FI62100C - FOERFARANDE FOER FRAMSTAELLNING AV 1-N-SUBSTITUERADE DERIVAT AV 4,6-DI- (AMINOGLYCOSYL) -1,3-DIAMINOCYCLITOLERNA GENTAMICIN B GENTAMICIN C1 GENTAMICIN C1A SISOMICIN VERDAMICIN ANTIBIOTIC JIB - Google Patents

Description

RSr ^ l ΓΒΐ «« KUULUTUSJULKAISU, 01nn

jjgfA IBJ (11) PUBLISHING LETTERS OZlUU

c (45fr tontti n yö nn: · l Ly 10 11 1932 (51) Q.lk?/lnt.CI.3 C 07 H 15/22 SUOMI — FINLAND (21) Pttenulh * kenHi «- PMmcmeknlni 230) + / 7h (22) H * k * ml »p * lvl - AnaBknlnftday 01 _ Qg. y] t '' (23) Alkupllvt — GHtlfhatadag 01. () 8.7) 1 (11) Tullut Julkliakil— Bllvlt offantllg. ...

Patentti · ja rekisterihallitut .... 'B i' (41) Nlhtlvikilpenon Ja kuuLJulkalsun pvm. -

Patent and Registration Board Antttkan posted and published ) 389751, i + 52600, i + 52586 (71) Oy Kolster Ab, Lönnrotsg. 19 B, Helsinki, Suomi-Finland (FI) (72) Marvin Joseph Weinstein, East Brunswick, New Jersey, Peter John Lovell Daniels, Cedar Grove, New Jersey, Gerald uoward Wagman,

East Brunswick, New Jersey, Raymond Testa, Verona, New Jersey,

Alan Keith Mallams, West Orange, New Jersey, John Jessen Wright,

Orange, New Jersey, Tattanahalli Lakshminarayan Nagabhushan,

East Orange, New Jersey, USA (US) (5) +) Process for preparing 1-N-substituted derivatives of) +, 6-di - (aminoglycosyl) -1,3-diaminocyclitols gentamicin B, gentamicin C- | , gentamicin C ^α, sisomicin, verdamicin, antibiotic JI-20B, antibiotic G-52, mutamicin 2 and mutamicin 6 - Menetelmä L, 6-di- (amino-glycosyl) -1,3-diaminocyclitolien gentamisiini B, gentamisiini The present invention relates to a process for the production of 1-N, C substituted derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclycols gentamicin B, gentamicin C, gentamicin C, sisomicin, verdamicin, X1 cl antibiotic JI-20B, antibiotic G-52, mutamicin 2 and mutamicin 6, wherein the 1-N substituent is -ch 2x where X is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl, or tolyl containing aliphatic groups -7 carbon atoms, and if substituted with amino and hydroxy, these substituents are present on various carbon atoms, as well as pharmaceutically acceptable acid addition salts of these compounds.

2 62100

The novel 1-N-substituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol derivatives have the all-new formula b a-C-c NH 2. (5- µg in 0H L · * -o 0 4 "(nhch, iy \ h34_ / i- 3 | 3" 2 "

OH

where X has the meaning given above and substituents a - in the dotted line in the 4 '- 5' position are as defined in the following table: (6 ') (6') (6 ') (41) (3') (2 ') (5) (5) 4' - 5 'ANTIBIOTIC abcdfghi *)

SISOMICIN H H NH H H NH H OH

2 2

'/ ERDAMIC IN H CH3 NH2 H H NH2 H OH

ANTIBIOTIC G-52 H H NHCH3 H H NH2 H OH

MUTAMICIN 2 H H NH „H H NH H OH

2 2

MUTAMICIN 6 H H NH2 H H NH2 OH H

GENTAMICIN C, H H NH_ H H NH_ H OH

la 2 2

GENTAMICIN ^ CH3NH CH ^ H H H NH2 H OH

GENTAMICIN B H H NH2 OH OH OH H OH

ANTIBIOTIC JI-20B H CH3 NH2 OH OH NH2 H OH

*) = double bond - single bond 3 62100

The aforementioned 1-N-substituted antibiotics and their acid addition salts are well-known very active broad-spectrum antibiotics. It has now surprisingly been found that by inserting the substituent -CH2 X1 amino group at position 1 of the general formula I, the general activity is improved; In particular, it may be mentioned that the new 1-N-substituted compounds (and their addition salts) are also effective against such bacteria that have become resistant to the known 1-N-unsubstituted antibiotics.

Included among the substituents proposed for the moiety CH 2 X are straight and branched alkyl groups, such as ethyl, n-propyl, n-butyl, 3-methylpropyl, n-pentyl, 3-methylbutyl, 3-methylbutyl and / dimethylpropyl; n-hexyl, S-methyl-pentyl, 3-ethylbutyl, Y-ethylbutyl, n-heptyl, 6-methylheptyl, / 3-ethylpentyl, Y-ethylpentyl, S-ethylpentyl, Y-propylbutyl, n-octyl, iso-octyl ,? -ethylhexyl, ό-ethylhexyl, ω-ethylhexyl, / 6-propylpentyl, Y * -propylpentyl; alkenyl groups such as β-propenyl, -methylpropenyl, / 4-butenyl, β-methyl-γ-butenyl, ethyl-β-hexenyl, cyclic groups such as cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cyclopentylethyl; aromatic groups such as o-, m-, and p-methyl-benzyl hydroxy-substituted straight and branched alkyl groups, such as β-hydroxy-pentyl, β-hydroxy-Y-methylbutyl> / 3-hydroxy-r / 3-methylpropyl, < 6-hydroxybutyl, / 3-hydroxypropyl, Y-hydroxypropyl, CO-hydroxyoctyl; amino-substituted straight and branched alkyl groups, such as 6-aminopentyl, -aminopropyl, γ'-aminopropyl, 6-aminobutyl, S-amino-γ'-methylbutyl and CO-aminoctyl and mono-N-alkylated derivatives thereof, such as N-methyl , N-ethyl, and the N-propyl derivatives, for example β-methylaminopentyl, β-methylaminopropyl, β-methylaminopropyl, 6-methylaminobutyl, β-methylamino-Y-methylbutyl and C C>-methylaminobutyl; amino and hydroxy substituted straight and branched alkyl groups, such as / S -hydroxy-β-aminopentyl, Y'-hydroxy-V-methyl-6-aminobutyl, β-hydroxy-S-aminobutyl, / d-hydroxy-Y * -aminopropyl and / 3-hydroxy-6-methyl-γ-aminopropyl; and mono-N-alkylated derivatives thereof, such as /? -hydroxy-amin-methylaminopentyl, Y ^-hydroxy-Y-methyl-ό-methylaminobutyl, β-hydroxy-6-methylaminobutyl, / O-hydroxy-Y-ethylaminopropyl and A-Hydroxy-β-methyl-Y-methylaminopropyl,

The pharmaceutically acceptable acid addition salts of the 1NC 2 X 6, 6-di- (aminoglycosyl) -1,3-diaminocyclitols are customary, prepared by known methods, such as by neutralizing the free base with the acid to about pH 5. Suitable acids for for this purpose, such as hydrochloric, sulfuric, phosphoric, nitric, hydrobromic, acetic, propionic, malic, ascorbic, citric and the like are included. These acid addition salts are white solids, which are soluble in water, soluble in most polar solvents, and insoluble in non-polar organic solvents.

1- N-CH 2 X 6, 6-di- (aminoglycosyl) -1,3-diaminocyclitols of formula I and their non-toxic, pharmaceutically acceptable acid addition salts generally exhibit broad-spectrum antibacterial activity. In particular, the 1-N (lower alkyl) derivatives exhibit improved antibacterial activities compared to the antibiotic compounds, which is specifically evident by the enhanced activity of the compounds against organisms resistant to the parent compound. Thus, for example, the compounds are more active against organisms that inactivate the parent antibiotic compounds by acylation of the 3-amino group and / or by adenylation of the 2 "hydroxy group. Some of these also exhibit anti-protocellal, anti-amoebic and antelmintic properties.

In the 1-N substituent, X is preferably hydrogen, alkyl, aminoalkyl and hydroxyalkyl having up to 7 carbon atoms and aminohydroxyalkyl having up to 3 carbon atoms and carrying the substituents on various carbon atoms.

Particularly useful compounds of the invention are those wherein X is hydrogen, methyl, ethyl or propyl, especially methyl or ethyl. A particularly valuable class is the 1N-CH 2 X 2 -aminoglycosyl-β-garosaminyl 3 -deoxyseptoramines of Formula I, wherein X is lower alkyl of 1-3 carbon atoms, especially the 1N (lower alkyl) derivatives of gentamicin C, gentamicin C, sisomicin and verdamicin, which in turn are antibacterial broad-spectrum agents active against gram-positive bacteria (such as Staphylococcus aureus) and gram-negative bacteria (such as Escherichia coli and Pseudomonas aeruginosa), as determined by standard dilution. bacteria resistant to 1-N unsubstituted precursors. Particularly valuable are 1-N-ethyl-verdamicin and 1N- (lower alkyl) -isomicins, such as 1-N-methylsisomicin, 1-N- (n-propyl) -isomicin, 1-N- (n-rbutyl) -isomicin and preferably 1-N-ethylsisomicin, which exhibit activity against Gram-negative organisms resistant to their 1-N unsubstituted precursors. Other particularly valuable compounds are 1-N-ethyl-gentamicin C ^, 1-N-ethylgentamicin, 1-N-ethyl antibiotic G-52, 1-N- (n-propyl) -verdamicin, 1-N (-aminobutyl) ) -sisomicin, 1-N-methylverdamicin, 1-N- (n-butyl) verdamicin, 1-N- (S-2-hydroxy-4-amino-butyl) -gentamicin, 1-N- (S-2- hydroxy-4-aminobutyl) -isomicin and 1-N- (8-2-hydroxy-4-aminobutyl) -verdamicin.

The majority of the 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol antibiotics from which the 1-N-substituted derivatives of the invention can be prepared are known in the art. The starting compound described herein as gentamicin is isolated and characterized according to Preparation 1.

5 621 00

It herein as gentamicin c. designated the starting compound, isolated 2b and characterized in Preparation 2 and exhibiting the structural formula herein, referred to in some literature gentamicin C.

The isolation, properties and planar configuration of gentamicin C are described in U.S. Patent No. 3,651,042.

Antibiotics 66-40B and 66-40D, their preparation, isolation, properties and configuration are described in Belgian patent 811 370. Said antibiotics are produced together with sisomicin, the main product in the fermentation of Micromonosprora inyoensis (described in British patent 1,274,518). and is separated from the fermentation medium using special chromatographic separation technique.

The mutamicins 2 and 6, the configuration of which is shown above, can be prepared by culturing a strain of Micromonospora inyoensis, herein designated Micromono-spora inyoensis 1550F-1G, in a nutrient aqueous medium. This mutant strain is unable to produce an antibiotic in culture under Submer's aerobic conditions in a nutrient-water medium in the absence of the 1,3-diaminocyclitol building block. However, when certain of such compounds are added to the fermentation medium, the mutamicins are produced. When 2-deoxypertyptamine is added to the fermentation, the known antibiotic sisomicin is produced.

The required 1,3-diaminocyclitols which must be present during the fermentation to produce mutamicins are as follows: 2,5-dideoxyistreptamine for mutamicin 2, and 5-epi-2-deoxysterptamine for mutamicin 6.

The preparation of mutamicin has been described in the Swedish patent 7 409 945-8.

According to a suitable process according to the invention, 1-N-substituted derivatives of the 4,6-di (aminoglycosyl) -1,3-diaminocyclitols gentamicin B, gentamicin C, gentamicin C, sisomicin, verdamicin, antibiotic are prepared.

X

JI-20B, antibiotic G-52, mutamicin 2 and mutamicin 6, wherein the 1-N substituent is -ch 2x where X has the meaning previously defined. and pharmaceutically acceptable acid addition salts thereof, thus treating any of said 4,6-di (aminoglycosyl) -1,3-diaminocyclitols which may have amino protecting groups at any position other than the 1 position, with an aldehyde of the formula

X'-CHO

Wherein X 'is the same as X, however any amino or hydroxy group present may be protected, in the presence of a hydride-donor reducing agent, and if so required, all protecting groups present in the molecule are removed, whereafter the derivative is isolated as usual or in the form of a pharmaceutically acceptable acid addition salt.

This process, whereby the 1-amino function of a 1-N unsubstituted 4,6-di (aminoglycosyl) -1,3-diaminocyclitol is selectively condensed with an aldehyde and then reduced in situ to form a 1-N-alkyl 1-4, 6-di (aminoglycosyl) -1,3-diaminocyclitol antibacterial agent is usually carried out at room temperature in the presence of air, although it may be advantageous to perform it under an inert atmosphere (such as argon or nitrogen). The reaction is advantageously completed in a short period of time, usually less than 30 minutes, as determined by thin-layer chromatography.

Hydride donor reducing agents useful in this process include dialkylaminoboranes (such as dimethylaminoborane, diethylaminoborane and preferably morpholinoborane), tetralkylammonium cyanoborohydride (such as tetrabutylammonium anhydride, anhydride, anhydride, anhydride,

The process is conveniently carried out in an inert solvent. By "inert solvent" is meant any organic or inorganic solvent in which the U, 6-di- (aminoglycosyl) -1,3-diaminocyclitol starting compounds and the reactants are soluble and do not interfere with the process under the reaction conditions, thus reducing the competing side reactions. a minimum. Although anhydrous aprotic solvents can sometimes be used advantageously in the process (such as tetrahydrofuran when using morpholinoborane as hydride donor reducing agent), the process is usually carried out in protic solvents, such as in a lower alkanol, or preferably in water or in water / lower alkanol (as diluted). methanol or ethanol), although other water-miscible co-solution systems can be used, such as water / dimethylformamide, water / hexamethylphosphoramide, water / tetrahydrofuran and water / ethyl glycol dimethyl ether.

The process is conveniently carried out at a pH in the range 1-11, preferably 2-5, and proceeds best in the range 2.5-3.5. The particularly suitable acidic medium can be obtained by adding an organic or inorganic acid to 6,6-di (aminoglycosyl) -1,3-diaminocyclitol. In this way, the acid addition salts are formed by the compounds. Any organic acid, such as acetic acid, trifluoroacetic acid or p-loluenesulfonic acid, or inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid or salicylic acid can be used. Sulfuric acid is particularly suitable. According to a particularly suitable embodiment of the process, it is also convenient to prepare the acid addition salt starting compound in situ by adding the desired acid (such as sulfuric acid) to a solution or suspension of> 4,6-di (amino-glycosyl) -1. 3-diaminocyclitole (such as sisomicin) in a protic solvent (such as water) until the pH of the solution is adjusted to the desired pH.

Typical aldehydes of the formula X'CHO, wherein X 'is as defined, useful in the process include straight and branched alkyl aldehydes such as formaldehyde, acetaldehyde, n-propanol, n-butanal, 2-methylpropanal, n-pentanal, 2-methylbutanal, 3-methylbutanal, 2,2-dimethylpropanal, n-hexanal, 2-ethylbutanal, n-heptanal and n-octanal; alkenyl aldehydes, such as propenal, 2-methylpropenal, 2-butenal, 2-methyl-2-butenal, 2-ethyl-2-hexanalicyclic aldehydes, such as cyclopropanecarbaldehyde, cyclopentanecarbaldehyde, cyclopentane acetaldehyde, cyclohexanecarhydride, , m and p-toluene carbaldehydes and phenylacet aldehydes hydroxy substituted straight and branched alkyl aldehydes such as 5-hydroxy pentanal, 2-hydroxy-3-methylbutanal, 2-hydroxy-3 ~ methylbutanal, 2-hydroxy-2-methylpropanal hydroxybutanal, 2-hydroxy-propanal and 8-hydroxyoctanal; amino substituted straight and branched alkyl aldehydes such as 5-aminopentanal, 2-aminopropanal, 3-amino-propanal, U-aminobutanal, 2-amino-3-methylbutanal, 8-aminoctanal and mono-N-alkyl derivatives thereof; and amino and hydroxydisubstituted, straight and branched alkyl aldehydes, such as 2-hydroxy-5-aminopentanal, 3-hydroxy-3-methyl-U-aminobutanal, 2-hydroxy-3-aminobutanal, 2-hydroxy-3-aminopropanal, 2-hydroxy-2-methyl-3-aminopropanal, 2-amino-3-aminopropanal, 2-amino ~ 3-hydroxy-octanal and mono-N-alkyl derivatives thereof.

If the aldehyde has a chiral center, in this process, enantiomers can be used separately or together as the racemate and the respective diastereoisomers or a mixture thereof are obtained.

The aldehyde reactants useful in the process are either known compounds or they can also be readily prepared from known compounds using methods well known in the art. Thus, for example, alkyl aldehydes substituted with both hydroxy and amino functions (such as 2-hydroxy-5-aminopentanal) can be prepared from an aminoaldehyde acetal (such as li-amino-butanal diethyl acetal) by protecting the amino function therein in the form of one acetamido or phthalimido group using known methods and subsequent elimination of acetal function by acid hydrolysis, thereby obtaining an N-protected aminoaldehyde (such as by transfer of U-aminobutane-diethyl acetal to the corresponding N-phthalimido derivative, which in acidic hydrolysis yields U-phthalimido-butal). Treatment of the N-protected amino aldehyde with cyanic acid gives the corresponding N-protected aminoalkylhydroxinitrile (such as 2-hydroxy-5-phthalimido-valeronitrile), as in catalytic reduction (such as hydrogen in the presence of palladium) or by hydride reduction (such as hydride reduction). ) is an N-protected amino-hydroxy aldehyde (such as 2-hydroxy-5-phthalimidopentanal) which is an agent useful in this process.

When carrying out the procedure, a 1-N-unsubstituted 1+, 6-di (aminoglycosyl) -1,3-di aminocyclitol with a hydride donor to form equivalent 1-N-substituted derivatives of a 1+, 6-di ( to minimize competing side reactions when using an aminoaldehyde as a reactant, it is particularly desirable to protect the amino function of the aldehyde, such as with an acyl blocking group such as acetamido, phthalimido or the like. before carrying out the procedure and then removing the N-protecting group in the compound thus formed. It may also be advantageous to protect the hydroxy group in hydroxy-containing aldehydes in carrying out the process, but this is generally not necessary.

It is also possible to use the acetal or hemiacetal of the aldehyde reactant in acidic medium which gives rise to in situ formation of the required aldehyde.

A suitable method for carrying out the process comprises preparing an antibacterially active 1-N-unsubstituted U, 6-di (aminoglyhosyl) -1,3-diamino-cyclitol (such as sisomicin or verdamicin) in a protic solvent (preferably water). the pH of the solution is changed from about 2 to 5 with an acid (usually dilute sulfuric acid) to obtain the required acid addition salt of the starting compound. When the pH of the solution is about 5, the acid addition salt formed contains about 1 equivalent acid per amino function in the 1,6-di (aminoglycosyl) -1,3-diaminocyclitole (such as 2.5 moles of sulfuric acid per mole of sisomicin). After preparing the acid addition salt solution, at least 1 molar equivalent is added, preferably a large molar excess of the desired aldehyde (such as acetaldehyde, propanal or butanal), and within a short period of time (usually about five minutes), about 1 molar equivalents di (aminoglycosyl) -1,3-diaminocyclitol starting compound 7 of a hydride donor reducing agent, preferably an alkali metal cyanoborohydride, usually sodium cyanoborohydride. The reaction is often completed in less than 30 minutes, as determined by thin layer chromatography, to give the corresponding 1-N-substituted derivatives of H, 6-di (aminoglycosyl) -1,3-diaminocyclitol (such as 1-N-ethylsisomicin or 1-N -etylverdamicin). Isolation and purification of the formed derivative are carried out using known techniques, such as precipitation, extraction and preferably chromatography.

Thus, the process involves a novel convenient single vessel method wherein an amino glycoside is reacted in situ with an aldehyde (preferably in excess) and with a hydride donor reducing agent to form a major product in the form of a mono-N-substituted derivative (such as 1- N-ethylsisomicin), in which the process 1-amino group attached to a secondary carbon atom is usually alkylated primarily in competition with other amino groups attached to primary and other secondary carbon atoms κ, 6-di (aminoglycosyl) -1,3-amine. diaminocyklitol-utgängsmaterialet.

It is also possible to use partially N-protected h, 6-di (aminoglycosyl) -1,3-diaminocyclitols as starting material in the process. The) *, 6-di (aminoglycosyl) -1,3-diaminocyclitols which exhibit an H 2 NCH Sisomicin can be protected in the 6 'positions, in the 2' and 6 'positions or the 2', 3 'and 6' positions. Other non-protecting groups may be bridging groups at the 3 "and 3" positions, such as carbonyl, in the k, 6-di (aminoglycosyl) -1,3-diaminocyclitols which have the 3 "amino and 1 +" amino group of cis configuration in relation to each other. Thus, for example, as a starting compound, a 1-N-unsubstituted 1+, 6-di (aminoglycosyl) -1,3-diaminocyclitol, where the amino function at the 6 'position is N-protected (such as 6'-Nt-butoxycarbonyl isomicin), or a 1-N unsubstituted U, 6-di (aminoglycosyl) -1,3-diaminocyclitol, wherein the amino functions of C-2 'and C-3 are N-protected (such as 2', 3-di-N-trifluoroacetylgentamicin C ) and corresponding, partially N-protected 1-N-substituted derivatives (such as 1-N-ethyl-6'-Nt-butoxycarbonylsisomicin and 1-N-ethyl-2 ', 3-di-N-trifluoroacetylgentamicin C ), which upon elimination of the N-protecting groups by known methods yields the 1-N-CH 2 X compounds of the invention, such as 1-N-ethyl isomicin and 1-N-ethyl-gentamicin C.

The required starting materials, wherein the amino group is protected, can be prepared by methods similar or identical to those described in the following Preparation 3. The term "blocking group" or "protecting group" refers to groups which make the blocked or protected amino groups inert to subsequent nucleic acid. Examples of such amino-protecting groups are benzyl, U-nitrobenzyl, triphenylmethyl, 2, U-dinitrophenyl; acyl groups, such as acetyl, propionyl and benzoyl, alkoxycarbonyl groups, such as methoxycarbonyl, such as methoxycarbonyl. 2-trichloroethoxycarbonyl, t-butoxycarbonyl and 2-iodoethoxycarbonyl; and arylalkoxycarbonyl groups such as carbobenzyloxy and U-methoxybenzyloxycarbonyl groups.

In the blocking process, the protecting group is usually used in the form of an acid imidazole derivative, and acid azide or as active esters, for example, ethyl thiol trifluoroacetate, N-benzyloxycarbonyloxysuccinimide or p-nitrophenyl trichloroethyl carbonate. Blocking groups can thus be described as derived from a compound BgLg, where Bg is the blocking group, such as the acid moiety of an active ester, and I45 is a leaving group, such as imidazole.

Said process can alternatively be carried out in such a way that a 1-N Schiff base derivative is formed, after which it is reduced. The process for the preparation of the 1-N-substituted derivatives of the aforementioned k, 6-di (aminoglycosyl) -1,3-diamino cyclitols, wherein the substituent is -CH reducing the N, C double bond of a 1-NeCHX 'substituted derivative of any of the said U, 6-di (aminoglycosyl) -1,3-diamino-cyclitols, all of which NH ^ groups are protected and NHCH groups can be protected with X ', as defined above, after which all protecting groups present in the molecule are removed and the desired derivative isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

The 1,6-di (aminoglycosyl) -1,3-diaminocyclitols, wherein the 6-aminoglycosyl group is garosaminyl, typically exhibits a 3 '' - N-it '-O-protecting group which is identical to 1-N the substituent in the starting material, since the oxazolidine ring is formed simultaneously with the 1-N-Schiff base group. Thus, for example, 2 ', 3-di-N-trifluoroacetylgentamicin C is reacted with an aldehyde (such as benzaldehyde, phenylacetaldehyde or acetaldehyde) to the corresponding 3' Oxazolidin-1-ylidene Schiff base starting material in this process (such as 1-N-3 "-NV-O-di-benzylidene-2'3-di-N-trifluoroacetylgentamicin, 1-N-3" -NH " -O-di-phenethylidene-2 ', 3-di-N-trifluoroacetylgentamicin and 1-N-3 "NU" -O-diethylidene-2', - 3-di-N-trifluoroacetylgentamicin (C) sodium borohydride and sodium methoxide / methanol give the corresponding 1N-CH 2 XS ", + +" - oxazolidine (e.g. -O-phenethylidene-gentamicin and 1-N-ethyl-3 "-NU" -O-ethylidene-gentamicin C In treatment with acids, a 1-N-CH 2 X compound of the invention (e.g. 1-N-benzylgentamicin C, 1-N-phenethylgentamicin C 1-N-ethylgentamicin C

The novel 1-N-substituted derivatives of the U, 6-di (aminoglycosyl) -1,3-diamino-cyclitols, defined by formulas I, III, IV and V, may also be prepared by a process comprising treating a 1- N-substituted derivative of any of said k, 6-di (aminoglycosyl) -1,3-diaminocyclitols, wherein one or more amino groups can be protected and the 1-N substituent is 0 f? -CX ", where X" is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl, tolyl or hydroxycarbyloxy, which contains aliphatic groups T carbon atoms and, if substituted by amino or hydroxy, carry the substituents on different carbon atoms and from which each amino or hydroxy group present may be protected, with an amide-reducing hydride reactant, whereupon all present in the molecule are removed protecting group the final process step is followed by isolation of the desired derivative as usual or in the form of a pharmaceutically acceptable acid addition salt.

The process is usually carried out in a non-reactionable organic solvent which is assumed to be a solvent in which the starting compounds and the amide reducing reactant are soluble and which do not react with the reactant, by which means reducing the occurrence of competing side reactions to a minimum. Non-reactionable organic solvents which are particularly useful in the reduction process include ethers such as dioxane, tetrahydrofuran, diethylene glycol dimethyl ether and the like.

Particularly suitable amide-reducing hydride reactants are aluminum hydrides and boron hydrides, incl. lithium aluminum hydride, lithium trimethoxy aluminum hydride, aluminum hydride, diborane, diisoamyl borane and 9-borabicyclo / 3.3.1.

It is generally particularly suitable to use diborane as an amide-reducing reactant, except when the starting compound exhibits a double bond, such as in 1-N-acylsisomicin, 1-N-acylverdamicin, 1-N-acyl antibiotic 66-1 + 013, 1-N -acyl antibiotic 66-1 + OD and 1-N-acyl antibiotic G-52, which compounds are conveniently reduced with lithium aluminum hydride.

When X "is hydrocarbyloxy, such as t-butoxy, and the amide is subjected to reduction, the corresponding 1-N-methyl compound is formed.

0 tl

In this process, wherein a 1-NCX "-U, 6-di (aminoglycosyl) -1,3-diaminocyclitol is reduced to the corresponding 1-N-CH 2 X derivative of the invention, if the acyl side chain of 1-N the acyl intermediate exhibits a chiral center using each stereoisomer separately or a mixture thereof, and counterbalanced stereoisomer and mixture thereof are obtained.

Another process for preparing 1-N-substituted derivatives of the above-listed U, 6-di (aminoglycosyl) -1,3-diaminocyclitols, the substituent is a straight chain alkyl group having up to 5 carbon atoms, and of the ceutically acceptable acid addition salts thereof, include the reaction of any of these U, 6-di (aminoglycosyl) -1,3-diaminocyclitols which have amino protecting groups at any other position at the 1 position and wherein the 1-amino group may be activated , with an alkylating agent containing the straight chain alkyl group having up to 5 carbon atoms and a leaving group, elimination of the protecting group and optionally of the activating group or groups present in the molecule and isolation of the derivative as usual or in the form of a pharmaceutically acceptable acid addition salt.

Examples of alkylating agents which can be advantageously used in this process are alkyl iodide, alkyl bromide, dialkyl sulfate, alkyl fluorosulfonate and alkyl p-toluenesulfonate, the alkyl group being the required straight chain alkyl group of up to 5 carbon atoms. Other alkylating agents, wherein the alkyl group preferably contains 1 or 2 carbon atoms, are trialkylanilinium hydroxide, trialkyloxonium fluorborate, trialkylsulfonium fluorborate or trialkylsulfonium fluoroborate. All of these alkylating agents contain a suitable leaving group, such as Br, I, OSO 2 F, dialkylaniline or dialkyl ether.

The amino group at the 1-position of It, 6-di (aminoglycosyl) -1,3-diaminocyclitol may be free or activated. An example of an activating group is trifluoromethylsulfonyl. These activating groups can be introduced into the molecule by reacting an It, 6-di (aminoglycosyl) -1,3-diaminocyclitol, which exhibits amino protecting groups at any position other than the 1 position, such as 3 "-N-lt" -O- carbonyl-2 ', 3,6'-tri-Nt-butoxycarbonyl-isomicin, with a compound providing the activating group, such as trifluoromethylsulfonyl chloride.

The 1-amino group may also be alkylated by the corresponding di (2-cyanoethyl) derivative obtained by treatment with the acrylonitrile of lt, 6-di (aminoglycosyl) ~ 1,3 ~ diaminoeyclitol, which contains protecting groups in a position other than 1 position.

The resulting 1-N-di (2-cyanoethyl) derivative is then alkylated with some of the above alkylating agents, after which the cyanoethyl groups are removed.

The process of the invention is carried out under conditions similar to those used in the well known direct alkylation methods for amines.

Further processes for preparing 1-N-substituted derivatives of the above-mentioned 1,6-di (aminoglycosyl) -1,3-diaminocyclitols wherein the substituent is methyl and of the pharmaceutically acceptable acid addition salts thereof comprise reacting some of these 1,6-di (aminoglycosyl) -1,3-diaminocyclitols, which have amino protecting groups at any one other than the 1 position, either with formaldehyde and a cyclic imide, preferably succinimide, and treatment of the obtained compound with a hydride donor reducing agent, preferably sodium borohydrate , or with formaldehyde in the presence of formic acid, removing all protecting groups in the molecule and isolating the derivative as usual or in the form of a pharmaceutically acceptable acid addition salt. The formation of the 1-N-methyl substituent with formaldehyde and formic acid is widely known as the Eschweiler-Clarke reaction.

A process for preparing 1-N-substituted derivatives of the above-mentioned 1,6-di (aminoglycosyl) -1,3-diaminocyclitols, wherein the substituent is 2-hydroxyethyl, and of the pharmaceutically acceptable acid addition salts thereof, comprises reacting some of the these U, 6-di (aminoglycosyl) -1,3-diaminocyclitols, which have amino protecting groups at any one other than the 1 position, with ethylene-62100 oxide, elimination of all protecting groups in the molecule and isolation of the derivatives as such or in the form of a pharmaceutically acceptable acid addition salt.

The compounds of the present invention are broad-spectrum antibacterial agents which advantageously exhibit activity against many organisms, especially gram-negative organisms, which are resistant to their 1-N unsubstituted precursors. Thus, the compounds obtained according to the invention can be used separately or in combination with other anti-biotic agents to prevent the growth or reduction of the number of bacteria in different environments. For example, they can be used directly to disinfect laboratory glass, dental and medical apparatus contaminated with Staphylococcus aureus or other bacteria, can be inhibited by the compounds obtained by the invention. The activity of the compounds against gram-negative bacteria makes these compounds useful for controlling infections caused by gram-negative bacteria, such as species of Proteus and Pseudomonas. The 1-N-substituted derivatives of the β, 6-di (aminoglycosyl) -1,3-diaminocyclitols, such as 1-N-ethylsisomicin and 1-N-ethylverdamicin, have veterinary use, especially in the treatment of bovine mastitis and Salmonella -induced diarrhea in pets, such as in dogs and cats.

Results of pharmacological tests Table I shows the minimum inhibitory concentration (MIC) of some compounds obtained according to the invention. The tests were performed in Mueller-Hinton culture medium (pH 7.2) using standard methods. The following compounds were tested: a) New compounds prepared according to the invention Compound 1: 1-N-ethylgentamicin Compound 2: 1-N-ethylgentamicin Compound 3 '. 1-N-Ethyl Antibiotic G-52 Compound U: 1-N- (1 * -amino-2 (S) -hydroxybutane) -gentamicin C1 Compound 5: 1-N-hydroxyethylgentamicin Compound 6: 1-N-phenethylgentamicin Compound 7: 1-N-ethylverdamicin Compound 8: 1-N-ethylsisomicin Compound 9s 1-N- (2-ethylbutyl) -isomicin Compound 10: 1-N- (U-aminobutyl) -isomicin

Compound 11: 1-N-ethylgentamicin B

Compound 12: 1-N-Ethyl Antibiotic JI-20B

Compound 13: 1-N-ethylmutamicin 2 Compound 1U: 1-N-ethylmutamicin 6 1U 62100 b) Known Compounds

Compound 15: 1- (1MS-1 + -amino-2-hydroxybutyl) -gentamicin C

-Lei (US Patent 3,796,699) Compound 16: 1- N- (S-1 * -amino-2-hydroxybutyrryl) -gentamicin C. (U.S. Patent 3,780,010) Compound 17: Sisomicin Compound 18: Verdamicin

Test Method

Prepare and sterilize 2000 ml Mueller-Hinton nutrient liquid with pH = 7.2. Transfer 5 ml of the sterile medium to 120 sterile test tubes (16 x 150 mm) with cotton stoppers. Arrange the test tubes in 10 groups pl, 12 tubes each. ...... li

Added to each test cell 10 cells of the hake strain to be used. Add to each batch of test tube an aqueous solution of the antibiotic to be tested; use the following concentrations: 50, 25, 10, 5, 2, 1, 0, 0.5, 0.2, 0.1, 0.05, 0.01, 0.005 and 0.000 (control) yug / ml. Incubate the test tubes for 2k hours at 37 ° C. Visually determine for each group of test tubes the lowest concentration of antibiotic that inhibits bacterial growth and the highest concentration that still causes growth. Determine the minimum inhibitory concentration (MIC) by calculating the mean of these two values.

Table I

Compound no

Escherichia coli _1_ 2_ 3. Ji W677 / R55 - 3.0 0.3 0.03 JR 66 3.0 3.0 0.3 3.0 JR 88 3.0 7.5 3.0 3.0 JR 90 0.75 17.5 7.5 3.0 LA290 R55 0.3 0.75 0.3 3.0 R5 / W677 - 0.3 17.5 0.75 HL97 / W677 - 3.0 0.75 3 , 0

Swidinsky U195 0.75 3.0 3.0 7.5

St. Michael 589 0.3 0.75 0.75 3.0

Baker 2 0.3 0.3 0.3 3.0 F1U-BK 0.075 0.3 0.3 3.0 157U-1 0.075 0.3 3.0 3.0 ATCC 10536 0.075 0.3 0.3 0 , 75

Pseudomonas

Travers 1 3.0 7.5 3.0 17.5

Stone 130 3.0 - 7.5 17.5

Stone 138 7.5 17.5 7.5 7.5

Capetown 18 3.0 17.5 7.5 3.0

Shreveport 3796> 25 17.5> 25> 25

Stone 20 0.05 0.3 0.03 0.3

Stone 39 0.3 3.0 3.0 3.0

St. Michael 762 0.3 3.0 3.0 3.0 1395 0.75 17.5 3.0 17.5 NRRL 3223 0.3 3.0 3.0 3.0 GN 315 - 3.0 17.5 3.0

Klebsiella

Georgetown 369 ^ 3.0 0.75 3.0 7.5 3020 3.0 0.075 0.3 0.3

Oklahoma 6 - 3.0 0.75 3.0 AD 17 0.075 0.75 3.0 3.0 AD 18 0.075 0.3 3.0 3.0

Providencia 16U 17.5> 25> 25> 25

Proteus mirabillis

Harding 0.3 3.0 7.5 3.0 Straightening Membel 0.75 3.0 17.5 17.5 Straightening Anderson -> 25> 25> 25

Serratia

Dalton 0.75 0.3 0.3 3.0

Salmonella

Group B typhim 0.3 3.0 3.0 3.0

Table I, cont.

62100 ι6

Association no

Escherichia coli jj 6_] _ Q

W0T7 / R55 3.0 0.75 0.3 0.3 JR 66 3.0 0.75 0.3 0.3 JR 88 3.0 0.75 3.0 0.8 JR 90 3.0 0, 75 3.0 0.3 LA290 R55 3.0 0.75 0.3 0.3 R5 / W677 3.0 7.5 3.0 17.5 HL97 / W677 0.01 -> 25 725

Swidinsky U195 3.0 17.5 3.0 3.0

St. Michael 589 3.0 7.5 0.8 0.3

Baker 2 3.0 17.5 0.3 0.3 F1U-BK 3.0 0.75 0.3 0.3 157U-1 3.0 7.5 0.3 0.3 ATCC 10536 0.75 0 , 75 0.1 0.08

Pseudomonas

Travers 1 7.5 - 3.0 0.8

Stone 130 17.5 - 3.0 0.8

Stone 138 17.5 - 3.0 0.8

Capetown 18 7.5 - 3.0 0.3

Shreveport 3796 17.5 - 3.0> 25

Stone 20 0.3 0.03 0.03

Stone 39 3.0 - 0.8 0.3

St. Michael 762 3.0 - 3.0 0.3 1395 17.5 - 3.0 0.3 NRRL 3223 3.0 - 0.8 0.3 GN 315 3.0

Klebsiella

Georgetown 369 ^ 3.0 - 0.3 0.3 3020 0.75 - 0.3 0.3

Oklahoma 6 7.5 - 0.3 0.3 AD 17 3.0 - 0.3 0.1 AD 18 3.0 - 0.3 0.1

Providencia 16U> 25 - 17.5 17.5

Proteus mirabillis

Harding 3.0 - 0.8 0.3 Straightening Membel 17.5 - 0.8 0.3 Straightening Anderson> 25 -> 25> 25

Serratia

Dalton 3.0 - 0.3 0.9

Salmonella

Group B typhim 3.0 - 0.8 3.0

Table I, cont.

Compound no

Escherichia coli 9. 10 11 12 W67T / R55 - 0.3 3.0 3.0 JR 66 0.075 0.3 3.0 7.5 JR 88 - 0.3 3.0 17.5 JR 90 0.75 0 , 3 3.0 LA290 R55 0.3 0.3 3.0 7.5 R5 / W677 17.5 3.0> 25 17.5 HL97 / W677 17.5

Swidinsky U195 0.75 3.0> 25> 25

St. Michael 589 0.3 0.75 7.5 17.5

Baker 2 0.3 0.3 3.0 17.5 F1Ϊ + -ΒΚ 0.3 0.3 3.0 0.75 157 * 1-1 0.3 0.3 3.0 0.75 ATCC 10536 0 , 3 0.03 0.3 0.3

Pseudomonas T-ravers 1 17.5 0.3 3.0 3.0

Stone 130 17.5 0.3 7.5

Stone 138 17.5 0.3 7.5

Capetown 18 7.5 0.3 3.0

Shreveport 3796> 25> 25> 25 17 »5

Stone 20 0.03 0.03 0.3 0.075

Stone 39 3.0 0.3 3.0 3.0

St. Michael 762 7.5 0.3 3.0 3.0 1395 17.5 0.3 3.0 7.5 NRRL 3223 3.0 0.03 3.0 3.0 GN 315> 25> 25> 25> 25

Klebsiella

Georgetown 369 * 1 7.5 0.03 3.0 7.5 3020 0.3 0.03 3.0 0.75

Oklahoma 6 0.75 0.03 3.0 7.5 AD 17 7.5 0.3 - 0.75 AD 18 7.5 0.3 - 0.75

Providencia 16U -> 25 7.5> 25

Proteus mirabillis

Harding 3.0 0.75 17.5 17.5 Straightening Membel - 3.0 3.0 17.5 Straightening Anderson -> 25> 25> 25

Serratia

Dalton 17.5 3.0 7.5 17.5

Salmonella

Group B typhim 3.0 0.75 3.0 3.0

Table I, cont.

Compound no

Escherichia coli 13 1¾ 15 16 W677 / R55 0.075 0.125 17.5 7.5 JR 66 0.075 1 7.5 7.5 JR 88 0.075 0.25 17.5 17.5 JR 90 0.3 0.5 17.5 7.5 LA290 R55 0.075 0.25 7.5 7.5 R5 / W677 17.5> 16 HL97 / W677 -8--

Swidinsky U195 0.3 1 - 7.5

St. Michael 589 0.3 2 7.5 7.5

Baker 2 0.3 1 7.5 17.5 F11 + -BK 0.075 0.125 3.0 7.5 157 ^ -1 0.075 0.25 - 7.5 ATCC 10536 0.03 0.125 3.0 17.5

Pseudomonas

Travers 1 0.3 0.125 17.5 7.5

Stone 130 0.3 0.5> 25 17.5

Stone 138 0.3 0.5> 25 17.5

Capetown 18 0.3 0.25> 25 17.5

Shreveport 3796 7.5 b -> 25

Stone 20 <0.01 0.06 3.0 7.5

Stone 39 0.03 0.25 17.5 17.5

St. Michael 762 0.3 0.25> 25 7.5 1395 0.3 0.25> 25 7.5 NRRL 3223 0.075 0.25 ·> 25 17.5 GN 315> 25> 16

Klebsiella

Georgetown 369 * + 0.3 0.125 3.0 7.5 3020 0.3 0.06 3.0 7.5

Oklahoma 6 0.3 0.25 AD 17 0.075 0.25 3.0 7.5 AD 18 0.075 0.06 3.0 7.5

Providencia 16U 3.0 8

Proteus mirabillis

Harding 3.0 0.5 - 17.5 Straightening Membel 0.3 0.5> 25> 25 Straightening Anderson> 25 16

Serratia

Dalton 0.75 0.5 3.0 7.5

Salmonella

Group B typhim 0.3 0.25 3.0 7.5

Table I, cont.

19 621 00

Escherichia coli 17 18 W677 / R55 17.5 17.5 JR 66 17.5 17.5 JR 88 17.5,> 25 JR 90 17.5> 25 LA290 R55 17.5 17.5 R5 / W677 3.0 0.1 HL97 / W677> 25 3.0

Swidinsky 4195 3.0 7.5

St. Michael 589 0.8 0.8

Baker 2 0.3 0.3 F14-BK 0.3 0.3 1574-1 0.3 0.3 ATCC 10536 0.1 0.1

Pseudomonas

Travers 1> 25> 25

Stone 130> 25> 25

Stone 138> 25> 25

Capetown 18 17.5> 25

Shreveport 3796> 25> 25

Stone 20 0.8 0.8

Stone 39 0.1 0.3

St. Michael 762 0.1 0.5 1395 0.3 0.3 NRRL 3223 0.1 0.3 GN 315

Klebsiella

Georgetown 3694 17.5 17.5 3020 17.5 17.5

Oklahoma 6> 25. > 25 AD 17 0.3 0.1 AD 18 0.3 0.1

Providencia 164 7.5 17.5

Proteus mirabillis

Harding 0.8 0.8 straightening Mernbel 0.3 0.3 straightening Anderson> 25 17.5 * Serratia

Dalton »0.3 0.3 S a_.l monella

Group B typhim 0.3 1.0 20 621 00

The dosage administered of the present compounds depends on the age and weight of the animal, the mode of administration and the type and severity of the infection intended to inhibit or reduce. Generally, a dosage of derivatives of U, 6-di- (aminoglycosyl) -1,3-diaminocyclitols is used to control a given bacterial infection of the same order required by the corresponding 1-N unsubstituted U, 6-di- (aminoglycosyl) ) -1,3-diaminocyclitols.

The compounds obtained according to the invention can be administered orally.

They can also be used topically in the form of ointments, both hydrophilic and hydrophobic, and in the form of lotions, which may be aqueous, anhydrous or emulsion-type or in the form of creams. Pharmaceutical carriers useful in the preparation of such compositions include, for example, water, oils, fats, polyesters, polyols and the like.

For oral administration, the present compounds may be formulated in the form of tablets, capsules, tinctures and mixtures or may also be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective in treating bacterial infections in the gastrointestinal tract, which infections cause diarrhea.

Topical preparations contain about 0.1-3.0 of the compounds obtained according to the invention per 100 g ointment, cream or lotion. The topical preparations are usually applied gently, approximately 2-5 times daily.

The antibacterial agents obtained according to the invention can be used in liquid form, as in the form of solutions, suspensions and the like for otic and optical use and can also be administered parenterally via intramuscular injection. The injection solution or suspension is usually administered in an amount of about 1-15 mg of active substance per kg of body weight per day, divided into about 2-k doses. The exact dose depends on the stage and severity of the infection, the sensitivity of the infected organism to the antibacterial agent, and the individual characteristics of the treated animal.

The following preparations illustrate the preparation of a number of required starting materials, while the invention is further illustrated in the following examples. All temperatures indicated are Celsius degrees.

Preparation 1. Gentamicin C2a

Separation of gentamicin from co-produced antibiotics.

Dissolve 96 g of gentamicin base (prepared from the sulfate salt obtained according to Example 1+ in US Patent 3,091,572) in UOO ml of the upper phase obtained when methanol, chloroform and 17 # -ig ammonium hydroxide are mixed in the volume ratio 1: 2: 1 . Put 1/10 of the solution into each of the first 10 62100 tubes in a 600 x 80 ml tube counter current extractor. Fill all pipes, incl. the first 10, to their capacity with the lower phase of said solvent mixture. Install the solvent container to dispense 1 + 0 ml of the upper phase to the tube (1) for each transfer. Install the device on 500 transmissions. When the transfers are completed, remove each eighth tube for chromatography (in duplicate) on a Schleicher and Schuell paper No. 589 using the lower phase of said solvent mixture. Allow the chromatograms to develop for about 16 hours and dry the paper. Place a paper on an agar plate yinpad with Staphylococcus aureus (ATCC 6538P), spread the duplicate with the conventional ninhydrin solution and heat to develop. Incubate the agar plate at 37 ° overnight and combine the solution from the tubes, containing the material that migrates in the same manner as gentamicin (i.e., tubes 290-360).

Replace tubes 290-360 with new tubes containing 1 + 0 ml of the upper phase and 1 + 0 ml of the lower phase. Install the device for an additional 2800 transfers and repeat the chromatographic procedure. Combine tubes 1 - 16 and concentrate in vacuo to give 1.3 g of gentamicin having the following properties: (a) a molecular weight of 1 + 63, determined by mass spectrometry, which is in accordance with the empirical formula ^ 20 ^ 1 + 1 ^ (B) a specific optical rotation, as measured by the D-line of sodium at 26 ° of + 11 + + + + 5 ° in water at a concentration of 0.3% and (c) a proton magnetic resonance spectrum (PMR) of the following: PMR (ppm) (D 2 O): δ 0.99 (3H, d, J = 6.5 Hz, CH-CH ^; 1.17 (3H, s, C-CH ^; 2.1 + 7) 3H, s, N-CH 3); 2.51 (1H, d, J-10.5 Hz, H ~ 3 "h 3.75 (1H, q, J-10.5, * + Hz, II-2 "); 1 +, 00 (1H, d, J = 12 Hz, H-5" -eqh 5.0U (1H, d, J + 1 H +, H-1 ") ·, 5.13 (lii, d , J = 3.5 Hz, H-1 ').

Irradiation of the secondary methyl group at £ 0.99 ppm reveals H-6 'as doublet (j = 6.5 Hz) at ^ 2.81 ppm.

Preparation 2. Gentamicin

Separation of gentamicin from co-produced antibiotics.

Separate the predominantly gentamicin C components (C, C „and C) presently in U.S. Pat. No. 3,651,01 + 2, Example 2, 1c. 1Θ.

and combining the fractions containing significant amounts of free gentamicin and C2 base (500 g gentamicin mixture gives 53.1+ g overlap). transfer 1.5 g of this gentamicin and C3 mixture to a column containing 50 g of silica gel prepared in a solvent system comprising chloroform / methanol / 15 ammonium hydroxide (1: 2: 1). Elute the column with the same solvent system and monitor the eluted fractions by thin layer chromatography 1 in 62100 Pa, silica gel plates using the solvent system chloroform / methanol / 22% ammonium hydroxide (1: 2: 1) as the evaporator. Combine the fractions containing a mixture of gentamicins and Cg together with gentamicin (fractions 39-57 Aio mg7) · Chromatograph the fractions 39_57 over silica gel using the solvent system chloroform / methanol / 7% ammonium hydroxide (1: 2: 1) and combine the fractions (98-130) containing pure gentamicin C, as determined by thin layer chromatography (yield &wt; 2 6 o 1 * 5 mg) with the following constants: + 165.5 (c = 0.3 #, HgO); mass spectrum m / e 1 * 63 (M + 1) +, kk61 1 * 1 * 5, 1 * 33, 350, 332, 322, 30l +, 333, 305, 287, 191, 173, 163, 1U5, 160, 1 1 * 2, 118, 1U3; PMR (ppm) (DgO): δ1.25 (3H, s, C-CH2) 2.1 * 0 (3H, s, N-CH3) in 2.55 (3H, s, N-CHg); 5.12 (1H, d, J = 1 * Hz, Η-Γ); 5.22 (1H, d, J = 3 Hz, H-1 ')

Pure gentamicin can be differentiated from gentamicin and Cg by its mobility on thin layer chromatograms using silica gel plates and the chloroform / methanol / 22% ammonium hydroxide (1: 2: 1) solution system as a coolant. The approximate R 2 values in this system are as follows: gentamicin 0.1 * 7 gentamicin Cg 0.1 * 7 gentamicin Cg 0.35

Preparation 3. Selectively blocked di (aminoglycosyl) -1,3-diaminocyclitols.

A. 2 ', 3,6'-tri-N-butoxycarbonyl-3 "-N-1 *" - 0-carbonylsisomicin 1. Penta-N-carbobenzoxyisomycin Dissolve 25 g of sisomicin and 13 g of sodium carbonate in 625 ml of distilled water.

Add 100 ml of carbobenzoxy chloride to the stirred solution at 25 ° and stir the mixture for 16 hours. Filter off the solid, wash thoroughly with water, dry in vacuo and wash with hexane to give 62 g of penta-N-carbobenzoxy isomycin as a colorless amorphous solid. Melting point = 165-173 °, + 96.2 ° (CH 3 OH) IR + max (CHCl 3) 31 + 00, 1720, 1515, 1215, 1050, 695 cm -1. NMR: δ (CDCl 3) 1.03 (3H, broad singlet, 1 + - C-CH 2); 3.02 (3H, broad singlet, 3 "-NCH 3); 5.02 (10H, broad singlet, CH 2 Cl 2 H 2; 3.28, 3.30 ppm (25H, broad singlet, -CH 2 Cl 2 H 2)).

2. Tetra-N-carbobenzoxy - 3 MNl Filter and add 2 ml of glacial acetic acid to the filtrate, which is then concentrated in vacuo. of t a ra-N-carbobenzoxy-3 "-N-V'-Ok ar bonyl-r. in the form of an amorphous powder (3.5 g), m.p. 210-213 °, / o <7 + 68.8 ° (c = 0.22).

IR: γ max (nujol) 3550, 181 + 0.1760, 15Ö0 cm -1. NMR: δ (CDCl 3) 1.3 ** (3H, singlet, 2.68 (3H, singlet, 3 "-N-Me); 5.0¾ (8H, broad singlet, -CH 2

3. 3 "-N-1 +" - O-carbonyl-sisomicin Add a solution of 10.1 g of 1,3,2 ', 6'-tetra-N-benzyloxycarbonyl-3 "-N-1 +" - 0-carbonyl -sisomicin in 200 ml of tetrahydrofuran with 1 liter of liquid ammonia (redistilled over sodium). Add 6 g of sodium in small pieces to the stirred solution. After three hours of stirring, the excess sodium destroys by the addition of ammonium chloride. Allow the solvents to evaporate under a stream of nitrogen. Dissolve the residue (s) in water and allow the solution to pass through a medium of Amberlite IRC-50 resin (H + form), wash the resin carefully with water and elute with 2N ammonium hydroxyl solution. Evaporate the ammonium eluate in water to give the title compound in a yield of 1+ g. IR: V (nujol) 171 * 5 cm 1. The product can be used in the maximum subsequent step without further purification. However, a very pure sample can be obtained by chromatographing the product over silica gel using the lower phase of the solvent system chloroform / methanol / cone. ammonium hydroxide (1: 1: 1) as eluent.

1+. 2 ', 3,6'-tri-Nt-butoxycarbonyl-3 "-NU" -O-carbonylsisomicin Dissolve 1.1+ g (3 mmol) 3β-N-1 + "- 0-carbonyl-isomicin in 10 ml 50% methanol containing 3.5 mmol of triethylamine While stirring, add dropwise 3.5 mmol of t-butoxycarbonyl azide, stirring the mixture for two days at room temperature Add 5 ml of Amberlite® IRA-1 + 01S (OH) ion exchange resin together with 5 ml of methanol and stir for 30 minutes. Remove the resin by filtration and wash with methanol. Concentrate the filtrate and chromatograph the residue on a column of 20.0 g of silica gel (60-100 mesh) using chloroform / methanol / ammonium hydroxide (30: 10: 0 , 1 +) as solvent system. Combine the homogeneous fractions containing the title material and remove the solvent by evaporation in vacuo. Dissolve the residue in methanol and precipitate with excess ether. Isolate the solid product by filtration and dry.

B. 2 ', 3-di-N-trifluoroacetyl-gentamicin 1. 2'-N-trifluoroacetyl-gentamicin Dissolve 1.7 g of gentamicin in 20 ml of methanol, cool the mixture to 1 + ° and add 0.1 + 6 ml (0.563 g) ethyl thiol trifluoroacetate with stirring. Allow the reaction to continue for two hours and concentrate the solution to a residue in vacuo. Chromatograph the product on 80 g of silica gel G using the lower phase 2h 62100

of a mixture of chloroform / methanol / water / ammonium hydroxide in the volume ratio as an eluent. Combine the fractions containing the major component and concentrate to give 1.1+ g of the title compound, m.p. 108-111 ° C, [<* f + = + 128 ° (c = 0.3%, Hg0). Analysis for C ^H₂ ^N OgFFO

Calculated: C 1 + 6.69 H 7.1 + 0 N 11.81 + F 9.63%

Found: C 1 + 6.66 H 7.65 N 11, 60 F 9.2l +%.

2. 2 ', 3-di-N-trifluoroacetyl-gentamicin

Dissolve 0.66 g of the product from step 1 in 10 ml of methanol, cool the mixture to 1 + ° and add 0.1 U8 ml (0.182 g) of ethyl thiol trifluoroacetate, dissolved in 3 ml of methanol. Stir the mixture for about 16 hours and concentrate to a residue in vacuo. Chromatograph the product on 30 g of silica gel according to step 1. Monitor the column by thin layer chromatography, combine the intended fractions and concentrate to give 0.32 g of the title compound, mp 121-129 ° / wf = 121 ° (c = 0.3%, HgO). Analysis for C ^ O ^ g.

Calculated: C 1 + 1 +, 81 + H 6.17 N 10.1 + 6%

Found: C 1 + 1 +, 9 * + H 6.35 N 10.17% C. 6'-N-trifluoroacetyl-sisomicin Dissolve 20 g of sisomicin in 1.2 liters of anhydrous methanol and add dropwise a solution under three hours of stirring. of 6 ml of ethyl thiol trifluoroacetate in 6θ ml of methanol. Allow the reaction to proceed for 18 hours at room temperature and remove the solvent in vacuo to give a residue of 23.8 g of product of a purity of about 95% and having the following physicochemical properties: tnass spectral data: m / e 5 3 M +, other definite peaks at m / e 1 + 13, 395, 385, 362, 223 and 126. NMR (60MHz, D20) δ 5.37 (doublet, J = 2Hz, H-1'h 5.12 (doublet, J = 1 + Hz, H-1 "); 1 +, 96 (broad singlet, H-U '); 2.57 (singlet, N-CH 2); 1.26 (singlet, c-ch 3).

D. 6'-N-t-butoxycarbonyl-gentamicin C

I Q.

Dissolve 2.69 g of gentamicin C, in 60 ml of methanol / water (1: 1), cool to 5 and add 1.815 ml of triethylamine. While stirring, add 1.91 g of t-butoxy-carbonylazide dropwise. Stir the mixture for 18 hours at 5 °. Add 20 ml of Amberlite® IRA-1 + 01S resin (OH form), stir 30 minutes, filter and evaporate the filtrate to dryness in vacuo. Chromatograph the crude product over 350 g of silica gel using the lower phase of the solvent system chloroform / methanol / concentrated ammonium hydroxide (2: 1: 1) as eluent. Take out 3 ml fractions and monitor their contents with TLC. Combine the fractions containing the essential reaction product and evaporate to give the title compound (0.1 + 2 g, 13%), + 137 ° (CH 3 OH), PMR + 1.23 (3H, s, C-CH , 1 + 5 (9H, s, 62100 C-iCHg) +) 2.53 (3H, s, N-CH3); Δ 5.08 (2H, overlapping duplicates, H-1 'and H-1 ") ppm; mass spectrum m / e 550 [(Μ + ϊ) + 2 and m / e 5 ^ 9 (M +).

N, N-t-butoxycarbonyl-gentamicin B

Dissolve 1 g of gentamicin B in 30 ml of 50% methanol in water and cool to 5 ° · Add dropwise 0.297 g of t-butoxycarbonyl azide with stirring and then 0.186 ml of triethylamine and stir the obtained solution for 18 hours. Evaporate the reaction mixture in vacuo to an ether and chromatograph the residue on 100 g of silica gel using the lower phase of the chloroform / methanol / concentrated ammonium hydroxide solvent system as the elution model. Collect 2 ml fractions and monitor the column outflow with TLC. Combine fractions containing the intended material (fractions 180-230) and evaporate to give 0.830 g of N-t-butoxycarbonylgentamicin B with the following physical constants: PMR (60MHz, DgO) 1 1.21 (3H, s, C-CH); 1, 1 * 2 (9H, s, CiCH 2); 2.53 (3H, s, N-CH 3); 5.2 (1H, d, J = 1.5 *, 5Hz, H-175) 5.23 (1H, d, J = 3, ÖHz, H-11) ppm.

Preparation h. 1-N-acetylsisomicin A. Dissolve 1.25 g of sisomicin sulfate in 200 ml of methanol / water (2: 3, volume) and cool the solution. Add 1.5 ml of acetic anhydride and after about 10 minutes 0.125 ml of triethylamine in 10 ml of methanol over a period of 15 minutes. Allow the reaction mixture to reach room temperature for two hours and then evaporate the solvent in vacuo. Dissolve the ether residue in water and transfer the product to the free base by passing an aqueous solution thereof through Amberlite® IRA-1 + 01S resin into the hydroxide ion cycle. Lyophilize the column eluate and chromatograph the residue on 50 g of silica gel using the lower phase of the chloroform / methanol / 7% ammonium hydroxide (2: 1: 1) system as eluent. Monitor the fractions via TLC and combine equal fractions to form the title compound. Yield: 0.185 g, mp 128-130 °, = 159 ° (0.3%, H O), NMR: (D 0): / 1.22 (3H, s, -C-CH); 2.02 (3H, s, NH-CO-CH2; 2.53 (3H, s, N-CH2) M8 (1H, m, = CH-); 5.08 (1H, d, J = 1 * Hz, Η ^ Ή 5.35 (1H, d, J = 2Hz, H +); mass spectrum: (M + 1) + m / e 1 * 90, M + m / e 1 * 89.

Preparation 5. Preparation of Aldehyde Intermediates A. 2-Acetamido-3-hydroxy octanal

Protect the amino function of the 2-amino-3-hydroxy-octanoic acid by transferring it to an acetamido function, esterifying the formed 2-acetamido-3-hydroxy-octanoic acid with methanol, reducing the resulting 2-acetamido-3-hydroxyoctanoic acid methyl ester with di-ishydcbutyl acid methods for forming 2-acetamido-3-hydroxy octanal.

B. 62-Acetoxy-1 + - (N-methylacetamido) -but anal

Treat the diethyl acetal of 2-hydroxy-1 + -aminobutanal with acetic anhydride in pyridine and then treat the formed diethyl acetal of 2-acetoxy-U-acetamidobutanal with sodium hydride and methyl iodide to form the diethyl acetal of 2-acetoxy-1 * - (N-methyl) butanal. Remove the protective acetal group using an acid to form 2-acetoxy-1 * - (N-methylacetamido) -butanal.

Example 1 1-N-substituted sisomicin A. 1-N-ethylsisomicin

A solution of 5 g of sisomicin in 250 ml of water is added with 1N sulfuric acid until the pH of the solution is adjusted to about 5. The resulting solution of sisomicin sulfuric acid addition salt is added with 2 ml of acetaldehyde, stirred for 10 minutes, and 0.85 g of sodium cyanobhydride is added. . Continue stirring for 15 minutes at room temperature, concentrate the solution in vacuo to a volume of about 100 ml, treat the solution with a basic ion exchange resin / such as Amberlite®IRA UoiS (OH) J and lyophilize to an ether residue comprising 1-N. ethylsisomicin.

Purify by chromatography on 200 g of silica gel, eluting with the lower phase of the chloroform / methanol / T% ammonium hydroxide (2: 1: 1) system. Combine equal eluate as determined by thin layer chromatography and concentrate the combined eluate of the main component in vacuo to an ether, comprising 1-N-ethyl isomicin (yield 1.25 g). Purify by chromatography on 100 g of silica gel and elute with the system chloroform / methanol / 3.5 µg ammonium hydroxide (1: 2: 1). Let the combined, similar eluates (as determined by thin layer chromatography) pass through a column of basic ion exchange resin and lyophilize the eluate to give 1-N-ethyl isomicin (yield 0.5 µg); 16U +

(0.3%, H 2 O); PMR (ppm) (DgO): δ 1.05 (3H, t, J = 7Hz, -CH 2 CH) in 1.19 (3H, s, -C-CH 3); 2.5 (3H, s, N-CH 3 U M 5 (1H, m, -CH-); 95 (1H, d, J = UHz, H 2 ') · 5.33 (1H, d, J = 2, Mass spectrum: (M + 1) + m / e 1 + 76 as well as m / e 127, 15 * +, 160, 173, 191, 201, 219, 256, 299, 317, 332, 31 * 5, 350, 360, 378, 390 and 1 * 00.

B. 1-N-methylsisomicin

A solution of 1 +, 6 * + g of sisomicin in 180 ml of water is added with 1N hydrochloric acid until the solution is adjusted to pH about 5 · Add 1, 2 ml of 37% formaldehyde, stir for 10 minutes and then add U60 mg sodium cyanoborohydride. Pass the reaction solution through a column of a basic ion exchange resin (such as Amberlite® IRA 1 * 01 S (OH) form and lyophilize. Chromatograph the formed ether residue on silica gel in the lower phase of the chloroform / methanol / 7% ommonium hydroxide system (2 : 1: 1) Combine equal eluate, containing essentially 27 621 00 1-N-methylsisomicin, as determined by thin layer chromatography, evaporate 26 o in vacuo to a residue of 1-N-methylsomicin; +153 (0.3%, H Mass spectrum: (M + 1) + m / e 1 + 62 also m / e 127, 1U0, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 ( w), 3I + 6, 376, 386.

C. 1-N- (n-propyl) -isomicin

In the manner of Example 1A, treat the sulfuric acid addition salt of sisomicin in the can with propanol and sodium cyanoborohydride. Isolate and purify the formed product in the manner described above to form 1-N- (n-propyl) -isomicin; Zo <7p + + 1 ° 0 ° (0.3%, H 2 O); PMR (ppm) (DO): δ 0.83, (3H, t, J = 7Hz, -CH 2 CH 3); 1, U (3H, s, -C-CH); 2.1 * 5 (3H, s, -N-CH 3); 1+, 82 (1H, m, = CH-); 1 +, 90 (1H, d, J = 1 + Hz, H +); 5.78 (1H, d, J = 2Hz, H +) j mass spectrum: (M + 1) + m / e 1 + 90 also 127, 160, 168, 187, 205, 215, 233, 256, 313, 331, 31 + 6, 359, 361+, 37! +, 1 + 01 +, 1 + 11 +.

D. 1-N- (n-butyl) -isomicin

A solution of 3 g of sisomicin in 200 ml of water is added with 1N sulfuric acid until the solution has a pH of about 3.5. Add 1.5 ml of n-butanol, stir for 10 minutes and then add 1 + 50 mg of sodium cyanoborohydride. Continue stirring for one hour and then concentrate the solution in vacuo to about 100 ml.

Let the solution pass through a column of a basic ion exchange resin (such as Amberlite IRA 1 + 01S (OH)) and lyophilize. Chromatograph the residue on 11 + 0 g silica gel in the lower phase of the chloroform / methanol / 7% ammonium hydroxide (2: 1: 1) system. Combine equal fractions containing 1-N- (n-butyl) -isomicin as determined by thin-layer chromatography and evaporate the combined eluates in vacuo to an ether, comprising 1-N- (n-butyl) -isomicin; IcA₂ + 129 ° (0.3%, H₂O), PMR (ppm) (DgO) 0 0.82 (3H, t, J = 7Hz, -CHgCH); 1.15 (3H, s, C-CH); 2.1 + 6 (3H, s, -N-CH 3); 1 +, 82 (1H, m, = CH-); 1 +, 92 (1H, d, J = 1 + Hz, H +); 5.29 (1Η, d, J = 2Hz, H +), mass spectrum: (M + 1) + M / e 50U also m / e 127, 1Ö0, 182, 201, 219, 229, 21 + 7, 256, 327, 3I + 5, 360, 373, 388, 1 + 18, 1 + 28.

E. Other 1-N-alkyl and 1-N-alkenyl isomicins

In the experiment of Example 1A, instead of using acetaldehyde, use equivalent amounts of each of the following alkyldehydes: 2-ethylbutanal and propenal.

In each case, perform the reaction in the same manner as in Example 1A and isolate and purify the formed products in the same manner to form respectively: 1-N- (/ 6-ethylbutyl) -isomicin + 121 ° (c = 0.1 +%, H 2 O); PMR (ppm) (O): 0.75 (6H, t, 6.5Hz, CH2 ~ CH3h 2.1+ (3H, s, N-CH2; 1 +, 78 (1H, m, = CH +) + 1, 88 (1H, d, 1+, 0 Hz, lf ") in 5.22 (li, d, 2.0 Hz, 11 +) * mass spectrum: (M + 1) + m / e 532 also m / e 127, 1bO, 210, 220, 256, 275, 355, 375, ^ 88, 1 + 01, U06,> + l6, I + 1 + 6, 1 + 56; 28 62100 1-N - (6-propenyl) -isomicin, + 1 7 (0, U, MeOH); NMR (D 2 O) J 1.18 (s, 3H, C-CH 3) in 2, U8 (s, 3H, N-CH3) ·, 3.75 (1H, dd, J = U, 11 Hz, H-2 "); 3.95 (1H, d, J = T3 ~ Hz, H-5e); U.8U OFTm , H-U'h (1H, d, J = U Hz, H-1 "); 5.30-5.0 (3H, m, H-1 ', = CH 2); 5.8 (1H, m Mass spectrum m / e in + 88 (M +) F. 1- N- (3-Aminobutyl) -isomicin Add 1N sulfuric acid dropwise to a solution of 3 g of sisomicin in 120 ml of water, until Adjust the pH of the solution to about 5. Add the aqueous solution of the formed sulfuric acid addition salt of sisomicin with 60 ml of dimethylformamide and then with 2 g of it-phthalimidobutanal in 10 ml of dimethylformamide, continue stirring for 10 minutes and then add in + 20 mg sodium cyanide. After about 20 minutes sat The reaction solution was added to 1 liter of anhydrous methanol with stirring and collected by filtration the precipitate formed, comprising the sulfuric acid addition salt of 1-N- (ζ-phthalimidobutyl) -isomicin.

Purify by dissolving the precipitate in water and allow the aqueous solution to pass over a basic ion exchange resin. Evaporate in vacuo to an ether, chromatograph the ether over silica gel, elute with the lower phase of the solvent system chloroform / methanol / 7% ammonium hydroxide (2: 1: 1) and evaporate the combined, similar eluates to an ether, comprising 1 -W- (3-phthalimido-butyl) -isomicin.

Add 0.5 g of -1-N- (3-phthalimidobutyl) -isomicin with 5 ml of 5 # -hydrazine hydrate in ethanol and reflux for three hours. Pour the reaction mixture into a large volume of tetrahydrofuran and collect by filtration the precipitate formed, comprising 1-N- (3-aminobutyl) -isomicin.

The compound of this example may alternatively be prepared in the following manner: (1) it-acetamidobutyraldehyde Dissolve 5 g of -acetamidobutyraldehyde diethyl acetal in 75 ml of distilled water and 5 ml of 1N sulfuric acid. Store the solution at room temperature until the hydrolysis is complete, as determined by thin layer chromatography. Neutralize the solution with sodium bicarbonate, carpet the solution with sodium chloride and extract with chloroform. Distill the combined chloroform extracts to form an ether residue, comprising H-acetamidobutyraldehyde, which can be used without further purification by the following procedure.

(2) 1- N- (3-Acetemidobutyl) -sisomicin Add 3 g of sisomicin in 120 ml of distilled water with 0.1N sulfuric acid until the solution has a pH of about 5 · Add 6 g of racetamidobutyraldehyde, prepared according to the previous procedure, and after 10 minutes 600 g of solid sodium cyanoborohydride. After two hours, concentrate the solution to a small volume and pour it into methanol. Collect the formed precipitate by filtration,. (R) dissolve in water and pass the solution through a column of Amberlite IRA U01-C '(OH) ion exchange resin. Evaporate the eluant, chromatograph the residue formed on silica gel and elute with the system chloroform / methanol / 7% ammonium hydroxide. Evaporate the eluent to an ether, comprising * - ** - (3-acetamidobutyl) -s isomic in.

(3) 1- N- (3-Aminobutyl) -isomicin

Treat the 1-N - (3'-acetamidobutyl) isomicine obtained in the previous example with 10% potassium hydroxide solution for three hours at 100 °, neutralize with Amberlite IRC-5O ion exchange resin and elute with 2N ammonium hydroxide.

Concentrate the eluent, dissolve the formed residue in water, and lyophilize to form 1-N- (3-aminobutyl) -sisomi cin. + 109 ° (c = 0.3%, HoO) ·, mesh spectrum: (M + 1) + m / e 519 also 127, 160, 197, 216, 23 * +, 2 * + * +, 256, 262, 3 * + 2, 36 (), 375, 388, 393, * + 03, * + * + 3.

G. 1-N- (-aminoethyl) -sisomicin and 1-N - (3-aminopropyl) -isomicin Treat according to the alternative procedure of Example 1-1 an aqueous solution of sisomicin, which is added with 0.1N sulfuric acid, until the solution is adjusted to about pH 5, with γ-acetamidoacetaldehyde and then with solid sodium cyanoborohydride. Isolate and purify the resulting product in the manner described to form 1-N- (3-acetamidoethyl) -isomicin. Hydrolyze 1-N- (5-acetamidoethyl) -isomicin with 10% potassium hydroxide solution and isolate and purify the resulting product at step (3) in the alternative procedure of Examples 1-1 described for the formation of 1-N- (β-Aminoethyl) -isomicin. Mass spectrum: (M + 1) m / e * + 91, also 160, 169, 187, 206, 216, 23 * +, 256, 283, 31 * +, 325, 33 * +, 3 * + 7, 360, 370, 375, * + 05 and * + 15-

Using β-acetamidopropanal instead of β-acetamidoacetaldehyde in that procedure gives 1N-1β-acetamidopropyl) -isomicin, which after hydrolysis with 10% potassium hydroxide solution, isolation and purification in the manner described gives 1-N-1 (γ'-aminopropyl) -isomicin, fcA₂ + 127 ° (H₂O), NMR (D₂O): δ 1.2, (3H, s, -C-CH h 2.5 (3H, s, N -CH) in 3.8 (1H, dd, J = * +, 10.5 Hz, H2 ") j * t, 0 (1H, d, J = 12.5 Hz, H" e); * +, 85 (1H, m, -C = CH-); * +, 95 (1H, d, J = * + Hz, H1 "); 5.3 * + (1H, d, J = 2.5 Hz, H M + + 1 m / e 50 * + also 160, 202, 220, 230, 2 * + 8, 256, 328, 3 * + 6, 361, 37 * +, 379, 389, * +07, * + 19 and * + 29.

Example 2 1-N-substituted gentamicin C.

1 a A. 1-N-ethylgentamicin Add 5 g of gentamicin C in 125 ml of water with 1N sulfuric acid until the pH of the solution is about 5.2. Then add 2 ml of acetaldehyde. Stir the solution for five minutes and then add 1 g of sodium cyanoborohydride. Continue stirring for 30 minutes at room temperature, concentrate the solution in vacuo to a volume of about 75 ml, for the solution through a column of a basic ion exchange resin / such as Amberlite®IRA 1 + 01S (OH) J and lyophilize to an ether residue comprising 1-N ethylgentamicin C.

I &

Purify by chromatography on 200 g of silica gel, eluting with the lower phase of the system chloroform / methanol / 7 ammonium hydroxide (2: 1: 1). Combine the same eluate as determined by thin layer chromatography and concentrate the combined eluate of the main component in vacuo to an ether, comprising 1-N-ethylgentamicin C. (yield 0.95 g) · Purify further by chromatography 1-N-ethylgentamicin C. of 100 g of silica gel and elute with the system chloroform /

I SI

methanol / 3.5 $ ammonium hydroxide (1: 2: 1). Treat the combined similar eluates (as determined by thin layer chromatography) with a basic ion exchange resin and lyophilize the eluate to give 1-N-ethylgentamicin C, (0.1 + 2g), / es7p + 118 ° (c = 0.3% , H 2 O) in PMR (ppm) (DgO): 1.06 (3H, t, J = 7 Hz, -CH2 CH3h 1.19 (3H, s, -C-CH3); 2.51 (3H, s N-CH 2); U, 97 (1H, d, JU Hz, H, +) in 5.16 (1H, d, J = 3.5 Hz, H +); mass spectrum (M + 1) + m / e U78 also m / e 129, 15 ^, 160, 173, 191, 201, 219, 258, 301, 317, 319, 329, 332, 3I + 7, 350, 360, 378 and 1 + 02.

Example 3 1-N-substituted verdamicin A. 1-N-ethylverdamicin 0.5 g of verdamicin in 65 ml of water is added with 1N sulfuric acid until the pH of the solution in ns is about 1 *, 9, and then with 0.2 ml of aeetaldehyde.

Stir the solution for minutes, add 65 mg of sodium cyanoborohydride, concentrate the solution in vacuo to a volume of about 10 ml and pour the solution into 50 ml of methanol with stirring. Recover by filtration the precipitate formed, comprising 1-N-ethylverdamicin. Purify by chromatography on 100 g of silica gel and elute with the system chloroform / methanol / 3.5% ammonium hydroxide (1: 2: 1). Collect similar fractions, as determined by thin layer chromatography and evaporate in vacuo the combined fractions containing the major component, to an ether, comprising 1-N-ethylverdamicin. Purify further by chromatography on 7 g of silica gel and elute with the lower phase of the system chloroform / methanol / 7% ammonium hydroxide (2: 1: 1). Combine the same eluates and evaporate in vacuo to a residue of 1-N-ethylverdamicin (yield 50 mg). Mass spectrum: (M + 1) + m / e 1 + 90 also m / e IUl, 15I +, 160, 173, 191, 201, 219, 270, 313, 317 (v), 331, 332, 3U1, 350 (w), 357, 359, 360, 378, 390, 1 * 1U.

B. Use in the procedure of Example 3A instead of acetaldehyde other aldehyde intermediates. Isolate and purify each of the obtained products in a similar manner to form respectively: 1-N- (n-propyl) -verdamicin, + 122 ° (c = 0.3% T 2 O) -, PMR (ppm) (d 2 O): δ 0.88 (3H, t, 1 = 7 Hz, CH 2 CH 3) 1, 1.19 (3H, s, C-CH 3) in 1.16 (3H, d, 1 = 6 Hz, CH-CH ); U, 81 (1H, m, = CH-) ·, i +, 97 (1H, d, 1 = ¼ Hz, H1 ") ^ 5.30 (1H, d, J = 2.0 Hz, = H ') mass spectrum: (M + 1) m / e 528 as well as m / e 1, 160, 168, 187, 205, 215, 233, 2? 0, 3 ^ 6, 355, 373, 50¼. N- (n-butyl) -verdamicin, Cc ^T ^ + 121 ° (c = 0.3%, H₂O); PMR (ppm) (D₂O): δ8.8 (3H, t, J = 6.5 Hz, CHg-CH); 2.1 + 5 (3H, s, NCH); 1 +, 8 (1H, m, C-CH-); 1 +, 92 (1H, d, J = 1 +, 0 Hz ), 5.25 (1H, d, J = 2.0 Hz, H 2 '); mass spectrum: (M + 1) + m / e 518 as well as m / e 1U1, 160, 182, 201, 219, 229, 2U7, 270, 3, 360, 378, 387, 388, 1 + 18, 1 + 1 + 2nd

Example 1+ 1-N-substituted gentamicin A. 1-N-ethylgentamicin

Treat in the manner described in Example 1A 5 g of gentamicin in 250 ml of water with 1N sulfuric acid until the pH of the solution is about 5, then treat the acidified solution with acetaldehyde and sodium cyanoborohydride in the manner indicated and isolate the resulting product to form 1-N ethyl gentamicin C1 + 11¼ (c = 0.3%, HgOh PMR (ppm) (DO): $ 1.03 (3H, t, 7 Hz, -CH2 CH3); 1.03 (3H, d, 1 = 6.5 Hz, -CH-CH); 1.17 (3H, s, C-CH 3); 2.32 (3H, s, 6'N-CH 3); 2 9 (3H, s, 3 "- NHCH3); ¼, 9¼ (1H, d, 1 = ¼ Hz, H, ") * 5.13 (1H, d, J = 3.5 Hz, H 2 ') in mass spectrum: (M + 1 ) + m / e 506 also m / e 15 ^ 157, 16Ο, 173, 191, 201, 219, 286, 317, 329 (v), 3¼7, 350, 360 375 and ¼30.

B. Repeat the experiment of Example ¼A, but use other aldehyde reactants such as formaldehyde, n-propanal, n-butanal, n-octanal, hydroxyacetaldehyde, ¼-bydroxybutanal and phenylacetaldehyde instead of acetaldehyde. Isolate and purify each of the products in the manner indicated to form the respective 1-N-methylgentamicin C1, δ (DgO) 1.0¼ (3H, d, J = 6.5 Hz, 6'-CH3); 1.18 (3H, s, V-CH 3); 2.29 (3H, s, 1-WCH 3) in 2.32 (3H, s, 6'-NCH 3) in 2, U9 (3H, s, 3 "-HCH) in ¼, 95 (1H, d," , 2 ^ = ¼ Hz, H1; and 5.13 ppm (1H, d, 1 ^, 2 ^ 3.5 Hz, H1 '); M + m / e ¼91 also ¼16, 38¼. 36¼. 361, 3¼6, 3¼3, 336, 333, 318, 315, 303, 286, 205, 187, 177, 160, 159, 157i 1-N- (β-hydroxyethyl) -gentamicin O + + 98.0 ° (c = 0.3 %, Η,, Ο); PMR (ppm) (D 2 O): δ 0.99 (3H, d, 1 = 6.5 Hz, 6'-CH 3); 1.13 (3H, s, V'-CH 2; 2.28 (3H, s, 6'-NCH 3), 2, U 5 (3H, s, 3 "-NCH 3) in ¼, 97 (1H, d, 1 = ¼ Hz, H 2 ') and 5, 11 (1H, d, 1 = 3.5 Hz, H 2 '); mass spectrum: (M + 1) + m / e 522 also m / e ^ 6, ¼0¼, 39¼, 391, 32 621 00 376, 373, 366, 363, 3.1 + 8, 3.1 + 5, 333, 286, 235, 217, 207, 189, 160 and 157; Ymax (KBR) 3300, 1060 cm -1, 1-N- (phenylethyl) -gentamicin + 99.1 + ° (c = 9.3%, H 2 O); PMR (ppm) (D 2 O): 0.99 (3H, d, J = 6.5 Hz, 6 '-CH ; 1.10 (3H, s, 1 + "- CH 3); 2.28 (3H, s, ό'-ΝΟΗ 2; 2.1 + 3 (3H, s, 3" -NCH 3); 1 +, 88 ( 1 H, d, J = 1 + Hz, "); 5.08 (1H, d, J = 3.5Hz, H1") and 7.33 (5H, s, -CgH 2); mass spectrum: +1) + m / e 582 also m / e 506, 1 + 61 +, 1 + 51 +, 1 * 51, 1 * 36, 1 + 33, 1 * 26, 1 + 23, Uo8, l + 05 (v), 393, 295, 286, 277, 267, 2l + 9, I60 and 157; Ϋ (KBR) 3300, 1050, 1030 cm

Example 5 1-N-Substituted Antibiotic G-52 A. 1-N-Ethyl Antibiotic G-52 Dissolve 87U mg of antibiotic G-52 in 1 + 0 ml of distilled water and add 1N sulfuric acid until the pH of the solution is adjusted to about 3 5. Add 0.7 ml of acetaldehyde, stir the reaction mixture for 10 minutes and add 100 mg of sodium cyanoborohydride. Monitor the reaction solution by thin layer chromatography and, when the starting material antibiotic G-52 appears to have fully reacted (i.e., in about 10 minutes), concentrate the solution in vacuo at about 35-1 + 0 ° to a volume of about 10 ml. Then, for the concentrated solution through a basic ion exchange resin, lyophilize to an ether, comprising 1-N-ethyl antibiotic G-52. Purify chromatographically on a silica gel column (120 x 1.25 cm) and elute with the lower phase of the system chloroform / methanol / 7% ammonium hydroxide (2: 1: 1). Combine the same eluates as determined by thin layer chromatography and concentrate the combined eluates of the main component in vacuo to an ether, comprising 1-N-ethyl antibiotic G-52 (yield 60 mg). Purify the additional overlap eluates from the prior chromatography on silica gel and elute with the lower phase of the chloroform / methanol / 7 Antibiotic G-52. Bring the combined ester sites of 1-N-ethyl antibiotic G-52 to pass through a column of basic ion exchange resin (such as Amberlite®IRA 1 + 01S) and lyophilize the eluate to form 1-N-ethyl antibiotic G-52 (yield 90 mg); + 122.1 ° (c = 0.3%, H 2 O), PMR (ppm) (D 2 O): δ 1.06 (3H, t, J = 6.5 Hz, 1N-CH 2 CH 3); 1.21 (3H, s, 1 + "- C-CH 3 H 2.30 (3H, s, 3" -N-CH 3); 2.50 (3H, s, 6'-N-CH 3); 1 +, 91 + (1H, m, H 2 '); 1 +, 97 (1H, d, J = 1 +, 0 Hz, H 2'); 5.3 ^ (1H, d, J = 2.5 Hz, H mass spectrum: (M + 1) + m / e 1 + 90 also m / e 1U1, 15l +, 160, 173, 191, 201, 219, 270, 313, 317 (w), 331, 332, 350, 359, 360, 378, 390 and l (ll +.

33 621 00

Example 6 A. In the manner described in Example 1A, treat the following it, 6-diaminoglycosyl-1,3-diaminocyclitols in water with sulfuric acid and then with acetaldehyde and sodium cyanoborohydride: gentamicin B, antibictic JI-20B, mutamicin 2, mutamicin 6.

Isolate and purify each of the obtained products as described in Example 1A to form the corresponding 1-N-ethyl compound, namely: 1-N-ethylgentamicin B, [ck₂ ^ + 119.7 ° (c_, 1 in water) ; mass spectrum m / e / MH / + 380, 352, 33U, 378, 350, 332, 219, 191, 173, NMR (60 MHz DgO): δ 5.55 (H-1 *, J1, 3.0 Hz) 5.05 (H-1 ", Jg" 2 "= b Hz), 2.9 (N-CH2, 1.05-1.5 (2C-Cl2)); 1-N ethyl antibiotic JI-20B, + 112.5 (HgO) NMR (D O): δ 1.1 (3H, t, J = 7 Hz, CH 2 -CH 3) in 1.22 (3H, s, C CH3) 1.3 (3H, d, -CH-CH4); 1 *, 95 (1H, d,

Hz, H₂); 5.35 (1H, J = 3.5 Hz, H₂); mass spectrum: M + + 1 m / e 52 U, also 15Π, 160, 173, 175, 191, 201; 219, 30U, 317, 332, 333, 350, and 360; 1-N-ethylmutamide 2, mp 8o-8i + ° C, PMR (DgO): 1.06 (t, J = 7 Hz, 3 , 1.19 (s, 3, Um-CH 3), 2.50 (s, 3, 3 "-N-CH 3), 2.53 (d, J 2" = 10.5 Hz, 1, H 3 "), 3.75 (dd, J 2" 2 "= 1+ Hz; 3" = 10.5 Hz, 1, H 2,), 3.83 (d, H 2, t eq, _ "Ax 12 Hz, 1, H5"), it, 86 (m, 1, H 2), 1 +, 98 (d, H 2 "1" = it Hz, 1, H 2), and 5.10 ( d,, gr = 2.5 Hz, 1, H +, mass spectrum: m / e 1 + 59, 38 U, 329, 316, 311, 301, 283, 203, 1Ö5, 175, 160, 11+ 2 and 127.

1-N-ethylmutamicin 6, sp. 118-122 ° C (decomposes); mass spectrum (M) + m / e 1 + 75, (M + 1) m / e 1 + 76 + monosaccharides: m / e 160, 127; 2-deoxyceptamines: m / e 219, 201, 191, 171; disaccharides: m / e 355, 317, 299, m / e 378, 350, 322 PMB (/) D20

5.1 * + d, J = 2.5 Hz 1'-H

5.00 d, J = 1 +, 1 Hz 1 "-H

i +, 9 broad singlet it'-H

i +, 38 wide singlet 5-H

3.93 d, J = 12, 5Hz 5 "e-H

3.78 q. 2 "-H

3.38 d, J = 12.5 Hz 5 "a-H

3.21 (1H) broad singlet 6'-H

2.65 d, J = 11.0 Hz 3 "-H

2.52 singlet 3 "-N-CH3 1.22 singlet i + MC-CH3 1.07 triplet 1-N-CH2-CH3 CMR (DgO): su 62100 PPM: β1 + 9.8, 102.9 , 97.1 *, 97.0, 83.9, 80.5, 73.2, 70.1, 69.6, 68.5, 63.9, 5.1 *, 5, 1 * 7.1, 1 * 7.0, 1 * 3.1, 1 * 0.8, 37.5, 33.0, 25.6, 22, U and IU, 6.

Example 7

Preparation of 1-N-substituted-U, 6-di- (aminoglycosyl) -1,3-diamino-cyclitols via selectively blocked intermediates A. 1-N-ethylgentamicin via a 2 ', 3-di-N-substituted intermediate 3U0 mg of 2 ', 3-di-N-trifluoroacetylgentamicin in 10 ml of water / methanol (2: 1) and install the pH of the solution in about 3.5 by the addition of 1N sulfuric acid. Add 0.19 ml of acetaldehyde, stir for 10 minutes, add 27 mg of sodium cyanoborohydride and stir the reaction mixture for another 10 minutes. Evaporate the reaction mixture in vacuo to an ether stage, comprising 1-N-ethyl-2 ', 3-di-N-trifluoroacetylgentamicin C Dissolve said iterate in 50 ml of concentrated ammonium hydroxide and store at room temperature for 2 hours. Evaporate the mixture in vacuo and chromatograph the obtained mixture over 12 g of silica gel and elute with the lower phase of the system chloroform / methanol / 10% ammonium hydroxide (2: 1: 1). Combine equal fractions (as determined by thin layer chromatography) and evaporate the combined eluates in vacuo to an ether column, comprising 1-N-ethylgentamicin (yield 80 mg); data same as in Example 1 + B.

B. 1-N-ethylsisomicin via a 6'-N-substituted intermediate

Treat pi in Example 7A described method 6'-N-t-butoxycarbonyl isomicin (prepared according to Preparation 3D) in water / methanol with sulfuric acid and directly with acetaldehyde and sodium cyanoborohydride. Store the mixture for 30 minutes at room temperature and evaporate in vacuo to give 1-N-ethyl-6'-N-t-butoxycarbonyl isomicin. Dissolve the obtained iterate in trifluoroacetic acid and store for 10 minutes. Add an excess of anhydrous methanol, filter off the formed precipitate of the trifluoroacetic acid salt of 1-N-ethylsomicin and chromatograph over silica gel using the lower phase of the chloroform / methanol / ammonium hydroxide pi system of Example 7A ; data same as in Example 1A.

C. 1-N-methylsisomicin from 3 "-N-1 *" -O-carbonylsisomicin 5 g of 3 "-N-U" -O-carbonylsomomicin in 300 ml of distilled water is treated with 1N sulfuric acid until the pH of the solution is 2.5. 2 ml of 37 µg formaldehyde is added and after 10 minutes a solution of 500 mg sodium borohydride is added dropwise in 5 ml of water. After one hour, the volume of the solution is reduced to half by evaporation in vacuo, the pH of the concentrated solution is adjusted to 11 with the addition of 1N sodium hydroxide solution and the solution is evaporated to dryness. The residue is extracted with 3 x 100 ml of methanol and the combined methanol extracts are diluted with an equal volume of chloroform, filtered and evaporated to give crude 1-N-methyl-3M-N-1 + - 0-carbonylsisomicin.

The crude product is treated for five hours with 10% potassium hydroxide solution in water at 100 °. The cooled solution is passed through an Amberlite® IRC-50 (H +) ion exchange resin and eluted with 2N ammonium hydroxide and the eluent is concentrated and lyophilized to give crude 1-N-methyl-isomicin.

Chromatography of the crude material on 300 g of silica gel in chloroform / methanol / T% ammonium hydroxide (2: 1: 1) gives 1-N-methyl-sisomicin. Zc + 7D + 153 (0.3%, HgO); mass spectrum: (M + 1) + m / e 1 + 62 also m / e 127, 11 + 0, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331 and 336 (w) , 31 + 6, 376, 386.

D. 1-N-methylverdamicin via a 3 ", 1 +" - N-O-oxizolidone intermediate (1) Add aqueous 1 g of verdamicin with sodium carbonate until the pH is in the range of 8-9. Add, during three hours of stirring, a solution of 5 g of p-nitrophenylchlorocarbonate in 25 ml of dimethylamide, while maintaining the pH of the reaction mixture at about 8-9 by adding an additional amount of sodium carbonate solution. After the addition is complete, stirring is continued for 16 hours at pH 8-9. Evaporate the mixture in vacuo several times with hot chloroform, combine and evaporate the extracts and chromatograph the residue formed over 100 g of silica gel, eluting with the lower phase of the system chloroform / methanol / concentrated ammonium hydride (2: 1: 1). Combine and evaporate like fractions as determined by thin layer chromatography to give 3 ", 1 +" - N, O-oxazolidone.

(2) Treat according to Example ID the oxazolidone derivative in water with sulfuric acid, formaldehyde and sodium cyanoborohydride. Isolate and purify the resulting product in the manner described to form 1-N-methylverdamicin-3 ", 1 +" - N, 0-oxazolidone.

(3) Dissolve 0.2 g of 1-N-methylverdamicin-3 ", 1 +" - ~ N, 0-oxizolidone in 10 ml of 2N sodium hydroxide solution. Reflux for five hours, neutralize the reaction solution with acetic acid and evaporate to an ether residue. Chromatograph over 10 g of silica gel and elute with the lower phase of the system chloroform / methanol / 15% ammonium hydroxide (2: 1: 1). Combine and evaporate similar fractions, as determined by thin layer chromatography to a residue to form 1-N-methylverdamicin.

Example 8 1-N-benzylgentamicin via a tri-N protected 1-N-Schiff base intermediate (1) Dissolve 0.3 g of 2 ', 3-di-N-trifluoroacetylgentamicin in 12 ml of ethanol and add 0.9 ml of benzaldehyde. Stir the reaction mixture for three hours and evaporate 3β 62100 in vacuo. Dissolve the ether residue in 0.8 ml of chloroform and drop the solution dropwise into 25 ml of hexane / ether (3: 1) · Separate the formed precipitate by filtration and dry in vacuo to give 1-N-3 "-N-V'-0 -bis-benzylidene-2 ', 3-di-N-trifluoroacetylgentamicin C ^ (Yield = 0.38g); mp 128-13 ° C; + 7 °, 6 ° (c = 0.26 ethanol) .

(2) Dissolve 1.37 g of the product obtained in 100 ml of ethanol and add to a stirred mixture of 1.37 g of sodium nitoxide and 1.9 * g of sodium borohydride in 100 ml of ethanol. Stir for three hours, acidify the mixture to a pH of about 3 with hydrochloric acid and stir for an additional 16 hours. Extract the solution with ether, separate and discard the ether layer. Add ammonium hydroxide to the aqueous phase until it is basic and evaporated in vacuo to an ether. Extract the ether with 35 ml of warm ethanol. Combine the extracts and evaporate in vacuo. Chromatograph the formed ether over 75 g of silica gel and elute with the lower phase of the system chloroform / methanol / ammonium hydroxide / water (2: 1: 0.2: 0.8). Combine similar fractions as determined by thin layer chromatography and evaporate to an ether residue, comprising 1-N-benzyl-gentamicin C., mp 83-88 °, + 90 ° (c = 0.3%, H 2 O); PMR (ppm) (D 2 O): δ 1.03 (3H, d, J = 7 Hz, HC-CH 3); 1.16 (3H, s, C-CH 3); 2.27 (3H, s, N-CH 3); 2.50 (3H, s, H-CH)} k J (DgO + PhCHgN-); U, 92 (1H, d, J + U Hz, H-1 '); 5.08 (1H, d, J = 3.5 Hz, H-1'); 7, 3 (5H, s, aromatic H) mass spectrum: (M + 1) + m / e 568 as well as m / e UUo, U37, U12, 393, 379, 281, 263 »253, 235, 160 and 157.

Example 9 1-N-Substance-1 +, 6-Diamonoglycosyl-1,3-diaminocyclitols

Prepare by hydride reduction of the corresponding 1-N-acyl derivative A. 1-N- (S - 3-amino-3-hydroxybutyl) -gentamicin (1) Suspend 1-N- (S- 3 -amino-β-hydroxybutyr1) -gentami in 8 ml of tetrahydrofuran. Add 1U ml of 1N diborane to tetrahydrofuran and reflux heated for six hours under a nitrogen atmosphere. Carefully add 2 ml of water to decompose any excess diborane and evaporate. Dissolve the formed ether in hydrazine hydrate and reflux heating under a nitrogen atmosphere for 16 hours. Evaporate the solution and extract the ether with warm ethanol in water. Evaporate the combined ethanol extracts and chromatograph the formed ether over 10 ml of silica gel and elute with the lower phase of the chloroform / methanol / concentrated ammonium hydroxide system (2: 1: 1). Combine and evaporate like fractions, as determined by thin layer chromatography to give 1-N- (S + 72.4 ° (c = 0.35 $% H2 O); PMR (ppm) (DgO): δ.18 (3H, d, J = 7 Hz, 37 621 00 CH-CH 3 H 1.2U (3Η, s, C-CHg); , N-CH 2 in 2.5 »+ (3H, s, N-CH 2 ·, 5.07 (1H, d, J = 3.5 Hz, H-1 d, J = 3.5 Hz, H-1 '); mass spectrum: (M + 1) + m / e 565 as well as m / e 528, 516, 1 + 90, 1 + 37, 1 + 31 +, 1 + 10, 397, 278, 250, 232, 160 and 157. Prepare, by analogy, 1N-1S-γ'-methylamino- [5-hydroxypropyl) -gentamicin B, PMR: D 3H, s, C-CH 3); 2.33 (3H, s, 3 ", - N-CH 3); 2.62 (3H, s, 3" -N-CH 3); J = U, 5 HzTh-1 '); 5.12 (1H, d, J = 3 0 Hz, H-1').

Example 10 1-N-methylsisomicin from 3 "-N-1 +" - 0-carbonyl-2 ', 3,6' -tri-t-butoxycarbonyl-isomicin Dissolve 0.77 g of 3 "-N-1 +" - 0- carbonyl-2'3,6'-tri-Nt-butoxycarbonyl-isomicin in 20 ml of tetrahydrofuran and cool in an ice bath. Add 0.12 g of methyl fluorosulfonate and allow the temperature to rise to room temperature. Remove the solvent and dissolve the residue in trifluoroacetic acid. After five minutes at room temperature, remove the trifluoroacetic acid in vacuo and treat the ether with 10% sodium hydroxide solution for five hours at 100 °.

For the cooled solution through a column of Amberlite® IRC-50 (H +) ion exchange resin and elute with 2N ammonium hydroxide. Concentrate the eluent and lyophilize to give the crude title product.

Chromatograph the crude silica gel material in the lower phase of the chloroform / methanol / 7 $ ammonium hydroxide (2: 1: 1) system to form 1-N-methyl-sisomicin. + 153 ° (0.3- $, H 2 O); mass spectrum: (M + 1) + m / e 1 + 62, also m / e 127, 1 * + 0, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 ( v), 31 + 6, 376 and 386.

Example 11 1-N-methylsisomycin 5 g of 2 ', 3,6, -tri-Nt-butoxycarbonyl-3 "-N-1 + + - 0-carbonylsisomicin in 100 ml of ethanol is treated with 1 ml of 37g formaldehyde and 5 g of ammonium formate and the solution is heated for 2k hours under reflux. The solution is diluted with 200 ml of water and extracted with 3 x 100 ml of chloroform. The combined extracts are reduced to dryness and the residue containing 2 ', 3,6'-tri-Nt-butoxy-carbonyl-3 "-N-1 +" - O-carbonyl-1-N-methyl-isomicin is dissolved in trifluoroacetic acid and after five minutes at room temperature, the solvent is removed in vacuo.

The residue is treated with 25 ml of 10 $ potassium hydroxide solution at 100 ° for five hours. The cooled solution is passed through a column of Amberlitev IRC 50 (H +) ion exchange resin and the crude product is eluted with 2N ammonium hydroxide.

The combined eluent is evaporated to dryness in vacuo and the ether is chromatographed on 200 g of silica gel in the lower phase of the chloroform / methanol / 7 $ ammonium hydroxide (2: 1: 1) to give 1-N-methylsisomicin. (0.3%, H 2 O) \ mass spectrum: (M + 1) + m / e 1 + 62, also m / e 122, 1 Uo, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (v), 3I + 6, 376 and 386.

Example 12 1-N-methylsilines

Treat 0.77 g of 2 ', 3.6, -tri-Nt-butoxycarbonyl-3 "-N-1 +" - 0-kajibonyl-isomicin in 25 ml of tetrahydrofuran with 101 mg of methylamine and 290 mg of trifluoromethylsulfonic anhydride at 0 ° for 18 hours. Evaporate the solution to dryness and dissolve the residue in 10 ml of dimethylformamide and stir together with 300 mg of methyl iodide and 130 mg of potassium carbonate for an additional 18 hours. Remove the solvent by evaporation and treat the ether with 10 M potassium hydroxide solution at 100 ° for 12 hours. For the cooled solution through a column of Amberlite IRC 50 (H) ion exchange resin. The crude product is eluted with 2N ammonium hydroxide. The combined eluent is evaporated to dryness and the residue is chromatographed on 200 g of silica gel in the lower phase of the chloroform / methanol / 7% ammonium hydroxide (2: 1: 1) system to give 1-N-methylsisomicin.

+ 153 ° (0.3%, HgO); mass spectrum (M + 1) + m / e 1 + 62 also m / e 127, 1 Uo, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 3I +6, 376 and 386.

Example 13 1-N-methylsisomicin 0.77 g of 2 ', 3,6'-tri-Nt-butoxycarbonyl-3 "-N-1 +" - 0-carbonylsisomicin in 20 ml of THF is treated with 3 ml of 37 # -ig formaldehyde and 170 mg succinimide at room temperature for 18 hours. The solution is immersed in a mixture of diethyl ether and hexane (1: 1) and the precipitate is collected by filtration. This material is treated with 200 mg of sodium borohydride in 20 ml of ethanol at room temperature for four hours.

The ethanol is removed in vacuo and the ethereal is treated with 10% potassium hydroxide solution for 12 hours at 100 °. The cooled solution is passed through a column of

Amberlite (S) IRC 50 (H) ion exchange resin and the crude product are eluted with 2N

ammonium hydroxide. The combined eluent is evaporated to dryness in vacuo and the residue is chromatographed on 200 g of silica gel in the system chloroform / methanol / 7% ammonium hydroxide to form 1-N-methylsisomicin. / <X -7D + 153 ° (0.3 Jf, H2 O) in mass spectrum: (M + 1) + m / e 1 + 62, also m / e 127, 11 + 0, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (v), 3I + 6, 376, 386.

Example 1U

1-N-methylsisomicin Dissolve 0.77 g of 2 ', 3,6'-tri-Nt-butoxycarbonyl-3' '- N-1 +' - 0-carbonylsisomicin in 100 ml of dichloromethane together with 0.25 g of acrylonitrile for 2+ hours .

In vacuo, remove the solvent to form an ether residue, which is dissolved in dimethylformamide (3962100), and treat with 180 mg of methyl iodide at 50 ° for 12 hours. Remove the solvent and treat the residue with 10% potassium hydroxide solution at 100 ° for eight hours. The cooled solution is passed through a column of Amberlite® IRC 50 (H +) ion exchange resin and the crude product is eluted with 2N ammonium hydroxide. The concentrated eluent is evaporated to dryness in vacuo and the residue is chromatographed on 400 g of silica gel in the lower phase of the chloroform / methanol / 7β ammonium hydroxide (2: 1: 1) system to give 1-N-methylsisomycin. + 153 (0.3%> HgO); mass spectrum: (M + 1) m / e 1 + 62 also & m / e 127, 11 * 0, 159, 160, -177, 187, 205, 256, 285, 303, 318, 331, 336 (v) , 31 + 6, 376, 386.

Claims (2)

ko 62100 Process for the preparation of 1-N-substituted derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols gentamicin B, gentamicin, gentamicin C, sisomicin, verdamicin, antibiotic JI-20B, antibiotic G-52, 1 is mutamicin 2 and mutamicin 6, wherein the 1-N substituent is -ch 2x where X is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl containing aliphatic groups containing from 1 to 7 carbon atoms, and if substituted with amino and hydroxy, these substituents are on different carbon atoms, and from the pharmaceutically acceptable acid addition salts of these compounds, characterized in that a) treating any of said 4, 6-di (aminoglycosyl) -1,3-diaminocyclitols, which may have amino protecting groups in any position other than the 1-position, with an aldehyde of formula X'-CHO where X 'is the same as X, however, optionally present amino-el or hydroxy group may be protected, in the presence of a hydride-donor reducing agent, and, if required, remove all protecting groups present in the molecule, or b) reduce the N, C double bond in a 1-N = CHX + - substituted derivatives of it, 6-di- (aminoglycosyl) -1,3-diaminocyclitol, wherein X 'is the same as before, and all NH NH groups are protected, and NHCH₂ groups can be protected, removing all present protecting groups or c) treats a 1-N-substituted derivative of said U, 6-di- (aminoglycosyl) -1,3-diaminocyclitol, wherein one or more amino groups may be protected and the 1-N substituent is 0H - CX "where X" is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl, tolyl, or hydrocarbyloxy, which contains aliphatic groups, and if substituted by amino and hydroxy, these substituents are p various carbon atoms, and any amino or hydride 62100 oxy group may be protected, with an amide-reducing hydride reactant, and if necessary, remove all protecting groups, or d) to prepare 1-N-substituted derivatives of 4.6 -di (aminoglycosyl) - 1,3- diaminocyclitols gentamicin B, gentamicin C, gentamicin C, sisomicin, 1α t verdamicin, antibiotic JI-20B, antibiotic G-52f mutamicin 2 and mutamicin 6, the substituent being a straight or branched alkyl group of 1 5 carbon atoms, a said 4,6-di (aminoglycosyl) -1,3-diaminocyclitol, which has amino protecting groups at any other than the 1 position, and wherein the 1-amino group may be activated, with an alkylating agent having a straight chain alkyl group having 1-5 carbon atoms, as well as a leaving group, removing the protecting groups and, if desired, also the activating groups or groups present in the molecule, or e) for the preparation of 1-N-substituted derivatives of 4,6-di (aminoglycosyl) - 1.3 - the diaminocyclitole gentamicin B, gentamicin C, gentamicin C, sisomicin, X id verdamicin, antibiotic JI-20B, antibiotic G-52, mutamicin 2 and mutamicin 6, where the substituent is methyl, any of the said 4,6-di (aminoglycosyl) - The 1,3-diaminocyclitols, which have amino-protecting groups in anyone other than the 1-position, with formaldehyde and a cyclic imide, treat the compound formed with a hydride-donor reducing agent and remove all protecting groups present in the molecule, or f) for preparation of the compounds listed in point (e), some of the said 4,6-di (aminoglycosyl) -1,3-diaminocyclitols, which have amino protecting groups in any other than the 1 position, react with formaldehyde in the presence of formic acid, and remove all protecting groups present in the molecule, isolating the derivative obtained in paragraphs a) to f) as such or in the form of a pharmaceutically acceptable acid addition salt. "2 62100 Patent Tivatimus: Menetelmä 4,6-di (aminoglycosylyl) -1,3-diaminocyclitoline, gentamisiini B, gentamisiini C, gentamisiini C, sisomisiini, verdamisiini, antibiootti 1 Id. mutamisiin 6 1-N-substituent thehdannaiste valmistamisexi, joissa 1-N-substituent on -ch2x jossa X on vety, alkyl, alkenyyl, cycloalkyl, cycloalkylalkylalkyl, hydroxyalkylyl, aminoalkylalkyl, N-alkylalkyl, N-alkyl hydroxyalkyl, phenyyli, bentsyyli tolyyli, jotka alifaattiset ryhmät sisältävät 1-7 hiiliatomia, ja mikäli ne ovat substituoituja aminolla ja hydroksilla, mainituista 4,6-di (aminoglycosyl) -1,3-diamino-cyclitoleista, joissa voi olla amino-suojaryhmia jossakin muussa kuin 1-ash mass, aldehydillä, young kaava on X'-CHO jossa. X'merkitsee samaa kuin X, jolloin kuitenkin mahdollisesti läsnäoleva amino-tai hydroxyiryhmä voi olla suojattu, hydridi-donaattori-rpelkistimen läsnäollessa, ja tarvittaessa poistetaan kaikki molecyylissä läsnäolevat , 3-diaminocyclitolin lN = CHX'-substituoidus johdannaisessa N, C-kaksoissidos, jolloin X 'merkitsee samaa kuin edellä, ja jolloin kaikki NH2-ryhmät 4, β-di (aminoglycosyl) -1,3-diaminocyclitolin 1-N substituent, jolloin yxy to aminory ammonium amine or suojattu and 1-N substituent to 0 ft -CX "jossa X" on fatty, alkyl, alkenyl , cycloalkylyl, cycloalkylalkylalkyl, hydroxyalkylyl, aminoalkylyl, N-alkylylaminoalkylyl, aminohydroxyalkylyl, N-alkyylamino-hydroxyalkylalkyl, phenylyl, bentsyloxy, tolyylalkyl hydrocarbylalkoxy, etc. älvävät 1-7 hiiliatomia, ja jos ne ovat substituoituja aminolla ja hydroxilla, substituentit sijaitsevat eri hiiliatomeilla, ja mah-