Synthesis, cytotoxicity, DNA binding and topoisomerase II inhibition of cassiarin A derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.04.107

Four series of cassiarin A derivatives with alkanoyl (3a-3d), aroyl (4a-4d), hydroxy/amino-substituted ethylene glycol (5a-5c) and selenium-containing (6a) side chains were synthesized. Their antitumor activities were evaluated against BT474, CHAGO, HepG2

Full text of DOI:10.1016/j.bmcl.2014.04.107

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cytotoxicity, DNA binding and topoisomerase II inhibition
of cassiarin A derivatives
Urarika Luesakul a, Tanapat Palaga b, Kuakarun Krusong c, Nattaya Ngamrojanavanich a, Tirayut Vilaivan a,
Songchan Puthong d, Nongnuj Muangsin a,
⇑
a Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
d Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Four series of cassiarin A derivatives with alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted
ethylene glycol (5a–5c) and selenium-containing (6a) side chains were synthesized. Their antitumor
activities were evaluated against BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki cancer cell lines.
Preliminary results demonstrated that 5b had moderate activities against HepG2 and KATO-III cell lines,
while 5c showed moderate to high cytotoxicity against most tested cell lines. In addition, 6a exhibited
moderate cytotoxicity against cervical cells, CaSki. DNA-binding and ethidium bromide displacement
experiments suggested that 5c and 5b binds to DNA via an intercalative mode, whereas 6a did not.
However, the selenium-containing cassiarin A derivative 6a inhibited topoisomerase II with more than
Received 20 December 2013
Revised 17 March 2014
Accepted 25 April 2014
Available online xxxx
Keywords:
Cassiaria A
Amonafide
Selenium compound
DNA-binding
Topoisomerase II
95% inhibition at the concentration of 50 lM. These cassiarin A derivatives showed lower toxicity to
normal cells, WI-38 than amonafide therefore they are potential lead compounds to be further developed
as new anticancer agents.
Ó 2014 Elsevier Ltd. All rights reserved.
Cancer is one of the major causes of death worldwide,
accounted for 7.6 million deaths in 2007 and projected to continue
rising.1 Many effective compounds used for treatment of cancer are
natural products or related to them. The first plant-derived agents
were used as clinical drugs, the so-called vinca alkaloids vinblas-
tine and vincristine were isolated from the Madagascar periwinkle.
Other well-known anticancer drugs approved for clinical use
include etoposide from Mayapple (Podophyllum pellatum),2 doxo-
rubicin from bacterium Streptomyces peucetius,3 and camptothecin
from Camptotheca acuminata.4 In addition to these well-known
examples, there are more than 3000 compounds which can be
potentially useful for cancer treatment.5
Cassia siamea is a common plant in Southeast Asia. This herb has
been used as food and for treatmentof insomnia. Themajor chemical
constituent extracted from C. siamea leaves and flowers is barakol
(1).6 In 2007, Morita et al. discovered cassiarin A (2)—an aromatic
alkaloid with a tricyclic skeleton—in C. siamea but is less abundant
than barakol.7 In 2010, Thamyongkit and co-workers were
interested in their potent antiplasmodial activity and transformed
barakol into cassiarin A. Due to the structural similarity between
the tricyclic aromatic core of cassiarin A and the anticancer agent
amonafide (Fig. 1), it was proposed that derivatives of cassiarin A
may exhibit cytotoxicity against cancer cells.8 Cassiarin A deriva-
tives including ester, ether, nitrogen-substituted and dehydroxy
derivatives had been synthesized and evaluated for their antimalar-
ial activities. Among them, the parent compound, cassiarin A
showed more potent antimalarial activity and less toxicity to human
cells, MCF-7.9
Amonafide is a naphthalimide analog that exhibits excellent
antitumor activity and was tested in clinical trials for treatment
of cancer. This compound binds to DNA by intercalation and also
inhibits Topoisomerase II.10 Topoisomerase II is the key enzyme
that regulates the topological state of DNA. It plays important roles
in transcription and replication process by breaking and rejoining
of phosphodiester backbone of DNA stands.11 However, amonafide
has failed to enter clinical phase III12 because it is easily metabo-
lized to N-acetyl-amonafide by enzyme N-acetyl transferase 2
(NAT2), resulting in unpredictable toxicity.13,14 Accordingly, many
derivatives such as alkyl, aryl and alkylamino substituted amona-
fide analogues have been investigated. These compounds showed
similar or more potent cytotoxicity than amonafide but could sur-
vive drug-metabolized enzymes. Moreover, DNA-binding studies
⇑
Corresponding author. Tel./fax: +66 2 218 7635.
E-mail addresses: nongnuj.j@chula.ac.th, nongnuj.ms@gmail.com (N. Muangsin).
http://dx.doi.org/10.1016/j.bmcl.2014.04.107
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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U. Luesakul et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
for development of anticancer agent due to (i) the molecule con-
tains a tricyclic planar aromatic moiety which is an important fea-
ture of DNA intercatator,24 (ii) the molecule has a amonafide-like
aromatic core but exhibited low toxicity against human cell,9 and
(iii) it showed potent biological activities including antiplasmodial
and antimalarial activities.7,9 In this work, the C7–OH position of
cassiarin A was modified which can be classified into four different
groups namely alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-
substituted ethylene glycol (5a–5c) and selenium-containing (6a)
as shown in Figure 2. The compounds were synthesized by acyla-
tion of cassiarin A or by nucleophilic substitution of appropriate
electrophiles (alkyl tosylate for 5b and 5c and phenylselenyl chlo-
ride for 6a) with cassiarin A under basic conditions. The chemical
structures were confirmed by spectroscopic methods (FT-IR, 1H
NMR, 13C NMR and MALDI-TOF MS). All compounds were prelimi-
narily evaluated for their cytotoxicity and active compounds were
further explored for their possible functional mechanisms such as
DNA-binding activity and topoisomerase II inhibition.
Cytotoxicity of the cassiarin A derivatives were determined by
MTT assay against six human cancer cell lines including breast car-
cinoma (BT474), lung carcinoma (CHAGO), liver carcinoma
(HepG2), gastric carcinoma (KATO-III), colon carcinoma (SW620),
cervical carcinoma (CaSki) cell lines comparison with amonafide
as positive control. All compounds were subjected to preliminary
screening for their cytotoxic activity at high concentration
Figure 1. Chemical structures of barakol, cassiarin A and amonafide.
reveal that they can interact with DNA by a similar mode of action
of amonafide.15,16
Selenium is an essential trace mineral that is important for
human health. Selenium compounds have been proven as potent
anticarcinogenic agents.17,18 The mechanism of selenium action
is unclear. Some selenium compounds effects on important tumor
suppressor protein p53 activity and different selenium forms reg-
ulate p53 in the different ways.19–21 Selenomethionine and
methyl-seleninic acid may modify p53 for DNA repair, while
sodium selenite may modify p53 for apoptosis.19 Selenite acts as
a topoisomerase II poison by inducing topoisomerase II cleavage
complexes.22 In addition, selenium analogues of naphthalimide
showed good cytotoxicity against many cell lines with IC50 ranging
10–20 l
M.23
(100 lM) as shown in Figures 3 and S1 in Supplementary data
and divided into two groups as active (less than 20% cell viability)
and inactive (more than 20% cell viability). Subsequently, IC50
In this research, new analogues of amonafide were synthesized
by modification of cassiarin A.8 Cassiarin A is a potential candidate
Figure 2. Cassiarin A derivatives with four groups of side chains: alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and selenium-
containing (6a) side chains.
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U. Luesakul et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Figure 4. Toxicity of amonafide, 5b, 5c and 6a to normal cell line, WI-38.
Figure 3. Preliminary screening of 5b, 5c and 6a at concentration 100 lM against
six human cancer cell lines compared to amonafide as the reference.
CaSki cell line with the IC50 value of 29.74 lM. It is likely that
glycol and selenium-containing side chain had major influence
on the cytotoxic activities. While amonafide is quite toxic to nor-
mal cells (WI-38), compounds 5b, 5c and 6a showed relatively
lower toxicity (Fig. 4). However, amonafide was used as the refer-
ence due to the structural similarity between the tricyclic aromatic
core of cassiarin A and amonafide. Based on structure of amonafide,
its chromophore can intercalate between base pairs of DNA, while
dimethylamino-ethyl group of the side chain interacts with nega-
tively-charged DNA resulting in increased antitumor potency.27
Based on the structural similarities between 5c and amonafide (tri-
cyclic planar chromophore with dimethylamino-ethyl side chain),
we proposed that 5c and (as well as other cassiarin A derivatives)
and amonafide might share the same mechanisms for their cyto-
toxic activities, that is, as DNA intercalators and topoisomerase II
inhibitors.
To test this hypothesis, compounds 5b, 5c and 6a which showed
reasonable cytotoxicity against some cell lines, were subjected to
further study on anticancer mechanism related to DNA binding
such as thermal denaturation of ct-DNA, ethidium bromide dis-
placement assay and topoisomerase II.
DNA-binding studies: Thermal denaturation of ct-DNA: To inves-
tigate the mechanism of cytotoxicity of cassiarin A derivatives, the
interaction between the compounds and ct-DNA was examined by
measuring the DNA melting temperature or Tm, which is defined as
the temperature at which 50% of base pairs in unpaired. The melting
curves of ct-DNA in the absence and presence of each compound are
illustrated in Figure 5 and Table 1. In this experiment, a solution
values were determined for active compounds and these were clas-
sified into three categories as high (IC50 <10 M), moderate
(IC50 = 10–30 M) and low (IC50 = 30–50 M) activities. The IC50
values are summarized in Table 1.
l
l
l
The parent compound, cassiarin A, showed no activity with all
cell lines tested. All tested compounds showed low activities
against breast cancer (BT474) cell line. Substitution of –OH group
with different alkanoyl side chain lengths, 3a–3d and different aro-
matic rings, 4a–4d did not increase any cell growth inhibitory
activity, and this may due to unfavorable steric effects. In contrast,
replacement of the alkanoyl side chain with glycol groups in 5a
and 5b to increase cell permeability showed that compound 5b
with diethylene glycol group exhibited low to moderate activities
against liver cancer (HepG2) and gastric cancer (KATO-III) cell
lines, which is better than 5a indicating that cytotoxic effect
enhances with the increase of side chain length. According to pre-
vious study suggested that the –OH group of glycol side chain can
form hydrogen bonds with biomacromolecules which may
increase the binding affinity between compounds and DNA.25
Among cassiarin A derivatives with glycol side chains, compound
5c carrying a dimethylamino-ethyl group exhibited high cytotoxic-
ity against KATO-III and SW620 cell lines with IC50 values of 9.82
and 9.85 lM, respectively, and moderate cytotoxicity against cer-
vical cancer (CaSki), lung cancer (CHAGO) and HepG2 cell lines.
These results correspond well with the higher cytotoxicity of ami-
noalkyl-substituted derivatives of amidoanthraquinones against
several cell lines compared to the alkyl/aryl-substituted deriva-
tives.26 In addition, replacement of the aroyl side chain with phe-
nylselenyl group, 6a showed only moderate activities against
ct-DNA at a concentration of 60
lM (by nucleotide) and 30
lM
of the tested compounds were prepared to give
a
ratio of
[compound]/[nucleotide] equal to 1:2. The concentration of the
ct-DNA in terms of nucleotide was measured from Beer’s Law
Table 1
The IC50 (lM) values against human cancer cells and the melting temperature data of cassiarin A derivatives compared to amonafide as the reference
Compound
R
Cytotoxicity, as the IC50
(l
M) value, of each compound on the cancer cell linesa
The melting temperature data
BT474
CHAGO
SW620
HepG2
KATO-III
CaSki
Tm (°C)
D
Tm (°C)
ct-DNA
Cassiarin A
70.7
70.7
—
—
Inactiveb
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
O
N
5b
5c
29.81 0.13
10.12 0.02
75.7
>86
4.8
O
O
O
OH
Inactive
29.54 0.10
9.85 0.04
10.11 0.02
9.82 0.09
30.15 0.28
>15.3
Se
6a
Inactive
Inactive
Inactive
Inactive
Inactive
29.74 0.03
9.96 0.10
70.0
78.9
—
Amonafide
37.80 6.05
1.05 0.05
0.32 0.07
1.71 0.14
9.24 0.09
8.2
a
IC50, as drug concentration that caused 50% reduced the viable cell population determined by MTT assay.
Inactive is defined as % cell viability more than 20% at high concentration (100 lM).
b
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U. Luesakul et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Ethidium bromide displacement assay: Ethidium bromide dis-
placement assay has been widely used to confirm the intercalative
binding mode between a compound and DNA. The principle of the
assay based on the displacement of the intercalating dye, ethidium
bromide (EtBr) from ct-DNA.34 The emission spectra of EtBr bound
to ct-DNA in the absence and the presence of amonafide, 5b and 5c
are given in Figure 6. Ethidium bromide is barely fluorescent in
aqueous solution but exhibits intense fluorescence emission at
595 nm when bound to ct-DNA, due to the strong intercalation
with DNA base pairs. If addition of the test compound to ct-DNA
pretreated with EtBr in the ratio of EtBr/DNA equal to 1:2.5 in
terms of base pairs results in quenching of the emission intensity,
one can conclude that the compound can displace the EtBr from its
intercalative site. The efficiency of the EtBr displacement was
investigated by determining the Stern–Volmer constant (KSV
)
value.35 The data were plotted as Stern–Volmer equation: I0/
I = 1 + KSV[Q] where I0 and I denoted the fluorescence emission
intensity in the absence and presence of the quencher, KSV is the
Stern–Volmer quenching constant and Q is the concentration of
the quencher (in this case, 5b, 5c and 6a). The compound 5c
Figure 5. Thermal denaturation profiles of ct-DNA (60
absence and presence of amonafide, cassiarin A, 5b, 5c and 6a (30
l
M by nucleotide) in the
M) in 10 mM
phosphate buffer pH 7.4 (A) Tm curves and (B) derivative curves of Tm at 260 nm.
l
equation expressed as A =
ct-DNA, in the range of 0.38–0.39,
260 nm (6600 MÀ1 cmÀ1
and is the cuvette path length
ebc where A is UV absorbance of
e
= extinction coefficient at
)
b
(1 cm).28,29 The Tm values of the ct-DNA were found to be 70.7,
70.7 and 70.0 °C for free DNA, with cassiarin A and with 6a,
respectively. The insignificant change of Tm indicated that cassiarin
A and 6a did not interact with DNA by intercalation or groove bind-
ing, which should result in stabilization of the DNA duplex. On the
other hand, amonafide and 5b can stabilize the DNA duplex as
shown by small Tm increase to 78.9 °C (
Tm = 4.8 °C), for amonafide and 5b, respectively. Importantly,
the compound 5c caused a significant increase of the Tm of
ct-DNA (Tm >86 °C and Tm >15.3 °C) which is comparable to com-
mon organic intercalator such as ethidium bromide ( Tm = 13 °C).
The large Tm indicated that 5c strongly interacted with and
DTm = 8.2 °C) and 75.7 °C
(D
D
D
D
stabilized the double helix of DNA. It is well known that many
anti-tumor agents containing positively charged group such as
dimethylamino-ethyl showed the excellent cytotoxic activity.30–32
The terminal nitrogen of dimethylamino-ethyl group of 5c
which has pKa values around 9 should be readily protonated and
electrostatically bound to the anionic phosphate group of DNA. This
favorable electrostatic interaction precedes subsequent intercala-
tive binding by the aromatic core of cassiarin A that results in the
increase of Tm of the ct-DNA.15,33 This experiment suggested that
compounds 5b and 5c can interact with ct-DNA, while compound
6a cannot. There are three types of interaction between
a
DNA-binding compound and double stranded DNA including
electrostatic interactions with the phosphate groups, binding to
minor or major grooves of DNA and intercalation between the
DNA based pairs. In order to confirm the intercalation ability of
these compounds, ethidium bromide displacement assay was
studied using fluorescence spectrophotometry.
Figure 6. Emission spectra of EtBr (10
the presence of compound (A) amonafide, (B) 5b and (C) 5c (0–70
phosphate buffer pH 7.4. The arrow shows the direction of change of fluorescence
intensity of EtBr bound to ct-DNA upon increasing amounts of tested compound.
Inserts show Stern–Volmer curve for quenching of ct-DNA.
l
M) bound to ct-DNA, 25
l
M base pairs in
M) in 10 mM
l
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U. Luesakul et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
showed the highest KSV value of 2.1 Â 104 MÀ1, while the 5b and
glycol derivatives (5b and 5c) showed moderate to strong
cytotoxicity against several cell lines including HepG2, KATO-III
and SW620 cell lines (with IC50 values around 10–30 lM).
amonafide showed smaller values of 4.3 Â 103 and 1.5 Â 104 MÀ1
,
respectively, which is comparable to DNA-intercalator such as
acridine derivative (KSV = 1.5 Â 104 MÀ1).36 From these results, it
can be concluded that the compounds 5b and 5c bind to ct-DNA
via the intercalation mode. Furthermore, the apparent binding
Moreover, the selenium-containing derivative (6a) of cassiarin A
exhibited moderate cytotoxicity against cervical cell line line, CaSki.
The compound 5c with positively charged dimethylamino-ethyl
side chain displayed a strong DNA intercalating property, as dem-
onstrated by its ability to stabilize ct-DNA and by ethidium bromide
displacement assay. The compound 5b, but not 6a, also acts
through intercalative DNA binding mode. The kDNA decatenation
experiments showed that 6a strongly inhibited topoisomerase II.
This suggests that a plausible explanation for cytotoxicity of the
selenium-containing cassiarin A derivative 6a. All three active
compounds showed lower toxicity to normal cell line (WI-38) than
amonafide, which made them a good candidate for new anticancer
agents.
constants (Kapp
)
can be calculated using the equation;
Kapp[compound] = KEtBr[EtBr], where KEtBr = 1.0 Â 107 MÀ1 and
[EtBr] = 10 l
M.34 Compound 5c also showed the highest apparent
binding constant (2.1 Â 106 MÀ1), which confirmed that the inter-
calative binding occurs between compound 5c and ct-DNA. The
DNA-binding studies correlate well with the cytotoxicity to cancer
cell lines, the strongest DNA binding compound (5c) is the most
cytotoxic compound. As 6a showed only weak DNA binding, its
moderate cytotoxicity against CaSki cell line may arise from a
different mode of action. Many reports showed that selenium
compound could play multi-roles such as cancer prevention, DNA
damage or topoisomerase II poison.22,23 Subsequently, compound
6a was further explored for other plausible mechanisms of action
such as the inhibition of topoisomerase II.
The evaluation of cytotoxicity was based on the reduction of
MTT dye by viable cells to give purple formazan products, which
can be measured spectrophotometrically at 540 nm. One hundred
microliters of cancer cells were seeded into 96-well plates at
Topoisomerase II inhibition: Topoisomerase II is the key cellular
enzyme that regulates the topological state of the DNA in cells. It
plays an important role in replication and transcription process
by breaking and rejoining of phosphodiester backbone of DNA
stands.11 Amonafide is a known DNA intercalator and can inhibit
topoisomerase II by stabilizing the enzyme–DNA complex.10 In this
work, we also determined the ability of compound 5b, 5c and 6a to
inhibit topoisomerase II decatenation of kDNA.37 Both forms of
kDNA including (i) catenated kDNA (C) and (ii) nicked open circular
decatenated kDNA (NOC) which is formed by the action of topoiso-
merase II were monitored. Catenated kDNA appears at the top and
cannot enter into the gel because of its size, while decatenated
product can readily move into the gel.38
5 Â 103 cell/well and incubated at 37 °C. After 24 h, 1
ll of each
compound (200, 100, 10 and 1 lM) was mixed into triplicate wells
and incubated as above for 3 days. Then, 10
(5 mg/ml) was added and incubated again for 4 h. After removal
of media and solubilization of forman crystals in 150 L of DMSO,
ll of MTT solution
l
the absorbance was measured at 540 nm. The cytotoxicity activity
was measured as % cell viability. The IC50 values were calculated
using software GraphPad Prism 5 (GraphPad. Software Inc.),
using a nonlinear regression of ‘log(inhibitor) versus response’:
Y = bottom + (top À bottom)/(1 + 10^((X À LogIC50))).
Thermal denaturation of ct-DNA: The thermal denaturation
experiments were performed to study the effect of each compound
on the stability of ct-DNA by determination of melting temperature
(Tm) value. The ct-DNA was prepared in phosphate buffer (pH 7.4).
The Tm of ct-DNA were measured using CARY 100 Bio UV–visible
The results shown in Figure 7 demonstrated that at a concentra-
tion of 100 lM, the selenium-containing compound 6a displayed a
strong topoisomerase II inhibitory activity with more than 95%
inhibition. The compound 5c exhibited a weak inhibitory effect
(30.1%), whereas compound 5b did not inhibit topoisomerase II.
The positive control, amonafide, showed a moderate activity of
61.8%. Moreover, 6a inhibited topoisomerase II with 31.5%, 75.7%
spectrophotometer with a temperature control attachment.
Absorption at 260 nm of DNA complements was measured with
rate 1 °C/min.
Ethidium bromide displacement assay: DNA binding studies were
performed with fluorescent intercalator displacement assay using
Ethidium bromide (EtBr) as intercalator.34 The experiments were
and 95.7% inhibition 10, 30 and 50 lM, respectively.
In conclusion, cassiarin A derivatives were synthesized and their
cytotoxicity against six cancer cell lines (BT474, CHAGO, HepG2,
KATO-III, SW620 and CaSki) were investigated. The preliminary
results demonstrated that alkanoyl (3a–3d), aroyl (4a–4d) and eth-
yleneglycol (5a) derivatives showed low cytotoxic activity against
all tested cell lines. Two hydroxy/amino-substituted ethylene
performed by adding the compound (0–70
l
M) into a solution of
ct-DNA (50 M base pairs) and ethidium bromide (10
l
l
M) in
phosphate buffer (pH 7.4). The fluorescence spectra (kex = 520 nm)
were recorded at room temperature. The data were plotted as
Stern–Volmer equation: I0/I = 1 + KSVQ where I0 and I denoted the
fluorescence emission intensity (at kem = 595 nm) in the absence
and presence of the quencher, KSV is the Stern–Volmer quenching
constant and Q is the concentration of the quencher.35 The
apparent binding constant (Kapp) was calculated from the equation
KEtBr[EtBr] = Kapp[compound],
where
KEtBr = 1.0 Â 107 MÀ1
,
[EtBr] = ethidium bromide concentration and [compound] is the
compound concentration at 50% reduction of fluorescence
intensity of complex EtBr-ct-DNA was observed.39
The kDNA decatenation assay was performed in order to study
the ability of topoisomerase II decationation of kDNA by using
amonafide as a positive control.
Stock solutions of the cassiarin A derivatives (10 mM) were
prepared in DMSO. The reaction mixture contain 250 ng kDNA
(TopoGEN), 4 units of topo IIa (USB Affymetrix) and the indicated
concentrations of compounds were incubated for 1 h at 37 °C in
10Â reaction buffer (USB Affymetrix) in final volume of 20
l
L.
The reaction was stopped by adding 3
l
L of 6Â loading dye (USB
Figure 7. Inhibitory effect of the tested compounds on human DNA topoisomarase
II decatenation. Lane 1, decatenated kDNA marker; lane 2, kDNA; lane 3, kDNA + 4
units of Topo II; lanes 4–7, kDNA + 4 units of Topo II + amonafide, 5b, 5c and 6a
Affymetrix). Samples were electrophoresed in 1% SDS in TBE buffer
for 90 min at 75 V. The gel was stained with ethidium bromide and
photographed under a UV transilluminator.
(100 lM); lanes 8–10, 6a at 10, 30 and 50 lM.
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U. Luesakul et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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The authors gratefully acknowledge funding from the
Ratchadaphiseksomphot Endowment Fund of Chulalongkorn
University to N.M. (RES560530064-FW).
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Supplementary data
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Please cite this article in press as: Luesakul, U.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.107