HK1082000B - Method for producing cephalexin - Google Patents

Description

Process for preparing cephalexin

The invention relates to a method for producing cephalexin by means of penicillin amidase immobilized on bead-shaped, cross-linked, hydrophilic copolymers having binding activity to ligands having nucleophilic groups.

Prior Art

The synthesis of semi-synthetic β -lactam antibiotics by acylation of β -lactam residues (β -lactam nucleus) with activated side chains such as amides or esters using penicillin acylases (penicillin amidases) is well known to the person skilled in the art.

In most cases, the enzyme is bound to a solid, water-insoluble support and subsequently contacted with the β -lactam nucleus and the activated side chain in aqueous solution.

The disadvantage of the processes disclosed so far is that the hydrolysis of the desired compounds by enzymes to the non-valuable side chain acids with respect to the activated side chain and the ratio of the hydrolysis of the desired products, the so-called S/H value, is often disadvantageous and economical production is difficult to achieve.

It is known from WO93/12250 that in the synthesis of semisynthetic beta-lactam antibiotics, cefadroxil and cefalexin by E.coli penicillin amidase immobilized on Eupergit ® (R ö hm GmbH & Co. KG, Damstat, Germany, see also comparative example 1), the S/H value can be influenced favourably by the choice of the reaction conditions. However, no influence of the properties of the support material is taught. The disadvantages of the process taught in WO93/12250 are, inter alia, the need to isolate cephalexin complexed with beta-naphthol from the reaction mixture, so that a subsequent purification step is necessary and a reduction in yield occurs.

Therefore, there are attempts to develop an optimal carrier material. For example, WO97/04086 discloses E.coli penicillin amidase immobilized on a support material consisting of a swelling agent and a polymer containing free amino groups, and its use for the preparation of beta-lactam derivatives. However, a disadvantage of this disclosed process for the preparation of cephalexin is that a 3-fold molar excess of the β -lactam core 7-aminodesacetoxycephalosporanic acid (7-ADCA) is used compared to the activated side chain D-Phenylglycinamide (PGA). If a stoichiometric excess of beta-lactam cores is used, these cores must be recovered on an industrial scale in order to be able to operate economically. This is expensive and results in a reduced yield. In addition, the environment also results in impure products since the nucleus is unstable.

Likewise, EP 0730035 teaches a process for preparing cephalexin in acceptable yields on a specific support. However, the carrier material used (Emphaze)^TM) Has a particle size of only 60 to 80 μm. This has a great disadvantage for industrial applications. For example, a chromatography column packed with this material has only a low flow rate.

Support materials for enzymes are described in DE 19804518. It is mentioned that this material can be used for the enzymatic synthesis of amoxicillin and ampicillin. There is no mention of a process suitable for the synthesis of cephalexin by means of immobilized penicillin amidase.

Objects and solutions

In view of the prior art discussed above, it is therefore an object of the present invention to provide an improved process for the synthesis of cephalexin, which overcomes the above disadvantages.

The method according to the invention should in particular enable an advantageous S/H value to be achieved. Furthermore, the support material used should have a particle size of 120-250 μm, which is advantageous for industrial processes.

This object is achieved by the method defined in claim 1. Preferred embodiments of the process of the invention are defined in the dependent claims depending on claim 1.

In particular, the above object was successfully achieved in an unforeseeable, technically simple manner by means of bead-form, crosslinked, hydrophilic support polymer materials having binding activity for ligands carrying nucleophilic groups, which polymer materials can be prepared by phase-inversion bead polymerization of a monomer phase consisting of monomers and diluents, wherein the monomers involved are:

(a)5 to 40% by weight of hydrophilic, free-radically polymerizable monomers having vinyl groups which form at least 10% aqueous solutions at room temperature,

(b) 30-50% by weight of a radically polymerizable monomer having a vinyl group and an additional functional group capable of generating covalent bonds during a polymer-like reaction with nucleophilic groups of a ligand,

(c)20 to 60% by weight of a crosslinkable, free-radically polymerizable monomer having two or more ethylenically unsaturated polymerizable groups,

with the proviso that the sum of a), b) and c) is 100% by weight, the diluent used is a mixture of methanol and water in a ratio of 1: 1.0 to 1: 4.0, the monomer phase being distributed in the form of droplets in a continuous phase consisting of an aliphatic hydrocarbon organic solvent having from 5 to 7 carbon atoms, the monomer phase being present in a ratio of 1: 2.0 to 1: 4.0 to the continuous phase, and the free-radical polymerization being carried out in this form in the presence of a polymerization initiator and a protective colloid, with the proviso that the ratio of monomer to diluent is 1: 1.7 to 1: 2.4, coating with penicillinase, and bringing the coated support into contact with an aqueous solution containing

(i) 7-aminodesacetoxycephalosporanic acid and

(ii) d-phenylglycinamide is used as the raw material,

the ratio is from 1: 2 to 2: 1, preferably from 1.5: 1 to 1: 1.5, particularly preferably approximately equimolar, i.e.from 1.2: 1 to 1: 1.2.

The support materials used and their preparation are described in DE 19804518.

Practice of the invention

Preparation of the support Material

Monomer

In order to ensure the hydrophilicity of the monomer mixture, it must consist essentially of hydrophilic monomers. Hydrophilic monomers are those which form at least a 10% aqueous solution at room temperature and preferably contain no ionic groups or groups which can be ionized by the addition of acids or bases.

The monomers a) are 5 to 40% by weight, 8 to 35% by weight, in particular 9 to 12% by weight, of hydrophilic, vinyl-bearing, free-radically polymerizable monomers which form at least 10% aqueous solutions at room temperature.

Particularly suitable monomers a) are acrylamide and/or methacrylamide, with methacrylamide being preferred here. Further examples are hydroxyalkyl esters of unsaturated polymerizable carboxylic acids, such as hydroxyethyl acrylate and hydroxyethyl methacrylate or N-vinylpyrrolidone.

The monomers b) are from 30 to 50% by weight, preferably from 35 to 45% by weight, of free-radically polymerizable monomers having a vinyl group and an additional functional group, preferably an oxirane group (epoxy group), which is capable of producing a covalent bond in a polymer-analogous reaction with the nucleophilic groups of the ligand. Ethylene oxide groups are particularly suitable to bind the ligands and obtain their biological activity.

Preferred monomers b) are glycidyl methacrylate and/or allyl glycidyl ether. It is particularly preferred that both monomers have been used together in approximately equal amounts.

The monomers c) are from 20 to 60% by weight, in particular from 25 to 55% by weight, particularly preferably from 40 to 55% by weight, of hydrophilic, crosslinkable, free-radically polymerizable monomers having two or more ethylenically unsaturated polymerizable groups. Preferred monomers c) are N, N '-methylenebis (acrylamide) or N, N' -methylenebis (methacrylamide). N, N' -methylenebis (methacrylamide) is particularly preferred. Optionally, from 0 to 10% by weight of other crosslinkable, free-radically polymerizable monomers having two or more ethylenically unsaturated polymerizable groups can also be used. Suitable are hydrophilic di (meth) acrylates, for example polyethylene oxide di (meth) acrylates.

The sum of the monomers a), b) and c) is in each case 100% by weight.

Diluent

The monomer phase consists of the monomers a) to c) dissolved in a diluent which must be a mixture of methanol and water in a ratio of 1: 1.0 to 1: 4.0. A particularly advantageous mixing ratio of methanol and water is from 1: 1.2 to 1: 2.5, in particular from 1: 1.3 to 1: 1.7.

Monomer to diluent ratio

The monomer to diluent ratio is particularly critical. It must be in the range from 1: 1.7 to 1: 2.4, particularly preferably in the range from 1.9 to 2.1.

Continuous phase

Suitable as the continuous phase are aliphatic hydrocarbon organic solvents having from 4 to 7 carbon atoms. N-heptane is preferred, and cyclohexane is particularly preferred.

Ratio of monomer phase/continuous phase

The ratio of the monomer phase to the continuous phase formed by the organic solvent must be from 1: 2.0 to 1: 4.0, preferably from 1: 2.8 to 1: 3.3.

Other process conditions

As further components, the suspended monomer phase contains, in a known manner, a polymerization initiator, preferably a sulfur-free initiator, particularly preferably 4, 4' -azobis (4-pentanoic acid), and also a protective colloid (emulsifier), for example a copolymer of 95 parts of n-butyl methacrylate and 5 parts of 2-trimethylammoniumethyl methacrylate having a weight-average molecular weight of 30000-80000.

Bead polymerization (also known as suspension polymerization) is furthermore carried out in a known manner by, for example, initially charging the continuous phase and the protective colloid, distributing the monomer phase, also in the presence of initiator, in the organic phase, for example at from 40 to 60 ℃ with stirring, and then heating to from 60 to 70 ℃. The water/methanol mixture can be removed, for example, near completely azeotropically over a period of 6 hours. The mixture was allowed to react for about 3-5 hours, and then cooled to room temperature. The beads formed are filtered off with suction and dried in vacuo for, for example, 12 hours. Alternatively, the bead polymer is filtered off and washed with water. Drying is preferably carried out in a fluidized-bed dryer, since in this way solvent residues can be removed particularly effectively. The resulting polymer beads (═ carrier polymer material) have a size of from 50 to 500. mu.m, in particular from 120 to 250. mu.m. By binding capacity is meant the enzymatic activity which can be achieved at maximum loading of the support polymer material with an enzyme. The binding capacity is expressed as penicillin amidase activity in units per gram of support polymer beads [ U/g wet ]. The binding capacity of the carrier polymer beads of the invention is measured as at least 220[ U/g wet ].

The swellability of the polymer beads in water is indicated by the swelling index [ ml wet/ml dry ]. The carrier polymer beads of the present invention have a swell index of no greater than 1.5.

Coating of the support materials that can be used according to the invention

In the examples, the support material was coated in potassium phosphate buffer at pH 7.5. However, it is clear to the person skilled in the art that there are a very large number of other ways of ensuring satisfactory coating.

Synthesis of cefalexin

The precursors (i) 7-aminodesacetoxycephalosporanic acid and (ii) D-phenylglycinamide are used in a concentration of 10 to 500mM, preferably 50 to 300mM and particularly preferably 150 mM and 250 mM.

The invention has the advantages of

The process of the invention enables the synthesis of cephalexin with an advantageous S/H ratio (synthesis/hydrolysis). Advantageously in the present invention, this is achieved by using support materials having a particle size of from 50 to 500. mu.m, in particular 120-250. mu.m. This results in better performance properties, for example higher flow rates in fixed-bed reactors. The higher flow rates lead to better space-time yields. Larger support particles are also advantageous in batch processes because they can be filtered out more quickly. This again increases the space-time yield and thus the economics of the process.

Examples

(the following assay methods are common to those skilled in the art of porous support polymer materials and are mentioned for completeness only)

Determination of the binding Capacity of penicillin amidase (═ penicillin G-acylase) in E.coli (EC 3.5.1.11)

a) Covalent bonding of penicillin amidase on carrier polymer materials

1g of the carrier polymer material was added to 1530 units of penicillin amidase in 5ml of sterile 1M potassium phosphate buffer (pH7.5) and incubated for 48 hours at 23 ℃.

The polymer beads were then loaded onto a sintered glass funnel (porosity 2 or 3) and washed twice with deionized water, followed by 0.1M potassium phosphate buffer (pH7.5) containing 0.05% ethyl 4-hydroxybenzoate, by suction filtration on the funnel. The wet weight of the resulting beads loaded with penicillin acylase was determined.

b) Determination of binding Capacity

250-300mg of wet penicillin amidase coupled carrier polymer material (polymer beads) were added to a solution of 20ml of 2% penicillin G in 0.05M, pH7.5 potassium phosphate buffer containing 0.05% ethyl 4-hydroxybenzoate at 37 ℃. Under uniform stirring, the liberated phenylacetic acid is titrated with 0.5M NaOH, keeping the pH constant at 7.8 for about 10 minutes, where the consumption of NaOH is recorded.

The polymer beads were then obtained as in a) from 20ml of 0.05M, pH7.5 potassium phosphate buffer containing 0.05% ethyl 4-hydroxybenzoate by aspiration through a glass funnel, and this measurement was repeated twice.

c) Calculation of binding Capacity

The linear range of the curve (typically this range is 1-5 minutes) is measured as the basis for the calculation and extrapolated for 10 minute intervals. The binding capacity is expressed as penicillin amidase units per gram of wet carrier polymer material (U/g wet state). One unit corresponds to 1. mu. mol of hydrolyzed penicillin G per minute (. mu. mol/min); here 1 liter of 0.5M NaOH corresponds to 500mol of hydrolyzed penicillin G (the water content of the carrier polymer material is approximately constant and therefore negligible).

Comparative example 1

1530 units of penicillin amidase derived from E.coli were dissolved in 6ml of sterilized 1M potassium phosphate buffer (pH 7.5). This solution was added to 1g of Eupergit ® C (R ö hmGmbH & co. kg, dalmstadt, germany) and the resulting suspension was incubated at 23 ℃ for 72 hours. The polymer beads were collected on a sintered glass funnel and washed with 0.1M potassium phosphate buffer. The following synthesis and determination of cephalexin S/H values were carried out according to example 4 or 5.

Eupergit ® C (a copolymer of N, N' -methylenebismethacrylamide, allyl glycidyl ether and methacrylamide) and a process for its preparation are described in DE-C2722751, U.S. Pat. No. 3, 490713 or U.S. Pat. No. 4511694.

Comparative example 2

Cephalexin was synthesized as described in example 4 according to WO97/04086 using the type A or type B enzymes disclosed in this document.

Comparative example 3

The E.coli penicillin amidase was immobilized on Sepabeads ® FP-EP or Sepabeads ® FP-EP/G (resolution S.R.I., Milan, Italy) as described in comparative example 1. The following synthesis of cephalexin and determination of the S/H value were carried out according to example 4 or 5.

Sepabeads ® FP-EP or Sepabeads ® FP-EP/G is a highly polymerized, crosslinked acrylic copolymer with oxirane groups as in Eupergit ®. The average particle size is 150 μm to 300 μm according to the manufacturer's information.

Comparative example 4

Emphaze for measurement^TMOr the flow rate of a chromatography column packed with a material that can be used according to the invention.

Materials: emphaze Ultralink^TMBio-supportive media, Lot # DC53515, Pierce, with an average particle size of 50-80 μm, according to the manufacturer's information; the average particle size of the materials which can be used according to the invention is 208 μm.

Comparable flow rates were determined empty on borosilicate glass chromatography columns (column size 1X 20cm) with glass funnel bottoms and polypropylene end caps. The support material was suspended in water overnight and then washed with water into each column, and the liquid was allowed to settle and slowly flow out to give a packed bed 6.5cm in height. Possible voids are driven away by tapping. The upper open column formed a constant water column of 23cm by the inflow of water. The flow rate is obtained by means of hydrostatic pressure only. The flow rate was determined by means of a stopwatch and a 10ml measuring cylinder.

The flow rate determined for the formulation usable according to the invention was 4.25 ml/min. For Emphaze^TMThe flow rate was determined to be 0.71 ml/min. The higher the flow rate (higher space-time yield), the better the suitability of the spherical particles for operation in a fixed bed reactor. The pressure drop in a fixed bed reactor can also be mathematically calculated as: buchholz and B.G ö delmann, "characterization of immobilized biocatalysts", the Dechema monograph, vol 84, compiled as K.Buchholz, VCH Weinheim, 1979, p 128-129).

It is clearly seen that^TMThe materials which can be used according to the invention have better application skills for use in fixed-bed reactors than the materials which can be used according to the inventionPerformance of the operation.

Examples 1 to 3

Test conditions consistent in examples 1-3:

in a 2 l stirred flask with thermometer, water separator, reflux condenser and nitrogen inlet, 3g of a copolymer of 95 parts of n-butyl methacrylate and 5 parts of 2-trimethylammonioethyl methacrylate as protective colloid and 5g of dry ice were introduced. A monomer phase consisting of water and methanol in a methanol to water ratio of 1: 1.5 (example 1) or of formamide (examples 2 and 3) as diluent at 50 ℃ with stirring and introduction of nitrogen, and

10g of a methacrylic acid amide,

20g of an allyl glycidyl ether and, in each case,

20g of glycidyl methacrylate and

50g of a methylene-bis-methacrylamide,

and

2g of 4, 4' -azobis (4-cyanovaleric acid) (as polymerization initiator),

distributed in the organic phase and then heated to boiling at 65-70 ℃. The mixture was incubated for about 6 hours and cooled to room temperature. The resulting polymer beads were suction filtered, washed and dried in a fluid bed dryer. The binding capacity [ U/g wet ] and swelling index [ ml wet/ml dry ] of the penicillin amidase were then determined.

The table below lists the important test parameters and results for examples 1-3

	Example 1 (according to the invention)	Example 2 (comparative example)	Example 3 (comparative example)
				Organic solvent (continuous phase)	952g cyclohexane	669g of cyclohexane	530g of n-heptane +530g of tetrachloroethylene
Total amount of monomer	100g	100g	100g
				Diluent	80g of methanol +120g of water (═ 1: 1.5)	263g formamide	264g of formamide
Monomer + diluent (monomer phase)	300g	363g	364g
				Monomer/diluent ratio	1∶2	1∶2.63	1∶2.64
Ratio of monomer phase/continuous phase	1∶3.2	1∶1.8	1∶2.9
				Binding Capacity of penicillin amidase (1530U) (U/g Wet State)]	252	194	192
Swelling index [ ml wet/ml dry]	1.3	4.0	3.9

Example 4

Synthesis of cefalexin

The reaction was carried out in a fixed bed reactor at 25 ℃ and pH 7.5. 10ml of an aqueous solution of a mixture consisting of 0.2M of 7-ADCA and 0.2M of D-phenylglycinamide were led through a column containing 0.5ml of immobilized penicillin amidase. During the reaction, the pH was kept constant by adding HCl in a storage vessel under stirring with an attached stirrer. Samples were taken at regular intervals and analyzed by HPLC as shown in example 5.

Example 5

The reaction products of example 4 and comparative examples 1 to 3 were analyzed by HPLC using an RP-8 column (MerckkKGaA, Dammstat, Germany). The mobile phase used was a sterile 67mM potassium phosphate buffer pH 7.5. Cephalexin was eluted with 30% (v/v) aqueous methanol.

Synthesis Rate of Cefalexin (V)_Ceph) And the hydrolysis rate (V) of D-phenylglycinamide_D-PhG) From the HPLC analysis determination, the S/H value (synthesis rate/hydrolysis rate ratio (V) was calculated_Ceph/V_D-PhG)). The results are listed in the following table.

Enzyme source	Carrier/stationary phase	VCeph/VD-PhG
			Escherichia coli	According to the invention	4.6
Escherichia coli	Type A in WO97/04086 (see comparative example 2)	3.3
			Escherichia coli	Type B in WO97/04086 (see comparative example 2)	3.3
Escherichia coli	Comparative example 1	3.5
			Escherichia coli	Comparative example 3(Sepabeads FP-EP)	3.2
Escherichia coli	Comparative example 3(Sepabeads FP-EP/G)	2.9

The values of the process of the invention consist of 7 test series carried out in parallel, which give the following S/H values: 5.3, 4.7, 3.6, 4.6, 4.3, 5.2, 4.7;

it is clear that a significant increase in S/H value of about 30% can be achieved with the process of the invention compared with the comparative examples.

Claims (5)

1. Process for the preparation of cephalexin, characterized in that a beaded, crosslinked, hydrophilic carrier polymer material having binding activity for ligands having nucleophilic groups, which carrier polymer material can be prepared by phase-inversion bead polymerization of a monomer phase consisting of monomers and a diluent, wherein the monomers contained therein are:

(a)5 to 40% by weight of hydrophilic, free-radically polymerizable monomers having vinyl groups which form at least 10% aqueous solutions at room temperature,

(b) 30-50% by weight of a radically polymerizable monomer having a vinyl group and an additional functional group capable of generating covalent bonds during a polymer-like reaction with nucleophilic groups of a ligand,

(c)20 to 60% by weight of a crosslinkable free-radically polymerizable monomer having two or more ethylenically unsaturated polymerizable groups,

with the proviso that the sum of a), b) and c) is 100% by weight, the diluent used is a mixture of methanol and water in a methanol-to-water ratio of from 1: 1.0 to 1: 4.0, the monomer phase being distributed in the form of droplets in a continuous phase consisting of an aliphatic hydrocarbon organic solvent having from 5 to 7 carbon atoms, the monomer phase and the continuous phase having a ratio of from 1: 2.0 to 1: 4.0, and in this form the free-radical polymerization is carried out in the presence of a polymerization initiator and a protective colloid, with the proviso that the ratio of monomer to diluent is from 1: 1.7 to 1: 2.4, and the coated carrier polymer material is brought into contact with an aqueous solution containing

(i) 7-aminodesacetoxycephalosporanic acid and

(ii) d-phenylglycinamide is used as the raw material,

the ratio of the components is 1: 2-2: 1.

2. A process as claimed in claim 1, wherein the monomers used are

a) (ii) acrylamide and/or methacrylamide,

b) glycidyl methacrylate and/or allyl glycidyl ether,

c) n, N '-methylenebis (acrylamide) or N, N' -methylenebis (methacrylamide).

3. A process according to claim 1 or 2, characterized in that cyclohexane is used as organic solvent.

4. A method according to claim 1 or 2, characterized in that the penicillin amidase is derived from e.

5. Use of a carrier polymeric material as defined in the process according to any one of claims 1 to 4 for the synthesis of cephalexin.