Antitumor agents 294. Novel E-ring-modified camptothecin–4b-anilino-40- O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents

Bioorganic Medicinal Chemistry 20(2012)4489-4494 Contents lists available at SciVerse Science Direct Bioorganic Medicinal Chemistry ELSEVIER journalhomepagewww.elsevier.com/locate/bmc Antitumor agents 294. Novel E-ring- modified camptothecin -4B-anilino O-demethyl-epipodophyllotoxin conjugates as DNa topoisomerase I inhibitors and cytotoxic agents Deyong Ye a, Qian Shia, * Chung-Hang Leung b . Seung-Whan Kim b, Shin-Young Park b, Elizabeth A. Gullen Zao Li Jiang Hao Zhu, Susan L. Morris-Natschke Yung-Chi Cheng b, * f Kuo-Hsiung Lee,,* tural Products Research Laboratories, UNC Eshelman School of pharmacy, University of North Carolina at Chapel Hill, Chapel Hill NC 27599-7568, epartment of pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA Department of chemistry, The Rutgers Center for Computational and integrative Biology, Rutgers University. 315 Penn St, Camden, N 08102, USA Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung Taiwan ARTICLE INFO ABSTRACT Two conjugates (I and 2)of camptothecin(CPT)and 4B-anilino-4'-0-demethylepipodophyllotoxin were eceived 3 February 2012 previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this cur rent study, two novel conjugates(lE and 2E) with an open E-ring in the CPr moiety were first synthe- Available online 19 May 2012 sized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT-derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo l-linked DNA(TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analog his lower religation potency resulted in decreased formation of protein-linked DNa breaks(PLDBs nd hence, less cell growth inhibition. In addition to their impact on topo l, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo Il-induced DNA cleavage. The novel structures of 1E and 2E pharmacological profiles and physicoche p ment of dual function topo I and ll inhibitors with improved may present scaffolds for further devel 2012 Elsevier Ltd. All rights reserved. 1 Introduction CPT-resistance can result from various cellular events, including inadequate accumulation of the drug, alteration of topo I resulting Camptothecin(CPT), an alkaloid isolated from the Chinese bush in decreased formation of protein-linked DNa breaks(PLDB), and Camptotheca acuminata(Nyssaceae) by Wall and co-workers in alterations in the cellular response to the topo l-CPT interaction. 1966, was the first identified specific inhibitor of DNa topoisc merase 1.2 Topoisomerase I(Topo I)cuts one strand of double- ten accompanied by a concomitant rise in the level of topo ll stranded DNA, relaxes the strands, and then re-anneals the expression and vice versa, which leads to the failure of clinical strands. CPT exhibits its cytotoxic activity by stabilizing the to therapies. I-DNA complex, which prevents religation and causes single Topoisomerase inhibitors with dual target specificity could pos- stranded breaks, a process that eventually leads to cell death. Sev- sibly overcome CPT-resistance. Dual target inhibitors would likely cral CPT-derivatives including topotecan and irinotecan( CPt- retain cytotoxic activity when resistance was acquired due to alter 11).5.6 have been used clinically for the treatment of colorectal, ation of only one drug target. However, the physical combination of ovarian and small cell lung cancers. However, the development CPT with drugs such as amsacrine and doxorubicin, which also of resistance to these compounds is still a critical clinical problem. interfere with DNA topoisomerases, often caused antagonistic cyto- toxic effects rather than synergistic activity -In this regards, single compound able to inhibit both topo I and topo ll may present ng authors.Te:+19198436325(Qs.J.tel+19199620066 he advantage of improving anti-topoisomerase activity with re- fax:+19199663893(KHL): tel:+12037857118: fax: +1 203 7129 (Y C C.). duced or avoiding adversities respect to the combination of two E-mail addresses: qshil@emailuncedu(Q Shi) yacheng@yale.edu(Y-C. Cheng lee@unc.edu(K-H. Lee). nhibitors. Many efforts have been made to discover and develop Leung, CH and Kim, S.w. contributed equally to this wor a powerful'magic bullet targeted to both enzymes, such as f Fellow of the National Foundation for Cancer Research. Tafluposide, Batracylin, 5 and other synthetic or natural products -0896/s- see front matter o 2012 Elsevier Ltd. All right l/dx doiorg/10. 1016 j. bmc. 2012.05.03

Antitumor agents 294. Novel E-ring-modified camptothecin–4b-anilino-40 - O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents Deyong Ye a , Qian Shi a,⇑ , Chung-Hang Leung b, , Seung-Whan Kim b, , Shin-Young Park b , Elizabeth A. Gullen b , Zao Li Jiang b , Hao Zhu c , Susan L. Morris-Natschke a , Yung-Chi Cheng b,⇑, , Kuo-Hsiung Lee a,d,⇑ aNatural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7568, USA bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA cDepartment of Chemistry, The Rutgers Center for Computational and Integrative Biology, Rutgers University, 315 Penn St., Camden, NJ 08102, USA d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan article info Article history: Received 3 February 2012 Revised 4 May 2012 Accepted 12 May 2012 Available online 19 May 2012 Keywords: Topoisomerase Cytotoxicity Camptothecin (CPT) Etoposide (VP-16) Epipodophyllotoxin Conjugates abstract Two conjugates (1 and 2) of camptothecin (CPT) and 4b-anilino-40 -O-demethylepipodophyllotoxin were previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this cur￾rent study, two novel conjugates (1E and 2E) with an open E-ring in the CPT moiety were first synthe￾sized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT-derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo I-linked DNA (TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analogs. This lower religation potency resulted in decreased formation of protein-linked DNA breaks (PLDBs), and hence, less cell growth inhibition. In addition to their impact on topo I, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo II-induced DNA cleavage. The novel structures of 1E and 2E may present scaffolds for further development of dual function topo I and II inhibitors with improved pharmacological profiles and physicochemical properties. 2012 Elsevier Ltd. All rights reserved. 1. Introduction Camptothecin (CPT), an alkaloid isolated from the Chinese bush Camptotheca acuminata (Nyssaceae) by Wall and co-workers in 1966,1 was the first identified specific inhibitor of DNA topoiso￾merase I.2 Topoisomerase I (Topo I) cuts one strand of double￾stranded DNA, relaxes the strands, and then re-anneals the strands.3 CPT exhibits its cytotoxic activity by stabilizing the topo I-DNA complex, which prevents religation and causes single stranded breaks, a process that eventually leads to cell death.4 Sev￾eral CPT-derivatives, including topotecan and irinotecan (CPT- 11),5,6 have been used clinically for the treatment of colorectal, ovarian and small cell lung cancers. However, the development of resistance to these compounds is still a critical clinical problem. CPT-resistance can result from various cellular events, including inadequate accumulation of the drug, alteration of topo I resulting in decreased formation of protein-linked DNA breaks (PLDB), and alterations in the cellular response to the topo I-CPT interaction.7 Furthermore, the acquisition of resistance to topo I inhibitors is of￾ten accompanied by a concomitant rise in the level of topo II expression and vice versa, which leads to the failure of clinical therapies. Topoisomerase inhibitors with dual target specificity could pos￾sibly overcome CPT-resistance. Dual target inhibitors would likely retain cytotoxic activity when resistance was acquired due to alter￾ation of only one drug target. However, the physical combination of CPT with drugs such as amsacrine and doxorubicin, which also interfere with DNA topoisomerases, often caused antagonistic cyto￾toxic effects rather than synergistic activity.8–11 In this regards, a single compound able to inhibit both topo I and topo II may present the advantage of improving anti-topoisomerase activity, with re￾duced or avoiding adversities respect to the combination of two inhibitors. Many efforts have been made to discover and develop a powerful ‘magic bullet’ targeted to both enzymes,12,13 such as Tafluposide,14 Batracylin,15 and other synthetic or natural products. 0968-0896/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2012.05.030 ⇑ Corresponding authors. Tel.: +1 919 843 6325 (Q.S.); tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.H.L.); tel.: +1 203 785 7118; fax: +1 203 785 7129 (Y.C.C.). E-mail addresses: qshi1@email.unc.edu (Q. Shi), yccheng@yale.edu (Y.-C. Cheng), khlee@unc.edu (K.-H. Lee). Leung, C.H. and Kim, S.W. contributed equally to this work. Fellow of the National Foundation for Cancer Research. Bioorganic & Medicinal Chemistry 20 (2012) 4489–4494 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

4490 D Ye et aL/ Bioorg. Med. Chem. 20(2012)4489-4494 But, to date, none has been used in the clinic. Conjugation of two 2. Chemistry drug molecules through covalent chemical bonds to generate a sin- gle drug molecule with dual functional topoisomerase inhibitory The lactone E-ring conjugates 1 and 2 were synthesized as activity has not been fully explored. Chemical conjugation of CPt scribed previously, and were the starting materials to synthesize with another chemotherapeutic topoisomerase inhibitor could still the open E-ring conjugates lE and 2E( Fig. 1). Conjugate 1 or 2 metabolism profile of a conjugate(as a monomolecule) may be flux for 22 h under nitrogen, and monitored by TLC(CH2Cl2 independent from those of the parent compounds alone or the MeOH=20: 1). The solvent was removed under vacuum, and the simultaneous physical (rather than chemical)combination of residue was purified by silica gel chromatography(CH2Cl2: MeOH the two parents. We hope through this strategy to develop new 100: 0-95: 5 as eluent)to afford the target compounds lE and 2E, drug candidates potentially inhibiting proliferation of CPT-resistant respectively, in 51-70% yield. We originally suspected that the five-membered lactone ring in the epipodophyllotoxin unit might Etoposide(VP-16), a prototypical epipodophyllotoxin, is another also undergo ring ning through amide formation. However widely used anticancer drug, which functions as a topoisomerase ll the major products obtained under the described conditions were nhibitor Topoisomerase ll (topo In)incises double-stranded dNa to the desired products. The structures of the final products were facilitate the passage of an intact duplex through the gap before identified from spectroscopic and other analytical data. Additional rejoining the cut DNA. Etoposide inhibits cell growth by stabiliz- physicochemical properties, such as logP and logS values, of the ing the topo II-DNA complex, resulting in double-stranded dna newly synthesized conjugates 1E and 2E were also evaluated to breaks and subsequent cell death. 7, 18 Previously, numerous epip- further understand the SAR of the new compounds odophyllotoxin derivatives were synthesized and reported as active po ll inhibitors.9,20 The design of compounds that could also 3 Results and discussion interact with topo ll, in addition to topo l, was attempted throug the chemical combination of topo I and topo ll inhibitors. Two The effects of the new conjugates(1 and 2)in which the topo I inhibitor camptothecin on the growth of KB and CPT-sensitive and resistant KB cell lines nd the topo ll inhibitor 4B-anilino-4-0-demethylepipodophyllo- were studied in a growth inhibition assay. 2 As shown in Table spectrum of cytotoxic activity against drug-resistant cell lines i d potent than C Podophyllotoxin conjugates(1,1E.2,2E)were less toxin were linked via a covalent imine bond were synthesized the four cpt- and evaluated for biochemical and biological activities." gainst KB cells; thus, conjugation reduced pe In earlier studies, the conjugates 1 and 2 displayed a broad tency Interestingly, an open E-ring did not significantly affect Both compounds inhibited topo I, but only 2 was active against were either as potent as or only slightly less potent than their lac- ompounds induced PLDB in KB cells and had similar in vitro the para-position of the aniline moiety in the epipodophyllotoxin, cytotoxicity. These results warrant the further molecular design were more potent than 2 and 2E, conjugated at the ortho-positic of CPT-epipodophyllotoxin conjugates as antitumor agents Prior structure-activity relationship(SAR)studies on CPT-deriv- resistance against etoposide-resistant KB-7D cells, which over atives indicated that the lactone E-ring is essential for both antitu- express MRP and down-regulate topo ll. 29 However, as expected mor and topo I inhibitory activities. The 20-hydroxy group of CPTs CPT-resistant KBCPT100 cells, which down-regulate topo Iand ntact lactone E-ring forms a hydrogen bond with the topo l-linked up-regulate X-ray repair cross-complementing gene I protein DNA(TLD)and, thus, stabilizes the complex 23-25 CPT can be rap (XRCC1). nd the conjugates. Th idly inactivated through E-ring lactone reversible hydrolysis at cytotoxic effects were partially restored in CPT partial reverting physiological conditions, leading to an inactive water soluble car- KBCPT100REV cells, because the XRCCl over-expression is re- oxylate, which binds readily to human serum albumin making versed. 29) The ICso of CPT increased by 48-fold in CPT-resistant the compound inaccessible for cellular uptake.2627 KBCPT100 cells compared with the sensitive cells. However, the However, E-ring-modified ester-amide CPT-derivatives have ICso values of the conjugates increased by only 2.8-to 3.9-fold. been reported to show high potency against L1210 tumors. The Thus, CPT-resistance was overcome by some extent by the conju to be necessary for significant activity. The isopropyl amide of a ative resistance over the parental cell line. The data suggest that CPT open E-ring analog was reported to be more stable under the topo I could be the primary molecular target of these conjugate experimental conditions and less likely than other amide analogs compounds in KB cells to convert to the E-ring closed lactone form(e.g, CPr)or subse- quently to the hydroxy-acid (E-ring open form). Consequently, PLDB formation was studied according to the method described it might bind less readily to human serum album. Thus, the biolog previously Consistent with previous find CPr induced ical activity derived from the isopropyl amide analog could be con- threefold greater PLDB compared with conjugates 1 and 2 sidered as the open E-rings, though it was not so reported (Fig. 2). The open E-ring conjugates lE and 2 showed fourfold less In addition, CPT-epipodophyllotoxin conjugates with an open induction compared with their intact E-ring congeners 1 and 2, E-ring isopropyl amide moiety have not been studied, and their respectively. Increasing the concentration of conjugate 2E from biological activity, especially anti-tumor activity through inhibi- 2. 5 to 22.5 uM induced a sevenfold increase in PLDB in 30 min, tion of topoisomerases, mechanism of action, as well as metabo- which is equivalent to 60% of the PLDB induced by 2.5 uM of CPt lism profile have not been evaluated In this regard, in order to (data not shown ). understand the role of the lactone E-ring in the conjugated com- DNA relaxation by topo I is a process th pounds, E-ning-modified derivatives(lE and 2E)of l and 2, respec of DNA to topoisomerase l, followed by cleavage and subsequent tively, were synthesized in this study and evaluated in comparison religation of the DNA. The impact of the CPt conjugate with their intact E-ring congeners for cytotoxicity against KB cells, individual steps was investigated. A DNA duplex, which including resistant cells, and for inhibition of topo I and topo Il. a 25-mer(ON4)and a 14-mer(ON5), was used as the

But, to date, none has been used in the clinic. Conjugation of two drug molecules through covalent chemical bonds to generate a sin￾gle drug molecule with dual functional topoisomerase inhibitory activity has not been fully explored. Chemical conjugation of CPT with another chemotherapeutic topoisomerase inhibitor could still be a relevant approach to generate a new monomolecule with possible dual target specificity. The mechanism of action and metabolism profile of a conjugate (as a monomolecule) may be independent from those of the parent compounds alone or the simultaneous physical (rather than chemical) combination of the two parents. We hope through this strategy to develop new drug candidates potentially inhibiting proliferation of CPT-resistant cells. Etoposide (VP-16), a prototypical epipodophyllotoxin, is another widely used anticancer drug, which functions as a topoisomerase II inhibitor. Topoisomerase II (topo II) incises double-stranded DNA to facilitate the passage of an intact duplex through the gap before rejoining the cut DNA.16 Etoposide inhibits cell growth by stabiliz￾ing the topo II-DNA complex, resulting in double-stranded DNA breaks and subsequent cell death.17,18 Previously, numerous epip￾odophyllotoxin derivatives were synthesized and reported as active topo II inhibitors.19,20 The design of compounds that could also interact with topo II, in addition to topo I, was attempted through the chemical combination of topo I and topo II inhibitors. Two conjugates (1 and 2) in which the topo I inhibitor camptothecin and the topo II inhibitor 4b-anilino-40 -O-demethylepipodophyllo￾toxin were linked via a covalent imine bond were synthesized and evaluated for biochemical and biological activities.21,22 In earlier studies, the conjugates 1 and 2 displayed a broad spectrum of cytotoxic activity against drug-resistant cell lines.21,22 Both compounds inhibited topo I, but only 2 was active against topo II, although at a higher concentration (100 lM). Both compounds induced PLDB in KB cells and had similar in vitro cytotoxicity. These results warrant the further molecular design of CPT–epipodophyllotoxin conjugates as antitumor agents. Prior structure–activity relationship (SAR) studies on CPT-deriv￾atives indicated that the lactone E-ring is essential for both antitu￾mor and topo I inhibitory activities. The 20-hydroxy group of CPT’s intact lactone E-ring forms a hydrogen bond with the topo I-linked DNA (TLD) and, thus, stabilizes the complex.23–25 CPT can be rap￾idly inactivated through E-ring lactone reversible hydrolysis at physiological conditions, leading to an inactive water soluble car￾boxylate, which binds readily to human serum albumin making the compound inaccessible for cellular uptake.26,27 However, E-ring-modified ester–amide CPT-derivatives have been reported to show high potency against L1210 tumors.28 The re-conversion of these derivatives to CPT in vivo was considered to be necessary for significant activity. The isopropyl amide of a CPT open E-ring analog was reported to be more stable under the experimental conditions and less likely than other amide analogs to convert to the E-ring closed lactone form (e.g., CPT) or subse￾quently to the hydroxy-acid (E-ring open form).28 Consequently, it might bind less readily to human serum album. Thus, the biolog￾ical activity derived from the isopropyl amide analog could be con￾sidered as the open E-ring’s, though it was not so reported. In addition, CPT-epipodophyllotoxin conjugates with an open E-ring isopropyl amide moiety have not been studied, and their biological activity, especially anti-tumor activity through inhibi￾tion of topoisomerases, mechanism of action, as well as metabo￾lism profile have not been evaluated. In this regard, in order to understand the role of the lactone E-ring in the conjugated com￾pounds, E-ring-modified derivatives (1E and 2E) of 1 and 2, respec￾tively, were synthesized in this study and evaluated in comparison with their intact E-ring congeners for cytotoxicity against KB cells, including resistant cells, and for inhibition of topo I and topo II. 2. Chemistry The lactone E-ring conjugates 1 and 2 were synthesized as de￾scribed previously,21 and were the starting materials to synthesize the open E-ring conjugates 1E and 2E (Fig. 1). Conjugate 1 or 2 (22.2 mg, 0.262 mmol) was dissolved in CHCl3 (2 mL), and then isopropylamine (2 mL) was added. The solution was heated to re- flux for 22 h under nitrogen, and monitored by TLC (CH2Cl2: MeOH = 20:1). The solvent was removed under vacuum, and the residue was purified by silica gel chromatography (CH2Cl2:MeOH = 100:0–95:5 as eluent) to afford the target compounds 1E and 2E, respectively, in 51–70% yield. We originally suspected that the five-membered lactone ring in the epipodophyllotoxin unit might also undergo ring-opening through amide formation. However, the major products obtained under the described conditions were the desired products. The structures of the final products were identified from spectroscopic and other analytical data. Additional physicochemical properties, such as logP and logS values, of the newly synthesized conjugates 1E and 2E were also evaluated to further understand the SAR of the new compounds. 3. Results and discussion The effects of the new conjugates, as well as CPT and etoposide, on the growth of KB and CPT-sensitive and resistant KB cell lines were studied in a growth inhibition assay.22 As shown in Table 1, the four CPT-epipodophyllotoxin conjugates (1, 1E, 2, 2E) were less potent than CPT against KB cells; thus, conjugation reduced po￾tency. Interestingly, an open E-ring did not significantly affect the inhibitory activity, based on the observation that 1E and 2E were either as potent as or only slightly less potent than their lac￾tone E-ring congeners, 1 and 2. Compounds 1 and 1E, conjugated at the para-position of the aniline moiety in the epipodophyllotoxin, were more potent than 2 and 2E, conjugated at the ortho-position. CPT and all four CPT conjugates showed no significant cross￾resistance against etoposide-resistant KB-7D cells, which over￾express MRP and down-regulate topo II.29 However, as expected, CPT-resistant KBCPT100 cells, which down-regulate topo I and up-regulate X-ray repair cross-complementing gene I protein (XRCC1), were less susceptible to CPT and the conjugates. [The cytotoxic effects were partially restored in CPT partial reverting KBCPT100REV cells, because the XRCC1 over-expression is re￾versed.29] The IC50 of CPT increased by 48-fold in CPT-resistant KBCPT100 cells compared with the sensitive cells. However, the IC50 values of the conjugates increased by only 2.8- to 3.9-fold. Thus, CPT-resistance was overcome by some extent by the conju￾gation. Also, the presence of the open E-ring did not affect the rel￾ative resistance over the parental cell line. The data suggest that topo I could be the primary molecular target of these conjugate compounds in KB cells. After exposing KB cells to 2.5 lM of test compound for 30 min, PLDB formation was studied according to the method described previously.30 Consistent with previous findings,22 CPT induced threefold greater PLDB compared with conjugates 1 and 2 (Fig. 2). The open E-ring conjugates 1E and 2 showed fourfold less induction compared with their intact E-ring congeners 1 and 2, respectively. Increasing the concentration of conjugate 2E from 2.5 to 22.5 lM induced a sevenfold increase in PLDB in 30 min, which is equivalent to 60% of the PLDB induced by 2.5 lM of CPT (data not shown). DNA relaxation by topo I is a process that involves the binding of DNA to topoisomerase I, followed by cleavage and subsequent religation of the DNA. The impact of the CPT conjugates on these individual steps was investigated. A DNA duplex, which contains a 25-mer (ON4) and a 14-mer (ON5), was used as the substrate 4490 D. Ye et al. / Bioorg. Med. Chem. 20 (2012) 4489–4494

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

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

D Ye et al /Bioorg. Med. Chem. 20 (2012)4489-4494 4493 and eightfold, respectively, more potent than etoposide in sup- 5. Experimental pressing KB cell growth. However, the two compounds induced 5.1. Chemistry tency in the potencies between the two assays indicates that mechanism(s)independent of topo I and topo ll could be involved Melting points (with decomposition) were determined on an in the growth inhibition. In addition, KBCPT100 cells were more electrothermal MEL-TEMP 3.0 apparatus and are uncorrected FT- susceptible to the conjugates than to CPT. Our previous study indi- IR was conducted on a Shimadzu IR Prestige 21 instrument. H cated that one factor that leads to CPT-resistance in KBCPT100 cells NMR spectra were measured on an Inova 500 spectrometer with is the up-regulation of XRCC1, which plays an important role in tetramethylsilane(TMS)as the internal standard Chemical shifts repairing single-strand breaks generated by CPT action Based on are reported in 5(ppm). Mass spectra(MS)were obtained on an rudies on mechanism of action are ongoing. Si conjugates(100-200 mesh). Precoated silica gel plates(Kieselgel 60, F254 also showed cytotoxicity to etoposide resistant ), addi- 0.25 mm)were used for thin layer chromatography(TLc)analysis tional mechanism could be involved and merits further investiga- CPT and etoposide were purchased from the Sigma Chemical Co tion Multi-target actions of these conjugates could render cancer Log P and log S values were calculated with the methods published cells less susceptible to develop resistance. andrecommendedonhttp://www.vcclab.org/lab/alogps/ he assay results suggest that the conjugate molecule remain intact under the cell culture conditions, because the activities of 5.2. Conjugate IE the conjugates and their individual parent compounds are distin- guishable. This observation is consistent with the reported stability Red-brown crystalline powder; mp >300C(decomp ) ESI MS of the imine linkage and previous experimental observation.21,22 m/z 908.3(M+1)'; FT-IR(ATR crystal plate)3311(broad, OH, NH) Finally, related physicochemical properties, such as logP and 2968, 2935(alkyl CH), 1754(lactone carbonyl), 1649, 1583(amide S values, were evaluated as listed in Table 1 to further under- carbonyl)cm- H NMR(CDCl3):81.01(t, J=7.0 Hz, 3H, 18-H stand the SAR of the conjugates. In spite of the heavy mass and lim- 1.21(d, J=7.0 Hz, 6H. (CH3)). 2.30-2.38(m, 2H, 19"-H). 2.94- ited solubility of the conjugates, other molecular properties in 2.96(m, IH, 3-H), 3. 20(dd, J=5.0. 14.0 Hz, 1H, 2-H), 3.80(5, 6H The E-ring open compounds 1E and 2E have slightly improved par- 11-H), 4.55(d, J=4.0 Hz, 1H, 1-H). 4.91(s, 2H, 22M-H). 4.93(d tition coefficient property (log P) relative to their E-ring intact J =6.0 Hz, 1H, 4-H), 5.00(s, 2H, 5"-H) 5.96 (s, 2H, OCH20). 6.11(s counterparts(5.091 vs 5.37). These preliminary data suggest that 1H, 4-0H), 6.64(s, 2H, 2, 6-H). 6.55(s, 1H, 8-H). 6.79(d, structural modifications of the conjugates merit further investiga- J=10.0 Hz, 2H, 2. 6" -H). 6.89(s, IH, 5-H). 7.35. 742(m, each 1H. better pharmaceutical drug-like molecules. 10"-H, 11-H). 7.44(s, 1H, 14"-H). 7.46(d, J=5.5 Hz, 2H, 3".5" Continuing research in the authors'laboratory includes structural H). 7.75(d, J=9.0 Hz, 1H, 9-H). 7.98(d, J=8.5 Hz, 1H, 12 m-H). modifications to generate more E-ring open CPT es with 9.01(d, J=8.0 Hz, 1H, N=CH). C NMR(400 MHZ, CDCl3):87.45 various amides/esters, with different linkers, and t conju(CH3);23.56( isopropyl CH3×2):3412(CH2):37.85(CH);42.65 ted partners. Additional mechanism of action (such as (CH): 44.85(CH): 45.54 (isopropyl CH): 51.60(camptothecin C-ring locking experiments), as well as metabolism studies, are also CH2): 5656(OCH3 x 2): 58.27(CH2 OH): 6892(epipodophyllotoxin ongoing. The results will be reported in due course C-ring CHNH): 70.90 (lactone CH2): 95.56(-COH): 100.73(campto- thein D-ring CH): 101.55(OCH20): 107.38(epipodophyllotoxin 4. Conclusion E-ring CHX 2): 110.52 (epipodophyllotoxin B-ring CH): 110.52 (epipodophyllotoxin B-ring CH): 116.60(aniline CH): 116.82(ani- Current preliminary study results show that CPT- line CH): 123.56(camptothecin D-ring C): 125. 25(aniline CH); pipodophyllotoxin conjugates are potent inhibitors of the growth 125. 58(camptothecin A-ring CH): 125.58(aniline CH): 126. 28 f both CPT-sensitive and-resistant cancer cells. Conjugation of a (camptothecin A-ring CH): 127. 85 (quinolone C): 128.42(campto- well-known topo-I inhibitor and topo-ll inhibitor generated thein A-ring CH): 129.76(quinolone C): 129.28(camptothecin new single molecule and provided a possible approach to over- A-ring CH): 130.67(epipodophyllotoxin B-ring C): 131.37(epipod come CPT-resistance to tumor cells from the current results, one ophyllotoxin B-ring C): 134. 22 (epipodophyllotoxin E-ring c) of the major mechanisms involved is the inhibition of topoisomer- 134.78(epipodophyllotoxin E-ring C): 136.81(aniline C): 136.39 ase I-mediated religation. The intact E-ring was critical, but not (camptothecin A-ring C): 142.58(quinolone C): 146.58 (aniline C) essential, for the inhibitory activity. Conjugates with an open E- 146.65(camptothecin C-ring C): 147. 54(epipodophyllotoxin B-ring ring were active, although they were less potent than the closed C): 147.54(epipodophyllotoxin B-ring C): 148.94 (epipodophyllo ring analogs. In addition to their impact on topo l these new com- toxin E-ring C): 149.67(epipodophyllotoxin E-ring C): 151.37(cam- unds exhibited full cytotoxic activity against both etoposide- ptothecin D-ring C): 152.70 (quinolone C): 154.77(amide c=o); ensitive(KB)and-resistant(KB-7D )cell proliferation, while they 161.79(imine C=N-): 173. 22 (lactone C=0): 176.35(amide hibited minor inhibitory activity against topo I-induced DNA C=O); HRMS(m/z): C5:HsoNsO11 Calcd: 908 3501 [M+H. Found: cleavage. The findings also indicated that mechanisms indepen- 908.3513[M+H] dent of topo I and ll could exist. The lactone E-ring has been long considered as critical to CPT's therapeutic application; however, 5.3. Conjugate 2E the present study first reports that open E-ring CPT-epipodophyllo- xin conjugates with an isopropyl amide group retain antitumo Red crystalline powder; mp 242C(decomp ) my9084 proliferation activity in both CPT-sensitive and resistant cells. (M+1) FT-IR(ATR crystal plate)3311 (broad, OH, NH), 2968, 2935 The novel structures of 1E and 2E may serve as scaffolds for further (alkyl CH), 1766(lactone carbonyl), 1649, 1589 evelopment of topo I and ll inhibitors with improved pharmaco- cm: H NMR (CDCl3) :8 1.01(t, J-7.5 Hz, 3H, 18 -H). 1.21(d, logical profiles and physicochemical properties to, ultimately, J=6.5 Hz, 6H,(CH3)2), 2.35(m, 2H, 19" -H), 3.09(m, 1H, 3-H). overcome cpt-resistance 3.60(dd,J=5.5,14.5Hz,1H,2-H).3.81(s,6H,OCH3×2).408

and eightfold, respectively, more potent than etoposide in sup￾pressing KB cell growth. However, the two compounds induced significantly lower PLDB compared with etoposide. The inconsis￾tency in the potencies between the two assays indicates that mechanism(s) independent of topo I and topo II could be involved in the growth inhibition. In addition, KBCPT100 cells were more susceptible to the conjugates than to CPT. Our previous study indi￾cated that one factor that leads to CPT-resistance in KBCPT100 cells is the up-regulation of XRCC1,29 which plays an important role in repairing single-strand breaks generated by CPT action. Based on the increased potency of the conjugates relative to CPT in this cell line, XRCC1 could be a cellular target of the conjugates.29 Further studies on mechanism of action are ongoing. Since the conjugates also showed cytotoxicity to etoposide resistant cell (KB-7D), addi￾tional mechanism could be involved and merits further investiga￾tion. Multi-target actions of these conjugates could render cancer cells less susceptible to develop resistance. The assay results suggest that the conjugate molecule remains intact under the cell culture conditions, because the activities of the conjugates and their individual parent compounds are distin￾guishable. This observation is consistent with the reported stability of the imine linkage33 and previous experimental observation.21,22 Finally, related physicochemical properties, such as logP and logS values, were evaluated as listed in Table 1 to further under￾stand the SAR of the conjugates. In spite of the heavy mass and lim￾ited solubility of the conjugates, other molecular properties in regard to Lipinski’s five rules are still marginally acceptable.34 The E-ring open compounds 1E and 2E have slightly improved par￾tition coefficient property (logP) relative to their E-ring intact counterparts (5.091 vs 5.37). These preliminary data suggest that structural modifications of the conjugates merit further investiga￾tion to generate better pharmaceutical drug-like molecules. Continuing research in the authors’ laboratory includes structural modifications to generate more E-ring open CPT conjugates with various amides/esters, with different linkers, and different conju￾gated partners. Additional mechanism of action studies (such as docking experiments), as well as metabolism studies, are also ongoing. The results will be reported in due course. 4. Conclusion Current preliminary study results show that CPT￾epipodophyllotoxin conjugates are potent inhibitors of the growth of both CPT-sensitive and -resistant cancer cells. Conjugation of a well-known topo-I inhibitor and topo-II inhibitor20 generated a new single molecule and provided a possible approach to over￾come CPT-resistance to tumor cells. From the current results, one of the major mechanisms involved is the inhibition of topoisomer￾ase I-mediated religation. The intact E-ring was critical, but not essential, for the inhibitory activity. Conjugates with an open E￾ring were active, although they were less potent than the closed ring analogs. In addition to their impact on topo I, these new com￾pounds exhibited full cytotoxic activity against both etoposide￾sensitive (KB) and -resistant (KB-7D) cell proliferation, while they exhibited minor inhibitory activity against topo II-induced DNA cleavage. The findings also indicated that mechanisms indepen￾dent of topo I and II could exist. The lactone E-ring has been long considered as critical to CPT’s therapeutic application; however, the present study first reports that open E-ring CPT-epipodophyllo￾toxin conjugates with an isopropyl amide group retain antitumor proliferation activity in both CPT-sensitive and resistant cells. The novel structures of 1E and 2E may serve as scaffolds for further development of topo I and II inhibitors with improved pharmaco￾logical profiles and physicochemical properties to, ultimately, overcome CPT-resistance. 5. Experimental 5.1. Chemistry Melting points (with decomposition) were determined on an electrothermal MEL-TEMP 3.0 apparatus and are uncorrected. FT￾IR was conducted on a Shimadzu IR Prestige 21 instrument. 1 H NMR spectra were measured on an Inova 500 spectrometer with tetramethylsilane (TMS) as the internal standard. Chemical shifts are reported in d (ppm). Mass spectra (MS) were obtained on an Agilent 1100 series LC-MSD-Trap or PE-Sciex API-300 spectrome￾ter. Flash column chromatography was performed on silica gel (100–200 mesh). Precoated silica gel plates (Kieselgel 60, F254, 0.25 mm) were used for thin layer chromatography (TLC) analysis. CPT and etoposide were purchased from the Sigma Chemical Co. LogP and logS values were calculated with the methods published and recommended on http://www.vcclab.org/lab/alogps/. 5.2. Conjugate 1E Red–brown crystalline powder; mp >300 C (decomp.); ESI MS m/z 908.3 (M+1)+ ; FT-IR (ATR crystal plate) 3311 (broad, OH, NH), 2968, 2935 (alkyl CH), 1754 (lactone carbonyl), 1649, 1583 (amide carbonyl) cm1 ; 1 H NMR (CDCl3): d 1.01 (t, J = 7.0 Hz, 3H, 18000-H), 1.21 (d, J = 7.0 Hz, 6H, (CH3)2), 2.30–2.38 (m, 2H, 19000-H), 2.94– 2.96 (m, 1H, 3-H), 3.20 (dd, J = 5.0, 14.0 Hz, 1H, 2-H), 3.80 (s, 6H, OCH3 2), 4.04–4.08 (m, 2H, 11-H, NCH), 4.36 (t, J = 8.5 Hz, 1H, 11-H), 4.55 (d, J = 4.0 Hz, 1H, l-H), 4.91 (s, 2H, 22000-H), 4.93 (d, J = 6.0 Hz, 1H, 4-H), 5.00 (s, 2H, 5000-H), 5.96 (s, 2H, OCH2O), 6.11 (s, 1H, 4´ -OH), 6.64 (s, 2H, 20 , 60 -H), 6.55 (s, 1H, 8-H), 6.79 (d, J = 10.0 Hz, 2H, 200, 600-H), 6.89 (s, 1H, 5-H), 7.35, 7.42 (m, each 1H, 10000-H, 11000-H), 7.44 (s, 1H, 14000-H), 7.46 (d, J = 5.5 Hz, 2H, 300, 500- H), 7.75 (d, J = 9.0 Hz, 1H, 9000-H), 7.98 (d, J = 8.5 Hz, 1H, 12000-H), 9.01 (d, J = 8.0 Hz, 1H, N@CH). 13C NMR (400 MHz, CDCl3): d 7.48 (CH3); 23.56 (isopropyl CH3 2); 34.12 (CH2); 37.85 (CH); 42.65 (CH); 44.85 (CH); 45.54 (isopropyl CH); 51.60 (camptothecin C-ring CH2); 56.56 (OCH3 2); 58.27 (CH2OH); 68.92 (epipodophyllotoxin C-ring CHNH); 70.90 (lactone CH2); 95.56 (–COH); 100.73 (campto￾thecin D-ring CH); 101.55 (OCH2O); 107.38 (epipodophyllotoxin E-ring CH 2); 110.52 (epipodophyllotoxin B-ring CH); 110.52 (epipodophyllotoxin B-ring CH); 116.60 (aniline CH); 116.82 (ani￾line CH); 123.56 (camptothecin D-ring C); 125.25 (aniline CH); 125.58 (camptothecin A-ring CH); 125.58 (aniline CH); 126.28 (camptothecin A-ring CH); 127.85 (quinolone C); 128.42 (campto￾thecin A-ring CH); 129.76 (quinolone C); 129.28 (camptothecin A-ring CH); 130.67 (epipodophyllotoxin B-ring C); 131.37 (epipod￾ophyllotoxin B-ring C); 134.22 (epipodophyllotoxin E-ring C); 134.78 (epipodophyllotoxin E-ring C); 136.81 (aniline C); 136.39 (camptothecin A-ring C); 142.58 (quinolone C); 146.58 (aniline C); 146.65 (camptothecin C-ring C); 147.54 (epipodophyllotoxin B-ring C); 147.54 (epipodophyllotoxin B-ring C); 148.94 (epipodophyllo￾toxin E-ring C); 149.67 (epipodophyllotoxin E-ring C); 151.37 (cam￾ptothecin 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. Calcd: 908.3501 [M+H]+ . Found: 908.3513 [M+H]+ . 5.3. Conjugate 2E Red crystalline powder; mp 242 C (decomp.); ESI MS m/z 908.4 (M+1)+ ; FT-IR (ATR crystal plate) 3311 (broad, OH, NH), 2968, 2935 (alkyl CH), 1766 (lactone carbonyl), 1649, 1589 (amide carbonyl) cm1 ; 1 H NMR (CDCl3): d 1.01 (t, J = 7.5 Hz, 3H, 18000-H), 1.21 (d, J = 6.5 Hz, 6H, (CH3)2), 2.35 (m, 2H, 19000-H), 3.09 (m, 1H, 3-H), 3.60 (dd, J = 5.5, 14.5 Hz, 1H, 2-H), 3.81 (s, 6H, OCH3 2), 4.08 D. Ye et al. / Bioorg. Med. Chem. 20 (2012) 4489–4494 4493

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. 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