SMMC-7721 cells with Fe2 , (C) MHCC97H cells,
þ
Fig. 5. Morphological changes of cells induced by hybrid 5a at a concentration of 25
m
2þ
2þ
(
D) MHCC97H cells with Fe , (E) HCCLM3 cells, and (F) HCCLM3 cells with Fe .
compounds were recorded on a Thermo Fisher LTQ Orbitrap XL. The
X-ray single-crystal data for the compound 5c was collected on a
Bruker AXS SMART 1000 CCD diffractometer using graphite mon-
mucobromic acid 3 (30 mmol), methyl alcohol or ethyl alcohol
(1 mL every 8 h), and 1,2-dichloroethane (90 mL) were added to a
flask and stirred for 10 min, then 0.5 mL concentrated sulphuric
acid added dropwise and stirred for another 20 min. Then a water
segregator was set up and heated to reflux for 36 h. After the
mixture was cooled to room temperature, saturated sodium car-
bonate (2 ꢁ 40 mL) and saturated salt water (40 mL) were used to
ochromated Mo K
tests were performed by Wuhan Bafeier Biotechnology Service Co.,
Ltd.
a
radiation (
l
¼ 0.71073 Å). The biological activity
2 4
wash the mixture, then dried over Na SO and the solvent removed
4.1.2. Synthesis of 1-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-
under diminished pressure. The residue was purified by chroma-
tography on a silica gel column to give product 4.
Method B (for 4c, 4d, 4g and 4h): mucochloric acid or muco-
bromic acid 3 (30 mmol), menthol (4.68 g, 30 mmol), or borneol
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]
isochromen-10-yl)piperazine (2)
Under the anhydrous condition, anhydrous piperazine (2.5 g,
2
9 mmol) and dry dichloromethane (DCM) (35 mL) were added to a
(4.63 g, 30 mmol) and cyclohexane (120 mL) were added to a flask
flask-3-neck (250 mL), then stirred until completely dissolved.
and stirred for 10 min, then 0.5 mL concentrated sulphuric acid
added dropwise and stirred for another 20 min. Then a water
segregator was set up and heated to reflux for 36 h. After the
mixture was cooled to room temperature, saturated sodium car-
bonate (2 ꢁ 40 mL), and saturated salt water (40 mL) were used to
Under the anhydrous condition, dry DCM (25 mL) and DHA (2 g,
7
mmol) were added to another flask-3-neck (200 mL) and stirred
at room temperature, within 1 min, DMSO (50e80 L) were added
m
and stirred for another 3 min. Oxalyl chloride (0.6e0.7 mL) was
slowly added until no gas was produced and stirred another
2 4
wash the mixture, then dried over Na SO and the solvent removed
7
e10 min. The mixture was slowly dropwise in the flask containing
under diminished pressure. The residue was recrystallised to give
product 4.
piperazine, allowing overnight reaction at room temperature.
Following the addition of saturated sodium carbonate (20 mL) and
AcOEt (30 mL), the organic phase was separated, and the aqueous
layer was extracted with AcOEt (2 ꢁ 30 mL). The combined extracts
4.1.3.1. 3,4-Dichloro-5-methoxyfuran-2(5H)-one
Compound 4a [31,32] was formed in 90% as a colourless oil. H NMR
(4a).
1
2 4
were washed with brine (20 mL), dried over anhydrous Na SO , and
1
3
concentrated under reduced pressure. The residue was purified by
chromatography on an ammonia-treated silica gel column. Get the
elution with 25:1 DCM-methanol. Compound 2 [30] was formed in
(400 MHz, Chloroform-d)
d 5.77 (s, 1H), 3.59 (s, 3H); C NMR
(101 MHz, CDCl 163.1, 147.2, 124.5, 101.4, 56.4.
3
) d
ꢂ
20
6
CHCl
8% yield as a yellow solid. m.p. 76.1e76.5 C; ½aꢃ þ19 (c 2.0,
4.1.3.2. 3,4-Dichloro-5-ethoxyfuran-2(5H)-one (4b). Compound 4b
D
1
1
3
); H NMR (400 MHz, Chloroform-d)
d
5.26 (s, 1H), 3.96 (d,
[31] was formed in 85% as a colourless oil. H NMR (400 MHz,
J ¼ 10.2 Hz, 1H), 2.90 (d, J ¼ 17.8 Hz, 6H), 2.57 (ddd, J ¼ 10.1, 7.3,
.3 Hz, 3H), 2.33 (ddd, J ¼ 14.5, 13.3, 4.0 Hz, 1H), 2.05e1.94 (m, 1H),
.72e1.64 (m, 2H),1.56e1.45 (m, 2H),1.38 (s, 3H),1.32e1.27 (m, 2H),
Chloroform-d)
d
5.81 (s, 1H), 3.86 (ddq, J ¼ 44.2, 9.3, 7.1 Hz, 2H),1.31
13
4
1
(t, J ¼ 7.1 Hz, 3H); C NMR (101 MHz, CDCl
3
)
d
163.3, 147.4, 124.2,
100.8, 66.1, 14.8.
1
3
9
.21 (td, J ¼ 11.2, 6.5 Hz, 1H), 1.07e0.97 (m, 1H), 0.93 (d, J ¼ 6.3 Hz,
13
H), 0.79 (d, J ¼ 7.2 Hz, 3H); C NMR (101 MHz, CDCl
0.9, 80.3, 51.6, 50.4, 48.3, 46.0, 45.7, 37.3, 36.2, 34.2, 28.2, 25.9, 24.7,
1.6, 20.2, 13.4.
3
)
d
103.8, 91.6,
4.1.3.3. 3,4-Dibromo-5-methoxyfuran-2(5H)-one
(4e).
Compound 4e [32] was formed in 80% as a colourless solid. m.p.
ꢂ
1
2
51.1e51.5 C; H NMR (400 MHz, Chloroform-d)
d
5.78 (s, 1H), 3.58
13
(
s, 3H); C NMR (101 MHz, CDCl ) d 163.9, 142.9, 119.0, 103.7, 56.1.
3
4
.1.3. General procedure for the furan ethers 4aꢀh
Method A (for 4a, 4b, 4e and 4f): mucochloric acid or
4.1.3.4. 3,4-Dibromo-5-ethoxyfuran-2(5H)-one (4f). Compound 4f
6

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
was formed in 78% as a faint yellow solid. m.p. 40.3e41.0 C; 1
NMR (400 MHz, Chloroform-d)
5.82 (s, 1H), 3.90 (dq, J ¼ 9.4,
.1 Hz, 1H), 3.80 (dq, J ¼ 9.4, 7.1 Hz, 1H), 1.32 (t, J ¼ 7.1 Hz, 3H);
NMR (101 MHz, CDCl 164.1, 143.2, 118.7, 103.1, 65.9, 14.9.
ꢂ
H
CHCl
); H NMR (400 MHz, Chloroform-d)
1
d
5.69 (d, J ¼ 2.5 Hz, 1H),
3
d
5.28 (s, 1H), 4.03 (dd, J ¼ 10.3, 3.3 Hz, 1H), 3.85 (s, 1H), 3.70 (s, 4H),
3.47 (d, J ¼ 4.9 Hz, 3H), 3.14e2.99 (m, 2H), 2.81e2.71 (m, 2H), 2.34
(td, J ¼ 14.0, 3.7 Hz, 1H), 2.03e1.97 (m, 1H), 1.87 (dq, J ¼ 10.2, 3.3 Hz,
1
3
7
C
3
) d
1
1
H), 1.71 (dq, J ¼ 10.7, 3.5 Hz, 2H), 1.60 (s, 3H), 1.58e1.44 (m, 1H),
.39 (d, J ¼ 1.6 Hz, 3H), 1.35 (s, 1H), 1.07e0.99 (m, 1H), 0.95 (d,
4
.1.3.5. (S)-3,4-Dichloro-5-(((1S,2S,5R)-2-isopropyl-5-
methylcyclohexyl)oxy)furan-2(5H)-one (4c). Compound 4c [33] was
13
J ¼ 6.2 Hz, 3H), 0.82 (d, J ¼ 7.1 Hz, 3H); C NMR (101 MHz, CDCl
3
)
d
168.3, 153.8, 104.1, 97.5, 91.8, 90.7, 86.7, 80.4, 54.5, 54.3, 51.8, 45.8,
crystallized from EtOH/PE/H
2
O in 31% as a colourless crystal. m.p.
ꢂ
20
1
37.5, 36.4, 34.4, 28.5, 26.1, 26.1, 24.9, 21.8, 20.4, 13.6; IR (KBr)
cm : 3455, 2928, 1763, 1635, 1449, 1207, 1122, 983, 880, 745;
n
/
1
12.4e113.4 C; ½aꢃ þ53 (c 1.0, CHCl
3
); H NMR (400 MHz, Chlo-
D
ꢀ1
roform-d)
d
5.85 (d, J ¼ 31.3 Hz, 1H), 3.63 (dtd, J ¼ 34.2, 10.7, 4.4 Hz,
HRMS m/z: calcd for C24
H35ClN
2
O
7
Na 521.2025, found 521.2001
1
1
0
3
H), 2.30e2.06 (m, 2H), 1.68 (dh, J ¼ 10.5, 2.7, 2.3 Hz, 2H), 1.57 (s,
þ
[
MþNa] .
H), 1.46e1.27 (m, 1H), 1.11e1.02 (m, 1H), 0.97 (d, J ¼ 6.6 Hz, 2H),
.93 (dd, J ¼ 6.8, 3.7 Hz, 2H), 0.89 (d, J ¼ 7.1 Hz, 3H), 0.82e0.77 (m,
13
4.1.4.2. 3-Chloro-5-ethoxy-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-
0-yl)piperazin-1-yl)furan-2(5H)-one (5b). Compound 5b was
3
H); C NMR (101 MHz, CDCl ) d 163.4, 147.6, 123.9, 102.2, 84.5,
47.9, 42.1, 33.9, 31.6, 25.3, 22.9, 22.0, 20.8, 15.8.
1
ꢂ
20
D
formed in 96% as a yellow solid. m.p. 67.0e68.0 C; ½aꢃ ꢀ4 (c 1.0,
4
.1.3.6. 3,4-Dichloro-5-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-
yl)oxy)furan-2(5H)-one (4d). Compound 4d [34] was crystallized
1
CH
3
OH); H NMR (400 MHz, Chloroform-d)
d
5.71 (d, J ¼ 3.4 Hz,
ꢂ
1H), 5.27 (d, J ¼ 8.9 Hz,1H), 4.02 (dd, J ¼ 10.3, 4.3 Hz, 1H), 3.85e3.61
from EtOH/H
2
O in 33% as a white crystal. m.p. 79.8e80.2 C;
2
0
1
(m, 4H), 3.05 (td, J ¼ 10.4, 4.8 Hz, 2H), 2.81e2.66 (m, 2H), 2.58e2.52
½
aꢃ ꢀ15 (c 1.0, CHCl
3
); H NMR (400 MHz, Chloroform-d)
d
5.80 (d,
D
(
m, 1H), 2.32 (td, J ¼ 14.1, 3.9 Hz, 1H), 2.02e1.96 (m, 1H), 1.85 (ddt,
J ¼ 9.5 Hz, 1H), 4.06 (dddd, J ¼ 37.4, 9.9, 3.3, 1.9 Hz, 1H), 3.69 (dp,
J ¼ 20.0, 6.8 Hz, 1H), 3.08 (q, J ¼ 7.4 Hz, 1H), 2.33e2.18 (m, 1H), 1.92
J ¼ 13.4, 6.7, 3.6 Hz, 1H), 1.77e1.66 (m, 2H), 1.53 (ddt, J ¼ 11.7, 7.6,
4
.4 Hz,1H),1.38e1.31 (m, 4H),1.29e1.20 (m, 8H),1.02e0.97 (m,1H),
(
3
ddd, J ¼ 14.0, 10.0, 4.3 Hz, 1H), 1.81e1.56 (m, 6H), 1.52e1.37 (m,
13
H), 1.33e1.21 (m, 3H), 0.93 (s, 2H), 0.90e0.84 (m, 9H); 1 C NMR
3
0.93 (d, J ¼ 6.2 Hz, 3H), 0.80 (d, J ¼ 7.0 Hz, 3H); C NMR (101 MHz,
CDCl 168.4, 154.1, 104.2, 96.9, 91.7, 90.7, 86.6, 80.4, 64.1, 63.7,
1.8, 45.8, 37.5, 36.4, 34.3, 28.5, 28.4, 26.1, 24.9, 21.8, 20.4, 15.1, 13.6;
3
) d
(
101 MHz, CDCl
3
)
d
163.4, 147.6, 124.1, 102.4, 100.5, 89.1, 86.1, 49.8,
9.4, 48.1, 47.7, 44.9, 44.8, 36.6, 36.1, 28.1, 27.9, 26.5, 26.3,19.6 ( ꢁ 2),
5
4
ꢀ
1
IR (KBr) n/cm : 2929, 2873, 1763, 1636, 1449, 1258, 1131, 978, 880,
45; HRMS m/z: calcd for
35.2156 [MþNa] .
18.8, 18.7, 13.6, 13.3.
7
5
C
25
H37ClN
2
O
7
Na 535.2182, found
þ
4
.1.3.7. (S)-3,4-Dibromo-5-(((1R,2S,5R)-2-isopropyl-5-
methylcyclohexyl)oxy)furan-2(5H)-one (4g). Compound 4g [35] was
4
.1. 4 . 3 . ( 5 S ) - 3 - c h l o ro - 5 - ( ( ( 1 S , 2 R , 5 S ) - 2 - i s o p ro p yl - 5 -
crystallized from EtOH/PE/H
2
O in 35% as a gray crystal. m.p.
methylcyclohexyl)oxy)-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-
ꢂ
20
1
1
40.2e140.8 C; ½aꢃ þ37 (c 1.0, CHCl
3
); H NMR (400 MHz, Chlo-
D
roform-d)
d
5.81 (s, 1H), 3.59 (td, J ¼ 10.7, 4.4 Hz, 1H), 2.32 (qt,
10-yl)piperazin-1-yl)furan-2(5H)-one (5c). Compound 5c was
J ¼ 7.0, 3.5 Hz, 1H), 2.29e2.21 (m, 1H), 1.74e1.63 (m, 2H), 1.56 (s,
ꢂ
20
D
formed in 97% as a yellow solid. m.p. 101.3e102.0 C; ½aꢃ ꢀ24 (c
4
1
1
H), 1.46e1.38 (m, 1H), 1.38e1.32 (m, 1H), 1.11 (td, J ¼ 12.4, 11.0 Hz,
1
1.0, CH
3
OH); H NMR (400 MHz, Chloroform-d)
d
5.77 (s, 1H), 5.28
H), 1.04e0.98 (m, 1H), 0.94 (dd, J ¼ 6.8, 2.3 Hz, 6H), 0.90e0.86 (m,
13
(s, 1H), 4.02 (d, J ¼ 10.3 Hz, 1H), 3.78 (s, 2H), 3.55 (dd, J ¼ 15.6,
H), 0.82 (d, J ¼ 6.9 Hz, 3H); C NMR (101 MHz, CDCl
3
) d 164.3,
4
.9 Hz, 2H), 3.11e3.03 (m, 2H), 2.78e2.70 (m, 2H), 2.56 (ddd,
143.4, 118.5, 104.5, 84.5, 77.2, 58.5, 48.0, 42.1, 33.9, 31.6, 25.2, 22.8,
J ¼ 10.8, 7.2, 4.4 Hz, 1H), 2.39e2.29 (m, 1H), 2.25e2.14 (m, 2H),
2
2.1, 20.9, 18.4, 15.8.
2
.04e1.83 (m, 2H), 1.76e1.27 (m, 14H), 1.23e0.98 (m, 3H),
.96e0.90 (m, 11H), 0.82 (d, J ¼ 7.1 Hz, 3H), 0.76 (d, J ¼ 6.9 Hz, 3H);
0
4
.1.3.8. (5S)-3,4-Dibromo-5-(((2S)-1,7,7-trimethylbicyclo[2.2.1]hep-
13
3
C NMR (101 MHz, CDCl ) d 168.5, 155.1, 104.2, 97.2, 91.7, 90.7, 87.2,
tan-2-yl)oxy)furan-2(5H)-one (4h). Compound 4h [34] was crys-
tallized from EtOH/H O in 14% as a colourless crystal. m.p.
5.0e95.6 C; ½aꢃ þ8 (c 1.0, CHCl
); 1H NMR (400 MHz, Chloro-
5.78 (d, J ¼ 1.3 Hz, 1H), 3.74 (ddd, J ¼ 31.8, 7.8, 3.4 Hz, 1H),
8
2
0.7, 80.4, 51.8, 48.3, 48.2, 45.8, 42.6, 37.5, 36.4, 34.4, 34.1, 31.8, 28.5,
2
6.1, 25.2, 24.9, 22.9, 22.4, 21.8, 21.3, 20.4, 15.9, 14.3, 13.6; IR (KBr)
n/
ꢂ
20
D
ꢀ1
9
3
cm : 2928, 2871, 1763, 1634, 1449, 1257, 1119, 973, 880, 746; HRMS
m/z: calcd for C33
þ
form-d)
d
H52ClN
2
O
7
623.3458, found 623.3436 [MþH] .
1
1
.96 (ddq, J ¼ 20.9,13.3, 3.6 Hz,1H),1.82e1.76 (m,1H),1.76e1.71 (m,
H), 1.71e1.63 (m, 1H), 1.55 (s, 2H), 1.08e1.00 (m, 3H), 1.00e0.92
4
.1.4.4. 3-Chloro-5-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)
oxy)-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-trimethyldecahydro-
H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)piperazin-1-yl)
13
(
m, 5H), 0.91e0.82 (m, 6H), 0.81e0.71 (m, 1H); C NMR (101 MHz,
CDCl 164.3, 143.7, 104.5, 102.1, 90.3, 87.6, 49.9, 49.2, 46.7, 46.7,
3
) d
3
4
5.0, 39.3, 38.5, 34.0, 27.0, 20.0, 12.0, 11.7.
furan-2(5H)-one (5d). Compound 5d was formed in 95% as a yellow
ꢂ
20
D
OH); 1H NMR
solid. m.p. 111.0e112.0 C; ½aꢃ
ꢀ6 (c 1.0, CH
3
4
.1.4. General procedure for the hybrids 5aꢀh
(400 MHz, Chloroform-d)
d
5.72e5.63 (m, 1H), 5.28 (d, J ¼ 1.9 Hz,
Compound 2 (40 mg, 1.14 mmol), furan ethers 4 (1.14 mmol),
1
9
3
1
4
H), 4.02 (d, J ¼ 10.2 Hz, 1H), 3.82e3.68 (m, 3H), 3.59 (dd, J ¼ 12.2,
DIPEA (0.2 mL) and DCM (15 mL) were added to a flask, and stirred
for 2e4 h at room temperature. The reaction mixture was evapo-
rated umder reduced pressure and the residue was purified by
chromatography on a silica gel column (5e6 cm, 20:1 to 5:1 pe-
troleum ethereethyl acetate) to give hybrids 5a¡h.
.4 Hz, 2H), 3.06 (q, J ¼ 10.8, 9.1 Hz, 3H), 2.77 (dt, J ¼ 15.5, 7.9 Hz,
H), 2.63e2.51 (m, 1H), 2.34 (td, J ¼ 14.0, 3.9 Hz, 1H), 2.03e1.98 (m,
H), 1.90e1.82 (m, 2H), 1.74e1.66 (m, 4H), 1.54 (dq, J ¼ 10.0, 5.3,
.7 Hz, 1H), 1.47e1.31 (m, 5H), 1.01 (q, J ¼ 7.0, 5.9 Hz, 3H), 0.97e0.93
13
(
m, 7H), 0.91e0.86 (m, 3H), 0.81 (td, J ¼ 7.0, 6.6, 2.6 Hz, 7H);
C
NMR (101 MHz, CDCl
3
)
d
168.5, 154.8, 104.1, 98.4, 96.2, 91.6, 90.6,
4
3
.1.4.1. 3-Chloro-5-methoxy-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-
,6,9-trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochro-
8
3
8.6, 86.1, 80.3, 51.7, 49.6, 49.3, 46.8, 45.8, 45.2, 39.9, 37.4, 36.3, 34.6,
4.3, 28.4, 27.1, 26.1, 24.8, 21.7, 20.3, 20.2, 13.5, 12.5, 12.0; IR (KBr)
n/
men-10-yl)piperazin-1-yl)furan-2(5H)-one (5a). Hybrid 5a was
ꢀ1
cm : 2953, 2873, 1766, 1635, 1453, 1121, 974, 880, 745; HRMS m/z:
calcd for C33
ꢂ
20
D
þ
formed in 98% as a yellow solid. m.p. 93.3e94.0 C; ½aꢃ þ8 (c 1.0,
H49ClN
2
O
7
Na 643.3121, found 643.3091 [MþNa] .
7

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
4
3
.1.4.5. 3-Bromo-5-methoxy-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-
,6,9-trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochro-
J ¼ 12.4, 4.2 Hz, 1H), 1.38 (d, J ¼ 10.2 Hz, 4H), 1.31 (t, J ¼ 3.8 Hz, 1H),
1.18 (t, J ¼ 7.1 Hz,1H),1.11e0.98 (m, 2H), 0.94 (d, J ¼ 6.5 Hz, 3H), 0.88
(dd, J ¼ 6.3, 2.4 Hz, 2H), 0.86 (d, J ¼ 1.7 Hz, 3H), 0.84 (d, J ¼ 1.7 Hz,
men-10-yl)piperazin-1-yl)furan-2(5H)-one (5e). Compound 5e was
13
ꢂ
20
D
3H), 0.83e0.82 (m, 3H), 0.80 (dd, J ¼ 6.2, 3.1 Hz, 2H); C NMR
formed in 98% as a yellow solid. m.p. 88.6e89.4 C; ½aꢃ ꢀ5 (c 1.0,
1
(101 MHz, CDCl )
3
d
168.9, 157.3, 104.2, 99.3, 91.7, 90.7, 87.1, 80.4,
CH
3
OH); H NMR (400 MHz, Chloroform-d)
d
5.74 (d, J ¼ 3.6 Hz,1H),
8
2
2
0.3, 51.8, 49.6, 47.7, 45.8, 45.0, 37.5, 37.3, 36.4, 34.4, 28.5, 28.4, 28.2,
5
(
2
1
.28 (s, 1H), 4.03 (d, J ¼ 10.2 Hz, 1H), 3.87e3.74 (m, 1H), 3.72e3.62
m, 1H), 3.12e3.02 (m, 1H), 2.82e2.71 (m, 2H), 2.61e2.53 (m, 1H),
.34 (td, J ¼ 13.9, 3.9 Hz, 1H), 2.04e1.98 (m, 2H), 1.91e1.83 (m, 1H),
.71 (dq, J ¼ 10.5, 3.5 Hz, 2H), 1.61 (s, 1H), 1.55 (dt, J ¼ 13.8, 4.3 Hz,
ꢀ
1
6.8, 26.1, 26.0, 24.9, 21.8, 20.4,19.8,19.0,14.2,13.6; IR (KBr)
n
/cm
:
952, 1763, 1628, 1452, 1206, 1127, 962, 880, 744; HRMS m/z: calcd
þ
for C33
4.2. Pharmacology
.2.1. Human liver cancer cell lines SMMC-7721
H50BrN
2
O
7
665.2796, found 665.2794 [MþH] .
1
H), 1.39 (d, J ¼ 6.6 Hz, 3H), 1.36e1.29 (m, 1H), 1.26 (td, J ¼ 7.1,
13
2
.2 Hz, 8H), 0.95 (d, J ¼ 6.2 Hz, 3H), 0.83e0.80 (m, 3H); C NMR
168.6, 156.7, 104.0, 97.7, 91.6, 90.5, 80.3, 63.9,
3.5, 60.4, 51.7, 45.7, 37.4, 36.3, 34.2, 28.3, 26.0, 24.7, 21.6, 20.3, 15.0,
(
3
101 MHz, CDCl ) d
4
6
4
ꢀ
1
SMMC-7721 cells (1 ꢁ 10 in 100
m
L) were seeded on 96 plates in
13.5; IR (KBr)
n
/cm : 2931, 1761, 1628, 1450, 1257, 1206, 962, 880,
35BrN K 581.1259, found 581.1750
ꢂ
triplicate. Following a 24 h culture at 37 C, the medium was
replaced with fresh medium at various concentrations (10, 25, 50,
744; HRMS m/z: calcd for C24
H
2 7
O
þ
[MþK] .
1
00, and 200
m
g/mL) of compounds 5a¡h, DHA, vincristine (VCR)
and cytosine arabinoside (ARA) in a final volume of 100
mL. At the
4
.1.4.6. 3-Bromo-5-ethoxy-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-
same time, the drug-free medium negative control well and the
solvent control well were set with the same volume of dimethyl
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-
10-yl)piperazin-1-yl)furan-2(5H)-one (5f). Compound 5f was
ꢂ
20
D
sulfoxide (DMSO). Cells were respectively incubated at 37 C for
ꢂ
formed in 99% as a yellow solid. m.p. 98.9e99.9 C; ½aꢃ ꢀ5 (c 1.0,
1
24 h. Then, 10 mL of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyl
CH
3
OH); H NMR (400 MHz, Chloroform-d)
d
5.74 (d, J ¼ 3.5 Hz,1H),
tetrazolium bromide (MTT) (2 mg/mL in a phosphate buffer solu-
tion (PBS)) was added to each well, incubated for an additional 4 h,
centrifuged at 1000 r/min for 10 min, and then the medium was
5
.27 (s, 1H), 4.02 (d, J ¼ 10.3 Hz, 1H), 3.87e3.62 (m, 5H), 3.06 (td,
J ¼ 11.2, 7.1 Hz, 2H), 2.83e2.69 (m, 2H), 2.56 (dddd, J ¼ 9.9, 7.0, 4.5,
.5 Hz, 1H), 2.33 (td, J ¼ 14.0, 4.0 Hz, 1H), 2.03e1.95 (m, 1H), 1.86
ddd, J ¼ 13.6, 6.6, 3.3 Hz, 1H), 1.74e1.65 (m, 2H), 1.55 (dt, J ¼ 13.6,
.2 Hz, 1H), 1.49e1.40 (m, 1H), 1.39e1.22 (m, 10H), 1.00 (ddd,
J ¼ 14.2,12.5, 9.8 Hz,1H), 0.94 (d, J ¼ 6.2 Hz, 3H), 0.82e0.79 (m, 3H);
2
(
4
removed. MTT formazan precipitates were dissolved in 150 mL of
DMSO, shaken mechanically for 10 min and then read immediately
at 568 nm using a plate reader (Multiskan MK3, Thermo Fisher
Scientific, USA).
13
3
C NMR (101 MHz, CDCl ) d 168.8, 156.7, 104.2, 97.7, 91.7, 90.7, 80.4,
7
2
2.7, 63.9, 63.5, 51.8, 45.8, 37.5, 36.4, 34.3, 28.5, 27.0, 26.1, 24.8, 21.7,
Cell inhibition rate ¼ [A568 (negative control well) ꢀ A568 (dosing
well)]/A568 (negative control well) ꢁ 100%
ꢀ
1
0.4, 15.1, 13.5; IR (KBr)
n
/cm : 2929, 2872, 1761, 1628, 1449, 1118,
38BrN 557.1857, found
1017, 880, 744; HRMS m/z: calcd for C25
H
2 7
O
þ
5
57.1860 [MþH] .
4
.1. 4 . 7 . ( 5 S ) - 3 - B ro m o - 5 - ( ( ( 1 S , 2 R , 5 S) -2 - i s o p ro p yl - 5 -
4
.2.2. Human normal liver cell lines LO2
The method was similar to that of SMMC-7721 cells described
methylcyclohexyl)oxy)-4-(4-((3R,5aS,6R,8aS,9R,10R,12aR)-3,6,9-
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-
above.
1
0-yl)piperazin-1-yl)furan-2(5H)-one (5g). Compound 5g was
formed in 98% as a yellow solid. m.p. 101.5e102.8 C; ½aꢃ 2D 0 ꢀ19 (c
ꢂ
4.2.3. The general method for testing the activity of compound 5a
1
1.0, CH
3
OH); H NMR (400 MHz, Chloroform-d)
d
5.78 (s, 1H), 5.27
Cells (SMMC-7721, MHCC97H, HCCLM3, HUH-7, HCT116,
(
s, 1H), 4.01 (d, J ¼ 10.2 Hz, 1H), 3.82 (s, 2H), 3.71e3.48 (m, 3H),
4
CCD18CO, MCF-7, and MCF-10A) (1 ꢁ 10 in 100
m
L) were respective
3
1
1
3
3
3
.10e3.02 (m, 2H), 2.73 (ddd, J ¼ 11.3, 7.3, 3.1 Hz, 2H), 2.58e2.51 (m,
H), 2.33 (td, J ¼ 13.9, 3.8 Hz, 1H), 2.23e2.12 (m, 2H), 2.05e1.94 (m,
H), 1.86 (ddt, J ¼ 13.5, 6.7, 3.6 Hz, 1H), 1.79 (s, 2H), 1.73e1.61 (m,
H), 1.54 (dt, J ¼ 13.8, 4.3 Hz, 1H), 1.48e1.26 (m, 6H), 1.22e0.97 (m,
H), 0.95e0.89 (m, 11H), 0.89e0.82 (m, 1H), 0.81 (dd, J ¼ 7.1, 3.1 Hz,
ꢂ
seeded on 96 plates in triplicate. Following a 24 h culture at 37 C,
the medium was replaced with fresh medium at various concen-
trations (0.01, 0.1, 1, 10, and 25
volume of 100 L. At the same time, the drug-free medium negative
control well and the solvent control well were set with the same
volume of dimethyl sulfoxide (DMSO). Cells were respectively
incubated at 37 C for 48 h. Then, 10
2
phosphate buffer solution (PBS)) was added to each well, incubated
for an additional 4 h, centrifuged at 1000 r/min for 10 min, and then
the medium was removed. MTT formazan precipitates were dis-
mg/mL) of compound 5a in a final
m
13
H), 0.75 (d, J ¼ 7.0 Hz, 3H); C NMR (101 MHz, CDCl
3
) d 168.9,
157.6, 156.7, 104.2, 98.1, 91.7, 90.6, 80.6, 80.4, 73.6, 72.8, 63.9, 51.8,
ꢂ
mL of 3-(4,5-dime-thylthiazol-
4
8.3, 45.8, 42.6, 37.5, 36.4, 34.4, 31.8, 28.5, 26.1, 24.9, 22.9, 22.4, 21.8,
-yl)-2,5-diphenyltetrazolium bromide (MTT) (2 mg/mL in a
ꢀ
1
21.3, 20.4, 15.9, 15.1, 13.5; IR (KBr)
n
/cm : 2928, 2871, 1761, 1627,
52BrN
1449, 1118, 961, 880, 744; HRMS m/z: calcd for C33
H
2 7
O
þ
6
67.2952, found 667.2955 [MþH] .
solved in 150 mL of DMSO, shaken mechanically for 10 min and then
read immediately at 568 nm using a plate reader (Multiskan MK3,
Thermo Fisher Scientific, USA).
4
2
.1.4.8. (5S)-3-Bromo-5-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-
- yl ) o x y ) - 4 - ( 4 - ( ( 3 R , 5 a S , 6 R , 8 a S , 9 R ,10 R ,12 a R ) - 3 , 6 , 9 -
trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-
0-yl)piperazin-1-yl)furan-2(5H)-one (5h). Compound 5h was
1
Cell inhibition rate ¼ [A568 (negative control well) ꢀ A568 (dosing
ꢂ
20
D
formed in 98% as a yellow solid. m.p.113.5e114.6 C; ½aꢃ ꢀ14 (c 1.0,
well)]/A
(negative control well) ꢁ 100%.
568
3
CH d 5.75e5.67 (m, 1H),
OH); 1H NMR (400 MHz, Chloroform-d)
5.26 (d, J ¼ 6.6 Hz, 1H), 4.02 (d, J ¼ 10.3 Hz, 1H), 3.92 (d, J ¼ 8.2 Hz,
1H), 3.71 (d, J ¼ 40.6 Hz, 4H), 3.12e3.02 (m, 2H), 2.83e2.71 (m, 2H),
2.62e2.51 (m, 1H), 2.40e2.28 (m, 1H), 2.24 (td, J ¼ 10.4, 5.5 Hz, 1H),
2.04e1.95 (m, 2H), 1.92e1.84 (m, 1H), 1.70 (td, J ¼ 8.8, 7.7, 3.7 Hz,
4H), 1.64 (t, J ¼ 4.6 Hz, 1H), 1.55 (dt, J ¼ 13.7, 4.2 Hz, 1H), 1.46 (dd,
4.2.4. Flow cytometry analysis
SMMC-7721 cells were seeded in on 96 plates in triplicate.
Following a 24 h culture at 37 C, the medium was replaced with
ꢂ
8

M.-X. Wei, J.-Y. Yu, X.-X. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113295
10.1016/j.ejmech.2017.08.005.
2] C.K. Peng, T. Zeng, X.J. Xu, Y.Q. Chang, W. Hou, K. Lu, H. Lin, P.H. Sun, J. Lin,
W.M. Chen, Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives
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[
dimethyl sulfoxide (DMSO). Cells were respectively cultivated at
ꢂ
37
C for 48 h then 0.25% trypsin without ethylene diamine tetra
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[
mg/mL) of compound
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a in a final volume of 100 L. At the same time, the drug-free
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Acknowledgments
We are thankful for the financial support from the National
Natural Science Foundation of China (No. 21462032), the Natural
Science Foundation of Ningxia (No. 2019AAC03018), the Third
Batch of Ningxia Youth Talents Supporting Program (No.
TJGC2018100), the Discipline Project of Ningxia (No.
NXYLXK2017A04), and the College Students’ Innovative and
Entrepreneurship Training Program of Ningxia University (No.
G2020107490003). Thanks to Wuhan Bafeier Biotechnology Service
Co., Ltd. for testing the biological activity.
(2013) 3791e3800, https://doi.org/10.1007/s13277-013-0963-0.
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