Exploring the Boundaries of âPracticalâ: De Novo Syntheses of

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Exploring the Boundaries of “Practical”: De Novo Syntheses of Complex Natural Product-Based Drug Candidates Tyler K. Allred,* Francesco Manoni, and Patrick G. Harran Department of Chemistry and Biochemistry, University of California−Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States ABSTRACT: This review examines the state of the art in synthesis as it relates to the building of complex architectures on scales sufficient to drive human drug trials. We focus on the relatively few instances in which a natural-product-based development candidate has been manufactured de novo, rather than semisynthetically. This summary provides a view of the strengths and weaknesses of current technologies, provides perspective on what one might consider a practical contribution, and hints at directions the field might take in the future.

CONTENTS 1. Introduction 2. Halichondrin B 2.1. Introduction 2.2. Discovery of Halichondrins 2.3. Synthetic Studies toward Halichondrin B 2.4. Truncated Halichondrins Reveal Pharmacophore 2.5. SAR Studies: The Discovery of Eribulin 2.6. Synthesis of Eribulin from Milligram to Gram Scale 2.7. Multikilogram Manufacturing Process of Halaven 3. Synthetic Tetracyclines 3.1. History of the Tetracyclines 3.2. Myers’ Synthetic Studies on the Tetracyclines 3.3. Tetraphase SAR Studies 3.4. Eravacycline Synthesis 4. Epothilone 4.1. Epothilone Background 4.2. Danishefsky’s Synthetic Approach 4.3. Nicolaou’s Synthesis 4.4. Schinzer’s Formal Synthesis 4.5. Epothilone Structure−Activity Relationships 4.6. Development of Sagopilone 4.7. Dehydelone, Fludelone, and Isofludelone 5. Cryptophycin 5.1. Discovery and Background 5.2. Tius−Moore’s Synthesis of Cryptophycin 1 5.3. Exploration of SAR 5.4. Eli Lilly’s Synthesis of Cryptophycin 52 6. PM060184 6.1. Discovery of PM050489 and PM060184 6.2. Synthetic Studies on PM060184 7. Discodermolide 7.1. Discovery and Background 7.2. Smith’s Gram-Scale Synthesis 7.3. Paterson’s Second-Generation Synthesis © 2017 American Chemical Society

7.4. Novartis’ Hybrid Synthesis 8. Diazonamide A 8.1. Background 8.2. Harran’s Construction of Diazonamide A 8.3. Development of DZ-2384 9. Ingenol 3-Angelate 9.1. Discovery and Development 9.2. Summary of Early Synthetic Work 9.3. Baran’s Synthesis of Ingenol 10. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References

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1. INTRODUCTION The laboratory synthesis of complex natural products and the manufacture of small-molecule drugs are chemistry compatriots that seldom meet. The former is typically an academic pursuit in which emerging methods are tested and assembly tactics are explored, whereas the latter are beautifully engineered exercises in brevity, efficiency, and scale. The two operate on common principles, of course, but thereafter, they focus on different goals with markedly different criteria for success. In fact, they diverge widely at the idea of practicality. However, “practical” is a nebulous concept that shifts with perspective and, ultimately, intention. According to the Oxford Dictionary, the colloquial definition of practical is “concerned with the actual doing or use of something rather than with theory and ideas”. What happens when a complex natural product is sufficiently compelling as a drug that a group attempts its manufacture? If the synthesis is Special Issue: Natural Product Synthesis Received: March 3, 2017 Published: June 12, 2017 11994

DOI: 10.1021/acs.chemrev.7b00126 Chem. Rev. 2017, 117, 11994−12051

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completed and there is a viable market for the product, is that not, by definition, practical? The most common means to access natural products on a large scale has been through semisynthesis. The sequential use of bioengineering and synthetic chemistry has allowed truly remarkable substances to be brought to market at reasonable cost.1 Examples from the recent literature include the Sanofi process to generate artemisinin, the Pharma Mar Trabectedin process, and the semisynthesis of paclitaxel by Bristol-Myers Squibb (Figure 1).2−7 In addition, microbial fermentation of

Figure 2. Microbial fermentation provides core structures utilized for peripheral functionalization.

such campaigns become more or less frequent in the future? It is with these questions in mind that this review was assembled.

2. HALICHONDRIN B 2.1. Introduction

This section describes academic and industrial explorations that led to the discovery and development of a commercialized synthetic route to eribulin mesylate, an anticancer agent sold by Eisai under the trade name Halaven. This molecule, inspired by the marine natural product halichondrin B, is the result of an intense drug-discovery program undertaken by the Eisai Research Institutes in Massachusetts and Japan. The synthetic path to the structure, far more complex than any other commercialized small-molecule drug,9 required significant optimization to secure a reliable manufacturing process. 2.2. Discovery of Halichondrins

In the early 1980s, a research team led by Uemura searched marine invertebrates for new biologically active natural substances.10 The group focused on the collection of a common sponge called Halichondria okadai Kadota, widely distributed along Japanese coastlines. Earlier studies of the same organism had led to the isolation of okadaic acid.12 The team suspected that the chemistry in the sponge was more extensive. Further fractionations of the extracts guided by hyperpotent antineoplastic activity resulted in the discovery of a new family of polyether macrolides, which they named halicondrins.10,11 From ∼600 kg of sponge, the group eventually purified eight small-molecule constituents that exhibited cytotoxic activities against several carcinoma cell lines (Figure 3).11 The most abundant of these was norhalichondrin A (1) with an isolated yield of (5.8 × 10−8)% (35 mg from 600 kg), whereas the most active proved to be halichondrin B (4), obtained in (2.1 × 10−8)% yield (12.5 mg) (Figure 3).11 Halichondrin B proved to be approximately 50 times more active than the more abundant norhalichondrin A (i.e., 1). These exciting results encouraged the team of isolation chemists to explore the in vivo activity of the compound. Halichondrin B was evaluated against three different types of human cancer cells in nude mice. The results are summarized in Figure 3. Halichondrin B displayed powerful activity at low concentration with both intraperitoneal and intravenous injections effectively doubling the life span of the diseased mice.11 The high potency exhibited

Figure 1. Commercial semisyntheses of complex natural products.

natural-product core structures has fueled an analogue industry, particularly valuable in the antimicrobial arena where serial drug resistance is a perennial challenge. Scores of β-lactam- and macrolide-derived antibiotics have been discovered, developed, and commercialized in this way (Figure 2).8 Semisynthetic methods are powerful and likely to become more so in the future. There are situations, however, in which these tools are not applicable. If an advanced intermediate cannot be sourced from nature or from heterologous expression systems or if earlier studies have identified structural analogues that are not accessible by peripheral functionalization, the remaining option becomes de novo total synthesis. In many cases, that goal has been deemed insurmountablethat is, hopelessly impractical. Nonetheless, the methods of synthesis are constantly being refined and expanded, providing new ways to solve complex problems. We were drawn to the relatively few examples in which audacious research groups bucked the trend and tackled the total synthesis of a natural product with the explicit intent of manufacturing the product. What can be learned from their efforts? Where does the state of the art stand in this context? Will 11995


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Figure 3. (A) Halichondrins isolated from Halichondria okadai Kadota sponge and cytotoxicity activity against B-16 melanoma. (B) Initial data on the in vivo activity of halichondrin B (4).11

became the main fragment-union technology employed on the industrial scale. Another defining feature of the approach utilized thermodynamically driven transformations to construct both the C38 spirocycle and ansa-bridged, caged C14 ketal late in the synthesis. This approach allowed large domains of the natural product to be evaluated biochemically as isolated entities and as part of increasingly elaborate architectures. Furthermore, each of the fragments was derived from readily available chiral poolderived raw materials (Figure 4). The synthesis of fragment 9 commenced with the transformation of the readily available 2-deoxy-L-arabinose (13) to generate 14 in four simple steps according to the known literature procedure.17 Further modification of the diethyldithioacetal 14 into 15 was then accomplished in 13 further steps.16 In the meantime, lactone 1618 was elaborated to give the phosphonate 17 in 10 steps16 and used for the Horner− Wadsworth−Emmons olefination of the aldehyde derived from

by halichondrin B both in vitro and in vivo coupled with its striking structure attracted considerable attention in the synthetic community. Shortly after the structure and the bioactivity were reported, several academic groups embarked on the challenge of producing synthetic samples of halichondrin B (4) to expand studies of its activity and address its extremely low natural availability. These efforts have been reviewed previously.13−15 This discussion focuses mainly on the efforts of Kishi’s group and the Eisai Research Institute that led to the industrial manufacturing of eribulin mesylate (Halaven). 2.3. Synthetic Studies toward Halichondrin B

The first total syntheses of halichondrin B (4) and norhalichondrin B (2) were reported by Kishi and co-workers in 1992.16 By design, the route was highly convergent and assembled the target molecules from four primary fragments (Figure 4); three of the four were equipped with the (E)-vinyl iodide motif, such that they could participate in Nozaki−Hiyama−Kishi coupling, which 11996


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Scheme 2. Synthesis of Fragment 10

starting from compound 25, readily produced from D-glucose diacetonide (24) in 10 steps.21 Modification of 25 through 11 steps generated intermediate 26, which, following nine further synthetic transformations, yielded fragment 11 for a total of 30 steps from 24 (Scheme 3).16

Figure 4. Retrosynthetic approach to the synthesis of halichondrin B by Kishi and co-workers.16

alcohol 15, to give fragment 9 in five further steps with the longest linear sequence of 22 steps from 13 (Scheme 1).16


Scheme 1. Synthesis of Fragment 9 through Coupling of 15 with 17

Finally, the synthesis of the last required fragment of halichondrin B took place according to procedures established by the group.22 The synthesis commenced with the 11-step transformation of butenolide 27, readily produced from ascorbic acid,23 into compound 28. Eight additional steps then yielded fragment 12 in a total of 19 steps (Scheme 4). With the key fragments in hand, what remained was to controllably join these fragments en route to halichondrin B. Initial coupling of fragments 9 and 10 by a Nozaki−Hiyama− Kishi reaction between the vinyl iodide in 9 and the aldehyde functionality in 10 produced a 6:1 diastereomeric mixture favoring the desired allylic alcohol. Subsequent base-promoted cyclization yielded the desired tetrahydropyran 29 in moderate overall yields.16 Removal of the pivalate protecting group, oxidation of the alcohol, and Nozaki−Hiyama−Kishi reaction between the resulting aldehyde and fragment 11 furnished, upon oxidation of the obtained allylic alcohol, the enone intermediate 30 in good overall yield (Scheme 5).16 Compound 30 was then treated with 2,3-dichloro-5,6dicyano-p-benzoquinone (DDQ) for the removal of the p-

Fragment 10 was synthesized according to a route previously established by Kishi’s group.19 Starting from D-galactose glycal, a four-step transformation generated compound 19, which then underwent stereoselective Ireland−Claisen rearrangement and, upon basic hydrolysis, furnished 20 with high diastereoselectivity. Subsequent iodolactonization of 20 followed by reductive dehalogenation gave lactone 21. This molecule was transformed into aldehyde 22 through 10 further simple synthetic steps. Nozaki−Hiyami−Kishi coupling between 22 and methyl trans-βiodoacrylate, followed by three further functional-group manipulations, yielded the bicyclic structure 23, which, upon four additional transformative steps and a final Dess−Martin oxidation, generated aldehyde 10.16 This compound was synthesized in 27 steps from 18 (Scheme 2). Resorting to previous efforts aimed toward the synthesis of halichondrins,20,21 Kishi’s team was able to obtain fragment 11 11997


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Scheme 6. Generation of Intermediate 32

Scheme 5. Coupling of Fragments 9, 10, and 11 for the Formation of Intermediate 30

(4) was produced in a sequence of 47 longest linear steps (Scheme 7). Scheme 7. Completion of the Synthesis of Halichondrin B (4)

methoxybenzyl (PMB) protecting group, the methyl ester was hydrolyzed, and the resulting seco-acid was subjected to cyclization under Yamaguchi’s conditions to furnish the macrolactone 31 in good overall yield. Removal of the silyl protecting groups with tetrabutylammonium fluoride (TBAF) followed by exposure to pyridinium p-toluenesulfonate (PPTS) triggered initial Michael addition of the C9 alcohol functionality to the enone. This was then followed by ketalization to generate the polycyclic moiety present in the natural product. Finally, blocking of the primary alcohol as its p-nitrobenzoate, protection of the secondary alcohol, and subsequent hydrolysis yielded intermediate 32 (Scheme 6).16 Compound 32 was then oxidized, and the resulting aldehyde underwent Nozaki−Hiyama−Kishi coupling with fragment 12 to generate an allyl alcohol intermediate that, following oxidation to an enone, cleavage of the silyl ether protecting groups, PPTS treatment, PMB cleavage, and final exposure to camphorsulfonic acid (CSA), produced halichondrin B in good yield (20−30% over six steps).16 Following this synthetic route, halichondrin B

2.4. Truncated Halichondrins Reveal Pharmacophore

Once a synthetic pathway to the natural product had been established, the attainment of a pure material supply to expand the biological studies of the molecule seemed feasible. In March 1992, the Decision Network Committee at the National Cancer Institute (NCI) recommended that halichondrin B be further explored as a therapeutic chemotype. In the same year, Eisai Research Institute (ERI) was provided with synthetic samples of halichondrin B and several intermediates for evaluation both in vitro and in vivo as antineoplastic agents. Surprisingly, intermediate 33 displayed activity comparable to that of the parent halichondrin B against the growth of cancer cells, more specifically in DLD-1 human colon cancer cells (Figure 5).24 This discovery began the important process of identifying more synthetically accessible analogues having equivalent or greater anticancer activity. Additionally, the results suggested 11998


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Additional SAR studies aimed at resolving the in vivo issue began with the evaluation of macrolactone analogues that were different in the C30−C38 region. From initial studies, compound 34, which bears a shortened chain at C36 and is epimeric at C35, emerged as the first simplified analogue of halichondrin B capable of inducing irreversible CMB upon washout in the assay (Figure 6). Various structural simplifications that would allow easier access to the compound were examined. To this end, it was found that modifications of the macrocyclic lactone core and most of its peripheral substituents were generally deleterious for the biological activity. Thus, it was decided to explore modifications of the octahydropyrano[3,2-b]pyran ring moiety constituted by the C29−C36 sector, where alterations were well tolerated. A simplified version bearing only a tetrahydropyran ring (i.e., 35) in place of the bicyclic structure was found to exhibit a similar range of biological activities when compared to 34 (Figure 6). Further structural modification led to compounds 36 and 37. Studies revealed that the replacement of the C31 methyl group with a methoxy substituent was well tolerated and greatly simplified the construction of the system, which utilized easily sourced carbohydrate precursors (Figure 6). With simpler structures capable of retaining the same biological characteristics as the parent architecture, attention next turned to an alternative strategy for improving potency. On the basis of X-ray data on norhalichondrin A and NMR analysis of halichondrin B, it was hypothesized that the replacement of the tetrahydropyran ring with a smaller and more constrained tetrahydrofuran ring could stabilize the bioactive conformation of the macrolactone ring, giving less conformational freedom to the structure and effectively “locking” it into a more active conformation.27 This hypothesis was corroborated by the activity of 38, which displayed activity equivalent to that of 37 (Figure 6). Further structural and configurational optimization led to 39 in which the hydroxymethyl substituent at C32 was replaced with a stereodefined vicinal propanediol. This compound exhibited high activity and was capable of maintaining CMB. However, further evaluation of compounds 36 and 39 (most promising candidates from each of the pyran and furan analogues) in a LOX human melanoma xenograft model revealed the in vivo inactivity of these structures. Many hypotheses were put forward to explain this lack of activity, with the most likely being the hydrolytic instability of the embedded macrocyclic lactone to nonspecific esterases, which are present in high levels in mouse serum.

Figure 5. Intermediate 33, a promising candidate in anticancer drug discovery.

that the macrocyclic moiety was the main pharmacophore of halichondrin B and that the polyether appendage could be significantly truncated (Figure 5). Microtubules are an important target of anticancer drugs as they play a crucial role during cell replication. Other potent anticancer agents such as taxanes and Vinca alkaloids disrupt the polymerization process of microtubules during mitosis, thus inducing apoptosis. Halichondrin B and its simplified analogues belong to the family of microtubule-targeting drugs, which block the growth of microtubules and transform tubulin into functionally inactive aggregates.25 It was shown that 33 directly binds to tubulin and that the same mechanism of action is shared by the related structure halichondrin B, which cemented the macrocyclic core as the antineoplastic pharmacophore.26 2.5. SAR Studies: The Discovery of Eribulin

The discovery of the macrocycle’s central role in the antiproliferative properties of the halichondrins allowed structure−activity relationship (SAR) studies to focus on peripheral functionalization, with the aim of both maximizing the biological activity and simplifying the structure. In contrast with halichondrin B, macrolactone 33 was found to be inactive in vivo, proving to be incapable of maintaining a complete mitotic block (CMB) in vitro after washout.27

Figure 6. Progression from an active fragment of halichondrin B to a variant having potent efficacy in vivo. 11999


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Intense investigation eventually led to the evaluation of nonhydrolyzable bioisosteres such as amide, ether, and ketone alternatives to the labile macrocyclic ester. As a result, compound 40 was identified as the best candidate to further drive the in vivo biological studies, as it proved highly effective in a variety of human cancer models (Figure 6). Additional structure optimization finally led to compound 41, which bears a primary amino group in place of the primary alcohol at C35 (Figure 6). This compound, which was later dubbed eribulin, proved extremely active, and for the first time, a simplified analogue of halichondrin B showed reversibility ratio equal to 1, meaning that the molecule was capable of maintaining a CMB even after a 10-h washout (Table 1). Table 1. Growth Inhibition Activities and Complete Mitotic Block (CMB) Reversibility Ratios of Evaluated Compounds27 compound

growth inhibition potency (DLD-1) IC50 (nM)a

CMB reversibility ratio (U937 cells)

4 33 34 35 36 37 38 39 40 41

0.74 4.6 3.4 2.5 1.8 2.0 1.0 0.67 1.0 20

3 >29 24 17 30 22 33 10 13 1

Figure 7. Retrosynthetic approach to eribulin (41).

nucleophilic acetylene anion of 44,31 which afforded a 3:1 mixture of regioisomers favoring the desired one.29 The intermediate homopropargylic alcohol was then partially reduced under Lindlar conditions. Acetylation of the secondary alcohol then furnished compound 46. Dihydroxylation promoted by OsO4, and subsequent mesylation of the obtained diol yielded product 47. The acetyl protecting group was cleaved to afford an intermediate alcohol, which underwent a facile cyclization to provide an intermediate substituted tetrahydrofuran ring. This material was then converted to methyl ether 48 through deprotection and methylation steps. The dioxolane was then hydrolyzed and reprotected as the bis-tert-butyldimethylsilyl (TBS) derivative. The benzyl protecting group was removed by hydrogenolysis, and the resultant alcohol was oxidized to afford key aldehyde 42 (Scheme 8). Although the new route demonstrated its feasibility by reducing the number of steps and increasing the yield of the desired final product 42, the search for an improved and more scalable pathway continued. An alternative approach to aldehyde

a Growth inhibition against DLD-1 cells after 3−4 days of continuous exposure.

From Table 1, it is possible to compare the in vitro biological activities of the compounds described above. Although eribulin exhibited the highest half-maximal inhibitory concentration (IC50) among the series tested, the reversibility ratio, which was utilized as a proxy parameter for in vivo activity, led to its selection for clinical trials. Eventually, these studies culminated in the discovery of a new anticancer treatment commercialized as its mesylate salt under the name Halaven by Eisai.


2.6. Synthesis of Eribulin from Milligram to Gram Scale

At the Eisai Research Institute, the development of a reliable and facile route to this extremely complex molecule was necessary to facilitate the advancement of preclinical and clinical studies.9 The key part of the synthetic strategy to eribulin was already established in earlier studies by Kishi and co-workers during the total synthesis of halichondrin B. From the retrosynthetic diagram depicted in Figure 7, it can be seen that the synthesis of eribulin can be traced back to formation of three separate fragments, two of which are identical to those used in the synthesis of halichondrin B (i.e., 9 and 11). The western portion (i.e., fragment 42 and its eventual successor 43) is the only part of eribulin that was not studied and developed during the synthesis of halichondrin B or as part of the SAR studies that followed. The initial discovery strategy commenced with L-arabinose and required just over 20 steps.28 However, the route was not ideal because the isolation of the desired material was complicated by the presence of a major isomeric side product. Although this route was capable of providing sufficient material (ca. 600 μg) for initial in vitro assessment, an improved route was developed to procure more substantial quantities of 41.29 In this improved route, the synthesis commenced with the ring-opening reaction of epoxide 4530 by attack of the 12000


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43 employing D-(+)-glucurono-6,3-lactone (49) as the starting material was demonstrated (Scheme 9).32 An α-deoxygenation

Scheme 10. Third-Generation Synthesis of Fragment 11

Scheme 9. Synthesis of Intermediate 54 en Route to Fragment 43

was facilitated in three steps to convert 49 to 50. 32 Diisobutylaluminium hydride (DIBAL-H) reduction of the lactone 50 to lactol followed by a Wittig reaction produced 51. Benzylation of the resulting secondary alcohol and subsequent asymmetric Sharpless dihydroxylation gave a 3:1 diastereomeric mixture favoring the desired isomer. Following bis-benzoylation of the diol and a diastereoselective glycosidic allylation, compound 52 was generated. Moreover, the high crystallinity exhibited by the desired diastereomer of this compound allowed its isolation in high purity by crystallization. Subsequent oxidation of the alcohol at C30, formation of the unsaturated sulfone by Horner−Wadsworth−Emmons reaction at the same position, FeCl3-promoted debenzylation, and asymmetric reduction of the sulfone by the directing effect of the alcohol at C31 furnished 53 as a single diastereomer. This compound was then methylated at C31, the two benzoyl esters were hydrolyzed, and the resulting diol was protected as the acetonide. Finally, ozonolysis of the resulting intermediate furnished aldehyde 54.32 This compound could then be transformed into fragment 43 in two simple steps (Scheme 9). This new synthetic approach was studied to satisfy the demands of scalability and general efficiency and to dramatically reduce the required number of chromatography steps to one.32 A few years later, Kishi and co-workers reported a secondgeneration variant of this approach to make it even more practical and scalable with concomitant removal of chromatographic purification steps.33 The synthesis of fragment 11, which constitutes the C1−C13 segment of eribulin, has been the focus of continual improvement to reduce the steps and to facilitate the supply of the desired product with increasing stereocontrol. The original route to 11 employed in the studies for the synthesis of halichondrin B required 30 steps from D-glucose diacetonide (24, Scheme 3). Later, Kishi and co-workers were able to reduce the number of steps by commencing the synthesis with a different starting material, namely, commercially available L-mannoic-γ-lactone (55, Scheme 10). Following this second-generation route, the group was able to secure fragment 11 in 16 steps.34 However, only 3 years later, the team was capable of further reducing the number of required steps. Their third-generation synthetic approach to fragment 11 required 12 steps from the same starting

material (i.e., 55), was carried out on a 100-g scale, and is described below (Scheme 10).35 Starting with L-mannoic-γ-lactone (55), initial double cyclohexylidene ketal formation, DIBAL-H reduction of the lactone, and Witting olefination of the resultant lactol smoothly produced 56. Catalytic asymmetric Sharpless dihydroxylation followed by acetalization and double acetylation yielded intermediate 57. Allylation of the glycosidic position with allyl silane 58 was promoted by BF3·Et2O. Treatment of the obtained product with Triton-B triggered the hydrolysis of the acetate, isomerization of the olefin to the conjugated position, and conjugate oxy-Michael addition to selectively produce compound 59. Subsequently, selective deprotection of the more sterically accessible cyclohexylidene ketal and oxidative cleavage of the resulting 1,2-diol generated an aldehyde, which, upon organometallic addition of 60 through Nozaki−Hiyama−Kishi coupling, furnished 61. Onepot hydrolysis of the remaining ketal and bis-silylation of the resulting diol, followed by iododesilylation achieved with NIS, generated 11 in 12 steps and 11% overall yield from 55 (Scheme 10).35 The synthetic pathway to fragment 916 was already discussed (see Scheme 1) during the description of the synthesis of halichondrin B.24 Over the years, a number of accounts detailing attempts to optimize the synthetic route have been reported in the literature.15,32,36 Most of these alterations of the original route were only partially beneficial and did not constitute substantial improvements of the route. However, in 2002, Kishi’s group reported a new concise synthetic approach to fragment 9, as a culmination of an impressive body of work on Nozaki− Hiyama−Kishi coupling15,32 (Scheme 11).37 The route commenced with the epoxidation of protected alcohol 62 followed by Jacobsen’s hydrolytic kinetic resolution38 of the obtained epoxide to furnish 63. The epoxide was opened through nucleophilic addition of the trimethylsilylacetylene 12001


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Scheme 11. Improved Route to Fragment 9

Scheme 12. Coupling of Fragments 42 and 9 for the Synthesis of Intermediate 71

Scheme 13. Synthesis of seco-Ketone 73 through Coupling of 71 and 72

anion, followed by mesylation of the resultant secondary alcohol and vinyl iodide formation, generated 64.39 Asymmetric Nozaki−Hiyama−Kishi coupling between 64 and aldehyde 65, promoted by catalyst 66, preceded the acid-promoted tetrahydrofuran formation and the subsequent removal of the benzoyl group to generate 67 isolated in high yield as a 9:1 diastereomeric mixture. Finally, oxidation of the primary alcohol to aldehyde and coupling with 68 under the conditions depicted in Scheme 11 generated 69 in good yield and 5.3:1 stereoselectivity.37 This product could then be easily converted to fragment 9 in three steps. With all three fragments then in hand, the construction of the eribulin (41) could take place. Coupling between 42 and 9 under Nozaki−Hiyama−Kishi conditions followed by a base-promoted tetrahydropyran formation (dr 3:1) and subsequent PMB cleavage generated compound 70. The primary alcohol was converted to the mesylate derivative, which was subjected to a nucleophilic substitution with thiophenol. The intermediate thioether was then oxidized to the sulfone, and upon removal of the pivalic protecting group, intermediate 71 was formed (Scheme 12). Sulfone 71 was then treated with nBuLi and added to aldehyde 72, obtained by DIBAL-H reduction of fragment 11, to generate an inconsequential mixture of diastereomeric alcohols, which were promptly oxidized to ketone 73 in high yield (Scheme 13). Once the three fragments had been combined into intermediate 73, a three-step sequence was employed that involved a SmI2-promoted desulfonylation, an intramolecular Nozaki−Hiyama−Kishi coupling between the vinyl iodide and the aldehyde functionality in 73, and subsequent oxidation of the obtained secondary alcohol to produce enone 74. Removal of the TBS protecting groups by action of TBAF and exposure to PPTS triggered the formation of compound 75 in a manner analogous to the original approach of Kishi and colleagues described in Scheme 6. Finally, selective tosylation of the primary alcohol followed by treatment with ammonia yielded eribulin (41) in high yield (Scheme 14).

2.7. Multikilogram Manufacturing Process of Halaven

Following the success of eribulin in preclinical40 and early clinical40−45 trials and of the relatively condensed reproducible synthetic pathway to the molecule just described, Halaven (i.e., eribulin mesylate) was approved in late 2010 by the U.S. Food and Drug Administration (FDA) for the treatment of patients with metastatic breast cancer who had already received at least two other chemotherapeutic treatments. At Eisai, the synthetic approach adopted to supply the material in multikilogram scale mirrored that used to provide material in gram scale for the preclinical and clinical studies, discussed above. However, as often occurs in industrial-scale manufacturing, additional optimization and modification of the synthetic route was necessary to obtain a cost-effective synthesis of the desired molecule; to reduce the number of purifications; and in general, to improve the practicality of the process. The retrosynthetic approach taken for the kilogram-scale synthesis of eribulin (41) can be traced back to the same three fragments previously employed, with the only difference being the use of vinyl triflate 88 in place of vinyl iodide 9. The synthesis of fragment 8046 commenced with hydration of the readily available dihydrofuran 76 promoted by Amberlyst resin, followed by the organometallic allylation of the 12002


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Scheme 14. Final Steps toward Eribulin (41)

intermediate lactol, to generate 78 as a racemate. Selective protection of the primary alcohol, followed by simulatedmoving-bed (SMB) chiral chromatography,47,48 allowed separation of the two enantiomers. The desired isomer was then tosylated to furnish compound 80, whereas the configuration of the undesired enantiomer was inverted by Mitsunobu reaction and then subjected to tosylation (Scheme 15).46

Scheme 16. Synthesis of Fragment 88 at the Multikilogram Scale

Scheme 15. Synthesis of Vinyl Bromide 80

The other portion of fragment 88 began with Jacobsen hydrolytic kinetic resolution38 of epoxide 81 followed by ringopening by the enolate of diethyl malonate and subsequent cyclization, furnishing lactone 82. Selective hydrolysis of the ester, decarboxylation, and diastereoselective methylation generated 83 as a 6:1 mixture in favor of the desired isomer. Trimethylaluminum-promoted lactone opening by dimethyl hydroxylamine, followed by protection of the resulting secondary alcohol, OsO4-mediated dihydroxylation of the olefin, and oxidative cleavage of the resulting diol by NaIO4 furnished aldehyde 84 in high stereopurity (Scheme 16).46 The union of intermediates 80 and 84 was then achieved by an asymmetric Nozaki−Hiyama−Kishi coupling between the two, adopting the previously developed and highly optimized conditions.32,37,39,49 This set of conditions involved the use of the chiral ligand 85, which was selected to induce stereoselectivity. In addition, it was fortuitously found to increase the reaction rate. In this instance, the process team at Eisai further developed the conditions established for this reaction to minimize side-product formation and maximize the reproducibility of the process. To

avoid base-promoted tosylate elimination, compound 86 was instead cyclized to form the intermediate tetrahydrofuran ring as an 8:1 diastereomeric mixture at C20, using SiO2 as a promoting agent. This material was then treated with methyl Grignard to form the methyl ketone, the enolate of which was then trapped as triflate 87 by means phenyl triflimide. Upon removal of the silyl protecting groups, the diastereomeric mixture of compounds was purified by preparative high-performance liquid chromatography (HPLC) and the desired isolated product was then protected as pivalate at the C14 alcohol and mesylated at C23 to furnish 12003


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on the gram scale reported in Scheme 10, with some modification necessary to improve the scalability (Scheme 18).50 To reduce

fragment 88 in high stereochemical purity, although in relatively low yield in 15 steps (longest linear sequence) from 81 (Scheme 16). The route to fragment 43 is a direct evolution of the path employed for gram-scale construction (Scheme 9), with some improvements to the process that were deemed necessary for large-scale manufacturing.46 It began with the same starting material 49, which was converted to 50 in three simple steps without the use of tin hydride, which was employed in the original route (Scheme 17). Lactone 50 was transformed into a

Scheme 18. Kilogram-Scale Synthesis of Fragment 11

Scheme 17. Kilogram-Scale Synthesis of Fragment 43

lactol by DIBAL-H reduction. Addition of the organomagnesium reagent derived from (trimethylsilyl)methyl chloride to the lactol, followed by Peterson olefination and benzylation of the secondary alcohol, produced 89. This path allowed the team to avoid the use of Wittig chemistry to produce the allylic substituent, thus circumventing the production of triphenylphosphine oxide that would have required removal according in the original route. Conversion of 89 to 90 is procedurally identical to the route reported in Scheme 9, with the exception that the intermediate alcohol was oxidized to 90 using a Moffat oxidation in place of the Swern that was originally employed. Transformation of the ketone to an unsaturated sulfone, debenzylation, and directed asymmetric reduction of the conjugated olefin was achieved in the same manner as described in Scheme 9. However, the benzyl cleavage was accomplished through a homogeneous process employing trimethylsilyl iodide (TMSI) instead of FeCl3. The compound obtained was then treated with K2CO3 in MeOH to hydrolyze the benzoate groups, furnishing 91, which was purified by crystallization, avoiding chromatography. Protection of 91 as its acetonide followed by methylation of the secondary alcohol, subsequent hydrolysis of the ketal, and reprotection of the diol as the tert-butyldimethylsilyl ether generated the precursor to the final product 43. The methylation in this instance was performed without the use of the silver reagent originally employed (Scheme 9). Aldehyde 43 was then obtained by ozonolysis of the olefin, employing a mild hydrogenation as the reducing agent for the ozonide formed, yielding 43, which was purified by crystallization. Following this path, 43 was obtained in a chromatography-free 20-step process in a moderate overall yield (Scheme 17).46 The kilogram-scale manufacturing process to access fragment 11 was directly derived from the synthesis of the same molecule

the cost of the process and to overcome issues with the availability of the original starting material L-mannoic-γ-lactone (55, Scheme 10), it was decided to initiate the synthesis with the less expensive and easily accessible C11 epimeric material D(−)-gulono-1,4-lactone (92, Scheme 18). The configuration of the C11 alcohol was not relevant, as the stereocenter would be lost and re-formed during the route. The first three steps of the synthesis to furnish compound 93 proceeded in accordance with those described in Scheme 10. The only modifications were applied in the Wittig reaction to improve the purity while avoiding the need for chromatography. These modifications consisted of the addition of the substrate to the preformed ylide, removal of the unreacted triphenylphosphine with maleic anhydride, and removal of the formed triphenylphosphine oxide by precipitation. Dihydroxylation of 93 with subsequent hydroxypyran formation as reported in the gram-scale route (Scheme 10) was followed by treatment with ZnCl2/AcOH/ Ac2O to deliver compound 94. This last step consisted of a modification adopted by the team as a solution to difficulties encountered in the subsequent glycosylation when conducted on larger scale. In this manner, selective removal of one cyclohexylidene and peracetylation delivered crystalline compound 94 that smoothly underwent allylation with allyl silane 58, followed by global deacetylation and ring formation when treated with NaOMe to yield 95. This compound, upon oxidative cleavage of the diol, generated an aldehyde that was then transformed into 61 following the same procedure as reported in Scheme 10. The final three steps to fragment 11 proceeded as already described in Scheme 10 with some small modifications to avoid the use of a known sensitizer (i.e., chloroacetonitrile), which, at that scale, might have been dangerous for the production personnel (Scheme 18).50 12004


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Scheme 19. Final Steps for the Kilogram-Scale Synthesis of Eribulin (41) and Its Conversion to the Commercialized Mesylate Derivative Halaven (97)

(Scheme 19). The additional five steps necessary to access product 75 were consistent with the gram-scale process depicted in Scheme 14. However, the intramolecular Nozaki−Hiyama− Kishi coupling had never been performed in quantities above 6− 7 g and required days for completion. To increase the rate of the reaction, it was decided to apply the conditions for the asymmetric variant of the coupling, previously developed in the synthesis of fragment 88 (Scheme 16). Although there was no need for asymmetric induction, as subsequent oxidation cleared the newly formed stereocenter, application of this set of conditions was capable of producing the desired product in better yield and shorter time when compared to the ligand-free variant. Ultimately, the process team was able to apply the transformation to kilogram-scale batches (Scheme 19).51 Finally, transformation of 75 into eribulin upon purification by crystallization was easily achieved by converting 75 into the primary tosylate and then treating it with NH4OH, to generate an epoxide intermediate that was then opened by a second reaction with ammonia, furnishing 41. This was then converted to its mesylate salt by treatment with ammonium mesylate, to furnish Halaven (Scheme 19).51 The journey of halichondrin to the discovery of eribulin and its commercialization as Halaven is the perfect example of how challenging the development of a new drug candidate can be. Great effort was made by Kishi’s group in the development of new conditions and strategies for the Nozaki−Hiyama−Kishi coupling, which proved crucial for the construction of several C− C bonds in the process. In this regard, great admiration must be shown to the scientists at Eisai involved in this monumental project that developed an industrial manufacturing route to Halaven, which was approved by the FDA in 2010 for the

Coupling of fragments 43 and 88 through asymmetric Nozaki−Hiyama−Kishi coupling under the same conditions as developed for the formation of 86 in Scheme 16 were then applied in this instance, generating a 20:1 diastereomeric mixture (Scheme 19).46 This mixture was subsequently transformed into the intermediate pyran by the action of potassium hexamethyldisilazide (KHMDS) at low temperature, followed by DIBAL-H treatment to remove the pivalate to furnish 71, which was then purified by chromatography and crystallization (Scheme 19).46 Coupling between intermediate 71 and the aldehyde derived from 11 commenced with the DIBAL-H reduction of fragment 11 to the aldehyde, followed by nucleophilic addition of the anion derived from sulfone 71 upon deprotonation with n-BuLi (Scheme 19).51 This path is identical to that used in the gramscale route. However, several optimizations were necessary to improve the process for kilogram scale. One of the problems encountered was the ready oxidation of the aldehyde to the corresponding acid. Extensive experimentation revealed that the addition of small amount of butylated hydroxytoluene (BHT) to the aldehyde-containing fractions upon chromatography and before concentration would prevent this problem. Sulfone− aldehyde coupling also required optimization to maximize the product yield and solve irreproducible results. Upon intense interrogation, including deuterium-labeling studies, optimal conditions were developed and applied to the process, which furnished the desired aldehyde in high yield.51 This two-step transformation was recently successfully demonstrated under continuous-flow conditions.52 Subsequent Dess−Martin oxidation generated a 1:1 diastereomeric mixture of ketone−aldehyde 73. It was found that addition of a catalytic amount of water to the reaction mixture dramatically increased the yield, the rate, and the reproducibility of the reaction 12005


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treatment of metastic breast cancer and in 2016 for the treatment of inoperable liposarcoma.53

3. SYNTHETIC TETRACYCLINES 3.1. History of the Tetracyclines

In the early 1940s, a team of scientists led by the renowned botanist Benjamin Duggar made a seminal discovery at Lederle Laboratories.54 A soil sample retrieved from a field in Missouri, when cultured and extracted, exhibited extraordinary growth inhibition against all strains of bacteria in the preliminary screen.54 Subsequent tests of this extract continued to astonish Duggar and his co-workers, as the extract proved efficacious against several infectious bacteria for which there was no cure at the time.54 The unprecedented activity of this material, named Aureomycin (98) for the gold color of its cultures, prompted it to be one of the first labeled “broad-spectrum antibiotics”.55 Because of the unanticipated potency of Aureomycin, American Cyanamid utilized large-scale fermentation to obtain commercial quantities of the substance and quickly pushed it into the clinic within a year of its initial report.54 The effectiveness of Aureomycin against a broad spectrum of bacteria made it highly successful.54 Other companies were quick to start scouring the biosphere for other potential blockbuster antibiotics. Pfizer was one of the first to publish its findings on a new extract coined Terramycin (the producing organism was isolated from a soil sample from Terre Haute, IN).54,56 Pfizer rapidly capitalized on Terramycin after obtaining FDA approval in 1950, entering the new broad-spectrum antibiotic market in force and catapulting Pfizer toward being the pharmaceutical giant it is today.54 Even though Aureomycin (98) and Terramycin (99) were wildly successful, the molecular structures of the extracts’ active components were elusive to scientists for several years after their discoveries. Both Pfizer and American Cyanamid cooperated by sharing samples to solve the structures. Eventually, a Pfizer-led team aided by R. B. Woodward deciphered the enigmatic structure and in 1954 published their findings in a seminal article.57,58 They discovered that the structures of Aureomycin (98) and Terramycin (99) (Figure 8) shared the same tetracyclic napthacene core with differential functional ornamentation, a chlorinated D ring in Aureomycin and a B ring hydroxyl in Terramycin. The elucidation of the molecular skeletons of these compounds ushered in the age of semisynthetic derivatization of the tetracycline motif. However, during this time period, it was widely assumed that chemical alterations of the natural-product structures would result in lower bioactivity.54 In 1955, Lloyd Conover discovered that a simple hydrogenolysis of the chlorine of Aureomycin gave rise to a compound that was more potent, soluble, and pharmacologically active than Aureomycin. This novel compound became the namesake for this entire class of antibiotics: tetracycline (100).59,60 After the discovery that manipulations of the functionality around the skeletal core of tetracycline could potentially produce more potent compounds, several other tetracycline derivatives entered the clinic in relatively rapid succession, with minocycline (101) being the most recent in 1971.54 After minocycline, the development of novel tetracyclines remained stagnant for several decades until growing antibiotic resistance provoked Wyeth into reinvestigating the tetracycline scaffold. Introduction of a second amino group on the D ring of the minocycline skeleton and appending of a modified glycine residue resulted in tigecycline

Figure 8. Structures of Aureomycin, Terramycin, tetracycline, minocycline, and tigecycline, along with the numbering scheme for the tetracycline core.

(102).63,64 Tigecycline gained FDA approval in 2006.54 However, the field of tetracycline derivatization has remained a continual of cycle of isolation and peripheral modification.65 This tactic has its limitations and is unable to access core structural changes. X-ray crystallography studies have allowed examination of how the tetracyclines interact with the bacterial ribosome and indicate that there are sections of the D ring where modifications could be utilized (Figure 9).61,62 Unfortunately, semisynthetic methods are barred from accessing these sites of modification. However, the development of a practical synthetic route would allow for a more thorough exploration of the tetracycline SAR. The rich history of tetracyclines includes valiant attempts at its total synthesis. The dense array of stereocenters (e.g., five contiguous stereocenters in the case of 99) is a formidable task in itself for these deceptively simple polycycles. In addition, these compounds have proven to be sensitive to a variety of environments, which further complicates the modification of the parent structures (Scheme 20).66 In acidic media, the C6 hydroxyl group, which is both benzylic and tertiary, is poised for facile dehydration to produce anhydrotetracyclines (104).66 The C4 dimethylamino group is prone to epimerization in mildly acidic solutions, and this process is accelerated by certain counterions.66 In basic media, the C and B rings undergo a retro-Dieckmann reaction, generating γ-butyrolactone 108.66 Suffice it to say that developing a successful route to the tetracycline scaffold requires extensive forethought and finesse in the execution of each step. It is no surprise that, after collaborating to help unravel the skeleton of the tetracyclines, Woodward, in collaboration with a group from Pfizer, was the first to establish a route to the tetracycline core in 1962 (Scheme 21).67−69 The Woodward route focused on building the napthacene core one ring at a time starting with 12006


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the D ring and produced 6-demethyl-6-deoxytetracycline. Woodward’s synthesis was quickly followed by that of Shemyakin and colleagues in 1963, which adapted the same strategy as Woodward starting from juglone.70 An alternative approach that involved constructing multiple rings in a spectacular cascade of Dieckmann condensations was pioneered by Muxfeldt and co-workers in 1979.71,72 Tatsuta and co-workers established the first asymmetric route to tetracycline (100), utilizing a protected form of D-glucosamine as the starting material in 2000.73 Stork et al.’s approach to the tetracycline core also used a remarkable cascade of Dieckmann condensations to construct the tetracycline nucleus.74 Although each of these syntheses is elegant in its own approach to the synthetic tetracycline problem, the major drawback of all of them, with the exception of Stork et al.’s, lies in their efficiency, the highest being that of the Muxfeldt approach at 0.06% overall yield. However, the Stork synthesis constructs racemic 12adeoxytetracycline 124 in 18−25% yield over 16 steps. Unfortunately, the 12a-deoxy variants are plagued by a major reduction in potency, which means that derivatives constructed by this route would be handicapped from the start.75 In about 2005, semisynthesis was still the most viable method of obtaining and modifying tetracycline derivatives. However, that was soon to change. Andrew Myers and co-workers, armed with the knowledge of the inherent limitations of the semisynthetic preparations of tetracyclines, as well as the drawbacks of the current synthetic state of the art, set out to devise a practical synthesis that would allow manipulation of sites inaccessible by current means. 3.2. Myers’ Synthetic Studies on the Tetracyclines

The Myers tetracycline route commenced with unornamented benzoic acid, which was subjected to microbial-mediated dihydroxylation with a mutant strain of Acaligenes eutrophus (Scheme 22).76,77 This yielded diol 126 in 79% yield and >95% ee; in addition, this process was easily scaled up to 90-g batches in an academic setting.77 This process introduces the troublesome 12a hydroxyl group that was absent in the Stork synthesis in the first step, which allows derivatives from this approach to be equipotent with their natural congeners. Myers and co-workers

Figure 9. (A) X-ray crystal structure of tetracycline bound to a bacterial 30S ribosomal subunit and (B) pictorial representation of the key interactions between the tetracycline motif and its receptor61,62 [Protein Data Bank (PDB) accession number 1HNW]. (Reproduced with permission from ref 62. Copyright 2000 Elsevier.)

Scheme 20. Chemistry of Tetracycline under Various Conditions as an Example of the Sensitivity of These Systems

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Scheme 21. Summaries of the Woodward, Shemyakin, Muxfeldt, Tatsuta, and Stork Approaches to the Tetracycline Motif

Scheme 22. Myers Synthesis of the AB Ring System of the Tetracycline Core

then proceeded to build the rest of the core around what became the B ring. The diol was subjected to a hydroxyl-directed epoxidation with m-chloroperoxybenzoic acid (m-CPBA) to afford the α-epoxide 127 in 83% yield. The acid functionality was then esterified utilizing trimethylsilyldiazomethane. The allylic

epoxide was then isomerized when the molecule was treated with excess tert-butyldimethylsilyl triflate and trimethylamine to produce epoxy ester 128. For the next series of transformations, Myers and co-workers took inspiration from the work of Stork et al. by utilizing functionalized isoxazole 130 as a synthon for the 12008


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heated in the presence of trimethylphosphite to induce a Mislow−Evans rearrangement to afford allylic alcohol 142. Alcohol 142 was converted to a benzyl carbonate, and the system was subjected to a deprotection/oxidation/protection sequence similar to that described above to afford key enone 143. With these pivotal enones in hand, Myers and co-workers could construct the tetracycline skeleton with all of the desired stereochemistry and functionality in protected form in a masterstroke. The C ring was constructed in a deft introduction of the D ring into the system. Subjection of enones 139 and 143 to anionic D-ring precursors resulted in conjugate addition to the enone, followed by a Dieckmann condensation of the appended phenyl ester, closing the C ring and completing the skeleton of the tetracycline framework (Scheme 25).65 This transformation was essential for the development of novel tetracycline analogues, which is discussed below. In the case of doxycycline (149), Myers and co-workers deprotonated phenyl benzoate 144 with lithium diisopropyl amide (LDA) in the presence of tetramethylethylenediamine (TMEDA) under cryogenic conditions and then introduced enone 139 into the reaction mixture, which then beautifully executed the desired Michael−Dieckmann cascade to provide pentacycle 147 in 79% yield as a single diastereomer.65 Pentacycle 147 was then subjected to mild TBS/tert-butoxycarbonyl (Boc) deprotection and a triple hydrogenolysis of the benzyl carbonate, benzyl-protected imidate, and isoxazole N−O bond to provide doxycycline 149 in 90% yield. This approach provided doxycycline in a longest linear sequence of 18 steps, 22 overall, and 11% overall yield. However, Myers and co-workers did not only establish a practical route to doxycycline. To exhibit the power and practicality of their route, they proceeded to synthesize several novel tetracycline derivatives that are inaccessible by current semisynthetic means, as discussed below. In a subsequent publication, Myers and co-workers also utilized enone 139 to construct the parent tetracycline 100 (Scheme 26).81 The enone was subjected to α-bromination with pyridinium tribromide followed by bromide displacement with thiophenol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford vinyl sulfide 150.81 The selection of the phenylthio group was 2-fold: It served to activate the system for the ensuing [4 + 2] cycloaddition and functioned as a handle to introduce unsaturation later in the sequence. Vinyl sulfide 150 was then heated with triethylsiloxybenzocyclobutene derivative 151, generating the reactive o-quinone dimethide in situ to serve as the 4π component of the Diels−Alder cycloaddition.82−84 Pentacycle 153 was produced from the cycloaddition as a single diastereomer. In addition, lactone 154 was isolated as an interesting side product of the reaction, which is thought to be the result of a retro-Dieckmann of pentacycle 153.81 Pentacycle 153 was then subjected to TBS deprotection and oxidation to produce ketone 155. The second function of the phenylthio substituent then came into play. Subjection of 155 to TFA protonated the dimethylamino group serving as in situ protection, introduction of m-CPBA into the system then oxidized the sulfide to the sulfoxide, and gentle warming resulted in elimination to afford unsaturated tetracycline derivative 156.81 Unsurprisingly, the anhydrotetracycline derivative proved difficult to isolate, given that the facile stereoselective photooxidation of similar anhydrotetracyclines to the corresponding hydroperoxytetracyclines has been known for decades.66,85−87 Myers and co-workers used this effortless photo-oxidation to their advantage and simply stirred the crude anhydrotetracycline 156 in chloroform open to the air to produce hydroperoxide 157,

vinylogous carbamic acid present in the A ring of the tetracycline core.78 Starting from glyoxylic acid, isoxazole 130 was prepared in four steps and on a mole scale in an academic setting. Myers and co-workers intriguingly decided to modify the order of bond formations for the A ring, so that it was the reverse of that of Stork et al.’s approach. Treatment of isoxazole 130 with n-BuLi generated the lithiated isoxazole at C4, which was subsequently treated with epoxy ester 128. This process provided ketone 131 in 70% yield. To close the A ring, gentle warming of the ketone with catalytic amounts of lithium triflate facilitated a putative SN2′ opening of the allylic epoxide through the pendant dimethylamino group. The α-position of the resultant quaternized amine was then deprotonated to produce an ylide, which underwent a [2,3]-sigmatropic rearrangement to afford tricycle 135. This transformation was likened to the classic Sommelet−Hauser rearrangement. This process provides the desired cis fusion of the AB rings and the α-oriented dimethylamino group. The crude material was then treated with trifluoroacetic acid (TFA) in dichloromethane (DCM), to achieve a mild deprotection of the allylic silyl ether in the presence of the other silyl ether to afford tricycle 136. Tricycle 136 is a key branching point, from which Myers and co-workers could prepare either 5-deoxy- or 5-hydroxytetracycline derivatives. To prepare 5-deoxy derivatives, tricycle 136 was subjected to a reductive allylic transposition with triphenylphosphine, diethyl azodicarboxylate (DEAD), and o-nitrobenzensulfonyl hydrazide to afford cyclohexene 137 (Scheme 23).79 Cyclohexene 137 was then subjected to acid-mediated TBS deprotection, oxidation, and protection of the 12a-alcohol to provide key enone 139. Scheme 23. Elaboration to the Protected 5-Deoxy Precursor

The 5-hydroxy derivatives could be accessed by converting the allylic alcohol of tricycle 136 to the sulfide while retaining the desired stereochemistry (Scheme 24). The sulfide was then oxidized with chiral oxaziridine 141 in a diastereoselective fashion to the sulfoxide in 99:1 selectivity.80 The system was then Scheme 24. Elaboration to the 5-Hydroxy Precursor

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Scheme 25. Completion of the Total Synthesis of Doxycycline (149)

Scheme 26. Myers’ Elaboration of Enone 139 to Tetracycline (100)

which was subsequently hydrogenated in the presence of Pd black to afford tetracycline (100) in 42% yield over three steps. This route constructed tetracycline in a longest linear sequence of 18 steps and an overall yield of 1.4%. Once Myers and co-workers had a viable, convergent, and practical route to the tetracycline skeleton, the next step was to start exploring uncharted chemical space in terms of tetracycline derivatives. In the original communication, two tetracycline derivatives contained a nitrogen atom in the D-ring systems, which are now nominally referred to as azatetracyclines. Myers and co-workers prepared 7-aza- and 9-azatetracyclines, where the 7-azatetracycline exhibited modest activity in the small bacterial screen that Myers and co-workers performed.65 It is at this point that Tetraphase Pharmaceuticals began investigating these novel tetracycline derivatives.

Figure 10. Myers’ novel azatetracyclines, azatetracycline numbering notation, and Tetraphase’s most active azatetracycline.

3.3. Tetraphase SAR Studies

clines with substitution at the 7-position were tested in an in vitro assay with both Gram-positive (S. aureus and S. pneumoniae) and Gram-negative (E. coli and K. pneumoniae) bacterial strains, each as the wild type or expressing a tetracycline resistance gene. It was determined that substitution at this position was well tolerated

Tetraphase quickly determined that moving the nitrogen from the 7- to the 8-position led to compounds that were comparable to the parent tetracycline and its semisynthetic derivative minocycline (Figure 10).88,89 Initially, a variety of 8-azatetracy12010


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and that the compounds retained or had improved activity against the tetracycline-susceptible strains, with the 7-fluoro analogue being the most potent.88 Unfortunately, all of the derivatives tested at this stage were substrates for the tet(A) efflux pump.88 To make a direct comparison to tigecycline (102), Tetraphase proceeded to prepare the corresponding 9-glycylamido-8azatetracycline analogues. However, this modification also proved to be unsuccessful, and these derivatives exhibited poor activity in the whole-cell assays; however, when subjected to a cell-free transcription/translation assay, they showed activity comparable to that of tigecycline (102). This indicated that these derivatives had difficulty getting to the target in the whole-cell assay, ostensibly due to poor permeability.88 In an effort to increase the permeability of the compounds, 9-amino-8-azatetracycline derivatives were explored. In this series of compounds, it was found that activity against the elusive tet(A) efflux pump began to emerge. 9Amino-8-azatetracycline 161 proved to be the most potent in the whole-cell assays and showed activity commensurate with that of tigecycline (102) in cell-free translation/transcription assays.88 Further investigation into the pharmacokinetic profile of compound 161 in Sprague−Dawley rats revealed that it had a 4-fold higher area under the curve (AUC) and an almost 5-fold lower clearance than tigecycline (102), but the oral bioavailability was functionally identical to that of tigecycline.88 In the S. aureus septicemia models, 161 and tigecycline (102) were comparable in protection, even though tigecycline (102) was more potent in the initial minimum inhibitory concentration (MIC) screens. In the E. coli septicemia model, 161 required a higher dosing for protection equivalent to that provided by tigecycline (102). However, Tetraphase was not satisfied with the activity profile of 161 and decided to investigate other variations to the tetracycline core. At the same time as the company was investigating the 8azatetracyclines, Tetraphase also explored another series of novel tetracyclines first constructed by Myers and co-workers, the pentacyclines (Figure 11).90−92 These compounds contain an

tigecycline (102), with several showing efficacy comparable to that of tigecycline (102) in vivo with septicemia mouse models.91 The third variation on the tetracycline framework explored by Tetraphase was the introduction of novel functionality at C7 of the traditional scaffold accompanied by an array of C9 substituents.93−95 Tetraphase examined several novel substituents and found that extremely potent compounds resulted from fluorine substitution at C7, which Tetraphase has designated as “fluorocyclines”.93,94 Although a C7 fluorotetracycline had been synthesized prior to Tetraphase’s investigations, the conditions for fluorine introduction were harsh and not amenable to industrial processing.96 When coupled with a C9 glycylamido substituent, the fluorocyclines displayed a range of activities. However, one particular analogue, 164, also referred to as TP434, proved to be exceptionally potent, beating or matching tigecycline (102) in the initial antibacterial screen (Figure 12).93 Attempted modification of the appended pyrrolidine ring of 164 resulted in activity loss.93

Figure 11. Myers’ novel pentacycline and the pentacycline numbering system.

3.4. Eravacycline Synthesis

Figure 12. Tetraphase’s hyperpotent, completely synthetic fluorocycline, TP-434, aka Eravacycline.

Examination of the in vivo activity of TP-434 in tetracyclineresistant mouse septicemia models revealed excellent efficacy with 50% protective dose (PD50) values lower than that of tigecycline (102).93 The pharmacokinetic (PK) properties of TP-434 were also found to be favorable, having a larger AUC value than tigecycline and a half-life similar to those of tigecycline and tetracycline.93 Further investigations into TP-434 revealed that it was active not only against strains carrying the three main tetracycline-specific efflux pumps but also against ribosomal protection mechanisms and enzymes that target tetracyclines.97 TP-434 retained activity against strains resistant to multiple antibiotic classes, such as those with β-lactamases.98 One intriguing result disclosed by Tetraphase was that TP-434 showed potent activity in vitro against established biofilms.99 The extremely promising results for TP-434 induced Tetraphase to explore it in higher animals, where the PK data indicated that it had an acceptable bioavailability of ∼25% in chimpanzees and >10% in rats, mice, dogs, and cynomolgus monkeys.97 These promising preliminary results prompted Tetraphase to move forward into clinical trials. To provide an adequate material supply for the upcoming clinical trials, the process division of Tetraphase began the development of a route that would improve upon Myers’ initial synthesis that could be utilized on an industrial scale. Fortunately, Brubaker and Myers reported an improved route to enone 139 that Tetraphase employed to full advantage by modifying it to suit their needs (Scheme 27).100 Tetraphase’s route commenced with the bromination of dimethyl maleate (165), followed by treatment with potassium tert-butoxide and hydroxyurea to afford isoxazole 168.101,102 Subsequent benzyl protection of the hydroxyl group and DIBAL-H reduction furnished aldehyde 170.101 Aldehyde 170 was condensed with the (S)-variant of the Ellman auxiliary

additional ring appended to the tetracycline scaffold at C8 and C9, aptly named the E ring. The new ring introduced several new derivatization sites for the tetracycline scaffold to be explored.91 Tetraphase explored substitution at all four of the new substitution sites and found that most retained some degree of activity. However, those that proved most potent contained a methoxy group at C7 and a small alkylaminomethyl group at the new C10-position prototype structure 163. However, all of the novel pentacyclines proved to be less potent in vitro than 12011


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Scheme 27. Construction of Chiral Amine 175

Scheme 28. Completion of the Industrial Synthesis of Eravacycline, the First Fully Synthetic Tetracycline To Be Taken into the Clinic

171, providing sulfonyl imine 172. A diastereoselective addition of vinylmagensium chloride was facilitated by catalytic amounts of dimethyl zinc and lithium chloride generated in situ.101 It was postulated that a highly reactive triorganic zincate was generated in situ and that activation of the imine by lithium/magnesium complexation led to an exacerbation of the stereochemical effect of the auxiliary. The crude sulfonylamine 173 was then telescoped into a one-pot deprotection/reductive methylation by first treatment with HCl followed by addition of sodium acetate, paraformaldehyde, and picoline−borane to afford allylamine 175. The purity of this chiral amine could be upgraded by salt formation with L-tartaric acid to afford a crystalline solid that was easily manufactured, stored, and transported.101 The salt could be converted back to the free amine by simple treatment with aqueous NaOH. Next, the 4-position of the isoxazole 175 was deprotonated with the magnesium salt of 2,2,6,6-tetramethylpiperidine (TMP) and then treated with furfural derivative 177 to afford alcohol 178 as a mixture of diastereomers (Scheme 28).100,103 Alcohol 178 was then refluxed in dimethyl sulfoxide (DMSO) with Hünig’s base to effectuate an intramolecular Diels−Alder reaction, affording a mixture of four diastereomers with the endoproduct being favored in a 45:1 ratio from the (S)-alcohol and a 1:1 ratio from the (R)-alcohol.100,103 After the reaction mixture had been allowed to cool, sulfur trioxide pyridine was added to furnish oxidation of the alcohol to provide ketone 179.103 A Lewis-acid-mediated cleavage of the oxabicycle with tandem enol ether demethylation followed by TBS protection of the tertiary alcohol afforded enone 139 after recrystallization.100,103 Enone 139 was then combined with aniline derivative 181 by means of Myers’ Michael−Dieckmann cascade under conditions that were optimized by Tetraphase.104 The entire system was then deprotected in two steps, first treatment with aqueous HF followed by a powerful global hydrogenolysis in the presence of HCl to prevent epimerization of the C4 dimethylamino group.104 Aniline 184 was then acylated with acid chloride 185 to afford the

free base of eravacycline (164), which was then converted to the bis-hydrochloride salt 186.104 This route was utilized to provide ample material supply for the early clinical studies and is currently still being optimized. The overall process has a longest linear sequence of 17 steps and an overall yield of 16.2%. With the issue of supply addressed, investigations of the safety, tolerability, and PK properties of TP-434 in humans went smoothly, revealing no adverse effects and establishing the oral bioavailability at 28% with oral doses being well tolerated.97 The effectiveness of eravacycline against complicated intra-abdominal infection was then studied in phase II clinical trials.105 TP-434 was shown to have an efficacy commensurate with that carbapenem and continued into phase III.105 In the first portion of phase III, eravacycline was compared to ertapenem, and it was disclosed that there were no significant differences between the treatments.106 Phase III trials for eravacycline are currently ongoing and look promising. To date, Tetraphase has constructed over 3000 tetracyclines with novel functionality, many of which are potent antibiotics (see Figure 13).107 These analogues were constructed from the 12012


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activity of epothilones in 1987. However, their cytotoxicity made them less suitable as antifungal agents, and there were also questions about their selectivity for other applications.112 For these reasons, the epothilones had lain dormant in the literature for several years until Merck’s rediscovery in 1995. Merck determined that epothilone B was more active than paclitaxel in tubulin polymerization assays and, more importantly, that it retained potency against the multidrug- (including paclitaxel-) resistant cell lines that were beginning to surface.109 These findings ignited a flurry of research in the pharmaceutical industry and in academic laboratories. GBF quickly worked to optimize fermentation methods for the production of epothilones and also to determine the absolute configurations of the epothilones.112,113 Kosan Biosciences and Novartis identified and cloned the biosynthetic gene cluster from the original GBF strain, which allowed these companies to explore genetic engineering to modify the epothilone core.112,114 Bristol-Myers Squibb developed a partnership with GBF to exploit fermentation for the production of epothilones and improve the pharmalogical profile by semisynthetic means.112,115 Novartis utilized the technology it developed to produce large amounts of epothilone B as their prime pharmaceutical contender and utilized formulations to mitigate the toxic side effects associated with the parent structure.112,116 The epothilones also piqued the interest of many academic laboratories, which viewed the structures as viable candidates for industrial total synthesis because of their simplified structures relative to paclitaxel. In addition, even though the epothilones were accessible through fermentation, the ability for structural modifications around the macrolide was somewhat limited.109,112 For these reasons, there was intense competition to establish a fully synthetic route to the epothilones to facilitate thorough SAR explorations. By the beginning of 1997, the Danishefsky, Nicolaou, and Schinzer laboratories had established routes to epothilone A, which is less potent than epothilone B by an order of magnitude.117−119 However, epothilone B quickly succumbed to synthetic efforts by the same three groups as well.120−122 Each of these synthetic approaches was influential in the production of synthetic epothilones as pharmaceutical candidates.

Figure 13. Next-generation synthetic tetracyclines from Tetraphase.

practical and enabling technology developed by Myers and coworkers. Recently, another novel fluorocycline from Tetraphase, TP-271 (187), entered phase I clinical trials.108 Tetraphase has also reported that another novel tetracycline derivative, which contains a methoxy substituent at C7 and is referred to as TP2758 (188), is approaching clinical trials.107 It is now quite clear that a renaissance is occurring within the realm of tetracycline chemistry and that the development of a practical, convergent total synthesis was decisively enabling of these efforts.

4. EPOTHILONE 4.1. Epothilone Background

In the early 1990s, Merck established a program to identify compounds that exhibited taxane-like stabilization of microtubules in an effort to discover more synthetically or biosynthetically accessible alternatives to paclitaxel.109,110 After a screen of 7000 plant extracts, a confirmed hit revealed the structures of epothilones A and B, which surprisingly had been known for several years in the patent literature, although the absolute stereochemistries of the structures remained unassigned (Figure 14). The compounds were patented by Gesellschaft für Biotechnoligsiche Forschung (GBF, now Hemholtz-Zentrum für Infektionsforschung, HZI) and Ciba-Geigy; however, the structures were protected only under German law, as international patent filings had languished due to an initial lack of interest.111,112 Ciba-Geigy had noticed exceptional fungicidal

4.2. Danishefsky’s Synthetic Approach

The Danishefsky group established the first route to epothilone A, and this route hinged on two key bond formations to unite fragments 200 and 208. Construction of fragment 200 commenced with the cycloaddition of aldehyde 193 to oxygenated diene 194 (Scheme 29).123 The resultant dihydropyranone 195 was then reduced to the allylic alcohol, which was utilized to direct a subsequent Simmons−Smith cyclopropanation to the α-face of the pyran ring to afford 196. The newly constructed cyclopropane was then oxidatively ring-opened with N-iodosuccinimide to afford glycoside 197. This compound was then dehalogenated and differentially protected (over several steps) to afford dithiane 198. The system was then homologated by two carbons to afford fragment 199, whereupon the dithiane was oxidatively degraded with concomitant acetal formation to provide 200. The other component, 208, was constructed from tetrahydropyranyl- (THP-) protected (R)-glycidol (201). (Trimethylsilyl)acetylide opening of the epoxide afforded homopropargyl alcohol 202, which was subsequently protected and deprotected to provide 203 (Scheme 30). A three-step sequence converted primary alcohol 203 to methyl ketone 204.

Figure 14. Structures of the parent epothilones (A and B) and their desoxy derivatives (C and D), along with the numbering scheme for the epothilone core. 12013


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Scheme 29. Danishefsky’s Synthesis the “Southern” Portion of Epothilone A

Scheme 31. Danishefsky’s Union of Fragments to Furnish Epothilone A

Scheme 30. Danishefsky’s Preparation of the “Western” Portion of Epothilone A

Danishefsky and co-workers quickly adapted this sequence for the construction of epothilone B, utilizing fragment 217 as the coupling partner instead of fragment 208 (Scheme 32).120,124 Epothilone B (192) was produced in a longest linear sequence of 23 steps (28 overall) in 6.6% yield. Scheme 32. Danishefsky’s Adaption of Epothilone A Synthesis for the Construction of Epothilone B

The ketone was then subjected to an Emmon’s homologation using phosphine oxide 205 to provide olefin 206 as an 8:1 mixture of regioisomers. The pendant alkyne was then oxidized to an intermediate iodoalkyne, which was subjected to hydroboration−protonolysis to afford Z-iodoalkene 207. The homoallylic alcohol was revealed under methoxymethyl (MOM) cleavage conditions and capped with an acetyl group to afford fragment 208. Danishefsky and co-workers then proceeded to assemble fragments in an elegant and succinct manner (Scheme 31). Hydroboration of 200 to 9-borabicyclo[3.3.l]nonane (9-BBN) afforded an intermediate trialkyl borane (209), which was crosscoupled with cis-vinyl iodide 208 employing low-valent palladium catalysis to afford 210. Treatment of acetal 210 with p-toluenesulfonic acid (p-TsOH) revealed the corresponding aldehyde. In a bold undertaking, Danishefsky and co-workers were successful at closing the macrolactone core of epothilone A through intramolecular aldolization. A short sequence of steps produced epothilone C, which, upon subjection to dimethyldioxirane (DMDO), afforded epothilone A. This approach provided epothilone A in a longest linear sequence of 23 steps (33 steps total) in 2.7% overall yield.

4.3. Nicolaou’s Synthesis

The second route to the epothilone core was developed by Nicolaou and co-workers and also utilized a short sequence to bring together two main fragments 226 and 229 in a highly convergent manner.121 Access to northern segment 226 commenced with partial reduction of thiazole carboxylate 218 followed by homologation with ylide 219 to afford 212 (Scheme 33). Treatment with allyl 12014


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Scheme 33. Nicolaou’s Access to “Northern” Portion of Epothilone B

Scheme 34. Nicolaou’s Combination of Fragments to Produce Epothilone B

convergent fashion. This approach proved to be highly practical because of a synergistic combination of fragments that were of roughly equal complexity (i.e., each contained one stereocenter) to construct the epothilone architecture. Synthesis of the first fragment commenced with allylation of oxazolidinone 233 followed by reductive cleavage of the auxiliary and protection to afford olefin 235 (Scheme 35).122 The system was then hydroborated/iodinated to produce iodide 236.

diisopinocampheylborane [(+)-Ipc2B-allyl] then gave homoallylic alcohol 214 in high enantiomeric excess. Protection of the alcohol and oxidative degradation of the olefin afforded aldehyde 220, which was subsequently treated with phosphorane 221 to afford ester 222 as a single regio- and stereoisomer. The ester moiety was net reduced (over several steps) to a methyl group, and the α-olefin was converted into primary iodide 223. The iodide was then displaced with the carbanion derived from (S)-1amino-2-methoxymethylpyrrolidine (SAMP) hydrazone 224 to give 225. Oxidative cleavage of the auxiliary by magnesium monoperoxyphtalate followed by partial reduction of the resultant nitrile afforded key aldehyde 226. The other fragment was prepared from ketoaldehyde 227 (Scheme 34). The aldehyde was subjected to Brown’s asymmetric allylation to afford alcohol 228, which was manipulated over several steps to provide protected diol 229. Deprotonation of 229 followed by treatment with aldehyde 226 afforded aldol adduct in 85% yield as a 3:1 mixture of the two anti diastereomers, preferring the desired. The system was then manipulated over several steps to afford 231. The hydroxyacid was then subjected to Yamaguchi macrolactonization conditions to provide the core of epothilone D 232. The system was then subjected to global deprotection under acidic conditions to afford epothilone D (190). The natural product was then converted by treatment with DMDO to epothilone B (192), as a 5:1 mixture of desired to undesired diastereomers. The Nicolaou approach provides epothilone B in 23 steps, 28 overall, in an overall yield of 3.8%.

Scheme 35. Schinzer’s Assembly of the Eastern Portion of Epothilone B

The next fragment was derived from (S)-malic acid (not shown), whose cyclohexylidene ketal (237, Scheme 36). The cyclohexylidene acetal of malic acid was subjected to a selective reduction of the carboxylic acid that provided lactone 238 upon treatment with acid and subsequent protection.122 Addition of methyllithium to the lactone and subsequent protection of the primary alcohol provided methyl ketone 239. The ketone was then reacted with Horner−Wadsworth−Emmons (HWE) reagent 240 to afford the trisubstituted alkene 241.125 The primary silyl ether was then selectively deprotected, oxidized, and olefinated to provide vinyl iodide 243. The last fragment was produced starting with α-bromoester 244 and was originally used in Schinzer et al.’s second-generation synthesis of epothilone A

4.4. Schinzer’s Formal Synthesis

The formal route to epothilone B developed by Schinzer and coworkers involved the combination of three major fragments in a 12015


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Scheme 36. Schinzer’s Method of Producing the Northern Portion of Epothilone B

Scheme 38. Schinzer’s Completed Formal Synthesis of Epothilone B

(Scheme 37).125 A Reformatsky reaction with 3-pentanone (245) followed by dehydration afforded olefin 246. The ester Scheme 37. Schinzer’s Route to the Southern Portion of Epothilone B

production of synthetic epothilones for therapeutic development. 4.5. Epothilone Structure−Activity Relationships

With viable routes to the epothilones established, both the Danishefsky and Nicolaou laboratories extensively explored structure−activity relationships.128−131 Several pharmaceutical companies also contributed significantly to investigations of epothilone SARs, namely, Novartis, Bristol-Myers Squibb, and Schering AG.132−138 During the course of these experiments, convenient subdivisions of the epothilone core were identified to delineate areas of modification. Danishefsky and co-workers presented the structure as three distinct zones, whereas Nicolaou and co-workers described four domains (Figure 15).129−131 An enormous amount of work has been invested into the SARs of the epothilones, and the discussion below reflects a small portion of that effort while showing the progression from the natural core to the modified synthetic epothilone drug candidates.112,128,129

was then converted to an aldehyde and subjected to an aldol reaction with the enolate of chiral acetate 248. The ester was then reduced to the primary alcohol, and the diol moiety was protected as the acetonide to give 250. The olefin was then oxidatively cleaved by ozonolysis to provide ethyl ketone 251. Fragments 236 and 243 were then stitched together by a Negishi coupling to afford thiazole 253 (Scheme 38). The primary alcohol protecting group was then cleaved and oxidized to afford aldehyde 225, an intermediate seen in the Nicolaou route. Deprotonation of fragment 251 followed by treatment with aldehyde 225 afforded the aldol product as a 9:1 mixture of the desired and undesired diastereomers. The acetonide was then cleaved, and the diol was protected as the bis-silyl ether to intercept intermediate 229 in the Nicolaou route. This construction of epothilone B required a longest linear sequence of 21 steps, 34 overall, and delivered the natural product in 3% yield. Over time, there have been numerous additional creative contributions to the area of epothilone synthesis.112,126−128 However, the routes detailed above had the most impact on the

Figure 15. Sections of the epothilone B structure used to summarize/ generalize SAR data. 12016


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Region A consists of the aliphatic alcohol on the eastern portion of the molecule. Examination of either ring contraction or ring expansion in this area with methylene units resulted in substantial activity loss.129,139 However, introducing unsaturation in this region improved the activity by 4-fold for the 9,10dehydro-Epo D as compared to Epo D, which led to the development of dehydelone (vide infra).140,141 Region B harbors the epoxide in the northern portion of the molecule. One crucial initial observation was that the deoxy variants, namely, Epo C and Epo D, retained the high levels of potency found in their parent compounds.128 This finding suggested that the epoxide was not necessary for biological activity. Nicolaou and co-workers constructed analogues with a cyclopropane in place of the epoxide and found them to be equipotent to their natural cogeners.142,143 Because of these findings, it is believed that the epoxide, rather than having a reactivity role in the biological activity, has a conformational role, whereby it stabilizes the active conformer of the macrolide.128 Installation of substituents larger than methyl on C12 were tolerated.129 Region C consists of the highly functionalized, stereochemically rich southern portion of the molecule. This region is relatively intolerant of modification, with most changes leading to losses in potency. One alteration of the epothilone B core exploited by Bristol-Myers Squibb was the conversion of the macrolactone to a macrolactam, also known as Ixabepilone, which won FDA approval for the treatment of breast cancer.115,128,144 However, this modification decreased the potency by an order of magnitude and rendered the compound susceptible to multidrug resistance mechanisms.145−147 Elimination of the C3 hydroxyl group to form an α,β-unsaturated macrolactone resulted in a structure with reduced potency compared to the parent.131 Reduction of the C5 ketone or alteration of the gem-dimethyl C4 result in significant loss of bioactivity.129,131 One particular modification that not only is tolerated but actually enhances the potency is the extension of the C6 methyl to larger aliphatic chains; this approach was used by Schering AG to explore potential differences in the mode of action of Epo B versus Epo D through radiolabeling experiments.135,147 The last remaining segment of the molecule is the heteroaryl side chain referred to as region D. Modifications in this area have been extensively investigated.128 Deletion of the olefinic linker leads to a significant loss of activity, as does elimination of the aryl group.129 Replacement of the thiazole ring with a phenyl results in a large drop in potency; however, more conservative heteroaromatic surrogates are tolerated.128,133,135,148 A collaboration between the Nicolaou group and Novartis generated a large library of epothilone B derivatives with a plethora of heterocyclic side chains.148,149 When the Lewis-basic nitrogen was converted to its N-oxide, the system retained potency against tumorigenic cells, but its activity was completely ablated in multidrug-resistant (MDR) cells.135 In terms of the location of the thiazole nitrogen in the epothilone structure, the system can adopt two rotameric forms (Figure 16). To determine whether one is more responsible for the bioactivty, Schering AG orchestrated several key experiments. They constructed three derivatives of epothilone D, in which the thiazole moiety was replaced with 2-pyridyl, 3-pyridyl, and 6benzothiazyl.135 Whereas the 3-pyridyl derivative showed a substantial loss of activity, the 2-pyridyl and 6-benzothiazyl derivatives of epothilone D showed marked increases in potency, approaching that of epothilone B.135 These findings support the

Figure 16. Thiazole rotamers in 192.

idea that the potency of the epothilones rests on the thiazole nitrogen being oriented as in rotamer I (Figure 16). 4.6. Development of Sagopilone

Although a tremendous amount of work has been done with synthetic epothilones, in terms of exploring SARs both academically and industrially, many of the structures chosen as the industrial clinical contenders are semisynthetic in form. However, Schering AG considered epothilones to be viable targets for industrial total synthesis from the outset and never seriously pursued semisynthetic means to access the bioactive material.112 Instead, Schering AG relied solely on total synthesis to explore the nature of the epothilone core and to develop drug candidates.112,147 A major goal of the company’s agenda was to expand the therapeutic safety window of these structures while maintaining their potency.147 A secondary goal was to capitalize on the inherent activity against tumor lines resistant to paclitaxel and other frontline cancer treatment regimens.112,147 A small portion of Schering AG’s SAR studies have been discussed (vide supra). The company discovered that two minor modifications to the structure of epothilone B created a compound with a superior therapeutic window and activity surpassing that of epothilone B. This compound was kept secret until it was revealed by Schering AG in 2006.112,147 Schering AG had extended the C6 methyl group into an allyl and had converted the vinyl thiazole side chain into a benzothiazole. As stated above, this structure was discovered and composed through total synthesis in a pharmaceutical setting. Klar and co-workers at Schering AG were highly cognizant of the synthetic efforts toward epothilone B and D discussed above and used this work to their advantage. They identified the strengths of these routes and orchestrated a highly convergent hybrid approach that allowed them to modify nearly every position of the macrolactone scaffold.147 The discovery research route involved the combination of three roughly equal-sized fragments, each with one stereocenter, in a manner analogous to the Schinzer route to construct the lead compound ZK-Epo. The convergency coupled with the relatively simple fragments made this a powerful approach for producing epothilone structures. Synthesis of the first fragment, which is essentially region C of the epothilone core, began with the protection of (−)-pantolactone (256), an abundant optically active building block (Scheme 39).150 Reduction to the lactol followed by olefination afforded enantiopure allylic ether 258. The primary alcohol was then benzylated, and the alkene was hydroborated to afford alcohol 259 following oxidative workup. Ketal interchange with 2,2-dimethoxypropane, hydrogenolysis, and oxidation then gave aldehyde 261. Treatment with butenyl magnesium bromide and subsequent oxidation provided fragment 262, which is very similar to 251 from the Schinzer synthesis.150,151 Although this sequence was somewhat lengthy, it was preferred by the discovery division because each step could be easily scaled (to approximately 80 g of material per batch) and was highly flexible to facilitate SAR studies. 12017


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aldehyde 273. A subsequent Evans aldol reaction with oxazolidinone 274 afforded alcohol 275 as a 4:1 mixture of diastereomers that were separated by recrystallization.147 βHydroxyimide 275 was then protected and transesterified with ethanol. A subsequent reduction afforded alcohol 276, which was then converted to phosphonium salt 277 in two steps. Fragments 270 and 277 were combined through a Wittig reaction, reminiscent of both the Schinzer and Nicolaou approaches, followed by acid-mediated cleavage of the THP protecting group to afford olefin 278, unfortunately as a 1:1 mixture of E and Z isomers (Scheme 42).147 After separation, the undesired E isomer could be converted to achieve an E-to-Z ratio of 6:4 by photochemical means.147 Oxidation of 278 afforded aldehyde 279.

Scheme 39. Schering AG’s Construction of the Southern Fragment

Scheme 42. Schering AG’s Combination of the Three Fragments of Sagopilone Fragment 270, which became region A of the epothilone core, was constructed from Roche ester (265), another abundant optically active building block (Scheme 40). Roche ester was Scheme 40. Schering AG’s Access to the Eastern Fragment of Sagopilone

derivatized as the THP adduct and then reduced to a primary alcohol, which was then converted to tosylate 267.147,152 Tosylate 267 was coupled to Grignard 268 using Li2CuCl4. The olefin was then oxidatively cleaved to provide fragment 270. The last fragment was synthesized from benzoic acid derivative 271 (Scheme 41). A one-pot reduction, benzothiazole formation, provided acid 272, which was then converted to Scheme 41. Schering AG’s Route to the Western Portion of Sagopilone

Fragments 262 and 279 were combined through an aldol reaction, mirroring the Schinzer and Nicolaou approaches. Deprotonation of fragment 262 followed by transmetalation with ZnCl2 and treatment with aldehyde 279 afforded antialcohol 280 as a 12:1 mixture of diastereomers. The acetonide was removed, and the resulting diol was subjected to a protection/deprotection sequence to afford primary alcohol 281. A two-stage oxidation of the alcohol provided acid 283, 12018


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original manufacturing sequence of 292 began with a crossClaisen condensation of tert-butyl acetate (286) and nitrile 287 to afford β-keto ester 288. The ketone was then reduced, and the ester was saponified to provide acid 289. The acid was then subjected to a sequence of crystallizations with (R)-(+)-N-(phydroxybenzyl)phenyl ethylamine (290) to afford an amine salt, which was then converted to the enantioenriched ester 291. The ester was then reduced to the primary alcohol, and the diol moiety was protected as acetonide 292.155 An alternative approach to acetonide 292 was developed by Schering AG and became the preferred route (Scheme 44).156,157

which was then subjected to conditions that cleaved the allylic silyl ether preferentially. Access to the macrolide 284 was achieved under Yamaguchi conditions. Global deprotection of the silyl groups followed by epoxidation with DMDO afforded ZK-Epo (285) as a 7:1 mixture of diastereomeric epoxides, favoring the desired form. This route was utilized by the discovery division to produce 36 g of compound for conducting extensive preclinical evaluations.147 Initial tests on ZK-Epo indicated that it was able to outmatch paclitaxel and several other chemotherapeutics, in terms of IC50, against a panel of tumor cell lines.147 One particularly important finding was that ZK-Epo showed a very low toxic potential on normal, nondividing cells.147 In addition, it retained its potency against MDR cell lines and proved not to be susceptible to cellular efflux pumps, such as P-gp.147,153 Finally, ZK-Epo exhibited activity against several cell lines resistant to normal chemotherapy regimens.153 These exciting preclinical results accelerated ZK-Epo’s trajectory into clinical trials. In 2003, sagopilone, the name chosen for ZK-Epo, entered phase I studies.153 These studies indicated that sagopilone was welltolerated and that the major dose-limiting toxicity was peripheral neuropathy. With phase II on the horizon, Schering AG’s process group embarked on a route optimization to ensure an adequate supply of sagopilone. The challenge of adapting a 22-step discovery sequence (38 steps overall) to an industrial scale was daunting to say the least, but Schering AG’s process division took the challenge head on. Production had to start with several hundred kilograms of material at the start of the process to manufacture kilograms of sagopilone.154 For the construction of fragment 262, the discovery route proved difficult to scale up, so another route was devised (Scheme 43).154 Compound 292 was made commercially available during development, and Schering AG’s process chemists capitalized on the availability of this material.155 The

Scheme 44. Schering AG’s Optimized Biocatalysis Route to Intermediate 292

It involved deprotonation of nitrile 294 and treatment with aldehyde 295 to afford alcohol 296. The alcohol was then acetylated, and the racemic mixture was subjected to enzymatic hydrolysis to give enantioenriched acetate 298, which was easily scaled to 22-kg batches.156,157 This material was then saponified, hydrogenolyzed, and protected to give acetonide 292 (Scheme 43). This material was subjected to MeLi followed by an acidic workup to afford methyl ketone 293. The ketone was then converted to the β-keto ester, allylated, and decarboxylated to afford fragment 262 after distillation.154 Fragment 270 was constructed from Roche ester in the process route as well (Scheme 45). The same sequence of

Scheme 43. Sumika Fine Chemical’s Manufacturing Route to 292 and Schering AG’s Process Division Elaboration to Fragment 262

Scheme 45. Process Route to Fragment 270

protection, reduction, and tosylation was used. However, at this stage, the process division chose instead displacement of the tosylate with lithium acetylide followed by a deprotonation and N,N-dimethylaniline (DMA) quench to afford methyl ketone 299. This system was then hydrogenated to afford fragment 270 after distillation. This alternative route precluded the use of toxic OsO4 and was easily scaled to 50-kg batches. Optimization for process-scale synthesis of fragment 304 took a very different form as compared to the discovery route (Scheme 46). Benzothiazole 272 was converted to the β-keto ester through activation, displacement, and decarboxylation. The β12019


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The discovery division’s sequence of steps to close the ring system was adopted for process scale. The workup for the global deprotection step required modification because extended exposure to alkaline conditions generated the ring-opened product. This was remedied through the use of mild aqueous potassium borate solutions. For the final epoxidation, a methyltrioxorhenium/hydrogen peroxide system was preferred over DMDO because of scalability concerns.133 This change in oxidation procedure increased the selectivity for the epoxidation from 7:1 to 23:1. After chromatography and recrystallization, this process provided the desired material in a 21-step longest linear sequence, 35 steps overall, in 7.2% overall yield and has been used to make multiple kilograms of sagopilone (285). This route reliably supplied sagopilone through Schering AG’s clinical studies. It has shown efficacy against several forms of cancer.159−161 However, the clinical development of sagopilone has been halted for reasons unknown, and Bayer, who acquired Schering AG, has been quiet for some time on the fate of this drug.162

Scheme 46. Schering AG’s Process Route for Fragment 304

keto ester was then subjected to microbial reduction conditions with Pichia wickerhamii yeast.157,158 This approach precluded the use of a chiral auxiliary, which simplified the purification immensely. The alcohol was then protected, and the ester was reduced to the primary alcohol to afford 276. The primary alcohol was then converted to iodide 304. The iodide was converted to the phosphonium salt in situ, which was then treated with sodium hexamethyldisilazide (NaHMDS) and fragment 270 to give olefin 278 as a 1:1 mixture of E and Z isomers (Scheme 47). Unfortunately, this step could not be easily circumvented by other means, and the material had to be separated by chromatography after deprotection. Fortunately, the undesired isomer could be photorecycled on scale, and after two iterations of irradiation and separation, 278 was obtained in 75% yield. Alcohol 278 was then oxidized and carefully isolated to afford the easily epimerizable aldehyde 279. Fragments 279 and 262 were combined through an aldol reaction that gave 280 as a 10:1 mixture of diastereomers on scale, which, unfortunately, required chromatography for separation.

4.7. Dehydelone, Fludelone, and Isofludelone

The other major players in the field of synthetic epothilones are dehydelone, fludelone, and isofludelone, compounds championed by Danishefsky and co-workers. The story of these structures is one of judicious modifications to the structure of epothilone and, in the case of fludelone, one fortunate and cursory decision to introduce a trifluoromethyl group at C26.163 After completing their synthetic approach to epothilone B (192), Danishefsky and co-workers were intrigued that epothilone D (190, referred to as dEpoB by Danishefsky) retains the tubulin-stabilization properties, albeit less potent, as well as the MDR tumor activity of its oxygenated counterpart.124,163 In addition, the therapeutic window of epothilone D is much larger than that of epothilone B, leading to the conclusion that the C12−C13 epoxide could be a source of nonselective toxicity.163 During this time, Kosan Biosciences began to collaborate with Sloan-Kettering by providing epothilone D through fermentation, which also lead to the discovery of epothilone 490 (Figure 17).164 This system contains a diene obtained by introducing additional unsaturation at C10−

Scheme 47. Process Route to Complete Sagopilone

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Scheme 49. Danishefsky’s Route to Fragment 317

Figure 17. Structures of epothilone 490 and dehydelone.

C11 of 190. However, despite its potency in vitro, the in vivo activity was fairly lackluster in mouse models because of metabolic instability.163 Synthetic studies of epothilone 490 led Danishefsky and coworkers to construct the skipped diene analogue of epothilone 490, with a methylene group inserted between C11 and C12 to provide the expanded 17-membered ring (not shown).140,163 Surprisingly, this expanded ring system was exceptionally active in vitro, even though its saturated analogue is inactive. This led Danishefsky and co-workers to ponder whether this skipped diene motif imparted rigidity to the molecule and could be utilized in the traditional 16-membered scaffold.163 A concise route to access this skipped diene analogue of epothilone D (307), also referred to as KOS-1584 or dehydelone, was pursued. Unlike Danishefsky’s initial access to epothilone B, where two fragments were brought together, the discovery route to dehydelone adopted a three-fragment approach.140,165 Construction of the first fragment commenced with an aldol reaction between ketone 308 and Roche ester derivative 309 to afford the syn product 310 in a 5.7:1 ratio of diastereomers (Scheme 48). The system was then protected, and

dimethylhydroxylamine to afford Weinreb amide 318 after reprotection. The system was then subjected to Stille coupling with allyltributyltin followed by treatment with MeMgBr to afford an intermediate methyl ketone. Mild deprotection was effected by treatment with aqueous acetic acid to provide 319.165 Ester 314 was then subjected to acidic hydrolysis conditions and then coupled with hydroxyketone 319 to afford seco precursor 320 (Scheme 50). The system was then subjected to Scheme 50. Completion of Danishefsky’s Discovery Route to Dehydelone

Scheme 48. Danishefsky’s Discovery Route to Fragment 312 of Dehydelone

ring-closing metathesis using Grubbs second-generation catalyst. The last fragment was installed by a Wittig reaction with macrolide 321 and fragment 322. This installed the aryl side chain as a 9:1 mixture of E and Z regioisomers that could be separated. A global deprotection was then effected by treatment with pyridine hydrofluoride. With a concise route to dehydelone (307) developed, examination of its activity revealed an approximate 10-fold increase in drug potency against MDR tumors.140 In vivo and in vitro experiments showed that 307 was more cytotoxic than epothilone B (192) and more metabolically stable than epothilone D (190).140,168 Even though 307 was found to be superior to the parent epothilones, it exhibited significant nontumor-specific toxicity, which restrained the ability to push the target tumor into a nonrelapsable state. However, dehydelone 307 was explored as a drug candidate by a collaboration between Kosan Biosciences and Hoffmann-La Roche and has completed a phase II trial for the treatment of metastic nonsmall cell lung cancer.169,170 To provide material for the clinical trials, Kosan Biosciences and Hoffmann-La Roche modified Danishefsky’s approach to

the acetal was cleaved to obtain aldehyde 311. The aldehyde was reacted with the chiral titanium enolate of tert-butyl acetate to afford alcohol 313 in greater than 20:1 dr.166 The system was then subjected to a protection, deprotection, oxidation, and olefination sequence to provide ester 314. The next fragment was constructed from oxazolidinone 315 and allyl iodide 316 (Scheme 49).165,167 The system was then deprotected, and the oxazolidinone was displaced with 12021


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still being optimized when Bristol-Myers Squibb acquired Kosan Biosciences. Currently, there are no active clinical studies on dehydelone (KOS-1584), and it is not known by us whether the program has been halted or terminated.177 Danishefsky and co-workers have continued their work on dehydelone because of dissatisfaction with the toxicology profile. They considered the incorporation of fluorine atoms into the structure as a means to temper the cytotoxicity of the system. The C26 methyl group was contemplated as an area to integrate the fluorine atoms because of earlier studies indicating polar functionality being tolerated in that position.129,168 Although the incorporation of the fluorine mitigated the toxicity of the structure to a small degree, fluorinated analogue 332, referred to as fludelone, proved to be quite remarkable (Figure 18).

dehydelone. Fragment 319 was constructed from (R)(+)-glycidol (Scheme 51).171 The alcohol was oxidized by in Scheme 51. Kosan Biosciences’ Optimized Route to Fragment 317

situ-generated RuO4 and subsequently converted to Weinreb amide 324. Some intriguing organometallic chemistry was then utilized to install the lower portion of fragment 319. A carboalumination of propyne with an in situ-generated allylalane facilitated by Cp2ZrCl2 produced an intermediate vinylalane, which, upon treatment with 324, opened the epoxide to afford homoallylic alcohol 325. Finally, addition of MeMgBr afforded methyl ketone 319. This sequence was easily scaled to 100-g batches.171 For fragment 330, Kosan Biosciences explored the option of biosynthesis to produce the polyketide-like moiety.172,173 The company was able to establish a fermentation method to construct 326 (Scheme 52). The system was then methylated,

Figure 18. Structures of fludelone and isofludelone.

Treatment of mouse models with fludelone not only suppressed the growth of MX-1 xenograft tumors on mouse models but shrank and eliminated the tumors for as long as 64 days.168 In addition, the therapeutic window was quite broad for fludelone, where the decrease in mouse model body weight due to treatment was rarely lethal and, after suspension of treatment, weight quickly increased to near pretreatment control levels.168 Fludelone 332 proved to be quite stable toward metabolic processes as compared to 307, and the introduction of the fluorines imparted an increase in hydrophilicity, increasing bioavailability.168 Examination of fludelone’s effect on human myeloma models indicated a rapid induction of apoptosis, even in paclitaxel-resistant cell lines.178 Fludelone’s therapeutic spectrum was found to encompass leukemia, breast, colon, and lung carcinomas and ovary and prostate adenocarcinomas.179 In the hopes of optimizing fludelone further and regaining some lost potency during its transition from dehydelone, Danishefsky and co-workers modified the thiazole moiety into an isoxazole to afford isofludelone (333).180 The desire for an increase in potency was achieved, as this modification gave rise to a remarkably potent compound that is able to achieve complete remission and cures in xenograft mouse models. In addition to enhanced potency, isofludelone (333) has greater metabolic stability than fludelone. After extensive preclinical investigations, isofludelone has recently entered phase I clinical trials that are examining dose escalation and pharmacokinetics.162 Currently, the only epothilone analogue approved for clinical use by the FDA is Bristol-Myers Squibb’s semisynthetic derivative Ixabepilone.181,182 However, the monumental amount of work done in this area was driven by total synthesis. The lessons learned by developing highly convergent and modular synthetic constructions toward this class of natural products made it possible for fully synthetic analogues to make it to clinical trials. The beautiful work by Schering AG to develop a viable process capable of delivering kilograms of sagopilone, an epothilone B-derived active pharmaceutical ingredient (API), is nothing short of remarkable. The elegant studies by Danishefsky

Scheme 52. Kosan Biosciences and Hoffmann-La Roche’s Route to Complete Dehydelone

subjected to a Claisen condensation, and reduced under Noyori conditions to afford 329.174 Protection of the secondary alcohol followed by cleavage of the chiral auxiliary afforded fragment 330.174 The two moieties were then coupled to furnish ester 331.174,175 The seco precursor was then converted to dehydelone by ring-closing metathesis, deprotection, and a Wittig olefination to install the heterocyclic side chain.170,174−176 This route was 12022


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(vide supra), these compounds were viewed by many as a prime opportunity for total synthesis to drive further research and development of potential clinical candidates. In fact, cryptophycins succumbed to synthesis within a year of their rediscovery by Tius, Moore, and Kitagawa and their co-workers.188,193 These cryptophycin constructions were followed by many other approaches to the depsipeptides.194−200 Although each of these syntheses is elegant in its own way, the Tius−Moore construction proved to be the most influential in the advancement of a cryptophycin-based pharmaceutical. The Tius−Moore synthesis of cryptophycin 1 utilizes several fragments that are stitched together over a few steps in a highly convergent manner. This approach allows for flexibility in peripheral functionalization of the molecule or for changes in the core architecture. The construction of the first fragment commenced with an HWE homologation of dihydrocinnamaldehyde to afford ester 338 (Scheme 53).188 This material was then reduced to the allylic

and co-workers, which led to the development of several synthetic epothilone candidates, is equally inspiring. Several synthetic epothilones are still viable pharmaceutical candidates, and the hope for a fully synthetic epothilone-based drug is very much alive.

5. CRYPTOPHYCIN 5.1. Discovery and Background

Amid investigations aimed at traditionally “overlooked” microorganisms in the early 1990s, a team of researchers led by Gregory Patterson at the University of Hawaii discovered antineoplastic activity in the extracts of cultured blue-green algae.183 This discovery presented itself after the screening of more than 1000 cyanobacteria extracts. In addition to potent cytotoxicity, it was determined that the extracts were more selective for solid-tumor-derived cell lines than for leukemias as measured by the Corbett assay.184,185 The cytotoxicity of this particular strain of Nostoc sp. was pinpointed to a group of macrocyclic depsipeptides (Figure 19).184 Surprisingly, the elucidated structure of the most potent

Scheme 53. Tius−Moore Synthesis of Cryptophycin’s Northern Fragment

Figure 19. Structures of cryptophycin 1 (aka cryptophycin A, 334) and cryptophycin 3 (aka cryptophycin C, 335).

depsipeptide matched one already existing in the literature denominated as cryptophycin. The structure was originally discovered in another Nostoc sp. strain by Merck, which investigated its potential as an antifungal agent.186,187 However, cryptophycin 1’s overt toxicity was problematic in this regard.184 Merck originally communicated a skeletal structure of cryptophycin 1, without assigning stereochemistry. In contrast, Moore and co-workers assigned both absolute and relative configurations, as well as the structures of the other newly isolated cryptophycin family members.184,188 Meanwhile, the mechanism of action of cryptophycin 1 was explored by several groups. Cryptophycin 1 was found to interact with tubulin at picomolar concentrations and arrest the mitotic cycle by inhibiting tubulin polymerization and promoting depolymerization in vitro.189−192 Cryptophycin reversibly binds in the Vinca domain on the ends of microtubules with high affinity.190,191 In addition to hyperpotency, cryptophycin retained activity against cell lines that overexpressed P-glycoprotein.189 This characteristic was perhaps its most exciting because of the potential for treating P-gp-mediated multiple-drug-resistant cancer types.189

alcohol 339, which was then subjected to a Sharpless asymmetric epoxidation with (+)-diethyl tartrate [(+)-DET] to provide epoxide 340.201 The epoxide was then opened by the action of AlMe3 with complete regioselectivity to afford diol 341. The system was then converted to a styrene derivative by protection of the diol moiety as its acetonide, benzylic bromination, and elimination to afford acetonide 342. The acetonide then underwent cleavage followed by monotosylation and protection of the secondary alcohol to provide 343.202 The tosylate was then displaced by cyanide to afford an intermediate nitrile, which was then reduced and homologated to ester 344. The ester was then saponified to afford fragment 345. The second main fragment was constructed from Roche ester (Scheme 54). The ester was converted by heating in the presence of ammonia to an amide, which was then reduced to amino alcohol 346 isolated by distillation.188 The amine was derivatized

5.2. Tius−Moore’s Synthesis of Cryptophycin 1

The material utilized for the mechanism of action and other preclinical studies was supplied primarily by algae cultures. However, this did not stop the synthetic community from taking notice of these powerful structures. Similarly to the epothilones 12023


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methyl substituent on the southern β-amino acid fragment of the structure.207 This fortuitous decision led to the creation of the more robust cryptophycin 52 (372; see Scheme 59 below). Cryptophycin 52 proved to be a potent antiproliferative structure that surpassed the activity of the parent structure.190,207 It was the most potent suppressor of microtubule dynamics known at the time.210 The cryptophycin 52−tubulin complex has a dissociation rate constant (Kd) of 47 nM and a very low dissociation rate.210 As a result, a large fraction of intracellular cryptophycin is thought to be adsorbed by its receptor and is unavailable to efflux machinery.211

Scheme 54. Construction of the Southern Portion of Cryptophycin

5.4. Eli Lilly’s Synthesis of Cryptophycin 52

These results intrigued the scientists at Eli Lilly and convinced them to initiate a collaboration with Moore aimed at taking this powerful structure into clinical trials.212 To provide an adequate supply of material necessary for clinical trials, the chemists at Eli Lilly launched an effort to refine the Tius−Moore synthesis into a form that could be reliably executed on industrial scales. The first fragment was constructed in a manner similar to that employed in the Tius−Moore approach.213−215 Dihydrocinnamaldehyde was homologated with trimethylphosphonoacetate, and the product was reduced to afford allylic alcohol 339 (Scheme 56). This material was then subjected to asymmetric epoxidation followed by AlMe3-mediated ring opening and methylation to give an intermediate diol.213,214 The diol was selectively tosylated at the primary position through an intermediate dibutylstannylene acetal followed by TBS protection of the secondary alcohol.216 The system was then subjected to a benzylic bromination and elimination to provide styrene derivative 343.214 The tosylate was converted to the nitrile by treatment with KCN followed by partial reduction with DIBAL and HWE homologation to provide ester 344. This material was then saponified to afford acid 345. An alternative approach to this fragment was also explored (Scheme 57). Beginning with enantioenriched alcohol 356, conversion to its propargyl ether followed by lithiation initiated a [2,3]-Wittig rearrangement to afford alcohol 358 as a 9:1 mixture of diastereomers.217 These were separated by chromatography. The alcohol was then protected as its TBS derivative, and the alkyne was subjected to a hydroboration/oxidation to provide aldehyde 359. The aldehyde was then homologated to an acrylate derivative through an HWE reaction, and the pendant olefin was cleaved by ozonolysis. The resultant aldehyde was then converted to styrenyl intermediate 344 by a Wittig reaction.

with Boc2O, and the alcohol was oxidized to afford acid 347. The acid was then coupled to leucic acid derivative 348 to provide ester 349. The allyl ester was then cleaved by the action of catalytic Pd to provide acid 350.203 Fragment 345 was then coupled to D-tyrosine derivative 351 and deprotected to afford alcohol 352 (Scheme 55). This alcohol was then coupled to acid 350, which gave bis-protected seco precursor 353. The protecting groups were cleaved by treatment with Zn and AcOH followed by neat TFA to provide amino acid 354. Macrocyclization was then effected with pentafluorophenyl diphenyl phosphinate (FDPP) to provide cryptophycin C (335).204 This natural product could then be converted to cryptophycin 1 (334) by treatment with m-CPBA to afford the natural product as a roughly 2:1 mixture of diastereomers favoring the desired form.188,205 This route constructed cryptophycin 1 in a longest linear sequence of 21 steps, 27 overall, in 3% yield. 5.3. Exploration of SAR

Having developed a modular route to the cryptophycin motif, Moore and Tius set to work exploring the SAR of the structure. It was determined that very few alterations of the parent were tolerated.206 However, from preliminary studies on fermented cryptophycins, it was noted that the ester linkages were fairly sensitive to mildly alkaline media, leading to facile ring opening and degradation.184,207−209 Moore and Tius hypothesized that this susceptibility could be abated by the installation of a second Scheme 55. Completion of Cryptophycin 1

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Scheme 56. Eli Lilly’s Construction of the Northern Fragment

Scheme 58. Eli Lilly’s Process for the Southern Portion of Cryptophycin 52

serve as a masked form of the epoxide that could be unveiled at the end of the synthesis.222 The system was then deprotected to afford the seco precursor. It was discovered during investigations at Eli Lilly that the trichloroethyl ester was susceptible to intramolecular aminolysis by the pendant amine, which allows for the avoidance of coupling reagents.223,224 This susceptibility was enhanced by the action of 2-hydroxypyridine, which was exploited in this case to afford macrocycle 370. The diol moiety was then converted to an orthoester by exchange with trimethyl orthoformate. The orthoformate was then treated with acetyl bromide, which ionized the orthoformate and produced vicinal formyloxy bromide 371 upon isolation.212 This material could then be converted to cryptophycin 52 upon treatment with base. This approach produced the API in a longest linear sequence of 17 steps, 22 overall, in a 9.4% yield. In an alternative approach, a screen of epoxidizing agents on intermediates revealed that styrene 351 was epoxidized in high selectivity under Shi conditions (Scheme 60).205,225 This epoxidation afforded a 6.5:1 mixture of epoxides, which was upgraded to 10.3:1 after coupling to fragment 366 to afford seco precursor 374.205 The bis-protected seco precursor was then subjected to 9-fluorenylmethyloxycarbonyl (Fmoc) cleavage conditions, and surprisingly, it was discovered that the free secoamine slowly converted to cryptophycin 52 under the reaction conditions. Therefore, the seco-amine was not isolated, but instead was allowed to proceed to the final product, which could be chromatographed to afford a 9.4:1 mixture of diastereomeric epoxides. These isomers could be separated by reverse-phase HPLC.205 This alternative route produced cryptophycin 52 in a longest linear sequence of 13 steps, 20 steps overall, in a 16% yield. These routes were both being explored and optimized as cryptophycin 52 entered clinical trials. Early preclinical trials indicated that an intermittent dosing schedule would be optimal for treatment regimens.226 Phase I trials found that cryptophycin 52 had dose-limited neurotoxicity that could be reversed after treatment ended.226 In addition, there was evidence of antitumor activity against nonsmall-cell lung cancer, renal cancer, and head and neck cancer, which warranted further investigations.226 Unfortunately, cryptophycin 52 had to be withdrawn from phase II human clinical trials due to peripheral neuropathy.227,228 This is a perennial dose-limiting toxicity of tubulin-binding anti-

Scheme 57. Alternative Approach to the Northern Fragment of Cryptophycin

Both of these routes could be effectively scaled to afford large amounts of the first fragment. Synthesis of the second fragment commenced with the double methylation of ethyl cyanoacetate (360) with MeI to provide 361 after distillation (Scheme 58).218 The nitrile was then reduced to the amine in the presence of Boc2O to provide the βcarbamoyl ester 362, which was subsequently saponified to provide acid 363. The acid was coupled with 348 to afford ester 364, which was then deallylated and subjected to a protectinggroup exchange to afford 366.218,205 Fragment 345 was then coupled to the tyrosine derivative 351 to afford amide 367 (Scheme 59).218 This material was then deprotected to afford alcohol 351.215,219 Because of the issues of diastereoselectivity with late-stage epoxidation, the researchers explored epoxidations and other transformations at various stages in the synthesis.205,212,220 One approach that was explored coupled alcohol 351 with fragment 365 to provide 368 and then subjected the system to a Sharpless asymmetric dihydroxylation.212,221 The diol would 12025


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Scheme 59. Eli Lilly’s Completion of Cryptophycin 52

Scheme 60. Alternative Endgame for Cryptophycin 52 Developed by Eli Lilly

mitotics, which, to date, seems to be absent only for the diazonamide class of structures (vide infra). The cryptophycins are an intriguing class of compounds with potency that is difficult to match. This unprecedented activity led to the development of cryptophycin 52, a compound that enthralled Eli Lilly and Co. The efforts by Eli Lilly to adapt the Tius−Moore route into a scalable form are meritorious, and much can be learned from their achievements. To our knowledge, there are no active trials examining cryptophycin 52, which was the only member of this chemical class to reach the clinic.

6. PM060184 6.1. Discovery of PM050489 and PM060184

A marine natural products discovery campaign by the PharmaMar corporation in Spain recently uncovered a new set of mixed-lineage polyenes with extremely potent antiproliferative activity.229,230 The structures were isolated from a Lithoplocamia sp. sponge collected near Madagascar and named PM050489 and PM060184 (Figure 20).229 Both molecules were obtained in trace quantities from sponge samples: 0.002 wt % in the case of PM050489 and 0.00003 wt % in the case of PM060184.229 These molecules are composed of a valerolactone with a large polyunsaturated tail at the δ-position. Initial expeditions

Figure 20. (A) Structures of PM050489 and PM060184. (B) Crystal structure of PM060184 bound to the T2R-TTL (stathmin-like protein RB3−tubulin tyrosine ligase) complex231 (PDB accession number 4TV9). (Reproduced with permission from ref 231. Copyright 2014 National Academy of Sciences.)

provided sufficient material to assign the constitution and a majority of the relative chemistry. 12026


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In addition to their intriguing structures, PM050489 and PM060184 showed subnanomolar activities against several tumor cell lines, which was exciting to PharmaMar. However, these preliminary tests exhausted the material supply, and the C6 and C21 stereocenters had yet to be assigned.229 Given these constraints, the chemists at PharmaMar embarked on a synthetic sequence to deliver more of these intriguing polyenes, a maneuver seldom seen in the private sector.

Scheme 62. PharmaMar Production of Fragment 400

6.2. Synthetic Studies on PM060184

PharmaMar constructed the two compounds by similar routes; however, PM060184 was chosen as the prime clinical candidate based on its activity, safety profile, and distinct mechanism of action (vide infra).229 The syntheses utilized several stereoretentive sp2-atom-based cross-coupling methodologies to install key Z olefinic linkages. This allowed the structures to be broken into two fragments. The route to the first fragment commenced with 1,3-propanediol (375), which was subjected to TBS protection and oxidation to afford aldehyde 376 (Scheme 61).232 An Evans aldol reaction between aldehyde 376 and Scheme 61. PharmaMar’s Construction of Fragment 387

CPBA to give a racemic mixture of epoxide 389.234 This mixture was then resolved by Jacobsen’s hydrolytic kinetic resolution to afford the enantiopure (R)-epoxide 391.38 The epoxide was then opened by nucleophilic addition of lithiated propyne to afford homopropargylic alcohol 392. This was then protected and partially hydrogenated under Lindlar conditions to provide alkene 393. The primary alcohol was then deprotected, oxidized, and converted to the vinyl iodide in a manner analogous to that used for the first fragment. Vinyl iodide 395 was then coupled to Boc-Tle-NH2 using copper catalysis developed by Buchwald and co-workers.235 This procedure established Z-configured enamide 397 in a stereoretentive manner. The system was then pyrolyzed for Boc removal because acid treatment proved problematic for the t-butyldiphenylsilyl (TBDPS) protecting group and for the enamide. Free amine 398 was then coupled with acid 399 to afford stannane 400, the second main fragment. The two pieces were then stitched together in a process mediated by copper(I) thiophene-2-carboxylate (CuTC) (Scheme 63).236 The system was then deprotected, and the primary urethane was installed by conventional means to afford PM060184 (374). This route provided PM060184 in a longest linear sequence of 17 steps, 29 overall, in 5.5% yield. This synthetic material drove further clinical investigations and mode-of-action studies. PM060184 showed potent activity against a variety of cancer cell lines.237,238 In addition, it proved effective at treating subcutaneous xenografted tumors in nude mice with no major toxic side effects observed at the maximum tolerated dose.238 Further studies indicated that activity was retained against cells and xenografted tumors overexpressing P-

oxazolidinone 233 afforded β-hydroxyimide 377.233 This system was then TBS-protected, and the auxiliary was reductively cleaved to provide alcohol 378. The alcohol was then oxidized and subjected to a Wittig reaction with phosphorane 380 to afford acrylate derivative 381. The ester was then converted to an aldehyde and subjected to a subsequent olefination reaction to afford vinyl iodide 383. This intermediate was then partially deprotected to reveal the primary alcohol, which was oxidized and homologated through Horner− Wadsworth−Emmons olefination with phosphonate 385. Upon treatment with dilute HCl, the other TBS group was cleaved, and the system was lactonized to provide key fragment 387. The second main fragment was produced from 3-buten-1-ol (388) (Scheme 62). The alcohol was derivatized as the tertbutyldimethylsilyl ether, and the olefin was epoxidized by m12027


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Scheme 63. PharmaMar’s Completion of PM060184

gp efflux pumps.238 Examinations of PM060184’s effects on microtubules demonstrated that it suppresses their dynamic instability, which increased the amount of time microtubules spent in pause.238 X-ray crystallographic studies with a tubulin complex (specifically two αβ-tubulin dimers, stathmin-like protein RB3 and tubulin tyrosine ligase, aka T2R-TTL) were utilized to determine the mode of action of PM060184, which binds to a novel region of tubulin denominated as the maytansine site (Figure 20).231 PM060184 shares its binding region with maytansine (whose antibody drug conjugate is FDA-approved for the treatment of advanced breast cancer) and rhizoxin (which advanced to phase II).231 The key binding points between PM060184 and tubulin involve hydrogen bonding at the C1 and C13 carbonyls and a hydrophobic interaction of the C27 methyl with a pocket on the protein (Figure 20). Binding to this region results in the blocking of tubulin addition to the microtubule plus ends, which prevents longitudinal growth of microtubules.231 These findings provided the momentum to carry PM060184 into phase I clinical trials. PharmaMar has completed the adaption of this route for the industrial scale.239−242 Thus far, PM060184 has shown a reasonable safety profile, with the only observed dose-limiting toxicity being noncumulative peripheral neuropathy, which could be managed by a modification of the administration schedule. In addition, clinical antitumor activity could be observed in these studies, which bodes well for the future of PM060184 and its impending phase II trials. Although PM060184 has only recently arrived on scene, PharmaMar’s endeavors have taken this compound further in development than most marine natural products. The potent activities of PM050489 and PM060184 not only captured the attention of the PharmaMar discovery division, but also prompted them into the bold decision of developing a convergent and scalable route to these molecules. This decision is an exquisite example of how establishing a fully synthetic approach to complex natural products is not exclusive to those in academia, making the efforts of PharmaMar highly commendable. The tale of PM060184 is far from over, and it is exciting to see what the future will hold for this polyketide.

exhibited potent antitumor properties against P388 leukemia cells and suppressed a two-way mixed lymphocyte reaction.243,244 In addition, the compound, termed discodermolide, appeared to be mildly selective toward tumorigenic cell lines versus normal murine splenocytes.243 In addition to its potent anticancer activity, discodermolide also exhibited a powerful immunosuppressive activity.245−247 The structure was assigned as a multiply substituted valerolactone harboring an extended highly substituted, unsaturated appendage at its δ-position (Figure 21).243,244 The absolute stereochemistry of discodermo-

Figure 21. Structure of (+)-discodermolide.

lide was not determined in the original structural elucidation.243,247 Further studies on the biological profile of this compound revealed it to be a microtubule-stabilizing agent, similar to paclitaxel and the epothilones (vide supra).247,248 Like the epothilones, discodermolide showed efficacy against several multidrug-resistant cancer lines.243,249 In addition, discodermolide was reported to exhibit synergistic effects when combined with paclitaxel in various cancer cell lines.247,250 These preliminary findings increased enthusiasm for designating discodermolide as a formal development candidate. However, as with many marine natural products, supply was a concern. Only 0.002% of the sponge mass was discodermolide. Other avenues clearly had to be explored as a means to access the quantities needed for clinical trials.243,244,247 The two paths considered were fermentation/isolation and total synthesis. It was then unclear whether discodermolide was biosynthesized by the Discodermia sponge from which it was isolated or produced by a commensal microorganism present within the sponge.243 Some efforts have been pursued to produce discodermolide by aquaculture of a particular Discodermia sp. and subsequent isolation; however, this process is still being explored and has not been validated on a larger scale.251 In addition, the biosynthesis of discodermolide has yet to be fully elucidated, although the compound is believed to be constructed from a polyketide synthase gene cluster.252 These constraints have effectively extinguished the ability to manufacture discodermolide by current fermentation methods. The above reasons have left chemical synthesis as the only viable method of production, and many chemists have chosen to

7. DISCODERMOLIDE 7.1. Discovery and Background

During the late 1980s, the Harbor Branch Oceanographic Institute in Florida was conducting expeditions aimed at discovering prospective marine-derived therapeutics through bioassay-guided screening of extracts.243 A Discodermia sp. sponge collected in March of 1987 yielded a compound that 12028


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answer the call for a discodermolide preparation. The first to achieve a route to the enantiomer of natural discodermolide were Stuart Schreiber and co-workers, which allowed for the absolute assignment of natural discodermolide.253 The Schreiber synthesis was quickly followed by efforts by Smith et al., who also had constructed ent-discodermolide.254 Schreiber and co-workers established a route to natural discodermolide in 1996.255 Discodermolide and its enantiomer then succumbed to several more syntheses by the groups of Myles, Marshall, Smith, and Paterson.256−266 These campaigns have been reviewed previously. The current discussion focuses on the syntheses that drove large-scale production of discodermolide for use in clinical investigations.247,267,268

Scheme 65. Elaboration of Common Precursor 408 to Fragment 411

7.2. Smith’s Gram-Scale Synthesis

Scheme 66. Construction of Fragment 415 from Precursor 408

The repeating nature of substituents on polypropionate chains invites synthetic strategies that use common fragments multiple times during assembly. This theme was beautifully exploited in the Smith synthesis.256−258 When the strategy is executed well, both total operations and the longest linear sequence can be significantly minimized. This synthesis commenced with p-methoxybenzylation of (S)methyl-(3-hydroxy-2-methyl)proprionate (aka Roche ester) followed by reduction to afford alcohol 404 (Scheme 64). The Scheme 64. Smith’s Preparation of Common Precursor 408

Scheme 67. Formation of the Third Fragment 421 from Precursor 408

alcohol was then oxidized and engaged in a diastereoselective aldol reaction with the dibutylboron enolate derived from oxazolidinone 406. After recrystallization, the product was converted to Weinreb amide 408, which Smith and co-workers designated as their “common precursor” or CP. This intermediate was then elaborated in three different ways to construct the three main fragments of discodermolide. The path to the first fragment began by oxidizing 408 under anhydrous conditions to provide the p-methoxybenzylidene acetal (Scheme 65). The Weinreb amide was then reduced to an aldehyde and subjected to an aldol addition using the dibutylboron enolate derived from oxazolidinone 233 to afford β-hydroxyimide 410. This molecule was then silylated, reduced, and then iodinated to deliver 411. The second fragment was constructed by first subjecting 408 to TBS protection conditions followed by a controlled reduction to afford aldehyde 413 (Scheme 66). The aldehyde was then converted to vinyl iodide 415, the second main fragment, by the Zhao−Wittig protocol.269 To construct the final fragment, 408 was TBS-protected, hydrogenolyzed, and oxidized to afford aldehyde 416 (Scheme 67). A TiCl4-catalyzed Mukaiyama aldol was utilized with silyl enol ether 417 to afford the anti-Felkin product, which, upon

subsequent treatment with acid, cyclized to afford lactone 418. The enone functionality was chemo- and diastereoselectively reduced to allylic alcohol 419 by K-selectride. This alcohol was then protected, and the pendant olefin was oxidatively cleaved to afford aldehyde 421. Fragments 411 and 415 were joined by means of a Negishi coupling (Scheme 68). Lithiation and transmetalation of iodide 411 followed by treatment with catalytic Pd and vinyl iodide 415 afforded olefin 423. The p-methoxybenzyl group was then cleaved and replaced by a trityl protecting group to provide ether 424. The p-methoxybenzylidene acetal was then reduced to relinquish the primary alcohol and form the secondary ether, which was oxidized to afford aldehyde 425. The aldehyde was then converted to a diene under Yamamoto conditions.270 The mixture of olefin isomers could be carried on together, because of the undesired isomer being cleared in a subsequent step, in which it underwent an unanticipated Diels−Alder cycloaddition with DDQ. The primary alcohol was then deprotected and converted 12029


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Scheme 68. Smith’s Combination of Key Fragments for the Construction of (+)-Discodermolide 403

to primary iodide 429. The iodide was converted to the phosphonium salt under extremely high-pressure conditions. This proved to be necessary because of competing cyclopentannulation processes involving the proximal trisubstituted alkene. The hygroscopic phosphonium salt was then treated with NaHMDS and aldehyde 421 to afford olefin 430. The homoallylic p-methoxybenzyl ether was then oxidatively cleaved to provide the free secondary alcohol, which was converted to primary urethane 431. The tetrasilylated discodermolide was deprotected by treatment with dilute HCl to afford discodermolide (403). The repeated use of an advanced intermediate in this synthesis was an excellent strategy that resulted in (relatively) high overall efficiency. Smith and co-workers exploited their route to produce over 1 g of discodermolide, which clearly caught the attention of chemists at Novartis Pharmaceuticals (vide infra).

Scheme 69. Paterson’s Route to Fragment 436

7.3. Paterson’s Second-Generation Synthesis

The other route that would eventually influence large-scale production of discodermolide was Paterson’s “second-generation” synthesis.264 Paterson and co-workers have also published additional variations of the second-generation route, and these are described elsewhere.265 The Paterson route, similarly to the Smith synthesis, utilizes three main fragments, two of which are constructed from the same chiral pool material. The first fragment was constructed from Roche ester, which was protected and converted to ethyl ketone 432 (Scheme 69). The system was then converted to the boron enolate and treated with acetaldehyde to give an intermediate borylated aldol product, which was then reduced in situ with LiBH4 to provide diol 433. The diol was double-TBS-protected then subjected to a mild selective deprotection of the more sterically accessible secondary alcohol to afford 434. The benzyl ether was then hydro-

genolyzed, and the diol was oxidized over several steps to yield ketoester 436, the first fragment. The second fragment was constructed from ethyl (S)-lactate (437), a chiral pool material (Scheme 70). This material was converted to ethyl ketone 438, which was then subjected to a boron-mediated aldol with Roche ester derivative 439 to afford β-hydroxy ketone 440.271 The alcohol was then protected to give ether 441. The ketone then underwent reduction to an inconsequential mixture of alcohols followed by cleavage of the benzoate to give an intermediate diol, which was subsequently oxidatively cleaved to afford aldehyde 442. The aldehyde was then subjected to a Nozaki−Hiyama−Kishi reaction with allyl silane 443, followed by a base-mediated Peterson olefination to provide the terminal 12030


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Scheme 70. Paterson’s Synthesis of the Second Major Fragment

Scheme 71. Construction of the Third Fragment and Completion of the Synthesis of Discodermolide

diene moiety.272 The system was then deprotected and oxidized to afford aldehyde 445. Roche ester was the starting material for the final fragment as well (Scheme 71). The system was converted to ethyl ketone

446. Formation of the boron enolate and treatment with methacrolein afforded aldol product 447. The ketone was reduced by internal hydride delivery using Me4HB(OAc)3 to provide anti-1,3-diol 448. The diol was then converted to 12031


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dioxane 450 by acetal exchange. The system was oxidized and then heated with base to initiate a Claisen rearrangement.273 This elegant rearrangement created eight-membered unsaturated lactone 452, which established the trisubstituted olefin that is centrally located on the discodermolide tail. This lactone was then saponified, coupled with a phenol, and protected to afford the second main fragment ester 455. Ester 455 and aldehyde 445 were then combined by aldol chemistry followed by reduction of the ester to afford diol 457. The more sterically accessible primary alcohol was then converted to a sulfonate, which was then reductively cleaved with lithium aluminium hydride (LAH) to afford alcohol 459. The system was then subjected to TBS protection and DDQ-mediated double PMB deprotection to give diol 460. The primary alcohol was then selectively oxidized and subjected to Still−Gennari olefination conditions to produce β-substituted acrylate 462.274 The primary urethane was then installed by means of Cl3CCONCO followed by basic hydrolysis. The acrylate was then converted to acrolein derivative 464. Fragment 436 was then converted to the (+)-diisopinocampheylboryl enolate and combined with 464 to produce aldol product 465. These conditions were utilized to overpower the inherent re-face preference of the enolate approach to instead attack the si-face of the aldehyde, to obtain the correct diastereomer of 465. With the carbon skeleton of discodermolide intact, all that remained was a reduction of ketone 465, accomplished by an internal hydride delivery, and a tandem global deprotection/lactonization facilitated by HF. This process delivered discodermolide in 27 steps, 48 overall, in 13.6% yield. This approach was used to construct several derivatives for preliminary SAR studies.

Scheme 72. Novartis’ Process for the Preparation of Smith’s Common Precursor 408

protected derivative and subjected to reduction with LiBH4, which was much more amenable to scale than LAH, to afford alcohol 404. The alcohol was then converted to unstable aldehyde 405 by a catalytic 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)/stoichiometric bleach oxidation. The aldehyde was then subjected to an Evans aldol reaction with oxazolidinone 233 to give alcohol 468. At this point, a departure was needed from the Smith route because the pyrophoric nature of AlMe3 made it unsuitable for large-scale plant use.275 Mickel and co-workers cleaved the oxazolidinone with a basic peroxide solution followed by acidification and recrystallization with (R)-1-phenylethylamine, which was the first purification of the sequence. The acid was then liberated and coupled to N,O-dimethylhydroxylamine by means of an in situ-generated mixed anhydride. This process was used to produce almost 29 kg of key precursor 408 in one batch. Elaboration to the first discodermolide fragment was initiated by TBS protection of 408 followed by partial reduction of the Weinreb amide moiety to aldehyde 413 (Scheme 73).276 One of the more problematic reactions of the route was the conversion of aldehyde 413 to the vinyl iodide 415. A variety of methods and procedures were attempted to improve the yield of this reaction

7.4. Novartis’ Hybrid Synthesis

By 1998, Novartis Pharmaceuticals had become enamored with discodermolide because of its potent microtubule stabilization activity and promising biological profile. It proceeded to license discodermolide from Harbor Branch Oceanographic Institute for the development of a novel anticancer therapeutic and took the structure into clinical trials.247,275 However, total synthesis was and remains the only avenue to access meaningful quantities of this compound. Therefore, Novartis commissioned Stuart Mickel and a team of chemists to utilize synthetic efforts to construct discodermolide for ongoing clinical trials. After extensive examination of all academic endeavors toward discodermolide by about 2001, Mickel and his team had trouble deciding between the Smith and Paterson routes. The Smith synthesis was valued for its high convergency with fragment 408, as well as its ability to deliver a gram of material. The reagentcontrolled, chiral boron enolate end game and the superb overall yield of the Paterson synthesis aligned attractively with the strategy they were contemplating. The inherent practicality of both approaches made them both extremely attractive. However, not quite satisfied with the ability of either the Smith or Paterson approach to effectively be translated to scale, Mickel and his team of chemists at Novartis proceeded to extract the advantageous components of each, to orchestrate an elegant hybrid of the two strategies that could be used to obtain meaningful amounts of material. The initial synthetic plan was to use Smith’s common intermediate 408, because of its extremely practical nature, to construct three main fragments of discodermolide and then to stitch them together in a concise manner (Scheme 72).275 Mickel and co-workers embarked on their hybrid synthesis with Roche ester and planned to convert it into Smith’s common intermediate 408. Roche ester was converted to the PMB-

Scheme 73. Novartis’ Conversion of Precursor 408 to Fragment 415

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afforded alcohol 475. The alcohol was then converted to lightsensitive primary iodide 411 by means of an Appel reaction. These fragments were then assembled in an elegant fashion over several operations (Scheme 76).278 Mickel and co-workers found that Smith’s Negishi coupling of vinyl iodide 415 and primary iodide 411 was problematic at a large scale, leading to several side products. However, crossing over into a Suzuki manifold similar to Marshall’s approach resulted in a cleaner reaction profile, and recrystallization afforded olefin 423.261 The Novartis approach thus transitioned from Smith’s strategy to a Paterson-type end game. Reductive cleavage of the pmethoxybenzylidene acetal followed by oxidation of the newly liberated primary alcohol afforded aldehyde 447. This aldehyde was then subjected to the two-step, one-pot Nozaki−Hiyama− Kishi allylation/Peterson olefination process that was developed by Paterson, with a minor modification of KOH as the base over KH for ease of operation, to provide diene 478. Oxidative cleavage of the two PMB protecting groups followed by a selective oxidation of the primary alcohol with a TEMPO/ diacetoxy iodobenzene procedure provided aldehyde 479. The aldehyde was then subjected to the Still−Gennari modification of the Horner−Wadsworth−Emmons reaction to provide ester 462, an intermediate in the Paterson synthesis. The primary urethane was then installed by conventional means, and the ester was converted to aldehyde 464. The last fragment was incorporated into the system by means of the latestage Paterson chiral boron enolate aldol reaction.279 Unfortunately, this step proved to be extremely problematic at scale. The solid (+)-B-chlorodiisopinocampheylborane proved to be difficult to handle at scale and unstable. This problem could be remedied by utilizing a commercial hexane solution of this compound. However, these modified conditions required a large excess (6.6 equiv) of fragment 473 for the reaction to proceed in viable yields. Several other issues with this particular step almost caused the Novartis campaign to end in failure.279 Fortunately, a workable procedure emerged, and the Novartis team pushed forward. The next step was an internal hydride delivery to diastereoselectively reduce ketone 480 by means of Me4NHB(OAc)3 to give 1,3-anti-diol 481. The final global deprotection/ lactonization was conducted under carefully controlled conditions: Aqueous HCl was added in portions, and the walls of the reaction vessel were washed with MeOH to prevent oiling out of various intermediates. Discodermolide was obtained after chromatography and recrystallization. The Novartis construction of discodermolide required 26 steps in the longest route, 34 overall, in 0.9% yield. This route produced more than 60 g of discodermolide for use in clinical investigations. Although this route was successful in delivering discodermolide, it is not without issues that would need to be addressed to establish a routine process. The construction of vinyl iodide 415 would have to be optimized, and the arduous late-stage boron aldol reaction would need to be modified. In addition, the low yield of the route would be a huge problem in the later stages of development and would not be practical for long-term production. With the synthetic supply temporarily addressed, phase I clinical investigations of discodermolide in patients with solid tumors started well, with no obvious dose-limiting toxicities and no signs of neuropathy.243,280 However, the trial had to be discontinued as a result of significant pulmonary toxicity developing in the later stages of the clinical studies.281,282 Despite this setback, interest in discodermolide has not waned. Several laboratories are currently exploring the construction of discodermolide analogues or discodermolide−dictyostatin hy-

at scale to no avail, and researchers were forced to leave this step as is. This route was used to produce 415 in kilogram batches. The second main fragment was constructed from 408 through TBS protection and hydrogenolysis of the PMB group to afford alcohol 471 (Scheme 74). This system had a propensity to Scheme 74. Construction of Fragment 473

lactonize, so handling was minimized by isolating the intermediate alcohol as a solution in t-BuOH. The alcohol was immediately oxidized using a TEMPO/diacetoxy iodobenzene system. The resultant aldehyde was then subjected to MeMgBr followed by an immediate oxidation to avoid lactonization to provide methyl ketone 473 in kilogram batches. The final main fragment was elaborated from 408 by first oxidizing p-methoxybenzyl ether under anhydrous conditions to afford dioxane 474 after recrystallization (Scheme 75).277 The Weinreb amide was then reduced with LAH to afford aldehyde 409, which was then subjected to an Evans aldol reaction to afford β-hydroxyimide 410 after recrystallization. The system was then TBS-protected and subjected to reductive cleavage of the chiral auxiliary, after which a chromatographic purification Scheme 75. Novartis’ Preparation of Fragment 411

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Scheme 76. Novartis’ Combination of Discodermolide Fragments and Completion of Synthesis

co-workers then interpreted this result in a way they felt best explained the entirety of their spectroscopic analyses and designated diazonamides A−D as 483−486, wherein the core acetal existed in hydrated form and the C2 amine was either free or conjugated to L-valine through either an amidyl or amidinyl linkage.288 In addition to being a beautifully intricate structure, diazonamide A possessed potent cytostatic activity towards human cancer cell lines.287 Disclosure of these structures immediately captured the attention of the synthetic community. By the end of the 1990s, nearly a dozen groups were attempting to prepare diazonamides in the laboratory.289,290 These efforts were confronted with a very unexpected finding when, in late 2001, Harran and co-workers completed the first synthesis of 483 only to discover that it was not the natural product.291,292 In fact, synthetic 483 proved difficult to handle and characterize because of its facile degradation. Careful re-examination of spectroscopic data collected on diazonamides as well as reinterpretation of the crystallographic data used to assign structure 482 led to the conclusion that the natural products were, in fact, 487 and 488.292,293 The phenolic hemiacetals thought to be present in natural diazonamides were actually diaryl aminal motifs, and as a compensatory change, diazonamide A contained a hydroxyl group on its side chain rather than an amine. These assignments were fully consistent with available data and also had the satisfying feature of showing how diazonamides were uniformly peptide-derived, rather than a hybrid of peptidyl and type II polyketide origin that one

brids to determine whether the harmful side effects of discodermolide can be attenuated.283−286 Discodermolide synthesis presents a particularly acute challenge to methods for acyclic diastereocontrol. Smith and Paterson’s multiply convergent strategies that relied upon repetitive use of flexible raw materials were exquisitely designed and highly effective. These academic forays set the stage for a remarkable campaign of refinement and scale-up at Novartis. While these efforts faced significant challenges in handling and purifying diastereomeric mixtures at multiple points, the result was nonetheless a remarkable achievement that was able to assemble a dauntingly complex structure on sufficient scale to drive human clinical trials.

8. DIAZONAMIDE A 8.1. Background

In the late 1980s, Fenical and co-workers extracted four related antimitotic agents from the colonial ascidian Diazona angulata collected off the coast of the Phillipines.287 The molecules, named diazonamides A−D, were purified to homogeneity on milligram scales. After extensive spectroscopic analyses, it was evident a new type of polyheterocyclic structure had been discovered; yet a complete assignment for any of the four substances remained elusive. Eventually, a p-bromobenzamide derivative of diazonamide B was coaxed to crystallize in a form amenable to X-ray diffraction, and data were collected by Clardy and Van Duyne at Cornell University, who assigned its structure as the polycyclic diarylacetal 482 (see Figure 22).287 Fenical and 12034


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At this stage, the full constituency of the western region was present, being accessed by simple peptide couplings and a benzylic oxidation. What remained was to oxidatively form the macrocycle, which was achieved by subjecting the molecule to Kita oxidation conditions using iodobenzene diacetate. The remarkable transformation that followed involved oxidation of the tyrosine moiety, capture of the incipient phenoxenium ion by the pendant indole, and ring closure to aminal 494. This process installed the challenging C10 quaternary center early in the synthesis and afforded macrocycle 494 as a 3:1 mixture of diastereomers favoring the desired isomer. Although the yield of 494 was less than ideal because of competing spirocyclohexadienone formations, the reaction was uniquely enabling. It converted a modified linear peptide into an advanced polycycle in a single operation. Intermediate 494 was then subjected to a protecting-group exchange and saponification to provide acid 495. The acid was then coupled to 7-hydroxytryptamine (496) to provide amide 497. Acetylation followed by a two-step benzylic oxidation/cyclodehydration provided bis-oxazole 498. At that point, the stage was set to form the eastern macrocycle of diazonamide A. Harran and co-workers had discovered during their synthesis of the initially proposed diazonamide A structure that difficulties with atropisomerism in the eastern macrocycle formation could be subverted by the presence of an established western macrocycle.291,302 This discernment served them well when 498 was irradiated under basic conditions to afford bicycle 499 in high yield. The irradiation initiated an electron transfer between the two indole moieties, leading to a radical ion pair, which underwent lithium bromide elimination followed by radical recombination and tautomerization to afford the target diazonamide core 499.302,307,308 The added phenol was subsequently reduced through its triflate derivative. The aminal nitrogen was then protected as the (trimethylsilyl)ethoxycarbonyl (Teoc) derivative. The western macrocycle was then twice chlorinated by the action of perchloro-2,4-cyclohexadien-1-one (503) and subjected to global deprotection to afford des-bromo diazonamide B 504.309 Final appending of (S)α-hydroxy isovaleric acid (505) to the core produced diazonamide A. This approach to diazonamide A (487) completes the molecule in 19 steps and 1% yield.

Figure 22. Original structures proposed for diazonamides A and B and revised variants elucidated by Harran and co-workers.

seemingly needed to invoke for the initial assignments to be correct.292 Following the reports by Harran and co-workers, synthetic efforts shifted toward the new targets. The Nicolaou group adapted their ongoing route to complete the synthesis of 487.294,295 This same group established a second pathway to diazonamide A some time later.296 Interest in diazonamide synthesis has continued unabated over the years.297−301 Many laboratories have made important and creative contributions to the area. These efforts have been reviewed.290 The tactics most pertinent here, namely towards the clinical development of diazonamides as anticancer drugs, are those first outlined in Harran and co-workers’ total synthesis of diazonamide A published in 2003.302

8.3. Development of DZ-2384

Harran and co-workers wasted no time and began to explore the mode of action of the diazonamides and also to construct diazonamide analogues.310−313 As indicated above, diazonamide showed potent antiproliferative activity in human cancer cell lines. However, the cellular target of diazonamide had been elusive. Early studies implicated microtubules as a potential target, whereas others have indicated no direct interaction.311,314 The target of diazonamide A has also been tracked to the mitochondrial enzyme ornithine δ-amino transferase, which, surprisingly, has a role in regulating mitotic cell division in human cancer cells.311,312 Although the target remained unknown, diazonamide treatment of human tumor xenografts in nude mice showed regression with little evidence of overt toxicity.311,312 This lack of toxicity was an exciting result, which led to the development of Joyant Pharmaceuticals, which continued the development of diazonamide SAR utilizing Harran’s route to construct analogues.313,315−318 It was determined that the eastern macrocycle was not necessary for activity, nor was the ansa-bridged indole moiety.318 In terms of the northern bisoxazole motif, a large number of oxazole−heterocycle combinations were explored.318 However,

8.2. Harran’s Construction of Diazonamide A

Inspired by their contemplated biosynthesis of the diazonamides, Harran and co-workers decided to build the structures in a related manner. The approach was conceptually simple and consisted of assembling a seco-peptidyl precursor and installing the central diarylaminal motif by direct oxidative annulation of tethered tyrosine and tryptophan side chains (Scheme 77). The synthesis commenced with racemic 7-bromotryptophan (489).303,304 This compound was acylated with Z-L-valine to afford dipeptide 491 as an inconsequential mixture of diastereomers.305 This material was subjected to Yonemitsu oxidation with DDQ to produce a single 3-oxazolylindole intermediate, which was subsequently treated with HBr in AcOH to afford amine salt 492.306 This material was then acylated with nosyl-protected tyrosine to afford seco precursor 493. 12035


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Scheme 77. Harran’s Construction of Diazonamide A

the best system retained the native bisoxazole moiety appended with a primary carbinol. In addition, conversion of the northernmost isopropyl group to a tert-butyl group and installation of a fluorine atom on the southern indoline ring improved the pharmacokinetic profile of the system. The product of these extensive investigations was the structure designated as DZ-2384 (522, Scheme 78), which was chosen as the development candidate in 2012.318,319 The same general approach of oxidatively modifying a polypeptide was developed to supply DZ-2384 for preclinical development and the upcoming clinical investigations. The sequence began with L-tert-leucine (506, Scheme 78).319 Derivatization with carboxybenzyl chloride (Cbz-Cl) followed by a conventional coupling with L-serine methyl ester and saponification afforded dipeptide 509. The serine moiety was then activated in the presence of 5-fluoroindole 510 to afford tryptophan derivative 511. Another standard coupling of L-serine methyl ester afforded tripeptide 512. This material was then subjected to a sequence of oxidations. First, a DDQ-mediated

oxidation of the system produced oxazolylindole 513. Subsequently, a one-pot cyclodehydration with Deoxo-Fluor (514) produced oxazoline 515, which was then oxidized in situ under a modified variant of Wipf and William’s conditions to provide bisoxazole 516.320,321The oxidation product was then deprotected by treatment with strong acid to afford amine salt 517. The amine was then acylated with Boc-Tyr-OH under standard coupling conditions to afford 518, which thereafter was deprotected by treatment with acid to give amine salt 519. This compound was subjected to a simple coupling reaction with (S)-2-hydroxyisovaleric acid followed by a reduction of the pendant ester to afford seco precursor 521. The final step would be akin to the Kita oxidation that Harran and co-workers used in the initial diazonamide A synthesis. However, this operation proved to be a bottleneck during SAR investigations, where side products complicated the purification and lowered yields. Harran and the chemists at Joyant Pharmaceuticals searched for a means to eliminate byproducts inherent to the Kita protocol.319 They discovered a unique solution using electrochemistry wherein the 12036


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Scheme 78. Harran and Diazon/Paraza Collaborative Development of a Refined DZ-2384 Synthesis

observed only upon extended treatments at the maximum tolerated dose, but this resolved following a recovery period. This lack of toxicity and the wide safety margin led to the hypothesis that these characteristics were derived from DZ-2384’s interaction with its receptor. A genome-wide RNA interference screen allowed Diazon Pharmaceuticals to identify the cell cycle and mitosis as potential targets for DZ-2384’s mechanism of action.322 H1299 cells treated with DZ-2384 accumulated in the G2-M phase of the cell cycle.322 Further investigations revealed that preincubating tubulin with vinorelbine led to an inhibition of binding to biotinylated DZ-2384 surfaces in a dose-responsive manner, which suggested that DZ-2384 interacted with the Vinca domain.322 X-ray crystallographic analysis of DZ-2384 bound to a tubulin complex (specifically two αβ-tubulin dimers, namely, stathmin-like protein RB3 and tubulin tyrosine ligase, abbreviated as T2R-TTL) indicated that DZ-2384 does bind to the Vinca domain, but in a manner that is unique.322 Closer investigations of the binding mode and a superimposed atomic model with vinblastine revealed that the two structures share similar binding interactions at the interdimer interface.322 However, DZ-2384 lacks the bulky catharanthine moiety that is characteristic of the vinca alkaloids, allowing the interdimer interface to be more compact than both the apo- and vinblastinebound T2R-TTL complexes.322 This change caused a large shift

substrate was controllably oxidized at an anode surface at constant potential. This allowed for an indolic oxidation (rather than a phenolic oxidation) to form a radical cation that captured the pendant phenol and then underwent internal cyclization and oxidation to give diazonamide macrolactams free from spirocyclohexadienone contaminants.313 This beautiful process was utilized for the final step in the construction of DZ-2384 (522), affording the target as a 2.3:1 mixture of diastereomers. This route constructs the drug candidate in 13 steps and 4.8% overall yield. DZ-2384 performed superbly in extensive preclinical investigations. Studies with subcutaneous xenograft models (including pancreatic ductal adenocarcinoma) revealed that DZ-2384 caused complete regression within 3 months of treatment.322 In addition, DZ-2384 administration to a model for metastic triple negative breast cancer (MDA-MB-231-LM2) in immunocompromised mice caused regression of all metastases at the lowest dose tested.322 An analysis of the safety profile for DZ2384 indicated that it has a much wider safety margin than vinorelbine. Cumulative peripheral neuropathy is a major toxicity issue for many microtubule-targeting agents. An examination of extended DZ-2384 treatment on nerve function revealed that it had no electrophysiological or microscopic signs of peripheral neuropathy at circulating concentrations 13 times higher than those needed for antitumor activity in mice.322 Neurotoxicity was 12037


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in the orientation of the two tubulin dimers.322 Further investigations with helical superassemblies of T2R-TTL complexes indicated that the addition of DZ-2384 increased the radii, leading to the hypothesis that DZ-2384 straightens protofilaments.322 The ability of DZ-2384 to interrupt the cell cycle coupled with its safety profile suggested that sensitivity to the drug was increased in molecules actively undergoing mitosis.322 Microtubule sedimentation assays on H1299 cells revealed that DZ2384-treated cells retained higher concentrations of microtubule polymer mass than cells treated with vinorelbine.322 Further studies with HeLa cells synchronized in late G2 showed stunted microtubules and misaligned chromosomes.322 These results indicate that the cytoskeletons of interphase cells are not as susceptible to DZ-2384-mediated apoptosis as those actively undergoing apoptosis.322 To determine how DZ-2384 affects the dynamic instability of microtubules, Diazon Pharmaceuticals analyzed growing ends of microtubules in U-2 OS (osteosarcoma) cells. It was determined that DZ-2384 decreased the number of microtubule growths from the centrosome and their frequency.322 In addition, DZ-2384-treated cells, when compared to vinorelbine-treated cells, had an increased rescue frequency relative to the controls.322 These studies indicated that DZ-2384 slows microtubule growth, but allows for a sufficient amount of rescue events to maintain polymeric tubulin in interphase cells.322 The journey to DZ-2384 has been intensely interestingand full of twists and turns. Moreover, the enabling synthetic methods advanced by Harran and Joyant/Diazon Pharmaceuticals now permit the construction of diazonamide structures in an almost trivial manner. Linear peptidyl motifs are directly oxidized at a graphite surface to give structurally complex polyheterocycles at room temperature. Extensive exploration of the diazonamide pharmacophore has produced DZ-2384, which is poised to enter clinical trials as a next-generation antimitotic with an unprecedented safety profile. The coming years will be a truly exciting time for the diazonamide project.

Figure 23. Structures of ingenol 3-hexadecanoate; ingenol; and Picato, the topical agent used to treat actinic keratosis.

effects.330 Notably, ingenol 3-angelate is easily degraded by esterases to reveal ingenol, making systemic administration difficult.326 Nevertheless, ingenol 3-angelate has advanced to clinical trials as a topical agent and is ab effective therapy for actinic keratosis and basal cell carcinoma.331,332 In fact, the FDA approved 525 as a first-in-class molecular entity for the topical treatment for actinic keratosis.326,327,333 Leo Pharma developed ingenol 3-angelate as a pharmaceutical candidate and markets the drug as Picato.334−341 The supply of ingenol 3-angelate for all preclinical studies and clinical trials was produced either from natural sources or through derivatization from ingenol.342−344 Although these routes were firmly established at the time of FDA approval, they were laborious, costly, and inefficient, which hampered progress in terms of practicality.326,327 To potentially address some of these issues and to greatly expand access to analogues and structural variants, Leo Pharma began a collaboration with the Baran laboratory at the Scripps Research Institute to evaluate fully synthetic materials.326,327 One goal of this collaboration was to develop a practical total synthesis of 525 that was cost-effective and more amenable to industrial scales.326,327 9.2. Summary of Early Synthetic Work

There had been several prior syntheses of ingenol at the onset of the Leo Pharma and Baran laboratory collaboration (Scheme 79).345−348 These works proved invaluable to the Baran laboratory for exploring the reactivity of various ingenane-type frameworks, information that would be exploited in their own synthesis. The first synthesis was established by Winkler et al., who employed a spectacular de Mayo-type photocycloaddition− retroaldol fragmentation cascade to establish the trans-bridged bicycle.345 Shortly thereafter, Tanino, Kuwajima, and co-workers reported an approach based on a Nicholas ion-mediated alkylative cyclization/semipinacolic rearrangement sequence.346 The elegant work of Funk and co-workers toward an asymmetric entry to the ingenane skeleton proved invaluable for the efforts of Kigoshi and Wood, who simultaneously explored similar routes to ingenol.349,350 Utilizing Funk and co-workers’ intermediate 535 derived from (+)-3-carene, Kigoshi and co-workers established the first asymmetric entry to the ingenol ring system.348 Kigoshi and co-workers utilized ring-closing metathesis to construct the B ring, demonstrating its utility in producing strained carboskeletons, which after subsequent oxidation intercepted aldehyde 537 from Winkler’s synthesis.348 Shortly after Kigoshi and co-workers’ reports, Wood and coworkers established a synthesis of ingenol also employing ringclosing metathesis for B ring formation on functionalized diene precursor 538.347,351 In addition to these advanced efforts, there have been numerous creative syntheses of ingenol-type carbon frameworks.352,353

9. INGENOL 3-ANGELATE 9.1. Discovery and Development

In the late 1960s, Hecker purified several new compounds from various plants of the Euphorbia genus.323 One of the molecules isolated showed strong phorbol-like tumorigenic activity. When this compound was saponified, hexadecanoic acid and a complex alcohol were produced.323 The identity of the latter, named ingenol, remained a puzzle until it was resolved by X-ray crystallographic analysis two years later.324 Ingenol’s structure revealed a fascinating trans-bridged bicyclo[4.4.1]undecane ring system (aka in/out configured) that introduces significant strain into the system (Figure 23).325,326 Numerous biological activities have been ascribed to ingenol and its derivatives since their discovery.325−327 Among these, the antiproliferative nature of the 3-angelate ester has proved to be most valuable in medicinal terms.326,327 Ingenol 3angelate can be isolated from several Euphorbia species, which have a long history of use as traditional topical treatments.328 Angelate 525 proved to be effective against a wide range of tumor cell lines, inducing mitochondrial swelling and necrosis, and when administered topically, it was found to be able to regress tumor xenografts in rodent models.328,329 The activity 525 is thought to derive from perturbation of protein kinase C signaling, resulting in antiproliferative and proapoptotic 12038


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biosynthetic stages leading to oxygenated polycyclic hydrocarbons in general. Similarly to Funk, Kigoshi, and Wood, Baran chose to begin the synthesis of ingenol with (+)-3-carene (534), an abundant optically active monoterpene (Scheme 80). This choice allowed Baran and co-workers to begin with a molecular topography useful for establishing further stereocenters in a controlled manner. (+)-3-Carene was subjected to an allylic chlorination followed by ozonolysis of the transposed alkene to afford ketone 541. The α-chloroketone could then be reductively methylated by sequential treatment with lithium naphthalenide and methyl iodide. Isolation of this sensitive intermediate ketone proved difficult. However, it was discovered that, after methylation, in situ treatment with lithium hexamethyldisilazide (LiHMDS) followed by aldehyde 542 afforded adduct 543 as a single diastereomer. 542 was constructed from propargyl alcohol (557) and triethyl orthopropionate (558) by a Claisen rearrangement followed by reduction with LAH to afford racemic alcohol 560 (Scheme 81).354 The alcohol was then subjected to a lipasemediated transesterification using vinyl acetate, which provided the desired (R)-alcohol in 98.7% ee.354 The alcohol was then oxidized with 2-iodoxybenzoic acid (IBX) to afford 542. The aldol product was then treated with ethynylmagnesium bromide to afford the intermediate propargyl alcohol as a 10:1 mixture of diastereomers, which could be separated after double silylation to provide crystalline 545. This material was then subjected to an allene-yne Pauson−Khand cyclization under high-dilution conditions, which constructed the tetracyclic tigliane skeleton 546. This key process markedly increased the complexity of the system in a single step that was both elegant and practical. The resultant ketone was then treated with methylmagnesium bromide to afford tertiary alcohol 547. This procedure marked the end point of the “cyclase phase”, because all carbons for the ingenol framework were in place. The synthesis then transitioned into the “oxidase phase”, where the periphery of the ingenol framework was waoxidatively decorated.326,327 The first step was a dihydroxylation of the trisubstituted olefin in the southern portion of the molecule. The hindered nature of the olefin precluded catalytic dihydroxylations, but stoichiometric dioxoosmylation was successful.327 The diol subsequently formed by hydrolysis was then masked as its carbonate. The system was now poised for a vinylogous pinacol rearrangement, which was accomplished by treatment with BF3 at low temperature followed by gentle warming and quenching with Et3N/MeOH. Structure 550 proved to be a kinetic product. Resubjection at warmer temperatures resulted in a retro-pinacol rearrangement to give elimination product 562 (Scheme 82). The oxidase phase continued with a SeO2-mediated allylic oxidation followed by in situ acetylation to afford acetate 551. The structure was then deprotected and dehydrated and subjected to a basic workup to cleave the carbonate to afford olefin 554. This system was subjected to an additional allylic oxidation with SeO2 to provide ingenol. To convert ingenol into Picato, Leo Pharma devised the following route: Ingenol was derivatized as the crystalline acetonide 555 according to conditions developed by Hecker.342,355 555 was then deprotonated with LiHMDS and treated with angelic anhydride to afford ester 556.342 The acetonide was then cleaved by treatment with phosphoric acid, and the crude material was recrystallized to afford ingenol 3-angelate. This process constructs Picato in a longest linear sequence of 19 steps,

Scheme 79. Summary of Previous Synthetic Constructions of Ingenol

Although all of these efforts were significant accomplishments, the shortest completed route to ingenol exceeded 30 steps, which would present significant challenges on manufacturing scales. 9.3. Baran’s Synthesis of Ingenol

Baran and co-workers carefully evaluated previous approaches to identify both their advantages and their drawbacks. They were drawn to the approaches of Tanino−Kuwajima and Cha and Epstein, which utilized semipinacol rearrangements to construct the ingenane skeleton from a tigliane core.353 It was thought that this transformation might be related to the biosynthesis of ingenol, wherein a pinacol rearrangement has been postulated. In addition, Baran and co-workers sought a preparation that could be dissected into cyclase and oxidase “phases”, analogous to the 12039


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Scheme 80. Baran’s Preparation of Ingenol and Leo Pharma’s Elaboration to Ingenol 3-Angelate, Picato

action.356 In addition to the construction of analogues, Leo Pharma scaled up the synthesis and has made more than 1 kg of ingenol by the Baran route.357,358 Although this route is wonderfully elegant, some steps remain problematic on a process scale, such as the stoichiometric use of OsO4 and SeO2. However, these issues are being addressed by the scientists at Leo Pharma. Despite the success of this synthesis, efforts have been made by Phyton Biotech to establish a more efficient fermentation protocol of ingenol 3-angelate using plant-cell-culture-based technology.359,360 The ingenol story is clearly far from complete. Extensive SAR studies and core structural modifications are ongoing. Hopefully, further insight into the biological mechanism of action could provide a rational basis for designing new derivatives for systemic administration. All of this work is being made possible by Baran’s highly enabling and concise new synthesis of ingenol.

Scheme 81. Construction of Aldehyde 542

Scheme 82. Formation of the More Stable Elimination Product by Resubjection to Vinylogous Pinacol Conditions at a Higher Temperature

10. CONCLUSIONS In this review, we have described remarkable efforts to convert complex synthetic problems into manageable chemical processes amenable to manufacturing scales. Whether or not the solutions presented reach a level one could term practical is certainly open for debate and undoubtedly influenced by cost-of-goods, process timelines, and market size/demand, data that were not readily available to us. However, in terms of synthetic strategy, traditional hurdles are amplified. By that we mean that, without exception, what makes the structures discussed particularly challenging are their stereochemical features and the need to install multiple contiguous asymmetric centers with exquisite

21 steps total, with an overall yield of 1.9% from propargyl alcohol. With a concise synthetic route established to ingenol, Leo Pharma wasted no time in exploiting its investment. This synthesis was utilized to construct a variety of analogues that allowed for a more thorough investigation of Picato’s mode of 12040


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REFERENCES

control while avoiding long linear sequences in the process. Convergency remains king, in this regard, and the execution of high-yielding, flexible fragment couplings are powerfully enabling strategies. There is definitely room for new discoveries in this area. As stated at the outset, it is unlikely that de novo synthesis will supplant semisynthetic methods when a biological source of advanced raw material is available. However, in those cases where such a source is not available or where deep-seated structural alterations of a natural-product lead are sought, we look forward to ever more exciting, ambitious, and refined synthesis campaigns. Our field has come a long waybut there is still so much farther to go.

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AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): PGH is a founder and shareholder in Diazon Pharmaceuticals, which is attempting to commercialize DZ2384. Biographies Tyler K. Allred obtained his B.S. in chemistry in 2012 at the University of California, Davis, where he synthesized designed guanosine analogues to investigate their effect on the MUTY/MutY DNA repair pathway with Professor Sheila David. In 2013, Allred joined Professor Patrick Harran’s group at the University of California, Los Angeles, where his research is focused on the synthesis of complex marine-derived alkaloids. Francesco Manoni obtained his B.S. and M.S. degrees in chemistry at the University of Bologna, Bologna, Italy, with Professor Diego Savoia. During his Ph.D. research, Manoni worked with Professor Stephen J. Connon developing new organocatalytic processes for the enantioselective addition of enolizable cyclic anhydrides to various electrophiles towards the formation of bioactive compounds. During this time, he also collaborated on other projects, including the organocatalytic dynamic kinetic resolution of azlactones paired with a ligation-inspired coupling process and the development of the first catalytic thiolate-catalyzed cross-Tishchenko reaction between aldehydes and ketones. In 2014, he joined Professor Patrick Harran’s group at the University of California, Los Angeles, where his postdoctoral research has resulted in a unique total synthesis of the marine-derived macrolide callyspongiolide. Professor Patrick Harran received his B.A. degree from Skidmore College in New York. He obtained a Ph.D. from Yale University in 1995 (with F. E. Zeigler) and completed an NIH-sponsored postdoctoral fellowship at Stanford University (with P. A. Wender) in 1997. That fall, he joined the faculty at the University of Texas Southwestern Medical Center. In 2005, he was promoted to Full Professor and named the Mar Nell & F. Andrew Bell Distinguished Chair in Biochemistry. He joined the faculty at the University of California, Los Angeles, in July 2008 as the inaugural D. J. and J. M. Cram Chair in Organic Chemistry.

ACKNOWLEDGMENTS This work was supported by the Donald J. & Jane M. Cram Endowment, a National Institutes of Health/National Cancer Institute grant (R01CA184772-01) and a postdoctoral fellowship provided to F.M. by the Human Frontier Science Program. 12041


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