SALT OF PHENYLGLYCINE METHYL ESTER

Abstract

The present invention relates to the hemi sulfuric acid salt of D-phenylglycine methyl ester, to a method for the preparation of said salt and to the use of said salt in the enzymatic synthesis of antibiotics and of D-phenylglycine methyl ester free base.

Claims

1. The hemi sulfuric acid salt of D-phenylglycine methyl ester.

2. The hemi sulfuric acid salt according to claim 1 having an XRD powder diffraction pattern comprising peaks at 6.1±0.2 degrees 2-theta, 12.1±0.2 degrees 2-theta, 18.8±0.2 degrees 2-theta and 24.1±0.2 degrees 2-theta.

3. The hemi sulfuric acid salt according to claim 2 further comprising peaks at 7.9±0.2 degrees 2-theta, 14.4±0.2 degrees 2-theta, 15.6±0.2 degrees 2-theta, 16.7±0.2 degrees 2-theta, 19.5±0.2 degrees 2-theta and 25.6±0.2 degrees 2-theta.

4. A method for the preparation of the hemi sulfuric acid salt of D-phenylglycine methyl ester comprising the steps of: (a) contacting a solution of D-phenylglycine methyl ester in an organic solvent with sulfuric acid; (b) isolating the hemi sulfuric acid salt of D-phenylglycine methyl ester from the mixture obtained in step (a), wherein the molar amount of sulfuric acid in step (a) is from 0.4 to 0.6 relative to the molar amount of D-phenylglycine methyl ester.

5. The method according to claim 4 wherein step (a) is followed by separation of the aqueous phase and step (b) is carried out on said aqueous phase.

6. The method according to claim 5 wherein said aqueous phase obtained after step (a) is subjected to crystallization.

7. The method according to claim 6 wherein said crystallization is carried out by lowering the temperature of said aqueous phase obtained after step (a).

8. The method according to claim 6 wherein said crystallization is carried out at a temperature of from −5 to 15° C.

9. The method according to claim 5 wherein the aqueous phase remaining after said isolating in step (b) is added to the mixture of step (a).

10. The method according to claim 4 wherein the solubility in water of said organic solvent is from 0% (w/w) to 25% (w/w) and the polarity index of said organic solvent is from 1 to 5.

11. The method according to claim 10 wherein said polarity index is from 2 to 3.

12. The method according to claim 10 wherein said solvent is chosen from the group consisting of butyl acetate, diethyl ether, ethyl acetate, methyl isobutyl ketone, methyl tert-butyl ether and mixtures thereof.

13. A method for preparing one or more of ampicillin, cefaclor or cephalexin comprising contacting the hemi sulfuric acid salt of D-phenylglycine methyl ester with 6-aminopenicillanic acid, 7-amino-3-chloro-3-cephem-4-carboxylate or 7-aminodeacetoxycephalosporanic acid, respectively in the presence of a penicillin acylase.

14. (canceled)

Description

LEGEND TO THE FIGURES

[0023] FIG. 1 is the XRD spectrum of the hemi sulfuric acid salt of D-phenylglycine methyl ester. X-axis: 2-theta value (deg). Y-axis: intensity (cps). The following distinct peaks can be discerned:

[0024]

TABLE-US-00001 Peak no. 2-Theta (deg) Flex width d-Value Intensity I/Io 1 6.102 0.107 144.744 24164 100 2 7.866 0.128 112.307 739 3 3 12.081 0.104 73.199 1445 6 4 14.428 0.122 61.340 1251 5 5 15.623 0.136 56.677 762 3 6 16.683 0.134 53.098 972 4 7 18.772 0.158 47.234 1367 6 8 19.459 0.131 45.580 967 4 9 24.138 0.138 36.841 2997 12 10 25.577 0.163 34.791 1219 5

EXAMPLES

General

X-Ray Powder Diffraction Analysis

[0025] A sample was loaded onto a closed sample holder with inner knife (to minimize background scattering) and cavity (diameter 2 cm). The loading was carried out in a fume hood without grinding, in order to minimize dust formation during the sample preparation. Samples were analyzed on an X-ray powder diffractometer D2 Phaser from Bruker. It uses a LynxEye detector with 1° opening angle, a 0.1 mm receiving slit and a nickel filter. The diffraction angle 2∂ ranges from 2° to 60°, with step (in 2θ)˜0.008° and the count time 4 s/step. The sample rotates at 15 rpm during the measurement (for good statistics) and the data are approximately background subtracted.

HPLC Analysis

[0026] Column: HPLC column Crownpak CR(-) (DAICEL), length 150 mm, diameter 4 mm, diameter of particles 5 μm. [0027] Eluent: Solution of HClO.sub.4, pH=2.0. Weigh 1.43 g HClO4 (70%, 1.43 g) was diluted with water for chromatography to 1000 ml and the pH of the solution was checked.
Chromatographic conditions: [0028] Eluent: HClO.sub.4, pH=2 [0029] Isocratic conditions [0030] Flow: 1.0 ml.Math.min.sup.−1 [0031] Injection volume: 20 μl [0032] Wavelength: 220 nm [0033] Temperature of column: room temperature, 20-25° C. [0034] Time of chromatogram: 30 min [0035] Retention times (approximately): [0036] L-phenylglycine: 2.7 min [0037] D-phenylglycine: 8.7 min [0038] L-phenylglycine methyl ester: 9.3 min [0039] D-phenylglycine methyl ester: 21.0 min

Preparation of an Aqueous Solution of D-Phenylglycine Methyl Ester (See Also WO 2008/110527, the Similar Procedure of Example 8 of U.S. Pat. No. 8,541,199, with Different Amounts Also Leads to the Same Product and was Used for Example 4)

[0040] D-phenylglycine (PG; 135 g) was suspended in methanol (252 mL) and concentrated sulfuric acid (98%, 107 g) was added. The mixture was kept at reflux for 2 hours at approximately 73° C. and concentrated at a reduced pressure using a vacuum pump. The pressure dropped from atmospheric to 20 mbar while at the same time the temperature of the reaction mixture increased from 40 to 80° C. Methanol (126 mL, 100 g) was added and the mixture was kept at reflux for 1 hour at approximately 81° C. and concentrated as described before. The procedure was repeated for another four times (addition of methanol, reflux and concentrating). Finally, methanol (126 mL) was added and the solution was refluxed for another hour and cooled to ambient temperature. Ammonia (15 mL) was dosed with constant rate in 35 min up to pH 2.3-2.4. Water (75 mL) was added and methanol was distilled off at reduced pressure and a temperature below 50° C. The pH of the final D-phenylglycine methyl ester (PGM) solution was 2.0 and the conversion was 99.0%.

Example 1

Preparation of Seed of (PGMH).SUB.2.SO.SUB.4

[0041] An aqueous solution of D-phenylglycine methyl ester, obtained as described in the General section (1800 g) was added to a mixture of methyl tert-butyl ether (900 ml) and water (25 ml) at 5-10° C. while the pH was maintained at 9.2 by addition of 8 M NaOH. The phases were separated. The aqueous phase was extracted with methyl tert-butyl ether (600 ml). Both organic phases were combined and added to water (5 mL) while maintaining the pH at 4.2 by addition of 48% (w/w) H.sub.2SO.sub.4. The phases were separated. A viscous, oily water phase (turbid) was obtained. Part of the mixture was evaporated under vacuum (2 mbar) at 20° C. until the weight did not decrease anymore. A viscous oil was obtained. Upon storage at 20°, in the course of days, crystals formed in the oil. Some of these crystals were used to seed the rest of the aqueous phase (in the meantime stored at 3° C.). Very slow crystallization at 3° C. was observed. The crystal suspension was filtered. The crystals were analyzed with HPLC. It turned out that the crystals were contaminated with D-phenylglycine. In the filtrate, crystals formed again upon standing overnight at room temperature. These crystals were isolated, and used as seed in subsequent experiments.

Example 2

Preparation of (PGMH).SUB.2.SO.SUB.4

[0042] An aqueous solution of D-phenylglycine methyl ester, obtained as described in the General section (1800 g) was added to a mixture of methyl tert-butyl ether (900 ml) and water (25 ml) at 5-10° C. while the pH was maintained at 9.2 by addition of 8 M NaOH. The phases were separated. The aqueous phase was extracted with methyl tert-butyl ether (600 ml). Both organic phases were combined. The organic phase was determined by HPLC to contain 350.4 g of D-phenylglycine methyl ester. The organic phase was added to water (5 mL) while maintaining the pH at 4.2 by addition of 48% (w/w) H.sub.2SO.sub.4. The consumption of 48% (w/w) H.sub.2SO.sub.4 was 201.7 g. The molar ratio of D-phenylglycine methyl ester (350.4 g, 2.1 mol) and H.sub.2SO.sub.4 added (201.7*.48=96.8 g, 1.0 mol) was 2:1. Phases were separated. A viscous, oily water phase (turbid) was obtained. Seed, obtained as described in Example 1 was added to the aqueous phase. Massive crystallization started, in the course of less than one minute the mixture was a solid cake of white crystals. The wet cake of crystals was dried in vacuum at 20° C. The assay of D-phenylglycine methyl ester in the crystals was 73% (w/w), theoretical assay of D-phenylglycine methyl ester in the hemi sulfuric acid salt of D-phenylglycine methyl ester is 100*2*165.2/(2*165.2+98)=77%.

Example 3

Solubility of (PGMH).SUB.2.SO.SUB.4 .in Water as a Function of Temperature

[0043] In the preparation of (PGMH).sub.2SO.sub.4 as described in Example 2 separation of the organic phase at pH=4.2 is done while (PGMH).sub.2SO.sub.4 is supersaturated. At some point in time, crystallization may start before the organic layer is separated from the aqueous phase. In order to design a process that will avoid crystallization of (PGMH).sub.2SO.sub.4 in the presence of organic solvent, and controlled crystallization after separation of the organic phase, solubility of (PGMH).sub.2SO.sub.4 as a function of temperature was investigated. The hemi sulfuric acid salt of D-phenylglycine methyl ester (1 g), obtained as described in Example 2 was mixed with water (2 g) at 20° C. and the solid material dissolved. Additional hemi sulfuric acid salt of D-phenylglycine methyl ester (1 g) was added and the mixture was agitated at 20° C. for 25 minutes. Not all solid was dissolved. An aliquot of approximately 0.5 mL of supernatant was filtered, and in the filtrate the concentration of hemi sulfuric acid salt of D-phenylglycine methyl ester was determined by HPLC. The rest of the mixture was stirred at 3° C. Water (2 mL) was added to allow mixing. Additional hemi sulfuric acid salt of D-phenylglycine methyl ester (0.5 g) was added and the mixture was agitated for 30 minutes. Not all solid was dissolved. An aliquot of approximately 0.5 mL of supernatant was filtered and in the filtrate the concentration of hemi sulfuric acid salt of D-phenylglycine methyl ester was determined by HPLC. The results of HPLC analysis are presented in Table 1.

TABLE-US-00002 TABLE 1 Solubility of hemi sulfuric acid salt of D-phenylglycine methyl ester in water as a function of temperature Hemi sulfuric T (° C.) acid salt of D-phenylglycine methyl ester (g)/kg of solution 20 478 3 268

[0044] The solubility at 20° C. should allow phase separation after mixing D-phenylglycine methyl ester in organic solvent plus aqueous H.sub.2SO.sub.4 at pH=4.2 at 20° C. Subsequent cooling to 3° C. of the aqueous phase will result in crystallization of about 478-268=210 g of hemi sulfuric acid salt of D-phenylglycine methyl ester per kg of mixture. After isolation of hemi sulfuric acid salt of D-phenylglycine methyl ester from the crystal suspension at 3° C., the mother liquor can be re-used for extraction of D-phenylglycine methyl ester in organic solvent with water/H.sub.2SO.sub.4/mother liquor.

Example 4

Preparation of Cephalexin Using (PGMH).SUB.2.SO.SUB.4 .vs PGM in Solution

[0045] 7-Aminodeacetoxycephalosporanic acid (7-ADCA, 55.4 g) was suspended in water (237 mL) and the temperature was controlled at 20° C. The mixture was stirred for 5 min while maintaining the pH at 7.0 by the addition of an aqueous solution of ammonia (25%). Immobilized enzyme (comprising mutant 1 as described in U.S. Pat. No. 8,541,199; 18.7 g) was added together with water (25 mL). Next, solid (PGMH).sub.2SO.sub.4 (61.5 g) was dosed at a constant rate in 200 min. whilst the pH was maintained at 7.0 by the addition of an aqueous solution of ammonia (25%) or with an aqueous solution of sulfuric acid (30%) once all (PGMH).sub.2SO.sub.4 was added. After 230 min., the pH was adjusted to 5.8 by addition of an aqueous solution of sulfuric acid (30%). During the course of the reaction samples were taken and analyzed by HPLC with the results as outlined in Table 2.

TABLE-US-00003 TABLE 2 Formation of cephalexin from 7-ADCA using solid (PGMH).sub.2SO.sub.4 Cepha- Conver- Time PG 7-ADCA PGM lexin sion (min) (%) (%) (%) (%) (%) Ratio S/H 120 0.34 3.5 0.62 12.74 69.2 0.805 16.3 150 0.43 2.65 0.75 15.63 78.4 0.913 15.8 180 0.44 1.83 0.63 17.87 85.8 0.970 17.7 201 0.53 0.67 0.28 19.68 94.8 1.035 16.2 230 0.58 0.5 0 20.03 96.1 1.025 15.0 235 0.59 0.45 0 20.42 96.6 1.030 15.1 Components are given in weight % Conversion: 100*moles cephalexin/(moles cephalexin + 7-ADCA) Ratio: (moles cephalexin + PGM + PG)/(moles cephalexin + 7-ADCA) S/H: Synthesis/Hydrolysis ratio, or moles cephalexin/moles PG

[0046] For comparative reasons the above cephalexin protocol was repeated however using PGM solution (as obtained in by Example 8 of U.S. Pat. No. 8,541,199; 100.7 g; assay PGM: 44%) instead of solid (PGMH).sub.2SO.sub.4. In addition the initial suspension of 7-ADCA was in 187 mL of water instead of 237 mL During the course of the reaction samples were taken and analyzed by HPLC with the results as outlined in Table 3.

TABLE-US-00004 TABLE 3 Formation of cephalexin from 7-ADCA using PGM in solution Cepha- Conver- Time PG 7-ADCA PGM lexin sion (min) (%) (%) (%) (%) (%) Ratio S/H 120 0.49 2.72 0.41 14.13 76.2 0.869 12.6 150 0.55 2.54 0.23 15.54 79.1 0.879 12.3 180 0.64 2.08 0.46 17.52 83.9 0.955 11.9 205 0.72 1.36 0.47 18.26 89.2 1.021 11.0 230 0.77 0.86 0.07 19.08 93.2 1.026 10.8 235 0.79 0.75 0.02 19.67 94.2 1.031 10.8 Legend: As in Table 2

[0047] Inspection of Tables 2 and 3 revealed that the use of solid (PGMH).sub.2SO.sub.4 resulted in significantly better results over the use of PGM in solution, in terms of maximum cephalexin formation, maximum conversion and overall S/H ratio.