Exploring the Boundaries of Practical : De Novo Syntheses ... · Exploring the Boundaries of “Practical”: De Novo Syntheses of Complex Natural Product-Based Drug Candidates Tyler

Exploring the Boundaries of “Practical”: De Novo Syntheses ofComplex Natural Product-Based Drug CandidatesTyler 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 thebuilding of complex architectures on scales sufficient to drive human drug trials. Wefocus on the relatively few instances in which a natural-product-based developmentcandidate has been manufactured de novo, rather than semisynthetically. This summaryprovides a view of the strengths and weaknesses of current technologies, providesperspective on what one might consider a practical contribution, and hints at directionsthe field might take in the future.

CONTENTS

1. Introduction 119942. Halichondrin B 11995

2.1. Introduction 119952.2. Discovery of Halichondrins 119952.3. Synthetic Studies toward Halichondrin B 119962.4. Truncated Halichondrins Reveal Pharmaco-

phore 119982.5. SAR Studies: The Discovery of Eribulin 119992.6. Synthesis of Eribulin from Milligram to Gram

Scale 120002.7. Multikilogram Manufacturing Process of

Halaven 120023. Synthetic Tetracyclines 12006

3.1. History of the Tetracyclines 120063.2. Myers’ Synthetic Studies on the Tetracyclines 120073.3. Tetraphase SAR Studies 120103.4. Eravacycline Synthesis 12011

4. Epothilone 120134.1. Epothilone Background 120134.2. Danishefsky’s Synthetic Approach 120134.3. Nicolaou’s Synthesis 120144.4. Schinzer’s Formal Synthesis 120154.5. Epothilone Structure−Activity Relationships 120164.6. Development of Sagopilone 120174.7. Dehydelone, Fludelone, and Isofludelone 12020

5. Cryptophycin 120235.1. Discovery and Background 120235.2. Tius−Moore’s Synthesis of Cryptophycin 1 120235.3. Exploration of SAR 120245.4. Eli Lilly’s Synthesis of Cryptophycin 52 12024

6. PM060184 120266.1. Discovery of PM050489 and PM060184 120266.2. Synthetic Studies on PM060184 12027

7. Discodermolide 120287.1. Discovery and Background 120287.2. Smith’s Gram-Scale Synthesis 120297.3. Paterson’s Second-Generation Synthesis 12030

7.4. Novartis’ Hybrid Synthesis 120328. Diazonamide A 12034

8.1. Background 120348.2. Harran’s Construction of Diazonamide A 120358.3. Development of DZ-2384 12035

9. Ingenol 3-Angelate 120389.1. Discovery and Development 120389.2. Summary of Early Synthetic Work 120389.3. Baran’s Synthesis of Ingenol 12039

10. Conclusions 12040Author Information 12041

Corresponding Author 12041Notes 12041Biographies 12041

Acknowledgments 12041References 12041

1. INTRODUCTION

The laboratory synthesis of complex natural products and themanufacture of small-molecule drugs are chemistry compatriotsthat seldom meet. The former is typically an academic pursuit inwhich emerging methods are tested and assembly tactics areexplored, whereas the latter are beautifully engineered exercisesin brevity, efficiency, and scale. The two operate on commonprinciples, of course, but thereafter, they focus on different goalswith markedly different criteria for success. In fact, they divergewidely at the idea of practicality. However, “practical” is anebulous concept that shifts with perspective and, ultimately,intention. According to the Oxford Dictionary, the colloquialdefinition of practical is “concerned with the actual doing or useof something rather than with theory and ideas”. What happenswhen a complex natural product is sufficiently compelling as adrug that a group attempts its manufacture? If the synthesis is

Special Issue: Natural Product Synthesis

Received: March 3, 2017Published: June 12, 2017

Review

pubs.acs.org/CR

© 2017 American Chemical Society 11994 DOI: 10.1021/acs.chemrev.7b00126Chem. Rev. 2017, 117, 11994−12051

pubs.acs.org/CR

http://dx.doi.org/10.1021/acs.chemrev.7b00126

completed and there is a viablemarket for the product, is that not,by definition, practical?Themost commonmeans to access natural products on a large

scale has been through semisynthesis. The sequential use ofbioengineering and synthetic chemistry has allowed trulyremarkable substances to be brought to market at reasonablecost.1 Examples from the recent literature include the Sanofiprocess to generate artemisinin, the Pharma Mar Trabectedinprocess, and the semisynthesis of paclitaxel by Bristol-MyersSquibb (Figure 1).2−7 In addition, microbial fermentation of

natural-product core structures has fueled an analogue industry,particularly valuable in the antimicrobial arena where serial drugresistance is a perennial challenge. Scores of β-lactam- andmacrolide-derived antibiotics have been discovered, developed,and commercialized in this way (Figure 2).8

Semisynthetic methods are powerful and likely to becomemore so in the future. There are situations, however, in whichthese tools are not applicable. If an advanced intermediate cannotbe sourced from nature or from heterologous expression systemsor if earlier studies have identified structural analogues that arenot accessible by peripheral functionalization, the remainingoption becomes de novo total synthesis. In many cases, that goalhas been deemed insurmountablethat is, hopelessly imprac-tical.Nonetheless, the methods of synthesis are constantly being

refined and expanded, providing new ways to solve complexproblems. We were drawn to the relatively few examples in whichaudacious research groups bucked the trend and tackled the totalsynthesis of a natural product with the explicit intent ofmanufacturing the product. What can be learned from theirefforts? Where does the state of the art stand in this context? Will

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

2. HALICHONDRIN B

2.1. Introduction

This section describes academic and industrial explorations thatled to the discovery and development of a commercializedsynthetic route to eribulin mesylate, an anticancer agent sold byEisai under the trade name Halaven. This molecule, inspired bythe marine natural product halichondrin B, is the result of anintense drug-discovery program undertaken by the EisaiResearch Institutes in Massachusetts and Japan. The syntheticpath to the structure, far more complex than any othercommercialized small-molecule drug,9 required significantoptimization to secure a reliable manufacturing process.2.2. Discovery of Halichondrins

In the early 1980s, a research team led by Uemura searchedmarine invertebrates for new biologically active naturalsubstances.10 The group focused on the collection of a commonsponge called Halichondria okadai Kadota, widely distributedalong Japanese coastlines.Earlier studies of the same organism had led to the isolation of

okadaic acid.12 The team suspected that the chemistry in thesponge was more extensive. Further fractionations of the extractsguided by hyperpotent antineoplastic activity resulted in thediscovery of a new family of polyether macrolides, which theynamed halicondrins.10,11 From ∼600 kg of sponge, the groupeventually purified eight small-molecule constituents thatexhibited 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 activethan the more abundant norhalichondrin A (i.e., 1). Theseexciting results encouraged the team of isolation chemists toexplore the in vivo activity of the compound. Halichondrin B wasevaluated against three different types of human cancer cells innude mice. The results are summarized in Figure 3. HalichondrinB displayed powerful activity at low concentration with bothintraperitoneal and intravenous injections effectively doublingthe life span of the diseased mice.11 The high potency exhibited

Figure 1. Commercial semisyntheses of complex natural products.

Figure 2. Microbial fermentation provides core structures utilized forperipheral functionalization.

Chemical Reviews Review

DOI: 10.1021/acs.chemrev.7b00126Chem. Rev. 2017, 117, 11994−12051

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by halichondrin B both in vitro and in vivo coupled with itsstriking structure attracted considerable attention in thesynthetic community. Shortly after the structure and thebioactivity were reported, several academic groups embarkedon the challenge of producing synthetic samples of halichondrinB (4) to expand studies of its activity and address its extremelylow natural availability. These efforts have been reviewedpreviously.13−15 This discussion focuses mainly on the effortsof Kishi’s group and the Eisai Research Institute that led to theindustrial manufacturing of eribulin mesylate (Halaven).

2.3. Synthetic Studies toward Halichondrin B

The first total syntheses of halichondrin B (4) and norhalichon-drin B (2) were reported by Kishi and co-workers in 1992.16 Bydesign, the route was highly convergent and assembled the targetmolecules from four primary fragments (Figure 4); three of thefour were equipped with the (E)-vinyl iodide motif, such thatthey could participate in Nozaki−Hiyama−Kishi coupling, which

became the main fragment-union technology employed on theindustrial scale. Another defining feature of the approach utilizedthermodynamically driven transformations to construct both theC38 spirocycle and ansa-bridged, caged C14 ketal late in thesynthesis. This approach allowed large domains of the naturalproduct to be evaluated biochemically as isolated entities and aspart of increasingly elaborate architectures. Furthermore, each ofthe fragments was derived from readily available chiral pool-derived raw materials (Figure 4).The synthesis of fragment 9 commenced with the trans-

formation of the readily available 2-deoxy-L-arabinose (13) togenerate 14 in four simple steps according to the knownliterature procedure.17 Further modification of the diethyldi-thioacetal 14 into 15was then accomplished in 13 further steps.16

In the meantime, lactone 1618 was elaborated to give thephosphonate 17 in 10 steps16 and used for the Horner−Wadsworth−Emmons olefination of the aldehyde derived from

Figure 3. (A) Halichondrins isolated fromHalichondria okadaiKadota sponge and cytotoxicity activity against B-16 melanoma. (B) Initial data on the invivo activity of halichondrin B (4).11



11996


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

Fragment 10 was synthesized according to a route previouslyestablished by Kishi’s group.19 Starting from D-galactose glycal, afour-step transformation generated compound 19, which thenunderwent stereoselective Ireland−Claisen rearrangement and,upon basic hydrolysis, furnished 20 with high diastereoselectiv-ity. Subsequent iodolactonization of 20 followed by reductivedehalogenation gave lactone 21. This molecule was transformedinto aldehyde 22 through 10 further simple synthetic steps.Nozaki−Hiyami−Kishi coupling between 22 andmethyl trans-β-iodoacrylate, followed by three further functional-groupmanipulations, yielded the bicyclic structure 23, which, uponfour additional transformative steps and a final Dess−Martinoxidation, generated aldehyde 10.16 This compound wassynthesized 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

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

Finally, the synthesis of the last required fragment ofhalichondrin B took place according to procedures establishedby the group.22 The synthesis commenced with the 11-steptransformation of butenolide 27, readily produced from ascorbicacid,23 into compound 28. Eight additional steps then yieldedfragment 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 aldehydefunctionality in 10 produced a 6:1 diastereomeric mixturefavoring the desired allylic alcohol. Subsequent base-promotedcyclization yielded the desired tetrahydropyran 29 in moderateoverall yields.16 Removal of the pivalate protecting group,oxidation of the alcohol, and Nozaki−Hiyama−Kishi reactionbetween the resulting aldehyde and fragment 11 furnished, uponoxidation of the obtained allylic alcohol, the enone intermediate30 in good overall yield (Scheme 5).16

Compound 30 was then treated with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) for the removal of the p-

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

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

Scheme 2. Synthesis of Fragment 10




11997


methoxybenzyl (PMB) protecting group, the methyl ester washydrolyzed, and the resulting seco-acid was subjected tocyclization under Yamaguchi’s conditions to furnish themacrolactone 31 in good overall yield. Removal of the silylprotecting groups with tetrabutylammonium fluoride (TBAF)followed by exposure to pyridinium p-toluenesulfonate (PPTS)triggered initial Michael addition of the C9 alcohol functionalityto 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, protectionof the secondary alcohol, and subsequent hydrolysis yieldedintermediate 32 (Scheme 6).16

Compound 32 was then oxidized, and the resulting aldehydeunderwent Nozaki−Hiyama−Kishi coupling with fragment 12 togenerate an allyl alcohol intermediate that, following oxidation toan enone, cleavage of the silyl ether protecting groups, PPTStreatment, PMB cleavage, and final exposure to camphorsulfonicacid (CSA), produced halichondrin B in good yield (20−30%over six steps).16 Following this synthetic route, halichondrin B

(4) was produced in a sequence of 47 longest linear steps(Scheme 7).

2.4. Truncated Halichondrins Reveal Pharmacophore

Once a synthetic pathway to the natural product had beenestablished, the attainment of a pure material supply to expandthe biological studies of the molecule seemed feasible. In March1992, the Decision Network Committee at the National CancerInstitute (NCI) recommended that halichondrin B be furtherexplored as a therapeutic chemotype. In the same year, EisaiResearch Institute (ERI) was provided with synthetic samples ofhalichondrin B and several intermediates for evaluation both invitro and in vivo as antineoplastic agents. Surprisingly,intermediate 33 displayed activity comparable to that of theparent halichondrin B against the growth of cancer cells, morespecifically in DLD-1 human colon cancer cells (Figure 5).24

This discovery began the important process of identifyingmore synthetically accessible analogues having equivalent orgreater anticancer activity. Additionally, the results suggested


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

Scheme 6. Generation of Intermediate 32

Scheme 7. Completion of the Synthesis of Halichondrin B (4)



11998


that the macrocyclic moiety was the main pharmacophore ofhalichondrin B and that the polyether appendage could besignificantly truncated (Figure 5).Microtubules are an important target of anticancer drugs as

they play a crucial role during cell replication. Other potentanticancer agents such as taxanes and Vinca alkaloids disrupt thepolymerization process of microtubules during mitosis, thusinducing apoptosis. Halichondrin B and its simplified analoguesbelong to the family of microtubule-targeting drugs, which blockthe growth of microtubules and transform tubulin intofunctionally inactive aggregates.25 It was shown that 33 directlybinds to tubulin and that the same mechanism of action is sharedby the related structure halichondrin B, which cemented themacrocyclic core as the antineoplastic pharmacophore.26

2.5. SAR Studies: The Discovery of Eribulin

The discovery of the macrocycle’s central role in theantiproliferative properties of the halichondrins allowedstructure−activity relationship (SAR) studies to focus onperipheral functionalization, with the aim of both maximizingthe biological activity and simplifying the structure. In contrastwith halichondrin B, macrolactone 33was found to be inactive invivo, proving to be incapable of maintaining a complete mitoticblock (CMB) in vitro after washout.27

Additional SAR studies aimed at resolving the in vivo issuebegan with the evaluation of macrolactone analogues that weredifferent in the C30−C38 region. From initial studies, compound34, which bears a shortened chain at C36 and is epimeric at C35,emerged as the first simplified analogue of halichondrin Bcapable of inducing irreversible CMB upon washout in the assay(Figure 6). Various structural simplifications that would alloweasier access to the compound were examined. To this end, it wasfound that modifications of the macrocyclic lactone core andmost of its peripheral substituents were generally deleterious forthe biological activity. Thus, it was decided to exploremodifications of the octahydropyrano[3,2-b]pyran ring moietyconstituted by the C29−C36 sector, where alterations were welltolerated. A simplified version bearing only a tetrahydropyranring (i.e., 35) in place of the bicyclic structure was found toexhibit a similar range of biological activities when compared to34 (Figure 6).Further structural modification led to compounds 36 and 37.

Studies revealed that the replacement of the C31 methyl groupwith a methoxy substituent was well tolerated and greatlysimplified the construction of the system, which utilized easilysourced carbohydrate precursors (Figure 6).With simpler structures capable of retaining the same

biological characteristics as the parent architecture, attentionnext turned to an alternative strategy for improving potency. Onthe basis of X-ray data on norhalichondrin A and NMR analysisof halichondrin B, it was hypothesized that the replacement ofthe tetrahydropyran ring with a smaller and more constrainedtetrahydrofuran ring could stabilize the bioactive conformationof the macrolactone ring, giving less conformational freedom tothe structure and effectively “locking” it into a more activeconformation.27 This hypothesis was corroborated by the activityof 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 witha stereodefined vicinal propanediol. This compound exhibitedhigh activity and was capable of maintaining CMB. However,further evaluation of compounds 36 and 39 (most promisingcandidates from each of the pyran and furan analogues) in a LOXhuman melanoma xenograft model revealed the in vivo inactivityof these structures. Many hypotheses were put forward to explainthis lack of activity, with the most likely being the hydrolyticinstability of the embedded macrocyclic lactone to nonspecificesterases, which are present in high levels in mouse serum.

Figure 5. Intermediate 33, a promising candidate in anticancer drugdiscovery.

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



11999


Intense investigation eventually led to the evaluation ofnonhydrolyzable bioisosteres such as amide, ether, and ketonealternatives to the labile macrocyclic ester. As a result, compound40was identified as the best candidate to further drive the in vivobiological studies, as it proved highly effective in a variety ofhuman cancer models (Figure 6).Additional structure optimization finally led to compound 41,

which bears a primary amino group in place of the primaryalcohol at C35 (Figure 6). This compound, which was laterdubbed eribulin, proved extremely active, and for the first time, asimplified analogue of halichondrin B showed reversibility ratioequal to 1, meaning that the molecule was capable of maintaininga CMB even after a 10-h washout (Table 1).

From Table 1, it is possible to compare the in vitro biologicalactivities of the compounds described above. Although eribulinexhibited the highest half-maximal inhibitory concentration(IC50) among the series tested, the reversibility ratio, which wasutilized as a proxy parameter for in vivo activity, led to itsselection for clinical trials. Eventually, these studies culminated inthe discovery of a new anticancer treatment commercialized as itsmesylate 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 andfacile route to this extremely complex molecule was necessary tofacilitate the advancement of preclinical and clinical studies.9

The key part of the synthetic strategy to eribulin was alreadyestablished in earlier studies by Kishi and co-workers during thetotal synthesis of halichondrin B. From the retrosyntheticdiagram depicted in Figure 7, it can be seen that the synthesis oferibulin can be traced back to formation of three separatefragments, two of which are identical to those used in thesynthesis of halichondrin B (i.e., 9 and 11). The western portion(i.e., fragment 42 and its eventual successor 43) is the only part oferibulin that was not studied and developed during the synthesisof 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 notideal because the isolation of the desired material wascomplicated 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 wasdeveloped to procure more substantial quantities of 41.29

In this improved route, the synthesis commenced with thering-opening reaction of epoxide 4530 by attack of the

nucleophilic acetylene anion of 44,31 which afforded a 3:1mixture of regioisomers favoring the desired one.29 Theintermediate homopropargylic alcohol was then partiallyreduced under Lindlar conditions. Acetylation of the secondaryalcohol then furnished compound 46. Dihydroxylation pro-moted by OsO4, and subsequent mesylation of the obtained diolyielded product 47. The acetyl protecting group was cleaved toafford an intermediate alcohol, which underwent a facilecyclization to provide an intermediate substituted tetrahydrofur-an ring. This material was then converted to methyl ether 48through deprotection and methylation steps. The dioxolane wasthen hydrolyzed and reprotected as the bis-tert-butyldimethyl-silyl (TBS) derivative. The benzyl protecting group was removedby hydrogenolysis, and the resultant alcohol was oxidized toafford key aldehyde 42 (Scheme 8).Although the new route demonstrated its feasibility by

reducing the number of steps and increasing the yield of thedesired final product 42, the search for an improved and morescalable pathway continued. An alternative approach to aldehyde

Table 1. Growth Inhibition Activities and Complete MitoticBlock (CMB) Reversibility Ratios of Evaluated Compounds27

compoundgrowth inhibition potency (DLD-1)

IC50 (nM)aCMB reversibility ratio

(U937 cells)

4 0.74 333 4.6 >2934 3.4 2435 2.5 1736 1.8 3037 2.0 2238 1.0 3339 0.67 1040 1.0 1341 20 1

aGrowth inhibition against DLD-1 cells after 3−4 days of continuousexposure.

Figure 7. Retrosynthetic approach to eribulin (41).




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

was facilitated in three steps to convert 49 to 50.32

Diisobutylaluminium hydride (DIBAL-H) reduction of thelactone 50 to lactol followed by a Wittig reaction produced 51.Benzylation of the resulting secondary alcohol and subsequentasymmetric Sharpless dihydroxylation gave a 3:1 diastereomericmixture favoring the desired isomer.Following bis-benzoylation of the diol and a diastereoselective

glycosidic allylation, compound 52was generated. Moreover, thehigh crystallinity exhibited by the desired diastereomer of thiscompound allowed its isolation in high purity by crystallization.Subsequent oxidation of the alcohol at C30, formation of theunsaturated sulfone by Horner−Wadsworth−Emmons reactionat the same position, FeCl3-promoted debenzylation, andasymmetric reduction of the sulfone by the directing effect ofthe alcohol at C31 furnished 53 as a single diastereomer. Thiscompound was then methylated at C31, the two benzoyl esterswere hydrolyzed, and the resulting diol was protected as theacetonide. Finally, ozonolysis of the resulting intermediatefurnished aldehyde 54.32 This compound could then betransformed 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 dramaticallyreduce the required number of chromatography steps to one.32 Afew years later, Kishi and co-workers reported a second-generation variant of this approach to make it even morepractical and scalable with concomitant removal of chromato-graphic purification steps.33

The synthesis of fragment 11, which constitutes the C1−C13segment of eribulin, has been the focus of continual improve-ment to reduce the steps and to facilitate the supply of the desiredproduct with increasing stereocontrol. The original route to 11employed in the studies for the synthesis of halichondrin Brequired 30 steps from D-glucose diacetonide (24, Scheme 3).Later, Kishi and co-workers were able to reduce the number ofsteps by commencing the synthesis with a different startingmaterial, namely, commercially available L-mannoic-γ-lactone(55, Scheme 10). Following this second-generation route, thegroup was able to secure fragment 11 in 16 steps.34 However,only 3 years later, the team was capable of further reducing thenumber of required steps. Their third-generation syntheticapproach to fragment 11 required 12 steps from the same starting

material (i.e., 55), was carried out on a 100-g scale, and isdescribed below (Scheme 10).35

Starting with L-mannoic-γ-lactone (55), initial double cyclo-hexylidene ketal formation, DIBAL-H reduction of the lactone,andWitting olefination of the resultant lactol smoothly produced56. Catalytic asymmetric Sharpless dihydroxylation followed byacetalization and double acetylation yielded intermediate 57.Allylation of the glycosidic position with allyl silane 58 waspromoted by BF3·Et2O. Treatment of the obtained product withTriton-B triggered the hydrolysis of the acetate, isomerization ofthe olefin to the conjugated position, and conjugate oxy-Michaeladdition to selectively produce compound 59. Subsequently,selective deprotection of the more sterically accessible cyclo-hexylidene ketal and oxidative cleavage of the resulting 1,2-diolgenerated an aldehyde, which, upon organometallic addition of60 throughNozaki−Hiyama−Kishi coupling, furnished 61. One-pot hydrolysis of the remaining ketal and bis-silylation of theresulting diol, followed by iododesilylation achieved with NIS,generated 11 in 12 steps and 11% overall yield from 55 (Scheme10).35

The synthetic pathway to fragment 916 was already discussed(see Scheme 1) during the description of the synthesis ofhalichondrin B.24 Over the years, a number of accounts detailingattempts to optimize the synthetic route have been reported inthe literature.15,32,36 Most of these alterations of the originalroute were only partially beneficial and did not constitutesubstantial improvements of the route. However, in 2002, Kishi’sgroup 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).37The 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 openedthrough nucleophilic addition of the trimethylsilylacetylene

Scheme 9. Synthesis of Intermediate 54 en Route to Fragment43

Scheme 10. Third-Generation Synthesis of Fragment 11



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anion, followed by mesylation of the resultant secondary alcoholand vinyl iodide formation, generated 64.39 AsymmetricNozaki−Hiyama−Kishi coupling between 64 and aldehyde 65,promoted by catalyst 66, preceded the acid-promotedtetrahydrofuran formation and the subsequent removal of thebenzoyl group to generate 67 isolated in high yield as a 9:1diastereomeric mixture. Finally, oxidation of the primary alcoholto aldehyde and coupling with 68 under the conditions depictedin Scheme 11 generated 69 in good yield and 5.3:1 stereo-selectivity.37 This product could then be easily converted tofragment 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 underNozaki−Hiyama−Kishi conditions followed by a base-promotedtetrahydropyran formation (dr 3:1) and subsequent PMBcleavage generated compound 70. The primary alcohol wasconverted to the mesylate derivative, which was subjected to anucleophilic substitution with thiophenol.The intermediate thioether was then oxidized to the sulfone,

and upon removal of the pivalic protecting group, intermediate71was formed (Scheme 12). Sulfone 71 was then treated with n-BuLi and added to aldehyde 72, obtained by DIBAL-H reductionof fragment 11, to generate an inconsequential mixture ofdiastereomeric alcohols, which were promptly oxidized to ketone73 in high yield (Scheme 13).Once the three fragments had been combined into

intermediate 73, a three-step sequence was employed thatinvolved a SmI2-promoted desulfonylation, an intramolecularNozaki−Hiyama−Kishi coupling between the vinyl iodide andthe aldehyde functionality in 73, and subsequent oxidation of theobtained secondary alcohol to produce enone 74. Removal of theTBS protecting groups by action of TBAF and exposure to PPTStriggered the formation of compound 75 in a manner analogousto the original approach of Kishi and colleagues described inScheme 6. Finally, selective tosylation of the primary alcoholfollowed by treatment with ammonia yielded eribulin (41) inhigh yield (Scheme 14).

2.7. Multikilogram Manufacturing Process of Halaven

Following the success of eribulin in preclinical40 and earlyclinical40−45 trials and of the relatively condensed reproduciblesynthetic pathway to the molecule just described, Halaven (i.e.,eribulin mesylate) was approved in late 2010 by the U.S. Foodand Drug Administration (FDA) for the treatment of patientswith metastatic breast cancer who had already received at leasttwo other chemotherapeutic treatments. At Eisai, the syntheticapproach adopted to supply the material in multikilogram scalemirrored that used to provide material in gram scale for thepreclinical and clinical studies, discussed above. However, asoften occurs in industrial-scale manufacturing, additionaloptimization and modification of the synthetic route wasnecessary to obtain a cost-effective synthesis of the desiredmolecule; to reduce the number of purifications; and in general,to improve the practicality of the process. The retrosyntheticapproach taken for the kilogram-scale synthesis of eribulin (41)can be traced back to the same three fragments previouslyemployed, with the only difference being the use of vinyl triflate88 in place of vinyl iodide 9.The synthesis of fragment 8046 commenced with hydration of

the readily available dihydrofuran 76 promoted by Amberlystresin, followed by the organometallic allylation of the

Scheme 11. Improved Route to Fragment 9 Scheme 12. Coupling of Fragments 42 and 9 for the Synthesisof Intermediate 71

Scheme 13. Synthesis of seco-Ketone 73 through Coupling of71 and 72



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intermediate lactol, to generate 78 as a racemate. Selectiveprotection of the primary alcohol, followed by simulated-moving-bed (SMB) chiral chromatography,47,48 allowed separa-tion of the two enantiomers. The desired isomer was thentosylated to furnish compound 80, whereas the configuration ofthe undesired enantiomer was inverted by Mitsunobu reactionand then subjected to tosylation (Scheme 15).46

The other portion of fragment 88 began with Jacobsenhydrolytic kinetic resolution38 of epoxide 81 followed by ring-opening by the enolate of diethyl malonate and subsequentcyclization, furnishing lactone 82. Selective hydrolysis of theester, decarboxylation, and diastereoselective methylationgenerated 83 as a 6:1 mixture in favor of the desired isomer.Trimethylaluminum-promoted lactone opening by dimethylhydroxylamine, followed by protection of the resulting secondaryalcohol, OsO4-mediated dihydroxylation of the olefin, andoxidative cleavage of the resulting diol by NaIO4 furnishedaldehyde 84 in high stereopurity (Scheme 16).46

The union of intermediates 80 and 84was then achieved by anasymmetric Nozaki−Hiyama−Kishi coupling between the two,adopting the previously developed and highly optimizedconditions.32,37,39,49 This set of conditions involved the use ofthe chiral ligand 85, which was selected to induce stereo-selectivity. In addition, it was fortuitously found to increase thereaction rate.In this instance, the process team at Eisai further developed the

conditions established for this reaction to minimize side-productformation and maximize the reproducibility of the process. To

avoid base-promoted tosylate elimination, compound 86 wasinstead cyclized to form the intermediate tetrahydrofuran ring asan 8:1 diastereomeric mixture at C20, using SiO2 as a promotingagent.This material was then treated with methyl Grignard to form

the methyl ketone, the enolate of which was then trapped astriflate 87 by means phenyl triflimide. Upon removal of the silylprotecting groups, the diastereomeric mixture of compounds waspurified by preparative high-performance liquid chromatography(HPLC) and the desired isolated product was then protected aspivalate at the C14 alcohol and mesylated at C23 to furnish

Scheme 14. Final Steps toward Eribulin (41)

Scheme 15. Synthesis of Vinyl Bromide 80

Scheme 16. Synthesis of Fragment 88 at the MultikilogramScale



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fragment 88 in high stereochemical purity, although in relativelylow yield in 15 steps (longest linear sequence) from 81 (Scheme16).The route to fragment 43 is a direct evolution of the path

employed for gram-scale construction (Scheme 9), with someimprovements to the process that were deemed necessary forlarge-scale manufacturing.46 It began with the same startingmaterial 49, which was converted to 50 in three simple stepswithout the use of tin hydride, which was employed in theoriginal route (Scheme 17). Lactone 50 was transformed into a

lactol by DIBAL-H reduction. Addition of the organomagnesiumreagent derived from (trimethylsilyl)methyl chloride to thelactol, followed by Peterson olefination and benzylation of thesecondary alcohol, produced 89. This path allowed the team toavoid the use of Wittig chemistry to produce the allylicsubstituent, thus circumventing the production of triphenyl-phosphine oxide that would have required removal according inthe original route. Conversion of 89 to 90 is procedurallyidentical to the route reported in Scheme 9, with the exceptionthat the intermediate alcohol was oxidized to 90 using a Moffatoxidation in place of the Swern that was originally employed.Transformation of the ketone to an unsaturated sulfone,

debenzylation, and directed asymmetric reduction of theconjugated olefin was achieved in the same manner as describedin Scheme 9. However, the benzyl cleavage was accomplishedthrough a homogeneous process employing trimethylsilyl iodide(TMSI) instead of FeCl3. The compound obtained was thentreated with K2CO3 in MeOH to hydrolyze the benzoate groups,furnishing 91, which was purified by crystallization, avoidingchromatography.Protection of 91 as its acetonide followed by methylation of

the secondary alcohol, subsequent hydrolysis of the ketal, andreprotection of the diol as the tert-butyldimethylsilyl ethergenerated the precursor to the final product 43. The methylationin this instance was performed without the use of the silverreagent originally employed (Scheme 9). Aldehyde 43 was thenobtained by ozonolysis of the olefin, employing a mildhydrogenation as the reducing agent for the ozonide formed,yielding 43, which was purified by crystallization. Following thispath, 43 was obtained in a chromatography-free 20-step processin a moderate overall yield (Scheme 17).46

The kilogram-scale manufacturing process to access fragment11 was directly derived from the synthesis of the same molecule

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

the cost of the process and to overcome issues with theavailability of the original starting material L-mannoic-γ-lactone(55, Scheme 10), it was decided to initiate the synthesis with theless expensive and easily accessible C11 epimeric material D-(−)-gulono-1,4-lactone (92, Scheme 18). The configuration ofthe C11 alcohol was not relevant, as the stereocenter would belost and re-formed during the route. The first three steps of thesynthesis to furnish compound 93 proceeded in accordance withthose described in Scheme 10. The only modifications wereapplied in the Wittig reaction to improve the purity whileavoiding the need for chromatography. These modificationsconsisted of the addition of the substrate to the preformed ylide,removal of the unreacted triphenylphosphine with maleicanhydride, and removal of the formed triphenylphosphineoxide by precipitation. Dihydroxylation of 93 with subsequenthydroxypyran 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 amodification adopted by the team as a solution to difficultiesencountered in the subsequent glycosylation when conducted onlarger scale. In this manner, selective removal of one cyclo-hexylidene and peracetylation delivered crystalline compound 94that smoothly underwent allylation with allyl silane 58, followedby global deacetylation and ring formation when treated withNaOMe to yield 95. This compound, upon oxidative cleavage ofthe diol, generated an aldehyde that was then transformed into61 following the same procedure as reported in Scheme 10. Thefinal three steps to fragment 11 proceeded as already described inScheme 10 with some small modifications to avoid the use of aknown sensitizer (i.e., chloroacetonitrile), which, at that scale,might have been dangerous for the production personnel(Scheme 18).50

Scheme 17. Kilogram-Scale Synthesis of Fragment 43

Scheme 18. Kilogram-Scale Synthesis of Fragment 11



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Coupling of fragments 43 and 88 through asymmetricNozaki−Hiyama−Kishi coupling under the same conditions asdeveloped for the formation of 86 in Scheme 16 were thenapplied in this instance, generating a 20:1 diastereomeric mixture(Scheme 19).46 This mixture was subsequently transformed intothe intermediate pyran by the action of potassium hexamethyl-disilazide (KHMDS) at low temperature, followed by DIBAL-Htreatment to remove the pivalate to furnish 71, which was thenpurified by chromatography and crystallization (Scheme 19).46

Coupling between intermediate 71 and the aldehyde derivedfrom 11 commenced with the DIBAL-H reduction of fragment11 to the aldehyde, followed by nucleophilic addition of theanion derived from sulfone 71 upon deprotonation with n-BuLi(Scheme 19).51 This path is identical to that used in the gram-scale route. However, several optimizations were necessary toimprove the process for kilogram scale. One of the problemsencountered was the ready oxidation of the aldehyde to thecorresponding acid. Extensive experimentation revealed that theaddition of small amount of butylated hydroxytoluene (BHT) tothe aldehyde-containing fractions upon chromatography andbefore concentration would prevent this problem. Sulfone−aldehyde coupling also required optimization to maximize theproduct yield and solve irreproducible results. Upon intenseinterrogation, including deuterium-labeling studies, optimalconditions were developed and applied to the process, whichfurnished the desired aldehyde in high yield.51

This two-step transformation was recently successfullydemonstrated under continuous-flow conditions.52 SubsequentDess−Martin oxidation generated a 1:1 diastereomeric mixtureof ketone−aldehyde 73. It was found that addition of a catalyticamount of water to the reaction mixture dramatically increasedthe yield, the rate, and the reproducibility of the reaction

(Scheme 19). The additional five steps necessary to accessproduct 75 were consistent with the gram-scale process depictedin 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, previouslydeveloped in the synthesis of fragment 88 (Scheme 16).Although there was no need for asymmetric induction, assubsequent oxidation cleared the newly formed stereocenter,application of this set of conditions was capable of producing thedesired product in better yield and shorter time when comparedto the ligand-free variant. Ultimately, the process team was ableto apply the transformation to kilogram-scale batches (Scheme19).51 Finally, transformation of 75 into eribulin uponpurification by crystallization was easily achieved by converting75 into the primary tosylate and then treating it with NH4OH, togenerate an epoxide intermediate that was then opened by asecond reaction with ammonia, furnishing 41. This was thenconverted to its mesylate salt by treatment with ammoniummesylate, to furnish Halaven (Scheme 19).51

The journey of halichondrin to the discovery of eribulin and itscommercialization as Halaven is the perfect example of howchallenging the development of a new drug candidate can be.Great effort was made by Kishi’s group in the development ofnew conditions and strategies for the Nozaki−Hiyama−Kishicoupling, which proved crucial for the construction of several C−C bonds in the process. In this regard, great admiration must beshown to the scientists at Eisai involved in this monumentalproject that developed an industrial manufacturing route toHalaven, which was approved by the FDA in 2010 for the

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



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

3. SYNTHETIC TETRACYCLINES

3.1. History of the Tetracyclines

In the early 1940s, a team of scientists led by the renownedbotanist Benjamin Duggar made a seminal discovery at LederleLaboratories.54 A soil sample retrieved from a field in Missouri,when cultured and extracted, exhibited extraordinary growthinhibition against all strains of bacteria in the preliminaryscreen.54 Subsequent tests of this extract continued to astonishDuggar and his co-workers, as the extract proved efficaciousagainst several infectious bacteria for which there was no cure atthe time.54 The unprecedented activity of this material, namedAureomycin (98) for the gold color of its cultures, prompted it tobe one of the first labeled “broad-spectrum antibiotics”.55

Because of the unanticipated potency of Aureomycin, AmericanCyanamid utilized large-scale fermentation to obtain commercialquantities of the substance and quickly pushed it into the clinicwithin a year of its initial report.54

The effectiveness of Aureomycin against a broad spectrum ofbacteria made it highly successful.54 Other companies were quickto start scouring the biosphere for other potential blockbusterantibiotics. Pfizer was one of the first to publish its findings on anew extract coined Terramycin (the producing organism wasisolated from a soil sample from Terre Haute, IN).54,56 Pfizerrapidly capitalized on Terramycin after obtaining FDA approvalin 1950, entering the new broad-spectrum antibiotic market inforce and catapulting Pfizer toward being the pharmaceuticalgiant it is today.54

Even though Aureomycin (98) and Terramycin (99) werewildly successful, the molecular structures of the extracts’ activecomponents were elusive to scientists for several years after theirdiscoveries. Both Pfizer and American Cyanamid cooperated bysharing samples to solve the structures. Eventually, a Pfizer-ledteam aided by R. B. Woodward deciphered the enigmaticstructure and in 1954 published their findings in a seminalarticle.57,58 They discovered that the structures of Aureomycin(98) and Terramycin (99) (Figure 8) shared the same tetracyclicnapthacene core with differential functional ornamentation, achlorinated D ring in Aureomycin and a B ring hydroxyl inTerramycin.The elucidation of the molecular skeletons of these

compounds ushered in the age of semisynthetic derivatizationof the tetracycline motif. However, during this time period, it waswidely assumed that chemical alterations of the natural-productstructures would result in lower bioactivity.54 In 1955, LloydConover discovered that a simple hydrogenolysis of the chlorineof Aureomycin gave rise to a compound that was more potent,soluble, and pharmacologically active than Aureomycin. Thisnovel compound became the namesake for this entire class ofantibiotics: tetracycline (100).59,60

After the discovery that manipulations of the functionalityaround the skeletal core of tetracycline could potentially producemore potent compounds, several other tetracycline derivativesentered the clinic in relatively rapid succession, with minocycline(101) being the most recent in 1971.54 After minocycline, thedevelopment of novel tetracyclines remained stagnant for severaldecades until growing antibiotic resistance provoked Wyeth intoreinvestigating the tetracycline scaffold. Introduction of a secondamino group on the D ring of the minocycline skeleton andappending of a modified glycine residue resulted in tigecycline

(102).63,64 Tigecycline gained FDA approval in 2006.54

However, the field of tetracycline derivatization has remained acontinual of cycle of isolation and peripheral modification.65 Thistactic has its limitations and is unable to access core structuralchanges. X-ray crystallography studies have allowed examinationof how the tetracyclines interact with the bacterial ribosome andindicate that there are sections of the D ring where modificationscould be utilized (Figure 9).61,62 Unfortunately, semisyntheticmethods are barred from accessing these sites of modification.However, the development of a practical synthetic route wouldallow 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., fivecontiguous stereocenters in the case of 99) is a formidable task initself for these deceptively simple polycycles. In addition, thesecompounds have proven to be sensitive to a variety ofenvironments, which further complicates the modification ofthe parent structures (Scheme 20).66 In acidic media, the C6hydroxyl group, which is both benzylic and tertiary, is poised forfacile dehydration to produce anhydrotetracyclines (104).66 TheC4 dimethylamino group is prone to epimerization in mildlyacidic solutions, and this process is accelerated by certaincounterions.66

In basic media, the C and B rings undergo a retro-Dieckmannreaction, generating γ-butyrolactone 108.66 Suffice it to say thatdeveloping a successful route to the tetracycline scaffold requiresextensive forethought and finesse in the execution of each step. Itis no surprise that, after collaborating to help unravel the skeletonof the tetracyclines, Woodward, in collaboration with a groupfrom Pfizer, was the first to establish a route to the tetracyclinecore in 1962 (Scheme 21).67−69 The Woodward route focusedon building the napthacene core one ring at a time starting with

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



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the D ring and produced 6-demethyl-6-deoxytetracycline.Woodward’s synthesis was quickly followed by that ofShemyakin and colleagues in 1963, which adapted the samestrategy as Woodward starting from juglone.70 An alternativeapproach that involved constructing multiple rings in aspectacular cascade of Dieckmann condensations was pioneeredbyMuxfeldt and co-workers in 1979.71,72 Tatsuta and co-workersestablished the first asymmetric route to tetracycline (100),utilizing a protected form of D-glucosamine as the startingmaterial in 2000.73 Stork et al.’s approach to the tetracycline corealso used a remarkable cascade of Dieckmann condensations toconstruct the tetracycline nucleus.74

Although each of these syntheses is elegant in its own approachto the synthetic tetracycline problem, the major drawback of allof them, with the exception of Stork et al.’s, lies in their efficiency,the highest being that of the Muxfeldt approach at 0.06% overallyield. However, the Stork synthesis constructs racemic 12a-deoxytetracycline 124 in 18−25% yield over 16 steps.Unfortunately, the 12a-deoxy variants are plagued by a majorreduction in potency, which means that derivatives constructedby this route would be handicapped from the start.75 In about2005, semisynthesis was still the most viable method of obtainingand modifying tetracycline derivatives. However, that was soonto change. Andrew Myers and co-workers, armed with theknowledge of the inherent limitations of the semisyntheticpreparations of tetracyclines, as well as the drawbacks of thecurrent synthetic state of the art, set out to devise a practicalsynthesis that would allow manipulation of sites inaccessible bycurrent means.

3.2. Myers’ Synthetic Studies on the Tetracyclines

The Myers tetracycline route commenced with unornamentedbenzoic acid, which was subjected to microbial-mediateddihydroxylation 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 inan academic setting.77 This process introduces the troublesome12a hydroxyl group that was absent in the Stork synthesis in thefirst step, which allows derivatives from this approach to beequipotent with their natural congeners. Myers and co-workers

Figure 9. (A) X-ray crystal structure of tetracycline bound to a bacterial30S ribosomal subunit and (B) pictorial representation of the keyinteractions between the tetracycline motif and its receptor61,62 [ProteinData Bank (PDB) accession number 1HNW]. (Reproduced withpermission 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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then proceeded to build the rest of the core around what becamethe B ring. The diol was subjected to a hydroxyl-directedepoxidation with m-chloroperoxybenzoic acid (m-CPBA) toafford the α-epoxide 127 in 83% yield. The acid functionality wasthen esterified utilizing trimethylsilyldiazomethane. The allylic

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

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



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vinylogous carbamic acid present in the A ring of the tetracyclinecore.78 Starting from glyoxylic acid, isoxazole 130 was preparedin 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 reverseof that of Stork et al.’s approach. Treatment of isoxazole 130withn-BuLi generated the lithiated isoxazole at C4, which wassubsequently treated with epoxy ester 128. This processprovided ketone 131 in 70% yield. To close the A ring, gentlewarming of the ketone with catalytic amounts of lithium triflatefacilitated a putative SN2′ opening of the allylic epoxide throughthe pendant dimethylamino group. The α-position of theresultant quaternized amine was then deprotonated to producean ylide, which underwent a [2,3]-sigmatropic rearrangement toafford tricycle 135. This transformation was likened to the classicSommelet−Hauser rearrangement. This process provides thedesired cis fusion of the AB rings and the α-orienteddimethylamino group. The crude material was then treatedwith trifluoroacetic acid (TFA) in dichloromethane (DCM), toachieve a mild deprotection of the allylic silyl ether in thepresence of the other silyl ether to afford tricycle 136. Tricycle136 is a key branching point, from which Myers and co-workerscould prepare either 5-deoxy- or 5-hydroxytetracycline deriva-tives.To prepare 5-deoxy derivatives, tricycle 136was subjected to a

reductive allylic transposition with triphenylphosphine, diethylazodicarboxylate (DEAD), and o-nitrobenzensulfonyl hydrazideto afford cyclohexene 137 (Scheme 23).79 Cyclohexene 137 wasthen subjected to acid-mediated TBS deprotection, oxidation,and protection of the 12a-alcohol to provide key enone 139.

The 5-hydroxy derivatives could be accessed by converting theallylic alcohol of tricycle 136 to the sulfide while retaining thedesired stereochemistry (Scheme 24). The sulfide was thenoxidized with chiral oxaziridine 141 in a diastereoselectivefashion to the sulfoxide in 99:1 selectivity.80 The system was then

heated in the presence of trimethylphosphite to induce aMislow−Evans rearrangement to afford allylic alcohol 142.Alcohol 142was converted to a benzyl carbonate, and the systemwas subjected to a deprotection/oxidation/protection sequencesimilar 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 desiredstereochemistry and functionality in protected form in amasterstroke. The C ring was constructed in a deft introductionof the D ring into the system. Subjection of enones 139 and 143to anionic D-ring precursors resulted in conjugate addition to theenone, followed by a Dieckmann condensation of the appendedphenyl ester, closing the C ring and completing the skeleton ofthe tetracycline framework (Scheme 25).65 This transformationwas essential for the development of novel tetracyclineanalogues, which is discussed below. In the case of doxycycline(149), Myers and co-workers deprotonated phenyl benzoate 144with lithium diisopropyl amide (LDA) in the presence oftetramethylethylenediamine (TMEDA) under cryogenic con-ditions and then introduced enone 139 into the reaction mixture,which then beautifully executed the desired Michael−Die-ckmann cascade to provide pentacycle 147 in 79% yield as asingle diastereomer.65 Pentacycle 147was then subjected to mildTBS/tert-butoxycarbonyl (Boc) deprotection and a triplehydrogenolysis of the benzyl carbonate, benzyl-protectedimidate, and isoxazole N−O bond to provide doxycycline 149in 90% yield. This approach provided doxycycline in a longestlinear sequence of 18 steps, 22 overall, and 11% overall yield.However, Myers and co-workers did not only establish a practicalroute to doxycycline. To exhibit the power and practicality oftheir route, they proceeded to synthesize several noveltetracycline derivatives that are inaccessible by current semi-synthetic 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 withpyridinium tribromide followed by bromide displacement withthiophenol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) toafford vinyl sulfide 150.81 The selection of the phenylthio groupwas 2-fold: It served to activate the system for the ensuing [4 + 2]cycloaddition and functioned as a handle to introduceunsaturation later in the sequence. Vinyl sulfide 150 was thenheated with triethylsiloxybenzocyclobutene derivative 151,generating the reactive o-quinone dimethide in situ to serve asthe 4π component of the Diels−Alder cycloaddition.82−84

Pentacycle 153 was produced from the cycloaddition as a singlediastereomer. In addition, lactone 154 was isolated as aninteresting side product of the reaction, which is thought to bethe result of a retro-Dieckmann of pentacycle 153.81 Pentacycle153 was then subjected to TBS deprotection and oxidation toproduce ketone 155. The second function of the phenylthiosubstituent then came into play. Subjection of 155 to TFAprotonated the dimethylamino group serving as in situprotection, introduction of m-CPBA into the system thenoxidized the sulfide to the sulfoxide, and gentle warming resultedin elimination to afford unsaturated tetracycline derivative 156.81

Unsurprisingly, the anhydrotetracycline derivative proveddifficult to isolate, given that the facile stereoselective photo-oxidation of similar anhydrotetracyclines to the correspondinghydroperoxytetracyclines has been known for decades.66,85−87

Myers and co-workers used this effortless photo-oxidation totheir advantage and simply stirred the crude anhydrotetracycline156 in chloroform open to the air to produce hydroperoxide 157,

Scheme 23. Elaboration to the Protected 5-Deoxy Precursor

Scheme 24. Elaboration to the 5-Hydroxy Precursor



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which was subsequently hydrogenated in the presence of Pdblack to afford tetracycline (100) in 42% yield over three steps.This route constructed tetracycline in a longest linear sequenceof 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 tostart exploring uncharted chemical space in terms of tetracyclinederivatives. In the original communication, two tetracyclinederivatives contained a nitrogen atom in the D-ring systems,which are now nominally referred to as azatetracyclines. Myersand co-workers prepared 7-aza- and 9-azatetracyclines, where the7-azatetracycline exhibited modest activity in the small bacterialscreen that Myers and co-workers performed.65 It is at this pointthat Tetraphase Pharmaceuticals began investigating these noveltetracycline derivatives.

3.3. Tetraphase SAR Studies

Tetraphase quickly determined that moving the nitrogen fromthe 7- to the 8-position led to compounds that were comparableto the parent tetracycline and its semisynthetic derivativeminocycline (Figure 10).88,89 Initially, a variety of 8-azatetracy-

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

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

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

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



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and that the compounds retained or had improved activityagainst the tetracycline-susceptible strains, with the 7-fluoroanalogue being the most potent.88 Unfortunately, all of thederivatives tested at this stage were substrates for the tet(A)efflux pump.88

To make a direct comparison to tigecycline (102), Tetraphaseproceeded to prepare the corresponding 9-glycylamido-8-azatetracycline analogues. However, this modification alsoproved to be unsuccessful, and these derivatives exhibited pooractivity in the whole-cell assays; however, when subjected to acell-free transcription/translation assay, they showed activitycomparable to that of tigecycline (102).This indicated that these derivatives had difficulty getting to

the target in the whole-cell assay, ostensibly due to poorpermeability.88 In an effort to increase the permeability of thecompounds, 9-amino-8-azatetracycline derivatives were ex-plored. In this series of compounds, it was found that activityagainst the elusive tet(A) efflux pump began to emerge. 9-Amino-8-azatetracycline 161 proved to be the most potent in thewhole-cell assays and showed activity commensurate with that oftigecycline (102) in cell-free translation/transcription assays.88

Further investigation into the pharmacokinetic profile ofcompound 161 in Sprague−Dawley rats revealed that it had a4-fold higher area under the curve (AUC) and an almost 5-foldlower clearance than tigecycline (102), but the oral bioavail-ability was functionally identical to that of tigecycline.88 In the S.aureus septicemia models, 161 and tigecycline (102) werecomparable in protection, even though tigecycline (102) wasmore potent in the initial minimum inhibitory concentration(MIC) screens. In the E. coli septicemia model, 161 required ahigher dosing for protection equivalent to that provided bytigecycline (102). However, Tetraphase was not satisfied withthe activity profile of 161 and decided to investigate othervariations to the tetracycline core.At the same time as the company was investigating the 8-

azatetracyclines, Tetraphase also explored another series of noveltetracyclines first constructed by Myers and co-workers, thepentacyclines (Figure 11).90−92 These compounds contain an

additional ring appended to the tetracycline scaffold at C8 andC9, aptly named the E ring. The new ring introduced several newderivatization sites for the tetracycline scaffold to be explored.91

Tetraphase explored substitution at all four of the newsubstitution sites and found that most retained some degree ofactivity. However, those that proved most potent contained amethoxy group at C7 and a small alkylaminomethyl group at thenew C10-position prototype structure 163. However, all of thenovel pentacyclines proved to be less potent in vitro than

tigecycline (102), with several showing efficacy comparable tothat of tigecycline (102) in vivo with septicemia mouse models.91

The third variation on the tetracycline framework explored byTetraphase was the introduction of novel functionality at C7 ofthe traditional scaffold accompanied by an array of C9substituents.93−95 Tetraphase examined several novel substitu-ents and found that extremely potent compounds resulted fromfluorine substitution at C7, which Tetraphase has designated as“fluorocyclines”.93,94 Although a C7 fluorotetracycline had beensynthesized prior to Tetraphase’s investigations, the conditionsfor fluorine introduction were harsh and not amenable toindustrial processing.96 When coupled with a C9 glycylamidosubstituent, the fluorocyclines displayed a range of activities.However, one particular analogue, 164, also referred to as TP-434, proved to be exceptionally potent, beating or matchingtigecycline (102) in the initial antibacterial screen (Figure 12).93

Attempted modification of the appended pyrrolidine ring of 164resulted in activity loss.93

Examination of the in vivo activity of TP-434 in tetracycline-resistant mouse septicemia models revealed excellent efficacywith 50% protective dose (PD50) values lower than that oftigecycline (102).93 The pharmacokinetic (PK) properties ofTP-434 were also found to be favorable, having a larger AUCvalue than tigecycline and a half-life similar to those of tigecyclineand tetracycline.93 Further investigations into TP-434 revealedthat it was active not only against strains carrying the three maintetracycline-specific efflux pumps but also against ribosomalprotection mechanisms and enzymes that target tetracyclines.97

TP-434 retained activity against strains resistant to multipleantibiotic classes, such as those with β-lactamases.98 Oneintriguing result disclosed by Tetraphase was that TP-434showed potent activity in vitro against established biofilms.99 Theextremely promising results for TP-434 induced Tetraphase toexplore it in higher animals, where the PK data indicated that ithad an acceptable bioavailability of ∼25% in chimpanzees and>10% in rats, mice, dogs, and cynomolgus monkeys.97 Thesepromising preliminary results prompted Tetraphase to moveforward into clinical trials.3.4. Eravacycline Synthesis

To provide an adequate material supply for the upcoming clinicaltrials, the process division of Tetraphase began the developmentof a route that would improve upon Myers’ initial synthesis thatcould be utilized on an industrial scale. Fortunately, Brubaker andMyers reported an improved route to enone 139 that Tetraphaseemployed to full advantage by modifying it to suit their needs(Scheme 27).100 Tetraphase’s route commenced with thebromination of dimethyl maleate (165), followed by treatmentwith potassium tert-butoxide and hydroxyurea to afford isoxazole168.101,102 Subsequent benzyl protection of the hydroxyl groupand DIBAL-H reduction furnished aldehyde 170.101 Aldehyde170 was condensed with the (S)-variant of the Ellman auxiliary

Figure 11. Myers’ novel pentacycline and the pentacycline numberingsystem.

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



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171, providing sulfonyl imine 172. A diastereoselective additionof vinylmagensium chloride was facilitated by catalytic amountsof dimethyl zinc and lithium chloride generated in situ.101 It waspostulated that a highly reactive triorganic zincate was generatedin situ and that activation of the imine by lithium/magnesiumcomplexation led to an exacerbation of the stereochemical effectof the auxiliary. The crude sulfonylamine 173 was thentelescoped into a one-pot deprotection/reductive methylationby first treatment with HCl followed by addition of sodiumacetate, paraformaldehyde, and picoline−borane to affordallylamine 175. The purity of this chiral amine could beupgraded by salt formation with L-tartaric acid to afford acrystalline solid that was easily manufactured, stored, andtransported.101

The salt could be converted back to the free amine by simpletreatment with aqueous NaOH. Next, the 4-position of theisoxazole 175 was deprotonated with the magnesium salt of2,2,6,6-tetramethylpiperidine (TMP) and then treated withfurfural derivative 177 to afford alcohol 178 as a mixture ofdiastereomers (Scheme 28).100,103

Alcohol 178 was then refluxed in dimethyl sulfoxide (DMSO)with Hunig’s base to effectuate an intramolecular Diels−Alderreaction, affording a mixture of four diastereomers with theendoproduct being favored in a 45:1 ratio from the (S)-alcoholand a 1:1 ratio from the (R)-alcohol.100,103 After the reactionmixture had been allowed to cool, sulfur trioxide pyridine wasadded to furnish oxidation of the alcohol to provide ketone179.103 A Lewis-acid-mediated cleavage of the oxabicycle withtandem enol ether demethylation followed by TBS protection ofthe tertiary alcohol afforded enone 139 after recrystalliza-tion.100,103

Enone 139 was then combined with aniline derivative 181 bymeans of Myers’Michael−Dieckmann cascade under conditionsthat were optimized by Tetraphase.104 The entire system wasthen deprotected in two steps, first treatment with aqueous HFfollowed by a powerful global hydrogenolysis in the presence ofHCl to prevent epimerization of the C4 dimethylamino group.104

Aniline 184was then acylated with acid chloride 185 to afford the

free base of eravacycline (164), which was then converted to thebis-hydrochloride salt 186.104 This route was utilized to provideample material supply for the early clinical studies and iscurrently still being optimized. The overall process has a longestlinear 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 wentsmoothly, revealing no adverse effects and establishing the oralbioavailability at 28% with oral doses being well tolerated.97 Theeffectiveness of eravacycline against complicated intra-abdominalinfection was then studied in phase II clinical trials.105 TP-434was shown to have an efficacy commensurate with thatcarbapenem and continued into phase III.105 In the first portionof phase III, eravacycline was compared to ertapenem, and it wasdisclosed that there were no significant differences between thetreatments.106 Phase III trials for eravacycline are currentlyongoing 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

Scheme 27. Construction of Chiral Amine 175 Scheme 28. Completion of the Industrial Synthesis ofEravacycline, the First Fully Synthetic Tetracycline To BeTaken into the Clinic



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practical and enabling technology developed by Myers and co-workers. Recently, another novel fluorocycline from Tetraphase,TP-271 (187), entered phase I clinical trials.108 Tetraphase hasalso reported that another novel tetracycline derivative, whichcontains a methoxy substituent at C7 and is referred to as TP-2758 (188), is approaching clinical trials.107 It is now quite clearthat a renaissance is occurring within the realm of tetracyclinechemistry and that the development of a practical, convergenttotal synthesis was decisively enabling of these efforts.

4. EPOTHILONE

4.1. Epothilone Background

In the early 1990s, Merck established a program to identifycompounds that exhibited taxane-like stabilization of micro-tubules in an effort to discover more synthetically or biosyntheti-cally accessible alternatives to paclitaxel.109,110 After a screen of7000 plant extracts, a confirmed hit revealed the structures ofepothilones A and B, which surprisingly had been known forseveral years in the patent literature, although the absolutestereochemistries of the structures remained unassigned (Figure14). The compounds were patented by Gesellschaft furBiotechnoligsiche Forschung (GBF, now Hemholtz-Zentrumfur Infektionsforschung, HZI) and Ciba-Geigy; however, thestructures were protected only under German law, as interna-tional patent filings had languished due to an initial lack ofinterest.111,112 Ciba-Geigy had noticed exceptional fungicidal

activity of epothilones in 1987. However, their cytotoxicity madethem less suitable as antifungal agents, and there were alsoquestions about their selectivity for other applications.112 Forthese reasons, the epothilones had lain dormant in the literaturefor several years until Merck’s rediscovery in 1995. Merckdetermined that epothilone B was more active than paclitaxel intubulin polymerization assays and, more importantly, that itretained potency against the multidrug- (including paclitaxel-)resistant cell lines that were beginning to surface.109

These findings ignited a flurry of research in thepharmaceutical industry and in academic laboratories. GBFquickly worked to optimize fermentation methods for theproduction of epothilones and also to determine the absoluteconfigurations of the epothilones.112,113 Kosan Biosciences andNovartis identified and cloned the biosynthetic gene cluster fromthe original GBF strain, which allowed these companies toexplore genetic engineering to modify the epothilone core.112,114

Bristol-Myers Squibb developed a partnership with GBF toexploit fermentation for the production of epothilones andimprove the pharmalogical profile by semisynthetic means.112,115

Novartis utilized the technology it developed to produce largeamounts of epothilone B as their prime pharmaceuticalcontender and utilized formulations to mitigate the toxic sideeffects associated with the parent structure.112,116

The epothilones also piqued the interest of many academiclaboratories, which viewed the structures as viable candidates forindustrial total synthesis because of their simplified structuresrelative to paclitaxel. In addition, even though the epothiloneswere accessible through fermentation, the ability for structuralmodifications around themacrolide was somewhat limited.109,112

For these reasons, there was intense competition to establish afully synthetic route to the epothilones to facilitate thorough SARexplorations. By the beginning of 1997, the Danishefsky,Nicolaou, and Schinzer laboratories had established routes toepothilone A, which is less potent than epothilone B by an orderof magnitude.117−119 However, epothilone B quickly succumbedto synthetic efforts by the same three groups as well.120−122 Eachof these synthetic approaches was influential in the production ofsynthetic epothilones as pharmaceutical candidates.

4.2. Danishefsky’s Synthetic Approach

The Danishefsky group established the first route to epothiloneA, and this route hinged on two key bond formations to unitefragments 200 and 208.Construction of fragment 200 commenced with the cyclo-

addition of aldehyde 193 to oxygenated diene 194 (Scheme29).123 The resultant dihydropyranone 195 was then reduced tothe allylic alcohol, which was utilized to direct a subsequentSimmons−Smith cyclopropanation to the α-face of the pyranring to afford 196. The newly constructed cyclopropane was thenoxidatively ring-opened with N-iodosuccinimide to affordglycoside 197. This compound was then dehalogenated anddifferentially protected (over several steps) to afford dithiane198. The system was then homologated by two carbons to affordfragment 199, whereupon the dithiane was oxidatively degradedwith concomitant acetal formation to provide 200.The other component, 208, was constructed from tetrahy-

dropyranyl- (THP-) protected (R)-glycidol (201).(Trimethylsilyl)acetylide opening of the epoxide affordedhomopropargyl alcohol 202, which was subsequently protectedand deprotected to provide 203 (Scheme 30). A three-stepsequence converted primary alcohol 203 to methyl ketone 204.

Figure 13. Next-generation synthetic tetracyclines from Tetraphase.

Figure 14. Structures of the parent epothilones (A and B) and theirdesoxy derivatives (C and D), along with the numbering scheme for theepothilone core.



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The ketone was then subjected to an Emmon’s homologationusing phosphine oxide 205 to provide olefin 206 as an 8:1mixture of regioisomers. The pendant alkyne was then oxidizedto an intermediate iodoalkyne, which was subjected tohydroboration−protonolysis to afford Z-iodoalkene 207. Thehomoallylic alcohol was revealed under methoxymethyl (MOM)cleavage conditions and capped with an acetyl group to affordfragment 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 cross-coupled with cis-vinyl iodide 208 employing low-valentpalladium catalysis to afford 210. Treatment of acetal 210 withp-toluenesulfonic acid (p-TsOH) revealed the correspondingaldehyde. In a bold undertaking, Danishefsky and co-workerswere successful at closing the macrolactone core of epothilone Athrough intramolecular aldolization. A short sequence of stepsproduced epothilone C, which, upon subjection to dimethyldiox-irane (DMDO), afforded epothilone A. This approach providedepothilone A in a longest linear sequence of 23 steps (33 stepstotal) in 2.7% overall yield.

Danishefsky and co-workers quickly adapted this sequence forthe construction of epothilone B, utilizing fragment 217 as thecoupling partner instead of fragment 208 (Scheme 32).120,124

Epothilone B (192) was produced in a longest linear sequence of23 steps (28 overall) in 6.6% yield.

4.3. Nicolaou’s Synthesis

The second route to the epothilone core was developed byNicolaou and co-workers and also utilized a short sequence tobring together two main fragments 226 and 229 in a highlyconvergent manner.121

Access to northern segment 226 commenced with partialreduction of thiazole carboxylate 218 followed by homologationwith ylide 219 to afford 212 (Scheme 33). Treatment with allyl

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

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

Scheme 31. Danishefsky’s Union of Fragments to FurnishEpothilone A

Scheme 32. Danishefsky’s Adaption of Epothilone A Synthesisfor the Construction of Epothilone B



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diisopinocampheylborane [(+)-Ipc2B-allyl] then gave homo-allylic alcohol 214 in high enantiomeric excess. Protection of thealcohol and oxidative degradation of the olefin afforded aldehyde220, which was subsequently treated with phosphorane 221 toafford ester 222 as a single regio- and stereoisomer. The estermoiety was net reduced (over several steps) to a methyl group,and the α-olefin was converted into primary iodide 223. Theiodide was then displaced with the carbanion derived from (S)-1-amino-2-methoxymethylpyrrolidine (SAMP) hydrazone 224 togive 225. Oxidative cleavage of the auxiliary by magnesiummonoperoxyphtalate followed by partial reduction of theresultant nitrile afforded key aldehyde 226.The other fragment was prepared from ketoaldehyde 227

(Scheme 34). The aldehyde was subjected to Brown’sasymmetric allylation to afford alcohol 228, which wasmanipulated over several steps to provide protected diol 229.Deprotonation of 229 followed by treatment with aldehyde 226afforded aldol adduct in 85% yield as a 3:1 mixture of the two antidiastereomers, preferring the desired. The system was thenmanipulated over several steps to afford 231. The hydroxyacidwas then subjected to Yamaguchi macrolactonization conditionsto provide the core of epothilone D 232. The system was thensubjected to global deprotection under acidic conditions toafford epothilone D (190). The natural product was thenconverted by treatment with DMDO to epothilone B (192), as a5:1 mixture of desired to undesired diastereomers. The Nicolaouapproach provides epothilone B in 23 steps, 28 overall, in anoverall yield of 3.8%.4.4. Schinzer’s Formal Synthesis

The formal route to epothilone B developed by Schinzer and co-workers involved the combination of three major fragments in a

convergent fashion. This approach proved to be highly practicalbecause of a synergistic combination of fragments that were ofroughly 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 auxiliaryand protection to afford olefin 235 (Scheme 35).122 The systemwas then hydroborated/iodinated to produce iodide 236.

The next fragment was derived from (S)-malic acid (notshown), whose cyclohexylidene ketal (237, Scheme 36). Thecyclohexylidene acetal of malic acid was subjected to a selectivereduction of the carboxylic acid that provided lactone 238 upontreatment with acid and subsequent protection.122 Addition ofmethyllithium to the lactone and subsequent protection of theprimary alcohol provided methyl ketone 239. The ketone wasthen reacted with Horner−Wadsworth−Emmons (HWE)reagent 240 to afford the trisubstituted alkene 241.125 Theprimary silyl ether was then selectively deprotected, oxidized, andolefinated to provide vinyl iodide 243. The last fragment wasproduced starting with α-bromoester 244 and was originally usedin Schinzer et al.’s second-generation synthesis of epothilone A

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

Scheme 34. Nicolaou’s Combination of Fragments to ProduceEpothilone B

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



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(Scheme 37).125 A Reformatsky reaction with 3-pentanone(245) followed by dehydration afforded olefin 246. The ester

was then converted to an aldehyde and subjected to an aldolreaction with the enolate of chiral acetate 248. The ester was thenreduced to the primary alcohol, and the diol moiety wasprotected as the acetonide to give 250. The olefin was thenoxidatively 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). Theprimary alcohol protecting group was then cleaved and oxidizedto afford aldehyde 225, an intermediate seen in the Nicolaouroute. Deprotonation of fragment 251 followed by treatmentwith aldehyde 225 afforded the aldol product as a 9:1 mixture ofthe desired and undesired diastereomers. The acetonide was thencleaved, and the diol was protected as the bis-silyl ether tointercept intermediate 229 in the Nicolaou route. Thisconstruction of epothilone B required a longest linear sequenceof 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

production of synthetic epothilones for therapeutic develop-ment.4.5. Epothilone Structure−Activity Relationships

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

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

Scheme 37. Schinzer’s Route to the Southern Portion ofEpothilone B

Scheme 38. Schinzer’s Completed Formal Synthesis ofEpothilone B

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



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Region A consists of the aliphatic alcohol on the easternportion of the molecule. Examination of either ring contractionor ring expansion in this area with methylene units resulted insubstantial activity loss.129,139 However, introducing unsatura-tion in this region improved the activity by 4-fold for the 9,10-dehydro-Epo D as compared to Epo D, which led to thedevelopment of dehydelone (vide infra).140,141

Region B harbors the epoxide in the northern portion of themolecule. One crucial initial observation was that the deoxyvariants, namely, Epo C and Epo D, retained the high levels ofpotency found in their parent compounds.128 This findingsuggested that the epoxide was not necessary for biologicalactivity. Nicolaou and co-workers constructed analogues with acyclopropane in place of the epoxide and found them to beequipotent to their natural cogeners.142,143 Because of thesefindings, it is believed that the epoxide, rather than having areactivity 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 weretolerated.129

Region C consists of the highly functionalized, stereochemi-cally rich southern portion of the molecule. This region isrelatively intolerant of modification, with most changes leadingto losses in potency. One alteration of the epothilone B coreexploited by Bristol-Myers Squibb was the conversion of themacrolactone to a macrolactam, also known as Ixabepilone,which won FDA approval for the treatment of breastcancer.115,128,144 However, this modification decreased thepotency by an order of magnitude and rendered the compoundsusceptible to multidrug resistance mechanisms.145−147 Elimi-nation of the C3 hydroxyl group to form an α,β-unsaturatedmacrolactone resulted in a structure with reduced potencycompared to the parent.131 Reduction of the C5 ketone oralteration of the gem-dimethyl C4 result in significant loss ofbioactivity.129,131 One particular modification that not only istolerated but actually enhances the potency is the extension ofthe C6 methyl to larger aliphatic chains; this approach was usedby Schering AG to explore potential differences in the mode ofaction of Epo B versus Epo D through radiolabeling experi-ments.135,147

The last remaining segment of the molecule is the heteroarylside chain referred to as region D. Modifications in this area havebeen extensively investigated.128 Deletion of the olefinic linkerleads to a significant loss of activity, as does elimination of the arylgroup.129 Replacement of the thiazole ring with a phenyl resultsin a large drop in potency; however, more conservativeheteroaromatic surrogates are tolerated.128,133,135,148 A collabo-ration between the Nicolaou group and Novartis generated alarge library of epothilone B derivatives with a plethora ofheterocyclic side chains.148,149 When the Lewis-basic nitrogenwas converted to itsN-oxide, the system retained potency againsttumorigenic cells, but its activity was completely ablated inmultidrug-resistant (MDR) cells.135 In terms of the location ofthe thiazole nitrogen in the epothilone structure, the system canadopt two rotameric forms (Figure 16). To determine whetherone is more responsible for the bioactivty, Schering AGorchestrated several key experiments.They constructed three derivatives of epothilone D, in which

the thiazole moiety was replaced with 2-pyridyl, 3-pyridyl, and 6-benzothiazyl.135 Whereas the 3-pyridyl derivative showed asubstantial loss of activity, the 2-pyridyl and 6-benzothiazylderivatives of epothilone D showed marked increases in potency,approaching that of epothilone B.135 These findings support the

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

Although a tremendous amount of work has been done withsynthetic epothilones, in terms of exploring SARs bothacademically and industrially, many of the structures chosen asthe industrial clinical contenders are semisynthetic in form.However, Schering AG considered epothilones to be viabletargets for industrial total synthesis from the outset and neverseriously pursued semisynthetic means to access the bioactivematerial.112 Instead, Schering AG relied solely on total synthesisto explore the nature of the epothilone core and to develop drugcandidates.112,147 A major goal of the company’s agenda was toexpand the therapeutic safety window of these structures whilemaintaining their potency.147 A secondary goal was to capitalizeon the inherent activity against tumor lines resistant to paclitaxeland other frontline cancer treatment regimens.112,147 A smallportion of Schering AG’s SAR studies have been discussed (videsupra). The company discovered that twominormodifications tothe structure of epothilone B created a compound with a superiortherapeutic window and activity surpassing that of epothilone B.This compound was kept secret until it was revealed by ScheringAG in 2006.112,147 Schering AG had extended the C6 methylgroup into an allyl and had converted the vinyl thiazole side chaininto a benzothiazole. As stated above, this structure wasdiscovered and composed through total synthesis in apharmaceutical setting.Klar and co-workers at Schering AG were highly cognizant of

the synthetic efforts toward epothilone B and D discussed aboveand used this work to their advantage. They identified thestrengths of these routes and orchestrated a highly convergenthybrid approach that allowed them to modify nearly everyposition of the macrolactone scaffold.147 The discovery researchroute involved the combination of three roughly equal-sizedfragments, each with one stereocenter, in a manner analogous tothe Schinzer route to construct the lead compound ZK-Epo. Theconvergency coupled with the relatively simple fragments madethis 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 (−)-pan-tolactone (256), an abundant optically active building block(Scheme 39).150 Reduction to the lactol followed by olefinationafforded enantiopure allylic ether 258. The primary alcohol wasthen benzylated, and the alkene was hydroborated to affordalcohol 259 following oxidative workup. Ketal interchange with2,2-dimethoxypropane, hydrogenolysis, and oxidation then gavealdehyde 261. Treatment with butenyl magnesium bromide andsubsequent oxidation provided fragment 262, which is verysimilar to 251 from the Schinzer synthesis.150,151 Although thissequence was somewhat lengthy, it was preferred by thediscovery division because each step could be easily scaled (toapproximately 80 g of material per batch) and was highly flexibleto facilitate SAR studies.

Figure 16. Thiazole rotamers in 192.



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Fragment 270, which became region A of the epothilone core,was constructed from Roche ester (265), another abundantoptically active building block (Scheme 40). Roche ester was

derivatized as the THP adduct and then reduced to a primaryalcohol, 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, benzothiazoleformation, provided acid 272, which was then converted to

aldehyde 273. A subsequent Evans aldol reaction withoxazolidinone 274 afforded alcohol 275 as a 4:1 mixture ofdiastereomers that were separated by recrystallization.147 β-Hydroxyimide 275 was then protected and transesterified withethanol. A subsequent reduction afforded alcohol 276, which wasthen converted to phosphonium salt 277 in two steps.Fragments 270 and 277 were combined through a Wittig

reaction, reminiscent of both the Schinzer and Nicolaouapproaches, followed by acid-mediated cleavage of the THPprotecting group to afford olefin 278, unfortunately as a 1:1mixture of E and Z isomers (Scheme 42).147 After separation, theundesired E isomer could be converted to achieve an E-to-Z ratioof 6:4 by photochemical means.147 Oxidation of 278 affordedaldehyde 279.

Fragments 262 and 279 were combined through an aldolreaction, mirroring the Schinzer and Nicolaou approaches.Deprotonation of fragment 262 followed by transmetalationwith ZnCl2 and treatment with aldehyde 279 afforded anti-alcohol 280 as a 12:1 mixture of diastereomers. The acetonidewas removed, and the resulting diol was subjected to aprotection/deprotection sequence to afford primary alcohol281. A two-stage oxidation of the alcohol provided acid 283,

Scheme 39. Schering AG’s Construction of the SouthernFragment

Scheme 40. Schering AG’s Access to the Eastern Fragment ofSagopilone

Scheme 41. Schering AG’s Route to the Western Portion ofSagopilone

Scheme 42. Schering AG’s Combination of the ThreeFragments of Sagopilone



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which was then subjected to conditions that cleaved the allylicsilyl ether preferentially. Access to the macrolide 284 wasachieved under Yamaguchi conditions. Global deprotection ofthe silyl groups followed by epoxidation with DMDO affordedZK-Epo (285) as a 7:1 mixture of diastereomeric epoxides,favoring the desired form. This route was utilized by thediscovery division to produce 36 g of compound for conductingextensive preclinical evaluations.147

Initial tests on ZK-Epo indicated that it was able to outmatchpaclitaxel and several other chemotherapeutics, in terms of IC50,against a panel of tumor cell lines.147 One particularly importantfinding was that ZK-Epo showed a very low toxic potential onnormal, nondividing cells.147 In addition, it retained its potencyagainst MDR cell lines and proved not to be susceptible tocellular efflux pumps, such as P-gp.147,153 Finally, ZK-Epoexhibited activity against several cell lines resistant to normalchemotherapy regimens.153 These exciting preclinical resultsaccelerated ZK-Epo’s trajectory into clinical trials. In 2003,sagopilone, the name chosen for ZK-Epo, entered phase Istudies.153 These studies indicated that sagopilone was well-tolerated and that the major dose-limiting toxicity was peripheralneuropathy. With phase II on the horizon, Schering AG’s processgroup embarked on a route optimization to ensure an adequatesupply 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 ofmaterial at the start of the process to manufacture kilograms ofsagopilone.154

For the construction of fragment 262, the discovery routeproved difficult to scale up, so another route was devised(Scheme 43).154 Compound 292 was made commerciallyavailable during development, and Schering AG’s processchemists capitalized on the availability of this material.155 The

original manufacturing sequence of 292 began with a cross-Claisen condensation of tert-butyl acetate (286) and nitrile 287to afford β-keto ester 288. The ketone was then reduced, and theester was saponified to provide acid 289. The acid was thensubjected to a sequence of crystallizations with (R)-(+)-N-(p-hydroxybenzyl)phenyl ethylamine (290) to afford an amine salt,which was then converted to the enantioenriched ester 291. Theester was then reduced to the primary alcohol, and the diolmoiety was protected as acetonide 292.155

An alternative approach to acetonide 292 was developed bySchering AG and became the preferred route (Scheme 44).156,157

It involved deprotonation of nitrile 294 and treatment withaldehyde 295 to afford alcohol 296. The alcohol was thenacetylated, and the racemic mixture was subjected to enzymatichydrolysis to give enantioenriched acetate 298, which was easilyscaled to 22-kg batches.156,157 This material was then saponified,hydrogenolyzed, and protected to give acetonide 292 (Scheme43). This material was subjected to MeLi followed by an acidicworkup to afford methyl ketone 293. The ketone was thenconverted to the β-keto ester, allylated, and decarboxylated toafford fragment 262 after distillation.154

Fragment 270 was constructed from Roche ester in theprocess route as well (Scheme 45). The same sequence of

protection, reduction, and tosylation was used. However, at thisstage, the process division chose instead displacement of thetosylate with lithium acetylide followed by a deprotonation andN,N-dimethylaniline (DMA) quench to afford methyl ketone299. This system was then hydrogenated to afford fragment 270after distillation. This alternative route precluded the use of toxicOsO4 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 (Scheme46). Benzothiazole 272 was converted to the β-keto esterthrough activation, displacement, and decarboxylation. The β-

Scheme 43. Sumika Fine Chemical’s Manufacturing Route to292 and Schering AG’s Process Division Elaboration toFragment 262

Scheme 44. Schering AG’s Optimized Biocatalysis Route toIntermediate 292

Scheme 45. Process Route to Fragment 270



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keto ester was then subjected to microbial reduction conditionswith Pichia wickerhamii yeast.157,158

This approach precluded the use of a chiral auxiliary, whichsimplified the purification immensely. The alcohol was thenprotected, and the ester was reduced to the primary alcohol toafford 276. The primary alcohol was then converted to iodide304.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 1:1mixture of E and Z isomers (Scheme 47). Unfortunately, this stepcould not be easily circumvented by other means, and thematerial had to be separated by chromatography afterdeprotection. Fortunately, the undesired isomer could bephotorecycled on scale, and after two iterations of irradiationand separation, 278 was obtained in 75% yield. Alcohol 278 wasthen oxidized and carefully isolated to afford the easilyepimerizable aldehyde 279. Fragments 279 and 262 werecombined through an aldol reaction that gave 280 as a 10:1mixture of diastereomers on scale, which, unfortunately, requiredchromatography for separation.

The discovery division’s sequence of steps to close the ringsystem was adopted for process scale. The workup for the globaldeprotection step required modification because extendedexposure to alkaline conditions generated the ring-openedproduct. This was remedied through the use of mild aqueouspotassium borate solutions. For the final epoxidation, amethyltrioxorhenium/hydrogen peroxide system was preferredover DMDO because of scalability concerns.133 This change inoxidation procedure increased the selectivity for the epoxidationfrom 7:1 to 23:1. After chromatography and recrystallization, thisprocess provided the desired material in a 21-step longest linearsequence, 35 steps overall, in 7.2% overall yield and has beenused 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 ofcancer.159−161 However, the clinical development of sagopilonehas been halted for reasons unknown, and Bayer, who acquiredSchering AG, has been quiet for some time on the fate of thisdrug.162

4.7. Dehydelone, Fludelone, and Isofludelone

The other major players in the field of synthetic epothilones aredehydelone, fludelone, and isofludelone, compounds champ-ioned by Danishefsky and co-workers. The story of thesestructures is one of judicious modifications to the structure ofepothilone and, in the case of fludelone, one fortunate andcursory decision to introduce a trifluoromethyl group at C26.163

After completing their synthetic approach to epothilone B(192), Danishefsky and co-workers were intrigued thatepothilone D (190, referred to as dEpoB by Danishefsky)retains the tubulin-stabilization properties, albeit less potent, aswell as the MDR tumor activity of its oxygenated counter-part.124,163 In addition, the therapeutic window of epothilone Dis much larger than that of epothilone B, leading to theconclusion that the C12−C13 epoxide could be a source ofnonselective toxicity.163 During this time, Kosan Biosciencesbegan to collaborate with Sloan-Kettering by providingepothilone D through fermentation, which also lead to thediscovery of epothilone 490 (Figure 17).164 This system containsa diene obtained by introducing additional unsaturation at C10−

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

Scheme 47. Process Route to Complete Sagopilone



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C11 of 190. However, despite its potency in vitro, the in vivoactivity was fairly lackluster in mouse models because ofmetabolic instability.163

Synthetic studies of epothilone 490 led Danishefsky and co-workers to construct the skipped diene analogue of epothilone490, with a methylene group inserted between C11 and C12 toprovide the expanded 17-membered ring (not shown).140,163

Surprisingly, this expanded ring system was exceptionally activein vitro, even though its saturated analogue is inactive. This ledDanishefsky and co-workers to ponder whether this skippeddiene motif imparted rigidity to the molecule and could beutilized in the traditional 16-membered scaffold.163

A concise route to access this skipped diene analogue ofepothilone D (307), also referred to as KOS-1584 ordehydelone, was pursued. Unlike Danishefsky’s initial access toepothilone B, where two fragments were brought together, thediscovery route to dehydelone adopted a three-fragmentapproach.140,165 Construction of the first fragment commencedwith an aldol reaction between ketone 308 and Roche esterderivative 309 to afford the syn product 310 in a 5.7:1 ratio ofdiastereomers (Scheme 48). The system was then protected, and

the acetal was cleaved to obtain aldehyde 311. The aldehyde wasreacted with the chiral titanium enolate of tert-butyl acetate toafford alcohol 313 in greater than 20:1 dr.166 The system wasthen subjected to a protection, deprotection, oxidation, andolefination sequence to provide ester 314.The next fragment was constructed from oxazolidinone 315

and allyl iodide 316 (Scheme 49).165,167 The system was thendeprotected, and the oxazolidinone was displaced with

dimethylhydroxylamine to afford Weinreb amide 318 afterreprotection. The system was then subjected to Stille couplingwith allyltributyltin followed by treatment with MeMgBr toafford an intermediate methyl ketone. Mild deprotection waseffected by treatment with aqueous acetic acid to provide 319.165

Ester 314 was then subjected to acidic hydrolysis conditionsand then coupled with hydroxyketone 319 to afford secoprecursor 320 (Scheme 50). The system was then subjected to

ring-closing metathesis using Grubbs second-generation catalyst.The last fragment was installed by a Wittig reaction withmacrolide 321 and fragment 322. This installed the aryl sidechain as a 9:1 mixture of E and Z regioisomers that could beseparated.A global deprotection was then effected by treatment with

pyridine hydrofluoride. With a concise route to dehydelone(307) developed, examination of its activity revealed anapproximate 10-fold increase in drug potency against MDRtumors.140 In vivo and in vitro experiments showed that 307 wasmore cytotoxic than epothilone B (192) and more metabolicallystable than epothilone D (190).140,168 Even though 307 wasfound to be superior to the parent epothilones, it exhibitedsignificant nontumor-specific toxicity, which restrained theability to push the target tumor into a nonrelapsable state.However, dehydelone 307 was explored as a drug candidate by acollaboration between Kosan Biosciences and Hoffmann-LaRoche and has completed a phase II trial for the treatment ofmetastic nonsmall cell lung cancer.169,170

To provide material for the clinical trials, Kosan Biosciencesand Hoffmann-La Roche modified Danishefsky’s approach to

Figure 17. Structures of epothilone 490 and dehydelone.

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

Scheme 49. Danishefsky’s Route to Fragment 317

Scheme 50. Completion of Danishefsky’s Discovery Route toDehydelone



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dehydelone. Fragment 319 was constructed from (R)-(+)-glycidol (Scheme 51).171 The alcohol was oxidized by in

situ-generated RuO4 and subsequently converted to Weinrebamide 324. Some intriguing organometallic chemistry was thenutilized to install the lower portion of fragment 319. Acarboalumination of propyne with an in situ-generated allylalanefacilitated by Cp2ZrCl2 produced an intermediate vinylalane,which, upon treatment with 324, opened the epoxide to affordhomoallylic alcohol 325. Finally, addition of MeMgBr affordedmethyl ketone 319. This sequence was easily scaled to 100-gbatches.171

For fragment 330, Kosan Biosciences explored the option ofbiosynthesis to produce the polyketide-like moiety.172,173 Thecompany was able to establish a fermentation method toconstruct 326 (Scheme 52). The system was then methylated,

subjected to a Claisen condensation, and reduced under Noyoriconditions to afford 329.174 Protection of the secondary alcoholfollowed by cleavage of the chiral auxiliary afforded fragment330.174 The two moieties were then coupled to furnish ester331.174,175 The seco precursor was then converted to dehydeloneby ring-closing metathesis, deprotection, and aWittig olefinationto install the heterocyclic side chain.170,174−176 This route was

still being optimized when Bristol-Myers Squibb acquired KosanBiosciences. Currently, there are no active clinical studies ondehydelone (KOS-1584), and it is not known by us whether theprogram has been halted or terminated.177

Danishefsky and co-workers have continued their work ondehydelone because of dissatisfaction with the toxicology profile.They considered the incorporation of fluorine atoms into thestructure as a means to temper the cytotoxicity of the system. TheC26 methyl group was contemplated as an area to integrate thefluorine atoms because of earlier studies indicating polarfunctionality being tolerated in that position.129,168 Althoughthe incorporation of the fluorine mitigated the toxicity of thestructure to a small degree, fluorinated analogue 332, referred toas fludelone, proved to be quite remarkable (Figure 18).

Treatment of mouse models with fludelone not only suppressedthe growth of MX-1 xenograft tumors on mouse models butshrank and eliminated the tumors for as long as 64 days.168 Inaddition, the therapeutic window was quite broad for fludelone,where the decrease in mouse model body weight due totreatment 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 metabolicprocesses as compared to 307, and the introduction of thefluorines imparted an increase in hydrophilicity, increasingbioavailability.168 Examination of fludelone’s effect on humanmyeloma models indicated a rapid induction of apoptosis, evenin paclitaxel-resistant cell lines.178 Fludelone’s therapeuticspectrum was found to encompass leukemia, breast, colon, andlung carcinomas and ovary and prostate adenocarcinomas.179

In the hopes of optimizing fludelone further and regainingsome lost potency during its transition from dehydelone,Danishefsky and co-workers modified the thiazole moiety intoan isoxazole to afford isofludelone (333).180 The desire for anincrease in potency was achieved, as this modification gave rise toa remarkably potent compound that is able to achieve completeremission and cures in xenograft mouse models. In addition toenhanced potency, isofludelone (333) has greater metabolicstability than fludelone. After extensive preclinical investigations,isofludelone has recently entered phase I clinical trials that areexamining dose escalation and pharmacokinetics.162

Currently, the only epothilone analogue approved for clinicaluse by the FDA is Bristol-Myers Squibb’s semisyntheticderivative Ixabepilone.181,182 However, the monumental amountof work done in this area was driven by total synthesis. Thelessons learned by developing highly convergent and modularsynthetic constructions toward this class of natural productsmade it possible for fully synthetic analogues to make it to clinicaltrials. The beautiful work by Schering AG to develop a viableprocess capable of delivering kilograms of sagopilone, anepothilone B-derived active pharmaceutical ingredient (API), isnothing short of remarkable. The elegant studies by Danishefsky

Scheme 51. Kosan Biosciences’Optimized Route to Fragment317

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

Figure 18. Structures of fludelone and isofludelone.



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

5. CRYPTOPHYCIN

5.1. Discovery and Background

Amid investigations aimed at traditionally “overlooked” micro-organisms in the early 1990s, a team of researchers led byGregory Patterson at the University of Hawaii discoveredantineoplastic activity in the extracts of cultured blue-greenalgae.183 This discovery presented itself after the screening ofmore than 1000 cyanobacteria extracts. In addition to potentcytotoxicity, it was determined that the extracts were moreselective for solid-tumor-derived cell lines than for leukemias asmeasured by the Corbett assay.184,185

The cytotoxicity of this particular strain of Nostoc sp. waspinpointed to a group of macrocyclic depsipeptides (Figure19).184 Surprisingly, the elucidated structure of the most potent

depsipeptide matched one already existing in the literaturedenominated as cryptophycin. The structure was originallydiscovered in another Nostoc sp. strain by Merck, whichinvestigated 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 ofcryptophycin 1, without assigning stereochemistry. In contrast,Moore and co-workers assigned both absolute and relativeconfigurations, as well as the structures of the other newlyisolated cryptophycin family members.184,188 Meanwhile, themechanism of action of cryptophycin 1 was explored by severalgroups. Cryptophycin 1 was found to interact with tubulin atpicomolar concentrations and arrest the mitotic cycle byinhibiting tubulin polymerization and promoting depolymeriza-tion in vitro.189−192 Cryptophycin reversibly binds in the Vincadomain on the ends of microtubules with high affinity.190,191 Inaddition to hyperpotency, cryptophycin retained activity againstcell lines that overexpressed P-glycoprotein.189 This character-istic was perhaps its most exciting because of the potential fortreating P-gp-mediated multiple-drug-resistant cancer types.189

5.2. Tius−Moore’s Synthesis of Cryptophycin 1

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

(vide supra), these compounds were viewed by many as a primeopportunity for total synthesis to drive further research anddevelopment of potential clinical candidates. In fact, cryptophy-cins succumbed to synthesis within a year of their rediscovery byTius, Moore, and Kitagawa and their co-workers.188,193 Thesecryptophycin constructions were followed by many otherapproaches to the depsipeptides.194−200 Although each of thesesyntheses is elegant in its own way, the Tius−Mooreconstruction proved to be the most influential in the advance-ment 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 highlyconvergent manner. This approach allows for flexibility inperipheral functionalization of the molecule or for changes in thecore architecture.The construction of the first fragment commenced with an

HWE homologation of dihydrocinnamaldehyde to afford ester338 (Scheme 53).188 This material was then reduced to the allylic

alcohol 339, which was then subjected to a Sharpless asymmetricepoxidation with (+)-diethyl tartrate [(+)-DET] to provideepoxide 340.201 The epoxide was then opened by the action ofAlMe3 with complete regioselectivity to afford diol 341. Thesystem was then converted to a styrene derivative by protectionof the diol moiety as its acetonide, benzylic bromination, andelimination to afford acetonide 342. The acetonide thenunderwent cleavage followed by monotosylation and protectionof the secondary alcohol to provide 343.202 The tosylate was thendisplaced by cyanide to afford an intermediate nitrile, which wasthen reduced and homologated to ester 344. The ester was thensaponified to afford fragment 345.The second main fragment was constructed from Roche ester

(Scheme 54). The ester was converted by heating in the presenceof ammonia to an amide, which was then reduced to aminoalcohol 346 isolated by distillation.188 The amine was derivatized

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

Scheme 53. Tius−Moore Synthesis of Cryptophycin’sNorthern Fragment



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with Boc2O, and the alcohol was oxidized to afford acid 347. Theacid was then coupled to leucic acid derivative 348 to provideester 349. The allyl ester was then cleaved by the action ofcatalytic Pd to provide acid 350.203

Fragment 345 was then coupled to D-tyrosine derivative 351and deprotected to afford alcohol 352 (Scheme 55). This alcoholwas then coupled to acid 350, which gave bis-protected secoprecursor 353. The protecting groups were cleaved by treatmentwith Zn and AcOH followed by neat TFA to provide amino acid354. Macrocyclization was then effected with pentafluorophenyldiphenyl phosphinate (FDPP) to provide cryptophycin C(335).204 This natural product could then be converted tocryptophycin 1 (334) by treatment with m-CPBA to afford thenatural product as a roughly 2:1 mixture of diastereomersfavoring the desired form.188,205 This route constructedcryptophycin 1 in a longest linear sequence of 21 steps, 27overall, 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. Itwas determined that very few alterations of the parent weretolerated.206 However, from preliminary studies on fermentedcryptophycins, it was noted that the ester linkages were fairlysensitive to mildly alkaline media, leading to facile ring openingand degradation.184,207−209 Moore and Tius hypothesized thatthis susceptibility could be abated by the installation of a second

methyl substituent on the southern β-amino acid fragment of thestructure.207

This fortuitous decision led to the creation of the more robustcryptophycin 52 (372; see Scheme 59 below). Cryptophycin 52proved to be a potent antiproliferative structure that surpassedthe activity of the parent structure.190,207 It was the most potentsuppressor of microtubule dynamics known at the time.210 Thecryptophycin 52−tubulin complex has a dissociation rateconstant (Kd) of 47 nM and a very low dissociation rate.210 Asa result, a large fraction of intracellular cryptophycin is thought tobe adsorbed by its receptor and is unavailable to effluxmachinery.211

5.4. Eli Lilly’s Synthesis of Cryptophycin 52

These results intrigued the scientists at Eli Lilly and convincedthem to initiate a collaboration with Moore aimed at taking thispowerful structure into clinical trials.212 To provide an adequatesupply of material necessary for clinical trials, the chemists at EliLilly launched an effort to refine the Tius−Moore synthesis into aform that could be reliably executed on industrial scales. The firstfragment was constructed in a manner similar to that employedin the Tius−Moore approach.213−215

Dihydrocinnamaldehyde was homologated with trimethyl-phosphonoacetate, and the product was reduced to afford allylicalcohol 339 (Scheme 56). This material was then subjected toasymmetric epoxidation followed by AlMe3-mediated ringopening and methylation to give an intermediate diol.213,214

The diol was selectively tosylated at the primary position throughan intermediate dibutylstannylene acetal followed by TBSprotection of the secondary alcohol.216 The system was thensubjected to a benzylic bromination and elimination to providestyrene derivative 343.214 The tosylate was converted to thenitrile by treatment with KCN followed by partial reduction withDIBAL and HWE homologation to provide ester 344. Thismaterial 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 mixtureof diastereomers.217 These were separated by chromatography.The alcohol was then protected as its TBS derivative, and thealkyne was subjected to a hydroboration/oxidation to providealdehyde 359. The aldehyde was then homologated to anacrylate derivative through an HWE reaction, and the pendantolefin was cleaved by ozonolysis. The resultant aldehyde wasthen converted to styrenyl intermediate 344 by aWittig reaction.

Scheme 54. Construction of the Southern Portion ofCryptophycin

Scheme 55. Completion of Cryptophycin 1



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Both of these routes could be effectively scaled to afford largeamounts of the first fragment.Synthesis of the second fragment commenced with the double

methylation of ethyl cyanoacetate (360) with MeI to provide361 after distillation (Scheme 58).218 The nitrile was thenreduced to the amine in the presence of Boc2O to provide the β-carbamoyl ester 362, which was subsequently saponified toprovide acid 363. The acid was coupled with 348 to afford ester364, which was then deallylated and subjected to a protecting-group exchange to afford 366.218,205

Fragment 345 was then coupled to the tyrosine derivative 351to afford amide 367 (Scheme 59).218 This material was thendeprotected to afford alcohol 351.215,219 Because of the issues ofdiastereoselectivity with late-stage epoxidation, the researchersexplored epoxidations and other transformations at variousstages in the synthesis.205,212,220

One approach that was explored coupled alcohol 351 withfragment 365 to provide 368 and then subjected the system to aSharpless asymmetric dihydroxylation.212,221 The diol would

serve as a masked form of the epoxide that could be unveiled atthe end of the synthesis.222 The system was then deprotected toafford the seco precursor. It was discovered during investigationsat Eli Lilly that the trichloroethyl ester was susceptible tointramolecular aminolysis by the pendant amine, which allowsfor the avoidance of coupling reagents.223,224 This susceptibilitywas enhanced by the action of 2-hydroxypyridine, which wasexploited in this case to afford macrocycle 370. The diol moietywas then converted to an orthoester by exchange with trimethylorthoformate. The orthoformate was then treated with acetylbromide, which ionized the orthoformate and produced vicinalformyloxy bromide 371 upon isolation.212 This material couldthen be converted to cryptophycin 52 upon treatment with base.This approach produced the API in a longest linear sequence of17 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 highselectivity under Shi conditions (Scheme 60).205,225 Thisepoxidation afforded a 6.5:1 mixture of epoxides, which wasupgraded to 10.3:1 after coupling to fragment 366 to afford secoprecursor 374.205 The bis-protected seco precursor was thensubjected to 9-fluorenylmethyloxycarbonyl (Fmoc) cleavageconditions, and surprisingly, it was discovered that the free seco-amine slowly converted to cryptophycin 52 under the reactionconditions. Therefore, the seco-amine was not isolated, butinstead was allowed to proceed to the final product, which couldbe chromatographed to afford a 9.4:1 mixture of diastereomericepoxides. These isomers could be separated by reverse-phaseHPLC.205 This alternative route produced cryptophycin 52 in alongest 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 trialsindicated that an intermittent dosing schedule would be optimalfor treatment regimens.226 Phase I trials found that cryptophycin52 had dose-limited neurotoxicity that could be reversed aftertreatment ended.226 In addition, there was evidence of antitumoractivity against nonsmall-cell lung cancer, renal cancer, and headand neck cancer, which warranted further investigations.226

Unfortunately, cryptophycin 52 had to be withdrawn from phaseII human clinical trials due to peripheral neuropathy.227,228 Thisis a perennial dose-limiting toxicity of tubulin-binding anti-

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

Scheme 57. Alternative Approach to the Northern Fragmentof Cryptophycin

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



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mitotics, which, to date, seems to be absent only for thediazonamide class of structures (vide infra).The cryptophycins are an intriguing class of compounds with

potency that is difficult to match. This unprecedented activity ledto the development of cryptophycin 52, a compound thatenthralled Eli Lilly and Co. The efforts by Eli Lilly to adapt theTius−Moore route into a scalable form are meritorious, andmuch can be learned from their achievements. To ourknowledge, there are no active trials examining cryptophycin52, which was the only member of this chemical class to reach theclinic.

6. PM060184

6.1. Discovery of PM050489 and PM060184

A marine natural products discovery campaign by thePharmaMar corporation in Spain recently uncovered a new setof mixed-lineage polyenes with extremely potent antiproliferativeactivity.229,230 The structures were isolated from a Lithoplocamiasp. sponge collected nearMadagascar and named PM050489 andPM060184 (Figure 20).229 Both molecules were obtained intrace quantities from sponge samples: 0.002 wt % in the case ofPM050489 and 0.00003 wt % in the case of PM060184.229 Thesemolecules are composed of a valerolactone with a largepolyunsaturated tail at the δ-position. Initial expeditions

provided sufficient material to assign the constitution and amajority of the relative chemistry.

Scheme 59. Eli Lilly’s Completion of Cryptophycin 52

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

Figure 20. (A) Structures of PM050489 and PM060184. (B) Crystalstructure of PM060184 bound to the T2R-TTL (stathmin-like proteinRB3−tubulin tyrosine ligase) complex231 (PDB accession number4TV9). (Reproduced with permission from ref 231. Copyright 2014National Academy of Sciences.)



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In addition to their intriguing structures, PM050489 andPM060184 showed subnanomolar activities against severaltumor cell lines, which was exciting to PharmaMar. However,these preliminary tests exhausted the material supply, and the C6and C21 stereocenters had yet to be assigned.229 Given theseconstraints, the chemists at PharmaMar embarked on a syntheticsequence to deliver more of these intriguing polyenes, amaneuver seldom seen in the private sector.6.2. Synthetic Studies on PM060184

PharmaMar constructed the two compounds by similar routes;however, PM060184 was chosen as the prime clinical candidatebased on its activity, safety profile, and distinct mechanism ofaction (vide infra).229 The syntheses utilized several stereo-retentive sp2-atom-based cross-coupling methodologies to installkey Z olefinic linkages. This allowed the structures to be brokeninto two fragments. The route to the first fragment commencedwith 1,3-propanediol (375), which was subjected to TBSprotection and oxidation to afford aldehyde 376 (Scheme61).232 An Evans aldol reaction between aldehyde 376 and

oxazolidinone 233 afforded β-hydroxyimide 377.233 This systemwas then TBS-protected, and the auxiliary was reductivelycleaved to provide alcohol 378. The alcohol was then oxidizedand subjected to a Wittig reaction with phosphorane 380 toafford acrylate derivative 381.The ester was then converted to an aldehyde and subjected to

a subsequent olefination reaction to afford vinyl iodide 383. Thisintermediate was then partially deprotected to reveal the primaryalcohol, which was oxidized and homologated through Horner−Wadsworth−Emmons olefination with phosphonate 385. Upontreatment with dilute HCl, the other TBS group was cleaved, andthe 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 tert-butyldimethylsilyl ether, and the olefin was epoxidized by m-

CPBA to give a racemic mixture of epoxide 389.234 This mixturewas then resolved by Jacobsen’s hydrolytic kinetic resolution toafford the enantiopure (R)-epoxide 391.38 The epoxide was thenopened by nucleophilic addition of lithiated propyne to affordhomopropargylic alcohol 392. This was then protected andpartially hydrogenated under Lindlar conditions to providealkene 393. The primary alcohol was then deprotected, oxidized,and converted to the vinyl iodide in a manner analogous to thatused for the first fragment. Vinyl iodide 395 was then coupled toBoc-Tle-NH2 using copper catalysis developed by Buchwald andco-workers.235 This procedure established Z-configured enamide397 in a stereoretentive manner. The system was then pyrolyzedfor Boc removal because acid treatment proved problematic forthe t-butyldiphenylsilyl (TBDPS) protecting group and for theenamide. Free amine 398 was then coupled with acid 399 toafford 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 theprimary urethane was installed by conventional means to affordPM060184 (374). This route provided PM060184 in a longestlinear 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 activityagainst a variety of cancer cell lines.237,238 In addition, it provedeffective at treating subcutaneous xenografted tumors in nudemice with no major toxic side effects observed at the maximumtolerated dose.238 Further studies indicated that activity wasretained against cells and xenografted tumors overexpressing P-

Scheme 61. PharmaMar’s Construction of Fragment 387

Scheme 62. PharmaMar Production of Fragment 400



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gp efflux pumps.238 Examinations of PM060184’s effects onmicrotubules demonstrated that it suppresses their dynamicinstability, which increased the amount of time microtubulesspent in pause.238

X-ray crystallographic studies with a tubulin complex(specifically two αβ-tubulin dimers, stathmin-like protein RB3and tubulin tyrosine ligase, aka T2R-TTL) were utilized todetermine the mode of action of PM060184, which binds to anovel region of tubulin denominated as the maytansine site(Figure 20).231 PM060184 shares its binding region withmaytansine (whose antibody drug conjugate is FDA-approvedfor the treatment of advanced breast cancer) and rhizoxin (whichadvanced to phase II).231 The key binding points betweenPM060184 and tubulin involve hydrogen bonding at the C1 andC13 carbonyls and a hydrophobic interaction of the C27 methylwith a pocket on the protein (Figure 20). Binding to this regionresults in the blocking of tubulin addition to the microtubule plusends, which prevents longitudinal growth of microtubules.231

These findings provided the momentum to carry PM060184 intophase I clinical trials.PharmaMar has completed the adaption of this route for the

industrial scale.239−242 Thus far, PM060184 has shown areasonable safety profile, with the only observed dose-limitingtoxicity being noncumulative peripheral neuropathy, whichcould be managed by a modification of the administrationschedule. In addition, clinical antitumor activity could beobserved in these studies, which bodes well for the future ofPM060184 and its impending phase II trials.Although PM060184 has only recently arrived on scene,

PharmaMar’s endeavors have taken this compound further indevelopment than most marine natural products. The potentactivities of PM050489 and PM060184 not only captured theattention of the PharmaMar discovery division, but alsoprompted them into the bold decision of developing aconvergent and scalable route to these molecules. This decisionis an exquisite example of how establishing a fully syntheticapproach to complex natural products is not exclusive to those inacademia, making the efforts of PharmaMar highly commend-able. The tale of PM060184 is far from over, and it is exciting tosee what the future will hold for this polyketide.

7. DISCODERMOLIDE

7.1. Discovery and Background

During the late 1980s, the Harbor Branch OceanographicInstitute in Florida was conducting expeditions aimed atdiscovering prospective marine-derived therapeutics throughbioassay-guided screening of extracts.243 A Discodermia sp.sponge collected in March of 1987 yielded a compound that

exhibited potent antitumor properties against P388 leukemiacells and suppressed a two-way mixed lymphocyte reac-tion.243,244 In addition, the compound, termed discodermolide,appeared to be mildly selective toward tumorigenic cell linesversus normal murine splenocytes.243 In addition to its potentanticancer activity, discodermolide also exhibited a powerfulimmunosuppressive activity.245−247 The structure was assignedas a multiply substituted valerolactone harboring an extendedhighly substituted, unsaturated appendage at its δ-position(Figure 21).243,244 The absolute stereochemistry of discodermo-

lide was not determined in the original structural elucida-tion.243,247 Further studies on the biological profile of thiscompound revealed it to be a microtubule-stabilizing agent,similar to paclitaxel and the epothilones (vide supra).247,248 Likethe epothilones, discodermolide showed efficacy against severalmultidrug-resistant cancer lines.243,249 In addition, discodermo-lide was reported to exhibit synergistic effects when combinedwith paclitaxel in various cancer cell lines.247,250 Thesepreliminary findings increased enthusiasm for designatingdiscodermolide 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. Otheravenues clearly had to be explored as a means to access thequantities needed for clinical trials.243,244,247

The two paths considered were fermentation/isolation andtotal synthesis. It was then unclear whether discodermolide wasbiosynthesized by the Discodermia sponge from which it wasisolated or produced by a commensal microorganism presentwithin the sponge.243 Some efforts have been pursued to producediscodermolide by aquaculture of a particularDiscodermia sp. andsubsequent isolation; however, this process is still being exploredand has not been validated on a larger scale.251

In addition, the biosynthesis of discodermolide has yet to befully elucidated, although the compound is believed to beconstructed from a polyketide synthase gene cluster.252 Theseconstraints have effectively extinguished the ability tomanufacture 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

Scheme 63. PharmaMar’s Completion of PM060184

Figure 21. Structure of (+)-discodermolide.



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answer the call for a discodermolide preparation. The first toachieve a route to the enantiomer of natural discodermolide wereStuart Schreiber and co-workers, which allowed for the absoluteassignment of natural discodermolide.253 The Schreiber syn-thesis was quickly followed by efforts by Smith et al., who alsohad constructed ent-discodermolide.254 Schreiber and co-work-ers established a route to natural discodermolide in 1996.255

Discodermolide and its enantiomer then succumbed to severalmore syntheses by the groups of Myles, Marshall, Smith, andPaterson.256−266 These campaigns have been reviewed pre-viously. The current discussion focuses on the syntheses thatdrove large-scale production of discodermolide for use in clinicalinvestigations.247,267,268

7.2. Smith’s Gram-Scale Synthesis

The repeating nature of substituents on polypropionate chainsinvites synthetic strategies that use common fragments multipletimes during assembly. This theme was beautifully exploited inthe Smith synthesis.256−258 When the strategy is executed well,both total operations and the longest linear sequence can besignificantly 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

alcohol was then oxidized and engaged in a diastereoselectivealdol reaction with the dibutylboron enolate derived fromoxazolidinone 406. After recrystallization, the product wasconverted to Weinreb amide 408, which Smith and co-workersdesignated as their “common precursor” or CP. Thisintermediate was then elaborated in three different ways toconstruct the three main fragments of discodermolide.The path to the first fragment began by oxidizing 408 under

anhydrous conditions to provide the p-methoxybenzylideneacetal (Scheme 65). The Weinreb amide was then reduced to analdehyde and subjected to an aldol addition using thedibutylboron enolate derived from oxazolidinone 233 to affordβ-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 reductionto afford aldehyde 413 (Scheme 66). The aldehyde was thenconverted to vinyl iodide 415, the second main fragment, by theZhao−Wittig protocol.269

To construct the final fragment, 408 was TBS-protected,hydrogenolyzed, and oxidized to afford aldehyde 416 (Scheme67). A TiCl4-catalyzed Mukaiyama aldol was utilized with silylenol 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 diastereoselectivelyreduced to allylic alcohol 419 by K-selectride. This alcohol wasthen protected, and the pendant olefin was oxidatively cleaved toafford aldehyde 421.Fragments 411 and 415 were joined by means of a Negishi

coupling (Scheme 68). Lithiation and transmetalation of iodide411 followed by treatment with catalytic Pd and vinyl iodide 415afforded olefin 423. The p-methoxybenzyl group was thencleaved and replaced by a trityl protecting group to provide ether424. The p-methoxybenzylidene acetal was then reduced torelinquish the primary alcohol and form the secondary ether,which was oxidized to afford aldehyde 425. The aldehyde wasthen converted to a diene under Yamamoto conditions.270 Themixture of olefin isomers could be carried on together, because ofthe undesired isomer being cleared in a subsequent step, in whichit underwent an unanticipated Diels−Alder cycloaddition withDDQ. The primary alcohol was then deprotected and converted

Scheme 64. Smith’s Preparation of Common Precursor 408

Scheme 65. Elaboration of Common Precursor 408 toFragment 411

Scheme 66. Construction of Fragment 415 from Precursor408

Scheme 67. Formation of the Third Fragment 421 fromPrecursor 408



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to primary iodide 429. The iodide was converted to thephosphonium salt under extremely high-pressure conditions.This proved to be necessary because of competing cyclo-pentannulation processes involving the proximal trisubstitutedalkene. The hygroscopic phosphonium salt was then treated withNaHMDS and aldehyde 421 to afford olefin 430. Thehomoallylic p-methoxybenzyl ether was then oxidatively cleavedto provide the free secondary alcohol, which was converted toprimary urethane 431. The tetrasilylated discodermolide wasdeprotected by treatment with dilute HCl to afford discodermo-lide (403).The repeated use of an advanced intermediate in this synthesis

was an excellent strategy that resulted in (relatively) high overallefficiency. Smith and co-workers exploited their route to produceover 1 g of discodermolide, which clearly caught the attention ofchemists at Novartis Pharmaceuticals (vide infra).

7.3. Paterson’s Second-Generation Synthesis

The other route that would eventually influence large-scaleproduction of discodermolide was Paterson’s “second-gener-ation” synthesis.264 Paterson and co-workers have also publishedadditional variations of the second-generation route, and theseare described elsewhere.265 The Paterson route, similarly to theSmith synthesis, utilizes three main fragments, two of which areconstructed 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 treatedwith acetaldehyde to give an intermediate borylated aldolproduct, which was then reduced in situ with LiBH4 to providediol 433.The diol was double-TBS-protected then subjected to a mild

selective deprotection of the more sterically accessible secondaryalcohol to afford 434. The benzyl ether was then hydro-

genolyzed, and the diol was oxidized over several steps to yieldketoester 436, the first fragment.The second fragment was constructed from ethyl (S)-lactate

(437), a chiral pool material (Scheme 70). This material wasconverted to ethyl ketone 438, which was then subjected to aboron-mediated aldol with Roche ester derivative 439 to affordβ-hydroxy ketone 440.271 The alcohol was then protected to giveether 441.The ketone then underwent reduction to an inconsequential

mixture of alcohols followed by cleavage of the benzoate to givean intermediate diol, which was subsequently oxidatively cleavedto afford aldehyde 442. The aldehyde was then subjected to aNozaki−Hiyama−Kishi reaction with allyl silane 443, followedby a base-mediated Peterson olefination to provide the terminal

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

Scheme 69. Paterson’s Route to Fragment 436



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diene moiety.272 The system was then deprotected and oxidizedto 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 withmethacrolein afforded aldol product 447. The ketone wasreduced by internal hydride delivery using Me4HB(OAc)3 toprovide anti-1,3-diol 448. The diol was then converted to

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

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



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dioxane 450 by acetal exchange. The system was oxidized andthen heated with base to initiate a Claisen rearrangement.273 Thiselegant rearrangement created eight-membered unsaturatedlactone 452, which established the trisubstituted olefin that iscentrally located on the discodermolide tail. This lactone wasthen saponified, coupled with a phenol, and protected to affordthe second main fragment ester 455. Ester 455 and aldehyde 445were then combined by aldol chemistry followed by reduction ofthe ester to afford diol 457. The more sterically accessibleprimary alcohol was then converted to a sulfonate, which wasthen reductively cleaved with lithium aluminium hydride (LAH)to afford alcohol 459. The system was then subjected to TBSprotection and DDQ-mediated double PMB deprotection togive diol 460. The primary alcohol was then selectively oxidizedand subjected to Still−Gennari olefination conditions to produceβ-substituted acrylate 462.274 The primary urethane was theninstalled bymeans of Cl3CCONCO followed by basic hydrolysis.The acrylate was then converted to acrolein derivative 464.Fragment 436 was then converted to the (+)-diisopinocam-pheylboryl enolate and combined with 464 to produce aldolproduct 465. These conditions were utilized to overpower theinherent re-face preference of the enolate approach to insteadattack the si-face of the aldehyde, to obtain the correctdiastereomer of 465. With the carbon skeleton of discodermolideintact, all that remained was a reduction of ketone 465,accomplished by an internal hydride delivery, and a tandemglobal deprotection/lactonization facilitated by HF. This processdelivered discodermolide in 27 steps, 48 overall, in 13.6% yield.This approach was used to construct several derivatives forpreliminary SAR studies.

7.4. Novartis’ Hybrid Synthesis

By 1998, Novartis Pharmaceuticals had become enamored withdiscodermolide because of its potent microtubule stabilizationactivity and promising biological profile. It proceeded to licensediscodermolide fromHarbor BranchOceanographic Institute forthe development of a novel anticancer therapeutic and took thestructure into clinical trials.247,275 However, total synthesis wasand remains the only avenue to access meaningful quantities ofthis compound. Therefore, Novartis commissioned StuartMickel and a team of chemists to utilize synthetic efforts toconstruct discodermolide for ongoing clinical trials. Afterextensive examination of all academic endeavors towarddiscodermolide by about 2001, Mickel and his team had troubledeciding between the Smith and Paterson routes. The Smithsynthesis was valued for its high convergency with fragment 408,as well as its ability to deliver a gram of material. The reagent-controlled, chiral boron enolate end game and the superb overallyield of the Paterson synthesis aligned attractively with thestrategy they were contemplating. The inherent practicality ofboth approaches made them both extremely attractive. However,not quite satisfied with the ability of either the Smith or Patersonapproach to effectively be translated to scale, Mickel and his teamof chemists at Novartis proceeded to extract the advantageouscomponents of each, to orchestrate an elegant hybrid of the twostrategies that could be used to obtain meaningful amounts ofmaterial. The initial synthetic plan was to use Smith’s commonintermediate 408, because of its extremely practical nature, toconstruct three main fragments of discodermolide and then tostitch them together in a concise manner (Scheme 72).275

Mickel and co-workers embarked on their hybrid synthesiswith Roche ester and planned to convert it into Smith’s commonintermediate 408. Roche ester was converted to the PMB-

protected derivative and subjected to reduction with LiBH4,which was much more amenable to scale than LAH, to affordalcohol 404. The alcohol was then converted to unstablealdehyde 405 by a catalytic 2,2,6,6-tetramethylpiperidin-1-oxyl(TEMPO)/stoichiometric bleach oxidation. The aldehyde wasthen subjected to an Evans aldol reaction with oxazolidinone 233to give alcohol 468. At this point, a departure was needed fromthe Smith route because the pyrophoric nature of AlMe3 made itunsuitable for large-scale plant use.275 Mickel and co-workerscleaved the oxazolidinone with a basic peroxide solution followedby acidification and recrystallization with (R)-1-phenylethyl-amine, which was the first purification of the sequence. The acidwas then liberated and coupled to N,O-dimethylhydroxylamineby means of an in situ-generated mixed anhydride. This processwas used to produce almost 29 kg of key precursor 408 in onebatch.Elaboration to the first discodermolide fragment was initiated

by TBS protection of 408 followed by partial reduction of theWeinreb amide moiety to aldehyde 413 (Scheme 73).276 One ofthe more problematic reactions of the route was the conversionof aldehyde 413 to the vinyl iodide 415. A variety of methods andprocedures were attempted to improve the yield of this reaction

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

Scheme 73. Novartis’ Conversion of Precursor 408 toFragment 415



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at scale to no avail, and researchers were forced to leave this stepas 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 affordalcohol 471 (Scheme 74). This system had a propensity to

lactonize, so handling was minimized by isolating theintermediate alcohol as a solution in t-BuOH. The alcohol wasimmediately oxidized using a TEMPO/diacetoxy iodobenzenesystem. The resultant aldehyde was then subjected to MeMgBrfollowed by an immediate oxidation to avoid lactonization toprovide methyl ketone 473 in kilogram batches.The final main fragment was elaborated from 408 by first

oxidizing p-methoxybenzyl ether under anhydrous conditions toafford dioxane 474 after recrystallization (Scheme 75).277 TheWeinreb amide was then reduced with LAH to afford aldehyde409, which was then subjected to an Evans aldol reaction toafford β-hydroxyimide 410 after recrystallization. The systemwas then TBS-protected and subjected to reductive cleavage ofthe chiral auxiliary, after which a chromatographic purification

afforded alcohol 475. The alcohol was then converted to light-sensitive 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-workersfound that Smith’s Negishi coupling of vinyl iodide 415 andprimary iodide 411 was problematic at a large scale, leading toseveral side products. However, crossing over into a Suzukimanifold similar to Marshall’s approach resulted in a cleanerreaction profile, and recrystallization afforded olefin 423.261 TheNovartis approach thus transitioned from Smith’s strategy to aPaterson-type end game. Reductive cleavage of the p-methoxybenzylidene acetal followed by oxidation of the newlyliberated primary alcohol afforded aldehyde 447. This aldehydewas then subjected to the two-step, one-pot Nozaki−Hiyama−Kishi allylation/Peterson olefination process that was developedby Paterson, with a minor modification of KOH as the base overKH for ease of operation, to provide diene 478. Oxidativecleavage of the two PMB protecting groups followed by aselective oxidation of the primary alcohol with a TEMPO/diacetoxy iodobenzene procedure provided aldehyde 479. Thealdehyde was then subjected to the Still−Gennari modification ofthe Horner−Wadsworth−Emmons reaction to provide ester462, an intermediate in the Paterson synthesis.The primary urethane was then installed by conventional

means, and the ester was converted to aldehyde 464. The lastfragment was incorporated into the system by means of the late-stage Paterson chiral boron enolate aldol reaction.279 Unfortu-nately, this step proved to be extremely problematic at scale. Thesolid (+)-B-chlorodiisopinocampheylborane proved to bedifficult to handle at scale and unstable. This problem could beremedied by utilizing a commercial hexane solution of thiscompound. However, these modified conditions required a largeexcess (6.6 equiv) of fragment 473 for the reaction to proceed inviable yields. Several other issues with this particular step almostcaused the Novartis campaign to end in failure.279 Fortunately, aworkable procedure emerged, and the Novartis team pushedforward. The next step was an internal hydride delivery todiastereoselectively 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 con-ditions: Aqueous HCl was added in portions, and the walls of thereaction vessel were washed with MeOH to prevent oiling out ofvarious intermediates. Discodermolide was obtained afterchromatography and recrystallization. TheNovartis constructionof discodermolide required 26 steps in the longest route, 34overall, in 0.9% yield. This route produced more than 60 g ofdiscodermolide for use in clinical investigations.Although this route was successful in delivering discodermo-

lide, it is not without issues that would need to be addressed toestablish a routine process. The construction of vinyl iodide 415would have to be optimized, and the arduous late-stage boronaldol reaction would need to be modified. In addition, the lowyield of the route would be a huge problem in the later stages ofdevelopment and would not be practical for long-termproduction. With the synthetic supply temporarily addressed,phase I clinical investigations of discodermolide in patients withsolid tumors started well, with no obvious dose-limiting toxicitiesand no signs of neuropathy.243,280 However, the trial had to bediscontinued as a result of significant pulmonary toxicitydeveloping 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 ofdiscodermolide analogues or discodermolide−dictyostatin hy-

Scheme 74. Construction of Fragment 473

Scheme 75. Novartis’ Preparation of Fragment 411



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brids to determine whether the harmful side effects ofdiscodermolide can be attenuated.283−286

Discodermolide synthesis presents a particularly acutechallenge to methods for acyclic diastereocontrol. Smith andPaterson’s multiply convergent strategies that relied uponrepetitive use of flexible raw materials were exquisitely designedand highly effective. These academic forays set the stage for aremarkable campaign of refinement and scale-up at Novartis.While these efforts faced significant challenges in handling andpurifying diastereomeric mixtures at multiple points, the resultwas nonetheless a remarkable achievement that was able toassemble a dauntingly complex structure on sufficient scale todrive human clinical trials.

8. DIAZONAMIDE A

8.1. Background

In the late 1980s, Fenical and co-workers extracted four relatedantimitotic agents from the colonial ascidian Diazona angulatacollected off the coast of the Phillipines.287 The molecules,named diazonamides A−D, were purified to homogeneity onmilligram scales. After extensive spectroscopic analyses, it wasevident a new type of polyheterocyclic structure had beendiscovered; yet a complete assignment for any of the foursubstances remained elusive. Eventually, a p-bromobenzamidederivative of diazonamide B was coaxed to crystallize in a formamenable to X-ray diffraction, and data were collected by Clardyand Van Duyne at Cornell University, who assigned its structureas the polycyclic diarylacetal 482 (see Figure 22).287 Fenical and

co-workers then interpreted this result in a way they felt bestexplained the entirety of their spectroscopic analyses anddesignated diazonamides A−D as 483−486, wherein the coreacetal existed in hydrated form and the C2 amine was either freeor conjugated to L-valine through either an amidyl or amidinyllinkage.288 In addition to being a beautifully intricate structure,diazonamide A possessed potent cytostatic activity towardshuman cancer cell lines.287

Disclosure of these structures immediately captured theattention of the synthetic community. By the end of the 1990s,nearly a dozen groups were attempting to prepare diazonamidesin the laboratory.289,290 These efforts were confronted with a veryunexpected finding when, in late 2001, Harran and co-workerscompleted the first synthesis of 483 only to discover that it wasnot the natural product.291,292 In fact, synthetic 483 proveddifficult to handle and characterize because of its faciledegradation.Careful re-examination of spectroscopic data collected on

diazonamides as well as reinterpretation of the crystallographicdata used to assign structure 482 led to the conclusion that thenatural products were, in fact, 487 and 488.292,293 The phenolichemiacetals thought to be present in natural diazonamides wereactually diaryl aminal motifs, and as a compensatory change,diazonamide A contained a hydroxyl group on its side chainrather than an amine. These assignments were fully consistentwith available data and also had the satisfying feature of showinghow diazonamides were uniformly peptide-derived, rather than ahybrid of peptidyl and type II polyketide origin that one

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



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seemingly needed to invoke for the initial assignments to becorrect.292

Following the reports by Harran and co-workers, syntheticefforts shifted toward the new targets. The Nicolaou groupadapted their ongoing route to complete the synthesis of487.294,295 This same group established a second pathway todiazonamide A some time later.296 Interest in diazonamidesynthesis has continued unabated over the years.297−301 Manylaboratories have made important and creative contributions tothe area. These efforts have been reviewed.290 The tactics mostpertinent here, namely towards the clinical development ofdiazonamides as anticancer drugs, are those first outlined inHarran and co-workers’ total synthesis of diazonamide Apublished in 2003.302

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 arelated manner. The approach was conceptually simple andconsisted of assembling a seco-peptidyl precursor and installingthe central diarylaminal motif by direct oxidative annulation oftethered 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 toafford dipeptide 491 as an inconsequential mixture ofdiastereomers.305 This material was subjected to Yonemitsuoxidation with DDQ to produce a single 3-oxazolylindoleintermediate, which was subsequently treated with HBr in AcOHto afford amine salt 492.306 This material was then acylated withnosyl-protected tyrosine to afford seco precursor 493.

At this stage, the full constituency of the western region waspresent, being accessed by simple peptide couplings and abenzylic oxidation. What remained was to oxidatively form themacrocycle, which was achieved by subjecting the molecule toKita oxidation conditions using iodobenzene diacetate. Theremarkable transformation that followed involved oxidation ofthe tyrosine moiety, capture of the incipient phenoxenium ion bythe pendant indole, and ring closure to aminal 494. This processinstalled the challenging C10 quaternary center early in thesynthesis and afforded macrocycle 494 as a 3:1 mixture ofdiastereomers favoring the desired isomer. Although the yield of494 was less than ideal because of competing spirocyclohex-adienone formations, the reaction was uniquely enabling. Itconverted a modified linear peptide into an advanced polycyclein a single operation. Intermediate 494 was then subjected to aprotecting-group exchange and saponification to provide acid495. The acid was then coupled to 7-hydroxytryptamine (496)to provide amide 497. Acetylation followed by a two-stepbenzylic oxidation/cyclodehydration provided bis-oxazole 498.At that point, the stage was set to form the eastern macrocycle ofdiazonamide A. Harran and co-workers had discovered duringtheir synthesis of the initially proposed diazonamide A structurethat difficulties with atropisomerism in the eastern macrocycleformation could be subverted by the presence of an establishedwestern macrocycle.291,302 This discernment served them wellwhen 498 was irradiated under basic conditions to afford bicycle499 in high yield. The irradiation initiated an electron transferbetween the two indole moieties, leading to a radical ion pair,which underwent lithium bromide elimination followed byradical recombination and tautomerization to afford the targetdiazonamide core 499.302,307,308 The added phenol wassubsequently reduced through its triflate derivative. The aminalnitrogen was then protected as the (trimethylsilyl)-ethoxycarbonyl (Teoc) derivative. The western macrocycle wasthen twice chlorinated by the action of perchloro-2,4-cyclo-hexadien-1-one (503) and subjected to global deprotection toafford des-bromo diazonamide B 504.309 Final appending of (S)-α-hydroxy isovaleric acid (505) to the core produceddiazonamide A. This approach to diazonamide A (487)completes the molecule in 19 steps and 1% yield.

8.3. Development of DZ-2384

Harran and co-workers wasted no time and began to explore themode of action of the diazonamides and also to constructdiazonamide analogues.310−313 As indicated above, diazonamideshowed potent antiproliferative activity in human cancer celllines. However, the cellular target of diazonamide had beenelusive. Early studies implicated microtubules as a potentialtarget, whereas others have indicated no direct interaction.311,314

The target of diazonamide A has also been tracked to themitochondrial enzyme ornithine δ-amino transferase, which,surprisingly, has a role in regulatingmitotic cell division in humancancer cells.311,312 Although the target remained unknown,diazonamide treatment of human tumor xenografts in nude miceshowed regression with little evidence of overt toxicity.311,312

This lack of toxicity was an exciting result, which led to thedevelopment of Joyant Pharmaceuticals, which continued thedevelopment of diazonamide SAR utilizing Harran’s route toconstruct analogues.313,315−318

It was determined that the eastern macrocycle was notnecessary for activity, nor was the ansa-bridged indole moiety.318

In terms of the northern bisoxazole motif, a large number ofoxazole−heterocycle combinations were explored.318 However,

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



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the best system retained the native bisoxazole moiety appendedwith a primary carbinol. In addition, conversion of thenorthernmost isopropyl group to a tert-butyl group andinstallation of a fluorine atom on the southern indoline ringimproved the pharmacokinetic profile of the system. Theproduct of these extensive investigations was the structuredesignated as DZ-2384 (522, Scheme 78), which was chosen asthe development candidate in 2012.318,319

The same general approach of oxidatively modifying apolypeptide was developed to supply DZ-2384 for preclinicaldevelopment and the upcoming clinical investigations. Thesequence began with L-tert-leucine (506, Scheme 78).319

Derivatization with carboxybenzyl chloride (Cbz-Cl) followedby a conventional coupling with L-serine methyl ester andsaponification afforded dipeptide 509. The serine moiety wasthen activated in the presence of 5-fluoroindole 510 to affordtryptophan derivative 511. Another standard coupling of L-serinemethyl ester afforded tripeptide 512. This material was thensubjected to a sequence of oxidations. First, a DDQ-mediated

oxidation of the system produced oxazolylindole 513. Sub-sequently, a one-pot cyclodehydration with Deoxo-Fluor (514)produced oxazoline 515, which was then oxidized in situ under amodified variant of Wipf and William’s conditions to providebisoxazole 516.320,321The oxidation product was then depro-tected by treatment with strong acid to afford amine salt 517. Theamine was then acylated with Boc-Tyr-OH under standardcoupling conditions to afford 518, which thereafter wasdeprotected 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 thependant ester to afford seco precursor 521. The final step wouldbe akin to the Kita oxidation that Harran and co-workers used inthe initial diazonamide A synthesis. However, this operationproved to be a bottleneck during SAR investigations, where sideproducts complicated the purification and lowered yields. Harranand the chemists at Joyant Pharmaceuticals searched for a meansto eliminate byproducts inherent to the Kita protocol.319 Theydiscovered a unique solution using electrochemistry wherein the

Scheme 77. Harran’s Construction of Diazonamide A



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substrate was controllably oxidized at an anode surface atconstant potential. This allowed for an indolic oxidation (ratherthan a phenolic oxidation) to form a radical cation that capturedthe pendant phenol and then underwent internal cyclization andoxidation to give diazonamide macrolactams free fromspirocyclohexadienone contaminants.313 This beautiful processwas 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 inves-

tigations. Studies with subcutaneous xenograft models (includingpancreatic ductal adenocarcinoma) revealed that DZ-2384caused complete regression within 3 months of treatment.322

In addition, DZ-2384 administration to a model for metastictriple negative breast cancer (MDA-MB-231-LM2) in immuno-compromised mice caused regression of all metastases at thelowest dose tested.322 An analysis of the safety profile for DZ-2384 indicated that it has a much wider safety margin thanvinorelbine. Cumulative peripheral neuropathy is a major toxicityissue for many microtubule-targeting agents. An examination ofextended DZ-2384 treatment on nerve function revealed that ithad no electrophysiological or microscopic signs of peripheralneuropathy at circulating concentrations 13 times higher thanthose needed for antitumor activity in mice.322 Neurotoxicity was

observed only upon extended treatments at the maximumtolerated dose, but this resolved following a recovery period. Thislack of toxicity and the wide safety margin led to the hypothesisthat these characteristics were derived from DZ-2384’sinteraction with its receptor.A genome-wide RNA interference screen allowed Diazon

Pharmaceuticals to identify the cell cycle and mitosis as potentialtargets for DZ-2384’s mechanism of action.322 H1299 cellstreated with DZ-2384 accumulated in the G2-M phase of the cellcycle.322 Further investigations revealed that preincubatingtubulin with vinorelbine led to an inhibition of binding tobiotinylated DZ-2384 surfaces in a dose-responsive manner,which suggested that DZ-2384 interacted with the Vincadomain.322 X-ray crystallographic analysis of DZ-2384 boundto a tubulin complex (specifically two αβ-tubulin dimers, namely,stathmin-like protein RB3 and tubulin tyrosine ligase, abbre-viated as T2R-TTL) indicated that DZ-2384 does bind to theVinca domain, but in a manner that is unique.322 Closerinvestigations of the binding mode and a superimposed atomicmodel with vinblastine revealed that the two structures sharesimilar binding interactions at the interdimer interface.322

However, DZ-2384 lacks the bulky catharanthine moiety thatis characteristic of the vinca alkaloids, allowing the interdimerinterface to be more compact than both the apo- and vinblastine-bound T2R-TTL complexes.322 This change caused a large shift

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



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in the orientation of the two tubulin dimers.322 Furtherinvestigations with helical superassemblies of T2R-TTL com-plexes indicated that the addition of DZ-2384 increased the radii,leading to the hypothesis that DZ-2384 straightens protofila-ments.322

The ability of DZ-2384 to interrupt the cell cycle coupled withits safety profile suggested that sensitivity to the drug wasincreased in molecules actively undergoing mitosis.322 Micro-tubule sedimentation assays on H1299 cells revealed that DZ-2384-treated cells retained higher concentrations of microtubulepolymer mass than cells treated with vinorelbine.322 Furtherstudies with HeLa cells synchronized in late G2 showed stuntedmicrotubules and misaligned chromosomes.322 These resultsindicate that the cytoskeletons of interphase cells are not assusceptible to DZ-2384-mediated apoptosis as those activelyundergoing apoptosis.322 To determine how DZ-2384 affects thedynamic instability of microtubules, Diazon Pharmaceuticalsanalyzed growing ends of microtubules in U-2 OS (osteosarco-ma) cells. It was determined that DZ-2384 decreased the numberof microtubule growths from the centrosome and theirfrequency.322 In addition, DZ-2384-treated cells, when com-pared to vinorelbine-treated cells, had an increased rescuefrequency relative to the controls.322 These studies indicated thatDZ-2384 slows microtubule growth, but allows for a sufficientamount of rescue events to maintain polymeric tubulin ininterphase cells.322

The journey to DZ-2384 has been intensely interestingandfull of twists and turns. Moreover, the enabling syntheticmethods advanced by Harran and Joyant/Diazon Pharmaceut-icals now permit the construction of diazonamide structures in analmost trivial manner. Linear peptidyl motifs are directly oxidizedat a graphite surface to give structurally complex polyhetero-cycles at room temperature. Extensive exploration of thediazonamide pharmacophore has produced DZ-2384, which ispoised to enter clinical trials as a next-generation antimitotic withan unprecedented safety profile. The coming years will be a trulyexciting time for the diazonamide project.

9. INGENOL 3-ANGELATE

9.1. Discovery and Development

In the late 1960s, Hecker purified several new compounds fromvarious plants of the Euphorbia genus.323 One of the moleculesisolated showed strong phorbol-like tumorigenic activity. Whenthis compound was saponified, hexadecanoic acid and a complexalcohol were produced.323 The identity of the latter, namedingenol, remained a puzzle until it was resolved by X-raycrystallographic analysis two years later.324

Ingenol’s structure revealed a fascinating trans-bridgedbicyclo[4.4.1]undecane ring system (aka in/out configured)that introduces significant strain into the system (Figure23).325,326 Numerous biological activities have been ascribed toingenol and its derivatives since their discovery.325−327 Amongthese, the antiproliferative nature of the 3-angelate ester hasproved to be most valuable in medicinal terms.326,327 Ingenol 3-angelate can be isolated from several Euphorbia species, whichhave a long history of use as traditional topical treatments.328

Angelate 525 proved to be effective against a wide range oftumor cell lines, inducing mitochondrial swelling and necrosis,and when administered topically, it was found to be able toregress tumor xenografts in rodent models.328,329 The activity525 is thought to derive from perturbation of protein kinase Csignaling, resulting in antiproliferative and proapoptotic

effects.330 Notably, ingenol 3-angelate is easily degraded byesterases to reveal ingenol, making systemic administrationdifficult.326 Nevertheless, ingenol 3-angelate has advanced toclinical trials as a topical agent and is ab effective therapy foractinic keratosis and basal cell carcinoma.331,332 In fact, the FDAapproved 525 as a first-in-class molecular entity for the topicaltreatment for actinic keratosis.326,327,333 Leo Pharma developedingenol 3-angelate as a pharmaceutical candidate andmarkets thedrug as Picato.334−341

The supply of ingenol 3-angelate for all preclinical studies andclinical trials was produced either from natural sources orthrough derivatization from ingenol.342−344 Although theseroutes were firmly established at the time of FDA approval,they were laborious, costly, and inefficient, which hamperedprogress in terms of practicality.326,327 To potentially addresssome of these issues and to greatly expand access to analoguesand structural variants, Leo Pharma began a collaboration withthe Baran laboratory at the Scripps Research Institute to evaluatefully synthetic materials.326,327 One goal of this collaboration wasto develop a practical total synthesis of 525 that was cost-effectiveand 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 ofthe Leo Pharma and Baran laboratory collaboration (Scheme79).345−348 These works proved invaluable to the Baranlaboratory for exploring the reactivity of various ingenane-typeframeworks, information that would be exploited in their ownsynthesis.The first synthesis was established by Winkler et al., who

employed a spectacular de Mayo-type photocycloaddition−retroaldol fragmentation cascade to establish the trans-bridgedbicycle.345 Shortly thereafter, Tanino, Kuwajima, and co-workersreported an approach based on a Nicholas ion-mediatedalkylative cyclization/semipinacolic rearrangement sequence.346

The elegant work of Funk and co-workers toward an asymmetricentry to the ingenane skeleton proved invaluable for the efforts ofKigoshi and Wood, who simultaneously explored similar routesto ingenol.349,350 Utilizing Funk and co-workers’ intermediate535 derived from (+)-3-carene, Kigoshi and co-workersestablished the first asymmetric entry to the ingenol ringsystem.348 Kigoshi and co-workers utilized ring-closing meta-thesis to construct the B ring, demonstrating its utility inproducing strained carboskeletons, which after subsequentoxidation intercepted aldehyde 537 from Winkler’s synthesis.348

Shortly after Kigoshi and co-workers’ reports, Wood and co-workers established a synthesis of ingenol also employing ring-closing metathesis for B ring formation on functionalized dieneprecursor 538.347,351 In addition to these advanced efforts, therehave been numerous creative syntheses of ingenol-type carbonframeworks.352,353

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



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

Baran and co-workers carefully evaluated previous approaches toidentify both their advantages and their drawbacks. They weredrawn to the approaches of Tanino−Kuwajima and Cha andEpstein, which utilized semipinacol rearrangements to constructthe ingenane skeleton from a tigliane core.353 It was thought thatthis transformation might be related to the biosynthesis ofingenol, wherein a pinacol rearrangement has been postulated. Inaddition, Baran and co-workers sought a preparation that couldbe dissected into cyclase and oxidase “phases”, analogous to the

biosynthetic stages leading to oxygenated polycyclic hydro-carbons in general.Similarly to Funk, Kigoshi, and Wood, Baran chose to begin

the synthesis of ingenol with (+)-3-carene (534), an abundantoptically active monoterpene (Scheme 80). This choice allowedBaran and co-workers to begin with a molecular topographyuseful for establishing further stereocenters in a controlledmanner. (+)-3-Carene was subjected to an allylic chlorinationfollowed by ozonolysis of the transposed alkene to afford ketone541. The α-chloroketone could then be reductively methylatedby sequential treatment with lithium naphthalenide and methyliodide. Isolation of this sensitive intermediate ketone proveddifficult. However, it was discovered that, after methylation, insitu treatment with lithium hexamethyldisilazide (LiHMDS)followed by aldehyde 542 afforded adduct 543 as a singlediastereomer. 542was constructed from propargyl alcohol (557)and triethyl orthopropionate (558) by a Claisen rearrangementfollowed by reduction with LAH to afford racemic alcohol 560(Scheme 81).354 The alcohol was then subjected to a lipase-mediated transesterification using vinyl acetate, which providedthe desired (R)-alcohol in 98.7% ee.354 The alcohol was thenoxidized 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 10:1mixture of diastereomers, which could be separated after doublesilylation to provide crystalline 545. This material was thensubjected to an allene-yne Pauson−Khand cyclization underhigh-dilution conditions, which constructed the tetracyclictigliane skeleton 546.This key process markedly increased the complexity of the

system in a single step that was both elegant and practical. Theresultant ketone was then treated with methylmagnesiumbromide to afford tertiary alcohol 547. This procedure markedthe end point of the “cyclase phase”, because all carbons for theingenol framework were in place.The synthesis then transitioned into the “oxidase phase”,

where the periphery of the ingenol framework was waoxidativelydecorated.326,327 The first step was a dihydroxylation of thetrisubstituted olefin in the southern portion of the molecule. Thehindered nature of the olefin precluded catalytic dihydroxyla-tions, but stoichiometric dioxoosmylation was successful.327 Thediol subsequently formed by hydrolysis was then masked as itscarbonate. The system was now poised for a vinylogous pinacolrearrangement, which was accomplished by treatment with BF3at low temperature followed by gentle warming and quenchingwith Et3N/MeOH. Structure 550 proved to be a kinetic product.Resubjection at warmer temperatures resulted in a retro-pinacolrearrangement to give elimination product 562 (Scheme 82).The oxidase phase continued with a SeO2-mediated allylicoxidation followed by in situ acetylation to afford acetate 551.The structure was then deprotected and dehydrated andsubjected to a basic workup to cleave the carbonate to affordolefin 554. This system was subjected to an additional allylicoxidation with SeO2 to provide ingenol.To convert ingenol into Picato, Leo Pharma devised the

following route: Ingenol was derivatized as the crystallineacetonide 555 according to conditions developed by Heck-er.342,355 555 was then deprotonated with LiHMDS and treatedwith angelic anhydride to afford ester 556.342 The acetonide wasthen cleaved by treatment with phosphoric acid, and the crudematerial was recrystallized to afford ingenol 3-angelate. Thisprocess constructs Picato in a longest linear sequence of 19 steps,

Scheme 79. Summary of Previous Synthetic Constructions ofIngenol



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21 steps total, with an overall yield of 1.9% from propargylalcohol.With a concise synthetic route established to ingenol, Leo

Pharma wasted no time in exploiting its investment. Thissynthesis was utilized to construct a variety of analogues thatallowed for a more thorough investigation of Picato’s mode of

action.356 In addition to the construction of analogues, LeoPharma scaled up the synthesis and has made more than 1 kg ofingenol by the Baran route.357,358 Although this route iswonderfully elegant, some steps remain problematic on a processscale, 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 byPhyton Biotech to establish a more efficient fermentationprotocol of ingenol 3-angelate using plant-cell-culture-basedtechnology.359,360

The ingenol story is clearly far from complete. Extensive SARstudies and core structural modifications are ongoing. Hopefully,further insight into the biological mechanism of action couldprovide a rational basis for designing new derivatives for systemicadministration. All of this work is being made possible by Baran’shighly enabling and concise new synthesis of ingenol.

10. CONCLUSIONS

In this review, we have described remarkable efforts to convertcomplex synthetic problems into manageable chemical processesamenable to manufacturing scales. Whether or not the solutionspresented reach a level one could term practical is certainly openfor debate and undoubtedly influenced by cost-of-goods, processtimelines, and market size/demand, data that were not readilyavailable to us. However, in terms of synthetic strategy,traditional hurdles are amplified. By that we mean that, withoutexception, what makes the structures discussed particularlychallenging are their stereochemical features and the need toinstall multiple contiguous asymmetric centers with exquisite

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

Scheme 81. Construction of Aldehyde 542

Scheme 82. Formation of the More Stable EliminationProduct by Resubjection to Vinylogous Pinacol Conditions ata Higher Temperature



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control while avoiding long linear sequences in the process.Convergency remains king, in this regard, and the execution ofhigh-yielding, flexible fragment couplings are powerfully enablingstrategies. There is definitely room for new discoveries in thisarea. As stated at the outset, it is unlikely that de novo synthesiswill supplant semisynthetic methods when a biological source ofadvanced rawmaterial is available. However, in those cases wheresuch a source is not available or where deep-seated structuralalterations of a natural-product lead are sought, we look forwardto ever more exciting, ambitious, and refined synthesiscampaigns. Our field has come a long waybut there is still somuch farther to go.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Notes

The authors declare the following competing financialinterest(s): PGH is a founder and shareholder in DiazonPharmaceuticals, which is attempting to commercialize DZ-2384.

Biographies

Tyler K. Allred obtained his B.S. in chemistry in 2012 at the University ofCalifornia, Davis, where he synthesized designed guanosine analogues toinvestigate their effect on the MUTY/MutY DNA repair pathway withProfessor Sheila David. In 2013, Allred joined Professor Patrick Harran’sgroup at the University of California, Los Angeles, where his research isfocused on the synthesis of complex marine-derived alkaloids.

FrancescoManoni obtained his B.S. andM.S. degrees in chemistry at theUniversity 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 enantiose-lective addition of enolizable cyclic anhydrides to various electrophilestowards the formation of bioactive compounds. During this time, he alsocollaborated on other projects, including the organocatalytic dynamickinetic resolution of azlactones paired with a ligation-inspired couplingprocess and the development of the first catalytic thiolate-catalyzedcross-Tishchenko reaction between aldehydes and ketones. In 2014, hejoined Professor Patrick Harran’s group at the University of California,Los Angeles, where his postdoctoral research has resulted in a uniquetotal synthesis of the marine-derived macrolide callyspongiolide.

Professor Patrick Harran received his B.A. degree from SkidmoreCollege in New York. He obtained a Ph.D. from Yale University in 1995(with F. E. Zeigler) and completed an NIH-sponsored postdoctoralfellowship at Stanford University (with P. A. Wender) in 1997. That fall,he joined the faculty at the University of Texas Southwestern MedicalCenter. In 2005, he was promoted to Full Professor and named the MarNell & F. Andrew Bell Distinguished Chair in Biochemistry. He joinedthe faculty at the University of California, Los Angeles, in July 2008 asthe inaugural D. J. and J. M. Cram Chair in Organic Chemistry.

ACKNOWLEDGMENTS

This work was supported by the Donald J. & Jane M. CramEndowment, a National Institutes of Health/National CancerInstitute grant (R01CA184772-01) and a postdoctoral fellow-ship provided to F.M. by the Human Frontier Science Program.

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