Semi-synthesis and biological evaluation of 1,2,3-triazole-based podophyllotoxin congeners as potent antitumor agents inducing apoptosis in HepG2 cells

Article abstract of DOI:10.1002/ardp.201100438

A series of 4β-[(4-substituted)-1,2,3-triazol-1-yl]podophyllotoxin congeners were synthesized by employing click chemistry and further evaluated for their antitumor activity by MTT assay. Among them, six congeners (10, 11, 12, 13, 22, and 24) exhibited ap

Full text of DOI:10.1002/ardp.201100438

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
1
Full Paper
Semi-Synthesis and Biological Evaluation of
1,2,3-Triazole-Based Podophyllotoxin Congeners as
Potent Antitumor Agents Inducing Apoptosis in HepG2 Cells
Jinying Chen1y, Liang Ma1y, Ronghong Zhang1, Jie Tang3, Huijun Lai2, Jun Wang1,
Guangcheng Wang1, Qinyuan Xu1, Tao Chen1, Fei Peng1, Jingxiang Qiu1, Xiaolin Liang1,
Dong Cao1, Yan Ran1, Aihua Peng1, Yuquan Wei1, and Lijuan Chen1
1 State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University,
Chengdu, Sichuan, P. R. China
2 Institute of Chemical Engineering, Sichuan University, Chengdu, P. R. China
3 West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, P. R. China
A series of 4b-[(4-substituted)-1,2,3-triazol-1-yl]podophyllotoxin congeners were synthesized by
employing click chemistry and further evaluated for their antitumor activity by MTT assay.
Among them, six congeners (10, 11, 12, 13, 22, and 24) exhibited approximately 100-fold more
potent inhibitory activity against four tumor cell lines (HepG2, MKN-45, NCI-H1993, and B16) than
etoposide as positive control. Docking studies on binding in the ATPase domain of topoisomerase II
revealed perfect docking of four congeners in the active site. Furthermore, the podophyllotoxin
congeners 10, 11, 12, and 13 induced cell cycle arrest of HepG2 cells at the G2/M phase in a
concentration-dependent manner, assessed by flow cytometric analysis, highlighting that they
exert their antitumor activity via HepG2 cell apoptosis.
Keywords: Antitumor activity / Cell cycle / Docking study / G2/M phase / Podophyllotoxin congeners
Received: December 2, 2011; Revised: July 13, 2012; Accepted: July 19, 2012
DOI 10.1002/ardp.201100438
Supporting information available online
:
Introduction
covery of potent and low-cytotoxic podophyllotoxin-based
drugs. These drugs, including etoposide (1b) [6–8] and
teniposide (1c) [9, 10], were widely used in the clinic for
the treatment of malignancies such as small-cell lung cancer,
Kaposi’s sarcoma, lymphoma, glioblastoma multiforme,
and leukemia [11–14]. Structural modification of the podo-
phyllotoxin scaffold resulted in an essential change in the
mechanism of action: from the antimicrotubule activity of
podophyllotoxin to the anti-topoisomerase II (Topo-II) activity
of etoposide [15, 16]. The anti-microtubule agent podophyl-
lotoxin promotes cell death by interfering with the
function of the mitotic spindle and induces cell apoptosis
by promoting mitotic arrest, while the Topo-II inhibitor
etoposide inhibits DNA Topo-II by stabilizing the covalent
Topo-II–DNA cleavable complex.
Podophyllotoxin (1a, Fig. 1), a well-known naturally occur-
ring aryltetralin lignan isolated from the root of Podophyllum
hexandrum, possesses potent antiproliferative activity against
several tumor cell lines and has also been used as specific
antimicrotubule agent acting at the colchicine binding site
of tubulin [1–5]. However, severe toxicity and gastrointestinal
side effects of podophyllotoxin limited its application as
an antitumor drug in cancer chemotherapy. Hence, with
podophyllotoxin as lead compound, chemical disconnec-
tions and structural modifications were undertaken to
develop novel semi-synthetic congeners, leading to the dis-
Correspondence: Lijuan Chen, State Key Laboratory of Biotherapy, West
China Hospital, West China Medical School, Sichuan University, Keyuan
Road 4, Gaopeng Street, Chengdu, 610041, P. R. China.
E-mail: lijuan17@hotmail.com
Fax: 86 28 85164060
y These two authors contributed equally to this paper.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Figure 1. Structures of several podophyllo-
toxin derivatives.
Although etoposide is widely used in the clinic, many studies Results and discussion
have focused on its side effects, such as potential drug resist-
ance, metabolic inactivation, myelosuppression, poor water
solubility, and even induction of secondary tumors [17, 18].
Hence, structural modifications of etoposide have been devel-
oped, thus exploring for more effective congeners. Previous
studies have proven that the trans-lactone, 4b-substituted,
and 40-demethyl moieties were crucial for the antitumor
activity [19–21]. Therefore, the preparation of 4b-substituted
podophyllotoxin congeners might be an effective strategy to
improve the anti-Topo-II and antitumor potency [19–21].
According to the reported works, podophyllotoxin linked
with different aliphatic side chains by an 1,2,3-triazole ring
exhibited significant binding affinity to Topo-II, which gave
us the inspiration to focus on other substitutions at this
position of the scaffold.
Chemistry
The synthesis of 4b-(4-aryl)-1,2,3-triazole-1-yl)podophyllotoxin
congeners is illustrated in Scheme 1. Congeners 3 and 4
were synthesized according to the previously reported
method [22]. The key intermediate, 4b-azidopodophyllotoxin
2 was obtained by treating podophyllotoxin 1 with TFA
and NaN3 in dichloromethane, with good yield. The O-pro-
pargylated (thio)phenols b1–b14 were prepared through
the condensation of propargyl bromide or 3-bromo-3-
dimethyl-1-butyne with commercially available (thio)phenols
in the presence of KOH as base and NaI as a valid catalyst.
The acylation of the appropriate benzoic acid with
propargyl amine led to the corresponding N-(prop-2-yn-1-yl)-
benzamides (b15 and b16) by employing dicyclohexyl-
carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP).
Finally, the condensation of 2 with b1–b16 afforded the
target congeners though click chemistry, with excellent
yields. The click chemistry was accomplished using
CuSO4 ꢀ 5H2O, sodium ascorbate, and t-butyl ammonium
bromide (TBAB) in THF/water (1:1). Click chemistry in
this study enabled a modular approach to generate the
pharmacophores by utilizing the collection of reliable
chemical reactions and provided near-perfect properties
including high yields, few byproducts and mild conditions
[23, 24].
In this study, a series of 4b-[(4-substituted)-1,2,3-triazol-1-
yl]podophyllotoxin congeners with various aromatic sub-
stituents were synthesized by click chemistry. Twenty-seven
derivatives were subsequently screened against four human
tumor cells, and most of these compounds showed compar-
able or superior antiproliferative activity compared to etopo-
side. The docking results of binding in the ATPase domain of
Topo-II revealed perfect docking of the four congeners in the
active site. Additionally, the podophyllotoxin congeners 10,
11, 12, and 13 concentration-dependently induced cell
cycle arrest of HepG2 cells at the G2/M phase, highlighting
that they exert their antitumor potency via HepG2 cell
apoptosis.
The intermediates c2–c11 were obtained by aldol conden-
sation of c1 with appropriate benzaldehydes using 50% KOH
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Podophyllotoxin Congeners as Potent Antitumor Agents
3
Scheme 1. Synthesis of 4b-[(4-aryl)-1H-1,2,3-triazol-1-yl)podophyllotoxin congeners. Reagents and conditions: (a) NaN3, trifluoro-
acetic acid, CH2Cl2, 24 h, 95.0%; (b) KOH, NaI, acetone, 258C, 8 h, 95.0%; (c) CuSO4 ꢀ 5H2O, sodium ascorbate, TBAB, THF/H2O
(1:1 v/v); (d) 2-propynylamine, DCC, DMAP, CH2Cl2, r.t., 12 h, 80.0%.
and methanol [25]. Subsequent cycloaddition of c2–c11
with 2 was achieved by the above-mentioned synthesis
methods (Scheme 2). At this stage, all 27 congeners were
fully characterized by NMR, MS, and HPLC before being sub-
mitted to biological screening, and the analysis results are
summarized in Table 1.
reference. The results are summarized in Table 2. From the
collected data, compounds 11, 12, and 13 were shown to be
highly potent against all the cell lines. Similarly, 10 exhibited
high in vitro anticancer activity, and comparable cytotoxic
effects were observed for 22 and 24. Among the six represen-
tative congeners, compound 11 containing a biphenyl ring
showed the highest antiproliferative activity against the four
cell lines (especially an IC50 value of 150 nM at 48 h against
HepG2 cells, which was 100-fold more active than the positive
controls). The introduction of a sulfur atom in place of
the oxygen atom showed a great improvement in inhibitory
potency (12 vs. 5). Importantly, the attachment of a fluorine
atom in the phenyl moiety (e.g., 24) was optimal for the
Biological evaluation
The anticancer efficacy of the 27 compounds was evaluated
by measuring their antiproliferative activity on well-estab-
lished cancer cells, namely, HepG2, MKN-45, NCI-H1993, and
B16, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay. Etoposide was selected as a
Scheme 2. Synthesis of 4b-[(4-chalcone)-1H-1,2,3-triazol-1-yl)podophyllotoxin congeners. Reagents and conditions: (a) NaH, DMF,
258C, 12 h, 98.0%; (b) 50% KOH (aq.), CH3OH, r.t., 24 h, 70.0%.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Table 1. Structural profiles of the podophyllotoxin congeners.
Compounds
3
X
R1
R2
R3
R0
O
H
H
4
O
O
O
O
O
O
O
O
S
H
H
H
H
5
6
H
H
7
H
H
8
H
H
9
H
H
10
11
12
13
14
15
16
17
18
H
H
H
H
H
H
S
H
H
O
O
O
CH3
CH3
CH3
H
CH3
CH3
CH3
H
H
H
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Table 1. (continued )
Podophyllotoxin Congeners as Potent Antitumor Agents
5
Compounds
19
X
R1
R2
R3
R0
O
H
H
20
O
H
H
21
22
O
O
H
H
H
H
23
24
25
26
27
28
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
29
O
H
H
chalcone-containing analogues. In addition, two methoxyl
groups at the meta or para position of the phenyl ring were
also tolerated as 22 displayed distinct increases in antitumor
activity with an average IC50 value of 1.58 mM.
activity than the corresponding electron-withdrawing group
(EWG; e.g., 3, 4, 8, and 9). However, the placement of the
amide bond between the triazole and the phenyl ring showed
negative effects on the antitumor potency (17 and 18 were
almost fivefold less potent than podophyllotoxin). However,
it was found that replacement of the hydrogen atoms on the
linker between the triazole ring and the phenyl ring with the
As demonstrated in the biological evaluation, an electron-
donating group (EDG; e.g., 5, 7, 10, 12, 13) at the meta or para
position of the aromatic ring tended to possess much better
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Table 2. IC50 values of various podophyllotoxin congeners against selected tumor cells.
Compounds
IC50 at 48 h (mM)a)
IC50 at 72 h (mM)
HepG2
MKN-45
NCI-H1993
B16
HepG2
MKN-45
NCI-H1993
B16
Etoposideb)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
>10.00
6.34 ꢁ 0.89
6.10 ꢁ 0.98
0.99 ꢁ 0.01
1.05 ꢁ 0.06
7.62 ꢁ 0.87
4.26 ꢁ 0.65
3.11 ꢁ 0.22
1.45 ꢁ 0.13
0.93 ꢁ 0.04
0.22 ꢁ 0.01
0.13 ꢁ 0.02
0.44 ꢁ 0.04
0.31 ꢁ 0.02
1.72 ꢁ 0.09
3.14 ꢁ 0.78
4.20 ꢁ 0.98
3.10 ꢁ 0.81
>10
7.93 ꢁ 0.77
59.60 ꢁ 2.34
54.50 ꢁ 4.78
10.70 ꢁ 1.26
64.00 ꢁ 4.76
8.25 ꢁ 0.89
4.13 ꢁ 0.34
6.92 ꢁ 0.55
0.85 ꢁ 0.05
0.24 ꢁ 0.03
0.49 ꢁ 0.05
1.45 ꢁ 0.12
0.29 ꢁ 0.05
3.00 ꢁ 0.14
2.04 ꢁ 0.43
7.25 ꢁ 0.98
3.90 ꢁ 0.56
8.73 ꢁ 0.75
1.63 ꢁ 0.07
>10.00
>10.00
1.85 ꢁ 0.15
3.21 ꢁ 0.21
0.87 ꢁ 0.01
0.80 ꢁ 0.01
8.35 ꢁ 0.78
3.16 ꢁ 0.16
2.88 ꢁ 0.17
4.17 ꢁ 0.61
0.20 ꢁ 0.01
0.13 ꢁ 0.02
0.14 ꢁ 0.01
0.23 ꢁ 0.01
0.17 ꢁ 0.01
1.11 ꢁ 0.33
2.64 ꢁ 0.67
6.75 ꢁ 0.99
4.25 ꢁ 0.87
4.99 ꢁ 0.66
2.57 ꢁ 0.43
20.87 ꢁ 1.04
0.67 ꢁ 0.04
8.48 ꢁ 1.07
0.53 ꢁ 0.05
2.17 ꢁ 0.44
2.27 ꢁ 0.34
1.09 ꢁ 0.14
4.33 ꢁ 0.89
2.15 ꢁ 0.42
6.05 ꢁ 0.65
3.40 ꢁ 0.19
0.84 ꢁ 0.01
0.91 ꢁ 0.06
5.93 ꢁ 0.66
4.10 ꢁ 0.24
2.76 ꢁ 0.17
1.23 ꢁ 0.11
0.64 ꢁ 0.02
0.13 ꢁ 0.01
0.31 ꢁ 0.02
0.36 ꢁ 0.04
0.26 ꢁ 0.01
0.92 ꢁ 0.04
3.30 ꢁ 0.23
3.23 ꢁ 0.15
1.70 ꢁ 0.16
6.03 ꢁ 0.67
0.92 ꢁ 0.06
>10.00
0.69 ꢁ 0.04
5.16 ꢁ 0.97
0.48 ꢁ 0.02
2.24 ꢁ 0.41
1.78 ꢁ 0.31
0.85 ꢁ 0.11
3.21 ꢁ 0.93
1.03 ꢁ 0.04
4.40 ꢁ 0.25
37.80 ꢁ 2.22
15.80 ꢁ 2.87
4.00 ꢁ 0.56
19.70 ꢁ 1.89
5.20 ꢁ 0.33
3.68 ꢁ 0.22
6.63 ꢁ 0.56
0.50 ꢁ 0.05
0.17 ꢁ 0.01
0.40 ꢁ 0.02
0.85 ꢁ 0.04
0.26 ꢁ 0.01
2.33 ꢁ 0.15
1.61 ꢁ 0.11
5.80 ꢁ 0.67
3.17 ꢁ 0.11
5.09 ꢁ 0.23
1.26 ꢁ 0.15
>10.00
0.67 ꢁ 0.06
6.67 ꢁ 1.02
0.49 ꢁ 0.05
2.45 ꢁ 0.43
1.95 ꢁ 0.33
1.00 ꢁ 0.30
3.27 ꢁ 0.76
0.98 ꢁ 0.15
1.35 ꢁ 0.15
3.60 ꢁ 0.18
0.98 ꢁ 0.02
1.05 ꢁ 0.04
32.00 ꢁ 2.67
>80.00
5.75 ꢁ 0.43
2.10 ꢁ 0.17
0.30 ꢁ 0.02
0.40 ꢁ 0.04
0.27 ꢁ 0.01
0.68 ꢁ 0.04
0.27 ꢁ 0.01
2.19 ꢁ 0.48
5.16 ꢁ 0.78
3.70 ꢁ 0.34
2.30 ꢁ 0.24
5.60 ꢁ 0.44
2.02 ꢁ 0.17
>20.00
1.14 ꢁ 0.17
8.09 ꢁ 0.91
0.55 ꢁ 0.07
1.98 ꢁ 0.19
2.63 ꢁ 0.22
0.93 ꢁ 0.06
6.29 ꢁ 0.87
1.93 ꢁ 0.12
3.87 ꢁ 0.23
1.05 ꢁ 0.04
0.98 ꢁ 0.04
19.00 ꢁ 1.89
4.18 ꢁ 0.56
3.80 ꢁ 0.19
7.78 ꢁ 0.77
0.25 ꢁ 0.01
0.15 ꢁ 0.01
0.26 ꢁ 0.02
0.31 ꢁ 0.08
0.18 ꢁ 0.01
1.72 ꢁ 0.09
3.14 ꢁ 0.19
8.55 ꢁ 0.98
4.85 ꢁ 0.56
9.88 ꢁ 1.00
2.80 ꢁ 0.09
>80.00
1.61 ꢁ 0.23
9.65 ꢁ 0.98
0.81 ꢁ 0.04
2.39 ꢁ 0.11
2.20 ꢁ 0.15
1.64 ꢁ 0.11
11.95 ꢁ 1.01
2.26 ꢁ 0.44
30.00 ꢁ 1.87
10.00 ꢁ 1.89
2.85 ꢁ 0.67
66.60 ꢁ 4.56
>80.00
4.53 ꢁ 0.55
4.35 ꢁ 0.65
2.93 ꢁ 0.32
0.54 ꢁ 0.09
2.52 ꢁ 0.33
0.90 ꢁ 0.35
0.57 ꢁ 0.01
2.50 ꢁ 0.34
3.35 ꢁ 0.12
8.25 ꢁ 0.65
4.10 ꢁ 0.45
8.72 ꢁ 0.99
2.48 ꢁ 0.32
>80.00
1.05 ꢁ 0.02
>10.00
1.01 ꢁ 0.05
6.80 ꢁ 0.66
0.69 ꢁ 0.04
2.34 ꢁ 0.22
2.33 ꢁ 0.19
1.04 ꢁ 0.08
>10.00
2.14 ꢁ 0.07
1.56 ꢁ 0.09
>10.00
>20.00
0.85 ꢁ 0.11
8.20 ꢁ 0.99
4.37 ꢁ 0.76
2.23 ꢁ 0.19
>10.00
0.53 ꢁ 0.01
3.45 ꢁ 0.45
2.18 ꢁ 0.88
1.01 ꢁ 0.02
>10.00
0.91 ꢁ 0.04
2.04 ꢁ 0.17
2.93 ꢁ 0.22
a)
Half-maximum inhibitory concentration.
Positive control.
b)
methyl groups led to a moderate increase in inhibitory
activity (methyl in 14 and 16, hydrogen in 3 and 7). 21 and
23 showed weaker antiproliferative activity than the corre-
sponding compounds 22 and 24, because of an ortho-methoxyl
group close to the double bond of a,b-unsaturated ketone.
Additionally, the replacement of the phenyl ring by a
pyrimidine group could reduce the inhibitory potency
(e.g., 28 with all IC50 values >10.0 mM at 48 h).
and Asn91, while 11 exhibited four strong H-bonds
with Arg98, Thr215, Ser149, and Asn91. Compound 12 (hydro-
phobic interaction with Arg98, and Thr215) and 13 (five
H-bonds with residues: one methoxyl group with Gly164,
Asn163, and Arg162; S and N atom with Ala167 and Asn150,
respectively) also showed strong affinity to the receptor.
Cell cycle analysis
The cell cycle of a eukaryotic cell is a crucial checkpoint for
chemotherapeutic drugs killing tumor cells, involving a
variety of fundamental processes such as cell division and
duplication (replication), etc. [28]. As shown in Table 3, all
HepG2 cells were accumulated in the G2/M phase, with
a concomitant decrease of cells in the G1 and S phases,
demonstrating that the congeners 10–13 dose-dependently
blocked the progression of the cell cycle in the G2/M phase.
Importantly, compound 11, superior to the other compounds
(10, 12, and 13), significantly induced apoptosis of the
HepG2 cells in a dose-dependent manner (Fig. 3), highlight-
ing that the four congeners exerted their antitumor potency
by inducing cell apoptosis.
Docking study
To study the molecular interaction and affinity of binding of
the podophyllotoxin congeners, all the ligands were docked
in the ATPase domain of Topo-II, which has been proven to
be an active binding site of etoposide (see the data in the
Supporting Information) [26, 27]. The high-ranked (by com-
putational scoring) compounds 10–13 are depicted in Fig. 2.
The scaffold occupied the active site of the enzyme, deeply
located inside the cavity and surrounded by the residues,
and formed hydrogen bond (H-bond) interactions with
several residues. Compound 10 possessed three H-bonds with
Gly164, Asn163, and Arg162 and two H-bonds with Ala167
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Podophyllotoxin Congeners as Potent Antitumor Agents
7
Figure 2. Docking models of podophyllotoxin congeners in the ATPase domain of Topo-II. Interactions of compounds 10 (A), 11 (B), 12 (C),
and 13 (D) with residues within the ATPase domain of Topo-II.
Table 3. Effects of four podophyllotoxin congeners on the cell cycle distribution in HepG2 cells.
mM
Compounds
% G1
% G2
% S
Compounds
% G1
% G2
% S
0
10
50.66
53.77
20.88
0.43
50.66
17.63
25.14
0.50
15.60
17.33
63.77
95.64
15.60
57.18
49.62
94.66
33.74
28.90
15.34
3.93
33.74
25.19
25.25
4.84
11
50.66
41.06
1.83
15.60
39.53
70.85
94.95
15.60
21.00
63.03
85.89
33.74
19.41
27.32
7.34
33.74
29.34
25.72
13.46
0.1
0.2
0.4
0
0.1
0.2
0.4
0.24
12
13
50.66
49.66
11.25
0.64
The cells were exposed to various concentrations of podophyllotoxin derivatives for 24 h, and then the DNA content was analyzed
by flow cytometry.
Conclusion
Further, the four congeners 10–13 were validated as specific
G2/M phase blockers by cell cycle analysis.
In summary, a series of 1,2,3-triazole-based podophyllotoxin
congeners were synthesized and evaluated for their antitumor
activity against four tumor cell lines. Among them, compound
11, featuring a biphenyl motif substituted at the 4-position of
the 1,2,3-triazole ring, exhibited the most potent antitumor
activity. Meanwhile, 10, 12, and 13 also exerted strong anti-
cancer potency. The docking study revealed perfect docking of
the four congeners in the active site of the ATPase domain.
Experimental
Chemistry
Melting points were recorded on an SGW X-4 micro melting
point apparatus. 1H NMR spectra were recorded on a Varian
spectrometer (Varian, Palo Alto, CA, USA) model Gemini 400;
chemical shifts are expressed in ppm, relative to internal tetra-
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Figure 3. Flow cytometric analysis showing DNA histograms of HepG2 cells. (A) Control; (B) treated with 0.1 mM 11; (C) treated with
0.2 mM 11; (D) treated with 0.4 mM 11.
methylsilane (TMS), where TMS (d) ¼ 0.00 ppm. The multiplicity
of the signal is indicated as: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet, defined as all multipeak signals where
overlap or complex coupling of signals makes definitive descrip-
tions of peaks difficult. Reactions were monitored by analytical
TLC on 0.20 mm silica gel F254 plates (Qingdao Ocean Chemical
Factory, Shandong, China). Low-resolution mass spectra were
obtained by a Q-TOF Premier mass spectrometer utilizing electro-
spray ionization (ESI; Micromass, Manchester, UK). HPLC was
performed using a photodiode array detector (Waters, Milford,
MA, USA) and the chromatographic column was an Atlantis C18
(150 mm ꢂ 4.6 mm, id 5 mm) (Waters, Milford, Ireland) at a flow
rate of 1 mL/min for 30 min.
(305.8 mg, 4.70 mmol), and 10 mL CH2Cl2 in an ice bath.
The mixture was stirred overnight at room temperature.
Water was added and the mixture was extracted with CH2Cl2,
washed with brine and dried over anhydrous Na2SO4, followed
by evaporation to afford a brown solid. The crude was purified
by silica gel (300–400 mesh) column chromatography using
petroleum ether/ethyl acetate (4:1) to yield a brown solid
(422.5 mg, 96.0%).
General procedure for the preparation of chalcones c2–c11
To a solution of 1-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)etha-
none (c1) (2.45 mmol) and the appropriate benzaldehydes
(4.90 mmol) in methanol was added 40 mL of 50% KOH aqueous
solution. The reaction mixture was stirred under nitrogen atmo-
sphere at room temperature for 24 h. After completion, the
mixture was neutralized with 1 N HCl, and the formed solid
was collected by filtration. The crude was purified through
recrystallization with methanol to yield the product.
General procedure for the synthesis of 40-O-demethyl-4-
azido-4-desoxypodophyllotoxin 2
Of TFA/CH2Cl2 (1:1), 4 mL was slowly added to a stirred solution
of podophyllotoxin (1) (400.0 mg, 0.96 mmol), sodium azide
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Podophyllotoxin Congeners as Potent Antitumor Agents
9
General procedure for click chemistry
4b-[(4-(4-Ethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 7
To a solution of 40-O-demethyl-4-azido-4-desoxypodophyllotoxin (2)
(0.22 mmol) in THF/water (1:1, 4 mL) was added CuSO4 ꢀ 5H2O
(0.22 mmol), sodium ascorbate (0.5 mmol), and TABA (0.5 mmol).
The mixture was stirred at 608C for 20 h. After completion, the
mixture was diluted with water and extracted with ethyl acetate,
washed with brine, dried over anhydrous Na2SO4, and evaporated
in vacuo. The crude was purified by silica gel column chromatog-
raphy (300–400 mesh) with petroleum ether/ethyl acetate (1:1) to
yield the solid products 3–29.
Yield 60.45%; colorless oil; mp 123–1258C; 1H NMR (400 MHz,
CDCl3) d 7.13 (s, 1H), 7.11 (s, 1H), 6.90 (s, 1H), 6.87 (s, 1H), 6.65
(s, 1H), 6.62 (s, 1H), 6.32 (s, 2H), 6.10 (d, 1H, J ¼ 3.6Hz), 6.03–6.00
(m, 2H), 5.15 (s, 2H), 4.75 (d, 1H, J ¼ 4.8 Hz), 4.40 (s, 1H), 3.82 (s, 3H),
3.77 (s, 6H), 3.23 (t, 2H, J ¼ 6.0 Hz), 3.06–3.01 (m, 1H), 3.80 (s, 3H),
3.77 (s, 6H), 3.25–3.22 (m, 2H), 3.06–3.01 (m, 1H), 2.60 (q, 2H,
J ¼ 7.2 Hz), 1.21 (t, 3H, J ¼ 7.6 Hz). MS (ESI), m/z: 622.59 [MþNa]þ.
4b-[(4-(4-Methyl-3-nitrophenoxy)methyl)-1H-1,2,3-triazol-
Podophyllotoxin 1
1-yl)]-4-desoxypodophyllotoxin 8
1H NMR (400 MHz, CDCl3) d 7.26 (s, 1H), 7.11 (s, 1H), 6.60 (s, 1H),
6.36 (s, 2H), 5.98 (d, 2H, J ¼ 8.0 Hz), 4.77 (d, 1H, J ¼ 8.8 Hz), 4.60
(t, 2H, J ¼ 8.4 Hz), 4.08 (t, 1H, J ¼ 8.8 Hz), 3.80 (s, 3H), 5.76 (s, 3H),
2.82–2.77 (m, 2H). MS (ESI), m/z: 437.41 [MþNa]þ.
Yield 55.77%; yellow solid; mp 123–1248C; 1H NMR (400 MHz,
CDCl3) d 7.63 (s, 1H), 7.20 (s, 2H), 6.67 (s, 1H), 6.69 (s, 1H), 6.31
(s, 2H), 6.04 (d, 2H, J ¼ 7.2 Hz), 5.30 (s, 2H), 4.78 (s, 1H), 4.42
(s, 1H), 3.82 (s, 3H), 3.77 (s, 6H), 3.28 (s, 2H), 3.00 (s, 1H), 2.54 (s, 3H).
MS (ESI), m/z: 653.21 [MþNa]þ.
4b-N3-Desoxypodophyllotoxin 2
Yield 62.14%; 1H NMR (400 MHz, CDCl3) d 6.79 (s, 1H), 6.65 (s, 1H),
6.24 (s, 2H), 5.99 (d, 2H, J ¼ 5.2 Hz), 4.78 (d, 1H, J ¼ 3.6 Hz), 4.61
(d, 1H, J ¼ 5.2 Hz), 4.33–4.27 (m, 2H), 3.79 (s, 3H), 3.70 (s, 6H), 3.23
(d, 1H, J ¼ 5.2 Hz), 2.95–2.92 (m, 1H). MS (ESI), m/z: 462.22
[MþNa]þ.
4b-[(4-((2-Oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)-1H-1,2,3-triazol-1-yl)]-4-desoxypodophyllotoxin 9
Yield 45.65%; white solid; mp 122–1258C; 1H NMR (400 MHz,
CDCl3) d 7.63 (s, 1H), 7.60 (s, 1H), 7.33–7.26 (m, 2H), 6.64 (s, 1H),
6.67 (s, 1H), 6.39 (s, 2H), 6.03 (d, 2H, J ¼ 10.0 Hz), 5.30 (s, 2H), 4.76
(s, 2H), 4.39 (s, 1H), 3.81 (s, 3H), 3.76 (s, 6H), 3.24 (s, 2H), 3.06 (s, 1H).
MS (ESI), m/z: 662.59 [MþNa]þ.
4b-[(4-((4-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 3
Yield 57.04%; yellow solid; mp 119–1208C; 1H NMR (400 MHz,
CDCl3) d 7.89 (s, 1H), 7.87 (s, 1H), 7.34–7.32 (m, 2H), 6.68 (s, 1H),
6.67 (s, 1H), 6.33 (s, 2H), 6.05 (d, 2H, J ¼ 7.2 Hz), 5.25 (s, 2H), 4.79
(d, 1H, J ¼ 5.2 Hz), 4.45 (t, 1H, J ¼ 7.2 Hz), 4.15–4.10 (m, 1H), 3.82
(s, 3H), 3.77 (s, 6H), 3.34–3.25 (m, 2H), 3.03 (m, 1H, J ¼ 9.2 Hz). MS
(ESI), m/z: 639.56 [MþNa]þ.
4b-[(4-(Benzo[d][1,3]dioxol-5-yloxy)methyl)-1H-1,2,3-
triazol-1-yl]-4-desoxypodophyllotoxin 10
Yield 51.43%; white solid; mp 112–1148C; 1H NMR (400 MHz,
CDCl3) d 7.55–7.52 (m, 2H), 7.43–7.39 (m, 1H), 7.03 (d, 1H,
J ¼ 8.0 Hz), 6.62 (d, 1H, J ¼ 10.4 Hz), 6.31 (s, 1H), 6.01–5.97
(m, 2H), 5.21 (s, 2H), 4.75 (d, 1H, J ¼ 4.0 Hz), 4.41 (m, 1H), 3.81
(s, 3H), 3.76–3.73 (m, 6H), 3.32–3.23 (m, 2H), 3.08–3.04 (m, 1H).
MS (ESI), m/z: 638.07 [MþNa]þ.
4b-[(4-((3-Nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 4
4b-[(4-(([1,10-Biphenyl]-4-yloxy)methyl)-1H-1,2,3-triazol-1-
Yield 68.24%; yellow solid; mp 120–1238C; 1H NMR (400 MHz, CDCl3)
d 7.87 (d, 1H, J ¼ 8.0 Hz), 7.82 (m, 1H), 7.46 (t, 1H, J ¼ 8.4Hz), 7.37–
7.31 (m, 2H), 6.70 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H), 6.13 (d, 1H,
J ¼ 4.8 Hz), 6.02 (d, 2H, J ¼ 6.4 Hz), 5.24 (s, 2H), 4.76 (d, 1H,
J ¼ 5.2 Hz), 4.43–4.41 (m, 1H), 3.80 (s, 3H), 3.77–3.74 (m, 6H),
3.36–3.25 (m, 2H), 3.05–3.00 (m, 1H). MS (ESI), m/z: 639.52 [MþNa]þ.
yl]-4-desoxypodophyllotoxin 11
Yield 46.67%; colorless oil; mp 126–1308C; 1H NMR (400 MHz,
CDCl3) d 7.68–7.52 (m, 2H), 6.71–6.62 (m, 3H), 6.63–6.62 (d, 2H,
J ¼ 8.4 Hz), 6.40–6.33 (m, 1H, J ¼ 6.0 Hz), 6.32–6.31 (m, 2H), 6.10
(d, 1H, J ¼ 4.0 Hz), 6.05–6.01 (m, 4H), 5.92 (s, 2H), 5.10 (s, 2H), 4.75
(d, 1H, J ¼ 5.2 Hz), 4.42–4.39 (m, 1H), 4.30 (t, 1H, J ¼ 6.4 Hz),
4.14–4.09 (m, 1H), 3.82–3.74 (m, 9H), 3.28–3.19 (m, 2H), 3.04–3.01
(m, 1H). MS (ESI), m/z: 670.36 [MþNa]þ.
4b-[(4-(4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 5
Yield 51.33%; white solid; mp 124–1258C; 1H NMR (400 MHz,
CDCl3) d 7.22–7.18 (m, 1H), 6.66 (s, 2H), 6.61–6.60 (m, 3H), 6.37–
6.28 (m, 2H), 6.10 (d, 2H, J ¼ 9.6 Hz), 4.78 (s, 1H), 4.40 (s, 1H), 3.82–
3.74 (m, 12H), 3.29 (s, 2H), 3.29 (s, 2H), 2.97 (s, 1H). MS (ESI), m/z:
624.57 [MþNa]þ.
4b-[(4-((4-Methoxyphenyl)thio)methyl)-1H-1,2,3-triazol-1-
yl)]-4-desoxypodophyllotoxin 12
Yield 53.76%; white solid; mp 124–1268C; 1H NMR (400 MHz,
CDCl3) d 7.54–7.53 (m, 1H), 7.43–7.39 (m, 2H), 6.82 (s, 2H), 6.64–
6.31 (m, 2H), 6.62 (s, 1H), 6.65–6.30 (m, 2H), 5.29 (s, 2H), 4.73–4.70
(d, 1H, J ¼ 10.0 Hz), 4.60 (s, 1H), 3.84–3.74 (m, 12H), 3.25 (s, 1H),
2.98–2.82 (m, 1H). MS (ESI), m/z: 640.37 [MþNa]þ.
4b-[(4-(4-(Tert-pentyl)phenoxy)methyl)-1H-1,2,3-triazol-
1-yl]-4-desoxypodophyllotoxin 6
Yield 46.47%; white solid; mp 122–1248C; 1H NMR (400 MHz,
CDCl3) d 7.00–6.91 (m, 2H), 6.69 (s, 1H), 6.69 (s, 1H), 6.31 (s, 2H),
6.03–5.98 (m, 3H), 5.15 (s, 2H), 4.78 (s, 1H), 4.01 (s, 1H), 3.82 (s, 3H),
3.78 (s, 6H), 3.27 (s, 2H), 3.04 (s, 1H), 1.55 (q, 2H, J ¼ 7.2 Hz), 0.84–
0.82 (m, 6H), 0.69–0.65 (t, 3H, J ¼ 7.2 Hz). MS (ESI), m/z: 664.64
[MþNa]þ.
4b-[4-(3,4-Dimethoxyphenyl)thio)methyl)-1H-1,2,3-triazol-
1-yl)]-4-desoxypodophyllotoxin 13
Yield 67.87%; white solid; mp 127–1308C; 1H NMR (400 MHz,
CDCl3) d 7.33–7.21 (m, 3H), 6.74 (s, 1H), 6.62 (s, 1H), 6.61 (s, 1H),
6.28 (s, 2H), 5.98–5.95 (m, 3H), 4.69 (s, 2H), 4.31 (s, 1H), 3.85–3.84
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
(m, 6H), 3.81(s, 3H), 3.75 (s, 6H), 3.32–3.19 (m, 2H), 2.78 (s, 1H). MS
(ESI), m/z: 670.43 [MþNa]þ.
(s, 2H), 6.70 (s, 1H), 7.05 (s, 1H), 7.03 (s, 1H), 6.30 (s, 1H), 6.02 (d, 2H,
J ¼ 10.8 Hz), 5.37 (s, 2H), 4.74 (d, 1H, J ¼ 3.6 Hz), 4.40 (s, 1H),
3.94–3.90 (m, 9H), 3.86 (s, 3H), 3.81–3.73 (m, 9H), 3.26 (s, 1H), 3.03
(d, 1H, J ¼ 5.2 Hz). MS (ESI), m/z: 844.56 [MþNa]þ.
4b-[(4-(2-(4-Ethylphenoxy)propan-2-yl)-1H-1,2,3-triazol-1-
yl)]-4-desoxypodophyllotoxin 14
Yield 59.41%; white solid; mp 126–1278C; 1H NMR (400 MHz,
CDCl3) d 7.32–7.27 (m, 3H), 6.63 (s, 1H), 6.68–6.61 (m, 1H), 6.30–
6.28 (m, 2H), 6.03–5.97 (m, 3H), 4.77 (s, 1H), 4.40 (s, 1H), 3.82
(s, 3H), 3.76 (s, 6H), 3.24 (s, 2H), 2.99–2.97 (m, 1H), 1.27–1.18
(m, 9H). MS (ESI), m/z: 650.79 [MþNa]þ.
4b-[(4-((2-Methoxy-4-(3-(2,4,5-trimethoxyphenyl)-
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin 21
Yield 60.67%; yellow solid; mp 135–1398C; 1H NMR (400 MHz,
CDCl3) d 8.10 (d, 1H, J ¼ 15.6 Hz), 7.65 (t, 1H, J ¼ 8.0 Hz), 7.51
(s, 1H), 7.34 (s, 1H), 7.11 (s, 1H), 7.64 (s, 1H), 7.59 (s, 1H), 6.63 (d, 1H,
J ¼ 5.2 Hz), 6.31 (s, 2H), 6.09 (d, 1H, J ¼ 3.6 Hz), 6.02 (d, 2H,
J ¼ 9.6 Hz), 5.35 (s, 1H), 5.27 (s, 1H), 4.74 (d, 1H, J ¼ 4.4 Hz),
4.40 (d, 1H, J ¼ 6.4 Hz), 3.95–3.67 (m, 21H), 3.28 (d, 2H,
J ¼ 8.4 Hz), 3.04 (t, 1H, J ¼ 8.8 Hz). MS (ESI), m/z: 844.59 [MþNa]þ.
4b-[(4-(2-(4-Methoxyphenoxy)propan-2-yl)-1H-1,2,3-
triazol-1-yl)]-4-desoxypodophyllotoxin 15
Yield 65.12%; white solid; mp 135–1398C; 1H NMR (400 MHz,
CDCl3) d 7.73–7.54 (m, 4H), 6.62 (s, 1H), 6.68 (d, 1H, J ¼ 5.2 Hz),
6.31 (s, 2H), 6.02 (d, 2H, J ¼ 6.0 Hz), 4.41 (s, 1H), 4.30 (m, 1H,
J ¼ 6.4 Hz), 3.87–3.80 (m, 12H), 3.24 (s, 2H), 2.01–1.99 (m, 1H),
1.47 (s, 6H). MS (ESI), m/z: 652.52 [MþNa]þ.
4b-[(4-((4-(3-(3,4-Dimethoxyphenyl)acryloyl)-2-
methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin 22
Yield 65.23%; yellow solid; mp 140–1448C; 1H NMR (400 MHz,
CDCl3) d 7.79 (d, 1H, J ¼ 15.6 Hz), 7.64 (t, 1H, J ¼ 8.8 Hz), 7.61
(s, 1H), 7.36 (d, 1H, J ¼ 6.8 Hz), 7.19 (d, 1H, J ¼ 3.6 Hz), 7.15 (s, 1H),
7.05 (d, 1H, J ¼ 8.8 Hz), 6.90 (t, 1H, J ¼ 8.0 Hz), 6.64 (s, 1H),
6.60 (s, 1H), 6.31 (s, 2H), 6.09 (s, 1H), 6.02 (d, 2H, J ¼ 10.4 Hz),
5.36 (s, 2H), 4.74 (d, 1H, J ¼ 4.4 Hz), 4.40 (s, 1H), 3.96–3.70 (m,
18H), 3.40–3.24 (m, 1H), 3.03 (t, 1H, J ¼ 4.0 Hz). MS (ESI), m/z: 814.66
[MþNa]þ.
4b-[(4-(2-(3-Nitrophenoxy)propan-2-yl)-1H-1,2,3-triazol-
1-yl)]-4-desoxypodophyllotoxin 16
Yield 71.90%; white solid; mp 139–1418C; 1H NMR (400 MHz,
CDCl3) d 7.65–7.43 (m, 4H), 6.62 (d, 2H, J ¼ 8.0 Hz), 6.31 (s, 2H),
6.07–6.02 (m, 3H), 4.75 (d, 1H, J ¼ 4.8 Hz), 4.41 (s, 1H), 3.87 (s, 3H),
3.71 (s, 6H), 3.25 (s, 2H), 3.06–3.03 (m, 1H), 1.36 (s, 6H). MS (ESI),
m/z: 667.54 [MþNa]þ.
4b-[((2-Chlorobenzamide)methyl)-H-1,2,3-triazol-1-yl)]-
4b-[4-((2-Methoxy-4-(3-(2-methoxyphenyl)-
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 17
Yield 66.67%; white solid; mp 131–1358C; 1H NMR (400 MHz,
CDCl3) d 7.77 (s, 2H), 7.41 (s, 2H), 6.65 (s, 1H), 6.66 (s, 1H), 6.30
(s, 2H), 6.03 (d, 2H, J ¼ 9.6 Hz), 4.87 (s, 1H), 4.86 (s, 2H), 4.38 (t, 1H,
J ¼ 7.0 Hz), 4.15–4.09 (m, 2H), 3.82 (s, 3H), 3.76 (s, 6H), 3.25 (s, 2H),
3.00 (m, 1H). MS (ESI), m/z: 655.51 [MþNa]þ.
4-desoxypodophyllotoxin 23
Yield 68.55%; white solid; mp 139–1418C; 1H NMR (400 MHz,
CDCl3) d 8.13 (d, 1H, J ¼ 16. 0 Hz), 7.66–7.62 (m, 3H), 7.40 (d, 1H,
J ¼ 2.4 Hz), 7.02–6.94 (m, 2H), 6.78–6.74 (m, 1H), 6.64 (s, 1H), 6.69
(t, 1H, J ¼ 5.2 Hz), 6.30 (s, 2H), 6.09 (s, 1H), 6.02 (d, 2H, J ¼ 9.0 Hz),
5.35 (s, 2H), 4.75 (d, 1H, J ¼ 4.4 Hz), 4.40 (s, 1H), 3.97–3.81 (m, 8H),
3.76 (s, 6H), 3.27 (m, 2H), 3.02 (t, 1H, J ¼ 8.4 Hz). MS (ESI), m/z:
784.59 [MþNa]þ.
4b-[((4-Chlorobenzamide)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 18
Yield 59.88%; white solid; mp 134–1398C; 1H NMR (400 MHz,
CDCl3) d 7.77 (s, 2H), 7.41 (s, 2H), 6.65 (s, 1H), 6.66 (s, 1H), 6.30
(s, 2H), 6.03 (d, 2H, J ¼ 9.6 Hz Hz), 4.77 (s, 2H), 4.79 (d, 1H,
J ¼ 5.2 Hz), 4.45 (s, 1H), 3.81 (s, 3H), 3.76 (s, 6H), 3.27 (s, 2H),
3.04 (s, 1H). MS (ESI), m/z: 655.51 [MþNa]þ.
4b-[(4-(4-(3-(4-Fluorophenyl)acryloyl)-2-
methoxyphenoxy)methyl]-4-desoxypodophyllotoxin 24
Yield 71.12%; yellow solid; mp 145–1508C; 1H NMR (400 MHz,
CDCl3) d 7.80 (d, 1H, J ¼ 15.6 Hz), 7.67–7.63 (m, 3H), 7.55 (s, 2H),
7.12 (t, 2H, J ¼ 8.4 Hz), 6.64 (s, 1H), 6.69 (s, 1H), 6.30 (s, 2H), 6.09
(s, 1H), 6.02 (d, 2H, J ¼ 10.4 Hz), 5.36 (d, 2H, J ¼ 10.4 Hz), 4.75
(d, 1H, J 4.4 Hz), 4.40 (s, 1H), 3.94–3.76 (m, 12H), 3.26 (s, 1H), 3.02
(d, 1H, J ¼ 9.2 Hz). MS (ESI), m/z: 772.63 [MþNa]þ.
4b-[((4-(4-Acetyl-2-methoxyphenoxy)methyl)-1H-1,2,3-
triazol-1-yl))]-4-desoxypodophyllotoxin 19
Yield 75.44%; white solid; mp 145–1508C; 1H NMR (400 MHz,
CDCl3) d 7.59 (d, 2H, J ¼ 7.2 Hz), 7.33 (s, 1H), 7.10 (s, 1H), 6.64
(s, 1H), 6.69 (s, 1H), 6.30 (d, 2H), 6.10 (d, 1H, J ¼ 4.4 Hz), 6.03 (d, 2H,
J ¼ 10.4 Hz), 5.34 (s, 2H), 4.74 (d, 1H, J ¼ 5.2 Hz), 4.40 (s, 1H), 3.91
(s, 3H), 3.82 (s, 3H), 3.77 (s, 6H), 3.50 (d, 1H, J ¼ 5.2 Hz), 3.25 (t, 1H,
J ¼ 4.8 Hz), 2.57 (s, 3H). MS (ESI), m/z: 666.59 [MþNa]þ.
4b-[4-((2-Methoxy-4-(3-(4-(trifluoromethyl)phenyl)-
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin 25
Yield 67.33%; white solid; mp 131–1358C; 1H NMR (400 MHz,
CDCl3) d 8.14 (d, 1H, J¼ 16.0 Hz), 7.83–7.60 (m, 6H), 7.35 (s, 1H),
7.16 (d, 1H, J ¼ 8.4 Hz), 6.65 (s, 1H), 6.60 (s, 1H), 6.30 (s, 2H), 6.10
(d, 1H, J ¼ 4.4 Hz), 6.02 (d, 2H, J ¼ 10.4 Hz), 5.36 (s, 2H), 4.74
(d, 1H, J ¼ 4.8 Hz), 4.40 (s, 1H), 3.87–3.95 (m, 3H), 3.81 (s, 3H), 3.76
(s, 6H), 3.26–3.23 (m, 2H), 3.04–3.03 (d, 1H, J ¼ 5.2 Hz). MS (ESI),
m/z: 822.58 [MþNa]þ.
4b-[(4-((2-Methoxy-4-(3-(3,4,5-trimethoxyphenyl)-
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 20
Yield 66.33%; yellow solid; mp 122–1248C; 1H NMR (400 MHz,
CDCl3) d 7.52 (s, 1H), 7.13 (s, 2H), 7.56 (t, 1H, J ¼ 7.6 Hz), 7.45
(d, 1H, J ¼ 15.6 Hz), 7.34 (s, 1H), 7.16 (d, 1H, J ¼ 7.6 Hz), 6.88
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Podophyllotoxin Congeners as Potent Antitumor Agents
11
3 min, the cells were fixed in 70% ethanol at 48C in the dark. The
cells were then incubated with RNAase (0.1 mg/mL) at 378C for
30 min and stained with propidium iodide (2 mg/mL) for 30 min
in the dark. Finally, flow cytometric analysis was performed with
a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA,
USA) with excitation at 488 nm and emission at 630 nm. The
percentage of cell cycle distribution was determined using the
MODFIT software (Becton-Dickinson).
4b-[(4-((2-Methoxy-4-(3-(thiophen-2-yl)acryloyl)phenoxy)-
methyl)-1H-1,2,3-triazol-1-yl)-]-4-desoxypodophyllotoxin 26
Yield 60.98%; yellow solid; mp 124–1268C; 1H NMR (400 MHz,
CDCl3) d 7.96 (d, 1H, J ¼ 15.6 Hz), 7.65–7.61 (m, 2H), 7.42 (d, 1H,
J ¼ 5.2 Hz), 7.42–7.32 (m, 2H), 7.14–7.08 (m, 2H), 6.64 (s, 1H), 6.69
(s, 1H), 6.30 (s, 2H), 6.09 (d, 1H, J ¼ 4.4 Hz), 6.01 (d, 2H,
J ¼ 9.2 Hz), 5.33 (s, 2H), 4.74 (d, 1H, J ¼ 4.8 Hz), 4.40 (t, 1H,
J ¼ 8.0 Hz), 3.93 (s, 3H), 3.81 (s, 3H), 3.75 (s, 6H), 3.31–3.23
(m, 2H), 3.04 (d, 1H, J ¼ 8.8 Hz). MS (ESI), m/z: 760.40 [MþNa]þ.
Molecular docking
The simulation system was built based on the X-ray crystal
structure of the human Topo-II ATPase-AMP-PNP complex,
which was obtained from the Protein Data Bank (PDB – 1ZXM;
www.rcsb.org) and further modified for docking calculations.
Water molecules were removed and H atoms were added to
the structure. 3D structures of the podophyllotoxin derivatives
were generated and optimized by the Discovery Studio 2.1 pack-
age (Accelrys, San Diego, CA, USA). The receptor-grid files were
carried out using a grid-receptor generation program using
default settings after ensuring that the ligands and the protein
are in correct form. The GOLD program in the Discovery Studio
software was used to perform the docking simulations, which
allows full flexibility of the ligand.
The structures of the synthesized compounds were drawn in
chem3D with standard lengths and angles. The Gasteiger–
Huckel charge, with a distance-dependent dielectric function,
and AM1 docking calculations were applied for the minimiz-
ation of the molecules. To modify the structure of Topo-II, miss-
ing atoms, bonds, and contacts were checked, hydrogen atoms
were added to the enzyme structure, and water molecules were
removed. Intercalation models were optimized using the
CHARMm forcefield with the added parameters. After perform-
ing the docking simulation, the scores of the docked conformers
were ranked and the best four compounds (10–13) were specu-
lated regarding the detailed binding modes in the cavity.
4b-[(4-((2-Methoxy-4-(3-(thiophen-3-yl)acryloyl)phenoxy)-
methyl)-1H-1,2,3-triazol-1-yl)]-4-desoxypodophyllotoxin 27
Yield 66.09%; yellow solid; mp 134–1398C; 1H NMR (400 MHz,
CDCl3) d 7.82 (d, 1H, J ¼ 15.6 Hz), 7.63–7.58 (t, 3H, J ¼ 8.0 Hz),
7.44–7.43 (d, 1H, J ¼ 4.8 Hz), 7.38–7.34 (m, 2H), 7.13 (d, 1H,
J ¼ 8.4 Hz), 6.63 (s, 1H), 6.69 (s, 1H), 6.30 (s, 2H), 6.09 (d, 1H,
4.4 Hz), 6.01 (d, 2H, J ¼ 10.0 Hz), 5.31 (s, 2H), 3.90 (s, 3H), 3.81
(s, 3H), 3.79 (s, 6H), 3.29–3.17 (m, 2H), 3.04–2.99 (m, 1H,
J ¼ 9.2 Hz). MS (ESI), m/z: 760.43 [MþNa]þ.
4b-[(4-((2-Methoxy-4-(3-(pyridin-4-yl)acryloyl)phenoxy)-
methyl)-1H-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin 28
Yield 60.48%; white solid; mp 136–1408C; 1H NMR (400 MHz,
CDCl3) d 7.55 (s, 2H), 7.40–7.33 (m, 1H), 7.10–7.08 (d, 2H,
J ¼ 8.4 Hz), 6.64 (s, 1H), 6.69 (s, 1H), 6.30 (s, 2H), 6.08 (d, 1H,
J ¼ 4.4 Hz), 6.03 (s, 1H), 6.00 (s, 1H), 5.33 (s, 2H), 4.73 (d, 1H,
J ¼ 5.2 Hz), 4.40 (s, 1H), 3.93 (s, 3H), 3.82 (s, 3H), 3.76 (s, 6H), 3.25
(s, 2H), 3.02–2.97 (m, 1H). MS (ESI), m/z: 756.78 [MþNa]þ.
4b-[4-((4-(3-(4-(Dimethylamino)phenyl)acryloyl)-
2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)]-
4-desoxypodophyllotoxin 29
Yield 65.97%; brown solid; mp 146–1508C; 1H NMR (400 MHz,
CDCl3) d 7.84 (d, 1H, J ¼ 15.6Hz), 7.55–7.54 (m, 2H), 7.40–7.33
(m, 1H), 7.08 (d, 1H, J ¼ 8.4 Hz), 6.64 (s, 1H), 6.63 (s, 1H), 6.30
(s, 1H), 6.08 (d, 1H, J ¼ 3.6 Hz), 6.03 (d, 2H, J ¼ 10.8 Hz), 5.33
(s, 2H), 4.73 (d, 1H, J ¼ 5.2 Hz), 4.40 (s, 1H), 3.94–3.91 (m, 3H), 3.82
(s, 3H), 3.76 (s, 6H), 3.28–3.25 (m, 2H), 3.02–2.97 (m, 1H), 1.24–0.86
(m, 6H). MS (ESI), m/z: 797.46 [MþNa]þ.
We are grateful to the National Key Programs of China during the 12th
Five-Year Plan period (2012ZX09103101-033).
The authors have declared no conflict of interest.
Antiproliferative activity
References
MTT assays were carried out using HepG2, MKN-45, NCI-H1993,
and B16 tumor cell lines. The four cell lines were maintained in
RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin,
and 100 mg/mL streptomycin in a 95% air/5% CO2 humidified
atmosphere at 378C. The cells were seeded into 96-well culture
plates at a density of 1 ꢂ 104 cells/well with 500 mL culture
medium. The cells were pretreated with the compounds 3–29,
washed with PBS and incubated in 10 mL MTT (5 mg/mL) dis-
solved in DMSO for 4 h. The optical density was recorded on an
ELISA reader at 570 nm. Cell growth was calculated by subtract-
ing the mean OD value of the respective blank from the mean OD
value of the experimental set.
[1] A. D. Kinghorn, M. F. Balandrin, in The Human Medicinal Agents
from Plants, American Chemical Society, Washington, DC
1993, p. 170.
[2] T. Imbert, Biochemie 1998, 80, 207–222.
[3] D. M. Reddy, J. Sriniva, G. Chashoo, A. K. Saxena, H. M.
Kumar, Eur. J. Med. Chem. 2011, 46, 1983–1991.
[4] A. Kamal, B. A. Kumar, P. Suresh, N. Shankaraiah, M. S.
Kumar, Bioorg. Med. Chem. Lett. 2011, 21, 350–353.
[5] A. Knmal, P. Suresh, A. Mallareddy, B. A. Kumar, P. V. Reddy,
P. Raju, J. R. Tamboli, T. B. Shaik, N. Jain, S. V. Kalivendi,
Bioorg. Med. Chem. 2011, 19, 2349–2358.
[6] A. M. Wilstermann, R. P. Bender, M. Godfrey, S. Choi,
C. Anklin, D. B. Berkowitz, N. Osheroff, D. E. Graves,
Biochemistry 2007, 46, 8127–8225.
Cell cycle analysis
The HepG2 cell line was used by incubating the cells at
4 ꢂ 105 cells/well with the tested compounds (0.1, 0.2, and
0.4 mM) for 24 h and then harvesting by trypsinization. The cells
were washed twice with ice-cold PBS. After centrifugation for
[7] 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. 1990, 33, 1364–1368.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

12
J. Chen et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
[8] H. Stahelin, A. Von Wartburg, Prog. Drug Res. 1989, 33, 169–
173.
[19] M. Gordaliza, M. Angels-Castro, J. M. Miguel del Corral,
´ ´ ´
M. L. Lopez-Vazquez, P. A. Garcıa, A. San Feliciano, M. D.
´
´
Garcıa-Gravalos, H. Broughton, Tetrahedron 1997, 53, 15743–
[9] K. J. Chavez, X. H. Feng, J. A. Flanders, E. Rodriguez, F. C.
Schroeder, J. Nat. Prod. 2011, 74, 1293–1297.
15760.
[20] M. A. Castro, J. M. Corral, M. Gordaliza, P. A. Carcia, M. A.
Zurita, A. S. Feliciano, Bioorg. Med. Chem. 2007, 15, 1670–
1678.
[10] B. You, B. Trabchand, P. Girard, C. Falandry, B. Ribba,
S. Chabaud, P. J. Souquet, C. F. Isabelle, T. L. Veronique,
C. Fournel, M. Tod, F. Freyer, Lung Cancer. 2008, 62, 261–272.
[21] D. Imperio, T. Pirali, U. Galli, F. Pagliai, L. Cafici, P. L.
Canonico, G. Sorba, A. A. Genazzani, G. C. Tron, Bioorg.
Med. Chem. 2007, 15, 6748–6757.
[11] C. M. Das, P. E. Zage, P. Taylor, D. Aguilera, E. A. Wolff, D. Lee,
V. Gopalakrishnan, Eur. J. Cancer 2010, 46, 2771–2780.
[12] J. H. Li, Y. Chen, J. S. Wang, W. Kong, Y. H. Jin, Chem. Res. Chin.
Univ. 2008, 24, 597–602.
[22] W. H. Binder, R. Sachsenhofer, Macromol. Rapid Commun.
2007, 28, 15–54.
[13] A. Longhi, E. Mariani, J. J. Kuehn, Eur. J. Integr. Med. 2009, 1,
27–33.
[23] P. B. Reddy, S. K. Agrawal, S. Singh, B. A. Bhat, A. K. Saxena,
H. M. Kumar, G. N. Qazi, Chem. Biodivers. 2008, 5, 1792–
1802.
[14] L. S. Thiago, E. S. Costa, D. V. Lopes, I. B. Otazu, A. E. Nowill,
F. A. Mendes, D. M. Portilho, J. G. Abreu, C. S. Mermelstein,
A. Orfao, M. I. D. Rossi, R. Borojevic, Biomed. Pharmacother.
2010, 64, 63–72.
[24] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed.
2001, 40, 2004–2021.
[25] P. Boeck, C. B. Falcao, P. C. Leal, R. A. Yunes, V. C. Filho, E. C.
Santos, B. R. Bergmann, Bioorg. Med. Chem. 2006, 14, 1538–
1545.
[15] S. L. Pitts, M. J. Jablonksy, M. Duca, D. Dauzonne,
C. Monneret, P. B. Adrimondo, C. Anklin, D. E. Graves,
N. Oscheroff, Biochemistry 2011, 50, 5058–5066.
[26] D. Leroy, A. V. Kajava, C. Frei, S. M. Gasser, Biochemistry 2001,
40, 1624–1634.
[16] D. Passarellla, A. Giardini, B. Petretto, F. Gabriele, A.
Sacchetti, A. Silvani, C. Ronchi, G. Cappelletti, D. Caetelli,
C. Blorlak, B. Danieli, Med. Chem. 2008, 16, 6269–6285.
[27] A. K. Patel, S. Patel, P. K. Naik, A. T. Vihar, P. K. Distt, Curr. Res.
Biol. Sci. 2010, 2, 13–23.
[17] J. Shah, J. R. Chen, D. Chow, Int. J. Pharm. 1995, 113, 103–111.
[28] A. C. Nagy, D. Battogtokh, K. C. Chen, B. Novak, J. J. Tyson,
Biophys. J. 2006, 90, 4361–4379.
[18] K. Kobayashi, M. J. Ratain, Cancer Chemother. Pharmacol. 1994,
34, S64–S68.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com