Synthesis of 5-azacytidine

Abstract

The present invention provides a method for the preparation of 5-azacytidine, wherein 5-azacytidine is represented by the structure: The method involves the silylation of 5-azacytosine, followed by the coupling of silylated 5-azacytosine to a protected β-D-ribofuranose derivative. The coupling reaction is catalyzed by trimethylsilyl trifluoromethanesulfonate (TMS-Triflate).

1 - 30 . (canceled) 
     
     
         31 . 5-azacytidine prepared by a method comprising the steps of:
 a) reacting 5-azacytosine with at least one silylating reagent to yield a silylated 5-azacytosine having the structure:   
       
         
           
           
               
               
           
         
         wherein each R 1  is an optionally substituted C 1 -C 20  alkyl group independently selected from the group consisting of straight chain alkyl groups, branched alkyl groups, and cyclic alkyl groups; 
         b) isolating the silylated 5-azacytosine by removing the silylating reagent using vacuum distillation or by filtration; 
         c) coupling the isolated silylated 5-azacytosine with a compound of the structure: 
       
       
         
           
           
               
               
           
         
         wherein the coupling is carried out in the presence of trimethylsilyl trifluoromethanesulfonate (TMS-Triflate) in at least one dry organic solvent; 
         d) quenching the reaction mixture of Step c) with an aqueous quenching composition comprising a bicarbonate salt and extracting the quenched reaction mixture; 
         e) replacing substantially all of the solvent in the extract of Step d) with methanol; 
         f) deprotecting the product in the extract of Step d) with sodium methoxide in methanol to yield 5-azacytidine; and 
         g) recrystallizing the 5-azacytidine product of Step f) from hot dimethylsulfoxide. 
       
     
     
         32 . The 5-azacytidine of  claim 31  wherein each said silylating reagent is a trimethylsilyl (TMS) reagent. 
     
     
         33 . The 5-azacytidine of  claim 31  wherein each said silylating reagent is selected from the group consisting of hexamethyldisilizane (HMDS) and chlorotrimethylsilane (TMSCl). 
     
     
         34 . The 5-azacytidine of  claim 33  wherein said silylating reagent is HMDS. 
     
     
         35 . The 5-azacytidine of  claim 33  wherein said silylating reagents are HMDS and TMSCl. 
     
     
         36 . The 5-azacytidine of  claim 31  wherein said silylation reaction in step a) is carried out in the presence of ammonium sulfate. 
     
     
         37 . The 5-azacytidine of  claim 31  wherein each said dry organic solvent is selected from the group consisting of dichloromethane and 1,2-dichloroethane. 
     
     
         38 . The 5-azacytidine of  claim 31  wherein Step g) comprises:
 i) dissolving the product from Step f) in dimethylsulfoxide; 
 ii) adding methanol to the solution of i); and 
 iii) isolating the recrystallized product. 
 
     
     
         39 . The 5-azacytidine of  claim 31  wherein R 1  is methyl. 
     
     
         40 . The 5-azacytidine of  claim 31  wherein the silylated 5-azacytosine from Step a) is isolated by removing the silylating reagent using vacuum distillation. 
     
     
         41 . The 5-azacytidine of  claim 31  wherein the silylated 5-azacytosine from Step a) is isolated by filtration. 
     
     
         42 . The 5-azacytidine of  claim 41  wherein the filtration is performed in the presence of heptane. 
     
     
         43 . The 5-azacytidine of  claim 41  wherein the filtration is performed under inert atmosphere. 
     
     
         44 . The 5-azacytidine of  claim 31  wherein the aqueous quenching composition of Step d) comprises an aqueous solution of sodium carbonate and sodium bicarbonate. 
     
     
         45 . The 5-azacytidine of  claim 44  wherein the sodium carbonate and sodium bicarbonate have a weight ratio of about 1:1. 
     
     
         46 . The 5-azacytidine of  claim 31  wherein the aqueous quenching composition of Step d) is cooled to a temperature of between about 0° C. and about 5° C. 
     
     
         47 . The 5-azacytidine of  claim 31  wherein the reaction of Step c) is performed in dichloromethane. 
     
     
         48 . The 5-azacytidine of  claim 47  wherein the quenched reaction mixture of Step d) is extracted with dichloromethane. 
     
     
         49 . The 5-azacytidine of  claim 48  wherein the dichloromethane extract is washed with cooled sodium bicarbonate solution. 
     
     
         50 . The 5-azacytidine of  claim 48  wherein the dichloromethane extract is dried over MgSO 4  and filtered. 
     
     
         51 . The 5-azacytidine of  claim 48  wherein Step e) comprises:
 1) removing at least some of the dichloromethane using vacuum distillation; 
 2) adding methanol; and 
 3) continuing vacuum distillation until substantially all of the dichloromethane is removed. 
 
     
     
         52 . The 5-azacytidine of  claim 31  wherein the reaction of Step f) is performed at ambient temperature. 
     
     
         53 . The 5-azacytidine of  claim 31  wherein the product of Step f) forms a solid and separates from the reaction mixture. 
     
     
         54 . The 5-azacytidine of  claim 31  wherein the product of Step 0 is isolated by filtration. 
     
     
         55 . The 5-azacytidine of  claim 31  wherein Step g) comprises:
 1) dissolving the 5-azacytidine product of step f) in hot dimethylsulfoxide; and 
 2) adding methanol to the solution of 1) and cooling the mixture. 
 
     
     
         56 . The 5-azacytidine of  claim 55  wherein the 5-azacytidine product of step t) is dissolved in dimethylsulfoxide heated to a temperature of about 85° C. to about 90° C. 
     
     
         57 . The 5-azacytidine of  claim 55  wherein the mixture of Step 2) is cooled to ambient temperature. 
     
     
         58 . 5-azacytidine prepared by a method comprising the steps of:
 a) reacting 5-azacytosine with at least one silylating reagent in a dry organic solvent to yield a silylated 5-azacytosine having the structure:   
       
         
           
           
               
               
           
         
         wherein each R 1  is an optionally substituted C 1 -C 20  alkyl group independently selected from the group consisting of straight chain alkyl groups, branched alkyl groups, and cyclic alkyl groups; 
         b) contacting the reaction mixture of Step a) with trimethylsilyl trifluoromethanesulfonate and a compound having the structure; 
       
       
         
           
           
               
               
           
         
         wherein each R 2  is an optionally substituted C 1 -C 20  acyl group independently selected from the group consisting of straight chain acyl groups, branched acyl groups, and benzoyl group, to yield a compound having the structure: 
       
       
         
           
           
               
               
           
         
         c) contacting the reaction mixture of Step b) with an aqueous quenching composition and extracting the quenched reaction mixture; 
         d) removing substantially all of the solvent in the extract of Step c); 
         e) deprotecting the product in the extract of Step c) with sodium methoxide in methanol to yield 5-azacytidine; and 
         f) recrystallizing the 5-azacytidine product of Step e) from a solvent system comprising dimethylsulfoxide. 
       
     
     
         59 . The 5-azacytidine of  claim 58 , wherein R 1  is methyl. 
     
     
         60 . The 5-azacytidine of  claim 58 , wherein each said silylating reagent is a trimethylsilyl (TMS) reagent. 
     
     
         61 . The 5-azacytidine of  claim 60 , wherein each said silylating reagent is selected from the group consisting of hexamethyldisilizane and chlorotrimethylsilane. 
     
     
         62 . The 5-azacytidine of  claim 61 , wherein the silylating reagent is hexamethyldisilizane. 
     
     
         63 . The 5-azacytidine of  claim 61 , wherein the silylating reagents are hexamethyldisilizane and chlorotrimethylsilane. 
     
     
         64 . The 5-azacytidine of  claim 58 , wherein the reaction of Step a) is carried out in the presence of ammonium sulfate. 
     
     
         65 . The 5-azacytidine of  claim 58 , wherein the dry organic solvent of Step a) is a polar solvent. 
     
     
         66 . The 5-azacytidine of  claim 65 , wherein the dry organic solvent of Step a) is acetonitrile. 
     
     
         67 . The 5-azacytidine of  claim 66 , wherein the reaction of Step a) is carried out in acetonitrile at refluxing temperature. 
     
     
         68 . The 5-azacytidine of  claim 66 , wherein the reaction of Step b) is carried out at ambient temperature. 
     
     
         69 . The 5-azacytidine of  claim 66 , wherein the reactions of Step a) and Step b) are carried out under an inert atmosphere. 
     
     
         70 . The 5-azacytidine of  claim 58 , wherein Compound (B) is: 
       
         
           
           
               
               
           
         
       
     
     
         71 . The 5-azacytidine of  claim 58 , wherein Compound (B) is: 
       
         
           
           
               
               
           
         
       
     
     
         72 . The 5-azacytidine of  claim 66 , wherein the method further comprises, between Step b) and Step c), the following steps:
 1) removing substantially all of the acetonitrile; and   2) dissolving the product of Step b) in a non-polar organic solvent.   
     
     
         73 . The 5-azacytidine of  claim 72 , wherein the non-polar organic solvent is dichloromethane or 1,2-dichloroethane. 
     
     
         74 . The 5-azacytidine of  claim 72 , wherein the non-polar organic solvent is dichloromethane. 
     
     
         75 . The 5-azacytidine of  claim 58 , wherein the aqueous quenching composition of Step c) comprises an aqueous solution of sodium bicarbonate. 
     
     
         76 . The 5-azacytidine of  claim 58 , wherein the aqueous quenching composition of Step c) comprises an aqueous solution of sodium carbonate and sodium bicarbonate. 
     
     
         77 . The 5-azacytidine of  claim 76 , wherein the sodium carbonate and sodium bicarbonate have a weight ratio of about 1:1. 
     
     
         78 . The 5-azacytidine of  claim 58 , wherein the aqueous quenching composition of Step c) is at a temperature of between about 0° C. and about 5° C. 
     
     
         79 . The 5-azacytidine of  claim 58 , wherein the quenched reaction mixture in Step c) is extracted with dichloromethane. 
     
     
         80 . The 5-azacytidine of  claim 79 , wherein the dichloromethane extract is washed with a cooled solution comprising sodium bicarbonate. 
     
     
         81 . The 5-azacytidine of  claim 80 , wherein the dichloromethane extract is further washed with brine, dried over MgSO 4 , and filtered. 
     
     
         82 . The 5-azacytidine of  claim 58 , wherein the reaction of Step e) is carried out at ambient temperature. 
     
     
         83 . The 5-azacytidine of  claim 58 , wherein the 5-azacytidine product of Step e) forms a solid and separates from the reaction mixture. 
     
     
         84 . The 5-azacytidine of  claim 58 , wherein the 5-azacytidine product of Step e) is isolated by filtration. 
     
     
         85 . The 5-azacytidine of  claim 58 , wherein Step 0 comprises:
 i) dissolving the 5-azacytidine product of Step e) in dimethylsulfoxide;   ii) adding methanol to the solution of i); and   iii) isolating the recrystallized product.   
     
     
         86 . The 5-azacytidine of  claim 58 , wherein Step 0 comprises:
 i) dissolving the 5-azacytidine product of Step e) in hot dimethylsulfoxide;   ii) adding methanol to the solution of i) and cooling the mixture; and   iii) isolating the recrystallized product.   
     
     
         87 . The 5-azacytidine of  claim 86 , wherein the 5-azacytidine product of Step e) is dissolved in dimethylsulfoxide heated to a temperature of from about 85° C. to about 90° C. 
     
     
         88 . The 5-azacytidine of  claim 86 , wherein the mixture of Step ii) is cooled to ambient temperature.