NO175207B - - Google Patents

Description

The present invention relates to the production of a therapeutically active teicoplanin compound which has the formula:

where Y represents a group

where

R<1> represents hydrogen, (C^-C^)alkyl, hydroxy(C2-C4)-alkyl, halo(C2-C4)alkyl, (C^C^)alkoxy(C2-C4)alkyl, amino(C2 -C4)alkyl, (C^C^)alkylamino(C2-C4)alkyl, dl(C1-C4)alkylamino(C2-C4)alkyl,

R** represents hydrogen, (C^-Cf, )alkyl, hydroxy(C2-C4 )-alkyl, halo(C2-C4)alkyl, (C1-C4)alkoxy(C2-C4)alkyl, a nitrogen-containing 5-6 -membered heterocyclic ring which is completely saturated and where nitrogen atoms can be substituted with a benzyl residue or a bridging C2 alkylene chain attached to one of the C atoms in the heterocyclic ring;

a group -alk-W where "alk" represents a linear alkylene chain with 1-8 carbon atoms which is optionally

substituted with a substituent selected from (C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, aminocarbonyl, (C1-C4)alkylaminocarbonyl, di(C1-C4)alkylaminocarbonyl, (C1-C4)alkoxycarbonyl, phenyl(C1-C4) alkoxycarbonyl, and W represents carboxy, (C1-C4) alkoxycarbonyl, phenyl(C1-C4) alkoxycarbonyl, aminocarbonyl, (C±- C4) aminocarbonyl, di(C1-C4) aminocarbonyl, pentosaminocarbonyl, hexosaminocarbonyl , ureido, guanidino, pyrrolidine where the N atom may be (C1-C4) alkyl substituted, pyridine or morpholine, a group of the formula

where R<3> and R<4> each independently represent hydrogen, (cl~c6)alkyl, hydroxy<y>(C2~C4)alkyl and halo(C2-C4)alkyl, or R<4> represents phenylmethyloxycarbonyl and R <3> represents hydrogen; a group of the formula: where r<6>, r<7> and R<8> each independently represent a (C^-C4)alkyl, or R<*> and R^ together with the adjacent nitrogen atom represent piperazino, substituted in 4-position with a methyl group or a pyridinyl group, morpholino or thiomorpholino; A represents hydrogen or -N[(C^q-Ch)aliphatic acyl]-P-D-2-deoxy-2-amino-glucopyranosyl; B represents hydrogen or N-acetyl-p<->D-2-deoxy-2-amino-glucopyranosyl; M represents hydrogen or α-D-mannopyranosyl; provided that B represents hydrogen only when A and M are simultaneously hydrogen and M represents hydrogen only when A is hydrogen, and with the further proviso that when W represents a group a group

, ureido, guanidino

or a nitrogenous 5-6 membered heterocyclic ring as defined above directly connected to the "alk" moiety through a bond with a ring nitrogen atom, then the linear alkylene "alk" moiety must have at least two carbon atoms;

as well as acid addition salts thereof.

Teicoplanin is the international non-proprietary name (INN) of the antibiotic substance formerly known as teichomycin which is obtained by culturing the strain Actinoplanes teichomyceticus nov. sp. ATCC 31121 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts (see US patent 4,239,751). According to the method described in said patent, an antibiotic complex containing Teichomycin A^, A2 and A3 is recovered from the separated fermentation medium by extraction with a suitable water-insoluble organic solvent and precipitation from the extracting solvent according to usual methods. Teichomycin A2, which is the main factor in the isolated antibiotic complex, is then separated from the other factors by column chromatography on Sephadex®. Published GB Patent Application 2,121,401 states that the antibiotic Teichomycin A2 is actually a mixture of five closely related co-produced main components.

According to recent structural studies, it is possible to represent teicoplanin A2 (formerly Teichomycin A2) major components 1, 2, 3, 4 and 5 by the above formula I in which R is hydrogen, Y is hydroxy, A represents -N[(C^ q-C^^ )-aliphatic acyl]-p-D-2-deoxy-2-amino-glucopyranosyl, B represents N-acetyl-g<->D-2-deoxy-2-amino-glucopyranosyl, M represents oc-D-manno -pyranosyl.

More particularly, in teicoplanin A2 component 1 the t (c10~^ll)~aliphatic acyl] substituent represents Z-decenoyl, in teicoplanin A2 component 2 it represents 8-methyl-nona-noyl, in teicoplanin A2 component 3 it represents decanoyl, in teicoplanin A2 component 4 it represents 8-methyldecanoyl, in teicoplanin A2 component 5 it represents 9-methyldecanoyl.

All sugar moieties, when present, are bound to the teico-planin core through O-glycosidic bonds.

In addition, it has been found that it is possible to convert teicoplanin, a pure factor thereof or a mixture of any of said factors in any amount ratio, into antibiotic unit products by selective hydrolysis of one or two sugar moieties or - groups. They are designated antibiotic L 17054 and antibiotic L 17046 are known respectively from EP patent applications publ. no. 119.574 and 119.575. Preferred hydrolysis conditions for the production of antibiotic L 17054 are in the aforementioned EP patent application, publ. no. 119,575 stated to be: 0.5N hydrochloric acid at a temperature between 70 and 90°C and for a time which is generally between 45 and 90 min. Antibiotic L 17054 is represented by the above formula I in which Y is hydroxy, R and A represent hydrogen, B represents N-acetyl-p<->D-2-deoxy-2-amino-glucopyranosyl, M represents _a-D- mannopyranosyl, where the sugar moieties are linked to the peptidic core through an O-glycosidic bond.

Preferred hydrolysis conditions stated in the aforementioned EP patent application, publ. no. 119,574 for the production of antibiotic L 17046 is: 1-3N hydrochloric acid, at a temperature between 50 and 90°C and a time which is generally between 40 and 60 min.

Antibiotic L 17046 is represented by formula I above in which Y is hydroxy, R, A and M represent hydrogen atoms, and B is N-acetyl-p<->D-2-deoxy-2-amino-glucopyranosyl, where the sugar moiety is attached to the peptidic core through an O-glycosidic bond.

The complete selective cleavage of all the sugar moieties or groups in the teicoplanin compounds yields an aglycone molecule designated antibiotic L 17392 or deglucoteicoplanin, and is represented by the above formula I wherein Y is hydroxy, and R, A, B and M each indi -dual represents a hydrogen group. This selective hydrolysis process is known from NO patent 170,019.

A substance which has the same structural formula is known from EP patent application, publ. no. 0090578 and is designated antibiotic A 41030 factor B. This substance is obtained by means of a microbiological process which involves fermentation of the strain Streptomyces virginiae NRRL 12525 or Streptomyces virginiae NRRL 15156 in a suitable medium, isolation, purification and separation into its components of antibiotic A 41030 , an antibiotic complex of at least seven factors, antibiotic A 41030 factor B, incl.

All of the above named compounds, i.e. teicoplanin, teicoplanin A2 complex, teicoplanin A2 component 1, teicoplanin A2 component 2, teicoplanin A2 component 3, teicoplanin A2 component 4, teicoplanin A2 component 5, antibiotic L 17054, antibiotic L 17046, antibiotic L 17392 and any mixture thereof in any proportion, are suitable starting materials for the preparation of the amide derivatives of formula I.

In the present context, "teicoplanin compound" or "teicoplanin starting material" is used to indicate one of the above-mentioned starting materials, i.e. teicoplanin as obtained according to US patent 4,239,751, any further purification thereof, teicoplanin Å2 complex, a compound with the above formula I in which R is hydrogen, Y is hydroxy, A represents hydrogen -N[(C^o~ Cu)aliphatic acyl]-p<->D-2-deoxy-2-amino-glucopyranosyl, B represents hydrogen or N-acetyl-g<->D-2-deoxy-2-amino-glucopyranosyl, M represents hydrogen or α-D-manno-pyranosyl, provided that B can be hydrogen only when A and M are simultaneously hydrogen, and M can represent hydrogen only when A is hydrogen, a salt thereof, or a mixture thereof in any proportion.

The term "alkyl" used here includes, either alone or in combination with other substituents, both straight and branched hydrocarbon groups; more particularly, "(Ci-Cfc)alkyl" represents a straight or branched aliphatic hydrocarbon chain of 1-6 carbon atoms such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 1,1-dimethylethyl, pentyl, 1- methylbutyl, 2-methylbutyl, 1-hexanyl, 2-hexanyl, 3-hexanyl, 3,3-dimethyl-1-butanyl, 4-methyl-1-pentanyl and 3-methyl-1-pentanyl; likewise, "(C1-C4)alkyl" represents a straight or branched hydrocarbon chain of 1-4 carbon atoms such as the alkyl groups of 1-4 carbon atoms exemplified above. The term "halogen" represents a halogen atom selected from fluorine, chlorine, bromine or iodine.

The pentosamino moieties in the pentosaminocarbonyl substituent are 2- or 3-amino (2- or 3-deoxy) either D- or L- or D,L-pentose groups in either anomeric form or in an anomeric mixture such as 2- or 3- amino(2- or 3-deoxy)-ribose, 2- or 3-amino(2- or 3-deoxy)arabinose, 2- or 3-amino(2- or 3-deoxy)xylose and 2- or 3-amino (2-or 3-deoxy)lyxose.

The hexosamino moieties in the hexosaminocarbonyl substituent are either D or L, or D,L 2- or 3-amino (2- or 3-deoxy)hexose group 1 either in anomeric form or in an anomeric mixture such as 2- or 3-amino( 2- or 3-deoxy)allose, 2- or 3-amino(2- or 3-deoxy)altrose, 2- or 3-amino(2- or 3-deoxy)glucose, 2- or 3-amino(2- or 3-deoxy)mannose, 2- or 3-amino(2- or 3-deoxy)gulose, 2- or 3-amino(2- or 3-deoxy)galactose and 3- or 4-amino(2- or 3 -deoxy)fructofuranose.

The definition "linear alkylene chains of 1-8 carbon atoms" is straight alkylene chains of 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. Representative examples of linear alkylene chains with 1-6 carbon atoms are:

These linear alkylene chains may optionally have substituents as described above.

The term "a nitrogenous 5-6 membered heterocyclic ring" as used herein includes ring systems such as pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, pyrrolinyl and imidazolidinyl.

The definition "nitrogen-containing 5-6-membered heterocyclic ring" also includes those heterocyclic rings having two ring members bridged by a C2-alkylene chain. Examples of said bridged rings are the following: l-azabicyclo[2,2,2]octane, 1,4-diazabicyclo[3,2,0]nonane, l-azabicyclo[2,2,1]heptane, l-azabicyclo[ 3,2,1]octane, 8-azabicyclo[3,2,1]octane, 3-azabicyclo[3,2,1]octane, 1-azabicyclo[3,3,l]nonane, 9-azabicyclo[3, 3,l]nonane, 3,8-diazabicyclo[3,2,1]octane, 2-azabicyclo[2,2,1]heptane, 2-azabicyclo[2,2,2]octane, 3-azabicyclo[3, 2,2]nonane. Representative compounds of formula I accordingly include those of the above general formula wherein the symbol represents a substituent derived from one of the following groups: l-azabicyclo[2,2,2]octan-3-amine, l-azabicyclo[2,2,2]octan-2-amine, l-azabicyclo[2,2, 2]octan-3-amine, 6-methyl l-azabicyclo[2,2,2]octan-3-amine, N-methyl l-azabicyclo[2,2,2]octane-3-ethanamine, l-azabicyclo[ 2,2,2]octan-4-amine, l-azabicyclo[2,2,2]octan-4-amine, N-methyl l-azabicyclo[2,2,2]octane-2-methanamine, l-azabicyclo [2,2,l]heptan-3-amine, l-azabicyclo[3,2,l]octane-3-methanamine, 8-azabicyclo[3,2,l]octan-3-amine, 8-methyl 8- azabicyclo[3,2,l]octane-3-amine, 8-ethyl 8-azabicyclo[3,2,l]octane-2-methanamine, 3-azabicyclo[3,2,1]octane-3-ethanamine, l -azabicyclo[3,3,1]nonan-4-amine,

1-azabicyclo[3,3,1]nonane-3-methanamine,

9-azabicyclo[3,3,1]nonan-3-amine, 9-methyl

2-azabicyclo[2,2,1]heptan-5-amine, 2-methyl

2-azabicyclo[2,2,2]octan-5-amine, 2-methyl.

A preferred group of compounds are the compounds of formula I where R<1> represents hydrogen and the other substituents have the meanings indicated above.

A further preferred group of compounds of formula I includes those compounds where R<1> represents hydrogen and R<**> represents a group -alk-W where "alk" is a linear alkylene chain of 2-8 carbon atoms, W represents pyrroli-dino where the N atom may be (C^-C^)alkyl substituted, morpholino or piperidino, or W represents a group of the formula

where R<3> and R<4> each independently represent hydrogen, a (cl~c6 )alkyl group and A, B and M have the meanings given above, as well as acid addition salts thereof. Preferred compounds of formula I are also represented by those compounds where R<1>, A, B and M represent hydrogen atoms and R<2> represents a group

where "alk" is a linear alkylene chain with 2-6 carbon atoms and R<3> and R<4> represent (C^-C^, )alkyl groups, as well as pharmaceutically acceptable addition salts thereof.

Another group of preferred compounds of formula I is represented by the compounds in which A, B and M either represent the sugar moieties as defined above or each simultaneously represents a hydrogen atom.

Other most preferred compounds are those of formula I where A, B and M either simultaneously represent the sugar moieties as defined above or simultaneously represent hydrogen atoms, and NR1!?2 represents a group -HN(alk)W where "alk" represents a linear alkylene chain with 2, 3, 4, 5, 6, 7 or 8 units and W represents a group selected from: -NH2, -NHCH3, -NHC2H5,

-N(CH3)2, -N(C2H5)2 and -N(CH3)(C2H5), or a group -HNCH(COOCH3)(CH2)4NH2 or Representative examples of the compounds of formula I include those compounds where A, B and M have the meanings given above, and

represents: -NH2, -NHC4H9, -NH(CH2)4~<0>H, -NHCH2C00H, -NHCH2COOCH2<C>5H5, -NHCH2COOC2H5, -NH-CH2C0NH2,

-NH-CH2-C0N(C2H5)2, where m represents a whole number, 1,2, 3 or 4, -NH-(CH2)n-NH2, -NH-(CH2)n<N>HCH3, -NH( CH2)n-N(CH3)2, -NH-(CH2)nN(C2H5), -HN(CH2)nN(CH3)(C2H5)

where n represents 2, 3, 4, 5, 6, 7 or 8

-N(CH3)2, N(CH2CH2OH)2, -N(CH2CH2NH2)2, <->N(CH2CH2NHCH3)2, -N[CH2CH2N(CH3)2]2, -N(CH3)(CH2CH2NH2 ), - N(CH3)[(CH2)NHCH3], -N(CH3)[(CH2)2N(CH3 )2], -N(C2H5)[(CH2)2-NHCH3], -N[CH2CH2CH2N(C2H5)2] 2,

The compounds of formula I can form salts according to conventional methods. In particular, the compounds of formula I in which the group -NR1!?2 contains additional amine functions will form acid addition salts.

In addition, the compounds of formula I which contain acid functions in the -NR<1>R<2> part can also form base addition salts.

In general, the compounds of formula I containing acidic and basic functions can form internal salts. For the scope of the invention, "internal salts" are included in the definition for the "non-salt" form. Preferred addition salts of the compounds of formula I are the pharmaceutically acceptable acid and/or base addition salts.

The term "pharmaceutically acceptable acid and/or base addition salts" means those salts with acids and/or bases which, from a biological production and formulation point of view, are compatible with pharmaceutical practice and with the use in animal growth enhancement.

Representative and suitable acid addition salts of the compounds of formula I include the salts formed by standard reaction with both organic and inorganic acids such as e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, succinic acid, citric acid, ascorbic acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, mucoid acid, glutamic acid, camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid and similar acids.

Representative examples of said bases are: alkali metal or alkaline earth metal hydroxide such as sodium, potassium and calcium hydroxide;_ ammonia and organic aliphatic, alicyclic or aromatic amines such as methylamine, dimethylamine, trimethylamine and picoline.

When the compounds of formula I contain a (C^-C^)alkyl-pyridine moiety or a -<+>NR<5>R<7>R8 moiety where R<6>, R7 and R<8> have the above meanings, the respective anion is an anion derived from a pharmaceutically acceptable acid. Representative examples of said anion are those that arise from the above-mentioned acids.

The conversion of the free amino compounds or non-salt compounds of formula I to the corresponding acid addition salts, and vice versa, i.e. the conversion of an addition salt of a compound of formula I to the non-salt form or the free amine form, is known to a person skilled in the art and is covered by the invention.

A compound of formula I can e.g. is converted to the corresponding acid or base addition salt by dissolving the non-salt form in an aqueous solvent and adding a small molar excess of the selected acid or base. The resulting solution or suspension is then lyophilized to recover the desired salt. Instead of lyophilizing, it is in some cases possible to recover the final salt by extraction with an organic solvent, concentration to a small volume of the separated organic phase and precipitation by addition of a non-dissolving solvent.

In the event that the final salt is insoluble in an organic solvent where the non-salt form is soluble, it is recovered by filtration from the organic solution of the non-salt form after addition of the stoichiometric amount or a small molar excess of the selected acid or base.

The non-salt form can be prepared from a corresponding acid or base salt dissolved in an aqueous solvent which is then neutralized to the free non-salt form. This is then extracted, e.g. by extraction with an organic solvent or converted into another base or acid addition salt by addition of the selected acid or base and processing as indicated above.

When desalination is necessary after neutralization, a normal desalination method can be used. E.g. column chromatography on polydextran resins with regulated pore size (such as "Sephadex L H 20") or silanated silica gel can be suitably used. After elution of the undesired salts with an aqueous solution, the desired product is eluted using a linear gradient or step gradient of a mixture of water and a polar or apolar organic solvent, such as acetonitrile/water from 50:50 to 100# acetonitrile .

As is known in the field, salt formation either with pharmaceutically acceptable acids (bases) or non-pharmaceutically acceptable acids (bases) can be used as an appropriate purification technique. After formation and isolation, the salt form of a compound of formula I can be converted to the corresponding non-salt or to a pharmaceutically acceptable salt.

In some cases, the acid addition salt of a compound of formula I is more soluble in water and hydrophilic solvents and has an increased chemical stability.

In view of the similarity in the properties of the compounds of formula I and their salts, however, what has been said in the present context in connection with the biological activities of the compounds of formula I also applies to their pharmaceutically acceptable salts and vice versa.

The compounds of formula I are useful as semi-synthetic antibacterial agents mainly active against gram-positive bacteria, but also active against gram-negative bacteria.

The therapeutically active compounds of formula I as defined

above is prepared according to the present invention by reacting a teicoplanin starting material with the formula:

where R is hydrogen or a protecting group for the amide function; Y is hydroxy; A represents hydrogen or -N[(C10-C11)-aliphatic acyl]-ε-D-2-deoxy-2-amino-gluco-pyranosyl; B represents hydrogen or N-acetyl-ø-D-2-deoxy-2-amino-glucopyranosyl, M represents hydrogen or α-D-manno-pyranosyl, provided that B can represent hydrogen only when A and M are simultaneously hydrogen, and M can represent hydrogen only when A is hydrogen; a salt thereof or a mixture thereof in any proportion;

with a molar excess of an amine of the formula HNR^R<2>, where R<1> and R<2> have the meanings given above and where the reactive functions, other than the amino function, which are to be reacted with the carboxyl part of the teicoplanin starting material, is protected in an inert organic solvent and in the presence of a small molar excess of a condensing agent selected from (C1-C4 )alkyl, phenyl or heterocyclic phosphorazidates such as diphenyl phosphorazidate (DPPA), diethyl phosphorazidate, di(4-nitrophenyl) phosphorazidate, dimorpholyl phosphorazidate and diphenyl phosphorochloridate and remove the protecting groups if such are present.

A general method for the preparation of a compound of formula I is represented by the reaction (amidation) of a suitable teicoplanin starting material (II) as defined above, with the selected amine of formula HNR^-R<2> where R<1> and R<2> has the meanings given above, in an inert organic solvent in the presence of a condensing agent. When teicoplanin or teicoplanin Å2 complex is used as starting material, the relative amide of formula I obtained according to the amidation reaction according to the present method is a mixture of five amide derivatives corresponding to the five main components of teicoplanin A2 as mentioned above. Said mixture can be separated into five individual amide derivatives according to analogous known techniques (see e.g. published GB patent application 2.121.401). For the sake of clarity, both the actual mixture obtained from the amidation reaction and each of the five amide derivatives shall form part of the present invention as herein defined, the meaning of A representing -N[(Cio~cll)aliphatic acyl]- ε-D-2-deoxy-2-amino-gluco-pyranosyl. Conversely, the individual pure amide derivatives of each teicoplanin A2 component are obtained by following the present method starting from the individual component itself instead of starting with the complex.

In carrying out the amidation to prepare the compounds of formula I, it is sometimes, and especially when at least one of A, B and M in the teicoplanin starting material represents hydrogen, convenient to protect the primary amino function in the teicoplanin starting material to reduce possible undesirable side reactions.

When the amine HNR<*>R<2> contains additional reactive functions such as amino or carboxy groups, which may adversely interfere with the course of the amidation, they are also protected by means of per se known methods such as those described in textbooks such as T.W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, 1981, and M. Mc. Omie, "Protecting Groups in Organic Chemistry", Plenum Press, New York, 1973. These protecting groups must be stable under the conditions of the reaction process, they must not adversely interfere with the main amidation reaction, and must be easily cleaved and removed from the reaction medium at the end of the reaction without changing the newly formed amide bond.

Representative examples of N-protecting groups that can be advantageously used in the present process for protecting an amino function both in the teicoplanin starting material and, when appropriate, in the R<1-> and R<2-> part of the amine HNR^R< 2>, carbamate-forming reagents are characterized by the following oxycarbonyl groups: 1,1-dimethylpropynyloxycarbonyl, t-butyloxycarbonyl, vinyloxycarbonyl, aryloxycarbonyl, cinnamyloxycarbonyl, benzyloxycarbonyl, p-n i trobenzyloxycarbonyl-3,4-dimethoxy-6-nitrobenzyloxycarbonyl , 2,4-dichlorobenzyloxycarbonyl, 5-benzisoxazolylmethyloxycarbonyl, 9-anthanylmethyloxycarbonyl, diphenylmethyloxycarbonyl, isonicotlnyloxycarbonyl, diphenylmethyloxycarbonyl, isonicotlnyloxycarbonyl, S-benzyloxycarbonyl and the like.

Other suitable N-protecting agents are aldehydes or ketones, or derivatives thereof which can form Schiff bases with the amino group in the teicoplanin core to be protected.

Preferred examples of such Schiff base forming agents are benzaldehydes and particularly preferred is 2-hydroxybenzaldehyde (salicylaldehyde).

An appropriate method of protection in the case where the amine reactant HNR^-R2 contains a primary amino function as a substituent for R<1> and/or R<2>, is in some cases the formation of a benzylidene derivative which can be prepared by reacting the amine HNR<* >R<2> with benzaldehyde in a lower alkanol, such as ethanol, preferably at room temperature. After the reaction with the selected teicoplanin starting material has been completed, the benzylidene protecting group can be removed as known in the art, e.g. by treatment with dilute mineral acid, preferably hydrochloric acid, at room temperature.

When the final compound of formula I contains groups which are labile under acidic conditions, e.g. when A, B or M represent sugar moieties or groups as defined above which can be hydrolysed in an acidic medium, obviously other removal conditions must be used, such as catalytic hydrogenation using e.g. of palladium on carbon as a catalyst to remove the appropriate protecting group. In this case, however, attention should be paid to the presence of groups that can be modified by catalytic hydrogenation. A typical consequence of the catalytic hydrogenation of a derivative of formula I, in which A represents a group as defined above whose acyl part is Z-decenoyl (ie a teicoplanin A2 component 1 derivative or a mixture containing this), is that at least is partially converted to the corresponding decanoyl derivative (ie a derivative of teicoplanin A2 component 3).

One skilled in the art can, also on the basis of what is stated herein, determine which functions in the amine HNE^-R2 must be protected, how they must be protected, and the correct deprotection reaction necessary to release the end compound. A suitable protection for reactive carboxylic acid function is e.g. formation of an ester function. As will be understood by one skilled in the art, the final choice of the specific protecting group depends on the properties of the particular amide derivative desired. This amide function in the final compound should indeed be stable under the conditions of removal of the protecting group^) .

Since the conditions for removal of the various protecting groups are known, a person skilled in the art can select the correct protecting group. For example, when the final compound also has a benzyl ester function or N-benzyl function, the protecting groups that can usually be removed by catalytic hydrogenation, such as the benzyloxycarbonyl group, should be avoided, while the protecting groups that can be removed under acidic conditions, such as t-butoxycarbonyl, should be appropriately can be used. In contrast, catalytic hydrogenation can be suitably used in a case similar to the above when it is desired to convert a compound of formula I containing said N-benzyl or benzyl ester function I -NR<1>R<2> part into the corresponding compound in which said N -benzyl or benzyl ester function is replaced by a hydrogen atom.

Inert organic solvents useful for the condensation reaction are the organic aprotic solvents which do not adversely interfere with the course of the reaction and which can at least partially solubilize the teicoplanin starting material. Examples of said inert organic solvents are organic amides, alkyl ethers, ethers of glycols and polyols, phosphoramides, sulphoxides and aromatic compounds. Preferred examples of inert organic solvents are: dimethylformamide, dimethoxyethane, hexamethylphosphoramide, dimethylsulfoxide, benzene, toluene and mixtures thereof.

The condensation agent in the present method is one which is suitable for the formation of amide bonds in organic compounds and especially in peptide synthesis. Representative and preferred examples of condensing agents are (C1-C4 )alkyl-, phenyl- or heterocyclic phosphorazidates such as diphenyl phosphorazidate (DPPA), diethyl phosphorazidate, di(4-nitrophenyl)phosphorus azidate, dimorpholyl phosphorazidate and diphenyl phosphorochloridate. The preferred condensing agent is diphenyl phosphorazidate (DPPA).

In the present process, the amine reactant HNR^R<2> is normally used in a molar excess. In general, a 2- to 6-fold molar excess is used, while a 3- to 4-fold molar excess is preferred.

For the amidation to proceed, it is necessary that the amine HNr<I>r<2> can form a salt with the carboxy function in the teicoplanin starting material. In the case that HNR<1>R<2> is not strong enough to form a salt in the chosen reaction medium, it is necessary to add a salt-forming base to the reaction sb land i ngen at least in an equimolecular amount with the teicoplanin starting material .

Examples of said salt-forming bases are tertiary organic aliphatic or alicyclic amines such as trimethylamine, triethylamine, N-methylpyrrolidine or heterocyclic bases such as picoline and the like.

The condensing agent is generally used in a small molar excess such as from 1.2 to 1.7 and is preferably 1.5 times the teicoplanin starting compound. In addition, the amine reactant ENR^-R2 can also suitably be introduced into the reaction medium as a corresponding acid addition salt, e.g. the hydrochloride. In this case, at least a double molar amount and preferably a 2-4 times molar excess of a strong base which can liberate the HNR<1>R<2>amine from its salts is used. Also in this case the suitable base is a tertiary organic aliphatic or alicyclic amine such as those exemplified above. The use of the salt of the amine HNr<I>r<2>, which is then liberated in situ with the above-mentioned bases, is actually, at least in some cases, highly preferred especially when the salt is more stable than the corresponding free amine.

The reaction temperature will vary significantly depending on the specific starting materials and reaction conditions. It is generally preferred to carry out the reaction at temperatures between 0 and 20°C. The reaction time also varies significantly depending on other reaction parameters. The condensation reaction is usually completed within 24-48 hours.

In all cases, the course of the reaction is monitored by TLC or preferably by means of HPLC according to methods known per se. On the basis of the results of these analyses, an expert in the field will be able to judge the course of the reaction and decide when the reaction should be stopped and when the preparation of the reaction mass should be started according to per se known techniques such as e.g. includes extraction with solvent, precipitation by addition of non-solvents, etc., in conjunction with further separations and purifications by column chromatography.

When protection of the HNR^R<2->reactant or the teicoplanin starting material, or both of these compounds, is required, as already mentioned, the protection is removed from the protected end compound according to methods known per se and these depend mainly on the protecting group that is involved. In the case that both the amine HNR 2 -R 2 and the teicoplanin starting material are protected, it may be convenient to use a similar type of protection that can be removed under the same conditions so that only one deprotection step is required to release both functions.

It is also obvious that in many cases a compound of formula I can be prepared in more than one way and that a compound of formula I can be converted into another by means of reactions known per se. When e.g. The HNR^-R2-amine is a diamine compound such as HN(R<1>)-alk-NR<3>R<4> as defined above, the desired amine compound of formula I can be prepared either directly by condensation of said amine, appropriately protected if necessary, with the selected starting material or it can be prepared by reacting an amide of formula I in which the substituent R<2> is alk-halogen, where halogen is preferably a chlorine or bromine atom, with an amine of formula HNR<3> R<4>. Moreover, an amide compound of formula I containing a carboxy function on the -NR^-R^ moiety can be converted into the corresponding amide or substituted amide derivative by conventional techniques.

Said carboxy function can furthermore also be converted into the corresponding ester of acyl halide function using common techniques. More particularly, an ester function is generally formed by reacting the carboxy-containing product with a preparation of an alcohol in the presence of an acid catalyst at a temperature varying between room temperature and the boiling point of the reaction mixture. The acid is preferably a mineral acid and the alcohol contains the part to be bound to the carboxylic function in the ester derivative. An inert solvent can also be used. A compound of formula I containing a carboxylic ester function on the -NR<1>R<2->substituent can obviously be converted into the corresponding carboxylic compound by hydrolysis.

A preferred hydrolysis technique involves an aqueous solution of an alkali metal carbonate such as sodium or potassium carbonate, at a temperature from room temperature to the boiling point of the reaction mixture. A compound of formula I containing an -NH2~ function on the -NR<1>R<2> part can be converted into the corresponding monoalkylamino derivative by means of a "reductive alkylation" which involves reaction of the compound with the selected carbonyl derivative (which can provide the desired alkyl substituent by reduction) to form the corresponding Schiff base intermediate which is then reduced in the presence of a suitable reducing agent such as sodium or potassium borohydride.

When a free amino group is present in the -NR<1>R<2> moiety of formula I, it can be alkylated as known in the art, e.g. in that it is reacted, or optionally that the corresponding compound in which the primary amino group in the teicoplanin part has been protected with an alkyl halide (bromide, chloride or iodide). Likewise, a secondary amino function can be converted into a tertiary function or a tertiary amino function can be quaternized.

In the following table (Table I), the structural formulas for representative examples of compounds of formula I are indicated.

The following table (Table II) sets forth the preparation procedures, starting materials and reaction yields of representative examples of compounds of formula I:

HPLC analysis

The following table sets forth the R 2 value of representative examples of the compounds of formula I. The analyzes were performed with a Varian model 5000 LC pump equipped with a 20 µl loop injector. Rheodyne model 7125 and a Perkin-Elmer LC 15 UV detector at 254 pm.

Columns: pre-column (1.9 cm) Hibar LiChro Cart 25-4 Merck pre-packed with LiChrosorb RP-8 (20-30 pm) followed by a column Hibar RT 250-4 Merck pre-packed with LiChrosorb RP-8 (10 pm) .

Eluents: A, 0.2% aqueous HC00NH4 and B, CH3CN

In. 1 ex. 1on: 20 pl - Flow rate: 2 ml/min.

The reaction is monitored by injecting, at fixed times, samples of the solutions (or suspensions) diluted with the solvent mixture (CH3CN : H2O, 6:4 (v/v)) enough to achieve final concentrations of either 1, 2 or 3 mg/ml.

Method A: Linear step gradient from 5 to 75$ of B in A for 35 min. according to the program below:

Method B: Linear gradient from 5 to 6056 of B in A for 30 min.

Method C: Linear gradient from 20 to b0% of B in A for 30 min.

Method D: Suitable chromatographic conditions for comparing all the teicoplanin amides with deglucoteicoplanin.

HPLC automatic apparatus: Hewlett-Packard model 1084

Column: Hibar (Merck) LiChrosorb RP-8 (7 pm) Flow rate: 1.5 ml/min.

Eluents: A, 0.02 M aqueous NaH 2 PO 4 /CH 3 CN

25/75 (w/w)

B, 0.02 M aqueous NaH 2 PO 4 /CH 3 CN

95/5 (w/w)

Elution: linear step gradient from 8 to 6056 of B in A for 48 min., according to the program below:

TABLE III

a) HPLC analysis of amides of teicoplanin A2 (formula I wherein A is N[(<C>i<g>~<c>ll)aliphatic acyl]-p<->D-2-deoxy-2-amino- glucopyranosyl, B is N-acetyl-P-D-2-deoxy-2-amino-glucopyranosyl, M is oc-D-mannopyranosyl)

(method A)

62

b) HPLC analysis of amides of antibiotic L 17054 (formula I in which A represents hydrogen, B is N-acetyl-g-D-2-deoxy-2-aminoglucopyranosyl, M is α-D-mannopyranosyl) (Method B)

c) HPLC analysis of amides of antibiotic L 17046 (formula I in which A and M represent hydrogen, B represents N—acetyl-p<->D-2-deoxy-aminoglucopyranosyl) (Method B) d) HPLC analysis of amides of deglucoteicoplanin (formula I in which A, B and M represent hydrogen atoms) (Method C) e) HPLC analysis according to method D (K' = tjj/tjj for deglucoteicoplanin)

The K' values for the complex derivatives refer to component 2.

The following table (Table IV) sets forth acid-base titration data for some representative compounds of the invention. The analyzes were carried out in Methylcellosolve <R>/H 2 O, 4:1 (v/v). A sample (10 pmol in ca. 20 mL) then 0.01N HCl (2 mL) is added and the mixture titrated with 0.01N KOH in the same solvent mixture.

Table VII sets forth % NMR data obtained at 250 MHz with a Bruker AM-250 spectrometer 1 DMSO-d^, at 20°C, at a sample concentration of 20 mg/ml (internal standard: TMS, = 0.00 ppm).

Isoelectric point (pl)

The isol electrofocusing (IEF) technique coupled with bioautography detection has been used to determine the pl value of representative compounds of formula I using the following materials: Ampholine carrier ampholytes (40% w/v) were purchased from LKB Produketer AB, Bromma, Sweden. Acrylamide, N,N'-methylene-bisacrylamide (BIS), N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate were from Bio Rad Laboratorie, Richmond, California, USA. Glycerol and antibiotic agar N. 1 (Grove and Randall medium N. 1) were from E. Merck Darmstadt FRG. Gel Fix polyester sheets were purchased from Serva Feinbiochemica, Heidelberg. Phenolindo (2,6-dichlorophenol) was from BDH Chemicals Ltd., Poole, England.

Isoelectric focusing

IEF was performed on gel plates using an LKB Multiphor 2117 cell and a Bio-Rad Power Supply Model 1420A. Plates of dimensions 24.5 x 11.5 cm and 1 mm thickness were prepared on a sheet of Gel Fix.

Polyacrylamide gels with a concentration of 8% T and with a crosslink of 4% C (30% T stock solution was prepared by dissolving 28.8 g of acrylamide and 1.2 g of bis-acrylamide in 100 ml of distilled water), glycerol, 3, 5% v/v, 2% Ampholine, 0.05% ammonium persulfate as catalyst and 0.05$ Temed as accelerator.

The carrier ampholyte composition for 35 ml gel-forming solution was as follows: 1) pH 3.5-10: 1.6 ml Ampholine 3.3-10, 0.05 ml Ampholine 4-6, 0.05 ml Ampholine 7.9 and 0, 05 ml Ampholine 8-9.5. 2) pH 2.5-6: 0.4 ml Ampholine 2.5-4; 1.1 ml Ampholine 4-6, 0.2 ml Ampholine 3-10.

3) pH 7-10; 0.5 Ampholine 7-9, 0.8 ml Ampholine 8-9.5, 0.4 ml Ampholine 9-11.

The electrode solutions, as recommended by LKB for the respective pH range, were:

Test conditions

The gel was cooled to 4°C using an LKB 2209 refrigerated constant temperature circulator. After prefocusing for 30 minutes at 5 W, the samples (20 µl containing 0.2-2.5 µg of antibiotic) were placed in the slot on the cathodic side. Electrofocusing was performed using 10 W constant power and was completed after 3-3Yt hour with a final potential of 1400 V.

pl determination

The pH values were determined by dividing a portion of the gel into 1 cm sections and eluting the individual pieces at room temperature with 1 ml of 10 mM CK1 prior to pB readings.

The isoelectric point for each antibiotic was determined by interpolation on a curve obtained by plotting pH values against the distance from the anode. The results obtained at the two separate pE ranges are given in Table VIII below.

Microbiological development

The antibiotic substances were detected by bioautography. Polyacrylamide gel was placed on a 3 mm layer of agar medium N.

1 inoculated with 1% Bacillus subtils ATCC 6633 spore (0.5 OD at 600 nm). After 10 min. the gel was removed and the plate was incubated overnight at 37°C and examined for zones of inhibition. The contrast between the lysis area and that of bacterial growth was increased by using Phenolindo (2,6-dichlorophenol) 1% w/v (oxidation-reduction indicator).

The antibacterial activity of the compounds of formula I can be demonstrated in vitro by means of standard agar dilution tests.

Isosennsitest medium (Oxoid) and Todd-Hewitt medium (Difco) are used for growing staphylococci and streptococci respectively. Medium cultures are diluted so that the final inoculum is approx. IO<4> colony forming units/ml (CFTJ/ml). Minimal inhibitory concentration (MIC) is considered the lowest concentration that shows no visible growth after 18-24 hours of incubation at 37 °C. The results of the antibacterial testing of representative compounds of formula I are summarized in Table IX below.

Table IXa below reports MIC data for a significant number of the compounds prepared according to the invention on a clinical isolate of S.haemolyticus compared to data for teicoplanin (ie the structurally "equivalent" known compounds where the group Y in formula I represents OH). The experiments have been carried out essentially as described herein.

In addition, the following compounds which fall under the definition for formula I where A, B and M are the sugar groups defined therein, in the same experiments have shown improved MIC compared to teicoplanin:

As regards the compounds of formula I where A represents hydrogen and B and M represent the defined sugars, they show improved activity on S. epidermidis (see above with a view to the pathological relevance of this microorganism) and S. pyogenes, which usually occurs in connection with infections of the respiratory tract and may be responsible for rheumatoid fever and diseases. In this case too, the frequency of resistance to commonly used broad-spectrum antibiotics such as penicillins and cephalosporins is increasing.

Data for the exemplified compounds are given in Table IX of the specification, while data for the reference compound, i.e. "antibiotic L 17054" is 0.25 for S. epidermidis ATCC 12228 and 0.5 for S. pyogenes C203. Data for the defined compounds is consistently found to be better. In vivo data on a small number of such compounds confirm this improvement.

A similar type of improvement is also found for the compounds of formula I where the groups A and M represent hydrogen and B represents the defined sugar groups compared to the corresponding compound where Y is OH, i.e. "antibiotic L 17046". In addition to the improved activity on S.pyogenes and S.epidermidis, these compounds also show improvements on S.aureus and S.faecalis. The data for examples of the defined compounds are reported in Table IX of the description, while the reference compound (antibiotic L 17046) shows the following MIC in pg/ml under the same experimental conditions:

The overall significance with regard to improved activity will be apparent from the above stated and discussed data.

The EDso values (mg/kg) for representative compounds of formula I found by means of in vivo tests in mice experimentally infected with S. pyogenes L 49 according to the method described by V. Arioli et al., Journal of Antibiotics .29 , 511

(1976), is set out in Table X below:

In view of the antimicrobial activity indicated above, the compounds of formula I of the invention can be effectively used as an active ingredient in antimicrobial preparations used in human and veterinary medicine for the relief and treatment of infectious diseases caused by pathogenic bacteria which are susceptible to said active ingredients.

In such treatment, these compounds can be used as such or in the form of mixtures in any quantity ratio. The compounds of formula I can be administered orally, topically or parenterally, parenteral administration being however preferred. Depending on the method of administration, these compounds can be formulated into different dosage forms. Preparations for oral administration can be in the form of capsules, tablets, liquid solutions or suspensions. As is known in the art, the capsules and tablets can, in addition to the active ingredient, contain conventional excipients such as diluents, e.g. lactose, calcium phosphate, sorbitol and the like, lubricants e.g. magnesium stearate, talc, polyethylene glycol, binders, e.g. polyvinylpyrrolidone, gelatin, sorbitol, tragacanth, acacia, flavorings and acceptable disintegrants and wetting agents. The liquid preparations, which are generally in the form of aqueous or oily solutions or suspensions, may contain conventional additives such as suspending agents. For topical use, the compounds of formula I can also be prepared in suitable forms for absorption through the mucous membrane of the nose and pharynx or bronchial tissue and can conveniently take the form of liquid sprays or inhalants, pastilles or throat-brushing materials.

For medicating the eyes or ears, the preparation can be in the form of liquid or semi-liquid. Topical applications can be formulated in hydrophobic or hydrophilic bases such as ointments, creams, lotions, brushes or powders.

For rectal administration, the compounds are administered in the form of suppositories mixed with conventional carriers such as e.g. coconut butter, wax, spermacetate or polyethylene glycols and their derivatives.

Preparations for injection can take such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and can contain formulation agents such as suspending, stabilizing and/or dispersing agents. The active ingredient can alternatively be in powder form for reconstitution at the time of delivery with a suitable carrier, such as sterile water.

The amount of active ingredient to be administered depends on various factors such as the size and condition of the subject to be treated, the route and frequency of administration, and the causative agent involved.

The compound of formula I is generally effective at a dosage between 0.5 and 30 mg of active ingredient per kg body weight, preferably divided into 2-4 administrations per day. Particularly desired preparations are those produced in the form of dosage units containing from 20-300 mg per unit.

Representative examples of the preparation of pharmaceutical preparations are as follows: A parenteral solution is prepared with 100 mg of compound No. 3 dissolved in 2 ml of sterile water for injection. A parenteral solution is prepared with 250 mg of Compound No. 19 hydrochloride dissolved in 3 ml of sterile water for injection.

A topical ointment is prepared with 200 mg of Compound No. 19.

3.6 g polyethylene glycol 4000 U.S.P.

6.2 g polyethylene glycol 400 U.S.P.

EXAMPLE 1: (Procedure : Reaction of unprotected teicoplanin starting material with the selected amine and preparation of the acetate salt of the final compound)

Preparation of compounds No. 1-6. 26. 34. 35. 82. 87. 88 and 95

To a stirred solution of 1 mmol of teicoplanin Ag complex prepared as described in US 4,239,751 and 2 mmol of the selected amine in 20 ml of dimethylformamide (DMF), a solution of 1.1 mmol of diphenylphosphoryl azide (DPPA) in 5 ml of DMF is added dropwise for 10 minutes while cooling to 0-5'C. The reaction mixture is stirred for approx. 6 hours at 5°C overnight at room temperature, after which a solution of 0.5 mmol DPPA in 2.5 ml DMF is added dropwise at 0-5°C. Stirring is continued at room temperature for a further 24 hours, then 125 ml of ethyl ether is added and the solid which separates is collected, washed with 100 ml of ether and redissolved in 100 ml of a mixture of water:acetonitrile, 8:2 (v/v), adjusted at pH 2.5 with IN HCl. The resulting solution is applied to a chromatographic column, prepared with 250 g of silanized silica gel (0.063-0.2 mm; Merck) pre-equilibrated with a mixture of water:acetone:trile 8:2 (v/v). The column is developed with a linear gradient elution from 20% CH3CN in 0.001N HC1 to 80* CH3CN in 0.01N HC1 for 20 hours at a rate of 250 ml/hour.

Fractions of 25 ml are collected and monitored by HPLC. Fractions containing the pure title compound are collected and the resulting solution is brought to pH 8.5 with 1N NaOH, and an equal volume (v/v) of water is then added. This mixture is then extracted with butanol (v/v) and the organic layer is separated, washed with water and concentrated under vacuum at 40°C until most of the water is removed. The cloudy butanol solution is filtered, ethyl acetate (0.5 v/v, i.e. half a volume of solvent per volume of solution) is added and the resulting suspension (or cloudy solution) is extracted with water (0.5 v/v). The organic layer is concentrated to a small volume, ethyl ether is added and the solid that separates is collected, washed with ether, then dried in vacuo at 50°C overnight to give the title compound as the corresponding free base which is then dissolved in methanol (typically 1 g in 50-100 ml). Glacial acetic acid (0.5 ml per g of the free base) is added and the resulting solution is stirred for a few minutes at room temperature. On addition of ethyl ether (300-500 ml), a solid separates which is collected, washed with ether (100 ml) and dried overnight at room temperature, and this gives the title compounds as the corresponding monoacetate salt.

Preparation of compounds No. 7. 8. 9. 10. 11. 12. 28. 43.

44. 47

The above-mentioned procedure is essentially followed by using antibiotic L 17054 instead of teicoplanin Ag.

Preparation of compounds No. 14. 17. 50

The above-mentioned procedure is essentially followed by using antibiotic L 17046 instead of teicoplanin Ag.

EXAMPLE 2: (Procedure Ag: Reaction of unprotected teicoplanin starting material with the selected amine and preparation of the hydrochloride salt of the final compound)

Preparation of compounds No. 13. 18. 76. 77. 80. 81. 89 and 91

The reaction between teicoplanin Ag complex and the selected amine is carried out as described in example 1. Once the crude product of the title compound has been precipitated with ethyl ether and separated as a solid, it is suspended in methanol (approx. 1 g of substance in 100 ml of solvent). Water is added (v/v) and the resulting solution (or suspension) is brought to pH 2.5 with IN EC1. Silanated silicon dioxide gel (0.063-0.2 mm, 5 g per g impure product - Merck) and n-butanol (200 ml) are then added. The resulting suspension is stirred for a few minutes at room temperature after which the solvents are completely evaporated and the residue is placed on top of a chromatographic column containing the same type of silanated silica gel (100 g) equilibrated with the mixture water:acetonitrile, 95:5 (v/v). The column is developed with linear gradient elutions from 5 to 40# (in the case of compound 13) or 15-60* (in the case of compound 18) of CH 3 CN in 0.001N HCl, for 20 hours at a rate of 100 ml/hour. Fractions of 10 ml are collected and analyzed using HPLC (method b). Fractions containing the title compound are collected and concentrated under vacuum at 45 °C and by adding suitable amounts of n-butanol, an anhydrous, butanolically cloudy final solution is obtained (approx. 200 ml). After addition of IN HCl (0.2 mL), the solution is concentrated to a small volume under vacuum at room temperature (below 25°C). Precipitation with ethyl ether, washing with ether and drying in vacuo at 40°C overnight gives the title compound (as equivalent dihydrochloride).

EXAMPLE 3: (Procedure A3: Reaction of an unprotected teicoplanin starting material with an acid addition salt of the selected amine in the presence of a base)

Preparation of compounds No. 16. 38. 75. 85. 92 and 105 A solution of 0.6 ml (2.8 mmol) of DPPA in 2 ml of DMF is added to a stirred solution of 2.8 g (2 mmol) of antibiotic L 17046 and 0.6 g (4.2 mmol) glycine ethyl ester, hydrochloride, in 100 ml DMF at 0-5° C. After addition of 1.1 ml (8 mmol) triethylamine (TEA) the reaction mixture is stirred for 2 hours at 5° C overnight at room temperature. The course of the reaction is monitored by HPLC (method b). The resulting solution is poured into 500 ml of ethyl ether and the precipitate that forms is collected and redissolved in 500 ml of a mixture of water:acetonitrile, 7:3 (v/v), while the pH is adjusted to 2.3 with 1N HCl. After addition of 600 ml of n-butanol and 200 ml of water, the mixture is brought to pH 8.2 with 1N NaOH with vigorous stirring. The organic layer is separated, washed with 400 ml (2 x 200 ml) of water, and then concentrated to a small volume (about 50 ml) at 50°C under vacuum. On addition of ethyl ether (200 ml) a solid separates (the title compound as the free base) which is collected and redissolved in 200 ml of methanolic

0.02 M HCl. On addition of ethyl ether (500 ml), a precipitate separates which is collected, washed with ether and dried in vacuo at 40°C overnight, and this gives 1.62 g of compound 16 (as the corresponding hydrochloride).

EXAMPLE 4; (Procedure A4: Reaction of an unprotected teicoplanin starting material with an HNR<1>R<2->amine having an additional amino group and/or additional carboxyl groups, all of which are protected, and subsequent deprotection by catalytic hydrogenation).

Preparation of compounds No. 29. 36. 37. 39. 40. 46. 51.

64. 71. 84 and 90

The procedure in the first part of example 1 (procedure Ai) is substantially followed.

Once the condensation product containing either protected additional amino or carboxy functions is obtained, the protection is removed by catalytic hydrogenation using palladium on carbon as described in the second part of Example 6 below, method ).

EXAMPLE 5: (Procedure A5: Reaction of an unprotected teicoplanin starting material with an HNR<1>R<2->amine having an additional amino group and/or additional carboxyl groups that are protected and subsequent deprotection in acidic medium).

Preparation of Compounds Nos. 48 and 57

The procedure in the first part of example 1 (procedure Ai) is essentially followed.

The amine chosen in this case is an amine compound containing additional carboxyl functions which are protected with groups which can be removed under anhydrous acidic conditions such as glutamic acid di-butyl ester. Once the condensation product containing the protected carboxy functions is obtained, the protection is removed in an acidic medium consisting of anhydrous trifluoroacetic acid (as described in the second part of Example 7 below, method Bg).

EXAMPLE 6: (Procedure Bj_: Reaction of an N-protected teicoplanin starting material and a selected amine followed by deprotection by catalytic hydrogenation).

Preparation of Compounds Nos. 9, 13, 22, 54, 55, 61 and 73

a) Preparation of the N-benzyloxycarbonyl-protected starting material (NCBzO-ST)

A solution of 0.45 ml of benzyl chloroformate in 10 ml of dry acetone is added dropwise, while cooling at 0-3°C, to a stirred solution of 2 mmol of the selected teicoplanin starting material and 0.5 g of NaHCO 3 in 150 ml of a mixture of acetone:water, 2:1 (v/v). After approx. 30 min. 500 ml of water are added and the resulting solution is extracted with 500 ml of ethyl ether. The aqueous layer is adjusted to approx. pH 3.5 with 1N HCl and then extracted with 500 ml of n-butanol. The organic layer is separated, washed with 400 ml of water (2 x 200 ml) and then concentrated to a small volume at 45°C under vacuum. Addition of ethyl ether separates a solid which is collected, washed with ether and dried at room temperature in vacuo overnight, and this gives the N-CBzO derivative of the teicoplanin starting material with a purity (HPLC titer > 90$, method c) which is sufficient for the next step (yield

> 80%).

b) Preparation of the N-CBzO derivative of the teicoplanin-amide compound

The condensation of the above N-benzoyloxycarbonyl starting material with the selected amine is carried out in DMF (HPLC, method c) in the presence of DPPA under the same reaction conditions as described in Example 1. The N-CBzO-teicoplanin amld compound is obtained as a solid which precipitates from the reaction mixture by adding ethyl ether.

c) Preparation of the teicoplanin-amide derivative of the title compound

The crude N-CBzO-teicoplanin amide (1

g) is dissolved in a mixture (100 ml) of methanol:0.1N hydrochloric acid, 7:3 (v/v) and the resulting solution is hydrogenated by

room temperature and pressure in the presence of 0.5 g 5* Pd/C. The reaction mixture is filtered and to the clear filtrates is added a mixture of silanated silica gel (0.063-0.2 mm; 4 g Merck) and n-butanol (60 ml). The solvents are then evaporated under vacuum at 45°C and the residue applied to a chromatographic column containing the same type of silanated silica gel (100 g) prepared in a mixture of water:acetonitrile, 95:5 (v/v).

The column is developed with a linear gradient elution from 5*

(compound 9), 10* (compound 13) and 20* (compound 22) CH3CN in 0.001N HCl to 30*. 40* and 55*, respectively, CH 3 CN in HgO for 15 hours at a rate of 120 ml/hour. Fractions of 12 ml each are collected and analyzed by HPLC. Fractions containing the pure compounds of the title compound are collected and to the resulting solution is added n-butanol (v/v) and 1N HCl (2 ml). After concentration to a small volume under vacuum at 40°C, the title compounds (as the corresponding dihydrochloride) are obtained by precipitation with ethyl ether from the butanol phase, washing and drying overnight in vacuum at 40°C.

EXAMPLE 7: (Procedure Bg: Reaction of an N-protected teicoplanin starting material with a selected amine followed by deprotection by acid hydrolysis). Preparation of compounds No. 11. 14. 18. 19. 20. 21. 23. 24. 25. 31. 52. 53. 78. 79. 83. 94. 97. 99. 100. 101 and 102

a) Preparation of the N-tert-butoxycarbonyl-protected teicoplanin starting material (N-t-BOC-ST)

A mixture of 4 mmol of the selected teicoplanin starting material, 2 ml (14.5 mmol) TEA and 2 g (~7 mmol) tert-butyl-2,4,5-trichlorophenylcarbonate in 100 ml DMF is stirred for 24 hours at room temperature . On addition of ether (900 ml), a solid is separated which is collected and redissolved in a mixture (1:1) of water:methanol, 7:3. The resulting solution is brought to pH 3.5 with 1N HCl, then extracted with ether (500 mL). The aqueous layer is extracted again with n-butanol (1 1). The butanol layer is washed with water (2 x 500 ml) and concentrated to a small volume under vacuum at 35°C. Addition of ethyl ether precipitates a solid which is collected, washed with ether and dried in vacuo at 40°C overnight to give (yields are always higher than 90*) the sufficiently pure N-t-BOC-protected teicoplanin starting material (HPLC titer > 90*, method c) for the next step.

b) Preparation of the N-t-BOC derivative of the teicoplanin-amide compound

The condensation of the above obtained N-t-BOC-protected teicoplanin starting material with the selected amine is carried out in DMF (HPLC, method C) in the presence of DPPA under the same conditions as described in Example 1. As is the case for N-CBzO-teicoplanin -amide (see Example 4 b), the impure N-t-BOC-teicoplanin amide obtained from the reaction mixture after precipitation with ethyl ether is pure enough for use in the deprotection step. c) Preparation of the teicoplanin-amide derivative of the title compound.

A solution of 1 mmol of N-t-BOC-teicoplanin-amide in 40 ml of 100* trifluoroacetic acid (TFA) is stirred for 10-20 min. at 5°C, after which the solvent is evaporated under vacuum at 25°C. The oily residue is triturated with ether, then collected and redissolved in 150 ml of methanol. Silanated silica gel (0.063-0.2 mm 5 g Merck) is added and the solvent is evaporated under vacuum at 40°C. The residue is placed on top of a column containing the same silanated silica gel (150 g) prepared in the mixture water:acetonitrile, 95:5 (v/v). Column chromatography is performed essentially according to the method described in Example 4 c. More specifically, the column is developed with a linear gradient elution from 5* CH3CN in 0.001N HCl to 30* CH3CN in H2O in the case of compound 9, with a linear gradient elution from 10* CH3CN in 0.001N HC1 to 40* CH3CN in H20 in the case of compound 14, and with a linear gradient from 20* CH3CN in 0.001N HC1 to 55* CH3CN in water in the case of compound 22. The flow rate is 120 ml/hr and the time is 15 hours. Fractions of 12 ml are collected, monitored by HPLC and worked up essentially as already described in example 4 c.

Fractions containing the pure title compounds are collected and to the resulting solution is added n-butanol (v/v) and 1N HCl (2 ml). After concentration to a small volume under vacuum at 40°C, the title compound is obtained (as the corresponding dihydrochloride, with the exception of compound No. 25 which is recovered as the mono-hydrochloride) by precipitation with ethyl ether from the butanol phase, washing and drying overnight under vacuum at 40°C.

EXAMPLE 8:

Preparation of the trifluoroacetate salts of the teicoplanin-amide compounds 18-25.

A teicoplanin amide compound (amides 18-25) is dissolved (1 g in 300 ml) in a mixture of water: acetonitrile, 8:2 (v/v). The resulting solution is brought to pH 8.5 with 0.1N NaOH and extracted (v/v) with n-butanol. The organic layer is separated, washed with water (v/v) and concentrated to a small volume. Upon addition of ether, the solid which separates is collected, washed with ether and dried overnight in vacuum at 35°C, and this gives the corresponding free base which is redissolved (1 g in 10 ml) and precipitated with ethyl ether (100-200 ml). After collecting the solid by filtration, washing with ether and drying in vacuo for 24 hours at room temperature, the title compounds (18-24, di-trifluoroacetates and 25 trifluoroacetates) are obtained.

EXAMPLE 9: (Procedure Fj: Transesterification and ester function-hydrolysis of a compound of formula I)

Preparation of compound 17

In a container protected by a soda-lime valve, add dropwise a solution of 3 mL of methanolic IM KOE (85* commercial pellets) at room temperature to a stirred solution of 1.05 g (~0.7 mmol) of compound 16 ( hydrochloride) in 60 ml of methanol. After one hour, a further 0.75 ml of 1 M KOE in methanol is added and stirring is continued for 30 min. (HPLC, method b). The reaction mixture is then cooled to approx. 5°C and 3.75 ml of IN EC1 are added. The resulting solution is diluted with 200 ml of H 2 O and 100 ml of CE 3 CN. Silanated silica gel (0.063-0.2 mm, 5 g; Merck) and n-butanol (400 ml) are then added and the solvents are evaporated under vacuum at 40°C.

The residue is placed on top of a column containing 200 g of the same silanized silica gel prepared in E20. The column is developed with a linear gradient from 1-60* CE3CN in E20 for 20 hours at a rate of 250 ml/hour and then with a linear gradient from 60* CE3CN in E20 to 70* CE3CN in 0.01N EC1 for 60 hours at a rate of 150 ml/hour. Fractions of 25 ml each are collected, analyzed by HPLC and the fractions containing compound 17 (241-254) are collected. 200 ml of n-butanol is added to the resulting solution which is then concentrated to a small volume under vacuum at 45°C to obtain a butanol suspension. On addition of ethyl ether, a solid separates which is collected, washed with ether and dried in vacuo at 30°C overnight, yielding 0.795 g (~78*) of pure compound 17.

By essentially following this method, but using larger amounts of methanolic KOH and/or by extending the reaction time as needed, the corresponding compound having a free carboxy function instead of the methoxycarbonyl function can be obtained.

EXAMPLE 10: (Procedure B3: Reaction of an unprotected or N^-protected teicoplanin starting material with an HNR^R<2->amine having an additional amino group and/or additional carboxyl groups, all of which are protected)

Preparation of compounds 68 and 72

A solution of 3 ml (approx. 14 mmol) of DPPA in 25 ml of DMF is added dropwise to a stirred solution of 12 mmol of teicoplanin Ag complex (in the case of the preparation of compound 68) or N<15->CBzO-deglucoteicoplanin (in the case of preparation of compound 72), 13 mmol N<c->CBzO-lysine methyl ester, hydrochloride and 24 mmol triethylamine (TEA) in 225 ml DMF, for 10 min. while maintaining the temperature at 0-5°C. After stirring for 4 hours at 0-5°C and 24 hours at 20°C, the reaction mixture is poured into 1.5 L of ethyl ether and the precipitate that forms is collected by filtration and redissolved in 500 ml of a mixture of methanol:water, 4:1 ( v/v). The resulting solution is cooled to 10°C and 800 ml of n-butanol is added thereto with stirring. After adjusting the pH at about 8.3 (with 1N NaOH), the organic layer is separated, washed with 800 ml (2 x 400 ml) of water, and then concentrated to a small volume (approx. 100 ml) under reduced pressure at 40° C. When ethyl ether (400 ml) is added, a solid is separated which is collected and dried in vacuo at 40 ° C overnight, and this gives the title compound.

By essentially repeating the same procedure, but by starting with teicoplanin Ag component 1, 2, 3, 4 or 5, the corresponding derivative of the pure individual components is obtained.

EXAMPLE 11: (Procedure Fg: Ester function hydrolysis of a compound of formula I)

Preparation of compounds 64, 69, 86 and 104

A solution of 5 g of KgCO3 in 500 ml of HgO is added with stirring at room temperature to a solution of 4 mmol of compound 63 (for the preparation of compound 64), 68 (for the preparation of compound 69), 85 (for the preparation of compound 86), and 105 (for the preparation of compound 104), in 500 ml of a mixture of methanol: water, 1:1 (v/v). After addition of 750 ml of n-butanol, the resulting mixture is stirred vigorously for 36 hours. The organic layer is separated, the aqueous phase is brought to pH 3.5 with 1N HCl and then extracted with 500 ml of n-butanol. The butanol solutions are combined, washed with 600 ml of HgO (2 x 300 ml) and concentrated to a small volume (50 ml) under vacuum at 40°C. On addition of ethyl ether (350 ml), a solid is separated which is collected and dried in vacuo at room temperature overnight, giving the title compound.

EXAMPLE 12: (Procedure F3: Esterification of a compound of formula I in which the group -NR^-R<2> contains carboxyl functions)

Preparation of compound 51

A stirred suspension of 4.1 g (~2 mmol) of compound 27 in 200 mL of 2.5 M HCl in absolute ethanol is refluxed for 5 h. The reaction mixture is then concentrated to a small volume (~40 ml) at 50° C. under vacuum. On addition of ethyl ether (~260 ml), a solid is separated which is collected by filtration and redissolved in 50 ml of a mixture of acetonitrile:water, 1:1 (v/v). After adding 150 ml of HgO, the resulting solution is placed on a column of 400 g of silanated silica gel (Merck) in HgO. The column is developed with a linear gradient from 20-70* of CH3CN in 0.001N HC1 for 20 hours at a rate of 200 ml/hour, while 20 ml fractions are collected and analyzed by HPLC. The fractions containing the pure title compound are combined and the resulting solution brought to pH 8.0 with 2* NaHCO 3 . After extraction with n-butanol (v/v), IN HC1 is added (2.5 ml IN HC1 per 100 ml of the butanol solution) and the resulting organic solution is concentrated to a small volume and this gives a dry butanol suspension which at addition of ethyl ether (v/v) gives a solid which is collected by filtration and dried in vacuo at 40°C overnight to give 0.97 g of pure compound 51 as the dihydrochloride.

EXAMPLE 13: (Procedure G: Separation of the amides of teicoplanin Ag complex into their components by reverse phase column chromatography)

Preparation of compounds 2b. 32b. 32c and 71

A solution of 5 mmol of the starting amide derivative of teicoplanin Ag complex in 250 ml of a mixture of acetonitrile:-water, 1:1 (v/v) is adjusted to pH 3.5 with IN HCl, after which most of the organic solvent is evaporated under vacuum at 20°C to obtain a slightly cloudy solution which is applied to a column of 1 kg silanated silica gel (Merck) in HgO. The column is developed with a linear gradient from 20* of CH 3 CN in HgO to 60* of CH 3 CN in 0.001 N HCl for 20 hours at a rate of 200 ml/hour, while 20 ml fractions monitored by HPLC are collected. The fractions containing the amide of teicoplanin Ag component

2 are collected. Appropriately, the fractions containing the amides of components 1-3 and 4 and 5 are also collected, respectively. Each solution is then concentrated to a small volume after addition of suitable amounts of n-butanol to obtain a dry butanol suspension from which the title compounds, like the free bases, are precipitated as usual with ethyl ether. The addition of a small excess of 1N HCl or trifluoroacetic acid before concentration gives the corresponding hydrochlorides or trifluoroacetates, respectively.

EXAMPLE 14: (Procedure A^,: Reaction of an unprotected teicoplanin starting material with the α-amino group of Nco-nitro-arginine, the methyl ester hydrochloride, followed by cleavage of the protecting nitro group in the resulting compound)

Preparation of compounds 33 and 42

The first part of the reaction, which started from 16 g (~8 mmol) teicoplanin Ag and 12 mmol Nco-nitro-arginine methyl ester hydrochloride, is carried out according to method A3 described in Example 3, which gives 14 g of compound 105.

A solution of 14 g (~6.5 mmol) of this compound in 200 ml of 90* aqueous acetic acid is treated with 3.6 g (~55 gatom) of zinc powder with vigorous stirring at room temperature. The resulting suspension is stirred for 30 minutes at room temperature and then filtered. By adding ethyl acetate (-800 ml) to the filtrate, a powder (~13 g) is separated which is collected by filtration and purified by reverse-phase column chromatography on 700 g of silanated silica gel according to the method described in example 1, and this gives 10.2 g of the title compound as the free base (yield of this reaction from compound 105 is about 75*).

EXAMPLE 15: (Procedure A7: Reaction of an N<15->protected or unprotected teicoplanin starting material with the α-amino group in Nco-nitro-arginine methyl ester or -benzyl ester, respectively, followed by removal of the N<15->t -B0C- or N^-CBzO- and benzyl protecting groups in acid medium according to method A^, or by catalytic hydrogenation, according to method A<4>, respectively, and finally displacement of the nitro group according to method A^,).

Preparation of compounds 32a. 32b. 32c. 41. 59. 60. 98. 103 and 104

In the first step, starting from teicoplanin Ag (complex or a single component thereof), N^-t-BOC-deglucoteicoplanin or N<15->CBzO-deglucoteicoplanin and the appropriate Nu-nitro-arginine deriv., the respective protected title compounds. By treatment with 100* trifluoroacetic acid the N^-^-t-BOC protecting group is removed and by catalytic hydrogenation over 5-10* Pd/C the N<15->CBzO and benzyl groups are also displaced.

The nu-nltro derivatives of compounds 32a, 59, 60 and 98 are thus obtained. The Nco-nitro group is then removed by following procedure A, as described in Example 14, yielding the title compounds.

EXAMPLE 16: (Procedure As: Reaction of teicoplanin with 4-morpholinetanamine followed by acid hydrolysis).

Teicoplanin Ag and 4-morpholinethanamine are reacted according to the method indicated in example 1 (method A^) and the product obtained is suspended in 50 ml of absolute trifluoroethanol (TFE), stirred at 75°C for 12-16 hours while bubbling dry HCl, after which the cooling to 10°C and settling overnight at this temperature. After addition of 20 ml of ethyl ether, the impure final compound is recovered from the reaction mixture as a dark yellow powder. Purification by column chromatography as indicated in Example 6c gives the pure compound.

Claims (1)

Analogous process for the preparation of a therapeutically active teicoplanin compound having the formula: where Y represents a group where R<1> represents hydrogen, (C1-C5)alkyl, hydroxy(C2-C4)- alkyl, halo(C2-C4)<a>lkyl, (C1-C4)alkoxy(C2-C4)alkyl, amino(C2-C4)alkyl, (C1-C4)alkylamino(C2-C4)alkyl, di(C1 -C4)alkylamino(C2-C4)alkyl, R<2> represents hydrogen, (C^-Cf, )alkyl, hydroxy(C2-C4 )- alkyl, halogeno(C2-C4)alkyl, (Ci~C4)alkoxy(C2-C4)alkyl, a nitrogen-containing 5-6 membered heterocyclic ring which is fully saturated and where nitrogen atoms may be substituted with a benzyl residue or a bridging C2- alkylene chain attached to one of the C atoms in the heterocyclic ring; a group -alk-W where "alk" represents a linear alkylene chain of 1-8 carbon atoms optionally substituted with a substituent selected from (C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, aminocarbonyl, ( cl~c4)alkylaminocarbonyl, di(C1-C4)alkylaminocarbonyl, (cl~c4 )alkyloxycarbonyl, phenyl(Ci~C4)alkyloxycarbonyl, and W represents carboxy, (C1-C4)alkylcarbonyl, phenyl(Ci~C4)alkylcarbonyl, aminocarbonyl , ()aminocarbonyl, di(C1-C4)aminocarbonyl, pentosaminocarbonyl, hexosaminocarbonyl, ureido, guanidino, pyrrolidine where the N atom may be (C1-C4) alkyl substituted, pyridine or morpholine, a group of the formula where R<3> and R<4> each independently represent hydrogen, (cl~c6)alkyl, hydroxy(C2-C4)alkyl and halo(C2~C4)alkyl, or R<4> represents phenylmethyloxycarbonyl and R<3> represents hydrogen; a group with the formula: where R*1, R<7> and R<8> each independently represent a (C 1 -C 4 )alkyl, or R<1> and R<2> together with the adjacent nitrogen atom represent piperazino, substituted in the 4-position with a methyl group or a pyridinyl group, morpholino or thiomorpholino; A represents hydrogen or -N[(C^o-Cn)aliphatic acyl]- 3-D-2-deoxy-2-amino-glucopyranosyl; B represents hydrogen or N-acetyl-e-D-2-deoxy-2- amino-glucopyranosyl; M represents hydrogen or α-D-mannopyranosyl; provided that B represents hydrogen only when A and M are simultaneously hydrogen and M represents hydrogen only when A is hydrogen, and with the additional proviso that when W represents a group a group ureido, guanidino or a nitrogenous 5-6 membered heterocyclic ring as defined above directly connected to the "alk" moiety through a bond with a ring nitrogen atom, then the linear alkylene "alk" moiety must have at least two carbon atoms; as well as acid addition salts thereof, characterized by reacting a teicoplanin starting material with the formula: where R is hydrogen or a protecting group for the amide function; Y is hydroxy; A represents hydrogen or -N[(<C>iø~<c>ll)-aliphatic acyl]-ε-D-2-deoxy-2-amino-gluco-pyranosyl; B represents hydrogen or N-acetyl-p<->D-2-deoxy-2-amino-glucopyranosyl, M represents hydrogen or α-D-manno-pyranosyl, provided that B can represent hydrogen only when A and M are simultaneously hydrogen, and M can represent hydrogen only when A is hydrogen; a salt thereof or a mixture thereof in any proportion; with a molar excess of an amine of the formula HNR^R<2>, where R<1> and R<2> have the meanings given above and where the reactive functions, other than the amlnofunction, which are to be reacted with the carboxyl part of the teicoplanin starting material, is protected in an inert organic solvent and in the presence of a small molar excess of a condensing agent selected from (C1-C4) alkyl, phenyl or heterocyclic phosphorazidates such as dlphenyl phosphorazidate (DPPA), diethyl phosphorazidate, di(4-nitrophenyl) phosphorazidate, dimorpholyl phosphorazidate and diphenyl phosphorochloridate and remove the protecting groups if such are present.