4β-[4′-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA topoisomerase i and IIα inhibitors and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmc.2013.06.033

A series of 4β-[4′-(1-(aryl)ureido)benzamide]podophyllotoxin congeners (11a-l) were synthesized and evaluated for their cytotoxic activity against six human cancer cell lines. Some of the compounds like 11a, 11h, 11k and 11l showed significant anti-prolif

Full text of DOI:10.1016/j.bmc.2013.06.033

Bioorganic & Medicinal Chemistry 21 (2013) 5198–5208
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4b-[40-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA
topoisomerase I and II
a inhibitors and apoptosis inducing agents
a
b
Ahmed Kamal a, , Paidakula Suresh , M. Janaki Ramaiah , T. Srinivasa Reddy a,c,d, Ravi Kumar Kapavarapu e,
⇑
Bolla Narasimha Rao c, Syed Imthiajali a, T. Lakshminarayan Reddy b, S. N. C. V. L. Pushpavalli b,
Nagula Shankaraiah c, Manika Pal-Bhadra b,
⇑
a Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
c Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
d RMIT-IICT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
e Centre for Neuroscience and Cell Biology, Institute for Interdisciplinary Research, University of Coimbra (IIIUC), 3004517 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
A series of 4b-[40-(1-(aryl)ureido)benzamide]podophyllotoxin congeners (11a–l) were synthesized and
evaluated for their cytotoxic activity against six human cancer cell lines. Some of the compounds like
11a, 11h, 11k and 11l showed significant anti-proliferative activity in Colo-205 cells and were superior
to etoposide. The flow-cytometric analysis studies indicated that these compounds show strong G1 cell
Article history:
Received 26 April 2013
Revised 12 June 2013
Accepted 13 June 2013
Available online 21 June 2013
cycle arrest, as well exhibited improved inhibitory activities on DNA topoisomerase I and II
a enzymes.
These compounds induce apoptosis by up regulating caspase-3 protein as observed by ELISA and Western
blotting analysis. In addition, a brief structure–activity relationship studies within the series along with
docking results of representative compounds 11a, 11h, 11k, 11l were presented.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Etoposide
Podophyllotoxin
Cell cycle
Cytotoxicity
Topoisomerase-IIa enzyme
Topoisomerase-I enzyme
1. Introduction
have been carried out by a number of researchers. This led to the
development of NK-611 (4) and GL-331 (5), which are in different
(ꢀ)-Podophyllotoxin (1) is a lignan of aryltetralin family found
in the plants like Podophyllum peltatum and Podophyllum emodi.1
Interest in podophyllotoxin has been heightened by its potent anti-
mitotic activity2,3 and it inhibits the assembly of tubulin protein
into microtubules through tubulin binding at the colchicine site.4,5
However, it failed to advance in human clinical trials because of
toxic side-effects. Extensive structural modifications have been
performed since 1950s, principally at Sandoz laboratories,6,7 which
led to the semi-synthetic derivatives, etoposide (2)8 and teniposide
(3, Fig. 1).9,10 Both these derivatives demonstrated significant activ-
ity with lower toxicity in clinical trials and they target DNA topoi-
stages of clinical studies and exhibit improved cytotoxicity as well
as DNA topoisomerase-II
a
inhibition.13 It has also been indicated in
the literature that bulky substitution at the C-4 position of the pod-
ophyllotoxin usually enhances the cytotoxicity and DNA topoiso-
merase-IIa
inhibition activity.14–19
Similarly, the urea derivatives such as bis-aryl ureas have been
most extensively investigated new chemical entities following the
success of Sorafenib20 for the treatment of advanced renal cell car-
cinoma and unresectable hepatocellular carcinoma.21,22 More
importantly, this class of urea derivatives have been found to be
potent inhibitors of human DNA topoisomerase-I and IIa 23–25
.
somerase-IIa 11
.
These are currently being used as frontline cancer
DNA topoisomerase-I and topoisomerase-IIa enzymes are ex-
chemotherapeutics against various cancers, including small-cell
lung cancer, testicular carcinoma, lymphoma, and Kaposi’s sar-
coma.12 However, their therapeutic use has encountered certain
limitations such as acquired drug resistance, and lower bioavail-
ability. To overcome such problems, extensive synthetic efforts
pressed at different levels in different cell types; the expression
of topoisomerase-I is prominent in colon cancer cell lines, while
topoisomerase-II is high in breast and ovarian cancer cell lines.26
The expression of either of the enzyme, sufficient to support cell
division and the development of resistance to topoisomerase-I
inhibitors is often accompanied by a concomitant rise in the level
of topoisomearse-II
a
and vice versa.27,28 Thus dual inhibitors of
may have advantages and
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.);
tel.: +91 40 27193236 (M.P.-B.).
topoisomerase-I and topoisomerase-II
a
there has been much interest in such compounds in clinical
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M. Pal-
Bhadra).
evaluation, including intoplicine and XR 500016.29,30
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.033

A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
5199
H
R1
O
O
OH
R1
R2
R3
H
O
R2
O
O
O
HO
CH3
OH
Etoposide (2)
O
O
O
O
OH
H
H
Teniposide (3)
NK-611( 4)
O
S
O
NMe2
NMe2
H3CO
OCH3
OCH3
H3CO
OCH3
OR3
Podophyllotoxin (1)
NO2
H3C
NH
CF3
Cl
HN
O
O
O
O
O
O
N
N
H
N
H
O
Sorafenib (6)
H3CO
OCH3
OH
GL-331 (5)
Figure 1. Representative chemical structures of bioactive podophyllotoxin (1), etoposide (2), teniposide (3), NK611 (4), GL-331(5) and sorafenib (6).
In our ongoing research efforts to design and develop new po-
tent anticancer agents, we have been involved for many years in
the synthesis and in vitro biological evaluation of podophyllotoxin
congeners.31–37 In continuation of these efforts, we have under-
taken the synthesis of some new analogues of podophyllotoxin
by linking it to a urea moiety through an aryl amino spacer at
C-4 position of the podophyllotoxin scaffold with a view to com-
bine the pharmacological characteristics of both these chromoph-
ores. In the present study, we report the synthesis and in vitro
cytotoxic activity of a series of novel 4b-[40-(1-(aryl)ureido)benz-
amide]podophyllotoxin derivatives (11a–l). Most of these podo-
phyllotoxin derivatives have exhibited better cytotoxicity than
etoposide. Based on their promising in vitro cytotoxicity, it was
considered of interest to investigate their role in the cell prolifer-
ation apart from their effect on DNA topoisomerase-I and II
inhibition.
a
2. Results and discussion
2.1. Chemistry
The new 4b-[40-(1-(aryl)ureido)benzamide]podophyllotoxin
congeners (11a–l) have been synthesized from the key intermedi-
ate (5R,5aR,8aS,9S)-9-amino-5-(3,4,5-trimethoxyphenyl)-5,5a,6,8,
8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]isobenzofuran-6-one
O
O
COCl
NO2
NH2
NH2
HN
HN
O
O
O
O
O
O
8
O
O
O
NO2
i
ii
O
O
O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
9
H3CO
OCH3
OCH3
10
7
iii
Cl
Cl
R =
O
Br
S
NH
Cl
H3C
CH3
Cl
O
Cl
HN
F
e
a
c
b
d
f
R
NH
O
O
O
O
Cl
Br
O
F3C
CF3
H3CO
OCH3
OCH3
11a l
OCH3
h
g
i
j
k
l
−
Scheme 1. Reagents and conditions: (i) Et3N, CH2Cl2, rt, 3 h; (ii) Pd/C, H2, EtOAc, rt, 12 h; (iii) R-N = C = O (a–l), CH2Cl2, rt, 1 h.

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A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
Table 1
In vitro cytotoxic activitya (GI50
l
M) of 4b-[40-(1-(aryl)ureido)benzamide] podophyllotoxin conjugates (11a–l)
Compound
Zr-75-1b
MCF7b
KBc
Guravc
DWDc
Colo 205d
A-549e
Hop62e
SiHaf
A-2780g
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
0.13 0.01
2.90 0.15
NA
2.40 0.14
2.9 0.31
2.91 0.22
2.22 0.12
0.18 0.03
0.11 0.001
2.6 0.29
NA
NA
1.97 0.15
2.01 0.11
NA
NA
NA
2.7 0.21
NA
0.24 0.17
2.90 0.17
NA
0.1 0.004
1.85 0.27
0.51 0.09
0.17 0.011
0.10 0.002
2.30 0.19
NA
0.1 0.002
2.61 0.15
1.6 0.13
NA
0.01 0.005
2.60 0.3
NA
NA
2.52 0.39
NA
2.14 0.219
NA
NA
1.40 0.125
1.5 0.42
NA
2.98 0.61
1.50 0.22
2.01 0.30
2.62 0.15
1.12 0.19
0.1 0.027
3.08 0.135
7.25 0.56
0.17 0.02
2.5 0.17
NA
2.6 0.31
2.71 0.66
NA
NA
0.12 0.014
NA
NA
NA
NA
2.9 0.13
NA
NA
NA
NA
2.3 0.09
NA
2.20
2.8 0.57
NA
2.1 0.15
NA
NA
2.3 0.39
1.35 0.04
2.22 0.37
2.3 0.09
1.3 0.015
0.14 0.01
1.31 0.27
0.16 0.015
0.15 0.012
0.12 0.004
0.12 0.03
0.15 0.004
2.7 0.1
1.1 0.055
0.11 0.007
2.9 0.22
0.1 0.002
0.14 0.021
2.11 0.024
0.17 0.018
2.8 0.25
2.5 0.011
2.01 0.25
2.31 0.17
2.6 0.15
2.31 0.4
NA
1.93 0.01
0.21 0.007
NA
NA
1.7 0.16
NA
0.02 0.001
2.23 0.41
2.34 0.35
0.01 0.009
0.01 0.031
0.13 0.017
0.14 0.041
2
0.11
2.6 0.15
2.9 0.31
0.16 0.017
0.2 0.015
1.79 0.14
2.5 0.33
1.6 0.27
0.17 0.02
0.62 0.014
0.1 0.015
NA
0.12 0.014
0.1 0.002
0.80 0.19
0.14 0.022
NA
0.21 0.03
3.11 0.11
0.17 0.011
Etoposideh
ADRi
0.31 0.051
0.17 0.025
a
b
c
d
e
f
50% Growth inhibition and the values are mean of three determinations.
Breast cancer.
Oral cancer.
Colon cancer.
Lung cancer.
Cervix cancer.
Ovarian cancer.
Etoposide.
ADR (adriamycin), NA = not active.
g
h
i
(7)31,32 as depicted in Scheme 1. The compound 7 was coupled
with 4-nitrobenzoyl chloride (8) in the presence of Et3N to afford
9, which upon nitro reduction with Pd/C provides the correspond-
ing amine 10. Finally, this intermediate was then reacted with a
variety of substituted phenyl isocyanates gave the desired 4b-[40-
(1-(aryl)ureido)benzamide]podophyllotoxins (11a–l) in overall
good yields.
demonstrated the GI50 in the range of 0.13–3.11
7.25
structure–activity relationship (SAR) was examined for these new
l
M and 0.10–
l
M, respectively. Based on the cytotoxicity data, the
podophyllotoxin congeners (11a–l). It is interesting to observe that
3. Biological activity evaluation
3.1. Anti-proliferative activity
All the compounds 11a–l were evaluated for their anti-prolifer-
ative activity in selected human cancer cell lines of breast, oral,
colon, lung and ovarian origin by using sulforhodamine B (SRB)
method.38 The compounds that exhibit GI50 610ꢀ5
M are
considered to be active in the respective cell lines and the results
are illustrated in Table 1. All the compounds 11a–l exhibit signifi-
cant anti-proliferative activity with GI50 values ranging from 0.01
to 2.90 lM, while the positive controls, etoposide and adriamycin
Figure 2B. The number of viable cells present after 24 h was measured in
compound treated [11a, 11h, 11k, 11l and etoposide (Eto)] Colo-205 cells.
Figure 2C. The effect of compounds on cellular energy (ATP) in Colo-205 cells was
Figure 2A. Colo-205 and MDA-MB-231 cells were treated with compounds 11a,
11h, 11k, 11l and etoposide (Eto) at 0.5, 1, 2 and 4 M concentration for 24 h. The
MTT cell viability assay was conducted and the optical density (O.D) was observed
measured by luminescence based assay. Compounds at 2 lM concentration have
l
strongly depleted the cellular ATP. The error bar in the graph represents the
standard deviation (S.D) obtained from three independent experiments. ⁄⁄⁄ indicate
P <0.001. Con: represents control untreated cells.
at 570 nm. DMSO indicates the untreated cells.

A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
5201
one of the compounds 11a with chloro substitution at para-posi-
11a, 11h, 11k, 11l and etoposide at 0.5, 1, 2 and 4
tions for 24 h. It was observed that below 0.5 or 1
l
l
M concentra-
M concentra-
tion of the phenyl ring showed similar or slightly reduced activity
in comparison to the methoxy substitution 11h. Both these com-
pounds 11a and 11h displayed potent cytotoxic activity against
different cell lines such as colon, breast and lung cancer cell lines,
which were similar or better than etopside. However, shifting the
chloro group from para-position (11a) to the meta-position (11b)
remarkably decreased the cytotoxicity, suggesting that the elec-
tron distribution pattern on benzoyl urea side chains probably play
an important role towards the activity profile. Moreover, the pres-
ence of different disubstitutions on phenyl ring such as 2,6-di-
chloro (11c), 3,5-dimethyl (11f), 3,5-ditrifluoromethyl (11g) and
3-flouro-4-chloro (11e) groups at different positions of the phenyl
ring determined a marked decrease or no inhibitory activity on
these cancer cell lines. In addition, the introduction of heterocyclic
motifs into urea side chain, such as furan (11d) and thiophene (11j)
exhibited marginal or no activity against the different human tu-
mor cell lines, while 11d showed selective activity towards MCF7
and DWD cell lines. It is also observed that the removal of the phe-
nyl ring and substitution with aliphatic side chains like 2-chloro
ethyl (11k) and 2-bromo ethyl (11l) derivatives resulted in
enhancement of activity and is comparable to that of etoposide.
Interestingly, all the synthesized compounds 11a–l showed potent
cytotoxic activity against MCF7 cell lines. The significant growth
inhibitory activity exhibited by promising compounds like 11a,
11h, 11k and 11l prompted us to evaluate their cell viability in
Colo-205 cells and MDA-MB-231 cells, with a view to study their
detailed biological effects. Both these Colo-205 as well as MDA-
MB-231 cells were treated with some of these compounds like
tions, there was no significant cytotoxicity; however at
concentrations 2 or 4 M, these compounds have exhibited effec-
tive cytotoxicity in Colo-205 and MDA-MB-231 cells as shown in
Figure 2A. It was also observed that Colo-205 cells responded bet-
ter than MDA-MB-231 cells. Thus, further studies were carried out
l
by employing Colo-205 cells at 2 lM concentration. Moreover, try-
pan blue and ATP assays39 clearly indicated that the effect of these
podophyllotoxins on the cell viability (Figs. 2B and C).
3.2. Effect on cell cycle
In order to understand the role of these new podophyllotoxin
conjugates 11a, 11h, 11k and 11l, on cell cycle regulation, flow-
cytometric analysis was conducted in colon cancer cells (Colo-
205) at 2 lM concentration for 24 h. It was observed that Colo-
205 cells showed 78%, 76%, 76% and 72% of G1 phase cell cycle ar-
rest in cells treated with 11a, 11h, 11k and 11l respectively,
whereas control (untreated cells) exhibited 65%. However, stan-
dard drug showed G2/M phase cell cycle arrest (Fig. 3). Interest-
ingly, DNA topoisomerase-II inhibitors were also known to cause
G1 cell cycle arrest.40
3.3. Effect on DNA topoisomerase enzyme
The DNA topoisomerase-II
ined in comparison to etoposide. The pBR322 plasmid was incu-
bated with DNA topoisomerase-II enzyme that causes relaxed
a enzyme inhibition assay was exam-
a
Figure 3. Colo-205 cells were treated with compounds 11a, 11h, 11k, 11l and etoposide (Eto) at 2
lM concentration for 24 h. The cells were further processed for FACS
analysis. Compounds 11a, 11h, 11k and 11l have shown G1 cell cycle arrest. Control indicates the untreated cells.

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A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
Figure 4A. pBR322 plasmid DNA and compounds 11a–l were added at 2
was prepared fresh using ATP, NaCl, MgCl2, EDTA, Tris pH-7.9 components. The reaction was performed at 37 °C for 30 min. V: pBR322 DNA alone, Eto:
pBR322 + etoposide + topoisomerase-II , 11a–k: pBR322 + compound + topoisomerase-II
lM concentration along with DNA topoisomerase-IIa enzyme. The buffer used for enzyme activity
a
a.
Figure 4B. pBR322 plasmid DNA and compounds 11a–l were added at 2 lM concentration along with DNA topoisomerase-I enzyme. The relaxation buffer (10 mM Tris–HCl
(pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol. The reaction was performed at 37 °C for 30 min. V: pBR322 DNA alone, T:
pBR322 + topoisomerase-I, 11a–l: pBR322 + compound + topoisomerase-I, CPT: pBR322 + camptothecin (CPT) + topoisomerase-I.
Figure 6. Colo-205 cells were incubated with effective compound 11a from the
series and etoposide (Eto) at 2 and 4 lM concentrations for 24 h. Compound treated
cells were stained with Hoechst nuclear dye. Arrow depicts the fragmented DNA
and blebbing nature.
Figure 5. A.The effect of compounds 11a, 11h, 11k, 11l and etoposide (Eto) on
caspase-3. Colo-205 cells were treated with compounds at 2 M concentration for
l
24 h. The cell lysates were subjected to fluorescence based caspase-3 assay. The
compounds caused increase of caspase-3 protein activity. Control indicates the
⁄⁄⁄
⁄⁄ ⁄
indicate P <0.01, indicate P <0.05, these
untreated cells.
indicate P <0.001,
comparison of compound treated cells were made against control untreated cells.
The error bar represents the standard deviation (SD) obtained from three
independent experiments. (B) The effect of compounds on activation of caspase-3
by Western blot analysis. (C) Control untreated cells. Actin was used as a loading
control.
DNA. The effect of these compounds 11a–l on topoisomerase-II
was compared with etoposide as standard topoisomerase II inhib-
itor at 2 M concentration as shown in Fig. 4A. These results were
clearly demonstrated that these new compounds in particular 11a,
11h, 11k, 11l displayed significant inhibitory activity on topoiso-
a
a
l
11a
merase-IIa. Further, we have also tested these compounds for
Figure 7A. Compound 11a docked in ATP binding pocket of human topoisomerase-
IIa, showing p interactions with magnesium cation and hydrogen-bonding inter-
actions observed between Arg98 and carbonyl moiety along with Ser148 and
Asn150 interacting with methoxy moiety of structure 11a.
inhibitory activity on DNA topoisomerase-I. To our surprise, 11a
exhibits topo-I inhibition and is comparable with respect to cam-
ptothecin (CPT), a standard topoisomerase-I inhibitor (Fig. 4B).

A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
5203
11h
11a
Figure 7C. Compound 11a docked in DNA sequence between A113 and TGP11.
was up-regulated with compound treated cells particularly in case
of 11a as well as etoposide. The order of performance with regard
to caspase-3 activation is 11a > etoposide > 11h > 11k > 11l as
shown in Figure 5A and B.
It was considered of interest to examine DNA fragmentation
and morphological changes associated with apoptosis as caspase-
3 is known to be responsible for causing apoptosis.43,44 Colo-205
cells were treated with 11a (most potent compound) and etopo-
11k
side (Eto) at 2 and 4 lM concentrations for 24 h, and nuclear stain-
ing was performed by using Hoechst stain. It was observed that the
compound treated cells showed DNA fragmentation and blebbing,
whereas control cells did not show such blebbing, thereby reveal-
ing that these compounds induce apoptosis as shown in Figure 6.
4. Molecular docking studies
To evaluate the binding modes and nature of interactions for
some of these potent compounds like 11a, 11h, 11k, and 11l with
active site residues of human DNA topoisomerase I as well as topo-
11l
isomerase IIa, docking studies were performed by using Chemical
Computing Group’s Molecular Operating Environment (MOE) soft-
ware 2008.10 versions. Human topoisomerase-I (1SC7) and topoi-
somerase-IIa (1ZXN) proteins were retrieved from PDB and refined
with protonation, addition of gasteiger partial charges and finally
energy minimized to relieve bad crystallographic contacts. ‘Active
site finder’ function of the MOE is used to denote potential docking
pockets within the protein crystal structure. These compounds
11a, 11h, 11k and 11l were placed in the active site pocket of
the protein by the ‘Triangle Matcher’ method, which generate
poses by aligning the ligand triplet of atoms with the triplet of al-
pha spheres in cavities of tight atomic packing. Dock scoring was
carried with London dG method and then finally retaining their
10 best poses of scoring. The preparation of the ligands for docking
simulation involves the energy minimization with Molecular
Mechanics Force-field MMFF94ꢁ (Merck Molecular Force Field
94ꢁ) and then molecules were subjected to conformational search
in MOE using the conformational stochastic search module to find
the lowest energy conformers. To validate the docking accuracy of
the program, camptothecin and etoposide were considered as stan-
dards for the docking in human topoisomerase-I and topoisomer-
Figure 7B. Molecular docking of compounds 11h, 11k and 11l in ATP binding
pocket of human topoisomerase-IIa enzyme.
Thus, 11a causes significant inhibition of both topoisomerase-I as
well as topoisomerase-IIa enzymes.
3.4. Effect on caspase-3
Caspases belong to cysteine protease family and plays a crucial
role in the induction of apoptosis.41 Previous studies have reported
ase-IIa
enzymes, respectively.45,46
that etoposide, a DNA topoisomerase-IIa inhibitor causes apoptosis
by elevating the levels of caspase-3 protein.42 These aspects were
Molecular docking of these compounds 11a, 11h, 11k, and 11l
showed similar interactions and orientations with active site resi-
examined by treating the compounds 11a, 11h, 11k and 11l with
dues in the binding pocket of topo-II
correlation with DNA topoisomerase-II
a
a
(Figs. 7A and B), and is in
inhibition assay. Methoxy
Colo-205 cells at 2 lM concentration for 24 h and compared with
etoposide. The lysates obtained were subjected to caspase-3 based
substituents on the trimethoxy phenyl ring of these compounds
ELISA and Western blot analysis. It was observed that caspase-3

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A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
show common
p
interactions with magnesium and hydrogen-
1H), 6.83 (s, 1H), 7.94 (d, 2H, J = 9.0 Hz), 8.14 (d, 2H, J = 9.0 Hz).
MS (ESI): m/z 563 [M+H]+.
bonding with Asn91 residue similar to etoposide in the active site.
Common arene–cation interactions were also observed with Arg98
residues which are considered to assist in DNA intercalation. Com-
6.2. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-aminobenzamide (10)
pound 11a shows interaction with adenine moiety, A113 and p–p
stacking interaction between trimethoxy phenyl moiety of 11a and
TGP11 in human DNA topoisomerase-I (Fig. 7C), confirming its
dual affinity towards both topoisomerase-I and topoisomerase-
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-nitrobenzamide (9, 1.54 g, 3.53 mmol) in
80 mL of EtOAc was added 300 mg, of 10% Pd/C. The mixture was
stirred overnight under hydrogen atmosphere, the reaction mix-
ture was filtered and then filtrate was evaporated. The crude prod-
uct was purified by column chromatography with ethyl acetate/
hexane (6:4) gave pure compound 10 in excellent yield (1.42 g,
98%). 1H NMR (200 MHz, CDCl3): d 2.93–3.01 (m, 2H), 3.60 (s,
3H), 3.69 (s, 6H), 3.80 (t, 1H), 4.01–4.10 (dd, 1H, J = 7.5, 6.7 Hz),
4.30–4.44 (m, 2H), 6.00 (d, 2H, J = 4.5 Hz), 6.23 (s, 2H), 6.43 (s,
1H), 6.80 (s, 1H), 7.90 (d, 2H, J = 9.0 Hz), 8.16 (d, 2H, J = 9.0 Hz).
MS (ESI): m/z 533 [M+H]+.
IIa enzymes.
5. Conclusion
In conclusion, a series of new 4b-[40-(1-(aryl)ureido)benzam-
ide]podophyllotoxin congeners were synthesized and evaluated
for their in vitro cytotoxicity, moreover detailed studies relating
to their biological implications were carried out. Some of the com-
pounds 11a, 11h, 11k and 11l showed significant anti-proliferative
activity in Colo-205 cells, and equipotent to that of etoposide.
These compounds were tested for their effect on cell viability in
Colo-205 and MDA-MB-231 cells. The flow cytometric analysis
indicated that these compounds showed strong G1 cell cycle arrest.
It was also observed that caspase-3 was up-regulated by these
compounds, particularly 11a being the most effective among the
series. Interestingly, 11a showed dual inhibition of DNA topoiso-
6.3. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((4-chloroanilino)carbonyl)
amino)benzamide (11a)
merase I and topoisomerase II
showed similar orientation and interactions at DNA topo-I and
topo-II active sites compared with standards, camptothecin
a enzymes. The docking results
a
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10, 200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 4-chlorophenyl isocyanate (a, 0.81 mL,
0.56 mmol) was added at 0 °C and the stirring was continued for
1 h at room temperature. Later, the formed solid was filtered and
washed thoroughly with CH2Cl2 to afford the pure compound
(CPT) and etoposide (Eto) respectively. Overall, these new findings
provide an insight for future direction of drug design and develop-
ment of such congeners of podophyllotoxin.
6. Experimental section
11a (220 mg, 81%). Mp 180–183 °C; ½a D25
= ꢀ45.9 (c 0.5 in CHCl3);
ꢂ
All chemicals and reagents were obtained from Aldrich (Sigma–
Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA) and were used without further
purification. Reactions were monitored by TLC, performed on silica
gel glass plates containing 60 F-254, and visualization on TLC was
achieved by UV light or iodine indicator. 1H and 13C NMR spectra
were recorded on INOVA (400 MHz) or Gemini Varian-VXR-unity
(200 MHz) or Bruker UXNMR/XWIN-NMR (300 MHz) instruments.
Chemical shifts (d) are reported in ppm downfield from internal
TMS standard. ESI spectra were recorded on Micro mass, Quattro
LC using ESI+ software with capillary voltage 3.98 kV and ESI mode
positive ion trap detector. Melting points were determined with an
Electrothermal melting point apparatus, and are uncorrected.
1H NMR (300 MHz, CDCl3): d 3.00–3.10 (m, 2H), 3.67 (s, 3H), 3.70
(s, 6H), 4.40–4.48 (m, 2H), 4.60 (d, 1H, J = 4.83 Hz), 5.50–5.53 (m,
1H), 6.01 (d, 2H, J = 6.44 Hz), 6.30 (s, 2H), 6.60 (s, 1H), 6.80 (s,
1H), 7.31 (d, 2H, J = 8.05 Hz), 7.50 (d, 2H, J = 8.05 Hz), 7.91 (d, 2H,
J = 8.19 Hz), 8.60 (d, 2H, J = 8.19 Hz). 13C NMR (75 MHz, CDCl3): d
40.7, 43.0, 47.5, 55.4, 59.8, 68.6, 100.9, 107.7, 108.5, 109.2, 116.3,
116.9, 123.7, 124.0, 126.1, 127.9, 129.7, 129.9, 130.5, 131.8,
134.8, 134.3, 137.4, 137.9, 142.3, 146.5, 151.7, 152.3, 166.6,
174.4. MS (ESI): m/z 709 [M+Na]+.
6.4. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]-
isobenzofuran-5-yl)-4-(((3-chloroanilino) carbonyl)-
amino)benzamide (11b)
6.1. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-nitrobenzamide (9)
To
a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trime-
thoxyphenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo
[40,50:4,5]benzo[f]isobenzofuran-5-yl)-4-aminobenzamide
(10,
To a solution containing (5R,5aR,8aS,9S)-9-amino-5-(3,4,5-tri-
methoxyphenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxol-
200 mg, 0.37 mmol) in dry CH2Cl2 (10 mL), 3-chlorophenyl isocy-
anate (b, 0.8 mL, 0.56 mmol) was added at 0 °C and the stirring
was continued for 1 h at the room temperature. Later, the formed
solid was filtered and washed thoroughly with CH2Cl2 to obtain
the pure compound 11b (210 mg, 76%). Mp 247–249 °C;
o[40,50:4,5]benzo[f]isobenzofuran-6-one (7, 2.0 g, 0.820 mmol),
Et3N (2.3 mL, 1.652 mmol) in 20 mL of CH2Cl2 and 4-nitrobenzoyl
chloride (8, 1.83 g, 0.991 mmol) in 10 mL of CH2Cl2 was added un-
der nitrogen atmosphere and stirred at room temperature for 3 h,
till the completion of the reaction as monitored by TLC. The reac-
tion mixture was washed with water and extracted with CH2Cl2,
dried over anhydrous Na2SO4 and the crude product was purified
by column chromatography with ethyl acetate/hexane (3:7) pro-
vided pure compound 9 in good yield (2.5 g, 92%). Mp 174–
177 °C; 1H NMR (200 MHz, CDCl3): d 2.95–3.01 (m, 2H), 3.65 (s,
3H), 3.70 (s, 6H), 3.81 (t, 1H), 4.06–4.13 (dd, 1H, J = 7.5, 6.7 Hz),
4.37–4.46 (m, 2H), 5.98 (d, 2H, J = 4.5 Hz), 6.22 (s, 2H), 6.45 (s,
½
a 2D5
ꢂ
= ꢀ61.1 (c 0.5 in CHCl3); 1H NMR (400 MHz, CDCl3): d
3.40–3.51 (m, 2H), 3.73 (s, 3H), 3.77 (s, 6H), 4.41 (t, 1H,
J = 8.08 Hz), 4.61–4.69 (m, 2H), 5.49–5.53 (m, 1H), 6.01 (d, 2H,
J = 6.6 Hz), 6.30 (s, 2H), 6.51 (s, 1H), 6.80 (s, 1H), 7.20 (d, 2H,
J = 6.61 Hz), 7.51 (d, 2H, J = 8.08 Hz), 7.70 (s, 1H), 7.91 (d, 2H,
J = 8.81 Hz) 8.41 (d, 1H, J = 8.08 Hz). 13C NMR (75 MHz, CDCl3): d
40.6, 43.1, 47.1, 55.4, 59.8, 68.1, 100.1, 107.5, 108.7, 109.2,
116.1, 116.7, 120.6, 122.9, 124.0, 125.0, 125.7, 128.2, 129.1,

A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
5205
130.8, 131.7, 134.9, 134.6, 137.7, 137.4, 137.9, 142.5, 146.6, 151.9,
60.2, 68.5, 100.9, 107.4, 108.8, 109.3, 116.5, 116.9, 118.5, 121.9,
122.1, 124.1, 123.9, 126.2, 128.3, 129.5, 131.5, 133.4, 134.8,
137.7, 137.9, 142.8, 146.9, 152.0, 152.3, 159.1, 166.1, 174.2. MS
(ESI): m/z 727 [M+Na]+.
152.2, 166.4, 174.3. MS (ESI): m/z 686 [M+H]+.
6.5. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((2,6-dichloroanilino)carbonyl)
amino)benzamide (11c)
6.8. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((3,5-dimethylanilino)carbonyl)amino)
benzamide (11f)
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo [40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol)
in dry CH2Cl2 (10 mL), 2,6-dichlorophenyl-1-isocyanate (c),
(106 mg, 0.56 mmol) was added at 0 °C and the stirring was contin-
ued for 1 h at the room temperature. After 1 h, the formed solid was
filtered and washed with CH2Cl2 thoroughly to obtain pure com-
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 3,5-dimethyl-phenyl isocyanate (f), (0.66 mL,
0.56 mmol) was added at 0 °C and the stirring was continued for
1 h at room temperature. After 1 h, the formed solid was filtered
and washed with CH2Cl2 thoroughly to obtain the pure compound
pound 11c in 190 mg, 80% yield. Mp 140–143 °C; ½a D25
= ꢀ70.6 (c
ꢂ
0.5 in CHCl3); 1H NMR (300 MHz, CDCl3): d 3.20–3.27 (m, 2H),
3.78 (s, 3H), 3.81 (s, 6H), 4.50–4.57 (m, 1H), 4.71–4.46 (m, 2H),
5.60–5.65 (m, 1H), 6.10 (s, 2H, J = 2.93 Hz), 6.40 (s, 2H), 6.71 (s,
1H), 7.01 (s, 1H), 7.40 (t, 1H, J = 7.34, 8.81 Hz), 8.00 (d, 2H,
J = 8.81 Hz). 13C NMR (75 MHz, CDCl3): d 40.8, 43.1, 47.2, 55.3,
60.1, 68.6, 100.9, 107.4, 108.6, 109.3, 116.2, 116.9, 123.9, 126.3,
127.8, 128.0, 129.2, 130.6, 131.4, 132.4, 134.6, 137.4, 137.9, 142.5,
146.9, 151.9, 152.4, 166.7, 174.3. MS (ESI): m/z 720 [M+H]+.
11f in 200 mg, 86% yield. Mp 176–179 °C; ½a D25
= ꢀ69.9 (c 0.5 in
ꢂ
CHCl3); 1H NMR (200 MHz, CDCl3): d 2.50 (s, 6H), 3.19–3.26 (m,
2H), 3.67 (s, 3H, -OCH3), 3.70 (s, 6H), 4.40 (t, 1H, J = 7.77,
8.32 Hz), 4.60–4.68 (m, 1H), 5.51–5.59 (m, 1H), 6.00 (d, 2H,
J = 4.90 Hz), 6.30 (s, 2H), 6.61 (s, 1H), 6.80 (s, 1H), 7.20 (d, 2H,
J = 3.96 Hz), 7.50 (d, 2H, J = 8.68 Hz), 7.80 (d, 2H, J = 8.68 Hz), 8.50
(s, 1H). 13C NMR (75 MHz, CDCl3): d 20.7, 40.8, 43.2, 47.3, 55.5,
60.0, 68.4, 100.8, 107.5, 108.7, 109.1, 116.0, 116.8, 123.8, 125.9,
128.0, 129.3, 131.6, 134.7, 137.8, 138.2, 142.6, 146.7, 147.3,
151.8, 152.2, 166.5, 174.3. MS (ESI): m/z 703 [M+Na]+.
6.6. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-((((5-bromo-2-thienyl)amino)
carbonyl)amino)benzamide (11d)
6.9. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((3,5-di(trifluoromethyl)
anilino)carbonyl)amino)benzamide (11g)
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 5-bromothiophenyl isocyanate (d), (76.5 mg,
0.37 mmol) was added at 0 °C and the stirring was continued for
1 h at room temperature. After 1 h, the formed solid was filtered
and washed with CH2Cl2 thoroughly to obtain the pure compound
To
thoxyphenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo
[f]isobenzofuran-5-yl)-4-aminobenzamide (10), (200 mg,
a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trime-
11d in 210 mg, 75% yield. Mp 168–170 °C; ½a D25
= ꢀ80.9 (c 0.5 in
ꢂ
0.37 mmol) in dry CH2Cl2 (10 mL), 3,5-bis(trifluoromethyl)phenyl
isocyanate (g), (115 mg, 0.56 mmol) was added at 0 °C and the
stirring was continued for 1 h at the room temperature. After
1 h, the formed solid was filtered and washed with CH2Cl2 thor-
oughly to obtain the pure compound 11g in 220 mg, 77% yield.
CHCl3); 1H NMR (200 MHz, CDCl3): d 3.03–3.17 (m, 1H), 3.50–
3.60 (m, 1H), 3.68 (s, 3H), 3.73 (s, 6H), 4.40 (t, 1H, J = 8.81,
7.34 Hz), 4.61–4.68 (m, 2H), 5.48–5.54 (m, 1H), 6.03 (d, 2H,
J = 2.94 Hz), 6.37 (s, 2H), 6.61 (s, 1H), 6.88 (s, 1H), 7.49–7.58 (m,
4H), 7.93 (d, 2H, J = 8.81 Hz). 13C NMR (75 MHz, CDCl3): d 41.0,
43.6, 47.4, 55.8, 60.6, 68.9, 101.0, 107.7, 108.6, 109.2, 116.4,
117.0, 119.3, 124.0, 124.1, 126.0, 128.3, 129.7, 131.9, 132.5,
135.0, 135.7, 137.6, 137.8, 142.5, 146.4, 151.7, 152.3, 166.0,
174.2. MS (ESI): m/z 759 [M+Na]+.
Mp 213–215 °C;
½
a 2D5
ꢂ
= ꢀ76.9 (c 0.5 in CHCl3); 1H NMR
(300 MHz, CDCl3): d 2.91–2.99 (m, 2H), 3.74 (s, 3H), 3.78 (s, 6H),
4.20–4.26 (m, 1H), 4.30–4.48 (m, 2H), 5.40–5.46 (m, 1H), 5.90
(d, 2H, J = 12.2 Hz), 6.30 (s, 2H), 6.71 (s, 1H), 7.30 (d, 2H,
J = 8.43 Hz), 7.50 (s, 1H), 7.62 (d, 2H, J = 8.43 Hz), 7.90 (d, 2H,
J = 8.10 Hz), 8.41 (s, 1H). 13C NMR (75 MHz, CDCl3): d 41.1, 43.3,
47.7, 55.6, 60.6, 68.5, 101.2, 107.6, 109.0, 109.5, 116.4, 116.9,
119.4, 121.9, 124.1, 125.6, 126.2, 128.2, 129.1, 131.9, 131.6,
134.4, 137.2, 137.5, 137.9, 142.4, 146.9, 151.8, 152.3, 166.1,
174.3. MS (ESI): m/z 788 [M+H]+.
6.7. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((3-chloro-4-fluoroanilino)
carbonyl)amino)benzamide (11e)
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 3-chloro-4-fluorophenyl isocyanate (e),
(63 mg, 0.37 mmol) was added at 0 °C and the stirring was contin-
ued for 1 h at room temperature. After 1 h, the formed solid was fil-
tered and washed with CH2Cl2 thoroughly obtained the pure
compound 11e in 220 mg, 83% yield. Mp 180–182 °C;
6.10. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((4-methoxyanilino)carbonyl) amino)
benzamide (11h)
To
thoxyphenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo
[f]isobenzofuran-5-yl)-4-aminobenzamide (10), (200 mg,
a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trime-
½
a 2D5
ꢂ
= ꢀ57.9 (c 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3): d 2.97–
0.37 mmol) in dry CH2Cl2 (10 mL), 4-methoxyphenyl-1-isocyanate
(h), (84 mg, 0.56 mmol) was added at 0 °C and the stirring was
continued for another 1 h at the room temperature. After 1 h,
the formed solid was filtered and washed with CH2Cl2 thoroughly
obtained the pure compound 11h in 180 mg, 75% yield. Mp 164–
3.14 (m, 1H), 3.48–3.55 (m, 1H), 3.63 (s, 3H), 3.67 (s, 6H), 4.33–
4.39 (m, 1H), 4.49–4.59 (m, 2H), 5.45–5.48 (m, 1H), 6.01 (s, 2H),
6.33 (s, 2H), 6.58 (s, 1H), 6.85 (s, 1H), 7.33 (d, 2H, J = 5.28 Hz),
7.54 (d, 1H, J = 6.79 Hz), 7.80–7.88 (m, 3H), 8.61 (d, 1H,
J = 6.79 Hz). 13C NMR (75 MHz, CDCl3): d 40.7, 43.0, 47.2, 55.6,
67 °C; ½a 2D5
ꢂ
= ꢀ35.7 (c 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3): d

5206
A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
3.41–3.48 (m, 2H), 3.73 (s, 12H), 4.30 (t, 1H, J = 8.05 Hz), 4.50–
4.59 (m, 2H), 5.50–5.58 (m, 1H), 5.90 (d, 2H, J = 1.79 Hz), 6.30
(s, 2H), 6.50 (s, 1H), 6.70 (t, 2H, J = 8.05, 8.95 Hz), 6.80 (s, 1H),
7.50 (d, 2H, J = 8.95 Hz), 7.80 (d, 2H, J = 8.95 Hz), 8.20 (d, 2H,
J = 7.10 Hz), 8.30 (d, 2H, J = 7.10 Hz). 13C NMR (75 MHz, CDCl3):d
40.8, 43.3, 47.3, 55.5, 57.0, 60.0, 68.4, 100.8, 107.5, 108.7, 109.1,
115.8, 116.3, 116.9, 123.5, 123.9, 125.9, 128.4, 128.9, 129.3,
131.6, 134.8, 137.1, 137.8, 142.6, 146.7, 151.7, 152.0, 158.2,
166.6, 174.5. MS (ESI): m/z 682 [M+H]+.
dry CH2Cl2 (10 mL), 2-chloroethyl isocyanate (k), (0.07 mL,
0.56 mmol) was added at 0 °C and the stirring was continued for
another 1 h at the room temperature. After 1 h, the formed solid
was filtered and washed with CH2Cl2 thoroughly to obtain the pure
compound 11k in 160 mg, 66% yield. Mp 201–203 °C;
½
1 a 2D5
ꢂ
= ꢀ86.7 (c 0.5 in CHCl3); 1H NMR (400 MHz, CDCl3): d 3.44–
3.56 (m, 2H), 3.59–3.65 (m, 2H), 3.72 (s, 3H), 3.73 (s, 6H), 3.78–
3.87 (m, 3H), 4.31–4.39 (m, 1H), 4.54 (d, 1H, J = 3.91 Hz), 5.44–
5.50 (m, 1H), 5.95 (s, 2H), 6.29 (s, 2H), 6.49 (s, 1H), 6.83 (s, 1H),
7.43 (d, 2H, J = 7.83 Hz), 7.80 (d, 2H, J = 7.83 Hz). 13C NMR
(75 MHz, CDCl3): d 41.0, 42.1, 43.3, 43.8, 47.2, 55.4, 59.8, 68.5,
100.9, 107.7, 109.0, 109.4, 116.1, 117.2, 124.0, 125.8, 128.0,
129.0, 131.1, 134.5, 137.2, 137.7, 142.5, 146.7, 151.8, 152.4,
166.3, 174.3. MS (ESI): m/z 661 [M+Na]+.
6.11. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-(((1-naphthylamino)carbonyl)
amino)benzamide (11i)
6.14. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro [1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-((((2-bromoethyl)amino)
carbonyl)amino)benzamide (11l)
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), naphthalene-1-isocyanate (i), (95 mg,
0.56 mmol) was added at 0 °C and the stirring was continued for
1 h at the room temperature. After 1 h, the formed solid was fil-
tered and washed with CH2Cl2 thoroughly to obtain the pure com-
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 2-bromoethyl isocyanate (l), (0.13 mL,
0.56 mmol) was added at 0 °C and the stirring was continued for
another 1 h at the room temperature. After 1 h, the formed solid
was filtered and washed with CH2Cl2 thoroughly to obtain the pure
pound 11i in 180 mg, 83% yield. Mp 170–174 °C; ½a D25
= ꢀ69.8 (c
ꢂ
0.5 in CHCl3); 1H NMR (300 MHz, CDCl3): d 3.23–3.29 (m, 2H),
3.61 (s, 3H), 3.70 (s, 6H), 4.10 (t, 1H, J = 7.93 Hz), 4.30–4.38 (m,
2H), 5.20–5.27 (m, 1H), 5.70 (d, 2H, J = 3.96 Hz), 6.00 (s, 2H), 6.40
(s, 1H), 6.61 (s, 1H), 7.30–7.59 (m, 7H), 7.61–7.78 (m, 2H), 7.91
(d, 1H, J = 7.93 Hz), 8.31 (d, 1H, J = 9.32 Hz). 13C NMR (75 MHz,
CDCl3): d 40.9, 43.4, 47.3, 55.5, 60.1, 68.3, 100.7, 107.4, 108.8,
109.2, 110.8, 116.1, 116.7, 120.1, 122.6, 123.7, 125.1, 125.7,
125.8, 126.6, 127.5, 128.3, 129.2, 129.6, 131.4, 134.6, 134.4,
137.7, 137.7, 142.1, 142.4, 146.5, 151.9, 152.3, 166.6, 174.4. MS
(ESI): m/z 702 [M+H]+.
compound 11l in 170 mg, 70% yield. Mp 212–214 °C; 1½a D25
= ꢀ76.2
ꢂ
(c 0.5 in CHCl3); 1H NMR (300 MHz, CDCl3): d 2.96–3.10 (m, 2H),
3.54–3.59 (m, 2H), 3.62 (s, 3H), 3.66 (s, 6H), 3.69–3.79 (m, 3H),
3.83–3.92 (m, 1H), 4.36 (t, 1H, J = 7.55, 7.17 Hz), 4.57 (d, 1H,
J = 4.91 Hz), 5.40–5.49 (m, 1H), 5.99 (d, 2H, J = 4.91 Hz), 6.32 (s,
2H), 6.57 (s, 1H), 6.83 (s, 1H), 7.46 (d, 2H, J = 8.49 Hz), 7.81 (d,
2H, J = 8.49 Hz). 13C NMR (75 MHz, CDCl3): d 40.1, 40.8, 43.2,
47.3, 50.2, 55.5, 60.0, 68.4, 100.8, 107.5, 108.7, 109.1, 116.0,
116.8, 123.8, 125.9, 128.0, 129.3, 131.6, 134.7, 137.3, 137.8,
142.6, 146.7, 151.8, 152.2, 166.5, 174.3. MS (ESI): m/z 705 [M+Na]+.
6.12. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-((((2-furylmethyl)amino)
carbonyl)amino)benzamide (11j)
7. Materials and methods
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), furfuryl isocyanate (j), (0.48 mL, 0.56 mmol)
was added at 0 °C and the stirring was continued for another 1 h
at the room temperature. After 1 h, the formed solid was filtered
and washed with CH2Cl2 thoroughly obtained the pure compound
7.1. Procedure of the SRB-assay
The synthesized compounds 11a–l have been evaluated for
their in vitro cytotoxicity in human cancer cell lines. A protocol
of 48 h continuous drug exposure has been used and a sulforhoda-
mine B (SRB) protein assay has been used to estimate cell viability
or growth. The cell lines were grown in DMEM medium containing
11j in 210 mg, 79% yield. Mp 210–213 °C;
½
1 a 2D5
ꢂ
= ꢀ29.7 (c 0.5 in
CHCl3); 1H NMR (400 MHz, CDCl3): d 3.40–3.45 (m, 2H), 3.70 (s,
3H), 3.73 (s, 6H), 4.25 (d, 1H, J = 5.85 Hz), 4.29 (d, 2H,
J = 5.12 Hz), 4.50–4.65 (m, 2H), 5.51–5.58 (m, 1H), 6.20 (d, 1H,
J = 2.92 Hz), 6.30 (s, 2H), 6.51 (s, 1H), 6.70 (s, 1H), 7.21 (d, 2H,
J = 7.35 Hz), 7.50 (d, 2H, J = 7.35 Hz), 7.80 (d, 1H, J = 8.04 Hz), 8.10
(d, 1H, J = 8.04 Hz). 13C NMR (75 MHz, CDCl3): d 40.1, 40.8, 43.1,
47.2, 55.6, 60.2, 68.5, 100.7, 107.1, 107.6, 108.9, 109.4, 111.2,
116.2, 116.9, 123.9, 126.3, 128.2, 129.0, 131.4, 134.3, 137.1,
137.7, 143.2, 142.5, 146.8, 149.7, 151.9, 152.3, 166.4, 174.1. MS
(ESI): m/z 655 [M+H]+.
10% fetal bovine serum and 2 mM L-glutamine and were inoculated
into 96 well microtiter plates in 90 microliter at plating densities
depending on the doubling time of individual cell lines. The micro-
titer plates were incubated at 37 °C, 5% CO2, 95% air, and 100% rel-
ative humidity for 24 h prior to addition of experimental drugs.
Aliquots of 10 microliter of the drug dilutions were added to the
appropriate microtiter wells already containing 90 microliter of
cells, sulting in the required final drug concentrations. For each
compound four concentrations (0.1, 1, 10 and 100 lM) were eval-
uated and done in triplicate wells. Plates were incubated further
for 48 h and assay was terminated by the addition of 50 microliter
of cold trichloro acetic acid (TCA) (final concentration, 10% TCA)
and incubated for 60 min at 4 °C. The plates were washed five
times with tap water and air dried. Sulforhodamine B (SRB) solu-
tion (50 mL) at 0.4% (w/v) in 1% acetic acid was added to each of
the cells, and plates were incubated for 20 min at room tempera-
ture. The residual dye was removed by washing five times with
1% acetic acid. The plates were air dried. Bound stain was subse-
quently eluted with 10 mM trizma base, and the absorbance was
6.13. N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-Trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]
isobenzofuran-5-yl)-4-((((2-chloroethyl)amino)
carbonyl)amino)benzamide (11k)
To a solution of N1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxy-
phenyl)-5,5a,6,8,8a,9-hexahydro[1,3]dioxolo[40,50:4,5]benzo[f]iso-
benzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in

A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
5207
read on an ELISA plate reader at a wavelength of 540 with 690 nm
reference wavelengths. Percent growth was calculated on a plate
by plate basis for test wells relative to control wells. The above
determinations were repeated three times. Percentage growth
was expressed as the (ratio of average absorbance of the test well
to the average absorbance of the control wells) ꢁ 100. Growth inhi-
extracts were obtained. After centrifugation at 12,000 rpm for
2 min at 4 °C to remove cell debris, we collected supernatants for
ATP measurement. The amount of ATP was determined by the
ATP monitoring kit.
7.6. Cell cycle analysis
bition of 50% (GI50) was calculated from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢁ 100
50, which is the drug concentration resulting in a 50% reduction
For flow cytometric analysis of DNA content, 5 ꢁ 105 Colo-205
1
4
in the net protein increase (as measured by SRB staining) in control
cells in exponential growth were treated at a concentration of
1
cells during the drug incubation. Where, Tz
Optical density at
2 lM of 11a, 11h, 11k, 11l and etoposide (Eto) for 24 h. After the
4
time zero, OD of control 1 C, and OD of test growth in the presence
incubation period, the cells were collected, centrifuged and fixed
with ice-cold 70% ethanol at 4 °C for 30 min. Then the cells were
incubated with 1 mg/mL RNAase A solution (Sigma) at 37 °C for
4
1
of drug
Ti.
4
7.2. Cell culture
30 min followed by staining with 250 lL of DNA staining solution
[10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and
0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ
water at room temperature for 30 min in the dark]. The DNA con-
tents of 20,000 events were measured by flow cytometer (DAKO
CYTOMATION, Beckman Coulter, and Brea, CA). Histograms were
analyzed using Summit Software.
Human colon (Colo-205) and breast (MDA-MB-231) carcinoma
cells were purchased from American Type Culture collection Cen-
tre (ATCC). Dulbecco’s modified Eagle’s medium (DMEM) (Invitro-
gen), supplemented with 2 mM glutamax (Invitrogen), 10% fetal
calf serum and 100 U/ml Penicillin and 100 mg/ml streptomycin
sulfate (Sigma) was used as growth media for MDA-MB-231 cells.
RPMI medium supplemented with 10% FCS with appropriate anti-
biotics (Penicillin, Streptomycin and Kanamycin) was used for
growing Colo-205 cells. These cell lines were maintained at 37 °C
in a humidified atmosphere containing 5% CO2 in the incubator.
7.7. DNA topoisomerase-IIa inhibition assay
The mixture of 200 ng of super coiled pBR322 plasmid DNA and
4 units of human DNA topoisomerase-II (Sigma, USA) was incu-
a
bated with the compounds [11a–11l and etoposide (Eto)] in the as-
7.3. MTT assay
say buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 5 mM
MgCl2, 1 mM EDTA, 1 mM ATP, and 15
min) for 30 min at 30 °C. The reaction in a final volume of 20
was terminated by the addition of 3 L of 7 mM EDTA. Reaction
products were analyzed on a 1% Agarose gel at 60 V for 1 h with
a running buffer of TAE. Gels were stained for 30 min in an aqueous
lg/mL bovine serum albu-
Individual wells of a 96-well tissue culture micro titer plate
lL
were inoculated with 100
l
L of complete medium containing
l
1 ꢁ 104 cells. The plates were incubated at 37 °C in a humidified
5% CO2 incubator for 18 h prior to the experiment. After medium
removal, 100
11a, 11h, 11k, 11l and etoposide (Eto) at different concentrations
such as 0.5, 1, 2 and 4 M were added to each well and incubated
at 37 °C for 24 h. Then the medium was discarded and replaced
with 10 L MTT dye. Plates were incubated at 37 °C for 2 h. The
resulting formazan crystals were solubilized in 100 L extraction
buffer. The optical density (O.D) was read at 570 nm with micro
plate reader (Multi-mode Varioskan Instrument-Themo Scientific).
The percentage of DMSO in the medium never exceeded 0.25%.
l
L of fresh medium containing the test compounds
solution of ethidium bromide (0.5 lg/mL). DNA bands were visual-
ized by transillumination with UV light.
l
7.8. DNA topoisomerase-I inhibition assay
l
l
The activity of DNA topoisomerase-I was determined by assess-
ing the relaxation of supercoiled DNA pBR322. The mixture of
500 ng of plasmid pBR322 DNA and 0.4 units of recombinant hu-
man DNA topoisomerase-I (TopoGEN INC., USA) was incubated
without and with the synthesized compounds (11a–11l) at 37 °C
for 30 min in the relaxation buffer (10 mM Tris–HCl (pH 7.9),
150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5%
7.4. Trypan blue exclusion test of cell viability
This method is used to determine the number of viable cells
present in cell suspension. This method is based on the principle
that live cells possess intact cell membranes that excludes trypan
blue; whereas dead cells are not capable of excluding trypan blue.
Here viable cells show a clear cytoplasm where as non viable cells
show blue colour cytoplasm. Compounds were treated for 24 h
time period. After compound treatment (11a, 11h, 11k, 11l and
glycerol). The reaction in the final volume of 10
lL was terminated
by adding 2.5 L of the stop solution containing 5% sarcosyl,
l
0.0025% bromophenol blue, and 25% glycerol. DNA samples were
then electrophoresed on a 1% agarose gel.
7.9. Caspase-3 assay
etoposide (Eto) at 1, 2 and 4
trypsinized and the dead and viable cells were counted. Each sam-
ple was assayed for triplicates. In this assay 10 L of 0.4% solution
of trypan blue was added to 100 L of cells. Once after mixing the
l
M concentrations, the cells were
The caspase-3 fluorescent assay kit (Clonetech, CA) was applied
to evaluate the caspase-3 activity, using the procedures provided
by the manufacturer. Colo-205 cells were treated with compounds
l
l
11a, 11h, 11k, 11l and etoposide (Eto) at 2 lM concentration for
sample was loaded onto haemocytometer and examined immedi-
ately. The percentage of dead cells was calculated.
24 h. Cell lysates were added to the 2ꢁ reaction buffer containing
DTT and caspase substrate was added and incubation was carried
out at 37 °C for 1 h. Readings were taken at excitation wavelength
400 nm and emission wavelength 505 nm.
7.5. ATP content bioluminescence assay
The content of intracellular ATP was measured by biolumines-
cent assay on the basis of the measurement of the light output of
the luciferin-luciferase reaction. The luciferin-luciferase was pur-
chased as a kit from Promega Company. Colo-205 cells were trea-
7.10. Protein extraction and Western blot analysis
Colo-205 human colon cancer cells were seeded in 60 mm
dishes and were allowed to grow to attain 80% confluency for
24 h. Compounds (11a, 11h, 11k, 11l and etoposide (Eto)) were
ted with concentration of 2
lM for 24 h, then cells were harvested
and lysed after treatment with ice cold RIPA buffer, and cell
added to the culture media for a final concentration of 2 lM, and

5208
A. Kamal et al. / Bioorg. Med. Chem. 21 (2013) 5198–5208
13. Jardine, I. In Anti-Cancer Agents Based on Natural Products Models, Cassady, J. M.;
Doures, J. Eds.; Academic: New York, 1980, p. 319.
14. Issell, B. F. Cancer Chemother. Pharmacol. 1982, 7, 73.
15. Budman, D. R. Semin. Oncol. 1996, 23, 8.
16. Greco, F. A.; Hainsworth, J. D. Semin. Oncol. 1996, 23, 45.
17. Lee, H. K.; Wang, K. H. J. Food Drug Anal. 1995, 3, 209.
18. Xiao, Z.; Xiao, Y.-D.; Golbraikh, A.; Tropsha, A.; Lee, K. H. J. Med. Chem. 2002, 45,
2294.
19. VanVilet, D. S.; Tachibana, Y.; Bastow, K. F.; Huang, E. S.; Lee, K. H. J. Med. Chem.
2001, 44, 1422.
the cells were incubated with compounds for 24 h. After 24 h, total
cell lysates from cultured colo-205 cells were obtained by lysing
the cells in ice-cold RIPA buffer (1ꢁ PBS, 1% NP-40, 0.5% sodium
deoxycholate, and 0.1% SDS) containing100 mg/mL PMSF, 5 mg/
mL aprotinin, 5 mg/mL leupeptin, 5 mg/mL pepstatin, and
100 mg/mL NaF. After centrifugation at 12,000 rpm (8064 g) for
10 min, protein in the supernatant was quantified by the Bradford
method (BioRad) using a Multimode VarioSkan instrument (Ther-
20. Smith, R. A.; Dumas, J.; Adnane, L.; Wilhelm, S. M. Curr. Top. Med. Chem. 2006, 6,
1071.
mo Scientific); 50 lg proteins per lane was loaded on a 12% SDS–
polyacrylamide gel. After electrophoresis, the protein was trans-
ferred to a polyvinylidine difluoride (PVDF) membrane (Amersham
Biosciences). The membrane was blocked at room temperature for
2 h in TBS containing 0.1% Tween 20 (TBST) with 5% blocking pow-
der (Santa Cruz Biotechnology). The membrane was washed with
TBST for 5 min and then primary antibody was added and incu-
bated at 4 °C overnight. Active caspase-3 and b-actin were pur-
chased from Imgenex Corporation (San Diego, CA, USA).
Membranes were washed three times with TBST for 15 min, and
the blots were visualized with chemiluminescent reagent (Thermo
Scientific). X-ray films were developed and fixed using solutions
from Kodak.
21. Montagut, C.; Settleman, J. Cancer Lett. 2009, 283, 125.
22. Ramnath, N.; Adjei, A. Update Cancer Ther. 2007, 2, 111.
23. Miyahara, M.; Kashiwada, Y.; Guo, X.; Chen, H. X.; Cheng, Y. C.; Lee, K. H.
Heterocycles 1994, 39, 361.
24. Peticlerc, E.; Deschesnes, R. G.; Cote, M. F.; Marques, C.; Janvier, R.; Lacroix, J.;
Miot-Noirault, E.; Legault, J.; Mounetou, E.; Madel-mont, J. C.; Gaudreault, R. C.
Cancer Res. 2004, 64, 4654.
25. (a) Esteves-Souza, A.; Pissinate, K.; Nascimento, M. G.; Grynberg, N. F.;
Echevarria, A. Bioorg. Med. Chem. 2006, 14, 492; (b) Denny, W. A.; Baguley, B.
C. Curr. Top. Med. Chem. 2003, 3, 339; (c) Salerno, S.; Da Settimo, F.; Taliani, S.;
Simorini, F.; La Motta, C.; Fornaciari, G.; Marini, A. M. Curr. Med. Chem. 2010, 17,
4270; (d) Sheng, C.; Miao, Z.; Zhang, W. Curr. Med. Chem. 2011, 18, 4389; (e)
Dong, G.; Wang, S.; Miao, Z.; Yao, J.; Zhang, Y.; Guo, Z.; Zhang, W.; Sheng, C. J.
Med. Chem. 2012, 55, 7593.
26. Heck, M. M. S.; Hittelman, W. N.; Earnshaw, W. C. Proc. Natl. Acad. Sci. 1998, 85,
1086.
27. Sugiomoto, Y.; Tsukahara, S.; Oh-Hara, T.; Isoe, T.; TsuRuo, T. Cancer Res. 1990,
50, 6925.
28. Whiteacre, C. M.; Zborowska, E.; Gordon, N. H.; Mackay, W.; Berger, N. A.
Cancer Res. 1997, 57, 1425.
7.11. Hoechst nuclear staining
Colo-205 cells were seeded on cover slips, treated with com-
pounds 11a and etoposide (Eto) for 24 h, washed with PBS and
fixed with 4% Para formaldehyde for 15 min at room temperature.
Fixed cells were incubated in PBS (pH 7.4) containing DNAse-free
RNase (Sigma) for 30 min at 37 °C and stained with Hoechst. Nu-
clear morphology of the cells was observed under confocal
microscope.
29. Poddevin, B.; Riou, J. F.; Lavelle, F.; Pommier, Y. Mol. Pharmacol. 1993, 44, 767.
30. Finlay, G. J.; Riou, J. F.; Baguley, B. C. Eur. J. Cancer 1996, 32A, 708.
31. Kamal, A.; Laxman, N.; Ramesh, G. Bioorg. Med. Chem. Lett. 2000, 10, 2059.
32. Kamal, A.; Kumar, B. A.; Arifuddin, M.; Sunanda, G. D. Bioorg. Med. Chem. 2003,
11, 5135.
33. Kamal, A.; Kumar, B. A.; Suresh, P.; Agrawal, S. K.; Chashoo, G.; Singh, S. K.;
Saxena, A. K. Bioorg. Med. Chem. 2010, 18, 8493.
34. Kamal, A.; Kumar, B. A.; Suresh, P.; Shankaraiah, N.; Kumar, M. S. Bioorg. Med.
Chem. Lett. 2011, 21, 350.
35. Kamal, A.; Suresh, P.; Mallareddy, A.; Kumar, B. A.; Reddy, P. V.; Raju, P.;
Tamboli, J. R.; Shaik, T. B.; Jain, N.; Kalivendi, S. V. Bioorg. Med. Chem. 2011, 19,
2349.
Acknowledgment
36. Kamal, A.; Kumar, B. A.; Suresh, P.; Juvekar, A.; Zingde, S. Bioorg. Med. Chem.
2011, 19, 975.
37. Kamal, A.; Suresh, P.; Ramaiah, M. J.; Mallareddy, A.; Kumar, B. A.; Raju, P.;
Gopal, J. V.; Pushpavalli, S. N. C. V. L.; Lavanya, A.; Sarma, P.; Bhadra, M. P.
Bioorg. Med. Chem. 2011, 19, 4589.
38. Skehn, P.; Storeng, R.; Scudiero, A.; Monks, J.; McMohan, D.; Vistica, D.;
Jonathan, W. T.; Bokesch, H.; Kenney, S.; Boyd, R. M. J. Natl. Cancer Inst. 1990,
82, 1107.
39. Hu, W. P.; Yu, H. S.; Sung, P. J.; Tsai, F. Y.; Shen, Y. K.; Chang, L. S.; Wang, J. J.
Chem Res. Toxicol. 2007, 20, 905.
P.S is thankful to UGC, New Delhi for the award of Senior Re-
search Fellowship.
References and notes
1. Li, T. K.; Liu, L. F. J. Antimicrob. Agents 1998, 42, 1022.
2. Kelly, M. G.; Hartwell, J. L.; Natl, J. Cancer Inst. 1954, 967–1010.
3. Imbert, T. Biochimie 1998, 80, 207.
4. Loike, J. D.; Brewer, C. F.; Sternlicht, H.; Gensler, W. J.; Horwitz, S. B. Cancer Res.
1978, 38, 2688.
5. Sackett, D. L. Pharmacol. Ther. 1993, 59, 163.
40. Cheng, M. H.; Yang, Y. C.; Wong, Y. H.; Chen, T. R.; Lee, C. Y.; Yang, C. C.; Chen, S.
H.; Yang, I. N.; Yang, Y. S.; Hunag, H. S.; Yang, C. Y.; Huang, M. S.; Chiu, H. F.
Anticancer Drugs 2012, 23, 191.
6. Cortese, F.; Bhattacharyya, B.; Wolff, J. J. Biol. Chem. 1977, 252, 1134.
7. Ter Haar, E.; Rosenkranz, H. S.; Hamel, E.; Day, W. D. Bioorg. Med. Chem. 1996, 4,
1659.
41. Kumar, S. Cell Death Differ. 2007, 14, 32.
42. Droin, N.; Dubrez, L.; Eymin, B.; Renvoize, C.; Bread, J.; Dimanche-Boitrel, M. T.;
Solary, E. Oncogene 1998, 16, 2885.
8. Stähelin, H.; von Wartburg, A. Cancer Res. 1991, 51, 5.
9. Stähelin, H. Eur. J. Cancer 1973, 9, 215.
10. Hande, K. R. Eur. J. Cancer 1998, 34, 1514.
43. Thornberry, N. A. Chem. Biol. 1998, 5, R97.
44. Janicke, R. U.; Sprengart, M. L.; Wati, M. R.; Porter, A. G. J. Biol. Chem. 1998, 273,
9357.
11. Stähelin, H. Eur. J. Cancer 1970, 6, 303.
45. Naik, P. K.; Dubey, A.; Soni, K.; Kumar, R.; Singh, H. J. Mol. Graphics Modell. 2010,
29, 546.
46. Xiao, X.; Cushman, M. J. Am. Chem. Soc. 2005, 127, 9960.
12. Macdonald, T. L.; Lehnert, E. K.; Loper, J. T.; Chow, K. C.; Ross, W. E. In DNA
Topoisomerases in Cancer, Potmesil, M.; Kohn, K. W. Eds.; Oxford University:
New York, 1991, p. 199.