Oxo-heterocyclic fused naphthalimides as antitumor agents: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.12.039

Three series of novel oxo-heterocyclic fused naphthalimide derivatives (8a-8f, 13a-13d, 17a-17d) were prepared. The newly-synthesized compounds, and their thio-heterocyclic fused analogs (1a-1c, 2a-2d, 3a-3c) exhibited potent antiproliferative activity co

Full text of DOI:10.1016/j.ejmech.2012.12.039

European Journal of Medicinal Chemistry 62 (2013) 130e138
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Oxo-heterocyclic fused naphthalimides as antitumor agents:
Synthesis and biological evaluation
,
,
,
d
,
a
,
b
,
,b,c,d,
Shaoying Tan a d, Hong Yin b d, Zhuo Chen c , Xuhong Qian d, Yufang Xu a
*
*
a State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
b Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237, China
c Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai 200237, China
d School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of novel oxo-heterocyclic fused naphthalimide derivatives (8ae8f, 13ae13d, 17ae17d) were
prepared. The newly-synthesized compounds, and their thio-heterocyclic fused analogs (1ae1c, 2ae2d,
3ae3c) exhibited potent antiproliferative activity correlated well with their structure. Further research
demonstrated that all the representative compounds 13a, 2a and 17a, 3a showed strong inhibition ac-
tivity to topo II similarly with amonafide, and also potent topo I inhibition activity, which was seldom
reported before for naphthalimide derivatives. Preliminary exploration proved their DNA sequence
preference. In all, dual topo I/topo II inhibition and DNA sequence preference might contribute to
enhancing tumor selectivity and overcoming drug resistance.
Received 27 August 2012
Received in revised form
23 November 2012
Accepted 23 December 2012
Available online 3 January 2013
Keywords:
Oxo-heterocyclic
Naphthalimides
Antitumor agents
Topo I
Ó 2013 Elsevier Masson SAS. All rights reserved.
Topo II
DNA intercalation
1. Introduction
Netropsin, could bind to the AT-rich sequences in double stranded
DNA and showed potent selectivity to tumor cells.
In the past decade, DNA targeted antitumor agents remain the
cornerstone for cancer therapy despite the emerging of drugs with
different mechanisms of action. Great efforts have been made to
explore novel agents with better specificity and efficacy, as non-
specific cytotoxicity was the Achilles’ heel of conventional DNA-
interactive drugs [1,2]. The specificity could be acquired by tar-
geting protein-DNA complex. For example, the clinical effective
drugs, like Doxorubicin and Etoposide, could stabilize topoisom-
erase II-DNA cleavable complex. Irinotecan (Camptosar, CPT-11)
and Topotecan (Hycamtin), derivatives of the plant alkaloid, act
on topoisomerase I [3]. Another strategy was to explore sequence
selective DNA binding agents. For example, derivatives of
Naphthalimide derivatives, first designed by Braña et al. in 1973
[4], are a typical type of intercalators, which normally contain a flat
aromatic or heteroaromatic moiety and basic side chains. Amona-
fide [5] is one of the most widely studied naphthalimides, which
binds DNA by intercalation and poisons Topoisomerase II as
reported, and shows excellent activity in clinical phase II breast
cancer trials [6e9]. But it failed in clinical phase III trials because of
its unpredictable side effects on dose-limiting bone marrow tox-
icity due to its metabolization to N-acetyl-amonafide. Accordingly,
many derivatives were synthesized. Remers group put forward an
effective modified method by fusing rings on the naphthalene
moiety and obtained novel naphthalimides with the IC50 values up
to 7 nM against L1210 [10,11]. Braña group have reported sulfo-
nated, pyrazine, furan, thiophene-fused naphthalimides, which
could induce 10 folds more potent cytotoxicity comparing with
amonafide [12e14]. Besides, N-substitution naphthalimides with
thiourea, amide and urea were synthesized by Kiss group and
increased the maximum tolerated dose to 3e4 folds [5].
Abbreviations: CD, circular dichroism; CPT, camptothecin; ctDNA, calf thymus
DNA; kDNA, kinetoplast DNA; SAR, structureeactivity relationship.
* Corresponding authors. School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China. Tel.: þ86 21 64251399; fax: þ86 21
64252603.
Our group also made significant attempts in synthesizing
heterocyclic-fused naphthalimides to enhance cytotoxicity. For
E-mail addresses: chenzhuo@ecust.edu.cn (Z. Chen), yfxu@ecust.edu.cn (Y. Xu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.039

S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
131
Fig. 1. Naphthalimides derivatives reported in our group.
example, linear and angular thiazonaphthalimides (Fig. 1 1ae1h)
were reported by Li et al. with IC50 values up to 0.8 nM against
A549 [15e17]. Xie et al. synthesized N-substitution amonafide de-
rivatives (Fig. 11ie1o), which showed improved antitumor activity
over amonafide and were supposed to avoid the side effects of
amonafide [18]. Besides, the thio-heterocyclic fused naph-
thalimides (Fig. 1 2ae2d, 3ae3d) were reported as high efficient
DNA photocleaver [19,20], and have not yet been characterized as
cytotoxic agents except for 3a.
compounds 13ae13d were obtained from corresponding aminos
condensed with 12.
A similar synthesis strategy was implicated for 17ae17d as
shown in Scheme 3 [23], only that the synthesis started from 4-
bromo-3-nitro-naphathalimide. The critical intermediate 16 was
mixed with corresponding aminos to give the target compounds
C1eC4.
3. Results and discussion
Recently, amonafide reentered phase III clinical trial against
secondary acute myeloid leukemia, which encouraged us to
develop naphthalimide derivatives as potential antitumor drugs.
Previously reported naphthalimide derivatives of our group
were normally thio-heterocyclic fused naphthalimides. In this
paper, oxo-heterocyclic fused naphthalimides (Fig. 2) were
synthesized for establishing better structureeactivity relation-
ship (SAR). These novel-synthesized compounds, as well as part
of their thio-heterocyclic fused analogs (1ae1c, 2ae2d, 3ae3d)
were evaluated of their antiproliferative activity from DNA
intercalation, inhibition of topo I and II in comparison with
amonafide.
3.1. Antitumor activity in vitro
The antitumor activities of oxo- and thio-heterocyclic fused
naphthalimides were evaluated in vitro against tumor cell lines
A549 (human lung cancer cell), P388 (murine leukemia cell) and
2. Chemistry
As shown in Scheme 1, the critical intermediates 7ae7f were
prepared as we have reported previously [21]. They were synthe-
sized from 4-bromo-3-nitro-naphathalimide 4 via substitution,
reduction as well as cyclization in the existence of PPA. The target
compounds 8ae8f were then obtained by 7ae7f condensed with
N0,N0-dimethylethane-1,2-diamine.
As for 13ae13d, the critical intermediate 12 were synthesized
from 4-bromo-naphathalimide as reported (see Scheme 2) [22,23].
Compound 12 was obtained by 4-bromo-naphathalimide 10
substituted with o-amino phenol, reduced with Fe/acetic acid, and
then cyclized by Diazo reaction and Pschorr reaction. The target
Fig. 2. CD spectra of ctDNA (100 mM) incubated with target compounds (20 mM).

132
S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
Scheme 1. Syntheses of target compounds 8ae8f. (a) NaOH, 80 ꢀC, 8 h; (b) SnCl2, HCl, 80 ꢀC 2 h (c) substituted carboxylic acids, PPA, 120 ꢀC, 5 h; (d) N0,N0-dimethylethane-1,2-
diamine, ethanol, reflux, 3 h.
Scheme 2. Syntheses of target compounds 13ae13d. (a) o-nitrophenol, NaOH, DMF, Cu, reflux 1 h; (b) Fe, acetic acid, reflux, 1 h. (c) HCl, acetic acid, NaNO2 0e5 ꢀC, CuSO4, HAc, H2O.
(d) R2NH2, ethanol, reflux 3 h.
normal cell lines LO2 (human liver cell), respectively, as listed in
Tables 1 and 2.
for their sulfur-containing analogs (2ae2d and 3ae3d), which
normally induced enhanced cytotoxicity. Compounds 2a and 3a
exhibited significant antiproliferative activity with IC50 values at
Among 8ae8f, it was found from Table 1 that compounds 8a, 8c
and 8f exhibited potent antiproliferative activities, which was
comparable with amonafide. Compounds with electron-donating
groups provided more antitumor efficacy, such as 8c vs 8e. Be-
sides, substitution on the ortho-position induced better cytotox-
icity than the substitution on the para-position, for example, 8f was
7.6 folds more potent than 8c against A549. Similar SAR were
observed for their sulfur-containing analogs, except that they are
more potent antitumor agents, e.g. 1c vs 8c.
As for 13ae13d and 17ae17d, the antiproliferative potency
varied from the substitution of the condensed aminos. N0,N0-
dimethylethane-1,2-diamine derivatives 13a, 17a were generally
more potent than their analogs, which was in consistence with
previous reported naphthalimide-derived DNA intercalators.
Extension of linker or increasing rigidity of amino group would
normally reduce the cytotoxicity. For example, 13b and 13d
exhibited much decreased antiproliferative activity against A549
0.14 and 0.007 mM for A549 respectively. Besides, compounds 2b
and 3b also possessed efficient antitumor activities. Moreover, 2c
and 3c induced potent antiproliferative activates against both A549
and P388.
Considering the shortcoming of cytotoxic compounds, the
newly-synthesed compounds (8ae8f, 13ae13d, 17ae17d) and
compounds (2a, 3a) were selected to evaluate their cytotoxicity
against the normal cell line LO2 (Table 1). Less cytotoxicity against
the normal cell line in contrast to tumor cell lines, or certain tumor
specificity was observed.
3.2. DNA intercalating studies by circular dichroism (CD) spectra
As 13a, 2a, 17a, 3a exhibited potent cytotoxicity and moderate
specificity in antiproliferative assay, their mechanism of action was
investigated. Firstly, the CD spectroscopy was applied for the
interaction with DNA, as typical intercalating agents could induce
calf thymus DNA’s (ctDNA) conformation changes [24]. As shown in
Fig. 2, amonafide could induce significant changes in a negative
peak around 245 nm caused by the helical B conformation and in
a positive peak around 275 nm due to base stacking, which was
consistent with literature. All the representative compounds could
with IC50 values at 98.26 mM and 28.95 mM, respectively. Besides, it
was quite interesting to observe compounds 13c and 17c without
amino side chain exhibited potent antiproliferative activities
against A549. Since they were not typical DNA intercalators in
structure, other mechanism of action was probably involved, which
would not be discussed in this article. Similar SAR were observed
Scheme 3. Syntheses of target compounds 17ae17d. (a) pyridine, phenol, NaH, Cu, reflux 5 h; (b) SnCl2, HCl, 80 ꢀC 2 h; (c) HCl, acetic acid, NaNO2 0e5 ꢀC, CuSO4, HAc, H2O (d)
R2NH2, ethanol, reflux 3 h.

S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
133
Table 1
Table 2
SAR of oxo-heterocyclic fused naphthalimides and their sulfur-containing
analogues.
Association constants (Ka’s, Mꢂ1) of 13a, 2a and 17a, 3a with polydeoxynucleotides.a,b
DNA/compds
13a
2a
17a
3a
Poly (dAdT)$Poly
(dAdT)
Poly (dGdC)$Poly
(dGdC)
1.32 ꢁ 105
4.48 ꢁ 105
5 ꢁ 106
8 ꢁ 106
6.89 ꢁ 105
4.76 ꢁ 106
6.36 ꢁ 106
7.6 ꢁ 107
a
A measurement by fluorospectrometric methods in 20 mM sodium phosphate
buffer (pH 7.0) at room temperature (20 ꢃ 1 ꢀC). The concentrations of compounds
and polydeoxynucleotides were 3 ꢁ 10ꢂ6 and 0e6.32 ꢁ 10ꢂ5 M, respectively.
b
These values were derived from the experimental data by nonlinear curve fit-
ting methods using Microcal Origin software (version 8.0).
3.3. Fluorescent spectra studies
Compounds
R¼
X
Cytotoxicity (IC50,
m
M)
LO2a
For further research for the interaction of the representative
compounds with DNA, the association steady constants (Kb) were
measured according to the quenching of fluorescence following the
increasing concentrations of ctDNA. Fig. 3 showed the fluorescence
of the compound before and after the addition of ctDNA (2a as an
example), which exhibited that the fluorescence intensity of 2a
quenched rapidly with the addition of ctDNA [25,26]. From the
analyses on the relationship between the fluorescence intensities
and the DNA concentrations by nonlinear curve fitting methods, the
association steady constants of the compounds (13a, 2a, 17a, 3a)
were available at 1.45 ꢁ 105, 9.70 ꢁ 104, 2.89 ꢁ 105 and
A549a
P388b
8a
8b
8c
8d
8e
8f
1a
1b
1c
13a
13b
13c
R1 ¼ H
O
O
O
O
O
O
S
S
S
O
O
O
0.53
2.50
3.0
6.7
3.4
R1 ¼ p-Me
34.70
6.60
19.30
1.29
R1 ¼ p-OMe
R1 ¼ p-Cl
72.40
17.80
0.89
20.10
100.00
1.30
>100
>100
1.9
R1 ¼ p-NO2
R1 ¼ o-OMe
R1 ¼ H
0.085
0.42
0.66
0.62
N.d.c
N.d.
N.d.
4.1
R1 ¼ p-Me
R1 ¼ p-OMe
R2 ¼ CH2CH2N(CH3)2
R2 ¼ CH2CH2CH2N(CH3)2
R2 ¼ CH2CH2CH2CH3
0.016
0.61
0.43
1.11
98.26
5.39
3.50
>100
10.3
>100
1.10 ꢁ 105
M
ꢂ1, respectively (Figure S1). It indicated that the thio-
heterocyclic analogs exhibited enhanced ability of DNA-
interaction (e.g. 13a vs 2a), which is consistent with cytotoxicity
and CD spectroscopy.
13d
O
28.95
4.52
11.5
2a
2b
2c
R2 ¼ CH2CH2N(CH3)2
R2 ¼ CH2CH2CH2N(CH3)2
R2 ¼ CH2CH2CH2CH3
S
S
S
0.14
0.19
0.87
1.58
0.63
1.20
4.1
N.d.
N.d.
3.4. Topo II inhibition assay
Topo II, the important target enzyme of antitumor chemo-
therapy, is related with mitotic chromosome pairing, chromosome
segregation, gene recombination and transcription, DNA manipu-
lation and restoration etc [27]. Amonafide was an important DNA
intercalator and could inhibit topo II by forming and stabilizing
a ternary drug-DNA-topoisomerase complex [28]. The kinetoplast
DNA (kDNA) assay was introduced based on specific decatenation
of kDNA by topo II. As shown in Fig. 4, all the representative com-
pounds decrease the amount of released decatenated kDNA mini-
circles, and thus were capable of inhibiting topo II activity.
Compounds 13a, 17a and 3a exhibited similar topo II inhibition
efficacy as amonafide at the tested concentration. Besides, all the
naphthalimides exhibited stronger activity than Etoposide (VP-16),
a famous topo II inhibitor.
2d
S
100.00
>100
N.d.
17a
17b
17c
R3 ¼ CH2CH2N(CH3)2
R3 ¼ CH2CH2CH2N(CH3)2
R3 ¼ CH2CH2CH2CH3
O
O
O
0.36
0.74
1.40
0.65
0.17
>100
5.0
6.6
>100
17d
O
>100
0.58
11.9
3a
3b
3c
R3 ¼ CH2CH2N(CH3)2
R3 ¼ CH2CH2CH2N(CH3)2
R3 ¼ CH2CH2CH2CH3
S
S
S
0.007
0.25
1.35
0.26
0.32
0.74
0.9
N.d.
N.d.
3d
S
11.75
1.10
72.44
0.20
N.d.
5.0
3.5. Topo I inhibition assay
Amonafide
a
Cytotoxicity (CTX) against human lung cancer cell (A549) and human liver cell
(LO2) was measured by sulforhodamine B dye-staining method.
Topo I, another member of topoisomerase family, was associated
with the process of DNA replication, transcription and recombinant
etc and caused a short DNA single-strand breaks via the formation
of intermediates in the catalytic process [3]. The research of topo I
inhibitory activity was implicated in conventional chemotherapy
agents. As shown in Fig. 5, the topo I inhibitor camptothecin (CPT)
could reduce topo I-induced pBR322 relaxation [29]. It is quite
exciting that all the naphthalimides including amonafide, exhibited
stronger inhibitory activity to topo I than camptothecin (CPT). Be-
sides, compound 2a was relatively weaker than its analogs.
b
CTX against murine leukemia cells (P388) was measured by microculture
tetrazolium-formazan method.
c
Not determined.
induce the conformational changes, which supported their inter-
calating capacity, but were much weaker than amonafide. In detail,
the thio-heterocyclic fused naphthalimides (2a, 3a) showed
stronger changes in the corresponding position than the oxo-
heterocyclic fused naphthalimides (13a, 17a). Thus, the sulfur-
containing derivatives were more potent DNA intercalators than
the oxygen-containing analogs, which was in consistence with
their cytotoxic superiority.
Existing topoisomerase-targeting clinical drugs, such as camp-
tothecin and podophyllotoxin derivatives, formed topo-DNA com-
plexes with either topo I or topo II. These drugs have a fatal flaw in
drug resistance which limited their clinical use. As reported, the

134
S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
Fig. 3. Fluorescence spectra of 2a (10
mM) with ctDNA. The concentrations of ctDNA were increased from 0 to 100
m
M in 20 mM triseHCl (pH 7.5) at 25 ꢀC. The insets indicated the
relationship between the fluorescent intensity and the concentration of ctDNA (
mM).
association constants of 2a and 3a with poly[dG-dC]$poly[dG-dC]
were 10 folds higher than poly[dA-dT]$poly[dA-dT]. The GC
sequence preference was also observed for 13a and 17a though not
so potent as their sulfur-containing analogs.
Preliminary exploration was carried out to provide further
insight into the precise sequence selectivity [32,33]. The association
constants of 13a and 2a with seven designed double-strand oligo-
deoxynucleotides were measured (Table S1). Compound 2a
exhibited 4e111 folds selectivity against the sequence 50-TGCGCA-
30/30-ACGCGT-50. Researches on selectivity for core sequences
associated with tumor progression are still going on.
4. Conclusions
Fig. 4. Inhibition of topo II-mediated kDNA decatenation by target compounds
(100 M). Lane 1, kDNA; lane 2, minicircles (no drug); lanes 3e8, compounds 13a, 2a,
17a, 3a, amonafide, and Etoposide (VP-16), respectively.
m
In this article, three series of novel oxo-heterocyclic fused
naphthalimides were synthesized and evaluated of their antitumor
potency in comparison with their thio-heterocyclic analogs. The
newly-synthesized compounds exhibited more potent anti-
proliferative activity than amonafide, though they were relatively
less potent than their sulfur-containing analogs. Representative
compounds 13a, 2a, 17a and 3a were then investigated of their
antitumor mechanism of action. These compounds acted as DNA
intercalators, but were much weaker than amonafide according to
the results of CD. The sulfur-containing analogs exhibited better
DNA affinity which was consistent with their antiproliferative po-
tency. Furthermore, the tested compounds could induce significant
inhibition activity to both topo II and topo I in cell-free system. The
tested compounds also exhibited GC sequence preference, and
certain sequence selectivity against designed double-strand oligo-
deoxynucleotides. In all, dual topo I/topo II inhibition and DNA
sequence preference might contribute to enhancing tumor selec-
tivity and overcoming drug resistance.
resistance could be abated in case of inhibiting both topo I and topo
II activity [30]. Thus, it is probably that naphthalimides have ad-
vantages in overcoming drug resistance.
3.6. DNA sequence preference
According to previous report, amonafide exhibited GC sequence
preference, which would contribute to its tumor selectivity [31].
Accordingly, we investigated the sequence preference of oxo-
heterocyclic fused compounds and their thio analogs. The binding
affinities of compounds 13a, 2a, 17a and 3a with poly[dA-dT]$poly
[dA-dT], poly[dG-dC]$poly[dG-dC] were listed in Table 2. The
5. Experimental section
All the solvents were of analytic grade. ctDNA and poly-
deoxynucleotides (poly[dG-dC]$poly[dG-dC] and poly[dA-dT]$poly
[dA-dT]) were products of Amersham Biosciences and Sigma
Chemicals Co., respectively. Calf thymus DNA, poly-
deoxynucleotides and oligodeoxynucleotides were used without
further purification. 1H NMR and 13C NMR were measured on
a Bruker AV-400 spectrometer with chemical shifts reported as
parts per million (in CD3OD/DMSO-d6/CDCl3, TMS as an internal
standard). Mass spectra were measured on a HP 1100 LC-MS
spectrometer. Melting points were determined by an X-6 micro-
melting point apparatus and uncorrected. Absorption spectra
Fig. 5. Inhibition of topo I-mediated DNA std (pBR322) decatenation by target com-
pounds (100 mM except for CPT 200 mM). Lane 1, DNA std; lane 2, topo I (no drug);
lanes 3e8, compounds 13a, 2a, 17a, 3a, camptothecin (CPT) and amonafide,
respectively.

S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
135
were determined on PGENERAL TU-1901 UVevis Spectropho-
tometer. Milli-Q water (Millipore Co.) was used throughout the
whole experiments.
5.1.5.2. N-(N0,N0-dimethylamine-ethyl)-9-(40-methyl-phenyl)-oxa-
zole[4,5-c] naphthalimide (8b). Pale yellow solid, yield: 84%, m.p.
230e231 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 9.00 (s, 1H), 8.66 (t,
J ¼ 8.8 Hz, 2H), 8.25 (d, J ¼ 7.6 Hz, 2H), 7.92 (t, J ¼ 8.0 Hz,1H), 7.41 (d,
J ¼ 7.6 Hz, 2H), 4.42 (t, J ¼ 5.6 Hz, 2H), 2.82 (t, J ¼ 5.6 Hz, 1H), 2.51 (s,
3H), 2.48 (s, 6H). 13C NMR (100 MHz, CDCl3) (ppm): 164.5, 164.2,
163.9, 150.0, 142.9, 139.6, 130.5, 129.9, 127.8, 127.6, 126.6, 126.4,
125.1, 123.6, 123.4, 120.1, 118.4, 56.7, 45.4, 37.9, 21.7. IR (KBr): 3287,
2820, 2774, 2369, 1660, 1330, 773 cmꢂ1. HR-MS: C24H21N3O3 cal-
culated: 399.1583, found: 399.1564.
5.1. Synthesis
5.1.1. 4-Bromo-3-nitro-1,8-naphthalic anhydride (4) [34]
Sodium nitrate (2.6 g, 0.03 mol) was added to 4-bromo-1,8-
naphthalic anhydride (7.0 g, 0.025 mol) in concentrated sulfuric
acid (37.5 mL) within 1 h. Stirred for 2 h in room temperature, the
solution was poured into water (500 mL) and the precipitate was
filtered, washed with water and recrystallized from acetic acid to
obtain compound 4. Yellow solid, yield: 79%; m.p. 233e235 ꢀC (m.p.
233e234 ꢀC in literature). 1H NMR (400 MHz, DMSO-d6) (ppm):
8.90 (s, 1H), 8.80 (d, J ¼ 8.4 Hz, 1H), 8.73 (d, J ¼ 7.2 Hz, 1H), 8.17 (t,
J ¼ 7.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) (ppm): 160.1, 159.5,
149.8, 135.7, 135.4, 131.4, 130.9, 126.0, 122.3, 121.4, 120.8.
5.1.5.3. N-(N0,N0-dimethylamine-ethyl)-9-(40-methoxy-phenyl)-oxa-
zole[4,5-c] naphthalimide (8c). Pale yellow solid, yield: 86%, m.p.
213e214 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 8.96 (s, 1H), 8.65 (d,
J ¼ 7.6 Hz,1H), 8.61 (d, J ¼ 8.0 Hz,1H), 8.27 (d, J ¼ 8.0 Hz, 2H), 7.90 (t,
J ¼ 7.6 Hz,1H), 7.08 (d, J ¼ 8.4 Hz, 2H), 4.39 (t, J ¼ 6.8 Hz, 2H), 3.95 (s,
3H), 2.74 (t, J ¼ 6.8 Hz, 2H), 2.42 (s, 6H). 13C NMR (100 MHz, CDCl3)
(ppm): 164.4, 164.2, 163.9, 162.8, 149.9, 139.7, 130.3, 129.6, 127.5,
126.4, 126.3, 124.9, 123.4, 120.0, 118.7, 118.3, 114.6, 56.9, 55.6, 45.7,
38.3. IR (KBr): 2950, 2771, 2349, 1690, 1655 cmꢂ1. HR-MS:
C24H21N3O4 calculated: 415.1532, found: 415.1538.
5.1.2. 4-Hydroxyl-3-nitro-1,8-naphthalic anhydride (5) [34]
4 (32.2 g, 0.1 mol) was stirred in 20% aqueous sodium hydroxide
(200 mL) for 8 h at 80e85 ꢀC. The dark red solution was cooled and
poured into ice-cold 10% hydrochloric acid (500 mL), stirred and
filtered to obtain pale yellow residue. The crude product was
washed and recrystallized from glacial acetic acid to give com-
pound 5. Pale yellow needle, yield: 85%; m.p. 289e290 ꢀC (m.p.
288e289 ꢀC in literature). 1H NMR (400 MHz, DMSO-d6) (ppm):
8.74 (s, 1H), 8.65 (d, J ¼ 8.0 Hz, 1H), 8.43 (d, J ¼ 7.2 Hz, 1H), 7.71 (t,
J ¼ 7.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) (ppm): 162.4, 161.4,
160.0, 134.7, 133.7, 132.8, 132.5, 131.2, 128.2, 126.9, 119.0, 103.5.
5.1.5.4. N-(N0,N0-dimethylamine-ethyl)-9-(40-chloro-phenyl)-oxazole
[4,5-c] naphthalimide (8d). Pale yellow solid, yield: 85%, m.p. 191e
192 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 9.03 (s, 1H), 8.70 (d,
J ¼ 7.2 Hz,1H), 8.66 (d, J ¼ 8.0 Hz,1H), 8.32 (d, J ¼ 8.4 Hz, 2H), 7.94 (t,
J ¼ 8.0 Hz,1H), 7.60 (d, J ¼ 8.4 Hz, 2H), 4.42 (t, J ¼ 5.6 Hz, 2H), 2.78 (t,
J ¼ 5.6 Hz, 2H), 2.45 (s, 6H). 13C NMR (100 MHz, CDCl3) (ppm): 164.1,
163.8, 163.3, 150.1, 139.5, 138.6, 130.7, 129.6, 129.0, 127.8, 126.8,
126.4, 125.1, 124.8, 123.5, 120.5, 118.4, 56.9, 45.6, 38.2. IR (KBr):
3081, 2931, 2758, 1703, 1657, 1341, 780 cmꢂ1. HR-MS: C23H18N3O3Cl
calculated: 421.1007, found: 421.0974.
5.1.3. 4-Hydroxyl-3-amino-1,8-naphthalic anhydride (6) [34]
A mixture of 5 (15 g, 0.058 mol), tin (II) chloride (50 g, 0.26 mol)
and concentrated hydrochloric acid (80 mL) was stirred for 2 h at
80 ꢀC. The suspension was cooled and filtered. The residue was
washed with 5% hydrochloric acid and water, dried and recrystal-
lized from pyridine. Orangeeyellow needles, yield: 66%; m.p.
>340 ꢀC (m.p. >340 ꢀC in literature). 1H NMR (400 MHz, DMSO-d6)
(ppm): 8.50 (d, J ¼ 8.4 Hz, 1H), 8.21 (d, J ¼ 6.8 Hz, 1H), 8.07 (s, 1H),
7.63 (t, J ¼ 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) (ppm): 162.4,
160.9, 129.8, 129.6, 125.5, 124.9, 124.4, 118.6.
5.1.5.5. N-(N0,N0-dimethylamine-ethyl)-9-(40-nitro-phenyl)-oxazole
[4,5-c] naphthalimide (8e). Pale yellow solid, yield: 94%, m.p. 205e
206 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 9.06 (s, 1H), 8.73 (d,
J ¼ 7.2 Hz, 1H), 8.70 (d, J ¼ 8.0 Hz, 1H), 8.58 (d, J ¼ 8.4 Hz, 2H), 8.48
(d, J ¼ 8.0 Hz, 2H), 7.98 (t, J ¼ 8.0 Hz,1H), 4.40 (t, J ¼ 5.6 Hz, 2H), 2.75
(t, J ¼ 5.6 Hz, 2H), 2.42 (s, 6H). 13C NMR (100 MHz, CDCl3) (ppm):
164.0, 163.6, 161.8, 150.4, 149.8, 139.4, 131.9, 131.1, 128.1, 127.2, 126.4,
125.3, 124.5, 123.6, 121.0, 118.4, 57.0, 45.7, 38.4. IR (KBr): 3065, 2945,
5.1.4. General procedure for the preparation of 7ae7f [34]
The PPA (35 mL), 6 (0.8 g, 0.0035 mol) and corresponding
benzoic acid (0.011 mol) was mixed and stirred at 135 ꢀC. After 4 h,
the solution was cooled and poured into the ice water (350 mL), the
precipitate was filtered, washed with water and dried for direct use
in the next step. Yellow solid, yield: 60.9e68.7%.
2777, 1696, 1334, 780 cmꢂ1
428.1277, found: 428.1277.
. HR-MS: C23H18N4O5 calculated:
5.1.5.6. N-(N0,N0-dimethylamine-ethyl)-9-(20-methoxy-phenyl)-oxa-
zole[4,5-c] naphthalimide (8f). Pale yellow solid, yield: 90%, m.p.
213e214 ꢀC. 1H NMR (400 MHz, CDCl3) (ppm): 9.09 (s, 1H), 8.68 (t,
J ¼ 7.6 Hz, 2H), 8.28 (d, J ¼ 8.0 Hz, 1H), 7.92 (t, J ¼ 8.0 Hz, 1H), 7.61 (t,
J ¼ 7.6 Hz,1H), 7.24e7.13 (m, 2H), 4.44 (t, J ¼ 5.6 Hz, 2H), 4.11 (s, 3H),
2.85 (t, J ¼ 5.6 Hz, 2H), 2.50 (s, 6H). 13C NMR (100 MHz, CDCl3)
(ppm): 164.3, 164.0, 162.8, 158.7, 149.8, 139.6, 133.5, 131.3, 130.5,
127.5, 126.7, 125.5, 123.3, 120.9, 119.9, 118.3, 115.3, 112.3, 56.7, 56.3,
45.4, 37.8. IR (KBr): 2953, 2774, 2357, 1699, 1657, 773 cmꢂ1. HR-MS:
C24H21N3O4 calculated: 415.1532, found: 415.1548.
5.1.5. General procedure for the preparation of 8ae8f
The corresponding intermediates 7ae7f (0.001 mol) were dis-
solved in 20 mL ethanol. After adding N,N-dimethylethylenedi-
amine (0.163 mL, 0.0015 mol), the mixture were stirred and
refluxed for 2e3 h, then the solution was evaporated in vacuum and
the residue was purified on silica gel chromatography.
5.1.5.1. N-(N0,N0-dimethylamine-ethyl)-9-phenyl-oxazole[4,5-c]
naphthalimide (8a). Pale yellow solid, yield: 92%, m.p. 172e173 ꢀC.
1H NMR (400 MHz, CDCl3) (ppm): 9.01 (s, 1H), 8.69e8.64 (m, 2H),
8.36 (d, J ¼ 5.6 Hz, 2H), 7.92 (t, J ¼ 8.0 Hz, 1H), 7.62 (d, J ¼ 5.6 Hz,
2H), 7.29 (s, 1H), 4.40 (t, J ¼ 7.2 Hz, 2H), 2.76 (t, J ¼ 6.8 Hz, 2H), 2.43
(s, 6H). 13C NMR (100 MHz, CDCl3) (ppm): 164.2, 164.1, 163.8, 150.1,
139.5, 132.2, 130.6, 129.2, 127.8, 127.7, 126.7, 126.4, 126.3, 125.1,
123.4, 120.3, 118.4, 56.9, 45.7, 38.2. IR (KBr): 2755, 2344, 1700, 1650,
1338, 773 cmꢂ1. HR-MS: C23H19N3O3 calculated: 385.1426, found:
385.1441.
5.1.6. 4-(2-Nitrophenoxy)-1,8-naphthalic anhydride (10) [35]
A
mixture of 4-bromonaphthalic anhydride 9 (1.02 g,
0.0037 mol), o-nitrophenol (0.34 g, 0.0024 mol), sodium hydroxide
(0.025 g) and copper powder (0.04 g) was mixed and refluxed in
DMF (45 mL) for 1 h. The solution was cooled and hydrochloric acid
(7.5 mL 20%) was added. Then the precipitate was filtered and
recrystallized from acetic acid. Pale yellow solid, yield: 82%, m.p.
268e269 ꢀC (m.p. 266e268 ꢀC in literature). 1H NMR (400 MHz,
DMSO-d6) (ppm): 8.79 (d, J ¼ 8.4 Hz, 1H), 8.63 (d, J ¼ 7.2 Hz, 1H),
8.45 (d, J ¼ 8.4 Hz, 1H), 8.29 (d, J ¼ 8.4 Hz, 1H), 8.00 (t, J ¼ 8.0 Hz,

136
S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
1H), 7.95 (t, J ¼ 7.6 Hz,1H), 7.67 (d, J ¼ 4.8 Hz, 2H), 7.07 (d, J ¼ 8.0 Hz,
1H). 13C NMR (100 MHz, DMSO-d6) (ppm): 161.3, 160.5, 159.3, 146.9,
142.2, 136.6, 134.9, 133.8, 131.9, 129.7, 128.3, 127.8, 127.0, 124.9,
123.4, 119.8, 114.2, 111.1.
129.9, 125.0, 123.7, 119.1, 118.8, 118.1, 114.8, 109.8, 40.2, 30.2, 20.5,
13.9. IR (KBr): 2947, 2867, 1855, 1386, 759 cmꢂ1; HR-MS: C22H17NO3
calculated: 343.1208, found: 343.1206.
5.1.9.4. N-(20-piperazidine-ethyl)-benzo[k,l]xanthene-3,4-
naphthalimide (13d). Yellow solid, yield: 82%, m.p. 198e200 ꢀC. 1H
NMR (400 MHz, CDCl3eCD3OD) (ppm): 8.58 (d, J ¼ 8.0 Hz, 1H), 8.53
(d, J ¼ 8.4 Hz,1H), 8.08 (d, J ¼ 7.6 Hz,1H), 7.96 (d, J ¼ 7.6 Hz,1H), 7.54
(t, J ¼ 7.2 Hz, 1H), 7.37 (brs, 2H), 7.28 (t, J ¼ 7.6 Hz, 1H), 4.31 (t,
J ¼ 6.8 Hz, 2H), 3.93e3.87 (m, 2H), 3.01 (brs, 4H), 2.74 (brs, 4H). 13C
NMR (100 MHz, CDCl3eCD3OD) (ppm): 164.2, 163.9, 156.1, 151.9,
134.0, 133.9, 133.4, 132.3, 130.0, 125.2, 123.8, 118.9, 118.7, 118.1, 115.0,
110.0, 55.6, 51.5, 44.3, 37.0. IR (KBr): 2921, 2855, 1688, 1660, 1590,
5.1.7. 4-(2-Aminophenoxy)-1,8-naphthalic anhydride (11) [35]
A mixture of compound 10 (0.25 g, 0.0008 mol) and iron powder
(0.12 g, 0.002 mol) was refluxed in glacial acetic acid (10 mL) for 1 h.
The brown solution was cooled and water (30 mL) was added. The
precipitate was filtered and washed with water. Yellow solid, yield:
94%, m.p. 172e174 ꢀC (m.p. 171e172 ꢀC in literature). 1H NMR
(400 MHz, DMSO-d6) (ppm): 8.89 (d, J ¼ 8.4 Hz, 1H), 8.60 (d,
J ¼ 5.2 Hz, 1H), 8.46 (d, J ¼ 8.0 Hz, 1H), 7.96 (t, J ¼ 7.2 Hz, 1H), 7.10 (t,
J ¼ 7.6 Hz, 1H), 7.05 (d, J ¼ 7.6 Hz, 1H), 6.93 (d, J ¼ 8.0 Hz, 1H), 6.84
(d, J ¼ 8.4 Hz, 1H), 6.68 (t, J ¼ 7.6 Hz, 1H), 5.19 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) (ppm): 161.6, 160.7, 160.6, 141.4, 139.6, 135.6,
133.7, 132.0, 130.8, 127.4, 127.4, 123.6, 122.2, 119.2, 117.1, 117.0, 112.0,
109.6.
765 cmꢂ1
399.1576.
. HR-MS: C24H21N3O3 calculated: 399.1583, found:
5.1.10. 4-Phenoxy-3-nitro-dicarboxylic anhydride (14) [23]
A mixture of 4 (9.8 g, 0.030 mol), phenol (1.8 mL, 0.0175 mol)
and sodium hydride (1.3 g, 0.054 mol) was refluxed in pyridine
(220 mL) for 5 h. The solution was concentrated, filtered, and
washed with water. Pale yellow solid, yield: 97%, m.p. 289.0e
289.9 ꢀC (m.p. 287 ꢀC in literature). 1H NMR (400 MHz, DMSO-d6)
(ppm): 8.99 (s, 1H), 8.73 (d, J ¼ 7.2 Hz, 1H), 8.52 (d, J ¼ 8.4 Hz, 1H),
8.04 (t, J ¼ 8.0 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 2H), 7.17 (t, J ¼ 7.2 Hz, 1H),
6.99 (d, J ¼ 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) (ppm): 160.3,
159.5, 158.4, 149.6, 140.7, 135.7, 133.1, 131.4, 130.8, 130.3, 128.0,
126.9, 124.3, 120.9, 118.1, 116.0.
5.1.8. Benzo[k,l]xanthene-3,4-dicarboxylic anhydride (12) [35]
11 (0.5 g, 0.0017 mol) was dissolved in glacial acetic acid (12 mL)
and then hydrochloric acid (1 mL) and sodium nitrite solution
(1.14 g, 0.016 mol in 4 mL water) were added at 0 ꢀC. After 1 h
copper sulfate solution (1.12 g, 0.007 mol in 20 mL water) was
added. The reaction mixture was refluxed further for 0.5 h and
cooled. The precipitate was filtered, washed with water and crys-
tallized from DMF. Yellow solid, yield: 93.2%. M.p. 156e159 ꢀC (m.p.
155e160 ꢀC in literature).
5.1.11. 4-Phenoxy-3-amino-dicarboxylic anhydride (15) [23]
14 (5.24 g, 0.016 mol) was added to SnCl2$2H2O (29 g, 0.129 mol)
in hydrochloric acid (36%,100 mL). The reaction mixture was stirred
at 85 ꢀC for 2 h. Then the reaction was cooled and filtered. The
residue was washed with water and recrystallized from pyridine.
Yellow solid, yield: 79.7%, m.p. 261.2e262.3 ꢀC (m.p. 258e259 ꢀC in
literature). 1H NMR (400 MHz, DMSO-d6) (ppm): 8.25 (s, 1H), 8.17
(d, J ¼ 6.8 Hz, 1H), 7.94 (d, J ¼ 8.0 Hz, 1H), 7.68 (t, J ¼ 7.6 Hz, 1H), 7.33
(t, J ¼ 7.2 Hz, 2H), 7.07 (t, J ¼ 6.0 Hz, 1H), 6.87 (d, J ¼ 7.6 Hz, 2H), 6.02
(s, 2H). 13C NMR (100 MHz, DMSO-d6) (ppm): 161.4, 160.8, 157.6,
141.0, 136.5, 130.4, 128.3, 128.0, 127.5, 127.1, 125.1, 124.3, 123.0, 119.7,
117.0, 115.6.
5.1.9. General procedure for the preparation of 13ae13d
12 (0.29 g, 0.001 mol) was dissolved in 20 mL ethanol. After
adding corresponding primary amine (0.003 mol), the mixture was
stirred and refluxed for 2e3 h, then the solution was evaporated in
vacuum and the residue was purified on silica gel chromatography.
5.1.9.1. N-(N0,N0-dimethylamine-ethyl)-benzo[k,l]xanthene-3,4-
naphthalimide (13a). Yellow solid, yield: 85%, m.p. 191e192 ꢀC. 1H
NMR (400 MHz, CDCl3) (ppm): 8.60 (d, J ¼ 8.0 Hz, 1H), 8.56 (d,
J ¼ 8.0 Hz,1H), 8.04 (d, J ¼ 8.0 Hz,1H), 7.89 (d, J ¼ 7.6 Hz,1H), 7.54 (t,
J ¼ 7.6 Hz,1H), 7.36 (d, J ¼ 8.0 Hz, 2H), 7.26 (d, J ¼ 8.4 Hz,1H), 4.37 (t,
J ¼ 7.2 Hz, 2H), 2.74 (t, J ¼ 7.2 Hz, 2H), 2.44 (s, 6H). 13C NMR
(100 MHz, CDCl3) (ppm): 164.0, 163.7, 155.8, 151.9, 133.8, 133.4,
133.2, 132.1, 130.0, 125.0, 123.7, 119.9, 118.8, 118.1, 114.9, 114.8, 109.9,
5.1.12. Benzo[b]furano[2,1-c] dicarboxylic anhydride (16) [23]
The solution of NaNO2 (0.545 g, 0.008 mol) in concentrated
sulfuric acid (32.7 mL) was added dropwise with glacial acetic acid
(4.87 mL) at ꢂ5 to 0 ꢀC and then added with 15 (2 g, 0.007 mol) in
batches. After 2 h, the solution was added dropwise to the mixed
solution (74 g copper sulfate, 100 mL glacial acetic acid, 800 mL
water) and then continued to reflux for 0.5 h. The resulting sus-
pension was cooled, filtered and washed with water. The solid was
purified on silica gel chromatography (petroleum ether/ethyl ace-
tate, 5:1). Yellow solid, yield: 61%, m.p. 147e149 ꢀC (m.p. 145e
146 ꢀC in literature). 1H NMR (400 MHz, DMSO-d6) (ppm): 9.43 (s,
1H), 8.93 (d, J ¼ 8.4 Hz, 1H), 8.63 (d, J ¼ 7.2 Hz, 1H), 8.52 (d,
J ¼ 7.6 Hz, 1H), 8.09 (t, J ¼ 8.0 Hz, 1H), 7.99 (d, J ¼ 8.4 Hz, 1H), 7.70 (t,
J ¼ 8.0 Hz, 1H), 7.59 (t, J ¼ 7.6 Hz, 1H).
56.9, 45.6, 37.9, 29.7. IR (KBr): 2937, 2770, 1645, 1594, 1380 cmꢂ1
HR-MS: C22H18N2O3 calculated: 358.1317, found: 358.1321.
.
5.1.9.2. N-(N0,N0-dimethylamine-propyl)-benzo[k,l]xanthene-3,4-
naphthalimide (13b). Yellow solid, yield: 78%, m.p. 194e195 ꢀC. 1H
NMR (400 MHz, DMSO-d6) (ppm): 8.31 (d, J ¼ 8.0 Hz, 2H), 8.18 (d,
J ¼ 8.4 Hz, 1H), 8.03 (d, J ¼ 7.6 Hz,1H), 7.59 (t, J ¼ 7.6 Hz, 1H), 7.36 (d,
J ¼ 8.4 Hz, 2H), 7.25 (d, J ¼ 8.4 Hz,1H), 4.05 (t, J ¼ 6.4 Hz, 2H), 3.12 (t,
J ¼ 4.0 Hz, 2H), 2.73 (s, 6H), 2.05 (t, J ¼ 7.6 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) (ppm): 163.5, 163.2, 155.3, 151.5, 133.7, 133.1,
133.0, 129.4, 125.8, 124.8, 119.5, 118.7, 118.2, 118.1, 116.1, 114.6, 110.3,
54.9, 42.5, 37.4, 23.4. IR (KBr): 2929, 2766, 1588, 1657, 1382,
769 cmꢂ1
372.1473.
; HR-MS: C23H20N2O3 calculated: 372.1474, found
5.1.13. General procedure for the preparation of 17ae17d
16 (0.29 g, 0.001 mol) was dissolved in 20 mL ethanol. After
adding corresponding primary amine (0.003 mol), the mixture was
stirred and refluxed for 2 h. Then the solution was evaporated in
vacuum and the residue was purified on silica gel chromatography.
5.1.9.3. N-(n-butyl)-benzo[k,l]xanthene-3,4-naphthalimide
(13c).
Yellow solid, yield: 75%, m.p. 181e182 ꢀC. 1H NMR (400 MHz, CDCl3)
(ppm): 8.62 (d, J ¼ 7.6 Hz, 1H), 8.58 (d, J ¼ 8.4 Hz, 1H), 8.06 (d,
J ¼ 7.6 Hz, 1H), 7.91 (d, J ¼ 7.2 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.35 (t,
J ¼ 8.0 Hz, 2H), 7.27 (d, J ¼ 10.0 Hz, 1H), 4.21 (t, J ¼ 7.2 Hz, 2H), 1.79e
1.71 (m, 2H), 1.53e1.44 (m, 2H), 1.01 (t, J ¼ 6.8 Hz, 3H). 13C NMR
(100 MHz, CDCl3) (ppm): 155.7, 151.9, 133.7, 133.3, 133.1, 132.0,
5.1.13.1. N-(N0,N0-dimethylamine-ethyl)-benzo[b]furano[2,1-c]naph-
thalimide (17a). Yellow solid, yield: 91%, m.p. 156e157 ꢀC. 1H NMR
(400 MHz, CDCl3) (ppm): 9.15 (s, 1H), 8.74 (d, J ¼ 8.4 Hz,1H), 8.67 (d,

S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
137
J ¼ 7.2 Hz, 1H), 8.10 (d, J ¼ 7.6 Hz, 1H), 7.89 (t, J ¼ 8.0 Hz, 1H), 7.77 (d,
J ¼ 8.0 Hz, 1H), 7.60 (t, J ¼ 8.0 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz, 1H), 4.42 (t,
J ¼ 7.2 Hz, 2H), 2.80 (t, J ¼ 6.4 Hz, 2H), 2.47 (s, 6H). 13C NMR
(100 MHz, CDCl3) (ppm): 164.3, 164.2, 156.5, 155.2, 130.9, 127.9,
127.8, 127.2, 127.1, 125.5, 124.2, 124.0, 123.2, 121.0, 120.7, 119.2, 118.0,
topoisomerase I and the indicated concentrations of compounds
were incubated for 30 min at 37 ꢀC in DNA topoisomerase I buffer
with 0.01% bovine serum albumin, in a total volume of 20
mL. The
reactions were stopped by the addition of 2 L 10% SDS and 2 mL
m
6 ꢁ loading dye solution (Fermentas). Samples were then electro-
phoresed in 0.8% agarose gel in TAE buffer for 35 min at 120 V. The
gel was stained with ethidium bromide at room temperature and
photographed with UV transilluminator.
112.2, 56.9, 45.6, 38.0. IR (KBr): 2925, 2848, 2361, 1696, 1337 cmꢂ1
.
HR-MS: C22H18N2O3 calculated: 358.1317, found: 358.1317.
5.1.13.2. N-(N0,N0-dimethylamine-propyl)-benzo[b]furano[2,1-c]
naphthalimide (17b). Yellow solid, yield: 85%, m.p. 185e186 ꢀC. 1H
NMR (400 MHz, CDCl3eCD3OD) (ppm): 9.12 (s, 1H), 8.77 (d,
J ¼ 8.0 Hz, 1H), 8.63 (d, J ¼ 7.6 Hz, 1H), 8.12 (d, J ¼ 7.6 Hz, 1H), 7.92 (t,
J ¼ 8.4 Hz, 1H), 7.77 (t, J ¼ 8.4 Hz, 1H), 7.61 (t, J ¼ 8.0 Hz, 1H), 7.52 (t,
J ¼ 6.8 Hz, 1H), 4.31 (t, J ¼ 6.4 Hz, 2H), 3.09 (t, J ¼ 7.6 Hz, 2H), 3.77 (s,
6H), 2.24e2.18 (m, 2H). 13C NMR (100 MHz, CDCl3eCD3OD) (ppm):
164.6, 164.4, 156.5, 155.4, 131.1, 128.1, 127.7, 127.6, 127.3, 125.7, 124.3,
123.6, 122.6, 120.9, 120.9, 119.2, 117.4, 112.2, 56.0, 43.3, 37.5, 29.6,
5.4. DNA intercalating assay by CD spectra
ctDNA was purchased from Sigma Aldrich and used without
further purification. The concentrations of compounds and calf
thymus DNA were 20 mM and 100 mM respectively in 20 mM Trise
HCl buffer, pH 7.5 and DMSO (1% by volume). The CD measurements
were performed on a ChirascanÔ CD Spectrometer (Applied Pho-
tophysics Ltd, England) using 1 cm quartz cell in the wavelength
range of 240e300 nm.
24.1. IR (KBr): 2921, 2844, 2754, 1657, 1345 cmꢂ1
.
HR-MS:
C23H20N2O3 calculated: 372.1474, found: 372.1465.
5.5. DNA intercalating assay by fluorescence quenching
5.1.13.3. N-(n-butyl)-benzo[b]furano[2,1-c]naphthalimide
(17c).
Yellow solid, yield: 81%, m.p. 151e152 ꢀC. 1H NMR (400 MHz, CDCl3)
(ppm): 9.21 (s, 1H), 8.78 (d, J ¼ 8.0 Hz, 1H), 8.71 (d, J ¼ 7.2 Hz, 1H),
8.15 (d, J ¼ 7.6 Hz, 1H), 7.92 (t, J ¼ 8.0 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H),
7.62 (t, J ¼ 8.0 Hz, 1H), 7.53 (t, J ¼ 7.6 Hz, 1H), 4.26 (t, J ¼ 7.6 Hz, 2H),
1.83e1.75 (m, 2H), 1.56e1.47 (m, 2H), 1.03 (t, J ¼ 7.6 Hz, 3H). 13C
NMR (100 MHz, CDCl3) (ppm): 164.3, 164.2, 156.5, 155.2, 130.9,
127.8, 127.2, 127.1, 125.4, 124.2,124.0, 123.3, 121.0, 120.8, 119.3, 118.2,
112.2, 40.4, 30.3, 20.4, 13.9. IR (KBr): 2921, 2830, 1700, 1650,
The fluorescence spectra were obtained using a Varian Cary
Eclipse fluorescence spectrophotometer at 25 ꢀC. The concentra-
tions of compounds and calf thymus DNA were 20
mM and 0e
150 M respectively in 20 mM TriseHCl buffer, pH 7.5 and DMSO
m
(1% by volume), the solution in a final volume of 10 mL, which
was used for fluorescence-quenching experiments. And an equi-
librium period of 1 h for constant stirring at 25 ꢀC in the dark of
the mixed solution was allowed before recording each spectrum.
The association constants (Ka’s) were derived according to
the equation I ¼ I0 þ {(IN ꢂ I0)/2[Q]0} ꢁ {([DNA]0 þ [Q]0 þ 1/
Ka) ꢂ {([DNA]0 þ [Q]0 þ 1/Ka)2 ꢂ 4[DNA]0[Q]0}1/2}, wherein I0, I and
IN represent the fluorescence intensities of compounds alone, the
sample and DNA totally bound, respectively. [DNA]0 and [Q]0 were
the initial analytical concentrations of DNA and the agents,
respectively.
1330 cmꢂ1
343.1303.
. HR-MS: C22H17NO3 calculated: 343.1208, found:
5.1.13.4. N-(20-piperazidine-ethyl)-benzo[b]furano[2,1-c]naph-
thalimide (17d). Yellow solid, yield: 76%, m.p. 195e196 ꢀC. 1H NMR
(400 MHz, CDCl3eCD3OD) (ppm): 9.14 (s, 1H), 8.77 (d, J ¼ 8.4 Hz,
1H), 8.64 (d, J ¼ 7.2 Hz, 1H), 8.11 (d, J ¼ 8.0 Hz,1H), 7.90 (t, J ¼ 8.0 Hz,
1H), 7.76 (d, J ¼ 8.4 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.49 (t, J ¼ 7.2 Hz,
1H), 4.36 (t, J ¼ 6.4 Hz, 2H), 2.97 (brs, 4H), 2.77 (t, J ¼ 6.8 Hz, 2H),
2.72 (brs, 4H). 13C NMR (100 MHz, CDCl3eCD3OD) (ppm): 164.6,
164.4, 156.5, 155.3, 131.0, 128.0, 127.7, 127.4, 127.3, 125.6, 124.3,
123.8, 122.9, 120.9, 120.9, 119.3, 117.7, 112.2, 55.8, 52.2, 44.5, 37.2. IR
(KBr): 2925, 2855, 2357, 1660, 1365 cmꢂ1. HR-MS: C24H21N3O3
calculated: 399.1583, found: 399.1588.
Acknowledgments
This work is financially supported by the National Basic
Research Program of China (973 Program, 2010CB126100), the
National High Technology Research and Development Program of
China (863 Program, 2011AA10A207), the China 111 Project (grant
B07023), the Fundamental Research Funds for the Central Univer-
sities, and the Shanghai Committee of Science and Technology
[grant 11DZ2260600]. We thank Chenghui Xu for antiproliferation
assay against A549 and P388 cancer cell lines.
5.2. kDNA decatenation assay
Topoisomerase II activity was measured by the ATP-dependent
decatenation of kDNA according to the manufacturer’s in-
structions (TopoGEN, Florida, USA). 0.1 mg kDNA, 1 unit of human
Appendix A. Supplementary data
topo IIa (TopoGEN) and the indicated concentrations of compounds
were incubated for 30 min at 37 ꢀC in 50 mM TriseHCl (pH 8),
150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM DTT in a total
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.039.
volume of 20
mL. The reactions were stopped by the addition of 2 mL
10% SDS and 2
m
L 6 ꢁ loading dye solution (Fermentas). Samples
References
were then electrophoresed in 0.8% agarose gel in TAE buffer for
50 min at 120 V. The gel was stained with ethidium bromide at
room temperature and photographed with UV transilluminator.
[1] J. Feigon, W.A. Denny, W. Leupin, D.R. Kearns, Interaction of antitumor drugs
with natural DNA: 1H NMR study of binding mode and kinetics, J. Med. Chem.
27 (1984) 450e465.
[2] L.H. Hurley, DNA and its associated processes as targets for cancer therapy,
Nat. Rev. Cancer 2 (2002) 188e200.
[3] C.H. Tseng, C.C. Tzeng, C.L. Yang, P.J. Lu, H.L. Chen, H.Y. Li, Y.C. Chuang,
C.N. Yang, Y.L. Chen, Synthesis and antiproliferative evaluation of certain
indeno[1,2-c]quinoline derivatives. Part 2, J. Med. Chem. 53 (2010) 6164e
6179.
[4] M.F. Braña, Butyramiden und verfahren zu dessen herstelling, Patent
DE2318136, 1973.
[5] E. Van Quaquebeke, T. Mahieu, P. Dumont, J. Dewelle, F. Ribaucour, G. Simon,
S. Sauvage, J.F. Gaussin, J. Tuti, M. El Yazidi, F. Van Vynckt, T. Mijatovic,
5.3. DNA relaxation assay
Topoisomerase I activity was assayed by relaxation of super-
coiled pBR322 DNA according to the manufacturer’s instructions
(Takara, Dalian, China). Topo I (calf thymus), buffer, BSA, loading
buffer and supercoiled pBR322 DNA were all from TaKaRa Bio-
technology CO., Ltd. 0.5 mg supercoiled pBR322 DNA, 0.1 units of

138
S. Tan et al. / European Journal of Medicinal Chemistry 62 (2013) 130e138
F. Lefranc, F. Darro, R. Kiss, 2,2,2-Trichloro-N-({2-[2-(dimethylamino)ethyl]-
[21] W. Yao, X. Qian, Oxazolonaphthalimides and their hydroperoxides: photo-
physical and photobiological properties, Dyes Pigm. 48 (2001) 43e47.
[22] H. Yin, W. Zhu, Y. Xu, M. Dai, X. Qian, Y. Li, J. Liu, Novel aliphatic N-oxide of
naphthalimides as fluorescent markers for hypoxic cells in solid tumor, Eur. J.
Med. Chem. 46 (2011) 3030e3037.
[23] X. Qian, J. Cui, R. Zhang, Intramolecular aromatic 1,5-hydrogen transfer in
preparation of oxacyclic naphthalic anhydride via unusual Pschorr cyclisation,
Chem. Commun. (2001) 2656e2657.
[24] Z. Chen, X. Liang, H.Y. Zhang, H. Xie, J.W. Liu, Y.F. Xu, W.P. Zhu, Y. Wang,
X. Wang, S.Y. Tan, D. Kuang, X.H. Qian, A new class of naphthalimide-
based antitumor agents that inhibit topoisomerase II and induce lysoso-
mal membrane permeabilization and apoptosis, J. Med. Chem. 53 (2010)
2589e2600.
[25] I. Ott, Y.F. Xu, J.W. Liu, M. Kokoschka, M. Harlos, W.S. Sheldrick, X.H. Qian,
Sulfur-substituted naphthalimides as photoactivatable anticancer agents:
DNA interaction, fluorescence imaging, and phototoxic effects in cultured
tumor cells, Bioorg. Med. Chem. 16 (2008) 7107e7116.
1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin5-yl} carbamoyl) acetamide
(UNBS3157), a novel nonhematotoxic naphthalimide derivative with potent
antitumor activity, J. Med. Chem. 50 (2007) 4122e4134.
[6] M. Lv, H. Xu, Overview of naphthalimide analogs as anticancer agents, Curr.
Med. Chem. 16 (2009) 4797e4813.
[7] S. Li, W. Zhong, Z. Li, X. Meng, Unprecedented synthesis, in vitro and in vivo
anti-cancer evaluation of novel triazolonaphthalimide derivatives, Eur. J. Med.
Chem. 47 (2012) 546e552.
[8] L. Ingrassia, F. Lefranc, R. Kiss, T. Mijatovic, Naphthalimides and azonafides as
promising anti-cancer agents, Curr. Med. Chem. 16 (2009) 1192e1213.
[9] V.K. Malviya, P.Y. Liu, D.S. Alberts, E.A. Surwit, J.B. Craig, E.V. Hanningan,
Evaluation of amonafide in cervical cancer, phase II study, Am. J. Clin. Oncol.
15 (1992) 41e44.
[10] S.M. Sami, R.T. Dorr, D.S. Alberts, W.A. Remers, 2-substituted 1,2-dihydro-3H-
dibenz[de,h]isoquinoline-1,3-diones. A new class of antitumor agent, J. Med.
Chem. 36 (1993) 765e770.
[11] S.M. Sami, R.T. Dorr, A.M. Solyom, D.S. Alberts, W.A. Remers, Amino-
substituted 2-[20-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h] iso-
quinoline-1,3-diones. Synthesis, antitumor activity, and quantitative struc-
tureeactivity relationship, J. Med. Chem. 38 (1995) 983e993.
[26] X.L. Li, Y.J. Lin, Y.K. Yuan, K. Liu, X.H. Qian, Novel efficient anticancer agents
and DNA-intercalators of 1,2,3-triazol-1,8-naphthalimides: design, synthesis,
and biological activity, Tetrahedron 67 (2011) 2299e2304.
[27] H. Zhu, M. Huang, F. Yang, Y. Chen, Z.H. Miao, X.H. Qian, Y.F. Xu, Y.X. Qin,
H.B. Luo, X. Shen, M.Y. Geng, Y.J. Cai, J. Ding, R16, a novel amonafide analogue,
induces apoptosis and G(2)-M arrest via poisoning topoisomerase II, Mol.
Cancer Ther. 6 (2007) 484e495.
[28] M.G. Ferlin, C. Marzano, V. Gandin, S. Dall’Acqua, L. Dalla Via, DNA binding
ellipticine analogues: synthesis, biological evaluation, and structureeactivity
relationships, ChemMedChem 4 (2009) 363e377.
[29] W. Liu, L. Zhu, W. Guo, C. Zhuang, Y. Zhang, C. Sheng, P. Cheng, J. Yao,
W. Wang, G. Dong, S. Wang, Z. Miao, W. Zhang, Synthesis and biological
evaluation of novel 7-acyl homocamptothecins as topoisomerase I inhibitors,
Eur. J. Med. Chem. 46 (2011) 2408e2414.
[30] M. López-Lázaro, E. Willmore, A. Jobson, K.L. Gilroy, H. Curtis, K. Padget,
C.A. Austin, Curcumin induces high levels of topoisomerase I- and II-DNA
complexes in K562 leukemia cells, J. Nat. Prod. 70 (2007) 1884e1888.
[31] C. Bailly, C. Carrasco, A. Joubert, C. Bal, N. Wattez, M.P. Hildebrand, A. Lansiaux,
P. Colson, C. Houssier, M. Cacho, A. Ramos, M.F. Braña, Chromophore-modified
bisnaphthalimides: DNA recognition, topoisomerase inhibition, and cytotoxic
properties of two mono- and bis-furonaphthalimides, Biochemistry 42 (2003)
4136e4150.
[32] J. Wang, A.B. Wu, Y.F. Xu, J.W. Liu, X.H. Qian, M(2)-A induces apoptosis and
G(2)-M arrest via inhibiting PI3 K/Akt pathway in HL60 cells, Cancer Lett. 283
(2009) 193e202.
[33] X. Liang, K. Xu, Y.F. Xu, J.W. Liu, X.H. Qian, B1-induced caspase-independent
apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53
binding to P2 promoter TATA box, Toxicol. Appl. Pharmacol. 256 (2011) 52e
61.
[12] M.F. Braña, M. Cacho, M.A. Garcia, B. de Pascual-Teresa, A. Ramos, N. Acero,
F. Llinares, D. Munoz-Mingarro, C. Abradelo, M.F. Rey-Stolle, M. Yuste, Syn-
thesis, antitumor activity, molecular modeling, and DNA binding properties of
a new series of imidazonaphthalimides, J. Med. Chem. 45 (2002) 5813e5816.
[13] M.F. Braña, M. Cacho, M.A. Garcia, B. de Pascual-Teresa, A. Ramos,
M.T. Dominguez, J.M. Pozuelo, C. Abradelo, M.F. Rey-Stolle, M. Yuste,
M. Banez-Coronel, J.C. Lacal, New analogues of amonafide and elinafide,
containing aromatic heterocycles: synthesis, antitumor activity, molecular
modeling, and DNA binding properties, J. Med. Chem. 47 (2004) 1391e1399.
[14] M.F. Braña, M. Cacho, A. Ramos, M.T. Dominguez, J.M. Pozuelo, C. Abradelo,
M.F. Rey-Stolle, M. Yuste, C. Carrasco, C. Bailly, Synthesis, biological evaluation
and DNA binding properties of novel mono and bisnaphthalimides, Bioorg.
Med. Chem. 1 (2003) 648e654.
[15] Y.G. Li, Y.F. Xu, X.H. Qian, B. Qu, Naphthalimide-thiazoles as novel photo-
nucleases: molecular design, synthesis, and evaluation, Tetrahedron Lett. 45
(2004) 1247e1251.
[16] X.H. Qian, Z.G. Li, Q. Yang, Highly efficient antitumor agents of heterocycles
containing sulfur atom: linear and angular thiazonaphthalimides against
human lung cancer cell in vitro, Bioorg. Med. Chem. 15 (2007) 6846e6851.
[17] X. Liang, A.B. Wu, Y.F. Xu, K. Xu, J.W. Liu, X.H. Qian, B1, a novel naphthalimide-
based DNA intercalator, induces cell cycle arrest and apoptosis in HeLa cells
via p53 activation, Investig. New Drug 29 (2011) 646e658.
[18] L.J. Xie, Y.F. Xu, F. Wang, J.W. Liu, X.H. Qian, J.N. Cui, Synthesis of new amo-
nafide analogues via coupling reaction and their cytotoxic evaluation and
DNA-binding studies, Bioorg. Med. Chem. 17 (2009) 804e810.
[19] X. Qian, Y. Li, Y. Xu, Y. Liu, B. Qu, Highly-efficient DNA photocleavers with long
wavelength absorptions: thio-heterocyclic fused naphthalimides containing
aminoalkyl side chains, Bioorg. Med. Chem. Lett. 14 (2004) 2665e2668.
[20] Y. Xu, B. Qu, X. Qian, Y. Li, Five-member thio-heterocyclic fused naph-
thalimides with aminoalkyl side chains: intercalation and photocleavage to
DNA, Bioorg. Med. Chem. Lett. 15 (2005) 1139e1142.
[34] Q. Yang, P. Yang, X.H. Qian, L.P. Tong, Naphthalimide intercalators with chiral
amino side chains: effects of chirality on DNA binding, photodamage and
antitumor cytotoxicity, Bioorg. Med. Chem. 13 (2008) 6210e6213.
[35] Y.Sh. Bcheshtiha, M.M. Heravi, F.A. Salehi, Synthesis of benzimid-
azoxanthenoisoquinolinones, new intermediates and dyes for synthetic-
polymer fibres, Indian J. Chem. 37B (1998) 1177e1179.