Folate salts for medical use

Abstract

The invention relates to amorphous folate salt. The salt consists of a folate anion and an organic cation. The folate anion is selected from the group consisting of 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, (6S)-tetrahydrofolic acid, 5,10-Methylene-(6R)-tetrahydrofolic acid, and its oxidized derivatives JK12A and Mefox, and the cation is an organic compound selected from the group arginine, choline, acetylcholine, 1,1-dimethyl-biguanidin and phenylethylbiguanidin. The cation is an organic compound with a complementary pharmacological activity.

Claims

1. An amorphous folate salt consisting of a tetrahydrofolic acid anion and an organic cation characterized in that the anion is 5-formyl-(6S)-tetrahydrofolic acid and the cation is an organic compound selected from the group arginine, choline, acetylcholine, 1,1-dimethyl-biguanidin, phenylethylbiguanidin, betaine-methylester and dimethylaminoethanol, wherein the folate salts have a high solubility in an apolar solvent, said apolar solvent having a relative polarity of 0.82 compared to water.

2. An amorphous folate salt according to claim 1 consisting of tetrahydrofolic acid anion and an organic cation characterized in that the anion is 5-formyl-(6S)-tetrahydrofolic acid and the cation is di-arginine.

3. An amorphous folate salt according to claim 1 consisting of tetrahydrofolic acid anion and an organic cation characterized in that the anion is 5-formyl-(6S)-tetrahydrofolic acid and the cation is mono-arginine.

4. An amorphous folate salt according to claim 2, characterized in that the tetrahydrofolic acid anion is 5-formyl-(6S)-tetrahydrofolic acid and the organic cation is di-arginine wherein the .sup.1 1-1-NMR shifts in D.sub.2O are TABLE-US-00044 δ (1H) in ppm Multiplicity Intensity 8.53/7.82 s/s 1H 7.56/7.53 d/d 2H 6.64/6.59 d/d 2H 4.75 m 1H 4.21 m 1H 3.66 t 2H 3.44 m 1H 3.30 m 2H 3.17 m 1H 3.11 s 4H 2.23 m 2H 2.07 m 1H 1.95 m 1H 1.79 m 4H 1.58 m  4H.

5. A pharmaceutical composition, comprising at least one folate salt according to claim 1, as a main active compound and at least a pharmaceutically acceptable excipient.

Description

EXAMPLES

Example 1

(1) Preparation of 5-formyl-(6S)-tetrahydrofolic acid di L-arginine salt 10.0 g (16.62 mmol) of calcium levofolinate pentahydrate under argon was dissolved in 230 ml water at 70° C. Then 5.79 g (33.24 mmol) L-arginine was added, followed by 2.09 g (16.62 mmol) oxalic acid di-hydrate. After cooling to room temperature (RT), the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with methanol, filtered and dried under vacuum at 60° C., to give 13.63 g of the title compound. Analytical data:

(2) TABLE-US-00019 δ (1H) in ppm Multiplicity Intensity 8.53/7.82 s/s 1H 7.56/7.53 d/d 2H 6.64/6.59 d/d 2H 4.75 m 1H 4.21 m 1H 3.66 t 2H 3.44 m 1H 3.30 m 2H 3.17 m 1H 3.11 s 4H 2.23 m 2H 2.07 m 1H 1.95 m 1H 1.79 m 4H 1.58 m 4H

(3) Optical rotation: α.sup.20D+1.75° (c=1 H.sub.2O)

Example 2

Preparation of 5-formyl-(6S)-tetrahydrofolic Acid Di Metformin Salt

(4) 1.86 g (3.09 mmol) of calcium levofolinate pentahydrate under argon was dissolved in 43 ml Water at 70° C. Then 0.80 g (6.18 mmol) metformin free base was added, followed by 0.39 g (3.09 mmol) oxalic acid di-hydrate. After cooling to RT, the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with ethanol, filtered and dried under vacuum at 60° C., to give 2.13 g of the title compound. Analytical data:

(5) TABLE-US-00020 δ (1H) in ppm Multiplicity Intensity 8.57/7.86 s/s 1H 7.60/7.56 d/d 2H 6.68/6.63 d/d 2H 4.80 m 1H 4.25 m 1H 3.48 m 1H 3.33 m 2H 3.22 m 1H 2.96 s 12H  2.25 m 2H 2.10 m 1H 1.96 m 1H

(6) Optical rotation: α.sup.20D−2.84° (c=1 H.sub.2O)

Example 3

Preparation of 5-formyl-(6S)-tetrahydrofolic Acid Di-Phenformin Salt

(7) 1.21 g (2.02 mmol) of calcium levofolinate pentahydrate under argon was dissolved in 43 ml water at 70° C. Then 0.83 g (4.04 mmol) phenformin free base was added, followed by 0.25 g (2.02 mmol) oxalic acid di-hydrate. After cooling to RT, the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 5.5) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60° C., to give 1.80 g of the title compound. Analytical data:

(8) TABLE-US-00021 δ (1H) in ppm Multiplicity Intensity 8.52/7.84 s/s 1H 7.60/7.56 d/d 2H 7.31 m 4H 7.23 m 6H 6.67/6.61 d/d 2H 4.77 m 1H 4.26/4.22 m 1H 3.48 m 1H 3.42 m 4H 3.28 m 2H 3.16 m 1H 2.79 m 4H 2.26 m 2H 2.10 m 1H 1.96 m 1H

(9) Optical rotation: α.sup.20D+1.39° (c=1 H.sub.2O)

Example 4

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Di-Choline Salt

(10) 1.92 g (3.19 mmol) of calcium levofolinate pentahydrate under argon was dissolved in 40 ml Water at 70° C. Then a filtered, aqueous solution of 0.79 g (6.38 mmol) choline fluoride (obtained from choline chloride and silver fluoride) was added. After cooling to 0° C., the precipitated calcium fluoride was filtered off through a Hyflo pad and the resulting clear solution (pH 6.5) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60° C., to give 1.89 g of the title compound.

(11) Analytical data:

(12) TABLE-US-00022 δ (1H) in ppm Multiplicity Intensity 8.60/7.88 s/s 1H 7.63/7.59 d/d 2H 6.71/6.66 d/d 2H 4.83 m 1H 4.27 m 1H 4.01 m 4H 3.48 m 1H 3.47 m 4H 3.37 m 2H 3.27 m 1H 3.14 s 18H  2.26 m 2H 2.11 m 1H 1.98 m 1H

(13) Optical rotation: α.sup.20D+1.14° (c=1 H.sub.2O)

Example 5

Preparation of 5-methyl-(6S)-tetrahydrofolic Acid Di L-Arginine Salt

(14) 10.0 g (17.67 mmol) of calcium levomefolate×3.8 hydrate under argon was suspended in 100 ml water at 70° C. Then 2.23 g (17.67 mmol) oxalic acid di-hydrate was added, followed by 6.15 g (35.33 mmol) L-arginine. After cooling to 0° C., the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with methanol, filtered and dried under vacuum at 60° C., to give 13.98 g of the title compound. Analytical data:

(15) TABLE-US-00023 δ (1H) in ppm Multiplicity Intensity 7.62 d 2H 6.70 d 2H 4.27 m 1H 3.72 t 2H 3.48 m 1H 3.28 m 1H 3.18 t 4H 3.15 m 1H 3.07 m 1H 2.98 m 1H 2.53 s 3H 2.28 m 2H 2.12 m 1H 1.99 m 1H 1.86 m 4H 1.64 m 4H

(16) Optical rotation: α.sup.20D+30.0° (c=1 H.sub.2O)

Example 6

Preparation of 5-methyl-(6S)-tetrahydrofolic Acid Di-Metformin Salt

(17) 1.49 g (2.63 mmol) of calcium levomefolate×3.8 hydrate under argon was suspended in 38 ml water at 70° C. Then 0.332 g (2.63 mmol) oxalic acid di-hydrate dissolved in water was added, followed by 0.68 g (5.26 mmol) metformin free base dissolved in water. After cooling to 0° C., the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60° C., to give 1.89 g of the title compound.

(18) Analytical data:

(19) TABLE-US-00024 δ (1H) in ppm Multiplicity Intensity 7.59 d 2H 6.67 d 2H 4.25 m 1H 3.44 m 1H 3.24 m 1H 3.10 m 1H 3.01 m 1H 2.95 s 12H  2.92 m 1H 2.48 s 3H 2.25 m 2H 2.10 m 1H 1.97 m 1H

(20) Optical rotation: α.sup.20D+17.95° (c=1 H.sub.2O)

Example 7

Preparation of 5-methyl-(6S)-tetrahydrofolic Acid Di Phenformin Salt

(21) 1.64 g (2.90 mmol) of calcium levomefolate×3.8 hydrate under argon was suspended in 35 ml Water at 70° C. Then 0.366 g (2.90 mmol) oxalic acid di-hydrate dissolved in water was added, followed by 0.68 g (5.26 mmol) phenformin free base dissolved in water. After cooling to 0° C., the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 5.5) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60° C., to give 2.54 g of the title compound. Analytical data:

(22) TABLE-US-00025 δ (1H) in ppm Multiplicity Intensity 7.58 d 2H 7.29 m 4H 7.21 m 6H 6.65 d 2H 4.24 m 1H 3.44 m 1H 3.40 m 4H 3.24 m 1H 3.10 m 1H 3.01 m 1H 2.91 m 1H 2.78 m 4H 2.49 s 3H 2.24 m 2H 2.09 m 1H 1.96 m 1H

(23) Optical rotation: α.sup.20D+16.06° (c=1 H.sub.2O)

Example 8

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono L-Arginine Salt

(24) 0.258 g (0.429 mmol) of calcium levofolinate pentahydrate under argon was dissolved in 6 ml water at 70°. Then 0.0747 g (0.429 mmol) L-arginine was added, followed by 0.0541 g (0.429 mmol) oxalic acid dihydrate dissolved in water. After cooling to rt, the precipitated calcium oxalate was filtered off through a Hyflo pad and the resulting clear solution (pH 4) evaporated to dryness. The crude product was then thoroughly digested with methanol, filtered and dried under vacuum at 60°, to give 0.274 g of the title compound. Analytical data:

(25) TABLE-US-00026 δ (1H) in ppm Multiplicity Intensity 7.59 d 2H 6.67 d 2H 4.25 m 1H 3.69 t 1H 3.58 m 1H 3.41 m 2H 3.19 m 5H 2.71 s 3H 2.27 m 2H 2.10 m 1H 1.96 m 1H 1.83 m 2H 1.60 m 2H

(26) Optical rotation: α.sup.20D+0.62° (c=1 H.sub.2O)

Example 9

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono Metformin Salt

(27) 100.0 mg (0.2112 mmol) of levofolinic acid and 28.1 mg (0.2175 mmol) metformin free base were suspended under argon in 2 ml Water and 4 ml methanol. After 15′ of stirring and ultrasonification, the resulting solution (pH 5) was evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60°, to give 122 mg of the title compound.

(28) Analytical data:

(29) TABLE-US-00027 δ (1H) in ppm Multiplicity Intensity 8.51/7.8  s/s 1H 7.54/7.50 d/d 2H 6.62/6.57 d/d 2H 4.73 m 1H 4.27 m 1H 3.42 m 1H 3.28 m 2H 3.16 m 1H 2.92 s 6H 2.32 m 2H 2.12 m 1H 1.95 m 1H

(30) Optical rotation: α.sup.20D−15.3° (c=1 H.sub.2O)

Example 10

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono Phenformin Salt

(31) 100.1 mg (0.2114 mmol) of levofolinic acid and 44.7 mg (0.2178 mmol) phenformin free base were suspended under argon in 4 ml water and 4 ml methanol. After 10′ of stirring and ultrasonification, the resulting solution (pH 5) was evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60°, to give 137 mg of the title compound.

(32) Analytical data:

(33) TABLE-US-00028 δ (1H) in ppm Multiplicity Intensity 8.50/7.80 s/s 1H 7.54/7.50 d/d 2H 7.27 m 2H 7.19 m 3H 6.62/6.57 d/d 2H 4.75 m 1H 4.29 m 1H 3.40 m 1H 3.38 m 2H 3.26 m 2H 3.15 m 1H 2.76 m 2H 2.33 m 2H 2.13 m 1H 1.96 m 1H

(34) Optical rotation: α.sup.20D+13.50° (c=1 H.sub.2O)

Example 11

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono Choline Salt

(35) 100.0 mg (0.2112 mmol) of levofolinic acid and 62.5 ul (0.2218 mmol) of a 45% methanolic solution of choline hydroxide were suspended under Argon in 2 ml Water and 4 ml Methanol. After 5′ of stirring, ultrasonification and heating to reflux, the resulting slightly turbid solution (pH 5) was filtered still warm through a syringe filter and evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 60°, to give 118 mg of the title compound. Analytical data:

(36) TABLE-US-00029 δ (1H) in ppm Multiplicity Intensity 8.50/7.80 s/s 1H 7.54/7.50 d/d 2H 6.62/6.57 d/d 2H 4.73 m 1H 4.27 m 1H 3.93 m 2H 3.43 m 1H 3.39 m 2H 3.27 m 2H 3.16 m 1H 3.07 s 9H 2.32 m 2H 2.12 m 1H 1.95 m 1H

(37) Optical rotation: α.sup.20D−16.3° (c=1 H.sub.2O)

Example 12

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Mono Metformin Salt

(38) 72.7 mg (0.1582 mmol) of Levomefolic acid and 21.5 mg (0.1661 mmol) Metformin free base were suspended under Argon in 10 ml Water and 20 ml Methanol. After stirring, ultrasonification and refluxing, the resulting solution was evaporated to dryness. The crude product was then thoroughly digested with Acetonitrile, filtered and dried under vacuum at 50°, to give 94.6 mg of the title compound.

(39) Analytical data:

(40) TABLE-US-00030 δ (1H) in ppm Multiplicity Intensity 7.59 d 2H 6.67 d 2H 4.25 m 1H 3.57 m 1H 3.38 m 2H 3.20 m 1H 3.13 m 1H 2.95 s 6H 2.84 m 1H 2.67 s 3H 2.26 m 2H 2.10 m 1H 1.96 m 1H

Example 13

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Mono Phenformin Salt

(41) 90.4 mg (0.1967 mmol) of levomefolic acid and 42.4 mg (0.2066 mmol) phenformin free base were suspended under argon in 5 ml water and 10 ml methanol. After stirring, ultrasonification and refluxing for some time, the resulting solution was evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 50°, to give 133 mg of the title compound. Analytical data:

(42) TABLE-US-00031 δ (1H) in ppm Multiplicity Intensity 7.56 d 2H 7.27 m 2H 7.19 m 3H 6.63 d 2H 4.23 m 1H 3.53 m 1H 3.38 m 2H 3.35 m 2H 3.12 m 1H 3.07 m 1H 2.76 m 2H 2.64 s 3H 2.25 m 2H 2.09 m 1H 1.96 m 1H

(43) Optical rotation: α.sup.20D−5.90° (c=0.235 H.sub.2O)

Example 14

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Mono Choline Salt

(44) 2.61 g (4.611 mmol) of calcium levomefolinate ×3.8hydrate under argon was suspended in 60 ml water at 95°. Then a filtered, aqueous solution of 568 mg (4.611 mmol) choline fluoride (obtained from choline chloride and silver fluoride) was added. Stirring was continued for 10′ at 95° and after cooling to RT, the precipitated calcium fluoride was filtered off through a Hyflo pad and the resulting clear solution (pH 6.5) evaporated to dryness to give 2.568 g of the title compound. The crude product was thoroughly digested with Ethanol at 50°, then cooled in an ice-bath and filtered, dried under vacuum at 60°, to give 1.67 g of the title compound. Analytical data:

(45) TABLE-US-00032 δ (1H) in ppm Multiplicity Intensity 7.56 d 2H 6.65 d 2H 4.20 m 1H 3.95 m 2H 3.43 m 1H 3.40 t 2H 3.20 d 1H 3.08 s 9H 3.04 m 1H 2.99 m 1H 2.89 m 1H 2.42 s 3H 2.20 m 2H 2.04 m 1H 1.91 m 1H

(46) Optical rotation: α.sup.20D+0.1° (c=1 H.sub.2O)

Example 15

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Di Acetylcholine Salt

(47) 330.6 mg (0.5496 mmol) of calcium levofolinat pentahydrat under argon was dissolved in 6 ml water at 70°. Then a filtered, aqueous solution of 181.6 mg (1.099 mmol) acetylcholine fluoride (obtained from acetylcholine chloride and silver fluoride) was added at 30°. After cooling to 0°, the precipitated calcium fluoride was filtered off through a syringe filter and the resulting clear solution (pH 6) evaporated to dryness to give 407 mg of the title compound. Analytical data:

(48) TABLE-US-00033 δ (1H) in ppm Multiplicity Intensity 8.52/7.81 s/s 1H 7.56/7.59 d/d 2H 6.64/6.58 d/d 2H 4.75 m 1H 4.42 m 4H 4.20 m 1H 3.61 m 4H 3.45 m 1H 3.30 m 2H 3.18 m 1H 3.09 s 18H  2.20 m 2H 2.05 m 1H 2.02 s 6H 1.91 m 1H

(49) Optical rotation: α.sup.20D−3.4° (c=1 H.sub.2O)

Example 16

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Di Acetylcholine Salt

(50) 331.8 mg (0.5862 mmol) of calcium levomefolinate ×3.8hydrate under argon was suspended in 8 ml water at 70°. Then a filtered, aqueous solution of 193.6 mg (1.172 mmol) acetylcholine fluoride (obtained from acetylcholine chloride and silver fluoride) was added at 50°. After cooling to 0°, the precipitated calcium fluoride was filtered off through a syringe filter and the resulting clear solution (pH 6.5) evaporated to dryness to give 433 mg of the title compound. Analytical data:

(51) TABLE-US-00034 δ (1H) in ppm Multiplicity Intensity 7.55 d 2H 6.64 d 2H 4.42 m 4H 4.19 m 1H 3.60 m 4H 3.40 m 1H 3.19 m 1H 3.09 s 18H  3.02 m 1H 2.96 m 1H 2.87 m 1H 2.42 s 3H 2.19 m 2H 2.05 m 1H 2.02 s 6H 1.90 m 1H

(52) Optical rotation: α.sup.20D17.04° (c=1 H.sub.2O)

Example 17

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Di Betainmethylester Salt

(53) 50.6 mg (0.0736 mmol) of silver levofolinate under argon was suspended in 2 ml water. Then a solution of 38.1 mg (0.1473 mmol) betain-methylester iodide in 1 ml water was added and the mixture heated under stirring to 90° C. After cooling down, the suspension was filtered through a syringe filter and the resulting clear solution evaporated, dried under vacuum at 50° C., to give 56 mg of the title compound. Analytical data:

(54) TABLE-US-00035 δ (1H) in ppm Multiplicity Intensity 8.52/7.81 s/s 1H 7.56/7.52 d/d 2H 6.64/6.59 d/d 2H 4.75 m 1H 4.22 s 4H 4.21 m 1H 3.73 s 6H 3.44 m 1H 3.30 m 2H 3.22 s 18H  3.21 m 2H 2.22 m 2H 2.06 m 1H 1.93 m 1H

Example 18

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Di Betainmethylester Salt

(55) 520.1 mg (0.919 mmol) of calcium levomefolinate ×3.8hydrate under argon was suspended in 6 ml Water at 70°. Then a filtered, aqueous solution of 291.8 mg (1.930 mmol) betain methylester fluoride (obtained from betain methylester iodide and silver fluoride) was added at 20°. After cooling to 0° C., the precipitated calcium fluoride was filtered off through a syringe filter and the resulting clear solution (pH 6.5) evaporated, dried under vacuum at 40°, to give 682 mg of the title compound. Analytical data:

(56) TABLE-US-00036 δ (1H) in ppm Multiplicity Intensity 7.56 d 2H 6.64 d 2H 4.22 s 4H 4.20 m 1H 3.73 s 6H 3.42 m 1H 3.23 m 1H 3.21 s 18H  3.08 m 1H 3.00 m 1H 2.90 m 1H 2.46 s 3H 2.20 m 2H 2.05 m 1H 1.92 m 1H

(57) Optical rotation: α.sup.20D+10.5° (c=1 H.sub.2O)

Example 19

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Di 2-dimethylaminoethanol Salt

(58) 58.0 mg (0.1225 mmol) of levofolinic acid was suspended under argon in 0.5 ml water and treated with 24.7 μl (0.2450 mmol) of 2-dimethylaminoethanol (deanol).

(59) The mixture was stirred at room temperature (rt) until a clear solution is formed, evaporated and dried under vacuum at 45° C. to give 77 mg of the title compound.

(60) Analytical data:

(61) TABLE-US-00037 δ (1H) in ppm Multiplicity Intensity 8.50/7.80 s/s 1H 7.54/7.51 d/d 2H 6.62/6.57 d/d 2H 4.73 m 1H 4.18 m 1H 3.75 t 4H 3.43 m 1H 3.28 m 2H 3.17 m 1H 3.11 t 4H 2.75 s 12H  2.18 m 2H 2.03 m 1H 1.90 m 1H

(62) Optical rotation: α.sup.20D−8.5° (c=1 H.sub.2O)

Example 20

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono 2-dimethylaminoethanol Salt

(63) 71.6 mg (0.1512 mmol) of levofolinic acid was suspended under argon in 2 ml water and 5 ml methanol and treated with 15.7 μl (0.1558 mmol) of 2-dimethylaminoethanol (deanol). After ultrasonification and heating shortly to 60° C., a clear solution is formed (pH 5). This solution was evaporated and dried under vacuum at 45° C. to give 89.6 mg of the title compound. Analytical data:

(64) TABLE-US-00038 δ (1H) in ppm Multiplicity Intensity 8.58/7.87 s/s 1H 7.61/7.57 d/d 2H 6.69/6.64 d/d 2H 4.81 m 1H 4.33 m 1H 3.85 t 2H 3.50 m 1H 3.34 m 2H 3.24 t 2H 3.22 m 1H 2.87 s 6H 2.38 m 2H 2.17 m 1H 2.01 m 1H

(65) Optical rotation: α.sup.20D−13.8° (c=1 H.sub.2O)

Example 21

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Mono L-Arginine Salt

(66) 82.0 mg (0.1785 mmol) of levomefolic acid was suspended under argon in 10 ml water and 20 ml methanol. 31.1 mg (0.1785 mmol) of L-arginine were added and the mixture treated until a solution was formed. This was evaporated, thoroughly digested with acetonitrile, filtered and dried under vacuum at 50° to give 109.7 mg of the title compound. Analytical data:

(67) TABLE-US-00039 δ (1H) in ppm Multiplicity Intensity 7.59 d 2H 6.67 d 2H 4.25 m 1H 3.69 t 1H 3.58 m 1H 3.41 m 2H 3.19 m 5H 2.71 s 3H 2.27 m 2H 2.10 m 1H 1.96 m 1H 1.83 m 2H 1.60 m 2H

Example 22

Preparation of 5-Formyl-(6S)-tetrahydrofolic Acid Mono Acetylcholine Salt

(68) 656.0 mg (1.090 mmol) of Calcium levofolinat pentahydrat under argon was dissolved in 10 ml water at 70°. Then a filtered, aqueous solution of 180.1 mg (1.090 mmol) acetylcholine fluoride (obtained from acetylcholine chloride and silver fluoride) was added at 30°. After cooling to 0°, the precipitated calcium fluoride was filtered off through a syringe filter and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 50° C., to give 640 mg of the title compound. Analytical data:

(69) TABLE-US-00040 δ (1H) in ppm Multiplicity Intensity 8.58/7.86 s/s 1H 7.61/7.58 d/d 2H 6.69/6.64 d/d 2H 4.81 m 1H 4.49 m 2H 4.26 m 1H 3.67 m 2H 3.50 m 1H 3.57 m 2H 3.25 m 1H 3.15 s 9H 2.25 m 2H 2.10 m 1H 2.08 s 3H 1.97 m 1H

(70) Optical rotation: α.sup.20D−7.7° (c=1 H.sub.2O)

Example 23

Preparation of 5-Methyl-(6S)-tetrahydrofolic Acid Mono Acetylcholine Salt

(71) 625.6 mg (1.105 mmol) of calcium levomefolinate ×3.8hydrate under argon was suspended in 12 ml water at 70°. Then a filtered, aqueous solution of 182.6 mg (1.105 mmol) acetylcholine fluoride (obtained from acetylcholine chloride and silver fluoride) was added at 30°. After heating the mixture shortly to 60° C. and cooling again to 20° C., the precipitated calcium fluoride was filtered off through a syringe filter and the resulting clear solution (pH 6) evaporated to dryness. The crude product was then thoroughly digested with acetonitrile, filtered and dried under vacuum at 50° C., to give 637 mg of the title compound. Analytical data:

(72) TABLE-US-00041 δ (1H) in ppm Multiplicity Intensity 7.61 d 2H 6.69 d 2H 4.48 m 2H 4.26 m 1H 3.66 m 2H 3.45 dd 1H 3.25 m 1H 3.15 s 9H 3.09 m 1H 3.03 m 1H 2.93 m 1H 2.48 s 3H 2.25 m 2H 2.11 m 1H 2.08 s 3H 1.97 m 1H

(73) Optical rotation: α.sup.20D+29.7° (c=1 H.sub.2O)

(74) Solubility determination of selected folate salts was in general performed as follows:

(75) A quantity of the salt corresponding to the expected solubility (between 1-100 mg) was provided and mixed with increments of the respective solvent until a solution was formed. This may require longer times, especially with highly soluble salts where solutions of extreme viscosity resulted.

(76) The solubility in glycerol of the two di-arginine salts of 5-formyl-(6S)-tetrahydrofolic acid and 5-methyl-(6S)-tetrahydrofolic acid were subjected to a more detailed testing. The di-arginine salts were first finely ground in the agate mortar and then mixed with glycerine (50 mg in 1 ml solvent (5% m/v), under argon) and stirred with magnetic stirrer. After stirring overnight, a clear solution was achieved. After the 5% (m/v) solubility had already been confirmed, the same procedure was carried out at double concentration, resulting in solutions. Even at a concentration of 20% (m/v), the resulting solutions are very viscous.

(77) For comparison, the solubility of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid (Gelpell) in glycerine was performed. The salt was clearly insoluble at 5% (m/v), 2.5% (m/v) and 1.6% (m/v). At 1.25% (m/v) a solution was achieved, slightly milky at the beginning, but after stirring it was clearly solved over the weekend. This confirms the previous data of approximately 1% solubility of this calcium salt.

(78) Solubility of arginine (pestled) in glycerine: 100 mg in 500 μl, well soluble, 200 mg in 500 μl gives a solution after stirring overnight, a clear solution resulted after 36 h. (Quality glycerine: 99.5%, assay 99.99%, water but not specified. (Density: 1.26 g/ml))

(79) All solubility data are indicated in mass-% (m/m); solubility was determined at 20° C.:

(80) TABLE-US-00042 Glycerine MeOH EtOH n-PrOH i-PrOH DMSO Folate Counter ion % % % % % % FTHF di arginine 19.23 0.06 0.06 0.06 0.06 0.25 FTHF di metformin 7.35 55.80 0.63 0.62 0.63 0.25 FTHF di choline 28.41 >>50 >50 0.31 0.32 0.25 FTHF di acetylcholine 24.10 55.80 55.80 55.90 0.50 0.25 FTHF di deanol 0.79 55.80 0.63 0.31 0.32 0.25 MTHF di arginine 19.23 0.06 0.06 0.06 0.06 19.23 MTHF di metformin 7.35 55.80 0.63 0.62 0.63 31.25 MTHF di acetylcholine 24.10 >>50 50.00 50.00 50.00 47.62 Relative 0.812 0.762 0.654 0.617 0.546 0.444 Polarity Relative polarity data source: NIST site: http://webbook.nist.gov/chemistry/ FTHF = 5-Formyl-(6S)-tetrahydrofolic acid MTHF = 5-Methyl-(6S)-tetrahydrofolic acid

(81) Quantitative HPLC measurements (20.00 mg substance dissolved in 10.00 ml H.sub.2O, amount injected 1.0 μl, at 280 nm)

(82) TABLE-US-00043 theoretical % of content in theoretical solvent including Compound titer found value content solvent Ca-FTHF × 5H2O (titer) .sup. 100% — .sup. 100% — — FTHF × mono arginine 92.89% 92.79% 99.89% <0.1% 99.89% MeOH FTHF × di arginine 73.20% 69.00% 94.26% 3.7% 98.00% MeOH FTHF × mono metformin 99.83% 99.58% 99.75% 0.29% AcN 100.04%  FTHF × di metformin 82.21% 75.78% 92.18% 8.0% EtOH 100.18%  FTHF × mono phenformin 88.64% 90.18% 101.74%  <0.1% AcN 101.74%  FTHF × di phenformin 68.05% 66.94% 98.37% 4.0% AcN 102.37%  FTHF × mono cholin 104.33%  98.44% 94.36% 3.59% AcN 97.95% FTHF × di cholin 88.50% 89.31% 100.9% 0% AcN; 100.9%  H2O FTHF × mono acetylcholine 97.24% 93.45% 96.10% AcN; H2O — FTHF × di acetylcholine 78.76% 68.31% 86.73% AcN; H2O — FTHF × mono deanol 106.93%  100.05%  93.56% H2O — FTHF × di deanol 92.31% 91.84% 99.48% H2O 99.48% Ca-MTHF × 3.8H2O (titer) .sup. 100% — .sup. 100% — — MTHF × mono arginine 89.32% 81.12% 90.82% AcN; H2O — MTHF × di arginine 70.06% 67.41% 96.22% 2.3% 98.52% MeOH MTHF × mono metformin 96.15% 79.65% 82.84% 1.17% AcN 85.01% MTHF × di metformin 78.85% 74.38% 94.33% 0.2% AcN; 94.53% H2O? MTHF × mono phenformin 85.14% 68.57% 80.54% 3.86% AcN 84.40% MTHF × di phenformin 65.06% 63.43% 97.49% 0.47% AcN 97.96% MTHF × mono choline 100.60%  85.72% 85.22% 1.08% 86.30% EtOH MTHF × di cholin 85.01% 73.39%/ 86.33%/94.71% H2O; 86.33%/94.71% 80.51% <0.1% n- PrOH MTHF × mono acetylcholine 93.60% 80.13% 85.61% AcN; H2O — MTHF × di acetylcholine 75.48% 55.43% 73.44% H2O — MTHF × mono deanol 103.17%  102.58%  99.43% 0.3% IPOH 99.73% MTHF × di deanol 88.75% 81.62% 91.97% 0.67% n- 92.64% PrOH; H2O MTHF × di BetainOMe 78.41% 64.34% 82.05% H2O? — Deanol = dimethylaminoethanol