Process for preparing cephalosporin derivatives

Abstract

An enzymatic process for preparing 3-thiolated 7-aminocephalosporanic acid derivatives comprises the steps of enzymatically converting a 3-thiolated cephalosporin C of the formula I: to form a 3-thiolated-glutaryl-7-ACA of the formula II and enzymatically converting a compound of formula II to form a 3-thiolated-7-ACA of the formula III wherein R is a heterocyclic group comprising at least one nitrogen atom and R 1 and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.

1 . An enzymatic process for preparing 3-thiolated 7-aminocephalosporanic acid derivatives comprising the steps:—
 enzymatically converting a 3-thiolated cephalosporin C of the formula I:  
                     
 to form a 3-thiolated-glutaryl-7-ACA of the formula II  
                     
 and enzymatically converting a compound of formula II to form a 3-thiolated-7-ACA of the formula III  
                     
  wherein R is a heterocyclic group comprising at least one nitrogen atom and R 1  and R 2  are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.  
 
     
     
         2 . A process as claimed in  claim 1  wherein the 3-thiolated cephalosporin C of formula I is converted into a 3-thiolatedglutaryl-7-ACA of the formula II by:—
 reacting a compound of formula I with immobilised D-Amino acid oxidase in the presence of molecular oxygen;  
 separating the supported enzyme from the aqueous reaction mixture; and  
 adding hydrogen peroxide to convert the 3-thiolated-aketoadipyl cephalosporanic acid into a compound of formula II.  
 
     
     
         3 . A process as claimed in  claim 2  wherein the compound of formula I is reacted with immobilised D-Amino acid oxidase at a pressure of about 2 bar absolute, a pH of from 6.0 to 8.0, and a temperature of from 20° C. to 30° C. for a period of from 0.5 to 3 hours.  
     
     
         4 . A process as claimed in  claim 2  including the step of washing the supported enzyme with a concentrated salt solution and adding hydrogen peroxide preferably in an amount equivalent to 30 to 50 ppm to the solution thus formed.  
     
     
         5 . A process as claimed in  claim 1  comprising the step of eliminating excess hydrogen peroxide from the solution, preferably by adding a catalyst to the solution.  
     
     
         6 . A process as claimed in  claim 5  wherein the excess hydrogen peroxide is removed by adding catalase to the solution.  
     
     
         7 . A process as claimed in  claim 1  wherein a compound of formula II is converted into a compound of formula III by contacting a compound of formula II with immobilised glutaryl-7-ACA acylase.  
     
     
         8 . A process as claimed in  claim 7  wherein the reaction to form a compound of formula III from a compound of formula II is carried out at ambient pressure, at a pH of from 6.0 to 8.5 and at a temperature of from 20° C. to 35° C., for a period of from 0.5 to 3 hours under an inert atmosphere.  
     
     
         9 . A process as claimed in  claim 7  wherein the compound of formula III is precipitated by acidifying the reaction medium and the precipitate thus formed is subsequently washed and dried.  
     
     
         10 . A process as claimed in  claim 1  wherein the enzymes are immobilised using a suitable cross-linker agent in a suitable solid support.  
     
     
         11 . A process as claimed in  claim 10  wherein the enzymes are in the form of crystals of a size suitable for use as a biocatalyst.  
     
     
         12 . A process as claimed in  claim 1  wherein the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution.  
     
     
         13 . A process as claimed in  claim 1  wherein the or each enzymatic process is carried out in a column.  
     
     
         14 . A process as claimed in  claim 1  including the step of recovering the enzyme for reuse.  
     
     
         15 . A process as claimed in  claim 1  wherein crystallisation of a compound of formula III is carried out at an acidic pH.  
     
     
         16 . A process as claimed in  claim 1  wherein the enzymatic conversion of a 3 thiolated cephalosporin C of the formula I to form a 3 thiolated-7-ACA of the formula III is carried out in one pot.  
     
     
         17 . A process as claimed in  claim 1  wherein R is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom.  
     
     
         18 . A process as claimed in  claim 1  wherein R is a heterocyclic group selected from any one or more of the group comprising thienyl, diazolyl, tetrazolyl, thiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzo thiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5-methyl-1,3.4-thiadiazol-2-yl, 1-methyl-1H-tetrazol-5-yl or 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.  
     
     
         19 . A process as claimed in  claim 1 , wherein compounds of formula I are in a solid form or in the form of a non-toxic salt thereof.  
     
     
         20 . A 3-thiolated-7-ACA of the formula III whenever prepared by a process as claimed in  claim 1 .  
     
     
         21 . A process for the preparation of cephalosporin C antibiotics and derivatives thereof comprising forming a compound of formula III as defined in  claim 1  and subsequent enzymation.  
     
     
         22 . A process as claimed in  claim 21  wherein the antibiotic is cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam or ceftriaxone.  
     
     
         23 . An enzymatic process for preparing 3-thiolated 7-aminocephalosporanic acid derivatives as claimed in  claim 1  comprising the steps:—
 reacting cephalosporin C with a thiol compound of the general Formula IV  
 R—SH  (IV)  
 wherein R is a heterocyclic group comprising at least one nitrogen atom,  
 to form a compound of formula I  
 and, after formation of the compound of formula I removing excess thiol of Formula IV.  
 
     
     
         24 . A process as claimed in  claim 23  wherein the excess thiol is removed by adsorption on an anion exchange resin.  
     
     
         25 . A process as claimed in  claim 24  wherein the anion exchange resin is a microporous resin having a cross-linked acrylic copolymer structure.  
     
     
         26 . A process as claimed in  claim 25  wherein the anion exchange resin comprises an 8% cross-linking containing functional thialkyl benzyl ammonium group.  
     
     
         27 . A process as claimed in  claim 25  wherein the resin is in the chloride, hydroxy, phosphate or acetate cycle.  
     
     
         28 . A process as claimed in  claim 23  wherein the excess thiol is removed by crystallisation.  
     
     
         29 . A process as claimed in  claim 28  wherein crystallisation is carried out at an acidic pH.  
     
     
         30 . A process as claimed in  claim 23  wherein the excess thiol is removed by crystallisation followed by adsorption on an anion exchange resin.  
     
     
         31 . A process as claimed in  claim 23  wherein the cephalosporin C is in an aqueous medium.  
     
     
         32 . A process as claimed in  claim 23  wherein the cephalosporin C is in the form of a concentrated cephalosporin C solution.  
     
     
         33 . A process as claimed in  claim 23  wherein the reaction is carried out at a pH of between 5.5 and 8.0, at a temperature of from 60° C. to 80° C., for a period of from 1 to 12 hours.  
     
     
         34 . A process as claimed in  claim 33  wherein the reaction is carried out at a pH of approximately 6.0 and at a temperature of approximately 65° C.  
     
     
         35 . A process as claimed in  claim 23  wherein the thiol compound is present in an amount of between 1 and 5 mol/mol of cephalosporin C.  
     
     
         36 . A process as claimed in  claim 23  wherein R is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom.  
     
     
         37 . A process as claimed in  claim 23  wherein R is a heterocyclic group selected from any one or more of thienyl, diazolyl, thiazolyl, tetrazolyl, thiadiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-tetrazol-5-yl or 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.  
     
     
         38 . A process as claimed in  claim 23  wherein compounds of Formula I are in a solid form or in the form of a non-toxic salt thereof.  
     
     
         39 . A compound of formula:— 
       
         
           
           
               
               
           
         
         wherein R is a heterocyclic group comprising at least one nitrogen atom,  
         obtained by a process as claimed in any preceding claim.  
       
     
     
         40 . A compound of the formula:— 
       
         
           
           
               
               
           
         
         wherein in formula I R is 5-methyl-1,3,4-thiadiazol-2-yl.  
       
     
     
         41 . A compound of the formula:— 
       
         
           
           
               
               
           
         
         wherein in formula I, R is 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.  
       
     
     
         42 . A process for the preparation of cephalosporin C antibiotics and derivatives thereof comprising forming a compound of formula I as defined in  claim 1  and subsequent enzymation of the compound of formula I.  
     
     
         43 . A process as claimed in  claim 42  wherein the antibiotic is cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.