Synthesis and cytotoxicity evaluation of novel podophyllotoxin derivatives

Article abstract of DOI:10.1002/ardp.201100095

Seven benzylamino derivatives of podophyllotoxin 8a-8g were synthesized and their chemical structures were confirmed by IR, ¹H-NMR, ¹³C-NMR and ESI-MS spectral analyses. Their abilities to inhibit the growth of cancer cells A549, HCT-116 and HepG2, were investigated by MTT assay. Compound 8b possessed the highest cytotoxicity on cancer cell lines with average IC₅₀ values of 3.8 μM. All we synthetic compounds were cytotoxic against three cancer cell lines at the micromolar range, indicating podophyllotoxin derivatives with structural modification of benzylamino possess potent antitumor activity. Seven benzylamino derivatives of podophyllotoxin (8a-8g) were synthesized and screened for cytoxic activity. All seven compounds possess promising antitumor activity, especially compound 8b. Copyright

Full text of DOI:10.1002/ardp.201100095

Arch. Pharm. Chem. Life Sci. 2011, 344, 735–740
735
Full Paper
Synthesis and Cytotoxicity Evaluation of Novel
Podophyllotoxin Derivatives
Wang Chengniu1, Wu Zhonghua1, Zhao Yu1, Ni Chunyan1, Zhao Xiaodong2, and Zhu Li1
1 Institute of Nautical Medicine, Nantong University; Nantong, P.R. China
2 School of Electromechanical Engineering, Zhejiang Ocean University; Zhoushan, P.R. China
Seven benzylamino derivatives of podophyllotoxin 8a–8g were synthesized and their chemical
1
structures were confirmed by IR, H-NMR, 13C-NMR and ESI-MS spectral analyses. Their abilities to
inhibit the growth of cancer cells A549, HCT-116 and HepG2, were investigated by MTT assay.
Compound 8b possessed the highest cytotoxicity on cancer cell lines with average IC50 values of
3.8 mM. All we synthetic compounds were cytotoxic against three cancer cell lines at the micromolar
range, indicating podophyllotoxin derivatives with structural modification of benzylamino possess
potent antitumor activity.
Keywords: Benzylamino derivatives / Cytotoxicity / Podophyllotoxin
Received: March 16, 2011; Revised: April 16, 2011; Accepted: April 20, 2011
DOI 10.1002/ardp.201100095
Introduction
use, several limitations hindered their clinical use, such as
myelosuppression, neutropenia, nausea, drug resistance and
poor bioavailability [8, 9]. These problems have encouraged
further modifications. In order to get much more potent
and effective compounds, numerous derivatives have been
synthesized, even after the achievement of the synthesis of
etoposide. Several potent and promising derivatives such as
GL-331 (3) and NPF (4) reached phase I and phase II clinical
trials as antitumor agents [10–12]. Most of the derivatives of
podophyllotoxin are DNA topoisomerase II inhibitors pro-
ceeding through a mechanism of action entirely different
from that of their parent compound podophylloxin [13, 14].
New synthetic N-linked congeners such as GL-331 and NPF
exhibit improved cytotoxicity and topoisomerase II inhi-
bition activity [15, 16]. The 4b-amino substituted congeners
GL-331 have displayed a better pharmacological profile than
those of etoposide. Now GL-331 is currently in phase II of the
clinical trials against colon cancer, non-small cell carcinoma,
gastric carcinoma and etoposide resistant malignancies.
Interestingly, GL-331 was found to be effective in killing
cancer cells resistant to etoposide treatment [17, 18]. In
the literature, The 4b-amino substituted congeners NPF is
a promising source of new anticancer agents. It was found 10-
fold more potency in inhibiting human DNA topoisomerase II
and 100-fold more cytotoxic against various human tumor
cells when compared to etoposide [19, 20].
Podophyllotoxin (1, Fig. 1), extracted from the roots and
rhizomes of Podophyllum species such as P. hexandrum and
P. peltatum, is a naturally occurring cyclolignan with import-
ant antimitotic and antitumor properties [1]. The antimitotic
properties of podophyllotoxin are similar to those of colchi-
cine that inhibits assembly of microtubules into the mitotic
apparatus by binding to the tubulin dimer [2]. Because of the
side effects of podophyllotoxin, such as nausea, vomiting,
neutropenia and damage of normal tissues, the possible
clinical utility of podophyllotoxin as an antitumour agent
largely have been abandoned. Therefore, synthesis and acti-
vatity study of its analogs become a hotspot of anticancer
study [3].
Currently, several semisynthetic derivatives of podophyl-
lotoxin are used in clinics for anticancer chemotherapy.
The semisynthetic glucosidic cyclic acetal derivatives of
podophyllotoxin, etoposide or VP-16 (2) were obtained.
Etoposide is one of the most extensively used anticancer
drugs in the treatment of several types of tumors, including
small-cell lung cancer, testicular, leukemia, lymphoma and
Kaposi’s sarcoma [4–7]. Although etoposide has seen much
Correspondence: Zhu Li, Institute of Nautical Medicine, Nantong
University; Nantong 226001, P.R. China
E-mail: zhulili65@163.com
Fax: þ86 513 85051796
In order to obtain novel podophyllotoxin derivatives, we
induct one carbon atom between nitrogen atom and aryl
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

736
W. Chengniu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 735–740
H
NO2
F
O
O
Me
O
OH
O
HO
O
5
8
4
HN
HN
10
9
OH
6
3
2
O
O
O
O
O
O
O
O
O
7
1
1'
O
O
O
O
6'
5'
2'
O
O
3'
OMe
OMe
'
MeO
MeO
OMe
OMe
4OMe
OH
MeO
MeO
OH
OH
podophyllotoxin (1)
Etoposide (2)
GL-331 (3)
NPF (4)
Figure 1. Podophyllotoxin and its analogues.
group to increase chain length, on the basis of the two N-
linked congeners GL-331 and NPF. Then we examined their
anti-tumor activity in the hope of finding more efficient
agents. Herein, we report the synthesis and cytotoxicity
activity of seven benzylamino derivatives of podophyllotoxin
(8a–8g, Scheme 1). We found that all the seven compounds
possess promising antitumor activity, especial compound 8b.
gave a novel series of benzimidoyl derivatives (7a–7g) in the
yields of 70%ꢁ83%. Benzylamino derivatives of podophyllo-
toxin (8a–8g) were previously synthesized by reduction of
benzimidoyl derivatives (7a–7g) using sodium borohydride
(NaBH4) in the yield of 88%ꢁ93%.
Cytotoxicity of compounds 8a–8g against three cancer
cell lines
Results and discussion
The target compounds (8a–8g) were evaluated for their cyto-
toxic activities against three cancer cell lines including A549,
HCT-116 and HepG2, 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.
Synthesis of benzylamino derivatives of
podophyllotoxin
The synthetic route used to synthesize title compounds is
outlined in Scheme 1. In the presence of boron trifluoride
etherate (BF3 ꢀ Et2O), the reaction of triazoic acid (HN3) with
the primal material podophyllotoxin (1) gave 4b-azido-4-
deoxypodophyllotoxin (5) [21]. Amino intermediate was pre-
pared via reduction of the azide using Pd/C as a catalyst
according to the procedure reported previously [22]. 4b-
amino-4-deoxypodophyllotoxin (6) was prepared in 95% over-
all yield from podophyllotoxin. Benzaldehydes with amine 6,
respectively, in dehydrated alcohol over a 3 h reflux period
From the IC50 values, it is clear that the seven podophyllo-
toxin derivatives displayed strong cytotoxic activities against
three cancer cell lines. All of them demonstrated better
cytotoxicity when compared to etoposide. However, these
derivatives showed comparatively lesser activity against
HCT-116 cell line than other two cell lines. The average
IC50 of compound 8g, bearing phenyl group, is 5.3 mM.
The average IC50 values of the phenyl moiety, substituted
OH
N3
NH2
.
O
O
O
O
O
Pd/C, H2, EtOAc
HN3, BF3 Et2O
O
O
O
O
O
O
O
OMe
OMe
OMe
MeO
MeO
MeO
OMe
OMe
OMe
1
5
6
R
N
HN
R
O
8a: R=4-Br-Ph
8b: R=4-Cl-Ph
8c: R=4-F-Ph
8d: R=2-Cl-Ph
8e: R=4-Me-Ph
8f: R=4-MeO-Ph
8g: R=Ph
O
O
NaBH4
CH2Cl2/MeOH
R CHO
EtOH , reflux
O
O
O
O
O
OMe
OMe
MeO
MeO
OMe
OMe
7a-7g
8a-8g
Scheme 1. Synthesis of benzylamino deriva-
tives of podophyllotoxin 8a–8g.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 735–740
Synthesis and Cytotoxicity Evaluation of Novel Podophyllotoxin Derivatives
737
Table 1. The IC50 (mM) of Podophyllotoxin derivatives on cancer
cell lines.
progress was monitored by thin-layer chromatography (TLC)
using GF254 silica gel with fluorescent indicator on glass plates.
Flash chromatography was performed on silica gel (200–
300 mesh, Qingdao, China). 1H- 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
values. The high resolution spectra were obtained on a Q-TOF
Global Mass (ESI-MS).
Compound
IC50 (mM)
HepG2
A549
HCT-116
Averagea
8a
8b
8c
8d
8e
8f
8g
4.9
2.9
4.0
5.5
3.2
9.5
4.8
21.4
6.3
5.6
5.1
6.4
5.2
8.7
6.1
13.5
5.8
3.0
5.0
6.3
5.8
8.1
5.1
4.9
5.7
3.8
4.7
6.1
4.7
8.8
5.3
13.3
4b-(400-Bromo-benzimidoyl)-4-deoxypodophyllotoxin (7a)
1
White solid, yield 70%; H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H,
3-H), 3.72 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H,
40-OCH3), 4.22 (m, 2H, 11-H), 4.59 (d, J ¼ 3.9 Hz, 1H, 4-H), 4.69
(d, J ¼ 5.1 Hz, 1H, 1-H), 5.89 (s, 1H, -OCHO), 5.95 (s, 1H, -OCHO),
6.35 (s, 2H, 20,60-ArH), 6.54 (s, 1H, 5-ArH), 6.56 (s, 1H, 8-ArH), 7.52
(s, 1H, 300-ArH), 7.548 (s, 1H, 500-ArH), 7.59 (s, 1H, 200-ArH), 7.62
Etoposide
a
Average IC50 of three cancer cells IC50s.
(s, 1H, 6 -ArH), 8.38 (s, 1H, N CH); 13C-NMR (CDCl3, 300 MHz):
00
–
–
d 44.2, 44.7, 56.5, 60.5, 69.1, 70.1, 101.4, 102.9, 106.7, 107.9,
108.9, 125.0, 126.0, 128.5, 130.5, 131.7, 132.2, 132.9, 135.4,
136.7, 138.2, 139.6, 146.1, 146.2, 147.2, 153.5, 161.2, 179.6;
EIMS calcd. for C29H26BrNO7 (M þ H)þ 580.1, found 580.0.
by a halogen group, are in the range of 3.8 to 6.1 mM. These
results indicate that the halogen substitution had no effect
on their cytotoxicities. Compound 8b possessed the highest
growth inhibitory effect on A549, HCT-116 and HepG2 cancer
cell lines with average IC50 values of 3.8 mM. The cytotoxicity
of compound 8b is better than that of compound 8d. These
results indicate that compounds substituted with halogens
on para-position were more potent than that of ortho-position.
Compounds bearing a methyl group exhibited better cyto-
toxic activity. Cytotoxic activity decreased when the phenyl
moiety was substituted by methoxyl group. Compound 8f
possessed the least cytotoxicity with average IC50 values of
8.8 mM. The cytotoxic activities of compound 8e is stronger
than that of compound 8g indicate that the relative position
of methyl group on phenyl moiety contributed to the
cytotoxicity.
4b-(400-Chloro-benzimidoyl)-4-deoxypodophyllotoxin (7b)
1
White solid, yield 76%; H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H,
3-H), 3.7–3.73 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H,
40-OCH3), 4.22 (m, 2H, 11-H), 4.60 (d, J ¼ 4.2 Hz, 1H, 4-H), 4.69
(d, J ¼ 5.1 Hz, 1H, 1-H), 5.89 (s, 1H, -OCHO), 5.94 (s, 1H, -OCHO),
6.35 (s, 2H, 20, 6-ArH), 6.54 (s, 1H, 5-ArH), 6.56 (s, 1H, 8-ArH), 7.36
(s, 1H, 300-ArH), 7.39 (s, 1H, 500-ArH), 7.66 (s, 1H, 200-ArH), 7.69 (s, 1H,
6 -ArH), 8.39 (s, 1H, N CH); 13C-NMR (CDCl3, 300 MHz): d 44.2,
00
–
–
44.6, 56.5, 60.5, 69.2, 70.1, 101.4, 102.9, 106.7, 107.9, 108.9, 124.9,
126.1, 128.5, 130.5, 131.7, 132.2, 130.9, 133.4, 136.8, 138.2, 139.6,
146.1, 146.2, 147.8, 153.5, 159.8, 179.4; EIMS calcd. for
C29H26ClNO7 (M þ H)þ 536.2, found 536.1.
4b-(400-Fluoro-benzimidoyl)-4-deoxypodophyllotoxin (7c)
White solid, yield 80%; 1H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H, 3-H),
3.72 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H, 40-OCH3), 4.22
(m, 2H, 11-H), 4.59 (d, J ¼ 4.2 Hz, 1H, 4-H), 4.69 (d, J ¼ 5.1 Hz, 1H,
1-H), 5.89 (s, 1H, -OCHO), 5.95 (s, 1H, -OCHO), 6.36 (s, 2H, 20,60-ArH),
6.54 (s, 1H, 5-ArH), 6.57 (s, 1H, 8-ArH), 7.7100(s, 1H, 300-ArH), 7.73 (s, 1H,
In summary, we have prepared a series of benzylamino
derivatives of podophyllotoxin. The synthetic compounds
were cytotoxic against three cancer cell lines at the micro-
molar range.
00
00
–
5 -ArH), 7.74 (s, 1H, 2 -ArH), 7.76 (s, 1H, 6 -ArH), 8.40 (s, 1H, N CH);
–
13C-NMR (CDCl3, 300 MHz): d 44.3, 44.7, 56.5, 60.5, 69.1, 70.1, 101.4,
106.7, 107.9, 108.9, 116.0, 116.3, 126.0, 128.5, 130.9, 131.0, 131.9,
132.9, 133.0, 136.8, 138.2, 139.6, 146.1, 146.2, 147.2, 153.5, 160.8,
179.6; EIMS calcd. for C29H26FNO7 (M þ H)þ 520.1, found 520.2.
Experimental
Syntheses
General procedure for the synthesis of compounds 8a–8g
A solution of 6 (413 mg, 1 mmol) and 1 mmol of benzaldehyde in
EtOH 20 mL was refluxed on a water bath for 3 h. After the
reaction completed, the solution was evaporated under reduced
pressure. The residue was purified by recrystallization from
ethanol. Then, the obtained compound was dissolved in
20 mL MeOH/CH2Cl2 (1:1) and 4 mmol NaBH4 was added in
batch. After the reaction completed, the result solution was
evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel (EtOAc/petroleum ether,
1:3) to afford a solid.
4b-(200-Chloro- benzimidoyl)-4-deoxypodophyllotoxin (7d)
1
White solid, yield 74%; H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H,
3-H), 3.72 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H,
40-OCH3), 4.22 (m, 2H, 11-H), 4.61 (d, J ¼ 3.9 Hz, 1H, 4-H), 4.69
(d, J ¼ 5.1 Hz, 1H, 1-H), 5.88 (s, 1H, -OCHO), 5.94 (s, 1H, -OCHO),
6.35 (s, 2H, 20,60-ArH), 6.548 (s, 1H, 5-ArH), 6.554 (s, 1H, 8-ArH), 7.36
(d, J ¼ 7.5 Hz, 1H, 500-ArH), 7.42 (d, J ¼ 8.7 Hz, 1H, 400-H),
7.60 (d, J ¼ 7.2 Hz, 1H, 200-ArH), 7.756 (s, 1H, 600-ArH), 8.38
(s, 1H, N CH); 13C-NMR (CDCl3, 300 MHz): d 44.2, 44.6, 59.5,
–
–
60.5, 69.2, 70.1, 101.4, 106.6, 108.0, 108.9, 126.0, 127.5, 128.0,
128.6, 131.1, 131.2, 131.5, 132.9, 133.1, 134.0, 136.7, 138.2, 138.4,
139.7, 146.0, 146.25, 147.22, 153.5, 161.0, 179.6; EIMS calcd. for
C29H26ClNO7 (M þ H)þ 536.2, found 536.1.
Intermediate benzimidoyl derivatives (7a–7g) were character-
ized by 1H-NMR spectra. Synthesized target compounds 8a–8g
were characterized by m.p., 1H-NMR, 13C-NMR, IR and MS spectral
analyses. Solvents were purified in the usual way. Reaction
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W. Chengniu et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 735–740
4b-(400-Methyl-benzimidoyl)-4-deoxypodophyllotoxin (7e)
White solid, yield 83%; H-NMR (CDCl3, 300 MHz): d 2.37 (s, 3H,
4b-(400-Chloro-benzylamino)-4-deoxypodophyllotoxin (8b)
25
1
White solid, yield 90%; mp: 199ꢁ2018C from ethanol; ½aꢂD ꢃ62
CH3), 3.1 (m, 1H, 3-H), 3.72 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3),
3.80 (s, 3H, 40-OCH3), 4.22 (m, 2H, 11-H), 4.57 (d, J ¼ 4.2 Hz, 1H,
4-H), 4.68 (d, J ¼ 5.1 Hz, 1H, 1-H), 5.88 (s, 1H, -OCHO), 5.94 (s, 1H,
-OCHO), 6.36 (s, 2H, 20,60-ArH), 6.54 (s, 1H, 5-ArH), 6.58 (s, 1H,
8-ArH), 7.21 (s, 1H, 300-ArH), 7.24 (s, 1H, 500-ArH), 7.61 (s, 1H,
(c 0.5, CHCl3); 1H-NMR (DMSO, 300 MHz): d 2.04 (s, 1H,
1-NH), 2.37 (dd, J ¼ 2.4 Hz, 7.2 Hz, 1H, 2-H), 2.78 (m, 1H,
3-H), 3.56 (s, 3H, 40-OCH3), 3.58 (s, 6H, 30,50-OCH3), 3.75
(d, J ¼ 4.8 Hz, 2H, 13-H), 3.84 (d, J ¼ 4.8 Hz, 1H, 4-H), 4.28
(m, 2H, 11-H), 4.43 (d, J ¼ 4.8 Hz, 1H, 1-H), 5.93 (s, 2H,
-OCH2O), 6.19 (s, 2H, 20,60-ArH), 6.43 (s, 1H, 8-ArH), 6.67
(s, 1H, 5-ArH), 7.32 (d, J ¼ 9 Hz, 1H, 300-ArH), 7.34
(d, J ¼ 8.7 Hz, 1H, 500-ArH), 7.38 (d, J ¼ 5.7 Hz, 1H, 200-ArH),
7.41 (d, 1H, J ¼ 5.7 Hz, 600-ArH); 13C-NMR (CDCl3, 300 MHz):
d 37.8, 41.3, 41.4, 43.8, 56.2, 60.2, 60.3, 60.5, 66.6, 101.7, 108.6,
109.3, 110.2, 128.8, 129.0, 129.1, 131.3, 131.5, 131.6, 131.9,
136.3, 136.8, 136.9, 147.1, 147.6, 152.5, 161.3, 161.5, 176.4;
IR nmax (KBr, cmꢃ1): 3430, 2911, 2828, 1778, 1586, 1477,
1158, 1031; EIMS calcd. for C29H28ClNO7 (M þ Na)þ
560.1444, found 560.1441.
2 -ArH), 7.64 (s, 1H, 6 -ArH), 8.39 (s, 1H, N CH); 13C-NMR
00
00
–
–
(CDCl3, 300 MHz): d 21.6, 44.2, 44.7, 56.5, 60.5, 69.1, 70.1,
100.0, 101.4, 106.6, 107.8, 108.9, 126.0, 128.5, 128.7, 129.7,
132.0, 132.8, 133.8, 136.7, 138.1, 139.6, 141.3, 146.2, 147.1,
153.4, 161.9, 179.5; EIMS calcd. for C30H29NO7 (M þ H)þ 516.1,
found 516.2.
4b-(400-Methoxyl-benzimidoyl)-4-deoxypodophyllotoxin
(7f)
White solid, yield 79%; 1H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H,
3-H), 3.72 (m, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H, 40-OCH3),
3.82 (s, 3H, 500-OCH3) 4.22 (m, 2H, 11-H), 4.60 (d, J ¼ 4.2 Hz, 1H,
4-H), 4.69 (d, J ¼ 5.4 Hz, 1H, 1-H), 5.88 (s, 1H, -OCHO), 5.94 (s, 1H,
-OCHO), 6.36 (s, 2H, 20,60-ArH), 6.55 (s, 1H, 5-ArH), 6.58 (s, 1H, 8-ArH),
7.28 (d, J ¼ 1.2 Hz, 1H, 500-ArH), 7.30 (d, J ¼ 2.1 Hz, 1H, 400-H),
4b-(400-Fluoro-benzylamino)-4-deoxypodophyllotoxin (8c)
25
White solid, yield 92%; mp: 203ꢁ2058C from ethanol; ½aꢂD ꢃ65
(c 0.5, CHCl3); 1H-NMR (DMSO, 300 MHz): d 1.94 (s, 1H,
1-NH), 2.34 (dd, J ¼ 2.4 Hz, 7.2, 1H, 2-H), 2.71 (m, 1H,
3-H), 3.56 (s, 3H, 40-OCH3), 3.58 (s, 6H, 30,50-OCH3), 3.75
(d, J ¼ 4.8 Hz, 2H, 13-H), 3.87 (d, J ¼ 4.8 Hz, 1H, 4-H), 4.28
(m, 2H, 11-H), 4.43 (d, J ¼ 4.8 Hz, 1H, 1-H), 5.94 (s, 2H,
-OCH2O), 6.19 (s, 2H, 20,60-ArH), 6.42 (s, 1H, 8-ArH), 6.66
(s, 1H, 5-ArH), 7.11 (d, J ¼ 9 Hz, 1H, 300-ArH), 7.14
(d, J ¼ 8.7 Hz, 1H, 500-ArH), 7.40 (d, J ¼ 5.7 Hz, 1H, 200-ArH),
7.43 (d, 1H, J ¼ 5.7 Hz, 600-ArH); 13C-NMR (CDCl3, 300 MHz):
d 37.9, 41.4, 43.8, 56.2, 56.3, 60.2, 60.3, 60.4, 66.6, 68.4, 101.7,
108.7, 109.3, 110.2, 128.4, 128.8, 129.1, 131.3, 131.5, 131.9,
136.3, 136.8, 136.9, 147.1, 147.6, 152.4, 161.3, 161.5, 175.3;
IR nmax (KBr, cmꢃ1): 3433, 2936, 2840, 1780, 1591, 1482,
1159, 1037; EIMS calcd. for C29H28FNO7 (M þ Na)þ
544.1759, found 544.1754.
00
00
–
7.32 (s, 1H, 2 -ArH), 7.35 (s, 1H, 6 -ArH), 8.39 (s, 1H, N CH);
–
13C-NMR (CDCl3, 300 MHz): d 21.6, 44.2, 44.7, 56.5, 60.5, 69.1,
70.1, 100.0, 101.4, 106.6, 107.8, 108.9, 126.0, 128.5, 128.7, 129.7,
132.0, 132.8, 133.8, 136.7, 138.1, 139.6, 141.3, 146.2, 147.1, 153.4,
161.9, 179.5; EIMS calcd. for C30H29NO8 (M þ H)þ 532.1, found
532.2.
4b-(400-Benzimidoyl)-4-deoxypodophyllotoxin (7g)
1
White solid, yield 82%; H-NMR (CDCl3, 300 MHz): d 3.1 (m, 1H,
3-H), 3.72 (s, 1H, 2-H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H, 40-OCH3),
4.22 (m, 2H, 11-H), 4.60 (d, J ¼ 4.2 Hz, 1H, 4-H), 4.69 (d, J ¼ 5.1 Hz,
1H, 1-H), 5.88 (s, 1H, -OCHO), 5.94 (s, 1H, -OCHO), 6.36 (s, 2H,
20,60-ArH), 6.54 (s, 1H, 5-ArH), 6.58 (s, 1H, 8-ArH), 7.39 (s, 1H,
300-ArH), 7.42 (s, 1H, 500-ArH), 7.44 (s, 1H, 400-H), 7.73
(d, J ¼ 2.1 Hz, 1H, 200-ArH), 7.75 (d, J ¼ 1.5 Hz, 1H, 600-ArH),
4b-(200-Chloro-benzylamino)-4-deoxypodophyllotoxin (8d)
25
8.44 (s, 1H, N CH); 13C-NMR (CDCl3, 300 MHz): d 44.2, 44.7,
–
–
White solid, yield 91%; mp: 178ꢁ1808C from ethanol; ½aꢂD ꢃ60
(c 0.3, CHCl3); 1H-NMR (DMSO, 300 MHz): d 1.95 (s, 1H,
1-NH), 2.34 (dd, J ¼ 2.4, 7.2 Hz, 1H, 2-H), 2.71 (m, 1H,
3-H), 3.56 (s, 3H, 40-OCH3), 3.58 (s, 6H, 30,50-OCH3), 3.75
(d, J ¼ 4.8 Hz, 2H, 13-H), 3.87 (d, J ¼ 4.8 Hz, 1H, 4-H), 4.28
(m, 2H, 11-H), 4.44 (d, J ¼ 4.8 Hz, 1H, 1-H), 5.94 (s, 2H,
-OCH2O), 6.19 (s, 2H, 20,60-ArH), 6.43 (s, 1H, 8-ArH), 6.69
(s, 1H, 5-ArH), 7.28 (dd, J ¼ 1.5, 4.5 Hz, 1H, 600-ArH), 7.33
(s, 1H, 500-ArH), 7.35 (s, 1H, 200-ArH), 7.47 (s, 1H, 200-ArH);
13C-NMR (CDCl3, 300 MHz): d 31.2, 43.6, 43.7, 52.2, 54.9,
56.2, 56.3, 60.3, 60.4, 68.9, 101.5, 108.5, 109.6, 110.0, 127.1,
127.5, 128.5, 128.6, 130.5, 131.5 133.3, 133.4, 136.7, 136.9,
144.2, 146.7, 147.2, 152.4, 175.5; IR nmax (KBr, cmꢃ1): 3442,
2914, 2840, 1771, 1585, 1479, 1159, 1034; EIMS calcd.
for C29H28ClNO7 (M þ Na)þ 560.1451, found 560.1433.
56.5, 60.5, 60.7, 70.2, 70.5, 72.4, 101.4, 106.7, 107.9, 108.9,
126.0, 128.5, 128.7, 129.1, 131.4, 131.9, 132.9, 136.3, 136.7,
138.2, 139.6, 146.0, 146.2, 147.1, 153.5, 179.6; EIMS calcd. for
C29H27NO7 (M þ H)þ 502.1, found 502.2.
4b-(400-Bromo-benzylamino)-4-deoxypodophyllotoxin (8a)
25
White solid, yield 90%; mp: 202ꢁ2048C from ethanol; ½aꢂD ꢃ72
(c 0.7, CHCl3); 1H-NMR (DMSO, 300 MHz): d 2.04 (s, 1H,
1-NH), 2.39 (dd, J ¼ 2.4, 7.2 Hz, 1H, 2-H), 2.71 (m, 1H, 3-H),
3.56 (s, 3H, 40-OCH3), 3.58 (s, 6H, 30,50-OCH3), 3.75
(d, J ¼ 4.8 Hz, 2H, 13-H), 3.87 (d, J ¼ 4.8 Hz, 1H, 4-H), 4.28
(m, 2H, 11-H), 4.43 (d, J ¼ 4.8 Hz, 1H, 1-H), 5.94 (s, 2H,
-OCH2O), 6.19 (s, 2H, 20,60-ArH), 6.43 (s, 1H, 8-ArH), 6.69
(s, 1H, 5-ArH), 7.32 (d, J ¼ 9 Hz, 1H, 300-ArH), 7.37
(d, J ¼ 8.7 Hz, 1H, 500-ArH), 7.48 (d, J ¼ 5.7 Hz, 1H, 200-ArH),
7.52 (d, 1H, J ¼ 5.7 Hz, 600-ArH); 13C-NMR (CDCl3, 300 MHz):
d 37.9, 41.4, 43.8, 56.2, 56.3, 60.2, 60.3, 60.5, 66.6, 68.4, 101.7,
108.7, 109.3, 110.2, 128.4, 128.8, 129.1, 131.3, 131.5, 131.9,
136.3, 136.8, 136.9, 147.1, 147.6, 152.5, 161.3, 161.5, 175.4;
IR nmax (KBr, cmꢃ1): 3354, 2961, 2389, 1781, 1586, 1479,
1161, 1034; EIMS calcd. for C29H28BrNO7 (M þ Na)þ
604.1172, found 604.1175.
4b-(400-Methyl-benzylamino)-4-deoxypodophyllotoxin (8e)
25
White solid, yield 88%; mp: 208ꢁ2108C from ethanol; ½aꢂD ꢃ64
(c 0.3, CHCl3); 1H-NMR (DMSO, 300 MHz): d 1.95 (s, 1H,
1-NH), 2.26 (s, 3H, 400-OCH3), 2.37 (dd, J ¼ 2.4, 7.2 Hz, 1H,
2-H), 2.74 (m, 1H, 3-H), 3.56 (s, 3H, 40-OCH3), 3.58 (s, 6H,
30,50-OCH3), 3.71 (d, J ¼ 4.8 Hz, 2H, 13-H), 3.80 (d, J ¼ 4.8 Hz,
1H, 4-H), 4.30 (m, 2H, 11-H), 4.43 (d, J ¼ 4.8 Hz, 1H, 1-H),
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Arch. Pharm. Chem. Life Sci. 2011, 344, 735–740
Synthesis and Cytotoxicity Evaluation of Novel Podophyllotoxin Derivatives
739
5.93 (s, 2H, -OCH2O), 6.18 (s, 2H, 20,60-ArH), 6.42 (s, 1H,
8-ArH), 6.62 (s, 1H, 5-ArH), 7.11 (s, 1H, 300-ArH), 7.13
(s, 1H, 500-ArH), 7.25 (s, 1H, 200-ArH), 7.28 (s, 1H, 600-ArH);
13C-NMR (CDCl3, 300 MHz): d 37.9, 39.2, 40.8, 41.3, 41.4,
43.8, 56.2, 56.3, 60.3, 60.5, 66.5, 68.4, 101.7, 108.6, 109.3,
110.2, 114.5, 129.2, 130.4, 131.6, 131.8, 136.8, 136.9, 147.1,
147.5, 152.5, 160.5, 160.7, 167.0, 175.4; IR nmax (KBr, cmꢃ1):
3445, 2939, 2843, 1781, 1585, 1476, 1149, 1034; EIMS calcd.
for C30H31NO7 (M þ Na)þ 540.1991, found 540.1992.
into multiwell culture plates at 5 ꢄ 104ꢁ1 ꢄ 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 378C 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
4b-(400-Methoxyl-benzylamino)-4-deoxypodophyllotoxin
(8f)
Data were presented as means ꢅ SE and analyzed by SPSS soft-
ware. Mean values were obtained from at least thrice independ-
ent experiments.
25
White solid, yield 89%; mp: 215ꢁ2178C from ethanol; ½aꢂD ꢃ56
(c 0.3, CHCl3); 1H-NMR (DMSO, 300 MHz): d 1.94 (s, 1H,
1-NH), 2.24 (dd, J ¼ 2.4, 7.2 Hz, 1H, 2-H), 2.71 (m, 1H,
3-H), 3.57 (s, 3H, 40-OCH3), 3.59 (s, 6H, 30,50-OCH3),
3.75 (s, 3H, 400-OCH3), 4.00 (d, J ¼ 4.8 Hz, 2H, 13-H), 4.20
(d, J ¼ 4.8 Hz, 1H, 4-H), 4.35 (m, 2H, 11-H), 4.65
(d, J ¼ 4.8 Hz, 1H, 1-H), 6.04 (s, 2H, -OCH2O), 6.16 (s, 2H,
20,60-ArH), 6.58 (s, 1H, 8-ArH), 6.99 (s, 1H, 5-ArH), 7.00
(d, J ¼ 9 Hz, 1H, 300-ArH), 7.01 (d, J ¼ 8.7 Hz, 1H, 500-ArH),
7.49 (d, J ¼ 4.8 Hz, 1H, 200-ArH), 7.52 (d, 1H, J ¼ 3 Hz,
600-ArH); 13C-NMR (CDCl3, 300 MHz): d 35.6, 39.1, 40.8,
41.2, 43.5, 49.9, 55.0, 55.3, 55.7, 56.2, 56.4, 60.3, 60.4,
67.7, 102.2, 108.5, 110.3, 110.6, 114.4, 123.8, 124.5,
133.1, 134.2, 136.1, 136.8, 147.0, 148.9, 152.5, 160.3, 174.1;
IR nmax (KBr, cmꢃ1): 3436, 2923, 2850, 1774, 1581, 1479, 1159,
1027; EIMS calcd. for C30H31NO8 (M þ Na)þ 556.1947, found
556.1941.
The studies in the laboratories were supported by Natural Science
Foundation of Nantong City (K2010024), Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD), Zhejiang
Provincial Natural Science Foundation of China (Y4090065). And the
Priority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD).
The authors have declared no conflict of interest.
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White solid, yield 93%; mp: 193ꢁ1958C from ethanol; ½aꢂD ꢃ58
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www.archpharm.com