0 SYNTHETIC STUDIES Camptothecin and Its Analogs

III. SYNTHETIC STUDIES

Given the finite supplies of natural CPT and the need to develop additional analogs, much effort has focused on practical synthetic routes to CPT and its analogs. The following are descriptions of several of the synthetic approaches to CPT and of the semisynthesis of topotecan (3) and irinotecan (4).

A. SYNTHESIS OF RACEMIC CPT

Following the initial publication of the structure of CPT, a number of synthetic strategies for the preparation of CPT were reported. The first total synthesis of (R,S)-CPT was described by Stork and Schultz in 1971.36 Numerous successful synthetic approaches to 20(R,S)-CPT have since been published and reviewed.37,38

Given the early availability of methods for racemic synthesis, relative to asymmetric approaches, research also focused on the chiral resolution of the racemates or key synthetic intermediates. In 1975, Corey and coworkers were the first to report the successful resolution of a chiral intermediate, leading to the preparation of 20(S)-CPT.39 Wani et al. and Teresawa et al. have also reported the successful resolution of the intermediates in the synthesis of CPT, using (R)-(+)-α-methyl-benzylamine.40,41

B. ASYMMETRIC SYNTHESIS

Ejima et al. reported the first stereocontrolled synthesis of CPT via a diastereoselective ethylation process using N-tosyl-(R)-proline (6) (Scheme 2.1).42 Indolizine 5, the CD-ring precursor of the parent alkaloid, was employed in an approach to construction of ring E. Bromination of 5 followed by treatment with N-tosyl-(R)-proline (6) in the presence of base afforded compound 7. A diaste- reomeric mixture of (S,R)-8 and (R,R)-9 was quantitatively prepared by facial differentiated ethy- lation of 7 in 82% de; selective recrystallization provided pure 8 in 56% yield. Following Raney Ni catalyzed reduction of 8 and subsequent treatment with NaNO2, the optically pure ester 10 was obtained in 74% yield. The triester 10 was hydrolyzed using LiOH and lactonized to provide hydroxy lactone 11 having the proper 20(S)-configuration in 90% yield. Hydrolysis of the ketal functionality gave optically pure key intermediate 12, which was converted to 20(S)-CPT by Friedländer reaction with 2-aminobenzaldehyde derivative 13 in 84% yield.

Comins and coworkers have successfully employed chiral auxiliaries to establish the correct stereochemistry for the 20(S)-hydroxyl group (Scheme 2.2).43 Refinement of their method has culminated in the asymmetric synthesis of 20(S)-CPT in only six steps, using the α-ketobutyric ester derived from (-)-trans-2-(α-cumyl)cyclohexanol [(-)-TCC] (17) as chiral auxiliary (Scheme 2.2).44 Treatment of commercially available 2-methoxypyridine (14) with mesityllithium, followed by addition of N-formyl-N,N′,N′-trimethylethylenediamine, effected the alkylation of the aromatic ring at C3. Addition of n-BuLi followed by iodine and workup with aqueous NaBH4/CeCl3 provided alcohol 15 in 46% yield via a one-pot process. Conversion of 15 directly to 1,3-dioxane 16 using NaI/TMSCl/paraformaldehyde was accomplished in 87% yield. The DE ring precursor 18 was fashioned via another one-pot process involving lithium–halogen exchange effected with n-BuLi, followed by addition of chiral auxiliary 17. Addition of HCl effected protonation, acetal hydrolysis, and lactonization to give intermediate 18 in 60% yield (93% ee). Coupling of 18 with the quinoline intermediate 19 was accomplished via displacement of the primary iodide to provide enantiopure 20 in 81% yield. The C-ring was closed using a Heck reaction through treatment of 20 with Pd(II) and potassium acetate to provide 20(S)-CPT in 64% yield.

Two other research groups have employed chiral auxiliaries to establish the S-configuration at the C20 position of CPT. Tagami et al. used a Davis reagent, (2R, 8aS)-(+)-(camphorylsulfonyl)oxaziridine), to asymmetrically hydroxylate 20-deoxycamptothecin.45 In 2002, Bennasar et al. made use of (2R,5R)- 2-tert-butyl-5-ethyl-1,3-dioxolan-4-one to establish C20 asymmetry and synthesize 20(S)-CPT.46

To control absolute stereochemistry at C20, Ciufolini and Roschangar reported a synthesis of 20(S)-CPT that made use of an aldehyde intermediate obtained by an enzymatic desymmetrization of a corresponding malonate.47 In 1998, Imura and coworkers described the first asymmetric synthesis of a key chiral intermediate, using enzyme-catalyzed resolution.48

Fang et al. applied the first chiral catalytic method to prepare 20(S)-CPT (Scheme 2.3).49 Reductive etherification of aldehyde 21 followed by intramolecular Heck reaction gave the cyclic enol ether 22 in 45% yield (Scheme 2.3). Sharpless asymmetric dihydroxylation of 22 using (DHQD)2-Pyr as the chiral catalyst followed by oxidation with iodine and CaCO3 was performed to synthesize the DE ring precursor 23 with 94% ee (90% yield). Treatment of 23 with HCl provided the enatiomerically pure pyridone 18 in 74% yield. The authors completed the synthesis of 20(S)- CPT using the Comins route (cf Scheme 2.2).43,44

Jew et al. made further use of the catalytic asymmetric Sharpless dihydroxylation using (DHQD)2-Pyr as chiral catalyst, resulting in stereocontrolled oxidation of carbon 20 in greater than 90% ee in the total synthesis of 20(S)-CPT.50 In 2002, Blagg and Boger established the configuration of the C20 (S) tertiary alcohol through a Sharpless asymmetric dihydroxylation reaction, using a 3,4,5-trimethoxyphenyl-derived DHQ dimer ligand in 86% ee.51

Curran and coworkers have reported a synthesis of 20(S)-CPT based on a 4+1 radical cascade annulation (Scheme 2.4).52 Lactone 24 was obtained in a fashion similar to the synthesis developed by Fang et al.49 Exchange of the TMS group in 24 for iodine, followed by demethylation, provided 25 in 33% yield (Scheme 2.4). N-Propargylation of lactone 25 provided alkyne 26 in 88% yield followed by isonitrile treatment under irradiation to provide 20(S)-CPT in 63% yield. Curran and coworkers have subsequently reported improvements in the synthesis of the enantiopure DE ring precursor 25, using a samarium catalyst.53 Radical methods of this type have been shown to tolerate A- and B-ring substituents and have, therefore, been used for the synthesis of numerous CPT analogs.52

C. SEMISYNTHETIC METHODS

In addition to these total syntheses, many synthetic efforts have been focused on producing analogs of CPT. The majority of these efforts have involved the semisynthetic manipulation of CPT. The approaches to irinotecan and topotecan are described.

Sawada et al. first reported the synthesis of irinotecan (4) in 1991 (Scheme 2.5).54 Hydrogen peroxide was added to a solution of 20(S)-CPT in aqueous sulfuric acid in the presence of ferrous sulfate and propionaldehyde to afford 7-ethyl CPT (27) in 77% yield (Scheme 2.5). 7-Ethyl CPT (27) was converted to the corresponding N-oxide 28 using hydrogen peroxide in acetic acid. Irradiation of 28 in acidic media furnished the active metabolite of irinotecan (SN-38) (29) in 49% yield. Treatment of SN-38 (29) with 4-(1-piperidino)-1-(piperidino)-chloroformate then provided irinotecan (4) in 80% yield.

The second currently marketed CPT derivative, topotecan, was synthesized in 1991 by Kings- bury et al. in two steps starting from 20(S)-CPT (Scheme 2.6).55 Conversion of 20(S)-CPT to 10- hydroxy CPT (30) was accomplished through a reduction-oxidation sequence in 71% yield (Scheme 2.6). Treatment of 30 with dimethylamine in aqueous formaldehyde and acetic acid provided topotecan (3) in 62% yield.

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12. Yang, D. et al., DNA interactions of two clinical camptothecin drugs stabilize their active lactone forms, J. Am. Chem. Soc., 120, 2979, 1998.

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14. Pommier, Y. et al., Interaction of an alkylating camptothecin derivative with a DNA base at topoi- somerase I-DNA cleavage sites, Proc. Natl. Acad. Sci. USA, 92, 8861, 1995.

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16. Kerrigan, J.E. and Pilch, D.S., A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin, Biochemistry, 40, 9792, 2001.

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20. Fujimori, A. et al., Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin, Cancer Res., 55, 1339, 1995.

21. Jensen, A.D., and Svejstrup, J.Q., Purification and characterization of human topoisomerase I mutants, Eur. J. Biochem., 236, 389, 1996.

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23. Husain, I. et al., Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity and implications for cancer chemotherapy, Cancer Res., 54, 539, 1994.

24. Kauh, E.A. and Bjornsti, M.-A., SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I, Proc. Natl. Acad. Sci. USA, 92, 6299, 1995.

25. Kawato, Y. et al., Inhibitory activity of camptothecin derivatives against acetylcholinesterase in dogs and their binding activity to acetylcholine receptors in rats, J. Pharm. Pharmacol., 45, 444, 1993.

26. Siddoo-Atwal, C., Haas, A.L., and Rosin, M.P., Elevation of interferon beta-inducible proteins in ataxia telangiectasia cells, Cancer Res., 56, 443, 1996.

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30. Kretzschmar, M., Meisterernst, M., and Roeder, R.G., Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II, Proc. Natl. Acad. Sci. USA, 90, 11508, 1993.

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35. Walowsky, C. et al., The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin, J. Biol. Chem., 274, 7302, 1999.

36. Stork, G. and Schultz, A.G., The total synthesis of dl-camptothecin, J. Am. Chem. Soc., 93, 4074, 1971.

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38. Thomas, C.J., Rahier, N.J., and Hecht, S.M., Camptothecin: current perspectives, Bioorg. Med. Chem., 12, 1585, 2004.

39. Corey, E.J., Crouse, D.N., and Anderson, J.E., A total synthesis of natural 20(S)-camptothecin, J. Org. Chem., 40, 2140, 1975.

40. Wani, M.C., Nicholas, A.W., and Wall, M.E., Plant antitumor agents 28. Resolution of a key tricyclic synthon, 5′(RS)-1,5-dioxo-5′-ethyl-5′-hydroxy-2′H,5′H,6′H-6′-oxopyrano[3′,4′-f]∆6,8-tetrahydro- indolizine: total synthesis and antitumor activity of 20(S)- and 20(R)-camptothecin, J. Med. Chem., 30, 2317, 1987.

41. Terasawa, H. et al., Antitumor agents III. A novel procedure for inversion of the configuration of a tertiary alcohol related to camptothecin, Chem. Pharm. Bull., 37, 3382, 1989.

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43. Comins, D.L., Hong, H., and Jianhua, G., Asymmetric synthesis of camptothecin alkaloids: A nine- step synthesis of (S)-camptothecin, Tetrahedron Lett., 35, 5331, 1994.

44. Comins, D.L. and Nolan, J.M., A practical six-step synthesis of (S)-camptothecin, Org. Lett., 3, 4255, 2001.

45. Tagami, K. et al., Asymmetric synthesis of (+)-camptothecin and (+)-7-ethyl-10-methoxycamptothe- cin, Heterocycles, 53, 771, 2000.

46. Bennasar, M.-L. et al., Addition of ester enolates to N-alkyl-2-fluoropyridinium salts: total synthesis of (±)-20-deoxycamptothecin and (+)-camptothecin, J. Org. Chem., 67, 7465, 2002.

47. Ciufolini, M.A. and Roschangar, F., Practical synthesis of (20 S)-(+)-camptothecin: the progenitor of a promising group of anticancer agents, Targets Heterocyclic Syst., 4, 25, 2000.

48. Imura, A., Itoh, M., and Miyadera, A., Enantioselective synthesis of 20(S)-camptothecin using an enzyme-catalyzed resolution, Tetrahedron: Asymmetry, 9, 2285, 1998.

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55. Kingsbury, W.D. et al., Synthesis of water-soluble (aminoalkyl) camptothecin analogs: inhibition of  topoisomerase I and antitumor activity, J. Med. Chem., 34, 98, 1991.

56. Lackey, K. et al., Rigid analogues of camptothecin as DNA topoisomerase I inhibitors, J. Med. Chem., 38, 906, 1995.

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58. Kurihara, T. et al., Synthesis of C-nor-4,6-secocamptothecin and related compounds, J. Heterocycl. Chem., 30, 643, 1993.

59. Subrahmanyam, D. et al., Novel C-ring analogues of 20(S)-camptothecin Part 2. Synthesis and in vitro cytotoxicity of 5-C-substituted 20(S)-camptothecin analogues, Bioorg. Med. Chem. Lett., 9, 1633, 1999.

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Soure: Anticancer Agents from Natural Products edited by Gordon M. Cragg, David G. I. Kingston, David J. Newman

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0 MECHANISM OF ACTION Camptothecin and Its Analogs

II. MECHANISM OF ACTION 

A. POISONING OF TOPOISOMERASE I

In 1985, it was reported that CPT stabilized the DNA–topoisomerase I covalent binary complex.3 It had previously been shown that CPT is capable of inhibiting DNA synthesis, thereby causing cell death during the S-phase of the cell cycle.4 The S-phase specific cytotoxicity is directly correlated to the occurrence of irreversible DNA cleavage when the replication fork encounters the covalent DNA–enzyme binary complex (Figure 2.2).5 It has been demonstrated that deletion of the gene for topo I from Saccharomyces cerevisiae results in viable cells that are fully resistant to CPT.6 Further, a number of CPT-resistant cell lines have been identified, each containing mutations within topo I.7 These studies clearly support a mechanism of action for CPT involving topo I–mediated DNA cleavage.

The topoisomerases (type I and type II) are a class of enzymes that mediate the relaxation of chromosomal DNA prior to DNA replication and transcription.8 Mechanistically, topo II effects this relaxation via transient double-strand cleavage, DNA strand passage, and religation of the phosphodiester backbone. This process requires ATP and alters the DNA linking number by multiples of two. The topo I mechanism involves energy-independent single-strand DNA cleavage, followed by strand passage and religation.9 The mechanism of topo I–mediated DNA relaxation is known to involve an active site tyrosine that cleaves DNA by nucleophilic attack of the active site tyrosine phenolic OH group on the phosphodiester backbone (Figure 2.3). The resulting DNA–topo I intermediate is a covalent binary complex. The cleaved DNA strand can be passaged around the unbroken DNA strand; the intact duplex is reformed on religation of the phosphodiester bond, with the concomitant release of topo I.

Although topo I may be capable of DNA cleavage at a number of sites, it exhibits a strong preference for the nucleoside thymidine as the nucleobase directly upstream (the -I position) (cf. Figure 2.3). Stabilization of the covalent binary complex by CPT has been noted to involve an additional preference for guanosine at the +1 position.

Most of the agents that poison type I and type II topoisomerases are characterized by their ability to inhibit the religation step during DNA relaxation (Figure 2.3). Topo II poisons include a number of well-characterized clinical agents such as amsacrine and etopside.10 Several inhibitors of topo I have also been identified; however, CPT remains the most widely studied of this class of medicinal agents.

The development of a precise understanding of the way in which CPT stabilizes the DNA–topo I covalent binary complex, and thereby inhibits the religation of duplex DNA, is an important current goal. CPT has no binding affinity for topo I, and only the positively charged CPT analog topotecan (3) has shown any DNA binding.11,12 However, the ability of CPT to bind to the covalent binary complex formed between topo I and DNA is sufficient to inhibit religation. There is compelling evidence that CPT is capable of interacting with the covalent binary complex at or near the interface between DNA and topo I. Hertzberg and coworkers have established that a CPT analog containing a bromoacetamide at carbon 10 was, on prolonged exposure, capable of forming a drug–enzyme crosslink.13 Pommier and coworkers later showed that a 7-chloromethylated CPT analog alkylated N3 of the guanine at the +1 cleavage site of DNA.14

A number of computational models have been formulated depicting the interaction between CPT and the covalent binary complex.15–17 These models posit different energy-minimized inter- actions between CPT, the DNA 5-TpG-3 base pair, and selected topo I amino acid residues known to be important based on biochemical studies or their proximity to the putative CPT binding site.18–21 The analysis of several CPT-resistant cell lines has provided important information regarding the amino acid residues that play a role in CPT binding.7 For example, mutational analysis of Asp 533, Arg364, Asn722, and Lys532 has revealed that each of these amino acid residues likely play a role in CPT binding.19–21

Staker et al. have recently reported the X-ray crystal structure of a ternary complex formed between DNA, topo I, and topotecan (3).22 The reported complex used a DNA oligonucleotide containing a 5-bridging phosphothioate to facilitate crystal formation and indicated that topotecan bound the covalent binary complex in an intercalative fashion (Figure 2.4). The only direct drug–enzyme hydrogen bond interaction was between Asp533 and the 20-hydroxyl group of topotecan. In addition, one hydrogen bond was observed with a water molecule. Carbons 7, 9, and 10 of CPT were positioned in a manner that situated them in the vicinity of the major groove.

B. OTHER BIOCHEMICAL EFFECTS OF CPT

It is generally accepted that the basis for CPT-induced cytotoxicity is contingent on CPT acting as a topo I poison (as opposed to an inhibitor of enzyme activity, per se).3–7 However, the antitumor selectivity of CPT is somewhat surprising given that topo I is an enzyme found in all cell types. Elevated levels of topo I are present in tumors of the colon, ovary, and prostate, which may explain the therapeutic index of CPT.23 Deficiencies in DNA repair capabilities in some cancer cells may provide another possible basis for cancer cell selectivity. The selective inhibition of all dividing cell populations represents another possible source of antitumor selectivity. Further, other effects of CPT exposure have been noted and merit discussion.

Kauh and Bjornsti, using a genetic screen, have identified six dominant suppressors of camp- tothecin toxicity at a single genetic locus (SCT1).24 Mutant SCT1 cells were shown to express wild- type topo I, indicating additional factors in the overall cellular response to CPT. One report indicated that irinotecan, but not topotecan, inhibits acetylcholinesterase activity.25 Additional reports indicate that CPT activates the transcription factor NFκB, which has been implicated in numerous activities in vivo.26 Importantly, activation of NFκB has been implicated in the overproduction of interferons, triggering several cellular responses.26–29

In addition to its role in relaxation of supercoiled DNA, topo I is able to regulate transcription,30 recognize and cleave mismatched nucleotides at intrinsic cleavage sites,31 and associate with numerous proteins in vivo. Tazi and coworkers have reported that topo I is capable of influencing gene splicing by acting as a phosphorylating enzyme for SR proteins.32 This kinase activity is inhibited by CPT despite the fact that it has been shown to be unconnected to its DNA relaxation activity through mutational studies.33 Analysis of a family of topoisomerase I related function proteins (TRFp) demonstrate that one member of this family, TRF4p, plays a critical role in mitotic chromosome condensation during the S phase of the cell cycle.34 TRF4p is associated with the DNA binding protein Smc1p during chromosomal condensation, and reports detailing mutations to TRF4p have produced cell lines with unexpected hypersensitivity to CPT.35

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Soure: Anticancer Agents from Natural Products edited by Gordon M. Cragg, David G. I. Kingston, David J. Newman

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