WO2012147900A1 - Microreactor process for halichondrin b analog synthesis - Google Patents

Abstract

The present invention provides methods for producing eribulin, intermediates useful for the synthesis of eribulin, or pharmaceutically acceptable salts thereof, e.g., eribulin mesylate using microreactors.

Description

DESCRIPTION TITLE OF INVENTION MICROREACTOR PROCESS FOR HALICHONDRIN B ANALOG SYNTHESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application no. 61/480,098, filed April 28, 2011 , which is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to methods for the preparation of compounds useful as intermediates in the synthesis of pharmaceutically active macrolide compounds. BACKGROUND ART

Halichondrin B is a potent anticancer agent originally isolated from the marine sponge Halichondria okadai, and subsequently found in Axinella sp., Phakellia carteri, and

Lissodendoryx sp. A total synthesis of halichondrin B was published in 1992 (Aicher, T. D. et al., J. Am. Chem. Soc. 1 14:3162-3164). Eribulin (also called B 1939), a nontaxane microtubule dynamics inhibitor, is a structurally simplified, synthetic analog of halichondrin B. The mesylate salt of eribulin is also known as Halaven® or E7389. Methods and intermediates for the synthesis of eribulin mesylate and other halichondrin B analogs are described in International Publication Nos. WO 2005/1 18565, WO 2009/046308, WO 2009/064029, and WO 2009/124237 and U.S. Patent No. 6,214,865. New methods for the synthesis of halichondrin B analogs, in particular eribulin, are desirable. DISCLOSURE OF INVENTION

The present invention provides methods for producing eribulin, intermediates useful for synthesis of eribulin, or pharmaceutically acceptable salts thereof, e.g., eribulin mesylate:

eribulin.

In one aspect, the invention features a method of producing a compound of Formula (II) from a compound of Formula (I) by contacting the compound of Formula (I):

with a reducing agent in a microreactor for a time sufficient to produce the compound of Formula (II):

wherein each of PG₃, PG₄, and PG₅ is independently a hydroxyl protecting group; Ri is CI - C6 alkyl; and is a leaving group. In certain embodiments, one, two, or three of PG₃, PG₄, and PG₅ of Formula (I) or

Formula (II), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers. For example, one, two, or three of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II) are t-butyldimethylsilyl (TBS) or benzyl, or all of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II) are t-butyldimethylsilyl (TBS). In other embodiments, LGi is a halogen, (Cl-C6)alkylsulfonate,

(C6-C10 aryl or CI -C6 heteroaryl)sulfonate, (C6-C15)aryl(Cl -C6)alkylsulfonate, or (C l-

C6)heteroaryl(Cl-C6)alkylsulfonate. Specific examples of LGi include iodide, mesylate, toluenesulfonate, isopropylsulfonate, phenylsulfonate, and benzylsulfonate.

The compounds of Formula (I) and Formula (II) can have the absolute stereochemistry:

(D-A (II)-A.

A specific compound of Formula (I) for use in the present methods is

E -803895.

A specific compound of Formula (II) for use in the present methods

ER-803896.

Exemplary reducing agents include an aluminum hydride reagent or a borohydride reagent, e.g., lithium aluminum hydride, sodium aluminum hydride, diisobutylaluminum hydride (DIBAL-H), sodium bis(2-methoxyethoxy)aluminum hydride (Red Al), sodium borohydride, potassium borohydride, rubidium borohydride, cesium borohydride, or sodium

cyanoborohydride .

In certain embodiments, the temperature of the microreactor is between -80 and -20 °C. The residence time of the compound of Formula I in the microreactor is, e.g., 0.01 sec to 1 sec.

In another aspect, the invention features a method of producing a compound of Formula (IV) from a compound of Formula (II) and a compound of Formula (III), by contacting the compound of Formula (I

with a base in a microreactor for a time sufficient to deprotonate the compound of formula (III), wherein each of PGi and PG₂ is independently a hydroxyl protecting group, and PG₆ is hydrogen or a hydroxyl protecting group; and contacting the deprotonation product with a compound of Formula (II):

a microreactor for a time sufficient to produce the compound of formula (IV)

wherein each of PG₃, PG₄, and PG₅ is independently a hydroxyl protecting group; and LG] is a leaving group.

In certain embodiments, one or both of PGi and PG₂ of Formula (III), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers. For example, one or both of PG, and PG₂ are TBS or benzyl, or both PGi and PG₂ are TBS.

In certain embodiments, the compound of Formula (III) has the absolute stereochemistry:

A specific compound of Formula III) for use in the methods is ER-804028:

ER-804028 In certain embodiments, the compound of Formula(II) has the absolute stereochemistry of Formula (II)-A, or the compound is ER-803896. The method of producing the compound of Formula (IV) may further include synthesizing the compound of Formula (II) using the methods described herein.

In certain embodiments, the compound of Formula (IV) has the stereochemistry:

(IV)-A.

A specific compound of Formula IV that can be produced by the methods is ER-804029:

ER-804029.

The base can be an organometallic reagent, such as a Grignard's reagent, potassium bis(trimethylsilyl) amide, sodium bis(trimethylsilyl)amide, lithium bis(trimethylsilyl)amide, lithium diisopropylamide, «-butyl lithium, sec-butyl lithium, or ter/-butyl lithium.

In other embodiments, the temperature of the microreactor in the deprotonation and/or coupling step is between -80 to 20 °C. The residence time of the compound of Formula (III) in the deprotonation step is, e.g., 0.1 sec to 20 sec. The residence time of the compound of Formula (II) and the compound of Formula (III) in the coupling step is, e.g., 0.1 sec to 20 sec.

The invention further features a method of producing eribulin, or a pharmaceutically acceptable salt thereof, by producing a compound of Formula (II), e.g., ER-803896, as described herein and reacting the compound of Formula (II) under suitable conditions to produce eribulin, or a pharmaceutically acceptable salt thereof, e.g., eribulin mesylate. In these methods, the compound of Formula (II) can be coupled to a compound of Formula (III), e.g., ER-804028, to produce a compound of Formula (IV), e.g., ER-804029, and the compound of Formula (IV) can be reacted to produce eribulin, or the pharmaceutically acceptable salt thereof.

In another aspect, the invention features a method of producing eribulin, or a

pharmaceutically acceptable salt thereof, by producing a compound of Formula (IV), e.g., ER- 804029, as described herein; and reacting the compound of Formula (IV) under suitable conditions to produce eribulin, or a pharmaceutically acceptable salt thereof, e.g., eribulin mesylate.

Asymmetric or chiral centers exist in the compounds of the invention. The present invention includes the various stereoisomers of the compounds and mixtures thereof, unless otherwise specified. Individual stereoisomers of the compounds of the present invention are prepared synthetically from commercially available starting materials that contain asymmetric or chiral centers or by preparation of mixtures of compounds followed by resolution, as is well known in the art. These methods of resolution are exemplified by direct separation of the mixture of diastereomers on chiral chromatographic columns or by chiral HPLC methods.

Methods of chiral separation have been described previously (G.B. Cox (ed.) in Preparative Enantioselective Chromatography, 2005, Blackwell Publishing). Alternatively, chiral compounds can be prepared by an asymmetric synthesis that favors the preparation of one diastereomer over another. Geometric isomers may also exist in the compounds of the present invention. The present invention includes the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond, such as isomers of the Z or E configuration. It is also recognized that for structures in which tautomeric forms are possible, the description of one tautomeric form is equivalent to the description of both, unless otherwise specified. In certain embodiments, a diastereomer of a compound of the invention is present in a mixture at a ratio of 10:1 , 20:1, 30:1, 50:1, or greater as compared to other diastereomers.

Compounds useful in the invention may be isotopically labeled compounds. Useful isotopes include hydrogen, carbon, nitrogen, and oxygen (e.g., ²H, ³H, ^,3C, ¹⁴C, ^I5N, ^{I 8}0, and ¹⁷0). Isotopically-labeled compounds can be prepared by synthesizing a compound using a readily available isotopically-labeled reagent in place of a non-isotopically-labeled reagent.

For any of the following chemical definitions, a number following an atomic symbol indicates that total number of atoms of that element that are present in a particular chemical moiety. As will be understood, other atoms, such as hydrogen atoms, or substituent groups, as described herein, may be present, as necessary, to satisfy the valences of the atoms. For example, an unsubstituted C2 alkyl group has the formula -CH₂CH₃. When used with the groups defined herein, a reference to the number of carbon atoms includes the divalent carbon in acetal and ketal groups but does not include the carbonyl carbon in acyl, ester, carbonate, or carbamate groups. A reference to the number of oxygen, nitrogen, or sulfur atoms in a heteroaryl group only includes those atoms that form a part of a heterocyclic ring.

By "acetal" is meant -OCH O-, wherein R is H, alkyl, alkenyl, aryl, or arylalkyl.

By "acyl" is meant -C(0)R, wherein R is H, alkyl, alkenyl, aryl, or arylalkyl. In exemplary acyl groups, R is H, C1-C12 alkyl (e.g., C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and 307

C3-C6 alkyl), C2-C12 alkenyl (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), C6- C20 aryl (e.g., C6-C15, C6-C10, C8-C20, and C8-C15 aryl), monocyclic C1 -C6 heteroaryl (e.g., monocyclic C1-C4 and C2-C6 heteroaryl), C4-C19 heteroaryl (e.g., C4-C10 heteroaryl), (C6- C15)aryl(Cl -C6)alkyl, (Cl -C6)heteroaryl(Cl-C6)alkyl, or (C4-C19)heteroaryl(Cl-C6)alkyl. As defined herein, any heteroaryl group present in an acyl group has from 1 to 4 heteroatoms selected independently from O, N, and S.

By "alkyl" is meant a straight or branched chain saturated cyclic (i.e., cycloalkyl) or acyclic hydrocarbon group of from 1 to 12 carbons, unless otherwise specified. Exemplary alkyl groups include C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl. Specific examples include methyl, ethyl, 1 -propyl, 2-propyl, 2 -methyl- 1 -propyl, 1 -butyl, 2-butyl, and the like.

Unless otherwise noted, alkyl groups, used in any context herein, are optionally substituted with halogen, alkoxy, aryloxy, arylalkyloxy, oxo, alkylthio, alkylenedithio, alkylamino,

[alkenyl]alkylamino, [aryl] alkylamino, [arylalkyl] alkylamino, dialkylamino, silyl, sulfonyl, cyano, nitro, carboxyl, or azido.

By "alkylamino" is meant -NHR, wherein R is alkyl. By "[alkenyl]alkylamino" is meant

-NRR', wherein R is alkyl, and R' is alkenyl. By "[aryl] alkylamino" is meant -NRR', wherein R is alkyl, and R' is aryl. By "[arylalkyl]alkylamino" is meant -NRR', wherein R is alkyl, and R' is arylalkyl. By "dialkylamino" is meant -NR₂, wherein each R is alkyl, selected

independently.

By "alkylene" is meant a divalent alkyl group. Alkylene groups, used in any context herein, are optionally substituted in the same manner as alkyl groups. For example, a CI alkylene group is -CH₂-.

By "alkylenedithio" is meant -S-alkylene-S-.

By "alkylthio" is meant -SR, wherein R is alkyl. By "alkenyl" is meant a straight or branched chain cyclic or acyclic hydrocarbon group of, unless otherwise specified, from 2 to 12 carbons and containing one or more carbon-carbon double bonds. Exemplary alkenyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl. Specific examples include ethenyl (i.e., vinyl), 1-propenyl, 2-propenyl (i.e., allyl), 2-methyl- 1-propenyl, 1-butenyl, 2-butenyl (i.e., crotyl), and the like. Alkenyl groups, used in any context herein, are optionally substituted in the same manner as alkyl groups.

Alkenyl groups, used in any context herein, may also be substituted with an aryl group.

By "alkoxy" is meant -OR, wherein R is alkyl.

By "aryl" is meant a monocyclic or multicyclic ring system having one or more aromatic rings, wherein the ring system is carbocyclic or heterocyclic. Heterocyclic aryl groups are also referred to as heteroaryl groups. A heteroaryl group includes 1 to 4 atoms selected

independently from O, N, and S. Exemplary carbocyclic aryl groups include C6-C20, C6-C15, C6-C10, C8-C20, and C8-C15 aryl. A preferred aryl group is a C6-10 aryl group. Specific examples of carbocyclic aryl groups include phenyl, indanyl, indenyl, naphthyl, phenanthryl, anthracyl, and fluorenyl. Exemplary heteroaryl groups include monocylic rings having from 1 to 4 heteroatoms selected independently from O, N, and S and from 1 to 6 carbons (e.g., C1-C6, C1-C4, and C2-C6). Monocyclic heteroaryl groups preferably include from 5 to 9 ring members. Other heteroaryl groups preferably include from 4 to 19 carbon atoms (e.g., C4-C10). Specific examples of heteroaryl groups include pyridinyl, quinolinyl, dihydroquinolinyl, isoquinolinyl, quinazolinyl, dihydroquinazolyl, and tetrahydroquinazolyl. Unless otherwise specified, aryl groups, used in any context herein, are optionally substituted with alkyl, alkenyl, aryl, arylalkyl, halogen, alkoxy, aryloxy, arylalkyloxy, oxo, alkylthio, alkylenedithio, alkylamino,

[alkenyl]alkylamino, [aryl]alkylamino, [arylalkyl]alkylamino, dialkylamino, silyl, sulfonyl, cyano, nitro, carboxyl, or azido. 7

By "arylalkyl" is meant -R'R", wherein R' is alkylene, and R" is aryl.

By "arylalkyloxy" is meant -OR, wherein R is arylalkyl.

By "aryloxy" is meant -OR, wherein R is aryl.

By "carbamate" is meant -OC(0)NR₂, wherein each R is independently H, alkyl, alkenyl, aryl, or arylalkyl.

By "carbonate" is meant -OC(0)OR, wherein R is alkyl, alkenyl, aryl, or arylalkyl.

By "carboxyl" is meant -C(0)OH, in free acid, ionized, or salt form.

By "cyclic boronate" is meant -OBRO-, wherein R is alkyl, alkenyl, aryl, arylalkyl, alkoxy, or 2,6-diacetamidophenyl.

By "cyclic carbonate" is meant -OC(0)0-.

By "cyclic silylene" is meant -OSiR₂0-, wherein each R is independently alkyl, alkenyl, aryl, arylalkyl, or alkoxy. By "dialkylsilylene" is meant a cyclic silylene, wherein each R is alkyl.

By "ester" is meant -OC(0)R, where -C(0)R is an acyl group, as defined herein, that is bound to the oxygen atom of a protected hydroxyl, as defined below.

By "ether" is meant -OR, wherein R is alkyl, alkenyl, arylalkyl, silyl, or 2- tetrahydropyranyl .

By "halogen" is meant fluoro, chloro, bromo, or iodo.

By "ketal" is meant -OCR₂0-, wherein each R is independently alkyl, alkenyl, aryl, or arylalkyl, or both R groups are together alkylene.

By "oxo" or (O) is meant =0.

By "silyl" is meant -SiR₃, wherein each R is independently alkyl, alkenyl, aryl, or arylalkyl. Examples of silyl groups include tri(Cl-C6 alkyl)silyl, tri(C6-C10 aryl or C1-C6 heteroaryl)silyl, di(C6-C10 aryl or C1-C6 heteroaryl)(Cl-C6 alkyl)silyl, and (C6-C10 aryl or Cl- C6 heteroaryl)di(C 1 -C6 alkyl)silyl. It will be understood that, when a silyl group includes two or more alkyl, alkenyl, aryl, heteroaryl, or arylalkyl groups, these groups are independently selected. As defined herein, any heteroaryl group present in a silyl group has from 1 to 4 heteroatoms selected independently from O, N, and S.

By "sulfonate" is meant -OS(0)₂R, wherein R is alkyl, alkenyl, aryl, or arylalkyl. In exemplary sulfonates, R is C1-C12 alkyl (e.g., C1 -C8, C1 -C6, C1-C4, C2-C7, C3-C12, and C3- C6 alkyl), C2-C12 alkenyl (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), carbocyclic C6-C20 aryl (e.g., C6-C15, C6-C10, C8-C20, and C8-C15 aryl), monocyclic C1-C6 heteroaryl (e.g., C1-C4 and C2-C6 heteroaryl), C4-C19 heteroaryl (e.g., C4-C10 heteroaryl), (C6-C 15)aryl(C 1 -C6)alkyl, (C4-C 19)heteroaryl(C 1 -C6)alkyl, or (C 1 -C6)heteroaryl(C 1 -C6)alkyl. As defined herein, any heteroaryl group present in a sulfonate group has from 1 to 4 heteroatoms selected independently from O, N, and S.

By "sulfonyl" is meant -S(0)₂R, wherein R is alkyl, alkenyl, aryl, arylalkyl, or silyl. Preferred R groups for sulfonyl are the same as those described above for sulfonates.

By "hydro xyl protecting group" is meant any group capable of protecting the oxygen atom to which it is attached from reacting or bonding. Hydroxyl protecting groups are known in the art, e.g., as described in Wuts, Greene's Protective Groups in Organic Synthesis, Wiley- Interscience, 4^th Edition, 2006. Exemplary protecting groups (with the oxygen atom to which they are attached) are independently selected from esters, carbonates, carbamates, sulfonates, and ethers.

In exemplary ester hydroxyl protecting groups, R of the acyl group is C1-C12 alkyl (e.g., C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl), C2-C12 alkenyl (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), carbocyclic C6-C20 aryl (e.g., C6-C15, C6-C10, C8-C20, and C8-C15 aryl), monocyclic C1-C6 heteroaryl (e.g., C1-C4 and C2-C6 heteroaryl), C4-C19 heteroaryl (e.g., C4-C10 heteroaryl), (C6-C15)aryl(Cl-C6)alkyl, (C4-C19)heteroaryl(Cl- C6)alkyl, or (Cl-C6)heteroaryl(Cl-C6)alkyl. Specific examples of acyl groups for use in esters include formyl, benzoylformyl, acetyl (e.g., unsubstituted or chloroacetyl, trifluoroacetyl, methoxyacetyl, triphenylmethoxyacetyl, and p-chlorophenoxyacetyl), 3-phenylpropionyl, 4- oxopentanoyl, 4,4-(ethylenedithio)pentanoyl, pivaloyl (Piv), vinylpivaloyl, crotonoyl, 4- methoxy-crotonoyl, naphthoyl (e.g., 1- or 2-naphthoyl), and benzoyl (e.g., unsubstituted or substituted, e.g., p-methoxybenzoyl, phthaloyl (including salts, such a triethylamine and potassium), p-bromobenzoyl, and 2,4,6-trimethylbenzoyl). As defined herein, any heteroaryl group present in an ester group has from 1 to 4 heteroatoms selected independently from O, N, and S.

In exemplary carbonate hydroxyl protecting groups, R is C1-C12 alkyl (e.g., C1-C8, Cl- C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl), C2-C12 alkenyl (e.g., C2-C8, C2-C6, C2-C4, C3- C12, and C3-C6 alkenyl), carbocyclic C6-C20 aryl (e.g., C6-C15, C6-C10, C8-C20, and C8-C15 aryl), monocyclic C1-C6 heteroaryl (e.g., C1-C4 and C2-C6 heteroaryl), C4-C19 heteroaryl (e.g., C4-C10 heteroaryl), (C6-C15)aryl(Cl-C6)alkyl, (C4-C19)heteroaryl(Cl -C6)alkyl, or (Cl-

C6)heteroaryl(Cl-C6)alkyl. Specific examples include methyl, 9-fluorenylmethyl, ethyl, 2,2,2- trichloroethyl, 2-(trimethylsilyl)ethyl, 2-( henylsulfonyl)ethyl, vinyl, allyl, t-butyl, p-nitrobenzyl, and benzyl carbonates. As defined herein, any heteroaryl group present in a carbonate group has from 1 to 4 heteroatoms selected independently from O, N, and S.

In exemplary carbamate hydroxyl protecting groups, each R is independently H, CI -CI 2 alkyl (e.g., C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl), C2-C12 alkenyl (e.g., C2- C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), carbocyclic C6-C20 aryl (e.g., C6-C15, C6- C10, C8-C20, and C8-C15 aryl), monocyclic C1-C6 heteroaryl (e.g., C1 -C4 and C2-C6 heteroaryl), C4-C19 heteroaryl (e.g., C4-C10 heteroaryl), (C6-C15)aryl(Cl-C6)alkyl, (C4- C19)heteroaryl(Cl-C6)alkyl, or (Cl-C6)heteroaryl(Cl-C6)alkyl. Specific examples include N- phenyl and N-methyl-N-(o-nitrophenyl) carbamates. As defined herein, any heteroaryl group present in a carbamate group has from 1 to 4 heteroatoms selected independently from O, N, and S.

Exemplary ether hydroxyl protecting groups include C1-C12 alkylethers (e.g., C1 -C8,

C1 -C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl), C2-C12 alkenylethers (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), (C6-C15)aryl(Cl-C6)alkylethers, (C4-C19)heteroaryl(Cl- C6)alkylethers, (Cl-C6)heteroaiyl(Cl-C6)alkylethers, (Cl-C6)alkoxy(Cl-C6)alkylethers, (Cl - C6)alkylthio(Cl -C6)alkylethers, (C6-C10)aryl(Cl-C6)alkoxy(Cl-C6)alkylethers, and silylethers (e.g., tri(Cl-C6 alkyl)silyl, tri(C6-C10 aryl or C1-C6 heteroaryl)silyl, di(C6-C10 aryl or C1-C6 heteroaryl)(Cl-C6 alkyl)silyl, and (C6-C10 aryl or C1-C6 heteroaryl)di(Cl-C6 alkyl)silyl). Specific examples of alkylethers include methyl and t-butyl, and an example of an alkenyl ether is allyl. Examples of alkoxyalkylethers and alkylthioalkylethers include methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, and P-(trimethylsilyl)ethoxyrnethyl. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, triphenylmethyl (trityl), o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, naphthylmethyl, and 2- and 4-picolyl ethers. Specific examples of silylethers include trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), and triphenylsilyl (TPS) ethers. An example of an arylalkyloxyalkylether is benzyloxymethyl ether. As defined herein, any heteroaryl group present in an ether group has from 1 to 4 heteroatoms selected independently from O, N, and S.

Adjacent hydroxyl groups may be protected with a diol protecting group, such as acetal (e.g., C1-C6 alkyl), ketal (e.g., C3-C6 alkyl or C3-C6 cycloalkyl), cyclic silylene, cyclic carbonate, and cyclic boronate. Examples of acetal and ketal groups include methylene, ethylidene, benzylidene, isopropylidene, cyclohexylidene, and cyclopentylidene. An example of a cyclic silylene is di-t-butylsilylene. Another diol protecting group is 1 ,1,3,3- tetraisopropylsiloxanediyl. Examples of cyclic boronates include methyl, ethyl, phenyl, and 2,6- diacetamidophenyl boronates.

Protecting groups may be substituted as is known in the art; for example, aryl and arylalkyl groups, such as phenyl, benzyl, naphthyl, orpyridinyl, can be substituted with C1 -C6 alkyl, C1-C6 alkoxy, nitro, cyano, carboxyl, or halogen. Alkyl groups, such as methyl, ethyl, isopropyl, n-propyl, t-butyl, n-butyl, and sec -butyl, and alkenyl groups, such as vinyl and allyl, can also be substituted with oxo, arylsulfonyl, halogen, and trialkylsilyl groups. Preferred, protecting groups are TBS and Piv. Protecting groups that are orthogonal are removed under different conditions, as in known in the art.

By "leaving group" is meant a group that is displaced during a chemical reaction.

Suitable leaving groups are well known in the art, e.g., see, Advanced Organic Chemistry, March, 4th Ed., pp. 351-357, John Wiley and Sons, N.Y. (1992). Such leaving groups include halogen, C1-C12 alkoxy (e.g., C1-C8, C1-C6, C1-C4, C2-C7, and C3-C6 alkoxy), C1-C12 alkylsulfonate (e.g., C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkylsulfonate), C2-C12

alkenylsulfonate (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenylsulfonate), carbocyclic C6-C20 arylsulfonate (e.g., C6-C15, C6-C10, C8-C20, and C8-C15 arylsulfonate), C4-C19 heteroarylsulfonate (e.g., C4-C10 hetero arylsulfonate), monocyclic C1 -C6 heteroarylsulfonate (e.g., C 1-C4 and C2-C6 heteroarylsulfonate), (C6-C 15)aryl(Cl -C6)alkylsulfonate, (C4-

C19)heteroaryl(C l-C6)alkylsulfonate, (Cl -C6)heteroaryl(Cl -C6)alkylsulfonate, and diazonium. Alkylsulfonates, alkenylsulfonates, arylsulfonates, heteroarylsulfonates, arylalkylsulfonates, and heteroarylalkylsulfonates can be optionally substituted with halogen (e.g., chloro, iodo, bromo, or fluoro), alkoxy (e.g., C1-C6 alkoxy), aryloxy (e.g., C6-C15 aryloxy, C4-C19 heteroaryloxy, and C1-C6 heteroaryloxy), oxo, alkylthio (e.g., C1-C6 alkylthio) , alkylenedithio (e.g., C1-C6 alkylenedithio), alkylamino (e.g., C1-C6 alkylamino), [alkenyl]alkylamino (e.g., [(C2- C6)alkenyl](Cl-C6)alkylamino), [aryljalkylamino (e.g., [(C6-C10)aryl](Cl-C6)alkylamino, [(C l -C6)heteroaryl](Cl -C6)alkylamino, and [(C4-C19)heteroaryl](C l-C6)alkylamino),

[arylalkyl]alkylamino (e.g., [(C6-C10)aryl(Cl -C6)alkyl](Cl-C6)alkylamino, [(Cl-

C6)heteroaryl(C 1 -C6)alkyl](C 1 -C6)alkylamino, and [(C4-C19)heteroaryl(C 1 -C6)alkyl](C 1 - C6)alkylamino), dialkylamino (e.g., di(Cl-C6 alkyl)amino), silyl (e.g., tri(Cl -C6 alkyl)silyl, tri(C6-C10 aryl or C1 -C6 heteroaryl)silyl, di(C6-C10 aryl or C1-C6 heteroaryl)(Cl-C6 alkyl)silyl, and (C6-C10 aryl or C1 -C6 heteroaryl)di(Cl-C6 alkyl)silyl), cyano, nitro, or azido. Alkenylsulfonates can be optionally substituted with carbocyclic aryl (e.g., C6-C15 aryl), monocyclic C1-C6 heteroaryl, or C4-C19 heteroaryl (e.g., C4-C10 heteroaryl). Arylsulfonates can be optionally substituted with alkyl (e.g., C1 -C6 alkyl) or alkenyl (e.g. C2-C6 alkenyl). As defined herein, any heteroaryl group present in a leaving group has from 1 to 4 heteroatoms selected independently from O, N, and S.

Specific examples of suitable leaving groups include chloro, iodo, bromo, fiuoro, methanesulfonate (mesylate), 4-toluenesulfonate (tosylate), trifluoromethanesulfonate (triflate, OTf), nitro-phenylsulfonate (nosylate), and bromo-phenylsulfonate (brosylate). Leaving groups may also be further substituted as is known in the art.

The term "microreactor" used in the present specification refers to a reaction vessel in which at least two fluids are combined and allowed to react, wherein the vessel has at least one interior dimension of 1 mm or less, e.g., the diameter of tubing or a transverse dimension of a fluidic chip.

By "pharmaceutically acceptable salt" is meant a salt within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example,

pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences

66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. A preferred salt is the mesylate salt.

By "residence time" is meant the time during which a mixture of reactants passes through the volume of a micro reactor lying between the points at which two or more reactants first mix completely and the addition of a further reactant or quenching agent.

Other features and advantages of the invention will be apparent from the following description and the claims. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a schematic depiction of a microreactor system for converting ER-803895 to ER-803896.

Figure 2 is a schematic depiction of a microreactor system for converting ER-803895 to ER-803896. In Figure 2, the lines from Pumps A, B and C are SUS (φ 0.8 mm x 2 m); the line from Pump D is Teflon (φ 0.8 mm); Pump A is Shimadzu HPLC LCIOAD; Pumps B and C are Shimadzu HPLC LC8A; and Pump D is EYELA VSP2200.

Figure 3 is a schematic depiction of a microreactor system for producing ER-804029 from ER-804028 and ER-803896.

BEST MODE FOR CARRYING OUT THE INVENTION

Halichondrin B analogs, e.g., eribulin or pharmaceutically acceptable salts thereof, can be synthesized by coupling the C1 -C13 and C14-C35 fragments as described in U.S. Patent No. 6,214,865 and International Publication No. WO 2005/118565. A key step in this synthesis is the coupling of an anion or dianion of a C 14-35 fragment with an aldehyde of a Cl-13 fragment. In one example described in these references, the C14-C35 portion, e.g., ER-804028, of the molecule is coupled to the C1 -C13 portion, e.g., ER-803896, to produce ER-804029, and additional reactions are carried out to produce eribulin (Scheme 1):

ER-804029

Scheme 1

Lithiation of the C14-C35 sulfone fragment followed by coupling to the C1-C13 aldehyde fragment furnishes a mixture of diastereomeric alcohols (ER-804029). Typically, these steps occur at low temperature, e.g., -75 °C. Additional protecting group manipulation and oxidation followed by removal of the sulfonyl group and an intramolecular Nozaki-Hiyama- Kishi (NHK) reaction affords an intermediate, which, when oxidized and treated with tetrabutylammonium fluoride, undergoes intramolecular oxy-Michael ring closure. Pyridinium />-toluenesulfonate mediated ketal formation and conversion of the terminal alcohol to an amine furnishes eribulin.

For example, as described in WO 2005/118565 (Example 6), ER-804029 is reacted to produce ER-804030; ER-804030 is reacted to produce ER-118049; ER-118049 is reacted to produce mixture ER-118047/118048; the mixture ER-1 18047/118048 is reacted to produce ER- 118046; ER-118046 is reacted to produce ER-811475; ER-811475 is reacted to produce ER- 076349; and ER-076349 is reacted to produce eribulin.

Pharmaceutically acceptable salts of eribulin, e.g., eribulin mesylate, can be formed by methods known in the art, e.g., in situ during the final isolation and purification of the compound or separately by reacting the free base group with a suitable organic acid. In one example, eribulin is treated with a solution of MsOH and NH₄OH in water and acetonitrile. The mixture is concentrated. The residue is dissolved in DCM-pentane, and the solution is added to anhydrous pentane. The resulting precipitate is filtered and dried under high vacuum to provide eribulin mesylate, as shown in Scheme 2.

eribulin

eribulin mesylate 307

Scheme 2

The present invention provides new methods for the production of a CI- 13 fragment, e.g., ER-803896, and for a CI -35 fragment, e.g., ER-804029, using microreactors. Microreactors allow for accurate control of reaction temperature and/or reaction time with the benefit of using higher temperatures compared to batch processing and providing greater safety for

manufacturing processes. In addition, because the microreactor reactions occur as continuous flow processes, they offer the ability to sample the process stream for monitoring the progress of the reaction and, for example, the level of byproducts generated.

Synthesis of Cl-13 Fragments

CI -13 fragments have been made by reducing a carboalkoxyester, e.g., ER-803895, to provide an aldehyde, e.g., ER-803896, as described in U.S. Patent No. 6,214,865. The invention provides a method of reducing a compound of Formula I to an aldehyde of Formula II in a microreactor:

wherein each of PG₃, PG₄, and PG₅ is an independently selected hydroxyl protecting group; R] CI - C6 alkyl; and LGi is a leaving group. In certain embodiments, one, two, or three of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers. In yet other embodiments, one, two, or three of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II) are t-butyldimethylsilyl (TBS) or benzyl. In still other embodiments, all of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II) are t- butyldimethylsilyl (TBS). PG and PG₄ can also combine to form a diol protecting group. In certain embodiments, LG] is a halogen, such as iodide. In other embodiments, LG] is (Cl- C6)alkylsulfonate, (C6-C10 aryl or C1-C6 heteroaryl)sulfonate, (C6-C15)aryl(Cl- C6)alkylsulfonate, or (CI -C6)heteroaryl(C 1 -C6)alkylsulfonate. Specific leaving groups include mesylate, toluenesulfonate, isopropylsulfonate, phenylsulfonate, or benzylsulfonate

In some embodiments, the compounds of Formula (I) and Formula (II) have the absolute stereochemistries depicted below:

(I)-A (II)-A.

In some embodiments, the compound of Formula (I) is ER-803895. In some

embodiments, the compound of Formula (II) is ER-803896.

ER-803895 ER-803896.

The present invention provides methods for producing aldehydes of Formula II by the reduction of carboalkoxyesters of Formula I in a microreactor, which allows the reaction to proceed at temperatures higher than those previously disclosed for this transformation. Reducing agents useful in this transformation include an aluminum hydride reagent, a borohydride reagent, or the like. Examples of reducing agents include lithium aluminum hydride, sodium aluminum hydride, diisobutylaluminum hydride (DIBAL-H), sodium bis(2-methoxyethoxy)aluminum hydride (Red Al), sodium borohydride, potassium borohydride, rubidium borohydride, cesium borohydride, sodium cyanoborohydride, or the like. A preferred reducing agent is DIBAL-H.

The reaction can be carried out in solvents purged with nitrogen, argon, or another such inert gas. Examples of the solvents used in this synthesis include halogen solvents such as dichloromethane, chloroform, or 1,2 dichloroethane; ether solvents such as tetrahydrofuran, 1 ,2-dimethoxyethane, methyl-ter -butyl ether, cyclopentyl methyl ether, diethyl ether,

diisopropyl ether, dibutyl ether, or dicyclopentyl ether; aromatic hydrocarbon solvents such as benzene or toluene; and aliphatic hydrocarbon solvents such as heptane or hexane; or mixtures thereof. A preferred solvent is toluene.

The reaction temperature is preferably between -80 and -20 °C, e.g., between -60 and -20 °C, such as approximately -50 °C. The concentration of the solution containing a compound of Formula I is preferably 0.10 g/mL to 0.30 g/mL, e.g., 0.15 g/mL to 0.25 g/mL, such as approximately 0.185 g/mL. A residence time of the compound of Formula (I) is the time sufficient to produce the compound of Formula (II), preferably 0.01 sec to 1 sec, e.g., 0.1 sec to 0.5 sec. In other embodiments, the ratio of equivalents of reducing agent, e.g., DIBAL-H, to ester is 1.0 eq to 1.5 eq, e.g., 1.215 eq to 1.485 eq, such as approximately 1.35 eq.

Compounds of Formula (I) and ER-803895 can be produced by methods known in the art, e.g., as described in U.S. Patent No. 6,214,865 and International Publication No.

WO 2005/118565.

Synthesis of CI -35 Fragments

Cl -35 fragments have been made by coupling a Cl-13 fragment, e.g., ER-803896, to a C14-34 fragment, e.g., ER-804028, as described in U.S. Patent No. 6,214,865 and International Publication No. WO 2005/118565. The invention provides a method of coupling an anion or 07

dianion of a compound of Formula III to an aldehyde of Formula I, in a microreactor, to form a compound of Formula IV:

wherein PG₃, PG₄, PG₅, and LGi are as defined above; each of PGi and PG₂ is independently hydrogen or a hydroxyl protecting group; and PG₆ is hydrogen or a hydroxyl protecting group.

In certain embodiments, one or both of PGi and PG₂ of Formula (III), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers. In yet other embodiments, one or both of PGi and PG₂ are TBS or benzyl. In still other embodiments, both PGi and PG₂ are TBS. In other embodiments, PGi and PG₂ are combined to form a diol protecting group. In some embodiments, PG₆ is hydrogen.

In some embodiments, the compound of Formula (III) has the absolute stereochemistry depicted below:

some embodiments, the compound of Formula (III) is ER-804028

ER-804028

In some embodiments, the compound of Formula (IV) has the stereochemistry depicted below:

(IV)-A.

In some embodiments, the compound of Formula IV is ER-804029: 07

ER-804029.

A compound of Formula (III) is deprotonated with a base, e.g., an organometallic reagent, to produce an anion or dianion, which is reacted with a compound of Formula (II) to obtain a compound of Formula (IV). Example of organometallic reagents include a Grignard's reagent, potassium bis(trimethylsilyl) amide, sodium bis(trimethylsilyl)amide, lithium

bis(trimethylsilyl)amide, lithium diisopropylamide, ra-butyllithium, ^ec-butyllithium, and tert- butyllithium, preferably, n-butyllithium. The reaction can be carried out in solvents purged with nitrogen, argon, or another such inert gas. Examples of the solvent used in this synthesis include halogen solvents such as dichloromethane, chloroform, or 1,2 dichloroethane; ether solvents such as tetrahydrofuran, 1,2-dimethoxyethane, methyl- rt-butyl ether, cyclopentyl methyl ether, diethyl ether, diisopropyl ether, dibutyl ether, or dicyclopentyl ether; aromatic hydrocarbon solvents such as benzene or toluene; and aliphatic hydrocarbon solvents such as heptane or hexane; or a mixture thereof. A preferred solvent is tetrahydrofuran or heptane. The

temperature is preferably between -80 and 20 °C, e.g., between -50 to 10 °C , more preferably between -10 to 10 °C. The concentration of the solution containing the compound of Formula (II) or (III) is preferably 0.05 g/mL to 0.30 g/mL, e.g., 0.05 g/mL to 0.15 g/n L. A residence time for deprotonation of the compound of Formula (III) is the time sufficient to deprotonate the compound of formula (III), preferably 0.1 sec to 20 sec, e.g., 1 sec to 5 sec. A residence time for the coupling reaction between the compound of Formula (II) and the compound of Formula (HI) is the time sufficient to produce the compound of formula (IV), preferably 0.1 sec to 20 sec, e.g., 1 sec to 5 sec. For deprotonation, the ratio of equivalents of base, e.g., n-butyllithium, to compound of Formula (III) is preferably 2.0 eq to 2.5 eq, e.g., 2.05 eq to 2.25 eq, such as approximately 2.15 eq. The coupling of a compound of Formula III with a compound of Formula II, to produce a compound of Formula IV, in a microreactor, may proceed at temperatures higher than those previously disclosed for this transformation.

Compounds of Formula (III) and ER-804028 can be prepared using methods known in the art, e.g., as described in International Publication Nos. WO 2005/118565 and WO

2009/046308 and U.S. Patent No. 6,214,865.

Microreactors

Any suitable microreactor can be used in the present invention. Examples of

microreactors include a small fluidized bed reactor and a static microreactor. A microreactor can be fabricated as a single component or be made up of separate components that are connected, e.g., by tubing. Components of a microreactor can include mixers, fluidic chips, and other devices for the combination of two or more fluids. Microreactors can be designed to mix two or more fluids and combine the resulting mixture with additional fluids. Commercially available chips can be employed. Examples of commercially available chips include COMET X- 01 (Techno Applications Co., Ltd.), CYTOS Series (YMC Co., Ltd), micro high Mixer (Toray engineering Co., Ltd.), LLMR(ITEC Co., Ltd.), and Micro Process Server (Hitachi Plant

Technologies, Ltd.). Additional microreactors are described, for example, in Japanese Patent Laid-Open Publication No. JP 2006-241065, International Publication Nos. WO 01/70649 and WO 2009/003661 , Hessel et al. Recent Patents on Chemical Engineering 2008, 1 :1-16, and Tetsu Miyazawa., Monthly Fine Chemicals 2007, 36(7):5-8, each of which is hereby incorporated by reference.

A microreactor used in the present invention can be used with any desired peripheral devices, such as heaters or coolers, temperature sensors, pressure sensors, fluid pumps, mixers, or the like. Microreactors can also be connected to analytical instrumentation, e.g., mass spectrometers or HPLC, for in-line measurement of reactants and products. Microreactors can be connected to fluid pumps or collection vessels by appropriately sized tubing. Multiple microreactors can be employed in parallel to increase rate of production of products, e.g., for use in a commercial manufacturing process.

In the invention, the flow rate of the reactants is varied according to the microreactor used in the flow reaction, and the residence time is adjusted by changing the flow rate of reactants provided to the microreactor or changing a length or internal dimension of components of the microreactor. The flow rate of the fluids provided separately to a microreactor may be the same or different.

The present invention is advantageous in that higher temperatures of reaction can be employed, compared to previous batch processing. For example, typical batch processing temperatures for the production of ER-803896 are between -60 and -80 °C, compared to -50 °C or higher, for example from -50°C to -30°C, in the present invention.

The deprotonation and coupling of ER-804028 typically occurs at between -20 and - 70 °C in batch processing, compared to -10 °C or higher, for example from -10°C to 10°C, in the present invention.

Use of microreactors can also provide products that can be employed in further reactions without chromatographic purification. Furthermore, higher concentrations of the reactants can be employed in microreactors to decrease the volume of solvents used in the reaction. Example 1

Synthesis of ER-803896

A micro flow reaction was carried out in the system of Figure 1. The reduction of ER- 803895 using diisobutylaluminum hydride (DIBAL-H) was initiated in a CMPS-aOl (Hitachi Plant Technologies, Ltd., Japan) or CMPS-a02 (Hitachi Plant Technologies, Ltd.) chip. The reaction was quenched by addition of acetone to the reduction product in a CMPS-γΙΟΙΗ (P N:767808-02) chip (Hitachi Plant Technologies, Ltd). Inlets of the CMPS chips were connected by PTFE tubing (internal diameter: 1.5 mm, length: 2.0 m) to a Micro Process Server MPS- 200 (Hitachi Plant Technologies, Ltd.) having injection syringes. The outlet of the CMPS-aOl or -a02 chip was connected to an inlet of the CMPS-γΙΟΙΗ chip by SUS316 (stainless steel) tubing (internal diameter: 0.8 mm, length: 0.1 m), and the outlet of the CMPS- γΙΟΙΗ chip was connected by PTFE (polytetrafluoroethylene) tubing (internal diameter: 1.0 mm, length: 0.6 m) to a collection vessel.

A 25 mL injection syringe was filled with a solution of 9.12 g (1 1.83 mmol) of ER-

803895, 65.2 mg (0.30 mmol) of dibutylhydroxytoluene (BHT), and 49.2 mL of toluene; a 10 mL injection syringe was filled with a 1.0 M solution of DIBAL-H in toluene; and a further 25 mL injection syringe was filled with acetone (11.7 mL) in toluene (46.6 mL) as quenching reagent.

The contents of the three syringes were transferred to the CMPS chips using the

MPS-a.200 system as shown in Figure 1. Flow rates ofER-803895, DIBAL-H, and quenching reagent were set in various ranges, and the reactions were carried out at -30 °C to -50 °C for various residence times. The resultant reaction mixture was acidified using IN HC1 and extracted with methyl tert-butyl ether (MTBE). The purity of the organic layer was analyzed by HPLC. ER-803896 was obtained with high selectivity under Run 9 or Run 1 1 compared with batch reaction conditions (Table 1). For Runs 1-11, residence times were determined based on the time required for reactants to pass through the tubing connecting the outlet of the CMPS-aOl or -a02 chip and the inlet of the CMPS-γΙΟΙΗ chip.

Example 2

Synthesis of ER-803896

The reduction of ER-803895 was initiated in another chip, COMET X-01 (internal diameter: 0.5mm, internal volume 29.38 μί; Techno Applications Co., Ltd., Japan) as shown in Figure 2. A solution of 330 g (428 mmol) of ER-803895, 2.36 g (10.7 mmol) of BHT, and

1780 mL of toluene was prepared in a 5 L bottle, which was placed in the line of plunger pump B. 571 mL of 1.0 M solution of DIBAL-H in toluene in a 1 L bottle was placed in the line of plunger pump A. Acetone (464 mL) in toluene (1856 mL) was prepared in a 5 L bottle and placed in the line of plunger pump C. 3300 mL of 1 N HCl in a 5 L bottle was placed in the line of plunger pump D.

The contents of the bottles were transferred to COMET chips using the plunger pump systems under the conditions of Run 9 in Table 1. The resultant reaction mixture flowed continuously for 86 min 45 sec into a workup vessel containing toluene (1687 mL) and MTBE (2310 mL). The aqueous layer was drained. The organic layer was washed sequentially with 1 N HCl (3300 mL), water (3300 mL), 5% aqueous sodium bicarbonate (3300 g), and brine (3300 g) and then concentrated under reduced pressure to afford ER-803896 without column chromatography (96.5% wt/wt assay yield, Run 12). For Run 12, the residence times was determined based on the time required for reactants to pass through the SUS316 tubing connecting the outlet of one COMET-XOl -T chip and the inlet of the other COMET-XOl -T chip. 07

Reference of batch reaction example 1 (Run 13)

Synthesis of ER-803896

ER-803895 (1.00 g, 1.30 mmol) and BHT (7.1 mg, 0.032 mmol) were dissolved in toluene (16.2 mL) and cooled to <-80°C under a nitrogen atmosphere. DIBAL-H (1.0 M in toluene, 1.54 ml, 1.55 mmol, 1.2 eq) was added at a rate to maintain the internal reaction temperature at <-80 °C. The resulting mixture was stirred for 1 hour and then quenched sequentially with anhydrous acetone (0.30 mL) in toluene (0.69 mL) and anhydrous methanol (0.17 mL) in toluene (0.69 mL), maintaining the internal reaction temperature at <-75 °C. The reaction mixture was allowed to warm to -35 °C, and then MTBE (5.0 mL) and 1 N HC1

(10.0 mL) were added to the reaction mixture. The mixture was stirred for 30 minutes, and the aqueous layer was drained. The organic layer was washed sequentially with 1 N HC1 (10 mL), water (10 mL), 5% aqueous NaHC0₃ (10 g), and brine (10 g) and then concentrated under reduced pressure to afford ER-803896 (99% wt/wt assay yield, Run 13).

Reference of batch reaction example 2 (Run 14)

Synthesis of ER-803896

ER-803895 (1.00 g, 1.30 mmol) and BHT (7.2 mg, 0.032 mmol) were dissolved in toluene (16.2 mL) and cooled to <-70 °C under a nitrogen atmosphere. DIBAL-H (1.0 M in toluene, 1.54 ml, 1.55 mmol, 1.2 eq) was added at a rate to maintain the internal reaction temperature at <-70 °C. The resulting mixture was stirred for 1 hour and then quenched sequentially with anhydrous acetone (0.31 mL) in toluene (0.70 mL) and anhydrous methanol (0.17 mL) in toluene (0.70 mL), maintaining the internal reaction temperature at <-65 °C. The reaction mixture was allowed to warm to -30 °C, and then MTBE (5.0 mL) and 1 N HC1 (10.0 mL) were added to the reaction mixture. The mixture was stirred for 30 minutes, and the aqueous layer was drained. The organic layer was washed sequentially with 1 N HCl (10 mL), water (10 mL), 5% aqueous NaHC0₃ (10 g), and brine (10 g) and then concentrated under reduced pressure to afford ER-803896 (96% wt/wt assay yield, Run 14).

Reference of batch reaction example 3 (Run 15)

Synthesis of ER-803896

ER-803895 (1.00 g, 1.30 mmol) and BHT (7.2 mg, 0.032 mmol) were dissolved in toluene (16.2 mL) and cooled to <-60 °C under nitrogen atmosphere. DIBAL-H (1.0 M in toluene, 1.54 ml, 1.55 mmol, 1.2 eq) was added at a rate to maintain the internal reaction temperature at <-60 °C. The resulting mixture was stirred for 1 hour and then quenched sequentially with anhydrous acetone (0.31 mL) in toluene (0.70 mL) and anhydrous methanol (0.17 mL) in toluene (0.70 mL), maintaining the internal reaction temperature at <-65 °C. The reaction mixture was allowed to warm to -30 °C, and then MTBE (5.0 mL) and 1 N HCl (10.0 mL) were added to the reaction mixture. The mixture was stirred for 30 minutes, and the aqueous layer was drained. The organic layer was washed sequentially with 1 N HCl (10 mL), water (10 mL), 5% aqueous NaHC0₃ (10 g), and brine (10 g) and then concentrated under reduced pressure to afford ER-803896 (91% wt/wt assay yield, Run 15).

Table 1

Example 3

Synthesis of ER-804029

A micro flow reaction was carried out in the system of Figure 3. The coupling reaction of ER-804028 and ER-803896 using n-butyl lithium (n-BuLi) was initiated in a series of CMPS- a02 chips. Inlets of the CMPS chips were connected by PTFE tubing (internal diameter: 1.5 mm, length: 2.0 m) to a Micro Process Server MPS-a200 having injection syringes. The outlet of the first chip (A) was connected to an inlet of the second chip (B) via SUS316 tubing (internal diameter: 0.8 mm, length: 50 cm) or PTFE tubing (internal diameter: 1.0 mm, length: 32 cm or 64 cm or 96 cm). The outlet of the second chip was connected by PTFE tubing (internal diameter: 1.0 mm, length: 2.0 m or 4.0 m or 6.0 m) to a collection vessel.

A 25 mL injection syringe was filled with a solution of 8.00 g (9.397 mmol) of ER- _L 804028 and 80 mL of anhydrous THF; a 10 mL injection syringe was filled with a 1.63 M solution of n-BuLi in hexane; and an additional 25 mL injection syringe was filled with a solution of 7.659 g (10.337 mmol) of ER-803896 and 76.6 mL of n-heptane.

The contents of the three syringes were transferred to the microreactor using the MPS- a200 system. Flow rates of ER-804028, ER-803896, and n-BuLi were set in various ranges, and the reactions were carried out at -10 °C to 10 °C for various residence times. The resultant reaction mixture was quenched with 14% aqueous ammonium chloride and extracted with MTBE. The purity of the organic layer was analyzed by HPLC. ER-804029 was obtained with high conversion under Run 12 compared with batch reaction condition (Table 2). For Table 2, residence times were determined based on the time required for reactants to pass through the tubing connecting the outlet of the first CMPS-a02 chip and the point in the second CMPS- 02 chip where ER-803896 is added.

Table 2

Other Embodiments

All publications, patents, and patent application publications mentioned herein are hereby incorporated by reference. Various modifications and variations of the described compounds of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant art are intended to be within the scope of the invention.

Claims

1 . A method of producing a compound of Formula (II) from a compound of Formula (I), said method comprising the steps of contacting the compound of Formula (I):

with a reducing agent in a microreactor for a time sufficient to produce the compound of Formula (II):

wherein each of PG₃, PG₄, and PG₅ is independently a hydroxyl protecting group; i is C I - alkyl; and LGi is a leaving group.

2. The method of claim 1 , wherein one, two, or three of PG₃, PG₄, and PG5 of Formula (I) or Formula (II), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers.

3. The method of claim 1, wherein one, two, or three of PG₃, PG₄, and PG₅ of Formula (I) or Formula (II) are t-butyldimethylsilyl (TBS) or benzyl.

4. The method of claim 1 , wherein all of PG₃, PG₄, and PG5 of Formula (I) or Formula (II) are t-butyldimethylsilyl (TBS).

5. The method of claim 1 , wherein LGi is a halogen, (C 1 -C6)alkylsulfonate, (C6- C10 aryl or C1 -C6 heteroaryl)sulfonate, (C6-C15)aryl(Cl -C6)alkylsulfonate, or (C l - C6)heteroaryl(C 1 -C6)alkylsulfonate.

6. The method of claim 1 , wherein LGi is mesylate, toluenesulfonate,

isopropylsulfonate, phenylsulfonate, or benzylsulfonate.

7. The method of claim 1 , wherein LGi is iodide.

8. The method of claim 1 , wherein the compounds of Formula (I) and Formula (II) have the absolute stereochemistry:

(I)-A (II)-A.

9. The method of claim 1 , wherein the compound of Formula (I) is

ER-803895.

10. The method of claim 1 , wherein the compound of Formula (II) is

ER-803896.

1 1. The method of claim 1 , wherein the reducing agent is an aluminum hydride reagent or a borohydride reagent.

12. The method of claim 1, wherein the reducing agent is lithium aluminum hydride, sodium aluminum hydride, diisobutylaluminum hydride (DBAL-H), sodium bis(2- methoxyethoxy)aluminum hydride (Red Al), sodium borohydride, potassium borohydride, rubidium borohydride, cesium borohydride, or sodium cyanoborohydride.

13. The method of claim 1, wherein the temperature of the microreactor is between -80 and -20 °C.

14. The method of claim 1, wherein the residence time of the compound of Formula I is 0.01 sec to 1 sec.

15. A method of producing a compound of Formula (IV) from a compound of Formula (II) and a compound of Formula (III), said method comprising the steps of

(a) contacting the compound of Formula (III):

with a base in a microreactor for a time sufficient to deprotonate the compound of formula (III), wherein each of PGi and PG₂ is independently a hydroxyl protecting group, and PG₆ is hydrogen or a hydroxyl protecting group; and

(b) contacting the product of step (a) with a compound of Formula (II):

wherein each of PG₃, PG4, and PG₅ is independently a hydroxyl protecting group; and LGi is a leaving group.

16. The method of claim 15, wherein one or both of PGi and PG₂ of Formula (III), taken with the oxygen atom(s) to which they are bound, are silyl ethers or arylalkyl ethers.

17. The method of claim 15, wherein one or both of PGi and PG₂ are TBS or benzyl.

18. The method of claim 15, wherein both PG, and PG₂ are TBS.

19. The method of claim 15, wherein the compound of Formula (III) has the absolute stereochemistry:

The method of claim 15, wherein the compound of Formula (III) is ER-804028

ER-804028

21. The method of claim 15, wherein the compound of Formula (I) has the absolute stereochemistry:

The method of claim 15, wherein the compound of Formula (II)

ER-803896.

23. The method of claim 15, wherein the compound of Formula (IV) has the stereochemistry:

(IV)-A.

The method of claim 15, wherein the compound of Formula IV is ER-804029

ER-804029.

25. The method of claim 15, wherein the base is an organometallic reagent.

26. The method of claim 15, wherein the base is a Grignard's reagent, potassium bis(trimethylsilyl) amide, sodium bis(trimethylsilyl)amide, lithium bis(trimethylsilyl)amide, lithium diisopropylamide, ra-butyl lithium, sec-butyl lithium, or tert-butyl lithium.

27. The method of claim 15, wherein the temperature of the microreactor in step (a) and/or step (b) is between -80 to 20 °C.

28. The method of claim 15, wherein the residence time of the compound of Formula (III) in step (a) is 0.1 sec to 20 sec.

29. The method of claim 15, wherein the residence time of the compound of Formula (II) and the compound of Formula (III) in step (b) is 0.1 sec to 20 sec.

30. A method of producing eribulin, or a pharmaceutically acceptable salt thereof, said method comprising the steps of:

(a) producing a compound of Formula (II) by the method of any one of claims 1-14;

(b) reacting the compound of Formula (II) under suitable conditions to produce eribulin, or the pharmaceutically acceptable salt thereof.

31. The method of claim 30, wherein in step (b) the compound of Formula (II) is coupled to a compound of Formula (III) to produce a compound of Formula (IV), and said compound of Formula (IV) is reacted to produce eribulin, or the pharmaceutically acceptable salt thereof.

32. The method of claim 30, wherein eribulin mesylate is formed in step (b).

33. A method of producing eribulin, or a pharmaceutically acceptable salt thereof, said method comprising the steps of:

(a) producing a compound of Formula (IV) by the method of any one of claims 15-29;

(b) reacting the compound of Formula (IV) under suitable conditions to produce eribulin, or the pharmaceutically acceptable salt thereof.

34. The method of claim 33, wherein eribulin mesylate is formed in step (b).