DESC:PURIFICATION PROCESS FOR PREPARATION OF ERIBULIN AND INTERMEDIATES THEREOF

INTRODUCTION
Aspects of the present application relate to a purification process for preparation of halichondrin B analogues such as eribulin or a pharmaceutically acceptable salts thereof.
The drug compound having the adopted name eribulin, is a synthetic analogue of halichondrin B, and is represented by structure of formula I.

I
Eribulin is a microtubule inhibitor indicated for the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. U.S. Patent No. 6,214,865 discloses eribulin and its pharmaceutically acceptable salts.
Process for preparation of Eribulin is described in U.S. Patent document No. 6,214,865, PCT publication No.s WO 2005/118565A1, WO 2009/124237A1, WO 2015/000070A1 and WO 2015/085193A1,
The present application provides pure eribulin or pharmaceutically acceptable slats or intermediates thereof. The present application also provide improved purification processes for preparation of Eribulin and intermediates thereof, which are simple and commercially viable.

SUMMARY
In the first embodiment, the present application provides an improved process for preparation of Eribulin of formula I or a pharmaceutically acceptable salt thereof comprising purification of crude compound according to formula (II) or formula (III) or formula (IV) or formula (V) or formula (VI) or formula (VII) or formula (VIII) or formula (IX) or formula (X) by one or more methods selected from isolation, slurrying in a suitable solvent, acid-base treatment, liquid-liquid extraction, chromatography and treating with adsorbent.

In the second embodiment, the present application provides an improved process for preparation of Eribulin or a pharmaceutically acceptable salt comprising purification of crude Eribulin or crude Eribulin salt by one or more methods selected from isolation, slurrying in a suitable solvent, acid-base treatment, liquid-liquid extraction, chromatography and treating with adsorbent.
In the third embodiment, the present application provides an improved process for preparation of Eribulin of formula I or a pharmaceutically acceptable salt thereof comprising:
a) purification of crude compound of formula ((VIII), and
b) converting without isolation the pure compound of formula ((VIII) obtained in step(a) in to Eribulin of formula I or a pharmaceutically acceptable salt thereof.
In the fourth embodiment, the present application provides Eribulin or a salt there of substantially free from one or more impurities as measured by HPLC.
In the fifth embodiment, the present application provides substantially pure compound of formula (VIII) having a purity of not less than 98.0% by HPLC obtained by a process of the present application.
In the sixth embodiment, the present application provides compound of formula (VIII) substantially free from one or more impurities as measured by HPLC obtained by a process of the present application.
In the seventh embodiment, the present application provides substantially pure compound according to formula (II) or formula (III) or formula (IV) or formula (V) or formula (VI) or formula (VII) or formula (VIII) or formula (IX) or formula (X) having a purity of not less than 90% by HPLC obtained by a process of the present application.
In the eighth embodiment, the present application provides substantially pure Eribulin or a salt there of having a purity of not less than 98.5% by HPLC obtained by a process of the present application.
DETAILED DESCRIPTION
In an aspect, the present application provides an improved process for preparation of Eribulin of formula I or a pharmaceutically acceptable salt thereof comprising purification of one or more intermediates thereof mentioned herein by one or more methods selected from isolation, slurrying in a suitable solvent, acid-base treatment, liquid-liquid extraction, chromatography and treating with adsorbents.
In another aspect, the present application provides an improved process for preparation of Eribulin of formula I or a pharmaceutically acceptable salt thereof comprising purification of one or more intermediates thereof by chromatographic techniques selected from column chromatography, flash chromatography, ion exchange chromatography, supercritical fluid chromatography, high performance liquid chromatography (both reverse phase and normal phase), expanded bed adsorption chromatography and simulated moving bed chromatography or a combination thereof.
Suitable isolation methods that may be used for purification of one or more compounds provided in the present application include decantation or filtration or precipitation from a solvent or precipitation by adding an anti-solvent to a solution or by evaporation of solution and the like or any other suitable isolation techniques known in the art. Optionally the said precipitation may result in a crystalline compound including solvates and hydrates thereof. Suitable solvents that may be used for said isolation include water, alcohols, ketones, hydrocarbons, halogenated hydrocarbons, esters, ethers, polar aprotic solvents, nitriles or any mixtures thereof.
Suitable solvents that may be used for purification of one or more compounds provided in the present application by slurrying in a suitable solvent include water, alcohols, ketones, hydrocarbons, halogenated hydrocarbons, esters, ethers, polar aprotic solvents, nitriles or any mixtures thereof.
Purification of one or more of the compounds provided in the present application may be carried out using acid-base treatment. Acid-base treatment may be carried out by treating compounds provided in the first embodiment with a suitable acid or a suitable base to form respective acid or base addition salt of the compounds. The resultant acid or base addition salts of the compounds may be optionally purified by recrystallization or washing with a suitable solvent or slurrying in a suitable solvent. The resulting acid or base addition salts of the compounds are treated with suitable desaltification agents to get the purified compounds. Suitable solvents that may be used for acid-base treatment include water, alcohols, ketones, hydrocarbons, halogenated hydrocarbons, esters, ethers, polar aprotic solvents, nitriles or any mixtures thereof.
Purification of one or more compounds provided in the present application may be carried out by liquid-liquid extraction. In the said process, the compound is dissolved in a suitable first solvent to obtain a solution and the resulting solution is washed with a second solvent that is immiscible with the solution and the pure compound is isolated from the solution obtained after said washing.
Purification of one or more compounds provided in the present application may be carried out using chromatography. Suitable chromatography techniques that may be used for purification of compounds provided in the first and second embodiments include column chromatography, flash chromatography, ion exchange chromatography, supercritical fluid chromatography, high performance liquid chromatography (both reverse phase and normal phase), expanded bed adsorption chromatography, simulated moving bed chromatography or any other chromatographic techniques known in the art.
Suitable solvents that may be used in the chromatographic techniques include water, alcohols, ketones, hydrocarbons, halogenated hydrocarbons, esters, ethers, polar aprotic solvents, nitriles or any mixtures thereof.
Suitable mobile phases including buffers such as trifluoroacetic acid, sulfonate, phosphate, chloroacetate, formate, acetate, ammonium formate, ammonium bicarbonate, borate, Potassium hydrogen phosphate and the like or supercritical gases such as carbon dioxide (CO2), xenon (Xe), nitrous oxide (N2O), sulfur hexafluoride (SF6), ammonia (NH3), water (H2O), ethane (C2H6), propane (C3H8), n-butane (C4H10) and the like in combination with suitable solvents as outlined above may be used in chromatography techniques for separation of impurities from the crude compounds which in turn give rise to substantially pure compounds.
Suitable resins that may be used as adsorbents in the chromatographic techniques include cation exchange resins, anion exchange resins, chelated resins, synthetic adsorbents, non-ionic resins or combinations thereof. The resins may be lipophilic, hydrophilic and/or hydrophobic in nature.
Purification of one or more compounds provided in the present application may be carried out by treating with adsorbents in a batch mode. Suitable adsorbents that may be used for purification of compounds provided in the first and second embodiments include silica gel, activated alumina, molecular sieves, magnesium silicate, synthetic resin, and the like; or any other suitable adsorbents known in the art.
Optionally one or more of the purified intermediates obtained in the process of the present application may be converted without isolation in to Eribulin or a pharmaceutically acceptable salt thereof.
Eribulin or a pharmaceutically acceptable salt or intermediates thereof obtained in the process of the present application are substantially pure as measured by HPLC method.
Eribulin or a pharmaceutically acceptable salt thereof or intermediates thereof are substantially free from one or more impurities as measured by HPLC method.
The HPLC methods that may be followed to measure the purity of compounds obtained in the process of the present application involve the use of columns selected from Torus, Restek Biphenyl, YMC Pro C18, Princeton Diol, Acquity CSH Phenyl Hexyl, ZORBAX Rx-SIL, or any other suitable chromatography columns.
DEFINITIONS
The following definitions are used in connection with the present application unless the context indicates otherwise. In general, the number of carbon atoms present in a given group or compound is designated “Cx-Cy”, where x and y are the lower and upper limits, respectively. For example, a group designated as “C1-C6” contains from 1 to 6 carbon atoms. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions or the like.
An “alcohol” is an organic compound containing a carbon bound to a hydroxyl group. “C1-C6 alcohols” include methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropyl alcohol, ethylene glycol, 1-propanol, 2-propanol (isopropyl alcohol), 2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, cyclohexanol, phenol, glycerol and the like.
A “hydrocarbon solvent” is a liquid hydrocarbon compound, which may be linear, branched, or cyclic and may be saturated or have as many as two double bonds or aromatic. Examples of “C5-C15 aliphatic or aromatic hydrocarbons” include n-pentane, isopentane, neopentane, n-hexane, isohexane, 3-methylpentane, 2,3-dimethylbutane, neohexane, n-heptane, isoheptane, 3-methylhexane, neoheptane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, n-octane, isooctane, 3-methylheptane, neooctane, cyclohexane, methylcyclohexane, cycloheptane, petroleum ethers, benzene toluene, ethylbenzene, m-xylene, o-xylene, p-xylene, indane, naphthalene, tetralin, trimethylbenzene, chlorobenzene, fluorobenzene, trifluorotoluene, anisole, C6-C12 aromatic hydrocarbons and the like.
An “ether” is an organic compound containing an oxygen atom –O- bonded to two other carbon atoms. “C2-C6 ethers” include diethyl ether, diisopropyl ether, methyl t-butyl ether, glyme, diglyme, tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane, dibutyl ether, dimethylfuran, 2-methoxyethanol, 2-ethoxyethanol, anisole and the like.
A “halogenated hydrocarbon” is an organic compound containing a carbon bound to a halogen. Halogenated hydrocarbons include dichloromethane, 1,2-dichloroethane, trichloroethylene, perchloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, chloroform, carbon tetrachloride and the like.
An “ester” is an organic compound containing a carboxyl group -(C=O)-O- bonded to two other carbon atoms. “C3-C10 esters” include ethyl acetate, n-propyl acetate, n-butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, methyl acetate, methyl propanoate, ethyl propanoate, methyl butanoate, ethyl butanoate and the like.
A “ketone” is an organic compound containing a carbonyl group -(C=O)- bonded to two other carbon atoms. “C3-C10 ketones” include acetone, ethyl methyl ketone, diethyl ketone, methyl isobutyl ketone, ketones and the like.
A “nitrile” is an organic compound containing a cyano -(C=N) bonded to another carbon atom. “C2-C6 Nitriles” include acetonitrile, propionitrile, butanenitrile and the like.
A “polar aprotic solvents” include N, N-dimethylformamide, N, N-dimethylacetamide, dimethylsulfoxide, sulfolane, N-methylpyrrolidone and the like;
Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the application in any manner. Reasonable variations of the described procedures are intended to be within the scope of the present application. While particular aspects of the present application have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this application.
“Crude” as used herein refers to a compound having a purity less than the corresponding purified compound.
“Substantially pure” as used herein refers to a compound having a purity of not less than 98.5 % or not less than 99.0% or not less than 99.5 % or not less than 99.7 % or not less than 99.8 % or not less than 99.9 % as measured by HPLC method.
“Substantially free” as used herein refers to a compound that is having one or more individual impurities less than about 0.15% or less than about 0.10% or less than about 0.05% or less than about 0.03% or less than about 0.02% or less than about 0.01% as measured by HPLC method.
EXAMPLES
Example-1: Preparation of 3-{(2S,5S)-5-[2-((2S,4R,6R)-6-{[(2S,3S,4R,5R)-3-[(1RS,2RS)-3-[(2R,4aS,6S,7R,8S,8aS)-7,8-Bis{[tert-butyl(dimethyl)silyl]oxy}-6-((1S,2E)-1-{[tert-butyl(dimethyl)silyl]oxy}-3-iodoprop-2-en-1-yl)octahydropyrano [3,2-b]pyran-2-yl]-2-hydroxy-1-(phenylsulfonyl)propyl]-5-((2S)-2,3-bis{[tert-butyl (dimethyl)silyl]oxy}propyl)-4-methoxytetrahydrofuran-2-yl]methyl}-4-methyl-5-methylenetetrahydro-2H-pyran-2-yl)ethyl]-4-methylenetetrahydrofuran-2-yl}propan-1-ol

3-((2S,5S)-5-(2-((2S,4R,6R)-6-(((2S,3S,4R,5R)-5-((S)-2,3-bis((tert-butyldimethyl silyl)oxy)propyl)-4-methoxy-3-((phenylsulfonyl)methyl)tetrahydro furan-2-yl)methyl) -4-methyl-5-methylenetetrahydro-2H-pyran-2-yl)ethyl)-4-methylenetetrahydrofuran-2-yl)propan-1-ol (4.85 g) is dissolved in anhydrous THF (50 mL) under a nitrogen atmosphere and cooled to 0-4 °C. n-BuLi (1.6 M in hexanes, 7.30 mL) was added to the reaction mass slowly at 0-4 °C and stirred at 0-4°C for 45 minutes. The solution was then cooled to -72°C. A solution of 2-((2R,4aS,6S,7R,8S,8aS)-7,8-bis((tert-butyldimethylsilyl)oxy)-6-((S,E)-1-((tert-butyldimethylsilyl)oxy)-3-iodoallyl)octahydro pyrano[3,2-b]pyran-2-yl)acetaldehyde (5.30 g) in anhydrous n-heptane (50 mL) was slowly added to the reaction mass at -72 to -68 °C and stirred at -72 to -70 °C for 1 hour 45 minutes. 14 wt% aq. NH4Cl (30 mL) was added at below 0 °C. The mixture was then warmed to 18 °C and was diluted with water (30 mL) and MTBE (50 mL). Layers were separated and the aqueous was extracted with MTBE (30 mL). The combined organic layer was washed with water (30 mL) and then concentrated in vacuo. The obtained crude compound was purified using chromatography on a combiflash system using a silica cartridge, eluting with a mixture of EtOAc and heptane to afford the title compound.
Purity by HPLC of crude: 75.1%, Purity by HPLC after combiflash: 98.2%
Example-2: Preparation of 3-((2S,5S)-5-(2-((2S,4R,6R)-6-(((2S,3S,4R,5R)-3-((S)-3-((2R,4aS,6S,7R, 8S,8aS)-7,8-bis((tert-butyldimethylsilyl)oxy)-6-((S,E)-1-((tert-butyl dimethylsilyl)oxy)-3-iodoallyl)octahydropyrano[3,2-b]pyran-2-yl)-2-oxo-1-(phenyl sulfonyl)propyl)-5-((S)-2,3-bis((tert-butyldimethylsilyl)oxy)propyl)-4-methoxy tetrahydrofuran-2-yl)methyl)-4-methyl-5-methylenetetrahydro-2H-pyran-2-yl) ethyl)-4-methylene tetrahydrofuran-2-yl) propanal

Dess-Martin periodinane (1.97 g) was added to a mixture of compound of formula II (1.55 g), NaHCO3 (780 mg) and anhydrous dichloromethane (16 mL) at 15-20 °C. After stirring at 15-20 °C for 2 hours, iso-propanol (500 µL) was added and after stirring for a further 15 min, heptane (30 mL) was added. The reaction suspension was filtered through a sinter funnel, washed with heptane (2 x 10 mL) and concentrated. The resultant compound was purified by chromatography on a silica gel cartridge using a Combiflash system, eluting with a mixture of EtOAc and heptane.
Purity by HPLC of crude: 86%, Purity by HPLC after combiflash: 94%
Example-3: Preparation of 3-((2S,5S)-5-(2-((2S,4R,6R)-6-(((2S,3S,4R,5R)-3-(3-((2R,4aS,6S,7R,8S,8aS)-7,8-bis((tert-butyldimethylsilyl)oxy)-6-((S,E)-1-((tert-butyl dimethylsilyl)oxy)-3-iodoallyl)octahydropyrano[3,2-b]pyran-2-yl)-2-oxopropyl)-5-((S)-2,3-bis((tert-butyldimethylsilyl)oxy)propyl)-4-methoxy tetrahydrofuran-2-yl)methyl)-4-methyl-5-methylenetetrahydro-2H-pyran-2-yl)ethyl)-4-methylene tetrahydrofuran-2-yl)propanal

A solution of samarium iodide (0.08 M in THF; 215 mL) was transferred to a reaction flask under nitrogen, anhydrous THF (135 mL) was added and the solution was cooled to 72°C. A compound of formula III (10.2 g) was dissolved in a mixture of anhydrous THF (50 mL) and anhydrous MeOH (100 mL) under nitrogen and this solution was added to the samarium iodide solution at -65 to -72 °C. The solution was stirred at 72°C for 30 minutes. MTBE (50 mL) and a mixture of K2CO3/Rochelle’s salt/water (1:1:10, 180 mL) was simultaneously added to the reaction mass at below -50 °C. The mixture was diluted with aqueous K2CO3/Rochelle’s salt/water (1:1:10, 190 mL), MTBE (200 mL), and BHT (100 mg) at 16-21 °C and then separated. The aqueous layer was extracted with MTBE (200 mL). The combined organic layer was diluted with heptane (115 mL) before washing with water (150 mL). The organic layer was concentrated in vacuo. The crude material was purified by chromatography on a Combiflash system using a silica cartridge, eluting with a mixture of EtOAc and heptane to afford the title compound.
Purity by HPLC of crude: 76%, Purity by HPLC after combiflash: 96%
Example-4: Preparation of (1R,3S,5R,6R,7S,11R,14S,15S,16S,17R,18S,19S,20E, 22RS,25S,28S,31S,33R)-5-((2S)-2,3-Bis{[tert-butyl(dimethyl)silyl]oxy}propyl)-16, 17,19-tris{[tert-butyl(dimethyl)silyl]oxy}-22-hydroxy-6-methoxy-33-methyl-27,34-bis(methylene)-4,35,36,37,38-pentaoxahexacyclo[29.3.1.111,15.114,18.125,28.03,7]octa triacont-20-en-9-one.

A solution of (S)-N-(2-(4-isopropyl-4,5-dihydrooxazol-2-yl)-6-methylphenyl) methanesulfonamide (4.66 g) in anhydrous acetonitrile (63 mL) was added to chromium(II) chloride (1.93 g) under an inert atmosphere and the resulting suspension was warmed to 28-34 °C . Anhydrous triethylamine (2.2 mL) was added at below 35 °C. The resulting mixture was stirred at 30-35 °C for 60 minutes and then cooled to 15 °C. In a separate flask, the compound of formula IV (2.28 g) was dissolved in a mixture of anhydrous acetonitrile (12 mL) and anhydrous THF (24 mL) under an inert atmosphere. To the suspension containing the chromium chloride and ligand mixture was added NiCl2 (204 mg) followed immediately by the solution of compound of formula IV which was added over 1 hour. After stirring the mixture at 13-15 °C for 3 hours, it was warmed to 20°C and diluted with heptane (58 mL). The mixture was then filtered through a sinter funnel containing a short pad of Celite. The Celite was washed with heptane (46 mL) and acetonitrile (46 mL). The acetonitrile layer was removed and extracted with heptane (69 mL). The heptane layers were combined and then concentrated in vacuo. The crude compound was chromatographed using the combiflash system on a silica cartridge eluting with a mixture of EtOAc and heptane to afford the title compound.
Purity by HPLC of crude: 83%, Purity by HPLC after combiflash: 92.5%
Example-5:Preparation of (1R,3S,5R,6R,7S,11R,14S,15S,16S,17R,18S,19S,20E, 25S,28S,31S,33R)-5-((2S)-2,3-Bis{[tertbutyl(dimethyl)silyl]oxy}propyl)-16,17,19-tris {[tert-butyl(dimethyl)silyl]oxy}-6-methoxy-33-methyl-27,34-bis(methylene) -4,35,36,37,38-pentaoxahexacyclo[29.3.1.111,15.114,18.125,28.03,7]octatriacont-20- ene-9,22-dione

To a solution of compound of formula (V) (4 g) in dichloromethane (45 mL) was added Dess-Martin periodinane (1.48 g) at 18 °C and the resultant suspension was stirred at 20 °C for 1hour 30 minutes. Mixture of 5 wt% of NaHCO3 (30 mL) and 20 wt% Na2SO3 aq. (30 mL) were added and stirred for 15 minutes. Additional 5wt% aq NaHCO3 (30 mL) was added and the mixture was stirred for 5 min. The organic phase was separated and the aqueous layer extracted with dichloromethane (30 mL). The combined organic phase was filtered through a MgSO4 pad (5 g), and the pad was washed with dichloromethane (15 ml) and heptane (15 ml). The filtrate was then concentrated. The crude material was purified on a silica cartridge using a combiflash, eluting with a mixture of MTBE and heptane. The desired fractions were combined, BHT (4 mg) was added and then the solution was concentrated in vacuo to afford the title compound.
Purity by HPLC after combiflash: 97.8%
Example-6:Preparation of (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

A solution of compound of formula VI (3.28 g) in THF (150 mL) was cooled to 10°C. Acetic acid (410 mg) was added, followed immediately TBAF (1 M in THF, 13.6 mL) was added and the resultant reaction mixture was stirred at 10 °C for 49 hours. The reaction was warmed to 18°C and CaCO3 (2.73 g) and Dowex 50WX8-400 (8.19 g) were added. The resulting suspension was stirred at 18 °C for a further 1 h 15 minutes. The mixture was filtered through a pad of Celite (5 g), washing the flask and pad with ethyl acetate (2 x 60 mL). The resulting solution was partially concentrated in vacuo to afford the title compound as a solution in ethyl acetate which was used directly in the next stage.
Example-7:Preparation of (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

To a solution of compound of formula VII and VII(i) (150 mg) in dichloromethane (3 mL) was added PPTS solution (226 mg dissolved in 1 mL dichloromethane) at 25 °C and the resultant reaction mixture was stirred at 25 °C for 6 hours. The reaction mixture is filtered through silica, washed with acetonitrile and concentrated. The concentrated solution in acetonitrile was purified using SFC.
The concentrated solution in acetonitrile of compound of formula VIII (Purity by HPLC: 93.4%) was purified using supercritical fluid chromatography on a Princeton Diol column in acetonitrile. Fractions containing the product were combined and concentrated to give purified compound of formula VIII (purity by HPLC: 99.7%).
Example-8: Preparation of Eribulin.
Pyridine (103 µl of a 0.33M solution prepared in dichloromethane under nitrogen) and collidine (350 µl) were then added to a solution of compound of formula VIII (484 mg) in dichloromethane (9.1 mL) under nitrogen at -20 to -10 °C. A solution of toluenesulfonic anhydride (238 mg dissolved in 5 mL dichloromethane under nitrogen) was added slowly at -10 °C and stirred for 90 minutes at -20 to -10 °C. Water (1.9 mL) was added and the solution was warmed to room temperature. IPA (48 mL) and ammonium hydroxide (53 mL of a 28% solution) was added and the mixture stirred for 23 hours. Further ammonium hydroxide (5.3 mL of a 28% solution) was added and stirred for 17 hours. The resultant mixture was concentrated in vacuo to a total volume of ~30 mL. Dichloromethane (15 ml) was added and washed with a mixture of sodium bicarbonate/sodium carbonate/water (9:9:182 w/w/w, 4.5 g). The aqueous phase was re-extracted with dichloromethane (12 mL) and the combined organic phase was concentrated in vacuo at < 30 °C. Acetonitrile (6 mL) was added to the residue and concentrated in vacuo at < 30 °C. The residue was purified by flash column chromatography (methanol/dichloromethane; methanol/dichloromethane/28% ammonium hydroxide) to afford title compound (352 mg).
The obtained compound was dissolved in dichloromethane (20 mL) and washed with sodium bicarbonate/sodium carbonate/water (9:9:182 w/w/w, 25 mL). The aqueous phase was re-extracted with dichloromethane (10 mL). The combined organic extracts were concentrated in vacuo at < 30 °C. The residue was nitrogen purged and then dissolved in anhydrous dichloromethane/pentane (3:1 v/v, 8.5 mL) prepared under nitrogen. The resulting solution was filtered via a nitrogen-flushed cannula under nitrogen and then washed with in anhydrous dichloromethane/pentane (3:1 v/v, 8.5 mL).The filtrate was concentrated in vacuo at < 30 °C. The residue was dissolved in acetonitrile (2 mL) and concentrated in vacuo at < 35 °C to provide the title compound as a glassy white solid (311 mg, 65%).
Purity by HPLC of crude: 53%
Purity by HPLC after flash chromatography: 98%
Purity by HPLC of final compound: 98.6%
Example-9: Preparation of Eribulin mesylate.
A portion (5.33 g containing 40.1 mg methanesulfonic acid) of a stock solution prepared using methanesulfonic acid (201 mg), water (21.0 g) and 28% ammonium hydroxide (5.53 g) was added to eribulin (311 mg) in acetonitrile (3.9 mL). After 20 minutes stirring, the solution was concentrated in vacuo at <30 °C to ~2 mL. Fresh acetonitrile (10 mL) was added and the solution was concentrated in vacuo at <30 °C to ~1 ml. Fresh acetonitrile (10 mL) was added and the solution was concentrated in vacuo at <30 °C to dryness. This process was then repeated a further three times and for the final concentration. The residue was nitrogen purged and then dissolved in anhydrous dichloromethane/pentane (3:1 v/v, 7.3 mL) prepared under nitrogen. The resulting solution was filtered via a nitrogen-flushed cannula under nitrogen and then washed with anhydrous dichloromethane/pentane (3:1 v/v, 3 mL). The filtrate was concentrated in vacuo at < 30 °C. The residue was nitrogen purged and then dissolved in anhydrous dichloromethane/pentane (1:1 v/v, 7.4 mL) prepared under nitrogen. The resulting solution was filtered via a nitrogen-flushed cannula under nitrogen into anhydrous pentane (33.4 mL). The resultant suspension was stirred for 25 hours and then filtered via a nitrogen flushed PTFE cannula under vacuum/nitrogen and washed with further anhydrous pentane (15 mL). The material was dried under vacuum under nitrogen flow for 46 hours to provide the title compound.
Purity by HPLC: 98.3%
,CLAIMS:Claims:
1. A process for purification of Eribulin of Formula I or a salt thereof comprising:

I

a) purifying one or more compounds according formula II through formula X or salts thereof,

by methods selected from isolation, slurrying in a suitable solvent, acid-base treatment, liquid-liquid extraction, chromatography and treating with adsorbents, and
b) converting the pure compounds according formula II through formula X obtained in step (a) to Eribulin or a pharmaceutically acceptable salt thereof.

2. A process according to claim 1, wherein isolation is selected from decantation, filtration, precipitation from a solvent, precipitation by adding an anti-solvent to a solution and by evaporation of a solution.

3. A process according to claim 1, wherein the solvent for slurrying is selected from water, alcohols, ketones, hydrocarbons, halogenated hydrocarbons, esters, ethers, polar aprotic solvents, nitriles or any mixtures thereof.

4. A process according to claim 1, wherein the purification method is acid-base treatment.

5. A process according to claim 1, wherein chromatography method is selected from column chromatography, flash chromatography, ion exchange chromatography, supercritical fluid chromatography, high performance liquid chromatography, expanded bed adsorption chromatography and simulated moving bed chromatography.

6. A process according to claim 5, wherein the chromatography method is selected from flash chromatography, supercritical fluid chromatography and high performance liquid chromatography.

7. An improved process for preparation of Eribulin of formula I or a pharmaceutically acceptable salt thereof comprising:
a) purification of crude compound of formula ((VIII), and
b) converting without isolation the pure compound of formula ((VIII) obtained in step (a) in to Eribulin of formula I or a pharmaceutically acceptable salt thereof.