D Ye et aL/ Bioorg. Med. Chem. 20(2012)4489-4494 (m, 2H, 11-H and NCH), 4.45(t,J=8.0 HZ, 1H, 11-H), 4.81 (d, plex generated by cleavage reaction by adding 5 pmol of oN6 J=5.0 Hz, 1H, I-H), 4.93(s, 2H, 22-H, 4-H), 5.20(s, 2H, 5-H).(Fig 4A). The reaction was performed at 37C for 30 min prior to 5.82(s, 2H, OCH2 O), 6.41(5, 2H, 2, 6-H). 6. 47(S, 1H, 8-H), 6.70 urea/polycrylamide gel electophoresis (s,1H,5-H).6.76(d,J=8.0Hz,1H,2"-H),6.89(m,1H,5"-H).7.36 (m, 2H, 7.59, 3"-H, 4"-H), 7.59, 7.65(t, J-8.0 Hz, each 1H, 10-H, 5.4.5. DNA cleavage by topoisomerase ll 11m-H,744(s,1H,14-H,7.99(d,J=85Hz,1H,9"-H).8.03 Uniquely end-labeled pBR322 DNA, prepared as described pre- (m, 1H, 14-H),8.22(d, J-85Hz, 1H, 12 -H), 9.20(s, 1H, N=CH). viously. 2 was incubated with topoisomerase l(USB, Cleveland, C NMR(400 MHZ, CDCI3):87.89(CH3): 22.76(isopropyl OH)and compounds in 1x reaction buffer(USB, Cleveland, OH)at CH3x 2): 32.42(CH2): 38.91(CH): 41.65(CH): 43.85(CH): 46.74 37C for 15 min. The reaction was terminated with 1% SDS and (isopropyl CH): 52. 50(CH2): 5656(OCH3 x 2): 58.07( CH2OH): subjected to proteinase K digestion before gel thein D-ring CH): 101.55(OCH 20): 108.38(epipodopart(campto- The gel was dried and autoradiographed for 48h ring CHx 2): 110.52 (epipodophyllotoxin B-ring CH): 110.89 (epipodophyllotoxin B-ring CH): 117. 80(aniline CH): 122.76(cam- ptothecin D-ring C): 124.59(aniline CH): 125.25 (aniline CH); This work was partly supported by NIH grant 125.58(camptothecin A-ring CH): 126. 28(camptothecin A-ring awarded to K.H. L and an National Foundation for Cancer thecin a-ring CH): 129.76(quinolone C): 130.26(camptothecin as well as NIh grants CA63477 and CA154295-01A1 fror rom the ni A-ring CH): 130.67(epipodophyllotoxin B-ring C): 131.37(epipod- the national foundation for cancer research ophyllotoxin B-ring C): 132.81(aniline C): 134.22(epipodophyllo toxin E-ring C): 134.78(epipodophyllotoxin E-ring C): 137.39 B-ring C): 148.36(epipodophyllotoxin B-ring C): 148.94(epist References and notes ): 146.65(camptothecin C-ring C): 147. 54 (epipodophyllot 1. Wall, M. E: Wani, M. C. Cook, C. E: Palmer, K H. McPhail, A T: Sim, G..Am 2. Hsiang, Y H. Hertzberg R: Hecht, S; Liu, L F.. BioL Chem. 1985, 260, 14873. 152.37(camptothecin D-ring C): 152.70(quinolone C): 154.77 3. Wang. J. C.J. MoL BioL 1971, 55 R P: Kingsbury, w.D. 176.35 (amide C=0): HRMs (m/z): C51HsoNsO11 calculated Boehm, J. C: Caranfa, M.-; Holden, K. G. Proc. Am. Assoc Cancer Res. 1989, 30, 9083501M+H; found:908.3515[M+H]' 6. Fukuoka, M: Niitani, H, Suzuki, A; Motomiya. M. Hasegawa, K; Nishiwaki, Kuriyam, T; Ariyoshi. Y: Negoro J. Clin OncoL. 1992, 10, 16. 5.4. Biolog 7. Rasheed, Z. A: Rubin, E H Oncogene 2003, 22. herfellah, D: Richard, S: Robert, ]-: Montaudon, D. British J 5. 4.1. Cell lines Beardmore. C: Liu. F. Cancer Res. 1990. 50. 6919. cell line KB CPT 100 were maintained in growth medium supple- 11 R: OConner, P. M. Kerrigan, D; Pommier, V. Eur J. Cancer 1992, 28, mented with 7 HM of etoposide and 100 nM of CPT, respectively Denny, W. A: Baguley, B C. Curr. Top Me 2003.3,1349 Revertant KB Cpt 100 ev cells also were used for studies, all cell lines were maintained in RPMI 1640 medium containing 10% fetal Marini, A M. Curr. Med. Chem 2010, 17 bovine serum 14. Perrin, D: van Hille, B: Barret. J. M. Kruczynski, A; Etievant, C. Imtert, L; Hill, T. Biochem. pharmacol 2 Pommier. Y. Cancer Res. 2007.. 9971 5. 4.2. Growth inhibition assay 6. Gellert, M.: Mizuuchi, K: O'Dea, M. H: Nash, H. A. Proc. Natl. Acad. Sci. U.S.A. 976.73.3872 Cells in logarithmic phase were cultured at a density of 17. Chen, G L; Yang, L; Rowe, T C Halligan, B D: Tewey K M: Liu, L FJBioL 5000 cells/mL in 24-well plates. The cells were exposed to various 18. Ross, W:Rowe, T: Glisson,B: Yalowich,J: Liu, L FCancer Res 1 oncentrations of drugs for72 h. The effect on cell growth was192xKCn出上Mdm测S:m evaluated using the ethylene blue dye assay, as described previ- C: Gurwith. M: Lee ously. Drug concentrations that inhibited 50% of cell growth 20. Lee, K.H.; Xiao, z In Anticancer Agents from Natural Products: Cragg. G. M (Cso) were determined 21. Bastow, K F; Wang, H K; Cheng. Y C: Lee, K H. Bioorg. Med. Chem. 1997, 5, 5.4.3. Measurement of protein-linked dNa breaks(PLDBs) 22. Chang, J C Guo. X; Chen, H. X; Jiang, Z; Fu, Q; Wang, H K: Bastow, K. F: Zhu, were treated with drugs for one h before the analysis for PLDBs 24. Hertzberg. R. P: Caranfa, M ]: Holden, K G. Jakas. D. R: Gallaghe, R. G by potassium-SDS CO-precipitation metho Mattern, M.R. Mong. S M.: Bartus, ]. O : Johnson, R K: Kingsbury, w. D. J. Med. 25. Li, Q. Y: Zu, Y G. Shi, R. Z; Yao, L P. Curr. Med. Chem. 2006. 13, 2021 5.4.4. Oligonucleotide cleavage and religation by topoisomerase 26. Fassbery. ]: Stella,V].).Pharm. Sci.1992.81,676 Purified recombinant te rase i was used to study the pact of drugs on the cleavage and religation steps, as described pre- 28. Sawada, S. Yaegashi, T; Furuta, T- Yokokura, T. Miyasaka, T. Chem Pha. 1993 ously. Briefly, 50 fmols of radiolabled ON5-ON4 oligoduplex 29. ParkSY (Fig. 3A)was incubated with topoisomerase I(220 fmol)in the 30. Rowe. T C: Chen, GL; Hsiang. Y. H. Liu, L F Cancer Res1986,46,2021. presence or absence of drugs at 37C for 15 min and stopped by 31. Park, S.Y. cheng Y C. cancer Res. 2005, 65. 389 adding SDS to final concentration 0.58. Covalent-linked Topo I 32. Daidieau x adaveaeo Ix, A: Leonce, S: Kraus-Berthier, L: Bal-Mahieu, C: Mazinghien, R: M. H: Hautefaye, P: Lavielle, G. Bailly. C was digested by proteinase K(1 mg/mL final concentration) fol lowed by denaturing urea/polyacrylamide gel electrophoresis. 33. Crugeiras, J-: Rios . Arm. Cherm Soc. 2009, 13, 1 The gel was analyzed by autoradiography and Phospholmaging 34. bon ks A. Lombardo, F; Bominy, B W. Feeney, P J. Adv Drug Deliv. Res. creen(Molecular Dynamics-Amersham Bioscience, Piscataway, 35. Montecucco, A: Pedrali-Noy, G: Spadari, S: Zanolin,E: Ciarrocchi, GNucleic ND). Religation reaction was started with the suicide cleavage com-(m, 2H, 11-H and NCH), 4.45 (t, J = 8.0 Hz, 1H, 11-H), 4.81 (d, J = 5.0 Hz, 1H, l-H), 4.93 (s, 2H, 22000-H, 4-H), 5.20 (s, 2H, 5000-H), 5.82 (s, 2H, OCH2O), 6.41 (s, 2H, 20 , 60 -H), 6.47 (s, 1H, 8-H), 6.70 (s, 1H, 5-H), 6.76 (d, J = 8.0 Hz, 1H, 200-H), 6.89 (m, 1H, 500-H), 7.36 (m, 2H, 7.59, 300-H, 400-H), 7.59, 7.65 (t, J = 8.0 Hz, each 1H, 10000-H, 11000-H), 7.44 (s, 1H, 14000-H), 7.99 (d, J = 8.5 Hz, 1H, 9000-H), 8.03 (m, 1H, 14000-H), 8.22 (d, J = 8.5 Hz, 1H, 12000-H), 9.20 (s, 1H, N@CH). 13C NMR (400 MHz, CDCl3): d 7.89 (CH3); 22.76 (isopropyl CH3 2); 32.42 (CH2); 38.91(CH); 41.65 (CH); 43.85 (CH); 46.74 (isopropyl CH); 52.50 (CH2); 56.56 (OCH3 2); 58.07 (CH2OH); 68.92 (CHNH); 76.70 (lactone CH2); 99.54 (–COH); 100.73 (campto￾thecin D-ring CH); 101.55 (OCH2O); 108.38 (epipodophyllotoxin E￾ring CH 2); 110.52 (epipodophyllotoxin B-ring CH); 110.89 (epipodophyllotoxin B-ring CH); 117.80 (aniline CH); 122.76 (cam￾ptothecin D-ring C); 124.59 (aniline CH); 125.25 (aniline CH); 125.58 (camptothecin A-ring CH); 126.28 (camptothecin A-ring CH); 127.85 (quinolone C); 128.28 (aniline CH); 129.42 (campto￾thecin A-ring CH); 129.76 (quinolone C); 130.26 (camptothecin A-ring CH); 130.67 (epipodophyllotoxin B-ring C); 131.37 (epipod￾ophyllotoxin B-ring C); 132.81 (aniline C); 134.22 (epipodophyllo￾toxin E-ring C); 134.78 (epipodophyllotoxin E-ring C); 137.39 (camptothecin A-ring C); 142.58 (quinolone C); 144.58 (aniline C); 146.65 (camptothecin C-ring C); 147.54 (epipodophyllotoxin B-ring C); 148.36 (epipodophyllotoxin B-ring C); 148.94 (epipodo￾phyllotoxin E-ring C); 149.67 (epipodophyllotoxin E-ring C); 152.37 (camptothecin D-ring C); 152.70 (quinolone C); 154.77 (amide C@O); 161.79 (imine C@N–); 173.22 (lactone C@O); 176.35 (amide C@O); HRMS (m/z): C51H50N5O11 calculated: 908.3501 [M+H]+ ; found: 908.3515 [M+H]+ . 5.4. Biology 5.4.1. Cell lines The etoposide-resistant cell line KB/7D and the CPT-resistant cell line KB CPT 100 were maintained in growth medium supple￾mented with 7 lM of etoposide and 100 nM of CPT, respectively. Revertant KB CPT 100rev cells also were used for studies. All cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum. 5.4.2. Growth inhibition assay Cells in logarithmic phase were cultured at a density of 5000 cells/mL in 24-well plates. The cells were exposed to various concentrations of drugs for 72 h. The effect on cell growth was evaluated using the ethylene blue dye assay, as described previ￾ously.22 Drug concentrations that inhibited 50% of cell growth (IC50) were determined. 5.4.3. Measurement of protein-linked DNA breaks (PLDBs) Cells were labeled with [14C]thymidine for 24 h. Labeled cells were treated with drugs for one h before the analysis for PLDBs by potassium-SDS co-precipitation method.35 5.4.4. Oligonucleotide cleavage and religation by topoisomerase I Purified recombinant topoisomerase I was used to study the im￾pact of drugs on the cleavage and religation steps, as described pre￾viously.35 Briefly, 50 fmols of radiolabled ON5-ON4 oligoduplex (Fig. 3A) was incubated with topoisomerase I (220 fmol) in the presence or absence of drugs at 37 C for 15 min and stopped by adding SDS to final concentration 0.5%. Covalent-linked Topo I was digested by proteinase K (1 mg/mL final concentration) fol￾lowed by denaturing urea/polyacrylamide gel electrophoresis. The gel was analyzed by autoradiography and PhosphoImaging screen (Molecular Dynamics-Amersham Bioscience, Piscataway, NJ). Religation reaction was started with the suicide cleavage com￾plex generated by cleavage reaction by adding 5 pmol of ON6 (Fig. 4A). The reaction was performed at 37 C for 30 min prior to urea/polycrylamide gel electophoresis. 5.4.5. DNA cleavage by topoisomerase II Uniquely end-labeled pBR322 DNA, prepared as described pre￾viously,22 was incubated with topoisomerase II (USB, Cleveland, OH) and compounds in 1x reaction buffer (USB, Cleveland, OH) at 37 C for 15 min. The reaction was terminated with 1% SDS and subjected to proteinase K digestion before gel electrophoresis. The gel was dried and autoradiographed for 48 h. Acknowledgments This work was partly supported by NIH grant CA17625, awarded to K.H.L. and an National Foundation for Cancer Research as well as NIH grants CA63477 and CA154295-01A1 from the Na￾tional Cancer Institute awarded to Y.C.C. Dr. Cheng is a Fellow of the National Foundation for Cancer Research. References and notes 1. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. J. Am. Chem. Soc. 1966, 88, 3888. 2. Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem. 1985, 260, 14873. 3. Wang, J. C. J. Mol. Biol. 1971, 55, 523. 4. Hsiang, Y. H.; Lihou, M. G.; Liu, L. F. Cancer Res. 1989, 49, 5077. 5. Johnson, P. K.; McCabe, F. L.; Faucette, L. F.; Hertzberg, R. P.; Kingsbury, W. D.; Boehm, J. C.; Caranfa, M. J.; Holden, K. G. Proc. Am. Assoc. Cancer Res. 1989, 30, 623. 6. Fukuoka, M.; Niitani, H.; Suzuki, A.; Motomiya, M.; Hasegawa, K.; Nishiwaki, Y.; Kuriyam, T.; Ariyoshi, Y.; Negoro, S.; Masuda, N. J. Clin. Oncol. 1992, 10, 16. 7. Rasheed, Z. A.; Rubin, E. H. Oncogene 2003, 22, 7296. 8. Pavillard, V.; Kherfellah, D.; Richard, S.; Robert, J.; Montaudon, D. British J. Cancer 2001, 85, 1077. 9. Pommier, V. Cancer Chem. Pharmacol. 1993, 32, 103. 10. D’Arpa, P.; Beardmore, C.; Liu, F. Cancer Res. 1990, 50, 6919. 11. Bertrand, R.; O’Conner, P. M.; Kerrigan, D.; Pommier, V. Eur. J. Cancer 1992, 28, 743. 12. Denny, W. A.; Baguley, B. C. Curr. Top. Med. Chem. 2003, 3, 1349. 13. Salerno, S.; Da Settimo, F.; Taliani, S.; Simorini, F.; La Motta, C.; Fornaciari, G.; Marini, A. M. Curr. Med. Chem 2010, 17, 4270. 14. Perrin, D.; van Hille, B.; Barret, J. M.; Kruczynski, A.; Etievant, C.; Imtert, I.; Hill, B. T. Biochem. Pharmacol. 2000, 59, 807. 15. Rao, V. A.; Agama, K.; Holbeck, S.; Pommier, Y. Cancer Res. 2007, 67, 9971. 16. Gellert, M.; Mizuuchi, K.; O’Dea, M. H.; Nash, H. A. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3872. 17. Chen, G. L.; Yang, L.; Rowe, T. C.; Halligan, B. D.; Tewey, K. M.; Liu, L. F. J. Biol. Chem. 1984, 259, 13560. 18. Ross, W.; Rowe, T.; Glisson, B.; Yalowich, J.; Liu, L. F. Cancer Res. 1984, 44, 5857. 19. Zhu, X. K.; Guan, J.; Tachibana, Y.; Bastow, K. F.; Cho, S. J.; Cheng, H. H.; Cheng, Y. C.; Gurwith, M.; Lee, K. H. J. Med. Chem. 1999, 42, 2441. 20. Lee, K. H.; Xiao, Z. In Anticancer Agents from Natural Products; Cragg, G. M.; Kingston, D.; Newman, D. J., Eds.; CRC Press, 2005; p 71. 21. Bastow, K. F.; Wang, H. K.; Cheng, Y. C.; Lee, K. H. Bioorg. Med. Chem. 1997, 5, 1481. 22. Chang, J. C.; Guo, X.; Chen, H. X.; Jiang, Z.; Fu, Q.; Wang, H. K.; Bastow, K. F.; Zhu, X. K.; Guan, J.; Lee, K. H.; Cheng, Y. C. Biochem. Pharmacol. 2000, 59, 497. 23. Adamovics, J. A.; Hutchinson, C. R. J. Med. Chem. 1979, 22, 310. 24. Hertzberg, R. P.; Caranfa, M. J.; Holden, K. G.; Jakas, D. R.; Gallaghe, R. G.; Mattern, M. R.; Mong, S. M.; Bartus, J. O.; Johnson, R. K.; Kingsbury, W. D. J. Med. Chem. 1989, 32, 715. 25. Li, Q. Y.; Zu, Y. G.; Shi, R. Z.; Yao, L. P. Curr. Med. Chem. 2006, 13, 2021. 26. Fassbery, J.; Stella, V. J. J. Pharm. Sci. 1992, 81, 676. 27. Burke, T. G.; Mi, Z. Anal. Biochem. 1993, 212, 285. 28. Sawada, S.; Yaegashi, T.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chem. Pharm. Bull. 1993, 41, 310. 29. Park, S. Y.; Lam, W.; Cheng, Y. C. Cancer Res. 2002, 62, 459. 30. Rowe, T. C.; Chen, G. L.; Hsiang, Y. H.; Liu, L. F. Cancer Res. 1986, 46, 2021. 31. Park, S. Y.; Cheng, Y. C. Cancer Res. 2005, 65, 3894. 32. Lansiaux, A.; Léonce, S.; Kraus-Berthier, L.; Bal-Mahieu, C.; Mazinghien, R.; Didier, S.; David-Cordonnier, M. H.; Hautefaye, P.; Lavielle, G.; Bailly, C.; Hickman, J. A.; Pierré, A. Mol. Pharmacol. 2007, 72, 311. 33. Crugeiras, J.; Rios, A.; Riveiros, E.; Richard, J. P. J. Am. Chem. Soc. 2009, 13, 15815. 34. Lipinski, C. A.; Lombardo, F.; Bominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Res. 2001, 46, 3. 35. Montecucco, A.; Pedrali-Noy, G.; Spadari, S.; Zanolin, E.; Ciarrocchi, G. Nucleic Acids Res. 1988, 16, 3907. 4494 D. Ye et al. / Bioorg. Med. Chem. 20 (2012) 4489–4494