from micrograms to grams: scale-up synthesis of eribulin mesylate
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From micrograms
Fig. 1 The chemical structures of eribuli
Eisai Inc., Andover, MA, USA. E-mail: Melvi
Cite this: Nat. Prod. Rep., 2013, 30,1158
Received 30th May 2013
DOI: 10.1039/c3np70051h
www.rsc.org/npr
1158 | Nat. Prod. Rep., 2013, 30, 115
to grams: scale-up synthesis oferibulin mesylate
Melvin J. Yu,* Wanjun Zheng and Boris M. Seletsky
Covering: 1993 to 2002
The synthesis of eribulin mesylate from microgram to multi-gram scale is described in this Highlight. Key
coupling reactions include formation of the C30a to C1 carbon–carbon bond and macrocyclic ring
closure through an intramolecular Nozaki–Hiyama–Kishi reaction.
1 Introduction
Eribulin mesylate (Halaven�, 1, Fig. 1) is a rst-in-class, non-taxane microtubule dynamics inhibitor that is currentlyapproved for clinical use in over 40 countries (including Japan,the USA, and the European Union) for treatment of certainpatients with late-stage metastatic breast cancer. To date, thisagent is the only chemotherapeutic drug to have demonstratedan increase in overall survival in this patient population.1
Inspired by the marine natural product halichondrin B (HB, 2,Fig. 1),2 eribulin is a macrocyclic ketone derivative3 that repre-sents the most structurally complex, non-peptidic and fullysynthetic drug on the market today. For example, nineteen ofthe thirty-six atoms that comprise its carbon skeleton are
n mesylate (1) and halichondrin B (2).
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stereogenic, rendering the molecule rich in stereochemical andarchitectural features.
Preclinically, eribulin mesylate is a potent antimitotic agentthat exhibits considerable in vivo anticancer efficacy against awide variety of human tumor xenogra models in nude mice.Eribulin mesylate has a mechanistically novel mode of actioninvolving high affinity binding to a small number of sites atmicrotubule plus ends. As a result, it blocks the growth phase ofmicrotubule dynamics, leading to irreversible mitotic arrest andcell death by apoptosis. Importantly, both preclinical and clin-ical studies indicate that eribulin mesylate has a reducedpotential for severe peripheral neuropathy relative to severalother anticancer drugs in this mechanistic class. Of the nearly200 analogues synthesized in connection with this program,eribulin emerged as the most promising candidate for clinicaldevelopment based on its remarkable in vitro and in vivo bio-logical prole. However, as the program advanced increasingamounts of material were needed to support downstreamresearch activities.
While the challenges associated with producing chemicallycomplex molecules through total synthesis on a commercialscale are signicant, they are not insurmountable. Nevertheless,the pharmaceutical industry's acceptance of structurallycomplex natural products or natural product derivativessupplied commercially through total synthesis has historicallybeen low for a variety of reasons, but the situation is changing.For example, the supply problem has been solved through semi-synthesis (e.g., Taxol�, Yondelis�, Ixempra�) for an increasingnumber of synthetically challenging drugs that are currently onthe market. With respect to total synthesis, however, the supplyof an active pharmaceutical ingredient (API) with halichon-drin's structural and stereochemical complexity on a commer-cial scale was unprecedented and currently stands alone. It tooklong-term vision from upper management, unwaveringcommitment from individuals, and teamwork across disci-plines as well as geographical distances to push the halichon-drin project from initiation through early discovery, into clinical
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development and nally onto the marketplace. As more ster-eochemically complex leads (e.g., via natural product, DOS4,5 orBIOS6 strategies, for example) nd their way into the discoverypipeline and eventually enter clinical development (e.g., Apli-din�), the number of approved APIs with increasingly complexstructures supplied through total synthesis may be expected torise in the years to come.
At the time the halichondrin program was initiated atEisai there were a number of natural products known tointeract with tubulin at the vinca domain.7 Halichondrin Bstood out, however, as the only one that was both reported toexhibit salutary effects in an in vivo cancer model2 andpossess a solution8 to the material supply problem that wouldallow structure optimization to proceed in a rational andhypothesis-driven manner. The decision to pursue thisparticular class of marine natural product as the inspirationfor a drug discovery program was made based on syntheticand biological knowledge available at the time balanced
Wanjun Zheng received a Ph.D.in organic chemistry fromWesleyan University in 1994under the direction of ProfessorPeter A. Jacobi working onsynthetic methodology develop-ment and its application innatural product synthesis. Hespent over two years as a post-doctoral research fellow in Har-vard University under ProfessorYoshito Kishi working on thecomplete structure determina-
tion of maitotoxin. He joined Eisai in 1996 and has since beencontributing and leading many drug discovery projects includingproject in the discovery of Halaven�.
Melvin Yu received his B.S. fromMIT, and both his M.A. and Ph.D.degrees from Harvard Universitywhile studying under ProfessorYoshito Kishi. In 1985, he joinedEli Lilly, and in 1993 he relocatedto Eisai Inc. where he led thechemistry team that discoveredHalaven�. He was then respon-sible for the initial route ndingand synthesis scale-up effort thatultimately provided the rstmulti-gram batch of eribulin
mesylate. Mel retains a strong interest in natural products as theinspiration of new chemotherapeutic agents, and in this contextrecently expanded his area of research to include cheminformaticsand compound library design.
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against future chemical uncertainty and expectations. Thediscovery of eribulin mesylate has recently been reviewedwhere details regarding the optimization strategy can befound.9,10 This paper describes the path from initial synthesison microgram scale to the rst multigram scale-up campaignof eribulin mesylate, highlighting the challenges and riskstaken along the way.
2 Milligram scale synthesis
Two of the three key fragments (C1–C13 and C14–C26, hal-ichondrin numbering) for the synthesis of eribulin are basedupon intermediates used in the synthesis of HB that weredeveloped by the Kishi group and reported in 1992.8 Synthesisof one key fragment (C27–C35) and the nal assembly strategy,however, had to be modied from the Kishi route to accom-modate formation of a macrocyclic ketone in place of themacrolactone moiety.
The rst synthesis of the C27–C35 fragment proceededfrom stock intermediates that were already in place to supportthe preparation of macrolactone analogues that were previ-ously targeted in the program (Fig. 2).11,12 In this regard, stockintermediate 3 prepared from L-arabinose in 9 steps was con-verted to the C30a modied derivative 4 in 14 synthetictransformations (Fig. 2). In total, this particular route tocompound 4 required 23 steps from commercial startingmaterial and involved an unfavorable isomer mixture that wasdifficult to separate early in the sequence. Nevertheless, with
Fig. 2 The conversion of stock intermediate 3 to C27–C35 fragment 4.
Boris M. Seletsky earned his PhDin 1987 from Shemyakin Insti-tute of Bioorganic Chemistry inMoscow, Russia working on newmethods in steroid synthesisunder direction of Dr GeorgeSegal and Professor Igor Torgov.Aer several years of naturalproduct research at the sameInstitute, he moved on to post-doctoral studies in stereo-selective synthesis withProfessor Wolfgang Oppolzer at
the University of Geneva, Switzerland, and Professor James A.Marshall at the University of South Carolina. Boris joined Eisai in1994, and has contributed to many oncology drug discoveryprojects with considerable focus on natural products as chemicalleads, culminating in the discovery of Halaven�.
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Fig. 3 The key coupling reaction in the original synthesis of eribulin (X ¼ I).
Fig. 4 The starting materials for synthesis of the C14–C35 fragment 5.
Fig. 5 The synthesis of the C31–C.35 and C27–C30a subfragments.
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an overall yield of 1.3%, the route was sufficient as a startingpoint for accessing eribulin and other C1 ketone analoguesduring the discovery phase.
To accommodate the C1 ketone, coupling and cyclization ofthe two major C1–C13 and C14–C35 fragments (Fig. 3) wereenvisioned to take place in reverse order from that of the orig-inal synthesis of HB reported by Kishi. Whereas synthesis of themacrolactone analogue series involved coupling the two frag-ments at C13 and C14 followed by a Yamaguchi macro-lactonization reaction at C1, coupling of the fragments in theketone series was envisioned to rst occur between C30a andC1. Closure of the macrocyclic ketone ring was then planned totake place between C13 and C14 through an intramolecularNozaki–Hiyama–Kishi reaction.
Using this strategy, the rst total synthesis of eribulinafforded 600 mg of nal material, which was sufficient tosupport chemical characterization and in vitro biological eval-uation. However, as the halichondrin program advancedthrough the discovery milestone system at Eisai and eventuallyentered preclinical development, increasing amounts of eribu-lin were needed to support the studies necessary for internaldecision-making and ultimately investigational new drug(IND) ling.
In the rst generation eribulin synthesis, the key couplingreaction between fragments 5 and 6 utilized a halogen–metalexchange reaction as illustrated in Fig. 3. Although thisapproach successfully provided the amount of materialrequired for initial biological evaluation, the key couplingreaction was not sufficiently reliable and robust to run on ascale much above a few hundred milligrams. The rstattempt to scale the reaction proceeded stepwise, ultimatelyreaching 2 grams of 5 (X ¼ I). The resulting disastrously lowyield of coupled product, however, immediately prompted usto place on hold any further attempts to use this route.Under these conditions, quenching of the primary organo-lithium intermediate to the C30a methyl group from 5 was amajor by-product. Since gram quantities of eribulin would beneeded for preclinical toxicology studies, identifying analternative method of coupling the two advanced intermedi-ates represented a priority for the program. Unfortunately,however, at that point in time there was no processchemistry research support that could be formally assignedto the project. Thus, the initial gram scale synthesis routending and execution were performed by the discoverychemistry team subsequent to entering formal preclinicaldevelopment.
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3 First multi-gram scale synthesis
We envisioned the rst scale-up synthesis of eribulin to proceedthrough the two main fragments 5 (X ¼ OMs) and 6 although atthe time of scale-up initiation, alternatives to the halogen–metalexchange reaction had not been identied. Work to solve thisproblem began while synthesis of the intermediates proceeded,since we anticipated nding a solution to the problem by thetime the key intermediates became available on scale. Althoughsomewhat of a huge gamble, the need to balance time againstuncertainty was driven by internal timelines to initiatepreclinical toxicology studies and an NCI sponsored Phase Iclinical trial as soon as possible. Derivative 5 (X ¼ OMs)was prepared from the commercial starting materials depictedin Fig. 4.
Commercially available compound 10 was converted in astraightforward manner to the C31–C35 acetylenic fragment 13(Fig. 5). Its C27–C30a coupling partner, compound 14, was
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Fig. 6 The synthesis of the C27–C35 tetrahydrofuran fragment.
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prepared in ve steps from commercially available 1-butyn-4-ol8. These two subfragments were coupled under Lewis acidconditions to afford the desired alcohol 15 (Fig. 6). The acety-lenic moiety was hydrogenated under Lindlar's conditions toafford the corresponding cis olen, which was then stereo-selectively dihydroxylated with osmium tetroxide. Activation ofthe resulting bis-diol with methanesulfonyl chloride allowed a5-exo-tet ring closure reaction to proceed smoothly. Separationof isomers and functional group manipulation subsequentlyafforded the key C27–C35 tetrahydrofuran intermediate 17.Although still lengthy, this alternative route was shorter thanthe original and involved fewer isomer separations.13
From commercially available L-(+)-erythrulose (12) allylicbromide 19 was prepared in ve straightforward trans-formations (Fig. 7). This material was coupled with aldehyde 18in the presence of zinc to afford amixture of secondary alcohols.This was oxidized under Swern conditions and stereoselectivelyreduced with L-Selectride to generate the desired stereoisomer20 as the major product. Aer purication, this material was
Fig. 7 The synthesis of the C14–C21 aldehyde subfragment.
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transformed in three steps to aldehyde subfragment 21. Itscoupling partner 25 was prepared from butenolide 9 following amodication of Kishi's synthesis (Fig. 8). The resulting productwas converted to fragment 26 according to the original Kishiroute (Fig. 9).
By this time, the nal coupling sequence to replace thehalogen–metal exchange reaction had been worked out anddemonstrated on a milligram scale. Despite the seemingsensitivity of the functional groups in both the starting mate-rials and product, the new sequence appeared to be reliable andtherefore ready for scale-up.
Following the original Kishi synthesis of HB, aldehyde 17and vinyl iodide 26 were coupled under Nozaki–Hiyama–Kishiconditions to furnish a 3 : 1mixture of C27-isomers favoring thedesired product. Separation of isomers was not practical at thisstage, but was successfully accomplished by standard ashchromatography aer cleavage of the MPM-group, providingintermediate 27 in 59% yield (Fig. 10). Based on thepilot coupling studies that were conducted in tandem with the
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Fig. 8 The synthesis of the C22–C26 ketophosphonate subfragment.
Fig. 9 The synthesis of the C14–C26 fragment.
Fig. 10 The synthesis of the key C14–C35 fragment.
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scale-up campaign, the C30a alcohol needed to be transformedto a phenyl sulfone for coupling with the C1–C13 aldehyde 6.This was accomplished by activation of the primary alcohol,displacement with thiophenol and subsequent oxidation of theresulting sulde under TPAP/NMO conditions. Removal of thepivaloate protecting group was then cleanly achieved by treat-ment with DIBAL to afford the key C14–C35 intermediate 28.Preparation of this material required a total of 59 syntheticsteps with the longest linear sequence being 26 steps from
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readily available commercial starting materials. However, inaddition to the number of synthetic transformations a numberof chromatographic separations were required, which unfortu-nately further reduced the throughput and scalability of thismedicinal chemistry route.
Nevertheless, despite the limitations and challenges thediscovery project team prepared over 65 g of the C14–C35phenyl sulfone intermediate 28. Consistent with what wasobserved on milligram scale pilot studies, coupling with the
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C1–C13 aldehyde 6 proceeded smoothly to afford a mixture ofalcohols that was oxidized with Dess–Martin periodinane andtreated with SmI2 to remove the sulfone group. Scale-up thenproceeded in a stepwise manner starting with 2.28 g of 28(Fig. 11). The largest scale involved 55.8 g, which provided thedesired product 29 in 55% combined overall yield for thethree steps.
In a further demonstration of the Nozaki–Hiyama–Kishireaction's exibility and compatibility with highly functional-ized systems, compound 29 underwent a smooth macrocyclicring closure reaction in 95% yield. Oxidation of the resultingallylic alcohols to the corresponding enone, removal of the silylprotecting groups followed by Michael addition in the presenceof TBAF buffered with imidazole hydrochloride, and closure ofthe “cage” structure using PPTS furnished diol 30 in 69% overallyield for the three steps following a recycling sequence ofundesired isomeric intermediate materials. Assembly of threekey fragments 17, 26 and 6 provided 18.0 g of diol 30, a three-fold increase from the originally projected quantity. In the nalconversion, the C35 alcohol was transformed to the primaryamino group by activation with methanesulfonic anhydride andtreatment with ammonium hydroxide. Purication by columnchromatography, ltration and precipitation from hexanesultimately afforded 8.9 g of eribulin mesylate 1 as a white solid(17% overall yield from sulfone 28), thereby completing the rstmulti-gram total synthesis of this structurally complex chemo-therapeutic agent.
While the rst generation scale-up synthesis campaign oferibulin mesylate successfully delivered the target amount of
Fig. 11 The final coupling sequence.
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material, the route involved multiple chromatographic puri-cations with few crystalline intermediates. Tremendous processresearch improvement has since been realized, providing eri-bulin mesylate in much greater quantity, higher overall yieldand at a considerably reduced cost.14–16
Given the structural complexity of eribulin and the lack ofintermediates from naturally occurring sources to support asemisynthetic strategy, total synthesis represented the onlyoption to solve the material supply problem, thereby allowingthe halichondrin program to move forward from laboratorycuriosity through clinical development, and ultimately into thehands of physicians for the benet of cancer patients world-wide. In this regard, contemporary organic synthesis has boththe capacity and the potential to solve a wide array of drugdiscovery and development problems. Structural complexityneed not necessarily limit what may be possible in the quest toidentify, develop, and manufacture new drug candidates wherecost per dose rather than cost per kilogram can be considered.Furthermore, the discovery and development of eribulinmesylate serves to highlight the important role of naturalproducts as both a source and inspiration of novel pharma-cotherapeutic agents in areas of unmet medical need for thebenet of patients and their families. Agents such as eribulintherefore have the potential to change our perception of what adruggable chemical lead must look like and expand thehorizon of screening libraries with regard to new or under-exploited scaffolds with increasing levels of structural andstereochemical complexity against difficult to target biologicalsystems.
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4 Acknowledgements
This was very much a team effort and the manuscript is writtenon their behalf. In that light, we wish to thank the followingscientists and managers for their contributions and support tomake this work possible: Michael Alaimo, Roch Boivin, TrevorCalkins, Charles Chase, William J. Christ, Heather Davis, BruceDeCosta, Rulin Fan, Lynn Hawkins, Yoshito Kishi, Charles-Andre Lemelin, Bryan Lewis, Michael D. Lewis, Kechun Li,Xiang-Yi Li, Lily Lu, Paul Lydon, Zhaoyang Meng, ThomasNoland, John Orr, Monica Palme, John Roberts, Bhavdeep Shah,Yongchun Shen, Lori Singer, Lynda Tremblay, Thomas Varick,and Hu Yang.
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