Synthesis and biological evaluation of a series of podophyllotoxins derivatives as a class of potent antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2012.09.009

A series of eight novel podophyllotoxin derivatives were designed, synthesized and evaluated for biological activities. The antiproliferative activities were tested against a panel of human cancer cell lines (K562, SGC, Hela and HepG) and the inhibition of tubulin polymerization was also evaluated. Compound 8e displayed significant antiproliferative activities for all four cell lines and strong levels of tubulin polymerization inhibition effect. Combined with cell apoptosis and cell cycle analysis, it demonstrated that compound 3e that effectively interfere with tubulin dynamics prevent mitosis in cancer cells, leading to cell cycle arrest and, eventually dose dependent apoptosis. All experimental measurements were also supported by molecular docking simulations of colchicine binding site, which revealed the governing forces for the binding behavior and a good relationship with anti-tubulin activity and antiproliferative activities. The synthesis and biological studies provided an interesting new class of antitubulin agents for development of lead compounds and also a direction for further structure modification to obtain more potent anti-cancer drugs.

Full text of DOI:10.1016/j.bmc.2012.09.009

Bioorganic & Medicinal Chemistry 20 (2012) 6285–6295
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a series of podophyllotoxins derivatives
as a class of potent antitubulin agents
Yingqian Liu a, Dongfeng Wei b, Yonglong Zhao a, Weidong Cheng b, Yan Lu a, Yaqiong Ma a, Xin Li c,
Chao Han a, Yanxia Wei b, Huiming Cao a, Chunyan Zhao a,
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b School of Basic Medicine, Lanzhou University, Lanzhou 730000, China
c College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2012
Revised 4 September 2012
Accepted 6 September 2012
Available online 13 September 2012
A series of eight novel podophyllotoxin derivatives were designed, synthesized and evaluated for biolog-
ical activities. The antiproliferative activities were tested against a panel of human cancer cell lines (K562,
SGC, Hela and HepG) and the inhibition of tubulin polymerization was also evaluated. Compound 8e
displayed significant antiproliferative activities for all four cell lines and strong levels of tubulin
polymerization inhibition effect. Combined with cell apoptosis and cell cycle analysis, it demonstrated
that compound 3e that effectively interfere with tubulin dynamics prevent mitosis in cancer cells, leading
to cell cycle arrest and, eventually dose dependent apoptosis. All experimental measurements were also
supported by molecular docking simulations of colchicine binding site, which revealed the governing
forces for the binding behavior and a good relationship with anti-tubulin activity and antiproliferative
activities. The synthesis and biological studies provided an interesting new class of antitubulin agents
for development of lead compounds and also a direction for further structure modification to obtain more
potent anti-cancer drugs.
Keywords:
Podophyllotoxin derivatives
Microtubules
Antiproliferative
Docking
Molecular dynamic
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
are strongly associated with their stability, which is often regu-
lated by the interaction of various small molecules and tubulin
Microtubules (MTs), constructed from a heterodimer of two
highly homologous proteins known as - and b-tubulin, are ubiq-
proteins. The dynamics interference of tubulin induced by some
natural or synthetic substances has been proven to be clinically
useful for designing anticancer agents.7,8 The application of poly-
merization or depolymerization agents can normalize abnormal
tumor vasculature and leads to more efficient need to develop
new tubulin-targeted compounds for chemotherapy.9–11
a
uitous, essential cytoskeletal polymers in all eukaryotic cells.1 The
microtubule system plays a crucial role in a broad range of cellular
processes, including the mitosis, exocytosis, maintenance of cellu-
lar morphology and active transport of cellular components
throughout the cytoplasm.2,3 Especially, it has an essential role in
cell division because microtubules are the key component for for-
mation of the mitotic spindle apparatus, providing the mechanical
force required for chromosome separation during mitosis.4 It made
microtubules an attractive and successful target to treat many
types of malignancies. In this regard, tubulin, as the main compo-
nent of the microtubules represents an important target to anti-
cancer therapy. The discovery for new compounds with stabilize
or destabilize microtubules activity is considered one of the most
promising ways for treating cancer disease.5
Now there were a large number of chemically different tubulin
binding agents targeting the vascular system of tumors and these
exogenous ligands did dramatic effects on microtubules, often
interacting directly with tubulin to either stabilize (such as
paclitaxel)12,13 or destabilize microtubules (such as colchicine).14,15
These ligands can disrupt the dynamics of polymerization for
microtubules involved in cell division.16 The specific effects on the
microtubules dynamics were complex and depended on the differ-
ent binding site, including taxol site, vinblastine site and colchicine
sites.17 For example, colchicine generally showed potent inhibition
effects of polymerization by blocking colchicine’s binding site and
rapidly shutting down existing tumor vasculature.13,18 And there
is conclusive evidence that microtubule disruption affected by
colchicine caused cell cycle arrest mainly at the G2/M phase, and
subsequently induced apoptosis, a cell suicide mechanism.19
Indeed, the findings that some tubulin binding agents targeting
the vascular system of tumors has extended their use.20–22 In this
Microtubules are a dynamic cellar compartment in both neo-
plastic and normal cells. The dynamicity is characterized by the
continuous turnover of a,b-tubulin heterodimers in the polymeric
microtubules.6 Generally, most of the biological functions of MTs
⇑
Corresponding author. Tel.: +86 931 8915686; fax: +86 931 8915685.
E-mail address: zhaochy07@lzu.edu.cn (C. Zhao).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.009

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Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
regard, podophyllotoxin behaves in the same way as colchicine, the
binding of which to tubulin results in partial unfolding of the sec-
ondary structure of b-tubulin at the carboxy terminus and prevents
polymerization.23,24 Podophyllotoxins (1), as naturally aryl tetralin
lignan which has been found in a variety of plants, including Podo-
phyllum peltatum and Juniperus Sabina25,26 are generally believed
to be an effect agent which is able to inhibit tubulin assembly by
binding with its colchicine domain. Furthermore, the microtubule
disruption affected by podophyllotoxins leads to cell cycle arrest
and, eventually dose dependent apoptosis caused cell cycle ar-
rest.27,28 For now, research has identified podophyllotoxin and
their derivatives as clinically useful drugs against various cancers,
including small cell lung cancer, testicular carcinoma, lymphoma,
and Kaposi’s sarcoma.29,30 Considering the cyclolignanskeleton,
nearly every ring of the molecule (A to E) has been modified. Mod-
ification of the podophyllotoxin structure has revealed structural
features critical for anticancer activity: (a)the 4b-configuration is
essential with various substituents at C(4); (b) the trans-lactone
polymerization inhibitors interacting with the colchicine-binding
site. The synthesis and biological studies helped to develop a class
of antitubulin agents for probing lead compounds and also a direc-
tion for further structure modification to obtain more potent anti-
tumor drugs.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, starting from podophyllotoxin 1, propar-
gyl group was firstly introduced in the 4-position of 1 upon reac-
.
tion with propargyl alcohol in the presence of BF3 Et2O to give
intermediate 4b-O-propargylpodophyllotoxin 7 in 87% yield. Sub-
sequently, we applied a highly efficient copper-catalyzed three-
component coupling reaction, in which 7 react with methanesulfo-
nyl azide and a wide range of amines to afford twelve novel 4b-O-
Sulfonyl amidine derivatives of 4-deoxypodophyllotoxin 3a–h in
D ring with 2a,3b-configuration is very important; (c) the dioxo-
lane A ring is optimal; and (d) the free rotation of ring E is required.
It was demonstrated that an important role of various C-4 substi-
tutions in the activity profiles of podophyllotoxin analogues and
the feasibility of optimizing this compound class through rational
C-4 modification.31–34
Therefore, we introduced the sulfonylamidine groups to podo-
phyllotoxin via a Cu-catalyzed reaction in the present study. All
the synthesized podophyllotoxin derivatives were then screened
for anticancer activity against a panel of four human cancer cell
lines. Biological evaluations including cell cycle, cell apoptosis
analysis and the direct assay for inhibition of polymerization were
constructed to probe the interference with tubulin dynamics. All
experimental measurements were also supported by computa-
tional research including docking and molecular dynamics, indi-
cated that the podophyllotoxin derivatives can act as tubulin
Table 1
In vitro Activity of sythesized Compounds (3a–3h) and positive controls (Colchicine
and Podophyllotoxin) against the K562, SGC, HeLa and HepG Cell Lines
IC50
SGC-7901
2.87 0.03
(
l
M)
HeLa
2.63 0.03
K562
HepG2
3a
3b
3c
3d
3e
3f
3g
3h
2.96 0.03
2.14 0.04
12.07 0.04 19.16 0.02 11.23 0.02 17.88 0.02
9.01 0.03 18.20 0.03 10.37 0.02 57.14 0.01
8.34 0.02 11.21 0.04
5.99 0.03 20.79 0.02
1.01 0.02
2.61 0.02
3.48 0.03
5.18 0.02
0.55 0.02
4.51 0.03
1.36 0.01
3.21 0.02
4.31 0.01
3.71 0.03
0.05 0.02
0.34 0.02
0.75 0.03
4.33 0.01
3.75 0.02
2.58 0.02
0.10 0.02
0.14 0.04
0.79 0.01
1.95 0.02
2.87 0.03
4.17 0.02
0.02 0.03
0.51 0.02
Colchicine
Podophyllotoxin
Scheme 1.

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
6287
suitable yields. The structures of compounds 3a–h were well char-
48 h. For screening potential anti-proliferation effects, colchicine
and podophyllotoxin were treated as positive controls. As shown
in Table 1, the IC50 values derived from in vitro screening studies
of the synthesized compounds and the controls were summarized.
The term of IC50 was defined as the compound concentration
required to inhibit tumor cell proliferation by 50% and data are ex-
pressed as the Mean SE from the dose-response curves of at least
three independent experiments. As shown, compound 3e pos-
sessed highest antiproliferative activities against all four cell lines
acterized by using 1H NMR, 13C NMR, IR and HR-MS.
2.2. Effects on in vitro proliferation of the tumor cell lines
The effect of the podophyllotoxin derivatives on the prolifera-
tion of tumor cancer cell lines was evaluated using four human tu-
mor cell lines: human leukemia cell line K562, human gastric
cancer cell line SGC-7901, human cervical carcinoma cell line HeLa
and Human hepatocellular liver carcinoma cell line HepG2, which
with IC50 values of 1.01, 1.36, 0.75 and 0.79
pounds 3b, 3c and 3d showed poor activities.
lM, while the com-
were treated with the range of 0 to 100 lM of test compounds for
Figure 1. (a) Apoptosis effect on human leukemia K562 cell line induced by compound 3e. The cells were incubated in media containing various concentration of 3e (0,
0.375 M, 1.2 M and 3 M) for 24 h. Cells and the amount of apoptotic cells was determined by flow cytometry using FITV-Annexin V/PI staining. (b) The rate of apoptotic
cells as detected by flow cytometry. Cells in B1 and B2 quadrants were considered to be apoptotic cells. The experiment was repeated three times.
l
l
l

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Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
2.3. Effects on apoptosis
and 60 lM) on tubulin polymerization were monitored with col-
chicine and podophyllotoxin as reference compounds for compar-
ison (Fig. 3b). The inhibition assay on the tubulin polymerization
for the synthesized compounds was performed and 3e showed
the highest activity, which was consistent with the data in the cell
proliferation assay. As expected, the compound 3e on different
concentrations caused a dose-dependent inhibition of tubulin
polymerization in a similar way with positive control colchicine
and podophyllotoxin as shown in Figure 3c. Compound 3e, colchi-
cine and podophyllotoxin.showed 50% tubulin inhibition effects at
To probe whether compounds exhibiting high antiproliferative
activity induced apoptosis of K562 cells, the cells were treated with
the most potent antiproliferative compound 3e to determine the
percentage of cells. Flow cytometry was used to generate an apop-
totic cell scatterplot of the control groups treated with compound
3e (0, 0.375 lM, 1.2 lM and 3 lM) for 24 h (Fig. 1a). The cell scat-
ter could be composed of four subgroups as follows: B1quadrant
was the late apoptotic cells, B2 quadrant was the early apoptotic
cells, B3quadrant was the living cells, and B4 quadrant was the ne-
crotic cells. For the different concentration of compound 3e, the
cells count in B1 and B2 quadrants were considered to be apoptotic
cells and the total percentages were 0.98%, 22.19%37.04% and
66.0%, respectively (Fig. 1b). The results showed that the effect
was observed in a dose-dependent manner and the apoptotic rates
of K562 cells increased with the increase of concentration of 3e
(Fig. 1b). It demonstrated that the most active podophyllotoxin
derivatives 3e induced apoptosis of cells and thus as expected,
caused the anticancer effects.
18.19, 23.35 and 24.03 lM (IC50), respectively. The results strongly
implicated a direct interaction between the compound 3e and
tubulin. It can be concluded that the antiproliferative activity of
the compounds may derive from an interaction with tubulin and
an interference with microtubule disassembly.
In addition, as we know, the classic tubulin binding agents, such
as podophyllotoxin and their derivates, were recognized to have
damaging effects on vasculature. Thus, the effect of 3e on the puri-
fied microtubule protein was also examined using transmission
electron microscope. The structures of microtubules treated with
DMSO (control) and 3e were visualized. The normal interphase
microtubules with DMSO solution exhibited typical reticular for-
mation (Fig. 4a) on polymerization state at 37 °C, while the poly-
merization of microtubule was disturbed after treatment with 3e
2.4. Analysis of cell cycle
From the in vitro tests it was found that 3e significantly inhib-
ited the growth of four human cancer cell lines. To determine
whether the high anticancer effects of the compounds were caused
by cell cycle accumulate at a certain phase, the cell cycle progres-
sion were therefore analyzed in K562 cells cultured for 36 h in the
at a concentration of 3 lM. The complete inhibition effect of micro-
tubules polymerization was illustrated in Figure 4b and the dis-
crete club shaped cytoskeleton of tubulin was observed. The
phenomena, combined with the cellular microtubule depolymeriz-
ing effects and the ability of 3e to directly inhibit purified tubulin
assembly, provided strong evidence that loss of function of micro-
tubules in interphase cells treated with 3e may prevent these cells
from progressing into mitosis.
In summary, the relation of activities for the inhibition of micro-
tubule polymerization and inhibition of proliferation provided an
indication of tight linkage of antiproliferative effects and the com-
pound’s tubulin-dependent mechanisms of action. The antiprolif-
erative activity of the compounds may derive from an interaction
with tubulin and an interference with microtubule assembly. Com-
pounds that effectively interfere with tubulin dynamics prevent
mitosis in cancer cells, leading to cell cycle arrest and, eventually,
apoptosis.
presence of increasing concentration of compound 3e (0, 0.375
lM,
1.2 M and 3 M) (Fig. 2a). As we known, the podophyllotoxin
l
l
compounds were reported to exhibit strong activity on tubulin
binding, which should induce the alteration of cell cycle parame-
ters leading to a preferential G2/M blockade.27,28,35 It can be seen
from Fig. 2b, the cells accumulations in G2 phase of 10.87%,
50.91% and 82.49% were observed with 0.375
M treatment of 3e, respectively, compared with 4.87% in un-
treated cultures. Moreover, the greatest accumulation of cells ar-
rested in the G2/M stage occurred at M, and same
lM, 1.2 lM and
3
l
3
l
a
concentration led to the greatest apoptosis. In parallel to the G2/
M block (Fig. 2b), the cell cycle analysis showed a clear increase
of proportion of sub-G1 cells, which always suggested the exten-
sive DNA breakage. As shown, 1.17% of the cells were in sub-G1
phase for untreated cultures, whereas 2.26%, 7.43% and 13.73% of
2.6. Analysis of molecular docking
cells were in the sub-G1 phase with 0.375 lM, 1.2 lM and 3 lM,
respectively. In addition, a simultaneous sharp decrease in S and
G1 cells occurred (Fig. 2b). All effect was observed in a dose-depen-
dent manner. Thus, by two independent methods, it demonstrated
that compound 3e can obviously inhibit the proliferation of cancer
cells K562 by arresting the cell cycle in a certain phase G2/M and
inducing apoptosis.
As shown above, it could be hypothesized that the activity of
podophyllotoxin-derived compounds could be due to their poten-
tial effects on tubulin depolymerisation. In order to strengthen
the hypothesis, compound 3e, the most active compound, was
used as the representative one to investigate the binding modes
of synthesized podophyllotoxin derivatives. Docking modes be-
tween 3e and colchicine binding sites has been constructed and re-
2.5. Analysis of the inhibition effects of microtubule
polymerization
fined using molecular dynamics (MD) approach to offer a
molecular level explanation with the binding behavior and to ob-
tain more precise ligand–receptor models in the state close to nat-
ural conditions.
The average structure of compound 3e-tubulin complex within
the last 1 ns of the molecular dynamics trajectories was employed
as refined binding model. As shown in Figure 5a, compound 3e was
exactly located within the colchicine binding pocket, the position
To further explore whether the growth-inhibitory effect of the
novel prodophyllotoxin compounds was related to an interaction
with the tubulin system, an inhibition assay on microtubule poly-
merization in vitro was performed. As shown in Figure 3a, the
tubulin protein extracted from fresh goat brain tissue by two cycle
polymerization-depolymerizations (as expressed in Experimental
section) was purified to test the activity based on their polymeriza-
tion and polymerization inhibition curve on different temperature
(0 °C and 37 °C) by the fluorescent intensity. As indicated by the
shape of curve, the purified protein showed perfect activities on
polymerization for further assay. The inhibition effects of the most
activity compound 3e with different concentrations (0, 10, 20, 40
of which was between the interfaces of
as blue circle. The trimethoxyphenyl ring (E ring) of 3e was in-
serted in bigger hydrophobic cavity formed by b:Cys241,
a- and b-subunit described
a
b:Leu248, b:A0la250, b: Lys254, b:Asn258, b:Ala317, b:Lys352
and b:Thr353, the other was suited in a less hydrophobic cavity
formed by
a:Val177, a:Ser178, a:Thr179, a:Tyr224 (Fig. 5a and
Fig. 5b).This result indicated that the hydrophobic property was

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
6289
Figure 2. (a) Effects of compound 3e on the cell cycle distribution of K562 cells. The cells were cultured for 36 h without the compound or with compound 3e of 0.375
1.2 M, and 3 M inhibition of cell growth; (b) Quantitative analysis of cell cycle phase.
lM,
l
l
one of the main forces governing the interaction between 3e and
tubulin. On the other hand, the interaction was not exclusively
hydrophobic. It should be noted that there were several ionic res-
Figure 5d, it can be found that the docking pocket was electro neg-
ativity. In addition, amino residues b: Cys241 were just in the suit-
able position to form a hydrogen bond with O atom of 3e. Hence,
the amino acid residues with benzene ring can match that of 3e
in space to firm the conformation of the complex.
idues (b:Lys254,b:Lys352), and polar residues
(a:Ser178a:-
Thr179, :Tyr224,b:Asn258, b:Thr353) in the proximity to 3e,
a
which played important roles in stabilizing the tubulin-3e complex
via electrostatic interaction and hydrogen bonds (Fig. 5a and
Fig. 5c). The hydrogen-bonding or electrostatic interaction acted
as an ‘anchor’, intensely determining the 3D space position of 3e
in the binding pocket and facilitating the hydrophobic interaction
of the ligand 3e with the side chain of protein. As shown in
2.7. Analysis of binding free energy
In order to gain insight into the stability and dynamics proper-
ties of the complexes, further information about the forces in-
volved in ligand binding can be obtained by analyzing the free

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Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
Figure 3. (a) Polymerization and inhibition of polymerization curve for tubulin pProtein; (b) Pattern of various concentrations of compound 3e on in vitro tubulin
polymerization assay; (c) Inhibition of tubulin polymerization by Compound 3e, Podophyllotoxin (Control) and Colchicine (Control). All compounds were tested at different
concentrations (0, 10, 20, 40 and 60 lM). IC50 values were calculated based on the tubulin polymerization assay.
Figure 4. (a) Normal interphase microtubules; (b) Loss of interphase microtubules induced by compound 3e.

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
6291
Figure 5. Binding mode between compound 3e and tubulin. (a) 2D view; (b) Hydrophobic potentials plot on molecular surfaces (the colors of blue, green, white and red
represent decreasing of hydrophobic potentials); (c) Electrostatic potentials plot on molecular surfaces (the colors of blue, white and red represent decreasing of electrostatic
potentials); (d) 3D view.
energy contributions based on a more physically rigorous method
MM-GBSA.
In addition, the binding affinity of 3e with tubulin was further
calculated. The calculated free energy contributions for tubulin-3e
complex were presented in Table 2. As shown, the term of
Firstly, some properties were examined by means of root-
mean-square deviations (RMSD) values of different system with
respect to the initial structure. As shown in Figure 6, the evolution
of the RMSD values versus time for tubulin-3e complex using ini-
tial docking mode as a reference structure was obtained from the
MD simulation. The calculated average RMSD fluctuation values
of the complex was 2.5 Å after equilibrium indicating that the
tubulin-3e complex structure was stable after 4 ns of simulation
time.
D
Emm, ele was negative, which generally means electrostatic inter-
actions lead to favorable binding, whereas the term of Gsol, pol
D
was positive, which means the electrostatic component of the
solvation free energies is consistently unfavorable for binding.
The data of non-polar component (
negative indicating that the contribution is favorable. In addition,
from the quantitatively analysis of the non-polar ( Emm, vdw +
Gsol, npol) and the electrostatic ( Emm, ele + Gsol, pol) contribu-
DEmm, vdw + DGsol, pol) was
D
D
D
D

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Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
3.5
3
2.5
2
1.5
1
0.5
0
0
1000
2000
3000
4000
5000
Time
Figure 6. The RMSD values obtained during 5 ns of MD simulation for tubulin-3e
complex.
tions for 3e-tubulin complex, we came up with the conclusion that
the interaction between 3e and tubulin was dominated by more
favorable nonpolar interactions, which was common for non-cova-
lent association, rather than by electrostatic interactions.
Furthermore, to probe the importance of each residue, the en-
ergy contribution difference analysis was performed (Fig. 7). As
shown, the most important residues were obtained with
a:Thr179, a:Tyr224, b:Leu248, b:Leu255,b:Cys241, and b:Ala250,
for which their contributions to the binding free energy were more
than ꢀ10 kcal/mol. Interestingly, these residues were found to
form the core region of the colchicine binding site. Here, as ex-
pected, strongly favorable contributions to the binding were asso-
ciated with the residues located in the active site. In addition,
residues
b:Ala354 contributed weakly to the binding and
:Tyr224, b:Leu248, b:Leu255,b:Cys241, and b:Ala250 made strong
contributions (Fig. 7a). To represent the results more intuitively,
the energy contributions of 6 key residues ( :Thr179, :Tyr224,
b:Leu248, b:Leu255,b:Cys241, and b:Ala250) were plotted in the
active site for the 3e-tubulin systems (Fig. 7b). It can be found that
the non-polar residues (b: Leu248, b: Leu255, b: Ala250) contrib-
a
:Val177, b:Asn258, b:Ala317, b:Asn349, b:Lys352,
Figure 7. (a) Energy difference of each residue contribution to the binding of
compounds 3e; (b) Comparison of interactions between compound 3e for key
residues in the active site: black bar: Van der Waals and nonpolar solvation energy
a
:Thr179,
a
(
(
D
D
Emm, vdw,
Emm, ele,
D
Gsol, npol) and red bar: electrostatic and polar solvation energy
D
Gsol, pol). The negative values are favorable and positive values are
a
a
unfavorable for binding.
inhibition assay on the inhibition of polymerization for all of the
series compounds demonstrated that 3e exhibited the strong
inhibition levels of polymerization for tubulin assemblage. All
above results indicated that the synthesized compounds which
effectively interfered with tubulin dynamics can prevent mitosis
in cancer cells, thus leading to cell cycle arrest and, eventually a
dose dependent apoptosis. In addition, the analysis of docking
and molecular dynamic was consistent with the observed
biological analysis for 3e. The binding behavior of 3e to tubulin
was mainly stabilized by hydrophobic interaction, together with
hydrogen-bonding interactions with Cys241residue of b-tubulin.
The synthesis and biological studies provided a class of lead
compounds worthy of further investigation to obtain more potent
anti-cancer drugs.
uted more to the binding free energy than the polar residues (
Thr179, : Tyr224, b: Cys241). Moreover, for all of the 6 key resi-
dues, hydrophobic interactions (black bar in the Figure) did favor
for the binding behavior, while for residues : Thr179 and b:
a:
a
a
Leu248, the electrostatic interactions (red bar in the Figure) did
disfavor for binding. From these results, we can conclude that
hydrophobic interactions played the most important role in the
binding affinity with 3e. Thus, we believed that the hydrophobic
interaction was the driven forces for the interaction between com-
pound 3e and tubulin protein although there were hydrogen bonds
and electrostatic interactions.
3. Conclusion
In the present work, we synthesized a series of podophyllotoxin
derivatives. The growth inhibition activities against various human
cancer cell lines K562, SGC-7901, HeLa and HepG2 were tested and
their biological mechanisms of activity of these synthetic com-
pounds were investigated. Through the cell cycle and apoptosis
analysis, compound 3e which also showed the best proliferation
activities exhibited strong ability to inhibit the proliferation of can-
cer cells K562 by arresting a massive accumulation in a in a certain
phase G2/M and inducing a high levels of apoptosis. The direct
4. Experiments
4.1. Chemistry
Melting points were taken on a Kofler melting point apparatus
and are uncorrected; IR spectrawere obtained on NIC-5DX spectro-
photometer; mass spectral analysis was performed on a ZAB-HS
and BrukerDaltonics APEXII49e instrument. 1H NMR and 13C
NMR spectra were recorded at 400 MHz and 100 MHz on a Bruker
Table 2
The binding free energy components for tubulin-3e complex
Component
D
Emm, ele
D
Emm, vdw
D
Gsol, pol
D
Gsol, npol
D
Gele, tol
D
Gnp, tol
D
Gbind
Energy (kal/mol)
ꢀ23.20
ꢀ83.56
68.77
ꢀ9.59
45.57
ꢀ93.15
ꢀ47.58

Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
6293
AM-400 spectrometer using TMS as reference (Bruker Company,
USA). Podophyllotoxin1 was isolated from a Chinese medicinal
herb Juniperus Sabina Linnaeus and served as the starting material
for the preparations of all the derivatives (shown in Scheme 1).
H-8), 6.24 (s, 2H, H-20, 60),5.98 (d, J = 9.6 Hz, 2H, OCH2O), 4.61 (d, J =
4.8 Hz, 1H, H-4), 4.54 (m, 1H, 11a-H), 4.37–4.22 (m, 2H, 11b, 1-H),
3.79 (s, 3H, 40-OCH3), 3.72 (s, 6H, 30,50-OCH3), 3.57 (m, 2H, 100-H),
3.25-3.20 (m, 2H, 2,3-H), 3.02 (s, 3H, Ms-CH3), 2.83-2.16 (m, 4H,
piperidine-CH2),1.74-1.25 (m, 6H, 200-Hand piperidine); 13C NMR
(100 MHz, CDCl3) d: 174.8, 163.7, 152.6 (2C, C-30, 50), 148.4,
146.9, 137.2, 135.1, 131.7, 129.5, 109.6, 108.3, 106.9 (2C, C-20, 60),
101.5, 75.2, 67.8, 66.1, 60.7, 56.2 (2C, C-30, 50-OCH3), 48.2 (2C, R1-
10,50), 45.4, 43.9, 43.7, 38.2, 26.6, 25.3(2C, R1-20, 40), 24.0;HRMS
m/z calcd for C31H38N2O10S:631.2320 [M+H]+, found: 631.2327
[M+H]+.
4.1.1. Experimental procedure for the synthesis of
podophyllotoxin derivatives 3a–3h (Scheme 1) 4-(prop-2-
ynyloxy) podophyllotosxin
BF3.Et2O (0.36 ml) was added to a mixture of podophyllotoxin
(1) (500 mg, 1.21 mmol) and Propargyl alcohol (135 mg, 2.4 mmol)
in CH2Cl2 (10 ml) at ꢀ20–0 °C with stirring. After 2 h, the reaction
was quenched with pyridine (0.35 ml) and the mixture was ex-
tracted with EtOAc (100 ml). The extract was washed consecu-
tively with cold dilute HCl and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography with EtOAc-Petroleum ether (1:4), gave 2
(470 mg 90.9%).
4.1.2.4. Compound 3d.
69% yield, white solid, mp 93–
105 °C;IR cmꢀ1: 3425, 2928, 1776, 1589, 1551, 1483, 1269, 1125,
1039, 967, 776, 591; 1H NMR (400 MHz, CDCl3) d: 6.88 (s, 1H, H-
5), 6.52 (s, 1H, H-8), 6.23 (s, 2H, H-20, 60), 5.97 (d, J = 10.0 Hz, 2H,
OCH2O), 4.60 (d, J = 4.8 Hz, 1H, H-4), 4.38 (m, 1H, 11a-H), 4.26–
4.09 (m, 2H, 11b, 1-H), 3.81 (s, 3H, 40-OCH3), 3.73 (s, 6H, 30,50-
OCH3), 3.38 (m, 2H, 100-H), 3.31-16 (m, 2H, 2,3-H,), 3.03 (s, 3H,
Ms-CH3), 2.82-2.16 (m, 4H, pyrrolidine-CH2), 1.89–1.23 (m, 6H,
200-H and pyrrolidine-CH2);13C NMR (100 MHz, CDCl3) d: 174.9,
163.7, 152.6 (2C, C-30, 50), 148.4, 146.9, 137.2, 135.0, 131.9, 129.6,
110.5, 109.6, 108.1 (2C, C-20, 60), 101.4, 75.2, 68.4, 67.5, 60.7, 56.2
(2C, C-30, 50-OCH3), 48.7 (2C, R1-10, 40), 48.6, 45.3, 43.6, 41.1, 38.2,
26.7, 25.7 (2C, R1-20, 30); HRMS m/z calcd for C30H36N2O10S:
617.2163, [M+H]+, found: 617.2156 [M+H]+.
4.1.2. General synthetic procedure for compounds 3a–3h
To a stirred mixture of azide (0.6 mmol), 4b-O-propargylpodo-
phyllotoxin2 (0.5 mmol), and CuI (0.05 mmol) in CH2Cl2 (10 mL)
was slowly added the various amine nucleophile (0.6 mmol) at
room temperature under an N2 atmosphere. Triethylamine
(0.6 mmol) was added prior to the addition of nucleophiles. After
the reaction was completed, which was monitored with TLC, the
reaction mixture was diluted by adding CH2Cl2 (5 mL) and aqueous
NH4Cl solution (6 mL). The mixture was stirred for an additional
30 min and two layers were separated. The aqueous layer was ex-
tracted with CH2Cl2 (6 mL ꢁ 3). The combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo. The crude
residue was purified by flash column chromatograph with an
appropriate eluting solvent system.36
4.1.2.5. Compound 3e.
26% yield, white solid, mp 132–
134 °C; IR cmꢀ1: 3415, 2924, 1774, 1557, 1505, 1481, 1265,
1124,1041, 926, 761, 516; 1H NMR (400 MHz, CDCl3) d: 7.10 (s,
1H, pyrrole-H), 7.00 (s, 1H, H-5), 6.52 (s, 1H, H-8), 6.34 (s, 2H, pyr-
role-H), 6.21 (s, 2H, H-20, 60), 5.96 (d, J = 9.6 Hz, 2H, OCH2O), 4.64 (d,
J = 5.2 Hz, 1H, H-4), 4.54 (m, 1H, 11a-H), 4.22–4.12 (m, 2H, 11b, 1-
H), 3.80 (s, 3H, 40-OCH3), 3.72 (s, 6H, 30,50-OCH3), 3.48 (m, 2H, 100-
H), 3.27–3.10 (m, 5H, 2, 3-H, Ms-CH3), 1.68 (m, 2H, 200-H); 13C
NMR (100 MHz, CDCl3) d: 174.9, 152.6 (2C, C-30, 50), 148.4, 147.5,
137.2, 135.4, 132.3, 130.6, 129.1, 127.0, 112.3, 110.8, 109.5, 108.2
(2C, C-20, 60), 107.0, 101.4, 74.7, 68.0, 67.3, 60.7, 56.2 (2C, C-30, 50-
OCH3), 45.4, 44.0, 43.1, 40.8, 38.3, 29.7; HRMS m/z calcd for
4.1.2.1. Compound 3a.
69% yield, white solid, mp 99–101 °C;
IR cmꢀ1: 3425, 2928, 1776, 1589, 1551,1483, 1269, 1125, 1039,
967, 776, 591; 1H NMR (400 MHz, CDCl3) d: 6.92 (s, 1H, H-5),
6.51 (s, 1H, H-8), 6.24 (s, 2H, H-20,60), 5.96 (d, J = 10.0 Hz, 2H,
OCH2O), 4.60 (d, J = 4.8 Hz, 1H, H-4), 4.38 (m, 1H, 11a-H), 4.26–
4.09(m, 2H, 11b, 1-H), 3.81 (s, 3H, 40-OCH3), 3.73 (s, 6H, 30,50-
OCH3), 3.38 (m, 2H, 100-H), 3.31–3.16(m, 2H, 2,3-H,), 3.09 (s, 3H,
Ms-CH3), 1.64-1.50 (m, 6H, 200-H and N(CH2CH3)2), 0.90–0.78
(m,6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3) d:174.9, 164.6,
152.6 (2C, C-30, 50), 148.4, 146.9, 137.2, 135.2, 132.0, 129.6, 109.7,
108.2(2C, C-20, 60), 101.5, 75.2, 68.2, 67.6, 60.7, 56.2 (2C, C-30, 50-
OCH3), 51.4 (2C, R1-10, R2-1a0), 45.3,43.9, 43.7, 41.1, 38.2, 31.7,
22.2 (2C, R1-20, R2-2a0), 11.1 (2C, R1-30, R2-3a0); HRMS m/z calcd
for C32H42N2O10S: 647.2633 [M+H]+, found: 647.2639 [M+H]+.
C
30H32N2O10S: 612.178 [M+H]+, found: 612.182 [M+H]+.
4.1.2.6. Compound 3f. 50% yield, white solid, mp 122–
124 °C; IR cmꢀ1: 3448, 2931, 1778, 1541, 1504,1481, 1269, 1127,
1038, 932, 767, 515;1H NMR (400 MHz, CDCl3) d: 6.99 (s, 1H, H-
5), 6.50 (s, 1H, H-8), 6.36 (s, 2H, H-20, 60),5.96 (s, 2H, OCH2O),
4.64–4.63 (m, 2H, 4-H, 1-H), 4.57 (m, 1H, 11a), 4.00 (m, 1H,11b),
3.81 (s, 3H, 40-OCH3), 3.75 (s, 6H, 30,50-OCH3), 3.37 (m, 2H, 100-H),
3.28-3.19 (m, 2H,2,3-H), 3.00 (s, 3H, Ms-CH3), 2.89–2.80 (m, 2H,
cyclohexyl-CH), 1.78-1.24 (m, 22H, 200-H andcyclohexyl-CH2); 13C
NMR (100 MHz, CDCl3) d: 174.9, 163.0, 152.1 (2C, C-30, 50), 148.4,
147.1137.3, 135.4, 131.7, 130.5, 109.8, 108.5 (2C, C-20, 60), 106.8,
101.4, 75.2, 71.3, 65.6 (2C, R1-10, R2-1a0), 60.7, 59.7, 56.4 (2C, C-
30,50-OCH3), 45.4, 43.9, 43.6, 37.8, 30.9 (4C, R1-20, 60, R2-2a0, 6a0),
29.4 (2C, R1-40, R2-4a0), 25.0 (4C,R1-30, 50, R2-3a0, 5a0);HRMS m/z
4.1.2.2. Compound 3b.
67% yield, white solid, mp 107–
109 °C; IRcmꢀ1: 3478, 2934, 1776, 1588, 1482,1265, 1126, 1038,
935, 785, 582;1H NMR (400 MHz, CDCl3) d: 7.34–7.12 (m, 5H,
Ph), 6.79 (s, 1H, H-5), 6.46 (s, 1H, H-8), 6.17 (s, 2H,H-20, 60), 5.96
(d, J = 9.6 Hz, 2H, OCH2O), 4.47 (d, J = 4.8 Hz, 1H, H-1), 4.38 (m,
1H, 11a-H),4.12–4.06 (m, 2H, 11b, 4-H), 3.94 (s, 2H, PhCH2), 3.80
(s, 3H, 40-OCH3), 3.73 (s, 6H, 30,50-OCH3), 3.18 (m, 2H, 100-H),
3.16–3.01 (m, 5H, 2,3-H, Ms-CH3), 1.67 (m, 2H,200-H);13C NMR
(100 MHz, CDCl3) d: 174.3, 165.5, 152.6 (2C, C-30, 50), 148.5,
146.9, 137.2, 136.1, 135.1, 134.9, 131.9, 129.8, 129.0, 128.2,
127.9, 110.5, 109.6, 108.1 (2C, C-20, 60), 101.5, 75.4, 67.2, 66.6,
60.7, 56.2 (2C, C-30, 50-OCH3), 46.1, 45.3, 43.8, 43.7, 41.0, 37.4,
34.4; HRMS m/z calcd for C33H36N2O10S: 653.2163 [M+H]+, found:
653.2147 [M+H]+.
calcd for
C
38H50N2O10S: 727.3259 [M+H]+, found: 727.3239
[M+H]+.
4.1.2.7. Compound 3g. 72% yield, white solid, mp 116–118 °C; IR
cmꢀ1: 3421, 2927, 1776, 1566,1503, 1483, 1267, 1124, 1038, 932,
770, 574; 1H NMR (400 MHz, CDCl3) d: 6.89 (s, 1H, H-5), 6.53 (s,
1H, H-8), 6.24 (s, 2H, H-20,60), 5.97 (d, J = 8.8 Hz, 2H, OCH2O),
4.60 (d, J = 5.2 Hz, 1H, H-4), 4.38 (m, 1H, 11a-H),4.24–4.12 (m,
2H, 11b, 1-H), 3.81 (s, 3H, 40-OCH3), 3.74 (s, 6H, 30,50-OCH3), 3.33
(m, 2H, 100-H), 3.28–3.10 (m, 2H, 2,3-H), 3.09 (s, 6H, N(CH3)2),
3.04 (s, 3H, Ms-CH3), 1.64 (m, 2H, 200-H);13C NMR (100 MHz,
CDCl3) d: 174.8, 165.7, 152.6 (2C, C-30, 50), 148.4, 146.9, 137.2,
4.1.2.3. Compound 3c. 56% yield, white solid, mp 114–116 °C; IR
cmꢀ1: 3478, 2934, 1776, 1548, 1504, 1483, 1265, 1125, 1036, 931,
765, 531;1H NMR (400 MHz, CDCl3) d: 6.95 (s, 1H, H-5), 6.51 (s, 1H,

6294
Y. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6285–6295
135.1, 132.0, 129.3, 109.6, 108.2 (2C, C-20, 60),106.8, 101.4, 75.2,
67.9, 67.4, 60.7, 56.2 (2C, C-30, 50-OCH3), 45.4, 43.9, 43.6, 41.1,
the cells, and then 5
ll of the FITC-Annexin V mix was added. Next,
5
ll of the PI mix was added, and the suspension was mixed and
39.3
(2C,
R1-10,R2-1a0),
38.2;
HRMS
m/z
calcd
for
kept at room temperature in the dark for 15 min of reaction. Flow
cytometry was performed using a FACSCalibur Xow cytometer (BD
Biosciences, CA, USA).
C
28H34N2O10S:591.2007 [M+H]+, found: 591.2017 [M+H]+.
4.1.2.8. Compound 3h.
74% yield, white solid, mp 111–
113 °C; IR cmꢀ1: 3418, 3333, 2928, 1774, 1585,1505, 1482, 1273,
1124, 1037, 932, 787, 586; 1H NMR (400 MHz, CDCl3) d: 6.82 (s,
1H, H-5), 6.55 (s, 1H, H-8), 6.32 (s, 2H, H-20,60), 5.99 (s, 2H,
OCH2O),4.60-4.51 (m, 2H, H-4 and 11a-H), 4.23–4.15 (m, 2H,
11b,1-H), 3.80 (s, 3H, 40-OCH3), 3.74 (s, 6H, 30,50-OCH3), 3.34 (m,
2H, 100-H), 2.98–2.95 (m, 5H,2,3-H, Ms-CH3), 1.58 (m, 2H, 200-H);
13C NMR (100 MHz, CDCl3) d:174.6, 166.4152.6 (2C, C-30, 50),
148.7, 146.9, 137.3, 135.2, 132.3, 128.5, 110.9, 109.2, 108.2,
101.6, 75.3, 67.2, 66.4, 60.7, 56.2 (2C, C-30, 50-OCH3),45.3, 43.9,
42.0, 41.1, 38.1; HRMS m/z calcd for C26H30N2O10S:563.1694
[M+H]+, found: 653.1679 [M+H]+.
4.4. Separation of active microtubule protein
The goat brains (1.8 kg) were homogenized in a Waring blender
and then centrifuged in the cold at 32 800 rpm for 1 h. The super-
natant was accumulated and divided into four aliquots. ATP and
GTP were added to aliquot at 37 °C, incubated and centrifuged.
The supernatants were discarded, and the pellets were homoge-
nized. After 30 min on ice, the very viscous suspension was centri-
fuged in the cold for 30 min. The pellet was re-homogenized
sequentially 2 times and the cold centrifugation was repeated each
time. All supernatants were pooled and the cold pellets were set
aside. To each 100 mL of the combined supernatant were added
55.3 g of glycerol (4 M) and 5 mL of a solution containing Mes
(pH 6.4), EGTA, MgC12, 2-mercaptoethanol, and EDTA to maintain
the concentrations of these components and ATP and GTP for final
concentrations of 1 and 0.3 mM. The mixture was incubated at
37 °C for 1 h and centrifuged in the warm for 1 h at 32,000 rpm
and the warm supernatant was set aside. The suspension was left
on ice for 30 min and centrifuged in the cold for 30 min. Very small
cold pellets are obtained and then were combined with the previ-
ous cold pellets. Preparation of the microtubule protein was out-
lined in reference.38
4.2. Cell culture and inhibition of the cell growth
Four independent human tumor cell lines: human leukemia cell
line K562, human gastric cancer cell line SGC-7901, human cervical
carcinoma cell line HeLa and Human hepatocellular liver carci-
noma cell line HepG2 were obtained from key lab of preclincical
study for new drugs of Gausu province of Lanzhou university. All
test compounds were dissolved in dimethyl sulfoxide (DMSO) at
1 mg/mL immediately before use and diluted in the medium before
addition to the cells. All cell lines were cultured in an RPMI 1640
medium (GIBCO) supplemented with 10% bovine fetal calf serum,
2 mM
of penicillin, and 100
in 100 mL medium) in their log phase of growth were seeded in
96-well microtitre plates. After 24 h of incubation at 37 °C and
5% CO2 to allow cell attachment, cultures were treated with vary-
L-glutamine (GIBCO), 10 mM b-mercaptoethanol, 100U/mL
4.5. In vitro tubulin polymerization analysis
l
g/mL of streptomycin. Cells (2 ꢁ 105 cells
To evaluate the effect of the compounds on tubulin assembly
in vitro the test compounds were incubated across a concentration
range with 10 lM tubulin together with buffer containing 20%
ing concentrations (0–100 lM) of test compounds. For each con-
glycerol and 1 mM ATP at 37 °C and then cooled to 0 °C. The effect
of the test compounds on tubulin polymerization was observed
turbidimetrically using a fluorescent plate reader.39 The IC50 value
was defined as the compound concentration that inhibited the ex-
tent of assembly by 50%.
To observe the effect of polymerization inhibition of the tubulin
protein, electron micrographs were used. Tubulins were incubated
with or without Compound 3e in assay buffer (80 mM PIPES (pH
6.9), 1 mM MgCl2, and 1 mM EDTA) containing 30% glycerol and
1 mM GTP for 30 min at 37 °C. Samples were fixed by dilution
(1:20 –1:50 fold) into assay buffer containing 30% glycerol and
0.5% glutaraldehyde at 37 °C, and then 10 ml of each sample were
spotted onto Formavar-coated grids and stained with uranyl ace-
tate for 30 s. Electron micrographs were obtained using a Philips
CM12 transmission electron microscope with an accelerating volt-
age of 100 kV.
centration, five replicate wells were used. Considering the
possible antiproliferative effects of DMSO, the concentration of
DMSO in the array was less than 0.1% and will not affect the cell
growth of the cell lines. The cell growth was calculated by sub-
tracting mean OD value of the respective blank from the mean
OD value of experimental set. Percentage of growth in the presence
of test compounds was calculated considering the growth in the
absence of any test compounds as 100% and the results are re-
ported in terms of IC50 (concentration causing 50% inhibition rela-
tive to untreated controls) values.
4.3. Cell cycle analysis and apoptosis detection
Flow cytometry was used to measure cell cycle profile and
apoptosis. For cell cycle analysis, K-562 cells (1 ꢁ 105) treated with
compounds 3e with various concentration (0 lM, 0.375 lM,1.2 lM
and 3 lM) were washed twice with ice-cold PBS, harvested, fixed
with ice cold PBS in 70% ethanol and stored at –20 °C for 30 min.
4.6. Molecular modeling
After fixation, these cells were incubated with RNase A (0.1 mg/
The docking mode was based on the podophyllotoxin-
colchicine complex structure (PDB code: 1SA1). Water molecules,
co-crystallized ligand and ions were removed and the missing
hydrogens and Kollman partial charges were added. The structure
of compound 3e was build with Gview and optimized at the B3LYP/
6–31++G⁄⁄ level by Gaussian 98 software. Docking simulations
were performed with AutoDock4.040 using a Lamarkian genetic
algorithm. The large enough grid volumes were considered and
determined by the need to maintain a small grid spacing (0.375)
for the search box and at the same time to perform the docking
calculation without neglecting any portion of the protein surface.
The others docking parameters were set as default. During docking,
mL) at 37 °C for 30 min, stained with propidium iodide (50 lg/
mL) for 30 min on ice in dark. Cells were harvested by centrifuga-
tion and further stained with DNA staining solution (10 mg). The
DNA contents were then detected by flow sytometer and the cell
cycle profile was analyzed from the DNA content histograms.37
Flow cytometry with FITC-Annexin V/PI double staining Trypsin
(Sigma) without EDTA was used to digest and collect the control
group and the cells treated with 0.375 lM, 1.2 lM, and 3 lM
compound 3e. Flow cytometry was performed according to the
apoptosis detection kit procedures. The K562 cells were washed
with PBS twice, centrifuged at 300ꢁg for 5 min, and 5 ꢁ 105 cells
were collected. Binding buffer suspension (500 ll) was added to

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Acknowledgments
This work was financially supported by the Fundamental
Research Funds for the Central Universities (lzujbky-2010-134);
National Natural Science Foundation of China (NO. 31000017)
(NO. 21102065) (NO. 21207056) and the Natural Foundation of
Gansu Province (1104WCGA187).
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