DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION
(Section 10 and Rule 13)

A PROCESS FOR THE PREPARATION OF L-GLUTAMINE

AUROBINDO PHARMA LTD HAVING CORPORATE OFFICE AT
THE WATER MARK BUILDING,
PLOT NO.11, SURVEY NO.9,
HITECH CITY, KONDAPUR,
HYDERABAD, 500 084,
TELANGANA, INDIA
AN INDIAN ORGANIZATION

The following specification particularly describes and ascertains the nature of this invention and the manner in which the same is to be performed.
FIELD OF INVENTION

The present invention relates to a process for the preparation of L-Glutamine of Formula I.

Formula I
BACKGROUND OF THE INVENTION

L-Glutamine (I) is chemically known as 2 L-glutamic acid-5-amide. L-Glutamine (I) is an amino acid indicated to reduce the acute complications of sickle cell disease in adult and pediatric patients. L-Glutamine (I) is being marketed in the US under the brand name Endari®.

Several processes are available in the literature for synthesis of L-Glutamine and is synthesized by chemical methods and enzymatic methods.

US 2,762,841 discloses a chemical process for preparing L-Glutamine (I), which involves esterification of L-glutamic acid (II) using MeOH/H2SO4 to produce 5-methyl-L-glutamate (III), which is condensed with allyl chloroformate to produce 5-methyl-N-carboallyloxy-L-glutamate (IV), which is further treated with amination and ammonia hydroxide to produce N-carboallyloxy-L-glutamine (V). The compound (V) undergoes deprotection using AcOH/HBr to produce L-Glutamine (I).

The process is as shown in scheme-I below:

Scheme-I

The major drawback associated with the above process is that it involves more number of steps, usage of genotoxic reagents like allyl chloroformate and hazardous chemicals like highly corrosive and irritating to inhalation reagent “Hydrogen bromide”.

US 2,788,370 discloses a process for the preparation of L-Glutamine (I). The process involves heating of L-glutamic acid to produce L-pyrrolidone carboxylic acid (VI), which is treated with absolute hydrazine to produce L-Glutamyl hydrazide (VII), followed by hydrogenation to produce L-Glutamine (I).

The process is as shown in scheme-II below:

Scheme-II

The disadvantage of the above process is the use of genotoxic reagent hydrazine in absolute form at higher temperature and use of Raney nickel in excess quantity as well as hydrogen sulfide gas, which is a highly flammable, explosive and can cause possible life-threatening situations if not properly handled.

L-Pyrrolidone carboxylic acid synthesis involves longer hours at higher temperature in neat condition, which is not an industrially feasible process.

US 2,846,470 discloses a process for the preparation of L-Glutamine (I) involves treating ?-methyl-L-glutamate (III) to produce L-?-glutamylhydrazide (VII), followed by hydrogenated with Raney nickel to produce L-Glutamine (I).

The process is as shown in scheme-III below:

Scheme-III

The main disadvantage of the above process is the use of genotoxic reagent hydrazine hydrate and use of Raney nickel in excess quantity as well as ammonium sulfide, which has a very strong and unpleasant smell and is a very toxic chemical. It is explosive and highly flammable also. Hence, not an industrially viable process.

US 2,883,399 discloses a process for the preparation of L-Glutamine (I) by treating dibenzyl -L(+)-glutamine HCl (VIII) with trityl chloride to produce dibenzyl N-trityl-L(+)-glutamate (IX), which is treated with sodium/MeOH to give diester (X), followed by debenzylation to give methyl ester compound (XI). The Compound (XI) is treated with ammonia to give trityl –(L)-Glutamine (XII), followed by deprotected with aq. acetic acid to produce (L)-Glutamine (I).

The process is as shown in scheme-IV below:

Scheme-IV
The main disadvantage of the above process is the use of sodium metal, which is difficult to handle in large scale as its corrosive nature and is highly water reactive metal. The use of Palladium carbon, which is more expensive reagent and the lengthy synthetic process.

US 3,979,449 of discloses a process for the preparation of L-Glutamine (I), wherein 5-methyl-L-glutamate (III) is reacted with acetic anhydride to produce a-(N-acetyl)-L-glutamate (XIV) followed by treated with HCl to produce L-Glutamine (I).

The process is as shown in scheme-V below:

Scheme-V

The major disadvantage of above processes is mainly the de-acetylation stage, which is generally performed by enzymatic process. In chemical process during the de-acetylation with hydrochloric acid, the amide functional group also hydrolyzed and eventually gives L-Glutamic acid as a major product, which ends up with lowest yield.

However, there is always a need for alternative preparative routes, which for example, involve fewer steps, selection of protecting groups, usage of reagents that are less expensive and/or easier to handle and raw materials which are readily/commercially available.

Hence, there is a need to develop cost effective and commercially viable process involves minimum number of steps for the preparation of L-Glutamine (I).

The present invention is directed towards a process for the preparation of L-Glutamine (I), wherein amination of N-protected-5-methyl L-glutamate (XV) to produce N-protected -L-glutamine (XVI), followed by de-protection to produce L-Glutamine (I).

OBJECTIVE OF INVENTION

The main objective of the present invention is to provide a simple and cost effective process for the preparation of L-Glutamine (I) which is industrially viable.

SUMMARY OF THE INVENTION

The main embodiment of the present invention is to provide a process for the preparation of L-Glutamine of Formula (I).

Formula I

which comprises:
(i) amination of N-protected-5-methyl-L-glutamate (XV),

Formula (XV)

wherein p is a amine protecting group selected from phthaloyl, tert-butoxycarbonyl, and trifluoromethoxy carbonyl and trifluoromethyl carbonyl;
to produce N-protected-L-glutamine (XVI),

Formula (XVI)

(ii) deprotection of N-protected-L-glutamine (XVI) using suitable acid or an organic solvent in an acid to produce L-Glutamine (I).

DETAILED DESCRIPTION OF THE INVENTION

The main embodiment of present invention provides a process for the preparation of L-Glutamine (I).

The process comprises, amination of N-protected-5-methyl-L-glutamate (XV) to produce N-protected-L-glutamine (XVI).

The above reaction is carried out using aqueous ammonia in presence/absence of a solvent. The solvent comprises an ether selected from tetrahydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether, methyl tert-butyl ether, diglyme or monoglyme; an alcohol selected from C1-C10 straight or branched chain alcohol such as methanol, ethanol, isopropyl alcohol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 2,2-dimethyl-1-propanol, 2,2,2-trimethyl ethanol, 1-decanol, benzyl alcohol; hydrocarbon solvent selected from the group toluene, benzene, o-xylene, m-xylene, p-xylene; water; acetone; acetonitrile; ethyl acetate; methylene chloride; DMF; an aliphatic hydrocarbon selected from C1-8 carbon atoms containing straight chain or branched chain or cycloalkane substituted cycloalkane or mixture thereof.

Deprotection of N-protected-L-glutamine (XVI) using suitable acid or an organic solvent in an acid to produce crude L-Glutamine or acid (I).
The acid comprises an organic acid selected from acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, citric acid, benzoic acid, carbonic acid, methane sulfonic acid and p-toluene sulfonic acid, dichloroacetic acid, trichloroacetic acid and trifluoroacetic acid (TFA), or a inorganic acid selected from hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid and hydroiodic acid or mixtures thereof.
The organic solvent in an acid comprises an organic solvent in acid selected from IPA-HCl, Dioxane-HCl, Methanolic HCl and Ethanolic HCl or mixtures thereof.

The obtained crude L-Glutamine is isolated as acid may be further purified by crystallization from a suitable solvent, such as methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethyl acetate or mixtures thereof.

The above reaction is carried out in the presence of suitable base. The suitable base selected from but not limited to organic and inorganic base. The organic base is selected from a group comprising of triethylamine, diethylamine, diethyl aminopyridine, pyridine or the like. The suitable inorganic base is selected from a group comprising of lithium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, caesium carbonate, ammonia hydroxide, sodium bicarbonate or mixture thereof.

The above reaction is carried out in the presence/ absence of a solvent. The solvent comprises an ether such as tetrahydrofuran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofuran, cyclopentyl methyl ether, methyl tert-butyl ether, diglyme or monoglyme; an alcohol selected from C1-C10 straight or branched chain alcohol such as methanol, ethanol, isopropyl alcohol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 2,2-dimethyl-1-propanol, 2,2,2-trimethyl ethanol, 1-decanol, benzyl alcohol; hydrocarbon solvent selected from the group toluene, benzene, o-xylene, m-xylene, p-xylene; water; acetone; acetonitrile; ethyl acetate; methylene chloride; DMF; an aliphatic hydrocarbon selected from C1-8 carbon atoms containing straight chain or branched chain or cycloalkane substituted cycloalkane or mixture thereof.

The L-Glutamine (I) is isolated as a solid (Free base). Optionally, Compound (I) is subjected to purification either by column chromatography or by crystallization by known methods, for example by dissolving in a solvent comprises, methanol, ethanol, propanol, isopropanol, ethyl acetate, methylene chloride, hexane, heptane, cyclohexane, acetone, THF, acetonitrile, methyl-tert-butyl ether, diisopropyl ether, diethyl ether, water or mixtures thereof; and precipitating pure compound by cooling the solution or by adding an anti solvent comprises, cyclohexane, n-hexane, n-heptane or water etc.

Other embodiment of the present invention provides N-protected-5-methyl-L-glutamate (XV) used in the present invention is produced by known methods reported in the literature.

Still another embodiment of the present invention provides a pharmaceutical composition comprising, (L)-Glutamine (I) prepared by the above process and one of more pharmaceutical excipient(S) thereof.

The following example(s) illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.

EXAMPLE-1:

Preparation of L-Glutamine:

N-Boc-L-Glutamine preparation:

Aqueous ammonia (6 vol) was added to the N-Boc 5-methyl-L-glutamate aqueous layer at 25-35°C and was stirred for overnight. After completion of the reaction, the reaction mass was distilled under reduced pressure to get the gummy mass of the titled compound.

L-Glutamine preparation:

Trifluoroacetic acid (4 vol) was added to the above gummy mass (obtained from the above reaction) at 0-10°C and the reaction mass was allowed to heat at 25-35°C and stirred for 2-3 hrs. After completion of the reaction, the excess TFA was distilled under reduced pressure and aqueous ammonia (4 vol) was added then distilled ~ 2 vol under reduced pressure at below 55°C. Methanol (6 vol) was added to the aqueous mass at 25-35°C and cooled to 0-10°C then stirred for 1 hr. The obtained white solid was filtered and washed with methanol (2 vol). The titled compound was dried under reduced pressure at 70-75°C for ~5 hrs to get the 40% of the theoretical yield.

Example-2
Preparation of N-Boc-5-Methyl-L-Glutamate:
5-Methyl-L-Glutamate preparation:
L-Glutamic acid and methanol (8 vol) were mixed and cooled to 10-15°C. Sulfuric acid (0.5 volumes) was slowly added to this suspension at 10-15°C. The clear solution obtained was allowed to heat at 25-35°C and stirred for 3-4 hrs. After completion of the reaction, pH of the reaction mass was adjusted to 6-7 with triethylamine. The white solid obtained was stirred for 2 hrs at 25-35°C and filtered, washed with methanol (4 vol). Compound obtained was dried at 60°C under reduced pressure. The titled compound with 90% of theoretical yield was obtained.

N-Boc-5-Methyl-L-Glutamate preparation:

5-Methyl-L-Glutamate was taken in 1:1 ratio of 1,4 Dioxane & DM Water (6 Vol) and cooled the reaction mass to 5-10°C then Boc anhydride (1.2 mole equivalents) and triethylamine (3 mole equivalents) were added. The obtained clear solution was allowed to heat at 25-35°C and stirred for overnight. After completion of the reaction, the reaction mass of the titled compound was washed with methyl tert butyl ether (MTBE, 2 x 3 vol) to remove non-polar impurities.
Example-3
Preparation of L-Glutamine:
STAGE-I: 5-Methyl L-Glutamate preparation
L-Glutamic acid and methanol (9 vol) were cooled to 10-15°C, sulfuric acid (0.5 volumes) was added slowly to this suspension at 10-15°C. The obtained clear solution was allowed to 25-35°C and stirred for 3-4 hrs. After completion of the reaction, pH of the reaction mass was adjusted to 6-7 with triethylamine. Obtained white solid was stirred for 2 hrs at 0-10°C and filtered, washed with methanol (2 vol). The product was dried at 50-55°C under reduced pressure. Obtained yield is 85% of theoretical yield.

STAGE-II PART A: N-Boc 5-Methyl L-Glutamate preparation
Stage-I compound was taken in 1:1 ratio of methanol & DM water (8 Vol) at 25-35°C and added sodium bicarbonate (2 mole equivalents) followed by Boc anhydride (1.2 mole equivalents). The obtained suspension stirred at 25-35°C for overnight. After completion of the reaction, the pH of the reaction mass was adjusted to 3-4 with 50% aq. solution of citric acid (6 vol). Reaction mass was extracted with dichloromethane (1 x 4 vol). This DCM layer contains Stage-II Part A product, which was taken as such for Amide preparation.

STAGE-II PART B: N-Boc L-Glutamine preparation
Aqueous ammonia (6 vol) was added to the above Stage-II Part-A, DCM layer at 25-35°C and stirred for overnight at 25-35°C. After completion of the reaction, DCM layer was separated and aqueous layer was distilled under reduced pressure to get the gummy mass, which contains Stage-II Part B product.

STAGE-II PART C: L-Glutamine Hydrochloride Salt
To the above gummy mass (Stage-II Part B) was dissolved in IPA (5 Vol) at 25-35°C and added to 5 volumes of IPA. HCl solution (~25% w/w assay) at 10-20°C. Maintained the reaction mass for 4 hrs at 25-30°C. After completion of the reaction, filtered the solid (L-Glutamine hydrochloride salt) and wash with IPA (0.5 vol). The wet material was purified from methanol (4 vol) by slurry at 40-50°C & filtered at 0-10°C after 1 hr maintenance.

STAGE-II: L-Glutamine
The above wet material (Stage-II Part C) was dissolved in DM water (4 Vol) at 25-35°C & pH was adjusted to 6.5-7.0 with aq. ammonia. Then methanol (6 Vol) was added at 25-35°C and stirred at 0-10°C for 2 hrs. Filtered the obtained product and wash with methanol (0.5 Vol). Wet material was dried at 60-65°C under reduced pressure. ,CLAIMS:WE CLAIM:

1. A process for the preparation of L-Glutamine of Formula (I),

Formula I
which comprises:
(i) amination of N-protected-5-methyl-L-glutamate (XV),

Formula (XV)
wherein p is a amine protecting group selected from phthaloyl, tert-butoxycarbonyl, and trifluoromethoxy carbonyl and trifluoromethyl carbonyl; to produce N-protected-L-glutamine (XVI),

Formula (XVI)
(ii) deprotection of N-protected-L-glutamine (XVI) using suitable acid or organic solvent in acid to produce L-Glutamine (I).

2. The process as claimed in claim 1, wherein the reaction of step (i) is carried out in presence of aqueous ammonia in presence/absence of a solvent, comprises methanol, ethanol, isopropyl alcohol, tetrahydrofuran, dioxane, diisopropylether, diethylether, toluene, benzene, xylene, water; acetone; acetonitrile; ethyl acetate; methylene chloride; DMF or mixture thereof.
3. The process as claimed in claim 1, wherein the reaction of step (ii) is carried out in presence of suitable acid, comprises an organic acid selected from acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, citric acid, benzoic acid, carbonic acid, methane sulfonic acid and p-toluene sulfonic acid or a inorganic acid selected from hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid and hydroiodic acid or mixtures thereof.

4. The process as claimed in claim 1, wherein the reaction of step (ii) is carried out in presence of organic solvent in acid, comprises IPA-HCl, Dioxane-HCl, Methanolic HCl and Ethanolic HCl or mixtures thereof.

5. The process as claimed in claim 1, wherein L-Glutamine is isolated as an acid salt in step (ii).

6. The process as claimed in claim 5, the acid comprises acetic acid, propionic acid, hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid and hydroiodic acid or mixtures thereof.

7. A process for the purification of L-Glutamine acid salt, comprising:
a) adding a suitable solvent to crude L-Glutamine acid salt;
b) heating the reaction mixture to a suitable temperature;
c) cooling the reaction mixture to a suitable temperature; and
d) isolating pure L-Glutamine acid salt.

8. The process as claimed in claim 7, wherein L-Glutamine acid salt further converts to L-Glutamine (I) by using suitable base.

9. The process as claimed in claim 8, suitable base comprises organic and inorganic base. The organic base is selected from a group comprising of triethylamine, diethylamine, diethyl aminopyridine, pyridine or the like. The suitable inorganic base is selected from group comprising of lithium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, caesium carbonate, ammonia hydroxide, sodium bicarbonate or mixture thereof.

10. The process as claimed in claims 7 and 8, wherein the reaction is carried out in the presence/ absence of a solvent, comprises C1-C10 straight or branched chain alcohol such as methanol, ethanol, isopropyl alcohol, 1-butanol, 2-butanol; hydrocarbon solvent selected from the group toluene, benzene, o-xylene, m- xylene, p-xylene; water; acetone; acetonitrile; ethyl acetate; methylene chloride; DMF; dioxane, THF; ether solvent selected from diethyl ether, cyclopentyl methyl ether, MTBE or mixture thereof.