D Ye et aL/ Bioorg. Med. Chem. 20(2012)4489-4494 form the TLd complex. Therefore, the E-ring lactone of CPT-deriv- ON5 P-GAAAAAAGACTTGG CTTTTTTCTGGGCCTTTTTAAAAAT-p However, in a recent study, a CPt analog with a stable five-mem bered E-ring ketone, not lactone, moiety retained the ability to generate topo I-mediated DNA breaks. .In addition, in the pres ent study, activity was inhibited by conjugates lE and 2E bearing modified open E-ring. although they were less potent than CPT conjugates with an intact E-ring, suggesting that an open E-ring moiety in CPT-epipodophylloxin conjugates enables to retain inhibition of topo I religation. When the religation process is com- pleted, topo I dissociates from the DNA. Conceivably, the open E-ring CPT conjugates could have lower affinity towards the en- Substrate zyme. Therefore, the conjugate-enzyme complex could dissociate more readily, and hence the religation process could be facilitated. S C CPT 1 1E 2 2E VP However, further investigation including extensive Sar study is merited and other modes of action cannot be ruled out 100% topo ll activity, a previously described in vitro DNA cleavage assay was performed, with etoposide(vp-16)as the positive control.At concentrations up to 25 HM, none of the conjugates induced DNA cleavage(Fig. 5). However, at a high concentration(250 HM),con- jugates 1E, 2, and 2E interrupted topo Il-mediated religation to di ferent degrees. Thus, the conjugates could also interact with topo ll to cause PLDB and cytotoxicity. Given that etoposied resistant cells (KB-7D)were sensitive to CPt as well as the conjugates, it is antic Figure 3. Impact of CPr-e xin conjugates on topoisomerase I cleav- ipated that further structural modification of these compound ge activity. (A) Schematic diagram of the cleavage assay (B).1rPl-ATP-labelet could lead to the development of topoisomerase ll inhibitors that 37.C for 15 min. Covalent-lin! merase I was digested by proteinase k and can overcome resistance due to the expression of MRP he cleavage products(containing topoisomerase I amino acid residues on the 3 Overall, our current and prior findings indicate that this novel end of the cleaved strand)were separated by 20% denaturing urea/polyacrylamide class of CPT-epipodophyllotoxin conjugates exhibit antitumor ef- fects against bot inhibiting topo I-mediated, and possibly topo ll-mediated, relega tion. However, additional mechanisms are anticipated based on p-GAAAAAAGACTTGG GGAAAAATTTTTA ON6 TCTGGGCCTTTTTAAAAAT-p 7525025752502575250257525025conc.uM) 2583280.9 52 ligation product 01g §e06 。2 CPT CPT CPT CPT Conc(uM)2583280.92525252525 Figure 4. Inhibition of TLD religation by CPT-epi 5 pmol of oN6 to the suicide cleavage complex in the presence of indicated mpounds at 37C for 30 min. Samples were analyzed by 20% urea/polyacryl amide gel electrophoresis and quantified by phosphoimager open E-ring conjugates(1Eand 2E)were approximately 9-and 2 fold lower, respectively. Thus, an intact E-ring was not essential for the inhibition of TLD relegation, but did affect the degree of inhibi tion. The results from the tld assay are consistent with those in Figure the PLDB assay, indicating that dna breaks induced by the conju- induced DNA cleavage. Linearized an eled pBr322 was gates could be due to inhibition of the tld religation step with 10U topoisomerase Il and th CPT's indolizinoquinoline moiety is believed to be involved in Samples were proteinase K-treated, s DNA interaction, while the lactone E-ring interacts with topo I to ography. The same figure with higher contrast is shown in the lower panel.open E-ring conjugates (1Eand 2E) were approximately 9- and 25- fold lower, respectively. Thus, an intact E-ring was not essential for the inhibition of TLD relegation, but did affect the degree of inhibi￾tion. The results from the TLD assay are consistent with those in the PLDB assay, indicating that DNA breaks induced by the conju￾gates could be due to inhibition of the TLD religation step. CPT’s indolizinoquinoline moiety is believed to be involved in DNA interaction, while the lactone E-ring interacts with topo I to form the TLD complex. Therefore, the E-ring lactone of CPT-deriv￾atives is commonly thought to be essential for topo I inhibition. However, in a recent study, a CPT analog with a stable five-mem￾bered E-ring ketone, not lactone, moiety retained the ability to generate topo I-mediated DNA breaks.25,32 In addition, in the pres￾ent study, activity was inhibited by conjugates 1E and 2E bearing a modified open E-ring, although they were less potent than CPT conjugates with an intact E-ring, suggesting that an open E-ring moiety in CPT-epipodophylloxin conjugates enables to retain inhibition of topo I religation. When the religation process is com￾pleted, topo I dissociates from the DNA. Conceivably, the open E-ring CPT conjugates could have lower affinity towards the en￾zyme. Therefore, the conjugate-enzyme complex could dissociate more readily, and hence, the religation process could be facilitated. However, further investigation including extensive SAR study is merited and other modes of action cannot be ruled out. To determine whether the conjugate compounds could impact topo II activity, a previously described in vitro DNA cleavage assay was performed, with etoposide (VP-16) as the positive control.22 At concentrations up to 25 lM, none of the conjugates induced DNA cleavage (Fig. 5). However, at a high concentration (250 lM), con￾jugates 1E, 2, and 2E interrupted topo II-mediated religation to dif￾ferent degrees. Thus, the conjugates could also interact with topo II to cause PLDB and cytotoxicity. Given that etoposied resistant cells (KB-7D) were sensitive to CPT as well as the conjugates, it is antic￾ipated that further structural modification of these compounds could lead to the development of topoisomerase II inhibitors that can overcome resistance due to the expression of MRP. Overall, our current and prior findings indicate that this novel class of CPT-epipodophyllotoxin conjugates exhibit antitumor ef￾fects against both CPT-sensitive and -resistant cells, in part, by inhibiting topo I-mediated, and possibly topo II-mediated, relega￾tion. However, additional mechanisms are anticipated based on the assay data. For example, conjugates 1E and 2E were 10-fold 1 1E 2 2E VP 0 25 75 250 25 75 250 25 75 250 25 75 250 25 Conc. (µM) Substrate Cleavage Products Cleavage Products Figure 5. The impact of CPT-epipodophyllotoxin conjugates on topoisomerase II￾induced DNA cleavage. Linearized and [a- 32P]dCTP-labeled pBR322 was incubated with 10U topoisomerase II and the indicated compounds at 37 C for 30 min. Samples were proteinase K -treated, substrates and cleavage products (as indicated with arrows) were electrophoresed in 1.5% agarose gel and detected by autoradi￾ography. The same figure with higher contrast is shown in the lower panel. (A) (B) Cleavage Products Substrate S C CPT 1 1E 2 2E VP 0% 50% 100% 150% CPT 1 1E 2 2E VP Inhibition of cleavage (Percent of control) Figure 3. Impact of CPT-epipodophyllotoxin conjugates on topoisomerase I cleav￾age activity. (A). Schematic diagram of the cleavage assay. (B). [c32P]-ATP-labeled ON4/ON5 was incubated with topoisomerase I and indicated compounds (25 lM) at 37 C for 15 min. Covalent-linked topoisomerase I was digested by proteinase K and the cleavage products (containing topoisomerase I amino acid residues on the 30 end of the cleaved strand) were separated by 20% denaturing urea/polyacrylamide gel electrophoresis. (A) (B) Conc (µM) 25 8.3 2.8 0.9 25 25 25 25 25 C CPT 1 1E 2 2E VP Conc (µM) 25 8.3 2.8 0.9 25 25 25 25 25 Religation product 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CPT CPT CPT CPT 1 1E 2 2E VP Religation product (percent of control) Figure 4. Inhibition of TLD religation by CPT-epipodophyllotoxin conjugates. (A). Schematic diagram of the religation assay. (B). Religation was done by incubating 5 pmol of ON6 to the suicide cleavage complex in the presence of indicated compounds at 37 C for 30 min. Samples were analyzed by 20% urea/polyacryl￾amide gel electrophoresis and quantified by phosphoimager. 4492 D. Ye et al. / Bioorg. Med. Chem. 20 (2012) 4489–4494