Combinatorial production of nucleotide and nucleoside (XiTP) analogues

Abstract

Nucleotide analogues comprising a reactive hydrazide function (formula II) used as initial synthons for preparing compounds (formula I) capable of inducing mutations or capable of inhibiting a DNA polymerase or a kinase provided. Nucleic acids comprising the nucleotide and nucleoside analogues are also provided. Methods of using the compounds are also provided.

1 . Compounds of general formula I: 
       
         
           
           
               
               
           
         
       
       in which:
 the R1 group represents a hydrogen or a group chosen from the phosphate, diphosphate or triphosphate groups and the protective groups such as DMT. 
 R2 and R2′ are chosen independently from one another from H, OH, phosphoramidite and its derivatives, H-phosphonate and an elongation terminator T, 
 R3 and R3′ are chosen independently from one another from H, OH, the halogens (F, Br, Cl, I), a linear or branched alkyl group comprising 1 to 6 carbon atoms and an O—R5 group, R5 representing a linear or branched alkyl group comprising 1 to 6 carbon atoms, 
 R4 represents a substituted heterocycle (symbolised by a circle), of formula 
 
       
         
           
           
               
               
           
         
       
       in which E designates a spacer arm made up of a saturated or unsaturated carbon chain of 0 to 20 carbon atoms, which may or may not comprise heteroatoms and/or is optionally substituted; said heterocycle being selected from: 
       c) the 5-atom heterocycles of formula: 
       
         
           
           
               
               
           
         
       
       in which X represents independently from one another a CH, C—R6 or N group, R6 being chosen from the halogens (F, Br, Cl, I), a linear or branched alkyl group comprising 1 to 6 carbon atoms and an O—R7 or S—R7 group, R7 representing a hydrogen or a linear or branched alkyl group comprising 1 to 6 carbon atoms, said heterocycles belonging in particular to the azole family, such as the following rings 
       
         
           
           
               
               
           
         
       
       b) 6-atom rings, in particular pyrimidines of formula: 
       
         
           
           
               
               
           
         
       
       c) purine analogues of formula: 
       
         
           
           
               
               
           
         
       
       in which:
 Y1 represents NH 2  (designated by the synthon X 0 ) or a: 
 
       
         
           
           
               
               
           
         
       
       group
 Y2 et Y3 are chosen independently from one another from H, OH, O, NH 2 , the halogens (F, Br, Cl, I), SCH 3 , SH, an amine, an amide (in direction —NH—CO—R) or a branched or linear alkyl (direction —OR) comprising 1 to 6 carbon atoms, 
 
       Z1 and Z2 being chosen independently from one another from H and an organic group. 
     
     
         2 . Compounds according to  claim 1  characterised in that the R1 group is a triphosphate group. 
     
     
         3 . Compounds according to one of  claims 1  to  2  characterised in that at least one of the R1 and R2′ groups represents an OH group. 
     
     
         4 . Compounds according to one of  claims 1  to  3  characterised in that T is preferably selected from F, Br, Cl, I, N 3 , and a linear or branched alkyl group comprising 1 to 6 carbon atoms. 
     
     
         5 . Compounds according to one of  claims 1  to  4  characterised in that Y1 is NH 2  corresponding to the synthons of general formula II (synthon X 0 ). 
       
         
           
           
               
               
           
         
       
       in which R1, R2, R2′, R3, R3′ and E correspond to the groups of formula I. 
     
     
         6 . Compounds according to one of  claims 1  to  4  characterised in that Y1 represents a group chosen from the: 
       
         
           
           
               
               
           
         
       
       group 
       Z1 and Z2 representing independently from one another a hydrogen or an organic group (synthons X 1  and X 1 TP.). 
     
     
         7 . Compounds according to one of  claims 1  to  4  characterised in that Y1 represents a group chosen from the 
       
         
           
           
               
               
           
         
         group, Z1 and Z2 representing independently from one another a hydrogen or an organic group (synthons X 2  et X 2 TP.). 
       
     
     
         8 . Compounds according to one of  claims 6  and  7  characterised in that at least one of groups Z1 and Z2 is selected from the aromatic groups, in particular from the groups of formula: 
       
         
           
           
               
               
           
         
       
     
     
         9 . Compounds according to one of  claims 6  and  7  characterised in that at least one of groups Z1 and Z2 is selected from the thiol, alkyl, carbonyl, amine, alcohol, aryl, and amino acid groups. 
     
     
         10 . Compounds according to one of  claims 6  and  7  characterised in that Z1 or Z2 forms a ring with Y3. 
     
     
         11 . Compounds according to one of  claims 6  and  7  characterised in that Z1 or Z2 comprises a fluorescent or phosphorescent marker, in particular fluorescamine. 
     
     
         12 . Compounds according to one of the preceding claims characterised in that they are substrates of a polymerase, a reverse transcriptase and or a nucleotide kinase. 
     
     
         13 . Compounds according to one of the preceding claims characterised in that they form pairs with the natural bases. 
     
     
         14 . Compounds according to  claim 12  characterised in that they inhibit a polymerase, in particular a retrovirus reverse transcriptase and/or a kinase. 
     
     
         15 . Method for preparation of compounds of general formula II according to  claim 5  characterised in that it includes the following steps:
 a) preparation of the nucleoside having for heterocycle a 5-atom heterocycle of formula:   
       
         
           
           
               
               
           
         
         in which X represents independently from one another a CH, C—R6 or N group, R6 being chosen from the halogens (F, Br, Cl, I), a linear or branched alkyl group comprising 1 to 6 carbon atoms and an O—R7 or S—R7 group, R7 representing a hydrogen or a linear or branched alkyl group comprising 1 to 6 carbon atoms, said heterocycles belonging in particular to the azole family, such as the 
       
       
         
           
           
               
               
           
         
         rings 
         b) protection of the alcohol in position 5′ and conversion of the ethyl ester function into a carbohydrazide function by the action of hydrazine, 
         c) protection of the amine function of the carbohydrazide group by a protective group such as the benzyloxycarbonyl group, acetylation of the alcohol in position 3′, 
         d) phosphorylation after deprotection of the alcohol in position 5′, 
         e) and hydrogenolysis of the benzyloxycarbonyl group. 
       
     
     
         16 . Method of preparation according to  claim 15  characterised in that step a) is carried out by means of an N-transdeoxyribosylase. 
     
     
         17 . Method of preparation according to one of  claims 15  and  16  characterised in that step d) consists of the phosphorylation of the nucleoside by cyanoethyl phosphate in the presence of dicyclohexylcarbodiimide; concomitant release in a basic medium of the alcohol in position 3′ and of the cyanoethyl group of the 5′ phosphate; then condensation of pyrophosphate to 5′ activated phosphate in the form of morpholidate. 
     
     
         18 . Method of preparation of a compound of general formula I according to one of  claims 6  to  11 , characterised in that a compound of general formula II according to  claim 5  is condensed with an aldehyde or a ketone. 
     
     
         19 . Method according to  claim 18  characterised in that one or more compounds of general formula II according to  claim 5  are reacted with a library of aldehydes and/or ketones, optionally followed by a reduction. 
     
     
         20 . Compounds capable of being obtained using a method according to one of  claims 15 - 19 . 
     
     
         21 . Libraries of compounds of general formula I according to one of  claims 1  to  11  capable of being obtained using a method according to  claim 19 . 
     
     
         22 . Method for identification of compounds capable of introducing a mutation into a nucleic acid comprising the steps consisting of i) incorporating into a synthetic oligonucleotide, a compound according to  claim 1  in which Y1 is an NH 2  group, ii) reacting said compound in accordance with the method according to  claim 18  or  19 , optionally followed by a reduction, iii) replicating said oligonucleotide using a polymerase and determining whether a mutation has been introduced into the newly synthesised strand. 
     
     
         23 . Method for localised mutation of a nucleic acid characterised in that i) a synthetic oligonucleotide is prepared, comprising at least one compound according to one of  claims 1  to  11 , ii) said oligonucleotide is replicated using a polymerase. 
     
     
         24 . Method for random mutation of a nucleic acid comprising the steps consisting of using a reaction mixture comprising at least one compound according to  claims 1  to  11  for the amplification of a nucleic acid. 
     
     
         25 . Method according to one of  claims 22  to  24 , characterised in that a DNA exo-polymerase is used. 
     
     
         26 . Method for identification of a compound capable of inhibiting an enzyme selected from a polymerase, in particular a reverse transcriptase and a kinase, characterised in that the activity of said enzyme is tested in the present of at least one compound according to one of  claims 1  to  11 . 
     
     
         27 . Method for identification of a compound capable of inhibiting a reverse transcriptase of a retrovirus characterised in that the effect of at least one compound according to one  claims 1  to  11  is tested on cells infected by a retrovirus, said compound being found in the form of a nucleoside analogue. 
     
     
         28 . Method according to one of  claims 23  to  27 , characterised in that a library according to  claim 21  is used. 
     
     
         29 . Nucleic acid comprising at least one compound according to one of  claims 1  to  11 . 
     
     
         30 . Use of a compound according to one of  claims 1  to  11  in a random mutagenesis process. 
     
     
         31 . Use of a compound according to one of  claims 1  to  11  as an ambiguous base. 
     
     
         32 . Use of a compound according to one of  claims 1  to  11  in a primer or probe for the amplification and/or detection of a target nucleic acid. 
     
     
         33 . Use according to  claim 32  characterised in that said compound is marked. 
     
     
         34 . Kit for the amplification and/or detection of a target nucleic acid comprising a compound according to one of  claims 1  to  11  or a nucleic acid according to  claim 29 . 
     
     
         35 . Use of a nucleic acid according to  claim 29  for the preparation of a medicament. 
     
     
         36 . Use of a nucleic acid according to  claim 29  as ribozyme for the preparation of a medicament. 
     
     
         37 . Use of a nucleic acid according to  claim 29  as an antisense for the preparation of a medicament. 
     
     
         38 . Use of a compound according to one of  claims 1  to  5  in which Y1 is NH 2  as an initial synthon for the preparation of a library of nucleotide and/or nucleoside analogues. 
     
     
         39 . Use of a compound according to one of  claims 1  to  11  for the manufacture of a medicament. 
     
     
         40 . Use according to  claim 39  for the manufacture of a medicament intended for the treatment of retroviral infections. 
     
     
         41 . Use according to  claim 39  for the manufacture of a medicament intended for the treatment of cancer.