Design, synthesis, and anticancer evaluation of novel indole derivatives of ursolic acid as potential topoisomerase II inhibitors

Article abstract of DOI:10.3390/ijms21082876

In this study, a series of new indole derivatives of ursolic acid bearing different N-(aminoalkyl)carboxamide side chains were designed, synthesized, and evaluated for their in vitro cytotoxic activities against two human hepatocarcinoma cell lines (SMMC-7721 and HepG2) and normal hepatocyte cell line (LO2) via MTT assay. Among them, compound 5f exhibited the most potent activity against SMMC-7721 and HepG2 cells with IC50 values of 0.56 ± 0.08 μM and 0.91 ± 0.13 μM, respectively, and substantially lower cytotoxicity to LO2 cells. A follow-up enzyme inhibition assay and molecular docking study indicated that compound 5f can significantly inhibit the activity of Topoisomerase IIα. Further mechanistic studies performed in SMMC-7721 cells revealed that compound 5f can elevate the intracellular ROS levels, decrease mitochondrial membrane potential, and finally lead to the apoptosis of SMMC-7721 cells. Collectively, compound 5f is a promising Topoisomerase II (Topo II) inhibitor, which exhibited the potential as a lead compound for the discovery of novel anticancer agents.

Full text of DOI:10.3390/ijms21082876

International Journal of
Molecular Sciences
Article
Design, Synthesis, and Anticancer Evaluation of
Novel Indole Derivatives of Ursolic Acid as Potential
Topoisomerase II Inhibitors
A-Liang Li, Yun Hao, Wen-Yan Wang, Qing-Song Liu, Yue Sun and Wen Gu *
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Jiangsu Key Lab of
Biomass-Based Green Fuels and Chemicals, Co-Innovation Center for Efficient Processing and Utilization of
Forest Products, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China;
lialiang673@gmail.com (A-L.L.); nfuhg2209@gmail.com (Y.H.); wwy1914898594@gmail.com (W.-Y.W.);
liuqings673@gmail.com (Q.-S.L.); sunyue0620@gmail.com (Y.S.)
*
Correspondence: njguwen2020@gmail.com
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 29 February 2020; Accepted: 15 April 2020; Published: 20 April 2020
Abstract: In this study, a series of new indole derivatives of ursolic acid bearing different
N-(aminoalkyl)carboxamide side chains were designed, synthesized, and evaluated for their in vitro
cytotoxic activities against two human hepatocarcinoma cell lines (SMMC-7721 and HepG2) and
normal hepatocyte cell line (LO2) via MTT assay. Among them, compound 5f exhibited the most
potent activity against SMMC-7721 and HepG2 cells with IC50 values of 0.56
.13 M, respectively, and substantially lower cytotoxicity to LO2 cells. A follow-up enzyme inhibition
assay and molecular docking study indicated that compound 5f can significantly inhibit the activity
of Topoisomerase II . Further mechanistic studies performed in SMMC-7721 cells revealed that
±
0.08
µM and 0.91
±
0
µ
α
compound 5f can elevate the intracellular ROS levels, decrease mitochondrial membrane potential,
and finally lead to the apoptosis of SMMC-7721 cells. Collectively, compound 5f is a promising
Topoisomerase II (Topo II) inhibitor, which exhibited the potential as a lead compound for the
discovery of novel anticancer agents.
Keywords: ursolic acid; indole derivatives; anticancer activity; topoisomerase II; apoptosis
1
. Introduction
Cancer has become a major cause of death in human beings. In 2018, there were estimated 18.1
million new cancer cases and 9.6 million cancer deaths worldwide [
new cancer cases and 2.29 million cancer deaths occurred in 2018, both ranked first among countries
worldwide [ ]. Despite their indispensable role in the treatment of cancer, chemotherapies often cause
less satisfactory results due to severe side effects and rapidly occurring drug resistance [ ]. Therefore,
1]. In China, about 3.8 million
1
2
efforts to develop low-toxic and high-efficacy anticancer agents have still been the primary task of
medicinal chemists.
Topoisomerase, which are generally classified as types I and II, are critical nuclear enzymes
essential for cell proliferation by solving the topological hurdles in the process of DNA replication.
Topoisomerase I (Topo I) handles the breaking and rejoining of only a single strand of a DNA duplex
to relieve the topological tension. In the case of topoisomerase II (Topo II), it relaxes the DNA double
helices by scissoring and religating both strands [
Mg(II) for DNA relaxation, while homodimeric enzyme Topo II requires Mg(II) and ATP hydrolysis
for the enzyme’s function and rapid kinetics [ ]. The Topo II family has two isozymes—Topo II and
II . Only the level of Topo II protein among human topo isozymes (Topo I, Ii , and II ) increases in
proliferative stage dependent manner. In addition, Topo II is frequently overexpressed in different
3–5]. The monomeric enzyme Topo I does not require
3
α
β
α
α
β
Int. J. Mol. Sci. 2020, 21, 2876; doi:10.3390/ijms21082876
www.mdpi.com/journal/ijms

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types of tumors, so the expression level of Topo II
α
has been used as a useful diagnostic marker for
chemotherapy in diverse cancers [
Topo II , can be a good strategy for aiming at actively proliferating cancer cells rather than normal cells.
A number of Topo II-targeted chemotherapy drugs, such as etoposide (VP-16), doxorubicin (DOX), and
mitoxantrone have been approved by the FDA for clinical use [ ]. However, most Topo II inhibitors
exhibited rebarbative side effects including cardiotoxicity, development of secondary malignancies
6,7]. These findings provide insight that targeting Topo II, especially
α
8
and multidrug resistance [9]. Hence, it is still a necessity to develop novel Topo II inhibitors with high
efficacy and selectivity.
Natural products have long been used to treat various diseases due to their wide range of
pharmacological activities [10]. Since the structural modified derivatives of natural products
with anticancer activities generally have fewer side effects, a simple and effective method to
obtain anticancer agents is the structural modification of bioactive natural products [11]. Ursolic
acid (UA,
1) is a natural pentacyclic triterpenoid abundant in many vegetables, fruits, and
herbs. The compound and its derivatives exhibit a wide variety of biological activities, such as
antibacterial [12], anticancer [13], antiviral [14], anti-inflammatory [15], antidiabetic [16], antiulcer [17],
antiarrhythmic [18], anti-osteoporosis [19], anti-atherosclerotic [20], and anti-neurodegenerative
activities [21]. In addition, some pentacyclic triterpenoids including UA were reported to inhibit Topo I
and II
α
by contending with DNA for topoisomerase binding sites, thus preventing topoisomerase-DNA
24]. Therefore, UA could be a promising starting material for the
cleavable complex formation [22
–
discovery of new topoisomerase inhibitors as anticancer agents.
In an effort to discover novel anticancer agents, we have previously designed and synthesized a
series of indole derivatives of UA bearing N-aminoalkyl side chains. In the in vitro cytotoxic assay,
some derivatives showed significant cytotoxic activity against two human hepatocarcinoma cell lines
(
SMMC-7721 and HepG2). Among them, HY-1 (compound 5b) exhibited the strongest activity with
IC50 values of about 1 M [25]. Moreover, in a preliminary screening, HY-1 showed considerable
inhibitory activity to Topo I and II . Based on these findings, this compound was chosen as the starting
µ
α
material for further structure modifications. Since, in our preliminary study, the carbon chain and
N-containing moiety of C-28 side chain played an important role in the cytotoxic activity of derivatives,
we focused on the modification of the side chain in this work (Figure 1). As a result, a series of new UA
derivatives were designed and synthesized in order to find derivatives with better anticancer activities.
Furthermore, the anticancer mechanisms of the active compounds were also extensively explored.
Figure 1. The structural modification of HY-1 (5b).
2
. Results
2
.1. Chemistry
Based on the method reported earlier [25], a series of new indole derivatives of ursolic acid
with different amine side chains were synthesized. Synthetic routes for the target compounds were
summarized in Schemes 1 and 2. Briefly, the starting material UA ( ) was oxidized by Jones reagent
to afford 3-oxo-ursolic acid ( ) in 76% yield. Subsequently, compound was converted to the
indole derivative ( ) in 69% yield by reacting with p-tolylhydrazine hydrochloride through Fisher
indole reaction. Further, compound
1
2
2
3
1
1
3
was reacted with different N ,N -dialkyl-1,2-ethanediamine,

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1
1
1
1
N ,N -dialkyl-1,3-propanediamine, or N ,N -dialkyl-1,4-butanediamine to afford the corresponding
amide derivatives 4a 5a , and 6a in 72–93% yields (Scheme 1). Interestingly, when compound
was reacted with 1,2-ethanediamine, 1,3-propanediamine, or 1,4-butanediamine in the presence
of HOBt and DCC, the dimeric byproducts (7a ) could always be detected. However, if we treat
compound with 0.5 eq of 1,2-ethanediamine, 1,3-propanediamine or 1,4-butanediamine, the dimeric
derivatives 7a became the main products with the yield of 61%, 65%, 63%, respectively (Scheme 2).
All the synthesized compounds were purified by recrystallization or silica gel column chromatography,
–
f,
–
f
–f
3
–
c
3
–c
1
13
and the structures of these compounds were characterized through their HRMS, IR, H NMR and
C
1
13
NMR spectra ( H and C NMR spectra: Figure S1–S42 in the Supplementary Materials).
Scheme 1. Synthetic route for the indole derivatives (4a
–
f
,
5a
) p-methyl phenylhydrazine hydrochloride,
) N,N-dialkylethane-1,2-diamine, N,N-dialkylpropane-1,3-diamine,
or N,N-dialkylbutane-1,4-diamine (1.2 eq), HOBt, DCC, CH Cl , rt, 12 h.
–f, 6a–f) from ursolic acid. Reagents
◦
and conditions: (
EtOH, conc. HCl, reflux for 3 h; (
a) Jones reagent, acetone, 0 C, 5 h; (b
c
2
2
Scheme 2. Synthesis of compounds 7a–c from 3. Reagents and conditions: (a) 1,2-ethylenediamine,
1
,3-propanediamine and 1,4-butanediamine (0.5 eq), HOBt, DCC, CH Cl , rt, 12 h.
2
2
2
.2. Cytotoxic Assay
The in vitro cytotoxic activities of synthesized compounds were evaluated against two human
hepatocarcinoma cell lines (SMMC-7721 and HepG2) and the human normal hepatocyte cell line (LO2)
via MTT assay. All tested compounds were dissolved in DMSO and the stock solutions were diluted
by DMEM medium before treatment of the cultured cells. The IC50 values of the tested compounds
against four cell lines were shown in Table 1.

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Table 1. The cytotoxicity Topo I and II inhibitory activities of the target compounds.
IC50 Values (µM)
Topo I Inhibition (%)
Topo II Inhibition (%)
SMMC-7721
HepG2
LO2
100 µM
20 µM
100 µM
20 µM
4
a
3.50 ± 0.61
4.42 ± 0.71
2.10 ± 0.21
1.75 ± 0.11
7.82 ± 0.78
1.24 ± 0.16
2.02 ± 0.21
1.08 ± 0.22
1.15 ± 0.14
0.89 ± 0.11
3.75 ± 0.37
0.56 ± 0.08
1.21 ± 0.19
2.43 ± 0.21
3.62 ± 0.23
2.72 ± 0.31
4.55 ± 1.56
0.65 ± 0.07
>50
3.31 ± 0.35
4.11 ± 0.55
2.24 ± 0.32
1.47 ± 0.12
10.72 ± 2.09
1.35 ± 0.17
2.45 ± 0.38
1.26 ± 0.17
1.20 ± 0.24
1.04 ± 0.09
5.27 ± 0.67
0.91 ± 0.13
1.32 ± 0.21
2.91 ± 0.18
3.45 ± 0.25
2.31 ± 0.21
7.84 ± 2.31
1.01 ± 0.12
>50
29.59 ± 2.12
31.06 ± 2.63
26.39 ± 1.32
16.03 ± 1.07
>50
15.1
3.7
0
13.0
0
5.6
-
-
0
-
-
12.4
9.0
-
-
-
-
13.7
0
0
-
-
-
-
-
69.7
68.0
68.7
71.7
63.3
73.7
72.3
72.5
75.8
81.1
70.8
83.5
71.1
69.0
70.0
72.5
65.6
81.8
23.8
17.8
16.9
-
25.9
36.9
38.3
32.7
26.8
31.3
31.2
38.2
38.9
41.3
31.5
43.2
27.0
31.7
35.2
32.8
28.2
39.8
9.7
1
4
b
4
c
4
d
4
e
4
f
18.27 ± 0.78
21.32 ± 1.59
20.65 ± 1.73
15.85 ± 1.06
12.82 ± 0.83
28.62 ± 0.76
10.58 ± 0.52
15.23 ± 1.15
18.19 ± 1.64
28.42 ± 2.98
18.65 ± 1.36
35.39 ± 3.13
13.72 ± 1.28
>50
0
5
a
24.5
34.2
0
0
0
5
b
5
c
5
d
5
e
5
f
0
6
a
28.8
15.1
16.8
0
0
0
0
2.0
0
75.1
-
-
6
b
6
c
6
d
6
e
6
f
7
a
7
b
>50
>50
>50
>50
1.12 ± 0.26
0.57 ± 0.16
0.81 ± 0.19
2
>50
>50
8.93 ± 0.52
5.92 ± 0.29
5.32 ± 0.67
9.8
8.1
-
47.8
-
7
CPT
VP-16
DOX
c
-
2
0.56 ± 0.18
0.36 ± 0.10
0.68 ± 0.11
1
38.2
-
-
88.5
-
-: Not tested. CPT: camptothecin; VP-16: etoposide; DOX: doxorubicin.
As shown in Table 1, most synthesized compounds showed considerable cytotoxic activity against
two cancer cell lines. Among them, compounds 4d 4f 5b 5f 6a and 6f displayed strong inhibition
with IC50 values of around 1 M. Especially, compound 5f exhibited the most potent anticancer activity
with IC50 values of 0.56 0.08 and 0.91 0.13 M, respectively, which were comparable to those of
reference drugs. Notably, compound 5f showed significantly lower cytotoxicity to human normal
hepatocyte cell line LO2 (IC : 10.58 0.52 M), indicating that the compound had good selectivity
between hepatocarcinoma and noncancerous hepatocyte cells. Compounds 5d and 6f also exhibited
significant activities against SMMC-7721 cells with IC50 values of 0.89 0.11 and 0.65 0.07 M,
respectively. In addition, compounds 4a 4e 5a 5e, and 6b showed good inhibitory activity to
the two cancer cell lines. On the contrary, compounds 7a were inactive to all three cell lines (IC50
0 µM).
The results indicated that the anticancer activities of the synthesized compounds were affected by
the N-containing moieties anchored on the side chain. For Series 4a 5a , and 6a , compounds 4f
, and 6f with N-methylpiperazine moieties all exhibited the strongest activities in their own series.
Compounds 4a 5a , and 6a containing NH , N(Me) , N(Et) , and piperidine moieties generally
showed strong activities. However, compounds 4e
,
,
–
d,
,
µ
±
±
µ
±
µ
50
±
±
µ
–
c,
,
,
–e
–c
>
5
–
f,
–
f
–f
,
5
f
–
d,
–
d
–d
2
2
2
,
5e, and 6e with morpholine rings displayed
relatively weaker inhibition to cancer cells compared with their analogs in the same series. In general,
the order of effects to the cytotoxicity of these derivatives could be summarized as N-methylpiperazine
>
NH , N(Me) , N(Et) and piperidine > morpholine. On the other hand, the length of amide side
2 2 2
chain also affected the cytotoxicity to some extent. Generally, compounds 5af exhibited stronger
cytotoxic activities than compounds 4a and 6a , which suggested that the 3C alkyl linker could be
more beneficial to the cytotoxicity than 2C and 4C alkyl linkers. In addition, three dimeric derivatives
were all inactive to tested cancer cells (IC50 > 50 M), which indicated that such dimerization by
–f
–f
7
a
–c
µ
alkyl linkers might hinder the interaction between these compounds and their potential targets, thus
significantly decreasing their anticancer activities.

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2
.3. Topoisomerase Inhibition Assay
The conversion of supercoiled pBR322 plasmid DNA to relaxed DNA by Topo I and II was
examined in the presence of the prepared compounds 4a 5a 6a , and 7a . Camptothecin and
–f,
–f,
–f
–c
etoposide, well-known Topo I and II inhibitors, respectively, were used as positive controls. The effect
of the tested compounds on Topo I and II activities were evaluated by Topo I/II relaxation assays. The
reaction products, relaxed DNA could be distinguished from supercoiled DNA by gel electrophoresis
and visualized using gel imaging system.
As shown in Figure 2 and Table 1, compounds 5a
inhibitory activities against Topo I at concentrations of 100
compounds 4a 4b 4d 6b, and 6c also showed mild Topo I inhibitory activities at 100
the other compounds, especially compounds with potent anticancer activity such as 5d
were not inhibitory to Topo I even at 100 M. Therefore, it is evident that the Topo I inhibition of the
title compounds only have minor correlations with their anticancer activities.
,
5b, and 6a displayed moderate and weak
M and 20 M, respectively. In addition,
M. However,
5f, and 6f,
µ
µ
,
,
,
µ
,
µ
Figure 2. Topoisomerase I inhibitory activity of the prepared compounds 4a
) The DNA relaxation assay of the prepared compounds at 100 M; ( ) The DNA relaxation assay of
the selected active compounds at 20 µM.
–f, 5a–f, 6a–f, and 7a–c.
(
a
µ
b
Concerning Topo II inhibitory activity, it was observed that most compounds exhibited considerable
inhibitory activity at concentrations of 100 and 20
µM (Figure 3 and Table 1). Among them, compound
5f with the strongest cytotoxic activity exhibited the most potent inhibitory activity with inhibition rates
of 83.5% and 43.2% at concentrations of 100 and 20
µM, respectively. Compounds 5d and 6f also showed
significant activities comparable to positive control etoposide. On the contrary, three dimer derivatives
with poor cytotoxic activities only showed weak inhibitory activities at 100 M and 20 M. The
7
a
–c
µ
µ
close correlations between the Topo II inhibitory activity and cytotoxic activity suggested that Topo II
inhibition of the title compounds probably played an important role in their anticancer activity.
2
.4. Molecular Docking
To gain more understanding between target compounds and Topo II, the binding modes
of compound 5f with Topo II were explored by molecular docking based on the reported Topo
II /DNA/inhibitor complex structure (PDB: 5GWK). The docking studies were performed by using
the GLIDE docking module of Schrodinger suite 2015-1 and the docking results were analyzed and
α
visualized by Discovery Studio 2016 Client. The binding modes of compound 5f with Topo IIα/DNA
complex are shown in Figure 4.

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Figure 3. Topoisomerase II inhibitory activity of the prepared compounds 4a–f, 5a–f, 6a–f, and 7a–c.
(
a) The DNA relaxation assay of the prepared compounds at 100
µM; (b) The DNA relaxation assay of
the prepared compounds at 20 µM.
Figure 4. Binding mode of compound 5f with human topoisomerase II
GWK). ( ) Molecular structure of compound 5f. ( ) Binding pose of compound 5f within the active
site of Topo II . Ligand is presented as stick models and colored by atom type, whereas the protein
is presented as ribbons and DNA is presented as arrows (for backbone) and ladders (for base pairs).
) Detailed docked view of compound 5f in the active site. The amino acid residues and base pairs
are presented as sticks and colored by atom type, while the interactions are presented as dash lines.
d) Two-dimensional projection drawing of compound 5f docked into the active site of Topo IIα.
α in complex with DNA (PDB:
5
a
b
α
(
c
(

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It was observed that compound 5f could be suitably docked into the binding site of Topo
(Figure 4b,c), affording a significant docking score ( 8.240). Specifically, compound 5f was
deeply embedded into the major groove of DNA. The planar 5-methyl-indole moiety of 5f was
intercalated into the gap of DNA double strand and formed stacking interactions with Cytosine
IIα
−
π–π
DC8, Thymine DT9 and Guanine DG13. The methyl group on the indole ring formed hydrophobic
alkyl interactions with Arg487. Other hydrophobic alkyl interactions were also detected between the
methyl group (C40), methylene groups (C7 and C8) and MET762, methyl group (C32) and MET766.
The N-(3-(4-methylpiperazin-1-yl)propyl)carboxamide side chain of compound 5f also played an
important role in the molecular docking. The nitrogen atom (N4) on the piperazine ring formed an
electrostatic attractive charge interaction with the phosphate group of guanosine monophosphate
residue (DG13), and the hydrogen on N4 formed a salt bridge with the phosphate group of adenosine
monophosphate residue (DA12). In addition, compound 5f also formed van der Waals interactions
with residues Gly488, Glu506, Ser763, Pro803, Ser802, Ala801, Ser800, and Guanine DG10 (Figure 4d).
Taken together, the molecular docking results in combination with topoisomerase inhibition assay data
indicated that compound 5f could be a promising Topo IIα inhibitor worthy of further investigations.
2
.5. Annexin V-FITC/PI Staining Assay
To further investigate the role of apoptosis in the antitumor activity of compound 5f, SMMC-7721
cells were treated with different concentrations of compound 5f (0, 0.3, 0.6, and 1.2 M) for 48 h. Then
µ
the treated cells were stained with Annexin V-FITC/PI followed by flow cytometry analysis. As shown
in Figure 5, the percentage of early and late apoptotic cells (early apoptosis: Lower right quadrant,
AV+/PI
to 9.13% (0.3
compound 5f could trigger the apoptosis of SMMC-7721 cells in a dose-dependent manner.
−
; late apoptosis: Upper right quadrant, AV+/PI+) considerably increased from 4.41% (0
µM)
µ
M), 21.36% (0.6 M), and 30.33% (1.2 M), respectively. These results indicated that
µ
µ
Figure 5. Apoptotic assay by flow cytometry. SMMC-7721 cells were treated with compound 5f at 0,
.3, 0.6, and 1.2 M for 48 h. The cells were stained with Annexin V-FITC/PI and were detected by
flow cytometry.
0
µ
2
.6. AO/EB Staining Assay
Apoptosis induced by compound 5f was further evaluated using acridine orange / ethidium
bromide (AO/EB) double staining. AO, as a vital dye showing green fluorescence, can pass through
cell membrane of living or dead cells, while EB only stains apoptotic or necrotic cells that have lost
their integrity and appear red fluorescence. Therefore, the normal cells will be stained only by AO with
bright green while apoptotic cells will be stained by AO and EB with red-orange [26,27]. As shown in
Figure 6, the cell population in control appeared in green color with usual morphology. Fluorescence
images of cells treated with various concentrations of 5f displayed the increase of cells with red-orange

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fluorescence, the reduced number of normal cells and also displayed distorted structural characteristics
such as chromatin condensation, cell shrinkage and membrane blebbing, which further indicated that
compound 5f induced apoptosis in SMMC-7721 cells.
Figure 6. AO/EB staining of compound 5f in SMMC-7721 cells. SMMC-7721 cells were treated with
compound 5f at 0, 0.3, 0.6, and 1.2 µM for 48 h. The cells were stained with AO/EB and were detected
by fluorescence microscope. White arrow: apoptotic cells.
2
.7. ROS Generation Assay
Reactive oxygen species (ROS), which can exert oxidative stress to cells and lead to disturbances
in mitochondrial membrane potential, are important mediators for the induction of apoptosis in many
cancer cells [28]. To determine whether the apoptosis induced by compound 5f was associated with
intracellular ROS level, SMMC-7721 cells treated with different concentrations of 5f were stained with
the fluorescent probe DCFH-DA and analyzed by flow cytometry. As shown in Figure 7, the cells with
elevated ROS levels increased from 3.97% (Control) to 9.89% (0.3
1.2 M), which indicated that compound 5f could induce ROS generation in SMMC-7721 cells in a
dose-dependent manner.
µM), 19.84% (0.6 µM) and 34.61%
(
µ
Figure 7. Effects of compound 5f on the intracellur reactive oxygen species (ROS) level in SMMC-7721
cells. SMMC-7721 cells were treated with different concentrations of compound 5f (0, 0.3, 0.6, and
1
.2 µM) for 48 h, stained with DCFH-DA and analyzed using flow cytometry.

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2
.8. Mitochondrial Membrane Potential Assay
It has been widely believed that intracellular ROS accumulation could decrease mitochondrial
membrane potential (∆Ψm), which is a characteristic phenomenon of early apoptosis [29]. To investigate
the correlation between ∆Ψm and apoptosis induced by compound 5f, the measurement of cells with
decreased ∆Ψm were carried out by JC-1 assay kit on the instructions of the manual. As shown in
Figure 8, the percentage of cells with low ∆Ψm (Lower right quadrant) increased from 1.29% (Control)
to 7.43% (0.3 µM), 16.28% (0.6 µM), and 32.34% (1.2 µM). Therefore, the observations clearly showed
that compound 5f could decrease mitochondrial membrane potential (∆Ψm) of SMMC-7721 cells in a
dose-dependent manner, and the compound probably induced the apoptosis through ROS-mediated
mitochondrial pathway.
Figure 8. Compound 5f-induced loss of the mitochondrial membrane potential (∆Ψm). SMMC-7721
cells were treated with compound 5f (0, 0.3, 0.6, and 1.2 µM) for 24 h, incubated with JC-1, and analyzed
using flow cytometry.
3
. Discussion
A series of new indole derivatives of ursolic acid bearing N-(aminoalkyl)carboxamide side chains
were designed, synthesized, and screened for their in vitro cytotoxic activity against two human
hepatocarcinoma cell lines (SMMC-7721, HepG2) and a normal human hepatocyte cell line (LO2). A
number of derivatives showed considerable cytotoxic activities in MTT assay. Especially, compound 5f
exhibited the most potent inhibitory activity against SMMC-7721 and HepG2 cells among all derivatives
and significantly lower cytotoxicity to LO2 cells. Concerning structure-activity relationships, the order
of effects of the N-containing moieties to the cytotoxicity could be summarized as N-methylpiperazine
>
NH , N(Me) , N(Et) , and piperidine > morpholine. In addition, 3C linker on the side chain could
2 2 2
be more beneficial to the cytotoxicity than 2C and 4C alkyl linkers. In vitro pharmacological studies
showed that compound 5f could significantly inhibit Topoisomerase II activity and thus hinder the
relaxation of supercoiled DNA. In addition, it could induce intracellular ROS generation, decrease
mitochondrial membrane potential, and finally lead to the apoptosis of SMMC-7721 cells. Due to its
strong Topoisomerase II inhibitory activity and anticancer activity, this compound exhibited potential
as an anticancer drug candidate. In addition, this class of derivatives could expand the structural
diversity of anticancer agents and affirm the perspectives of further studies. More investigations on
the structural modification and the structure–activity relationship of this scaffold will be carried out in
the future.

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4
. Materials and Methods
4
.1. General
Melting points were measured on an XT-4 apparatus (Taike Corp., Beijing, China) and were
uncorrected. IR spetra were measured on a Nexus 870 FT-IR spectrometer (Thermo Fisher Scientific,
−1
Waltham, MA, USA), and the absorption bands were expressed in cm . The HRMS spectra were
recorded on a high-resolution mass spectrometer equipped with electrospray (ESI) and nanospray
sources, and a quadrupole-time of flight hybrid analyzer (Q-TOF Premier/nanoAquity, Waters, Milford,
1
13
MA, USA). H and C NMR spectra were obtained in CDCl on a Bruker DRX-600 NMR spectrometer
3
(Billerica, MA, USA) using TMS as internal standard. Reactions and the resulted products were
monitored by TLC which was carried out on TLC Silica gel 60 F254 Aluminum sheets from Merck
KGaA, Darmstadt, Germany, and visualized in UV light (254 nm). Silica gel (300–400 mesh) for
column chromatography was purchased from Qingdao Marine Chemical Factory (Qingdao, China).
The reagents (chemicals), all being of A.R. grade, were purchased from Shanghai Chemical Reagent
Company (Shanghai, China) and Energy Chemical (Shanghai, China). Ursolic acid (95%) was bought
from Jingzhu Biological Technology Co., Ltd. (Nanjing, China).
4
.2. Procedure for the Synthesis of Compound 3
To a solution of ursolic acid (1.0 g, 2.2 mmol) in acetone (150 mL) was added dropwise to 1 mL of
◦
Jones reagent (0.27 g CrO dissolved in 0.23 mL concentrated H SO and 0.73 mL H O) at 0 C. The
3
2
4
2
mixture was then stirred at room temperature for 5 h (TLC control). Subsequently, isopropanol (45 mL)
was added and the mixture was stirred for additional 0.5 h. Then the mixture was filtered, the filtrate
was concentrated in vacuo to remove solvent. The residue was then recrystallized in MeOH to afford
compound
2 as a white solid (0.56 g, yield 76%). The spectral data of compound 2 were in accordance
with the records of previous literature [30].
To a solution of compound
p-tolylhydrazine hydrochloride and 0.5 mL concentrated HCl. The mixture was refluxed at 85 C for
2
(0.44 g, 1 mmol) in 10 mL EtOH was added 0.33 g (2.1 mmol) of
◦
3
h. After cooling, the mixture was poured into 100 mL of ice-cold water and extracted with CH Cl (3
2 2
×
50 mL). The organic layer was combined, washed with water, saturated NaHCO solution and brine,
3
dried over anhydrous Na SO and concentrated to give a crude product, which was purified by silica
2
4
gel column chromatography (petroleum ether–acetone 15:1, v/v) to afford compound
69%, 0.37 g) [25].
3 as a white solid
(
4
.3. General Procedure for the Synthesis of Compounds 4a–f, 5a–f, and 6a–f
To a solution of compound (54 mg, 0.1 mmol) in CH Cl (5 mL) were added HOBt (19 mg,
3
2
2
0
0
.12 mmol) and DCC (24 mg, 0.12 mmol). The reaction mixture was stirred at room temperature for
.5 h and different amine (0.12 mmol) was added. The mixture was stirred for 12 h at room temperature.
At the end of reaction, the mixture was filtered, and the filtrate concentrated in vacuo. The obtained
residue was purified by silica gel column chromatography (petroleum ether–acetone 30:1, v/v) to
afford the corresponding product 4a–f, 5a–f, and 6a–f with 2-, 3- and 4-carbon side chains, respectively
(Scheme 1).
0 0
.3.1. N-(2-Aminoethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (4a)
4
◦
1
Yellow solid; Yield: 72%; mp. 228–230 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.93 (d,
J = 6.2 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.29–1.90 (m,
5H), 1.90–2.08 (m, 2H), 2.08–2.25 (m, 3H), 2.32 (brs, 2H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 2.83 (brs,
H), 3.12 (m, 1H), 3.42 (m, 1H), 5.45 (brs, 1H), 6.46 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.2 Hz,
H), 7.21 (s, 1H), 7.87 (brs, 1H); 13C NMR (150 MHz, CDCl3):
15.92, 16.95, 17.36, 19.39, 21.35, 21.60,
3
1
2
1
2
4
δ
3.35, 23.37, 23.66, 25.01, 28.07, 31.09, 31.12, 32.46, 34.15, 37.23, 37.50, 38.10, 39.19, 39.80, 39.99, 41.19,
1.77, 42.77, 46.38, 48.07, 53.30, 54.10, 106.32, 110.20, 117.81, 122.44, 126.17, 128.10, 128.54, 134.59, 139.51,

Int. J. Mol. Sci. 2020, 21, 2876
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1
41.19, 178.82; IR (KBr, cm 1): 3311, 2924, 2854, 1635, 1519, 1457, 1378, 1305, 1276, 794, 737; HRMS (ESI):
−
+
m/z [M + H] calcd. for C H N O: 584.4580; found: 584.4584.
39
58
3
0 0
.3.2. N-(2-(Dimethylamino)ethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (4b)
4
◦
1
Yellow solid; Yield: 89%; mp. 166–168 C; H NMR (600 MHz, CDCl ):
δ
0.90 (s, 3H), 0.93 (d,
3
J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.2 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.30 (s, 3H), 1.35–2.00
(
(
7
2
4
m, 16H), 2.10–2.23 (m, 3H), 2.25 (s, 6H), 2.30–2.42 (m, 3H), 2.43 (s, 3H), 2.78 (d, J = 14.8 Hz, 1H), 3.19
m, 1H), 3.35 (m, 1H), 5.41 (brs, 1H), 6.59 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H),
.20 (s, 1H), 7.87 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ 15.91, 17.00, 17.34, 19.43, 21.34, 21.59, 23.31,
3.37, 23.69, 25.02, 28.12, 31.08, 31.12, 32.60, 34.18, 36.98, 37.28, 37.41, 38.13, 39.23, 39.86, 40.02, 42.79,
5.35, 46.44, 48.01, 53.40, 54.30, 57.75, 106.33, 110.20, 117.81, 122.41, 126.24, 128.09, 128.59, 134.64, 139.25,
−
1
1
41.22, 178.33; IR (KBr, cm ): 3298, 2923, 2854, 1633, 1510, 1457, 1380, 1307, 1186, 1054, 793; HRMS
+
(ESI): m/z [M + H] calcd. for C H N O: 612.4893; found: 612.4887.
41 62 3
0 0
.3.3. N-(2-(Diethylamino)ethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (4c)
4
◦
1
Yellow solid; Yield: 88%; mp. 163–165 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.93 (d,
J = 6.3 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.3 Hz, 3H), 1.04 (t, J = 6.9 Hz, 6H), 1.15 (s, 3H), 1.21 (s, 3H), 1.30
s, 3H), 1.31–2.00 (m, 16H), 2.12 (m, 1H), 2.18–2.25 (m, 2H), 2.43 (s, 3H), 2.53 (m, 4H), 2.56–2.61 (m, 3H),
3
(
2
.78 (d, J = 14.8 Hz, 1H), 3.10 (m, 1H), 3.42 (m, 1H), 5.41 (brs, 1H), 6.61 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H),
13
7
1
3
.19 (d, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.96 (brs, 1H); C NMR (150 MHz, CDCl3):
7.42, 19.40, 21.34, 21.59, 23.34, 23.36, 23.55, 24.96, 28.05, 31.08, 31.09, 32.43, 34.14, 36.87, 37.17, 37.51,
8.10, 39.29, 39.77, 39.95, 42.66, 46.38, 46.64, 47.92, 51.40, 53.32, 54.15, 106.25, 110.19, 117.76, 122.34,
δ 11.79, 15.82, 16.85,
−1
1
1
6
25.99, 128.01, 128.53, 134.59, 139.24, 141.23, 178.03; IR (KBr, cm ): 3393, 3301, 2924, 2854, 1634, 1508,
457, 1378, 1306, 1185, 1083, 795; HRMS (ESI): m/z [M + H] calcd. for C H N O: 640.5206; found:
+
43
66
3
40.5203.
0
0
4
.3.4. N-(2-(Piperidin-1-yl)ethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (4d)
◦
1
Yellow solid; Yield: 90%; mp. 195–197 C; H NMR (600 MHz, CDCl ):
δ
0.89 (s, 3H), 0.94 (d,
J = 6.5 Hz, 3H), 0.95 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.16 (s, 3H), 1.21 (s, 3H), 1.30 (s, 3H), 1.35–2.10 (m,
3H), 2.10–2.26 (m, 3H), 2.36–2.42 (m, 6H), 2.43 (s, 3H), 2.78 (d, J = 14.8 Hz, 1H), 3.23 (m, 1H), 3.37
m, 1H), 5.44 (brs, 1H), 6.62 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.21 (s, 1H),
7
2
4
3
2
(
.85 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ 15.87, 16.90, 17.45, 19.44, 21.36, 21.59, 23.35, 23.37, 23.63,
4.48, 25.02, 26.18, 28.09, 31.13, 31.15, 32.48, 34.18, 36.08, 37.24, 37.51, 38.15, 39.27, 39.84, 40.03, 42.75,
6.43, 48.00, 53.37, 54.25, 54.44, 57.15, 106.34, 110.20, 117.79, 122.42, 126.04, 128.09, 128.58, 134.64, 139.29,
41.22, 178.08; IR (KBr, cm ): 3312, 2926, 2853, 1633, 1508, 1456, 1379, 1305, 1127, 794, 736; HRMS (ESI):
m/z [M + H] calcd. for C H N O: 652.5206; found: 652.5212.
−
1
1
+
44
66
3
0 0
.3.5. N-(2-Morpholinoethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (4e)
4
◦
1
Yellow solid; Yield: 82%; mp. 104–105 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.93 (d,
J = 6.5 Hz, 3H), 0.94 (s, 3H), 0.98 (d, J = 6.5 Hz, 3H), 1.16 (s, 3H), 1.20 (s, 3H), 1.34 (s, 3H), 1.36–2.35 (m,
2H), 2.43 (s, 3H), 2.50 (brs, 4H), 2.77 (d, J = 14.8 Hz, 1H), 3.26 (m, 1H), 3.43 (m, 1H), 3.76 (brs, 4H), 5.44
3
2
(
brs, 1H), 6.53 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.76 (brs, 1H); 13
C
NMR (150 MHz, CDCl3):
δ
15.92, 16.97, 17.45, 19.44, 21.36, 21.60, 22.82, 23.41, 23.69, 25.05, 25.79, 28.10,
3
5
0.36, 31.16, 31.58, 34.18, 37.24, 37.53, 38.16, 39.30, 39.85, 40.05, 42.83, 46.40, 48.10, 53.36, 53.54, 54.32,
7.14, 67.01, 106.37, 110.19, 117.87, 122.51, 125.89, 128.21, 128.59, 134.64, 139.63, 141.14, 178.22; IR (KBr,
−
1
cm ): 3313, 2954, 2923, 2852, 1635, 1498, 1458, 1379, 1303, 1266, 1187, 1119, 1081, 967, 792, 739; HRMS
+
(ESI): m/z [M + H] calcd. for C H N O : 654.4999; found: 654.4995.
43
64
3
2

Int. J. Mol. Sci. 2020, 21, 2876
12 of 20
0
0
4
.3.6. N-(2-(4-Methylpiperazin-1-yl)ethyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-
carboxamide (4f)
◦
1
Yellow solid; Yield: 87%; mp. 188–190 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.94 (d,
3
J = 6.5 Hz, 3H), 0.95 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.31–2.05 (m,
1
1
1
2
4
6H), 2.10–2.25 (m, 3H), 2.31 (s, 3H), 2.43 (s, 3H), 2.48–2.60 (m, 12H), 2.77 (d, J = 14.8 Hz, 1H), 3.23 (m,
H), 3.37 (m, 1H), 5.43 (brs, 1H), 6.53 (brs, 1H), 6.92 (d, J = 8.2 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.21 (s,
H), 7.97 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ
15.96, 16.87, 17.43, 19.37, 21.34, 21.58, 23.27, 23.32,
3.66, 25.01, 28.01, 31.08, 31.09, 32.39, 34.13, 36.00, 37.22, 37.43, 38.09, 39.22, 39.75, 39.98, 42.75, 46.13,
6.34, 47.99, 52.88, 53.27, 54.21, 55.28, 56.46, 106.17, 110.21, 117.70, 122.37, 125.91, 128.01, 128.46, 134.58,
−1
1
1
39.44, 141.20, 178.16; IR (KBr, cm ): 3404, 3311, 2924, 2868, 2853, 2791, 1635, 1507, 1457, 1377, 1301,
+
165, 1094, 1013, 795, 736; HRMS (ESI): m/z [M + H] calcd. for C H N O: 667.5315; found: 667.5309.
44
67
4
0 0
.3.7. N-(3-Aminopropyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (5a)
4
◦
1
Yellow solid; Yield: 73%; mp. 205–207 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.91 (d,
J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.1 Hz, 3H), 1.14 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.32–2.10 (m,
9H), 2.10–2.30 (m, 3H), 2.42 (s, 3H), 2.62 (brs, 2H), 2.76 (d, J = 14.6 Hz, 1H), 2.78 (m, 2H), 3.13 (m, 1H),
.44 (m, 1H), 5.42 (brs, 1H), 6.39 (brs, 1H), 6.93 (d, J = 8.2 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.20 (s, 1H),
.82 (brs, 1H); 13C NMR (150 MHz, CDCl3):
15.91, 16.91, 17.39, 19.40, 21.35, 21.60, 23.36, 23.39, 23.65,
3
1
3
7
2
4
δ
5.02, 26.67, 28.04, 31.08, 31.14, 32.42, 34.16, 37.22, 37.56, 38.12, 39.23, 39.65, 39.79, 40.00, 42.79, 46.38,
7.99, 53.30, 54.07, 55.34, 106.34, 110.19, 117.82, 122.46, 126.05, 128.15, 128.55, 134.59, 139.73, 141.19,
78.87; IR (KBr, cm ): 3294, 2924, 2854, 1637, 1524, 1457, 1379, 1306, 1187, 1078, 963, 794, 738; HRMS
−1
1
(
+
ESI): m/z [M + H] calcd. for C H N O: 598.4736; found: 598.4730.
40 60 3
0 0
.3.8. N-(3-(Dimethylamino)propyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (5b)
4
◦
1
Yellow powder solid; Yield: 81%; mp. 186–188 C; H NMR (600 MHz, CDCl ):
δ 0.88 (s, 3H), 0.92
3
(
(
(
1
2
3
d, J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 7.4 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.30 (s, 3H), 1.35–2.00
m, 18H), 2.10–2.25 (m, 3H), 2.26 (s, 6H), 2.30–2.40 (m, 3H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 3.07
m, 1H), 3.48 (m, 1H), 5.39 (t, J = 3.2 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.96 (brs, 1H), 7.18 (d, J = 8.2 Hz,
H), 7.20 (s, 1H), 7.81 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ
15.86, 16.94, 17.49, 19.44, 21.36, 21.59,
3.38, 23.49, 23.61, 24.92, 26.35, 28.08, 31.06, 31.14, 32.51, 34.17, 37.22, 37.70, 38.15, 39.21, 39.39, 39.83,
9.94, 42.69, 45.68, 46.44, 47.82, 53.38, 54.08, 58.76, 106.41, 110.19, 117.83, 122.44, 125.91, 128.13, 128.60,
−
1
1
1
34.65, 139.45, 141.24, 177.93; IR (KBr, cm ): 3411, 3298, 2946, 2924, 2866, 1636, 1522, 1459, 1380, 1305,
+
185, 1050, 794; HRMS (ESI): m/z [M + H] calcd. for C H N O: 626.5049; found: 626.5053.
42
64
3
0 0
.3.9. N-(3-(Diethylamino)propyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (5c)
4
◦
1
Yellow solid; Yield: 87%; mp. 147–149 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.93 (d,
J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.06 (t, J = 7.1 Hz, 6H), 1.15 (s, 3H), 1.21 (s, 3H),
.30 (s, 3H), 1.39–1.85 (m, 16H), 1.95–2.05 (m, 3H), 2.13 (m, 1H), 2.15–2.25 (m, 2H), 2.43 (s, 3H), 2.51 (t,
J = 6.4 Hz, 2H), 2.58 (q, J = 7.1 Hz, 4H), 2.77 (d, J = 14.8 Hz, 1H), 3.10 (m, 1H), 3.42 (m, 1H), 5.41 (brs,
3
1
13
1
H), 6.65 (brs, 1H), 6.93 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.85 (brs, 1H); C NMR
(
150 MHz, CDCl3): 11.28, 15.85, 16.95, 17.39, 19.44, 21.37, 21.59, 23.37, 23.46, 23.61, 24.92, 26.29, 28.10,
δ
3
5
1.11, 31.13, 32.51, 34.17, 37.22, 37.60, 38.15, 39.00, 39.25, 39.82, 39.93, 42.70, 46.44, 46.86, 47.78, 51.54,
3.38, 53.97, 106.36, 110.20, 117.81, 122.41, 125.93, 128.09, 128.58, 134.64, 139.56, 141.21, 177.97; IR (KBr,
−1
cm ): 3411, 3304, 2962, 2925, 2855, 1636, 1520, 1456, 1380, 1306, 1188, 1084, 794, 737; HRMS (ESI): m/z
+
[M + H] calcd. for C H N O: 654.5362; found: 654.5358.
44
68
3
0 0
.3.10. N-(3-(Piperidin-1-yl)propyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (5d)
4
◦
1
Yellow solid; Yield: 88%; mp. 183–185 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.93 (d,
3
J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.29 (s, 3H), 1.30–1.90 (m,

Int. J. Mol. Sci. 2020, 21, 2876
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2
(
(
2
3
3H), 1.90–2.06 (m, 2H), 2.07–2.25 (m, 3H), 2.30–2.40 (m, 6H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 3.10
m, 1H), 3.41 (m, 1H), 5.42 (brs, 1H), 6.66 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.20
s, 1H), 7.82 (brs, 1H); 13C NMR (150 MHz, CDCl3):
15.87, 17.01, 17.38, 19.45, 21.38, 21.59, 23.39, 23.52,
3.67, 24.37, 24.87, 25.64, 25.88, 28.07, 31.09, 31.15, 32.54, 34.18, 37.23, 37.62, 38.16, 39.12, 39.23, 39.84,
9.92, 42.69, 45.40, 46.45, 47.78, 53.38, 53.97, 54.87, 106.41, 110.19, 117.83, 122.44, 125.90, 128.13, 128.60,
δ
−
1
134.64, 139.58, 141.21, 178.01; IR (KBr, cm ): 3300, 2925, 2854, 1635, 1522, 1455, 1379, 1306, 1188, 1125,
+
7
94, 737; HRMS (ESI): m/z [M + H] calcd. for C H N O: 666.5362; found: 666.5369.
45
68
3
0
0
4
.3.11. N-(3-Morpholinopropyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (5e)
◦
1
Yellow solid; Yield: 90%; mp. 108–110 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.93 (d,
3
J = 6.3 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.33 (s, 3H), 1.36–2.35 (m,
2
3
7
2
4
2H), 2.42 (s, 3H), 2.43–2.50 (m, 2H), 2.51 (brs, 4H), 2.77 (d, J = 14.8 Hz, 1H), 3.09 (m, 1H), 3.44 (m, 1H),
.77 (brs, 4H), 5.41 (brs, 1H), 6.45 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.20 (s, 1H),
.89 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ 15.87, 16.97, 17.39, 19.40, 21.35, 21.59, 23.37, 23.43, 23.65,
4.93, 25.47, 28.03, 31.04, 31.11, 32.46, 34.15, 37.20, 37.58, 38.11, 38.49, 39.25, 39.78, 39.93, 42.73, 46.37,
7.85, 53.30, 53.79, 54.08, 57.37, 66.71, 106.27, 110.19, 117.79, 122.41, 125.89, 128.09, 128.51, 134.59, 139.72,
−
1
141.17, 178.21; IR (KBr, cm ): 3313, 2954, 2923, 2853, 1638, 1520, 1459, 1378, 1305, 1186, 1119, 1082, 968,
+
7
95, 739; HRMS (ESI): m/z [M + H] calcd. for C H N O : 668.5155; found: 668.5161.
44
66
3
2
0
0
4
.3.12. N-(3-(4-Methylpiperazin-1-yl)propyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-
carboxamide (5f)
◦
1
Yellow solid; Yield: 88%; mp. 171–173 C; H NMR (600 MHz, CDCl ):
δ
0.86 (s, 3H), 0.93 (d,
3
J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.4 Hz, 3H), 1.14 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.30–1.90 (m,
1
1
1
2
3
7H), 1.95–2.25 (m, 5H), 2.30 (s, 3H), 2.40 (m, 4H), 2.42 (s, 3H), 2.42–2.52 (m, 6H), 2.77 (d, J = 14.8 Hz,
H), 3.07 (m, 1H), 3.45 (m, 1H), 5.41 (brs, 1H), 6.51 (brs, 1H), 6.92 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.1 Hz,
H), 7.20 (s, 1H), 7.95 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ
15.85, 16.92, 17.37, 19.38, 21.32, 21.58,
3.34, 23.44, 23.63, 24.86, 25.77, 28.01, 31.03, 31.09, 32.44, 34.13, 37.18, 37.53, 38.09, 38.89, 39.21, 39.76,
9.91, 42.68, 46.04, 46.36, 47.78, 53.29, 53.36, 54.02, 55.07, 57.24, 106.23, 110.18, 117.75, 122.36, 125.86,
−
1
1
28.02, 128.49, 134.58, 139.68, 141.19, 178.02; IR (KBr, cm ): 3295, 2924, 2870, 2847, 2796, 1637, 1520,
+
1458, 1376, 1303, 1283, 1186, 1014, 794, 737; HRMS (ESI): m/z [M + H] calcd. for C H N O: 681.5471;
45
69
4
found: 681.5475.
0 0
.3.13. N-(4-Aminobutyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (6a)
4
◦
1
Yellow solid; Yield: 72%; mp. 256–258 C; H NMR (600 MHz, CDCl ):
δ
0.87 (s, 3H), 0.92 (d,
J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.4 Hz, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.30–2.10
m, 21H), 2.10–2.30 (m, 3H), 2.43 (s, 3H), 2.45 (brs, 2H), 2.76 (t, J = 5.2 Hz, 2H), 2.77 (d, J = 15.2 Hz,
3
(
1
H), 3.03 (m, 1H), 3.36 (m, 1H), 5.43 (brs, 1H), 6.09 (t, J = 5.3 Hz, 1H), 6.93 (dd, J = 8.2, 1.1 Hz, 1H),
13
7
1
3
.18 (d, J = 8.2 Hz, 1H), 7.20 (s, 1H), 7.86 (brs, 1H); C NMR (150 MHz, CDCl3):
9.39, 21.36, 21.61, 23.31, 23.39, 23.66, 25.08, 26.87, 28.05, 31.09, 31.13, 31.56, 32.44, 34.16, 37.23, 37.40,
8.12, 39.23, 39.37, 39.78, 40.03, 41.52, 42.84, 46.38, 47.95, 53.31, 54.21, 106.30, 110.19, 117.80, 122.44,
δ 15.93, 16.93, 17.39,
−1
1
25.98, 128.11, 128.53, 134.58, 139.96, 141.18, 178.41; IR (KBr, cm ): 3309, 2924, 2855, 1638, 1525, 1456,
+
1381, 1305, 1185, 1078, 967, 792, 738; HRMS (ESI): m/z [M + H] calcd. for C H N O: 612.4893;
41
62
3
found: 612.4887.
0 0
.3.14. N-(4-(Dimethylamino)butyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (6b)
4
◦
1
Yellow solid; Yield: 90%; mp. 163–165 C; H NMR (600 MHz, CDCl ):
δ
0.86 (s, 3H), 0.91 (d,
3
J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.96 (d, J = 6.0 Hz, 3H), 1.14 (s, 3H), 1.20 (s, 3H), 1.29 (s, 3H), 1.35–2.00
(
m, 20H), 2.10–2.25 (m, 3H), 2.32 (s, 6H), 2.30–2.42 (m, 3H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 3.03
m, 1H), 3.35 (m, 1H), 5.41 (t, J = 3.1 Hz, 1H), 6.15 (t, J = 5.3 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 7.18 (d,
(
J = 8.2 Hz, 1H), 7.20 (s, 1H), 7.99 (brs, 1H); 13C NMR (150 MHz, CDCl3):
δ 15.89, 16.87, 17.36, 19.38,

Int. J. Mol. Sci. 2020, 21, 2876
14 of 20
2
3
1.34, 21.60, 23.32, 23.35, 23.63, 24.49, 24.99, 27.15, 28.02, 31.05, 31.11, 32.41, 34.15, 37.21, 37.42, 38.09,
9.16, 39.20, 39.76, 39.98, 42.78, 44.93, 46.36, 47.89, 53.30, 54.09, 58.86, 106.22, 110.22, 117.76, 122.38,
−1
1
1
6
25.95, 128.04, 128.50, 134.59, 139.84, 141.22, 178.35; IR (KBr, cm ): 3287, 2924, 2855, 1637, 1523, 1459,
306, 1276, 1187, 1083, 794, 737; HRMS (ESI): m/z [M + H] calcd. for C H N O: 640.5206; found:
40.5201.
+
43
66
3
0
0
4
.3.15. N-(4-(Diethylamino)butyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (6c)
◦
1
Yellow solid; Yield: 88%; mp. 132–134 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.92 (d,
J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 1.02 (t, J = 7.1 Hz, 6H), 1.15 (s, 3H), 1.21 (s, 3H),
.30 (s, 3H), 1.36–2.05 (m, 21H), 2.13 (m, 1H), 2.18–2.25 (m, 2H), 2.43 (s, 3H), 2.45 (m, 2H), 2.53 (q,
J = 7.1 Hz, 4H), 2.77 (d, J = 14.8 Hz, 1H), 3.04 (m, 1H), 3.37 (m, 1H), 5.43 (brs, 1H), 6.01 (brs, 1H), 6.93 (d,
J = 8.2 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.21 (s, 1H), 7.88 (brs, 1H); 13C NMR (150 MHz, CDCl3):
3
1
δ
11.61, 15.90, 16.91, 17.38, 19.40, 21.34, 21.58, 23.31, 23.34, 23.66, 24.73, 25.08, 27.54, 28.07, 31.09, 31.11,
3
2.45, 34.17, 37.26, 37.37, 38.12, 39.26, 39.53, 39.80, 40.04, 42.86, 46.41, 46.93, 47.94, 52.60, 53.35, 54.27,
−1
1
06.26, 110.21, 117.77, 122.41, 125.97, 128.07, 128.54, 134.64, 140.03, 141.21, 178.22; IR (KBr, cm ): 3304,
+
2
964, 2926, 2869, 1637, 1524, 1456, 1379, 1306, 1262, 1086, 1051, 796; HRMS (ESI): m/z [M + H] calcd.
for C H N O: 668.5519; found: 668.5523.
45
70
3
0 0
.3.16. N-(4-(Piperidin-1-yl)butyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (6d)
4
◦
1
Yellow solid; Yield: 90%; mp. 121–123 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.93 (d,
J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.30 (s, 3H), 1.37–2.08 (m,
7H), 2.08–2.25 (m, 3H), 2.25–2.42 (m, 6H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 3.03 (m, 1H), 3.35 (m,
H), 5.43 (brs, 1H), 6.02 (brs, 1H), 6.93 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.85 (brs,
3
2
1
1
13
H); C NMR (150 MHz, CDCl3):
4.30, 25.08, 25.64, 27.51, 28.08, 31.11, 31.14, 32.47, 34.17, 37.27, 37.41, 38.13, 39.25, 39.45, 39.82, 40.04,
2.86, 46.43, 47.94, 53.35, 54.22, 54.46, 58.76, 106.34, 110.21, 117.81, 122.46, 125.99, 128.12, 128.57, 134.65,
δ 15.91, 16.93, 17.38, 19.41, 21.35, 21.59, 23.33, 23.37, 23.67, 24.18,
2
4
1
−1
40.01, 141.22, 178.28; IR (KBr, cm ): 3298, 2924, 2853, 1637, 1526, 1456, 1379, 1306, 1188, 1082, 963, 793,
+
7
38; HRMS (ESI): m/z [M + H] calcd. for C H N O: 680.5519; found: 680.5516.
4
6
70
3
0
0
4
.3.17. N-(4-Morpholinobutyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide (6e)
◦
1
Yellow solid; Yield: 86%; mp. 221–223 C; H NMR (600 MHz, CDCl ):
δ
0.88 (s, 3H), 0.92 (d,
J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.29 (s, 3H), 1.30–2.05 (m,
3H), 2.13 (m, 1H), 2.22 (m, 2H), 2.35 (brs, 4H), 2.43 (s, 3H), 2.77 (d, J = 14.8 Hz, 1H), 3.04 (m, 1H), 3.37
m, 1H), 3.71 (m, 4H), 5.43 (brs, 1H), 5.99 (t, J = 5.5 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 7.18 (d, J = 8.0 Hz,
H), 7.21 (s, 1H), 7.93 (brs, 1H); 13C NMR (150 MHz, CDCl3):
15.91, 16.90, 17.39, 19.37, 21.34, 21.59,
3.29, 23.35, 23.64, 24.18, 25.08, 27.39, 28.04, 31.07, 31.10, 32.42, 34.14, 37.23, 37.37, 38.09, 39.26, 39.48,
9.77, 40.01, 42.85, 46.37, 47.93, 53.28, 53.81, 54.27, 58.65, 67.01, 106.22, 110.20, 117.78, 122.42, 125.93,
3
2
(
1
2
3
δ
−
1
1
28.07, 128.50, 134.60, 140.08, 141.17, 178.27; IR (KBr, cm ): 3314, 2953, 2925, 2853, 1635, 1530, 1456,
376, 1305, 1274, 1118, 1084, 788, 732; HRMS (ESI): m/z [M + H] calcd. for C H N O : 682.5312;
+
1
45
68
3
2
found: 682.5317.
0
0
4
.3.18. N-(4-(4-Methylpiperazin-1-yl)butyl)-5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-
carboxamide (6f)
◦
1
Yellow solid; Yield: 91%; mp. 146–148 C; H NMR (600 MHz, CDCl ):
δ 0.87 (s, 3H), 0.92 (d,
3
J = 6.1 Hz, 3H), 0.96 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.29 (s, 3H), 1.30–2.10 (m,
0H), 2.10–2.25 (m, 3H), 2.28 (s, 3H), 2.35 (brs, 4H), 2.43 (s, 3H), 2.44–2.51 (m, 7H), 2.77 (d, J = 14.8 Hz,
H), 3.04 (m, 1H), 3.35 (m, 1H), 5.42 (brs, 1H), 5.99 (brs, 1H), 6.93 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
H), 7.20 (s, 1H), 7.88 (brs, 1H); 13C NMR (150 MHz, CDCl3):
15.94, 16.91, 17.38, 19.39, 21.33, 21.57,
3.30, 23.35, 23.66, 24.51, 25.08, 27.43, 28.06, 31.08, 31.11, 32.44, 34.16, 37.26, 37.37, 38.11, 39.25, 39.53,
9.80, 40.03, 42.86, 46.01, 46.40, 47.94, 53.13, 53.33, 54.27, 55.07, 58.16, 106.27, 110.20, 117.78, 122.42,
2
1
1
δ
2
3

Int. J. Mol. Sci. 2020, 21, 2876
15 of 20
1
25.95, 128.08, 128.54, 134.62, 140.04, 141.19, 178.26; IR (KBr, cm 1): 3289, 2925, 2854, 2799, 1638, 1522,
−
+
1457, 1375, 1305, 1282, 1165, 1013, 794, 737; HRMS (ESI): m/z [M + H] calcd. for C H N O: 695.5628;
46 71 4
found: 695.5620.
4
.4. General Procedure for the Synthesis of Compounds 7a–c
To a solution of compound (98 mg, 0.18 mmol) in CH Cl (5 mL) were added HOBt (36 mg,
.24 mmol) and DCC (48 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 0.5
3
2
2
0
h and then ethane-1,2-diamine, propane-1,3-diamine, or butane-1,4-diamine (0.09 mmol) was added.
The mixture was stirred for 12 h at room temperature. At the end of reaction, the mixture was worked
up as above to afford the corresponding dimer product 7a–c with 2-, 3- and 4-carbon chain, respectively
(Scheme 2).
0
0
0
4
.4.1. N,N -(ethane-1,2-diyl)-bis(5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide) (7a)
◦
1
Yellow solid; Yield: 61%; mp. 301–303 C; H NMR (600 MHz, CDCl ):
δ
0.86 (s, 6H), 0.93 (d,
J = 6.2 Hz, 6H), 0.95 (s, 6H), 0.98 (s, 6H), 1.16 (s, 6H), 1.19 (s, 6H), 1.29 (s, 6H), 1.30–2.10 (m, 34H),
.10–2.28 (m, 6H), 2.44 (s, 6H), 2.77 (d, J = 14.8 Hz, 2H), 3.14 (m, 2H), 3.48 (m, 2H), 5.48 (brs, 2H), 6.58
3
2
(
brs, 2H), 6.93 (d, J = 7.9 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.21 (s, 2H), 7.86 (brs, 1H), 7.92 (brs, 1H); 13
C
NMR (150 MHz, CDCl3):
δ
15.88, 16.85, 17.39, 19.38, 21.39, 21.61, 23.35, 23.39, 23.64, 25.02, 25.07, 28.08,
3
1.11, 32.37, 34.07, 34.14, 37.19, 37.65, 38.11, 39.22, 39.77, 39.97, 42.68, 46.38, 47.86, 53.30, 53.73, 106.30,
−
1
110.18, 117.84, 122.42, 126.33, 128.10, 128.54, 134.60, 139.29, 141.20, 179.39; IR (KBr, cm ): 3415, 3322,
+
2
923, 2853, 1639, 1522, 1456, 1379, 1304, 1237, 1083, 963, 792, 732; HRMS (ESI): m/z [M + H] calcd. for
C H N O : 1107.8394; found: 1107.8387.
76
107
4
2
0
0
0
4
.4.2. N,N -(propane-1,3-diyl)-bis(5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide) (7b)
◦
1
Yellow solid; Yield: 65%; mp. 276–278 C; H NMR (600 MHz, CDCl ):
δ 0.87 (s, 6H), 0.92–0.98
3
(
(
m, 18H), 1.15 (s, 6H), 1.16 (s, 3H), 1.21 (s, 3H), 1.28 (s, 3H), 1.33 (s, 3H), 1.30–2.10 (m, 36H), 2.10–2.26
m, 6H), 2.42 (s, 3H), 2.43 (s, 3H), 2.77 (m, 2H), 3.13 (m, 2H), 3.27 (m, 2H), 5.37 (brs, 1H), 5.48 (brs, 1H),
6
(
(
.92 (d, J = 7.1 Hz, 1H), 6.93 (d, J = 7.1 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.20
s, 2H), 7.93 (brs, 2H); 13C NMR (150 MHz, CDCl3):
15.91 (15.94), 17.03 (17.05), 17.34 (17.36), 19.66
19.68), 21.35 (21.37), 21.57 (21.61), 23.30 (23.33), 23.34 (23.37), 23.63 (23.65), 24.86 (24.99), 26.59 (26.69),
δ
2
3
8.00 (28.09), 31.05 (31.11), 32.50, 34.12 (34.15), 34.24 (34.26), 36.38 (36.41), 37.63 (37.66), 38.10 (38.11),
9.09 (39.11), 39.15 (39.16), 39.80 (39.85), 42.63 (42.72), 46.37 (46.38), 46.86 (46.89), 47.98 (48.00), 53.86
(53.91), 55.29 (55.35), 106.24 (106.27), 110.17 (110.21), 117.76 (117.80), 122.36 (122.42), 126.08 (126.11),
1
28.01 (128.06), 128.50 (128.52), 134.60 (134.61), 139.41 (139.47), 141.15 (141.18), 178.37 (178.57); IR (KBr,
−
1
+
cm ): 3318, 2924, 2853, 1634, 1521, 1455, 1379, 1305, 1083, 963, 796; HRMS (ESI): m/z [M + H] calcd.
for C H N O : 1121.8551; found: 1121.8558.
77
109
4
2
0
0
0
4
0
.4.3. N,N -(butane-1,4-diyl)-bis(5 -methyl-1 H-ursa-2,12-dieno[3,2-b]indol-28-carboxamide) (7c)
◦
1
Yellow solid; Yield: 63%; mp. 255–258 C; H NMR (600 MHz, CDCl ):
δ
0.86 (s, 3H), 0.87 (s, 3H),
.89–0.92 (m, 12H), 0.94 (s, 3H), 0.96 (s, 3H), 1.07 (s, 3H), 1.08 (s, 3H), 1.13 (s, 3H), 1.15 (s, 3H), 1.24 (s, 3H),
.25 (s, 3H), 1.30–2.08 (m, 38H), 2.08–2.25 (m, 6H), 2.31 (s, 3H), 2.32 (s, 3H), 2.68 (d, J = 14.7 Hz, 2H), 2.99
m, 4H), 5.31 (brs, 2H), 6.78 (d, J = 8.2 Hz, 2H), 7.06 (s, 2H), 7.08–7.15 (m, 4H), 10.50 (d, J = 7.1 Hz, 2H);
3
1
(
1
3
C NMR (150 MHz, CDCl3):
23.01), 23.18 (23.19), 23.52 (23.64), 26.33 (26.41), 27.37 (27.41), 30.55 (30.56), 33.66, 33.80 (33.82), 36.12,
6.82, 37.05, 37.57, 38.45, 41.72 (41.81), 45.76, 46.04, 46.53, 46.65, 52.20, 53.01, 54.19, 104.23, 110.16, 117.08,
21.34, 124.76, 125.91, 127.77, 134.54, 138.45, 141.21, 176.04; IR (KBr, cm ): 3313, 2925, 2853, 1635, 1523,
455, 1379, 1306, 1087, 792, 732; HRMS (ESI): m/z [M + H] calcd. for C H N O : 1135.8707; found:
δ 15.52, 16.72 (16.74), 17.12, 19.03 (19.10), 21.10, 21.28, 22.75 (22.76), 23.00
(
3
1
1
1
−
1
+
7
8
111
4
2
135.8715.

Int. J. Mol. Sci. 2020, 21, 2876
16 of 20
4
.5. Biological Evaluation
4
.5.1. In Vitro Anti-Proliferation Assay
Compounds 4a
–
f,
5a–
f
,
6a
–
f
, and 7a
–
c
were evaluated for their antitumor activity against two
human hepatocarcinoma cells (HepG2 and SMMC-7721) and the human normal hepatocyte cell line
LO2) using the typical MTT assay according to the literature [31]. Cell lines were obtained from
American Tissue Culture Collection (ATCC). Briefly, different tumor cells were grown in DMEM medium
supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (50 g/mL).
Cells were harvested at log phase of growth and seeded in 96-well plates (100 L/well at a density
10 cells/mL). After 24 h incubation at 37 C and 5% CO to allow cell attachment, cultures
(
µ
µ
5
◦
of 2
×
2
were exposed to various concentrations of the isolated compounds for 48 h. Finally, MTT solution
2.5 mg/mL in PBS) was added (40
◦
(
µ
L/well). Plates were further incubated for 4 h at 37 C, and the
formazan crystals formed were dissolved by adding 150
µL/well of DMSO. Absorption at 570 nm was
measured with an ELISA plate reader. The results were expressed as IC50 values, which was defined
as the concentration at which 50% survival of cells was obtained. Doxorubicin, camptothecin, and
etoposide (VP-16) were co-assayed as positive controls.
4
.5.2. Topoisomerase I Inhibition Assay
The activity of DNA topoisomerase I (TaKaRa, Kyoto, Japan) was determined by measuring the
relaxation of supercoiled DNA pBR322 [32]. Camptothecin was used as a positive control. The reaction
◦
mixture was prepared according to the manufacture’s protocol, and incubated at 37 C for 30 min. The
reactions were terminated by adding dye solution containing 1% SDS, 0.02% bromophenol blue and
5
0% glycerol. The mixtures were applied to 1% agarose gel and subjected to electrophoresis for 1 h, in
TAE buffer (40 mM Tris-acetate, 1 mM EDTA). Gels were stained for 30 min in 60 mL 1 TAE buffer
with ethidium bromide (0.5 g/mL). DNA bands were visualized by transillumination with UV light
and then photographed by a Gel Doc XR+ System (BIO-RAD, Hercules, CA, USA).
1
×
×
µ
4
.5.3. Topoisomerase II Inhibition Assay
Human Topoisomerase II activity was detected by measuring the relaxation of supercoiled
pBR322 plasmid DNA [22]. Reaction mixture contained 10 mM Tris (pH 6.9), 50 mM NaCl, 50 mM KCl,
mM MgCl , 1 mM ATP, pBR322 DNA (100 ng), and 2 units of Topo II (USB, Affymetrix, Waltham,
L. For inhibition studies, the compounds were pre-incubated with
and DNA for 15 min. Compounds were used at the appropriate concentrations
α
5
α
2
MA, USA) in a final volume of 20
human Topo II
µ
α
by dissolving in 2% (v/v) DMSO. DMSO didn’t show detrimental effect on the enzyme activity at
◦
concentration up to 2% (v/v). Reaction mixture was incubated at 37 C for 30 min and stopped by the
addition of 7 mM EDTA. Reaction product was mixed with DNA loading dye and electrophoresed on
1
% TAE-Agarose. The gel was stained with ethidium bromide (0.5
in 1 TAE buffer, and then visualized using a BIO-RAD Gel Doc XR+ System. Etoposide (VP-16) was
co-assayed as a positive control.
µg/mL) for 30 min, destained twice
×
4
.5.4. Molecular Docking
The molecular modeling of compound 5f was performed with Schrödinger Suite 2015-1
Schrödinger LLC., New York, NY, USA). The crystal structure of Topo II (PDB ID: 5GWK) was
(
α
downloaded from Protein Data Bank (PDB) and prepared using the Protein Preparation Wizard
workflow from Schrödinger Suite, including the optimization of hydrogen bond network and a short
energy minimization with position restraints on heavy atoms using OPLS_2005 force field. The docking
grid was generated according to the initial ligand etoposide. Then, the target compounds were freely
docked into the designated binding site using the standard protocol implemented in Maestro v 10.1
(
0
Schrödinger LLC, Cambridge, MA, USA). Van der Waals (vdW) scaling of 0.8 and partial cut-off of
.15 were set to soften the potential for non-polar sites, and no constraints were specified. The best

Int. J. Mol. Sci. 2020, 21, 2876
17 of 20
docked pose ranked by Glide Score value was recorded and saved for the ligand. The structures of
complexes were analyzed for interaction modes, and the binding pose of compound 5f with Topo II
α
was displayed using the Discovery Studio 2016 client (Accelrys Software, San Diego, CA, USA).
4
.5.5. Cell Apoptosis Analysis
The extent of apoptosis was quantitatively measured using Annexin V-FITC/PI dual staining
5
assay [33]. SMMC-7721 cells were seeded into a six-well plate at 5
×
10 cells per well in 10% fetal
calf serum (FBS)-DMEM into six-well plates and treated with different concentrations of the indicated
compound 5f for 48 h. The cells were detached with 0.25% trypsin, washed with ice-cold PBS for twice
and then resuspended in 1
The cells were stained with 5
×
Binding buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2).
L of Annexin V-FITC and 5 L of PI (propidium indole) to each tube.
µ
µ
The cells were gently vortexed and incubated in the dark at room temperature for 15 min and then kept
◦
at 4 C. The samples were analyzed by a flow cytometer (Becton-Dickinson FACSCalibur, Franklin
Lakes, NJ, USA), and data were analyzed using the FlowJo software (Becton-Dickinson Biosciences,
Franklin Lakes, NJ, USA).
4
.5.6. Acridine Orange/Ethidium Bromide (AO/EB) Staining
SMMC-7721 cells were seeded into six-well plates and incubated at 37 C with 5% CO for 24
◦
2
h. Then, the cells were exposed to different concentrations of compound 5f (0, 0.3, 0.6, and 1.2
µM)
for 48 h. After that, 10 L of each fluorescent dye cantaining AO and EB (10 g/mL) were added to
µ
µ
each well in equal volumes respectively and incubated for 5 min in the dark. The cells were visualized
through an inverted fluorescence microscope (Olympus 1X71 Inverted System Microscope, Olympus,
Tokyo, Japan) [26].
4
.5.7. ROS Generation Assay
ROS generation assay was performed by using the reactive oxygen species assay kit (Beyotime
Biotech., Shanghai, China). Intracellular ROS generation was tested through dichlorodihydro
fluorescein diacetate (DCFH-DA) assay [34]. DCFH-DA is taken up by SMMC-7721 cells, and
then activated by esterase-mediated cleavage of acetate to form dichlorodihydro fluorescein (DCFH),
which is trapped in the cells. DCFH is converted to fluorescein DCF in the presence of ROS. SMMC-7721
cells were seeded in six-well plates and incubated with different concentrations of compound 5f for
◦
48 h. After removing the compound solution, cells were treated with 10
µ
M of DCFH-DA at 37 C
for 20 min. Subsequently, the cells were washed with PBS for three times and then exposed to light.
Immediately after light exposure, the fluorescence intensity of dichlorofluorescein (DCF) was measured
with excitation at 488 nm and emission at 525 nm by a flow cytometry (Becton-Dickinson FACSCalibur,
Franklin Lakes, NJ, USA).
4
.5.8. JC-1 Mitochondrial Membrane Potential Assay
The JC-1 mitochondrial membrane potential assay kit (Keygene Biotech., Nanjing, China) was
employed to measure mitochondrial depolarization in SMMC-7721 cells. The fluorescence probe JC-1
was used to detect the change of mitochondrial membrane potential [35]. Briefly, cells cultured in
six-well plates after indicated treatments by compound 5f were incubated with an equal volume of JC-1
◦
staining solution (5
µ
g/mL) at 37 C for 20 min and rinsed twice with PBS. Mitochondrial membrane
potentials were monitored by determining the relative amounts of dual emissions from mitochondrial
JC-1 monomers or aggregates using flow cytometry (Becton-Dickinson FACSCalibur, Franklin Lakes,
NJ, USA). Mitochondrial membrane depolarization is indicated by an increase in the percentage of
cells with low ∆Ψm (green fluorescence, lower right quadrant) compared with cells with high ∆Ψ
red fluorescence, upper right quadrant).
m
(

Int. J. Mol. Sci. 2020, 21, 2876
18 of 20
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/8/2876/
s1.
Author Contributions: W.G. conceived and designed the experiments and wrote the manuscript; A-L.L. and Y.H.
synthesized and characterized the compounds; W.-Y.W. and Y.H. determined the biological activities; A-L.L.,
Q.-S.L., and Y.S. analyzed the experimental data and wrote the experimental part. All authors gave approval to
the final version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (31770616), the Natural
Science Foundation for Colleges and Universities in Jiangsu Province (17KJA220002), and the Top-Notch Academic
Programs Project of Jiangsu Higher Education Institutions (PPZY2015C221).
Acknowledgments: The authors would thank the Modern Analysis and Testing Center, Nanjing Forestry
University, for the measurement of spectroscopic data.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
Topo
UA
Topoisomerase
Ursolic acid
HOBt
DCC
MTT
PI
1-Hydroxybenzotriazole
N,N -Dicyclohexylcarbodiimide
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
Propidium Iodide
0
AO
Acridine orange
EB
Ethidium bromide
ROS
EDTA
PBS
SDS
Reactive oxygen species
Ethylene diamine tetraacetic acid
Phosphate buffer saline
Sodium dodecyl sulfate
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(