Combinatorial production of nucleotide and nucleoside(xitp)analogues

• US 20040023240A1
• Filed: 06/18/2003
• Published: 02/05/2004
• Est. Priority Date: 06/14/2000
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Inventors: Marliere, Philippe, Pochet, Sylvie
• Granted Patent: US 7,319,148 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 31/7052 having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/044 Pyrrole radicals
• CPC Class Codes: C07H 19/052 Imidazole radicals
• CPC Class Codes: C07H 19/056 Triazole or tetrazole radicals
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention concerns novel nucleotide analogues comprising a reactive hydrazide function used as initial synthons for preparing compounds (formula I) capable of inducing mutations or capable of inhibiting a DNA polymerase or a kinase. The invention also concerns nucleic acids comprising said nucleotide and nucleoside analogues. Among such compounds, can be cited in particular the compound of formula (I).

Claims

1. Compounds of general formula I:

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, N₃, 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 NH₂ corresponding to the synthons of general formula II (synthon X₀).

6. Compounds according to one of claims 1 to 4 characterised in that Y1 represents a group chosen from the:

7. Compounds according to one of claims 1 to 4 characterised in that Y1 represents a group chosen from the

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 NH₂ 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 NH₂ 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.