Synthesis and biological evaluation of novel 4β-(1,3,4-oxadiazole-2- amino)-podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.05.059

A series of new 4β-(1,3,4-oxadiazole-2-amino)-podophyllotoxin derivatives were designed and synthesized. Their cytotoxicity in vitro against six tumor cell lines (DU-145, SGC-7901, A549, SH-SY5Y, HepG2 and HeLa) were evaluated by standard MTT assay. The p

Full text of DOI:10.1016/j.bmcl.2012.05.059

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4778–4782
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 4b-(1,3,4-oxadiazole-2-amino)-
podophyllotoxin derivatives
⇑
⇑
Jie Ren, Lin Wu, Wen Qun Xin, Xin Chen , Kun Hu
School of Pharmaceutical Engineering & Life Science, Changzhou University, 1 Gehu Road, Changzhou, Jiangsu 213164, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 December 2011
Revised 1 May 2012
Accepted 15 May 2012
Available online 23 May 2012
A series of new 4b-(1,3,4-oxadiazole-2-amino)-podophyllotoxin derivatives were designed and synthe-
sized. Their cytotoxicity in vitro against six tumor cell lines (DU-145, SGC-7901, A549, SH-SY5Y, HepG2
and HeLa) were evaluated by standard MTT assay. The pharmacological results showed that most of the
newly synthesized podophyllotoxin derivatives displayed potent cytotoxicity against at least one of the
tested tumor cells; and among the new derivatives, 11b was more potent than podophyllotoxin against
HepG2 and Hela cell lines. Furthermore, 11b exhibited much better selectivity toward the normal cell
lines L929 and Vero than etoposide, 5-Fu and podophyllotoxin. The possible antitumor mechanism of
11b is to inhibit the activity of DNA topoisomerase II, result in the S-phase arrest, and then cause apop-
totic cell death.
Keywords:
Podophyllotoxin
1,3,4-Oxadiazole
Antitumor activity
Topoisomerase II
Mechanism
Ó 2012 Elsevier Ltd. All rights reserved.
Podophyllotoxin 1, a naturally occurring cyclolignan which is the
main component of podophyllum resin, shows strong cytotoxic
activity against various cancer cell lines.1 Although the therapeutic
application of 1 is limited to topical use due to its high toxicity,2
the unique cyclolignan scaffold of 1 has drawn a lot of attention
for the discovery and development of new anticancer agents. Exten-
sive structural modifications, particularly at the C4 position of
podophyllotoxin have been reported in the literature.3–5 For exam-
ple, two semisynthetic derivatives of 1, etoposide 2 and teniposide 3
(Fig. 1), are currently used in the chemotherapy for a variety of
malignancies, including germ-cell malignancies, small-cell lung
cancer, non-Hodgkin’s lymphoma, leukemia, Kaposi’s sarcoma,
neuroblastoma, and soft tissue sarcoma due to their ability to inhibit
the enzyme DNA topoisomerase II.6–9 Despite of their wide clinic
application, there are several limitations for these semisynthetic
podophyllotoxin derivatives, which has inspired to further search
for new effective antitumor agents based on this scaffold.10
Previous structure–activity relationship (SAR) results indicate
that bulky groups are well-tolerated at C4, and the resulting
derivatives based on C4 modification could significantly affect the
X
O
O
R
O
OH
OH
O
HN
HO
O
O
O
O
O
O
O
O
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OH
OH
1
2
R = CH3
4
X = F
3 R =
5 X = NO2
S
Figure 1. The structure of Podophyllotoxin 1, Etoposide 2, Teniposide 3, NPF 4 and GL-331 5.
⇑
Corresponding authors. Tel./fax: +86 519 86334598.
E-mail addresses: xinchen@cczu.edu.cn (X. Chen), hukun1979@163.com (K. Hu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.059

J. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4778–4782
4779
S
S
C
OH
NH2
N
HN
SH
N3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
c
d
b
a
O
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OMe
OMe
OMe
OMe
9
8
7
1
6
4''
N
3''
N
S
5''
H
R
N
R
2''
3
HN
N
H
O
1''
11
HN
4
15
5
8
O
10
9
O
O
O
6
7
H
10a,11a:
10g,11g:
R=
R=
10d,11d:
R=
12
O
O
16
O
13
OMe
NO2
f
e
O
2
1
17
10b,11b: R=
10h,11h: R=
10i, 11i: R=
10e,11e: R=
10f,11f: R=
CH3
O
O14
1'
2'
3'
6'
5'
N
10c,11c:
R=
MeO
OMe
MeO
OMe
4'
N
S
OMe
OMe
10a-h
11a-h
Scheme 1. Reagents and conditions: (a) NaN3, CF3COOH, CHCl3, rt, 6 h; 90%; (b) H2, Pd/C, MeOH, rt, 12 h; 75%; (c) Et3N, CS2, THF, rt, 1 h; (d) MsCl, THF, rt, 0.5 h; 81%; (e)
RCONHNH2, THF, rt, 2–6 h; 85%–90%; (f) pyridine, TsCl, THF, reflux,12–24 h; 64–77%.
Table 1
The cytotoxicity of compounds 11a–11i and 12 against DU-145, SGC-7901, A549, SH-SY5Y, HepG2, HeLa, L929 and Vero cell lines
a
Compd
Cytotoxicity (IC50
SH-SY5Y
, l )
mol Lꢀ1
DU-145
SGC-7901
A549
HepG2
HeLa
L929
Vero
11a
11b
11c
11d
11e
11f
11g
11h
11.34
>100
>100
76.12
>100
>100
35.42
>100
>100
60.65
>100
64.26
12.32
>100
38.11
>100
>100
>100
56.58
ND
>100
>100
9.76
>100
>100
ND
53.66
ND
>100
69.22
>100
>100
>100
13.62
10.12
8.83
25.81
14.66
26.88
7.39
16.58
20.02
29.48
22.47
41.53
4.61
11.85
1.51
>100
2.68
43.29
19.62
>100
18.15
>100
70.85
>100
54.56
20.47
4.82
15.37
5.49
>100
7.43
74.20
27.95
>100
38.82
38.81
72.77
96.56
17.94
2.27
85.34
17.66
>100
42.53
58.85
>100
>100
18.82
7.58
ND
>100
>100
>100
>100
46.09
1.78
11i
12
Podophyllotoxin
Etoposide
5-Fu
2.95
12.09
14.49
2.54
5.37
1.09
1.37
1.00
1.38
5.48
32.22
96.26
15.99
31.14
>100
ND: not determined.
a
Data are the mean of three independent experiments.
antitumor spectra of podophyllotoxin.11 One of the major break-
throughs in this field underlines that the sugar moiety of etoposide
is not essential for topoisomerase II inhibition.7 4b-Non-sugar-
substituted, especially amino group substituted podophyllotoxin
derivatives, such as NPF 4 and GL-331 5, have exhibited improved
cytotoxicity and DNA topoisomerase II inhibition activity.12,13 SAR
studies suggest that 4b-stereochemistry and 4-N-linkage are the
essential structural features for topoisomerase II inhibitory activity.
Moreover, oxadiazoles are common bioisosteres for biologically
active amides, esters and ureas. In particular, 2-amino-1,3,4-oxadi-
azoles are present in a wide range of pharmaceutical substances,
such as antimicrobial agents, antitumor agents, anti-inflammatory
agents and anticonvulsant agents.14
urthermore, the possible mechanism of HepG2 cell growth inhibi-
tion by 11b was also investigated in the present study.
Nine 4b-(1,3,4-oxadiazole-2-amino)-podophyllotoxin deriva-
tives were synthesized starting from 1 through a convenient
six-step procedure as described in Scheme 1. In the first step, the
4a-OH group in 1 was efficiently converted to 4b-azide (compound
N
N
O
HN
O
O
O
Our objective was to discover more potent and selective antitu-
mor agents based on podophyllotoxin scaffold. Therefore, we have
designed and synthesized a series of novel N-substituted deriva-
tives 11a–11i, in which 2-amino-1,3,4-oxadiazole moieties have
been introduced at the C4 position of podophyllotoxin, and the
new products were evaluated for their cytotoxicity against
DU-145, SGC-7901, A549, SH-SY5Y, HepG2 and HeLa cell lines.
O
MeO
OMe
OH
12
Scheme 2. The structure of C-40 demethylated derivative 12.

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J. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4778–4782
Figure 2. Effects of compound 11b on cell cycle of HepG2 cells. Cells were treated with compound 11b (0.25, 0.5 and 1 lM) for 48 h, and the DNA content of 10,000 events
was analyzed by flow cytometry. The profiles showed the cell cycle (A) and the proportions (%) in each phase (B) of HepG2 cells treated with 11b. ⁄p <0.05, ⁄⁄p <0.01 compared
to control.
6) via nucleophilic substitution with NaN3 and TFA (CF3CO2H) at
room temperature. The corresponding 4b-amino compound 7
was prepared by reducing the azide 6 with catalytic hydrogeno-
sis.15,16 A dithiocarbamate salt 8 was readily generated at room
temperature in THF by slow addition of 1 equiv of CS2 to a solution
of amine 7 and 3.5 equiv of Et3N. Then MsCl was used to facilitate
the elimination of H2S from the dithiocarbamate to afford the iso-
thiocyanate 9.17 In the final phase of the synthesis, 9 was treated
with various hydrazides at room temperature to generate key
intermediates 10a–10i in 85–90% yields. Upon treatment with
tosyl chloride (TsCl) and Et3N, thiosemicarbazides 10a–10i were
cyclized and produced the corresponding 4b-(1,3,4-oxadiazole-2-
Figure 3. Effects of compound 11b on cell apoptosis of HepG2 cells. Induction of apoptosis were measured by Annexin-V/PI double-staining assay after treatment with
compound 11b (0.25, 0.5, 1 and 2 M) for 48 h.
l

J. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4778–4782
4781
Figure 4. Effects of compound 11b on morphological changes of HepG2 cells. HepG2 cells were incubated with various concentrations of 11b (0.25, 0.5 and 1
lM) for 48 h
and stained by Hoechst 33258 for morphology observation.
amino) podophyllotoxin derivatives 11a–11i, respectively.18 The
structures of the intermediates and final products were confirmed
by their MS, IR, 1H NMR and 13C NMR spectral properties (Supple-
mentary data). Purity estimation was done with HPLC, and only
those with purity around 95% were tested for biological activity.
The biological activities of the podophyllotoxin derivatives
11a–11i were evaluated by an in vitro cytotoxicity test carried
out with a panel of six human tumor cell lines (human prostate
carcinoma cell line DU-145, human gastric cell line SGC-7901,
human lung cancer cell line A549, human neuroblastoma cell line
SH-SY5Y, human hepatoma cell line HepG2 and human cervical
carcinoma cell line HeLa) and two normal cell lines (mouse fibro-
blast cell line L929 and Africa green monkey kidney cell line Vero)
by MTT method.19 Podophyllotoxin, etoposide and 5-fluorouracil
(5-Fu) were selected as the positive controls. The results expressed
as IC50 values are summarized in Table 1.
As illustrated in Table 1, all the newly synthesized podophyllo-
toxin derivatives (11a–11i) exhibited much lower cytotoxicity
toward the tested normal cell lines (L929 and Vero) than Podophyl-
lotoxin. The results in Table 1 also shown that the derivatives with
alkyl groups at C500 position (11a and 11b) had relative better
activity profile than those with aryl groups (e.g., 11c–11i). Among
the tested compounds, 11b displayed the most potent cytotoxicity
against SH-SY5Y, HepG2 and HeLa, with IC50 values of 14.66
lM,
1.29 M and 2.68 M, respectively. The dose-and time-dependent
l
l
curve of 11b in HepG2 hepatoma cells was further observed (data
not shown). The cytotoxicity selectivity against the tumor cells and
normal cells is the most important characteristic property of the
new podophyllotoxin derivatives. According to the data in Table
1, 11b has much better cytotoxicity selectivity against the tumor
cells and normal cells than etoposide, 5-Fu and podophyllotoxin
itself. For example, the IC50 values of etoposide against tumor cell
HepG2 and normal cells L929 are 5.48
tively; whereas, for 11b, the IC50 values for the two cell lines are
1.51 M and 19.62 M. The results indicate that 11b is less cyto-
lM and 1.00 lM, respec-
l
l
toxic to the normal cells.
For some known podophyllotoxin derivatives, such as etoposide
2, teniposide 3, NPF 4 and GL-331 5, the C40 position is a free
hydroxyl group, instead of a methoxy group. In order to investigate
the effect of a free hydroxyl group on the antitumor activity of 11b,
a demethylated derivative 12 (Scheme 2) at C40 position has been
synthesized (the detailed experimental procedure is described in
the Supplementary data). The assay data in Table 1 shown that
12 demonstrated stronger inhibitory activity against SGC-7901
and SH-SY5Y cell lines (9.76
weaker activity against HepG2 and HeLa cell lines (17.94
18.82 M). On the other hand, 12 did not show good selectivity to-
ward the normal cell L929 (4.82 M). Therefore, we might con-
lM and 4.61
lM, respectively), but
lM and
l
l
clude that for the 4b-(1,3,4-oxadiazole-2-amino)-podophyllotoxin
derivatives, a free hydroxyl group at the C40 position does not have
obvious positive effects on their antitumor activity.
Compound 11b was chose to further evaluate its antitumor
activity in human hepatoma cell line (HepG2), and study was car-
ried out to determine whether or not its antitumor mechanism is
related to DNA topoisomerase, cell cycle arrest and apoptosis.
The HepG2 cells were treated with 11b (0.25, 0.5 and 1 lM) for
48 h, and the DNA content of 10,000 events was analyzed by flow
cytometry (Fig. 2). The profiles shown the cell cycle (Fig. 5A) and
the proportions (%) in each phase (Fig. 5B) of HepG2 cells treated
with 11b. Flow cytometric analysis of DNA profile in the HepG2
cells shown that 11b treatment produced a dose-dependent shift
of the cell population from G0/G1 to S-phase, revealing a signifi-
cant accumulation of cells in the S-phase. At the same time,
Figure 2 demonstrated a dose-dependent increase of apoptosis
induction which was indicated by percentage of sub-diploid DNA
content.
Figure 5. Effects of compound 11b on topoisomerase I, topoisomerase II
a and
topoisomerase IIb gene expressions in HepG2 cells. HepG2 cells were treated with
various concentrations of compound 11b. Control cells were incubated with vehicle
alone. (A) Topoisomerase mRNA expressions were assessed by RT-PCR in HepG2
cells exposed to compound 11b for 6 h. (B) The amount of RNA loaded in each lane
was confirmed by GAPDH mRNA. ⁄p <0.05,⁄⁄p <0.01 compared to control.

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J. Ren et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4778–4782
target DNA topoisomerase II
on the DNA topoisomerase II
isomerase II protein expression was assessed by western blot in
cells treated with different concentrations of 11b (0.5 and 1 M)
a, we investigated the effect of 11b
a
protein expression (Fig. 6). DNA topo-
a
l
for 48 h (Fig. 6A), and the relative protein expression levels were
measured by scanning densitometry of the band intensities
(Fig. 6B). As shown in Figure 6, 11b significantly inhibited the pro-
tein expression of DNA topoisomerase IIa. The results indicated that
11b could act as a topoisomerase II inhibitor.
In conclusion, we have designed, synthesized and evaluated for
the antitumor activity of ten novel 4b-(1,3,4-oxadiazole-2-amino)-
podophyllotoxin derivatives. Among these new compounds, 11b
exhibited promising antiproliferative activity in a selective HepG2
cell line. The mechanism study showed that 11b inhibits the gene
and protein expressions of DNA topoisomerase IIb, suspends the
cell cycle at S-phase, and eventually leads the tumor cells to apop-
tosis. Further biological evaluation is in progress to define its
effects on tubulin polymerization.
Acknowledgments
We thank the financial support from 2010 Industry for Attract-
ing Ph. D. Scientists program of Jiangsu Province, Changzhou key
technology R&D program (society development) and the Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
Figure 6. Effects of compound 11b on topoisomerase II
a protein expression in
HepG2 cells. HepG2 cells were treated with various concentrations of compound
11b. Control cells were incubated with vehicle alone. (A) Topoisomerase II protein
expression was monitored 48 h after treatment of cells with compound 11b. (B) The
a
relative Topoisomerase IIa protein levels were measured by scanning densitometry
of the band. ⁄p <0.05 compared to control.
Supplementary data
In order to explore whether the tested compounds-mediated
inhibition in cell growth is related to the induction of apoptosis,
Annexin V/PI staining was used to detect the apoptotic ratio of
compound 11b treated cells. Induction of apoptosis was measured
by Annexin-V/PI double-staining assay after treatment with 11b
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
059.
References and notes
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l
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Finally, we investigated the effects of 11b on the DNA topoiso-
merase I, DNA topoisomerase II
expression (Fig. 5). Topoisomerase I, topoisomerase II
merase IIb mRNA expression were assessed by RT-PCR in cells trea-
ted with different concentrations of 11b (0.5, 1, 2, 4 and 8 M) for
a
and DNA topoisomerase IIb gene
a
and topoiso-
l
6 h (Fig. 5A), and the relative gene expression levels were measured
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