DESC:RELATED PATENT APPLICATION
This application claims the priority to and benefit of Indian Provisional Patent Application No. 202041003386 filed on January 24, 2020; the disclosures of which are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to an improved purification process for the pharmaceutically acceptable salt of folinic acid thereof, pereferably calcium and sodium.

BACKGROUND OF THE INVENTION
Calcium folinate also known as Leucovorin calcium in USA, is a mixture of calcium salt of diastereoisomers 5-formyl derivative of tetrahydrofolic acid. Calcium folinate is chemically known as Calcium N-[p-[[[(6RS)-2-amino-5-formyl-5,6,7,8-tetrahydro-4-hydroxy-6-pteridinyl]methyl]amino]benzoyl]-L-glutamate (1:1) having the formula-I as mentioned below.

The given IUPAC name is not matching with the structure. Please clarify. IUPAC name cannot be “5,6,7,8-tetrahydro-4-hydroxy” – at 5th position there cannot be a hydrogen and in 4th position “OXO GROUP” is present but not “HYDROXY GROUP”.

Leucovorin Calcium has the IUPAC name as given is “calcium;(2S)-2-[[4-[(2-amino-5-formyl-4-oxo-3,6,7,8-tetrahydropteridin-6yl) methylamino]benzoyl]amino]pentanedioate
with the structural formula as;

Calcium folinate is used to diminish the toxicity methotrexate and also for the treatment of megaloblastic anemias due to folic acid deficiency.

The US Patent No. 2741608 first discloses the product calcium folinate and process of preparing the same as mentioned below.

The essential steps of the aforementioned preparation of calcium folinate are: (1) the reduction of 10-formylpteroyl glutamic acid; (2) neutralization steps after reduction; and (3) isolation of the final product. The Publication Analytical Drug Substances 1979, 8, Pages 316-350 discloses the calcium folinate containing 10 to 15% of water.

The US Patent No.4500711 discloses the use of water soluble organic bases in opening of the heterocylic imidazolinium chloride for maintain the pH that is favorable for the formation of N5-formyl derivative. This Patent discloses the purification of calcium folinate by washing the precipitated calcium folinate with different solvents such as acetone, ethyl acetate and ether.

The Patent application CN101863889 discloses the process for purification of folinic acid involving the steps of: dissolving the crude product of folinic acid in water; adjusting the pH to 6.0-7.5; precipitating the folinate by the addition of ethanol; crystallizing the product in ethanol and washing the crystalline product with a mixture of water and ethanol.

The PCT publication WO1995033749 discloses the process for purification of folinic acid involving the washing the crystalline product with ethanol.

Besides the availability of method for the purification of calcium folinate in state of the art, there is a need for an improved process for the purification of calcium folinate that is economically significant.
How the present invention is economically significate over prior art is to be stated here by way of comparison.

SUMMARY OF THE INVENTION
An aspect of the present invention is to provide an improved purification process for pharmaceutically acceptable salt of folinic acid thereof, preferably calcium and sodium comprising the steps of washing solid folinic acid salt with a mixture of cyclohexane and alcohol.

Another aspect of the present invention is to provide a preparation of pure calcium folinate comprising the steps of:
(i) dissolving or suspending a solid form of sodium folinate in water;
(ii) adding a calcium source to suspension or solution obtained in step (i);
(iii) maintaining the reaction mixture obtained in step (ii) till formation of calcium folinate; and
(iv) isolating calcium folinate from the reaction mass obtained in step (iii).

DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention provides a purification process for pharmaceutically acceptable salt of folinic acid thereof comprising the steps of washing solid folinic acid salt with a mixture of cyclohexane and alcohol.

According to the present invention, the alcohol is selected from the group comprising of C1 to C3 alcohols, preferably ethanol and methanol.

Unexpectedly the washing of pharmaceutically acceptable salt of folinic acid with a mixture of cyclohexane and alcohol decreases the amount of water in the final API. As the folinic acid and its salts are sensitive to oxygen, moisture and light, the decrease in water content of folinic acid salts decreases the degradation, thereby increases the stability of final API during storage.

The USP limits the water content of calcium folinate (calcium leucoverin) is not more than 17%. The prior art methods achieve the water content of the calcium folinate above 12%, whereas the present invention achieves the water content of the calcium folinate below 12%.

The present inventors also observed that the water content in calcium folinate obtained after washing with a mixture of cyclohexane and ethanol was about 7.77% w/w, whereas the water content in calcium folinate obtained after washing with ethanol alone was about 12.44% w/w.

Another embodiment of the present invention is to provide a preparation of pure calcium folinate comprising the steps of:
(i) dissolving or suspending a solid form of sodium folinate in water;
(ii) adding a calcium source to suspension or solution obtained in step (i);
(iii) maintaining the reaction mixture obtained in step (ii) till formation of calcium folinate; and
(iv) isolating calcium folinate from the reaction mass obtained in step (iii);
(v) purifying the calcium foliate of step (iv) by washing with cyclohexane and ethanol.

The sodium folinate employed in the present invention may be prepared in the methods known in the state of art.
The preparation of sodium folinate in the solid form for the present invention may be prepared by the crystallization or recrystallization procedures known in the state of art.

According to the present invention, the calcium source may be preferably calcium chloride.

The step (iv) isolating calcium folinate according to the present invention may be done known in the state of art, preferably by the addition of non-solvent to the concentrated mixture or by seeding crystalline form to the concentrated mixture or by cooling the concentrated mixture.

The present invention is explained in detail with reference to the following examples described below, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

EXAMPLES
Example-1: Preparation of 5,10-methenyl-5,6,7,8-tetrahydrofolic acid hydrochloride
A mixture of formic acid (1000 ml) and folic acid (200 gm) was heated to 38°C and stirred for 10 hours at the same temperature. The progress of the formation of 10-formyl folic acid in the reaction was monitored by HPLC. After completion of the reaction, the reaction mass was cooled to 30°C. To the cooled reaction mass, platinum oxide (4 gm) at 30°C and trifluoroacetic acid (200 ml) at 25°C were added in a nitrogen atmosphere; followed by passing hydrogen gas at a pressure of 1 Kg; and then stirred for 4 hours at 25°C. The progress of the reaction was monitored by HPLC. After the completion of the reaction, the hydrogen gas pressure with the nitrogen gas pressure applied during the reaction was removed and then filtered. To the filtrate, ascorbic acid (4 gm) was added at 30°C and concentrated at 50°C under vacuum. To the concentrated mass, water (400 ml) and aqueous hydrochloric acid (93.5 gm of HCl in 200 ml of water) were added and stirred for 1 hour at 50°C. The stirred reaction mass was cooled to 25°C and stirred for 4 hours at 25°C. The resultant solid was filtered under nitrogen atmosphere, washed with water (400 ml) and acetone (400 ml) and dried under nitrogen atmosphere. %Yield: 80.6%

Example-2: Preparation of Sodium folinate
To a mixture of compound 5,10-methylene tetrahydrofolate prepared in Example-1 (100 gm), ascorbic acid (10 gm) and water (500 ml); sodium hydroxide solution (24.4 gm of Sodium hydroxide in 100 ml of water) was slowly added at 30°C and then heated to 95°C. The heated reaction mixture was stirred for 10 hours at 95°C. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction mass was cooled to 25°C; the pH was adjusted 8.5 to 9.5 by using sodium hydroxide solution (10 gm of Sodium hydroxide in 100 ml of water) and then stirred for 2 hours at 25°C. The reaction mass was concentrated at 60°C under vacuum. To the concentrated mass, methanol (1000 ml) was added at 60°C; cooled to 25°C; and stirred for 3 hours at 25°C. The resultant solid was filtered under nitrogen atmosphere, washed with acetone (100 ml) and dried under nitrogen atmosphere. %Yield: 42%.

Example-3: Preparation of Calcium folinate
To a mixture of the Sodium folinate prepared in Example-2 (1 gm) and water (3 ml), calcium chloride solution (0.168 gm of Calcium chloride in 1 ml of water) was added at 30°C and stirred for 1 hour at the same temperature. To the reaction mass, activated charcoal (0.1 gm) was added and stirred for 25 minutes at 30°C. The reaction mass was filtered. To the filtrate, ethanol (10 ml) was slowly added at 30°C and stirred for 3 hours at the same temperature. The resultant wet solid was filtered under nitrogen atmosphere; washed with a mixture of ethanol (1 ml) and cyclohexane (1 ml); and dried in vacuum under nitrogen atmosphere. % Yield: 20.2%
Example-4 Purification of Calcium folinate
The crude Calcium folinate obtained in Example-3 (40 gm) was suspended in water (120 ml) at 30°C and heated to 60°C. The heated contents were then stirred for 1 hour at 60°C and filtered at 50°C. The filtrate was cooled to 25°C and stirred for 15 hours at the same temperature. The resultant solid was filtered under nitrogen atmosphere, washed with ethanol (80 ml), washed with a mixture of ethanol (40 ml) and cyclohexane (40 ml) and dried under nitrogen atmosphere. Yield: 60%; Water content: 10.64 % w/w

Example-5: Preparation of Calcium folinate
To a mixture of compound 5,10-methylene tetrahydrofolate prepared in Example-1 (150 gm), ascorbic acid (15 gm) and water (1500 ml); N-ethyldiisopropylamine (138 gm) was slowly added at 30°C and then heated to 95°C. The heated reaction mixture was stirred for 10 hours at 95°C. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction mass was cooled to 30°C. To the cooled reaction mass, ethyl acetate (750 ml) was added and stirred for 20 minutes at 30°C, then allowed to settle down to a biphasic mixture. The aqueous layer was separated, washed with ethyl acetate (750 ml) and heated to 50°C. The pH of the aqueous layer was adjusted to 8.5 to 9.5 by using 10% sodium hydroxide solution (15 gm of Sodium hydroxide in 150 ml of water) and stirred for 1 hours at 50°C. To the aqueous layer, calcium chloride solution (72 gm of calcium chloride in 150 ml of water) was added at 50°C and stirred for 1 hour at the same temperature. Ethanol (450 ml) was added to the reaction mass at 50°C and stirred for 30 minutes at the same temperature. The reaction mass was cooled to 25°C and stirred for 1 hour at the same temperature. The resultant solid was filtered. To the filtered solid, ethanol (1500 ml) was added at 25°C and stirred for 2 hours at the same temperature. The contents were further cooled to 5°C and stirred for 8 hours at the same temperature. The resultant solid was filtered under nitrogen atmosphere, washed with a mixture of cyclohexane (300 ml) and ethanol (300 ml) and dried under nitrogen atmosphere. To the wet solid, water (450 ml) was added and stirred for 30 minutes at 30°C followed by the addition of seeding material (1.5 gm). The contents were then cooled to 25°C and stirred for 8 hours at the same temperature. The resultant solid was filtered under nitrogen atmosphere, washed with ethanol (300 ml) and a mixture of cyclohexane (150 ml) and ethanol (150 ml) and dried under vacuum in a nitrogen atmosphere. %Yield: 27%.
,CLAIMS:1. A purification process for pharmaceutically acceptable salt of folinic acid preferably calcium and sodium salts comprising the step of washing solid folinic acid salt with a mixture of cyclohexane and alcohol.
2. The purification process according the claim-1, wherein the alcohol is selected from the group comprising of C1 to C3 alcohols.
3. The purification process according to claim-2, wherein the C1 to C3 alcohol is preferably ethanol and methanol.
4. The purification process according the claim-1, the pharmaceutically acceptable salt of folinic acid is calcium folinate.
5. The purification process as claimed in claim 1 wherein the washing solid folinic acid salt with a mixture of cyclohexane and alcohol is carried out wherein cyclohexane and alcohol are preferably in 1:1 ratio.
6. A process for the preparation of pure calcium folinate comprising the steps of:
(i) dissolving or suspending a solid form of sodium folinate in water;
(ii) adding a calcium source to suspension or solution obtained in step (i);
(iii) maintaining the reaction mixture obtained in step (ii) till formation of calcium folinate; and
(iv) isolating calcium folinate from the reaction mass obtained in step (iii);
(v) purifying the calcium foliate of step (iv) by washing with cyclohexane and ethanol.