Design, synthesis, and antitumor activity of novel podophyllotoxin derivatives as potent anticancer agents

Article abstract of DOI:10.1080/10286020.2013.802688

In order to find novel synthetic compounds with superior antitumor activity and overcome multidrug resistance, a series of novel 4β-N-substituted podophyllotoxin derivatives were synthesized under mild conditions with satisfactory yield. Nine novel podophyllotoxin derivatives were synthesized by linking 4β-amino-podophyllotoxin with aldehydes via the formation of a Schiff's base, and imines were reducted to secondary amines. These novel derivatives have been evaluated for cytotoxicity against human cancer cell lines Hela, K562, and K562/AO2. The results indicated that these compounds possess superior bioactivity (IC50 values were found at the range of 10 ^-6-10^-8 mol/l) and weak multidrug resistance.

Full text of DOI:10.1080/10286020.2013.802688

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Design, synthesis, and antitumor
activity of novel podophyllotoxin
derivatives as potent anticancer agents
Jing Liua, Bo Caobc, Ying Gaobc, Mei Baia, Xin Meia, Hong Chenbc,
Yun-Gen Jianga & Da-Jiang Huanga
a General Unit of Chinese People's Armed Police Force Hospital,
JiangxiNanchang330001, China
b Pharmacognosy Division, Logistics Universty of Chinese People's
Armed Police Force, Tianjin, 300162, China
c Tianjin Key Laboratory for Biomarkers of Occupational and
Environmental Hazard, Tianjin, 300162, China
Published online: 16 Jul 2013.
To cite this article: Jing Liu, Bo Cao, Ying Gao, Mei Bai, Xin Mei, Hong Chen, Yun-Gen Jiang & Da-
Jiang Huang (2013) Design, synthesis, and antitumor activity of novel podophyllotoxin derivatives
as potent anticancer agents, Journal of Asian Natural Products Research, 15:9, 985-992, DOI:
10.1080/10286020.2013.802688
To link to this article: http://dx.doi.org/10.1080/10286020.2013.802688
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 9, 985–992, http://dx.doi.org/10.1080/10286020.2013.802688
Design, synthesis, and antitumor activity of novel podophyllotoxin
derivatives as potent anticancer agents
Jing Liua, Bo Caobc, Ying Gaobc, Mei Baia, Xin Meia, Hong Chenbc,
Yun-Gen Jianga and Da-Jiang Huanga*
aGeneral Unit of Chinese People’s Armed Police Force Hospital, Jiangxi, Nanchang 330001,
China; bPharmacognosy Division, Logistics Universty of Chinese People’s Armed Police Force,
Tianjin 300162, China; cTianjin Key Laboratory for Biomarkers of Occupational and Environmental
Hazard, Tianjin 300162, China
(Received 28 September 2012; final version received 1 May 2013)
In order to find novel synthetic compounds with superior antitumor activity and
overcome multidrug resistance, a series of novel 4b-N-substituted podophyllotoxin
derivatives were synthesized under mild conditions with satisfactory yield. Nine novel
podophyllotoxin derivatives were synthesized by linking 4b-amino-podophyllotoxin
with aldehydes via the formation of a Schiff’s base, and imines were reducted to
secondary amines. These novel derivatives have been evaluated for cytotoxicity
against human cancer cell lines Hela, K562, and K562/AO2. The results indicated that
these compounds possess superior bioactivity (IC50 values were found at the range of
1026–1028 mol/l) and weak multidrug resistance.
Keywords: podophyllotoxin derivatives; antitumor; multidrug resistance
1. Introduction
tinal reactions, and are easy to induce
multidrug resistance (MDR). The mech-
anism of MDR is not well understood, and
it was associated with the overexpression
of P-glycoprotein and/or multidrug resist-
ance protein 1 (MDR1), the overproduc-
tion of specific tubulin isotypes, and the
decreased expression of topoisomerases
[9]. The problem of increasingly severe
MDR of tumor is the main reason to cause
abortive chemotherapy.
Podophyllotoxin (1), which is a naturally
occurring aryltetralin lignan extracted
from the roots of Podophyllum peltatum,
has very strong antiviral and cytotoxic
properties [1]. In particular, its semisyn-
thetic derivatives such as etoposide (VP-
16, 2), teniposide (VM-26, 3), and the
water-soluble prodrug, etopofos (4) have
been widely used in the chemotherapy of
various cancers such as small cell lung
cancer, testicular carcinoma, and lym-
phoma [2–5]. It exerts its antitumor
activity by inhibiting tubulin through
binding with part of its colchicine domain
or inhibiting DNA topoisomerase-II (topo-
II) by stabilizing the covalent topo-II
DNA cleavable complex [6–8]. However,
the existing chemotherapeutic medicines
have shortcomings of narrow spectrum
anti-cancer, poor water solubility, severe
bone marrow depression, and gastrointes-
To overcome such problems and
obtain better therapeutic agents, more
complex and diverse analogues have
been synthesized either to get more potent
compounds or to overcome drug resistance
by their structure–activity relationship
(SAR) studies. Some of them entered the
I, II clinical studies as anti-cancer drugs,
such as azatoxin (5), GL331 (6), and TOP-
53 (7), as shown in Figure 1. During the
course of researching new C-4-nonsugar
*Corresponding author. Email: 916202839@qq.com
q 2013 Taylor & Francis

986
J. Liu et al.
Figure 1. Structures of podophyllotoxin and related compounds.
derivatives of podophyllotoxin, the C-4
nonsugar substitution which can be linked
through O-, S-, or N-linkage has proven to
be significant in overcoming the drug
resistance [10,11]. In general, the N-linked
derivatives show higher activity in com-
parison to the O-linked and S-linked
congeners [12,13].
and indole ring have been synthesized, not
only with good activity but also some
drawbacks such as poor stability existed
[14–18]. Based on the SAR of podophyl-
lotoxin, a series of 4b-N-substituted
podophyllotoxin derivatives which have
one carbon atoms between nitrogen atom
and heterocyclic were prepared as potent
anticancer agents, and 4b-N-substituted
heterocyclic podophyllotoxin derivatives
exhibited higher antitumor activity com-
pared with 4b-N-substituted benzene, as
illustrated in Chart 1, structurally con-
firmed by 1H NMR, time-of-flight MS.
These derivatives have been evaluated for
antitumor activity in vitro against tumor
cell lines (Hela, K562 and K562/AO2).
A p-nitroanilino group at position 4b
of analogue GL331 was shown to be active
in many multidrug-resistant cancer cell
lines, help for good stability, biocompat-
ibility, and its favorable pharmacokinetic
profiles; the compound has no carbon atom
between nitrogen atom and aryl. In recent
years, our group has been engaged in the
chemical transformation of the structure of
podophyllotoxin and aiming at the dis-
covery of new podophyllotoxin derivatives
with improved bioactivities, lower tox-
icity, and overcoming drug resistance
effects. In order to lengthen carbon
chain, a dozen series of hundreds of
podophyllotoxin derivatives which have
two carbon atoms between nitrogen atom
2. Results and discussion
The five-substituted indole-3-carboxalde-
hyde derivatives were synthesized by a
Vilsmeier–Hacck reaction [19]. 4b-
Amino-podophyllotoxin and aldehydes in
95% EtOH containing a catalytic amount
of glacial acetic acid were stirred for 3–

Journal of Asian Natural Products Research
987
Chart 1. Design of the novel podophyllotoxin derivatives.
12 h, to give the corresponding products
compounds a–i. The reaction mixture was
filtered and washed with 95% EtOH. The
compounds a–g were reducted in anhy-
drous methanol by NaBH4 at 08C for 1–
3 h, as described in Scheme 1. Five percent
HCl was added to the mixture to end the
reaction. Saturated NaHCO3 was added to
adjust pH .7. The solvent was evaporated
under vacuum. Finally, the residue was
dissolved in anhydrous CH2Cl2 and
chromatographed on silica gel plate using
petroleum ether–ethyl acetate to give the
corresponding secondary amines 2a–i.
These derivatives were evaluated for
cytotoxicity against human cancer cell
lines HeLa (cervical cancer cells), K562
(human erythroleukemia cell), K562/AO2
(multidrug-resistant human erythroleuke-
mia cells induced by doxorubicin) using
the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay
in vitro with comparison to the parent
compounds VP-16 and doxorubicin
(ADM). The experimental data of com-
pounds 2a–i in vitro are shown in Table 1.
As shown in Table 1, the derivatives 2a,
2b, 2d, and 2f exhibited superior anticancer
activity compared with VP-16, against
Hela in vitro, while the derivatives 2b, 2c,
2e, 2g, and 2h exhibited superior anticancer
activity compared with VP-16 against
K562 in vitro. Moreover, all of them
showed significant capability in overcom-
ing MDR compared with VP-16 against
K562/AO2 in vitro. Especially, the MDR

988
J. Liu et al.
Scheme 1. Synthetic route of compounds 2a–g: (a) CH2Cl2, NaN3, CF3COOH, 508C , r.t., 4 h;
(b) CH2Cl2, 10% Pd/C, HCOONH4, 458C , r.t., 4 h; (c) 95% C2H5OH, acetic acid glacial, r.t., 3–
12 h; (d) anhydrous methanol, NaBH4, 08C, 1–3 h.
of the compounds 2a, 2f, 2h was obviously
lower than that of VP-16. In the prelimi-
nary SAR study, we speculated that the 4b-
N-substituted heterocyclic were the key
groups contributing much to anti-tumor
activity compared with benzene. Some
one-carbon atoms between nitrogen atom
and heterocyclic derivatives exhibited
superior anticancer activity compared to
no- or two-carbon atoms between nitrogen
atom and aryl [20]. We are taking up
further research that will explicit the SAR
to seek potential anti-tumor candidates.
In order to determine the configuration
of above compounds, using compound 2i
as the example of target compounds, it was
resolved that methanol solution upon slow
evaporation over 2 weeks at room
temperature (r.t.) gave transparent crystals
suitable for X-ray analysis [21]. X-ray
structure clearly showed that four-amino-
substituted congeners and one-substituted
benzene were the cis-configuration in the
Figure 2, N-substituted product was b
configuration, as characterized before
[22]. The substituted CvN bond of the
Table 1. The experimental data of compounds 2a–i in vitro.
IC50 (1026 mol/l)
Compounds
Hela
K562
K562/AO2
Resistant factor
2a
2b
2c
2d
2e
2f
2g
2h
2i
VP-16
ADM
0.10 ^ 0.24
0.33 ^ 0.48
1.19 ^ 0.33
0.46 ^ 1.27
0.95 ^ 0.58
3.13 ^ 0.21
0.32 ^ 0.18
2.01 ^ 0.44
1.85 ^ 0.25
1.98 ^ 0.20
1.13 ^ 0.27
0.68 ^ 0.52
0.64 ^ 0.46
1.31 ^ 2.32
0.46 ^ 0.59
2.62 ^ 0.94
0.64 ^ 0.87
0.53 ^ 0.20
3.00 ^ 0.33
2.02 ^ 0.83
0.26 ^ 0.10
2.23 ^ 1.23
3.49 ^ 3.46
2.61 ^ 0.24
6.20 ^ 2.71
4.21 ^ 0.49
5.53 ^ 0.76
1.99 ^ 1.21
1.04 ^ 0.53
11.78 ^ 2.11
22.81 ^ 4.23
28.62 ^ 4.27
1.97
5.12
4.11
4.73
9.25
2.11
3.13
1.97
3.93
11.29
110.08
Note: Cytotoxicity against HeLa, K562 and K562/AO2 cell lines was measured by the standard MTT assay
method, each value represents the mean ^ SD of three independent experiments.

Journal of Asian Natural Products Research
989
Figure 2. X-ray structure of compound 2i (hydrogen atoms are omitted for clarity).
product was cis-configuration, different
from the previous research [22].
5-Fluoroindole (1.0mmol) was dissolved in
DMF, which was added in dropwise to the
mixture at 08C. The mixture was stirred at
708C for 3 h. The reaction solution was
poured into icy water (50 ml) and continued
stirring until precipitation appeared. The
solid was filtered and washed with water.
Step 2: 5-fluoro-indole-3-carboxaldehyde
(1.2 mmol), 4b-amino-podophyllotoxin
(413 mg, 1.0 mmol), and two drops of
glacial acetic acid in 95% C2H5OH
(10.0 ml) were stirred at r.t. for 12h until
precipitation appeared. The solid was
filtered and washed with glacial acetic
acid. Step 3: the compound b (1.0mmol)
was reducted in anhydrous methanol by
NaBH4 (4.0mmol) at 08C for 3 h, 5% HCl
was added to the mixture to end the reaction.
The saturated NaHCO3 was add to adjustpH
.7. The solvent was evaporated under
vacuum. Finally, the residue was dissolved
in anhydrous CH2Cl2 and chromatographed
on silica gel plate using petroleum ether and
ethyl acetate (v/v 1:1) to give the corre-
sponding secondary amine 2b (0.31 g, 56%)
3. Experimental
3.1. General experimental procedures
Melting points were determined on an X-4
digital visual melting point apparatus and
uncorrected (Yuhua Instrument Corporation
1
in Henan, Gongyi, China). The H NMR
spectrawererecordedonaBrukerARX-300
spectrophotometer (Bruker Corporation,
Zurich, Switzerland), withtetramethylsilane
as the internal standard. Mass spectral data
were obtained on an Agilent 6210 TOP-MS
(Agilent Technologies Corporation, Santa
Clara, CA, USA). X-ray crystal data were
obtained on a Rigaku Saturn CCD area-
detector diffractometer (Rigaku Corpor-
ation, Tokyo, Japan). All materials and
reagents were used without further purifi-
cation unless otherwise stated. Podophyllo-
toxin with 98% purity was purchased from
the Qingze Corporation of Nanjing province
in China. CH2Cl2 was refluxed over P2O5
and distilled.
1
as white solid. For H NMR spectral data
3.2. General synthetic methods
(CDCl3) (see Table 2). HR-ESI-MS
m/z: 561.2033 [M þ H]þ (calcd for
C31H30FN2O7, 561.2037).
Compound 2b was obtained by four steps of
reactions. Step 1: POCl3 (10.0 mmol) was
added into DMF (N,N-dimethylformamide;
10.0mmol) and stirred at 158C for 15min.
Compounds 2a and 2c–i were pre-
pared as described above.

990
J. Liu et al.
Table 2. 1H NMR spectral data for the target compounds 2a–i.
Compounds
1H NMR (d/ppm)
2a
2.14–2.34(m, 1H, 4-NH), 2.76–2.83(m, 1H, 2-H), 3.33–3.40(m, 1H, 3-H), 3.73(s,
6H, 30,50-OCH3), 3.79(s, 3H, 40-OCH3), 3.98–3.40(m, 1H, 11b-H), 4.06(d, 2H,
J ¼ 6.0 Hz, 4-NH-CH2), 4.31–4.45(m, 2H, 11a,1-H), 4.52(d, 1H, J ¼ 4.3 Hz, 4-H),
5.89(s, 1H, b-OCH2O), 5.90(s, 1H, a-OCH2O), 6.25(s, 2H, 20,60-H), 6.44(s, 1H, 8-
H), 6.50(s, 1H, 5-H), 7.13–7.24(m, 3H, 500,600,700-H), 7.40(d, 1H, J ¼ 6.0 Hz, 400-H),
7.63(d, 1H, J ¼ 6.0 Hz, 200-H), 8.12–8.16(s, 1H, 100-NH)
2b
2c
2.05–2.24(m, 1H, 4-NH), 2.77–2.88(m, 1H, 2-H), 3.31–3.37(m, 1H, 3-H), 3.73(s,
6H, 30,50-OCH3), 3.79(s, 3H, 40-OCH3), 3.97–4.04(m, 2H, 4-NH-CH2), 4.02–
4.06(m, 1H, 11b-H), 4.29–4.38(m, 2H, 11a,1-H), 4.53(d, 1H, J ¼ 5.2 Hz, 4-H),
5.90(s, 1H, b-OCH2O), 5.92(s, 1H, a-OCH2O), 6.25(s, 2H, 20,60-H), 6.45(s, 1H, 8-
H), 6.51(s, 1H, 5-H), 6.94–7.01(m, 2H, 600,700-H), 7.28–7.33(m, 2H, 200,400-H),
8.16–8.18(s, 1H, 100-NH)
2.15–2.20(m, 1H, 4-NH), 2.80–2.87(m, 1H, 2-H), 3.34–3.40(m, 1H, 3-H), 3.73(s,
6H, 30,50-OCH3), 3.79(s, 3H, 40-OCH3), 3.86(s, 3H, 500-OCH3), 3.99–4.00(m, 1H,
11b-H), 4.04(d, 2H, J ¼ 8.8 Hz, 4-NH-CH2), 4.34–4.43(m, 2H, 11a,1-H), 4.53(d,
1H, J ¼ 5.3 Hz, 4-H), 5.89(d, 1H, J ¼ 1.2 Hz, b-OCH2O), 5.91(d, 1H, J ¼ 1.2 Hz,
a-OCH2O), 6.26(s, 2H, 20,60-H), 6.44(s, 1H, 8-H), 6.55(s, 1H, 5-H), 6.88(dd, 1H,
J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 600-H), 7.06(d, 1H, J ¼ 2.4 Hz, 700-H), 7.15–7.18(m, 1H,
400-H), 7.27–7.30(m, 1H, 200-H), 8.00–8.03(s, 1H, 100-NH)
2d
2e
2.18–2.31(m, 1H, 4-NH), 2.73–2.85(m, 1H, 2-H), 3.30–3.37(m, 1H, 3-H), 3.72(s,
6H, 30,50-OCH3), 3.76–3.80(m, 2H, 4-NH-CH2), 3.79(s, 3H, 40-OCH3), 3.90(d, 1H,
J ¼ 3.8 Hz, 11b-H), 4.28–4.38(m, 2H, 11a,1-H), 4.52(d, 1H, J ¼ 5.2 Hz, 4-H),
5.92(s, 1H, b-OCH2O), 5.93(s, 1H, a-OCH2O), 6.25(s, 2H, 20,60-H), 6.45(s, 1H, 8-
H), 6.54(s, 1H, 5-H), 6.82(d, 2H, J ¼ 8.3 Hz, 300,500-ArH), 7.20(d, 2H, J ¼ 8.3 Hz,
200,600-ArH)
2.16–2.29(m, 1H, 4-NH), 2.94–3.04(m, 1H, 2-H), 3.16–3.23(m, 1H, 3-H), 3.72(s,
6H, 30,50-OCH3), 3.79(s, 3H, 40-OCH3), 4.02(d, 1H, J ¼ 4.1 Hz, 11b-H), 4.09–
4.12(m, 2H, 4-NH-CH2), 4.39–4.42(m, 2H, 11a,1-H), 4.60(d, 1H, J ¼ 5.4 Hz, 4-
H), 5.97(d, 1H, J ¼ 1.2 Hz, b-OCH2O), 5.98(d, 1H, J ¼ 1.2 Hz, a-OCH2O), 6.21(s,
2H, 20,60-H), 6.50(s, 1H, 8-H), 6.74(s, 1H, 5-H), 6.83–6.91(m, 2H, 300,500-ArH),
7.09–7.12(m, 1H, 400-ArH), 7.19–7.22(m, 1H, 600-ArH)
2f
2.10–2.23(m, 1H, 4-NH), 2.74–2.88(m, 1H, 2-H), 3.24–3.38(m, 1H, 3-H), 3.72(s,
6H, 30,50-OCH3), 3.78(s, 3H, 40-OCH3), 3.81(s, 3H, 400-OCH3), 3.88–3.97(m, 2H, 4-
NH-CH2), 4.06–4.15(m, 1H, 11b-H), 4.27–4.35(m, 2H, 11a,1-H), 4.51(d, 1H,
J ¼ 4.5 Hz, 4-H), 5.98(s, 2H, a,b-OCH2O), 6.23(s, 2H, 20,60-H), 6.46(s, 1H, 8-H),
6.53(s, 1H, 5-H), 6.89–6.92(m, 2H, 300,500-ArH), 7.24–7.32(m, 2H, 200,600-ArH)
2.74–2.88(m, 1H, 2-H), 3.24–3.38(m, 1H, 3-H), 3.72(s, 6H, 30,50-OCH3), 3.78(s,
3H, 40-OCH3), 3.81(s, 3H, 400-OCH3), 3.88–3.97(m, 2H, 4-NH-CH2), 4.06–4.15(m,
1H, 11b-H), 4.27–4.35(m, 2H, 11a,1-H), 4.51(d, 1H, J ¼ 4.5 Hz, 4-H), 5.98(s, 2H,
a,b-OCH2O), 6.23(s, 2H, 20,60-H), 6.46(s, 1H, 8-H), 6.53(s, 1H, 5-H), 6.89–6.92(m,
2H, 300,500-ArH), 7.24–7.32(m, 2H, 200,600-ArH)
2.07–2.10(m, 1H, 4-NH), 2.72–2.83(m, 1H, 2-H), 2.95(s, 6H, 400-NCH3), 3.32–
3.38(m, 1H, 3-H), 3.72(s, 6H, 30,50-OCH3), 3.75–3.80(m, 2H, 4-NH-CH2), 3.79(s,
3H, 40-OCH3), 3.92–3.93(m, 1H, 11b-H), 4.26–4.42(m, 2H, 11a,1-H), 4.52(d, 1H,
J ¼ 5.2 Hz, 4-H), 5.94(s, 2H, a,b-OCH2O), 6.24(s, 2H, 20,60-H), 6.44(s, 1H, 8-H),
6.54(s, 1H, 5-H), 6.72(d, 2H, J ¼ 8.6 Hz, 300,500-ArH), 7.19–7.24(m, 2H, 200,600-ArH)
2.04–2.12(m, 1H, 4-NH), 2.74–2.87(m, 1H, 2-H), 3.34–3.40(m, 1H, 3-H), 3.73(s,
6H, 30,50-OCH3), 3.79(s, 3H, 40-OCH3), 3.81(s, 3H, 400-OCH3), 3.90–3.95(m, 3H, 4-
NH-CH2, 11b-H), 4.37–4.39(m, 2H, 11a,1-H), 4.53(d, 1H, J ¼ 5.3 Hz, 4-H),
5.93(s, 2H, a,b-OCH2O), 6.25(s, 2H, 20,60-H), 6.38(s, 1H, 8-H), 6.45(s, 1H, 5-H),
7.27–7.30(m, 2H, 400,600-ArH), 7.40–7.46(m, 2H, 300,500-ArH)
2g
2h
2i

Journal of Asian Natural Products Research
991
Compound 2a: White solid, overall
yield 44% in three steps of reactions,
indole was dissolved in DMF instead of 5-
fluoro indole in step 1, mp 126–1298C, 1H
NMR spectral data (CDCl3) (see Table 2).
HR-ESI-MS m/z: 543.2132 [M þ H]þ
(calcd for C31H31N2O7, 543.2131).
dimethylaminobenzaldehyde reacted with
4b-amino-podophyllotoxin in step 2, mp
203–2058C, 1H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS m/z:
547.2440
C31H35N2O7, 547.2444).
[M þ H]þ
(calcd
for
Compound 2i: White solid, overall
yield 67% in two steps of reactions, 2-
chlorobenzaldehyde reacted with 4b-
amino-podophyllotoxin in step 2, mp
194–1978C, 1H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS m/z:
Compound 2c: Light yellow solid,
overall yield 42% in three steps of
reactions, 5-methoxyindole was dissolved
in DMF instead of 5-fluoro indole in step
1
1, mp 148–1508C, H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS m/z:
538.1625
[M þ H]þ
(calcd
for
573.2231
C32H33N2O8, 573.2237).
[M þ H]þ
(calcd
for
C29H29ClNO7, 538.1633). This compound
has been characterized before [22].
Compound 2d: White solid, overall
yield 68% in two steps of reactions, 4-
hydroxybenzaldehyde reacted with 4b-
amino-podophyllotoxin in step 2, mp
115–1178C, 1H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS m/z:
4. Conclusion
In summary, a series of 4b-N-substituted
podophyllotoxin derivatives described here
were synthesized and evaluated for their
antitumor activities. Some of podophyllo-
toxin derivatives showed highly significant
antitumor effect and overcame MDR. It
takes up further synthesis and evaluation of
their new derivatives as potential candi-
dates. The mechanism studies and further
biological studies are being conducted.
520.1953
C29H30NO8, 520.1971).
[M þ H]þ
(calcd
for
Compound 2e: White solid, overall
yield 65% in two steps of reactions, 2-
hydroxybenzaldehyde reacted with 4b-
amino-podophyllotoxin in step 2, mp
129–1318C, 1H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS m/z:
520.1967
C29H30NO8, 520.1971).
[M þ H]þ
(calcd
for
Acknowledgments
Compound 2f: White solid, overall
yield 64% in two steps of reactions, 4-
methoxybenzaldehyde reacted with 4b-
amino-podophyllotoxin in step 2, mp
206–2088C, 1H NMR spectral data
(CDCl3) (see Table 2). HR-ESI-MS
m/z: 534.2145 [M þ H]þ (calcd for
C30H32NO8, 534.2128). This compound
has been characterized before [22].
This work was financially supported by the
National Natural Science Foundation of China
(No. 30873363), Program of Science Foun-
dation of Tianjin (08JCYBJC070000), and
Major Program of Science Foundation of
Tianjin (09ZCKFSH01700).
References
[1] A. Kamal, E. Laxman, and G.B.R.
Khanna, Bioorg. Med. Chem. 12, 4197
(2004).
[2] M. Gordaliza, P.A. Garcia, and J.M.
Miguel, Toxicon 44, 441 (2004).
[3] I. Wessel, P. Jensen, and J. Falck, Cancer
Res. 57, 4451 (1997).
[4] K.H. Lee, Y. Imakura, and M. Haruna,
J. Nat. Prod. 52, 606 (1989).
Compound 2g: White solid, overall
yield 72% in two steps of reactions, 2-
furaldehyde reacted with 4b-amino-podo-
1
phyllotoxin in step 2, mp 201–2038C, H
NMR spectral data (CDCl3) (see Table 2).
HR-ESI-MS m/z: 494.1808 [M þ H]þ
(calcd for C27H28NO8, 494.1815).
[5] X.M. Zhou, Z.Q. Wang, and J.Y. Chang,
J. Med. Chem. 34, 3346 (1991).
[6] K.H. Lee, J. Nat. Prod. 67, 273 (2004).
Compound 2h: White solid, overall
yield 53% in two steps of reactions, 4-

992
J. Liu et al.
[7] Y. Zhao, C.W. Ge, and Z.H. Wu, Eur.
[16] H. Chen, W.C. Bi, and B. Cao, Eur.
J. Pharmacol. 627, 69 (2010).
[17] H. Shang, H. Chen, D.M. Zhao, X.W.
Tang, Y.F. Liu, L. Pan, and M.S. Cheng,
Arch. Pharm. 345, 43 (2012).
[18] P.F. Yu, H. Chen, J. Wang, C.X. He,
B. Cao, M. Li, N. Yang, Z.Y. Lei, and
M.S. Cheng, Chem. Pharm. Bull. 56, 831
(2008).
[19] Z.L. Min, F.C. Jiang, and Q. Zhang, Chin.
J. Med. Chem. 15, 129 (2005).
[20] H. Chen, W.C. Bi, B. Cao, Z.X. Yang,
S. Chen, H. Shang, P.F. Yu, and J. Yang,
Eur. J. Pharmacol. 627, 69 (2010).
[21] T.L. Zhu, J.R. Jin, H. Chen, L.T. Chen,
and J. Liu, Acta Crystallogr. Sect. E. E67,
o1491 (2011).
[22] C.N. Wang, Z.H. Wu, Y. Zhao, C.Y. Ni,
X.D. Zhao, and L. Zhu, Arch. Pharm.
Chem. Life Sci. 344, 735 (2011).
J. Med. Chem. 46, 901 (2011).
[8] G.M. Cragg and D.J. Newman, J. Nat.
Prod. 67, 232 (2004).
[9] C. Ferlini, G. Raspaglio, and S. Mozzeti,
Cancer Res. 65, 2397 (2005).
[10] M. Gordaliza, Clin. Transl. Oncol. 9, 767
(2007).
[11] S.Y. Liu, B.D. Hwang, and M. Haruna,
Mol. Pharmacol. 36, 78 (1989).
[12] D.S. Van Vilet, Y. Tachibana, and K.F.
Bastow, J. Med. Chem. 44, 1422 (2001).
[13] T. Terada, K. Fujimoto, and M. Nomura,
J. Med. Chem. 36, 1689 (1993).
[14] Y.E. Guo, H. Chen, S. Zuo, D.L. Liu, Y.L.
Lu, J.J. Lv, S.P. Wen, and T.C. Zhang,
J. Asian Nat. Prod. Res. 13, 417 (2011).
[15] Y.J. Sun, Z.Y. Li, H. Chen, X.Q. Liu,
W. Zhou, and H.M. Hua, Planta Med. 78,
480 (2012).