Synthesis and biological evaluation of 4β-sulphonamido and 4β-[(4′-sulphonamido)benzamide]podophyllotoxins as DNA topoisomerase-IIα and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmc.2012.01.039

A series of new 4β-sulphonamido and 4β-[(4′-sulphonamido) benzamide] conjugates of podophyllotoxin (11a-j and 15a-g) were synthesized and evaluated for anticancer activity against six human cancer cell lines and found to be more potent than etoposide. Som

Full text of DOI:10.1016/j.bmc.2012.01.039

Bioorganic & Medicinal Chemistry 20 (2012) 2054–2066
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4b-sulphonamido and 4b-[(40-sulpho-
namido)benzamide]podophyllotoxins as DNA topoisomerase-II
inducing agents
a and apoptosis
Ahmed Kamal a, , Paidakula Suresh a, M. Janaki Ramaiah b, Adla Mallareddy a, Syed Imthiajali a,
⇑
S.N.C.V.L. Pushpavalli b, A. Lavanya b, Manika Pal-Bhadra b,
⇑
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Centre for Chemical Biology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
A series of new 4b-sulphonamido and 4b-[(40-sulphonamido)benzamide] conjugates of podophyllotoxin
(11a–j and 15a–g) were synthesized and evaluated for anticancer activity against six human cancer cell
lines and found to be more potent than etoposide. Some of the compounds 11b, 11d and 11e that showed
significant antiproliferative activity in Colo-205 cells, were superior to etoposide. The flow cytometric
analysis indicates that these compounds (11b, 11d and 11e) showed G2/M cell cycle arrest and among
them 11e is the most effective. It is observed that this compound (11e) caused both single-strand DNA
Article history:
Received 28 November 2011
Revised 23 January 2012
Accepted 24 January 2012
Available online 9 February 2012
Keywords:
Etoposide
Cell cycle
Anticancer activity
Comet assay
breaks as observed by comet assay as well as double-strand DNA breaks as indicated by
c-H2AX. Further
11e showed inhibition of topo-II as observed from Western blot analysis and related studies. Com-
a
pounds caused activation of ATM as well as Chk1 protein indicating that the compound caused effective
DNA damage. Moreover activation of caspase-3, p21, p16, NF-kB and down regulation of Bcl-2 protein
suggests that this compound (11e) has apoptotic cell death inducing ability, apart from acting as a
Topo-IIa enzyme
topo-IIa inhibitor.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Etoposide is widely used in clinic, alone or in association with other
chemotherapeutics for the treatment of testicular carcinomas and
small cell lung cancer.3 However its current therapeutic use is
somewhat limited by myelosuppression, particularly neutropenia.
Unlike podophyllotoxin, etoposide and teniposide do not inhibit
tubulin polymerization but induce dose-dependent DNA-strand
breakage associated with inhibition of DNA-topoisomerase II.4,5
In an ongoing effort to develop new and more potent antican-
cer agents, we have been involved in the last few years towards
the development of new molecules based on podophyllotoxins.6,7
In the present investigation, we report the synthesis and cyto-
toxic evaluation of 4b-sulphonamido and 4b-[(40-sulphonami-
do)benzamide] derivatives of 4-desoxypodophyllotoxin. The
choice of the sulphonamide group was dictated by the fact that
it constitutes an important class of drugs, with several types of
pharmacological agents possessing antitumor, anti-carbonic
anhydrase, or protease inhibitory activity.8 A host of structurally
novel sulphonamide derivatives have recently been reported to
show substantial antitumor activity. Some classical clinical anti-
cancer agents that comprise of sulphonamide groups are aceta-
zolamide (7), methazolamide (8) and ethoxzolamide (9)8a as
shown in Figure 1.
Chemotherapy is one of the main method to treat cancer. How-
ever during the course of cancer chemotherapy, it has been found
that prolonged treatment of carcinoma patients with certain anti-
cancer medicines can result in an acquired resistance toward mul-
tiple drugs, that is MDR.1 The problem of increasingly severe MDR
of tumor is a significant obstacle in providing effective chemother-
apy to many patients. Development of novel antitumor agents with
cytotoxicity against MDR cancer cell lines has currently become
the focus of oncology.
Podophyllotoxin (1), a well-known naturally occurring aryltetr-
alin lignan, is extracted as the main component from the roots and
rhizomes of Podophyllum species such as Podophyllum hexandrum
and Podophyllum peltatum.2 The biological activity of podophyllo-
toxin has led to several structural modifications resulting in many
useful compounds such as etoposide (2), teniposide (3) and the
water-soluble prodrug, etoposide phosphate (4). In addition, some
newly developed derivatives (e.g., NPF (5) and GL-331 (6)) display
a better pharmacological profile and are currently in clinical trials.
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.).
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.039

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2055
H
R1
O
OH
O
R2
O
O
O
HO
R1
R2
R3
H
O
O
O
CH3
O
OH
Etoposide (2)
O
O
Teniposide (3)
Etopophos (4)
H
H3CO
OH
OH
OCH3
S
OCH3
OH
P
H3CO
OCH3
HO
OR3
CH3
O
Podophyllotoxin (1)
R
HN
O
O
O
O
H3CO
OCH3
OH
R = F; NPF (5)
R = NO2; GL 331 (6)
O
N
O
N
N
N
N
EtO
S
SO2NH2
N
SO2NH2
Methazolamide (8)
N
H
SO2NH2
Acetazolamide (7)
S
S
Ethaxzolamide (9)
Figure 1. Structures of podophyllotoxin derivatives.
us to evaluate their cell viability in the Colo-205 cells, with a view
to study their detailed biological effects in this cell line. Colo-205
cells were treated with compounds 11b, 11d, 11e and etoposide
2. Chemistry
The synthesis of new 4b-sulphonamido and 4b-[(40-sulphonam-
ido)benzamide] congeners of 4-desoxypodophyllotoxin (11a–j and
15a–g) have been carried out from podophyllotoxin as shown in
Scheme 1. The key intermediates for the preparation of these pod-
ophyllotoxin derived compounds are 4b-amino podophyllotoxin
(10)6b,7a and 4b-[(40-amino)benzamide]podophyllotoxin (14). 4b-
Aminopodophyllotoxin is coupled with 4-nitrobenzoyl chloride
(12) in presence of Et3N in CH2Cl2 to afford 13, which upon reduc-
tion with Pd/C affords the intermediate 14. These intermediates,
4b-aminopodophyllotoxin (10) and 4b-[(40-amino)benzam-
ide]podophyllotoxin (14) are then coupled with different heteroar-
omatic sulphonylchlorides in presence of Et3N in CH2Cl2 to provide
the desired sulphonamide podophyllotoxins (11a–j and 15a–g). All
the synthesized compounds were characterized by 1H NMR, 13C
NMR, and mass spectral data.
(Eto) at 2, 4 and 8
lM concentration for 24 h and it was observed
that at 4 M concentration (Fig. 2) the cell death was reasonably
l
effective. All these compounds showed significant cell death, with
compound 11e being the most effective.
3.2. Effect of podophyllotoxin conjugates on the cell cycle
In order to understand the role of these compounds (11b, 11d,
11e and etoposide) in cell cycle, FACS analysis was conducted in
colon cancer cells (Colo-205).10 The cells were treated for 24 h with
compounds 11b, 11d, 11e and etoposide (Eto) at 4 lM concentra-
tion. It was observed that Colo-205 cells showed 74%, 28%, 37% and
40% of G2/M cell cycle arrest by etoposide (Eto), 11b, 11d and 11e
respectively, whereas control (untreated cells) exhibited 26%
(Fig. 3a).
Another independent experiment was carried out to observe
the influence of HDAC inhibitor like trichostatin A (TSA) on cell cy-
cle regulation caused by 11e and etoposide (Eto). TSA produces
dramatic change in cell cycle arrest from G2/M phase to G0 phase
as indicated by profound cell death. The cell death observed was
57% and 56% in case of 11e and etoposide (Eto), respectively
(Fig. 3b), whereas control cells cause 4% of cell death. This observa-
tion was supported by the fact that HDAC inhibitors stabilize the
DNA-topo-II complexes and cause cell death.11,12
3. Biological evaluation
3.1. Invitro cytotoxicity assay
The synthesized compounds 11a–j and 15a–g were evaluated
for their anticancer activity in selected human cancer cell lines of
breast, oral, colon, lung and ovarian origin by using sulforhoda-
mine B (SRB) method.9 The compounds that exhibit GI50 6 10ꢀ5
M
are considered to be active on the respective cell lines and the re-
sults are illustrated in Table 1. All the compounds (11a–j and 15a–
g) exhibit significant anticancer activity with GI50 values ranging
3.3. Effect of 11e on DNA damage
from 0.04 to 2.90
adriamycin demonstrated the GI50 in the range of 0.13–3.08
and 0.10–7.25 M, respectively. The significant anticancer activity
showed by the promising compounds 11b, 11d and 11e prompted
lM, while the positive controls, etoposide and
Recent studies have indicated that many podophyllotoxin based
compounds act as DNA topoisomerase-II (topo-II) inhibitors which
stabilize the DNA-topo-II complexes causing the DNA double-
lM
l

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A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
O
O
NH2
NO2
NH2
HN
HN
COCl
O
O
O
O
O
O
ii
i
O
O
O
O
O
O
NO2
12
H3CO
OCH3
H3CO
OCH3
OCH3
14
H3CO
OCH3
OCH3
13
OCH3
10
OH
O
O
O
S
S
R2
Cl
R1
Cl
i
i
O
R2
O
O
S
O
O
R1
S
NH
HN
HN
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
11a-j
15a-g
O
O
OCH3
CH3
H3C
N
N
R1
=
Br
Cl
NH
S
d
S
S
e
Cl
S
f
c
a
b
O
O
N
N
S
S
S
O
j
g
i
h
N
S
N
N
R2 =
Br
O
S
f
a
e
c
d
b
OCH3
Scheme 1. Reagents and conditions: (i) Et3N, CH2Cl2, rt, 3 h; (ii) Pd/C, H2, EtOAc, rt, 12 h.
g
strand breaks.13 Muslimovic and co-workers have shown that eto-
poside mainly cause single-strand DNA breaks up to 90%.14 Broom-
berg and co-workers have shown that etoposide induces single-
strand breaks (SSB) apart from double-strand breaks (DSB)
depending upon the binding to monomer.15 Thus we attempted
to examine the effect of these podophyllotoxin conjugates for
SSB as well as DSB. Hence Colo-205 cells were treated with the
most effective compound (11e) of the series and compared with
3.4. Effect of 11e on topoisomerase-IIa
DNA topoisomerase-II protein exists as two isoforms. It is well
established that throughout the cell cycle of rapidly proliferating
cells, topo-II
a
is expressed relatively higher than topo-IIb.16,17 To
further confirm the nature of topo-II inhibition of compound 11e,
Colo-205 cells were treated with 11e and etoposide (Eto) at
4 lM concentration for 24 h. The topo-IIa down regulated in case
etoposide (Eto) at 4
to 60% of SSB as indicated by the comet assay and DSB as indicated
by -H2AX foci formation in the compound 11e treated cells. Com-
l
M concentration for 24 h. We observed up
of 11e whereas topo-IIb was down regulated in case of etoposide
(Eto) treated Colo-205 cells (Fig. 5a). Our observations are in con-
junction with the previous studies wherein etoposide is known to
c
pound 11e caused SSB almost similar to etoposide in Colo-205 can-
cer cells. However the efficiency of causing DSB was only 50%. The
inhibit topo-IIb (100%) and to a lesser extent of topo-II
as observed by Western blot studies.13 Attempts were made to ob-
serve the effect of TSA on topo-II inhibition caused by 11e, etopo-
side (i.e., topo-II inhibitor) and camptothecin (i.e., topo-I inhibitor)
compounds. The expression of topo-II was increased in the com-
binational treatments of TSA with 11e/or etoposide (Eto). But not
a inhibition
decreased DSB gave a thought that 11e may function as topo-II
inhibitor as the formation of DSB (i.e., -H2AX foci) needs the
, but not the topo-IIb (Figs. 4a
a
a
c
endogenous expression of topo-II
a
a
and 4b).

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2057
Table 1
Cytotoxic activity (GI50 l
Ma) of compounds 11a–j and 15a–g
Compd
Zr-75-1b
MCF7b
KBc
DWDc
Colo 205d
A549e
Hop62e
A2780f
11a
11b
11c
11d
11e
11f
11g
11h
11i
2.10
0.08
2.50
0.18
0.06
0.17
2.30
2.31
2.11
2.51
2.90
2.23
2.81
nd
0.09
0.12
0.11
0.13
0.18
0.15
2.61
2.21
0.14
2.21
2.71
0.17
2.32
2.90
2.30
nd
2.30
0.06
2.10
0.04
0.04
0.17
nd
2.31
2.11
nd
2.00
0.17
0.15
0.05
0.04
0.14
2.21
2.91
2.61
nd
2.50
2.80
nd
0.14
2.88
2.10
2.02
0.62
0.10
2.10
0.04
0.15
0.04
0.04
0.11
2.71
nd
nd
nd
nd
nd
2.12
2.31
2.80
nd
2.51
0.13
0.14
0.16
0.04
0.14
0.12
0.15
0.12
2.00
2.11
nd
2.71
nd
nd
2.60
2.51
nd
2.70
nd
2.40
0.06
2.00
0.04
0.04
0.16
2.01
nd
1.91
0.08
0.16
0.06
0.05
0.15
2.11
2.40
2.30
2.311
2.11
2.11
2.00
2.42
2.51
nd
nd
11j
2.21
2.01
2.91
2.22
2.61
nd
2.00
2.71
0.80
0.14
15a
15b
15c
15d
15e
15f
15g
Etog
ADRh
nd
nd
2.00
nd
2.50
2.01
nd
2.11
2.90
2.12
0.20
1.79
0.16
2.11
0.17
nd
1.31
0.16
0.31
0.17
3.08
7.25
a
50% Growth inhibition and the values are mean of three determinations.
b
c
d
e
f
Breast cancer.
Oral cancer.
Colon cancer.
Lung cancer.
Ovarian cancer.
Eto (etoposide).
ADR (adriamycin), nd = not determined.
g
h
pounds 11e and etoposide (Eto) treated Colo-205 cells at 4 lM
concentration. Interestingly the activation of ATM was observed
in both 11e and etoposide (Eto) treated cells (Fig. 6).
3.6. Effect of 11e on Chk1 activity
Check point protein (Chk1) is a G2/M specific serine threonine
kinase and is activated by phosphorylation on ser 345.22,23 To fur-
ther investigate the G2/M arrest and DNA damage caused by 11e
and etoposide (Eto) Colo-205 cells were analyzed for Chk1-phos-
phorylation 24 h after the compounds (11e and Eto) treatment. In-
crease in Chk1 phosphorylation in both 11e as well as etoposide
(Eto) treated cells was observed (Fig. 7). A similar kind of observa-
tion was made by Guo and co-workers,24 wherein benzophenan-
thridine (NK-314) caused the Chk1 activation during DNA damage.
Figure 2. Colo-205 cells were treated with compounds 11b, 11d, 11e and etoposide
(Eto) at 2, 4 and 8 M concentration for 24 h. The MTT cell viability assay was
conducted and the optical density (O.D) was observed at 570 nm. Control indicates
the untreated cells.
l
3.7. Effect of 11e on apoptosis
Previous studies established that the inhibition of DNA topo-II
enzyme caused the replicating cells to undergo apoptosis.25 That
is upon receiving an apoptotic signal the precursor caspase under-
goes proteolytic cleavage and generates an active subunit.26 Thus
as a part of understanding apoptosis, we examined the role of cas-
pase-3, the effector caspase in compound 11e as well as etoposide
(Eto) treated cells. It was observed that there was an increase of
caspase-3 activation in both ELISA and Western blot analysis (
Fig. 8). In previous studies a similar observation was reported for
deoxypodophyllotoxins wherein G2/M cell cycle arrest and apop-
tosis are caspase-dependent.10
in cells treated with TSA and camptothecin (CPT) combination, this
confirms the previous observations that HDAC inhibitors stabilize
topo-II protein expression18 and HDAC proteins interact with
topo-II but not topo-I.19 Thus we conclude that the cytotoxic event
caused by compound 11e and TSA is an additive effect on apoptosis
(Figs. 5b and 5c). To further confirm the effectiveness of 11e to-
wards topo-II
say using topoisomerase-II
compounds (11e and etoposide) have shown inhibitory effect on
a
inhibition we carried out the topo-II
a inhibition as-
a
enzyme and PBR322 DNA.20 The
Further we were interested to see the involvement of mitochon-
dria as central key organelle in the apoptotic process. Bcl-2 is the
oncoprotein that play a major role in apoptosis.27 Thus an ELISA
based assay against Bcl2 protein was conducted by treating Colo-
205 cells with 11e, etoposide (Eto) and camptothecin (CPT). We
have observed strong down regulation of Bcl2 in compound treated
cells confirming the role of mitochondria in this apoptotic event (
Fig. 9).
topoisomerase-II
tion (Fig. 5d).
a mediated DNA digestion at 50 lM concentra-
3.5. Effect of 11e on ATM protein
Ataxia telengiectasia mutated (ATM) protein plays a key role as
signal transducer in response to induction of DSB.21 It was consid-
ered of interest to examine the activated ATM protein in com-

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A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
Figure 3a. Colo-205 cells were treated with compounds 11b, 11d, 11e and etoposide (Eto) at 4 lM concentration for 24 h. The cells were further processed for FACS analysis.
Compounds have shown G2/M cell cycle arrest. Control indicates the untreated cells.
Figure 3b. Effect of trichostatin A (TSA), the known HDAC inhibitor on the cell cycle arrest caused by 11e and etoposide (Eto) in Colo-205 cells at 4 lM concentration for 24 h.
Addition of TSA to the cells shifted cells from G2/M arrest towards G0 phase (cell death). Control indicates the untreated cells.
3.8. Effect of 11e on p21, p16, NF-kB and caspase-3 proteins
biological effects in this cell line. The treatment of Colo-205 cells
with these conjugates (11b, 11d, 11e) and etoposide (Eto) at 2, 4
It is known that tumor suppressor proteins such as p21 blocks
the formation of tumor,28 whereas p16 is another protein that
causes senescence. Further it is well established that NF-kB plays
an important role in topoisomerase-II inhibition by inducing the
Fas ligand. Thus in view of the above aspects Colo-205 cells were
treated with 11e as well as etoposide (Eto) and the levels of p21,
p16 and NF-kB proteins were analyzed. To our surprise we ob-
served the upregulation of p21, p16 and NF-kB proteins,29 thus
suggesting the role of podophyllotoxin conjugate such as 11e as
and 8
l
M concentration for 24 h showed that the optimum con-
centration for effective cell death was 4
lM. FACS studies indicated
the occurrence of G2/M cell cycle arrest in all the compounds stud-
ied with 11e being the most effective. These compounds caused
both single-strand DNA breaks (SSB) as studied by comet assay
as well as double strand DNA breaks (DSB) as indicated by
H2AX in Colo-205 cells. Surprisingly there was reduction in the
c-
number of cells that express double-stranded breaks in 11e com-
pared to etoposide, as indicated by number of cells with
staining. This compound showed inhibitory effect on topoisomer-
ase-II as observed from both Western blot analysis as well as
topoisomerase-II enzyme inhibition studies. Similarly 11e caused
c-H2AX
on effective DNA topo-IIa inhibitor that possesses effective apopto-
tic inducing ability (Fig. 10).
a
a
DNA damage with the activation of key signaling molecules such as
ATM and Chk1. The data generated also indicate concrete evidence
for the fact that rapidly replicating cells undergo apoptosis with
4. Conclusion
In conclusion, a series of new 4b-sulphonamido and 4b-[(40-sul-
phonamido)benzamide] conjugates of podophyllotoxin (11a–j and
15a–g) were synthesized. These conjugates (11a–j and 15a–g)
were tested for anticancer activity against six human cancer cell
lines and were found to be more potent than etoposide. The signif-
icant anticancer activity showed by some of the promising com-
pounds like 11b, 11d and 11e prompted us to evaluate the cell
viability in the Colo-205 cells, with a view to study their detailed
the inhibition of topoisomerase-II
pase-3 and down regulation of Bcl-2 proteins. Thus this study pro-
vides the development of new class of podophyllotoxin
conjugates as effective topoisomerase-II inhibitors with apoptotic
a with the activation of cas-
a
a
death inducing ability. Moreover these findings provide an insight
for future direction in the design and development of such conju-
gates of podophyllotoxin.

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2059
Figure 4a. The comet assay was conducted to observe the induction of single-strand DNA breaks due to compound 11e and etoposide (Eto) at 4
lM concentration for 24 h.
SYBR green-I dye was used to stain the DNA in both control and compound treated cells (11e, Eto). Etoposide has been employed as the positive control. Single-strand DNA
breaks were identified due to the bright head and tail formation up on compound treatment. 11e and etoposide (Eto) has shown single-strand DNA breaks. Whereas control
cells have very negligible level of single-strand breaks as shown in this comet assay.
Figure 4b.
c
-H2AX staining and Western blot analysis was carried out to observe the induction of double-stranded DNA breaks due to compound 11e and etoposide (Eto)
M concentration for 24 h.
using antibody against ser139 residue of H2AX protein, the indicator of double stranded DNA breaks at 4
l
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
5. Experimental section
All chemicals and reagents were obtained from Aldrich (Sigma–
Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey

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A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
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.
5.1. 4b-[Naphthalene-1-sulphonamido]podophyllotoxin (11a)
The compound 4b-aminopodophyllotoxin (10) (200 mg,
0.483 mmol) was dissolved in 10 mL of dried dichloromethane, fol-
lowed by addition of naphthalene-1-sulphonylchloride (131 mg,
0.579 mmol), and Et3N (1.34 mL, 0.966 mmol). The reaction mix-
ture was stirred at room temperature for 3 h, till the completion
of the reaction as monitored by TLC. The reaction mixture was
washed with water and extracted with dichloromethane, dried
over anhydrous Na2SO4 and the crude product was purified by col-
umn chromatography with ethyl acetate/hexane (1:1) to obtain
pure compound 11a in 260 mg, 93% yield. Mp: 139–142 °C,
Figure 5a. The effect of compounds 11e and etoposide (Eto) in Colo-205 cells on
topo-II
etoposide (Eto) at 4
Western blot analysis. b-Actin was used as loading control.
a
and b protein expression. Cells were treated with compounds 11e and
l
M for 24 h. Topo-II and b protein levels were examined in
a
½
a 2D5
ꢁ
= ꢀ30.0 (c = 0.5 in CHCl3), 1H NMR (200 MHz, CDCl3): d
2.71–2.88 (m, 1H), 2.91–3.00 (dd, 1H, J = 4.9, 4.6 Hz), 3.69 (s, 6H),
3.97 (s, 3H), 4.20–4.27 (m, 3H), 4.45–4.51 (m, 1H), 4.88 (d, 1H,
J = 6.6 Hz), 5.14 (s, 1H), 5.75 (d, 2H, J = 5.8 Hz), 6.16 (s, 2H), 6.36
(s, 1H), 7.63–7.70 (m, 3H), 8.06 (t, 1H, J = 6.6, 3.3 Hz), 8.24 (d, 1H,
J = 7.4 Hz), 8.41 (d, 1H, J = 7.4 Hz), 8.58 (t, 1H, J = 3.3, 5.8 Hz), 13C
NMR (75 MHz, CDCl3): d 36.7, 40.3, 43.0, 48.7, 51.0, 55.4, 59.8,
67.9, 100.7, 107.5, 107.9, 108.8, 123.9, 124.3, 126.5, 127.2, 127.8,
128.7, 128.9, 132.1, 133.6, 133.9, 134.8, 135.5, 136.1, 146.4,
147.2, 151.7; MS (ESI): 621 [M+Na]+.
Figure 5b. The effect of compounds TSA on topo-II
cancer cells (colo-205) were treated with compounds 11e and (11e + TSA)
combination at 4 M for 24 h. Topo-II protein level was examined by conducting
Western blot analysis. b-Actin was used as loading control. TSA with combination of
11e induced expression of topo-II in this study.
a protein expression. Colon
l
a
a
5.2. 4b-[Quinoline-8-sulphonamido]podophyllotoxin (11b)
This compound 11b was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg,
0.483 mmol)
with
quinoline-8-sulphonylchloride
(131 mg, 0.579 mmol), and Et3N (1.34 mL, 0.966 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (4:6) to afford pure compound 11b in 250 mg, 85%
yield. Mp: 170–174 °C, ½a D25
ꢁ
= +56.0 (c = 0.5 in CHCl3), 1H NMR
(300 MHz, CDCl3): d 2.68–2.83 (m, 1H), 3.02–3.11 (dd, 1H, J = 5.2,
5.2 Hz), 3.61 (s, 6H), 3.68 (s, 3H), 4.35 (t, 1H, J = 7.5, 9.0 Hz),
4.44–4.50 (m, 4H), 4.56 (t, 1H, J = 3.7, 4.5 Hz), 4.95 (s, 1H), 5.62
(d, 2H, J = 6.7 Hz), 6.06 (s, 2H), 6.29 (s, 1H), 6.36 (d, 1H,
J = 4.5 Hz), 7.44–7.49 (dd, 1H, J = 4.5, 4.5 Hz), 7.70 (t, 1H, J = 7.5,
7.5 Hz), 8.09 (d, 1H, J = 8.3 Hz), 8.25 (d, 1H, J = 8.3 Hz), 8.45 (d,
1H, J = 7.5 Hz), 8.77 (d, 1H, J = 3.0 Hz), 13C NMR (75 MHz, CDCl3):
d 36.8, 42.9, 51.7, 55.7, 59.8, 68.3, 101.0, 109.2, 122.4, 125.7,
128.7, 129.4, 130.7, 131.8, 133.9, 135.7, 136.2, 137.1, 137.5,
142.6, 145.9, 146.9, 151.4, 151.9, 174.4; MS (ESI): 627 [M+Na]+.
Figure 5c. The effect of combination of TSA and various DNA damaging agents on
topo-II protein expression. Colon cancer cells (colo-205) were treated with
compounds 11e + TSA, Eto + TSA and CPT + TSA at 4 M of each drug for 24 h. Topo-
II protein level was examined by conducting Western blot analysis. b-Actin was
used as loading control. TSA induced the expression of topo-II in the combination
(11e & TSA; Eto + TSA) drugs used in this study, but not in the topo-I inhibitor (CPT)
case. This shows the specificity of TSA towards topo-II induction.
a
l
a
a
a
5.3. 4b-[2,5-Dichlorothiophene-3-sulphonamido]
podophyllotoxin (11c)
This compound 11c was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 2,5-dichlorothiophene-3-sulphonyl-
chloride (0.145 mg, 0.579 mmol), and Et3N (1.34 mL, 0.996 mmol)
and the crude product was purified by column chromatography
with ethyl acetate/hexane (1:9) to afford pure compound 11c in
Figure 5d. The effect of compounds 11e and etoposide (Eto) on topoisomerase-II
enzyme inhibition. PBR322 plasmid DNA and compounds 11e and etoposide (Eto)
were added at 50, 25 and 10 M final concentration along in which topo-II
enzyme (4U/ L sigma company). The buffer used for enzyme activity was prepared
fresh in which ATP, KCL, MgCl2, DTT, Tris pH-8.0 components. The reaction was
performed at 37 °C for 30 min. Here T represents the topo-II digestion of DNA with
out any compound. U, represents the PBR322 DNA alone. Both 11e and etoposide
a
l
a
l
290 mg, 95% yield. Mp: 140–143 °C, ½a D25
= ꢀ38.0 (c = 0.5 in CHCl3),
ꢁ
1H NMR (300 MHz, CDCl3): d 2.92–3.03 (m, 2H), 3.75 (s, 6H), 3.81
(s, 3H), 4.36–4.40 (m, 2H), 4.58 (d, 1H, J = 4.5 Hz), 4.65–4.69 (m,
2H), 5.07 (d, 1H, J = 6.7 Hz), 5.98 (s, 2H), 6.05 (s, 1H), 6.24 (s, 2H),
6.51 (s, 1H), 7.28 (s, 1H), 13C NMR (75 MHz, CDCl3): d 37.1, 41.0,
43.4, 52.4, 56.1, 60.6, 68.5, 101.6, 107.7, 108.0, 110.1, 126.5,
127.7, 128.1, 129.9, 132.3, 134.6, 136.8, 137.0, 147.6, 148.4,
152.4, 174.1; MS (ESI): 650 [M+Na]+.
(Eto) inhibit the topo-IIa inhibition at 50 lM concentration.
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

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2061
Figure 6. ATM S1981 staining was carried out to observe the ATM activation and induction of double-stranded DNA breaks due to compounds 11e and etoposide (Eto) at
M concentration for 24 h.
4
l
Figure 7. The Chk1 phosphorylation as a consequence of DNA damage activity in compounds 11e and etoposide (Eto) treated cells.
5.4. 4b-[5-Bromothiophene-2-sulphonamido]podophyllotoxin
(11d)
ride (150 mg, 0.579 mmol), and Et3N (1.34 mL, 0.966 mmol) and
the crude product was purified by column chromatography with
ethyl acetate/hexane (3:7) to afford pure compound 11d in
This compound 11d was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 5-bromothiophene-2-sulphonylchlo-
280 mg, 90% yield. Mp: 130–132 °C, ½a D25
= ꢀ86.0 (c = 0.5 in CHCl3),
ꢁ
1H NMR (400 MHz, CDCl3): d 2.88–2.95 (m, 2H), 3.72 (s, 6H), 3.78
(s, 3H), 4.28–4.37 (m, 2H), 4.52 (d, 1H, J = 3.9 Hz), 4.62–4.64 (dd,

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A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
1H, J = 3.1, 3.9 Hz), 4.94 (d, 1H, J = 5.4 Hz), 5.86 (s, 1H), 5.93 (d, 2H,
J = 3.9 Hz), 6.20 (s, 2H), 6.45 (s, 1H), 7.22 (d, 1H, J = 3.9 Hz), 7.49 (d,
1H, J = 3.9 Hz), 13C NMR (75 MHz, CDCl3): d 37.0, 41.1, 43.4, 52.4,
56.1, 60.6, 68.5, 101.5, 107.7, 108.0, 110.0, 120.5, 127.7, 130.7,
132.2, 132.4, 134.6, 136.9, 142.1, 147.5, 148.3, 152.4, 174.1; MS
(ESI): 662 [M+Na]+.
5.5. 4b-[Methyl-3-(sulphonamido)thiophene-2-carboxylate]
podophyllotoxin (11e)
This compound 11e was prepared following the method de-
scribed for the preparation of the compound 11a, above employing
10 (200 mg, 0.483 mmol) with 2-carbomethoxy thiophene-3-sul-
phonylchloride (139 mg, 0.579 mmol), and Et3N (1.34 mL,
0.966 mmol) and the crude product was purified by column chro-
matography with ethyl acetate/hexane (1:9) to afford pure com-
pound 11e in 270 mg, 90% yield. Mp: 164–165 °C, ½a D25
= ꢀ40.0
ꢁ
(c = 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.86–2.98 (m,
1H), 3.08–3.14 (dd, 1H, J = 5.2, 5.2 Hz), 3.73 (s, 6H), 3.80 (s, 3H),
3.87 (s, 3H), 4.10–4.17 (dd, 1H, J = 6.7, 6.7 Hz), 4.40–4.52 (m, 2H),
4.61 (d, 1H, J = 5.2 Hz), 4.67 (t, 1H, J = 5.2, 3.7 Hz), 5.76 (s, 1H),
5.93 (s, 2H), 6.23 (s, 2H), 6.50 (s, 1H), 7.65–7.69 (m, 2H), 13C
NMR (75 MHz, CDCl3): d 37.62, 40.96, 43.58, 52.95, 56.21, 60.72,
60.69, 68.86, 101.59, 107.64, 108.22, 110.55, 127.60, 130.71,
131.09, 132.57, 148.43, 152.53, 174.09; MS (ESI): 640 [M+Na]+.
Figure 8. The effect of compounds 11e and etoposide (Eto) on caspase-3 activity.
The Colo-205 cells were treated with compounds 11e and etoposide (Eto) at 4 lM
concentration for 24 h. The cell lysates were subjected to a fluorescence based
caspase-3 assay. The compounds caused increase of caspase-3 protein. Control
indicates the untreated cells. Con + Inhi, indicates the caspase-3 activity in the
presence of caspase-3 inhibitor.
5.6. 4b-[2-Acetamido-4-methylthiazole-5-sulphonamido]
podophyllotoxin (11f)
This compound 11f was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 2-acetamido-4-methylthiazole-5-sul-
phonylchloride (147 mg, 0.579 mmol), and Et3N (1.34 mL,
0.966 mmol) and the crude product was purified by column chro-
matography with ethyl acetate/hexane (1:9) to afford pure com-
pound 11f in 280 mg, 91% yield. Mp: 164–167 °C, ½a D25
= ꢀ120.0
ꢁ
(c = 0.5 in CHCl3), 1H NMR (200 MHz, CDCl3): d 2.11 (s, 3H), 2.33
(s, 3H), 3.73 (s, 6H), 3.77 (s, 3H), 2.71–2.80 (dd, 1H, J = 4.6,
4.6 Hz), 2.85–2.94 (m, 1H), 4.03–4.12 (m, 1H), 4.35–4.43 (m, 2H),
4.53 (d, 1H, J = 4.6 Hz), 5.16–5.23 (m, 1H), 5.98 (d, 2H, J = 2.7 Hz),
6.22 (s, 2H), 6.50 (s, 1H), 6.76 (s, 1H), 13C NMR (75 MHz, CDCl3):
d 19.12, 25.73, 42.72, 45.33, 45.45, 57.14, 62.51, 64.38, 67.71,
101.04, 107.06, 110.15, 110.85, 111.59, 130.23, 133.76, 136.00,
138.40, 144.04, 147.31, 150.73, 161.11, 170.11; MS (ESI): 632
[M+H]+.
Figure 9. The Colo-205 cells were treated with compounds 11e and etoposide (Eto)
at 4 lM concentration for 24 h. The cell lysates were subjected to Bcl2 based ELISA
assay. The compounds (11e, Eto and CPT) caused decrease of Bcl2 protein. Control
indicates the untreated cells. Etoposide (Eto) and camptothecin (CPT) are employed
as positive controls wherein etoposide serves as the positive control for topo-II
inhibition and camptothecin as a positive control for the topo-I inhibition.
5.7. 4b-[Biphenyl-4-sulphonamido]podophyllotoxin (11g)
This compound 11g was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg,
0.483 mmol)
with
biphenyl-4-sulphonylchloride
(146 mg, 0.579 mmol), and Et3N (1.34 mL, 0.966 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (1:1) to afford pure compound 11g in 290 mg, 95
yield. Mp: 195–198 °C, ½a D25
ꢁ
= ꢀ64.0 (c = 0.5 in CHCl3), 1H NMR
(400 MHz, CDCl3): d 3.02–3.19 (m, 1H), 3.39–3.49 (dd, 1H, J = 3.7,
4.4 Hz), 3.71 (s, 3H), 3.73 (s, 6H), 3.98–4.10 (m, 2H), 4.36 (t, 1H,
J = 6.6, 6.6 Hz), 4.53 (d, 1H, J = 3.7 Hz), 5.49 (t, 1H, J = 5.1, 5.1 Hz),
5.95 (d, 2H, J = 6.1 Hz), 6.28 (s, 2H), 6.49 (s, 1H), 6.84 (s, 1H), 7.11
(d, 2H, J = 8.1 Hz), 7.43–7.53 (m, 4H), 7.69 (d, 2H, J = 6.6 Hz), 7.83
(d, 2H, J = 8.1 Hz), 7.93–8.00 (m, 3H), 13C NMR (75 MHz, CDCl3): d
37.2, 41.1, 43.4, 52.0, 56.1, 60.6, 68.7, 101.4, 107.8, 108.1, 110.0,
127.3, 127.6, 128.1, 128.7, 129.0, 132.2, 134.7, 137.0, 138.6,
138.9, 146.4, 147.4, 148.3, 152.4; MS (ESI): 652 [M+Na]+.
Figure 10. Treatment of Colo-205 cells with 11e and etoposide (Eto) at 4 lM
concentration for 24 h and subjected to western blot analysis using antibodies
against p21, p16, caspase-3 full length and NF-kB proteins. The less procaspase-3
protein expression indicates more active caspase-3 formation and more apoptosis.

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2063
5.8. 4b-[4-Phenoxybenzene-1-sulphonamido]podophyllotoxin
(11h)
the reaction as monitored by TLC. The reaction mixture was
washed with water and extracted with dichloromethane, dried
over anhydrous Na2SO4 and the crude product was purified by col-
umn chromatography with ethyl acetate/hexane (3:7) to obtain
pure compound 13 in 2.5 g, 92% yield. 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),
5.43 (d, 1H, J = 6.7 Hz), 5.98 (d, 2H, J = 4.5 Hz), 6.22 (s, 2H), 6.45
(s, 1H), 6.83 (s, 1H), 7.94 (d, 2H, J = 9.0 Hz), 8.14 (d, 2H,
J = 9.0 Hz); MS (ESI): 563 [M+H]+.
This compound 11h was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 4-phenoxybenzene-1-sulphonylchlo-
ride (155 mg, 0.579 mmol), and Et3N (1.34 mL, 0.966 mmol) and
the crude product was purified by column chromatography with
ethyl acetate/hexane (4:6) to afford pure compound 11h in
290 mg, 92% yield. Mp: 249–250 °C, ½a D25
= ꢀ96.0 (c = 0.5 in CHCl3),
ꢁ
1H NMR (300 MHz, CDCl3): d 2.65–2.77 (m, 1H), 2.81–2.87 (dd, 1H,
J = 5.2, 5.2 Hz), 3.63 (s, 6H), 3.65 (s, 3H), 4.16 (d, 2H, J = 9.0 Hz), 4.35
(d, 1H, J = 5.2 Hz), 4.40–4.43 (dd, 1H, J = 3.7 Hz), 5.10 (d, 1H,
J = 6.7 Hz), 5.57 (s, 1H), 5.83 (s, 2H), 6.08 (s, 2H), 6.27 (s, 1H),
7.03–7.09 (m, 4H), 7.14 (t, 1H, J = 6.7, 7.5 Hz), 7.33 (t, 2H, J = 7.5,
8.3 Hz), 7.81 (d, 2H, J = 9.0 Hz), 13C NMR (75 MHz, CDCl3): d 37.2,
41.1, 43.4, 51.9, 56.1, 60.6, 68.6, 101.4, 107.7, 108.1, 110.0, 118.1,
120.1, 125.0, 128.3, 129.2, 130.1, 132.2, 133.8, 134.7, 147.4,
148.3, 152.4, 155.1, 162.0, 174.4; MS (ESI): 668 [M+Na]+.
5.12. 4b-[(40-Amino)benzamide]podophyllotoxin (14)
To a solution of 4b-[(40-nitro)benzamide]podophyllotoxin (13)
(1.54 g, 3.53 mmol) in 80 mL of ethyl acetate was added 300 mg,
of 10% palladium on activated carbon. The mixture was stirred
overnight under hydrogen, the reaction mixture was filtered and
the filtrate was evaporated. The crude product was purified by col-
umn chromatography with ethyl acetate/hexane (6:4) to obtain
pure compound 14 in 1.42 g, 98% yield. 1H NMR (200 MHz, CDCl3):
d 2.93–3.01 (m, 2H), 3.601 (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), 5.49 (d, 1H,
J = 6.7 Hz), 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):
533 [M+H]+.
5.9. 4b-[5-(Phenylsulphonyl)thiophene-2-sulphonamido]
podophyllotoxin (11i)
This compound 11i was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 5-phenylsulphonylthiaphene-2-sul-
phonylchloride (186 mg, 0.579 mmol), and Et3N (1.34 mL,
0.966 mmol) and the crude product was purified by column chro-
matography with ethyl acetate/hexane (1:1) to afford pure com-
5.13. 4b-[40-(5-Bromothiophene-2-sulphonamido)benzamide]
podophyllotoxin (15a)
pound 11i in 300 mg, 88% yield. Mp: 210–213 °C, ½a D25
= ꢀ62.0
ꢁ
(c = 0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.75–2.85 (m,
2H), 3.67 (s, 6H), 3.71 (s, 3H), 4.03–4.18 (m, 2H), 4.40–4.44 (m,
1H), 4.56–4.60 (m, 1H), 5.40–5.47 (m, 1H), 5.78 (s, 1H), 5.87 (d,
2H, J = 3.7 Hz), 6.10 (s, 2H), 6.36 (s, 1H), 7.42–7.62 (m, 3H), 7.81
(d, 1H, J = 1.5 Hz), 7.91 (d, 2H, J = 6.7 Hz), 8.29 (d, 1H, J = 1.5 Hz),
13C NMR (75 MHz, CDCl3): d 37.0, 41.1, 43.4, 52.7, 56.1, 60.6,
68.5, 101.6, 107.6, 108.0, 110.1, 127.3, 127.6, 129.7, 129.9, 132.3,
134.0, 134.5, 136.2, 137.0, 140.1, 142.9, 145.0, 147.6, 152.5,
174.0; MS (ESI): 722 [M+Na]+.
This compound 15a was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg, 0.375 mmol) with 5-bromothiophene-2-sulphonylchlo-
ride (116 mg, 0.451 mmol), and Et3N (1.03 mL, 0.751 mmol) and
the crude product was purified by column chromatography with
ethyl acetate/hexane (1:1) to afford pure compound 15a in
270 mg, 95% yield. Mp: 213–215 °C, ½a D25
= ꢀ53.0 (c = 0.5 in CHCl3),
ꢁ
1H NMR (300 MHz, CDCl3): d 2.82–3.15 (m, 1H), 3.74 (s, 6H), 3.78
(s, 3H), 4.05–4.34 (m, 2H), 4.39–4.52 (m, 1H), 4.58 (d, 1H,
J = 4.4 Hz), 5.37–5.44 (m, 1H), 5.97 (d, 2H, J = 3.6 Hz), 6.28 (s, 2H),
6.53 (s, 1H), 6.79 (s, 1H), 7.12 (d, 1H, J = 4.4 Hz), 7.17 (d, 2H,
J = 8.0 Hz), 7.49 (d, 1H, J = 4.4 Hz), 7.78 (d, 2H, J = 8.0 Hz), 13C
NMR (75 MHz, CDCl3): d 37.2, 41.6, 43.6, 48.7, 60.6, 68.9, 101.5,
108.0, 108.9, 110.0, 123.6, 128.3, 129.8, 130.6, 131.4, 132.4,
134.5, 135.3, 136.0, 137.0, 138.7, 147.6, 148.3, 152.5, 166.1,
174.3; MS (ESI): 780 [M+Na]+.
5.10. 4b-[Benzo[c][1,2,5]thiadiazole-4-sulphonamido]
podophyllotoxin (11j)
This compound 11j was prepared following the method de-
scribed for the preparation of the compound 11a, employing 10
(200 mg, 0.483 mmol) with 1,2,5-thiadiazole-4-sulphonylchloride
(135 mg, 0.579 mmol), and Et3N (1.34 mL, 0.966 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (3:7) to afford pure compound 11j in 280 mg, 94%
5.14. 4b-[40-(Benzo[c][1,2,5]thiadiazole-4-sulphonamido)
benzamide]podophyllotoxin (15b)
yield. Mp: 176–179 °C, ½a D25
ꢁ
= +65.0 (c = 0.5 in CHCl3), 1H NMR
This compound 15b was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg, 0.375 mmol) with 1,2,5-thiadiazole-4-sulphonylchloride
(105 mg, 0.451 mmol), and Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (1:1) to afford pure compound 15b in 269 mg, 98%
(200 MHz, CDCl3): d 2.57–2.78 (m, 1H), 2.85–2.96 (dd, 1H, J = 5.1,
5.1 Hz), 3.54 (s, 6H), 3.60 (s, 3H), 4.15–4.26 (m, 2H), 4.33 (d, 1H,
J = 5.1 Hz), 4.49–4.56 (m, 1H), 4.89 (s, 1H), 5.50 (d, 1H, J = 7.3 Hz),
5.63 (s, 2H), 5.97 (s, 2H), 6.22 (s, 1H), 7.62–7.71 (m, 1H), 8.15–
8.22 (m, 2H), 13C NMR (75 MHz, CDCl3): d 37.4, 40.9, 43.4, 52.9,
56.1, 60.6, 68.9, 101.4, 107.1, 108.1, 110.2, 126.9, 127.2, 128.2,
129.6, 129.9, 130.7, 131.8, 132.4, 134.5, 137.1, 146.8, 148.2,
148.8, 152.4, 155.2, 174.0; MS (ESI): 634 [M+Na]+.
yield. Mp: 247–249 °C, ½a D25
ꢁ
= ꢀ78.0 (c = 0.5 in CHCl3), 1H NMR
(400 MHz, CDCl3): d 2.86–2.99 (m, 2H), 3.73 (s, 6H), 3.77 (s, 3H),
4.06–4.13 (dd, 1H, J = 6.0, 5.2 Hz), 4.24–4.38 (m, 2H), 4.46 (d, 1H,
J = 3.0 Hz), 5.33–5.37 (m, 1H), 5.93 (d, 2H, J = 9.8 Hz), 6.23 (s, 2H),
6.41 (s, 1H), 6.76 (s, 1H), 7.28 (d, 1H, J = 9.0 Hz), 7.65 (d, 2H,
J = 8.3 Hz), 7.73–7.79 (m, 3H), 8.34 (t, 1H, J = 8.3, 9.0 Hz), 13C
NMR (75 MHz, CDCl3): d 37.2, 41.5, 43.5, 48.6, 56.0, 60.7, 61.9,
68.9, 101.4, 108.0, 108.9, 109.9, 127.8, 128.0, 128.3, 128.4, 129.5,
129.8, 132.4, 132.8, 135.0, 135.8, 147.4, 148.2, 148.7, 152.4,
155.2, 166.1, 174.3; MS (ESI): 731 [M+H]+.
5.11. 4b-[(40-Nitro)benzamide]podophyllotoxin (13)
To a solution containing 4b-aminopodophyllotoxin (10) (2 g,
0.820 mmol), triethylamine (2.3 mL, 1.652 mmol) in 20 mL of
dichloromethane and 4-nitrobenzoyl chloride (12) (1.83 g,
0.991 mmol) in 10 mL of dichloromethane was added under nitro-
gen and stirred at room temperature for 3 h, till the completion of

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A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
5.15. 4b-[40-(Napthalene-1-sulphonamido)benzamide]
5.18. 4b-[40-(4-Phenoxyphenylsulphonamido)benzamide]
podophyllotoxin (15c)
podophyllotoxin (15f)
This compound 15c was prepared following method de-
scribed for the preparation of the compound 11a, employing
14 (200 mg, 0.375 mmol) with napthalene-1-sulphonylchloride
(101 mg, 0.451 mmol), and Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with
ethyl acetate/hexane (4:6) to afford pure compound 15c in
This compound 15f was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg, 0.375 mmol) with 4-phenoxybenzene-1-sulphonylchlo-
ride (120 mg, 0.451 mmol), Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (1:1) to afford pure compound 15f in 278 mg, 96%
268 mg, 98% yield. Mp: 180–183 °C,
½
a 2D5
ꢁ
= ꢀ66.0 (c = 0.5 in
yield. Mp: 190–193 °C, ½a D25
ꢁ
= ꢀ83.0 (c = 0.5 in CHCl3), 1H NMR
CHCl3), 1H NMR (300 MHz, CDCl3): d 2.89–3.00 (m, 2H), 3.74
(s, 6H), 3.76 (s, 3H), 4.08–4.15 (dd, 1H, J = 6.7, 6.7 Hz), 4.26–
4.30 (m, 1H), 4.38–4.43 (m, 1H), 5.29–5.38 (m, 2H), 5.87 (d,
2H, J = 21.9 Hz), 6.20 (s, 2H), 6.22 (s, 1H), 6.74 (s, 1H), 6.99 (d,
1H, J = 8.3 Hz), 7.22 (t, 1H, J = 7.5, 8.3 Hz), 7.50–7.59 (m, 4H),
7.62 (d, 1H, J = 8.3 Hz), 7.92 (d, 1H, J = 8.3 Hz), 8.04 (d, 1H,
J = 8.3 Hz), 8.13 (d, 1H, J = 8.3 Hz), 8.24 (d, 2H, J = 7.5 Hz), 13C
NMR (75 MHz, CDCl3): d 37.2, 41.5, 43.4, 48.6, 56.0, 60.5, 68.9,
101.3, 107.9, 108.8, 109.8, 123.8, 124.0, 127.0, 127.9, 128.1,
128.2, 128.3, 129.0, 132.2, 132.3, 132.6, 133.0, 133.6, 134.6,
134.8, 136.2, 136.3, 147.3, 148.0, 152.3, 166.0, 174.3; MS (ESI):
740 [M+NH4]+.
(400 MHz, CDCl3): d 2.74–2.85 (m, 1H), 3.38–3.43 (dd, 1H, J = 5.3,
5.3 Hz), 3.73 (s, 6H), 3.75 (s, 3H), 3.88 (t, 2H, J = 9.8, 9.8 Hz), 4.39
(t, 1H, J = 8.0, 8.0 Hz), 4.55 (d, 1H, J = 4.4 Hz), 5.48–5.51 (dd, 1H,
J = 5.3, 5.3 Hz), 5.93 (d, 2H, J = 6.2 Hz), 6.28 (s, 2H), 6.50 (s, 1H),
6.83 (s, 1H), 7.02–7.10 (m, 6H), 7.24 (t, 1H, J = 7.1, 7,1 Hz), 7.42
(t, 2H, J = 7.1, 7.1 Hz), 7.78 (d, 2H, J = 8.9 Hz), 7.93 (d, 2H,
J = 7.1 Hz), 13C NMR (75 MHz, CDCl3): d 36.7, 40.6, 43.1, 47.5,
55.7, 59.8, 68.4, 101.2, 108.1, 109.1, 109.3, 117.4, 120.4, 125.4,
128.9, 130.0, 130.5, 130.8, 131.1, 131.8, 132.1, 135.6, 135.8,
136.1, 136.3, 146.6, 147.2, 151.9, 154.2, 162.3, 165.5, 174.5; MS
(ESI): 765 [M+H]+.
5.19. 4b-[40-(4-Methoxyphenylsulphonamido)benzamide]
podophyllotoxin (15g)
5.16. 4b-[40-(Quinoline-8-sulphonamido)benzamide]
podophyllotoxin (15d)
This compound 15g was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg, 0.375 mmol) with 4-methoxybenzene-1-sulphonylchlo-
ride (93 mg, 0.451 mmol), Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with ethyl
acetate/hexane (3:7) to afford pure compound 15g in 259 mg, 98%
This compound 15d was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg,
0.375 mmol)
with
quinoline-8-sulphonylchloride
(102 mg, 0.451 mmol), and Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with
ethyl acetate/hexane (3:7) to afford pure compound 15d in
yield. Mp: 147–150 °C, ½a D25
ꢁ
= ꢀ20.0 (c = 0.5 in CHCl3), 1H NMR
(200 MHz, CDCl3): d 2.88–3.09 (m, 2H), 3.75 (s, 3H), 3.80 (s, 3H),
3.91 (s, 6H), 4.12–4.30 (m, 2H), 4.47 (t, 1H, J = 6.6, 6.6 Hz), 4.59
(d, 1H, J = 2.9 Hz), 5.32–5.44 (m, 1H), 5.96 (d, 2H, J = 5.1 Hz), 6.30
(s, 2H), 6.52 (s, 1H), 6.79 (s, 1H), 7.02 (d, 2H, J = 8.8 Hz), 7.11 (d,
2H, J = 8.0 Hz), 7.76 (d, 2H, J = 8.0 Hz), 7.79 (d, 2H, J = 8.8 Hz), 13C
NMR (75 MHz, CDCl3): d 41.5, 43.5, 48.6, 55.6, 56.0, 60.5, 61.9,
68.7, 101.4, 107.9, 108.9, 109.9, 114.1, 128.0, 128.4, 129.5, 129.8,
130.2, 130.6, 131.7, 132.3, 134.5, 134.7, 137.5, 147.4, 148.2,
152.4, 163.9, 166.3, 174.3; MS (ESI): 703 [M+H]+.
268 mg, 98% yield. Mp: 190–193 °C, ½a D25
= ꢀ160.0 (c = 0.5 in
ꢁ
CHCl3), 1H NMR (300 MHz, CDCl3): d 2.86–3.08 (m, 2H), 3.74 (s,
6H), 3.79 (s, 3H), 4.04–4.21 (m, 2H), 4.224.44 (m, 1H), 4.57 (d,
1H, J = 4.4 Hz), 5.22–5.39 (m, 1H), 5.96 (d, 2H, J = 2.9 Hz), 6.25–
6.31 (m, 4H), 6.51 (s, 1H), 6.72 (s, 1H), 7.09 (d, 2H, J = 8.0 Hz),
7.50–7.66 (m, 4H), 8.02 (t, 1H, J = 8.0, 8.8 Hz), 8.31 (t, 1H,
J = 7.3, 7.3 Hz), 13C NMR (75 MHz, CDCl3): d 41.5, 43.6, 48.3,
56.0, 60.5, 61.9, 68.9, 101.3, 107.9, 108.8, 109.9, 120.3, 122.3,
125.4, 128.1, 128.5, 129.2, 129.4, 129.8, 131.8, 132.4, 133.9,
134.5, 137.1, 140.4, 142.7, 147.4, 151.2, 152.3, 174.2. MS (ESI):
724 [M+H]+.
6. Procedure of the SRB-assay
The synthesized compounds (11a–j and 15a–g) have been eval-
uated for their in vitro cytotoxicity in human cancer cell lines. A
protocol of 48 h continuous drug exposure has been used and a sul-
forhodamine B (SRB) protein assay has been used to estimate cell
viability or growth. The cell lines were grown in DMEM medium
containing 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 microtiter plates were incubated at 37 °C, 5% CO2, 95%
air, and 100% relative humidity for 24 h prior to addition of exper-
5.17. 4b-[40-(Biphenyl-4-sulphonamido)benzamide]
podophyllotoxin (15e)
This compound 15e was prepared following the method de-
scribed for the preparation of the compound 11a, employing 14
(200 mg,
0.375 mmol)
with
biphenyl-4-sulphonylchloride
(113 mg, 0.451 mmol), and Et3N (1.03 mL, 0.751 mmol) and the
crude product was purified by column chromatography with
ethyl acetate/hexane (3:7) to afford pure compound 15e in
271 mg, 96% yield. Mp: 205–207 °C,
½
a 2D5
ꢁ
= ꢀ83.0 (c = 0.5 in
imental drugs. Aliquots of 10
to the appropriate microtiter wells already containing 90
cells, resulting in the required final drug concentrations. For each
compound four concentrations (0.1, 1, 10 and 100 M) were eval-
uated and done in triplicate wells. Plates were incubated further
for 48 h and assay was terminated by the addition of 50 L of cold
lL of the drug dilutions were added
CHCl3), 1H NMR (200 MHz, CDCl3): d 3.39–3.49 (m, 2H), 3.73 (br
s, 9H), 3.96–4.16 (m, 2H), 4.27–4.46 (m, 1H), 4.53 (d, 1H,
J = 3.7 Hz), 5.41–5.60 (m, 1H), 5.95 (s, 2H), 6.24 (s, 2H), 6.50 (s,
1H), 6.80 (s, 1H), 7.14 (d, 2H, J = 7.9 Hz), 7.41–7.51 (m, 5H),
7.62 (d, 2H, J = 7.9 Hz), 7.75 (d, 2H, J = 7.9 Hz), 7.97 (d, 2H,
J = 8.3 Hz), 13C NMR (75 MHz, CDCl3): d 37.3, 41.7, 43.6, 48.7,
56.1, 60.6, 68.9, 101.5, 108.0, 108.9, 110.1, 127.3, 127.6, 128.1,
128.4, 128.8, 128.9, 129.0, 131.9, 132.5, 134.6, 134.8, 137.3,
137.4, 138.7, 147.0, 147.6, 148.4, 152.5, 166.2, 174.2; MS (ESI):
749 [M+H]+.
lL of
l
l
trichloro acetic acid (TCA) (final concentration, 10% TCA) and incu-
bated for 60 min at 4 °C. The plates were washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (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 temperature. The

A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
2065
residual dye was removed by washing five times with 1% acetic
acid. The plates were air dried. Bound stain was subsequently
eluted with 10 mM trizma base, and the absorbance was read on
an ELISA plate reader at a wavelength of 540 nm with 690 nm ref-
erence wavelengths. Percent growth was calculated on a plate by
plate basis for test wells relative to control wells. The above deter-
minations were repeated three times. Percentage growth was ex-
pressed as the (ratio of average absorbance of the test well to the
slides were incubated 2 h in lysis solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, pH 10, 10% DMSO, 1% Triton X-100) followed
by 3 washes in neutralizing buffer (0.4 M Tris–HCl, pH 7.5). Elec-
trophoresis was carried out for 20 min at 40 V in 0.5% TBE buffer
(pH 8). During electrophoresis the damaged DNA migrates away
from the nucleus towards anode. Slides were then stained with a
20 mg/mL Syber green solution for 30 min. Images from were ta-
ken in fluorescent microscope at 20ꢂ.
average absorbance of the control wells) ꢂ 100. Growth inhibition
1
of 50% (GI50) was calculated from [(Ti-Tz)/(C-Tz)] ꢂ 100
50,
4
11. Immunofluorescence
which is the drug concentration resulting in a 50% reduction in
the net protein increase (as measured by SRB staining) in control
Human colon cancer cells (Colo-205) cells were seeded on cover
slips and treated with podophyllotoxin conjugate, the effective
conjugate 11e and etoposide (Eto) at concentration of 4 lM for
24 h. After treatment, cover slips were fixed with a paraformalde-
hyde solution (4% in 1X PBS) for 20 min at room temperature. Cell
permeabilization was achieved by administration of a Triton X-100
solution (0.2% in 1X PBS) for 5 min. Further the cover slips were
kept overnight in 100% methanol at 4 °C. Subsequently, cover slips
were blocked with a 1% BSA solution for 60 min and then incu-
1
cells during the drug incubation. Where, Tz
Optical density at
4
time zero, OD of control 1 C, and OD of test growth in the presence
4
1
of drug
Ti.
4
7. Cell culture
Human colon cancer cell line Colo-205 cells were purchased
from American Type Culture Collection that were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen), supple-
mented with 2 mM glutamax (Invitrogen), 10% fetal calf serum and
100U/ml Pencillin and 100 lg/mL streptomycin sulfate (Sigma).
The cell line was maintained at 37 °C in a humidified atmosphere
bated with anti-c H2AX, phos-ATM, phos-CHK1 (1:100) antibody
at room temperature for 2 h. The slides were washed three times
each of 5 min with PBST. Then cover slips were incubated with a
FITC-conjugated anti-rabbit secondary antibody (Jackson Immuno
Research Laboratories Inc., Pennsylvania, USA) for one hour and
cover slips were washed three times with PBST solution and
mounted with DAPI/PI solution. Finally, cells were observed under
confocal microscope (Olympus FV1000). Images taken were pro-
cessed with the support of the flow view version 1.7c software
program.
containing 5% CO2 in the incubator.
8. In vitro evaluation of cytotoxicity (MTT assay)
Cell viability was assessed by the MTT assay, a mitochondrial
function assay. It is based on the ability of viable cells to reduce
the MTT to insoluble formazan crystals by mitochondrial dehydro-
genase. The Colo-205 cells were seeded in a 96-well plate at a den-
sity of 10,000 cells/well. After overnight incubation, cells were
treated with compounds 11e and etoposide (Eto) at 2, 4 and
12. Protein extraction and Western blot analysis
Total cell lysates from cultured Colo-205 cells treated with 11e
and etoposide (Eto) were obtained by lysing the cells in ice-cold
RIPA buffer (1XPBS, 1% NP-40, 0.5% sodium deoxycholate and
8
l
M concentration and incubated for 24 h. Medium was then dis-
carded 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
l
0.1% SDS) and containing 100
l
g/mL PMSF, 5
l
l
g/mL Aprotinin,
g/mL NaF. After
l
5
l
g/mL leupeptin, 5 g/mL pepstatin and 100
l
570 nm with micro plate reader (Multi-mode Varioskan instru-
ment-Themo Scientific).
centrifugation at 12,000 rpm for 10 min, the protein in supernatant
was quantified by Bradford method (BIO-RAD) using Multimode
Varioskan instrument (Thermo-Fischer Scientifics). Fifty micro-
grams of protein per lane was applied in 12% SDS-polyacrylamide
gel. After electrophoresis, the protein was transferred to polyvi-
nylidine difluoride (PVDF) membrane (GE Biosciences). The mem-
brane was blocked at room temperature for 2 h in TBS + 0.1%
Tween20 (TBST) containing 5% blocking powder (Santacruz). The
membrane was washed with TBST for 5 min, primary antibody
9. Cell cycle analysis
5 ꢂ 10ꢀ5 Colo-205 cells were seeded in 60 mm dish. Cells were
treated with 11b, 11d, 11e and etoposide (Eto) at 4 lM for 24 h.
Trichostatin A (TSA) at 4 lM concentration for 12 h was used in
this study. Immediately after incubation period the cells were
incubated for an additional 24 h. Cells were harvested with Tryp-
sin-EDTA, fixed with ice-cold 70% ethanol at 4 °C for 30 min,
washed with PBS and incubated with 1 mg/mL RNase solution
(Sigma) at 37 °C for 30 min. Cells were collected by centrifugation
was added and incubated at 4 °C overnight (O/N). Topo-II
a, and b
were purchased From LS biotech and -H2AX, p21, p16, pro-cas-
c
pase-3, NF-kB (p65) antibodies were from Cell Signalling, USA. b-
Actin antibody was purchased from Imgenex company. The mem-
brane was incubated with corresponding horseradish peroxidase-
labeled secondary antibody (1:2000) (Santa Cruz) at room temper-
ature for 1 h. Membranes were washed with TBST three times for
15 min and the blots were visualized with chemiluminescence re-
agent (Thermo Fischer Scientifics Ltd). The X-ray films were devel-
oped with developer and fixed with fixer solution.
at 2000 rpm for 5 min and further stained with 250 lL of DNA
staining solution [10 mg of Propidium Iodide (PI), 0.1 mg of triso-
dium 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 contents of 20,000 events were measured by
flow cytometer (DAKO CYTOMATION, Beckman Coulter, Brea,
CA). Histograms were analyzed using Summit Software.
13. Caspase-3 assay
10. Comet assay (single cell gel electrophoresis)
The caspase-3 fluorescence assay kit (Clonetech, CA) was ap-
plied to evaluate the caspase-3 activity, using the procedures pro-
vided by the manufacturer Colo-205 cells were treated with
The Colo-205 cells were seeded in 60 mm dishes and were trea-
ted with etoposide (Eto) and podophyllotoxin conjugate 11e at a
concentration for 4
lM concentration for 24 h. Briefly, 20,000 cells
compounds 11e and etoposide (Eto) at 4
lM concentration as ob-
were embedded in agarose and deposited in microscope slides. The
tained from FACS analysis. Cell lysates were added to the 2X reac-

2066
A. Kamal et al. / Bioorg. Med. Chem. 20 (2012) 2054–2066
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contains Bcl2 antibody. After the addition of lysates 100 L of bio-
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Acknowledgments
The author P.S. is thankful to UGC, India and, A.M.R. is thankful
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