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).408and 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