Synthesis and antitumor activity of novel aroylthiourea derivatives of podophyllotoxin

Article abstract of DOI:10.1007/s10637-010-9508-1

A novel series of 4β-[(4-substituted) aroylthiourea] derivatives of podophyllotoxin were synthesized and their abilities to inhibit the growth of cancer cells were investigated by MTT assay. Compound 4a possessed the highest cytotoxicity on HepG2, A549 an

Full text of DOI:10.1007/s10637-010-9508-1

Invest New Drugs (2012) 30:17–24
DOI 10.1007/s10637-010-9508-1
PRECLINICAL STUDIES
Synthesis and antitumor activity of novel aroylthiourea
derivatives of podophyllotoxin
Yu Zhao & Chengniu Wang & Zhonghua Wu &
Jinghuai Fang & Li Zhu
Received: 16 June 2010 /Accepted: 26 July 2010 /Published online: 10 August 2010
#
Springer Science+Business Media, LLC 2010
Summary A novel series of 4β-[(4-substituted) aroylth-
iourea] derivatives of podophyllotoxin were synthesized
and their abilities to inhibit the growth of cancer cells were
investigated by MTT assay. Compound 4a possessed the
highest cytotoxicity on HepG2, A549 and HCT-116 cancer
cell lines with the IC50 values of 0.1 μM. Apoptosis in
HCT-116 cells induced by compound 4a was observed by
Hoechst33342-Propidium iodide (PI) and acridine orange
(AO)-ethidium bromide (EB) double staining assays. DNA
flow cytometric analysis revealed that 4a induced cell cycle
arrest at G2/M phase and kDNA decatenation assay
indicated that 4a inhibited topoisomerase IIα-mediated
kDNA decatenation. Our results indicated that compound
4a possessed promising antitumor activity, which need to
be studied further.
analogues etoposide and teniposide are in clinical use as
antineoplastic agents due to their ability to inhibit the
enzyme DNA-topoisomerase II [3, 4]. Their clinical use has
encountered certain limitations such as drug resistance and
poor water solubility. In order to obtain better therapeutic
agents, a lot of derivatives of podophyllotoxin have been
synthesized. New synthetic N-linked congeners like GL-
331 and NPF exhibite improved cytotoxicity and topo-II
inhibition activity [5–10].
Aroylthioureas are known as good ligands for metal
ions, and these metal ion plays important role in tumori-
genesis. Moreover, thiourea derivatives possess good
inhibitory activity against protein tyrosine kinases (PTKs),
which play critical roles in many aspects of tumorigenesis
[11–13]. This prompted us to synthesize a library of
aroylthiourea derivatives of podophyllotoxin as novel
anticancer agents with the hope of optimizing the antican-
cer and cytotoxic activity of the podophyllotoxin. Herein,
we report the synthesis and cytotoxicity activity of these
aroylthiourea derivatives of podophyllotoxin (4a–4l
Scheme 1). We found that compound 4a possess promising
antitumor activity.
.
.
.
Keywords Podophyllotoxin Aroylthiourea Synthesis
Antitumor
Introduction
Natural occuring lignan lactones include compounds with
important anticancer and antiviral activities such as podo-
phyllotoxin and diphyllin [1, 2] (Fig. 1). Podophyllotoxin
exerts its antitumor activities through inhibits the assembly
of tubulin into microtubules. However, podophyllotoxin
Results and discussion
Synthesis of aroylthiourea derivatives of podophyllotoxin
The synthetic route used to synthesis title compounds is
outlined in Scheme 1. The reaction of hydrazoic acid with
podophyllotoxin in the presence of BF3-Et2O gave 4-β-
azido derivative 2 [6]. Nextly, the compound 4β-amino-4-
deoxypodophyllotoxin 3 was prepared by reduction of the
azide using Pd/C as a catalyst according to the procedure
reported previously [14]. Coupled corresponding aroyliso-
:
:
:
Y. Zhao C. Wang Z. Wu L. Zhu (*)
Institute of Nautical Medicine, Nantong University,
Nantong 226001, China
e-mail: zhulili65@163.com
J. Fang
School of Science, Nantong University,
Nantong 226001, China

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Invest New Drugs (2012) 30:17–24
Fig. 1 Podophyllotoxin and its
analogues
thiocyanates [15] with amine 3 respectively in dry
acetonitrile over a 2 h reflux period gave a novel series of
aroylthiourea derivatives 4a–4l in the yields of 51.6–
63.6%. However, the reaction of aroylisothiocyanates
bearing strong electron-withdrawing groups like nitro group
with 3 failed to give desired products.
comparatively lesser activity against A549 cell line than
other two cell lines. Compound 4a possessed the highest
growth inhibitory effect on HepG2, A549 and HCT-116
cancer cell lines with IC50 values of 0.1 μM. Cytotoxic
activity decreased when the benzoyl moiety was substituted
by a halogen group or a methyl group. Compound 4a was
found more potent than etoposide (VP-16) and podophyl-
lotoxin. Apparently, the relative position of halogens on
benzoyl moiety was critical for their cytotoxicities. Com-
pounds subsitituted with halogens on ortho-position were
found to be more potent than that of meta-position and
para-position. Compounds bearing a methyl group
exhibited better inhibitory properties than halogen-
substituted compounds with the IC50 values in the range
from 1.4 μM to 6.7 μM. The relative position of methyl
groups on benzoly moiety had no effect on their cytotox-
icities. Compound 4h, which bearing α-napthyl group, was
found less potent than 4a, which indicated bulky groups
contributed to weak cytotoxicities.
Cytotoxicities of compounds 4a–4l 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.
From the IC50 values, it is clear that most of these
derivatives displayed strong cytotoxic activities against
three cancer cell lines. However, these derivatives showed
S
O
N
R
NH2
HN
N3
OH
H
5
8
4
3
2
CH3CN, reflux
O
6
7
10
O
O
O
O
O
O
O
O
HN3, BF3·Et2O
Pd, H2, EtOAc
O
O
O
O
9
1
O
O
O
O
R
C NCS
1'
6'
2'
3'
OCH3
H3CO
OCH3
H3CO
5'
H3CO
OCH3
H3CO
OCH3
4'
OCH3
OCH3
OCH3
OCH3
3
2
1
4a-4l
4a: R=Ph
4b: R=2-Cl-Ph
4c: R=3-Cl-Ph
4d: R=4-Cl-Ph
4e: R=2-Br-Ph
4f: R=2-I-Ph
4g: R=4-F-Ph
4h: R=α-napthyl
4i: R=2-Me-Ph
4j: R=3-Me-Ph
4k: R=4-Me-Ph
4l: R=thiophene
Scheme 1 Synthesis of aroylthiourea derivatives of podophyllotoxin 4a–4l

Invest New Drugs (2012) 30:17–24
19
Table 1 Cytotoxicities against cancer cell lines of aroylthiourea
derivatives
cancer cell lines and compound 4a showed most potent
cytotoxicity.
Compd
IC50 (μM)
Induction of apoptosis in HCT-116 cells by compound 4a
HepG2
HCT-116
A549
On the basis of the above results, further biological
evaluations have been focused on compound 4a. HCT-116
cells were stained with Hoechst33342-PI and AO-EB to
verify the type of cell death. Without compound 4a
treatment, the nuclei of control cells showed uniform dark
blue fluorescence, which indicated the cells were healthy
and the nuclei were intact. However, after treatment with
compound 4a in 0.1 μM for 72 h, 42% of cells were stained
bright blue, which indicated the cells were apoptotic
(Fig. 2). These results indicated that cells death occurred
primarily through apoptosis.
The results obtained from the AO-EB staining are as
shown in Fig. 2. Viable cells with intact DNA and nucleus
showed homogeneous bright green. Early apoptotic cells
showed green nuclei, but perinuclear chromatin condensa-
tion was visible as bright green patches or fragments. Late
apoptotic cells DNA were fragmented and stained orange. It
was clear that almost 45% of cells treated with compound
4a in 0.1 μM for 72 h exhibited typical characteristics of
apoptosis. These results were similar to Hoechst33342-PI
4a
0.1
12.1
26.7
30.0
13.8
6.8
0.1
4.3
5.9
6.8
7.1
7.0
6.1
1.9
1.5
1.8
1.7
3.5
3.2
13.5
2.7
0.1
8.6
4b
4c
8.3
4d
10.3
2.9
4e
4f
0.9
4g
>50
7.4
9.5
4h
4.6
4i
2.1
6.7
4j
1.4
6.1
4k
1.8
4.7
4l
3.5
13.5
5.9
3
2.2
etoposide
4.9
21.4
6.0
podophyllotoxin
1.4
In summary, we have prepared novel aroylthiourea
derivatives of podophyllotoxin. The synthetic compounds
4a–4l were cytotoxic at the micromolar range against three
Fig. 2 Morphological study
of HCT-116 cells treated with
compound 4a in 0.05 μM,
0.1 μM respectively for 72 h.
Upper panel: Hoechst/PI
staining, dark blue cells showed
early apoptotic, bright blue cells
were later apoptotic. Middle
panel: AO/EB staining,
vacuolation and DNA
fragmentation was obvious,
orange cells indicate later
apoptosis. Lower panel: Bar
diagram for comparison of
percent cells in apoptosis as
revealed by AO/EB, Hoechst/PI
staining followed by manual
counting in different fields of
vision for 3 times

20
Invest New Drugs (2012) 30:17–24
Fig. 3 Effects of compound 4a on cell cycle of HCT-116 cells. Cells were incubated with 0, 1 μM, 5 μM, 10 μM, 20 μM 4a for 24 h and stained
with propidium iodide (PI). Their DNA content was analyzed by fluorescence flow cytometry
staining, which also indicated that cells death occurred
primarily through apoptosis.
Experimental
Syntheses
Induction of cell cyle arrest in HCT-116 cells by compound 4a
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 (100–200
To gain further insight into the mode of action of compound
4a, we examined the effects of compound 4a 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 domonstrated that
treatment of HCT-116 cells for 24 h with 1, 5, 10 and
20 μm compound 4a increased the percentage of cells at
G2/M phase from 40.34% to 97.70%. The flow cytometric
data clearly indicated that compound 4a causes HCT-116
cell cycle arrest at G2/M phase.
1
mesh, Qingdao, China). H NMR and 13C NMR spectra
were taken on a Bruker 300 MHz spectrometer with
tetramethylsilane (TMS) as an internal standard, and chem-
ical shifts were recorded in values. The high resolution
spectra were obtained on a Q-TOF Global Mass (ESIMS).
Inhibition of human topoisomerase IIα kDNA decatenation
by compound 4a
To further understand the action mechanisms of antitumor
activity of compound 4a, the effect on the catalytic activity
of human topoisomerase IIα was evaluated using kDNA
decatenation assay. As shown in Fig. 4, compound 4a
completely inhibited the catalytic activity of topoisomerase
IIα at 400 μM and 800 μM, equating to the effect of VP-16
at 500 μM. The results indicated that 4a exerted its
antitumor activities through inhibition of human topoiso-
merase IIα catalytic activity.
Fig. 4 Effects of 50 μM, 100 μM, 200 μM, 400 μM, 800 μM
compound 4a and etoposide on human topoisomerase IIα-mediated
decatenation activity of kDNA

Invest New Drugs (2012) 30:17–24
21
Typical synthetic procedure: 1 mmol of 3 was added to a
solution of 1.2 mmol of corresponding aroylisothiocyanate
in 10 ml of in dry acetonitrile. The mixture was refluxed on
a water bath for 2 h. After the reaction completed, the result
solution was evaporated under reduced pressure. The
residue was purified by flash chromatography (1:3,
EtOAc-petroleum ether) to afford a solid.
14.4 Hz, 1 H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′, 5′-
OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d, J=4.5 Hz, 1 H, 4-
H), 4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.37 (d, J=1.8 Hz, 5″-ArH), 7.42 (d,
J=1.8 Hz, 4″-ArH), 7.56 (d, J=2.1 Hz, 1 H, 6″-ArH), 7.63
(s, 1 H, 2″-ArH); 13C NMR (CDCl3): δ 37.5, 42.2, 43.8,
54.2, 56.3, 60.7, 68.7, 76.6, 77.0, 77.5, 101.7, 108.2, 109.1,
110.2, 127.9, 130.5, 132.3, 134.6, 135.5, 137.3, 138.9,
147.8, 148.8, 152.6, 162.0, 165.9, 173.8, 180.2; HRMS
calcd for (M+Na)+ C30H27ClN2O8SNa 633.1074, found
633.1068; mp 133–135ºC from ethanol; IR (KBr, cm−1):
3426, 3231, 2946, 1767, 1677, 1511, 1123, 1043.
4β-(Benzoyl-thioureido)-4-deoxypodophyllotoxin (4a)
white solid, yield 62.1%; [α]D25-73 (c 0.3, CH2Cl2); H
1
NMR (CDCl3): δ 2.01 (s, 1 H, 1-NH), 2.93 (dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.07 (m , 1 H, 3-H), 3.73 (s, 6 H, 3′ , 5′-
OCH3), 3.78 (s, 3 H, 4′-CH3), 3.80 (d, J=6.0 Hz, 1 H, 4-H),
4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.48 (d, J=7.5 Hz, 1 H, 3″-ArH), 7.51
(d, J=5.7 Hz, 1 H, 5″-ArH), 7.62 (t, J=5.7 Hz, 1 H,
4″-ArH), 7.79 (s, 1 H, 2″-ArH), 7.81 (s, 1 H, 6″-ArH); 13C
NMR (CDCl3): δ 37.6, 42.2, 43.8, 54.1, 56.3, 60.7, 68.7,
76.6, 77.0, 77.4, 101.7, 108.3, 109.1, 110.2, 127.5, 129.2,
131.2, 132.3, 133.9, 134.6, 137.4, 147.8, 148.8, 152.7,
167.1, 173.8, 180.5; HRMS calcd for (M+Na)+
C30H28N2O8SNa 599.1464, found 599.1445; mp 114–
116ºC from ethanol; IR (KBr, cm−1): 3413, 3237, 2930,
1774, 1668, 1504, 1114, 1030.
4β-(4″-Chloro-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4d)
white solid, yield 60.0%; [α]D25-72 (c 0.3, CH2Cl2); H
1
NMR (CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′, 5′-
OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d, J=4.5 Hz, 1H, 4-
H), 4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.45 (d, J=1.8 Hz, 1 H, 3″-ArH), 7.48
(d, J=1.8 Hz, 1 H, 5″-ArH), 7.74 (d, J=2.1 Hz, 1 H, 2″-
ArH), 7.77 (d, J=1.8 Hz, 1 H, 6″-ArH); 13C NMR (CDCl3):
δ 37.5, 42.2, 43.8, 54.1, 56.3, 60.7, 68.6, 76.6, 77.0, 77.5,
97.4, 101.7, 108.3, 109.1, 110.2, 111.6, 127.7, 128.9,
129.6, 132.4, 134.6, 140.5, 147.8, 148.8, 152.7, 166.1,
173.8, 180.3; HRMS calcd for (M+Na)+ C30H27
ClN2O8SNa 633.1074, found 633.1066; mp 128–129ºC
from ethanol; IR (KBr, cm−1): 3423, 3231, 2939, 1770,
1681, 1514, 1120, 1040.
4β-(2″-Chloro-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4b)
white solid, yield 51.0%; [α]D25-85 (c 0.3, CH2Cl2); 1H NMR
(CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8, 14.4 Hz, 1
H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′, 5′-OCH3), 3.78
(s, 3 H, 4′-OCH3), 3.80 (d, J=5.1 Hz, 1 H, 4-H), 4.32 (m, 2
H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH), 6.84 (s, 1 H, 5-ArH),
7.38 (m, 1 H, 5″-ArH), 7.42 (dd, J=1.8, 7.2 Hz, 1 H, 3″-
ArH), 7.56 (dd, J=2.1, 7.2 Hz, 1 H, 4″-ArH), 7.63 (d, J=
6.9 Hz, 1 H, 6″-ArH); 13C NMR (CDCl3): δ 37.6, 42.2, 43.8,
54.0, 56.3, 60.7, 68.7, 76.6, 77.1, 77.5, 101.7, 108.2, 109.2,
110.2, 127.5, 130.5, 130.9, 131.6, 132.4, 134.6, 137.3, 147.8,
148.8, 152.6, 166.3, 173.9, 180.1; HRMS calcd for (M+Na)+
C30H27ClN2O8SNa 633.1074, found 633.1075 ; mp 128–
130ºC from ethanol; IR (KBr, cm−1): 3461, 3247, 2933,
1774, 1687, 1511, 1127, 1040.
4β-(2″-Bromo-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4e)
white solid, yield 53.8%; [α]D25-71 (c 0.3, CH2Cl2); H
1
NMR (CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′, 5′-
OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d, J=4.8 Hz, 1 H, 4-
H), 4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.37 (m, 1 H, 5″-ArH), 7.42 (m, 1 H,
4″-ArH), 7.56 (dd, J=2.1, 7.2 Hz, 1 H, 3″-ArH), 7.63 (dd,
J=1.8, 7.2 Hz, 1 H, 6″-ArH); 13C NMR (CDCl3): δ 37.5,
42.2, 43.8, 54.1, 56.3, 60.7, 68.7, 76.6, 77.1, 101.7, 108.2,
109.2, 110.2, 119.5, 127.5, 128.0, 130.0, 132.4, 133.3,
134.1, 134.6, 137.3, 147.8, 148.8, 152.6, 167.2, 173.9,
180.0; HRMS calcd for (M+Na)+ C30H27BrN2O8SNa
4β-(3″-Chloro-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4c)
white solid, yield 63.6%; [α]D25-77 (c 0.3, CH2Cl2); H
1
NMR (CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8,

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Invest New Drugs (2012) 30:17–24
677.0569, found 677.0572; mp 143–145ºC from ethanol; IR
ArH), 8.05 (d, J=8.4 Hz, 1 H, 4″-ArH); 8.29(d, J=8.4 Hz,
1 H, 8″-ArH); 13C NMR (CDCl3): δ 37.6, 42.3, 43.8, 54.1,
56.3, 60.6, 68.8, 76.6, 77.1, 77.4, 101.7, 108.4, 109.3,
110.3, 124.5, 124.5, 126.7 , 127.1, 127.8, 128.2, 129.8,
132.4, 133.8, 134.7, 147.8, 148.8, 152.7, 169.3, 173.9,
180.7; HRMS calcd for (M+H)+ C34H31N2O8S 627.1812,
found 627.1802; mp 171–173ºC from ethanol; IR (KBr,
cm−1): 3243, 3051, 2923, 2363, 1774, 1674, 1508, 1127,
1037.
(KBr, cm−1): 3455, 3231, 2933, 1780, 1681, 1501, 1127, 1037.
4β-(2″-Iodo-benzoyl-thioureido)-4-deoxypodophyllotoxin (4f)
1
white solid, yield 51.6%; [α]D25-74 (c 0.3, CH2Cl2); H
NMR (CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8 Hz,
14.4 Hz, 1 H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′ , 5′-
OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d, J=4.8, 1.0 Hz, 4-
H), 4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.20 (m, 1 H, 5″-ArH), 7.22 (m, 1 H,
4″-ArH), 7.56 (d, J=2.1 Hz, 1 H, 3″-ArH), 7.63 (d, J=
1.8 Hz, 1 H, 6″-ArH); 13C NMR (CDCl3): δ 37.5, 42.2,
43.8, 54.1, 56.3, 60.7, 68.7, 76.6, 77.0, 101.7, 108.4, 109.1,
110.2, 127.5, 128.5, 128.8, 132.5, 133.0, 134.6, 137.5,
138.4, 140.6, 147.8, 148.8, 152.7, 168.8, 173.8, 180.1;
HRMS calcd for (M+Na)+ C30H27IN2O8SNa 725.0431,
found 725.0452; mp 139–141ºC from ethanol; IR (KBr,
cm−1): 3452, 3231, 2933, 1777, 1684, 1504, 1127, 1030.
4β-(2″-Methyl-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4i)
white solid, yield 74.6%; [α]D25-83 (c 1.0, CH2Cl2); H
1
NMR (CDCl3): δ 2.13 (s, 1 H, 1-NH), 2.45 (s, 3 H, CH3),
2.93 (dd, J=4.8, 14.4 Hz, 1 H, 2-H), 3.08 (m, 1 H, 3-H),
3.76 (s, 6 H, 3′, 5′-OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d,
J=5.1, 1 H, 4-H), 4.26 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.51 (s, 1 H,
8-ArH), 6.84 (s, 1 H, 5-ArH), 7.45 (m, 4 H, 3″, 4″, 5″, 6″-
ArH) ; 13C NMR (CDCl3): δ 37.6, 42.2, 43.8, 54.1, 56.3,
60.7, 68.7, 76.6, 77.1, 77.5, 101.7, 108.4, 109.2, 110.2,
126.3, 127.2, 127.8, 131.9, 132.3, 132.3, 134.4, 137.8,
147.8, 148.8, 152.7, 169.6, 173.9, 180.7; HRMS calcd for
(M+H)+ C31H31N2O8S 591.1802, found 591.1802; mp
112–114ºC from ethanol; IR (KBr, cm−1): 3243, 2920,
2366, 1777, 1674, 1508, 1127, 1034.
4β-(4″-Fluor-benzoyl-thioureido)-4-deoxypodophyllotoxin (4g)
white solid, yield 57.4%; [α]D25-73 (c 0.3, CH2Cl2); H
1
NMR (CDCl3): δ 2.00 (s, 1 H, 1-NH), 2.93 (dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.07 (m, 1 H, 3-H), 3.73 (s, 6 H, 3′ , 5′-
OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.80 (d, J=4.8 Hz, 1 H, 4-
H), 4.32 (m, 2 H, 11-H), 4.62 (d, J=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, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.84 (s, 1 H, 5-ArH), 7.18 (d, J=2.1 Hz, 1 H, 3″-ArH), 7.21
(d, J=2.1 Hz, 1 H, 5″-ArH), 7.82 (d, J=1.8 Hz, 1 H, 2″-
ArH), 7.84 (d, J=1.8 Hz, 1 H, 6″-ArH); 13C NMR (CDCl3):
δ 37.5, 42.2, 43.8, 54.1, 56.2, 60.7, 68.7, 76.6, 77.2, 77.4,
100.0, 101.7, 108.2, 109.1, 110.2, 127.4, 130.2, 130.3,
132.3, 134.6, 137.3, 147.8, 148.8, 152.7, 166.0, 173.9,
180.4; HRMS calcd for (M+Na)+ C30H27FN2O8SNa
617.1370, found 617.1385; mp 132–134ºC from ethanol;
IR (KBr, cm−1):3439, 3233, 2923, 1783, 1671, 1509, 1125,
1043.
4β-(3″-Methyl-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4j)
1
white solid, yield 67.8%; [δ]D25-83 (c 1.0, CH2Cl2); H
NMR (CDCl3): δ 2.03 (s, 1 H, 1-NH), 2.42 (s, 3 H, CH3),
2.95 (dd, J=4.8, 14.4 Hz, 1 H, 2-H), 3.10 (m, 1 H, 3-H),
3.75(s, 6 H, 3′ , 5′-OCH3), 3.80 (s, 3 H, 4′-OCH3), 3.98 (d,
J=5.1, 1 H, 4-H), 4.53 (m, 2 H, 11-H), 4.64 (d, J=4.8 Hz, 1
H, 1-H), 5.86 (brs, 1 H, 2-NH), 5.97 (s, 1 H, OCH2O), 5.99
(s, 1 H, OCH2O), 6.31 (s, 2 H, 2′, 6′-ArH), 6.54 (s, 1 H,
8-ArH), 6.85 (s, 1 H, 5-ArH), 7.43 (m, 4 H, 2″, 4″, 5″, 6″-
ArH) ; 13C NMR (CDCl3): δ 21.3, 37.6, 42.4, 43.8, 54.1,
56.3, 60.7, 68.7, 76.6, 77.1, 77.4, 101.7, 108.3, 109.1,
110.2, 124.6, 127.8, 128.1, 129.1, 131.2, 132.3, 134.6,
139.3, 147.8,, 148.8, 152.6, 167.3, 173.8, 180.6; HRMS
calcd for (M+H)+ C31H31N2O8S 591.1797, found
591.1795; mp 122–124ºC from ethanol; IR (KBr, cm−1):
3423, 3231, 2933, 2360, 1774, 1504, 1120, 1034.
4β-(α-naphthoyl-thioureido)-4-deoxypodophyllotoxin (4h)
1
white solid, yield 65.4%; [α]D25-72 (c 0.3, CH2Cl2); H
NMR (CDCl3): δ 2.15 (s, 1 H, 1-NH), 3.00(dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.11 (m, 1 H, 3-H), 3.76 (s, 6 H, 3′, 5′-
OCH3), 3.80 (s, 3 H, 4′-OCH3), 3.98 (d, J=5.1, 1 H, 4-H),
4.32 (m, 2 H, 11-H), 4.67 (d, J=4.8 Hz, 1 H, 1-H), 5.93
(brs, 1 H, 2-NH), 5.98 (s, 1 H, OCH2O), 6.00(s, 1H,
OCH2O), 6.32 (s, 2 H, 2′, 6′-ArH), 6.55 (s, 1 H, 8-ArH),
6.92 (s, 1 H, 5-ArH), 7.60 (m, 3 H, 3″, 6″, 7″-ArH), 7.80
(d, J=6.9 Hz, 1 H, 2″-ArH), 7.92 (d, J=7.5 Hz, 1 H, 5″-
4β-(4″-Methyl-benzoyl-thioureido)-
4-deoxypodophyllotoxin (4k)
white solid, yield 67.8%; [α]D25-82 (c 1.0, CH2Cl2); H
1
NMR (CDCl3): δ 2.16 (s, 1 H, 1-NH), 2.43 (s, 3 H, CH3),

Invest New Drugs (2012) 30:17–24
23
2.95 (dd, J=4.8, 14.4 Hz, 1 H, 2-H), 3.10 (m, 1 H, 3-H),
3.75 (s, 6 H, 3′, 5′-OCH3), 3.80 (d, J=5.1, 1 H, 4-H), 4.36
(m, 2 H, 11-H), 4.64 (d, J=4.8 Hz, 1 H, 1-H), 5.88 (brs, 1
H, 2-NH), 5.96 (s, 1 H, OCH2O), 5.98 (s, 1 H, OCH2O),
6.31 (s, 2 H, 2′, 6′-ArH), 6.53 (s, 1 H, 8-ArH), 6.85 (s, 1 H,
5-ArH), 7.50 (m, 4 H, 2″, 3″, 5″, 6″-ArH) ; 13C NMR
(CDCl3): δ 37.6, 42.2, 43.8, 54.1, 56.3, 60.7, 68.7, 76.6,
77.1, 77.5, 101.7, 108.3, 109.2, 110.2, 127.6, 127.8, 128.3,
132.3, 134.7, 137.3, 147.7, 148.7, 152.6, 167.1, 173.9,
180.6; HRMS calcd for (M+H)+ C31H31N2O8S 591.1786,
found 591.1788; mp 140–142ºC from ethanol; IR (KBr,
cm−1): 3423, 3231, 2933, 2372, 1780, 1671, 1508, 1127,
1040.
Fluorescence morphological examination
Hoechst 33342-PI staining was carried out according to the
procedure of literature [16]. Cells were treated with
compound 4a in 0.05 μM, 0.1 μM for 72 h. Then stained
by incubating in PBS containing 500 nM Hoechst 33342
and 500 nM propidium iodide (PI) at 37°C for 3 min in the
dark. Then washed cells with PBS three times. Stained cells
were viewed under a fluorescence microscope (Leica,
German) with 200× magnification.
Apoptotic morphology was studied by staining the cells
with acridine orange (AO) and ethidium bromide (EB) [17].
Cells were washed three times with PBS after being
incubated with compound 4a in 0.05 μM, 0.1 μM for
72 h, and were then stained with AO and EB for 3 min.
Stained cells were viewed under a fluorescence microscope
(Leica, German) with 200× magnification.
4β-(thienyl-thioureido)-4-deoxypodophyllotoxin (4l)
1
white solid, yield 65.3%; [α]D25-76 (c 0.3, CH2Cl2); H
NMR (CDCl3): δ 2.15 (s, 1H, 1-NH), 2.89 (dd, J=4.8,
14.4 Hz, 1 H, 2-H), 3.05 (m, 1 H, 3-H), 3.75 (s, 6 H,
OCH3), 3.78 (s, 3 H, OCH3), 3.79 (d, J=5.1, 1 H, 4-H),
4.25 (m, 2 H, 11-H), 4.63 (d, J=4.8 Hz, 1 H, 1-H), 5.83
(brs, 1 H, 2-NH), 5.94 (s, 1 H, OCH2O), 5.97 (s, 1 H,
OCH2O), 6.29 (s, 2 H, 2′, 6′-ArH), 6.52 (s, 1 H, 8-ArH),
6.82 (s, 1 H, 5-ArH), 7.45 (m, 3 H, 2″, 3″, 5″-ArH); 13C
NMR (CDCl3): δ 37.6, 42.2, 43.8, 54.2, 56.3 , 60.8, 68.7,
76.6, 77.0, 77.5, 101.7, 108.4, 109.2, 110.2, 127.7, 128.6,
130.9, 132.4, 134.6, 134.7, 146.5, 148.8, 152.7, 161.3,
173.8, 180.2; HRMS calcd for (M+H)+ C28H27N2O8S2 ,
583.1212, found 583.1209; mp 132–134ºC from ethanol;
IR (KBr, cm−1): 3433, 3202, 2927, 1786, 1511, 1127, 1030.
Cell cycle analysis
We used the human colon carcinoma HCT-116 cell line for
cell cycle analysis. Briefly, cells were grown in RPMI
supplemented with 10% (v/v) heat-inactivated fetal calf
serumm, 2 mML-gutamine, 100 units/mL penicillin, and
100 μg/mL streptomycin at 37ºC in a humidified atmo-
sphere of 5%CO2 and 95% N2. Untreated and drug treated
cells were centrifuged and fixed overnight in 70% ethanol
at 4ºC. Washed three times with PBS, incubated for 1 h
with 1 mg/mL Rnase A and 20 μg/mL propidium iodide at
room temperature, and analyzed with a FACSCalibur flow
cytometer (BD). Quantification of apoptotic cells was
calculated as the precentage of cells in the sub-G1 peak in
cell cycle analysis.
Cell culture
Three human cell lines HepG2 (Hepatocellular carcinoma),
A549 (lung), HCT-116 (colon) were cultured on RPMI-
1640 medium supplemented with fetal bovine serum (10%),
penicillin (100U/mL) and streptomycin (100 μg/mL) in
25 cm2 culture flasks at 37ºC in a humidified atmosphere
with 5% CO2.
Topoisomerase IIα -mediated kDNA decatenation assay
A gel assay was carried out as previously described [18] to
determine if compound 4a inhibited the catalytic decatena-
tion activitiy of topoisomerase IIα. kDNA consists of highly
catenated networks of circular DNA, which can be deca-
tenated by topoisomerase IIα in an ATP-dependent reaction.
Cell viability
Cell viability was assessed by the MTT assay. Cells were
harvested from the culture during the exponential growth
phase, and seeded into multiwell culture plates at 5×104–
1×105 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 37ºC for 4 h. The resulting
formazan crystals were solubilized in DMSO, and the
optical density was read at 570 nm with a microplate reader
(Biotek synergy 2).
Statistical analyses
Data were presented as means SE and analyzed by SPSS
software. Picture were processed with Photoshop software.
Mean values were obtained from at least thrice indepent
experiments.
Acknowledgements The studies in the laboratories were supported by
National Program on Key Basic Research Project (2009CB930300),
Foundation of Jiangsu Educational Committee (08KJD350004) and
Natural Science Foundation of Jiangsu Province (BG2007607).

24
Invest New Drugs (2012) 30:17–24
10. Chang H, Shyu KG, Lee CC, Tsai SC, Wang BW, Lee YH, Lin
SK (2003) GL331 inhibits HIF-1α expression in a lung cancer
model. Biochem Bioph Res Commun 302:95–100
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