CRYSTALLINE SALTS OF 5-METHYL-(6S)-TETRAHYDROFOLIC ACID AND L-LEUCINE ETHYL ESTER

Abstract

The present invention refers to a crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester is from 1:0.3 to 1:3.0 (in mol/mol) and/or hydrates and/or solvates thereof as well as to a process of obtaining the same.

Claims

1. A crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is from 1:0.3 to 1:3.0 (in mol/mol) and/or hydrates and/or solvates thereof.

2. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is from 1:0.5 to 1:2.5 (in mol/mol) and/or hydrates and/or solvates thereof.

3. The crystalline salt of claim 1, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is from 1:0.75 to 1:1.25 (in mol/mol) and/or hydrates and/or solvates thereof.

4. The crystalline salt of claim 1, wherein the ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-leucine ethyl ester is approximately 1:1 (in mol/mol) and/or hydrates and/or solvates thereof.

5. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) selected from the following peaks located at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7.

6. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with at least three characteristic peaks (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7.

7. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with at least one characteristic peak (expressed in 2θ±0.2° 2θ (CuKα radiation)) selected from the following peaks located at 5.6, 6.9, 8.4, 12.9, 13.8, 14.1, 15.0, 17.5, 18.2, 19.1, 19.7, 21.2, 21.4, 23.7, 26.1 and 27.6.

8. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern with characteristic peaks (expressed in 2θ±0.2° 2θ (CuKα radiation)) at 5.6, 6.9, 8.4, 12.9, 14.1, 17.5, 19.1, 21.2, 21.4 and 23.7.

9. The crystalline salt of claim 1, characterized in that the salt is the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester and has a PXRD pattern substantially as shown in FIG. 1.

10. The crystalline salt of claim 1, having at least 99 wt % or more chemical and/or stereoisomerical purity.

11. A process for obtaining the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester according to claim 1 comprising the steps of: i) providing a mixture of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester, optionally in a suitable solvent or a mixture of solvents ii) adding a base, optionally in a suitable solvent or a mixture of solvents, to dissolve the compounds; iii) heating the composition to at least 60° C. and optionally carrying out a clear filtration; iv) crystallizing and cooling the mixture to a temperature between 1° C. and 30° C., optionally adding more solvent or mixture of solvents; and v) isolating the obtained solid material and optionally drying the product.

12. The process of claim 11, characterized in that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester in step ii) is in the range of from 1:1 to 1:3.

13. The process of claim 11, characterized in that the solvent is water.

14. The process of claim 11, characterized in that in step iii) and/or iv) seed crystals are added.

15. The process of claim 11, characterized in that L-leucine ethyl ester is used as L-leucine ethyl ester hydrochloride.

16. A pharmaceutical composition, food additive and/or preparation comprising the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester according to claim 1 and optionally one or more acceptable excipients.

17. The pharmaceutical composition according to claim 16 in the form of tablets, capsules, oral liquid preparations, powders, lyophilisates, granules, lozenges, reconstitutable powders, injectable or infusable solutions or suspensions or suppositories.

18. The pharmaceutical composition according to claim 16 further comprising at least one additional therapeutic agent.

19. The pharmaceutical composition according to claim 16, which is a pharmaceutical composition for oral, parenteral, intramuscular, intraspinal, intrathecal, peridontal, topical or rectal administration.

20. A method which comprises incorporating of the crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester according to claim 1 as constituent and/or additive in the production of drugs and/or food.

21. The crystalline salt comprising 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester according to claim 1 for use in the treatment in homocysteine-lowering, of anemia, neural tube defects, cardiovascular diseases, depression, cognitive impairment, Alzheimer's disease and osteoporosis and/or dietary management of low plasma and/or low red blood cell and/or low cerebrospinal fluid and/or low peripheral or central nervous system folate.

Description

EXAMPLES

[0042] Powder X-Ray Diffraction Stoe Stadi P equipped with a Mythen1 K Detector; Cu-Kα1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; curved Ge monochromator; 0.02° 2θ step size, 48 s step time, 1.5-50.5° 2θ scanning range; detector mode: step scan; 1° 2θ detector step; standard sample preparation: 10 to 20 mg sample was placed between two acetate foils; sample holder: Stoe transmission sample holder; the sample was rotated during the measurement. All sample preparation and measurement was done in an ambient air atmosphere.

[0043] TG-FTIR

[0044] Thermogravimetric measurements were carried out with a Netzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with a pinhole, N.sub.2 atmosphere, heating rate 10 K/min).

[0045] DVS

[0046] DVS measurements are typically performed with an SPS11-100n “Sorptions Prilfsystem” from ProUmid (formerly “Projekt Messtechnik”), August-Nagel-Str. 23, 89079 Ulm (Germany).

Example 1: Preparation of the Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid and L-Leucine Ethyl Ester without Seeding

[0047] A mixture of 5.0 grams of 5-methyl-(6S)-tetrahydrofolic acid monohydrate ([6S]-diastereoisomer: 98.4%) and 13 ml of water was heated to about 80° C. under an argon atmosphere and 1.98 mL of an aqueous solution of sodium hydroxide (concentration 30% w/w) were added to form a solution. The solution was heated to about 80° C. and a solution of 4.20 grams of L-leucine ethyl ester hydrochloride in 13 mL of water was added. The heating bath was removed to allow the solution to cool to about 25° C. within about two hours. While cooling, the solution gradually changes into a concentrated suspension. The suspension was diluted with 15 ml of water and stirred at ambient temperature overnight. The reactor with the suspension was further cooled in an ice/water bath to about 1° C. within about half an hour. The suspension was then filtered with a fritted glass filter and the solid product was washed with five ml of cold water. The solid product was dried in a vacuum dryer at about 35° C./10 mbar overnight and examined by .sup.1H-NMR and identified as 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester 1:1 salt. Powder X-ray diffraction was carried out and a PXRD pattern of the crystalline L-leucine ethyl ester salt substantially as depicted in FIG. 1 was obtained. HPLC analysis showed that the purity was 98.7% area and the optical purity was increased to 99.7% [6S]-diastereoisomer.

Example 2: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic Acid and L-leucine Ethyl Ester with Seeding

[0048] A mixture of 5.0 grams of 5-methyl-(6S)-tetrahydrofolic acid monohydrate ([6S]-diastereoisomer: 98.4%) and 20 ml of water was heated to about 80° C. under a nitrogen atmosphere and 1.9 mL of an aqueous solution of sodium hydroxide (concentration 32% w/w) was added to form a solution. The solution was heated to about 61° C. and a solution of 4.20 grams of L-leucine ethyl ester hydrochloride in 20 mL of water was added. The heating bath was removed to allow the solution to cool to about 32° C. within about half an hour. While cooling, the solution was seeded at about 60° C. with a small amount of crystalline L-leucine ethyl ester salt that was prepared according to Example 1 and the solution gradually changed into a concentrated suspension. At about 49° C. the suspension was diluted with 12 ml of water. At about 32° C. the reactor with the suspension was further cooled in an ice/water bath and a clear solution formed. After stirring at ambient temperature overnight, 0.15 mL of a 37% (w/w) concentrated hydrochloric acid aqueous solution was added, followed by 0.5 mL hydrochloric acid in form of a 2.00 molar aqueous solution. At ambient temperature, the solution was seeded with a small amount of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-leucine ethyl ester that was prepared according to Example 1 and a weak suspension formed. The suspension was stirred at ambient temperature and an additional 0.35 mL of hydrochloric acid in form of a 2 molar aqueous solution was added. The suspension was cooled to about 2° C. and an additional 0.30 mL of hydrochloric acid in the form of a 2 molar aqueous solution was added. Stirring of the suspension was continued at about 2° C. for about 15 minutes and the suspension filtered with a fritted glass filter. The solid product was dried in a vacuum dryer at about 40° C./10 mbar and examined by .sup.1H-NMR and identified as 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt. Powder X-ray diffraction was carried out and a PXRD pattern of the L-leucine ethyl ester salt substantially as depicted in FIG. 1 was obtained; however, the PXRD pattern revealed that the sample contained a small amount of NaCl. HPLC analysis shows that the purity is 98.39% area and the optical purity was increased to 99.7% [6S]-diastereoisomer.

Example 3: Washing the Salt of 5-methyl-(6S)-tetrahydrofolic Acid and L-leucine Ethyl Ester

[0049] About 420 mg of the solid material obtained in Example 2 was weighed into a filter centrifuge device and 2.0 mL of water is added followed by centrifugation under ambient conditions. This wash step is repeated twice more using 0.5 mL of water. The wet filter cake is then transferred to a fritted glass filter and air dried by drawing ambient air (about 22° C./about 21% r.h.) through the glass filter for about 10 minutes. The dried material was examined by .sup.1H-NMR and identified as a 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester 1:1 salt. TG-FTIR analysis showed that the sample contained only about 0.7% of water. Powder X-ray diffraction was carried out and a PXRD pattern of the L-leucine ethyl ester salt substantially as depicted in FIG. 1 was obtained which exhibits peaks at 2-theta angles as listed in Table 1.

TABLE-US-00001 TABLE 1 2-theta angles, d-spacings and qualitative intensities for 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt according to example 3. angle °2Θ d-spacing [Å] qualitative intensity 2.8 31.7 m 5.6 15.7 s 6.9 12.9 vs 8.4 10.5 s 10.6 8.4 w 11.3 7.8 w 12.9 6.9 s 13.8 6.4 m 14.1 6.3 s 14.5 6.1 m 15.0 5.90 m 15.5 5.70 w 15.8 5.61 m 16.4 5.41 m 17.0 5.22 m 17.5 5.07 s 18.2 4.88 s 18.6 4.78 m 19.1 4.64 s 19.7 4.50 m 20.2 4.40 w 20.6 4.32 w 20.9 4.25 w 21.2 4.18 s 21.4 4.15 s 22.3 3.98 m 22.6 3.93 m 23.0 3.86 w 23.4 3.80 w 23.7 3.75 s 24.3 3.65 w 24.7 3.61 w 25.0 3.56 w 25.2 3.53 w 25.5 3.49 w 26.1 3.41 m 26.7 3.34 w 27.1 3.29 w 27.6 3.23 m 28.0 3.18 w 28.3 3.15 w 28.6 3.12 w Vs = very strong, s = strong, m = medium, w = weak, and vw = very weak in intensity. It should be noted that intensity values can vary substantially due to preferred orientation effects.

Example 4: Hygroscopicity and Water Content (DVS Experiments)

[0050] The water content of a sample of 5-methyl-(6S)-tetrahydrofolic acid calcium salt was measured and found to be 12.4%. TG-FTIR analysis of a sample of 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt according to Example 3 showed that the sample contained only about 0.7% of water. A sample of 5-methyl-(6S)-tetrahydrofolic acid calcium salt and a sample of 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt according to Example 3 (about 20 mg each) were examined by dynamic water vapor sorption analysis (DVS) within the relative humidity range from 0 to 75% r.h. DVS measurements were performed as follows: The sample was placed on an aluminum holder on top of a microbalance and allowed to equilibrate at 50% RH before starting the pre-defined humidity program:

[0051] (1) two hours kept at 50% constant relative humidity (RH) then

[0052] (2) reduced to 0% RH at a rate of 5% per hour

[0053] (3) maintained RH at 0% for five hours

[0054] (4) raised RH to 75% at a rate of 5% per hour

[0055] (5) maintained RH at 75% for five hours

[0056] (6) reduced to 0% RH at a rate of 5% per hour

[0057] (7) maintained RH at 0% for five hours

[0058] (8) raised RH to 75% at a rate of 5% per hour

[0059] (9) reduced to 50% RH at a rate of 5% per hour

[0060] (10) maintained RH at 50% for about one hour

[0061] Comparing the result for 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt with the result for the calcium salt shows that the water content of 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt changes about 2.6% within the tested range, while the water content for the calcium salt changes by about 7.4%. The results are illustrated in FIG. 2.

Example 5: Kinetic Solubility of the Crystalline Salt of 5-methyl-(6S)-tetrahydrofolic Acid and L-leucine Ethyl Ester

[0062] 43.1 mg of the anhydrous form of the crystalline 5-methyl-(6S)-tetrahydrofolic acid L-leucine ethyl ester salt according to Example 3 were weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified/de-ionized water (for instance water for chromatography) was added to the solid using an adjustable volumetric pipette. The mixture was vigorously agitated at room temperature for one minute. After one minute a turbid solution was observed suggesting that most of the sample was dissolved. The solution was filtered by centrifugal filtration and 1.50 mL of the aqueous solution was transferred into a tared glass vial (about 10 mL volume). The water was evaporated in an air dryer at 40° C. for about 15 hours, then at 50° C. for about eight hours, subsequently drying was completed at 50° C. under vacuum (10 to 20 mbar) for about 13 hours. The solubility was determined by gravimetric evaluation of the solid residue. The solubility was 15.1 mg of 5-methyl-(6S)-tetrahydrofolic acid per mL.

Reference Example 1: Kinetic Solubility of the Calcium Salt of 5-methyl-(6S)-tetrahydrofolic Acid

[0063] 42.5 mg of the anhydrous form of the crystalline 5-methyl-(6S)-tetrahydrofolic acid calcium salt were weighed into a 7 mL glass vial with a screw cap. 2.00 mL of purified/de-ionized water (for instance water for chromatography) was added to the solid using an adjustable volumetric pipette. The mixture was vigorously agitated at room temperature for one minute. After one minute a suspension was observed. The suspension was filtered by centrifugal filtration and 1.50 mL of the aqueous solution was transferred into a tared glass vial (about 10 mL volume). The water was evaporated in an air dryer at 40° C. for about 15 hours, then at 50° C. for about eight hours, subsequently drying was completed at 50° C. under vacuum (10 to 20 mbar) for about 13 hours. The solubility was determined by gravimetric evaluation of the solid residue. The solubility was 9.0 mg of 5-methyl-(6S)-tetrahydrofolic acid per mL.