Field of the invention

The present invention relates to a process for the preparation of O-formyl cefamandole, an intermediate in the preparation of cefamandole nafate.

Background of the invention

(1 ) Cefamandole H H

(2) O-Formyl cefamandole H

(3) Cefamandole nafate Na

Cefamandole ((1 ); CAS 34444-01 -4; (6R,7R)-7-[[(2R)-hydroxyphenyl- acetyl]amino]-3-[[(1 -methyl-1 /-/-tetrazol-5-yl)thio]methyl]-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid is a second generation broad-spectrum semi-synthetic cephalosporin antibiotic, the preparation of which was first disclosed in US 3,641 ,021 . Cefamandole nafate ((3); CAS 42540-40-9) is the sodium salt of the formate ester of cefamandole and is the clinically used form of cefamandole.

Various approaches towards the preparation of cefamandole nafate have been reported. In a first approach, the compound can be prepared by chemical N-acylation of the appropriate β-lactam nucleus, 7-amino-3-(1 -methyl-1 H-tetrazol-5-ylthiomethyl)-3- cephem-4-carboxylic acid (7-TMCA) as for instance described in Kaiser et al. (J. Infect. Dis. (1978) 137, 10-16). In a second and environmentally more benign approach, an enzymatically catalyzed kinetically controlled acylation of 7-TMCA has been proposed in more recent literature by, amongst others, Nierstrasz et al. (Biocatalysis and Biotransformation (1999) 17, 209-223), Terreni et al. (Appl. Microbiol. Biotechnol. (2007) 77, 579-587) and Serra et al. (Eur. J. Org. Chem. (2009), 1384-1389). Although direct kinetically controlled acylation of 7-TMCA with D,L-0-formyl methyl mandelate towards O-formyl cefamandole (2) has been suggested by Fuganti et al. (Biotechnol. Lett. (1992) 14, 543-546) using the enzyme penicillin G acylase from Escherichia coii immobilized on Eupergit C, the general opinion is that the active site of penicillin G acylase is only moderately suited to accommodate the D,L-0-formyl mandelate side chain. Unfortunately, an efficient approach leading from cefamandole (1 ) to O-formyl cefamandole (2) and ultimately to cefamandole nafate (3), has not been reported yet.

Detailed description of the invention

The current most promising approach for the preparation of cefamandole nafate (3) is kinetically controlled acylation of 7-TMCA with an ester of mandelic acid, such as methyl mandelate, to give cefamandole (1 ) followed by conversion into O- formyl cefamandole (2) and subsequently into cefamandole nafate (3).

It is an object of the present invention to provide a method for the preparation of O-formyl cefamandole (2) from cefamandole (1 ), optionally followed by conversion into cefamandole nafate (3).

In a first aspect of the invention there is provided a method for the preparation of O-formyl cefamandole (2) or a salt thereof wherein cefamandole (1 ) or a salt thereof is reacted with a formylating agent.

In general, formylation of alcohols is well studied, as for instance in a review by G.A. Olah et al. (Chem. Rev. (1987) 87, 671 -686) and can be carried out using a variety of reagents such as formic acid (M. Chakrabarty et al., Synth. Commun. (2000) 30:2, 187-200), formic anhydrides, formic esters, formic halides or N-formyl derivatives. Examples are N-formylbenzotriazole (A.R. Katritzky et al., Synthesis (1995), 503-505), ethyl formate (M.H. Habibi et al., Tetrahedron (2001 ) 57, 8333-8337), 2,2,2-trifluoroethyl formate (D.R. Hill et al., Org. Lett. (2002), 1 1 1 -1 13), acetic acid with ethyl formate (V. Mirkhani et al., Monatshefte fur Chemie (2004) 135, 1257-1263). However not much is known of formylation of the class of sensitive β-lactams. In US 4,877,783 formylation of a β-lactam is reported using acetic formic anhydride, however the formylation concerns reaction of an amino group at the carbon adjacent to the carbonyl of the β-lactam ring and the carboxyl group of the β-lactam is protected. If not for anything else this example stresses the ease of formation of unwanted side products in β-lactam formylation reactions since it suggests that other functionalities such as carbonyl groups must be protected and since it does not address formylation of a hydroxyl group, as is the subject of the present invention.

In one embodiment, the formylating agent is formic acid, a formic anhydride, a formic ester, a formic halide or an N-formyl derivative. Preferably, the formylating agent is a mixed anhydride such as acetic formic anhydride of formula CH ₃ C(0)OC(0)H, a compound that can conveniently be prepared from acetic anhydride and formic acid. Alternatively, a sterically hindered derivative of acetic formic anhydride may be used, such as butyric formic anhydride of formula CH ₃ CH2CHC(0)OC(0)H or 2-ethylbutyric formic anhydride of formula (C2H ₅ )2CHC(0)OC(0)H or isobutyric formic anhydride of formula (CH ₃ )2CHC(0)OC(0)H or 2-methylbutyric formic anhydride of formula CH ₃ CH ₂ CH(CH ₃ )C(0)OC(0)H or propionic formic anhydride of formula CH ₃ CH ₂ C(0)OC(0)l-l or mixtures thereof. These sterically hindered derivatives of acetic formic anhydride have the additional advantage of reduced formation of unwanted acylated products. For example, the presence of acetylated contaminations may lead to liberation of traces of acetate upon parenteral administration. Acetate is known (A. Amore et al., J. Amer. Soc. Nephrol. (1997), 1431 ) for its negative effects in man leading to acetate intolerance, a condition associated with arterial hypertension, nausea, vomiting and headaches. For the purpose of the present invention it is also advantageous to use the mixed anhydride in the presence of formic acid as this suppresses the formation of unwanted acetylated products. For example, next to acetic formic anhydride, formic acid is preferably present in amounts of from 0.05 to 100 mol% based on the amount of acetic formic anhydride, more preferably of from 0.1 to 50 mol%, most preferably of from 0.2 to 10 mol%. Alternatively, the formation of unwanted acetylated products can be avoided by isolating the acetic formic anhydride by means of distillation prior to reaction with cefamandole (1 ). Distillation may be carried out at normal pressure or reduced pressure. Advantageously, acetic formic anhydride is prepared shortly prior to use as the compound is labile (degradation to form carbon monoxide). Thus, in certain instances it may be advisable to use the acetic formic anhydride within 24h after its preparation, preferably within 12h after its preparation, more preferably within 6h after its preparation and most preferably within 3h of its preparation. Also in view of the instability of acetic formic anhydride, this formylating agent is preferably added in excess compared to the amount of cefamandole (1 ). Preferably the molar ratio acetic formic anhydride/cefamandole (1 ) is between 100/1 and 100/99, more preferably between 50/1 and 100/95, most preferably between 10/1 and 10/9.

In another embodiment, the formylation is carried out in the presence of a base. It has been found that improved conversions are obtainable, in particular when the formylation is carried out at lower temperatures such as from -78°C to 30°C when the base in question has a dissociation constant pK _a (at 25°C) of from 3.0 to 1 1 .0, preferably of from 6.0 to 9.0, more preferably of from 6.5 to 8.0. Examples are

1 - amino-2-methoxyethane, allantoin, p-anisidine, benzylamine, 2-benzylpyridine, benzimidazole, n-butylamine, sec-butylamine, ie f-butylamine, cyclohexylamine, Ν,Ν-diethylaniline, diethylmethylamine, dimethylamine, N,N,-dimethylaniline,

2.3- dimethylpyridine, 2,4-dimethylpyridine, 2,5-dimethylpyridine, 2,6-dimethylpyridine,

3.4- dimethylpyridine, 3,5-dimethylpyridine, ephedrine, ethylamine,

2- ethylbenzimidazole, N-ethylaniline, 1 -ethylpiperidine, 2-ethylpyridine, heptylamine, hexadecylamine, hexylamine, imidazole, isoquinoline, melamine, methoxypyridine, methylamine, p-methylaniline, 1 -methylimidazole, N-methylmorpholine, 1 -methylpiperidine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, N-methylpyrrolidine, 2-methylquinoline, morfoline, pentylamine, p-phenetidine, phenethylamine, propylamine, pyridine, 2-pyridinamine, 2,5-pyridinediamine, theobromine, 2-thiazolamine, triethylamine, trimethylamine, 2,4,6-trimethylpyridine. Preferred examples of bases to be used in the method of the present invention are imidazole and derivatives thereof such as 1 -methylimidazole, morpholine and derivatives thereof such as N-methylmorpholine, pyridine and derivatives thereof such as the mono-, di- and tri-methyl pyridines and triethylamine. In the context of the present invention the term dissociation constant pK _a is defined as the negative logarithm of the acid dissociation constant K _a .

The reaction may be carried out at a wide temperature range, i.e. -50°C to 60°C. Preferably however, the temperature range is from -5°C to 40°C as the balance between reaction speed, degradation rate and optimal conversion is best tuned within this range. The most optimal temperature range, where high conversions are obtained in combination with low product degradation, was found to be from 0°C to 30°C.

The reaction can be carried in the presence or absence of a solvent. Suitable solvents are amides, alkanes, halo-alkanes, ethers, esters and the like. Examples are dichloromethane, dimethylformamide, ethyl acetate, isopropyl acetate, methyl acetate, tetrahydrofuran and the like.

In a second aspect of the invention, O-formyl cefamandole (2) obtained in the first aspect of the invention is further converted to cefamandole nafate (3) by reaction with a sodium source. Suitable sodium sources are sodium halides or sodium salts of organic acids such as sodium acetate, sodium ethyl hexanoate or sodium formate or mixtures thereof. It is well known that mandelic acid is formed as a side product (usually in significant amounts) during the enzymatic synthesis of cefamandole (1 ) from 7-TMCA and an ester of mandelic acid. Like cefamandole (1 ), th is mandel ic acid is also formylated to give O-formyl mandelic acid. Surprisingly it is found that upon conversion of O-formyl cefamandole (2) to the sodium salt cefamandole nafate (3), only the latter crystallizes and can be isolated in high purity whereas the sodium salt of O-formyl mandelic acid remains dissolved in the mother liquor. This unexpected phenomenon results in a highly efficient purification step.

In a third aspect of the present invention, the cefamandole nafate obtained by the processes of the second aspect, is used for the manufacture of a medicament with antibacterial properties. The medicament thus obtained has the advantage of being produced in high purity and with low environmental burden as compared to prior art approaches.

Legend to the Figures

Figure 1 is the formylation of cefamandole (1 ) to O-formyl cefamandole (2) using various bases at room temperature and in isopropyl acetate (unless otherwise mentioned): imidazole without isopropyl acetate (0); imidazole (■); N-methylmorpholine

(A ); N-methylimidazole (·); triethylam ine at 50°C (□). X-axis: time (h) . Y-axis: conversion (mol%).

Figure 2 is the formylation of cefamandole (1 ) to O-formyl cefamandole (2) using imidazole: O-formyl cefamandole (■); O-acetyl cefamandole (A ). X-axis: time (h).

Y-axis: conversion (mol%). MATERIALS AND METHODS

Preparation of immobilized penicillin acylase

The production, isolation and purification of wild type and mutant penicillin G acylases may be carried out as described in WO 1996/005318 and WO 2003/055998. Alternatively, genes encoding mutant penicillin G acylases may be obtained by gene synthesis. Production of the mutant penicillin G acylase was achieved by cloning the genes encoding mutant penicillin G acylases into an appropriate expression vector, transforming a suitable host such as Escherichia coii with said vector and culturing the transformed host under conditions suitable for the production of the mutant penicillin G acylases and recovery and purification of the mutants was carried out as described in WO 2010/072765. Penicillin G acylase AA (the Escherichia coii wild type penicillin G acylase with mutations B:F24A and B:V148L) and penicillin G acylase mutant 1 (the Escherichia coii wild type penicillin G acylase with mutations V1 1 A, A:S3L, A:V192E, B:F24A, B:V148L and B:F460L) as disclosed in Example 1 of WO 2010/072765 were immobilized according to the method disclosed in EP 839192 and EP 222462.

Analytical HPLC

The reactions were followed by quantitative HPLC analysis. Samples were diluted (typically -50 mg sample was added up to 50 mL with buffer) in phosphate buffer pH 2.7 (50 mL 1 M H ₃ P04 + 40 mL 1 M NaOH per liter MilliQ water).

Instrument: HPLC Hewlett Packard 1 100, detection at 220 nm

Column: Intersil ODS-3, 5μπι 4.6x150mm C/N 5020-01731

Flow: 1 mL/min, stop time 37 min

Method: Eluens A: Methanol 99.9%

Eluens B: Acetontirile

Eluens C: KH ₂ P0 ₄ (2.72 g) and 1 M H ₃ P0 ₄ (6 mL), to 1 L with MilliQ water Time (min) Eluens A (%) Eluens B (%) Eluens C (%)

0 0.5 ¹ 98.5

2.5 3 ¹ 96

1 1 25 ¹ 74

15 40 ¹ 59

25 60 ¹ 39

28 80 ¹ 19

32 80 ¹ 19

32.1 0.5 ¹ 98.5

Response factors and retention times:

Measurement of pH values

The pH values referred to in the present invention were measured as follows.

The measurement is performed using 718 STAT Titrino from Metrohm. The pH electrode is from Metrohm, series number 6.0234.1 10. It contains 3M KCI. The pH meter calibration is performed at 20°C at pH 4 and pH 7 using standard solutions from Merck, using the calibration program present in the instrument. EXAMPLES

Example 1

Enzymatic preparation of cefamandole (1 )

The enzymatic reaction was carried out with R-methyl mandelate (20.9 g; 294 mmol), 7-TMCA (58.4 g; 165 mmol) and water (671 g) at 2°C and pH 8.5. The reaction was started by adding immobilized penicillin G acylase mutant 1 (82.4 g, see Materials and Methods). At a conversion of 87% the reaction was stopped by removing the enzyme by filtration . The mother liquor of the enzymatic reaction was extracted at room temperature twice with isopropyl acetate (850 g each time) to remove the remaining methyl mandelate. The organic layer was evaporated under vacuum resulting in methyl mandelate oil. Remaining solvents were removed from the aqueous layer by evaporation. The pH was slowly reduced by adding 25% H ₂ S0 ₄ solution. At pH values of 6.0 and 4.3 the solution was seeded with 0.1 g 7-TMCA and the resulting suspension was filtered at pH 4.7. The suspension was filtered and the organic and aqueous phases were separated. The water phase was extracted twice with ethyl acetate (210 g). The organic phases were combined and ethyl acetate was evaporated to completion (40°C). The remaining solid was analyzed by means of HPLC (see Materials and Methods):

Example 2

Formylation of cefamandole (1 ) to O-formyl cefamandole (2)

Preparation mixed anhydride

Acetic anhydride (10.0 mL; 105.8 mmol) and formic acid (4 ml_; 106.0 mmol) were mixed and stirred at 55°C for 2h. Formylation

2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.6 g) and mixed anhydride (6 g) after which imidazole (0.04 g) was added. The solution was stirred at room temperature for 8h and analyzed by HPLC. The conversion to formyl cefamandole was >97%. Next, isopropyl acetate (15 mL) and water (5 mL) were added and the pH was adjusted to 2 with 25% H ₂ S0 ₄ . The organic phase was separated and washed twice washed with water (5 mL, adjusted to pH 2). Yield on 7-TMCA after work-up: 95%.

Example 3

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using triethyl amine

2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.8 g) and mixed anhydride (6 g) prepared according to Example 2. Then 0.59 mmol of triethyl amine was added. The solution was stirred at 50°C and the reaction mixture was analyzed at various points in time.

The results are depicted in Figure 1 .

Example 4

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using imidazole

2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.8 g) and mixed anhydride (6 g) prepared according to Example 2. Then 0.59 mmol of imidazole was added. The solution was stirred at room temperature and the reaction mixture was analyzed at various points in time.

The results are depicted in Figure 1 .

Example 5

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using imidazole in the absence of solvent

2.6 g of cefamandole obtained in Example 1 was mixed with mixed anhydride (6 g) prepared according to Example 2. Then 0.59 mmol of imidazole was added. The solution was stirred at room temperature and the reaction mixture was analyzed in time. The results are depicted in Figure 1 . Example 6

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using

N-methylmorpholine

2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.8 g) and mixed anhydride (6 g) prepared according to Example 2. Then 0.59 mmol of N-methylmorpholine was added. The solution was stirred at room temperature and the reaction mixture was analyzed in time.

The results are depicted in Figure 1 .

Example 7

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using

1 -methylimidazole

2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.8 g) and mixed anhydride (6 g) prepared according to Example 2. Then 0.59 mmol of 1 -methylimidazole was added. The solution was stirred at room temperature and the reaction mixture was analyzed in time.

The results are depicted in Figure 1 .

Example 8

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using catalytic amounts of imidazole

Preparation mixed anhydride

Acetic anhydride (14.12 g; 13.0 mL; 138.3 mmol) and formic acid (6.38 g; 5.2 mL;

138.6 mmol) were mixed and stirred at 55°C for 2.5h.

Formylation

The mixed anhydride solution was added to a cefamandole mixture from an enzymatic reaction comprising cefamandole (5.290 g), methyl mandelate (0.124 g), mandelic acid (0.166 g) and isopropyl acetate (16.92 g). After that, imidazole was added as solution in mixed anhydride (10.3 mg imidazole in 500 μΙ_ mixed anhydride). Samples were taken over time and analyzed by means of HPLC. After 20h, 95 mg imidazole was added. The results are depicted in Figure 2. Isopropyl acetate (56 g) and water (50 g) were added, the pH of the aqueous phase was adjusted to 1 .5 using 25% H ₂ S0 ₄ and the phases were separated. The organic phase was washed with aqueous HCI (pH 1 .8, 2 x 20 mL). Approximately 2.2 mol% of cefamandole remained in the combined aqueous phases.

Example 9

Cefamandole nafate

The organic phase obtained in Example 2 was evaporated to dryness at 37°C using a rotavap. Isopropanol (15 mL) and acetone (15 mL) were added and the resulting mixture was titrated at room temperature to pH 5.6 with a 10% sodium 2-ethylhexanoate solution in acetone (718 STAT Titrino by Methrohm filled with 25% NH ₃ ). The resulting crystals were isolated by filtration in a yield of 82.6%. Apart from the title product, HPLC analysis indicated the presence of cefamandole and acetyl cefamandole but not of any mandelic acid derivatives.

Example 10

Cefamandole nafate

The organic phase obtained in Example 8 was evaporated to 23.4 g at 38°C using a rotavap. Acetone (21 g) was added and the resulting mixture was titrated at room temperature to pH 5.66 with a 10% sodium 2-ethylhexanoate solution in acetone (55 mL). The resulting crystals were isolated by filtration to give, after drying overnight under vacuum at 30°C, 2.36 g of white solid. ¹ H NMR (700 MHz, DMSO-d ₆ , CD ₃ CN) δ 9.25 (d, 1 H, J = 8.2 Hz), 8.27 (s, 1 H), 7.5 (m, 2H), 7.4 (m, 3H), 6.1 1 (s, 1 H), 5.55 (dd, 1 H, Ji = 4.7 Hz, J ₂ = 8.4 Hz), 4.85 (d, 1 H, J = 4.8 Hz), 4.43 (d, 1 H, J = 12.5 Hz), 4.19 (d, 1 H, J = 12.5 Hz), 3.88 (s, 3H), 3.50 (d, 1 H, J = 17.6 Hz), 3.32 (d, 1 H, J = 17.6 Hz). HPLC analysis showed the following composition: O-Formyl cefamandole (96.19 area%), cefamandole (1 .85 area%), acetyl cefamandole (1 .96 area%).

Example 11

Formylation of cefamandole (1 ) to O-formyl cefamandole (2) using isobutyric formic anhydride and 1 -methylimidazole

Preparation mixed anhydride

Isobutyric anhydride (35.0 mL; 212.0 mmol) and formic acid (16 mL; 425.8 mmol) were mixed and stirred at 55°C for 2h.

Formylation 0.2 g of cefamandole obtained in Example 1 was mixed with mixed anhydride (2 g) after which 1 -methylimidazole (0.024 g) was added. The solution was stirred at room temperature for 8h and analyzed by HPLC and showed, apart from the title product, -0.5 area% O-isobutyric cefamandole.