DESC:FIELD OF THE INVENTION
The invention relates to an improved process for reduction of the azide intermediate (EBM N-2A) and obtaining Eribulin mesylate (1a) having desired purity.

BACKGROUND OF THE INVENTION
Eribulin (1), chemically known as (2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS, 15S,18S,21S,24S,26R,28R,29aS)-2-[(2S)-3-amino-2-hydroxypropyl] hexacosahydro-3-methoxy-26-methyl-20,27-bis(methylene)-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]dioxacyclopenta- -cosin-5(4H)-one is a structurally simplified, fully synthetic, macrocyclic ketone analog of the marine natural product halichondrin B. The natural product was originally isolated from the marine sponge Halichondria okadai and was subsequently found in Axinella sp., Phakellia carteri and Lissondendryx sp.

Eribulin mesylate (1a)

Eribulin (coded as B1939) is a non-taxane microtubule dynamics inhibitor, containing multiple chiral centers on an extended carbon framework which has demonstrated in-vitro and in-vivo anti-cancer properties. Eribulin has both cytotoxic and non-cytotoxic mechanisms of action. Its cytotoxic effects are related to its antimitotic activities, wherein apoptosis of cancer cells is induced following prolonged and irreversible mitotic blockade. Eribulin also exerts complex effects on the biology of surviving cancer cells and residual tumors that appear unrelated to its antimitotic effects.
An intravenous solution of Eribulin mesylate salt (coded as E7389) with proprietary name Halaven® and strength of 0.5mg/ml, based on a NDA application filed by Eisai Inc., was approved by USFDA on November 15, 2010 for the treatment of metastatic breast cancer.

Eribulin mesylate was disclosed in US 6,214,865 and the total synthesis of Halichondrin B was first published in J. Am. Chem. Soc. 114, 3162-3164, 1992. Additional synthetic methods and the associated intermediates for eribulin and other halichondrin B analogs are described in WO2005/118565, WO2009/046308, WO 2009/064029 and WO2009/124237.

Recent publications such as WO2020110146, WO2020016847 disclose various methods to obtain pure Eribulin mesylate.

The process disclosed in WO2020110146 comprises, a) reacting Eribulin free base with a protected chiral acid such as N-(p-Toluene sulfonyl)-L-phenyl alanine to form corresponding acid addition salt, b) purifying the said salt by recrystallization using solvents such as acetonitrile, dichloromethane, n-pentane and hexane, and c) further treatment with a base to give eribulin.

WO2020016847 describes processes wherein crude Eribulin is purified using chromatographic techniques such as flash chromatography, ion exchange chromatography, supercritical fluid chromatography (SFC), high performance liquid chromatography (HPLC), Ultra performance liquid chromatography (UPLC) etc. The disclosed processes also include derivatizing Eribulin using 9-fluorenylmethyl chloroformate, followed by treatment with base, and purifying the obtained Eribulin and/or its salts using solvents like acetonitrile, anhydrous dichloromethane/pentane.
Owing to the limited availability of halichondrin B from natural sources, new and improved methods for the synthesis of Eribulin mesylate and its pharmaceutically active analogs are being tried out for fully exploiting its medicinal potential, and overcoming drug shortage issues. The processes reported in prior art suffer from various disadvantages such as poor yields and low purity of the intermediates as well as eribulin base and salts, owing to the formation of diastereomeric, dimeric and other impurities. Hence, there is a need to provide a technically superior process for the preparation of Eribulin mesylate having desired purity and conforming to regulatory norms.

Further, Eribulin mesylate being an active pharmaceutical ingredient (API) for the treatment of metastatic cancer, the regulatory norms regarding the purity, assay, impurity profile are very stringent.

The inventors, in their quest for developing a technically superior process for Eribulin, carried out extensive experimentation focused on suitable alternatives for the key synthetic steps like introduction of the amino group, wherein yield improvement could be achieved through control on formation of impurities. The inventors also aimed at development of a convenient, economical purification procedure for Eribulin and the mesylate salt which avoided use of chiral acids to prepare the corresponding salts, or expensive separation techniques such as UPLC, SFC etc. and provided the final compound possessing impurity profile complying with regulatory specifications. The inventors, while developing the purification procedures, also focused on avoiding the use of environmentally hazardous, toxic halogenated hydrocarbons as solvents in the purification processes.

A prominent feature of eribulin synthesis is the introduction of an amino group in the final stages of the synthesis. Prior art procedure mentions that the tosylate derivative of diol intermediate is treated with ammonium hydroxide to provide the required amino group through the intermediate formation of the epoxide. US 6,214,865 discloses that the amino group is introduced by reduction of the azide intermediate, using reducing agent such as trimethyl phosphine to provide Eribulin base.

The instant inventors surprisingly found that reduction of azide intermediate, (EBM-N-2A) using triphenyl phosphine had several drawbacks as the triphenyl phosphine oxide formed during reduction was very difficult to separate from Eribulin base, which affected its purity and required several lengthy purification steps. The present process is particularly advantageous as it is carried out in presence of bulky organic bases such as 1,8-diazabicyclo[5.4. 0]undec-7-ene (DBU) or diisopropylethyl amine (DIPEA or Hunig base). These organic bases provided significant control on formation of impurities, thus causing substantial improvement in the overall yield for synthesis of Eribulin.

Further, the inventors serendipitously found that when the Eribulin free base thus obtained was treated with an alcoholic solvent during the preparation of the mesylate salt, which was later isolated with a combination of organic ester and hydrocarbon solvents, followed by treatment with ethanol, water and lyophilization, Eribulin mesylate so obtained possessed desired purity conforming to regulatory specifications. Consequently, the process avoided prior art drawbacks related to lower purity, higher content of impurities, leading to successive purifications, resulting in low yields.

The present inventors were thus able to develop an economical and convenient process for synthesis of Eribulin mesylate (1a) overcoming the problems faced in prior art to provide the final product having desired purity in good yield.

OBJECT OF THE INVENTION

An objective of the present invention is to provide a process for introduction of amino group in Eribulin synthesis with significant control on associated impurities.
Another object of the invention relates to carrying out the reduction of the azide group in the intermediate (EBM N-2A) in presence of bulky organic bases to achieve control on the side products and impurities formed during the said reaction.
Yet another objective of the instant invention is to provide a convenient and effective method for preparation and purification of Eribulin mesylate (1a) wherein the final compound (1a) possessed desired purity, conforming to the regulatory norms.

SUMMARY OF THE INVENTION

An aspect of the invention relates to a synthetic process for Eribulin mesylate from the intermediate EBM N-2.

Another aspect of the invention relates to a process for preparation of Eribulin mesylate comprising, reaction of azide intermediate (EBM N-2A) with triphenyl phosphine in presence of an organic base selected from 1,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU) and diisopropylethyl amine (DIPEA) and solvent pyridine to give Eribulin free base (EBM N-1), treatment of (EBM N-1) with alcohol like ethanol, methanol, butanol, and methanesulfonic acid to give Eribulin mesylate, further treatment of Eribulin mesylate with a combination of organic solvents selected from esters like ethyl acetate, butyl acetate and hydrocarbons such as pentane, hexane, followed by treatment with ethanol, water and lyophilization to provide Eribulin mesylate conforming to regulatory specifications.

The objectives of the present invention will become more apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors, while carrying out meticulous and extensive experimentation aimed at designing a convenient, industrially applicable synthetic strategy for Eribulin mesylate, surprisingly found that the reduction of penultimate azide intermediate (EBM N-2A) was advantageously carried out in presence of strong, bulky organic bases such as DBU and DIPEA.
The reaction comprised reduction of the azide intermediate (EBM N-2A) using triphenyl phosphine in presence of aqueous organic base and organic solvent.

Reduction of (EBM N-2A) in presence of organic bases was unexpectedly found to show the following advantages.

1. Use of bases such as DBU, DIPEA provided significant control on side reactions and subsequent formation of various impurities. Undesired reactions such as deprotonation at the carbon adjacent to ketone were restricted when the reduction was carried out in presence of bulky organic bases like DBU, DIPEA.

2. Use of DBU, DIPEA also helped in avoiding formation of associated impurities like the Dimeric Impurity, Impurity-B, Impurity-C (labeled respectively as EBM-Dimer impurity, EBM-Imp B and EBM-Imp C, structures given below). When the azide reduction was carried out in presence of bases such as DBU, DIPEA, the dimer impurity was not formed, others were significantly controlled and thus, the total impurities were found below 0.1% as compared to around 0.5% in the similar reaction using ammonia. This control on formation of impurities having polarities similar to Eribulin ensured a cleaner reaction mixture, enabling easier and faster chromatographic separation, purification.

3. Consequently, significant yield improvement of 10 to 15 % was observed when the reduction of azide intermediate (EBM N-2A) was carried out in presence of bases like DBU, DIPEA. This resulted in 5-10% enhancement in overall yield, making the present process cost-effective and industrially viable.

4. The molar requirement of base during triphenyl phosphine reduction was considerably reduced when organic bases were used, which also provided control on undesired side reactions.

5. It was observed that removal of the by-product, triphenyl phosphine oxide was easier when the reduction of azide intermediate was carried out in presence of DBU, DIPEA. Otherwise, the phosphine oxide impurity interfered with the isolation of the eribulin base, thereby leading to low yields.

The inventors, most unexpectedly, further observed that when Eribulin free base obtained by the above process was treated with methanesulfonic acid in presence of alcohol, ammonia and water, followed by lyophilization, the mesylate salt was obtained in good yield. During the experimentation for preparation of the mesylate salt, the inventors observed that use of an alcohol enabled significant control on the impurity formation. Consequently, substantial yield improvement of around 15 to 20% was observed as compared to the prior art processes wherein polar aprotic solvents like acetonitrile were used for the preparation of mesylate.

Further treatment of the salt with solvents selected from esters, hydrocarbons such as ethyl acetate, hexanes, heptanes, pentanes, followed by treatment with ethanol and water and lyophilization provided eribulin mesylate having desired purity.

Thus, the base-mediated reduction, coupled with the afore-mentioned method for preparation and isolation of the mesylate salt furnished Eribulin mesylate (1a) possessing desired purity in good yield.

Scheme-1: Scheme for synthesis of Eribulin mesylate (1a) from EBM N-2

In an embodiment, the tosylated intermediate (EBM-N-2) was treated with sodium azide at 60 to 800C using dimethyl formamide (DMF) as a solvent to give the penultimate intermediate, (EBM-N-2A).
Reduction of (EBM-N-2A), using mild reducing agents such as trialkyl or triaryl phosphine compounds selected from triphenylphosphine and trimethylphosphine provided the amino functional group in Eribulin, (EBM-N-1).
The reaction was carried out at ambient temperature in presence of an organic base using solvent selected from pyridine, water, acetonitrile (ACN), tetrahydrofuran (THF) and combinations thereof.

The organic base was selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and diisopropylethyl amine (DIPEA or Hunig base). Aqueous solution of the organic base 10% aqueous solution in particular, was used during the reaction. After completion of reaction as monitored by HPLC, concentration of the reaction mass and purification of the obtained residue provided the amine, Eribulin free base (EBM-N-1), which was then converted to the methanesulfonate (mesylate) salt.

The amine, (EBM-N-1) was stirred in alcoholic solvent at room temperature, followed by addition of methanesulfonic acid, ammonia and water. The stirring was continued till completion of the methanesulfonate formation reaction, as monitored by HPLC. The alcoholic solvent was selected from ethanol, methanol, isopropyl alcohol, n-butanol, isobutanol and tertiary butanol.

Lyophilization of the reaction mixture provided crude Eribulin mesylate, which was further treated with a combination of solvents selected from esters such as ethyl acetate, butyl acetate, and hydrocarbons like hexanes, heptanes, and pentanes.
The combination of ethyl acetate and n-pentane was preferably used.
Further treatment with ethanol and water, followed by lyophilization provided eribulin mesylate having desired purity.
The following examples are meant to be illustrative of the present invention. These examples exemplify the invention and are not to be construed as limiting the scope of the invention.

EXAMPLES

Example 1: Synthesis of EBM-N-2A
Crude EBM-N-2 (2.81 g) was dissolved in dimethyl formamide (DMF, 28.1ml), followed by addition of sodium azide (0.40 g). The reaction mixture was heated at 750C till completion of reaction as monitored by HPLC. After completion, the reaction mixture was diluted with water and extracted with methyl tertiary butyl ether (MTBE). Separation and concentration of the organic layer, followed by column chromatographic purification of the obtained residue using MTBE, Heptane mixture provided the desired compound, (EBM-N-2A) as white solid.
Yield: 1.37 g, (54.1%)
Purity: 99.57 % (HPLC).

Example 2: Synthesis of EBM-N-1 (Eribulin)
A 1:1 mixture of pyridine (29 ml) and 10% aqueous DBU (29 ml) was added to the mixture of EBM-N-2A (1.16 g) and triphenylphosphine (1.0 g) which was placed in a round bottom flask. The resultant mixture was stirred at room temperature till completion of reaction as monitored by HPLC. After completion, concentration of the reaction mass and purification of the obtained residue by preparative HPLC using mixtures of acetonitrile and water provided EBM-N-1 as white solid.
Yield: 0.87 g, (78.3%)
Purity: 99.9 % (HPLC).

Example 3: Synthesis of EBM-N-1
A 1:1 mixture of pyridine (6.25 ml) and 10% aqueous DIPEA (6.25 ml) was added to the mixture of EBM-N-2A (0.250 g) and triphenylphosphine (0.130 g) which was placed in a round bottom flask. The resultant mixture was stirred at room temperature till completion of reaction as monitored by HPLC. After completion, concentration of the reaction mass and purification of the obtained residue by preparative HPLC using mixtures of acetonitrile and water provided EBM-N-1 as white solid.
Yield: 0.098 g, (40%)
Purity: 99.5 % (HPLC)

Example 4: Synthesis of Eribulin mesylate (1a)
Ethanol (1.27 ml) was added to Eribulin (EBM-N-1, 0.55g) and the mixture was stirred at room temperature. A solution of methane sulfonic acid (0.054 ml) and ammonia solution (1.98 ml) in water (24.3 ml) was added to it at room temperature and resulting reaction mixture was stirred till completion of the salt formation reaction as monitored by HPLC.
Further lyophilization of the reaction mixture provided crude Eribulin mesylate (0.603 g, 97 % yield), which was treated with ethyl acetate (8.07 ml) and n-Pentane (86 ml). The precipitated solid was filtered, treated further with a 5% water-ethanol mixture (25 ml) and lyophilized to provide Eribulin mesylate,
Yield: 0.50 g, 81.9%
Purity: 99.78 % (HPLC).

Example 5: Synthesis of Eribulin mesylate (1a)
Eribulin (0.455 g) was dissolved in acetonitrile (4.5 ml) and stirred at room temperature until a clear solution was obtained. A solution of methanesulfonic acid (0.039 ml) and ammonium hydroxide (1.62 ml) in water (6.11 ml) was added to it at room temperature and resulting reaction mixture was stirred
Further, lyophilization of the reaction mixture provided crude Eribulin mesylate (0.449 g,), which was treated with 75% Dichloromethane: n-Pentane (12.5 ml), filtered and concentrated. Residue was re-dissolved in 50% Dichloromethane: n-Pentane (10.4 ml) and n-Pentane (30 ml). The precipitated solid was filtered, and dried under vacuum to provide Eribulin mesylate,
Yield: 0.19 g, 40%
Purity: 99.05 % (HPLC)

,CLAIMS:We claim,

1. A process for the preparation of Eribulin mesylate (1a) comprising, reaction of the azide intermediate (EBM N-2A) with triphenyl phosphine in presence of an organic base and solvent to give Eribulin free base (EBM N-1), treatment of (EBM N-1) with alcohol, methanesulfonic acid to give Eribulin mesylate, further treatment of Eribulin mesylate with a combination of organic solvents selected from esters and hydrocarbons, and treatment with ethanol, water, followed by lyophilization, to provide Eribulin mesylate conforming to regulatory specifications.

2. The process as claimed in claim 1, wherein the organic base is selected from 1,8-diazabicyclo[5.4. 0]undec-7-ene (DBU) and diisopropylethyl amine (DIPEA).

3. The process as claimed in claims 1 and 2 wherein the organic base is used as an aqueous solution.

4. The process as claimed in claim 1, wherein the solvent is pyridine.

5. The process as claimed in claim 1, wherein the alcohol is selected from methanol, ethanol, isopropyl alcohol, n-butanol, isobutanol and tertiary butanol.

6. The process as claimed in claim 1, wherein the treatment of (EBM N-1) with methanesulfonic acid is carried out in presence of ammonia and water.

7. The process as claimed in claim 1, wherein the organic solvents are selected from a group of esters consisting of ethyl acetate, butyl acetate and a group of hydrocarbons consisting of n-pentane, n-hexane, n-heptane.

8. The process as claimed in claim 7 wherein the organic solvents are selected from ethyl acetate and n-pentane.

9. The process as claimed in claim 1, wherein the treatment with ethanol, water is carried out using 5% ethanol in water solution.