Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
  • C07D499/04—Preparation
  • C07D499/18—Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
  • C07D501/02—Preparation
  • C07D501/12—Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
  • C12P37/04—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by acylation of the substituent in the 6 position

Definitions

• the invention relates to a process for recovery of a ⁇ -lactam antibiotic from a mixture substantially containing ⁇ -lactam antibiotic and D- phenyl glycine (FG) in solution, with the mixture being brought to a pH between 3 and 8 at a concentration such that FG remains in solution, the solid ⁇ -lactam antibiotic obtained being recovered and the remaining liquid being subjected to a concentration step in which a slurry with solid ⁇ -lactam antibiotic and solid FG develops, the slurry is brought to a pH at which the ⁇ - lactam antibiotic dissolves, FG is separated as a solid and the ⁇ -lactam antibiotic present in the mother liquor is at least partially utilized.
• FG D- phenyl glycine
• the invention provides a new concept for recovery of ⁇ -lactam antibiotics whereby, in a simple process that can be applied on an industrial scale, the losses of ⁇ -lactam antibiotics are strongly reduced and also valuable D-phenyl glycine is recovered.
• the acylation agent hydrolyzes with the ⁇ -lactam antibiotic to form D-phenyl glycine (FG) .
• the mixtures obtained after an acylation reaction may contain, besides the ⁇ -lactam antibiotic and FG, for example as-yet unconverted ⁇ -lactam nucleus and/or acylation agent, for example FGA or FGM. It has been found that the exact compositions of the mixtures that may be applied in the process according to the invention are not particularly critical.
• Mixtures that may suitably be applied in the process according to the invention are preferably mixtures containing 10-1500 mM, in particular 50-1000 mM ⁇ -lactam antibiotic; 0- 1500 mM, in particular 0-1000 mM FG, 0-1000 mM, in particular 0-200 mM ⁇ -lactam nucleus and 0-1000, in particular 0-400 mM D-phenyl glycine derivative.
• a mixture containing ⁇ -lactam antibiotic and FG is brought to such concentration and pH that all components, in particular the components mentioned, optionally without the ⁇ -lactam antibiotic, are dissolved.
• the pH may be chosen to be either high, for example between 6.5 and 11, preferably between 7 and 9.5, or low, for example between 0 and 3, in particular between 0.3 and 2.
• a more or less continuous working-up process is preferably opted for.
• a continuous dissolving process allows a shorter residence time at relatively high or low pH.
• any solid components still present can be separated out by for example filtration or ultrafiltration.
• the mixture which may still contain solid ⁇ -lactam antibiotic, is first brought to a pH between 3 and 8, preferably between 4 and 7, with measures being taken, for example adding water, to ensure that the concentration of the reactants, in particular FG, is such that the reactants, with the exception of the ⁇ -lactam antibiotic, remain in solution, whether supersaturated or not.
• the concentration is so chosen that the FG is supersaturated in the solution after the solution has been brought to a pH between 3 and 8.
• the temperature is not particularly critical and is between for example -5 and 45°C, in particular between 0 and 25°C. If the FG concentration in the mixture is relatively high, a temperature preferably between 0 and 10°C is maintained.
• the liquid is subjected to concentration.
• concentration step should last a relatively short period whilst on the other hand a relatively high temperature and a relatively long, gradual concentration procedure should be chosen for obtaining a not-too-viscous slurry and large FG crystals that can readily be separated.
• the temperature at which the concentration is effected may be between for example 10 and 80°C, preferably between 20 and 60°C, in particular between 25 and 55°C.
• the duration of the concentration is between for example 10 in. and 24 hours, preferably between 0.5 and 10 hours, in particular between 1 and 5 hours.
• the temperature at which the concentration is effected and the duration of the concentration are so chosen that, as a rule, a relatively shorter duration is chosen in combination with a higher temperature, and conversely.
• Concentration may be effected by for example evaporation at reduced pressure or by nanofiltration. Evaporation may be effected in for example a thin-film evaporator. It has been found that the wall is easy to keep clean and that little degradation of the ⁇ -lactam antibiotic took place.
• a second possible embodiment of the concentration is an evaporator-crystallizer, for example a bypass evaporator or a number of cascaded bypass evaporators.
• concentration can also be very well effected by means of nanofiltration. Surprisingly, it has been found that the flux through the membrane remains relatively high even though crystallization occurs during nanofiltration.
• Membranes that may suitably be applied in concentration through nanofiltration are for example SeIRO MPT- 10 (Membrane Products Kiryat Weizmann) , WFN0505 (Stork Friesland) and Nanomax-50 (Millipore) .
• the concentration factor i.e. the volume ratio before and after concentration, may vary between wide limits and preferably is between 1 and 30, in particular between 3 and 20. The higher the concentration factor, the higher the efficiency will be.
• the concentration factor is chosen so that salts developing throughout the process remain dissolved.
• the slurry obtained after the concentration step preferably is first subjected to separation into a clear liquid stream and a concentrated slurry or into a clear liquid stream and a solid. Separation may be effected with any separation apparatus, for example a centrifuge or a filter. Since the solids need not necessarily be separated as solids, it is preferred for a decanter to be used.
• the clear liquid stream may for example be discharged; this affords a simple manner of preventing degradation products and salts from building up. Since this discharge stream is relatively small, only little ⁇ -lactam antibiotic is lost with it because of solubility losses.
• both FG and ⁇ -lactam antibiotic are formed as a solid. It has been found that the solid FG formed in the concentration step can readily be filtered. In consequence, due in part to the fact that the concentration of ⁇ -lactam antibiotic now is relatively low, it was now found to be possible to recover FG by bringing the slurry to a pH at which the ⁇ -lactam antibiotic dissolves and FG does not, for example a pH between 6.5 and 11, preferably between 7 and 9.5, or between 0 and 3 , preferably between 0.3 and 2, and next separating FG as a solid by for example filtration or centrifuging.
• the separation process involves relatively little degradation of the ⁇ -lactam antibiotic in spite of the high, or low, pH.
• the filtrate can subsequently be returned to the process or reused otherwise.
• the ⁇ -lactam antibiotic in the aforementioned filtrate can be recovered by for example crystallization via a pH shift.
• Another possibility is to return the filtrate as such to the mixture containing dissolved ⁇ -lactam antibiotic and FG.
• the process according to the invention can suitably be applied in the preparation of such ⁇ -lactam antibiotics as have a phenyl glycine side chain, for example cephalexin, ampicillin, cephaclor, pivampicillin, becampicillin, talampicillin and cefaloglycine.
• Any ⁇ -lactam nucleus can in principle be used, in particular a ⁇ -lactam nucleus with the general formula (1)
• R 0 represents H or an alkoxy group having 1-3 C atoms ;
• ⁇ Y represents CH 2 , 0, S or an oxidized form of sulphur; and
• ⁇ Z represents
• Ri represents for example H, OH, halogen, an alkoxy group having 1-5 C atoms, an alkyl group having 1-5 C atoms, a cycloalkyl group having 4-8 C atoms, an aryl or a heteroaryl group having 6-10 C atoms in which the groups may or may not be substituted with for example an alkyl, an aryl, a carboxy or an alkoxy group having 1-8 C atoms; and where the carboxylic acid group may be an ester group if so desired.
• ⁇ -lactam nuclei that may be employed in the process according to the invention are penicillin derivatives, for example 6- aminopenicillanic acid (6-APA) and cephalosporanic acid derivatives, for example a 7-aminocephalosporanic acid with or without a substituent at the 3 -site, for example 7-aminocephalosporanic acid (7-ACA) , 7- aminodesacetoxycephalosporanic acid (7-ADCA) and 7- amino-3-chloro-cef-3 -em-4 -carboxylic acid (7-ACCA) .
• penicillin derivatives for example 6- aminopenicillanic acid (6-APA) and cephalosporanic acid derivatives, for example a 7-aminocephalosporanic acid with or without a substituent at the 3 -site, for example 7-aminocephalosporanic acid (7-ACA) , 7- aminodesacetoxycephalosporanic acid (7-ADCA) and 7- amino-3-
• any enzyme that is suitable as a catalyst in the coupling reaction can be used as the enzyme.
• Such enzymes include the enzymes collectively referred to as penicillin amidase or penicillin acylase .
• penicillin amidase or penicillin acylase Such enzymes are described in for example J.G. Shewale et al . , Process Biochemistry, August 1989, pp. 146-154 and in J.G. Shewale et all, Process Biochemistry International, June 1990, pp. 97- 103.
• suitable enzymes are enzymes derived from Acetobacter. in particular Acetobacter pasteurianum. Aeromonas , Alcaligenes. in particular Alcaligenes faecalis. Aphanocladium, Bacillus sp ..
• an immobilized enzyme is used, since the enzyme can be easily isolated and re-used then.
• a suitable immobilization technology is described for instance in EP-A-222462.
• Another suitable technology consists in immobilizing the Penicillin G acylase on a carrier which contains a gelating agent, for instance gelatin, and a polymer with free amino groups, for instance alginate amine, chitosan or polyethylene imine .
• enzymes may also be utilized as a crystalline substance (CLECsTM).
• Particularly suitable enzymes among the immobilized enzymes that are commercially available are the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name Enzygel®, the immobilized Penicillin-G acylase from Recordati and the immobilized Penicillin-G acylase from Pharma Biotechnology Hannover.
• the acylation agent may be for instance a D-phenyl glycine in activated form, preferably a (primary, secondary or tertiary) amide or salt thereof, or a lower alkyl (1- 4C) ester, for instance a methyl ester.
• the temperature at which the enzymatic acylation reaction is effected usually is below 40°C, preferably between -5 and 35°C.
• the pH at which the enzymatic acylation reaction is effected usually is between 5.5 and 9.5, preferably between 6.0 and 9.0. The reaction preferably is stopped almost completely when maximum conversion has virtually been achieved.
• a suitable embodiment for stopping 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 on attaining the maximum conversion.
• a combination of the two embodiments is possible also.
• the reaction mixture usually is present in the form of a suspension comprising a plurality of solids, for example the antibiotic, D-phenyl glycine and, possibly, immobilized enzyme.
• the immobilized enzyme preferably is recovered in the interest of process economics. This can suitably be accomplished by for example filtering the reaction mixture on a sieve, while stirring, the stirrer's direction of rotation being chosen so that the suspension is pumped upwards at the centre of the stirrer. Subsequently, valuable components such as the antibiotic and FG can be recovered by the process according to the invention, with the solid components, possibly apart from solid antibiotic, being dissolved first, by means of for example a pH shift. O 99/15531 - _10 n - PCT/NL98/00538
• the pH may be lowered in several ways in the framework of the invention, for instance by adding an acid to the mixture.
• Suitable acids are for example mineral acids, in particular sulphuric acid, hydrochloric acid or nitric acid.
• hydrochloric acid is used.
• the pH can be raised by for example adding a base to the mixture.
• Suitable bases are for example inorganic bases, in particular ammonium hydroxide, potassium hydroxide or sodium hydroxide.
• ammonium hydroxide is used.
• the enzymatic acylation reaction and the working-up of the reaction mixture are usually effected in water.
• the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol . % .
• suitable organic solvents that can be used are alcohols having 1-7 C atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol, or a triol, in particular glycerol .
• the process according to the invention is particularly suited for being used in working up the reaction mixture obtained after the enzymatic acylation reaction in which 6-APA is acylated with an amide of D- phenyl glycine, for example FGA, or an ester of D- phenyl glycine, for example FGM.
• measures are taken to ensure that the concentration of dissolved 6-APA in the reaction mixture is kept relatively low so that a higher conversion can be achieved than when the concentration of dissolved 6-APA is chosen to be as high as possible. Moreover, it has been found that the stirrability of the reaction mixture is significantly higher when the concentration of dissolved 6-APA is kept low.
• 'conversion' refers to the molar ratio of the ampicillin formed and the amount of 6-APA used.
• the concentration of dissolved 6- APA is expressed as the amount of 6-APA in moles per kg of the reaction mixture; the total concentration, dissolved and undissolved, of 6-APA is expressed as the amount of 6-APA plus ampicillin in moles per kg of the total reaction mixture; the total reaction mixture may contain, besides the solution, a number of solids, for example 6-APA, ampicillin, phenyl glycine and immobilized enzyme.
• the molar ratio of acylation agent and 6- APA i.e. the total amount of phenyl glycine derivative added, divided by the total amount of 6-APA added, expressed in moles, is preferably less than 2.5. It is preferred for the molar ratio to be between 1.0 and 2.0, in particular between 1.2 and 1.8.
• the enzymatic acylation reaction is preferably carried out as a batch process. If desired, the reaction can also be carried out continuously, with in-line control of the concentration of dissolved 6- APA.
• the total concentration of 6-APA plus ampicillin (in dissolved and undissolved form) in the reaction mixture preferably is chosen to be higher than
• the concentration of dissolved 6-APA during the preparation of ampicillin is preferably kept below 300 mM, in particular below 250 mM.
• the concentration of dissolved 6-APA may optionally chosen to be higher than at a lower concentration. This is because the rate of reaction is higher at higher concentrations of the acylation agent, so that 6-APA is dissolved in a high concentration for only a relatively short period.
• the concentration of 6-APA dissolved in the reaction mixture can be kept low in various ways . One possibility of keeping the concentration of dissolved 6-APA low is to initially feed only a portion of the total amount of 6-APA and to meter in the balance during the reaction.
• a drawback of this is that in that case 6-APA needs to be metered in solid form, which presents practical problems. Therefore, it is preferred in a batch process for the total amount of 6-APA to be supplied at the start of the reaction, whereupon, during the enzymatic acylation reaction, the concentration of 6-APA in the reaction mixture will decrease and the concentration of ampicillin will increase.
• a suitable method of achieving a low concentration of dissolved 6-APA is for example to keep the pH at a lower value than that at which maximum solubility of the reactants is achieved.
• a particularly suitable method of keeping the dissolved 6-APA concentration low is for example to ensure that the concentration of the phenyl glycine derivative is kept low, for example by metering in the phenyl glycine derivative partly in the course of the reaction.
• a particularly suitable embodiment is obtained when FGA is added in the form of one of its salts, preferably the salt of FGA and a mineral acid, for example FGA.HCl, FGA.l/2H 2 S0 4 and FGA.HN0 3 .
• FGA.HCl, FGA.l/2H 2 S0 4 and FGA.HN0 3 a mineral acid
• FGA.l/2H 2 S0 is used inasmuch as this salt possesses extremely high solubility.
• the various components may be present in the reaction mixture in the free form or as salts.
• the pH values mentioned are in all cases the pH values measured at room temperature.
• the invention will be further elucidated by means of the following examples, without however being restricted thereto.
• AssemblaseTM is an immobilized Escherichia coli penicillin acylase from E. coli ATCC 1105 as described in WO-A-97/04086.
• the immobilization is effected as set out in EP-A-222462, with gelatin and chitosan being used as gelating agents and glutaraldehyde as crosslinking agent.
• the ultimate activity of the Escherichia coli penicillin acylase is determined by the amount of enzyme added to the activated spherules and amounted to
• ASU/g of dry weight 1 ASU (Amoxicillin Syhthesis Unit) being defined as the amount of enzyme capable of producing 1 g of Amoxicillin.3H 2 0 from 6-APA and FGHM per hour (at 20°C; 6.5% 6-APA and 6.5% FGHM) .
• net-wet assemblaseTM (the term net-wet refers to the mass of the enzyme obtained on separating the enzyme from an enzyme slurry with the aid of a glass filter) .
• the temperature was kept at 10°C all the time.
• 423.7 g of FGA. ⁇ H 2 S0 4 solution (0,800 mole) were added at a constant rate over a period of 233 minutes.
• the pH was kept constant at approx. 6.3 by titration with 6N H 2 S0 4 .
• At t 570 minutes the amount of AMPI was maximum and the pH was reduced to 5.0 by adding 6N H 2 S0 .
• the enzyme reactor now contained: 575 mmole AMPI 15 mmole 6-APA 50 mmole FGA 365 mmole FG
• the AMPI/FG slurry prepared as described in Example II was removed from the enzyme reactor via the sieve bottom by means of stirred filtration. This was done using a pitched-blade stirrer, which was positioned at 0.5 cm over the sieve. Stirring was in upward direction at approx. 500 rpm.
• the wash waters, too, were removed via the sieve bottom by means of stirred filtration.
• the resulting AMPI/FG slurry so obtained contained > 99.8% of the total amount of AMPI produced in the enzyme reactor and > 99.5% of the total amount of FG produced. After this stirred filtration, > 99.5% of the AssemblaseTM was present in the enzyme reactor.
• This mother liquor was concentrated by evaporation with the aid of a thin-film evaporator.
• the feed to the thin-film evaporator was supplied from a storage vessel, and the product from the thin- film evaporator was returned to the same storage vessel .
• the storage vessel was stirred.
• Example IV was filtered on a glass filter (diameter 10 cm, filtration time 10 minutes) .
• the mother liquor (1155 g) was discharged.
• the AMPI/FG wet cake was quantitatively transferred to a stirred reactor with the aid of 400 g of water.
• the slurry was filtered on a glass filter (diameter 13 cm, filtration time 5 minutes) .
• Recrystallization was effected in a rig consisting, in the order as stated, of a storage vessel (4 1), a dissolving vessel (0.25 1), a filter fitted with a Seitz filter plate, and a crystallization vessel (7 1) . All vessels were provided with a stirrer, a pH electrode and a thermometer.
• the contents of the storage vessel were added to the dissolving vessel in 1 hour while the contents of the dissolving vessel were metered into the crystallization vessel, the level in the dissolving vessel being kept constant.
• the storage vessel was empty and a total of 300 ml of 6N HC1 solution had been added to the dissolving vessel.
• the pH in the crystallization vessel was reduced to 6.0 with the aid of 6N HC1.
• AMPI.3H 2 0 (excl. nuclei), that is, 92% AMPI.3H 2 0 relative to 600 mmoles of 6-APA.

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• 1997
  • 1997-09-19 NL NL1007077A patent/NL1007077C2/nl not_active IP Right Cessation
• 1998
  • 1998-09-08 IN IN2016MA1998 patent/IN188409B/en unknown
  • 1998-09-18 EP EP98944337A patent/EP1017698A1/de not_active Withdrawn
  • 1998-09-18 AU AU91897/98A patent/AU9189798A/en not_active Abandoned
  • 1998-09-18 BR BR9812482-0A patent/BR9812482A/pt not_active IP Right Cessation
  • 1998-09-18 TR TR2000/00880T patent/TR200000880T2/xx unknown
  • 1998-09-18 WO PCT/NL1998/000538 patent/WO1999015531A1/en not_active Application Discontinuation
  • 1998-09-18 KR KR1020007002763A patent/KR20010024028A/ko not_active Application Discontinuation
  • 1998-09-18 CN CN98811310A patent/CN1279685A/zh active Pending

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