Synthesis and biological evaluation of 4β-acrylamidopodophyllotoxin congeners as DNA damaging agents

Article abstract of DOI:10.1016/j.bmc.2011.06.017

A series of new 4β-acrylamidopodophyllotoxin derivatives (13a-o) were synthesized by coupling of substituted acrylic acids (10a-l and 11m-o) to the 4β-aminopodophyllotoxin. The synthesized derivatives 13a-o were evaluated for their cytotoxicity against fi

Full text of DOI:10.1016/j.bmc.2011.06.017

Bioorganic & Medicinal Chemistry 19 (2011) 4589–4600
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4b-acrylamidopodophyllotoxin congeners
as DNA damaging agents
Ahmed Kamal a, , Paidakula Suresh a, M. Janaki Ramaiah b, Adla Mallareddy a, Banala Ashwini Kumar a,
⇑
Paidakula Raju a, J. Vinay Gopal b, S. N. C. V. L. Pushpavalli b, A. Lavanya b, Pranjal Sarma 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
Article history:
A series of new 4b-acrylamidopodophyllotoxin derivatives (13a–o) were synthesized by coupling of
substituted acrylic acids (10a–l and 11m–o) to the 4b-aminopodophyllotoxin. The synthesized deriva-
tives 13a–o were evaluated for their cytotoxicity against five human cancer cell lines (breast, oral, colon,
lung and ovarian). These podophyllotoxin conjugates have shown promising activity with GI50 values
Received 6 April 2011
Revised 2 June 2011
Accepted 3 June 2011
Available online 16 June 2011
ranging from <0.1 to 0.29 lM. Some of the compounds 13j, 13k and 13l that showed significant antipro-
liferative activity were also evaluated for related cytotoxic effects in MCF-7 cells, and compared to eto-
poside. These compounds (13j, 13k and 13l) showed G2/M cell cycle arrest and the apoptotic event
was found to be due to both the single-strand DNA breaks as observed by comet assay as well as dou-
ble-strand breaks as observed by the large accumulation of gamma H2AX foci.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Podophyllotoxin
Etoposide
Cell cycle analysis
Comet assay
c
-H2AX staining
DNA strand breaks
Anticancer activity
1. Introduction
linked to enzyme.8 This lead has stimulated a renewed interest
in the chemical and biochemical studies of podophyllotoxin de-
Podophyllotoxin (1), a well-known naturally occurring aryltetr-
alin lignan is extracted from the roots of Podophyllotoxin peltatum.
The pharmacological properties of podophyllotoxin have been
well-recognized for centuries.1–3 Podophyllotoxin inhibits the
assembly of tubulin polymerization, thus preventing the formation
of the achromatic spindle and arresting cell division in metaphase.4
However, the high toxicity and severe gastrointestinal side effect of
podophyllotoxin has limited its application as a drug in cancer che-
motherapy.5 The biological activity of podophyllotoxin has led to
extensive structural modifications resulting in several more potent
and less toxic anticancer agents such as etoposide6 (2) and tenipo-
side (3).7 Etoposide is the most widely used anticancer agent for
the treatment of leukemia, testicular cancer and small cell lung
cancer. Its clinical efficacy is due to its ability to inhibit the enzyme
DNA-topoisomerase II. These compounds 2 and 3 block the cata-
lytic activity of DNA-topoisomerase II by stabilizing a cleavable en-
zyme–DNA-complex in which the DNA is cleaved and covalently
rived antitumor agents.9 The replacement of the C-4 sugar moiety
of etoposide and teniposide with a non-sugar substituent has im-
proved the therapeutic value of etoposide.10 The C-4 non-sugar
substituent can be linked through O-, S- or N-linkage. In general,
the O-linked (ethers, esters) and S-linked (thioethers) compounds
are less active in comparison to the N-linked congeners7,11–13 like
GL-331(4) and NPF (5), as shown in Figure 1. These are proved to
be more potent than etoposide. GL-331 is also an inhibitor of
topo-II,14 and induces apoptotic cell death through independent
mechanism which contributes to its cytotoxicity. This compound
underwent phase II clinical trials for the treatment of various can-
cers,15 however this did not proceed further. A recent study on
molecular-area-oriented chemical modifications of podophyllo-
toxin has revealed certain structural features that are critical for
the topo-II inhibition.16 The comparative molecular field
analysis12,16 model further demonstrated that bulky substituents
at C-4 might be favorable for topo-II inhibition. It is observed that,
among the C4-N-substituted congeners of podophyllotoxin that
have been synthesized and developed, C4b-N-amidopodophyllo-
toxin derivatives have received less attention. In the past few
years, a variety of compounds have been prepared and evaluated
for their biological activity.17,18 It is observed from the results that
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.),
tel.: +91 40 27193236 (M.P.-B.).
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M. Pal-
Bhadra).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.017

4590
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
H
R1
O
OH
O
R2
O
O
O
HO
R1
R2
R3
H
O
O
O
CH3
O
Etoposide
OH
(2)
O
O
Teniposide
(3)
H3CO
OH
H
OCH3
OCH3
S
H3CO
OCH3
OR3
(1)
Podophyllotoxin
R
HN
O
O
H3CO
H3CO
H3CO
O
O
OCH3
OCH3
OH
OCH3
R
OCH3
H3CO
OCH3
(7a,b)
OH
Combretastatin A-4 (6)
a: R = NH3+Cl-
b: R = OH
R = NO2, GL-331 (4)
R = F, NPF (5)
Figure 1.
some of these compounds exhibit enhanced DNA topoisomerase-II
inhibition in comparison to etoposide.
toxin (12) has been coupled to various substituted acrylic acids
(10a–l and 11m–o) in presence of EDCI and HOBt to afford the cor-
responding 4b-acrylamidopodophyllotoxin derivatives (13a–o) as
shown in Scheme 2. All the synthesized compounds were charac-
terized by 1H NMR, 13C NMR and mass spectral data.
The naturally occurring cis-stilbene, combretastatin A-4 (6) was
isolated from the bark of the south African tree Combretum
caffrum.19 The combretastatin shows strong cytotoxicity against a
variety of cancer cells, including multidrug-resistant cell lines.20
Several active stilbene based compounds were identified, among
them, cis-3,40,5-trimethoxy-30-aminostilbene (7a) and cis-3,40,5-
trimethoxy-30-hydroxystilbene (7b) were found to induce HL60
apoptosis at nonomolar concentration.21
3. Biological evaluation
3.1. Invitro cytotoxicity assay
We have been involved in the development of new synthetic
procedures22 for the podophyllotoxin-based compounds and also
in the design and synthesis of new analogues of podophyllotoxin
as potential anticancer agents.23 Based on the available struc-
ture–activity relationship (SAR) studies, we decided to explore
some new podophyllotoxin analogues further by introducing an
amide functionality at 4b-position. In continuation of these efforts,
in the present study, we report the synthesis of a series conjugates
of 4b-acrylamidopodophyllotoxin congeners, wherein the podo-
phyllotoxin is linked to a stilbene moiety. All these congeners have
been evaluated for their anticancer activity against a panel of five
human cancer cell lines and promising compounds 13j, 13k and
13l were evaluated for their cell viability, cell cycle analysis, and
DNA strand breaks.
The synthesized compounds 13a–o were evaluated for their
anticancer activity in selected human cancer cell lines of breast,
oral, colon, lung and ovarian by using sulforhodamine B (SRB)
method.25 The compounds that exhibit GI50 6 10ꢀ5 M are consid-
ered to be active on the respective cell lines and the results are illus-
trated in Table 1. All the compounds (13a–o) exhibited significant
anticancer activity with GI50 values ranging from <0.1 to 2.9
while the positive controls, etoposide and adriamycin demon-
strated the GI50 in the range of <0.1 to 1.15 M and 0.13 to
3.08 M, respectively. The significant anticancer activity showed
lM,
l
l
by the promising compounds 13j, 13k and 13l prompted us to eval-
uate the cell viability in the MCF-7 cells, with a view to study their
detailed biological effects in the cell line. The MCF-7 cells were trea-
ted with compounds etoposide (Eto), combretastatin (CA-4), podo-
phyllotoxin (Podo), 10k (cis-stilbene intermediate), 13j, 13k and
2. Chemistry
13l at 2 and 4 lM for 24 h. The effective cytotoxicity observed after
the treatment of these conjugates can be deduced from the MTT as-
say which is based on mitochondrial function. Podophyllotoxin and
10k are the starting materials have shown less cytotoxicity. But hy-
brid molecules (13j, 13k, 13l) have shown pronounced cytotoxicity
and the increase was observed to be synergistic. The data suggests
that these conjugates (13j, 13k, 13l) have almost similar activity to
that of etoposide as shown in Figure 2.
Acrylic acids (10b, 10e, 10i, 10m and 10n) were synthesized by
means of a Perkin reaction, as previously reported.24 Synthesis of
acrylic acids (10a–l and 11m–o) starting from appropriate meth-
oxy substituted phenyl acetic acids with substituted aromatic alde-
hydes in the presence of Ac2O and Et3N and the mixture was
refluxed to afford the acids (10a–l) and acetates (10m–o). As
10m–o are in acetate form, these upon further treatment of a base
provide the required acids (11m–o), as shown in Scheme 1.
Synthesis of 4b-acrylamidopodophyllotoxin conjugates (13a–o)
was carried out from the key intermediate, 4b-aminopodophyllo-
toxin (12).22e,23a Further the intermediate 4b-aminopodophyllo-
3.2. Effect of podophyllotoxin conjugates on cell cycle
Previous studies26,27 have reported that etoposide was known
to cause G2/M cell cycle arrest and its action on cell cycle was

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
4591
CO2H
8a: R = 3,4,5-trimethoxy
CHO
8b: R = 2-methoxy
9a: R1 = 4-fluoro-3-methoxy
9b: R1 = 3-fluoro-4-methoxy
9c: R1 = 2-fluoro-5-methoxy
9d: R1 = 2-fluoro-4-methoxy
9e: R1 = 3-nitro-4-methoxy
9f: R1 = 4-hydroxy-3-nitro
9g: R1 = 4-nitro
9i: R1 = 4-methoxy
R1
9j: R1 = 3-methoxy
9k: R1 = 2-nitro
R
9a-n
9l: R1 = 4-hydroxy-3-methoxy
9m: R1 = 3-hydroxy-4-methoxy
9n: R1 = 4-hydroxy
8a,b
i
9h: R1 = 3-nitro
CO2H
CO2H
CO2H
ii
R1
R1
R1
R
R
R
10m-o
10a-l
11m-o
10a: R = 3,4,5-trimethoxy, R1 = 4-fluoro-3-methoxy
10b: R = 3,4,5-trimethoxy, R1 = 3-fluoro-4-methoxy
10c: R = 3,4,5-trimethoxy, R1 = 2-fluoro-5-methoxy
10d: R = 3,4,5-trimethoxy, R1 = 2-fluoro-4-methoxy
10e: R = 3,4,5-trimethoxy, R1 = 3-nitro-4-methoxy
10f: R = 3,4,5-trimethoxy, R1 = 4-hydroxy-3-nitro
10g: R = 3,4,5-trimethoxy, R1 = 4-nitro
10m: R = 3,4,5-trimethoxy, R1 = 4-O-acetyl-3-methoxy
10n: R = 3,4,5-trimethoxy, R1 = 3-O-acetyl-4-methoxy
10o: R = 3,4,5-trimethoxy, R1 = 4-O-acetyl
11m: R = 3,4,5-trimethoxy, R1 = 4-hydroxy-3-methoxy
11n: R = 3,4,5-trimethoxy, R1 = 3-hydroxy-4-methoxy
11o: R = 3,4,5-trimethoxy, R1 = 4-hydroxy
10h: R = 3,4,5-trimethoxy, R1 = 3-nitro
10i: R = 3,4,5-trimethoxy, R1 = 4-methoxy
10j: R = 3,4,5-trimethoxy, R1 = 3-methoxy
10k: R = 2-methoxy, R1 = 4-nitro
10l: R = 2-methoxy, R1 = 2-nitro
Scheme 1. Reagents and conditions: (i) Ac2O, Et3N, reflux, 140 °C; (ii) K2CO3, MeOH/H2O 3/1, rt.
R1
R
NH2
O
NH
CO2H
O
O
O
O
i
O
O
O
O
R1
R
H3CO
OCH3
OCH3
10a-l or 11m-o
H3CO
OCH3
OCH3
12
13a-o
13a: R = 3,4,5-trimethoxy, R1 = 4-fluoro-3-methoxy
13b: R = 3,4,5-trimethoxy, R1 = 3-fluoro-4-methoxy
13c: R = 3,4,5-trimethoxy, R1 = 2-fluoro-5-methoxy
13d: R = 3,4,5-trimethoxy, R1 = 2-fluoro-4-methoxy
13e: R = 3,4,5-trimethoxy, R1 = 3-nitro-4-methoxy
13f: R = 3,4,5-trimethoxy, R1 = 4-hydroxy-3-nitro
13g: R = 3,4,5-trimethoxy, R1 = 4-nitro
13j: R = 3,4,5-trimethoxy, R1 = 3-methoxy
13k: R = 2-methoxy, R1 = 4-nitro
13l: R = 2-methoxy, R1 = 2-nitro
13m: R = 3,4,5-trimethoxy, R1 = 4-hydroxy-3-methoxy
13n: R = 3,4,5-trimethoxy, R1 = 3-hydroxy-4-methoxy
13o: R = 3,4,5-trimethoxy, R1 = 4-hydroxy
13h: R = 3,4,5-trimethoxy, R1 = 3-nitro
13i: R = 3,4,5-trimethoxy, R1 = 4-methoxy
Scheme 2. Reagents and conditions: (i) EDCI, HOBt, CH2Cl2, rt, 3 h.
purely cell type dependent. Similar to etoposide, the compounds of
the present study possess a podophyllotoxin scaffold and it was
considered of interest to study the effect of some of these promis-
ing molecules on the cell cycle regulatory mechanism in MCF-7
cells. Therefore, MCF-7 cells were treated by these conjugates at
intermediate) has shown G1/S phase cell cycle arrest and where
as podophyllotoxin (Podo) has shown very high G2/M cell cycle ar-
rest. Similar kind of observation was made by Nijmeijer et al., with
respect to 10k.28 Thus these results from FACS analysis indicate
G2/M cell cycle arrest is caused by these podophyllotoxin conju-
gates in MCF-7 cells as shown in Figure 3.
2 lM concentration for 24 h, and FACS analysis was carried out.
These conjugates 13j, 13k and 13l show 36%, 39% and 35% of cells
in G2/M phase, respectively, with concomitant decrease of cells in
G1 phase. Whereas, the untreated cells, positive control (Etopo-
side), CA-4 (6), podophyllotoxin (Podo), 10k (cis-stilbene interme-
diate) have shown 26%, 62%, 84%, 86% and 20% of cells in G2/M
phase, respectively. Interestingly one of conjugate 10k (stilbene
3.3. Effect of podophyllotoxin conjugates on single-strand DNA
breaks
Cancer can be treated with drugs that can induce double-strand
DNA breaks (DSBs) that preferentially kills the fast growing tumor

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
Table 1
Cytotoxic activity (GI50
l
M) of compounds 13a–o
Compound
Breast
Oral
Colon
Lung
Ovarian
A2780
Zr-75-1
MCF7
Gurav
DWD
Colo 205
A-549
Hop62
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
Etoposide
ADR
2.2
—
—
2.7
—
2.4
—
2.7
—
2.7
2.9
<0.1
<0.1
<0.1
—
—
—
2.1
0.13
2.4
—
—
—
—
—
2.4
—
—
—
—
2.1
—
—
—
2.4
—
2.7
2.7
2.8
2.4
2.9
2.3
3.08
<0.1
2.5
—
—
—
—
—
2.7
2.5
2.2
0.18
2.2
—
2.9
0.18
2.1
2.2
2.9
—
2.5
0.18
2.6
0.19
2.1
2.1
2.2
2.1
2.1
2.2
—
0.19
2.9
<0.1
2.4
<0.1
2.3
2.2
2.3
2.3
—
—
0.17
2.3
0.19
2.7
0.17
—
0.18
2.0
2.1
2.5
2.9
2.6
0.8
0.15
2.7
2.5
2.2
2.6
2.5
2.5
<0.1
2.3
2.2
—
2.7
2.0
2.1
—
0.17
—
—
—
—
—
—
—
—
—
1.3
<0.1
2.7
0.2
0.11
2.7
0.5
<0.1
2.7
0.6
<0.1
—
0.13
<0.1
ADR = adriamycin is one of the control drug.
(i.e.,
c-H2AX) is a direct indicator of double-strand DNA breaks
in cells that occur during DNA damage.32,33 As these podophyllo-
toxin congeners are closely related molecules to etoposide, it was
considered of interest to examine the effect of double-strand
DNA breaks. Thus MCF-7 cells were treated with etoposide, CA-4
(6), podophyllotoxin (Podo), 10k (cis-stilbene intermediate) and
13k at 2 lM for 24 h. Interestingly a large number of c-H2AX foci
were observed in etoposide (70%), CA-4 (6) (50%), 10k (30%) and
13k treated cells when compared to the control (DMSO) cells. Pod-
ophyllotoxin treated cells have shown very negligible effect on
double-strand DNA breaks. This was strongly supported by the fact
that podophyllotoxin cannot act as topo II inhibitor.34 The percent-
age of cells with double-strand breaks was observed to be 70% in
case of 13k as shown in Figure 5.
4. Conclusion
Figure 2. Cell viability assay was observed after the treatment with compounds
etoposide (Eto), CA-4 (6), Podophyllotoxin (Podo), 10k (cis-stilbene intermediate),
13j, 13k and 13l at 2 and 4 lM concentration by MTT assay for 24 h in 96 well
plates at 10,000 cells per well. Etoposide (Eto) was used as standard control. Control
indicates the untreated cells.
These new 4b-acrylamidopodophyllotoxin derivatives (13a–o)
were synthesized by coupling substituted acrylic acids (10a–l
and 11m–o) to the 4b-aminopodophyllotoxin. These compounds
(13a–o) were evaluated for their cytotoxic activity in five human
cancer cell lines. Some of these podophyllotoxin conjugates like
13j, 13k and 13l have shown enhanced cytotoxicity at 2 lM con-
centration and caused the G2/M cell cycle arrest. Among these
compounds, 13k has slightly better effectiveness in causing cell cy-
cle arrest. Interestingly, the conjugate 13k causes DNA damage at
both single strand (30%) as well as double-strand (70%) and even-
tually killing the cancerous cells. Where as etoposide has shown
10% of single strand and 70% of double strand specific DNA dam-
age. Thus our hybrid molecules are acting on par with etoposide.
These results are supported by previous findings on podophyllo-
toxin conjugates exhibiting dual inhibitory activities on (topo-I
and topo-II).35 Based on the studies, it seems that the effect of
these compounds is more on DNA double-strand breaks compared
to single-strand breaks. Thus these compounds can be considered
as leads for the effective treatment against cancer.
cells by the inhibition of topoisomerase-II (topo-II). During this
event there is a possibility that drugs can induce single-strand
breaks (SSBs).29–31 Further to examine the single-strand breaks co-
met assay has been performed wherein MCF-7 cells were treated
with compounds etoposide (positive control), CA-4 (6), podophyl-
lotoxin (Podo), 10k (cis-stilbene intermediate) and 13k at 2 lM for
24 h followed by agarose gel electrophoresis of slides on which
cells were plated. Surprisingly, single-strand DNA breaks have
been observed and are visualized as cells having bright head and
tail in the cells treated with etoposide, 10k and 13k in comparison
to the control cells and the percentage of cells with single-strand
(ssDNA) breaks was found to be 10%, 20% and 30%, respectively,
for as shown in Figure 4.
3.4. Effect of podophyllotoxin conjugates on double-strand DNA
breaks
5. Experimental section
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
Etoposide is well-known drug that causes double-strand DNA
breaks and is
documented that phosphorylation at Ser-139 on H2AX protein
a
topo-II inhibitor.26,27,31 It has been well

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
4593
Figure 3. FACS analysis of cell cycle distribution of MCF-7 breast carcinoma cells after treatment with compounds etoposide, CA-4 (6), podophyllotoxin (Podo), 10k
(cis-stilbene intermediate), 13j, 13k and 13l at 2 M concentration for 24 h. Etoposide was used as a standard control.
l
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 Gemini Varian-VXR-unity (200, 400 and
500 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.
reaction mixture was acidified with 35% aq HCl. The reaction mix-
ture was left overnight and the precipitated product was filtered.
The precipitate was recrystallized from absolute ethanol afford
the pure compound 10a, 130 mg in 40% yield. 1H NMR (400 MHz,
DMSO-d6): d 3.48 (s, 3H), 3.79 (s, 6H), 3.83 (s, 3H), 6.43 (s, 2H),
6.49 (s, 2H), 6.58 (d, 1H, J = 8.4 Hz), 6.80–6.81 (m, 1H), 6.93 (t, 1H,
J = 7.6, 9.3 Hz), 7.79 (s, 1H); MS (ESI): 376 [M+H]+.
5.2. (E)-3-(3-Fluoro-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10b)
5.1. (E)-3-(4-Fluoro-3-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10a)
This compound 10b was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9b (136 mg,
0.88 mmol) afford the pure compound 10b, 143 mg in 44% yield.
1H NMR (300 MHz, DMSO-d6): d 3.79 (s, 6H), 3.85 (s, 3H), 3.87 (s,
3H), 6.40 (s, 2H), 6.74–6.85 (m, 3H), 7.73 (s, 1H); MS (ESI): 382
[M+Na]+.
The compound 3,4,5-trimethoxyphenyl acetic acid (8a) (200 mg,
0.88 mmol) was dissolved in 10 mL of Ac2O, followed by addition of
substituted aldehyde 9a (136 mg, 0.88 mmol) and Et3N (1 mL). The
reaction mixture was heated at 140 °C for 12 h. After cooling, the

4594
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
Figure 4. Effect of podophyllotoxin conjugate (13k) on the single-strand DNA breaks. The compounds etoposide, CA-4 (6), podophyllotoxin (Podo), 10k (cis-stilbene
intermediate) and effective conjugate 13k were added to MCF-7 cells at 2 M concentration for 24 h. Sybr green I dye was used to stain the DNA in both control as well as
l
compound treated cells. Etoposide has been employed as the positive control. Single-strand DNA breaks were detected due to the bright head and tail formation in compound
treated cells. White colored arrows indicate the tail pattern that appears during single-strand DNA breaks. Etoposide, 10k and 13k has shown single-strand DNA breaks.
Where as podophyllotoxin (Podo) did not as shown in this comet assay.
5.3. (E)-3-(2-Fluoro-5-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10c)
0.88 mmol) afford the pure compound 10e, 156 mg in 45% yield.
1H NMR (300 MHz, DMSO-d6): d 3.80 (s, 6H), 3.89 (s, 3H), 3.94 (s,
3H), 6.40 (s, 2H), 6.66 (d, 1H, J = 7.3 Hz), 6.84–6.90 (m, 1H), 7.16
(d, 1H, J = 7.3 Hz), 7.77 (s, 1H); MS (ESI): 385 [M+Na]+.
This compound 10c was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9c (136 mg,
0.88 mmol) afford the pure compound 10c, 120 mg in 37% yield.
1H NMR (400 MHz, DMSO-d6): d 3.36 (s, 3H), 3.79 (s, 6H), 3.86 (s,
3H), 6.31–6.33 (m, 1H), 6.49 (s, 2H), 6.76–6.80 (m, 1H), 6.96 (t,
1H, J = 8.8, 9.6 Hz), 8.12 (s, 1H); MS (ESI): 385 [M+Na]+.
5.6. (E)-3-(4-Hydroxy-3-nitrophenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10f)
This compound 10f was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9f (147 mg,
0.88 mmol) afford the pure compound 10f, 121 mg in 67% yield.
1H NMR (300 MHz, DMSO-d6): d 4.31 (s, 6H), 4.37 (s, 3H), 6.94 (s,
2H), 7.48 (d, 1H, J = 8.8 Hz), 7.73–7.77 (dd, 1H, J = 2.2, 2.0 Hz),
8.20 (s, 1H), 8.33 (br s, 1H); MS (ESI): 382 [M+Na]+.
5.4. (E)-3-(2-Fluoro-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10d)
This compound 10d was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9d (136 mg,
0.88 mmol) afford the pure compound 10d, 145 mg in 45% yield.
1H NMR (500 MHz, DMSO-d6): d 3.66 (s, 6H), 3.70 (s, 3H), 3.72 (s,
3H), 6.42 (s, 2H), 6.55–6.58 (m, 1H), 6.70–6.75 (m, 1H), 6.80–6.85
(m, 1H), 7.82 (s, 1H), 12.57 (s, 1H); MS (ESI): 331 [M+H]+.
5.7. (E)-3-(4-Nitrophenyl)-2-(3,4,5-trimethoxyphenyl)acrylic
acid (10g)
This compound 10g was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9g (133 mg,
0.88 mmol) afford the pure compound 10g, 210 mg in 66% yield.
1H NMR (200 MHz, DMSO-d6): d 3.72 (s, 6H), 3.81 (s, 3H), 6.37 (s,
2H), 7.25 (d, 2H, J = 8.8 Hz), 7.25 (s, 1H), 8.00 (d, 2H, J = 8.8 Hz);
MS (ESI): 322 [M+Na]+.
5.5. (E)-3-(4-Methoxy-3-nitrophenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10e)
This compound 10e was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9e (160 mg,

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
4595
Figure 5. Effect of podophyllotoxin conjugate (13k) on the double-strand DNA breaks. The compounds etoposide, CA-4 (6), podophyllotoxin (Podo), 10k (cis-stilbene
intermediate) and effective conjugate (13k) were added to MCF-7 cells at 2 M concentration for 24 h. -H2AX antibody was used to detect serine 139 phosphorylation in
l
c
cells during double-strand DNA breaks. Etoposide has been employed as the positive control. Podophyllotoxin (Podo) has shown negligible effect on double-strand DNA
breaks. Whereas stilbene intermediate (10k) has shown considerable double-strand DNA breaks. CA-4 has shown increased foci size as well as effective double-strand DNA
breaks.
5.8. (E)-3-(3-Nitrophenyl)-2-(3,4,5-trimethoxyphenyl)acrylic
acid (10h)
0.88 mmol) afford the pure compound 10h, 196 mg in 61% yield.
1H NMR (300 MHz, DMSO-d6): d 3.65 (s, 6H), 3.69 (s, 3H), 6.48
(s, 2H), 7.51–7.62 (m, 2H), 7.81 (s, 1H), 7.85 (t, 1H, J = 1.7,
1.6 Hz), 8.06–8.09 (m, 1H), 12.85 (br s, 1H); MS (ESI): 391
[M+H]+.
This compound 10h was prepared by the method described
for 10a, employing 8a (200 mg, 0.88 mmol) and 9h (133 mg,

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
5.9. (E)-3-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic
5.15. (E)-3-(4-Acetoxyphenyl)-2-(3,4,5-
acid (10i)
trimethoxyphenyl)acrylic acid (10o)
This compound 10i was prepared by the method described for
10a, employing 8a (200 mg, 0.88 mmol) and 9i (0.12 mL,
0.88 mmol) afford the pure compound 10i, 176 mg in 57% yield.
1H NMR (300 MHz, DMSO-d6): d 3.77 (s, 3H), 3.78 (s, 6H), 3.89 (s,
3H), 6.44 (s, 2H), 6.72 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 9.0 Hz),
7.85 (s, 1H); MS (ESI): 362 [M+Na]+.
This compound 10o was prepared by the method described for
10m, employing 8a (200 mg, 0.88 mmol) and 9n (107 mg,
0.88 mmol) afford the pure compound 10o, 191 mg in 58% yield.
1H NMR (500 MHz, DMSO-d6): d 3.35 (s, 3H), 3.70 (s, 6H), 3.74 (s,
3H), 6.49 (s, 2H), 7.06 (d, 2H, J = 8.3 Hz), 7.19 (d, 2H, J = 8.3 Hz),
7.76 (s, 1H); MS (ESI): 373 [M+H]+
.
5.10. (E)-3-(3-Methoxyphenyl)-2-(3,4,5-
5.16. (E)-3-(4-Hydroxy-3-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10j)
trimethoxyphenyl)acrylic acid (11m)
This compound 10j was prepared by the method described 10a,
employing 8a (200 mg, 0.88 mmol) and 9j (0.12 mL, 0.88 mmol) af-
ford the pure compound 10j, 157 mg in 51% yield. 1H NMR
(300 MHz, DMSO-d6): d 3.76 (s, 3H), 3.78 (s, 6H), 3.87 (s, 3H),
6.41 (s, 2H), 6.65–6.70 (m, 2H), 6.86 (t, 1H, J = 7.1, 7.3 Hz), 7.04
(d, 1H, J = 8.8 Hz), 7.83 (s, 1H); MS (ESI): 383 [M+Na]+.
A solution of K2CO3 (202 mg, 1.47 mmol) in water (0.5 mL) was
added to solution of 10m (200 mg, 0.49 mmol) in methanol
(1.5 mL). The reaction mixture was stirred overnight at room tem-
perature and then neutralized with 1 N aqueous HCl. After concen-
tration, the resulting solution was extracted with ethyl acetate. The
combined organic layers were washed with water, brine and dried
over anhydrous MgSO4, filtered and evaporated to dryness. The
crude product was purified by column chromatography with ethyl
acetate/hexane (1:1) afford the pure compound 11m, 147 mg in
82% yield. 1H NMR (200 MHz, DMSO-d6): d 3.99 (s, 3H), 4.32 (s,
3H), 4.33 (s, 6H), 6.94–7.00 (m, 3H), 7.23 (s, 2H), 8.18 (s, 1H),
9.35 (br s, 1H); MS (ESI): 604 [M+H]+.
5.11. (E)-2-(2-Methoxyphenyl)-3-(4-nitrophenyl)acrylic acid
(10k)
This compound 10k was prepared by the method described for
10a, employing 8b (200 mg, 1.20 mmol) and 9k (181 mg,
1.20 mmol) afford the pure compound 10k, 161 mg in 44% yield.
1H NMR (200 MHz, DMSO-d6): d 3.74 (s, 3H), 6.83–6.98 (m, 3H),
7.21 (d, 2H, J = 8.5 Hz), 7.29–7.38 (m, 1H), 7.76 (s, 1H), 7.97 (d,
2H, J = 8.5 Hz); MS (ESI): 604 [M+H]+.
5.17. (E)-3-(3-Hydroxy-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (11n)
This compound 11n was prepared by the method described for
11m, employing 10n (200 mg, 0.49 mmol) and K2CO3 (202 mg,
1.47 mmol) afford the pure compound 11n, 139 mg in 77% yield.
1H NMR (200 MHz, DMSO-d6): d 3.75 (s, 6H), 3.80 (s, 3H), 3.81 (s,
3H), 6.40 (s, 2H), 6.52–6.65 (m, 3H), 7.62 (s, 1H); MS (ESI): 383
[M+Na]+.
5.12. (E)-2-(2-Methoxyphenyl)-3-(2-nitrophenyl)acrylic acid
(10l)
This compound 10l was prepared by the method described for
10a, employing 8b (200 mg, 1.20 mmol) and 9k (181 mg,
1.20 mmol) afford the pure compound 10l, 152 mg in 42% yield.
1H NMR (500 MHz, DMSO-d6): d 3.66 (s, 3H), 6.72–6.95 (m, 4H),
7.18 (t, 1H, J = 7.3, 7.3 Hz), 7.27–7.32 (m, 2H), 7.98 (d, 1H,
J = 7.3 Hz), 8.05 (s, 1H); MS (ESI): 322 [M+Na]+.
5.18. (E)-3-(4-Hydroxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (11o)
This compound 11o was prepared following the method de-
scribed for 11m, employing 10o (200 mg, 0.54 mmol) and K2CO3
(223 mg, 1.62 mmol) afford the pure compound 11o, 127 mg in
71% yield. 1H NMR (300 MHz, DMSO-d6): d 3.70 (s, 6H), 3.78 (s,
6H), 3.74 (s, 3H), 6.49 (s, 2H), 7.04 (d, 2H, J = 8.3 Hz), 7.19 (d, 2H,
J = 8.3 Hz), 7.76 (s, 1H); MS (ESI): 345 [M+H]+.
5.13. (E)-3-(4-Acetoxy-3-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10m)
The compound 3,4,5-trimethoxyphenyl acetic acid (8a)
(200 mg, 0.88 mmol) was dissolved in 4 mL of Ac2O, followed
by addition of aromatic aldehyde 9l (134 mg, 0.88 mmol) and
Et3N (2 mL). The reaction mixture was heated at 140 °C for
24 h. After cooling, the reaction mixture was acidified with 35%
aqueous HCl and kept at room temperature overnight. Then
the forming O-acetylated precipitated was collected by filtration,
recrystallized from ethanol afford the pure compound 10m,
170 mg in 50% yield. 1H NMR (200 MHz, DMSO-d6): d 1.93 (s,
3H), 3.42 (s, 3H), 3.76 (s, 3H), 3.77 (s, 6H), 6.40–644 (m, 2H);
5.19. 4b-(E)-3-(4-Fluoro-3-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13a)
The compound 4b-aminopodophyllotoxin (12) (200 mg,
0.48 mmol) was dissolved in 10 mL of dry CH2Cl2, followed by
addition of acrylic acid 10a (205 mg, 0.57 mmol), EDCI (108 mg,
0.57 mmol) and HOBt (88 mg, 0.57 mmol). The reaction mixture
was stirred at room temperature for 3 h. The reaction mixture
was washed with saturated solution of NaHCO3 and extracted with
CH2Cl2, dried over anhydrous Na2SO4. The crude product was puri-
fied by column chromatography with ethyl acetate/hexane (4:6)
afford the pure compound 13a, 330 mg in 90% yield. Mp: 144–
MS (ESI): 403 [M+H]+
.
5.14. (E)-3-(3-Acetoxy-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (10n)
This compound 10n was prepared by the method described for
10m, employing 8a (200 mg, 0.88 mmol) and 9m (134 mg,
0.88 mmol) afford the pure compound 10n, 175 mg in 52% yield.
1H NMR (200 MHz, DMSO-d6): d 1.96 (s, 3H), 3.76 (s, 6H), 3.81 (s,
3H), 3.82 (s, 3H), 6.41 (s, 2H), 6.53–6.66 (m, 3H); MS (ESI): 403
146 °C, ½a 2D5
ꢁ
ꢀ78.0 (c 0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d
2.52–2.63 (dd, 1H, J = 5.1, 5.1 Hz), 2.85–3.01 (m, 1H), 3.53 (s, 3H),
3.74 (s, 6H), 375 (s, 6H), 3.77 (s, 3H), 3.82 (s, 3H), 3.88 (t, 1H),
4.41–4.50 (dd, 2H, J = 6.6, 5.8 Hz), 5.23–5.30 (m, 1H), 5.73 (d, 1H,
J = 6.6 Hz), 5.95 (d, 2H, J = 3.6 Hz), 6.22 (s, 2H), 6.43 (s, 2H), 6.44
(s, 1H), 6.48–6.54 (dd, 1H, J = 1.4, 1.4 Hz), 6.72–6.76 (m, 2H),
[M+H]+
.

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
4597
6.87–6.98 (dd, 1H, J = 8.0, 8.0 Hz), 7.70 (s, 1H); 13C NMR (75 MHz,
5.23. 4b-(E)-3-(4-Methoxy-3-nitrophenyl)-2-(3,4,5-
CDCl3): d 36.7, 40.3, 43.1, 47.8, 55.1, 55.6, 55.7, 59.7, 59.8, 68.5,
101.1, 107.0, 108.0, 109.1, 109.2, 114.3, 115.4, 115.6, 123.1,
123.2, 130.0, 130.8, 131.8, 132.1, 133.5, 135.7, 135.8, 136.2,
137.2, 146.5, 147.1, 149.4, 151.9, 153.1, 167.4, 174.5; MS (ESI):
758 [M+H]+.
trimethoxyphenyl)acrylamido podophyllotoxin (13e)
This compound 13e was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10e (221 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
5.20. 4b-(E)-3-(3-Fluoro-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13b)
pound 13e, 310 mg in 79% yield. Mp: 140–143 °C, ½a D25
ꢀ23.9 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.56–2.63 (dd, 1H,
J = 5.3, 5.3 Hz), 2.87–3.00 (m, 1H), 3.36 (t, 1H, J = 7.0, 7.0 Hz), 3.73
(s, 6H), 3.75 (s, 6H), 3.76 (s, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 4.39–
4.46 (m, 2H), 5.25 (t, 1H, J = 5.3, 6.2 Hz), 5.81 (d, 1H, J = 6.2 Hz),
5.94 (d, 2H, J = 5.3 Hz), 6.20 (s, 2H), 6.38 (s, 2H), 6.71 (s, 1H), 6.91
(d, 1H, J = 7.9 Hz), 7.25 (s, 1H), 7.27 (d, 1H, J = 7.9 Hz), 7.34 (s,
1H), 7.68 (s, 1H); 13C NMR (75 MHz, CDCl3): d 36.7, 43.1, 46.5,
47.8, 48.5, 55.7, 55.8, 56.7, 59.8, 60.0, 101.2, 106.9, 108.1, 109.2,
109.3, 114.0, 125.8, 127.4, 130.0, 130.4, 131.8, 132.1, 135.7,
136.2, 136.3, 137.6, 138.6, 146.5, 147.1, 151.6, 151.9, 153.3,
167.4, 169.1, 174.4; MS (ESI): 785 [M+H]+.
This compound 13b was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10b (205 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
pound 13b, 350 mg in 95% yield. Mp: 159–160 °C, ½a D25
ꢁ
+13.9 (c
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.54–2.63 (dd, 1H,
J = 5.2, 5.2 Hz), 2.87–2.97 (m, 1H), 3.38 (t, 1H), 3.74 (s, 6H), 3.75
(s, 6H), 3.77 (s, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 4.41–4.49 (m,
2H), 5.26 (t, 1H), 5.74 (d, 1H, J = 6.7 Hz), 5.95 (d, 2H, J = 6.0 Hz),
6.22 (s, 2H), 6.40 (s, 2H), 6.44 (s, 1H), 6.62 (d, 1H, J = 12.8 Hz),
6.73 (s, 1H), 6.76–6.87 (m, 2H), 7.66 (s, 1H); 13C NMR (75 MHz,
CDCl3): d 36.7, 40.4, 43.1, 47.7, 55.6, 55.7, 59.7, 60.0, 68.5,
101.1, 106.9, 108.0, 109.1, 109.2, 113.2, 116.2, 116.5, 126.9,
127.8, 130.0, 130.7, 132.0, 133.1, 134.9, 135.7, 136.2, 137.3,
146.5, 147.1, 151.9, 152.2, 153.1, 167.5, 174.5; MS (ESI): 758
[M+H]+.
5.24. 4b-(E)-3-(4-Hydroxy-3-nitrophenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13f)
This compound 13f was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10f
(214 mg, 0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt
(88 mg, 0.57 mmol). The crude product was purified by column
chromatography with ethyl acetate/hexane (3:7) to afford pure
5.21. 4b-(E)-3-(2-Fluoro-5-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13c)
compound 13f, 330 mg in 88% yield. Mp: 184–187 °C, ½a D25
ꢀ84.9
ꢁ
(c 0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.55–2.61 (dd, 1H,
J = 4.5, 4.5 Hz), 2.88–3.02 (m, 1H), 3.74 (s, 6H), 3.76 (s, 6H), 3.77
(s, 3H), 3.88 (s, 3H), 4.38–4.44 (m, 2H), 5.21–5.31 (m, 3H), 5.96
(d, 2H, J = 4.5 Hz), 6.22 (s, 2H), 6.40 (s, 2H), 6.43 (s, 1H), 6.73 (s,
1H), 7.00 (d, 1H, J = 9.0 Hz), 7.28 (d, 1H, J = 9.0 Hz), 7.70 (s, 1H),
7.74 (s, 1H); 13C NMR (75 MHz, CDCl3): d 36.7, 40.4, 43.1, 47.7,
55.6, 55.8, 59.8, 60.0, 101.1, 106.9, 108.0, 109.1, 109.2, 118.7,
126.1, 126.2, 130.0, 130.4, 132.1, 135.7, 136.3, 136.4, 137.5,
146.5, 147.1, 151.9, 153.2, 167.4, 174.5; MS (ESI): 794 [M+Na]+.
This compound 13c was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10c (205 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
pound 13c, 350 mg in 95% yield. Mp: 123–126 °C, ½a D25
ꢀ12.0 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.67–2.74 (dd, 1H,
J = 5.2, 5.2 Hz), 2.95–3.08 (m, 1H), 3.40 (s, 3H), 3.74 (s, 6H), 375
(s, 6H), 3.80 (s, 3H), 3.84 (s, 3H), 3.85–3.95 (m, 2H), 4.49–4.55
(dd, 2H, J = 60, 6.0 Hz), 5.32–5.37 (dd, 1H, J = 4.5, 4.5 Hz), 5.96 (d,
2H, J = 9.0 Hz), 5.99 (d, 1H, J = 7.5 Hz), 6.20–6.25 (m, 1H), 6.28 (s,
2H), 6.45 (s, 2H), 6.48 (s, 1H), 6.72–6.77 (m, 1H), 6.78 (s, 1H),
6.93 (t, 1H), 7.94 (s, 1H); MS (ESI): 758 [M+H]+.
5.25. 4b-(E)-3-(4-Nitrophenyl)-2-(3,4,5-trimethoxyphenyl)
acrylamido podophyllotoxin (13g)
This compound 13g was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10g (204 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
5.22. 4b-(E)-3-(2-Fluoro-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13d)
This compound 13d was prepared by the method described
for 13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10d
(205 mg, 0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt
(88 mg, 0.57 mmol). The crude product was purified by ethyl
acetate/hexane (3:7) afford the pure compound 13d, 340 mg in
pound 13g, 350 mg in 95% yield. Mp: 187–190 °C, ½a D25
ꢁ
+71.0 (c
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.56–2.68 (dd, 1H,
J = 5.1, 5.1 Hz), 2.91–3.10 (m, 1H), 3.73 (s, 6H), 3.75 (s, 6H), 381
(s, 3H), 3.91 (s, 3H), 4.25–4.35 (m, 1H), 4.46–4.57 (m, 2H), 5.35
(t, 1H), 5.82 (d, 1H, J = 7.3 Hz), 5.97 (d, 2H, J = 5.1 Hz), 6.27 (s,
2H), 6.38 (s, 2H), 6.51 (s, 1H), 6.73 (s, 1H), 7.22 (d, 2H, J = 8.8 Hz),
7.86 (s, 1H), 8.07 (d, 2H, J = 8.8 Hz); 13C NMR (75 MHz, CDCl3): d
36.6, 43.0, 47.7, 55.6, 59.8, 60.0, 68.5, 101.2, 107.0, 109.0, 109.2,
109.3, 123.1, 129.9, 130.5, 131.7, 132.1, 135.7, 136.2, 137.5,
138.0, 139.9, 142.5, 146.2, 146.5, 147.1, 147.5, 151.9, 152.9,
167.5, 174.5; MS (ESI): 777 [M+Na]+.
92% yield. Mp: 123–125 °C, ½a D25
ꢁ
+48.9 (c 0.5 in CHCl3), 1H
NMR (400 MHz, CDCl3): d 2.61–2.66 (dd, 1H, J = 4.7, 4.7 Hz),
2.94–3.03 (m, 1H), 3.73 (s, 6H), 3.75 (s, 6H), 3.77 (s, 3H), 3.81
(s, 3H), 3.89 (s, 3H), 3.90–3.92 (m, 1H), 4.49–4.55 (m, 2H),
5.30–5.34 (m, 1H), 5.73 (d, 1H, J = 6.2 Hz), 5.96 (d, 2H,
J = 11.7 Hz), 6.27 (s, 2H), 6.37–6.41 (m, 1H), 6.42 (s, 2H), 6.49
(s, 1H), 6.56–6.61 (m, 2H), 6.74 (s, 1H), 7.96 (s, 1H); 13C NMR
(75 MHz, CDCl3): d 36.7, 40.2, 40.3, 43.1, 47.8, 55.9, 59.8, 59.9,
68.6, 101.0, 101.2, 101.4, 106.6, 107.0, 108.0, 109.3, 110.3,
114.8, 115.0, 125.9, 130.1, 130.4, 130.7, 132.1, 135.7, 136.2,
137.2, 146.5, 147.1, 151.9, 152.9, 160.8, 162.8, 167.6, 174.5;
MS (ESI): 758 [M+H]+.
5.26. 4b-(E)-3-(3-Nitrophenyl)-2-(3,4,5-trimethoxyphenyl)
acrylamido podophyllotoxin (13h)
This compound 13h was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10h (204 mg,

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
(s, 3H), 4.05–4.14 (m, 2H), 4.24–4.30 (m, 1H), 4.40–4.49 (m, 1H),
5.61 (d, 1H, J = 6.7 Hz), 5.95 (s, 2H), 6.19 (s, 2H), 6.42 (s, 1H), 6.68
(s, 1H), 6.92–7.07 (m, 3H), 7.12 (d, 2H, J = 8.8 Hz), 7.36–7.45 (m,
1H), 7.83 (s, 1H), 8.02 (d, 2H, J = 8.8 Hz); 13C NMR (75 MHz, CDCl3):
d 36.6, 43.1, 47.1, 55.2, 55.6, 59.8, 64.5, 68.3, 101.1, 108.0, 108.9,
109.3, 111.5, 119.2, 120.7, 123.3, 124.0, 130.3, 131.3, 132.0,
132.1, 135.7, 136.2, 137.9, 139.2, 142.4, 146.2, 146.5, 147.1,
151.9, 157.0, 174.6; MS (ESI): 696 [M+H]+.
pound 13h, 340 mg in 93% yield. Mp: 181–184 °C, ½a D25
ꢁ
+80.5 (c
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.54–2.62 (dd, 1H,
J = 5.0, 5.0 Hz), 2.90–3.03 (m, 1H), 3.73 (s, 6H), 3.74 (s, 6H), 3.77
(s, 3H), 3.88 (s, 3H), 3.89–3.90 (m, 1H), 4.43–4.50 (m, 2H), 5.25–
5.30 (m, 1H), 5.82 (d, 1H, J = 6.9 Hz), 5.94–5.98 (dd, 2H, J = 1.1,
1.1 Hz), 6.22 (s, 2H), 6.38 (s, 2H), 6.45 (s, 1H), 6.73 (s, 1H), 7.40–
7.43 (m, 2H), 7.77–7.81 (m, 2H), 8.04–8.09 (m, 1H); 13C NMR
(75 MHz, CDCl3): d 36.7, 40.4, 43.1, 47.7, 55.7, 55.8, 59.8, 60.0,
68.5, 101.2, 107.0, 108.1, 109.2, 109.3, 122.6, 123.6, 129.6, 130.0,
131.7, 132.1, 135.7, 136.1, 136.2, 137.0, 137.7, 138.7, 146.5,
147.2, 147.4, 151.9, 153.2, 167.4, 174.5; MS (ESI): 755 [M+H]+.
5.30. 4b-(E)-2-(2-Methoxyphenyl)-3-(2-nitrophenyl)acrylamido
podophyllotoxin (13l)
This compound 13l was prepared by the method described 13a,
employing 12 (200 mg, 0.48 mmol), acrylic acid 10l (170 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
5.27. 4b-(E)-3-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)
acrylamido podophyllotoxin (13i)
pound 13l, 320 mg in 95% yield. Mp: 187–190 °C, ½a D25
ꢀ59.9 (c
ꢁ
This compound 13i was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10i (196 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.47–2.54 (dd, 1H,
J = 5.2, 2.2 Hz), 2.83–3.02 (m, 1H), 3.73 (s, 6H), 3.73 (s, 6H), 3.76
(s, 3H), 3.83 (s, 3H), 4.02 (t, 1H, J = 9.8, 9.8 Hz), 4.40–4.50 (m,
2H,) 5.27–5.33 (m, 1H), 5.54 (d, 1H, J = 6.7 Hz), 5.94 (d, 2H,
J = 3.0 Hz), 6.20 (s, 2H), 6.39 (s, 1H), 6.70–6.92 (m, 4H), 7.17–7.33
(m, 5H), 8.03 (s, 1H); 13C NMR (75 MHz, CDCl3): d 36.6, 40.5,
43.1, 47.1, 55.2, 55.7, 59.8, 68.3, 101.2, 108.0, 108.9, 109.3, 111.0,
120.3, 124.3, 128.9, 129.8, 130.3, 131.2, 131.5, 131.6, 131.8,
132.0, 133.2, 135.7, 136.2, 136.4, 146.5, 147.1, 147.7, 151.9,
157.2, 167.6, 174.6; MS (ESI): 695 [M+H]+.
pound 13i, 320 mg in 94% yield. Mp: 128–130 °C, ½a D25
ꢀ26.4 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.55–2.66 (dd, 1H,
J = 4.4, 5.1 Hz), 2.87–3.07 (m, 1H), 3.74 (s, 12H), 3.78 (s, 3H), 380
(s, 3H), 3.91 (s, 3H), 4.25–4.34 (m, 1H), 4.45–4.55 (m, 2H), 5.32
(t, 1H), 5.69 (d, 1H, J = 6.6 Hz), 5.96 (d, 2H, J = 4.4 Hz), 6.26 (s,
2H), 6.43 (s, 2H), 6.48 (s, 1H), 6.69–6.74 (m, 3H), 6.99 (d, 2H,
J = 8.8 Hz), 7.78 (s, 1H); 13C NMR (75 MHz, CDCl3): d 36.7, 40.2,
40.3, 43.1, 47.7, 55.0, 55.6, 59.7, 59.9, 68.6, 101.1, 104.1, 107.0,
108.0, 109.2, 113.5, 127.2, 130.1, 131.2, 132.0, 133.6, 134.2,
135.7, 136.2, 137.0, 146.5, 147.1, 151.9, 153.0, 159.2, 167.8,
174.5; MS (ESI): 740 [M+H]+.
5.31. 4b-(E)-3-(4-Hydroxy-3-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13m)
This compound 13m was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 11m (220 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
5.28. 4b-(E)-3-(3-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)
acrylamido podophyllotoxin (13j)
pound 13m, 300 mg in 82% yield. Mp: 134–137 °C, ½a D25
ꢀ12.9 (c
ꢁ
This compound 13j was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10j (196 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.55–2.63 (dd, 1H,
J = 4.5, 4.5 Hz), 2.86–3.00 (m, 1H), 3.53 (s, 3H), 3.73 (s, 6H), 3.75
(s, 9H), 3.81 (s, 3H), 4.23–4.31 (m, 1H), 4.39–4.48 (m, 2H), 5.30
(t, 1H), 5.72 (d, 1H, J = 6.7 Hz), 5.95 (d, 2H, J = 3.0 Hz), 6.14–6.24
(m, 3H), 6.34–6.47 (m, 4H), 6.70–6.77 (m, 3H), 7.68 (s, 1H); 13C
NMR (75 MHz, CDCl3): d 36.8, 40.4, 43.1, 47.8, 54.6, 55.6, 55.8,
59.8, 60.0, 68.6, 101.1, 102.8, 107.2, 108.0, 109.1, 109.2, 109.4,
112.6, 115.0, 121.1, 124.7, 126.0, 130.1, 131.4, 131.6, 132.1,
132.4, 133.3, 135.2, 135.7, 136.2, 137.0, 146.5, 146.7, 147.1,
147.3, 151.9, 153.2, 167.6, 174.5; MS (ESI): 756 [M+H]+.
pound 13j, 310 mg in 88% yield. Mp: 187–190 °C, ½a D25
ꢀ69.9 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.51–2.58 (dd, 1H,
J = 4.8, 4.8 Hz), 2.85–3.01 (m, 1H), 3.71 (s, 3H), 3.72 (s, 6H), 3.75
(s, 6H), 3.76 (s, 3H), 3.86 (s, 3H), 4.40–4.48 (m, 2H), 5.22–5.37
(m, 3H), 5.93 (d, 2H, J = 8.0 Hz), 6.20 (s, 2H), 6.36–6.44 (m, 4H),
6.66 (d, 1H, J = 8.8 Hz), 6.72 (s, 1H), 6.93–7.02 (m, 2H), 7.70 (s,
1H); 13C NMR (75 MHz, CDCl3): d 36.8, 40.4, 43.1, 47.8, 55.0,
55.7, 55.8, 59.8, 60.0, 68.6, 101.2, 106.5, 107.0, 108.0, 109.2,
109.3, 113.5, 113.7, 127.3, 130.1, 131.2, 131.3, 132.0, 132.1,
132.2, 133.7, 134.2, 135.7, 136.2, 137.1, 138.5, 146.5, 147.1,
151.9, 153.1, 159.2, 159.9, 167.9, 174.6; MS (ESI): 740 [M+H]+.
5.32. 4b-(E)-3-(3-Hydroxy-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamido podophyllotoxin (13n)
This compound 13n was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 11n (220 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
5.29. 4b-(E)-2-(2-Methoxyphenyl)-3-(4-nitrophenyl)acrylamido
podophyllotoxin (13k)
pound 13n, 320 mg in 88% yield. Mp: 145–147 °C, ½a D25
ꢀ60.9 (c
ꢁ
This compound 13k was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 10k (170 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (2:8) afford the pure com-
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.52–2.63 (dd, 1H,
J = 4.6, 4.6 Hz), 2.85–3.02 (m, 1H), 3.75 (s, 9H), 3.78 (s, 3H), 389
(s, 6H), 4.24–4.33 (dd, 1H, J = 4.6, 4.6 Hz), 4.43–4.52 (dd, 2H,
J = 6.2, 6.2 Hz), 5.31 (t, 1H), 5.67 (d, 1H, J = 7.8 Hz), 5.97 (d, 2H,
J = 4.6 Hz), 6.24 (s, 2H), 6.42 (s, 2H), 6.47 (s, 1H), 6.52–6.70 (m,
3H), 6.75 (s, 1H), 7.68 (s, 1H); 13C NMR (75 MHz, CDCl3): d 36.7,
40.4, 43.1, 47.7, 55.3, 55.6, 55.7, 59.8, 60.0, 68.6, 101.1, 107.1,
108.0, 109.1, 109.2, 111.3, 116.6, 121.9, 127.5, 130.2, 131.1,
pound 13k, 320 mg in 95% yield. Mp: 181–183 °C, ½a D25
ꢀ42.5 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.42–2.51 (dd, 1H,
J = 4.7, 4.7 Hz), 2.86–3.01 (m, 1H), 3.74 (s, 6H), 3.77 (s, 3H), 383

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
4599
132.0, 133.5, 134.5, 135.7, 136.2, 137.1, 145.7, 146.5, 147.1, 148.0,
151.9, 152.9, 168.0, 174.5; MS (ESI): 778 [M+Na]+.
modified Eagle’s medium (DMEM) (Invitrogen), supplemented
with 10% fetal calf serum and 100 U/ml Pencillin and 100 g/ml
l
streptomycin sulfate (Sigma). The cell line was maintained at
37 °C in a humidified atmosphere containing 5% CO2 in the
incubator.
5.33. 4b-(E)-3-(4-Hydroxyphenyl)-2-(3,4,5-trimethoxyphenyl)
acrylamido podophyllotoxin (13o)
This compound 13o was prepared by the method described for
13a, employing 12 (200 mg, 0.48 mmol), acrylic acid 11o (188 mg,
0.57 mmol), EDCI (108 mg, 0.57 mmol) and HOBt (88 mg,
0.57 mmol). The crude product was purified by column chroma-
tography with ethyl acetate/hexane (3:7) afford the pure com-
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. In this assay MCF-7 cells were seeded in a 96-well plate at
a density of 10,000 cells/well. After overnight incubation cells were
treated with compounds etoposide, combretastatin (CA-4), podo-
pound 13o, 330 mg in 94% yield. Mp: 137–140 °C, ½a D25
ꢀ46.9 (c
ꢁ
0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.52–2.64 (dd, 1H,
J = 5.1, 5.1 Hz), 2.84–3.03 (m, 1H), 3.71 (s, 6H), 3.74 (s, 6H), 3.77
(s, 3H), 3.85 (s, 3H), 4.23–4.32 (m, 1H), 4.40–4.51 (m, 2H), 5.29
(t, 1H), 5.76 (d, 1H, J = 6.6 Hz), 5.96 (d, 2H, J = 2.9 Hz), 6.22 (s,
2H), 6.38 (s, 2H), 6.44 (s, 1H), 6.75 (s, 1H), 6.91 (d, 2H, J = 8.8 Hz),
7.03 (d, 2H, J = 8.8 Hz); 13C NMR (75 MHz, CDCl3): d 36.7, 40.4,
43.1, 47.7, 55.6, 55.7, 59.8, 60.0, 68.6, 101.2, 107.1, 108.1, 109.2,
109.3, 121.5, 130.1, 130.6, 132.1, 132.8, 133.3, 135.7, 136.2,
136.4, 137.2, 146.5, 147.1, 150.0, 151.9, 152.9, 167.8, 169.0,
174.5; MS (ESI): 764 [M+K]+.
phyllotoxin (Podo), 10k, 13j, 13k and 13l at 2 and 4
tion and incubated 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
lM concentra-
l
lL
extraction buffer. The optical density (OD) was read at 570 nm
with micro plate reader.
9. Cell cycle analysis
5 ꢃ 105 MCF-7 cells were seeded in 60 mm dish and were al-
6. Procedure of the SRB-assay
lowed to grow for 24 h, 2 lM concentration of etoposide, CA-4
The synthesized compounds (13a–o) 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 sulforhodamine
B (SRB) protein assay has been used to estimate cell viability or
growth. The cell lines were grown in DMEM medium containing
(6), podophyllotoxin (Podo), 10k and 13j, 13k, 13l compounds
were added to the culture media, and the cells were incubated
for an additional 24 h. Cells were harvested with Trypsin–EDTA,
fixed with ice-cold 70% ethanol at 4 °C for 30 min, washed with
PBS and incubated with 1 mg/ml RNAase solution (Sigma) at
37 °C for 30 min. Cells were collected by centrifugation at
10% fetal bovine serum and 2 mM L-glutamine and were inoculated
into96wellmicrotiterplatesin90 mLatplatingdensitiesdepending
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 experimental drugs. Aliquots of 10 mL of
the drug dilutions were added to the appropriate microtiter wells al-
ready containing 90 mL of cells, resulting in the required final drug
concentrations. For each compound four concentrations (0.1, 1, 10
2000 rpm for 5 min and further stained with 250 lL of DNA stain-
ing 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 contents of 20,000 events were measured by flow cytom-
eter (DAKO CYTOMATION, Beckman Coulter, Brea, CA). Histograms
were analyzed using Summit Software.
and 100 lM) were evaluated and each was done in triplicate wells.
Plates were incubated further for 48 h and assay was terminated
by the addition of 50 mL of cold trichloroacetic acid (TCA) (final con-
centration,10% TCA) and incubated for 60 min at 4 °C. The plates
were washed five times with tap water and air dried. Sulforhoda-
mine 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 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 absor-
bance was read on an ELISA plate reader at a wavelength of
540 nm 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. Per-
centage growth was expressed as the (ratio of average absorbance
of the test well to the average absorbance of the control wells) ꢂ 100.
Growth inhibition of 50% (GI50) was calculated from [(Ti ꢀ Tz)/
10. Comet assay (single cell gel electrophoresis)
The MCF-7 cells were seeded in 60 mm dishes and were treated
with compounds such as etoposide, CA-4 (6), podophyllotoxin
(Podo), 10k and the most effective hybrid (13k) at a concentration
for 2 lM concentration for 24 h. Here we have employed the pro-
tocol of Feridoun karimi Busheri et al., 2010 with slight modifica-
tion. Briefly, 20,000 cells were embedded in agarose and
deposited in microscope slides. The slides were incubated 45 min
in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10,
1% SDS, 1% Triton X-100) and incubated on ice for 30 min followed
by immersion in an alkaline solution (300 mM, 1 mM Na2EDTA) for
30 min. Electrophoresis was carried out for 20 min at 40 V in 0.5%
TBE buffer (pH 8). During electrophoresis the damaged DNA mi-
grates away from the nucleus towards anode. Slides were then
stained with a 20 mg/ml Sybr green I solution for 30 min. Images
from were taken in fluorescent microscope at 20ꢃ.
(C ꢀ Tz)] ꢂ 100 1 50, which is the drug concentration resulting in a
4
50% reduction in the net protein increase (as measured by SRB stain-
ing) in control cells during the drug incubation. Where, Tz1 4 Optical
density at time zero, OD of control1 4 C, and OD of test growth in the
11. c-H2AX staining
presence of drug 1 Ti.
4
MCF-7 breast cancer cells were seeded on cover slips and trea-
ted with as etoposide, CA-4 (6), podophyllotoxin (Podo), 10k and
the most effective hybrid (13k) at concentration of 2 M for 24 h.
7. Cell culture
l
After treatment, cover slips were fixed with paraformaldehyde
solution (4% in 1ꢃ PBS) for 20 min at room temperature. Cell per-
meabilization was achieved by administration of a TritonX-100
The human breast cancer cell line MCF-7 was purchased from
American type culture collection was maintained in Dulbecco’s

4600
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 4589–4600
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anti
c-H2AX (1:100) antibody at room temperature for 2 h. The
slides were washed three times each of 5 min with PBST and incu-
bated with a FITC-conjugated anti-rabbit secondary antibody
(Jackson Immuno Research Laboratories Inc., Pennsylvania, USA)
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Acknowledgments
One of the authors P.S. is thankful to UGC, New Delhi for the
award of Senior Research Fellowship.
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