Synthesis and biological evaluation of α-santonin derivatives as anti-hepatoma agents

Article abstract of DOI:10.1016/j.ejmech.2018.02.073

A series of α-santonin-derived compounds as potentially anti-hepatoma agents were designed and synthesized in an effort to find novel therapeutic agents. Among them, derivative 5h was more potent than the positive control 5-fluorouracil (5-Fu) on HepG-2, QGY-7703 and SMMC-7721 with IC₅₀ values of 7.51, 3.06 and 4.08 μM, respectively. The structure-activity relationships (SARs) of these derivatives were discussed. In addition, flow cytometry and western blot assay revealed that the derivatives induced hepatoma cells apoptosis by facilitating apoptosis-related proteins expressions. Our findings suggested that these α-santonin-derived analogues hold promise as chemotherapeutic agents for the treatment of human hepatocellular cancer.

Full text of DOI:10.1016/j.ejmech.2018.02.073

Accepted Manuscript
Synthesis and biological evaluation of α-santonin derivatives as anti-hepatoma agents
Hao Chen, Xiao Yang, Zongmin Yu, Ziying Cheng, Hu Yuan, Zeng Zhao, Guozhen
Wu, Ning Xie, Xing Yuan, Qingyan Sun, Weidong Zhang
PII:
S0223-5234(18)30210-1
10.1016/j.ejmech.2018.02.073
EJMECH 10250
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2017
Revised Date: 5 February 2018
Accepted Date: 21 February 2018
Please cite this article as: H. Chen, X. Yang, Z. Yu, Z. Cheng, H. Yuan, Z. Zhao, G. Wu, N. Xie, X.
Yuan, Q. Sun, W. Zhang, Synthesis and biological evaluation of α-santonin derivatives as anti-hepatoma
agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.073.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of
α-santonin derivatives as anti-hepatoma agents
a,c,†
a,†
a,†
a
b
a,c
Hao Chen , Xiao Yang , Zongmin Yu , Ziying Cheng , Hu Yuan , Zeng Zhao ,
a
c
a,*
b,*
a,b,*
Guozhen Wu , Ning Xie , Xing Yuan , Qingyan Sun , Weidong Zhang
a
School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
b
Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China.
c
State Key Laboratory of Innovative Natural Medicine and TCM Injections, Jiangxi
Qingfeng Pharmaceutical Co., Ltd. Ganzhou 341000, Jiangxi, China.
†
These authors contributed equally to this work.
*
Correspondence:
Weidong Zhang, phone/fax: +86-21-81871244, Email: wdzhangy@hotmail.com.
Qingyan Sun, phone/fax: +86-21-20572000-2030, Email: sqy_2000@163.com.
Xing Yuan, phone/fax: +86-21-81871251, Email: yuanxing2012@126.com.

ACCEPTED MANUSCRIPT
Abstract
A series of α-santonin-derived compounds as potentially anti-hepatoma agents were
designed and synthesized in an effort to find novel therapeutic agents. Among them,
derivative 5h was more potent than the positive control 5-fluorouracil (5-Fu) on
HepG-2, QGY-7703 and SMMC-7721 with IC values of 7.51, 3.06 and 4.08 µM,
5
0
respectively. The structure-activity relationships (SARs) of these derivatives were
discussed. In addition, flow cytometry and western blot assay revealed that the
derivatives induced hepatoma cells apoptosis by facilitating apoptosis-related proteins
expressions. Our findings suggested that these α-santonin-derived analogues hold
promise as chemotherapeutic agents for the treatment of human hepatocellular cancer.
Keywords:
α-Santonin-derived
analogues,
α-Methylene-γ-lactone,
Anti-hepatoma,
Structure-activity relationships, Apoptotic
1
. Introduction
Hepatocellular cancer (HCC) is the fifth most common malignancy worldwide
with more than 700,000 cases diagnosed yearly [1]. However, it is the third leading
cause of cancer death due to the high mortality and unsatisfactory treatment options
available [2,3]. At the initial diagnosis, surgical resection and transplantation are
considered a potentially curative modality for HCC [4]. For patients with localized
unresectable disease, systemic treatment is the therapeutic approach. Until recently,
the RAF inhibitor sorafenib is the only systemic treatment to demonstrate a
statistically significant effect. However, it only improves overall survival by

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approximately three months in patients with advanced HCC [5] and several side
effects are often observed [6]. Therefore, development of novel therapeutic targets
and chemotherapeutic agents is urgently needed.
Sesquiterpene lactones (SQLs) are a family of secondary metabolites derived
almost exclusively from Asteraceae/Compositae plants [7]. These natural products are
known for their potent bioactivities, such as anti-inflammatory [7-10], antiviral [11],
antimicrobial [12-13] and antiprotozoal properties [14], as well as cytotoxicity against
tumor
cells
including
HCC
[15-18].
Among
them,
artesunate,
dimethylaminoparthenolide, and L12ADT peptide prodrug, a derivative of
thapsigargin, have currently been evaluated in cancer clinical or preclinical trials
(Chart 1) [18]. SQLs’ anti-tumor properties have received considerable attention.
However, the exact mechanism of how SQLs play a role in inhibiting the proliferation
of tumor cells remains unclear [17]. Their cytotoxic activity has been ascribed as
dependent on the α-methylene-γ-lactone group, which is prone to react with suitable
nucleophiles [19-21]. Some researchers have found that the cytotoxic activities of
these compounds have a strong relationship with their inhibitory effect on many
thiol-containing enzymes involved in the synthesis and processing of proteins, RNA
and DNA [22,23]. In addition, SQLs exert their cytotoxic effects by triggering
apoptosis in many types of cell lines [24-27]. For instance, parthenolide (PTL) with an
active fragment α-methylene-γ-lactone skeleton was shown to inhibit proliferation and
induce apoptosis in various human cancers in vitro, including HCC, colorectal cancer,
cholangiocarcinoma and pancreatic cancer [28]. So it is very promising in searching

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for novel therapeutic agents for human hepatoma from SQLs.
Our previous studies of SQLs synthesized from α-santonin were limited to their
anti-inflammatory properties [29,30]. Combined with the preceding analysis, an
evaluation of the proliferation inhibition effect of these agents against HCC is
essential. The present study investigates their cytotoxic effects on HCC cells and the
correlations between the structures of the derivatives of guaiane-type sesquiterpene
lactone 4 and anti-HCC cells. A series of benzoic acids and cinnamon acids
substituted SQLs derivatives (4a-4p and 5a-5l) were synthesized, some from our
recent report. The effects of the prepared compounds on HCC cell apoptosis were
further evaluated. Herein, the synthesis and biological evaluation of these derivatives
are reported.
2
. Results and discussion
2
.1 Chemistry
A convenient method for synthesizing guaiane-type sesquiterpene lactone
(3αS,6R,8S,9βS)-8-hydroxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3α,4,5,6,6α,7,8,9β-d
ecahydroazuleno[4,5-β]furan-6-yl acetate (4) is shown in Scheme 1. First, we
intended to build the α-methylene group from α-santonin to get
(
3αS,5αS,9βS)-5α,9-dimethyl-3-methylene-3α,5,5α,9β-tetrahydronaphtho[1,2-β]furan-
,8(3H,4H)-dione (2). Then, compound
3αS,6R,9βS)-6,9-dimethyl-3-methylene-2,8-dioxo-2,3,3α,4,5,6,6α,7,8,9β-decahydroa
2
(
zuleno[4,5-β]furan-6-yl acetate (3) was obtained utilizing the photochemical reaction
of compound 2. Regrettably, compound 4 was not produced by reducing compound 3

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under NaBH , and eventually, α-methylene-γ-butyrolactone was reduced to
4
α-methyl-γ-butyrolactone,
3S,3αS,6R,8S,9βS)-8-hydroxy-3,6,9-trimethyl-2-oxo-2,3,3α,4,5,6,6α,7,8,9β-decahydr
oazuleno[4,5-β]furan-6-yl acetate (6).
producing
(
Following the method of our previous literature [27], compound 7 was treated
with t-butyldimethylsilyl triflate (TBSOTf) to protect the 2-hydroxyl group and
compound 8 was obtained. Then, a two-step procedure of selective dehydrogenation
of the α-methyl-γ-lactone moiety was used that included formation of
phenylseleno-substituted 8 followed by treatment with hydrogen peroxide (H O ).
2
2
Finally, deprotection of the TBS group of compound 9 using tetrabutylammonium
fluoride (TBAF), the compound 4 was obtained in excellent yield. The structures of
1
13
the synthesized target compounds were elucidated by H NMR, C NMR and HRMS.
All spectral data were in accordance with assumed structures.
2
.2 Pharmacology and structure-activity relationships
All newly synthesized compounds were tested in vitro and the percentage
inhibition of treated HepG-2 cells at a single dose of 10 µM compared to untreated
control cells was quantified. 5-Fu was used as the positive control. The results showed
in Table 1. Of the 28 compounds tested, most of them exhibited greater than 50%
inhibition of HepG-2 cell growth up to a concentration of 10 µM, except for
compounds 4c, 4g, 5c, 5e, 5f, 5j and 5l.
Compound 4 displayed 32.02% inhibition of HepG-2 cell growth at 10 µM
concentration. Compared with compound 4, the inhibition activities of all of the

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derivatives were enhanced. We hypothesized that the polar functional group hydroxyl
of compound 4 affected the permeability of membrane, which led to depressed
inhibition.
Compounds 4b-4h have methyl-, ethyl-, n-propyl-, n-butyl-, n-amyl-, isopropyl-
and tert-butyl- groups at the R position, respectively. All seven of these compounds
1
exhibited significant effects on inhibition of HepG-2 with the exception of
compounds 4c (42.33%) and 4g (37.86%). Compared to compound 4a, with a
hydrogen atom at the R1 position, compounds 4b-4h had lower activities.
Furthermore, the activities were reduced when branching of the linear carbon chains
were introduced into the R position [4d (62.47%) vs 4g (37.86%) and 4e (67.53%) vs
1
4
h (64.41%)]. These results indicated that a suitable length of the carbon chain could
increase the activity and steric hindrance affected the activity at R position.
1
Comparing analogues 4b (58.47%), 4k (72.42%) and 5b (72.03%) to analogues
4
i (53.03%), 4j (69.25%) and 5d (61.14%) with respect to structures, the latter have
an additional oxygen atom than the former, but the inhibition of HepG-2 cell growth
activities of the latter compounds was lower than that of the former compounds.
These results indicated that the activities of derivatives were affected by electron
cloud density at the R or R position; as the electron cloud density increased, the
1
2
activities decreased.
Among analogues 4m-4p, a halogen was introduced into the R position.
1
Compounds 4m (78.61%), 4n (72.69%), 4o (71.06%) and 4p (60.11%) contain
fluorine, chlorine, bromine and iodine, respectively. These results indicate that when

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the electronegativity of the halogen reduced, the activity with inhibition of HepG-2
decreased. Interestingly, this rule applies to cinnamic acid derivatives 5g-5i.
Compounds 5b-5f, methyl-, methylthio-, methoxy-, ethoxy- and propoxy- at the
R position. Among these five substituent groups, methylthio- was the strongest
2
electron donor, but compound 5c (36.24%) with methylthio- had the weakest activity
on inhibition of HepG-2 at 10 µM. According to the analysis of the structures and
activities of 5b-5f, the activity gradually decreased as electron-donating ability at R2
position enhanced.
Of the 28 derivatives, 21 tested compounds exhibited greater than 50% inhibition
of HepG-2 cell growth at a concentration of 10 µM, the exceptions being compounds
4
c, 4g, 5c, 5e, 5f, 5j and 5l. To further study the cytotoxic profile, the 21 compounds
were selected for further cytotoxicity investigation at different concentrations against
three different types of HCC cells (HepG-2, QGY-7703 and SMMC-7721). In these
tests, 5-Fu was used as the positive control. The IC values of individual compounds
50
for the 5-dose test against each HCC cell line are presented in Table 2. These
compounds showed more potent activity than 5-Fu and had lower cytotoxicity. Among
the derivatives tested, cinnamic acid derivatives 5h displayed the most potent
cytotoxicity against HepG-2, QGY-7703 and SMMC-7721 with IC values of 7.51,
5
0
3
1
.06, and 4.08 µM. For comparison, the positive control 5-Fu had IC values of 15.74,
50
4.20 and 12.47 µM, respectively.
As shown in Table 2, which compares IC values of compounds 4a, 4b, 4d, 4e
5
0
and 4f, the cytotoxicities against HepG-2 and SMMC-7721 gradually decreased with

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the extension of the benzoic acid para alkyl side chain. By contrast, the cytotoxicity
against QGY-7703 gradually enhanced. Comparing compounds 4b, 4k, and 5b to 4i,
4
j, and 5d, respectively, the former compounds showed more potent activities than the
latters against the three HCC cell lines. Beginning with structure analysis, the latter
group had one more oxygen atom than the former. It concluded that the introduction
of oxygen atoms might have a detrimental effect on inhibition potency.
Cytotoxic selectivity against HCC cells is the most important characteristic of
the newly synthesized derivatives. Thus, the inhibitory effects of 21 active compounds
on the proliferation of normal hepatic cells (QSG-7701) were further examined.
According to the data shown in Table 2, compared to 5-Fu, all target compounds
presented a weak cytotoxic activity toward QSG-7701. Among them, compounds 4a,
4
b, 4i, 4l, 5a, 5b, 5d, 5g, 5h and 5i exhibited better cytotoxic selectivity against the
tumor cells and normal cells than the others. So cinnamon acid substituted analogues
showed better cytotoxic selectivity than benzoic acid substituted analogues.
To explore the potential mechanisms of antiproliferative effect induced by
derivatives of α-santonin, annexin V-FITC and PI staining were used and flow
cytometry (FCM) was performed to quantify cell apoptosis (Fig. 1). HCC QGY-7703
cells were cultured with 4n and 5h at concentrations of 5, 10 and 15 µM, respectively.
Treatment with a low concentration (5 µM) of analogues, 4n and 5h did not alter the
number of apoptotic cells compared to the control group. In contrast, the apoptosis
percentage increased significantly after treatment with higher concentrations (10 and
1
5 µM) of compounds 4n and 5h. To further validate the mechanism of

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apoptosis-promoting activities, a western blotting assay was used to evaluate
apoptosis-related proteins expression, including PARP, pro-caspase 3, cleaved caspase
3
and pro-caspase 9 expression in QGY-7703. Compared to the DMSO control, the
expression levels of cleaved PARP and cleaved caspase 3 were increased, while the
pro-caspase 3 and pro-caspase 9 were decreased by 4n and 5h in dose-dependent
manner, which indicated 4n and 5h might up-regulate apoptotic-associated proteins
(
Fig. 2). Together, these observations demonstrated that derivatives of α-santonin
induced apoptosis by facilitating apoptosis-related proteins expressions.
. Conclusion
In conclusion, two series of 28 analogues in total were successfully synthesized
from α-santonin, and their activities on the inhibition of HCC cells were evaluated.
The main bioactive group in their molecular structures is
3
α-methylene-γ-butyrolactones. Thus, most of the analogues showed varying activities
against HCC cells. The SAR analysis proved that: (i) the introduction of halogen
atoms is beneficial to the activity against HCC cells, for instance, derivatives 4m-4p
(compared to 4a) and 4m-4p (compared to 5a); (ii) the introduction of oxygen atoms
at R or R position had a detrimental effect on inhibition potency such as derivatives
1
2
4
i, 5e, 5f and 5l; (iii) cinnamon acid substituted analogues showed better cytotoxic
selectivity than benzoic acid substituted analogues. Among these analogues, 4a, 4b, 4i,
l, 5a, 5b, 5d, 5g, 5h and 5i had selective inhibitory activity against HCC cells.
4
Furthermore, derivatives 4n and 5h induced significant apoptosis effects in human
HCC QGY-7703 cells. These derivatives, especially 5h, presented better cytotoxic

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selectivity for HCC cells, suggesting their potential in targeted chemotherapy for
HCC. Further lead optimization and mechanism studies are thus worth pursuing.
4
4
. Experimental section
.1 Chemistry
Nuclear magnetic resonance (NMR) spectra were recorded using TMS as the
internal standard in CDCl with a Bruker BioSpin GmbH spectrometer at 500 MHz.
3
The names of these compounds are all generated by Chemdraw Version 14.0
(CambridgeSoft, Cambridge, Mass.). When peak multiplicities are reported, the
following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet,
and dd = doublet of doublets. Melting points were determined using an X-4B
micromelting point apparatus. HRMS was recorded on an Agilent 6520
LC-Q-TOF/MS. Flash column chromatography was performed using silica gel (100–
2
00 and 200–300 mesh). All solvents and reagents were of analytical pure grade, and
no further purification needed. All starting materials were commercially available.
The spectroscopic data of intermediates 5 and 6 were given in Ref. 29. The
spectroscopic data of intermediates 4, 7, 8, 9 and derivatives 4a, 4b, 4i, 4l, 4n, 4o, 5a,
5
b, 5f, 5h, 5i, 5j were given in Ref. 30.
4
.1.1 (3R,3αR,5αS,9βS)-3,5α,9-Trimethyl-3-(phenylselanyl)-3α,5,5α,9β-tetrahydro
-
naphtho[1,2-β]furan-2,8(3H,4H)-dione (1)
To a stirred -78 ºC solution of α-santonin (0.246 g, 1.0 mmol, 1.0 equiv) in dry THF
5 mL) under argon, LDA (2.0 M, 0.6 mL, 1.2 equiv) was added dropwise via syringe
over 5 min. After 30 min, a solution of PhSeCl (0.211 g, 1.1 mmol, 1.1 equiv) in dry
(

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THF (10 mL) was added dropwise via syringe to the above mixture while keeping
internal temperature between -55 ̶ -78 ºC. The reaction solution was stirred at -78 °C
for 60 min and then at room temperature for overnight. The completion of the reaction
was detected by TLC and then quenched by addition of saturated aqueous NH Cl. The
4
mixture was extracted with EtOAc three times (3 × 10 mL) and washed with brine.
The combined organic layers were dried by anhydrous Na SO and evaporated under
2
4
vacuum to give a residue that was purified by silica-gel column chromatography
(PE/EtOAc=25:1
̶
20:1
̶
15:1), resulting in a white solid product 1 (0.30 g, 75%), mp
1
1
7
1
98.3−199.4 °C. H NMR (500 MHz, CDCl ) δ 7.65 (d, J = 7.3 Hz, 2H), 7.44 (d, J =
3
.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 2H), 6.68 (d, J = 9.9 Hz, 1H), 6.25 (d, J = 9.9 Hz,
H), 5.22 (d, J = 9.6 Hz, 1H), 2.14 (s, 3H), 2.02 – 1.95 (m, 4H), 1.60 (s, 3H), 1.53 (dd,
13
J = 13.4, 7.6 Hz, 1H), 1.33 (s, 3H); C NMR (125 MHz, CDCl ) δ 186.2, 174.7,
3
1
3
54.7, 150.9, 138.2 (2×C), 130.0, 129.3 (3×C), 126.0, 123.9, 79.3, 57.6, 48.8, 41.3,
7.5, 25.1, 22.3, 20.6, 11.0. The spectroscopic data are consistent with the values
reported in the literature [27].
.1.2 (3αS,5αS,9βS)-5α,9-Dimethyl-3-methylene-3α,5,5α,9β-tetrahydronaphtho[1,2
β]furan-2,8(3H,4H)-dione (2)
4
-
To a solution of compound 1 (0.4 g, 1.0 mmol, 1.0 equiv) and acetic acid (HAc) (0.13
g, 3.0 mmol, 3.0 equiv）in THF (5 mL) cooled to 0 ºC, 30% H O (0.74 mL, 6.5 mmol,
2
2
6
.5 equiv) was added dropwise. The reaction solution was stirred at 0 ºC for 20 min
and then at 20 ºC for 10 min. The reaction was complete detected by TLC and then
quenched by addition of saturated aqueous NaHCO . The mixture was extracted with
3

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EtOAc three times (3 × 10 mL) and washed with brine. The combined organic layers
were dried by anhydrous Na SO and evaporated under vacuum to give a residue that
2
4
was purified by silica-gel column chromatography (PE/EtOAc = 15:1 – 10:1 – 8:1),
1
resulting in a white solid product 2 (0.22 g, 90%); mp 200.6−201.4 °C. H NMR (500
MHz, CDCl ) δ 6.71 (td, J = 3.1, 9.9 Hz, 1H), 6.28 (d, J = 9.9 Hz, 1H), 6.14 (dd, J =
3
3
.1, 3.3 Hz, 1H), 5.50 (dd, J = 3.1, 3.3 Hz, 1H), 4.78 (d, J = 11.4 Hz, 1H), 2.80 (ttd, J
3.3, 7.2, 11.4 Hz, 1H), 2.17 (s, 3H), 2.18 (tdd, J = 6.9, 7.2, 14.1 Hz, 1H), 1.88 (dtd,
J = 3.1, 6.9, 13.8 Hz, 1H), 1.85 (tdd, J = 6.9, 7.2, 14.1 Hz, 1H), 1.68 (dtd, J = 3.1, 6.9,
=
1
3
1
1
3.8 Hz, 1H), 1.32 (s, 3H); C NMR (125 MHz, CDCl ) δ 185.9, 168.8, 155.4, 154.4,
3
38.0, 132.5, 126.1, 119.5, 81.3, 45.7, 40.2, 36.8, 25.0, 23.6, 11.3. The spectroscopic
data are consistent with the values reported in the literature [27].
.1.3 (3αS,6R,9βS)-6,9-Dimethyl-3-methylene-2,8-dioxo-2,3,3α,4,5,6,6α,7,8,9β-deca
hydroazuleno[4,5-β]furan-6-yl acetate (3)
4
-
A solution of compound 2 (10 g, 41.0 mmol) in glacial AcOH (400 mL) was
irradiated under argon at 17 °C for 12 h using a 500 W high-pressure mercury arc
lamp. After the AcOH was evaporated in vacuum, diethyl ether (20 mL) was added to
the resulting residue. Filtration of the precipitate afforded 3 (4.48 g, 36%) as a
1
colorless solid, mp 100.7−101.2 °C. H NMR (500 MHz, CDCl ) δ 6.34 (d, J = 3.2,
3
1
H), 5.61 (d, J = 3.2, 1H), 4.80 (d, J = 10.9, 1H), 4.21 (dd, J = 6.2, J = 2.6, 1H), 3.14
(m, 1H), 2.68 (m, 1H), 2.54 (dd, J = 19.3, J = 6.2, 1H), 2.43 (dd, J= 19.3, J= 2.6, 1H),
13
2
.20–2.34 (m, 2H), 2.01 (s, 3H), 1.95 (s, 3H), 1.46–1.56 (m, 1H), 1.13 (s, 3H); C
NMR (125 MHz, CDCl ): δ 206.8, 170.4, 168.8, 160.3, 143.2, 137.6, 120.8, 85.5,
3

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8
1.7, 47.3, 44.5, 37.5, 36.9, 24.5, 22.3, 20.0, 9.6; The spectroscopic data are
consistent with the values reported in the literature [27].
.1.4 General procedure for syntheses of derivatives 4a-4p and 5a-5l
To a solution of compound 4 (20 mg, 0.065 mmol) and acid (0.065 mmol) in DCM
10 mL) at room temperature was added DMAP (15.9 mg, 0.13 mmol). After stirring
4
(
for 10 min, EDCI (25.0 mg, 0.13 mmol) was added in one portion. The reaction was
allowed to stir overnight. Then, water (10 mL) was added. The reaction mixture was
extracted with DCM (3×10 mL). The organic layer was then washed with brine, dried
over Na SO , and concentrated in vacuum. Purification by flash column
2
4
chromatography (PE: EtOAc = 4:1) provided the derivatives of interest.
.1.4.1 (3 S,6R,8S,9 S)-6-Acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
7,8,9 -decahydroazuleno[4,5-
4
α
β
α,4,5,6,6α,
-
β
β
]furan-8-yl 4-ethylbenzoate (4c)
1
Colorless solid; mp 145.1−146.3 °C; yield 79%: H NMR (500 MHz, CDCl ) δ 7.97
3
(
d, J = 8.3 Hz, 2H), 7.28 (s, 1H), 7.27 (s, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.75 (d, J =
.6 Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.70 (dd, J = 11.0, 1.7 Hz, 1H), 4.30 (t, J = 6.7
Hz, 1H), 3.92 (s, 1H), 2.75 – 2.68 (m, 3H), 1.97 (s, 1H), 1.96 (s, 3H), 1.95 (s, 3H),
6
1
3
1
.27 (s, 1H), 1.26 (s, 3H), 1.25 (s, 2H), 1.24 (s, 4H); C NMR (125 MHz, CDCl ) δ
3
1
70.3, 169.6, 166.3, 141.2, 138.4, 133.2, 130.9, 129.7 (2×C), 128.8, 128.0 (2×C),
119.8, 86.2, 81.6, 80.4, 52.2, 45.1, 37.4, 32.2, 29.0, 24.2, 22.4, 20.0, 15.3, 12.9;
+
HRMS (ESI): m/z calcd for C H O [M+H] : 439.2115, found 439.2116.
2
6
31
6
4
.1.4.2 (3
α
S,6R,8S,9
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
]furan-8-yl 4-propylbenzoate (4d)
α,4,5,
6
,6
α
,7,8,9 -decahydroazuleno[4,5-β
β

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1
Colorless solid; mp 150.5−151.7 °C; yield 82%: H NMR (500 MHz, CDCl ) δ 7.96
3
(
d, J = 8.3 Hz, 2H), 7.26 – 7.26 (m, 1H), 7.24 (s, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.76 (t,
J = 6.1 Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.70 (dd, J = 10.9, 1.8 Hz, 1H), 3.92 (s, 1H),
.00 – 2.91 (m, 1H), 2.69 – 2.60 (m, 3H), 2.50 (td, J = 13.4, 4.2 Hz, 1H), 2.24 (ddd, J
18.3, 13.7, 3.6 Hz, 2H), 1.95 (d, J = 4.6 Hz, 6H), 1.66 (dd, J = 15.0, 7.5 Hz, 3H),
3
=
1
3
1
1
5
.24 (s, 4H), 0.94 (t, J = 7.4 Hz, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 166.3,
3
48.5, 141.2, 138.4, 133.2, 129.6 (2×C), 128.6 (2×C), 127.7, 119.8, 86.2, 81.6, 80.4,
2.2, 45.1, 38.0, 37.4, 32.2, 24.2, 24.2, 22.4, 20.0, 13.7, 12.9; HRMS (ESI): m/z calcd
+
for C H O [M+H] : 453.2272, found 453.2276.
2
7
33
6
4
.1.4.3 (3
α
S,6R,8S,9
βS)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3α,4,5,6,6α,
7
,8,9β
-decahydroazuleno[4,5-
β
]furan-8-yl 4-butylbenzoate (4e)
1
Colorless solid; mp 165.2−166.8 °C; yield 85%: H NMR (500 MHz, CDCl ) δ 7.96 (t,
3
J = 5.8 Hz, 2H), 7.25 (s, 2H), 6.27 (t, J = 2.8 Hz, 1H), 5.76 (t, J = 6.1 Hz, 1H), 5.53 (d,
J = 3.1 Hz, 1H), 4.70 (dd, J = 10.9, 1.7 Hz, 1H), 3.92 (s, 1H), 2.99 – 2.92 (m, 1H),
2
4
1
1
.69 – 2.61 (m, 3H), 2.50 (td, J = 13.4, 4.2 Hz, 1H), 2.30 – 2.18 (m, 2H), 1.95 (d, J =
.8 Hz, 6H), 1.78 – 1.73 (m, 1H), 1.63 – 1.59 (m, 2H), 1.35 (dd, J = 15.0, 7.5 Hz, 2H),
1
3
.25 (d, J = 6.5 Hz, 4H), 0.93 (t, J = 7.4 Hz, 3H); C NMR (125 MHz, CDCl ) δ
3
70.3, 169.6, 166.3, 148.7, 141.2, 138.4, 133.2, 129.6 (2×C), 128.5 (2×C), 127.7,
119.8, 86.2, 81.6, 80.4, 52.2, 45.1, 37.4, 35.7, 33.3, 32.2, 24.2, 22.3, 22.3, 20.0, 13.9,
+
1
2.9; HRMS (ESI): m/z calcd for C H O [M+H] : 467.2428, found 467.2435.
28 35 6
4
.1.4.4 (3
α
S,6R,8S,9
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
]furan-8-yl 4-pentylbenzoate (4f)
α,4,5,6,6α,
7
,8,9β
-decahydroazuleno[4,5-β

ACCEPTED MANUSCRIPT
1
Colorless solid; mp 170.2−172.1 °C; yield 77%: H NMR (500 MHz, CDCl ) δ 7.96
3
(d, J = 8.3 Hz, 2H), 7.24 (s, 2H), 6.26 (d, J = 3.3 Hz, 1H), 5.76 (t, J = 5.7 Hz, 1H),
5
.53 (d, J = 3.1 Hz, 1H), 4.70 (dd, J = 10.9, 1.7 Hz, 1H), 3.92 (s, 1H), 2.95 (dd, J =
0.8, 9.6 Hz, 1H), 2.66 (t, J = 7.8 Hz, 3H), 2.50 (td, J = 13.3, 4.2 Hz, 1H), 2.30 – 2.25
1
(m, 1H), 2.24 – 2.18 (m, 1H), 1.95 (d, J = 4.6 Hz, 6H), 1.79 – 1.72 (m, 1H), 1.65 –
1
0
1
5
.61 (m, 2H), 1.50 – 1.40 (m, 1H), 1.34 (d, J = 1.4 Hz, 1H), 1.25 (d, J = 6.5 Hz, 6H),
1
3
.89 (t, J = 9.6, 4.3 Hz, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 169.6, 166.3,
3
48.8, 141.2, 138.4, 133.2, 129.6 (2×C), 128.5 (2×C), 127.7, 119.8, 86.2, 81.6, 80.4,
2.2, 45.1, 37.4, 36.0, 32.2, 31.4, 30.8, 29.7, 24.2, 22.5, 20.0, 14.0, 12.9; HRMS (ESI):
+
m/z calcd for C H O [M+H] : 481.2585, found 481.2585.
2
9
37
6
4
.1.4.5 (3
,8,9
α
S,6R,8S,9
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3α,4,5,6,6α,
7
β
-decahydroazuleno[4,5-
β
]furan-8-yl 4-isopropylbenzoate (4g)
1
Colorless solid; mp 140.9−142.3 °C; yield 69%: H NMR (500MHz, CDCl ) δ 8.03 (d,
3
J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz,
1
1
1
1
1
1
6
1
H), 6.27 (dd, J = 3.1, 3.2 Hz, 1H), 5.85 (t, J = 14.0 Hz, 1H), 5.51 (dd, J = 3.0, 3.1 Hz,
H), 4.60 (d, J = 10.7 Hz, 1H), 3.49 (dd, J = 7.4, 10.6 Hz, 1H), 2.97 (spt, J = 6.9 Hz,
H), 2.95 (dddt, J = 3.0, 3.2, 10.7, 10.8 Hz, 1H), 2.40 (ddd, J = 7.4, 13.2, 14.0 Hz,
H), 2.24 (ddd, J = 8.3, 9.3, 13.9 Hz, 1H), 2.14 (dddd, J = 8.3, 9.3, 10.8, 12.6 Hz, 1H),
.97 (s, 3H), 1.83 (s, 3H), 1.86 (ddd, J = 9.3, 12.4, 13.9 Hz, 1H), 1.82 (ddd, J = 10.6,
3.2, 14.0 Hz, 1H), 1.64 (dddd, J = 9.3, 10.8, 12.4, 12.6 Hz, 1H), 1.24 (d, J = 6.9 Hz,
1
3
H), 1.22 (s, 3H); C NMR (125 MHz, CDCl ) δ 171.7, 168.1, 166.4, 152.0, 142.4,
3
39.7, 132.3, 129.4, 129.4, 126.9, 125.8, 125.8, 118.8, 86.8, 81.4, 81.3, 51.9, 45.5,

ACCEPTED MANUSCRIPT
3
7.3, 34.1, 32.4, 24.5, 23.3, 23.3, 22.5, 20.0, 12.2; HRMS (ESI): m/z calcd for
+
C H O Na [M+Na] : 475.2091, found 475.2108.
27
32
6
4
.1.4.6 (3
α
S,6R,8S,9
βS)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3α,4,5,6,6α,
7
,8,9β
-decahydroazuleno[4,5-
β
]furan-8-yl 4-(tert-butyl)benzoate (4h)
1
Colorless solid; mp 148.7−149.5 °C; yield 86%: H NMR (500 MHz, CDCl ) δ 7.99
3
(
d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 6.27 (t, J = 3.1 Hz, 1H), 5.80 – 5.72 (m,
H), 5.53 (d, J = 3.0 Hz, 1H), 4.70 (dd, J = 10.9, 1.7 Hz, 1H), 3.93 (s, 1H), 2.96 (dd, J
10.8, 9.7 Hz, 1H), 2.65 (dt, J = 14.7, 8.3 Hz, 1H), 2.50 (dt, J = 13.2, 6.6 Hz, 1H),
.29 – 2.19 (m, 2H), 1.95 (d, J = 4.7 Hz, 6H), 1.78 – 1.72 (m, 1H), 1.49 (s, 1H), 1.34
1
=
2
1
3
(
s, 9H), 1.24 (s, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 169.6, 166.2, 156.8, 141.2,
3
1
38.4, 133.2, 129.47 (2×C), 127.5, 125.40 (2×C), 119.7, 86.2, 81.5, 80.4, 52.2, 45.1,
3
7.4, 35.1, 32.2, 31.1 (3×C), 24.2, 22.4, 20.0, 12.9; HRMS (ESI): m/z calcd for
+
C H O Na [M+Na] : 489.2248, found 489.2252.
28
34
6
4
.1.4.7 (3
α
S,6R,8S,9
-decahydroazuleno[4,5-
Colorless solid; mp 138.2−139.9 °C; yield 76%: H NMR (500 MHz, CDCl ) δ 8.10
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α,4,5,6,6α,
7
,8,9β
β
]furan-8-yl 4-(trifluoromethoxy)benzoate (4j)
1
3
(d, J = 8.8 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 6.27 (d, J = 3.3 Hz, 1H), 5.77 (t, J = 5.7
Hz, 1H), 5.54 (d, J = 3.0 Hz, 1H), 4.69 (dd, J = 10.9, 1.6 Hz, 1H), 3.94 (s, 1H), 2.95 (t,
J = 10.3 Hz, 1H), 2.65 (dt, J = 14.7, 8.2 Hz, 1H), 2.51 (td, J = 13.3, 4.2 Hz, 1H), 2.30
–
1
1
2.21 (m, 2H), 1.96 (s, 3H), 1.94 (s, 3H), 1.79 – 1.72 (m, 1H), 1.51 – 1.41 (m, 1H),
1
3
.23 (s, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 169.5, 165.0, 152.7, 140.7, 138.3,
3
33.6, 131.6 (2×C), 128.6, 121.3, 120.3 (2×C), 119.9, 86.1, 81.5, 81.0, 52.2, 45.1,

ACCEPTED MANUSCRIPT
+
3
7.4, 32.2, 24.2, 22.4, 20.0, 12.9; HRMS (ESI): m/z calcd for C H F O [M+H] :
2
5
26
3
7
4
95.1625, found 495.1617.
S,6R,8S,9 S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
-decahydroazuleno[4,5-
4
.1.4.8 (3
α
β
α
,4,5,6,6α,
7
,8,9
β
β
]furan-8-yl 4-(trifluoromethyl)benzoate (4k)
1
Colorless solid; mp 129.7−131.5 °C; yield 84%: H NMR (500 MHz, CDCl ) δ 8.17
3
(d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 6.27 (d, J = 3.2 Hz, 1H), 5.80 (s, 1H),
5
1
3
.54 (d, J = 2.8 Hz, 1H), 4.70 (d, J = 11.0 Hz, 1H), 3.95 (s, 1H), 2.96 (t, J = 10.5 Hz,
H), 2.67 (dt, J = 16.1, 8.2 Hz, 1H), 2.58 – 2.47 (m, 1H), 2.31 – 2.18 (m, 2H), 1.96 (s,
13
H), 1.95 (s, 3H), 1.77 (d, J = 14.6 Hz, 1H), 1.52 – 1.39 (m, 1H), 1.24 (s, 3H); C
NMR (125 MHz, CDCl ) δ 170.3, 169.5, 165.0, 140.5, 138.3, 133.8, 133.4, 130.0
3
(3×C), 125.5 (3×C), 119.9, 86.1, 81.5, 81.3, 52.1, 45.1, 37.4, 32.1, 24.2, 22.4, 20.0,
+
1
2.9; HRMS (ESI): m/z calcd for C H F O Na [M+Na] : 501.1495, found
2
5
25
3
6
5
01.1514.
4
.1.4.9 (3
α
S,6R,8S,9
βS)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3α,4,5,6,6α,
7
,8,9β
-decahydroazuleno[4,5-
β
]furan-8-yl 4-fluorobenzoate (4m)
1
Colorless solid; mp 173.2−174.8 °C; yield 74%: H NMR (500 MHz, CDCl ) δ 8.07
3
(
dd, J = 8.2, 5.6 Hz, 2H), 7.12 (t, J = 8.5 Hz, 2H), 6.27 (d, J = 3.0 Hz, 1H), 5.76 (s,
H), 5.54 (d, J = 2.6 Hz, 1H), 4.70 (d, J = 10.6 Hz, 1H), 3.93 (s, 1H), 2.95 (t, J = 10.4
Hz, 1H), 2.72 – 2.45 (m, 2H), 2.33 – 2.17 (m, 2H), 1.96 (d, J = 8.9 Hz, 6H), 1.79 –
1
1
3
1
1
8
.70 (m, 1H), 1.49 – 1.42 (m, 1H), 1.24 (s, 3H); C NMR (125 MHz, CDCl ) δ 170.3,
3
69.5, 165.3, 140.9, 138.4, 133.4 (2×C), 132.1 (2×C), 119.8, 115.7, 115.5, 86.1, 81.5,
0.8, 52.1, 45.0, 37.4, 32.2, 29.7, 24.2, 22.4, 20.0, 12.9; HRMS (ESI): m/z calcd for

ACCEPTED MANUSCRIPT
+
C H FO [M+H] : 429.1708, found 429.1706.
24
26
6
4
.1.4.10 (3
α
S,6R,8S,9
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α
,4,5,6,6
α
,
7
,8,9β
-decahydroazuleno[4,5-
β
]furan-8-yl 4-iodobenzoate (4p)
1
Colorless solid; mp 190.2−191.1 °C; yield 89%: H NMR (500 MHz, CDCl ) δ 7.81
3
(
d, J = 8.6 Hz, 2H), 7.75 (d, J= 8.6 Hz, 2H), 6.26 (d, J = 3.3 Hz, 1H), 5.75 (t, J = 6.2
Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 4.69 (dd, J = 10.9, 1.7 Hz, 1H), 3.93 (s, 1H), 2.98 –
.91 (m, 1H), 2.64 (dt, J = 14.6, 8.2 Hz, 1H), 2.51 (td, J = 13.3, 4.2 Hz, 1H), 2.26 (dt,
J = 18.5, 4.4 Hz, 2H), 1.96 (s, 3H), 1.93 (s, 3H), 1.77 – 1.71 (m, 1H), 1.50 – 1.41 (m,
2
1
3
1
1
3
5
H), 1.23 (s, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 169.5, 165.8, 140.8, 138.3,
3
37.8 (2×C), 133.6, 131.0 (2×C), 129.7, 119.9, 100.9, 86.1, 81.5, 80.9, 52.1, 45.1,
+
7.4, 32.2, 24.2, 22.4, 20.0, 12.9; HRMS (ESI): m/z calcd for C H IO [M+H] :
2
4
26
6
37.0769, found 537.0767.
S,6R,8S,9 S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
-decahydroazuleno[4,5-
4
.1.4.11 (3
α
β
α
,4,5,6,6α,
7
,8,9
β
β
]furan-8-yl (E)-3-(4-(methylthio)phenyl)acrylate (5c)
1
Colorless solid; mp 150.3−151.7 °C; yield 84%: H NMR (500 MHz, CDCl ) δ 7.66
3
(d, J = 15.8 Hz, 1H), 7.48 – 7.41 (m, 2H), 7.23 (d, J = 8.4 Hz, 2H), 6.40 (t, J = 16.4
Hz, 1H), 6.26 (t, J = 3.1 Hz, 1H), 5.74 – 5.64 (m, 1H), 5.53 (d, J = 3.0 Hz, 1H), 4.72 –
4
2
1
1
.56 (m, 1H), 3.89 (s, 1H), 2.94 (t, J = 10.2 Hz, 1H), 2.60 (dt, J = 14.6, 8.3 Hz, 1H),
.51 (d, J = 1.6 Hz, 4H), 2.27 – 2.17 (m, 2H), 1.98 (s, 1H), 1.97 (s, 3H), 1.95 (s, 1H),
1
3
.92 (s, 2H), 1.85 (d, J = 16.6 Hz, 1H), 1.23 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
70.3, 169.6, 166.8, 144.6, 142.1, 141.2, 138.5, 133.0, 130.8, 128.5 (2×C), 126.0

ACCEPTED MANUSCRIPT
(2×C), 119.8, 116.9, 86.2, 81.6, 80.0, 52.1, 45.1, 37.5, 32.2, 24.2, 22.4, 20.0, 15.1,
+
1
2.9; HRMS (ESI): m/z calcd for C H O SNa [M+Na] : 505.1655, found 505.1648.
2
7
30
6
4
.1.4.12 (3
α
S,6R,8S,9
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α,4,5,6,6α,
7
,8,9β
-decahydroazuleno[4,5-β
]furan-8-yl (E)-3-(4-methoxyphenyl)acrylate (5d)
1
Colorless solid; mp 135.4−137.2 °C; yield 81%: H NMR (500 MHz, CDCl ) δ 7.67
3
(
d, J = 15.8 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 6.94 – 6.88 (m, 2H), 6.33 (d, J = 16.0
Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.66 (t, J = 6.7 Hz, 1H), 5.54 – 5.50 (m, 1H), 4.65
ddd, J = 33.0, 11.0, 1.7 Hz, 1H), 3.89 (dd, J = 5.4, 3.6 Hz, 1H), 3.84 (d, J = 2.0 Hz,
(
3
1
7
1
8
H), 2.94 (dd, J = 10.9, 9.5 Hz, 1H), 2.64 – 2.44 (m, 2H), 2.28 – 2.18 (m, 2H), 1.98 (s,
H), 1.97 (s, 2H), 1.95 (s, 1H), 1.92 (s, 2H), 1.72 – 1.69 (m, 1H), 1.44 (dd, J = 10.0,
1
3
.2 Hz, 1H), 1.23 (s, 2H), 1.14 (s, 1H); C NMR (125 MHz, CDCl ) δ 170.3, 169.6,
3
67.0, 161.5, 144.8, 138.5, 132.9, 129.8 (2×C), 127.1, 119.8, 115.4, 114.4 (2×C), 86.2,
1.6, 79.9, 55.4, 52.1, 45.1, 37.5, 32.3, 29.7, 24.2, 22.4, 20.0, 12.9; HRMS (ESI): m/z
+
calcd for C H O Na [M+Na] : 489.1883, found 489.1881.
2
7
30
7
4
.1.4.13 (3
α
S,6R,8S,9
-decahydroazuleno[4,5-
Colorless solid; mp 141.2−143.1 °C; yield 80%: H NMR (500 MHz, CDCl ) δ 7.66
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α,4,5,6,6α,
7
,8,9β
β
]furan-8-yl (E)-3-(4-ethoxyphenyl)acrylate (5e)
1
3
(
d, J = 15.8 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.91 – 6.86 (m, 2H), 6.32 (d, J = 16.0
Hz, 1H), 6.26 (d, J = 3.2 Hz, 1H), 5.73 – 5.63 (m, 1H), 5.52 (d, J = 3.1 Hz, 1H), 4.68
d, J = 12.5 Hz, 1H), 4.07 (q, J = 7.0 Hz, 2H), 3.87 (d, J = 16.5 Hz, 1H), 2.94 (t, J =
0.6 Hz, 1H), 2.62 – 2.55 (m, 1H), 2.30 – 2.27 (m, 1H), 2.20 (d, J = 4.6 Hz, 1H), 1.97
s, 3H), 1.94 (s, 1H), 1.92 (s, 2H), 1.87 (s, 1H), 1.73 – 1.67 (m, 1H), 1.64 – 1.58 (m,
(
1
(

ACCEPTED MANUSCRIPT
1
3
1
H), 1.44 – 1.41 (m, 3H), 1.23 (s, 3H); C NMR (125 MHz, CDCl ) δ 170.3, 169.6,
3
1
67.1, 160.9, 144.9, 141.3, 138.5, 132.9, 129.8 (2×C), 126.9, 119.8, 115.3, 114.8
(2×C), 86.2, 81.6, 79.8, 63.6, 52.1, 45.1, 37.5, 32.3, 29.7, 24.2, 22.4, 20.0, 14.7, 12.8;
+
HRMS (ESI): m/z calcd for C H O [M+H] : 481.2221, found 481.2220.
2
8
33
7
4
.1.4.14 (3
α
S,6R,8S,9
-decahydroazuleno[4,5-
Colorless solid; mp 129.7−131.5 °C; yield 91%: H NMR (500 MHz, CDCl ) δ 7.67
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α,4,5,6,6α,
7
,8,9β
β
]furan-8-yl (E)-3-(4-fluorophenyl)acrylate (5g)
1
3
(d, J = 16.0 Hz, 1H), 7.52 (dd, J = 8.7, 5.4 Hz, 2H), 7.08 (t, J = 8.6 Hz, 2H), 6.39 (d, J
=
16.0 Hz, 1H), 6.26 (d, J = 3.3 Hz, 1H), 5.69 (dt, J = 12.6, 6.1 Hz, 1H), 5.53 (d, J =
3
1
1
.0 Hz, 1H), 4.68 (dd, J = 10.9, 1.8 Hz, 1H), 3.90 (s, 1H), 2.94 (dd, J = 10.8, 9.5 Hz,
H), 2.50 (td, J = 13.3, 4.2 Hz, 1H), 2.29 – 2.17 (m, 2H), 1.97 (s, 3H), 1.94 (s, 1H),
13
.92 (s, 3H), 1.73 – 1.66 (m, 1H), 1.44 (dd, J = 10.9, 3.2 Hz, 1H), 1.23 (s, 3H); C
NMR (125 MHz, CDCl ) δ 170.3 (2×C), 169.6, 166.6, 165.0, 143.8, 141.0, 133.1,
3
1
3
4
30.0, 130.0 (2×C), 119.8 (2×C), 117.8, 116.2, 116.0, 86.2, 81.6, 80.1, 52.1, 45.1,
+
7.5, 32.2, 24.2, 22.4, 20.0, 12.8; HRMS (ESI): m/z calcd for C H FO [M+H] :
2
6
28
6
55.1864, found 455.1861.
S,6R,8S,9 S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
-decahydroazuleno[4,5-
4
.1.4.15 (3
α
β
α
,4,5,6,6α,
7
,8,9β
β
]furan-8-yl (E)-3-(4-(trifluoromethyl)phenyl)acrylate
(5k)
1
Colorless solid; mp 140.3−142.1 °C; yield 87%: H NMR (500 MHz, CDCl ) δ 7.75 –
3
7
.69 (m, 1H), 7.67 – 7.62 (m, 4H), 6.53 (t, J = 9.4 Hz, 1H), 6.28 – 6.24 (m, 1H), 5.75
5.64 (m, 1H), 5.52 (dd, J = 7.7, 3.0 Hz, 2H), 4.71 – 4.61 (m, 1H), 3.88 (d, J = 26.5
–

ACCEPTED MANUSCRIPT
Hz, 1H), 2.93 (dd, J = 22.6, 11.2 Hz, 1H), 2.61 (dt, J = 14.6, 8.2 Hz, 1H), 1.98 (s, 3H),
1
3
1
.92 (s, 3H), 1.25 (d, J = 4.8 Hz, 4H), 1.23 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
1
70.3, 169.5, 166.1, 143.2, 140.8, 138.4, 137.7, 133.3, 133.0, 128.2 (2×C), 125.9
(2×C), 120.6, 119.9, 86.2, 81.6, 80.4, 52.1, 45.1, 37.5, 32.2, 29.7, 24.2, 22.4, 20.0,
+
1
5
2.8; HRMS (ESI): m/z calcd for C H F O Na [M+Na] : 527.1652, found
2
7
27
3
6
27.1659.
4
.1.4.16 (3
α
S,6R,8S,9
-decahydroazuleno[4,5-
Colorless solid; mp 151.4−152.3 °C; yield 88%: H NMR (500 MHz, CDCl ) δ 8.26
β
S)-6-acetoxy-6,9-dimethyl-3-methylene-2-oxo-2,3,3
α,4,5,6,6α,
7
,8,9β
β
]furan-8-yl (E)-3-(4-nitrophenyl)acrylate (5l)
1
3
(d, J = 8.7 Hz, 2H), 7.78 – 7.66 (m, 3H), 6.57 (dd, J = 20.4, 16.0 Hz, 1H), 6.27 (t, J =
3
3
2
1
1
1
.2 Hz, 1H), 5.69 (t, J = 5.9 Hz, 1H), 5.54 (d, J = 3.0 Hz, 1H), 4.72 – 4.59 (m, 1H),
.92 (s, 1H), 2.62 (dt, J = 14.6, 8.2 Hz, 1H), 2.51 (td, J = 13.4, 4.2 Hz, 1H), 2.27 –
.18 (m, 2H), 1.99 – 1.97 (m, 3H), 1.96 (s, 1H), 1.93 (s, 3H), 1.74 – 1.68 (m, 1H),
1
3
.44 (ddd, J = 11.0, 8.5, 3.0 Hz, 1H), 1.23 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
70.3 169.5, 165.7, 148.6 142.2 140.7 140.4, 138.3, 133.5, 128.7 (2×C), 124.2 (2×C),
22.3, 119.9, 86.1, 81.5, 80.7 52.1, 45.1 37.4, 32.1, 24.2, 22.4, 20.0, 12.8; HRMS
+
(
ESI): m/z calcd for C H NO Na [M+Na] : 504.1629, found 504.1620.
2
6
27
8
4
4
.2 Biological assays
.2.1 Cell culture
Cells were cultured in media containing 10% FBS, 100 units/ml penicillin, 100 µg/ml
streptomycin at 37 °C in a humidified incubator containing 5% CO . HCC cell lines
2
QGY-7703, HepG-2 and SMMC-7721 and liver non-tumor cell lines QSG-7701 were

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obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
QGY-7703, SMMC-7721 and QSG-7701 cells were maintained in RPMI 1640
medium and HepG-2 cells were maintained in DMEM medium.
4
.2.2 Cell proliferation/viability assay
Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo,
Kumamoto, Japan) according to the manufacturer’s instructions. In brief, the cells (2
5
×
10 cells/well in 100 µl fresh medium) were cultivated in 96-well microplates for 24
h, treated with different concentrations of compounds or 1% DMSO as vehicle control.
After incubation with dose-related concentrations compounds or 1% DMSO as
vehicle control for 24 h, 10 µl of CCK-8 solution was added to each well for
additional 1 h incubation before the absorbance was measured at 450 nm by an epoch
microplate spectrophotometer.
4
.2.3 Flow cytometry analysis
5
QGY-7703 cells were seeded in 12-well plates (5 × 10 cells/well) for 24 h and then
treated with compounds at dose-related concentrations or DMSO for 24 h. The treated
cells were stained using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences,
San Jose, CA, USA) according to the manufacturer’s instructions. After incubation at
room temperature for 15 min in the dark, the apoptotic cells were immediately
analyzed by flow cytometry (Becton Dickinson FACS Vantage SE, San Jose, CA,
USA).
4
.2.4 Western blotting analysis
QGY-7703 cells were subcultured in a 6-well plate. After incubation for 24 h, cells

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were treated with compounds for 24 h. The cells were lysed in GLO lysis buffer
(Promega, USA) supplemented with 1x protease inhibitors (Roche Applied Sciences,
Germany). The concentration of protein in the supernatant fractions was measured
TM
using the Pierce
BCA Protein Assay Kit, according to the manufacturer’s
instructions. Equal amounts of protein were applied to 10 % SDS-polyacrylamide gel
and then transferred to PVDF membranes (Bio-Rad). After transfer, the membrane
was blocked in 5% fat-free milk for 1 h at RT and incubated with primary antibody
for PARP (1:1000; Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3,
pro-caspase 3, pro-caspase 9 (1:1000; Cell Signaling Technology) and GAPDH
(1:3000, Beyotime, Shanghai, China) at 4 ºC overnight，followed by incubation with
a donkey anti-rabbit or a donkey anti-mouse second antibodies (IRDye 800, LI-COR,
Biosciences) for 1 h at RT. The image was captured by the Odyssey Infrared Imaging
System (LI-COR, Biosciences), according to the manufacturer's instructions. Protein
densitometry was performed using Quantity One imaging software (Bio-Rad).
Acknowledgments
The work was supported by Professor of Chang Jiang Scholars Program, NSFC
(81230090, 81520108030, 21472238, 81602980), Shanghai Engineering Research
Center for the Preparation of Bioactive Natural Products (16DZ2280200), the
Scientific Foundation of Shanghai China (12401900801, 13401900103,
1
3401900101), National Major Project of China (2011ZX09307-002-03)
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Figures, Schemes and Tables
Chart 1. The chemical structures of artesunate, dimethylaminoparthenolide, and
L12ADT.
Figure 1. 5-Fu, 4n and 5h induce apoptosis of QGY-7703 cells in dose-dependent
manner.
Figure 2. 5-Fu, 4n and 5h induce apoptosis related proteins expression in QGY-7703
cells.
Scheme 1. Synthetic route for compound 4.
Scheme 2. Derivatives (4a-4p and 5a-5l) of compound 4.
Table 1. Inhibition rates of compounds 4, 4a-4p and 5a-5l against the HepG-2 cell
line (10 µM)
Table 2. Antiproliferative activities of target compounds against human HCC cells and
hepatocytes.