Methods useful in the synthesis of halichondrin B analogs

Abstract

In general, the present invention features improved methods useful for the synthesis of analogs of halichondrin B, such as eribulin and pharmaceutically acceptable salts thereof (e.g., eribulin mesylate).

Claims

1. A method of preparing an intermediate in the synthesis of eribulin, said method comprising reacting ER-076349: ##STR00031## with a sulfonylating reagent in the presence of a metal catalyst to produce the intermediate: ##STR00032## wherein R.sub.6 is sulfonyl, wherein sulfonyl is S(O).sub.2R, wherein R is alkyl, alkenyl, aryl, arylalkyl, or silyl.

2. The method of claim 1, wherein the sulfonylating reagent is tosyl chloride.

3. The method of claim 1, wherein the reacting occurs in acetonitrile.

4. The method of claim 1, wherein said metal catalyst is dibutyltin oxide.

5. The method of claim 1, wherein the reacting occurs above 0 C.

6. The method of claim 1, wherein ER-076349 is produced by reacting ER-811475: ##STR00033## with a conjugate acid of imidazole.

7. A method of producing eribulin, said method comprising the steps of: a) producing the intermediate ER-082892: ##STR00034## by reacting ER-076349: ##STR00035## with a sulfonylating reagent in the presence of a metal catalyst; and b) aminating ER-082892 to produce eribulin: ##STR00036##

8. The method of claim 7, further comprising salifying eribulin to produce a pharmaceutically acceptable salt of eribulin.

9. The method of claim 8, wherein said salt is the mesylate salt.

10. The method of claim 7, wherein the sulfonylating reagent is tosyl chloride.

11. The method of claim 7, wherein the reacting occurs in acetonitrile.

12. The method of claim 7, wherein said metal catalyst is dibutyltin oxide.

13. The method of claim 7, wherein the reacting occurs above 0 C.

14. The method of claim 7, wherein ER-076349 is produced by reacting ER-811475: ##STR00037## with a conjugate acid of imidazole.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides methods for the synthesis of halichondrin B analogs. In particular, the methods are useful for the synthesis of eribulin and pharmaceutically acceptable salts thereof:

(2) ##STR00007##
Synthesis of Compounds of Formula (I)

(3) Compounds of Formula (I):

(4) ##STR00008##
can be synthesized using methods known in the art (e.g., as described in U.S. Pat. Nos. 6,214,865, 6,365,759, 6,469,182, 7,982,060, and 8,148,554, International Publication Nos. WO 99/65894, WO 2005/118565, and WO 2011/094339, Chase et al. Syn. Lett. 2013; 24(3):323-326, Austad et al. Syn. Lett. 2013; 24(3):327-332, and Austad et al. Syn. Lett. 2013; 24(3):333-337, the syntheses of which are incorporated herein by reference). In one example, the C14-C35 portion (e.g., ER-804028) of the molecule is coupled to the C1-C13 portion (e.g., ER-803896) to produce the C1-C35 acyclic intermediate (e.g., ER-804029), and additional reactions are carried out to produce a compound of formula (I) (e.g., ER-118046) as shown in Scheme 1:

(5) ##STR00009##

(6) Other compounds of Formula I can be produced by using different protecting groups in the C1-C13 and/or C14-C35 fragments.

(7) In one specific example, deprotonation, e.g., by lithiation, of the C14-C35 sulfone fragment (i.e., ER-804028) followed by coupling to the C1-C13 aldehyde fragment (i.e., ER-803896) furnishes a mixture of diastereomeric alcohols (i.e., ER-804029). 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 furnishes a compound of formula (I) (i.e., ER-118046).

(8) Conversion of a Compound of Formula (I) to Eribulin

(9) A scheme for converting a compound of formula (I) to eribulin is as follows (Scheme 2).

(10) ##STR00010## ##STR00011##

(11) As outlined in Scheme 2, deprotection of the silyl ether hydroxyl protecting groups (i.e., R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5) of a compound of formula (I) followed by equilibration furnishes ER-811475 (Step A). Ketalization of ER-811475 provides ER-076349 (Step B). Activation of the C35 primary alcohol (e.g., as the C35 tosylate) resulting in a compound of formula (II), wherein X is a leaving group (e.g., halogen, mesylate, or tosylate) (Step C), followed by introduction of the amine functionality, provides eribulin (Step D). One skilled in the art would also understand that variations on the above scheme are possible.

(12) Step A: Conversion of a Compound of Formula (I) to ER-811475

(13) Method A1: Deprotection with Fluoride Source in THF

(14) One method for the conversion of a compound of formula (I) to ER-811475 is shown in Scheme 3:

(15) ##STR00012##

(16) Treatment of a compound of formula (I) with a fluoride source (e.g., tetrabutylammonium fluoride) and equilibration with a conjugate acid of imidazole (e.g., imidazole hydrochloride), in tetrahydrofuran as solvent, results in ER-811475 in a 4:1 mixture with its C12 stereoisomer ER-811474.

(17) Method A2: Deprotection with Fluoride Source in an Amide, e.g., DMAC

(18) An alternative method for the conversion of a compound of formula (I) to ER-811475 is shown in Scheme 4:

(19) ##STR00013##

(20) Treatment of a compound of formula (I) with a fluoride source (e.g., tetrabutylammonium fluoride) and equilibration with a conjugate acid of imidazole (e.g., imidazole hydrochloride), in an amide, e.g., N,N-dimethylacetamide (DMAC), as solvent (e.g., a mixture of tetrahyrofuran (THF) and DMAC), results in ER-811475. The addition of DMAC as co-solvent in the reaction results in improved selectivity at C12 (e.g., 18:1 vs. 4:1) and shortened reaction time (e.g., 1-2 days from 7-10 days). The addition of the mixture of acetonitrile and water increases the yield of ER-811475. Other amides include an N,N C1-C6 dialkyl C1-C6 alkyl amide or N C1-C6 alkyl C2-C6 lactam, such as N,N-dimethylformamide, N-methyl 2-pyrrolidone, N,N-diethylacetamide, or N,N-dimethylpropionamide may also be employed.

(21) Step 8: Ketalization of ER-811475 to ER-076349

(22) Method B1: Ketalization with Conjugate Acid of Pyridine

(23) A method for the ketalization of ER-811475 is shown in Scheme 5:

(24) ##STR00014##

(25) Ketalization of ER-811475 (e.g., in dichloromethane) with a conjugate acid of pyridine (e.g., pyridinium p-toluenesulfonate (PPTS)), followed by crystallization from acetonitrile and water, provides ER-076349.

(26) Method B2: Ketalization with Conjugate Acid of Imidazole

(27) An alternative method for the ketalization of ER-811475 to ER-076349 is shown in Scheme 6:

(28) ##STR00015##

(29) Conversion of ER-811475 to ER-076349 can be achieved through ketalization of ER-811475 (e.g., in ethanol) with a conjugate acid of imidazole (e.g., imidazole hydrochloride), followed by column chromatography. Replacing PPTS with imidazole hydrochloride results in a decrease of isomerization at C12 during post-processing (e.g., concentration of the reaction mixture). Changing of the solvent from dichloromethane to ethanol results in a more environmentally favorable process.

(30) Step C: Activation of ER-076349 to a Compound of Formula (II)

(31) Method C1: Activation with Tosyl Chloride and Pyridine

(32) A method for the activation of ER-076349 is shown in Scheme 7:

(33) ##STR00016##

(34) Reacting ER-076349 (e.g., in dichloromethane) with tosyl chloride and a base (e.g., pyridine) at 22 C. provides a compound of formula (II) (i.e., ER-082892).

(35) Method C2: Activation with Ts.sub.2O, Collidine, and Pyridine

(36) An alternative method for the activation of ER-076349 is shown in Scheme 8:

(37) ##STR00017##

(38) Treatment of ER-076349 (e.g., in dichloromethane) with 4-toluenesulfonic anhydride (Ts.sub.2O), and base (e.g., a combination of 2,4,6-collidine and pyridine) at 10 C. provides a compound of formula (II) (i.e., ER-082892).

(39) Method C3: Activation with Mesyl Chloride

(40) Another method for the activation of ER-076349 is shown in Scheme 9:

(41) ##STR00018##
Reacting ER-076349 (e.g., in dichloromethane) with mesyl chloride and a base (e.g., 2,4,6-collidine) at 0 C. provides a compound of formula (II) (i.e., B-2294).

(42) Method C4: Activation with Tosyl Chloride and Base

(43) Another method for the activation of ER-076349 is shown in Scheme 10:

(44) ##STR00019##

(45) The activation of ER-076349 (e.g., in acetonitrile) can be achieved by treatment (e.g., at 26 to 28 C.) with tosyl chloride and a base (e.g., a C1-C6 trialkylamine, such as triethylamine and N,N-diisopropylethylamine) in the presence of a catalyst (e.g., dibutyltin oxide). The use of dibutyltin oxide, for example, makes the process more robust (e.g., reduces reaction sensitivity to moisture) and improves process operational efficiency (e.g., by elimination of an azeotropic drying step). Replacing pyridine and/or collidine with N,N-diisopropylethylamine and the addition of dibutyltin oxide as a catalyst provide an improvement in selectivity for primary alcohol (e.g., the mono-tosylation:di-tosylation ratio improved from 96:4 to 99.8:0.2). The replacement of dichloromethane with acetonitrile as solvent results in a more environmentally favorable process, and the change in temperature from 10 C. to 26 C.-28 C. increases operational efficiency and yield.

(46) Step D: Amination of a Compound of Formula (II) to Eribulin

(47) Method D1: Staudinger Route

(48) A method for the amination of a compound of formula (II) is shown in Scheme 11, wherein X is a leaving group (e.g., OTs):

(49) ##STR00020##

(50) The amination of a compound of formula (II) (e.g., ER-082892) to eribulin can be achieved through treatment with sodium azide, followed by reduction of the resulting azide with trimethylphosphine under Staudinger reaction conditions.

(51) Method D2: Epoxide Opening Route

(52) An alternative method for the amination of a compound of formula (II) to eribulin is shown in Scheme 12, wherein X is a leaving group (e.g., OTs):

(53) ##STR00021##

(54) In this method, the amination of a compound of formula (II) (e.g., ER-082892) can be accomplished through treatment with alcoholic ammonium hydroxide resulting in cyclization to an epoxide in situ that reacts further with ammonia to provide eribulin. Replacement of the Staudinger route with the epoxide opening route results in the elimination of the use of hazardous reagents and an increase in operational efficiency.

(55) Salification of Eribulin

(56) 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 acid). In one example, eribulin is treated with a solution of methanesulfonic acid (i.e., MsOH) and ammonium hydroxide in water and acetonitrile. The mixture is concentrated. The residue is dissolved in dichloromethane-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 13.

(57) ##STR00022##

(58) Any combination of the methods described above for the synthesis of the various intermediates can be utilized to convert a compound of formula (I) to eribulin (e.g., Methods A1-B2-C1-D1, A1-B2-C2-D1, A1-B2-C1-D2, A2-B1-C1-D1, A2-B2-C1-D1, A2-B1-C2-D1, A2-B1-C1-D2, A2-B2-C2-D1, A2-B2-C1-D2, A2-B1-C2-D2, A2-B2-C2-D2, A2-B1-C3-D1, A1-B2-C3-D1, A2-B2-C3-D1, A2-B1-C3-D2, A1-B2-C3-D2, A2-B2-C3-D2, A1-B1-C4-D1, A2-B1-C4-D1, A1-B2-C4-D1, A1-B1-C4-D2, A2-B2-C4-D1, A2-B1-C4-D2, A1-B2-C4-D2, and A2-B2-C4-D2,).

(59) Experimental Procedures

(60) Step A: Conversion of ER-118046 to ER-811475

(61) Method A1:

(62) ER-811475: (1R,2S,3S,4S,5S,6RS,11S,14S,17S,19R,21R,23S,25R,26R,27S,31R,34S)-25-[(2S)-2,3-Dihydroxypropyl]-2,5-dihydroxy-26-methoxy-19-methyl-13,20-bis(methylene)-24,35,36,37,38,39-hexaoxaheptacyclo[29.3.1.13,6.14,34.111,14.117,21.023,27]nonatriacontane-8,29-dione

(63) ##STR00023##

(64) The solution of ER-118046 (0.580 kg, 0.439 mol, 1 eq) in n-heptane was concentrated in vacuo at 50 C. The residue was dissolved in anhydrous tetrahydrofuran (THF) (19.7 L) and treated with tetrabutyl ammonium fluoride (TBAF) (1.0 M solution in THF, 2.85 L, 2.85 mol, 6.5 eq) buffered with imidazole hydrochloride (0.142 kg, 1.36 mol, 3.1 eq) at 10-25 C. Upon confirmation of the level of C34/C35-diol (3%), toluene (7.6 kg) and water (8.7 kg) were added for extraction. The aqueous layer was separated and extracted with toluene (5.0 kg) and THF (5.2 kg). The aqueous layer was drained, and the organic layer was combined with the first extract. The combined organic layers were concentrated in vacuo at 35 C. During the concentration, the free pentaol was converted to ER-811475 and ER-811474. When the residual level of free pentaol was 5%, acetonitrile (ACN) (3.3 kg) and water (0.42 kg) were added and azeotroped in vacuo <35 C. until the level came down to <5%. Upon completion, the residue was further azeotroped in vacuo with acetonitrile (4.6 kg)<35 C. The residue was diluted with dichloromethane (7.7 kg) and azeotroped in vacuo <35 C. to give a mixture of ER-811475 and ER-811474 (4:1).

(65) Method A2:

(66) ##STR00024##

(67) The solution of enone ER-118046 (135 g) in n-heptane was concentrated in vacuo at 41 C. or below. The residue was dissolved in anhydrous tetrahydrofuran (THF) (2.03 L) and N,N-dimethylacetamide (675 mL) and then treated with tetrabutylammonium fluoride (TBAF) (0.97 mol/L solution in THF, 685 mL) buffered with imidazole HCl (31.5 g) at 16 C. to 18 C. The mixture was stirred at 16 C. to 18 C. for 47 hours, and the reaction progress was monitored by HPLC. After the residual level of the reaction intermediate C34/C35-diol reached 3% or below, acetonitrile (608 mL) and water (203 mL) were added. The mixture was stirred at 16 C. to 18 C. for 45 hours until the residual level of free pentaol came down to below 5%. The reaction mixture including ER-811475/ER-811474 (a mixture of two diastereomers 18:1) could be used for the next stage without further purification.

(68) Step B: Ketalization of ER-811475 to ER-076349

(69) Method B1:

(70) ER-076349: (1S,3S,6S,9S,12S,14R,16R,18S,20R,21R,22S,26R,29S,31R,32S,33R,35R,36S)-20-[(2S)- 2,3-Dihydroxypropyl]-21-methoxy-14-methyl-8,15-bis(methylene)-2,19,30,34,37,39,40,41-octaoxanonacyclo[24.9.2.13,32.13,33.16,9.112,16.018,22.029,36.031,35]hentetracontan-24-one

(71) ##STR00025##

(72) ER-811475 in a mixture with ER-811474 (0.329 kg, 0.439 mol, 1 eq) was dissolved in dichloromethane (DCM; 7.7 kg) and treated with a pyridinium p-toluenesulfonate (PPTS; 0.607 kg, 2.42 mol, 5.5 eq) solution in dichloromethane (1.7 kg) at 10-20 C. The resulting mixture was stirred at 10-20 C. The major diastereomer reacted to provide diol ER-076349, and the minor diastereomer ER-811474 remained unreacted. When the residual level of ER-811475 was >1%, additional PPTS (0.055 kg) in dichloromethane (0.15 kg) was added, and the reaction was continued at 10-20 C. Upon completion, the reaction mixture was directly loaded onto a silica gel column that was pre-equilibrated with methyl t-butyl ether (MTBE) (200 L). The reactor was further rinsed with dichloromethane (3.1 kg), and the rinse was loaded onto the column. The column was eluted sequentially with: (1) methyl t-butyl ether (125 L), (2) 96% v/v methyl t-butyl ether in acetonitrile (125 L), (3) 50% v/v methyl t-butyl ether in acetonitrile (250 L), and (4) acetonitrile (225 L). Desired fractions were combined, concentrated in vacuo <35 C., and azeotroped in vacuo with acetonitrile (4.6 kg) <35 C. The residue was dissolved in acetonitrile (0.32 kg) and water (0.54 kg) and subjected to crystallization with ER-076349 seed crystals (0.27 g, 0.36 mmol) and additional water (2.70 kg). The resulting crystals were filtered, and the weight of the filtrate was monitored until the recovery ratio to the crystallization solvent was reached 80%. The crystals were further washed with water (2.7 kg) and dissolved in dichloromethane (10.8 kg), and the solution was concentrated in vacuo at 25 C. The residue was diluted with acetonitrile (2.1 kg) and concentrated in vacuo at 40 C. to give ER-076349 (55-75% yield from ER-118046).

(73) Method B2:

(74) ##STR00026##

(75) To ER-811475 (in a mixture with ER-811474), a solution of imidazole HCl (85.5 g) in water (68 mL) was added. The solution was concentrated in vacuo at 28 C. or below. The residue was dissolved in EtOH (2.69 kg). The resulting mixture was stirred at 21 C. to 24 C. for 43 hours. The major diastereomer (ER-811475) reacted to provide diol ER-076349, and the minor diastereomer (ER-811474) remains unreacted. The reaction was monitored for a disappearance of ER-811475 by HPLC. After the residual level of ER-811475 reached below 1%, the solution was concentrated in vacuo at 37 C. or below. Toluene (1.35 L) was added, and the solution was azeotroped in vacuo at 37 C. or below. Tetrahydrofuran (THF) (4.20 kg), toluene (1.76 kg), and water (2.03 L) were added and extracted. The aqueous layer was separated, and the organic layer was washed with water (1.01 L). The aqueous layers were combined and extracted with toluene (1.18 kg) and THF (1.20 kg). The aqueous layer was drained, and the organic layer was combined with the first extract. The combined organic layers were concentrated in vacuo at 37 C. or below. Toluene (675 mL) was added, and the solution was azeotroped in vacuo at 38 C. or below. The concentrate was diluted with dichloromethane (1.01 L) and then loaded onto a silica gel column (5.511 kg) pre-equilibrated with methyl t-butyl ether (more than 55.1 L). The column was eluted sequentially with methyl t-butyl ether (40.8 L), 95% v/v methyl t-butyl ether in acetonitrile (24.9 L), 40% v/v methyl t-butyl ether in acetonitrile (83.6 L), and acetonitrile (76.3 L) to remove the unreacted intermediates, the reaction impurities, and the carryover impurities from ER-804028. Desired fractions were combined and concentrated in vacuo at 32 C. or below to give ER-076349 (assay 62.02 g, yield over two steps 84.0%). The residue was azeotroped in vacuo with acetonitrile (0.533 kg) at 29 C. or below and could be used for the next stage without further purification.

(76) Steps C and D: Conversion of ER-076349 to Eribulin:

(77) Methods C2+D2:

(78) eribulin: (1S,3S,6S,9S,12S,14R,16R,18S,20R,21R,22S,26R,29S,31R,32S,33R,35R,36S)-20-[(2S)-3-Amino-2-hydroxypropyl]-21-methoxy-14-methyl-8,15-bis(methylene)-2,19,30,34,37,39,40,41-octaoxanonacyclo[24.9.2.13,32.13,33.16,9.112,16.018,22.029,36.031,35]hentetracontan-24-one

(79) ##STR00027##

(80) ER-076349 (0.259 kg, 0.354 mol, 1 eq) was dissolved in toluene (4.7 kg) and azeotroped in vacuo at <25 C. The residue was diluted with toluene (4.5 kg) to give a toluene solution for monitoring of water content. Water content was measured by Karl-Fischer (KF) titration method. If the KF value was >125 ppm, the solution was azeotroped in vacuo at <25 C. and diluted with toluene (4.5 kg) until the water content came down to 125 ppm. If the KF value reached the target value, the solution was concentrated and dissolved in anhydrous dichloromethane (6.5 kg). 2,4,6-collidine (0.172 kg, 1.27 mol, 4 eq) and pyridine (0.0014 g, 0.018 mol, 0.05 eq) in anhydrous dichloromethane (84.1 g) were added, and the mixture was cooled. A solution of Ts.sub.2O (0.124 kg, 0.380 mol, 1.07 eq) in anhydrous dichloromethane (3.4 kg) was added to the reaction mixture at a rate to maintain the reaction temperature at 10 C., and the mixture was stirred at 10 C. When the residual amount of ER-076349 was <3% or the generation of corresponding bis-tosylate was more than 4%, the reaction mixture was quenched by the addition of water (1.0 kg). The mixture was warmed up, and then isopropyl alcohol (IPA) (20.5 kg) and ammonium hydroxide (NH.sub.4OH; 25.7 kg) were added consecutively at 10-30 C. Upon complete consumption of the epoxide (target0.85%; add extra NH.sub.4OH if necessary), the reaction mixture was concentrated in vacuo at <30 C. To the residue, dichloromethane (20.7 kg) and a sufficient amount of buffer solution of NaHCO.sub.3/Na.sub.2CO.sub.3/water (9/9/182 w/w/w; not more than 5.166 kg) were added and extracted. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (8.6 kg). The organic layer was separated and combined with the first extract. The combined organic layers were concentrated in vacuo at <30 C. The concentrate was diluted with acetonitrile (4.0 kg) and then loaded onto silica gel column which was preequilibrated with acetonitrile (200 L). The column was eluted sequentially with: (1) acetonitrile (100 L), (2) 90.0/7.5/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (152.4 L), (3) 85.8/11.7/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (152.4 L), (4) 83.5/14.0/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (152.6 L), and (5) 80.0/17.6/2.4 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (>100.2 L). Desired fractions were combined and concentrated in vacuo at 40 C. while maintaining the internal pH at 5.5-9.0 by adding NH.sub.4OH. To the residue, dichloromethane (13.9 kg) and a sufficient amount of buffer solution of NaHCO.sub.3/Na.sub.2CO.sub.3/water (9/9/182 w/w/w; not more than 15.51 kg) were added and extracted. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (8.7 kg). The organic layer was separated and combined with the first extract. The combined organic layers were concentrated in vacuo at <30 C. The residue was dissolved in 75% v/v anhydrous dichloromethane in n-pentane (6.12 kg) and filtered. The filtrate was concentrated in vacuo at <30 C., diluted with acetonitrile (2.1 kg), and concentrated in vacuo at 35 C. to give eribulin (75-95% yield).

(81) Methods C3+D1:

(82) ##STR00028##

(83) MsCl (0.3 M in CH.sub.2Cl.sub.2, 98 L, 0.030 mmol) was added dropwise over 40 min to a mixture of 2,4,6-collidine (7 L, 0.054 mmol), ER-076349 (20.8 mg, 0.028 mmol), and CH.sub.2Cl.sub.2 (1 mL) at 0 C. After 76 h at 4 C., the reaction was quenched with a 1:4 mixture of saturated aqueous NaHCO.sub.3-brine and extracted with CH.sub.2Cl.sub.2 (4). The combined extracts were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was dissolved in toluene (3 mL), concentrated, and purified by preparative TLC (1.5% MeOH-EtOAc) to afford mesylate B-2294 (21.4 mg, 95%). Tetra-n-butylammonium azide (0.2 M in dimethylformamide, 0.5 mL, 0.10 mmol) was added to a solution of mesylate B-2294 (21.4 mg, 0.026 mmol) in dimethylformamide (2 mL) at room temperature and warmed to 83 C. After stirring at 83 C. for 3.5 h, the reaction mixture was cooled to room temperature, diluted with toluene, concentrated and purified by preparative TLC (80% ethyl acetate-hexanes) to furnish B-1922 (18 mg, 92%). Me.sub.3P (1 M in tetrahydrofuran) and H.sub.2O (0.8 mL) were sequentially added to a solution of azide B-1922 (24.6 mg, 0.032 mmol) in THF (3.2 mL) at room temperature. The mixture was stirred for 22 h, diluted with toluene, concentrated and purified by flash chromatography [step gradient, 10% MeOH-EtOAc followed by MeOH-EtOAc-30% aqueous NH.sub.4OH (9:86:5)] to provide the desired primary amine (23.3 mg), which by .sup.1H-NMR contained 1% trimethylphosphine oxide. Lyophilization from benzene and standing under high vacuum for 2 d furnished eribulin (20.3 mg, 87%).

(84) Methods C4+D2:

(85) ##STR00029##

(86) The diol ER-076349 (58.3 g) was dissolved in acetonitrile (935 mL). A suspension of dibutyltin oxide (0.99 g) and N,N-diisopropylethylamine (28.5 mL) in acetonitrile (117 mL) were added. A solution of TsCl (30.5 g) in acetonitrile (117 mL) was added to the reaction mixture at a rate to maintain the reaction temperature at 26 C. to 28 C., and the mixture was stirred at 26 C. to 28 C. The reaction was monitored by HPLC for consumption of ER-076349. After the residual level of ER-076349 reached below 3% and reaction time passed over 27 hours, isopropyl alcohol (IPA) (4.58 kg) and ammonium hydroxide (5.82 kg) were added consecutively at 15 C. to 20 C. The mixture was stirred at 15 C. to 20 C. for 66 hours, and the reaction was monitored by HPLC for a consumption of reaction intermediate ER-809681. After the residual level of ER-809681 reached 0.85% or less, the reaction mixture was concentrated in vacuo at 29 C. or below. To the residue, dichloromethane (4.64 kg) and a sufficient amount of buffer solution of NaHCO.sub.3/Na.sub.2CO.sub.3/water (9/9/182 w/w/w) (530 mL) were added and extracted. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (1.94 kg). The organic layer was separated, and combined with the first extract. The combined organic layers were concentrated in vacuo at 25 C. or below. The concentrate was diluted with acetonitrile (1.17 L), and then loaded onto silica gel column (5.511 kg) which was pre-equilibrated with acetonitrile (more than 55.1 L). The column was eluted sequentially with acetonitrile (29.6 L), 90.0/7.5/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (46.2 L), 85.8/11.7/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (45.8 L), 83.5/14.0/2.5 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (46.5 L), 80.0/17.6/2.4 v/v/v acetonitrile/water/200 mM aqueous NH.sub.4OAc (29.8 L) to remove the unreacted intermediates and the reaction impurities. Desired fractions were combined and concentrated in vacuo at 36 C. or below while maintaining the internal pH at 5.5 to 9.0 by adding ammonium hydroxide. To the residue, dichloromethane (3.98 kg) and a sufficient amount of buffer solution of NaHCO.sub.3/Na.sub.2CO.sub.3/water (9/9/182 w/w/w) (2.02 kg) were added and extracted. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (2.48 kg). The organic layer was separated and combined with the first extract. The combined organic layers were concentrated in vacuo at 24 C. or below. The residue was dissolved in 75% v/v anhydrous dichloromethane in n-pentane (1.03 L) and filtered. The filtrate was concentrated in vacuo at 25 C. or below to give eribulin. The residue was diluted with acetonitrile (392 mL) and dichloromethane (69 mL) to give eribulin acetonitrile/dichloromethane solution (assay 49.11 g, corrected yield 85.3%). The solution was concentrated in vacuo at 29 C. or below and used for the next stage.

(87) Salification of Eribulin

(88) Salification to Eribulin Mesylate:

(89) eribulin mesylate: (2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-2-[(2S)-3-Amino-2-hydroxypropyl]-3-methoxy-26-methyl-20,27-dimethylidenehexacosahydro-11,15:18,21:24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-furo[3,2-i]furo[2,3:5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-one methanesulfonate

(90) ##STR00030##

(91) ER-086526-00 (46.68 g) was dissolved in acetonitrile (591 mL) and water (31 mL) and treated with a solution of methanesulfonic acid (MsOH, 4.09 mL) and NH.sub.4OH (187 mL) in acetonitrile (624 mL). The mixture was concentrated in vacuo at 24 C. or below and azeotroped repeatedly with anhydrous acetonitrile (234 mL) in vacuo at 24 C. or below to remove water. The residue was dissolved in 75% v/v anhydrous dichloromethane in n-pentane (1.10 L) and filtered. The filtrate was concentrated in vacuo at 24 C. or below. The residue was dissolved in 50% v/v anhydrous dichloromethane in n-pentane (1.16 L), and the solution was transferred through a filter to anhydrous pentane (3.26 kg) in the separate reactor. The resulting precipitate was stirred for 29 hours. The precipitates were filtered, washed with n-pentane (2.92 kg), and dried under nitrogen flow in vacuo until the residual solvent levels reached the target numbers: n-pentane25000 ppm; 2-methylbutane1000 ppm; 2,2-dimethylbutane1000 ppm; and cyclopentane1000 ppm. After drying, the precipitates were mixed in vacuo to give eribulin mesylate (gross 45.95 g, corrected yield 83.8%). The drug substance was filled in a polytetrafluoroethylene (PTFE) bottle. The PTFE bottle was packed in an aluminum laminate bag.

OTHER EMBODIMENTS

(92) Various modifications and variations of the described methods 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.