JPWO2020089443A5 - folate salts - Google Patents

Description

Furthermore, folate is known to be essential for the generation and maintenance of new cells. This is especially important during periods of rapid cell division and proliferation, such as infancy and pregnancy. Folate is required for DNA replication. Thus, folate deficiency interferes with DNA synthesis and cell division and has the greatest clinical impact on the bone marrow, a site of rapid cell turnover. Since RNA and protein synthesis are unimpeded, large red blood cells, megaloblasts, are produced, resulting in celiac disease and nutritionally induced anemia, i.e., during pregnancy, infancy or childhood. macrocytic anemia, such as megaloblastic anemia, occurs in early stages of life. Therefore, adults, especially the elderly and children, require folate to make normal red blood cells and prevent anemia. Folate also helps block changes in DNA that may cause cancer.

Derivatives of folate , such as various tetrahydrofolate derivatives, can also be used as a basis for making drugs or other derivatives. Moreover, tetrahydrofolic acid and its derivatives are known to have extreme instability, especially due to their susceptibility to oxidation. In particular, 5-formyltetrahydrofolic acid (folinic acid, leucovorin) and its physiologically active 5-formyl-(6S) form are primarily used as oncological drug moieties in combination therapy with methotrexate and 5-fluorouracil. It is also important for the treatment of folate deficiency anemia associated with pregnancy, antibiotic therapy, and the like. Among folates and reduced folates , the calcium salt can be said to be the most relatively stable derivative. Thus, US 5,817,659 and US 6,441,168 describe 5-methyl-(6R,S)-, (6S)- or (6R)-tetrahydros having at least one equivalent of water of crystallization per equivalent of said acid. Disclosed are crystalline salts of folic acid, preferably calcium salts . 5-Methyltetrahydrofolate is the only commercially available folic acid derivative that can directly penetrate the blood-brain barrier without further metabolism. Naturally occurring 5-methyltetrahydrofolic acid is exclusively in the 6S form. The 6R form is considered biochemically inactive and is excreted through the kidneys. In addition, some compositions for humans and animals containing various forms of folate and/or reduced folate , in various forms, also contain vitamins, arginine, lysine, thiamine and/or other active ingredients. Together, as a dietary supplement or for the treatment and prevention of various diseases such as neurological, pathophysiological, cardiovascular, arthritic and inflammatory conditions.

Various folate salts are known. Generally, these salts contain folate and inorganic cations such as calcium and magnesium. These alkaline earth metal cations are inert insofar as they themselves have no pharmacological effect on humans. The poor solubility of such salts in aqueous solutions is widely documented. Aqueous compositions with improved folate solubility and stability are disclosed, for example, in US 9,301,922 and US 9,642,853.

US 5,382,581 discloses diastereomeric separation of 5-methyl-tetrahydrofolic acid using ammonium salts. WO2018/178142 describes binary salts of 5-methyl-tetrahydrofolic acid containing sodium and an organic base. US2016/ 0207925 discloses salts of L-methyl-folate containing amino acids such as L-arginine and L-asparagine. These salts are lyophilized and appear amorphous. US 5,710,271 describes a process for the production, separation and purification of (6S) and (6R) diastereomers of folinic acid . Further, WO2009/103334 discloses freeze-dried or spray-dried glucosamine and galactosamine salts of L-methyl-tetrahydrofolate. WO2009/103333 describes (6R) and (6S) synthesis of 5-methyl-tetrahydrofolic acid using organic bases in the form of phenylethyl-amine compounds or naphthylethyl-amine compounds to obtain pure and stable diastereomers. ) describes the process of separating diastereomers. CN107304212 discloses a process for producing amorphous L-methyl-tetrahydrofolic acid amino acid salt. WO93/17022 discloses a process for separation of stereoisomers of folinic acid. This separation is accomplished by salifying the (R/S) folinic acid with a diamine or polyamine and then selectively crystallizing the (6S) diastereomer of the folinic acid.

In addition, a large number of folate compositions containing folate and compounds such as vitamins, lysine, thiamine, and other active ingredients have been described. However, stable salts of folate with good solubility in both water and organic solvents would find wider use in pharmaceutical compositions.

The object of the present invention is folate salts that are chemically bound to additional active compounds and exhibit good stability and good solubility in both water and non-polar solvents. to provide.

This object is achieved by a folate salt according to the invention as claimed in claim 1. Further preferred embodiments are according to the dependent claims.

Solubility in a particular organic or non-polar solvent, if the solubility of a particular folate salt in that solvent is greater than 2 percent by weight (w/w) of the total weight of the solution is considered high. Solubility is measured at 20°C. Nonpolar solvents or mixtures thereof are, for example, glycerol, methanol, ethanol, 1-propanol, 2-propanol and dimethylsulfoxide (DMSO). A non-polar solvent is understood to be a dry solvent that does not contain any water. The non-polar solvent can be a pure solvent comprising a single non-polar solvent or a mixture of at least two of said non-polar solvents. Polarity is compared to that of water.

High crystallinity means that the crystalline content of the folate salt is greater than 40% of the total amount of folate salt. A crystalline folate salt is thus understood to be a folate salt containing more than 40% crystalline. Crystallinity is determined by X-ray diffraction analysis (XRD).

A folate salt of the present invention consists of a tetrahydrofolate anion and an organic cation. This anion is folate, preferably 5-methyl-(6S)-tetrahydrofolic acid . Further, the cation is an organic compound, the organic compound being an alkanolamine selected from the group consisting of choline, N-methylaminoethanol, 2-amino-2-methylpropanol or 2-dimethyl-aminoethanol. .

A crystalline folate salt as claimed in claim 1, wherein the folate salts have a high solubility in organic solvents. High solubility in organic solvents is understood as a solubility of more than 2 percent by weight of the total weight of the solution.

In a further embodiment, the anion of the folate salt may also be 5-formyl-(6S)-tetrahydrofolic acid . Possible organic cations are selected from the same group, namely the group consisting of choline, N-methyl-aminoethanol, 2-dimethyl-aminoethanol and 2-amino-2-methyl-propanol.

The crystalline folate salts according to the invention show high stability and also high solubility in water and non-polar solvents.

A further counterion of the folate salt anion, 5-formyl-(6S)-tetrahydrofolic acid or 5-methyl-(6S)-tetrahydrofolic acid, can be the organic cation arginine .

In a preferred embodiment, the crystalline folate salt consists of a tetrahydrofolate anion and an organic cation. This anion is 5-methyl-(6S)-tetrahydrofolic acid. This cation is di-choline.

In another embodiment, the crystalline folate salt consists of a tetrahydrofolate anion and an organic cation, wherein the anion is 5-methyl-(6S)-tetrahydrofolate and the cation is mono-2-dimethylaminoethanol. be.

In another embodiment, the crystalline folate salt consists of a tetrahydrofolate anion and an organic cation, wherein the anion is 5-methyl-(6S)-tetrahydrofolate and the cation is N-methylaminoethanol.

In yet another embodiment, the crystalline folate salt consists of a tetrahydrofolic acid anion and an organic cation, wherein the anion is 5-methyl-(6S)-tetrahydrofolic acid and the cation is 2-amino-2-methyl is propanol.

In yet another embodiment, the crystalline folate salt comprises a tetrahydrofolate anion and an organic cation, wherein the tetrahydrofolate anion is 5-methyl-(6S)-tetrahydrofolic acid and the organic cation is di-choline. , where the 1H-NMR shifts in heavy water are shown in Table 1.

In yet another embodiment, the crystalline tetrahydrofolic acid salt consists of a tetrahydrofolic acid anion and an organic cation, wherein the tetrahydrofolic acid anion is 5-methyl-(6S)-tetrahydrofolic acid; The organic cation is di-2-dimethylaminoethanol, where the 1H-NMR shifts in heavy water are shown in Table 2.

In yet another embodiment, the crystalline tetrahydrofolic acid salt consists of a tetrahydrofolic acid anion and an organic cation, wherein the tetrahydrofolic acid anion is 5-methyl-(6S)-tetrahydrofolic acid; The organic cation is mono-2-dimethylaminoethanol, where the 1H-NMR shifts in heavy water are shown in Table 3.

In yet another embodiment, the crystalline tetrahydrofolic acid salt consists of a tetrahydrofolic acid anion and an organic cation, wherein the tetrahydrofolic acid anion is 5-methyl-(6S)-tetrahydrofolic acid; The organic cation is di N-methylaminoethanol, where the 1H-NMR shifts in heavy water are shown in Table 4.

In yet another embodiment, the crystalline tetrahydrofolic acid salt consists of a tetrahydrofolic acid anion and an organic cation, wherein the tetrahydrofolic acid anion is 5-methyl-(6S)-tetrahydrofolate; The organic cation is di 2-amino-2-methylpropanol, where the 1H-NMR shifts in heavy water are shown in Table 5.

In a further preferred embodiment, the pharmaceutical composition comprises at least one folate salt according to the invention as main active compound. The composition further contains at least a pharmaceutically acceptable excipient. The composition can include, for example, a buffering compound. Suitable and preferred buffer compounds are trometamol and HEPES. Additionally, an antioxidant compound can be present in the composition. Preferred antioxidant compounds are thioglycerol, dithiothreitol (DTT) and cysteine.

Furthermore, at least one folate salt according to the present invention as described above is used as a medicament, food additive or nutritional supplement for the prophylaxis and/or treatment of a deficiency or disorder that is beneficially affected by the administration of a folate salt. Used to produce food supplements. There are many conditions that are positively affected by compositions containing folate salts . Such diseases are, for example, pathophysiological, neurological and inflammatory diseases.

Further, the method of the present invention for producing a crystalline folate salt and a tetrahydrofolic acid salt consisting of a folate acid anion and an organic cation comprises oxalic acid and hydrofluoric acid instead. adding the compound salt, or directly from the free compound thereof, to an aqueous composition of folate earth alkaline metal salts or free folate acid .

Surprisingly, crystallization can be performed in organic solvents containing limited amounts of water, such as 1-propanol, for example containing less than 12% by weight of water, preferably containing less than 10% by weight of water. achieved from The weight percent of water is based on the total weight of the organic solvent and water. The minimum amount of water is the equivalent of 0.2% by weight of water in the organic solvent. This corresponds to a water of crystallization equivalent of 1 to 170 based on the molar amount of folate . 1.5 to 2 waters of crystallization have been detected in this isolated folate. This organic solvent or non-polar solvent is understood to be dry, ie anhydrous. Without adding water to the organic solvent in the various amounts mentioned above, all folate salts precipitate in amorphous form.

The crystalline folate salts according to the invention are further illustrated in the figures.

Under argon, 7.00 g (15.23 mmol) of levomefolic acid were suspended in 70 ml of 1-propanol and refluxed. Then 7.71 ml (31.99 mmol) of 47% aqueous solution of choline hydroxide was added and stirred under reflux until a clear solution was obtained. An additional 80 ml of 1-propanol was then added to give a water concentration of 3.2% w/w in the 1-propanol, seeding the solution with a crystalline sample of the product. After crystallization started at 65°C, the suspension was slowly cooled down from 65°C to 2-8°C. The suspension was gently agitated for another 2 hours at 2-8°C. The granular crystals were sucked off, washed with cold 1-propanol and dried to give 7.29 g of white crystalline crude title compound. Recrystallization under argon of 7.29 g of the crude title compound is carried out by dissolving in about 130 ml of methanol at 60°C and evaporating in vacuo at 60°C. The residue was taken up in about 60 ml of 1-propanol, dissolved at 60° C. and concentrated to a final mass of 40 g of solution. In a 70° C. oil bath, the solution is diluted with a mixture of 1-propanol and water (20:2% v/v) (about 4.0% water in 1-propanol) under argon at 70° C. On the other hand, crystallization started from the pale yellow solution after seed formation. The temperature of the suspension was lowered stepwise to 20°C and crystallization was completed by slow stirring at 2-8°C. The separated crystals were dried at 60° C./<1 mbar, yielding 5.76 g of MTHF di-choline. The analytical data are as follows.

The solubility of crystalline dicolin in organic solvents is highly variable and appears to depend on the degree of crystallinity. If the dicholine folate salt is highly crystalline, in other words if the crystalline content is high, the solubility of the dicholine folate salt in ethanol is low and the total weight of the solution is 0.3% by weight of With a high amorphous content, the solubility of the salt of dicholine folate in ethanol is very high (38.8% by weight). Furthermore, in mixtures of organic solvents, for example, in methanol at 5% by weight of the total weight of the solution or in mixtures of glycerol and ethanol, the solubility of highly crystalline dicholine folate salts is enhanced, up to 25% by weight. (of total solution weight). High crystallinity means that the crystalline content of the folate salt is greater than 40% of the total weight of the folate salt. High solubility in the solvent also means that the salt of folate can be dissolved in greater than 2% by weight of the total solution weight.

13.72 g (28.9 mmol) of levomefolic acid were suspended in 70 ml of methanol at 20-22° C. under argon. Then 160 ml (57.6 mmol, 0.360 mol/l in 1-propanol) of choline hydroxide solution was added at 20-22° C. for about 3 minutes and rinsed with 4 ml of 1-propanol to give a clear solution. Stirred at 20-22°C until An additional 13.5 ml of 1-propanol was then added and the reaction mixture was evaporated at 50° C./50 mbar and then co-evaporated twice with 11 ml of 1-propanol to give 43.6 g of A residue was formed. This residue was diluted with 140 ml of 1-propanol and heated to an external temperature of 65° C. under argon. Seed crystals were added and 5.25 ml of water was slowly added at 65° C. until crystallization started but turbidity persisted (approximately 3.7% w/w water). The mixture is stirred and the temperature is lowered to 50° C. for 20 minutes, then allowed to cool to 30° C., and the thick suspension thus formed is cooled to 2-8° C. for 70 minutes. was stirred with The crystals were separated, washed with a mixture of 4 ml of 1-propanol/water 31:1 (v/v) and 24 ml of 1-propanol, dried at 45-60° C./50-5 mbar and weighed 16.1 g of L -MTHF di-choline was produced. The analytical data are as follows.

2.00 g (4.353 mmol) of Levomefolic acid was placed under argon in 20 ml of water with a small amount of cysteine and heated to 70°C. This suspension was then treated with 876 μl (8.706 mmol, 2 eq) of 2-dimethylaminoethanol and stirred at 70° C. until a clear solution was obtained. Isopropanol was then added slowly at 70° C., a total of 150 ml. When turbidity was reached, an additional 70 ml of 2-propanol was added and the mixture was treated with seed crystals at about 60° C. to initiate crystallization (about 10.4% in 2-propanol). Water of). The mixture was then slowly and gradually cooled down from 60°C to 2-8°C. These crystals were aspirated, washed with chilled 2-propanol and dried under vacuum at 50° C. to give 1.96 g of the title compound as white crystals. The analytical data are as follows.

2.00 g (4.353 mmol) of Levomefolic acid was placed in 20 ml of 1-propanol under argon and heated to reflux while adding 800 μl of water. This suspension was then treated with 876 μl (8.706 mmol, 2 eq) of 2-dimethylaminoethanol, diluted with 3.2 ml of water and stirred at 100° C. until a clear solution was obtained. rice field. 1-Propanol (7 ml) was then added and the solvent was evaporated at 40° C. in vacuo. The residue (3.68 g) is dissolved in 30 ml 2-propanol/10 ml water and concentrated by distilling off the solvent in vacuo at 40° C. until crystallization begins. These crystals were sonicated in 1-propanol for 5 min at 20-25° C., suction filtered, washed 3 times with 1-propanol, dried at 60° C./<1 mbar and weighed 2.34 g. The title compound was obtained as white crystals. The analytical data are as follows.

3.00 g (5.30 mmol) of Levomefolinate Calcium were placed in 45 ml of water with a small amount of cysteine under argon and heated to 70°C. The resulting suspension was then treated with 848 μl (10.60 mmol) of N-methylaminoethanol and 668 mg (5.30 mmol) of oxalic acid dissolved in 5 ml of water. The thin suspension was stirred for an additional 5 minutes at 70° C., then cooled to 0° C. and stirred for 50 minutes. The reaction mixture was filtered and the clear solution was stabilized with a small amount of cysteine and concentrated at 45° C./<200 mbar. Seed crystals were added to the residue and the product slowly started to crystallize, and the crystallization was completed at 2-8°C. These crystals were dried at 50° C./<1 mbar to give 3.08 g of crude product. This crude product is dissolved in 25 ml of methanol, 1.21 ml (3 equivalents) of N-methylaminoethanol and 5 ml of 1-propanol. The cloudy liquid was filtered, washed with 10 ml of methanol and the clear filtrate was heated to 60°C. To this solution was then added 65 ml of 1-propanol, 150 μl of water and 1 equivalent of N-methylaminoethanol. The mixture was treated with seed crystals at 60°C to initiate crystallization, then slowly and gradually cooled down from 60°C to 0°C. These crystals were aspirated and washed with 1-propanol/methanol 2:1, then 1-propanol and ether. These separated crystals were dried at 50° C./<1 mbar to yield 2.60 g of the title compound as off-white crystals. The analytical data are as follows.

3.00 g (5.30 mmol) of Levomefolinate Calcium were placed in 45 ml of water with a small amount of cysteine under argon and heated to 70°C. This suspension was then treated with 1012 μl (10.60 mmol) of 2-amino-2-methylpropanol and 668 mg (5.30 mmol) of oxalic acid dissolved in 5 ml of water. The thin suspension was stirred at 70°C for 5 minutes and then cooled to 0°C. The dilute suspension was filtered and the solution was stabilized with cysteine and concentrated at 45° C./<200 mbar. The residue was treated with seed crystals and the crystallization was completed overnight at 2-8°C. These crystals were dried at 50° C./1 mbar. This crude product was dissolved in 6.5 ml of water and heated to 70°C. The solution was diluted with 70 ml of 1-propanol (10.4% water in 1-propanol), seeded and slowly cooled to 20-23°C. These crystals were separated, washed with 1-propanol/water 20:1 (v/v), 1-propanol and ether, dried at 50° C./<1 mbar, 2.79 g of off-white crystals of the title yielded the compound of The analytical data are as follows.

This solution was used as a stock form of choline which was used to produce high purity choline salts of folates .

Claims (12)

A crystalline folate salt comprising a tetrahydrofolate anion and an organic cation, wherein said anion is 5-methyl-(6S)-tetrahydrofolic acid, said cation is an organic compound, said organic compound is choline, 2- A salt of crystalline folate characterized by being an alkanolamine selected from the group consisting of dimethylaminoethanol, N-methylaminoethanol and 2-amino-2-methylpropanol. 2. The crystalline folate salt of claim 1, wherein said folate salt has a high solubility in organic solvents. 3. The crystalline folate salt according to claim 1 or 2, wherein the organic compound is di-choline. 3. The crystalline folate salt according to claim 1 or 2, wherein said organic compound is mono-2-dimethylaminoethanol. 3. The crystalline folate salt according to claim 1 or 2, wherein said organic compound is di-2-dimethylaminoethanol. 3. The crystalline folate salt according to claim 1 or 2, wherein said organic compound is di-N-methylaminoethanol. 3. The crystalline folate salt according to claim 1 or 2, wherein said organic compound is di-2-amino-2-methylpropanol. The acid salt of crystalline folate according to claims 1 or 2, characterized in that said tetrahydrofolic acid anion is 5-methyl-(6S)-tetrahydrofolic acid and said organic cation is di-choline. , an acid salt of said crystalline folate. The acid salt of crystalline folate according to claim 1 or 2, wherein said tetrahydrofolate anion is 5-methyl-(6S)-tetrahydrofolic acid and said organic cation is mono-2-dimethylaminoethanol. The acid salt of said crystalline folate, characterized in that: A pharmaceutical composition containing at least one folate salt according to any one of claims 1 to 9 as the main active compound and containing at least pharmaceutically acceptable excipients. thing. A salt of folate according to any one of claims 1 to 9, used as a medicament , food additive or dietary supplement, in deficiencies and disorders that are positively affected by the administration of tetrahydrofolic acid. A salt of said folate , which is used for the prevention and/or treatment of 10. A process for the production of a crystalline folate salt composed of a tetrahydrofolate anion and an organic cation according to any one of claims 1 to 9, the total weight of the mixture of organic solvent and water crystallizing said salt from an organic solvent containing water within the range of 0.2 to 12 weight percent of said method.