Formula I

BACKGROUND OF THE INVENTION

L-Glutamine (I) is chemically known as 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 Me0H/H ₂ S0 ₄ 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 g-methyl-L-glutamate (III) to produce L-y-glutamylhydrazide (VII), followed by hydrogenated with Raney nickel to produce L-Glutamine (I). The process is as shown in scheme-III below:

O o Aqueous o o hydrazine hydrate/ Methanol

NH ₂ NH ₂ g-Methyl L-glutamate (III) L-y-Glutamylhydrazide (VII)

Water/ alkaline Raney nickel/ Aq.ammonium sulfide/ acetic acid

L-Glutamine (I)

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 HC1 (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 HC1 to produce L- Glutamine (I).

The process is as shown in scheme- V below: i) Methanol/

Acetic anhydride/ Cone, ammonia ii) 50% sulphuric acid g-Methyl-L-glutamate (IP) a-(N-Acetyl)-L-glutamine (XIV)

L-Glutamine (I)

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. Acta Crystallographica (1952), 5, 644-53 reported single crystalline data of L- Glutamine (I) and its recrystallization from water at a temperature between 0 and 5°C.

US 2,762,841 discloses a purification process of L-Glutamine (I), wherein crude L-Glutamine (I) purified from acetone and water by recrystallization. The purity of L-Glutamine by HPLC maximum achieved to 96% with this recrystallisation.

US 3,105,852 discloses crystallization of L-Glutamine (I) from methanol and water.

The above two crystallization processes disclosed in US ‘852 and US ‘841 are capable to produce L-Glutamine (I) crystals with bulk density below 0.25 g/mL.

JP 04257551 discloses a purification of L-Glutamine, wherein crude L-Glutamine is dissolved in water, the pH was adjusted to 5.5 with dilute aqueous ammonia, followed by addition of carbon/seed crystal of L-Glutamine and addition of methanol to produce pure L-Glutamine.

DEI 124044 discloses purification of L-glutamine from warm water and precipitated by adding ethanol to produce pure L-Glutamine.

The above purification processes disclosed in above prior-art processes is capable to produce L-Glutamine (I) crystals with bulk density below 0.25 g/mL. L- Glutamine (I) obtained by above processes has uneven particle size distribution and low density of crystalline products resulting in the difficulty of separation of L-glutamine crystals from a liquid, granulation and constant volume packaging is difficult. The crystallization process in the production process is a key step that restricts product quality.

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).

The present invention also directed to a process for the purification of L- Glutamine (I) with spherical agglomerated crystals having specific bulk density and Hausner ratio, with improved flow properties.

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 an 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).

The other embodiment of the present invention is to provide a process for the purification of L-Glutamine (I) with spherical agglomerated crystals having bulk density 0.43-0.47 and Hausner ratio between 1.19-1.25, which comprises:

(i) dissolving L-Glutamine (I) in a first solvent;

(ii) heating the reaction mass at a suitable temperature;

(iii) adjusting pH by adding a base to step (ii); (iv) adding a second solvent; and

(v) isolating pure L-Glutamine (I) with spherical agglomerated crystals.

Another embodiment of the present invention is to provide L-Glutamine (I) with spherical agglomerated crystals having bulk density 0.43-0.47 and Hausner ratio between 1.19-1.25. 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.

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 Ci-Cio 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 Ci- 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 /Molucnc 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 IPA-HCl, Dioxane-HCl, Methanolic HC1 and Ethanolic HC1 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 tetrahydrof iran, dioxane, diisopropylether, diethylether, 2-methyltetrahydrofiiran, cyclopentyl methyl ether, methyl tert-butyl ether, diglyme or monoglyme; an alcohol selected from Ci-Cio 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 Ci-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.

Yet another embodiment of the present invention to provide a process for the purification of L-Glutamine (I) with spherical agglomerated crystals having bulk density 0.43-0.47 and Hausner ratio between 1.19-1.25.

Spherical crystallization is defined as an agglomeration process that transforms crystals directly into compact spherical forms during the crystallization. It also enables co-precipitation of drug and encapsulating particle size enlargement in the form of spherical particle. Spherical crystallization is an effective alternative to improve the dissolution rate of drugs. This can be achieved by various methods such as spherical agglomeration, quasi-emulsion solvent diffusion, Ammonia water method, Crystallo-co agglomeration and neutralization method. Agglomerates exhibit improved secondary characteristics, like flowability and compressibility, so that direct tableting or coating is possible without further processing (mixing, agglomerates, sieving, etc.). This improves the flowability property by Crystallo-co agglomeration technique.

Crystallo-co-agglomeration (CCA) technique involves simultaneous crystallization and agglomeration of drug from the suitable solvent and addition of an anti-solvent.

Spherical agglomeration is a process of formation of aggregates of crystals held together by liquid bridges. The agglomerates are formed by agitating the crystals in a liquid suspension and properties of the particles designed vary significantly as compared to the fine crystalline material. These agglomerates are found to have good flowability and compressibility. This technique can also be exploited to increase solubility, dissolution and hence bioavailability of poorly soluble drugs.

The process of the present invention comprises, L-Glutamine (I) is dissolved in a first solvent, the reaction mass is heated at a suitable temperature followed by adjusting pH by adding a base and a second solvent; then pure L-Glutamine (I) is isolated with spherical agglomerated crystals having bulk density 0.43-0.47 and Hausner ratio between 1.19- 1.25.

The first solvent comprises water or an alcohol selected from C1-C3 straight or branched chain alcohol such as methanol, ethanol, n-propanol and isopropyl alcohol.

The second 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 Ci- ₈ carbon atoms containing straight chain or branched chain or cycloalkane substituted cycloalkane or mixture thereof.

The reaction mass is heated at suitable temperature ranges from -10 to 65 °C.

The suitable base comprises aqueous ammonia, triethylamine, diethylamine, diethyl aminopyridine, pyridine lithium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate or mixture thereof.

The pH of the reaction mass is adjusted to 5 to 8.

Still another embodiment, the present invention provides L-Glutamine (I) having a bulk density not less than 0.30 g/mL, preferably not less than 0.35 g/mL, more preferably not less 0.40 g/mL.

Still another embodiment of the present invention provides L-Glutamine (I) having Hausner ratio between 1.19-1.25.

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: /V-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: A-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. HC1 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.

Example-4:

L-Glutamine (10 g) was dissolved in water (10 vol, 100 ml) into the reactor at 55- 60°C, the reaction mixture was added to pre-cooled methanol (40 vol, 400 ml) at - 10 to -5 °C and the solid was filtered and washed with methanol 1 vol, (10 ml). The product was dried at 60-65 °C under reduced pressure (NLT 650 mmHg) till LOD is < 0.5% w/w it took 5-6 h to achieve this result.

Yield: 9.6 g [0.96 w/w, based on L-Glutamine input weight)

Bulk density: 0.32 g/mL

Example -5:

L-Glutamine (75 g) was dissolved in water (10 vol, 750 ml) into the reactor at 55- 60°C and adjusted the reaction mass pH to 7.01 with aqueous ammonia. Then reaction mixture was added to precooled methanol (40 vol, 3000 ml) at -10 to -5 °C and filter the solid and washed with methanol 1 vol, (75 ml). The product was dried at 60-65°C under reduced pressure (NLT 650 mmHg) till LOD is < 0.5% w/w it took 5-6 h to achieve this result.

Yield: 65 g [0.86 w/w, based on L-Glutamine input weight)

Bulk density: 0.43 g/mL Example -6:

L-Glutamine (40 g) was dissolved in water (10 vol, 400 ml) into the reactor at 55- 60°C and adjusted the reaction mass pH to 6.93 with aqueous ammonia. Then reaction mixture was added to methanol (40 vol, 1600 ml) at 25-30 °C and the solid was filtered and washed with methanol 1 vol, (40 ml). The product was dried at 60-65°C under reduced pressure (NLT 650 mmHg) till LOD is < 0.5% w/w it took 5-6 h to achieve this result.

Yield: 36.5 g [0.91 w/w, based on L-Glutamine input weight)

Bulk density: 0.42 g/mL

Example -7:

L-Glutamine (20 g) was dissolved in water (10 vol, 200 ml) into the reactor at 55- 60°C and adjusted the reaction mass pH to 6.95 with aqueous ammonia. Then reaction mixture was added to methanol (40 vol, 800 ml) at 40-45 °C and the solid was filtered and washed with methanol 1 vol, (20 ml). Dried the product at 60- 65°C under reduced pressure (NLT 650 mmHg) till LOD is <0.5% w/w it took 5- 6 hr to achieve this result.

Yield: 14 g [0.70 w/w, based on L-Glutamine input weight)

Bulk density: 0.48 g/mL