Design, synthesis and bioactivity study of evodiamine derivatives as multifunctional agents for the treatment of hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bioorg.2021.105154

Topoisomerase has been found extremely high level of expression in hepatocellular carcinoma (HCC) and proven to promote the proliferation and survival of HCC. Cancer-associated fibroblasts (CAFs) as a kind of key reactive stromal cell that abundantly present in the microenvironment of HCC, could enhance the metastatic ability and drug resistance of HCC. Therefore, developing new drugs that address the above conundrums would be of the upmost significant in the fight against HCC. Evodiamine, as a multi-target natural product, has been found to exert various biological activities such as anti-cancer and anti-hepatic fibrosis via blocking topoisomerase, NF-κB, TGF-β/HGF, and Smad2/3. Inspired by these facts, 15 evodiamine derivatives were designed and synthesized for HCC treatment by simultaneously targeting Topo I and CAFs. Most of them displayed preferable anti-HCC activities on three HCC cell lines and low cytotoxicity on one normal hepatic cell. In particular, compound 8 showed the best inhibitory effect on HCC cell lines and a good inhibition on Topo I in vitro. Meanwhile, it also induced obvious G₂/M arrest and apoptosis, and significantly decreased the migration and invasion capacity of HCC cells. In addition, compound 8 down-regulated the expression of type I collagen in the activated HSC-T6 cells, and induced the apoptosis of activated HSC-T6 cells. In vivo studies demonstrated that compound 8 markedly decreased the volume and weight of tumor (TGI = 40.53%). In vitro and in vivo studies showed that its effects were superior to those of evodiamine. This preliminary attempt may provide a promising strategy for developing anti-HCC lead compounds taking effect through simultaneous inhibition on Topo I and CAFs.

Full text of DOI:10.1016/j.bioorg.2021.105154

Bioorganic Chemistry 114 (2021) 105154
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and bioactivity study of evodiamine derivatives as
multifunctional agents for the treatment of hepatocellular carcinoma
,
,
,
Xiaohong Fanb 1, Jiedan Dengb 1, Tao Shib *, Huaixiu Wenc, Junfang Lib, Ziyi Liangd,
Fang Leib, Dan Liub, Honghua Zhangb, Yan Liangb, Xiangyong Haoe *, Zhen Wanga
,
,
b,
d
,
*
a School of Pharmaceutical Science, University of South China, Hengyang 421001, China
b School of Pharmacy, Lanzhou University, Lanzhou 730000, China
c Key Laboratory of Tibetan Medicine Research, Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, China
d State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
e Department of General Surgery, Gansu Provincial Hospital, Lanzhou 730000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Topoisomerase has been found extremely high level of expression in hepatocellular carcinoma (HCC) and proven
to promote the proliferation and survival of HCC. Cancer-associated fibroblasts (CAFs) as a kind of key reactive
stromal cell that abundantly present in the microenvironment of HCC, could enhance the metastatic ability and
drug resistance of HCC. Therefore, developing new drugs that address the above conundrums would be of the
upmost significant in the fight against HCC. Evodiamine, as a multi-target natural product, has been found to
exert various biological activities such as anti-cancer and anti-hepatic fibrosis via blocking topoisomerase, NF-κB,
TGF-β/HGF, and Smad2/3. Inspired by these facts, 15 evodiamine derivatives were designed and synthesized for
HCC treatment by simultaneously targeting Topo I and CAFs. Most of them displayed preferable anti-HCC ac-
tivities on three HCC cell lines and low cytotoxicity on one normal hepatic cell. In particular, compound 8
showed the best inhibitory effect on HCC cell lines and a good inhibition on Topo I in vitro. Meanwhile, it also
induced obvious G2/M arrest and apoptosis, and significantly decreased the migration and invasion capacity of
HCC cells. In addition, compound 8 down-regulated the expression of type I collagen in the activated HSC-T6
cells, and induced the apoptosis of activated HSC-T6 cells. In vivo studies demonstrated that compound 8
markedly decreased the volume and weight of tumor (TGI = 40.53%). In vitro and in vivo studies showed that its
effects were superior to those of evodiamine. This preliminary attempt may provide a promising strategy for
developing anti-HCC lead compounds taking effect through simultaneous inhibition on Topo I and CAFs.
Hepatocellular carcinoma
Topo I
Evodiamine
Cancer-associated fibroblasts (CAFs)
1. Introduction
most suitable option [3]. However, most of the traditional chemo-
therapy agents have a lower efficacy on HCC for a widely various rea-
Primary liver cancer is one of the most aggressive and refractory
malignancies, and ranks the sixth (4.7%) in morbidity and the third
(8.2%) in mortality [1]. Hepatocellular carcinoma (HCC) is the pre-
dominant form of primary liver cancer, and carries commonly a poor
prognosis [2]. Most HCC patients are usually diagnosed as the inter-
mediate stage when symptoms start to appear, losing the opportunity for
radical treatment and conservative treatment strategy is usually the
sons [4]. For one thing, most of the advanced stage HCC patients are
accompanied by hepatic injury and cirrhosis resulting in the damage of
liver function, which limits the use of some systemic chemotherapy
drugs. For another thing, liver, as a crucial metabolic organ in the body,
possesses a specific group of cytochromes P-450 enzymes, which convert
most drugs to metabolites or inactive forms leading to the chemotherapy
resistance. Besides, the presence of a inflammatory and fibrotic
Abbreviations: TGF-β, transforming growth factor-β; Topo I, topoisomerase I; Topo II, topoisomerase II; HSC, hepatic stellate cell; EVO, Evodiamine; TME, tumor
microenvironment; HCC, Hepatocellular carcinoma; CAFs, Cancer-associated fibroblasts; Col-1, type I collagen; FBS, fetal bovine serum; m, multiplet (spectral); IC50
,
half-maximum inhibitory concentration; i.p., Intraperitoneal injection; HE, Hematoxylin and eosin stain; DMSO, dimethyl sulphoxide; SD, standard deviation; SEM,
standard error of mean; PBS, Phosphate-buffered saline.
* Corresponding authors at: School of Pharmaceutical Science, University of South China, Hengyang 421001, China (Z. Wang).
E-mail addresses: shit18@lzu.edu.cn (T. Shi), haoxy-2008@163.com (X. Hao), zhenw@lzu.edu.cn (Z. Wang).
1
These authors made equal contributions to this work.
https://doi.org/10.1016/j.bioorg.2021.105154
Received 2 March 2021; Received in revised form 16 June 2021; Accepted 4 July 2021
Available online 7 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
microenvironment in HCC could induce chemotherapy resistance by
releasing cytokines and extracellular matrix [5]. Based on the above
considerations, fewer choices exist for systemic chemotherapy on
advanced HCC [6]. Sorafenib as an anti-angiogenic and anti-
proliferation drug is the only first-line systemic therapy agent for
advanced HCC [7]. Whereas, it minimally provides survival improve-
ments, and thus developing more effective therapeutic strategies for
advanced HCC is urgently needed.
possesses lower inhibitory activity on Topo I/II and is easily transformed
into inactive metabolites in the liver [21–23]. These characteristics of
EVO may cause unsatisfactory in vivo anticancer activity and sorely
limits its further application in HCC treatment [22,24]. Therefore, many
excellent researches aim for improve its anticancer efficacy and reduce
its toxicity through various molecular modeling and drug design tech-
niques [25–29]. Admirably, Sheng and co-workers [25,27–28,30] has
done pioneering works in the development of novel agents based on
evodiamine.
According to the references, on the one hand, topoisomerase I and II
(Topo I and II) genes are highly expressed in the HCC tissue, and
correlate positively with the occurrence and development of HCC [8].
Numerous Topo inhibitors, including topotecan, irinotecan and doxo-
rubicin, are now widely used in the clinical setting. However, they have
limited efficacy in hepatocellular cancer, and one of the main reasons for
this is underlying chemoresistance in HCC microenvironment. On the
other hand, 90% of HCC cases always occur in the setting of cirrhosis
and chronic liver diseases,[9] and the fibrotic microenvironment plays
an important role in the occurrence, metastasis and chemoresistance of
HCC [4,10]. In addition, multiple interactive processes among hetero-
geneous cell types are extremely complex and could influence HCC
progression [11–12]. Among them, cancer-associated fibroblasts (CAFs)
as a key and abundant type of stromal cells which are thought to
probably derive from activated hepatic stellate cells (HSCs), could pro-
vide a favorable setting by secreting various immunosuppressive and
cancer-supportive soluble factors into the microenvironment [13].
These secreted components could promote multiple aspects of HCC
progression and growth by remodeling the TME in favor of cancer
expansion and therapeutic resistance [14]. Therefore, developing multi-
functional drugs holds a promise for combating this highly lethal cancer.
Natural products are an important source of therapeutic drugs [15].
More than half of cancer chemotherapeutic drugs come from natural
products and their derivatives [15]. Evodiamine (EVO), an indolequi-
none alkaloid extracted from Evodia, is a new promising natural anti-
cancer lead compound against a broad spectrum of cancers by dual in-
hibition on topoisomerase I and II (Topo I and II) proteins which have
long been considered as one of the most effective chemotherapeutic
targets [8,16–17]. Notably, some reports have indicated that EVO could
significantly reverse the CAFs phenotype and remodel tumor microen-
vironment (TME) via blocking the Smad 2/3 signaling pathway [18].
Moreover, it also exerts a favorable anti-hepatic fibrosis activity via
inhibiting TGF-1β/Smad pathway [19–20]. In view of its multi-target
and broad-spectrum anti-cancer activities as well as the potency of
remodeling TME, EVO could be regarded as an excellent anti-HCC
candidate. Unfortunately, previous studies have stated that EVO
In view of the above-mentioned points, firstly, topoisomerase in-
hibitors, a class of broad-spectrum chemotherapy agents, yet in fact they
have limited efficacy in hepatocellular cancer and one of the main
reasons for this is underlying chemoresistance in HCC microenviron-
ment. Development of highly potential and multi-functional topoisom-
erase inhibitors that address this critical challenge would therefore be of
the most importance in the fight against HCC. Secondly, EVO, as a multi-
target natural molecule, possesses anti-cancer activity and modulation
of inflammatory and fibrotic microenvironment (Scheme 1), and it has
the potential to be developed into anti-HCC agents with higher efficient
and lower toxic. Thirdly, EVO possesses unsatisfactory inhibitions on
Topo I/II and is easily transformed into extensive metabolism in the
liver, among them, N-demethylation metabolic pathways will lead to
inactivity [28,31]. We hypothesized that replacing methyl group in N14
position with more bigger groups or introducing various isosteres may
improve the anti-HCC efficiency of EVO (Scheme 1). To achieve these
goals, we rationally designed and synthesized a series of EVO derivatives
(Scheme 2) based on our previously reported synthetic processes [32].
In this protocol, we were able to modify different atoms or substituents
on the original N14 position to improve anti-cancer potency. Inhibiting
Topo I and reducing CAFs activation to show excellent anti-liver cancer
efficiency in vitro and in vivo was proven by the subsequent structur-
e–activity relationships study (SARs) and biological evaluation. This
study may provide a guidance for further optimizing the structure of
evodiamine and developing multi-functional anti-HCC agents.
2. Results and discussion
2.1. Chemistry
To explain and predict the SARs of EVO, 15 compounds were syn-
thesized by the one-step construction of three chemical bonds and two
heterocyclic-fused rings according to our previously reported method
[32]. The purity of all compounds was monitored by HPLC and
was>95.0%, which established solid foundation for the further biology
Scheme 1. Rational design of evodiamine derivatives and its anti-HCC mechanism.
2

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Scheme 2. Synthetic route to evodiamine derivatives. (a) EDCI, HOBt, TEA, DCM, r.t., 16 h; (b) AlMe3, DCM, r.t., 8 h; (c) HC(OEt)3, BF3⋅Et2O, DMF, 135 ◦C; (d) HC
(OEt)3, BF3⋅Et2O, DCM, r.t; (e) HC(OEt)3, BF3⋅Et2O, DMF, 100 ◦C.
activity exploration. Compounds 1 [32], 2 [27], 3 [27], 4 [32–33], 5
[32], 6 [32], 7 [32], 8 [32], 9 [32], 10 [32] have been reported pre-
viously, while compounds 11 to 15 are new.
the 14 position of EVO with oxygen atom would decrease the anti-
proliferation activity on MHCC-LM9 and Huh7 cells. Introducing
methyl group in R2-4 position, the anti-proliferation activity dramati-
cally decreased (compound 2), and introducing methyl group in R2-3
position, the anti-proliferation activity slightly increased (compound 3)
on three HCC cell lines. In the thio-evodiamine derivatives (compounds
4 and 5), an increasing trend of activity was observed on HepG2 and
Huh7 cells, and introducing methoxy group in 10 position (compound 5)
showed lower activity against MHCC-LM9 cells than compound 4. In the
N-phenyl-derivatives, the inhibition order was 8 > 9 > 7 > 10 on MHCC-
LM9 and Huh7 cells. The anti-proliferation activity of the compounds
substituted with methoxy decreased slightly, but the activity on HepG2
cell line enhanced. Besides, the replacement of phenyl rings by pyridine
rings and quinoxaline ring caused reduction of anti-proliferative activity
2.2. Biology
2.2.1. In vitro Anti-proliferation activity and SARs
The anti-proliferation activity of all compounds against human
normal liver cell line (HL-7702) and HCC cell lines (HepG2, MHCC-LM9,
Huh-7) were evaluated by Cell Counting Kit-8 (CCK-8) assay, with
evodiamine as the parent compound and Sorafenib as the positive con-
trol. As shown in Table 1, the toxicity of evodiamine derivatives on
normal human liver cells HL-7702 was lower than evodiamine and
sorafenib, and the IC50 values of synthesized compounds were higher
(compounds 11 and 15, IC50 > 40
μM). In general, N-phenyl and sulfur
than 40
μM, except for compound 1 (37.75 ± 4.72 μM). In the oxo-
atom substitutions on 14 position were more active than oxygen atom
evodiamine derivatives (compounds 1–3), replacing nitrogen atom on
3

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
2.2.3. Anti-proliferative effect of compound 8 on MHCC- LM9 and Huh7
Cells
Table 1
Anti-proliferation activity of compounds on different liver cell lines.
IC50 ± SDa
(μM)
To further verify the anti-proliferative effect of compound 8, EdU
and colony formation assay were employed to more accurately observe
the cell proliferation of MHCC-LM9 and Huh7 cells treated by compound
Compd.
MHCC-LM9
Huh7
HepG2
HL-7702
1
37.71 ± 0.47
>40
9.92 ± 0.66
>40
21.10 ± 1.53
>40
37.75 ± 4.72
>40
8 and EVO (0.1, 0.3, 0.9 μM). As shown in the Figs. 2 and 3, both
2
compound 8 and EVO exerted markedly antiproliferative activity in
MHCC- LM9 and Huh7 cells and the inhibitory activity of compound 8 at
tested concentrations was superior to that of evodiamine in both ex-
periments. To our delight, the relative colony formation ratios in MHCC-
3
10.21 ± 1.07
0.34 ± 0.10
3.76 ± 1.18
1.63 ± 0.28
9.82 ± 2.13
0.24 ± 0.05
0.53 ± 0.01
>40
1.06 ± 0.28
0.28 ± 0.05
0.65 ± 0.12
0.78 ± 0.22
0.74 ± 0.10
0.44 ± 0.12
0.72 ± 0.28
4.86 ± 0.56
>40
10.06 ± 3.12
2.30 ± 0.23
2.45 ± 0.38
21.52 ± 2.14
2.39 ± 0.45
0.92 ± 0.20
1.11 ± 0.27
0.54 ± 0.16
>40
>40
4
>40
5
>40
6(EVO)
26.32 ± 5.45
>40
7
LM9 of compound 8 at the 0.1, 0.3, 0.9 μM<10%, thus confirmed its
8
>40
outstanding antiproliferative capacities.
9
>40
10
>40
11
>40
>40
2.2.4. Compound 8 induced cell cycle G2/M arrest in Huh7 and MHCC-
LM9 cells
12
>40
>40
>40
>40
13
>40
>40
>40
>40
To confirm the effect of compound 8 on cell cycle progression in
Huh7 cells and MHCC- LM9 cells, the flow cytometric assay was per-
formed. The cell cycle distribution in Huh7 cells and MHCC- LM9 were
detected after a 48 h-exposure to compound 8 and EVO at different
concentrations. As shown in Fig. 4, the G2/M phase arrests were
significantly in a trend of dose-dependent change and compound 8
showed remarkably better activity than EVO. During the process of
14
>40
>40
>40
>40
15
>40
>40
>40
>40
Sorafenib
7.74 ± 0.80
10.12 ± 0.03
5.58 ± 0.46
5.61 ± 0.58
a
CCK-8 assay; cells exposed to tested compounds at the concentrations of 0,
0.156, 0.625, 2.5, 10, 40 μM for 48 h; the data were presented as mean ± SD (n
≥ 3).
increasing the concentration of compound 8 from 0.1 μM to 0.9 μM, the
substitutions for HCC cell lines. Finally, the substitution of different
atoms on 10 position has a certain effect on the activity.
proportion of G2/M phase in MHCC-LM9 and Huh7 cells increased from
25.53% to 78.75% and 20.87% to 63.32%, respectively. In contrast, the
ratios of untreated Huh7 and MHCC- LM9 cells in G2/M cell cycle were
13.86% and 23.21%, respectively, which were lower than those in the
Although different compounds displayed various activity against
diverse tested cell lines. Fortunately, compounds 4 and 8 displayed
extremely preferable anti-proliferation on three HCC cells and they were
less toxic to normal liver cells than both sorafenib and evodiamine.
Therefore, compounds 4 and 8 were selected for Topo I inhibition assay.
compound 8 group. After incubating with EVO at 0.1, 0.3 and 0.9 μM,
the proportion of G2/M phase in MHCC-LM9 and Huh7 cells were
changed gently (16.97% to 44.34% and 19.37% to 46.81%, respec-
tively). The cell cycle G2/M arrest of compound 8 at tested concentra-
tions still was superior to that of EVO.
2.2.2. Topo I inhibitory effect of compounds 4 and 8
According to the report, evodiamine has been proven to be an in-
hibitor of Topo I/II [17], therefore, target verification experiments of
the selected compounds 4 and 8 on Topo I were carried out. The results
were shown in Fig. 1. Topo I-mediated relaxation of supercoiled DNA
was significantly inhibited by compounds 8 and 4 at a concentration of
2.2.5. Compound 8 promoted cell apoptosis on Huh7 cells
Anti-cancer activity of Topo inhibitors is often related with induced
cell apoptosis [34]. To further assess the anti-HCC effect of compound 8,
firstly Giemsa staining assay was performed on Huh7 cells by treating
100
concentration. Then, as the concentration gradiently decreased from
100 to 10 M, the inhibitory activity of compounds 8 and 4 on Topo I
also significantly weakened, while camptothecin (CPT) still had strong
inhibitory activity at the lowest concentration of 10 M. At a concen-
tration of 50 M, compound 8 still retained potent inhibitory activity,
μM, while evodiamine showed no inhibitory activity at the same
the cells with compound 8 at gradient concentrations (0.15, 0.3, 0.6 μM)
for 24 h. As depicted in Fig. 5A, cells treated with compound 8 showed
classic apoptosis (marked by green arrows), such as cell swelling, kar-
yopyknotic, nuclear fragmentation or agglutination, and apoptotic body
formation compared to the normal cells. To further confirm the
apoptosis effects of compound 8, flow cytometry analysis was per-
formed. The apoptosis of cells exposed to compound 8 and EVO at
different concentrations after 48 h, stained with Annexin V-FITC and PI.
As showed in Fig. 5B, the number of late apoptotic cells in the compound
8 and EVO treatment groups were remarkedly increased from 29.4% to
μ
μ
μ
but compound 4 showed no inhibitory activity. These results demon-
strated that compounds 8 and 4 could inhibit Topo I and the activity of
compound 8 is a little better than that of compound 4. Therefore, we
chose compound 8 for the following anti-tumor experiments in vitro and
in vivo.
Fig. 1. Topo I inhibitory activity of compounds 8 and 4 (A) Inhibition activity on Topo I relaxation at 100
I; lane 3, DNA + Topo I + CPT; lane 4, DNA + Topo I + compound 8; lane 5, DNA + Topo I + EVO. (B) Inhibition on Topo I relaxation at different concentrations:
lane 1, supercoiled plasmid DNA; lane 2, DNA + Topo I; lanes 3–5, DNA + Topo I + CPT (100, 50 and 10 M); lanes 6–8, DNA + Topo I + compound 8 (100, 50 and
10 M). (C) Topo I inhibitory activity of compound 4. Lane 1, supercoiled plasmid DNA; lane 2, DNA + Topo I; lane 3, DNA + Topo I + CPT; lanes 4–6, DNA + Topo I
+ compound 4 (100, 50 and 10 M).
μM. Lane 1, supercoiled plasmid DNA; lane 2, DNA + Topo
μ
μ
μ
4

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 2. Compound 8 weakened markedly antiproliferative activities in Huh7 and MHCC-LM9 cells detected by EdU (red) staining. MHCC-LM9 and Huh7 cells
exposed to EVO and compound 8 at different concentrations for 24 h before EdU staining. Images showed representative pictures of inhibitions of compound EVO and
compound 8 on Huh7 (A) and MHCC-LM9 (C) cells proliferation were determined by EdU proliferation assays (magnification × 10, scale bar = 100 μm). The
statistical data of the EdU-positive cell ratio to DMSO groups in Huh7 (B) and MHCC-LM9 cells (D). Data are presented as mean ± SD from three independent
experiments. *P < 0.05; **P < 0.01, ***P < 0.001 compared with the DMSO group. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
40.3% and 14.8% to 28.0%, respectively. Induced apoptosis activity of
compound 8 at tested concentrations was more effective than EVO
treatment. These results demonstrate that compound 8 effectively in-
duces the apoptosis of Huh7 cells.
it can be activated, and the activated HSC will transform into contractive
and highly proliferative myofibroblast-like cells, and produce a large
amount of extracellular matrix, leading to liver fibrosis, and then to
cirrhosis, and ultimately to liver cancer [38]. It is the main source of
Cancer-associated fibroblasts (CAFs), involved in the tumor microenvi-
ronment with diverse functions, including remodeling and matrix
deposition, interacting closely with cancer cells and crosstalk with other
stromal cells [38–39]. Some evidences show that hepatic stellate cells
secrete type I collagen (Col-1) to promote the invasion and metastasis
ability of hepatoma cells by induction of the epithelial-mesenchymal
transition (EMT) [40]. Therefore, CAFs have become a potential target
for optimizing therapeutic strategies against HCC. To investigate the
effect of compound 8 on CAFs, HSC-T6 cell lines were exposed to the
2.2.6. Compound 8 reduced the invasion and migration activity of MHCC-
LM9 and Huh7 cells
The migration and invasion of cancer cells can explain their malig-
nancy to some extent. It has been reported that Topo I/II inhibitors could
down-regulate MMP9 mRNA to reduce the invasion ability of cells and
further alleviate cancer metastasis [35]. Some researches has shown that
EVO could suppress the invasion, migration and VEGF-induced angio-
genesis of HCC in vitro and vivo via reducing the expression of β-catenin
and VEGF [36]. Herein, to explore the effect of compound 8 on the in-
vasion and migration of MHCC-LM9 and Huh7 cells, the chamber
transwell and scratch assay were employed. In the transwell assay, as
shown in Fig. 6, MHCC-LM9 had a weak invasion and migration ability
for 24 h, compared to Huh7 cells. To our delight, compound 8 dose-
dependently obvious inhibited the migration and invasion of both
HCC cell lines. In the scratch wound healing assay, the horizontal
migration distance of MHCC-LM9 and Huh7 cells that were exposed to
compound 8 and EVO were significantly reduced compared with the
control cells (Fig. 7). Similarly, compound 8 performed better than EVO
in both assays.
compound 8 and EVO at concentrations of 0.1, 0.3 and 0.9 μM for 24 h in
the presence of TGF-β (10 ng/mL) to induce HSC-T6 cells activating. The
Col-1 level of HSC-T6 cells was determined by ELISA assay after treat-
ment for 24 h. As illustrated in Fig. 8 A, the Col-1 level of activated HSC-
T6 cell increased significantly after being stimulated by TGF-β (10 ng/
mL). But after compound 8 and EVO treatment at gradient concentra-
tions (0.1, 0.3, 0.9 μM) for 24 h, the Col-1 level significantly reduced,
indicating that compound 8 and EVO could suppress the activation of
HSC. In order to further explore the effect of compound 8 on apoptosis
and proliferation of activated HSC-T6, firstly, cells were stimulated by
TGF-1β (10 ng/mL) for 24 h, and then cells were treated with compound
8 and EVO at concentrations of 0.1, 0.3 and 0.9 μM for 24 h. The results
2.2.7. Compound 8 inhibited the activation of HSC-T6 cells, inhibited the
proliferation and induced the apoptosis of activated HSC-T6 cells
are shown in Fig. 8 B and C, indicating that compound 8 and EVO
markedly suppressed proliferation and induced apoptosis of activated
HSC-T6 cells, and EVO displayed lower activity than compound 8.
Hepatic stellate cell (HSC), also known as lipid storage cells, is
mainly located in the space around the hepatic sinus, accounting for 5%
~23% of hepatic stromal cells [12]. Normally, HSC had low prolifera-
tive activity and weak ability to synthesize collagen [37]. When HSC is
stimulated by various physical and chemical factors and viral infection,
2.3. Compound 8 inhibited MHCC-LM9 tumor xenograft growth in vivo
Based on significant anti-HCC activity of compound 8 in vitro, the
5

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 3. Compound 8 decreased colony formation of Huh7 and MHCC-LM9 cells. Huh7 cells seeded at low density and exposed to EVO and compound 8 (0.1, 0.3 and
0.9 mol/L) for 14 d. Representative images of plate clone formation assays of Huh 7 (A) and MHCC-LM9 cells (C) stained by 1% crystal violet. The statistical data of
μ
the relative cell colony ratio to control groups (0 μM) in Huh 7 (B) and MHCC-LM9 cells (D). All statistical data are presented as the mean ± SD of three experiments.
*P < 0.05; **P < 0.01, ***P < 0.001 compared with the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Fig. 4. Compound 8 caused cell cycle G2/M arrest in Huh7 and MHCC-LM9 cells. Flow cytometric analysis of DNA tested using PI staining after exposed to EVO and
compound 8 (0.1, 0.3, 0.9
μ
M) for 48 h in Huh7(A) and MHCC-LM9 (B). The illustration in the upper right corner represented percentage of each cell phase.
6

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 5. Compound 8 changed morphology and induced apoptosis of Huh7 cells. Cell morphological changes of Huh7 cells stimulated by the treatment of compound 8
and EVO at gradient concentrations (0.15, 0.3, 0.6 M) for 24 h and stained by Giemsa reagents and detected by microscopy (magnification 200×, scale bar = 500
μ
μ
m). Green arrows point apoptotic cells characterized by cell swelling, karyopyknotic, nuclear fragmentation or agglutination, and apoptotic body formation (A).
Apoptosis analysis of Huh7 cells induced by compound 8 and EVO using flow cytometric after cells stained by Annexin V/PI double staining (B). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
preliminary efficacy experiments on the MHCC-LM9 tumor xenograft
model in mice was prepared. MHCC-LM9 cells were injected into the
subcutaneous right forelimb armpit. Mice were randomly divided into
four groups after the tumors had grown to an average size of about 100
mm3. Sorafenib at the dose of 10 mg/kg as the positive control (twice a
day, intraperitoneal injection), Compound 8 and EVO at the dose of 50
mg/kg were injected intraperitoneally for 3 weeks (twice a day). As
shown in the Fig. 9, compound 8 obviously inhibited tumor growth and
decreased the weight and volume of tumor with a tumor growth inhi-
bition rate of 40.53%. More importantly, the body weight of mice in
treatment groups had no significant difference compared to control
group. Besides, the anti-tumor activity of the sorafenib was better even
at a lower dose, while that of the compound 8 was improved to a certain
extent compared with that of EVO. Subsequently, the tumor tissue was
harvested for hematoxylin and eosin stain (HE) staining. The results
displayed that high density of new blood vessels was observed in the
control group and EVO treatment group (highlighted by black arrows)
(Fig. 10). By contrast, compound 8 administration treatment reduced
formation of new blood vessels and did not show any significant changes
in the heart, lung, liver, kidney or spleen (Fig. 10). In addition, no sig-
nificant change was observed in simple routine blood test and blood
biochemistry between compound 8 group and control group (Table S1
and S2).
3. Conclusion
Hepatocellular carcinoma (HCC) is an intractable malignant cancer
that usually occurs in the setting of chronic liver inflammation and
7

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 6. Compound 8 attenuated migration and invasion capacities of Huh7 and MHCC-LM9 cells in vitro. (A and B): represented pictures of invasion and migration
assays after treated by compound 8 in Huh7 cells (magnification × 200, scale bar = 100 m). The illustration in the upper right corner represented the statistical data
of the invasive and migrative Huh 7 cells counted by Image J. (C and D) represented pictures of invasion (magnification × 200, scale bar = 100 m) and migration
μ
μ
(magnification × 200, scale bar = 500 μm) assays after treated by compound 8 in MHCC-LM9 cells. The illustration in the upper right corner represented the
statistical data of the invasive and migrative MHCC-LM9 cells counted by Image J. All statistical data are presented as the mean ± SD of three experiments. *P < 0.05,
***P < 0.001 compared with the control group; ##P < 0.01, ###P < 0.001 compared with the EVO group.
fibrosis. The growth of HCC is dependent on remodeling “stroma”,
which is comprised of CAFs and extracellular matrix (ECM). Of the two,
CAFs play an essential role in the progression, invasion, and chemo-
therapeutic resistance of HCC. On the other hand, Topo I have been
revealed to remarkedly express in many cancer cells, including HCC.
Therefore, CAFs and Topo I are two kinds of lucrative targets for novel
anti-HCC agents, and the development of novel multi-functional anti-
HCC agents taking effect through simultaneously abrogating tumor
growth and fibrotic microenvironment is expected to be a promising
strategy. In this study, we disclosed 15 evodiamine (EVO) derivatives as
Topo I inhibitors for HCC treatment. In comparison with evodiamine,
five compounds were proved to possess distinctly anti-proliferation ef-
fect in vitro. Among them, compound 8 with 10-methyl and N14-phenyl
substitution was found to be an effective agent for anti-HCC in vitro and
displayed lower cytotoxicity to normal hepatic cell than both sorafenib
and evodiamine. In subsequent research, compound 8 was verified
possessing inhibitory potency on Topo I in vitro, which was more potent
than that of EVO. Meanwhile, it greatly inhibited the proliferation,
significantly alleviated the migration and invasion capacity, induced
obvious G2/M arrest of Huh7 and MHCC-LM9 cells, promoted apoptosis
of HCC cells, down-regulated the expression of Col-1 in activated HSC-
T6 cells and attenuated the infinitive proliferative capacity and
induced-apoptosis of activated HSC cells. Further in vivo studies revealed
that compound 8 significantly suppressed the growth of xenograft HCC
tumors, reduced the weight of GBM tumors (TGI = 40.53%) and dis-
played low toxicity in-vivo. In vitro and in vivo studies showed that the
anti-CAFs and anti-HCC effects of compound 8 were superior to those of
EVO. On the basis of these preliminary studies, compound 8 was proved
to be an effective anti-HCC agent, which provided a new sight for the
development of multi-target anti-HCC agents through simultaneously
inhibiting Topo I and CAFs.
4. Experiment
4.1. Chemistry
Proton (1H) and carbon (13C) NMR spectra were respectively recor-
ded on a 400 or 300 MHz spectrometer (Bruker Company, Germany) and
a 100 MHz spectrometer. NMR spectra used DMSO‑d6, CDCl3 or
(CD3)2CO-d6 as solvent (TMS as the internal standard). Carbon chemical
shifts are reported relative to a residual solvent peak (CDCl3 at 77.00
ppm, DMSO‑d6 at 39.96 ppm, (CD3)2CO-d6 at 29.70 ppm). Proton
chemical shifts are reported relative to a residual solvent peak (CDCl3 at
7.26 ppm, DMSO‑d6 at 2.50 ppm, (CD3)2CO-d6 at 2.05 ppm). The values
of the chemical shifts are expressed in ppm and the coupling constants
(J) in hertz. The following abbreviations were used to designate multi-
plicities: s = singlet, d = doublet, t = triplet, q = quartet, quint =
quintet, m = multiples, br = broad. High-resolution mass spectrometry
(HRMS) was obtained on an Agilent UPLC-IM-QTOF spectrometer
(Agilent 6560, USA). Purity of all final compounds was>95% (Agilent
1260 Infinity II; USA).
4.2. HPLC analysis
HPLC analysis was run on an Agilent 1260 Infinity II HPLC chro-
matogram equipped with an Eclipse Plus C18 column (4.6 × 150 mm, 4
µm) and examined at 254 nm by DAD detector. Mobile phase: 0–7 min,
MeOH: H2O = 20: 80; 7–16 min MeOH: H2O = 95: 5, 16–25 min MeOH:
H2O = 20: 80. Flow rate is 0.5 mL/min and temperature are maintained
at 23 ◦C.
4.3. General procedure for the preparation of compounds and the
characterization data of new compounds
For compounds with oxygen or nitrogen atoms substituted at 14
position, the synthetic procedure is shown as follows: a test tube
equipped with a magnetic stir bar was charged with tryptamine (1
8

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 7. Compound 8 and EVO decreased the migration rate of MHCC-LM9 and Huh7 cells. Cell monolayers were wounded by scraping with a sterile 200
μL pipette
after exposed to compound 8 and EVO for 24 h. Images of horizontal migration of MHCC-LM9 (A) and Huh7 (B) cells at 0 h and 24 h (magnification × 10, scale bar =
100 μm). Analysis of wound closure ratio (C and D). All statistical data are presented as the mean ± SD of three experiments. *P < 0.05; **P < 0.01, ***P < 0.001
compared with the control group.
mmol), 2-(phenylamino) benzoic acid (1 mmol), EDCl (1.2 mmol), HOBt
(1.2 mmol), triethylamine (2.5 mmol) and DCM (10 mL). The resulting
mixture was stirred for 10 h at room temperature, then added 25 mL of
water and 10 mL of saturated brine solution and extracted with DCM for
3 times (3 × 25 mL). The combined organic extracts were concentrated
and the resulting residue was purified by column chromatography on
silica gel (petroleum ether/ethyl acetate = 3/1) to give intermediates as
white solid. For compounds with sulfur atom substituted at 14 position,
a test tube equipped with a magnetic stir bar was charged with trypt-
amine (1.2 mmol), methyl 2-mercaptobenzoate (1 mmol), AlMe3 (1.2
mmol), DCM (10 mL) at 0 ◦C. The resulting mixture was moved to room
temperature and stirred for 12 h, then quenched with HCl (5 M) aqueous
solution, and extracted with DCM for 3 times (3 × 25 mL), the combined
organic extracts were concentrated and the resulting residue was puri-
fied by column chromatography on silica gel (petroleum ether/ethyl
acetate = 2/1) to give intermediates as white solid. The next step is that
a mixture of the above intermediates (0.1 mmol) was triethyl ortho-
formate (0.30 mmol), BF3•Et2O (0.05 mmol, 0.5 eq) and DMF (1.0 mL)
were added and stirred at 100 ◦C for 5 h under an argon atmosphere.
Then, the mixture was added 25 mL of water and 10 mL of saturated
brine solution and extracted with ethyl acetate for 3 times (3 × 25 mL).
The extract was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude product was purified by column chroma-
tography (petroleum ether/ ethyl acetate = 4/1) to afford the products
as white solid. (compounds 1–3 were obtained at 135 ◦C; compounds 4 –
5 were obtained at room temperature in DCM.)
4.3.1. 14-(pyridin-3-yl)-8,13,13b,14-tetrahydroindolo[2′,3′:3,4]pyrido
[2,1-b]quinazolin-5(7H)-one (11)
yellow solid, yield 64%; mp 292.2–294.6 ◦C.1H NMR (400 MHz,
CDCl3) δ 8.44 (s, 1H), 8.36 (s, 1H), 8.33 (s, 1H), 8.14 (d, J = 1.6 Hz, 1H),
8.12 (d, J = 1.6 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.43 – 7.32 (m, 3H),
7.24 – 7.08 (m, 4H), 7.01 (dd, J = 8.2, 1.1 Hz, 1H), 4.92 – 4.81 (m, 1H),
3.25 (m, 1H), 2.93 – 2.82 (m, 1H), 2.78 (m, 1H). 13C NMR (100 MHz,
CDCl3) δ 164.9, 150.2, 146.6, 146.3, 144.8, 138.6, 136.2, 133.4, 132.2,
129.2, 129.1, 126.6, 124.2, 123.1, 123.0, 121.6, 120.1, 118.9, 114.3,
111.4, 71.9, 42.3, 19.7. MS (ESI+) m/z calcd for C23H19N4O+ (M + H)+
367.2, found 367.1. The purity was 96.52% by HPLC analysis.
4.3.2. 14-(6-methylpyridin-3-yl)-8,13,13b,14-tetrahydroindolo [2′,3′:3,4]
pyrido [2,1-b] quinazolin-5(7H)-one (12)
yellow solid, yield 58%; mp 261.4–262.8 ◦C. 1H NMR (400 MHz,
CDCl3) δ 8.95 (s, 1H), 8.36 (d, J = 2.7 Hz, 1H), 8.12 (dd, J = 7.9, 1.6 Hz,
1H), 7.46 (d, J = 7.9 Hz, 1H), 7.38 – 7.27 (m, 3H), 7.22 – 7.04 (m, 3H),
6.97 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.31 (s, 1H), 4.86 (m,
1H), 3.24 (m, 1H), 2.94 – 2.74 (m, 2H), 2.42 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 165.0, 155.5, 146.3, 145.6, 139.9, 136.3, 133.3, 133.3, 129.1,
129.0, 126.5, 123.9, 123.4, 123.0, 122.8, 121.5, 119.9, 118.8, 114.0,
111.4, 71.6, 42.0, 23.8, 19.8. MS (ESI+) m/z calcd for C24H21N4O+ (M +
H)+ 381.2, found 381.2. The purity was 95.20% by HPLC analysis.
9

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 8. Compound 8 and EVO suppressed TGF-β-induced the activation of HSC-T6 cells and their effects on antiproliferation and induced-apoptosis of activated HSC-
T6 cells in 24 h. HSC-T6 cells were exposed to compound 8 at different concentrations for 24 h in the presence of TGF-β (10 ng/mL). Col-1 as a biomarker protein
expressed by activated HSC-T6 cells was measured by ELISA assay(A). HSC-T6 cells were stimulated by TGF-β (10 ng/mL) for 24 h before compound 8 at different
concentrations were added into each well and incubated for 24 h, except for the control well. CCK-8 assay was first performed to evaluate antiproliferation of
compound 8 and EVO on TGF-β-induced activation of HSC-T6 cell. (B): the cell viability of compounds at the indicated concentrations (0.70, 2.22, 6.67, 20 and 60
μ
M). (C): Apoptosis analysis of activated HSC-T6 cells by flow cytometry using Annexin V/PI double staining. All statistical data are presented as the mean ± SD of
three experiments. *P < 0.05; **P < 0.01, ***P < 0.001 compared with the TGF-β group; ##P < 0.01, ###P < 0.001 compared with the control group.
4.3.3. 14-(6-chloropyridin-3-yl)-8,13,13b,14-tetrahydroindolo [2′,3′:3,4]
z calcd for C26H20N5O+ (M + H)+ 418.2, found 418.1. The purity was
pyrido[2,1-b]quinazolin-5(7H)-one (13)
97.03% by HPLC analysis.
yellow solid, yield 66%; mp 277.4–278.1 C. 1H NMR (400 MHz,
◦
CDCl3) δ 8.83 (s, 1H), 8.15 (dd, J = 7.9, 1.6 Hz, 1H), 8.10 – 7.98 (d, 1H),
7.48 – 7.44 (d, 1H), 7.40 (m, 1H), 7.35 (d, J = 8.3, 0.9 Hz, 1H), 7.25 –
7.17 (m, 3H), 7.15 – 7.06 (m, 2H), 6.97 (d, J = 8.2, 1.1 Hz, 1H), 6.30 (s,
4.4. Cell lines and culture
HL-7702, HepG2, MHCC-LM9, Huh7 cell lines were purchased from
Institute of Biochemistry and Cell Biology of the Chinese Academy of
Sciences (Shanghai, China). All cell lines were cultivated in a humidified
environment containing 95% air and 5% CO2 at 37 ◦C, grown in DMEM
media supplemented with 10% FBS (fetal bovine serum, Gemini Foun-
J = 1.5 Hz, 1H), 4.82 (m, 1H), 3.22 (m, 1H), 2.85 – 2.66 (m, 2H). 13
C
NMR (100 MHz, CDCl3) δ 164.8, 147.5, 146.6, 145.2, 141.2, 136.4,
135.6, 133.5, 129.2, 127.9, 126.3, 124.8, 124.2, 123.7, 123.3, 122.1,
120.1, 118.9, 114.5, 111.5, 71.3, 41.7, 19.8. MS (ESI+) m/z calcd for
C
23H18ClN4O+ (M + H)+ 401.1, found 401.1. The purity was 95.60% by
dational), 100 U/mL penicillin and 100 μg/mL streptomycin.
HPLC analysis.
4.5. Cell viability assay
4.3.4. 14-(5-bromopyridin-3-yl)-8,13,13b,14-tetrahydroindolo[2′,3′:3,4]
pyrido[2,1-b]quinazolin-5(7H)-one (14)
MHCC-LM9, Huh7, HL-7702 and HepG2 (2–5 × 103 cells/well) cells
were separately plated into 96-well microplates and cultured 24 h. The
cellular viability was assessed using a Cell Counting Kit-8 kit (Meilunbio,
MA0218-5-Apr-01F), after cells were treated with target compounds at
yellow solid, yield 71%; mp 282.5–283.1 C. 1H NMR (400 MHz,
◦
CDCl3) δ 8.44 (s, 1H), 8.36 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 2.3 Hz, 1H),
8.14 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 – 7.41 (m, 3H), 7.36 (d, J = 8.3, 1.0
Hz, 1H), 7.22 (m, 2H), 7.12 (m, 1H), 7.04 (d, J = 8.2, 1.1 Hz, 1H), 6.35
the concentration 0, 0.156, 0.625, 2.5, 10, 40 μM for 48 h. The absor-
(s, J = 1.6 Hz, 1H), 4.86 (m, 1H), 3.25 (m, 1H), 2.95 – 2.71 (m, 2H). 13
C
bance value (OD) was measured using a microplate reader (TECAN,
spark, Switzerland) at 450 nm. The IC50 values were calculated by
inhibitory curves using SPSS. All assays were in triplicate and repre-
sented in mean ± SD values.
NMR (100 MHz, CDCl3) δ 164.7, 147.1, 144.2, 144.0, 143.5, 137.0,
136.2, 134.3, 133.6, 132.1, 129.4, 128.6, 126.6, 125.0, 123.4, 121.88,
120.3, 119.0, 114.6, 111.4, 72.0, 42.3, 19.8. MS (ESI+) m/z calcd for
C23H18BrN4O+ (M + H)+ 445.1, found 445.0. The purity was 96.74% by
HPLC analysis.
4.6. Enzyme inhibition assay
4.3.5. 14-(quinoxalin-6-yl)-8,13,13b,14-tetrahydroindolo[2′,3′:3,4]
EVO, CPT, VP16 and compound 8 were dissolved in DMSO and were
examined at final concentrations of 100, 50, 10 in Topo I inhibition
assay (TaKaRa Biotechnology). The Topo I reaction mixture contained
pyrido[2,1-b]quinazolin-5(7H)-one (15)
◦
yellow solid, yield 66%; mp 315.2–316.6 C. 1H NMR (400 MHz,
CDCl3) δ 8.86 (s, 1H), 8.76 – 8.69 (m, 2H), 8.07 (dd, J = 8.1, 1.7 Hz, 1H),
7.99 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 2.5 Hz, 1H), 7.70 (dd, J = 9.1, 2.6
Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.35 (td, J = 7.7, 1.6 Hz, 1H), 7.28 (d,
J = 8.6 Hz, 1H), 7.19 – 7.06 (m, 4H), 6.56 (s, 1H), 4.89 (dd, J = 12.9, 5.4
Hz, 1H), 3.30 – 3.06 (m, 2H), 2.68 (dd, J = 15.4, 4.4 Hz, 1H).13C NMR
(100 MHz, CDCl3) δ 164.7, 148.0, 145.4, 144.0, 143.7, 142.4, 140.4,
136.0, 133.3, 130.8, 130.4, 129.3, 127.1, 126.1, 124.0, 122.9, 121.8,
120.5, 120.0, 119.4, 118.7, 113.7, 111.4, 73.3, 43.8, 19.6. MS (ESI+) m/
Topo I (0.5 units, 0.5
mM Tris-HCl, 72 mM KCl, 5 mM MgCl2, 5
spermidine, 2 L), 0.1% bovine serum albumin (BSA, 2
plasmid DNA (0.125 g, 0.5 L), and distilled water in a final volume of
20
L. Reactions were incubated for 30 min at 37 ◦C. All the reactions
μL), tested compounds (0.2
μ
L), buffer (pH 8.0 35
M dithiothreitol, 5 mM
L), pBR322
μ
μ
μ
μ
μ
μ
were vortexed for 10 s and centrifuged for 2 min. Then, 6 μL of 6 × gel
loading buffer (0.25% bromophenol blue and 50% glycerol) was added.
Products were analyzed by gel electrophoresis in a 0.8% agarose gel
10

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 9. Compound 8 inhibited tumor growth in the xenograft nude mice model. (A) Pictures of nude mice and separated entire MHCC-LM9 tumor tissue. (B) Growth
curve of implanted MHCC-LM9 xenograft in nude mice measured by volume. (C) The body weight change of nude mice treated with different compounds. (D) The
tumor weight of each group. Data are presented as the mean ± SEM (n = 5); *P < 0.05; **P < 0.01, ns: no significant, compared with the control group.
without EtBr. To visualize the DNA band, soaking the gel in EtBr work
4.8. Colony formation assay
solution (0.5 μg/mL) for 30 min after DNA electrophoresis. The DNA
bands were visualized under UV light.
Huh7 (1 × 103 cells/well), and MHCC-LM9 (1 × 103 cells/well) cells
were seeded onto 6-well plates and cultured 24 h. After being incubated
4.7. EdU assay
with tested compounds at indicated concentrations (0.1, 0.3 ,0.9 μM) for
7 days, changed medium every 3 days, cells were fixed with methanol
and stained with 1% crystal violet. Colonies of ≥ 50 cells were counted
under microscope at 4 × lens.
MHCC-LM9, and Huh7 cells were seeded at a density of 1 × 103 cells/
well onto 96-well plates and incubated until the cells attached, then
treated with different various of compound 8 and EVO at the final
concentrations of 0.1, 0.3, and 0.9
μ
M. EdU assay was performed ac-
4.9. Cell cycle analysis
cording to the manufacturer’s instructions (Cat.#: C10310-1, Ribobio,)
after treatment for 24 h. In short, containing 50
μM/100
μL of EdU
MHCC-LM9 (2–5 × 104 cells/well), and Huh7 (2–5 × 104cells/well)
cells were separately seeded into 6-well microplates and cultured 24 h
and then co-cultured with compound 8 and EVO at indicated concen-
DMEM was added and cultured for another 2 h. Removing the medium
before cells were washed with PBS three times. And then, 4% para-
formaldehyde was added into each well to fix cells at room temperature
trations (0.1, 0.3, and 0.9 μM) for 24 h. Then the cells were harvested
for 30 min. 0.5% TritonX-100 solution was added after adding 50
μL of 2
and fixed with 70% pre-cooled ethanol at 4 ◦C for overnight. After
removing ethanol by centrifugation, the fixed cells were resuspended in
PBS and treated with RNase A followed by staining with PI for 10 min.
The cell cycle analysis was determined by Flow cytometry (Becton
Dickinson, USA). Data were analyzed by Modfit software.
mg/mL glycine and incubated min on shaker for 5 min, following by
washed away with PBS. Cells were stained with Apollo in the dark for 30
min. Subsequently, staining with Hoechst 33,342 (Solarbio, Cat.#:
B8030) for 5 min in the dark, cells were observed by a fluorescence
microscope (Moticam Pro 282B, China).
11

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
Fig. 10. Histopathological images of mice tissues (heart, lung, liver, spleen, kidney and tumor, magnification × 100, scale bar = 300
μ
m), The black arrow marked
vessels in the tumor.
O
O
N
A
R1
AlMe3
0oC
H
O
DCM,
HS
O
N
Lewis acid
HC(OEt)3
N
HS
R1
H
NH2
Ar1
+
O
sol.,
temp.
O
N
N
Ar2
X
triethylamine
HOBt
H
H
N
H
R1
H
EDCI,
DCM,
HO
R2
rt
XH
R2
N
XH
X=
H
O
N-R,
4.10. Cell apoptosis and Giemsa staining assays
the chamber, were fixed with 4% paraformaldehyde for 3 min and
stained with crystal violet (0.1%). Five random views were chosen for
cell counting (magnification × 200).
Huh7 (2–5 × 104 cells/mL) cells were seeded into 6-well plates and
incubated until the cell attached, then treated with tested compounds at
0.1, 0.3 and 0.9 μM for 48 h. After that, the cells were collected by
4.12. Scratch assay
trypsinization, washed with PBS twice, and stained with PI and FITC-
Annexin-V for 0.5 h in the dark. Then, the flow cytometry (BD Biosci-
ence, USA) was used for determined the percentages of apoptotic cells.
Data were analyzed by Flow J software. Huh7 cells at the density of 2–5
× 104 were plated into 6-well plates and incubated for 24 h. Cells were
treated with evo and compound 8 at the concentrations of 0.1, 0.3 and
MHCC-LM9 and Huh7 cells were cultured in 6 well plates to 90%
confluence in DMEM supplemented with 10% FBS. Cell monolayers
were wounded by scraping with a sterile 200 μL pipet tip. The cells were
removed the medium and washed with PBS three times before treated
with EVO and compound 8 at the concentrations of 0.03, 0.1 and 0.3 μM.
0.9 μM. After cultured for 48 h, removed medium and cells were washed
After cultured for 24 h. Images were captured of each scratch at time
points 0 h and 24 h. Measurements were taken from edge to edge at time
0 h and compared to measurements from 24 h using ImageJ software.
with PBS and fixed with 4% paraformaldehyde for 3 min. Next, cells
were stained with Giemsa reagent (Solarbio, Cat. #: G1010) for 30 min.
The reagent was rinsed away with water and observed under inverted
microscope.
4.13. The activation of HSC-T6 cells stimulated by TGF-β
4.11. Cell invasion and migration assays
HSC-T6 (2–5 × 103 cells/mL) cells were seeded into 96-well micro-
plate at 37℃, 5% CO2 for overnight, and then were treated with TGF-β
(10 ng/mL) for 24 h, equal volume of water was added into control well.
Next, TGF-β induced cells exposed to various concentrations (0, 0.7, 2.2,
The vitro cell invasion and migration assays were employed using the
upper chamber with or without Matrigel (BD) of a 24-well Transwell
plate (Costar, Cambridge, MA). Huh7 and MHCC-LM9 cells suspended
with FBS-free medium at a density of 5 × 104/mL were added into the
upper chamber and the outer chamber contained 700 µL medium (15%
FBS). At the same time, the various concentrations of compound 8 (0.1,
6.6, 20, 60 μM) of tested compound. After incubating for 24 h, the
cellular viability was measured by CCK-8 method. The absorbance value
(OD) was tested using a microplate reader (TECAN, spark, Switzerland)
at 450 nm. the cellular viability was calculated using the following
equation: [(ODControl-ODBlank)/(ODControl-ODBlank)] × 100%. All assays
were in triplicate and represented in mean ± SD values. HSC-T6 cells
were exposed to compound 8 at different concentrations for 24 h in the
0.3 and 0.9 μM) and EVO (0.9 μM) were added into the chambers and
incubated at 37˚C for 24 h. Next, cells in upper chamber were removed
with cotton swabs. Invaded and migrated cells on the outer surface of
12

X. Fan et al.
Bioorganic Chemistry 114 (2021) 105154
presence of TGF-β (10 ng/mL). At the same time, equal volume of DMSO
was added into TGF-β treated well and control group without TGF-β.
Cells were collected after washing with PBS, were lysed by freeze–thaw
cycles three time, followed by centrifugation at 5000 rpm at 4 ◦C for 15
min. The protein concentrations were determined by BCA kit (Beyotime,
China), adjusted by PBS to the same concentration. Samples was added
in ELISA microplates precoated with polyclonal Col-1 antibodies
(shanghai mlbio, China), incubated for 45 min at 37 ◦C. After washed
with ELISA Wash buffer five times, Horseradish Peroxidase (HRP) so-
lution was added into to each microplate well and incubated for 30 min.
Subsequently, Solution A and solution B added into each well before
washed with ELISA Wash buffer five times, incubated for 15 min at
37 ◦C, terminated the reaction by adding the Stop solution and the
absorbance value (OD) was measured by a microplate reader at 450 ± 2
nm. The sample OD value is interpolated onto formula for the standard
curve to determine their content of Col-1. The results were calculated
from at least three independent experiments. HSC-T6 (2–5 × 104 cells/
mL) cells were seeded into 6-well plates at 37 ◦C, 5% CO2 for overnight,
and then were treated with TGF-β (10 ng/mL) for 24 h. TGF-β induced
Acknowledgements
We thank Dr. Na Yan for HRMS analysis (UPLC-IM-QTOF, 6560, ion
source: ESI or APCI). This work was technically supported by University
of South China, Lanzhou University, and Gansu Provincial Hospital. This
work was financially supported by the Gansu Province Science Foun-
dation for Distinguished Young Scholars (20JR5RA304) and Recruit-
ment Program of Global Experts (1000 Talents Plan). Dr. Xiangyong Hao
thanks the financially support from the Gansu Province Youth Innova-
tive Talents Project (2021, Xiangyong Hao), Health Industry Scientific
Research Program of Gansu Province (GSWSKY2020-06), the Natural
Science Foundation for Young Scientists and the Science & Technology
Planning Project of Gansu Province (18JR3RA058), and Research Pro-
jects of Gansu Provincial Hospital (Grant No.: 19SYPYB-7, 18GSSY3-1).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105154.
cells exposed to various concentrations (0, 0.1 0.3 and 0.9 μM) of tested
compound. After incubating for 24 h, apoptosis analysis was measured
by flow cytometry using Annexin V/PI double staining. All statistical
data are presented as the mean ± SD of three experiments. *P < 0.05;
**P < 0.01, ***P < 0.001 compared with the TGF-β group; ##P < 0.01,
###P < 0.001 compared with the control group.
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