Antiproliferative and apoptosis-inducing activities of novel naphthalimide-cyclam conjugates through dual topoisomerase (topo) I/II inhibition

Article abstract of DOI:10.1016/j.bmc.2015.07.011

A novel series of naphthalimide-cyclam conjugates were designed and synthesized. Among them, compounds 4c, 4d, 8c and 8d which bearing long lipophilic alkyl chains, displayed comparable or more potent cytotoxic activities against human tumor cell lines than amonafide. Furthermore, the four compounds were proved to possess strong inhibition against both topoisomerase I and II. The representative compound 8c exhibited moderate DNA intercalation activity. Molecular modeling studies identified the possible interaction of compound 8c with the molecular target by forming topoisomerase/DNA/drug ternary complex. Finally, derivatives with long lipophilic alkyl chains could efficiently induce apoptosis.

Full text of DOI:10.1016/j.bmc.2015.07.011

Bioorganic & Medicinal Chemistry 23 (2015) 5672–5680
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiproliferative and apoptosis-inducing activities of novel
naphthalimide–cyclam conjugates through dual topoisomerase
(topo) I/II inhibition
Shaoying Tan , Deheng Sun , Jiankun Lyu , Xiao Sun, Fangshu Wu, Qiang Li, Yiqi Yang, Jianxu Liu,
⇑
⇑
⇑
Xin Wang, Zhuo Chen , Honglin Li , Xuhong Qian, Yufang Xu
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, Shanghai Key Laboratory of Chemical Biology, 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:
A novel series of naphthalimide–cyclam conjugates were designed and synthesized. Among them, com-
pounds 4c, 4d, 8c and 8d which bearing long lipophilic alkyl chains, displayed comparable or more potent
cytotoxic activities against human tumor cell lines than amonafide. Furthermore, the four compounds
were proved to possess strong inhibition against both topoisomerase I and II. The representative com-
pound 8c exhibited moderate DNA intercalation activity. Molecular modeling studies identified the pos-
sible interaction of compound 8c with the molecular target by forming topoisomerase/DNA/drug ternary
complex. Finally, derivatives with long lipophilic alkyl chains could efficiently induce apoptosis.
Ó 2015 Published by Elsevier Ltd.
Received 29 May 2015
Revised 8 July 2015
Accepted 9 July 2015
Available online 15 July 2015
Keywords:
Naphthalimide–cyclam conjugates
Antitumor agents
Topoisomerase I
Topoisomerase II
DNA intercalation
Apoptosis
1. Introduction
Inhibition of topoisomerases could be classified by two cate-
gories: one is topoisomerase poisons which stabilizing the DNA–
Cancers, which figure among the leading causes of death world-
wide, account for 8.2 million deaths in 2012, and a proposed rise to
13.1 million deaths in 2030.1,2 Developing new anticancer drugs is
an important goal of research nowadays. DNA targeted antitumor
agents act as a mainstay of cancer chemotherapeutic agents by
forming covalent complexes and/or declining in the function of
DNA-processing enzymes.3–5 DNA topoisomerases are omnipre-
sent nuclear enzymes as active participant in the topological rear-
rangement of DNA during cellular processes like replication and
transcription.6 Topo I and topo II are the two main types of topoi-
somerases. Both of them could relax DNA supercoiling through a
cleavage/relegation mechanism, only that topo I generates DNA
single strand breaks and topo II breaks both strands of DNA.7
Topoisomerases are over-expressed in cancer cells. To interfere
with the enzymes or generate enzyme-mediated damages are clin-
ically significant strategy in anticancer therapy.
enzyme cleavable complex and causing an accumulation of broken
DNA in the cell.8 Most of these agents possess the structure compo-
nents of a planar aromatic scaffold and a hydrogen bond donor side
chain. Catalytic inhibitors, on the other hands, block the enzyme
pocket before DNA cleavage or inhibit any steps of the catalytic
cycle.9–12 Compounds which modulate topo I or topo II specifically
were used clinically in the treatment of various cancers. For exam-
ple, camptothecin (CPT) analogues (topotecan and irinotecan) as
topo I poisons and anthracycline derivatives (daunorubicin and
doxorubicin) which inhibits topo II. New research also found that
topo II were promising therapeutic target for the treatment
of colon cancers with defective decatenation checkpoint.13
However, it is found that selective topo I inhibition can induce a
concomitant rise in the level of topo II expression, and vice versa.
This phenomenon would lead to drug resistance and failure of clin-
ical therapies ultimately. In this regard, compounds which have the
potential to simultaneously inhibit topoisomerase I and II attracted
great attention to improve antitumor activities and to overcome
drug resistance problem.8,14–17
⇑
Corresponding authors. Tel./fax: +86 21 64250823 (Z.C.).
E-mail addresses: chenzhuo@ecust.edu.cn (Z. Chen), hlli@ecust.edu.cn (H. Li),
yfxu@ecust.edu.cn (Y. Xu).
Macrocyclic derivatives, especially tetraazamacrocycles are
applied in medical use. Cyclam (1,4,8,11-tetraazamacrocycle) is
one of the most important members in the tetraazamacrocycles
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.07.011
0968-0896/Ó 2015 Published by Elsevier Ltd.

S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
5673
family.18,19 Normally, the medical use of tetraazamacrocycles is
2. Chemistry
owing to their characters of binding various transition metal ions
with high kinetic and thermodynamic stability.20–22 Metal ion
complexes of cyclam are applied in diagnosis and the treatment
of AIDs.19 Recently, cyclam–metal ion complexes were reported
of their antitumor activities. Furthermore, introduction of lipophi-
lic substitution were proved to be able to improve the ability of
antiporliferative activity.23,24 According to Sibert et al., two new
lipophilic cyclam derivatives exhibited potent cytotoxic activity
The target molecules 4a–4d were synthesized in four steps from
6-bromobenzo[de]isochromene-1,3-dione as shown in Scheme 1.
Firstly, the 6-bromobenzo[de]isochromene-1,3-dione was refluxed
in ethanol with corresponding primary amine to obtain 1a–1d.
Then, 1a–1d reacted with ethanol amine under Ullmann’s
condition to get 2a–2d, which further converted to 3a–3d in the
existence of 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ), tetra-
butyl ammonium bromide and triphenylphosphine. Then the tar-
get molecules 4a–4d were finally obtained by cyclam condensed
with 3a–3d in CHCl3. 8a–8d were synthesized in a similar process
with 4a–4d as shown in Scheme 2.
with IC50 values below 10 l
M against cancer cell line L1210.23
Naphthalimide antitumor agents normally possess structural
features of DNA intercalators, namely a flat aromatic or heteroaro-
matic moiety and basic side chains.25 As shown in Figure 1, amon-
afide, which is the most famous one of them, could intercalate into
DNA and strongly inhibited topo II. Amonafide reenters phase III
clinical trial against secondary acute myeloid leukemia recently.26
Herein, due to the antitumor potency of azomacrocycles, cyclam
were introduced at the 2- or 6-position of naphthalimide core
instead of flexible basic side chain. Besides, lipophilic long alkyl
chains were introduced to naphthalimide–cyclam conjugates in
comparison with methyl and butyl substitution (see Fig. 1). The
newly-synthesized compounds were evaluated of their antiprolif-
erative activity against solid tumor cell lines compared with amon-
afide and cyclam. Representative compounds were evaluated of
their topo II/I inhibitory, DNA intercalation and collectively apop-
tosis inducing activity.
3. Results and discussions
3.1. In vitro cytotoxic activity
The antiproliferative activity of compounds 4a–4d, 8a–8b were
evaluated against human cancer cell lines A549 (human non-small
cell lung tumor cell), HeLa (human cervical carcinoma cell line),
and HCT116 (human colon cancer cell line). Amonafide and cyclam
were tested as reference compounds.
As show in the Table 1, all of the synthesized compounds dis-
played better antiporliferative than cyclam. Compounds 4c–4d,
8c–8d exhibited comparable or even more potent antiproliferative
Figure 1. Structure of cyclam, amonafide and the design strategy of naphthalimide–cyclam conjugates.
R
N
R
N
R
N
R
N
O
O
O
O
O
O
O
O
O
O
O
a
d
b
c
HN
3a-3d
HN
4a-4d
Br
Br
HN
2a-2d
N
H
HN
Br
N
1a-1d
OH
H
N
R=CH3, C4H9, C8H17, C12H25
Scheme 1. Syntheses of target compounds 4a–4d. (a) Corresponding primary amines, ethanol, reflux, 3 h; (b) ethanol amine, methoxyethanol, reflux, 4 h; (c) DDQ, PPh3,
(n-butyl)4NBr, CH2Cl2, rt, 12 h; (d) cyclam, KI, CHCl3, rt, 72 h.

5674
S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
N
H
HN
Br
O
N
O
OH
O
OH
O
H
N
O
N
O
O
O
O
N
O
N
O
N
a
d
b
c
HN
Br
Br
HN
6a-6d
HN
8a-8d
R
R
R
5
7a-7d
R=CH3, C4H9, C8H17, C12H25
Scheme 2. Syntheses of target compounds 8a–8d. (a) n-Propylamine, ethanol, reflux, 3 h; (b) corresponding primary amines, methoxyethanol, reflux, 4 h; (c) DDQ, PPh3,
(n-butyl)4NBr, CH2Cl2, rt, 12 h; (d) cyclam, KI, CHCl3, rt, 72 h.
Table 1
SAR of cyclam fused naphthalimides
R1
N
NH HN
O
O
HN
N
O
N
O
HN
N
HN
HN
NH
HN
R2
4a-4d
8a-8d
Figure 2. Inhibition of topo II-mediated kDNA decatenation by target compounds
(100 M). Lane 1, kDNA; lane 2, minicircles (no drug); lanes 3–8, compounds 8c, 8d,
l
4c, 4d, doxorubicin and amonafide, respectively.
Compd
R, X=
Cytotoxicitya (IC50
HeLa
,
l
M)
HCT116
A549
4a
4b
4c
4d
8a
8b
8c
8d
R
R
R
R
R
R
R
R
1 = –CH3,
34.33 2.04
38.89 4.50
15.77 2.47
18.41 6.71
>50
13.80 2.47
19.22 0.20
10.14 2.51
11.85 4.00
>50
34.12 2.47
29.22 3.64
16.73 0.20
18.18 0.80
>50
1 = n-C4H9,
1 = n-C8H17
1 = n-C12H25
2 = –CH3,
2 = n-C4H9
2 = n-C8H17
2 = n-C12H25
>50
>50
>50
11.58 4.40
15.84 7.52
>50
8.40 2.62
9.36 1.85
>50
8.07 1.05
13.00 3.82
>50
Cyclam
Amonafide
n.d.b
9.52 1.27
n.d.
a
Cytotoxicity (CTX) against all of the human cancer cell were measured by
sulforhodamine B dye-staining method.
b
Not determined.
Figure 3. Inhibition of topo I-mediated DNA std (pBR322) decatenation by target
compounds (100 lM except for CPT 200 lM). Lane 1, DNA std; lane 2, topo I (no
drug); lanes 3–8, compounds 8c, 8d, 4c, 4d, camptothecin (CPT) and amonafide,
respectively.
activity than amonafide, which stand for the rationale of naphthal-
imide–cyclam conjugates. Besides, introduction of long alkyl chain
could improve the cytotoxic activity. For example, 8c and 8d was
4–6 fold more potent than 8a and 8b on HeLa and HCT116.
accordance with the antiproliferation activity tendency that
derivatives with octyl chain (8c and 4c) were more potent than
dodecyl derivative (8c vs 8d, and 4c vs 4d). However, no obvious
difference on topo II inhibition were observed between compounds
8 and compounds 4. Therefore, other mechanism of action was
supposed to exist due to the advantages of compounds 8c and 8d
than 4c and 4d on their antiporliferative properties.
Due to the similar mechanism of topo I and II by relaxation of
DNA supercoiling, we tested the target compounds if they could
also affect topoisomerase I. According to Figure 3, compounds 4c
and 4d could inhibit topo I activity less potent than the reference
compounds camptothecin and amonafide. What’s more, com-
pounds 8c and 8d, were more efficient topo I inhibitors than camp-
tothecin and amonafide. This result matched the cytotoxic
activities that compound 8 were more potent than the correspond-
ing compound 4 analogues. (8c vs 4c, and 8d vs 4d).
3.2. Effect on topoisomerases
According to previous report, amonafide, the most active naph-
thalimide antitumor derivative in clinical trial could intercalate
into DNA and form a stable complex of drug–DNA–topoisomerase
II. Since the newly-synthesized compounds also have similar struc-
ture feature of amonafide by containing a flat aromatic moiety and
basic side chain, they were tested of their topo II inhibition activi-
ties with kinetoplast DNA (kDNA) assay.
As shown in Figure 2, decatenation kDNA by topo II were
observed in lane 2. Addition of topo II inhibitor, such as doxoru-
bicin (lane 7), would decrease the amount of released decatenated
kDNA minicircles. Compounds 8d and 4d showed similar topo II
inhibition activity as amonafide. Furthermore, compounds 8c and
4c were more potent than amonafide. This result were in

S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
5675
3.3. DNA intercalating studies by circular dichroism (CD) spectra
changes in a negative peak around 245 nm and a positive peak
around 275 nm due to helical B conformation and base stacking,
respectively (see Fig. 4). Compound 8c could also induce weaker
conformational changes in comparison with amonafide.
Therefore, compound 8c was moderate DNA intercalator.
Furthermore, the most active compound 8c were confirmed of
its DNA intercalation activity by CD spectra as intercalating agents
could induce calf thymus DNA’s (ctDNA) conformation changes. In
accordance with literature, amonafide could induce significant
3.4. Molecular modeling
To better understand the molecular basis for the inhibition of
topoisomerases induced by this series of compounds, molecular
docking was adopted to predict the binding modes of the represen-
tative compound 8c. The structures of the complex topoisomerase
I/DNA/topotecan (PDB ID: 1K4T) and of the complex topoisomerase
II/DNA/mitoxantrone (PDB ID: 4G0V) were used as receptor during
the respective simulation. The lowest-energy poses predicted by
Glide software are shown in Figure 5.
In both binding modes proposed by docking simulations, the
nucleus of compound 8c was intercalated at the site of double
strand DNA cleavage and formed base-stacking interactions with
base pairs upstream and downstream. In the case of enzyme topoi-
somerase I (Fig. 5A and B), compound 8c interacted with nearby
ASP533 and ARG364 residues, forming two H-bonds, respectively.
Particularly, the carbonyl oxygen of the scaffold established an
H-bond with the side chain of arginine, while the other H-bond
was between the nitrogen atom of cyclam ring and the carboxylate
ion of aspartate. In contrast, the case of topoisomerase II
(Fig. 5C and D), compound 8c formed multiple H-bonds with both
enzyme and DNA. The carbonyl oxygens on the scaffold were con-
tacted with DNA base pairs and GLN778 residue. The alkane side
chain established an H-bond with the backbone oxygen atom of
Figure 4. CD spectra of CT DNA (100
8c (20 M).
lM) incubated with representative compound
l
Figure 5. The binding poses for the compound 8c. (A) docking model with the topo I/DNA complex; (B) binding mode with contacting residues and base pairs of topo I/DNA
complex; (C) docking model with the topo II/DNA complex; (D) binding mode with contacting residues and base pairs of topo II/DNA complex. In docking models, the
enzymes are depicted as transparent cartoons. In binding modes, the non-contact parts of enzyme and DNA are hidden for clarity, whereas the residues of DNA and enzyme
are reported in different colored sticks. In all the cases, the H-bonds are highlight as red dashes, the compound is shown as green carbon colored sticks.

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S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
Figure 6. Apoptotic cells were detected with Annexin V/PI double staining after incubation with compound 8c–8d and 4c–4d for 24 h.
ARG503, as well as, the cyclam ring interacted with the backbone
polar atoms of DNA strand.
5.1. Synthesis
5.1.1. General procedure for the preparation of 1a–1d
6-Bromobenzo[de]-isochromene-1,3-dione (1.94 g, 7.00 mmol)
was dissolved in 20 ml ethanol. Then corresponding primary amine
(7.70 mmol) was added, and the mixture was stirred at 60 °C for
5–6 h. The mixture was cooled to room temperature and evapo-
rated in vacuum to obtain the residue. Then the residue was puri-
fied on silica gel chromatography (PE:EA = 10:1, V/V) to provide
1a–1d.
3.5. Apoptosis assay by Annexin V binding
As DNA intercalation and topoisomerase poison could induce
apoptosis in cancer cell lines, an Annexin V/PI assay was used to
quantitate the apoptotic cells of HeLa induced by compounds with
antitumor potency (compounds 4c, 4d and 8c, 8d). As illustrated in
Figure 6, all the tested compounds could efficiently induce apopto-
sis of Hela cells at the concentration of 10 lM. After incubated for
24 h, compounds 8c and 4c induce the majority of HeLa cells into
the late apoptotic stage (98.46%, 60.48%, respectively), while com-
pound 8d and 4d induced the major amount of HeLa cells into the
early apoptotic stage (59.52%, 86.40%, respectively). In all, all the
tested compounds induce more than 90% apoptosis of HeLa cells.
5.1.1.1. 6-Bromo-2-methyl-1H-benzo[de]isoquinoline-1,3(2H)-
dione (1a).
CDCl3): d 8.68 (d, J = 7.2 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.44 (d,
J = 8.0 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.86 (t, J = 8.4 Hz, 1H), 3.57
(s, 3H); MS(ESI) calcd for C13H9BrNO2 [M+H]+ 289.0, found: 289.0.
White solid, yield: 90%. 1H NMR (400 MHz,
5.1.1.2.
dione (1b).
6-Bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-
White solid, yield: 80%. 1H NMR (400 MHz,
4. Conclusion
CDCl3) d 8.66 (d, J = 7.2 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.41 (d,
J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 4.19
(t, J = 7.6 Hz, 1H), 1.77–1.69 (m, 2H), 1.51–1.42 (m, 2H), 1.00 (t,
J = 7.2 Hz, 3H); MS(ESI) calcd for C16H15BrNO2 [M+H]+ 331.0,
found: 331.0.
In this paper, a series of naphthalimide–cyclam conjugates were
designed and synthesized. The novel compounds could inhibit the
growth of cancer cell lines comparable or even more potent than
amonafide. Representative compounds 8c, 8d, 4c, 4d were investi-
gated of their antitumor mechanism of action. They could potently
inhibit both topo II and topo I in cell-free system. We also chose the
most active compound 8c and confirmed of its DNA intercalation
activity. The molecular interactions between 8c and its intracellu-
lar targets were investigated by molecular modeling simulations.
Finally, the inhibition of HeLa cell growth by representative com-
pounds 8c, 8d, 4c, 4d were associated with induction of apoptosis.
5.1.1.3.
dione (1c).
6-Bromo-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-
White solid, yield: 85%. 1H NMR (400 MHz,
CDCl3): d 8.68 (d, J = 7.2 Hz, 1H), 8.59 (d, J = 7.6 Hz, 1H), 8.44 (d,
J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 4.18
(t, J = 8.0 Hz, 2H), 1.78–1.71 (m, 2H), 1.47–1.29 (m, 10H), 0.89 (t,
J = 7.2 Hz, 3H); MS(ESI) calcd for C20H23BrNO2 [M+H]+ 387.1,
found: 387.1.
5. Experimental section
5.1.1.4. 6-Bromo-2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-
dione (1d).
All the chemical regents and solvents were analytic grade. All
the synthesized cyclam fused naphthalimide derivatives were ver-
ified by 1H NMR, 13C NMR and HRMS, while the metal complexes
were verified by HRMS and HPLC. 1H NMR and 13C NMR measured
on a Bruker AV-400 spectrometer (in CDCl3 and DMSO-d6, TMS as
an internal standard). HRMS were collected in the Center of
White solid, yield: 85%. 1H NMR (400 MHz,
CDCl3): d 8.68 (d, J = 7.2 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.44 (d,
J = 7.6 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 4.18
(t, J = 7.6 Hz, 2H), 1.78–1.71 (m, 2H), 1.47–1.27 (m, 18H), 0.90 (t,
J = 7.2 Hz, 3H); MS(ESI) calcd for C24H31BrNO2 [M+H]+ 443.1,
found: 443.2.
Analysis
&
Test of East China University of Science and
Hewlett-
Technology. Analytical HPLC was performed on
a
5.1.2. General procedure for the preparation of 2a–2d
Packard 1100 system chromatograph equipped with photodiode
array detector. Amonafide and camptothecin were purchased from
Selleck chemicals (Houston, TX, USA). All medium and FBS were
purchased from Gibco (Grand Island, NY, USA).
1a–1d (6 mmol) were dissolved in 50 ml 2-methoxyethanol,
respectively. Then ethanolamine (24 mmol) was added and the
mixture was refluxed for 6 h. 150 ml water was added and solid

S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
5677
was precipitated immediately. The crude production was obtained
by suction filtration and was purified on silica gel chromatography
(CH2Cl2/MeOH = 50:1–20:1, V/V).
5.1.3.3.
quinoline-1,3(2H)-dione (3c).
NMR (400 MHz, CDCl3):
6-((2-Bromoethyl)amino)-2-octyl-1H-benzo[de]iso-
Orange solid, yield: 85%. 1H
d
8.63 (d, J = 6.4 Hz, 1H), 8.50 (d,
J = 8.4 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 6.77
(d, J = 8.4 Hz, 1H), 5.60 (br, 1H), 4.17 (t, J = 7.6 Hz, 2H), 3.89 (q,
J = 4.4 Hz, 2H), 3.77 (t, J = 6.0 Hz, 2H), 1.78–1.70 (m, 2H), 1.47–
1.28 (m, 10H), 0.89 (t, J = 7.2 Hz, 3H); MS(ESI) calcd for
5.1.2.1. 6-(2-Hydroxyethylamino)-2-methyl-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (2a).
NMR (400 MHz, DMSO-d6): d 8.68 (d, J = 8.4 Hz, 1H), 8.43 (d,
J = 7.2 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 6.81
(d, J = 8.4 Hz, 1H), 3.69 (t, J = 6.0, 2H), 3.46 (t, J = 6.0, 2H); MS(ESI)
calcd for C15H15N2O3 [M+H]+ 270.1, found: 270.1.
Yellow solid, yield: 90%. 1H
C
22H28BrN2O2 [M+H]+ 431.1, found: 431.1.
5.1.3.4. 6-((2-Bromoethyl)amino)-2-dodecyl-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (3d).
NMR (400 MHz, CDCl3):
Orange solid, yield: 83%. 1H
8.63 (d, J = 7.2 Hz, 1H), 8.50 (d,
d
5.1.2.2.
quinoline-1,3(2H)-dione (2b).
NMR (400 MHz, CDCl3):
6-(2-Hydroxyethylamino)-2-butyl-1H-benzo[de]iso-
Yellow solid, yield: 87%. 1H
J = 8.4 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.4 Hz, 1H), 6.77
(d, J = 8.4 Hz, 1H), 5.60 (br, 1H), 4.17 (t, J = 7.6 Hz, 2H), 3.89 (t,
J = 5.6 Hz, 2H), 3.77 (t, J = 6.0 Hz, 2H), 1.78–1.70 (m, 2H), 1.45–
1.27 (m, 18H), 0.90 (t, J = 7.2 Hz, 3H), MS(ESI) calcd for C26H36
BrN2O2 [M+H]+ 487.2, found: 487.2.
d
8.58 (d, J = 7.2 Hz, 1H), 8.46 (d,
J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 8.4 Hz, 1H), 6.79
(d, J = 8.4 Hz, 1H), 4.17 (t, J = 7.6 Hz, 2H), 4.09 (t, J = 5.2 Hz, 2H),
3.60 (t, J = 5.2 Hz, 1H), 1.74–1.70 (m, 2H), 1.49–1.43 (m, 2H), 0.99
(t, J = 7.6 Hz, 3H). MS(ESI) calcd for C18H21N2O3 [M+H]+ 312.1,
found: 312.1.
5.1.4. General procedure for the preparation of 4a–4d
1,4,8,11-Tetraazacyclotetradecane (0.10 g, 0.50 mmol) was
dissolved in 10 ml dry CHCl3. Then the mixture of 3a–3d
(0.20 mmol, respectively) and 15 ml dry CHCl3 was dropped in rt
under the argon. 3 days later, the solvent was concentrated by vac-
uum and 4a–4d was purified on silica gel chromatography
(CH2Cl2/MeOH/ammonium hydroxide = 100:15:2).
5.1.2.3.
quinoline-1,3(2H)-dione (2c).
NMR (400 MHz, CDCl3):
J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 6.74
(d, J = 8.4 Hz, 1H), 4.16 (t, J = 8.0 Hz, 2H), 4.09 (t, J = 5.2 Hz, 2H),
3.60 (t, J = 5.2 Hz, 2H), 1.77–1.70 (m, 2H), 1.47–1.28 (m, 10H),
0.89 (t, J = 7.2 Hz, 3H); MS(ESI) calcd for C22H29N2O3 [M+H]+
369.2, found: 369.2.
6-(2-Hydroxyethylamino)-2-octyl-1H-benzo[de]iso-
Yellow solid, yield: 84%. 1H
d
8.57 (d, J = 7.2 Hz, 1H), 8.45 (d,
5.1.4.1.
mino)-2-methyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(4a).
Orange solid, yield: 50%. 1H NMR (400 MHz, CD3OD): d
6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)a-
5.1.2.4. 6-(2-Hydroxyethylamino)-2-dodecyl-1H-benzo[de]iso-
8.24–8.18 (m, 2H), 8.05 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H),
6.59 (d, J = 8.4 Hz, 1H), 3.50 (t, J = 7.2, 2H), 3.34 (s, 3H), 2.78–2.53
(m, 18H), 1.79–1.71 (m, 4H); 13C NMR (100 MHz, CD3OD): d
164.9, 164.4, 150.9, 134.3, 130.7, 129.6, 127.8, 124.0, 121.8,
120.4, 107.9, 103.7, 54.4, 53.5, 50.7, 50.5, 49.2, 48.3, 46.7, 40.2,
quinoline-1,3(2H)-dione (2d).
NMR (400 MHz, CDCl3):
Yellow solid, yield: 84%. 1H
8.53 (d, J = 7.2 Hz, 1H), 8.41(d,
d
J = 8.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 6.71
(d, J = 8.4 Hz, 1H), 4.15 (t, J = 7.6 Hz, 2H), 4.09 (t, J = 5.2 Hz, 2H),
3.58 (t, J = 5.2 Hz, 2H), 1.76–1.69 (m, 2H), 1.45–1.26 (m, 18H),
0.89 (t, J = 7.2 Hz, 3H); MS(ESI) calcd for C26H37N2O3 [M+H]+
425.3, found: 425.3.
27.4, 25.3, 24.1; HRMS (ESI) calcd for
453.2978, found: 453.2968.
C
25H37N6O2 [M+H]+
5.1.4.2.
mino)-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(4b).
Orange solid, yield: 66%. 1H NMR (400 MHz, CDCl3): d
6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)a-
5.1.3. General procedure for the preparation of 3a–3d
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
(0.41 g,
1.80 mmol) and triphenyl phosphine (0.47 g, 1.80 mmol) were dis-
solved in 10 ml dry CHCl3. Then the mixture of tetrabutyl ammo-
nium bromide (0.58g, 1.80 mmol), 2a–2d (1.50 mmol,
respectively) and 10 ml dry CHCl3 was dropped. 6 h later, the sol-
vent was concentrated by vacuum and 3a–3d were purified on sil-
ica gel chromatography (CH2Cl2).
9.09 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 6.8 Hz, 1H), 8.43 (d, J = 8.4 Hz,
1H), 7.80 (t, J = 6.0 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 6.68 (d,
J = 8.8 Hz, 1H), 4.17 (t, J = 7.6 Hz, 2H), 3.79 (d, J = 5.6 Hz, 2H),
3.09–3.03 (m, 4H), 2.97 (br, 2H), 2.89 (br, 2H), 2.73 (t, J = 4.8 Hz,
2H), 1.77–1.69 (m, 2H), 1.49–1.43 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H).
13C NMR (100 MHz, CDCl3): d 164.8, 164.2, 150.4, 134.5, 131.3,
130.1, 128.9, 124.4, 122.8, 120.7, 109.2, 103.5, 51.2, 51.0, 50.8,
50.6, 47.2, 46.8, 46.4, 46.0, 45.1, 40.1, 40.0, 30.3, 25.8, 25.2, 20.5,
13.9; HRMS (ESI) calcd for C28H43N6O2 [M+H]+ 495.3448, found:
495.3454.
5.1.3.1.
6-((2-Bromoethyl)amino)-2-methy-1H-benzo[de]iso-
Orange solid, yield: 75%. 1H
quinoline-1,3(2H)-dione (3a).
NMR (500 MHz, CDCl3): d 8.63 (d, J = 7.3 Hz, 1H), 8.50 (d, J =
7.2 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 6.75
(d, J = 6.9 Hz, 1H), 3.89 (t, J = 5.8 Hz, 2H), 3.76 (t, J = 5.7 Hz, 2H),
3.54 (s, 3H); MS(ESI) calcd for C15H14BrN2O2 [M+H]+ 332.0, found:
332.0.
5.1.4.3.
mino)-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(4c).
Orange solid, yield: 65%. 1H NMR (400 MHz, CD3OD): d
6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)a-
8.31–8.28 (m, 2H), 8.16 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H),
6.66 (d, J = 8.8 Hz, 1H), 3.99 (t, J = 7.2 Hz, 2H), 3.50 (t, J = 6.4 Hz,
2H), 2.77–2.51 (m, 18H), 1.77–1.62 (m, 6H), 1.31–1.23 (m, 10H),
0.85 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD): d 164.4,
163.8, 150.7, 134.3, 130.6, 129.6, 127.7, 123.9, 121.9, 120.3,
108.1, 103.6, 54.5, 53.7, 50.8, 50.6, 49.3, 48.5, 46.8, 40.3, 39.7,
31.6, 29.1, 29.1, 27.9, 27.6, 26.9, 25.4, 22.4, 13.2; HRMS (ESI) calcd
for C32H51N6O2 [M+H]+ 551.4074, found: 551.4070.
5.1.3.2.
quinoline-1,3(2H)-dione (3b).
NMR (400 MHz, CDCl3):
J = 8.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 6.76
(d, J = 8.4 Hz, 1H), 4.18 (t, J = 7.6 Hz, 2H), 3.89 (t, J = 6.0 Hz, 2H),
3.77 (t, J = 6.0 Hz, 1H), 1.77–1.69 (m, 2H), 1.51–1.41 (m, 2H), 0.99
(t, J = 7.2 Hz, 3H). MS (ESI) calcd for C18H20BrN2O2 [M+H]+ 375.1,
found: 375.0.
6-((2-Bromoethyl)amino)-2-butyl-1H-benzo[de]iso-
Orange solid, yield: 68%. 1H
d
8.62 (d, J = 7.2 Hz, 1H), 8.49 (d,

5678
S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
5.1.4.4.
6-((2-(1,4,8,11-Tetraazacyclotetradecan-1-yl)ethyl)a-
1.56–1.29 (m, 18H), 0.90 (t, J = 6.4 Hz, 3H); MS (ESI) calcd for
mino)-2-dodecyl-1H-benzo[de]isoquinoline-1,3(2H)-dione
(4d).
C27H38N2O3Na [M+Na]+ 461.3, found: 461.3.
Orange solid, yield: 62%. 1H NMR (400 MHz, CD3OD): d
8.33–8.29 (m, 2H), 8.18 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H),
6.67 (d, J = 8.8 Hz, 1H), 4.00 (t, J = 7.2 Hz, 2H), 3.52 (t, J = 6.4 Hz,
2H), 2.78–2.53 (m, 18H), 1.77–1.63 (m, 6H), 1.31–1.19 (m, 18H),
0.85 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD): d 164.4,
163.8, 150.7, 134.3, 130.6, 129.6, 127.8, 123.9, 121.9, 120.3,
108.1, 103.6, 54.5, 53.7, 50.8, 50.6, 49.3, 48.5, 46.8, 40.3, 39.7,
31.7, 29.4, 29.2, 29.2, 27.9, 27.5, 26.9, 25.4, 22.4, 13.3; HRMS
(ESI) calcd for C36H59N6O2[M+H]+ 607.4700, found: 607.4688.
5.1.7. General procedure for the preparation of 7a–7d
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
(0.41 g,
1.80 mmol) and triphenyl phosphine (0.47 g, 1.80 mmol) were
dissolved in 10 ml dry CHCl3. Then the mixture of tetrabutyl
ammonium bromide (0.58 g, 1.80 mmol), 6a–6d (1.50 mmol,
respectively) and 10 ml dry CHCl3 was dropped. 6 h later, the sol-
vent was concentrated by vacuum and 7a–7d were purified on sil-
ica gel chromatography (CH2Cl2).
5.1.5. 6-Bromo-2-(3-hydroxypropyl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (5)
5.1.7.1. 2-(3-Bromopropyl)-6-(methylamino)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (7a).
NMR (400 MHz, CDCl3):
Orange solid, yield: 73%. 1H
6-Bromobenzo[de]-isochromene-1,3-dione (1.94 g, 7.00 mmol)
was dissolved in 20 ml ethanol. Then 3-amino-1-propanol
(7.70 mmol) was added, and the mixture was refluxed for 5–6 h.
The mixture was cooled to room temperature and evaporated in
vacuum to obtain the residue. Then the residue was recrystallized
by EtOH to obtained 5. White solid, yield: 90%. 1H NMR (400 MHz,
CDCl3): d 8.68 (d, J = 7.2 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.43 (d,
J = 7.6 Hz, 1H), 8.07 (d, J = 8 Hz, 1H), 7.87 (t, J = 8.0 Hz, 2H), 4.36
(t, J = 6.0 Hz, 2H), 3.62 (t, J = 6.0 Hz, 2H), 2.63 (br, 1H), 2.04–1.98
(m, 2H); MS (EI) calcd for C15H12BrNO3 [M+] 333.0, found: 333.0.
d
8.62 (d, J = 6.8 Hz, 1H), 8.53 (d,
J = 8.4 Hz, 1H), 8.13 (d, J = 9.2 Hz, 1H), 7.67 (t, J = 8.4 Hz, 1H),
6.82–6.80 (m, 1H), 4.33 (t, J = 7.2 Hz, 2H), 3.52 (t, J = 6.8 Hz, 2H),
3.18 (s, 3H), 2.39–2.32 (m, 2H); MS (ESI) calcd for C16H14BrN2O2
[MÀH]À 345.0, found: 345.0.
5.1.7.2.
quinoline-1,3(2H)-dione (7b).
NMR (400 MHz, CDCl3):
2-(3-Bromopropyl)-6-(butylamino)-1H-benzo[de]iso-
Orange solid, yield: 77%. 1H
d
8.58 (d, J = 7.2 Hz, 1H), 8.46 (d,
J = 8.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 6.73
(d, J = 8.0 Hz, 1H), 5.32 (br, 1H), 4.31 (t, J = 6.4 Hz, 2H), 3.52–3.49
(m, 2H), 3.45–3.41 (m, 2H), 2.37–2.31 (m, 2H), 1.86–1.79 (m,
2H), 1.60–1.51 (m, 2H), 1.04 (t, J = 7.6 Hz, 3H); MS (ESI) calcd for
5.1.6. General procedure for the preparation of 6a–6d
5 (2.0 g, 6 mmol) were dissolved in 50 ml 2-methoxyethanol.
Then corresponded amines (24 mmol) was added and the mixture
was refluxed for 6 h. 150 ml water was added and solid was
precipitated immediately. The crude production was obtained by
suction filtration and was purified on silica gel chromatography
(CH2Cl2/MeOH = 50:1–20:1, V/V).
C
19H20BrN2O2 [MÀH]À 387.1, found: 387.1.
5.1.7.3.
quinoline-1,3(2H)-dione (7c).
NMR (400 MHz, CDCl3):
2-(3-Bromopropyl)-6-(octylamino)-1H-benzo[de]iso-
Orange solid, yield: 70%. 1H
d
8.60 (d, J = 7.2 Hz, 1H), 8.48 (d,
J = 8.8 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 6.74
(d, J = 8.4 Hz, 1H), 5.32 (br, 1H), 4.32 (t, J = 7.2 Hz, 2H), 3.51 (t,
J = 7.2 Hz, 2H), 3.42 (t, J = 7.2 Hz, 2H), 2.38–2.31 (m, 2H), 1.87–
1.80 (m, 2H), 1.55–1.32 (m, 10H), 0.91 (t, J = 6.8 Hz, 3H); MS (ESI)
calcd for C23H30BrN2O2 [M+H]+ 445.1, found: 445.1.
5.1.6.1. 2-(3-Hydroxypropyl)-6-(methylamino)-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (6a).
Yellow solid, yield: 79%.
1H NMR (400 MHz, CDCl3): d 8.63 (d, J = 8.4 Hz, 1H), 8.54 (d,
J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 6.79
(d, J = 8.0 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 5.6 Hz, 2H),
3.19 (s, 3H), 2.03–1.97 (m, 2H); MS (ESI) calcd for C16H17N2O3
[M+H]+ 285.1, found: 285.1.
5.1.7.4. 2-(3-Bromopropyl)-6-(dodecylamino)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (7d).
NMR (400 MHz, CDCl3):
Orange solid, yield: 70%. 1H
8.60 (d, J = 7.6 Hz, 1H), 8.48 (d,
d
5.1.6.2. 2-(3-Hydroxypropyl)-6-(butylamino)-1H-benzo[de]iso-
J = 8.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 6.75
(d, J = 8.4 Hz, 1H), 5.30 (br, 1H), 4.32 (t, J = 6.8 Hz, 2H), 3.51 (t,
J = 7.2 Hz, 2H), 3.43 (t, J = 7.2 Hz, 2H), 2.38–2.31 (m, 2H), 1.87–
1.80 (m, 2H), 1.55–1.29 (m, 18H), 0.90 (t, J = 6.8 Hz, 3H); MS (ESI)
calcd for C27H37N2O2 [MÀBr]+ 421.3, found: 421.3.
quinoline-1,3(2H)-dione (6b).
NMR (400 MHz, CDCl3):
Yellow solid, yield: 87%. 1H
d
8.61 (d, J = 6.4 Hz, 1H), 8.50 (d,
J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 6.78
(d, J = 8.4 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H),
3.45 (t, J = 7.2 Hz, 2H), 2.03–1.97 (m, 2H), 1.87–1.80 (m, 2H),
1.61–1.52 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H); MS (ESI) calcd for
5.1.8. General procedure for the preparation of 8a–8d
C
19H23N2O3 [M+H]+ 285.1, found: 285.1.
1,4,8,11-Tetraazacyclotetradecane (0.10 g, 0.50 mmol) was
dissolved in 10 ml dry CHCl3. Then the mixture of 7a–7d
(0.20 mmol, respectively) and 15 ml dry CHCl3 was dropped in rt
under the argon. 3 days later, the solvent was concentrated by vac-
uum and 8a–8d was purified on silica gel chromatography
(CH2Cl2/MeOH/ammonium hydroxide = 100:15:2).
5.1.6.3. 2-(3-Hydroxypropyl)-6-(octylamino)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (6c).
NMR (400 MHz, CDCl3):
Yellow solid, yield: 90%. 1H
8.62 (d, J = 8.0 Hz, 1H), 8.50 (d,
d
J = 8.4 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 6.75
(d, J = 8.4 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H),
3.43 (t, J = 7.2 Hz, 2H), 2.02–1.97 (m, 2H), 1.87–1.80 (m, 2H),
1.56–1.32 (m, 10H), 0.91 (t, J = 6.8 Hz, 3H); MS (ESI) calcd for
5.1.8.1. 2-(3-(1,4,8,11-Tetraazacyclotetradecan-1-yl)propyl)-6-
(methylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(8a).
Orange solid, yield: 47%. 1H NMR (400 MHz, CDCl3): d
C
23H31N2O3 [M+H]+ 383.2, found: 383.2.
8.42 (d, J = 7.2 Hz, 1H), 8.31–8.28 (m, 2H), 7.49 (t, J = 8.0 Hz,1H),
6.50 (d, J = 8.4 Hz, 1H), 6.32 (br, 1H), 4.22 (t, J = 8.0 Hz, 2H), 3.15–
3.10 (m, 5H), 2.94 (m, 10H), 2.76 (br, 2H), 2.64–2.58 (m, 4H),
1.98–1.91 (m, 6H); 13C NMR (100 MHz, CDCl3): d 164.8, 164.1,
151.1, 134.5, 131.0, 129.5, 127.6, 124.2, 122.1, 120.2, 108.8,
103.5, 53.2, 52.5, 51.0, 50.7, 49.3, 48.3, 48.0, 46.9, 46.4, 38.5, 30.2,
25.8, 25.4, 24.9; HRMS (ESI) calcd for C26H38N6O2Na [M+Na]+
489.2948, found: 489.2976.
5.1.6.4. 2-(3-Hydroxypropyl)-6-(dodecylamino)-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (6d).
Yellow solid, yield: 90%.1H
NMR (400 MHz, CDCl3): 8.62 (d, J = 6.4 Hz, 1H), 8.50 (d,
J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 6.76
(d, J = 8.4 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H),
3.44 (t, J = 7.2 Hz, 2H), 2.03–1.97 (m, 2H), 1.87–1.80 (m, 2H),
d

S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
5679
5.1.8.2. 2-(3-(1,4,8,11-Tetraazacyclotetradecan-1-yl)propyl)-6-
(butylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(8b).
Orange solid, yield: 52%. 1H NMR (400 MHz, CDCl3): d
ATP, 0.5 mM DTT in a total volume of 20
and 2
the reaction. 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.
l
L. Then 2
l
L 10% SDS
l
L 6Â loading dye solution (Fermentas) were added to stop
8.57 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.25 (t, J = 8.0 Hz,1H), 6.20 (d, J = 8.8 Hz, 1H), 4.24 (br, 2H),
3.78 (br s, 2H), 3.60–3.31 (m, 10H), 3.03–2.80 (m, 6H), 2.25–2.16
(m, 4H), 1.88–1.79 (m, 4H), 1.54–1.45 (m, 4H), 1.00 (t, J = 7.6 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 164.8, 163.7, 151.4, 133.9,
130.7, 129.7, 129.3, 123.6, 120.4, 119.8, 106.6, 103.4, 54.2, 51.3,
51.2, 49.8, 48.6, 47.1, 44.7, 44.6, 43.2, 37.9, 30.5, 24.5, 23.7, 22.2,
20.5, 13.9; HRMS (ESI) calcd for C29H45N6O2 [M+H]+ 509.3599,
found: 509.3595.
5.5. DNA relaxation assay
Topoisomerase I activity was assayed by relaxation of super-
coiled pBR322 DNA according to the manufacturer’s instructions
(Takara, Dalian, China). TopoI (calf thymus), buffer, BSA, loading
buffer and supercoiled pBR322 DNA were all from TaKaRa
5.1.8.3. 2-(3-(1,4,8,11-Tetraazacyclotetradecan-1-yl)propyl)-6-
Biotechnology CO., Ltd 0.5 lg supercoiled pBR322 DNA, 0.1 units
(octylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(8c).
of topoisomerase I and the indicated concentrations of compounds
were incubated for 30 min at 37 °C in DNA topoisomerase I buffer
Orange solid, yield: 55%. 1H NMR (400 MHz, CD3OD): d
8.34–8.29 (m, 2H), 8.14 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 8.0 Hz,1H),
6.54 (d, J = 8.8 Hz, 1H), 4.08 (t, J = 7.6 Hz, 2H), 3.33 (t, J = 6.8 Hz,
2H), 2.88–2.53 (m, 18H), 1.87–1.71 (m, 8H), 1.49–1.29 (m, 10H),
0.89 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD): d 164.6,
163.9, 151.0, 134.3, 130.5, 129.6, 127.9, 123.8, 121.7, 120.2,
107.6, 103.4, 53.3, 53.0, 50.4, 50.1, 49.4, 46.8, 46.5, 43.2, 38.1,
31.7, 29.2, 29.1, 28.2, 27.0, 26.7, 24.9, 24.5, 22.4, 13.2; HRMS
(ESI) calcd for C33H53N6O2 [M+H]+ 565.4230, found: 565.4228.
with 0.01% bovine serum albumin, in a total volume of 20
lL. The
reactions were stopped by the addition of 2 L 10% SDS and 2 lL
l
6Â loading dye solution (Fermentas). Samples were then elec-
trophoresed in 0.8% agarose gel in TAE buffer for 35 min at
120 V. The gel was stained with ethidium bromide at room tem-
perature and photographed with UV transilluminator.
5.6. DNA intercalating assay by CD spectra
5.1.8.4. 2-(3-(1,4,8,11-Tetraazacyclotetradecan-1-yl)propyl)-6-
ct-DNA was purchased from Sigma Aldrich and used without
further purification. A solution of ct-DNA in 20 mM Tris–HCl buffer
(pH = 7.4) was stored at 4 °C. The concentration of ct-DNA was
determined spectrophotometrically from the molar absorption
coefficient (6600 MÀ1 cmÀ1). 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
(dodecylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(8d).
Orange solid, yield: 58%. 1H NMR (400 MHz, CD3OD): d
8.38–8.31 (m, 2H), 8.17 (d, J = 8.8 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H),
6.58 (d, J = 8.8 Hz, 1H), 4.10 (s, 2H), 3.35–3.33 (m, 2H), 2.89–2.54
(m, 18H), 1.85–1.78 (m, 8H), 1.46–1.24 (m, 18H), 0.88 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD): d 164.6, 163.9,
151.1, 134.4, 130.6, 129.6, 127.9, 123.8, 121.7, 120.2, 107.6,
103.45, 53.4, 52.9, 50.4, 50.1, 49.4, 46.8, 46.4, 43.2, 38.1, 31.7,
29.4, 29.1, 28.2, 27.0, 26.6, 24.8, 24.5, 22.4, 13.2; HRMS (ESI) calcd
for C37H61N6O2 [M+H]+ 621.4856, found: 621.4857.
performed on
England) using 1 cm quartz cell in the range of 200–400 nm. ct-
DNA (100 M) interacted with drugs (20 M) in Tris–HCl buffer
a Chirascan spectrophotometer (Photophysics,
l
l
for 2 h at 25 °C. CD spectra in the range of 230–300 nm were
analyzed.
5.2. Cell lines and cell culture
5.7. Computational methods
HeLa was purchased from Cell Bank of Type Culture Collection
of Chinese Academy of Sciences (Shanghai, China). A549 and
HCT116 were purchased form American Type Culture Collection
(ATCC, Manassas, VA, USA). They were maintained in strict accor-
dance with the supplier’s instructions and established procedures
The crystallographic structure of the complex topoisomerase
I/DNA/topotecan (PDB ID: 1K4T) and of complex topoisomerase
II/DNA/mitoxantrone (PDB ID: 4G0V) were fetched from Protein
Data Bank (PDB, http://www.pdb.org). The protein/DNA/drug
structures were prepared with protein preparation wizard in
Maestro 9.0. Hydrogens were added and water molecules were
deleted from the proteins. The final structures for following dock-
ing were minimized in Impref with OPLS-2005 force field.
Compound 8c was converted to 3D structure from 2D structure
by LigPrep 2.3. The protonation state of the molecule was also
assigned at physiological pH. Grids were automatically generated
using Glide 5.5 with standard procedure recommended by
Schrödinger. The centers of intercalation sites were defined by geo-
metric centers of native ligands. Compound 8c was docked using
Glide in extra precision mode with default parameters. The docked
poses discussed in this paper were selected accounting for both
proper energy score and reasonable binding mode.
5.3. In vitro cytotoxicity assays of the 4a–4d and 8a–8d
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 24 h. Cells were then fixed
with 10% trichloroacetic acid and stained with sulforhodamine
B (Sigma Aldrich, St. Louis, MO, USA). Sulforhodamine B in the
cells was dissolved in 10 mM Tris–HCl and was measured at
515 nm using
a
multiwell spectrophotometer (MAX190™,
Molecular Devices, Sunnyvale, USA). The inhibition rate on
cell proliferation was calculated as follows: inhibition rate =
(1 À A515 treated/A515 control) Â 100%.
5.4. kDNA decatenation assay
5.8. Annexin V-FIFC apoptosis detection assay
Topoisomerase II activity was measured by the ATP-dependent
decatenation of kDNA, which was provided by the manufacturer’s
instructions (TopoGEN, Florida, USA). 0.1 mg kDNA, 1 unit of
human topo IIa (TopoGEN) and the corresponding concentrations
of the measured compounds were incubated for 30 min at 37 °C
in 50 mM TrisHCl (pH 8), 150 mM NaCl, 10 mM MgCl2, 2 mM
Annexin V-FITC apoptosis detection kit (Invitrogen, USA) was
used in this assay. Briefly, HeLa cells (1 Â 106/1.5 ml per well) were
seeded in six-well plates and treated with the measured com-
pounds at 10
resuspended in 400
of annexin V-FITC, incubated at 4 °C in the dark for 15 min. Then
l
M. 24 h later, cells were treated with cold PBS,
lL of binding buffer, and then added to 5
lL

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S. Tan et al. / Bioorg. Med. Chem. 23 (2015) 5672–5680
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10 lL of PI was added and incubated same condition for 5 min. The
samples were analyzed by a FACScan flow cytometer (Becton
Dickinson, USA).
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This work is financially supported by the Fundamental Research
Funds for the Central Universities, the National Natural Science
Foundation of China (Grants 21302054, 81222046, 21173076 and
81230076) (Z.C., H.L.), the Shanghai Committee of Science and
Technology (Grants 14431902100, and 13ZR1453100) (Y.X., Z.C.),
the Opening Fund of Shanghai Key Laboratory of Chemical
Biology (Grant SKLCB-2012-05) (Z.C.), the National S&T Major
Project of China (Grant 2013ZX09507004) and the 863 Hi-Tech
Program of China (Grant 2012AA020308) (H.L.). H.L. is also spon-
sored by Shanghai Rising-Star Tracking Program (Grant
13QH1401100), Specialized Research Fund for the Doctoral
Program of Higher Education (Grant 20130074110004) and Fok
Ying Tung Education Foundation (Grant 141035).
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