J. Guan et al./Bioorg. Med. Chem. 6 (1998) 2127±2131
2131
[a]2d5 239.1 ꢀ (c 0.88, MeOH); 1H NMR (CD3OD) d
1.97 (3H, s, COCH3), 1.85±2.40 (4H, m, H-5,6), 4.53
(1H, m, H-7), 6.26 (1H, s, H-4), 7.26 (2H, d, J=7.9 Hz,
H-30,50), 7.46 (1H, s, H-8), 7.55 (1H, d, J=10.8 Hz, H-
11), 7.98 (2H, d, J=7.9 Hz, H-20,60), 9.07 (1H, d,
J=10.8 Hz, H-12); Anal. calcd (found) for C25H21
versus HTCL. HTCL panel cultured in RPMI-1640,
FBS 10% (v/v), and kanamycin (100 mg/mL). Sulforho-
damine B assays, triplicate dose treatment and three day
exposure were standard procedures. Based on pre-
screening results, compounds were further tested versus
HTCL to establish EC50 values.
.
N2O6 2H2O: C, 61.79 (61.59); H, 5.29 (4.92); N, 5.76
(5.50).
Acknowledgement
Tridemethyl-N-(40-cyanobenzoyl)-colchiceinamide (10a).
Yield 20% (starting from 513 mg of 10); amorphous;
[a]d25 295.9 ꢀ (c 1.2, MeOH); 1H NMR (CD3OD) d 1.97
(3H, s, COCH3), 1.8±2.4 (4H, m, H-5,6), 4.5 (1H, m, H-
7), 6.25 (1H, s, H-4), 7.45 (1H, s, H-8), 7.54 (1H, d,
J=10.9 Hz, H-11), 7.86 (2H, d, J=8.0 Hz, H-30,50), 8.05
(2H, d, J=8.0 Hz, H-20,60), 9.05 (1H, d, J=10.9 Hz, H-
This investigation was supported in part by grant CA-
17625 from the National Cancer Institute awarded to
K. H. Lee.
References and Notes
1. Guan, J.; Zhu, X. K.; Tachibana, Y.; Bastow, K. F.; Brossi,
A.; Hamel, E.; Lee, K. H. J. Med. Chem. 1998, 41(11), 1956.
.
12); Anal. calcd (found) for C26H21N3O6 1CH3
CO2C2H5: C, 64.02 (64.07); H, 5.37 (5.55); N, 7.22
(7.55).
2. Brossi, A.; Yeh, H. J. C.; Chrzanowska, M.; Wol, J.;
Hamel, E.; Lin, C. M.; Quin, F.; Suness, M.; Silverton. J.
Med. Res. Rev. 1988, 8, 77.
3. Lery, M.; Spino, M.; Read, S. E. Pharmacotherapy 1991, 11,
196.
DNA topoisomerase II catalytic assay
Inhibition of topoisomerase II catalytic activity was
examined using the standard P4 DNA unknotting
assay.17 The reaction mixture (20 mL) which contained
50 mM Hepes (pH 6.7), 50 mM KCl, 100 mM NaCl,
nuclease-free bovine serum albumin (50 mg/mL), 10 mM
MgCl2, 0.1 mM EDTA, 0.1 mM ATP, P4 DNA sub-
strate (0.26 mg), 1.25 U enzyme was incubated with or
without drugs as the initial stage of screening for
activity. The reaction mixture was incubated at 37 ꢀC
for 30 min and terminated by adding a stop solution
(2% SDS, 20% glycerol and 0.05% bromophenol blue).
These samples were loaded onto a 1% agarose gel
and electrophoresed at 50 V overnight with an ele-
trophoresis buer which contained 90 mM Tris boric
acid (pH 8.3) and 2.5 mM EDTA. At completion, the
gel was stained in a 0.5 mg/mL of ethidium bromide.
Then a photograph (Polaroid Type 667 ®lm) was taken
of the DNA bands visualized with ¯uorescence induced
by a long wavelength UV-lamp. One unit was de®ned as
the amount of enzyme which converted 50% of the
knotted DNA on the gel to the unknotted form in
30 min.
4. Caplan, Y. H.; Orlo, K. G.; Thompson, B. C. J. Anal.
Toxicol. 1980, 4, 153.
5. Staretz, M. E.; Hastie, S. B. J. Med. Chem. 1993, 36, 758.
6. Muzaar, A.; Brossi, A.; Lin, C. M.; Hamel, E. J. Med.
Chem. 1990, 33, 567.
7. Kerekes, P.; Brossi, A.; Flippen-Anderson, J. L.; Chignell,
C. F. Helv. Chim. Acta. 1985, 68, 571.
8. Li, D. H.; Zhang, S. K.; Hao, X. G.; Ma, K. S.; Tan, X. R.;
Wang, Z. L.; Li, N. K. Chinese. Med. J. 1980, 93, 188.
9. Bastow, K. F.; Tatematsu, H.; Sun, L.; Fukushima, Y.; Lee,
K. H. Bioorg. Med. Chem. 1993, 3, 227.
10. Bastow, K. F.; Tatematsu. H.; Bori, I. D.; Fukushima, Y.;
Sun, L.; Goz, B.; Lee, K. H. Bioorg. Med. Chem. Lett. 1993, 3,
1045.
11. Roca, J. TIBS 1995, 20, 156.
12. For a review on topoisomerase, see: Wang, J. C. Advances
in Pharmacology 1994, 29, 1.
13. Smith, P. J.; Soues, S. Br. J. Cancer-Suppl. 1994, 23, S47.
14. Ringel, I.; Bakshi, O.; Mellado, W.; Rawu, A.; Gibson, D.;
Katzhendler, J. Biochem. Pharmco. 1988, 37, 2489.
15. Esbolaev, E. O.; Aleksandrova, L. A.; Toibaeva, K. A.;
Aitkhozhina, N. A. Chem. Nat. Compd. 1992, 28, 325.
16. Skehan, P.; Storeng, R.; Scudiero, D.; Morks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Boyd,
M. R. J. Nat. Cancer Inst. 1990, 82, 1107.
Cytotoxicity assay
For testing, samples diluted in culture medium immedi-
ately prior to use were prescreened at 4, 2, and 1 mM
17. Bastow, K. F.; Tatematsu, H.; Sun, L.; Fukushima, Y.;
Lee, K. H. Bioorg. Med. Chem. 1993, 3, 227.