Synthesis and evaluation of aroylthiourea derivatives of 4-β-amino-4'-O-demethyl-4-desoxypodophyllotoxin as novel topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.01.001

A novel series of aroylthiourea derivatives of 4-β-amino-4′-O- demethyl-4-desoxy- podophyllotoxin were synthesized. Their cytotoxicities against three cancer cell lines were investigated by MTT assay. The kDNA decatenation assay indicated that compounds 5a, 5f, 5h and 5l inhibited topoisomerase II-mediated kDNA decatenation. DNA flow cytometric analysis revealed that compound 5a induced cell cycle arrest at G2/M phase in HCT-116 cell line.

Full text of DOI:10.1016/j.ejmech.2011.01.001

European Journal of Medicinal Chemistry 46 (2011) 901e906
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of aroylthiourea derivatives of 4-b-amino-4’-O-
demethyl-4-desoxypodophyllotoxin as novel topoisomerase II inhibitors
,
Yu Zhao a, Cun Wang Ge b, Zhong Hua Wu a, Cheng Niu Wang a, Jing Huai Fang c, Li Zhu a
*
a Institute of Nautical Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, China
b School of Chemistry and Chemical Engineering, Nantong University, 19 Qixiu Road, Nantong 226001, China
c School of Science, Nantong University, 19 Qixiu Road, Nantong 226001, China
a r t i c l e i n f o
a b s t r a c t
A novel series of aroylthiourea derivatives of 4-b
-amino-40-O-demethyl-4-desoxy- podophyllotoxin were
Article history:
Received 19 September 2010
Received in revised form
22 December 2010
Accepted 4 January 2011
Available online 11 January 2011
synthesized. Their cytotoxicities against three cancer cell lines were investigated by MTT assay. The kDNA
decatenation assay indicated that compounds 5a, 5f, 5h and 5l inhibited topoisomerase II-mediated
kDNA decatenation. DNA flow cytometric analysis revealed that compound 5a induced cell cycle arrest at
G2/M phase in HCT-116 cell line.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
4-b
-Amino-40-O-demethyl-4-
desoxypodophyllotoxin
Aroylthiourea
Anticancer agents
Topoisomerase II
1. Introduction
sugar moiety of etoposide is not essential for topoisomerase II
inhibition [7,8].
DNA topoisomerases solve the topological problems associated
with DNA replication, transcription, recombination, and other
nuclear processes by introducing temporary single or double
strand breaks in the DNA [1]. DNA topoisomerase II controls DNA
topology by transient cleavage of the DNA double helix. This
enzyme has been established as important molecular target of
anticancer drug [2].
Recently, new synthetic N-linked congeners, as the known
antitumor agents GL-331 and NPF, exhibit improved cytotoxicity
and DNA topoisomerase II inhibition activity [9e13]. Moreover,
literature reported thiourea derivatives [14] and carbamate deriv-
atives [13,15] possess potent anti-HIV and antitumor activities. To
our best knowledge, there is no such effort addressed to synthesize
aroylthiourea derivatives of 4-
b
-amino-40-O-demethyl-4-desoxy-
Podophyllotoxin (Fig. 1) is a well known naturally occurring
antitumor lignan lactone isolated from the genus Podophyllum [3].
Semisynthetic podophyllotoxin derivatives etoposide (VP-16) and
teniposide are in clinical use as antineoplastic agents due to their
ability to inhibit the enzyme DNA topoisomerase II [4,5]. However,
their clinical use has encountered certain limitations such as drug
resistance and poor water solubility [6]. In order to obtain better
therapeutic agents, a great number of etoposide analogs have been
synthesized. The numerous synthesized analogs have allowed the
improvement of the knowledge of structureeactivity relationships.
One of the major breakthroughs in this field underlining that the
podophyllotoxin. Structureeactivity relationships (SAR) studies
suggested that the essential structural features for topoisomerase II
inhibitory activity are 40-hydroxyl group, 4-
b-stereochemistry and
4-N-linkage.
We previously discovered a series of aroylthiourea derivatives of
b-amino-4- desoxypodophyllotoxin as anticancer agents [16].
4-
Representative compound HY-1 (Fig. 1) possessed potent DNA
topoisomerase II inhibitory activity and cytotoxicity. These results
prompted us to further synthesize a library of aroylthiourea
derivatives of 4-b
-amino-40-O-demethyl-4-desoxypodophyllotoxin
as novel DNA topoisomerase II inhibitors.
Herein, we report the synthesis and antitumor activity of these
aroylthiourea derivatives of 4-b
-amino-40-O-demethyl-4-desoxy-
podophyllotoxin (5ae5l, Scheme 1). It is found that compound 5a
possesses promising DNA topoisomerase II inhibitory activity.
* Corresponding author. Tel./fax: þ86 513 85051796.
E-mail address: zhulili65@163.com (L. Zhu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.001

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Y. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 901e906
Fig. 1. Podophyllotoxin, etoposide and HY-1.
2. Results and discussion
of 3 using Pd/C as a catalyst according to the procedure reported
previously [18]. Coupled corresponding aroylisothiocyanates [19]
with 4 in dry acetonitrile over a 2 h reflux period gave a series of
aroylthioureas 5ae5l in the yields of 53%e70%.
2.1. Synthesis of aroylthiourea derivatives of 4-
demethyl-4-desoxypodophyllotoxin
b
-amino-40-O-
The synthetic route used to synthesize title compounds is out-
lined in Scheme 1. 40-demethylepipodophyllotoxin (2) was prepared
in 79% overall yield bytreatmentofpodophyllotoxin (1) with TMSIin
methylene chloride at 0 ꢀC followed by weak basic hydrolysis with
barium carbonate [17]. In the presence of boron trifluoride etherate,
the reaction of hydrazoic acid with 40-demethylepipodophyllotoxin
2.2. Cytotoxicities of compounds 5ae5l against three cancer cell
lines
The title compounds were evaluated for their cytotoxic activities
against three cancer cell lines including HepG2, A549 and HCT-116
using a MTT growth inhibition assay. IC50 values were summarized
in Table 1 and represented the concentration inducing a 50%
decrease of cell growth after 3 days incubation.
(2) gave 4-b-azido derivatives (3) [13]. Nextly, 4-b
-amino-40-O-
demethyl-4-desoxypodophyllotoxin (4) was prepared by reduction
Scheme 1. Synthesis of aroylthiourea derivatives of 4-
b
-amino-40-O-demethyl-4-desoxypodophyllotoxin 5ae5l.

Y. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 901e906
903
Table 1
5l, without halogen moiety possessed the highest cytotoxicity on
three cancer cell lines. Moreover, 5a and 5l possessed better
cytotoxicity than etoposide. These results indicate that the
halogen-substitution did not contribute to the cytotoxic potency of
Cytotoxicities against three cancer cell lines of 5ae5l.
Compd
IC50 (mM)
HepG2
HCT-116
A549
these compounds. Compound 5h, bearing a-naphthyl group, was
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6.1
7.7
7.6
7.6
5.1
6.7
8.3
18.7
5.2
4.6
3.0
4.4
1.5
4.9
6.3
7.7
6.8
7.2
6.2
6.7
6.8
2.2
4.7
5.5
6.8
5.0
2.1
13.5
9.8
10.3
17.9
11.5
11.4
15.1
10.8
6.0
12.2
13. 3
11.3
9.5
found a potent effect on A549 and HCT-116 cell lines, but less
potent on HepG2 cell line, which indicated a different mode of
action involved.
In summary, we have prepared a series of aroylthiourea deriv-
atives of 4-b
-amino-40-O-demethyl-4-desoxypodophyllotoxin. The
synthetic compounds were cytotoxic against three cancer cell lines
at the micromolar range.
2.3. Inhibition of human topoisomerase II kDNA decatenation by
compounds 5a, 5f, 5h and 5l
4
5.2
21.4
Etoposide
To further understand the action mechanisms of antitumor
activity of these compounds, the effect on the catalytic activity of
human topoisomerase II was evaluated using kDNA decatenation
assay. As shown in Fig. 2, compounds 5a, 5f, 5h and 5l completely
inhibited the catalytic activity of topoisomerase II at 200 mM, which
was much better than the effect of etoposide at same concentration.
The results indicated that these compounds exerted their anti-
tumor activities through inhibition of human topoisomerase II
catalytic activity.
2.4. Induction of cell cycle arrest in HCT-116 cells by compound 5a
On the basis of the above results, further biological evaluations
have been focused on compound 5a. To gain further insight into the
mode of action of compound 5a, we examined the effects of
compound 5a on cell cycle by flow cytometry in HCT-116 cells.
Interestingly, a concentration dependent change was observed in the
cell cycle pattern (Fig. 3). Our results demonstrated that treatment of
Fig. 2. Effects of 200
oisomerase II-mediated decatenation activity of kDNA.
mM compound 5a, 5f, 5h, 5l and etoposide on human top-
From the IC50 values, it is clear that most of these derivatives
displayed potent cytotoxic activities against three cancer cell lines.
However, these derivatives showed comparatively lesser activity
against A549 cell line than other two cell lines. Compound 5a and
HCT-116 cells for 24 h with 0, 1.25, 2.5, 5 and 10 mM 5a increased the
percentage of cells at G2/M phase from 35.67% to 96.45%. The flow
cytometric data clearly indicated that 5a caused HCT-116 cell cycle
arrest at G2/M phase follow the same pattern of etoposide.
Fig. 3. Effects of compound 5a on cell cycle of HCT-116 cells. Cells were incubated with 0, 1.25
content was analyzed by fluorescence flow cytometry.
mM, 2.5 mM, 5 mM, 10 mM 5a for 24 h and stained with propidium iodide (PI). Their DNA

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Y. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 901e906
3. Conclusion
(m,1 H, 3-H), 3.76 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 4.5,1 H, 4-H), 4.32
(m, 2 H, 11-H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.86 (brs, 1 H, 2-NH),
5.94 (d, J ¼ 1.2, 1 H, OCH2O), 5.96(d, J ¼ 1.2, 1 H, OCH2O), 6.29 (s, 2 H,
ArH), 6.50 (s, 1 H, ArH), 6.81 (s, 1 H, ArH), 7.43 (d, J ¼ 1.8 Hz, ArH),
7.56 (d, J ¼ 1.8 Hz, ArH), 7.69 (d, J ¼ 2.1 Hz, 1 H, ArH), 7.82 (s, 1 H,
The present work lead to the development of aroylthiourea
derivatives of 4-
as novel DNA topoisomerase II inhibitors. All of these results confirm
that the replacement of the glycoside moiety of etoposide with 4-
b
-amino-40-O-demethyl-4-desoxypodophyllotoxin
b-
ArH); 13C NMR (CDCl3):
d 37.5, 42.3, 43.6, 54.1, 56.5, 68.7, 76.6, 77.0,
N-aroylthiourea residue is favorable to topoisomerase II inhibitory
activity. Two of the novel agents 5a and 5l have shown higher growth
inhibitory effect on HepG2, A549 and HCT-116 cancer cell lines than
all the other tested compounds. Their mode of action could be
interpreted as inhibitory effect on the catalytic activity of DNA top-
oisomerase II, which caused HCT-116 cell cycle arrest at G2/M phase.
77.5, 101.7, 107.9, 109.1, 110.2, 125.6, 127.6, 128.1, 130.1, 130.4, 132.5,
133.1, 133.8, 134.2, 135.4, 146.5, 147.7, 148.8, 166.1, 173.9, 180.5;
HRMS calcd for C29H25N2O8NaSCl 619.0918, found 619.0916; mp
161e163 ꢀC from ethanol.
4.1.4. 4b
-[3-(4-Chlorobenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5d)
25
4. Experimental
Yellow solid, yield 54%; [
a
]
-76 (c 0.3, CH2Cl2); 1H NMR
D
(CDCl3):
d
2.13 (s, 1 H, 1-NH), 2.81 (dd, J ¼ 5.1 Hz, 14.4 Hz, 1 H, 2-H),
4.1. Syntheses
2.97 (m,1 H, 3-H), 3.65 (s, 6 H, 30, 50-OCH3), 3.71 (d, J ¼ 4.5,1 H, 4-H),
3.88 (m, 2 H,11-H), 4.37 (d, J ¼ 4.8 Hz,1H,1-H), 5.86 (brs,1 H, 2-NH),
5.94 (s, 1 H, OCH2O), 5.96(s, 1 H, OCH2O), 6.18 (s, 2 H, ArH), 6.40 (s,
1 H, ArH), 6.71(s, 1 H, ArH), 7.32(s, 1 H, ArH), 7.35 (s, 1 H, ArH), 7.75
Solvents were purified in the usual way. TLC was performed on
precoated Merck silica Gel 60 F254 plates. Flash chromatography
was performed on silica gel (100e200 mesh, Qingdao, China). 1H
NMR and 13C NMR spectra were taken on a Bruker 300 MHz
spectrometer with tetramethylsilane (TMS) as an internal standard,
and chemical shifts were recorded in ppm values. The high reso-
lution spectra were obtained on a Q-TOF Global Mass (ESIMS).
General procedure for the synthesis of compounds 5ae5l:
(s, 1 H, ArH), 7.78 (s, 1 H, ArH); 13C NMR (CDCl3):
d 37.4, 42.2, 43.5,
53.7, 56.4, 68.7, 76.8, 77.2, 77.6,101.5,108.0,109.0,110.0,127.8,129.0,
129.6, 129.9, 130.0, 132.4, 134.4, 139.9, 146.7, 147.6, 148.6, 167.1,
173.9, 181.1; HRMS calcd for C29H25N2O8NaSCl 619.0918, found
619.0923; mp 157e158 ꢀC from ethanol.
1 mmol of 4-
b
-amino-40-O-demethyl-4-desoxypodophyllotoxin (4)
4.1.5. 4b
-[3-(2-Bromobenzoyl)thioureido]-40-O-demethyl-4-
was added to a solution of 1.2 mmol of corresponding aroylisothio-
cyanate [19] in 10 mL of dryacetonitrile. The mixturewas refluxed on
a water bath for approximately 2 h (monitored by TLC). After the
reaction completed, the result solution was evaporated under
reduced pressure. The residue was purified by flash chromatography
on silica gel (1:3, EtOAc-petroleum ether) to afford a solid.
desoxypodophyllotoxin (5e)
25
Yellow solid, yield 57%; [
a
]
-76 (c 0.3, CH2Cl2); 1H NMR
D
(CDCl3):
d
2.13 (s,1 H,1-NH), 2.93 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07
(m,1 H, 3-H), 3.76 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 4.8,1 H, 4-H), 4.32
(m, 2 H, 11-H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.86 (brs, 1 H, 2-NH),
5.94 (s, 1 H, OCH2O), 5.96 (s, 1 H, OCH2O), 6.29 (s, 2 H, ArH), 6.51 (s,
1 H, ArH), 6.84 (s, 1 H, ArH), 7.38 (m, 1 H, ArH), 7.42 (m, 1 H, ArH),
7.61 (dd, J ¼ 2.1, 7.2 Hz, 1 H, ArH), 7.64 (dd, J ¼ 1.8, 7.2 Hz, 1H, ArH);
4.1.1. 4
desoxypodophyllotoxin (5a)
White solid, yield 54%; [
b
-(3-Benzoylthioureido)-40-O-demethyl-4-
13C NMR (CDCl3):
d 37.5, 42.3, 43.6, 54.1, 56.5, 68.7, 76.6, 77.0, 77.4,
a
]
25 -79 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
101.6, 108.0, 109.1, 110.2, 119.5, 127.6, 127.9, 130.1, 130.1, 132.6, 133.2,
134.1, 134.2, 146.6, 147.7, 148.8, 167.2, 173.9, 180.0; HRMS calcd for
C29H25N2O8NaSBr 663.0413, found 663.0406; mp 162e163 ꢀC from
ethanol.
D
d
2.14 (s,1 H,1- NH), 2.92 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07(m,1 H, 3-
H), 3.77 (s, 6 H, 30, 50-OCH3), 3.99 (d, J ¼ 5.4,1 H, 4-H), 4.32 (m, 2 H,11-
H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.86 (brs, 1 H, 2-NH), 5.94 (s, 1 H,
OCH2O), 5.96 (s,1 H, OCH2O), 6.30 (s, 2 H, ArH), 6.52 (s,1 H, ArH), 6.83
(s,1 H, ArH), 7.48 (d, J ¼ 7.5 Hz,1H, ArH), 7.51 (d, J ¼ 5.7 Hz,1H, ArH),
7.62 (t, J ¼ 5.7 Hz, 1 H, ArH), 7.80 (s, 1 H, ArH), 7.83 (s, 1 H, ArH); 13C
4.1.6. 4b
-[3-(2-Iodobenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5f)
NMR (CDCl3):
d
37.5, 42.3, 43.6, 54.1, 56.5, 68.7, 76.6, 77.0, 77.4,101.6,
Yellow solid, yield 55%; [a]
25 -81 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
D
108.6, 109.1, 110.2, 127.5, 127.8, 129.2, 130.1, 132.5, 133.9, 146.5, 147.7,
148.8, 167.1, 173.9, 180.6; HRMS calcd for C29H26N2O8NaS 585.1308,
found 585.1299; mp 156e158 ꢀC from ethanol.
d
2.13 (s,1 H,1-NH), 2.93 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07 (m,1 H, 3-
H), 3.76 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 4.8,1H, 4-H), 4.32 (m, 2 H,11-
H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.86 (brs, 1 H, 2-NH), 5.94 (s, 1 H,
OCH2O), 5.97(s,1 H, OCH2O), 6.29 (s, 2 H, ArH), 6.51 (s,1 H, ArH), 6.84
(s, 1 H, ArH), 7.20 (m, 1 H, ArH), 7.22 (m, 1 H, ArH), 7.56 (d, J ¼ 2.1 Hz,
4.1.2. 4b
-[3-(2-Chlorobenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5b)
1 H, ArH), 7.63 (d, J ¼ 1.8 Hz,1 H, ArH); 13C NMR (CDCl3):
d 37.5, 42.3,
25
Yellow solid, yield 55%; [
a
]
-84 (c 0.3, CH2Cl2); 1H NMR
43.6, 54.1, 56.5, 68.7, 76.6, 77.0, 77.4, 92.1, 101.6, 108.0, 109.1, 110.2,
127.6,128.5,128.8,130.1,132.6,132.9,138.5,140.2,146.6,147.7,148.8,
168.9, 173.9, 180.2; HRMS calcd for C29H25N2O8NaSI 711.0274, found
711.0267; mp 165e167 ꢀC from ethanol.
D
(CDCl3):
d
2.13 (s,1 H,1-NH), 2.93 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07
(m, 1 H, 3-H), 3.76 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 5.1, 1H, 4-H), 4.32
(m, 2 H,11-H), 4.62 (d, J ¼ 4.8 Hz,1H,1-H), 5.86 (brs,1 H, 2-NH), 5.94
(s, 1 H, OCH2O), 5.96 (s, 1 H, OCH2O), 6.28 (s, 2 H, ArH), 6.52 (s, 1 H,
ArH), 6.84 (s, 1 H, ArH), 7.38 (m, 1 H, ArH), 7.42 (dd, J ¼ 1.8, 7.2 Hz,
1 H, ArH), 7.56 (dd, J ¼ 2.1, 7.2 Hz, 1 H, ArH), 7.63 (d, J ¼ 6.9 Hz, 1 H,
4.1.7. 4b
-[3-(4-Fluorbenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5g)
ArH); 13C NMR (CDCl3):
d
37.5, 42.3, 43.6, 54.1, 56.5, 68.7, 76.6, 77.0,
Yellow solid, yield 53%; [a]
25 -82 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
D
77.4, 101.6, 108.0, 109.1, 110.2, 127.6, 130.1, 130.6, 132.6, 133.4, 146.6,
147.7, 148.8, 166.2, 173.9, 180.1; HRMS calcd for C29H25N2O8NaSCl
619.0918, found 619.0931; mp 161e163 ꢀC from ethanol.
d
2.12 (s,1 H,1-NH), 2.90 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07 (m,1 H, 3-
H), 3.76 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 4.8,1 H, 4-H), 4.32 (m, 2 H,11-
H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.86 (brs, 1 H, 2-NH), 5.94 (s, 1 H,
OCH2O), 5.96(s,1 H, OCH2O), 6.28 (s, 2 H, ArH), 6.51 (s,1 H, ArH), 6.82
(s,1 H, ArH), 7.18 (d, J ¼ 2.1 Hz,1 H, ArH), 7.21(d, J ¼ 2.1 Hz,1 H, ArH),
7.82 (d, J ¼ 1.8 Hz, 1 H, ArH), 7.84 (d, J ¼ 1.8 Hz, 1 H, ArH); 13C NMR
4.1.3. 4b
-[3-(3-Chlorobenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5c)
25
Yellow solid, yield 55%; [
a
]
-75 (c 0.3, CH2Cl2); 1H NMR
(CDCl3): d 37.5, 42.3, 43.6, 54.1, 56.5, 68.7, 76.6, 77.0, 77.4,101.6,107.9,
D
(CDCl3):
d
2.13 (s,1 H,1-NH), 2.93 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.07
109.1, 110.2, 116.3, 116.6, 127.7, 130.1, 130.3, 130.4, 132.5, 134.2, 146.5,

Y. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 901e906
905
147.7, 148.8, 166.1, 173.9, 180.5; HRMS calcd for C29H25N2O8NaSF
OCH2O), 5.97 (s, 1 H, OCH2O), 6.30 (s, 2 H, 20, 60-ArH), 6.51 (s, 1 H, 8-
ArH), 6.81(s, 1 H, 5-ArH), 7.50 (m, 3 H, 200, 300, 500-ArH), 13C NMR
603.1213, found 603.1219; mp 157e159 ꢀC from ethanol.
(CDCl3): d 37.5, 42.3, 43.7, 54.2, 56.5, 68.7, 76.6, 77.0, 77.5,101.7,108.0,
4.1.8. 4
desoxypodophyllotoxin (5h)
b
-(3-
a
-Naphthoylthioureido)-40-O-demethyl-4-
109.1, 110.2, 127.7, 128.6, 130.2, 131.0, 132.5, 134.3, 134.7, 135.7, 146.6,
147.8, 148.8, 167.4, 173.9, 180.2; HRMS calcd for C27H25N2O8S2,
569.1059 found 569.1073; mp 134e136 ꢀC from ethanol.
Yellow solid, yield 65%; [a]
25 -80 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
D
d
2.16 (s,1 H,1- NH), 3.00 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.11 (m,1 H, 3-
H), 3.79 (s, 6 H, 30, 50-OCH3), 3.80 (d, J ¼ 5.1,1 H, 4-H), 4.32 (m, 2 H,11-
H), 4.67 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.91 (brs, 1 H, 2-NH), 5.98 (s, 1 H,
OCH2O), 6.00(s, 1 H, OCH2O), 6.33 (s, 2 H, 20, 60-ArH), 6.55 (s, 1 H, 8-
ArH), 6.92 (s,1 H, 5-ArH), 7.60 (m, 3 H, 300, 600, 700-H), 7.80 (d, J ¼ 6.9 Hz,
1 H, 200-H), 7.92 (d, J ¼ 7.5 Hz,1 H, 500-H), 8.05 (d, J ¼ 8.4 Hz,1 H, 400-H);
4.2. Cell culture
Three human cell lines, HepG2 (Hepatocellular carcinoma), A549
(lung carcinoma), HCT-116 (colon carcinoma) were cultured on
RPMI-1640 medium supplemented with fetal bovine serum (10%),
8.30 (d, J ¼ 8.4 Hz,1 H, 800-H); 13C NMR (CDCl3):
d 37.5, 42.3, 43.7, 54.2,
penicillin (100 U/mL) and streptomycin (100
m
g/mL) in 25 cm2
culture flasks at 37 ꢀC in a humidified atmosphere with 5% CO2.
56.53 68.8, 76.6, 77.5, 101.7, 107.9, 109.2, 110.2, 124.5, 126.7, 127.1,
127.7,128.8,129.8,130.2,132.6,133.4,133.8,134.2,146.5,147.8,148.8,
169.3, 174.1, 180.7; HRMS calcd for C33H28N2O8S 613.1660, found
613.1665; mp 148e150 ꢀC from ethanol.
4.3. Cell viability
Cell viability was assessed by the MTT assay. Cells were har-
vested from the culture during the exponential growth phase, and
seeded into multiwell culture plates at 5 ꢁ 104e1 ꢁ 105 cells/mL
in fresh medium. After overnight growth, cells were treated with
compounds (predissolved in DMSO) at selected concentrations for
a period of 3 days. The medium was then discarded and replaced
with MTT dye. Plates were incubated at 37 ꢀC for 4 h. The
resulting formazan crystals were solubilized in lysis buffer
(sodium dodecyl sulfate (SDS) 10 g, N, N-dimethylformamide
(DMF) 25 mL, H2O 25 mL, Acetic acid 1 mL, pH 4.7), and the
optical density was read at 570 nm with a microplate reader
(Biotek synergy 2).
4.1.9. 4b
-[3-(2-Methylbenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin (5i)
25
Yellow solid, yield 62%; [
a
]
-90 (c 0.3, CH2Cl2); 1H NMR
D
(CDCl3):
d
2.15 (s, 1 H, 1-NH), 2.47 (s, 3 H, CH3), 2.93 (dd, J ¼ 4.8,
14.4 Hz, 1 H, 2-H), 3.08 (m, 1 H, 3-H), 3.78 (s, 6 H, 30, 50-OCH3), 3.80
(d, J ¼ 5.1, 1 H, 4-H), 4.29 (m, 2 H, 11-H), 4.63 (d, J ¼ 4.8 Hz, 1 H, 1-H),
5.86 (brs, 1 H, 2-NH), 5.96 (s, 1 H, OCH2O), 5.98 (s, 1 H, OCH2O), 6.31
(s, 2 H, 20, 60-ArH), 6.53 (s, 1 H, 8-ArH), 6.86 (s, 1 H, 5-ArH), 7.43 (m,
4 H, 300, 400, 500, 600-ArH); 13C NMR (CDCl3):
d 20.2, 29.7, 37.5, 42.3,
43.7, 54.1, 56.5, 68.8, 76.6, 77.0, 77.5, 101.7, 108.0, 109.2, 110.3, 126.3,
127.2, 127.8, 130.2, 132.0, 132.3, 132.6, 134.3, 137.9, 146.6, 147.8,
148.8, 169.5, 174.0, 180.7; HRMS calcd for C30H29N2O8S 577.1644,
found 577.1645; mp 144e146 ꢀC from ethanol.
4.4. Topoisomerase II-mediated kDNA decatenation assay
4.1.10. 4b
-[3-(3-Methylbenzoyl)thioureido]-40-O-demethyl-4-
A gel assay was carried out as previously described [20] to
determine if compound 5a inhibited the catalytic decatenation
activity of topoisomerase II. kDNA consists of highly catenated
networks of circular DNA, which can be decatenated by top-
oisomerase II in an ATP-dependent reaction.
desoxypodophyllotoxin (5j)
25
Yellow solid, yield 69%; [
a]
-76 (c 0.3, CH2Cl2); 1H NMR
D
(CDCl3):
d
2.03 (s, 1 H, 1-NH), 2.42 (s, 3 H, CH3), 2.92 (dd, J ¼ 4.8,
14.4 Hz, 1 H, 2-H), 3.08 (m, 1 H, 3-H), 3.76(s, 6 H, 30, 50-OCH3), 3.96
(d, J ¼ 5.1, 1 H, 4-H), 4.50 (m, 2 H, 11-H), 4.62 (d, J ¼ 4.8 Hz, 1 H, 1-H),
5.85 (brs, 1 H, 2-NH), 5.94 (s, 1 H, OCH2O), 5.97 (s, 1 H, OCH2O), 6.31
(s, 2 H, 20, 60-ArH), 6.52 (s, 1 H, 8-ArH), 6.83 (s, 1 H, 5-ArH), 7.55(m,
4.5. Cell cycle analysis
4 H, 200, 400, 500, 600-ArH), 13C NMR (CDCl3):
d 21.7, 31.7, 37.5, 42.3, 43.7,
We used the human colon carcinoma HCT-116 cell line for
cell cycle analysis. Briefly, cells were grown in RPMI-1640 supple-
mented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM
54.1, 56.5, 61.9, 71.1, 76.6, 77.1, 77.4, 77.5, 101.7, 108.0, 109.2, 110.2,
127.6, 127.8, 128.3, 130.0, 130.2, 132.5, 134.2, 145.1, 146.5, 147.7,
148.8, 167.1, 174.0, 180.6; HRMS calcd for C30H29N2O8S, 577.1642,
found 577.1645; mp 123e125 ꢀC from ethanol.
L
-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin
at 37 ꢀC in a humidified atmosphere with 5% CO2. Untreated and
drug treated cells were centrifuged and fixed overnight in 70%
ethanol at 4 ꢀC. Washed three times with PBS, incubated for 1 h
4.1.11. 4b
-[3-(4-Methylbenzoyl)thioureido]-40-O-demethyl-4-
desoxypodophyllotoxin(5k)
with 1 mg/mL RNase A and 20 mg/mL propidium iodide at room
temperature, and analyzed with a FACSCalibur flow cytometer (BD).
Yellow solid, yield 69%; [a]
25 -89 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
D
d
2.14 (s,1 H,1- NH), 2.41 (s, 3 H, CH3), 2.90 (dd, J¼ 4.8,14.4 Hz,1 H, 2-H),
3.05 (m,1 H, 3-H), 3.75 (s, 6 H, 30, 50-OCH3), 3.77 (d, J¼ 5.1,1 H, 4-H), 4.40
(m, 2 H,11-H), 4.63 (d, J ¼ 4.8 Hz,1 H,1-H), 5.87 (brs,1 H, 2-NH), 5.94 (s,
1 H, OCH2O), 5.97 (s,1H, OCH2O), 6.31 (s, 2 H, 20, 60-ArH), 6.52 (s,1 H, 8-
ArH), 6.84 (s, 1 H, 5-ArH), 7.50 (m, 4 H, 200, 300, 500, 600-ArH), 13C NMR
4.6. Statistical analysis
Data were presented as means ꢂ SE and analyzed by SPSS
software. Pictures were processed with Photoshop software. Mean
values were obtained from at least three independent experiments.
(CDCl3):
d 21.4, 37.5, 42.3, 43.7, 54.1, 56.5, 68.7, 76.6, 77.1, 77.5, 101.7,
108.0, 109.2, 110.2, 124.6, 127.8, 128.1, 129.2, 130.2, 131.2, 132.5, 134.2,
134.8, 139.4, 146.5, 147.8, 148.8, 167.4, 174.0, 180.6; HRMS calcd for
C30H29N2O8S, 577.1643 found 577.1645; mp 134e136 ꢀC from ethanol.
Acknowledgments
The studies in the laboratories were supported by National
Program on Key Basic Research Project (2009CB930300), Natural
Science Foundation of Nantong City (K2010024).
4.1.12. 4b
-(3-Thienylthioureido)-40-O-demethyl-4-
desoxypodophyllotoxin (5l)
Yellow solid, yield 70%; [a]
25 -81 (c 0.3, CH2Cl2); 1H NMR (CDCl3):
D
d
2.02 (s,1 H,1-NH), 2.90 (dd, J ¼ 4.8,14.4 Hz,1 H, 2-H), 3.08 (m,1 H, 3-
References
H), 3.74 (s, 6 H, 30, 50-OCH3), 3.77 (d, J ¼ 5.1,1 H, 4-H), 4.40 (m, 2 H,11-
H), 4.61 (d, J ¼ 4.8 Hz, 1 H, 1-H), 5.83 (brs, 1 H, 2-NH), 5.93 (s, 1 H,
[1] J.J. Champoux, Annu. Rev. Biochem. 70 (2001) 369e413.

906
Y. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 901e906
[2] Y. Pommier, E. Leo, H.L. Zhang, C. Marchand, Chem. Biol. 17 (2010) 421e433.
[3] C.R. Hauser, J.K. Lindsay, J. Org. Chem. 21 (1956) 381e382.
[12] B.A. Bhat, P.B. Reddy, S.K. Agrawal, A.K. Saxena, H.M.S. Kumar, G.N. Qazi, Eur. J.
Med. Chem. 43 (2008) 2067e2072.
[4] K.R. Hande, Eur. J. Cancer 34 (1998) 1514e1521.
[5] P. Meresse, E. Dechaux, C. Monneret, E. Bertounesque, Curr. Med. Chem. 11
(2004) 2443e2466.
[6] A. Kamal, N.L. Gayatri, D.R. Reddy, M.M. Reddy, M. Arifuddin, S.G. Dastidar,
A.K. Kondapi, M. Rajkumar, Bioorg. Med. Chem. 13 (2005) 6218e6225.
[7] H. Xu, M. Lv, X. Tian, Curr. Med. Chem. 16 (2009) 327e349.
[8] Y. You, Curr. Pharm. Des. 11 (2005) 1695e1717.
[13] S.W. Chen, Y.H. Wang, Y. Jin, X. Tian, Y.T. Zheng, D.Q. Luo, Y.Q. Tu, Bioorg. Med.
Chem. Lett. 17 (2007) 2091e2095.
[14] X. Tian, Y.G. Wang, M.G. Yang, Y.Z. Chen, Life Sci. 60 (1997) 511e517.
[15] M. Duca, P.B. Arimondo, S. Leonce, A. Pierre, B. Pfeiffer, C. Monneret,
D. Dauzonne, Org. Biomol. Chem. 3 (2005) 1074e1080.
[16] Zhao Y., Wang C.N., Wu Z.H., Fang J.H., Zhu L. Invest. New Drugs, in press, doi:
10.1007/s10637-010-9508-1.
[9] M.A. Castro, J.M. Corral, M. Gordaliza, P.A. Garcia, M.A. Zurita, A.S. Feliciano,
J. Med. Chem. 47 (2004) 1214e1222.
[17] L. Daley, P. Meresse, E. Bertounesque, C. Monneret, Tetrahedron Lett. 38
(1997) 2673e2676.
[10] T.S. Huang, C.H. Shu, Y.L. Shih, H.C. Huang, Y.C. Su, Y. Chao, W.K. Yang,
J.W. Peng, Cancer Lett. 110 (1996) 77e85.
[11] T.S. Huang, C.C. Lee, Y. Chao, C.H. Shu, L.T. Chen, L.L. Chen, M.H. Chen,
C.C. Yuan, J.W. Peng, Pharm. Res. 16 (1999) 997e1002.
[18] X.M. Zhou, Z.Q. Wang, J.Y. Chang, J. Med. Chem. 34 (1991) 3346e3352.
[19] A. Manaka, T. Ishii, K. Takahashi, M. Sato, Tetrahedron Lett. 46 (2005) 419e422.
[20] B.B. Hasinoff, H. Kazlowska, A.M. Creighton, W.P. Allan, P. Thampatty,
J.C. Yalowich, Mol. Pharmacol. 52 (1997) 839e845.