Salt of Dihydrophenylglycine Methyl Ester

Abstract

The present invention relates to the hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate, also trivially referred to as the hemi sulfuric acid salt of D-dihydrophenylglycine methyl ester, to a method for the preparation of said salt and to the use of said salt in the enzymatic synthesis of antibiotics.

Claims

1. The hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate.

2. The hemi sulfuric acid salt according to claim 1 having an XRD powder diffraction pattern comprising peaks at 5.90.2 degrees 2-theta, 11.80.2 degrees 2-theta, 19.20.2 degrees 2-theta and 23.80.2 degrees 2-theta.

3. The hemi sulfuric acid salt according to claim 2 further comprising peaks at 16.40.2 degrees 2-theta, 22.00.2 degrees 2-theta and 25.30.2 degrees 2-theta.

4. An aqueous solution comprising the hemi sulfuric acid salt according to claim 1.

5. Method for the preparation of the hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate comprising the steps of: (a) contacting methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate free base with sulfuric acid; (b) isolating the hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate from the mixture obtained in step (a), characterized in that in step (a) the molar amount of sulfuric acid is from 0.4 to 0.6 relative to the molar amount of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate.

6. Method according to claim 5 wherein said isolating in step (b) is achieved by centrifugation, filtration or sedimentation of crystalline hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate.

7. Method according to claim 5 wherein step (b) is preceded by lowering of the temperature to 5 C. to 15 C.

8. A method of preparing epicillin, cephradine or cefroxadine comprising contacting said hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate with 6-aminopenicillanic acid, 7-aminodeacetoxycephalosporanic acid or 7-amino-3-methoxy-3-cephem-4-carboxylic acid, respectively, in the presence of a penicillin acylase.

Description

LEGEND TO THE FIGURES

[0028] FIG. 1 is the XRD spectrum of the hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate, unambiguously showing the product to be in the crystalline state. X-axis: 2-theta value (deg). Y-axis: intensity (cps). The following distinct peaks can be discerned:

TABLE-US-00001 Intensity Peak no. 2-Theta (deg) d-Value (Angstrom) (Count) I/Io 1 5.89 15.00 210356 100 2 11.80 7.49 17012 8 3 16.41 5.40 5463 3 4 19.16 4.63 12259 6 5 21.95 4.05 8293 4 6 23.77 3.74 36247 17 7 25.29 3.52 5477 3

EXAMPLES

General

X-Ray Powder Diffraction Analysis

[0029] A sample was loaded onto a sample holder in a fume hood without grinding. The sample was 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 theta ranges from 5, to 40, with step (in 20) 0.008, and the count time 1 s/step. The sample rotates at 15 rpm during the measurement (for good statistics) io and the data were approximately background subtracted.

HPLC Analysis

[0030] Equipment: high pressure liquid chromatograph Hewlett Packard model 1100 [0031] Column:Inertsil ODS 15 cm4.6 mm, 5 m [0032] Mobile phase, solution pH=3.0: Phosphoric acid 85% (4.6 mL) was dissolved in water (1800 mL). The pH was adjusted to 3.0 using 5M NaOH and the final volume was adjusted to 2 L using water. A gradient with methanol was used (99.5% of the above solution with pH 3.0 and 5.0% of methanol). [0033] Sample preparation and analysis: approximately 150 mg of sample was weighed and diluted to 100 mL with phosphate buffer pH=5.0 (buffer: KH.sub.2PO.sub.4 (5.44 g) was diluted to 2 L with water and the pH was adjusted to 5.0 using 1M KOH or H.sub.3PO.sub.4).

Chromatographic Conditions:

[0034] Flow: 1 mL.min.sup.1 [0035] Injection volume: 25 L [0036] Wavelength: 220 nm [0037] Temperature of column: room temperature, 25 C. [0038] Retention times (approximately):

[0039] D-phenylglycine methyl ester (PGM): 10.0 min

[0040] Unknown 1: 10.9 min

[0041] Unknown 2: 11.9 min

[0042] methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate (DHPGM): 12.4 min

[0043] Unknown 3: 13.2 min

[0044] tetrahydro-D-phenylglycine methyl ester (THPGM): 17.5 min

Preparation of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate solution (DHPGM)

[0045] Under nitrogen atmosphere (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetic acid (D-dihydrophenylglycine, DHPG; about 1250-1500 kg) was suspended in methanol, at a ratio of 1 kg/1.1-2.0 L, while cooling down. The suspension was mixed with a mixture of methanol (about 0.7 L/kg DHPG) and 98% sulfuric acid (about 0.4 L/kg DHPG), while keeping temperature below 70 C. Subsequently, the mixture was heated to reflux and maintained for about 1-3 hours, then cooled concentrated under reduced pressure, until the temperature is less than 60 C. After cooling, methanol (about 0.3-1.5 L/kg DHPG) was added, and the mixture was heated to reflux and then distilled, as described above. This addition-reflux-distillation profile was repeated until a conversion of not less than 97% was reached. The mixture was then cooled. Ammonia (25%) was dosed until a pH of 1.7-3.4, maintaining temperature. Water was added (about 1 L/kg DHPG) and the solution was distilled under vacuum to remove methanol. Finally, the mixture comprising methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate (34.64%, pH=2) was cooled and stored for further use.

Preparation of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate free base (DHPGM free base; see also WO 2008/110527)

[0046] An aqueous solution of Na.sub.2SO.sub.4 (25%) was kept at 31 C. for later use. An aqueous solution of 2 M NaOH/5.3 M NaCl was pre-cooled in ice.

[0047] A vessel was pre-charged with a 5.3 M NaCl solution, sufficient to make contact with the agitator. The vessel was cooled in ice. The mixture (1002.5 g) comprising methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate obtained above was added drop wise into the vessel with co-current addition of 2 M NaOH/5.3 M NaCl, to maintain the pH at 9.2 while keeping the temperature <5 C. After all methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate was added, the mixture was heated to 25 C. and transferred to a separation funnel after which the aqueous phase was removed. The remaining organic phase was washed with 25% Na.sub.2SO.sub.4 twice (100 g and 96 g). The weight of the organic phase, being the free base of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate (DHPGM free base) was 296.0 g.

Example 1

Preparation of the hemi sulfuric acid salt of methyl (R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetate ((DHPGMH).SUB.2.SO.SUB.4.)

[0048] A vessel with an agitator, cooled in an ice-bath, was pre-charged with DHPGM free base obtained as described in the General section (50 g). Pre-cooled H.sub.2SO.sub.4 (20% w/w) was added into the vessel with agitation until the pH was 4.2. After precipitates were observed, agitation was continued for another 30 min. The remainder of the DHPGM free base (246 g) was added to the vessel and co-currently also H.sub.2SO.sub.4 (20% w/w) so as to maintain the pH at 4.2 while keeping the temperature <5 C. Afterwards, agitation was maintained at 4 C. for 60 min. The crystals were isolated by filtration and washed with water. The wet cake (crop 1) thus obtained was dried under vacuum at 30 C. overnight to give 72 g of (DHPGMH).sub.2SO.sub.4 with an assay of 89.7%. The mother liquor was stored at 4 C. overnight after which additional crystals formed. The crystals were isolated by filtration and washed with water. The wet cake (crop 2) thus obtained was dried under vacuum at 30 C. overnight to give 41 g of (DHPGMH).sub.2SO.sub.4 with an assay of 90.6%.

[0049] Based on the HPLC peak areas, the ratios of the impurities to DHPGM (as 100%) in the different phases along the preparation of the (DHPGMH).sub.2SO.sub.4 salt are as follows.

TABLE-US-00002 DHPGM Unknown 1 Unknown 2 Unknown 3 PGM THPGM % based on HPLC peak area Starting Material 100 0.7 0.1 0.3 8 3 DHME free base 100 0.7 0.1 0.3 8 3 Aqueous phase 100 0.8 0.1 0.4 8 3 Mother liquor 100 0.8 0.1 0.4 8 4 Wet cake 100 0.2 0.0 0.2 7 2 Crystals crop1 100 0.2 0.1 0.2 9 2 Crystals crop2 100 0.2 0.0 0.2 8 2

[0050] It was observed that, comparing with starting material, there were no extra impurities formed (DHPGM degradation) during the preparation of DHPGM free base, even with a large pH shift from 2 to 9. Unknown 1 was significantly reduced by the crystallization procedure, Unknown 3 and THPGM were slightly reduced.

[0051] In a separate analogous experiment, 16 g of (DHPGMH).sub.2SO.sub.4 with an assay of 93% was prepared.

Example 2

Preparation of cephradine using (DHPGMH).SUB.2.SO.SUB.4 .vs DHPGM in solution

[0052] 7-Aminodeacetoxycephalosporanic acid (7-ADCA, 50.0 g) was suspended in water (153 g) and the temperature was controlled at 20 C. The mixture was stirred for 5 min while maintaining the pH at 6.9 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; 55 g) was added together with water (60.5 g). Next, solid (DHPGMH)2504 (54.9 g) was dosed is at a constant rate in 150-180 min. whilst the pH was maintained at 6.9 by the addition of an aqueous solution of ammonia (25%) or with an aqueous solution of sulfuric acid (30%) once all (DHPGMH)2504 was added. After 210-240 min., the conversion as >93.5%, the suspension was cooled to 5 C. in 20 min. while maintaining the pH at 6.9. Subsequently the pH was lowered to 6.0 with sulfuric acid (30%). During the course of the reaction samples were taken and analyzed by HPLC with the results as outlined in Table 1.

TABLE-US-00003 TABLE 1 Formation of cephradine from 7-ADCA using solid (DHPGMH).sub.2SO.sub.4 Time DHPGM 7-ADCA DHPG Cephradine Cephalexin PG Conversion (min) (%) (%) (%) (%) (%) (%) (%) Ratio S/H 90 0.30 3.16 0.13 7.98 0.21 0.02 69.9 0.686 26.9 120 0.31 2.44 0.18 10.32 0.18 0.03 76.5 0.803 25.1 150 0.41 1.86 0.22 13.28 0.24 0.04 82.0 0.904 26.5 180 0.48 0.81 0.27 16.14 0.31 0.04 92.1 1.022 26.2 210 0.11 0.47 0.34 15.97 0.27 0.06 95.4 1.022 20.6 270 0.12 0.63 0.41 17.40 0.31 0.07 93.9 1.017 18.6 (5 C.) pH 6.0 0.11 0.61 0.36 15.10 0.23 0.08 94.1 1.015 18.4 Components are given in weight % Conversion: 100*moles cephradine/(moles cephradine + 7-ADCA) Ratio: (moles cephradine + DHPGM + DHPG)/(moles cephradine + 7-ADCA) S/H: Synthesis/Hydrolysis ratio, or moles cephradine/moles DHPG

[0053] For comparative reasons the above cephradine protocol was repeated however using DHPGM solution (34.64%, pH=2, as obtained in the above General section) instead of solid (PGMH).sub.2SO.sub.4, and less water as outlined in WO 2005/003367 to compensate for the water present in the DHPGM solution. During the course of the reaction samples were taken and analyzed by HPLC with the results as outlined in Table 2.

TABLE-US-00004 TABLE 2 Formation of cephradine from 7-ADCA using DHPGM in solution Time DHPGM 7-ADCA DHPG Cephradine Cephalexin PG Conversion (min) (%) (%) (%) (%) (%) (%) (%) Ratio S/H 90 0.78 6.68 0.13 9.19 0.17 0.03 38.8 0.561 31.0 120 0.33 5.09 0.16 11.19 0.32 0.02 53.1 0.637 30.7 150 0.80 3.32 0.22 13.10 0.40 0.03 69.2 0.832 26.1 181 0.47 1.24 0.29 15.29 0.44 0.03 88.5 0.983 23.1 210 0.16 0.76 0.33 16.22 0.44 0.04 92.9 0.997 21.5 240 0.10 0.62 0.32 16.55 0.45 0.03 94.2 1.000 22.7 300 0.06 0.56 0.31 16.24 0.48 0.04 94.7 1.001 23.0 (5 C.) pH 6.0 0.07 0.56 0.40 16.11 0.44 0.05 94.7 1.015 17.7 Legend: As in Table 1

[0054] Inspection of Tables 1 and 2 revealed that the use of solid (DHPGMH).sub.2SO.sub.4 resulted in better results over the use of DHPGM in solution, in terms of speed of conversion, formation of (unwanted) cephalexin and overall S/H ratio end of reaction.

Prophetic Example 3

Preparation of cefradoxine using (DHPGMH).SUB.2.SO.SUB.4 .vs DHPGM in solution

[0055] 7-Amino-3-methoxy-3-cephem-4-carboxylic acid (7-AMOCA, 234 mmol) is suspended in water (153 g) and the temperature is controlled at 20 C. The mixture is stirred for 5 min while maintaining the pH at 6.7 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; 55 g) is added together with water (60.5 g). Next, solid (DHPGMH).sub.2SO.sub.4 (54.9 g) is dosed at a constant rate in 150-180 min. whilst the pH was maintained at 6.9 by the addition of an aqueous solution of ammonia (25%) or with an aqueous solution of sulfuric acid (30%) once all (DHPGMH).sub.2SO.sub.4 is added. After the conversion is >93.5%, the suspension is cooled to 5 C. in 20 min. while maintaining the pH at 6.9. Subsequently the pH is lowered to 6.0 with sulfuric acid (30%). During the course of the reaction samples are taken and analyzed by HPLC.

[0056] For comparative reasons the above cephradine protocol was repeated however using DHPGM solution (34.64%, pH=2, as obtained in the above General section) instead of solid (PGMH).sub.2SO.sub.4, and less water as outlined in WO 2005/003367 to compensate for the water present in the DHPGM solution.

Prophetic Example 4

Preparation of epicillin using (DHPGMH).SUB.2.SO.SUB.4 .vs DHPGM in solution

[0057] 6-Aminopenicillanic acid (6-APA, 234 mmol) is suspended in water (153 g) and the temperature is controlled at 20 C. The mixture is stirred for 5 min while maintaining the pH at 6.7 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; 55 g) is added together with water (60.5 g). Next, solid (DHPGMH).sub.2SO.sub.4 (54.9 g) is dosed at a constant rate in 150-180 min. whilst the pH was maintained at 6.9 by the addition of an aqueous solution of ammonia (25%) or with an aqueous solution of sulfuric acid (30%) once all (DHPGMH).sub.2SO.sub.4 is added. After the conversion is >93.5%, the suspension is cooled to 5 C. in 20 min. while maintaining the pH at 6.9. Subsequently the pH is lowered to 6.0 with sulfuric acid (30%). During the course of the reaction samples are taken and analyzed by HPLC.

[0058] For comparative reasons the above cephradine protocol was repeated however using DHPGM solution (34.64%, pH=2, as obtained in the above General section) instead of solid (PGMH).sub.2SO.sub.4, and less water as outlined in WO 2005/003367 to compensate for the water present in the DHPGM solution.