4β-[(4-Alkyl)-1,2,3-triazol-1-yl] podophyllotoxins as anticancer compounds: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.02.016

A series of 4β-[(4-alkyl)-1,2,3-triazol-1-yl] podophyllotoxin derivatives were designed in silico, synthesised by employing click chemistry approach, and evaluated for cytotoxicity against a panel of human cancer cell lines (SF-295, A-549, PC-3, Hep-2, HC

Full text of DOI:10.1016/j.ejmech.2011.02.016

European Journal of Medicinal Chemistry 46 (2011) 1983e1991
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4b-[(4-Alkyl)-1,2,3-triazol-1-yl] podophyllotoxins as anticancer compounds:
Design, synthesis and biological evaluation
,b,
Doma Mahendhar Reddy b, Jada Srinivas a, Gousia Chashoo b, Ajit K. Saxena b, H.M. Sampath Kumar a
*
a Organic Division-I, Indian Institute of Chemical Technology, Hyderabad 500007, India
b Synthetic and Biological Chemistry Division, Indian Institute of Integrative Medicine, Canal road, Jammu 180001, 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 4b-[(4-alkyl)-1,2,3-triazol-1-yl] podophyllotoxin derivatives were designed in silico, synthesised
Received 11 November 2010
Received in revised form
11 February 2011
Accepted 11 February 2011
Available online 24 February 2011
by employing click chemistry approach, and evaluated for cytotoxicity against a panel of human cancer cell
lines (SF-295, A-549, PC-3, Hep-2, HCT-15 and MCF-7). Majority of the compounds proved to be more
potent than etoposide and select compounds exhibited significant anticancer activity with IC50 values in
the range of 0.001e1 mM. DNA fragmentation and flow-cytometric results reveals that 4b-[(4-alkyl)-1,2,3-
triazol-1-yl] podophyllotoxin derivatives induce dose dependent apoptosis. Docking experiments showed
a good correlation between their calculated interaction energies with the topoisomerase-II and the
observed IC50 values of all these compounds.
Keywords:
Podophyllotoxin
Anticancer activity
Cell cycle analysis
Docking study
Topoisomerase-II
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapeutic use of 2 and 3 is often hindered by problems such as
acquired drug-resistance and poor water solubility. To get more
Podophyllotoxin (1), is a most abundant naturally occurring
cyclolignan, mainly isolated from Podophyllum peltatum and podo-
phyllum hexandrum [1,2]. Podophyllotoxin has cathartic, antirheu-
matic and antiviral properties but its antimitotic activity has proved
to be the most attractive for researchers [3]. Podophyllotoxin is
known as an antimicrotubule agent acting at the colchicine-binding
site on tubulin [4]. Due to severe toxicity of 1, it is not being used as
an anticancer drug, but it’s semi synthetic derivatives etoposide
and teniposide (Fig. 1) are clinically useful drugs against various
cancers, including small cell lung cancer, testicular carcinoma,
lymphoma, and Kaposi’s sarcoma [5e12]. The chemical modifica-
tions that led to etoposide, teniposide and other derivatives, also
lead to the change in the mechanism of action of these ligands
wherein podophyllotoxin act as antimicrotubule agent whereas its
aforementioned derivatives act as topoisomerase-II inhibitors [13].
These derivatives block the catalytic activity of DNA topoisomerase-
II by stabilizing a cleavage enzymeeDNA complex in which the DNA
is cleaved and covalently linked to the enzyme. However, the
potent analogues and to overcome drug-resistance recently several
complex and more diverse analogues like Etopophos (4), GL-331,
TOP-53, NK-611, NPF etc. have been synthesised (Fig. 1). Etopophos,
is a water-soluble prodrug of 2, is readily converted in vivo to the
active drug, 2 and exhibits similar pharmacological and pharma-
cokinetic profiles that of 2. NK-611, NPF and GL-331 are presently
under clinical trial. According to structureeactivity relationship
(SAR) of podophyllotoxin, trans-lactone, 4
demethyl moieties were essential to maintain the anticancer
activity as topoisomerase-II inhibitors [14]. Particularly 4 -N-
b
-substituted and 40-
b
substituted derivatives of podophyllotoxin gained much impor-
tance owing to their improved cytotoxicity.
In recent years, we have been working on the chemical trans-
formation of podophyllotoxin and focused libraries of potent aniline,
phenol, thiophenol and carbohydrate based 1,2,3-triazole deriva-
tives have been generated, some of which exhibited significant
anticancer activity [15e17]. Even though aromatic substitution on
the triazole moiety yielded podophyllotoxin analogues with good
cytotoxicity, our recent docking studies revealed that 1,2,3-triazole
derivatives with various aliphatic substituent’s in triazole moiety
showed better binding ability to topoisomerase-II enzyme than
* Corresponding author. Synthetic and Biological Chemistry Division, Indian
Institute of Integrative Medicine, Canal road, Jammu 180001, India. Tel.: þ91
9912901010; fax: þ91 191 2569333.
etoposide. This prompted us to synthesise a series of 4b-[(4-alkyl)-
1,2,3-triazol-1-yl] podophyllotoxin derivatives using click reaction.
Click chemistry enables a modular approach to generate these novel
E-mail address: hmskumar@gmail.com (H.M. Sampath Kumar).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.016

1984
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
R2
R1
O
HO
N
O
O
N
N
N3
OH
CuSO4.5H2O
Sodium ascorbate
O
OR2
O
O
O
O
R2
O
O
O
O
O
O
O
t-BuOH:H2O (1:2)
O
11
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
MeO
OMe
OR1
OR1
OMe
OR3
9. R1 = CH3
10. R1 = H
12 to 29
1. Podophyllotoxin
R3
H
R1
R2
Scheme-1. Click-chemistry strategy for the synthesis of 4b-[(4-alkyl)-1,2,3-triazol-1-
yl] podophyllotoxin derivatives.
Etoposide
Teniposide
Etopophos
CH3
2.
H
H
H
H
3.
4.
S
hydrolysis with H2O/Me2CO/BaCO3 and reaction with NaN3 in TFA.
The azides 9 and 10 obtained were allowed to react with various
terminal alkynes, in the presence of CuSO4$5H2O and sodium
ascorbate, in t-butyl alcohol and water (1:2) at room temperature
O
P
HO
CH3
OH
5.
CH3
H
NMe2
NK-611
to yield selectively 4b-[(4-alkyl)-1,2,3-triazol-1-yl] derivatives in
excellent yields (>96%). Using this click-chemistry protocol
a focused library of analogues with different substitution has been
generated (Table 1).
R
HN
All the products were characterized by 1H NMR, 13C NMR, IR,
ESI-MS. In the 1H NMR spectra, the formation of triazoles was
confirmed by the resonance of HeC(5) of the triazole ring in the
aromatic region. The structure was further supported by the 13C
NMR spectra, which showed the C-atom signals corresponding to
triazole derivatives. ESI-MS of all compounds showed [M þ Na] or
[M þ 1].
N
Me
O
O
O
O
O
O
O
O
MeO
OMe
MeO
OMe
OH
OH
8. TOP-53
6. R = NO2 GL-331
7. R = F NPF
2.2. Evaluation of biological activity
2.2.1. Anticancer activity
Fig. 1. Structures of some podophyllotoxin derivatives.
The in vitro cytotoxicity of all the compounds was evaluated
against a panel of six human cancer cell lines viz., SF-295 (Neuro-
blastoma), A-549 (Lung), PC-3 (Prostate), Hep-2 (Liver), HCT-15
(Colon) and MCF-7 (Breast). Etoposide was taken as reference
compound. The IC50 values derived from in vitro screening studies
revealed that all the compounds posses significant cytotoxicity
against SF-295, PC-3, Hep-2, HCT-15 and MCF-7 cancer cell lines
(Table 2). However, a lesser activity was observed against A-549
cancer cell line. Compounds 12, 13, 20, 21, 22 and 29 showed
pharmacophores utilizing collection of reliable chemical reactions
[18]. Of particular interest is the Huisgen [3 þ 2] cycloaddition
between a terminal alkyne and an azide to generate substituted
1,2,3-triazoles [19]. All the 4b-[(4-alkyl)-1,2,3-triazol-1-yl] podo-
phyllotoxin derivatives were screened for anticancer activity against
a panel of six human cancercell lines. Most of the triazole derivatives
exhibited better cytotoxicity than etoposide. Podophyllotoxin
derivatives are known to exert its anti-tumour effect by promoting
programmedcell death (apoptosis) [20]. DNA fragmentation and cell
cycle analysis have also been evaluated on MCF-7 cell line. From the
IC50 values and docking score it is clear that compounds which
revealed good binding interaction with topoisomerase-II exhibited
better cytotoxicity. Induction of apoptosis by these ligands has been
confirmed by cell cycle and DNA fragmentation analysis.
Table 1
Various 4
score.
b-[(4-alkyl)-1,2,3-triazol-1-yl] podophyllotoxin derivatives and its docking
Entry
R1
R2
Yield%a
Glide score
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
Decyl
CH2OH
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
96
97
98
98
98
98
99
96
99
98
96
97
99
96
98
96
99
97
ꢀ3.53
ꢀ3.56
ꢀ3.25
ꢀ3.21
ꢀ3.38
ꢀ3.21
ꢀ3.35
ꢀ3.41
ꢀ4.08
ꢀ4.32
ꢀ3.45
ꢀ3.16
ꢀ3.24
ꢀ3.37
ꢀ3.36
ꢀ3.33
ꢀ3.23
ꢀ4.64
ꢀ3.02
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme-1,
podophyllotoxin derivatives were synthesised by the cycloaddition
reaction of C4 -azido podophyllotoxin, 9 and C4
-azido-40-O-
4b-[(4-alkyl)-1,2,3-triazol-1-yl]
b
b
demethyl podophyllotoxin, 10 with various terminal aliphatic
alkynes, 11. Compounds 9 and 10 were synthesised according to
literature procedures [21,22]. Compound 9 was obtained by the
reaction of podophyllotoxin with CH3SO3H and NaI in acetonitrile
followed by hydrolysis with H2O/Me2CO/BaCO3 and reaction with
NaN3 in TFA. Compound 10 was obtained by the reaction of podo-
phyllotoxin with CH3SO3H and NaI in dichloromethane followed by
Decyl
CH2OH
a
Isolated yields.

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
1985
Table 2
IC50 values
derivatives.
(
m
M) of various
4
b
-[(4-alkyl)-1,2,3-triazol-1-yl] podophyllotoxin
Entry
SF-295
A-549
PC-3
Hep-2
0.06
HCT-15
MCF-7
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2
1.8
3.6
8.1
24
10
25
35
39
82
39
35
0.03
5.1
6.6
8.4
6.4
23
0.4
0.03
11
11
27
45
>100
29
0.4
1.4
9.8
9.5
8.6
6.4
9.3
5.7
0.01
0.04
5.7
20
14
19
13
11
11
0.6
19
1.6
9.6
7.7
10
9.8
18
36
14
>100
>100
>100
17
27
35
26
26
45
>100
30
17
6.2
2.1
4.8
2.3
21
4.3
18
10
5.5
0.06
0.06
0.2
12
4.7
8.7
5.8
5.1
19
5.8
0.06
0.05
2.9
15
1.9
0.8
34
2.1
11
15
13
15
18
17
21
>100
6
3.8
19
6.7
2.15
Fig. 2. Agarose gel electrophoresis of DNA extracted from MCF-7 cells. The figure
represents MCF-7 cells treated with compounds 12 and 13 for 24 h. DNA from the cells
was extracted and electrophoresed in 1% agarose gel and visualized by ethidium
bromide staining under UV illumination. Lane 1, 2, and 3 are represent compound 12 at
5.7
13
15
18
5.62
8.2
17.5
13.5
7.15
0, 1, 5 mM. Lane 4: treated with compound 13 (5 mM).
DNA fragmentation analysis revealed that compound 12 and 13
induced a discrete ladder pattern in MCF-7 cell line at 1 M after
24 h of incubation (Fig. 2). The cell cycle analysis of compound 12
and 13 showed dose dependent increases in the sub-G1
(apoptotic) population (Fig. 3) in MCF-7 cell line after 24 h. Cell
cycle analysis showed that the ratio of apoptosis in MCF-7 cells for
significant cytotoxicity against PC-3, Hep-2, HCT-15 and MCF-7 cell
lines. Compound 12 showed more potent cytotoxicity against SF-
m
295 and PC-3 cell lines with IC50 values of 1.8 and 0.03
mM
a
respectively. Compound 13 was found to show highest cytotoxicity
against HCT-15 cell line with an IC50 value of 0.03. Compound 20
and 21 on the other hand showed highest cytotoxicity against HEP-
compound 12 at 1 and 5 mM was 69.34% (24 h) and 80.10% (24 h)
2 cell line with IC50 values of 0.05 mM both. The IC50 values of the
respectively, and for compound 13 at 1 and 5 mM it was found to
compounds revealed that methyl, ethyl and hydroxyl groups in
triazole moiety increases the anticancer activity, further, it was
found that increasing alkyl chain length of the compounds
decreases the anticancer activity.
be 55.34% and 78.16% respectively.
2.3. Docking studies
To study the molecular basis of interaction and affinity of
binding of the podophyllotoxin analogues, all the ligands were
docked into the ATPase domain of Topoisomerase-II. ATPase
domain is the probable binding site for etoposide as reported
earlier [23,24]. Docking was done using Glide module of Schro-
dinger software. Docking results of these ligands are given in
Table 1. Compounds 12, 13, 20, 21, 22 and 29 showed good inter-
action with TP-II and these results were matching with wet lab
2.2.2. DNA fragmentation and cell cycle analysis
DNA fragmentation, which is
a typical hallmark of the
apoptotic cell death was analysed in human breast cancer cell line
MCF-7. From the in vitro cytotoxicity studies it was found that
compounds 12 and 13 significantly inhibits the growth of human
breast cancer cell line MCF-7, and was therefore studied further to
determine the mechanism of cell death in the same cell line. The
Fig. 3. DNA Cell Cycle analysis for compound 12 and 13 (1 and 5 mM).

1986
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
findings. Compound 20 was found to have a score of ꢀ4.08 with 3
strong Hbonds with Arg98 and 2 Hbonds with Ser149 with
strengths of 2.14 Å and 1.83 Å. Also they were found to have strong
hydrophobic contacts with the residues of active site. Compound 21
was having single strong hydrogen bond with strength of 2.01 Ao
with Lys131 along with strong hydrophobic contacts; this signifies
a strong binding of the molecules to the receptor. The slight vari-
ation in dock score of compound 12 and 13 was observed with
scores of ꢀ3.53 and ꢀ3.56 although both were having 2 hydrogen
bonds with Arg98 with almost same strengths but the difference
lies in their hydrophobic contacts. Compound 13 was having 231
contacts whereas 12 was having 220 contacts. The orientation of
both the molecules was almost identical just the hydrophobic
interactions were less in 12 as compared to 13. The ligand receptor
interactions of compound 29 and 21 are shown in Fig.4 and the
interactions of compound 12 and 13 are shown in Fig. 5.
Fig. 4. Interaction of compound 29 and 21 with the residues within 5A of the receptor cavity.

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
1987
Fig. 5. Interactions of compound 12 and 13 with the residues within 5A of the receptor cavity.

1988
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
Fromtheresultsitwasapparentthattheinsilicofindings werewell
correlated with the data obtained through in vitro cytotoxicity assay.
137.60, 134.33, 133.06, 125.11, 110.66, 109.05, 108.90, 101.90, 67.51,
60.68, 58.70, 56.77, 43.81, 42.00, 37.55, 31.23, 27.66, 22.45, 13.74; IR
(KBr): 3424.55, 2926.56, 1779.20, 1589, 1505.97, 1485.27, 1236.15,
1125.92, 1036, 1001.95, 931.84 cmꢀ1; ESI-MS: 530 (M þ Na).
3. Conclusions
In conclusion, a series of 4
b
-[(4-alkyl)-1,2,3-triazol-1-yl] podo-
4.1.3. 4b-[(4-Butyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin (14)
phyllotoxin derivatives were designed, synthesised and screened
for anticancer activity against a panel of six human cancer cell lines.
Most of the compounds exhibited improved anticancer activity as
compared to etoposide and found to be pro-apoptotic molecules.
White solid; mp: 120e121 ꢂC; ½a D25
ꢃ
ꢀ33.3 (c 0.66, CHCl3); 1H NMR
(500 MHz, CDCl3) d 7.02 (s, 1H), 6.66 (s, 1H), 6.62 (s, 1H), 6.34 (s, 2H),
6.07 (d, J ¼ 4.66 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.77 (d, J ¼ 4.83 Hz,
1H), 4.4e4.37 (t, J¼ 7.69 Hz,1H), 3.83(s, 3H), 3.78(s, 6H), 3.25e3.2(m,
2H), 3.11e3.09 (dd, J ¼ 8.99 Hz, 4.88 Hz, 1H), 2.7e2.67 (t, J ¼ 7.71 Hz,
2H), 1.68e1.65 (p, J ¼ 7.5 Hz, 2H), 1.60e1.56 (m, 2H), 0.95e0.93 (t,
4. Experimental
J ¼ 7.41 Hz, 3H); 13C NMR (CDCl3):
d 173.30, 152.80, 149.31, 148.73,
Melting points were recorded on Buchi Melting point apparatus
D-545 and IR spectra (KBr) on Bruker Vector 22 instrument. NMR
spectra were recorded on Bruker DPX500 instrument in CDCl3 with
TMS as an internal standard. Chemical shift values are reported in
137.53, 134.34, 133.10, 124.99, 110.42, 108.90, 108.01, 101.80, 67.51,
60.75, 58.40, 56.21, 43.68, 41.73, 37.22, 31.38, 25.38, 22.41, 14.05; IR
(KBr): 3424.90, 2926.66, 1779.90, 1590, 1505.97, 1484.97, 1236.15,
1125.90, 1036.11, 1002.01, 931.84 cmꢀ1; ESI-MS: 522 (M þ 1).
d
(ppm) and coupling constants in hertz. Mass spectra were
recorded on ESI-esquire 3000 Bruker Daltonics instrument. The
progress of all reactions was monitored by TLC on 2 ꢁ 5 cm pre-
coated silica gel 60 F254 plates of thickness 0.25 mm (Merck). The
chromatograms were visualized under UV 254e366 nm and iodine.
Purity was checked with Waters analytical HPLC.
4.1.4. 4b-[(4-Pentyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
(15)
Whitesolid; mp: 153e155 ꢂC; ½a D25
ꢃ
ꢀ36.3 (c 0.56, CHCl3); 1H NMR
(500 MHz, CDCl3) d 6.98 (s,1H), 6.65 (s,1H), 6.62 (s,1H), 6.32 (s, 2H),
6.06 (d, J ¼ 4.72 Hz,1H), 6.03 (s, 1H), 6.01 (s,1H), 4.73 (d, J ¼ 4.91 Hz,
1H), 4.40e4.38 (t, J ¼ 7.8 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 6H), 3.21e3.16
(m, 2H), 3.10e3.07 (dd, J ¼ 9.03, 4.92 Hz, 1H), 2.65e2.62 (t,
J ¼ 7.71 Hz, 2H), 1.64e1.60 (m, 2H), 1.31e1.24 (m, 4H), 0.87e0.84 (t,
4.1. Click chemistry-general procedure
To a solution of 1-pentyne (0.46 mmol) in t-butyl alcohol and
water (1:2, 8 mL) was added CuSO4$5H2O (0.46 mmol), sodium
J ¼ 7.45 Hz, 3H); 13C NMR (CDCl3):
d 173.31, 152.79, 149.29, 148.05,
137.51, 134.35, 133.08, 125.03, 110.42, 108.90, 108.19, 101.96, 67.52,
60.75, 58.38, 56.31, 43.68, 41.74, 37.22, 31.91, 29.35, 25.68, 22.33,
14.12; IR (KBr): 3424.95, 2926.54,1779.90,1589.81,1505.77,1485.27,
1236.45, 1126.02, 1036, 1001.99, 931.95 cmꢀ1; ESI-MS: 536 (M þ 1).
ascorbate (1.1 mmol) followed by
4b-azido-podophyllotoxin
(0.23 mmol). The reaction mixture was stirred at room temperature
for 8 h (to synthesise compound 12 and 21 reaction mixture was
stirred at 0 ꢂC for 1 h, after 1 h reaction was allowed to reach room
temperature). After completion, the reaction mixture was diluted
with 80 mL of water and extracted with chloroform (3 ꢁ 30 mL). The
combined extracts were washed with brine, dried over anhydrous
Na2SO4 and evaporated in vacuo. The crude product obtained was
washed with n-hexane to remove excess alkyne and recrystallized
with ether to yield the pure product (12e29). All the compounds
were >98% pure, purity as confirmed by HPLC analysis (RP-18
column 4 ꢁ 250 mm, Merck, with a UV/vIS detector, mobile phase
gradient mixture of methanol and water, flow rate of 0.8 mL/min).
4.1.5. 4b-[(4-Hexyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
(16)
White solid; mp: 116 ꢂC; ½a D25
ꢃ
ꢀ25 (c 0.44, CHCl3); 1H NMR
(500 MHz, CDCl3) d 6.90 (s,1H), 6.64 (s,1H), 6.61 (s,1H), 6.32 (s, 2H),
6.06 (d, J ¼ 4.72 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.71 (d, J ¼ 4.54 Hz,
1H), 4.42e4.39 (t, J ¼ 7.42 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 6H),
3.23e3.18 (m, 2H), 3.12e3.09 (dd, J ¼ 8.091 Hz, 4.81 Hz, 1H),
2.66e2.63 (t, J ¼ 7.90 Hz, 2H), 1.66e1.61 (m, 2H), 1.30e1.23 (m, 6H),
0.87e0.83 (t, J ¼ 7.63 Hz, 3H); 13C NMR (CDCl3):
d 173.08, 153.18,
149.55, 148.30, 137.60, 134.32, 133.50, 125.31, 110.63, 109.22, 108.98,
102.00, 67.53, 60.78, 58.77, 56.71, 43.95, 42.02, 37.50, 31.88, 29.37,
29.21, 25.87, 22.65, 14.00; IR (KBr): 3425.27, 2926.64, 1779.63,
1590.09, 1505.77, 1485.27, 1236.01, 1125.81, 1036.27, 1001.90,
931.91 cmꢀ1; ESI-MS: 550 (M þ 1).
4.1.1. 4b-[(4-Ethyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
(12)
White solid; mp: 149e150 ꢂC; ½a D25
ꢃ
ꢀ24.3 (c 0.45, CHCl3); 1H
NMR (500 MHz, CDCl3) d 6.93 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32
(s, 2H), 6.07 (d, J ¼ 4.79 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.76 (d,
J ¼ 4.98 Hz, 1H), 4.41e4.38 (t, J ¼ 7.77 Hz, 1H), 3.82 (s, 3H), 3.75 (s,
6H), 3.28e3.16 (m, 2H), 3.08e3.04 (dd, J ¼ 8.97 Hz, 4.98 Hz, 1H),
2.75e2.70 (t, J ¼ 7.59 Hz, 2H), 1.28e1.25 (t, J ¼ 7.54 Hz, 3H); 13C
4.1.6. 4b-[(4-Heptyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
(17)
White solid; mp: 112 ꢂC; ½a D25
ꢃ
ꢀ66.3 (c 0.5, CHCl3); 1H NMR
NMR (CDCl3):
d
173.33, 152.80, 149.32, 148.06, 137.52, 134.35, 133.11,
(500 MHz, CDCl3): d 6.81 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32 (s,
124.99, 110.44, 108.93, 108.20, 101.96, 67.54, 60.75, 58.46, 56.32,
43.69, 41.74, 37.22, 31.21, 29.68, 13.46; IR (KBr): 3424.95, 2926.66,
1779.70, 1589.47, 1505.77, 1485.17, 1236.15, 1125.91, 1036.07,
1001.91, 931.94 cmꢀ1; ESI-MS: 494.1 (M þ 1).
2H), 6.07 (d, J ¼ 4.63 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.82 (d,
J ¼ 4.42 Hz, 1H), 4.45e4.42 (t, J ¼ 7.28 Hz, 1H), 3.82 (s, 3H), 3.77 (s,
6H), 3.28e3.24 (m, 2H), 3.12e3.09 (dd, J ¼ 9.01 Hz, 4.72 Hz, 1H),
2.76e2.73 (t, J ¼ 7.66 Hz, 2H), 1.76e1.72 (m, 2H),1.30e1.25 (m, 10H),
0.86e0.83 (t, J ¼ 7.51 Hz, 3H); 13C NMR (CDCl3):
d 173.79, 152.04,
4.1.2. 4
b
-[(4-Propyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
149.41, 148.14, 137.80, 134.25, 133.23, 124.94, 110.50, 108.91, 108.40,
101.91, 67.51, 60.77, 58.62, 56.43, 43.73, 41.81, 37.25, 31.46, 29.70,
29.25, 28.94, 25.77, 22.49, 13.99; IR (KBr): 3428.5, 2926.36, 1780.27,
1589.77, 1505.27, 1485.55, 1236.01, 1126.06, 1036.27, 1001.90,
932.03 cmꢀ1; ESI-MS: 564 (M þ 1).
(13)
White solid; mp: 145 ꢂC; ½a D25
ꢃ
ꢀ28.8 (c 0.50, CHCl3); 1H NMR
(500 MHz, CDCl3) d 6.95 (s,1H), 6.65 (s,1H), 6.62 (s,1H), 6.32 (s, 2H),
6.07 (d, J ¼ 4.42 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.76 (d, J ¼ 4.89 Hz,
1H), 4.41e4.39 (t, J ¼ 7.28 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 6H),
3.26e3.18 (m, 2H), 3.08e3.05 (dd, J ¼ 9.06 Hz, 4.82 Hz, 1H),
2.68e2.65 (t, J ¼ 7.6 Hz, 2H), 1.70e1.66 (m, 2H), 0.97e0.94 (t,
4.1.7. 4b-[(4-Octyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin (18)
White solid; mp: 125 ꢂC; ½a D25
ꢃ
ꢀ49.2 (c 0.60, CHCl3); 1H NMR
J ¼ 7.33 Hz, 3H); 13C NMR (CDCl3):
d
173.28, 153.09, 149.48, 148.45,
(500 MHz, CDCl3): d 6.96 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32 (s,

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
1989
2H), 6.08 (d, J ¼ 4.81 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.81 (d,
J ¼ 4.72 Hz, 1H), 4.42e4.39 (t, J ¼ 7.21 Hz, 1H), 3.82 (s, 3H), 3.77 (s,
6H), 3.26e3.19 (m, 2H), 3.08e3.04 (dd, J ¼ 8.72 Hz, 4.72 Hz, 1H),
2.71e2.67 (t, J ¼ 7.36 Hz, 2H), 1.75e1.63 (m, 4H), 1.42e1.26 (m, 8H),
1765.9, 1611.09, 1515.57, 1484.21, 1228.05, 1108.97, 1034.63, 995.01,
926.64 cmꢀ1; ESI-MS: 494 (M þ 1).
4.1.12. 40-O-Demethyl-4
b-[(4-butyl)-1,2,3-triazol-1-yl]-4-
0.89e0.84 (t, J ¼ 7.54 Hz, 3H); 13C NMR (CDCl3):
d
173.52, 152.11,
desoxypodophyllotoxin (23)
149.39, 148.22, 137.68, 134.33, 133.63, 124.99, 110.45, 109.00, 108.60,
101.95, 67.55, 60.71, 58.66, 56.40, 43.76, 41.79, 37.30, 31.51, 29.79,
29.29, 29.11, 28.90, 25.69, 22.52, 13.98; IR (KBr): 3425.90, 2926.03,
1779.79, 1589.87, 1505.99, 1485.01, 1236.54, 1120.24, 1036.57,
1001.51, 932.06 cmꢀ1; ESI-MS: 590 (M þ Na).
White solid; mp: 124e125 ꢂC; ½a D25
ꢃ
ꢀ25.2 (c 0.5, CHCl3); 1H NMR
(500 MHz, CDCl3) d 7.00 (s,1H), 6.65 (s,1H), 6.62 (s,1H), 6.34 (s, 2H),
6.06 (d, J ¼ 4.90 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.74 (d, J ¼ 4.66 Hz,
1H), 4.40e4.37 (t, J ¼ 7.82 Hz, 1H), 3.78 (s, 6H), 3.23e3.18 (m, 2H),
3.10e3.07 (dd, J ¼ 8.72 Hz, 4.65 Hz, 1H), 2.69e2.66 (t, J ¼ 7.51 Hz,
2H), 1.66e1.63 (p, J ¼ 7.33 Hz, 2H), 1.58e1.54 (m, 2H), 0.94e0.92 (t,
4.1.8. 4
b
-[(4-Decyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
J ¼ 7.41 Hz, 3H); 13C NMR (CDCl3):
d 173.38, 149.31, 147.99, 146.66,
(19)
134.46, 133.34, 129.81, 125.05, 110.89, 109.55, 108.89, 101.93,67.46,
58.59, 56.27, 43.50, 41.85, 37.05, 31.28, 25.41, 22.68, 13.99; IR (KBr):
3387.96, 2927.66, 1766.29, 1611.23, 1515.97, 1483.39, 1227.95,
1109.67, 1034.93, 995.45, 926.99 cmꢀ1; ESI-MS: 508 (M þ 1).
White solid; mp: 108 ꢂC; ½a D25
ꢃ
ꢀ55.2 (c 0.4, CHCl3); 1H NMR
(500 MHz, CDCl3)
d 6.90 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32 (s,
2H), 6.07 (d, J ¼ 4.81 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.75 (d,
J ¼ 4.63 Hz, 1H), 4.42e4.39 (t, J ¼ 7.21 Hz, 1H), 3.82 (s, 3H), 3.77
(s, 6H), 3.26e3.19 (m, 2H), 3.08e3.04 (dd, J ¼ 8.72 Hz, 4.72 Hz,
1H), 2.71e2.67 (t, J ¼ 7.36 Hz, 2H), 1.75e1.63 (m, 4H), 1.42e1.26
(m, 10H), 0.89e0.84 (t, J ¼ 7.54 Hz, 3H); 13C NMR (CDCl3):
4.1.13. 40-O-Demethyl-4
b-[(4-pentyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (24)
White solid; mp: 168 ꢂC; ½a D25
ꢃ
ꢀ29.1 (c 0.45, CHCl3); 1H NMR
d
173.30, 152.82, 149.33, 148.07, 137.60, 134.37, 133.14, 125.09,
(500 MHz, CDCl3) d 7.0 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H),
110.43, 108.92, 108.29, 101.96, 67.54, 60.74, 58.49, 56.34, 43.70,
41.77, 37.22, 31.89, 29.69, 29.57, 29.36, 29.11, 28.77, 25.75, 22.68,
18.45, 14.11; IR (KBr): 3425.54, 2926.77, 1779.70, 1589.54, 1505.90,
1485.03, 1236.45, 1125.81, 1036.07, 1001.91, 932.09 cmꢀ1; ESI-MS:
606 (M þ 1).
6.06 (d, J ¼ 4.51 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.75 (d, J ¼ 4.72 Hz,
1H), 4.41e4.38 (t, J ¼ 7.91 Hz, 1H), 3.76 (s, 6H), 3.20e3.17 (m, 2H),
3.09e3.06 (dd, J ¼ 8.72 Hz, 4.88 Hz, 1H), 2.63e2.60 (t, J ¼ 7.79 Hz,
2H), 1.64e1.60 (m, 2H), 1.31e1.24 (m, 4H), 0.88e0.85 (t, J ¼ 7.45 Hz,
3H); 13C NMR (CDCl3):
d 173.35, 149.35, 148.02, 146.67, 134.50,
133.36, 129.82, 125.10, 110.92, 109.54, 108.90, 101.94, 67.55, 58.61,
56.57, 43.80, 41.90, 37.09, 31.554, 29.69, 25.72, 22.35, 13.99; IR
(KBr): 3388.16, 2927.67, 1765.30, 1610.99, 1515.52, 1483.60, 1228.71,
1109.47, 1034.53, 994.41, 927.07 cmꢀ1; ESI-MS: 522 (M þ 1).
4.1.9. 4b-[(4-Hydroxy methyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (20)
White solid; mp: 148e150 ꢂC; ½a D25
ꢃ
ꢀ45 (c 0.6, CHCl3); 1H NMR
(500 MHz, CDCl3) d 7.24 (s, 1H), 6.65 (s,1H), 6.62 (s, 1H), 6.32 (s, 2H),
6.07 (d, J ¼ 4.69 Hz,1H), 6.03 (s,1H), 6.01 (s, 1H), 4.75 (d, J ¼ 4.97 Hz,
1H), 4.41e4.38 (t, J ¼ 6.74 Hz, 1H), 3.82 (s, 3H), 3.75 (s, 6H), 3.74 (s,
2H), 3.28e3.16 (m, 2H), 3.08e3.04 (dd, J ¼ 8.97 Hz, 4.98 Hz, 1H), 13C
4.1.14. 40-O-Demethyl-4
b-[(4-hexyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (25)
White solid; mp: 124e126 ꢂC; ½a D25
ꢃ
ꢀ21.6 (c 0.5, CHCl3); 1H NMR
NMR (CDCl3):
d
173.04, 153.11, 149.62, 148.14, 137.75, 134.29, 133.28,
(500 MHz, CDCl3) d 6.93 (s,1H), 6.65 (s,1H), 6.61 (s,1H), 6.32 (s, 2H),
124.74, 110.64, 109.19, 108.83, 102.03, 67.47, 59.85, 58.94, 56.69,
43.88, 41.83, 37.39, 29.71; IR (KBr): 3330.21, 2926.75, 1763.60,
1613.10, 1517.54, 1483.50, 1233.36, 1110.90, 1033.63, 997.10,
930.11 cmꢀ1; ESI-MS: 496 (M þ 1).
6.06 (d, J ¼ 4.46 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.71 (d, J ¼ 4.63 Hz,
1H), 4.43e4.40 (t, J ¼ 7.75 Hz, 1H), 3.76 (s, 6H), 3.21e3.16 (m, 2H),
3.11e3.08 (dd, J ¼ 8.64 Hz, 4.89 Hz, 1H), 2.65e2.62 (t, J ¼ 8.10 Hz,
2H), 1.65e1.60 (m, 2H), 1.31e1.24 (m, 6H), 0.88e0.84 (t, J ¼ 7.77 Hz,
3H); 13C NMR (CDCl3):
d 173.28. 149.14, 148.17, 146.99, 135.05,
4.1.10. 40-O-Demethyl-4
b
-[(4-ethyl)-1,2,3-triazol-1-yl]-4-
133.54, 130.02, 125.41, 110.55, 108.96, 108.57, 101.95, 67.57, 58.65,
56.80, 43.75, 42.06, 37.38, 31.51, 29.24, 28.95, 25.78, 22.51, 13.93; IR
(KBr): 3388.21, 2927.12, 1765.42, 1610.90, 1515.54, 1483.33, 1228.91,
1109.45, 1035.03, 994.45, 926.97 cmꢀ1; ESI-MS: 558 (M þ 23).
desoxypodophyllotoxin (21)
White solid; mp: 160 ꢂC; ½a D25
ꢃ
ꢀ19.2 (c 0.54, CHCl3); 1H NMR
(500 MHz, CDCl3) d 6.92 (s,1H), 6.64 (s, 1H), 6.61 (s,1H), 6.31 (s, 2H),
6.06 (d, J ¼ 4.90 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.72 (d, J ¼ 4.81 Hz,
1H), 4.40e4.37 (t, J ¼ 7.72 Hz, 1H), 3.76 (s, 6H), 3.26e3.15 (m, 2H),
3.07e3.03 (dd, J ¼ 9.03 Hz, 4.72 Hz, 1H), 2.73e2.68 (t, J ¼ 7.66 Hz,
4.1.15. 40-O-Demethyl-4
b-[(4-heptyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (26)
2H), 1.26e1.23 (t, J ¼ 7.82 Hz, 3H); 13C NMR (CDCl3):
d
173.39,
White solid; mp: 118 ꢂC; ½a D25
ꢃ
ꢀ51 (c 0.75, CHCl3); 1H NMR
149.35,148.00,146.65,134.46,133.36,129.82,125.09,110.40,108.93,
108.80, 101.94, 67.72, 58.51, 56.45, 43.51, 41.89, 37.04, 31.22, 29.62,
13.34; IR (KBr): 3388.06, 2927.36, 1766.09, 1610.99, 1515.77,
1483.69, 1228.15, 1109.47, 1034.63, 995.41, 926.94 cmꢀ1; ESI-MS:
480 (M þ 1).
(500 MHz, CDCl3) d 6.85 (s,1H), 6.65 (s,1H), 6.62 (s,1H), 6.32 (s, 2H),
6.07 (d, J ¼ 4.88 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.78 (d, J ¼ 4.66 Hz,
1H), 4.44e4.41 (t, J ¼ 7.54 Hz, 1H), 3.78 (s, 6H), 3.27e3.23 (m, 2H),
3.10e3.07 (dd, J ¼ 8.72 Hz, 4.81 Hz,1H), 2.75e2.72 (t, J ¼ 7.51 Hz, 2H),
1.75e1.71 (m, 2H),1.30e1.25 (m,10H), 0.87e0.84 (t, J ¼ 7.90 Hz, 3H);
13C NMR (CDCl3):
d 173.28, 149.14, 148.17, 146.99, 135.05, 133.54,
4.1.11. 40-O-Demethyl-4
b
-[(4-propyl)-1,2,3-triazol-1-yl]-4-
130.02, 125.41, 110.55, 108.96, 108.57, 101.95, 67.57, 58.69, 56.66,
43.75, 41.96, 37.29, 31.51, 29.77, 29.30, 28.99, 25.70, 22.55, 13.96; IR
(KBr): 3388.01, 2927.77, 1766.3, 1610.99, 1515.54, 1482.9, 1228.99,
1109.15, 1034.93, 994.55, 926.90 cmꢀ1; ESI-MS: 550 (M þ 1).
desoxypodophyllotoxin (22)
White solid; mp: 154 ꢂC; ½a D25
ꢃ
ꢀ24.0 (c 0.46, CHCl3); 1H NMR
(500 MHz, CDCl3) d 6.90 (s,1H), 6.64 (s,1H), 6.61 (s,1H), 6.32 (s, 2H),
6.07 (d, J ¼ 4.63 Hz,1H), 6.03 (s,1H), 6.01 (s, 1H), 4.71 (d, J ¼ 4.66 Hz,
1H), 4.40e4.38 (t, J ¼ 7.36 Hz, 1H), 3.78 (s, 6H), 3.25e3.17 (m, 2H),
3.08e3.05 (dd, J ¼ 8.90 Hz, 4.91 Hz, 1H), 2.67e2.64 (t, J ¼ 7.81 Hz,
2H), 1.69e1.65 (m, 2H), 0.98e0.95 (t, J ¼ 7.21 Hz, 3H); 13C NMR
4.1.16. 40-O-Demethyl-4
b-[(4-octyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (27)
White solid; mp: 128e130 ꢂC; ½a D25
ꢃ
ꢀ45 (c 0.60, CHCl3);
d 6.94 (s,
(CDCl3):
d
173.12, 149.29, 148.02, 146.86, 134.95, 133.41, 129.90,
1H), 6.64 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H), 6.07 (d, J ¼ 4.91 Hz, 1H),
6.03 (s, 1H), 6.01 (s, 1H), 4.80 (d, J ¼ 4.46 Hz, 1H), 4.43e4.40 (t,
J ¼ 7.51 Hz, 1H), 3.78 (s, 6H), 3.25e3.18 (m, 2H), 3.08e3.04 (dd,
125.29, 110.40, 108.80, 108.49, 101.79, 67.42, 58.50, 56.66, 43.61,
41.92, 37.25, 31.16, 27.63, 22.38, 13.60; IR (KBr): 3388.54, 2926.96,

1990
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
J ¼ 8.96, 4.79 Hz, 1H), 2.70e2.66 (t, J ¼ 7.66 Hz, 2H), 1.74e1.63 (m,
4H), 1.41e1.25 (m, 8H), 0.88e0.83 (t, J ¼ 7.77 Hz, 3H); 13C NMR
with compound 12 and 13 at 1 mM for 24 h. Cells were harvested,
washed with PBS, pellets were dissolved in lysis buffer (10 mM
EDTA, 50 mM tris pH 8.0, 0.5% w/v) SDS and proteinase K (0.5 mg/
mL) and incubated at 50 ꢂC for 1 h. Finally the DNA obtained was
heated rapidly to 70 ꢂC, supplemented with loading dye and
immediately resolved on to 1.5% agarose gel at 50 V for 2e3 h.
(CDCl3):
d 173.40, 149.35, 147.99, 146.70, 134.52, 133.39, 129.86,
125.22, 110.40, 108.92, 107.98, 101.94, 67.56, 58.60, 56.57, 43.52,
41.88, 37.00, 31.81, 29.68, 29.26, 29.17, 28.76, 25.73, 22.63, 13.93; IR
(KBr): 3388.15, 2928.27, 1765.60, 1612.03, 1515.27, 1483.9, 1230.01,
1109.55, 1034.63, 995.12, 927.30 cmꢀ1; ESI-MS: 564 (M þ 1).
4.4. Cell cycle analysis
4.1.17. 40-O-Demethyl-4
b-[(4-decyl)-1,2,3-triazol-1-yl]-4-
desoxypodophyllotoxin (28)
Effect of Compound 12 and 13 on DNA content by cell cycle
White solid; mp: 115e116 ꢂC; ½a D25
ꢃ
ꢀ46.2 (c 0.4, CHCl3); 1H NMR
phase distribution was assessed using MCF-7 cells by incubating
(500 MHz, CDCl3) d 6.93 (s, 1H), 6.65 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H),
the cells 1 ꢁ106 mL/well with compound 12 and 13 (1 & 5
mM each)
6.06 (d, J ¼ 4.91 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.74 (d, J ¼ 4.72 Hz,
1H), 4.43e4.40 (t, J ¼ 7.33 Hz, 1H), 3.78 (s, 6H), 3.25e3.18 (m, 2H),
3.08e3.04 (dd, J ¼ 9.01, 4.79 Hz, 1H), 2.70e2.66 (t, J ¼ 7.46 Hz, 2H),
1.76e1.64 (m, 4H), 1.41e1.25 (m, 10H), 0.90e0.85 (t, J ¼ 7.84 Hz, 3H);
for 24 h. The cells were then washed twice with ice-cold PBS,
harvested, fixed with ice-cold PBS in 70% ethanol and stored
at ꢀ20 ꢂC for 30 min. After fixation, these cells were incubated with
RNase A (0.1 mg/mL) at 37 ꢂC for 30 min, stained with propidium
13C NMR (CDCl3):
d 173.23, 149.48, 148.19, 147.02, 135.09, 133.61,
iodide (50 mg/mL) for 30 min on ice in dark, and then measured for
130.03,125.36,110.56,108.96,108.64,101.96, 67.55, 58.71, 56.81, 43.76,
42.06, 37.36, 31.94, 29.60, 29.35, 29.15, 28.83, 25.87, 22.68, 18.52,
14.02; IR (KBr): 3388.18, 2927.42, 1765.60, 1610.59, 1515.24, 1483.99,
1228.99,1109.45,1035.03, 994.45, 926.91 cmꢀ1; ESI-MS: 592 (M þ 1).
DNA content using BD-LSR flow cytometer (Becton Dickinson, USA)
equipped with electronic doublet discrimination capability using
blue (488 nm) excitation from argon laser. Data were collected in
list mode on 10,000 events for FL2-A vs. FL2-W.
4.1.18. 40-O-Demethyl-4
b-[(4-hydroxymethyl)-1,2,3-triazol-1-yl]-
4.5. Molecular docking
4-desoxypodophyllotoxin (29)
White solid; mp: 160e162 ꢂC; ½a D25
ꢃ
ꢀ45 (c 0.60, CHCl3); 1H NMR
A compound library of 18 podophyllotoxin analogues were built
and minimized in Schrödinger using the parental structure of
podophyllotoxin as a template. Each structure was assigned an
appropriate bond order using ligprep script shipped by Schrödinger
and optimized by means of the OPLS-2005 [25] force field using
default settings. Different conformations were generated for input
ligands to cover every possibility of best conformations using
confgen module (ConfGen, version 2.1, Schrodinger, LLC, New York,
NY, 2009) of Schrodinger. The starting coordinates of the human
topoisomerase-II ATPase-AMP-PNP complex [PDB: 1ZXM] was
taken from the Protein Data Bank (www.rcsb.org) and further
modified for docking calculations. For Glide (Schrödinger) calcu-
lations, TP-II complex was imported to Maestro (Schrödinger), the
co-crystallized ligands were identified and removed from the
structure and the protein was minimized using the protein prepa-
ration wizard (Schrödinger) by applying OPLS-2001 force field [26].
Water molecules were removed and H atoms were added to the
structure. Minimizations were performed until the average root
mean square deviation of the non hydrogen atoms reached 0.3.
Docking was performed using Glide. After ensuring that the protein
and ligands are in correct form for docking, the receptor-grid files
were generated using a grid-receptor generation program using
default settings. The ligands were docked with the binding site
using the ‘extra precision’ Glide algorithm in Schrodinger.
(500 MHz, CDCl3) d 7.25(s, 1H), 6.66 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H),
6.06 (d, J ¼ 4.89 Hz,1H), 6.03 (s,1H), 6.01 (s,1H), 4.76 (d, J ¼ 4.77 Hz,
1H), 4.42e4.39 (t, J ¼ 6.79 Hz,1H), 3.77 (s, 6H), 3.74 (s, 2H), 3.28e3.17
(m, 2H), 3.09e3.05 (dd, J ¼ 8.57 Hz, 4.88 Hz, 1H); 13C NMR (CDCl3):
d
173.01, 149.54, 148.10, 137.63, 134.27, 133.21, 124.60, 110.18, 109.07,
108.72,101.90, 67.36, 59.81, 58.91, 56.65, 43.86, 41.81, 37.36, 29.68; IR
(KBr): 3384.21, 2926.74, 1763.60, 1611.10, 1516.99, 1483.59, 1232.06,
1109.97, 1033.67, 997.06, 928.11 cmꢀ1; ESI-MS: 482 (M þ 1).
4.2. Evaluation of anticancer activity
The effect of 4b-[(4-alkyl)-1,2,3-triazol-1-yl] podophyllotoxin
derivatives on the growth of cancer cell lines was evaluated
according to the procedure adopted by the National Cancer Institute
for in vitro anticancer drug screening that uses the protein-binding
dye sulforhodamine B to estimate cell growth. Briefly, cells in their
log phase of growth were harvested, counted and seeded (104 cells/
well in 100 mL medium) in 96-well microtitre plates. After 24 h of
incubation at 37 ꢂC and 5% CO2 to allow cell attachment, cultures
were treated with varying concentrations (0.1e100 mM) of test
samples made with 1:10 serial dilutions. Four replicate wells were
set up for each experimental condition. Test samples were left in
contact with the cells for 48 h under same conditions. Thereafter
cells were fixed with 50% chilled TCA and kept at 4 ꢂC for 1 h, washed
and air-dried. Cells were stained with sulforhodamine B dye. The
adsorbed dye was dissolved in tris-buffer and the plates were gently
shaken for 10 min on a mechanical shaker. The optical density (OD)
was recorded on ELISA reader at 540 nm. The cell growth was
calculated by subtracting mean OD value of the respective blank
from the mean OD value of experimental set. Percentage of growth
in the presence of test material was calculated considering the
growth in the absence of any test material as 100% and the results are
reported in terms of IC50 values. Etoposide was taken as positive
control.
Acknowledgement
Authors, DMR, JS and GC are grateful to CSIR (India) for Senior
Research Fellowships.
References
[1] M.G. Kelly, J.L. Hartwell, J. Natl. Cancer Inst. 14 (1954) 967e1010.
[2] T. Imbert, Biochimie 80 (1998) 207e222.
[3] L. Bohlin, B. Rosen, Drug Discov. Today 1 (1996) 343e351.
[4] A. Jordan, J.A. Hadfield, N.J. Lawrence, A.T. McGown, Med. Res. Rev. 18 (1998)
259e296.
4.3. DNA fragmentation assay
[5] I. Jardine, in: J.M. Cassady, J. Douros (Eds.), Anticancer Agents Based on Natural
Product Models, Academic Press, New York, 1980, pp. 319e351.
[6] K.H. Lee, S.A. Beers, M. Mori, Z. Wang, Y. Kuo, L. Li, S. Liu, Y. Cheng, F. Han,
Y. Cheng, J. Med. Chem. 33 (1990) 1364e1368.
DNA fragmentation was determined by electrophoresis of
extracted genomic DNA from breast cancer cell line MCF-7. Cells
(2 ꢁ 106/6 mL medium/60 mm tissue culture plate) were treated
[7] H. Stahelin, A. Von Wartburg, Prog. Drug Res. 33 (1989) 169e267.

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 1983e1991
1991
[8] T.W. Doyle, Etoposide (VP-16) Current Status and New Developments.
Academic, New York, 1984.
[17] P.B. Reddy, D.V. Paul, S.K. Agrawal, A.K. Saxena, H.M.S. Kumar, G.N. Qazi, Arch.
Pharm. (Weinheim) 341 (2) (2008) 126e131.
[9] Z. Wang, Y. Kuo, J. Schnur, J. Bowen, S. Liu, F. Hen, J. Chang, Y. Cheng, K.H. Lee,
J. Med. Chem. 33 (1990) 2660e2666.
[18] H.C. Kolb, M.G. Finn, K.B. Sharpless, Angew. Chem. Int. Ed. 40 (2001) 2004e2021.
[19] R. Huisgen, Proc. Chem. Soc. 6 (1961) 357e369.
[10] B.F. Issell, Cancer Chemother. Pharmacol. 7 (1982) 73e80.
[11] D.R. Budman, Semin. Oncol. 23 (1996) 8e14.
[20] N.O. Karpinich, M. Tafani, R.J. Rothman, M.A. Russo, J.L. Farer, J. Biol. Chem. 277
(2002) 16547e16552.
[12] F.A. Greco, J.D. Hainsworth, Semin. Oncol. 23 (1996) 40e47.
[13] T.L. Macdonald, E.K. Lehenert, J.T. Loper, K.C. Chow, W.E. Ross, DNA Top-
oisomerase in Cancer. Oxford University, New York, 1991, pp. 119.
[14] D.L. Sackett, Pharmacol. Ther. 59 (1993) 163e228.
[15] B.A. Bhat, P.B. Reddy, S.K. Agrawal, A.K. Saxena, H.M.S. Kumar, G.N. Qazi, Eur. J.
Med. Chem. 43 (2008) 2067e2072.
[21] A. Kamal, B.A. Kumar, M. Arifuddin, Tetrahedron Lett. 44 (2003) 8457e8459.
[22] A. Kamal, N. Laxman, G. Ramesh, Bioorg. Med. Chem. Lett. 10 (2000) 2059e2062.
[23] A.K. Patel, S. Patel, P.K. Naik, Curr. Res. J. Biol. Sci. 2 (1) (2010) 13e23.
[24] C. Frei, S.M. Gasser, A.V. Kajava, D. Leroy, Biochemistry 40 (2001) 1624e1634.
[25] G.A. Kaminski, R.A. Friesner, J. Tirado-Rives, W.J. Jorgensen, J. Phys. Chem. B
105 (2001) 6474e6487.
[16] P.B. Reddy, S.K. Agrawal, S. Singh, B.A. Bhat, A.K. Saxena, H.M.S. Kumar,
G.N. Qazi, Chem. Biodivers. 5 (2008) 1792e1802.
[26] W.L. Jorgensen, D.S. Maxwell, J. Tirado-Rives, J. Am. Chem. Soc. 118 (1996)
11225e11236.