Design, synthesis and antitumor evaluation of new 1,8-naphthalimide derivatives targeting nuclear DNA

Article abstract of DOI:10.1016/j.ejmech.2020.112951

Four series of new 3-nitro naphthalimides derivatives, 4(4a?4f), 5(5a?5i), 6(6a?6e) and 7 (7a?7j), were designed and synthesized as antitumor agents. Methyl thiazolyl tetrazolium (MTT) screening assay results revealed that some compounds displayed effecti

Full text of DOI:10.1016/j.ejmech.2020.112951

Journal Pre-proof
Design, synthesis and antitumor evaluation of new 1,8-naphthalimide derivatives
targeting nuclear DNA
Gui-Bin Liang, Jian-Hua Wei, Hong Jiang, Ri-Zhen Huang, Jing-Ting Qin, Hui-Ling
Wang, Heng-Shan Wang, Ye Zhang
PII:
S0223-5234(20)30923-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112951
EJMECH 112951
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2020
Revised Date: 15 October 2020
Accepted Date: 15 October 2020
Please cite this article as: G.-B. Liang, J.-H. Wei, H. Jiang, R.-Z. Huang, J.-T. Qin, H.-L. Wang,
H.-S. Wang, Y. Zhang, Design, synthesis and antitumor evaluation of new 1,8-naphthalimide
derivatives targeting nuclear DNA, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112951.
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Graphical abstract
1 / 1

Design, synthesis and antitumor evaluation of new 1,8-naphthalimide
derivatives targeting nuclear DNA
*
Gui-Bin Lianga,b,†, Jian-Hua Weia,†, , Hong Jianga,†, Ri-Zhen Huanga,b, Jing-Ting Qina, Hui-Ling
*
Wanga, Heng-Shan Wangb, Ye Zhanga,c,d,
aSchool of Pharmacy, Guilin Medical University, Guilin 541004, China
bState Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School
of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
cDepartment of Chemistry & Pharmaceutical Science, Guilin Normal College, Guilin 541001,
China
dCollege of Chemistry and Food Science, Yulin Normal University, Yulin 537000, China
†These authors contributed equally to this work.
*Corresponding author. School of Pharmacy, Guilin Medical University, Guilin 541004, China
Tel: (086) 773−2120998; fax: (086) 773−21209958
E-mail addresses: zhangye81@126.com (Y. Zhang), 352142863@qq.com (J.-H Wei)
1 / 25

Abstract
Four series of new 3-nitro naphthalimides derivatives, 4(4a‒4f), 5(5a‒5i), 6(6a‒6e) and 7(7a‒
7j), were designed and synthesized as antitumor agents. Methyl thiazolyl tetrazolium (MTT)
screening assay results revealed that some compounds displayed effective in vitro antiproliferative
activity on SMMC-7721, T24, SKOV-3, A549 and MGC-803 cancer cell lines in comparison with
5-fluorouracil (5-FU), mitonafide and amonafide. Nude mouse xenotransplantation model assay
results indicated that compounds 6b and 7b exhibited good in vivo antiproliferative activity in
MGC-803 xenografts in comparison with amonafide and cisplatin, suggesting that compounds 6b
and 7b could be good candidates for antitumor agents. Gel electrophoresis assay indicated that
DNA and Topo I were the potential targets of compounds 6b and 7b, and comet assay confirmed
that compounds 6b and 7b could induce DNA damage, while the further study showed that the 6b-
and 7b-induced DNA damage was accompanied by the upregulation of p-ATM, P-Chk2, Cdc25A
and p-H2AX. Cell cycle arrest studies demonstrated that compounds 6b and 7b arrested the cell
cycle at the S phase, accompanied by the upregulation of the expression levels of the antioncogene
p21 and the down-regulation of the expression levels of cyclin E. Apoptosis assays indicated that
compounds 6b and 7b caused the apoptosis of tumor cells along with the upregulation of the
expression of Bax, caspase-3, caspase-9 and PARP and the downregulation of Bcl-2. These
mechanistic studies suggested that compounds 6b and 7b exerted their antitumor activity by
targeting to DNA, thereby inducing DNA damage and Topo I inhibition, and consequently causing
S stage arrest and the induction of apoptosis.
Keywords: naphthalimide derivatives; antitumor activity; DNA damage; Topo I inhibition; cell
cycle arrest and apoptosis
2 / 25

1. Introduction
Carcinoma is a disease of global importance and is caused by abnormal and uncontrolled cell
division and growth in an undesired manner. DNA plays an essential role in the uncontrolled cell
division and growth and is thus considered as a viable target for chemotherapy[1]. Many
chemotherapy drugs exert their anticancer activities by targeting DNA, thereby causing DNA
damage and DNA topoisomerase (Topo I and/or II) inhibition and consequently activating cell
cycle checkpoint and apoptosis responses[1-4]. Mitonafide is a typical DNA-targeting
naphthalimide chemotherapy agent and has entered clinical trials[5]. However, serious adverse
toxic side effects have limited further clinical trials[5,6]. In order to improve this problem, many
structural modifications of the naphthalimide skeleton, including the modification at the
N-position and positions 3 and 4 in the naphthalimide moiety, have been described in the
literature[5,7-11]. And the introduced nitro moiety at 3 position has been proved to be
a typical substituent preferably for the antitumor activity[5-11].
Characterized by a planar π-deficient chromophore system, naphthalimides are a promising
class of DNA-targeted anticancer compounds that exhibit potent antitumor activities and
effectively inhibit tumor growth[6,12]. Mitonafide (Fig. 1) is a leading member of this family that
effectively targets DNA and has entered clinical trials, though its severe toxicity has seriously
hampered further application. In addition, mitonafide has been proven to be metabolized into
amonafide (Fig. 1) in physiological environments[13], while amonafide could be finally acetylated
and converted to a toxic amino-acetyl metabolite by N-acetyl-transferase 2[6,12]. To improve its
antitumor activity and reduce its adverse toxicity, much attention has been paid to the structural
modification of mitonafide. It is recognized that modifications at the N-position in the cyclic imide
ring and at the 4-position in the mitonafide moiety have considerable influence on the antitumor
effect, toxicity and intercalation with DNA, according to the study of structure-activity
relationships of numerous 1,8-naphthalimide derivatives, while in some cases even a small
modification may lead to a vital effect on their antitumor activity[5,7,9,10,11,14].
Piperazine and its substitution derivatives are considered as privileged scaffolds in drug
discovery and widely introduced in many antitumor drugs, such as palbociclib, nintedanib esylate,
olmutinib and entrectinib. So, it is believed that piperazine derivatives may exhibit potent
anticancer activities; our previous work has demonstrated that the introduction of piperazine
scaffolds to some pharmacy core could improve the antitumor activity and the toxicity of its
analogs[15,16]. Therefore, the modification of antitumor pharmacy cores with piperazine
structures has attracted much attention among medicinal chemists.
Encouraged by the antitumor and DNA-targeting activity of mitonafide and the virtues of
piperazine structures, it is expected that the combination of piperazine scaffolds and mitonafide
may improve antitumor efficiency and toxicity. Therefore, as the continuation of our previous
work[17], different substituent groups fused were introduced in the functional piperazine moiety
3 / 25

at the 4-position of the mitonafide moiety in the present work, while some other substituents at the
N-position of the imide ring were also designed and used for comparison (Fig. 1). We expected
that the introduction of piperazine scaffolds at 4-position of mitonafide could trigger steric
hindrance effect and slow down its metabolism into amonafide, thereby improving the antitumor
efficiency and toxicity. With this expectation in mind, the target compounds, their antiproliferative
activity and the antitumor mechanisms related to DNA-targeting were investigated.
[Fig. 1]
2. Results and discussion
2.1. Chemistry
The synthetic route leading to the target 1,8-naphthalimide derivatives 4−7 is outlined in
Scheme 1 and Scheme 2. First, as shown in Scheme 1, the benzoylation and sulfonylation of
N-Boc piperazine (NBP) with benzoyl chloride and p-toluenesulfonyl chloride at room
temperature yielded N-boc-N-benzoyl piperazine (NBNBZP) and N-boc-N-p-toluenesulfonyl
piperazine (NBNTP), respectively, in accordance with previous studies[18,19], while the
treatment of NBP with carbon disulfide, potassium carbonate and benzyl bromide in the presence
of dichloromethane for 2 h offered N-boc-N-benzyl dithioformyl piperazine (NBNBDP) in good
yield, in an ice-water bath. These three intermediates NBNBZP, NBNTP and NBNBDP were
then hydrolyzed with trifluoroacetica acid (TFA) to offer N-benzoyl piperazine (NBZP),
N-p-toluenesulfonyl piperazine (NTP) and N-benzyl dithioformyl piperazine (NBDP) at good
yield, respectively[18,19]. Secondly, as shown in Scheme 2, the treatment of
4-bromo-1,8-naphthalic anhydride (1) with NaNO3 and H2SO4 at 0°C offered
3-nitro-4-bromo-naphthalic anhydride (2) in good yield, in accordance with our previous work[17].
Then the four N-substituted piperazines NBP, NBDP, NBZP and NTP were treated with
compound
2
in the presence of 2-methoxyethanol at 110°C to provide
4-piperazine-3-nitro-1,8-naphthalic anhydride 3a‒3d in moderate yield, respectively. Finally, the
desired 1,8-naphthalimides 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j were obtained in moderate yield, by the
condensation of primary amines with 3a‒3d at 80°C in the presence of ethanol, respectively.
[Scheme 1]
[Scheme 2]
The chemical structures of these desired compounds 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j were then
confirmed by 1H NMR, 13C NMR and high resolution mass spectrometry (HR-MS) (see Part 2 of
Supplementary Data). In the 1H NMR spectra for compounds 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j, the
chemical shift (δ) around 8.85−6.48 was attributed to the aromatic hydrogens (H-Ar) fused in
naphthalimide and aromatic rings, while δ in the range of 4.09−3.21 was mainly ascribed to the
4 / 25

hydrogens of the piperazine ring. In 13C NMR spectra, δ in the range of 160−165 was mainly
attributed to the carbons in carbonyl groups, and δ around 46 and 51 was ascribed to the carbons
in the piperazine moiety, while δ in the range of 185−189 was attributed to the carbon in the
thiocarbonyl group. In addition, HR-MS results for 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j were also
consistent with their chemical structures in Scheme 2. Hence, the chemical structures of
compounds 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j were identified.
2.2. Antitumor activity
2.2.1. In vitro antiproliferative activity
With these target compounds at hand, we investigated their antiproliferative activity by
MTT[15-17] against human liver cancer SMMC-7721 cells, human bladder cancer T24 cells,
human ovarian cancer SKOV3 cells, human hepatoma A549 cells and human gastric cancer
MGC-803 cells, using 5-FU, mitonafide and amonafide as the positive controls.
As shown in Table 1, most of the target compounds exhibited evident in vitro antiproliferative
activity on these five cell lines. Table 1 shows that in the inhibition assay with SMMC-7721 cells,
most compounds (except for compounds 4e, 5a, 5e, 6e, 7a, 7c, 7d and 7j) exhibited better
antiproliferative activity than the commercial anticancer drug 5-FU (IC50 = 15.32 ± 0.67 μM), and
compounds 4a, 4b, 5b, 6a, 6b and 7b displayed higher antiproliferative activity than amonafide
(IC50 = 6.93 ± 0.26 μM). Importantly, compounds 4b, 6b and 7b even showed more potent
antiproliferative activity than mitonafide (IC50 = 3.22 ± 0.26 μM), with IC50 values of 1.05 ± 0.09
μM, 1.57 ± 0.19 μM and 2.71 ± 0.13 μM, respectively, indicating that compounds 4b, 6b and 7b
exhibited potent antiproliferative activity against this cell line. In the antiproliferation assay with
T24 cells, all the target compounds 4‒7 exhibited better antiproliferative activity than 5-FU (IC50
= 40.14 ± 2.14 μM), and compounds 4a, 4b, 5b, 6a, 6b and 7b demonstrated better
antiproliferative activity than amonafide (IC50 = 5.01 ± 0.47 μM), while 4b, 6b and 7b showed
more potent antiproliferation than mitonafide (IC50 = 1.11 ± 0.31 μM), with IC50 values of 0.83 ±
0.15 μM, 0.42 ± 0.05 μM and 0.33 ± 0.07 μM, respectively, indicating their favorable
antiproliferation on this cell line. In the SKOV3 assay, many compounds (except for 5a, 5c, 5d,
5g‒5i, 7d and 7g‒7j) exhibited better antiproliferative activity than 5-FU (IC50 = 26.34 ± 0.57
μM), and compounds 4b, 5b, 6a, 6b and 7b displayed stronger antiproliferative activity than
amonafide (IC50 = 6.31 ± 0.46 μM), while compounds 4b and 7b exhibited better antiproliferation
than mitonafide (IC50 = 1.38 ± 0.51 μM), with IC50 values of 0.65 ± 0.13 μM and 0.32 ± 0.03 μM,
respectively, demonstrating the strong antiproliferative effects of compounds 4b and 7b against
SKOV3 cells. In the A549 test, except for compounds 4c‒4e, 5a, 5h, 5i, 6d, 7d and 7f‒7j, other
compounds showed stronger antiproliferation activity than 5-FU (IC50 = 34.47 ± 1.90 μM), while
compounds 4a, 4b, 5b, 6a, 6b and 7b displayed stronger antiproliferation activity than amonafide
5 / 25

(IC50 = 7.94 ± 0.61 μM), with IC50 values of 1.08 ± 0.15 μM, 7.52 ± 0.36 μM, 1.98 ± 0.11 μM,
6.66 ± 0.23 μM, 0.41 ± 0.12 μM and 0.27 ± 0.15 μM, respectively. Unfortunately, no compound
showed better antiproliferation activity than mitonafide in this cell line. In the MGC-803 cell assay,
all these compounds exhibited better antiproliferative activity than 5-FU (IC50 = 34.85 ± 1.75 μM),
and compounds 4a, 4b, 4e, 5b, 6a, 6b and 7b exhibited higher antiproliferative activity than
amonafide (IC50 = 9.06 ± 0.45 μM), while compounds 4a, 4b, 5b, 6b and 7b even showed more
potent antiproliferative activity than mitonafide (IC50 = 6.80 ± 0.45 μM), with IC50 values of 1.33
± 0.23 μM, 6.44 ± 0.12 μM, 6.08 ± 0.21 μM, 0.868 ± 0.16 μM and 1.42 ± 0.11 μM, respectively,
indicating that compounds 4a, 4b, 5b, 6b and 7b exhibited potent antiproliferative activity against
this cell line. On the basis of the above MTT results, compounds 6b and 7b displayed the best
antiproliferative activity on these five cell lines, and were thus chosen as representative
compounds for further investigation of the in vivo antiproliferative activity.
[Table 1]
In addition, based on the MTT results, some interesting structure-activity relationships may be
concluded: (1) both of the substituent groups at the N-position in the cycloamidite moiety and the
4-position had important but irregular influences on the antiproliferative activity; (2) importantly,
the fatty chain amine N,N-dimethylaminoethyl at the N-position in the cycloamidite ring showed
especially positive effects on the antiproliferative activity; (3) the comparison of 4 with 5, 6 and 7,
demonstrated that substitution in the piperazine moiety exhibited important but irregular effect on
their antiproliferative activity, while the aliphatic chain hydrocarbon may have positive effects on
the antiproliferative activity. This result indicated that in some cases even a small structural
transformation in the 1,8-naphthalimide skeleton may have a major effect on their antitumor
activity, well consistent with previous literature[6,17].
2.2.2 In vivo antiproliferative activity
To further evaluate the antitumor activity of 6b and 7b, in vivo antiproliferation assays were
performed. The MGC-803 xenograft model was chosen in this study, based on the in vitro
antiproliferation experimental result. Specific pathogen-free BALB/c nude mice were divided
randomly into five groups, i.e., the vehicle control group, the low-dose and high-dose
administration of compounds 6b and 7b groups and two positive control groups. Saline, low dose
(5 mg/kg) and high dose (15 mg/kg) compounds 6b and 7b, cisplatin (2 mg/kg) and amonafide (5
mg/kg) were administrated by intraperitoneal injection. The relative tumor increment rate (T/C)
and tumor inhibitory rate (IR) were then calculated to assess their in vivo antitumor activity, where
T/C values of less than 60.0% and IR values of more than 40.0% indicated potent and effective
antitumor activity[20].
6 / 25

[Fig. 2]
As shown in Fig. 2A, administration of 6b and 7b, at both low (5 mg/kg) and high doses (15
mg/kg), had strong antiproliferative effects on MGC-803 tumor growth; and the T/C values were
59.8% (6b at low dose), 47.1% (6b at high dose), 52.0% (7b at low dose) and 38.4% (7b at high
dose). In comparison, cisplatin (2 mg/kg) and amonafide (5 mg/kg) had T/C values of 42.3% and
34.4%, respectively. The tumors of the mice were collected (Fig. 2B) and weighed on day 21 to
assess the antiproliferative rates based on the increase in tumor weight. As shown in Fig. 2D,
compounds 6b and 7b displayed obvious antitumor activity in the MGC-803 model with
inhibitory rates of 40.1% (6b at low dose), 52.8% (6b at high dose), 41.9% (7b at low dose) and
61.7% (7b at high dose), while those of cisplatin (2 mg/kg) and amonafide (5 mg/kg) were 57.5%
(P < 0.01) and 65.5% (P < 0.01), respectively. Compounds 6b and 7b at both low and high doses
displayed T/C values of less than 60.0% and IR values of more than 40.0% and strong in vivo
antiproliferative effects in comparison with cisplatin and amonafide, demonstrating that
compounds 6b and 7b displayed effective antitumor activity in MGC-803 xenografts. Interestingly,
as shown in Fig. 2C, no obvious adverse effects and changes in body weight were found in the
mice treated with compounds 6b and 7b, indicating that 6b and 7b had no significant toxicity to
mice within the 21-day period of treatment and thus could be good candidates for antitumor drug
design. The high administration doses of 6b and 7b may also indirectly indicate their lower
toxicity in comparison with cisplatin and amonafide.
2.3 Antitumor mechanism investigation
2.3.1. DNA intercalation
Because a planar chromophore portion is considered as the common feature of
DNA-intercalating anticancer drugs, it is reasonable that DNA is and has been proven as the
potential target for naphthalimide derivatives[5]. To obtain insight into the mechanism of
naphthalimide derivatives targeting DNA, a gel electrophoresis assay was carried out, using
mitonafide and amonafide as positive controls. Gel electrophoresis assay results (Fig. 3) indicated
that 6b and 7b tightly bound to the supercoiled circular plasmid pBR322 and hindered its
migration in the gel at concentrations from 10 μM to 100 μM, while they displayed no
photocleavage activity or photoreactivity in this assay, confirming that compound 6b and 7b
exhibited important intercalation effects on pBR322.
[Fig. 3]
To better understand the intercalation effects of compounds 6b and 7b with DNA, we
performed molecular docking studies using SYBYL-X 2.0 software. As shown in Fig. S1-1,
compounds 6b and 7b stacked with the regions of the DNA minor groove by surface binding
7 / 25

interactions and adjunction with the target DNA double helix, implying their good binding affinity
with DNA. It was obvious that compounds 6b and 7b intercalated with nucleotide and forming
hydrogen bonds with NH group presented at DNA. Therefore, the results could confer the
anticancer activity of compounds 6b and 7b to their abilities to bind to DNA minor groove.
2.3.2. DNA damage
To investigate whether compounds 6b and 7b could lead to DNA damage, a comet assay, which
was proven to be a sensitive, simple but useful tool for DNA damage studies[21], was performed.
As shown in Fig. 4, long DNA tails were found after treatment with compounds 6b and 7b,
indicating compounds 6b and 7b could induce DNA damage[21].
[Fig. 4]
It is believed that PIKK kinases (ATM, ATR and DNA-PK), mediator or signal transducer
proteins (Chk1 and Chk2 signaling kinases) and effector proteins (phosphatases Cdc25A/B/C and
cyclin-dependent kinases [CDKs]) are main regulators of the DNA damage response and play an
important role in the course of DNA damage[2], while the increased levels of human p-H2AX are
considered as important evidence of DNA damage[22,23]. Hence, the expression levels of
p-ATM, P-Chk2, Cdc25A and p-H2AX in MGC-803 cells were examined by western blots. The
results (Fig. 5) show that compared to the control, the expression levels of p-ATM, P-Chk2,
Cdc25A and p-H2AX were significantly upregulated in MGC-803 cells treated with 6b and 7b,
directly verifying the presence of damaged DNA.
[Fig. 5]
2.3.3. Topo I inhibition
To study whether the inhibition of Topo 1 activity was related to the antiproliferative and
DNA-damaging effects of 6b and 7b, the DNA relaxation assay was carried out using amonafide,
mitonafide and the Topo I inhibitor camptothecin (CPT) as positive controls. The results (Fig. 6
and Fig. S1-2) indicate that, accompanying the DNA damage, compound 7b exhibited obvious
Topo I inhibition (0.1 U/L) at a concentration of 20 μM, while compound 6b exhibited evident
Topo I inhibition (0.1 U/L) at a concentration of 10 μM, implying that in addition to DNA, Topo I
was also a potential target of compounds 6b and 7b. To further determine the DNA relaxation
induced by compounds 6b and 7b, an unwinding assay was then carried out. The results (Fig. S1-3
and Fig. S1-4) show that compounds 6b and 7b exhibited obvious Topo I inhibition at low
concentrations (< 20 μM), while they led to the supercoiling of DNA at high concentrations (> 40
μM), confirming that DNA and Topo I are indeed potential targets of compounds 6b and 7b.
[Fig. 6]
8 / 25

2.3.4 Cell cycle arrest analysis
Because DNA-targeted drugs can usually induce cell cycle arrest[2], the effects of compounds
6b and 7b on cell cycle progression were investigated. As shown in Fig. 7, in MGC-803 cells,
compounds 6b and 7b mainly arrested the cell cycle at the S phase, causing an evident increase in
the S phase population (for 6b: 37.80% at 0.25 μM, 69.18% at 0.5 μM and 71.16% at 1 μM; for 7b:
40.44% at 0.25 μM, 43.96% at 0.5 μM and 75.18% at 1 μM) in comparison with the control group
(20.23%).
[Fig. 7]
The regulatory proteins CDK and cyclin E and the antioncogene p21 play an important role in
the regulation of the S phase checkpoint[23]. Therefore, the expression of CDK2, cyclin E and
p21 in MGC-803 cells was examined by western blots, using glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as a control. As shown in Fig. 8, treatment with compounds 6b and 7b
increased p21 expression, decreased cyclin E expression and only slightly affected CDK2
expression, indicating that compounds 6b and 7b induced S phase cell cycle arrest by inhibiting
the S phase-promoting CDK2–Cyclin E complex in MGC-803 cells[22].
[Fig. 8]
2.3.5 Apoptosis study
To further assess the antitumor mechanism of compounds 6b and 7b, apoptosis levels were
investigated. As shown in Fig. 9, in MGC-803 cells, 6b increased the percentage of apoptotic cells
to 21.7%, 28.7% and 52.5% (including the early and late apoptosis) at 0.25, 0.5 and 1 μM,
respectively, while 7b increased the percentage of apoptotic cells to 17.3%, 22.9% and 27% at the
same concentrations, indicating that compounds 6b and 7b could induce apoptosis in MGC-803
cells.
[Fig. 9]
Bcl-2/Bax family proteins, caspase proteases and poly(ADP-ribose) polymerase (PARP) play
important mediating roles in apoptosis[24-26]. To further study the mechanisms underlying 6b-
and 7b-induced apoptosis, the expression levels of Bax, Bcl-2, caspase-9, caspase-3 and PARP in
MGC-803 cells treated with compounds 6b and 7b were measured by western blot. The results
(Fig. 10) indicated that compounds 6b and 7b could upregulate the expression levels of Bax,
caspase-3, caspase-9 and PARP and downregulate Bcl-2 levels, implying that compounds 6b and
7b may exert proapoptotic effects through a mitochondrial-mediated pathway and a caspase
cascade.
9 / 25

[Fig. 10]
3. Conclusion
In this study, we designed and synthesized four sets of naphthalimide derivatives and evaluated
their antitumor activity against five cancer cell lines, i.e., SMMC-7721, T24, SKOV-3, A549 and
MGC-803. We identified some compounds displaying high in vitro antitumor activity in
comparison with 5-FU, mitonafide and amonafide, while compounds 6b and 7b displayed good in
vivo antitumor activity in the MGC-803 xenograft in comparison with amonafide and cisplatin.
Our gel electrophoresis assay indicated that DNA and Topo I are potential targets of compounds
6b and 7b, and our comet assay confirmed that compounds 6b and 7b could induce DNA damage,
while the further study showed that the 6b- and 7b-induced DNA damage was accompanied by the
upregulation of p-ATM, P-Chk2, Cdc25A and p-H2AX. Cell cycle arrest studies demonstrated that
compounds 6b and 7b trigger cell cycle arrest at the S phase through inhibition of CDK2–Cyclin
E complex activity, while apoptosis assays indicated that compounds 6b and 7b could cause the
apoptosis of tumor cells along with the upregulation of the expression Bax, caspase-3, caspase-9
and PARP and downregulation of Bcl-2. Based on these observations, it could be concluded that
compounds 6b and 7b may mainly exert their antitumor effects by targeting to DNA, thereby
inducing DNA damage and inhibiting Topo I, and consequently causing to S phase arrest and
inducing apoptosis.
4. Experimental procedures
4.1 Materials and instruments
All chemicals and solvents were of reagent grade and purchased from Aladdin (Shanghai) and
used without further purification. The materials used for biological experiments with pBR322
DNA and cell lines were purchased from Aladdin, Topo I was purchased from TaKaRa
Biotechnology Co., Ltd. (Dalian), and antibodies were purchased from Cell Signaling Technology.
Cell cycle and apoptosis assays were performed by BD FACSAria III flow cytometry (Becton
Dickinson) and the results were analyzed by ImageJ software. The NMR spectra were measured
on a BRUKER AVANCE AV 400/600 instrument, while the mass spectra were examined on a
BRUKER ESQUIRE HCT spectrometer.
4.2. Chemistry: general synthesis procedure for compounds 4−7
NBP was purchased form Aladdin and the intermediate compounds ZBZP, NTP and NBDP
were synthesized as previously described[18,19], using NBP as the starting material. The mixture
of compound 2 (1.5 mmol), the prefabricated 1-tosylpiperazine (NBP, ZBZP, NTP and NBDP)
10 / 25

(1.8 mmol) and ethylene glycol methyl ether (100 mL) was reacted at 110°C for 8 h. After the
reaction, the reaction solution was cooled naturally overnight and then filtered to provide desired
compounds 3 (3a‒3d). Compounds 3 (3a–3d) were then purified by silica column
chromatography, using dichloromethane–methanol (v:v = 10:1) solution as the eluent. Finally,
compounds 3 (3a‒3d) (1.0 mmol) were treated with the primarily amines (1.0 mmol) in hot
ethanol at 80°C for 6 h. The reaction solutions were cooled naturally overnight and then filtered to
offer the target compounds 4a‒4f, 5a‒5i, 6a‒6e and 7a‒7j. Compounds 4‒7 were purified by
silica column chromatography, using dichloromethane–methanol (v:v = 8:1) solution as the eluent.
Tert-butyl
4-(2-(3-(methylamino)propyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
1
benzo[de]isoquinolin-6-yl)piperazine-1-carboxylate (4a): Yellow solid, yield 42.7%. H NMR
(400 MHz, DMSO-d6) δ 8.67 – 8.62 (m, 1H, H-Ar), 8.56 (dd, J = 7.2, 0.7 Hz, 1H, H-Ar), 8.52 (s,
1H, H-Ar), 7.99 – 7.92 (m, 1H, H-Ar), 4.04 (t, J = 7.2 Hz, 2H, -CH2), 3.66 (s, 4H, 2-CH2), 3.18 (d,
J = 4.6 Hz, 5H, 2-CH2), 2.60 – 2.57 (m, 2H, -CH2), 2.29 (s, 3H, -CH3), 1.82 – 1.71 (m, 2H, -CH2),
1.45 (s, 9H, 3-CH3).13C NMR (100 MHz, DMSO-d6) δ 162.95 (C=O), 161.84 (C=O), 153.98,
147.01, 142.23, 132.80, 131.45, 129.58, 129.06, 128.48, 126.30, 122.98, 117.75, 79.30 (C-O),
51.06 (C-N), 48.60 (CH2), 45.41(C-N), 38.11(CH3), 35.43(CH2), 28.03(CH3), 27.02(CH2). HRMS
(m/z) (ESI): C25H31N5O6 [M+1]+ calcd for: 498.2269, found: 498.2335.
Tert-butyl
4-(2-(2-(dimethylamino)ethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
1
benzo[de]isoquinolin-6-yl)piperazine-1-carboxylate (4b): Yellow solid, yield 47.4%. H NMR
(400 MHz, DMSO-d6) δ 8.67 (d, J = 8.5 Hz, 1H, H-Ar), 8.59 (d, J = 7.2 Hz, 1H, H-Ar), 8.56 (s,
1H, H-Ar), 8.02 – 7.94 (m, 1H, H-Ar), 4.13 (t, J = 6.8 Hz, 2H, -CH2), 3.66 (s, 4H, 2-CH2), 3.19 (d,
J = 4.0 Hz, 4H, 2-CH2), 2.29 (s, 2H, -CH2), 2.20 (s, 6H, 2-CH3), 1.45 (s, 9H, 3-CH3).13C NMR
(100 MHz, DMSO-d6) δ 162.92 (C=O), 161.79 (C=O), 153.99, 147.09, 142.29, 132.91, 131.55,
129.60, 129.14, 128.51, 126.44, 122.93, 117.68, 79.30 (C-O), 56.35 (CH3), 51.06 (C-N), 45.36
(CH2), 37.71 (CH2), 28.04 (CH3). HRMS (m/z) (ESI): C25H31N5O6[M+1]+ calcd for: 498.2347,
found: 498.2330.
Tert-butyl 4-(2-(2-hydroxyethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
1
yl)piperazine-1-carboxylate (4c): Yellow solid, yield 48.1%. H NMR (400 MHz, DMSO-d6) δ
8.60 (dd, J = 8.4, 0.8 Hz, 1H, H-Ar), 8.51 (dd, J = 7.4, 0.8 Hz, 1H, H-Ar), 8.47 (s, 1H, H-Ar), 7.92
(dd, J = 8.4, 7.4 Hz, 1H, H-Ar), 4.79 (t, J = 5.9 Hz, 1H, -OH), 4.08 (t, J = 6.5 Hz, 2H, -CH2), 3.66
(s, 4H, 2-CH2), 3.61 – 3.56 (m, 2H, -CH2), 3.22 – 3.08 (m, 4H, 2-CH2), 1.46 (s, 9H, 3-CH3).13C
NMR (100 MHz, DMSO-d6) δ 162.90 (C=O), 161.79 (C=O), 153.98, 146.90, 142.13, 132.71,
131.30, 129.47, 128.94, 128.42, 126.18, 122.95, 117.71, 79.30 (C-O), 57.64 (CH2-OH), 51.05
(C-N), 41.91(CH2), 28.03 (CH3).HRMS (m/z) (ESI):C23H26N4O7[M+Na]+ calcd for: 493.1694,
found: 493.1679.
11 / 25

Tert-butyl 4-(2-(4-chlorobenzyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
1
yl)piperazine-1-carboxylate (4d): Yellow solid, yield 46.4%. H NMR (400 MHz, DMSO-d6) δ
8.66 (d, J = 8.4 Hz, 1H, H-Ar), 8.58 (d, J = 7.2 Hz, 1H, H-Ar), 8.55 (s, 1H, H-Ar), 7.96 (dd, J =
8.4, 7.2 Hz, 1H, H-Ar), 7.35 (q, J = 8.7 Hz, 4H, H-Ar), 5.18 (s, 2H, -CH2), 3.66 (s, 4H, 2-CH2),
3.23 – 3.11 (m, 4H, 2-CH2), 1.45 (s, 9H, 3-CH3).13C NMR (100 MHz, DMSO-d6) δ 162.97 (C=O),
161.86 (C=O), 153.97, 147.24, 142.21, 136.01, 133.07, 131.73, 129.71, 129.51, 129.08, 128.49,
128.27, 126.67, 122.81, 117.47, 79.29 (C-O), 51.10 (C-N), 42.41 (CH2), 28.02 (CH3). HRMS
(m/z) (ESI): C28H27ClN4O6[M+Na]+ calcd for: 573.1511, found: 573.1490.
Tert-butyl
4-(2-(3,4-dihydroxyphenethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
1
benzo[de]isoquinolin-6-yl)piperazine-1-carboxylate (4e): Yellow solid, yield 44.4%. H NMR
(400 MHz, DMSO-d6) δ 8.79 (s, 1H, -OH), 8.70 – 8.61 (m, 2H, H-Ar), 8.57 (d, J = 0.7 Hz, 1H,
H-Ar), 8.53 (s, 1H,-OH), 7.95 (dd, J = 8.4, 7.4 Hz, 1H, H-Ar), 6.63 (dd, J = 11.0, 5.0 Hz, 2H,
H-Ar), 6.47 (dd, J = 8.0, 2.0 Hz, 1H, H-Ar), 4.12 (dd, J = 8.9, 6.8 Hz, 2H, -CH2), 3.66 (s, 4H,
2-CH2), 3.25 – 3.08 (m, 4H, 2-CH2), 2.77 – 2.63 (m, 2H, -CH2), 1.46 (s, 9H, 3-CH3).13C NMR
(100 MHz, DMSO-d6) δ 162.73 (C=O), 161.61 (C=O), 153.98, 147.06, 145.16, 143.73, 142.20,
132.82, 131.49, 129.54, 129.27, 129.07, 128.48, 126.33, 122.91, 119.21, 117.64, 115.93, 115.58,
79.30 (C-O), 51.07 (C-N), 41.48 (CH2), 32.80 (CH2), 28.04 (CH3). HRMS (m/z) (ESI):
C29H30N4O8[M+Na]+ calcd for: 585.1956, found: 585.1934.
Tert-butyl
4-(2-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-yl)piperazine-1-carboxylate (4f): Yellow solid, yield 48.3%. 1H NMR
(400 MHz, DMSO-d6) δ 8.68 (d, J = 8.5 Hz, 1H, H-Ar), 8.60 – 8.51 (m, 2H, H-Ar), 7.97 (t, J =
7.9 Hz, 1H, H-Ar), 6.83 (s, 1H, H-Ar), 6.78 (t, J = 6.8 Hz, 1H, H-Ar), 6.67 (d, J = 8.0 Hz, 1H,
H-Ar), 5.96 (s, 2H, CH2-2O), 4.23 – 4.13 (m, 2H, -CH2), 3.66 (s, 4H, 2-CH2), 3.19 (s, 4H, 2-CH2),
2.86 – 2.77 (m, 2H, -CH2), 1.46 (s, 9H, 3-CH3).13C NMR (100 MHz, DMSO-d6) δ162.81(C=O),
161.69 (C=O), 153.98, 147.25, 147.10, 145.68, 142.27, 132.87, 132.36, 129.62, 129.13, 128.52,
126.38, 122.96, 121.90, 121.50, 117.70, 108.98, 108.18, 100.70 (C-2O), 79.30 (C-O), 51.07
(C-N), 41.25 (CH2), 33.09 (CH2), 28.03 (CH3). HRMS (m/z) (ESI): C30H30N4O8[M+1]+ calcd for:
575.2136, found: 575.1059.
Benzyl
4-(2-(4-methoxybenzyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
1
yl)piperazine-1-carbodithioate (5a): Yellow solid, yield 42.1%. H NMR (600 MHz, CDCl3) δ
8.72 (t, J = 3.4 Hz, 2H, H-Ar), 8.54 (d, J = 8.4 Hz, 1H, H-Ar), 7.88 (t, J = 7.9 Hz, 1H, H-Ar), 7.49
(d, J = 8.6 Hz, 2H, H-Ar), 7.41 (d, J = 7.4 Hz, 2H, H-Ar), 7.34 (t, J = 7.4 Hz, 2H, H-Ar), 7.29 (d,
J = 7.3 Hz, 1H, H-Ar), 6.82 (d, J = 8.7 Hz, 2H, H-Ar), 5.29 (s, 2H, -CH2), 4.63 – 4.19 (m, 6H,
3-CH2), 3.76 (s, 3H, -OCH3), 3.40 (s, 4H, 2-CH2).13C NMR (150 MHz, CDCl3) δ 198.23 (C=S),
163.46 (C=O), 162.35 (C=O), 159.22, 146.30, 143.86, 135.55, 133.67, 130.83, 130.60, 130.36,
12 / 25

129.86, 129.55, 129.12, 128.81, 128.51, 127.85, 127.19, 124.00, 119.61, 113.93, 55.37 (OCH3),
50.90 (C-N), 43.32 (CH2), 42.51 (C-S). HRMS (m/z) (ESI): C32H28N4O5S2[M+1]+ calcd for:
613.1574, found: 613.1552.
Benzyl
4-(2-(2-(dimethylamino)ethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-yl)piperazine-1-carbodithioate (5b): Yellow solid, yield 49.3%. 1H NMR
(400 MHz, DMSO-d6) δ 8.73 (d, J = 8.4 Hz, 1H, H-Ar), 8.65 – 8.48 (m, 2H, H-Ar), 7.98 (t, J =
7.8 Hz, 1H, H-Ar), 7.43 (d, J = 7.0 Hz, 2H, H-Ar), 7.37 – 7.19 (m, 3H, H-Ar), 4.59 (s, 4H,
2-CH2), 4.30 (s, 2H, -CH2), 4.18 – 4.05 (m, 2H, -CH2), 3.32 (s, 4H, 2-CH2), 2.50 (s, 2H, -CH2),
2.20 (s, 6H, 2-CH3).13C NMR (100 MHz, DMSO-d6) δ 195.74 (C=S), 162.89 (C=O), 161.77
(C=O), 146.31, 142.52, 136.02, 132.98, 131.70, 129.59, 129.25, 129.15, 128.63, 128.49, 127.43,
126.33, 122.94, 118.15, 56.31 (CH3), 50.64 (C-N), 45.32 (CH2), 40.91 (C-S), 37.69 (CH2). HRMS
(m/z) (ESI): C28H29N5O4S2[M+1]+ calcd for: 564.1734, found: 564.1711.
Benzyl
4-(2-(2-hydroxyethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
1
yl)piperazine-1-carbodithioate (5c): Yellow solid, yield 52.4%. H NMR (600 MHz, CDCl3) δ
8.72 (d, J = 7.1 Hz, 1H, H-Ar), 8.69 (s, 1H, H-Ar), 8.56 (d, J = 8.4 Hz, 1H, H-Ar), 7.90 (t, J = 7.9
Hz, 1H, H-Ar), 7.40 (d, J = 7.3 Hz, 2H, H-Ar), 7.33 (t, J = 7.4 Hz, 2H, H-Ar), 7.29 (d, J = 7.3 Hz,
1H, H-Ar), 4.66 – 4.19 (m, 8H, 4-CH2), 3.95 (t, J = 5.3 Hz, 2H, -CH2), 3.41 (s, 4H, 2-CH2).13C
NMR (150 MHz, CDCl3) δ 198.23 (C=S), 164.22 (C=O), 163.08 (C=O), 146.52, 143.74, 135.52,
133.77, 130.84, 130.39, 129.82, 129.52, 128.78, 128.55, 127.82, 127.31, 123.68, 119.16, 61.42
(CH2-O), 50.94 (C-N), 42.95 (CH2), 42.48 (C-S). HRMS (m/z) (ESI): C26H24N4O5S2[M+1]+ calcd
for: 537.1261, found: 537.1245.
Benzyl
4-(2-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-yl)piperazine-1-carbodithioate (5d): Yellow solid, yield 48.7%. 1H NMR
(600 MHz, CDCl3) δ 8.76 – 8.69 (m, 2H, H-Ar), 8.59 (d, J = 8.4 Hz, 1H, H-Ar), 7.96 – 7.88 (m,
1H, H-Ar), 7.43 (d, J = 7.4 Hz, 2H, H-Ar), 7.36 (t, J = 7.5 Hz, 2H, H-Ar), 7.31 (d, J = 7.3 Hz, 1H,
H-Ar), 6.84 (s, 1H, H-Ar), 6.80 – 6.72 (m, 2H, H-Ar), 5.94 (s, 2H, CH2-2O), 4.80 – 4.07 (m, 8H,
4-CH2), 3.44 (s, 4H, 2-CH2), 2.98 – 2.87 (m, 2H, -CH2).13C NMR (150 MHz, CDCl3) δ 198.21
(C=S), 163.26 (C=O), 162.10 (C=O), 147.75, 146.28, 143.83, 135.53, 133.50, 132.20, 130.60,
130.32, 129.88, 129.52, 128.78, 128.53, 127.82, 127.01, 123.86, 121.96, 119.46, 109.48, 108.40,
100.98 (C-2O), 50.87 (C-N), 42.48 (CH2), 42.19 (C-S), 33.97 (CH2). HRMS (m/z) (ESI):
C33H28N4O6S2[M+1]+ calcd for: 641.1523, found: 641.1502.
Benzyl
4-(2-(3,4-dihydroxyphenethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-yl)piperazine-1-carbodithioate (5e): Yellow solid, yield 35.3%. 1H NMR
(600 MHz, DMSO-d6) δ 8.85 – 8.71 (m, 3H), 8.61 – 8.50 (m, 2H, H-Ar), 8.02 – 7.93 (m, 1H,
H-Ar), 7.43 (d, J = 7.3 Hz, 2H, H-Ar), 7.34 (t, J = 7.5 Hz, 2H, H-Ar), 7.28 (t, J = 7.3 Hz, 1H,
13 / 25

H-Ar), 6.67 – 6.57 (m, 2H, H-Ar), 6.47 (dd, J = 8.0, 1.9 Hz, 1H, H-Ar), 4.71 – 4.54 (m, 4H,
2-CH2), 4.28 (s, 2H, -CH2), 4.14 – 4.07 (m, 2H, -CH2), 3.32 (s, 4H, 2-CH2), 2.73 – 2.68 (m, 2H,
-CH2).13C NMR (150 MHz, DMSO-d6) δ 195.73 (C=S), 162.76 (C=O), 161.64 (C=O), 146.33,
145.21, 143.78, 142.45, 136.04, 132.96, 129.60, 129.31, 129.28, 129.11, 128.66, 128.54, 127.48,
126.29, 122.97, 119.24, 118.18, 115.97, 115.64, 50.70 (C-N), 45.55 (C-N), 41.56 (C-S), 40.96
(CH2), 32.84 (CH2). HRMS (m/z) (ESI): C32H28N4O6S2[M+1]+ calcd for: 629.1523, found:
629.1503.
Benzyl
4-(5-nitro-1,3-dioxo-2-(phenylamino)-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)piperazine-1-carbodithioate (5f):. Yellow solid, yield 38.1%.1H NMR (400 MHz, DMSO-d6) δ
8.82 (d, J = 8.3 Hz, 1H, H-Ar), 8.73 – 8.60 (m, 3H, H-Ar), 8.08 – 7.99 (m, 1H, H-Ar), 7.44 (d, J =
7.1 Hz, 2H, H-Ar), 7.37 – 7.25 (m, 3H, H-Ar), 7.14 (t, J = 7.9 Hz, 2H, H-Ar), 6.77 (t, J = 8.3 Hz,
3H, H-Ar), 4.72 – 4.23 (m, 6H, 3-CH2), 3.51 – 3.35 (m, 4H, 2-CH2).13C NMR (100 MHz,
DMSO-d6) δ 195.74 (C=S), 162.44 (C=O), 161.37(C=O), 147.08, 146.61, 142.58, 136.02, 133.43,
132.11, 129.73, 129.38, 129.25, 128.79, 128.69, 128.49, 127.42, 126.81, 123.33, 119.52, 118.54,
112.53, 50.69 (C-N), 45.64 (C-N), 40.92 (C-S). HRMS (m/z) (ESI): C30H25N5O4S2[M+1]+ calcd
for: 584.1421, found: 584.1400.
Benzyl 4-(2-benzyl-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazine-1-
1
carbodithioate (5g): Yellow solid, yield 43.5%. H NMR (600 MHz, CDCl3) δ 8.74 (d, J = 7.0
Hz, 2H, H-Ar), 8.57 (d, J = 8.4 Hz, 1H, H-Ar), 7.93 – 7.88 (m, 1H, H-Ar), 7.54 (d, J = 7.4 Hz, 2H,
H-Ar), 7.43 (d, J = 7.4 Hz, 2H, H-Ar), 7.36 (t, J = 7.5 Hz, 2H, H-Ar), 7.31 (q, J = 7.4 Hz, 3H,
H-Ar), 7.29 – 7.26 (m, 1H, H-Ar), 5.37 (s, 2H, -CH2), 4.75 – 4.21 (m, 6H, 3-CH2), 3.43 (s, 4H,
2-CH2).13C NMR (150 MHz, CDCl3) δ 198.20 (C=S), 163.42 (C=O), 162.32 (C=O), 146.34,
143.80, 136.82, 135.53, 133.71, 130.66, 130.35, 129.85, 129.52, 129.14, 128.78, 128.62, 128.51,
127.83, 127.82, 127.22, 123.90, 119.46, 50.89 (C-N), 43.86 (CH2), 42.48 (C-S). HRMS (m/z)
(ESI): C31H26N4O4S2 [M+1]+ calcd for: 583.1468, found: 583.1444.
Benzyl
4-(2-(furan-2-ylmethyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)piperazine-1-carbodithioate (5h): Yellow solid, yield 33.7%. 1H NMR (600 MHz, DMSO-d6) δ
8.75 (d, J = 8.5 Hz, 1H, H-Ar), 8.64 – 8.58 (m, 2H, H-Ar), 7.99 (t, J = 7.9 Hz, 1H, H-Ar), 7.55 –
7.51 (m, 1H, H-Ar), 7.43 (d, J = 7.4 Hz, 2H, H-Ar), 7.34 (t, J = 7.5 Hz, 2H, H-Ar), 7.28 (t, J = 7.3
Hz, 1H), 6.37 (dd, J = 8.0, 2.3 Hz, 2H), 5.22 (s, 2H, -CH2), 4.65 – 4.52 (m, 4H, 2-CH2), 4.29 (s,
2H, -CH2), 3.34 – 3.26 (m, 4H, 2-CH2).13C NMR (150 MHz, DMSO-d6) δ 195.72 (C=S), 162.60
(C=O), 161.49 (C=O), 149.98, 146.56, 142.47, 142.27, 136.02, 133.22, 131.99, 129.72, 129.30,
129.17, 128.67, 128.53, 127.47, 126.69, 122.80, 117.91, 110.61, 108.21, 50.73 (C-N), 40.94
(C-S), 36.34 (CH2). HRMS (m/z) (ESI): C29H24N4O5S2[M+1]+ calcd for: 573.1261, found:
573.1237.
14 / 25

Benzyl
4-(2-(4-chlorobenzyl)-5-nitro-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)piperazine-1-carbodithioate (5i): Yellow solid, yield 47.3%. 1H NMR (400 MHz, DMSO-d6) δ
8.76 (d, J = 8.8 Hz, 1H, H-Ar), 8.66 – 8.56 (m, 2H, H-Ar), 8.03 – 7.96 (m, 1H, H-Ar), 7.43 (d, J =
7.1 Hz, 2H, H-Ar), 7.39 – 7.32 (m, 6H, H-Ar), 7.29 (d, J = 7.2 Hz, 1H, H-Ar), 5.20 (s, 2H, -CH2),
4.59 – 4.30 (m, 6H, 3-CH2), 3.33 (s, 4H,2 -CH2).13C NMR (100 MHz, DMSO-d6) δ 195.74 (C=S),
163.02 (C=O), 161.92 (C=O), 146.49, 142.53, 136.02, 133.18, 131.94, 131.73, 129.80, 129.51,
129.25, 129.16, 128.65, 128.49, 128.29, 127.43, 126.59, 122.90, 118.09, 50.68 (C-N), 42.45
(CH2), 40.91 (C-S). HRMS (m/z) (ESI): C31H25ClN4O4S2[M+1]+ calcd for: 617.1079, found:
617.1059.
6-(4-Benzoylpiperazin-1-yl)-2-(3-(methylamino)propyl)-5-nitro-1H-benzo[de]isoquinoline-1,3
1
(2H)-dione (6a): Yellow solid, yield 46.0%. H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 8.5
Hz, 1H, H-Ar), 8.55 (s, 1H, H-Ar), 8.49 (s, 1H, H-Ar), 8.03 – 7.91 (m, 1H, H-Ar), 7.49 (s, 5H,
H-Ar), 4.09 – 3.66 (m, 6H, 3-CH2), 3.23 (d, J = 5.3 Hz, 5H), 3.03 – 2.87 (m, 2H, -CH2), 2.51 (s,
3H, -CH3), 2.04 – 1.91 (m, 2H, -CH2).13C NMR (100 MHz, DMSO-d6) δ 169.54 (C=O), 163.07
(C=O), 161.97 (C=O), 146.81, 142.18, 135.59, 132.85, 131.55, 129.76, 129.60, 128.93, 128.57,
127.08, 126.24, 122.94, 117.76, 51.36 (C-N), 46.07 (CH2), 37.20 (CH3), 32.31 (CH2), 24.30
(CH2). HRMS (m/z) (ESI): C27H27N5O5 [M+1]+ calcd for: 502.2085, found: 502.2064.
6-(4-Benzoylpiperazin-1-yl)-2-(2-(dimethylamino)ethyl)-5-nitro-1H-benzo[de]isoquinoline-1,
1
3(2H)-dione (6b): Yellow solid, yield 45.1%. H NMR (400 MHz, DMSO-d6) δ 8.63 (d, J = 8.1
Hz, 1H, H-Ar), 8.52 (d, J = 7.2 Hz, 1H, H-Ar), 8.49 (s, 1H, H-Ar), 7.93 (dd, J = 8.4, 7.5 Hz, 1H,
H-Ar), 7.49 (s, 5H, H-Ar), 4.08 (t, J = 6.8 Hz, 2H, -CH2), 3.99 – 3.58 (m, 4H, 2-CH2), 3.24 (s, 4H,
2-CH2), 2.50 – 2.45 (m, 2H, -CH2), 2.19 (s, 6H, 2-CH3).13C NMR (100 MHz, DMSO-d6) δ 169.50
(C=O), 162.75 (C=O), 161.63 (C=O), 146.78, 142.20, 135.58, 132.82, 131.45, 129.69, 129.45,
128.99, 128.51, 128.44, 127.03, 126.34, 122.79, 117.59, 56.29 (CH3), 51.25 (C-N), 45.33 (CH2),
37.68 (CH2). HRMS (m/z) (ESI): C27H27N5O5[M+1]+ calcd for: 502.2085, found: 502.2073.
6-(4-Benzoylpiperazin-1-yl)-2-(2-hydroxyethyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-di
1
one (6c): Yellow solid, yield 53.2%. H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.5 Hz, 1H,
H-Ar), 8.50 (d, J = 7.2 Hz, 1H, H-Ar), 8.46 (s, 1H, H-Ar), 7.92 (t, J = 7.9 Hz, 1H, H-Ar), 7.49 (s,
5H, H-Ar), 4.80 (s, 1H, -OH), 4.07 (t, J = 6.4 Hz, 2H, -CH2), 4.00 – 3.64 (m, 4H, 2-CH2), 3.61 –
3.55 (m, 2H, -CH2), 3.24 (s, 4H, 2-CH2).13C NMR (100 MHz, DMSO-d6) δ 169.53 (C=O), 162.87
(C=O), 161.76 (C=O), 146.67, 142.17, 135.60, 132.71, 131.31, 129.71, 129.46, 128.91, 128.53,
128.43, 127.05, 126.16, 122.94, 117.77, 57.65 (CH2), 51.29 (C-N), 41.92 (CH2). HRMS (m/z)
(ESI): C25H22N4O6[M+1]+ calcd for: 475.1612, found: 475.1611.
6-(4-Benzoylpiperazin-1-yl)-2-(4-chlorobenzyl)-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-di
1
one (6d): Yellow solid, yield 46.6%. H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 8.4 Hz, 1H,
15 / 25

H-Ar), 8.65 – 8.57 (m, 2H, H-Ar), 7.99 (dd, J = 8.3, 7.6 Hz, 1H, H-Ar), 7.49 (s, 5H, H-Ar), 7.36
(q, J = 8.7 Hz, 4H, H-Ar), 5.20 (s, 2H, -CH2), 4.02 – 3.60 (m, 4H, 2-CH2), 3.27 (s, 4H, 2-CH2).13C
NMR (100 MHz, DMSO-d6) δ 169.52 (C=O), 163.03 (C=O), 161.92 (C=O), 147.03, 142.34,
136.04, 135.61, 133.11, 131.80, 131.73, 129.80, 129.70, 129.50, 129.14, 128.53, 128.29, 127.04,
126.68, 122.91, 117.69, 51.33 (C-N), 42.44 (CH2). HRMS (m/z) (ESI): C30H23ClN4O5[M+1]+
calcd for:555.1430, found: 555.1408.
6-(4-Benzoylpiperazin-1-yl)-2-(3,4-dihydroxyphenethyl)-5-nitro-1H-benzo[de]isoquinoline-1,
1
3(2H)-dione (6e): Yellow solid, yield 42.1%. H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H,
-OH), 8.65 (d, J = 7.8 Hz, 2H, H-Ar), 8.57 – 8.48 (m, 2H), 8.00 – 7.90 (m, 1H, H-Ar), 7.49 (s, 5H,
H-Ar), 6.63 (dd, J = 9.9, 5.0 Hz, 2H, H-Ar), 6.46 (dd, J = 8.0, 1.9 Hz, 1H, H-Ar), 4.15 – 4.06 (m,
2H, -CH2), 3.94 – 3.70 (m, 4H, 2-CH2), 3.25 (s, 4H, 2-CH2), 2.75 – 2.63 (m, 2H, -CH2).13C NMR
(100 MHz, DMSO-d6) δ 169.52 (C=O), 162.67 (C=O), 161.55 (C=O), 146.82, 145.16, 143.73,
142.21, 135.61, 132.80, 131.47, 129.71, 129.51, 129.27, 129.02, 128.54, 128.48, 127.05, 126.29,
122.88, 119.21, 117.69, 115.94, 115.59, 51.30 (C-N), 41.48 (CH2), 32.79 (CH2). HRMS (m/z)
(ESI): C31H26N4O7[M+1]+ calcd for:567.1874, found: 567.1860.
2-(4-Methoxybenzyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dio
ne (7a): Yellow solid, yield 34.2%. 1H NMR (600 MHz, CDCl3) δ 8.67 (s, 2H, H-Ar), 8.34 (d, J =
8.5 Hz, 1H, H-Ar), 7.78 (t, J = 7.8 Hz, 1H, H-Ar), 7.74 (d, J = 8.1 Hz, 2H, H-Ar), 7.48 (d, J = 8.5
Hz, 2H, H-Ar), 7.42 (d, J = 8.0 Hz, 2H, H-Ar), 6.82 (d, J = 8.6 Hz, 2H, H-Ar), 5.27 (s, 2H, -CH2),
3.76 (s, 3H, -OCH3), 3.45 – 3.29 (m, 8H, 4-CH2), 2.50 (s, 3H, -CH3).13C NMR (150 MHz, CDCl3)
δ 163.41 (C=O), 162.31 (C=O), 159.18, 146.55, 144.31, 133.48, 133.05, 130.79, 130.56, 130.19,
130.11, 129.69, 129.10, 128.77, 128.26, 127.93, 127.09, 123.86, 119.41, 113.88, 55.34 (OCH3),
50.87 (C-N), 46.32 (C-N), 43.25 (CH2), 21.77 (CH3). HRMS (m/z) (ESI): C31H28N4O7S[M+1]+
calcd for: 601.1751, found: 601.1741.
2-(2-(Dimethylamino)ethyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2
H)-dione (7b): Yellow solid, yield 62.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.54 – 8.47 (m, 2H,
H-Ar), 8.44 (d, J = 8.5 Hz, 1H, H-Ar), 7.88 (dd, J = 8.4, 7.5 Hz, 1H, H-Ar), 7.72 (d, J = 8.2 Hz,
2H, H-Ar), 7.51 (d, J = 8.1 Hz, 2H, H-Ar), 4.08 (t, J = 6.7 Hz, 2H, -CH2), 3.27 (s, 8H, 4-CH2),
2.52 (d, J = 6.8 Hz, 2H, -CH2), 2.45 (s, 3H, -CH3), 2.21 (s, 6H, 2-CH3). 13C NMR (100 MHz,
DMSO-d6) δ 162.78(C=O), 161.67(C=O), 146.43, 143.82, 142.72, 132.94, 132.81, 131.33,
130.06, 129.36, 128.94, 128.57, 127.51, 126.14, 122.79, 118.12, 56.21 (CH2), 50.53 (C-N), 45.94
(C-N), 45.20 (CH2), 37.54 (CH3), 21.07 (CH3). HRMS (m/z) (ESI): C27H29N5O6S[M+1] + calcd
for: 552.1911, found: 552.1894.
2-(2-Hydroxyethyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
1
(7c): Yellow solid, yield 57.9%. H NMR (400 MHz, DMSO-d6) δ 8.56 – 8.48 (m, 2H, H-Ar),
16 / 25

8.45 (d, J = 8.5 Hz, 1H, H-Ar), 7.94 – 7.81 (m, 1H, H-Ar), 7.72 (d, J = 8.2 Hz, 2H, H-Ar), 7.52 (d,
J = 8.1 Hz, 2H, H-Ar), 4.78 (s, 1H, -OH), 4.08 (t, J = 6.4 Hz, 2H, -CH2), 3.58 (q, J = 6.3 Hz, 2H,
-CH2), 3.27 (s, 8H, 4-CH2), 2.45 (s, 3H, -CH3).13C NMR (100 MHz, DMSO-d6) δ 162.93 (C=O),
161.82 (C=O), 146.30, 143.82, 142.76, 132.94, 132.70, 131.21, 130.07, 129.43, 128.92, 128.57,
127.52, 125.97, 123.03, 118.42, 57.63 (CH2-O), 50.53 (C-N), 45.96 (C-N), 41.93 (CH2), 21.08
(CH3). HRMS (m/z) (ESI): C25H24N4O7S[M+1]+ calcd for: 525.1438, found: 525.1420.
2-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinol
ine-1,3(2H)-dione (7d): Yellow solid, yield 64.3%. 1H NMR (600 MHz, CDCl3) δ 8.69 (t, J = 3.4
Hz, 2H, H-Ar), 8.38 (d, J = 8.4 Hz, 1H, H-Ar), 7.83 (t, J = 7.9 Hz, 1H, H-Ar), 7.76 (d, J = 8.1 Hz,
2H, H-Ar), 7.43 (d, J = 8.0 Hz, 2H, H-Ar), 6.85 (s, 1H, H-Ar), 6.78 –6.74 (m, 2H, H-Ar), 5.94 (s,
2H, CH2-2O), 4.37 – 4.28 (m, 2H, -CH2), 3.46 – 3.25 (m, 8H, 4-CH2), 2.99 – 2.89 (m, 2H, -CH2),
2.52 (s, 3H, -CH3).13C NMR (150 MHz, CDCl3) δ 163.32 (C=O), 162.15 (C=O), 147.79, 146.56,
146.32, 144.33, 144.05, 133.39, 133.15, 132.23, 130.60, 130.26, 130.14, 129.82, 128.33, 127.98,
127.01, 123.85, 122.00, 119.44, 109.52, 108.43, 101.01 (C-2O), 50.92 (C-N), 46.35 (C-N), 42.22
(CH2), 34.00 (CH2), 21.81 (CH3). HRMS (m/z) (ESI): C32H28N4O8S[M+1]+ calcd for: 629.1701,
found: 629.1676.
2-(3,4-Dihydroxyphenethyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2
1
H)-dione (7e): Yellow solid, yield 54.7%. H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H, -OH),
8.66 (s, 1H, -OH), 8.49 (d, J = 5.1 Hz, 2H, H-Ar), 8.43 (d, J = 8.5 Hz, 1H, H-Ar), 7.87 (dd, J =
8.3, 7.6 Hz, 1H, H-Ar), 7.71 (d, J = 8.2 Hz, 2H, H-Ar), 7.51 (d, J = 8.1 Hz, 2H, H-Ar), 6.61 (d, J =
8.2 Hz, 2H, H-Ar), 6.44 (dd, J = 8.0, 1.9 Hz, 1H, H-Ar), 4.10 – 3.99 (m, 2H, -CH2), 3.27 (s, 8H,
4-CH2), 2.73 – 2.60 (m, 2H, -CH2), 2.45 (s, 3H, -CH3).13C NMR (100 MHz, DMSO-d6) δ 162.59
(C=O), 161.47 (C=O), 146.41, 145.14, 143.82, 143.72, 142.71, 132.93, 132.74, 131.30, 130.07,
129.33, 129.23, 128.94, 128.58, 127.51, 126.05, 122.80, 119.18, 118.16, 115.90, 115.56, 50.54
(C-N), 45.94 (C-N), 41.45 (CH2), 32.76 (CH2), 21.08 (CH3). HRMS (m/z) (ESI):
C31H28N4O8S[M+1]+ calcd for: 617.1701, found: 617.1675.
5-Nitro-2-(phenylamino)-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
1
(7f): Yellow solid, yield 45.1%. H NMR (600 MHz, CDCl3) δ 8.72 (d, J = 7.6 Hz, 1H, H-Ar),
8.41 (d, J = 8.3 Hz, 1H, H-Ar), 7.87 – 7.82 (m, 1H, H-Ar), 7.73 (d, J = 8.2 Hz, 2H, H-Ar), 7.41 (d,
J = 8.0 Hz, 2H, H-Ar), 7.21 (t, J = 7.9 Hz, 2H, H-Ar), 6.95 (t, J = 7.4 Hz, 1H, H-Ar), 6.90 (s, 1H,
H-Ar), 6.86 (d, J = 7.8 Hz, 2H, H-Ar), 3.41 –3.35 (m, 8H, 4-CH2), 2.49 (s, 3H, -CH3).13C NMR
(150 MHz, CDCl3) δ 162.53 (C=O), 161.41 (C=O), 147.23, 145.92, 144.38, 143.89, 134.24,
133.05, 131.32, 130.14, 130.02, 129.97, 129.37, 128.45, 127.95, 127.89, 123.54, 122.77, 118.87,
115.10, 51.02 (C-N), 46.28 (C-N), 21.79 (CH3). HRMS (m/z) (ESI): C29H25N5O6S[M+1]+ calcd
for: 572.1598, found: 572.1576.
17 / 25

2-Butyl-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7g):
Yellow solid, yield 44.9%. 1H NMR (600 MHz, CDCl3) δ 8.68 – 8.62 (m, 2H, H-Ar), 8.34 (d, J =
8.3 Hz, 1H, H-Ar), 7.80 (d, J = 7.7 Hz, 1H, H-Ar), 7.72 (d, J = 8.2 Hz, 2H, H-Ar), 7.40 (d, J = 8.0
Hz, 2H, H-Ar), 4.16 – 4.07 (m, 2H, -CH2), 3.41 – 3.24 (m, 8H, 4-CH2), 2.49 (s, 3H, -CH3), 1.71 –
1.61 (m, 2H, -CH2), 1.44 – 1.35 (m, 2H, -CH2), 0.95 (t, J = 7.4 Hz, 3H, -CH3).13C NMR (150
MHz, CDCl3) δ 163.42 (C=O), 162.26 (C=O), 146.42, 144.31, 144.00, 133.31, 133.06, 130.47,
130.20, 130.11, 129.74, 128.29, 127.93, 126.87, 123.90, 119.54, 50.86 (C-N), 46.34 (C-N), 40.55
(CH2), 30.21 (CH3), 21.78 (CH2), 20.41 (CH2), 13.92 (CH3). HRMS (m/z) (ESI):
C27H28N4O6S[M+1]+ calcd for: 537.1802, found: 537.1805.
2-Benzyl-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7h):
Yellow solid, yield 61.4%. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (t, J = 3.3 Hz, 2H, H-Ar), 8.44
(d, J = 8.4 Hz, 1H, H-Ar), 7.85 (dd, J = 8.3, 7.6 Hz, 1H, H-Ar), 7.71 (d, J = 8.2 Hz, 2H, H-Ar),
7.50 (d, J = 8.1 Hz, 2H, H-Ar), 7.32 – 7.20(m, 5H, H-Ar), 5.16 (s, 2H, -CH2), 3.28 (s, 8H, 4-CH2),
2.43 (s, 3H, -CH3).13C NMR (100 MHz, DMSO-d6) δ 162.82 (C=O), 161.73 (C=O), 146.57,
143.80, 142.67, 136.90, 132.97, 132.90, 131.47, 130.04, 129.45, 128.92, 128.55, 128.29, 127.61,
127.50, 127.09, 126.39, 122.70, 117.96, 50.57 (C-N), 45.93 (C-N), 42.95 (CH2), 21.05 (CH3).
HRMS (m/z) (ESI): C30H26N4O6S[M+1]+ calcd for: 571.1646, found: 571.1630.
2-(Furan-2-ylmethyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dio
ne (7i): Yellow solid, yield 39.2%. 1H NMR (600 MHz, CDCl3) δ 8.68 (d, J = 6.5 Hz, 2H, H-Ar),
8.34 (d, J = 8.4 Hz, 1H, H-Ar), 7.79 (t, J = 7.9 Hz, 1H, H-Ar), 7.72 (d, J = 8.1 Hz, 2H, H-Ar), 7.40
(d, J = 8.0 Hz, 2H, H-Ar), 7.30 (d, J = 0.9 Hz, 1H), 6.42 (d, J = 3.1 Hz, 1H), 6.28 (dd, J = 3.1, 1.8
Hz, 1H), 5.34 (s, 2H, -CH2), 3.41 – 3.28 (m, 8H, 4-CH2), 2.48 (s, 3H, -CH3).13C NMR (150 MHz,
CDCl3) δ 163.10 (C=O), 161.97 (C=O), 149.94, 146.68, 144.32, 143.93, 142.35, 133.65, 133.07,
130.73, 130.28, 130.12, 129.78, 128.30, 127.95, 127.25, 123.70, 119.23, 110.54, 109.58, 50.90
(C-N), 46.32 (C-N), 36.58 (CH2), 21.78 (CH3). HRMS (m/z) (ESI): C28H24N4O7S[M+1]+ calcd
for: 561.1438, found: 561.1430.
2-(4-Chlorobenzyl)-5-nitro-6-(4-tosylpiperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7j): Yield 55.3%, yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (t, J = 3.4 Hz, 2H, H-Ar),
8.47 (d, J = 8.3 Hz, 1H, H-Ar), 7.93 – 7.85 (m, 1H, H-Ar), 7.71 (d, J = 8.2 Hz, 2H, H-Ar), 7.51 (d,
J = 8.2 Hz, 2H, H-Ar), 7.32 (dd, J = 8.0, 5.7 Hz, 4H, H-Ar), 5.16 (s, 2H, -CH2), 3.28 (s, 8H,
4-CH2), 2.44 (s, 3H, -CH3).13C NMR (100 MHz, DMSO-d6) δ 162.89 (C=O), 161.80 (C=O),
146.61, 143.80, 142.72, 135.95, 133.01, 132.92, 131.72, 131.57, 130.05, 129.55, 129.50, 128.97,
128.59, 128.25, 127.50, 126.41, 122.76, 118.05, 50.58 (C-N), 45.93 (C-N), 42.40 (CH2), 21.06
(CH3). HRMS (m/z) (ESI): C30H25ClN4O6S[M+1]+ calcd for: 605.1256, found: 605.1235.
4.3. Biological assays
18 / 25

The biological experimental procedures including antiproliferative activity assay, gel
electrophoresis experiment, cell cycle analysis, cell apoptosis study, comet assay and western blot
assay were carried out according to our previous work[17, 27-30] and described in the
Supplementary Data (Part 1).
4.4. Molecular docking
All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation and
described in the Supplementary Data (Part 1). The crystal structure of the DNA were retrieved
from the RCSB Protein Data Bank (DNA: 452D) [31].
Notes. The authors declare no competing financial interest.
Acknowledgments. This study was supported by the Innovative Team & Outstanding Talent
Program of Colleges and Universities in Guangxi (2017-38), Guangxi New Century Ten, Hundred
and Thousand Talents Project ([2017]42), Guangxi Natural Science Foundation (Nos.
2016GXNSFAA380300, 2014GXNSFBA118050 and 2018GXNSFAA138165), Guangxi Key
Laboratory of Agricultural Resources Chemistry and Biotechnology (2019KF02), and Middle-
aged and Young Teachers&apos; Basic Ability Promotion Project of Guangxi (2019KY0542).
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[30] W. -B. Kuang, R. -Z. Huang, J. -L. Qin, X. Lu, Q. -P. Qin, B. -Q. Zou, Z. -F. Chen, H. Liang,
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Design,
synthesis
and
pharmacological
evaluation
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new
3-(1Hbenzimidazol-2-yl)quinolin-2(1H)-one derivatives as potential antitumor agents, Eur. J. Med.
Chem. 157 (2018) 139−150.
[31] A. K. Todd, A. Adams, J. H. Thorpe, W. A. Denny, L. P. G. Wakelin, C. J. Cardin, Major
groove binding and 'DNA-induced' fit in the intercalation of a derivative of the mixed
topoisomerase I/II poison N-(2-(dimethlyamino)ethyl)acridine-4-carboxamide (DACA) into DNA:
X-ray structure complexed to d(CG(5Br-U)ACG)2 at 1.3-angstrom resolution. J. Med. Chem. 42
(1999) 536-540.
22 / 25

Tables
Table 1. In vitro antiproliferative activity of compounds 4‒7.
23 / 25

Figure Captions
Scheme 1. Synthetic route of the intermediate compounds N-substituted piperazines. Reagents and
conditions: (a) CS2, benzyl bromide, K2CO3, CH2Cl2, 0‒5°C; (b) TFA, CH2Cl2, RT; (c)
p-toluenesulfonyl chloride, RT; (d) benzoyl chloride, RT.
Scheme 2. Synthetic route of 3-nitro-1,4-naphthalimides derivatives 4‒7. Reagents and conditions:
(a’) HNO3, HAc, 0‒5°C; (b’) N-substituted piperazines, 2-methoxyethanol, 110°C; (c’) R-NH2,
ethanol, 80°C.
Fig. 1. Chemical structures of amonafide and mitonafide and their designed compounds.
Fig. 2. In vivo antitumor activity of compounds 6b and 7b in MGC-803 xenograft. (A) 6b and 7b
(at 5 and 15 mg/kg doses), cisplatin (at 2 mg/kg dose), amonafide (at 5 mg/kg dose), or vehicle
(5% DMSO in saline, v/v) was administered by intraperitoneal injection to inhibiting the tumor
growth. Tumor growth was monitored by the mean tumor volume (mm3) ± SD (n = 6) and
calculated as the relative tumor increment rate (T/C, %). (B) Photographs of the harvested tumors
from the mice. (C) Body weight change of the mice treated with compounds 6b and 7b. (D)
Tumor weight of the mice. The tumors were collected in the mice at day 21. The “**” signs
represent p < 0.01 (versus the vehicle control group)
Fig. 3. Gel electrophoresis assay results of pBR322 DNA treated with compounds 6b and 7b. R:
linear form DNA, Sc: supercoiled form DNA, Nc: circular form DNA.
Fig. 4. Representative images of nuclei in the comet assay. MGC-803 cells were treated with 1.0
µM of compounds 6b and 7b for 24 h.
Fig. 5. Effect of compounds 6b and 7b on the expression levels of relevant proteins in MGC-803
cells line.
24 / 25

Fig. 6. Gel electrophoresis result of topoisomerase I treated with 6b and 7b. Compounds was
added to the mixture of Topo I and pBR322 DNA; Lane 1and 9, pBR322 DNA + Topo І + CPT
(5μM); Lane 2−6 and 10−14, pBR322 DNA + Topo І + 6a/7b; Lane 7 and 15, pBR322 DNA +
Topo І ; Lane 8 and16, pBR322 DNA only.
Fig. 7. Cell cycle analysis results of compounds 6b and 7b treated MGC-803 cells. MGC-803
cells were treated with compounds 6b and 7b at different concentrations (0, 0.25, 0.5 and 1 μM)
for 24 h.
Fig. 8. Effect of compounds 6b and 7b on the expression levels of CDK2, cyclin E, and p21.
Fig. 9. Apoptosis ratio of the MGC-03 cells. MGC-803 cells were treated with compounds 6b and
7b at different concentrations (0, 0.5, 1 and 2 μM) for 24 h.
Fig. 10. The expression levels of Bax, Bcl-2, caspase-9, caspase-3 and PARP in the MGC-803
cells treated with compounds 6b and 7b.
25 / 25

Tables
Table 1. In vitro antiproliferative activity of compounds 4‒7.
IC50(μM)
Compd.
SMMC-7721
5.91±0.23
1.05±0.09
13.94±0.54
7.76±0.35
27.46±0.31
14.38±0.33
17.90±0.05
3.17±0.09
14.31±0.31
12.94±0.41
22.47±0.27
14.81±0.28
13.51±0.32
11.64±0.31
8.83±0.36
4.73±0.24
1.57±0.19
11.99±0.43
13.00±0.21
19.72±0.24
30.15±0.47
2.71±0.13
15.79±0.18
16.60±0.34
12.28±0.55
14.38±0.33
12.48±0.45
14.85±0.35
14.68±0.18
17.59±0.72
15.32±0.67
3.22±0.26
6.93±0.26
T24
SKOV3
A549
MGC-803
6.44±0.12
1.33±0.23
27.42±0.47
16.10±0.41
8.77±0.10
25.16±0.21
31.06±0.43
6.08±0.21
15.40±0.53
20.67±0.37
25.43±0.53
18.35±0.57
21.72±0.25
21.16±0.25
20.69±0.38
8.22±0.32
0.87±0.16
12.88±0.43
19.85±0.65
9.31±0.16
16.26±0.42
1.42±0.11
15.99±0.37
12.81±0.15
21.80±0.28
16.16±0.34
21.31±0.31
10.78±0.24
29.92±0.28
28.93±0.52
34.85±1.75
6.80±0.45
9.06±0.35
3.05±0.21
0.83±0.15
12.78±0.38
9.52±0.32
10.99±0.45
9.71±0.38
10.81±0.51
2.06±0.13
13.19±0.27
9.16±0.25
10.71±0.13
13.34±0.45
13.13±0.34
18.35±0.22
10.31±0.21
4.65±0.17
0.42±0.05
9.93±0.56
20.94±0.31
14.56±0.25
14.12±0.32
0.33±0.07
6.80±0.14
9.17±0.45
11.38±0.29
10.74±0.42
10.92±0.39
12.66±0.26
8.43±0.39
6.37±0.21
40.14±2.14
1.11±0.15
5.01±0.17
8.65±0.18
0.65±0.13
13.44±0.22
12.67±0.43
8.87±0.51
7.48±0.35
27.02±0.18
2.40±0.18
31.38±0.37
44.77±0.61
26.26±0.31
>50
7.52±0.36
1.08±0.15
45.62±0.48
40.37±0.50
42.14±0.63
16.51±0.22
38.59±0.35
1.98±0.11
>50
4a
4b
4c
4d
4e
4f
5a
5b
5c
33.36±0.49
17.36±0.21
>50
5d
5e
5f
34.20±0.41
40.35±0.63
29.65±0.41
4.11±0.31
1.88±0.08
16.81±0.42
15.94±0.35
19.91±0.14
24.07±0.71
0.32±0.03
12.24±0.57
28.92±0.57
20.43±0.17
23.19±0.56
33.62±0.45
38.16±0.56
29.52±0.34
30.39±0.51
26.34±0.57
1.38±0.19
6.31±0.36
>50
5g
44.52±0.48
39.96±0.36
6.66±0.23
0.41±0.12
31.33±0.51
47.74±0.34
22.53±0.42
26.91±0.28
0.27±0.15
20.77±0.29
41.76±0.27
24.68±0.41
38.39±0.26
36.84±0.43
42.91±0.69
47.20±0.62
>50
5h
5i
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
34.47±1.90
0.012±0.01
7.94±0.21
5-FU
Mitonafide
Amonafide
αIC50 values are presented as mean ± SD (standard error of the mean) from three repeating experiments.
1 / 1

Figures
H
N
Boc
N
Boc
N
Boc
N
H
N
b
b
c
a
N
S
N
N
N
H
NBP
N
O
O
O
S O
S
S
S
S
NBDP
NBNBDP
d
NTP
Boc
N
H
N
NBNTP
b
N
O
N
O
NBZP
NBNBZP
Scheme 1. Synthetic route of the intermediate compounds N-substituted piperazines. Reagents and
conditions: (a) CS2, benzyl bromide, K2CO3, CH2Cl2, 0‒5°C; (b) TFA, CH2Cl2, RT; (c)
p-toluenesulfonyl chloride, RT; (d) benzoyl chloride, RT.
NO2
NO2
O
N
O
S
S
O
N
O
4a: R2 = N-methylaminopropyl
4b: R2 = N, N-dimethylamino ethyl
4c: R2 = hydroxyethyl
4d: R2 = p-chlorobenzyl
4e: R2 = 3,4-dihydroxyphenethyl
N
N
N
N
Boc
R2
R2
4a-4f
5a-5i
4f: R2 = 3,4-methylenedioxyphenethyl
5a: R2 = p-methoxybenzyl
5b: R2 = N, N-dimethylamino ethyl
5c: R2 = hydroxyethyl
c'
c'
5d: R2 = 3,4-methylenedioxyphenethyl
5e: R2 = 3,4-dihydroxyphenethyl
5f: R2 = phenylamino
5g: R2 = benzyl
5h: R2 = furan-2-methylene
5i: R2 = p-chlorobenzyl
NO2
O
O
O
O
O
O
O
N
N
R1
a'
b'
O
O
NO2
c'
1
3
2
NO2
Br
Br
3a: R1 = boc
3b: R1 = benzyl dithioformyl
3c: R1 = benzoyl
O
N
N
R2
N
O
O
3d: R1 = p-toluenesulfonyl
6a-6e
7a: R2 = p-methoxybenzyl
7b: R2 = N, N-dimethylamino ethyl
7c: R2 = hydroxyethyl
7d: R2 = 3,4-methylenedioxyphenethyl
7e: R2 = 3,4-dihydroxyphenethyl
7f: R2 = phenylamino
7g: R2 = butyl
7h: R2 = benzyl
7i: R2 = furan-methylene
7j: R2 = p-chlorobenzyl
c'
6a: R2 = N-methylaminopropyl
6b: R2 = N, N-dimethylamino ethyl
6c: R2 = hydroxyethyl
6d: R2 = p-chlorobenzyl
6e: R2 = 3,4-dihydroxyphenethyl
NO2
O
O
N
O
N
N
S
R2
O
7a-7j
Scheme 2. Synthetic route of 3-nitro-1,4-naphthalimides derivatives 4‒7. Reagents and conditions:
1 / 6

(a’) HNO3, HAc, 0‒5°C; (b’) N-substituted piperazines, 2-methoxyethanol, 110°C; (c’) R-NH2,
ethanol, 80°C.
R2
N
N
O
N
O
O
N
O
O
N
O
Modification
NO2
N
NH2
NO2
N
4-7
Amonafide
Mitonafide
R1
Fig. 1. Chemical structures of amonafide and mitonafide and their designed compounds.
Fig. 2. In vivo antitumor activity of compounds 6b and 7b in MGC-803 xenograft. (A) 6b and 7b
(at 5 and 15 mg/kg doses), cisplatin (at 2 mg/kg dose), amonafide (at 5 mg/kg dose), or vehicle
2 / 6