Synthesis of rotenoid derivatives with cytotoxic and topoisomerase II inhibitory activities

Article abstract of DOI:10.1016/j.bmcl.2011.06.052

6-Deoxyclitoriacetal (1) and a series of 11 further derivatives of it (2-12) were synthesized and evaluated for their cytotoxic and topoisomerase IIα inhibitory activities. Compounds bearing epoxide (2), morpholine (6) and benzylamine (10) moieties showed

Full text of DOI:10.1016/j.bmcl.2011.06.052

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4813–4818
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of rotenoid derivatives with cytotoxic and topoisomerase II
inhibitory activities
Supranee Sangthong a, Kuakarun Krusong b, Nattaya Ngamrojanavanich c, Tirayut Vilaivan c,
Songchan Puthong d, Supajittra Chandchawan e, Nongnuj Muangsin c,
⇑
a Program of Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand
b Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand
c Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand
d The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand
e Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Deoxyclitoriacetal (1) and a series of 11 further derivatives of it (2–12) were synthesized and evaluated
Received 10 March 2011
Revised 26 May 2011
Accepted 13 June 2011
Available online 7 July 2011
for their cytotoxic and topoisomerase II
line (6) and benzylamine (10) moieties showed promising in vitro cytotoxic activities against four cancer
cell lines, with IC50 values ranging from 0.38 to 0.73 M. These three compounds also strongly inhibited
a inhibitory activities. Compounds bearing epoxide (2), morpho-
l
topoisomerase II activity at 68.3–93.5% and showed a moderately high DNA intercalating property.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Rotenoids
DNA-binding
Topoisomerase II inhibition
Anticancer activity
Cancer is one of the most common causes of premature death
and morbidity. However, the rise in resistance to available chemo-
therapeutic agents, combined with their adverse side affects and
high cost drives the search for new alternative anticancer com-
pounds from natural products. These can then form the templates
for further sequential chemical modifications to them or analogous
compounds for improvement of their biological activities. This is
an area of increasing interest, especially since some of the current
effective commercial anticancer drugs currently available are de-
rived from natural products,1 such as psorospermin,2 taxol3 and
vinblastine.4
Rotenoids consist of a four-fused tetrahydrochromeno[3,4b]
chromene ring core (Fig. 1) from which specific modifications are
built for each member. They are isolated from a fairly diverse array
of medicinal plants in tropical countries often with a medical folk-
lore tradition, and were originally applied as pesticides but now
have also been investigated for conventional medical bioactivities,
such as antibacterial, antimalarial, antifungal and anticancer prop-
erties.5,6 The structural and biological interest in this group of com-
pounds has encouraged attempts to isolate or modify the
compounds for newly developed drugs.7
6-Deoxyclitoriacetal (1) (Fig. 1) is one of the rotenoid com-
pounds extracted from roots of Stemona collinsae Craib. It exhibits
a strong in vitro cytotoxicity against various human cancer cell
lines, including the colon carcinoma (SW620), gastric carcinoma
(KATO), lung carcinoma (CHAGO), breast carcinoma (BT474) and
hepato carcinoma (HEP-G2).8–10
Chemical modifications of this compound in order to increase its
cytotoxicity and to investigate the possible anticancer mechanism
are our focus. A structure-based consideration of this compound
found some of the interesting features of 6-deoxyclitoriacetal that
appear to make it a good candidate anticancer agent that include
(i) the molecule contains a planar moiety similar to doxorubicin
(c) (Fig. 1), which is an important part for intercalating with
DNA11 and (ii) the molecule has a chromone ring system, like the
flavonoids and coumarins, which has been shown to be an impor-
tant feature for anticancer activity.12 Therefore, one possible mech-
anism of its anticancer activity could be mediated via DNA
intercalation or inhibition of the topoisomerase II process, like that
reported for doxorubicin.11
Previous investigations have introduced different types of func-
tional groups onto rotenoids, including doxyclitoriacetal, in an at-
tempt to enhance the cytotoxic activity and develop more potent
analogs. Firstly, coumarin-based compounds containing an epox-
ide group, for example, psorospermin (b), have been reported to
display a good cytotoxic activity, exerting a good anticancer and
⇑
Corresponding author. Tel.: +82 2 218 7635; fax: +82 2 218 7635.
E-mail address: nongnuj.j@chula.ac.th (N. Muangsin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.052

4814
S. Sangthong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4813–4818
Figure 1. Chemical structures of the (a) rotenoid core (1) 6-deoxyclitoriacetal, (b) psorospermin and (c) doxorubicin hydrochloride.
topoisomerase II inhibitor activity. This is due to the intercalation
of the psorospermin’s xanthone group between the DNA helix base
pair and that the epoxide ring undergoes alkylation reaction with
the N7-guanine in the presence of topoisomerase II.2 Secondly, sev-
eral publications have revealed that the ring-opened epoxide with
a good leaving group can enhance the cytotoxicity.13 Introduction
of either morpholine14 or benzylamine15 in part of the molecule
also resulted in a higher potent cytotoxicity than the original com-
pound. Finally, several studies suggested that the biological activ-
ities depended on the length of the side chain.16 Therefore, we
also studied the effect of the side chain length on the cytotoxic
activity by varying the aliphatic chain spacer length in both the
second and the third series.
In this Letter, we describe the synthesis, cytotoxicity and topo-
isomerase inhibitory activity of 11 substituted derivatives of 6-
deoxyclitoriacetal with different functional groups at the C11-OH
position (2–12) in an attempt to enhance the anticancer activity
and potentially gain insight into the mechanism of action of 6-
deoxyclitoriacetal and its derivatives. The reaction scheme for
the modification of 6-deoxyclitoriacetal is shown schematically
in Figure 2.
–Cl (3), –OH (4) and –OMe (5) to give the first series (3–5) to study
the effect of the electron donor and acceptor on the cytotoxic
activity. A similar reaction was performed using an amine in
refluxing ethanol at 80 °C for 6–12 h leading to the second series
(6–9) of compounds that consisted of cyclic and acyclic aliphatic
amines. Lastly, compounds (10–12) were prepared by reacting 2
with a nucleophile containing an aromatic group. It should be
noted that since the epichlorohydrin used was racemic, all
products obtain were a mixture of two diastereomers and were
evaluated as such.
The structures of the synthesized compounds were determined
on the basis of their 1H NMR, 13C NMR and MS spectral data
(shown in the Supplementary data), plus X-ray single crystal anal-
ysis for 3.17
The in vivo activities of 6-deoxyclitoriacetal (1) against the co-
lon carcinoma (SW620), gastric carcinoma (KATO), lung carcinoma
(CHAGO), BT474 (breast carcinoma) and hepato carcinoma (HEP-
G2) cell lines in tissue culture have already been reported.9 In this
work, all 6-deoxyclitoriaceal derivatives were tested for cytotoxic
activity against the aforementioned five cell lines plus additionally
the human oral carcinoma (KB), breast cancer (MCF-7) and human
small lung (NCI-H187) for the evaluation of growth inhibition. Cell
line growth was monitored using the MTT assay as reported,16,18
and compared with doxorubicin as a positive control. IC50 values
Initially the epoxy ring was introduced onto the C11-OH of the
6-deoxyclitoriacetal core by treating with epichlorohydrin in the
presence of sodium hydride in dimethylformamide (DMF) at
80 °C, leading to compound 2. Subsequently, the epoxide in the
intermediate 2 was then ring-opened with small nucleophiles
were classified by category as having a very high (IC50 6 1
lM),
high (1 6 IC50 6 10 M), moderate (10 6 IC50 6 50 M), low
l
l
Figure 2. Schematic chart showing the synthesis route of the 11 derivatives from 6-deoxyclitoriacetal.21

S. Sangthong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4813–4818
4815
Table 1
Cytotoxicity (as the inhibition of cell growth) and % topoisomerase II activity for compounds 1–12
H
H
MeO
HO
O
O
MeO
O
O
O
2
O
O
OH
OH
O
O
OMe
OMe
OMe
OMe
*R/S
*R/S
R
Compounds
R
% Topoisomerase II inhibition (100
lM)
IC50
(l
M)a
KATO
KB
MCF7
NCl-H187
SW620
CHAGO
BT474
HEP-G2
Etopb
—
—
—
—
–Cl
–OH
–OMe
68.7
72.1
72.1
68.3
58.3
38.8
58.4
—
2.01
—
—
—
—
—
—
—
Doxc
42.52
23.65
5.63
24.91
>100
>100
0.03
3.12
27.82
6.61
13.94
11.69
0.23
>100
0.38
1.09
8.04
>100
0.33
>100
0.63
1.09
>100
>100
0.83
>100
0.57
>100
>100
>100
0.07
>100
0.45
1.57
>100
>100
0.26
>100
0.55
0.81
>100
>100
1
2
3
4
5
13.28
32.87
14.77
13.36
46.92
O
6
93.5
26.51
31.71
2.93
0.73
>100
0.61
0.34
0.61
N
O
N
7
77.1
26.31
36.82
4.70
0.54
1.94
40.23
0.45
0.56
HN
O
8
54.2
>100
48.63
>100
>100
>100
>100
>100
>100
N
9
N
49.1
81.7
>100
>100
>100
>100
0.59
>100
0.67
>100
>100
0.62
>100
0.72
HO
OH
NH
10
32.29
34.63
35.53
31.04
NH
11
12
66.7
60.4
23.53
46.44
3.05
13.62
22.08
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
OH
R/S*
NH
>100
a
b
c
Data shown are derived from three independent trials.
Etop = etoposide.
Dox = doxorubicin.
(50 6 IC50 6 100
l
M) or no (IC50 more than 100
lM) sensitivity to
for one, high for four and medium for two cell lines), with IC50 val-
ues ranging from 0.85 to 24.91 M, but with no significant activity
the test compound.
l
The IC50 values of the substituted compounds 2–12 are summa-
rized in Table 1, along with that of the parental 6-deoxyclitoracetal
(1) and doxorubicin for comparison. The parent 6-deoxyclitorac-
etal (1) exhibited a moderate cytotoxic activity against the KB
and MCF-7 cell lines and a high activity against the NCI-H187 cell
line but showed no significant activity against the other five tested
cell lines. Interestingly, though, is the variation in response across
the cell lines to the same compound, a trend which was observed
with all of the 12 compounds screened. For the intermediate deriv-
atives, the results showed that the compound 2 with the epoxide
ring displayed the highest cytotoxicity, being active against all
eight cell lines and at a higher level (lower IC50) than the parent
compound.
against the CHAGO cell line. The –OH bearing compound 4 as only
an electron withdrawing group displays a lower cytotoxicity than
that seen with compound 3, with no significant activity against
six cell lines and only moderate activity against the KB and NCI-
H187 cell lines. However, this is slightly higher than that seen
for the compounds having electron donor substituted groups of –
NH2 (4) and –OMe (5), which, although only showing moderate
activity against the same two cell lines as compound 3, they do
so at a higher IC50 value. These results tentatively indicate that
the ability of Cl as a leaving group is an important influence on
the cytotoxicity, although the difference in electron withdrawing
ability of the substituent group may also be important. However,
all of these analogs exhibit lower activities than that of the epoxide
containing compound 2. There are examples in the literature that
the replacement of the unstable epoxide ring with a leaving group
resulted in moderate activities because of the electron withdraw-
ing property of the substituents.13
Concerning the epoxide ring-opened derivatives with small
substituent (3–5), the presence of –Cl group (3) as both a strong
electron withdrawing and a good leaving group resulted in the
highest cytotoxicity against seven of the eight cell lines (very high

4816
S. Sangthong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4813–4818
Among the compounds with aliphatic amine substituents (6–9),
spacers. Further characterization may prove useful in allowing
composition of a better cell line panel for screening new com-
pounds against particular cancer types or for understanding resis-
tance mechanisms.
some fairly consistent variation was seen. The compound with a
morpholine substituent (6) showed the highest cytotoxic activity
(four very high, one high, two moderate and one cell line with no
significant activity), with IC50 values in the range of 0.33–
The DNA replication process appears to be the key target involved
in the mechanism of action of anticancer drugs that act as intercala-
tors and/or topoisomerase II inhibitors, including doxorubicin and
psorospermin. In order to study the possibly anticancer mechanism
of 6-deoxyclitoriacetal (1) and its derivatives (2–11), we performed
31.71 lM for the seven responsive cell lines. This finding is in
agreement with the literature, where the introduction of a mor-
pholine core in part of the molecule was found to enhance its cyto-
toxic activity.14 When the oxygen group is replaced with a
carbonyl group, the piperidone (8) displays very poor cytotoxicity
with only moderate activity against one (MCF-7) cell line and no
significant activities against other seven cell lines, suggesting that
the oxygen ether group is preferred in this position.
As the length of the aliphatic chain spacer is increased, as seen
in comparing the morpholinoethyl derivative (7) to 6, the cytotoxic
activities in general are slightly decreased. Exceptions include the
significantly large decrease and increase in the cytotoxicity level
seen in the CHAGO and KATO cell lines, respectively. However,
the presence of the bis(hydroxyethyl)amino group (9) lead to a
dramatic decrease in the cytotoxicity, with no significant activity
seen against all eight cell lines. These results tentatively indicate
that the steric effect is not favored at this position. This also corre-
sponds with previous work where longer and branched side chains
were reported to reduce the intercalation ability of the compounds
into DNA base pairs and hence decreased the observed level of
cytotoxicity due to the steric effects.19
With respect to compounds with an aromatic substituent with
different spacer lengths, (10–12), the compound with a benzyl-
amine core (10) showed the highest cytotoxicity, with a very high
cytotoxicity against four cell lines and moderate cytotoxicity
against the other four cell lines. However, in contrast, compounds
11 and 12 with longer spacers displayed lower cytotoxicities than
compound 10, showing no significant toxicity against five (11) or
six (12) cell lines and only moderate cytotoxicity against two cell
lines (12), although compound 11 did show high cytotoxicity
against one cell line. The result is in agreement with the previously
mentioned unfavorable steric effects.19 Moreover, when a polar hy-
droxyl-substituent was introduced, a lower cytotoxic activity
resulted.
Doxorubicin is clinically used but has some resistance and so
new compounds are required. Although most of the 11 derivatives
of 1 evaluated here were found to mostly be significantly less effec-
tive than doxorubicin, some were seen to be more effective. Thus,
considering the% inhibition level of topoisomerase II, compound 1
was as effective as doxorubicin, whilst compounds 6, 7, and 10
were more effective. Considering the IC50 value for the inhibition
of the MTT assay, compounds 1–3, 6, 7, 10, and 11 showed lower
IC50 values than doxorubicin for the MCF7 cell line, as did com-
pounds 2 and 6 for the CHAGO cell line. However, importantly,
the rather ineffective compound 1 was significantly improved in
some of the derivatives paving the way for further future modifica-
tions to enhance the activity or specificity of new compounds or
overcome resistance mechanisms.
a single topoisomerase II
relaxation assay was carried out using human topoisomerase II
health care) and using etoposide as the reference (100
a
inhibition assay.20 The topoisomerase II
a
a
(GE
l
M) standard
and positive control. As shown in Figure 3, the% inhibition was clas-
sified into strongly and weakly active, depending on whether its%
inhibition was lower or higher than the activity of the reference con-
trol, etoposide (68% inhibition).
The ranked topoisomerase IIa inhibitory activity for all deriva-
tives was broadly consistent with the observed cell line cytotoxic
activity in Table 1. That is the derivatives with a strong cytotoxic
activity (2, 6, 7, and 10) also displayed a strong topoisomerase II
inhibitory activity with more than 80% inhibition at 100 M. Com-
pounds 4, 5, 8, and 9, which were inactive against almost all of the
eight cell lines tested, were also inactive against topoisomerase II
showing no inhibitory activity at 100 M. Indeed, compound 6
showed the strongest topoisomerase II inhibition (over 90%) com-
pared to etoposide as the reference (68.7%), the parent compound
(1) (72.1%) at 100 M. However, note that the concentration used
in the topoisomerase II inhibition assay (100 M) may well be
a
l
a
l
a
l
a
l
above the actual IC50 values for this inhibition, and certainly is high-
er than that reported for the inhibition of cell proliferation for many
of these compound-cell line combinations. Reducing the concentra-
tion of the strongest topoisomerase II
to 20 M caused an almost 2-fold decrease in the% topoisomerase
II inhibition (Fig. 4). Although, the correlation of the cytotoxic
and topoisomerase II inhibitory activity is not perfectly clear, in
terms of the IC50 especially, compound 6 exhibited a strong topoiso-
merase II inhibitory activity and showed nearly as good a cytotoxic
a inhibitor (6) 5-fold from 100
l
a
a
a
activity compared to doxorubicin, as the reference standard and po-
sitive control. These results could suggest that these series of 6-
deoxyclitoriacetal derivatives may not act via the same mechanism
as doxorubicin, and may be differentially modified by cell metabo-
lism. However, these results may provide advanced opportunities
to design new chemotherapeutic agents.
DNA intercalation is an important mode of control of gene
expression and DNA replication, as well as mutagenesis.2,11 Gener-
ally, the local Tm will increase when intercalative binding occurs be-
cause of the stabilization of the base stacking, and this can be used to
indicate the intercalating affinity of small molecules into the double
helix, and so potential cytotoxicity. The 6-deoxclitoriacetal deriva-
tives of 2, 6, 7, and 10 were selected to evaluate their effect upon
the ct-DNA Tm as they showed a high cytotoxic activity and topoiso-
merase IIainhibition compared to the parental 1 (Table 1). The melt-
ing temperature curves of ct-DNA in the absence and presence of 6-
deoxclitoriacetal derivatives 1, 2, 6, 7, and 10 are summarized in Ta-
ble 2 and Figure 5. The Tm of ct-DNA in the presence of the tested
compounds was about 57.3–68.0 °C compared with the absence
(56.4 °C). A slight increase in the Tm (0.9 °C increase to 57.3 °C) is ob-
served in the presence of the parent compound 1, indicating a low
binding affinity in DNA intercalation, 22 while the Tm of the ct-DNA
increased further upon the addition of the compounds 2, 6, 7, and
With respect to the cell lines, some dramatic variations in their
different susceptibilities to each compound were evident. This
serves as a warning to the use of just a few cell lines for the screen-
ing of cytotoxicity, and potentially alludes to the cancer type as
well as acquired drug resistance in culture. For example, the SW
620, KATO, CHATO, BT474 and HEP-G2 cell lines were typically
highly sensitive to compounds with epoxide rings, but gained
resistance upon the addition of the electron donor groups, or an
aromatic substituent with longer spacers. However, the same ef-
fect was not observed with the KB and NCI-H187 cell lines that,
in general, showed a lower sensitivity to compounds with epoxide
rings but retained some sensitivity to those with electron donor
groups, or to compounds with aromatic substituents with longer
10 (Table 2). The highest change in Tm
served in the presence of the benzylamine bearing compound 10,
and this may be due to the non-covalent interaction of the
(DTm = +10.7 °C) was ob-
p–p
DNA bases by the benzene ring, in addition to the intercalation.
For the other derivatives (epoxide 2 and heterocyclic 6 and 7), the
electrostatic attraction between the epoxide (2) and protonable

S. Sangthong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4813–4818
4817
Figure 3. Inhibitory effect of the tested compounds on human DNA topoisomerase II activity. Supercoiled pBR322 plasmid DNA was mixed with 0.3 U topoisomerase II
(TopoII) and the test compound (100 M) dissolved in DMSO (lanes 4–16). Lane 1: marker; lane 2, TopoII + plasmid DNA; lane 3, DNA only; lane 4, TopoII + plasmid
l
DNA + etoposide and lanes 5–16, TopoII + plasmid DNA + test compounds 1–12, respectively. Gels shown are representative of two independent assays.
Figure 4. Inhibitory effects of the morpholine derivative (6) at two different
concentrations on human DNA topoisomerase II activity. Supercoiled PBR322
plasmid DNA was mixed with 0.3 U topoisomerase II (TopoII) and the test
compounds. Lane 1, marker; lane 2, plasmid DNA only; lane 3, TopoII + plasmid
DNA; lanes 4 and 5, TopoII + plasmid DNA + compound 6 at (lane 4) 100 and (lane 5)
20
lM, respectively. Gels shown are representative of two independent assays.
Table 2
The melting temperature data for compounds 1, 2, 6, 7,
and 10
Compounds
Tm (°C)
D
Tm (°C)a
CT only
1
2
6
7
10
56.4
57.3
59.3
65.1
66.1
68.0
—
—
2.0
7.8
8.8
10.7
a
Difference between Tm values of ct-DNA in the
presence of the tested compound and 1.
Figure 5. Effects of five different 6-deoxyclitoriacetal derivatives (1, 2, 6, 7, and 10)
on the thermal denaturation of calf thymus DNA, showing the (a) Tm curves and (b)
derivative curves of Tm at 260 nm. Data shown are representative of two
independent assays.
nitrogen (6 and 7) with the anionic DNA may also be important. The
intercalative ability of the tested compounds is in the order of aro-
matic > heterocyclic > epoxy substituents by comparing the
DTm.
the different sensitivities and especially resistant levels of the dif-
ferent cell lines. Likewise, there are still a diverse number of other
compounds in this range still to be screened, and others yet to be
made, to further test the preliminary findings reported here.
It’s possible that the functional groups attached at C-11 of 6-deoxy-
clitoriacetal may enhance the DNA binding affinity. These results are
also consist with the trends of cytotoxic and topoisomerase II inhib-
itory activities. To conclude, the11 investigated 6-deoxyclitoriacetal
derivatives may show DNA-intercalation and be important targets
for cancer chemotherapy.
Acknowledgments
These results show that 6-deoxyclitoriacetal (1) and its deriva-
tives bearing epoxide (2), morpholine (6) and benzylamine (10)
moieties showed promising cytotoxic activities against seven can-
This work as supported by The Thailand Research Fund (TRF-
RSA4680016), The National Research University Project of CHE
and the Ratchadaphiseksomphot Endowment Fund (FW657A) to
N.M., the Thai Government Stimulus Package 2 (TKK2555), under
the Project for Establishment of Comprehensive Center for Innova-
tive food, Health Products and Agriculture, and the Center for
Petroleum Petrochemicals and Advanced Materials, Chulalongkorn
University. The comments of two anonymous referees are grate-
fully appreciated. Thanks are also given to Dr. Robert Butcher of
the PCU, Faculty of Science, Chulalongkorn University, for the con-
structive comments and English corrections.
cer cell lines with IC50 values ranging from 0.45 to 35.53 lM,
strongly inhibited topoisomerase II activity and displayed a rea-
sonably strong intercalating property, as determined by the in-
creased Tm of the ct-DNA. The anticancer activities of these
compounds may occur via inhibition of topoisomerase II
a and
DNA intercalation. However, this preliminary correlation certainly
requires further work for conformation, including to extensively
further investigate the structure–activity relationships of the 6-
deoxyclitoriacetal derivatives. It also becomes of interest as to

4818
S. Sangthong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4813–4818
14. Fish, V. P.; Deur, C.; Gan, X.; Greene, K.; Hoople, D.; Mackenny, M.; Para, K. S.;
Supplementary data
Reeves, K.; Ryckmans, T.; Stiff, C. Bioorg. Med. Chem. Lett. 2008, 18, 2562.
15. Lanetka, W. J.; Almeida, L.; Ashwell, S.; Brassil, J. P.; Daly, K.; Deng, C.; Gero, T.;
Glynn, E. R.; Horn, L. C.; Ioannidis, S.; Lyne, P.; Newcombe, N. J.; Oza, B. V.; Pass,
M.; Springer, K. S.; Su, M.; Toader, D.; Vasbinder, M.; Yu, U.; Zabludoo, D. S.
Bioorg. Med. Chem. Lett. 2008, 18, 4242.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.052.
16. Kuhajda, F. P.; Pizer, S. E.; Li, N. J.; Mani, S. N.; Frehywot, L. G.; Townsend, A. C.
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18. Cytotoxicity assay: Cytotoxic activity, as inhibition of cell line growth, was
evaluated by the MTT assay. Cell lines were cultured according to the supplier’s
instructions at 2–4 Â 104 cells per well in media containing 10% (v/v) fetal
bovine serum and incubated under 5% (v/v) CO2 at 37 °C, overnight. The
compounds were added in each well and three days later the MTT solution was
added to a final concentration of 0.5 mg/mL, incubated for 4 h and then the
medium in each well was discarded and replaced with 150
25 L of glycine pH 10.5. The absorbance of each well was determined by an
Automatic Elisa Reader System at 540 nm.
lL of DMSO and
l
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20. In vitro topoisomerase II assay: The reaction mixture contained 10 mM Tris–HCl
(pH 7.9), 175 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 2.5% (v/v) glycerol, 1 mM
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ATP, 0.5 mM dithiothreitol, 30
0.6 U DNA topoisomerase II
20 L. The reactions were incubated for 30 min at 37 °C and terminated by the
addition of 3 L of solution containing 0.77% (w/v) sodium dodecyl sulfate and
77 mM EDTA. Samples were mixed with 2 L of solution containing 30% (w/v)
l
g/mL bovine serum albumin, 0.2
lg pBR322,
a
and the test compounds in a final volume of
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l
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by transillumination with UV light and quantified using an image analyzer and
Syngene software.
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21. Experimental procedures and spectral data for compounds 2–12 can be found
in the supplementary data.
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