Process for the preparation of poly-alpha-glutamic acid and derivatives thereof

Abstract

The invention relates to an improved process for the preparation of poly-α-glutamic acids which comprises the polymerization of tertiary γ-esters of α-glutamic acid N-carboxy anhydride with appropriate solvents and initiators, followed by acid hydrolysis of the resulting poly-α-glutamic acid-γ-ester. The process is particularly advantageous in that it allows one to carefully control the molecular weight of the resulting poly-α-glutamic acid. The invention also relates to poly-α-glutamic acids capped at the amino terminus with carboxylic acids or amino acids and to a process for the preparation thereof.

1 . A process for the preparation of poly-α-glutamic acid of formula (I)  
     
       
         
         
             
             
         
       
     
     wherein the symbol * indicates a chiral center and n is between 60 and 310, so that the poly-α-glutamic acid has a molecular weight ranging from 8,000 to 40,000 Da 
 said process comprising the steps of:  
 polymerization of a tertiary γ-ester of α-glutamic acid N-carboxy anhydride of formula (II)  
                     
 wherein the symbol * is a defined above and R is selected from t-butyl, 1,1-dimethylpropyl and 1,1-dimethylbutyl  
 in water or in an organic solvent selected from: tetrahydrofuran, 1,4-dioxane, dimethylformamide, 1,4-dioxane/DMF and 1,4-dioxane/tetrahydrofuran mixture with an initiator selected from potassium tert-butoxide, sodium methoxide, diisopropylethylamine, 1,8-diazabicyclo[5,4,0]undec-7-ene, dimethylaminopyridine and L-glutamic acid-γ-tert-butylester, to give a compound of formula (III)  
                     
 wherein * and R are as defined above and R′ is hydrogen when the initiator is selected from diisopropylethylamine, 1,8-diazabicyclo[5,4,0]undec-7-ene, 4-dimethylaminopyridine, glutamic acid dimethyl ester and glutamic acid-γ-tert-butyl ester or R′ is a t-butyl or methyl group when the initiator is potassium tert-butoxide and sodium methoxide respectively; and followed by  
 b) acid hydrolysis of the γ- and α-ester groups to give a compound of formula (I).  
 
   
   
       2 . The process according to  claim 1  wherein the solvent is 1,4-dioxane and the initiator is 1,8-diazabicyclo[5,4,0]undec-7-ene.  
   
   
       3 . The process according to  claim 1  wherein the temperature ranges from 10 to 50° C.  
   
   
       4 . The process according to  claim 2  wherein the temperature ranges from 10 to 50° V.  
   
   
       5 . The process according to  claim 1  wherein the concentration of the tertiary γ-ester of α-glutamic acid N-carboxy anhydride ranges from 0.1 to 0.3 M.  
   
   
       6 . The process according to  claim 2  wherein the concentration of the tertiary γ-ester of α-glutamic acid N-carboxy anhydride ranges from 0.1 to 0.3 M.  
   
   
       7 . The process according to  claim 3  wherein the concentration of the tertiary γ-ester of α-glutamic acid N-carboxy anhydride ranges from 0.1 to 0.3 M.  
   
   
       8 . The process according to  claim 4  wherein the concentration of the tertiary γ-ester of α-glutamic acid N-carboxy anhydride ranges from 0.1 to 0.3 M.  
   
   
       9 . The process according to  claim 1  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       10 . The process according to  claim 2  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       11 . The process according to  claim 3  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       12 . The process according to  claim 4  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       13 . The process according to  claim 5  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       14 . The process according to  claim 6  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       15 . The process according to  claim 7  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       16 . The process according to  claim 8  wherein step b) is carried out in an acid selected from trifluoroacetic acid, formic acid and water/formic acid mixtures at a temperature ranging from 20 to 60° C.  
   
   
       17 . The process according to  claim 1  wherein R is t-butyl.  
   
   
       18 . The process according to any one of  claims 1  to  17  wherein the molecular weight of the poly-α-glutamic acid (I) ranges from 13,000 to 16,000 Da and the polydispersity index is ≦1.5.  
   
   
       19 . A process for the preparation of a poly-α-glutamic acid derivative of formula (IV)  
     
       
         
         
             
             
         
       
     
     wherein the symbol * indicates a chiral center and n is an integer comprised between 60 and 310 and R 1 CO— is selected from: 
 (C 1 -C 10 )alkylcarbonyl;  
 (C 4 -C 8 )cycloalkylcarbonyl;  
 (C 2 -C 6 )carboxyalkylcarbonyl;  
 (C 6 -C 10 )arylcarbonyl;  
 (C 6 -C 10 )aryl(C 1 -C 10 )alkylcarbonyl;  
 (C 1 -C 10 )alkyl(C 6 -C 10 )arylcarbonyl;  
 (C 5 -C 10 )heteroarylcarbonyl and (C 5 -C 10 )heteroaryl(C 1 -C 10 )alkylcarbonyl wherein the heteroaromatic ring contains one or more nitrogen, oxygen or sulphur atoms; and  
 D- or L-amino acid and non-natural amino acid residues;  
 said process comprising the steps of:  
 a) polymerization of a tertiary γ-ester of an α-glutamic acid N-carboxy anhydride of formula (II)  
                     
 wherein * is as defined above and R is selected from t-butyl, 1,1-dimethylpropyl and 1,1-dimethylbutyl  
 in water or in an organic solvent selected from: tetrahydrofuran, 1,4-dioxane, dimethylformamide, 1,4-dioxane/DMF and 1,4-dioxane/tetrahydrofuran mixtures with an initiator selected from potassium tert-butoxide, sodium methoxide, diisopropylethylamine, 1,8-diazabicyclo[5,4,0]undec-7-ene, dimethylaminopyridine and L-glutamic acid-γ-tert-butylester, to give a compound of formula (III);  
 b) reaction of a compound of formula (III)  
                     
 obtained according to step a) above with a carboxylic acid R 1 COOH, or an acyl chloride R 1 COCl or an anhydride (R 1 CO) 2 O wherein R 1  is as defined above, in the presence of a dehydrating agent to give a compound of formula (V)  
                     
 and  
 c) the hydrolysis of the compound of formula (V) to give a compound of formula (IV).  
 
   
   
       20 . The process according to  claim 19  wherein (C 1 -C 10 )alkylcarbonyl is acetyl or butyryl.  
   
   
       21 . The process according to  claim 19  wherein (C 4 -C 8 )cycloalkylcarbonyl is cyclopropylcarbonyl, cyclobutanecarbonyl, or cyclohexylcarbonyl.  
   
   
       22 . The process according to  claim 19  wherein (C 2 -C 6 )carboxyalkylcarbonyl is succinyl.  
   
   
       23 . The process according to  claim 19  wherein (C 6 -C 10 )arylcarbonyl is benzoyl, 1-naphthoyl or 2-naphthoyl.  
   
   
       24 . The process according to  claim 19  wherein (C 6 -C 10 )aryl(C 1 -C 10 )alkylcarbonyl is phenylacetyl or phenylbutyryl.  
   
   
       25 . The process according to  claim 19  wherein (C 1 -C 10 )alkyl(C 6 -C 10 )aryl carbonyl is o-, m- or p-tolyl.  
   
   
       26 . The process according to  claim 19  wherein (C 5 -C 10 )heteroarylcarbonyl is nicotinoyl, N-methylpyrrole-3-carbonyl, 3-thiophenecarbonyl or 3-quinolinecarbonyl.  
   
   
       27 . The process according to  claim 19  wherein (C 5 -C 10 )heteroaryl(C 1 -C 10 )alkylcarbonyl is 3-pyridylacetyl.  
   
   
       28 . The process according to  claim 19  wherein D- or L-natural amino acid residues are those deriving from glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, pyroglutamic acid, phenylalanine, tryptophan and cysteine.  
   
   
       29 . The process according to  claim 28  wherein the amino acid residue is a phenylalanine residue.  
   
   
       30 . The process according to  claim 19  wherein D or L non-natural amino acid residues are those deriving from β-alanine, α,α-dimethylglycine, α-phenylglycine, homophenylalanine, 3-amino-3-(4-methylphenyl)propionic acid, and 2-(1-aminocyclopentyl)acetic acid.  
   
   
       31 . A poly-α-glutamic acid derivative of formula (IV)  
     
       
         
         
             
             
         
       
     
     wherein the symbol * indicates a chiral center and n is an integer comprised between 60 and 310 and R 1 CO— is selected from: 
 (C 1 -C 10 )alkylcarbonyl;  
 (C 4 -C 8 )cycloalkylcarbonyl;  
 (C 6 -C 10 )arylcarbonyl;  
 (C 6 -C 10 )aryl(C 1 -C 10 )alkylcarbonyl;  
 (C 1 -C 10 )alkyl(C 6 -C 10 )arylcarbonyl;  
 (C 5 -C 10 )heteroarylcarbonyl and (C 5 -C 10 )heteroaryl(C 1 -C 10 )alkylcarbonyl wherein the heteroaromatic ring contains one or more nitrogen, oxygen or sulphur atoms; and  
 D- or L-amino acid and non-natural amino acid residues as well as their salts with inorganic acids or bases.  
 
   
   
       32 . The compound according to  claim 31  wherein R 1 CO is a D- or L-pyroglutamic acid residue and the content of the N-terminus free amine is lower than 1% w/w, preferably lower than 0.2% w/w.  
   
   
       33 . A poly-α-glutamic acid derivative of formula (V)  
     
       
         
         
             
             
         
       
     
     wherein the symbol * indicates a chiral center and n is an integer comprised between 60 and 310, R is selected from t-butyl, 1,1-dimethylpropyl and 1,1-dimethylbutyl, R′ is hydrogen or a t-butyl or methyl group and R 1 CO is selected from: 
 (C 1 -C 10 )alkylcarbonyl;  
 (C 4 -C 8 )cycloalkylcarbonyl;  
 (C 6 -C 10 )arylcarbonyl;  
 (C 6 -C 10 )aryl(C 1 -C 10 )alkylcarbonyl;  
 (C 1 -C 10 )alkyl(C 6 -C 10 )arylcarbonyl;  
 (C 5 -C 10 )heteroarylcarbonyl and (C 5 -C 10 )heteroaryl(C 1 -C 10 )alkylcarbonyl wherein the heteroaromatic ring contains one or more nitrogen, oxygen or sulphur atoms; and  
 D- or L-amino acid and non-natural amino acid residues.  
 
   
   
       34 . The poly-α-glutamic acid derivative according to  claim 31  or  claim 33  substituted at the —NH 2  terminus with a group which is able to be further functionalized with a moiety suitable for modulating the pharmacokinetic properties of poly-α-glutamic acid.