Synthesis, anticancer evaluation and mechanism studies of novel indolequinone derivatives of ursolic acid

Article abstract of DOI:10.1016/j.bioorg.2021.104705

A series of novel indolequinone derivatives of ursolic acid bearing ester, hydrazide, or amide moieties were designed, synthesized, and screened for their in vitro antiproliferative activities against three cancer cell lines (MCF-7, HeLa, and HepG2) and a normal gastric mucosal cell line (Ges-1). A number of compounds showed significant activity against tested cancer cell lines. Among them, compound 6t exhibited the most potent activity against three cancer cell lines with IC₅₀ values of 1.66 ± 0.21, 3.16 ± 0.24, and 10.35 ± 1.63 μM, respectively, and considerably lower cytotoxicity to Ges-1 cells. Especially, compound 6t could arrest cell cycle at S phase, suppress the migration of MCF-7 cells, elevate intracellular reactive oxygen species (ROS) level, and decrease mitochondrial membrane potential. Western blot analysis showed that compound 6t upregulated Bax, cleaved caspase-3/9, cleaved PARP levels and downregulated Bcl-2 level of MCF-7 cells. All these results indicated that compound 6t could significantly induce the apoptosis of MCF-7 cells. Meanwhile, compound 6t markedly decreased p-AKT and p-mTOR expression, which revealed that compound 6t probably exerted its cytotoxicity through targeting PI3K/AKT/mTOR signaling pathway. Therefore, compound 6t could be a promising lead for the discovery of novel anticancer agents.

Full text of DOI:10.1016/j.bioorg.2021.104705

Bioorganic Chemistry 109 (2021) 104705
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, anticancer evaluation and mechanism studies of novel
indolequinone derivatives of ursolic acid
Wen-Yan Wang, Wen-Yi Wu, A-Liang Li, Qing-Song Liu, Yue Sun, 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-Inovation Center
for Efficient Processing and Utilization of Forest Products, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel indolequinone derivatives of ursolic acid bearing ester, hydrazide, or amide moieties were
designed, synthesized, and screened for their in vitro antiproliferative activities against three cancer cell lines
(MCF-7, HeLa, and HepG2) and a normal gastric mucosal cell line (Ges-1). A number of compounds showed
significant activity against tested cancer cell lines. Among them, compound 6t exhibited the most potent activity
against three cancer cell lines with IC50 values of 1.66 ± 0.21, 3.16 ± 0.24, and 10.35 ± 1.63 µM, respectively,
and considerably lower cytotoxicity to Ges-1 cells. Especially, compound 6t could arrest cell cycle at S phase,
suppress the migration of MCF-7 cells, elevate intracellular reactive oxygen species (ROS) level, and decrease
mitochondrial membrane potential. Western blot analysis showed that compound 6t upregulated Bax, cleaved
caspase-3/9, cleaved PARP levels and downregulated Bcl-2 level of MCF-7 cells. All these results indicated that
compound 6t could significantly induce the apoptosis of MCF-7 cells. Meanwhile, compound 6t markedly
decreased p-AKT and p-mTOR expression, which revealed that compound 6t probably exerted its cytotoxicity
through targeting PI3K/AKT/mTOR signaling pathway. Therefore, compound 6t could be a promising lead for
the discovery of novel anticancer agents.
Ursolic acid
Indolequinone
Anticancer
PI3K/AKT/mTOR signaling pathway
Apoptosis
1. Introduction
a variety of biological activities such as antimicrobial [9], antiviral [10],
anti-inflammatory [11], antidiabetic [12], antiulcer [13], anti-
The incidence and mortality of cancer are rapidly growing world-
wide, which causes tremendous threat to human life and health. Ac-
cording to Annual World Cancer Report, there were approximately 18.1
million new cases and 9.6 million cancer deaths worldwide in 2018 [1].
Moreover, cancer cases are expected to rise to almost 20 million by 2025
[2]. Despite chemotherapy was considered as one of the most effective
methods for cancer treatment in the past few decades, the severe side
effects and rapidly occurred drug-resistance have dramatically reduced
its clinical efficacy [3,4]. Therefore, there is still an urgent demand in
developing new anticancer agents with a better therapeutic index.
Natural products have been considered as a rich source of the basic
scaffold for drug discovery. The majority of anticancer drugs currently
used are derived from natural product scaffolds [5]. Triterpenoids
contains a large number of natural products with diverse structures. In
particular, many triterpenes exhibit various biological activities without
significant toxicity even at high concentrations [6–8]. Ursolic acid (UA,
1) is a natural ursane-type triterpenoid abundant in many traditional
Chinese medicines. UA and its derivatives have been reported to exhibit
osteoporosis [14] and anti-neurodegenerative activities [15]. In addi-
tion, many UA derivatives have demonstrated prominent anticancer
activities through different mechanisms including regulation of
apoptosis, inhibition of DNA polymerase, modulation of signal trans-
ductions and interfere with angiogenesis and metastasis [16–18].
Therefore, UA could be a promising starting material for the develop-
ment of novel anticancer agents.
Heterocycles are considered as privileged scaffolds in drug discovery
and widely used in medicinal chemistry [19,20]. Connecting nitrogen-
containing heterocyclic structures to certain drug precursors can often
enhance biological activity [21]. Concerning the drugs approved by the
Food and Drug Administration, 59% of unique small molecule drugs
contain nitrogen heterocyclic compounds [22]. Indolequinone is a kind
of important heterocycle that is widely present in many drugs or
bioactive molecules. Previous studies have shown that indolequinone
derivatives have multiple functions such as antibacterial, anti-
inflammatory, anti-cancer, and antioxidant properties [23,24]. Some
natural products (Fig. 1) bearing indolequinone pharmacophore are
* Corresponding author.
E-mail address: njguwen@163.com (W. Gu).
https://doi.org/10.1016/j.bioorg.2021.104705
Received 15 September 2020; Received in revised form 15 December 2020; Accepted 28 January 2021
Available online 9 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
powerful anticancer agents such as mitomycin C (MMC) [25], mur-
rayaquinone A and B [26], exiguamine A [27], zyzzyanone B [28], and
BE-10988 [29]. Inspired by these natural molecules, a large number of
synthetic indolequinones have been developed, including the MMC
analogue apaziquone (EO9) (Fig. 1), which is currently in advanced
clinical trials as a treatment for bladder cancer [30]. Due to their potent
biological activities, it is speculated that the fusion of indolequinone
moiety with the molecule of UA would probably change its physico-
chemical properties and lead to better biological activities.
acylhydrazine derivatives 6e-j in 72 ~ 76% yields. On the other hand,
compounds 5 were converted to amide derivatives 6k–t in 67 ~ 78%
yields by reacting with the corresponding amine, 1-hydroxybenzotria-
zole (HOBt) and dicyclohexylcarbodiimide (DCC). All the synthesized
title compounds were purified by recrystallization or silica gel column
chromatography.
The structures of these compounds were characterized by their
HRMS, 1H NMR and 13C NMR spectra. As for a typical example 6t, its
molecular formula was determined to be C43H63N3O4 through its HRMS
spectrum (m/z [M+H]+ calcd for C43H64N3O4: 686.4897; found:
686.4901). IR spectrum of 6t exhibited broad bands at 3417 and 3223
On the other hand, previous studies indicated that the incorporation
of polar moiety onto the C-3 or C-28 position of UA might improve its
solubility and bioavailability [31,32]. In a former literature, UA was
conjugated with different amino acids on the C-28 carboxyl groups.
Some derivatives with amino groups exhibited more potent cytotoxic
activity than UA and the derivatives bearing carboxyl groups [33]. The
hydrazide moiety is also an important pharmacophore in drug devel-
opment. Many compounds containing hydrazide groups exhibit potent
antiviral, anti-inflammatory, and anti-cancer biological activities
[34–36]. Therefore, the introduction of amide and hydrazide moieties to
the molecule of UA might also produce derivatives with promising
anticancer activities. In continuation of our research on novel antitumor
derivatives of UA [37,38], we would like to report the synthesis and
cytotoxic activities of a new series of indolequinone derivatives of UA. In
addition, the preliminary studies on the anticancer mechanisms of
representative compound 6t were also presented.
cmꢀ 1 corresponding to the N H stretching of the amide group and
–
indolequinone ring. It also showed strong to medium absorptions
3
around 3000 ~ 2850 cmꢀ 1 contributing to the C H stretching of sp
–
carbon atoms. Moreover, the strong absorption bands at 1662 and 1641
cmꢀ 1 were due to the C O stretching vibrations of amide group and
–
–
quinone ketones. In its 1H NMR spectrum, five singlets at δ 0.84, 0.91,
1.11, 1.20 and 1.28 ppm could be assigned to five methyl groups at C23
~ C27, while the doublet at δ 0.89 ppm and the broad singlet at δ 0.95
ppm were due to the two adjacent methyl groups at C29 and C30.
Concerning the amide side chain, the singlet with six protons at δ 2.30
ppm could be attributed to the two methyl groups at C7′ and C8′. The
multiplets at δ 3.04 and 3.35 ppm were due to the methylene protons at
C2′, while the multiplet at δ 2.40 ppm could be assigned to the methy-
lene at C5′. The singlet at δ 3.81 ppm could be attributed to the methoxyl
group connected to the quinone moiety. The two singlets at δ 5.39 and
5.65 ppm were the signals of the olefinic hydrogen at C12 and the ar-
omatic proton at C35, respectively. In addition, the triplet at δ 6.12 ppm
2. Results and discussion
–
2.1. Chemistry
and the broad singlet at δ 9.51 ppm could be assigned to N H signals in
the amide group and indolequinone moiety, respectively. In the 13C
NMR spectrum of 6t, there were 42 peaks appearing in the range of δ 10
~ 185 ppm corresponding to 43 carbon atoms. Specifically, eight peaks
at δ 100 ~ 160 ppm were due to two olefinic carbons at C12 and C13,
and six aromatic carbons in the indolequinone moiety. Among them,
The peaks at δ 160.16 and 107.09 ppm could be assigned to C34 and its
adjacent carbon (C35), respectively. In addition, the peaks at δ 170.40
and 185.09 ppm were the signals of two carbonyl groups in the quinone
ring (C33 and C36, respectively), while the signal at δ 178.14 ppm could
be attributed to the amide carbonyl group (C28). In the range δ 10 ~ 60
ppm, the peak at δ 44.89 ppm (2C) could be assigned to the two methyl
groups at C7′ and C8′. The assignments of the signals in the 1H and 13C
NMR spectra of compound 6t were in good accordance with its
structure.
The title derivatives (6a-t) were designed and synthesized according
to the procedure described in Scheme 1. First, the starting material UA
(1) was oxidized by Jones reagent to form 3-oxo-ursolic acid (2) in 77%
yield. Subsequently, the indole derivative 3 was synthesized from
compound 2 and 3,5-dimethoxyphenyl hydrazine hydrochloride in 57%
yield through Fisher indole reactions. Further, compound 3 was treated
with POCl3 and DMF, then hydrolyzed to obtain compound 4 in 74%
yield, which was oxidized with H2O2 to obtain the indolequinone de-
rivative 5 in 71% yield. Concerning the modification of carboxyl group
in compound 5, the carboxyl groups were treated with thionyl chloride
to give the 28-acylchloride derivatives, which were then reacted with
different alcohols in the presence of Et3N to afford the corresponding
ester products 6a-d in 80 ~ 89% yields. Further, compounds 5 were
reacted with different hydrazides in similar procedure to obtain the
Fig. 1. Representative indolequinone derivatives with antitumor activities.
2

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
Scheme 1. Synthesis of the title indolequinone derivatives. Reagents and experimental conditions were as follows: (a) Jones reagent, acetone, 0 ◦C, 5 h; (b) (3,5-
dimethoxyphenyl)hydrazine, EtOH, conc. HCl, reflux for 4 h; (c) POCl3, DMF, rt, 3 h. (d) H2O2, HCl, MeOH, 0 ◦C, 3 h; (e) i. SOCl2, benzene, reflux for 3 h; ii. ROH or
RCONHNH2, Et3N, CH2Cl2/ether, rt, 8 ~ 12 h for compounds 6a-j; (f) the corresponding amine, HOBt, DCC, CH2Cl2, rt, 12 h, for compounds 6 k-t.
2.2. Cytotoxic assay
Table 1
Antiproliferative activities of compounds 1, 5 and 6a-t.
The in vitro cytotoxic activity of the title compounds were evaluated
Compd
IC50 value (µM)
by MTT assay against human breast cancer cell line (MCF-7), cervical
carcinoma cell line (HeLa), hepatocarcinoma cell line (HepG2) and
human normal gastric mucosal cell line (Ges-1). Etoposide was co-
assayed as the positive control. 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 three cell lines were shown in Table 1.
MCF-7
HeLa
HepG2
Ges-1
1
>50
>50
>50
NTa
5
18.03 ± 1.38
11.65 ± 1.21
3.19 ± 0.27
3.31 ± 0.26
3.39 ± 0.72
9.64 ± 0.63
10.79 ± 0.72
18.32 ± 0.20
37.70 ± 0.82
11.70 ± 1.22
22.78 ± 1.32
18.39 ± 1.97
11.84 ± 0.06
6.13 ± 0.24
9.04 ± 1.21
30.15 ± 0.17
18.76 ± 1.22
15.00 ± 1.12
2.52 ± 0.07
2.23 ± 0.19
1.66 ± 0.21
5.14 ± 0.32
23.72 ± 3.69
19.33 ± 1.61
3.39 ± 0.34
4.25 ± 0.07
4.23 ± 0.46
13.15 ± 1.93
11.08 ± 0.64
26.47 ± 1.53
21.56 ± 2.82
12.12 ± 1.92
16.28 ± 0.59
22.46 ± 1.22
10.85 ± 0.62
14.89 ± 0.83
>50
21.54 ± 2.12
>50
>50
6a
6b
6c
>50
18.50 ± 1.23
14.26 ± 1.31
22.18 ± 1.01
40.65 ± 2.37
>50
22.43 ± 0.31
23.17 ± 0.66
6d
6e
6f
23.69 ± 0.54
Among ester derivatives 6a-d, compounds 6b-d exhibited strong
cytotoxic activities against MCF-7 and HeLa cells at low µM levels (IC50
< 5 µM) and moderate activities against HepG2 cells. As for acylhy-
drazine derivatives 6e-j, most compounds showed moderate activities
against MCF-7 cells and HeLa cells, while they were almost inactive to
HepG2 cells lines (IC50 > 50 µM). Regarding amide derivative 6 k-t,
compounds 6 k-q generally displayed considerable activities against
MCF-7 cells and relatively weaker activities against HeLa cells, while
they only showed mild or no cytotoxicity against HepG2 cell lines.
Compounds 6r-t with dimethylamino group on amide side chain showed
potent cytotoxicity against MCF-7 and HeLa cells (IC50 < 5 µM). It is
worth noting that compound 6t exhibited the most potent cytotoxic
activity against MCF-7 and HeLa cells with IC50 values of 1.66 ± 0.21
and 3.16 ± 0.24 µM, respectively, stronger than positive control eto-
poside. Especially, the compound showed substantially lower cytotox-
icity against Ges-1 cells (IC50: 20.74 ± 0.61 µM), indicating good
selectivity of cytotoxicity (12.5) to MCF-7 and Ges-1 cells, which was
also better than that of etoposide (4.0) (Fig. 2). Hence, compound 6t was
selected for further investigations on its anticancer mechanisms.
From above-mentioned results, some preliminary structure–activity
relationships (SAR) could also be deduced. Most title derivatives bearing
the indolequinone heterocycles exhibited substantially stronger cyto-
toxic activity than compound 1, indicating that the introduction of
indolequinone moiety will improve the cytotoxicity of UA derivatives.
>50
NT
6g
6h
6i
>50
NT
>50
NT
>50
NT
6j
>50
NT
6k
6l
>50
NT
>50
NT
6m
6n
6o
6p
6q
6r
42.16 ± 1.78
>50
>50
>50
31.17 ± 1.37
>50
35.09 ± 1.23
>50
NT
NT
33.89 ± 0.72
3.47 ± 0.16
3.38 ± 0.60
3.16 ± 0.24
5.74 ± 0.42
30.38 ± 1.83
21.27 ± 2.01
14.90 ± 1.79
10.35 ± 1.63
8.73 ± 1.21
NT
21.63 ± 0.27
20.89 ± 0.45
20.74 ± 0.61
20.71 ± 0.49
6s
6t
Etoposide
a
NT: Not tested.
The substituents derived from the carboxyl group of UA also markedly
affected the cytotoxic potencies. Generally, compounds 6a-d with ester
groups showed substantially stronger cytotoxicity to three cancer cell
lines than compound 5 with carboxyl group, compounds 6e-j with
acylhydrazine moieties and compounds 6k-q with amide moieties.
Among amide derivatives, compounds 6o-q showed considerably
weaker cytotoxicity, which suggested that the introduction of N,N-
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W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
2.4. Compound 6t inhibited cell migration of MCF-7 cells
Metastasis is a multistep process involving cancer cell motility and
invasion which represents a key difficulty for cancer therapy. Thus, the
inhibition of metastasis is vital for efficient cancer treatment. Therefore,
the effect of compound 6t on the migration of cancer cells was investi-
gated by the wound healing assay. As shown in Fig. 4, there was almost
complete healing of wound in control group after 48 h, which indicated
the considerable migration of cancer cells. However, in 1
migration was significantly suppressed compared with the control
group. In 2 M group, the wound was only slightly healed after 48 h,
while in 4
μM group, the
μ
μ
M group, wound healing could not be observed even after 48
h. The results indicated that compound 6t could markedly suppress the
Fig. 2. The selectivity of cytotoxicity for some active compounds to MCF-7 and
migration of MCF-7 cells in a concentration-dependent manner.
Ges-1 cells.
2.5. Compound 6t induced apoptosis of MCF-7 cells
dialkylamide group will decrease the activity. On the contrary, com-
pounds 6r-t with dimethylamino groups on the amide side chain dis-
played much stronger cytotoxic activities than all of the other
derivatives, indicating that such moieties on the amide side chain were
beneficial to their cytotoxic activity. These preliminary SAR analyses
could give useful prompt for the further research on this class of UA
derivatives.
In order to evaluate whether compound 6t could induce the
apoptosis of cancer cells, the Annexin V-FITC/propidium iodide (PI)
dual staining assay on MCF-7 cells treated with compound 6t was car-
ried out through flow cytometry. As shown in Fig. 5, the percentage of
early and late apoptotic cells (early apoptosis: Lower right quadrant, AV
＋/ PI—; late apoptosis: Upper right quadrant, AV＋/PI＋) significantly
increased from 3.24 (0
(4
μM) to 13.22% (1 μM), 27.18 (2 μM) and 34.74%
2.3. Compound 6t induced cell cycle arrest in MCF-7 cells
μ
M), respectively, after treating with different concentrations of 6t (0,
1, 2 and 4 µM) for 48 h. These results indicated that compound 6t could
trigger the apoptosis of MCF-7 cells in a concentration-dependent
manner.
To further investigate the antitumor mechanism of action, cell cycle
distribution in MCF-7 cells was examined to determine whether or not
compound 6t inhibited the proliferation of cancer cells through cell
cycle arrest. MCF-7 cells were treated with different concentrations of
compound 6t (0, 1, 2 and 4 µM) for 48 h. Cell cycle distribution was
investigated by flow cytometric analysis after staining the DNA of the
treated cells by PI. Cell cycle analysis suggested that treatment of
compound 6t concentration-dependently decreased the population of
cells in G0/G1 phase. This phenomenon was accompanied by an in-
crease in the population of cells in S phase. As shown in Fig. 3, the
population of MCF-7 cells in the G0/G1 phase decreased from 51.80%
(control group) to 36.82% (4 µM group), while the percentage of cells in
the S phase increased from 20.85% (control group) to 36.22% (4 µM
group). These data indicated that compound 6t could arrest the cell
cycle of MCF-7 cells at the S phase.
2.6. Compound 6t induced the generation of intracellular ROS
Sufficient evidence revealed that apoptosis induction of cancer cells
could be mediated by reactive oxygen species (ROS) in many cellular
systems [39]. As a kind of metabolic by products of aerobic respiration,
ROS can exert oxidative stress to cells and result in severe damage to
organelles [40]. To determine whether the apoptosis was induced by the
title compounds, the intracellular ROS level in compound 6t-treated
MCF-7 cells was also examined by using 2′,7′-dichlorodihydro-
fluorescein diacetate (DCFH-DA), which could be converted into a green
fluorescent DCF by ROS oxidation [41]. The MCF-7 cells were treated
with different concentrations of compound 6t for 48 h, and the intra-
cellular fluorescence intensity was quantitatively analysed by flow
cytometry. As shown in Fig. 6, upon treatment with different
Fig. 3. Cell cycle distribution of MCF-7 cells. (a) MCF-7 cells were treated with 0, 1, 2 and 4 µM of compound 6t for 48 h, stained with propidium iodide (PI) and
analyzed for cell cycle by flow cytometry. (b) The column diagram for the cell cycle distribution of MCF-7 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs.
control group.
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W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
Fig. 4. Effect of compound 6t on in vitro migration potential of MCF-7 cells. Scratches were created with sterile 200
μ
L pipette and images were captured using phase
contrast microscopy at 0, 24 and 48 h after treatment with 0, 1, 2 and 4 μM of compound 6t.
Fig. 5. Annexin V-FITC/PI dual staining assay. (a) MCF-7 cells were treated with compounds 6t (0, 1, 2 and 4 µM) for 48 h, stained with Annexin V-FITC/PI and
analyzed by flow cytometry. (b) The bar diagram for the percentage of early and late apoptotic cells. **, p < 0.01; ***, p < 0.001 vs. control group.
concentrations of compound 6t, the percentage of cells with elevated
ROS level increased from 3.99% (Control) to 43.23% (4 µM). These re-
sults demonstrated that compound 6t could induce a significant increase
of ROS generation in MCF-7 cells in a dose-dependent manner. The
elevated intracellular ROS levels indicated that cell apoptosis was
correlated with the disruption of the balance between ROS generation
and elimination [42]. Therefore, ROS production was responsible for the
apoptosis induced by compound 6t.
dysfunction is one of the most profound features of apoptosis and in-
volves the process, such as mitochondrial permeability transition or the
reduction of mitochondrial membrane potential (MMP, ΔΨm) [44]. In
order to determine whether compound 6t-induced apoptosis was
involved in a disruption of mitochondrial membrane integrity, we
analyzed the ΔΨm changes by staining MCF-7 cells with JC-1 and
analyzing the cells by flow cytometry.
As shown in Fig. 7, the green fluorescence showing the loss of ΔΨm
(Lower right quadrant) in compound 6t-treated MCF-7 cells increased
from 5.51% (control group) to 15.55% (1 µM group), 20.78% (2 µM
group) and 31.19% (4 µM group), which indicated that compound 6t
could cause the decrease of mitochondrial membrane potential in a
concentration-dependent manner. Therefore, it could be concluded that
elevated ROS level and ΔΨm loss led to mitochondrial damage, which
2.7. Compound 6t reduced the mitochondrial membrane potential (MMP)
It is known that mitochondria play an important role in regulating
cellular functions, and the cell apoptosis cannot be reversed when the
mitochondrial transmembrane potential collapsed [43]. Mitochondrial
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W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
Fig. 6. ROS generation of MCF-7 cells induced by compound 6t. (a) Effect of compound 6t on the intracellular ROS level in MCF-7 cells. MCF-7 cells were treated
with different concentrations of 6t (0, 1, 2 and 4 μM) for 48 h, incubated with DCFH-DA and analyzed by flow cytometry. (b) The column diagram for the percentage
of relative ROS raised cells. ***, p < 0.001 vs. control group.
Fig. 7. (a) Effect of compound 6t on mitochondrial membrane potential (ΔΨm). MCF-7 cells were treated with compound 6t in 0, 1, 2 and 4 µM for 48 h, incubated
with JC-1 and analyzed by flow cytometry. (b) The bar diagram for the percentage of cells with low ΔΨm (Green fluorescence). ***, p < 0.001 vs. control group. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
was an important factor responsible for compound 6t-induced apoptosis.
2.8. Compound 6t regulated the expression of apoptosis-related proteins
of caspases which cleaves and deactivates PARP during the process of
apoptosis [48]. Thus the cleavage of PARP is also a hallmark of
apoptosis. As shown in Fig. 8e, the expression level of cleaved PARP was
dose-dependently elevated as well. All these results indicated that
compound 6t could induce the apoptosis of MCF-7 cells through the
ROS-mediated intrinsic mitochondrial pathway.
To further explore whether compound 6t induces apoptosis through
the mitochondrial signaling pathway, several key protein markers
involved in mitochondria-mediated apoptosis were examined by West-
ern blot analysis. Bcl-2 family proteins, mainly including Bax (pro-
apoptosis protein) and Bcl-2 (anti-apoptosis protein), are key regulators
of mitochondrial integrity [45]. The ratio of Bax/Bcl-2 is vital to
determine whether cells will undergo apoptosis. As shown in Fig. 8, the
expression level of Bax was obviously upregulated while that of Bcl-2
was decreased after treatment with compound 6t. Therefore, the ratio
of Bax/Bcl-2 increased in a dose-dependent manner (Fig. 8b).
2.9. Inhibitory effect of compound 6t on the PI3K/AKT/mTOR signaling
pathways
PI3K/AKT/mTOR signaling pathway plays an important role in
occurrence and progression of tumor and drug resistance by inducing
survival, proliferation, differentiation and vascularization of cancer
cells. Thus, this signaling pathway has become an attractive target for
cancer treatment [49,50]. Previous literatures demonstrated that ursolic
acid displayed anticancer activity by inhibiting the PI3K/AKT/mTOR
signaling pathway [51,52]. Therefore, the effects of compound 6t on the
PI3K/AKT/mTOR signaling pathway were explored by Western blot
analysis. As shown in Fig. 9, the treatment with compound 6t down-
regulated the levels of phosphorylated-AKT (p-AKT) and
phosphorylated-mTOR (p-mTOR) in MCF-7 cells in a concentration-
dependent manner, which are key proteins inducing cell proliferation
In addition, the collapse of MMP leads to the release of cytochrome c
from mitochondria to cytosol, which will activate the caspase cascade
pathway [46]. Therefore, the expression of cleaved caspase-3 and
cleaved caspase-9 were also examined. As shown in Fig. 8c and d, the
expression levels of cleaved caspase-3 and cleaved caspase-9 were
increased in a concentration-dependent manner. Moreover, poly ADP-
ribose polymerase (PARP) plays an essential role in DNA damage
repair and maintenance of genomic stability [47]. PARP is the substrate
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Bioorganic Chemistry 109 (2021) 104705
Fig. 8. (a) Western blot analysis of the level of Cleaved PARP, Bax, Bcl-2, Cleaved Caspase-3 and Cleaved Caspase-9 in MCF-7 cells treated with compound 6t (0, 1, 2
and 4 µM) for 48 h. (b) The rate changes of Bax/Bcl-2 in MCF-7 cells. (c) The expression level of Cleaved Caspase-3 in MCF-7 cells. (d) The expression level of Cleaved
Caspase-9 in MCF-7 cells. (e) The expression level of Cleaved PARP in MCF-7 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control group.
Fig. 9. Effect of compound 6t on PI3K/AKT/mTOR signaling pathway through Western blot analysis. (a) The levels of p-AKT, AKT, p-mTOR and mTOR in MCF-7
cells treated with compound 6t (0, 1, 2 and 4 µM) for 48 h. (b) The expression levels of p-AKT and AKT in MCF-7 cells. (c) The expression level of p-mTOR and mTOR
in MCF-7 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control group.
and apoptosis inhibition in this pathway. The results indicated that
compound 6t probably caused the apoptosis of MCF-7 cells by inhibiting
the PI3K/AKT/mTOR signaling pathway.
3. Conclusions
In present study, a series of novel indolequinone derivatives of
ursolic acid were designed, synthesised, and evaluated for their in vitro
antiproliferative activities against three human cancer cell lines (MCF-7,
HeLa, and HepG2) and a normal gastric mucosal cell line (Ges-1). As a
result, some indolequinone derivatives containing N-(dimethylamino)
alkyl amide moiety exhibited considerable antiproliferative activity.
Among them, compound 6t showed the most potent inhibitory activity
against three cancer cell lines, with IC50 values equivalent to or better
than those of positive control etoposide. More importantly, compound
6t displayed substantially lower cytotoxicity to Ges-1 cells. In addition,
compound 6t could arresting cell cycle of MCF-7 cells at the S phase,
suppress the migration of MCF-7 cells, enhance the intracellular ROS
level, decrease the mitochondrial membrane potential, up-regulate Bax,
cleaved caspase-3, cleaved caspase-9 and cleaved PARP levels, reduce
Bcl-2 level in a dose-dependent manner, and induce the apoptosis of
MCF-7 cells. Western blot analysis also indicated that compound 6t
2.10. Cytotoxic mechanisms of compound 6t on noncancerous Ges-1 cells
To explore if compound 6t could inhibit the growth of noncancerous
Ges-1 cells through the above-mentioned mechanisms, similar western
blot analyses were also carried out on compound 6t-treated Ges-1 cells.
As shown in Fig. 10, the ratio of Bax/Bcl-2 and the expression level of
cleaved caspase-3 were increased concentration-dependently, while the
expression levels of p-AKT and p-mTOR were decreased in a dose-
dependent manner. These results indicated that compound 6t could
inhibit the PI3K/AKT/mTOR signaling pathway and induce the
apoptosis of Ges-1 cells, which could be the possible cytotoxic mecha-
nisms of compound 6t to Ges-1 cells.
7

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
Fig. 10. (a) Western blot analysis of the level of Bax, Bcl-2, Cleaved Caspase-3, p-AKT and p-mTOR in Ges-1 cells treated with compound 6t (0, 1, 2 and 4 µM) for 48
h. (b) The rate changes of Bax/Bcl-2 in Ges-1 cells. (c) The expression level of Cleaved Caspase-3 in Ges-1 cells. (d) The expression level of p-AKT in Ges-1 cells. (e)
The expression level of p-mTOR in Ges-1 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control group.
markedly decreased the expression levels of p-AKT and p-mTOR, which
revealed that compound 6t displayed anticancer activity via interfering
with PI3K/AKT/mTOR signaling pathway. All these results demonstrate
the potential of this compound as promising candidate for the discovery
of novel anticancer agents. Other related pathways and protein targets
presumably involved in the anticancer effect of this series of UA de-
rivatives will be investigated in future investigations.
removed by concentration in vacuo. The corresponding products were
dissolved in ether (8 mL) and the mixture of corresponding alcohol or
acylhydrazine (0.225 mmol), Et3N (45 μL), and CH2Cl2 (2 mL) were
slowly added. The reaction mixture was stirred at room temperature for
8–12 h, and monitored by TLC. At the end of reaction, the mixture was
poured into 30 mL of ice-cold water and extracted with CH2Cl2 (3 × 40
mL). The organic layer was combined, washed with water, saturated
NaHCO3 and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo to obtain a crude product, which was purified by silica gel column
chromatography (CH2Cl2-MeOH 200:1 ~ 50:1, v/v) to afford com-
pounds 6a-j in 72 ~ 89% yields.
4. Experimental
4.1. General
4.2.1. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-oic acid (5)
Melting points were measured on an XT-4 apparatus (Taike Corp,
Beijing, China) and were uncorrected. The ESI-MS spectra were recorded
on a Mariner System 5304 mass spectrometer. 1H and 13C NMR spectra
were obtained in CDCl3 on Bruker AV-400 and DRX-600 NMR spec-
trometers (Billerica, MA, USA) using TMS as internal standard. Re-
actions and the resulted products were monitored by TLC which was
carried out on TLC Silica gel 60 F254 Aluminium sheets from Merck
KGaA, Darmstadt, Germany and visualised in UV light (254 nm). Silica
gel (300 ~ 400 mesh) for column chromatography was purchased from
Qingdao Marine Chemical Factory, China. The reagents (chemicals), all
being of A.R. grade, were purchased from Shanghai Chemical Reagent
Company (Shanghai, China) and Energy Chemical (Shanghai, China).
UA (95%) was bought from Jingzhu Biological Technology Co, Ltd.
(Nanjing, China).
Red Powder Solid; M.p. 297 ~ 299 ◦C; Yield: 82%; 1H NMR (600
MHz, CDCl3): δ 0.82 (s, 3H), 0.87 (s, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.95
(d, J = 6.3 Hz, 3H), 1.10 (s, 3H), 1.15 (m, 1H), 1.19 (s, 3H), 1.30 (m,
1H), 1.33 (s, 3H), 1.29～2.15 (m, 18H), 2.24 (d, J = 11.2 Hz, 1H), 3.06
(d, J = 16.6 Hz, 1H), 3.80 (s, 3H), 5.32 (brs, 1H), 5.65 (s, 1H), 10.63 (s,
1H); 13C NMR (150 MHz, CDCl3): δ 15.83, 16.87, 16.96, 19.07, 21.16,
23.23, 23.43, 23.45, 24.18, 28.03, 30.59, 30.70, 32.33, 34.22, 36.73,
37.81, 38.03, 38.85, 39.14, 39.53, 42.20, 45.96, 48.05, 52.60, 52.77,
56.57, 107.12, 119.47, 124.57, 125.92, 128.47, 137.70, 147.21, 160.30,
170.50, 182.24, 185.01; IR (KBr, cmꢀ 1): 3434, 3239, 2926, 2868, 1695,
1644, 1597, 1490, 1451, 1385, 1231, 1115, 1034, 842, 791; HRMS
(ESI): m/z [M+H]+ calcd for C37H50NO5: 588.3689; found: 588.3686.
4.2.2. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-oic acid methyl ester (6a)
4.2. General procedures for the synthesis of compounds 6a-j.
Red powder solid; M.p. 307 ~ 309 ◦C; Yield: 82%; 1H NMR (400
MHz, CDCl3): δ 0.81 (s, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.90 (s, 3H), 0.95
(d, J = 6.1 Hz, 3H), 1.10 (s, 3H), 1.20 (s, 3H), 1.25 (m, 2H), 1.27 (s, 3H),
1.32 (m, 1H), 1.35 ~ 1.75 (m, 11H), 1.80 (dd, J = 13.7, 4.4 Hz, 1H), 2.02
(m, 2H), 2.16 (m, 2H), 2.27 (d, J = 11.3 Hz, 1H), 3.07 (d, J = 16.5 Hz,
1H), 3.61 (s, 3H), 3.81 (s, 3H), 5.34 (s, 1H), 5.65 (s, 1H), 9.45 (s, 1H);
The key intermediate 5 was synthesized from ursolic acid (1) ac-
cording to the procedure reported before [53], which was further
treated as shown in Scheme 1 to afford the title compounds 6a-j. To the
solution of compounds 5 (0.15 mmol) in benzene (5 mL) was added
dropwise 200 μL of SOCl2 (1.5 mmol). The reaction mixture was refluxed
at 80 ◦C for 3 h. After cooling, the solvent and excess SOCl2 were
8

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
13C NMR (100 MHz, CDCl3): δ 15.79, 16.82, 17.00, 19.10, 21.18, 23.43,
23.45, 23.47, 24.29, 28.04, 30.73, 30.88, 32.37, 34.05, 36.63, 37.84,
37.97, 38.88, 39.17, 39.53, 42.22, 45.99, 48.24, 51.49, 52.55, 53.03,
56.54, 107.25, 118.95, 124.24, 125.72, 128.55, 137.87, 146.59, 160.02,
170.56, 178.10, 184.85; IR (KBr, cmꢀ 1): 3434, 3239, 2926, 2868, 1695,
1644, 1597, 1490, 1451, 1385, 1231, 1115, 1034, 842, 791; HRMS
(ESI): m/z [M+H]+ calcd for C38H52NO5: 602.3845; found: 602.3840.
MHz, CDCl3): δ 0.79 (s, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.89 (s, 3H), 0.94
(s, 3H), 1.11 (s, 3H), 1.24 (s, 3H), 1.26 (m, 2H), 1.30 (s, 3H), 1.31 (m,
1H), 1.36 (d, J = 10.6 Hz, 1H), 1.43 ~ 1.60 (m, 7H), 1.66 ~ 1.76 (m,
4H), 1.85 (d, J = 12.7 Hz, 1H), 2.03 (s, 3H), 2.07 (m, 2H), 2.14 (d, J =
16.9 Hz, 1H), 2.19 (m, 1H), 3.06 (d, J = 16.5 Hz, 1H), 3.78 (s, 3H,
OCH3), 5.52 (brs, 1H), 5.65 (s, 1H), 9.00 (d, J = 4.7 Hz, 1H), 9.85 (m,
1H), 10.91 (m, 1H); 13C NMR (150 MHz, CDCl3) δ 15.91, 16.43, 17.06,
19.04, 20.62, 21.15, 23.04, 23.24, 23.52, 24.84, 27.77, 30.59, 30.79,
32.15, 34.30, 37.11, 37.73, 38.17, 38.85, 39.58, 39.65, 42.49, 45.98,
47.58, 52.65, 53.45, 56.53, 107.12, 119.08, 124.39, 127.21, 128.64,
138.35, 146.85, 160.25, 166.29, 170.57, 173.72, 185.04; IR (KBr,
cmꢀ 1): 3257, 2928, 2870, 1662, 1639, 1596, 1487, 1456, 1385, 1263,
1231, 1115, 1031, 842, 792; HRMS (ESI): m/z [M+H]+ calcd for
4.2.3. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-oic acid ethyl ester (6b)
Red powder solid; M.p. 290 ~ 292 ◦C; Yield: 89%; 1H NMR (400
MHz, CDCl3): δ 0.83 (s, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.90 (s, 3H), 0.94
(d, J = 5.8 Hz, 3H), 1.10 (s, 3H), 1.20 (s, 3H), 1.22 (t, J = 7.3 Hz, 3H),
1.25 (m, 2H), 1.28 (s, 3H), 1.29 (m, 1H), 1.33 (m, 2H), 1.40 ~ 1.75 (m,
9H), 1.80 (td, J = 13.7, 4.5 Hz, 1H), 2.02 (m, 2H), 2.14 (m, 2H), 2.28 (d,
J = 11.2 Hz, 1H), 3.07 (d, J = 16.5 Hz, 1H), 3.81 (s, 3H, OCH3), 4.07 (q,
J = 7.1 Hz, 2H), 5.33 (t, J = 3.6 Hz, 1H), 5.65 (s, 1H), 9.52 (s, 1H); 13C
NMR (100 MHz, CDCl3): δ 14.26, 15.82, 16.99, 17.02, 19.10, 21.19,
23.36, 23.38, 23.44, 24.25, 28.00, 30.78, 30.87, 32.47, 34.06, 36.66,
37.83, 38.01, 38.88, 39.22, 39.61, 42.29, 46.00, 48.01, 52.57, 53.04,
56.54, 60.04, 107.24, 119.00, 124.26, 125.68, 128.55, 137.88, 145.88,
160.03, 170.55, 177.56, 184.85; IR (KBr, cmꢀ 1): 3458, 3275, 2925,
2858, 1727, 1667, 1642, 1597, 1494, 1455, 1385, 1231, 1115, 1033,
842, 806; HRMS (ESI): m/z [M+H]+ calcd for C39H54NO5: 616.4002;
found: 616.3996.
C39H54N3O5: 644.4063; found: 644.4066.
4.2.7. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-[2-(1- oxobutyl)hydrazide] (6f)
Red powder solid; M.p. 265 ~ 267 ◦C; Yield: 72%; 1H NMR (600
MHz, CDCl3): δ 0.79 (s, 3H), 0.85 ~ 1.92 (m, 9H), 0.95 (s, 3H), 1.12 (s,
3H), 1.23 (s, 3H), 1.29 (m, 2H), 1.30 (s, 3H), 1.31 (m, 1H), 1.38 (d, J =
10.1 Hz, 1H), 1.44 (m, 2H), 1.50 ~ 1.60 (m, 5H), 1.65 (m, 3H), 1.74 (m,
3H), 1.88 (d, J = 13.1 Hz, 1H), 2.09 (m, 2H), 2.15 (d, J = 17.0 Hz, 1H),
2.20 (m, 1H), 2.23 (t, J = 7.3 Hz, 2H), 3.07 (d, J = 16.5 Hz, 1H), 3.80 (s,
3H, OCH3), 5.57 (brs, 1H), 5.66 (s, 1H), 9.06 (d, J = 6.0 Hz, 1H), 9.49
(m, 1H), 10.47 (m, 1H); 13C NMR (150 MHz, CDCl3): δ 14.09, 15.83,
16.32, 17.06, 18.97, 19.04, 21.12, 22.67, 23.27, 23.54, 24.87, 27.79,
30.66, 30.78, 32.14, 34.24, 35.85, 37.04, 37.75, 38.13, 38.89, 39.62,
39.66, 42.51, 46.02, 47.55, 52.90, 53.39, 56.48, 107.18, 118.98,
124.36, 127.35, 128.65, 138.42, 146.45, 160.19, 168.72, 170.59,
173.41, 184.90; IR (KBr, cmꢀ 1): 3241, 2926, 2854, 1644, 1595, 1489,
1457, 1334, 1231, 1115, 1030, 842, 792; HRMS (ESI): m/z [M+H]+
calcd for C41H58N3O5: 672.4376; found: 672.4380.
4.2.4. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-oic acid propyl ester (6c)
Red Powder Solid; M.p. 260 ~ 262 ◦C; Yield: 80%; 1H NMR (600
MHz, CDCl3): δ 0.81 (s, 3H), 0.86 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.94
(d, J = 6.3 Hz, 3H), 1.09 (s, 3H), 1.14 (d, J = 13.5 Hz, 1H), 1.19 (s, 3H),
1.20 ~ 1.28 (m, 3H), 1.29 (s, 3H), 1.31 ~ 1.45 (m, 5H), 1.50 (m, 2H),
1.56 (s, 1H), 1.57 (s, 1H), 1.65 (dd, J = 13.3, 4.2 Hz, 1H), 1.68 ~ 1.79
(m, 4H), 1.88 (td, J = 13.4, 3.8 Hz, 1H), 2.00 (m, 2H), 2.13 (m, 2H), 2.24
(d, J = 11.3 Hz, 1H), 2.88 (s, 1H), 2.96 (s, 1H), 3.05 (d, J = 16.6 Hz, 1H),
3.80 (s, 3H), 5.29 (s, 1H), 5.31 (t, J = 4.1 Hz, 1H), 5.65 (s, 1H), 10.92 (s,
1H); 13C NMR (150 MHz, CDCl3) δ 15.84, 16.87, 16.97, 19.06, 21.19,
23.16, 23.35, 23.42, 24.19, 28.04, 30.54, 30.71, 31.54, 32.33, 34.25,
36.63, 37.79, 38.03, 38.84, 39.13, 39.51, 42.19, 45.94, 48.02, 52.59,
52.76, 53.47, 56.61, 107.09, 119.51, 124.57, 125.88, 128.47, 137.70,
147.40, 160.32, 170.47, 182.92, 185.09; IR (KBr, cmꢀ 1): 3476, 3232,
2928, 2855, 1737, 1667, 1626, 1597, 1452, 1202, 1107, 1029, 967, 856,
780; HRMS (ESI): m/z [M+H]+ calcd for C40H56NO5: 630.4158; found:
630.4149.
4.2.8. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-[2-(1- oxopentyl)hydrazide] (6g)
Red powder solid; M.p. 276 ~ 279 ◦C; Yield: 76%; 1H NMR (600
MHz, CDCl3): δ 0.78 (s, 3H), 0.87 ~ 0.91 (m, 9H), 0.96 (s, 3H), 1.13 (s,
3H), 1.22 (s, 3H), 1.28 (m, 2H), 1.29 (s, 3H), 1.32 (m, 3H), 1.38 (m, 1H),
1.46 (m, 2H), 1.50 ~ 1.61 (m, 6H), 1.68 (dd, J = 13.5, 3.8 Hz, 2H), 1.75
(m, 3H), 1.91 (d, J = 14.1 Hz, 1H), 2.01 (d, J = 5.9 Hz, 1H), 2.06 (m,
1H), 2.15 (d, J = 16.4 Hz, 1H), 2.21 (m, 1H), 2.24 (t, J = 7.4 Hz, 2H),
3.08 (d, J = 16.4 Hz, 1H), 3.80 (s, 3H), 5.58 (brs, 1H), 5.65 (s, 1H), 9.03
(d, J = 6.8 Hz, 1H), 9.18 (d, J = 6.7 Hz, 1H), 10.19 (s, 1H); 13C NMR
(150 MHz, CDCl3): δ 13.68, 15.82, 16.27, 17.06, 19.03, 21.12, 22.21,
22.66, 23.23, 23.54, 24.87, 25.61, 27.50, 30.72, 31.91, 32.12, 33.80,
34.18, 36.96, 37.76, 38.09, 38.91, 39.61, 39.68, 42.53, 46.02, 47.53,
52.60, 52.93, 56.49, 107.20, 118.94, 124.35, 127.37, 128.62, 138.54,
146.31, 160.13, 168.50, 170.60, 173.28, 184.82; IR (KBr, cmꢀ 1): 3417,
3413, 2924, 2853, 1652, 1632, 1598, 1488, 1456, 1385, 1231, 1116,
1031, 844, 792; HRMS (ESI): m/z [M+H]+ calcd for C42H60N3O5:
686.4533; found: 686.4514.
4.2.5. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-oic acid butyl ester (6d)
Red powder solid; M.p. 288 ~ 290 ◦C; Yield: 82%; 1H NMR (600
MHz, CDCl3): δ 0.83 (s, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.91 (s, 3H), 0.93
(t, J = 7.4 Hz, 3H), 0.95 (d, J = 6.2 Hz, 3H), 1.10 (s, 3H), 1.19 (s, 3H),
1.27 (m, 4H), 1.29 (s, 3H), 1.33 (m, 2H), 1.46 ~ 1.52 (m, 4H), 1.55 ~
1.75 (m, 8H), 1.81 (td, J = 13.4, 4.3 Hz, 1H), 1.97 ~ 2.05 (m, 2H), 2.15
(m, 2H), 2.28 (d, J = 11.2 Hz, 1H), 3.07 (d, J = 16.5 Hz, 1H), 3.81 (s,
4.2.9. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28- (2-benzoylhydrazide) (6h)
3H), 4.00 (m, 2H), 5.33 (t, J = 3.5 Hz, 1H), 5.65 (s, 1H), 9.28 (s, 1H); 13
C
NMR (150 MHz, CDCl3): δ 13.73, 15.79, 16.97, 17.02, 19.10, 19.26,
21.16, 23.38, 23.42, 23.48, 24.26, 28.00, 30.68, 30.89, 31.61, 32.46,
34.02, 36.71, 37.82, 37.96, 38.88, 39.20, 39.58, 42.27, 45.98, 48.17,
52.54, 53.04, 56.52, 63.98, 107.23, 118.95, 124.23, 125.69, 128.58,
137.90, 145.75, 159.99, 170.54, 177.63, 184.80; IR (KBr, cmꢀ 1): 3444,
3236, 2926, 2872, 1725, 1666, 1646, 1597, 1491, 1452, 1032, 1230,
1261, 1115, 843, 794; HRMS (ESI): m/z [M+H]+ calcd for C41H58NO5:
644.4315; found: 644.4314.
Red powder solid; M.p. 288 ~ 290 ◦C; Yield: 75%; 1H NMR (600
MHz, CDCl3): δ 0.80 (s, 3H), 0.89 (s, 3H), 0.94 (d, J = 5.9 Hz, 3H), 0.98
(d, J = 5.2 Hz, 3H), 1.15 (s, 3H), 1.17 (s, 3H), 1.26 (s, 3H), 1.33 (m, 4H),
1.38 (d, J = 13.2 Hz, 1H), 1.42 (d, J = 9.1 Hz, 1H), 1.50 (m, 5H), 1.71
(m, 1H), 1.78 (m, 2H), 1.96 (d, J = 13.7 Hz, 1H), 2.01 (m, 1H), 2.14 ~
2.21 (m, 3H), 2.27 (m, 1H), 3.08 (d, J = 16.5 Hz, 1H), 3.81 (s, 3H), 5.65
(s, 1H), 5.67 (brs, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.53 (t, J = 7.4 Hz, 1H),
7.79 (d, J = 8.0 Hz, 2H), 9.23 (d, J = 6.8 Hz, 1H), 9.29 (d, J = 7.2 Hz,
1H); 13C NMR (150 MHz, DMSO‑d6): δ 16.06, 17.52, 17.57, 19.13,
21.60, 22.89, 23.44, 23.54, 23.99, 27.87, 30.34, 31.05, 32.69, 34.38,
37.42, 37.71, 38.27, 38.85, 39.28, 42.36, 45.93, 47.15, 52.27, 52.84,
55.34, 56.81, 107.01, 117.63, 123.44, 125.10, 127.96, 128.70, 128.97,
4.2.6. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28- (2-acetylhydrazide) (6e)
Red powder solid; M.p. 279 ~ 281 ◦C; Yield: 72%; 1H NMR (600
9

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
131.92, 133.46, 138.44, 146.30, 160.45, 165.97, 170.08, 176.03,
184.92; IR (KBr, cmꢀ 1): 3444, 3340, 2925, 2869, 1641, 1595, 1632,
1490, 1452, 1382, 1334, 1268, 1231, 1115, 1030, 843, 792; HRMS
(ESI): m/z [M+H]+ calcd for C44H56N3O5: 706.4220; found: 706.4211.
1H), 6.71 (s, 2H), 12.43 (s, 1H); 13C NMR (100 MHz, DMSO‑d6): δ 16.00,
17.26, 17.56, 19.12, 21.23, 21.60, 22.53, 22.91, 23.46, 23.55, 24.10,
26.81, 27.91, 30.33, 34.37, 37.53, 37.70, 38.13, 38.95, 39.42, 42.31,
45.79, 47.16, 52.69, 52.72, 56.84, 107.01, 117.60, 121.44, 123.40,
124.90, 128.95, 138.89, 160.44, 170.09, 179.18, 184.95; IR (KBr,
cmꢀ 1): 3558, 3232, 2926, 2854, 1662, 1641, 1596, 1489, 1450, 1385,
1334, 1264, 1231, 1114, 1029, 842, 792; HRMS (ESI): m/z [M+H]+
calcd for C37H51N2O4: 587.3849; found: 587.3850.
4.2.10. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-[2-(4- methylbenzoyl)hydrazide] (6i)
Red powder solid; M.p. 295 ~ 297 ◦C; Yield: 72%; 1H NMR (600
MHz, DMSO‑d6): δ 0.82 (s, 3H), 0.83 (s, 3H), 0.87 (d, J = 6.4 Hz, 3H),
0.93 (d, J = 5.8 Hz, 3H), 1.08 (s, 3H), 1.16 (s, 3H), 1.23 (m, 3H), 1.25 (s,
3H), 1.29 (m, 1H), 1.38 (m, 3H), 1.45 ~ 1.57 (m, 3H), 1.63 ~ 1.75 (m,
3H), 1.78 (d, J = 8.4 Hz, 1H), 1.95 (m, 2H), 2.01 ~ 2.05 (m, 3H) 2.30 (d,
J = 11.0 Hz, 1H), 2.35 (s, 3H), 2.93 (d, J = 16.3 Hz, 1H), 3.73 (s, 3H),
5.25 (t, J = 3.1 Hz, 1H), 5.68 (s, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.77 (d, J
= 8.1 Hz, 2H), 9.32 (s, 1H), 10.01 (s, 1H), 12.43 (s, 1H); 13C NMR (150
MHz, DMSO‑d6): δ 16.08, 17.54, 17.59, 19.12, 21.46, 21.62, 22.91,
23.43, 23.53, 23.95, 27.85, 30.34, 31.03, 31.62, 32.68, 34.38, 37.43,
37.70, 38.24, 38.86, 39.25, 42.35, 45.89, 47.12, 52.25, 52.79, 56.84,
107.01, 117.64, 123.41, 125.05, 127.98, 129.25, 130.61, 138.44,
141.88, 146.31, 147.24, 160.45, 165.83, 170.09, 176.07, 184.96; IR
(KBr, cmꢀ 1): 3418, 3257, 2925, 2855, 1664, 1641, 1597, 1490, 1448,
1385, 1262, 1231, 1114, 1027, 802, 745; HRMS (ESI): m/z [M+H]+
calcd for C45H58N3O5: 720.4376; found: 720.4362.
4.3.2. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-methyl-1′H-ursa-2,12-dieno
[3,2-b]indol-28-amide (6l)
Red powder solid; M.p. 287 ~ 289 ◦C; Yield: 74%; 1H NMR (600
MHz, CDCl3): δ 0.82 (s, 3H), 0.88 (dd, J = 6.4, 1.9 Hz, 3H), 0.90 (s, 3H),
0.94 (s, 3H), 1.11 (s, 3H), 1.23 (s, 3H), 1.25 ~ 1.29 (m, 2H), 1.30 (s, 3H),
1.32 (m, 1H), 1.36 ~ 1.50 (m, 5H), 1.51 ~ 1.64 (m, 3H), 1.77 (m, 2H),
1.87 (d, J = 10.8 Hz, 1H), 1.91 (m, 1H), 1.96 (m, 1H), 2.02 (m, 1H), 2.07
(m, 1H), 2.15 (d, J = 16.4 Hz, 1H), 2.22 (m, 1H), 2.73 (d, J = 4.6 Hz,
3H), 3.06 (d, J = 16.4 Hz, 1H), 3.79 (s, 3H, OCH3), 5.39 (brs, 1H), 5.65
(s, 1H) , 5.98 (m, 1H), 10.13 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 15.81,
16.38, 17.25, 19.07, 21.23, 23.18, 23.23, 23.50, 25.00, 26.28, 27.85,
30.66, 30.92, 32.00, 34.16, 36.90, 37.78, 38.01, 39.13, 39.48, 39.90,
42.66, 45.98, 47.83, 52.57, 53.97, 56.50, 107.12, 118.63, 124.16,
125.71, 128.71, 139.95, 146.05, 160.18, 168.52, 178.83, 185.12; IR
(KBr, cmꢀ 1): 3445, 3209, 2935, 2852, 1667, 1644, 1594, 1493, 1448,
1384, 1231, 1113, 1029, 843, 792; HRMS (ESI): m/z [M+H]+ calcd for
4.2.11. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-[2-(3- pyridinylcarbonyl)hydrazide] (6j)
C38H53N2O4: 601.4005; found: 601.4005.
Red powder solid; M.p. 285 ~ 287 ◦C; Yield: 74%; 1H NMR (600
MHz, CDCl3): δ 0.80 (s, 3H), 0.89 (s, 6H), 0.95 (s, 3H), 1.12 (s, 3H), 1.22
(s, 3H), 1.24 ~ 1.29 (m, 3H), 1.31 (s, 3H), 1.36 (m, 1H), 1.38 ~ 1.61 (m,
7H), 1.65 ~ 1.77 (m, 3H), 1.85 (m, 1H), 1.98 ~ 2.12 (m, 3H), 2.15 (d, J
= 16.6 Hz, 2H), 2.22 (m, 1H), 3.07 (d, J = 16.4 Hz, 1H), 3.76 (s, 3H),
5.59 (brs, 1H), 5.63 (s, 1H), 7.34 (t, J = 5.9 Hz, 1H), 8.13 (s, 1H), 8.72 (s,
1H), 9.06 (s, 1H), 9.25 (s, 1H), 10.51 (brs, 1H); 13C NMR (150 MHz,
CDCl3) δ 15.92, 16.43, 17.09, 19.18, 21.14, 22.69, 23.24, 23.58, 24.93,
27.83, 30.19, 30.73, 31.43, 32.11, 34.21, 37.75, 38.07, 38.89, 39.59,
39.68, 42.57, 45.98, 47.86, 52.57, 52.96, 56.56, 107.15, 119.00,
119.09, 124.36, 127.39, 128.59, 128.83, 130.90, 135.96, 138.56,
146.52, 147.98, 160.15, 161.58, 170.61, 174.60, 184.91; IR (KBr,
cmꢀ 1): 3451, 3248, 2928, 2856, 1654, 1641, 1593, 1488, 1450, 1334,
1312, 1231, 1115, 1029, 842, 705; HRMS (ESI): m/z [M+H]+ calcd for
4.3.3. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-ethyl-1′H-ursa-2,12-dieno
[3,2-b]indol-28-amide (6m)
Red powder solid; M.p. 279 ~ 281 ◦C; Yield: 71%; 1H NMR (600
MHz, CDCl3): δ 0.86 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.91 (s, 3H), 0.94
(s, 3H), 1.09 (t, J = 7.3 Hz, 3H), 1.11 (s, 3H), 1.22 (s, 3H), 1.28 (m, 1H),
1.30 (s, 3H), 1.31 (m, 2H), 1.38 ~ 1.49 (m, 4H), 1.57 (m, 2H), 1.67 (m,
2H), 1.75 (m, 2H), 1.89 (m, 3H), 1.97 (dd, J = 13.6, 3.8 Hz, 1H), 2.05
(m, 1H), 2.15 (d, J = 14.4 Hz, 1H), 2.21 (dt, J = 18.4, 5.0 Hz, 1H), 3.06
(d, J = 4.4 Hz, 1H), 3.13 (m, 1H), 3.29 (m, 1H), 3.79 (s, 3H, OCH3), 5.39
(t, J = 3.7 Hz, 1H), 5.65 (s, 1H), 5.88 (t, J = 5.1 Hz, 1H), 10.21(s, 1H);
13C NMR (150 MHz, CDCl3): δ 14.53, 15.86, 16.73, 17.23, 19.07, 21.22,
23.09, 23.20, 23.53, 24.98, 27.89, 30.66, 30.97, 32.21, 34.18, 34.36,
37.10, 37.78, 38.07, 39.12, 39.57, 39.92, 42.77, 45.98, 47.68, 52.61,
54.14, 56.51, 107.12, 118.74, 124.17, 125.70, 128.70, 139.79, 146.12,
160.17, 170.44, 177.98, 185.08; IR (KBr, cmꢀ 1): 3333, 3208, 2930,
2851, 1665, 1629, 1595, 1490, 1449, 1259, 1232, 1115, 1031, 841, 792;
HRMS (ESI): m/z [M+H]+ calcd for C39H55N2O4: 615.4162; found:
615.4161.
C
43H55N4O5: 707.4172; found: 707.4172.
4.3. General procedures for the synthesis of compounds 6k-t
The indoloquinone derivative 5 (0.1 mmol) was dissolved in 2 mL of
dichloromethane, HOBT (18 mg, 0.12 mmol) and DCC (24 mg, 0.12
mmol) were added to the reaction system and the mixture was stirred at
room temperature for 30 min. Then different 1,2-diaminoalkanes (0.12
mmol) were added, and the mixture was stirred at room temperature
overnight. After the reaction was completed, DCU was filtered off. The
filtrate was concentrated under reduced pressure. The acetonitrile was
dissolved and left overnight. The DCU was further filtered off and
concentrated under reduced pressure to remove the solvent. The residue
was purified by silica gel chromatography (dichloromethane-methanol
200:1 ~ 50:1, v/v) to afford the corresponding amide derivatives 6k-t in
67 ~ 78% yields.
4.3.4. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-propyl-1′H-ursa-2,12-dieno
[3,2-b]indol-28-amide (6n)
Red powder solid; M.p. 285 ~ 287 ◦C; Yield: 71%; 1H NMR (600
MHz, CDCl3): δ 0.84 (s, 3H), 0.88 ~ 0.91 (m, 9H), 0.94 (s, 3H), 1.11 (s,
3H), 1.22 (s, 3H), 1.23 ~ 1.28 (m, 3H), 1.30 (s, 3H), 1.31 (m, 1H), 1.38
~ 1.51 (m, 7H), 1.56 (m, 2H), 1.66 (td, J = 13.4, 3.3 Hz, 1H), 1.75 (m,
2H), 1.88 (d, J = 16.5 Hz, 2H), 1.96 (td, J = 13.7, 3.8 Hz, 1H), 2.05 (m,
1H), 2.15 (d, J = 16.5 Hz, 1H), 2.21 (dt, J = 18.2, 5.3 Hz, 1H), 2.98 (m,
1H), 3.07 (d, J = 16.4 Hz, 1H), 3.28 (m, 1H), 3.79 (s, 3H), 5.38 (t, J =
3.6 Hz, 1H), 5.65 (s, 1H), 5.94 (d, J = 5.2 Hz, 1H), 10.17 (s, 1H); 13
C
NMR (150 MHz, CDCl3) δ 11.58, 15.86, 16.76, 17.22, 19.07, 21.22,
22.52, 23.10, 23.23, 23.52, 24.90, 27.88, 30.68, 30.98, 32.23, 34.17,
37.20, 37.78, 38.05, 39.12, 39.56, 39.92, 41.23, 42.77, 45.98, 47.85,
52.59, 54.22, 56.51, 107.13, 118.77, 124.20, 125.66, 128.67, 139.82,
146.13, 160.17, 170.46, 178.03, 185.07; IR (KBr, cmꢀ 1): 3431, 3220,
2927, 2869, 1667, 1642, 1597, 1490, 1451, 1385, 1335, 1231, 1115,
1031, 842, 792; HRMS (ESI): m/z [M+H]+ calcd for C40H57N2O4:
629.4318; found: 629.4318.
4.3.1. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-1′H-ursa-2,12-dieno[3,2-b]
indol-28-amide (6k)
Red powder solid; M.p. 278 ~ 280 ◦C; Yield: 72%; 1H NMR (400
MHz, DMSO‑d6): δ 0.82 (s, 3H), 0.83 (d, J = 4.3 Hz, 3H), 0.85 (d, J = 6.0
Hz, 3H), 0.92 (s, 3H), 1.07 (s, 3H), 1.15 (s, 3H), 1.24 (m, 2H), 1.25 (s,
3H), 1.28 ~ 1.36 (m, 4H), 1.40 ~ 1.48 (m, 4H), 1.59 (m, 3H), 1.69 (t, J
= 8.6 Hz, 1H), 1.82 (dd, J = 12.6, 2.2 Hz, 1H), 1.88 (dd, J = 13.2, 2.6 Hz,
1H), 1.97 (m, 2H), 2.04 (d, J = 16.5 Hz, 1H), 2.15 (d, J = 10.8 Hz, 1H),
2.92 (d, J = 16.3 Hz, 1H), 3.73 (s, 3H), 5.29 (t, J = 3.6 Hz, 1H), 5.68 (s,
10

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
4.3.5. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N,N-dimethyl-1′H-ursa-2,12-
dieno[3,2-b]indol-28- amide (6o)
658.4584; found: 658.4579.
Red powder solid; M.p. 269 ~ 271 ◦C; Yield: 67%; 1H NMR (400
MHz, CDCl3): δ 0.81 (s, 3H), 0.89 (m, 6H), 0.94 (d, J = 6.4 Hz, 3H), 1.10
(s, 3H), 1.20 (s, 3H), 1.26 (m, 2H), 1.28 (s, 3H), 1.32 (m, 2H), 1.38 ~
1.53 (m, 4H), 1.64 (m, 1H), 1.75 (m, 2H), 1.85 (d, J = 14.1 Hz, 2H), 2.00
(m, 2H), 2.09 ~ 2.25 (m, 4H), 2.54 (d, J = 11.0 Hz, 1H), 3.00 (s, 6H),
3.06 (d, J = 16.5 Hz, 1H), 3.80 (s, 3H), 5.31 (t, J = 3.6 Hz, 1H), 5.65 (s,
1H), 9.73 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 15.74, 16.65, 17.35,
19.11, 21.26, 23.40, 23.78, 27.22, 28.01, 29.32, 30.61, 30.81, 32.37,
34.02, 34.08, 37.88, 37.94, 38.72, 38.87, 39.36, 42.38, 42.71, 46.11,
48.74, 52.22, 52.63, 56.53, 107.21, 119.07, 124.29, 128.55, 129.91,
137.47, 146.05, 160.05, 170.54, 176.39, 184.89; IR (KBr, cmꢀ 1): 3418,
3217, 2924, 2850, 1667, 1643, 1594, 1490, 1447, 1384, 1259, 1229,
1114, 1029, 842, 792; HRMS (ESI): m/z [M+H]+ calcd for C39H55N2O4:
615.4162; found: 615.4161.
4.3.9. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-[3-(dimethylamino)
propyl]-1′H-ursa-2,12-dieno [3,2-b]indol-28-amide (6s)
Red powder solid; M.p. 285 ~ 287 ◦C; Yield: 75%; 1H NMR (400
MHz, CDCl3): δ 0.81 (s, 3H), 0.87 ~ 0.91 (m, 6H), 0.94 (d, J = 6.4 Hz,
3H), 1.10 (s, 3H), 1.21 (s, 3H), 1.27 (m, 2H), 1.29 (s, 3H), 1.38 ~ 1.60
(m, 8H), 1.67 ~ 1.81 (m, 4H), 1.87 (m, 2H), 1.96 ~ 2.07 (m, 4H), 2.14
(d, J = 16.8 Hz, 1H), 2.20 (m, 1H), 2.56 (s, 6H), 2.74 (m, 2H), 3.06 (d, J
= 16.5 Hz, 1H), 3.15 (m, 1H), 3.42 (m, 1H), 3.80 (s, 3H), 5.41 (t, J = 3.5
Hz, 1H), 5.64 (s, 1H), 6.85 (t, J = 5.0 Hz, 1H), 9.96 (s, 1H); 13C NMR
(100 MHz, CDCl3): δ 15.81, 16.81, 17.18, 19.08, 21.21, 23.27, 23.36,
23.44, 24.65, 25.20, 27.85, 30.78, 30.96, 32.21, 34.12, 37.21, 37.60,
37.80, 37.96, 39.00, 39.57, 39.74, 42.51, 43.87, 45.96, 47.80, 52.55,
53.50, 56.46, 56.53, 107.16, 118.84, 124.23, 125.76, 128.59, 139.00,
146.09, 160.10, 170.51, 178.63, 184.95; IR (KBr, cmꢀ 1): 3433, 3224,
2925, 2853, 1662, 1644, 1597, 1489, 1449, 1260, 1230, 1113, 1029,
842, 804; HRMS (ESI): m/z [M+H]+ calcd for C42H62N3O4: 672.4740;
found: 672.4742.
4.3.6. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N,N-diethyl-1′H-ursa-2,12-
dieno[3,2-b]indol-28- amide (6p)
Red powder solid; M.p. 270 ~ 272 ◦C; Yield: 76%; 1H NMR (600
MHz, CDCl3): δ 0.85 (s, 3H), 0.89 (d, J = 5.9 Hz, 3H), 0.90 (s, 3H), 0.93
(d, J = 6.2 Hz, 3H), 1.10 (s, 3H), 1.12 (brs, 6H), 1.19 (s, 3H), 1.25 (m,
2H), 1.28 (s, 3H), 1.30 (m, 2H), 1.35 ~ 1.60 (m, 8H), 1.66 ~ 1.90 (m,
6H), 2.00 ~ 2.20 (m, 4H), 3.07 (d, J = 16.5 Hz, 1H), 3.21 (brs, 2H), 3.80
(s, 3H, OCH3), 5.33 (s, 1H), 5.65 (s, 1H), 9.58 (s, 1H); 13C NMR (150
MHz, CDCl3): δ 14.08, 15.87, 16.85, 17.46, 19.09, 21.01, 23.25, 23.41,
23.54, 24.94, 28.43, 30.57, 30.86, 32.58, 33.93, 34.11, 36.70, 37.86,
38.11, 38.74, 39.18, 39.77, 42.49, 45.98, 49.24, 52.70, 53.18, 56.49,
107.22, 118.74, 124.20, 125.76, 128.79, 139.13, 145.77, 160.06,
170.53, 173.34, 184.84; IR (KBr, cmꢀ 1): 3431, 3222, 2928, 2868, 1666,
1644, 1597, 1490, 1452, 1385, 1231, 1114, 1030, 841, 792; HRMS
(ESI): m/z [M+H]+ calcd for C41H59N2O4: 643.4475; found: 643.4479.
4.3.10. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-[4-(dimethylamino)
butyl]-1′H-ursa-2,12-dieno [3,2-b]indol-28-amide (6t)
Red powder solid; M.p. 307 ~ 309 ◦C; Yield: 72%; 1H NMR (600
MHz, CDCl3): δ 0.84 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.91 (s, 3H), 0.95
(brs, 3H), 1.11 (s, 3H), 1.20 (s, 3H), 1.25 (m, 2H), 1.28 (s, 3H), 1.31 (m,
2H), 1.41 ~ 1.60 (m, 11H), 1.67 (td, J = 13.3, 3.2 Hz, 1H), 1.75 (m, 2H),
1.86 (d, J = 12.8 Hz, 1H), 1.91 (d, J = 10.4 Hz, 1H), 1.97 (m, 1H), 2.06
(m, 1H), 2.16 (d, J = 16.5 Hz, 1H), 2.22 (dt, J = 18.4, 4.0 Hz, 1H), 2.30
(s, 6H), 2.40 (m, 2H), 3.04 (m, 1H), 3.07 (d, J = 16.4 Hz, 1H), 3.35 (m,
1H), 3.81 (s, 3H), 5.39 (s, 1H), 5.65 (s, 1H), 6.12 (t, J = 4.9 Hz, 1H), 9.51
(s, 1H); 13C NMR (150 MHz, CDCl3): δ 15.84, 16.72, 17.19, 19.07, 21.21,
23.14, 23.19, 23.50, 24.39, 24.81, 27.03, 27.86, 30.64, 30.94, 32.19,
34.19, 37.26, 37.76, 38.05, 39.02, 39.06, 39.55, 39.85, 42.67, 44.89,
45.96, 47.77, 52.62, 53.95, 56.50, 58.78, 107.09, 118.72, 124.20,
125.67, 128.69, 139.61, 144.00, 160.16, 170.40, 178.14, 185.09; IR
(KBr, cmꢀ 1): 3417, 3223, 2925, 2854, 1662, 1641, 1596, 1491, 1452,
1230, 1261, 1114, 1030, 842, 793; HRMS (ESI): m/z [M+H]+ calcd for
C43H64N3O4: 686.4897; found: 686.4901.
4.3.7. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N,N-dipropyl-1′H-ursa-2,12-
dieno[3,2-b]indol-28- amide (6q)
Red powder solid; M.p. 278 ~ 280 ◦C; Yield: 72%; 1H NMR (600
MHz, CDCl3): δ 0.85 (s, 3H), 0.88 ~ 0.91 (m, 12H), 0.94 (s, 3H), 1.11 (s,
3H), 1.23 (s, 3H), 1.28 (m, 2H), 1.30 (s, 3H), 1.31 (m, 2H), 1.37 ~ 1.60
(m, 10H), 1.68 (m, 2H), 1.76 (m, 2H), 1.80 ~ 2.00 (m, 4H), 2.05 (m,
2H), 2.15 (d, J = 16.5 Hz, 1H), 2.21 (dt, J = 18.3, 4.9 Hz, 1H), 3.00 (m,
1H), 3.07 (d, J = 16.4 Hz, 1H), 3.30 (m, 1H), 3.79 (s, 3H, OCH3), 5.38
(brs, 1H), 5.65 (s, 1H), 10.19 (s, 1H); 13C NMR (150 MHz, CDCl3): δ
11.56, 15.84, 16.76, 17.21, 19.08, 21.19, 22.52, 23.11, 23.18, 23.53,
24.91, 27.90, 30.65, 30.98, 32.25, 34.19, 37.22, 37.79, 38.08, 39.12,
39.58, 39.92, 41.24, 42.77, 46.00, 47.86, 52.65, 54.23, 56.47, 107.13,
118.72, 124.18, 125.66, 128.72, 139.82, 146.10, 160.18, 170.43,
177.98, 185.05; IR (KBr, cmꢀ 1): 3417, 3217, 2937, 2868, 1667, 1648,
1594, 1491, 1452, 1385, 1232, 1115, 1032, 841, 790; HRMS (ESI): m/z
[M+H]+ calcd for C43H63N2O4: 671.4788; found: 671.4797.
4.4. MTT assay
Human breast cancer cell line (MCF-7), human cervical carcinoma
cell line (HeLa), human hepatocarcinoma cell line (HepG2) and human
normal gastric mucosal cell line (Ges-1) were maintained in Dulbecco
Modified Eagle Medium (DMEM) containing 4.0 mM L-Glutamine and
4500 mg/l Glucose supplemented with 10% (v/v) foetalbovine serum
(FBS) and 100 unites/mL penicillin/streptomycin at 37 ◦C in humidified
atmosphere of 5% CO2 and 95% air.
Cytotoxic activities of the indolequinone Derivative of UA were
determined in vitro cytotoxic activities against the aforementioned cell
lines via the typical MTT colorimetric method. Briefly, exponentially
growing cells were seeded into 96-well (100 mL/well at a density of 2 ×
105 cells/mL). After 24 h incubation at 37 ◦C and 5% CO2 to allow cell
attachment, and treated with different concentrations of the synthetic
compounds for 72 h, and then 100 mL of MTT (10 mg/mL) was added
and incubation for 3 ~ 4 h at 37 ◦C, the purple formazan crystals (a
reduced form of MTT) generated in viable cells were dissolved by adding
100 mL DMSO to each well. The plates were swirled gently for 5 min to
dissolve the precipitate and quantified by measuring the OD of the plates
at the wavelength of 540 nm. Each concentration was repeated in three
wells and the same experimental conditions were maintained for all
testing procedures. The MTT assays were repeated three times for each
cell line. The results were expressed as IC50 values (mean, n = 3) with
standard deviations, which was defined as the concentration at which
50% survival of cells was discerned. Etoposide was co-assayed as posi-
tive control.
4.3.8. 6′-Methoxy-4′,7′-dioxo-4′,7′-dihydro-N-[2-(dimethylamino)ethyl]-
1′H-ursa-2,12-dieno [3,2-b]indol-28-amide (6r)
Red powder solid; M.p. 332 ~ 335 ◦C; Yield: 78%; 1H NMR (600
MHz, CDCl3): δ 0.87 (s, 3H), 0.89 (d, J = 7.1 Hz, 3H), 0.90 (s, 3H), 0.94
(d, J = 6.1 Hz, 3H), 1.11 (s, 3H), 1.22 (s, 3H), 1.30 (s, 3H), 1.32 (m, 2H),
1.37 ~ 1.51 (m, 6H), 1.56 (m, 2H), 1.69 (t, J = 13.14 Hz, 1H), 1.76 (m,
2H), 1.89 (m, 2H), 1.92 ~ 2.08 (m, 3H), 2.17 (m, 2H), 2.21 (s, 6H), 2.36
(dd, J = 7.2 Hz, 5.5 Hz, 2H), 3.08 (d, J = 16.4 Hz, 1H), 3.16 (m, 1H),
3.31 (m, 1H), 3.79 (s, 3H), 5.36 (s, 1H), 5.64 (s, 1H), 6.51 (s, 1H), 10.20
(s, 1H); 13C NMR (150 MHz, CDCl3): δ 15.82, 16.80, 17.12, 19.10, 21.13,
23.10, 23.22, 23.55, 24.84, 27.96, 30.66, 31.02, 32.35, 34.17, 36.91,
37.23, 37.81, 38.09, 39.10, 39.65, 39.88, 42.68, 45.22, 46.05, 47.89,
52.74, 54.25, 56.41, 57.64, 107.13, 118.75, 124.22, 125.94, 128.73,
139.05, 146.12, 160.19, 170.47, 178.02, 184.95; IR (KBr, cmꢀ 1): 3390,
3227, 2924, 2857, 1649, 1642, 1596, 1489, 1450, 1385, 1231, 1115,
1030, 842, 792; HRMS (ESI): m/z [M+H]+ calcd for C41H60N3O4:
11

W.-Y. Wang et al.
Bioorganic Chemistry 109 (2021) 104705
4.5. Cell cycle analysis
after indicated treatments by compound 6t were incubated with an
equal volume of JC-1 staining solution (5 µg/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, New York, NY, USA). Mitochondrial
membrane depolarisation is indicated by an increase in the percentage
of cells with low ΔΨm (green fluorescence, lower right quadrant)
compared with cells with high ΔΨm (red fluorescence, upper right
quadrant).
Cell cycle distributions in MCF-7 cells were determined through PI
staining and analysed by flow cytometry [54]. MCF-7 cells were seeded
into a six-well plate at 5 × 105 cell/mL and treated with different con-
centrations of compound 6t for 48 h. After treatment, cells were de-
tached with 0.25% trypsin, harvested by centrifugation, washed twice
with ice-cold PBS and then fixed and permeabilised with ice-cold 70%
ethanol at 4 ◦C overnight. Ethanol was removed and the cells were
washed twice with ice-cold PBS. After this, the cells were treated with
100 µL of RNase (100 µg/mL) at 37 ◦C for 30 min, followed by incu-
bation with 400 µL of DNA staining solution propidium iodide (PI) (1
mg/mL) in the dark at 4 ◦C for 30 min. The samples were analysed by a
flow cytometer (BectonDickinson FACSCalibur, Totowa, NJ, USA) and
data were analysed using the FlowJo software (Becton-Dickinson & Co,
Totowa, NJ, USA).
4.10. Western blot analysis
MCF-7 cells were seeded at a density of 5 × 106 cells per well and
attached for 8 h, and then treated with different concentrations of
compound 6t for 48 h. After the treatment, the cells were harvested and
washed twice with PBS. The harvested cells were lysed with radio-
immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech.,
Nantong, China) with 1% cocktail (Sigma-Aldrich, St. Louis, MO, USA).
Whole-cell protein lysates were prepared and centrifuged at 12,000 rpm
for 10 min at 4 ◦C. The total proteins were determined using Bradford
reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Exactly 40 µg of
protein per lane was separated through sodium dodecyl sulpha-
te–polyacrylamide gel electrophoresis and then transferred onto a pol-
yvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were incubated with each antibody and detected through
immunoblot analysis. All of the antibodies were purchased from Cell
signalling Technology, Inc. (Boston, MA, USA) and diluted in accor-
dance with the manufacturer’s instruction. Proteins were visualised
using a C-Digit® imaging system (LI-COR, Lincoln, NE, USA).
4.6. Wound healing assay
MCF-7 cells (5 × 105 cells/well) were grown in a 12-well plate for 24
h. Scratches were made in confluent monolayers using 200 μL pipette
tip. Then, wounds were washed twice with PBS to remove non-adherent
cell debris. The media containing different concentrations (0, 2, 4 and 6
μ
M) of compound 6t were added to the corresponding wells. Cells which
migrated across the wound area were photographed under the phase
contrast microscope (Olympus 1X71 Inverted System Microscope,
Olympus, Japan) after 24 and 48 h treatment.
4.7. Cell apoptosis analysis
The extent of apoptosis was quantitatively measured using Annexin
V-FITC/PI dual staining assay [55]. MCF-7 cells were seeded into a six-
well plate at 5 × 105 cells per well in 10% foetal calf serum (FBS)-DMEM
into six-well plates and treated with different concentrations of the
indicated compound 6t for 48 h. The cells were detached with 0.25%
trypsin, washed with ice-cold PBS for twice and then resuspended in 1 ×
Binding buffer (0.1 M Hepes/ NaOH (pH 7.4), 1.4 M NaCl, 25 mM
CaCl2). The cells were stained with 5 µL of Annexin V-FITC and 5 µL of PI
(propidium indole) to each tube. The cells were gently vortexed and
incubated in the dark at room temperature for 15 min and then keep
them at 4 ◦C. The samples were analysed by a flow cytometer (Becton-
Dickinson FACSCalibur, Totowa, NJ, USA) and data were analysed using
the FlowJo software.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (31770616), the Natural Science Foundation for Colleges and
Universities in Jiangsu Province (17KJA220002) and Top-notch Aca-
demic Programs Project of Jiangsu Higher Education Institutions
(PPZY2015C221). The authors are grateful to the Advanced Analysis &
Testing Center of Nanjing Forestry University for the measurements of
spectroscopic data.
4.8. ROS generation assay
ROS generation assay was performed by using the reactive oxygen
species assay kit (Beyotime Biotech., Nantong, China). Intracellular ROS
generation was tested through dichlorodihydro fluorescein diacetate
(DCFH-DA) assay [56]. DCFH-DA is taken up by MCF-7 cells, and then
activated by esterase-mediated cleavage of acetate to form DCFH, which
is trapped in the cells. DCFH is converted to fluorescein DCF in the
presence of ROS. MCF-7 cells were seeded in six-well plates and incu-
bated with different concentrations of compound 6t for 48 h. After
removing the compound solution, cells were treated with 10 µM 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, Totowa, NJ, USA).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104705.
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13