Synthesis and biological evaluations of N′-substituted methylene-4-(quinoline-4-amino) benzoylhydrazides as potential anti-hepatoma agents

Article abstract of DOI:10.1016/j.bioorg.2020.103592

In the effort to develop novel quinoline derivatives for the treatment of liver cancer, we synthesized a series of N′-Substituted methylene-4-(quinoline-4-amino) benzoylhydrazides and evaluated their biological activities as anticancer agents. Compounds 5

Full text of DOI:10.1016/j.bioorg.2020.103592

Journal Pre-proofs
Synthesis and biological evaluations of N'-Substituted methylene-4-(quino-
line-4-amino) benzoylhydrazides as potential anti-hepatoma agents
Baicun Li, Feifeng Zhu, Fengming He, Qingqing Huang, Tong Wu, Taige
Zhao, Yingkun Qiu, Zhen Wu, Yuhua Xue, Meijuan Fang
PII:
S0045-2068(19)31970-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.103592
YBIOO 103592
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Bioorganic Chemistry
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Revised Date:
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20 November 2019
16 January 2020
18 January 2020
Please cite this article as: B. Li, F. Zhu, F. He, Q. Huang, T. Wu, T. Zhao, Y. Qiu, Z. Wu, Y. Xue, M. Fang,
Synthesis and biological evaluations of N'-Substituted methylene-4-(quinoline-4-amino) benzoylhydrazides as
potential anti-hepatoma agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103592
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Synthesis and biological evaluations of N'-Substituted
methylene-4-(quinoline-4-amino) benzoylhydrazides as
potential anti-hepatoma agents
Baicun Li,1, 2, # Feifeng Zhu,1, # Fengming He,1, # Qingqing Huang,1 Tong Wu,1 Taige
Zhao,1 Yingkun Qiu,1 Zhen Wu,1 Yuhua Xue,1, * Meijuan Fang1, *
1. Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key
Laboratory of Cellular Stress Biology, School of Pharmaceutical Sciences, Xiamen
University, Xiamen 361102, China
2. State Key Laboratory of Medical Molecular Biology, Department of Physiology, Institute of
Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine,
Peking Union Medical College, Beijing 100005, China
#These authors contributed equally to this work.
*Corresponding author. Fujian Provincial Key Laboratory of Innovative Drug Target
Research and State Key Laboratory of Cellular Stress Biology, School of
Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China
E-mail addresses: fangmj@xmu.edu.cn (M.-J. Fang), xueyuhua@xmu.edu.cn (Y.-H.
Xue).

Abstract
In the effort to develop novel quinoline derivatives for the treatment of liver cancer,
we synthesized a series of N'-Substituted methylene-4-(quinoline-4-amino)
benzoylhydrazides and evaluated their biological activities as anticancer agents.
Compounds 5h and 5j were found to be the potent antiproliferative agents against
HepG2 cell line with an IC50 value of 12.6±0.1 μM and 27.3±1.7 μM, respectively. The
most effective compound 5h also exhibited potent cytotoxicity against SMMC-7721
and Huh7 cells with IC50 values of 9.6±0.7 M and 6.3±0.2 M, respectively.
Inspiringly, both 5h and 5j exhibited lower cytotoxic property in normal cells than
hepatic carcinoma cells. Compounds 5h and 5j could down-regulate mRNA level of c-
Myc and expression level of c-Myc. Meanwhile, they decreased expression level of
anti-apoptotic protein Bcl-2 and increased expression levels of pro-apoptotic protein
Bax and cleaved PARP with reference to tubulin. So various assays including cell
colony formation, cell cycle distribution, as well as cell apoptosis and migration were
performed to understand their antitumor role. It was confirmed that 5h and 5j inhibited
the growth of HepG2 cells due to their anti-survival effect, induction of cell cycle arrest
and cell apoptosis, and inhibition of cell migration. These results demonstrated that 5h
might be as potential lead compounds to develop anticancer agents for the treatment of
hepatocellular carcinoma.
Keywords: Liver cancer; Methylene-4-(quinoline-4-amino) benzoylhydrazide;
Anticancer; c-Myc inhibitor; Apoptosis.

1. Introduction
Cancer is a major public health problem and consumes a lot of social resources[1].
Liver cancer is the fifth most common malignant tumor in the world. The incidence of
liver cancer has been increasing all over the world in recent years [2-5]. Hepatocellular
carcinoma is the most common type of liver cancer and also is the most common
primary malignant tumor of the liver. The morbidity and mortality of hepatocellular
carcinoma continued increased in recent years make it urgent. Therefore, it is necessary
to speed up the development of the target drugs for hepatocellular carcinoma. C-Myc
protein is a transcription factor which targets genes involved in cell growth, cell-cycle
progression, transcription, differentiation, apoptosis, and cell motility. Dysregulation of
c-Myc is commonly found in many cancer [6-8]. Clinical research has been shown that
c-Myc is overexpressed in most hepatocellular carcinoma patients and correlated with
poor prognosis [9]. Further down-regulating of c-Myc in HepG2 cells by RNAi could
significantly inhibit migration, invasion and proliferation of HepG2 cells. Therefore, c-
Myc is thought to be a potential therapeutic target for hepatocellular carcinoma [10].
Quinoline is one of the most ubiquitous heterocycles and appears as an important
structural component in many natural products and synthetic compounds that are
associated with a wide spectrum of bioactivities such as antifungal [11], antibacterial
[12], and anticonvulsant [13]. Quinoline derivatives play an important role in the
development of anticancer drugs. For example, camptothecin bearing quinolone motif
is the first natural topoisomerase inhibitor with broad-spectrum anti-tumor activity, and
it is mainly used for the treatment of colon cancer, ovarian cancer, liver cancer, bone
cancer and leukemia [14]. Semisynthetic camptothecin derivatives, such as topotecan
and irinotecan, have been approved to treat ovarian and colon cancer, respectively[14].
Neratinib (LNTN) with a quinoline pharmacophore is a double inhibitor of
HER2/EGFR, approved as an orally available drug that is used in the extended adjuvant
therapy of early-stage breast cancer [15]. Besides, 8-hydroxyquinoline and its

derivatives isolated from the phytotoxic activity of T. chinensis roots [16], exhibit a
relatively promising anticancer effect in vitro and in vivo, being reported as potential
anti-tumor agent [17]. 83b1 is a novel quinoline derivative which can inhibit cancer
growth in human esophageal squamous cell carcinoma (ESCC) [18]. Additionally,
William Kemnitzer et al. identified a series of 1-benzoyl-3-cyanopyrrolo [1,2-a]
quinoline derivatives that are more active in human breast cancer cell line T47D, human
colon cancer cell HCT116 and hepatocellular carcinoma cell line SNU398 [19].
To develop new type quinoline derivatives for the treatment of hepatocellular
carcinoma, we first synthesized a series of N'-Substituted methylene-4-(quinoline-4-
amino) benzoylhydrazides and evaluated their antiproliferative activities against
HepG2 cells using MTT assay. Compounds 5h and 5j demonstrated more potent
inhibiting activity against HepG2 cells compared with other synthesized compounds.
In vitro cell cytotoxicity of 5h and 5j was then investigated against the other two
hepatocellular carcinoma cell lines (SMMC-7721 and Huh7) and normal cell lines
(LO2 and MRC-5) by MTT assay using Neratinib (LNTN) as a positive control.
Subsequently, 5h and 5j as representative compounds were examined for their effects
on alterations in the expression of oncogene c-Myc and apoptotic proteins, representing
Bcl-2, Bax, as well as cleaved PARP. Additionally, further experiments into an
antitumor role behind the antitumor activity of compounds 5h and 5j were also carried
out, including cell colony formation, cell cycle progression, apoptotic induction, and
cell migration. Taken together, we found quinoline derivatives 5h might be as potential
lead compounds to develop anticancer agents for the treatment of hepatocellular
carcinoma.
2. Results and discussion
2.1. Chemistry
The
synthesis
of
N'-Substituted
methylene-4-(quinoline-4-amino)
benzoylhydrazides (5a-5p) were delineated in Scheme 1. 4-Hydroxyquinoline (1) was

firstly halogenated to form 4--chloroquinoline (2) using phosphorus oxychloride[20].
Ethyl 4-(quinolin-4-ylamino) benzoate (3) was then prepared from benzocaine by N-
alkylation using 4--chloroquinoline (2) in the presence of hydrochloric acid as a catalyst
in refluxing butyl alcohol. Compound 3 was treated with hydrazine hydrate in refluxing
ethanol, followed by condensation with different substituted aldehydes to give the
corresponding target compounds 5a-5p in 49−92% yields after isolation.
All new target compounds were characterized by proton nuclear magnetic resonance
(1H NMR), carbon nuclear magnetic resonance (13C NMR), and electrospray ionization
mass spectrometry (ESI-MS). The C=N double bonds of N-acylhydrazones were in the
E form based on reported studies [21-24]. Furthermore, a single E geometrical isomer
of the representative compound 5j was identified by H-9 (-NH-) signal observed as a
1
single peak (δ 10.46, H-9) in H NMR spectra and the cross-peak (10.46, 8.62)
intensities of -CONH- and =CH-(H-9/H-22) observed in 2D NOESY spectrum (Figure
S4, see Supporting Information). Therefore, we concluded that the stereochemistry of
compounds 5a-5p as a single E geometrical isomer.
Insert Scheme 1 here.
Scheme 1. Preparation of target compounds 5a-5p
2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity of the target compounds 5a-5p against HepG2 cells
The synthesized compounds were evaluated for their cytotoxic activity against
HepG2 cells by MTT assay using Neratinib (LNTN) as a positive control. The cytotoxic
activity of compounds against HepG2 cells was first tested at the concentration of 25
μM (Figure S1). As shown in Figure S1, most target compounds showed very weak
cytotoxic activity (<50%) against HepG2 cells at the concentration of 25 μM, while 5h
and 5j displayed significant inhibitory effect (100% and 90.70%, respectively) on the

proliferation of HepG2 cells at the same tested concentration. To clarify the effect of
different substitutions (R) on the cytotoxic activity, the IC50 values of these compounds
for the cytotoxic activity against HepG2 cells were further determined and listed in
Table 1. Considering the IC50 results in HepG2 cells, we attempted to establish the
structure-activity relationship (SAR) among the tested compounds. Firstly, we found
that alkyl substitutions (5a & 5b) at nitrogen atom (N’) of N-acylhydrazone had little
effect on the cytotoxic activity. However, different substituted groups of the phenyl ring
introduced at the N′-methylene position displayed an important relationship with
cytotoxic activity. Compounds 5d, 5e, and 5k possessing carboxyl (COOH) or nitro
(NO2) group at phenyl ring were inactive. Compound 5l which had 3,5-dibromo and 4-
hydroxyl groups at phenyl ring significantly decreased cellular cytotoxic activity.
However, except compound 5i, three compounds (5g, 5h, and 5j) with 2 hydroxy (OH)
or Methoxy (OCH3) substitutions at phenyl enhanced cytotoxic activity. Compared with
compound 4, the introduction of aromatic heterocycles at the N′- methylene (5m, 5n,
5o, and 5p) was unhelpful for cytotoxic activity. Above all, 5h and 5j displayed better
inhibition against HepG2 cells than the other synthesized compounds.
Insert Table 1 here.
Table 1. In vitro cytotoxic activity of the target compounds 5a-5p against HepG2 cells
Both anti-hepatoma effect and safety of 5h and 5j were further determined by MTT
assay in vitro. Three different human hepatic carcinoma cell lines such as HepG2,
SMMC-7721, and Huh7 were used to evaluate the anti-hepatoma effect of these two
selected compounds. Meanwhile, the safety of two selected compounds toward normal
cells such as LO2 and MRC-5 was evaluated. The activities of 5h, 5j, and the reference
drug Neratinib (LNTN) were shown in Table 2. As shown in Table 2, 5h exerted
superior antiproliferative effects against HepG2, SMMC-7721, and Huh7 cell lines,
while 5j had lower antitumor activity in all three cancer cell lines compared to 5h,

especially SMMC-7721. Inspiringly, both 5h and 5j exhibited lower cytotoxic property
in normal cells than hepatic carcinoma cells. So compounds 5h and 5j were selected
for further study in more detail.
Insert Table 2 here.
Table 2. The anti-hepatoma effect and safety of 5h and 5j
2.2.2 Modulating effect of 5h and 5j on the key proteins of cancer
Inhibition of the expression of oncogene c-Myc. c-Myc plays an important role in
tumorigenesis and the development of cancer, being a potential therapeutic target for
hepatocellular carcinoma. So the represented compounds 5h and 5j were first evaluated
their regulating effect on the proto-oncogene c-Myc. As shown in Figure 1A, the
expression of protein c-Myc was significantly inhibited by 15 M 5h and 20 M 5j.
We also detected that the expression of the c-Myc oncogene mRNA was inhibited by
compounds 5h and 5j (Figure 1B), especially 5h. It suggests that compounds 5h and
5j may inhibit cell proliferation and induce cell cycle arrest and eventual apoptosis.
Regulation of apoptosis-related protein expression. The BCL-2 protein family
having pro-apoptotic (e.g. Bax) and anti-apoptotic proteins (e.g. Bcl-2) are crucial for
the regulation cell apoptosis, tumorigenesis and cellular responses. So we checked the
levels of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in HepG2 cells
treated with or without compounds 5h and 5j. The expression of Bcl-2 and Bax proteins
was first determined by Western Blotting analysis. Treatment of HepG2 cells with
compound 5h and 5j resulted in the down-regulation of anti-apoptotic protein Bcl-2
levels and the up-regulation of the pro-apoptotic protein Bax (Figure 1C, visually
represented by grayscale analysis). Meanwhile, the mRNA levels of Bcl-2 and Bax
were examined using qRT-PCR. The results showed that anti-apoptotic protein Bcl-2
decreased, while pro-apoptotic protein Bax increased after 5h or 5j treatment (Figure

1D). It was consistent with Western Blotting analysis. Next, the immunofluorescence
assay (Figure 1E) showed that Bax in the cytoplasm significantly increased after 5h
treatment at indicated doses, whereas there is a little increase after 5j treatment at
indicated doses. All the above results imply that 5h and 5j may induce apoptosis of
HepG2 cells through mitochondrial apoptosis. Moreover, the increase in cleaved PARP
(c-PARP, a marker for apoptosis) following treatment of cells by 5h and 5j indicates
cell death via apoptosis (Figure 1C).
Insert Figure 1 here.
Figure 1. Modulating effect of 5h and 5j on the key proteins of cancer. (A). The expression
level of c-MYC in HepG2 cells treated with 5h or 5j for 24 h, determined via Western blot. (B).
mRNA level of c-MYC in HepG2 cells treated with 5h or 5j for 24 h determined by RT-PCR.
(C). The expression level of Bcl-2, Bax, and c-PARP proteins in HepG2 cells treated with 5h
or 5j, determined via Western blot. (D). mRNA levels of Bcl-2 and Bax in HepG2 cells treated
with 5h or 5j for 24 h determined by RT-PCR. (E). Immunofluorometric images of Bax, DAPI
staining and merge in untreated or treated HepG2 cells with 5h and 5j for 24 h. (∗) P < 0.05,
(∗∗) P < 0.01, (∗∗∗) P < 0.001 compared with control group.
2.2.3 Anticancer activity of 5h and 5j in HepG2
Anticancer drugs usually target oncogenes and inhibit the growth of tumor cells by
regulating cell cycle, apoptosis, and cell migration to achieve the anticancer effect. To
confirm the anticancer activity and explore the anticancer role of 5h and 5j, we first
evaluated their anti-survival effect on HepG2 cells using colony formation assays and
examined their cell cycle effect. Then, we studied whether 5h and 5j also induced
cellular apoptosis to produce an antitumor effect. At last, wound healing assay was
carried out to examine whether they reduced the migratory capability of cancer cells.
Colony formation analysis. Colony formation assays for compounds 5h and 5j (two

C-Myc inhibitors) were carried out to evaluate their influence on the cancer cell survival
and proliferation abilities. As shown in Figure 2A, the colonies formed by HepG2 cells
were reduced on exposure to 5h or 5j at indicated doses. Comparing to 5j, 5h has better
efficacies against HepG2 cells. It indicates that compound 5h has a strong anti-survival
effect on liver cancer cells.
Cell cycle analysis. Since c-Myc inhibitors may induce cell cycle arrest and eventual
apoptosis, the effect of 5h and 5j on cell cycle progression using propidiumiodide (PI)
staining in HepG2 cells was examined. As illustrated in Figure 2B, the G2/M phase
cell cycle arrest was significantly increased with HepG2 cells treated with 5h compared
with the control group. When HepG2 cells were treated with 5h at concentrations of
7.5 and 15 μM for 24 h of incubation, the percentage of cells in the G2/M phase dose-
dependently increased from 22.58% to 37.77%. While the incubation of HepG2 cells
with increasing concentrations of 5j (10 and 20 μM) only increased the percentage of
cells in the G2/M phase from 22.65% to 27.58% compared to the control cells incubated
with DMSO (18.56%). It implies that 5h has a better cell cycle arrest effect on HepG2
cells than 5j.
Insert Figure 2 here.
Figure 2. Cell colony formation and cell cycle phase distribution of HepG2 cells with or
without treatment of compounds 5h and 5j. (A) Compounds 5h or 5j suppressed cell colony
formation of HepG2 cells. HepG2 cells were incubated with varying concentrations of
compounds 5h or 5j for 7 days and dyed with 0.1% crystal violet. (B) Compounds 5h or 5j
induced cell cycle arrest at the G2/M phase in HepG2 cells. HepG2 cells were plated in six-
well plates for 24 h and treated with or without compound 5h and 5j and at different
concentrations for 24 h of incubation. Then, the cells were harvested to determine the PI-stained
DNA content via flow cytometry.
Cell apoptosis analysis. According to the above-mentioned apoptosis-related

proteins expression and cell cycle analysis, we supposed that compounds 5h and 5j
might prime the mitochondria for cytochrome-c release and promote the induction of
apoptosis. To confirm their effects on apoptotic induction, HepG2 cells were treated
with 5h or 5j at different concentrations (5, 10, and 15 μM) for 24 h, and then, the cells
were harvested and doubly stained with annexin V/PI (propidium iodide). As shown in
Figure 3, at the 5 μM concentration 5h causes 11.15% apoptotic cells, and the
percentage increases to 19.65% at 10 μM and 42.6% at 15 μM. Meanwhile, 5j also
displayed a clear dose-dependent apoptotic effect in HepG2 cells, with the percentages
of apoptosis cells increasing from 13.09 at 5 μM up to 85.7% at 20 μM. These results
confirmed that compound 5h and 5j effectively induced cell apoptosis in HepG2 cells
in a dose-dependent manner.
Insert Figure 3 here.
Figure 3. Apoptosis analysis of 5h and 5j in HepG2 cells. Cells were treated with 5h or 5j at
distinct concentrations for 24 h, and the cell apoptosis induction of these cells was analyzed by
flow cytometry.
Cell migration analysis. Cell migration is a key feature of cancer progression and
metastasis and a good anticancer should suppress cell migration. So we also examined
whether 5h and 5j can inhibit the migration of HepG2 cancer cells at the different
concentrations using a wound-healing assay. As shown in Figure 4A, 5h inhibited the
migration of the HepG2 cells in a dose-dependent manner. Compound 5f also reduces
the migratory capability of the HepG2 cells as evident from the wound healing assays
(Figure 4B).
Insert Figure 4 here.
Figure 4. The effects of compounds 5h and 5j on the migration of HepG2 cells (A&B).
Compounds 5h (A) and 5j (B) suppressed the migration of HepG2 cells in a dose-dependent

manner.
3. Conclusion
In present work, new N'-substituted methylene-4-(quinoline-4-amino)
benzoylhydrazide derivatives were synthesized as anticancer agents. These derivatives
were initially evaluated for their antiproliferative activities against HepG2 cells.
Compounds 5h and 5j exhibited higher antiproliferative activities towards HepG2 cells
with IC50 values of 12.6±0.1 µM and 27.3±1.1 µM, respectively. Compound 5h also
exhibited potent antiproliferative activity with the IC50 values of lower than 10 M
against SMMC-7721 and Huh7cells (two human hepatic carcinoma cell lines).
Remarkably, 5h and 5j lower cytotoxic properties in normal cells. Further bioassays
revealed that 5h and 5j were found to be two c-Myc inhibitors and could induce the
down-regulation of anti-apoptotic protein Bcl-2 and the up-regulation of the pro-
apoptotic protein Bax. The antitumor effects of 5h and 5j in HepG2 cells resulted from
anti-survival effects, cell cycle arrest, the induction of apoptotic cell death, and
inhibition of cell migration.
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from commercial sources and were used
without further purification unless otherwise indicated. All reactions were magnetically
stirred and monitored by thin-layer chromatography (TLC) on (Qingdao Haiyang
Chemical, China) silica gel 60F-254 by fluorescence. The target compounds were
purified by silica gel column chromatography. Melting points (mp) were determined
using a Shanghai Jingke SGW X-4 microscope melting point apparatus and were
uncorrected. 1H-NMR, 13C-NMR, and NOE spectra were obtained using a Bruker AV2
13
600 Ultra shield spectrometer, at 600 MHz for 1H NMR and 150 MHz for C NMR.
Chemical shifts were given in ppm () relative to tetramethylsilane (TMS) as an internal

standard. Splitting patterns are designated as follows: s (singlet), br. s (broad singlet), d
(doublet), t (triplet), q (quartet), dd (double doublet), m (multiplet). Mass spectra were
measured on a Single Quadrupole Mass Spectrometer (Shimadzu, LCMS-2020). The
purities of synthesized compounds were detected by high performance liquid
chromatography (HPLC) with a COSMOSIL 5C18-MS-II column (4.6 ID x 250 mm).
The HPLC analysis was performed on an Agilent Technologies 1100 Series HPLC
system. The mobile phase was water (A) and acetonitrile (B) in a linear gradient mode
as follows: (A) from 95% to 0% and (B) from 5% to 100% during 0-30 min. The flow
rate was 1 mL min-1, and the detection wavelengths were 254 nm and 365 nm.
4.1.1. The synthesis of 4-chloroquinoline (2)
A mixture of 4-hydroxyquinoline 1 (2.90 g, 0.020 mol) and phosphorus oxychloride
(30 mL) was stirred at 120℃ for 12 hours. Then, the mixture was cooled to room
temperature and the solvent was removed by reduced pressure distillation. The residue
was dissolved in 50 mL of code water, the aqueous was adjusted pH to 8-9 with 10%
NaOH under cooling in an ice-water bath, and extracted with dichloromethane (DCM,
3 x 30mL). The combined organic layer was dried over anhydrous MgSO4, filtered, and
1
concentrated in vacuo to get the colorless liquid (2.80 g, 87%). H NMR (600 MHz,
DMSO-d6): δ 8.87-8.92 (m, 1H), 8.22-8.25 (m, 1H), 8.13-8.17 (m, 1H), 7.91-7.94 (m,
1H), 7.80-7.84 (m, 2H); ESI-MS: positive mode m/z 164.3 [M+H]+.
4.1.2. The synthesis of ethyl 4-(quinolin-4-ylamino)benzoate (3)
4-chloroquinoline 2 (1.60 g, 0.010 mol), benzocaine (1.70 g, 0.010 mol), n-butanol
(35 mL), and 2 drops of concentrated hydrochloric acid were added to the dry 100 mL
round bottom flask, and the reaction mixture was stirred at reflux for 15 hours. Then,
the mixture was cooled to room temperature and the resulting solid was filtered off,
washed with Hexane, and dried to give a yellowish solid with a yield of 81%. 1H NMR
(600 MHz, DMSO-d6): δ 11.20(s, 1H), 8.88-8.93 (m, 1H), 8.59-8.62 (m, 1H), 8.14-8.17
(m, 1H), 8.10-8.14 (m, 2H), 8.04-8.08 (m, 1H), 7.81-7.85 (m, 1H), 7.67-7.71 (m, 2H),
7.06-7.10 (m, 1H), 4.33-4.39 (m, 2H), 1.33-1.38 (m, 3H);13C NMR (151 MHz, DMSO-
d6): δ 165.5, 154.7, 143.5, 142.4, 138.8, 134.5, 131.3(2C), 128.3, 127.8, 125.1(2C),

124.4, 120.8, 118.1, 101.2, 61.4, 14.7. ESI-MS: positive mode m/z 293.1 [M+H]+.
4.1.3. The synthesis of 4-(quinolin-4-ylamino) benzohydrazide (4)
A suspension of ethyl 4-(quinolin-4-ylamino)benzoate 3 (2.90 g, 0.010 mol) and
hydrazine hydrate (20 mL) in ethanol (15 mL) was stirred and refluxed for 24 h. The
reaction mixture was then cooled to room temperature, and the resulting solid was
filtered, washed with water, and dried. White solid (2.20 g, 78%); HPLC purity: 99.23%;
mp: 272-274℃.1H NMR (600 MHz, DMSO-d6): δ 9.70 (s, 1H), 9.16 (s, 1H), 8.56 (d,
J=5.1Hz, 1H), 8.36-8.39 (m, 1H), 7.90-7.95 (m, 1H), 7.87-7.90 (m, 2H), 7.73 (dt, J=1.1,
7.6 Hz, 1H), 7.55-7.59 (m, 1H), 7.42 (d, J=8.6 Hz, 2H), 7.16 (d, J=5.1 Hz, 1H), 4.48
(br. s, 2H); 13C NMR (151 MHz, DMSO-d6): δ 166.0, 151.2, 149.5, 146.9, 144.3, 129.9,
129.7, 128.8(2C), 127.7, 125.4, 122.7, 120.8, 120.3(2C), 103.9. ESI-MS: positive
mode m/z 279.1 [M+H]+, 301.1 [M+Na]+; negative mode m/z 277.1 [M-H]-.
4.1.4. General procedure for the synthesis of compounds 5a-5p
A mixture of the appropriate substituted aldehydes (0.2 mol) and 4-(quinolin-4-
ylamino) benzohydrazide 4 (55.0 mg, 0.2 mmol) in absolute ethanol (5 mL) containing
two drops of trifluoroacetic acid was stirred and refluxed for 8h. Then, the reaction
mixture was cooled to room temperature, and the solvent was removed by reduced
pressure distillation. The residue was purified by column chromatography
(DCM/MeOH/Triethylamine, 200:10:1 (v/v/v)) to get (E)-N'-Substituted methylene-4-
(Quinoline-4-Amino) benzoylhydrazides 5a-5p.
4.1.4.1. (E)-N'-((1H-imidazol-2-yl)methylene)-4-(quinolin-4-ylamino)benzohydrazide
(5a)
According to the general procedure, compound 5a was obtained by using
acetaldehyde. Grey solid; yield 50%; HPLC purity: 96.98%; mp: 279-281℃. 1H NMR
(600 MHz, DMSO-d6): δ 11.80 (br. s, 1H), 9.84 (br. s, 1H), 8.57 (d, J=5.7 Hz, 1H),
7.97-8.00 (m, 2H), 7.91-7.93 (m, 1H), 7.77-7.85 (m, 2H), 7.58-7.67 (m, 2H), 7.52 (d,
13
J=8.4 Hz, 2H), 7.14 (d, J=5.5 Hz, 1H), 1.96 (d, J=5.5 Hz, 3H). C NMR (151 MHz,
DMSO-d6): δ 162.9, 149.1, 147.0, 144.1, 131.1, 131.0, 129.6, 128.8, 128.1, 127.5,
125.9, 123.5, 121.6, 121.2, 121.2, 120.3, 103.4, 18.9. ESI-MS: positive mode m/z 305.1

[M+H]+, 327.1 [M+Na]+, 609.3 [2M+H]+, 631.1 [2M+Na]+; negative mode m/z 303.1
[M-H]-.
4.1.4.2. (E)-N'-butylidene-4-(quinolin-4-ylamino)benzohydrazide (5b)
According to the general procedure, compound 5b was obtained by using
butyraldehyde. Yellowish solid; yield 92%; HPLC purity: 95.53%; mp 147-149 ℃. 1H
NMR (600 MHz, DMSO-d6): δ 11.57 (br. s., 1H), 8.69 (d, J = 8.4Hz, 1H), 8.60-8.64
(m, 1H), 8.02-8.06 (m, 4H), 7.76-7.84 (m, 2H), 7.59-7.63 (m, 2H), 7.03 (d, J = 6.8 Hz,
13
1H), 2.21-2.30 (m, 2H), 1.49-1.59 (m, 2H), 0.94 (t, J = 1.0 Hz, 3H). C NMR (151
MHz, DMSO-d6): δ 162.4, 152.9, 144.8, 141.3, 133.9, 131.7, 129.7, 129.1, 127.5, 124.5,
124.4, 123.7, 122.2, 118.3, 101.3, 34.4, 19.9, 14.1. ESI-MS: positive mode m/z 333.2
[M+H]+, 355.1 [M+Na]+, 687.2 [2M+Na]+.
4.1.4.3.
(E)-4-(quinolin-4-ylamino)-N'-(2-(trifluoromethyl)benzylidene)
benzohydrazide (5c)
According to the general procedure, compound 5c was obtained by using 2-
(trifluoromethyl)benzaldehyde. Yellowish solid; yield 77%; HPLC purity: 98.37%; mp:
214-216 ℃. 1H NMR (600 MHz, DMSO-d6): δ 12.26 (br. s, 1H), 8.89 (br. s, 1H), 8.63
(d, J = 6.1 Hz, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 7.2 Hz, 1H), 8.09 (d, J = 7.9
Hz, 2H), 8.01 (d, J = 8.3 Hz, 1H), 7.92 (t, J = 7.6 Hz, 1H), 7.83 (d, J = 7.2 Hz, 1H),
7.76-7.82 (m, 1H), 7.71-7.76 (m, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H),
7.16 (d, J = 6.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 163.0, 147.5, 143.3, 143.0,
133.3, 133.1, 132.7, 132.3, 130.5, 130.1, 129.9, 129.2, 127.3, 127.1, 126.7, 126.4, 126.4,
125.6, 125.3, 123.8, 123.4, 122.5, 119.5, 102.7. ESI-MS: positive mode m/z 435.1
[M+H]+, 457.1[M+Na]+, 891.2 [2M+Na]+.
4.1.4.4.
(E)-2-((2-(4-(quinolin-4-ylamino)benzoyl)hydrazineylidene)methyl)benzoic
acid (5d)
According to the general procedure, compound 5d was obtained by using 2-
formylbenzoic acid. Yellow solid; yield 75%; HPLC purity: 97.94%; mp: 227-229℃.
1H NMR (600 MHz, DMSO-d6): δ 12.12 (br. s, 1H), 10.17 (br. s, 1H), 9.24 (br. s, 1H),
8.60-8.65 (m, 1H), 8.57 (d, J=8.4 Hz, 1H), 8.03-8.21 (m, 3H), 8.00 (d, J=8.3 Hz, 1H),

7.89-7.96 (m, 2H), 7.70-7.75 (m, 1H), 7.62-7.69 (m, 1H), 7.51-7.61 (m, 3H), 7.14 (d,
J=6.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 168.6, 163.1, 150.9, 147.4, 147.0,
144.1, 143.0, 135.1, 132.4(2C), 131.3, 130.8, 130.1, 129.9(2C), 129.6, 127.1, 126.7,
125.3, 123.3, 122.5(2C), 119.4, 102.7. ESI-MS: positive mode m/z 411.1 [M+H]+,
433.1 [M+Na]+; negative mode m/z 409.0 [M-H]-.
4.1.4.5.
(E)-3-((2-(4-(quinolin-4-ylamino)benzoyl)hydrazineylidene)methyl)benzoic
acid (5e)
According to the general procedure, compound 5e was obtained by using 3-
formylbenzoic acid. Yellow solid; yield 74%; HPLC purity: 96.63%; mp: 236-238℃.
1H NMR (600 MHz, DMSO-d6): δ 12.08 (s, 1H), 10.56 (br. s, 1H), 8.60-8.68 (m, 2H),
8.56 (s, 1H), 8.35 (br. s, 1H), 8.06-8.15 (m, 2H), 7.95-8.04 (m, 4H), 7.75-7.84 (m, 1H),
7.59-7.67 (m, 3H), 7.10 (d, J=6.4 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 167.4,
162.9, 152.8, 147.3, 145.7, 142.1, 141.6, 135.3, 133.4, 132.0, 131.9, 131.1, 130.7,
129.9(2C), 129.8, 127.8, 127.2, 123.8(2C), 123.6, 123.3, 118.8, 101.8. ESI-MS:
positive mode m/z 411.1 [M+H]+, 433.1[M+Na]+; negative mode m/z 409.0 [M-H]-.
4.1.4.6.
(E)-4-((2-(4-(quinolin-4-ylamino)benzoyl)hydrazineylidene)methyl)phenyl
acetate (5f)
According to the general procedure, compound 5f was obtained by using 4-
formylphenyl acetate. Yellow solid; yield 66%; HPLC purity: 96.53%; mp: 229-
231℃.1H NMR (600 MHz, DMSO-d6): δ 11.90 (br. s, 1H), 9.66 (br. s, 1H), 8.60 (d,
J=5.5 Hz, 1H), 8.46-8.52 (m, 2H), 8.02 (d, J=8.4Hz, 2H), 7.97 (d, J=8.4 Hz, 1H), 7.77-
7.86 (m, 3H), 7.63-7.66 (m, 1H), 7.53 (d, J=8.6 Hz, 2H), 7.20-7.28 (m, 2H), 7.19 (d,
13
J=5.5 Hz, 1H), 2.30 (s, 3H). C NMR (151 MHz, DMSO-d6): δ 169.6, 163.0, 158.6,
152.1, 149.5, 148.5, 147.1, 147.0, 144.3, 144.3, 132.5, 131.0, 129.7, 128.7, 128.2, 127.8,
126.0, 123.1, 122.9, 122.9, 121.1, 120.3, 116.2, 103.6, 21.3. ESI-MS: positive
mode m/z 425.1 [M+H]+, 447.0 [M+Na]+, 849.1 [2M+H]+, 871.1 [2M+Na]+; negative
mode m/z 423.0 [M-H]-.
4.1.4.7. (E)-N'-(2,6-dimethoxybenzylidene)-4-(quinolin-4-ylamino)benzohydrazide (5g)
According to the general procedure, compound 5g was obtained by using 2,6-

dimethoxybenzaldehyde. Yellow solid; yield 92%; HPLC purity: 97.16%; mp: 220-
223 ℃. 1H NMR (600 MHz, DMSO-d6): δ 11.81 (s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.67
(s, 1H), 8.60-8.65 (m, 1H), 8.13 (d, J = 8.3 Hz, 2H), 8.03-8.09 (m, 2H), 7.85 (ddd, J =
2.7, 5.4, 8.3 Hz, 1H), 7.63-7.68 (m, 2H), 7.33-7.40 (m, 1H), 7.03 (d, J = 6.8 Hz, 1H),
6.74 (d, J = 8.4 Hz, 2H), 3.83 (s, 6H). 13C NMR (151 MHz, DMSO-d6): δ 159.2, 158.8,
158.6, 154.8, 144.2, 144.0, 140.8, 139.1, 134.5, 132.4, 131.8, 129.9, 127.7, 125.0, 123.9,
121.2, 118.0, 111.5, 104.9, 101.0, 56.5. ESI-MS: positive mode m/z 427.1 [M+H]+,
449.3 [M+Na]+ ,.
4.1.4.8. (E)-N'-(3,4-dimethoxybenzylidene)-4-(quinolin-4-ylamino)benzohydrazide (5h)
According to the general procedure, compound 5h was obtained by using 3,4-
dimethoxybenzaldehyde. Yellow solid; yield 84%; HPLC purity: 99.36%; mp:162-
164 ℃．1H NMR (600 MHz, DMSO-d6): δ 11.80 (s, 1H), 8.60 (d, J = 6.1 Hz, 1H), 8.58
(d, J = 8.4 Hz, 1H), 8.42 (s, 1H), 8.05 (d, J = 8.4Hz, 2H), 8.00 (d, J = 9.0 Hz, 1H), 7.93
(t, J = 7.6 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 7.22
(d, J = 7.5 Hz, 1H), 7.11 (d, J = 6.1 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 3.84 (s, 3H), 3.82
(s, 3H)13C NMR (151 MHz, DMSO-d6): δ 162.7, 161.6, 158.3, 151.2, 149.6, 148.4,
147.1, 142.7, 136.7, 132.5, 130.2, 129.7, 127.5, 126.8, 123.4, 122.9, 122.4, 119.3, 112.0,
108.7, 102.4, 56.1, 55.9 ESI-MS: positive mode m/z 427.1 [M+H]+, 875.1 [2M+Na]+.
4.1.4.9. (E)-N'-(3,4-dihydroxybenzylidene)-4-(quinolin-4-ylamino)benzohydrazide (5i)
According to the general procedure, compound 5i was obtained by using 3,4-
dihydroxybenzaldehyde. Yellow solid; yield 63%; HPLC purity: 94.89%; mp: 196-
198℃. 1H NMR (600 MHz, DMSO-d6): δ 11.74 (s, 1H), 10.91 (br. s, 1H), 9.46 (br. s,
1H), 9.31 (br. s, 1H), 8.73 (d, J=8.4 Hz, 1H), 8.63 (d, J=6.8 Hz, 1H), 8.31 (s, 1H), 8.11
(d, J=8.4 Hz, 2H), 8.03-8.07 (m, 2H), 7.86 (ddd, J=1.6, 6.6, 8.4 Hz, 1H), 7.65 (d, J=8.4
Hz, 2H), 7.26 (d, J=1.7 Hz, 1H), 7.01-7.04 (m, 1H), 6.96 (dd, J=1.7, 8.1 Hz, 1H), 6.81
(d, J=7.9 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 162.4, 158.7, 154.8, 149.1, 148.6,
146.2, 143.9, 140.8, 139.0, 134.5, 132.4, 129.8(2C), 127.8, 126.2, 125.0(2C), 123.9,
121.1(2C), 118.0, 116.1, 113.2, 101.0. ESI-MS: positive mode m/z 399.1 [M+H]+,
421.1[M+Na]+; negative mode m/z 355.0 [M-H]-.

4.1.4.10. (E)-N'-(2,5-dihydroxybenzylidene)-4-(quinolin-4-ylamino)benzohydrazide (5j)
According to the general procedure, compound 5j was obtained by using 2,5-
dihydroxybenzaldehyde. Yellow solid; yield 82%; HPLC purity: 98.80%; mp: 245-
247 ℃. 1H NMR (600 MHz, DMSO-d6): δ 12.12 (s, 1H), 10.46 (s, 1H), 9.07 (br. s., 1H),
8.65 (d, J = 8.4 Hz, 1H), 8.61-8.64 (m, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.03-8.11 (m, 2H),
7.82-7.88 (m, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 6.8 Hz, 1H), 7.00 (d, J = 2.6
13
Hz, 1H), 6.75-6.77 (m, 2H). C NMR (151 MHz, DMSO-d6): δ 162.4, 158.9, 154.5,
150.7, 150.4, 148.2, 144.2, 141.3, 139.4, 134.3, 131.4, 129.9, 127.7, 124.8, 123.8, 121.5,
119.5, 118.1, 117.5, 114.2, 101.2. ESI-MS: positive mode m/z 399.0 [M+H]+, 421.1
[M+Na]+ , 819.2 [2M+Na]+.
4.1.4.11.
(E)-N'-(2-hydroxy-5-nitrobenzylidene)-4-(quinolin-4-ylamino)
benzohydrazide (5k)
According to the general procedure, compound 5k was obtained by using 2-hydroxy-
5-nitrobenzaldehyde. Yellow solid; yield 71%; HPLC purity: 99.40%; mp: 225-227℃.
1H NMR (600 MHz, DMSO-d6): δ 12.38 (br. s, 1H), 10.92 (br. s, 1H), 8.77-8.84 (m,
1H), 8.73 (d, J=8.4 Hz, 1H), 8.53-8.68 (m, 2H), 8.12-8.26 (m, 3H), 8.04-8.09 (m, 2H),
7.86 (ddd, J=1.3, 6.8, 8.4 Hz, 1H), 7.69 (d, J=8.4 Hz, 2H), 7.11-7.18 (m, 1H), 7.06 (d,
J=6.6 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ 163.1, 162.8, 154.7, 144.7, 144.0,
141.3, 140.4, 139.1, 134.5, 131.3, 130.0(2C), 127.8, 127.1, 125.0(2C), 123.9(2C),
121.2, 120.6, 118.1, 117.6, 101.1 ESI-MS: positive mode m/z 428.0 [M+H]+, 450.0
[M+Na]+; negative mode m/z 426.0 [M-H]-.
4.1.4.12.
(E)-N'-(3,5-dibromo-4-hydroxybenzylidene)-4-(quinolin-4-ylamino)
benzohydrazide (5l)
According to the general procedure, compound 5l was obtained by using 3,5-
dibromo-4-hydroxybenzaldehyde. Yellow solid; yield 70%; HPLC purity: 99.40%; mp:
267-269℃. 1H NMR (600 MHz, DMSO-d6): δ 12.08 (s, 1H), 10.91 (br. s, 1H), 8.72 (d,
J=8.4 Hz, 1H), 8.63 (d, J=6.8 Hz, 1H), 8.35 (s, 1H), 8.08-8.17 (m, 2H), 8.01-8.08 (m,
2H), 7.94 (s, 2H), 7.86 (ddd, J=1.5, 6.7, 8.3 Hz, 1H), 7.65 (s, 2H), 7.02-7.06 (m, 1H).
13C NMR (151 MHz, DMSO-d6): δ 162.8, 154.8, 152.7, 145.4, 144.0, 141.0, 139.0,

134.5, 131.9, 131.1(2C), 129.9(2C), 129.3, 127.8, 125.0(2C), 123.9, 121.1, 118.0,
112.7(2C), 101.0. ESI-MS: positive mode m/z 540.8 [M+H]+; negative mode m/z 538.8
[M-H]-.
4.1.4.13.
(E)-N'-((1H-imidazol-2-yl)methylene)-4-(quinolin-4-ylamino)
benzohydrazide (5m)
According to the general procedure, compound 5m was obtained by using 1H-
imidazole-2-carbaldehyde. Grey solid; yield 50%; HPLC purity: 95.32%; mp: 279-
281℃.1H NMR (600 MHz, DMSO-d6): δ 14.64 (br. s, 1H), 13.20 (br. s, 1H), 10.92 (br.
s, 1H), 8.73 (d, J=8.4 Hz, 1H), 8.63 (d, J=6.8 Hz, 1H), 8.11-8.19 (m, 2H), 8.04-8.09
(m, 2H), 7.87 (ddd, J=1.6, 6.6, 8.4 Hz, 1H), 7.74 (d, J=8.1 Hz, 2H), 7.56-7.62 (m, 1H),
7.46-7.53 (m, 1H), 7.42 (br. s, 1H), 7.11 (d, J=6.8 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6): δ 162.9, 154.3, 144.3, 142.6, 139.6, 134.3, 131.5, 130.0(2C), 129.4,
127.7(2C), 125.2, 124.8(2C), 123.8(2C), 121.6, 118.2, 101.2. ESI-MS: positive
mode m/z 357.0[M+H]+, 379.0 [M+Na]+, 713.0 [2M+H]+, 735.1 [2M+Na]+; negative
mode m/z 355.0[M-H]-.
4.1.4.14. (E)-N'-((1H-pyrazol-5-yl)methylene)-4-(quinolin-4-ylamino)benzohydrazide
(5n)
According to the general procedure, compound 5n was obtained by using 1H-
pyrazole-5-carbaldehyde. Grey solid; yield 52%; HPLC purity: 92.85%; mp：203-
205℃．1H NMR (600 MHz, DMSO-d6): δ 13.96 (br. s, 1H), 13.52 (br. s, 1H), 10.95
(br. s, 1H), 8.73 (d, J=8.4 Hz, 1H), 8.63 (d, J=6.8 Hz, 1H), 8.12 (d, J=7.4 Hz, 2H), 8.07
(br. s, 2H), 8.04 (br. s, 1H), 7.86 (ddd, J=2.6, 5.6, 8.2 Hz, 1H), 7.67-7.76 (m, 3H), 7.09
(d, J=6.8 Hz, 1H), 6.76 (br. s, 1H)．13C NMR (151 MHz, DMSO-d6): δ 162.7, 154.8,
143.9, 140.9, 139.0, 134.5, 132.1, 130.7, 130.1, 129.8(2C), 127.8(2C), 125.4,
125.1(2C), 123.9, 121.1, 118.1, 101.0. ESI-MS: positive mode m/z 357.1 [M+H]+,
379.0 [M+Na]+, 713.1 [2M+H]+, 735.1 [2M+Na]+; negative mode m/z 355.0 [M-H]-.
4.1.4.15.
(E)-N'-((4-methylthiazol-5-yl)methylene)-4-(quinolin-4-ylamino)
benzohydrazide (5o)

According to the general procedure, compound 5o was obtained by using 4-
methylthiazole-5-carbaldehyde. Yellow solid; yield 68%; HPLC purity: 98.12%; mp:
250-252℃.1H NMR (600 MHz, DMSO-d6): δ 12.07 (s, 1H), 10.86-11.00 (m, 1H), 9.06-
9.13 (m, 1H), 8.79-8.83 (m, 1H), 8.71-8.75 (m, 1H), 8.61-8.66 (m, 1H), 8.10-8.14 (m,
2H), 8.04-8.08 (m, 2H), 7.84-7.88 (m, 1H), 7.66-7.69 (m, 2H), 7.04-7.06 (m, 1H), 2.52-
2.54 (m, 3H). 13C NMR (151 MHz, DMSO-d6): δ 162.5, 158.8, 155.5, 154.8, 143.9,
141.5, 141.1, 139.1, 134.5, 131.8, 129.8(2C), 128.1, 127.8, 125.0(2C), 123.9, 121.1,
118.0, 101.1, 15.8. ESI-MS: positive mode m/z 388.1 [M+H]+, 410.0 [M+Na]+, 797.0
[2M+Na]+.
4.1.4.16.
(E)-N'-((1H-benzo[d]imidazol-2-yl)methylene)-4-(quinolin-4-ylamino)
benzohydrazide (5p)
According to the general procedure, compound 5p was obtained by using 1H-
benzo[d]imidazole-2-carbaldehyde. Yellow solid; yield 72%; HPLC purity: 94.47%;
mp: 330-332℃. 1H NMR (600 MHz, DMSO-d6): δ 15.08 (br. s, 1H), 13.54 (br. s, 1H),
10.92 (br. s, 1H), 8.75 (d, J=8.6 Hz, 1H), 8.66 (d, J=6.8 Hz, 1H), 8.19-8.30 (m, 2H),
8.06-8.09 (m, 2H), 7.90-7.95 (m, 1H), 7.83-7.89 (m, 2H), 7.83 (br. s, 2H), 7.71 (d, J=7.9
Hz, 1H), 7.41-7.46 (m, 1H), 7.35-7.41 (m, 1H), 7.19 (d, J=5.9 Hz, 1H). 13C NMR (151
MHz, DMSO-d6): δ 163.1, 154.5, 148.4, 144.2, 141.5, 139.8, 139.5, 139.4, 134.4, 131.3,
130.1(2C), 127.7, 125.3, 124.9(3C), 123.9(2C), 123.4, 121.5, 121.4, 118.2, 101.2. ESI-
MS: positive mode m/z 407.1 [M+H]+, 429.0 [M+Na]+, 835.1 [2M+Na]+; negative
mode m/z 405.0 [M-H]-.
4.2. Biological activity
4.2.1. Cell lines and reagents.
The human hepatocellular carcinoma cell line HepG2 was purchased from the
American Type Culture Collection (Manassas, VA, USA). The other two human
hepatocellular carcinoma cell lines (SMMC-7721 and Huh7) and two human normal
cell lines (LO2 and MRC-5) were purchased from the American type culture collection
(ATCC, Shanghai China). Cells were maintained in DMEM at 37˚C with 5% CO2 and

10% fetal bovine serum (FBS) (from Gibco, Waltham, MA, USA). The primary
antibodies of α-Tubulin (T6199, Sigma-Aldrich), c-Myc (Abcam, ab39688),
PARP(Cell Signaling Technology, #9542), Bcl-2 (60178-1-Ig, Protein tech), Bax
(60267-1-Ig, Protein tech) and tubulin (10094-1-AP, Protein Tech ). Goat and anti-
rabbit secondary antibodies were obtained from Thermo Fisher Scientific, Inc.
4.2.2. RT-qPCR.
HepG2 cells were treated with 5h and 5j at different concentrations as indicated.
24hours after treatment, total RNA from HepG2 was isolated using TRIzol® reagent
(Life technologies ambion, Inc.). qPCR was conducted using the Hieff™ qPCR
SYBR® Green Master Mix (YEASEN, Inc.) and the StepOnePlus Real-Time PCR
system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The conditions of PCR
were listed as follows: 95˚C for 5 min, then following 40 cycles of 95˚C for 10 sec,
56˚C for 30 sec, 72˚C for 20 sec, 95˚C for 15 sec. Expression levels were normalized
to that of GAPDH; the relative gene expression was determined using the 2-ΔΔCt method.
Primers were synthesized by Sangon Biotech (Shanghai, China) and were presented in
Table 3.
Insert Table 2 here.
Table 2. Reverse transcription-quantitative polymerase chain reaction primer sequences.
4.2.3. Western blotting
HepG2 cells were lysed with whole-cell lysis buffer containing PMSF to extract total
proteins after indicated drug treatment. Equal amounts of proteins were separated via
10% polyacrylamide gel electrophoresis, and proteins were transferred onto
polvinylidene difluoride (PVDF) membranes. Blocking was performed in Tris-buffered
saline and 0.05% Tween-20 containing 5% nonfat milk for 1 h at 37˚C. The PVDF
membranes were washed with TBST three times, then incubated with rabbit polyclonal

primary antibodies at 4˚C overnight. Subsequently, the membrane was incubated with
secondary antibodies (1:10,000) for 2 h at 37˚C. Lastly, an enhanced
chemiluminescence kit (Advansta, lnc.) was used to analyze the membranes according
to the instruction of the manufacturer. Protein bands were scanned with Molecular
Imager® ChemiDoc™ XRS+ Imaging System (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). The optical densities of a selection of bands were detected using ImageJ
(National Institutes of Health, Bethesda, MD, USA).
4.2.4. Cell proliferation.
HepG2 cells were first plated onto a 96 well plate at a density of 5,000 cells/100 μL
per well and incubated overnight in an incubator at 37˚C. Following quinolone
compounds (2.5, 5, 10, 20, 40 and 80 μM) or DMSO treatment for 24 hours, 20 μL
MTT reagents were added to each well, and cells were incubated in the dark for 4 h at
37˚C. Untreated cells served as a control. A spectrophotometer was used to measure the
absorbance of the generated optical density (OD) value at 490 nm. The half-maximal
inhibitory concentration (IC50) values were determined using GraphPad Prism 6
(GraphPad Software, Inc., La Jolla, CA, USA).
4.2.5. Colony formation.
HepG2 cells were seeded in 6-well plates with 1,000 cells per well, followed
treatment with 5h and 5j at different concentrations as indicated for 7 days. Then, the
cells were washed with 1×PBS, stained with a 0.5% crystal violet solution for 15
minutes at room temperature, and then washed with PBS, and photographed under a
microscope.
4.2.6. Wound healing.
HepG2 cells were seeded in 6-well plates and incubated in complete medium
(DMEM medium with 10% FBS) overnight. A 20 μL sterile pipette tip was scraped
across the wells and cell debris was removed via washing with 1×PBS. The supernatant

was subsequently replaced with the serum-free medium. Cells were treated with 5h and
5j as indicated. Finally, the scratch wounds were analyzed at various time points (0, 24
and 48 h) under the microscope.
4.2.7. Flow cytometry analysis of the cell cycle.
HepG2 cells were cultured in 6-well dishes. Following attachment, cells were treated
with 5h and 5j. Cell suspensions were harvested using trypsin at 37˚C and placed in
precooled 70% ethanol for fixation at -20˚C overnight. Then, a ribozyme (50 μg/mL)
and propidium iodide (50 μg/mL, PI; BD Biosciences, San Jose, CA, USA) were added
to the cells, which were incubated in the dark at room temperature for 30 min. Samples
were subsequently evaluated with a flow cytometer (ATTUNE NXT, Life tech, lnc.)
and data were analyzed by ModFit LT 2.0 software (Verity Software House, Inc.,
Topsham, ME, USA).
4.2.8. Flow cytometry analysis of apoptosis.
HepG2 cells were plated in 6-well dishes. Following attachment, cells were treated
with 5h and 5j. Cell suspensions were harvested via trypsin at 37˚C without EDTA,
washed with 1×PBS, and resuspended in 250 mL binding buffer. Cell suspensions were
stained with 5 μL PI and 5 μL Annexin V-fluorescein isothiocyanate solution (Yeasen,
lnc.) and incubated at room temperature for 15 min in the dark. Samples were
subsequently evaluated with a flow cytometer (ATTUNE NXT, Life tech, lnc.). All
assays were performed in triplicate.
4.2.9. Immunofluorescence.
After adding slides to the 24-well plate, HepG2 cells were plated in dishes, and the
culture was continued after 5h and 5j treatment. The culture solution was aspirated and
washed twice with 1×PBS, 500 μL of 4% paraformaldehyde was added to each well,
fixed at room temperature for 10 minutes, 500 μL of permeabilizing solution was added
to each well for 30 minutes, and 300 μL of 5 mg/mL BSA was added at room

temperature four 1 hour. Add 25μL of 5 mg/mL BSA 1:100 with a primary antibody at
room temperature for 3 hours or overnight, add 25 μL of 5 mg/mL BSA 1:100-500 with
secondary antibody, avoid light at room temperature for 1 hour, add 4-5 μL slide
mounting medium, seal the corners with nail polish, and stay away from light overnight.
Finally, find the field of view and take a picture under a laser confocal microscope.
4.3. Statistical analysis.
GraphPad Prism software (version 6) or SPSS 20.0 (IBM Corp., Armonk, NY, USA)
were used for statistical analysis. Data are presented as the mean ± standard deviation.
All experiments were performed in triplicate. A Student's t-test, χ2 or one-way analysis
of variance were used to evaluate whether statistically significant differences were
present. A Bonferroni post-hoc test for multiple comparisons was also applied. P<0.05
was considered to indicate a statistically significant difference.
Declaration of Competing Interest
Authors have no conflict of interest to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
81773600 and 81672955), the National Key R&D Program of China
(2018YFA0107303), the Natural Science Foundation of Fujian Province of China (No.
2018J01132), and the Fundamental Research Funds for the Central Universities (No.
20720180051).

Appendix A. Supplementary material
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Scheme 1. Preparation of target compounds 5a-5p

Figure 1. Modulating effect of 5h and 5j on the key proteins of cancer. (A). The expression
level of c-Myc in HepG2 cells treated with 5h or 5j for 24 h, determined via Western blot. (B).
mRNA level of c-Myc in HepG2 cells treated with 5h or 5j for 24 h determined by RT-PCR.
(C). The expression level of Bcl-2, Bax, and c-PARP proteins in HepG2 cells treated with 5h
or 5j, determined via Western blot. (D). mRNA levels of Bcl-2 and Bax in HepG2 cells treated
with 5h or 5j for 24 h determined by RT-PCR. (E). Immunofluorometric images of Bax, DAPI
staining and merge in untreated or treated HepG2 cells with 5h and 5j for 24 h. (∗) P < 0.05,
(∗∗) P < 0.01, (∗∗∗) P < 0.001 compared with control group.

Figure 2. Cell colony formation and cell cycle phase distribution of HepG2 cells with or
without treatment of compounds 5h and 5j. (A) Compounds 5h or 5j suppressed cell colony
formation of HepG2 cells. HepG2 cells were incubated with varying concentrations of
compounds 5h or 5j for 7 days and dyed with 0.1% crystal violet. (B) Compounds 5h or 5j
induced cell cycle arrest at the G2/M phase in HepG2 cells. HepG2 cells were plated in six-
well plates for 24 h and treated with or without compound 5h and 5j and at different
concentrations for 24 h of incubation. Then, the cells were harvested to determine the PI-stained
DNA content via flow cytometry.