Naphthalimides exhibit in vitro antiproliferative and antiangiogenic activities by inhibiting both topoisomerase II (topo II) and receptor tyrosine kinases (RTKs)

Article abstract of DOI:10.1016/j.ejmech.2013.05.002

Novel naphthalimide derivatives were designed and synthesized to modulate both topoisomerase II (topo II) and receptor tyrosine kinases (RTKs). Most target compounds exhibited effective and selective antiproliferative activities against three cancer cell lines by inhibiting topo II. The IC₅₀ values ranged from 1.5 to 19.1 μM. Moreover, compounds 8d and 12d moderately inhibited various angiogenesis-related RTKs, including FGFR1, VEGFR2 and PDGFRα. The representative compound 8d was then proved to possess antiangiogenic activity, which was evidenced by the inhibition of migration and tube formation activities of HMEC-1 cells. To our knowledge, it is the first time naphthalimides were identified as tyrosine kinases inhibitors (TKIs) besides their conventional cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2013.05.002

European Journal of Medicinal Chemistry 65 (2013) 477e486
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Naphthalimides exhibit in vitro antiproliferative and antiangiogenic
activities by inhibiting both topoisomerase II (topo II) and receptor
tyrosine kinases (RTKs)
,
b
,
b
a
a
b
Xin Wang a, Zhuo Chen a , Linjiang Tong , Shaoying Tan , Wei Zhou , Ting Peng ,
**
*
Kun Han b, Jian Ding b, Hua Xie b , Yufang Xu
,
a,
***
a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel naphthalimide derivatives were designed and synthesized to modulate both topoisomerase II
(topo II) and receptor tyrosine kinases (RTKs). Most target compounds exhibited effective and selective
antiproliferative activities against three cancer cell lines by inhibiting topo II. The IC50 values ranged from
Received 28 March 2013
Received in revised form
30 April 2013
Accepted 3 May 2013
Available online 14 May 2013
1.5 to 19.1
m
M. Moreover, compounds 8d and 12d moderately inhibited various angiogenesis-related
. The representative compound 8d was then proved to
RTKs, including FGFR1, VEGFR2 and PDGFR
a
possess antiangiogenic activity, which was evidenced by the inhibition of migration and tube formation
activities of HMEC-1 cells. To our knowledge, it is the first time naphthalimides were identified as
tyrosine kinases inhibitors (TKIs) besides their conventional cytotoxicity.
Keywords:
Angiogenesis
Multitarget
Ó 2013 Elsevier Masson SAS. All rights reserved.
Naphthalimide
Topoisomerase II
Tyrosine kinase
1. Introduction
number of topo II-targeted chemotherapy drugs, such as Etoposide
(VP-16), Doxorubicin (DOX) and Mitoxantrone, have been approved
DNA topoisomerase II (topo II) is a kind of nuclear enzyme that
modulates the topology of chromosomal DNA by causing transient
double-stranded break. The enzyme plays key roles in a number of
DNA-related processes [1]. Topo II is frequently overexpressed in
different types of tumors, and has been proved as one of the ideal
molecular targets for widely prescribed chemotherapy agents [2]. A
by FDA for clinical use [3]. Despite the emergence of kinase in-
hibitors, topo II inhibitors still make great contributions in clinic for
cancer therapy [4].
Receptor tyrosine kinases (RTKs) are primary mediators of the
signaling networks that transmit extracellular signals into the cells.
They are vital in many cellular processes including proliferation,
differentiation, migration and angiogenesis [5,6]. There are more
than 20 subfamilies of receptor tyrosine kinases that have been
discovered so far, including epidermal growth factor receptor
(EGFR) family, vascular endothelial growth factor receptor (VEGFR)
family, platelet-derived growth factor receptor (PDGFR) family,
fibroblast growth factor receptor (FGFR) family [7]. Aberrant RTKs
activities are implicated with tumor growth and development, and
thus provide popular targets in cancer therapy [8]. Numerous RTKs
inhibitors have been developed and several of them have been
approved for clinical use.
Abbreviations: EGFR, epidermal growth factor receptor; FGFR, fibroblast growth
factor receptor; HMEC-1, Human Microvascular Endothelial Cells; kDNA, kineto-
plast DNA; PDGFR, platelet-derived growth factor receptor; RTK, Receptor Tyrosine
Kinase; Topo II, topoisomerase II; TKIs, tyrosine kinases inhibitors; VEGFR, vascular
endothelial growth factor receptor.
* Corresponding author. State Key Laboratory of Bioreactor Engineering, Shanghai
Key Laboratory of Chemical Biology, Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and Technology, Shanghai
200237, China. Tel./fax: þ86 21 64250823.
** Corresponding author. Tel.: þ86 21 50805897; fax: þ86 21 50806722.
*** Corresponding author. Tel.: þ86 21 64251399; fax: þ86 21 64252603.
E-mail addresses: chenzhuo@ecust.edu.cn (Z. Chen), hxie@simm.ac.cn (H. Xie),
yfxu@ecust.edu.cn (Y. Xu).
Angiogenesis is essential in tumor progression by providing
both oxygen and nutrition for tumors beyond the size of 1e2 mm3
[9]. Antiangiogenesis has been proved to be a promising strategy
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.002

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X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
pharmacodynamic relationships and toxicology profiles [18],
discovering single agents with both antiangiogenic and cytotoxic
activities is a desirable strategy for cancer therapy.
Naphthalimides, first discovered by Brana and co-workers [19],
are a class of DNA intercalators showing potent antitumor activities
against various cancers preclinically and clinically [20e23]. Amo-
nafide is one of the most active naphthlimide-based topo II poisons
and is in phase III clinical trials for the treatment of acute myeloid
leukemia. Heterocyclic fused naphthalimides and bisnaph-
thalimides showed great DNA binding affinity and cytotoxic activ-
ity. Several of them also entered clinical trials [20,24]. Besides
modulating DNA-related processes, new mechanisms of action
have been verified for naphthalimide skeleton, which suggests
novel therapeutic strategies with naphthalimide derivatives. For
example, UNBS5162 could depress expression of the chemokines,
and induce antiangiogenic effects in vivo [21].
According to our previous research, some of the naphthalimide
derivatives with long alkyl chains and polyamines exhibited more
potent antiproliferative activity than Amonafide. For example,
compounds 7c and 7d could inhibit topo II and induce lysosomal
membrane permeabilization (LMP) and apoptosis (see Fig. 1) [25].
Compounds 8d and 12d were analogs of 7c and 7d with long alkyl
chains at 2-position and polyamines at the 6-position. They also
showed potent antiproliferative activity by inhibiting topo II.
Herein, we designed and synthesized the derivatives of 8d and 12d
with different lengths of alkyl chains. These newly-synthesized
compounds inhibited topo II activity and exhibited structureeac-
tivity relationship in proliferation of different types of cancer cells.
Furthermore, tyrosine kinases were proved to be the antitumor
targets of naphthalimide derivatives for the first time. The repre-
sentative compound 8d exhibited potent antiangiogenesis activity
in vitro.
Fig. 1. Representative compounds of naphthalimides.
for the treatment of cancers with few side effects [10,11]. Many
investigations have proved that angiogenesis is regulated by cyto-
kines and specific tyrosine kinases like VEGFR, PDGFR and FGFR
[12]. Tyrosine kinases inhibitors (TKIs) which inhibit angiogenesis
have been developed for clinical use. For example, the VEGFR and
PDGFR inhibitor Sunitinib has been approved in the treatment of
renal cell carcinoma which is a highly vascularized tumor [13].
As tumors involve complex biological networks, it is not easy to
provide long-term effects by targeting a single target in most pa-
tients [14,15]. Therefore, multitarget TKIs are desirable for
enhancing antitumor potency and overcoming possible resistance
mechanism. Antiangiogenic TKIs in clinical use are frequently
multitarget kinase inhibitors, such as Sunitinib and Sorafenib [16].
As antiangiogenesis is generally cytostatic rather than cytoreduc-
tive, recent studies have indicated that combination of anti-
angiogenic agents with cytotoxic chemotherapy agents is more
effective in cancer treatment [17]. While direct damage of endo-
thelial cells could improve antiangiogenic effects, the anti-
angiogenic agents will normalize vessels and improve delivery of
cytotoxic drugs to tumor [7]. As combinational therapy used in
clinic usually exhibited complex pharmacokinetic and
2. Results and discussions
2.1. Chemistry
As shown in Scheme 1, the target compounds 1de16d were
prepared in a facile four-step sequence with good yields [25].
Commercially available 6-bromobenzo[de]-isochromene-1,3-dione
was condensed with corresponding amines to yield 1ae16a, which
were converted to 1be16b under Ullmann’s condition. The alcohol
were then converted to the corresponding bromides 1ce16c using
a system of Ph3P, 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ)
Scheme 1. Reagents and conditions: a) 1.1 equiv of corresponding primary amines, ethanol, reflux, yield 81e95%, (for 1a and 2a corresponding 70% amines aqueous were used as
solvent, and reacted at r.t., yield 90e95%); b) 3.0 equiv of ethanolamine, 2-methoxyethanol, reflux, yield 72e88%; c) 1.2 equiv of DDQ, 1.2 equiv of PPh3, 1.2 equiv of (n-butyl)4NBr,
CH2Cl2, r.t., yield 78e83%; d) 5.0 equiv of polyamines, 1.2 equiv of KI, CHCl3, reflux, yield 62e71%.

X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
479
Fig. 2. Inhibition of topo II-mediated kDNA decatenation by target compounds (100
mM). (A) Lane 1, kDNA; lane 2, minicircles (no drug); lanes 3e7, compounds 1d, 2d, 4d, 6d and
Amonafide, respectively. (B) Lane 1, kDNA; lane 2, minicircles (no drug); lanes 3e6, compounds 8d, 12d, 16d, and Amonafide, respectively.
and tetrabutyl ammonium bromide [26]. Finally, the reactions be-
tween 1ce16c and N’,N’-dimethylethane-1,2-diamine were
accomplished in chloroform to provide the target compounds 1de
16d.
with their potent topo II inhibitory activity and balanced solubility
and membrane penetrability (see Table 1). Whereas, compounds
1de4d and 16d showed much weaker antiproliferative activity
with IC50 values mainly at double digit micromolar. The less potent
antitumor activity of 2d and 4d might be owing to their weaker
topo II inhibitory potency, while poor membrane penetrability of
1d and poor aqueous solubility of 16d might underlie their weaker
antiproliferative activities (see Table 1). Besides, the tested com-
pounds did not intercalate in ct-DNA as effectively as conventional
naphthalimide derivative according to CD spectra and fluorescent
spectra studies (Fig. S2 and S3). We further investigated the anti-
proliferative effect on normal cell lines-LO2 and GES-1. In com-
parison with tumour cell lines, less cytotoxicity was observed on
2.2. Topo II inhibition
As naphthalimides could inhibit the activity of topo II by
forming a complex of drug-DNA-topoisomerase [27,28], the kinet-
oplast DNA (kDNA) decatenation assay was taken to investigate the
topo II inhibitory effect in cell-free system [29]. As illustrated in
Fig. 2, topo II catalyzed the decatenation of kDNA to minicircles in
the presence of ATP (lane 2 vs. lane 1). All the tested compounds
could reduce the kDNA minicircles, which indicated their topo II
inhibition capacity. The images were quantitated by software Gel-
Pro Analyzer (Fig. S1 and Table S1). Compounds 1d and 6de16d
exhibited similar topo II inhibitory potency as Amonafide at the
tested concentration. Meanwhile, compounds 2d and 4d were less
potent topo II inhibitors in comparison with Amonafide.
normal cells, with IC50 values mainly higher than 50 mM. Therefore,
the target compounds exhibited good selectivity against tumor cell
lines.
2.4. RTK inhibition
2.3. In vitro antiproliferative activity
According to recent reports, the naphthalimides can modulate
AKT/mTOR signaling pathway [30,31]. As AKT/mTOR is one of the
representative downstream signal pathways of tyrosine kinases, we
assumed that naphthalimides might target tyrosine kinases and
thereby down-regulate AKT/mTOR pathway. We investigated the
tyrosine kinases inhibition activity of the derivatives using ELISA
method. As shown in Table 2, 8d inhibited FGFR1 and KDR
The antiproliferative activity of compounds 1de16d was eval-
uated against three human cancer cell lines and two normal cell
lines: HL-60 (human promyelocytic leukemia cell line), MDA-MB-
231 (human breast cancer line), A549 (human lung adenocarci-
noma epithelial cell line), LO2 (human liver cell line) and GES-1
(human gastric epithelial mucosa cell line).
As shown in Table 1, the length of alkyl chains significantly
influenced the antiproliferative activities. Compounds 6de12d
exhibited significant antitumor activity with IC50 values mainly at
single digital micromolar range, which were comparable with the
reference compound Amonafide. These results were in accordance
(VEGFR2) kinase activities with IC50 at 18.06 and 48.94
respectively. 12d also inhibited FGFR1 and KDR at micromolar
range. Besides, 12d inhibited PDGFR with IC50 value at 26.17 M.
Their analogs exhibited no inhibition activity against the tested
kinases at the concentration of 50 M or higher. Though the kinases
mM
a
m
m
inhibition activity of these compounds was still much weaker than
Table 1
Antiproliferative activities and physical properties of compounds 1de16d.
No.
Antiproliferative activities (IC50,
mM)
Physical properties
HL60
MDA-MB-231
A549
LO2
GES-1
Solubility (
mM)
LogP
1d
2d
4d
6d
7.8 ꢀ 1.0
17.0 ꢀ 1.1
5.8 ꢀ 0.3
1.5 ꢀ 0.5
2.1 ꢀ 0.4
1.8 ꢀ 0.2
24.5 ꢀ 8.0
0.9 ꢀ 0.3
7.9 ꢀ 1.0
24.3 ꢀ 3.2
10.5 ꢀ 0.9
6.1 ꢀ 0.2
8.6 ꢀ 1.8
6.6 ꢀ 2.2
17.7 ꢀ 1.1
4.4 ꢀ 1.0
24.5 ꢀ 5.6
31.4 ꢀ 7.0
21.4 ꢀ 8.1
9.8 ꢀ 4.0
7.8 ꢀ 1.2
19.1 ꢀ 7.6
31.3 ꢀ 2.0
3.2 ꢀ 1.8
13.6 ꢀ 0.7
>50
>50
>50
>50
>50
>50
13.8 ꢀ 1.0
>50
>50
>50
78.4 ꢀ 1.9E3
46.7 ꢀ 5.0E3
15.4 ꢀ 2.5E3
430 ꢀ 20
264 ꢀ 13
63 ꢀ 7
0.15
0.68
1.74
2.80
3.86
5.99
8.12
ND
46.0 ꢀ 3.5
8d
>50
12d
16d
Am
>50
>50
21.6 ꢀ 1.6
16 ꢀ 2
NDa
a
ND: not determined.

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X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
Table 2
BIBF1120, a multitarget kinase inhibitor in clinical trial, it is the first
time naphthalimides were verified to be tyrosine kinase inhibitors.
Receptor tyrosine inhibition of the target compounds 1de16d.
Compd.
IC50 (mM)
KDR
PDGFR
a
PDGFR
b
FGFR1
2.5. Antiangiogenic activity of compound 8d
1d
2d
4d
6d
>100
>100
>100
>100
48.94 ꢀ 0.41
27.64 ꢀ 0.93
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
>50
>50
0.002
>100
>100
>100
>100
18.06 ꢀ 2.42
28.79 ꢀ 7.52
>100
Angiogenesis is a complex process which includes the prolifer-
ation, migration and tube formation of endothelial cells [32]. As
KDR, FGFR1 and PDGFR are highly related with angiogenesis pro-
cess, we evaluated antiangiogenic activity of 8d which exhibited
more balanced solubility and penetration activity than 12d, by
examining migration and tube formation activities of human
microvascular endothelial cells (HMEC-1).
8d
12d
16d
BIBF1120
26.17 ꢀ 2.67
>50
<0.001
0.004
0.060
2.5.1. Inhibition of proliferation of HMEC-1
We first examined the growth inhibition effect of compound 8d
on HMEC-1 cells by SRB assay. We found that treatment with
compound 8d for 72 h at the concentrations of 20 mM and 40 mM
caused a dose-dependent inhibition of HMEC-1 cell proliferation.
As shown in Fig. 3, after 72 h treatment, compound 8d could inhibit
around 68% and 99% cell proliferation at 20
mM and 40 mM,
respectively, while 10 M or lower concentrations of 8d did not
m
induce appreciable changes in cell growth. Subsequently, 8d was
used in the following antiangiogenesis assay with highest con-
centration at 10 mM.
2.5.2. Inhibition of migration
Endothelial cell migration, which occurs through chemotaxis, is
necessary for angiogenesis. To investigate the effect of 8d on the
migration of HMEC-1 cells in vitro, a transwell Boyden chamber
assay was performed. We found that treatment with 8d for 6 h
could reduce the number of migrated cells in a dose-dependent
manner. As illustrated in Fig. 4A, after exposed to 5
mM of 8d,
Fig. 3. The antiproliferative activity of 8d on HMEC-1endothelial cells.
about 55% of the migrated HMEC-1 cells were inhibited. Moreover,
Fig. 4. Inhibitory effect of 8d on HMEC-1 cells migration and tube formation activities. (A) Representative images and summary data (bar graphs on right hand side) showing
inhibition of migration; (B) Representative images and summary data (bar graphs on right hand side) showing inhibition of migration tube formation. Results are expressed as
mean ꢀ SD; n ¼ 3; *P < 0.01 vs. control.

X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
481
70% of cell migration was inhibited when the concentration
reached 10 M.
3. Conclusion
m
In this study, we designed and synthesized a series of naph-
thalimides with different lengths of alkyl chains and examined their
antitumor activity in vitro. Compounds 1d, 6de16d could inhibit
topoIIactivityaspotentasAmonafideinkDNAdecatenationassay. In
accordancewith their topo II inhibition activity, compounds 6de12d
inhibited the growth of cancer cell lines comparably with Amona-
fide. Besides, compounds 1d and 16d exhibited less potent anti-
proliferative activity because of their poor membrane penetration
or aqueous solubility. Meanwhile, the compounds displayed good
selectivity to tumour cell lines comparing with normal cell lines.
We further proved that compounds 8d and 12d were moderate
RTK inhibitors against angiogenesis-related FGFR1, KDR and
2.5.3. Inhibition of tube formation
Tube formation is one of the most important traits of endothelial
cells on matrigel substratum. To further characterize the anti-
angiogenesis activity of 8d, we investigated the inhibitory effect of
the formation of functional tubes by plating HMEC-1 cells on
matrigel substratum. As shown in Fig. 4B, in the control group,
stimulation of 20% serum resulted in a rapid alignment of HMEC-1s
and formation of tube-like structures within 8 h. Compound 8d
could effectively inhibit serum-stimulated tube formation of
HMEC-1 cells in a dose-dependent manner. Treatment with 5 and
10 mM 8d could induce 30% and 70% inhibition of tube-like struc-
tures respectively.
PDGFRa. To our knowledge, this is the first time naphthalimides
were reported as TKIs. We evaluated the antiangiogenesis activity of
compound 8d by investigating the inhibition of proliferation,
migration and tube formation of endothelial cells. Compound 8d
could inhibit HMEC-1 cell proliferation at high concentrations
2.6. Down-regulation of FGFR1 phosphorylation and downstream
signaling pathways by compound 8d
(>10
significantly inhibit HMEC-1 cells migration and tube formation
activities at low concentrations (less than 10 M) after 6e8 h
mM) after 72 h treatment. Besides, compound 8d could
We further evaluated the effect of 8d on the phosphorylation of
FGFR1 and its downstream signaling pathways in HMEC-1 cells
[33]. Serum-starved HMEC-1 cells were treated with different
concentrations of 8d for 10 h, followed by the addition of
50 ng mLꢁ1 bFGF (10 min), then assayed by Western blotting. The
results showed that 8d treatment resulted in a dose-dependent
inhibition of FGFR1 phosphorylation induced by bFGF. As shown
in Fig. 5, phospho-FGFR1 expression declined after exposure to 1.25
m
exposure. Moreover, we proved that the phosphorylation of FGFR1
induced by bFGF, and the phosphorylation FGFR downstream mol-
ecules AKT and Erk could be inhibited by 8d treatment. According to
the desirable results, compound 8d exhibited effective anti-
proliferative and antiangiogenic activities by targeting topo II and
tyrosine kinases. In all, we provided evidence for further investiga-
tion of naphthalimides as multitarget antitumor candidates.
and 2.5 mM 8d and became undetectable at exposure to 5 mM 8d.
Moreover, the phosphorylation of the key molecules downstream
of FGFR, including AKT and Erk, were obviously decreased. Together
with the data described above, we concluded that 8d inhibited
angiogenesis by blocking the activation of tyrosine kinases and
subsequent downstream signaling.
4. Experimental section
All chemical reagents and solvents were purchased from com-
mercial sources and used without further purification. Thin-layer
chromatography (TLC) was performed on silica gel plates. Column
chromatography was performed using silica gel (Hailang, Qingdao)
300e400 mesh. Biological agents: SRB, MTT and so on were pur-
chased from Sigma Aldrich (St. Louis, MO, USA); all medium and
FBS from Gibco (Grand Island, NY, USA); bFGF from R&D systems
(Minneapolis, MN, USA); Matrigel from BD Biosciences (San Jose,
CA, USA); antibodies to phosphorylated form of the FGF receptor
FGFR1, Erk and AKT from Cell Signalling Technology (Danvers, MA,
USA); antibody to GAPDH (glyceraldehyde-3-phosphate dehydro-
genase) from KangChen Bio-tech (Shanghai); secondary antibodies
from Calbiochem (San Diego, CA, USA). The melt points were tested
by WRS-1B-digital melting point apparatus. 1H and 13C NMR
spectra were recorded employing a Bruker AV-400 spectrometer
with chemical shifts expressed in parts per million. The mass
spectra were collected at the Mass Instrumentation Facility of the
Analysis and Research Center of ECUST. The purities of 1de16d
were analyzed by HPLC, with the purity all being higher than 95%
(Table S2). Analytical HPLC was performed on a HewlettePackard
1100 system chromatograph equipped with photodiode array de-
tector using a Zorbax Bonus-RP 5
m
M 250 mm ꢂ 4.6 mm column
(reverse phase) to detect the purity of the products. The mobile
phase was a gradient of 70e100% methanol (solvent 1) and 10 mM
NH4OAc in water (pH 6.0) (solvent 2) at a flow rate of 1.0 mL/min
(0e1.0 min, 0e70% solvent 1; 1.0e15.0 min, 70% solvent 1; 15.0e
25.0 min, 70e100% solvent 1).
4.1. Synthesis
4.1.1. General procedure for the preparation of 1a and 2a
To a solution of corresponding 70% amine aqueous, bromobenzo
[de]- isochromene-1,3-dione (2.00 g, 7.94 mmol) was added.
Fig. 5. 8d down-regulated the phosphorylation FGFR1 (Tyr 653/654) and downstream
Erk1/2 (Thr 202/204) and AKT (Ser 473) signaling pathways induced by bFGF in HMEC-
1 cells.

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X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
The mixture was stirred overnight at room temperature. After
completion, the solid was filtered and washed with water and dried
in infra-ray oven. Then the product was purified by column chro-
matography on silica gel (PE:EA ¼ 10:1, v/v) to provide 1a and 2a.
(d, J ¼ 8.8 Hz, 1H, ArH), 8.58 (d, J ¼ 7.2 Hz, 1H, ArH); 13C
NMR (100 MHz, CDCl3):
d 14.1 (C15H30CH3), 22.7 (C14H28CH2CH3),
27.1 (C13H26CH2CH2CH3), 28.1 (C2H4C11H22C2H4CH3), 29.4 (C2H4C11
H22C2H4CH3), 29.6 (C2H4C11H22C2H4CH3), 29.7 (C2H4C11H22C2
H4CH3), 31.9 (CH2CH2C13H26CH3), 40.6 (CH2CH2C11H22C2H4CH3),
122.3 (ArC), 123.2 (ArC), 128.0 (ArC), 128.9 (ArC), 130.1 (ArC), 130.5
(ArC), 131.0 (ArC), 131.1 (ArC), 131.9 (ArC), 133.1 (ArC), 163.4 (C]O),
163.5 (C]O); MS (EI) calcd for C28H38BrNO2 [M]þ: 499.2, found:
499.0.
4.1.1.1. 6-Bromo-2-methyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1a). White solid (95.0% yield); mp: 182.4e183.4 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
3.57 (s, 3H, CH3), 7.86 (t, J ¼ 8.4 Hz, 1H, ArH),
8.06 (d, J ¼ 7.6 Hz, 1H, ArH), 8.44 (d, J ¼ 8.0 Hz, 1H, ArH), 8.59
(d, J ¼ 8.4 Hz,1H, ArH), 8.68 (d, J ¼ 7.2 Hz,1H, ArH); MS (EI) calcd for
C
13H8BrNO2 [M]þ: 289.0, found: 289.0.
4.1.3. General procedure for the preparation of 1be16b
To a stirred solution of the amination intermediate (5.00 mmol)
in 2-methoxyethanol (15 mL), ethanolamine (15.00 mmol) was
added. The mixture refluxed for 6e12 h and monitored by TLC.
After completion, the reaction mixture was cooled to room tem-
perature and concentrated. The residue was further purified.
4.1.1.2. 6-Bromo-2-ethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(2a). White solid, (90.2% yield); mp: 164.5e164.7 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
1.34 (t, J ¼ 7.1 Hz, 3H, CH3), 4.24 (q, J ¼ 7.1 Hz,
2H, CH2CH3), 7.85 (dd, J1 ¼ 7.4 Hz, J2 ¼ 8.5 Hz, 1H, ArH), 8.04
(d, J ¼ 7.9 Hz, 1H, ArH), 8.41 (d, J ¼ 7.9 Hz, 1H, ArH), 8.56 (d,
J ¼ 8.5 Hz,1H, ArH), 8.66 (d, J ¼ 7.4 Hz,1H, ArH); 13C NMR (100 MHz,
4.1.3.1. 6-(2-Hydroxyethylamino)-2-methyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (1b). The residue was purified by column
chromatography on silica gel (DCM:MeOH ¼ 30:1, v/v) to provide
1b. Yellow solid (88.6% yield); mp: 223.7e224.5 ꢃC; 1H NMR
CDCl3):
d 13.3 (CH2CH3), 35.7 (CH2CH3), 122.3 (ArC), 123.2 (ArC),
128.0 (ArC), 128.9 (ArC), 130.2 (ArC), 130.6 (ArC), 131.1 (ArC), 131.9
(ArC), 133.2 (ArC), 163.3 (C]O), 163.4(C]O); MS (ESI) calcd for
C
14H11BrNO2 [M þ H]þ: 304.0, found: 304.1.
(400 MHz, CDCl3):
d
3.46 (t, J ¼ 6.0, 2H, NHCH2CH2OH), 3.54 (s, 3H,
CH3), 3.69 (t, J ¼ 6.0, 2H, NHCH2CH2OH), 6.81 (d, J ¼ 8.4 Hz,1H, ArH),
7.67 (t, J ¼ 8.0 Hz, 1H, ArH), 8.25 (d, J ¼ 8.8 Hz, 1H, ArH), 8.43 (d,
J ¼ 7.2 Hz, 1H, ArH), 8.68 (d, J ¼ 8.4 Hz, 1H, ArH); MS (ESI) calcd for
4.1.2. General procedure for preparation of 4a, 6a, and 16a
To a stirred solution of 6-bromobenzo[de]isochromene-1,3-
dione (2.00 g, 7.22 mmol) in EtOH (20 mL) was added the corre-
sponding primary amine (7.94 mmol). The resulting mixture was
heated and refluxed for 2e10 h and monitored by TLC. After
completion, the reaction mixture was cooled to room temperature
and concentrated under vacuum. The crude products were washed
with EtOH, and then recrystallized from EtOH.
C
15H13N2O3 [M ꢁ H]ꢁ: 269.1, found: 269.1.
4.1.3.2. 6-(2-Hydroxyethylamino)-2-ethyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (2b). The residue was purified by column
chromatography on silica gel (DCM:MeOH ¼ 30:1, v/v) to provide
2b. Yellow solid (73.8% yield); mp: 212.9e213.0 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
1.34 (t, J ¼ 7.2 Hz, 3H, CH3), 3.60 (t, J ¼ 4.8 Hz,
4.1.2.1. 6-Bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
2H, NHCH2CH2OH), 4.10 (t, J ¼ 4.8 Hz, 2H, NHCH2CH2OH), 4.24 (q,
J ¼ 7.2 Hz, 2H, CH2CH3), 6.76 (d, J ¼ 8.4 Hz, 1H, ArH), 7.64 (t,
J ¼ 8.0 Hz, 1H, ArH), 8.15 (d, J ¼ 8.4 Hz, 1H, ArH), 8.46 (d, J ¼ 8.4 Hz,
1H, ArH), 8.58 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, DMSO-
(4a). White solid (81.3% yield); mp: 104.7e105.4 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
0.98 (t, J ¼ 7.2 Hz, 3H, CH3), 1.40e1.50 (m, 2H,
CH2CH3), 1.68e1.75 (m, 2H, CH2CH2CH2CH3), 4.17 (t, J ¼ 7.2 Hz, 2H,
CH2CH2CH2CH3), 7.83 (t, J ¼ 8.0 Hz, 1H, ArH), 8.02 (d, J ¼ 7.6 Hz, 1H,
ArH), 8.40 (d, J ¼ 7.2 Hz, 1H, ArH), 8.55 (d, J ¼ 8.4 Hz, 1H, ArH),
d6):
d 13.8 (CH2CH3), 34.7 (CH2CH3), 46.0 (NHCH2CH2OH), 59.2
(NCH2CH2OH), 104.3 (ArC), 108.1 (ArC), 120.6 (ArC), 122.4 (ArC),
124.7 (ArC), 129.1 (ArC), 129.9 (ArC), 131.1 (ArC), 134.7 (ArC), 151.3
(ArC), 163.2 (C]O), 164.0 (C]O); MS (ESI) calcd for C16H17N2O3
[M þ H]þ: 285.1, found: 285.1.
8.64 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
d 13.8
(C2H6CH3), 20.3 (C2H4CH2CH3), 30.2 (CH2CH2CH2CH3), 40.4
(CH2C2H4CH3), 122.3 (ArC), 123.2 (ArC), 128.1 (ArC), 128.9 (ArC),
130.1 (ArC), 130.6 (ArC), 131.0 (ArC), 131.2 (ArC), 131.9 (ArC), 133.2
(ArC), 163.6 (C]O); MS (EI) calcd for C16H14BrNO2 [M]þ: 331.0,
found: 331.0.
4.1.3.3. 6-(2-Hydroxyethylamino)-2-butyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (4b). The residue was purified by column
chromatography on silica gel (DCM:MeOH ¼ 40:1, v/v) to provide
4b. Yellow solid (82.0% yield); mp: 171.7e172.3 ꢃC; 1H NMR
4.1.2.2. 6-Bromo-2-hexyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(6a). White solid (84.6% yield); mp: 66.5e66.7 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
0.97 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH2CH3), 1.40e
(400 MHz, CDCl3):
d
0.89 (t, J ¼ 6.4 Hz, 3H, CH3), 1.33e1.43 (m, 6H,
1.49 (m, 2H, CH2CH2CH2CH3), 1.69e1.74 (m, 2H, CH2CH2CH2CH3),
3.58 (t, J ¼ 5.2 Hz, 2H, NHCH2CH2OH), 4.07 (t, J ¼ 4.8 Hz, 2H,
NHCH2CH2OH), 4.16 (t, J ¼ 7.6, 2H, CH2CH2CH2CH3), 6.78 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.62 (t, J ¼ 8.4 Hz, 1H, ArH), 8.15 (d, J ¼ 8.4 Hz,
1H, ArH), 8.44 (d, J ¼ 8.4 Hz, 1H, ArH), 8.56 (d, J ¼ 7.2 Hz,
CH2CH2C3H6CH3), 1.68e1.75 (m, 2H, CH2CH2C3H6CH3), 4.14 (t,
J ¼ 7.6 Hz, 2H, CH2CH2C3H6CH3), 7.80 (t, J ¼ 8.0 Hz, 1H, ArH), 7.98 (d,
J ¼ 8.0 Hz, 1H, ArH), 8.35 (d, J ¼ 7.6 Hz, 1H, ArH), 8.49 (d, J ¼ 8.4 Hz,
1H, ArH), 8.60 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
d
14.1 (C5H10CH3), 22.6 (C4H8CH2CH3), 26.8 (C3H6CH2CH2CH3), 28.0
1H, ArH); 13C NMR (100 MHz, DMSO-d6):
d 14.2 (C3H6CH3),
(C2H4CH2C2H4CH3), 31.5 (CH2CH2C3H6CH3), 40.6 (CH2C4H8CH3),
122.2 (ArC), 123.1 (ArC), 128.0 (ArC), 128.8 (ArC), 130.1 (ArC), 130.5
(ArC), 131.0 (ArC), 131.1 (ArC), 131.9 (ArC), 133.1 (ArC), 163.4 (C]O),
163.5 (C]O); MS (ESI) calcd for C18H19BrNO2 [M þ H]þ: 360.1,
found: 360.0.
20.3 (C2H4CH2CH3), 30.3 (CH2CH2C2H5), 39.4 (CH2C3H7), 45.8
(NHCH2CH2OH), 59.1 (NCH2CH2OH), 104.3 (ArC), 108.1 (ArC), 120.5
(ArC), 122.3 (ArC), 124.7 (ArC), 129.1 (ArC), 129.9 (ArC), 131.2 (ArC),
134.7 (ArC), 151.2 (ArC), 163.4 (C]O), 164.2 (C]O); MS (ESI) calcd
for C18H21N2O3 [M þ H]þ: 313.2, found: 313.1.
4.1.2.3. 6-Bromo-2-cetyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
4.1.3.4. 6-(2-Hydroxyethylamino)-2-hexyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (6b). The residue was purified by column
chromatography on silica gel (DCM:MeOH ¼ 50:1, v/v) to provide
6b. Yellow solid (72.4% yield); mp: 146.5e146.8 ꢃC; 1H NMR
(16a). White solid (85.8% yield); mp: 81.2e81.7 ꢃC; 1H NMR
(400 MHz, CDCl3):
d
0.80 (t, J ¼ 6.4 Hz, 3H, CH3), 1.17e1.34 (m, 26H,
CH2CH2C13H26CH3), 1.61e1.80 (m, 2H, CH2CH2C13H26CH3), 4.08 (t,
J ¼ 7.6 Hz, 2H, CH2CH2C13H26CH3), 7.77 (t, J ¼ 7.2 Hz，1H, ArH), 7.96
(d, J ¼ 8.0 Hz, 1H, ArH), 8.33 (d, J ¼ 7.6 Hz, 1H, ArH), 8.48
(400 MHz, CDCl3):
d 0.90 (br, 3H, CH2CH2C3H6CH3), 1.25e1.42
(m, 6H, CH2CH2C3H6CH3), 1.69e1.74 (m, 2H, CH2CH2C3H6CH3), 3.58

X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
483
(t, J ¼ 4.4 Hz, 2H, NHCH2CH2OH), 4.07 (t, J ¼ 4.4 Hz, 2H,
NHCH2CH2OH), 4.13 (t, J ¼ 7.6 Hz, 2H, CH2CH2C3H6CH3), 6.70 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.58 (t, J ¼ 7.6 Hz, 1H, ArH), 8.10 (d,
J ¼ 8.0 Hz, 1H, ArH), 8.40 (d, J ¼ 8.4 Hz, 1H, ArH), 8.53 (d, J ¼ 7.2 Hz,
4.1.4.3. 6-((2-Bromoethyl)amino)-2-butyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (4c). Orange solid (83.0% yield); mp: 155.0e
156.0 ꢃC; 1H NMR (400 MHz, CDCl3):
d
0.99 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH2CH3), 1.41e1.51 (m, 2H, CH2CH2CH2CH3), 1.69e1.77 (m,
2H, CH2CH2CH2CH3), 3.77 (t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH), 3.89 (t,
1H, ArH); 13C NMR (100 MHz, DMSO-d6):
d 14.4 (C5H10CH3), 22.5
(C4H8CH2CH3), 26.7 (C3H6CH2C2H5), 28.1 (C2H4CH2C3H7), 31.5
(CH2CH2C4H9), 39.6 (CH2C5H11), 46.0 (NHCH2CH2OH), 59.1
(NCH2CH2OH), 104.3 (ArC), 108.1 (ArC), 120.6 (ArC), 122.3 (ArC),
124.7 (ArC), 129.0 (ArC), 129.9 (ArC), 131.1 (ArC), 134.7 (ArC), 151.3
(ArC), 163.3 (C]O), 164.2 (C]O); MS (ESI) calcd for C20H25N2O3
[M þ H]þ: 341.2, found: 341.1.
J
¼
6.0 Hz, 2H, NHCH2CH2OH), 4.18 (t,
J
¼
7.6 Hz, 2H,
CH2CH2CH2CH3), 6.76 (d, J ¼ 8.4 Hz, 1H, ArH), 7.68 (t, J ¼ 8.0 Hz,
1H, ArH), 8.15 (d, J ¼ 8.4 Hz, 1H, ArH), 8.49 (d, J ¼ 8.4 Hz, 1H,
ArH), 8.62 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
d
13.9 (C3H6CH3), 20.4 (C2H4CH2CH3), 30.3 (CH2CH2C2H5), 30.9
(NHCH2CH2Br), 40.1 (NHCH2CH2Br), 44.6 (CH2C3H7), 104.6 (ArC),
111.8 (ArC), 120.6 (ArC), 123.4 (ArC), 125.2 (ArC), 125.8 (ArC),
129.7 (ArC), 131.3 (ArC), 134.1 (ArC), 148.2 (ArC), 164.1 (C]O), 164.6
(C]O); MS (ESI) calcd for C18H20BrN2O2 [M þ H]þ: 375.1, found:
375.0.
4.1.3.5. 6-(2-Hydroxyethylamino)-2-cetyl-1H-benzo[de]isoquinoline-
1,3 (2H)-dione (16b). The residue was purified by column chro-
matography on silica gel (DCM:MeOH ¼ 50:1, v/v) to provide 16b.
Yellow solid (86.7% yield); mp: 142.7e143.0 ꢃC; 1H NMR (400 MHz,
CDCl3):
d
0.90 (t, J ¼ 6.0 Hz, 3H, CH2CH2C13H26CH3), 1.27e1.44 (m,
4.1.4.4. 6-((2-Bromoethyl)amino)-2-hexyl-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (6c). Orange solid (78.5% yield); mp: 148.2e
26H, CH2CH2C13H26CH3),1.70e1.77 (m, 2H, CH2CH2C13H26CH3), 3.61
(t, J ¼ 5.2 Hz, 2H, NHCH2CH2OH), 4.10 (t, J ¼ 5.2 Hz, 2H,
NHCH2CH2OH), 4.17 (t, J ¼ 7.6 Hz, 2H, CH2CH2C13H26CH3), 6.86 (br,
1H, ArH), 7.67 (t, J ¼ 8.0 Hz, 1H, ArH), 8.19 (d, J ¼ 8.4 Hz, 1H, ArH),
8.48 (d, J ¼ 8.4 Hz, 1H, ArH), 8.61 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR
148.7 ꢃC; 1H NMR (400 MHz, CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H,
CH2CH2C3H6CH3), 1.31e1.41 (m, 6H, CH2CH2C3H6CH3), 1.67e1.75
(m, 2H, CH2CH2C3H6CH3), 3.74 (t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH),
3.86 (t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH), 4.14 (t, J ¼ 7.6 Hz, 2H,
CH2CH2C3H6CH3), 6.73 (d, J ¼ 8.4 Hz, 1H, ArH), 7.64 (t, J ¼ 7.6 Hz,
1H, ArH), 8.13 (d, J ¼ 8.4 Hz, 1H, ArH), 8.45 (d, J ¼ 8.4 Hz, 1H,
ArH), 8.58 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
(100 MHz, DMSO-d6):
(C13H26CH2C2H5), 28.1 (C12
d
14.4 (C15H30CH3), 22.6 (C14H28CH2CH3), 27.0
H24CH2C3H7), 29.1 (C2H4C11H22C3H7),
29.2 (C2H4C11H22C3H7), 29.3 (C2H4C11H22C3H7), 29.4 (C2H4C11H22
C3H7), 29.5 (C2H4C11H22C3H7), 31.8 (CH2CH2C14H29), 39.6 (CH2C15
H131), 46.0 (NHCH2CH2OH), 59.2 (NCH2CH2OH), 104.3 (ArC), 108.0
(ArC), 120.6 (ArC), 122.3 (ArC), 124.6 (ArC), 129.1 (ArC), 129.9 (ArC),
131.1 (ArC), 134.6 (ArC), 151.3 (ArC), 163.3 (C]O), 164.2 (C]O); MS
(ESI) calcd for C30H45N2O3 [M þ H]þ: 481.3, found: 481.3.
d
14.1 (C5H10CH3), 22.6 (C4H8CH2CH3), 26.9 (C3H6CH2C2H5), 28.2
(C2H4CH2C3H7), 30.9 (CH2CH2C4H9), 31.6 (NHCH2CH2Br), 40.3
(NHCH2CH2Br), 44.7 (CH2C5H11), 104.6 (ArC), 111.7 (ArC), 120.6
(ArC), 123.3 (ArC), 125.2 (ArC), 125.8 (ArC), 129.7 (ArC), 131.3
(ArC), 134.1 (ArC), 148.2 (ArC), 164.0 (C]O), 164.5 (C]O); MS
(ESI) calcd for C20H24BrN2O2 [M þ H]þ: 403.1, found: 403.1.
4.1.4. General procedure for the preparation of 1ce16c
To a stirred solution of DDQ (545 mg, 2.40 mmol) and PPh3
(629 mg, 2.40 mmol) in dry CH2Cl2, (n-butyl)4NBr (773 mg,
2.40 mmol) was added at room temperature. 1be16b
(2.00 mmol) was then added to this mixture, which immediately
turned the yellow color of the reaction mixture to deep red. The
mixture was stirred at room temperature for 12e24 h. The sol-
vent was then removed under vacuum to fifth of the volume. The
residue was purified by column chromatography on silica gel
using pure CH2Cl2 as eluant to give the corresponding bromide
product.
4.1.4.5. 6-((2-Bromoethyl)amino)-2-cetyl-1H-benzo[de]isoquinoline-
1,3 (2H)-dione (16c). Orange solid (78.7% yield); mp: 121.6e
121.9 ꢃC; 1H NMR (400 MHz, CDCl3):
d
0.88 (t, J ¼ 6.4 Hz, 3H,
CH2CH2C13H26CH3), 1.24e1.43 (m, 26H, CH2CH2C13H26CH3), 1.68e
1.75 (m, 2H, CH2CH2C13H26CH3), 3.75 (t, 6.0 Hz, 2H,
J
¼
NHCH2CH2OH), 3.87 (t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH), 4.15 (t,
J ¼ 7.6 Hz, 2H, CH2CH2C13H26CH3), 6.73 (d, J ¼ 8.4 Hz, 1H, ArH), 7.65
(t, J ¼ 7.6 Hz, 1H, ArH), 8.14 (d, J ¼ 8.4 Hz, 1H, ArH), 8.46
(d, J ¼ 8.4 Hz, 1H, ArH), 8.59 (d, J ¼ 6.8 Hz, 1H, ArH); 13C NMR
(100 MHz, CDCl3):
d 14.1 (C15H30CH3), 22.7 (C14H28CH2CH3), 27.2
(C2H4C12H24C2H5), 28.2 (C2H4C12H24C2H5), 29.4 (C2H4C12H24C2H5),
29.4 (C2H4C12H24C2H5), 29.6 (C2H4C12H24C2H5), 29.7 (C2H4C12H24
C2H5), 29.7 (C2H4C12H24C2H5), 30.8 (C2H4C12H24C2H5), 31.9 (NHCH2
CH2Br), 40.3 (NHCH2CH2Br), 44.7 (CH2C15H31), 104.5 (ArC), 111.6
(ArC), 120.5 (ArC), 123.3 (ArC), 124.1 (ArC), 125.8 (ArC), 129.7 (ArC),
131.2 (ArC), 134.0 (ArC), 148.3 (ArC), 164.0 (C]O), 164.5 (C]O); MS
(ESI) calcd for C30H44BrN2O2 [M þ H]þ: 543.2, found: 543.2.
4.1.4.1. 6-((2-Bromoethyl)amino)-2-methy-1H-benzo[de]isoquino-
line-1,3 (2H)-dione (1c). Orange solid (78.0% yield); mp: 208.8e
210.8 ꢃC; 1H NMR (400 MHz, CDCl3):
d
3.54 (s, 3H, CH3), 3.76 (t,
J
¼
5.8 Hz, 2H, NHCH2CH2OH), 3.89 (t, 5.8 Hz, 2H,
J
¼
NHCH2CH2OH), 6.75 (d, J ¼ 6.9 Hz, 1H, ArH), 7.69 (t, J ¼ 8.0 Hz, 1H,
ArH), 8.15 (d, J ¼ 8.4 Hz, 1H, ArH), 8.50 (d, J ¼ 7.2 Hz, 1H, ArH), 8.63
(d, J ¼ 7.2 Hz, 1H, ArH); MS (EI) calcd for C15H13BrN2O2 [M]þ: 332.0,
found: 332.0.
4.1.5. General procedure for the preparation of 1de16d
To a stirred solution of the bromide precursor (0.225 mmol) and
KI (45 mg, 0.271 mmol) in dry CHCl3 was added the N1,N1-dime-
thylethane-1,2-diamine (1.12 mmol) under argon. The mixture was
heated and refluxed for 48 h. The reaction mixture was then cooled
to room temperature and concentrated under vacuum.
4.1.4.2. 6-((2-Bromoethyl)amino)-2-ethy-1H-benzo[de]isoquinoline-
1,3 (2H)-dione (2c). Orange solid (80.2% yield); mp: 209.1e
209.2 ꢃC; 1H NMR (400 MHz, CDCl3):
d
1.32 (t, J ¼ 7.2 Hz,
3H, CH2CH3), 3.76 (t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH), 3.87
(t, J ¼ 6.0 Hz, 2H, NHCH2CH2OH), 4.23 (q, J ¼ 7.2 Hz, 2H, CH2CH3),
6.75 (d, J ¼ 8.4 Hz, 1H, ArH), 7.68 (t, J ¼ 8.0 Hz, 1H, ArH), 8.14
4.1.5.1. 6-((2-((2-(dimethylamino)ethyl)amino)ethyl)amino)-2-
methyl-1H-benzo[de] isoquinoline-1,3(2H)-dione (1d). The crude
product was purified by column chromatography on silica gel
(DCM:MeOH:Et3N ¼ 100:20:1, v/v/v) to provide 1d. Orange solid
(d,
J
¼ 8.4 Hz, 1H, ArH), 8.48 (d,
J
¼ 8.4 Hz, 1H, ArH),
8.61 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3):
d 13.4
(CH2CH3), 30.9 (NHCH2CH2Br), 35.3 (NHCH2CH2Br), 44.6
(CH2CH3), 104.6 (ArC), 111.8 (ArC), 120.6 (ArC), 123.4 (ArC),
125.8 (ArC), 129.7 (ArC), 131.2 (ArC), 134.0 (ArC), 148.2 (ArC),
163.9 (C]O, 164.4 (C]O); MS (ESI) calcd for C16H15BrN2O2
[M þ H]þ: 347.0, found: 347.0.
(62.0% yield); mp: 93.4e94.7 ꢃC; 1H NMR (400 MHz, CD3OD):
d 2.31
(s, 6H, N(CH3)2), 2.57 (t, J ¼ 6.8 Hz, 2H, NCH2CH2NHCH2CH2N), 2.85
(t, J ¼ 6.8 Hz, 2H, NCH2CH2NHCH2CH2N), 2.98 (t, J ¼ 6.4 Hz, 2H,
NCH2CH2NHCH2CH2N), 3.22 (s, 3H, CH3), 3.42 (t, J ¼ 6.4 Hz, 2H,

484
X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
NCH2CH2NHCH2CH2N), 6.35 (d, J ¼ 8.4 Hz, 1H, ArH), 7.21 (t,
J ¼ 8.0 Hz, 1H, ArH), 7.77 (d, J ¼ 8.4 Hz, 1H, ArH), 7.93 (d, J ¼ 6.8 Hz,
1H, ArH), 8.01 (d, J ¼ 8.0 Hz, 1H, ArH); 13C NMR (100 MHz, CD3OD):
13C NMR (100 MHz, CDCl3):
d 14.1 (C5H10CH3), 22.6 (C4H10CH2CH3),
26.9 (C3H6CH2C2H5), 28.2 (C2H4CH2C3H7), 31.6 (CH2CH2C4H9), 40.2
(CH2C5H11), 42.2 (NHCH2CH2N(CH3)2), 45.4 (NHCH2CH2N(CH3)2),
46.2 (NHCH2CH2NH), 47.3 (NHCH2CH2NH), 58.6 (NHCH2CH2
N(CH3)2), 104.3 (ArC), 110.1 (ArC), 120.5 (ArC), 123.0 (ArC), 124.5
(ArC), 126.7 (ArC), 129.8 (ArC), 131.0 (ArC), 134.5 (ArC), 149.8 (ArC),
164.2 (C]O), 164.7 (C]O); HRMS (ESI) calcd for C24H35N4O2
[M þ H]þ: 411.2760, found: 411.2755.
d
25.6 (CH3), 42.3 (NHCH2CH2N(CH3)2), 44.2 (NHCH2CH2N(CH3)2),
46.1 (NHCH2CH2NH), 48.5 (NHCH2CH2NH), 58.0 (NHCH2CH2
N(CH3)2), 103.5 (ArC), 108.1 (ArC), 120.2 (ArC), 121.5 (ArC), 123.8
(ArC), 127.6 (ArC), 129.2 (ArC), 130.4 (ArC), 134.1 (ArC), 150.7 (ArC),
164.2 (C]O), 164.7 (C]O); HRMS (ESI) calcd for C19H25N4O2
[M þ H]þ: 341.1978, found: 341.1974.
4.1.5.5. 6-((2-((2-(dimethylamino)ethyl)amino)ethyl)amino)-2-
cetyl-1H-benzo[de] isoquinoline-1,3(2H)-dione (16d). The resulting
mixture was diluted with water (10 mL) containing 10% NaCl and
1% Na2CO3 and extracted with CH2Cl2 (3 ꢂ 15 mL). The organic layer
was then concentrated under vacuum and then further purified.
The crude product was purified by column chromatography on
silica gel (DCM:MeOH:Et3N ¼ 100:10:1, v/v/v) to provide 16d. Or-
ange solid (70.0% yield); mp: 128.6e130.2 ꢃC; 1H NMR (400 MHz,
4.1.5.2. 6-((2-((2-(dimethylamino)ethyl)amino)ethyl)amino)-2-
ethyl-1H-benzo[de] isoquinoline-1,3(2H)-dione (2d). The crude
product was purified by column chromatography on silica gel
(DCM:MeOH:Et3N ¼ 100:20:1, v/v/v) to provide 2d. Orange solid
(65.0% yield); mp: 93.0e94.1 ꢃC; 1H NMR (400 MHz, CD3OD):
d 1.24 (t,
J ¼ 6.8 Hz, 3H, CH2CH3), 2.32 (s, 6H N(CH3)2), 2.59 (t, J ¼ 6.8 Hz, 2H,
NCH2CH2NHCH2CH2N), 2.86 (t, J ¼ 6.8 Hz, 2H NCH2CH2NHCH2CH2N),
3.03 (t, J ¼ 6.4 Hz, 2H NCH2CH2NHCH2CH2N), 3.51 (t, J ¼ 6.4 Hz, 2H
NCH2CH2NHCH2CH2N), 4.04 (q, J ¼ 7.2 Hz, 2H, CH2CH3), 6.55
(d, J ¼ 8.8 Hz,1H, ArH), 7.38 (t, J ¼ 8.0 Hz,1H, ArH), 8.03 (d, J ¼ 8.4 Hz,
1H, ArH), 8.17 (d, J ¼ 7.6 Hz, 1H, ArH), 8.20 (d, J ¼ 8.4 Hz, 1H, ArH);
CDCl3):
d
0.88 (t, J ¼ 6.4 Hz, 3H, CH2CH2C13H26CH3), 1.30e1.43 (m,
26H, CH2CH2C13H26CH3), 1.67e1.74 (m, 2H, CH2CH2C13H26CH3), 2.36
(s, 6H), 2.70 (t, J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N), 2.92 (t,
J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N), 3.18 (t, J ¼ 5.6 Hz, 2H,
NCH2CH2NCH2CH2N), 3.59 (br, 2H, NCH2CH2NCH2CH2N), 4.13 (t,
J ¼ 7.6 Hz, 2H, CH2CH2C13H26CH3), 6.63 (d, J ¼ 8.4 Hz, 1H, ArH), 6.93
(s, 1H, ArH), 7.61 (t, J ¼ 7.6 Hz, 1H, ArH), 8.40 (d, J ¼ 8.4 Hz, 1H, ArH),
13C NMR (100 MHz, CD3OD):
d 13.75 (CH2CH3), 36.02 (CH2CH3),
43.59 (NHCH2CH2N(CH3)2), 45.59 (NHCH2CH2N(CH3)2), 47.4 (NHCH2
CH2NH), 48.5 (NHCH2CH2NH), 59.3 (NHCH2CH2N(CH3)2),104.9 (ArC),
109.6 (ArC),121 (ArC),123.0(ArC),125.2(ArC),128.9(ArC),130.6(ArC),
131.7 (ArC), 135.3 (ArC), 152.0 (ArC), 165.1 (C]O), 165.60 (C]O);
HRMS (ESI) calcd for C20H27N4O2 [M þ H]þ: 355.2134, found:
355.2134.
8.53e8.57 (m, 2H, ArH); 13C NMR (100 MHz, CDCl3):
d
14.1
(C15H33CH3), 22.7 (C14
H28CH2CH3), 27.3 (C2H4C12H24C2H5), 28.3
(C2H4C12H24C2H5), 29.4 (C2H4C12H24C2H5), 29.5 (C2H4C12H24C2H5),
29.6 (C2H4C12H24C2H5), 29.7 (C2H4C12H24C2H5), 29.7 (C2H4C12
H24C2H5), 31.9 (CH2CH2C14H29), 40.2 (CH2C15H31), 41.9 (NHCH2CH2
N(CH3)2), 44.8 (NHCH2CH2N(CH3)2), 45.4 (NHCH2CH2NH), 47.0
(NHCH2CH2NH), 57.4 (NHCH2CH2N(CH3)2), 104.9 (ArC), 110.2 (ArC),
120.7 (ArC), 122.8 (ArC), 124.7 (ArC), 127.7 (ArC), 129.8 (ArC), 131.2
(ArC), 134.3 (ArC), 149.7 (ArC), 164.2(C]O), 164.8 (C]O); HRMS
(ESI) calcd for C34H55N4O2 [M þ H]þ: 551.4325, found: 551.4329.
4.1.5.3. 6-((2-((2-(dimethylamino)ethyl)amino)ethyl)amino)-2-
butyl-1H-benzo[de] isoquinoline-1,3(2H)-dione (4d). The crude
product was purified by column chromatography on silica gel
(DCM:MeOH:Et3N ¼ 100:15:1, v/v/v) to provide 4d. Orange solid
(67.0% yield); mp: 90.1e92.0 ꢃC; 1H NMR (400 MHz, CDCl3):
d 0.97
(t,
J
¼
7.2 Hz, 3H, CH2CH2CH2CH3), 1.40e1.49 (m, 2H,
CH2CH2CH2CH3), 1.67e1.75 (m, 2H, CH2CH2CH2CH3), 2.27 (s, 6H,
N(CH3)2), 2.49 (t, J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N), 2.78 (t,
J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N), 3.10 (t, J ¼ 5.6 Hz, 2H,
NCH2CH2NCH2CH2N), 3.43 (q, J ¼ 5.2 Hz, 2H, NCH2CH2NCH2CH2N),
4.16 (t, J ¼ 7.6 Hz, 2H, CH2CH2CH2CH3), 6.43 (s, 1H, ArH), 6.67 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.60 (t, J ¼ 7.8 Hz, 1H, ArH), 8.22 (d, J ¼ 8.4 Hz,
1H, ArH), 8.44 (d, J ¼ 8.4 Hz, 1H, ArH), 8.57 (d, J ¼ 7.2 Hz, 1H, ArH);
4.2. kDNA decatenation assay [29]
Topo II activity was measured by the ATP-dependent decate-
nation of kinetoplast DNA (kDNA) according to the manufac-
turer’s instructions (TopoGEN, Florida, USA). 0.1 mg kDNA, 1 unit
of human topoIIa (TopoGEN) and the indicated concentrations of
compounds were incubated for 30 min at 37 ꢃC in 50 mM Trise
13C NMR (100 MHz, CDCl3):
d 13.9 (C3H7CH3), 20.5 (C2H4CH2CH3),
HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM DTT
30.3 (CH2CH2C2H5), 40.0 (CH2C3H7), 42.2 (NHCH2CH2N(CH3)2), 45.4
(NHCH2CH2N(CH3)2), 46.3 (NHCH2CH2NH), 47.3 (NHCH2CH2NH),
58.8 (NHCH2CH2N(CH3)2), 104.3, 110.1, 120.5, 123.0, 124.6, 126.6,
129.8, 131.1, 134.5, 149.7, 164.2, 164.8; HRMS (ESI) calcd for
in a total volume of 20
m
L. The reactions were stopped by the
L 6ꢂ loading dye solution
addition of 2 L 10% SDS and 2
m
m
(Fermentas). Samples 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.
C
22H31N4O2 [M þ H]þ: 383.2447, found: 383.2445.
4.1.5.4. 6-((2-((2-(dimethylamino)ethyl)amino)ethyl)amino)-2-
hexyl-1H-benzo[de] isoquinoline-1,3(2H)-dione (6d). The resulting
mixture was diluted with water (10 mL) containing 10% NaCl and
1% Na2CO3 and extracted with CH2Cl2 (3 ꢂ 15 mL). The organic layer
was then concentrated under vacuum and then further purified.
The crude product was purified by column chromatography on
silica gel (DCM:MeOH:Et3N ¼ 100:10:1, v/v/v) to provide 6d. Or-
ange solid (71.0% yield); mp: 83.2e85.1 ꢃC; 1H NMR (400 MHz,
4.3. DNA intercalating assay by CD spectra
ct-DNA was purchased from Sigma Aldrich and used without
further purification. A solution of ct-DNA in 20 mM Tris-HCl
buffer (pH ¼ 7.4) was stored at 4 ꢃC. The concentration of ct-
DNA was determined spectrophotometrically from themolar ab-
sorption coefficient (6600 Mꢁ1 cmꢁ1). The stock solution was
attested to be sufficiently free from protein, as it gave a UV
absorbance ratio at 260 and 280 nm of more than 1.8. The CD
measurements were performed on a Chirascan spectrophotom-
eter (Photophysics, England) using 1 cm quartz cell in the range
CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H, CH2CH2C3H6CH3), 1.31e1.42 (m, 6H,
CH2CH2C3H6CH3), 1.70e1.72 (m, 2H, CH2CH2C3H6CH3), 2.27 (s, 6H,
N(CH3)2), 2.51 (t, J ¼ 6.4 Hz, 2H, NCH2CH2NCH2CH2N), 2.80 (t,
J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N), 3.11 (t, J ¼ 6.0 Hz, 2H,
NCH2CH2NCH2CH2N), 3.44 (t, J ¼ 6.0 Hz, 2H, NCH2CH2NCH2CH2N),
4.14 (t, J ¼ 7.6 Hz, 2H, CH2CH2C3H6CH3), 6.49 (s, 1H, ArH), 6.65 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.59 (t, J ¼ 8.0 Hz, 1H, ArH), 8.25 (d, J ¼ 8.0 Hz,
1H, ArH), 8.42 (d, J ¼ 8.4 Hz, 1H, ArH), 8.55 (d, J ¼ 7.2 Hz, 1H, ArH);
of 200e400 nm ct-DNA (100 mM) interacted with drugs (20 mM)
in Tris-HCl buffer for 2 h at 25 ꢃC. CD spectra in the range of 230e
300 nm were analyzed.

X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
485
4.4. DNA intercalating assay by fluorescence spectra [34]
4.8. Protein kinase assays [36]
The fluorescence spectra were obtained using a Varian Cary
Eclipse fluorescence spectrophotometer at 25 ꢃC. The concentra-
Tyrosine kinase activity was determined by an enzyme-linked-
immunosorbent assay (ELISA) in 96-well plates pre-coated with
tions of compounds and calf thymus DNA were 10
mM and 0e80
mM
2.5
mg/well poly (Glu, Tyr)4:1 (Sigma) as a substrate. Fifty microliters
respectively in 20 mM Tris-HCl buffer (pH ¼ 7.5) and 1% DMSO.
And an equilibrium period of 1 h for constant stirring at 25 ꢃC in the
dark was allowed. The association constants (Kb) were derived
according to equation I ¼ I0þ{(IN ꢁ I0)/2[Q]0)} ꢂ {([DNA]0þ[Q]0 þ 1/
Ka) ꢁ {([DNA]0þ[Q]0 þ 1/Ka)2 ꢁ 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.
of 10
m
M ATP solution diluted in reaction buffer [50 mM HEPES, pH
7.4, 20 mM MgCl2, 0.1 mM Na3VO4, and 1 mM DTT] was added to
each well, and the reaction was initiated by the addition of
increasing concentrations of tyrosine kinase. After incubation for
1 h at 37 ꢃC, the plate was washed three times with phosphate
buffered saline containing 0.1% Tween20 (PBST). Next, 100 mL of
antiphosphotyrosine (PY99; 1:1000 dilution) antibody was added.
After 0.5 h incubation at 37 ꢃC, the plate was washed three times
and goat anti-mouse IgG horseradish peroxidase (100
mL of a
1:2000 dilution) diluted in PBST containing 5 mg/mL BSA was
4.5. Evaluation of solubility [35] and logP
added. The plate was reincubated at 37 ꢃC for 0.5 h and washed as
before. Finally, 100 mL of color development solution (0.03% H2O2
A Varian Cary100 Bio UVeVisible spectrophotometer was used
to detect the absorption spectra of the tested compounds. Stock
solutions of the tested compounds were prepared in DMSO at a
concentration of 10ꢁ3 M. The maximum absorbance wavelengths
of the compounds were all around 450 nm. Standard curves of
the compounds were tested by several concentrations in PBS
(20 mM, pH ¼ 7.4). The equations obtained from the standard
curves were used to calculate the corresponding solubility. In
detail, excessive compound was added to PBS, and shaken for
24 h to be fully dissolved. The mixture was centrifugated at
104 rpm for 10 min. Then the saturated supernatant was taken for
detection. The supernatant was diluted to the concentration
range of corresponding standard curve. The solubility was
calculated by the equation of standard curve from three inde-
pendent tests. The log P values were calculated with ACD/Labs
software V12.
and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.4)
was added and the plate was incubated at room temperature until
color emerged. The reaction was terminated by the addition
of 50 mL of 2 M H2SO4, and then the plate was read using a multi-
well spectrophotometer (MAX190Ô) at 490 nm. The inhibition
rate (%) was calculated using the following equation: inhibition
rate ¼ [1 ꢁ (A490 treated/A490control)] ꢂ 100%. IC50 values were
determined from the results of at least three independent tests and
calculated from the inhibition curves.
4.9. Cell migration assay
HMEC-1s migration was determined in a transwell Boyden
chamber (Costar, MA, USA) [37]. Briefly, cell suspension
(5 ꢂ 105 cells/mL) with different concentrations of 8d or control
was added to the upper compartment of the chamber. The lower
compartment contained 20% FBS MCDB131 medium and the same
concentrations of 8d or control. After incubation (6 h, 37 ꢃC),
the inhibition of migration was calculated: inhibition of
migration ¼ [1 ꢁ (Acompound ꢁ Ablank)/(Acontrol ꢁ Ablank)] ꢂ 100%.
4.6. Cell lines and cell culture
MDA-MB-231, A549, HMEC-1 were purchased from American
Type Culture Collection (ATCC, Manassas, VA, USA). HL60 and LO2
were from Shanghai Institute of Biochemistry and Cell Biology
(Shanghai, China). GES-1 was obtained from Renji Hospital
(Shanghai, China). They were maintained in strict accordance with
the supplier’s instructions and established procedures.
4.10. Tube formation assay
Matrigel (75 mL wellꢁ1) was used to coat 96-well plates, allowed
to solidify (37 ꢃC, 1 h) [38], prior to seeding with HMEC-1s
(1 ꢂ 105 cells/mL) that were then cultured (37 ꢃC, 8 h) in
MCDB131 medium with 20% FBS containing 8d or control. For
investigation of neovessel disruption, HMEC-1s were seeded on
Matrigel and left to align for 24 h. The formed capillary-like
structures or cords were exposed to 8d, and then examined with
an inverted phase contrast microscope (DP70, Olympus, Japan). The
number of the tubes was quantified from five random fields. The
inhibition of tube formation was calculated: inhibition of tube
formation ¼ [1 ꢁ (tubescompound/tubescontrol)] ꢂ 100%.
4.7. Cell proliferation assay
Cell proliferation was evaluated using the SRB or MTT assay.
Firstly, cancer cells were seeded into 96-well plates and cultured
overnight. The cells were then treated with increasing concentra-
tions of compounds for a further 72 h. For MDA-MB-231, A549, LO2,
GES-1, HMEC-1 cell lines, cells were then fixed with 10% tri-
chloroacetic acid and stained with sulforhodamine B (Sigma). Sul-
forhodamine B in the cells was dissolved in 10 mM TriseHCl and
was measured at 515 nm using a multiwell spectrophotometer
(MAX190Ô, Molecular Devices, Sunnyvale, USA). The inhibition
rate on cell proliferation was calculated as follows: inhibition
4.11. Western blot analysis [39]
HMEC-1 cells were plated into 6-well plate (3 ꢂ 105 cell/well).
After adherence, cells were starved and incubated in serum-free
medium for 24 h and then exposed to corresponding compounds
for 12 h. For analysis of FGFR1 phosphorylation and downstream
signal transduction pathways, cells were stimulated with 50 ng/ml
bFGF for 10 min at 37 ꢃC at the end of compounds treatment.
Whole-cell lysates were collected and boiled for 10 min in 2 ꢂ SDS
sample buffer, subjected to 10% SDS-PAGE, and transferred to
nitrocellulose (Amersham Life Sciences). The blot was blocked in
blocking buffer (5% non-fat dry milk/1% Tween-20 in TBS) for 1 h at
room temperature, and then incubated with primary antibodies
rate ¼ (1 ꢁ A515 treated/A515 control) ꢂ 100%. For HL60 cell line, 20
(5 mg/mL in 0.9% brine) of MTT (Sigma) was added to each well. The
cells were then incubated for an additional 4 h, after which 100
mL
mL
of “triplex solution” (10% SDS-5% isobutanol-HCl, 12 mM) was
added, and the cells were incubated overnight at 37 ꢃC. The plates
were read at 570 nm on the scanning multiwell spectrophotometer
(MAX190Ô). The inhibition rate on cell proliferation was calculated
as: inhibition rate ¼ (1 ꢁ A570 treated/A570 control) ꢂ 100%. The IC50
values were obtained by the Logit method. Each experiment was
repeated in triplicate.

486
X. Wang et al. / European Journal of Medicinal Chemistry 65 (2013) 477e486
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vascular endothelial growth factor receptor-2 and fibroblast growth factor
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(anti- phospho-FGFR1 (1:200), anti-phospho-AKT (1:500), anti-
phospho-Erk (1:2000) and anti-GAPDH (1:10000)) in blocking
buffer for 2 h at room temperature. The bands were then visualized
using horseradish peroxidase-conjugated secondary antibodies
(1:2000) followed by ECL (Pierce Biotech, Rockford, IL).
a
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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 National Natural Science
Foundation of China (Grant 81173080), the Shanghai Committee
of Science and Technology (Grant 10431902600 and Grant
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Supplementary data related to this article can be found at
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