The present invention relates to the resolution of tetrahydrofolic acid derivatives. More specifically, the present invention relates to a process that employs N-alkyl-D-glucamine to assist in the resolution of tetrahydrofolic acid derivatives. The invention further relates to the formation of the corresponding alkali metal or alkaline earth metals salts, such as the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid.

BACKGROUND OF THE INVENTION

Folic acid has the structural formula represented below as Formula (I):

(I)

Folic acid and its derivatives are especially important in cellular reproduction and therefore are important for fetal development, cancer treatment, and other occurrences of rapid cell division and growth. Folic acid and its derivatives are important substances in treating such diseases as cancer, opportunistic infections, autoimmune diseases, and megaloblastic anemia. These compounds are also important as assays in developing new antifolates.

Tetrahydrofolic acid has the structural formula represented below as Formula (II):

(Π) 5-methyl-(6S)-tetrahydrofolic acid is a derivative of folic acid that is used in the treatment of folic acid deficiencies. 5-methyl-(6S)-tetrahydrofolic acid is represented by the chemical structure of Formula (III):

(III)

Folinic acid, or 5-formyl-tetrahydrofolic acid, has the structural formula represented below as Formula (IV):

(IV)

Folinic acid is a formyl derivative of tetrahydrofolic acid. Folinic acid can be converted to numerous other folic acid derivatives and therefore can be used for treatment of many of the same ailments for which folic acid is prescribed. However, because folinic acid does not require dihydrofolate reductase for its conversion into its active derivatives, it is capable of functioning as a vitamin supplement in the presence of drugs that inhibit dihydrofolate reductase, such as methotrexate. This allows folinic acid to be used in co-therapy with methotrexate during cancer chemotherapy treatment. Additional therapies that employ folinic acid include co-therapy with 5-fluorouracil for treatment of colon cancer, and combination with the folic acid antagonists pyrimethamine and sulfadiazine for the treatment of toxoplasmosis retinitis. Only the levo configuration of folinic acid is active, and it is sold under the tradename FUSILEV ^® . Calcium 5-methyl-(6S)-tetrahydrofolate is represented by the chemical structure of Formula (V):

(V)

Calcium 5-methyl-(6S)-tetrahydrofolate is the only folic acid derivative which is able to directly penetrate the blood/brain barrier without further metabolism. Naturally occurring 5- methyltetrahydrofolic acid is solely in the S form; the R-form is biochemically inactive and is excreted through the kidneys.

In recent years, the pharmaceutical industry has become aggressive in investigating compounds that contain chiral centers to determine whether one of the individual enantiomers possesses greater efficacy, less side effects or both advantages. Because one isomer of a racemic mixture is often pharmacologically inactive or significantly less active, employing a racemic mixture of a chiral drug can be viewed as introducing an unnecessary impurity into a pharmaceutical product. One isomer of the racemic mixture may also produce unwanted side- effects. Isomeric molecules may also undergo different metabolic processes which further complicates pharmacokinetic issues. Accordingly, the U.S. Food and Drug Administration (FDA) often requires stringent investigation of active molecules that contain chiral centers to determine potential efficacy and safety issues.

EP 0 537 842 discloses a process for the separation of 5-methyl-(6RS)-tetrahydrofolic acid and formyl-5-methyl-(6RS)-tetrahydrofolic acid through a process that comprises precipitating a mixture of the racemic tetrahydrofolic acids and inorganic salts, preferably alkali metal or alkaline earth metal salts, and subsequent treatment of the reaction mixture with a strong acid, followed by adjusting the pH of the reaction mixture to neutrality.

U.S. Patent Nos. 5,194,611 and 5,382,581 disclose processes for the resolution of 5- methyl-(6RS)-tetrahydrofolic acid by fractional crystallization of ammonium salts of 5-methyl- (6RS)-tetrahydrofolic acid with cyclohexyl amine, diisopropyl amine, benzyl amine, ammonia, ethanolamine, triethanolamine, 2-dimethylamine, amino ethanol, tertbutyl amine, lysine or arginine. The processes disclose separation of the amine salts in the presence of a mixture of solvents containing water and at least one organic solvent. The resulting single enantiomer (6S or 6R) can then be prepared as an alkali metal or alkaline earth metal salt.

U.S. Patent No. 5,457,202 relates to a process for the resolution of 5-methyl-(6RS)- tetrahydrofolic acid. The racemic 5-methyl-(6RS)-tetrahydrofolic acid or a salt thereof is suspended in a water solution comprising N-ethyl-2-aminomethylpyrrolidine or one of its optical isomers, followed by precipitation of the resulting salt. The reaction mixture is then heated to 40-90°C, which results in dissolution of the 6S acid, which can then be separated from the salt of the 6R acid. The salt of the 6S acid is then precipitated by cooling of the solution. Suspension of the 6S acid in water and addition of a sodium hydroxide solution results in a sodium salt that can then be converted with an alkaline earth metal hydroxide or chloride into an alkaline earth metal salt.

IT 1254954 discloses a process for the preparation of the 6S diastereoisomer of folic acid, comprising reacting the racemic 5-N-formyl-(6RS)-tetrahydrofolic acid with an optically active amine, selected from S-(-)-([alpha])-methyl-p-nitrobenzylamine or (S)-(-)- [alpha] -phenyl ethylamine and subsequent crystallisation of the 6S salt. U.S. Patent No. 5,006,655 discloses a process for the preparation of 5,10-methylenyl- (6R)-, 5-formyl-(6S)-, or 5-methyl-(6S)-tetrahydrofolic acid or salts thereof by fractional crystallization of 5,10-methenyl-(6RS)-tetrahydrofolic acid or a salt thereof in a strong acid, to obtain 5,10-methenyl-(6R)-tetrahydrofolic acid, which can then be reduced to form 5-methyl- (6S)-tetrahydrofolic acid.

U.S. Patent No. 5,324,836 discloses a process for the preparation of 6S and 6R tetrahydrofolic acid by formation of salts of racemic (6RS)-tetrahydrofolic acid with a sulphonic or sulphuric acid, followed by fractional crystallization of the 6S and 6R isomers.

U.S. Patent No. 6,858,731 discloses a process for preparing and concentrating (6S, aS) or (6S, aR) tetrahydrofolic acid ester salts and (6S, aS) or (6S, aR) tetrahydrofolic acid comprising preparing and dissolving diastereomers of acid addition salts of tetrahydrofolic acid esters with aromatic sulphonic acids in organic solvents, and subsequent factional crystallization and hydrolization to yield the individual isomers.

U.S. Patent No. 5,489,684 discloses a process for the preparation of (6S)-tetrahydrofolic acid having a diastereomeric purity of at least 75% comprising the direct crystallization from an aqueous solution of a (6RS) diastereomeric mixture of an alkali metal salt of tetrahydrofolic acid with a strong acid and under acidic conditions, specifically at a pH between 4.8 and 5.3.

U.S. Patent No. 6,271,374 discloses crystalline (6S)-tetrahydrofolic acid having a purity higher than 98%, and a process for the preparation of (6S)-tetrahydrofolic acid or (6R)- tetrahydrofolic acid comprising adding the racemic mixture to a polar medium, generally a mixture of water and a water miscible organic solvent, and maintaining the pH at > 3.5 for crystallization of the (6S) compound, or maintaining the pH at > 2 for crystallization of the (6R) compound. The above processes provide poor yields and poor enantioselectivity. Additionally these processes are expensive due to the need to perform multiple purifications steps in order to achieve an enantioselectivity greater than 99% of the desired isomer. Further, the disclosed processes do not translate well to commercially and industrially effective scale up procedures.

U.S. Patent No. 5,698,693 discloses processes for separation of folic acid derivatives employing chiral chromatography or fractional crystallization carried out on a-monoesters of (6RS)-tetrahydrofolic acid derivatives, wherein the a-esters are subsequently removed from the tetrahydrofolic acid derivatives to yield the pure 6R or 6S stereoisomers.

EP 0 627 435 discloses chiral phase chromatography for the resolution of diastereoisomeric mixtures of tetrahydrofolates, yielding single enantiomers such as 5-mefhyl- (6S)-tetrahydrofolic acid or 5-formyl-(6S)-tetrahydrofolic acid.

Unfortunately, the processes disclosed in U.S. Patent No. 5,698,693 and EP 0 627 435 are restrictive and costly due to the expensive and time consuming nature of chromatographic separation, which requires highly sophisticated instrumentation, and therefore are commercially unfeasible for commercial scale up.

A need exists to develop a new process for resolving tetrahydrofolic acid derivatives in a cost efficient and easy process that may be used on a commercial scale. Additionally, the need exists for a process for the formation of alkali metal or alkaline earth metals salts of the pure chiral form of tetrahydrofolic acid derivatives, which is cost effective, uses easily available reagents, is scalable and is commercially viable. SUMMARY OF THE INVENTION

The present invention provides a process for the resolution of tetrahydrofolic acid derivatives of Formula (VI), wherein R is H, CH ₃ or CHO, and specifically, 5-methyl-(6RS)- tetrahydrofolic acid, using N-alkyl-D-glucamine.

(VI)

The present invention is a process for resolving racemic tetrahydrofolic acid derivatives into (6S)-tetrahydrofolic acid derivatives by diastereomeric crystallization of salts of (6RS)- tetrahydrofolic acid derivatives. More specifically, the present invention is a process for the formation of the 6S and 6R N-alkyl-D-glucamine salts of tetrahydrofolic acid derivatives, followed by separation of the 6S and 6R salts, and removal of the N-alkyl-D-glucamine cation to yield tetrahydrofolic acid derivatives. The preferred 6S isomer may be further transformed into an alkali earth or alkaline earth metal salt, such as the calcium salt, substantially free of the 6R isomer.

The three named R substituents correlate with (1) tetrahydrofolic acid when R=H; (2) 5- methyl tetrahydrofolic acid when R=CH ₃ , and (3) folinic acid when R=CHO.

One embodiment of the present invention employs a reaction of 5-methyl-(6RS)- tetrahydrofolic acid with N-alkyl-D-glucamine to yield the 6S and 6R N-alkyl-D-glucamine salts of 5 -methyl tetrahydrofolic acid. The 6S and 6R salts can then be separated to yield the 6S salt. The physical and chemical differences of the diastereomeric 6S and 6R compounds enables separation using less costly and less complex techniques than many of the known prior art methods. Removal of the N-alkyl-D-glucamine cation from the 5-methyl-(6S)-tetrahydrofolic acid anion yields the 5-methyl-(6S)-tetrahydrofolic acid. Alkali metal and alkaline earth metal salts can then be formed with the 5-methyl-(6S)-tetrahydrofolic acid.

An embodiment of the present invention for preparing 5-methyl-(6S)-tetrahydrofolic acid comprises the steps of:

i) reacting racemic 5-methyl-(6RS)-tetrahydrofolic acid of formula (XV):

(XV)

l N-alkyl-D-glucamine of Formula (VII):

(VII)

wherein n=l-20, preferably 2-15 and most preferably 3-12;

to form a mixture of the N-alkyl-D-glucamine salt of 5-methyl-(6S)-tetrahydrofolic acid of Formula (Villa):

and the N-alkyl-D-glucamine salt of 5-methyl-(6R)-tetrahydro folic acid of Formula

(Vlllb);

(Vlllb);

ii) separating the mixture of the N-alkyl-D-glucamine salt of 5-methyl-(6S)- tetrahydrofolic acid and the N-alkyl-D-glucamine salt of 5-methyl-(6R)-tetrahydrofolic acid, to yield the N-alkyl-D-glucamine salt of 5-methyl-(6S)-tetrahydrofolic acid of Formula (Villa); iii) removing the N-alkyl-D-glucamine cation from the 5-methyl-(6S)-tetrahydrofolic acid anion and forming a mixture of N-alkyl-D-glucamine and 5-methyl-(6S)-tetrahydrofolic acid of Formula (IX):

(IX)

and;

iv) separating the N-alkyl-D-glucamine and 5-methyl-(6S)-tetrahydrofolic acid mixture of Formula (IX), to yield 5-methyl-(6S)-tetrahydro folic acid of Formula (III):

(III).

The present invention further provides a process for the formation of alkali metal and alkaline earth metal salts of 5-methyl-(6S)-tetrahydrofolic acid as follows:

v) salification of the 5-methyl-(6S)-tetrahydrofolic acid of Formula (III) to form a crude salt of 5-methyl-(6S)-tetrahydrofolate of Formula (XIV):

(XIV);

wherein X is an alkali metal or an alkaline earth metal, and

vi) purification of the crude salt of 5-methyl-(6S)-tetrahydrofolate of Formula (XIV) to form the pure salt of 5-methyl-6S-tetrahydrofolate of Formula (XIV) of at least 90% purity. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for resolving racemic (6RS)-tetrahydrofolic acid derivatives vising N-alkyl-D-glucamine, and preferably N-octyl-D-glucamine.

The process for preparing tetrahydrofolic acid derivatives of Formula (VI)

(VI)

wherein R is H, CH ₃ , or CHO;

comprises the steps of: i) reacting the racemic tetrahydrofolic acid derivative of formula (X):

(X)

with an N-alkyl-D-glucamine of Formula (VII):

(VII)

wherein n=l-20, preferably 2-15, and most preferably 3-12;

to form a mixture of the N-alkyl-D-glucamine salt of the (6S)-tetrahydrofol derivative of Formula (XIa):

(XIa)

and the N-alkyl-D-glucamine salt of (6R)-tetrahydro folic acid derivative of Formula

(Xlb);

(Xlb);

ii) separating the mixture of the N-alkyl-D-glucamine salt of (6S)-tetrahydrofolic acid derivative and the N-alkyl-D-glucamine salt of (6R)-tetrahydrofolic acid derivative, to yield the N-alkyl-D-glucamine salt of (6S)-tetrahydrofolic acid derivative of Formula (XIa);

iii) removing the N-alkyl-D-glucamine cation from the (6S)-tetrahydrofolic acid derivative anion and forming a mixture of N-alkyl-D-glucamine and (6S)-tetrahydrofolic acid derivative of Formula (XII):

(VI). The present invention further provides a process for the formation of alkali metal and alkaline earth metal salts of tetrahydrofolic acid derivatives as follows:

v) salification of the (6S)-tetrahydrofolic acid derivative of Formula (VI) to form a crude salt of (6S)-tetrahydrofolate derivative of Formula (XIII):

(XIII);

wherein X is an alkali metal or an alkaline earth metal, and

vi) purification of the crude salt of (6S)-tetrahydrofolate derivative of Formula (XIII) to form the pure salt of (6S)-tetrahydro folate derivative of Formula (XIII).

The three named R substituents correlate with (1) tetrahydrofolic acid when R=H; (2) 5- methyl tetrahydrofolic acid when R=CH ₃ , and (3) folinic acid when R=CHO.

Methods of obtaining racemic tetrahydrofolic acid derivatives, the compound of Formula (X), are described in E. Khalifa et. al., Helv. Chim. AcTA, Vol. 63, p 2554 (1980), U.S. Patent Nos. 5,324,836 and 5,006,655 and EP 0 537 842, all of which are incorporated herein by reference. One such method is reduction and methylation of folic acid in the presence of a reducing agent, such as sodium borohydride, and a methylation agent, such as formaldehyde. Other suitable reducing agents include but are not limited to lithium aluminum hydride, hydrogen, nickel borohydride, diisobutylaluminum hydride, formic acid, hydrazine, sodium hydrosulfite, trichlorosilane, sodium hydroxymethanesulfinate, and mixtures of the foregoing. Other suitable methyktion agents include iodomethane, dimethyl sulfate, dimethyl carbonate, methyl triflate or methyl fluorosulfonate. One embodiment of the present invention involves reacting the racemic (6RS)- tetrahydrofolic acid derivative of Formula (X) with an N-alkyl-D-glucamine of Formula (VII) in an aqueous solution. The alkyl linkage in the N-alkyl-D-glucamine can be selected from the any C ₁ -C ₂ 0 alkyl. Suitable compounds include, but are not limited to, N-methyl-D-glucamine, N- ethyl-D-glucamine, N-butyl-D-glucamine, N-propyl-D-glucamine, N-pentyl-D-glucamine, N- hexal-D-glucamine, N-heptyl-D-glucamine, N-octyl-D-glucamine, N-nonyl-D-glucamine, N- decyl-D-glucamine, and N-dodecyl-D-glucamine. The preferred alkyl linkage is C ₂ -C ₁₅ , and most preferably C3-C ₁₂ . The preferred aqueous solution is distilled mineral water, however other aqueous solutions as well as combinations of aqueous solutions with organic solvents can be employed. The reaction mixture may be heated to assist in the formation of the 6S and 6R N- alkyl-D-glucamine salts of the tetrahydrofolic acid derivatives. In certain embodiments the reaction mixture of the N-alkyl-D-glucamine and the tetrahydrofolic acid derivatives is heated to about 50°C, preferably at least 60°C, and most preferably at least about 70°C. The N-alkyl-D- glucamine is present in the reaction mixture at least at one molar equivalent to the tetrahydrofolic acid derivatives, and can be present in a molar excess of 1.5 to 2.0 molar equivalents. Once the 6S and 6R N-alkyl-D-glucamine salts of the tetrahydrofolic acid derivatives have been formed, the reaction mass is cooled, filtered and washed to produce a solid. In certain embodiments the reaction mixture is cooled to about 25°C, preferably at least about 20°C. The reaction mixture is vacuum dried while maintaining the temperature for at least two (2) hours, preferably four (4) hours and most preferably six (6) hours. The reaction product is a mixture of the 6S and 6R N- alkyl-D-glucamine salts of the tetrahydrofolic acid derivatives. The mixture of 6S and 6R N-alkyl-D-glucamine salts of the tetrahydrofolic acid derivatives can then be separated by techniques known in the art, such as diastereomeric crystallization, solvent based separation, chromatographic methods, or combinations thereof.

One method of diastereomeric crystallization involves obtaining a racemic mixture of the 6S and 6R N-alkyl-D-glucamine salts of the tetrahydrofolic acid derivatives, preferably in a crystal form, and placing the racemic mixture into a solvent so that the N-alkyl-D-glucamine salt of the (6S)-tetrahydrofolic acid derivative is crystallized from the reaction mixture. The N-alkyl- D-glucamine salt of the 6S-tetrahydrofolic acid derivative is preferably crystallized at a chiral purity level of at least 80%. One embodiment of the diastereomeric crystallization of the present invention comprises placing a racemic mixture of the 6S and 6R powder into an aqueous solvent. The aqueous solvent may be any solvent, but distilled mineral water is the preferred aqueous solvent.

The reaction mixture of the aqueous solvent and racemic mixture of 6S and 6R N-alkyl- D-glucamine salts of the tetrahydrofolic acid derivatives may be heated to assist in the dissolution of the 6S and 6R salts. In certain embodiments the reaction mixture is heated to about 50°C, preferably at least 60°C, and most preferably to at least about 70°C. Once the 6S and 6R salts have been dissolved, the solution is cooled to selectively crystallize the 6S and 6R salts. In certain embodiments the reaction mixture is cooled to about 25°C, preferably at least about 20°C. The reaction mixture is vacuum dried while maintaining the temperature for at least one (1) hours, preferably one and a half (1.5) hours and most preferably two (2) hours.

In one embodiment of the present invention the N-alkyl-D-glucamine salt of (6S)- tetrahydrofolic acid derivative is isolated as a crystal from the reaction mass with a purity of at least 90% or greater, preferably at least 92% or greater, and most preferably at least 95% or greater. The purification process can be done in the presence of an aqueous solvent, wherein the aqueous solvent is present at a volume of 10 to 1 , preferably 3 to 8 and most preferably 4 to 6 based on the volume of the reaction mass. The purification process can also be conducted at 20- 40°C, and most preferably 25-30°C.

Subsequent separation reactions, via either diastereomeric crystallization, solvent based separation, or chromatographic separation, can produce the N-alkyl-D-glucamine salt of (6S)- tetrahydrofolic acid derivatives with a chiral purity (or enantiomeric excess) greater than 95%, preferably greater than 99% and most preferably greater than 99.5%.

Once the N-alkyl-D-glucamine salt of the (6S)-tetrahydrofolic acid derivative has been isolated the N-alkyl-D-glucamine cation may be removed from the (6S)-tetrahydrofolic acid derivative anion. The removal of the N-alkyl-D-glucamine cation can be accomplished by known techniques. One embodiment of the present invention comprises dissolving the N-alkyl- D-glucamine salt of the (6S)-tetrahydrofolic acid derivative in an aqueous solvent in the presence of a molar excess of an acid. The acid can be present in the reaction mixture at least at one molar equivalent to the tetrahydrofolic acid derivatives, and can be present in a molar excess of 1.5 to 2.0 molar equivalents. The acid is an inorganic or organic acid, such hydrochloric acid, sulfuric acid, hydroiodic acid, hydrobromic acid, perchloric acid, p-toluenesulfuric acid, fumaric acid, succinic acid, citric acid or malic acid. The preferred acid is an inorganic acid, such as hydrochloric acid.

The reaction solution should preferably be maintained at an acidic pH, i.e. below 7, preferably below 6, and most preferably below 5.5. If the reaction solution rises above 5.3, additional aliquots of the acid should be added to the reaction mixture to maintain the acidic pH. The reaction mixture can also be heated to assist in the completion of the reaction to about 20°C, preferably at least about 25°C, and most preferably at least about 30°C. The reaction mixture is then stirred while maintaining the temperature and pH for at least two (2) hours, preferably four (4) hours and most preferably six (6) hours.

The reaction solution is then filtered and washed to yield a mixture of (6S)- tetrahydrofolic acid derivative and N-alkyl-D-glucamine.

The (6S)-tetrahydrofolic acid derivative and N-alkyl-D-glucamine mixture is then separated by the addition of a base. The base employed can be alkyl amine, sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia, barium hydroxide, calcium hydroxide, lithium hydroxide, sodium amide, sodium hydride or other suitable bases known in the art. The preferred base is an alkyl amine, such as t-butyl amine. The base can be present in the reaction mixture at least at one molar equivalent to the tetrahydrofolic acid derivatives, and can be present in a molar excess of 1.5 to 2.0 molar equivalents. The base is preferably added to the reaction mixture of the (6S)-tetrahydrofolic acid derivative and N-alkyl-D-glucamine as a solution, and preferably as an aqueous solution. The reaction mixture may be heated to assist in the separation. In certain embodiments the reaction mixture is heated to about 20°C, preferably at least about 25°C, and most preferably at least about 30°C. The reaction mixture is stirred while maintaining the temperature for at least one (1) hour, preferably two (2) hours and most preferably three (3) hours.

Once the separation is completed, the solvents are removed and the (6S)-tetrahydrofolic acid derivative is collected. The (6S)-tetrahydrofolic acid derivative is collected by filtration, and may be washed and dried. The resulting (6S)-tetrahydrofolic acid derivative exhibits a chiral purity of at least 90%, preferably 92%, and most preferably 95%. The separation can be done in the presence of an aqueous solvent, wherein the aqueous solvent is present at a volume of 10 to 1 , preferably 3 to 8 and most preferably 4 to 6 based on the volume of the reaction mass. The purification process can also be conducted at 20-40°C, and most preferably 25-30°C.

The N-alkyl-D-glucamine can be collected and further processed, if necessary, for reuse in further separation procedures.

In a further embodiment, the (6S)-tetrahydrofolic acid derivative can then be reacted with an alkali metal or alkaline earth metal salt in an aqueous solvent to form the (6S)- tetrahydrofolate derivative alkali metal or alkaline earth metal salt. Alkali metal or alkaline earth metals that can be employed in the present invention include lithium, sodium, potassium, beryllium, magnesium, and calcium. A preferred alkaline earth metal is calcium.

One embodiment of the present invention comprises the reaction of an alkali earth metal such as calcium with the (6S)-tetrahydrofolic acid derivative to form calcium (6S)- tetrahydrofolate derivative. The (6S)-tetrahydrofolic acid derivative is reacted with a calcium source, such as calcium chloride anhydrous. Additional calcium sources that can be employed in the present invention are calcium fluoride, calcium iodide, and calcium bromide. These calcium sources are particularly useful in the present invention because they readily dissolve in aqueous solutions to form calcium cations, in contrast to many calcium compounds. The reaction preferably is performed in an aqueous solvent in the presence of a molar excess of a strong acid. The acid can be present in the reaction mixture at least at one molar equivalent to the tetrahydrofolic acid derivatives, and can be present in a molar excess of 1.5 to 2.0 molar equivalents. The strong acid can be selected from the group consisting of hydrochloric acid, sulfuric acid, hydroiodic acid, hydrobromic acid, perchloric acid, and p-toluenesulfuric acid. The reaction solution should preferably be maintained at an acidic pH, i.e. below 7, preferably below 6, and most preferably below 5.5. If the reaction solution rises above 5.5, additional aliquots of the strong acid should be added to the reaction mixture to maintain the acidic pH.

The calcium source is added to the acidic solution of the (6S)-tetrahydrofolic acid derivative, and the reaction solution is then adjusted to neutrality with the addition of a base. The base employed can be an alkyl amine, such as tert-butyl amine, sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia, barium hydroxide, calcium hydroxide, lithium hydroxide, sodium amide, sodium hydride or other suitable bases known in the art. The preferred base is an alkyl amine, such as tert-butyl amine. The base is preferably added as a solution to the mixture of the (6S)-tetrahydrofolic acid derivative and calcium, and preferably as an aqueous solution. The reaction mixture is then cooled to assist in crystallization. In certain embodiments the reaction mixture is cooled to about 10°C, preferably at least about 5°C, and most preferably at least about 0-5 °C. The reaction mixture is then stirred while maintaining the lowered temperature for at least one (1) hour, preferably two (2) hours and most preferably three (3) hours.

Once the salt formation is completed, the solvents are removed and the salt of the (6S)- tetrahydrofolate derivative is collected. The salt of the (6S)-tetrahydrofolate derivative is collected by filtration, and may be washed and dried. The purification process can be done in the presence of an aqueous solvent, wherein the aqueous solvent is present at a volume of 10 to 1 , preferably 3 to 8 and most preferably 4 to 6 based on the volume of the reaction mass. The purification process can also be conducted at 20-40°C, and most preferably 25-30°C. The resulting salt of the (6S)-tetrahydrofolate derivative exhibits a chiral purity of at least 90%, preferably 92%, and most preferably 95%. Additional purification steps can take place in aqueous solutions to achieve higher levels of purity of the salt of the (6S)-tetrahydrofolate derivative.

The chiral purity of the compounds described herein can be determined by any means known in the art. The chiral purity reported in the present application was determined by high performance liquid chromatography (HPLC).

The salt of the (6S)-tetrahydrofolate derivative, and other compounds prepared in accordance with the present invention may be mixed with at least one additional conventional pharmaceutical excipient to prepare a pharmaceutical dosage forms such as a tablets, capsules or solutions.

EXAMPLE

All of the below reactions were conducted in round bottom flasks equipped with a thermometer pocket, a nitrogen supply and a water condenser.

Step 1- Preparation of calcium 5-methyl-(6RS)-tetrahydrofoIate

A 20% solution of sodium hydroxide was prepared by dissolving 72.0 g of sodium hydroxide (NaOH) in 360 ml of water. The solution was then added to a 5.0 L round bottom flask and the temperate was maintained at 25-30°C. 280 g of sodium borohydride (NaBH ₄ ) was added into the reaction mass and the temperature was maintained at 25-30°C. 10 g of EDTA disodium salt was added into the reaction mass and stirred while the temperature was maintained at 25-30°C. The reaction mass was then heated to 60°C.

Folic acid was added to the reaction mass in three separate aliquots or lots.

1 ^st lot of folic acid - A suspension of 200 g of folic acid in 600 ml of water (1 ^st Lot), was added dropwise to the reaction mass. 2" lot of folic acid - The reaction mass was continuously stirred for 15 minutes and the temperature was maintained at 60°C. A second suspension of folic acid comprising 80 g of folic acid in 240 ml of water was prepared (2 ^nd Lot). The following additions were then made:

(a) 25% of the 2 ^nd lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 68°C, and the reaction mass was then cooled to 60°C.

(b) A second 25% of the 2 ^nd lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 80°C, and the reaction mass was then cooled to 60°C.

(c) A third 25% of the 2 ^nd lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 65°C, and the reaction mass was then cooled to 60°C.

(d) A fourth 25% of the 2 ^nd lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 62°C. The solution was stirred for 30 minutes, and then cooled to 60°C.

3 ^rd lot of folic acid - A 3 ^rd lot of folic acid was prepared by dissolving 120 g of folic acid in 360 ml of water (3 ^rd lot). The reaction mass was maintained at 60°C and the following additions were made:

(a) 50% of the 3 ^rd lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 73 °C, and the reaction mass was then cooled to 60°C.

(b) The second 50% of the 3 ^,u lot of folic acid was added dropwise to the reaction mass. The reaction mass increased in temperature to approximately 62°C. The reaction mass was stirred for 15 minutes, heated to 88-92°C, and stirred for an additional 20 minutes.

A clear solution with strong effervescence was formed. The reaction mass was then gradually cooled to 25-30°C (allowing up to 90 minutes for the reaction mass to reach 30°C). The reaction mass was analyzed using a high performance liquid chromatography (HPLC). Not more than (NMT) 15% of folic acid remained.

The reaction mass was cooled to 0°C (allowing up to 25 minutes for the reaction mass to cool to 0°C). The reaction mass exhibited a pH of approximately 12.9. The pH of the reaction mass was then adjusted to 9.0 using 317 ml of concentrated hydrochloric acid, while maintaining the reaction solution below 10°C (allowing up to 45 minutes for the pH adjustment).

200 ml of formaldehyde was added into the reaction mass while the reaction mass was maintained below 10°C (allowing up to 35 minutes for the addition of formaldehyde) until a yellow suspension was formed. The reaction mass was then stirred for 15 minutes while maintaining the temperature at 0-5°C.

A 0.2N NaOH solution was prepared by dissolving 1.6 g of NaOH in 200 ml water. A suspension was prepared by adding 140 g of NaBH ₄ into the 0.2N NaOH solution. The suspension was then added into the reaction mass while maintaining the reaction mass below 20°C.

The reaction mass was slowly heated to 60°C, allowing up to 1 hour for the reaction mass to reach 60°C. The reaction mass was then stirred for 15 minutes. The reaction mass was gradually cooled to 25-30°C, followed by additional cooling of the reaction mass to 0°C, allowing for up to 1 hour and 10 minutes for the temperature to drop to 0°C. The pH of the reaction mass was approximately 12.3. The pH of the reaction mass was then adjusted to 8.5 using 181 ml of concentrated HCl while maintaining the temperature of the reaction mass below 10°C (allowing up to 25 minutes for the pH adjustment). A yellow thick suspension was formed.

1600 ml of water was then added into the reaction mass and stirred for 30 minutes, while maintaining the reaction mass at 0-5 °C. The reaction mass was filtered and washed with two units of 200 ml of water.

The reaction mass was then filtered in a 5.0 L round bottom flask. The reaction mass was cooled to a temperature of approximately 0-5°C, and maintained at a pH of 8.6. A 4M calcium chloride (CaCl ₂ ) solution was prepared by dissolving 1 1 1 g of CaCl ₂ in 250 ml of water. The 4M CaCl ₂ solution was slowly added into the reaction mass over 30 minutes while maintaining the temperature at 0-5°C. The reaction mass exhibited a pH of approximately 8.2, and a solid was obtained in approximately 15 minutes. The reaction mass was then filtered and washed with two units of 200 ml of water, followed by vacuum filtration for approximately 30 minutes. The wet weight of the solid obtained (calcium 5 -methyl-(6RS)-tetrahydro folate) was approximately 1500.0 g. The solid was dried in a vacuum oven at 40°C, for 12 hours and then in fluid bed dryer at 40°C until it possessed a moisture content of NMT 35%.

Step II- Preparation of 5-methyl-(6RS)-tetrahydrofolic acid

1545 ml of water and 515 g of calcium 5-methyl-(6RS)-tetrahydrofolate (Step I product) were added to a 2.0 L round bottom flask and the temperature was maintained at 25-30°C. A thick off- white suspension with a pH of approximately 8.1 was formed. 150 ml of concentrated HCl was added into 300 ml of water forming a dilute solution of hydrochloric acid. The pH of the reaction mass was then adjusted to 3.2-3.4 using the dilute solution of hydrochloric acid, while maintaining the reaction mass at 25-30°C, until a light pick suspension was formed. The reaction mass was cooled to 5-10°C, followed by stirring for 1 hour. The reaction mass was filtered and washed with 2 units of 257.5 ml of water and a solid was obtained. The wet weight of the solid obtained (5-methyl-(6RS)-tetrahydrofolic acid) was approximately 945 g.

The solid was dried in a vacuum oven at 40°C, for 12 hours and then in fluid bed dryer at

40°C until it possessed a moisture content of NMT 15%.

Step 111(a)- Preparation of N-octyl-D-glucamine salt of 5-methyl-(6RS)-tetrahydrofolic acid

370 g of 5-methyl-(6RS)-tetrahydrofolic acid (the Step II product) and 1600 ml of water were added to a 3.0 L round bottom flask while maintaining the reaction solution at 25-30°C. 451.8 g of N-octyl-D-glucamine was added to the reaction mass, while maintaining the reaction mass at 25-30°C. A thick suspension was formed. The reaction mass was heated to 60-70°C forming a clear solution. After the clear solution was formed the reaction mass was gradually cooled to 25-30°C. A solid precipitated out of the reaction mass at approximately 50-58°C.

The reaction mass was then cooled to 20°C, followed by a very slow filtration. The solid obtained was washed with two units of 160 ml of water, followed by vacuum filtration for 5-6 hours. The wet weight of the solid obtained (crude N-octyl-D-glucamine salt of 5-mefhyl-(6RS)- tetrahydrofolic acid) was approximately 880 g.

The solid was dried in a vacuum oven at 40°C until it possessed a moisture content of NMT 20%. Step III(b)-Purifkation of crude N-octyl-D-glucamine salt of 5-methyl-(6S)-tetrahydrofolic acid

308 g of the crude N-octyl-D-glucamine salt of 5-methyl-(6RS)-tetrahydrofolic acid (Step 111(a) product) was added to 1356 ml of water in a 3.0 L round bottom flask and the temperature was maintained at 25-30°C.

The reaction mass was heated to 60-70°C and a clear solution formed. A solid precipitated at 55-65°C. The reaction was then gradually cooled to 25-30°C. The reaction mass was then cooled to 20°C, followed by filtration of the reaction mass. The solid obtained was washed with two units of 154 ml of water. The solid obtained (pure N-octyl-D-glucamine salt of 5-methyl-(6S)-tetrahydrofolic acid) was slowly vacuum dried for 2 hours. The wet weight of the solid was approximately 400 g.

The solid was dried in a vacuum oven at 40°C until it possessed a moisture content of NMT 15%. Step IV- 5-methyl-(6S)-tetrahydrofolic acid and N-octyl-D-glucamine mixture

204 g of pure N-octyl-D-glucamine salt of 5-methyl-(6S)-tetrahydrofolic acid (Step 111(b) product) and 612 ml of water was added to a 2.0 L round bottom flask and the temperature was maintained at 25-30°C. The reaction mass formed a yellow suspension with a pH of 7.6. 30 ml of concentrated HC1 was added to 90 ml of water to form an HC1 solution. The pH of the reaction mass was adjusted to 5.0-5.3 using 90 ml of the HC1 solution. The reaction mass was maintained at 25-30°C and stirred for 6 hr. If during stirring of the reaction suspension the pH increased beyond 5.0-5.3, the pH of the reaction mass was re-adjusted using the HC1 solution. The reaction mass was filtered under vacuum and the solid obtained (5-methyl-(6S)- tetrahydrofolic acid and N-octyl-D-glucamine mixture) was washed with two units of 102 ml of water. The wet weight of the solid obtained was approximately 570 mg.

The solid was dried in a vacuum oven at 40°C until it possessed a moisture content of NMT 15%.

Step V- Preparation of crude calcium 5-methyl-(6S)-tetrahydrofoIate

166 g of 5-methyl-(6S)-tetrahydrofolic acid and N-octyl-D-glucamine mixture (Step IV product) was added to 498 ml of water in a 1.0 L round bottom flask. The reaction mass was stirred and exhibited a pH of approximately 5.2. The pH of the reaction mass was adjusted to 9.0-9.5 using 31 ml solution of tert-butyl amine. The reaction mass was stirred for 3 hours and maintained at 25-30°C until an off white thick suspension was formed. The pH of the reaction mass was maintained at 9.0-9.5 for 3 hours. The reaction mass was filtered under vacuum and the solid obtained was washed with one unit of 83 ml of water. The undissolved solid precipitate was removed from the reaction mass.

The reaction mass, which was a clear yellow solution with a pH of 8.9, was added into a 1000 ml round bottom flask and the pH of the reaction mass was adjusted to 5.5 using 14 ml of a dilute solution of concentrated HC1 (prepared from 5 ml of concentrated HC1 in 15 ml of water). 35.07 g of calcium chloride anhydrous was added to 80 ml of water and the solution was added to the reaction mass until a clear solution was formed. The pH of the reaction mass was adjusted to 7.0 using 5.0 ml of t-butyl amine. A clear yellow solution was formed. The reaction mass was stirred and cooled to 0-5°C. A solid was obtained at 6°C. The reaction mass was then stirred for 2-3 hours, followed by filtration of the reaction mass under vacuum, and the solid obtained was washed with two units of 83 ml of chilled water. The solid (crude calcium 5- methyl-(6S)-tetrahydrofolate) was vacuum dried for 3-4 hours. The wet weight of the solid obtained was lOOg.

The solid was dried in vacuum oven at 40°C until it possessed a moisture content of

NMT 25%.

Step VI- Preparation of pure calcium 5-methyl-(6S)-tetrahydrofolate

68 g of crude calcium 5-methyl-(6S)-tetrahydrofolate (Step V product) and 269 ml of water was added to a 1.0 L round bottom flask and the reaction mass was maintained at 25-30°C.

The reaction mass was heated to 60-65 °C and a clear solution was formed. The reaction mass was stirred for 5 minutes, followed by cooling of the reaction mass to 25-30°C. A solid was obtained at approximately 58-64°C. The reaction mass was cooled to 5-10°C and stirred for 30 minutes. The reaction mass was filtered under vacuum and the solid obtained was washed with two units of 34 ml of chilled water, followed by vacuum filtration for 30 minutes. The solid had a wet weight of approximately 102 g.

102 g of the solid obtained above and 298 ml of water was added to a 1.0 L round bottom flask and the reaction mass was maintained at 25-30°C.

The reaction mass was heated to 68-72°C and stirred for 15 minutes. The reaction mass was cooled to 25-30°C, followed by additional cooling of the reaction mass to 5-10°C, and stirred for 30 minutes. The reaction mass was filtered under vacuum and the solid obtained was washed with two units of 17 ml of chilled water. The solid (pure calcium 5-methyl-(6S)- tetrahydrofolate) was vacuum dried for 30 minutes. A solid with a wet weight of approximately 68 g was formed. The solid was dried under vacuum at 40°C until the loss on drying (LOD) was NMT 16.5%. The dry weight of the product was approximately 28 g.

The compound was checked using a UV assay to ensure that not less than (NLT) 83.5% of the product was the pure calcium 5-methyl-(6S)-tetrahydrofolate product. If the product was present at less than 83.5% at this stage an additional purification with water was employed. The purification process can be done in the presence of an aqueous solvent, such as water, wherein the water is present at a volume of 10 to 1 , preferably 3 to 8 and most preferably 4 to 6 based on the volume of the reaction mass. The purification process can also be conducted at 20-40°C, and most preferably 25-30°C.

28 g of the pure calcium 5-methyl-(6S)-tetrahydrofolate product was added to 280 ml of water in a 1.0 L round bottom flask, and the reaction mass was maintained at 25-30°C. The reaction mass was heated to 68-72°C and stirred for 15 minutes. The reaction mass was cooled to 25-30°C, followed by additional cooling of the reaction mass to 5-10°C, and stirred for 30 minutes. The reaction mass was filtered under vacuum and the solid obtained was washed with two units of 14 ml of chilled water, followed by additional vacuum filtration for 30 minutes. The product obtained had a wet weight of approximately 58 g. The solid was dried under vacuum at 40°C until the LOD was NMT 16.5%. The dry weight of the final product, namely, pure calcium 5-methyl-(6S)-tetrahydrofolate, was approximately 23 g.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.