Novel panaxadiol triazole derivatives induce apoptosis in HepG-2 cells through the mitochondrial pathway

Article abstract of DOI:10.1016/j.bioorg.2020.104078

In this study, we introduced 1, 2, 4-triazole groups into panaxadiol (PD) to obtain 18 panaxadiol triazole derivatives. Five cancer cells and one normal cell were evaluated for cytotoxicity by MTT assay. The results showed that most of the derivatives could inhibit cancer cell proliferation, and the anti-proliferative activity of compound A1 was the most significant. For HepG-2 cells, the IC₅₀ value was 4.21 ± 0.54 μM, which was nearly 15 times higher than the activity of PD. Further studies showed that compound A1 could induce apoptosis in HepG-2 cells, and could enhance the expression of Cl-caspase-3, Cl-caspase-9 and Cl-PARP. Moreover, Western blot analysis showed that after treating HepG-2 cells with compound A1, the expression of p53 protein was increased and the ratio of Bax/Bcl-2 was gradually increased. The cytoplasmic Bax is then translocated to the mitochondria, causing the release of Cyt c protein. Therefore, the results indicate that compound A1 induces apoptosis through the mitochondrial pathway and can be used the potential to develop new anti-proliferative agents.

Full text of DOI:10.1016/j.bioorg.2020.104078

Journal Pre-proofs
Novel panaxadiol triazole derivatives induce apoptosis in HepG-2 cells
through the mitochondrial pathway
Shengnan Xiao, Xude Wang, Lei Xu, Tao Li, Jiaqing Cao, Yuqing Zhao
PII:
S0045-2068(20)31375-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104078
YBIOO 104078
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 April 2020
2 July 2020
4 July 2020
Please cite this article as: S. Xiao, X. Wang, L. Xu, T. Li, J. Cao, Y. Zhao, Novel panaxadiol triazole derivatives
induce apoptosis in HepG-2 cells through the mitochondrial pathway, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.104078
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Novel panaxadiol triazole derivatives induce apoptosis in HepG-2
cells through the mitochondrial pathway
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Shengnan Xiao a, †, Xude Wang a, †, Lei Xu a, Tao Li a, Jiaqing Cao a, **, Yuqing Zhao a, b, *
a
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang, 110016,
5
China
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b Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, Shenyang, 110016, China
*Corresponding author: Tel: +86 24 43520309, 43520303. Fax: +86 24 43520300; E-mail
address: zyq4885@126.com (Yuqing Zhao); jiaqingcao@163.com (Jiaqing Cao)
** Corresponding author
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† These authors contributed equally to this work.
Abstract
In this study, we introduced 1, 2, 4-triazole groups into panaxadiol (PD) to obtain
18 panaxadiol triazole derivatives. Five cancer cells and one normal cell were
evaluated for cytotoxicity by MTT assay. The results showed that most of the
derivatives could inhibit cancer cell proliferation, and the anti-proliferative activity of
compound A1 was the most significant. For HepG-2 cells, the IC50 value was 4.21 ±
0.54 μM, which was nearly 15 times higher than the activity of PD. Further studies
showed that compound A1 could induce apoptosis in HepG-2 cells, and could
enhance the expression of Cl-caspase-3, Cl-caspase-9 and Cl-PARP. Moreover,
Western blot analysis showed that after treating HepG-2 cells with compound A1, the
expression of p53 protein was increased and the ratio of Bax / Bcl-2 was gradually
increased. The cytoplasmic Bax is then translocated to the mitochondria, causing the
release of Cyt c protein. Therefore, the results indicate that compound A1 induces
apoptosis through the mitochondrial pathway and can be used the potential to develop
new anti-proliferative agents.
Keyword: Panaxadiol; Synthesis; 1, 2, 4-triazole; Anti-proliferative activity;

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Mitochondrial pathway.
1. Introduction
In recent years, more and more natural products have been used in the
development of anticancer drugs [1]. Some compounds of ginsenoside have gradually
become the candidate for potential anticancer drugs because of their good therapeutic
efficacy and minimum side effects [2]. Unfortunately, owing to the low
bioavailability and poor biological activity of most ginsenosides [3, 4], a great deal of
research on novel anticancer ginsenosides with better biological activity has been
highlighted.
In the research of ginsenosides, we are committed to the development and
application of dammarane-type ginsenosides. We first isolated two new ginsenosides
with anticancer activity [25-OCH3-PPD (AD-1) and 25-OH-PPD (AD-2)] [5, 6]. A
large amount of panaxadiol (PD) was obtained during the acid hydrolysis of
ginsenosides to produce AD-1 and AD-2. At the same time, in the extensive research
on AD-1, PD was isolated from AD-1 metabolites for the first time [7]. This also
makes the related research on PD more significant.
In the previous research, PD could be used as a potential anti-cancer drug, which
has been reported by different studies [8-10]. PD (shown in Fig 1.) is an artificial
saponin of ginsenoside derived from the protopanaxadiol (PPD) by acid hydrolysis or
metabolism [7, 11, 12]. Previous studies demonstrated that PD could inhibit cancer
cell proliferation. Our team has reported some methods of designing and synthesizing
a series of PD derivatives, Liu designed and synthesized PD fatty acid and amino acid
derivatives, which increased the anti-cancer activity of PD by 3-5 times [13]. Xiao
combined aromatic acids and acid chloride compounds with PD to obtain a series of
highly active derivatives. The IC50 value increased to about 10 μM [14, 15]. However,
in the derivatives obtained, the activity has not been significantly improved. At the
same time, the researches on PD derivatives did not provide deep insights on the
potential mechanisms, and the signaling pathways of PD and its derivatives to inhibit
cell proliferation or induce apoptosis have not been discussed in detail. Therefore, this

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study focuses on the synthesis and development of new PD derivatives and the
discussion of the possible molecular mechanisms.
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O
20
OH
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18
1
4
14
9
2
3
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15
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7
5
HO
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Fig 1. Chemical structure of panaxadiol (PD)
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Based on a large number of previous research results [16-19], we designed and
synthesized a large number of dammarane-type ginsenoside derivatives. Through
comparison of activity and structure-activity relationship, we found that PD has two
hydroxyl structures (C-3 and C-12), when the chemical group binds to the hydroxyl
group at the C-3 position, the activity was more potent than that of the substituted
product at the C-12 position. Therefore, it is very important to discover that the C-3
binding group improves the anticancer activity of PD. In this study, it is intended to
link the introduced group with the hydroxyl group at the C-3 position of PD to obtain
a compound with better activity.
It has been reported that the introduction of 1, 2, 4-triazole groups can
significantly enhance the anti-proliferative activities, and augment the bioavailability
and chemical stability. [20-23]. Mechanistic studies showed that the introduction of 1,
2, 4-triazole could induce apoptosis through the mitochondrial pathway [24], so we
hope that the anti-proliferative activity of PD would be increased by introducing 1, 2,
4-triazole groups.
Therefore, to find drugs with better anticancer activity, we aim to introduc 1, 2,
4-triazole structures into PD. The PD will be combined with chloroacetyl chloride to
obtain an intermediate and then combined with the synthesized 1, 2, 4-triazole group
to obtain the final product. After the completion of the cytotoxicity assay, the
structure-activity relationship has been discussed. The compounds with promising

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anti-proliferative activity have been further studied to clarify their mechanism of
action.
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2. Results and discussion
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2.1. Chemistry
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Compounds A1-A3 and B1-B15 were synthesized according to the synthesis
methods in Scheme 1. First, under the catalysis of N,N’-dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP), panaxadiol combined with
chloroacetic acid to obtain compound 1. The synthesis of the triazole compound was
divided into two parts, namely a linear carboxylic acid part and an aromatic acid part.
In the linear carboxylic acid reaction part, the thiosemicarbazide was refluxed in a
carboxylic acid solution to obtain an intermediate. The cyclization reaction was
performed under the action of a strong base of NaOH to obtain the linear carboxylic
triazole compound 2. Regarding the partial reaction of the aromatic acid, first, the
aromatic acid was formed into acid chloride under the action of thionyl chloride,
which was added to the solution of thiosemicarbazone dissolved in pyridine to obtain
an intermediate. Secondly, a cyclization reaction occurred under the action of a strong
base of NaOH, aromatic acid triazolyl intermediate 3 was obtained. Finally, under the
conditions of acetone and K2CO3, compound 1 and compound 2 or 3 underwent a
substitution reaction to obtain compounds A1-A3 and B1-B15.
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O
OH
A1: R1 = H
S
A2: R1 = CH3
A3: R1 = CH2CH3
H N
2
N
NH
2
H
O
H
N
R
S
1
O
i
A1-A3
N
N
H
N
S
R
H
1
ii
O
N
S
N
H
NH
2
vii
N
N
H
R
2
O
O
OH
OH
iii
O
Cl
O
HO
1
R
2
O
H
N
H
N
vi
NH
2
N
H
vii
S
R
2
N
S
N
H
3
v
B1: R2 = H
B9: R2 = 4-Cl
O
B2: R2 = 3-CH3
B3: R2 = 4-CH3
B4: R2 = 3,5-2CH3
B5: R2 = 2-F
B6: R2 = 3-F
B7: R2 = 4-F
B10: R2 = 2,4-2Cl
B11: R2 = 2-Br
B12: R2 = 4-Br
B13: R2 = 2-NO2
B14: R2 = 3-NO2
B15: R2 = 4-OCH3
O
OH
Cl
R
2
R
2
O
H
N
iv
S
O
N
COOH
N
B1-B15
B8: R2 = 2,4-2F
R
2
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Scheme 1. Synthesis of compounds A1-A3 and B1-B15. Reaction conditions: i: Formic acid,
acetic acid or propionic acid, reflux; ii: NaOH/H2O, reflux, 3h; iii: Chloroacetic acid,
EDC/DMAP, DCM, rt, 3h; iv: SOCl2, DCM/DMF, reflux, 4h; v: Thiosemicarbazone, Pyridine, rt,
16h; vi: NaOH/H2O, reflux, 3h; vii: acetone/K2CO3, reflux, 2h.
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2.2. In vitro anti-proliferative activity and SAR analysis
Five cancer cells (human lung cancer cell lines A549, human hepatoma cell lines
HepG-2, human cervical cancer cell lines Hela, human prostate cancer cell lines PC-3,
human breast cancer cell lines MCF-7) and a normal cell (human gastric epithelial
cell lines GES-1) were used to evaluate the anti-growth activity of the compounds by
MTT assay. The results showed that compound A1 exhibited the most potent
anti-proliferative activity with IC50 = 4.21 ± 0.54 μM for HepG-2 cells. All
compounds were evaluated for the activity on normal cells, and the results showed
that all compounds had low or non-toxic effects (IC50> 100 μM) (Table 1).
We designed and synthesized panaxadiol triazole derivatives to increase the
anti-proliferative activity of panaxadiol. Based on the results shown in Table 1, it was
found that the anti-proliferative activities of most compounds were significantly

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improved. Based on SAR analysis, it could be concluded that compounds A1-A3 were
significantly more active than compounds B1-B15. In compounds A1-A3, as the
carbon chain of the substituent at the C-5 position of the triazole was increased, the
steric hindrance gradually increased, and the anti-proliferative activity gradually
decreased. For example, in HepG-2 cells, the IC50 values of compounds A1-A3 were
4.21, 9.60, and 36.47 μM, respectively. Due to the increased steric hindrance of
substituents, the anti-proliferative activity of the compounds decreased; the
anti-proliferative activity of compounds B1-B15 was generally lower than that of
compounds A1-A3. Although some compounds did not show anti-proliferative
activity, it can be concluded from the results in Table 1 that the same substituent at
different positions can lead to the changes in the anti-proliferative activity. In
compounds B5-B7, the activity of the electron-withdrawing group F in the
meta-substituted compound was stronger than that of the ortho- and para-substituted
compounds, which was verified in the comparison of compounds B13 and B14. In
comparing the effects of different substituent groups on the anti-proliferative activity,
we found that the compounds with halogen atom F substitution had more potent
anti-proliferative activity than those with Cl or Br substitution. At the same time, the
electron-withdrawing group “nitro” had a significant effect on the anti-proliferative
activity; especially, the activity of compound B14 was stronger than that of
panaxadiol. Compared to compound B1, the anti-proliferative activities of the
derivatives have been significantly improved by introducing electron-withdrawing
groups (such as F, Cl, NO2) into the benzene ring. However, the addition of a methyl
group on the benzene ring significantly inhibited the anti-proliferative activity.
Table 1. The IC50 values of compounds A1-A3 and B1-B15 for A549, HepG-2, Hela,
PC-3, MCF-7 and GES-1.
IC50(μM)a
Compound
A549
HepG-2
Hela
PC-3
MCF-7
GES-1
>100
>100
>100
A1
A2
A3
10.54 ± 0.65
24.42 ± 1.10
58.49 ± 1.25
4.21 ± 0.54
9.60 ± 0.47
36.47 ± 1.36
8.58 ± 0.22
16.24 ± 1.41
50.26 ± 0.85
9.68 ± 1.23
15.64 ± 1.36
28.57 ± 1.44
11.03 ± 0.11
23.37 ± 1.02
38.56 ± 1.39

B1
45.52 ± 2.58
50.54 ± 1.14
48.58 ± 2.85
>100
57.56 ± 1.54
79.65 ± 2.51
47.69 ± 2.74
>100
40.85 ± 1.47
61.21 ± 1.26
39.41 ± 1.84
82.57±1.69
48.61 ± 2.49
18.66 ± 1.25
56.39 ± 1.74
23.71 ± 0.64
50.58 ± 1.28
36.14 ± 1.09
86.51 ± 2.45
65.68 ± 2.41
32.56 ± 2.34
21.54 ± 2.94
>100
36.99 ± 2.71
47.34 ± 1.59
40.59 ± 2.86
>100
40.91 ± 2.03
54.88 ± 1.85
36.27 ± 2.01
88.73±3.64
72.52 ± 1.5
24.25 ± 1.26
59.47 ± 2.59
16.35 ± 0.84
46.35 ± 2.40
27.51 ± 1.64
72.68 ± 2.37
51.59 ± 1.05
38.24 ± 1.59
26.35 ± 1.05
86.58 ± 2.36
61.96 ± 1.63
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
B2
B3
B4
B5
78.11 ± 3.10
23.19 ± 0.56
40.29 ± 1.21
30.52 ± 2.50
40.25 ± 3.14
29.54±1.20
90.21 ± 1.35
60.65 ± 1.33
50.69 ± 1.25
36.55 ± 2.31
68.31 ± 2.52
40.88 ± 2.37
69.51 ± 2.17
30.22 ± 1.68
45.31 ± 2.68
26.62 ± 1.39
52.64 ± 1.23
31.56 ± 0.98
78.69 ±2.84
70.31 ± 2.54
42.37 ± 2.64
30.84 ± 1.01
78.45 ± 2.54
63.20 ± 1.64
55.32 ± 1.94
20.55 ± 1.44
39.57 ± 2.79
20.14 ± 1.53
41.45 ± 1.98
25.89 ± 2.13
>100
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
PD
60.38 ± 1.11
40.21 ± 0.19
31.33 ± 1.84
>100
72.15 ± 0.52
57.95 ± 1.78
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a Each data represents mean ± S.D.
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2.3. Effects of compound A1 on cell apoptosis
To investigate whether compound A1 induces apoptosis in HepG-2 cells, we
treated the cells with compound A1 at a concentration of 5, 10 μM. The HepG-2 cells
treated with compound A1 appeared shrunken with apoptotic bodies indicating the
development of apoptosis (Fig 2.). In addition, DAPI staining showed that the nucleus
was shrinking, and the cells were fragmented, which are characteristics of cell
apoptosis (Fig 3.). The data indicates that compound A1 can induce HepG-2 cell
apoptosis. Compared with PD, the cell morphology did not change when the cells
were treated at the same concentration.
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Fig 2. Morphological observation of Compound A1 and PD inducing apoptosis in HepG-2 cells
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Fig 3. Effect of compound A1 on inducing apoptosis in HepG-2 cells with DAPI staining
Then, the Annexin V-FITC/PI detection was employed to further explore the
apoptosis triggered by compound A1 in HepG-2 cells (Fig. 4). The results showed
that compound A1 strongly induced apoptosis in HepG-2 cells in a
concentration-dependent manner. The apoptosis rates induced by compound A1 at
concentrations of 5 μM and 10 μM were 10.2% and 42.7%, respectively, compared
with that of the control group (5.3%). However, there was no change in the apoptotic

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rate after treating cells with PD at the same concentration.
Subsequently, we measured Cl-PARP, Cl-Caspase-3, Cl-Caspase-9 protein
expression. As the concentration of compound A1 increased, the corresponding
protein expression gradually increased. Compared with PD, the results of previous
studies showed that PD did not induce apoptosis in HepG-2 cells. In the detection of
apoptotic proteins, no expression of apoptotic proteins was detected, which also
confirmed the previous results. This data showed that compound A1 triggered the
caspase cascade in HepG-2, thereby inducing apoptosis (Fig 5.).
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Fig 4. Effects of compound A1 on HepG-2 cells apoptosis. The histogram was used to show the
comparison of the apoptosis rate. Data are expressed as means ± SDs of triplicate experiments
performed independently. *P<0.05, **P<0.01, ***P<0.001.
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Fig 5. Protein expression level in HepG-2 cells with compound A1 or PD. (A) The expression levels of
apoptosis-related proteins. (B) and (C) The data analysis of compound A1 and PD for protein
expression. Data are expressed as means ± SDs of triplicate experiments performed independently.
***P<0.001.
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2.4. Compound A1 induces apoptosis through mitochondrial pathway
The mitochondrial apoptosis pathway is a classic pathway for inducing apoptosis
[25, 26]. Due to the increased expression of Cl-caspase-3 and Cl-caspase-9, the
upstream expressed proteins may be regulated through the mitochondrial pathway, so
we hypothesize that compound A1 will induce apoptosis by triggering the
mitochondrial apoptosis pathway. Bcl-2 family proteins and p53 are key proteins of
the mitochondrial apoptotic pathway [27], mainly including Bcl-2 (anti-apoptotic
protein) and Bax (pro-apoptotic protein). And the ratio of Bax / Bcl-2 is a key
indicator of the passage through the mitochondrial apoptotic pathway [28]. Therefore,
we measured the expression levels of Bcl-2, Bax, and p53 proteins. The results

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showed that increasing the concentration of compound A1 induced up-regulation of
the expression of Bcl-2, Bax, and p53 proteins, and increased the ratio of Bax/Bcl-2
(Fig 6.).
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Fig 6. Protein expression level in HepG-2 cells with compound A1. (A) The expression levels of
apoptosis-related proteins. (B) Analysis of Bax, Bcl-2 and p53 protein expression data. (C)
Analysis of Bax / Bcl-2 ratio. Data are expressed as means ± SDs of triplicate experiments
performed independently. *P<0.05, **P<0.01, ***P<0.001.
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During the process of mitochondrial induction of apoptosis, under the influence
of compounds, the Bax protein in the cytoplasm is translocated to the mitochondrial
membrane, leading to the release of Cytochrome c (Cyt c) from the mitochondria into
the cytoplasm. The release of mitochondrial Cyt c is an important indicator of
apoptosis [29]. To further demonstrate that compound A1 induces apoptosis through
the mitochondrial pathway, we examined the expression of Bax and Cyt c proteins in
the cytoplasm and mitochondria, respectively. As depicted in Fig. 7, the expression of
cytoplasmic Bax protein in the cytoplasm was gradually reduced, and the expression
of mitochondrial Bax protein is gradually increased. Nevertheless, the cytoplasmic
Cyt c protein was gradually increased, and the mitochondrial Cyt c protein was
gradually decreased. Further, Fig. 7 showed that treating HepG-2 cells with
compound A1 caused the translocation of the Bax protein from the cytoplasm to the
mitochondria, leading to the release of mitochondrial Cyt c protein into the cytoplasm,
and triggering downstream proteins and inducing apoptosis.

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Fig 7. Protein expression level of Bax and Cyt c in HepG-2 cells after compound A1 treatment.
Data are expressed as means ± SDs of triplicate experiments performed independently.
***P<0.001.
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Therefore, based on the results of a series of protein expressions, we have
concluded that treating HepG-2 cells with compound A1 caused Bax protein
expression in large quantities, the inhibition of Bcl-2 protein expression, and
mitochondrial membrane changes. This leads to the release of Cyt c protein, which
further triggers the caspase cascade, causing the expression of Cl-PARP,
Cl-Caspase-3, and Cl-Caspase-9 proteins, and eventually apoptosis.
3. Conclusion
In summary, in this study, we designed and synthesized 18 new triazole
panaxadiol compounds and evaluated their anti-proliferative activities on A549,
HepG-2, Hela, PC-3, MCF-7, and GES-1 cells by MTT assay. The results showed that
the anti-proliferative activity of most of the compounds was significantly improved,
the activity of compound A1 was the most significant, and it had the strongest
anti-proliferative activity against HepG-2 cells, with an IC50 value of 4.21 ± 0.54 μM,
which was nearly 15 times higher than that the activity of PD. Further research
showed that compound A1 could induce apoptosis in HepG-2 cells. Compared with
the control group, the apoptosis rate of compound A1 treated cells at a concentration
of 10 μM was 42.7%. In the study of the mechanism implicated in inducing apoptosis,
we found that treating cells with compound A1, significantly increased the expression

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of Bax protein, but significantly decreased the expression of Bcl-2 protein. At the
same time, Cyt c protein gradually increased in the cytoplasm, which further activated
the protein expression of Cl-PARP, Cl-Caspase-3 and Cl-caspase-9. The results of
Western blot analysis showed that compound A1 induced apoptosis through the
mitochondrial pathway.
4. Experimental section
4.1. Chemistry
Panaxadiol was obtained through the separation of ginsenoside acid hydrolysate,
and its purity was greater than 99% by HPLC. Other chemical reagents purchased
from Sigma (St. Louis, MO, USA). All reactions detected using thin layer
chromatography (TLC).
The melting point is determined in an open capillary. Purity was measured by
high performance liquid chromatography-evaporation light scattering detector
(HPLC-ELSD) with a C18 reversed-phase column (4.6*250mm, 5μm). Elution was
1
carried out with MeOH-H2O (85:15) at a flow rate of 1 mL/min. H-NMR and
13C-NMR spectra were measured on an AV-400 spectrometer (Bruker, Karlsruhe,
Germany) using tetramethylsilane as an internal standard in a CDCl3 or pyridine-d5
solution. High-resolution accurate mass spectrometry (HRMS) determinations for
compounds were obtained on a Bruker micromass time of flight mass spectrometer
equipped with an electrospray ionization (ESI) detector.
4.1.1. Preparation of compound 1, 2 and 3.
The synthesis method of compound 1 was as follows [30, 31]: The PD (2 g, 4.3
mmol,
1
equiv)
was
dissolved
in
dichloromethane.
Then
N,
N’-dicyclohexylcarbodiimide (DCC, 1.79 g, 8.6 mmol, 2 equiv), chloroacetic acid
(0.41 g, 4.3 mmol, 1 equiv) and 4-dimethylaminopyridine (DMAP, 0.53 g, 4.3 mmol,
1 equiv) were added in sequence, stirred at room temperature for 3 h. Compound 1
was obtained by column chromatography (The silica gel is 300-400 mesh). The
separation conditions were petroleum ether: ethyl acetate = 10: 1.

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The synthesis method of compound 2 was as follows [32]: A suspension of
thiosemicarbazide (1 g, 10 mmol) in formic acid, acetic acid or propionic acid (5 mL)
was heated under reflux for 0.5 - 3 h to give a clear solution. After cooled to room
temperature, the reaction product gradually precipitated in the solution, filtered to
obtain the crude product, and recrystallized with ethanol to obtain an intermediate.
Dissolve the intermediate (0.3 g, 2.5 mmol, 1 equiv) in purified water (15 mL), add
sodium hydroxide (0.15 g, 3.75 mmol, 1.5 equiv), and reflux at 100 °C for 3 h, cool to
room temperature, adjust the pH with 10% HCl, compound 2 gradually precipitates,
filter, and rinse with a small amount of ethanol to obtain pure compound 2.
The synthesis method of compound 3 was as follows [33]: To a cold solution of
a catalytic amount of DMF in anhydrous benzoic acid (1.0 mmol, 1 equiv) in
anhydrous DCM (25 mL) was added thionyl chloride (1.5 mmol, 1.5 equiv) dropwise.
The reaction mixture was refluxed for 4 h. The solvent was removed under reduced
pressure. The acid chloride residue was added to a solution of thiosemicarbazone (1.5
mmol, 1.5 equiv) in pyridine (25 mL), and then stirred at room temperature for 16
hours. Pyridine was removed under reduced pressure. The crude residue was
dissolved in an aqueous solution (25 mL), sodium hydroxide (1.5mmol, 1.5 equiv)
was added, and the resulting mixture was reacted at reflux at 100 °C for 3 h. After the
reaction was completed, the mixture was cooled to room temperature and neutralized
with 10% HCl. The precipitate was filtered, washed with water, and dried to obtain
the compound
4.1.2. Procedure for the synthesis of compounds A1-A3 or B1-B15.
Compound 1 (50 mg, 0.09 mmol, 1 equiv) was dissolved in acetone, compound 2
°
or 3 (20-30 mg, 0.18 mmol, 2 equiv) and K2CO3 were added, reacted at 60 C under
reflux for 2 hours. Compounds A1-A3 or B1-B15 was separated by column
chromatography. The separation conditions were petroleum ether: Acetone = 5: 1.
4.1.2.1. 20(R)-Pananxadiol-12β-hydroxy-3β-yl-[4H-[1,2,4]triazol-3-yl]-thioacetate
°
(A1). White solid, yield: 43%. Melting point: 155-157 C. Purity: HPLC > 95%.

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1H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 6H), 0.88(s, 3H), 0.89(s, 3H), 0.98(s,
3H), 1.18(s, 3H), 1.21(s, 3H), 1.26(s, 3H), 1.30-1.97(m, 16H), 3.56(td, J1 = 12.0 Hz,
J2 = 8.0 Hz, 1H), 3.91(s, 2H), 4.54(dd, J1 = 8.0 Hz, J2 = 8.0 Hz, 1H), 6.56(s, 1H);
13C-NMR (CDCl3, 100 MHz, ppm): 169.49, 83.26, 77.36, 73.41, 70.24, 56.09, 54.83,
51.39, 49.97, 49.23, 39.96, 38.68, 38.18, 37.19, 36.58, 35.89, 34.92, 34.83, 33.17,
31.31, 30.65, 28.05, 27.35, 25.33, 23.72, 19.58, 18.29, 17.21, 16.54, 16.39, 16.32,
15.78. HR-MS (ESI) m/z calcd for C34H55N3O4S. [M+H]+ 602.3986, found 602.3994.
4.1.2.2.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-methyl-4H-[1,2,4]triazol-3-yl]-thioacetate
°
(A2). White solid, yield: 40%. Melting point: 156-158 C. Purity: HPLC > 95%.
1H-NMR (CDCl3, 400 MHz, ppm): δ 0.82(s, 6H), 0.87(s, 3H), 0.90(s, 3H), 0.97(s,
3H), 1.18(s, 3H), 1.22(s, 3H), 1.26(s, 3H), 1.40-1.92(m, 24H), 2.42(s, 2H), 3.53(td, J1
= 12.0 Hz, J2 = 4.0 Hz, 1H), 3.85(s, 2H), 4.54(dd, J1 = 8.0 Hz, J2 = 8.0 Hz, 1H),
6.33(s, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.50, 82.94, 77.37, 73.29, 70.08,
56.11, 54.87, 51.37, 49.98, 49.31, 39.98, 38.69, 38.19, 37.20, 36.61, 35.91, 34.94,
34.65, 33.18, 31.29, 30.71, 28.03, 27.34, 25.33, 23.73, 19.59, 18.31, 17.21, 16.54,
16.43, 16.30, 15.79. 12.80. HR-MS (ESI) m/z calcd for C35H57N3O4S. [M+H]+
616.4143, found 616.4156.
4.1.2.3.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-ethyl-4H-[1,2,4]triazol-3-yl]-thioacetate
°
(A3). White solid, yield: 40%. Melting point: 158-160 C. Purity: HPLC > 95%.
1H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 6H), 0.87(s, 3H), 0.89(s, 3H), 0.97(s,
3H), 1.18(s, 3H), 1.21(s, 3H), 1.25(s, 6H), 1.29-1.87(m, 24H), 2.76(q, J1 = 8.0 Hz,
2H), 3.54(td, J1 = 8.0 Hz, J2 = 4.0 Hz, 1H), 3.86(s, 2H), 4.54(dd, J1 = 8.0 Hz, J2 = 4.0
Hz, 1H), 6.36(s, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.52, 82.86, 76.83,
73.28, 70.10, 56.07, 54.84, 51.36, 49.95, 49.26, 39.94, 38.66, 38.16, 37.17, 36.58,
35.88, 34.91, 34.67, 33.17, 31.26, 30.68, 29.84, 28.03, 27.32, 20.61, 19.57, 18.29,
17.18, 16.54, 16.40, 16.29, 15.77, 12.16. HR-MS (ESI) m/z calcd for C36H59N3O4S.

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[M+H]+ 630.4299, found 630.4315.
4.1.2.4.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-phenyl-4H-[1,2,4]triazol-3-yl]-thioacetate
°
(B1). White solid, yield: 52%. Melting point: 154-156 C. Purity: HPLC > 95%.
1H-NMR (CDCl3, 400 MHz, ppm): δ 0.83(s, 6H), 0.87(s, 3H), 0.88(s, 3H), 0.97(s,
3H), 1.18(s, 3H), 1.21(s, 3H), 1.25(s, 3H), 1.46-1.91(m, 24H), 3.54(td, J1 = 8.0 Hz, J2
= 4.0 Hz, 1H), 3.90(s, 2H), 4.56(dd, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 6.37(s, 1H),
7.43-7.45(m, 3H), 7.98-8.00(m, 2H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.75,
129.97, 128.79, 126.38, 83.29, 77.22, 73.16, 69.95, 55.93, 54.72, 51.22, 49.82, 49.14,
39.81, 38.50, 38.04, 37.03, 36.45, 35.75, 34.77, 33.03, 31.14, 30.54, 29.71, 27.94,
27.16, 25.17, 23.61, 19.43, 18.15, 17.05, 16.42, 16.28, 16.16, 15.63. HR-MS (ESI)
m/z calcd for C40H59N3O4S. [M+H]+ 678.4299, found 678.4323.
4.1.2.5.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(3-methyl-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B2). White solid, yield: 51%. Melting point: 155-157 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.82(s, 3H), 0.83(s, 3H), 0.87(s, 3H),
0.97(s, 3H), 1.17(s, 3H), 1.20(s, 3H), 1.24(s, 3H), 1.25(s, 3H), 1.31-1.97(m, 20H),
2.39(s, 3H), 3.55(td, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 3.92(s, 2H), 4.56(dd, J1 = 12.0 Hz,
J2 = 8.0 Hz, 1H), 6.44(s, 1H), 7.22-7.24(m, 1H), 7.32(t, J1 = 16.0 Hz, J2 = 8.0 Hz, 1H),
13
4.75(d, J1 = 4.0 Hz, 1H), 7.81(s, 1H); C-NMR (CDCl3, 100 MHz, ppm): 169.60,
138.56, 130.79, 128.68, 128.47, 127.10, 123.42, 83.09, 77.22, 73.19, 70.03, 55.92,
54.69, 51.22, 49.80, 49.11, 39.80, 38.49, 38.02, 37.02, 36.45, 35.75, 34.75, 33.01,
31.13, 30.48, 29.70, 27.93, 27.16, 25.17, 23.61, 21.37, 19.44, 18.14, 17.04, 16.41,
16.26, 16.15, 15.63. HR-MS (ESI) m/z calcd for C41H61N3O4S. [M+H]+ 692.4456,
found 691.4478.
4.1.2.6.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(4-methyl-phenyl)-4H-[1,2,4]triazol-3-yl]-t

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hioacetate (B3). White solid, yield: 57%. Melting point: 159-161 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 3H), 0.82(s, 3H), 0.87(s, 6H),
0.97(s, 3H), 1.17(s, 3H), 1.20(s, 3H), 1.25(s, 3H), 1.36-1.97(m, 24H), 2.39(s, 3H),
3.55(td, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 2.39(s, 2H), 4.56(dd, J1 = 8.0 Hz, J2 = 4.0 Hz,
1H), 6.40(s, 1H), 7.23(d, J1 = 8.0 Hz, 2H), 7.84(d, J1 = 8.0 Hz, 2H); 13C-NMR
(CDCl3, 100 MHz, ppm): 169.75, 129.65, 126.45, 83.22, 77.37, 73.31, 70.13, 56.06,
54.84, 51.36, 49.95, 49.27, 39.95, 38.64, 38.17, 37.16, 36.60, 35.89, 34.90, 33.16,
31.27, 30.64, 29.84, 28.06, 27.31, 25.31, 23.74, 21.60, 19.58, 18.28, 17.19, 16.54,
16.41, 16.29, 15.77. HR-MS (ESI) m/z calcd for C41H61N3O4S. [M+H]+ 692.4456,
found 691.4478.
4.1.2.7.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(3,5-Dimethyl-phenyl)-4H-[1,2,4]triazol-3-
°
yl]-thioacetate (B4). White solid, yield: 50%. Melting point: 157-159 C. Purity:
1
HPLC > 95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 3H), 0.83(s, 3H), 0.87(s,
6H), 0.97(s, 3H), 1.17(s, 3H), 1.20(s, 3H), 1.24(s, 3H), 1.33-1.98(m, 19H), 2.35(s,
6H), 3.55(td, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 3.94(s, 2H), 4.56(dd, J1 = 12.0 Hz, J2 =
8.0 Hz, 1H), 6.50(s, 1H), 7.06(s, 1H), 7.61(s, 2H); 13C-NMR (CDCl3, 100 MHz,
ppm): 169.47, 138.51, 131.95, 127.79, 124.24, 83.06, 77.22, 73.21, 70.06, 55.92,
54.69, 51.22, 49.80, 49.09, 39.80, 38.49, 38.01, 37.01, 36.44, 35.74, 34.76, 34.73,
33.01, 31.13, 30.45, 27.93, 27.16, 25.17, 23.60, 21.25, 19.44, 18.14, 17.04, 16.41,
16.25, 16.15, 15.62. HR-MS (ESI) m/z calcd for C42H63N3O4S. [M+H]+ 706.4612,
found 706.4645.
4.1.2.8.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(2-Fluoro-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B5). White solid, yield: 45%. Melting point: 161-163 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.82(s, 3H), 0.86(s, 3H), 0.88(s, 6H),
0.96(s, 3H), 1.17(s, 3H), 1.20(s, 3H), 1.26(s, 3H), 1.37-1.97(m, 24H), 3.52(td, J1 = 8.0
Hz, J2 = 4.0 Hz, 1H), 3.97(s, 2H), 4.55(dd, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 6.27(s, 1H),

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7.19-7.24(m, 1H), 7.29(t, J1 = 8.0 Hz, 1H), 7.45(q, 1H), 8.21(td, J1 = 8.0 Hz, J2 = 4.0
Hz, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 167.94, 131.21, 131.12, 128.80, 124.16,
115.14, 114.93, 81.63, 76.20, 72.09, 68.85, 54.92, 53.70, 50.18, 48.79, 48.14, 38.79,
37.49, 36.99, 36.00, 35.43, 34.73, 33.76, 33.44, 32.00, 30.10, 29.53, 26.85, 26.13,
24.14, 22.58, 18.40, 17.13, 16.02, 15.36, 15.26, 15.12, 14.61. HR-MS (ESI) m/z calcd
for C40H58FN3O4S. [M+H]+ 696.4205, found 696.4236.
4.1.2.9.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(3-Fluoro-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B6). White solid, yield: 50%. Melting point: 158-160 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.82(s, 3H), 0.84(s, 3H), 0.88(s, 6H),
0.98(s, 3H), 1.18(s, 3H), 1.20(s, 3H), 1.25(s, 3H), 1.34-1.97(m, 20H), 3.58(td, J1 = 8.0
Hz, J2 = 4.0 Hz, 1H), 3.93(s, 2H), 4.56(dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 6.60(s, 1H),
7.10(td, J1 = 8.0 Hz, J2 = 4.0 Hz, 1H), 7.39(q, 1H), 7.74(d, J1 = 12.0 Hz, 1H), 7.83(d,
J1 = 8.0 Hz, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 168.62, 129.36, 121.09, 115.74,
115.53, 112.60, 112.37, 82.43, 76.20, 72.26, 69.10, 54.91, 53.66, 50.22, 48.79, 48.04,
38.78, 37.47, 37.01, 36.00, 35.42, 34.72, 33.85, 33.73, 32.00, 30.13, 29.45, 26.94,
26.15, 24.15, 22.59, 18.41, 17.12, 16.02, 15.39, 15.22, 15.15, 14.60. HR-MS (ESI)
m/z calcd for C40H58FN3O4S. [M+H]+ 696.4205, found 696.4236.
4.1.2.10.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(4-Fluoro-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B7). White solid, yield: 55%. Melting point: 162-164 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 3H), 0.83(s, 3H), 0.87(s, 6H),
0.97(s, 3H), 1.18(s, 3H), 1.20(s, 3H), 1.24(s, 3H), 1.35-1.97(m, 20H), 3.58(td, J1 =
12.0 Hz, J2 = 4.0 Hz, 1H), 3.97(s, 2H), 4.56(dd, J1 = 8.0 Hz, J2 = 4.0 Hz, 1H), 6.67(s,
1H), 7.11-7.15(m, 2H), 8.06(s, 2H); 13C-NMR (CDCl3, 100 MHz, ppm): 168.47,
127.69, 127.60, 114.99, 114.78, 82.37, 76.20, 72.30, 69.15, 54.93, 53.65, 50.23,
48.79, 48.01, 38.78, 37.49, 37.01, 36.00, 35.41, 34.72, 33.94, 33.73, 32.01, 30.13,
29.43, 26.97, 26.18, 24.16, 22.59, 18.43, 17.12, 16.04, 15.40, 15.21, 15.17, 14.61;

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HR-MS (ESI) m/z calcd for C40H58FN3O4S. [M+H]+ 696.4205, found 696.4236.
4.1.2.11.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(2,4-Difluoro-phenyl)-4H-[1,2,4]triazol-3-
°
yl]-thioacetate (B8). White solid, yield: 45%. Melting point: 163-165 C. Purity:
1
HPLC > 95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 6H), 0.86(s, 3H), 0.88(s,
3H), 0.96(s, 3H), 1.17(s, 3H), 1.21(s, 3H), 1.25(s, 3H), 1.36-1.94(m, 24H), 3.52(td, J1
= 12.0 Hz, J2 = 4.0 Hz, 1H), 3.95(s, 2H), 4.53(dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H),
6.31(s, 1H), 6.96(td, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 7.02(td, J1 = 12.0 Hz, J2 = 4.0 Hz,
1H), 8.20(q, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 167.98, 130.14, 111.79,
111.58, 103.54, 103.59, 103.33, 81.73, 76.20, 72.11, 68.88, 54.91, 53.69, 50.18,
48.79, 48.12, 38.78, 37.49, 36.99, 36.00, 35.43, 34.73, 33.75, 33.45, 31.99, 30.10,
29.50, 26.86, 26.12, 24.14, 22.57, 18.40, 17.13, 16.02, 15.37, 15.25, 15.13, 14.16;
HR-MS (ESI) m/z calcd for C40H57F2N3O4S (M+H) 714.4111, found 714.4124.
4.1.2.12.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(4-Chloro-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B9). White solid, yield: 50%. Melting point: 150-152 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.80(s, 3H), 0.82(s, 3H), 0.87(s, 6H),
0.97(s, 3H), 1.18(s, 3H), 1.19(s, 3H), 1.24(s, 3H), 1.30-1.99(m, 19H), 3.59(td, J1 = 8.0
Hz, J2 = 4.0 Hz, 1H), 3.97(s, 2H), 4.53(dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 6.72(s, 1H),
7.39(d, J1 = 8.0 Hz, 2H), 7.99(d, J1 = 8.0 Hz, 2H); 13C-NMR (CDCl3, 100 MHz, ppm):
169.46, 135.98, 129.02, 127.87, 83.39, 77.22, 73.33, 70.20, 55.94, 54.66, 51.26,
49.81, 49.02, 39.80, 38.49, 38.02, 37.02, 36.42, 35.73, 34.87, 34.74, 33.01, 31.15,
30.44, 27.95, 27.18, 25.17, 23.59, 19.44, 18.13, 17.05, 16.40, 16.22, 16.18, 15.62;
HR-MS (ESI) m/z calcd for C40H58ClN3O4S. [M+H]+ 712.3909, found 712.3914.
4.1.2.13.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(2,4-Dichloro-phenyl)-4H-[1,2,4]triazol-3-
°
yl]-thioacetate (B10). White solid, yield: 58%. Melting point: 152-154 C. Purity:

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HPLC > 95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.80(s, 6H), 0.86(s, 6H), 0.96(s,
3H), 1.17(s, 3H), 1.19(s, 3H), 1.24(s, 3H), 1.30-1.95(m, 20H), 3.54(td, J1 = 12.0 Hz,
J2 = 8.0 Hz, 1H), 3.96(s, 2H), 4.53(dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 6.74(s, 1H),
7.33(dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.47(d, J1 = 1.6 Hz, 1H), 8.02(dd, J1 = 8.0 Hz,
J2 = 4.0 Hz, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.01, 136.46, 132.35, 130.37,
128.32, 127.68, 126.90, 82.83, 77.26, 73.20, 70.05, 55.91, 54.69, 51.22, 49.80, 49.08,
39.79, 38.51, 38.00, 37.01, 36.44, 35.73, 34.76, 34.62, 33.00, 31.13, 30.47, 27.90,
27.15, 25.16, 23.59, 20.45, 19.43, 18.14, 17.04, 16.41, 16.25, 16.16, 15.63. HR-MS
(ESI) m/z calcd for C40H57Cl2N3O4S. [M+H]+ 746.3520, found 746.3538.
4.1.2.14.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(2-Bromo-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B11). White solid, yield: 60%. Melting point: 156-158 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 6H), 0.86(s, 6H), 0.96(s, 3H),
1.16(s, 3H), 1.19(s, 3H), 1.24(s, 3H), 1.32-1.99(m, 20H), 3.52(td, J1 = 12.0 Hz, J2 =
4.0 Hz, 1H), 3.96(s, 2H), 4.52(dd, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 6.37(s, 1H), 7.30(t,
J1 = 4.0 Hz, 1H), 7.41(t, J1 = 8.0 Hz, 1H), 7.65(d, J1 = 8.0 Hz, 1H), 8.04(d, J1 = 8.0
Hz, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.08, 133.91, 132.09, 131.39, 127.85,
120.57, 82.76, 77.23, 73.16, 69.96, 55.92, 54.70, 51.21, 49.80, 49.11, 39.80, 38.61,
38.01, 37.01, 36.44, 35.74, 34.77, 34.59, 33.01, 31.12, 30.50, 27.91, 27.15, 25.16,
23.59, 19.42, 18.14, 17.04, 16.42, 16.28, 16.16, 15.63. HR-MS (ESI) m/z calcd for
C40H58BrN3O4S. [M+H]+ 756.3404, found 756.3430.
4.1.2.15.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(4-Bromo-phenyl)-4H-[1,2,4]triazol-3-yl]-t
hioacetate (B12). White solid, yield: 60%. Melting point: 161-163 °C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 3H), 0.82(s, 3H), 0.87(s, 6H),
0.97(s, 3H), 1.18(s, 3H), 1.20(s, 3H), 1.24(s, 3H), 1.33-1.99(m, 20H), 3.56(td, J1 =
12.0 Hz, J2 = 4.0 Hz, 1H), 3.90(s, 2H), 4.56(dd, J1 = 10.4 Hz, J2 = 6.4 Hz, 1H), 6.52(s,
1H), 7.54(d, J1 = 8.0 Hz, 2H), 7.88(d, J1 = 8.0 Hz, 2H); 13C-NMR (CDCl3, 100 MHz,

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ppm): 168.63, 130.85, 127.46, 126.94, 122.93, 82.33, 76.20, 72.23, 69.08, 54.90,
53.66, 50.22, 48.79, 48.06, 38.78, 37.48, 37.01, 36.00, 35.42, 34.72, 33.78, 33.72,
31.99, 30.12, 29.45, 26.91, 26.15, 24.15, 22.58, 18.42, 17.12, 16.03, 15.38, 15.23,
15.15, 14.61; HR-MS (ESI) m/z calcd for C40H58BrN3O4S. [M+H]+ 756.3404, found
756.3430.
4.1.2.16.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(2-Nitro-phenyl)-4H-[1,2,4]triazol-3-yl]-thi
°
oacetate (B13). White solid, yield: 55%. Melting point: 165-167 C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.81(s, 3H), 0.82(s, 3H), 0.87(s, 6H),
0.97(s, 3H), 1.17(s, 3H), 1.20(s, 3H), 1.24(s, 3H), 1.36-1.94(m, 21H), 3.57(td, J1 =
12.0 Hz, J2 = 4.0 Hz, 1H), 3.95(s, 2H), 4.54(dd, J1 = 12.0 Hz, J2 = 8.0 Hz, 1H), 6.74(s,
1H), 7.53(t, J1 = 8.0 Hz, 1H), 7.62(t, J1 = 8.0 Hz, 1H), 7.75(d, J1 = 8.0 Hz, 1H),
7.94(d, J1 = 8.0 Hz, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.21, 148.99, 131.98,
131.07, 130.10, 123.84, 83.37, 77.23, 73.36, 70.21, 55.91, 54.65, 51.26, 49.80, 49.01,
39.80, 38.51, 38.00, 37.03, 36.43, 35.74, 34.93, 34.75, 32.98, 31.17, 30.46, 27.95,
27.14, 25.17, 23.57, 19.41, 18.13, 17.05, 16.41, 16.23, 16.19, 15.63; HR-MS (ESI)
m/z calcd for C40H58N4O6S. [M+H]+ 723.4150, found 723.4168.
4.1.2.17.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(3-Nitro-phenyl)-4H-[1,2,4]triazol-3-yl]-thi
°
oacetate (B14). White solid, yield: 50%. Melting point: 154-156 C. Purity: HPLC >
1
95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.80(s, 3H), 0.84(s, 3H), 0.88(s, 6H),
0.98(s, 3H), 1.18(s, 3H), 1.21(s, 3H), 1.25(s, 3H), 1.33-1.97(m, 20H), 3.62(td, J1 =
12.0 Hz, J2 = 8.0 Hz, 1H), 4.02(s, 2H), 4.55(dd, J1 = 8.0 Hz, J1,2 = 4.0 Hz, 1H), 6.94(s,
1H), 7.59(t, J1 = 8.0 Hz, 1H), 8.22(d, J1 = 8.0 Hz, 1H), 8.41(d, J1 = 8.0 Hz, 1H),
8.91(s, 1H); 13C-NMR (CDCl3, 100 MHz, ppm): 169.16, 148.58, 132.19, 131.78,
129.65, 123.97, 121.38, 83.44, 76.87, 73.46, 70.38, 55.92, 54.64, 51.29, 49.82, 48.96,
39.80, 38.51, 38.00, 37.03, 36.42, 35.72, 34.97, 34.73, 33.00, 31.18, 30.43, 29.70,
27.95, 27.17, 25.18, 23.61, 19.43, 18.13, 17.05, 16.39, 16.21, 15.62; HR-MS (ESI)

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m/z calcd for C40H58N4O6S. [M+H]+ 723.4150, found 723.4168.
4.1.2.18.
20(R)-Pananxadiol-12β-hydroxy-3β-yl-[5-(4-Methoxy-phenyl)-4H-[1,2,4]triazol-3-yl]
-thioacetate (B15). White solid, yield: 56%. Melting point: 158-160 °C. Purity: HPLC
1
> 95%. H-NMR (CDCl3, 400 MHz, ppm): δ 0.79(s, 3H), 0.82(s, 3H), 0.85(s, 3H),
0.87(s, 3H), 0.96(s, 3H), 1.17(s, 3H), 1.19(s, 3H), 1.23(s, 3H), 1.33-1.95(m, 19H),
3.56(td, J1 = 12.0 Hz, J2 = 4.0 Hz, 1H), 3.84(s, 3H, OCH3), 3.96(s, 2H), 4.52(dd, J1 =
12.0 Hz, J2 = 4.0 Hz, 1H), 6.58(s, 1H), 6.94(d, J1 = 8.0 Hz, 2H), 7.95(d, J1 = 8.0 Hz,
13
1H); C-NMR (CDCl3, 100 MHz, ppm): 169.30, 128.25, 120.31, 114.32, 107.36,
83.04, 77.61, 73.25, 70.15, 55.91, 55.43, 54.66, 51.24, 49.79, 49.05, 39.78, 38.47,
37.99, 36.99, 36.42, 35.73, 34.74, 33.01, 31.13, 30.44, 29.70, 27.93, 27.18, 25.17,
23.57, 19.44, 18.12, 17.04, 16.40, 16.23, 16.16, 15.61; HR-MS (ESI) m/z calcd for
C41H61N3O5S. [M+H]+ 708.4326, found 708.4339.
4.2. Cell culture
Five cancer cells (human lung cancer cell A549, human hepatoma cell HepG-2,
human cervical cancer cells Hela, human prostate cancer cells PC-3, human breast
cancer cell MCF-7) and a normal cell (human gastric epithelial cells GES-1) were
bought from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The
cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented
°
with 10% heat-inactivated fetal bovine serum (FBS) at 37 C in a humidified
atmosphere containing 5% CO2.
4.3. Cytotoxicity Assay
Cell viability was measured using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl-2H-tetrazo-lium bromide) assay. The cells were treated with the
compound (80, 40, 20, 10, 5 μM) for 48 h. Then, 10 μL of MTT (5 mg / mL) was
added to each well, and the cells were incubated at 37 °C for 4 hours in the dark. The
crystals were dissolved in DMSO (100 μL / well). The solution was stirred for 10

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minutes, and the absorbance was measured at 490 nm using a microplate reader to
calculate a 50% inhibitory concentration (IC50).
4.4. Morphological observation
For cell morphology analysis, HepG-2 cells were grown in 6-well plates (2 * 10 5
cells/well). After 24 h, 5 and 10 μM compound A1 and PD were incubated with the
cells for 12 h, respectively. 0.1% DMSO was used to culture control cells. The
cellular morphology was observed with a phase contrast microscopy (Olympus,
Tokyo, Japan).
4.5. DAPI staining analysis
HepG-2 cells were seeded on 6-well plate and treated with 5, 10 μM of
compound A1 or PD for 12 h, respectively. Then the cells were rinsed with PBS and
resuspended in 1 mL of 0.1% DAPI prepared in methanol for 15 min. PBS was used
to wash the cells for three times. Nuclear morphology was observed by fluorescent
microscope (Olympus BH-2, Osaka, Japan).
4.6. Apoptosis analysis
The annexin V-FITC and propidium iodide staining were carried out to explore
the apoptosis effect of compound A1. 6-well plate were used to seed HepG-2 cells (2
°
* 10 5 cells/well). Compound A1 and PD at doses of 5 and 10 μM for 12 h at 37 C
were added to the 6-well plate, respectively. PBS was used to wash the cells after they
were harvested. The 300 μL binding buffer was used to resuspend cells. 5 μL Annexin
V- FITC and 10 μL PI was added under shading conditions at room temperature after
that. Apoptosis was determined by flow cytometry.
4.7. Western blot detection
HepG-2 cells were incubated with various concentrations of compound A1 (0, 5,
10, 20 μM) and PD (0, 10, 20 μM) for 24 h. To extract the total protein, cells were
lysed in lysis bufer and phosphatase inhibitors. Proteins were separated by

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SDS-PAGE (8%, 12% gels) and then transmembrane was conducted. 5% skimmed
milk was used to block polyvinylidene fluoride membrane. Subsequently, the
membranes were incubated with anti-Cl-PARP, anti-Cl-caspase-3, anti-Cl-caspase-9,
anti-p53, anti-Bcl-2, anti-Bax, anti-Cyt c and anti- β -actin, respectively (1:1000) at 4
°C overnight. TBST was used to wash the membrane. Incubating the membranes with
goat anti-rabbit/anti-mouse secondary antibody (1:5000), X-ray film was employed to
develop the signals.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the Technology Platform of Industrialization
Chromatographic Preparation for Standard Extract of Traditional Chinese Medicine
(2010ZX09401-304-105B) and Liaoning (FGW) Engineering Technology Research
Center for industrial chromatographic preparation of natural innovative drugs
materials (2017-1007).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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693 Figure legends
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Fig 1. Chemical structure of panaxadiol (PD)
Fig 2. Morphological observation of Compound A1 and PD inducing apoptosis in
HepG-2 cells
Fig 3. Effect of compound A1 on inducing apoptosis in HepG-2 cells with DAPI