SK597A3 - Streptogramines and method for preparing same by mutasynthesis - Google Patents

Abstract

Streptogramin B derivs. of formula (I) are new, in which R2 and R4 = H or Me; R3 = H or OH; X = CO, CHOH or CH2; R1 = 2-thienylmethyl, 2-naphthylmethyl or a gp. of formula (i); for meta derivs., A,C,D,E = H; B = halo; mono- or di-alkylamino; ether or thioether gp.; 1-3C alkyl; or trihalomethyl; for para derivs., A, B, D, E = H; C = halo; NRaRb; opt. substd. N-pyrrolidinyl; ether or thioether gp.; acyl; alkoxycarbonyl; 1-6C alkyl; alkylthiomethyl; aryl; or trihalomethyl; Ra and Rb = H, 1-4C alkyl (if one is Me, the other must be Et), alkyl (3-4C cycloalkyl)methyl, opt. substd. 3-4C cycloalkyl or 1-4C alkenyl (if one is alkenyl the other cannot be Me or cycloalkyl); for meta-para derivs., A ,D, E = H, B is as defined for meta derivs. but not trihalomethyl; and C is halo; NH2 (opt. substd. by 1 or 2 alkyl, provided that B is not Cl, Br or opt. substd. allyl) ; ether or thioether gp.; 1-6C alkyl; or trihalomethyl; for ortho-para derivs., B, E, D = H; A=C=Me. Also claimed is prodn. of streptogramins by culturing an appropriate microorganism having a modification in a gene involved in biosynthesis of streptogramin gp. B precursors, in a medium contg. a new precursor (other than the one whose synthesis is being affected). Further claimed are (inter alia) (1) new DNA contg. specified genes from Streptomyces pristinaespiralis, and (2) certain specified new precursors.

Description

Streptogramins and method for the preparation of streptogramins by mutase synthesis

Technical field

In particular, the invention relates to novel compounds related to group B streptogramins and to a process for the preparation of streptogramins. The invention also relates to novel genes involved in the biosynthesis of Group B streptogramin precursors, as well as their use.

BACKGROUND OF THE INVENTION

Streptogramins form a homogeneous group of antibiotics consisting of a combination of two chemically different types of molecules, i. as polyunsaturated macrolactones (constituents of the

A) as well as depsipeptides (group B components). This class of antibiotics includes a number of antibiotics known under various names denoting their origin, including pristinamycins, micamycins and virginiamycins (Cocito 1979, 1983).

Components A and B exhibit synergistic antibacterial efficacy, which may be up to 100 times the efficacy of components A and B alone and which, in contrast to the efficacy of the individual components, are bactericidal (Cocito 1979). This activity is particularly effective against Gram-positive bacteria such as staphylococci and streptococci (Cocito 1979, Videau 1982). Components A and B inhibit protein synthesis by fixing to the 50S ribosome subunit (Cocito 1979, Di Giambattista et al. 1989).

Heretofore, the biosynthesis mechanisms of the formation of each of these components are only partially known, although the studies described in patent application PCT / FR93 / 0923 have made it possible to identify several proteins and genes of the corresponding structures that are involved in the biosynthesis of both types of said components.

In the B-group streptogramin biosynthesis process, the following two stages can be distinguished:

1) biosynthesis of precursors or their analogs, macrocycle: 3-hydroxypicolinic acid, L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine, L-pipecolic acid, L-phenylglycine.

2) formation of the macrocycle of L-threonine and L-proline or analogues thereof from the above precursors and any subsequent modification or possible subsequent modifications of the N-peptide methylation, epimerization, hydroxylation and oxidation type.

International patent application PCT / FR9 / 0923 relates to enzymes catalysing the incorporation of precursors into the peptide chain of streptogramins B during chain elongation, as well as their structural genes. These results made it possible to elucidate the peptide non-ribosomal character of the synthesis of the said type B compounds

In particular, the invention relates to novel compounds which are similar to group B streptogramins and more particularly to novel compounds of the pristinamycin I group (Figs. 1 and 2), hereinafter referred to as PI, or the virginiamycin S group (Fig. 3).

The major component of pristinamycin I (PI) is PI _a (Fig. 1), which represents about 94% of pristinamycin I, while the remaining about 6% are minor depsipeptide components (PI _B to PI), their structures are shown in Figure 1. 2. Pristinamycin I is essentially the result of the condensation of amino acids, some of which are essential for protein synthesis (threonine and proline) and others are native and considered as secondary metabolites (L-2-aminobutyric acid,

4-dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid and L-phenylglycine for PI _α , as well as the aromatic precursor of 3-hydroxypicolinic acid.

For the virginiamycin S derivatives, these derivatives are the result of condensation of the same amino acids reported for PI except for DMPAPA, which in this case is replaced by phenylalanine (see Fig. 3).

Such various compounds are produced by biosynthesis, i.e., pre-synthesis using a production strain of the above-mentioned precursors.

More precisely, the invention results from the original streptogramin preparation method, wherein a strain of microorganisms that is mutated to alter the biosynthesis of group B streptogramin precursors is used as the streptogramin production strain. According to this method, said mutant strain is cultured in an environment complemented by the original precursor which is different from a precursor whose biosynthesis is modified. Quite unexpectedly, this has resulted in the production of novel compounds which are structurally close to Group B streptogramin, which are of interest to the therapeutic field.

SUMMARY OF THE INVENTION

The invention relates to novel compounds of the general formula I

HO

In which

R ² and R ⁴ independently of one another are hydrogen or methyl;

R ³ represents a hydrogen atom or a hydroxyl group,

X is CO, CHOH or CH ₂ and

^R1 is

while

- for meta derivatives:

A, C, D and E are hydrogen and

B may mean:

- a halogen atom and preferably a fluorine atom,

a monoalkylamino group or a dialkylamino group, in which the alkyl radical preferably represents a methyl group or an ethyl group

- an ether group, in particular an OR group in which R preferably represents a methyl group, an ethyl group, a trifluoromethyl group or an allyl group,

- a thioether group, which is preferably an alkylthio group, in which the alkyl radical is preferably a methyl group,

an alkyl group having 1 to 3 carbon atoms, or

- a trihalomethyl group, preferably a trifluoromethyl group,

- for para derivatives:

A, B, D and E are hydrogen and

C can mean:

- halogen atom,

- NR ^X R ² in which R ¹ and R ^{2 are} independently of one another

- a hydrogen atom,

- a straight or branched (C 1 -C 4) alkyl group, where one of R ¹ or R ² is methyl, the other obligatory is ethyl,

- an alkylcycloalkylmethyl group in which the cycloalkyl radical contains 3 or 4 carbon atoms,

- an optionally substituted cycloalkyl group containing 3 or 4 carbon atoms,

straight or branched (C 1 -C 4) alkenyl, where in the case where one of R ¹ or R ² is an alkenyl group, the other is different from a methyl or cycloalkyl group containing 3 to 6 carbon atoms, preferably alkylthio, preferably alkyl substituted or unsubstituted N-pyrrolidinyl, ether, preferably OR, wherein R is preferably methyl, ethyl, optionally substituted with chlorine atoms, trifluoromethyl and alkenyl, thioether a group in which an alkyl radical of 1 to 3 carbon atoms, an acyl group or an alkoxycarbonyl group and in particular a COR group in which R preferably represents an alkyl group of 1 to 3 carbon atoms or an alkoxy group of 1 to 3 carbon atoms,

- a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group, an isopropyl group and a tert-butyl group,

- an alkylthiomethyl group and in particular a CH ₂ SR group in which R preferably represents an alkyl group having 1 to 3 carbon atoms,

an aryl group and preferably a phenyl group; or

- trihalomethyl and preferably trifluoromethyl,

- for disubstituted meta-para derivatives:

A, D and E are hydrogen and

B may mean:

- a halogen atom and preferably a fluorine atom,

- a monoalkylamino group or a dialkylamino group, in which the alkyl radical preferably represents a methyl group or an ethyl group,

- an ether group and preferably an OR group, in which R preferably represents a methyl group, an ethyl group and a trifluoromethyl group,

a thioether group and preferably an alkylthio group in which the alkyl moiety preferably represents an ethyl group, or

an alkyl group having 1 to 3 carbon atoms, and

C can mean:

- a halogen atom and preferably a fluorine atom,

- an amino group, a monoalkylamino group or a dialkylamino group, in which the alkyl radical preferably represents a methyl group, with the proviso that B is different from a bromine or chlorine atom, or a substituted or unsubstituted allyl group,

- an ether group, and preferably an OR group, in which R preferably represents a methyl group, an ethyl group or a trifluoromethyl group,

- a thioether group and preferably an alkylthio group in which the alkyl radical is a methyl group,

an alkyl group having 1 to 6 carbon atoms, or

a trihalomethyl group, preferably a trifluoromethyl group, and

- for disubstituted ortho-para derivatives:

B, E and D are hydrogen and A and C are methyl.

Preferred compounds can include: 4ζ-Γη6Γ / 1ίίο-άβ5 (4ζ- <ϋιτΐ6ίγΐ3ΐτπηο) ρπ5ΐίηαπινάη _IA ^ εΐν1ύο 4ζ-ά68 (4ζ- <1ίπΐ6ίγΐ3ηιΐηο) ρη8ϋη3πιγαη I _H 5y-hydroxy--rnetyltio ^ of ^ -dimethylamino) pristinamycin I _H-4u of methyl N-dimethylamino) pnstmamycín I _and 4u-methyl-des ^ -dimethylamino) pristinamycin I _H-4ζ ιη6ΐοχγ · 65 ^ (^ · 4ζ ίιη6ίγΐ3ππηο) ρΓί5ΐίη3ΐηγοίη _IA ^ εΐοχγ1ί3ΐ1 4ζ) οηγ1-ά63 (4ζ -άζίΐΐΐΐύύ)) ρή3ίιη3πιγοίη I _A 4ζ-εΜ0Γ ^ 63 (4ζ-ΐΓΓ6ΐγΐ3ΐηιηο) ρη3ΐιη3η'ΐγοίη I _A 4--bromo-des (4-dimethylamino) pristinamycin I _A

4ζ-Bromo-des (4ζ-dimethylamino) pristinaπlycine Ι _Η

4ζ-Iodo-des (4ζ-dimethylamino) pristinamycin Ι _Α

4ζ -) <^^ ε8 (4ζ ^ ίιη ^ 1αηιίηο) ρπ5ΐίηαπψ (; ίη Ιη

4ζ-ίτίΑυόπη ^ Μ65 (4ζ ^ πη ^ ΐΗηιίηο) ρΓί5ίίηΗπψ (; _ίη Ι Α 4ζ-ίιί1υόπτ ^ Μβ5 (4ζ ^ ιιτ ^ ΐΗΓηίηο) ρη5ίιηΗΐΓ ^ _οίη Ι Η 4ζ-ΐ6Γε-Βι ^ Με5 (4ζ ^ ίπ ^ ΐ3ΐηίηο) ρη5ίίη3π ^ <, ίη _Ι Α 4ζ-isopropyl-de (4ζ-dimetylanlino) pristinaInycín _Ι Α 4ζ-isopropyl-de (4ζdίmetylamino) pristinamycin Ιε 48-methylamino-des ^ dimethylamino) pnstinamycín _Ι Α-methoxy-48 of N-dimethylamino) pristinamycin _Ι Α 4s-methoxy-de (47-dimethylamino) pristinamycin _Ι Η-fluoro-4 s-41metyl of N-dimethylamino) pristmamycín _Ι Α 4ζ-8πυηο ^ 68 (4ζ ^ ^ ίηι ΐ8ΐηίηο) ρΓί5ΐίη3η · ^ οίη _Ι Α 4ζ- ethylamino-des (4ζ-dimethylamino) pristinamycin Ι _Α 4ζ-diethylamino-des (4ζ-dimethylamino) pristinamycin Ι _Α 4ζ-allylamino-des (4ζ-dimethylamino) pristinamycin _Α ζ 4ζ-Dialylamino-des (4ζ-dimethylamino) pristinamycin Ι _Α 4ζ-alyletylanlino-de (4ζ-dπnetylamlno) pristinamycin _Ι Α 4ζ-etylpropylamίno-de (4ζ-dimethylamino) pristinamycin _Ι Α 47-ethylisopropylaminocarbonyl amino-de (47-dimetylainmo) pristinamycin _Ι Α 4ζ-etylmetylcyklopropylamino-de (4ζ-dimethylamino) pristinamycin _Ι Α 4ζ- (l-pyrrolidinyl) des ^ -dimethylamino) pnstinamycín _Ι Α-4C4rifluórmetoxy of N-Dimethylamino) pristinaniycm _Ι Α 4ζ-allyloxy-de (4ζ-dimethylamino) pristinamycin _Ι Α 4ζ-ethoxy-de (4ζ-dimethylamino) pristinamycin _Ι Α 4ζ-ethylthio-de (4ζ-dimethylamino) pristinaInycín _Ι Α 4ζ-methylthiomethyl-de (4ζ-dimethylamino) pristinamycin _Ι Α 47- (2-chloroethoxy) des ^ -dirnetylamino) pristinamycin _Ι Α 4ζ-αο Με3 ^ (^ 4ς ίπι ^ ΐ3ΐηίηο) ρΓί5ΐίη3πψ <; ίη _Ι Α 4ζ-ethyl-de (dimethylamino 4ζ) pristlnamycín _Ι Α 4ζ -ethyl-des (4ζ-dimethylamino) pristinamycin Ιη 4e-dimethylamino-des (4-dimethylamino) pristinamycin Ι _4α -Methylthio-des (47-dimethylamino) pristinamycin Ι _4α -ethoxy-des (4-dimethylamino) pristinamycin Ι _Α

The invention also relates to a process, in particular suitable for the preparation of the compounds of formula I.

The invention relates to a process for the preparation of streptogramins, which comprises using a strain of streptogramin-producing microorganisms having at least one genetic modification affecting the biosynthesis of a Group B precursor, and wherein said mutant strain is cultivated in a culture medium which is adequately and complemented by at least one parent precursor other than the precursor whose biosynthesis has been altered and that said streptogramins are isolated.

Thus, the strains used within the scope of the invention are mutant strains that produce streptogramins. Said genetic modification or said genetic modifications may be located either at the level of the genes involved in the biosynthesis of said precursors or the coding region thereof, for example in the region responsible for expression and / or transcriptional or post-transcriptional regulation of said genes or in the region belonging to a transcript comprising said genes.

According to a preferred embodiment of the invention, the mutant strains have one or more genetic modifications at the level of at least one of their genes which are involved in the biosynthesis of group B streptogramins.

Said or said genetic modifications alter the expression of said gene, i.e., it makes this gene and optionally other genes which are involved in the biosynthesis of precursors, partially or wholly incapable of encoding a natural enzyme that is involved in the biosynthesis of at least one precursor.

The inability of said genes to encode natural proteins can be manifested either by the production of an inactive protein as a result of structural or conformational modifications, or by the absence or production of a protein having altered enzymatic activity, or also by the production of a natural protein to a reduced extent. A set of these possible manifestations results in a deterioration or even blockage of at least one of the group B streptogramins precursors at the synthesis level.

The genes which are capable of mutation are preferably those which are involved in the biosynthesis of the following precursors: L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid, L-phenylglycine and / or acid 3 -hydroxypicoline (3-HPA).

They are preferably papA genes. papM. papB (SEQ ID NO: 3), papC (SEQ ID NO: 2), hpaA (SEQ ID NO: 8), snbF (SEQ ID NO: 6), pipA (SEQ ID NO: 5), which are described below.

for the papA and papM genes. these genes are described in International Patent Application PCT / FR93 / 0923. These genes are present in the cosmid pOBV2. The papA gene appears to correspond to the 4-amino-L-phenylalanine biosynthesis gene from the chorismate. 4-amino-L-phenylalanine is then dimethylated by the product of the gene papM, which is N-methyltransferase, and to give 4-dimethylamino-L-phenylalanine (DMPAPA, which is then incorporated into pristinamycin IA _in. The two genes are therefore specific procedure related to the synthesis of the precursor indicating DMPAPA.

As for the other papB genes. papC. Pip. snbF and hpaA. these genes are defined and characterized within the scope of the invention. These genes are grouped with the snbA genes. papA and papM in the chromosomal region of about 10 kb (Fig. 7).

Sequence homologies shown for the PapB and PapC proteins show that these proteins also play a role in the biosynthesis of the precursor DMPAPA together with the PapA and PapM proteins. Both novel papB and papC co-genes were isolated and identified by subcloning made from the cosmid pIBV2 described in International Patent Application PCT / FR93 / 0923 and the plasmid pVRC900, which is derived from pIBV2 by the HindIII deletion and also described in the aforementioned PCT International Patent Application. / FR93 / 0,923th

Comparison of the protein encoded by the pape gene with the protein sequences contained in the Genpro bank shows 27% homology to the region of the predicate dehydrogenase activity of the bifunctional TyrA proteins from Escherichia coli (Hudson and Davidson, 1984) and Erwinia herbicola (EMBL data library, 1991). This region of TyrA catalyzes the aromatization of the prefenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. A similar aromatization starting from 4'-deoxy-4-aminoprephenate, which leads to -aminophenylpyruvate, is very likely to work in the synthesis of DMPAPA. It would be catalyzed by the PapC protein (SEQ ID No. 2).

As for the PapB protein, this protein has 24 to 30% homology to the region exerted in the chorizmate mutase activity of the bifunctional TyrA and PheA proteins of Escherichia coli (Hudson and Davidson, 1984) and TyrA y Erwinia herbicola. This region catalyzes the isomerization of chorismate to the prefenate in tyrosine and phenylalanine biosynthesis. The PapB protein (SEQ ID No. 3) appears to act at a similar isomerization level starting from 4-deoxy-4-aminochorismate and resulting in 4-deoxy-aminoprephenate in the synthesis of DMPAPA. Regarding pipA genes. snbF and hpaA, these genes were located in regions located between the snbA gene encoding the acid

3-hydroxypicolin / AMP ligase described in PCT / FR9 / 0923 and papA or snbR genes. These were precisely localized by subcloning performed from plasmid pVRC900 and cosmid pIBV2, which are described in International Patent Application PCT / FR93 / 0923.

Comparison of the protein encoded by the hpaA gene and the protein sequences found in the Genpro bank showed 30-40% homology to the protein family likely to be involved (Thorson et al., 1993) in the transamination of various biosynthesis intermediates of various antibiotics (DnrJ, EryCl, TylB, StrS, PrgL). Synthesis of the 3-HPA precursor, which is presumably derived from lysine, by another route, such as cyclodeamination (see Examples 1-2 and 2-1), probably requires a transamination step capable of catalysing the product of this gene called hpaA (SEQ ID NO 8). The results of the mutation made in this gene show a clear application of this gene in the synthesis of the precursor 3-HPA.

Comparison of the product encoded by the gene designated pipA with the protein sequences found on the Genpro bank shows 30% homology to ornithine cyclodeaminase from Agrobacterium tumefasciens (Schindler et al., 1989). This enzyme acts in the final stage of octopine catabolism and converts L-ornithine to L-proline by a cyclodeamination mechanism. The authors have shown by incorporation of labeled lysine that 4-oxopipecolic acid and 3-hydroxypicolinic acid, found in both PI _A and virginiamycin, are derived from lysine (Molinero et al., 1989, Reed et al., 1989. Cyclodeamination reaction of lysine A similar reaction to that described for ornithine would lead to the formation of pipecolic acid, and, taking this hypothesis into account, this product was designated PipA (SEQ ID No. 5.) The results of the mutation in the pipA gene shown in the examples below show that It should be noted that this mutation does not affect the biosynthesis of 3-hydroxypicolinic acid, which is also derived from lysine and whose precursor could be pipecolic acid.

Comparison of the snbF gene product with the protein sequences contained in the Genpro bank revealed 30-40% homology to several cytochrome P450-like hydroxylases involved in secondary metabolite biosynthesis (Omer et al., 1990, Trower et al., 1992). Some hydroxylations can be envisaged in the biosynthesis of pristinamycin I precursors, particularly at the level of 3-HPA (hydroxylation at the 3-position of picolinic acid) and 4-oxopipecolic acid (hydroxylation at the 4-position of pipecolic acid). The corresponding protein was designated as SnbF (SEQ ID No. 6).

The results of the mutation in the pipA gene with polar effects on snbF gene expression indicate the action of the snbF gene in hydroxylating the pipecolic acid residue of group B streptogramins.

Thus, the expression of the snbF gene is modified due to genetic modification at the pipA gene level.

Preferably, said genetic modification or said genetic modifications make said gene partially or wholly incapable of encoding a natural protein.

Genetic modification is to be understood in particular to mean any suppression, substitution, deletion or addition of one or more bases in a gene or genes of interest. Such modifications can be achieved in vitro (on isolated DNA) or in situ, for example using genetic engineering techniques, or also by exposing said microorganisms to mutagenic agents. Such mutagenic agents include, for example, physical agents such as energetic radiation (X-rays, gamma-radiation, ultraviolet radiation, etc.) or chemical agents that are capable of reacting with various functional bases of DNA bases, such as alkylating agents. [ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroquinoline-1-oxide (NQO)], bialkylating agents, intercalating agents, etc. Deletion means any suppression of some or all of the gene under consideration . In particular, it may be the coding region of said proteins and / or the entire promoter region or part of the transcriptional, translational or transcriptional promoter region.

Genetic modifications can also be achieved by a gene disorder, realized, for example, according to the protocol originally described by Rothstein (Meth. Enzymol. 101 (1983) 202) or preferably by double homologous recombination. In this case, the coding sequence will be interrupted in order to optionally allow replacement by homologous recombination of the wild-type genomic sequence with a non-functional or mutant sequence.

In another alternative embodiment of the invention, said genetic modification may consist in introducing a gene or genes encoding said proteins under the control of a regulated promoter.

The mutant strains of the microorganisms of the invention can be obtained from any streptogramin producing microorganism (see Table V). According to a specific embodiment of the invention, it is a strain derived from S. pristinaespiralis and in particular S. pristinaespiralis SP92.

A preferred mutant strain within the scope of the invention is, in particular, the strain SP92 :: pVRC508, mutated in DMPAPA precursor biosynthesis by disruption by the papA gene cross-over mechanism. or the SP212 strain mutated in DMPAPA precursor biosynthesis by disruption of the papA gene by a double homologous recombination mechanism. These strains no longer produce PI except when complemented by DMPAPA precursor. Unexpectedly, it has been found that when an original precursor different from DMPAPA and capable of potentially metabolizing incorporation by PI synthetase (the SnbD protein responsible for incorporating the residues L-proline and DMPAPA) is added to the production environment, both become capable of producing new pristinamycins. or virginiaamycins, or produce a majority component that is normally a minor component of PI, especially a component of PI _b (FIG. 2).

Two additional mutant strains were prepared within the scope of the invention. These are the SP92pipA :: Dam ^R strain disrupted in the pipA gene by homologous recombination and the SP92hpaA :: nam ^R strain disrupted in the hpaA gene. The SP92pipA: Dam ^R strain no longer produces PI under standard fermentation conditions and, in the presence of pipecolic acid, allows high production of the originally minor component of streptogramin components B, in which 4-oxopipecolic acid is replaced by L-pipecolic acid. As regards S. pristinaespiralis SPhpaA: flam ^R. this strain also no longer produces PI under standard fermentation conditions, but is able to produce novel group B streptogramins in the presence of the original precursors.

It has been shown that by complementing the culture medium containing the mutant strains according to the invention with at least one original precursor, it is possible to orient the biosynthesis either towards new streptogramins or one of the minor forms, or to privilege the formation of one of them.

Derivatives or analogs of amino acids, and in particular phenylalanine, as well as organic acids, in particular α-ketocarboxylic acids, and in particular phenylpyruvic acid derivatives, can be precursors used within the scope of the invention.

Of course, the original precursor is one which causes alteration or even blockade within the scope of the invention at the level of biosynthesis of one of the natural precursors of group B streptogramins, leading to the synthesis of streptogramins. Within the scope of a specific embodiment of the invention, the original precursor is selected to be close to the precursor whose synthesis is altered. Thus, in the specific case of the mutant blocked in DMPAPA biosynthesis, the parent prodrug is preferably a phenylalanine derivative.

Precursors which may be suitable for use within the scope of the invention include, but are not limited to, the following precursors:

4-dimethylaminophenylalanine,

4-metylaminofenylalanín,

4-amino phenylalanine.

4-dietylaminofenylalanín,

4-etylaminofenylalanín,

4-metyltiofenylalanín,

4-phenylglycine,

4-methoxyphenylalanine,

4-trifluórmetoxyfenylalanín,

4-metoxykarbonylfenylalanín,

4-chloro-phenylalanine,

4-bromophenylalanine,

4-iodophenylalanine,

4-trifluórmetylfenylalanín,

4-tert-butylfenylalanín,

4-izopropylfenylalanín,

3-metylaminofenylalanín,

3-methoxyphenylalanine,

3-metyltiofenylalanín,

3-fluoro-4-methylphenylalanine, L-pipecolic acid, 4-tert-butylphenylpyruvic acid 4-methylaminophenylpyruvic acid,

2- Naphthylphenylalanine

4-fluorophenylalanine,

3-fluorophenylalanine,

3-etoxyfenylalanín,

2,4-dimethylphenylalanine,

3.4- dimethylphenylalanine,

3-Methylphenylalanine

4-phenylphenylalanine,

4-butylfenylalanín,

2-Thienyl-3-alanine

3-trifluoromethylphenylalanine, hydroxyphenylalanine,

3-ethylaminophenylalanine,

4-allylaminophenylalanine,

4-dialylaminofenylalanín,

4-alyletylaminofenylalanín,

4-ethylpropylaminophenylalanine,

4-ethylisopropylaminophenylalanine, 4-ethylmethylcyclopropylphenylalanine,

4- (1-pyrrolidinyl) phenylalanine, 4-0-allyltyrosine,

4-O-ethyltyrosine,

4-etyltiofenylalanín,

4-etyltiometylfenylalanín,

4-0- (2-chloroethyl) tyrosine,

4-acetyl phenylalanine,

4-ethyl phenylalaninate,

3-dimetylaminofenylalanín,

3-etoxyfenylalanín,

3-Fluoro-4-methylphenylalanine a

4-aminomethylphenylalanine.

Of these precursors, 4-trifluoromethoxyphenylalanine,

3-methylthiophenylalanine, 3-fluoro-4-methylphenylalanine, acid

4-methylaminophenylpyruvinyl, 3-ethoxyphenylalanine, 4-allylaminophenylalanine, 4-dialylaminophenylalanine, 4-allylethylaminophenylalanine, 4-ethylpropylaminophenylalanine, 4-ethylmethylcyclopropylphenylalanine, 4- (1-pyrrolidinyl) thienyl, 4- (1-pyrrolidinyl) thienyl 3-Dimethylaminophenylalanine, 3-ethylaminophenylalanine, novel compounds which have been prepared and characterized within the scope of the invention, these precursors have proven particularly useful for the preparation of streptogramins of the invention.

The claimed method has proven to be of particular interest for the preparation of novel group B streptogramins or also for the preferred formation of some of them. This method is particularly suitable for preparing PIB.

The present invention also provides a nucleotide sequence selected from the group consisting of:

(a) all or part of the papC (SEQ ID No.3), pipA (SEQ ID No.5), snbF (SEQ ID No.6) and hpaA (SEQ ID No.8) genes,

(b) hybrid sequences with all or part of the genes of:

(c) sequences derived from sequences (a) and (b) as a result of the degeneracy of the genetic code.

In the specific case of hybrid sequences b), these sequences preferably encode a polypeptide useful in streptogramin biosynthesis.

Even more preferably, the invention provides nucleotide sequences represented by the genes papC (SEQ ID No. 2), papB (SEQ ID No. 3), PIPA (SEQ ID No. 5), snbF (SEQ ID No. 6), and hpaA (SEQ ID No. 2). .8).

The present invention further relates to recombinant DNA comprising the papC gene (SEQ ID NO: 2), papB (SEQ ID NO: 3), pipA (SEQ ID NO: 5), snbF (SEQ ID NO: 6) and hpaA (SEQ ID NO: 2). .8).

Of course, the above-defined nucleotide sequences may be part of an expression vector type vector, which may be a vector with autonomous or integrative replication or a suicide vector (vecteur suicide). The invention also relates to these vectors as well as to any use of the sequence of the invention or the corresponding vector, in particular for the preparation of interesting metabolites.

The invention also relates to any mutated S. pristinaespiralis strain having at least one genetic modification at the level of any of the genes papC (SEQ ID No. 2), papB (SEQ ID No. 3), PIPA (SEQ ID No. 5), snbF (SEQ ID NO. 6) and hpaA (SEQ ID NO. 8), and especially strains of SP92pipA :: Oam ^R. SP92hpa :: Dam ^R. as well as a S. pristinaespiralis strain having a genetic modification consisting in disruption of the papA gene by double homologous recombination, such as SP212.

The combination of the group A streptogramin component and the compound of formula I according to the invention forms compositions of particular interest in the therapeutic field. These compositions are used in particular for the treatment of diseases caused by Gram-positive bacteria (genera Staphylococcus, Streptococcus, Pneumococcus and Enterococcus) and Gram-negative bacteria (genera Haemophilus, Gonococcus, Meningococcus). The compounds of the invention thus synergize the antibacterial effect of pristinamycin IIB on Staphylococcus aureus IP8203 in vivo in mice at doses that are substantially between 30 mg / kg and 100 mg / kg for oral administration when combined in a PI / PII ratio. that equals 30: 70.

The invention relates to any pharmaceutical composition comprising at least one compound of the formula I, optionally in combination with group A streptogramin.

In the following, the invention will be described in more detail by way of examples of a specific embodiment, these examples being illustrative only and not limiting in any way the scope of the invention, which is clearly defined by the wording of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. FIG. FIG. FIG. FIG. FIG

In the accompanying drawings it shows the structure of pristinamycin I, shows the structure of minor components of pristinamycin I, shows examples of the structures of the streptogramin B components, shows the 2.9 kb Pst1-Xhol region, shows the 4.5 kb Xhol-PstI region, illustrates the HindIII region -BglII with 1.6 kb, shows the BglII-XhoI region of about 10 kb, shows the plasmid pVRC415, shows the plasmid pVRC420, shows the plasmid pVRC411, shows the plasmid pVRC414, shows the plasmid pVRC414, and shows the construction strategy SP212.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Sequencing and identification of genes involved in the biosynthesis of pristinamycin I and its precursors

Identification by sequencing of genes located downstream and upstream of the PapA coding gene described in PCT / FR93 / 0923, as well as the gene located behind the SnbA coding gene, also described in PCT / FR93 / 0923.

This example describes how it is possible for the pIBV2 cosmid described in PCT / FR93 / 0923 and containing the structural genes of PapA and PapM enzymes intervening in the synthesis of the precursor 4-dimethylamino-L-phenylalanine (DMPAPA) pristinamycin

I and the structural gene of the enzyme SnbA responsible for activation of the aromatic precursor pristinamycin I, which is 3-hydroxypicolinic acid (3-HPA), identified by sequencing around these genes and studies of corresponding mutants to identify other genes involved in biosynthesis of precursor DMPAPA precursor or other pristinamycin I.

For this purpose, subcloning was performed starting from the cosmid pIBV2 and the plasmid pVRC900, derived from pIBV2 by the deletion of HindIII and also described in patent PTC / FR93 / 0923.

This example illustrates how nucleotide sequences of fragments located downstream and upstream of the papa and snbA genes from S. pristinaespiralis can be obtained.

Techniques for Cloning of Contemplated DNA Fragments in M13mp18 / 19 Vectors (Messing et al., 1981) s) by classical cloning techniques from Escherichia coli as described in Maniatis et al. (1989).

1-1 Sequencing and analysis of the region downstream of the papA gene

For the purpose of sequencing this region between the papA and papM genes. fragments of 1.5 kb PstI-PstI, 0.7 kb PstI-XhoI, and 0.7 kb XhoI-XhoI were subcloned into the M13mpl8 and 13mpl9 vectors, starting from plasmid pVRC900. The cloning sites were traversed by sequencing on double stranded DNA using the plasmids pVRC409 described in PCT / FR93 / 0923. The cloning steps were performed as follows.

About 2µg of plasmid pVRC900 was digested with PstI restriction enzymes. and / or Xhol (New England Biolabs) under the conditions specified by the supplier. The thus obtained restriction fragments were separated on a 0.8% agarose gel, whereby the desired 1.5 kb Pst1-PstI, 0.7 kb Pst1-Xhol and 0.7 kb Hhol-Xhol fragments were isolated and purified by Geneclean (BiolO1). , La Jolla, California). For each cloning, about 10 ng of M13mpl9 and / or

M13mn18 digested with PstI and / or XhoI binds 100 ng of the fragment to cloning under the conditions described by Maniatis et al., 1989. After transformation of the TG1 strain (K12, delta (lac-pro) supE thi hsd deltaS traD36 for A ^ B * laclq lacZ deltaMIS, Gibson , 1984) and selecting lysis regions on LB + X-gal + IPTG media by the technique described by Maniatis et al. (1989) isolate phages that carry the desired fragments. The individual inserts were sequenced by the chain termination method using as a universal primer synthesis initiator or synthetic and complementary oligonucleotides of the 20 nucleotide sequence of the insert for sequencing. Reactions were performed using fluoroscopic dideoxynucleotides (Prism Ready Reactions DyeDeoxy terminator Cycle Sequncing Kit-Applied Biosystem) and analyzed on an Applied Biosystems Model 373A DNA sequencing machine. The cleavage between these different advertisers made it possible to assemble the entire nucleotide sequence that is present between the papA and papM genes (SEQ ID NO: 1).

From this nucleotide sequence, it is possible to determine the open reading frames and thus to identify genes involved in the biosynthesis of PI or its precursors in S. pristinaespiralis, as well as polypeptides encoded by these genes.

The presence of open reading frames in the 2.9 kb PstI-XhoI fragment containing the nucleotide sequence between the papA and papM genes was investigated, taking advantage of the fact that Streptomyces DNA contains a high percentage of G and C bases as well as the appropriate codon usage that they form coding phases (Bibb et al. 1984). The Staden and Mc Lachlan method (1982) allows the probability of coding phases to be calculated based on the use of Streptomyces codons already sequenced and summarized in a file containing 19673 codons obtained from the Bosance information base (Dessen et al.)

This method makes it possible to characterize the four very likely open reading phases in the 2.9 kb PstI-XhoI fragment shown in the following table (Table I). These phases are designated as phases 1 to 4 according to their location from the site

Pst. For each of these phases, its length is indicated as the number of bases and its position in the fragment (the PstI site is situated at position 1). The number of amino acids of the encoded polypeptide is also indicated for open reading phases 2 and 3. Phases 1,3 and 4 are encoded by the same strand and phase 2 is encoded by the complementary strand (Fig. 4). Phases 1 and 4 correspond to the C-terminal region of the PapA protein, respectively. The N-terminal regions of the PapM protein previously identified and described in PCT / FR / 00923.

Table I

phase number and / or mark gene location Count nucleotides Number of amino acids 1 (PapA) 1-684 684 - 2 (PapC) 949-1836 888 296 3 (PapB) 1873-2259 387 129 4 (PapM) 2259-2887 629 -

Comparison of the Phase 2 product (Table I) with the protein sequences contained in Genpro shows 27% homology with the region exerted in the Escherichia coli-derived prefenate-dehydrogenase activity of Escherichia coli-derived TyrA proteins (Hudson and Davidson, 1984) by Erwinia herbicola (EMBL bank) (1991). A similar aromatization starting from 4-deoxy-4-aminoprephenate and leading to 4-aminophenylpyruvate is very likely to occur in the synthesis of the precursor DMPAPA. This reaction would be catalyzed by the Phase 2 product referred to as PapC (SEQ ID No. 2).

Comparison of the Phase 3 product (Table I) with the protein sequences contained in the Genpro flask shows 24-30% homology with the region applicable to the Escherichia coli-derived chorismate mutase activity of the bifunctional TyrA and PheA proteins ((Hudson and Davidson, 1984) and Erwinia herbicola-derived TyrA protein catalyzes isomerization of chorizmate to prefenate in tyrosine and phenylalanine biosynthesis.A similar isomerization, starting from 4-deoxy-4-aminochorismate, leading to 4-deoxy-4-aminoprephenate, is most likely involved in DMP precursor synthesis This reaction would be catalyzed by a Phase 3 product referred to as PapB (SEQ ID No. 3).

In the case of TyrA and PheA, chorismate mutase and prefenate dehydratase or prefenate dehydrogenase activities are catalyzed by the same protein. In S. pristinaespiralis, the enzymatic activities of chorismate mutase and prefenate dehydrogenase are catalyzed by two different proteins, PapB and PapC.

Sequence homologies shown for the PapB and PapC proteins show that these two proteins are involved in the biosynthesis of the aromatic derivative DMPAPA, together with the PapA and PapM proteins. As with papA, disruption of the papB and papC genes must result in the construction of strains of S. pristinaespiralis that are unable to produce PI but are able to produce new PIs modified at the DMPAPA residue level in the presence of the original precursors.

1-2. Sequencing and analysis of the region upstream of the papA gene

This region is located between the snbA gene. which encodes the 3-hydroxypicolinic acid-AMP-ligase enzyme described in PCT / FR93 / 0923 and the papA gene.

Cloning was performed as described in the example

1-1. starting from plasmid pVRC900 and cosmid pIBV2 described in PCT / FR93 / 0923. 1.3 kb XhoI-XhoI fragments, 0.2 kb XhoI-XhoI, 3.3 kb XhoI-XhoI, 1.1 kb HindIII-PstI with 2.2 kb PstI-PstI were subcloned into the M13mpl8 vectors, and M13mpl9. These different cloning allowed to traverse all cloning sites. Individual inserts were sequenced as described in Example 1-1, using a universal primer or complementary synthetic oligonucleotides of the 20 nucleotide sequence of the insert for sequencing.

The coverage between the various inserts made it possible to determine the total nucleotide sequence present between the snbA and papA genes (SEQ ID NO: 4).

From this nucleotide sequence, open reading phases can be determined and genes that are involved in the biosynthesis of precursors of PI products from S. pristinaespiralis as well as polypeptides that are encoded by these genes can be identified.

The presence of open reading phases within the 4.5 kb XhoI-PstI containing the nucleotide sequence between the snbA and papA genes was investigated as described in Example 1-1. This method made it possible to characterize within the 4.5 kb XhoI-PstI fragment four very likely open reading phases, which are shown in the following table (Table II). These phases are referred to as phases 1-4 according to their position from the XhoI site. For each of these phases, its length in number of bases and its position within the fragment (where the XhoI site is located at position 1) are shown. The number of amino acids of the encoded polypeptide is also indicated for open reading phases 2 and 3. Phases 2 and 4 are encoded by the same strand and phase 1 is encoded by the complementary strand (Fig. 5). Phases 1 and 4 correspond to the N-terminal region of the SnbA and PapA proteins previously identified and described in PCT / FR93 / 0923.

Table II

phase number and / or mark gene location Count nucleotides Number of amino acids 2 (SnbA) (inv) 1-329 329 - 2 (PipA) 607-1671 1065 355 3 (SnbF) 1800-2993 1194 398 4 (PapA) 3018-4496 1479 -

Comparison of the Phase 2 product (Table II) with the protein sequences found in the Genpro flask shows 30% homology to ornithine-cyclodeaminase derived from Agrobacterium tumefasciens (Schindler et al 1989). This enzyme acts at the last stage of octopine catabolism, converting L-ornithine to L-proline by a cyclodeamination mechanism. The authors have shown by incorporation of labeled lysine that 4-oxopipecolic acid and 3-hydroxypipecolinic acid found in both PI _A and virginiaamycin SI are derived from lysine (Molinero et al., 1989, Reed et al., 1989). A cyclodeamination reaction of lysine, similar to that described for ornithine, would lead to the formation of pipecolic acid. Upon accepting this hypothesis, the Phase 2 product was designated PipA (SEQ ID No. 5). Results of mutation in pipA gene. shown in 2-1, show the action of the popA gene in the pipecolic acid synthesis itself, since this mutation does not affect the biosynthesis of 3-hydroxypicolinic acid, from which lysine is also derived and whose precursor could be pipecolic acid.

Comparison of the Phase 2 product (Table II) with protein sequences found in the Genpro bank shows 30-40% homology with several cytochrome P450-like hydroxylases involved in the biosynthesis of secondary metabolites (Omer et al., 1990, Tower et al., 1992) . several hydroxylations are contemplated in the biosynthesis of pristinamycin precursors, particularly at the levels of 3-HPA biosynthesis (hydroxylation at the 3-position of picollic acid) and 4-oxopipecolic acid (hydroxylation at the 4-position of pipecolic acid). Results of mutation in pipA gene. shown in 2-1-3 show the action of the Phase 3 product in hydroxylating the pipecolic acid residue of PI _e . Thus, the corresponding gene was designated as snbF and the corresponding protein as SnbF (SEQ ID NO: 6).

1-3. Sequencing downstream of the snbA gene

This region is located between the snbA gene encoding the 3-hydroxypicolinic-adenylate ligase enzyme and the snbR gene encoding the membrane protein, which is probably responsible for transport and resistance to PI, both of which are described in PCT / FR93 / 0923. Sequences were carried out from the isolated fragment of cosmid pIBV2 as described in Example 1-1.

The 1.6 kb HindIII-BglII fragment was subcloned into the M13mpl8 and M13mpl9 vectors from cosmid pIBV2. The insert was sequenced as described in Example 1-1, using a universal primer or complementary synthetic oligonucleotides of the sequence of 20 nucleotides of the insert at sequence 26 as the initiator of the synthesis. From the nucleotide sequence thus obtained (SEQ ID No. 7), it is possible to determine the open reading phases and to identify the genes involved in the biosynthesis of precursors of the S. pristinaespiralis PO product, as well as the polypeptides encoded by these genes. The presence of open reading phases within the 1.6 kb HindIII-BglII fragment corresponding to the end of the snbA genes in the region downstream of this gene was investigated, as described in the example.

1-1. A complete open phase coded with the same strand as the snbA gene was shown (Fig. 6). With respect to position 1 corresponding to the HindIII site. this phase begins at nucleotide 249, i. 30 nucleotides after the snbA gene end, and ends at nucleotide 1481. Its size is 1233 nucleotides corresponding to the 411 amino acid protein.

Comparison of the product of this open phase with protein sequences found in the Genpro bank shows 30-40% homology with a group of proteins likely to be involved (Thorson et al., 1993) in the transamination of various biosynthesis intermediates of various antibiotics (DnrJ, EryCl, TylB, Strs, PrgL) ). The synthesis of the 3-HPA precursor, which appears to be derived from lysine by a mechanism other than cyclodeamination (see Examples 1-2 and 2-1, could require a transamination stage capable of catalyzing the product of said phase 1, referred to as HpaA (SEQ ID NO. The mutation results in this gene, as shown in 2-2, unequivocally confirm the action of this gene in the synthesis of the precursor 3-HPA and confirm the above hypothesis.

PapB genes. pape, pipA. The snbF and hpaA described within the scope of the invention are grouped with the snbA genes. papA and papM on the chromosomal region of about 10 kb (Fig. 7) This confirms the presence of a cluster of genes involved in the biosynthesis of PI and its precursors. Studying the regions before and after this cluster of genes should allow the identification of other genes involved in the biosynthesis of precursors of the PI product, in particular L-phenylglycine and L-2-aminobutyric acid.

Example 2

Construction of strains recombined by disruption of identified genes

This example illustrates how it is possible to demonstrate the action of the genes described in Example 1 in the biosynthesis of pristinamycin precursors, as well as to construct strains s. pristinaespiralis capable of producing new pristinamycins. These strains are obtained by disrupting the genes involved in the biosynthesis of the residue to be substituted, wherein the new pristinamycins are produced by complementing these mutants with the original precursors.

The strain SP92 :: pVRC508 used within the scope of the invention for the production of new PI derivatives by replacing the DMPAPA precursor with other

I molecules are described in PCT / FR93 / 0923. This strain results from a disruption by the cross-over mechanism of the papA gene involved in the biosynthesis of the DMPAPA precursor and appearing at the early stage of chorismatate transamination. This disruption is polar because the expression of the papM gene (PCT / FR93 / 0923), located 1.5 kb downstream of the papA gene, involved in the double methylation of 4-amino-L-phenylalanine to DMPAPA is greatly reduced in this mutant. In fact, determining the activity of the 4-amino-L-phenylalanine methylation enzyme (PAPA) on DMPAPA, indicates for SP92 :: pVRC508 mutant only activity equal to 5% of wild-type activity.

Within the scope of the invention, this strain SP92 :: pVRC508 allows, under adequate complementation and fermentation conditions, the production of novel pristinamycins modified at the DMPAPA residue level as shown in Example 3. Mutants of the same phenotype can be obtained by disrupting the papB or papC genes. described within the scope of the invention.

In a similar manner, another type of S. pristinaespiralis strain, disrupted in the papA gene and having the same phenotype as the SP92 :: pVRS508 strain, can be obtained by disrupting the crossing mechanism of the papA gene. This construct was performed using a 4.6 kb DphI-HindIII starting fragment isolated from the pIBV cosmid and containing the 3 'region of the pipA gene. the snbF and papA genes as a whole, as well as part 3 'of the papC gene. This fragment was cloned into the suicide gene pDH5, capable of replicating only in Escherichia coli, but carrying a resistance marker (marquer de resistance) expressing in Streptomyces (thiostrepton or thiheptide resistance gene, tsr). This vector pDH5 was developed by Wohlebben et al. (1991 Nucleic Acid Res. 19: 727-731). The 1.1 kb Bcl1-Bcl1 deletion was then performed in the papA gene, and the 2.2 kb HindIII-HindIII fragment carrying the amR gene (genecithin and apramycin resistance) was introduced after the cohesive ends were added. The recombinant vector was designated pVRC414 and is shown in FIG. After transformation of the pristinamycin-producing strain with plasmid pVRC414, geneticin resistant and thiostrepton sensitive transformants were isolated and analyzed. These clones result from double recombination between the DNA regions of the S. pristinaespiralis strain pVRC414 and the corresponding chromosomal region of the S. pristinaespiralis strain as described in FIG. 13. One of these clones was designated SP212. Its phenotype is identical to the phenotype of strain SP92 :: pVRC508 in terms of the inability to produce new antibiotics in the presence of the original precursors. Preferably, this type of strain obtained by a double crossing-over mechanism has better stability compared to strains obtained by a simple crossing-over mechanism.

2-1. Construction of a mutant derived from S. pristinaespiralis SP92 disrupted in the pipA gene

This example illustrates how, by disrupting the pipA gene, it is possible to construct a S. pristinaespiralis strain that no longer produces PI under standard fermentation conditions and which is capable of producing novel pristinamycins modified at the level of the 4-oxopipecolic acid residue of PIA when fed to the fermentation. environment will add the original precursors.

It was constructed using a suicide vector replicating only in Escherichia coli, the vector being pUC1318. This vector does not carry a resistance marker expressed in Streptomyces. Its presence in the Streptomyces genome can only be detected by colony hybridization.

2-1-1. Construction of plasmid pVRC420

This example illustrates how a non-replicating plasmid in S. pristinaespiralis SP92 can be constructed that can be used to disrupt the pipA gene by a double homologous recombination mechanism.

Plasmid pVRC420 was constructed to carry out the chromosomal mutant SP92 disrupted in the pipA gene, starting from the cosmid pIBV2 described in PCT / FR93 / 0923. The pIBV2 cosmid was digested with the restriction enzyme PstI, and after separation of the thus generated fragments by agarose gel electrophoresis (0.8%), a 2.8 kb PstI-PstI fragment containing the start of the snbA and snbF genes and the entire pipA gene was isolated and purified. Geneclean (BiolOl, La Jolla, CA). 50 ng of the vector pUC1318 linearized by PstI digestion. was bound with 200 ng of a 2.8 kb fragment as described in the Example

A clone carrying the desired fragment was isolated after transformation of the TG1 strain and selection in LB + ampicillin 150 gg / ml + X-gal + IPTG medium. This recombinant plasmid was designated as pVRC415 (FIG. 8). Am ^R gene cassette. coding for resistance to apramycin or geneticin (Kuhstoss et al., 1991) was thus introduced at the exclusive HindIII site of plasmid pVRC415, which site is located downstream of the pipA gene. This construction was performed as follows. A 2.5 kb DNA fragment containing the am ^R gene, the PermE promoter (Bibb et al., 1985) as well as the 158 first amino acids of the erythromycin resistance gene, ermE was isolated by double digestion of SalI-BcrlII from a plasmid derived from plasmids pIJ4026 (carrier plasmid) the ermE gene under the control of the PermE promoter) and pHP45Dam ^R. After filling the cohesive ends 5 exiting SalI and BglII

By the Klenow enzyme according to the protocol described by Maniatis et al., 1989, the fragment containing the am ^R gene is cloned into the HindIII site of plasmid pVRC415, whose protruding 5 'cohesive ends were also filled with the Klenow enzyme, as described above. The recombinant plasmid thus obtained was named pVRC420. A restriction map is shown in FIG. 9th

2-1-2. Isolation of the SP92pipA :: flam ^R mutant disrupted in the pipA gene by homologous recombination

This example illustrates how to construct a mutant from

S. pristinaespiralis interrupted in the pipA gene.

This mutant was isolated by transformation of the SP92 strain with the suicide plasmid pVRC420.

The preparation of protoplasts, their transformation, as well as the extraction of total DNA of the recombinant strains were performed according to the methods described by Hopwood et al. (1985).

The strain SP92 was cultured in a medium YEME (Hopwood et al., 1985): 34% sucrose, 5 mM MgCl _and 0.25% glycine - for 40 hours at 30 ° C. The mycelium was protoplasted in the presence of lysozyme and 5 x 1 µg of pVRC420 was used to transform (using the PEG method) protoplasts. After a one night stage, when the protoplasts were regenerated to R2YE (D. Hopwood et al., 1985), the recombinants were selected by digesting 3 ml of SNA medium (D. Hopwood et al., 1985) containing 1500 g / ml geneticin.

100 geneticin resistant clones were isolated for 5 transformations performed. These recombinants result from integration by single or double homologous recombination between the pipA gene carried by the SP92 chromosome and portions of the pipA gene contained in the 5.3 kb fragment carried by the suicide plasmid pVRC420. For the selection of recombinants obtained by the double crossing-over mechanism (ie not containing part of the pUC1318 plasmid pVRC420 in their genome) hybridizations were carried out on colonies of 90 clones using pUC19 labeled in (and ³² P) dCTP as a probe, as described by Maniatis and al. (1989). Selection yielded 10 clones resistant to geneticin but not hybridizing to the pUC19 vector. Spores of the recombinants were isolated by digestion and growth in HT7 + 10 µg geneticin and re-digestion in the same medium to obtain isolated colonies. To verify the integration area of plasmid pVRC420, several Southern blots were performed with total DNA of several recombinant clones, purified as described by Hopwood et al. (1985), and hybridized with a 2.8 kb PstI-PstI fragment used as a probe after labeling (? ³² P) dCTP. The results obtained confirm that these recombinants were obtained by a double crossing-over mechanism between the pVCR420 vector and the chromosome of SP92, resulting in the replacement of the 2.8 kb PstI-PstI fragment containing the pipA gene with the 5.3 kb PstI-PstI fragment containing the pipA gene disrupted introduction of the am ^R gene. One of these mutants was designated SP92pipA :: nam ^R.

2-1-3. Production of pristinamycins by the SP92pipA :: nam ^R mutant

This example illustrates how the mutant strain is determined

S. pristinaespiralis SP92 disrupted in the pipA gene by integration of plasmid pVCR420, however, no longer produces PI under standard fermentation conditions and, on the other hand, allows high production of the minor form of Streptogramin components B in which the oxopipecolic acid is replaced by pipecolic acid.

The SP92pipA :: Dam ^R mutant as well as the SP92 strain as a reference strain were cultured in a production liquid medium. Fermentation was carried out as follows: 0.5 ml of the spore suspension of the above strain was added under sterile conditions to a 40 ml inoculation medium in a 300 ml Erlenmeyer flask. The inoculation medium comprises 10 g / l corn steep, 15 g / l sucrose, 10 g / l ammonium sulfate, 1 g / l potassium hydrogen phosphate, 3 g / l sodium chloride, 0.2 g / l sulfate heptahydrate magnesium and 1.25 g / l calcium carbonate. The pH is adjusted to 6.9 with sodium hydroxide prior to the introduction of the calcium carbonate. The contents of the Erlenmeyer flasks are stirred for 44 hours at 27 ° C in a rotary mixer at 325 rpm. 2.5 ml of the above-mentioned 44-hour medium is sterile added to a 30 ml production medium in an Erlenmeyer flask containing 300 ml. The production environment consists of 25 g / l soybean meal, 7.5 g / l starch, 22.5 g / l glucose, 3.5 g / l feed yeast, 0.5 g / l zinc sulfate and 6 g / l calcium carbonate . The pH was adjusted to 6.0 with hydrochloric acid before the calcium carbonate was introduced. The Erlenmeyer flasks are stirred for 24, 28 and 32 hours at 27 ° C. At the end of each time interval, 10 g of the medium is introduced into an Erlenmeyer flask, to which 20 ml of a mobile phase consisting of 34% acetonitrile and 66% 0.1 M potassium dihydrogen phosphate solution (whose pH is adjusted to 2.9 with concentrated acid) are added. phosphorous) and allowing the extraction of pristinamycins. After mixing, the entire aliquot is centrifuged and the pristinamycins contained in the supernatant are determined by high performance liquid chromatography, where a 150 μΐ centrifugation supernatant is loaded onto a 4.6 x 150 mm Nucleosil 5-C8 column and eluted with a 40% elution system. % acetonitrile and 60% 0.1M phosphate buffer pH 2.9. Pristinamycins I are detected by their absorbance in ultraviolet light with a wavelength of 206 nm.

The results obtained show that under the above standard fermentation conditions, the mutant strain SP92pipA :: Qam ^R did not produce PI even after 24, 28 and 32 hours of fermentation, while the reference strain SP92 produced a standard amount of PI at the three time stages. Both strains produced the same amount of Pil. The SP92pipA :: ham ^R mutant strain is thus effectively blocked at the PI biosynthesis step.

Complementary fermentation assays were also performed in which, after 16 cultures, various PI precursors were added to the production medium separately or together. The results of these complementations made it possible to show that if 100 mg / l pipecolic acid and 100 ng./l DMPAPA were added to the fermentation broth simultaneously, the mutant strain produced normally a minor PI derivative, namely PI _e (lower production) as 5% for strain SP92) in an amount equivalent to the amount of PI produced by the reference strain. Such production does not occur when pipecolic acid and DMPAPA are added separately. PI _e differs from PI (which is a major component of PI) in the absence of the ketone function at position 4 of pipecolic acid. The fact that the complementation of the SP92pipA :: nam ^R mutant strain can be accomplished by the simultaneous addition of pipecolic acid and DMPAPA shows that the papA and probably papB and papM genes were disrupted by the polar effect of the construct. In fact, all these genes are located downstream of the pipA gene and are probably counter-transcribed with pipA. Thus, disruption of the latter gene also causes disruption of the pap genes and hence the absence of DMPAPA. The fact that complementation of the SP92pipA :: Dam ^R mutant with pipecolic acid causes PI _e production and not PI production _and leads to two conclusions: the first of these is that the construction of the PI cycle is done by incorporating pipecolic acid and not 4-oxo-pipecolic acid and that for the hydroxylation providing the ketone function at the 4-position, it is only later. The second conclusion is that this hydroxylation is likely to be accomplished by the SnbF enzyme, whose structural gene is located directly downstream of the pipA gene. In fact, the apparent polarity of the ppa gene disruption on the pap genes probably implies a polar effect on the snbF gene, which is situated between the pipA gene and the pap genes. as a result, inhibition of the hydroxylating function of the pipecolic acid residue in PI to 4-hydroxypipecolic acid found in PI _e and PI _g (Fig. 2), which is then oxidized to 4-oxo-pipecolic acid in PI _A.

The realization of such a mutant strain made it possible to construct a strain of S. pristinaespiralis which is capable of producing PI only in the presence of precursors of the PI product, DMPAPA and pipecolic acid from which it is capable of producing, in an amount equivalent to that produced by the non-mutant strain. is normally a minor derivative in the pristinamycin mixture. Similarly, in the presence of the original precursors or a mixture of the original precursors and the precursors normally present in PI, this strain will be able to produce novel pristinamycins modified in either or both or both of DMPAPA and 4-oxopipecolic acid residues.

2-2. Construction of a mutant strain of S. pristinaespiralis SP92 disrupted in the hpaA gene

This example illustrates how, by disrupting the hpaA gene, a S. pristinaespiralis SP92 strain that does not produce PI under normal standard fermentation conditions and which is capable of producing new pristinamycins modified at the level of precursor 3-HPA can be constructed when original precursors are added to the fermentation environment. .

Construction of said strain was carried out using a plasmid that does not replicate in S. pristinaespiralis SP92 and which can be used to disrupt the hpaA gene by double homologous recombination.

2-2-1. Construction of suicide plasmid pVRC421

Construction of the plasmid pVRC421 was performed using a suicide vector that is only able to replicate in Escherichia coli, but which carries a resistance marker expressing in Streptomyces, i. thiostrepton or nosiheptide tsr resistance gene. This vector pDH5 was developed by Hillemann et al. (1991).

Plasmid pVRC421 was constructed to carry out the chromosomal mutant strain SP92 disrupted in the hpaA gene of cosmid pIBV2, described in PCT / FR93 / 0923. The cosmid pIBV2 was dispersed with the restriction enzyme SphI and after separation of the thus generated fragments by electrolysis on an agarose gel (0.6%), a 4.8 kb Sph1-Sph1 fragment containing the entire hpaA gene and almost the entire snbA gene was isolated. and this fragment was purified by the Geneclean system described above. 50 ng of SphI digested linear vector pDH5 was ligated with 200 ng of the 4 kb fragment described later. The clone carrying the desired fragment was isolated after transformation of the TG1 strain and selection to LB + ampicillin 150 gg / ml + I / TG + X-gal. The recombinant plasmid was designated as pVRC411 (FIG. 10). The cassette containing the am ^R gene encoding apramycin or geneticin resistance is then inserted into the Pflml exclusive site of plasmid pVRC411, which is located 610 bp downstream of the hpaA gene. This construction was carried out as follows. The 2.2 kb DNA fragment containing the am ^R gene was isolated after digestion of the plasmid pHP45 am ^R containing the am ^R gene. using HindIII. After filling the protruding cohesive ends with 5 'HindIII Klenow enzyme according to the protocol described by Maniatis et al. (1989), a fragment containing the am ^R gene was cloned into the Pflml site of plasmid pVRC411, whose protruding 3 'cohesive ends were released by the enzyme T4-polymerase as described by Maniatis et al., (1989). The recombinant plasmid thus obtained was designated as pVRC421. Its restriction map is shown in Fig. 11.

2-2-2. Isolation of strain SP92hpaA :: ham ^R interrupted in hpaA gene by homologous recombination

This example illustrates how to construct a mutant strain

S. pristinaespiralis disrupted in hpaA gene.

This mutant was isolated by transformation of the SP92 strain with the suicide plasmid pVRC421.

The protoplasts were prepared and transformed according to the methods described above.

The SP92 strain was cultured in ΥΕΜΕ (34M% sucrose, 5 mM MgCl 2, 0.25% glycine) for 40 hours at 30 ° C. The mycelium was protoplasted in the presence of lysozyme and 5 x 1 µg of plasmid pVRC421 was used to transform (using PEG method) protoplasts. During the night, when the protoplasts were regenerated in the R2YE medium, the recombinants were selected by digesting 3 ml of SNA medium containing 1500 gg / ml geneticin.

600 geneticin resistant clones were isolated for 5 transformations performed. These recombinants result from integration accomplished by single or double homologous recombination between the hpaA gene. carried by the SP92 chromosome and a 6 kb fragment of the suicide plasmid pVRC421. For the selection of recombinants obtained by the double crossing-over mechanism (ie clones that no longer contain part of the pDH5 plasmid pVRC421 in their genome), said clones were re-seeded in HT7 medium containing 40 Mg / ml thiostrepton. Selection yielded 6 clones resistant to geneticin but sensitive to thiostrepton. Recombinant spores were isolated by digestion and growth on HT7 + 10 gg / ml geneticin and re-digested in the same medium to obtain isolated colonies. To verify the integration position of plasmid pVRC421, various Southern blots of total DNA of 6 recombinant clones were purified as described by Hopwood et al. (1985), and a 4.8 kb hybridized SphI-SphI fragment used as a probe to label (? ³² P) dCTP. The results obtained confirm that these recombinants were obtained by a double crossing-over mechanism between the pVRC421 vector and the chromosome of SP92, resulting in the replacement of the 4.8 kb Sphl-Sphl fragment containing the hpaA gene with the 6 kb Sphl-Sphl fragment containing the hpaA gene disrupted by the am gene. ^R. One of these mutants was designated SP92hpaA :: Dam ^R.

2-2-3. Production of pristinamycins by SP92hpaA :: flam ^R mutant

This example illustrates how the mutant strain is determined

S. pristinaespiralis SP 92 disrupted in the hpaA gene by integration of plasmid pVRC421 no longer produces PI under standard fermentation conditions.

Mutant strain SP92hpaA :: nam ^R. as well as strain SP92 as a reference strain, were cultured in a liquid production medium. The fermentation was carried out as described in Example 2-1-3, then the pristinamycins were extracted and determined as above. The results show that under the fermentation conditions realized, the mutant strain SP92hpaA :: amam ^R does not produce PI even after 24, 28 and 32 hours of fermentation, whereas the reference strain produced a standard amount of PI in the three time stages. Both strains produced equal amounts of Pil. The mutant strain SP92hpaA :: amam ^R is thus effectively blocked at the PI biosynthesis step. Complementary fermentation tests were also performed in which different PI precursors were added separately or together after 16 hours of culture to the culture production medium. When 100 mg / L of 3-hydroxypicolinic acid is added to the fermentation broth, the mutant strain produces Pi in an amount equivalent to the amount of PI produced by the reference strain. The fact that complementation of the mutant strain SP92hpaA :: flam ^R can only be accomplished by the addition of 3-hydroxypicolinic acid shows that the hpaA gene is involved in the synthesis of this precursor.

The construction of this mutant made it possible to obtain a strain of S. pristinaespiralis mutated for PI production, but which, in the presence of the 3-HPA precursor, is capable of producing PI in an amount equivalent to that produced by the parent strain. As in the previous examples, it is expected that such a strain in the presence of the original precursors will produce new pristinamycins modified at the acid residue level

3-hydroxypicolinic.

Example 3

Production of compounds of formula I by mutant strain SP92 :: pVRC508

This example illustrates how a mutant strain of S. pristinaespiralis SP92 is disrupted in the papA gene by integration of plasmid pVRC508 capable of synthesizing novel streptogramins in the presence of precursors added to the production environment. These precursors may be derivatives of amino acids and, in particular, phenylalanine, but also α-ketocarboxylic acids and in particular phenylpyruvic acid.

The SP92 :: pVRC508 mutant strain was cultured in a liquid production medium. Fermentation was carried out as follows: 0.5 ml of the spore suspension of the above strain was added to a 40 ml inoculation medium in an Erlenmeyer flask equipped with a 300 ml septum. This inoculation medium consists of 10 g / l corn steep, 15 g / l sucrose, 10 g / l ammonium sulfate, 1 g / l potassium hydrogen phosphate, 3 g / l sodium chloride, 0.2 g / l sulfate heptahydrate magnesium and 1.25 g / l calcium carbonate. The pH is adjusted to 6.9 with sodium hydroxide before the addition of calcium carbonate. The Erlenmeyer flasks are shaken for 44 hours at 27 ^C, c in a rotary shaker at a speed of 325 rpm. 2.5 ml of the aforementioned 44 h culture are sterile added to 30 ml of production medium in a 300 ml Erlenmeyer flask. The production environment is 25 g / l soybean meal, 7.5 g / l starch, 22.5 g / l glucose,

3.5 g / l of feed yeast, 0.5 g / l of zinc sulfate and 6 g / l of calcium carbonate. The pH was adjusted to 6.0 with hydrochloric acid before the calcium carbonate was introduced. The Erlenmeyer flasks are stirred at 28 ° C in a rotary mixer at a rotation speed of 325 rpm. After 16 hours, 1 ml of a solution of one of the precursors listed in Table III below (generally 5 or 10 g / l) is added to the culture medium. The culture is then discontinued 8 or 24 hours later. The volume of the culture mash is measured, then 2 volumes of the mobile phase consisting of 34% acetonitrile and 66% 0.1 M potassium hydrogen phosphate solution (pH 2.9 adjusted with concentrated phosphoric acid) are added to allow the extraction of pristinamycins. After mixing, all the mixture is centrifuged and the pristinamycins contained in the supernate are extracted and purified according to the methods described in Example 4. These products are also determined by high performance liquid chromatography in which 150 μΐ of the above centrifugation supernatant is loaded onto a Nucleosil column.

The 5-C8, 4.6 x 150 mm, was eluted with a mixture of 40% acetonitrile and 60% 0.1M phosphate buffer pH 2.9. The novel pristinamycins are detected by their absorbance in ultraviolet radiation at 206 nm and optionally by their fluorescence emission (filter 370 nm, excitation at 306 nm).

Table III

precursor

Origin of phenylalanine

4-dimetylaminofenylalanín

4-metylaminofenylalanín

4-amino phenylalanine

4-dietylaminofenylalanín

4-etylaminofenylalanín

4-metyltiofenylalanín

4-methylphenylalanine

4-methoxyphenylalanine

4-trifluórmetoxyfenylalanín

4-methoxycarbonylphenylalanine

4-chlorophenylalanine

4-bromophenylalanine

4-iodophenylalanine

4-trifluórmetylfenylalanín

4-tert-butylfenylalanín

4-isopropylphenylalanine

3-metylaminofenylalanín

3-methoxyphenylalanine

3-metyltiofenylalanín

3-fluoro-4-methylphenylalanine 4-tert-butylphenylpyruvic acid 4-methylaminophenylpyruvic acid

Janssen

Example 33

Example 34-1

Janssen 22.794.96

Example 33

Example 33

Example 33

J.P.S101-312-4 / example 33 Janssen 16.975.97 Example 34-8 Example 33 Janssen 15.728.14 Janssen 22.779.81

Bachem F 1675 P.CR.Inc.12 445-3 Example 35-1 Example 36-1 Example 35-3 J.P.S.101-313-2 Example 34-11 Example 34-5

Example 33

Example 34-4

Table II (continued) 2-naftylenylalanín Bachem F ’1865 4-fluoro-phenylalanine Bachem F ’1535 3-fluorophenylalanine Bachem F ’2135 3-etoxyfenylalanín Example 37-1 2,4-dimethylphenylalanine Example 33 3,4-dimethylphenylalanine Example 33 3-methylphenylalanine Example 33 4-fenylfenylalanín Example 33 4-butylfenylalanín Example 36-3 2-thienyl-3-alanine Aldrich 28.728.8 3-trifluórmetylfenylalanín Example 33 3-dihydroxyphenylalanine Aldrich T 9.039.5 3-etylaminofenylalanín Example 35-6 4-aminomethylphenylalanine Example 33 4-alylaminofenylalanín Example 38-2 4-dialylaminofenylalanín Example 38-1 4-alyletylaminofenylalanín Example 39-4 4-ethylpropylaminophenylalanine Example 39-6 4-etylizopropylaminofenylalanín Example 39-1 4-etylmetylcyklopropylamino- phenylalanine Example 39-8 4- (1-pyrrolidinyl) phenylalanine Example 40-1 4-0-alyltyrozín Example 33 4-O-etyltyrozIn Example 33 4-etyltiofenylalanín Example 33 4-etyltiometylfenylalanín Example 41-1 4-O- (2-chloroethyl) tyrosine Example 42-1 4-acetylfenylfenylalanín Example 33 4-ethyl phenylalaninate Example 33 4-dimetylaminofenylalanín Example 35-10

The following table (Table IV) shows the retention times for new PI products using ΡΙ _α as the reference substance. Absolute retention times were determined at 25 ° C in a high performance liquid chromatography system as defined below. These retention times vary slightly from one injection to the column head to the other and depending on the temperature.

Table IV

precursor t (relative retention time R ' new PI (NeoPi) drunk A drunk H another neoPI 4-metylaminofenylalanín 0.85 4-amino phenylalanine 0.64 4-metyltiofenylalanín 1.93 2.73 1.63 4-metylfenylfenylalanín 1.77 2.65 4-methoxyphenylalanine 1.46 4-metoxykarbonylfenylalanín 1.49 4-chlorophenylalanine 2.04 4-bromophenylalanine 2.16 4-iodophenylalanine 2.42 4-trifluórmetylfenylalanín 2.56 3.74 4-tert-butylfenylalanín 3.34 4-izopropylfenylalanín 2.80 4.35 3-metylaminofenylalanín 1.15 3-methoxyphenylalanine 1.49 2.04 3-fluoro-4-methylphenylalanine 2.93 4-tert-Butylphenyl- pyruvic 4-methylaminophenyl- 3.34 pyruvic 0.85 4-etylaminofenylalanín 0.94 4-dietylaminofenylalanín 0.61

Table IV (continued)

4-alylaminofenylalanin 1.83 4-dialylaminofenylalanin 2.64 4-Allylethyl aminophenylalanine 2.4 4-ethylpropylaminophenylalanine 1.06 4-etylizopropylaminofenyl- alanine 0.89 4-etylmetylcyklopropylamino- phenylalanine 1.1 4- (1-pyrrolidinyl) phenylalanine 2.0 4-O-Trifluoromethyltyrosine 2.42 4-0-alyltyrozín 2.62 4-O-ethyl-tyrosine; 2.2 4-etyltiofenylalanin 1.96 4-metyltiometylfenylalanin 1.98 4-0- (2-chloroethyl) tyrosine 2.45 4-acetylphenylalanine 1.61 4-ethyl phenylalaninate 1.86 3-dimetylaminofenylalanin 1.49 4-metyltiofenylalanin 1.93 3-0-etyltyrozín 1.78

The new PI for 4-isopropylphenylalanine corresponds to the neoPI _E described in Example 14.

The new PI with t 1.63 for 4-methylthiophenylalanine corresponds to the 5r-hydroxy or PI described in Example 5.

The SP92 :: pVRC508 mutant strain was fermented in the presence

4-dimetylaminofenylalanínu. Under these complementation conditions, the mutant strain SP92 :: pVRC508 produces an amount of pristinamycin I equivalent to that produced by the SP92 strain.

Example 4

Preparation of pristinamycin I (4ζ-methylamino-des (4ζ-dimethylamino) pristinamycin I _a ) and 4ζ-amino-des (4ζ-dimethylamino) pristinamycin I

4.1: Preparation of pristinamycin I (4ζ-methylamino-des (4 £-dimethylamino) pristinamycin I)

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultivated in a production medium as described in Example 3, with 1 ml of an aqueous solution (10 g / l) added to the production medium after 16 hours of culture.

4-methylaminophenylalanine having the (R, S) configuration synthesized in Example 34-1. After 40 hours of culture, 1.8 liters of production medium from 60 Erlenmeyer flasks are extracted with 2 volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and loaded onto a silica gel column (30 g) in dichloromethane, then the column was eluted successively with an elution gradient of 0-10% methanol in dichloromethane. The fraction containing pristinamycin I was combined and evaporated. The dry residue is taken up in 6 ml of a mixture of 65% water and 35% acetonitrile and the resulting mixture is loaded onto a 10 × 250 nm Nucleosil 7 µ08 semiprep column (Macherey Nagel) and eluted with a 65% 100 mM phosphate buffer mixture. pH 2.9 and 35% acetonitrile. The fractions containing pristinamycin I are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and evaporated. 52 mg of pristinamycin _IB H-NMR (400 MHz, CDC1 _3, ppm s, ref.TMS): 0 71 (d J 16: 6 Hz, 1 H, β 5,), 0.92 (t, J = 7.5 Hz, 3H: _CH3 2 γ) cd 1.10m 1:40 (mt, 2H: 3 (¾ γ ₂ and 3), 1:34 (d, J = 7.5 Hz, 3H: _CH3 1 _7), ca. 1.50 m 1.85 ( mt, 3H: 3 γ _Ι and CH, 2 β), 2.03 (mt, 1 H, 3 β ₃ ), 2.22 (mt, 1 H, 5 δ ₂ ), 2.33 (broad d, J = 16 Hz, 1H: 5 δ,), 2:40 (d, J = 16 Hz, 1H: 5 β _3), 2.82 (mt, 1H: 5 ε.), 2.81 (s, 3H: 4 vgarakferylu NCH), 2.90 (dd,

J = 12 and 4 Hz, 1H: 4 & 3.29 (s, 3H: 4 NH3, from 3.20 3.45 to 3.60 (2 mts, 1H each), 3.40 (t, J = 12 Hz, 1H : 4 β _χ ), 4.57 (dd, J = 7 and 8 Hz, 1H, 3 a), 4.75 (dd, J = 13 and 7 Hz, 1H: 5 ej, 4.83 (nit, 1H: 2a), 4.89 (broad _d, J = 10 Hz, 1H: la), 5.24 (dd, J = 12 and 4 Hz, 1H: 4 a), 5:32 (broad d. J = 6 Hz, 1H: 5 a), 5:39 (d, J = 9 Hz, 1H: 6a), 5.90 (q broad, J = 7.5 Hz, 1H: 1β), 6.53 (d, J = 9 Hz, 1H: NH2), 6.53 (d,

J = 5Hz, 2H: 4a), 7.03 (d, J = 8Hz, 2H: 45), cd 7.10 0: 7.35 (mt, 5H: δ Arcmztic 6), 7.46 (mt, 2H: 1 H _s and 1 ') H, 7.85 (dd, J = 5.5 and 2 Hz, 1H: 1 Hj, 8.44 (d, J = 10 Hz, 1H: NH 1), 8.76 (d, J = 9 Hz, 1H: NH 6), 11.63 ( s, 1H: OH).

4.2: Preparation of 4ζ-amino-des (4ζ-dimethylamino) pristinamycin I

The strain SP92 :: pVRC508 was cultivated in a production medium as described in Example 3, to which 60 ml of Erlemeyer flasks were added, after 16 hours of cultivation, 1 ml of an aqueous solution of (5 g / l) (S) -4-aminophenylalanine was added. After 40 hours of cultivation, the production mash originating from 60 Erlenmeyer flasks (1.8 liters) is extracted with 2 volumes of a mixture of 66% 100 mM phosphate buffer with 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and loaded onto a column (30 g) of silica gel in dichloromethane, then the column is eluted with an elution gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. The dry residue is taken up in 6 ml of a mixture of 65% water and 35% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7µΟ8 semipreparative column (Macherey Nagel), which is then eluted with 65% 100 mM phosphate buffer 2 , 9 and 35% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 5 mg of 4-amino-of ζ (4ζ -dimethylamino) pristinamycin I, _and.

¹ H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): δ 72 (dd J = et 5.5 Hz, 1H, 5 b.), 0.90 (t, J = 7.5 Hz, 3H: CH3 ₎ 2 γ), l1.10 ° 1.40 (mt, 2H: 3 β.

and γ 3.), 1:33 (d, J = 7.5 Hz, 3H: _CH3 1 γ), σά 1.50c 1.85 (mt, 3H: 3 γ, and β 0¾ 2),

2.02 (mt, 1H, 3?), 2.19 (mt, 1H, 5? U, 2.33 (d broad, J = 16 Hz, 1H: 5? U, 2.42 (d,

J = 16 Hz, 1H: 5 β.), 2.81 (dt, J = 13 and 4 Hz, 1H: 5 ε,), 2.90 (dd, J = 12 and 4 Hz, 1H: 4 β-), 3.24 ( s, 3H: NCH ₃ 4), <± 3.20 <± 3.40 and 3.54 (2 bowls, 1H, CH3 3 δ), 3.30 (t, J = 12 Hz, 1K: 4 β,), 3.72 (mf, 2H: ArNH ., 4.54 (dd, J = 7.5 and 7 Hz, 1H, 3a),

4.73 (broad dd, J = 13.8 Hz, 1H: 5 ε.), 4.82 (mt, 1H: 2a), 4.89 (broad d, J = 10 Hz,

1K: 1a), 5.22 (dd, J = 12 a. 4 Hz, 1H: 4 a), 5.32 (d broad, J = 5.5 Hz, 1H: 5 a), 5.89 (mi, 2H: 6 a and 1 ( 3), 6.51 (d, J = 9.5Hz, 1H: ΝΉ 2), 6.61 (d, J = 8Hz, 2H: 4ε), 6.98 (d, J = 8Hz, 2K: 4δ), <± 7.15cb7 .35 (mt, 5H: aromatic 6), 7.45 (dd, J = 8.5 and 13 Hz, 1K: 1 H, 7.48 (dd, J = 8.5 and 4 Hz, 1H: 1 H,)), 7.82 (dd, J = 4 and 1.5 Hz, 1H: 1 H, 8.43 (d, J = 10 Hz, 1H: ΝΉ1), 8.76 (d, J = 9.5 Hz, 1H; ΝΉ6), 11.63 (s, 1H:

OH).

Example 5

Preparation of 4'-methylamino-des (4'-dimethylamino) pristinamycin I _a

On a 50-ml Erlemeyer flask, the strain SP92 :: pVRC508 is cultured in the production medium, as in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -4-methylthiophenylalanine in O 1N sodium hydroxide, wherein said (R, S) -4-methylthiophenylalanine was synthesized as described in Example 33. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlemeyer flasks were extracted with 2 volumes of a mixture of 66% 100mM phosphate buffer. pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane which was then eluted with an elution gradient of 0 to 10% methanol in dichloromethane. The fraction containing the new derivative of pristinamycin I is combined and evaporated. 65 mg of dry residue are obtained. This residue is taken up in 6 ml of a mixture of 60% water and 40% acetonitrile and the mixture is introduced in two aliquots onto a 10 x 250 mm Nucleosil 7 juC8 semiprep column (Macherey Nagel), which is then eluted with a mixture containing 55 % 100 mM phosphate buffer pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated.

45 mg of 4'-methylthio-des (4'-dimethylamino) pristinamycin I are obtained.

H ¹ H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): 0.68 (dd,

J = 16 and 5.5 Hz, 1H, 5 βύ, 0.93 (t, J = 7.5 Hz, 3H: CH ₃ 2 γ), 1.13 (mt, 1H: 3 β.), Cd 1.25-3.4 (mt, 1H; 3 γ ₂ ), 1.33 (d, J = 7.5 Hz, 3 H: CH ₃ 1 γ), cd 1.55b 1.85 (mt, 3 H: 3 γ ₃ and CH ₂ 2 β), 2.02 (mt, 1H, 3 β ₃ ), 2.18 (mt, 1H, δ ₂ ), 2.38 (broad d, J = 16.5 Hz, 1H:

δ _3), 2:46 (s, 3H, _SCH3), 2:48 (d, J = 16 Hz, 1 H, 5 β _3), 2.85 (dt, J = 13.5 and 4 Hz, 1H:

Δ, 3.00 (dd, J = 12 and 5 Hz, 1H: 4β,), 3.23 (s, 3H: NCH ₃ 4), 3.37 (t, J = 12 Hz, 1H:

β,), 3:37, and 3:58 (2 mts, lHkaždý CH ₂ 3 O), 4.55. (t, J = 7.5 Hz, 1 H, 3 a), 4.77 (broad dd., J = 13.5 and 8 Hz, 1H: 5 ε,), 4.86 (mt, 1H: 2a), 4.89 (dd, J = 10 and 1.5 Hz, 1H: flight), 5.30 (d broad, J = 5.5 Hz, 1H: 5a), 5.32 (dd J = 12 and 5 Hz, 1H: 4 (a), 5.90 (d, J =

9.5 Hz, 1H: 6 (a), 5.92 (dq, J = 7.5 and 1.5 Hz, 1Η; 1β), 6.55 (d, J = 9.5 Hz, 1H: NH2),

7.13 (d, J = 8Hz, 2H: 4δ), cd 7.15b7.35 (mt, 5H: aromatic H at position 6), 7.19 (d, J = 8)

Hz, 2H: 4ε), 7.45 (mt, 2H; 1 H ₄ and 1 H H, 7.76 (t, J = 5 Hz, 1H: 1 H H, 8.42 (d, J = 10 Hz, 1H: NH 1)), 8.76 (d, J = 9.5 Hz, 1H: NH6), 11.65 (s, 1H: OH).

Fractions leaving the above-described silica gel column containing the new derivative of pristinamycin I, _h are isolated 10 mg of 4-methylthio ζ-de (4-ζ-dimethylamino) pristinamycin X _h, where, by carrying out semi-preparative chromatography on a silica gel column, which is described above, , with the exception that the acetonitrile content of the eluent is 50%.

^X H-NMR (400 MHz, CDCl, δ ppm, ref.TMS): ^{,,: 3} 0.32 (mt, 1H, pp, 0.93 (t, J = 7.5 Hz, 3H, CH ₃ γ 2), 1.20 cd < bl.35 (mt, 2H, 3 β, γ ₂ and 3), 1.30 (d,

J = 7.5 Hz, 3H: _CH3 1 γ). cd 1.35b2.05 (mt, 9H: 3 γ ₃ - 3 β ₃ - 0¾ 2 p - CH. 5 δ - CH.

5γ a. 5 p _3), 2:44 (dt, J = 13.5 et 1.5 Hz, 1H: 5 ε,), 2:49 (s, 3H, _SCH3), 2.99 (dd, J = 12 and 5 Hz, 1H: 4 &). 3.09 (dd, J = 12.5 and 12 Hz, 1H: 4 p ₃ ), 3.54 and 3.64 (2 mts, 1H each / CH ₂ 3 δ), 4.17 (dd, J = 7 and 6 Hz, 1H: 3 and ), 4.49 (broad d, J = 13.5 Hz: 1H: 5 ε ₃ ), cd4.70cb4.80 (mt, 3H: 2a - 5 a and 4 a), 4.84 (dd, J = 10 and 1.5 Hz, 1H : 1a), 5.51 (d, J = 7Hz, 1H: 6a), 5.73 (mt, 1H: 1β), 6.65 (d, J = 9.5Hz, 1H: NH2), 7Π0 (d, J = 8) Hz, 2H: 4 $), 7.22 (d, J = 8Hz, 2H; 4ε), from. 7.20 <b7.40 (mt, 7H: H aromatic 6 - 1 H ₄ and 1 H 5 _s ), 7.87 (d, J = 4 Hz, 1H: 1 Hp, 8.55 (mf, 1H: NH 6), 8.55 (d J = 10 Hz, 1H:

NH 1), 11.70 (s, 1H: OH).

Μ

3 mg of 5'-hydroxy-4'-methylthio-des (4'-dimethylamino) pristinamycin I were isolated from the fractions leaving the above-described silica gel column containing the novel pristinamycin I derivative by performing the semi-preparative column chromatography described above. maintaining the acetonitrile content of the eluent at 45%.

1 H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): unequivocally the major isomer: -OH at 5v in axial position can be observed.

0.37 (d mt, J 16)

Hz, 1H, 5 β ..), 0.93 (t, J = 7.5 Hz, 3 H: CH ₃ 2 γ), * 1.20 * 1.45 (mt, 2H: 3 β and 3 γ ₂ ),

1.31 (d, J = 7.5 Hz, 3H: _CH3 1 γ) * * 1:40 1.85 (mt, 5H, 3 - CH. 2 (CH 3 a, δ 5).

1.98 (mt, 1H, 3β), 2.17 (d, J = 16Hz, 1H: 5ft), 2.50 (s, 3H: SCH3), 2.77 (dt, J =

13.5 and 2 Hz, 1H: 5 e.), 2.99 (dd, J = 12 and 4 Hz, 1H: 4 &amp;), 3.11 (t, J = 12 Hz, 1H: 4 b.), Cd 3.45 * 3.70 ( mt, 2H: CH, 3 δ), 3.73 (mt, 1H: 5 γ ^{at the} equatorial position), 4.13 (t, J = 7Hz, 1H, 3c / T 4.37 (dx), J = 13.5 Hz, 1H 4.75 * 4.95 (mt, 3H: 2a - 4 cc and 5a), 4.89 (dd, J = 10 and 1 Hz, 1H; 1a), 5.70 (d, J = 8 Hz, 1H); : 6 (a), 5.80 (dq,

J = 7.5 and 1 Hz, 1H; 1β), 6.37 (d, J = 5Hz, 1H: NH4), 6.71 (d, J = 10Hz, 1H: NH2),

7.10 (d, J = 8Hz, 2H: 4δ), 7.22 (d, J = 8Hz, 2H: 4e), cd 7.20dc7.40 (mt, 5H: H aromatic 6), 7.43 (dd, J = 8.5 and 1.5 Hz, 1H: 1 HJ, 7.47 (dd, J = 8.5 and 4 Hz, 1H; 1 H H), 7.89 (dd, J = 4 and 1.5 Hz, 1H; 1 H 4, 8.55 (d, J = 10 Hz, 1H: NH2), 9.15 (d, J =

Hz, 1H: NH 6), 11.70 (s, 1H: OH).

Example 6

Preparation of 4-methyl-ζ of (4 ^ -dimethylamino) pristinamycin I, _and a methyl 4-de (dimethylamino 4ς) pristinamycin I _h

On a 69-Erlenmeyer flask, the SP92 :: pVRC508 strain is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of a solution of (R, S-4-methylphenylalanine in O) is added. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then the mixture is centrifuged. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated.The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane and the column is eluted with elution. A gradient of 0-10% methanol in dichloromethane The fractions containing the new derivative of pristinamycin I were combined and evaporated to give 49 mg of a dry residue. the residue is taken up in 6 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is introduced in two aliquots onto a 10 × 250 Nucleosil Z008 semiprep column (Macherey Nagel), which is then eluted with 55% 100mM phosphate buffer pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 44 mg of 4-methyl-ζ of (4Z7-dimethylamino) pristinamycin I, _and.

H-NMR (400 MHz, CDC1 _3, δ in ppm, ref.TMS): 0.52 (dd,.

J = 16 and 6 Hz, 1H, 5 β.), 0.93 (t, J = 7.5 Hz, 3H: CH ₃ 2 γ), 1.15 (mt, 1H: 3 β.), Cdl. 031.40 (mt, 1H : 3 γ ₂ ), 1.35 (d, J = 7.5 Hz, 3 H; CH ₃ 1 γ), α 1.50 * 1.85 (mt, 3H: 3

CH-2 β), 2.04 (mt, 1 H, 3 β ₃ ), 2.18 (mt, 1 H, 5 δ ₂ ), cd 2.25 * 2.45 (mt, 2H: 5 δ ₃ and 5 β.), 2.36 (s, 3H: _ArCH3), 2.83 (dt, J = 13 and 4 Hz, 1H: 5 ej, 2.99 (dd, J = 13 and 4 Hz,

1H: 4β, 3.28 (s, 3H; NCH ₃ 4), 3.31 and 3.59 (2 mts, 1H δ # CH ₂ 3 Ô), 3.40 (t, J =

Hz, 1H: 4 ft), 4.59 (t, J = 7.5 Hz, 1H, 3 a), 4.74 (dd broad, J = 13 and 7 Hz, 1H: 5 zs, 4.85 (mt, 1H: 2a), 4.89 (broad, J = 10 Hz, 1H: 1a), from 5.25 * 5.35 (mt, 2H; 5a and 4a), CD 5.85 * 5.95 (mt, 2H; 6a and 1β), 6.52 (d, J) = 9.5 Hz, 1H: NH 2), 7.14 (AB limit., J = 9 Hz, 4H: 4δ and 4ε), from 7.15 * 7.35 (mt, 5H: aromatic H 6), 7.50 (mt,

2H: 1 H, 1 'H _s ), 7.81 (dd, J = 4 and 2 Hz, 1H: 1 H, 8.41 (d, J = 10 Hz, 1H: NH 1)),

8.74 (d, J = 9 Hz, 1H: NH6), 11.63 (s, 1H: OH).

21 mg of 4ajú-methyl-des (4-dimethylamino) pristinamycin 1 _H (mass spectroscopy: M + H + = 810) are isolated from the fractions leaving the above silica gel column containing the new derivative of pristinamycin _1H. chromatography on the semi-preparative column described above.

Example 7

Preparation of 4'-methoxy-des (4'-dimethylamino) pristinamycin I

The strain of SP92 :: pVRC508 is cultivated in a production medium as described in Example 3, to which 12 ml of (5 g / l) (R, S) -4-methoxyphenylalanine solution in 0.1N sodium hydroxide. After 40 hours of culture, 0.35 liters of mash produced from 12 Erlenmeyer flasks is extracted with two volumes of a mixture containing 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane and then eluted with a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. 14 mg of dry residue are obtained. This dry residue is taken up with 3 ml of a mixture containing 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is then eluted with a 60% 100 mM phosphate elution system. buffer pH 2.9 and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 12 mg of ^4-methoxy-1 ζ de (4 £ -dimethylamino) pristinamycin I, _and.

^X H-NMR (400 MHz, CDC1 _3, ppm doors, ref.TMS): θ ₆₃ (dd, J = 16 Hz A.5.5, 1 H, 5 FO), 0.96 (t, J = 7.5 Hz, 3H: CH ₃ 2 7), 1.17 (mt, 1H: 3 (¾)), 1.30dol.45 (mt, 1H: 372), 1-38 (d, J = 7.5 Hz, 3H: CH3 1 γ), αό 1.55o 1.85 (mt,

3H; 7 R 3 and β 2 CH _2), 2.5 (mt, 1H, 3 βχ). 2.20 (mt, 1H, 622), 2.40 (broad, J = 16 Hz, 1H: 5 δχ), 2.47 (d, J = 16 Hz, 1H; 5 βχ), 2.88 (dt, J = 13 and 4 Hz, 1H: δ 63), 2.99 (dd, J = 12.5 and 5 Hz, 1H: 4 fo), 3.30 (s, 3H; NCH 3 4), 3.32 and 3.60 (2 mts, 1H each CH ₂ 3 δ) 3.40 (t, J = 12.5Hz, 1H: 4βχ), 3.80 (s, 3H; OCH3), 4.60 (t, J = 7.5Hz, 1H, 3a), 4.80 (broad dd, J = 13 and 8.5 Hz, 1H: 5 εχ), 4.88 (mt, 1H: 2a), 4.92 (broad d, J = 10 Hz, 1H: 1a), 5.31 (dd, J = 12.5 and 5 Hz, 1H: 4 a) 5.34 (d Si, J = 5.5 Hz, 1H: 5 a), 5.90 (d, J = 9 Hz, 1H: 6 a). 5.93 (broad broad, J = 7.5 Hz, 1H: 1p), 6.54 (d, J = 9 Hz, 1H: NH2), 6.87 (d, J = 8 Hz, 2H: 4 &apos;), 7.16 (d, J = 8 Hz, 2H: 4Ô), cd 7.15-7.40 (mt, 5H: H aromatic 6), 7.50 (mt, 2H: 1 H ₅ and 1 H ₄ ), 7.80 (dd, J = 4 and 2.5 Hz) 1 H: 1 H ( ₆ ), 8.43 (d, J = 10 Hz, 1H: NH 1), 8.78 (d, J = 9 Hz, 1H:? 6), 11.65 (s, 1H: OH).

Example 8

Preparation of 4? -Methoxycarbonyl-des (4ζ-dimethylamino) pristinamycin I

A

On a 60-Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in the production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -4-methoxycarbonylphenylalanine solution was added. After 24 hours of culture, 1 liter of mash produced from 60 Erlenmeyer flasks was extracted with two volumes of a mixture containing 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane and then eluted with a gradient of 0 to 10% methanol in dichloromethane. Fractions containing the novel derivative of pristinamycin I _and were pooled and evaporated. 14 mg of dry residue are obtained. This dry residue is taken up with 3 ml of a mixture containing 60% water and 40% acetonitrile and the resulting mixture is loaded onto a 10 × 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is then eluted with 55% 100 mM phosphate buffer. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 9 mg 4ζ methoxycarbonyl-de (4ζ · di methylamino) pristinamycin I, _and.

H-NMR (400 MHz, _CDCl3, in ppm, ref.TMS): 0.70 (dd,

J = 16 and 6 Hz, 1H, δ (¾). 0.93 (t, J = 7.5 Hz, 3H: CH, γ 2), 1.8 (mt, 1H: 3 &), CD aol.40 1.30 (mt, 1H: 3 _γ Σ), 1:33 (d, J = 7.5 Hz , 3H: CH, 1 γ), ο5 1.55d.85 (mt, 3H: ₃₇₁ and CH, 2 β), 2.02 (mt 1H, 3 ft), 2.13 (mt, 1H, 5 δ ₂ ), 2.40 ( d broad, J = 16.5 Hz, 1H; 5 δ,), 2.48 (d, J = 16 Hz, 1H, 5 ft), 2.89 (dt, J = 14.5 and 4.5 Hz, 1H: 5 e,), 3.10 (dd, J =

13.5 and 6 Hz, 1H: 4β,), 3.24 (s, 3H: NCH, 4), 3.38 and 3.61 (2 mts, 1H each; Cl · 3 δ), 3.47 (t, J = 13.5 Hz, 1H 4.55 (t, J = 7.5 Hz, 1H, 3H, COOCH3), 4.96 (s, 3H: COOCH3);

a), 4.78 (broad d, J = 14.5, 8 Hz, 1H: 5 ε,), 4.86 (broad m, 1H; 2a), 4.89 (broad d, J = 10 Hz, 1H: flight), 5.33 ( broad d, J = 6 Hz, 1H: 5 a), 5.42 (dd, J = 13.5 and 6 Hz, 1H: 4 a), 5.92 (d (J = 9.5 Hz) and mt, 1H heavier; respectively 6 aa 1β), 6.52 (d, J = 10Hz, 1H: NH2), from 7.15 to 7.35 (mt, 5H: H aromatic 6), 7.28 (d, J = 8Hz, 2H; 4δ), 7.43 (dd, J = 9 and 1.5 Hz, 1 H, L H _4), 7:47 (dd, J = 9 and 5 Hz, 1H: L H _s), 7.66 (d, J = 5 and 1.5 Hz, 1H: 1 'Hj). 7.98 (d, J = 8Hz, 2H: 4ε), 8.38 (d, J = 10Hz, 1H: NH1), 8.76 (d, J = 9.5Hz, 1H; NH6), 11.70 (s, 1H: OH).

Example 9

4'-chloro-des (4'-dimethylamino) pristinamycin I _a

On a 60 Erlenmeyer flask, the strain SP92 :: pVRC508 is cultivated in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -4-chlorophenylalanine solution in 0.1N sodium hydroxide. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane and eluted with a gradient of 0 to 10% methanol in dichloromethane. Fractions containing the novel derivative of pristinamycin I _and were pooled and evaporated. The dry residue is taken up in 3 ml of a mixture of 60% water and 40% acetonitrile and the mixture is loaded onto a 10 × 250 mm Nucleosil 7 μϋδ semiprep column (Macherey Nagel), which is then eluted with a 60% 100 mM phosphate buffer mixture. pH 2.9 and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 1 mg of 4''-chloro-des (4'-dimethylamino) pristinamycin I is obtained.

¹ H-NMR (400 MHz, CDCl _a, d in ppm, ref.TMS): 0.93 (t, J =

7.5 Hz, 3H: _CH3 2 γ), 0.95 (d. J = 16 and 5 Hz, 1 H, 5 &amp;), 1.9 (mt, 1H: 3 β-) 20.1 ± <± 1:40 (mt, 1H: 3 γ _2), 1:35 (d, J = 7.5 Hz, 3H: _CH3 1 γ), from 1:50 ± 1.85 (mt, 3H: 3 and _{7 liters}

CH, 2 β), 2.02 (mt, 1 H, 3 β.), 2.17 (mt, 1 H, 5 δ ₂ ), 2.43 (broad d, J = 16 Hz, 1H: 5 δ,),

2.59 (d, J = 16 Hz, 1 H, 5 β ₃ ), 2.90 (dt, J = 13.5 and 4 Hz, 1H: 5 e), 3.04 (dd, J = 13 and 6 Hz, 1H: 4 β). ), 3.21 (s, 3H: 4 _NCH3), 3:36 (t, J = 13 Hz, 1H: 4 β,), 3:39, and 3:59 (2 mts, IHkaščz: CH 3 δ), 4:53 (t, J = 7.5 Hz, 1H, 3 a), 4.76 (dd sir., J = 13.5 and 8 Hz,

1H: 5 ε.), 4.86 (mt, 1H: 2a), 4.87 (broad d, J = 10 Hz, 1H: 1a), 5.38 (mt, 2H: 5 and 4)

6.52 (d, J = 10Hz, 1H: NH2), 7.12 (d, J = 8Hz, 2H);

4δ), ± 7.15 ± 7.35 (mt, 7H: aromatic ą 6 et 4ε), 7.38 (dd, J = 9 and 4.5 Hz, 1H: 1 H _s ), 7.43 (broad, J = 9 Hz, 1H) 7.68 (dd, J = 4.5 and 1 Hz, 1H: 1 H +, 8.36 (d, 1H);

J = 10 Hz, 1H: NH 1), 8.75 (d, J = 9 Hz, 1H: NH 6), 11.65 (s, 1H: OH).

Example 10

Preparation of 4'-bromo-des (4'-dimethylamino) pristinamycin I _A and 4% -bromo-des (4'-dimethylamino) pristinamycin I

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -4-bromophenylalanine solution was added. v 0, IN sodium hydroxide. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane and this column was then eluted with a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. The dry residue is taken up in 6 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is then loaded twice onto a 10 × 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is then eluted with a 60% 100 mM phosphate buffer mixture. pH 2.9 and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 6 mg of 4'-bromo-des (4'-dimethylamino) pristinamycin 1 'are obtained.

^X H-NMR (400 MHz, _CDCl3, in ppm, ref.TMS): 0.93 (t, J = 7.5

Hz, 3H: CH ₃ 2 7), 0.95 (dd, J = 16 and 5 Hz, 1H, 5 β.), 1.10 (mt, 1H: 3 β,), 1.35 (d, J =

7.5 Hz, 3H: CH ₃ i γ), 1.36 (mt, 1H: 3 γ ₂ ), cdl.50dol.85 (mt, 3H: 3 γ ₃ and CH, 2 β),

2.02 (mt, 1 H, 3 β ₃ ), 2.18 (mt 1 H, 5 δ.), 2.43 (broad d, J = 16 Hz, 1H: 5 δ ₃ ), 2.59 (d,

J = 16 Hz, 1H, 5? ₃ , 2.90 (dt, J = 13 and 4 Hz, 1H: 5 ey, 3.02 (dd, J = 13 and 5.5 Hz, 1H);

Λ), 3.21 (s, 3H: 4 NCH ₃ ), 3.33 (dd, J = 13-11 Hz, 1H: 4 β ₃ ), 3.39 and 3.59 (2 dishes,

1H težúv ·. CH 3 δ), 4.53 (t, J = 7.5 Hz, 1H, 3 a), 4.76 (broad d, J = 13 and 7 Hz, 1H):

ε ₃ ), 4.86 (broad m, 1H: 2a), 4.89 (broad d, J = 10 Hz, 1H: 1a), 5.37 (broad d, J = 5 Hz, 1H:

5.39 (dd, J = 11 and 5.5 Hz, 1H: 4a), 5.92 (mt, 2H; 6a and 1β), 6.56 (d, J = 9.5 Hz, 1H: NH2), 7.08 (d, J = 8 Hz, 2H: 4δ), from 7.15 to 7.35 (mt, 5H: H aromatic 6), 7.40 (mt, 4H: 1 H, - 1 H, and 4ε), 7.70 (broad d, J = 5 Hz, 1H: 1 'H, 8.40 (d, J =

Hz, 1H: NH 1), 8.77 (d, J = 9 Hz, 1H, NH 6), 11.68 (s, 1H: OH).

3 mg of 4? -Bromo-des (4? -Dimethylamino) pristinamycin I _h (mass spectroscopy: M + H + = 874) were isolated from the fractions leaving the silica gel column described above containing the new derivative of pristinamycin I. ) in the case of chromatography on the semi-preparative column described above.

Example 11

Preparation of 4-iodo-des ^ (4ζ-dimethylamino) pristinamycin IA _and £ iodo-de (4 ζ -dimethylamino) pristinamycin I, _R

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -4-iodophenylalanine solution is added. in

0, IN sodium hydroxide. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The chloromethylene phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane and this column was then eluted with a gradient of 0 to 10% methanol in dichloromethane. Fractions containing the novel derivative of pristinamycin I _and were pooled and evaporated. The dry residue is taken up in 6 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is then loaded twice onto a 10 × 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is then eluted with a 60% 100 mM phosphate buffer mixture. pH 2.9 and 40% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 12 mg of 4-iodo-of ζ (4 tons; -dimethylamino) pristinamycin I, _and.

^X H-NMR (400 MHz, CDCl, in ppm, ref.TMS):

³ 0.93 (t, J =

7.5 Hz, 3H: CH ₃ 2 γ), 0.95 (dd, J = 16 and 5.5 Hz, 1H, 5 (¾). 1.10 (mt, 1H: .3 β.), 1.35 (d, J = 7.5 Hz, 3H) : CH ₃ 1 γ), 1.38 (mt, 1 H: 3 γ ₂ ), cd 1.55 ldl.85 (mt, 3H: 3 γ, and CH ₂ 2 β), 2.02 (mt, 1 H, 3 β,), 2.17 (mt, 1H, 5 δ ₂ ), 2.43 (broad d, J = 16.5 Hz, 1H: 5 δ, 2.60 (d, J = 16 Hz, 1H, 5 β ₃ ), 2.89 (dt, J = 14 and 4.5 Hz, 1H: 5 a,), 3.02 (dd, J = 13 and 5.5 Hz,

1H: 4 β.), 3.21 (s, 3H: NCH ₃ 4), 3.31 (dd, J = 13 and 11 Hz, 1H: 4 β ₃ ), 3.39 and 3.59 (2 mts, 1H each: CH 3 δ) 4.53 (t, J = 7.5Hz, 1H, 3a), 4.75 (broad dd, J = 14 and 8Hz,

1H: 5e, 4.83 (mt, 1H: 2a), 4.88 (broad d, J = 10 Hz, 1H: 1a), 5.37 (broad d, J = 5.5)

Hz, 1H: 5a), 5.39 (dd, J = 11 and 5.5 Hz, 1H: 4a), 5.92 (mt, 2H: 6a and 1β), 6.54 (d,

J = 9.5 Hz, 1H: NH2), 6.94 (d, J = 7.5 Hz, 2H: 4δ), ad 7.15 * 7.50 (mt, 5H: aromatic H at position 6), 7.36 (dd, J = 9 and 4 Hz), 1H: 1 H ₅ ), 7.43 (broad d, J = 9 Hz, 1H: 1 H ₄ ),

7.62 (d, J = 7.5Hz, 2H: 4a), 7.68 (d, J = 4Hz, 1H: 1 H), 8.38 (d, J = 10Hz, 1H: NH 1), 8.76 (d, J = 9 Hz, 1H: NH6), 11.60 (s, 1H: OH).

6 mg of 4'-iodo-des (4'-dimethylamino) pristinamycin I (mass spectroscopy: M + H + = 922) was isolated from the fractions leaving the above silica gel column containing the new derivative of pristinamycin I, if performed. Chromatography on the semi-preparative column described above.

Example 12

Preparation ζ luórmetyl -trifluoromethyl-de (4 ζ -dimethylamino) pristinamycin I, _and a ξ * 4 trifluoromethyl-de (4 ζ -dimethylamino) pristinamycin I,

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVPC508 was cultured in a production medium to which, after 16 hours of culture, a solution of (5 g / l) (S) -4-trifluoromethylphenylalanine at 0.1N was added. Sodium hydroxide. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract was taken up in 20 ml of dichloromethane and the resulting mixture was loaded onto a silica gel column (30 g) in dichloromethane, eluting with a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. The dry residue is taken up with 3 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is then loaded twice onto a 10 × 250 mm Nucleosil 7 µ08 semiprep column (Macherey Nagel), which is then eluted with 55% 100 mM phosphate buffer. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 5 mg of 4'-trifluoromethyl-des (4'-dimethylamino) pristinamycin 1 'is obtained.

1 H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): 0.86 (dd,

J = 16 and 5.5 Hz, 1H, 5 β ₂ ), 0.91 (t, J = 7.5 Hz, 3H: CH ₃ 2 γ), 1.13 (mt, 1H: 3 β.), 1.31 (d, J = 7.5 Hz , 3H: CH ₃ 1 γ), 1.42 (mt, 1H: 3 γ ₂ ), c 1.55 <±> 1.80 (mt, 3H: 3 y, and CH, 2 β), 2.02 (mt, 1H, 3 β ₃ ), 2.15 (mt, 1H, δ ₂ ), 2.40 (broad d, J = 16.5 Hz, 1H: 5 δ), 2.55 (d, J = 16 Hz, 1H, 5, 2.88 (dt, J = 14 and 4 Hz, 1H: 5 ε,), 3.18 (s, 3H: NCH. 4), 3.20 and 3.31 (2 dd, respectively J = 13 and 6 Hz and J = 13 and 10 Hz, 1H each): 4 β and 4

β.), 3.42 and 3.60 (2 mts, 1H heavy: CH, 3 δ), 4.50 (t, J = 7.5 Hz, 1H, 3 c), 4.73 (broad d, J = 14 and 7.5 Hz, 1H): 5 ε,), 4.83 (broad m, 1H: 2a), 4.91 (broad d, J = 10 Hz, IH: 1c), 5.40 (broad d, J = 5.5 Hz, 1H: 5 c), 5.55 (dd, J = 10 and 6 Hz, 1H: 4 <z), 5.87 (d, J = 9.5Hz, 1H: 6a), 5.90 (q broad, J = 7.5 Hz, 1Η: 1β), 6.68 (d, J = 9.5 Hz, 1K: ΝΉ 2), cd 7.15 to7.40 (mt, 9 H: 45 - H Aromatic δ - 1 H _s and 1 H ₄ ), 7.52 (d, J = 8 Hz, 2H: 4ε), 7.68 (d, J = 4 and 1.5 Hz, 1H: 1 H, 8.43 (d, J = 10 Hz, 1H: ΝΉ1)), 8.76 (d, J = 9; 5 Hz, 1H: NH 6), 11.70 (s, 1H: OH).

4 mg of 4'-trifluoromethyl-des (4'-dimethylamino) pristinamycin I _k (mass spectroscopy: M + H + = 922) are recovered from the fractions leaving the above silica gel column containing the new derivative of pristinamycin I, if: carry out the chromatography on the semi-preparative column described above.

Example 13

Preparation of 4'-tert-butyl-des (4'-dimethylamino) pristinamycin I _a

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium to which, after 16 hours of culture, (5 g / l) (R, S) -4-tert-butylphenylalanine solution was added. v 0, IN sodium hydroxide. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, and the mixture is centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, eluting with a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. The dry residue is taken up with 7 ml of a mixture of 60% water and 40% acetonitrile and the mixture is loaded twice onto a 10 × 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is then eluted with 55% 100 mM phosphate buffer pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 30 mg of 4'-tert-butyl-des (4'-dimethylamino) pristinamycin I are obtained.

A 'H NMR (400 MHz, _CDCl3, in ppm, ref.TMS):

0.21 (dd, J = 16 and 5.5 Hz, IH, 5 β,), 0.91 (t, J = 7.5 Hz, 3 H; CH ₃ 2 γ), 1.17 (mt, IH; 3 β,), od.20dol .40 (mt, 1H: 3?), 1.33 (s, 9H: CH. T-butyl), 1.35 (d, J = 7.5 Hz,

3H; CH ₃ 1 (γ), αά. 1.50Ch.85 (mt, 3H; 3 γ ₃ and CH, 2 β), 2.04 (mt, IH, 3 β.), 2.13 (mt,

IH, 5 δ ₂ ), 2.30 (mt, 2H: 5 δ _χ and 5 ββ, 2.80 (dt, J = 13 and 4 Hz, IH: 5 e,), 3.00 (dd, J = 4 Hz, IH: 4 β,), 3.29 (s, 3H: NCH ₃ 4), 3.31 and 3.59 (2 bowls, IH sliding. CH, 3 δ), 3.40 (t, J = 12 Hz, IH: 4 β ₃ ), 4.57 ( t, J = 7.5 Hz, IH, 3 a), 4.74 (dd wide, J = 13 and 7 Hz, IH: 5 εβ, 4.85 (mt, IH: 2a), 4.90 (d wide, J = 10 Hz 1H: 1a), 5.21 (broad d, J =

5.5 Hz, 1H; 5 a), 5.25 (dd, J = 12 and 4 Hz, IH: 4 a), 5.87 (d, J = 9 Hz, IH; 6 a), 5.92 (q broad, J = 7.5 Hz, IH: 1

Example 14

Preparation of 4-isopropyl-ζ of (4 ^ -dimethylamino) pristinamycin I, _and a 4ζ-isopropyl-de (dimethylamino ^ 4Z) pristinamycin I _E

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium to which 1 ml of (R, S) -4-isopropylphenylalanine (10 g / l) solution was added after 16 hours of culture. in 0.1 N sodium hydroxide solution, wherein said (R, S) -4-isopropylphenylalanine was synthesized as described in Example 36-1. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. 61 mg of dry residue are obtained. This residue is taken up in 9 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is introduced three times on a 10 × 250 mm Nucleosil 7 µ08 semiprep column (Macherey Nagel), eluting with a 55% 100 mM phosphate buffer mixture. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 51 mg of 4ζ-isopropyl-des (4ζ-dimethylamino) pristinamycin I _and ¹ H-NMR (400 MHz, CDCl ₃ , 6 ppm, ref.TMS) were obtained:

0:31 (dd, J = 16 and 5.5 Hz, 1 H, β 5,), 0.91 (t, J = 7.5 Hz, 3H: _CH3 2 γ), cdl.00dol.45 (mt, 2H: 3 (3 ¾ γ _2), 1.25 (d, J = 7.5 Hz, 6H: _CH3 isopropyl.), 1:35 (d, J = 7.5 Hz, 3H: _CH3 1 7), from 1:50 * 1.85 (mt, 3H: 3 _γ χ and CH, 2 β), cd1.95do2.20 (mt, 2H, 3 β _χ and 5 δ ₂ ), 2.30 (mt, 2H: 5 δ _χ and 5 β _χ ), 2.80 (dt, J = 13 and 4 Hz, 1H: 5 (e), 2.88 (mt, 1H:

CH isopropyl), 2.98 (dd, J = 12 and 4 Hz, 1H: 4 β ₂ ), 3.30 (s, 3H: NCH ₃ 4), 3.32 and 3.55 (2 mts, 1H each = CH, 3 δ), 3.38 ( t, J = 12 Hz, 1H: 4 β _χ ), 4.55 (t, J = 7.5 Hz,

1H, 3 a), 4.72 (broad dd, J = 13 and 7 Hz, 1H: 5 ε _χ ), 4.85 (mt, 1H: 2a), 4.88 (broad d, J =

Hz, 1H: 1a), 5.21 (broad d, J = 5.5 Hz, 1H: 5 a), 5.25 (dd, J = 12 and 4 Hz, 1H: 4 a),

5.87 (d, J = 9Hz, 1H: 6a), 5.90 (q broad, J = 7.5Hz, 1H: 1β), 6.50 (d, J = 9.5Hz, 1H:

NH 2), ad 7.05 to 7.35 (mt, 9H: aromatic amide 6-4e and 4δ), 7.50 (mt, 2H: 1 H ₅ and 1 H ₄ ), 7.86 (dd, J = 4 and 1.5 Hz, 1H 8: 1H, 8.40 (d, J = 10Hz, 1H: NH1), 8.72 (d, J = 9Hz, 1H: NH6), 11.60 (s, 1H: OH).

For the same fraction, leaving the above-described silica gel column and containing the new derivative of pristinamycin I _E, 5 mg of the ζ 4-isopropyl-de (4 ζ -dimethylamino) pristinamycin I _E, where, by carrying out the chromatography described above semi-preparative column.

1 H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): 0.20 (mt, 1H, β,), 0.92 (t, J = 7.5 Hz, 3H: CH ₃ 2 γ), from * 1:40 1.15 (mt, 2H: 3 β, γ ₂ and 3), 1.24 (d,

J = 7.5 Hz, 6H: CH _{3 of} isopropyl), 1.34 (d, J = 7.5 Hz, 3H: CH ₃ 1 γ), from 1.35 * 2.05 (mt, 9H: 3 γ _χ - 3 β _χ - CH ₂ 2 β - CHj 5 δ - CH ₂ 5γ and 5 β _χ ), 2.45 (dt, J = 13 and 1.5 Hz,

1H: 5 (e,), 2.89 (mt, 1H: ArCH), 3.09 (dd, J = 14 and 7 Hz, 1H: 4β,), 3.17 (s, 3H:

4 _NCH3), 3.25 (dd, J = 14 and 9 Hz, 1H: 4 β>), 3.32 and. 3.52 (2 mts, 1H of CH, 3δ), 4.55 (mt, 2H: 3 and 5 εβ, 4.80 (mt, 1H: 2a), 4.89 (dd, J = 10 and 1.5 Hz, 1H: 1a), 4.90 (mt, 1H: 5a), 5.35 (dd, J = 9 and 7Hz, 1H: 4a), 5.60 (d, J = 8Hz, 1H: 6ct), 5.89 (dq, J = 7.5 and 1.5) Hz, 1H: 1 (3), 6.65 (d, J = 9.5Hz, 1H: NH2), 7.08 (d, J = 8Hz, 2H: 46),

7.14 (d, J = 8Hz, 2H: 4ε), ca 7.20 <± 7.40 (mt, 7H: H aromatic 6 - 1 H, 1 'H _s ), 7.77 (d, J = 4 Hz, 1H: 1 H 3), 8.46 (d, J = 10 Hz, 1H: NH 1), 8.48 (d, J = 8 Hz, 1H: NH 6), 11.70 (s, 1H: OH).

Example 15

Preparation of 4'-methylamino-des (4'-dimethylamino) pristinamycin I _a

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (10 g / l) (R, S) -3-methylaminophenylalanine solution was added. , which was synthesized as described in Example 35-3. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, and the mixture is centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane which is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. 19 mg of dry residue are obtained. This residue is taken up with 3 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is eluted using a 55% 100 mM phosphate buffer mixture. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 8 mg of 4ζ-methylamino-des (4ζ-dimethylamino) -60-tinamycin is obtained _. · _Α 1 H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): θ 93

J = 7.5 Hz, 3H: _CH3 2 γ), 1:00 (dd, J = 16 and 6 Hz, 1 H, β 5.), 1.17 (rat, 1 H: β 3.), CD 1.25ôol.40 (yarn, 2 H 3 γ _2), 1:35 (d, J = 7.5 Hz, 3H: _CH3 1 γ), äzl.80 to 1.55 (mt, 3H: 3 and _{7 liters.} 0¾ β 2), 3.2 (mt, 1H, 3 β,), 2.23 (mt, 1H, 5 δ,), 2.39 (d, J = 16 Hz, 1H: 5 Ô), 2.52 (d, J = 16 Hz, 1H: 5 β,), 2.78 (s, 3H: ArNCH, 4), 2.85 (dt, J = 13 and 4 Hz,

1H: 5?, 2.99 (dd, J = 13 and 4.5 Hz, 1H: 4? ₂ ), 3.23 (s, 3H: NCH ₃ 4), 3.25 (t, J = 13Hz, 1H: 4? ₃ ), 3:38 and 3:58 (2 mts, 1H teščz CH ₂ 3 δ), 5.4 (mf, 1H: ArNH), 4:58 (dd, J = 6.5 and 7.5 Hz, 1 H, 3 a), 4.76 (broad dd., J = 13 and 8 Hz, 1H: 5 ε,), 4.85 (mt, 1H:

2a), 4.87 (broad d, J = 10 Hz, 1H: 1a), 5.35 (broad d, J = 13 and 4.5 Hz, 1H: 4a), 5.38 (broad d, J = 6 Hz, 1H: 5) a), 5.90 (d, J = 9.5Hz, 1H: 6a), 5.91 (mt, ΙΗ: 1β), 6.36 (broad, 1H: H 2 aromatic at 4), cd 6.45 δo 6.55 (mt, 2H: H 4 and H 6 aromatic v. 4), 6.53 (d, J = 10 Hz, 1H: NH 2), 7.12 (t, J = 8 Hz, 1H: H 5 aromatic v. 4), from 7.15037 .45 (mt, 5H: aromatic 6), 7.35 (mt, 2H: 1 H ₄ and 1 H 3), 7.75 (t, J = 3 Hz, 1H: 1 H 3), 8.40 (d, J = 10 Hz, 1H: NH1), 8.78 (d, J = 9.5Hz,

Δ: NH 6), 11.60 (s, 1H: OH).

Example 16

Preparation of 4-methoxy-ζ de (4 ζ -dimethylamino) pristinamycin I, _{and the} ζ-methoxy and 4-de (4 ζ -dimethylamino) pristinamycin I,

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of a solution of (S) -3-methoxyphenylalanine (5 g / l) in 0 was added. 1N sodium hydroxide solution. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture consisting of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative istinamycínu I pr _and are combined and evaporated. 41 mg of dry residue are obtained. This residue is taken up in 6 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 gC8 semiprep column (Macherey Nagel) eluting with a 55% 100 mM phosphate buffer mixture. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 28 mg of 4-methoxy-ζ de (4 ζ -dimethylamino) pristinamycin I, _and.

¹ H-NMR (400 MHz, CDCl 3, δ in ppm, ref.TMS):

0.52 (dd)

J = 16 and 5.5 Hz, 1H, 5 β., 0.90 (t, J = 7.5 Hz, 3H: CH ₃ 2 γ), cd 1.10 * 1.34 (mt, 2H: 3 β, and 3 γ ₂ ), 1.34 (d, J = 7.5 Hz, 3H: _CH3 1γ), from 1:50 * 1.80 (mt, 3H: CH 3, β 2),

2.04 (mt, 1 H, 3 β,), 2.20 (mt, 1 H, 5 δ,), 2.35 (d broad, J = 16 Hz, 1H: 5 δ), 2.38 (d,

J = 16 Hz, 1H: 5 b), 2.83 (dt, J = 13 and 4 Hz, 1H: 5 e,), 2.97 (dd, J = 12 and 4 Hz, 1H: 4 b,), 3.28 (s , 3H: _NCH3 4), 3:28, and 3:56 (2 mts, _lHIoščz, CH. 3 δ), 3:40 (t, J = 12 Hz, 1H: 4 β), 3.80 (s, 3H, OCH), 4:58 (t J = 7.5 Hz, 1H, 3 a), 4.76 (broad d, J = 13 and 8 Hz, 1H: 5 ε), 4.85 (broad m, 1H: 2a), 4.90 (broad d, J = 10 Hz) 1H: 1a) 5.27 (dd, J =

a. 4 Hz, 1H: 4 a), 5.30 (broad d, J = 5.5 Hz, 1H: 5 a), 5.89 (broad d, J = 9.5 Hz, 1H: 6 a),

5.91 (broad q, J = 7.5 Hz, 1H: 1β), 6.51 (d, J = 10 Hz, 1H: ΝΉ 2), c 6.80 * 6.90 (mt,

3H: H 2 - H 4 and H 6 aromatife v 4), .cd 7.15 * 7.40 (mt, 6H: H 5 aromatics at 4 et H aromatic 6), 7.45 (broad, J = 9 Hz, 1H: 1 H) 7.50 (dd, J = et 4 Hz, 1H: 1 H), 7.80 (broad d, J = 4 Hz, 1H: 1 H), 8.40 (d, J = 10 Hz, 1H: NH 1), 8.73 (d, J = 9.5 Hz, 1H: NH6), 11.62 (s, 1H: OH).

7 mg of 4ζ-methoxy-des (4ζ-dimethylamino) pristinamycin I (mass spectroscopy: M + H + = 826) are isolated from the fractions leaving the above silica gel column containing the new derivative of pristinamycin I by chromatography on M + H + = 826. the semi-preparative column described above.

Example 17

Preparation 4 C-fluoro-4'-methyl-des (4'-dimethylamino) pristinamycin I

A

On a 60-ml Erlenmeyer flask, the SP92 :: pVRC508 strain was cultured in a production medium to which 1 ml of (10 g / l) (R, S) -3- solution was added after 16 hours of culture. fluoro-4-methylphenylalanine in 0.1 N sodium hydroxide solution, wherein said (R, S) -3-fluoro-4-methylphenylalanine was synthesized as described in Example 34-5. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. 15 mg of dry residue are obtained. This residue is taken up with 3 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is eluted using a 55% 100 mM phosphate buffer mixture. pH 2.9 and 45% acetonitrile. The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 9 mg of ζ-fluoro-4-methyl-des (4ζ-dimethylamino) pristinamycin I is obtained.

H-NMR (400 MHz, _CDCl3, in ppm, ref.TMS): 0.60 (dd

J = 16 and 5.5 Hz, 1H, δ (¾). 0.91 (t, J = 7.5 Hz, 3H: _CH3 2 γ), 1.12 (mt, 1H: 3 β.), The 1.25ÔO1.35 (mt, 1H: 3 γ _2), 1:33 (d, J = 7.5 Hz, 3H: CH ₃ 1 γ), cd 1.50m 1.85 (mt, 3H; 3 γ ₁ and CH 2 β), 2.02 (mt, 1H, 3 ft), 2.13 (mt, 1H, 5 Ô ₂ ) , 2.27 (s, 3H: _ArCH3), 2:36 (broad d. J = 16 Hz, 1H: 5 δ _3), 2:45 (d, J = 16 Hz, 1H: 5 β _3), 2.85 (dt, J = 13 and 4.5 Hz, 1H:

e,), 2.97 (dd, J = 12.5 and 4.5 Hz, 1H: 4 &), 3.23 (s, 3H: _NCH3 4), 3:30, and 3:56 (2 mts, 1H each: CH 3 δ), 3:37 (t J = 12.5 Hz, 1H: 4 fr), 4.55 (t, J = 7.5 Hz, 1H, 3 a),

4.75 (broad d, J = 13 and 8 Hz, 1H: 5 ej, 4.83 (broad m, 1H: 2a), 4.89 (broad d, J = 10 Hz,

1H: 1a), 5.29 (dd, J = 12.5 and 4.5 Hz, 1H: 4a), 5.32 (d broad, J = 5.5 Hz, 1H: 5a),

5.89 (d, J = 9.5Hz, 1H: 6a), 5.92 (mt, 1H: 1β), 6.49 (d, J = 10Hz, 1H: NH2). 6.90 (mt, 2H: H 2 and H 6 'aromatics at 4), 7.11 (t, J = 8 Hz, 1H: H 5 aromatics &apos;), cd 7.10 ° 7.30 (mt, 5H: H aromatic 6), 7.43 (dd, J = 8.5 and 1 Hz, 1H: 1 H ₄ ),

7.49 (dd, J = 8.5 and 4.5 Hz, 1H: 1 Hj), 7.75 (dd, J = 4.5 and 1Hz, 1H: 1 Hj), 8.48 (d, J =

Hz, 1H:? 1), 8.70 (d, J = 9.5 Hz, 1H: NH 6), 11.60 (s, 1H: OH).

Example 18

Preparation of 4 &apos; -ethylamino-des (4 &apos; -dimethylamino) pristinamycin I

A.

On a 50-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -4-ethylaminophenylalanine solution is added. hydrogen chloride, synthesized as described in Example 33, in 0.1 N sodium hydroxide solution. After 40 hours of culture, 1.5 liters of mash produced from 50 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4ζ-ethylamino-des (4C-dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in 7 ml of a mixture of 65% water and 35% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is eluted using a 60% 100 mM phosphate buffer mixture. pH 2.9 and 40% acetonitrile. Fractions containing 4ζ ethylamino-de (dimethylamino 4ζ) pristinamycin IA are combined _and extracted with one volume and dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 10 mg of 4-ethylamino-ζ of (4ζ-dimethylamino) pristinamycin I, _and · H-NMR (400 MHz, _CDCl3, δ in ppm, ref.TMS): 0.72 (dd, J = 6 Hz, and , 1H: 1H cdCH, [delta] b); 0.90 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.15 (mt.

1H: 1H (CH, v3b); cd 1.20 * 1.40 (mt, 1H: 1H cd of CH, at 3 γ); 1.27 (t, J = 7.5 Hz, 3H: _CH3 of ethyl); 1.33 (d, J = 7 Hz, 3H: _CH3 in 1 γ); from 1.50 * 1.65 (mt, 1H: second. Had CH, at 3 γ); 1.60 and 1.74 (2 mts, 1H each: CH, v2b); 2.02 (mt,

1H: second H cd CH, at 3β); 2.21 and 2.33 (respectively mt and d broad, J = 16.5 Hz,

1H each _: CH, v5 δ); 2.40 (d, J = 16 Hz, 1H: second H of CH at position 5b); 2.82 (dt,

J = 13 a. 4.5 Hz, 1H: 1H * CH, at 5 ε); 2.89 (dd, J = 12 and 4 Hz, 1H: 1H of CH at position 4b); 3.10 (mt, 2H: NCH, ethyl); from 3.20 and 3.35 (mt, 1H: 1H cd 0¾ at 3 δ); 3.26 (s, 3H: NCH3; 3.31 (t, J = 12 Hz, 1H: black Hen (3/4 at 4)); 3.54 (mt, 1H: broad H at CH- ^at 3δ); 3.67 (broad m, 1H: NH) 4.56 (dd, J = 6.5 and 7 Hz, 1H: 3)

a); 4.75 (broad dd, J = 13 and 8 Hz, 1H: ent H from CH3 at 5 ε); 4.84 (mt, 1H: 2a); 4.90 (d broad, J = 10 Hz, 1H: 1 a); 5.24 (dd, J = 12 at 4 Hz, 1H: 4a); 5.32 (broad d, J = 6 Hz, 1H: 5 a); 5.88 (d, J = 9.5 Hz, 1H: 6a); 5.90 (mt, 1H: 1β); 6.52 (d, J = 8 Hz, 3H: NH at 2 and H Aromatic at 4 ε); 7.00 (d, J = 8 Hz, 2H: H aromatic at 4 δ); ad 7.1 (δ 7.35 (mt, 5H: H aromatic at 6); 7.46 (AB limit ·, 2H: 1 H ₄ and 1 H ₆ ; 7.84 (dd, J = 4 and 1 Hz) 8.45 (d, J = 10 Hz, 1H: NH at position 1); 8.77 (d, J = 9.5 Hz, 1H: NH at position 6); 11.65 (d, J = 9 Hz); s, 1H: OH).

Example 19

Preparation 4 C-Diethylamino-des (4H-dimethylamino) pristinamycin I

On a 50-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultivated in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -4-diethylaminophenylalanine solution is added. , synthesized as described in Example 33, in 0.1N sodium hydroxide solution. After 40 hours of culture, 1.5 liters of mash produced from 50 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4ζ-diethylamino-des (4ζ-dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is loaded twice onto a 10 x 250 mm Nucleosil 7 / zC8 semiprep column (Macherey Nagel), which is then eluted using a 60 % 100 mM phosphate buffer at pH 2.9 and 40% acetonitrile. Fractions containing 4C-diethylamino-des (4'-dimethylamino) pristinamycin I are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 50 mg of 4'-diethylamino-des (4'-dimethylamino) pristinamycin I are obtained.

^X H-NMR (400 MHz, CDCl _p, in ppm, ref.TMS): 0.65 (dd, J = 6 Hz and, 1H: 1H of the CDC ^ v β 5); 0.90 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.14 (t, J = 7 Hz, 6H: _CH3 of ethyl); 1.15 (mt, 1H: 1H 'cd (3', 3 ')); 1.26 (mt, 1H: 1H: côCH, in 3'); 1.32 (d, J = 6.5 Hz, 3H: CH ₃ in 1 γ), 1.55 (mt, 1H: fume H d 0¾ in 3 γ), 1.63 and 1.75 (2 mts, 1H each: ¾ in 2 β), 2.02 (mt 1 H: δn H (CH 3 CH 3 in β), 2.22 and 2.31 (respectively · mt ad broad, J = 16.5 Hz, 1H each) _;

0¾ in 5 δ); 2.37 (d, J = 16 Hz, 1H td-tubular H cd (¾ v. 5 β); 2.80 (dt, J = 13 and 4.5)

Hz, 1H: 1H of CH, v4b): 2.89 (dd, J = 12.5 and 4 Hz, 1H: 1H of CH, vbeta); 3.20 to 3.40 (mt, 6H: NCH, ^et al - 1 H CH, v 3 δ and rough H of CH, ^v 4 β); 3.27 (s, 3H: NCH3); 3.55 (mt, 1H: dual H at the CH, at 3 DEG); 4.58 (dd, J = 8 and 6 Hz, 1H: 3 a); 76 (broad dd, J = 13 and 7.5 Hz, 1H: day Hod CH, at 5 ε); 4.84 (mt, 1H: 2 a); 4.89 (dd, J = 10 and 1 Hz) 5.21 (dd, J = 12.5 and 4 Hz, 1H: 4)

a); 5.28 (broad d, J = 6 Hz, 1H: 5 a); 5.87 (d, J = 9.5 Hz, 1H: 6a); 5.90 (mt, IH):

β); 6.52 (d, J = 9.5 Hz, 1H: NH at position 2); 6.60 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.02 (d, J = 8 Hz, 2H: H aromatic at 4 δ); from 7.10 to 7.35 (mt, 5H: aromatic H at position 6); 7.46 (AB limit, 2H: 1 H, 1 'H); 7.88 (dd, J = 4.5 and 2.5 Hz,

1H: 1 H; 8.43 (d, J = 10 Hz, 1H: NH at position 1); 8.76 (d, J = 9.5 Hz, 1H: NH at position 6); 11.62 (s, 1H: OH).

Example 20

Preparation 4 C-Dialylamino-des (4'-dimethylamino) pristinamycin I

On a 94-Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium to which, after 16 hours of culture, a solution (20 g / l) of (R, S) -4-dialylaminophenylalanine synthesized is added. as described in Example 38-1, in water. After 40 hours of culture, 2.8 liters of mash produced from 94 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4 ζ -diethylamino-de (4 ζ -dimethylamino) pristinamycin IA are combined _and evaporated and. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 pC8 semipreparative column (Macherey Nagel), which is eluted using a 5% 2% 100 mM phosphate eluent. buffer pH 2.9 and 48% acetonitrile. The fractions containing 4ζ-dialylamino-des (4ζ-dimethylamino) pristinamycin I are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 15 mg of 4'-dialylamino-des (4'-dimethylamino) pristinamycin 1 'are obtained.

¹ H-NMR (400 MHz, CDCl 3, δ in ppm, ref.TMS): _{n =} . _T ³ U, oo (dd, J = 6 Hz, 1H: 1Hcd 0¾ at 5 β); 0.93 (t, J = 7.5 Hz, 3H: CH3 at position 2); 1.18 (mt, 1H: 1H of CH 3 at position 3β); 1.25 (mt, 1H: 1H 02 (3¾ in 3 γ); 1.34 (d, J = 6.5 Hz, 3H: CH 3 in 1 γ); 1.59 (mt, 1H: rough H and d ( 3¾ v. 3 γ) 1,68 al, 78 (2 mts,

1H weekly: (3¾ in 2 β); 2.04 (mt, 1H: rough H cd (3¾ in 3 β); 2.25 and 2.34 (respectively mt ad broad, J = 16.5 Hz, 1H each: (3¾ ⁱⁿ 5 δ); 2 40 (d, J = 16 Hz, 1H: rough Hcd (3¾ in 5 β); 2.83 (dt, J = 13 and 4.5 Hz, 1H: 1H 00 (3¾ in 5 ε); 2.92 (dd, J = 12 and 4 Hz, 1H: 1H ¢ 6 (3¾ the β 4), of 3.20 * 3.30 (mt, 1H: 1H of the _CH2 at 3 δ); 3.29 (s, 3H: NCH3), 3.33 (t, J = 12 Hz, 1H: * _Dy Hcd _CH2 at 4 β); 3.57 (mt, 1H: H of Drdy 0¾ in. 3 δ); 3.93 ( AB limit, 4H: NCH 2 allyl) 4.60 (dd, J = 8 and 6.5 Hz, 1H: 3 a), 4.78 (dd broad, J = 13 and 7.5 Hz, 1H: day H) cd (3¾ in 5e); 4.87 (mt, 1H: 2a); 4.92 (dd, J = 10 and 1 Hz, 1H: 1a); ad: 5.10005.25 (mt, 5H) 5.28 (broad d, J = 6 Hz, 1H: 5 a);

5.85 (mt, 2H: CH-aryl) _; 5.92 (d, J = 9.5 Hz, 1H: 6a); 5.94 (mt, 1H: 1β);

6.54 (d, J = 10 Hz, 1H: NH at position 2); 6.65 (d, J = 8 Hz, 2H: H aromatic at 4 ε);

7.05 (d, J = 8 Hz, 2H: H aromatic at 4 δ); CD 7.10 * 7.35 (mt, 5H:

H Aromatic v 6); 7.51 (AB limit, 2H: 1 H 4 and 1 H 5); 7.88 (dd, J = 4 and 2 Hz,

1H: 1 (1H); 8.43 (d, J = 10 Hz, 1H: NH at position 1); 8.77 (d, J = 9.5 Hz, 1H: NH at position 6); 11.65 (s, 1H: OH).

Example 21

Preparation of 4 ζ -alylethylamino-des (4ζ-dimethylamino) pristinamycin I

A

On a 26 Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -4-allylethylaminophenylalanine solution is added. hydrochloride, synthesized as described in Example 39-4, in 0.1N sodium hydroxide solution. After 40 hours of culture, 0.78 liters of mash produced from 26 Erlenmeyer flasks is extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4'-allylethylamino-des (4'-dimethylamino) pristinamycin I are pooled and evaporated, the dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 7 μ08 Nucleosil semipreparative column. 10 x 250 mm (Macherey Nagel), eluting with 52% 100 mM phosphate buffer at pH 2.9 and 48% acetonitrile Fractions containing 4 &apos; -alylethylamino-des (4 &apos; -dimethylamino) pristinamycin I combined and extracted with one volume of dichloromethane. the authority nick phase was washed with water, dried over sodium sulphate and then evaporated. 20 mg of 4-ζ alyletylamino-de (4-dimethylamino) pristinamycin iA _and.

¹ H-NMR (400 MHz, CDCl 3, δ in ppm, ref.TMS):? __ _ ... U ^3, 3 ° (dd, J =

6 Hz, 1H: 1H and CH3 at [beta] _; of, 91 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.16 (d, J = 7 Hz, 3H, _CH3 of ethyl); 1.16 (mt, 1H: 1H by CH, at 3b); 1.25 (mt, 1H: 1H of CH at .delta.); 1.32 (d, J = 6.5 Hz, 3H; CH ₃ in 1 γ); 1.54 (mt, 1H; days Hod CH3 at 3 γ); 1.63 and 1.75 (2 mts, 1H each: CH, at 2β); 2.02 (mt, 1H: expensive H of CH, at 3β); 2.23 and 2.31 (respectively mt ad sir., J = 16.5 Hz, 1H cutters:

CH, at 5 δ); 2.37 (d, J = 16 Hz, 1H: rough H cd CH, at 5β); 2.80 (dt, J = 13 and 4.5)

Hz, 1H: 1H of CH2 -. 5 ε); 2.87 (dd, J = 12 and 4 Hz, 1H: 1H of 0¾ at 4 β); from

3.15 03.30 (mt, 1H; 1H cdCH3 at 3δ); 3.26 (s, 3H: NCH3; 3.30 (t, J = 12 Hz, 1H: clear H at the CH4 at position 4)); 3.36 (mt, 2H: NCH3 of ethyl); 3.54 (mt, 1H: κ K κ cd CH- δ at 3 Ô); 3.90 (AB limit, 2H; NCH, ^{α1 lu l} ); 4.57 (dd, J = 8 and 6 Hz, 1H;

a); 4.76 (broad d, J = 13 and 7.5 Hz, 1H: δ H at the CH, at 5 ε); 4.84 (mt, 1H: 2)

a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); 5.05 * 5.20 (mt, 3H: 4aa = CHayl) _; 5.27 (broad d J = 6 Hz, 1H: 5 a); 5.83 (mt, 1H: CH = alkyl); 5.58 (d, J = 9.5 Hz, 1H: 6a); 5.91 (mt, 1H: 1β); 6.50 (d, J = 10 Hz, 1H: NH at position 2); 6.60 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.02 (d, J = 8 Hz, 2H: H aromatic at 4 δ); cd 7.10 * 7.35 (mt, 5H: aromatic H at position 6); 7.47 (AB limit, 2H: 1 H ₄ and 1 H ₃ ;

7.88 (dd, J = 4 and 2 Hz, 1H: 1 HJ; 8.41 (d, J = 10 Hz, 1H: NH at 1); 8.75 (d, J =

9.5 Hz, 1H: NH at 6); 11.62 (s, 1H: OH).

Example 22

Preparation of 4'-1'-ethyl-propylamino-des (4''-dimethylamino) pristinamycin I

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -4-ethylpropylaminophenylalanine solution was added. hydrochloride, synthesized as described in Example 39-6, in 0.1N sodium hydroxide solution. After 40 hours of culture, 1.8 liters of mash originating from 26 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using an eluting gradient of 0-10% methanol in dichloromethane. The fractions containing 4'-ethylpropylamino-des (4'-dimethylamino) pristinamycin I are pooled and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 × 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is eluted using a 63% 100 mM phosphate buffer mixture. pH 2.9 and 37% acetonitrile. Fractions containing des-4ζ -alyletylamino (4ζ -dimethylamino) pristinamycin IA are combined _and extracted with one volume and dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 16 mg of 4 &apos; -ethylpropylamino-des (4 &apos; -dimethylamino) pristinamycin I _and &lt; 1 &gt; H-NMR (400 MHz, CDCl3, in ppm, ref. TMS) are obtained: [delta] 1H: 1H du CH, at 5b); 0.91 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 0.95 (t, J = 7.5 Hz, 3H: CH _third prcpyk); 1.14 (t, J = 7 Hz, 3H: _CH3 of ethyl) 1.15 (mt,

1H; 1H cd CH. v 3 β); 1.25 (mt, 1H: 1H of CH3 at position 3); 1.33 (d, J = 7Hz, 3H):

CH ₃ in 1 γ); cd 1.45 δ C, 65 (mt, 3H: aruryl H and CH at .delta. in CH3 of propyl); 1.63 and 1.75 (2 mts, 1H each: CH, at 2β); 2.02 (mt, 1H: Drury H of _CH2 at 3 β);

2.23 2.33 (respectively mt and broad, J = 16.5 Hz, 1H each; CH, v5 δ);

2.37 (d, J = 16 Hz, 1H: second H at the CH, at 5 β); 2.80 (dt, J = 13 and 5 Hz, 1H: 1H at CH, at .delta.); 2.89 (dd, J = 12 and 4 Hz, 1H: 1H cd, CH2 at position 4b); cd3.10 < 3.25 (mt, 3H: 1H cd of CH at .delta. and NCH. of propyl); 3.26 (s, 3H: NCH3; cd 3.25 ± 3.40 (mt, 2H: NCH3 ethyl); 3.34 (t, J = 12 Hz, 1H: onion H at the CH, at 4b) ;

3.54 (mt, 1H: second H cdCH3 at 3δ); 4.57 (dd, J = 7.5 and 6 Hz, 1H: 3a); 4.76 (broad dd, J = 13 and 7.5 Hz, 1H: no H at the CH, v. 5e); 4.84 (mt, 1H: 2a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); 5.21 (dd, J = 12 and 4 Hz, 1H: 4a); 5.28 (broad d, J =

Hz, 1H: 5 (a); 5.88 (d, J = 9.5 Hz, 1H: 6a); 5.91 (mt, 1H: 1β); 6.48 (d, J = 10 Hz, 1H: NH at position 2); 6.60 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.03 (d, J = 8Hz,

2H: H Aromatic at 4 δ); cd 7.10? 7.35 (mt, 5H: H aromatic at 6); 7.47 (AB limit, 2H: 1 H ₄ and 1 'H 3; 7.89 (mt, 1H: 1 H 3; 8.42 (d, J = 10 Hz, 1H: NH at 1); 8.76) (d, J = 9.5 Hz, 1H: NH and 6) 11.62 (s, 1H: OH).

Example 23

Preparation of 4 &apos; -trifluoromethoxy-des (4 &apos; -dimethylamino) pristinamycin I

A

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 was cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of a solution of (R, S) -4-O-trifluoromethyltyrosine synthesized as described in Example 34-8. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is then extracted with two volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up with 1 ml of dichloromethane and loaded onto a silica gel column (30 g) and the column is eluted using eluting gram-ethanol in dichloromethane. Fractions and the resulting mixture methylmorpholine, and DIENTE 0-10 ζ-trifluoromethoxy-de (4-ζ-dimethylamino) pristinamycin IA are combined _and evaporated and. The evaporation residue is taken up with 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 × 250 mm Nucleosil 7 µ08 semiprep column (Macherey Nagel), which is eluted using a 60% 100 mM phosphate eluent. buffer pH 2.9 and 40% acetonitrile. Fractions containing 4ζ-trifluoromethoxy-de (4 <ζ-dimethylamino) pristinamycin IA are combined _and extracted with one volume and dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 46.5 mg of 4'-trifluoromethoxy-des (4'-dimethylamino) pristinamycin I are obtained.

¹ H-NMR (400 MHz, CDCl 3, δ in ppm, ref.TMS):

0.77 (dd, J = et 5.5 Hz, 1H: 1H of CH at position 5b); 0.92 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.08 (mt, 1H: 1H of CH2, at 3 b); from 1.30 * 1.40 (mt, 1H: 1H of CH, * 3 y); 1.33 (d, J = 7 Hz, 3H, CH ₃ in the γ 1); ad 1.55 * 1.70 (mt, 1H: rough of H from CH, v. 3?); 1,65 a

1.76 (2 mts, 1H each: CH, v2b); 2.02 (mt, 1H: crude _: H of CH, at 3b); 2.11 and 2.40 (respectively mt and broad J = 16.5 Hz, 1H each: CH, v5 δ); 2.54 (d, J = 16 Hz, 1H: onion H to CH, [delta] β); 2.88 (dt, J = 13 and 4 Hz, 1H: 1H of CH at position 5e); 3.08 (dd, J = 12 and 5 Hz, 1H: 1H by CH at position 4b); 3.22 (s, 3H: NCR); from 3.30 * 3.45 (mt, 1H: 1H of CD at CH3 at position 3); 3.39 (t, J = 12 Hz, 1H: rough of H from CH, v4b); 3.59 (mt, 1H: clear H of CH, at 3δ); 4.53 (t, J = 7.5Hz, 1H: 3a); 4.75 (broad dd, J = 13 and 8 Hz, 1H - second H from CH, at 5 ε); 4.85 (mt, 1H: 2a); 4.89 (dd, J = 10 and 1.5 Hz, 1H: 1 α); 5.35 (broad d, J = 5.5 Hz, 1H: 5a); 5.41 (dd, J = 12 and 5 Hz, 1H: 4a); 5.92 (d, J = 10Hz, 1H: 6a); 5.93 (mt, 1H: 1β); 6.53 (d, J = 9.5 Hz, 1H: NH at position 2); ± 7.15 ± 7.35 (mt, 5H: aromatic H at position 6); 7.16 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.26 (d, J = 8 Hz, 2H: aromatic H at position 4); 7.37 (dd, J = 8.5 and 4 Hz, 1H: 1 'H'); 7.42 (dd, J = 8.5 and 1.5 Hz, 1H: 1 HJ; 7.70 (dd, J = 4 and 1.5 Hz, 1H: 1 H); 8.37 (d, J = 10 Hz, 1H: NH at 1), 8.75 (d, J = 10 Hz, 1H: NH at 6), 11.66 (s, 1H: OH).

Example 24

Preparation of 4'-allyloxy-des (4'-dimethylamino) pristinamycin I

In a 90-gauge Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (S) -4-O-allyltyrosine solution is added. , synthesized as described in Example 33, in a 0.1N hydrochloric acid solution. After 40 hours of culture, 2.7 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4'-allyloxy-des (4'-dimethylamino) pristinamycin I are pooled and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 × 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is eluted using a 52% 100 mM phosphate buffer elution system. pH 2.9 and 48% acetonitrile. The fractions containing 4 ζ allyloxy-de (4-dimethylamino ^) pristinamycin IA are combined _and extracted with one volume and dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated.

29 mg of 4ζ-allyloxy-des (4ζ-dimethylamino) pristinamycin are obtained

I.

^X 1 H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS): δ 63 (dd J 16 and 6 Hz, 1H: 1H at position 5 at β); 0.91 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.13 (mt,

1H: 1Hcd. CH ₂ <±>3β); 1.29 (mt, 1H: 1H of the _CH2 at 3 γ); 1.33 (d, J = 6.5 Hz, 3H):

_CH3 1 in _7); 1.57 (mt, 1H: day H at the CH, at 3 γ); 1.65 and 1.74 (2 dishes, 1H: 0¾ in 2β); 2.02 (mt, 1H: cis H at the position CH at .delta.); 2.14 et 2.34 (respectively, mt and broad d, et., J = 16.5 Hz, 1 H _{Lescol, CH2} at 5 δ); 2.43 (d, J = 16 Hz, 1H: the other H ±. 5 cc _CH2 β); 2.85 (dt, J = 13 and 4 Hz, 1H: 1H of CDCl 3 δ ° 5 ε); 2.95 (dd, J = 12 and 4 Hz, 1H: 1H of the _CH2 in 4 β); 3.25 (s, 3H: NCH3; 3.33 (mt, 1H: 1H cdCH, at 3δ); 3.36 (t, J = 12 Hz, 1H: second H from CH at 4b); 3 56 (mt, 1H: dual H at the CDCl3 at position 3); 4.51 (AB limit, 2H: OCH. Allyl); 4.56 (t, J = 7.5 Hz, 1H: 3a); 4.75 (broad dd, J = 13 and 8 Hz, 1H: double H at the CH at 5 ε); 4.84 (mt, 1H: 2a); 4.88 (dd, J = 10 and 1); Hz, 1H: 1 a) 5.27 (dd, J = 12 and 4 Hz, 1H: 4 a) 5.32 (broad d, J =

Hz, 1H: 5c): 5.30 and 5.40 (respectively mt and dd, J = 17 and 1.5 Hz, 1H each: = 0¾ alkyl); 5.89 (d, J = 9.5 Hz, 1H: 6a); 5.91 (mt, 1H: 1β); 6.02 (mt, IH):

CH = alkyl); 6.50 (d, J = 10 Hz, 1H: NH at position 2); 6.85 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.12 (d, J = 8 Hz, 2H: aromatic H at position 4); cd 7.10 to 7.35 (mt, 5H: H aromatic at position 6); 7.45 (dd, J = 8.5 and 1.5 Hz, 1H: 1 H); 7.57 (dd, J =

8.5 and 4Hz, 1H: 1 '; 7.77 (dd, J = 4 and 1.5 Hz, 1H H'H); 8.41 (d, J = 10 Hz, 1H: NH at position 1); 8.74 (d, J = 9.5 Hz, 1H: NH at position 6); 11.63 (s, 1H: OH).

Example 25

Preparation of 4'-ethoxy-des (4'-dimethylamino) pristinamycin I

In a 90-gauge Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (S) -4-O-ethyltyrosine solution is added. hydrochloride, synthesized Example 33, in a 0.1N hydrochloric acid solution. After 40 hours of cultivation, 2.7 liters of mash produced from 90 Erlenmeyer flasks are extracted with two volumes of a mixture containing 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant was extracted twice with 0.5 volume of dichloromethane / methane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4? -Alyloxy-des (4-dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 gC8 semipreparative column (Macherey Nagel), which is eluted using a 52% 100 mM phosphate buffer elution system. pH 2.9 and 48% acetonitrile. Fractions containing 4-ethoxy-des (4'-dimethylamino) pristinamycin I _A are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 29 mg of 4-ethoxy-ζ'de (4-dimethylamino) pristinamycin IA _and ¹ H-NMR (400 MHz, _{CDCl, and,} in ppm, ref.TMS):

0.64 (dd, J = and 5.5 Hz, 1H: 1H of _CH2 5 β); 0.90 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.12 (mt, 1H: 1H of the _CH2 at 3 β); 1.25 (mt, 1H: 1H of the _CH2 at 3 γ); 1.33 (d, J = 7Hz,

3H: CH ₃ in 1 γ); 1.42 (t, J = 7 Hz, 3H: _CH3 of ethyl); 1.57 (mt, 1H: species / Hcd CH, at 3 γ); 1.63 and 1.74 (2 mts, 1H same: 0¾ in 2β); 2.02 (mt, 1H: δ H cd (3¾ in 3 β); 2.16 and 2.35 (respectively m and d d, J = 16.5 Hz, 1H also:

0¾ in 5 δ); 2.43 (d, J = 16 Hz, 1H: the other H of the _CH2 in 5 β); 2.83 (dt, J = 13 and 4 Hz, 1H: 1H of CYL at position 5e); 2.93 (dd, J = 12 and 4 Hz, 1H: 1H of 0¾ in 4 β), 3.15 and 3.30 (mt, 1H: 1H cd (3 33 δ); 3.24 (s, 3H: 3.36 (t, J = 12 Hz, 1H: second H ^ (3¾ in 4 β); 3.55 (mt, 1H r ^ n Red 10¾ in 3 Ô); 3.95 (AB limit-,

2H: 00 ety ethyl); 4.56 (dd, J = 7.5 and 6 Hz, 1H: 3a); 4.75 (broad dd, J = 13 and 8 Hz, 1H: second Hcd, 0.5 'at 5 e); 4.84 (mt, 1H: 2a); 4.87 (dd, J = 10 and 1 Hz,

1H: 1 (a); 5.26 (dd, J = 12 and 4 Hz, 1H: 4a); 5.32 (broad d, J = 5.5 Hz, 1H: 5 a);

5.88 (d, J = 10 Hz, 1H: 6a); 5.92 (mt, 1H: 1β); 6.48 (d, J = 10 Hz, 1H: NH at position 2); 6.83 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.10 (d, J = 8 Hz, 2H: H aromatic at 4 δ); cd 7.10 to 7.35 (mt, 5H: H aromatic at position 6); 7.44 (dd, J =

8.5 and 1.5 Hz, 1H: 1 H; 7.57 (dd, J = 8.5 and 4.5 Hz, 1H: 1 'H'); 7.77 (dd, J = 4.5 and

1.5 Hz, 1H: 1H; 8.38 (d, J = 10 Hz, 1H: NH at position 1); 8.75 (d, J = 10 Hz, 1H: NH at position 6); 11.60 (s, 1H: OH).

Example 26

Preparation of 4ζ- (2-chloroethoxy) -des (4ζ-dimethylamino) pristinamycin I

A

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultivated in a production medium to which 1 ml of (20 g / l) (S) -4-O- solution is added after 16 hours of culture. 2-chloroethyl) tyrosine hydrochloride, synthesized as described in Example 42-1, in water. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture containing 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4 ζ - (2-chloroethoxy) -de (4 £ -dimethylamino) pristinamycin IA are combined _and evaporated and. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 / tC8 semipreparative column (Macherey Nagel), which is then eluted using a 60% 100 mM phosphate eluent. buffer pH 2.9 and 40% acetonitrile. The fractions containing 4- (2-chloroethoxy) -des (4'-dimethylamino) pristinamycin I are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 3.2 mg of 4 £ - (2-chloroethoxy) -de (4 '^ -dimethylamino) pristinamycin I, _and.

@ 1 H-NMR (400 MHz, CDCl3, .delta. In ppm, ref. TMS):. _T ³ υ, οο (, α α, J = and 5.5 Hz, IH: IH from CH, at 5 β); 0.91 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.13 (mt, 1H: 1H at position CH, at position 3b); 1.28 (mt, 1H: 1H at position CH, at position 3); 1.33 (d, J = 7Hz,

3H: CH ₃ in 1 γ); 1.57 (mt, 1H: day H at the CH, at 3 γ); 1.66 and 1.76 (2 mts, IH also #: CH, at 2β); 2.02 (mt, 1H; second H cd CH, at 3β); 2.16 and 2.37 (respectively mt ad broad, J = 16.5 Hz, IH each: CH, at 5δ); 2.47 (d, J = 16 Hz, 1H: rough H of CH, at 5 β); 2.86 (dt, J = 13 and 4 Hz, 1H: 1H cdCH, at 5 ε);

2.95 (dd, J = 12 and 4 Hz, 1H: 1H and CH3; 4b); 3.23 (s, 3H: NCH3; 3.32 (mt, 1H: 1H cd 0 'at 3 δ); 3.37 (t, J = 12 Hz, 1H: <± 1 H at 0¾ in 4 β) 3.57 (mt, 1H: second 1H of CH3 at position 3); 3.82 (t, J = 6 Hz, 2H: CH2Cl); 4.19 (AB limit, 2H: OCH, ethyl); 4.55 (dd, J = 7.5 and 7 Hz, 1H: 3 a); 4.75 (dd broad, J = 13 and 8 Hz, 1H: second H from CH, v, 5 ε); 4 84 (mt, 1H: 2a); 4.87 (broad J = 10Hz, 1H: 1a);

5.28 (dd, J = 12 and 4 Hz, 1H: 4a); 5.32 (broad d, J = 5.5 Hz, 1H: 5 a); 5.88 (d, J = 10Hz, 1H: 6a); 5.90 (mt, 1H: 1β); 6.50 (d, J = 10 Hz, 1H: NH at position 2); 6.86 (d, J = 8 Hz, 2H: H aromatic at 4 ε); 7.13 (d, J = 8 Hz, 2H: H aromatic at 4 δ); cd. 7.10 and 7.35 (mt, 5H: H of aromatic at position 6); 7.45 (AB limit, 2H: 1'H ₄ and 1 'H 3); 7.75 (dd, J = 4 and 2 Hz, 1H: 1 H); 8.38 (d, J = 10 Hz, 1H: NH at 1); 8.74 (d, J = 10 Hz, 1H: NH) [delta] 11.62 (s, 1H: OH).

Example 27

Preparation of 4? -Acetyl-des (4'-dimethylamino) pristinamycin I _a

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (S) -4-acetylphenylalanine synthesized solution is added. as described in Example 33, in a 0.1N sodium hydroxide solution. After 40 hours of cultivation, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, then centrifuged. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4α-acetyl-des (4 acet-dimethylamino) pristinamycin I are pooled and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 semipreparative column (Macherey Nagel), which is then eluted using a 60% 100 mM pH phosphate buffer eluent. 2, 9 and 40% acetonitrile. Fractions containing 4Z, 4-acetyl-des (4, 76)

(dimethylamino) pristinamycin I _and are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 4.2 mg of 4 ζ -acetyl-de (4 ζ -dimethylamino) pristinamycin I, _and.

^X H-NMR (400 MHz, CDCl, δ in ppm, ref.TMS):

0.73 (dd, J =

6 Hz, 1H: 1H and CH. v 5 β); 0.93 (t, J = 7.5 Hz, 3H: CH3 _at 2 γ); 1.12 (mt,

1H: 1H cd. CH. cn 1.25 b1, 45 (mt, 1H: 1H HC CH, at 3y); 1.33 (d, J = 7 Hz, 3H: CH3 / ₁₇ ); 1.62 (mt, 1H: scratch H at CH3 at position 37); 2.02 (mt, 1H: clear H cd, CH at position 3β); 2.20 and 2.42 (respectively: mts d broad, J = 16.5 Hz, 1K each: CH at 5 δ); 2.52 (d, J = 16 Hz, 1H: scratch H at CH at .delta.); 2.60 (s, 3H: ArCOCH3; 2.88 (dt, J = 13 and 4.5 Hz, 1H: 1H from CH, at 5 ε); 3.13 (dd, J = 13.5 and 5) .5 Hz, 1H: 1H cd of CH at [beta]; 3.21 (s, 3H: NCH [beta]; cd of 3.30 b3.50 (mt, 1H: scratch of H of CD of CH at 4 [beta]); 30b3.50 s 3.63 (2 mts, 1H each; CH at 3 δ); 4.53 (t, J = 7.5 Hz, 1H: 3 c); 4.75 (broad d, J = 30 Hz); 13 and 8 Hz,

1H: rugged K cd CH. v 5 ε); 4.84 (mt, 1H: 2a); 4.88 (dd, J = 10 and 1 Hz, 1H: 1)

a); 5.35 (broad d, J = 6 Hz, 1H: 5 a); 5.43 (dd, J = 10.5 and 4 Hz, 1H: 4a); 5.90 (d,

J = 9.5 Hz, 1H: 6 (a); 5.92 (mt, 1H: 1β); 6.56 (d, J = 9.5 Hz, 1H: NH at position 2); from 7.10 [beta], 35 (mt, 5H: H of the aromatic v. 6); 7.28 (d, J = 8 Hz, 2H: H aromatic at 4 δ); 7.38 (dd, J = 8.5 and 2 Hz, 1H: 1 'Hy; 7.42 (dd, J = 8.5 and 4.5 Hz, 1H: 1 Hp; 7.66 (dd, J) = 4.5 s 2 Hz, 1H: 1 EJ; 7.88 (d, J = 8 Hz, 2H: H aromatic v. 4 ε); 8.38 (d, J = 10 Hz, 1H; NH at 1) 8.74 (d, J = 9.5 Hz, 1H: NH at position 6); 11.65 (s, 1H: OH).

Example 28

Preparation 4 N-dimethylamino-des (4 ζ-dimethylamino) pristinamycin I

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -3-dimethylaminophenylalanine solution is added. hydrochloride, synthesized Example 35-10, in 0.1N sodium hydroxide solution. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34 L of acetonitrile, and centrifuged. The supernatant is then extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4? -Dimethylamino-des (4? -Dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in ml of a mixture of 60% water and 40% acetonitrile, and the resulting mixture is loaded onto a 10 × 250 mm Nucleosil 7 μ08 semiprep column (Macherey Nagel), which is then eluted using a 57% 100mM phosphate buffer at pH

2.9 and 43% acetonitrile. The fractions containing 4β-dimethylamino-des (4ζ-dimethylamino) pristinamycin I are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. To give 1.1 mg of £ -dimethylamino-de (4 ^ -dimethylamino) pristinamycin I, _and.

^X H-NMR (400 MHz, _CDCl3, δ in ppm, ref.TMS):

0.63 (dd,

J = 16 and 5 Hz, IH: IH cd CH3 at 5 β); 0.91 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.13 (mt, 1H: 1H at position CH, at position 3b); from 1.200 to 1.35 (mt, 1H: 1H of CH3 at position 3); 1.32 (d, J = 6.5 Hz, 3H: _CH3 in 1 γ); 1.57 (mt, 1H: given by H from CH, at 3 γ); 1.63 and 1.76 (2 mts, IH fractures: CH, at 2β); 2.02 (mt, IH: day H cdCH, v 3 (3); 2.08 and 2.31 (respectively: mt a. D sir., J = 16.5 Hz, IH each: CH, v 5 δ) 2.35 (d, J = 16);

Hz, IH: rough H cd CH, at 5 β); 2.81 (dt, J = 13 and 4 Hz, 1H: 1H and CH at 5 ε);

2.90 (s, 6H: N (CH3)); 2.97 (dd, J = 12 and 4 Hz, 1H: 1H cd CH, at 4β); cd 3.20-3.30 (mt, IH: IH from CH, at 3 δ), 3.28 (s, 3H: NCH,); 3.37 (t, J = 12 Hz, IH; δni Hod CH. At 4β); 3.57 (mt 1H: other H cd CH, at 3 δ) 4.58 (t, J = 7.5 Hz,

IH: 3 (a); 4.74 (broad d _:> J = 13 and 8 Hz, IH: dn ± Hod hr.CH, at 5 ε); 4.86 (mt, 1H: 2a); 4.89 (broad d, J = 10 Hz, 1H: 1 a); 5.27 (dd, J = 12 and 4 Hz, 1H; 4a); 5.29 (broad d, J = 5 Hz, 1H: 5a); 5.89 (d, J = 9.5 Hz, 1H: 6a); 5.90 (mt, 1H: 1β);

6.50 (d, J = 10 Hz, 1H: δ at 2); from 6,50036.70 (mt, 3H: H aromatic in ortho and para to dimethylamino); cd 7.15 DEG -7.35 (mt, 5H: H of the aromatic at position 6); 7.20 (t,

J =

Example 29

Preparation 4 of 6-methylthio-des (4'-dimethylamino) pristinamycin I

On a scale of 56 Erlenmeyer flasks, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -3-methylthiophenylalanine solution is added. hydrochloride synthesized as described in the Example

34-11, in Q, 1 N sodium hydroxide solution. After 40 hours of culture, 1.68 liters of mash is extracted with two volumes of a mixture of 66% 100mM phosphate buffer pH 2.9 and 34% acetonitrile, and the mixture is centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative of pristinamycin I are pooled and evaporated. The dry residue is taken up in 7 ml of a mixture of 54% water and 46% acetonitrile and loaded onto a 10 × 250 mm Nucleosil 7 µ08 semipreparative column (Macherey Nagel), which is then eluted using a 55% 100 mM phosphate eluent system. buffer pH 2.9 and 45% acetonitrile.

The fractions containing the new pristinamycin are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 20 mg of 4'-methylthio-des (4'-dimethylamino) pristinamycin I are obtained.

^X H-NMR (400 MHz, CDCl, δ ppm, ref.TMS):

³ 0.5o (dd,

J-16 and 5.5 Hz, 1H: 1H of CH3 at [beta]; 0.90 (t, J = 7.5 Hz, 3H: _CH3 2 γ s); 1.13 (mt, 1H: 1H of Producer ₂ β 3); 1.28 (mt, 1H: 1Hcd at 3y); 1.32 (d, J = 6.5Hz,

3H: CH ₃ in 1 _γ ); 1.58 (mt, 1H: δ x Hod 0¾ at 3 γ); 1.62 and 1.74 (2 mts, 1H atz: CH, at 2β); 2.02 (mt, 1H: onium: H of CH3 at position 3b); 2.25 and 2.35 (respectively mt ad broad J = 16.5 Hz, 1H lying: 0¾ in 5 δ); 2.39 (d, J = 16 Hz, 1H: day H of C17 at position 5b); 2.43 (s, 3H: SCH2; 2.82 (dt, J = 13 and 4 Hz, 1H):

1Η cgCH ₂ * 5 ε); 2.98 (dd, J = 12 and 4.5 Hz, 1H: 1H of .alpha. At 4 &apos;); 3.26 (s, 3H: NCH); 3.30 (t, J = 12 Hz 1H: 1H of CH 3 at 3 δ); 3.38 (mt, 1H: the other H ** _CH2 at 4 β); 3.57 (mt, 1H: rough Hd CK3 at position 3); 4.56 (t, J = 7.5Hz, 1H: 3g); 4.74 (dd broad, J = 13 and 8 Hz, 1H: blue H at the CD 0¾ at 5 ε); 4.84 (mt, 1H: 2a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); 5.29 (dd, J = 12 and 4.5 Hz, 1H: 4a); 5.32 (broad d, J = 5.5 Hz, 1H: 5c); 5.88 (d, J = 9.5 Hz, 1H: 6c); 5.90 (mt, 1H: 1β); 6.51 (d, J = 10 Hz, 1H: NH at position 2); 6.99 (broad d, J = 8 Hz, 1H: H aromatic in methyl ket); 7.10 and 7.15 (respectively with aar. Ad sir., J = 8 Hz, 1H each: H aromatic ortho to ethylthio); cd 7.15 * 7.35 (mt, 6H: H aromatic v. 6 and H aromatic v in methylthio); 7.43 (broad d, J = 8 Hz, 1H: 1H); 7.52 (dd, J = 8 &apos; and 4 Hz, 1H: 1 H); 7.79 (broad d, J = 4Kz, 1H: 1 H); 8.38 (d, J = 10 Hz, 1H, NH at position 1); 8.73 (d, J = 9.5 Hz, 1H: NH at position 6); 11.62 (s, 1H: OH).

Example 30 '

Preparation of 4'-ethoxy-des (4'-dimethylamino) pristinamycin I

On a 60-ml Erlenmeyer flask, the strain SP92 :: pVRC508 is cultured in a production medium to which 1 ml (20 g / l) of (S) -3-O-ethyltyrosine solution is added after 16 hours of culture. hydrochloride synthesized as described in Example 37-1. After 40 hours of culture, 1.8 liters of mash produced from 60 Erlenmeyer flasks are extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, and centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing the new derivative I are combined and evaporated. 19 mg of dry residue are obtained. This residue is taken up in 3 ml of a mixture of 60% water and 40% acetonitrile and the resulting mixture is applied to a 10 × 250 mm Nucleosil 7 μϋ8 semiprep column (Macherey).

Nagel), which is eluted using a mixture of 60% 100 mM phosphate buffer pH 2.9 and 40% acetonitrile. The fractions containing the new pristinamycin were combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 15.8 mg of 4 E-O-ethoxy-des (4'-dimethylamino) pristinamycin I are obtained.

¹ H-NMR (400 MHz, CDCl 3, δ in ppm, ref.TMS):

0.55 (cd,

J = 16 and 5.5 Hz, 1H: 1K cdCH3 at 5β); 0.90 (t, J = 7.5 Hz, 3H: _CH3 in 2 γ); 1.12 (mt, 1H: 1H of ₂ CDCl 3 β); 1.20 (mt, 1H: 1 H CDCl 3 γ _2); 1.31 (d, J = 6.5Hz,

3H: _CH3 in 1 _7); 1.49 (t, J = 7 Hz, 3H: _CH3 of _ethyl); 1.54 (mt, 1H: pure H at the CH, at 37); 1.63 and 1.73 (2 mts, 1Hot: CH3 at 2β); 2.02 (mt, 1H: black H cd, 0 'at 3'); 2.22 and 2.33 (respectively mt ad broad, J = 16.5 Hz, 1H @ 1: CH3 at 5 δ); 2.46 (d, J = 16 Hz, 1H: rough H cdCl · 4 in 5β); 2.83 (dt, J = 13 d 4 Hz, 1H: 1H cnCH ₂ at 5 ε); 2.95 (dd, J = 12 and 4 Hz, 1H: 1H cdCH, at 4β); 3.22 (mt, 1H: 1H of CH at position 3δ); 3.27 (s, 3H: NCH3); 3.39 (t, J = 12 Hz, 1H: rough H cd, 0 'at 4'); 3.53 (mt, 1H: rough H at the CDCl3); 3.93 and 4.03 (2 mts, 1H each: 00¾ ethyl); 4.56 (dd, J = 7 and 5.5 Hz, 1H: 3a); 4.75 (broad dd, J = 8 Hz, 1H: dd H cd 0¾ at 5 ε); 4.82 (mt, 1H: 2a); 4.88 (dd, J = 10 and

Hz, 1H: 1 a); 5.23 (dd, J = 12 and 4 Hz, 1H: 4a); 5.23 (broad d, J = 5.5 Hz, 1H: 5)

a); 5.87 (d, J = 9.5 Hz, 1H: 6a); 5.92 (mt, 1H: 1β); 6.47 (d, J = 10 Hz, 1H: NH at position 2); 6.80 (mt, 3H: H aromatic in ortho and para to ethoxy); from 7.10 to 7.35 (mt, 6H: H aromatic at 6 et H aromatic at methoxy); 7.43 (dd, J = 8 and 1 Hz, 1H: 1 HJ; 7.50 (dd, J = 8 and 4 Hz, 1H: 1 Hj); 7.77 (dd, J = 4 and 1 Hz); 1H: 1 Hg), 8.38 (d, J = 10 Hz, 1H: NH at position 1), 8.70 (d, J = 9.5 Hz, 1H: NH at position 6), 11.60 (s, 1H: OH).

Example 31

Preparation of 4'-ethylthio-des (4'-dimethylamino) pristinamycin I

On a Erlenmeyer flask 2, the strain SP92 :: pVRC508 is cultured in a production medium to which 1 ml (20 g / l) of (S) -4-ethylthiophenylalanine hydrochloride solution is added after 16 hours of culture. , synthesized as described in Example 33, in 0.1N sodium hydroxide solution. After 40 hours of cultivation, 60 ml of the mash produced from 2 Erlenmeyer flasks is extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, and then the mixture is centrifuged. The supernatant is extracted twice with 0.5 volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4? -Ethylthio-des (4? -Dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in 7 ml of a mixture of 60% water and 40% acetonitrile and loaded onto a 10 x 250 mm Nucleosil 7 mC8 semipreparative column (Macherey Nagel), which is eluted using a 52% 100 mM phosphate buffer mixture. pH 2.9 and 48% acetonitrile. The fractions containing C 4-ethylthio-de (4-ζ-dimethylamino) pristinamycin IA are combined _and extracted with one volume and dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. There was obtained 4ζ-ethylthio-des (4 S-dimethylamino) pristinamycin ¹ H-NMR (400 MHz, CDCl ₃ , δ in ppm, ref.TMS):

0.68 (dd, J = 16 and 6 (mt, 5H: 1H ± CH3 at 3 β and 1H ± CH3 in 3 γ CH _{3 of} ethyl) · 1.32 (d, J = 7 Hz, 3H: CH ₃ δ, 45dol, 85 (mt, 3H: rough H cd CH 3 _ν 3γ CH 3 _V 2 β); 2.02 (mt, 1H H cd CH 3 in 3 β); 2.18 and 2.37 ( respectively mt ad broad, J = 16.5 Hz, 1H each: 01 ^ .50), 2.45 (broad, J = 16Hz, 1H: black H at 5 β); 2.85 (dt, J = 13 MHz, 1H: 1H (CH2 Cl2 at position 5); 2.90 (mt, 2H: ArSCH3 ethyl); 2.98 (dd, J = 12 and 4 Hz, 1H: 1H at the CDCl3 at position 4b); 3.25 (s, 3H: NCH3); 3.35 (mt, 1H: 1H at position 3); 3.39 (t, J = 12 Hz, 1H: second H cd)

0¾ in 4 β); 3.57 (mt, 1H: rough H cd of CH3 at 3δ); 4.55 (t, J = 7.5Hz, 1H: 3cc); 4.75 (broad dd, J = 13 and 7.5 Hz, 1H: pure CH3 at 5 ε); 4.85 (mt, 1H: 2a); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 a); cd 5.25 ± 5.40 (mt, 2H: 5 a and 4 a);

5.88 (d, J = 9.5 Hz, 1H: 6a); 5.91 (mt, 1H: 1β); 6.55 (d, J = 9.5 Hz, 1H; NH at position 2); 7.10 (d, J = 8 Hz, 2H: H aromatic at 4 δ); cd 7.10 d, 35 (mt, 7H: H)

Aromatic in $ 4 ε); 7.44 (AB limit, 2H: 1 H ₄ and 1 H 3); 7.74 (mt, 1H: 1 ')

Hj; 8.38 (d, J = 10 Hz, 1H: NH at position 6) _: 8.75 (d, J = 9.5 Hz, 1H: NH at position 6); 11.62 (s, 1H: OH).

Example 32

Preparation of 4'-ethyl-des (4'-dimethylamino) pristinamycin 1β

On a Erlenmeyer flask 2, the strain SP92 :: pVRC508 is cultured in a production medium as described in Example 3, to which, after 16 hours of culture, 1 ml of (20 g / l) (R, S) -4-ethylphenylalanine solution is added. Synthesized as described in Example 33. After 40 hours of cultivation, 60 ml of the mash produced from 2 Erlenmeyer flasks was extracted with two volumes of a mixture of 66% 100 mM phosphate buffer pH 2.9 and 34% acetonitrile, and then centrifuged. The supernatant was extracted with two volumes of dichloromethane. The dichloromethane phases are washed with water and then combined, dried over sodium sulphate and evaporated. The dry extract is taken up in 20 ml of dichloromethane and the resulting mixture is loaded onto a silica gel column (30 g) in dichloromethane, and this column is eluted using a gradient of 0 to 10% methanol in dichloromethane. The fractions containing 4 C-ethyl des (4 ζ-dimethylamino) pristinamycin I are combined and evaporated. The dry residue is taken up in 7 ml of a mixture of 52% water and 48% acetonitrile and loaded onto a 10 × 250 mm Nucleosil 7 μΟ8 semiprep column (Macherey Nagel), which is eluted using a 52% 100 mM phosphate buffer mixture. pH 2.9 and 48% acetonitrile. Fractions containing 4'-ethyl-des (4-dimethylamino) pristinamycin I _A are combined and extracted with one volume of dichloromethane. The organic phase is washed with water, dried over sodium sulphate and then evaporated. 0.50 mg of 4'-ethyl-des (4'-dimethylamino) pristinamycin I is obtained.

^X H-NMR (400 MHz, CDC1 _3, in ppm, ref.TMS): 0.42 (dd, J = 16 and 5.5 Hz, 1H: 1H of CHjdo β 5); 0.92 (ζ J = 7.5 Hz, 3H: CH, at 2 γ); from

1.10 dd, 40 (mt, 2H: 1H of CH, at 3β and 1H of CH, at 3γ); 1.23 (t, J = 7.5Hz,

3H: CH, ethyl j; 1.35 (d, J = 7 Hz, 3H: CH, v @ 1); od, 45 ad, 85 (mt, 3H: second K cd CH, at 3 γ and CH, at 2 β); 2.02 (mt, 1H: rough H at the CH, at 3b);

2.15 and cd 2.25 * 2.40 (2 mts, 1H each: CH, at 5δ); cd 2.25 * 2.40 (mt, 1H: second H cd CH, at 5β); 2.60 (q, J = 7.5 Hz, 2H: ArCH, ethyl) _; 2.83 (dt, J = 13 and 4 Hz, 1H: 1H and CH at 5 ε); 2.98 (dd, J = 12 and 4 Hz, 1H: 1H cd of CH at [beta]); from 3.25 * 3.35 (mt, 1H: 1H of CD at CH3 at position 3); 3.27 (s, 3H: NCH); 3.39 (t, J = 12 Hz, 1H: second H of CH at position 4b); 3.59 (mt, 1H: dirty H at the CH, at 3δ); 4.58 (dd, J = 7 and 6.5 Hz, 1H: 3a); 4.75 (broad dd, J = 138 Hz, 1H: dirty)

H cd CH, at 5 ε); 4.87 (mt, 1H: 2 c); 4.89 (dd, J = 10 and 1 Hz, 1H: 1 c); 5.24 (broad d, J = 5.5 Hz, 1H: 50 °); 5.29 (dd, J = 12 and 4 Hz, 1H: 4 _c ); 5.88 (d, J = 10Hz, 1H: 6c); 5.92 (mt, 1H: 1β); 6.73 (d, J = 10 Hz, 1H: trans 2); cd 7,10 *

7.35 (mt, 9H: H aromatic at 6 - 4 ε and 4 δ); 7.44 (dd, J = 8.5 and 1.5 Hz, 1H: 1 HJ; 7.50 (dd, J = 8.5 and 4.5 Hz, 1H: 1 H); 7.80 (dd J = 4.5 and 1.5 Hz, 1H: 1 Hg) 8.38 (d, J = 10 Hz, 1H: NH at 1), 8.75 (d, J = 10 Hz, 1H: NH) 11.66 (s, 1H: OH).

0.3 mg of 4'-ethyl-des (4'-dimethylamino) pristinamycin _1H is isolated from the same fractions leaving the silica gel column described above and also containing the derivative of pristinamycin I _h , if chromatographed on a semi-preparative column which is described above.

1 H-NMR (400 MHz, CDCl ₃ , 5 V ppm, ref.TMS):

0.04 (mt, 1H: 1H of the title compound);

CH, ^v 5 β); 0.92 (t, J = 7.5 Hz, 3H: CH ^at 2 γ); cd 1.10 * 1.40 (mt, 2H: 1H of CH ^at .delta. 1.18 (t, J = 7.5 Hz, 3H: CH, ethyl, 1.30 (d, J = 6? Hz, 3H: CH, vi _γ), of 1.45 04,85 (mt, 7H: rough H cd CH, v 5 γ second h oä CH, ^v 5 δ - 1H ^ d CH, v 3 β - CH, v 3 γ and CH, v 2 β) 1,81 (d, J = 13 Hz 2 H (2H, CD 3, β), 2.02 (dt, J = 13 Λ 4 Hz, 1H: 1 cd CH, * 5 ε) 2.65 (q, J = 7.5Hz, 2H)

Ar (CH, ethyl); 2.97 and 3.09 (respectively dd and t, J = 12 and 5 Hz and J = 12 Hz, 1H lying; CH, at 4β); 3.50 and 3.60 (2 mts, 1H heavy: CH, at 3 '); 4.13 (dd, J = 8 and 5 Hz, 1H, 3a); 4.49 (broad d, J = 13 Hz, 1H; rough H from CH, at 5 ε); 4.70 (mt, 2H; 5a and 4a); 4.77 (mt 1H: 2a); 4.83 (dd, J = 10 and 1 Hz, 1H):

a); 5.50 (d, J = 7Hz, 1H: 6a); 5.74 (mt, 1H: 1β); 6.09 (d, J = 4Hz, 1H)

NH in 4); 6.72 (broad m, 1H: NH at position 2); 7.07 (d, J = 8 Hz, 2H: aromatic H at .delta.); 7.15 (d, J = 8 Hz, 2H: aromatic H at position 4 &apos;); from 7.15 * 7.35 (mt, 5H: -)

H Aromatic v. 6); 7.40 (dd, J = 8 and 1 Hz, 1H: 1 HJ; 7.45 (dd, J = 8 and 4 Hz, 1H: 1 H 5); 7.92 (dd, J = 4 and 1 Hz, 1H: 1H (g), 8.40 (broad m, 1H: NH at position 6), 8.50 (d, J = 10 Hz, 1H: NH at position 1), 11.72 (s, 1H: OH).

Example 33

Preparation of the phenylalanine and phenylpyruvic acid derivatives described above

Phenylalanine and 4-methoxyphenylalanine, 4-bromophenylalanine derivatives,

4-chlorophenylalanine, 4-iodophenylalanine, 4-trifluoromethylphenylalanine, 4-aminophenylalanine, 3-methoxyphenylalanine are commercially available compounds.

The following phenylalanine derivatives can be prepared by methods described in the literature.

(R, S) -4-dimetylaminofenylalanín:

D.F. Elliot, A.T.Fuller, C.R. Harrington, J.Chem., 1948, 85-89, (R, S) -diethylaminophenylalanine:

Moldarev B.L., Pushkareva Z.V., Zhur. Obshchei Khim., 31, 1560-1469 (1961).

C.A.1961, 22226f, J.A.Stock, J. Chem. Soc., 1959, 90-97, (R, S) -4-ethylaminophenylalanine:

F. Bergel, J. A.Stock, J. Chem. Soc., 1959, 90-97, (R, S) -4-phenylalanine:

J.V.Braun, J.Nelles, Berichte, 66B, 1933, 1464-1470, (R, S) -4-methylphenylalanine:

R. R. Herr, T. Enjoki, J. P. Dailey, J. Am. Chem. Soc., 1957, 79,

4229-4231, (R, S) -4-methylthiophenylalanine and (R, S) -4-ethylthiophenylalanine: R.L.Colescott, R.R.Herr, J.P.Dailey, J.Am.Chem.Soc. 1957, 79, 4232-4235, (R, S) -4-methoxycarbonylphenylalanine:

H.Cleland, J. Org. Chem., 1969, 34, 747, (R, S) -2,4-dimethylphenylalanine:

R. R. Herr, T. Enjoki, J. P. Dailey, J. Am. Chem. Soc., 1957, 79,

4229-4231, (R, S) -3,4-Dimethylphenylalanine:

R. R. Herr, T. Enjoki, J. P. Dailey, J. Am. Chem. Soc., 1957, 79,

4229-4231, (R, S) -3-Trifluoromethylphenylalanine hydrochloride:

R. Filler and H.Novar, J. Org. Chem., 1960, 25, 733-736, (S) -4-aminomethylphenylalanine:

G.E.Stokker, W.F.Hoffman and C.F. Homnick, J. Org. Chem., 1993, 58, 5015-5017, (R, S) -3-methylphenylalanine:

J. H. Burckhalter, V.C.Stephens, J.A.C.S., 1951, 73, 56-58, (R, S) -4-acetylphenylalanine:

J.I.Degaw et al., J. Med. Chem., 1969, 11, 225-227, (S) -4-O-allyltyrosine:

A.Loffet, H.Zang, Int.J.Pept. Protein Res., 1993, 42, 346, (S) -4-O-ethyltyrosine:

Y.Sasaki et al., Chem. Pharm. Bull., 1982, 30, 4435, (R, S) -4-ethylphenylalanine:

A.Zhuze et al., Coll.Czech.Chem.Commun., 1965, 62, 26-48,

4-tert-Butylphenylpyruvic acid can be prepared according to R.Breslow, J.W.Canary, MVarney, S.T.Waddell and D.Yang, J.Am.Chem.Soc., 1990, 112, 5212-5219.

Other phenylalanine derivatives were prepared according to the methods described in Examples 34-42. In these examples, flash chromatography was performed at a mean nitrogen pressure of 50 kPa using silica gel with a granulometry of 40-53 μια. according to St11a et al., J. Org. Chem., 43, 2923 (1978).

Example 34

Preparation of phenylalanine and phenylpyruvic acid derivatives by Method

This example follows the following reaction scheme:

34-3: Methyl 4-methylamino-2-acetamidocinamate

5.75 g of methyl 2-acetylamidoacrylate, 0.185 g of palladium acetate, 0.81 tetrabutylammonium chloride and 6.03 of sodium bicarbonate are introduced into a three-necked flask under a nitrogen atmosphere, then 6.5 g of 4-iodo-N- are added. methylamine in solution in 200 ml of dimethylformamide. The mixture is heated at 82 ° C for 16 hours and 30 minutes, then poured into 1000 ml of distilled water. The mixture thus obtained is taken up in 250 ml of dichloromethane, then the organic phase is decanted and the aqueous phase is washed twice with 250 ml of dichloromethane. The organic phases are combined, dried over sodium sulphate, filtered and concentrated under reduced pressure (50 kPa) at 70 ° C to give a brown oil which is purified by flash chromatography using first a mixture of ethyl acetate and cyclohexane and then only pure ethyl acetate.

There was thus obtained 4 g of methyl 4-methylamino-2-acetamidocinamate as a yellow solid (Merck silica gel 5719, Rf = 0.48) which was used as such.

N-Methyl-p-iodo-aniline can be prepared according to:

Krishnamurthy, Tetrahedron Letters, 33, 3315-3318 (1982).

34-34: 4-Methylaminophenylpyruvic acid

2.4 g of methyl 4-methylamino-2-acetamidocinamate and 32 ml of 12N hydrochloric acid are introduced into a round-bottomed flask. The mixture was heated at reflux for 3 hours, then cooled and washed twice with 20 ml of diethyl ether. The aqueous phase is cooled to -10 ° C and the precipitate obtained is filtered off and then washed with a minimal amount of cold hydrochloric acid. The solid obtained is dried in a desiccator under reduced pressure to give 1.1 g of 4-methylaminophenylpyruvic acid as a pale beige solid, melting at 210 ° C.

34-5: (R, S) -3-Fluoro-4-methyl-phenylalanine hydrochloride

The procedure of Example 34-1 is followed except that 0.6 g of (R, S) -3 is obtained from 1.6 g of methyl N-acetyl- (3-fluoro-4-methyl) phenylalaninate. -fluoro-4-methylphenyl88 alanine in the form of white crystals melting above 260 ° C.

34-6: Methyl N-acetyl- (3-fluoro-4-methyl) phenylalaninate

The procedure of Example 34-2 is followed except that from 1.9 g of methyl (3-fluoro-4-methyl) -2-acetamidocinamate, 0.2 g of 10% palladium on carbon in 230 ml. ethanol gave 1.6 g of methyl N-acetyl- (3-fluoro-4-methyl) phenylalaninate as a colorless oil (Merck silica gel 5719, Rf = 0.46, 50:50 dichloromethane / ethyl acetate). 34-7: Methyl (3-fluoro-4-methyl) -2-acetamidocinamate

The procedure of Example 34-3 is followed except that from 3.6 g of methyl 2-acetamido acrylate, 0.12 g of palladium acetate, 5.2 g of tetrabutylammonium chloride, 3.8 g of sodium bicarbonate and 4 g of 2 of fluoro-4-bromotoluene in solution in 120 ml of dimethylformamide (anhydrous) gives 2.6 g of methyl (3-fluoro-4-methyl) -2-acetamidocinamate as a white solid, melting at 163 ° C.

34-8: (R, S) -4-Trifluoromethoxyphenylalanine hydrochloride or (R, S) -O-trifluoromethyltyrosine hydrochloride

The procedure is as in Example 34-1, except that 3 g of methyl N-acetyl- (4-trifluoromethoxy) phenylalaninate and 30 ml of 12N hydrochloric acid are obtained.

1.5 g of (R, S) -4-trifluoromethoxyphenylalanine hydrochloride as white crystals, melting at 260 ° C.

34-9: (R, S) -Methyl-N-acetyl- (4-trifluoromethoxy) phenylalaninate

The procedure is as in Example 34-2, except that from 3.1 g of methyl (4-trifluoromethoxy) -2-acet amidocinamate, 0.3 g of 10% palladium on carbon and 50 ml of ethanol, 3 g are obtained. g of methyl N-acetyl- (4-trifluoromethoxy) phenylalaninate as a white solid, melting at 80 ° C.

34-10: Methyl 1- (4-trifluoromethoxy) -2-acetamidocinamate

The procedure is as in Example 34-3 except that 4.3 g of methyl 2-acetamidoacrylate, 0.44 palladium acetate, 6.1 g tetrabutylammonium chloride, 4.6 g sodium bicarbonate and 5 g 4 are used. of trifluoromethoxybromobenzene in solution in 150 ml of anhydrous dimethylformamide gave 3.1 g of methyl (4-trifluoromethoxy) -2-acetamidocinamate as a white solid, melting at 135 ° C.

34-11: (R, S) -3-Methylthiophenylalanine hydrochloride

The procedure of Example 34-1 is followed except that 1.38 g of (R, S) -3- is obtained from 3.3 g of methyl N-acetyl-3-methylthiophenylalaninate and 40 ml of 12N hydrochloric acid. methyl thiophenylalanine as white crystals, melting at 190 ° C.

34-12: Methyl N-acetyl-3-methylthiophenylalaninate

3.72 g of methyl 3-methylthio-2-acetamidocinamate in solution in 100 ml of methanol and 30 ml of tetrahydrofuran are introduced into a round-bottomed flask and 1.4 g of magnesium are added. After 20 minutes, the reaction mixture is cooled on ice, 1.4 g of magnesium are added thereto, the reaction mixture is stirred at room temperature for 18 hours, then poured into 1.4 liters of distilled water and 300 ml of dichloromethane and the mixture is stirred at room temperature. filter using Clarcel ^R filter aid. The aqueous phase is adjusted to pH 6 by addition of 12N hydrochloric acid and the aqueous phase is then decanted and washed with 100 ml of dichloromethane. The organic phases are combined and dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure. To give 3.42 g of methyl N-acetyl-3-metyltiofenylalaninátu as a colorless oil (Merck, R _£ = 0.5, eluent: ethyl acetate).

34-13: Methyl 3-methylthio-2-acetamidocinamate

The procedure is as in Example 34-3 except that from 5.6 g of methyl 2-acetamido acrylate, 0.18 g of palladium acetate, 8.2 g of tetrabutylammonium chloride, 5.86 g of sodium bicarbonate, 5 g of 3-iodo-1-methylbenzene in solution in 160 ml of anhydrous dimethylformamide gave 4.8 g of methyl (3-methylthio) -2-acetamidocinamate as a white solid, melting at 139 ° C.

34-14: 3-Iodo-1-methylthiobenzene

20 ml of distilled water and 20 ml of 12N hydrochloric acid are introduced into the three-necked flask with stirring, followed by the addition of 10 ml of 3-methylthioaniline. The mixture is gently warmed to ensure the solids dissolve and then cooled to 5 ° C. 5.86 g of sodium nitrite in solution in 15 ml of water are then slowly added and the temperature of the reaction mixture is maintained between 5 and 8 ° C. 20 minutes after the addition, 13.57 g of potassium iodide in solution in 15 ml of water are added over 10 minutes and the mixture is then stirred at room temperature for 15 hours. The oil formed is separated from the aqueous phase by decantation, then an aqueous solution of sodium sulphite is added. The aqueous phase is decanted and the product is extracted with 100 ml of dichloromethane. The organic phase is washed with 100 ml of water, the pH of the aqueous phase is adjusted to 9 with concentrated sodium hydroxide solution and then decanted. The organic phase is washed twice with 100 ml of water, decanted, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure (50 kPa) at 40 ° C. The resulting product was purified by flash chromatography (eluent: cyclohexane) to give 13 g

3-iodo-1-methylthiobenzene as a yellow liquid (Merck silica gel 5719, Rf = 0.8, eluent: cyclohexane).

Example 35

Preparation of Phenylalanine Derivatives by Method B

35-1: (R, S) -4-tert-Butylphenylalanine

25 g of diethyl 4- (tert-butyl) benzylacetamidomalonate and 250 ml of 37% hydrochloric acid are introduced into a three-necked flask fitted with a condenser. The mixture was stirred and heated to reflux until no more gas was released. After cooling the reaction mixture, the precipitate formed is filtered off and then recrystallized from acetonitrile to give 25.6 g of (R, S) -4-tert-butylphenylalanine hydrochloride as a white solid, melting at 234 ° C ( see also Journal of the Takeda Research Laboratories, Vol. 43, No.3 / 4, December 1984, pp. 53-76).

35-2: Diethyl 4- (tert-butyl) benzylacetamidomalonan

25 g of 4-tert-butylbenzyl bromide, 50 ml of anhydrous toluene and 3.1 g of sodium hydride as an 80% suspension in oil and then 21.8 g of diethyl acetamidomalonate are introduced into a three-necked flask fitted with a condenser. The reaction mixture was then heated at 110 ° C for 17 hours. After cooling, 15 ml of absolute ethanol is added slowly, followed by ml of 50% alcohol and finally 50 ml of water. The organic phase is decanted and the aqueous phase is washed three times with 50 ml of diethyl ether each time. The organic phases are combined, washed with water and then dried over sodium sulphate. After filtration and concentration under reduced pressure, the product is crystallized from petroleum ether to give 25 g of diethyl 4- (tert-butyl) benzylacetamidomalonate as a white solid, melting at 80 ° C.

35-3: (R, S) -3-Methylaminophenylalanine dihydrochloride

The procedure of Example 35-1 was followed except that from 1.07 g of diethyl 3-methylaminobenzylacetamidomalonate and 20 ml of 12N hydrochloric acid, 1.0 g of a light beige solid was obtained. Dissolve this product in 20 ml of absolute ethanol and add 0.4 g of charcoal to the solution, filter the solution using Clarcel ^R filter aid and concentrate under reduced pressure (50 kPa). the same operation is carried out again with 1 g of charcoal and the solid obtained is triturated in 20 ml of ether. After filtration and drying under reduced pressure (2.7 kPa) at 50 ° C, 0.65 g of (R, S) -3-methylaminophenylalanine dihydrochloride is obtained in the form of a white powder, melting at 135 ° C. dec).

35-4: Diethyl-3-methylaminobenzylacetamidomalonan

To a 3-necked flask was charged with 3.11 mL acetic anhydride under a nitrogen atmosphere. Then, 1.51 ml of formic acid is added over 3 minutes at 0 ° C and the mixture is heated at 50 ° C for two hours. The reaction mixture is then allowed to cool to room temperature with stirring, over hours and 20 minutes, then 4 ml of tetrahydrofuran (anhydrous) are added under a nitrogen atmosphere. The reaction mixture was then cooled to 20 ° C. A solution of diethyl 3-aminobenzylacetamidomalonanane in a mixture of 15 ml of anhydrous tetrahydrofuran and 15 ml of anhydrous dichloromethane is added over 10 minutes. Stirring is continued for one hour and 10 minutes at -20 ° C and then for 16 hours at 20 ° C. the reaction mixture is concentrated to dryness under reduced pressure (50 kPa) at 30 ° C, then co-evaporated with 30 ml of anhydrous toluene. A white solid is obtained which is dissolved in a mixture of 10 ml of anhydrous tetrahydrofuran and 20 ml of anhydrous 1,2-dichloroethane and the solution obtained is introduced into a three-necked flask under a nitrogen atmosphere.

The reaction mixture was cooled to -5 ° C and then borane-dimethylsulfide complex (2M solution in tetrahydrofuran) was added over 10 minutes. The reaction mixture was then allowed to warm to room temperature and then refluxed for 3 hours. The reaction mixture is cooled to 0 ° C and then 10 ml of methanol are added over 25 minutes and the reaction mixture is stirred at 0 ° C for 45 minutes and then at room temperature for 30 minutes. The mixture is cooled to 0 ° C, then hydrogen chloride gas is bubbled through the mixture until a pH of 2 is reached. The mixture is then heated at reflux for one hour and then concentrated to dryness under reduced pressure at 30 ° C. 5 g of product are obtained, which is taken up in 30 ml of aqueous sodium hydrogen carbonate solution and 30 ml of dichloromethane, the organic phase is decanted and the aqueous phase is washed with 20 ml of water. The phases are combined, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure (2.6 kPa) to give 3.43 g of a yellow oil which is purified by flash chromatography using a mixture of v / v mixture 50:50. Thus, the woman was pressurized (2.7 kPa) at diethyl 3-methylaminobenzylacetamidomalonate as a light beige solid, melting at 122 ° C.

ethyl acetate and cyclohexane, after drying at 20 [deg.] C, 1.18 g

35-5: Diethyl-3-aminobenzylacetamidomalonate

Diethyl-3-aminobenzylacetamidomalonan may be prepared as described in: S. S. Odden, D. N. Ward, W. H. Chapman, and H. Rakoff, J. Am. Chem. Soc., 81, 1959, 3100-3102.

35-6: (R, s) -3-Ethylaminophenylalanine dihydrochloride

The procedure of Example 34-1 is followed except that 1.7 g of (R, S) is obtained from 2 g of (R, S) -ethyl N-acetyl-3-ethylaminophenylalaninate and 30 ml of 12N hydrochloric acid. -3-ethylaminophenylalanine dihydrochloride in the form of a light beige solid, which is hygroscopic and contains 10 mol. (R, S) -3-dietylaminofenylalanín dihydrochloride.

35-7: (R, S) -Ethyl-N-acetyl-3-ethylaminophenylalaninate

3 g of (R, S) -ethyl-N-acetyl-3-aminophenylalaninate, 40 ml of ethanol and 14 g of Raney nickel are pre-washed with distilled water and ethanol into a round-bottomed flask under a nitrogen atmosphere. The reaction mixture is heated at reflux for 19 hours, then cooled, filtered through a Clarcel ^R filter and then concentrated to dryness under reduced pressure (50 kPa). 3.07 g of a colorless oil are obtained which, after purification by flash chromatography, eluting with ethyl acetate. 2.1 g of (R, S) -ethyl N-acetyl-3-ethylaminophenylalaninate are obtained in the form of a colorless oil (Merck silica gel 5719, Rf = 0.6, ethyl acetate) containing 10% of (R, S) -ethyl-N acetyl-3-dietylaminofenylalaninátu.

35-8: (R, S) -Ethyl-N-acetyl-3-aminophenylalaninate g of a mixture of (R, S) -ethyl-N-acetyl-3-nitrophenylalaninate (75 mol% / mol) and diethyl 3-nitrobenzylacetamidomalonate (25 mol / mol) was introduced into the autoclave under a nitrogen atmosphere. 2.5 g of palladium on carbon are added, followed by 200 ml of dichloromethane. The mixture is subjected to a hydrogen pressure of 10 bar with stirring at 18 ° C for 4 hours, after transition to atmospheric pressure, the reaction mixture is filtered through a Clarcel ^R filter, washed with dichloromethane and then concentrated to dryness under reduced pressure (50 ml). kPa) to give a solid product which is recrystallized from 450 ml of distilled water, which was previously heated to reflux in the presence of 4 g of 3S charcoal. After filtering at 95 ° C over a Clarcel ^R filter, the product is crystallized at 4 ° C and the crystals are filtered off and then dried. 9.9 g of (R, S) -ethyl N-acetyl-3-aminophenylalaninate are obtained in the form of a pale beige solid which melts at 10 DEG C. and contains 5% of diethyl 3-aminobenzylacetamidomalonate.

35-9: (R, S) -Ethyl-N-acetyl-3-nitrophenylalaninate and diethyl 3-nitrobenzylacetamidomalonate

600 ml of absolute ethanol and then 7.9 g of sodium are introduced into a three-necked condenser with nitrogen. After complete dissolution, 74.5 g of diethyl acetamidomalonate and then 60 g of 4-nitrobenzyl chloride in 200 ml of anhydrous ethanol are added. The reaction mixture was then heated to reflux for 16 hours and 30 minutes. After cooling, the reaction mixture is concentrated under reduced pressure (50 kPa) and then taken up in a mixture of 500 ml of dichloromethane and 500 ml of water. The pH is adjusted to 7 by addition of 0.5 N sulfuric acid, then the organic phase is decanted and the aqueous phase is washed twice with 200 ml of dichloromethane. The organic phases are combined, washed with 200 ml of saturated aqueous sodium bicarbonate solution, decanted and then dried over magnesium sulphate. After filtration and concentration under reduced pressure (50 kPa), the product is recrystallized from 600 ml of refluxing ethanol to give, after crystallization at ambient temperature, filtration and drying, 70.4 of diethyl 3-nitrobenzylacetamidomalonate as white crystals. , melting at 156 ° C. The mother liquors were concentrated and then purified by flash chromatography (eluent: ethyl acetate) to give 25.6 g of a mixture of ethyl N-acetyl-3-nitrophenylalaninate (70 mol% / mol) and diethyl 3-nitrobenzylacetamidomalonate (25 g). % mol / mol) as a pale beige solid which is used as such in the next step.

35-10: (R, S) -3-Dimethylaminophenylalanine dihydrochloride

The procedure was as in Example 34-1 except that from the product (0.72 g of (R, S) -ethyl-N-acetyl-3-dimethylaminophenylalaninate and 8.6 ml of 10N hydrochloric acid) a solid product was obtained. This is triturated with 50 ml of acetone, filtered and dried under reduced pressure (2.7 kPa) at 40 ° C. 0.68 g (93%) of (R, S) -3-dimethylaminophenylalanine is obtained in the form of a white solid which melts at a temperature in the region of 120 DEG C. (with decomposition).

35-11: (R, S) -Ethyl-N-acetyl-3-dimethylaminophenylalaninate

4 g of (R, S) -ethyl-N-acetyl-3-aminophenylalaninate, prepared as described in Example 35-8, in 15 ml of dimethylformamide are introduced into a three-necked flask under a nitrogen atmosphere, followed by 5.5 ml of this mixture. triethylamine followed by 2.5 ml of methyl iodide and 4 ml of dichloromethane while maintaining a temperature close to 30 [deg.] C. with ice. The mixture was then heated to 35 ° C for 18 hours. 1 ml of methyl iodide in solution in 1 ml of dimethylformamide is then slowly added, keeping the temperature close to 30 ° C, then 2.2 ml of triethylamine are added and the mixture is heated at 35 ° C for 5 hours. The mixture is allowed to cool to room temperature, then extracted with 100 ml of ethyl acetate and 150 ml of distilled water. The aqueous phase is decanted and then washed twice more with 70 ml of ethyl acetate. The organic phases are combined, washed twice with 80 ml of distilled water and then with 50 ml of a saturated solution of sodium chloride in distilled water. The organic phase is decanted, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure to give 2.4 g of product which is purified by flash chromatography, eluting with a dichloromethane / methanol (90:10 by volume) mixture. . There was thus obtained (R, S) -ethyl N-acetyl-3-dimethylaminophenylalaninate (0.72 g, 16%) as yellow crystals.

Example 36

Preparation of Phenylalanine Derivatives by Method C

36-1: (R, S) -4-Isopropylphenylalanine

7 g of red phosphorus and 8 g of 4- (isopropylbenzylidene) -2-methyl-5-oxazole in 45 ml of acetic anhydride are introduced into a three-necked flask, then 35 ml of 57% hydroiodic acid are slowly added to this mixture. After the addition was complete, the mixture was heated at reflux for 3 hours 30 minutes, then left at room temperature for three days. The reaction mixture is filtered, the separated solid is washed twice with 10 ml of acetic acid and the filtrate is concentrated to dryness under reduced pressure. The residue is taken up in 100 ml of distilled water, concentrated to dryness under reduced pressure to give a solid which is taken up in 50 ml of distilled water and then extracted three times with 50 ml of diethyl ether each time after addition of 0.5 g of sodium sulfite. The ether is decanted and the aqueous phase is subjected to reduced pressure to remove traces of diethyl ether. 2 g of charcoal are added to the aqueous phase, the mixture is heated to 40-50 ° C, filtered on a Clarcel ^R filter and washed with a minimum of water. The pH is adjusted to 5 by adding 32% aqueous ammonium hydroxide solution at 4 ° C. The precipitate obtained is filtered in the cold, washed twice with 10 ml of water, with 10 ml of ethanol and then twice with 10 ml of ether, and after drying under reduced pressure at 20 DEG C., 3.97 g of (R, S) -4-isopropylphenylalanine is obtained. in the form of a white solid which melts above 260 ° C (see also Takeda Research Vol. 43, No. 3/4, December 1984, pp. 53-76).

36-2: 4- (Isopropylbenzilidene) -2-methyl-5-oxazolone

18.52 g of N-acetylglycine, 10.6 g of sodium acetate, 20 ml are introduced into a round-bottomed flask equipped with a condenser.

Of 4-isopropylbenzaldehyde and 57 ml of acetic anhydride. The mixture was stirred for 30 minutes, then stirred for one hour at 110 ° C and then for 15 hours at room temperature. The reaction mixture was then poured into 600 ml of water and 400 ml of petroleum ether, which had been preheated to 50 ° C. The organic phase is decanted and the aqueous phase is washed twice with 150 ml of petroleum ether. The organic phases are combined, dried over magnesium sulphate, filtered and concentrated under reduced pressure to a volume of 100 ml to obtain a precipitate. The precipitate was filtered off, washed twice with 50 ml of pentane to give

8.2 g of 4- (isopropylbenzilidene) -2-methyl-5-oxazolone as a yellow solid, melting at 77 ° C.

36-3: (R, S) -4-Butylphenylalanine

The procedure was as in Example 36-1 except that 1.49 g of red phosphorus, 1.8 g

4- (butylbenzilidene) -2-methyl-5-oxazolone in 9.23 ml of acetic anhydride and 7.39 ml of 57% hydroiodic acid gives 0.35 g of (R, S) -4-butylphenylalanine as a light beige solid, which melts above 260 ° C.

36-4: 4- (Butylbenzilidene) -2-methyl-5-oxazolone

The procedure of Example 36-2 is followed except that 1.89 g of 8.43 g of N-acetylglycine, 4.92 g of sodium acetate, 9.8 g of 4-butylbenzaldehyde and 26 ml of acetic anhydride are obtained. g of 4- (butylbenzilidene) -2-methyl-5-oxazolone as a yellow solid, melting at 74 ° C.

Example 37

Preparation of a phenylalanine derivative by Method D

37-1: (R, S) -3-Ethoxyphenylalanine hydrochloride (or (R, S) -3-O-ethyltyrosine hydrochloride)

1 g of (R, S) -tert-butoxycarbonyl-3-ethoxyphenylalanine in solution in 3.6 ml of dioxane saturated with hydrogen chloride gas are introduced into a round-bottomed flask and the resulting mixture is stirred for 5 hours at room temperature. The precipitate formed is filtered off, washed with dioxane and then with ether and then dried under reduced pressure (2.7 kPa) to give 0.65 g of (R, S) -3-ethoxyphenylalanine hydrochloride as a white solid, m.p. at 200 ° C.

37-2: (R, S) -N- tert -Butoxycarbonyl-3-ethoxyphenylalanine

1.33 g of (R, S) -ethyl-N-tert-butoxycarbonyl-3-ethoxyphenylalaninate in a solution of 8 ml of methanol are introduced into a round-bottomed flask, then 8 ml of 1N sodium hydroxide solution are added to the flask. After stirring at room temperature for 18 hours, the mixture was evaporated under reduced pressure, then acidified with 1N hydrochloric acid. The product is extracted twice with 10 ml of ethyl acetate, the organic phases are combined, washed twice with 10 ml of water, dried, filtered and then concentrated to dryness under reduced pressure to give 1 g of (R, S) -N-tert-butoxycarbonyl- 3-Ethoxyphenylalanine as a yellow oil (Merck silica gel 5719, Rf = 0.7, eluent: toluene, methanol, and diethylamine in an 80:10:10 by volume ratio).

37-3: (R, S) -Ethyl-N-tert-butoxycarbonyl-3-ethoxyphenylalaninate

It is introduced into a round-bottomed flask under a nitrogen atmosphere

1.5 g of (R, S) -N-tert-butoxycarbonyl-3-tyrosine in solution in 7.5 ml of dimethylformanide (anhydrous), then 0.508 g of 50% sodium hydride in oil are added to the flask. After stirring at room temperature for 2 hours, iodomethane (0.86 ml) was added followed by stirring

Stir 100 for 4 hours at room temperature. The mixture is filtered, and the resulting solid is washed three times with 10 ml of water and twice with 10 ml of petroleum ether to give, after drying under reduced pressure (2.7 kPa) at 30 ° C, 1.33 g of (R, S) -ethyl- N-tert-butoxycarbonyl-3-ethoxyphenylalaninate as a white solid.

37-4: (R, S) -N-tert-Butoxycarbonyl-3-tyrosine

18 g of (R, S) -3-tyrosine in solution in 180 ml of dioxane are introduced into the three-necked flask with stirring, then 99 ml of 1N sodium hydroxide solution are added to the flask, followed by 26 g of di-tert-butyl carbonate in solution in 160 ml of dioxane. . After stirring for 36 hours, the reaction mixture is concentrated under reduced pressure, taken up in 100 ml of distilled water, acidified with hydrochloric acid to pH, and then extracted twice with 200 ml of ethyl acetate. The organic phase is dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure at 30 ° C to give 30 g of (R, S) -N-tert-butoxycarbonyl-3-tyrosine as a white solid ( Merck silica gel 5719, Rf = 0.25, eluent: toluene / dimethylamine (80/10 by volume) at 30 ° C, IN acid

Example 38

Preparation of Phenylalanine Derivatives by Method E

38-1: (R, S) -4-Dialylaminophenylalanine dihydrochloride

The procedure is as in Example 35-1 except that 5.8 g of diethyl dialylaminobenzylacetamidomalononate and 48 ml of 10N hydrochloric acid are obtained after evaporation, which is triturated in 50 ml of acetone, filtered and. it is again triturated in 10 ml of dichloromethane, filtered and then washed three times with 10 ml of diethyl ether. After drying under reduced pressure (2.7 kPa) at 40 ° C, 4.41 g is obtained

101 (R, S) -4-Dialylaminophenylalanine dihydrochloride as a white solid, crisp, melting at near 135 ° C (dec.).

38-2: (R, S) -4-Allylaminophenylalanine dihydrochloride

The procedure of Example 35-1 is followed except that 3.27 g of diethyl 4-allylaminobenzylacetamidomalononate and 30 ml of 10N hydrochloric acid are obtained after evaporation which is triturated in 50 ml of acetone, filtered and then dried under reduced pressure (2.7 kPa) at 40 ° C. 2.3 g of (R, S) -4-allylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts at 134 DEG C. (decomposition).

38-3: Diethyl 4-dialylaminobenzylacetamidomalonate and diethyl 4-allylaminobenzylacetamidomalonate

In a three-necked flask equipped with an addition funnel and kept under a nitrogen atmosphere, 10 g of diethyl 4-aminobenzylacetamidomalonate in solution in 150 ml of dimethylformamide was introduced. 6.57 ml of allyl bromide are then slowly added at room temperature, followed by 10.76 ml of triethylamine while stirring. After stirring for 19 hours, 1.31 ml of allyl bromide and 2.15 ml of triethylamine were added again and the mixture was stirred for 26 hours. The reaction mixture was then poured into 1.5 L of distilled water and extracted with 1 L of ethyl acetate. The aqueous phase is decanted, washed twice with 500 ml of ethyl acetate. The organic phases are combined, washed with 500 ml of distilled water and then with 500 ml of saturated sodium chloride solution, decanted, dried over magnesium sulphate, filtered and then concentrated to dryness to give a brown oil which is purified by flash chromatography using elution. 90:10 mixture of dichloromethane and ethyl acetate to give 6.66 g of diethyl 4-dialylaminobenzylacetamidomalonate as a beige solid, melting at 94-96 ° C (Rf = 0.6, ethyl acetate / cyclohexane) 50:50 v / v and 3.49 g of diethyl 4-allylaminobenzylacetamidomalonate as a white solid, m.p. 104 DEG-106 DEG C. (Rf = 0.45, ethyl acetate / cyclohexane, 50:50 v / v).

Diethyl 4-allylaminobenzylacetamidomalonan may be prepared according to: J. Burckhalter, V.C.Stephens, J.Am.Chem.Soc. 56, 1951, 73.

Example 39

Preparation of Phenylalanine Derivatives by Method F

R

39-1. (R, S) -4-Etylizopropylfenylalanín dihydrochloride

The procedure is as in Example 35-1 except that from 2.9 g of diethyl 4-ethylisopropylbenzylacetamidomalonate and 24.6 ml of 10N hydrochloric acid, a solid is obtained by evaporation which is triturated in 20 ml of acetone, filtered. and then dried under reduced pressure (2.7 kPa) at

103 at 40 ° C. 2 g of (R, S) -4-ethylisopropylaminophenylalanine dihydrochloride are obtained in the form of a white solid which melts at a temperature in the region of 147 DEG C. (with decomposition).

39-2: Diethyl-4-ethylisopropylaminobenzylacetamidomalonan

15 g of diethyl 4-ethylaminobenzylacetamidomalononate in 70 ml of tetrahydrofuran was introduced into a three-necked flask, kept under nitrogen, then 6.4 ml of 2-iodopropane was added to the contents of the flask, followed by 8.4 ml of 1,5-diazabicyclo [4.3, Of O-non-5-ene and the mixture is then heated at 60 ° C for 24 hours. Then 2.13 ml of 2-iodopropane and then 8.4 ml of 1,5-diazabicyclo [4.3.0] non-5-ene are added, followed by heating at 60 ° C for a further 24 hours. The mixture was then cooled to room temperature and extracted with 50 mL of dichloromethane and 50 mL of distilled water. The aqueous phase is decanted and then washed twice more with 30 ml of dichloromethane. The organic phases are combined, washed with 60 ml of distilled water and then with 50 ml of a saturated solution of sodium chloride in distilled water, the organic phase is decanted, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure to give 16.3 g. product, which is purified by chromatography (flash chromatography eluting with a dichloromethane / methanol (90:10 by volume) mixture). This gives 4.59 g of product, which is recrystallized from 45 ml of cyclohexane to give 3.44 g of diethyl 4-etylizopropylaminobenzylacetamidomalonanu as white crystals melting at 80 ^e C.

39-3: Diethyl-4-ethylaminobenzylacetymidomalonan

Diethyl 4-ethylaminobenzylacetymidomalonan can be prepared as described in Example 35-7 except that 23.8 g diethyl-4-ethylaminobenzylacetymidomalonate is obtained from 22 g diethyl 4-aminobenzylacetamidomalonan, 500 ml ethanol and 70 g Raney nickel. as a white, brittle product melting at 136 ° C.

104

9-4: (R, S) -4-Allylethylaminophenylalanine hydrochloride

The procedure of Example 35-1 is followed except that from 4.54 g of diethyl 4-allylethylbenzylacetamidomalonate and 37.9 ml of 10N hydrochloric acid, a solid is obtained after evaporation which is dried under reduced pressure (2, 3). 7 kPa) at 40 ° C. 3.67 g of (R, S) -4-allylethylaminophenylalanine are obtained in the form of a brown solid which melts at a temperature in the region of 130 DEG C. (with decomposition).

39-5: Diethyl-4-allylethylaminobenzylacetamidomalonan

The procedure is as in Example 39-2 except that from 8 g of diethyl 4-ethylaminobenzylacetamidomalonan, 4 ml of allyl bromide, 5.82 ml of 1,5-diazabicyclo [4.3.0] non-5- Purification by flash chromatography, eluting with a dichloromethane / ethyl acetate mixture (90:10; v / v) gave 5.6 g of a solid which was recrystallized from 35 ml of cyclohexane. 5.43 g of diethyl 4-allylethylaminobenzylacetamidomalononate are thus obtained in the form of a white solid melting at 86 ° C.

39-6: (R, S) -4-Ethylpropylaminophenylalanine dihydrochloride

The procedure is as in Example 35-1 except that 2.5 g of diethyl 4-ethyl-propylamino-benzylacetamidomalonate and 21 ml of 10N hydrochloric acid are obtained after evaporation to give a solid which is dried under reduced pressure (2.7 g). kPa) at 40 ° C to give 2 g (97%) of (R, S) -4-ethylpropylaminophenylalanine dihydrochloride as a white solid melting at a temperature near 147 ° C (dec.).

39-7: Diethyl-4-ethylpropylaminobenzylacetamidomalonan

The procedure was as in Example 39-2 except that from 10 g of diethyl-4-ethylaminobenzylacetam105 domalonate, 5.6 ml of 1-iodopropane, 7.2 ml of 1,5-diazabicyclo [4.3.0.0] of non-ene in 70 ml of tetrahydrofuran, after 36 hours of reaction and then after flash chromatography using a 97: 3 mixture of dichloromethane and methanol, 2.8 g of solid product, which is recrystallized from 26 ml of cyclohexane. . This is obtained

2.9 g of diethyl 4-ethylpropylaminobenzylacetamidomalonate as a white solid, melting at 84-86 ° C.

39-8:

(R, S) -4-Etylmetylcyklopropylaminofenylalanín dihydrochloride

The procedure of Example 35-1 is followed except that 3 g of diethyl 4-ethylmethylcyclopropylaminobenzylacetamidomalonate and 25 ml of 10N hydrochloric acid are obtained after 3 days of reaction and then evaporated to give a solid which is triturated in 40 ml of acetone. , filtered, dried under reduced pressure (2.7 kPa) at 40 ° C. 2.24 g of (R, S) -4-ethylmethylcyclopropylaminophenylalanine dihydrochloride are obtained in the form of a white solid, melting at a temperature in the region of 140 DEG C. (with decomposition).

39-9: Diethyl-4-ethylmethylcyclopropylaminobenzylacetamidomalonan

The procedure was as in Example 39-2 except that from 8 g of diethyl 4-ethylaminobenzylacetamidomalonan, 2.6 ml of bromomethylcyclopropane, 2.97 ml of 1,5-diazabicyclo [4.3.0] non-5 -enene in 50 ml of tetrahydrofuran after 3 days of reaction and after flash chromatography using a 90:10 mixture of dichloromethane and ethyl acetate, 3.3 g of diethyl 4-ethylmethylcyclopropylaminobenzylacetamidomalonane as a white solid, m.p. 112-114 ° C.

106

Example 40

Preparation of Phenylalanine Derivatives by Method G

40-1: (R, S) -4- (Pyrrolidinyl) phenylalanine dihydrochloride

The procedure of Example 35-1 is followed except that 1.5 g of diethyl 4- (1-pyrrolidinyl) benzylacetamidomalonate and 40 ml of 5N hydrochloric acid are obtained after evaporation to give a solid which is triturated in 15 g. ml of acetone, filtered and dried under reduced pressure (2.7 kPa) at 40 ° C. 0.6 g of (R, S) -4- (pyrrolidinyl) phenylalanine dihydrochloride is obtained as a brittle white product.

40-2: Diethyl-4- (1-pyrrolidinyl) benzylacetamidomalonan

4 g of diethyl 4- (1-pyrrolyl) -benzylacetamidomalonan in solution in 100 ml of methanol and 1 g of 10% palladium on carbon are introduced into the autoclave. After purging with nitrogen three times, the product is hydrogenated at 1.4 MPa at 19 ° C. After stirring for 25 hours, the hydrogenation was discontinued, the hydrogenation catalyst was removed by filtration through Clarcel ^R , washed with dichloromethane, and the filtrate was concentrated under reduced pressure to give 3.85 g of a solid which was triturated in 50 ml of heptane and 10 ml. ml diethyl ether. Solid proportion

107 was filtered, dried and purified by flash chromatography, eluting with dichloromethane / acetone (90:10) to give 1.6 g of diethyl 4- (1-pyrrolidinyl) benzylacetamidomalonate as a white solid, m.p. at 132 ° C.

40-3: Diethyl 4- (1-pyrrolyl) benzylacetamidomalonan

4.6 g of diethyl 4-aminobenzylacetamidomalonan in 104 ml of acetic acid are introduced into a three-necked flask maintained under nitrogen. 7.02 g of sodium acetate are added, followed by 1.87 ml of 2,5-dimethoxytetrahydrofuran. The reaction mixture is heated to 65 ° C and maintained at this temperature for one hour and 15 minutes, then extracted with 100 ml of dichloromethane and 100 ml of distilled water. The aqueous phase is decanted and then washed with 100 ml of dichloromethane. The organic phases are combined, washed with 1.00 ml of water and then with 100 ml of saturated aqueous sodium chloride solution, decanted and then dried over magnesium sulphate, then filtered and evaporated to dryness under reduced pressure (50 kPa) to give 6.2 g. The solid was purified by flash chromatography using a 75/25 mixture of dichloromethane and acetone to give 3.57 g of diethyl 4- (1-pyrrolyl) benzylacetamidomalonate as a beige solid which was purified by flash chromatography. melting at 110 ° C.

Example of phenylalanine derivatives by method H

41-1: (R, S) -4-Ethylthiomethylphenylalanine

300 ml of anhydrous methanol are introduced into a three-necked flask, kept under a nitrogen atmosphere, then 1.72 g of sodium methoxide are added while stirring, followed by 5.55 ml of ethyl mercaptan. The solvent was concentrated under reduced pressure at 40 ° C to give 8.5 g of ethyl mercaptan sodium which was dissolved in 100 ml of anhydrous tetrahydrofuran. 3.6 g of (R, S) -4108 are added at room temperature

-chloromethylphenylalanine, then the mixture is heated at reflux for 10 hours. The solvent was evaporated under reduced pressure at 40 ° C and the residue was taken up in 100 ml of distilled water. The cloudy solution obtained is acidified with 5 ml of acetic acid. The precipitate formed is filtered off, washed with distilled water and then dried under reduced pressure at 60 ° C to give 3.6 g of a solid which is purified by flash chromatography, eluting with a mixture of ethyl acetate, acetic acid and water by volume. ratio 60:12:10. There was thus obtained (R, S) -4-ethylthiomethylphenylalanine (256 mg) as a white solid which melted at 251 ° C.

(R, S) -4-chloromethylphenylalanine can be obtained analogously to (S) -4-chloromethylphenylalanine according to: R.Gonzalez-Muniz,

F.Cornille, F.Bergeron, D. Ficheux, J.Pothier, C. Duriex and B. Rogues, Int.J.Pept.Protein.Res., 1991, 37 (41), 331-340.

Example 42

Preparation of Phenylalanine Derivatives by Method I

42-1: (S) -4-O- (2-Chloroethyl) tyrosine hydrochloride

5 g of (s) -N-tert-butoxycarbonyl-4-O- (2-chloroethyl) tyrosine in solution in 50 ml of dioxane saturated with hydrochloric acid are introduced into a round-bottomed flask. After stirring for 28 hours, the mixture was concentrated to dryness under reduced pressure. The residue is taken up in 50 ml of ether, stirred and then filtered. The solid is washed twice with 25 ml of diethyl ether, dried under reduced pressure to give 1.58 g of (S) -4-O- (2-chloroethyl) tyrosine as a white solid, m.p. .

109

42-2: (S) -N-tert-Butoxycarbonyl-4- [2- (2-chloroethyl) tyrosine

4 g of (S) -N-tert-butoxycarbonyltyrosine in solution in 140 ml of dimethylformamide were introduced into a three-necked flask under a nitrogen atmosphere. 4.8 g of 50% sodium hydride in oil are added slowly by means of a spatula. After 2 hours at room temperature, 16.87 g of 1-tosyl-2-chloroethanol. After stirring for 2 hours, 2.4 g of 50% sodium hydride in oil and 8.4 ml of an additional portion of 1-tosyl-2-chloroethanol were added. The same operation is performed after 24 h and stirring is continued for another 24 hours. The reaction is quenched by the addition of 100 ml of distilled water and the reaction mixture is concentrated to dryness under reduced pressure. The residue is taken up in 100 ml of distilled water and extracted three times with 100 ml of ethyl acetate. The aqueous phase is decanted, acidified with 50 ml of 1N hydrochloric acid until pH = 3 and the product is extracted three times with 100 ml of ethyl acetate. The organic phases are combined, washed twice with 50 ml of water, decanted, dried over magnesium sulphate, filtered and then concentrated to dryness under reduced pressure to give 13.51 g of (S) -N-tert-butoxycarbonyl-4-O- ( 2-chloroethyl) tyrosine as a brown oil (Rf = 0.5, 70:20:10 toluene / methanol / diethylamine) which was used as such in the next step.

110

Table V

Microorganisms antibiotics mushrooms Micromonosoora so. vernamycin STREPTOMYCES S.alborectus virginiamycin S..conganensis (ATCC13528) S.diastaticus F1370 A Plauracíny. streptogramins S. crraminof asciens streptogramins S.crriseus ÍNRRL2426) Viridogriséin (Etamycin) S. crriseovir idus Griseoviridln S.crriseoviridus ÍFERMP3562) Néoviridogriséiny S.íavendulae etamycin S.lo'idensis IATCC114151 S.mitakaensis IATCC152971 S.olivaceus IATCC12019) vernamycin Micamycins Synergistins (PA114 A, B) Osteogrycins S.ostréoariseus (ATCC27455) S. pristinaespiralis (ATCC254861) pristinamycin S.virainiae (ÄTCC13161) Virginiamycins (Staphylomycins) actinomycetes A.Auranticolor (ATCC31011) Plauracíny A.azureus (ATCC31157) Plauracíny A.daahestanicus etamycin A.phiippinens i s A-2315 A, B, C Actinoplanes sd. ÍATCC33002) A15104 Actinoplanes sd. A17002 A, B, C, F Actinomadura flava Madumycíny

111

Abbreviations used:

DNA deoxyribonucleic acid

AMP adenosine-5'-monophosphate dCTP deoxycytosine-5'-triphosphate

DMPAPA 4-dimethylamino-L-phenylalanine

HT7 Hickey-Tresner solid culture medium

3-HPA 3-hydroxypicolinic acid

IPTG isopropyl-β-D-thiogalactopyranoside kb kilobase

LB Luria culture medium (enriched growth medium for Escherichia coli

MMPAPA 4-methylamino-L-phenylalanine

PAPA 4-amino-L-phenylalanine

PEG polyethylene glycol

PI pristinamycin I

Saw pristinamycin II pb base pair

SAM S-adenosylmethionine

TE buffer or 10 mM Tris-HCl, 1 mM EDTA, pH 7.5

Tris amino-2-hydroxymethyl-2-propane-1,3-diol

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside

YEME culture medium based on yeast extract and malt extract (enriched growth medium for Streptomyces).

Bibliography:

- Bibb M. J., Findlay P. R. and Johnson M. W. (1984) Gene, 30: 157-166.

- Bibb M. J., Janssen G. R., and WardJ. M. (1985) Gene 38: 215-226.

- Cocito C. G. (1979) Microbiol Rev., 43: 145-198.

- Cocito C.G. (1983) In Antibiotics, 6: (Ed. F. E. Hahn), 296-332.

- Dessen P. C., Fondrat C., Valencien C. and Mugnier C. (1990) Comp. Appl. in Biosciences, 6: 355-356.

- Di Giambattista M., Chinali G. and Cocito C. G. (1989) J. Antim. Chemother., 24: 485-507.

- Gibson TJ. (1984) Ph.D. thesis, Cambridge University, England.

Hillemann D., Piilher A. and Wohlleben W. (1991) NucL Acids Res., 19: 727-731.

112

Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Scrempf, H. (1985).

Hudson G. S. and Davidson B. E. (1984) J. MoL Biol., 180: 1023-1051.

Kuhstoss S., Richardson M.A., and Rao R. N. (1991) Gene 97: 143-146.

- Maniatis T., Fritsh E. F. and Sambrook J. (1989) Molecular cioning: a laboratory manual. Cold Spring Harbor, N.Y.,

Messing J., Crea R. and Seeburg P. H. (1981) Nucleic Acids Res., 9: 309.

Molinero A.A., Kingston D, G.I., and Reed J.W. (1989) J. Nat. Prod., 52: 99108.

Omer C. A. Lenstra R., Litle P. J., Dean J., Tepperman J. M., Leto K. J., Romesser J. A., and O'Keefe D. P. (1990) J. Bact. 172: 3335-3345.

- Reed J.W., Purvis M.B., Kingston D.G. I, Biot A., and Gosselé F. (1989) J. Org. Chem. 54: 1161-1165.

Staden R. and McLachlan A. D. (1982) Nucleic Acids Res., 10: 141-156.

Schindler, U., Sans, N., and Schroder, J. (1989) J. Bact. 171: 847-854.

- Thorson J.S .; Lo S.F. and Liu H-W. (1993) J. Am. Chem. Soc. 115: 6993-6994.

- Videau, D. (1982) Path. Biol., 30: 529-534.

Sequence list

Information for SEQ ID No. 1:

sequence characteristics: length: 2888 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical not antisense: not origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID NO: 1:

20 30 40 50 60

CTGCAGTTCC CCGGGGCCAC CGTGCTCAGC TCCTCACCCG AACGGTTCCT GCGCATCGGC

80 90 100 Π0 120

GCGGACGGCT GGGCGGAGTC CAAACCCATC AAGGGCACCC GCCCCCGCGG CGCCGGCCCC

130 140 150 160 170 180

GCCCAGGACG CCGCCGTCAA GGCCTCCCTC GCCGCGGCCG AGAAGGACCG CAGGGAGAAC

113

190 CTGATGATCG 200 TCGACCTGGT 210 CCGCAACGAC 220 CTCGGCCAGG 230 TCTGCGACAT 240 CGGCTCCGTC 250 CACGTACCGG 260 GCCTGTTCGA 270 GGTGGAGACC 280 TACGCCACCG 290 TCCACCAGCT 300 CGTCAGCACG 310 GTCCGCGGCC 320 GCCTGGGGGC 330 CGACGTCTCC 340 CGCCCCCGCG 350 CGGTACGGGC 360 CGCCTTCCCC 370 380 390 GCCCAAGGTC 400 CGCACCATGC 410 AGTTCATCGA 420 CCGGCTCGAG 430 AAGGGCCCGC 440 GCGGCGTGTA 450 CTCGGGCGCG 460 CTGGGCTACT 470 TCGCCCTCAG 480 CGGCGCGGCC 490 GACCTCAGGA 500 TCGTCATCCG 510 CACCATCGTC 520 GCCACCGAGG 530 AGGCCGCCAC 540 CATCGGCGTG 550 GGCGGCGCCG 560 TCGTCGCCCT 570 GTCCGACCCC 580 GACGACGAGG 590 TCCGCGAAAT 600 GCTCCTCAAG 610 GCGCAGACCA 620 CCCTCGCCGC 630 CCTGCGCCAG 640 GCACACGCGG 650 GCGCCACCGC 660 CTCGGACCGT 670 GAACTCCTGG 630 CCGGCAGCCT 690 GCGGTGACCC 700 ACCCACCGCC 710 CCACCCCGGC 720 CACCGCAACC 730 CCGGCTCACC 740 CCCGGGGCGG 750 CCGCGCGCGG 760 TGGCGCCCGG 770 CGGCCGACCC 780 GGCGACGGGT 790 CCGCTCGCGG 800 ACCGuGTGAC 810 GGACCCGGCG 820 GCGuGGCCGG 830 CGGCGGGCCG 840 GGACGTGGGC 850 CGGGACGTGG 860 GCCCGGCGTC 870 CCCGGCGACC 880 GGCACGGCGG 890 CGGGCCCGGA 900 CGTGGGCCCG 910 GCGTGCCCGG 920 CGACCGGCAC 930 GGTGGCGGGG 940 950 GACGGTCAGT 960 GCAGGGCGGT 970 GAACATCCGC 980 GCGCACAGCC 990 GTTCCAGCTC 1000 CGCGCCGTGC 1010 TCGCCCAGCA 1020 CACCGCGCAG 1030 TTCGGCGAAC 1040 AGGGCGGCGA 1050 ACGTCTCCTC 1060 GTCGCCCCTC 1070 TCGACGGCCT 1080 GCCCCAGCCG 1090 CACCAGGCCG 1100 CGGCCCAGCG 1110 CCTGCCGCGC 1120 GGCCGGCGCG 1130 CCGGGGTTGG 1140 CGGCCTGGAT 1150 GTCGAAATAC 11 60 ACCTCCGGGG 1170 TCCCGCCGGC 1180 GATCCGGGCC 1190 AGCAGCGCCA 1200 GCATCGCCAG 1210 ATGCGGCGGC 1220 GGGGCACTGT 1230 CCCGCAGCGC 1240 CCCCACGTCC 1250 ACCGACAGCT 1260 CACCCAGGCC 1270 CAGCCCGAAG 1230 GCCAGCACCG 1290 CGGCATGCGT 1300 GGCGGCCTGC 1310 TGCGCGGCGG 1320 TCAGCTCGTC 1330 GTGCCGCCGC 1340 GCCGGCATCT 1350 CCACCACCCG 1360 GGCCCCCCAC 1370 CCGGCCACCA 1380 GCTCCACCAG 1390 GGCCCGCACA 1400 CCGGGCCCGT 1410 CGGTGACCAC 1420 CACCGCCGCC 1430 ACCGGCCGCC 1440 CCTGAAGACC 1450 CAGCGAGGGG 1460 GCGAACATCG 1470 GGTTCAGCCC 1480 CACCGCCTGC 1490 AGCCCCGGCG 1500 CCGCCTCACG 1510 CAGCCGCCCG 1520 GCGATCCGGC 1530 TCTTGACCGA 1540 CAAGGTGTCC 1550 GCGAGCACCG 1560 CACCGGGCCG 1570 CATCACCCCC 1580 GCCAGCACCT 1590 CCACCGCCTC 1600 CCACGCCACC 1610 GGCTCCGGCA 1620 CCGCCAGCAC

114

1630 CACCACGTCC 1640 GCCGCCGCCA 1650 GCGCCGCGAC 1660 CGCCTCCGGC 1670 CCCGGCCGCC 1680 GCACATCACC 1690 1700 1710 1720 1730 1740 GGCCACCACC CGCACCCCGT CCGCCGCACC GGCCCCGGCC ACGTCCAGCC AGGTCACCGC 1750 CACCCCCGAA 1760 CGCACCAGCC 1770 AGTGGCTGAA 1780 CATGCGGCCC 1790 ACCGCACCGG 1800 CCCCGCCCAC 1810 CACCACACAA 1820 CGCCCGAACA 1830 CCGAACCACC 1840 CCTCATCCGC 1850 GTTCCCGATC 1860 CCCCCGGTAC 1370 GGAGGAAGAA 1880 CCATGACCCC 1890 GCCCGCCATC 1900 CCCGCCGCCC 1910 CGCCCGCCAC 1920 ČGGGCCCGCC 1 930 CCCGCCACGG 1940 ACCCCCTCGA 1950 CGCGCTGCGC 1960 GCCCGCCTGG 1,970 ACGCCGCGGA 1980 CGCCGCCCTG 1990 CTGGACGCCG 2000 TCCGCACACG 2010 CCTGGACATC 2020 TGCCTGCGCA 2030 TCGGCGAGTA 2040 CAAGCGCCTC 2050 CACCAGGTGC 2060 CGATGATGCA 2070 GCCCCACCGG 2080 ATCGCCCAGG 2090 TCCACGCCAA 2100 CGCCGCCCGC 2110 TACGCCGCCG 2120 ACCACGGCAT 2130 CGACCCCGCC 2140 TTCCTGCGCA 2150 CCCTGTACGA 2160 CACGATCATC 21 70 ACCGAGACCT 2180 GCCGCCTCGA 2190 GGACGAGTGG 2200 ATCGCCTCCG 2210 GCGGCGCCCC 2220 CGTCCCCACG 2230 CCCGTGCACG 2240 CGTCCGCGTC 2250 CGCGCGGGGG 2260 GCCGTGTCGT 2270 GACCGCCGCC 2280 GCACCCACCC 2290 TCGCCCAGGC 2300 GCTGGACGAG 2310 GCCACCGGGC 2320 AGCTGACCGG 2330 CGCCGGGATC 2340 ACCGCCGACG 2350 CCGCCCGGGC 2360 CGACACCCGG 2370 CTGCTGGCCG 2380 CCCACGCCTG 2390 CCAGGTCGCC 2400 CCGGGGGACC 2410 TCGACACCTG 2420 CCTGGCCGGC 2430 CCGGTGCCGC 2440 CCCGGTTCTG 2450 GCACTACGTC 2460 CGGCGCCGTC 2470 TGACCCGCGA 2480 ACCCGCCGAA 2490 CGCATCGTCG 2500 GCCACGCCTA 2510 CTTCATGGGC 2520 CACCGCTTCG 2530 ACCTGGCCCC 2540 CGGCGTCTTC 2550 GTCCCCAAAC 2560 CCGAGACCGA 2570 GGAGATCACC 2530 CGGGACGCCA 2590 TCGCCCGCCT 2600 GGAGGCCCTC 2610 GTCCGCCGCG 2620 GCACCACCGC 2630 ACCCCTGGTC 2640 GTCGACCTGT 2650 GCGCCGGACC 2660 GGGCACCATG 2670 GCCGTCACCC 2680 TGGCCCGCCA 2690 CGTACCGGCC 2700 GCCCGCGTCC 2710 TGGGCATCGA 2720 ACTCTCCCAG 2730 GCCGCCGCCC 2740 GCGCCGCCCG 2750 GCGCAACGCC 2760 CGCGGCACCG 2770 GCGCCCGCAT 2780 CGTGCAGGGC 2790 GACGCCCGCG 2800 ACGCCTTCCC 2810 CGAACTGAGC 2820 GGCACCGTCG 2830 ACCTCGTCGT 2840 CACCAACCCG 2850 CCCTACATCC 2860 CCATCGGACT 2870 GCGCACCTCC 2880 GCACCCGAAG

TGCTCGAG

115

Information for SEQ ID No. 2:

sequence characteristics: length: 888 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical not antisense: not origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID No 2:

ATG AGG GGT GGT TCG GTG TTC GGG CGT TGT GTG GTG GTG GGC GGG GCC GGT G ^ u? 54 Met Arg Gly Gly Ser wall Phe Gly Arg Cys wall wall wall Gly Gly Ala Gly Ala ia GTG GGC CGC. ATG AGs. CAC TGG CTG GTG CGT TCG GGG GTG GCG GTG ACC m / - / - X 108 wall Gly Arg Met Phe Ser His Trp Leu wall Arg Ser Gly wall Ala wall Thr Trp 36 CTG GAC GTG GCC GGG GCC GGT GCG GCG GAC GGG GTG CGG GTG GTG GCC GGT GAT 1 62 Leu Asp wall Ala Gly Ala Gly Ala Ala Asp Gly wall Arg wall wall Ala Gly Asp 54 GTG CGG CGG CCG GGG CCG GAG GCG GTC GCG GCG CTG GCG GCG GCG GAC GTG GTG 21 6 wall Arg Arg for Gly for Glu Ala wall Ala Ala Leu Ala Ala Ala Asp wall wall 72 GTG CTG GCG GTG CCG GAG CCG GTG GCG TGG GAG GCG GTG GAG GTG CTG GCG GGG 270 wall Leu Ala wall for Glu for wall Ala Trp Glu Ala wall Glu wall Leu Ala Gly 90 GTG ATG CGG CCC GGT GCG GTG CTC GCG GAC ACC TTG TCG GTC AAG AGC CGG ATC 324 wall Met Arg for Gly Ala wall Leu Ala Asp Thr Leu Ser wall Lys Ser Arg Ile 108 GCC GGG CGG CTG CGT GAG GCG GCG CCG GGG CTG CAG GCG GTG GGG CTG AAC CCG 378 Ala Gly Arg Leu Arg Glu Ala Ala for Gly Leu gin Ala wall Gly Leu same time for 126

ATG Met TTC Phe GCC Ala CCC for TCG Ser CTG Leu GGT Gly CTT Leu CAG gin GGG Gly CGG Arg CCG for GTG wall GCG GCG Ala Ala GTG wall GTG wall GTC wall 432 1 44 ACC Thr GAC Asp GGG Gly CCC for GGT Gly GTG wall CGG Arg GCC Ala CTG Leu GTG wall GAG Glu CTG Leu GTG wall GCC GGG Ala Gly TGG Trp GGG Gly GCC Ala 486 1 62 CGG Arg GTG wall GTG wall GAG Glu ATG Met CCG for GCG Ala CGG Arg CGG Arg CAC His GAC Asp GAG Glu CTG Leu ACC GCC Thr Ala GCG Ala CAG gin CAG gin 540 1 80 GCC Ala GCC Ala ACG Thr CAT His GCC Ala GCG Ala GTG wall CTG Leu GCC Ala TTC Phe GGG Gly CTG Leu GGC Gly CTG GGT Leu Gly GAG Glu CTG Leu TCG Ser 594 198 GTG wall GAC Asp GTG wall GGG Gly GCG Ala CTG Leu CGG Arg GAC Asp AGT Ser GCC Ala CCG for CCG for CCG for CAT CTG His Leu GCG Ala ATG Met CTG Leu 648 216

GCG CTG CTG GCC CGG ATC GCC GGC GGG ACG CCG GAG GTG TAT TTC GAC ATC CAG 702 Ala Leu Leu Ala Arg Ala Ala Gly Gly Thr Pro Glu Val Tyr Phe Asp Gin 234

116

GCC GCC AAC CCC GGC GCG CGG GCC GCG CGG CAG GCG CTG GGC CGC GGC CTG GTG 756 Ala Ala same time for Gly Ala for Ala Ala Arg gin Ala Leu Gly Arg Gly Leu wall 252 CGG CTG GGG CAG GCC GTC GAG AGG GGC GAC GAG GAG ACG TTC GCC GCC CTG TTC 810 Arg Leu Gly gin Ala wall Glu Arg Gly Asp Glu Glu Thr Phe Ala Ala Leu Phe 270 GCC GAA CTG CGC GGT GTG CTG GGC GAG CAC GGC GCG GAG CTG GAA CGG CTG TGC 864 Ala Glu Leu Arg Gly wall Leu Gly Glu HÚS Gly Ala Glu Leu Glu Arg Leu Cvs 283 GCG CGG ATG rpqiQ ACC GGC CTG CAC 888 Ala Arg Mer Phe Thr Ala Leu His 296

SEQ ID NO. 3:

sequence characteristics: length: 387 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID No 3:

ATG ACC CCG CCC GCC ATC CCC GCC GCC CCG CCC GCC ACC GGG CCC GCC CCC GCC 54 Met Thr for for Ala Ile for Ala Ala for for Ala Thr Gly for Ala Ala Ala 18 ACC GAC CCC CTC GAC GCG CTG CGC GCC CGC CTG GAC GCC GCG GAC GCC GCC CTG 108 Thr Asp for Leu Asp Ala Leu Arg Ala Arg Leu Asp Ala Ala Asp Ala Ala Leu 36 CTG GAC GCC GTC CGC ACA CGC CTG GAC ATC TGC CTG CGC ATC GGC GAG TAC AAG 162 Leu Asp Ala wall Arg Thr Arg Leu Asp Ile Cys Leu Arg Ile Gly Glu Ty r Lys 54 CGC CTC CAC CAG GTG CCG ATG ATG CAG CCC CAC CGG ATC GCC CAG GTC CAC GCC 216 Arg Leu His gin wall for Met Met gin for His Arg Ile Ala gin wall His Ala 72

AAC GCC GCC CGC TAC GCC GCC GAC CAC GGC ATC GAC CCC GCC TTC CTG CGC ACC 270 same time Ala Ala Arg Tyr Ala Ala Asp His Gly Ile Asp for Ala Phe Leu Arg Thr 90 CTG TAC GAC ACG ATC ATC ACC GAG ACC TGC CGC CTC GAG GAC GAG TGG ATC GCC 324 Leu Tyr Asp Thr Ile Ile Thr Glu Thr Cys Arg Leu Glu Asp Glu Trp Ile Ala 108 TCC GGC GGC GCC CCC GTC CCC ACG CCC GTG CAC GCG TCC GCG TCC GCG CGG GGG 378 Ser Gly Gly Ala for wall for Thr for wall His Ala Ser Ala Ser Ala Arg Gly 126

GCC GTG TCG Ala Val Ser

387

129

117

Information for SEQ ID No. 4:

sequence characteristics: length: 4496 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID No 4:

10 CTCGAGCAGG 20 TGCCCCACCT 30 CGGCGGCACG 40 GTGCGCGGGC 50 AGCGCGAACA 60 CCGGCAGCGC 70 GCCCAGACGG 80 AACAGGGCGA 90 AGCACACCGC 100 GACGAACTCG 110 GGCGTGTTCG 1 20 GCAGCTGCAC 1 30 CAGCACCCGC 1 40 TCGCCGGCGC 150 CGATCCCGCG 1 60 CGCCGGGAAC 170 CCCGCCGCCA 180 GCCGGTCGCA 1 90 CCAGCGGTCC 200 AGGGCACGGT 210 AGGTGACACG 220 GGAGGACCCG 230 TCCGCGGCGA 240 CCAGCGCCTC 250 CCGCTCGCCG 260 TACTGCTCCG 270 CCCAGCGGCC 280 CAGCAGCATG 290 CCCAGCGGCT 300 CGCCCCGCCA 310 GTAGCCGGCC 320 GCCCGGTACT 330 TCGCGGCCAC 340 ATCCTCGGGC 350 CAGGGAACGC 360 _ATCCGT ccag 370 380 390 400 410 420 CATCGTTGGT CCTTTCCGGC TTCGTCCTCG CGTCGCGCCC agtgtcggca GCGCCGTTGA 430 CACGCCGCTG 440 atgcgccgcg 450 CCCGCGCGCC 460 GCCGCTCCGT 470 CAGGAGCCGA 480 TCAGGGCGGC 490 GTCAGCCGGG 500 ccggacagga 510 TGCCGCCCAC 520 GGGGCCCGGC 530 ACACCGGGCC 540 GCGGCGACAG 550 CGGGCCGGCG 560 ACCGGCAGGC 570 CGACACCACG 580 CACGGACGAG 590 AAGAAACAAC 600 ÁCAAGGGGAG 610 CACCCGATGG 620 AGACCTGGGT 630 CCTGGGCCGG 640 CGCGACGTCG 650 CCGAGGTGGT 660 GGCCGCCGTC 670 GGCCGCGACG 680 AACTCATGCG 690 CCGCATCATC 700 GACCGCCTCA 710 CCGGCGGACT 720 GGCCGAGATC 730 GGCCGCGGCG 740 AGCGGCACCT 750 GTCCCCGCTG 760 CGCGGCGGAC 770 TGGAACGCAG 780 CGAACCCGTG 790 CCCGGCATCT 800 GGGAATGGAT 810 GCCGCACCGC 820 GAACCCGGCG 830 ACCACATCAC 840 CCTCAAGACC 850 GTCGGCTACA 860 CCCCCGCCAA 870 CCCCGGCCGC 880 TTCGGCCTGC 890 CGACCATCCT 900 GGGCACCGTC 910 GCCCGCTACG 920 ACGACACCAC 930 CGGCGCCCTG 940 ACCGCCCTGA 950 TGGACGGCGT 960 GCTGCTCACC

118

970 980 990 GCCCTGCGCA CCGGCGCCGC CTCCGCCGTC 1030 1040 1050 CACACCCTGG GACTGATCGG CACCGGCGCC 1090 1100 1110 CTGGTACTGC CCCTGCAACG GGCCCTGGTG 1150 1160 1170 GCGCCGCGTT CACCGGCGTC 1 210 1220 1230 G ^ -CGGCGAGvj CCGACGTCAT CTCCACCGCC 1270 1230 1290 CTGCCCGACA CCGGCGTCCG CGAGCACCTG 1330 1340 1350 GG> _AAGACGG AACTGCCGCT CGGCCTGCTC

1000 1010 1020 GCCTCCCGCC TGCTGGCCCG CCCCGACAGC 1060 1070 1080 CAGGCCGTCA CCCAACTGCA CGCCCTGTCC 1 1 20 1 1 30 1 1 40 TGGGACACCG ACCCCGCCCA CCGGGAAAGC 1180 1190 1200 AGCGTCGAGA TCGCCGAGCC CGCCCGGATC 1240 1250 1260 ACGTCGGTAG CCGTCGGCCA GGGCCCGGTC 1300 1310 1320 CACATCAACG GGACCTCGTC 1 360 1370 1380 GAGCGGGCGT TCGTCACCGC CGACCACCCC

1 390 1 400 1410 1 420 1430 GAGCAG ^ jCGC TGCGCGAGGG CGAGTGCCAG CAACTCTCCG CCGACCGGCT 1450 1460 1470 1 480 1490 CTGGCCCACC TGTGCGGCGA CCCGGGGGCC GCCGCCGGCC GGCAGGACAC 1510 1520 1530 1540 1550 TTCGACTCCA CCGGCTTCGC CTTCGAGGAC GCCCTGGCGA TGGAAGTGTT 1570 1580 1590 1600 1610 GCCGCCGAAC GGGACCTGGG CATCCGGGTG GGCATCGAAC ACCACCCCGG 1 630 1 640 1650 1660 1670 GACCCCTACG CCCTCCAGCC CCTGCCCCTG CCCCTGGCCG CCCCCGCCCA 1690 1700 1710 1720 1730 CCTTTTTTCG GGACCCCCGG TCTTTTTCGA GACCCCCGCC CGGCCGGCCC 1750 1760 1770 1780 1790 CCGCCGGCCC CCATGCCCGG CCGGGCCGGG GCACCCACGA CGCCCTCGCG 1810 1820 1830 1840 1850 TGCCCCCCAC CCCCCGGCCC ACCACCGACG ACGGCGGCCG TGAACTGCTC 1870 1880 1890 1900 1910 GCGAGATGCG CCACCACCAC CCCGTCCACG AGGACGAATA CGGTGCCTTC 1930 1940 1950 1960 1970 GGCACGCCGA CGTCCTCACC GTCGCCTCCG ACCCCGGCGT CTACTCCTCC 1990 2000 2010 2020 2030 GGCTACGGCC CGGCTCCCAG GCGTTGAGCG AACAGATCCT GTCGGTCATC 2050 2060 2070 2080 2090 TGCACCGCAC CCTGCGCCGC CTGGTCAGCC AGGCCTTCAC CCCCCGCACC 2110 2120 2130 2140 2150 TCGAACCACG CGTCACCGAA CTGGCCGGGC 'AACTGCTCGA CGCCGTCGAC 2170 2180 2190 2200 2210 TCGACCTCGT CGCCGACTTC GCCTACCCGC TGCCCGTGAT CGTGATCGCC 2230 2240 22S0 2260 2270 GCGTGCCGCC CGCCGACCGC ACCCTGTTCC GCTCCTGGTC CGACCGGATG 2290 2300 2310 2320 2330 AGGTCGCCGA C.CCGGCGGAC ATGCAGTTCG GCGACGACGC CGACGAGGAC 2350 2360 2370 2380 2390 TCGTCAAAGA ACCCATGCGC GCCATGCACG • CCTACCTCCA CGACCACGTC

1440

CGGCCCGCAG

1500

CCTGAGGGTC

1560

CCTCGAGGGG

1620

1680

CTGACCCCCC

1740

GGCCCTCCTC

1800

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

19

2410 GCGCCCGCCC 2420 CGCGAACGAC 2430 CTGATCTCCG 2440 CACTCGTCGC 2450 CGCCCGCGTG 2460 GAGGGCGAAC 2470 GACTCACCGA 2480 CGAGCAGATC 2490 GTCGAATTCG 2500 GGGCGCTGCT 2510 GCTGATGGCC 2520 GGCCACGTCT 2530 CCACCTCCAT 2540 GCTGCTCGGC 2550 AACACCGTGC 2560 TGTGCCTGAA 2570 GGACCACCCC 2530 CGGGCCGAGG 2590 CCGCCGCCCG 2600 CGCCGACCGG 2610 TCCCTGATCC 2620 CCGCCCTGAT 2630 CGAAGAAGTA 2640 CTGCGGCTGC 2650 GGCCGCCGAT 2660 CACCGTCATG 2670 GGCCGCGTCA 2680 CCACCAAGGA 2690 CACCGTCCTC 2700 GCCGGCACCA 2710 CCATCCCCGC 2720 CGGACGCATG 2730 GTCGTGCCCT 2740 CCCTGCTGTC 2750 CGCCAACCAC 2760 GACGAACAGG 2770 TCTTCACCGA 2780 CCCCGACCAC 2790 CTCGACCTCG 2800 CCCGCGAAGG 2810 CCGCCAGATC 2320 GCCTTCGGCC 2830 ACGGCATCCA 2840 CTACTGCCTG 2850 GGCGCCCCGC 2860 TCGCCCGCCT 2870 GGAGGGCCGC 2830 ATCGCCCTGG 2390 AAGCCCTCTT 2900 CGACCGATTC 2910 CCCGACTTCT 2920 CGCCCACCGA 2930 CGGCGCAAAA 2940 CTGCGCTACC 2950 ACCGv-GACGG 2960 actgttcggc 2970 GTCAAGAACC 2980 TGCCGCTGAC 2990 CGTACGGCGC 3000 GGCTGACACA 3010 GACAAGGGGG 3020 CCACCTGGTG 3030 CGCACCGTGC 3040 GAACCCTGCT 3050 GATCGACAAC 3060 TACGACTCGT 3070 TCACCTACAA 3080 CCTCTTCCAG 3090 ATGCTGGCCG 3100 AGGTGAACGG 3110 CGCCGCTCCG 3120 CTCGTCGTCC 3130 GCAACGACGA 3140 CACCCGCACC 3150 TGGCAGGCCC 3160 TGGCGCCGGG 3170 CGACTTCGAC 3180 AACGTCGTCG 3190 TCTCACCCGG 3200 CCCCGGCCAC 3210 CCCGCCACCG 3220 ACACCGACCT 3230 GGGCCTCAGC 3240 CGCCGGGTGA 3250 TCACCGAATG 3260 GGACCTGCCG 3270 CTGCTCGGGG 3280 TGTGCCTGGG 3290 CCACCAGGCC 3300 CTGTGCCTGC 3310 TCGCCGGCGC 3320 CGCCGTCGTC 3330 CACGCACCCG 3340 AACCCTTTCA 3350 CGGCCGCACC 3360 AGCGACATCC 3370 GCCACGACGG 3380 GCAGGGCCTG 3390 TTCGCGAACA 3400 TCCCCTCCCC 3410 GCTGACCGTG 3420 GTCCGCTACC 3430 ACTCGCTGAC 3440 CGTCCGGCAA 3450 CTGCCCGCCG 3460 ACCTGCGCGC 3470 CACCGCCCAC 3480 ACCGCCGACG 3490 ggcagctgat 3500 GGCCGTCGCC 3510 CACCGCCACC 3520 TGCCCCGCTT 3530 CGGCGTGCAG 3540 TTCCACCCCG 3550 AATCGATCAG 3560 CAGCGAACAC 3570 GGCCACCGGA 3580 TGCTCGCCAA 3590 CTTCCGCGAC 3600 CTGTCCCTGC 3610 GCGCGGCCGG 3620 CCACCGCCCC 3630 CCGCACACCG 3640 AACGCATACC 3650 CGCACCCGCA 3660 CCCGCCCCCG 3670 CCCCCGCCCC 3680 CGCACCGGCA 3690 CCGCCCGCGT 3700 CCGCGCCGGT 3710 GGGGGAGTAC 3720 CGGCTGCATG 3730 TGCGCGAGGT 3740 CGCCTGCGTG 3750 CCCGACGCGG 3760 ACGCCGCGTT 3770 CACCGCCCTG 3780 TTCGCCGACG 3790 CCCCGGCCCG 3800 gttctggctc 3810 GACAGCAGCC 3820 GCGTCGAGCC 3830 GGGCCTCGCC 3840 CGCTTCACCT

120

3850 TCCTCGGCGC 3860 CCCCGCCGGC 3870 CCGCTCGGCG 3880 AACAGATCAC 3890 CTACGACGTC 3900 G-CGACCGGG 3910 CCGTGCGCGT 3920 CAAGGACGGT 3930 TCAGGCGGCG 3940 AGACCCGCCG 3950 GCCCGGCACG 3960 CTCTTCGACC 3970 ACCTGGAACA 3980 CGAACTGGCC 3990 GCCCGCGCCC 4000 TGCCCGCCAC 4010 CGGCCTGCCC 4020 TTCGAGTTCA 4030 ACCTCGGCTA 4040 CGTCGGCTAC 4050 CTCGGCTACG 4060 AGACCAAG \ jC 4070 CGACAGCGGC 4080 GG \ _GAG \ JACGAL 4090 CCCACCGCGG 4100 CGAACTGCCC 4110 GACGGCGCCT 40 20 TCATGTTCGC 4130 CGACCGGATG 4140 CTCGCCCTCG 4150 ACCACGAACA 4160 GGGGCGGGCC 4170 TGGCTCCTGG 4180 CACTGAGCAG 4190 CACCCGACGG 4200 CCCGCCACCG 4210 CACCCGCCGC 4220 CGAACGCTGG 4230 CTCACCGACG 4240 CCGCCCGGAC 4250 CCTCGCCACC 42SG ACCGCCCCCC 4270 GCCCGCCCTT 4280 CACCCTGCTG 4290 CCCGACGACC 4300 AACTGCCCGC 4310 CCTGGACGTC 4320 CACTACCGCC 4330 ACAGCCTGCC 4340 CCGCTACCGG 4350 GAACTGGTCG 4360 AGGAATGCCG 4370 CCGCCTGATC 4380 ACCGACGGCG 4390 4400 4410 4420 4430 4440 AGACCTACGA GGTGTGCCTG ACGAACATGC TCCGGGTGCC CGGCCGGATC GACGCGCTCA 4450 CCGCCTACCG 4460 CGCCCTGCGC 4470 ACCGTCAGCC 4480 CCGCCCCCTA 4490 CGCCGCCTAC CTGCAG

Information for SEQ ID No. 5:

sequence characteristics: length: 1065 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: Streptomyces pristinaespiralis organism sequence description: SEQ ID No 5:

ATG GAG ACC TGG GTC CTG GGC CGG CGC GAC GTC GCC GAG GTG GTG GCC GCC GTC 5 4 Met Glu Thr Trp wall Leu Gly Arg Arg Asp wall Ala Glu wall wall Ala Ala wall 1 3 GGC CGC GAC GAA CTC ATG CGC CGC ATC ATC GAC CGC CTC ACC GGC GGA CTG GCC 108 Gly Arg Asp Glu Leu Met Arg Arg Ile Ile Asp Arg Leu Thr Gly Gly Leu Ala 36 GAG ATC GGC CGC GGC GAG CGG CAC CTG TCC CCG CTG CGC GGC GGA CTG GAA CGC 162 Glu Ile Gly Arg Gly Glu Arg His Leu Ser for Leu Arg Gly Gly Leu Glu Arg 54 AGC GAA CCC GTG CCC GGC ATC TGG GAA TGG ATG CCG CAC CGC GAA CCC GGC GAC 216 Ser Glu for wall for Gly Ile Trp Glu Trp Met for His Arg Glu for Gly Aso 72

121

CAC ATC ACC CTC AAG ACC GTC GGC TAC AGC CCC GCC AAC CCC GGC CGC TTC GGC 270 His Ile Thr Leu Lys Thr wall Gly Tyr Ser for Ala same time for Gly Arg Phe Gly 90 CTG CCG ACC ATC CTG GGC ACC GTC GCC CGC TAC GAC GAC ACC ACC GGC GCC CTG 324 Leu for Thr Ile Leu Gly Thr wall Ala Arg Tyr Asp Asp Thr Thr Gly Ala Leu 103 ACC GCC CTG ATG GAC GGC GTG CTG CTC ACC GCC CTG CGC ACC GGC GCC GCC TCC 373 ? JJR Ala Leu Met Asp Gly wall Leu Leu Thr Ala Leu Arg Thr Gly Ala Ala Ser 125 GCC GTC GCC TCC CCC CTG CTG GCC CGC CCC GAC AGC CAC ACC CTG GGA CTG ATC 432 A 2. a. wall Ala Ser Arg Leu Leu Ala Arg for Asp Ser His Thr Leu Gly Leu Ile 144 G ^ C ACC GGC GCC CAG GCC GTC ACC CAA CTG CAC GCC CTG TCC CTG GTA CTG CCC 436 Gly Thr Gly Ala gin Ala wall Thr gin Leu His Ala Leu Ser Leu wall Leu for 1 62 CTG CAA CGvj GGC CTG GTG TGG GAC ACC GAC CCC GCC CAC CGG GAA AGC TTC GCC 540 Leu gin Arg Ala Leu wall Trp Asp Thr Asp for Ala His Arg Glu Ser Phe Ala 180 CGG CGG GCC GCG TTC ACC GGC GTC AGC GTC GAG ATC GCC GAG CCC GCC CGG ATC 594 Arg Arg Ala Ala Phe Thr Gly wall Ser wall Glu Ile Ala Glu for Ala Arg Ile 198 GCC GCC GAG GCC GAC GTC ATC TCC ACC GCC ACC TCG GTA GCC GTC GGC CAG GGC 643 Ala Ala Glu Ala Asp wall Ile Ser Thr Ala Thr Ser wall Ala wall Gly gin Gly 21 6 CCG GTC CTG CCC GAC ACC GGC GTC CGC GAG CAC CTG CAC ATC AAC GCC GTC GGC 702 for wall Leu for Asp Thr Gly wall Arg Glu His Leu His Ile same time Ala wall Gly 234 GCG GAC CTC GTC GGC AAG ACG GAA CTG CCG CTC GGC CTG CTC GAG CGG GCG TTC 756 Ala Asp Leu wall Gly Lys Thr Glu Leu for Leu Gly Leu Leu Glu Arg Ala Phe 2S2 GTC ACC GCC GAC CAC CCC GAG CAG GCG CTG CGC GAG GGC GAG TGC CAG CAA CTC 810 wall Thr Ala Asp His for Glu gin Ala Leu Arg Glu Gly Glu Cys gin gin Leu 270 TCC GCC GAC CGG CTC GGC CCG CAG CTG GCC CAC CTG TGC GCC GAC CCG GCG GCC 864 Ser Ala Asp Arg Leu Gly for gin Leu Ala His Leu Cys Ala Asp for Ala Ala 288 GCG GCC GGC CGG CAG GAC ACC CTG AGC GTC TTC GAC TCC ACC GGC TTC GCC TTC 918 Ala Ala Gly Arg gin Asp Thr Leu Ser wall Phe Asp Ser Thr Gly Phe Ala Phe 306 GAG GAC GCC CTG GCG ATG GAA GTG TTC CTC GAG GCC GCC GCC GAA CGG GAC CTG 972 Glu Asp Ala Leu Ala Met Glu wall Phe Leu Glu Ala Ala Ala Glu Arg Asp Leu 324 GGC ATC CGG GTG GGC ATC GAA CAC CAC CCC GGC GAC GCC CTG GAC CCC TAC GCC 1026 Gly Ile Arg wall Gly Ile Glu His His for Gly Asp Ala Leu Asp for Tyr Ala 342 CTG CAG CCC CTG CCC CTG CCC CTG GCC GCC CCC GCC CAC 1065 Leu gin for Leu for Leu for Leu Ala Ala for Ala His 355

122

Information for SEQ ID No. 6:

sequence characteristics: length: 1194 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID No 6:

ATG CCC CCC ACC CCC CGG CCC ACC ACC GAC GAC GGC GGC CGT GAA CTG CTC GCC 54 Met for for Thr for Arg for Thr Thr Asp Asp Gly Gly Arg Glu Leu Leu Ala 18 TGG CTG CGC GAG ATG CGC CAC CAC CAC CCC GTC CAC GAG GAC GAA TAC GGT GCC 108 Trp Leu Arg Glu Met Arg His His His for wall His Glu Asp Glu Tyr Gly Ala 36 TTC CAC GTC TTC CGG CAC GCC GAC GTC CTC ACC GTC GCC TCC GAC CCC GGC GTC 1 62 Phe His wall Phe Arg HiS Ala Asp wall Leu Thr wall Ala Ser Asp for Gly wall 54 TAC TCC TCC CAG CTC AGC CGG CTA CGG CCC GGC TCC CAG GCG TTG AGC GAA CAG 216 Ty r Ser Ser gin Leu Ser Arg Leu Arg for Gly Ser gin Ala Leu Ser Glu gin 72 ATC CTG TCG GTC ATC GAC CCG CCG ATG CAC CGC ACC CTG CGC CGC CTG GTC AGC 270 Ile Leu Ser wall Ile Asp for for Met His Arg Thr Leu Arg Arg Leu wall Ser 90 CAG GCC TTC ACC CCC CGC ACC GTC GCC GAC CTC GAA CCA CGC GTC ACC GAA CTG 324 gin Ala Phe Thr for Arg Thr wall Ala Asp Leu Glu for Arg wall Thr Glu Leu 108 GCC GGG CAA CTG CTC GAC GCC GTC GAC GGC GAC ACG TTC GAC CTC GTC GCC GAC 378 Ala Gly gin Leu Leu Asp Ala wall Asp Gly Asp Thr Phe Asp Leu wall Ala Asp 1 26 TTC GCC TAC CCG CTG CCC GTG ATC GTG ATC GCC GAA CTC CTC GGC GTG CCG CCC 432 Phe Ala Tyr for Leu for wall Ile wall Ile Ala Glu Leu Leu Gly wall for for 144 GCC GAC CGC ACC CTG TTC CGC TCC TGG TCC GAC CGG ATG CTG CAG ATG CAG GTC 486 Ala Asp Arg 'Thiz * Leu Phe Arg Ser Trp Ser Asp Arg Met Leu gin Met gin wall 1 62 GCC GAC CCG GCG GAC ATG CAG TTC GGC GAC GAC GCC GAC GAG GAC TAC CAA CGC 540 Ala Asp for Ala Asp Met gin Phe Gly Asp Asp Ala Asp Glu Asp Tyr gin Arg ISO CTC GTC AAA GAA CCC ATG CGC GCC ATG CAC GCC TAC CTC CAC GAC CAC GTC ACC 594 Leu wall Lys Glu for Met Arg Ala Met His Ala Tyr Leu His Asp His wall Thr 1 98 GAC CGC CGC GCC CGC CCC GCG AAC GAC CTG ATC TCC GCA CTC GTC GCC GCC CGC 648 Asp Arg Arg Ala Arg for Ala same time Asp Leu Ile Ser Ala Leu wall Ala Ala Arg 216 GTG GAG GGC GAA CGA CTC ACC GAC GAG CAG ATC GTC GAA TTC GGG GCG CTG CTG 702 wall Glu Gly Glu Arg Leu Thr Asp Glu gin Ile wall Glu Phe Gly Ala Leu Leu '234 CTG ATG GCC GGC CAC GTC TCC ACC TCC ATG CTG CTC GGC AAC ACC GTG CTG TGC 756 Leu Met Ala Gly His wall Ser Thr Ser Met Leu Leu Gly same time Thr wall Leu Cvs 252

123

CTG AAG GAC CAC CCC CGG GCC GAG GCC GCC GCC CGC GCC GAC CGG TCC CTG ATC 810 Leu Lys Asp His for Arg Ala Glu Aia Ala Ala Arg Ala Asp Arg Ser Leu Ile 270 CCC GCC CTG »/ *, / * · Λ _ -w GAA GAA GTA CTG CGkj CTG CGG CCG CCG ATC ACC GTC ATG GCC 364 ? rs Ala Leu Press Giu Giu wall Leu Arg Leu Arg for for Ile Thr wall Met Ala 233 CGC GTC ACC ACC AAG GAC ACC GTC CTC GCC ACC ACC ATC CCC GCC GUA CG <_ 913 Arg wall Thr Lys Asp Thr wall Leu Ala Gly Thr Thr Ile for Ala Gly Arg 306 ATG GTC GTG CCC m s * r * CTG CTG i'r * GCC AAC CAC GAC GAA CAG GTC TTC ACC GAC 972 mec wall wall for Ser Leu Leu Ser Ala same time HLS Asp Glu gin wall Phe *HERE _ Asp 324 ccc GAC CAC 'T' / - GAC CTC GCC CGC GAA GGC CGC CAG ATC GCC TTC GGC CAC GGC 1025 for Asp USU Leu Asp Leu Ala Arg Glu Gly Arg gin Ile AUA Phe Gly Hl s Gly 342 ATC CAC TAC «- «J'— CTG GGC GCC CCG CTC GCC CGC CTG GAG GGC CGC ATC GCC CTG 1080 Ile Hl s Tyr Cys Leu Gly Ala for Leu Ala Arg Leu Glu Gly Arg Ile Ala Leu 360 GAA GCC CTC GAC CGA ccc GAC ŕTľpQ TCG CCC ACC GAC GGC GCA AAA CTG i 1 34 Glu Ala Leu Phe Asp nr g Phe for Asp Phe Ser for Thr Asp Gly Ala Lys Leu 373 CGC TAC CAC U «J v- GAC GGA CTG TTC GGC GTC AAG AAC CTG CCG CTG ACC GTA CGG 1 1 33 Arg Tyr His Arg Asp Gly Leu Phe Gly wall Lys same time Leu for Leu Thr wall Arg 395 CGC GGC i 1 94 Arg Gly 393

Information for SEQ ID No. 7:

sequence characteristics: length: 1561 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: Streptomyces pristinaespiralis organism sequence description: SEQ ID No 7:

10 20 30 40 50 60 AAGCTTCCCG ACCGGGTGGA GGTCGTCGAC GCGTTCCCGC TGACCGGCCT CAACAAGGTC 70 80 90 100 110 120 GACAAGAAGG CCCTGGCGGC CGACATCGCC GCCAAGACCG CCCCCACCCG CCCCACCACC 130 140 150 160 170 180 GCCGGCCACG GCCCGACCAC GGACGGCGAT ACGGCCGGTG GGGGTGGGTC CGCGGGCGGG 190 200 210 220 230 240 GTGACGGCCG CCGGTGGCGkj GCGGGAGGAG GCGGCGTGAG CGGGCCCGGG CCCGAGGGCG

124

250 GCTACCGGGT 260 GCCGTTCGCG 270 CGACGCGGTT 280 CGGTGGTGGG 290 CGAGGCGGAC 300 CTGGCGGCGC 310 TGGGCGAACT 320 GGTCCGCTCG 330 GGCCGGTCGC 340 TGACGTCGGG 350 GGTGTGGCGG 360 GAGCGGTTCG 370 AGGAACAGTT 380 CGCCCGCCTG 390 ACCGGCGCCC 400 GGCACGCGCT 410 CAGTGTCACC 420 AGCGGCACCG 430 TCGCGCTGGA 440 ACTGGCGGTG 450 CGGATGCTGG 460 ACCTGGCGCC 470 GGGCGACGAG 480 GTGATCGCCA 490 CCCCGCAGAC 500 GTTCCAGGCG 510 ACGGTGCAGC 520 CGCTGCTCGA 530 CCACGACGTG 540 CGGCTGCGGT 550 TCTGCGACAT 560 CGACCCGGAC 570 ACCCTCAACC 580 TCGACCCGGC 590 GGTGCTGGAG 600 ACGCTGATCA 610 CCGACCGCAC 620 CCGGGCGATC 630 CTGCTCGTCC 640 ACTACGGCGG 650 CAACCCGGCC 660 GACATGGACC 670 GCATCATGGC 680 CCTGGCCCGC 690 AAGCGCGGCA 700 TCATCGTCGT 710 CGAGGACAGC 720 GCGCACGCGC 730 TGGGCGCCGT 740 GTACCGGGGG 750 CGGCGGCCGG 760 GGGCACTGGC 770 GGACATCGGC 780 TGCTTCACTT 790 TCCACTCCAC 800 GAAGAACATC 810 ACCACCCTCG 820 GCGAGGGCGG 830 CATGATCACC 840 CTGTCGCGTG 850 ACGAGTGGGC 860 CCAGCGGGTG 870 GGACGTATCC 880 GCGACAACGA 890 GGCCGACGGC 900 GTGTACGCGG 910 CGCTGCCGGA 920 CTCCGCGCGG 930 GCGGGTGCTC 940 CGGCGCTGCT 950 GCCGTGGATG 960 AAGTTCGCGG 970 AGGGTGTGTA 980 CGGTCACCGG 990 GCGGTCGGGG 1000 TCCGCGGGGC 1010 GGGCACGAAC 1020 GCGACGATGT 1030 CGGAGGCGGC 1040 GGCGGCGGTG 1050 GGCGTGGTGC 1060 AACTGGCGTC 1070 GCTGGAGCGG 1080 TTCGTGGCCC 1090 GGCGCCGGAG 1100 CATCGCGCAG 1110 CGGCTGGACG 1120 AGGCCGTGGC 1130 CTCGGTGGCC 1140 GGCACCCGGC 1150 TGCACCGGGC 1160 GGCGGCGGAC 1170 AGTCTGCACG 1180 CCTACCACCT 1190 GTACACGTTC 1200 TTCCTCACCG 1210 GCGGCCGGCA 1220 GGTGCGGGAG 1230 CGGTTCGTGC 1240 GCGCCCTGGA 1250 CCGGCTGGGT 1260 GTGGAGGTCC 1270 AGTTGCGGTA 1280 CTTCCCGCTC 1290 CATCTGTCGC 1300 CCGAGTGGCG 1310 GCTGCGCGGC 1320 CACGGGCCGG 1330 GCGAGTGTCC 1340 GACGGCCGAA 1350 CGGGTCTGGT 1360 TCGAGGAGCA 1370 CATGAACCTG 1,380 CCGTGCCATC 1390 CCGGTCTGAG 1400 TGACGGCCAG 1410 GTCGACTACA 1420 TGGTCGAGGC 1430 GGTCACCCGC 1440 GCCCTGCACG 1450 AGGCCCACGG 1460 CACGGGGACG 1470 CGGGTGGCGG 1480 CCGGGCACCT 1490 GTGACACCGT 1500 CCGCATCCGG 1510 CCGGTGGTTT 1520 TCCAAGACCG 1530 AGGGAGAGGC 1540 AGGCGTATGC 1550 CGTTCATCGA 1560 AGTGAAGATC

125

Information for SEQ ID No. 8:

sequence characteristics: length: 1233 base pairs type: nucleic acid number of chains: double-stranded configuration: linear molecule type: DNAc hypothetical: no antisense: no origin: organism Streptomyces pristinaespiralis sequence description: SEQ ID No 8:

GTG CCG TTC GCG CGA CGC GGT TCG GTG GTG GGC GAG GCG GAC CTG GCG GCG CTG 54 wall for Phe Ala Arg Arg Gly Ser wall wall Gly Glu Ala Asp Leu Ala Ala Leu 13 GGC GAA CTG GTC CGC TCG GGC CGG TCG CTG ACG TCG GGG GTG TGG CGG GAG CGG 108 Gly Glu. Leu wall Arg Ser Gly Arg Ser Leu Thr Ser Gly wall Trp Arg Glu Arg 36 TTC GAG GAA 'GAG TTC GCC CGC CTG ACC GGC GCC CGG CAC GCG CTC AGT GTC ACC 162 Phe Glu Glu gin Phe Ala i r-r. Leu Thr Gly Ala Arg His Ala Leu Ser Val. Thr 54 AGC GGC ACC GTC GCG CTG GAA CTG GCG GTG CGG ATG CTG GAC CTG GCG CCG GGC 216 Ser Gly Thr wall Ala Leu Glu Leu Ala wall Arg Met Leu Asp Leu Ala for Gly 72 GAC GAG GTG ATC GCC ACC CCG QG ACG TTC CAG GCG ACG GTG CAG CCG CTG CTC 270 Asp Glu wall Ile Ala Thr for gin Thr Phe gin Ala Thr wall gin for Leu Leu 90 GAC CAC GAC GTG CGG CTG CGG TGC GAC ATC GAC CCG GAC ACC CTC AAC CTC 324 Asp His Asp wall Arg Leu Arg Phe Cys Asp Ile Asp for Asp Thr Leu same time Leu 108 GAC CCG GCG GTG CTG GAG ACG CTG ATC ACC GAC CGC ACC CGG GCG ATC CTG CTC 373 Asp for Ala wall Leu Glu Thr Leu Ile Thr Asp Arg Thr Arg Ala Ile Leu Leu 126 GTC CAC TAC GGC GGC AAC CCG GCC GAC ATG GAC CGC ATC ATG GCC CTG GCC CGC 432 wall His Tyr Gly Gly Temporarily. for Ala Asp Met Asp Arg Ile Met Ala Leu Ala Arg 144 AAG CGC GGC ATC ATC GTC GTC GAG GAC AGC GCG CAC GCG CTG GGC GCC GTG TAC 486 Lys Arg Gly Ile Ile wall wall Glu Asp Ser Ala His Ala Leu Gly Ala wall Tyr 162 CGG GGG CGG CGG CCG GGG GCA CTG GCG GAC ATC GGC TGC TTC ACT TTC CAC TCC 540 Arg Gly Arg Arg for Gly Ala Leu Ala Asp Ile Gly Cys Phe Thr Phe His Ser 180 ACG AAG AAC ATC ACC ACC CTC GGC GAG GGC GGC ATG ATC ACC CTG TCG CGT GAC 594 Thr Lys same time Ile Thr Thr Leu Gly Glu Gly Gly Met Ile Thr Leu Ser Arg Asp 198 GAG TGG GCC CAG CGG GTG GGA CGT ATC CGC GAC AAC GAG GCC GAC GGC GTG TAC 648 Glu Trp Ala gin Arg wall Gly Arg Ile Arg Asp same time Glu Ala Asp Gly wall Tyr 216 GCG GCG CTG CCG GAC TCC GCG CGG GCG GGT GCT CCG GCG CTG CTG CCG TGG ATG 702 Ala Ala Leu for Asp Ser Ala Arg Ala Gly Ala for Ala Leu Leu for Trp Met 234

126

AAG OWTlQ GCG GAG GGT GTG TAC GGT CAC CGG GCG GTC GGG GTC CGC GGG GCG GGC 756 Lys Phe Ala Glu Gly wall Tyr Gly His Arg Ala wall Gly wall Arg Gly Ala Gly 252 ACG AAC GCG ACG ATG TCG GAG GCG GCG GCG GCG GTG GGC GTG GTG CAA CTG GCG 810 Thr same time Ala Thr Met Ser Glu Ala Ala Ala Ala wall Gly wall wall gin Leu Ala 270 TCG CCG GAG CGG TTC GTG GCC CGG CGC CGG AGC ATC GCG CAG CGG CTG GAC GAG 864 Ser Leu Glu Arg Phe wall Ala Arg Arg Arg Ser Ile Ala gin Arg Leu Asp Glu 288 GCC GTG GCC TCG GTG GCC GUC ACC CGG CTG CAC CGG GCG GCG GCG GAC AGT CTG 918 Ala wall Ala Ser wall Ala Gly Thr Arg Leu His Arg Ala Ala Ala Asp Ser Leu 306 CAC GCC TAC CAC CTG TAC ACG TTC TTC CTC ACC GGC GGC CGG CAG GTG CGG GAG 972 His Ala Ty r His Leu Tyr Thr Phe Phe Leu Thr Gly Gly Arg gin wall Arg Glu 324 CG \ j GTG CGC GCC CTG GAC CGG CTG GGT GTG GAG GTC CAG TTG CGG TAC TTC 1026 Arg Phe wall Arg Ala Leu Asp Arg Leu Gly wall Glu wall gin Leu Arg Tyr Phe 342 CCG CTC CAT CTG TCG CCC GAG TGG CGG CTG CGC GGC CAC GGG CCG GGC GAG TGT 1080 for Leu His Leu Ser for Glu Trp Arg Leu Arg Gly His Gly for Gly Glu Cys 360 CCG ACG GCC GAA CGG GTC TGG TTC GAG GAG CAC ATG AAC CTG CCG TGC CAT CCC 1 1 34 for Thr Ala Glu Arg wall Trp Phe · Glu Glu His Met same time Leu for Cys His for 378 GGT CTG AGT GAC GGC CAG GTC GAC TAC ATG GTC GAG GCG GTC ACC CGC GCC CTG 1188 GLV Leu Ser Asp Gly gin wall Asp Tyr Met wall Glu Ala wall Thr Arg Ala Leu 396 CAC GAG GCC CAC GGC ACG GGG ACG CGG GTG GCG GCC GGG CAC CTG 1233 His Glu Ala His Gly Thr Gly Thr Arg wall Ala Ala Gly His Leu 411

127

Claims (29)

PATENT CLAIMS A compound of formula I wherein: Ί> ϊ (I) denotes a hydrogen atom or a hydroxyl group, CHOH or CH a R ² and R ⁴ independently is a methyl group, ^R3 is hydrogen X is CO, R ¹ is wherein - for meta derivatives: A, C, D and E are hydrogen and B can be: - a halogen atom and preferably a fluorine atom, 128 a monoalkylamino group or a dialkylamino group, in which the alkyl radical is preferably a methyl group or an ethyl group - an ether group a thioether group an alkyl group having 1 to 3 carbon atoms, or - a trihalomethyl group, preferably a trifluoromethyl group, - for para derivatives: A, B, D and E are hydrogen and C can mean: - halogen atom, - NR ¹ R ² in which R ¹ and R ^{2 are} independently of one another - a hydrogen atom, - a straight or branched (C 1 -C 4) alkyl group, where one of the substituents R ¹ or R ² is a methyl group, the other obligatory is an ethyl group, - an alkylcycloalkylmethyl group in which the cycloalkyl radical contains 3 or 4 carbon atoms, - an optionally substituted cycloalkyl group containing 3 or 4 carbon atoms, straight or branched (C 1 -C 4) alkenyl, where in the case where one of R ¹ or R ² is an alkenyl group, the other is different from a methyl or cycloalkyl group containing 3 to 6 carbon atoms, - substituted or unsubstituted N-pyrrolidinyl, - an ether group a thioether group an acyl group or an alkoxycarbonyl group - a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group, an isopropyl group and a tert-butyl group, an alkylthiomethyl group 129 an aryl group and preferably a phenyl group; or - trihalomethyl and preferably trifluoromethyl, - for disubstituted meta-para derivatives: A, D and E are hydrogen and B may mean: - a halogen atom and preferably a fluorine atom, - a monoalkylamino group or a dialkylamino group, in which the alkyl radical preferably represents a methyl group or an ethyl group, - an ether group a thioether group an alkyl group having 1 to 3 carbon atoms, and C can mean: - a halogen atom and preferably a fluorine atom, - an amino group, a monoalkylamino group or a dialkylamino group, in which the alkyl radical preferably represents a methyl group, with the proviso that B is different from a bromine or chlorine atom, or a substituted or unsubstituted allyl group, - an ether group a thioether group an alkyl group having 1 to 6 carbon atoms, or a trihalomethyl group, preferably a trifluoromethyl group, and - for disubstituted ortho-para derivatives: B, E and D are hydrogen and A and C are methyl. A compound according to claim 1 of formula I selected from the group consisting of 4ζ-ιηβίγ1ϋο- <ΐ65 (4ζ- <ΗπιβΐγΙαπιίηο) ρη5ΐίη3ΐηγςίη _IA ^ βΐγ1ύο 4ζ-άε5 (4ζ-άΪΓη6ΐγ1αιηίηο) ρη5ΐιηαι ηγοίη · I _H 5y-hydroxy -metyltio- ^ <^ tes -dinwtyl -amino) pristinamycin Ih 4ζ-η7εΐγΜε3 ( 4ζ ^ ίπιείγΐ3ηΊΐηο) ρη3ΐίη3Γηγείη _IA-4ζ πιεΐγΜε3 (4ζ ΐπιείγ ^ ^ ίηο) pristinamycin I _H-4ζ ηιείοχν ε3 ^ (^ ίηιεΐγΐ3ΐηίηο 4ζ) ρπ3ΐίη3ΐηγείη _IA 130 ^ methoxycarbonyl-de (4 R-dimethylamino) pristinamycin _{IA-4ζ-εΗ10Γ} άε8 (4ζ-άίΓηείγΐ3ΐηίηο) ρη8ίιη3Γηγοίη _IA-4ζ ϋΓ0πι 65 ^ (^ ίΓηβίγ1αιηίηο 4ζ) ρη8ηΐΉπιγοίη _{IA-4ζ-0Γ0πι} άε8 (4ζ-άίΓηεΐνΐ3Γηίηο) ρπ8ϋηΗΓηγείη I 4ζ- _H] ό ^ -άε8 (4ζ ίηιεΐγΐ3ΐηίηο ^) · ρι ί8ΐίη3Γηγείη _IA-iodo-4C of N-dimethylamino) pnstinamycín I _H-4ζ ίηί1υόπηεΐγΜε8 (4ζ ^ ίΐ'ηείγΐ3Γηίηο) ρπ8ΐίη3ΐηγείη _IA-4ζ ύΠυόπηείγΜε8 (4ζ -ύίΓηεΐ7ΐ3η · ηηο) ρΐΊ8ηη3η'ΐγείη 4ζ-I _H-ίεΓΌ Ι} ΐιΐγ1-άε8 (4ζ-ύίιτιείγΐ3η'υηο) ρπ8ΐιη3ηιγα'η _{IA-4ζ-ιζορΐΌργ1} όε8 (4ζ-άίηιεΐγΐ3ΐτιιηο) ρπ8ΐιη3Γηνείη 4ζ-I _and des-izopΓopyl ( 4ζdimetylamino) pristinamycin le-4s-rnetylamino of -dirnetylarnino ^) I pnstinamycín _and 4E-methoxy-des-dimethylamino ^) I pristinainycín _A £ 4-methoxy-de (4-dimethylamino £) pristinarnycín I 4E-fluoro _H ^ me of ethyl-N-Dimethylamino) pristinamycin I _{A-4ζ-3πυηο} 4ε8 (4ζ ^ ίΓηείγΐ3ΐηιηο) ρη8ΐιη3ΐηγείη _IA-4ζ εΐγΐ3ΐηίηο ε8 ^ (^ ίπιείγΐ3ΐηίηο 4ζ) ρπ8ΐίη3Γηγείη _IA 4ζ ^ ^ ϊε1γΐ3ϊηίηο ε8 (4ζ ^ ίπιείγΐ3πιίηο) ρη8ΐίη3ΐηγείη _IA 4ζ -3ΐγΐ3πιίηο ε8 ^ (^ · 4ζ ΐπιείγΐ3πιίηο) ρπ8ΐίη3ΐηγα'η _IA 4ζ ^ ^ ΐ3ΐγΐ3πιίηο ε8 (4ζ ^ ίπιείγΐ3πύηο) ρη8ίίη3ΐηγΕίη _IA-4ζ α1γ1εΐγΐ3ΐηίηο ε8 ^ (^ ίιηείγΐ3ΐηίηο 4ζ) ρη8Ρη3ΐηγάη 4ζ-I _A ^ εΐν1ρΐΌργ1αιηιηο ε8 (4ζ-4ίιηεΐγΐ3Γηιηο ) ρη8ύη3Γηγα'η _IA-4ζ εΐγ1ίζορΓοργΐ3ηιίηο ε8 ^ (^ ίπιεΙγΐ3πιίηο 4ζ) ρη8ΐίη3ΐηγ (: ίη _IA-4ζ εΐγΐΓηεΐγ1εγ1 <1ορΓοργΐ3πιίηο ε8 ^ (^ ίπιείγΐ3ηιίηο 4ζ) ρη8ίίη3Γηγείη _IA 4ζ- (1-pyrrolidinyl) des ^ -dimethylamino) pristinamycin I _A 4ζ-ίήΑιιόπηείοχγ ^ ε8 (4ζ ^ ϊπιεΐγΐ3ΐηίηο) ρΓί5ύη3πιγάη _IA 4ζ-3ΐν1οχγ ^ ε8 (4ζ ^ ίπιείγΐ3ηιίηο) ρη8ΐίη3πιγ <: ίη _IA 4ζ-είοχγ-άε8 (4ζ-4ίιηείγΐ3ΐηίηο) ρη8ΐιη3Γηνείη _IA 4ζ-εΐγ1ίίο ^ ε8 (4ζ ^ ίπιεΐγΐ3ΐηίηο) ρΓί8ΐίιΐ3ΐηγείη _IA-4ζ πιεΐγ1ΐιοπιεΐγΜε8 (4ζ ^ ίπιεΐνΐ3ΐτιίηο) ρη8ΐιιΐ3Γηγείη _IA 4C- (2-chloroethoxy) des ^ -dimethylamino) pnstinamycín _{IA-4ζ-3εεΐγ1} όε8 (4ζ-άϊιτιεΐγΐ3Γηιηο) ρη8ΐιη3ϊηγάη _IA 131 4? -Ethyl-des (4? -Dimethylamino) pristinamycin I _? 4C-ethyl-des-4-dimethylamino) -pininacinin? 4? -Dimethylamino-des-4-dimethylamino) pristinaniycin I _? 4? -Methylthio-des (4? -Dimethylamino) pristamamycin I _A 4e-ethoxy-de (4 £ -dimethylamino) pristinamycin I _A 3. A method for the preparation of streptogramins, characterized in that a strain of a microorganism producing streptogramins having at least one genetic modification affecting the biosynthesis of a Group B precursor is used, said mutant strain is cultivated on an adequate culture medium complemented by at least one parent precursor different from the precursor. the biosynthesis is modified and that said streptogramins are isolated. The method of claim 3, wherein the mutant strain has at least one genetic modification located at the level of the genes involved in the biosynthesis of Group B streptogramin precursors. Method according to claim 4, characterized in that the gene or genes whose expression is modified are selected from the group of genes involved in the biosynthesis of L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine (DMPAPA), L-acid. -pipecolic, L-phenylglycine and / or 3-hydroxypicolinic acid. Method according to claim 4 or 5, characterized in that it is at least one gene selected from the group comprising papA genes. papM. papC (SEQ ID NO: 2), papB (SEQ ID NO: 3), PIPA (SEQ ID NO: 5), snbF (SEQ ID NO: 6) and hpaA (SEQ ID NO: 8). The method according to any one of claims 3 to 6, characterized in that the genetic modification conducted makes at least one of the genes involved in the biosynthesis of Group B streptogramin precursors partially or wholly incapable of encoding the natural enzyme. 132 The method according to claims 3 to 7, characterized in that the genetic modification consists in disrupting one of the genes involved in the biosynthesis of group B streptogramin precursors. Method according to any one of the preceding claims, characterized in that the mutant strain used is derived from a S. pristinaespiralis strain and preferably from a S. pristinaespiralis SP92 strain. Method according to claim 9, characterized in that it is preferably strain SP92: pVRC508. Method according to claim 9, characterized in that it is preferably an SP212 strain. Method according to claim 9, characterized in that it is preferably the strain SP92pipA :: nam ^R. Method according to claim 9, characterized in that it is preferably a strain SPhpaA :: Ham ^R. Method according to any one of the preceding claims, characterized in that the original precursor introduced into the culture medium is selected from the group consisting of derivatives or analogs of amino acids and α-ketocarboxylic acids. Method according to any one of the preceding claims, characterized in that the original precursor is preferably selected to be related to a precursor whose biosynthesis is modified. 16. The method according to claim 14 or 15, wherein the precursor is preferably a phenylalanine derivative when the gene whose expression is modified relates to DMPAPA biosynthesis. 133 Process according to any one of the preceding claims, characterized in that it is useful for preparing pristinamycin IB. 18. A nucleotide sequence selected from the group consisting of: a) all or part of the pape genes (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5), snbF (SEQ ID No. 6) and hpaA (SEQ ID No. 8) . (b) sequences hybridizing to all or part of the genes of (a); and c) sequences derived from sequences a) and b) due to the degeneracy of the genetic code. The nucleotide sequence of claim 18, characterized in that it is selected from the group consisting of pape (SEQ ID NO. 2), papB (SEQ ID NO. 3), pipA (SEQ ID NO. 5), snbF (SEQ. 6) and hpaA (SEQ ID NO. 8). 20. A recombinant DNA comprising a gene selected from the group consisting of papC (SEQ ID NO. 2), papB (SEQ ID NO. 3), pipA (SEQ ID NO. 5), snbF (SEQ ID NO. 6) genes. ) and hpaA (SEQ ID NO. 8). 21. A vector comprising the nucleotide sequence of claim 18 or 19 or the recombinant DNA of claim 20. Use of a sequence according to claim 18 or 19 and / or a vector according to claim 21 for the preparation of metabolites. A polypeptide as a result of the expression of the sequence of claim 18 or 19. 24. A mutant S. pristinaespiralis strain having at least one genetic modification at the level of one of its genes papC (SEQ ID No. 2), papB (SEQ ID No. 3), pipA (SEQ ID No. 5). , snbF (SEQ ID NO. 6), and / or hpaA (SEQ ID NO. 8). 134 25. The mutant strain of claim 24, wherein said mutant strain is SP92pipA :: nam ^R. 26. The mutant strain of claim 24, wherein said mutant strain is SP92hpaA :: amam ^R. 27. A mutant S. pristinaespiralis strain having genetic modification consisting in disrupting the papA gene by double homologous recombination, such as SP212. 28. A compound from the group consisting of 4-trifluoromethoxyphenylalanine, 3-methylaminophenylalanine, 4-methylaminophenylalanine, 4-methylaminophenylalanine, 4-methylaminophenylalanine, 4-methylaminophenylalanine, 4-trifluoromethoxyphenylalanine, 4-trifluoromethoxyphenylalanine, 4-methylaminophenylalanine, 4-methylaminophenylalanine. 4-ethylisopropylaminophenylalanine, 4-ethylmethylcyclopropylaminophenylalanine, 4- (1-pyrrolidinyl) phenylalanine, 4-ethylthiomethylphenylalanine, 4-O- (2-chloroethyl) tyrosine, 3-dimethylaminophenylalanine and 3-ethylaminophenylalanine. A pharmaceutical composition comprising at least one compound according to claim 1 or 2, optionally in combination with group A streptogramin.