Crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid
11185545 · 2021-11-30
Assignee
Inventors
- Rudolf Moser (Schaffhausen, CH)
- Viola Groehn (Dachsen, CH)
- Fritz Blatter (Reinach, CH)
- Martin Szelagiewicz (Basel, CH)
- Ruth Boehni Stamm (Stein Am Rhein, CH)
- Markus Ruettimann (Winterthur, CH)
- Giuseppe Lapadula (Basel, CH)
Cpc classification
A61K31/519
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
C07D475/04
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention is directed to a crystalline sodium salt of 5-methyl-(6S)-tetrahydrofolic acid wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to sodium is from 1:0.5 to 1:1.5 (in mol/mol) and/or hydrates and/or solvates thereof, as well as, a processes of obtaining the same.
Claims
1. A crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid of the following formula: ##STR00001## wherein the crystalline monosodium salt is characterized by a powder X-ray diffraction pattern comprising at least one characteristic peak (° 2θ) at 6.5°±0.2° 2θ, 10.2°±0.2° 2θ, 13.1°±0.2° 2 θ, 15.3°±0.2° 2θ, 17.9°±0.2° 2θ, 20.5°±0.2° 2θ, and 22.0°±0.2° 2θ, and further wherein the X-ray powder diffraction pattern is determined on a diffractometer using CuKα radiation.
2. The crystalline monosodium salt of claim 1, wherein the crystalline monosodium salt is further characterized by at least one additional characteristic peak (° 2θ) selected from the group consisting of 12.5°±0.2° 2θ, 14.8°±0.2° 2θ, 15.1°±0.2° 2θ, 16.1°±0.2° 2θ, 17.2°±0.2° 2θ, 20.4°±0.2° 2θ, 21.2°±0.2° 2θ, 22.5°±0.2° 2θ, and 27.2°±0.2° 2θ.
3. The crystalline monosodium salt of claim 1, wherein the crystalline monosodium salt is characterized by all of the following characteristic peaks (° 2θ) at 6.5°±0.2° 2θ, 10.2°±0.2° 2θ, 12.5°±0.2° 2θ, 13.1°±0.2° 2θ, 14.8°±0.2° 2θ, 15.1°±0.2° 2θ, 15.3°±0.2° 2θ, 16.1°±0.2° 2θ, 17.2°±0.2° 2θ, 17.9°±0.2° 2θ, 20.4°±0.2° 2θ, 20.5°±0.2° 2θ, 21.2°±0.2° 2θ, 22.0°±0.2° 2θ, 22.5°±0.2° 2θ, and 27.2°±0.2° 2θ.
4. The crystalline monosodium salt of claim 1, wherein the crystalline monosodium salt is further characterized by a powder X-ray diffraction pattern as shown in
5. A food additive comprising the crystalline monosodium salt of claim 1 and one or more acceptable excipients.
6. A vitamin composition comprising the crystalline monosodium salt of claim 1 and one or more acceptable excipients.
7. A pharmaceutical composition comprising the crystalline monosodium salt of claim 1 and one or more pharmaceutically acceptable excipients.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is a tablet, a capsule, an oral liquid preparation, a powder, a lyophilisate, a granule, a lozenge, an injectable solution, an infusible solution, a suspension, or a suppository.
9. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is formulated for oral, parenteral, intramuscular, intraspinal, intrathecal, periodontal, topical, or rectal administration.
10. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent.
11. A method for lowering homocysteine levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
12. A method for managing low central nervous system folate levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
13. A method for managing low peripheral nervous system folate levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
14. A method for managing low cerebrospinal fluid folate levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
15. A method for managing low plasma folate levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
16. A method for managing low red blood cell folate levels in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
17. A method for treating Alzheimer's disease in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
18. A method for treating anemia in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
19. A method for treating a cardiovascular disease in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
20. A method for treating a cognitive impairment in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
21. A method for treating depression in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
22. A method for treating a neural tube defect in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
23. A method for treating osteoporosis in a patient, wherein the method comprises administering to the patient in need thereof an effective amount of the crystalline monosodium salt of claim 1.
24. A process for synthesizing the crystalline monosodium salt of claim 1, wherein the process comprises the following steps: (a) providing 5-methyl-(6S)-tetrahydrofolic acid monohydrate, optionally dissolved in a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof; (b) adding sodium hydroxide to the composition provided in step (a), to provide a composition comprising 5-methyl-(6S)-tetrahydrofolic acid; (c) optionally adding a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof, to the composition provided in step (b); or (c) optionally adding a co-salt former selected from the group consisting of tert-butylamine, 2-(dimethylamino)ethanol, 1-(2-hydroxyethyl)pyrrolidine, 4-(2-hydroxyethyl)morpholine, and imidazole, or a mixture thereof, to the composition provided in step (b); or (c) optionally adding a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof, and a co-salt former selected from the group consisting of tert-butylamine, 2-(dimethylamino)ethanol, 1-(2-hydroxyethyl)pyrrolidine, 4-(2-hydroxyethyl)morpholine, and imidazole, or a mixture thereof, to the composition provided in step (b); or (c) adding the composition provided in step (b) to a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof; or (c) adding the composition provided in step (b) to a co-salt former selected from the group consisting of tert-butylamine, 2-(dimethylamino)ethanol, 1-(2-hydroxyethyl)pyrrolidine, 4-(2-hydroxyethyl)morpholine, and imidazole, or a mixture thereof; or (c) adding the composition provided in step (b) to a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof, and a co-salt former selected from the group consisting of tert-butylamine, 2-(dimethylamino)ethanol, 1-(2-hydroxyethyl)pyrrolidine, 4-(2-hydroxyethyl)morpholine, and imidazole, or a mixture thereof; (d) crystallizing by optionally adding a solvent selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, benzyl alcohol, acetonitrile, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and water, or a mixture thereof, or a mixture thereof, to the composition provided in step (c); and (e) isolating the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid of claim 1.
25. The process of claim 24, wherein the process further comprises adding seed crystals in step (a), step (b), step (c), or step (d), or a combination thereof.
26. The process of claim 24, wherein the solvent in step (a), step (c), or step (d), or a combination thereof, is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, and water, or a mixture thereof.
27. The process of claim 24, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid monohydrate to sodium hydroxide in the composition provided in step (b) is in the range of 1:0.5 to 1:1.5.
28. The process of claim 24, wherein the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to the co-salt former in the composition provided in step (c) is in the range of 1:0.5 to 1:3.
29. The process of claim 24, wherein the co-salt former in step (c) is selected from the group consisting of 2-(dimethylamino)ethanol, 4-(2-hydroxyethyl)morpholine, and imidazole, or a mixture thereof.
30. The process of claim 29, wherein the co-salt former in step (c) is 4-(2-hydroxyethyl)morpholine.
31. The process of claim 24, wherein the temperature in step (d) is at least 30° C.
32. A process for producing the food additive of claim 5, wherein the process comprises adding the crystalline monosodium salt of claim 1 to one or more acceptable excipients.
33. A process for producing the vitamin composition of claim 6, wherein the process comprises adding the crystalline monosodium salt of claim 1 to one or more acceptable excipients.
34. A process for producing the pharmaceutical composition of claim 7, wherein the process comprises adding the crystalline monosodium salt of claim 1 to one or more pharmaceutically acceptable excipients.
35. A crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid of the following formula: ##STR00002## wherein the crystalline monosodium salt is characterized by a powder X-ray diffraction pattern comprising all of the following characteristic peaks (° 2θ) at 6.5°±0.2° 2θ, 10.2°±0.2° 2θ, 13.1°±0.2° 2θ, 15.3°±0.2° 2θ, 17.9°±0.2° 2θ, 20.5°±0.2° 2θ, and 22.0°±0.2° 2θ, and further wherein the X-ray powder diffraction pattern is determined on a diffractometer using CuKα radiation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLE 1
Preparation of Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid with Imidazole as Co-Salt Former without Seeding
(11) To 467 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. 1.0 mL of sodium hydroxide standard solution 1.00 mol/L and 76 mg of imidazole were added. At ambient temperature, an essentially clear solution was obtained to which 7.0 mL of ethanol was added. The addition of ethanol led to a thick suspension at room temperature. The suspension was heated to 80° C. and stirred for two hours before the solid was separated by hot filtration using a fritted glass filter (porosity P4) and dried in air at room temperature. The filter cake was washed with 10 mL ethanol and let dry in air for about 30 minutes. 320 mg (63% yield, assay 5-methyltetrahydrofolic acid 89.38% w/w) of a fine, slightly yellow powder was obtained as the product and was characterized by powder X-ray diffraction (
(12) TABLE-US-00001 TABLE 1 Powder X-ray diffraction data for crystalline monosodium salt of Example 1 with 2-theta angles, d-spacing values in Ångstrom and qualitative intensity values as follows: vs = very strong, s = strong, m = medium, w = weak and vw = very weak. ° 2-theta d-spacings [Å] intensity (qualitative) 6.5 13.5 m 10.2 8.6 vs 12.5 7.1 vw 13.1 6.8 s 14.8 5.99 s 15.1 5.87 s 15.3 5.79 vs 15.8 5.61 w 16.1 5.50 s 17.2 5.15 s 17.9 4.94 vs 19.7 4.51 vw 20.0 4.44 w 20.4 4.35 s 20.5 4.32 vs 21.0 4.22 w 21.2 4.18 s 21.5 4.13 w 22.0 4.03 vs 22.5 3.95 vs 24.7 3.61 w 25.3 3.51 m 25.5 3.48 w 25.9 3.44 w 26.4 3.38 w 26.9 3.31 m 27.2 3.27 s 27.6 3.23 w 27.9 3.20 w 28.4 3.14 m 28.6 3.12 m 28.8 3.09 vw 29.3 3.04 vw 29.7 3.01 m 30.2 2.96 w 30.4 2.94 vw 30.9 2.89 w 31.2 2.86 w 31.9 2.81 m
(13) TABLE-US-00002 TABLE 2 Raman data for the crystalline monosodium salt of Example 1 with wavenumber in cm.sup.−1 and intensity values. It should be noted that the intensities vary with Laser power, sample amount and other factors. wavenumber (cm.sup.−1) intensity (arbitrary units) 3079 0.43 3036 0.49 2991 0.51 2962 0.82 2941 1.20 2927 0.90 2902 0.87 2852 0.62 1674 0.51 1608 7.98 1570 2.87 1527 1.06 1483 0.93 1466 0.99 1446 0.80 1415 0.81 1321 6.43 1302 2.22 1274 0.96 1243 0.55 1224 0.51 1193 0.97 1168 1.33 1059 0.35 1019 0.51 947 0.57 898 2.03 854 0.60 810 0.61 775 0.54 648 1.60 637 1.62 510 0.39 479 0.64 415 0.51 378 0.52 284 0.57 260 0.62 196 0.83
EXAMPLE 2
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid in Isopropanol with 4-(2-Hydroxyethyl)-Morpholine as Co-Salt Former and Seeding
(14) To 467 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. 1.0 mL of sodium hydroxide standard solution 1.00 mol/L and 140 μl of 4-(2-hydroxyethyl)-morpholine were added. At ambient temperature, an essentially clear solution was obtained to which 7.0 mL of isopropanol was added. The addition of isopropanol led to a thick suspension at room temperature. The suspension was heated to 80° C. and stirred for one hour then seed crystals of 5-methyl-(6S)-tetrahydrofolic acid monosodium salt according to example 1 were added and stirring at 80° C. was continued for one hour before the solid was separated by hot filtration using a fritted glass filter (porosity P4) and dried in air at room temperature. The filter cake was washed with 10 mL isopropanol and let dry in air at r.t. for about 30 minutes. 413 mg of a fine, slightly yellow powder was obtained as the product. Powder X-ray diffraction confirmed the identity of the monosodium salt.
EXAMPLE 3
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid with 4-(2-Hydroxyethyl)-Morpholine as Co-Salt Former and Seeding
(15) 4.694 g of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a 100 mL glass flask equipped with a magnetic stirrer bar. 10.00 mL of sodium hydroxide standard solution 1.00 mol/L and 1.470 mL of 4-(2-hydroxyethyl)-morpholine were added. After stirring at room temperature for 10 minutes a brown clear solution was formed. While stirring the solution at room temperature, 50 mL ethanol was added. A turbid solution was present after stirring for 6 minutes. The mixture was seeded with 56 mg of crystalline MTHF monosodium salt according to example 1 and after stirring for 5 minutes at room temperature a thick suspension formed, which was no longer stirrable using a magnetic stirrer bar. Thus the mixture was heated to 80° C. within about 6 minutes and a light brown suspension was formed which was easy to stir. The sample was seeded again with 36 mg of crystalline MTHF mono-Na salt. After stirring for 6 minutes at 80° C. a yellow suspension was present with some off-white solid material that adhered to the glass wall. After stirring the suspension at 80° C. for 75 minutes 20 mL of ethanol were added to the yellow suspension and stirring was continued for 70 minutes before the solid product was filtered off by hot filtration using a fritted glass filter (porosity P4, Ø 5 cm) and the filter cake was air dried for about 2 minutes. 16 mL of an ethanol-water mixture 7:1 (v/v) was added to the filter cake and the washing solution was pulled through the filter by vacuum suction. The wash step was repeated with another 16 mL of the same ethanol-water mixture. The filter cake was then air dried for 20 minutes (air was drawn through the fritted glass filter; 24° C., 41% relative humidity). After about 10 minutes the filter cake was broken into smaller pieces and lumps were slightly crushed using a spatula. After 20 minutes the solid material was transferred into a 40 mL glass bottle. 4.36 grams (about 86% yield, assay 5-methyltetrahydrofolic acid 89.94% w/w) of a fine, slightly yellow powder was obtained as the solid product and was characterized by powder X-ray diffraction, H-NMR spectroscopy and TG-FTIR. Light microscopy and PXRD confirmed the crystalline nature of the sample and H-NMR spectroscopy was in agreement with the chemical integrity of 5-methyl-(6S)-tetrahydrofolic acid and showed no significant amount of 4-(2-hydroxyethyl)-morpholine. Further investigation by TG-FTIR revealed a mass loss of about 1.6%, attributable to water. The sodium content found by ICP-OES was 4.59% which is in good agreement with a monosodium salt. HPLC showed a purity of 97.4%.
EXAMPLE 4
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid with 4-(2-Hydroxyethyl)-Morpholine as Co-Salt Former and Seeding
(16) The first step of the experiment was carried out in a separate vial in order to prevent seeds of crystalline MTHF free acid in the larger glass reactor.
(17) 9.3888 grams of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a 60 mL glass vessel equipped with a magnetic stirrer bar. 20.00 mL of sodium hydroxide standard solution 1.00 mol/L and 2.940 mL of 4-(2-hydroxyethyl)-morpholine were added. After stirring at room temperature for about 15 minutes a brown clear solution was formed with pH of about 6.9. This solution was transferred into a 350 mL glass reactor and stirred with a paddle stirrer. The gas phase was purged using a dry nitrogen flow and the mixture heated to 80° C. within about 20 minutes. At 80° C. a solution was still present and 20 mL of ethanol were added within about 6 minutes before 7 mg of crystalline MTHF monosodium salt according to example 1 was added as seed crystals. Within about 13 minutes an additional 50 mL of ethanol were added while stirring (about 340 rpm). During the addition of ethanol the solution was seeded several times using 5 mg to 7 mg of the same seed crystals but a turbid solution rather than a suspension was observed. Within about 85 minutes an additional 80 mL of ethanol (in total 150 mL) were added while stirring. During the addition of ethanol the seeding was performed several times each time using 3 mg to 9 mg of the same seeding crystals. However by visual observation, the system was judged as an emulsion with slightly brown material adhering to the glass wall of the reactor. The mixture was then stirred at 80° C. for 10 minutes and a small amount of the light brown material was scraped from the glass wall using a spatula. Over time, a weak suspension formed. While stirring some material that adhered to the glass wall was redispersed by scraping it from the glass wall using a spatula. After stirring at 500 rpm at 80° C. for about 2.5 hours a yellow suspension had formed that was easy to stir but still contained relatively small brown lumps. Hot filtration was carried out using a fritted glass filter (porosity P4, Ø 5 cm) and the filter cake was air dried for about 2 minutes. The glass reactor was washed twice with 30 mL of an ethanol-water mixture 7:1 (v/v) that was added to the filter cake and the wash solution was pulled through the filter by vacuum suction. The filter cake was then washed a third time using 30 mL using the same ethanol-water mixture. The filter cake was then air dried for 30 minutes (air was drawn through the fritted glass filter; 23° C., 59% relative humidity). After about 7 minutes the filter cake was broken into smaller pieces and lumps were slightly crushed using a spatula. After 30 minutes the solid product (8.28 grams) was transferred into a 60 mL glass container and a sample was drawn for analysis by PXRD, which confirmed the presence of crystalline MTHF mono-Na salt. The remaining solid material was vacuum dried at 80° C./about 10 mbar for 0.5 hour. The solid product was 8.05 g (yield about 78%, assay 5-methyltetrahydrofolic acid 89.12% w/w) of a fine, slightly yellow powder. Characterization by PXRD showed that drying did not lead to a change of the crystalline form. H-NMR spectroscopy confirmed the chemical integrity of 5-methyl-(6S)-tetrahydrofolic acid and showed no significant amount of 4-(2-hydroxyethyl)-morpholine. The sample was further investigated by TG-FTIR and HPLC. HPLC showed a purity of 96.5%.
EXAMPLE 5
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid in Ethanol with 2-Diethylaminoethanol as Co-Salt Former and Seeding
(18) To 467 mg of 5-methyl-(6S)-tetrahydrofolic acid monohydrate (assay 5-methyltetrahydrofolic acid 97.65% w/w) were weighed into a glass vial equipped with a magnetic stirrer bar. 1.0 mL of sodium hydroxide standard solution 1.00 M, 5.0 mL ethanol and 100 μl of 2-diethylaminoethanol were added. The mixture was heated to 80° C. under stirring and then seed crystals of 5-methyl-(6S)-tetrahydrofolic acid monosodium salt according to example 1 were added and stirring at 80° C. was continued for one hour before the solid was separated by hot filtration using a fritted glass filter (porosity P4) and dried in air at room temperature. 370 mg (55% yield, assay 5-methyltetrahydrofolic acid 68.2% w/w) of solid product was obtained and characterized by powder X-ray diffraction and HPLC. HPLC showed a purity of 93.7%.
EXAMPLE 6
Kinetic Solubility of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(19) Step 1: 23.8 mg of crystalline 5-methyl-(6S)-tetrahydrofolic acid monosodium salt are weighed into a 7 ml glass vial with screw cap. 2.38 ml of purified/de-ionized water (for instance water for chromatography) is added to the solid using an adjustable volumetric pipette. The mixture is vigorously agitated at room temperature and briefly sonicated and a clear slightly yellow solution is readily obtained. Thus the solubility is greater than 10 mg per 1 ml of water. The solution remains clear for more than 20 hours at r.t.
(20) Step 2: 62.3 mg of crystalline monosodium salt are weighed into a 7 ml glass vial with screw cap. 4.15 ml of purified/de-ionized water (for instance water for chromatography) is added to the solid using an adjustable volumetric pipette. The mixture is vigorously agitated at room temperature and briefly sonicated and a clear slightly yellow solution is readily obtained. Thus the solubility is greater than 15 mg per 1 ml of water. The solution remains clear for several hours at r.t.
(21) Step 3: 72.6 mg of crystalline MTHF monosodium salt are weighed into a 7 ml glass vial with screw cap. 2.9 ml of purified/de-ionized water (for instance water for chromatography) is added to the solid using an adjustable volumetric pipette. The mixture is vigorously agitated at room temperature and briefly sonicated and a clear slightly yellow solution is readily obtained. Thus the solubility is greater than 25 mg per 1 ml of water. The solution remains clear for about one hour at r.t.
REFERENCE EXAMPLE 7
Kinetic Solubility of the Calcium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(22) 27.9 mg of crystalline calcium salt of MTHF (containing about 11% of water, thus corresponding to a dry weight of about 25 mg) are weighed into a 4 ml glass vial with screw cap. 2.535 ml of purified/de-ionized water (for instance water for chromatography) is added to the solid using an adjustable volumetric pipette. The mixture is vigorously agitated at room temperature and briefly sonicated. No clear solution can be obtained and a fairly concentrated suspension persists; thus the solubility is considerable smaller than 10 mg per 1 ml of water.
EXAMPLE 8
Properties on Drying of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(23) A sample of crystalline monosodium salt according to at least one of the previous examples is investigated by thermogravimetry coupled with infrared spectroscopy (TG-FTIR). Thermogravimetric measurements were done with a sample mass of about 6 mg. The mass loss at 200° C. is about 1.3% which essentially corresponds to the water content of the crystalline monosodium salt after drying in air at room temperature. The same sample is now examined by dynamic vapor sorption analysis (DVS). About 20 mg of sample are placed put into an aluminum sample pan. Humidity change rates of 5% per hour were used. The applied measurement program is visualized in the figure (dashed trace) and described below. The presentation shows the effective water content with a correction based upon TG-FTIR result. 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 in the following way: (1) 2 hours at 50% relative humidity (2) 50.fwdarw.0% relative humidity (5%/hour); 10 hours at 0% relative humidity (3) 0.fwdarw.75% relative humidity (5%/hour); 5 hours at 75% relative humidity (4) 75.fwdarw.0% relative humidity (5%/hour); 10 hours at 0% relative humidity (5) 0.fwdarw.50% relative humidity (5%/hour); 2 hours at 50% relative humidity
(24) The result as shown in the
REFERENCE EXAMPLE 9
Drying of the Calcium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(25) A sample of crystalline calcium salt of MTHF is examined by Karl Fischer titration to evaluate the water content. The water content found is 12.4%. Thereafter the same sample is investigated by DVS according to the very same measurement program as for the monosodium salt of the example above. The results as shown in
EXAMPLE 10
Water Content of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid Under Changing Relative Humidity Conditions
(26) A sample of crystalline monosodium salt of MTHF according to at least one of the previous examples is investigated by thermogravimetry coupled with infrared spectroscopy (TG-FTIR). Thermogravimetric measurements were done with a sample mass of about 6 mg. The mass loss at 200° C. is about 1.3% which essentially corresponds to the water content of the crystalline monosodium salt after drying in air at room temperature. The same sample is now examined by dynamic vapor sorption analysis (DVS). About 20 mg of sample are placed put into an aluminum sample pan. Humidity change rates of 5% per hour were used. Where applicable, the applied measurement program is visualized in the figure (dashed trace) and described below. The presentation shows the effective water content with a correction based upon TG-FTIR result. The sample was placed on an aluminum or platinum holder on top of a microbalance and allowed to equilibrate at 50% RH before starting the pre-defined humidity program in the following way: (1) 2 hours at 50% relative humidity (2) 50.fwdarw.0% relative humidity (5%/hour); 10 hours at 0% relative humidity (3) 0.fwdarw.75% relative humidity (5%/hour); 5 hours at 75% relative humidity (4) 75.fwdarw.0% relative humidity (5%/hour); 10 hours at 0% relative humidity (5) 0.fwdarw.50% relative humidity (5%/hour); 2 hours at 50% relative humidity
(27) The result as shown in the
REFERENCE EXAMPLE 11
Water Content of the Calcium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid Under Changing Relative Humidity Conditions
(28) A sample of crystalline calcium salt of MTHF is subjected to the very same relative humidity program in a DVS instrument. The results as depicted in
EXAMPLE 12
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid with 2 Equivalents of 4-(2-Hydroxyethyl)-Morpholine as Co-Salt Former
(29) To a mixture of 40 g 5-methyl-(6S)-tetrahydrofolic acid (assay 5-methyltetrahydrofolic acid 95.4% w/w) and 86 g water, 3.46 g solid sodium hydroxide and 21.79 g 4-(2-hydroxyethyl) morpholine were added at room temperature under a nitrogen atmosphere while stirring. The mixture was added within 5.5 hours to a mixture of 472 g ethanol with 5% v/v 2-propanol and 0.4 g crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 75-78° C. under a nitrogen atmosphere while stirring. 3 g water were added and the mixture was stirred for an additional hour at 76° C. The solids were separated by filtration, washed three times with each 133 mL ethanol 5% 2-propanol/water 7:1 v/v and dried in vacuum at 40° C. for 16.5 hours to give 42.0 g of an off-white powder corresponding to 102% yield (assay 5-methyltetrahydrofolic acid 92.45% w/w). PXRD confirmed the crystalline nature of the sample and H-NMR spectroscopy was in agreement with the chemical integrity of 5-methyl-(6S)-tetrahydrofolic acid and showed no significant amount of 4-(2-hydroxyethyl)-morpholine. Further investigation by TGA (Thermogravimetric Analysis) revealed a mass loss of about 0.86%. The sodium content found by IC (Ion chromatography) was 4.94% which is in good agreement with a monosodium salt. HPLC showed a purity of 98.2%.
EXAMPLE 13
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid with a Purity of 98.6% by Recrystallization of Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid in the Presence of 1 Equivalent of 4-(2-Hydroxyethyl)-Morpholine
(30) 2 g crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid (assay 5-methyltetrahydrofolic acid 92.63% w/w) were added to a mixture of 10 g water and 0.51 g 4-(2-hydroxyethyl) morpholine under a nitrogen atmosphere. After stirring for 10 minutes at 25° C., the mixture was added to a mixture of 70 mL ethanol with 5% v/v 2-propanol and 0.05 g crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 72° C. within 2.5 hours under a nitrogen atmosphere while stirring. The mixture was stirred for additional 2.5 hours at 72° C. The solids were separated by filtration, washed with 20 mL ethanol 5% 2-propanol/water 7:1 v/v and dried in vacuum at 33° C. for 18 hours to give 1.75 g of an off-white powder corresponding to 87% yield (assay 5-methyltetrahydrofolic acid 92.5% w/w). PXRD confirmed the crystalline nature of the sample and H-NMR spectroscopy was in agreement with the chemical integrity of 5-methyl-(6S)-tetrahydrofolic acid and showed no significant amount of 4-(2-hydroxyethyl)-morpholine. Further investigation by TGA (Thermogravimetric Analysis) revealed a mass loss of about 1.48%. The sodium content found by IC (Ion chromatography) was 4.83% which is in good agreement with a monosodium salt. HPLC showed a purity of 98.6%.
EXAMPLE 14
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid Starting from a Crystalline 1:1:1 Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid, Sodium and 4-(2-Hydroxyethyl)-Morpholine
(31) 5 g of a 1:1:1 salt of 5-methyl-(6S)-tetrahydrofolic acid, sodium and 4-(2-hydroxyethyl)-morpholine (assay 5-methyltetrahydrofolic acid 70.60% w/w) were suspended in a mixture of 49.4 g ethanol 5% v/v 2-propanol and 12.5 g water under a nitrogen atmosphere. The mixture was stirred for 3 hours at 74° C. The solids were separated by filtration and washed with 17 mL of a mixture of ethanol/water 7:1 v/v. The solids were dried at 40° C. in vacuum to give 2.93 g of an off-white powder corresponding to 78% yield (assay 5-methyltetrahydrofolic acid 93.65% w/w). PXRD confirmed the crystalline nature of the sample and H-NMR spectroscopy was in agreement with the chemical integrity of 5-methyl-(6S)-tetrahydrofolic acid and showed no significant amount of 4-(2-hydroxyethyl)-morpholine. Further investigation by TGA (Thermogravimetric Analysis) revealed a mass loss of about 0.30%. The sodium content found by IC (Ion chromatography) was 4.56% which is in good agreement with a monosodium salt. HPLC showed a purity of 99.02%.
EXAMPLE 15
Preparation of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid in Larger Scale
(32) All operations were performed under a nitrogen atmosphere if nothing else is mentioned. Solvents in the reaction and crystallization vessel were condensed during heating.
(33) 9.6 kg of 4-(2-hydroxyethyl) morpholine were added to 101.7 kg of a mixture of water/ethanol (5% 2-propanol) 1:1 v/v in a reaction vessel at room temperature while stirring. Further 1.0 kg of water/ethanol (5% 2-propanol) 1:1 v/v were added followed by 28.8 kg 5-methyl-(6S)-tetrahydrofolic acid (assay 5-methyltetrahydrofolic acid 97.17% w/w) while stirring. Additional 2 kg of a mixture of water/ethanol (5% 2-propanol) 1:1 v/v were added. Then 8.7 kg of an aqueous solution of sodium hydroxide were added (assay sodium hydroxide 29.22% w/w) to the reaction vessel followed by 1 kg of a mixture of water/ethanol (5% 2-propanol) 1:1 v/v. The mixture was heated to 38° C. and the pH of the resulting solution was 7.56. The solution was transferred from the reaction vessel over a filter into a crystallization vessel. The transfer pipes and the filter were washed with 9.8 kg of a mixture of water/ethanol (5% 2-propanol) 1:1 v/v. The washings were added to the filtrate. The filtrate was heated to 75.1° C. while stirring and within 59 minutes 53.8 kg ethanol (5% 2-propanol) were added while maintaining the temperature between 75.1 and 80.4° C. The mixture was cooled to 64.8° C. and 0.29 kg of crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid were added for seeding. The mixture was again heated to 74.5° C. and 286.2 kg ethanol (5% 2-propanol) were added within 3 hours while maintaining the temperature between 70 and 80° C. The mixture was cooled to 71° C. and stirred for 13 hours and 37 minutes while keeping the temperature between 65 and 75° C. Then the mixture was cooled to 59.9° C. and the crystals were separated by centrifugation. The crystals on the centrifuge were washed with a mixture of 38.0 kg ethanol (5% 2-propanol) and 6.9 kg water via the crystallization vessel and directly via a CIP (cleaning in place) pipe with a mixture of 38.1 kg ethanol (5% 2-propanol) and 6.9 kg water followed by 43.8 kg ethanol (5% 2-propanol). After separation of the washing liquids by centrifugation, the crystals were transferred into a dryer and dried for 12 hours and 7 min. at 50° C. under vacuum (101 mbar) using a nitrogen flow of 500 L/min. A sample was taken to measure the content of residual solvents. The water content of the sample was 0.49% w/w and the content of ethanol was 0.04% w/w. No 2-propanol was detected. 22.98 kg of crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid (assay 5-methyltetrahydrofolic acid 97.9% w/w) were removed from the dryer, corresponding to an assay corrected yield of 75.9%.
EXAMPLE 16
Solubility of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(34) The solubility of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid in water was measured by determining the concentration of 5-methyl-(6S)-tetrahydrofolic acid in the supernatant liquid after having stirred a suspension of crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid in water for 30 minutes, always keeping an excess of solid material at the bottom of the flask.
(35) The solubility of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid in water at room temperature was 9.3 mg/ml (equivalent to 9.6 mg/ml of the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid).
(36) The solubility of the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid in water measured under the same conditions was 7.1 mg/ml.
REFERENCE EXAMPLE 17
Preparation of the Amorphous Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(37) 6.0 g of crystalline 5-methyl-(6S)-tetrahydrofolic acid monosodium salt were added at room temperature to 600 g water. Solids were removed by suction filtration. The filtrate was frozen in a bath with liquid nitrogen and lyophilized in vacuum at 0.07 mbar. After 2 days the vacuum was replaced by nitrogen gas. 6.0 g amorphous 5-methyl-(6S)-tetrahydrofolic acid monosodium salt were obtained with a purity of 95.2% area, water content 8.8% w/w, assay sodium 4.5% w/w.
EXAMPLE 18
Stability of the Crystalline Monosodium Salt of 5-Methyl-(6S)-Tetrahydrofolic Acid
(38) In order to compare the long-term stabilities of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid, the compound of the invention, to the long-term stability of the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid as prepared according to EP 1 044 975 B1, respective stability data has been generated at various temperatures and humidities.
(39) (a) Stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 25° C./60% rh
(40) Crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared according to literature procedures (EP 1 044 975 B1) and crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared as disclosed in Example 15, were stored at 25° C./60% rh. The content of 5-methyl-(6S)-tetrahydrofolic acid remaining in the samples was measured by HPLC at periodic intervals (% w/w). The results are shown in Table 3a and
(41) TABLE-US-00003 TABLE 3a Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 25° C./60% rh (% w/w) 5-methyl-(6S)-tetrahydrofolic acid (% w/w) 0 3 6 9 12 months months months months months crystalline 94.7 93.0 94.2 95.6 94.1 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 80.7 79.0 77.7 78.5 77.9 salt of 5-methyl- (6S)-tetrahydrofolic acid
(42) TABLE-US-00004 TABLE 3b Long-term stability of the crystalline monosodium salt of 5- methyl-(6S)-tetrahydrofolic acid at 25° C./60% rh (% rel.) 5-methyl-(6S)-tetrahydrofolic acid (% rel.) 0 3 6 9 12 months months months months months crystalline 100.0 98.3 99.5 101.0 99.4 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 100.0 97.9 96.3 97.3 96.5 salt of 5-methyl- (6S)-tetrahydrofolic acid
(43) TABLE-US-00005 TABLE 4 Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 25° C./60% rh (major degradation product [MeFox]) Pyrazino-s-triazine derivative of 4α-hydroxy-5- methyl-THF (MeFox) (% w/w) 0 3 6 9 12 months months months months months crystalline 0.06 0.04 0.09 0.11 0.05 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 0.20 0.84 0.90 0.85 0.86 salt of 5-methyl- (6S)-tetrahydrofolic acid
(44) (b) Stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 40° C./75% rh
(45) Crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared according to literature procedures (EP 1 044 975 B1) and crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared as disclosed in Example 15, were stored at 40° C./75% rh. The content of 5-methyl-(6S)-tetrahydrofolic acid remaining in the sample was measured by HPLC at periodic intervals (% w/w). The results are shown in Table 5a and
(46) TABLE-US-00006 TABLE 5a Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 40° C./75% rh (% w/w) 5-methyl-(6S)-tetrahydrofolic acid (% w/w) 0 3 6 9 12 months months months months months crystalline 94.7 92.7 (81.8).sup.1 94.8 92.9 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 80.7 78.5 78.0 78.8 77.8 salt of 5-methyl- (6S)-tetrahydrofolic acid .sup.1Most likely lab error
(47) TABLE-US-00007 TABLE 5b Long-term stability of the crystalline monosodium salt of 5- methyl-(6S)-tetrahydrofolic acid at 40° C./75% rh (% rel.) 5-methyl-(6S)-tetrahydrofolic acid (% rel.) 0 3 6 9 12 months months months months months crystalline 100.0 98.0 (86.4).sup.1 100.1 98.2 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 100.0 97.2 96.6 97.6 96.3 salt of 5-methyl- (6S)-tetrahydrofolic acid .sup.1Most likely lab error
(48) TABLE-US-00008 TABLE 6 Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 40° C./75% rh (major degradation product [MeFox]) Pyrazino-s-triazine derivative of 4α-hydroxy-5- methyl-THF (MeFox) (% w/w) 0 3 6 9 12 months months months months months crystalline 0.06 0.05 0.08 0.13 0.05 monosodium salt of 5-methyl-(6S)- tetrahydrofolic acid crystalline calcium 0.20 1.01 0.96 0.93 0.95 salt of 5-methyl- (6S)-tetrahydrofolic acid
(49) Tables 3 to 6 with the stability data of crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid as disclosed in in the present invention clearly shows that
(50) i) there is a remarkable difference in the stability of crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid compared to the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid and
(51) ii) the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid is much more stable over a long period of time than crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid.