Synthesis, and evaluation of in vitro and in vivo anticancer activity of 14-substituted oridonin analogs: A novel and potent cell cycle arrest and apoptosis inducer through the p53-MDM2 pathway

Article abstract of DOI:10.1016/j.ejmech.2019.04.005

A series of novel oridonin derivatives bearing various substituents on the 14-OH position were designed and synthesised. Their antitumour activity was evaluated in vitro against three human cancer cell lines (HCT116, BEL7402, and MCF7). Most tested derivatives showed improved anti-proliferative activity compared to the lead compound oridonin and the positive control drug 5-fluorouracil (5-Fu). Among them, compound C7 (IC₅₀ = 0.16 μM) exhibited the most potent anti-proliferative activity against HCT116 cells; it was about 43- and 155-fold more efficacious than that of oridonin (IC₅₀ = 6.84 μM) and 5-Fu (IC₅₀ = 24.80 μM) in HCT116 cancer cells. Interestingly, the IC₅₀ value of compound C7 in L02 normal cells was 23.6-fold higher than that in HCT116 cells; it exhibited better selective anti-proliferative activity and specificity than oridonin and 5-Fu. Furthermore, compound C7 possibly induced cell cycle arrest and apoptosis by regulating the p53-MDM2 signalling pathway. Notably, C7 displayed more significant suppression of tumour growth than oridonin in colon tumour xenograft models where the tumour growth inhibition rate was 85.82%. Therefore, compound C7 could be a potential lead compound for the development of a novel antitumour agent.

Full text of DOI:10.1016/j.ejmech.2019.04.005

Accepted Manuscript
Synthesis, and evaluation of in vitro and in vivo anticancer activity of 14-substituted
oridonin analogs: A novel and potent cell cycle arrest and apoptosis inducer through
the p53-MDM2 pathway
Qing-Kun Shen, Hao Deng, Shi-Ben Wang, Yu-Shun Tian, Zhen-Shan Quan
PII:
S0223-5234(19)30304-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.005
EJMECH 11240
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 December 2018
Revised Date: 1 April 2019
Accepted Date: 1 April 2019
Please cite this article as: Q.-K. Shen, H. Deng, S.-B. Wang, Y.-S. Tian, Z.-S. Quan, Synthesis, and
evaluation of in vitro and in vivo anticancer activity of 14-substituted oridonin analogs: A novel and
potent cell cycle arrest and apoptosis inducer through the p53-MDM2 pathway, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.005.
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ACCEPTED MANUSCRIPT
Synthesis, and evaluation of in vitro and in vivo anticancer activity of
1
4-substituted oridonins: A novel and potent cell cycle arrest and apoptosis
inducers through the p53-MDM2 pathway
1
1
2
1
1
Qing-Kun Shen , Hao-Deng , Shi-Ben Wang , Hao Deng , Yu-Shun Tian * and
1
Zhen-Shan Quan *
1
Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University,
Yanji, Jilin, 133002, China
2
School of Pharmacy, Liaocheng University, LiaoCheng, Shandong, 252059, China

ACCEPTED MANUSCRIPT
Synthesis, and evaluation of in vitro and in vivo anticancer activity of
1
4-substituted oridonin analogs: A novel and potent cell cycle arrest
and apoptosis inducer through the p53-MDM2 pathway
1
$
1$
2
1
Qing-Kun Shen , Hao-Deng , Shi-Ben Wang , Yu-Shun Tian * and Zhen-Shan
Quan *
1
1
Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University,
Yanji, Jilin, 133002, China
2
School of Pharmacy, Liaocheng University, LiaoCheng, Shandong, 252059, China
*
Corresponding author: Tel: + 86 433 243-6004; Fax: + 86 433 243-6004.
E-mail: ystian@ybu.edu.cn (Y. S. Tian).
*
Corresponding author: Tel: + 86 433 243-6020; Fax: + 86 433 243-6020.
E-mail: zsquan@ybu.edu.cn (Z. S. Quan).
$
These authors contributed equally

ACCEPTED MANUSCRIPT
Abstract
A series of novel oridonin derivatives bearing various substituents on the 14-OH
position were designed and synthesised. Their antitumour activity was evaluated in
vitro against three human cancer cell lines (HCT116, BEL7402, and MCF7). Most
tested derivatives showed improved anti-proliferative activity compared to the lead
compound oridonin and the positive control drug 5-fluorouracil (5-Fu). Among them,
compound C7 (IC = 0.16 µM) exhibited the most potent anti-proliferative activity
5
0
against HCT116 cells; it was about 43- and 155-fold more efficacious than that of
oridonin (IC50 = 6.84 µM) and 5-Fu (IC50 = 24.80 µM) in HCT116 cancer cells.
Interestingly, the IC50 value of compound C7 in L02 normal cells was 23.6-fold
higher than that in HCT116 cells; it exhibited better selective anti-proliferative
activity and specificity than oridonin and 5-Fu. Furthermore, compound C7 possibly
induced cell cycle arrest and apoptosis by regulating the p53-MDM2 signalling
pathway. Notably, C7 displayed more significant suppression of tumour growth than
oridonin in colon tumour xenograft models where the tumour growth inhibition rate
was 85.82%. Therefore, compound C7 could be a potential lead compound for the
development of a novel antitumour agent.
Key words: Oridonin; Antitumour; ; Apoptosis; Cell cycle arrest; In vivo

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1
. Introduction
Recently, malignant tumours have become a serious health issue as their
incidence has been increasing yearly [1]. So far, drug therapy is still an effective and
important method in the clinical treatment of cancer. One of the major obstacles to the
druggability of compounds with effective antitumour activity is their toxicity to
normal cells. Therefore, the development of novel anticancer agents with lower
toxicity to normal cells is needed. Natural products play a leading role in drug
discovery. About 65% of the 1211 drugs and small-molecule new chemical entities
approved for marketing in 1981-2014 are directly or indirectly derived from natural
products [2]. Thus, structural modifications of active natural products is an effective
way for discovering potentially active molecules, lead compounds, and new drugs.
Oridonin (Figure 1A), initially isolated from various Isodon species which are
commonly used as a home remedy herb in China and Japan, was proven to possess a
wide variety of pharmacological activities, such as antifibrosis [3-5], anticancer
effects [6-7]. In the past few years, a number of oridonin derivatives have been
designed and synthesised to yield better drug candidates with enhanced activities
[
8-19]. The analysis of the reported derivatives provided a clear structure activity
relationship. Briefly, α, β-unsaturated ketone is the active moiety of oridonin, and
reduction or opening of this moiety will significantly reduce the anti-proliferative
efficacy of oridonin [20-21]. Most derivatives with C-14 hydroxyl functional groups
exhibit improved anti-proliferative activity. For instance, compound J (Figure 1A),
oridonin-coupled nitrogen mustard conjugate at the C-14 hydroxyl, displayed
improved anti-proliferative activity in vitro (IC = 0.50 µM for BEL7402 cells),
5
0
which was 15-fold more potent than that of oridonin [8]. Similarly, Xu et al. prepared
a series of oridonin derivatives of a saturated, long-chain terminal acid in the C-14
position. Among them, compound K (Figure 1A) was identified as the most potent
analogue of this series with an enhanced anti-proliferative effect (IC = 2.06 µM
5
0
against BEL7402 cells) compared to oridonin (IC = 29.80 µM) [11]. In addition,
50
compound HAO472 (Figure 1A), an L-alanine-(14-oridonin) ester trifluoroacetate,
has been discovered and developed by Hengrui Medicine Co. Ltd in China for the

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treatment of acute myelogenous leukaemia and it is now in Phase I of human clinical
trials (CTR20150246; www.chinadrugtrials.org.cn) [22]. According to the examples
illustrated in Figure 1A, it was rational to introduce active groups at the C-14
hydroxyl group of the α, β-unsaturated ketone structure of oridonin.
Phenyl 1,2,3-triazole has a high dipole moment and can form a hydrogen bond
with drugs target, which is beneficial for the binding of the compound to the target
[
23]. Thus, it is an important heterocyclic structural unit distributed in a large number
of biologically active molecules as anti-bacterial [24-25], anti-malarial [26],
anti-tubercular [27], anti-inflammatory [28-29], and anticancer molecules [30]. In
recent years, natural active products and the phenyl 1, 2, 3 triazole molecular
fragment were combined to improve the antitumour activity, physicochemical
properties, and drug-forming properties of the natural products. The modification
strategy of natural products such as compounds L, M, N, O and P in Figure 1B has
attracted more attention [31-34]. Compound N is a synthetic xanthotoxin derivative
designed according to a strategy described in our previous studies, which exhibited
antitumour activity and low toxicity [32]. In particular, Zhou et al. utilized click
reaction to generate a series of oridonin-based 1,2,3-triazole derivatives by
introducing 1,2,3-triazoles into the C-1, C-2, or C-3 position of the A ring of oridonin.
Among them, the representative triazole-substituted oridonin analogue P exhibits a
6
1-fold increase in antiproliferative potency (EC50 = 0.48 µM) against the
MDA-MB-231 triple-negative breast cancer cell line relative to oridonin (EC = 29.4
50
µM)[34]. Therefore, a combination of 1,2,3-triazole groups with oridonin is an
effective strategy to enhance its antitumour activity.
Indole is a bicyclic heterocycle, which widely distributed in natural products
such as alkaloids, plants, animals, and microbial hormones [35,36]. As a special active
fragment, indole has become the focus of attention in drug discovery and
development [37]. Indole containing compounds showed anti-inflammatory [38,39],
antiviral [40], antioxidant [41,42], and anticancer activities [43-45]. Interestingly, the
molecular hybridisation of many natural products and indole significantly enhances
their anticancer activity such as compounds Q, R, and S in Figure 1C [46-48].

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Therefore, the introduction of indole active fragments in natural products is an
effective modification strategy.
In this study, we selected oridonin as a lead compound and successfully
introduced phenyl 1,2,3-triazoles and various indoles at the C-14 hydroxyl group of
oridonin. Consequently, we designed and synthesised oridonin derivatives containing
different phenyl 1, 2, 3-triazoles (A1-9). In order to complete the structure activity
relationship (SAR) study, we changed the linker between the two parts and designed
and synthesised the target compounds B1-B3. Moreover, we designed and synthesised
the oridonin derivatives C1-C9 and D1-D2 containing different substituted indoles.
Compounds E and F have been designed and synthesised according to the alternative
design of quinoline ring or isoquinoline ring because of their similar physicochemical
and biological properties to the indole ring (Figure 2). Subsequently, all the 25 new
compounds were screened against three different types of cancer cell lines. The
toxicity of potential compounds in normal human liver L02 cells was also tested. In
addition, the in vivo antitumour efficacy and mechanism of action of the most
promising compound C7 was also investigated.
2
. Results and discussion
2
.1 Chemistry
Preparation of the intermediates is shown in Scheme 1. Compounds 1a-1i was
obtained by diazotisation and azide reactions of various anilines [49]. Then, a click
reaction between intermediates 1a-1i and propionic acid or propylamine gave
compounds 2a-2i and compound 3a-3c, respectively with good yields [50].
Compounds 3a-3c was mixed with succinic anhydride in CH Cl at room temperature
2
2
to give compounds 4a-4c. Commercially available 4-substituted anilines were
replaced by hydrazine, reacted with pyruvic acid, cyclised, and then hydrolysed with
NaOH to obtain different 5-substituted indole-2-acids 8a-8c. Simultaneously, other
substituted indole-2-acid intermediates 8d-8i were obtained by condensation of
different substituted benzaldehydes with ethyl-2-azidoacetate followed by cyclisation
and hydrolysation of NaOH [51]. Indole-3-carboxylic acid mixed with CH I and
3

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catalysed by KOH in dimethylformamide (DMF) at room temperature produced
compound 9. When indole-3-carboxylic acid was mixed with di-tert-butyl dicarbonate
and catalysed by 4-dimethylaminopyridine (DMAP) in CH Cl at room temperature,
2
2
it produced compound 10 [52,53].
The general path to synthesise the target oridonin analogues A1-A9, B1-B3,
C1-C9, D1,D3, E, and F is shown in Scheme 2. Oridonin as a starting material and
target compounds A1-A9 and B1-B3 were obtained by an amide condensation
reaction
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and DMAP in
anhydrous CH Cl at 0℃ ; under these conditions, only the C-14 hydroxyl group is
with
different
acid
catalysed
by
1
2
2
condensed with different carboxylic acids [54-55]. All the synthesised compounds
1
13
were characterised by H NMR, C NMR, and HRMS approaches.
2
2
.2. Biological evaluation
.2.1. In vitro anti-proliferative activity and SAR study
The anti-proliferative activities against three human cancer cell lines including
HCT116 (human colorectal cancer cell line), MCF-7 (human breast cancer cell line),
and BEL7402 (human hepatocellular carcinoma cell line), were established for all the
target compounds by the MTT assay. The IC50 values (concentration required to
inhibit tumour cell proliferation by 50%) are listed in Table 1; oridonin and 5-Fu are
used as positive controls. As shown in Table 1, all oridonin derivatives A1-A9
containing different phenyl 1,2,3-triazoles exhibited stronger anti-proliferative
activities against all three selected cancer cell lines than oridonin and 5-Fu. Among
them, compound A6 with 4-methoxyphenyl 1,2,3-triazole, was the most potent
compound in this series with an IC value of 1.94 µM in HCT116 cell line. It was
5
0
about 3-fold more potent than oridonin in the cancer cell lines tested. Compound A5
containing 4-methylphenyl 1,2,3-triazole was the most potent compound in this series,
with an IC value of 3.01 µM in MCF-7 cell lines. It was about 6-fold more potent
5
0
than oridonin in the cancer cell lines tested. In order to explore the effect of the linker
on the anti-proliferative activity between oridonin and 1,2,3-triazole, we designed and

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synthesised compounds B1-B3 with methyl 4-(methylamino)-4-oxobutanoate instead
of ester. Interestingly, these compounds do not further enhance anti-proliferative
activity, exhibiting only similar activities as that of the lead compound oridonin,
which indicates that the change to the linker is a failure. Preliminary results suggested
that the phenyl 1,2,3-triazole groups would improve the anti-proliferative activities of
oridonin, highlighting the importance of the linker.
As we can see in Table 1, all the compounds C1-C9, with different substituted
indole-2-carboxylic acids, were slightly more potent against HCT116, MCF-7, and
BEL7402 cells than oridonin and 5-Fu. They exhibited better anti-proliferative
activities (IC = 2.64 µM, 1.64 µM, 0.81 µM, 0.29 µM, 0.82 µM, 0.68 µM, 0.16 µM,
5
0
1
.67 µM, and 0.55 µM, respectively) than oridonin (IC = 6.84 µM) in HCT116 cells.
50
The order of activity was 6-OCH > 6-F > 4,5,6-OCH > 6-Br > 5-OCH ≈ 6-Cl >
3
3
3
5
-Cl ≈ 5,6-OCH > 5-H. Similarly, compounds C1-C9 displayed lower IC values
3 50
(0.39-2.45 µM) than oridonin (17.56 µM) in MCF-7 cells. Moreover, they were more
potent than oridonin (IC = 9.59 µM) against BEL7402 cells with IC values ranging
5
0
50
from 0.83 µM to 3.03 µM. In order to explore the importance of the indole-2-yl group
for the anti-proliferative activity, we designed and synthesised compounds D1, D2, E,
and F by replacing it with N-methyl-indole-3-yl, indole-3-yl, quinoline ring, and
isoquinoline ring, respectively. The pharmacological results of these compounds are
shown in Table 1. Compounds D1, E, and F exhibited potent anti-proliferative
efficacy against HCT116, MCF-7, BEL7402 cancer cell lines, with IC50 values
ranging from 1.17 µM to 2.51 µM, 0.41 µM to 0.95 µM, and 2.30 µM to 3.95 µM,
respectively. Their anti-proliferative activity was similar to that of compound C1 with
IC values of 2.64 µM, 0.78 µM, and 3.12 µM, respectively. However, compound D2
5
0
showed lower anti-proliferative activity than C1 with IC values of 2.73 µM, 3.81
5
0
µM, and 5.31 µM, respectively. The results suggested that, compared with indole-2-yl,
N-methyl-indole-3-yl, quinoline ring and isoquinoline ring can also greatly improve
the anti-proliferative activity of oridonin. Therefore, we believe that compounds D1,
E, and F could be potential lead compounds for further optimisation. Based on the
preliminary results above, a general SAR was established (Figure 3). Among all target

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compounds, compounds C1, C3-C8, F, and G exhibited a remarkable
anti-proliferative efficacy with IC50 < 1.0 µM; we defined them as potential
compounds and conducted more in-depth research on them. Notably, compound C3,
with a 5-methoxyindole-2-acid substituent, exhibited potent anti-proliferative efficacy
against HCT116, MCF-7, and BEL7402 cancer cell lines with IC values of 0.81 µM,
5
0
0
.39 µM, and 0.83 µM, respectively, showing efficient and broad-spectrum
anti-proliferative activity. Regarding compound C7 with a 6-methoxyindole-2-acid
substituent, it exhibited the strongest activity against HCT116 cells in all compounds;
it was about 43- and 155-fold more efficacious than oridonin (IC = 6.84 µM) and
5
0
5
-Fu (IC = 24.80 µM).
50
2
.2.2 Selective inhibition of cancer cell growth by compounds C1, C3-C8, E, and F in
vitro
The lack of selectivity between normal and cancer cells is one of the main limitations
of antitumour drugs [56]. Therefore, we evaluated the cytotoxicity of compounds C1,
C3-C8, E, and F in normal cell lines L02 (human normal liver cells) in order to
determine the selectivity index (ratio of cytotoxicity in L02 cells compared to that in
cancer cells). As shown in Table 2, compound C7 exhibited a 23.6-fold higher
selectivity for HCT116 cells than for normal L02 cells; this selectivity was
significantly higher than that exhibited by oridonin and 5-Fu. Compound C7 not only
exhibited the strongest anti-proliferative activity against HCT116 cells, but selectively
inhibited tumour cells. Therefore, it was chosen for further biological studies.
2
.2.3 Compound C7 inhibited HCT-116 Cells colony formation
Malignant tumour cells have the ability to proliferate indefinitely. The colony
formation assay is used to determine the colony formation ability of cells [57].
Therefore, we investigated the anti-proliferative efficacy of compound C7 by
performing a colony formation assay. As shown in Figure 4, the exposure of HCT116
cells to compound C7 significantly decreased the number and size of colonies in a
concentration-dependent manner. These data demonstrated that C7 could efficiently

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inhibit colony formation in HCT116 cells.
2
.2.4 Compound C7 induced cell cycle arrest with a change in the expression of cyclin
A and cyclin B1
Cell cycle dysregulation, uncontrolled mitotic is an important cause of infinite
proliferation of cancer cells [58]. Therefore, blocking the cell cycle and inhibiting
mitotic division of cells are considered effective to prevent cell proliferation. In order
to explore the mechanism of compound C7 in the inhibition of cancer cell
proliferation, we investigated the effect of C7 on the cell cycle progression using flow
cytometry analysis. As illustrated in Figure 5A and 5B, C7 increased the percentage
of cell population in G2/M phase of the cell cycle in a concentration-dependent
manner from 11.90% to 39.42% after 24 h incubation compared to cells incubated in
DMSO as a vehicle control. Similarly, C7 increased the percentage of cell population
in the S phase of the cell cycle from 23.30% to 29.92%. These results suggested that
C7 could induce cancer cell cycle arrest at the S and G2/M phases.
It has been reported that cyclin A forms a complex with (Cyclin-dependent
kinase 1) CDK1 and (Cyclin-dependent kinase 2) CDK2 as a key regulator in the S
phase [59]. Cyclin B1, also called maturation or mitosis promoting factor, forms a
complex with CDK1. The reduced expression of cyclin B1 complex inhibits cell cycle
progression from the G2 phase to the M phase [60]. In order to determine the
mechanism of cell cycle arrest by C7, we performed western blotting. As shown in
Figure 5C and D, treatment with compound C7 or oridonin for 24 h inhibited the
expression of cyclin A and cyclin B1 in a dose-dependent manner compared with the
control group. At the same concentration (3 µM), the effect of compound C7 on the
expression of proteins was significantly stronger than that of oridonin (p < 0.001).
2
.2.5 Compound C7-induced morphological changes in HCT116 cells by
haematoxylin and eosin (H&E) staining
The mechanism of action of many anti-proliferative drugs is to induce apoptosis.
Cells that undergo apoptosis generally undergo morphological changes [61].

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Therefore, we used H&E staining to assess the morphological changes in cells. As
shown in Figure 6, HCT116 cells incubated with C7 or oridonin for 24 h displayed
significant apoptotic morphological changes such as loss of the original morphology,
unstructured state, volume swelling, cell condensed nuclei, cell lysis, and membrane
shrinkage. These results suggest that C7 has stronger effects on cell morphology than
oridonin at the same concentration (1 µM).
2
.2.6 Compound C7-induced cancer cell apoptosis with a change in the expression of
apoptosis-related proteins
To further confirm that C7 could induce apoptosis, vehicle-, C7-, or
oridonin-incubated HCT116 cells were stained with Annexin V and PI. As shown in
Figure 7A and B, incubation with C7 (0.03, 0.10, 0.30, 1.00, 3.00 µM) induced a
concentration-dependent increase in the percentage of total apoptotic (early and late
stage) cells from 14.02% to 94.8% (3.00 µM). This result indicates that compound C7
induces apoptosis.
Bcl-2 family proteins including pro-apoptotic (e.g., Bax) and anti-apoptotic
proteins (e.g., Bcl-2) are key regulators of apoptosis. The upregulation or
downregulation of apoptotic genes will promote or inhibit cell apoptosis [62]. The
caspase family exhibits important functions in the regulation process of cell apoptosis.
For example, caspase-9 is one of the initiators of apoptosis and caspase-3 is one of the
activators of apoptosis [62]. As shown in Figure 8C, the expression of Bax increased
after treatment with compound C7 for 24 h at concentrations ≥ 0.10 µM, while Bcl-2
expression significantly decreased in a concentration-dependent manner (Figure 7D).
Furthermore, pro-apoptotic proteins caspase-9 and caspase-3 increased in a
concentration-dependent manner after treatment with C7 for 24 h (Figure 7C and D).
These results indicate that C7 significantly inhibits the growth of HCT116 cells by
inducing apoptosis through the caspase-dependent pathway and increasing the
Bax/Bcl2 ratio. The effect of C7 on the expression level of Bcl-2, Bax, caspase-9 and
caspase-3 in HCT116 cells was more pronounced at the same concentration of 3.00
µM (p < 0.001, Figure 7D) than that of oridonin. This also explains that compound C7
has better antitumour activity than oridonin.

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2
.2.7 Effect of compound C7 on the expression of p53-related proteins
To date, there have been many reports on the mechanism of action of Rubescensine A.
The molecular mechanism of its antitumour activity involves reactive oxygen species
(ROS), Bcl-2/Bax, NF-κB, p53/p21, MAPK, PI3K, microRNA, and fatty acid
synthase pathways [63,64]. Among them, the p53/p21 signalling pathway attracted
our interest because it plays a key role in both cell cycle arrest and apoptosis. P53,
known as the “guardian of genome”, is a powerful tumour suppressor encoded by the
p53 gene. It promotes the expression of functional p53 and results in antitumour
effects [65]. It has previously been reported that p53 transactivates p21, directly
binding and inhibiting the expression of cyclin B1/CDK1 complex, thus inducing
G2/M phase arrest. It can also initiate apoptosis by upregulating Bax while
downregulating Bcl-2 expression, which contributes to the activation of caspase-9 and
caspase-3 [66]. In addition, ubiquitin ligase murine double minute chromosome 2
(MDM2) is a key negative regulator of p53 expression [65]. Recently, it has become
an important target for antitumour drugs and many MDM2 inhibitor molecules have
entered clinical research [67-70].
Preliminary results indicate that compound C7 can induce cell cycle arrest by
affecting cyclin family proteins and apoptosis through Bcl-2/Bax/Caspase signalling.
Therefore, we suspect that C7 may inhibit cell proliferation by regulating the p53/p21
signalling pathway. In order to explore its mechanism of action, western blotting was
performed to determine the expression of p21, p53, and MDM2. As shown in Figure
8
A and B, compound C7 inhibited the expression of MDM2 at concentrations greater
than 0.10 µM, upregulating the expression of p53 and p21 compared to that in the
control group. Moreover, it acts better on these proteins than oridonin at 3.00 µM. It
suggested that the p53-MDM2 pathway may be involved in C7 induced the arrest of
G2/M phase and apoptosis in HCT116 cancer cells.
2
.2.8. In vivo antitumour activity of compound C7
To evaluate the antitumour activity of compound C7 in vivo, a colon cancer xenograft

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model was established by subcutaneous inoculation of HCT116 cells into nude mice.
The mice were sorted into four experimental groups (six animals per group) and one
control group, oridonin-, C7-, and paclitaxel-treated mice received a vehicle solution,
2
5 mg/kg of oridonin, 25 mg/kg of compound C7, and 25 mg/kg of paclitaxel. The
tumour size and body weight of the mice were recorded every two days. At the end of
the treatment, the mice were sacrificed and the tumours were recovered and separately
weighed. As shown in Figure 9, average volume and weight of the tumour in
oridonin-, C7-, and paclitaxel-treated groups were significantly lower than those in
the control group. The tumour growth inhibition rates in mice treated with oridonin,
C7, and paclitaxel were 58.61%, 85.82%, and 90.57%, respectively. However, in this
group, the body weight of mice changed slightly compared to that in the control group
with no statistically significant difference. Based on the above pharmacological data,
the antitumour activity of compound C7 was significantly stronger than that of
oridonin and lower than that of the positive control paclitaxel in vivo.
3
. Conclusions
In this study, 25 novel C-14 derivatives of oridonin were designed, synthesised, and
biologically evaluated. Most of them displayed significantly enhanced
anti-proliferative efficacy in a panel of human cancer cell lines. SAR analysis
indicated that the target compounds containing an indole ring exhibited more
remarkable anti-proliferative activity than the target compounds containing phenyl 1,
2
, 3-triazole. Among these molecules, compound C7 with a 6-methoxyindole-2-acid
substituent, displayed the most potent inhibitory activity against HCT116 cell lines
with an IC value of 0.16 µM (43-fold more potent than oridonin). Particularly, the
5
0
IC value of C7 in L02 normal cells was 23.6-fold higher than that in HCT116 cells,
5
0
indicating its selective anti-proliferative effects. In addition, compound C7 can inhibit
the colony formation of HCT116 cells in a concentration-dependent manner.
Furthermore, cell cycle arrest and apoptosis induced by compound C7 may be
achieved through the p53-MDM2 signalling pathway; this involves inhibition of the

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expression of cyclin A and cyclin B1 and regulation of the expression of Bcl and
caspase family proteins. Moreover, compound C7 significantly suppressed the tumour
volume and reduced its weight by 85.82% at a dose of 25 mg/kg/day (iv) in an
HCT116 colon cancer xenograft model, compared to oridonin (58.61%).
In summary, compound C7 showed an outstanding antitumour activity both in
vivo and in vitro and has the potential to be further developed into a promising
antitumor compound.
4
. Experimental section
All chemicals were purchased from commercial source and used without further
purification unless otherwise stated. The reactions were monitored by TLC using
Merck Kieselgel 60 F 254 plates and visualized under UV light at 254 nm. Column
1
chromatography was generally performed on silica gel (200 mesh size). H-NMR and
1
3
C-NMR spectra were measured on an AV-300 (Bruker BioSpin, Switzerland) and
AV-500 (Bruker BioSpin, Switzerland), and all chemical shifts were given in ppm
relative to tetramethylsilane (TMS). High-resolution mass spectra (HRMS) were
measured with an Thermo Scientific LTQ Orbitrap XL in ESI mode.
4
.1 General procedure for the reaction of oridonin with different intermediates
containing-carboxyl
To a solution of oridonin (36.4 mg, 0.1 mmol) in 10 mL dry CH Cl and different
2
2
carboxyl compounds (0.15 mmol), the solution was treated with EDCI (0.3 mmol)
and 4-dimethylaminopyridine (DMAP) (10 mg). The mixture was stirred at 25 ℃ for
2
-8 h and monitored by TLC. After completion of the reaction, water was added and
the mixture was extracted with dichloromethane and the organic phase was washed
with saturated sodium bicarbonate solution and brine, and dried over Na SO . The
2
4
evaporation of the solvents gave the crude products, which were purified by silica gel
column (CH Cl /MeOH, 100:1) to afford compounds A1-9, B1-3, C1-9, D1, E, F.
2
2
4
.1.1 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-

ACCEPTED MANUSCRIPT
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
1
4-yl 1-phenyl-1H-1,2,3-triazole-4-carboxylate (A1)
25
1
White powder; yield 69%. [a]D -57 (c 0.09, CH OH); H NMR (300 MHz,
3
DMSO-d ppm) δ 9.14 (s, 1H), 7.93 (d, J = 7.7 Hz, 2H), 7.66-7.51 (m, 3H), 6.09 (d, J
6
,
=
10.1 Hz, 2H), 5.93 (d, J = 10.3 Hz, 1H), 5.88 (s, 1H), 5.68 (s, 1H), 4.46 (t, J = 7.4
Hz, 1H), 4.16 (d, J = 10.1 Hz, 1H), 3.87 (d, J = 10.0 Hz, 1H), 3.50 (dd, J = 9.8, 6.9 Hz,
1
1
2
2
1
3
5
H), 3.22 (d, J = 9.8 Hz, 1H), 2.59 (dd, J = 14.4, 6.8 Hz, 1H), 2.19 (ddd, J = 22.8,
5.3, 8.2 Hz, 1H), 1.94 (dd, J = 12.9, 5.8 Hz, 1H), 1.79-1.67 (m, 1H), 1.50 (dd, J =
1
3
6.4, 13.9 Hz, 3H), 1.34-1.14 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz, DMSO-d ) δ
6
07.12, 158.88, 150.35, 140.03, 135.98, 129.87 (2C), 129.37, 127.21, 120.76 (2C),
19.84, 95.78, 74.96, 74.01, 71.50, 62.68, 62.06, 59.15, 53.99, 41.19, 40.50, 38.30,
+
+
3.32, 32.65, 30.42, 29.32, 21.57, 19.63. ESI-HRMS calcd for C H N O ([M+H] ):
2
9
34
3
7
36.2391; found: 536.2390.
4
.1.2 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (A2)
1
25
1
White powder; yield 82%; [a]D -85 (c 0.11, CH OH); H NMR (300 MHz,
3
DMSO-d ppm) δ 8.91 (s, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.71-7.53 (m, 2H), 7.47 (d, J
6
,
=
7.5 Hz, 1H), 6.11 (s, 1H), 6.05 (s, 1H), 5.93-5.83 (m, 2H), 5.67 (s, 1H), 4.42 (d, J =
5
6
1
=
.0 Hz, 1H), 4.16 (d, J = 10.1 Hz, 1H), 3.87 (d, J = 9.9 Hz, 1H), 3.51 (dd, J = 10.2,
.9 Hz, 1H), 3.43-3.34 (m, 1H), 3.21 (d, J = 9.4 Hz, 1H), 2.26-2.10 (m, 1H),
.97-1.91 (d, 1H), 1.82-1.70 (m, 1H), 1.57-1.4 (m, 3H), 1.36-1.10 (m, 4H), 1.00 (d, J
1
3
2.2 Hz, 6H). C NMR (75 MHz, DMSO-d6 ppm) δ 207.54, 154.59 (d, J = 249.6
,
Hz, 1C), 150.90, 140.12, 132.51 (d, J = 8.49 Hz, 1H), 130.97, 126.92, 126.0170,
1
25.97, 124.55 (d, J = 10.88 Hz, 1C) 120.22, 117.54 (d, J = 19.16 Hz, 1C), 96.27,
7
5.33, 74.46, 72.02, 63.13, 62.54, 59.63, 54.45, 41.71, 40.99, 38.79, 33.78, 33.12,
+
+
3
0.86, 29.78, 22.05, 20.12. ESI-HRMS calcd for C H FN O ([M+H] ): 554.2297;
29 32 3 7
found: 554.2294.

ACCEPTED MANUSCRIPT
4
.1.3 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-o
xododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (A3)
1
25
1
White powder; yield 70%; [a]D -78 (c 0.12, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.12 (s, 1H), 7.99 (dd, J = 9.0, 4.7 Hz, 2H), 7.49 (d, J = 8.8 Hz,
6
2
4
1
1
2
H), 6.09 (d, J = 11.5 Hz, 2H), 5.91 (d, J = 10.2 Hz, 1H), 5.86 (s, 1H), 5.68 (s, 1H),
.45 (d, J = 5.0 Hz, 1H), 4.16 (d, J = 10.7 Hz, 1H), 3.87 (d, J = 9.8 Hz, 1H), 3.50 (s,
H), 3.42-3.38 (m, 1H), 3.21 (d, J = 9.7 Hz, 1H), 2.23-2.15 (s, 1H), 1.98-1.92 (m, 1H),
1
3
.78-1.73 (m, 1H), 1.55-1.47 (m, 3H), C NMR (126 MHz, DMSO-d , ppm) δ
6
07.91, 159.40, 150.47, 140.38, 132.81, 127.91, 123.84 (2C), 123.77, 120.80, 117.36
(2C), 117.17, 96.19, 75.77, 74.45, 72.08, 63.18, 62.51, 59.63, 54.41, 41.57, 40.97,
3
8.61, 33.67, 32.94, 30.71, 29.56, 21.82, 19.95. 1.31-1.17 (m, 4H), 1.00 (s, 6H).
+
7
+
ESI-HRMS calcd for C H FN O ([M+H] ): 554.2297; found: 554.2296.
2
9
32
3
4
.1.4 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(4-chlorophenyl)-1H-1,2,3-triazole-4-carboxylate (A4)
1
25
1
White powder; Yield 78%; [a]D -88 (c 0.08, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.17 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H),
6
6
.09 (d, J = 12.6 Hz, 2H), 5.91 (d, J = 10.3 Hz, 1H), 5.86 (s, 1H), 5.68 (s, 1H), 4.44 (d,
J = 4.9 Hz, 1H), 4.15 (d, J = 10.9 Hz, 1H), 3.87 (d, J = 10.6 Hz, 1H), 3.50 (dd, J =
0.2, 7.0 Hz, 2H), 3.21 (d, J = 9.4 Hz, 1H), 2.19 (s, 1H), 1.98-1.91 (m, 1H), 1.78-1.72
1
13
(
m, 1H), 1.53-1.45 (s, 3H), 1.30-1.16 (m, 4H), 1.00 (s, 6H). C NMR (126 MHz,
DMSO-d , ppm) δ 207.59, 159.30, 150.83, 140.62, 135.29, 134.22, 130.32 (2C),
6
1
27.81, 123.05 (2C), 120.36, 96.26, 75.45, 74.50, 72.00, 63.15, 62.55, 59.63, 54.49,
1.68, 41.00, 38.78, 33.79, 33.13, 30.91, 29.78, 22.05, 20.11. ESI-HRMS calcd for
4
+
7
+
C H ClN O ([M+H] ): 570.2002; found: 570.1999.
29
33
3
4
.1.5 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-

ACCEPTED MANUSCRIPT
1
4-yl 1-(p-tolyl)-1H-1,2,3-triazole-4-carboxylate (A5)
25
1
White powder; Yield 81%; [a]D -92 (c 0.11, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.08 (s, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H),
6
6
.08 (d, J = 8.7 Hz, 2H), 5.92 (d, J = 10.3 Hz, 1H), 5.87 (s, 1H), 5.68 (s, 1H), 4.44 (d,
J = 5.0 Hz, 1H), 4.16 (d, J = 10.1 Hz, 1H), 3.89 (s, 1H), 3.56-3.46 (m, 1H), 3.42-3.38
m, 1H), 3.22 (d, J = 9.8 Hz, 1H), 2.39 (s, 3H), 2.28-2.14 (m, 1H), 2.05-1.89 (m, 1H),
(
1
.74 (dd, J = 16.9, 8.9 Hz, 1H), 1.60-1.45 (m, 3H), 1.23-1.12 (m, 4H), 1.00 (s, 6H).
1
3
C NMR (126 MHz, DMSO-d , ppm) δ 207.63, 159.40, 150.86, 140.42, 139.60,
6
1
6
2
34.23, 130.70 (2C), 127.49, 121.10 (2C), 120.30, 96.27, 75.45, 74.50, 71.99, 63.16,
2.55, 59.66, 54.47, 41.68, 41.01, 38.80, 33.80, 33.13, 30.91, 29.80, 22.06, 21.08,
+
+
0.10. ESI-HRMS calcd for C H N O ([M+H] ): 550.2548; found: 550.2546.
3
0
36
3
7
4
.1.6 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carboxylate (A6)
1
25
1
White powder; yield 84%; [a]D -77 (c 0.07, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.03 (s, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.1 Hz, 2H),
6
6
.08 (d, J = 7.8 Hz, 2H), 5.92 (d, J = 10.1 Hz, 1H), 5.86 (s, 1H), 5.68 (s, 1H), 4.45 (d,
J = 4.6 Hz, 1H), 4.15 (d, J = 10.2 Hz, 1H), 3.86 (s, 1H), 3.84 (s, 3H), 3.49 (dd, J = 9.5,
6
1
1
1
6
2
5
.0 Hz, 1H), 3.22 (d, J = 9.6 Hz, 1H), 2.61 (dd, J = 11.8, 5.6 Hz, 1H), 2.24-2.14 (m,
H), 1.99-1.92 (m, 1H), 1.78-1.70 (m, 1H), 1.57-1.46 (m, 3H), 1.32-1.17 (m, 4H),
1
3
.00 (s, 6H). C NMR (75 MHz, DMSO-d , ppm) δ 207.64, 160.25, 159.43, 150.84,
6
40.28, 129.79, 127.57, 122.96 (2C), 120.30, 115.33 (2C), 96.27, 75.48, 74.47, 71.99,
3.16, 62.53, 59.66, 56.09, 54.46, 41.66, 40.99, 38.78, 33.79, 33.11, 30.89, 29.78,
+
+
2.03, 20.07. ESI-HRMS calcd for C H N O ([M+H] ): 566.2497; found:
3
0
36
3
8
66.2492.
4
.1.7 (1S,4aR,5S,6S,6aR,9S,11aS)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazole-4-carboxylate (A7)
1

ACCEPTED MANUSCRIPT
25
1
White powder; yield 75%; [a]D -66 (c 0.05, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.09 (s, 1H), 7.46 (dd, J = 10.7, 5.7 Hz, 2H), 7.13 (d, J = 8.7 Hz,
6
1
H), 6.09 (d, J = 11.4 Hz, 2H), 5.92 (d, J = 10.2 Hz, 1H), 5.83 (s, 1H), 5.69 (s, 1H),
.45 (d, J = 4.9 Hz, 1H), 4.16 (d, J = 10.0 Hz, 1H), 3.89 (s, 1H), 3.86 (s, 3H), 3.82 (d,
4
J = 4.0 Hz, 3H), 3.55-3.47 (m, 1H), 3.22 (d, J = 9.6 Hz, 1H), 2.54 (s, 1H), 2.21 (dd, J
16.3, 8.9 Hz, 1H), 1.96 (dd, J = 13.0, 6.1 Hz, 1H), 1.73 (dd, J = 14.7, 8.2 Hz, 1H),
=
1
3
1
.58-1.44 (m, 3H), 1.34-1.17 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz, DMSO-d ,
6
ppm) δ 207.64, 159.47, 150.83, 149.89, 149.75, 140.22, 129.78, 127.65, 120.34,
1
13.58, 112.39, 105.82, 96.29, 75.53, 74.43, 71.99, 63.17, 62.52, 59.69, 56.46, 56.32,
4.48, 41.65, 41.00, 38.77, 33.80, 33.11, 30.93, 29.79, 22.03, 20.07. ESI-HRMS calcd
5
+
+
for C H N O ([M+H] ): 596.2603; found: 596.2600.
3
1
38
3
9
4
.1.8 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole-4-carboxylate (A8)
1
2
5
1
White powder; yield 77%; [a]D -175 (c 0.02, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.18 (s, 1H), 7.22 (s, 2H), 6.10 (d, J = 15.9 Hz, 2H), 5.91 (d, J =
6
1
0.3 Hz, 1H), 5.82 (s, 1H), 5.69 (s, 1H), 4.45 (d, J = 4.7 Hz, 1H), 4.16 (d, J = 9.8 Hz,
H), 3.87 (s, 7H), 3.77 (d, J = 6.0 Hz, 1H), 3.71 (s, 3H), 3.51 (s, 1H), 3.23 (d, J = 8.9
1
Hz, 1H), 2.65 (d, J = 14.1 Hz, 1H), 2.24 – 2.12 (m, 1H), 1.97 (s, 2H), 1.79-1.71 (m,
1
3
1
H), 1.58-1.42 (m, 3H), 1.34-1.17 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz,
DMSO-d , ppm) δ 207.64, 159.47, 153.95, 150.81, 140.29, 138.44, 132.30, 127.90,
6
1
20.37, 99.51, 96.31, 75.58, 74.41, 71.98, 63.17, 62.51, 60.67, 59.70, 56.88, 54.51,
1.66, 41.01, 38.79, 33.80, 33.10, 31.14, 29.79, 24.21, 22.03. ESI-HRMS calcd for
4
+
10
+
C H N O ([M+H] ): 626.2708; found: 626.2707.
32
40
3
4
.1.9 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carboxylate (A9)
1

ACCEPTED MANUSCRIPT
25
1
White powder; yield 64%; [a]D -47 (c 0.08, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.35 (s, 1H), 8.46 (d, J = 9.1 Hz, 2H), 8.28 (d, J = 9.1 Hz, 2H),
6
6
.11 (d, J = 16.9 Hz, 2H), 5.91 (d, J = 10.4 Hz, 1H), 5.87 (s, 1H), 5.69 (s, 1H), 4.45 (d,
J = 4.9 Hz, 1H), 4.16 (d, J = 10.1 Hz, 1H), 3.92-3.85 (m, 1H), 3.52 (d, J = 6.9 Hz, 1H),
3
1
.22 (d, J = 8.8 Hz, 1H), 2.60 (s, 1H), 2.25-2.12 (m, 1H), 2.02-1.90 (m, 1H),
1
3
.79-1.71 (m, 1H), 1.58-1.44 (m, 3H), 1.37-1.14 (m, 4H), 1.01 (s, 6H). C NMR (126
MHz, DMSO-d , ppm) δ 207.52, 159.17, 150.86, 147.70, 140.99, 140.76, 128.18,
6
1
5
25.90 (2C), 122.03 (2C), 120.37, 96.28, 75.45, 74.53, 72.01, 63.13, 62.58, 59.61,
4.54, 41.71, 41.01, 38.80, 33.80, 33.16, 30.96, 29.80, 22.08, 20.13. ESI-HRMS calcd
+
+
for C H N O ([M+H] ): 581.2242; found: 581.2240.
2
9
33
4
9
4.1.10 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-
7-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
14-yl 4-oxo-4-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)butanoate (B1)
25
1
White powder; yield 43%; [a]D -57 (c 0.10, CH OH); H-NMR (300 MHz,
3
DMSO-d , ppm): δ 8.59 (s, 1H), 8.43 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.60 (t, J = 7.7
6
Hz, 2H), 7.49 (d, J = 7.4 Hz, 1H), 6.00 (s, 1H), 5.89 (t, J = 10.3 Hz, 2H), 5.60 (s, 1H),
4
.42 (d, J = 4.9 Hz, 1H), 4.37 (d, J = 5.0 Hz, 2H), 4.07 (d, J = 10.3 Hz, 1H), 3.82 (d, J
9.9 Hz, 1H), 3.59-3.46 (m, 2H), 2.95 (d, J = 9.9 Hz, 1H), 2.46-2.24 (m, 4H), 2.05 (s,
H), 1.91-1.79 (m, 1H), 1.69 (d, J = 13.9 Hz, 1H), 1.44 (dd, J = 21.3, 13.3 Hz, 4H),
=
1
1
1
9
3
1
3
.35-1.17 (m, 4H), 0.99 (s, 6H). C-NMR (75 MHz, DMSO-d , ppm): δ 207.64,
6
71.61, 171.38, 151.19, 146.72, 137.13, 130.36 (2C), 129.03, 121.47, 120.35 (2C),
6.16, 74.54, 73.88, 72.01, 62.99, 62.27, 59.53, 54.30, 41.73, 40.92, 38.80, 34.70,
3.78 (2C), 33.16, 30.73, 30.17 (2C), 29.79, 22.10, 20.15. ESI-HRMS calcd for
+
+
C H N O ([M+H] ): 621.2919; found: 621.2922.
33
41
4
8
4
.1.11 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-
-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen
7
-14-yl 4-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-4-oxobutanoate
(B2)

ACCEPTED MANUSCRIPT
25
1
White powder; yield 40%; [a]D -68 (c 0.09, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 8.62 (s, 1H), 8.42 (s, 1H), 7.93 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 8.9
6
Hz, 2H), 5.99 (s, 1H), 5.88 (dd, J = 11.5, 9.3 Hz, 2H), 5.59 (s, 1H), 4.41 (d, J = 5.0 Hz,
1
H), 4.36 (d, J = 5.3 Hz, 2H), 4.10 (d, J = 5.3 Hz, 1H), 3.82 (d, J = 10.1 Hz, 1H), 3.52
dd, J = 18.2, 11.1 Hz, 2H), 2.95 (d, J = 9.7 Hz, 1H), 2.38 (s, 4H), 2.11-1.97 (m, 1H),
.85 (s, 1H), 1.70 (dd, J = 18.8, 7.1 Hz, 1H), 1.61-1.35 (m, 4H), 1.35-1.12 (m, 4H),
(
1
0
1
7
3
13
.99 (s, 6H). C NMR (126 MHz, DMSO-d , ppm) δ 207.87, 171.96, 171.79, 150.89,
6
46.78, 135.75, 133.51, 130.36 (2C), 122.16 (2C), 121.64, 120.32, 96.08, 74.54,
4.13, 72.07, 63.02, 62.25, 59.55, 54.29, 43.17, 41.63, 40.89, 38.67, 34.63, 33.68,
+
3.02, 30.58, 30.13, 29.62, 21.92, 20.03. ESI-HRMS calcd for C H ClN O
8
3
3
40
4
+
(
[M+H] ): 655.2529; found: 655.2527.
4
7
1
.1.12 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-
-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 4-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-4-oxobutanoate
(B3)
25
1
White powder; yield 52%; [a]D -43 (c 0.07, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 8.47 (s, 1H), 8.40 (t, J = 5.5 Hz, 1H), 7.79 (d, J = 8.9 Hz, 2H), 7.13
6
(d, J = 9.0 Hz, 2H), 6.00 (s, 1H), 5.86 (d, J = 7.8 Hz, 2H), 5.60 (s, 1H), 4.41 (d, J =
5
.0 Hz, 1H), 4.35 (d, J = 5.3 Hz, 2H), 4.07 (d, J = 10.2 Hz, 1H), 3.82 (s, 4H),
.48-3.58 (m, 2H), 2.95 (d, J = 10.4 Hz, 1H), 2.37 (dd, J = 12.3, 6.5 Hz, 4H), 2.04 (d,
3
J = 7.9 Hz, 1H), 1.83 (d, J = 12.6 Hz, 1H), 1.71 (d, J = 7.9 Hz, 1H), 1.56-1.35 (m, 4H),
1
3
1
.19-1.33 (m, 4H), 0.99 (s, 6H). C NMR (75 MHz, DMSO-d , ppm) δ 207.80,
6
1
71.80, 171.78, 159.69, 152.18, 150.96, 146.41, 130.46, 122.13 (2C), 121.52, 115.36
(2C), 97.35, 96.10, 74.54, 74.05, 72.05, 63.01, 62.25, 61.99, 59.54, 55.99, 54.30,
4
3.11, 41.66, 40.90, 34.70, 33.71, 33.06, 30.63, 30.14, 29.66, 22.05, 21.98, 20.07.
+
9
+
ESI-HRMS calcd for C H N4O ([M+H] ): 651.3025; found: 651.3024.
3
4
43

ACCEPTED MANUSCRIPT
4
.2.13 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1H-indole-2-carboxylate (C1)
1
25
1
White powder; yield 81%; [a]D -70 (c 0.09, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.63 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H),
6
7
.33-7.19 (m, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.95 (s, 1H), 6.07 (d, J = 5.6 Hz, 2H),
.94 (d, J = 10.4 Hz, 1H), 5.91 (s, 1H), 5.67 (s, 1H), 4.45 (d, J = 4.9 Hz, 1H), 4.15 (d,
5
J = 10.0 Hz, 1H), 3.87 (d, J = 10.0 Hz, 1H), 3.52 (dd, J = 10.1, 6.9 Hz, 1H), 3.39 (d, J
5.0 Hz, 1H), 3.17 (d, J = 6.8 Hz, 1H), 2.15 (dd, J = 21.7, 13.2 Hz, 1H), 1.94 (dd, J =
2.4, 4.8 Hz, 1H), 1.84-1.71 (m, 1H), 1.57-1.44 (m, 3H), 1.41-1.13 (m, 4H), 1.01 (s,
=
1
6
1
6
2
1
3
H). C NMR (126 MHz, DMSO-d , ppm) δ 207.69, 160.67, 151.22, 137.73, 128.20,
6
27.09, 124.94, 122.49, 120.53, 120.01, 112.98, 108.58, 96.34, 74.68, 74.57, 72.04,
3.08, 62.51, 59.64, 54.48, 41.95, 41.01, 38.83, 33.82, 33.19, 30.83, 29.82, 22.11,
+
7
+
0.21. ESI-HRMS calcd for C H NO ([M+H] ): 508.2330; found: 508.2328.
2
9
34
4
.2.14 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 5-chloro-1H-indole-2-carboxylate (C2)
1
25
1
White powder; yield 83%; [a]D -40 (c 0.11, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.85 (s, 1H), 7.73 (s, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.24 (d, J =
6
8
.8 Hz, 1H), 6.92 (s, 1H), 6.08 (s, 2H), 5.93 (t, J = 5.0 Hz, 2H), 5.67 (s, 1H), 4.44 (d,
J = 4.9 Hz, 1H), 4.15 (d, J = 10.1 Hz, 1H), 3.87 (d, J = 10.0 Hz, 1H), 3.52 (dd, J = 9.9,
7
1
3
1
1
3
.0 Hz, 1H), 3.37 (d, J = 3.2 Hz, 1H), 3.24-3.09 (m, 1H), 2.16 (dt, J = 20.4, 10.5 Hz,
H), 1.93 (dd, J = 12.0, 5.2 Hz, 1H), 1.78 (dd, J = 12.7, 6.8 Hz, 1H), 1.57-1.39 (m,
13
H), 1.37-1.08 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz, DMSO-d , ppm) δ 207.59,
6
60.39, 151.17, 136.08, 129.69, 128.09, 125.04, 125.00, 121.53, 120.06, 114.64,
08.01, 96.32, 74.75, 74.54, 72.04, 63.05, 62.52, 59.62, 54.49, 41.93, 40.99, 38.82,
+
3.80, 33.19, 30.85, 29.79, 22.11, 20.25. ESI-HRMS calcd for C H ClNO
7
2
9
33
+
(
[M+H] ): 542.1940; found: 542.1945.

ACCEPTED MANUSCRIPT
4
.2.15 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 5-methoxy-1H-indole-2-carboxylate (C3)
1
25
1
White powder; yield 84%; [a]D -9 (c 0.10, CH OH); H NMR (300 MHz, DMSO-d ,
3
6
ppm) δ 11.46 (s, 1H), 7.34 (d, J = 8.9 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 6.89 (dd, J =
10.0, 3.4 Hz, 2H), 6.05 (d, J = 10.1 Hz, 2H), 5.94 (d, J = 10.3 Hz, 1H), 5.88 (s, 1H),
5.66 (s, 1H), 4.44 (d, J = 5.0 Hz, 1H), 4.15 (d, J = 10.1 Hz, 1H), 3.87 (d, J = 10.1 Hz,
1H), 3.73 (s, 3H), 3.52 (dd, J = 10.2, 6.8 Hz, 1H), 3.39 (d, J = 4.7 Hz, 1H), 3.16 (d, J
=
10.0 Hz, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.00-1.88 (m, 1H), 1.82-1.71 (m, 1H), 1.50
1
3
(
dd, J = 26.1, 13.4 Hz, 3H), 1.42-1.12 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz,
DMSO-d , ppm) δ 207.73, 160.59, 154.32, 151.16, 133.08, 128.35, 127.40, 120.03,
6
1
5
16.48, 113.87, 108.24, 102.53, 96.33, 74.66, 74.55, 72.05, 63.07, 62.48, 59.65, 55.67,
4.47, 41.93, 40.99, 38.79, 33.79, 33.14, 30.79, 29.79, 22.07, 20.18. ESI-HRMS calcd
+
8
+
for C H NO ([M+H] ): 538.2435; found: 538.2434.
3
0
36
4
.2.16
(1S,4aR,5S,6S,6aR,9S,11aS)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 6-fluoro-1H-indole-2-carboxylate (C4)
1
25
1
White powder; yield 89%; [a]D -71 (c 0.07, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.70 (s, 1H), 7.67 (dd, J = 8.8, 5.6 Hz, 1H), 7.16 (d, J = 8.5 Hz,
6
1
H), 6.97 (s, 1H), 6.95-6.87 (m, 1H), 6.07 (d, J = 3.1 Hz, 2H), 5.93 (d, J = 12.5 Hz,
H), 5.66 (s, 1H), 4.44 (d, J = 4.9 Hz, 1H), 4.13 (dd, J = 12.4, 7.7 Hz, 1H), 3.87 (d, J
2
=
10.1 Hz, 1H), 3.52 (dd, J = 10.1, 6.9 Hz, 1H), 3.18 (d, J = 5.0 Hz, 1H), 2.63-2.53 (m,
1
1
H), 2.15 (dd, J = 20.7, 13.2 Hz, 1H), 1.92 (dd, J = 12.6, 5.1 Hz, 1H), 1.83-1.70 (m,
1
3
H), 1.58-1.44 (m, 3H), 1.35-1.16 (m, 4H), 1.00 (s, 6H). C NMR (126 MHz,
DMSO-d , ppm) δ 207.65, 160.37, 151.19, 137.78, 137.60, 129.05, 124.17 (d, J =
6
1
0.73 Hz, 1C), 124.00, 120.00, 109.33 (J = 54.75 Hz, 1C), 108.87, 98.40 (J = 25.63
Hz, 1C), 96.29, 74.65, 74.55, 72.05, 63.06, 62.50, 59.64, 54.48, 41.94, 40.99, 38.82,
+
3
3.80, 33.17, 30.82, 29.81, 22.09, 20.21. ESI-HRMS calcd for C H FNO
29 33 7
+
(
[M+H] ): 526.2236; found: 526.2234.

ACCEPTED MANUSCRIPT
4
7
1
.2.17 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-
-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 6-chloro-1H-indole-2-carboxylate (C5)
25
1
White powder; yield 74%; [a]D -31 (c 0.04, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.78 (s, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.46 (s, 1H), 7.07 (dd, J =
6
8
.6, 1.8 Hz, 1H), 6.96 (s, 1H), 6.08 (s, 2H), 5.92 (t, J = 5.1 Hz, 2H), 5.67 (s, 1H), 4.44
(d, J = 5.0 Hz, 1H), 4.27-4.09 (m, 1H), 3.86 (d, J = 10.2 Hz, 1H), 3.58-3.40 (m, 1H),
3
1
.16 (d, J = 9.4 Hz, 2H), 2.21-2.11 (m, 1H), 1.95 (d, J = 4.1 Hz, 1H), 1.79-1.71 (m,
1
3
H), 1.51-1.45 (d, J = 8.3 Hz, 3H), 1.23-1.16 (d, J = 6.6 Hz, 4H), 1.00 (s, 6H). C
NMR (126 MHz, DMSO-d , ppm) δ 207.62, 160.42, 151.18, 137.93, 129.52, 129.27,
6
1
6
25.87, 124.15, 121.13, 120.04, 112.40, 108.67, 96.33, 74.76, 74.56, 72.05, 63.07,
2.52, 59.64, 54.48, 41.93, 41.00, 38.82, 33.81, 33.19, 30.84, 29.81, 22.11, 20.21.
+
+
ESI-HRMS calcd for C H ClNO ([M+H] ): 542.1940; found: 542.1936.
2
9
33
7
4
.2.18 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 6-bromo-1H-indole-2-carboxylate (C6)
1
25
1
White powder; yield 64%; [a]D -82 (c 0.05, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.79 (s, 1H), 7.68-7.57 (m, 2H), 7.18 (dd, J = 8.6, 1.6 Hz, 1H),
6
6
1
1
1
.95 (s, 1H), 6.08 (s, 2H), 5.92 (d, J = 9.3 Hz, 2H), 5.67 (s, 1H), 4.44 (d, J = 5.0 Hz,
H), 4.15 (d, J = 10.0 Hz, 1H), 3.87 (d, J = 10.2 Hz, 1H), 3.52 (s, 1H), 3.44-3.36 (m,
H), 3.17 (d, J = 9.3 Hz, 1H), 2.24-2.08 (m, 1H), 1.93 (dd, J = 12.7, 5.1 Hz, 1H),
13
.84-1.69 (m, 1H), 1.60-1.42 (m, 3H), 1.37-1.13 (m, 4H), 1.00 (s, 6H). C NMR (75
MHz, DMSO-d , ppm) δ 207.60, 160.41, 151.18, 138.37, 129.12, 126.08, 124.44,
6
1
5
23.61, 120.02, 117.75, 115.43, 108.67, 96.32, 74.77, 74.55, 72.05, 63.07, 62.52,
9.64, 55.37, 41.92, 40.99, 38.83, 33.80, 33.19, 30.84, 29.82, 22.10, 20.24.
+
+
ESI-HRMS calcd for C H BrNO ([M+H] ): 586.1435; found: 586.1433.
2
9
33
7

ACCEPTED MANUSCRIPT
4
.2.19 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 6-methoxy-1H-indole-2-carboxylate (C7)
1
25
1
White powder; yield 87%; [a]D -76 (c 0.08, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.40 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 6.88 (s, 2H), 6.75 – 6.66
6
(
m, 1H), 6.06 (s, 1H), 6.01 (s, 1H), 5.94 (d, J = 10.3 Hz, 1H), 5.86 (s, 1H), 5.65 (s,
H), 4.43 (d, J = 4.9 Hz, 1H), 4.15 (d, J = 10.0 Hz, 1H), 3.87 (d, J = 9.7 Hz, 1H), 3.77
s, 3H), 3.52 (dd, J = 10.1, 6.9 Hz, 1H), 3.39 (dd, J = 10.4, 5.4 Hz, 1H), 3.19-3.13 (m,
H), 2.25-2.12 (m, 1H), 1.92 (d, J = 12.6 Hz, 1H), 1.80-1.71 (m, 1H), 1.52-1.44
1
(
1
1
3
(
m,3H), 1.30-1.17 (dd, 4H), 1.00 (s, 6H). C NMR (75 MHz, DMSO-d , ppm) 207.75,
6
1
9
3
60.53, 158.29, 151.22, 138.83, 126.94, 123.33, 121.44, 119.95, 112.20, 109.09,
6.34, 94.42, 74.55, 72.09, 72.02, 63.09, 62.47, 59.66, 55.52, 54.44, 41.95, 41.00,
+
8.83, 33.80, 33.16, 30.79, 29.81, 22.08, 20.19. ESI-HRMS calcd for C H NO
8
30
36
+
(
[M+H] ): 538.2435; found: 538.2434.
4
.2.20
(1S,4aR,5S,6S,6aR,9S,11aS)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 5,6-dimethoxy-1H-indole-2-carboxylate (C8)
1
25
1
White powder; yield 80%; [a]D -85 (c 0.05, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.28 (s, 1H), 7.07 (s, 1H), 6.89 (s, 1H), 6.84 (s, 1H), 6.06 (s, 1H),
6
6.00 (s, 1H), 5.96 (d, J = 10.3 Hz, 1H), 5.84 (s, 1H), 5.65 (s, 1H), 4.44 (d, J = 4.8 Hz,
1H), 4.15 (d, J = 10.2 Hz, 1H), 3.87 (d, J = 10.3 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H),
3.56-3.49 (m, 1H), 3.41 (d, J = 6.2 Hz, 1H), 3.16 (d, J = 9.6 Hz, 1H), 2.20-2.14 (d,
1H), 1.96-1.90 (d, 1H), 1.79-1.73 (m, 1H), 1.56-1.45 (m, 3H), 1.37-1.14 (m, 4H), 1.01
1
3
(
s, 6H). C NMR (75 MHz, DMSO-d , ppm) δ 207.79, 160.41, 151.25, 149.80,
6
1
46.03, 133.01, 126.19, 120.05, 108.86, 103.06, 96.36, 94.88, 74.55, 74.51, 72.02,
3.10, 62.45, 59.68, 56.09, 55.83, 55.37, 54.44, 41.96, 40.99, 38.82, 33.81, 33.15,
6
+
+
3
0.78, 29.82, 22.08, 20.18. ESI-HRMS calcd for C H NO ([M+H] ): 568.2541;
31 38 9
found: 568.2540.

ACCEPTED MANUSCRIPT
4
.2.21 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 4,5,6-trimethoxy-1H-indole-2-carboxylate (C9)
1
2
5
1
White powder; yield 64%; [a]D -100 (c 0.05, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 11.44 (s, 1H), 6.91 (s, 1H), 6.67 (s, 1H), 6.07 (s, 1H), 5.96 (d, J =
6
12.7 Hz, 2H), 5.92 (s, 1H), 5.66 (s, 1H), 4.43 (d, J = 4.8 Hz, 1H), 4.14 (d, J = 10.4 Hz,
1H), 3.95 (s, 3H), 3.86 (d, J = 10.3 Hz, 1H), 3.80 (s, 3H), 3.69 (s, 3H), 3.52 (dd, J =
10.0, 7.0 Hz, 1H), 3.39 (dd, J = 10.8, 5.1 Hz, 1H), 3.16 (d, J = 5.1 Hz, 1H), 2.17 (dd,
J = 14.0, 8.8 Hz, 1H), 1.92 (d, J = 12.3 Hz, 1H), 1.81-1.70 (m, 1H), 1.58-1.44 (m, 3H),
1
3
1
1
9
4
.36-1.17 (m, 4H), 1.00 (s, 6H). C NMR (75 MHz, DMSO-d , ppm) δ 207.86,
6
60.38, 153.61, 151.20, 146.13, 135.73, 135.05, 126.25, 119.99, 114.86, 106.77,
6.34, 90.08, 74.67, 74.55, 72.02, 63.09, 62.47, 61.31, 60.88, 59.68, 56.19, 54.43,
1.91, 40.99, 38.80, 33.81, 33.15, 30.80, 29.81, 22.07, 20.16. ESI-HRMS calcd for
+
+
C H NO ([M+H] ): 598.2647; found: 598.2645.
32
40
10
4
.2.22 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1-methyl-1H-indole-3-carboxylate (D1)
1
25
1
White powder; Yield 81%; [a]D -89 (c 0.08, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 7.93 (s, 1H), 7.90 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.19 (dt, J =
6
1
5.0, 7.2 Hz, 2H), 6.06 (d, J = 5.9 Hz, 2H), 6.00 (d, J = 10.2 Hz, 1H), 5.74 (s, 1H),
.63 (s, 1H), 4.44 (d, J = 5.0 Hz, 1H), 4.16 (d, J = 10.0 Hz, 1H), 3.89 (s, 1H), 3.84 (d,
5
J = 6.2 Hz, 3H), 3.52 (dd, J = 10.0, 6.8 Hz, 1H), 3.45-3.35 (m, 1H), 3.14 (d, J = 9.6
Hz, 1H), 2.29-2.11 (m, 1H), 1.93 (dd, J = 12.6, 4.9 Hz, 1H), 1.83-1.70 (m, 1H),
1
3
1
.59-1.42 (m, 3H), 1.40-1.11 (m, 4H), 1.01 (s, 6H). C NMR (75 MHz, DMSO-d ,
6
ppm) δ 208.14, 163.33, 151.61, 137.37, 136.74, 126.48, 122.74, 121.86, 121.31,
1
19.71, 111.03, 106.35, 96.40, 74.52, 73.65, 72.07, 63.10, 62.36, 59.75, 54.38, 42.18,
1.01, 38.82, 33.82, 33.46, 33.16, 30.76, 29.84, 22.07, 20.21. ESI-HRMS calcd for
4
+
+
C H NO ([M+H] ): 522.2486; found: 522.2483.
30
36
7

ACCEPTED MANUSCRIPT
4
7
1
.2.23 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-
-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl quinoline-6-carboxylate (E)
25
1
White powder; yield 67%; [a]D -77 (c 0.13, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 9.02 (s, 1H), 8.55 (d, J = 15.0 Hz, 2H), 8.10 (q, J = 8.9 Hz, 2H),
6
7
.64 (s, 1H), 6.11 (d, J = 12.0 Hz, 2H), 5.98 (s, 1H), 5.76 (s, 1H), 5.67 (s, 1H), 4.18 (d,
J = 9.0 Hz, 1H), 3.88 (d, J = 9.2 Hz, 1H), 3.55 (s, 1H), 3.39 (s, 1H), 3.22 (d, J = 9.2
Hz, 1H), 2.69-2.55 (m, 1H), 2.32-2.09 (m, 1H), 1.96 (d, J = 11.9 Hz, 1H), 1.86-1.68
1
3
(
m, 1H), 1.63-1.40 (m, 3H), 1.35-1.19 (m, 4H), 1.01 (s, 6H). C NMR (75 MHz,
DMSO-d , ppm) δ 207.21, 164.05, 150.85, 150.68, 145.96, 140.89, 131.17, 130.28,
6
1
5
29.23, 127.28, 126.63, 122.50, 119.55, 95.89, 75.04, 74.12, 71.57, 62.67, 62.14,
9.29, 53.99, 41.41, 40.55, 38.37, 33.34, 32.68, 30.38, 29.34, 21.61, 19.72.
+
7
+
ESI-HRMS calcd for C H NO ([M+H] ): 520.2335; found: 520.2330.
3
0
34
4
.2.24
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl isoquinoline-1-carboxylate (F)
(1S,4aR,5S,6S,6aR,9S,11aS)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
1
25
1
White powder; yield 87%; [a]D -87 (c 0.11, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 8.69 (d, J = 8.5 Hz, 1H), 8.54 (d, J = 5.5 Hz, 1H), 8.18-8.03 (m,
6
2H), 7.91-7.84 (m, 1H), 7.82-7.76 (m, 1H), 6.43 (s, 1H), 6.13 (s, 1H), 6.00 (s, 1H),
5.92 (d, J = 9.7 Hz, 1H), 5.65 (s, 1H), 4.47 (d, J = 5.0 Hz, 1H), 4.20 (d, J = 10.2 Hz,
1H), 3.92 (d, J = 10.6 Hz, 1H), 3.55 (dd, J = 9.5, 6.3 Hz, 1H), 3.42 (d, J = 10.1 Hz,
1H), 2.71-2.58 (m, 1H), 2.38-2.23 (m, 1H), 2.05 (dd, J = 12.4, 6.0 Hz, 1H), 1.78-1.68
1
3
(
m, 1H), 1.60-1.43 (m, 3H), 1.39-1.18 (m, 4H), 1.02 (s, 3H), 0.99 (s, 3H). C NMR
(
75 MHz, DMSO-d , ppm) δ 207.41, 184.82, 163.82, 150.16, 147.19, 141.19, 136.54,
6
1
6
31.19, 129.29, 127.38, 125.77, 125.02, 119.76, 95.91, 77.34, 73.66, 71.48, 63.06,
2.05, 59.53, 53.77, 40.96, 40.60, 38.34, 33.38, 32.50, 30.39, 29.40, 21.44, 19.45.
+
7
+
ESI-HRMS calcd for C H NO ([M+H] ): 520.2335; found: 520.2331.
3
0
34
4
.3 Procedure for the preparation of compound D2

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CF COOH (3 mL) was added to a mixture of 9 (36.0 mg) in CH Cl (15 mL) The
3
2
2
reaction was stirred at 0℃for 3 h. After the completion of the reaction, ice water was
added, and the mixture was extracted with ethyl acetate three times. The organic
phase was washed with brine and dried over anhydrous sodium sulfate. The
evaporation of the solvents gave the crude products, which were purified by silica gel
column (CH Cl /MetOH, 100:1) to afford D2
2
2
4
.3.1 (1S,4aR,5S,6S,6aR,9S,11aS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-methylene-7-
oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-
4-yl 1H-indole-3-carboxylate (D2)
1
25
1
White powder; yield 34%; [a]D -57 (c 0.09, CH OH); H NMR (300 MHz,
3
DMSO-d , ppm) δ 12.34 (s, 1H), 8.24 (d, J = 40.7 Hz, 2H), 7.61 (s, 1H), 7.35 (s, 2H),
6
6
3
6
1
6
2
.40-5.79 (m, 4H), 5.63 (s, 1H), 4.44 (s, 1H), 4.19 (s, 1H), 3.90 (s, 1H), 3.54 (s, 1H),
.21 (s, 2H), 2.21 (s, 1H), 1.96 (s, 1H), 1.78 (s, 1H), 1.54 (s, 3H), 1.21 (s, 4H), 1.02 (s,
H). 13C NMR (126 MHz, DMSO-d , ppm) δ 207.96, 163.63, 151.45, 137.07, 127.40,
6
22.58, 121.09, 119.77, 115.46, 113.23, 111.65, 108.47, 96.36, 74.62, 74.29, 72.05,
3.13, 62.49, 59.73, 54.30, 42.08, 40.99, 38.82, 33.83, 33.15, 29.84, 22.26, 22.09,
+
7
+
0.21. ESI-HRMS calcd for C H NO ([M+H] ): 508.2330; found: 508.2329.
2
9
34
4
.4 Cell lines and cell culture
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was
3
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The propidium iodide (PI)
and Annexin V-FITC apoptosis detection kit were purchased from Invitrogen (Eugene,
OR, USA). All human cell lines were used in this study, colorectal cancer cell
HCT116, breast cancer cell MCF-7, liver hepatocellular carcinoma cell Bel-7402 and
normal liver cell L02 were initially purchased from American Type Culture Collection
(ATCC, Manassas, VA, USA). RPMI-1640 media, Dulbecco’s modified Eagle’s
medium (DMEM), and foetal bovine serum (FBS) were provided from Gibco
Company (Grand Island, NY, USA). Cell line MCF-7 was cultivated in DMEM
containing 10% (v/v) heat-inactivated FBS, 100 units/mL penicillin and 100 µg/mL

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streptomycin (Grand Island, NY, USA). Cell lines HCT116, Bel-7402, L02 were
cultivated in RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS, 100
units/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated at
3
7℃under a 5% CO and 90% relative humidity (RH) atmosphere.
2
4
.5 In vitro anticancer activity (MTT assay)
Cells were plated in 96-well plates at appropriate densities to ensure exponential
3
4
growth throughout the experimental period (8×10 ~ 1×10 cells per well) and then
allowed to adhere for 24 h. Cells were then treated for 48 h with serial concentrations
(DMSO, 0.3, 1, 3 and 10 µM) of each compound. 5-fluorouracil (5-Fu) was used as
positive control. After 48 h of incubation, 10 µL of MTT solution was added to each
well to a final concentration of 2 mg/mL. Plates were then incubated for a further 4 h.
After incubation, the MTT solution was removed and 150 µL of DMSO was added to
each well for coloration. The plates were shaken vigorously for 10 min at
roomtemperature to ensure complete solubilization. The optical density (OD) was
read on a microplate reader (ELx 800, BioTek, Highland Park, Winooski, VT, USA) at
a wavelength of 492 nm, and the data were subsequently analysed.
4
.6 Colony formation assay
Exponentially growing determined HCT-116 cells (600 per well) were plated in
-well plates with the Roswell Park Memorial Institute (RPMI) 1640 culture medium
6
containing with 10% foetal bovine serum (FBS). After 12 h of incubation, the culture
medium was removed and replaced fresh medium. Then, the cells were treated with
various concentrations of compound C7 (0.03, 0.06, and 0.12 µM) dissolved in
DMSO. Some cells were treated with DMSO only as a negative control. The cells
were then incubated for another 7 days. Finally, the cells were fixed with 4%
paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15 min at room
temperature, after which, the staining was washed with PBS until the colonies were
totally cleared. One colony was defined to be an aggregate of > 50 cells. At least three
independent experiments were performed for each assay. The numbers of cell colonies

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was calculated and analyzed as the ratio of the number of treated samples to untreated
samples.
4
.7 Analysis for cell cycle by flow cytometry
5
HCT-116 cells were plated in 6-well plates (5.0×10 cells per well) and incubated at
3
7℃ for 24 h. Exponentially growing cells were then incubated with compound C7 at
0
.03 µM, 0.1 µM, 0.3 µM and 1µM. After 24 h, untreated cells (control) or cells
treated with compound C7 were centrifuged at 1000 rpm for 10 min and then fixed in
0% ethanol at -20℃ for at least 24 h. The cells were subsequently resuspended in
7
phosphate-buffered saline (PBS) containing 0.1 mg/mL of RNase A and 5 µg/mL PI.
The cellular DNA content for the cell cycle distribution analysis was measured by
flow cytometry using a FACSalibur flow cytometer with Cell Quest software
(Becton-Dickinson, Franklin Lakes, NJ, USA), plotting at least 30,000 events per
sample. The percentage of cells in the G1, S, and G2/M phases of the cell cycle were
determined using the ModFit LT version 4.0 software package (Verity Software,
Topsham, ME, USA).
4
.8 Analysis of cell morphology by hematoxylin-eosin staining (HE) staining in vitro
6
Exponentially growing determined HCT-116 cells (5 × 10 per well) were plated in
6
-well plates and incubated at 37℃for 24 h. The original culture solution was
discarded, and the culture group was added with a culture solution containing
different concentrations of C7 (0.03, 0.1, 0.3, 1.0 µM). Following 24 h of incubation,
conventional HE staining was performed, and images were observed and collected
under an optical microscope.
4
.9 Analysis for apoptosis by flow cytometry
Apoptosis was detected using an Apoptosis Detection Kit (Invitrogen, Eugene, OR,
USA). In brief, cells were cultured in 6-well plates (5.0×105 cells per well) and
incubated at 37℃ for 24 h. Cells with exponential growth were then incubated with
compound C7 at 0.03, 0.1, 0.3, 1.0 and 3.0 µM. Following 24 h of incubation, the