NL1010506C2 - Production of beta-lactam antibiotics comprises enzyme-catalysed acylation with new D-p-hydroxyphenylglycinamide derivatives - Google Patents

Abstract

Process (A) for preparing beta-lactam antibiotics comprises enzyme-catalyzed reaction of a beta-lactam core compound with a D-p-hydroxyphenylglycinamide derivative (I) in which the amide group is monosubstituted. Independent claims are also included for: (1) D-p-hydroxyphenylglycinamide derivatives of formula (Ia) and their salts: (2) a process (B) for preparing (I) by reacting a p-hydroxyphenylglycine ester (II), or a salt of (II), with the corresponding amine. R = NH2 or alkyl substituted with one or more OH or alkoxy groups.

Description

- 1 - PN 9845 PROCESS FOR THE PREPARATION OF β-LACTAM ANTIBIOTICS 5

In the enzymatic preparation of β-lactam antibiotics by acylation of a β-lactam core with a D-p-hydroxyphenylglycine derivative, a lower alkyl ester or the amide of 10 D-p-hydroxyphenylglycine is often used as the acylating agent.

An important drawback of the use of the p-hydroxyphenylglycine amide (FGHA), however, is that the enzymatic coupling with a β-lactam core gives a low conversion. For example, for amoxicillin this results in the need to use a large excess of p-hydroxy-phenylglycine amide relative to the amount of β-lactam nucleus (6-aminopenicillanic acid; 6-APA) used to achieve a sufficiently high conversion, resulting in much acylating agent is lost through hydrolysis.

In addition, the reaction takes a relatively long time, as a result of which there is a relatively large amount of breakdown of the β-lactam nucleus and the β-lactam antibiotic. In the preparation of cefadroxil from FGHA and 7-aminodesacetoxycephalosporanic acid (7-ADCA), the conversion remains too low even at high acylating agent to β-lactam core ratios. The same applies to the preparation of cefprozil from FGHA and 7-amino-3 - [(Z) -1-propenyl] -3- (desacetoxymethyl) -cephalosporanoic acid (7-PACA).

It has now been found that when instead of the (unsubstituted) p-hydroxyphenylglycine amide a compound in which the amide group is monosubstituted is used, a much faster reaction is obtained, whereby a higher conversion is obtained.

J J * U 0 U O

- 2 -

In addition, Applicant has found a method by which the p-hydroxyphenylglycine amides whose amide group is monosubstituted can be prepared in high yield. An important additional advantage is therefore also that the p-hydroxyphenylglycine amides used according to the invention can be easily prepared and can be used without intermediate isolation in the enzymatic preparation of β-lactam antibiotics.

It is known from WO-A-98/04732 to use substituted esters of D-p-hydroxyphenylglycine as an acylating agent. However, it has been found that - in contrast to when, according to the invention, the D-p-hydroxyphenylglycide amides, the amide group of which is monosubstituted, are used, the enzymatic activity of the enzymes used in the acylation reaction decreases relatively quickly. Since the enzyme costs are relatively very high in these acylation processes, many times the enzymes are reused to achieve a commercially attractive process. Consequently, the use of substituted esters is not commercially interesting.

Preferably as substituted p-25 hydroxyphenylglycine amide, a p-hydroxyphenylglycine amide of formula 1

O

/ - \ II

HO- O - C-C-NHR (1) 30 / | NH 2 used, wherein R represents NH 2 or an aliphatic alkyl group substituted with one or more OH-1010506-3 and / or alkoxy groups, or a salt thereof. An alkyl group with 1-10 carbon atoms is preferably used as the aliphatic alkyl group; the alkoxy groups preferably have 1 to 5 C atoms. Particular preference is given to R = 2-hydroxyethyl because the ethanolamine required for its preparation is readily available, inexpensive and non-toxic.

For example, the D-p-hydroxyphenylglycine amides whose amide group is monosubstituted can be prepared by suspending an ester, for example, the methyl ester of D-p-hydroxyphenylglycine in a mixture of water and the corresponding amine. According to another method, from a (racemic) mixture of the enantiomers of an ester of p-hydroxyphenylglycine, a (racemic) mixture of the enantiomers of the p-hydroxyphenylglycine amide of which the amide group is monosubstituted is first prepared; then this mixture is then split into enantiomers, for example enzymatically, using an L-aminopeptidase, the L-amide being converted to the corresponding L-acid and the D-amide remaining. Suitable aminopeptidases are present, for example, in Pseudomonas putida, Ochrobactrum anthropi and Mycobacterium neoaurum.

Preferably, the reaction of the ester of p-hydroxyphenylglycine with the corresponding amine is carried out in the presence of water, for example 1-75% by volume, preferably 2-50% by volume, in particular 5-30% by volume, water calculated relative to the amount of amine. It has been found that the presence of water accelerates the reaction considerably. The optimum amount of water is easy to determine ï 0 * ï 05 0 6 - 4 - as a balance between the reaction speed on the one hand and the hydrolysis of the ester due to the presence of water on the other. The amount of amine to be used is preferably greater than the equimolar amount, in particular 2-10 equivalent, calculated with respect to the amount of ester of p-hydroxyphenylglycine.

The temperature at which the reaction with the amine takes place is preferably kept as low as possible without freezing occurring, for example between -5 and + 30 ° C, preferably between -2 and + 5 ° C.

The method of the invention can be suitably used in the preparation of β-lactam antibiotics with a p-hydroxyphenylglycine side chain, for example amoxicillin, cefadroxil, cefprozil, cefatrizine, cefoperazone and ceppyamide.

In principle, any β-lactam core can be used; in particular a β-lactam core with the general formula (1) 20

Ro Y

HjN Ï / \ 25 o- N γ ^ Ζ

Ci'OH

Wherein R0 represents H or an alkoxy group having 1-3 C atoms; Y represents CH2, O, S or an oxidized form of sulfur; and Z stands for / 1010506 - 5 -

Xch3 \ \ \ II / 1k CH3, / \ Ri, ^ \ Ri, Or / VCH2 where R3 is, for example, H, OH, halogen, an alkyl group with 1-5 C atoms, alkoxy group with 1-5 C atoms , an alkenyl group with 2-5 C atoms, a 10 (thio) ether group with 1-5 C atoms, an ester group with 2-10 C atoms, an amide group with 1-5 C atoms, a cycloalkyl group with 4- 8 C atoms, an aryl or a heteroaryl group with 6-10 C atoms, the groups optionally substituted, for example, with a halogen or a hydroxy, alkyl, an aryl, a carboxy, an alkoxy or a (substituted) amino group with 0-8 C atoms; and wherein the carboxylic acid group may optionally be an ester group.

Suitable examples of β-lactam nuclei that can be used in the method according to the invention are penicillin derivatives, for example 6-aminopenicillanic acid (6-APA), and cephalosporin derivatives, for example a 7-aminocephalosporanoic acid with or without a substituent on the 3- site, for example, 7-aminocephalosporanoic acid (7-ACA), 7-aminodesacetoxycephalosporanoic acid (7-ADCA), 7-amino-3 - [(Z) -1-propenyl] -3- (desacetoxymethyl) -cephalosporanoic acid (7-PACA) , 7-amino-3-chloro-cef-3-em-4-carboxylic acid (7-30 ACCA) and 7-amino-3-chloro-8-oxo-1-azabicyclo [4.2.0] oct-2-en -2-carboxylic acid.

In principle, any enzyme suitable as a catalyst in the coupling reaction can be used as the enzyme. Such enzymes are, for example, 1010506-6 - the enzymes known under the general term penicillin amidase or penicillin acylase. Such enzymes are described, for example, in J.G. Shewale et. al. Process Biochemistry, August 1989 p. 146-154 5 and in J.G. Shewale et. al. Process Biochemistry

International, June 1990, p. 97-103. Examples of suitable enzymes are enzymes derived from Acetobacter, in particular Acetobacter pasteurianum. Aeromonas. Alcaliaenes. in particular Alcaliaenes 10 faecalis. Aphanocladium. Bacillus sp .. in particular Bacillus megaterium. Cephalosporium. Escherichia, in particular Escherichia, coli. Flavobacterium, Fusarium. in particular Fusarium oxvsporum and F-usar iu.m_S.alani, KLayyara, Mycoplana, Erotamangbagter> 15 Proteus, in particular Proteus rettaari. Pseudomonas and Xanthomonas. in particular Xanthomonas citrii.

An immobilized enzyme is preferably used, since the enzyme can then be easily separated and reused.

For example, among the immobilized enzymes commercially available, the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the ena Engelgel®, has been found immobilized, Recordati's immobilized Penicillin-G acylase and the Immobilized Penicillin-G acylase from Pharma Biotechnology Hanover. In addition, enzymes can also be used in crystalline form (CLECs ™).

The temperature at which the enzymatic acylation reaction is carried out is usually below 40 ° C, preferably between -5 and 35 ° C. The pH at which the enzymatic acylation reaction is carried out is 101050fi-7, usually between 5.5 and 9.5, preferably between 6.0 and 9.0.

Preferably the reaction is stopped almost completely when the maximum conversion 5 has been reached. A suitable embodiment to stop the reaction is to lower the pH, preferably to a value between 4.0 and 6.3, in particular between 4.5 and 5.7. Another suitable embodiment is to lower the temperature of the reaction mixture 10 once the maximum conversion has been reached. A combination of both embodiments is also possible.

After the reaction has virtually stopped upon reaching maximum conversion, the reaction mixture is usually in the form of a suspension containing multiple solids, for example, the antibiotic, D-p-hydroxyphenylglycine (FGH) and optionally immobilized enzyme. Due to the process economy, the immobilized enzyme is preferably recovered. This can conveniently be done, for example, by filtering the reaction mixture over a screen while stirring, preferably choosing the direction of rotation of the stirrer such that the suspension is pumped up in the center of the stirrer. Valuable components, for example the antibiotic and FGH, can then be recovered, for example with the aid of a pH change.

Within the scope of the invention, a pH reduction can be effected, for example, by adding an acid. Suitable acids are, for example, mineral acids, in particular sulfuric, hydrochloric or nitric and carboxylic acids, for example acetic, formic and citric acid. For example, a pH increase can be accomplished by adding a base. Suitable bases are, for example, inorganic bases, in particular ammonia, caustic potash or caustic soda and organic bases, for example triethylamine, ethanolamine and quaternary ammonium hydroxides.

The enzymatic acylation reaction can be performed in water. If desired, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30% by volume. Examples of organic solvents that can be used are alcohols with 1-7 C atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, especially ethylene glycol or a triol, especially glycerol. The molar ratio of acylating agent to β-lactam core, i.e. the total amount of added acylating agent divided by the total amount of added β-lactam core, expressed in moles, is preferably less than 2.5. The molar ratio is preferably between 0.5 and 2.0, in particular between 0.7 and 1.8.

The enzymatic acylation reaction is preferably carried out as a batch process. If desired, it is also possible to carry out the reaction continuously.

The invention will be further elucidated on the basis of the examples without, however, being limited thereto.

Examples 30 7-ADCA: 7-aminodesacetoxycephalosporanic acid 6-APA: 6-aminopenicillanic acid CEDROX: cefadroxil 1010506 - 9 - AMOX: amoxicillin FGH: Dp-hydroxyphenylglycine FGHM: Dp-hydroxyphenylglycine methyl ester FGHA: Dp-hydroxyflyglyphHA: Dp-hydroxyphide glyphine: ethanolamide FGHH: Dp-hydroxyphenylglycine hydrazide

Assemblase ™ is an immobilized Escherichia coli penicillin acylase from E. coli ATCC 1105, as described in WO-A-97/04086. The immobilization was performed as described in EP-A-222462 using gelatin and chitosan as a gelling agent and glutaraldehyde as a crosslinker.

The final activity of the Escherichia coli penicillin acylase is determined by the amount of enzyme added to the activated beads and was 3 ASU / g dry weight with 1 ASU (Amoxicillin Synthesis Unit) defined as the amount of enzyme per hour Amoxicillin. 3H20 20 generates from 6-APA and FGHM (at 20 ° C; 6.5% 6-APA and 6.5% FGHM).

Comparative Experiment Synthesis of Cefadroxil with FGHA 25 An enzyme reactor (1.5 L, diameter 11 cm), equipped with a 175 µ mesh screen bottom, was charged with 400 g net wet Assemblase ™.

A production reactor (1.2 L) was charged with 108.9 g of 7-ADCA (0.500 mol), 100.1 g of FGHA (0.600 mol), 4.0 g of sodium bisulfite and 350 g of water (T = 8eC). This mixture was brought to pH = 8.3 using ca 32 ml concentrated ammonia. At t = o, the suspension 1010506 - 10 - was taken from the production reactor by means of 35 ml of water (T = 8 ° C) are transferred to the enzyme reactor. The stirrer was turned on in the enzyme reactor at t = 0. The temperature was kept at T = 8 ° C. The pH was kept at 8.3 by means of titration with concentrated H 2 SO 4. Using HPLC determined the concentrations of reactants and products. The measured concentrations and the calculated conversions and S / H ratios as a function of time are shown in Table 1: 10

Iabe.1 t (min) jCEDROX 17-ADCA IFGHA FGH conversion S / H j (mM) (mM) (mM) (mM) (%) TB == 20 ~ 481 58Ö 2 = 4 "= 10.0 ~ 6Ö 38 460 550 6 8 6 ^ 3

120 73 426 503 20 15 T7II I.

101 101 402 451 43 20 2 ~ / 3 |

240 TÏ7 375 4ÏÖ 73 23 ΪΤδ I

3ÖÖ Ï3Ö T? 5 36Ö TÖ5 26 ΐΤΪ I

36Ö 138 "35Ö ÏÏ2 143 28 Ï7Ö I

Example I

15 Synthesis of FGHEA

A production reactor (1.2 L) was charged with a mixture of 183 g of ethanolamine (3.00 mol) and 20 g of water. The liquid was cooled to T = 2 ° C. 109.1 g FGKM (0.600 mol) was added with stirring, after which the suspension was stirred at 2 ° C for 2 days. After 2 days

stirring had resulted in a clear solution. Using HPLC

1010506-11 - The concentrations of the reactants and products were determined:

[FGHM] = 0 mM

[FGHEA] = 1900 mM, about 0.59 mole [FGH] = 29 mM, about 0.009 mole

Example II

Synthesis of cefadroxil with FGHEA

An enzyme reactor (1.5 L, diameter 11 cm), equipped with a 175 µ mesh screen, was charged with 80 g net wet Assemblase ™. A production reactor (1.2 L) was charged with the FGHEA (0.59 mol) / FGH (0.009 mol) solution prepared according to Example I. 400 ml of water (T = 8 ° C) and 4.0 g of sodium bisulfite were added. Under cooling (T = 8 ° C), the pH was lowered to about 9.5 using e.g. concentrated H 2 SO 4. Then 108.9 g of 7-ADCA (0.500 mol) was added, the pH falling to about 8.5. At t = o, the solution was taken from the production reactor using 35 ml of water (T = 8 ° C) are transferred to the enzyme reactor. The stirrer was started in the enzyme reactor at t = 0. The temperature was kept at 8 ° C during the reaction. The pH was kept at 8.3 by means of titration with concentrated H 2 SO 4. Using HPLC determined the concentrations of 25 reactants and products. The measured concentrations and the calculated conversions and S / H ratios as a function of time are shown in Table 2.

This example shows that, compared to the comparative experiment, both the conversion and the S / H ratio are significantly higher, and the reaction proceeds faster despite the fact that less enzyme is used.

10) 0506 - 12 -

Table 2

ft (min) CEDROX 7-ADCA FGHEA FGH conversion S / H I.

(mM) (mM) (raM) (mM) (%) 0 = 0 503 59Ö 9 = Ö - “7Ö 147 35Ï 445 17 29 77β ~ 60 246 253 7fÖ 27 49 §71 ~ 90 702 703 2 * 66 33 60 977 Ï2Ö 345 149_ 2ÏÖ 43 69 WTÖ Ï5Ö 37Ï IÏ9 Ï6Ï 62 74 ëTÖ Ï8Ö T86 103 127 75 77 ËTl 240 798 91 80 108 80 7/7 T - - '- —5BSSS = SS - - = = S5aBBeea ™

Example III 5 Synthesis of FGHEA

Four solutions were made, A, B, C and D, with the following compositions: A) 50 ml ethanolamine and 50 ml water 10 B) 90 ml ethanolamine and 10 ml water C) 99 ml ethanolamine and 1 ml water D) 50 ml of ethanolamine and 50 ml of methanol

The four solutions were cooled to T = 2eC, after which 20 g FGHM (0.11 mol) was added. After 2 days of stirring at T = 2eC, the compositions were determined, see table 3 (the amounts are defined in mol%, relative to 0.11 mol FGHM).

1010506 - 13 -

Table 3 Sample FGHM (mol%) FGHEA (mol%) FGH (mol%) A 0 83 17 ____ B 0 98 2 C ï 97 ΓΤ5 D ~ 9 "Ö ~ ÏÖ Ö

y.Q <? da.eel, d_lV

5 Synthesis and Isolation of FGHEA

A clear FGHEA solution was prepared as described in Example 1. This solution was evaporated in vacuo (p 1 mm Hg) at a temperature of 35 ° C. The last residues of ethanolamine and water were removed by means of co-evaporation with m-xylene to give a light brown oil with a purity of about 95%; enantiomeric excess (eeD) = 98%.

* Η NMR (DMSO-de, 200 MHz): δ 2.1 (s, 2H), 3.41 (d, J = d 15 5.9 Hz, 2H) 3.67 (d, J = 5.9 Hz, 2H), 4.21 φ, 1H), 6.66 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 9.3 (s , 2H).

Example V Synthesis of FGHH

A mixture of 98 g of hydrazine (3.0 mol) and 400 g of water was cooled to T = 2 ° C. 181.9 g of FGHM (1.0 mol) was added over 15 min while the temperature was kept at 2 ° C. After 1 night, a suspension was formed, which was filtered through a glass filter. The solid was washed with 2 x 100 ml 1010506-14 acetone / water (8: 2, v / v) and dried. Yield: 136 g (75%). Purity: 99.6%: e.e.D = 99%.

: H NMR (DMSO-de, 200 Mhz): δ 2.06 (s, 2H), 4.18 (s, 3H), 5 6.67 (d, J = 8.5 Hz 2H), 7.17 (d, J = 8.5 Hz, 2H), 9.12 (S, 2H).

Example VI

Synthesis of Cefadroxil with FGHH 10 An enzyme reactor (1.5 L, diameter 11 cm), equipped with a sieve bottom with 175 µ g mesh, was charged with 200 g net-wet Assemblase ™.

A production reactor (1.2 L) was charged with a mixture of 108.9 g of 7-ADCA (0.500 mol), 109.0 g of FGHH (0.600 mol), 4.0 g of sodium bisulfite and 500 g of water. The temperature was brought to T = 8 ° C, and the pH to 8.0 using concentrated cimmonia. At t = 0, the solution was released from the production reactor using 50 ml of water (T = 8 ° C) are transferred to the enzyme reactor. The stirrer was started in the enzyme reactor at t = 0 20. The temperature was kept at 8 ° C during the reaction. The pH was kept at 8.0 by means of titration with concentrated H 2 SO 4. Using HPLC determined the concentrations of reactants and products. The measured concentrations and the calculated conversions and S / H ratios as a function of time are shown in Table 4.

1010506 - 15 -

Table .1

t (min) CEDROX 7-ADCA FGHH FGH conversion S / H

(mM) (mM) (mM) (mM) (%) “119 321 460 7 24 17 --------- 120 174 200 345 15 35 11.6 Ï8Ö Ï86 127 765 27 37 679 24Ö 17Ö 85 "705 39" 34 4.4 5 10 105 J 6

Claims (12)

A process for the preparation of a β-lactam 5 antibiotic in which a β-lactam core is acylated with a Dp-hydroxyphenylglycine amide in the presence of an enzyme, characterized in that a Dp-hydroxyphenylglycide amide or a salt thereof is used, the amide group of which is 10-fold is substituted. The method of claim 1 wherein the amide group is monosubstituted with NH 2 or with an aliphatic alkyl group substituted with one or more hydroxy or alkoxy groups. The method of claim 2 wherein the substituted alkyl group is 2-hydroxyethyl. 4. Process according to any one of claims 1-3, wherein as β-lactam antibiotic, amoxicillin, cefadroxil, cefprozil, cefatrizine, cefoperazone or ceppyamide is prepared. 5. D-p-hydroxyphenylglycine amide of the formula 1 O / - \ II HO - O-C-C-NHR (1) \ / I NH2 where R is NH2 or an aliphatic alkyl group substituted with one or more hydroxy or alkoxy groups, or a salt thereof. D-p-hydroxyphenylglycine amide according to claim 5, wherein R represents 2-hydroxyethyl. 10Ί05ΟΡ - 17 - 7. A process for the preparation of a p-hydroxyphenylglycine amide of which the amide group is monosubstituted, wherein an ester of p-hydroxyphenylglyine or a salt thereof is contacted with the corresponding amine. The method of claim 7, wherein the reaction mixture also contains water. 9. A method according to claim 8, wherein the amount of water is between 2 and 50% by volume calculated with respect to the amount of amine used. A method according to any one of claims 7-9, wherein the temperature is between -2 and + 5eC. A method according to any one of claims 6-9, wherein the p-hydroxy-phenylglycine amide is used without intermediate isolation in a method according to any one of claims 1-4. . 1010506 COOPERATION TREATY (PCT) REPORT ON NEWNESS RESEARCH OF INTERNATIONAL TYPE IDENTIFICATION OF OE NATIONAL APPLICATION Characteristic of the applicant or of the authorized representative 9845NL Dutch application no. rvpe Granted by the International Research Authority (ISA) to the request for an international type examination no. SN 31988 EN I. CLASSIFICATION OF THE SUBJECT (when using different classifications, indicate all classification symbols) Complies with International Classification (IPC ) Int.Cl.6: C 12 P 35/04, C 12 P 37/04, C 07 C 243/34, C 07 C 237/20 II. FIELDS OF TECHNIQUE EXAMINED Minimum Documentation Examined Classification System Claims Classification Int. Cl. 6: C 12 P, C 07 C Researched documentation other than minimum documentation to the extent that such documents are included in the areas examined III. I j CANNOT EXAMINE CERTAIN CONCLUSIONS (comments on supplementary sheet) IV. Kl LACK OF UNIT OF INVENTION (comments on supplement sheet) PCT / ISA / 20U *> 07.1979