Synthesis and biological evaluation of novel podophyllotoxin analogs as antitumor agents

Article abstract of DOI:10.1080/10286020.2014.913578

A series of 4β N-indole-substituted podophyllotoxin derivatives were synthesized. Nine target compounds were evaluated against human cancer cell lines (HeLa, K562, and K562/A02) using MTT assay including three imine derivatives 8, 9, and 10in vitro. The result showed that the three compounds had higher antitumor activity than their reduced forms. Among them, compounds 8, 9, 11, and 16 were superior to the positive control VP-16.

Full text of DOI:10.1080/10286020.2014.913578

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Synthesis and biological evaluation
of novel podophyllotoxin analogs as
antitumor agents
Zhong-Heng Zhangab, Li-Ming Zhangab, Gang Luoa, Shi Zhangb,
Hong Chenab & Jing Zhoua
a Tianjin Key Laboratory on Technologies Enabling Development of
Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin
Medical University, Tianjin 300070, China
b Tianjin Key Laboratory for Biomarkers of Occupational and
Environmental Hazard, Department of Pharmacognosy, Logistics
University of Chinese People's Armed Police Forces, Tianjin
300162, China
Click for updates
Published online: 14 May 2014.
To cite this article: Zhong-Heng Zhang, Li-Ming Zhang, Gang Luo, Shi Zhang, Hong Chen
& Jing Zhou (2014) Synthesis and biological evaluation of novel podophyllotoxin analogs
as antitumor agents, Journal of Asian Natural Products Research, 16:5, 527-534, DOI:
10.1080/10286020.2014.913578
To link to this article: http://dx.doi.org/10.1080/10286020.2014.913578
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 5, 527–534, http://dx.doi.org/10.1080/10286020.2014.913578
Synthesis and biological evaluation of novel podophyllotoxin analogs as
antitumor agents
Zhong-Heng Zhangab, Li-Ming Zhangab, Gang Luoa, Shi Zhangb, Hong Chenab* and
Jing Zhoua*
aTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China; bTianjin Key
Laboratory for Biomarkers of Occupational and Environmental Hazard,
Department of Pharmacognosy, Logistics University of Chinese People’s Armed Police Forces,
Tianjin 300162, China
(Received 13 November 2013; final version received 7 April 2014)
A series of 4b N-indole-substituted podophyllotoxin derivatives were synthesized.
Nine target compounds were evaluated against human cancer cell lines (HeLa, K562,
and K562/A02) using MTT assay including three imine derivatives 8, 9, and 10 in vitro.
The result showed that the three compounds had higher antitumor activity than their
reduced forms. Among them, compounds 8, 9, 11, and 16 were superior to the positive
control VP-16.
Keywords: podophyllotoxin; indole; antitumor; thiophene
1. Introduction
effects associated with myelosuppression,
neutropenia, nausea, drug resistance, and
poor water solubility [10,11]. To over-
come the disadvantages and obtain more
prodrugs, diverse analogs such as GL-331
(2), NPF(3), TOP-53(4), NK-611(5), and
so on (Figure 1) which are under clinical
trial have been developed.
Podophyllotoxin as an antitumor arylte-
tralin lignin is isolated from the roots of
the North American Podophyllum pelta-
tum Linnaeus and Tibetan Podophyllum
emodi Wall [1,2]. Due to its antineoplasc-
tic and antiviral properties, podophyllo-
toxin is concerned by more and more
researchers [3]. But due to severe cyto-
toxicity of podophyllotoxin (1) (Figure 1),
it has not been used as an anticancer drug
[4]. Some derivatives of C-4 position of
podophyllotoxin such as etoposide [5],
etoposide (VP-16) and teniposide used in
clinic have lower side effects than
podophyllotoxin as antitumor agent [6].
Chemical modifications lead to the change
in the mechanism of action of these
ligands, wherein podophyllotoxin acts as
an antimicrotubule agent, whereas its
aforementioned derivatives act as topoi-
somerase-II inhibitors [7–9]. However, its
current therapeutic use is hindered by side
Due to the fact that the chemical and
biological studies of heterocyclic com-
pounds have played an important role for a
long time in medicine, more and more
antitumor drugs are expected to be found.
However, compounds with indole group in
which the benzene and pyrrole rings are
fused through the 2- and 3-positions of the
pyrrole nucleus can also be found in
various natural products, such as indole
alkaloids.
In addition, indole derivatives are
found to possess several biological activi-
ties including antibiotic, anti-inflammatory,
analgesic, anticonvulsant, antimalarial,
*Corresponding authors. Email: chenhongtian06@163.com; zhou195620@126.com
q 2014 Taylor & Francis

528
Z.-H. Zhang et al.
Figure 1. Structures of podophyllotoxin, NPF, GL331, TOP 53, NK61, and VP-16.
anticancer, antiulcer, and so on [12,13].
Besides, thiophene has a similar construct
like furan, which is an important part of
ribose-5-phosphate [14,15]. For example,
thienyl derivatives of Meldrum’s acid
possess neurotropic activity comprising
both depriming and activating components
[16]. Therefore, it may be an important
construct for antitumor activity.
activities between imine and amine
products.
2. Results and discussion
In this project, nine novel podophyllotoxin
derivatives were designed and syn-
thesized. Then, the cytotoxicity of
compounds was tested in vitro by the
standard MTT assay using HeLa cells for
the first time.
In recent years, our group has been
engaged in structural modification of
podophyllotoxin in order to get high-
efficiency and low-toxicity antitumor
agents, and multiple compounds were
found by active screening, e.g., indole-3-
gloryl podophyllotoxin designed accord-
ing to the association strategy. And the
antitumor activity of imine derivatives was
first screened as intermediate. Finally, we
got nine podophyllotoxin derivatives and
we compared the different antitumor
The synthesis of these compounds
consisted of two parts. The first part was
the synthesis of N-substituted indole. In
the presence of NaH, indole should be put
into dimethylformamide (DMF) with
environmental temperature below 08C
[17]. In particular, attention needs to be
paid that the raw material should be added
separately in order to reduce secondary
reaction. From the result of the reaction

Journal of Asian Natural Products Research
529
Table 1. In vitro activity of synthesized
compounds (8–16) and positive controls (VP-
16) against HeLa, K562, and K562/A02 cell
lines.
activity with IC50 value of 1.45 mmol l21
against K562/A02.
Compared with compounds 8–13,
compounds 14–16 were supposed that
the C-4 position had many electrons,
which led to stronger activity. Except for
compound 14, other new compounds
showed moderate cytotoxic activity
against human tumor cell lines. Among
them, compound 16 showed superior
activity with an IC50 value of
1.15 mmol l21, compared with VP-16
with an IC50 value of 14.50 mmol l21
against HeLa cells. Compound 16 was
more potent than compounds 15 and 14.
This observation indicated the critical role
of the substituted group. An electron-
withdrawing group was detrimental to
cytotoxicity, as the activity of compound
14 drastically reduced with IC50 value of
more than 100 mmol l21. However, the
transfer of an electron-donating group was
benefitful to antitumor activity. This
suggested that the electron distribution
and substituents on thiophene played an
important role in cytotoxic activity of the
derivatives.
IC50 (1026 mol l21
)
a
No.
HeLa
K562
K562/A02
8
9
0.79
16.46
27.20
.100
.100
.100
.100
25.26
1.15
.100
4.77
3.64
60.94
76.56
76.56
86.88
80.30
.100
12.45
.100
4.23
2.43
10
11
12
13
14
15
16
VP-16
1.45
14.04
.100
90.54
93.32
.100
8.02
21.44
a IC50: concentration that causes a 50% reduction of
cell growth.
rate and absorptivity at different tempera-
tures, we found that we could get higher
reaction absorptivity and the product could
be used in the next reaction without further
purification when the temperature was
kept at 25–308C.
The second part was the synthesis of
N-substituted indole-4-imine-deoxypodo-
phyllotoxins; much glacial acetic acid was
needed in this reaction, and the compounds
must be sealed and stored in the dark as
they could decompose easily.
We can see that the series of
deoxypodophyllotoxin derivatives have
potential to be new drugs, and more work
is ongoing to confirm this.
The cytotoxicity of all compounds was
tested in vitro against HeLa, K562, and
K562/A02 cells using the standard MTT
assay. The result is shown in Table 1, and
all the imine derivatives showed stronger
cytotoxicity than their reduced analogs
against HeLa cells. The possible reason
could be that the imines with many
electrons showed superior antitumor cyto-
toxicity. Compounds 8 and 9 showed
stronger activity with IC50 values of 0.79
and 16.46 mmol l21, respectively, than 10
(IC50 27.20 mmol l21) against HeLa cells.
It was suggested that the substituted
phenyl group might enhance activity.
However, compound 11 showed superior
3. Experimental
3.1 General experimental procedures
1H NMR and 13C NMR spectra were
recorded on a Bruker Advance 2B/400
(Bruker Technologies, Bremen, Germany)
spectrometer (400 MHz) or Varian Mer-
cury Vx300 MHz (Varian, Palo Alto, CA,
USA) in CDCl3, with TMS as an internal
standard. Chemical shifts were recorded in
(ppm) values relative to TMS, and J values
are expressed in hertz. MS were recorded
using an Agilent-6210 LC/TOF-MS
spectrometer (Agilent Technologies,
Waldbronn, Germany). Podophyllotoxin
was purchased from Nanjing Qingze
Pharmaceutical Technology Development

530
Z.-H. Zhang et al.
Scheme 1. The synthesis route of target compounds; conditions and reagents: (a) NaN3,
CF3COOH, CH2Cl2; (b) HCOONH4, 10% Pd/C, CH3COOC2H5; (c) NaH, DMF; (d) HAc, CH3OH;
(e) NaBH4, CH3OH.
Co., Ltd (Nanjing, China). Other chemical
reagents were purchased from Alfa-
reagent (Tianjin, China).
filtered. The filtrate was washed with
saturated brine, then dried over anhydrous
Na2SO4, and rotary evaporated to get a
white compound 7 (3.63 g, 90% yield).
NaH (0.34 g, 10 mmol) was dissolved
in DMF (10 ml) in a 50-ml three-neck
bottle at 08C. Then indole (5 mmol) was
added to the mixture gradually. Upon
completion of the addition, the flask was
removed from the ice bath and the solution
was allowed to stir at room temperature.
After 5 h, R1X (6 mmol) was added at 08C.
Then, the reaction was kept at room
temperature and monitored by TLC until
the starting material was gone. With
stirring, the mixture was poured into cold
water until precipitation was observed.
The precipitate was collected on a Buchner
funnel, washed twice with distilled water,
and dried in air under reduced pressure.
Then the powder was used for reaction.
R2-CHO or (N)-R1-5-methoxyindole-
3-carboxaldehyde and acetic acid with
catalytic dosage were added to a stirred
solution of compound 7 in dry CH3OH at
3.2 General reaction
Podophyllotoxin (4.00 g, 10 mmol) dis-
solved in anhydrous CH2Cl2 was allowed
to stir around the ice bath. Then, NaN3
(2.06 g, 40 mmol) was added carefully.
CF3COOH (10 ml) must be dribbled in
30 min at 08C. After stirring for 1 h, the
mixture should be refluxed for 4 h at room
temperature. To make sure the pH value of
the mixture was adjusted to 7, saturated
aqueous NaHCO3 was needed. Then, the
organic extracts were dried over anhy-
drous Na2SO4, concentrated and crystal-
lized from CH2Cl2–CH3COOC2H5 (1:1)
to afford a white product (3.99 g, 91%
yield). And 10% Pd/C (1.00 g) and
HCOONH4 (2.25 g, 40 mmol) were added
to the solution of 4b-N3 podophyllotoxin
dissolved in 50 ml CH3COOC2H5. The
mixture was stirred under heat for 5 h and

Journal of Asian Natural Products Research
531
room temperature. After 4 h, appropriate
NaBH4 was added to the solution at 08C,
and after an additional 4 h, 5% HCl was
added to the mixture to adjust pH value to
7. The mixture was poured into a large
amount of water, and the precipitate was
filtered to get the product (Scheme1).
3.81 (6H, br s, 40, 500-OCH3), 4.21–4.32
(2H, m, H-11), 4.56 (1H, d, J ¼ 4.2 Hz, H-
4), 4.70 (1H, d, J ¼ 5.1 Hz, H-1), 5.24
(2H, s, CH2-1000), 5.87 (1H, s, H-13), 5.93
(1H, s, H-13), 6.37 (2H, s, H-20, 60), 6.53
(1H, s, H-5), 6.65 (1H, s, H-8), 6.88 (1H,
dd, J ¼ 2.4, 8.9 Hz, H-600), 7.06 (2H,
J ¼ 8.2 Hz, H-3000, 5000), 7.19 (1H, d,
J ¼ 8.9 Hz, H-700), 7.32 (2H, d,
J ¼ 8.2 Hz, H-2000,6000), 7.40 (1H, s, H-200),
7.74 (1H, d, J ¼ 2.4 Hz, H-400), 8.52 (1H, s,
H-1000); 13C NMR (300 MHz, CDCl3): d
175.6 (C-12), 152.5 (C-30, 50), 147.6 (C-7),
147.1 (C-6), 136.1 (C-1’), 133.3 (C-40),
131.8 (C-10), 133.2 (C-9), 104.3 (C-5),
110.8 (C-8), 108.5 (C-20, 60), 104.3 (C-13),
68.4 (C-11), 60.7 (C-4), 55.7 (C-40-OCH3),
56.3 (C-30, 50-OCH3), 50.3 (C-indol-
OCH3), 44.2 (C-1), 41.7 (C-2), 38.0
(C-3), 131.6 (C-200), 114.0 (C-300), 125.8
(C-400), 154.5 (C-500), 126.8 (C-700), 110.8
(C-600), 131.6 (C-800), 130.3 (C-900), 55.7
(100-CH2), 155.5 (CvN), 126.8 (C-2000,6000),
125.8 (C-3000,5000), 113.2 (C-4000), 133.4
(C-1000), 34.5 (4000-CH), 31.2 (C-(CH3)3);
HR-ESI-MS: m/z 717.3196 [M þ H]þ
(calcd for C43H45N2O8, 717.3176).
3.2.1 Compound 8: N0-4b-imino-[1-(2-
fluoro)-benzyl-5-methoxyl-1H-indol-3-
methylene]-4-deoxypodophyllotoxin
20
White powder; ½aꢀD 2 26.1 (c ¼ 0.52,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
3.01–3.20 (1H, m, H-3), 3.77 (6H, s, 30, 50-
OCH3), 3.81 (6H, s, 40, 500-OCH3), 3.83
(1H, d, J ¼ 4.8 Hz, H-2), 4.23–4.29 (2H,
m, H-11), 4.56 (1H, d, J ¼ 4.2 Hz, H-4),
4.70 (1H, d, J ¼ 4.8 Hz, H-1), 5.32 (2H, s,
CH2-1000), 5.87 (2H, s, H-13), 6.37 (2H, s,
H-20, 60), 6.53 (1H, s, H-5), 6.65 (1H, s, H-
8), 6.80–6.90 (2H, m, H-3000, 6000), 6.93–
7.05 (2H, m, H-4000,5000), 7.06–7.28 (2H, m,
H-600, 700), 7.74 (1H, d, J ¼ 2.4 Hz, H-200),
8.53 (1H, s, H-1000); 13C NMR (300 MHz,
CDCl3): d 174.6 (C-12), 160.6 (C-2000),
158.2 (CvN), 153.4 (C-500), 151.5 (C-30,
50), 146.7 (C-7), 146.1 (C-6), 135.1 (C-1’),
135.1 (C-40), 132.4 (C-10), 132.3 (C-800),
131.2 (C-900), 131.3 (C-200), 130.0 (C-5000),
128.1 (C-9), 125.6 (C-6000), 123.6(C-400),
123.5 (C-1000), 122.5 (C-4000), 122.4 (C-600),
114.5 (C-3000), 113.3 (C-300), 112.3 (C-700),
111.2 (C-8), 109.5 (C-5), 107.5 (C-20, 60),
100.5 (C-13), 67.5 (C-CH2), 67. 4 (C-11),
59.7 (C-4), 55.3 (C-30, 50-OCH3), 54.7 (C-
500-OCH3), 54.5 (C-40-OCH3), 43.2 (C-1),
43.3 (C-2), 37.0 (C-3); HR-ESI-MS: m/
3.2.3 Compound 10: N’-4b-imino(1-
benzyl-5-methoxyl-1H-indol-3-
methylene)-4-deoxypodophyllotoxin
20
White powder; ½aꢀD 2 35.9 (c ¼ 0.56,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
3.09–3.12 (2H, m, H-2, 3), 3.77 (6H, s, H-
30, 50-OCH3), 3.81 (6H, s, H-40, 500-OCH3),
4.22–4.32 (2H, m, H-11), 4.55 (1H, d,
J ¼ 3.9 Hz, H-4), 4.69 (1H, d, J ¼ 4.8 Hz,
H-1), 5.41 (2H, s, CH2-1000), 6.01 (1H, s, C-
13), 5.94 (1H, s, C-13), 6.37 (2H, s, H-20,
60), 6.65 (1H, s, H-5), 6.53 (1H, s, H-8),
6.87 (1H, d, J ¼ 2.1 Hz, H-600), 7.06 (2H,
J ¼ 6.6 Hz, H-3000, 5000), 7.74 (1H, d,
J ¼ 2.1 Hz, H-700), 7.31 (2H, d,
J ¼ 6.6 Hz, H-2000, 6000), 7.39 (1H, s, H-
400), 7.16 (1H, s, H-200), 8.53 (1H, s, H-1000);
13C NMR (300 MHz, CDCl3): d 175.6 (C-
12), 152.6 (C-30, 50), 147.7 (C-7), 136.1
(C-10), 136.3 (C-40), 128.9 (C-9), 132.5 (C-
z
C39H36FN2O8, 679.2456).
679.2468 [M þ H]þ (calcd for
3.2.2 Compound 9: N’-4b-imino[1-(4-t-
butyl)-benzyl-5-methoxyl-1H-indol-3-
methylene]-4-deoxypodophyllotoxin
20
White powder; ½aꢀD 2 65.8 (c ¼ 0.49,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
1.29 (9H, s, 4000-CH(CH3)3), 3.09–3.12
(2H, m, H-2, 3), 3.77 (6H, s, 30, 50-OCH3),

532
Z.-H. Zhang et al.
10), 108.9 (C-5), 110.4 (C-8), 108.9 (C-20,
60), 101.3 (C-13), 68.5 (C-11), 60.8 (C-4),
55.7 (C-40-OCH3), 56.3 (C-30, 50-OCH3),
50.7 (C-indol-OCH3), 44.2 (C-2), 41.7 (C-
1), 38.0 (C-3), 128.9 (C-3000, 5000), 127.0 (C-
2000, 6000), 126.7 (C-4000), 155.6 (CvN),
137.2 (C-200), 113.3 (C-300), 128.0 (C-400),
154.5 (C-500); HR-ESI-MS: m/z 661.2627
[M þ H]þ (calcd for C39H37N2O8,
661.2570).
1.29 (9H, s, 4000-CH(CH3)3), 2.64–3.00
(2H, m, H-3, 2), 3.44 (2H, s, CH2-N), 3.72
(6H, s, H-30, 50-OCH3), 3.77 (6H, s, H-40,
5000-OCH3), 3.90–4.12 (2H, m, H-11), 4.31
(1H, d, J ¼ 1.5 Hz, H-4), 4.50 (1H, d,
J ¼ 4.9 Hz, H-1), 5.25 (2H, s, CH2-1000),
5.97 (2H, s, H-13), 6.24 (2H, s, H-20, 60),
6.33 (1H, s, H-8), 6.45 (1H, s, H-5), 6.87
(1H, dd, J ¼ 2.2, 8.9 Hz, H-600), 7.01–7.12
(2H, m, H-3000, 5000), 7.21 (1H, d,
J ¼ 2.2 Hz, H-700), 7.50–7.54 (2H, m, H-
2000, 6000), 7.70 (1H, d, J ¼ 8.9 Hz, H-400);
13C NMR (300 MHz, CDCl3): d 174.6 (C-
12), 151.6 (C-30, 50), 146.6 (C-7), 146.1
(C-6), 136.1 (C-10), 136.3 (C-40), 135.1 (C-
9), 132.3 (C-10), 109.4 (C-5), 111.3 (C-8),
107.9 (C-20, 60), 103.3 (C-13), 67.4 (C-11),
59.7 (C-4), 54.7 (C-40-OCH3), 55.3 (C-30,
50-OCH3), 49.3 (C-indol-OCH3), 42.0 (C-
1), 43.2 (C-2), 40.7 (C-3), 33.5 (4000-CH),
30.2 (C-(CH3)3), 126.3 (C-2000, 6000), 124.8
(C-3000, 5000), 113.1 (C-4000), 125.8 (C-1000),
135.7 (C-200), 112.2 (C-300), 125.5 (C-400),
153.5 (C-500), 66.8 (N-CH2), 67.4 (100-
CH2Ar); HR-ESI-MS: m/z 719.3383
[M þ H]þ (calcd for C43H47N2O8,
719.3332).
3.2.4 Compound 11: N0-4b-[1-(2-
fluoro)-benzyl-5-methoxyl-1H-indol-3-
methylene]-amino-4-
deoxypodophyllotoxin
20
White powder; ½aꢀD 2 53.3 (c ¼ 0.39,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
4.70 (1H, d, J ¼ 4.8 Hz, H-1), 3.83 (1H, d,
J ¼ 4.8 Hz, H-2), 3.01–3.20 (1H, m, H-3),
4.56 (1H, d, J ¼ 4.2 Hz, H-4), 6.65 (1H, s,
H-5), 6.53 (1H, s, H-8), 4.23–4.29 (2H, m,
H-11), 6.37 (2H, s, H-20, 60), 5.93 (2H, s,
H-13), 6.80–6.90 (2H, m, H-3000, 6000),
6.93–7.05 (2H, m, H-4000, 5000), 7.06–7.28
(3H, m, H-600, 700, 400), 7.74 (1H, s, H-200),
3.80 (2H, s, CH2-N); 13C NMR (300 MHz,
CDCl3): d 177.3 (C-12), 161.6 (C-2000),
154.4 (C-500), 153.4 (C-30,50), 147.4 (C-7),
146.7 (C-6), 136.4 (C-10), 135.1 (C-40),
132.0 (C-9), 129.3 (C-800), 127.9 (C-900),
132.3 (C-200), 117.6 (C-5000), 124.5 (C-10000),
127.7 (C-6000), 115.6 (C-400), 121.6 (C-1 ),
120.3 (C-4000), 110.2 (C-600), 109.7 (C-3000),
113.9 (C-300), 107.3 (C-700), 106.2 (C-8),
105.9 (C-5), 104.9 (C-20,60), 100.9 (C-13),
67.5 (C-CH2), 67.4 (C-11), 60.9 (C-4),
55.8 (C-N), 56.3 (C-30, 50-OCH3), 56.3 (C-
indol-OCH3), 55.9 (C-40-OCH3), 43.8 (C-
1), 41.5 (C-2), 38.8 (C-3); HR-ESI-MS:
m/z 681.2661 [M þ H]þ (calcd for
C39H38FN2O8, 681.2612).
3.2.6 Compound 13: N0-4b-(1-benzyl-5-
methoxyl-1H-indol-3-methylene)-amino-
4-deoxypodophyllotoxin
20
White powder; ½aꢀD 2 31.6 (c ¼ 0.49,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
2.66–2.90 (2H, m, H-2, 3), 3.39 (2H, s, N-
CH2), 3.73 (6H, s, 30, 50-OCH3), 3.79 (3H,
s, 40-OCH3), 3.85 (3H, s, 500-OCH3), 4.05–
4.10 (2H, m, H-11), 4.35 (1H, t,
J ¼ 7.7 Hz, H-4), 4.53 (1H, d,
J ¼ 5.2 Hz, H-1), 5.27 (2H, s, CH2-1000),
5.93 (2H, s, C-13), 6.25 (2H, s, H-20, 60),
6.45 (1H, s, H-8), 6.58 (1H, s, H-5), 6.85
(1H, dd, J ¼ 8.9, 2.3 Hz, H-600), 7.17 (1H,
3.2.5 Compound 12: N0-4b-[1-(4-t-
butyl)-benzy-5-methoxyl-1H-indol-3-
methylene]-amino-4-
d, J ¼ 2.3 Hz, H-700), 7.27 (1H,
d
J ¼ 8.9 Hz, H-400), 7.00–7.15 (3H, m, H-
3000, 4000, 5000), 7.25 (2H, br s, H-6000, 2000), 7.69
(1H, s, H-200); 13C NMR (300 MHz,
CDCl3): d 174.6 (C-12), 154.6 (C-30, 50),
146.7 (C-7), 146.1 (C-6), 132.4 (C-10),
deoxypodophyllotoxin
20
White powder; ½aꢀD 2 41.7 (c ¼ 0.46,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d

Journal of Asian Natural Products Research
533
135.3 (C-40), 127.8 (C-9), 127.1 (C-10),
107.9 (C-5), 110.4 (C-8), 107.8 (C-20, 60),
103.8 (C-13), 67.5 (C-11), 59.7 (C-4), 55.3
(C-40-OCH3), 54.8 (C-30, 50-OCH3), 49.6
(C-N), 49.2 (C-indol-OCH3), 43.2 (C-2),
40.7 (C-1), 37.0 (C-3), 132.3 (C-1000), 127.9
(C-2000, 6000), 126.0 (C-3000, 5000), 125.7
(C-4000), 135.2 (C-200), 112.3 (C-300), 128.0
(C-400), 154.0 (C-500), 112.2 (C-600), 109.4
(C-700), 128.7 (C-800); HR-ESI-MS: m/z
4), 4.30–4.42 (2H, m, N-CH2), 4.54 (1H,
d, J ¼ 5.0 Hz, H-1), 5.93 (2H, d,
J ¼ 5.1 Hz, H-13), 6.24 (2H, s, H-20, 60),
6.46 (1H, s, H-8), 6.52 (1H, s, H-5), 6.98
(2H, br s, H-300, 400), 7.25 (1H, br s, H-200);
13C NMR (300 MHz, CDCl3): d 174.9 (C-
12), 152.3 (C-30, 50), 147.6 (C-7), 147.1
(C-6), 125.5 (C-500), 137.0 (C-10), 135.6
(C-40), 132.2 (C-9), 131.5 (C-10), 126.9
(C-200), 125.4 (C-300), 124.9 (C-400), 110.5
(C-8), 109.9 (C-5), 108.5 (C-20, 60), 101.6
(C-13), 69.1 (C-11), 61.4 (C-4), 56.9 (C-30,
50-OCH3), 55.7 (C-40-OCH3), 49.4 (C-N),
44.6 (C-1), 42.2 (C-2), 39.5 (C-3); HR-
ESI-MS: m/z 510.1624 [M þ H]þ (calcd
for C27H28NO7S, 510.1586).
663.2747
C39H39N2O8, 663.2706).
[M þ H]þ
(calcd
for
3.2.7 Compound 14: N0-4b-(5-bromo-
thiophene-2-methylene)-amino-4-
deoxypodophyllotoxin
20
White powder; ½aꢀD 2 61.4 (c ¼ 0.55,
3.2.9 Compound 16: N0-4b-(5-ethyl-
thiophene-2-methylene)-amino-4-
deoxypodophyllotoxin
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
2.75–2.90 (1H, m, H-3), 3.20–3.30 (1H,
m, H-2), 3.73 (6H, s, H-30, 50-OCH3), 3.79
(3H, s, H-40-OCH3), 3.95 (2H, s, CH2-N),
4.09 (1H, d, J ¼ 14.2 Hz, H-4), 4.34 (2H,
d, J ¼ 8.2 Hz, H-11), 4.54 (1H, d,
J ¼ 5.1 Hz, H-1), 5.96 (2H, s, H-13),
6.24 (1H, s, H-20, 60), 6.48 (1H, s, H-8),
6.62 (1H, s, H-5), 6.74 (1H, s, H-400), 6.92
(1H, s, H-300); 13C NMR (300 MHz,
CDCl3): d 173.4 (C-12), 151.1 (C-30, 50),
146.6 (C-7), 145.8 (C-6), 143.8 (C-500),
134.2 (C-10), 135.9 (C-40), 130.4 (C-9),
128.4 (C-10), 124.3 (C-300, 400), 110.5
(C-8), 109.4 (C-5), 107.3 (C-20, 60), 100.5
(C-13), 67.7 (C-11), 60.1 (C-4), 55.7 (C-30,
50-OCH3), 54.6 (C-40-OCH3), 48.4 (C-N),
43.3 (C-1), 40.9 (C-2), 38.1 (C-3); HR-
ESI-MS: m/z 588.0733 [M þ H]þ (calcd
for C27H27BrNO7S, 588.0692).
20
White powder; ½aꢀD 2 57.5 (c ¼ 0.46,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
1.31 (3H, t, J ¼ 7.4 Hz, 200-CH2CH3),
2.65–2.90 (3H, m, H-3, 200-CH2), 3.30
(1H, dd, J ¼ 4.8, 13.7 Hz, H-2), 3.73 (6H,
s, H-30, 50-OCH3), 3.79 (3H, s, 40-OCH3),
4.00 (3H, br s, H-4, 11), 3.43–4.37 (2H, m,
N-CH2), 4.53 (1H, d, J ¼ 4.8 Hz, H-1),
5.93 (2H, d, J ¼ 3.8 Hz, C-13), 6.25 (2H,
s, H-20, 60), 6.45 (1H, s, H-8), 6.54 (1H, s,
H-5), 6.65 (1H, s, H-300), 6.76 (1H, s, H-
400); 13C NMR (300 MHz, CDCl3): d 174.3
(C-12), 151.6 (C-30, 50), 147.5 (C-7), 146.3
(C-6), 139.6 (C-500), 135.0 (C-10), 136.4
(C-40), 131.8 (C-9), 130.9 (C-10), 124.6
(C-300), 122.4 (C-400), 109.8 (C-8), 107.9
(C-20, 60), 107.5 (C-5), 101.0 (C-13), 68.5
(C-11), 60.8 (C-4), 56.3 (C-30, 50-OCH3),
55.0 (C-40-OCH3), 49.1 (C-N), 43.9 (C-1),
41.6 (C-2), 38.9 (C-3), 24.0 (C-200-CH2),
16.5 (C-200-CH2CH3); HR-ESI-MS: m/z
3.2.8 Compound 15: N0-4b-(thiophen-2-
methylene)-amino-4-
deoxypodophyllotoxin
538.1941
C29H32NO7S, 538.1899).
[M þ H]þ
(calcd
for
20
White powder; ½aꢀD 2 46.2 (c ¼ 0.51,
1
CH2Cl2); H NMR (300 MHz, CDCl3): d
2.70–2.83 (1H, m, H-3), 3.31 (1H, dd,
J ¼ 5.0, 13.6 Hz, H-2), 3.73 (6H, s, 30, 50-
OCH3), 3.79 (3H, s, 40-OCH3), 4.00–4.10
(2H, m, H-11), 4.09 (1H, d, J ¼ 3.1 Hz, H-
Acknowledgments
This work was financially supported by the
National Natural Science Foundation of China
(No. 30873363), the Great Program of Science

534
Z.-H. Zhang et al.
Foundation of Tianjin (No. 09ZCKFNC01200),
and the Program of Science Foundation of
Tianjin (No. 08JCYBJC070000).
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