Description:FORM 2

THE PATENTS ACT,
(39 OF 1970)
THE PATENT RULES, 2003.

COMPLETE SPECIFICATION
(SECTION 10 AND RULE 13)

SYNTHESIS OF L-ECTOINE FROM L-2,4-DIAMINOBUTYRIC ACID AND PURIFICATION THEREOF

PURVIEW LIFESCIENCES P LTD
3rd floor, Trendz Solitaire, Near Westin Hotel, Vittalrao Nagar, Hitech City, HYDERABAD-500 081

The following specification particularly describes the invention and the manner in which it is to be performed.
SYNTHESIS OF L-ECTOINE FROM L-2,4-DIAMINOBUTYRIC ACID AND PURIFICATION THEREOF

FIELD OF THE INVENTION:
The present invention relates to an improved process for synthesis of L-Ectoine of Formula (I) from L-2,4-Diaminobutyric acid. The present invention further relates to a process for purification to obtain highly pure L-Ectoine having purity greater than 99.5%.

Formula (I)

BACKGROUND OF THE INVENTION:

L-Ectoine is a natural compound found in several species of bacteria. It is a compatible solute which serves as a protective substance by acting as an osmolyte and thus helps organisms survive extreme osmotic stress. L-Ectoine is used as an active ingredient in skin care and sun protection products which stabilizes proteins and other cellular structures and protects the skin from stresses like UV irradiation and dryness.

Formula (I)
CN101698662 discloses a process for preparation of 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid (L-Ectoine) by the reaction of L-2,4-diaminobutyric acid and 4-nitrophenyl acetate in the presence of inorganic alkali in aqueous solution via N-acetylation to give the mixture of 2-acetylamino-4-aminobutyric acid and 2-amino-4-acetylaminobutyric acid, then cyclization in the presence of triethylamine in polar organic solvent to give L-Ectoine. Purification of final compound by column chromatography leads to increase the cost of the project and also not suitable for large scale synthesis. Our impugned invention avoids the final column chromatography and provides process for the preparation of L-Ectoine which is a simple, less time consuming and commercially scalable; and provides highly pure form of L-Ectoine.
US patent document US 2011/0178292 discloses a method for synthesizing 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid using 2,4-diaminobutyric acid and trimethyl orthoformate as raw materials. The last few steps of the process need to be prepared by liquid phase separation, which involves the disadvantages of high cost and difficult industrialization. Present invention avoids tedious and time consuming liquid separation methods and achieves high purity levels by a simple new and inventive purification process.
The inventors of the present application have attempted to prepare L-Ectoine from L-2,4-Diaminobutyric acid using the process disclosed in prior arts, but the compound has resulted lower yields which further increases the manufacturing cost of the final product. The inventors are also tried various purification techniques and experiments but ended up with doing number of experiments using different solvents in different ratios and under different conditions. Finally, the present inventors after performing various permutations and combinations surprisingly found a new and an inventive purification process which produces L-Ectoine at high purity greater than 99.5% in an easily reproducible, more economical, and highly scalable at commercial levels.
The inventors of the present invention have surprisingly found and emulated the process for the preparation L-Ectoine from L-2,4-Diaminobutyric acid and made it very much possible at large scale level which could be reproduced at economical price and in a very short run time and also with higher yields.

Advantages of the process described in present invention over the prior arts:
1. Synthesis of pure L-Ectoine with higher purities and yields.
2. Avoid of purification by column chromatography.
3. Avoid of liquid phase separation methods.
4. Easily operable, reproducible and non-tedious process.
5. Suitable for large scale synthesis.

SUMMARY OF THE INVENTION:
An embodiment of the present invention is to provide a process for the preparation of L-Ectoine, comprising the steps of:
a) reaction of L-2,4-Diaminobutyric acid with trimethylortho acetate in presence of a suitable organic solvent,
b) removal of organic solvent from reaction mixture after completion of reaction,
c) addition of another suitable solvent to the material obtained in step (b),
d) carbon treatment to a solution obtained in step (c) followed by filtration,
e) pH adjustment of filtrates obtained in step (d) with a suitable reagent followed by evaporation,
f) treating the material obtained in step (e) with DMSO (Dimethyl sulfoxide) solvent at a suitable temperature,
g) treatment for product obtained in step (f) with another suitable solvent.

Another embodiment of present invention is to provide an improved process for the purification of L-Ectoine, comprising the steps of:
1) providing a solution of L-Ectoine in a suitable solvent,
2) pH adjustment of the solution obtained in step (1) with a suitable reagent,
3) carbon treatment to a solution obtained in step (2) followed by filtration,
4) evaporation of filtrates obtained in step (3),
5) treating the material obtained in step (4) with a suitable solvent,
6) isolation of the product obtained in step (5),
7) treatment for product obtained in step (6) with another suitable solvent to obtain highly pure L-Ectoine having purity greater than 99.0%, preferably greater than 99.3%, more preferably greater than 99.5%,
8) isolation of final product obtained in step (7).
9) Another embodiment of the present invention is to provide a process for preparation of L-Ectoine from L-2,4-diaminobutyric acid.
Another embodiment of the present invention provides a process which is economical at commercial scales.
Another embodiment of the present invention provides a process which is reproducible, with higher yields.
Another embodiment of the present invention provides a process which is reproducible, with higher yields and in a short run time.

DETAILED DESCRIPTION OF THE INVENTION
The present invention will be further described below in conjunction with specific embodiments. The examples are only preferred embodiments of the present invention and are not intended to limit the present invention.
In a preferred embodiment, the present invention provides a process for the preparation L-Ectoine of Formula (I) starting from L-2,4-Diaminobutyric acid, comprising the steps of:
a) reaction of L-2,4-Diaminobutyric acid optionally free base or corresponding salt with trimethylortho acetate in presence of a suitable organic solvent,

b) removal of organic solvent from reaction mixture after completion of reaction,
c) addition of another suitable solvent to the material obtained in step (b),
d) carbon treatment to a solution obtained in step (c) followed by filtration,
e) pH adjustment of filtrates obtained in step (d) with a suitable reagent followed by evaporation,
f) treating the material obtained in step (e) with DMSO (Dimethyl sulfoxide) solvent at a suitable temperature,
g) treatment for product obtained in step (f) with another suitable solvent.

Another embodiment of present invention further provides an improved process for the purification of L-Ectoine of Formula (I), comprising the steps of:
1) providing a solution of L-Ectoine in a suitable solvent,
2) pH adjustment of the solution obtained in step (1) with a suitable reagent,
3) carbon treatment to a solution obtained in step (2) followed by filtration,
4) evaporation of filtrates obtained in step (3),
5) treating the material obtained in step (4) with a suitable solvent,
6) isolation of the product obtained in step (5),
7) treatment for product obtained in step (6) with another suitable solvent to obtain highly pure L-Ectoine having purity greater than 99.0%, preferably greater than 99.3%, more preferably greater than 99.5%;
8) isolation of final product obtained in step (7).
Another embodiment of the present invention provides a process which is economical at commercial scales.
Another embodiment of the present invention provides a process which is reproducible, with higher yields.
Another embodiment of the present invention provides a process which is reproducible, with higher yields and in a short run time.
In aspects, conversion of L-2,4-Diaminobutyric acid into L-Ectoine may carried out using trimethylortho acetate at a suitable temperature using a suitable solvent. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be Methanol. In aspects, the reaction may be carried out for about one hour to about 24 hours at about 0 °C to about 80 °C. Preferably, reaction may be carried out for about 2 hours to about 6 hours at about 40 °C to about 80 °C. More specifically, reaction may be carried out for about 3 hours at about 50 °C to 55°C.
In aspects, L-Ectoine synthesized under the said conditions was further dissolved in a suitable solvent and subjected to carbon treatment at a suitable temperature. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be Water. In aspects, the carbon treatment may carry at a temperature about 10 °C to 40 °C. Preferably, at about 25 °C to 35 °C.
In aspects, the pH of filtrates obtained after carbon treatment (in step d) may further adjusted with a suitable reagent at a range about 5.0 to 8.0. The reagents used for PH adjustment include but not limited to alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, ammonia, ammonium hydroxide and like or mixture thereof. Preferably, ammonium hydroxide. The PH of the filtrates may adjust at a range between 6.0 to 7.0. Preferably, at a range between 6.0 to 6.5.
In aspects, the product obtained from step (e) may further purified using Dimethyl sulfoxide solvent at a temperature range about 60 °C to 90 °C. Preferably, between 60 °C to 70 °C. In aspects, the product obtained in step (f) further purified using a suitable solvent. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, acetone, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be acetone.
In aspects, purification of L-Ectoine may carried out by providing a solution of L-Ectoine in a suitable solvent. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be water.
In aspects, the pH of the solution obtained in step (1) may further adjusted with a suitable reagent at a range about 5.0 to 8.0. The reagents used for pH adjustment include but not limited to alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, ammonia, ammonium hydroxide and like or mixture thereof. Preferably, ammonium hydroxide. The PH of the solution may adjust at a range between 6.0 to 7.0. Preferably, at a range between 6.0 to 6.5.
In aspects, the material obtained in step (4) may further treated with a suitable solvent. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be Methanol. In aspects, method for isolation of the product obtained in step (5) and (7) is either filtration, filtration under vacuum, decantation, centrifugation or a combination thereof.
In aspects, the material obtained in step (6) may further treated with a suitable solvent. The solvents used include but not limited to methanol, toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, acetone, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc, water and like or a mixture thereof. Specifically, the solvent may be acetone.
The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
Brief Manufacturing process:
Example 1:
Conversion of L-2,4-Diamnobutyric acid into L-Ectoine:

To a stirred solution of trimethylortho acetate (500 mL) in methanol (3.5 lit) was added L-2,4-Diaminobutyric acid (250.0 g) at 50 °C to 55 °C and stirred for 3.0 hours. On completion of reaction, distilled off methanol completely under vacuum and resulting residue was dissolved in water (500 mL) and added activated carbon (10.0 g) at room temperature and stirred for 10 to 15 minutes. Filtered the reaction mass through a hyflo bed. Adjusted the pH of the filtrates to 6.18 with 10% ammonium hydroxide solution. Distilled off filtrates under vacuum and diluted with DMSO solvent (600 mL) at 70 °C. Stirred reaction mass at 70 °C to 75 °C for 30 minutes. Cooled the reaction mass to 65 °C and filtered the product at 450C. The product was further diluted with acetone (200 mL) and stirred for 30 minutes. Filtered the product and washed with acetone (wet weight: 81.5 g).

Purification of L-Ectoine:

To a stirred solution of wet L-Ectoine (150.0 g) in water (600.0 mL) was added 10% ammonium hydroxide solution slowly and adjusted the pH to 6.1; charged activated carbon (10.0 g) to the reaction mass and stirred for 30 minutes at room temperature. Filtered the reaction mass through a hyflo bed and washed with water (30.0 ml). Filtrates are further filter on micron filter paper. Distilled off the filtrates completely under vacuum at a temperature below 60 °C. Diluted the resulting product with methanol (100.0 mL) and stirred for 10 minutes at room temperature. Reaction mass was further cooled to 15 °C and stirred for 20 minutes. Filtered the product and washed with methanol (50.0 mL). The resulting wet material was transferred to RBF and diluted with acetone (350.0 mL). Heated the reaction mass to 50 °C to 55 °C and stirred for 10 minutes. Filtered the product and washed with acetone (150. 0 mL). The resulting product was dried under vacuum at 80 °C to 85 °C.
Without wishing to be bound to a theory, the process described in the present invention is believed to be an improved process for the preparation of L-Ectoine or pharmaceutical acceptable salts and purification process thereof which is stable, economical, and commercially scalable.
The process described in the present invention also discloses the process for the preparation of L-Ectoine or pharmaceutical acceptable salts and purification process thereof which is stable, economical, and commercially scalable, results in higher yields in a short run time.
While the illustrative embodiments of the invention have been described with particularity, it will be understood that various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the examples and descriptions set forth hereinabove but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present invention, including all features which would be treated as equivalents thereof by those skilled in the art to which the invention pertains.

Dated this Day of 21st May 2023
, Claims:We Claim:
1. A process for preparation of pure L-Ectoine having purity greater than 99.5% comprising,
step (1): providing a solution of L-Ectoine in a suitable solvent,
step (2): adjusting the pH of the solution obtained in step (1) with a suitable reagent in a range of 6.0 to 6.5,
step (3): subjecting the solution obtained in step (2) to carbon treatment followed by filtration,
step (4): evaporation of filtrates obtained in step (3),
step (5): treating the material obtained in step (4) with a suitable solvent,
step (6): isolation of the product obtained in step (5),
step (7): treatment for product obtained in step (6) with another suitable solvent,
step (8): isolation of final product obtained in step (7),
wherein, the reaction comprises,
step (a): reaction of L-2,4-Diaminobutyric acid optionally free base or corresponding salt with trimethylortho acetate in presence of methanol solvent at a temperature 50 °C to 55 °C,
step (b): removal of methanol solvent from reaction mixture after completion of reaction,
step (c): addition of another suitable solvent to the material obtained in step (b),
step (d): carbon treatment to a solution obtained in step (c) followed by filtration,
step (e): pH adjustment of filtrates obtained in step (d) with a suitable reagent followed by evaporation,
step (f): treating the material obtained in step (e) with a water miscible polar aprotic solvent,
step (g): further treatment of product obtained in step (f) with another suitable solvent.

2. The process as claimed in claim 1, wherein the suitable solvent used in step (1) and step (c) is water.
3. The process as claimed in claim 1, wherein the suitable reagent used for pH adjustment in step (2) and step (e) is ammonium hydroxide.
4. The process as claimed in claim 1, wherein the water miscible polar aprotic solvent used in step (f) is Dimethyl sulfoxide.
5. The process as claimed in claim 1, wherein the suitable solvent used in step (7) and step (g) is acetone.
6. The process as claimed in claim 1, wherein the suitable solvent used in step (5) is methanol.

Dated this Day of 21st May 2023

Signature:
Dr. VURE PRASAD
Patent Agent Reg. No.: IN/PA-1636