Process for the synthesis of pyrazolopyrimidines

Abstract

The present invention provides a nucleoside comprising a pyrazolopyrimidine base and a process for producing the same. In particular, the processes of the present invention comprises using a halogenated pyrazolopyrimidine base and removing the halogen after the base is coupled to a sugar moiety. The presence of the halogen on the nucleoside base allows facile and economical production of a large quantity of nucleosides.

1 . A PPG phosphoramidite comprising a photolabile hydroxy protecting group, wherein said phosphoramidite nucleoside is of the formula:  
     
       
         
         
             
             
         
       
     
     wherein 
 R 1  is selected from the group consisting of hydrogen and alkyl;  
 R 2  is selected from the group consisting of hydrogen, alkyl, and an amine protecting group, or R 1  and R 2  together from an amine protecting group;  
 each of Z 1 , Z 2 , Z 4 , and Z 6  is independently selected from the group consisting of hydrogen halide, alkyl, —OR 11 , wherein each R 11  is independently selected from the group consisting of hydrogen, alkyl, and a hydroxy protecting group or two R 11  groups form a diol protecting group, or Z 2  and Z 4  together with the carbon atoms to which they are attached and C-3 carbon atom of the carbohydrate ring form a five-to seven membered ring; and  
 one of Z 3  or Z 5  is —OR 12  and the other is —OR 13 , where R 12  is a photolabile hydroxy protecting group and R 13  is a phosphoramidite.  
 
   
   
       2 . The PPG phosphoramidite according to  claim 1  of the formula:  
     
       
         
         
             
             
         
       
     
     wherein 
 R 1 , R 2 , Z 3  and Z 5  are those defined in  claim 1 .  
 
   
   
       3 . The PPG phosphoramidite according to  claim 2 , wherein Z 3  is —OR 13  and Z 5  is —OR 12 , where R 12  and R 13  are those defined in  claim 1 .  
   
   
       4 . The PPG phosphoramidite according to  claim 3 , wherein the photolabile hydroxy protecting group is selected from the group consisting of α-methyl-6-nitropiperonyloxycarbonyl, 2-(2-nitrophenyl)-2-methylethoxycarbonyl, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl, and 3′,5′-dimethoxybezoinoxycarbonyl.  
   
   
       5 . The PPG phosphoramidite according to  claim 4 , wherein R 1  and R 2  together form an amine protecting group.  
   
   
       6 . The PPG phosphoramidite according to  claim 5 , wherein R 1  and R 2  together form an amine protecting group of the formula: ═CH—N(CH 3 ) 2 .  
   
   
       7 . A process for producing a non-halogenated nucleoside base containing nucleoside comprising: 
 (a) contacting a halogenated nucleoside base with an activated sugar under conditions sufficient to produce a halogenated nucleoside base containing nucleoside; and    (b) reducing said halogenated nucleoside base containing nucleoside under conditions sufficient to produce said non-halogenated nucleoside base containing nucleoside.    
   
   
       8 . The process of  claim 7 , wherein said non-halogenated nucleoside base containing nucleoside is purified by recrystallization.  
   
   
       9 . The process of  claim 7 , wherein the yield of said non-halogenated nucleoside base containing nucleoside from said halogenated nucleoside base is at least about 50%.  
   
   
       10 . The process of  claim 7 , wherein said halogenated nucleoside base containing nucleoside reducing step comprises hydrogenation of said halogenated nucleoside base containing nucleoside in the presence of a hydrogenation catalyst.  
   
   
       11 . The process of  claim 7 , wherein said non-halogenated nucleoside base containing nucleoside is used in a synthesis of a phosphoramidite nucleoside.  
   
   
       12 . The process of  claim 11 , wherein said phosphoramidite nucleoside is used in a synthesis of an oligonucleotide or an oligonucleotide.  
   
   
       13 . A process for producing a nucleoside comprising a hydropyrazolopyrimidine nucleoside base, said process comprising hydrolyzing and reducing or reducing and hydrolyzing an iodopyrazolopyrimidine nucleoside of the formula:  
     
       
         
         
             
             
         
       
     
     under conditions sufficient to produce a hydropyrazolopyrimidine nucleoside of the formula:  
     
       
         
         
             
             
         
       
     
     wherein 
 R 1  is selected from the group consisting of hydrogen and alkyl;  
 R 2  is selected from the group consisting of hydrogen, alkyl, and an amine protecting group, or R 1  and R 2  together form an amine protecting group;  
 R 3  is selected from the group consisting of alkyl, and a hydroxy protecting group; and  
 each of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6  is independently selected from the group consisting of hydrogen, halide, alkyl, —OR 4 , wherein each R 4  is independently selected from the group consisting of hydrogen, alkyl, and a hydroxy protecting group or two R 4  groups form a diol protecting group, or Y 2  and Y 4  together with the carbon atoms to which they are attached to and C-3 carbon atom of the carbohydrate ring form a five-to seven membered ring.  
 
   
   
       14 . The process of  claim 13 , wherein R 1 , R 2 , Y 1 , Y 2 , Y 4 , and Y 6  are hydrogen, and Y 3  and Y 5  are —OR 4 .  
   
   
       15 . The process of  claim 14 , wherein R 4  are hydrogen.  
   
   
       16 . The process of  claim 15  further comprising producing a PPG phosphoramidite of the formula:  
     
       
         
         
             
             
         
       
     
     from said hydropyrazolopyrimidine nucleoside,  
     wherein 
 R 1  is hydrogen and R 2  is an amine protecting group or R 1  and R 2  together form an amine protecting group; and  
 one of R 9  and R 10  is a phosphoramidite and the other is a hydroxy protecting group,  
 said PPG phosphoramidite producing step comprises:  
 (a) (i) contacting said hydropyrazolopyrimidine nucleoside with an amine protecting reagent under conditions sufficient to produce an amine-protected nucleoside of the formula:  
                     (ii) contacting said amine-protected nucleoside with a hydroxy protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula:                          
 or 
 (i) contacting said hydropyrazolopyrimidine with a hydroxy protecting reagent under conditions sufficient to produce a monohydroxy protected nucleoside of the formula:  
                     
 (ii) contacting said monohydroxy protected nucleoside with an amine protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula:  
                     
 
 wherein 
 R 1  is hydrogen and R 2  is an amine protecting group or R 1  and R 2  together form an amine protecting group; and  
 one of R 7  and R 8  is hydrogen and the other is a hydroxy protecting group; and  
 
 (b) contacting said amine/monohydroxy protected nucleoside with an activated phosphoramidite under conditions sufficient to produce said PPG phosphoramidite.  
 
   
   
       17 . The process of  claim 16 , wherein said amine protecting reagent is selected from the group consisting of N,N-dialkylformamide dialkylacetal, and N,N-dialkylacetamide dialkylacetal.  
   
   
       18 . The process of  claim 16 , wherein said hydroxy protecting reagent is a photolabile hydroxy protecting reagent.  
   
   
       19 . The process of  claim 18 , wherein said photolabile hydroxy protecting reagent is selected from the group consisting of 1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate, 2-(2-nitrophenyl)-2-methylethyl chloroformate, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl chloride and 3′,5′-dimethoxybezoinoxyl chloroformate.  
   
   
       20 . The process of  claim 16 , wherein said hydroxy protecting reagent is an acid labile hydroxy protecting reagent.  
   
   
       21 . The process of  claim 20 , wherein said acid labile hydroxy protecting reagent is selected from the group consisting of trityl halide, monomethoxytrityl halide and dimethoxytrityl halide.  
   
   
       22 . The process of  claim 16 , wherein said activated phosphoramidite is of the formula:  
     
       
         
         
             
             
         
       
     
     wherein 
 X 2  is a leaving group.  
 
   
   
       23 . The process of  claim 22 , wherein x is selected from the group consisting of halide and diisopropylamino.  
   
   
       24 . The process of  claim 22 , wherein R 9  is dimethoxytrityl and R 10  is a phosphoramidite moiety of the formula —P[N(i-Pr) 2 ]OCH 2 CH 2 CN.  
   
   
       25 . The process of  claim 13  further comprising producing said nucleoside of Formula I, wherein said nucleoside of Formula I producing step comprises: 
 contacting an iodopyrazolopyrimidine of the formula:                          with an activated sugar of the formula:                          under conditions sufficient to produce said nucleoside of Formula I, wherein    R 1 , R 2 , R 3 , Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6  are those defined  claim 13;  and    X 1  is a leaving group.    
   
   
       26 . The process of  claim 25  further comprising producing said iodopyrazolopyrimidine nucleoside of Formula I from a pyrimidinone of the formula:  
     
       
         
         
             
             
         
       
     
     said iodopyrazolopyrimidine nucleoside producing process comprising: 
 (i) contacting said pyrimidinone with a halogenating agent and a formylating agent under conditions sufficient to produce a dihalopyrimidine carboxyaldehyde of the formula:  
                     
 wherein  
 each X 3  is independently selected from the group consisting of F, Cl, Br and I;  
 (ii) contacting said dihalopyrimidine carboxyaldehyde with hydrazine under conditions sufficient to produce a halopyrazolopyrimidine of the formula:  
                     
 (iii) contacting said halopyrazolopyrimidine with an alkoxide of the formula R 3 —OM, wherein R 3  is alkyl and M is a metal, to produce an alkoxypyrazolopyrimidine of the formula:  
                     
 and  
 (iv) iodinating said alkoxypyrazolopyrimidine with an iodinating agent under conditions sufficient to produce said iodopyrazolopyrimidine.  
 
   
   
       27 . The process of  claim 26 , wherein said halogenating agent is selected from the group consisting of POCl 3 , iodine monochloride, N-iodosuccinamide and SOCl 2 .  
   
   
       28 . The process of  claim 26 , wherein said formylating agent is a compound comprising a formyl group attached to a secondary amino group.  
   
   
       29 . The process of  claim 28 , wherein said formylating agent is selected from the group consisting of dimethyl formamide, 1-formylpiperidine, 1-formylmorpholine and triformamide.  
   
   
       30 . The process of  claim 26 , wherein said iodinating agent is selected from the group consisting of iodine monochloride and N-iodosuccinimide.  
   
   
       31 . A process for producing a nucleoside comprising: 
 (a) contacting an iodopyrazolopyrimidine of the formula:                          with an activated sugar of the formula:                          under conditions sufficient to produce an deoxy iodopyrazolopyrimidine nucleoside of the formula:                          (b) producing an amino dihydro hydropyrazolopyrimidine nucleoside from said deoxy iodopyrazolopyrimidine nucleoside, wherein said amino dihydro hydropyrazolopyrimidine nucleoside is of the formula:                          wherein    R 3  is alkyl;    R 5  and R 6  are hydroxy protecting groups; and    X 1  is a leaving group.    
   
   
       32 . The process of  claim 31 , wherein said step of producing said amino dihydro hydropyrazolopyrimidine nucleoside comprises removing said hydroxy protecting groups R 5  and R 6 ; hydrolyzing —OR 3  group; and reducing the iodine.  
   
   
       33 . The process of  claim 31  further comprising: 
 (c) contacting said amino dihydro hydropyrazolopyrimidine nucleoside with an amine protecting reagent under conditions sufficient to produce an amine protected nucleoside of the formula:                          (d) contacting said amine protected nucleoside with a hydroxy protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula:                          and    (e) contacting said amine/monohydroxy protected nucleoside with an activated phosphoramidite of the formula:                          under conditions sufficient to produce a PPG phosphoramidite of the formula:                          wherein    R 1  is hydrogen;    R 2  is an amine protecting group;    or R 1  and R 2  together form an amine protecting group;    R 4  is a hydroxy protecting group; and    X 2  is a leaving group.    
   
   
       34 . The process of  claim 33 , wherein X 2  is selected from the group consisting of halide, and —N(i-Pr) 2 .  
   
   
       35 . The process of  claim 33 , wherein R 1  and R 2  together form a nitrogen protecting group of the formula: ═CH—N(CH 3 ) 2 .  
   
   
       36 . The process of  claim 35 , wherein R 4  is selected from the group consisting of an acid labile hydroxy protecting group and a photolabile hydroxy protecting group.  
   
   
       37 . The process of  claim 36 , wherein R 4  is selected from the group consisting of dimethoxytrityl, trityl, pixyl, 1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl, α-methyl-6-nitropiperonyloxycarbonyl, 2-(2-nitrophenyl)-2-methylethoxycarbonyl, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl and 3′,5′-dimethoxybezoinoxycarbonyl.  
   
   
       38 . The process of  claim 31 , wherein said step (b) comprises reducing the iodide by hydrogenation.  
   
   
       39 . The process of  claim 31 , wherein said iodopyrazolopyrimidine is produced from a pyrimidinone of the formula:  
     
       
         
         
             
             
         
       
     
     said iodopyrazolopyrimidine producing step comprising: 
 (i) contacting said pyrimidinone with a halogenating agent and a formylating agent under conditions sufficient to produce a dihalopyrimidine carboxyaldehyde of the formula:  
                     
 wherein each X 3  is independently selected from the group consisting of F, Cl, Br and I;  
 (ii) contacting said dihalopyrimidine carboxyaldehyde with hydrazine under conditions sufficient to produce a halopyrazolopyrimidine of the formula:  
                     
 (iii) contacting said halopyrazolopyrimidine with an alcohol of the formula R 3 —OH to produce an alkoxypyrazolopyrimidine of the formula:  
                     
 and  
 (iv) iodinating said alkoxypyrazolopyrimidine with an iodinating agent under conditions sufficient to produce said iodopyrazolopyrimidine.  
 
   
   
       40 . The process of  claim 39 , wherein said halogenating agent is selected from the group consisting of POCl 3 , iodine monochloride, N-iodosuccinamide and SOCl 2 .  
   
   
       41 . The process of  claim 40 , wherein said halogenating agent is selected from the group consisting of POCl 3  and SOCl 2 .  
   
   
       42 . The process of  claim 39 , wherein said formylating agent is selected from the group consisting of dimethyl formamide, 1-formylpiperidine, 1-formylmorpholine and triformamide.  
   
   
       43 . The process of  claim 39 , wherein said iodinating agent is selected from the group consisting of iodine monochloride and N-iodosuccinimide.