Design, synthesis and biological evaluation of 3-nitro-1,8-naphthalimides as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2020.127051

A series of 3-nitro-naphthalimides 1(1a–1h) were designed and synthesized as antitumor agents. MTT assay results showed that all these compounds exhibited obvious antiproliferative activity against SKOV3, HepG2, A549, T-24 and SMMC-7721 cancer cell lines, while compound 1a displayed the best antiproliferative activity against HepG2 and T-24 cell lines in comparison with mitonafide, with IC₅₀ of 9.2 ± 1.8 and 4.133 ± 0.9 μM, respectively. In vivo antiproliferative activity assay results showed that compound 1a exhibited good antiproliferative activity in the HepG2 and T-24 models, compared with mitonafide. Action mechanism results showed that compound 1a could induced the damage of DNA and the inhibition topo I, accompanying by inducing the G2-stage arresting and the apoptosis of T-24 cancer cells through up-regulating expression levels of cyclin B1, cdc 2-pTy, Wee1, γH2AX, p21, Bax and cytochrome c and down-regulating expression of Bcl-2.

Full text of DOI:10.1016/j.bmcl.2020.127051

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Design, synthesis and biological evaluation of 3-nitro-1,8-naphthalimides as
potential antitumor agents
Mao Xin, Jian-Hua Wei, Chen-Hui Yang, Gui-Bin Liang, Dan Su, Xian-Li
Ma, Ye Zhang
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DOI:
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https://doi.org/10.1016/j.bmcl.2020.127051
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Please cite this article as: Xin, M., Wei, J-H., Yang, C-H., Liang, G-B., Su, D., Ma, X-L., Zhang, Y., Design,
synthesis and biological evaluation of 3-nitro-1,8-naphthalimides as potential antitumor agents, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127051
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Bioorganic & Medicinal Chemistry Letters
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Design, synthesis and biological evaluation of 3-nitro-1,8-naphthalimides as potential
antitumor agents
a†
a†
a
a
a
a,
a,b,
Mao Xin , Jian-Hua Wei , Chen-Hui Yang , Gui-Bin Liang , Dan Su , Xian-Li Ma *, Ye Zhang
a
School of Pharmacy, Guilin Medical University, Guilin 541004, China.
Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Guangxi 541001, China
b
ARTICLE INFO
ABSTRACT
†
Co-first author: these authors contributed equally to this work.

Corresponding author. E-mail: mxl78@glmc.edu.cn (X. L. Ma), zhangye81@126.com (Y. Zhang)
Article history:
Received
2009 Elsevier Ltd. All rights reserved.
A series of 3-nitro-naphthalimides 1(1a–1h) were designed and synthesized as antitumor agents.
MTT assay results showed that all these compounds exhibited obvious antiproliferative activity
against SKOV3, HepG2, A549, T-24 and SMMC-7721 cancer cell lines, while compound 1a
displayed the best antiproliferative activity against HepG2 and T-24 cell lines in comparison
with mitonafide, with IC50 of 9.2±1.8 and 4.133±0.9 μM, respectively. In vivo antiproliferative
activity assay results showed that compound 1a exhibited good antiproliferative activity in the
HepG2 and T-24 models, compared with mitonafide. Action mechanism results showed that
compound 1a could induced the damage of DNA and the inhibition topo I, accompanying by
inducing the G2-stage arresting and the apoptosis of T-24 cancer cells through up-regulating
expression levels of cyclin B1, cdc 2-pTy, Wee1, γH2AX, p21, Bax and cytochrome c and
down-regulating expression of Bcl-2.
Revised
Accepted
Available online
Keywords:
3
-Nitro naphthalimides
Antitumor activity
Cell cycle arrest and Apoptosis
DNA damage
Topo I
As the disease caused by uncontrolled cell division in
undesired manner, cancer has become a major health concern
today and represented the second leading cause of death
worldwide. It is well known that DNA plays a vital role in the
growth and survival of tumor. Consequently, DNA is considered
the structure–activity relationships of 1,8-naphthalimide
5
-9
derivatives, it could be found that the main modification of 1,8-
naphthalimide derivatives including the alteration in N-position
in imide and positions 3 and 4 in the aromatic moiety have
important effect on the antitumor activity and binding strength
with DNA, while in some cases even small changes in the
structure of 1,8-naphthalimide could have a major effect on their
1
as one of the major targets for anticancer drugs. Some anticancer
agents usually exert their antitumor effect by targeting to DNA,
thus leading to the damage of DNA and inhibition of
topoisomerase (topo I or/and II) and consequently inducing the
cell cycle arrest and apoptosis.1-4 Mitonafide is a famous DNA-
targeting naphthalimide anticancer agent that has been tested in
5
-10
antitumor activity.
N
HN
O
N
O
Demethylation
O
N
O
5
clinic trials for the treatment of some solid tumors and has
gained considerable attraction in antitumor medicinal chemistry.
However, the unexpected central neurotoxicity and
hematotoxicity limit the further application of mitonafide in the
NO2
NO2
Mitonafide
1a
5
clinical trial. To improve these issues, a great deal of effort,
Fig. 1. The possible metabolism of Mitonafide.
containing the alternative modification of the side chain, the
The discovery of the poly ADP-ribose polymerase inhibitor
aromatic ring system, and the substituents on the ring, has been
1
1
explored in the past four decades.5-10
rucaparib (AG014699) with better efficiency and much lower
toxicity from its fore runner AG14361, indicated that the
replacement of N, N-dimethylamine group with N-methylamine
may lead to better efficiency and lower toxicity. The USA Food
and Drug Administration has granted the approval to rucaparib
for use as maintenance therapy for recurrent ovarian cancer. It is
well known that mitonafide contains a N, N-dimethylimine
ethylenediamine group. Therefore, in the present work, motivated
by promising antitumor activities of naphthalimide and the
success of rucaparib, the N-methylamine ethylenediamine moiety
were designed to replace the N, N-dimethylamine
ethylenediamine group in mitonafide to improve the toxicity (Fig.
1), while some aromatic moiety were also designed and used for
Since the discovery of mitonafide and amonafide in 1970s, the
design and development of the naphthalimide compounds as
anticancer drugs has attracted a great deal of antitumor medicinal
chemists during the last 40 years. To improve the therapeutic
potency of naphthalamides, a large number of naphthalamide
derivatives have been designed and synthesized by the modifying
of naphthalimide skeleton.5-10 The synthesized naphthalamides
generally feature with a planar chromophore portion capable of
intercalation into DNA and consequently could bind to double-
stranded DNA by intercalation and induce topoisomerase
inhibition, cell cycle arrest and apoptosis.5-10 By the summarizing
1
/ 9

comparison. To ascertain our expectation, the in vitro and in vivo
antitumor activities, as well as the in vitro cytotoxicity against
the normal human cell line were evaluated. Furthermore, the
preliminary action mechanisms based on the DNA-targeting and
topo I inhibition, as well as cell cycle arrest and apoptosis assays
were also performed.
result indicated that compounds 1a and 1b may be cytotoxic
agents. In addition, by comparing of the antiproliferative activity
of compound 1a with 1b, it may be concluded that the N-
methylamine ethylenediamine part in imide was more beneficial
to antitumor activity than N, N-dimethylamine ethylenediamine.
In vivo antiproliferation experiment was then carried out to
further assess the antitumor activity of compound 1a. The HepG-
R
O
O
O
O
O
O
O
N
O
2
and T-24 xenograft models were consequently selected in this
NaNO3
H2SO4
R-NH2
study according to the in vivo antiproliferation results, and
mitonafide was used as the positive control for comparison. The
relative tumor increment rates (T/C) and tumor inhibitory rate (IR)
were then determined to evaluate the in vivo antitumor activity.
Generally, T/C value of lower than 60.0% and IR value of higher
than 40.0% indicated effective and good activity in antitumor
NO2
NO2
NA
NNA
1(1a-1h)
Scheme 1. Synthetic routes of compounds 1(1a−1h). Reagents and
conditions: (a) NaNO -H SO ; (b) R-NH , ethanol.
3
2
4
2
The target 3-nitro-1,8-naphthalimide analogues 1(1a−1h)
were synthesized as shown in Scheme 1. Firstly, 3-nitro-1,8-
naphthalene anhydride (NNA) was succinctly obtained in good
15
effect.
As shown in Fig. 2A, compound 1a at the dose of 5 mg/kg
and 15 mg/kg displayed obvious antiproliferation in HeGp-2
xenograft model assay, with the T/C values of 66.7% and 38.6%,
respectively, based on the tumor volume. In comparison,
mitonafide at the 5 mg/kg dose exhibited a T/C value of 28.8%.
In addition, Fig. 2B and Fig. 2D demonstrated that compound 1a
showed important antiproliferative activity in the HeGp-2 model
with IR of 26.8% (5 mg/kg) and 60.0% (15 mg/kg), respectively,
compared with that of mitonafide (68.3%, P < 0.01). It was
obvious that compound 1a at the dose of 15 mg/kg exhibited the
T/C value of lower than 60.0% and IR value more than 40.0%
and similar in vivo antiproliferative activity with mitonafide,
indicating compound 1a exhibited good antitutmor activity in
HeGp-2 xenograft model. Moreover, Fig. 2C showed that no
significant change in body weight and no other adverse effects
were observed among the mice treated with 1a, suggesting that
yields by the treatment of 1,8-naphthalic anhydride (NA) with the
o
mixture of NaNO
3
-H
2
SO
4
at the 0 C for 1h, according to the
previous literature.12 Secondly, the combination of NNA and a
series of primary amines in hot ethanol, including chained fatty
and aromatic amines offered the target 3-nitro-naphthalimides
1
(1a−1h) in good yields, respectively. The synthetic procedure
1
3
and experimental data were described in reference. It was worth
noting that the synthetic procedure and after-treatment of
1
(1a−1h) was very simple and convenient, which was beneficial
to the industrial production. The chemical structures of
naphthalimides 1 were confirmed by NMR spectroscopy and
high-resolution mass spectrometry (HR-MS) (Part
supplmentary materials).
2 in
The in vitro antiproliferative activity of naphthalimides
(1a−1h) were assayed against the SKOV3 (human ovarian
1
1
a exhibited no significant toxicity to the mice within the period
cancer cells), HepG2 (human liver cancer cells), A549 (human
lung cancer cells), T-24 (bladder cancer cells), SMMC-7721
of treatment. Fig. S-1 showed that compound 1a at the dose of 5
mg/kg and 15 mg/kg also exhibited important in vivo
antiproliferation in T-24 xenograft assay, with T/C and IR values
of 62.9%, 38.4% and 30.1%, 58.7%, respectively, in comparison
with that (29.0% and 67.0%) of mitonafide in 5mg/kg dose. It
was evident that compound 1a exhibited the T/C value of lower
than 60.0% and IR value more than 40.0% and similar in vivo
antiproliferative activity with mitonafide, indicating compound
(human liver cancer cells) and HL-7702 (human liver normal
cells) cell lines, using the methylthiazoltetrazolium (MTT)
1
4
assay. Mitonafide was used as positive control for comparison.
As shown in Table 1, naphthalimides 1(1a−1h) showed
obvious in vitro antiproliferative activity against these five tumor
cell lines, while they exhibited lower cytotoxicity on the HL-
1
a exhibited good antitutmor activity in T-24 xenograft model.
7
702 normal cell line, in comparison with mitonafide. It was
Further investigation (Fig. S-1C) also revealed that no significant
change in body weight and no other adverse effects were
observed among the mice treated with 1a. On the basis of the
observation in above HeGp-2 and T-24 xenograft model assay, it
could be concluded that 1a should be good candidate for
potential antitumor agents.
important to note that compound 1a exhibited better
antiproliferative activity than mitonafide against HepG2 and T-24
cell lines, with IC50 of 9.2±1.8μM and 4.133±0.9μM, respectively,
while compound 1b displayed equal antiproliferative activity
with mitonafide against HepG2 cell line, with IC50 of
1
0.321±2.2μM, indicating that compound 1a showed the best in
vitro antiproliferative activity among these target compounds.
The above MTT assay results confirmed our expectation that
compound 1a may exhibit effective activity and lower toxicity
and thus possibly better therapeutic potency than mitonafide.
Since that compound 1a (15mg/kg) exhibited similar in vivo
antiproliferation with mitonafide and the three times dose of
mitonafide (5mg/kg) for compound 1a do not lead to adverse
effects in HepG2 and T-24 models, it may be concluded that
compound 1a exhibited lower in vivo toxicity than mitonafide,
consistent with the result in MTT assay. Due to its best in vitro
and in vivo antitumor activity among the compounds 1a-1h, the
action mechanism of compound 1a was thus investigated.
The structure-activity relationship investigation of 3-nitro
naphthalimides 1 was then carried out. By the comparison of the
antiproliferative activity of compounds 1a and 1b with 1c, 1d, 1e,
1
f, 1g and 1h, respectively, it could be concluded that the
flexious chained substituents in N-position of naphthalimide
exhibited positive effect on the antiproliferative activity, while
the presence of aromatic substituents displayed negative effect.
However, the flexious chained substituents may also lead to
increased cytotoxicity on normal cell line HL-7702, while the
As a series of tightly regulated molecular events controlling
DNA replication and mitosis, cell cycle happen in a cell leading
to its division and duplication and play important mediated role
in the occurrence and formation cancer. Moreover, DNA is
known as the potential target for naphthalamides.
2
aromatic substituents resulted to decreased cytotoxicity. The
α
Table 1. IC50 values of compounds 1(1a−1h) towards the selected tumor cell lines and a normal cell line.
Compounds
IC50α(μM)
SK-OV-3
HepG2
A-549
T-24
SMMC-7721
HL-7702
2
/ 9

NO2
O
N
O
2
2
0.71±2.1
8.16±2.4
9.20±1.8
10.32±2.2
30.53±3.1
50.38±5.0
12.60±2.1
19.82±2.4
31.59±3.1
36.90±3.6
4.13±0.9
9.03±1.6
33.23±3.2
54.87±5.2
13.96±2.8
26.60±3.1
81.36±7.1
54.09±4.6
18.67±2.1
14.35±1.9
62.93±5.4
＞100
HN
1
a
NO2
O
N
O
NH
1
b
NO2
O
N
N
O
HN
77.15±6.4
N
H
1
c
NO2
O
N
O
N
6
4
0.47±5.2
1.43±4.1
N
H
1
d
NO2
H
N
O
O
N
O
HN
23.95±2.4
38.74±3.4
44.75±4.2
32.07±3.1
58.02±5.4
15.08±2.4
54.72±4.5
＞100
1
e
NO2
O
HN
N
O
6
4
1.15±5.4
8.16±4.4
＞100
59.09±4.9
1f
NO2
N
O
N
O
HN
42.69±4.1
29.99±2.6
57.65±5.4
＞100
82.43±7.1
1g
NO2
N
O
S
HN
N
O
58.80±5.4
6.26±1.1
51.30±5.4
10.88±2.3
44.13±4.0
7.94±1.3
31.18±3.4
5.01±1.1
＞100
65.26±5.4
8.51±1.7
1h
Mitonafide
6.94±1.2
αIC50 values are presented as mean ± SD (standard error of the mean) from three repeating experiments.
Fig. 2. In vivo anticancer activity of 1a in HepG2 xenograft model. (A) Effect of 1a (at the doses of 15 and 5 mg/kg), Mitonafide (at the dose of 5 mg/kg), or
3
vehicle (5% DMSO in saline, v/v) on tumor growth. Tumor growth was monitored by the mean tumor volume (mm ) ± 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 1a. (D) Tumor weight
of the mice. The tumors were collected in the mice at day 14. The “**” signs represent p < 0.01 (versus the vehicle control group).
So the action mechanism of the antitumor effect of 1a was
investigated by the cell cycle distribution of T-24 cells for 24 h
percentage of cells in S and G1 phases decreased concomitantly.
The result indicated that 1a specifically arrested the cell cycle of
T-24 cells in the G2/M stage.
through treatment with 0, 2.06(IC50/2), 4.13(IC50
)
and
8
.26(2×IC50) μM of 1a, respectively. As shown in Fig. 3, treating
of T-24 cells with compound 1a at different concentrations
induced cell cycle arrest at the G2 phase, leading to concomitant
increment in the percentage in the G2/M phase (to 92.24%) in
comparison with the control cells (10.16%), whereas the
3
/ 9

of the T-24 cells with compound 1a at different concentrations
0, 2.06(IC50/2), 4.13(IC50) and 8.26(2×IC50) μM) led to obvious
(
increment (0.9%, 5.4% and 23.4%) of the apoptotic cell
population in a concentration-dependent manner, in comparison
with the control (0.7%), indicating that compound 1a induced the
apoptosis of T-24 cells in a dose-dependent manner.
Fig. 3. Cell cycle analysis of compound 1a treated T-24 cells. T-24 cells were
treated with different concentrations (0, 2.06, 4.13 and 8.26 μM) of
compound 1a for 24 h to determine DNA fluorescence and cell cycle phase
distribution.
Fig. 5.Apoptosis ratio of the T-24 cells. (a) Negative control; (b) treated with
As an important cell cycle checkpoint in eukaryotic organisms
ranging from yeast to mammals, G2-M DNA damage checkpoint
ensures that cells don't generate mitosis before they have a
chance to repair damaged DNA after replication and usually
2
.06 μM 1a, (c) treated with 4.13 μM 1a, (d) treated with 8.26 μM 1a. The
cells were analyzed using the Annexin V/PI assay in a flow cytometer.
It is known that the famous anti- and pro-apoptotic proteins,
Bcl-2/Bax family proteins play important mediated roles in the
inhibition or promotion of cell death and the ratio of Bcl-2/Bax
2
,16-
accompanies with the increased expression of γH2AX protein.
1
8
G2/M transition is mainly regulated by timed activation of
3
,20-22
2
expression are very important to the regulation of apoptosis,
distinct cyclin B1/CDK1 (aka CDC2) complexes. It is generally
considered that Cdc2 activity is regulated by phosphorylation of
while the release of cytochrome c is considered as the signal of
apoptosis. To obtain insight into the mechanism of compound 1a
inducing apoptosis of T-24 cells, the expression levels of Bax,
Bcl-2 and cytochrome c in T-24 cells were investigated by
western blot assay. As shown in Fig. 6, in comparison with the
control, the lysates of the T-24 cells treating with 1a up-regulated
the expression levels of Bcl-2 and cytochrome c, while down-
regulated the expression level of Bax, confirming that compound
its tyrosine-15 residue by the kinase wee1, while phosphorylation
of tyrosine-15 would inhibit cdc2 activity.2,19 Moreover, in
addition to the anti-oncogene p53 and p27, p21 also plays an
important role in G
2
/M arrest through promoting accumulation of
the inactive cyclin B-CDK complex before the regulation to
1
7
2
G /M transition. To obtain insight into the mechanism of
compound 1a arresting the cell cycle of T-24 cells in the G2/M
stage, the expression levels of cyclin B1, cdc 2-pTy, Wee1,
γH2AX and p21 in T-24 cells were thus investigated using
western blot assay. As shown in Fig. 4, in comparison with the
control, the lysates of the T-24 cells treating with 1a up-regulated
the expression levels of cyclin B1, cdc 2-pTy, Wee1, γH2AX and
1
a could indeed induce and promote the apoptosis of T-24 cells.
2
p21, demonstrating that compound 1a induced G /M phase cell
cycle arrest through increasing the expression levels of cyclin B1,
cdc 2-pTy, Wee1, γH2AX and p21.
Fig. 6. Effect of compound 1a on the expression levels of Bax, Bcl-2 and
cytochrome c in the HepG2 cells. GAPDH was used as a loading standard.
Con: Control.
It is reasonable that the flexible chains in N-position in
naphthalimide moiety and the plane naphthalimide-structure may
be attributed to the intercalation of DNA. In addition, it is known
5
that DNA is the potential targets of naphthalimide derivatives.
So the interaction of DNA for compound 1a was investigated by
gel electrophoresis.
As shown in Fig. 7, compound 1a exhibited obvious
increment to the electrophoresis mobility of supercoiled circular
plasmid pbr322-DNA at the concentrations from 20μM to 100μM,
while it showed no evident cleavage ability in this assay,
indicating that compound 1a showed an important intercalation
effect on pbr322-DNA. Based on the DNA intercalation effect of
compound 1a, it should be hypothesized that compounds 1a
Fig. 4. Effect of compound 1a on the expression levels of relevant proteins in
HepG2 cells. Western blot analysis was performed with antibodies against
cyclin B1, cdc 2-pTy, Wee1, γH2AX and p21. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as the loading standard.
It is well known that apoptosis is an important mechanism in
3
cancer treatment and a key pathway leading to cell death. So the
apoptosis-inducing effect of compound 1a was investigated
AO/EB staining assay. In the AO/EB staining assay, as shown in
Fig. S-2, the morphology of 1a-treated T24 cells exhibited
obvious change. The cell nuclei were stained as yellow/orange,
whereas the morphology changes indicated pycnosis, membrane
blebbing and cell budding, implying that compound 1a could
induce the apoptosis of T-24 cells.
could induce DNA damage, cause
a series of signal
transductions, and eventually lead to cell cycle arrest and
apoptosis. It is clear that the cell cycle arrest and apoptosis of 1a
has been approved by our above cell cycle arrest and apoptosis
assays. To verify the DNA-damage of 1a, a Click-iT 5-ethynyl-
2
′-deoxyuridine (EdU) assay was thus performed to assess the
ability of cells to complete DNA replication. As shown in Fig. 8,
EdU was incorporated into DNA staining as green in the control.
However, when the T24 cells were treated with 10 μM of
The apoptosis ratios of T-24 cells treating with 1a were then
determined by flow cytometry. As shown in Fig. 5, the treatment
4
/ 9

compound 1a for 24 h, just a little green fluorescence was
observed, indicating that 1a inhibited the DNA replication
process and confirming the occurrence of the damage of DNA.23
lines and low toxicity against normal HL-7702 cells, while it
exhibited good in vivo antiproliferative activity in the HepG2 and
T24 model in comparison with mitonafide. Mechanistic studies
showed that compound 1a may exert the antitumor effect mainly
by the damage of DNA and the inhibition topo I, and
consequently leading to the G2-stage arresting and the inducing
apoptosis of cancer cells.
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
Fig. 7. Gel electrophoresis mobility shift assay of pBR322 DNA treated with
1
a (lane 0: DNA alone; lanes 1–6: DNA + 1a, with increasing concentrations
of 5, 10, 20, 40, 60, 80 and 100 μM, respectively). R: linear form DNA, Sc:
supercoiled form DNA, Nc: circular form DNA.
Foundation
(Nos.
2016GXNSFAA380300,
2
014GXNSFBA118050 and 2018GXNSFAA138165) and
Previous work had revealed that topo I inhibition is one of the
potential targets of naphthalimide derivatives.10 In addition, we
have indicated the naphthalimide derivatives exerted the
antitumor effect mainly by the covalent binding with DNA and
the inhibition of topo I.24 In the present work, the inhibition of
topo 1 activity by compound 1a was thus measured using the
DNA relaxation assay to investigate whether it was related to the
cytotoxicity of the 1a and the 1a-inducing DNA damage.
Guangxi Key Laboratory of Agricultural Resources Chemistry
and Biotechnology (2019KF02).
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μM, respectively).
0. Tan, S.; Sun, D.; Liu, J.; Sun, X.; Wu, F.; Li, Q.; Yang, Y.; Liu, J.;
Wang, X.; Chen, Z.; Li, H.; Qian, X.; Xu, Y. Antiproliferative and
apoptosis-inducing activities of novel naphthalimide-cyclam
conjugates through dual topoisomerase (topo) I/II inhibition.
Bioorg. Med. Chem. 2015, 23, 5672−5680.
As shown in Fig. 9, compound 1a exhibited obvious in vitro
inhibition on topo I. It was worth noting that compound 1a
displayed good inhibition of topo I (0.1U/L) at the concentration
of 20μM, indicating that compound 1a may exert the antitumor
activity by the inhibition of topo I at molecular level. Previous
work had described that camptothecin was a classical topo I
11. Plummer, R.; Lorigan, P.; Steven, N.; Scott, L.; Middleton, M. R.;
Wilson, R. H.; Mulligan, E.; Curtin, N.; Wang, D.; Dewji, R.;
Abbattista, A.; Gallo, J.; Calvert, H. A phase II study of the potent
PARP inhibitor, Rucaparib (PF-01367338, AG014699), with
temozolomide in patients with metastatic melanoma
demonstrating evidence of chemopotentiation. Cancer Chemother.
Pharmacol., 2013, 71, 1191–1199.
inhibitor with the effective inhibited concentration of about
5
2
0μM.2 By the comparison of the effective inhibited
concentrations of camptothecin with compound 1a (20μM), it
could be concluded that compound 1a exhibited similar topo I
inhibition activity with camptothecin. In the DNA relaxation
assay (Fig. S-3), compared with camptothecin (5μM), it was
evident that compound 1a exhibited obvious inhibition on topo I
at 20μM concentration, accompanying by the inhibition of DNA
1
2. Li, Z.; Yang, Q.; Qian, X. Novel 2-aminothiazonaphthalimides as
visible light activatable photonucleases: effects of intercalation,
heterocyclic-fused area and side chains. Bioorg. Med. Chem. Lett.
2005, 15, 1769–1772.
replication. The result indicated that the inhibited topo I activity
may accelerate the 1a-induced DNA damage.23
13.
General procedure for the preparation of 1(1a-1h): The mixture of
primary amine (1.1 mmol), ethanol (50 mL) and NNA (1 mmol)
o
In summary, we designed and synthesized a serial of 3-nitro
naphthalimides 1a–1h and evaluated their antiproliferative
activity against five cancer cell lines (SKOV3, HepG2, A549, T-
was reacted at 80 C for 4h. After reaction, the reaction solution
was filtrated to afford yellow powder of 1,8-naphthalimides 1(1a-
1
1
h), respectively. 1a: Yield 75.1%, H NMR (400 MHz, DMSO-
d
=
6
) δ: 9.33 (d, J = 2.3 Hz, 1H), 8.81 (d, J = 2.3 Hz, 1H), 8.64 (d, J
7.8 Hz, 1H), 8.57 (dd, J = 7.3, 0.9 Hz, 1H), 7.99 – 7.91 (m, 1H),
2
4, SMMC-7721). We identified compound 1a exhibiting good
in vitro sensitivity and selectivity against HepG2 and T24 cell
5
/ 9

4
6
1
1
.15 (t, J = 6.4 Hz, 2H), 3.00 (dd, J = 11.4, 5.7Hz, 1H), 2.84 (t, J =
.4 Hz, 2H), 2.35 (s, 3H). C NMR (101 MHz, DMSO) δ 162.79,
62.29, 145.55, 136.08, 133.72, 130.62, 129.42, 129.31, 129.04,
131.04, 130.39, 129.33, 129.21, 125.97, 123.75, 122.38, 122.27,
121.59. ESI-HRMS, calculated m/z for C19H N O [M+H] :
10 4 4
391.0456, found: 391.0599.
1
3
+
23.92, 122.57, 122.48, 56.01, 48.37, 35.27. ESI-HRMS,
14. Kuang, W.; Huang, R.; Qin, J.; Lu, X. Qin, Q.; Zou, B.; Chen, Z.;
Liang, H.; Zhang, Y. Design, synthesis and pharmacological
evaluation of new 3-(1Hbenzimidazol-2-yl)quinolin-2(1H)-one
derivatives as potential antitumor agents. Eur. J. Med. Chem. 2018,
157, 139–150.
+
calculated m/z for
C
15
H
13
N
3
O
4
[M+H] : 300.0940, found:
1
3
9
8
6
00.0938. 1b: Yield 50.3%, H NMR (400 MHz, DMSO-d ) δ:
.34 (d, J = 2.3 Hz, 1H), 8.82 (d, J = 2.3Hz, 1H), 8.65 (d, J =
.1Hz, 1H), 8.58 (dd, J = 7.3, 0.9 Hz, 1H), 8.01-7.89 (m, 1H), 4.16
(
t, J = 6.4 Hz, 2H), 3.44 (d, J = 7.0 Hz, 1H), 2.98 (d, J = 6.5 Hz,
H), 2.85 (t, J = 6.4 Hz, 2H), 2.35 (s, 3H), 1.06 (t, J = 7.0 Hz, 1H).
C NMR (101 MHz, DMSO-d ) δ: 163.27, 162.77, 146.03,
6
36.57, 134.21, 131.10, 129.90, 129.80, 129.52, 124.41, 123.06,
15. Technical guidelines for non-clinical research of cytotoxic
antitumor drugs, State Food and Drug Administration, 2006.
16. Dighe, S. U.; Khan, S.; Soni, I.; Jain, P.; Shukla, S.; Yadav, R.;
Sen, P.; Meeran, S. M.; Batra, S. Synthesis of β-carboline-Based
N-heterocyclic carbenes and their antiproliferative and
antimetastatic activities against human breast cancer cells. J. Med.
Chem. 2015, 58, 3485−3499.
1
1
3
1
5
C
6.49, 48.85, 35.75, 19.02. ESI-HRMS, calculated m/z for
+
16
H
15
N
3
1
O
4
[M+H] : 314.1096, found: 314.1100. 1c: Yield
6
4.1%, H NMR (400 MHz, DMSO-d
6
) δ: 14.49 (s, 1H), 9.54 (d, J
=
1.9Hz, 1H), 8.97 (d, J = 2.1 Hz, 1H), 8.88-8.75 (m, 2H), 8.70 (d,
17. Boutros, R.; Lobjois, V.; Ducommun, B. CDC25 phosphatases in
cancer cells: key players? Good targets? Nat. Rev. Cancer. 2007, 7,
495−507.
1
3
J = 6.9 Hz, 1H), 8.07 (dd, J = 23.5, 15.9 Hz, 1H). C NMR (101
MHz, DMSO-d
6
) δ: 163.16, 162.68, 149.87, 146.36, 145.69,
1
1
3
44.18, 137.65, 134.92, 131.65, 130.99, 129.82, 127.38, 123.96,
18. Belluti, F.; Fontana, G.; Bo, L. D.; Carenini, N.; Giommarelli, C.;
Zunino, F. Design, synthesis and anticancer activities of stilbene-
coumarin hybrid compounds: Identification of novel proapoptotic
agents. Bioorg. Med. Chem. 2010, 18, 3543−3550.
19. Wright, G.; Golubeva, V.; Rix, L. L. R.; Berndt, N.; Luo, Y.;
Ward, G. A.; Gray, J. E.; Schönbrunn, E.; Lawrence, H. R.
Monteiro, A. N. A.; Rix, U. Dual targeting of WEE1 and PLK1 by
AZD1775 elicits single agent cellular anticancer activity. ACS
Chem. Biol. 2017, 12, 7,1883−1892.
20. Tsujimoto, Y.; Cossman, J.; Jaffe, E.; Croce, C. M. Involvement
of the bcl-2 gene in human follicular lymphoma. Science, 1985,
228, 1440−1443.
21. Hockenbery, D. M.; Zutter, M.; Hickey, W.; Nahm, M.;
Korsmeyer, S. J. Bcl2 protein is topographically restricted in
tissues characterized by apoptotic cell death. Proc. Natl. Acad. Sci.
USA. 1991, 88, 6961−6965.
22. Qiao, S.; Nagasaka, T.; Harada, T.; Nakashima, N. p53, Bax and
Bcl-2 expression, and apoptosis in gestational trophoblast of
complete hydatidiform mole. Placenta. 1998, 19, 361−369.
23. Wei, J.; Chen, Z.; Qin, J.; Liu, Y.; Li, Z.; Khan, T.; Wang, M.;
Jiang, Y.; Shen, W.; Liang, H. Water-soluble oxoglaucine-Y(III),
Dy(III) complexes: in vitro and in vivo anticancer activities by
triggering DNA damage, leading to S phase arrest and apoptosis.
Dalton T. 2015, 44, 11408−11419.
+
22.57. ESI-HRMS, calculated m/z for C14
10.0532, found: 310.0630. 1d: Yield 62.6%, H NMR (400 MHz,
H
7
N
5
O
4
[M+H] :
1
DMSO-d
1
8
6
) δ 11.01 (s, 1H), 9.49 (d, J = 2.1 Hz, 1H), 8.99 (d, J =
.9 Hz, 1H), 8.80 (d, J = 8.2 Hz, 1H), 8.71 (d, J = 7.2 Hz, 1H),
.06 (t, J = 7.8 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.8
1
3
Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H).
NMR (101 MHz, DMSO-d
1
1
C
6
) δ 161.13, 160.74, 145.82, 137.06,
34.69, 131.04, 130.39, 129.33, 129.21, 125.97, 123.75, 122.38,
22.27, 121.59. ESI-HRMS, calculated m/z for
C
H
19 10
1
N
4
O
4
+
[M+H] : 359.0736, found: 359.0899. 1e: Yield 61.0%, H NMR
6
(400 MHz, DMSO-d ) δ 9.95 (s, 2H), 8.65 (s, 1H), 8.23 – 7.66 (m,
1
3
3
1
1
1
3
H), 7.22 (s, 1H), 6.17 (s, 3H). C NMR (101 MHz, DMSO-d
6
) δ
63.31, 162.71, 155.53, 145.76, 136.17, 133.78, 130.88, 129.85,
29.24, 128.24, 124.77, 123.36, 122.64, 121.08, 121.04, 109.39,
+
08.26. ESI-HRMS, calculated m/z for C19
H
10
1
N
4
O
5
[M+H] :
75.0685, found: 375.0803. 1f: Yields 56.2%, H NMR (400
) δ 9.49 (d, J = 1.7 Hz, 1H), 8.98 (d, J = 1.8 Hz,
H), 8.80 (d, J = 8.2 Hz, 1H), 8.71 (d, J = 9.3 Hz, 2H), 8.07 (t, J =
MHz, DMSO-d
1
7
NMR (101 MHz, DMSO-d
1
1
6
1
3
.8 Hz, 1H), 7.16 (t, J = 7.7 Hz, 2H), 6.85 – 6.74 (m, 3H).
) δ 162.23, 161.81, 147.03, 145.77,
36.68, 134.37, 131.02, 130.00, 129.52, 129.24, 128.78, 124.33,
23.31, 122.83, 119.56, 112.61. ESI-HRMS, calculated m/z for
C
6
+
C
18
H
11
N
3
1
O
4
[M+H] : 334.0783, found: 334.0896. 1g: Yields
4.4%, H NMR (400 MHz, DMSO-d ) δ 9.48 (d, J = 2.8 Hz, 2H),
6
8
7
7
5
1
1
6
24. Liang, G.; Yu, Y.; Wei, J.; Kuang, W.; Chen, Z.; Zhang, Y.
.96 (d, J = 2.3 Hz, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.67 (dd, J =
.3, 0.9 Hz, 1H), 8.10 – 8.01 (m, 1H), 7.96 (d, J = 4.1 Hz, 1H),
.65 – 7.58 (m, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 6.5,
Design,
synthesis
and
biological
evaluation
of
naphthalenebenzimidizole platinum (II) complexes as potential
antitumor agents. Eur. J. Med. Chem. 2020, 188, 112033. doi:
10.1016/j.ejmech.2019.112033.
1
3
6
.2 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ 161.99, 161.58,
56.94, 147.38, 145.84, 137.57, 136.76, 134.44, 131.03, 130.11,
25. Zenker, A.; Galanski, M.; Bereuter, T. L.; Keppler, B. K.;
Lindner, W. Kinetics of binding properties of 59-GMP with
cisplatin under simulated physiological conditions by capillary
electrophoresis. J. Chromatogr. B. 2000, 745, 211–219.
29.31, 124.02, 123.48, 122.58, 115.58, 108.11. ESI-HRMS,
+
calculated m/z for
C
17
H
10
N
4
1
O
4
[M+H] : 335.0736, found:
3
1
8
6
35.0732. 1h: Yields 76.4%, H NMR (400 MHz, DMSO-d ) δ
1.01 (s, 1H), 9.49 (d, J = 2.1 Hz, 1H), 8.99 (d, J = 1.9 Hz, 1H),
.80 (d, J = 8.2 Hz, 1H), 8.71 (d, J = 7.2 Hz, 1H), 8.06 (t, J = 7.8
Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.24 (t,
1
3
J = 7.6 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H). C NMR (101 MHz,
DMSO-d ) δ 166.08, 161.13, 160.74, 145.82, 137.06, 134.69,
6
Graphical Abstract
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To create your abstract, type over the instructions in the
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influencing the position presented in, or the review of, the
Design, synthesis and biological evaluation of
-nitro-1,8-naphthalimides as potential
antitumor agents
Leave this area blank for abstract info.
3
Mao Xin, Jian-Hua Wei, Chen-Hui Yang, Gui-Bin Liang, Dan Su,Xian-Li Ma, Ye Zhang
Fm oa nn t us so cr r ai pb ts tr ea nc tt i dt li emd en “s iDo ne ss i sg hn o, ul ds y nn ot th eb se i sc ha anng de d bo ir o al lo t ge ri ec da l.
Highlights
evaluation of 3-nitro-1,8-naphthalimides as potential
antitumor agents”.




3-nitro-naphthalimides 1a-1h were designed
and synthesized as antitumor agents
1a exhibited good in vitro and in vivo
antiproliferation activity.
Tables
Table 1. αIC50 values of compounds 1(1a−1h) towards the selected
tumor cell lines and a normal cell line.
Compounds
IC50α(μM)
SK-OV-3
20.71±2.1
HepG2
A-549
T-24
SMMC-
7721
1a induced cell cycle arrest and apoptosis of T-
NO2
O
N
O
2
4.
9.20±1.8
12.60±2.1
4.13±0.9
13.96±2.8
26.60±3.1
HN
1a
NO2
1a induced DNA damage and inhibition of
O
N
O
2
8.16±2.4 10.32±2.2 19.82±2.4
9.03±1.6
NH
topo I.
1b
NO2
O
N
N
O
HN
77.15±6.4 30.53±3.1 31.59±3.1 33.23±3.2 81.36±7.1
N
H
1c
NO2
O
N
O
N
6
4
0.47±5.2 50.38±5.0 36.90±3.6 54.87±5.2 54.09±4.6
1.43±4.1 23.95±2.4 44.75±4.2 58.02±5.4 54.72±4.5
N
H
1
d
NO2
H
N
O
O
N
O
Declaration of Interest Statement
HN
1e
NO2
O
HN
N
O
6
4
1.15±5.4 38.74±3.4 32.07±3.1 15.08±2.4
8.16±4.4 42.69±4.1 29.99±2.6 57.65±5.4
＞100
＞100
We declare that we have no financial and personal
relationships with other people or organizations that can
inappropriately influence our work, there is no professional
or other personal interest of any nature or kind in any
product, service and/or company that could be construed as
1f
NO2
N
O
N
O
HN
1g
1
/ 1

mice. (C) Body weight change of the mice treated with 1a. (D)
NO2
N
O
N
O
S
Tumor weight of the mice. The tumors were collected in the mice at
HN
58.80±5.4 51.30±5.4 44.13±4.0 31.18±3.4
＞100
65.26±5.4
day 14. The “**” signs represent p < 0.01 (versus the vehicle control
1h
group).
8.51±1.7
Mitonafide
6.26±1.1
10.88±2.3
7.94±1.3
5.01±1.1
6.94±1.2
αIC50 values are presented as mean ± SD (standard error of the mean) from
three repeating experiments.
Figures
R
O
O
O
O
O
O
O
N
O
NaNO3
H2SO4
R-NH2
NO2
NO2
NA
NNA
1(1a-1h)
Scheme 1. Synthetic routes of compounds 1(1a−1h). Reagents and
conditions: (a) NaNO
3
-H
2
SO
4
; (b) R-NH
2
, ethanol.
Fig. 3. Cell cycle analysis of compound 1a treated T-24 cells. T-24
cells were treated with different concentrations (0, 2.06, 4.13 and
8.26 μM) of compound 1a for 24 h to determine DNA fluorescence
and cell cycle phase distribution.
N
HN
metabolism
O
N
O
O
N
O
NO2
NO2
Mitonafide
1a
Fig. 1. The possible metabolism of Mitonafide.
Fig. 4. Effect of compound 1a on the expression levels of relevant
proteins in HepG2 cells. Western blot analysis was performed with
antibodies against cyclin B1, cdc 2-pTy, Wee1, γH2AX and p21.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
the loading standard.
Fig. 2. In vivo anticancer activity of 1a in HepG2 xenograft model
.
(A) Effect of 1a (at the doses of 15 and 5 mg/kg), Mitonafide (at the
dose of 5 mg/kg), or vehicle (5% DMSO in saline, v/v) on tumor
growth. Tumor growth was monitored by the mean tumor volume
Fig. 5.Apoptosis ratio of the T-24 cells. (a) Negative control; (b)
treated with 2.06 μM 1a, (c) treated with 4.13 μM 1a, (d) treated
3
(
mm ) ± SD (n = 6) and calculated as the relative tumor increment
rate (T/C, %). (B) Photographs of the harvested tumors from the
1
/ 1

with 8.26 μM 1a. The cells were analyzed using the Annexin V/PI
assay in a flow cytometer.
Fig. 6. Effect of compound 1a on the expression levels of Bax, Bcl-2
and cytochrome c in the HepG2 cells. GAPDH was used as a loading
standard. Con: Control.
Fig. 7. Gel electrophoresis mobility shift assay of pBR322 DNA
treated with 1a (lane 0: DNA alone; lanes 1–6: DNA + 1a, with
increasing concentrations of 5, 10, 20, 40, 60, 80 and 100 μM,
respectively). R: linear form DNA, Sc: supercoiled form DNA, Nc:
circular form DNA.
Fig. 8. T-24 cells were cultured with compound 1a at 10 μM for 24
h. Cells were labeled by EdU incorporation (green).
Fig. 9. Gel electrophoresis assay of topoisomerase I treated with 1a
(lanes 1–6: Topo I + 1a, with increasing concentrations of 5, 10, 20,
4
0, 60, 80 and 100 μM, respectively).
1
/ 1