Oridonin derivatives as potential anticancer drug candidates triggering apoptosis through mitochondrial pathway in the liver cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.06.006

The biological function of the natural ent-kaurene diterpenoid isolated from genus Isodon, oridonin, has been intensively studied. However, its mechanism studies and clinical applications were hampered by its moderate biological activities. In order to en

Full text of DOI:10.1016/j.ejmech.2019.06.006

European Journal of Medicinal Chemistry 178 (2019) 365e379
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Oridonin derivatives as potential anticancer drug candidates
triggering apoptosis through mitochondrial pathway in the liver
cancer cells
,
,
Dongdong Luo a 1, Yujiao Yi a 1, Kai Peng a, Tangrong Liu a, Jiayu Yang a, Shan Liu a,
Wanzhou Zhao b, Xianjun Qu c, Wengong Yu a, Yuchao Gu a **, Shengbiao Wan a
,
, *
a Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Marine Drugs,
Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, and Yushan Road 5, Qingdao, 266003, China
b The Nanjing Han & Zaenker Cancer Institute (NHZCI), Nanjing OGpharma Co. Ltd., Nanjing, 210036, China
c Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological function of the natural ent-kaurene diterpenoid isolated from genus Isodon, oridonin, has
been intensively studied. However, its mechanism studies and clinical applications were hampered by its
moderate biological activities. In order to enlarge the applied range of oridonin and explore its mech-
anism of action, a series of derivatives were designed and synthesized based on the structure of oridonin.
Some of the derivatives were significantly more potent than oridonin against four cancer cell lines.
Especially, the most potent compound 20 markedly inhibited the proliferation of well differentiated
Received 5 April 2019
Received in revised form
20 May 2019
Accepted 3 June 2019
Available online 4 June 2019
HepG2 and poorly differentiated PLC/PRF/5 cells, with IC50 values as low as 1.36
mM and 0.78 mM
Keywords:
Oridonin derivatives
Liver cancer
Anticancer activity
Apoptosis
Mouse xenograft model
respectively, while the IC50 values of oridonin are 8.12 M and 7.41 M. We found that compound 20
m
m
inhibited liver cancer cell proliferation via arresting cell cycle at G1 phase. Moreover, it induced liver
cancer cell apoptosis by decreasing the mitochondrial membrane potential, increasing intracellular
reactive oxygen species level and inducing the expression of apoptosis-related proteins. Furthermore,
compound 20 significantly inhibited growth of PLC/PRF/5 xenograft tumors in nude mice and had no
observable toxic effect. Altogether, these results indicated that compound 20 is a promising lead for liver
cancer therapeutics.
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
[20e25]. The most prominent achievement was made by Hengrui
Medicine Co. Ltd. (Lianyungang, China), with the -alanine-(14-
L
Oridonin, one of the main active compounds isolated from
Chinese herb medicine Rabdosia rubescens [1,2], has aroused
considerable attention for its potential antibacterial, anti-
inflammatory, and antitumor applications [2e14], and is widely
used to treat inflammation and cancer for many years [15]. How-
ever, its relatively low biological activity and low bioavailability
hinder the development of oridonin as a clinical drug [16e19].
Some significant structural optimizations have been made and
more potent antitumor and antibacterial derivatives were found
oridonin)ester trifluoroacetate (HAO472, 2, Fig. 1) approved to
carry out phase I human clinical trial (CTR20150246) in China for
the treatment of acute myelogenous leukemia [26]. It was also re-
ported some 14-O oridonin derivatives and a ring-opening reaction
producing ent-6,7-seco-oridonin derivatives by using NaIO4 and
Pb(OAc)4, could markedly inhibit the proliferation of cancer cells
[27,28]. It was then concluded that the modification of 14-OH and
B-ring were effective methods for the synthesis of novel potential
derivatives. So in previous study [29], we synthesized several 14-O
derivatives and novel 6,20-epoxy ent-kaurane diterpenoid de-
rivatives from oridonin, in which 6,20-endo ring was formed via
DAST promoted rearrangement. CCK-8 assay showed that 14-O
derivative 4 exhibited superior antitumor activities compared to
6,20-epoxy derivative 3 against HepG2 cell line (Fig. 1).
* Corresponding author.
** Corresponding author.
E-mail addresses: guych@126.com (Y. Gu), biaowan@ouc.edu.cn (S. Wan).
These authors contribute equally to this work.
In this study, three acetylated derivatives and a series of novel
1
https://doi.org/10.1016/j.ejmech.2019.06.006
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
Fig. 1. The structures of oridonin, HAO472, 6,20-epoxy derivatives and 14-O derivatives.
14-O derivatives of oridonin were synthesized. Biological evalua-
tions including antitumor activity against human hepatic carci-
noma HepG2, lung cancer A549, multiple myeloma RPMI-8226 cell
lines were carried out. Some of them exhibited potent anti-
proliferative potency against HepG2 cell line. Subsequently, we
evaluated the potency of these oridonin derivatives against PLC/
PRF/5 (another human hepatic carcinoma) cell line and the repre-
sentative compound 20 demonstrated more potent inhibiting ac-
tivity against PLC/PRF/5 cell line compared with HepG2 cell line.
Additionally, further experiments into in vivo antitumor efficacy
and mechanisms behind antitumor activity of compound 20 were
also performed, including cell cycle progression, apoptotic induc-
tion, mitochondria membrane potential disruption and the
expression of apoptotic proteins. Overall, a novel 14-O derivative 20
with potent cytotoxic activity against PLC/PRF/5 cell line was
developed as a promising lead compound for cancer therapeutics.
fluorobenzoic acid 11 in anhydrous methanol with the concen-
trated sulfuric acid which was added dropwise afforded the corre-
spondent ester 12. The nitro group of compound 12 is reduced
through hydrogenation to obtain methyl 2-amine-4-fluorobenzoate
13. The amide 14, 16, 18 were obtained through the esterification of
the primary amine 13 with three different carboxylic acids, respec-
tively. The final deprotections were succeeded with methanolic so-
diumhydroxidetoyield parafluorobenzoic acidderivatives 15, 17, 19,
respectively. A series of 14-O derivatives 20-39 were obtained, as
shown in Scheme 3, through the combination of oridonin 1 and
compounds 15, 17, 19 or other commercially available carboxylic
acids under the condition of 4-dimethylaminopyridine (DMAP) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCI) in dichloromethane (DCM).
2.2. Biology assays
2. Results and discussion
2.2.1. Cytotoxic activity and structure-activity relationships
analysis
2.1. Chemistry
The cytotoxicity of the oridonin derivatives was determined by
CCK-8 assay in vitro. Three different cell lines were used: HepG2
(human hepatic carcinoma), RPMI-8226 (human multiple
myeloma) and A549 (human lung carcinoma). The activities of
derivatives were shown in Table 1 with Paclitaxel (PTX) as positive
control.
Among all the molecules, acetylation of the hydroxyl group at
the C-1 produced compound 8 to generate a moderate increase of
antitumor activity on all the three cell lines compared to oridonin.
Consequent acetylation of the hydroxyl group at the C-6 provided
compound 9 to exhibit the diminished antitumor activity. Sur-
prisingly, oridonin tetraacetate compound 10 exerted superior
antiproliferative effects to oridonin against HepG2 and A549 cell
lines. Inspiringly, acylation of hydroxyl group at the C-14 by
Acetylation of oridonin was investigated and shown in Scheme 1
[30,31]. Oridonin was reacted with 2,2-dimethoxypropane in pres-
ence of p-TsOH in acetone to give a protected intermediate 5.
Consequent acetylation provided the known compound 6 and the
newcompound7. Compound7wasunambiguouslydeterminedbyX-
ray crystallography (see CIF file and Fig. S1 of Supporting Informa-
tion). Deprotection of 6 and 7 produced the known compound 1-O-
acetyl oridonin 8 and a new compound 1, 6-O-diacetyl oridonin 9,
respectively. Direct acetylation of oridonin catalyzed by HClO4 affor-
ded oridonin tetraacetate 10, inwhich the hemiketal ring was broken.
The synthesis procedure of the parafluorobenzoic acid de-
rivatives was outlined in Scheme 2. The reflux of 3-nitro-4-
Scheme 1. Synthesis of acetylated derivatives. Reagents and conditions: (a) 2,2-Dimethoxypropane, acetone, p-TsOH, 56 ꢀC, 90%; (b) Ac2O, pyridine, rt; (c) 2% HCl/THF, rt, 85%; (d)
Ac2O, HClO4, rt, 88%.

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
367
Scheme 2. Synthesis of parafluorobenzoic acid derivatives. Reagents and conditions: (a) MeOH, H2SO4, reflux; (b) Pd/C, H2, MeOH; (c) EDCI, DMAP, DCM; (d) NaOH, MeOH/H2O.
Scheme 3. Synthesis of 14-O derivatives. Reagents and conditions: (a) corresponding acids, EDCI, DMAP, DCM, 0 ꢀC, overnight.
methoxy substituted cinnamic acids afforded compounds 20 (with
a 4-methoxycinnamyl group), 21 (with a 3,4,5-trimethoxycinnamyl
group), and 22 (with a 3,4-dimethoxycinnamyl group) which have
enhanced antitumor activity in all the three cell lines compared to
oridonin. Remarkably, compound 20, being the most potent anti-
substituted benzoic acid moieties, substituted phenylacetic acid
moiety, substituted phenylpropionic acid moieties, did not provide
potent cytotoxicity as compound 20.
2.2.2. Cytotoxicity of derivatives against PLC/PRF/5 cell line
tumor agent with IC50 value of 1.36
suggested that the cinnamyl structure are more preferable. Com-
M in
mM against HepG2 cell line,
Different liver cancer cells may have different responses to the
bioactive compounds due to various etiological factors. HepG2 cells
are well differentiated, which does not have any genomic loss.
However, some liver cancer cells are poorly differentiated and have
gene defects, such as PLC/PRF/5, they may have different responses
to compound 20 compared with HepG2. Three most potent ori-
donin compounds 4 (Fig. 1), 20 and 22 against HepG2 were further
tested against PLC/PRF/5 and normal L-O2 cells (Table 2), and all the
pound 31, 14-parafluorobenzoylated oridonin (IC50 ¼ 3.10
m
HepG2), has potent inhibiting activity on HepG2 cell line. However,
compounds 26, 27, 28 contained p-fluorobenzoyl moiety at the 14-
O position have exhibited less potent antitumor activity against
HepG2 cell line than compound 31. The remaining compounds 32-
39, containing hydrophobic moieties such as lipoic acid moiety,

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D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
Table 1
Therefore, the most promising compound 20 was selected to
investigate the underlining mechanisms of its antitumor efficacy
against PLC/PRF/5 and HepG2 liver cancer cells.
The cytotoxicity data of oridonin derivatives in three cancer cell lines.
Compds
Cytotoxicity (IC50
, mM)
HepG2
RPMI-8226
A549
2.2.4. Compound 20 induced cell cycle arrest at G1 phase in PLC/
PRF/5 and HepG2 cells
PTX
1
8
0.18 0.35
8.12 0.19
5.67 0.55
12.34 0.23
5.40 0.44
1.36 0.45
5.98 0.44
1.98 0.66
8.99 0.86
9.63 0.67
12.56 0.87
6.54 0.18
8.20 0.65
3.76 0.78
27.46 1.10
32.33 0.54
3.10 0.44
26.01 0.99
16.04 1.56
15.40 1.43
36.60 1.00
5.66 0.49
44.56 2.77
9.76 2.02
8.79 0.98
1.53 0.44
9.25 0.32
11.03 0.22
12.98 0.57
13.86 0.19
8.56 0.43
5.98 0.88
5.77 0.65
9.42 0.90
10.09 0.41
12.44 0.19
10.45 0.90
6.71 0.67
18.99 1.26
15.77 0.14
˃100
14.35 1.88
12.21 0.10
17.54 1.32
14.65 1.54
35.21 3.11
5.01 1.32
16.63 0.37
8.65 1.65
10.88 1.89
0.39 0.54
30.40 1.34
10.90 0.61
10.54 0.25
10.45 0.90
12.01 0.43
8.80 0.78
24.91 1.44
13.98 2.13
22.81 1.36
11.03 1.86
20.90 1.33
11.03 0.46
17.19 0.21
31.13 1.22
˃100
14.66 1.15
10.66 1.77
13.98 0.89
12.44 1.23
17.42 2.00
16.74 1.87
22.55 1.76
11.54 1.54
37.54 0.65
To explore whether the antitumor effect of compound 20 in PLC/
PRF/5 and HepG2 cells is resulted from cell cycle arrest, we detected
the cell cycle progression by flow cytometry. After incubation with
compound 20 for 24 h, the cell cycle of PLC/PRF/5 cells was signif-
icantly arrested at G1 phase and the percentage of cells in G1 stage
increases from 34.78 to 45.41 (Fig. 3). The cell cycle of HepG2 cells
was also arrested at G1 phase. The percentage of cells in G1 stage
increases from 37.06 to 42.33 (Fig. 3). These results indicated that
compound 20 induced cell cycle arrest at G1 phase in PLC/PRF/5
and HepG2 cells.
9
10
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
2.2.5. Compound 20 induced apoptosis of PLC/PRF/5 and HepG2
cells
For normal cells, apoptosis is a common process, with an or-
dered, genetically controlled cell death procedure, maintaining
homeostasis. However, cancer cells could avoid apoptosis and enjoy
division. Therefore, induction of apoptosis is a common way for
cancer therapeutic. To investigate whether compound 20 decreases
liver cancer cell survival by inducing apoptosis, several methods
were used to detect cell apoptosis. Hoechst 33258 staining was
used to assess nuclear changes in PLC/PRF/5 and HepG2 cells. We
found that the chromatin is markedly shrunk after incubation with
compound 20 for 24 h (Fig. 4A), suggesting cell apoptosis. To further
confirm whether compound 20 could induce apoptosis, PLC/PRF/5
and HepG2 cells were cultured in medium contained different
Antitumor activity was measured by determining IC50 in HepG2, RPMI-8226 and
A549 cells. All compounds were dissolved in DMSO and used at different concen-
trations. Each experiment was repeated at least three times independently and IC50
value is presented as mean standard error of mean. Paclitaxel (PTX) was used as a
positive control.
three compounds exhibit strongly effective cytotoxicity against
PLC/PRF/5 cells, especially the compounds 20, 22. The results
showed that these compounds might have good activities against
liver cancer cells.
concentrations of compound 20 (0.25, 0.5, 1 mM) for 24 h, respec-
tively. Then the cells were stained with the Muse apoptosis kit and
measured by the Muse cell analyzer. The results showed that
compound 20 induced a concentration-dependent (0.25, 0.5, 1 mM)
increase (10.46e66.05%) in both the early and late stage apoptosis
of PLC/PRF/5 cells (Fig. 4B&C). Similarly, the results showed the
significant increase of apoptotic cells in HepG2 cells after com-
pound 20 treated (Fig. 4 B&C). These results demonstrated com-
pound 20 had a severe effect on cellular apoptosis, indicating that
PLC/PRF/5 and HepG2 cells apoptosis induced by compound 20
contributes to its antitumor effects.
2.2.3. Compounds inhibited tumor cell colony formation
Colony formation assays are often used to detect the cancer
cell survival and proliferation abilities. The colony number rep-
resents the cell survival ability, while the colony size shows the
cell proliferation ability. To detect the antitumor roles of the
compounds 4, 20 and 22, colony formation assays were carried
out. Comparing to the parent compound oridonin, all the three
derivatives have better efficacies against HepG2 cells (Fig. 2A&B).
Additionally, the compounds have similar anti-colony formation
effects by using PLC/PRF/5 cells, and compound 20 could
2.2.6. Compound 20 induced the dissipation of mitochondrial
membrane potential
There are two main pathways to induce apoptosis, including
extrinsic pathway mediated by death receptors and intrinsic
pathway induced by mitochondria. Mitochondrial function is a
critical indicator of cell survival and most of antitumor drugs induce
cell apoptosis through mitochondria-mediated intrinsic pathway
[32]. To investigate whether compound 20 induced apoptosis
through mitochondria pathway, the mitochondrial membrane po-
tential (MMP) was detected by using a fluorescent dye JC-1. In living
cells, JC-1 exists in the form of aggregates in the mitochondria and
fluoresces red. While cells undergo apoptosis or necrosis, JC-1 ex-
ists as a monomer and fluoresces green. We treated PLC/PRF/5 and
HepG2 cells with different concentrations of compound 20 for 24 h
and then stained cells with JC-1, respectively (Fig. 5). Compared
with the control cells, incubation of PLC/PRF/5 cells with compound
20 resulted in decrease of fluorescence ratio of red to green fluo-
rescence in a concentration-dependent manner. The effect was
similar in HepG2 cells. It is evident that compound 20 could cause
dissipation of mitochondrial membrane potential and result in
mitochondrial dysfunction in PLC/PRF/5 cells and HepG2 cells.
completely eliminate the colonies at 0.5 mM (Fig. 2C&D). These
results indicated that compound 20 has strong anti-survival effect
on liver cancer cells. We also monitored the colony size by mi-
croscope, and found that the compounds could also decrease the
colony sizes (Fig. 2E&F), indicating the compounds also play anti-
proliferation roles.
Table 2
The cytotoxicity data of oridonin derivatives in cancer cell lines and normal cell lines.
Compounds
Cytotoxicity (IC50, mM)
PLC/PRF/5
L-O2
SI
1
4
20
22
7.41 0.10
2.67 0.04
0.78 0.04
1.03 0.10
17.47 0.93
6.30 0.21
1.47 0.05
1.85 0.04
2.4
2.4
1.8
1.8
IC50 was measured in PLC/PRF/5 and L-O2 cells. SI: selectivity index (IC50 on L-O2
cells/IC50 on PLC/PRF/5 cells).

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
369
Fig. 2. Colony formation of HepG2 and PLC/PRF/5 cells were inhibited by compounds 1, 4, 20 and 22. (A, B) HepG2 cells were incubated with varying concentrations of compounds
1, 4, 20 and 22 (0, 0.1, 0.5, 1 M) and dyed with 0.1% crystal violet, and the relative colony numbers were showed. (C, D) PLC/PRF/5 cells were incubated with varying concentrations
of compounds 1, 4, 20 and 22 (0, 0.1, 0.5, 1 M) and dyed with 0.1% crystal violet, and the relative colony numbers were showed. (E, F) The micrographic differences between the
m
m
colonies of PLC/PRF/5 cells. Data are shown as the mean SD of three independent experiments. ***p < 0.001 vs control group (Solvent group). (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)

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D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
Fig. 3. Compound 20 induced G1 arrest in PLC/PRF/5 and HepG2 cancer cells. (A) PLC/PRF/5 and HepG2 cells were incubated respectively with varying concentrations of compound
20 (0, 0.1, 0.5, 1 M) and analyzed by flow cytometry. (B) Histograms displaying the percentage of cell cycle distribution. *p < 0.05 vs control group.
m
2.2.7. Compound 20 induced reactive oxygen species generation
The mitochondrial membrane depolarization is related to the
production of reactive oxygen species (ROS) [33], which is a strong
apoptosis inducer. Based on this, we detected the ROS level of PLC/
PRF/5 and HepG2 cells by fluorescence probe DCFH-DA after
treated with different concentrations of compound 20. We found
that compound 20 could induce intracellular ROS generation in a
dose-depended manner in both kinds of cells (Fig. 6A). The ROS
determined by Western Blotting. Treatment of PLC/PRF/5 and
HepG2 cells with compound 20 resulted in the down-regulation of
anti-apoptotic protein Bcl-2 levels and the up-regulation of the pro-
apoptotic protein Bax in a dose-dependent manner (Fig. 7).
Cytochrome c is released to cytosol during mitochondrial
membrane depolarization, which plays a key role in mitochondrial-
dependent apoptotic pathway by activating caspase-3. Then,
caspase-3 will activate the apoptotic caspase cascade and cleave
many important cellular substrates (such as PARP). Therefore cy-
tochrome c and caspase-3 were detected and it was found that
cytochrome c was released from mitochondria to cytoplasm after
the treatment of compound 20 in both PLC/PRF/5 and HepG2 cells
(Fig. 7). Meanwhile, the up-regulations of cleaved caspase-3 and
cleaved PARP were also observed in a dose-depended manner
(Fig. 7). The p53, which is often mutants in tumor cells, plays
important role in the major apoptosis signaling pathways. HepG2
have wild type p53 gene which was reported to be involved in
oridonin bioactivities in other studies [36], while PLC/PRF/5 har-
bors a mutant p53 that lose its transcriptional function. Therefore,
the expression of p53 was also detected in HepG2 cells, and no
significant change was observed (Fig. 7B). Combined with the
generation has almost tripled in cells incubated with 1 mM of
compound 20 compared to that in control cells (Fig. 6B). The results
suggested that compound 20 might induce cell apoptosis by the
generation of ROS in PLC/PRF/5 and HepG2 cells.
2.2.8. Compound 20 regulates the expression of apoptosis-related
proteins
The mitochondrial membrane depolarization was regulated by
Bcl-2 family proteins [34,35]. The Bcl-2 family includes pro-
apoptotic (e.g. Bax) and anti-apoptotic proteins (e.g. Bcl-2). To
investigate whether the dissipation of mitochondrial membrane
potential induced by compound 20 is through the regulation of Bcl-
2 family protein expression, the expression of Bax and Bcl-2 was

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
371
Fig. 4. Compound 20 induced apoptosis of PLC/PRF/5 and HepG2 cells. (A) PLC/PRF/5 and HepG2 cell morphological changes after incubation with compound 20. (B) Different
concentrations of compound 20 (0.25, 0.5 and 1.0
**p < 0.01 and ***p < 0.001 vs control group.
mM) induced apoptosis in PLC/PRF/5 cells and HepG2 cells. (C) Histograms display the percentage of cell distribution. *p < 0.05,

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D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
Fig. 5. Fluorescence photomicrograph of changes in the MMP after incubated with different concentrations of compound 20 (0, 0.25, 0.5, 1
mM) for 24 h in PLC/PRF/5 cells and
HepG2 cells. (B) Histograms display the ratio of cell distribution. Data are represented as mean SD of three independent experiments. *p < 0.05, ***p < 0.001 vs control group.
results that compound 20 also induces cell apoptosis in the p53
mutant PLC/PRF/5 cells, we concluded that p53 might not be
essential for the apoptosis induced by compound 20, indicating that
compound 20 induced liver cancer cells apoptosis via regulating the
expression of Bcl-2 family proteins.
stability of compound 20 was evaluated (plasma stability graph
shown in Supporting Information) and the results showed that
compound 20 had an approximate half-life of 2 h in human
plasma at 37 ꢀC. Besides stability experiments, the antitumor effect
of combination of oridonin and cinnamic acid derivative was also
determined by CCK-8 assay in vitro (IC50 table shown in Sup-
porting Information), and IC50 value was similar to oridonin. These
results illustrated that compound 20 was relatively stable in the
plasma and itself had potent antitumor activity in vivo rather than
through hydrolyzing rapidly into oridonin and a cinnamic acid
derivative in plasma to exert effect. Overall, these findings indicate
that compound 20 is effective in inhibiting the growth of cancer
in vivo and represents a promising drug candidate for clinical
therapeutics.
2.2.9. Compound 20 suppressed growth of xenograft tumors in
nude mice
In the above studies, we showed that compound 20 have
strong antitumor activity in vitro. Then, the hepatic carcinoma PLC/
PRF/5 xenograft model was used to evaluate its antitumor activity
in vivo due to compound 20 showed lower IC50 value against this
cell line. The mice were treated intravenously with compound 20
(10 mg/kg), oridonin (10 mg/kg) and DMSO (dissolved in sodium
chloride, control) once every three days for 4 weeks. The tumor
sizes and body weights of the mice were monitored and recorded
every day. The mice treated with 10 mg/kg of compound 20
showed a significant effect in inhibiting tumor growth and com-
pound 20 could reduce tumor weight by 38.1% at a dose of 10 mg/
kg once every three days. In contrast, the mice treated with the
same dose of oridonin almost has no effect (Fig. 8A&B). Mean-
while, compound 20 was found to be tolerated during the tests
and showed no significant loss of body weight (Fig. 8C). In addi-
tion, in order to verify whether compound 20 could be hydrolyzed
quickly into oridonin and a cinnamic acid derivative in plasma to
produce in vivo antitumor activity. The preliminary plasma
3. Conclusion
In summary, a series of novel 14-O oridonin derivatives were
synthesized and evaluated for their antitumor activity. Some of the
oridonin derivatives displayed potent efficacy, especially com-
pounds 20 and 22 bearing a trans-cinnamic acid moiety. The most
effective compound 20 exhibited potent cytotoxicity against well
differentiated HepG2 and poorly differentiated PLC/PRF/5 cells with
IC50 values of 1.36 mM and 0.78 mM, respectively. Further biological
studies demonstrated that compound 20 inhibited liver cancer cell
proliferation by arresting cells at the G1 phase of the cell cycle.

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
373
Fig. 6. Compound 20 increased levels of ROS in PLC/PRF/5 and HepG2 cells. (A) ROS levels of PLC/PRF/5 and HepG2 cells after incubated with different concentrations of compound
20 (0, 0.25, 0.5, 1 M) were detected by flow cytometry. (B) Histograms showed ROS generation levels. Data are represented as means SD of three independent experiments.
m
***p < 0.001 compared with control group.
Fig. 7. Western blot of apoptosis-related proteins in PLC/PRF/5 and HepG2 cells after incubated with the indicated concentrations of compound 20 (0 mM, 0.25 mM, 0.5 mM, 1.0 mM
and 1.25 mM) for 24 h.
Furthermore, compound 20 also induced liver cancer cell apoptosis
through mitochondrial pathway, which was mediated by
decreasing the mitochondrial membrane potential, increasing
intracellular ROS level and inducing the expression of apoptosis-
related proteins. More importantly, compared to its parent com-
pound oridonin, which showed no significant inhibitory activity
against PLC/PRF/5 cells in vivo at same dose, the antitumor efficacy
of compound 20 with low micromolar was verified in liver cancer
xenograft mouse model with no notable toxicity. Compound 20
showed a significant effect in inhibiting tumor growth and could
reduce tumor weight by 38.1% at a dose of 10 mg/kg once every
three days in the PLC/PRF/5 xenograft nude mice model. Therefore,
our study demonstrated that compound 20 showed significant
antitumor efficacy both in vitro and in vivo and represents a
promising drug candidate for anticancer therapeutics.
4. Experimental section
4.1. Chemistry
All chemicals were acquired commercially. Unless otherwise
stated, all commercial reagents were used without additional pu-
rification and solvents were distilled prior to use. 1H and 13C NMR
spectra were recorded on an Agilent DD2 500 NMR spectrometer.
1H NMR and 13C NMR spectra were recorded in CDCl3 or DMSO‑d6,
using CDCl3 as a reference standard (
d
¼ 7.26 ppm) for 1H NMR and
(
d
¼ 77.00 ppm) for 13C NMR or DMSO‑d6 as a reference standard
(d
¼ 2.50 ppm) for 1H NMR and (
d
¼ 39.52 ppm) for 13C NMR.
Melting points were measured with a Laboratory Device MEL-TEMP
and were uncorrected. TLC was performed using commercially
prepared silica gel plates (GF254), and visualization was effected at

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D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
Fig. 8. Compound 20 inhibits PLC/PRF/5 xenograft growth in vivo. (A) The mice were randomly divided into 3 groups with 6 mice in each group. Mice were treated intravenously
with compound 20 (10 mg/kg), oridonin (10 mg/kg) and control (10%N-Methyl pyrrolidone/12% Tween-80/78% saline) once every three days for 4 weeks. (B) Visible tumor for-
mation and photographs of representative tumors removed from mice at 28 days after initiation of treatment. (C) Body weight changes of mice during treatment. (D) Compound 20
treatment resulted in significantly lower tumor weight compared with oridonin and control. ***p < 0.001.
254 nm and 365 nm. High-resolution mass spectra were obtained
by using a Q-TOF Ultima GLOBAL spectrometer and were recorded
on the Exactive Mass Spectrometer equipped with ESI ionization
source.
(500 MHz, CDCl3)
d
5.99 (s, 1H), 5.33 (s, 1H), 5.00 (d, J ¼ 6.1 Hz, 1H),
4.78 (d, J ¼ 0.9 Hz, 1H), 4.66 (dd, J ¼ 11.4, 5.6 Hz, 1H), 4.28 (d,
J ¼ 10.1 Hz, 1H), 4.18 (dd, J ¼ 10.5, 1.2 Hz, 1H), 2.99 (d, J ¼ 9.8 Hz, 1H),
2.41 (dt, J ¼ 13.7, 9.0 Hz, 1H), 2.28 (s, 3H), 1.99 (s, 3H), 1.91 (dt,
J ¼ 12.6, 4.3 Hz, 1H), 1.84e1.76 (m, 1H), 1.62 (d, J ¼ 6.0 Hz, 1H), 1.53
(ddd, J ¼ 16.4,13.9, 8.9 Hz, 2H),1.45 (dt, J ¼ 13.6, 3.2 Hz,1H),1.32 (td,
4.1.1. General procedures to synthesize acetylated derivatives 8-10
4.1.1.1. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6,14-trihydroxy-4,4-
J ¼ 14.1, 2.8 Hz, 1H), 1.27e1.18 (m, 2H), 1.16 (s, 3H), 0.89 (s, 3H). 13
C
NMR (126 MHz, CDCl3) d 201.19, 175.38, 169.77, 151.56, 117.33, 98.01,
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
75.72, 75.08, 72.99, 63.33, 60.31, 55.45, 50.63, 41.82, 40.16, 37.48,
33.54, 31.63, 29.82, 25.12, 21.51. 17.59. HRESIMS m/z 449.2168
[MþH]þ (calcd for C24H33O8 449.2170).
ymethano)-6a,9-methanocyclohepta[a]naphthalen-1-yl acetate (5).
Treatment of oridonin with 2,2-dimethoxypropane in presence of
p-TsOH in acetone afforded compound 5 in 87% yield. Compound 5
upon reaction with Ac2O/C5H5N/Et3N/DMAP/CH2Cl2, yielded cor-
responding compound 6 in 95% yield. Deprotection of compound 6
with 1 M HCl gave the corresponding compound 8 in quantitative
4.1.1.3. (1S,4aR,5S,6aR,9S,11aS,11bS,12R)-11b-(acetoxymethyl)-4,4-
dimethyl-8-methylene-6,7- dioxotetradecahydro-6a,9-
yield: mp 249.3e250.6 ꢀC; 1H NMR (500 MHz, CDCl3)
d 6.17 (s, 1H),
methanocyclohepta[a]naphthalene-1,5,12-triyl
triacetate
(10).
5.55 (s, 1H), 4.84 (s, 1H), 4.59 (dd, J ¼ 11.2, 5.5 Hz, 1H), 4.25 (d,
J ¼ 10.4 Hz, 1H), 4.17 (d, J ¼ 10.3 Hz, 1H), 3.79 (d, J ¼ 7.4 Hz, 1H), 3.05
(d, J ¼ 9.6 Hz, 1H), 2.47e2.38 (m, 1H), 1.97 (s, 3H), 1.89e1.82 (m, 2H),
1.78e1.71 (m, 1H), 1.61e1.52 (m, 1H), 1.45 (dd, J ¼ 15.6, 8.1 Hz, 2H),
Oridonin (100 mg, 0.27 mmol) was dissolved into acetic anhy-
dride(5 mL), HClO4(2 drops) was added into the solution. The
mixture was stirred at room temperature overnight. The mixture
was poured into saturated NaHCO3 solution, and extracted with
dichloromethane. The organic layer was combined and washed
with water and saturated NaCl solutions. Then organic layer was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by column chromatography to give
the compound 7 (85%) as a white solid: mp 237.5e239.2 ꢀC; 1H
1.36e1.28 (m, 2H), 1.20 (t, J ¼ 7.6 Hz, 1H), 1.13 (d, J ¼ 5.9 Hz, 6H). 13
C
NMR (126 MHz, CDCl3) d 207.31, 169.87, 150.93, 121.03, 96.98, 75.59,
74.10, 72.37, 63.57, 61.76, 59.28, 53.08, 42.55, 39.79, 38.15, 33.46,
32.65, 29.83, 29.66, 25.06, 21.94, 21.48, 18.17. HRESIMS m/z
407.2058 [MþH]þ (calcd for C22H31O7 407.2064).
NMR (500 MHz, CDCl3)
d
6.18 (s, 1H), 6.04 (d, J ¼ 12.9 Hz, 1H), 5.93
4.1.1.2. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-6,14-dihydroxy-4,4-
(s, 1H), 5.43 (s, 1H), 4.83 (d, J ¼ 13.5 Hz, 1H), 4.70 (dd, J ¼ 8.8, 5.0 Hz,
1H), 4.56 (d, J ¼ 13.5 Hz,1H), 3.12 (s,1H), 2.38 (d, J ¼ 6.1 Hz,1H), 2.21
(s, 3H), 2.14 (s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.92 (d, J ¼ 12.9 Hz, 1H),
1.82e1.75 (m, 2H), 1.73e1.68 (m, 1H), 1.67e1.52 (m, 4H), 1.50e1.41
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalene-1,5-diyl
oxododecahydro-1H-6,11b-(epox-
diac-
etate (9). Compound 5 upon reaction with Ac2O/C5H5N/Et3N/
DMAP/CH2Cl2, yielded corresponding compound 7 in 95% yield.
Deprotection of compound 7 with 1 M HCl gave the corresponding
compound 9 in quantitative yield: mp 236.6e238.4 ꢀC; 1H NMR
(m,1H),1.10 (s, 3H), 0.95 (s, 3H). 13C NMR (126 MHz, CDCl3)
d 199.61,
197.65, 170.55, 170.42, 169.56, 169.35, 145.65, 119.43, 81.78, 75.36,
73.81, 66.03, 63.26, 54.17, 53.17, 45.90, 42.73, 38.91, 34.42, 33.88,

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
375
30.94, 26.03, 21.94, 21.52, 21.19, 21.02, 20.83, 18.75. HRESIMS m/z
533.2377 [MþH]þ (calcd for C28H37O10 533.2381).
73.86, 73.52, 63.38, 61.84, 59.94, 55.97, 55.95, 54.55, 41.37, 41.24,
38.65, 33.77, 32.54, 30.50, 30.12, 21.63, 19.78. HRESIMS m/z
555.2595 [MþH]þ (calcd for C31H39O9 555.2589).
4.1.2. General procedures to synthesize acetylated derivatives 20-39
4.1.2.1. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
4.1.2.4. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(4-
methoxyphenyl)propanoate (23). White solid (87%): mp
80.0e82.6 ꢀC; 1H NMR (500 MHz, CDCl3)
7.04 (d, J ¼ 8.6 Hz, 2H),
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl (E)-3-(4-
methoxyphenyl)acrylate (20). Oridonin (100 mg, 0.27 mmol) was
mixed with 4-methoxycinnamic acid (48 mg, 0.27 mmol),
EDCI(157 mg, 0.82 mmol) and DMAP(101 mg, 0.82 mmol) in dry
dichloromethane(10 mL) and stirred at room temperature over-
night. The mixture was poured into 1 M HCl solution, and extracted
with dichloromethane. The organic layer was combined, washed
with water and saturated NaCl solutions, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by column chromatography to give the compound 20
(122 mg, 86%) as a white solid: mp 234.6e236.1 ꢀC; 1H NMR
d
6.78 (d, J ¼ 8.6 Hz, 2H), 6.29 (d, J ¼ 10.4 Hz, 1H), 6.08 (s, 1H), 5.82 (s,
1H), 5.40 (s, 1H), 4.34 (s, 1H), 4.29 (d, J ¼ 10.4 Hz, 1H), 4.05 (d,
J ¼ 10.3 Hz, 1H), 3.80e3.71 (m, 4H), 3.46 (dd, J ¼ 11.3, 5.6 Hz, 1H),
3.05 (d, J ¼ 9.9 Hz, 1H), 2.89e2.80 (m, 2H), 2.62e2.50 (m, 3H),
2.32e2.20 (m, 1H), 1.93 (dd, J ¼ 12.9, 5.8 Hz, 1H), 1.76e1.47 (m, 5H),
1.44 (dt, J ¼ 13.5, 2.9 Hz, 1H), 1.23 (d, J ¼ 6.7 Hz, 1H), 1.08 (d,
J ¼ 4.1 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 206.71, 171.42, 158.08,
149.72, 131.87, 129.20, 129.16, 120.13, 113.90, 96.12, 76.50, 73.94,
73.22, 63.46, 61.85, 59.70, 55.24, 54.57, 41.27, 41.24, 38.63, 36.28,
33.71, 32.53, 30.48, 29.86, 29.72, 21.60, 19.71. HRESIMS m/z
527.2637 [MþH]þ (calcd for C30H39O8 527.2639).
(500 MHz, CDCl3)
d
7.59 (d, J ¼ 15.9 Hz, 1H), 7.43 (d, J ¼ 8.7 Hz, 2H),
6.88 (d, J ¼ 8.7 Hz, 2H), 6.20(d, J ¼ 15.9 Hz, 1H), 6.16(s, 1H), 5.92 (s,
1H), 5.49 (s, 1H), 4.74 (s, 1H), 4.33 (d, J ¼ 10.4 Hz, 1H), 4.08 (d,
J ¼ 10.4 Hz, 1H), 3.82 (s, 3H), 3.79 (d, J ¼ 6.6 Hz, 1H), 3.50 (dd,
J ¼ 11.2, 5.7 Hz, 1H), 3.27 (d, J ¼ 9.8 Hz, 1H), 2.65 (dt, J ¼ 14.1, 9.1 Hz,
1H), 2.33 (ddd, J ¼ 27.5, 13.2, 8.3 Hz, 1H), 2.01 (dd, J ¼ 12.8, 5.8 Hz,
1H), 1.79e1.71 (m, 1H), 1.71e1.64 (m, 1H), 1.64e1.55 (m, 2H),
1.50e1.44 (m, 1H), 1.33e1.22 (m, 3H), 1.12 (s, 6H), 0.87 (dd, J ¼ 13.3,
4.1.2.5. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(3,4-
dimethoxyphenyl)propanoate (24). White solid (81%): mp
6.5 Hz, 1H).13C NMR (126 MHz, CDCl3)
d
206.71, 165.45, 161.84,
93.4e94.1 ꢀC; 1H NMR (500 MHz, CDCl3)
d
6.76 (d, J ¼ 7.9 Hz, 1H),
149.84, 146.35, 130.08, 126.51, 120.20, 114.40, 113.97, 96.22, 73.64,
73.31, 63.39, 61.79, 59.87, 55.39, 54.55, 41.32, 41.24, 38.63, 33.75,
32.51, 30.48, 29.97, 21.59, 19.73. HRESIMS m/z 525.2487 [MþH]þ
(calcd for C30H37O8 525.2483).
Following the procedure described for preparation of compound
20, compounds 21e39 was prepared from oridonin.
6.67 (d, J ¼ 8.9 Hz, 2H), 6.13e6.07 (m, 2H), 5.83 (s, 1H), 5.39 (s, 1H),
4.30 (d, J ¼ 10.4 Hz,1H), 4.20 (s,1H), 4.07 (d, J ¼ 10.4 Hz,1H), 3.84 (d,
J ¼ 2.2 Hz, 6H), 3.75 (dd, J ¼ 10.5, 6.7 Hz, 1H), 3.48 (dd, J ¼ 11.0,
5.4 Hz, 1H), 3.07 (d, J ¼ 9.8 Hz, 1H), 2.90e2.79 (m, 2H), 2.65e2.52
(m, 3H), 2.32e2.21 (m, 1H), 1.96 (dd, J ¼ 13.0, 5.9 Hz, 1H), 1.80e1.50
(m, 6H), 1.46 (d, J ¼ 13.6 Hz, 1H), 1.37 (s, 1H), 1.11 (s, 6H). 13C NMR
(126 MHz, CDCl3)
d 206.48, 171.27, 149.64, 148.87, 147.54, 132.32,
4.1.2.2. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
120.12, 120.01, 111.54, 111.30, 96.19, 76.70, 73.97, 73.38, 63.40, 61.80,
59.74, 55.91, 55.83, 54.55, 41.31, 41.18, 38.62, 36.23, 33.73, 32.53,
30.48, 30.23, 30.01, 21.62, 19.72. HRESIMS m/z 557.2747 [MþH]þ
(calcd for C31H41O9 557.2745).
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl
(3,4,5-trimethoxyphenyl)acrylate (21). White solid (81%): mp
147.3e149.1 ꢀC; 1H NMR (500 MHz, CDCl3)
oxododecahydro-1H-6,11b-(epox-
(E)-3-
d
7.53 (d, J ¼ 15.9 Hz,1H),
6.70 (s, 2H), 6.25 (d, J ¼ 15.9 Hz, 1H), 6.19e6.11 (m, 1H), 5.94 (s, 1H),
5.51 (s, 1H), 4.39e4.30 (m, 1H), 4.09 (d, J ¼ 10.6 Hz, 1H), 3.87 (s, 6H),
3.79 (dd, J ¼ 10.3, 6.8 Hz, 1H), 3.55e3.47 (m, 1H), 3.28 (d, J ¼ 9.8 Hz,
1H), 2.66 (dt, J ¼ 13.9, 9.1 Hz, 1H), 2.33 (dd, J ¼ 13.5, 8.0 Hz, 1H), 2.02
(dd, J ¼ 12.8, 5.6 Hz, 1H), 1.80e1.72 (m, 1H), 1.65e1.56 (m, 2H), 1.47
(d, J ¼ 13.6 Hz, 1H), 1.33e1.22 (m, 2H), 1.12 (s, 4H). 13C NMR
4.1.2.6. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(3,4,5-
trimethoxyphenyl)propanoate (25). White solid (87%): mp
89.1e91.5 ꢀC; 1H NMR (500 MHz, CDCl3)
d 6.34 (s, 2H), 6.11 (d,
J ¼ 10.5 Hz, 1H), 6.07 (s, 1H), 5.83 (s, 1H), 5.39 (s, 1H), 4.28 (d,
J ¼ 10.4 Hz, 1H), 4.17 (s, 1H), 4.05 (d, J ¼ 10.4 Hz, 1H), 3.80 (d,
J ¼ 9.4 Hz, 9H), 3.72 (dd, J ¼ 10.5, 6.7 Hz, 1H), 3.47 (dd, J ¼ 11.2,
5.6 Hz, 1H), 3.05 (d, J ¼ 9.8 Hz, 1H), 2.83 (t, J ¼ 7.5 Hz, 2H), 2.66e2.51
(m, 3H), 2.31e2.19 (m, 1H), 1.94 (dd, J ¼ 12.9, 5.8 Hz, 1H), 1.77e1.68
(m, 1H), 1.68e1.48 (m, 4H), 1.48e1.41 (m, 1H), 1.28e1.17 (m, 1H),
(126 MHz, CDCl3)
d 206.67, 165.10, 153.41, 149.83, 146.43, 140.53,
129.23, 120.32, 115.94, 105.43, 96.34, 77.22, 73.94, 73.45, 63.43,
61.88, 60.96, 59.87, 56.20, 54.60, 41.32, 38.64, 33.76, 32.55, 30.52,
30.07, 21.64, 19.79. HRESIMS m/z 585.2699 [MþH]þ (calcd for
C
32H41O10 585.2694).
1.09 (s, 6H). 13C NMR (126 MHz, CDCl3)
d 206.49, 171.20, 153.16,
4.1.2.3. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7- oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl (E)-3-(3,4-
dimethoxyphenyl)acrylate (22). White solid (80%): mp
144.5e146.7 ꢀC; 1H NMR (500 MHz, CDCl3)
7.59 (d, J ¼ 15.9 Hz,
149.62, 136.40, 135.50, 120.17, 105.09, 96.16, 76.66, 74.01, 73.27,
63.44, 61.82, 60.79, 59.70, 56.06, 54.56, 41.29, 41.23, 38.61, 36.08,
33.70, 32.53, 30.96, 30.49, 29.93, 21.61, 19.71. HRESIMS m/z
587.2860 [MþH]þ (calcd for C32H43O10 587.2851).
d
1H), 7.08 (dd, J ¼ 8.3, 1.9 Hz, 1H), 7.00 (d, J ¼ 1.9 Hz, 1H), 6.85 (d,
J ¼ 8.3 Hz, 1H), 6.22 (d, J ¼ 15.9 Hz, 1H), 6.18 (s, 1H), 6.15 (d,
J ¼ 10.5 Hz, 1H), 5.95 (s, 1H), 5.51 (s, 1H), 4.43 (s, 1H), 4.35 (d,
J ¼ 10.4 Hz,1H), 4.10 (dd, J ¼ 10.4,1.4 Hz,1H), 3.91 (d, J ¼ 2.4 Hz, 6H),
3.82 (dd, J ¼ 10.5, 6.6 Hz,1H), 3.56e3.50 (m,1H), 3.30 (d, J ¼ 10.0 Hz,
1H), 2.72e2.63 (m, 1H), 2.41e2.28 (m, 1H), 2.04 (dd, J ¼ 13.0, 5.1 Hz,
1H), 1.81e1.73 (m, 1H), 1.73e1.62 (m, 3H), 1.49 (dt, J ¼ 13.5, 3.3 Hz,
1H), 1.34e1.28 (m, 3H), 1.14 (d, J ¼ 2.4 Hz, 6H), 1.11 (d, J ¼ 4.0 Hz,
4.1.2.7. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 4-fluoro-3-
(4-(trifluoromethoxy)benzamido)benzoate (26). White solid (83%):
mp 149.1e151.3 ꢀC; 1H NMR (500 MHz, CDCl3)
d
8.87 (dd, J ¼ 7.5,
2.0 Hz, 1H), 8.08 (d, J ¼ 2.4 Hz, 1H), 7.80 (d, J ¼ 7.8 Hz, 1H), 7.77 (s,
1H), 7.72 (ddd, J ¼ 8.5, 4.9, 2.2 Hz, 1H), 7.55 (t, J ¼ 8.0 Hz, 1H),
7.45e7.41 (m, 1H), 7.15 (dd, J ¼ 10.1, 8.7 Hz, 1H), 6.20 (s, 1H), 6.16 (d,
J ¼ 10.9 Hz, 1H), 6.08 (d, J ¼ 0.7 Hz, 1H), 5.53 (s, 1H), 4.35 (d,
J ¼ 10.4 Hz, 1H), 4.08 (d, J ¼ 10.4, 1.3 Hz, 1H), 4.05 (s, 1H), 3.73 (dd,
1H).13C NMR (126 MHz, CDCl3)
d
206.69, 165.32, 151.60, 149.86,
149.26, 146.53, 126.77, 123.23, 120.25, 114.22, 110.97, 109.50, 96.37,

376
D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
J ¼ 10.9, 6.9 Hz, 1H), 3.51 (dd, J ¼ 5.8, 3.5 Hz, 1H), 3.31 (d, J ¼ 9.9 Hz,
1H), 2.74e2.62 (m, 1H), 2.40e2.25 (m, 1H), 2.00 (dd, J ¼ 12.8, 5.5 Hz,
1H), 1.83e1.78 (m, 1H), 1.77 (s, 1H), 1.70e1.58 (m, 3H), 1.47 (dt,
J ¼ 13.5, 3.2 Hz,1H),1.42 (s,1H),1.32e1.26 (m, 2H),1.10 (d, J ¼ 7.5 Hz,
0.99 (d, J ¼ 6.0 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 207.22, 162.54,
152.61, 150.99, 148.77, 141.11, 127.24, 123.50, 120.29, 96.25, 75.43,
74.47, 72.00, 63.05, 62.51, 59.45, 54.40, 41.81, 40.95, 38.78, 33.78,
33.21, 30.79, 29.77, 22.15, 20.24. HRESIMS m/z 504.1774 [MþH]þ
(calcd for C26H31O7NCl 504.1774).
6H). 13C NMR (126 MHz, CDCl3)
d 206.77, 164.15, 163.91, 157.24,
149.95, 149.59, 136.14, 130.42, 126.73, 125.20, 124.70, 124.54, 120.41,
120.34, 115.46, 115.29, 96.08, 76.67, 74.57, 73.44, 63.44, 62.17, 59.54,
54.76, 41.49, 41.37, 38.65, 33.71, 32.58, 30.52, 29.98, 21.69, 19.92.
HRESIMS m/z 690.2327 [MþH]þ (calcd for C35H36O9NF4 690.2321).
4.1.2.11. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 5-(1,2-
dithiolan-3-yl)pentanoate (30). Yellow solid (84%): mp
104.7e106.1 ꢀC;1H NMR (500 MHz, CDCl3)
6.16 (s, 1H), 6.08 (dd,
oxododecahydro-1H-6,11b-(epox-
4.1.2.8. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
d
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(3-(ben-
oxododecahydro-1H-6,11b-(epox-
J ¼ 10.5, 2.0 Hz, 1H), 5.85 (s, 1H), 5.51 (s, 1H), 4.31 (dd, J ¼ 7.6, 4.0 Hz,
1H), 4.23 (d, J ¼ 2.1 Hz, 1H), 4.08 (dd, J ¼ 10.4, 1.5 Hz, 1H), 3.78 (dd,
J ¼ 10.5, 6.7 Hz, 1H), 3.57e3.48 (m, 2H), 3.22e3.14 (m, 2H), 3.11 (dt,
J ¼ 11.0, 6.9 Hz,1H), 2.66e2.59 (m, 1H), 2.45 (td, J ¼ 12.5, 6.1 Hz, 1H),
2.36e2.23 (m, 3H), 1.99 (dd, J ¼ 12.8, 5.7 Hz, 1H), 1.90 (dtd, J ¼ 13.9,
7.0, 1.2 Hz, 1H), 1.78e1.54 (m, 9H), 1.51e1.35 (m, 3H), 1.31 (dd,
J ¼ 12.5, 9.0 Hz, 1H), 1.30e1.27 (m, 1H), 1.14 (d, J ¼ 7.7 Hz, 6H). 13C
zyloxy)-4-methoxybenzamido)-4-fluorobenzoate (27). White solid
(82%): mp 148.0e150.3 ꢀC; 1H NMR (500 MHz, CDCl3)
d 8.82 (dd,
J ¼ 7.5, 2.0 Hz, 1H), 8.09 (d, J ¼ 2.5 Hz, 1H), 7.64 (ddd, J ¼ 8.4, 4.8,
2.1 Hz, 1H), 7.51 (d, J ¼ 2.0 Hz, 1H), 7.45 (dd, J ¼ 7.7, 3.0 Hz, 3H), 7.36
(t, J ¼ 7.4 Hz, 2H), 7.30 (t, J ¼ 7.3 Hz, 1H), 7.10 (dd, J ¼ 9.9, 8.9 Hz, 1H),
6.91 (d, J ¼ 8.5 Hz, 1H), 6.24 (d, J ¼ 10.7 Hz, 1H), 6.15 (s, 1H), 6.04 (s,
1H), 5.49 (s, 1H), 5.17 (s, 1H), 4.32 (d, J ¼ 10.4 Hz, 1H), 4.24 (s, 1H),
4.04 (d, J ¼ 10.2 Hz, 1H), 3.91 (s, 3H), 3.71 (dd, J ¼ 10.7, 6.8 Hz, 1H),
3.46 (dd, J ¼ 11.2, 5.6 Hz, 1H), 3.26 (d, J ¼ 9.8 Hz, 1H), 2.61 (dt,
J ¼ 13.7, 9.2 Hz, 1H), 2.32e2.22 (m, 1H), 2.04 (d, J ¼ 18.2 Hz, 1H), 1.95
(dd, J ¼ 13.0, 5.7 Hz, 1H), 1.79e1.72 (m, 1H), 1.68e1.55 (m, 2H),
1.44e1.38 (m, 2H), 1.27e1.21 (m, 2H), 1.05 (d, J ¼ 8.8 Hz, 6H). 13C
NMR (126 MHz, CDCl3)
d
206.46,171.68 (d, J ¼ 3.0 Hz),149.81,120.14
(d, J ¼ 5.8 Hz), 96.24, 73.91, 73.47, 63.38, 61.78, 59.79, 56.22 (d,
J ¼ 3.6 Hz), 54.54, 41.33, 41.21, 40.20, 38.55 (d, J ¼ 20.2 Hz), 34.46 (d,
J ¼ 3.6 Hz), 34.34 (d, J ¼ 4.8 Hz), 33.75, 32.53, 30.47, 30.09, 29.69,
28.53 (d, J ¼ 6.6 Hz), 24.37 (d, J ¼ 7.5 Hz), 21.64, 19.75. HRESIMS m/z
553.2277 [MþH]þ (calcd for C28H41O7S2 553.2288).
NMR (126 MHz, CDCl3)
d
206.94, 165.05, 164.28,
d
156.28 (d,
4.1.2.12. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
J ¼ 252.5 Hz), 153.09, 149.99, 148.19, 136.46, 128.61, 128.08, 127.60,
126.71, 126.63, 126.54, 126.29, 124.64, 120.68, 120.33, 115.37, 115.20,
113.12, 110.96, 96.11, 77.28, 76.76, 74.49, 73.21, 71.08, 63.50, 62.18,
59.61, 56.09, 54.73, 41.50, 41.31, 38.66, 33.67, 32.58, 30.52, 26.90,
21.64,19.85. HRESIMS m/z 742.3019 [MþH]þ (calcd for C42H45O10NF
742.3022).
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 4-
fluorobenzoate (31). White solid (82%): mp 265.6e267.1 ꢀC; 1H
NMR (500 MHz, CDCl3)
d
7.95 (dd, J ¼ 8.5, 5.6 Hz, 2H), 7.07 (t,
J ¼ 8.6 Hz, 2H), 6.20 (s, 1H), 6.13e6.08 (m, 2H), 5.52 (s, 1H), 4.37 (d,
J ¼ 10.4 Hz, 1H), 4.10 (d, J ¼ 10.7 Hz, 1H), 3.94 (s, 1H), 3.76 (dd,
J ¼ 10.8, 7.0 Hz, 1H), 3.57e3.49 (m, 1H), 3.29 (d, J ¼ 9.8 Hz, 1H),
2.72e2.64 (m, 1H), 2.39e2.30 (m, 1H), 2.02 (dd, J ¼ 13.1, 5.8 Hz, 1H),
1.84e1.77 (m, 1H), 1.73e1.64 (m, 2H), 1.53e1.46 (m, 1H), 1.33e1.27
4.1.2.9. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(3,4-
(m, 2H), 1.13 (d, J ¼ 8.0 Hz, 6H).13C NMR (126 MHz, CDCl3)
d 206.62,
dimethoxybenzamido)-4-fluorobenzoate (28). White solid (83%):
165.98 (d, J ¼ 254.8 Hz), 164.29, 149.98, 132.34, 132.26, 125.89,
120.30, 115.82, 115.64, 96.11, 76.52, 74.46, 73.52, 63.43, 62.10, 59.59,
54.74, 41.51, 41.39, 38.65, 33.74, 32.61, 30.54, 30.06, 21.71, 19.94.
HRESIMS m/z 487.2132 [MþH]þ (calcd for C27H32O7F 487.2127).
mp 219.3e221.0 ꢀC; 1H NMR (500 MHz, CDCl3)
d
8.75 (d, J ¼ 6.3 Hz,
1H), 7.64e7.55 (m, 1H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.04e6.95 (m, 1H),
6.86 (s, 2H), 6.83 (s, 1H), 6.18 (d, J ¼ 10.8 Hz, 1H), 6.16 (s, 1H), 6.03 (s,
1H), 5.49 (s, 1H), 4.31 (d, J ¼ 10.4 Hz, 1H), 4.10 (s, 1H), 4.04 (d,
J ¼ 10.2 Hz, 1H), 3.87 (s, 6H), 3.74e3.65 (m, 3H), 3.46 (dd, J ¼ 11.1,
5.4 Hz, 1H), 3.24 (d, J ¼ 9.8 Hz, 1H), 2.61 (dt, J ¼ 13.9, 9.1 Hz, 1H),
2.36e2.22 (m, 2H), 2.16 (s, 1H), 1.96 (dd, J ¼ 13.0, 5.7 Hz, 1H), 1.91 (s,
1H), 1.80e1.71 (m, 1H), 1.69e1.52 (m, 3H), 1.42 (dd, J ¼ 10.6, 3.0 Hz,
1H), 1.23 (s, 1H), 1.06 (d, J ¼ 9.6 Hz, 6H).13C NMR (126 MHz, CDCl3)
4.1.2.13. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 2,5-
difluorobenzoate (32). White solid (83%): mp 230.5e232.0 ꢀC; 1H
NMR (500 MHz, DMSO)
d
7.56e7.46 (m, 2H), 7.36 (td, J ¼ 9.5, 4.2 Hz,
d
206.81, 169.52, 164.20, 155.76 (d, J ¼ 252.4 Hz), 149.96, 149.47,
1H), 6.22 (s, 1H), 6.05 (d, J ¼ 9.6 Hz, 2H), 5.89 (d, J ¼ 8.5 Hz, 1H), 5.63
(s, 1H), 4.43 (s, 1H), 4.13 (d, J ¼ 10.2 Hz, 1H), 3.86 (d, J ¼ 10.2 Hz, 1H),
3.54 (s, 1H), 3.14 (d, J ¼ 9.7 Hz, 1H), 2.54 (dt, J ¼ 13.7, 9.1 Hz, 1H), 2.13
(dt, J ¼ 21.3,13.4 Hz,1H),1.90 (dd, J ¼ 12.8, 5.3 Hz,1H),1.80e1.71 (m,
1H), 1.48 (ddd, J ¼ 35.9, 19.1, 9.5 Hz, 3H), 1.31 (d, J ¼ 13.3 Hz, 1H), 1.18
(dt, J ¼ 20.7, 12.6 Hz, 3H), 0.98 (t, J ¼ 13.3 Hz, 6H). 13C NMR
148.62, 126.70, 126.47, 126.27, 124.25, 121.74, 120.32, 115.21, 115.05,
112.39, 111.70, 96.07, 76.70, 74.49, 73.25, 63.48, 62.15, 59.57, 55.94,
55.92, 54.74, 44.13, 41.48, 41.32, 38.66, 33.67, 32.60, 30.51, 29.83,
21.66, 19.85. HRESIMS m/z 680.2882 [MþH]þ (calcd for
C
37H43O10NF 680.2882).
(126 MHz, DMSO)
d
207.37, 160.97, 158.70 (d, J ¼ 11.9 Hz), 156.74,
4.1.2.10. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7- oxododecahydro-1H-6,11b-(epox-
151.01, 121.98 (dd, J ¼ 24.3, 9.3 Hz), 120.50 (dd, J ¼ 11.5, 7.8 Hz),
120.11, 119.19 (dd, J ¼ 24.6, 8.4 Hz), 118.32, 118.12, 96.27, 75.21,
74.44, 72.02, 63.07, 62.47, 59.56, 54.34, 41.78, 40.95, 38.79, 33.78,
33.18, 30.75, 29.78, 22.10, 20.20. HRESIMS m/z 505.2018 [MþH]þ
(calcd for C27H31O7F2 505.2032).
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 2-
chloronicotinate (29). White solid (88%): mp 249.3e251.9 ꢀC; 1H
NMR (500 MHz, DMSO)
d
8.53 (dd, J ¼ 4.8, 2.0 Hz, 1H), 8.13 (dd,
J ¼ 7.7, 1.9 Hz, 1H), 7.52 (dd, J ¼ 7.7, 4.8 Hz, 1H), 6.26 (s, 1H), 6.11 (s,
1H), 6.01 (s, 1H), 5.85 (d, J ¼ 10.4 Hz, 1H), 5.63 (s, 1H), 4.43 (d,
J ¼ 5.0 Hz, 1H), 4.13 (d, J ¼ 10.2 Hz, 1H), 3.86 (d, J ¼ 10.2 Hz, 1H), 3.53
(dd, J ¼ 10.4, 7.1 Hz, 1H), 3.36 (dd, J ¼ 10.0, 4.3 Hz, 1H), 3.13 (d,
J ¼ 9.7 Hz, 1H), 2.55 (dt, J ¼ 13.6, 9.1 Hz, 1H), 2.18e2.06 (m, 1H), 1.90
(dd, J ¼ 12.9, 5.4 Hz, 1H), 1.80e1.70 (m, 1H), 1.55e1.38 (m, 3H), 1.30
(d, J ¼ 13.3 Hz, 1H), 1.21 (d, J ¼ 10.9 Hz, 1H), 1.13 (t, J ¼ 8.2 Hz, 1H),
4.1.2.14. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 4-(benzy-
loxy)-2-fluorobenzoate (33). White solid (80%): mp
264.0e266.0 ꢀC; 1H NMR (500 MHz, DMSO)
7.74 (t, J ¼ 8.8 Hz, 1H),
7.44 (d, J ¼ 7.4 Hz, 2H), 7.39 (t, J ¼ 7.4 Hz, 2H), 7.34 (t, J ¼ 7.1 Hz, 1H),
oxododecahydro-1H-6,11b-(epox-
d

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
377
6.93 (dd, J ¼ 27.8, 11.0 Hz, 2H), 6.01 (s, 2H), 5.95 (s, 1H), 5.89 (d,
J ¼ 10.2 Hz, 1H), 5.61 (s, 1H), 5.18 (d, J ¼ 10.2 Hz, 2H), 4.41 (d,
J ¼ 4.0 Hz, 1H), 4.12 (d, J ¼ 10.1 Hz, 1H), 3.85 (d, J ¼ 10.2 Hz, 1H), 3.51
(dd, J ¼ 9.8, 7.1 Hz, 1H), 3.36 (d, J ¼ 10.7 Hz, 1H), 3.10 (d, J ¼ 9.7 Hz,
1H), 2.58e2.50 (m, 1H), 2.13 (dt, J ¼ 21.4, 13.5 Hz, 1H), 1.89 (dd,
J ¼ 12.8, 5.4 Hz, 1H), 1.79e1.69 (m, 1H), 1.57e1.39 (m, 3H), 1.31 (d,
J ¼ 13.6 Hz, 1H), 1.22 (dd, J ¼ 22.8, 12.2 Hz, 2H), 1.15 (d, J ¼ 6.8 Hz,
1H), 0.97 (dd, J ¼ 19.9, 7.0 Hz, 6H). 13C NMR (126 MHz,DMSO)
(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl)
(4S,5R)-2-(4-methoxyphenyl)-4-phenyloxazolidine-3,5-dicarboxylate
(37). White solid (87%): mp 144.0e146.3 ꢀC; 1H NMR (500 MHz,
CDCl3)
d
7.38e7.27 (m, 7H), 6.88 (d, J ¼ 8.4 Hz, 2H), 6.32 (s, 1H), 6.07
(s, 1H), 5.96 (d, J ¼ 10.6 Hz, 1H), 5.86 (s, 1H), 5.37 (d, J ¼ 34.4 Hz, 2H),
4.50 (d, J ¼ 3.3 Hz, 1H), 4.27 (d, J ¼ 10.4 Hz, 1H), 4.04 (d, J ¼ 10.4 Hz,
1H), 3.84 (s, 3H), 3.67 (dd, J ¼ 10.3, 7.2 Hz, 1H), 3.48e3.41 (m, 1H),
2.48 (s, 1H), 2.20 (d, J ¼ 13.0 Hz, 1H), 1.88 (d, J ¼ 11.7 Hz, 1H),
1.75e1.67 (m, 1H), 1.64 (s, 3H), 1.59e1.41 (m, 4H), 1.25e1.16 (m, 4H),
d
207.56, 163.62 (d, J ¼ 11.6 Hz), 151.25, 133.78, 128.96, 128.60,
128.37, 119.82, 111.65, 96.29, 74.43 (d, J ¼ 6.4 Hz), 72.03, 70.45,
63.03, 59.61, 54.31, 41.91, 40.95, 38.80, 33.79, 33.17, 30.73, 29.80,
22.09, 20.21. HRESIMS m/z 593.2539 [MþH]þ (calcd for C34H38O8F
593.2545).
1.07 (d, J ¼ 10.9 Hz, 15H). 13C NMR (126 MHz, CDCl3)
d 205.91,
160.10, 149.53, 128.72, 128.39, 127.91, 126.33, 120.19, 113.65, 95.83,
92.02, 80.69, 74.40, 73.45, 63.30, 62.84, 61.98, 59.25, 55.32, 54.61,
41.27, 40.95, 38.62, 33.66, 32.55, 30.49, 30.00, 29.69, 27.84, 21.70,
19.81, 1.01. HRESIMS m/z 744.3380 [MþH]þ (calcd for C42H50O11
N
4.1.2.15. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
744.3378).
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 2,3,4-
4.1.2.19. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7- oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 3-(2-
trifluorobenzoate (34). White solid (80%): mp 245.1e247.0 ꢀC; 1H
NMR (500 MHz, DMSO)
d
7.64 (dd, J ¼ 14.2, 6.8 Hz, 1H), 7.41 (dd,
J ¼ 16.9, 8.9 Hz, 1H), 6.10 (s, 1H), 6.04 (s, 1H), 5.86 (s, 1H), 5.63 (s,
1H), 4.42 (s, 1H), 4.13 (d, J ¼ 10.2 Hz, 1H), 3.85 (d, J ¼ 10.2 Hz, 1H),
3.52 (s, 1H), 3.14 (d, J ¼ 9.7 Hz, 1H), 2.54 (dt, J ¼ 13.6, 9.1 Hz, 1H),
2.18e2.05 (m, 1H), 1.89 (dd, J ¼ 12.9, 5.4 Hz, 1H), 1.80e1.72 (m, 1H),
1.56e1.39 (m, 3H), 1.31 (d, J ¼ 13.3 Hz, 1H), 1.19 (dd, J ¼ 23.6,10.5 Hz,
1H), 1.13 (t, J ¼ 8.2 Hz, 1H), 1.05 (t, J ¼ 7.0 Hz, 2H), 0.97 (dd, J ¼ 18.2,
bromoethoxy)-4-methoxybenzoate (38). White solid (83%): mp
132.2e134.6 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.54 (dd, J ¼ 8.5, 1.9 Hz,
1H), 7.47 (d, J ¼ 1.9 Hz, 1H), 6.85 (d, J ¼ 8.5 Hz, 1H), 6.20e6.16 (m,
2H), 6.03 (s, 1H), 5.49 (s, 1H), 4.35e4.30 (m, 2H), 4.17 (d, J ¼ 2.9 Hz,
1H), 4.08 (d, J ¼ 10.4 Hz, 1H), 3.90 (d, J ¼ 7.3 Hz, 3H), 3.75 (dd,
J ¼ 10.6, 6.7 Hz, 1H), 3.65 (t, J ¼ 6.4 Hz, 2H), 3.51 (dd, J ¼ 11.1, 5.4 Hz,
1H), 3.29 (d, J ¼ 9.9 Hz, 1H), 2.70e2.62 (m, 1H), 2.34 (ddd, J ¼ 27.4,
13.1, 8.1 Hz, 1H), 2.01 (dd, J ¼ 12.9, 5.7 Hz, 1H), 1.83e1.74 (m, 2H),
1.66 (dd, J ¼ 11.0, 5.7 Hz, 1H), 1.63e1.55 (m, 3H), 1.46 (dt, J ¼ 13.4,
3.1 Hz, 1H), 1.26 (d, J ¼ 8.2 Hz, 2H), 1.10 (d, J ¼ 5.8 Hz, 6H). 13C NMR
6.3 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 207.24, 160.64, 151.07,
138.89, 127.17 (d, J ¼ 4.9 Hz), 120.03, 117.30, 113.40e112.93 (m),
96.26, 75.18, 74.45, 72.03, 63.02, 62.50, 59.52, 56.48, 54.37, 41.82,
40.94, 38.80, 33.78, 33.18, 30.76, 29.78, 22.11, 20.23, 18.98. HRESIMS
m/z 523.1929 [MþH]þ (calcd for C27H30O7 F3 523.1938).
(126 MHz, CDCl3)
d 206.80, 164.69, 154.22, 150.01, 147.26, 124.73,
121.88, 120.16, 115.03, 111.18, 96.19, 74.24, 73.38, 69.19, 63.48, 62.05,
59.75, 56.15, 54.66, 41.46, 38.65, 33.73, 32.56, 30.49, 29.98, 29.68,
28.68, 21.65, 19.85. HRESIMS m/z 621.1692 [MþH]þ (calcd for
4.1.2.16. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 4-
C30H38O9Br 621.1694).
cyanobenzoate (35). White solid (80%): mp 258.9e260.1 ꢀC; 1H
NMR (500 MHz, DMSO)
d
7.99e7.92 (m, 4H), 6.06 (d, J ¼ 17.3 Hz,
4.1.2.20. (1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7- oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl 2-(3,4,5-
trimethoxyphenyl)acetate (39). White solid (82%): mp
87.7e89.1 ꢀC; 1H NMR (500 MHz, CDCl3)
6.39 (s, 2H), 6.10 (d,
3H), 5.88 (d, J ¼ 10.2 Hz, 1H), 5.63 (s, 1H), 4.42 (s, 1H), 4.12 (d,
J ¼ 10.2 Hz, 1H), 3.84 (d, J ¼ 10.2 Hz, 1H), 3.54e3.47 (m, 1H), 3.35 (s,
1H), 3.14 (d, J ¼ 9.8 Hz, 1H), 2.54 (dt, J ¼ 13.7, 9.2 Hz, 1H), 2.19e2.08
(m, 1H), 1.91 (dd, J ¼ 12.8, 5.5 Hz, 1H), 1.78e1.70 (m, 1H), 1.55e1.39
(m, 3H),1.30 (d, J ¼ 13.6 Hz,1H),1.24e1.19 (m,1H),1.14 (d, J ¼ 6.8 Hz,
d
1H), 0.99 (d, J ¼ 15.8 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 207.49,
J ¼ 10.4 Hz, 1H), 6.05 (s, 1H), 5.82 (s, 1H), 5.36 (s, 1H), 4.28 (d,
J ¼ 10.4 Hz, 1H), 4.09 (s, 1H), 4.05 (d, J ¼ 10.5 Hz, 1H), 3.80 (d,
J ¼ 4.2 Hz, 9H), 3.73 (dd, J ¼ 10.4, 6.7 Hz, 1H), 3.51e3.43 (m, 3H),
3.09 (d, J ¼ 9.9 Hz, 1H), 2.60e2.50 (m, 1H), 2.28e2.17 (m, 1H), 1.95
(dd, J ¼ 12.9, 5.8 Hz, 1H), 1.87 (s, 1H), 1.77e1.69 (m, 1H), 1.64 (ddd,
J ¼ 13.6, 8.8, 4.7 Hz, 1H), 1.58 (d, J ¼ 14.1 Hz, 1H), 1.55e1.48 (m, 2H),
163.84, 151.04, 134.78, 133.03, 130.39, 120.09, 115.57, 96.28, 75.37,
74.44, 72.00, 63.05, 62.53, 59.62, 54.31, 41.77, 40.92, 38.78, 33.79,
33.15, 30.74, 29.78, 22.08, 20.20 HRESIMS m/z 494.2162 [MþH]þ
(calcd for C28H32O7N 494.2173).
1.48e1.41 (m, 1H), 1.10 (s, 6H). 13C NMR (126 MHz, CDCl3)
d 206.33,
4.1.2.17. (1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
169.85, 153.25, 149.72, 137.21, 128.39, 119.90, 106.30, 96.15, 74.00,
73.31, 63.41, 61.87, 60.81, 59.66, 56.10, 54.49, 41.97, 41.27, 41.06,
38.60, 33.70, 32.52, 30.41, 29.95, 21.62, 19.74. HRESIMS m/z
590.2965 [MþH]þ (calcd for C31H44O10N 590.2960).
dimethyl-8-methylene-7-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl
omethyl)benzoate (36). White solid (80%): mp 251.1e253.6 ꢀC; 1H
NMR (500 MHz, CDCl3)
oxododecahydro-1H-6,11b-(epox-
4-(chlor-
d
7.92 (d, J ¼ 8.3 Hz, 2H), 7.43 (d, J ¼ 8.2 Hz,
2H), 6.20 (s, 1H), 6.14e6.07 (m, 2H), 5.51 (s,1H), 4.58 (s, 2H), 4.37 (d,
J ¼ 10.4 Hz, 1H), 4.10 (d, J ¼ 10.3 Hz, 1H), 3.97 (s, 1H), 3.77 (dd,
J ¼ 10.8, 6.9 Hz, 1H), 3.57e3.50 (m, 1H), 3.30 (d, J ¼ 9.9 Hz, 1H),
2.74e2.64 (m, 1H), 2.41e2.28 (m, 1H), 2.03 (dd, J ¼ 13.1, 5.7 Hz, 1H),
1.86e1.76 (m, 1H), 1.73e1.59 (m, 4H), 1.49 (dt, J ¼ 13.6, 3.2 Hz, 1H),
1.13 (d, J ¼ 8.8 Hz, 6H), 1.10 (d, J ¼ 3.9 Hz, 1H). 13C NMR (126 MHz,
4.2. Materials for biological studies
Paclitaxel (PTX) was obtained from PTX injection. 96-well plates
were purchased from Costar company. Roswell Park Memorial
Institute (RPMI) 1640, Dulbecco's modified Eagle's medium
(DMEM) and Fetal bovine serum (FBS) were purchased from Gibco
company. DMSO was purchased from Sigma company. Cell Count-
ing Kit-8 (CCK-8), HepG2 (human hepatic carcinoma), RPMI-8226
(human multiple myeloma) and A549 (human lung carcinoma)
cell strains were purchased from EnoGene company and PLC/PRF/
5 cell strain was kindly provided by Stem Cell Bank, Chinese
Academy of Sciences.
CDCl3)
d 206.56, 164.66, 149.95, 142.84, 130.10, 129.48, 128.66,
120.27, 96.15, 74.37, 73.54, 63.41, 62.08, 59.64, 54.73, 45.20, 41.48,
41.39, 38.66, 33.75, 32.61, 30.53, 30.09, 21.71, 19.93. HRESIMS m/z
517.1990 [MþH]þ (calcd for C28H34O7 Cl 517.1988).
4.1.2.18. 3-(tert -butyl)-5-((1S,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-
trihydroxy-4,4-dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-

378
D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
4.2.1. Cell culture
4.2.7. Mitochondrial membrane potential assay
RPMI-8226 and A549 cells were cultured in supplemented RPMI
1640 media containing 10% Fetal bovine serum (FBS) and 1%
penicillin-streptomycin. HepG2, PLC/PRF/5 and L-O2 were cultured
in Dulbecco's Modified Eagle's Medium (DMEM, HyClone,
SH30243.01B) which contained 10% fetal bovine serum (FBS) and
1% penicillin-streptomycin. The condition of culture is at 37 ꢀC in a
humid incubator with 5% CO2.
PLC/PRF/5 and HepG2 cells were seeded into 12-well plates.
After culturing 12 h, the cells were treated with different concen-
trations of compound 20 for 24 h. Following, the depolarization of
mitochondria was detected by mitochondrial membrane potential
detection kit (JC-1) (Beyotime Biotechnology, China). After treated
with drugs, removing the medium and wash with PBS for one time.
Then the cells were incubated in medium which contained 5 mg/mL
JC-1 for 20 min at 37 ꢀC and washed three times before detection.
Two different forms of JC-1 (monomer and aggregate) were
detected by fluorescence microscope. (OLYMPUS, Tokyo, Japan)
4.2.2. Cell proliferation assay
10,000 cells of HepG2, RPMI-8226, A549 or L-O2 were prepared
into 100
m
L cell suspension in each well of 96-well plates and the
plates were incubated for 24 h at 37 ꢀC with 5% CO2. 100
mL Medium
4.2.8. Detection of ROS production
with compounds was mixed into each well of 96-well plates,
respectively. And the negative control group, the solvent control
group, the positive control group were established, respectively.
Intracellular ROS generation was detected by the Reactive Ox-
ygen Species Assay Kit (Beyotime Biotechnology, China). After
treatment of different concentrations of compound 20 and then
stained with 10
three times, the cells were collected and suspended in 400 mL PBS.
The plates were incubated for 72 h at 37 ꢀC with 5% CO2. Then 10
mL
m
M DCHF-DA for 20 min at 37 ꢀC. After washing
CCK-8 solution was mixed into each well of 96-well plates and the
plates were incubated for 4 h. Optical absorbance at 450 nm was
then determined with microplate absorbance reader (Bio-Rad). IC50
values were calculated from the doseeresponse curves of the assay
(Prism 7.0).
The fluorescence was measured by flow cytometer (ACEA
NoVoCyte).
4.2.9. Western blot analysis
Cells were cultured in DMEM medium containing different
concentrations of compound 20. After 24 h incubation, the
adherent and floating cells were collected and lysed in RIPA lysis
buffer on ice for 30 min, samples were centrifuged at 12,000 rpm at
4 ꢀC for 10 min. The amount of protein was measured by the BCA
protein assay kit. Same amounts of protein were separated using
7.5e12.5% SDS polyacrylamide gels and transferred to PVDF mem-
brane (#IPVH00010, Merck Millipore). The membrane was blocked
with 5% skim milk in PBST (PBS with 0.05% Tween-20) for 30 min at
room temperature, and then probed with primary antibody dilu-
tion overnight at 4 ꢀC. Cleaved caspases-3 (1:1000, #CST9661, Cell
Signaling Technology), cleaved PARP (1:1000, #CST5625, Cell
Signaling Technology), Tubulin(1:2000, #M20005, Abmart), cyto-
chrome c (1:2000, ab133504, Abcam), p53 (1:2000, # CST2527, Cell
Signaling Technology). After washing three times, the PVDF mem-
brane was covered with secondary antibody for 1 h at room tem-
perature. After three washes, protein bands were captured by a
chemiluminescence (ECL) kit (Super ECL Prime, US EverbrightRInc).
4.2.3. Colony formation assay
Five hundred PLC/PRF/5 and HepG2 cells were plated in 30 mm
plates. After 12 h incubation, all the cells were fed with various
concentrations of compound 20. Here was a negative group that the
cells in this group were treated with DMSO. The cells were allowed
to grow for 10 days. Then, the cells were fixed with methanol for
15 min and then stained with 0.1% crystal violet for 15 min. After
washing away thecrystal violet, the plates were photographed and
counted by ImageJ software. At least three independent experi-
ments were performed for each result.
4.2.4. Hoechst 33258 staining
About 2 ꢁ 104 cells were plated in twelve-well plates, after
overnight incubation, the cells were then cultured with 0, 0.25, 0.5,
1 mM compound 20 for 24 h. After incubation, removing the me-
dium and washing cells with PBS, cells were fixed in 4% para-
formaldehyde for 30 min at room temperature, and then stained
with 20 m
g/mL Hoechst 33258 for 20 min at an 37 ꢀC incubator. Cells
were assessed by fluorescence microscope (OLYMPUS, Tokyo,
Japan).
4.2.10. Determination of the antitumor effect of compound 20
in vivo
In order to explore the in vivo antitumor effect of compound 20,
we built a mouse liver cancer xenograft model by inoculating PLC/
PRF/5 cells into nude mice. After about 1 week of acclimatization,
cells (2 ꢁ 107) concentrated to 0.2 mL saline were inoculated sub-
cutaneously in nude mice's right forelimb. The lump sizes and body
weights of the mice were measured every day. Tumor volumes
were calculated by a formula: tumor volume (mm3) ¼ a ꢁ b2/2, (a:
the longest diameter, b: the shortest diameter). When the tumors
reached a size of about 120 mm3, mice were randomly divided into
three groups: vehicle, oridonin and compound 20. Mice were
treated intravenously with compound 20 (10 mg/kg), oridonin
(10 mg/kg) and vehicle (10%N-Methyl pyrrolidone/12% Tween-80/
78% saline) once every three days for 4 weeks. Finally, all of the
mice were sacrificed by dislocation, tumors were dissected,
weighed and stored at ꢂ80 ꢀC.
4.2.5. Detection of apoptosis
The liver cells were cultured in six-well plates (2 ꢁ 105 cells/
well).Then the cells were treated with DMSO and various concen-
trations of compound 20 for 24 h.After incubation, collecting
adherent and floating cells and washing them with PBS for one
time. Finally, the cells were measured by Muse™ Annexin V & Dead
Cell Kit (Muse TM Cell Analyzer, Millipore(catalog no.
MCH100105)), according to the instructions.
4.2.6. Cell cycle analysis
PLC/PRF/5 and HepG2 cells were seeded into six-well plates
(5 ꢁ 104 cells/well) and incubated for 12 h, respectively. Then, cells
were treated with different concentrations of compound 20 for
24 h. The cancer cells were harvested, washed with cold PBS for
three times, and then fixed with ice-cold 70% ethanol at ꢂ20 ꢀC for
at least 12 h. Subsequently, collecting all of the cells and washing
three times with PBS to remove extra ethanol.then the cells were
4.2.11. Stability of compound 20 in human plasma at 37 ꢀC
HPLC grade methanol were obtained from Merck (Darmstadt,
Germany), and formic acid was from Sigma-Aldrich Chemical Co.
LLC. (St. Louis, USA). In vitro stability of compound 20 was carried
out with initial concentration of 5000 ng/mL in human plasma at
resuspended in PBS which containing 100 mg/mL RNase and 100 mg/
mL PI and incubated in the dark for 30 min at room temperature.
The samples were determined by flow cytometry on an FC500 cy-
tometer (Beckman Coulter)
37 ꢀC. A 100
mL of plasma was extracted from the incubation

D. Luo et al. / European Journal of Medicinal Chemistry 178 (2019) 365e379
379
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gation and filtration. Then 5 L of the extract was injected into the
LC-MS/MS system. Mass spectrometry was carried out on an A
Agilent 6460 Triple Quad mass spectrometer with ESI source. The
signal of compound 20 was monitored through MS2 Scan mode.
Reverse phase liquid chromatographic separation was performed
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