Synthesis and biological evaluations of novel indenoisoquinolines as topoisomerase I inhibitors

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1276–1281

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Synthesis and biological evaluations of novel indenoisoquinolines as topoisomerase I inhibitors Xiaoyun Zhang, Rubing Wang, Li Zhao, Na Lu, Jubo Wang, Qidong You, Zhiyu Li ⇑, Qinglong Guo ⇑ Department of Medicinal Chemistry, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 11 August 2011 Revised 4 October 2011 Accepted 7 October 2011 Available online 24 October 2011 Keywords: Top1 inhibitor Anticancer Indenoisoquinolone Synthesis Indenoisoquinolone derivate

a b s t r a c t A series of novel indenoisoquinoline derivatives were synthesized. The anticancer activities of these molecules were tested in human cancer cell lines A549, HepG2, and HCT-116. These compounds were also tested for their activity of topoisomerase I (top1) inhibition. Among them, compound 25 was found to be 10-times more potent in cell-killing activity for both cell lines HepG2 and HCT-116 than reported compound 11, with IC50 of 0.019 and 0.093 lM, respectively. Compound 25 was also found to have stronger top1 inhibition activity than 11 in our inhibition assay. Further in vivo evaluations of compound 25 are in progress and will be reported in due course. Ó 2011 Elsevier Ltd. All rights reserved.

DNA-topoisomerase I (top1) is widely considered as an important enzyme to relax supercoiled DNA for transcription, replication, and mitosis.1 Thus, DNA-topoisomerase I (top1) has been identified as a promising cancer therapeutic target.2 Two camptothecin (CPT) derivatives, irinotecan (1, Camptosar) and topotecan (2, Hycamptin), are the only topoisomerase I (top1) inhibitors approved by the FDA as anticancer drugs3,4 (Fig. 1). However, their clinical utility was limited by several disadvantages such as easy hydrolysis of the lactone ring to a hydroxy carboxylate which binds to human serum albumin,5 and the rapid reversibility of ternary DNA–enzyme–camptothecin complex after drug removal, necessitating long infusion time for maximum activity.6 Moreover, some cancer cells developed resistance to camptothecin.7 Thus, these problems recommended further development of other non-camptothecin top1 inhibitors with better pharmacokinetic features. In 19788,9 indenoisoquinoline 3 (NSC 314622) was first synthesized, and was found to be a novel top1 inhibitor. Its cytotoxicity

Abbreviations: DNA, deoxyribonucleic acid; Top1, DNA-topoisomerase I; CPT, camptothecin; FDA, food and drug administration; 1H NMR, hydrogen-1 nuclear magnetic resonance spectroscopy; 13C NMR, 13C nuclear magnetic resonance spectroscopy; MS, mass spectrometry; IR, infrared; A549, non-small cell lung carcinoma; HepG2, human hepatocellular liver carcinoma cell line; HCT-116, human colon cancer cell line; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IC50, half maximal inhibitory concentration; TopoGEN, topoisomerase I drug screening kit. ⇑ Corresponding authors. Tel./fax:+86 25 83271017. E-mail addresses: [email protected], [email protected] (Z. Li). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.10.019

profile revealed a strong resemblance with camptothecin derivatives (irinotecan and topotecan) in twenty years later (Fig. 1). Indenoisoquinoline, unlike the camptothecins, is hydrolytically stable but alternatively suffered from intrinsically low biological activity. As a result, considerable effort had been devoted to improve the biological activity of indenoisoquinolines.10–27,32–34 Novel insights had been gleaned regarding the contributions of the indenone ring, isoquinoline ring, and lactam side chain toward the biological activity.15,16,20–27 Previous investigation4 demonstrated that the biological activity of indenoisoquinolines (4–6) was improved by introducing a nitro substituent on the isoquinoline ring and a methylenedioxy-substitute on the indenone ring. After that,24 a detailed study indicated that compounds with a methoxy group at the 9-position of indenoisoquinolines (7–9) afforded superior biological activity. Further modifications on the lactam side chain yielded new lead compounds (10–13) with potent biological activity.25 The chemical structures of compounds 4–13 are provided in Figure 2. The present work was undertaken to further improve the biological activity of previously reported indenoisoquinolines with a nitro group on the isoquinoline ring and a 9-methoxygroup on the indenone ring. Two series of compounds were synthesized. In the first series, the nitro substituent on the isoquinoline ring remains unchanged and modification were made to the indenone ring with the electron-donating methoxy substituent replaced by the electron-withdrawing substituent fluorine and chlorone, the other series of analogues were prepared by reducing the strong

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Figure 1. Structure of irinotecan, topotecan and NSC314622.

From the data in Table 1, the biological activity is greatly improved, for both cytotoxicity and top1 inhibition, with the utilization of the nitro group at 3-position of indenoisoquinolines. As the lead compound 10 and 11, two molecules displayed superb cytotoxicity (0.023 lM, 0.16 lM, 0.168 lM, and 4 nM, 0.27 lM, 0.84 lM, respectively) and potent top1 inhibition (++++ and +++ respectively). Like compound 10 and 11, analogues 23 and 36–38 also contain a methoxy substituent on the indenone ring. Compound 36 (0.014 lM, 0.024 lM, 0.043 lM, top1 inhibition ++++) with a methylpiperazinyl lactam side chain, was more potent than reported compound 11 in the biological assays. Compound 38 (4 nM, 0.29 lM, 0.27 lM) with a propylamino lactam side chain showed similar cytotoxicity to that of the lead compound 11. The results were identical to the previously identified that methoxy substituent was optimally suited for enhancing the biological activity of the indenoisoquinoline 9-position. Furthermore, utilizing optimal lactam side chains (such as a morpholine, an imidazole and a methylpiperazinyl group) were identified to improve the biological activity of the indenoisoquinolines. Although the methoxy group was the only substituent identified in the previous study to enhance both the cytotoxicity and the top1 inhibition of these analogues, the electron-withdrawing fluorine atom at the 9-position (analogues 25–27, showed in Table 1) provided compounds equally as cytotoxic as camptothecin. Furthermore, when the 9-position fluorine atom was combined with a methylpiperazinyl-substituted lactam nitrogen, the resulting analogue 25 (top1 inhibition +++++) showed the strongest activity of top1 inhibition among them, which was 10-times more potent than that of the lead compound 11. In addition, Compound 26, 27 and 29 all possessed submicromolar IC50 values and moderate or potent top1 inhibition. The results demonstrated that modification of a methoxy group at the 9-position to the

electron-withdrawing nitro substituent to the corresponding aniline in an effort to further explore the effect of the nitro group. The synthesis of advanced indenoisoquinoline analogues 10, 11, 21–38 was described in Scheme 1. Condensation of homophthalic anhydrides 1428 with Schiff bases 15–1729 provided carboxylic acids 18–20. The cis-configuration was established by the observed coupling constant of 6 Hz for the two methine protons.30 These carboxylic acids were subjected to oxidative Friedel–Crafts ring closure with thionyl chloride7 and aluminum chloride15,24 to provide indenoisoquinolines 21–23. Compounds 21–23 were treated with appropriate amines in refluxing 1,4-dioxane to give target compounds 24–38 (Scheme 1). The chemical data including 1H NMR, 13C NMR, MS, IR, and elemental analysis of compounds 25 and 36 were provided in references and notes.31 In order to explore the importance of the electron-withdrawing nitro group on the isoquinoline ring, compounds 52–54 were synthesized (Scheme 2) with the nitro substituent replaced with an electron-donating aniline-type amino group. Compounds 21–23 were treated with appropriate amines in refluxing 1,4-dioxane to provide target compounds 39–51 (Scheme 2). All the synthesized indenoisoquinoline derivatives were screened for cytotoxicity on three human cancer cell lines A549 (non-small cell lung carcinoma), HepG2 (human hepatocellular liver carcinoma cell line) and HCT-116 (human colon cancer cell line) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. These compounds were also screened for their ability to stabilize the ternary DNA-enzyme-camptothecin complex using a topoisomerase I drug screening kit (TopoGEN, Inc.). From the biological data presented in Tables 1 and 2, the substitution pattern at the indenoisoquinoline 3-position, 9-position, and the lactam side chain has a pronounced effect on the biological activities of the molecules.

O O

O

O

O

O O

N

O2N

R

O 4:R=Cl 5:R=N3 6:R=NH2.HCl

O2N

N

R

N

O2N

R

O O 7:R=Cl 8:R=N3 9:R=NH2.HCl

10:R=

N

11:R=

N

12:R=

H N

O

N

13:R= N(CH3)2 Figure 2. Structure of the mentioned indenoisoquinolines.

OH

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Scheme 1. The synthesis indenoisoquinolines.

R

O

R

O a

N

O2N

Cl

N

H2N

O

Cl

O 52: R=F 53: R=Cl 54: R=OCH3

21: R=F 22: R=Cl 23: R=OCH3

R

O b

N

H2N

O R1

39: R=F; R1=

N

O

40: R=F; R1=

N

N

41: R=F; R1= 42: R=F; R1= 1

CH2CH2OH N CH2CH2OH NHCH2CH2OH

43: R=F; R =

N

44: R=F; R1=

N

N

45: R=Cl; R1=

N

O

47: R=OCH3; R1=

N

O

46: R=Cl; R1=

N

N

48: R=OCH3; R1=

N

N

49: R=OCH3; R1=

CH2CH3 N CH2CH3

50: R=OCH3; R1=

N

51: R=OCH3; R1=

N

Scheme 2. The synthesis indenoisoquinolines.

N

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X. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1276–1281 Table 1 Cytotoxicities and topoisomerase I inhibitory activities of indenoisoquinoline analogues

Compound

R

R1

A549 (lM)

HepG2 (lM)

HCT-116 (lM)

Top1 Cleavagea

TCPT TPT

N N

N N

0.064 0.19

2.24 2.21

66.3 88.1

++++ ++++

10

OCH3

0.023

0.16

0.168

++++

11

OCH3

21 22 23

F Cl OCH3

24

F

N

25

F

N

26 27 28

F F F

29

F

30

F

31

Cl

32

Cl

33 34

Cl Cl

35

Cl

N

36

OCH3

N

37

OCH3

38

OCH3

N N

O

0.0042

0.27

0.84

+++

22.4 0.749 3.88

>100 25.2 25.8

>100 54.0 5.72

+++ +++ ++

O

>100

>100

>100

+++

N

0.021

0.019

0.0932

+++++

0.068 0.031 3.52

0.54 0.49 2.77

0.162 0.207 0.741

++ +++ ++

0.035

0.91

0.159

+++

N

Cl Cl Cl

–N(CH2CH2OH)2 –NHCH2CH2OH –N(CH2CH3)2 N

N

–N(CH(CH3)2)2

18.5

14.8

2.68

++

N

O

3.03

>100

>100

+++

N

N

4.03

7.18

1.13

+++

3.28 1.18

9.42 1.38

1.79 1.13

++++ ++

0.00482

2.21

1.23

+++++

0.014

0.024

0.043

++++

–N(CH2CH3)2 –NHCH2CH2CH3 N

N

N

–NHCH2CH2CH3

0.61

6.83

12.3

0/+

0.00427

0.29

0.27

++

a The compounds were tested at concentrations ranging up to 100 nM. The activity of the compounds to produce top1-mediated DNA cleavage was expressed semiquantitatively as follows: +: weak activity; ++: modest activity; +++: similar activity as 100 nM camptothecin; ++++ or +++++: greater activity than 100 nM camptothecin.

Table 2 Cytotoxicities and topoisomerase I inhibitory activities of indenoisoquinoline analogues

R1

Compound

R

39

F

40

F

41 42

F F

43

F

N

44

F

N

A549 (lM)

HepG2 (lM)

HCT-116 (lM)

Top1 Cleavagea +

N

O

1.56

16.3

0.747

N

N

0.93

1.24

3.82

++

0.75 0.16

40.7 1.21

0.233 1.08

++ ++

2.69

61.8

1.85

++++

0.51

0.86

1.03

++++

–N(CH2CH2OH)2 –NHCH2CH2OH N

(continued on next page)

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Table 2 (continued) R1

A549 (lM)

HepG2 (lM)

HCT-116 (lM)

Top1 Cleavagea

O

7.55

27.3

2.97

++

N

N

1.69

0.29

1.95

++

N

O

1.63

12.3

6.82

++

N

N

0.63

12.9

13.7

+++

0.25

0.71

0.672

+

0.038

39.7

41.5

+

Compound

R

45

Cl

N

46

Cl

47

OCH3

48

OCH3

49

OCH3

50

OCH3

51

OCH3

52 53 54

F Cl OCH3

–N(CH2CH3)2 N N

Cl Cl Cl

N

0.045

0.53

0.870

+

11.1 6.24 1.85

55.5 30.7 >100

0.654 >100 >100

++++ ++ ++

a The compounds were tested at concentrations ranging up to 100 nM. The activity of the compounds to produce top1-mediated DNA cleavage was expressed semiquantitatively as follows: +: weak activity; ++; modest activity; +++: similar activity as 100 nM camptothecin; ++++ or +++++: greater activity than 100 nM camptothecin.

electron-withdrawing fluorine atom led to enhancement of top1 inhibition, especially combined with a methylpiperazinyl-substituted lactam nitrogen. Further, above results inspired us to synthesize a new series of compounds with the fluorine atom substituted with a chlorine atom. Six compounds were provided in Table 1, five of them showed potent top1 inhibitory activity (top1 inhibition +++ or greater). For example, compound 35 showed an IC50 value of 4 nM against A549 and it showed strong top1 inhibition with a resemblance to that of compound 25. It displayed that a chlorine atom at the 9-position also conferred potent biological activity. In an effort to further define the effect of the substituents with different electronic effects on the isoquinoline ring, a series of novel analogues possessing an amino substituent on the isoquinoline ring were synthesized, in conjunction with a methoxy, fluoro, or chloro substituent on the indenone ring. In Table 2, compound 43, 44 and 52 exhibited potent top1 inhibition (top1 inhibition ++++, ++++, ++++). Other compounds were found to have weak top1 inhibition. It was possible to make a conclusion that the fluoro group was important for top1 inhibition of these compounds, especially when combined with optimal lactam side chains. In conclusion, a series of novel indenoisoquinoline derivatives were synthesized based upon previously reported lead compound 11. Our work has led to the discovery of more potent compounds than 11. For example, compound 25 was found to be 10-times more potent in cell-killing activity for both cell lines HepG2 and HCT-116 than reported compound 11, with IC50 of 0.019 and 0.093 lM, respectively. Compound 25 was also found to have stronger top1 inhibition activity than 11 in our inhibition assay. Further in vivo evaluations of compound 25 are in progress and will be reported in due course. Acknowledgments This work was supported by, the National Natural Science Foundation of China (No. 21072232), and the National NaturalScience Foundation of China (No. 91029744). Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.10.019.

References and notes 1. Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. Science 1998, 279, 1504. 2. Rasheed, Z. A.; Rubin, E. H. Oncogene 2003, 22, 7296. 3. Garcia-Carbonero, R.; Supko, J. G. Clin. Cancer Res. 2002, 8, 641. 4. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. Lett. 2004, 14, 3659. 5. Burke, T. G.; Mi, Z. J. Med. Chem. 1994, 37, 40. 6. van Warmerdam, L. J.; Creemers, G. J.; Rodenhuis, S.; Rosing, H.; de BoerDennert, M.; Schellens, J. H.; ten Bokkel Huinink, W. W.; Davies, B. E.; Maes, R. A.; Verweij, J.; Beijnen, J. H. Cancer Chemother. Pharmacol. 1996, 38, 254. 7. Li, T. K.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 53. 8. Mark, C.; Cheng, L. J. Org. Chem. 1978, 43, 3781. 9. Kohlhagen, G.; Paull, K. D.; Cushman, M.; Nagafuji, P.; Pommier, Y. Mol. Pharmacol. 1998, 54, 50. 10. Ioanoviciu, A.; Antony, S.; Pommier, Y.; Staker, B. L.; Stewart, L.; Cushman, M. J. Med. Chem. 2005, 48, 4803. 11. Strumberg, D.; Pommier, Y.; Paull, K.; Jayaraman, M.; Nagafuji, P.; Cushman, M. J. Med. Chem. 1999, 42, 446. 12. Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.; Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. J. Med. Chem. 2000, 43, 3688. 13. Jayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2002, 45, 242. 14. Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Staker, B. L.; Stewart, L.; Cushman, M. J. Med. Chem. 2003, 46, 3275. 15. Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2003, 46, 5712. 16. Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2004, 47, 5651. 17. Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. 2004, 12, 5147. 18. Antony, S.; Kohlhagen, G.; Agama, K.; Jayaraman, M.; Cao, S.; Durrani, F. A.; Rustum, Y. M.; Cushman, M.; Pommier, Y. Mol. Pharmacol. 2005, 67, 523. 19. Xiao, X.; Miao, Z. H.; Antony, S.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. Lett. 2005, 15, 2795. 20. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. Lett. 2006, 16, 1846. 21. Xiao, X.; Antony, S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2005, 48, 3231. 22. Morrell, A.; Jayaraman, M.; Nagarajan, M.; Fox, B. M.; Meckley, M. R.; Ioanoviciu, A.; Pommier, Y.; Antony, S.; Hollingshead, M.; Cushman, M. Bioorg. Med. Chem. Lett. 2006, 16, 4395. 23. Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.; Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 6283. 24. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 7740. 25. Morrell, A.; Placzek, M.; Parmley, S.; Antony, S.; Dexheimer, T. S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 4419. 26. Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 2040. 27. Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 4388. 28. Whitmore, W. F.; Cooney, R. C. J. Am. Chem. Soc. 1944, 66, 1237. 29. Yus, M.; Soler, T.; Foubelo, F. J. Org. Chem. 2001, 66, 6207. 30. Cushman, M.; Cheng, L. J. Org. Chem. 1978, 43, 286.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1276–1281 31. 6-(3-[(1-Methylpiperazinyl)-1-propyl])-9-fluoro-3-nitro-5,6-di-hydro-5,11dioxo-11H-indeno[1,2-c]isoquinoline (25) mp 236–239 °C; EI-MS (m/ z):450[M]+; IR(KBr): 3415, 2816, 1667, 1613, 1500, 1347, 795 cm 1; Anal. calcd for C24H23FN4O4: C 63.99, H 5.15, N 12.44 Found: C 64.11, H 5.09, N 12.29; 1H NMR (300 MHz, CDCl3): d 9.16(s, 1H, Ar-H), 8.82(d, 1H, J = 8.7 Hz, ArH), 8.49(d, 1H, J = 8.7 Hz, Ar-H), 7.94(m, 1H, Ar-H), 7.43(m, 1H, Ar-H), 7.21(m, 1H, Ar-H), 4.64(t, 2H, J = 7.8 Hz, CON–CH2–), 2.61(br s, 13H, piperazine-H), 2.04(m, 2H, –CH2–CH2–CH2–).6-(3-[(1-Methylpiperazinyl)-1-propyl])-3-nitro9-methoxy-5,6-dihydro5,11-dioxo-11H-indeno[1,2-c]isoquinoline (36) mp 197–199 °C; MS-EI (m/z):462[M]+; IR(KBr): 3466, 3417, 2840, 1675, 1609, 1506, 1332, 1121, 853, 791 cm 1; Anal. calcd for C25H26N4O5: C 64.92, H 5.67, N

1281

12.11 Found: C 64.88, H 5.69, N 12.08; 1H NMR (300 MHz, CDCl3): d 9.16(d, 1H, J = 2.4 Hz, Ar-H), 8.78(d, 1H, J = 9 Hz, Ar-H), 8.44(dd, 1H, J1 = 2.4 Hz, J2 = 9 Hz, Ar-H), 7.76(d, 1H, J = 8.4 Hz, Ar-H), 7.27(d, 1H, J = 2.7 Hz, Ar-H), 6.92(dd, 1H, J1 = 2.7 Hz, J2 = 8.4 Hz, Ar-H), 4.60(t, 2H, J = 7.5 Hz, CON–CH2–), 3.94(s, 3H, – OCH3), 2.51(br s, 13H, piperazine-H), 2.06(m, 2H, –CH2–CH2–CH2–). 32. Kiselev, E.; Dexheimer, T. S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2010, 53, 8716. 33. Peterson, K. E.; Cinelli, M. A.; Morrel, A. E.; Metha, A.; Dexheimer, T. S.; Agama, K.; Antony, S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2011, 54, 4937. 34. Khadlka, D. B.; Cho, W. J. Bioorg. Med. Chem. 2011, 19, 724.