Design and synthesis of C-19 isosteviol derivatives as potent and highly selective antiproliferative agents

Article abstract of DOI:10.3390/molecules24010121

Six series of novel isosteviol derivatives; modified in the C-19 position; were synthesized; and their antiproliferative activity was evaluated against three human cancer cell lines (HCT-116; BEL-7402; HepG2) and the human L02 normal cell line in vitro. M

Full text of DOI:10.3390/molecules24010121

molecules
Article
Design and Synthesis of C-19 Isosteviol
Derivatives as Potent and Highly Selective
Antiproliferative Agents
Tian Luan 1,†, Li-Hua Cao 2,†, Hao Deng 1, Qing-Kun Shen 1, Yu-Shun Tian 1,* and
Zhe-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 133002, China;
sapphire0614@163.com (T.L.); 2017010676@ybu.edu.cn (H.D.); 2016001065@ybu.edu.cn (Q.-K.S.)
Department of Pharmacology, College of Medicine, Yanbian University, Yanji 133002, China;
lhcao@ybu.edu.cn
2
*
Correspondence: ystian@ybu.edu.cn (Y.-S.T.); zsquan@ybu.edu.cn (Z.-S.Q.); Tel.: +86-433-243-6020 (Z.-S.Q.);
Fax: + 86-433-243-6020 (Z.-S.Q.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 17 December 2018; Accepted: 24 December 2018; Published: 30 December 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Six series of novel isosteviol derivatives; modified in the C-19 position; were synthesized;
and their antiproliferative activity was evaluated against three human cancer cell lines (HCT-116;
BEL-7402; HepG2) and the human L02 normal cell line in vitro. Most of the derivatives tested
here exhibited improved antiproliferative activity with high selectivity when compared with the
parent compound isosteviol and the positive control drug 5-fluorouracil. Among these derivatives;
compound 5d exhibited the most potent antiproliferative activity and commendable selectivity
between cancer and normal cells. In addition; compound 5d inhibited the colony formation of
HCT-116 cells in a concentration-dependent manner. Further studies revealed that compound 5d
arrested the HCT-116 cell cycle in the S phase; and western blot analysis demonstrated the mechanism
may be correlated with a change in the expression of cyclin A; cyclin B1; and cyclin E1. Furthermore;
the results of a docking study that involved placing compound 5d into the CDK2/cyclin A binding
site revealed that its mode of action was possibly as a CDK2/cyclin A inhibitor.
Keywords: isosteviol; derivatives; antiproliferation; HCT-116
1. Introduction
Cancer is one of the most dangerous, fast propagating diseases of the present century. Despite the
enormous advances in cancer treatment, it remains the second most common cause of death worldwide
because of ineffective chemotherapy, caused by drug resistance and the inability of many drugs to
differentiate between cancerous cells and normal cells [1,2]. In this background, natural products
have proven the utility as the core sources of novel composition, the percentage of natural product
based drugs into the market was increasing from the past years, which makes it an attractive source of
cancer drug discovery [3]. Therefore, the discovery of potent and highly selective derivatives through
structural modification of natural products in the development of anticancer drugs is promising.
The diterpene, isosteviol (Figure 1), bearing the distinctive tetracyclic skeleton of kaurene, exhibits
wide biological activities including blood lipid control [
protection [ ], blood glucose reduction [ ] and antibacterial [10] and antiinflammation [11
effects [13 16]. It can be obtained as a metabolite of stevioside isolated from the leaves of the
natural stevia plant [17]. Furthermore, the cytotoxic activities of isosteviol derivatives have attracted
4
], anti-hypertension [5], heart-brain cell
6,7
8
,9
,12]
–
Molecules 2019, 24, 121; doi:10.3390/molecules24010121
www.mdpi.com/journal/molecules

Molecules 2019, 24, 121
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much attention in recent years. In the previous study, the C-15 and C-16 functionalized isosteviol
derivatives, obtained by means of group-conversion or structural modification exhibited good cytotoxic
activities [17
–
24]. In view of the low cytotoxicity of isosteviol, it is suitable for the development of
16]. In addition, to the best of our
highly selective anticancer drugs by chemical modification [14
–
knowledge, the antiproliferative mechanism of isosteviol derivatives such as cell cycle, cell apoptosis
and related markers have not been reported. Therefore, in the present work, we introduced different
crucial fragment in the C-19 of isosteviol in order to obtain some compounds with significantly
improved antiproliferative activity and highly selectivity.
17
12
20
13
14
11
O
16
1
2
3
9
10
8
15
4
7
5
6
18
HO
O
19
Figure 1. The chemical structure and atom number of isosteviol.
Aniline and heterocyclic fragments are common pharmacophores that display diverse biological
functions, especially antiproliferative activities. Many reports have shown that introducing aniline
and heterocyclic fragments in different natural products can improve their antiproliferative activity.
As shown in Figure 2, compound
against the HeLa and HT24 cell lines with IC50 values of 1.63
A
and
B
exhibited significant in vitro antiproliferative potency
M and 1.3 M, respectively [25 28].
µ
µ
–
Amino acids are important organic compounds that have many functions in metabolism and are
defined by their unique side chain. Due to their structural diversity, amino acids provide a balance
between hydrophilicity and hydrophobicity which is necessary for the cell membrane solubility
and permeability [29]. Our previous study investigated the structure-activity relationship of the
antiproliferative effects of celastrol analogues. Compound
introduced in the 20th carboxylic acid position, inhibited cell proliferation of AGS cells with an IC50
value of 0.44 M [23]. Similarly, a drug with a phosphonate introduced can display enhanced solubility
C which has a tryptophan methyl ester
µ
and drug-like properties by regulating the distribution coefficient. Researchers have combined a variety
of different natural products with these pharmacophores and found that their derivatives play an
important role in antiproliferative activity [30,31]. Among them, compound D exhibited significantly
improved antiproliferative activity when compared with the parent compound asiatic acid [30].
Interestingly, there are many drug discovery initiatives where triazole has been successfully conjugated
with biologically active cores, showing potent antiproliferative activity. Khaybullin et al. reported that
a series of isosteviol derived triazole D ring conjugates facilitated the development of potential
antiproliferative agents and as the result, some derivatives showed promising antiproliferative
activities against different types of cancer cell lines such as compound E [20,32].
Based on the combination principles of drugs, the aforementioned findings stimulated our interest
in designing and synthesizing six series of novel isosteviol derivatives, linking these pharmacophores
in the C-19 position. The antiproliferative activity of the target compounds was evaluated on human
colorectal cancer (HCT-116), human hepatocellular carcinoma (BEL-7402), human liver cancer (HepG2)
and human normal liver cells (L02). Furthermore, we chose the antiproliferative activity of the
derivative with the strongest antiproliferative activity and investigated its possible mechanism of
action. Finally, molecular docking analysis has also been performed to support the effective binding of
the compound at the active site of the protein.

Molecules 2019, 24, 121
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H
N
O
O
20
N
O
O
O
H
O
NH
O
O
HN
O
O
N
HO
Compound A
Compound B
Compound C (Celastrol derivative)
IC50 = 1.63 μM against HeLa cells IC50 = 1.30 μM against HT29 cells
IC50 = 0.44 μM against AGS cells
N
N
N
O
H
O
P
AcO
AcO
HN
H
OEt
H
OEt
Cl
H
H
O
AcO
O
Compound D
IC50 = 6.59 μM against T24 cells
Compound E
IC50 = 25.6 μM against HL-60 cells
O
O
Molecular
Hybridization
H
H
H
O
H
O
HO
Pharmacophores
Figure 2. Design of target compounds based on the combination principles (The black part represents
the parent compound and the red part represents the pharmacophore).
2. Results and Discussion
2.1. Chemistry
The synthetic procedure adopted to obtain the target compounds is shown in Scheme 1. The
reaction occurred at the C-19 position of isosteviol◦. Compounds 1a
–
1c are products of the reaction
3d were obtained by an
of isosteviol chloride with different amines at 30 C and compounds 3a
–
amide condensation reaction with different amino acid esters, catalyzed by EDC
·
HCl, HOBt and
◦
Et3N in anhydrous CHCl3 at 60 C. The yields of 1a
–
1c were in the range of 66–71% and those of
2e 4a 4j 5a 5e and
6e) were generated from various chlorinated derivatives via nucleophilic substitution in good
3a
6a
–
–
3d were in the range of 70–76%. All of the other isosteviol derivatives (2a
–
,
–
,
–
to excellent yields (78–90%). We chose methylenecarbonyl group as a linker between isosteviol
and these pharmacophores. While the carbonyl group of the linker can be used to introduce into
a hydrogen bond receptor, finishing with a nucleophilic substitution reaction can make full use of
starting material isosteviol and significantly increase the yield of products compared to condensation
reaction. Before biological evaluation, all target compounds were characterized via 1H-NMR, 13C-NMR
and high-resolution mass spectrometry. 1H and 13C-NMR spectra of these compounds are available in
the supplementary materials.

Molecules 2019, 24, 121
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O
a
b
c
d
e
f
H
1a-1c
H
O
R
O
H
H
O
R
2a-2e
O
O
O
H
H
3a-3d
R
N
O
O
H
H
O
H
O
H
HO
H
H
N
O
O
R
4a-4j
O
O
R
H
N
H
H
O
N
N
5a-5e
N
O
O
O
H
O
H
C2H5O
C2H5O
H
P
N
O
O
6a-6e
O
R
Compound
R
Compound
3a
R
C6H5
Compound
4e
R
Compound
5c
R
H
N
1a
1b
1c
2‐F
3‐F
4‐F
4‐OCH3
N
O
O
CH2C6H5
CH(CH3)2
H
N
3b
3c
4f
5d
5e
4‐CH3
3,4‐Cl
O
O
N
O
O
N
O
4g
N
N
2a
3d
4h
4‐NO2
6a
‐H
O
O
N
H
Scheme 1. Cont.

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N
4‐OCH
2b
2c
4a
‐H
4i
4j
6b
6c
4‐Cl
3
N
C2H5
N
2,5‐OC
H3
4b
2‐Cl
4‐OCH3
N
C6H5
N
N
2d
2e
4c
3‐Cl
4‐Cl
5a
‐H
6d
6e
4‐CH3
3,4‐Cl
CH2C6H5
N
4d
5b
4‐Cl
◦
Scheme 1. Reagents and conditions: (
a
) (i) oxalyl chloride, CHCl3, 60 C; (ii) IIa-IIe, Et3N, CH2Cl2, 30
◦
◦
C. (
EDC
b
) different heterocyclic chloroacetamides, K2CO3, CH3CN, 80 C. (
c
) different amino acid esters,
◦
◦
·
HCl, HOBt, Et3N, CHCl3, 60 C. (
d
◦
) IVa-IVj, K2CO3, CH3CN, 80 C. (
e) Va-Ve, K2CO3, CH3CN,
◦
80 C. (f) VIa-VIe, K2CO3, CH3CN, 80 C.
2.2. In Vitro Antiproliferative Activity
As shown in Table 1, thirty-two target compounds were evaluated for their antiproliferative
activities in vitro against HCT-116, BEL-7402 and HepG2 cell lines. The activity of isosteviol was used
as reference. With the exception of compounds 1c
exhibited stronger antiproliferative activity against three different human cancer cell lines than the
lead compound isosteviol at 100 M. However, some compounds lost their antiproliferative activity at
, 2b, 2c, 3d, 4b, 4c, 4e, 4i and 4j, all target compounds
µ
lower concentrations and half of the target compounds had a lower IC50 value (<100
lead compound isosteviol against the cancer cell lines.
µM) than the
Table 1. The antiproliferative activity of compounds (% Growth Inhibition at 100 µM).
% Growth Inhibition at 100 µM
Compound
HCT-116
BEL-7402
HepG2
1a
1b
1c
2a
2b
2c
2d
2e
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
65.22 ± 2.52
76.61 ± 1.78
NA
87.10 ± 2.37
88.51 ± 2.02
31.34 ± 1.22
65.04 ± 2.62
64.36 ± 1.66
40.46 ± 0.87
70.86 ± 1.05
64.87 ± 1.01
80.01 ± 0.95
61.76 ± 0.96
66.62 ± 1.90
35.78 ± 2.37
50.78 ± 2.31
NA
35.68 ± 2.56
52.92 ± 2.11
38.66 ± 1.52
54.58 ± 3.11
62.96 ± 2.13
71.38 ± 2.36
45.62 ± 1.47
31.30 ± 1.76
66.02 ± 1.33
56.96 ± 2.35
NA
62.06 ± 1.02
60.37 ± 1.69
15.71 ± 0.61
75.37 ± 1.06
85.61 ± 0.96
86.26 ± 0.59
70.75 ±0.98
54.71 ± 0.59
19.66 ± 1.11
60.44 ± 3.11
NA
62.34 ± 3.55
56.85 ± 2.35
27.87 ± 0.44
68.74 ± 2.67
65.64 ± 3.01
55.35 ± 2.55
53.14 ± 2.56
NA
40.61 ± 0.70
NA
NA
70.63 ± 0.89
50.36 ± 0.57
75.25 ± 0.56
81.15 ± 2.66
23.87 ± 0.85
NA
31.18 ± 1.19
NA
NA
31.91 ± 1.31
NA
49.12 ± 0.44
39.11 ± 1.13
76.46 ± 1.00
NA
4g
4h
4i
4j
NA

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Table 1. Cont.
% Growth Inhibition at 100 µM
Compound
HCT-116
BEL-7402
HepG2
5a
5b
5c
5d
5e
6a
6b
6c
6d
81.87 ± 2.55
85.60 ± 1.34
84.64 ± 1.55
84.61 ± 0.96
71.14 ± 0.65
50.05 ± 2.27
50.34 ± 2.31
74.07 ± 2.45
76.63 ± 2.33
43.45 ± 0.56
34.09 ± 1.89
90.02 ± 1.92
90.49 ± 1.04
75.56 ± 1.76
86.46 ± 0.87
67.96 ± 1.17
33.66 ± 0.91
50.36 ± 1.63
50.87 ± 2.68
51.46 ± 0.68
49.88 ± 1.26
28.04 ± 1.79
87.03 ± 1.06
82.10 ± 1.82
82.85 ± 2.02
82.77 ± 0.86
66.10 ± 0.97
28.02 ± 0.99
40.22 ± 2.56
41.40 ± 1.86
80.64 ± 0.76
39.47 ± 1.37
11.79 ± 0.88
6e
Isosteviol
NA: antiproliferative activity < 10%. % Growth Inhibition are presented as the mean
mean) from three separated experiment.
±
SD (standard error of the
As shown in Table 2, all of these compounds showed moderate to significant activity against all
three cancer cell lines, improved potency up to one-two digits µM compared with isosteviol. Among
the compounds 1a
higher antiproliferative activity against the HCT-116 cell line (IC50 = 24.04
with the positive-control drug 5-FU. Compounds 2a 2e are products of a reaction between isosteviol
and heterocyclic chloroacetamides. In this series of compounds, compound 2d exhibited higher
antiproliferative activity against the HCT-116 and HepG2 cell lines (IC50 value of 18.18 1.26 M and
11.05 0.55, respectively) and compound 2e exhibited higher antiproliferative activity against the
BEL-7402 and HepG2 cell lines (IC50 value of 13.22 0.32 M and 20.51 0.46, respectively) when
compared with 5-FU. Compounds 3a 3d were generated from amino acid esters. Only compound
3b, which has a D-phenylalanine methyl ester introduced in the C-19 position, displayed similar
antiproliferative activity against the three cancer cell lines (IC50 = 22.78 0.81 M against HCT-116
cells, IC50 = 36.49 0.67 M against BEL-7402 cells and IC50 = 24.40 1.75 M against HepG2 cells).
Compounds 4a 4j are products of a reaction between isosteviol and various chloroacetanilides. Among
them, compound 4f showed selective cytotoxic effects against the HCT-116 cell line, with an IC50
value of 17.22 0.43 M, while compound 4h displayed better antiproliferative activity against the
BEL-7402 and HepG2 cell lines (IC50 = 12.14 0.12 M and IC50 = 14.11 0.27 M, respectively). Thus,
compounds with an electron-withdrawing group (–F, –NO2) at the phenyl moiety displayed better
antiproliferative activity. Compounds 5a 5e have different phenyl 1,2,3-triazole chloroacetamides
–
1c, which react with amines compounds, only compound 1b exhibited slightly
±
1.20 M) compared
µ
–
±
µ
±
±
µ
±
–
±
µ
±
µ
±
µ
–
±
µ
±
µ
±
µ
–
compounds substituted. All of these compounds showed considerably higher antiproliferative activity
against the HCT-116 and HepG2 cell lines. Perhaps the triazole acts as a hydrogen bond acceptor and
binds to some key enzymes involved in cancer cell metabolism, inhibiting their expression. Compound
5a and 5b exhibited significant in vitro antiproliferative potency against the HepG2 and HCT-116 cell
lines with IC50 values of 9.87
methyl group introduced at the para position of the benzene ring, had the strongest antiproliferative
activity among all the target compounds (IC50 value of 5.38 0.26 M, 15.91 0.41 M and 8.92
0.44 M against HCT-116, BEL-7402 and HepG2 cell lines, respectively). It was 4.6-fold (against
±
0.13
µM and 7.44
±
0.37 µM, respectively. Compound 5d, which has a
±
µ
±
µ
±
µ
HCT-116 cells), 1.3-fold (against BEL-7402 cells) and 2.6-fold (against HepG2 cells) more active than
5-FU. Also, it was 18.6-fold (against HCT-116 cells), 6.3-fold (against BEL-7402 cells) and 11.2-fold
(against HepG2 cells) more active than lead compound isosteviol at least. Compounds 6a
generated by reactions with different phosphonic acid [[(chloroacetyl) amino] phenylmethyl]-diethyl
esters. Compound 6c showed higher antiproliferative activity against the HCT-116 cell line (IC50
15.15 0.11 M) and compound 6d showed better antiproliferative activity against the HCT-116 and
HepG2 cell lines (IC50 = 18.70 0.17 and 13.19 0.86 M, respectively). However, compounds 6a 6b
–6e, were
=
±
µ
±
±
µ
,

Molecules 2019, 24, 121
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and 6e lost their antiproliferative activity. It seems that the antiproliferative ability was enhanced after
the introduction of electron-donating groups to the para position of the benzene ring.
It is worth mentioning that all the target compounds exhibited low cytotoxic activities against
human normal liver cells (L02), with remarkable selectivity (IC50 > 100 µM), which supported the
rationale of the drug design. Above all, we chose to investigate the mechanism of action for compound
5d because it had the strongest antiproliferative activity against HCT-116 cell line.
Table 2. IC50 values (µM) of several active compounds.
IC50 Values (µM) a
Compound
HCT-116
BEL-7402
HepG2
L02
1b
2b
2d
2e
3a
3b
4a
4f
4h
5a
5b
5c
5d
5e
6c
6d
24.04 ± 1.20
30.71 ± 1.98
18.18 ± 1.26
>100
40.41 ± 0.97
22.78 ± 0.81
56.56 ± 1.56
17.22 ± 0.43
>100
14.63 ± 0.51
7.44 ± 0.37
14.40 ± 1.31
5.38 ± 0.26
14.32 ± 0.15
15.15 ± 0.11
18.70 ± 0.17
>100
>100
>100
52.98 ± 1.97
13.22 ± 0.32
>100
>100
>100
11.05 ± 0.55
20.51 ± 0.46
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
36.49 ± 0.67
24.40 ±1.75
>100
>100
>100
>100
12.14 ± 0.12
27.10 ± 0.87
22.50 ± 0.58
>100
14.11 ± 0.27
9.87 ± 0.13
11.67 ± 0.23
12.27 ± 0.77
8.92 ± 0.44
12.82 ± 0.09
>100
15.91 ± 0.41
>100
92.61 ± 1.96
60.93 ± 1.37
>100
21.30 ± 2.43
13.19 ± 0.86
Isosteviol
5-FU
>100
24.80 ± 2.08
23.45 ± 0.37
19.12
±
1.01
a
IC50: concentration that inhibits 50% of cell growth. The values are presented as the mean
the mean) from three separated experiments.
±
SD (standard error of
2.3. Selective Inhibition of Cancer Cell Growth by Compounds 5d
Lack of selective cytotoxicity is the main factor that restricts the conventional chemotherapeutic
agents [29 33]. Thus, to evaluate the selective antiproliferative activity of the compound 5d, the
,
selectivity index (SI) between cancer and normal cells was calculated and the results are summarized
in Table 3. The SI was calculated by the dividing the IC50 values in normal cells by the IC50 values in
cancer cells. 5-FU had similar effects and toxic doses, with SI values of 0.77 (HCT-116), 0.90 (BEL-7402)
and 0.82 (HepG2), respectively, indicating that both normal cells and cancer cells would be killed.
Whereas, compound 5d exhibited a 38.17-fold, 11.24-fold and 22.01-fold higher selectivity for HCT-116,
BEL-7402 and HepG2 cells, respectively. It means that 5d has much better therapeutic activity and
specificity than 5-FU.
Table 3. In vitro antiproliferative activities of compound 5d against normal cell line (L02).
(IC50, µM)
Selectivity Index a
Compound
L02
HCT-116
BEL-7402
HepG2
5d
5-FU
a
161.04 ± 1.96
29.93
0.77
10.12
0.90
18.05
0.82
19.12 ± 1.01
SI: selective index (IC50 on normal cells/IC50 on cancer cells).

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2.4. Compound 5d Inhibited HCT-116 Cells Colony Formation
Colony formation represents the malignant potential, which is a basic characteristic of cancer cells.
Colony formation assay is used to test cells for their ability to form colonies and undergo unlimited
division [34]. The combined results of three independent experiments are depicted in Figure 3. The
exposure of HCT-116 cells to compound 5d resulted in a significant suppression of colony formation in
a concentration-dependent manner. It led to fewer and smaller colonies compared to the control with
an inhibition rate of 84% at 15 µM and colony formation was almost completely suppressed. These
results demonstrate that compound 5d can significantly inhibit the proliferation of HCT-116 cells.
Figure 3. The colony formation of HCT-116 cells after treatment with different concentration (5, 10 and
15 µM) of compound 5d for 7 days; The rate of inhibition induced by compound 5d was expressed as
the mean
±
SD of three independent experiments, *** p < 0.001 vs control group (untreated cells); The
clonogenicity assay was quantified using Image J software.
2.5. Analysis for Cell Cycle and Apoptosis by Flow Cytometry
Cell cycle dysregulation resulting in mitogenic signaling and leading to uncontrolled proliferation
is one of the hallmarks of cancer. Also, inducing apoptosis is one of the protective mechanisms used
against cancer initiation and progression, which is a significant goal of anticancer therapy. Many
cytotoxic compounds exert their antiproliferative effect by the arrest of the cell cycle at a particular
cell cycle checkpoint, the induction of apoptosis or by a combination of these two effects. This type of
mechanism is considered to be an effective anticancer strategy [35,36]. Therefore, in order to identify
whether compound 5d inhibited cell proliferation through cell cycle arrest or apoptosis, we assayed
its ability to trigger cell cycle arrest and apoptosis in HCT-116 cells by flow cytometry. As shown in
Figure 4A), the proportion of cells in the G1 phase decreased from 70.67% in the control to 51.67% and
41.32% when treated with 5 and 15
phase dramatically increased in a concentration-dependent manner, from 18.64% (control) to 25.70% (5
M) and 42.31% (15 M), this implies that the cell is unable to duplicate its DNA. The results indicate
that compound 5d arrests cells in the S phase of the cell cycle.
µM compound 5d, respectively and the proportion of cells in the S
µ
µ

Molecules 2019, 24, 121
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To determine whether antiproliferation caused by compound 5d was due to increasing of
apoptosis, annexin V-FITC/propidium iodide assay was used to investigate apoptosis. As shown
in Figure 4B), at 5
inconsiderably, from 5.98% (control) to 7.67%. After increasing the concentration of the drug to 15
the percentage of total apoptotic cells increased to 24.94%. However, we have found that the number
of dead cells (UL) also increased from 8.84% (control) to 49.22% (15 M). It is likely that compound 5d
µM, the percentage of total apoptotic cells (right quadrants, UR + LR) increased
µM,
µ
causes cell death by inducing apoptosis in combination with other ways. These results indicate that
compound 5d have a weaker influence on cell apoptosis.
Figure 4.
compound 5d (5 and 15
HCT-116 cells after treatment of compound 5d (5 and 15
(
A
): Flow cytometry analyses of cell cycle distribution of HCT-116 cells after treatment of
M) and no treatment (Ctrl) as a reference for 24 h; ( ): Apoptosis induction in
M) and no treatment (Ctrl) as a reference for
µ
B
µ
24 h.

Molecules 2019, 24, 121
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2.6. Western Blot Analysis
It is known that cell proliferation is generally regulated by controlling cell cycle progression
through inhibition or promotion of activities of cyclins and their associated proteins. Cyclins play a
significant role in cell cycle progression by activation of ATP binding site [37]. Cyclin E helps in the
Rb/E2F transcription via formation of a complex with the CDK2 and controls G1 phase [38]. Cyclin
A forms a complex with CDK2 and helps in the regulation of S phase [37]. Cyclin B is also called as
maturation or mitosis promoting factor as it forms complex with CDK1 and controls the M phase of the
cell cycle [39]. Analysis for cell cycle by flow cytometry confirmed that compound 5d induced arrest
in S phase of the HCT-116 cells. However, it had a weaker influence on cell apoptosis. Therefore, the
further research on apoptosis-related proteins was abandoned. Since entry into the S phase in the cell
cycle requires the accumulation of cell cycle activation-related cyclins, we further examined the effect
of compound 5d on cell cycle regulating proteins such as cyclin A, cyclin B1 and cyclin E1 to determine
whether the decreased cell proliferation by 5d involved these proteins. As shown in Figure 5, in a
concentration-dependent manner, compound 5d decreased cyclin A and cyclin E1 protein levels, while
increasing the level of cyclin B1. It has been reported that expression of cyclin B1 is increased by
exposure to anticancer agents [40,41]. Hence, these results suggest that the up-regulation of cyclin B1
and down-regulation of cyclins A and E1 are related to the decrease in cell proliferation and S phase
arrest by 5d treatment on HCT-116 cells.
Figure 5. Western blot analysis: Effects of Cyclin A, Cyclin E1 and Cyclin B1 in HCT-116 cells after 24 h
treatment with compound 5d at 5, 10 and 15 µM. β-actin served as an internal control.
2.7. Molecular Docking Analysis
Our cell cycle study revealed compound 5d induced arrest in S phase of the HCT-116 cells and
western blot analysis demonstrated the mechanism of action may be correlated with down-regulation
of cyclin A, in addition, cyclin A and CDK2 form a complex that plays a key role in the regulation
of the S phase. Therefore, in order to better understand the potency of compound 5d and to gain
further understanding of the probable binding model, molecular docking studies with CDK2/cyclin A
(PDB ID: 3my5) were performed. The studies were performed as a crucial step towards understanding
the specific position of interaction between compound 5d and CDK2/cyclin A complex [42]. The
docking results revealed that compound 5d was held in the active pocket by a combination of various
hydrogen bonds and alkyl interactions with CDK2/cyclin A (Figure 6). The carbonyl group of
isosteviol interacted with the -NH2 group of Lys-A56 via hydrogen bond, while the carbonyl group of
1,2,3-triazole chloroacetamides interacted with the -NH- moiety of Lys-A56 via hydrogen bond, which
supported the preceding drug design. Meanwhile, the 1,2,3-triazole moiety formed one pi-donor
hydrogen bond with Gly-A1 residue, as well as the methylenecarbonyl group and the carbonyl
group of C-19 formed three carbon hydrogen bond with Ile-C70, Val-C69 and Lys-B300 amino acids.
Additionally, the methyl group at the para position of the benzene ring formed a favorable alkyl
interaction with Val-D301 residue. It was also observed that the B and C ring of isosteviol showed alkyl

Molecules 2019, 24, 121
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interaction with amino acid residue Ala-B303. The CDOCKER interaction energy score was 41.3422.
These indicate that compound 5d has a strong binding affinity for the CDK2/cyclin A complex and
may play a crucial role in inhibiting its activity.
Figure 6. Computer modelling of compound 5d binding to CDK2/cyclin A (3my5). Compound 5d
was colored in yellow.
3. Materials and Methods
3.1. General Procedures
Melting points were determined in open capillary tubes and were uncorrected. Reactions were
1
monitored by thin-layer chromatography (TLC) on silica gel plates. H-NMR and 13C-NMR spectra
were measured on an AV-300 (Bruker BioSpin, Fällanden, Switzerland) and AV-500 (Bruker BioSpin,
Fällanden, Switzerland) and all chemical shifts were given in ppm relative to tetramethylsilane (TMS).
High-resolution mass spectra were measured using a matrix-assisted laser desorption/ionization
(MALDI)-time of flight (TOF)/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). The
major chemicals were purchased from Aldrich Chemical Corporation (Milwaukee, WI, USA). All other
chemicals were of analytical grade.
3.2. General Procedure for the Synthesis of Compound (1a–1c)
Isosteviol (63.6 mg, 0.20 mmol) was placed in CHCl3 (10 mL), oxalyl chloride (38.1 mg, 0.30 mmol)
◦
was added and the mixture was stirred at 60 C for 2 h. Upon completion, the solvent was evaporated
in vacuo to obtain the crude product, a colorless crystal, which was used in the next step without
further purification. Next, the crude product and different amines (0.21 mmol) and Et3N (22.2 mg,

Molecules 2019, 24, 121
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◦
0.22 mmol) were added to CH2Cl2 (10 mL) and the resulting mixture was stirred at 30 C for 2 h.
Then, the solvent was evaporated in vacuo, water was added (10 mL), the mixture was filtered and the
residue was washed with water to obtain the target compounds (1a–1c) as white powders.
(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxo-N-(2-(pyrrolidin-1-yl)ethyl)tetradecahydro-6a,9-
◦
methanocyclohepta[a]naphthalene-4-carboxamide (1a). White powder; yield, 71%; m.p. 136–137 C; 1H-NMR
(CDCl3, 300 MHz, ppm):
δ 6.34 (brs, 1H, -CO-NH-), 3.31 (q, J = 6 Hz, 2H, -CO-NH-CH2-), 2.70–2.51 (m,
7H, N(CH2)3, 15-He), 2.10 (d, J = 12 Hz, 1H, 3-He), 2.00–1.95 (m, 1H), 1.89–1.86 (m, 1H), 1.83–1.75 (m,
8H), 1.71–1.64 (m, 2H), 1.60–1.59 (m, 1H), 1.55 (s, 1H), 1.50–1.49 (m, 1H), 1.45–1.44 (m, 1H), 1.41–1.38
(m, 1H), 1.35–1.23 (m, 2H), 1.19 (s, 3H, 18-CH3), 1.16–1.07 (m, 2H), 0.99 (s, 3H, 17-CH3), 0.95–0.89 (m,
1H), 0.79 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.40, 176.70, 57.49, 54.75, 54.26, 53.78,
53.61(2C), 48.71, 48.38, 43.61, 41.80, 40.26, 39.50, 38.11(2C), 37.92, 37.31, 30.21, 23.63(2C), 22.01, 20.33,
19.86, 19.04, 13.41. HRMS (m/z): calcd for C26H43N2O2+ [M + H]+: 415.3319, found: 415.3316.
(4R,4aS,6aR,9S,11aR,11bS)-N-(2-(dimethylamino)ethyl)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-
◦
methanocyclohepta[a]naphthalene-4-carboxamide (1b). White powder; yield, 66%; m.p. 102–103 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 6.30 (brs, 1H, -CO-NH-), 2.29 (q, J = 6 Hz, 2H, -CO-NH-CH2-),
2.67 (dd, J = 18, 3 Hz, 1H, 15-He), 2.41 (t, J = 6 Hz, 2H, -CO-NH-CH2-), 2.29–2.19 (m, 6H, N(CH2)3),
2.09 (d, J = 12 Hz, 1H, 3-He), 2.02–1.94 (m, 1H, 6-He), 1.88–1.78 (m, 3H), 1.76–1.68 (m, 3H), 1.64–1.59
(m, 2H), 1.55–1.48 (m, 2H), 1.45–1.38 (m, 3H), 1.36–1.31 (m, 1H), 1.27 (s, 3H, 18-CH3), 1.16–1.09 (m,
2H), 0.99 (s, 3H, 17-CH3), 0.95–0.90 (m, 1H), 0.79 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ
222.43, 176.71, 57.55(2C), 54.75, 54.28, 48.71, 48.37, 45.09(2C), 43.62, 41.79, 40.25, 39.51, 38.09(2C), 37.32,
+
36.73, 30.18, 22.04, 20.34, 19.86, 19.11, 13.44. HRMS (m/z): calcd for C24H41N2O2 [M + H]+: 389.3163,
found: 389.3166.
(4R,4aS,6aR,9S,11aR,11bS)-N-methoxy-N,4,9,11b-tetramethyl-8-oxotetradecahydro-6a,9-methanocyclohepta
◦
1
[a]naphthalene-4-carboxamide (1c). White powder; yield, 68%; m.p. 144–145 C; H-NMR (CDCl3,
300 MHz, ppm): 3.66 (s, 3H, -O-CH3), 3.12 (s, 3H, -N-CH3), 2.73 (dd, J = 18, 3 Hz, 1H, 15-He), 2.53 (d,
δ
J = 12 Hz, 1H, 3-He), 2.16–2.02 (m, 1H, 6-He), 1.90–1.78 (m, 2H), 1.74–1.58 (m, 6H), 1.54–1.34 (m, 5H),
1.28 (s, 3H, 18-CH3), 1.22–1.17 (m, 1H), 1.13–1.04 (m, 2H), 0.99 (s, 3H, 17-CH3), 0.96–0.90 (m, 1H), 0.83
(s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.81, 178.30, 60.94, 60.46, 55.71, 54.43, 48.72,
48.51, 46.21, 42.36, 40.91, 39.66, 38.55, 38.44, 37.40, 34.38, 27.22, 22.31, 20.43, 20.13, 19.90, 15.48. HRMS
(m/z): calcd for C22H36NO3+ [M + H]+: 362.2690, found: 362.2987.
3.3. General Procedure for the Synthesis of Compound (2a–2e)
A mixture of isosteviol (63.6 mg, 0.20 mmol), K2CO3 (41.5 mg, 0.30 mmol) and chlorinated
derivatives (0.21 mmol) in CH3CN (10 mL) was stirred at 80 ◦C for 4 h. After confirming the reaction
progress by thin-layer chromatography, the solvent was evaporated in vacuo, the mixture was dissolved
with 15 mL ethyl acetate and then washed with saline (5 mL
silica gel column chromatography and eluted with petroleum ether:ethyl acetate (5:1) to obtain the
×
3). The mixture was then purified using
target compound 2a–2e.
2-(4-methylpiperazin-1-yl)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (2a). White powder; yield, 87%; m.p. 112–113 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 4.82 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.63 (d, J = 15 Hz, 1H,
-O-CHa-CO-), 3.65 (s, 2H, -CO-N-CH2-), 3.45 (s, 2H, -CO-N-CH2-), 2.65 (dd, J = 12, 3 Hz, 1H, 15-He),
2.45–2.26 (m, 8H, 3-He, -(CH2)2-N-CH3), 1.97–1.84 (m, 3H), 1.78–1.65 (m, 4H), 1.60–1.55 (m, 2H),
1.50–1.40 (m, 4H), 1.32 (s, 3H, 18-CH3), 1.28–1.21 (m, 3H), 1.16–1.07 (m, 1H), 0.99 (s, 3H, 17-CH3),
0.95–0.88 (m, 1H), 0.75 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 125 MHz, ppm):
δ 222.50, 176.89, 165.02,
60.69, 57.16, 54.77, 54.70, 54.38, 54.33, 50.90, 48.73, 48.51, 45.85, 44.24, 44.08, 41.54, 39.82, 39.48, 38.11,
+
37.98, 37.33, 29.06, 21.65, 20.34, 19.87, 18.99, 13.57. HRMS (m/z): calcd for C27H43N2O4 [M + H]+:
459.3217, found: 459.3221.

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2-(4-ethylpiperazin-1-yl)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
9-methanocyclohepta[a]naphthalene-4-carboxylate (2b).White crystal; yield, 84%; m.p. 115–117 ◦C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 4.81 (d, J = 9 Hz, 1H, -O-CHe-CO-), 4.62 (d, J = 9 Hz, 1H,
-O-CHa-CO-), 3.64 (s, 2H, -CO-N-CH2-), 3.42 (s, 2H, -CO-N-CH2-), 2.63 (d, J = 12 Hz, 1H, 15-He),
2.45–2.43 (m, 6H, -(CH2)2-N-CH2-), 2.27 (d, J = 9 Hz, 1H, 3-He), 1.95–1.92 (m, 1H), 1.87–1.78 (m, 3H),
1.73–1.65 (m, 3H), 1.61 (d, J = 9 Hz, 1H), 1.55 (d, J = 6 Hz, 1H), 1.51–1.34 (m, 4H), 1.31 (s, 3H, 18-CH3),
1.26–1.15 (m, 4H), 1.11–1.06 (m, 3H, -NCH2C
H
3), 0.97 (s, 3H, 17-CH3), 0.94–0.89 (m, 1H), 0.74 (s, 3H,
20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ
222.57, 176.85, 164.93, 60.72, 57.13, 54.74, 54.30, 52.62,
52.20, 52.13, 48.70, 48.48, 44.40, 44.05, 41.72, 41.51, 39.80, 39.46, 38.08, 37.96, 37.31, 29.04, 21.63, 20.32,
19.85, 18.97, 13.55, 11.77. HRMS (m/z): calcd for C28H45N2O4+ [M + H]+: 473.3374, found: 473.3379.
2-oxo-2-(4-phenylpiperazin-1-yl)ethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (2c). White crystal; yield, 81%; m.p. 143–144 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 7.34–7.30 (m, 2H, Ar-H), 6.96–6.92 (m, 3H, Ar-H), 4.88 (d,
J = 15 Hz, 1H, -O-CHe-CO-), 4.70 (d, J = 15 Hz, 1H, -O-CHa-CO-), 3.80 (s, 2H, -CO-N-CH2-), 3.59 (s,
2H, -CO-N-CH2-), 3.21 (s, 4H, ph-N-(CH2)2-), 2.67 (dd, J = 18, 3 Hz, 1H, 15-He), 2.30 (d, J = 12 Hz, 1H,
3-He), 1.99–1.94 (m, 1H), 1.90–1.77 (m, 3H), 1.74–1.66 (m, 3H), 1.56–1.50 (m, 2H), 1.46–1.39 (m, 2H),
1.34 (s, 3H, 18-CH3), 1.27 (s, 2H), 1.23–1.04 (m, 4H), 1.00 (s, 3H, 17-CH3), 0.95–0.89 (m, 1H), 0.77 (s,
3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.44, 176.85, 165.09, 150.82, 129.28(2C), 120.77,
116.80(2C), 60.73, 57.16, 54.77, 54.32, 49.66, 49.39, 48.71, 48.50, 44.56, 44.08, 41.80, 41.53, 39.81, 39.47,
38.11, 37.99, 37.32, 29.07, 21.65, 20.34, 19.87, 18.99, 13.58. HRMS (m/z): calcd for C32H45N2O4+ [M +
H]+: 521.3374, found: 521.3370.
2-(4-benzylpiperazin-1-yl)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (2d). White powder; yield, 83%; m.p. 149–150 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 7.38–7.31 (m, 5H, Ar-H), 4.82 (d, J = 15 Hz, 1H, -O-CHe-CO-),
4.63 (d, J = 15 Hz, 1H, -O-CHa-CO-), 3.64 (s, 2H, -CO-N-CH2-), 3.54 (s, 2H, -N-CH2-ph), 3.41 (s, 2H,
-CO-N-CH2-), 2.66 (dd, J = 18, 3 Hz, 1H, 15-He), 2.46 (s, 4H, -N-(CH2)2-), 2.29 (d, J = 12 Hz, 1H, 3-He),
1.97–1.92 (m, 1H), 1.85 (s, 1H), 1.78–1.70 (m, 4H), 1.66–1.59 (m, 3H), 1.55 (s, 1H), 1.50–1.40 (m, 3H), 1.27
(s, 3H, 18-CH3), 1.22–1.16 (m, 3H), 1.13–1.07 (m, 1H), 0.99 (s, 3H, 17-CH3), 0.94–0.85 (m, 1H), 0.76 (s,
3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.57, 176.85, 164.94, 137.47, 129.10(2C), 128.37(2C),
127.35, 62.84, 60.74, 57.15, 54.76, 54.32, 52.84, 52.53, 48.72, 48.49, 44.53, 44.06, 41.90, 41.53, 39.81, 39.47,
38.09, 37.97, 37.32, 29.06, 21.63, 20.33, 19.86, 18.99, 13.56. HRMS (m/z): calcd for C33H47N2O4+ [M +
H]+: 535.3530, found: 535.3525.
2-(azepan-1-yl)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,◦9-
methanocyclohepta[a]naphthalene-4-carboxylate (2e). White powder; yield, 83%; m.p. 110–112 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 4.84 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.62 (d, J = 15 Hz, 1H,
-O-CHa-CO-), 3.55 (t, J = 6 Hz, 2H, -CO-N-CH2-), 3.39 (t, J = 6 Hz, 2H, -CO-N-CH2-), 2.67 (dd, J = 18,
3 Hz, 1H, 15-He), 2.31 (d, J = 12 Hz, 1H, 3-He), 1.99–1.82 (m, 3H), 1.79–1.71 (m, 7H), 1.66–1.59 (m, 7H),
1.55 (s, 1H), 1.50–1.38 (m, 4H), 1.34 (s, 3H, 18-CH3), 1.12–1.07 (m, 1H,), 0.99 (s, 3H, 17-CH3), 0.94–0.84
(m, 3H), 0.76 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.60, 176.92, 166.05, 60.69, 57.18,
54.76, 54.32, 48.71, 48.50, 46.46, 45.99, 44.02, 41.55, 39.85, 39.47, 38.10, 37.98, 37.33, 29.08, 28.93, 27.47,
27.22, 26.89, 21.64, 20.32, 19.87, 19.01, 13.56. HRMS (m/z): calcd for C28H44NO4+ [M + H]+: 458.3265,
found: 458.3261.
3.4. General Procedure for the Synthesis of Compound (3a–3d)
A mixture of isosteviol (63.6 mg, 0.20 mmol), EDC
0.22 mmol), Et3N (102
·
HCl (42.2 mg, 0.22 mmol), HOBt (29.7 mg,
L, 0.30 mmol) and various amino acid esters (0.30 mmol) in CHCl3 (10 mL)
µ
was stirred at 60 ◦C for 8 h. After confirming the reaction progress by thin-layer chromatography, the
solvent was evaporated in vacuo, the mixture was dissolved with 15 mL ethyl acetate and then washed

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with saline (5 mL
×
3). The mixture was then purified using silica gel column chromatography and
eluted with a gradient of petroleum ether:ethyl acetate (10:1–5:1) to obtain the target compound 3a
Methyl(S)-2-phenyl-2-((4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-
methanocyclohepta[a]naphthalene-4-carboxamido)acetate (3a). White crystal; yield, 70%; m.p. 143–145 ◦C;
1H-NMR (CDCl3, 300 MHz, ppm):
7.40–7.33 (m, 5H, Ar-H), 6.67 (d, J = 9 Hz, 1H, -CO-NH-), 5.56 (d,
J = 9 Hz, 1H, -NH-C -ph), 3.75 (s, 3H, -OCH3), 2.59 (dd, J = 18, 3 Hz, 1H, 15-He), 2.13 (d, J = 15 Hz, 1H,
–3d.
δ
H
3-He), 1.99–1.85 (m, 2H), 1.81–1.66 (m, 5H), 1.61 (s, 4H), 1.54–1.51 (m, 1H), 1.48–1.39 (m, 2H), 1.24–1.20
(m, 5H), 1.15–1.13 (m, 1H), 0.99 (s, 3H, 17-CH3), 0.94–0.87 (m, 1H), 0.65 (s, 3H, 20-CH3). 13C-NMR
(CDCl3, 75 MHz, ppm):
δ 222.40, 175.88, 171.76, 136.77, 128.94(2C), 128.47, 127.30(2C), 57.49, 56.36,
54.73, 54.25, 52.77, 48.70, 48.32, 43.70, 41.67, 40.14, 39.47, 38.10, 38.08, 37.28, 29.93, 22.11, 20.30, 19.85,
19.04, 13.40. HRMS (m/z): calcd for C29H40NO4+ [M + H]+: 466.2952, found: 466.2947.
Methyl((4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]
naphthalene-4-carbonyl)-D-phenylalaninate (3b). White crystal; yield, 76%; m.p. 147–148 ◦C; 1H-NMR
(CDCl3, 300 MHz, ppm):
-CO-NH-), 4.84 (dd, J = 15, 9 Hz, 1H, -NH-C
δ
7.33–7.25 (m, 3H, Ar-H), 7.15–7.13 (m, 2H, Ar-H), 5.97 (d, J = 6 Hz, 1H,
-ph), 3.75 (s, 3H, -OCH3), 3.19 (dd, J = 15, 6 Hz, 1H,
H
ph-CHe-), 3.06 (dd, J = 15, 6 Hz, 1H, ph-CHa-), 2.63 (dd, J = 18, 3 Hz, 1H, 15-He), 2.05 (d, J = 15 Hz, 1H,
3-He), 1.82 (s, 2H), 1.76–1.73 (m, 2H), 1.68–1.64 (m, 5H), 1.59–1.56 (m, 2H), 1.53–1.48 (m, 1H), 1.45–1.33
(m, 4H), 1.27 (s, 3H, 18-CH3), 0.98 (s, 3H, 17-CH3), 0.92–0.81 (m, 2H), 0.61 (s, 3H, 20-CH3). 13C-NMR
(CDCl3, 75MHz, ppm):
δ 222.44, 176.48, 172.43, 136.09, 129.14(2C), 128.65(2C), 127.17, 57.33, 54.71,
54.20, 53.00, 52.29, 48.70, 48.34, 43.74, 41.66, 40.16, 39.49, 38.17, 38.08, 37.82, 37.29, 29.84, 21.88, 20.32,
19.84, 18.89, 13.38. HRMS (m/z): calcd for C30H42NO4+ [M + H]+: 480.3108, found: 480.3100.
Methyl((4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]
naphthalene-4-carbonyl)-L-valinate (3c). White powder; yield, 71%; m.p. 96–98 ◦C; 1H-NMR (CDCl3,
300 MHz, ppm):
δ 5.99 (d, J = 6 Hz, 1H, -CO-NH-), 4.68–4.61 (m, 1H, -NH-CH-), 3.74 (s, 3H, -OCH3),
2.66 (dd, J = 18, 3 Hz, 1H, 15-He), 2.10–1.96 (m, 2H), 1.89–1.84 (m, 2H), 1.80–1.75 (m, 2H), 1.72–1.58 (m,
7H), 1.55–1.48 (m, 3H), 1.45–1.38 (m, 2H), 1.27 (s, 2H), 1.25–1.20 (m, 5H), 1.18–1.14 (m, 1H), 0.99 (s, 3H,
17-CH3), 0.97-0.92 (m, 5H), 0.77 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.39, 176.41,
173.84, 57.54, 54.68, 54.25, 52.16, 50.36, 48.69, 48.34, 43.76, 41.70, 41.61, 40.17, 39.48, 38.08(2C), 37.28,
+
30.11, 25.06, 22.85, 22.17, 21.91, 20.31, 19.84, 19.00, 13.53. HRMS (m/z): calcd for C27H44NO4 [M +
H]+: 446.3265, found: 446.3260.
Methyl((4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]
◦
1
naphthalene-4-carbonyl)-L-tryptophanate (3d). White crystal; yield, 74%; m.p. 165–166 C; H-NMR
(CDCl3, 300 MHz, ppm): 8.36 (s, 1H, -NH-), 7.59 (d, J = 6 Hz, 1H, Ar-H), 7.40 (d, J = 9 Hz, 1H, Ar-H),
7.26–7.12 (m, 2H, Ar-H), 7.05 (s, 1H, -NH-C =), 6.03 (d, J = 6 Hz, 1H, -CO-NH-), 4.85 (q, J = 6 Hz, 1H,
-NH-C -), 3.75 (s, 3H, -OCH3), 3.38–3.20 (m, 2H, Ar-CH2-), 2.40 (dd, J = 18, 3 Hz, 1H, 15-He), 1.93
δ
H
H
(d, J = 15 Hz, 1H, 3-He), 1.75–1.67 (m, 5H), 1.63–1.56 (m, 3H), 1.51–1.43 (m, 2H), 1.37–1.31 (m, 5H),
1.28 (s, 3H, 18-CH3), 1.10–1.08 (m, 1H), 0.97 (s, 3H, 17-CH3), 0.92–0.77 (m, 2H), 0.52 (s, 3H, 20-CH3).
13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.60, 176.60, 172.94, 136.12, 127.60, 122.48(2C), 119.84, 118.50,
111.43, 110.41, 57.35, 54.62, 54.18, 53.15, 52.29, 48.69, 48.26, 43.53, 41.61, 40.07, 39.32, 37.96, 37.92, 37.24,
+
29.85, 27.23, 21.80, 20.24, 19.83, 18.84, 13.08. HRMS (m/z): calcd for C32H43N2O4 [M + H]+: 519.3217,
found: 519.3221.
3.5. General Procedure for the Synthesis of Compound (4a–4j)
A mixture of isosteviol (63.6 mg, 0.20 mmol), K2CO3 (41.5 m◦g, 0.30 mmol) and different
chloroacetanilides (0.21 mmol) in CH3CN (10 mL) was stirred at 80 C for 2 h. After confirming
the reaction progress by thin-layer chromatography, the solvent was evaporated in vacuo, the mixture
was dissolved with 15 mL ethyl acetate and then washed with saline (5 mL
×
3). The mixture was then

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purified using silica gel column chromatography and eluted with a gradient of petroleum ether:ethyl
acetate (4:1–5:1) to obtain the target compound 4a–4j.
2-oxo-2-(phenylamino)ethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,9-
◦
methanocyclohepta[a]naphthalene-4-carboxylate (4a). White powder; yield, 86%; m.p. 154–156 C;
1H-NMR (CDCl3, 300 MHz, ppm):
δ 7.92 (s, 1H, -CO-NH-), 7.51 (d, J = 6 Hz, 2H, Ar-H), 7.35 (t,
J = 6 Hz, 2H, Ar-H), 7.15 (t, J = 6 Hz, 1H, Ar-H), 4.75 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.58 (d, J = 15 Hz,
1H, -O-CHa-CO-), 2.62 (dd, J = 18, 3 Hz, 1H, 15-He), 2.28 (d, J = 12 Hz, 1H, 3-He), 2.02 (d, J = 15 Hz,
1H, 6-He), 1.84 (s, 1H), 1.79–1.70 (m, 5H), 1.64–1.54 (m, 4H), 1.51–1.40 (m, 3H), 1.34 (s, 3H, 18-CH3),
1.23–1.20 (m, 3H), 1.17–1.11 (m, 1H), 0.99 (s, 3H, 17-CH3), 0.74 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75
MHz, ppm):
δ 221.95, 175.76, 165.03, 136.83, 129.22(2C), 124.94, 119.74(2C), 63.36, 57.00, 54.71, 54.19,
48.68, 48.39, 44.03, 41.39, 39.61, 39.43, 38.06, 37.99, 37.20, 28.99, 22.01, 20.34, 19.83, 18.93, 13.50. HRMS
(m/z): calcd for C28H38NO4+ [M + H]+: 452.2795, found: 452.2790.
2-((2-chlorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-
6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (4b). White powder; yield, 83%; m.p. 107–108 ◦C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 8.46–8.43 (m, 1H, -CO-NH-), 7.39 (d, J = 10 Hz, 1H, Ar-H),
7.33–7.29 (m, 2H, Ar-H), 7.09 (t, J = 10 Hz, 1H, Ar-H), 4.79 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.62 (d,
J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (dd, J = 20, 5 Hz, 1H, 15-He), 2.31 (d, J = 15 Hz, 1H, 3-He), 2.02
(d, J = 15 Hz, 1H, 6-He), 1.91–1.85 (m, 1H), 1.82–1.68 (m, 4H), 1.63–1.60 (m, 3H), 1.55–1.49 (m, 4H),
1.44–1.40 (m,1H), 1.35 (s, 3H, 18-CH3), 1.27–1.22 (m, 3H), 1.20–1.13 (m, 1H), 0.98 (s, 3H, 17-CH3), 0.73 (s,
3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.03, 175.62, 165.27, 133.74, 129.12, 127.95, 125.25,
122.65, 121.67, 63.41, 57.10, 54.73, 54.23, 48.67, 48.39, 44.06, 41.31, 39.59, 39.40, 38.02, 37.98, 37.22, 29.01,
+
21.97, 20.33, 19.83, 19.08, 13.43. HRMS (m/z): calcd for C28H37ClNO4 [M + H]+: 486.2406, found:
486.2401.
2-((3-chlorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (4c). White powder; yield, 80%; m.p. 120–122 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 7.80 (s, 1H, -CO-NH-), 7.60 (s, 1H, Ar-H), 7.36 (d, J = 5 Hz, 1H,
Ar-H), 7.31–7.28 (m, 1H, Ar-H), 7.14 (t, J = 5 Hz, 1H, Ar-H), 4.74 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.57
(d, J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (dd, J = 20, 5 Hz, 1H, 15-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.00
(d, J = 10 Hz, 1H, 6-He), 1.87–1.71 (m, 5H), 1.63–1.53 (m, 5H), 1.45–1.38 (m, 2H), 1.33 (s, 3H, 18-CH3),
1.26–1.21 (m, 3H), 1.20–1.13 (m, 1H), 0.98 (s, 3H, 17-CH3), 0.95–0.84 (m, 1H), 0.73 (s, 3H, 20-CH3).
13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.02, 175.84, 165.17, 137.98, 134.86, 130.21, 124.98, 119.83, 117.67,
63.28, 56.99, 54.70, 54.18, 48.69, 48.40, 44.04, 41.36, 39.59, 39.43, 38.06, 37.97, 37.20, 28.99, 21.99, 20.33,
19.81, 18.92, 13.50. HRMS (m/z): calcd for C28H37ClNO4+ [M + H]+: 486.2406, found: 486.2403.
2-((4-chlorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (4d). White powder; yield, 84%; m.p. 140–142 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 7.80 (s, 1H, -CO-NH-), 7.46 (d, J = 5 Hz, 2H, Ar-H), 7.32-7.30
(m, 2H, Ar-H), 4.73 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.57 (d, J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (dd,
J = 20, 5 Hz, 1H, 18-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.00 (d, J = 15 Hz, 1H, 6-He), 1.84–1.69 (m,
6H), 1.63–1.62 (m, 2H), 1.56–1.52 (m, 2H), 1.49–1.38 (m, 2H), 1.32 (s, 3H, 18-CH3), 1.26–1.23 (m, 3H),
1.20–1.13 (m, 1H), 0.98–0.88 (m, 4H), 0.73 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 221.95,
175.84, 165.08, 135.42, 129.95, 129.25(2C), 120.94(2C), 63.33, 56.99, 54.69, 54.18, 48.69, 48.39, 44.03,
41.37, 39.58, 39.42, 38.06, 37.96, 37.19, 28.99, 21.97, 20.33, 19.82, 18.92, 13.51. HRMS (m/z): calcd for
C28H37ClNO4+ [M + H]+: 486.2406, found: 486.2401.
2-((2-fluorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (4e). White crystal; yield, 81%; m.p. 168–169 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 8.42–8.39 (m, 1H, Ar-H), 8.18 (s, 1H, -CO-NH-), 7.18–7.10 (m, 3H,
Ar-H), 4.78 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.60 (d, J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (d, J = 20 Hz,
1H, 15-He), 2.28 (d, J = 15 Hz, 1H, 3-He), 2.03 (d, J = 10 Hz, 1H, 6-He), 1.89–1.71 (m, 5H), 1.63–1.59

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(m, 2H), 1.54–1.51 (m, 2H), 1.44–1.38 (m, 2H), 1.33 (s, 3H, 18-CH3), 1.26–1.23 (m, 4H), 1.18–1.13 (m,
1H), 0.99 (s, 3H, 17-CH3), 0.94–0.84 (m, 1H), 0.74 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ
222.03, 175.64, 165.16, 125.58, 124.92, 124.84, 121.43, 114.95, 114.70, 63.28, 56.95, 54.74, 54.21, 48.68,
48.36, 44.05, 41.35, 39.64, 39.43, 38.05(2C), 37.21, 28.04, 22.00, 20.34, 19.83, 18.84, 13.40. HRMS (m/z):
calcd for C28H37FNO4+ [M + H]+: 470.2701, found: 470.2704.
2-((3-fluorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (4f). White powder; yield, 80%; m.p. 144–145 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 7.84 (s, 1H, -CO-NH-), 7.47 (d, J = 10 Hz, 1H, Ar-H), 7.31–7.28 (m,
1H, Ar-H), 7.13 (d, J = 5 Hz, 1H, Ar-H), 6.88–6.84 (m, 1H, Ar-H), 4.74 (d, J = 15 Hz, 1H, -O-CHe-CO-),
4.58 (d, J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (dd, J = 20, 5 Hz, 1H, 18-He), 2.27 (d, J = 15 Hz, 1H, 3-He),
2.01 (d, J = 15 Hz, 1H, 6-He), 1.87–1.69 (m, 5H), 1.63–1.61 (m, 1H), 1.54-1.52 (m, 5H), 1.43 (dd, J = 9,
3 Hz, 1H), 1.40–1.35 (m, 1H), 1.33 (s, 2H), 1.27–1.20 (m, 3H), 1.19–1.13 (m, 1H), 0.98 (s, 3H, 17-CH3),
0.95–0.86 (m, 1H), 0.73 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 221.98, 175.81, 165.14,
164.65, 138.32, 130.31, 114.84, 111.67, 107.31, 63.30, 56.98, 54.69, 54.17, 48.69, 48.39, 44.03, 41.36, 39.58,
+
39.42, 38.06, 37.96, 37.19, 28.98, 21.99, 20.32, 19.81, 18.92, 13.50. HRMS (m/z): calcd for C28H37FNO4
[M + H]+: 470.2701, found: 470.2699.
2-((4-fluorophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
◦
9-methanocyclohepta[a]naphthalene-4-carboxylate (4g). White powder; yield, 90%; m.p. 140–141 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 7.76 (s, 1H, -CO-NH-), 7.48–7.45 (m, 2H, Ar-H), 7.05 (t, J = 10 Hz,
2H, Ar-H), 4.74 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.58 (d, J = 15 Hz, 1H, -O-CHa-CO-), 2.61 (d, J = 20 Hz,
1H, 18-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.00 (d, J = 15 Hz, 1H, 6-He), 1.87–1.70 (m, 5H), 1.63–1.61 (m,
2H), 1.55–1.52 (m, 3H), 1.44–1.36 (m, 2H), 1.33 (s, 3H), 1.26–1.23 (m, 3H), 1.19–1.13 (m, 1H), 0.98 (s, 3H),
0.95–0.86 (m, 1H), 0.73 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.01, 175.86, 165.06,
161.29, 132.87, 121.63, 121.53, 116.06, 115.75, 63.29, 56.99, 54.69, 54.18, 48.69, 48.40, 44.02, 41.37, 39.59,
+
39.42, 38.06, 37.96, 37.19, 28.99, 21.96, 20.32, 19.82, 18.92, 13.51. HRMS (m/z): calcd for C28H37FNO4
[M + H]+: 470.2701, found: 470.2707.
2-((4-nitrophenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-6a,
9-methanocyclohepta[a]naphthalene-4-carboxylate (4h). Pale yellow solid; yield, 78%; m.p. 189–190 ◦C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 8.24 (d, J = 10 Hz, 2H, Ar-H), 8.08 (s, 1H, -CO-NH-), 7.69 (d,
J = 10 Hz, 2H, Ar-H), 4.77 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.62 (d, J = 15 Hz, 1H, -O-CHa-CO-), 2.60 (d,
J = 20 Hz, 1H, 18-He), 2.27 (d, J = 10 Hz, 1H, 3-He), 2.00 (d, J = 15 Hz, 1H, 6-He), 1.83–1.76 (m, 3H),
1.74–1.70 (m, 2H), 1.63–1.59 (m, 2H), 1.45–1.37 (m, 3H), 1.34 (s, 3H), 1.30 (s, 1H), 1.23-1.10 (m, 4H), 0.98
(s, 3H), 0.95–0.84 (m, 2H), 0.73 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 125 MHz, ppm):
δ 222.00, 175.99,
165.48, 144.11, 142.50, 125.27(2C), 119.17(2C), 63.42, 56.99, 54.69, 54.18, 48.70, 48.41, 44.10, 41.36, 39.55,
+
39.44, 38.08, 37.97, 37.19, 29.01, 21.98, 20.34, 19.81, 18.94, 13.55. HRMS (m/z): calcd for C28H37N2O6
[M + H]+: 497.2646, found: 497.2640.
2-((4-methoxyphenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-
◦
6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (4i). White powder; yield, 81%; m.p. 86–88 C;
1H-NMR (CDCl3, 500 MHz, ppm):
δ 7.70 (s, 1H, -CO-NH-), 7.40 (d, J = 15 Hz, 2H, Ar-H), 6.88 (d,
J = 10 Hz, 2H, Ar-H), 4.73 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.57 (d, J = 15 Hz, 1H, -O-CHa-CO-), 3.80 (s,
3H, ph-O-CH3), 2.62 (d, J = 15 Hz, 1H, 18-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.00 (d, J = 10 Hz, 1H,
6-He), 1.88–1.70 (m, 5H), 1.63–1.58 (m, 3H), 1.56–1.52 (m, 2H), 1.44–1.39 (m, 2H), 1.32 (s, 3H, 18-CH3),
1.25–1.23 (m, 3H), 1.18–1.12 (m, 1H), 0.98 (s, 3H), 0.94–0.88 (m, 1H), 0.74 (s, 3H, 20-CH3). 13C-NMR
(CDCl3, 75 MHz, ppm):
δ 222.01, 175.81, 164.88, 156.85, 129.90, 121.60(2C), 114.35(2C), 63.35, 57.01,
55.52, 54.71, 54.20, 48.70, 48.39, 44.02, 41.39, 39.61, 39.42, 38.05, 37.98, 37.20, 28.99, 21.98, 20.33, 19.81,
18.93, 13.50. HRMS (m/z): calcd for C29H40NO5+ [M + H]+: 482.2901, found: 482.2905.
2-((2,5-dimethoxyphenyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-8-oxotetradecahydro-
6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (4j). White powder; yield, 79%; m.p. 166–168 ◦C;

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1H-NMR (CDCl3, 500 MHz, ppm):
δ
8.55 (s, 1H, -CO-NH-), 8.14 (d, J = 3 Hz, 1H, Ar-H), 6.81 (d,
J = 5 Hz, 1H, Ar-H), 6.61 (dd, J = 10, 5 Hz, 1H, Ar-H), 4.80 (d, J = 15 Hz, 1H, -O-CHe-CO-), 4.54 (d,
J = 15 Hz, 1H, -O-CHa-CO-), 3.83 (s, 3H, ph-O-CH3), 3.79 (s, 3H, ph-O-CH3), 2.61 (dd, J = 15, 5 Hz, 1H,
18-He), 2.31 (d, J = 15 Hz, 1H, 3-He), 2.01 (d, J = 15 Hz, 1H, 6-He), 1.88 (qt, J = 9, 3Hz, 1H) 1.82–1.74 (m,
3H), 1.71–1.67 (m, 2H), 1.63–1.60 (m, 1H), 1.55–1.47 (m, 3H), 1.44–1.37 (m, 2H), 1.35 (s, 3H, 18-CH3),
1.27–1.22 (m, 3H), 1.18–1.10 (m, 1H), 0.98–0.93 (m, 4H), 0.73 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75
MHz, ppm):
δ 222.00, 175.50, 164.97, 153.94, 142.05, 127.36, 110.68, 109.07, 106.08, 63.14, 57.09, 56.04,
55.83, 54.74, 54.24, 48.66, 48.39, 43.99, 41.39, 39.63, 39.41, 38.02, 37.88, 37.22, 28.83, 21.76, 20.32, 19.83,
18.96, 13.38. HRMS (m/z): calcd for C30H42NO6+ [M + H]+: 512.3007, found: 512.3001.
3.6. General Procedure for the Synthesis of Compound (5a–5e)
A mixture of isosteviol (63.6 mg, 0.20 mmol), K2CO3 (41.5 mg, 0.30 mmol) and different phenyl
◦
1,2,3-triazole chloroacetamides (0.21 mmol) in CH3CN (10 mL) was stirred at 80 C for 2 h. After
confirming the reaction progress by thin-layer chromatography, the solvent was evaporated in vacuo,
the mixture was dissolved with 15 mL ethyl acetate and then washed with saline (5 mL
mixture was then purified using silica gel column chromatography and eluted with petroleum
×
3). The
ether:ethyl acetate (1:1) to obtain the target compound 5a–5e.
2-oxo-2-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl
-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (5a). White solid; yield, 81%;
m.p. 83–84 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ 8.01 (s, 1H, triazole-H), 7.74-7.71 (m, 2H, Ar-H),
7.56–7.42 (m, 3H, Ar-H), 6.91 (brs, 1H, -NH-CO-), 4.74–4.46 (m, 4H, -O-CH2-, -CH2-NH-), 2.56 (dd,
J = 18, 3 Hz, 1H, 15-He), 2.22 (d, J = 12 Hz, 1H, 3-He), 1.95 (d, J = 12 Hz, 1H, 6-He), 1.85–1.66 (m, 6H),
1.62–1.47 (m, 4H), 1.42–1.37 (m, 2H), 1.27 (s, 3H, 18-CH3), 1.20–1.13 (m, 3H), 1.10–1.04 (m, 1H), 0.96
(s, 3H, 17-CH3), 0.93–0.88 (m, 1H), 0.67 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.09,
176.00, 167.37, 144.64, 136.87, 129.81(2C), 128.93, 120.52(2C), 120.44, 62.98, 56.98, 54.66, 54.17, 48.64,
48.31, 43.95, 41.28, 39.61, 39.39, 37.99, 37.94, 37.20, 34.69, 28.91, 21.90, 20.29, 19.82, 18.87, 13.42. HRMS
(m/z): calcd for C31H41N4O4+ [M + H]+: 533.3122, found: 533.3119.
2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b
-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (5b). White solid; yield,
82%; m.p. 80–82 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ 8.00 (s, 1H, triazole-H), 7.71–7.68 (m, 2H,
Ar-H), 7.54–7.51 (m, 2H, Ar-H), 6.81 (brs, 1H, -NH-CO-), 4.74–4.47 (m, 4H, -O-CH2-, -CH2-NH-), 2.57
(dd, J = 18, 3 Hz, 1H, 15-He), 2.23 (d, J = 12 Hz, 1H, 3-He), 1.96 (d, J = 12 Hz, 1H, 6-He), 1.82–1.68 (m,
6H), 1.60–1.39 (m, 6H), 1.28 (s, 3H, 18-CH3), 1.22–1.15 (m, 3H), 1.12–1.06 (m, 1H), 0.99 (s, 3H, 17-CH3),
0.95–0.87 (m, 1H), 0.68 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 222.09, 176.02, 167.44,
144.90, 135.35, 134.76, 130.01(2C), 121.69(2C), 120.44, 62.99, 56.98, 54.67, 54.18, 48.66, 48.33, 43.96, 41.30,
39.61, 39.40, 38.00, 37.94, 37.21, 34.64, 28.91, 21.91, 20.30, 19.82, 18.87, 13.42. HRMS (m/z): calcd for
C31H40ClN4O4+ [M + H]+: 567.2733, found: 567.2729.
2-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,
11b-trimethyl-8-oxotetrade◦cahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (5c). White solid;
1
yield, 83%; m.p. 71–72 C; H-NMR (CDCl3, 300 MHz, ppm):
δ
7.92 (s, 1H, triazole-H), 7.62 (d,
J = 6 Hz, 2H, Ar-H), 7.04 (d, J = 6 Hz, 2H, Ar-H), 6.87 (brs, 1H, -NH-CO-), 4.74–4.47 (m, 4H, -O-CH2-,
-C 2-NH-), 3.88 (s, 3H, -O-C 3), 2.57 (dd, J = 18, 3 Hz, 1H, 15-He), 2.23 (d, J = 12 Hz, 1H, 3-He), 1.96
H
H
(d, J = 12 Hz, 1H, 6-He), 1.80–1.68 (m, 6H), 1.62–1.48 (m, 4H), 1.45–1.38 (m, 3H), 1.23–1.19 (m, 3H),
1.16–1.05 (m, 2H), 0.98 (s, 3H, 17-CH3), 0.94–0.87 (m, 2H), 0.68 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75
MHz, ppm):
δ 222.11, 175.97, 167.33, 159.95, 144.37, 130.30, 122.18(2C), 120.55, 114.84(2C), 63.00, 56.98,
55.64, 54.67, 54.17, 48.65, 48.30, 43.95, 41.28, 39.61, 39.39, 38.00, 37.94, 37.21, 34.70, 28.91, 21.92, 20.29,
19.82, 18.87, 13.41. HRMS (m/z): calcd for C32H43N4O5+ [M + H]+: 563.3228, found: 563.3225.

Molecules 2019, 24, 121
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2-oxo-2-(((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl
-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (5d). White solid; yield, 83%; m.p.
66–68 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ 7.96 (s, 1H, triazole-H), 7.60 (d, J = 9 Hz, 2H, Ar-H),
7.33 (d, J = 9 Hz, 2H, Ar-H), 6.84 (brs, 1H, -NH-CO-), 4.74–4.47 (m, 4H, -O-CH2-, -CH2-NH-), 2.56 (dd,
J = 21, 3 Hz, 1H, 15-He), 2.43 (s, 3H, Ar-CH3), 2.23 (d, J = 15 Hz, 1H, 3-He), 1.95 (d, J = 12 Hz, 1H,
6-He), 1.80–1.66 (m, 6H), 1.62–1.48 (m, 4H), 1.43–1.38 (m, 2H), 1.28 (s, 3H, 18-CH3), 1.21–1.15 (m, 3H),
1.10–1.05 (m, 1H), 0.97 (s, 3H, 17-CH3), 0.93–0.88 (m, 1H), 0.68 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75
MHz, ppm):
δ 222.15, 175.97, 167.34, 144.42, 139.10, 134.58, 130.31(2C), 120.44(2C), 120.41, 62.99, 56.97,
54.66, 54.16, 48.65, 48.30, 43.94, 41.27, 39.60, 39.38, 37.99, 37.94, 37.20, 34.71, 28.92, 21.91, 21.11, 20.29,
19.83, 18.87, 13.41. HRMS (m/z): calcd for C32H43N4O4+ [M + H]+: 547.3279, found: 547.3276.
2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,
11b-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (5e). White solid;
yield, 79%; m.p. 96–97 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ 8.02 (s, 1H, triazole-H), 7.92–7.91 (m,
1H, Ar-H), 7.65–7.59 (m, 2H, Ar-H), 6.81 (brs, 1H, -NH-CO-), 4.73–4.47 (m, 4H, -O-CH2-, -CH2-NH-),
2.56 (dd, J = 18, 3 Hz, 1H, 15-He), 2.23 (d, J = 15 Hz, 1H, 3-He), 1.98–1.93 (m, 1H, 6-He), 1.82-1.67 (m,
6H), 1.63–1.47 (m, 4H), 1.45–1.35 (m, 2H), 1.28 (s, 3H, 18-CH3), 1.22–1.15 (m, 3H), 1.12–1.06 (m, 1H),
0.98 (s, 3H, 17-CH3), 0.95–0.89 (m, 1H), 0.69 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ
222.08, 176.06, 167.50, 145.20, 135.87, 134.08, 133.07, 131.55, 122.26, 120.43, 119.39, 63.00, 56.98, 54.66,
54.19, 48.66, 48.34, 43.96, 41.31, 39.60, 39.40, 38.01, 37.94, 37.21, 34.64, 28.92, 21.89, 20.31, 19.82, 18.88,
13.44. HRMS (m/z): calcd for C31H39Cl2N4O4+ [M + H]+: 601.2343, found: 601.2340.
3.7. General Procedure for the Synthesis of Compound (6a–6e)
A mixture of isosteviol (63.6 mg, 0.20 mmol), K2CO3 (41.5 mg, 0.30 mmol) and different
phosphonic acid [[(chloroacetyl) amino] phenylmethyl]-diethyl esters (0.21 mmol) in CH3CN (10
◦
mL) was stirred at 80 C for 3 h. After confirming the reaction progress by thin-layer chromatography,
the solvent was evaporated in vacuo, the mixture was dissolved with 15 mL ethyl acetate and then
washed with saline (5 mL
and eluted with a gradient of petroleum ether:ethyl acetate (3:1-1:1) to obtain the target compound
6a–6e.
×
3). The mixture was then purified using silica gel column chromatography
2-(((diethoxyphosphoryl)(phenyl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl
-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (6a). White powder; yield, 81%;
◦
1
m.p. 156–157 C; H-NMR (CDCl3, 300 MHz, ppm):
δ 7.41–7.35 (m, 5H, Ar-H), 7.20–7.12 (m, 1H,
-NH-CO-), 5.48 (dd, J = 18, 9 Hz, 1H, -P-C -NH-), 4.76–4.64 (m, 1H, -O-CHe-CO-), 4.53–4.40 (m, 1H,
H
-O-CHa-CO-), 4.19–4.07 (m, 2H, -O-CHe-), 4.00–3.87 (m, 1H, -O-CHa-), 3.77–3.62 (m, 1H, -O-CHa-),
2.65–2.56 (m, 1H, 15-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.09–1.99 (m, 1H, 6-He), 1.89–1.69 (m, 6H),
1.65–1.50 (m, 4H), 1.45–1.41 (m, 2H), 1.35–1.29 (m, 6H), 1.27–1.19 (m, 3H), 1.15–1.07 (m, 4H), 1.00–0.99
(m, 3H), 0.95–0.87 (m, 1H), 0.70 (s, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz, ppm):
δ 221.94, 175.72,
166.43, 134.77, 128.78(2C), 128.38, 127.91(2C), 63.60, 63.12, 62.74, 56.91, 54.73, 54.21, 50.82, 48.68, 48.40,
43.97, 41.41, 39.64, 39.41, 38.02(2C), 37.23, 28.86, 21.92, 20.32, 19.82, 18.93, 16.42, 16.10, 13.38. HRMS
(m/z): calcd for C33H49NO7P+ [M + H]+: 602.3241, found: 602.3238.
2-(((4-chlorophenyl)(diethoxyphosphoryl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-
trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (6b). White powder;
yield, 83%; m.p. 149–150 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ
7.46–7.33 (m, 4H, Ar-H), 7.27–7.16
(m, 1H, -NH-CO-), 5.44 (ddd, J = 21, 9, 3 Hz, 1H, -P-C
H-NH-), 4.68 (t, J = 15 Hz, 1H, -O-CHe-CO-), 4.46
(t, J = 15 Hz, 1H, -O-CHa-CO-), 4.18–4.09 (m, 2H, -O-CHe-), 4.03–3.90 (m, 1H, -O-CHa-), 3.84–3.70 (m,
1H, -O-CHa-), 2.60 (d, J = 18 Hz, 1H, 15-He), 2.25 (d, J = 12 Hz, 1H, 3-He), 2.05–1.98 (m, 1H, 6-He),
1.89–1.65 (m, 7H), 1.60–1.53 (m, 3H), 1.45–1.40 (m, 2H), 1.35–1.29 (m, 6H), 1.26–1.22 (m, 4H), 1.16–1.11
(m, 3H), 1.00–0.99 (m, 3H), 0.95–0.91 (m, 1H), 0.70–0.69 (m, 3H, 20-CH3). 13C-NMR (CDCl3, 75 MHz,
ppm):
δ 221.99, 175.77, 166.59, 134.39, 133.40, 129.30, 129.19, 128.99(2C), 63.72, 63.30, 62.78, 56.96, 54.72,

Molecules 2019, 24, 121
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54.21, 50.31, 48.68, 48.41, 43.97, 41.36, 39.64, 39.41, 38.02, 37.98, 37.22, 28.89, 21.99, 20.32, 19.82, 18.92,
16.45, 16.16, 13.38. HRMS (m/z): calcd for C33H48ClNO7P+ [M + H]+: 636.2851, found: 636.2846.
2-(((diethoxyphosphoryl)(4-methoxyphenyl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-
trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (6c). White powder;
yield, 81%; m.p. 134–135 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ
7.37–7.34 (m, 2H, Ar-H), 7.17–7.15
(m, 1H, -NH-CO-), 6.91–6.87 (m, 2H, Ar-H), 5.48 (dd, J = 21, 9 Hz, 1H, -P-C -NH-), 4.72–4.62 (m, 1H,
H
-O-CHe-CO-), 4.51–4.39 (m, 1H, -O-CHe-CO-), 4.18–4.07 (m, 2H, -O-CHe-), 4.00–3.90 (m, 1H, -O-CHa-),
3.80 (s, 3H, ph-OCH3), 3.76–3.66 (m, 1H, -O-CHa-), 2.60 (d, J = 18 Hz, 1H, 15-He), 2.25 (d, J = 12 Hz,
1H, 3-He), 2.05–1.98 (m, 1H, 6-He), 1.88–1.73 (m, 5H), 1.68–1.53 (m, 5H), 1.44–1.40 (m, 2H), 1.35–1.29
(m, 6H), 1.26–1.17 (m, 4H), 1.13–1.08 (m, 3H), 0.99–0.98 (m, 3H), 0.96–0.90 (m, 1H), 0.69 (s, 3H, 20-CH3).
13C-NMR (CDCl3, 75 MHz, ppm):
δ 221.86, 175.71, 166.29, 159.63, 129.24(2C), 126.75, 114.19(2C), 63.57,
62.98, 62.75, 56.92, 55.31, 54.73, 54.21, 50.14, 48.67, 48.40, 43.96, 41.41, 39.64, 39.42, 38.02(2C), 37.21,
28.86, 21.95, 20.32, 19.82, 18.93, 16.39, 16.24, 13.31. HRMS (m/z): calcd for C34H51NO8P+ [M + H]+:
632.3347, found: 632.3344.
2-(((diethoxyphosphoryl)(p-tolyl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b-trimethyl-
8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (6d). White powder; yield, 84%;
◦
m.p. 119–120 C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ
7.32–7.29 (m, 2H, Ar-H), 7.18–7.11 (m, 3H, Ar-H,
-NH-CO-), 5.44 (dd, J = 21, 9 Hz, 1H, -P-C
H-NH-), 4.74–4.62 (m, 1H, -O-CHe-CO-), 4.51–4.38 (m, 1H,
-O-CHe-CO-), 4.18–4.06 (m, 2H, -O-CHe-), 4.00–3.87 (m, 1H, -O-CHa-), 3.78–3.64 (m, 1H, -O-CHa-),
2.60 (dq, J = 18, 3 Hz, 1H, 15-He), 2.34 (s, 3H, ph-CH3), 2.26 (d, J = 15 Hz, 1H, 3-He), 2.07–1.99 (m,
1H, 6-He), 1.89–1.78 (m, 3H), 1.73–1.69 (m, 3H), 1.64–1.54 (m, 4H), 1.49–1.40 (m, 3H), 1.35–1.29 (m,
6H), 1.26–1.22 (m, 3H), 1.13–1.08 (m, 3H), 1.00–0.99 (m, 3H), 0.95–0.91 (m, 1H), 0.69 (s, 3H, 20-CH3).
13C-NMR (CDCl3, 75 MHz, ppm):
δ 221.98, 175.68, 166.28, 138.26, 131.68, 129.48(2C), 127.86(2C), 63.48,
63.08, 62.79, 57.00, 54.74, 54.24, 50.53, 48.68, 48.41, 43.97, 41.36, 39.65, 39.42, 38.02(2C), 37.24, 28.96,
22.16, 21.14, 20.32, 19.82, 18.95, 16.42, 16.13, 13.30. HRMS (m/z): calcd for C34H51NO7P+ [M + H]+:
616.3398, found: 616.3393.
2-(((3,4-dichlorophenyl)(diethoxyphosphoryl)methyl)amino)-2-oxoethyl(4R,4aS,6aR,9S,11aR,11bS)-4,9,11b
-trimethyl-8-oxotetradecahydro-6a,9-methanocyclohepta[a]naphthalene-4-carboxylate (6e). White powder;
yield, 80%; m.p. 168–169 ◦C; 1H-NMR (CDCl3, 300 MHz, ppm):
δ
7.50–7.44 (m, 2H, Ar-H), 7.26–7.24
(m, 1H, Ar-H), 7.08–7.03 (m, 1H, -NH-CO-), 5.40 (ddd, J = 21, 9, 3 Hz, 1H, -P-C -NH-), 4.78–4.65 (m,
H
1H, -O-CHe-CO-), 4.54–4.41 (m, 1H, -O-CHa-CO-), 4.18–4.10 (m, 2H, -O-CHe-), 4.07–3.99 (m, 1H,
-O-CHa-), 3.92–3.81 (m, 1H, -O-CHa-), 2.66–2.58 (m, 1H, 15-He), 2.27 (d, J = 15 Hz, 1H, 3-He), 2.06–1.97
(m, 1H, 6-He), 1.89–1.66 (m, 6H), 1.61 (s, 2H), 1.58–1.50 (m, 2H), 1.46–1.41 (m, 2H), 1.36–1.30 (m, 6H),
1.27–1.11 (m, 7H), 1.01–1.00 (m, 3H), 0.98–0.92 (m, 1H), 0.72–0.71 (m, 3H, 20-CH3). 13C-NMR (CDCl3,
75 MHz, ppm):
δ 221.98, 175.83, 166.68, 135.15, 132.96, 132.62, 130.75, 129.67, 127.25, 63.81, 63.50, 62.81,
56.95, 54.78, 54.27, 50.01, 48.67, 48.39, 44.05, 41.40, 39.70, 39.48, 38.02, 37.91, 37.29, 28.92, 21.99, 20.41,
19.91, 18.90, 16.42, 16.24, 13.39. HRMS (m/z): calcd for C33H47Cl2NO7P+ [M + H]+: 670.2462, found:
670.2458.
3.8. General Procedure for the Synthesis of Intermediates (IIa-IIe, IVa-IVj, Va-Ve, VIa-VIe)
Chloroacetyl chloride (1.23 g, 11 mmol), Et3N (1.11 g, 11 mmol) and different amines (10 mmol)
were added to CH2Cl2 (20 mL) and the resulting mixture was stirred at 30 ◦C for 2–8 h. Then, the
solvent was evaporated in vacuo, water was added (15 mL), the mixture was filtered and the residue
was washed with water to obtain the different intermediates (Scheme 2).

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Scheme 2. Reagents and conditions:
(
a
) chloroacetyl chloride, Et3N, CH2Cl2, 30 ◦C; (
b) (i)
.
◦
propynylamine, CuSO4 5H2O, sodium ascorbate, t-BuOH/H2O (1:1), 30 C; (
c) chloroacetyl chloride,
◦
◦
Et3N, CH2Cl2, 30 C; (
CH2Cl2, 30 C.
d) diethyl phosphite, CH3COONH4, 90 C; (
e
) chloroacetyl chloride, Et3N,
◦
3.9. Biological Evaluation Materials
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma
Chemical Co. (St. Louis, MO, USA). The propidium iodide (PI) and Annexin V-FITC apoptosis
detection kit was purchased from BD Pharmingen (San Diego, CA, USA).
3.10. In Vitro Antiproliferative Activity
The antiproliferative activity of the target compounds against a normal L02 cell line and the
three different human cancer cell lines viz. colorectal (HCT-116), liver (BEL-7402) and liver (HepG2)
were evaluated using a standard MTT-based colorimetric assay. All cell lines were obtained from the
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain (Yanbian
University, Jilin, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI
Media 1640 (RPMI1640), supplemented with 10% fetal bovine serum (FBS) at 37 ◦C in a humidified
atmosphere containing 5% CO2.
Cells were plated in 96-well plates at appropriate densities to ensure exponential growth
throughout the experimental period (9
were then treated for 48 h with four serial concentrations (1, 10, 50 and 100
5-fluorouracil (5-FU) was used as a positive control. After 48 h of incubation, 10
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 L of DMSO was added to each well
×
103 cells per well) and then allowed to adhere for 24 h. Cells
µM) of each compound.
µL of MTT solution
µ
for coloration. The plates were shaken vigorously for 10 min at room temperature to ensure complete
solubilization. The optical density (OD) was read on a microplate reader (ELx800, BioTek, Highland

Molecules 2019, 24, 121
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Park, Winooski, VT, USA) at a wavelength of 492 nm and the data were subsequently analyzed. The
percentage of cell growth inhibition was calculated from the following equation:
Inhibitory rate (%) = [(1 − (ODtreated-ODblank)/(ODcontrol-ODblank)] × 100.
3.11. Colony Formation Assay
Exponentially growing determined HCT-116 cells (6
with the Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco, USA) containing
with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). After an overnight incubation, the
culture medium was removed and replaced fresh medium. Then, the cells were treated with various
(1)
×
102 per well) were plated in 6-well plates
concentrations of compound 5d (5, 10 and 15 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.
3.12. Analysis for Cell Cycle and Apoptosis by Flow Cytometry
◦
HCT-116 cells were plated in 6-well plates (5.0
h. Exponentially growing cells were then incubated with compound 5d at 5 and 15
untreated cells (control) or cells treated with compound 5d were centrifuged at 1000 rpm for 10 min
and then fixed in 70% ethanol at
20 ◦C for at least 24 h. The cells were subsequently resuspended in
phosphate-buffered saline (PBS) containing 0.1 mg/mL of RNase A and 5 g/mL propidium iodide
×
105 cells per well) and incubated at 37 C for 24
µM. After 24 h,
−
µ
(PI). The cellular DNA content for the cell cycle distribution analysis was measured by flow cytometry
using a FACSCalibur 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 phases
of the cell cycle were determined using the ModFit LT version 4.0 software package (Verity Software,
Topsham, ME, USA).
Apoptosis was detected using an Apoptosis Detection Kit (Invitrogen, Eugene, OR, USA). In brief,
cells were cultured in 6-well plates (5.0
with exponential growth were then incubated with compound 5d at 5 and 15
incubation, the cells were collected, washed twice with PBS and once with 1 binding buffer and then
stained with 5 M of annexin V-FITC and 2.5 M of PI 5mg/mL in 1 binding buffer for 30 min at
×
105 cells per well) and incubated at 37 ◦C for 24 h. Cells
µM. Following 24 h of
×
µ
µ
×
room temperature in the dark. Apoptotic cells were enumerated using a FACSCalibur flow cytometer
with Cell Quest software (Becton–Dickinson, Franklin Lakes, NJ, USA).
3.13. Western Blotting
HCT-116 cells were cultured with different concentrations of compound 5d for 24 h. Then, the
floating cells was collected and washed two times with ice cold PBS. The pellet was resuspended in lysis
◦
buffer. After the cells were lysed on ice for 20 min, lysates were centrifuged at 12,000 rpm at 4 C for 15
min. The protein concentration in the supernatant was determined using BCA protein assay reagents.
Equal amounts of protein (50
µg) were resolved using sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) (8
−
12% acrylamide gels) and transferred to a PVDF Hybond-P
membrane. Membranes were blocked with PBS containing 5% non-fat milk for 1 h at room temperature.
Membranes were then incubated with primary antibodies against Cyclin A (ENT1167, Elabscience,
Wuhan, China), Cyclin E1 (ENT1176, Elabscience, Wuhan, China), Cyclin B1 (ENT1169, Elabscience,
Wuhan, China) and β-actin (ENM0028, Elabscience, Wuhan, China), with gentle rotation overnight at
4 ◦C. Membranes were next incubated with fluorescent secondary antibodies for 2 h. Proteins were
detected by electrochemiluminescence (Bio-Rad, Hercules, CA, USA).

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3.14. Molecular Docking Study
The molecular docking study was performed using Discovery Studio (DS) 2017. The protein and
ligand were prepared, water molecules were deleted and a DS Server added hydrogen. The docking
result was treated with DS Client. In this study, the crystal structure of the CDK2/cyclin A complex
(3my5) was chosen for docking. The xyz coordinates (
−
25.8518, 4.7751,
−
23.0309, radius 34.6 Å) of
CDK2/cyclin A complex residues were defined as the binding site sphere. The protocol, CDOCKER
was used to perform the docking. The output poses of the ligands generated were analyzed based on
the LibDockScore function.
4. Conclusions
In the present study, thirty-two novel isosteviol derivatives were designed, synthesized and
evaluated for their antiproliferative effects against three cancer cell lines and the normal human L02
cell line. Half of these prepared compounds displayed some cytotoxic activity against the three cancer
cell lines, with low cytotoxicity against the normal L02 line. In particular, compound 5d exhibited
the most potent inhibitory activity against the three cancer cell lines. In addition, compound 5d can
inhibit the colony formation of HCT-116 cells in a concentration-dependent manner. Cell cycle analysis
revealed that compound 5d inhibited cell growth via the induction of S phase arrest in HCT-116 cells.
The possible mechanism of action may be correlated with down-regulation of cyclin A and cyclin E1
expression with the up-regulation of cyclin B1 expression. Our docking study also revealed the amino
acids His-C71 and Lys-A56 are playing a crucial role in the binding of compound 5d within the active
site of the CDK2/cyclin A complex. Hence, compound 5d may be a valuable candidate for further
studies aimed at the development of effective and harmless anticancer drugs.
Supplementary Materials: The supplementary materials are available online, 1H and 13C-NMR spectra of these
compounds are available in the supplementary materials.
Author Contributions: T.L. performed the experiments and wrote the paper. T.L. and Z.-S.Q. designed the
experiments. All authors took part in data analysis and discussion. All authors read and approved the
final manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (No. 21662036) and
research grants from the Health and Family Planning in Jilin Province science and technology backbone training
program for youth (No. 2016Q047).
Conflicts of Interest: The authors declare no conflict of interest.
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