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).
References
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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).
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4b-((5-Methoxy-indol-3-yl)-glyoxylamide)-4-desoxypodophyllotoxin
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(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).
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Jꢃ8.7 Hz), 7.93 (1H, d, Jꢃ7.8 Hz), 8.22 (1H, d, Jꢃ8.8 Hz), 8.92 (1H, d,
1064—1066 (2000).