New streptogramin, the method of production thereof, nucleotide sequences, polypeptides and pharmaceutical composition

 

The present invention relates to new streptogramins formula I and to a method for streptogramins by metasynthesis when using the mutant microorganism to change the biosynthesis of at least one of the predecessors of streptogramins. Also described nucleotide sequences and polypeptides involved in the biosynthesis of these precursors. Also described pharmaceutical composition on the basis of the claimed compounds with antibacterial activity. 13 C. and 27 C.p. f-crystals, 13 ill., 5 table.

Description text in facsimile form (see graphic part).

Claims

1. The compound of General formula (I)in which R2and R4independently from each other represent a hydrogen atom or methyl group, except for the case when R2= R4is methyl, X= CO; R3is a hydrogen atom; X is a group WITH, SNON; R1indicatesor

where substituted in the meta-position derivatives of a, C, D and E is a hydrogen atom and may osoznatelno methyl or ethyl group; simple ester group; a simple thioester group; substituted in the para-position derivatives of a, b, D, and E is a hydrogen atom and may indicate a halogen; a group NR1R2where R1and R2independently of one another denote a group selected from hydrogen; linear or branched alkyl group with 1-4 carbon atoms, where one of the substituents R1or R2denotes a methyl group, the other necessarily denotes ethyl group; a linear or branched alkenylphenol group with 1-4 carbon atoms, where one of the substituents R1or R2means alkenylphenol group differs from the other methyl group; or cycloalkyl group with 3-6 carbon atoms; ether group; a simple thioester group; acyl or alkoxycarbonyl group; a linear or branched alkyl group with 1-6 carbon atoms and preferably selected from methyl, isopropyl and tert-butilkoi groups; or trihalomethyl group and preferably trifluoromethyl.

2. Connection on p. 1, selected from the following group of compounds:
4-methylthio-de (4-dimethylamino) pristinamycin 1A;
4-hydroxy-4-methylthio-de (4-dimethylamino) pristinamycin 1H;
4-tert-de (4-dimethylamino) pristinamycin 1A;
4-tert-de (4-dimethylamino) pristinamycin 1H;
4-methoxy-de (4-dimethylamino) pristinamycin 1A;
4-methoxycarbonyl-de (4-dimethylamino) pristinamycin 1A;
4-chlorine-de (4-dimethylamino) pristinamycin 1A;
4-bromo-de (4-dimethylamino) pristinamycin 1A;
4-bromo-de (-dimethylamino) pristinamycin 1H;
4-iodine-de(4-dimethylamino)pristinamycin 1A;
4-iodine-de (4-dimethylamino)pristinamycin 1H;
4-trifluoromethyl-de (4-dimethylamino) pristinamycin 1A;
4-trifluoromethyl-de (4-dimethylamino) pristinamycin 1A;
4-isopropyl-de (4-dimethylamino) pristinamycin 1A;
4-isopropyl-de (4-dimethylamino) pristinamycin 1E;
4-methylamino-de (4-dimethylamino) pristinamycin 1A;
4-methoxy-de (4-dimethylamino) pristinamycin 1A;
4-methoxy-de (4-dimethylamino) pristinamycin 1H;
4-fluoro-4-tert-de (4-dimethylamino) pristinamycin 1A;
4-amino-de (4-dimethylamino) pristinamycin 1A;
4-ethylamino-de (4-dimethylamino) pristinamycin 1A;
4-diethylamino-de (4-dimethylamino) pristinamycin 1A;
4-allylamino-de (4-dimethylamino) pristinamycin 1A;
4-diallylamine-de (4-dimethy is thylamino) pristinamycin 1A;
4-ethylpropylamine-de (4-dimethylamino) pristinamycin 1A;
4-ethylisopropylamine-de (4-dimethylamino) pristinamycin 1A;
4-ethylethylenediamine-de (4-dimethylamino) pristinamycin 1A;
4-(1-pyrrolidinyl) de (4-dimethylamino) pristinamycin 1A;
4-triptoreline-de (4-dimethylamino) pristinamycin 1A;
4-allyloxy-de (4-dimethylamino) pristinamycin 1A;
4-ethoxy-de (4-dimethylamino) pristinamycin 1A;
4-ethylthio-de (4-dimethylamino) pristinamycin 1A;
4-methylthiomethyl-de (4-dimethylamino) pristinamycin 1A;
4-(2 chloroethoxy-de (4-dimethylamino) pristinamycin 1A;
4-acetyl-de (4-dimethylamino) pristine the ub>;
4-ethyl-de (4-dimethylamino) pristinamycin 1H;
4-dimethylamino-de (4-dimethylamino) pristinamycin 1A;
4-methylthio-de (4-dimethylamino) pristinamycin 1Aand
4-ethoxy-de (4-dimethylamino) pristinamycin 1A.

3. The method of receiving streptogramins, characterized in that use strain of S. pristinaespiralis, having at least one genetic modification, localized at the level of one of the genes selected from the genes Rara, rarm, rars (sequence 2), RRV (sequence 3), pipA (sequence 5), snbF (sequence 6), and hpaA (sequence 8), the fact that this mutant strain is cultured in an appropriate culture medium, optionally including at least one original precursor selected from derivatives or analogues of amino acids and alpha-ketocarboxylic acids, different from its predecessor, the biosynthesis of which change and allocate the above streptogramin.

4. The method according to p. 3, characterized in that the above predvaritelnym to encode natural enzyme.

5. The method according to p. 3 or 4, wherein the genetic modification consists in the destruction of one of the foregoing genes.

6. The method according to any of paragraphs. 3-5, characterized in that a strain of S. pristinaespiralis is preferably a strain of S. pristinaespiralis SP92.

7. The method according to p. 6, characterized in that preferably the mentioned strain is SP92: pVRC508.

8. The method according to p. 6, characterized in that preferably the mentioned strain is SP212.

9. The method according to p. 6, characterized in that preferably the mentioned strain is SP92pipA:amR.

10. The method according to p. 6, characterized in that preferably the mentioned strain is SP92hpaA: :amR.

11. The method according to any of paragraphs. 3-10, characterized in that the original precursor is chosen so that it was related to the predecessor, the biosynthesis of which change.

12. The method according to p. 11, characterized in that the original precursor is preferably a derivative of phenylalanine, and a gene whose expression change associated with the biosynthesis of DMAPA.

13. The method according to any of paragraphs. 3-12, wherein the received streptogramins is pristinamycin IB.

14. Nucleotide posledniot 2) or derived from this sequence due to the degeneracy of the genetic code.

15. The nucleotide sequence under item 14, characterized in that it is a gene rars (sequence 2).

16. The nucleotide sequence for p. 15, characterized in that it is part of the recombinant DNA.

17. The nucleotide sequence under item 14 or 15, characterized in that it is embedded in the vector.

18. The nucleotide sequence encoding a polypeptide involved in the biosynthesis of DMAPA, the corresponding gene of RRV (sequence 3) or derived from this sequence due to the degeneracy of the genetic code.

19. The nucleotide sequence under item 18, characterized in that it is a gene rarw (sequence 3).

20. The nucleotide sequence under item 19, characterized in that it is part of the recombinant DNA.

21. The nucleotide sequence under item 18 or 19, characterized in that it is embedded in the vector.

22. The nucleotide sequence encoding a polypeptide involved in the biosynthesis pipecolinic acid, the corresponding gene pipA (sequence 5) or derived from this sequence due to the degeneracy of the genetic code.

23. The nucleotide sequence for p. 22, tlicho p. 23, characterized in that it is part of the recombinant DNA.

25. The nucleotide sequence under item 22 or 23, characterized in that it is embedded in the vector.

26. The nucleotide sequence encoding a polypeptide that participates in the hydroxylation pipecolinic acid, the corresponding gene snbF (sequence 6) or derived from this sequence due to the degeneracy of the genetic code.

27. The nucleotide sequence under item 26, characterized in that it is a gene snbF (sequence 6).

28. The nucleotide sequence for p. 27, characterized in that it is part of the recombinant DNA.

29. The nucleotide sequence under item 26 or 27, characterized in that it is embedded in the vector.

30. The nucleotide sequence encoding a polypeptide involved in the biosynthesis of 3-hydroxypyridones acid, the corresponding gene hpaA (sequence 8) or derived from this sequence due to the degeneracy of the genetic code.

31. The nucleotide sequence for p. 30, characterized in that it is an hpaA gene (sequence 8).

32. The nucleotide sequence for p. 31, characterized in that it that it is embedded in the vector.

34. The polypeptide of Rars involved in the biosynthesis of DMAPA resulting from the expression of the nucleotide sequence 2.

35. The polypeptide of Rarv involved in the biosynthesis of DMAPA resulting from the expression of the nucleotide sequence of the 3.

36. Polypeptide PipA involved in the biosynthesis pipecolinate acids formed as a result of expression of the nucleotide sequence 5.

37. Polypeptide SnbF involved in the hydroxylation pipecolinate acids formed as a result of expression of the nucleotide sequence 6.

38. The polypeptide of NRA involved in the biosynthesis of 3-hydroxypyridones acids formed as a result of expression of the nucleotide sequence 8.

39. A compound selected from the group consisting of 4-cryptomaterial, 3-methylenedianiline, 3-methyldiphenylamine, 3-fluoro-4-methylphenylamine, 4-methylaminoanthraquinone acid, 3-ethoxypropylamine, 4-allylaminogeldanamycin, 4-diallylmethylamine, 4-allylaminogeldanamycin, 4-ethylpropylamine, 4-ethylisopropylamine, 4-ethylethylenediamine, 4-(1-pyrrolidinyl) phenylalanine, 4-ethyldiethanolamine, 4-0-(2-chloroethyl)ment antibacterial activity, characterized in that it contains at least one connection under item 1 or 2 in combination with streptogramin group or without him.

 

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