DESC:FIELD OF THE INVENTION
The present invention relates to a process for the preparation of micafungin sodium and intermediates thereof. In particular, the present invention relates to a process for the preparation and purification of micafungin sodium and its echinocandin intermediate. The present invention more particularly relates to a process for the preparation of compound FR-179642, an echinocandin intermediate of micafungin sodium and purification thereof.

BACKGROUND OF THE INVENTION
The following discussion of the prior art is intended to present the invention in an appropriate technical context and allow its significance to be properly appreciated. Unless clearly indicated to the contrary, however, reference to any prior art in this specification should be construed as an admission that such art is widely known or forms part of common general knowledge in the field.
Micafungin is an echinocandin antifungal drug which inhibits the production of beta-1,3-glucan, an essential component of fungal cell walls. Micafungin is mainly indicated for treatment of candidemia, acute disseminated candidiasis, Candida peritonitis, abscesses and esophageal candidiasis. Micafungin sodium, also known as FK-463 is chemically known as a 1-[(4R5R)-4,5-dihydroxy-N2-[4-[5-[4-(pentyloxy)phenyl]-3-isoxazolyl]benzoyl]-L-ornithine]-4-[(4S)-4-hydroxy-4-[-4-hydroxy-3-(sulfooxy)phenyl]-L-threonine]sodium, represented by Formula (I).

(I)
Echinocandin family includes the following members: WF11899A, echinocandin, cilofungin, Pneumocandin, aculeacins, and mulundocandin, with micafungin, echinocandin and Pneumocandin being actively investigated and currently applied clinically.
Micafungin sodium (I) is reported to be prepared by removing the side-chain of the precursor compound of Formula III (FR-901379) through an enzyme reaction to obtain a compound of Formula II (FR-179642). The compound of Formula II is further reacted with side chain compound of Formula (IV) to obtain Micafungin. Therefore, obtaining compound of Formula (II) with high purity is one of the important aspect of the invention.

International PCT Publication No. WO 96/11210 discloses the process for the preparation of compound (IV) as a starting compound for a lipopeptide antifungal agent micafungin sodium and a process for obtaining micafungin sodium by using cation-exchange resin (DOWEX-50WX4).
U.S. Patent Nos. US 8,981,055 discloses a process that involves activation of micafungin side chain of Formula (IV) and the coupling of this activated micafungin side chain of Formula (V) with FR-179642 of Formula (II) in a one-pot procedure to obtain micafungin or a salt.
International PCT Publication No. WO 2013/034670 A1 discloses a process for preparation of micafungin sodium from micafungin TEA using ion-exchange resin (CG 120 I; Counter ion-sodium). Further, WO ‘670 discloses a process for preparation of micafungin TEA by reacting 4-[5-[(4-pentyloxyphenyl) isoxazol-3-yl]benzoic acid (PPIB) of Formula (IV) with FR-179642 of Formula (II) in presence of pivalic acid chloride, TEA and dimethyl formamide. The process is as illustrated in following reaction scheme-1.

Scheme-1

U.S. Patent No. 7,199,248 B2 discloses a process for preparation of micafungin sodium from micafungin DIPEA using ion-exchange resin (UBK510L). Further, US ‘248 discloses a process for preparation of micafungin DIPEA salt by reacting 1-[({4-[5-(4-pentyloxyphenyl)isoxazol-3-yl]phenyl}carbonyl)oxy]-1H-benzotriazole (micafungin side chain) of Formula (III) with FR-179642 of Formula (II) in the presence of DIPEA and dimethyl formamide. The process is as illustrated in following reaction scheme-2.

Scheme-2

U.S. Patent No. 9,115,177 B2 discloses process for preparation of micafungin sodium from micafungin by using weak base, water and organic solvent.
U.S. Patent No. 5,376,634 discloses a method for purifying the compound of Formula (II), by using active coal column and silica gel column, under reduced pressure.
U.S. Patent No. 6,506,726 discloses a method for purifying the echinocandin cyclopeptide compounds by using resins.
U.S. Patent No. 6,207,434 discloses isolated acylase enzymes obtained from Streptomyces anulatus No. 4811, Streptomyces anulatus No. 8703, or Streptomyces No. 6907, wherein said enzymes catalyze deacylation of the acyl group of a cyclic lipopeptide or a salt thereof to produce a cyclic peptide or a salt thereof and a method of producing a cyclic peptide or a salt thereof using a cyclic lipopeptide or a salt thereof Streptomyces No. 6907.
U.S. Patent No. 7,241,866 discloses a method of purifying a peptide or a lipopeptide by using a mobile phase modifier in a normal phase chromatography system to improve the selectivity and/or productivity of the purification, wherein the mobile phase modifier is selected from a group consisting of an amino acid and an amino acid ester, the normal phase chromatography system includes a mobile phase and a stationary phase, the mobile phase is a solvent system comprising one or more solvents, and the stationary phase is selected from silica gel and alumina, except that when the lipopeptide is Pneumocandin B0, then the mobile phase modifier is not L-proline.
U.S. Patent No. 8,101,712 discloses a process for purifying an echinocandin-type compound by extracting an echinocandin-type compound from a fermentation broth or from a filtered mycelium obtained from a fermentation broth, with a water-immiscible organic solvent.
International PCT Publication No. WO 2014/044803 discloses a method for producing a cyclic peptide compound or a salt thereof which comprises contacting a cyclic lipopeptide compound or a salt thereof with an acylase derived from a microorganism belonging to the genus Pseudomonas to deacylate said cyclic lipopeptide.
U.S. Patent No. 8,911,968 B2 discloses a mutagenized strain of Coleophoma empetri, deposited in China General Microbiological Culture Collection Center and method for its preparation.
U.S. Patent No. 8,927,690 discloses a method for purifying the compound of Formula (II) by using a macroporous adsorption resin and washing the resin the macroporous adsorption resin using water, an organic solvent or a mixed solution of an organic solvent and water as the washing liquid, wherein said organic solvent comprises a C1-C4alcohol or a C1-C4ketone.
U.S. Patent No. 8,877,777 discloses a method of purifying FR-901379 or the salts thereof by mixing a fermentation liquid comprising FR-901379 or the salts thereof with an organic solvent followed by sequential steps and adsorption onto a macroporous adsorption resin and eluting FR-901379 from the macroporous adsorption resin using water, an organic solvent, or a mixed solution of an organic solvent and water as eluent, wherein the organic solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, acetone and butanone.
In view of the above, it is therefore, desirable to provide an efficient, more economical, less cumbersome and eco-friendly process for the purification of compound of Formula (II) and the preparation of micafungin sodium with high purity.

SUMMARY OF THE INVENTION
In one general aspect, there is provided a process for the preparation of micafungin sodium, the process comprising:
(a) treating a sodium source with one or more solvents to obtain a mixture;
(b) reacting the mixture with highly pure compound FR-179642 of Formula (II)

(II)
and the compound of Formula (V),

(V)
in one or more solvents to obtain micafungin sodium.

In another general aspect, there is provided a process for the preparation of highly pure micafungin sodium, the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;

(II)
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as washing liquid, wherein the solvent comprises of C1-C4nitrile;
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as an eluting liquid to obtain highly pure compound of Formula (II) (FR-179642), wherein the solvent comprises of C1-C4nitrile; and
(d) reacting the highly pure compound of Formula (II) with a compound of Formula (V),

(V)
in the presence of a sodium source in one or more solvents to obtain a reaction mixture;
(e) adding one or more solvents into the reaction mixture to obtain micafungin sodium;and
(f) purifying the micafungin sodium to obtain highly pure micafungin sodium.

In another general aspect, there is provided a process for the preparation of compound of Formula II,

(II)
the process comprising:
(a) adding one or more first solvents into fermentation liquid comprising a compound of Formula (III) or salt thereof,

(III)
(b) extracting the fermentation liquid by adjusting pH to obtain the compound (III) by filtration or centrifugation of the first solvents;
(c) adding one or more second solvents into the extract followed by adjusting the pH to obtain the compound of Formula (III);
(d) converting the compound of Formula (III) to a crude compound of Formula (II) by an action of an acylase enzyme;
(e) loading the crude compound of Formula (II) on a macroporous adsorption resin;
(f) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as a washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(g) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as an eluting liquid, wherein the solvent comprises of C1-C4nitrile.
In another general aspect, there is provided a process for the purification of compound of Formula II;

the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as the washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as the eluting liquid, wherein the solvent comprises of C1-C4nitrile to obtain a highly pure compound of Formula (II); and
(d) optionally, converting the highly pure compound of Formula (II) to micafungin or its pharmaceutically acceptable salts thereof.

In another general aspect, there is provided process for purification of micafungin sodium using column chromatography, preparative HPLC, selective precipitation, recrystallization etc.
In another general aspect, there is provided a pharmaceutical composition comprising highly pure micafungin sodium and one or more of pharmaceutically acceptable excipients, diluents or carriers.

DETAILED DESCRIPTION OF THE INVENTION
The present invention features methods of preparation of micafungin sodium directly from echinocandin-type compound (FR-179642), without preparing micafungin TEA or DIPEA salts as reported in the prior art. The present invention herein possesses advantage over the cited prior arts by avoiding formation of organic amine salts and thereby obtaining better yield and purity of micafungin sodium.
Further object of the invention provides methods for preparation and purification of echinocandin-type compound (FR-179642), respectively and use thereof for the preparation of micafungin sodium with high purity. The improvement over the prior art involves use of substrate compound of Formula (III) i.e. FR-901379 as such without purification for the preparation and purification of echinocandin-type compound (FR-179642).
The present invention is based on the process disclosed herein possesses advantage over the cited prior art for avoiding the use of ion-exchange resin which is beneficial for scale-up and provides simple process.
The above and other objects of the present invention are achieved by the process of the present invention, which leads a process for the preparation of highly pure micafungin sodium.
Optionally, the solution, prior to any solids formation, can be filtered to remove any undissolved solids, solid impurities prior to removal of the solvent. Any filtration system and filtration techniques known in the art can be used.
All ranges recited herein include the endpoints, including those that recite a range “between” two values. Terms such as “about”, “generally”, “substantially,” and the like are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.
As used herein the term, “highly pure”, may be interchangeably used with “high-purity” and “HPLC purity”, all referring to the percentage of the peak area of micafungin sodium and echinocandin-type compound (FR-179642) wherever applicable, over the sum of all peak areas as measured under the detecting conditions of high performance liquid chromatography (HPLC) provided by the invention.
As used herein the term “highly pure” refers to the level of purity of compound. In particular, the term “highly pure” means the compound having a purity of about 99% or more, 99.5% or more, 99.8% or more, 99.9% or more when measured by area percentage of HPLC.
As used herein the term “highly pure micafungin sodium” refers to micafungin sodium having a purity of about 99% or more, 99.5% or more, 99.8% or more, 99.9% or more when measured by area percentage of HPLC.
As used herein the term “substantially free” refers to highly pure micafungin sodium comprising one or more of each of impurity-1 (VI), impurity-2 (VII), impurity-3 (VIII), impurity-4 (IX), impurity-5 (X), impurity-6 (XI) and impurity-7 (XII) in a concentration of about 0.15% or less. In particular, each of impurity-1 (VI) to impurity-7 (XII) is from about 0.02% to about 0.15%, when measured by area percentage of HPLC.
As used herein the term, “crude compound of Formula II”, means a solution containing <80% of compound of Formula II as measured by HPLC.
As used herein the term, “loading” refers to the process of adsorbing the extract containing the crude compound II onto a macroporous adsorption resin so that the compound II is adsorbed.
As used herein the term, “washing the adsorption resin” means that a suitable solution is allowed to pass through or over the adsorption resin.
As used herein the term, “washing” means the macroporous adsorption resin means that a suitable buffer solution is allowed to pass through or over the macroporous adsorption resin.
As used herein the term, “eluting” means the molecules are removed from the macroporous adsorption resin by changing the polarity of the buffer solution around the macroporous adsorption resin. Due to the polarity, the buffer solution can compete with the molecules for the adsorption sites on the macroporous adsorption resin.
The term, “pharmaceutically acceptable salt” means salts formed from the following bases: inorganic base, such as sodium, potassium, magnesium, calcium, aluminium, etc.; organic base, such as methylamine, ethylamine, ethanolamine, diethanolamine, triethanolamine, cyclohexanolamine, lysine, ornithine, etc., or other bases relevant to the pharmaceutically acceptable salts.
The term, "pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable, and includes that which is acceptable for veterinary use and/or human pharmaceutical use.
The term, "pharmaceutical composition" is intended to encompass a drug product including the active ingredient(s), pharmaceutically acceptable excipients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients. Accordingly, the pharmaceutical compositions encompass any composition made by admixing the active ingredient, active ingredient dispersion or composite, additional active ingredient(s), and pharmaceutically acceptable excipients.
In one general aspect, there is provided a process for the preparation of micafungin sodium, the process comprising:
(a) treating a sodium source with one or more solvents to obtain a mixture;
(b) reacting the mixture with highly pure compound FR-179642 of Formula (II)

(II)
and compound of Formula (V),

(V)
in one or more solvents to obtain micafungin sodium.
The prior art discloses the preparation of micafungin sodium from the compound of Formula (II) by reacting with the compound (V) in the presence of organic amines, for example triethylamine (TEA) or diispropylethylamine (DIPEA) and using ion-exchange resin. The present invention advantageously provides the simple method to provide micafungin sodium of Formula (I) with purity of at least about 95% or more by area percentage of HPLC and high yield of about 90% or more.
The micafungin sodium of Formula (I) obtained by the process with purification as per the scope of present invention provides the highly pure micafungin sodium.
In general, the solvents in step (a) is selected from one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof. In particular, dimethylformamide is used.
In general, the sodium source is selected from one or more of disodium hydrogen citrate, sodium citrate, sodium acetate, sodium propionate, sodium butyrate, sodium isobutyrate, sodium tartrate, sodium oxalate, sodium benzoate, sodium sorbate, sodium malate, monosodium succinate, disodium succinate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium borate, sodium sulfite, and sodium hydrosulfide. In particular, sodium source may be sodium acetate.
In general, the step (a) of reaction is performed at temperature about -10°C to 30°C. In particular, the temperature is about -10°C to about 10°C.
In general, the step (b) of reaction is performed at temperature about -10°C to 30°C. In particular, the temperature is about -10°C to about 10°C for 1-10 hours, particularly 4 hours.
In another general aspect, there is provided a process for the preparation of highly pure micafungin sodium, the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;

(II)
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as washing liquid, wherein the solvent comprises of C1-C4nitrile;
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as an eluting liquid to obtain highly pure compound of Formula (II) (FR-179642), wherein the solvent comprises of C1-C4nitrile;
(d) reacting the highly pure compound of Formula (II) with a compound of Formula (V),

(V)
in the presence of a sodium source in one or more solvents to obtain a reaction mixture;
(e) adding one or more solvents into the reaction mixture to obtain micafungin sodium; and
(f) purifying the micafungin sodium to obtain highly pure micafungin sodium.
In general, the loading of crude compound of Formula (II) in step (a) comprises the loading of solution containing the crude compound of Formula II on the chromatographic column, prefilled with macroporous adsorption resin thereby loading the compound.
In general, the macroporous adsorption resin is dissolved in one or more solvents and packed into chromatographic column, thereby providing the chromatographic column.
In general, the macroporous adsorption resin in step (a) is nonionic polystyrene-divinylbenzene polymer resin. In particular, the adsorption resin comprises one or more of SP-207, SP-207ss, or mixture thereof.
The embodiment comprises dissolving the SP-207ss resin in C1-C4alcohol comprises of methanol, ethanol, isopropanol and butanol to form slurry, and packing the chromatographic column under pressure.
In general, the washing of the macroporous adsorption resin in step (b) comprises washing the chromatographic column containing crude compound of Formula II using water, one or more solvents or mixed solution of one or more solvents. In particular, the chromatographic column containing crude compound of Formula II was washed with water followed by gradient wash with acetonitrile of 1-5%, particularly, 1-3%.
In general, the solvent comprises C1-C4nitrile selected from acetonitrile, propionitrile.
In general, the concentration of the solvent in the eluent is 0-10%; particularly 5%. The solvent for the eluent comprises C1-C4nitrile selected from acetonitrile, propionitrile. The flow rate for elution is 1-5 ml/minute, In particular, 1-2 ml/minute.
In general, the solvents in step (d) is selected from one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof. In particular, dimethylacetamide or dimethylformamide may be used.
In general, the sodium source comprises one or more of disodium hydrogen citrate, sodium citrate, sodium acetate, sodium propionate, sodium butyrate, sodium isobutyrate, sodium tartrate, sodium oxalate, sodium benzoate, sodium sorbate, sodium malate, monosodium succinate, disodium succinate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium borate, sodium sulfite, and sodium hydrosulfide. In particular, sodium source may be sodium acetate.
In general, the mole equivalent of sodium source in step (d) is about 0.5 mole to 5 mole equivalents with respect to compound FR-179642 of formula (II). In particular, sodium source is about 1 mole equivalent to 2 mole equivalents.
In general, the step (d) of reaction is performed at temperature about -10°C to 30°C. In particular, the temperature is about -10°C to about 10°C for 1-10 hours, particularly 4 hours.
In general, the solvents in step (e) is selected from one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof. In particular, dimethylacetamide or dimethylformamide may be used.
In general, the process for purification of micafungin sodium in step (f) comprising one or more of conventional method selected from pulverization, recrystallization, column-chromatography, preparative HPLC and re-precipitation. In particular, the column-chromatography or preparative HPLC may be used for purification of crude micafungin sodium.
In general, the process for purification of micafungin sodium in step (f) comprises purifying micafungin sodium having purity of about 95% or less by area percentage of HPLC comprising purification by preparative HPLC using Knauer preparative HPLC instrument with Grace (YMC AQ ODS Media or equivalent) Column having 250mm X 50mm column size, flow rate between 75-85 ml/min and mobile phase of aqueous ammonium formate (buffer solution) and acetonitrile.
In general, the highly pure micafungin sodium obtained after purification process of the present invention has purity of about 99% or more, 99.5% or more, or 99.9% or more by area percentage of HPLC.
In general, the highly pure micafungin sodium obtained is substantially free from one or more of each impurity-1 (VI), impurity-2 (VII), impurity-3 (VIII), iimpurity-4 (IX), impurity-5 (X), impurity-6 (XI) and impurity-7 (XII) by area percentage of HPLC.
In general, the impurity-1 to impurity-7 as herein above are characteritized, respectively by the following structures:

Impurity-1 (VI)


Impurity-2 (VII)

Impurity-3 (VIII)

Impurity-4 (IX)

Impurity-5 (X)

Impurity-6 (XI)

Impurity-7 (XII)

In another general aspect, the purification by column chromatography comprises of column filled with HP20SS resin and then eluted with solution of organic solvent with water. Further, the crude micafungin sodium is dissolved in the mixture of water and organic solvents, and subjected to column. Then, the eluting mixtures of organic solvent and water passed through column and fractions are collected. The collected fractions containing micafungin sodium are combined and concentrated under vacuum to obtain pure micafungin sodium.
In general, the column chromatography may be performed with one or more organic solvents selected from methanol, ethanol, propanol, isopropanol, acetone, and acetonitrile. In particular, the organic solvent may be methanol.
In another general aspect, the invention provides highly pure compound of Formula (II) used for the preparation of highly pure micafungin sodium.
In another general aspect, there is provided a process for the preparation of compound of Formula II;

(II)
the process comprising:
(a) adding one or more first solvents into the fermentation liquid comprising the compound of Formula (III) or salt thereof,

(III)
(b) extracting the fermentation liquid by adjusting the pH to obtain compound (III) by filtration or centrifugation of first solvents;
(c) adding one or more second solvents into the extract followed by adjusting the pH to obtain the compound of Formula (III);
(d) converting the compound of Formula (III) to a crude compound of Formula (II) by an action of acylase enzyme;
(e) loading the crude compound of Formula (II) on a macroporous adsorption resin;
(f) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as the washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(g) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as the eluting liquid, wherein the solvent comprises of C1-C4nitrile.
In general, the first solvent in step (a) comprises one or more of C1-C4alcohols selected from methanol, ethanol, isopropanol, butanol, and isobutanol; C1-C4ketones selected from acetone, methyl ethyl ketone, and methyl isobutyl ketone; C1-C4nitrile selected from acetonitrile and propionitrile.
In general, the extraction of compound (III) from fermentation in one or more first solvents is performed at the pH of 5.0 to 6.5. In particular, the pH is 6.0.
The embodiments further comrpises addition of one or more second solvents. The second solvent comprises one or more of non-polar solvent selected from n-hexane, n-heptane, cyclohexane, toluene, xylene, ethylbenzene, and methylene dichloride. In particular, the solvent is n-hexane or n-heptane.
In general, the addition of non-polar solvents results in phase separation and extraction of compound of Formula III into aqueous layer by adjusting to neutal pH. The aqueous layer may be concentrated to reduce the content of solvent.
The compound of Formula (III) i.e. FR-901379 obtained may be converted to crude comopund of Formula (II) by the action of acylase enzyme. In particular, the acylase enzyme is selected from one or more of Coleophoma empetri, Actinoplanes utahensis IFO-13244, Actinoplanes utahensis ATCC 12301, and Actinoplanes missourienses NRRL 15053.
In general, the embodiments of the process comprises use of crude compound of Formula (II) obtained after deacylation of compound of Formula (III) without subjecting to purification.
In general, the loading of crude compound of Formula (II) in step (e) comprises the loading of solution containing crude compound of Formula II on the chromatographic column, prefilled with macroporous adsorption resin thereby loading the compound.
In general, the macroporous adsorption resin is dissolved in one or more solvents and packed into chromatographic column, thereby providing the chromatographic column.
In general, the macroporous adsorption resin in step (e) is nonionic polystyrene-divinylbenzene polymer resin. In particular, the adsorption resin comprises one or more of SP-207, SP-207ss, or mixture thereof.
The embodiment comprises dissolving the SP-207ss resin in C1-C4alcohol comprising of methanol, ethanol, isopropanol and butanol to form slurry, and packing the chromatographic column under pressure.
In general, the washing of the macroporous adsorption resin in step (f) comprises washing the chromatographic column containing crude compound of Formula II using water, one or more solvents or mixed solution of one or more solvents. In particular, the chromatographic column containing crude compound of Formula II was washed with water followed by gradient wash with acetonitrile of 1-5%, particularly, 1-3%.
In general, the solvent C1-C4nitrile is selected from acetonitrile, and propionitrile.
In general, the concentration of the solvent in the eluent is 0-10%; particularly 5%. The solvent for the eluent comprises C1-C4nitrile selected from acetonitrile, propionitrile. The flow rate for elution is 1-5 ml/minute, In particular, 1-2 ml/minute.
In another general aspect there is provided a process for the purification of compound of Formula II;

the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as the washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as the eluting liquid, wherein the solvent comprises of C1-C4nitrile to obtain a highly pure compound of Formula (II); and
(d) optionally, converting the highly pure compound of Formula (II) to micafungin or its pharmaceutically acceptable salts thereof.
In another general aspect there is provided a pharmaceutical composition comprising highly pure micafungin sodium together with one or more pharmaceutically acceptable excipients, diluents and carriers.
In general, the pharmaceutical compositions comprising highly pure micafungin sodium of the invention may be prepared by using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, surface active agents, and lubricants. Various modes of administration of the pharmaceutical compositions of the invention can be selected depending on the therapeutic purpose, for example tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, or injection preparations.
In another general aspect, there is provided a process for preparation of micafungin sodium as depicted in scheme-3.

Scheme-3
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any way, to limit the scope thereof. Plausible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

Example 1: Preparation of the crude compound of Formula II (Method 1)
Into a 50 mL three necked round bottom flask, 5.3 L solution containing crude compound of Formula II was obtained according to Example 1 of U.S. Pat. No. 5,376,634, having the content of the compound of Formula II was 1.4 g/L. Solution was freeze dried to get title compound as white powder (12.5 g, 74.25% HPLC purity).

Example 2: Preparation of the crude compound of Formula II (Method 2)
Into a 25 L container, 5 L of fermentation broth containing 30 g compound of formula III (Broth activity: 6 g/L) and 15 L isobutanol were added. The pH of resulting mixture was adjusted to 6.0 under stirring with sodium bicarbonate. The reaction mixture was stirred for 2 hours and the compound of Formula III was extracted from fermentation broth to isobutanol. The isobutanol layer was centrifuged. 1.41 g/L of compound of Formula III was obtained in 17 L extract 1. 2 L n-heptane was added to the extract 1. The pH of the resulting mixture was adjusted to 7.0 with stirring for 30 min. The separated aqueous layer comprising 20 g compound of Formula III and residual heptane was removed by evaporation using rotavapour. The aqueous layer was charcoalized, stirred for 2 hours and filtered through hyflow bed. The filtrate was quantified for the amount of compound of Formula III in aqueous form and taken up as starting material for deacylation to obtain crude compound of Formula II.

Example 3: Purification of compound of Formula II.
Into a 50 mL three necked round bottom flask, 12.5 g of crude powder obtained according to Example 2 was dissolved in to 300 ml DM water and pH was adjusted to 6.0 using 0.25mM KH2PO4 buffer. Resulting solution was loaded on a chromatographic column filled with SP207ss resin column with peristaltic pump having flow rate of 2 ml/min. 2CBV WFI wash to the column was given subsequently. Afterwards, gradient washing of 2CBV each to the column having flow rate 1.8ml/min with acetonitrile solution was given. 5% acetonitrile with flow rate of 1.2 ml/minute was used to elute the title compound. Fractions containing pure title compound were evaporated and lyophilized for 24-48 Hrs to obtain pure title compound (4.46 g, 95% HPLC purity).

Example 4: Preparation of micafungin sodium
Into a 50 mL three necked round bottom flask, 16 mg of sodium acetate (0.117 mmol) and 2 ml of dimethyl formamide were stirred and cooled to 0°C. To the above mixture, 100 mg of FR-179642 (0.106 mmol) added at 0°C and stirred for 5 minutes followed by addition of 50 mg 1-[({4-[5-(4-pentyloxyphenyl)isoxazol-3-yl]phenyl}carbonyl)oxy]-1H-benzotriazole (0.106 mmol, Micafungin side chain). Reaction mixture was stirred for 2 hours. To the reaction mass, mixture of 0.5 ml of methanol and 1 ml of acetone were added, and temperature was raised to 10°C. Reaction mixture was stirred for 30 minutes followed by addition of 10 ml ethyl acetate within 1 hour. Again reaction mixtures was stirred for 16 hours, solid obtained was filtered and washed with 15 ml of ethyl acetate. Resultant material was dried under vacuum at room temperature for 4 hours to obtained 110 mg of micafungin sodium salt (Yield = 79.7%)

Example 5: Purification of micafungin sodium
The column was filled with 1.6 ml of ion exchange resin HP20SS and eluted with solution of 10% methanol in water. The crude micafungin sodium (100 mg) obtained from example-1 was dissolved in the mixture of 1 ml of water and 0.1 ml of methanol, and subjected to column chromatography. Then the column was passed with following solvent mixtures,
5 ml 10% methanol in water
20 ml 25% methanol in water
20 ml 50% methanol in water
20 ml 60% methanol in water;
50 ml 70% methanol in water
The micafungin sodium solution was eluted in initial 19 column volumes i.e. 30 ml 70% methanol in water. Fractions containing pure micafungin sodium were combined and concentrated under vacuum at 25°C to obtain 55 mg of pure micafungin sodium in solid form (Yield = 55%, Purity = 95.22%).

Example 6: Preparation of micafungin sodium
Into a 250 mL three necked round bottom flask, 160 mg of sodium acetate trihydrate (1.174 mmol) and 15 mL of dimethyl formamide were stirred and cooled to -5 to 0°C. To the above mixture, 1 g of FR-179642 (1.07 mmol) and 500 mg 1-[({4-[5-(4-pentyloxyphenyl)isoxazol-3-yl]phenyl}carbonyl)oxy]-1H-benzotriazole (1.067 mmol, micafungin side chain) were added. The reaction mixture was stirred for 4 hours and progress of the reaction was monitored by HPLC. After the completion of the reaction, ethyl acetate was added to the reaction mixture at -5 to 0° C and stirred for 1.5 hours. The reaction mixture was filtered and washed with ethyl acetate. The product micafungin sodium was dried U/V for 4 hours to obtain micafungin sodium (Yield 95%, purity 95.10%).

Example 7: Purification of micafungin sodium by preparative HPLC
Instrument : Knauer preparative HPLC
Column : Grace (YMC AQODS Media or equivalent)
Column Size : 250mm X 50mm
Flow rate : 75-85 ml/min
Mobile Phase A : Aqueous ammonium formate (buffer solution)
Mobile Phase B : Acetonitrile
Process: The column was saturated with buffer solution and sample solution was loaded into the column. Buffer solution was flushed for 5 min and run for gradient from 0 to 120 min with different concentrations of mobile phase B. The highly pure fractions of micafungin sodium were collected and combined. The combined fractions were lyophilized for 24 hours at -60° to -65°C under 250 mT vacuum to obtain highly pure micafungin sodium having purity of 99.95%.
While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
,CLAIMS:We Claim:
1. A process for the preparation of micafungin sodium, the process comprising:
(a) treating a sodium source with one or more solvents to obtain a mixture;
(b) reacting the mixture with highly pure compound FR-179642 of Formula (II),

(II)
and compound of Formula (V),

(V)
in one or more solvents to obtain the micafungin sodium.
2. The process according to claim 1, wherein the solvents in step (a) is selected from one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof.
3. The process according to claim 1, wherein the sodium source is selected from one or more of disodium hydrogen citrate, sodium citrate, sodium acetate, sodium propionate, sodium butyrate, sodium isobutyrate, sodium tartrate, sodium oxalate, sodium benzoate, sodium sorbate, sodium malate, monosodium succinate, disodium succinate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium borate, sodium sulfite, and sodium hydrosulfide.
4. A process for the preparation of highly pure micafungin sodium, the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;

(II)
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as washing liquid, wherein the solvent comprises of C1-C4nitrile;
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as an eluting liquid to obtain highly pure compound of Formula (II) (FR-179642), wherein the solvent comprises of C1-C4nitrile;
(d) reacting the highly pure compound of Formula (II) with a compound of Formula (V),

(V)
in the presence of a sodium source in one or more solvents to obtain a reaction mixture;
(e) adding one or more solvents into the reaction mixture to obtain micafungin sodium; and
(f) purifying the micafungin sodium to obtain highly pure micafungin sodium.
5. The process according to claim 4, wherein the macroporous adsorption in step (a) is nonionic polystyrenedivinylbenzene polymer resin.
6. The process according to claim 4, wherein the washing in step (b) comprises washing the chromatographic column containing crude compound of Formula II.
7. The process according to claim 4, wherein the solvent C1-C4nitrile is selected from acetonitrile, and propionitrile.
8. The process according to claim 4, wherein the solvent in step (d) comprises one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof.
9. The process according to claim 4, wherein the sodium source in step (d) comprises one or more of disodium hydrogen citrate, sodium citrate, sodium acetate, sodium propionate, sodium butyrate, sodium isobutyrate, sodium tartrate, sodium oxalate, sodium benzoate, sodium sorbate, sodium malate, monosodium succinate, disodium succinate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium borate, sodium sulfite, and sodium hydrosulfide.
10. The process according to claim 4, wherein the sodium source in step (d) is about 0.5 mole to 5 mole equivalent with respect to compound FR-179642 of Formula (II).
11. The process according to claim 4, wherein the solvent in step (e) comprises one or more of alcohols comprises methanol, ethanol, isopropanol, n-butanol and t-butanol; ketones comprises acetone, methyl ethyl ketone and methyl isobutyl ketone; esters comprises ethyl acetate, isopropyl acetate, n-butyl acetate and t-butyl acetate; hydrocarbons comprises toluene, xylene, and ethyl benzene; halogenated solvents comprises methylene dichloride, ethylene dichloride, chloroform, and chlorobenzene; nitriles comprises acetonitrile and propionitrile; amides comprises formamide, dimethylformamide, and dimethylacetamide, or a mixture thereof.
12. The process according to claim 4, wherein the purification of micafungin sodium in step (f) comprises purifying micafungin sodium having purity of about 95% or less by area percentage of HPLC comprising purification by preparative HPLC using Knauer preparative HPLC instrument with Grace (YMC AQ ODS Media or equivalent) Column having 250mmX50mm column size, flow rate of about 75-85 ml/min and mobile phase of aqueous ammonium formate (buffer solution) and acetonitrile.
13. The highly pure micafungin sodium according to claim 4, which is having a purity of about 99% or more by area percentage of HPLC.
14. The highly pure micafungin sodium according to claim 13, which is substantially free from one or more of each impurity-1 (VI), impurity-2 (VII), impurity-3 (VIII), iimpurity-4 (IX), impurity-5 (X), impurity-6 (XI) and impurity-7 (XII) by area percentage of HPLC.
15. The highly pure micafungin according to claim 14, wherein one or more of each impurity-1 (VI), impurity-2 (VII), impurity-3 (VIII), iimpurity-4 (IX), impurity-5 (X), impurity-6 (XI) and impurity-7 (XII) is from about 0.02% to about 0.15% by HPLC.
16. A process for the preparation of compound of Formula II;

(II)
the process comprising:
(a) adding one or more first solvents into the fermentation liquid comprising the compound of Formula (III) or salt thereof,

(III)
(b) extracting the fermentation liquid by adjusting the pH to obtain compound (III) by filtration or centrifugation of first solvents;
(c) adding one or more second solvents into the extract followed by adjusting the pH to obtain the compound of Formula (III);
(d) converting the compound of Formula (III) to crude compound of Formula (II) by an action of acylase enzyme;
(e) loading the crude compound of Formula (II) on a macroporous adsorption resin;
(f) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as the washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(g) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as the eluting liquid, wherein the solvent comprises of C1-C4nitrile.
17. The process according to claim 16, wherein the first solvent in step (a) comprises one or more of C1-C4alcohols selected from methanol, ethanol, isopropanol, butanol, and isobutanol; C1-C4ketones selected from acetone, methyl ethyl ketone, and methyl isobutyl ketone; C1-C4nitrile selected from acetonitrile and propionitrile.
18. The process according to claim 16, wherein the second solvent in step (c) comprises one or more of non-polar solvent selected from n-hexane, n-heptane, cyclohexane, toluene, xylene, ethylbenzene, and methylene dichloride.
19. The process according to claim 16, wherein the acylase enzyme in step (d) is selected from one or more of Coleophoma empetri, Actinoplanes utahensis IFO-13244, Actinoplanes utahensis ATCC 12301, and Actinoplanes missourienses NRRL 15053.
20. The process according to claim 16, wherein the solvent C1-C4nitrile is selected from acetonitrile, and propionitrile.
21. A process for the purification of compound of Formula II,

the process comprising:
(a) loading a crude compound of Formula (II) on a macroporous adsorption resin;
(b) washing the macroporous adsorption resin with water, one or more solvents or mixed solution of one or more solvents and water as the washing liquid, wherein the solvent comprises of C1-C4nitrile; and
(c) eluting the compound of Formula (II) from the macroporous adsorption resin by using water, one or more solvents or mixed solution of one or more solvents and water as the eluting liquid, wherein the solvent comprises of C1-C4nitrile to obtain a highly pure compound of Formula (II); and
(d) optionally, converting the highly pure compound of Formula (II) to micafungin or its pharmaceutically acceptable salts thereof.
22. The highly pure compound of Formula (II) according to claim 21, which is having purity of about 99% or more by area percentage of HPLC.
23. The highly pure micafungin sodium according to claim 14, prepared by using highly pure compound of Formula (II).
24. A pharmaceutical composition comprising highly pure micafungin sodium according to claim 14 together with one or more pharmaceutically acceptable excipients, diluents and carriers.

Dated this 28th day of February 2017.

[AAYSU MAHLA]
(IN/PA-1490)
Of SUBRAMANIAM & ASSOCIATES
Attorneys for the applicants