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 theBut, 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