D Ye et al /Bioorg. Med. Chem. 20 (2012)4489-4494 H3C HcO OCH3 Camptothecin(CPT) Etoposide(VP-16) op reflux. 22 h H3 OCH3 HcO OCH 1:S para 二 Figure 1. Structures of CPT, etoposide, and CPT-epipodophyllotoxin conjugates. hibition of tumor cell lines and physicochemical properties of CPt and conjugates ICso(nM BCPT100 KBCPT100 evb KB-7D 423(48) 24±0.5 08973° 24.5±4.5 4±6 5.37 42.5±2.5 120±20(28) 57±27 37±0 06±34(34) 531 475±25 336±55 374±62 55000 Cell lines: KB, nasopharyngeal; KBCPT100, CPT resistant KB; KBCPT100, partial revertant KBCPT100: KB-7D, MRP-expressing VP-16 resistant KB Values in parenthesis reflect the relative resistance over the parental cell line c Values are the average of two experiments. Data obtained from ChemBiodraw Ultra 12.0 f Not available (Fig 3A). The presence of two nucleotides downstream of the topo I cutting site prevents the religation process and allows the forma tion of a suicide tld complex after cleavage Similar amounts of formed in the presence of CPT and the conjugated derivatives at a concentration of 25 HM( Fig. 3B). This result implies that these compounds do not interfere with the dna cleavage activity of topo or by implication, with the binding of dNa to the enzym Religation was then initiated by the addition of a 13-mer (oN6 to the pre-generated suicide cleavage complex( Fig 4A). The topo I religation activity was determined by measuring the amount of the religation product (a 25-mer)formed, as previously described. re 2. Inductio B [CH-thymidine-labeled KB cells were incubated with CPT inhibited TLD religation in a dose HM of the indicated compounds at 37C for 1h. PLDB was analyzed by the reached >95% inhibition at 25 HM(Fig. depent mparison, the potassium/SDS method as described. inhibitory potencies of the intact E-ring conjugates(1 and 2)and(Fig. 3A). The presence of two nucleotides downstream of the topo I cutting site prevents the religation process and allows the forma￾tion of a suicide TLD complex after cleavage. Similar amounts of cleavage products (measured as described previously31) were formed in the presence of CPT and the conjugated derivatives at a concentration of 25 lM (Fig. 3B). This result implies that these compounds do not interfere with the DNA cleavage activity of topo I, or by implication, with the binding of DNA to the enzyme. Religation was then initiated by the addition of a 13-mer (ON6) to the pre-generated suicide cleavage complex (Fig. 4A). The topo I religation activity was determined by measuring the amount of the religation product (a 25-mer) formed, as previously described.31 CPT inhibited TLD religation in a dose-dependent manner and reached >95% inhibition at 25 lM ( Fig. 4B). In comparison, the inhibitory potencies of the intact E-ring conjugates (1 and 2) and O O O O OH H3CO OCH3 HN N N O O O HO N 1 4 5 8 3" 2' 2" 9'" 12'" 5'" 14'" 22'" 19'" 18'" S O O O O OH H3CO OCH3 HN N N O O HO N 1 4 5 8 3" 2' 2" 9'" 12'" 5'" 14'" 22'" 19'" 18'" S isopropylamine, CHCl3 reflux, 22 h H N OH 1:S= para- 2:S= ortho- 1E:S= para- 2E:S= ortho￾O O O O OH H3CO OCH3 O Etoposide (VP-16) O O O H3C HO OH Camptothecin (CPT) N N O O O HO A B C D E 1 2 3 4 6 5 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 14 17" A E A Figure 1. Structures of CPT, etoposide, and CPT-epipodophyllotoxin conjugates. Table 1 Growth inhibition of tumor cell lines and physicochemical properties of CPT and conjugates IC50 (nM)a LogPd LogSd KBb KBCPT100b KBCPT100revb KB-7Db CPT 8.8 ± 1.2 423 (48) 24 ± 0.5 6.8 ± 1.9 0.8973e —f 1 24.5 ± 4.5 95 ± 7 (3.9) 44 ± 6 37.5 ± 12 5.37 5.04 1E 42.5 ± 2.5 120 ± 20 (2.8) 57 ± 27 50c 5.091 5.2 2 37 ± 0 112 ± 22 (3.0) 70 ± 0 70 ± 10 5.37 5.04 2E 60 ± 21 206 ± 34 (3.4) 141 ± 31 61 ± 2 5.091 5.2 VP-16 475 ± 25 336 ± 55 374 ± 62 55,000c 0.02982e —f a Mean ± S.D. b Cell lines: KB, nasopharyngeal; KBCPT100, CPT resistant KB; KBCPT100rev, partial revertant KBCPT100; KB-7D, MRP-expressing VP-16 resistant KB. Values in parenthesis reflect the relative resistance over the parental cell line. c Values are the average of two experiments. d http://www.vcclab.org/lab/alogps/. e Data obtained from ChemBioDraw Ultra 12.0. f Not available. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 C CPT VP 1 1E 2 2E PLDB (Fold) Figure 2. Induction of PLDB. [14C]-thymidine-labeled KB cells were incubated with 2.5 lM of the indicated compounds at 37 C for 1 h. PLDB was analyzed by the potassium/SDS method as described. D. Ye et al. / Bioorg. Med. Chem. 20 (2012) 4489–4494 4491