Design, synthesis and cytotoxicity of novel podophyllotoxin derivatives

Article abstract of DOI:10.1248/cpb.56.831

A series of novel podophyllotoxin derivatives were designed using association strategy and synthesized by coupling either podophyllotoxin (1) or 4β-amino podophyllotoxin (3) with substituted indol-3-yl-glyoxyl chlorides. Their structures were identified using spectroscopic techniques. These novel derivatives have been evaluated for cytotoxicity in vitro against four human cancer cell lines with comparison to the parent compounds 1, 3 and indibulin (2). Some of the compounds (7a, 7c) showed comparable cytotoxicity to that of podophyllotoxin.

Full text of DOI:10.1248/cpb.56.831

June 2008
Notes
Chem. Pharm. Bull. 56(6) 831—834 (2008)
831
Design, Synthesis and Cytotoxicity of Novel Podophyllotoxin Derivatives
Peng-Fei YU,a,b Hong CHEN,b Jing WANG,b Chun-Xian HE,a Bo CAO,b Min LI,b Na YANG,a
b
,a
*
Zhi-Yong LEI, and Mao-Sheng CHENG
a Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University; Shenyang 110016, China: and b Staff Room of Pharmacognosy, Medical College of Chinese
People’s Armed Police Forces; Tianjin 300162, China.
Received November 6, 2007; accepted March 11, 2008; published online March 12, 2008
A series of novel podophyllotoxin derivatives were designed using association strategy and synthesized by
coupling either podophyllotoxin (1) or 4b-amino podophyllotoxin (3) with substituted indol-3-yl-glyoxyl chlo-
rides. Their structures were identified using spectroscopic techniques. These novel derivatives have been evalu-
ated for cytotoxicity in vitro against four human cancer cell lines with comparison to the parent compounds 1, 3
and indibulin (2). Some of the compounds (7a, 7c) showed comparable cytotoxicity to that of podophyllotoxin.
Key words podophyllotoxin; indibulin; cytotoxicity; multidrug resistance
During the course of cancer chemotherapy treatment, it mors in vitro and in vivo. Furthermore, indibulin demon-
has been found that prolonged treatment of carcinoma pa- strates efficacy toward MDR tumor cells and no systemic
tients with certain anticancer medicines can result in an ac- toxicity in the treatment of rat tumor models in vivo, which
quired resistance toward multiple drugs, which is known as might be related to its unknown tubulin-binding mode.10)
multidrug resistance (MDR).1) The mechanism of MDR is
It has been hypothesized that tubulin-targeting agents
not well understood. Until now, it was known that MDR is possessing a novel binding mode could be effective to MDR
often associated with the overexpression of P-glycoprotein cell lines.11,12) Both podophyllotoxin and indibulin are potent
(P-gp) and/or multidrug resistance protein 1 (MDR1), the microtubulin inhibitors, but the tubulin-binding sites of the
overproduction of specific tubulin isotypes,2) and the de- two compounds are different. This intrigues us and has
creased expression of topoisomerases,3) etc. The develop- prompted us to design hybrids of indibulin and podophyllo-
ment of MDR is one of the major problems encountered in toxin with the aim of discovering novel tubulin inhibitors that
chemotherapy. Hence, the need to overcome MDR has fueled can both overcome MDR and lower podophyllotoxin’s toxic-
considerable interest in the development of novel antitumor ity by binding to a novel tubulin-binding domain. The novel
agents with cytotoxicity against MDR cancer cell lines.
podophyllotoxin derivatives based on indol-3-yl-glyoxyl sub-
A variety of natural products, such as Taxanes and Vincas, stituents were generated by replacing the pyridin-4-yl-amino
have proven to be successful in the clinical treatment of can- moiety of indibulin with the structure of podophyllotoxin (1)
cers. A well known cytotoxic natural product, aryltetralin lig- or 4b-amino podophyllotoxin (3), as illustrated in Chart 1.
nan podophyllotoxin (1), can exert antitumor activity by in- Incorporating an indole structure could provide a field for
hibiting tubulin through binding with part of its colchicine structural modification, and various commercially available
domain. Moreover, podophyllotoxin-derived antitumor agents scaffolds could be exploited to manipulate the substituents
can also inhibit human DNA topoisomerase II.4,5) Extensive on the indole ring. More significantly, the target compounds
structure modifications have been carried out on podophyllo- are expected to inhibit human DNA topoisomerase II, just as
toxin to reduce its high toxicity, which makes it unsuitable some other podophyllotoxin derivatives do.
for clinical use as an anticancer medicine. As shown in Fig.
1, two less toxic semisynthetic podophyllotoxin analogues,
etoposide and teniposide, have been used for the treatment of
a broad spectrum of tumors.6,7) In addition, some newly de-
veloped derivatives (e.g. NPF and GL-331) display a better
pharmacology profile and are currently in clinical trials.8,9)
However, some intrinsic shortcomings, such as MDR and
toxicity, make these natural products faulty for clinical can-
cer treatment, which has stimulated researchers to discover
novel tubulin inhibitors.
Indibulin (D-24851, N-(pyridin-4-yl)-[1-(4-chlorobenzyl)-
indol-3-yl]-glyoxylamide, 2) is a synthetic small molecule
tubulin inhibitor discovered by Baxter Oncology. This clini-
cal candidate was identified based on its ability to destabilize
microtubules in tumor cells, thus exerting antitumor activity.
The binding site of indibulin does not overlap with the tubu-
lin-binding sites of well-characterized microtubule-destabi-
lizing agents, such as colchicine or vincristine, suggesting
that it may bind to a novel domain of tubulin. Indibulin
shows significant antitumor activity against a variety of tu-
Fig. 1
∗ To whom correspondence should be addressed. e-mail: mscheng@syphu.edu.cn
© 2008 Pharmaceutical Society of Japan

832
Vol. 56, No. 6
Table 1. Structures of Synthesized Compounds
Compound C-4 configuration
X
Y
R
7a
7b
7c
7d
7e
7f
7g
7h
7i
a
a
a
b
b
b
b
b
b
b
O
O
H
4-Chlorobenzyl
H
H
H
H
3,4-Dichlorobenzyl
O
Benzo[1,3]dioxol-5-ylmethyl
NH
NH
4-Chlorobenzyl
Benzo[1,3]dioxol-5-ylmethyl
NH 5-Bromo
NH 2-Methyl
NH 6-Fluoro
NH 5-Methoxy
NH 6-Methoxy
H
H
H
H
H
Chart 1. Design of the Novel Podophyllotoxin Derivatives
7j
7a—c demonstrates their 4a-configuration. The structures of
1
the resulting compounds were identified by H-NMR and
mass spectroscopy.
Results and Discussion
The biological activity of this series of podophyllotoxin
derivatives was evaluated by an in vitro cytotoxicity test,
which was carried out with a panel of four human cancer
cell lines including HeLa (cervix), SKOV3 (ovary), K562
(leukemia) and K562ADR (adriamycin resistant leukemia),
using podophyllotoxin (1), indibulin (2), 4b-aminopodophyl-
lotoxin (3) and adriamycin as reference compounds. The
screening procedure was based on the standard MTT or SRB
method. The IC50 and GI50 values are shown in Table 2.
As can be seen from the results, most of the derivatives
presented substantial in vitro cytotoxic activity, even against
the adriamycin resistant leukemia cell line (K562ADR). The
potency of some of the compounds (7b, 7c, 7f, 7i, 7j) against
K562ADR (the resistant multiple was 31.19) was comparable
Reagents and conditions: (a) NaN3, CF3COOH, CHCl3, reflux, 90%; (b) HCOONH4,
CH3OH, rt, 70%; (c) NaH, RX, DMF, 0 °C—rt, 80—95%; (d) oxalyl chloride, Et2O,
0 °C, 70—95%; (e) 1 or 3, Et3N, DMAP, DMF, 0 °C—rt, 43—56%.
Chart 2. Synthesis of Compounds 7a—j
We report herein the synthesis and cytotoxicity of a series to those against the common K562 cell line. However, two
of 4a-O- and 4b-N-indol-3-yl-glyoxyl-substituted derivatives derivatives (7g, 7h) were unable to inhibit the growth of
of podophyllotoxin. The biological activity of these deriva- K562ADR cells, though they displayed appreciable cytotoxi-
tives was evaluated by in vitro cytotoxicity study.
city against the three other common tumor cell lines.
Chemistry The novel podophyllotoxin derivatives 7a—j
In general, the O-linked derivatives (esters) of podophyllo-
were prepared according to the general synthetic approach as toxin showed lower activity in comparison to the N-linked
depicted in Chart 2. The N-substituted indoles 5a—c were congeners (amides). However, as far as these analogues are
readily synthesized by the reaction of indole 4 with an ar- concerned, the activities of ester derivatives are more potent
alkyl chloride in the presence of NaH. Treatment of substi- than the amide derivatives (compare 7a with 7d and 7c with
tuted indoles 5 with oxalyl chloride in diethyl ether gave sub- 7e), which might not only correlate with the hetero-atom at
stituted indol-3-yl-glyoxyl chlorides 6 with a 70—95% yield. podophyllotoxin’s C-4 position, but also with the configura-
Subsequently, 6 was coupled with podophyllotoxin or 4b- tion of the C-4 carbon atom.
amino podophyllotoxin in the presence of triethylamine and
Another finding was that a number of compounds exhib-
4-(N,N-dimethylamino)-pyridine (DMAP) to generate the ited improved cytotoxicity particularly with N-substituted
products listed in Table 1. As illustrated in Chart 2, 4b- group of the indole ring. Compound 7a was found to be
amino podophyllotoxin was prepared from podophyllotoxin more potent than 7b, which was substituted by an additional
according to known procedures.5,13) Acylation of podophyllo- chlorine atom at the R group compared with 7a. Compound
toxin retained the a-configuration of its C-4 carbon atom, 7c with benzo[1,3]dioxol-5-ylmethyl group substituted at
1
which could be deduced from the H-NMR spectral data of position 1 of the indole ring is the most potent compound
1
7a—c. In their H-NMR spectra, the J value (8.3 Hz) of the among all the synthetic derivatives against the four cancer
hydrogen atom (d 6.05 or 6.06 ppm) at the C-4 position of cell lines. This compound inhibited the growth of K562ADR

June 2008
833
Table 2. The IC50 and the GI50 Values of the Novel Podophyllotoxin Derivatives
IC50 (mM)
GI50 (mM)
Compound
HeLa
SKOV3
K562
K562ADR
7a
7b
7c
7d
7e
7f
7g
7h
0.55ꢀ0.06
0.65ꢀ0.08
0.40ꢀ0.06
11.05ꢀ1.21
5.72ꢀ0.69
4.65ꢀ0.54
3.62ꢀ0.43
1.34ꢀ0.12
0.94ꢀ0.10
2.45ꢀ0.27
0.10ꢀ0.02
1.27ꢀ0.14
0.06ꢀ0.02
—
0.31ꢀ0.04
3.34ꢀ0.26
0.24ꢀ0.03
13.49ꢀ1.17
9.50ꢀ1.08
46.20ꢀ4.13
7.96ꢀ0.88
4.82ꢀ0.59
3.04ꢀ0.37
7.85ꢀ0.64
0.17ꢀ0.02
0.85ꢀ0.06
0.30ꢀ0.04
—
0.03ꢀ0.01
0.10ꢀ0.02
0.07ꢀ0.02
5.42ꢀ0.63
0.94ꢀ0.13
1.55ꢀ0.12
0.89ꢀ0.10
1.95ꢀ0.16
0.21ꢀ0.02
3.04ꢀ0.27
0.03ꢀ0.01
0.36ꢀ0.03
0.12ꢀ0.02
2.73ꢀ0.31
0.30ꢀ0.05
0.22ꢀ0.01
0.18ꢀ0.02
7.61ꢀ0.68
33.85ꢀ2.76
1.51ꢀ0.19
ꢅ50
ꢅ50
7i
7j
0.42ꢀ0.06
3.09ꢀ0.28
0.17ꢀ0.02
0.72ꢀ0.05
0.28ꢀ0.02
85.15ꢀ8.07
Podophyllotoxin
4b-Aminopodophyllotoxin
Indibulin
Adriamycin
a) Cytotoxicity against HeLa and SKOV3 cell lines was measured by the standard MTT assay method. b) Cytotoxicity against K562 and K562ADR cell lines was measured
by the standard SRB assay method. c) Each value represents the meanꢀS.D. of three independent experiments.
time. After 3—5 h stirring at room temperature, the mixture was added to
30 g ice water and stirred until precipitation. The product was filtered off,
washed with water and dried under vacuum for 12 h at 25—30 °C. The title
compounds 5a—c were obtained respectively and could be used directly for
carcinoma cells with a GI50 value of 0.18ꢀ0.02 mM, essen-
tially comparable to those of podophyllotoxin and indibulin.
We also evaluated the effects of substitutents at other posi-
tions of the indole ring. Replacing the bromine atom of com-
pound 7f with a lipophilic and electron-releasing methoxyl
group in situ afforded derivative 7i, resulting in an increased
cytotoxicity by approximately one order of magnitude. Chang-
ing the position of the methoxyl group of compound 7i from
C-5 to C-6 of the indole ring conferred decreased cytotoxic-
ity on isomer 7j. The mechanism of action of these com-
pounds will be further elucidated by biochemical investiga-
tions currently in progress.
In conclusion, a series of podophyllotoxin derivatives have
been synthesized via a concise synthesis route and have been
observed to act as novel cytotoxic agents against a series of
four human cancer cell lines, including one that expresses the
MDR phenotype. Therefore, the results obtained with the
newly synthesized podophyllotoxin derivatives showed both
that our association design strategy was applicable and that
these derivatives are candidates worthy of additional re-
search. Further studies are being conducted to determine the
actions of these compounds on tubulin and human DNA
topoisomerase II. Additional synthesis and in vivo antitumor
activity studies are also currently in progress and will be re-
ported in the near future.
the next step without further purification.
General Procedure for the Synthesis of 6a—c and 6f—j Substituted
indole 5 (5.0 mmol) was dissolved in 20 ml Et2O and cooled to 0 °C. To this
mixture, 0.58 ml oxalyl chloride (6.5 mmol) was then added drop wise with
stirring so that the temperature did not rise above 5 °C. After an additional
3—6 h stirring at room temperature, the resulting solid was filtered, washed
with 10 ml Et2O twice and dried under vacuum for 24 h at 25—30 °C. The
title compounds 6a—c and 6f—j were obtained respectively and could be
used directly for the next step without further purification.
General Procedure for the Synthesis of 7a—c 0.42 g podophyllotoxin
1 (1.0 mmol), 0.28 ml Et3N (2.0 mmol) and 5 mg DMAP were dissolved in
5 ml anhydrous DMF and cooled to 0 °C. A solution of 6 (2.0 mmol) in
15 ml anhydrous DMF was added drop wise with stirring so that the temper-
ature stayed below 5 °C. The mixture was stirred at room temperature for
36 h, and then extracted with dichloromethane (40 mlꢁ3) after 30 ml water
was added. The organic phase was washed with water, dried over Na2SO4
and concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel using petroleum ether/ethyl acetate as eluent to produce
7a—c.
4a-((1-(4-Chlorobenzyl)-indol-3-yl)-glyoxyl)-podophyllotoxin (7a): Yield
was found to be 51%, mp 147.8 °C (d). [a]D25 ꢂ69.0° (cꢃ0.10, CHCl3). H-
1
NMR (CDCl3) d ppm: 2.99—3.02 (2H, m), 3.77 (6H, s), 3.81 (3H, s), 4.29
(1H, t, Jꢃ9.5 Hz), 4.44—4.48 (1H, m), 4.65 (1H, d, Jꢃ3.6 Hz), 5.38 (2H, s),
5.98 (2H, d, Jꢃ1.3 Hz), 6.06 (1H, d, Jꢃ8.3 Hz), 6.44 (2H, s), 6.58 (1H, s),
6.89 (1H, s), 7.12 (2H, d, Jꢃ8.4 Hz), 7.30—7.39 (5H, m), 8.39 (1H, s), 8.44
(1H, d, Jꢃ7.1 Hz). ESI-MS m/z: ꢄESI 732.2 (MꢄNa). HR-ESI-MS m/z:
732.1597 (Calcd for C39H32NO10ClNa: 732.1612).
4a-((1-(3,4-Dichlorobenzyl)-indol-3-yl)-glyoxyl)-podophyllotoxin (7b):
Experimental
Yield was found to be 47%, mp 116.8 °C (d). [a]D25 ꢂ79.0° (cꢃ0.10,
General All materials and reagents were obtained from commercial
sources and used without further purification unless stated. Podophyllotoxin
was purchased from the Huike Corporation of Shanxi province, China. Et2O
was distilled from sodium-benzophenone. Melting points were determined
on a BÜCHI Melting Point B-540 (Büchi Labortechnik) and were uncor-
rected. The 1H-NMR spectra were recorded using a BRUKER ARX-300 in-
strument at 300 MHz spectrophotometer with tetramethylsilane (TMS) as
the internal standard. The coupling constants (J) are reported in Hz. Mass
spectra data were obtained on a Quattro MicroTM API mass spectrometer
(Waters, Milford, MA, U.S.A.) and a high resolution ESI-FTICR mass spec-
trometry (Ionspec 7.0T). Optical rotations were measured at room tempera-
ture with a Perkin-Elmer 241 MC polarimeter at the sodium D line.
General Procedure for the Synthesis of 5a—c Indole 4 (1.19 g,
10.0 mmol) in anhydrous 5 ml DMF was added slowly to a solution of 70%
NaH (0.69 g, 20 mmol) in anhydrous 10 ml DMF at 0 °C, and the mixture
was subsequently stirred for 5 h at room temperature. The mixture was cooled
to 0 °C and the aralkyl chloride (12.0 mmol) was added to the mixture at one
1
CHCl3). H-NMR (CDCl3) d ppm: 2.94—3.10 (2H, m), 3.77 (6H, s), 3.81
(3H, s), 4.29 (1H, t, Jꢃ9.5 Hz), 4.45—4.51 (1H, m), 4.64 (1H, d, Jꢃ3.7 Hz),
5.36 (2H, s), 5.98 (2H, s), 6.06 (1H, d, Jꢃ8.3 Hz), 6.44 (2H, s), 6.57 (1H, s),
6.89 (1H, s), 6.98 (1H, dd, Jꢃ1.8, 6.5 Hz), 7.27—7.43 (5H, m), 8.40 (1H, s),
8.45 (1H, d, Jꢃ7.2 Hz). ESI-MS m/z: ꢄESI 766.1 (MꢄNa). HR-ESI-MS
m/z: 766.1215 (Calcd for C39H31NO10Cl2Na: 766.1223).
4a-((1-(Benzo[1,3]dioxol-5-ylmethyl)-indol-3-yl)-glyoxyl)-podophyllo-
toxin (7c): Yield was found to be 53%, mp 110.3 °C (d). [a]D25 ꢂ80.0°
1
(cꢃ0.10, CHCl3). H-NMR (CDCl3) d ppm: 2.94—3.07 (2H, m), 3.76 (6H,
s), 3.80 (3H, s), 4.27 (1H, t, Jꢃ9.5 Hz), 4.40—4.47 (1H, m), 4.64 (1H, d,
Jꢃ3.7 Hz), 5.28 (2H, s), 5.94 (2H, s), 5.97 (2H, d, Jꢃ1.4 Hz), 6.05 (1H, d,
Jꢃ8.3 Hz), 6.43 (2H, s), 6.56 (1H, s), 6.65 (1H, s), 6.69—6.78 (2H, m), 6.90
(1H, s), 7.34—7.40 (3H, m), 8.30 (1H, s), 8.40—8.44 (1H, m). ESI-MS
m/z: ꢄESI 742.2 (MꢄNa). HR-ESI-MS m/z: 742.1891 (Calcd for
C40H33NO12Na: 742.1900).
General Procedure for the Synthesis of 7d—j The title compounds

834
Vol. 56, No. 6
were synthesized by adopting the same procedure as for 7a—c from 4b-
amino podophyllotoxin 3.
Jꢃ2.9 Hz), 9.20 (1H, s), 9.40 (1H, s). ESI-MS m/z: ꢄESI 637.2 (MꢄNa).
HR-ESI-MS m/z: 637.1787 (Calcd for C33H30N2O10Na: 637.1798).
Cell Assay Cells were grown in RPMI-1640 medium supplemented
4b-((1-(4-Chlorobenzyl)-indol-3-yl)-glyoxylamide)-4-desoxypodophyllo-
toxin (7d): Yield was found to be 56%, mp 150.9 °C (d). [a]D25 ꢂ80.5° with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin and were
1
(cꢃ0.10, CHCl3). H-NMR (CDCl3) d ppm: 3.03—3.09 (2H, m), 3.73 (6H, incubated in a 5% CO2 humidified atmosphere.
s), 3.79 (3H, s), 3.88 (1H, t, Jꢃ9.7 Hz), 4.39—4.46 (2H, m), 5.24 (1H, dd,
The cytotoxicity of compounds 7a—j against HeLa and SKOV3 cell lines
Jꢃ4.0, 7.8 Hz), 5.38 (2H, s), 5.98 (2H, s), 6.19 (1H, s), 6.24 (2H, s), was evaluated by the MTT method in vitro. Cells were plated at a density of
6.78 (1H, s), 7.15 (2H, d, Jꢃ8.3 Hz), 7.28—7.36 (4H, m), 7.42 (1H, d, 4000 cells per well in a 96-well plate and incubated for 24 h. The cells were
Jꢃ7.8 Hz), 8.03 (1H, d, Jꢃ7.9 Hz), 8.39 (1H, d, Jꢃ7.8 Hz), 9.01 (1H, s).
ESI-MS m/z: ꢄESI 731.2 (MꢄNa). HR-ESI-MS m/z: 731.1765 (Calcd for
C39H33N2O9ClNa: 731.1772).
then treated with increasing concentrations of tested compounds and grown
for 4 d. 100 ml MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bro-
mide) at a concentration of 1.0 mg/ml in nutrient medium was added and
4b-((1-(Benzo[1,3]dioxol-5-ylmethyl)-indol-3-yl)-glyoxylamide)-4-des- cells were allowed to incubate for a further 4 h. Then, 150 ml DMSO was
oxypodophyllotoxin (7e): Yield was found to be 58%, mp 151.2 °C (d). added to each well and the absorbance of samples were measured at 490 nm.
[a]D25 ꢂ92.0° (cꢃ0.10, CHCl3). H-NMR (CDCl3) d ppm: 3.01—3.08 (2H, The IC50 values were calculated according to Logit method after getting the
1
m), 3.75 (6H, s), 3.81 (3H, s), 3.83—3.89 (1H, m), 4.41—4.47 (1H, m), 4.58 inhibitory rate.
(1H, d, Jꢃ3.6 Hz), 5.24 (1H, dd, Jꢃ3.6, 8.0 Hz), 5.31 (2H, s), 5.91 (2H, s),
The quantitative SRB (sulphorhodamine B) colorimetric assay was used
5.98 (2H, d, Jꢃ4.6 Hz), 6.30 (2H, s), 6.45 (1H, s), 6.68 (1H, s), 6.72—6.80 to determine the cytotoxic effect of compounds 7a—j on K562 and
(3H, m), 7.31—7.40 (3H, m), 7.82 (1H, d, Jꢃ7.9 Hz), 8.39 (1H, d, K562/ADR cell lines. Cells were seeded at 8000 per well in a 96-well plate
Jꢃ7.1 Hz), 9.00 (1H, s). ESI-MS m/z: ꢄESI 741.2 (MꢄNa). HR-ESI-MS and grown for 24 h. After treating with increasing concentrations of tested
m/z: 741.2048 (Calcd for C40H34N2O11Na: 741.2060).
compounds, cells were incubated for 4 d. At the end of the incubation, cells
4b-((5-Bromo-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin (7f): were fixed with 80% trichloracetic acid (1 h at 4 °C) and then stained for
Yield was found to be 43%, mp 179.3 °C (d). [a]D25 ꢂ91.5° (cꢃ0.10, 30 min at room temperature with 100 ml SRB solution (0.4% w/v) in 1%
1
CHCl3). H-NMR (CDCl3) d ppm: 3.05—3.07 (2H, m), 3.75 (6H, s), 3.81 acetic acid. SRB was then removed and cells were quickly rinsed five times
(3H, s), 3.84—3.92 (1H, m), 4.43—4.48 (1H, m), 4.57 (1H, d, Jꢃ3.8 Hz),
5.25 (1H, d, Jꢃ6.4 Hz), 5.98 (2H, s), 6.31 (3H, s), 6.77 (1H, s), 7.32 (1H, d,
Jꢃ8.6 Hz), 7.42 (1H, dd, Jꢃ1.8, 8.6 Hz), 7.87 (2H, d, Jꢃ7.7 Hz), 8.49 (1H,
with 1% acetic acid. After air-drying, protein-bound dye was dissolved in
150 ml of 10 mM unbuffered Tris base (pH 10.5) for 5 min. The pink SRB
was quantified by measuring the optical density at 540 nm. Then, values of
d, Jꢃ1.4 Hz), 9.00 (1H, d, Jꢃ3.2 Hz), 9.63 (1H, s). ESI-MS m/z: ꢄESI GI50 were calculated.
685.1 (MꢄNa). HR-ESI-MS m/z: 685.0790 (Calcd for C32H27N2O9BrNa:
685.0798).
Acknowledgements The authors gratefully thank the Great Program of
4b-((2-Methyl-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin (7g): Science Foundation of Tianjin (06YFJZJCO2700) and the Science Founda-
Yield was found to be 46%, mp 175.1 °C (d). [a]D25 ꢂ105.5° (cꢃ0.10, tion of Chinese People’s Armed Police Forces (WKH2005-6) for financial
1
CHCl3). H-NMR (CDCl3) d ppm: 2.76 (3H, s), 2.93—3.09 (2H, m), 3.76 support of this research.
(6H, s), 3.82 (3H, s), 3.96 (1H, t, Jꢃ9.6 Hz), 4.45—4.52 (1H, m), 4.61 (1H,
d, Jꢃ4.0 Hz), 5.34 (1H, dd, Jꢃ4.3, 7.7 Hz), 5.98 (2H, d, Jꢃ6.9 Hz), 6.32
(2H, s), 6.54 (1H, s), 6.80 (1H, s), 7.20—7.33 (4H, m), 8.17 (1H, d,
Jꢃ6.9 Hz), 8.75 (1H, s). ESI-MS m/z: ꢄESI 621.2 (MꢄNa). HR-ESI-MS
m/z: 621.1842 (Calcd for C33H30N2O9Na: 621.1849).
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3) Wessel I., Jensen P., Falck J., Mirski S., Cole S., Sehested M., Cancer
Res., 57, 4451—4454 (1997).
4b-((6-Fluoro-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin (7h):
Yield was found to be 48%, mp 185.1 °C (d). [a]D25 ꢂ93.0° (cꢃ0.10,
1
CHCl3). H-NMR (CDCl3) d ppm: 3.02—3.06 (2H, m), 3.75 (6H, s), 3.82
(3H, s), 3.86—3.92 (1H, m), 4.43—4.49 (2H, m), 5.26 (1H, d, Jꢃ6.7 Hz),
5.98 (2H, s), 6.28 (2H, s), 6.34 (1H, s), 6.78 (1H, s), 7.09—7.17 (2H, m),
7.84 (1H, d, Jꢃ7.8 Hz), 8.30 (1H, dd, Jꢃ5.5, 8.1 Hz), 9.02 (1H, d,
Jꢃ2.9 Hz), 9.35 (1H, s). ESI-MS m/z: ꢄESI 625.2 (MꢄNa). HR-ESI-MS
m/z: 625.1589 (Calcd for C32H27N2O9FNa: 625.1598).
4) Lee K. H., Imakura Y., Haruna M., Beers S. A., Thurston L. S., Dai H.
J., Chen C. H., Liu S. Y., Cheng Y. C., J. Nat. Prod., 52, 606—613
(1989).
5) Zhou X. M., Wang Z. Q., Chang J. Y., Chen H. X., Cheng Y. H., Lee
K. H., J. Med. Chem., 34, 3346—3350 (1991).
4b-((5-Methoxy-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin
6) Lee K. H., Beers S. A., Mori M., Wang Z., Kuo Y., Li L., Liu S.,
Cheng Y., Han F., Cheng Y., J. Med. Chem., 33, 1364—1368 (1990).
7) Wang Z., Kuo Y., Schnur D., Bowen J. P., Liu S., Hen F., Chang J.,
Cheng Y., Lee K. H., J. Med. Chem., 33, 2660—2666 (1990).
8) Tawa R., Takami M. J., Imakura Y. J., Lee K. H., Sakuri H., Bioorg.
Med. Chem. Lett., 7, 489—494 (1997).
9) Xiao Z., Xiao Y. D., Golbraikh A., Tropsha A., Lee K. H., J. Med.
Chem., 45, 2294—2309 (2002).
10) Bacher G., Nickel B., Emig P., Vanhoefer U., Seeber S., Shandra A.,
Klenner T., Beckers T., Cancer Res., 61, 392—399 (2001).
(7i): Yield was found to be 45%, mp 174.6 °C (d). [a]D25 ꢂ100.5° (cꢃ0.10,
1
CHCl3). H-NMR (CDCl3) d ppm: 2.95—3.09 (2H, m), 3.76 (6H, s), 3.82
(3H, s), 3.84—3.92 (1H, m), 3.89 (3H, s), 4.43—4.49 (1H, m), 4.61 (1H, d,
Jꢃ4.1 Hz), 5.26 (1H, dd, Jꢃ4.0, 7.7 Hz), 5.98 (2H, d, Jꢃ4.4 Hz), 6.31 (2H,
s), 6.48 (1H, s), 6.78 (1H, s), 6.97 (1H, dd, Jꢃ2.4, 8.8 Hz), 7.35 (1H, d,
Jꢃ8.8 Hz), 7.76 (1H, d, Jꢃ7.8 Hz), 7.88 (1H, d, Jꢃ2.3 Hz), 9.00 (2H, s).
ESI-MS m/z: ꢄESI 637.2 (MꢄNa). HR-ESI-MS m/z: 637.1791 (Calcd for
C33H30N2O10Na: 637.1798).
4b-((6-Methoxy-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin
(7j): Yield was found to be 50%, mp 170.2 °C (d). [a]D25 ꢂ102.5° (cꢃ0.10, 11) Dumontet C., Exp. Opin. Invest. Drugs, 9, 779—788 (2000).
1
CHCl3). H-NMR (CDCl3) d ppm: 3.03—3.07 (2H, m), 3.74 (6H, s), 3.81 12) Burkhart C. A., Kavallaris M., Horwitz S. B., Biochim. Biophys. Acta
(3H, s), 3.86 (3H, s), 3.89—3.95 (1H, m), 4.39—4.47 (2H, m), 5.24 (1H, d,
Rev. Cancer, 1471, O1—O9 (2001).
Jꢃ4.5 Hz), 5.97 (2H, s), 6.26 (2H, s), 6.77 (1H, s), 6.94 (1H, s), 7.03 (1H, d, 13) Chen S. Y., Yu Y. P., You J. Z., Chen Y. Z., Chem. J. Chin. Univ., 7,
Jꢃ8.7 Hz), 7.93 (1H, d, Jꢃ7.8 Hz), 8.22 (1H, d, Jꢃ8.8 Hz), 8.92 (1H, d,
1064—1066 (2000).