Patent ID: 12221446

EXAMPLES

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.

TG-FTIR

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, N2atmosphere, heating rate 10 K/min).

DVS

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

Raman Spectroscopy

FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman or a Bruker RFS 100 FT-Raman system with a near infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. 64 scans with a resolution of 2 cm−1were accumulated in the range from 3500 to −50 cm−1; however, only data above 100 cm−1are evaluated due to filter cutoff effects. Nominal laser powers are typically 100 or 300 mW.

Example 1: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A without Seeding

A suspension of 2.00 grams of 5-methyl-(6S)-tetrahydrofolic acid (assay: 95.6% w/w) in 20.0 ml water was heated to 70° C. and 2.13 grams of L-isoleucine ethyl ester hydrochloride is added. The temperature in the suspension was 65° C. and sodium hydroxide was added in form of a 30% (w/w) concentrated aqueous solution. In total, 0.95 g of the 30% sodium hydroxide solution were added. Addition of sodium hydroxide leaded to an essentially clear solution that gradually changed into a concentrated suspension. The suspension was diluted with 20.0 ml of water and the heater was switched off to allow the mixture to cool to ambient temperature within about two hours. The reactor with the suspension was further cooled in an ice/water bath to about 10° C. within half an hour, then filtered with a fritted glass filter and washed with five ml of cold water. The solid product was dried in a vacuum dryer at 35° C. for about 20 hours and examined by powder X-ray diffraction and identified as 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A. The powder X-ray diffraction pattern of Form A is depicted inFIG.1and exhibits peaks at 2-theta angles as listed in Table 1. HPLC analysis showed that the purity is 98.65% area.

TABLE 12-theta angles, d-spacings and qualitativeintensities for Form A.angle °2Θd-spacing [Å]qualitative intensity5.815.34m6.912.81vs8.510.39w10.78.27w11.67.64w12.57.05m12.96.88m14.06.34vs14.95.96m15.35.80w15.85.62w16.25.47w17.55.07s17.94.94m18.44.81w18.94.69m19.24.62m19.54.54w19.94.45w21.04.23w21.54.14vw22.24.01m22.53.95w22.73.91w23.23.83w23.93.72w24.13.68w24.43.65m24.73.60w25.13.55w25.43.51m25.83.45m26.93.31vw27.73.22w28.23.16w28.53.13w28.93.09vw29.23.06w29.73.01vw29.92.98w30.52.93vwVs = 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 2: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A with Seeding

To 15 g of 5-methyl-(6S)-tetrahydrofolic acid (assay: 95.4% w/w, 6S-diastereoisomer: 97.74%) were added 225 g water under a nitrogen atmosphere. The pH was adjusted to 6.5 by addition of aqueous sodium hydroxide solution (30% w/w). The mixture was heated to 66° C. and further aqueous sodium hydroxide solution (30% w/w) was added to keep the pH at 6.5. A solution of 15.2 g L-isoleucine ethyl ester hydrochloride in 75 g water was added at 66° C. After seeding with 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A at 66° C. the mixture was stirred for 16 hours at 66° C. The mixture was cooled to 1° C. while the pH was adjusted to 5.7 by addition of aqueous 1 molar hydrochloric acid. After stirring for 1 hour at 1° C. the crystalline material was isolated by suction filtration and washed with 44 g water that was pre-cooled to 1° C. The material was dried at 36° C. in vacuum for 22 hours to give 14.6 g of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt corresponding to 75% of theoretical yield (assay corrected). (Analytical data of isolated product: purity: 98.9% area, assay: 73.95% w/w 5-methyl-(6S)-tetrahydrofolic acid corresponding to the 1:1 salt, loss on drying (residual water): 1.2% w/w, (6S)-diastereoisomer: 99.8%. TG-FTIR analysis showed that the obtained solid product is essentially free of water as the mass loss at 150° C. is not more than about 0.2%. H-NMR spectroscopic analysis shows that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-isoleucine ethyl ester is about 1:1. The powder X-ray diffraction pattern as shown inFIG.1corresponds to Form A.

Example 3: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid an L-isoleucine ethyl ester Salt Form A

1.92 g of 5-methyl-(6S)-tetrahydrofolic acid (4 mmol) were dissolved in 8.0 ml 1 N NaOH. The solution was heated to 80° C. 1.28 g L-isoleucine ethyl ester hydrochloride (˜6.5 mmol) were dissolved in 6.0 ml water and added to the solution with the 5-methyl-(6S)-tetrahydrofolic acid. The solution was cooled to 60° C. within about 15 minutes and seeded with about 10 to 20 mg of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A obtained according to Example 1. A test of the pH typically showed a pH of 7, then 2.0 ml of 1 N HCl were added dropwise. A suspension formed and the mixture was cooled to 5° C. at a rate of 15 degrees C. per hour. After about 30 minutes stirring at 5° C., the suspension was filtered and the solid product dried 24 hours under vacuum at 40° C. Powder X-ray diffraction of the sample showed that 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A was obtained.

Example 4: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form B

To a mixture of 6.0 ml water and 452 mg of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A were added 6.0 ml of a 0.5 M solution of L-isoleucine ethyl ester hydrochloride in water and 2.0 ml ethanol at r.t. The pH was adjusted to pH˜6-7 by addition of 0.250 ml 1 N NaOH and then the mixture was heated to 75° C. The temperature of the slightly yellow suspension was kept at 75° C. for 30 minutes. Then the mixture was allowed to cool to 25° C. within about three hours. Stirring at 25° C. was continued during about 4 hours and a solid sample, termed PP555-P40a, was recovered by centrifugal filtration and examined by PXRD without drying. The solid product was identified as 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form B.

TABLE 22-theta angles, d-spacings and qualitativeintensities for Form B.angle °2Θd-spacing [Å]qualitative intensity5.316.6m7.012.6vs8.410.5m10.38.6w13.06.8m13.26.7m14.06.3vs14.56.1w14.76.0s15.35.80w15.95.56m16.55.36w16.95.24w17.45.09m17.75.00s18.14.91s18.74.75m19.94.46w20.24.40w20.54.34w20.74.28m21.24.19s21.74.09w22.33.98m22.93.88m23.23.84w23.33.81w23.73.75w24.03.70w24.53.64s24.93.57m25.43.51m25.83.46m26.23.40w26.73.34w27.23.28w27.43.25w27.93.20w28.13.17w28.93.08w29.33.04w29.73.01wVs = 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 5: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form C

To 81 mg of amorphous 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt, produced by freeze drying of a solution in water-dioxane 4:1, were added 1.0 ml ethanol-water 2:1 (volume ratio) and the mixture was stirred at r.t. for two days. Then the suspension was filtered and the solid submitted to powder X-ray diffraction. Powder X-ray diffraction showed that a crystalline form, designated as Form C was obtained.1H-NMR spectroscopic analysis showed that the molar ratio of 5-methyl-(6S)-tetrahydrofolic acid to L-isoleucine ethyl ester was about 2.8:1

TABLE 32-theta angles, d-spacings and qualitativeintensities for Form C.angle °2Θd-spacing [Å]qualitative intensity5.316.6m7.012.6vs8.410.5m8.810.1s10.38.6s10.78.3w11.27.9s12.27.2m13.06.8s13.36.7m13.66.5m14.06.3s14.76.00m15.15.88s15.95.57s16.85.28s17.45.10s17.65.02vs18.14.91m18.74.75m19.04.68w19.54.54m20.24.39vs20.54.34m20.84.27s21.24.19m21.44.14s22.33.98m22.63.94m22.93.87m23.43.80vs23.73.74w24.13.69w24.53.63s25.03.56m25.43.51m25.83.45m26.23.40m26.73.34m26.93.31s27.33.26m28.03.18w28.53.13w29.03.08w29.23.05m30.42.93w30.92.90mVs = 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 6: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Out of In Situ Formed 5-Methyl-(6S)-tetrahydrofolic acid from (6S)-tetrahydrofolic acid benzenesulfonate

To a mixture of 20 g of [6S]-tetrahydrofolic acid benzenesulfonate (prepared according to EP 0 495 204, assay tetrahydrofolic acid: 72.2% w/w, (6S)-diastereoisomer: 95.9%) and 50 g water were added at 20-30° C. 9.9 mL of an aqueous solution of sodium hydroxide (30% w/w sodium hydroxide) while stirring. Then 0.83 mL of an 1 molar aqueous solution of sodium hydroxide were added and the mixture was cooled to 0-5° C. At 0-5° C. 3.13 mL of an aqueous solution of formaldehyde (concentration: 36.9% w/w) were added and after stirring for 1 hour 0.31 mL of an aqueous solution of sodium hydroxide (30% w/w sodium hydroxide) were added followed by a mixture of 14 g water, 1.7 g of an aqueous solution of sodium hydroxide (30% w/w sodium hydroxide) and 3.07 g sodium borohydride. The mixture was heated to about 63° C. and stirred for 90 minutes. After cooling to ambient temperature 7.9 mL of aqueous hydrochloric acid (37% w/w) were added followed by addition of 4.1 mL of an aqueous solution of sodium hydroxide (30% w/w sodium hydroxide). A small amount of sodium tetraborate was added and the mixture was cooled to 0-5° C. and stirred for about 22 hours. The solids were removed by filtration (suction) and washed with 5 g of water. To the filtrate was added a mixture of 15.86 g of L-isoleucine ethyl ester hydrochloride and 30 g water at ambient temperature while stirring. The mixture was heated to about 66° C. and a small amount of crystalline 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A was added for seeding. At about 66° C. 27.1 mL of an 1 molar aqueous solution of hydrochloric acid were added. A suspension was formed. Within about 90 minutes the mixture was cooled to about 20° C. while further 33.2 mL of an 1 molar aqueous solution of hydrochloric acid were added. The crystallized product was isolated by filtration (suction) and washed with 84 mL of water that was pre-cooled in an ice bath. The product was dried for about 60 hours at room temperature in vacuum (10 mbar) to give 17.2 grams of crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A (assay 5-methyl-(6S)-tetrahydrofolic acid: 74.0% w/w, purity: 98.6% area, (6S)-diastereoisomer: 99.5%) corresponding to 88.1% assay corrected chemical yield.

Example 7: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester

To 1670 g of water were added 250 g of 5-methyl-(6S)-tetrahydrofolic acid (assay: 96.2% w/w) and 256.1 g L-isoleucine ethyl ester hydrochloride at ambient temperature while stirring. 500 g water were added and the pH was adjusted to pH=7.3 by addition of 134.6 mL of an aqueous sodium hydroxide solution (30% w/w sodium hydroxide). The mixture was heated to approximately 66° C. while stirring and a small amount of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester salt Form A was added for seeding. At 66° C. 385 mL of an aqueous 1 molar solution of hydrochloric acid was added within about 30 minutes. Within about 90 minutes the mixture was cooled to about 20° C. while further 365 mL of an aqueous 1 molar solution of hydrochloric acid were added. The crystallized product was isolated by filtration (suction) and washed with 733 g of water that was pre-cooled in an ice bath. The product was dried for about 60 hours at about 40° C. in vacuum (10 mbar) to give 300.8 grams of crystalline salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A (assay 5-methyl-(6S)-tetrahydrofolic acid: 74.3% w/w, purity: 98.7% area) corresponding to 92.9% assay corrected chemical yield.

Example 8: Preparation of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester

To 250.5 kg of water in a reaction vessel were added 31.0 kg of 5-methyl-(6S)-tetrahydrofolic acid (assay: 96.8% w/w, 6S-diastereoisomer: 98.3%) and 32.0 kg L-isoleucine ethyl ester hydrochloride at ambient temperature while stirring. 16.5 kg water were added and the pH was adjusted to pH=7.3 by addition of 18.6 kg aqueous sodium hydroxide solution (30% w/w sodium hydroxide). The mixture was heated to approximately 66° C. while stirring and solids were removed by filtration. The filter was washed with 20.3 kg water via the reaction vessel. The filtrate and washing were combined in a crystallization vessel and heated to approximately 65° C. 37.5 grams of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A were added for seeding. 64.5 kg of aqueous 1 M hydrochloric acid were added at approximately 65° C. within 30 minutes. The mixture was cooled to 20° C. within 90 minutes and the pH was adjusted to pH=5.4 by addition of 1.5 kg of aqueous 1 M hydrochloric acid. The mixture was stirred for 1 hour at approximately 20° C. and the crystallized product was isolated by centrifugation. The product was washed with 45 kg of water that was pre-cooled to approximately 3° C. The product was then dried for 17 hours at 50° C. in vacuum to give 23.63 kg 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester Form A (assay 5-methyltetrahydrofolic acid 77.2% w/w, 6S-diastereoisomer: 99.5%) corresponding to an assay corrected yield of 60.8%. The yield of this example is not representative since part of the crystallized product was lost during centrifugation due to a technical defect.

Example 9: Hygroscopicity and Water Content (DVS Experiments)

The water content of a sample of 5-methyl-(6S)-tetrahydrofolic acid calcium salt was measured and the water content was found to be 12.4%. TG-FTIR analysis of a sample of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A prepared according to example 1 or 2 showed that the sample is essentially free of water.

A sample of 5-methyl-(6S)-tetrahydrofolic acid calcium salt and a sample of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A prepared according to example 1 or 2 (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 with an SPS11-100n “Sorptions Prüfsystem” from ProUmid (formerly “Projekt Messtechnik”), August-Nagel-Str. 23, 89079 Ulm (Germany). Measurements were conducted as follows: 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 programs:(1) five hours at 50% relative humidity (RH) then(2) scan to 50→0% RH at a rate of 5% per hour(3) maintain constant RH at 0% for five hours(4) raise RH to 75% at a rate of 5% per hour(5) maintain constant RH at 75% for five hours(6) scan to 50% RH at a rate of 5% per hour
Comparing the result for 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A with the result for the calcium salt shows that the water content of 5-methyl-(6S)-tetrahydrofolic acid L-isoleucine ethyl ester salt Form A changes less than 0.8% within the tested range while the water content for the calcium salt of 5-methyl-(6S)-tetrahydrofolic acid changes by more than 6%. The results are visually illustrated inFIG.5.

Example 10: Stability of the Salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine Ethyl Ester

In order to compare the long-term stabilities of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester, the compounds 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.

(a) Stability of the Salt of 5-methyl-(6S)-Tetrahydrofolic acid and L-isoleucine Ethyl Ester at 25° C./60% Rh

Crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared according to literature procedures (EP 1 044 975 B1) and the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A 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. The results are shown in Table 4a andFIG.6. The content of 5-methyl-(6S)-tetrahydrofolic acid remaining was also compared to the initial value at the time of preparation (% rel.). The results are shown in Tables 4b. Additionally the content of the pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-THF (MeFox), a major degradation product, was measured by HPLC at periodic intervals and disclosed as absolute values (% w/w). The results are shown in Table 5 andFIG.7.

TABLE 4aLong-term stability of the salt of 5-methyl-(6S)-tetrahydrofolicacid and L-isoleucine ethyl ester at 25° C./60% rh (% w/w)5-methyl-(6S)-tetrahydrofolic acid (% w/w)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-75.172.873.874.374.1(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium82.482.580.880.6n/asalt of 5-methyl-(6S)-tetrahydrofolicacid

TABLE 4bLong-term stability of the salt of 5-methyl-(6S)-tetrahydrofolicacid and L-isoleucine ethyl ester at 25° C./60% rh (% rel.)5-methyl-(6S)-tetrahydrofolic acid (% rel.)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-100.097.098.398.998.7(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium100.0100.198.197.9n/asalt of 5-methyl-(6S)-tetrahydrofolicacid

TABLE 5Long-term stability of the salt of 5-methyl-(6S)-tetrahydrofolicacid and L-isoleucine ethyl ester at 25° C./60% rh (majordegradation product [MeFox])Pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-THF (MeFox) (% w/w)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-0.010.030.020.030.03(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium0.200.590.710.68n/asalt of 5-methyl-(6S)-tetrahydrofolicacid
(b) Stability of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine Ethyl Ester at 40° C./75% rh

Crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid, prepared according to literature procedures (EP 1 044 975 B1) and the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester Form A 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. The results are shown in Table 6a andFIG.8. The content of 5-methyl-(6S)-tetrahydrofolic acid remaining was also compared to the initial value at the time of preparation (% rel.). The results are shown in Tables 6b. Additionally the content of the pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-THF (MeFox), a major degradation product, was measured by HPLC at periodic intervals and disclosed as absolute values (% w/w). The results are shown in Table 7 andFIG.9.

TABLE 6aLong-term stability of the salt of 5-methyl-(6S)-tetrahydrofolicacid and L-isoleucine ethyl ester at 40° C./75% rh (% w/w)5-methyl-(6S)-tetrahydrofolic acid (% w/w)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-75.172.773.473.074.7(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium82.481.380.581.2n/asalt of 5-methyl-(6S)-tetrahydrofolicacid

TABLE 6bLong-term stability of the salt of 5-methyl-(6S)-tetrahydrofolicacid and L-isoleucine ethyl ester at 40° C./75% rh (% rel.)5-methyl-(6S)-tetrahydrofolic acid (% rel.)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-100.096.897.897.299.5(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium100.098.797.698.5n/asalt of 5-methyl-(6S)-tetrahydrofolicacid

TABLE 7Long-term stability of the crystalline monosodium salt of 5-methyl-(6S)-tetrahydrofolic acid at 40° C./75% rh (majordegradation product [MeFox])Pyrazino-s-triazine derivative of 4α-hydroxy-5-methyl-THF (MeFox) (% abs.)036912monthsmonthsmonthsmonthsmonthssalt of 5-methyl-0.010.030.030.040.06(6S)-tetrahydrofolicacid andL-isoleucine ethylestercrystalline calcium0.200.720.800.68n/asalt of 5-methyl-(6S)-tetrahydrofolicacid

Tables 4 to 7 with the stability data of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester as disclosed in in the present invention clearly are showing thati) there is a remarkable difference in the stability of the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester compared to the crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid andii) the salt of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester is much more stable over a long period of time than crystalline calcium salt of 5-methyl-(6S)-tetrahydrofolic acid.

Example 11: Isomeric Enrichment of the Salts of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine Ethyl Ester

When preparing salts of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester starting with 5-methyl-(6S)-tetrahydrofolic acid the following isomeric enrichment can be obtained (measured by HPLC)

(6S) content of the(6S) content of theresulting salt ofstarting material5-methyl-(6S)-(5-methyl-(6S)-tetrahydrofolic acidExperimenttetrahydrofolic acid)and L-isoleucineno[%]ethyl ester [%]198.099.6298.099.7398.099.7498.099.6598.099.6698.099.6798.099.7

When preparing salts of 5-methyl-(6S)-tetrahydrofolic acid and L-isoleucine ethyl ester starting with in situ prepared 5-methyl-(6S)-tetrahydrofolic acid benzenesulfonate the following isomeric enrichment can be obtained (measured by HPLC)

(6S) content of(6S) content of thethe startingresulting salt ofmaterial ((6S)-5-methyl-(6S)-tetrahydrofolic acidtetrahydrofolic acidExperimentbenzenesulfonate)and L-isoleucineno[%]ethyl ester [%]895.999.5