Description:FORM 2

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

COMPLETE SPECIFICATION
(SECTION 10 AND RULE 13)

AN IMPROVED PROCESS FOR SYNTHESIS OF ENANTIOMERICALLY PURE L-2,4-DIAMINOBUTYRIC ACID

PURVIEW LIFESCIENCES PVT 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.
AN IMPROVED PROCESS FOR SYNTHESIS OF ENANTIOMERICALLY PURE L-2,4-DIAMINOBUTYRIC ACID
FIELD OF THE INVENTION:
The present invention relates to process for synthesis of enantiomerically pure L-2,4-diaminobutyric acid of Formula-I. The invention further relates to synthesis of L-Ectoine using L-2,4-diaminobutyric acid.

Formula (I)

BACKGROUND OF THE INVENTION:

L-2,4-diaminobutyric acid of Formula-I is an important intermediate used for synthesis of L-Ectoine. 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)
In 1901, Fischer reported earliest preparation of racemic 2,4-diaminobutyric acid via β-phthalimidoethylmalonic ester. The racemic 2,4-diaminobutyric acid was isolated in low yield.

(Scheme-1)
In 1939, Akabori and Numano adopted a method for 2,4-diaminobutyric acid preparation which involves reaction of Glutamic acid with highly toxic potassium cyanate to form α-carbamyl glutamic acid. The resultant product was further converted into 2,4-diaminobutyric acid (scheme-2) by multiple steps and tedious process and results the final product in less than 20% yield.


Synthesis of 2,4-diaminobutyric acid from glutamic acid by Akabori and Numano
(Scheme-2)
Later, Akinson and Poppelsdorf reported another method (scheme-3) which involves condensation of Acrolein with phthalimide in Triton B to form β-phthalimidopropionaldehyde. Further the resulting product was converted into 2,4-diaminobutyric acid hydrochloride in 43% yield. The present process involves the usage of toxic Acrolein and sodium cyanide reagents. Usage of these reagents is not suggestable for large scale synthesis and also it leads to harm the environment.


Synthesis of 2,4-diaminobutyric acid from acrolein by Atkinson and Poppelsdorf
(Scheme-3)
CN110804634 discloses a process for preparing 2,4-diaminobutyric acid by enzyme catalysis method comprises carrying out enzyme catalysis reaction on raw materials of L-aspartic acid, ATP or its salt, pyridoxal phosphate, alanine, NADPH or its salt, aspartokinase ASK, aspartate semialdehyde oxidase ASADH and transaminase AMT in a reaction medium with pH value of 6.0-9.0 to generate 2,4-diaminobutyric acid. Use of enzyme catalysis leads to increase the cost of the project and also not suitable for large scale synthesis.
Synthesis of 2,4-diaminobutyric acid from corresponding Glutamic acid is disclosed in Journal of the Chemical Society (1939), 1564-8 using highly toxic Hydrazoic acid. Usage of Hydrazoic acid is not suggestable for large scale synthesis and also it leads to harm the environment. The present invention avoids the usage of highly toxic Hydrazoic acid and is suitable for commercial scale production.
Most of the known methods to synthesize both racemic 2,4-diaminobutyric acid and enantiomeric pure L-2,4-diaminobutyric acid were quite tedious and the yield obtained in most cases did not seem to justify labor. Although, procedures mentioned in above schemes were very general, operational difficulties in handling the toxic reagents and lower yields hindered their employments.
The inventors of the present invention have surprisingly found and emulated the process for the preparation enantiomerically pure 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.
In view of above prior arts, the inventors of the present invention have found a process for the preparation enantiomerically pure L-2,4-diaminobutyric acid at high purity level by starting with enantiomerically pure L-Glutamic acid in an easily reproducible, more economical, and highly scalable at commercial levels.

Advantages of the process described in present invention over the prior arts:
1. Synthesis of enantiomerically pure L-2,4-diaminobutyric acid with higher purities and yields.
2. Avoid of toxic chemicals (Acrolein, sodium cyanide, Hydrazoic acid etc.,)
3. Avoid of expensive enzyme catalysis reactions.
4. Easily operable, reproducible and non-tedious process.
5. Lower in steps and higher in yields and purities.
6. Suitable for large scale synthesis.

SUMMARY OF THE INVENTION:
An embodiment of the present invention is to provide an improved process for the preparation enantiomerically pure L-2,4-diaminobutyric acid of Formula (I),

Formula (I)
Which comprises,
(a) conversion of L-Glutamic acid into sulphate salt of L-2,4-diaminobutyric acid,
(b) conversion of sulphate salt of L-2,4-diaminobutyric acid obtained in step (a) optionally with or without isolation into picrate salt of L-2,4-diaminobutyric acid,
(c) conversion of picrate salt of L-2,4-diaminobutyric acid obtained in step (b) into hydrochloride salt of L-2,4-diaminobutyric acid,
(d) Optionally, converting the hydrochloride salt into L-2,4-diaminobutyric acid free base.
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 is relating to an improved process for the preparation of enantiomerically pure L-2,4-diaminobutyric acid of Formula (I),

Formula (I)
Which comprises,
(a) conversion of L-Glutamic acid into sulphate salt of L-2,4-diaminobutyric acid by reaction with sodium azide and sulphuric acid at a suitable temperature using a suitable solvent.

(b) conversion of sulphate salt of L-2,4-diaminobutyric acid obtained in step (a) optionally with or without isolation into picrate salt of L-2,4-diaminobutyric acid.

(c) conversion of picrate salt of L- 2,4-diaminobutyric acid obtained in step (b) into hydrochloride salt of L-2,4-diaminobutyric acid in presence of a suitable solvent.

(d) optionally, conversion of hydrochloride salt of L- 2,4-diaminobutyric acid obtained in step (c) into freebase of L-2,4-diaminobutyric acid.

Another embodiment of the present invention is to provide a process for preparation of L-Ectoine from L-2,4-diaminobutyric acid.

In aspects, conversion of L-Glutamic acid into sulphate salt of L-2,4-diamino butyric acid may carried out using sodium azide and sulphuric acid at a suitable temperature using a suitable solvent. The solvents used include but not limited to toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc and water or a mixture thereof. Specifically, the solvent may be Chloroform. In aspects, the reaction (step a) may be carried out for about 2 hours to about 24 hours at about 0 °C to about 80 °C. Preferably, reaction may be carried out for about 7 hours to about 9 hours at about 40 °C to about 50 °C. More specifically, reaction may be carried out for about 8 hours at about 45°C.
In aspects, conversion of sulphate salt of L-2,4-diaminobutyric acid obtained in step (a) into corresponding picrate salt may carried out using picric acid at a suitable temperature. In aspects, the reaction (step b) may be carried out for about 10 minutes to about one hour at about 30 °C to about 100 °C. Preferably, reaction may be carried out for about 30 minutes at about 70 °C to about 80 °C.
In aspects, conversion of picrate salt of L-2,4-diamino butyric acid into corresponding hydrochloride salt may carried out using hydrochloric acid at a suitable temperature using a suitable solvent. The solvents used include but not limited to toluene, xylene, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, isopropyl ether, ethyl acetate, isopropyl acetate, acetonitrile, THF, DMF, DMAc and water or a mixture thereof. Specifically, the solvent may be Ethyl acetate. In aspects, the reaction (step c) may be carried out for about 30 minutes to about 6 hours at about 0 °C to about 40 °C. Preferably, reaction may be carried out for about 30 minutes to about 3 hours at about 20 °C to about 40 °C. More specifically, reaction may be carried out for about 2 hours at about 25°C to 30°C.
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:
Preparation of L-2,4-diaminobutyric acid hydrochloride:
Example 1: Conversion of L-Glutamic acid into L-2,4-Diamnobutyric acid sulphate salt:
To a stirred solution of H2SO4 (2.5 lit) and CHCl3 was added L-Glutamic acid (750 g) at room temperature. Heated the reaction mixture to 45°C. Added slowly sodium azide (412 g) lot wise over a period of 4 to 5 hours. Heated the reaction mixture to reflux temperature and maintained at same temperature for 8 hours. Cooled the reaction mass to room temperature and quench the reaction mass in ice-cold water. Adjusted the PH to 2.8 to 3.0 with sodalime. Filtered the resulting salts. Filtrates are carried over to next step without further isolation.

Example 2: Conversion of L-2,4-Diamnobutyric acid sulphate salt into corresponding Picrate salt:
Charged picric acid (1.2 kg) to the above filtrates slowly lot wise at 70 °C to 80 °C and maintained at same temperature for 30 minutes. Cooled the reaction mixture to room temperature and stirred for 30 minutes. Filtered the resulting precipitate and washed with water to obtain 2,4-diaminobutyric acid picric acid salt (1.6 kg).

Example 3: Preparation of L-2,4-diaminobutyric acid hydrochloride:
To a stirred solution of picrate salt of L-2,4-Diaminobutyric acid (1.2 kg) in Ethyl acetate (6. 2 lit) was added hydrochloric acid slowly drop wise at 25 °C to 30 °C. Stirred the reaction mass at same temperature for 2 hours. Filtered the resulting precipitate and washed with excess of ethyl acetate. Filtrates were evaporated under vacuum and diluted the resulting product with water (1.0 lit) and stirred for one hour at room temperature. Filtered the resulting precipitate and dried under vacuum to obtain the desired product (1.06 kg, yield: 82.3%).

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-2,4-diaminobutyric acid hydrochloride or pharmaceutical acceptable salts thereof which is stable, economical, and commercially scalable.
The process described in the present invention also discloses the process for the preparation of L-2,4-diaminobutyric acid hydrochloride or pharmaceutical acceptable salts 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 14th May 2023
, Claims:We Claim:
1. A process for the preparation of enantiomerically pure L-2,4-diaminobutyric acid hydrochloride comprising:
(a) conversion of L-Glutamic acid into sulphate salt of L-2,4-diaminobutyric acid by reaction with sodium azide in presence of sulphuric acid at a temperature 40 °C to 50 °C followed by usage of sodalime in neutralizing the reaction medium,

(b) conversion of sulphate salt of L-2,4-diaminobutyric acid obtained in step (a) optionally with or without isolation into picrate salt of L-2,4-diaminobutyric acid at a suitable temperature,

(c) conversion of picrate salt of L-2,4-diaminobutyric acid obtained in step (b) optionally with or without isolation into hydrochloride salt of L-2,4-diaminobutyric acid in presence of hydrochloric acid and a solvent followed by purification with a suitable solvent.

(d) optionally, conversion of hydrochloride salt of L- 2,4-diaminobutyric acid obtained in step (c) into freebase of L-2,4-diaminobutyric acid.

2. The process as claimed in claim 1, wherein the suitable temperature in step (b) is 70 °C to 80 °C.
3. The process as claimed in claim 1, wherein the solvent used in step (c) is Ethyl acetate.
4. The process as claimed in claim 1, wherein the solvent used for purification in step (c) is water.
5. A process for preparation of L-Ectoine from L-2,4-Diaminobutyric acid hydrochloride, prepared as claimed in claim 1.