NO139483B - ANALOGICAL PROCEDURE FOR PREPARING NEW PSEUDOTRISACCHARIDES - Google Patents

Description

The present invention relates to a method for the production of active 1-N-substituted derivatives of 4>6-di-(aminoglycosyl)-1,3-diaminocyclitols.

A broad spectrum of antibacterial agents is known in the art

real agents that can be classified chemically as 4>°-di-(aminoglycosyl)-1,3-diaminocyclitols. Valuable antibacterial agents of this group are those in which the aminocyclitol is 2-deoxy-streptarain or a derivative thereof with amino functions in the 1- and 3-positions.

Particularly valuable antibacterial agents of 4,6-di-(aminoglycosyl)-2-deoxystreptamines are those in which the aminoglycosyl group in the 6-position is a garosaminyl radical. Within the class of 4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines are antibiotics such as gentamicin-B, B-jy C-^, C^a, C2a' C2b °^ X2' s^-soinicin' verdamicin, Antibiotic G- 418, Antibiotic G-52, Antibiotic JI-20A and Antibiotic JI-20B.

The present invention relates to the preparation of compounds where the amino group in the 1-position of the 2-deoxystreptamine or its derivative in a 4>6-di-(aminoglycosyl)-1,3-diaminocyclitol is selectively N-substituted. The 1-N-substituted derivatives of 4»6-di-(aminoglycosyl)-2-deoxystreptamines or derivatives: thereof are valuable broad-spectrum antibacterial agents. : More specifically, the invention relates to the production of 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols: gentamicin B, gentamicin B, , gentamicin C, , gentamicin C, ,

X X X 3 gentamicin C2, gentamicin C2a, gentamicin C^, sisomicin, verdamicin, Antibiotic G-4l8, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4in mutamicin 5 and mutamicin 6, where the substituent is -CH2X where X is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl or benzyl, which aliphatic radicals have up to 7 carbon atoms and , if substituted with amino and hydroxy, bear the substituents on different carbon atoms and of pharmaceutically acceptable acid addition salts thereof.

Included among the substituents considered for the group CH^X in the new compounds are straight chain and branched alkyl groups such as ethyl, n-propyl, n-butyl, g-methylpropyl, n-pentyl, g-methylbutyl, Y-methylbutyl and B,3-dimethylpropyl, n-hexyl, S-methylpentyl, g-ethylbutyl, yethylbutyl, n-heptyl, e-methylheptyl, g-ethylpentyl, yethylpentyl, 6-ethylpentyl, Y-propylbutyl, n-octyl, iso-octyl, 3-ethylhexyl, 6-ethylhexyl, e-ethylhexyl, g-propylpentyl, y-propylpentyl, alkenyl groups such as (3-propenyl, g-methylpropenyl, g-butenyl, g-methyl-g-butenyl, p-ethyl-p- hexenyl, hydroxy-substituted straight chain and branched alkyl groups such as e-hydroxypentyl, 6-hydroxy-Y-methylbutyl, 6-hydroxy-8-methylpropy1, 6-hydroxybutyl, g-hydroxypropyl, Y~hydroxypropyl, u-hydroxyoctyl, amino-substituted straight chain bg branched alkyl groups such as "e-aminoperityl, - g-aminopropyl, Y_aminopropyl, 6-aminobutyl, g-amino-Y-methylbutyl and u)-aminooctyl and mono-N-alkylated derivatives thereof such as N-methyl, N-ethyl and N-propyl derivatives, e.g. ermethylaminopentyl, g-methylaminopropyl, g-ethylaminopropyl, 6-methylaminobutyl, g<->methylamino-Y-methylbutyl and aj-methylaminobutyl, amino- and hydroxy-disubstituted straight chain and branched alkyl groups such as 3-hydroxy-e-aminopentyl,Y- hydroxy-Y-niethyl-6-aminobutyl, <g>~hydroxy-6-aminobutyl, B-hydroxy-Y-aminopropyl and g<->hydroxy-g<->methyl-Y-aminopropyl and mono-N-alkylated derivatives thereof such as g<->hydroxy-e-methylaminopentyl, Y-hydroxy-Y-methyl-6-methylaminobutyl, <g->hydroxy-S-methylaminobutyl, g-hydroxy-Y-ethylaminopropyl and &-hydroxy-g<- >methyl-Y-methylaminopropyl.

The compounds are preferably 1-N-CH 2 X derivatives containing garosaminyl as 6-aminoglycoside radical and preferably 2-deoxystreptamine as 1,3-diaminocyclitol.

The 2-deoxystreptamine is present in all the above compounds except the mutamicins. The 1,3-diamino-cyclitol core in each of l-N-CH2X-mutamicin 1, 2, 4, 5 and 6 is streptamine, 2,5-dideoxystreptamine, 2-epi-streptamine, 5-amino-2,5-dideoxystreptamine and 5-epi-2-deoxystreptamine.

The l-N-CH2X-4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines are the derivatives of gentamicin B, genta-

micin B^, gentamicin C^, gentamicin C^a, gentamicin C2, gentamicin <C>2a' 9entamicin C2b' 9entam^c^-n x2' sisomic:i-n' verdamicin, Antibiotic G-418, Antibiotic JI-20A, Antibiotic JI-20B and Antibiotic G-52 which compounds are defined by the following structural formula I:

wherein X is as previously indicated, and wherein Y is an aminoglycosyl function selected from the group consisting of:

Other useful 1-N-CH2X-4,6-di-(aminoglycosyl)-2-. deoxy-streptamines include 1-N-CH2X-Antibiotic 66-40D of the following formula III (which is among the preferred compounds prepared according to the invention):

where X is as previously stated,

and 1-N-CH2X-Antibiotic 66-40B of formula IV:

where X is as previously stated,

The 1-N-C^X-mutamicins include 1-N-CH2X-4-aminoglycosyl-6-garosaminyl-1,3-diaminocyclitols of formula V:

wherein X is as previously indicated, and wherein 1-N-CH2X-mutamicin 1, V2 and W5 are hydrogen and W2 and V5 are hydroxyl,

wherein 1-N-CH2X-mutamicin 2, W2, B2, W5 and V& are hydrogen,

wherein l-N-CH2X-mutamicin 4, W2 and W5 are hydrogen and V2 and Vg are hydroxyl,

wherein 1-N-CH2X-mutamicin 5, V*2, V"2 and Wg are hydrogen and V5 is amino and

wherein 1-N-CH2X-mutamicin 6, W2, V2 and V5 are hydrogen while W5 is hydroxyl.

(In the structural formulas given above, the unspecified substituents at the bonds are hydrogen atoms).

Also included within the invention is the preparation of pharmaceutically acceptable acid addition salts of the 1-N-CH2X-4,6-di-(aminoglycosyl)-1,3-diaminocyclitols as indicated by the formulas I, III, IV and V, which salts are prepared according to known methods as follows as by neutralizing the free base with a suitable acid usually to pH 5. Suitable acids for this purpose include acids such as hydrochloric, sulfuric, phosphoric, nitric, hydrobromic, acetic, propionic, maleic, ascorbic, citric and the like.

As physical embodiments of the acid addition salts of the 1-N-CH2X-4,6-di-(aminoglycosy1)-1,3-diaminocyclitols are characterized by being white solids which are soluble in water, slightly soluble in most polar solvents and insoluble in non-polar organic solvents.

1-N-CH2X-4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols as indicated by formulas I, III, IV and

V and their non-toxic pharmaceutically acceptable acid addition salts exhibit broad-spectrum antibacterial activity. In particular, the 1-N-lower alkyl derivatives exhibit improved antibacterial activities compared to the parent antibiotics, which is particularly indicated by an increased activity of the compounds against organisms that are resistant to the parent compound. The connections are, for example; more active against organisms that inactivate the parent antibiotics by acetylation of the 3-amino group and/or by adenylation of the 2"-hydroxyl group. Some of these also exhibit anti-protozoal, anti-amoebic and anthelmintic properties.

A preferred group of compounds are the 1-N-substituted derivatives of the 4-arninoglycosyl-6-garosaminyl-2-deoxy-streptamines: gentamicin B, gentamicin B^ , gentamicin , gentamicin C, X 3 , gentamicin C £~, sisomicin, verdamicin, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52 and Antibiotic G-4l8>of which the derivatives of gentamicin CX . , gentamicin CX . 3, sisomicin, verdamicin and Antibiotic G-52 are preferred. Other particularly useful compounds are the 1-N-substituted derivatives of Antibiotic 66-40D.

In the 1-N-substituent, X is preferably selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, aminohydroxyalkyl, phenyl or benzyl, which aliphatic radicals have up to 7 carbon atoms, and if they are substituted with amino and hydroxyl, the substituents are carried on different carbon atoms . Of these, the preferred radicals are hydrogen, alkyl, aminoalkyl and hydroxyalkyl with up to 7 carbon atoms and aminohydroxyalkyl with up to 3 carbon atoms and with the substituents on different carbon atoms.

Particularly useful compounds are those

wherein X is hydrogen, methyl, ethyl and propyl and preferably methyl and ethyl. A particularly valuable group are the 1-N-CH2X-4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines of formula I where X is lower alkyl of 1-3 carbon atoms, in particular the 1-N-lower alkyl derivatives of gentamicin C^, gentamicin Cla , gentamicin C2, gentamicin C2a, gentamicin C^, sisomicin, verdamicin and Antibiotic G-52 as well as 1-N-lower alkyl Antibiotic 66-40D of formula III, which derivatives are broad-spectrum antibacterial agents, which are active against gram-positive bacteria (eg, Staphylococcus aureus) and Gram-negative bacteria (eg, Escherichia coli and Pseudomonas aeruginosa) as determined by standard dilution tests, including bacteria resistant to the 1-N-unsubstituted precursors. Particularly useful are 1-N-ethylverdamicin, and 1-N-lower alkylsisomycins, e.g. 1-N-methylsisomicin, 1-N-(n-propyl)-sisomicin, 1-N-(n-butyl)-sisomicin and preferably 1-N-ethylsisomicin which exhibit activity against gram-negative organisms resistant to their 1 -N-unsubstituted precursors. Other particularly useful compounds are 1-N-ethylgentamicin C^a, 1-N-ethylgentamicin C^, 1-N-ethylantibiotic G-52, 1-N-(n-propyl)-verdamicin, 1-N-(6- aminobutyl)-sisomicin, 1-N-methylverdamicin, 1-N-(n-butyl)-verdamicin, 1-N-(S-2-hydroxy-4-aminobutyl)-gentamicin Clt 1-N-(S-2- hydroxy-4-aminobutyl)-sisomicin and 1-N-(S-2-hydroxy-

4-aminobutyl)-verdamicin.

Most of the above-mentioned 1-N-unsubstituted 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol antibiotics from which the 1-N-substituted derivatives are prepared are known, the starting compound indicated here as gentamicin ^3 , is isolated and characterized as indicated in preparation 1.

The starting compound indicated here as gentamicin C2b, is isolated and characterized as described in preparation 2, and which has the structural pattern shown here, is described in some of the known publications as gentamicin C„.

The isolation, properties and configuration of gentamicin C2 is described in US Patent No. 3,651,042.

Antibiotic 66-40B. and 66-40D, their preparation, isolation, properties and configuration are described in Belgian Patent Document No. 811 370. The antibiotics are co-produced with sisomicin which is the main product of fermentation of Micromonospora invoensis (described in British Patent Document No. 1 274 518) and can be separated from the fermentation medium by subjecting it to special chromatographic separation techniques.

Mutamicin 1, 2, 4, 5 and 6 whose configuration is shown above can be produced by growing a mutant strain of Micromonospora invoensis, here designated as Micromonospora invoensis strain 1550F-1G in an aqueous nutrient medium. This mutant strain is unable to produce an antibiotic when grown under submerged aerobic conditions in an aqueous nutrient medium lacking the 1,3-diaminocyclitol building block. However, when certain such compounds are added to the fermentation medium, mutamicins are formed. When 2-deoxystreptamine is added to the fermentation; the well-known antibiotic sisomicin is formed.

The necessary 1,3-diaminocyclitols that must be present

during the fermentation to obtain the mutamicins^ are as follows:

streptamine for mutamicin 1

2,5-dideoxystreptamine for mutamicin 2

2-epistreptamine for mutamicin 4

2,5-dideoxy-5-aminostreptamine for mutamicin 5 5-epi-2-deoxystreptamine for mutamicin 6.

The production of the mutamicins is described in widely available Swedish patent application 7409945-8-

The present invention thus relates to an analogous method for the preparation of 1-N-substitu-

pea derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols gentamicin B, gentamicin,B^, gentamicin C^, gentamicin C^Q, gentamicin ,C2> gentamicin; G2a,: .gentamicin £2%)' sisomicin, verdamicin, Antibiotic G-418, Antibiotic-66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-5 2, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is

wherein X is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl,. aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl or benzyl, which aliphatic radicals have up to 7 carbon atoms and, if X is substituted by amino and hydroxy, the substituents carry different carbon atoms,

and pharmaceutically acceptable acid addition salts thereof,

which method is characterized by the use of one of the following procedures A to G,

A) one of the above-mentioned 4,6-di(aminoglycosyl)-1,3-diaminocyclitols which may have amino-protecting groups in any position other than the 1-position is treated with an aldehyde of the formula

wherein X' is a group as defined for X and wherein any amino or hydroxy group present may be protected, in the presence of a hydride donor reducing agent, and, if necessary, that any protecting groups present in the molecule are removed,

B) reduction of the N,C double bond in a 1-N=CHX'-substituted

derivative of one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diamihocyclitols, in which all NH^ groups are protected and. NHCH^ groups may be protected, where X' is a group as defined for X, in which any amino or hydroxy group present may be protected, and that all protecting groups present in the molecule are removed,

C) a 1-N-substituted derivative of one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, in which one or more amino groups may be protected and the 1-N substituent is 0

II

-C-X"

where X" is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or hydrocarbyloxy, which aliphatic radicals have up to 7 carbon atoms, and if X is substituted with amino and hydroxy, carries the substituents on different carbon atoms, and in which any amino or hydroxy group present may be protected, is treated with an amide-reducing hydride reagent, and if desired, that all present protecting groups in the molecule are removed, D) that for the preparation of the above indicated 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols in which the substituent is straight-chain alkyl with up to 5 carbon atoms, that one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, exhibiting anino-protecting groups in any position other than the 1-position, and in which the 1-amino group may be activated, is reacted with an alkylating agent containing a straight-chain alkyl group with up to 5 carbon atoms and a leaving group, and that the protective groups and, if desired, the activating group or groups present in the molecule are removed,

E) that for the preparation of the above-mentioned 1-N-substituted

derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols in which the substituent is methyl, that one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which exhibit amino-protecting groups in any position other than the 1-position, is reacted with formaldehyde and a cyclic imide, after which the

the compound thus obtained is treated with a hydride-donor-reducing agent, and that all protective groups present in the molecule are removed,

F) that for the production of the same compounds as indicated in

point E, that one of the above-mentioned 4,6-di(aminoglycosyl)-1,3-diaminocyclitols exhibiting amino-protecting groups in any position other than the 1-position, is reacted with formaldehyde in the presence of formic acid and that all protective groups present in the molecule are removed,

which procedure alternatives A to F are followed by

isolation of the 'derivative- as such or as a pharmaceutically acceptable acid addition salt.

Process alternative A) whereby the 1-amino function i

a 1-N-unsubstituted-4, 6-di-(aminoglycosyl)-1, 3-diaminocyclitol is selectively condensed -with an eth- aldehyde and in connection therewith in situ reduced to form a 1-N-alkyl-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 carry this out under an inert atmosphere (e.g. argon or nitrogen). Preferably, the reaction is completed within a short time, usually less than 30 minutes as determined by thin layer chromatography.

The hydride donor reducing agents useful in this process include dialkylaminoboranes (e.g. dimethylaminoborane, diethylaminoborane and preferably morpholinoborane), tetraalkylammonium cyanoborohydride (e.g. tetrabutylammonium cyanoborohydride), alkali metal borohydride (e.g. sodium borohydride) and preferably alkali metal cyanoborohydride (eg lithium cyanoborohydride and sodium cyanoborohydride).'

The procedure is suitably carried out in an inert solvent. By "inert solvent" is meant any organic or inorganic solvent in which the 4,6-di-(aminoglycosyl 1-1, 3-diaminocyclitol starting materials and reagents are soluble, and which will not affect the process under the reaction conditions used such that a minimum of competing side reactions is formed. Although anhydrous aprotic solvents can sometimes be advantageously used in the process (such as tetrahydrofuran when morpholinoborane is used as hydride donor-reducing agent), the process is usually carried out in protic solvents, e.g. .in a lower alkanol or preferably in

water or in an aqueous lower alkanol (eg aqueous methanol, aqueous ethanol), although other water-miscible co-solvent systems may be used such as aqueous dimethylformamide, aqueous hexamethyl phosphoramide, aqueous tetrahydofuran and aqueous ethylene glycol dimethyl ether.

The method is conveniently carried out at a pH within the range from 1 to 11, preferably from 2 to 5 and proceeds best within the range from 2.5 to 3.5. The preferred acidic medium can be obtained by adding an organic or inorganic acid to 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol. Acid addition salts of the compounds are thereby formed. Any organic acid such as acetic acid, trifluoroacetic acid or p-toluenesulfonic acid or inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid or nitric acid can be used. It is most appropriate to use sulfuric acid. In a preferred embodiment of the method, it is also appropriate to prepare the acid addition salt of the starting compound in situ by adding the desired acid (e.g. sulfuric acid) to a solution or suspension of 4,6-di-(aminoglycosyl)-1,3- diamlnocyclitol (eg sisomicin) in a protic solvent (eg water) until the pH of the solution is adjusted to the desired pH.

Typical aldehydes of formula X'CHO wherein X' is as previously indicated which are useful in the process include straight chain and branched alkyl aldehydes such as formaldehyde, acetaldehyde, n-propanol, n-butanol, 2-methylpropanol, 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-hexenal, benzaldehyde and phenylacetaldehyde,

hydroxy-substituted straight chain and branched alkyl aldehydes such as 5-hydroxypentanal, 2-hydroxy-3-methylbutanal, 2-hydroxy-2-methylpropanal, 4-hydroxybutanal, 2-hydroxypropanal and 8-hydroxy-octanal, amino-substituted straight chain and branched alkyl aldehydes such as 5-aminopentanal',' 2-aminopropanal, 3-aminopropanal, '"4-- ~~ aminobutanal, 2-amino-3-methylbutanal, 8-aminooctanal and mono-N-alkyl derivatives thereof, and amino- and hydroxyl- disubstituted straight chain and branched alkyl aldehydes such as 2-hydroxy-5-aminopentanal, 3-hydroxy-3-methyl-4-aminobutanal, 2-hydroxy-4-aminobutanal, 2-hydroxy-3-aminopropanal, 2-hydroxy-2-methyl -3-aminopropanal, 2-amino-3-hydroxyoctanal and mono-N-alkyl derivatives thereof.

If the aldehyde exhibits a chiral center, in this method each enantiomer can be used separately or together as a racemate whereby the respective diastereoisomers or a mixture thereof are obtained.

The aldehyde reagents which are applicable in the method,

are either known compounds or can be easily prepared from known compounds using well-known methods. Thus, e.g. alkyl aldehydes substituted with both hydroxyl and amino functions (eg 2-hydroxy-5-aminopentanal) are prepared from an amino aldehyde acetal (eg 4-aminobutanaldiethyl acetal) by protecting the amino function therein as an acetamido or phthalimido group using known methods, followed by removal of the acetal function by acid hydrolysis whereby an N-protected aminoaldehyde is obtained (e.g. by converting 4-aminobutanaldiethyl acetal to the corresponding N-phthalimido derivative which on acid hydrolysis gives 4-phthalimidobutanal). The treatment of the N-protected aminoaldehyde with hydrocyanic acid gives the corresponding N-protected-aminoalkylhydroxynitrile (e.g. 2-hydroxy-5-phthalimidovaleronitrile) as by catalytic reduction (e.g. hydrogen in the presence of palladium) or by hydride reduction (e.g. with di-isobutylaluminum hydride) gives an N-protected amine p-hydroxyaldehyde (e.g. 2-hydroxy-5-phthalimidopenta-nal) which is an aldehyde reagent used in this method.- -

When the process is carried out by treating a 1-N-unsubstituted-4,6-di-(aminoglycosyl)-1, 3-diaminocy eli tol with a hydride-^ donor and an aldehyde to give the corresponding 1-N-substituted derivative of a 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol, in order to reduce competing side reactions when an aminoaldehyde is used as a reagent, it is preferred to protect the amino function in the aldehyde.

e.g. with an acyl-blocking group such as acetamido, phthalimido or the like before carrying out the process, and then removing the N-protecting group in the compound thus formed. It can also be advantageous to protect the hydroxyl group in hydroxy1-containing aldehydes when carrying out the method, however, this is not necessary.

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

A suitable method for carrying out the method comprises preparing a solution of a 1-N-unsubstituted-4,6-di-(aminoglycosyl)-1,3-diaminocyclitol antibacterial agent (e.g. sisomicin or verdamicin) in a protic solvent ( preferably water) and adjust the pH of the solution from pH 2 to pH 5 with an acid (usually dilute sulfuric acid) thereby preparing the necessary acid addition salt of the starting compound. When the pH of the solution is approx. pH 5, the resulting acid addition salt usually contains approx. one equivalent acid for each amino function in 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol (e.g. per mole of sisomicin there are 2.5 moles of sulfuric acid). After the acid addition salt solution is prepared, at least one molar equivalent, and preferably a larger molar excess of the desired aldehyde (eg acetaldehyde, propanal or butanal) is added followed within a short time (usually within 5 minutes) of addition of approx. one molar equivalent (based on starting 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol) of a hydride donor reducing reagent, preferably an alkali metal cyanoborohydride, usually sodium cyanoborohydride. The reaction is usually completed within at least 30 minutes as determined by thin-layer chromatography, and the corresponding 1-N-substituted derivative of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol is obtained (e.g. . 1-N-ethylsisomicin or 1-N-ethylverdamicin). Isolation and purification of the resulting derivative takes place using known methods such as precipitation, extraction and preferably chromatography.

Process alternative A) according to the invention thus provides a new, handy, one-reaction vessel process where an aminoglycoside is reacted in situ with an aldehyde (preferably in excess amounts) and with a hydride donor

reducing agent during the formation of as the main product,

a mono-N-substituted derivative (e.g. 1-N-ethyl-sisomicin),

in which method the 1-amino group bound to a

secondary carbon atom is usually alkylated preferentially in relation to other, amino groups attached to primary and other secondary carbon atoms .in the 4 , 6.-*di-(aminoglycosyl)-1,3-diaminocyclitol starting material.

It is also possible to use partially N-protected 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols as starting materials in this method. In general, 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols exhibiting a -CH^NI^ group such as the 6<1-> group can be N-protected in this position as this group is most reactive in the blocking reaction . Sisomicin can protect at position 6' or in positions and 6' or in positions 2', 3 and 6'. Other amino-protecting groups can be bridging groups in positions 3", 4", such as carbonyl in the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which have the 3"-amino and the 4"-hydroxyl group in cis -position to each other. Thus, it can e.g. is used as starting material a 1-N-unsubstituted 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol where the amino function at the 6-carbon is N-protected (e.g. 6 *-N-t-butoxycarbonylsisomicin) or a 1-N- unsubstituted-4,6-di-(aminoglycosyl)-1,3-diaminocyclitol where the amino functions at C-2' and C-3 are N-protected (e.g. 2',3-di-N-trifluoroacetylgentamicin C-^ and the corresponding partially N-protected 1-N-substituted derivative will be formed (e.g. 1-N-ethyl-6<1->N-t-butoxycarbonylsisomicin and 1-N-ethyl-2',3-di-N-trifluoroacetylgentamicin C-^) which, on removal of the N-protecting groups according to known methods, gives 1-N-CH^X compounds,

e.g. 1-N-ethylsisomicin and 1-N-ethylgentamicin C-^.

The necessary starting compounds in which amino groups are protected can be prepared by methods little or identical to those described in Preparation 3. As used herein, the terms "blocking group" or "protecting group" denote groups which render the blocked or protected amino groups inert to subsequent chemical treatment, but which can be easily removed after the chemical treatment has been carried out. Examples of such amino-protecting groups are benzyl, 4-nitrobenzyl, triphenylmethyl, 2,4-dinitrophenyl, acyl groups such as acetyl, propionyl and benzoyl, alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, t -butoxycarbonyl and 2-iodoethoxycarbonyl, and arylalkoxycarbonyl groups such as carbobenzyloxy and 4-methoxy-benzyloxycarbonyl 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 such as ethylthioltrifluoroacetate, N-benzyloxycarbonyl-

oxy-succinimide or p-nitrophenyltrichloroethylcarbonate. Thus

the blocking groups can be described as being derived from a compound BgLg, where Bg is the blocking group such as the acid moiety of an active ester, and Lg is a leaving group such as imidazole.

.... Procedure option B) out-

is carried out in a manner which allows the formation of a 1-N-Schiff's base derivative followed by reduction of the Schiff's base thus obtained. The process for preparing the 1-N-substituted derivatives of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols where the substituent is -CH2X and X is as defined above, comprises reduction of the N,C double bond in a 1-N=CHX'-substituted derivative of one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, where all NH2 groups are protected and NHCH3~ groups may be protected, where X ' is as previously indicated, after which all present jprotecting groups in the molecule are removed and the desired derivative is isolated as such or as a pharmaceutically acceptable acid addition salt.

The 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols in which the 6-aminoglycosyl radical is garosaminyl usually exhibit a 3"-N-4"-O protecting group identical to the 1-N substituent in the the starting material because the oxazolidine ring is formed simultaneously with the 1-N-Schiff base group. Thus, e.g. 2',3-di-N-trifluoroacetylgentamicin C-, by reaction with an aldehyde (e.g. benzaldehyde, phenylacetaldehyde or acetaldehyde) to the corresponding 3",4"-oxa-zolidin-1-ylidene Schiff base starting material according to this method (e.g. l-N-3",N-4"-0-di-benzylidene-2',3-di-N-trifluoroacetylgentamicin C-jy l-N-3 "-N-4"-0-di-f enethylideneS' , 3-di-N-trifluoroacetylgentamicin C1 and 1-N-3"-N-4"-G-diethylidene-2',3-di-N-trifluoroacetylgentamicin C^), which by reduction with sodium borohydride and methanolic sodium methoxide gives the corresponding l-N-CH2X-3",4"-oxa-zolidine (e.g. l-N-benzyl-3",N-4"-0-benzylidengentamicin C±, 1-N-phenethyl-3"-N -4"-0-phenethylidengentamicin and 1-N-ethyl-3"-N-4"-O-ethylidengentamicin c^) which on treatment with acid gives a 1-N-CH2X compound according to the invention (e.g. 1-N -benzylgentamicin C1# 1-N-phenethylgentamicin and 1-N-ethylgentamicin C^).

The new 1-N-substituted derivatives of 4>6-di-(aminoglycosyl)-1,3-diaminocyclitols as defined by the formulas I, III, IV and V can also be prepared yed, f process alternative C) which comprises

.treatment of a 1-rN-substituted derivative of one of the above

4,6-di-(aminoglycosyl)-ri,3-diaminocyclitols, in which one or more amino groups may be protected and where the 1-N-substituent is

0

II

-C^X" where X" is hydrogen, alkyl, alkenyl,

hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or hydrocarbyloxy, which aliphatic radicals have up to 7 carbon atoms, and if substituted with amino and hydroxyl, the substituents are carried on different carbon atoms, and where which any amino or hydroxyl group present may be protected, with an amide-reducing hydride reagent, and, if necessary, removal of any protecting groups present in the molecule, the final step being followed by isolation of the desired derivative as such or as a pharmaceutically acceptable acid addition salt.

The process is usually carried out in a non-reactive organic solvent which is considered to be a solvent in which the starting compounds and the amide-reacting reagent are soluble and which will not react with the reagent so that a minimum of competing side reactions is formed. Non-reactive organic solvents useful in the reduction process are ethers such as dioxane, tetrahydrofuran, diethylene glycol dimethyl ether and the like.

Preferred amide reducing hydride reagents are aluminum hydrides and borohydrides including lithium aluminum hydride, lithium trimethoxy aluminum hydride, aluminum hydride, diborane, diisoamylborane and 9-borabicyclo(3,3,3)-nonane.

In general, it is preferred to use diborane as the amide-reducing agent except when the starting compound exhibits a double bond, as e.g. in 1-N-acylsisomicin, 1-N-acylverdamicin, 1-N-acyl-Antibiotic 66-40B, 1-N-acyl-Antibiotic 66-40D and 1-N-acyl-Antibiotic G-52, which compounds are appropriately reduced using lithium aluminum hydride.

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

ii

If in this method where a 1-N-C-X"-4,6-di-(aminoglycosyl-1,3-diaminocyclitol is reduced to the corresponding 1-N-CH^X derivative, the acyl side chain of the 1-N-acyl intermediate exhibits a chiraite center, each of the stereoisomers can be used separately or a mixture thereof, whereby the corresponding diastereoisomers or a mixture thereof are obtained.

Procedure option D) for the

: position of 1-N-substituted derivatives of the time-

4,6-di-(aminoglycosyl)-1,3-diaminocyclitols specified above. where the substituent is straight-chain alkyl with up-

to 5 carbon atoms and pharmaceutically acceptable acid addition salts thereof including conversion of one of. disse-4,6-di-(aminoglycosyl)-1,3-. diaminocyclitols exhibiting amino-protecting groups in any position other than position 1, and where the 1-amino group may be activated, with an alkylating agent containing

the straight-chain alkyl group with up to 5 carbon atoms and a leaving group, after which the protective groups are removed, and if necessary, the activating group or groups present in the molecule, after which the derivative is isolated as such or as a pharmaceutically acceptable acid addition salt. ..Examples of alkylating agents that are preferably used in this method are alkyl iodide, alkyl bromide, dialkyl sulfate, alkyl fluorosulfonate and alkyl p-toluenesulfonate where the alkyl group is the required straight-chain alkyl group with up to 5 carbon atoms.

,,, Other alkylating agents where the alkyl group preferably has one or two carbon atoms are trialkyl anilimim hydroxide, trialkyloxonium fluoroborate, trialkylsulfoniunifluoroborate or trialkylsulfoxonium-. fluoroborate. All of these alkylating agents contain a good leaving group, such as Br", I~, 0SO 2 F"", di-alkylaniline or dialkyl ether.

The amino group in the 1-position in 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol can be free or activated. An example of a

activating group is trifluoromethylsulfonyl. These activating groups can be introduced into the molecule by reacting a 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol exhibiting amino-protecting groups in any position other than position 1, e.g. 3"-N-4"-O-carbonyl-2',3,6'-tri-N-t-butoxycarbonyl-sisomicin with a compound that provides the activating group, such as trifluoromethylsulfonyl chloride.

The 1-amino group can also be alkylated by means of the corresponding di-(2-cyanoethyl) derivative derived by treatment with acrylonitrile of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol exhibiting amino-protecting groups in any position other than position 1. The 1-N-di-(2-cyanoethyl)-derivative thus prepared is then alkylated with one of the above alkylating agents followed by removal of the cyanoethyl groups.

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

Process eight alternatives E and F for the production of 1-N-substituted derivatives of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols where the substituent is methyl, and pharmaceutically acceptable acid addition salts thereof include conversion of one of these 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols exhibiting amino-protecting groups in any position other than position 1, either with formaldehyde and a cyclic imide, preferably succinimide, and treating the thus obtained compound with a hydride-donor-reducing agent, preferably sodium borohydride, or with formaldehyde in the presence of formic acid, followed by removal of all protective groups present in the molecule and isolation of the derivative as such or as a pharmaceutically acceptable acid addition salt. The formation of the 1-N-methyl substituent with formaldehyde and formic acid is well known as the Eschweiler-Clarke reaction.

The subsequent preparations exemplify the preparation of a plurality of necessary starting materials and the subsequent examples illustrate the invention.

Production 1

Gentamicin C~

2a

Separation of Gentamicin C^a from co-produced antibiotics

Dissolve 96 g of gentamicin base (prepared from the sulfate salt obtained according to the method described in example 4 in US patent document 3,091,572) in 400 ml of the upper phase that appears when methanol, chloroform and 17% ammonium hydroxide are mixed in the volume ratio 1:2: 1. Add one-tenth of the solution to each of the first ten tubes in a 500 x 80 mL countercurrent extractor. Fill all tubes including the first ten completely with the lower phase of the solvent mixture described above. Adjust the solvent reservoir to deliver 40 mL of the upper phase to tube #1 (1) for each transfer. Set the equipment to 500 transmissions. When the transfers are complete, collect every eighth tube for chromatography (in duplicate) on Schleicher and Schuell No. 590 paper using the

lower phase of the solvent mixture described above. Allow the chromatograms to develop for 16 hours and then dry the papers. Place a paper on an agar plate inoculated with Staphylococcus aureus (A.T.C.C. 6538P1, spray the duplicate with normal ninhydrin solution and heat for development. Incubate the agar plate at 37° C overnight and combine the solution from the tubes' containing the material that migrates as gentamicin Ci (ie-, tubes 290 - 360).

Replace tubes 290 - 360 with fresh tubes containing 40 ml of upper phase and 40 ml of lower phase. Set up the equipment for

-another 2800 transfers and repeat the previously performed chromatographic procedure. Combine tubes 1 - 16 and concentrate in vacuo to 1.3 g of gentamicin C0_ having the following properties: (a) a molecular weight of 463 as determined by mass spectrometry

which is consistent with the empirical formula of C20<H>41N5°7'

(b) a specific optical rotation as measured at the D line of sodium at 26°C of +114° + 5° in water at 0.3% concentration and (c) a proton magnetic resonance (pmr) spectrum as follows: pmr(ppm ) (D2O), 6 0.99 (3H, d, J=6.5Hz, CH-CH3), 1.17 (3H, s, C-CH3), 2.47 (3H, s, N-CH3) , 2.51 (1H, d, J=10.5 Hz, H-3"),

3.75 (1H, q, J=10.5, 4Hz, H-2"), 4.00 (1H, d, J=12Hz, H-5" eq), 5.04 (1H, d, J =4Hz, H-1"), 5.13 (1H, d, J=3.5 Hz, H-1').

Irradiation of the secondary methyl group at 6 0.99 ppm shows H-6' as a doublet (J=6.5 Hz) at <S 2.81 ppm.

Manufacturing 2

Gentamicin Cg^

Separation of gentamicin from co-produced antibiotics

Separate the major gentamicin C components (C₁, C₂, and C₂₂) as described in US Patent 3,651,042, Example 2, and combine the fractions containing dominantly overlapping amounts of gentamicin C₂ and C₂ free base (500 g of gentamicin C mixture gives 53.4 g overlap). Add 1.5 g of this gentamicin C-1 and C2 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 on silica gel plates at use of the solvent system chloroform:-methanol:22% ammonium hydroxide (1:2:1) as developer. Combine the fractions containing a mixture of gentamicin C1 and C2 together with gentamicin (fractions 39 - 57 (410 mg)). Rechromatograph fractions 39 - 57 over silica gel using a chloroform:methanol:7% ammonium hydroxide (1:2:1) solvent system and combine fractions (38 - 1301 containing pure gentamicin C , as determined by thin layer chromatography (yield 45 mg) with the following constants (: , i;,.(a^D,; 165.5o1;(c=0.3%, H2O) , mass spectrum m/e'463 (M+1)+, 446 , 445, 433, 350, 332, 322, 304, 333, 305, 287, 191, 173,

163, 145, 160, 142, 118, 143, pmr(ppm)(D2O): 6 1.25 (3H, s, C-CH3), ,2.40 (3H, s, N-CH3), 2, 55 3H, s, N-CH3), 5.12 (1H, d, J=4Hz, H-1"), 5.22 (1H, d, J, 3Hz, H-1').

Pure gentamicin C-^ can be separated from gentamicin C-^ and C2 due to its mobility by thin-layer chromatography using silica gel plates and a chloroform:methanol:22% ammonium hydroxide (1:2:) developing solvent system. The approximate Rf values in this system are as follows:

Manufacturing 3

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

A. 2', 3, 6'- trl- N- butoxycarbonyl- 3"- N- 4"- O- carbonylsisomicin

1. Penta-N-carbobenzoxysomicin

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°C and stir the mixture for 16 hours. Filter off the solid, wash thoroughly with water, dry in vacuo and then wash with hexane to give 62 g of penta-N-carbobenzoxysomicin (62 g) as a colorless solid, m.p. = 165 - 173° C (a)-<26> + 96/2° (CH3OH) IR: y max (CHCl3) 3400, 1720, 1515, 1215, 1050, 695, cm"<1> NMR: 6 (CDCl ) 1.03 (3H, broad singlet, 4"-C-CH3) , 3.02 (3H, broad singlet, 3"-NCH3), 5.02 (10H, broad singlet, C<H>2CgH5), 3 .28, 3.30 ppm (25H, broad singlet, -CH2CgH5).

2. Tetra- N- carbobenzoxy- 3"- N- 4"- O- carbonyl- sisomicin

Dissolve 5 g of penta-N-carbobenzoxy-sisomicin in 50 ml of dimethylformaraide, add 250 mg of sodium hydride to the stirred solution and stir the reaction mixture under argon at room temperature for 2 hours., Filter and add glacial acetic acid (2 ml) to the filtrate which then ^ is concentrated in, vacuum. Extract the residue with chloroform; (20.0 ml previously passed through basic aluminum oxide), wash the extract with water and dry over sodium sulphate. The solution is evaporated to give 3.5 g of tetra-N-carbobenzoxy-3"-N-4"-O-carbonyl-sisomicin as an amorphous powder. SSi.p. = 210 - 213° C (α}^6 + 68.8 (C=0.22) IR: y.max (Nujol) . 3500, 184.0, 1760, 1580 cm"1 NMR: <5(CDC13) 1.34 (3H, singlet, 4"-CH3), 2.68 (3H, singlet, 3"-N-Me), 5.04 (8H, broad singlet, -CH2CgH5) . • "

3. 3"- N- 4"- O- carbonyl- sisomicin

. To a solution of 10.1 g of 1,3,2',6'-tetra-N-benzyloxycarbonyl-3"-N-4"-0-carbonyl-sisomicin in 200 ml of tetrahydrofuran is added 1 1 liters of liquid ammonia (redistilled from sodium) . Add 6 g of sodium in small pieces to the stirred solution. After stirring

for 3 hours, excess sodium is destroyed by the addition of ammonium chloride. Allow solvents to evaporate under a stream of nitrogen. Dissolve the residue in water and pass this through Amberlite IRC-50 resin (H form) and wash the resin well with water and elute the product with 2N ammonium hydroxide solution. Evaporate the ammonium eluate in vacuo to give the title compound (3",N-4"-carbonyl-sisomicin). Yield approx. 4g.

IR: y.max (Nujol) 1745 cm 1. The product can be used in the following steps without further purification. However, a very pure sample can be obtained by chromatography of the product over silica gel using the lower phase of a chloroform:methanol:concentrated ammonium hydroxide (1:1:1) solvent system as eluent. 4. 2', 3, 6'- tri- N- t- butoxycarbonyl- 3"-N-4"- O- carbonyl- sisomicin Dissolve 1.4 g, 3 mmol, 3"-N-4"-0- carbonyl-sisomicin in 10 ml of 50% aqueous methanol containing 3.5 mmol of triethylamine. Add 3.5 mmol of t-butoxycarbonylazide dropwise while stirring. Stir the mixture for 2 days at room temperature. Add 5 ml of Amberlite IRA-401S (OH ) ion exchange resin together with 5 ml of methanol and stir for 1 hour. Remove the resin by filtration and wash with methanol. Concentrate the filtrate and chromatograph the residue on a column of silica gel (60 - 100 mesh, 20.0 g) using chloroform:methanol:ammonium hydroxide (30:10:0.4) as the solvent system. Combine the homogeneous fractions containing the title compound 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- trifTuoracetyT- gentamicin C^

1. • 2'-N-trifluoroacetyl-qentamicin C ±

Dissolve 1.7 g of gentamicin C1 in 20 ml of methanol, cool

.the mixture to 4°C and add 0.46 mL (0.563 g) of ethyl thiol trifluoroacetate with stirring. Allow the reaction to proceed for 2 hours and concentrate the solution to a residue in vacuo. The product is chromatographed on 80 g of silica gel G using a lower phase of a mixture of chloroform:methanol:water:ammonium hydroxide in the volume ratio 10:5:4:1 as eluent. Combine the fractions containing the -major component and concentrate to give 1.4 g of the title compound, m.p. 108 - 111° C, (a).^6 = +128°

(c = 0.3%, H 2 O). Analysis for C^H^NgOgF^R^O

Calculated: C = 46.69%, H = 7.50%, N = 11.84%, F = 9.63%.

Found : C = 46.66%, H = 7.65%, N = 11.60%, F = 9.24%

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

Dissolve 0.66 g" of the product from step 1 in 10 ml of methanol, cool the mixture to 4°C and add 0.148 ml (0.182 g) of ethyl thiol trifluoroacetate dissolved in 3 ml of methanol. Stir the reaction mixture for 16 hours and concentrate to a residue in vacuo. Chromatograph the product on 30 g of silica gel as described in step 1. Monitor the column by thin layer chromatography, combine the appropriate reactions and concentrate to give 0.32 g of the title compound, mp 121 - 129° C (cOq<6> 121° ( c = 0.3%, H20). Analysis for <C>25<H>41<N>5°9F6* Calculated: C = 44.84%, H = 6.17%, N = 10.46%

Found : C = 44.94%, 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 of 6 ml of ethylthioltrifluoroacetate in 60 ml of methanol over the course of 3 hours, while stirring. Allow the reaction to proceed for 18 hours at room temperature and remove the solvent in vacuo to leave a residue of 23.8 g of the product with a purity of approx. 95% with the following physiochemical properties: .Mass spectral data: m/e 543 M<+>, other peaks at m/e 413, 395, 385, 362, 223 and 126.

NMR (60MHz, D 2 O 1 55.37 (doublet, J = 2Hz, H-1'), 5.22 (doublet, J=4Hz, H-1"), 4.96 (broad singlet, H-4'), 2, 57 (singlet, N-CH 2 )', 1.26 (singlet, C-CH 2 ).

D. 6'-N-t-butoxycarbonylT-qentamicin C^a

Dissolve 2.69 g of gentamicin C^a in 60 ml of methanol:water (1:1), cool to 5° C and add 1.815 ml of triethylamine. Add, while stirring, 1.91 g of t-butoxycarbonyl azide dropwise. Stir the mixture

at 5° C for 18 hours. Add 20 ml of Amberlite IRA^-401S resin (OH—form), stir for 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 a 2:1:1 chloroform:methanol unconcentrated ammonium hydroxide solvent system as eluent. Take 3 ml fractions and monitor the contents by thin-layer chromatography. Combine fractions containing the main reaction product and evaporate to give the title compound of this example (0.42 g, 13%) , (a)£<6> + 137° (CH 3 OH), PMR 61.23 (3H, s, C- CH3), 1.45 (9H, s, C-(CH3)3), 2.53 (3H, s, N-CH3), 65.08 (2H, overlapping doublets, J-3.5Hz, H-lV and H-1") PPM. Mass spectrum m/e 550 [(M+l)^] and m/e 549 (M+).

E. 6'-N-t-butoxycarbonyl-gentamicin B

Dissolve 1 g of gentamicin B in 30 ml of 50% aqueous methanol and cool to 5° C. Add 0.297 g of t-butoxycarbonyl azide dropwise with stirring followed by 0.186 ml of triethylamine and stir the resulting solution for 18 hours. Evaporate the reaction mixture in vacuo to a residue and chromatograph the residue on 100 g silica gel using the lower phase of a 2:1:1 chloroform:methanol:concentrated ammonium hydroxide solvent system as eluent. Collect 2 ml fractions and monitor the column effluent by thin layer chromatography. Combine fractions containing equal material (fractions 180 - 230) and evaporate to give 0.830 g of 6'-N-t-butoxycarbonyl-gentamicin B with the following physical constants: PMR (60 MHz, D20) , 6 1.21 (3H, s, C -CH3) , 1.42 (9H, s, C(CH3)3), 2.53 (3H, s, N-CHg), 5.2 (1H, d, J=4.5Hz H-1"), 5 .23 (1H, d, J=3.0 Hz, H-1') PPM.

Manufacturing 4

1- N- acetylsisomicin

(A) Dissolve 1.25 g of sisomicin sulfate in 200 ml of methanol-water (2:3, v/v) and cool the solution. Add 1.5 ml of acetic anhydride

and after approx. 10 minutes, add Q.125 ml triethylamine in 10 ml methanol over a 15 minute period. Allow the reaction mixture to warm to room temperature over 2 hours and then evaporate the solvent in vacuo. Dissolve the residue in water and convert the product to the free base by passing an aqueous solution of it

through Amberlite IRA-401S resin in hydroxide ion form. Lyophilize the column eluate and chromatograph the residue on 50 g silica gel using the lower phase (2:1:1) chloroform, methanol, 7% ammonium hydroxide solvent system as eluent. Monitor the fractions by thin-layer chromatography and combine equal fractions to obtain the title compound.

Yield - 0.185g, m.p. 128° - 130° C, (a)^<6> = 159°

(0.3% H 2 O), NMR: (D 2 O) <<$> 1.22 (3H, s, -C-CH 3 ); 2.02 (3H, s, NH-CO-CH3); 2.53 (3H, s, N-CH3); 4.88 (1H, m, =CH-); 5.08 (1H, d, J=4Hz, H-<l>"); 5.35 (1H, d, J=2Hz, Hy) .

Mass spectrum: (M + 1)<+> m/e 490, M+ m/e 489.

Manufacturing 5

Preparation of aldehyde intermediates

A. 2~acetam£do-3-hydroxyoctanal

Protect the amino function in the 2-amino-3-hydroxyoctanoic acid by converting it to an acetamido function, then esterify the resulting 2-acetamido-3-hydroxyoctanoic acid with methanol, reduce the resulting 2-acetamido-3-hydroxyoctanoic acid methyl ester with diisobutylaluminum hydride according to known methods methods during the formation of 2-acetamido-3-hydroxyoctanal.

B. 2-acetoxy-4-(N-methylacetamido)-butanal

Treat the diethyl acetal of 2-hydroxy-4-aminobutanal with acetic anhydride in pyridine followed by treatment of the resulting diethyl acetal of 2-acetoxy-4-acetamidobutanal with sodium hydride and methyl iodide to form the diethyl acetal of 2-acetoxy-4-(N-methylacetamido)- butanal. Remove the acetal protecting groups with acid to form 2-acetoxy-4-(N-methylacetamido)-butanal.

Example 1

1- N- substituted sisomycin

A. 1-N-ethylsisomicin

To a solution of 5 g of sisomicin in 250 ml of water, IN sulfuric acid is added until the pH of the solution is adjusted to approx. 5. To the solution of sisomycin sulfuric acid addition salt, add 2 ml of acetaldehyde, stir for 10 minutes, and add 0.85 g of sodium cyanoborohydride. Continue stirring at room temperature for 15 minutes, then concentrate the solution in vacuo to a volume of approx. 100 ml, treat the solution with a basic ion exchange resin (e.g. Amberlite IRA 401S (OH ) ) and lyophilize to a residue comprising 1-N-ethyl-sisomicin.

Purify by chromatography on 200 g silica gel, eluting with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) system. Combine the equal eluates as determined by thin-layer chromatography and concentrate the combined eluates of the major component in vacuo to a residue comprising 1-N-ethylsisomicin (yield 1.25 g). Purify further by chromatography on 100 g of silica gel and elute with a chloroform-methanol-3.5% ammonium hydroxide (1:2:1) system. Pass the combined, equal eluates (as determined by thin layer chromatography) through a column of a basic ion exchange resin and lyophilize the eluate to give 1-N-ethylsisomicin (yield 0.54 g),

(a)^<6> + 164° (0.3%, H 2 O), pmr (ppm) (D 2 O): δ 1.05 (3H, t, J=7Hz, -CH 2 C<H> 3 ); 1.19 (3H, s, -C-CH 3 ), 2.5 (3H, s, N-CH 3 ); 4.85 (1H, m, =CH-); 4.95 (1H, d, J=4Hz, Hy) ; 5.33 (1H, d, J=2.5 Hz, H-^) .

Mass spectrum: (M+1)<+> m/e 476

and m/e 127, 154, 160, 173, 191, 201, 219, 256, 299, 317, 332, 345, 350, 360, 378, 390, 400.

B. 1-N-methylsisomicin

To a solution of 4.6 4 g sisomicin in 180 ml water add IN sulfuric acid until the solution has a pH of 5. Add 1.2 ml

37% aqueous formaldehyde, stir for 10 minutes and then add

460 ml of sodium cyanoborohydride. Pass the reaction solution through a column of a basic ion exchange reagent (e.g. Amberlite IRA 401S (OH-~form) and lyophilize. Chromatograph the resulting residue on silica gel and elute with the lower phase of a chloroform-methanol-7% dilute ammonium hydroxide. (.2:1:1) solvent mixture. Combine the equal eluates containing mainly 1-N-methylsisomicin as determined by thin-layer chromatography. Evaporate in vacuo to a residue of 1-N-methylsisomicin} (a)D 2 6 + 153° (0 .3%, H 2 O);

Maasespectxum: (M+l)<+> m/e 462

and w/e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336(w), 346, 376, 386.

C. 1-N-(h-propyl)-sisomycin

=^^^^^^^^ .B^ similarly described in example la' the sulfuric acid addition salt of sisomicin in water with propanal and sodium cyanoborohydride. Isolate and purify the resulting product in a similar manner as described to give 1-N-(n-propyl)-sisomycin; (α}^<6> +140° (0.3%, H 2 O);

pmr (ppm) (D 2 O): δ 0.83, (3H, t, J=7Hz, -CH 2 CH 3 ); 1.14 (3H, s, -C-CH3); 2.45 (3H, s, -N-CH 2 ); 4.82 (1H, m, =CH-); 4.90 (1H, d, J=4Hz, Hi"); 5.78 (1H, d, J=2Hz, );

Mass spectrum: (M+1)+ m/e 490

and 127, 160, 168, 187, 205, 215, 233, 256, 313, 331, 346, 359, 364, 374, 404, 414.

D» 1-N-(n-butyl)-sisomycin

Add to a solution of 3 g of sisomicin in 200 ml of water IN sulfuric acid until the solution has a pH of 3.5. Add 1.5 ml of n-butanal, stir for 10 minutes and then add 450 mg of sodium cyanoborohydride. Continue stirring for 1 hour and then concentrate the solution in vacuo to a volume of approx. 100 ml. Pass this solution through a column of a basic ion exchange resin (e.g. Amberlite IRA 401S (QH~)) and lyophilize^ Chromatograph the resulting residue on 140 g of silica gel and elute in the lower phase with a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) solvent-agent mixture. Combine the equal fractions containing 1-N-(n-fautyl)-sisomycin as determined by thin layer chromatography and evaporate the combined eluates in vacuo to a residue comprising 1-N-(n-butyl)-sisomycin; (ct)^<6> + 129° (0.3%, H2O) , pmr (ppm) (D2O)

5:0.82 (3H, t, J=*7Hz, -CH 2 CH 3 ); 1.15 (3H, s, C-CH 3 ); 2.46 (3H, s, -N-CH 3 ); 4.82 (1H, m, =CH-); 4.92 (1H, d, J=4Hz, H^");

5.29 (1H, d, J=2Hz, %') .

Mass spectrum: (M+1)+ m/e 504;

also w/e 127, 160, 182, 201, 219, 229, 247, 256, 327, 345, 360, 373, 388, 418, 428.

E. Other 1-N-alkyl, 1-N-alkenyl and 1-N-aralkyl sisomycins

,. Proceed as described in example IA but instead of acetaldehyde use equivalent amounts of each of the following alkyl aldehydes:

1. 2-ethylbutanal,

2. propenal.

In each case carry out the reaction in the same way as described in example .IA and isolate and xenize each of the resulting

end products in a similar way as described, whereby it

is obtained

1. 1-N-(g-ethylbutyl)-sisomicin {ol)* 6 + 121°

(c=0.4%, H2O) pmr (ppm) (D2O) : 6 0.75 (6H,

t, 6.5 Hz, CH2-CH3); 2.4 (3H, s, N-CH3);

4.78 (1H, m, =CH-); 4.88 (1H, d, 4.0 Hz, H^')

5.22 (1H, d, 2.0 Hz, H-, ') ;

Mass spectrum: (M+1) m/e 532

also, m/e 127, 160, 210, 229,

256, 275, 355, 373, 388, 401, 406, 416, 446, 456,

2. 1-N-(P-propenyl)-sisomicin [α]£° + 147° (0.4, MeOH); nmr (D20) fi 1.18

(s, 3H, C-CH3); 248 (p, 3H, N-CH 3 ); 3.75 (1H,

dd, J=4,H Hz, H-2"); 3.95 (1H, d, J=13<Hz,> H<->5<e>);

4.84 (1H, m, H-4<*>); 4.94 (1H, d, j=4hz, h-1”);

5.30-5.0 (3H,+m, H-11, =CH2); 5.8 (1H, m, -CH=CH2);

MS m/e 488 •

F. 1-N-(5-aminobutyl)-sisomycin

Add IN sulfuric acid dropwise to a solution of 3 g of sisomicin in 120 ml of water until the pH of the solution is adjusted to approx. 5. To the aqueous solution of the sulfuric acid addition salt of sisomycin, add 60 ml of dimethylformamide followed by a solution of 2 g of 4-phthalimidobutanal in 10 ml of dimethylformamide. Continue stirring for 10 minutes and add 420 sodium cyanoborohydride. After 20 minutes, add the reaction solution to 1 liter of anhydrous methanol with stirring and collect by filtration the resulting precipitate comprising the sulfuric acid addition salt of 1-N-(6-phthalimidobutyl)-sisomycin.

Clean by dissolving the precipitate in water and passing the aqueous solution over a basic ion exchange resin. Evaporate the solution in vacuo to a residue, chromatograph the residue over silica gel and elute with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) solution mixture, and evaporate the combined,

equal eluates to a residue comprising 1-N-(6-phthalimidobutyl)-sisomycin.

To 0.5 g of 1-N-(6-phthalimidobutyl)-sisomicin add 5 ml of 5% ethanolic hydrazine hydrate and heat under reflux for 3 hours. Pour the reaction solution into a large volume of tetrahydrofuran and collect by filtration the resulting precipitate comprising 1-N-(6-aminobutyl)-sisomicin.

Alternatively, the compound according to this example can be prepared as follows:

(1) 4-acetamidobutyraldehyde

...<,>'„. r,,/,v .. -.-O<p>løs-.5 g of 4-racetamidobutyraldehyddiethylacetal in 75 ml of distilled water and 5 ml of IN sulfuric acid. Allow the solution to stand at room temperature until hydrolysis is complete as determined by thin layer chromatography i. Neutralize the solution with sodium bicarbonate and then saturate the solution with sodium chloride and extract with chloroform. Distill the combined chloroform extracts to a residue comprising 4-acetamidobutyraldehyde, which can be used without further

purification in the subsequent procedure.

(2) 1-N-(6-acetamidobutyl)-sisomycin

To 3 g of sisomicin in 120 ml of distilled water, 0.1 N sulfuric acid is added until the solution has a pH of 5. Add 6 g of 6-acetamidobutyraldehyde prepared as described above, and after 10 minutes 600 g of solid sodium cyanoborohydride. After 2 hours, the solution is concentrated to a small volume and poured into methanol. Collect the resulting precipitate by filtration, dissolve in water and pass the aqueous solution through a column of Amberlite IRA 401-S (OH ) ion exchange resin. Evaporate the eluent and chromatograph the resulting residue on silica gel, eluting with the lower phase of a chloroform:methanol:7% ammonium hydroxide solvent mixture. Evaporate the eluent to a residue comprising 1-N-(6-acetamido-butyl)-sisomicin.

(3) 1-N-(6-aminobutyl)-sisomycin

. ; v ^ Treat: 1-N-(6-acetamidobutyl)-sisomicin obtained in the previous example with 10% aqueous potassium hydroxide at 100°C for 3 hours and then neutralize with Amberlite IRC-50 ion exchange resin and elute with 2N aqueous ammonium hydroxide. Concentrate the eluate and dissolve the resulting residue in water and lyophilize to form 1-N-(6-aminobutyl)-sisomicin. (a) DO CL + 109° (c=0.3%, H2O)j Mass spectrum: (M + 1)* m/e 519

in;:;: , in ; also 127, 160, 197, 216, 234, 244, 256, 262, 342, 360, 375, 388, 393, 403, 443.

G. 1-N-(γ-aminoethyl)-sisomicin and 1-N-(γ-aminopropyl) ,

sisomicin

In a similar manner as described in the alternative procedure in Example 1-F, treat an aqueous solution of sisomycin to which 0.1 N sulfuric acid has been added until the solution has reached a pH of 5, with γ-acetamidoacetaldehyde followed by solid sodium cyanoborohydride . Isolate and purify the resulting product as previously described, whereby 1-N-(g-acetamidoethyl)-sisomicin is obtained. Hydrolyze 1-N-(g-acetamidoethyl)-sisomicin with 10% aqueous potassium hydroxide and isolate and purify the resulting product as described in section (3) of. the alternative method according to example 1-F whereby 1-N-(g-aminoethyl)-sisomicin is obtained.

Mass spectrum: (M + 1)<+> m/e 491,

also 160, 169, 187, 206, 216, 234, 256, 283, 314, 325, 334, 347, 360, 370, 375, 405, 415.

If γ-acetamidopropanol is used in the above-mentioned method instead of γ-acetamidoacetaldehyde, 1-N-(Y-acetamidopropyl)rhisomicin is formed which, on hydrolysis with 10% aqueous potassium hydroxide followed by isolation and purification, gives 1-N-(y< ->aminopropy 1 Jrs isomycin.

[a]<26> + 127° (H20)

nrar (D 2 O): 1.2 (3H, s, -C-CH 3 ); 2.5

(3H, s, N-CH3); 3.8 (1H, dd, J = 4, 10.5 Hz,

<H>2"); 4.0(1H, d, J = 12.5 Hz, H^' e);

4.85 (1H, m, -C=CH-); 4.95 (1H, d, J = 4 Hz,

H-,/'); 5.34 (1H, d, J = 2.5 Hz, I-y).

Mass spectrum M<+> •♦• 1 m/e 5o4 also l6o, 202 , 220, 230, 248, 256, 328, 346, 361, 374, 379, 389,

407, 419 and 429.

Example 2

1- N- substituted gentamicin C^^

A. 1-N-ethylgentamicin

Add to 5 g of gentamicin Cla in 125 ml of water 1 N sulfuric acid until the pH of the solution is 5.2. Then add 2 ml of acetaldehyde. Stir the solution for 5 minutes, then add 1 g of sodium cyanoborohydride. Continue stirring at room temperature for 30 minutes, concentrate the solution in vacuo to a volume of approx. 75 ml, pass the solution through a column of basic ion exchange resin (e.g. Amberlite IRA 401S (0H~)), and then lyophilize a residue comprising 1-N-ethylgentamicin c^a.

Purify by chromatography on 200 g silica gel and elute the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) system. Combine the equal eluates as determined by thin-layer chromatography and concentrate the combined eluates with the major component in vacuo to a residue comprising 1-N-ethylgentamicin C^a (yield 0.95 g). Purify further by chromatograph! of 1-N-ethylgentamicin C^a on 100 g of silica gel and elute with chloroform-methanol-3.5% ammonium hydroxide (1:2:1) system. Treat the combined, equal eluates (as determined by thin layer chromatography) with a basic ion exchange resin and lyophilize the eluate to give 1-N-ethylgentamicin CX - cl (0.42 g) , { ol)U* 6 + 118° (c=0 .3%, H 2 O) ; pmr (ppm) (D 2 O): 61.06 (3H, g, J=7Hz, -CH 2 CH 3 ); 1.19 (3H, s, -C-CH3);

2.51 (3H, s, -N-CH3); 4.97 (1H, d, J=4Hz, R^"); 5.16 (1H, d, J=3.5Hz, H1').

Mass spectrum (M + 1)<+> m/e 478

also w/e 129, 154, 160, 173, 191, 201, 219, 258, 301, 317, 319, 329, 332, 347, 350, 360, 378 and 402.

Example 3

1- N- substituted- v erdamicin

A. 1-N-ethylverdamicin

Add to 0.5 g of verdamicin in 65 ml of water IN sulfuric acid until the pH of the solution is adjusted to 4.9 and then add 0.2 ml of acetaldehyde. Stir the solution, add 65 mg of sodium cyanoborohydride, concentrate the solution in vacuo to a volume of approx. 10 ml and pour the solution into 50 ml of methanol while stirring. Collect by filtration the resulting precipitate comprising 1-N-ethylverdamicin. Purify by chromatograph! on 100 g of silica gel and elute with a chloroform-methanol-3.5% ammonium hydroxide (1:2:1) system. Collect equal fractions as determined by thin layer chromatography and evaporate in vacuo the combined fractions containing the major component to a residue comprising 1-N-ethylverdamicin. Purify further by chromatograph! on 7 g of silica gel and elute with the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) system. Combine the equal eluates and evaporate in vacuo to a residue of 1-N-ethylverdamicin (yield 50 mg),

Mass spectrum: (M + 1)<+> m/e 490

also w/e 141, 154, 160, 173, 191, 201, 219, 270, 313, 317(w), 331, 332, 341, 350(w), 357, 359, 360, 378, 390, 414.

,B> : Repeat the procedure according to example<p>pel 3A but instead of acetaldehyde use other aldehyde intermediates. Isolate and purify each of the resulting products in the same manner as previously described to give 1-N-(n-propyl)-verdamicin, [α]p6 + 122° (c=0.3%, H2O); pmr (ppm ) (D 2 O); £0.88 (3H, X, J=7Hz, CH2CH3); 1.19 (3H, s, C-CH^; 1.16 (3H, d,

J=6Hz, CH-CH3); 4.81 (1H, m, =CH-); 4.97 (1H, d, J=4.OHz, H^') ;

5.30 (1H, d, J=2.OHz, ^'J;

Mass spectrum: (M + 1) m/e 528

also w/e 141, 160, 168, 187, 205, 215, 233, 270, 346, 355, 373, 504.

1-N-(n-butyl)verdamicin (a}^<6> + 121° j(c=0.3%, H20) ; pmr (ppm)

(D 2 O): 60.8 (3H, t, J=6.5 Hz, CH 2 -CH 3 ); 2.45 (3H, s, NCH-j); 4.8 (1H, m, C=CH-); 4.92 (1H, d, J=4.0 Hz, E^')} 5.25 (1H, d, J=2.oHz, ) ;

Mass spectrum: (M + 1><+> m/e 518

also w/e 141, 160, 182, 201, 219, 229, 247, 270, 341, 360, 378, 387, 388, 418, 442.

Example 4

1- N- substituted- gentamicin

A. 1-N-ethylgentamicin C^

Treat in the same way as described in example IA 5 g of gentamicin C-, in 250 ml of water with 1N sulfuric acid until the pH of the solution is approx. 5. Then treat the acidified solution with acetaldehyde and sodium cyanoborohydride in the manner described and isolate and purify the resulting product, thereby obtaining 1-N-ethylgentamicin Cl' (°0d<6> + 114° (c=0>3%' H2O) ; pmr (ppm) (D2O) : 61.03 (3H, t, 7Hz, -CH2CH3); 1.03 (3H, d, J=6.5Hz, -CH-CH.^ ; 1.17 ( 3H, s, C-CH3);

2.32 (3H, s, 6'N-CH3); 2.49 (3H, s, 3"-NHCH3); 4.94 (1H, d, J=4Hz, H-,"); 5.13 (1H, d, J=3+.5Hz, H-,').

Mass spectrum: (M +1) m/e 506

also w/e 154, 157, 160, 173, 191, 201, 219, 286, 317, 329(w), 347, 350, 360, 375, 430.

B. Repeat the procedure according to Example 4A, but instead of acetaldehyde, use other aldehyde reagents. Insulate

and purify each of the resulting products as previously described, whereby 1-N-methylgentaraicin is obtained,

.5 (D2p) 1,p4 (3H, d, J=6.5Hz, 6.V-CH3) ; 1.18 (3H, s, 4"-CH3); ! !-2.29 (3H, s, 1-HCH3); 2.32 (3H, s, 6'-HCH3 ); 2.49 (3H, s,

3"-HCH3); 4.95 (1H, d, J1",2"=4Hz, H^'); and 5.13 ppm (1H, d, Jj.' ,2'=3.5Hz, ^ • ) ; M+ m/e 491 also 4l6, 384, 364, 36l, 346,

343, 336, 333, 318, 315, 303, 286, 205, 187, 177, 160, 159, 157-l-N-Y (3-hydroxyethyl )-gent amicin c^

+ 98.0° (c=0.3%, H 2 O); pmr (ppm) (D 2 O); £0.99 (3H,

d, J=»&,5Hz, 6'-CE3)/ 1.13 (3H, s, 4"-CH3), 2.28 (3H, s, 6*-NCH3)f. 2.45 (3H, a, 3"-WCH^l,- 4.97 (1H, d, J~4Hz, E^') and 5.11 (1H, d, J=*3.5HZ, R^ U

Mass spectrum: (M + 1)+ m/e 522

also w/e 446, 404, 394, 391, 376, 373, 366, 363, 348, 345, 333, 286, 235, 217, 207, 189, 160, 157; v max (KBR) 3300, 1060 cm<-1>.

1-N-(phenylethyl)-gentamicin C_

(a) D° + 99.4° (c=0.3%, H 2 O); pmr (ppm) (D2O): 6 0.99 (3H, d, J=6.5Hz, 6'-CH3), 1.10 (3H, s, 4"-CH3), 2.28 (3H, s , 6'-NCH3), 2.43 (3H, s, 3"-NCH3), 4.88 (1H, d, J=4Hz, Hy), 5.08 (1H, d, J=3.5HZ, H^) and 7.33 (5H, s, -CgH 5 );

Mass spectrum: (M + 1)<+> m/e 582

also with 506, 464, 454, 451, 436, 433, 426,

423, 408, 405(w), 393, 295, 286, 277, 267, 249, 160, 157,

v max (KBR) 3300, 1050, 1030 cm"<1>.

Example 5

1- N- substituted- Antibiotic G- 52

A. 1- N- ethyl- Antibiotic G- 52

Dissolve 875 mg of Antibiotic G-52 in 40 ml of distilled water and add 1 N sulfuric acid until the pH of the solution is adjusted to approx. 3.5. Add 0.7 ml of acetaldehyde, stir the reaction mixture for 10 minutes and then add 100 mg of sodium cyanoborohydride. Monitor the reaction solution by thin-layer chromatography and when the starting Antibiotic G-52 appears to have reacted completely (ie, in about 10 minutes), then concentrate the solution in vacuo at 35 to 40° C. to a volume of about 10 ml. Pass the concentrated solution through a basic ion exchange resin and then lyophilize to a residue comprising 1-N-ethyl-Antibiotdkum G-52. Purify by chromatography on a silica gel column and elute with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) system. Combine the equal eluates as determined by thin layer chromatography and concentrate the combined eluates with the major component in vacuo to a residue comprising 1-N-ethyl-Antibiotic G-52 (yield 60 mg). Purify further the overlapped eluates from the preceding chromatography by chromatography on silica gel and elute with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (1:2:1) system whereby 35 mg of a residue comprising 1-N- ethyl Antibiotic cumin G-52. Pass the combined residues of 1-N-ethyl-Antibiotic G-52 through a column of basic ion exchange resin (e.g. Amberlite IRA 401S) and lyophilize the eluate to obtain 1-N-ethyl-Antibiotic G-52 (yield 90 mg );

{ a}* 6 + 122.1° (c~0.3%, H2O), pmr (ppm) (D2O) : <S 1.06 (3H, t, J= 6.5 Hz, 1N-CH2CH3) ; 1.21 (3H, s, 4"-C-CH3); 2.30 (3H, s, 3"-N-CH3)j 2.50 (3H, s, 6'-N-CH3); 4.94 (1H, m, H4'); 4.97 (1H, d, J=»4.0Hz ^"1; 5.^34 (1H, d, J=2.5Hz, R±') ).

Mass spectrum: (M + 1)<+> m/e 490

also-m/e, 141, 154, 160, 173, 191, 201, 219,

270, 313, 317(w), 331, 332, 341, 350, 359, 360, 378, 390, 414.

Example 6

A. In the same manner as described in Example IA, treat each of the following 4,6-diaminog.lycosyl-1,3-diaminocyclitols in water with sulfuric acid followed by acetaldehyde and sodium cyanoborohydride:

1. gentamicin B,

2. Antibiotic JI-20B,

3-mutamicin 2,

4. mutamicin 6.

Isolate and purify each of the resulting products in the same manner as described in Example IA, whereby the corresponding 1-N-ethyl compounds are obtained, i.e.

1. 1-N-ethylgentamicin B,

[a]<*6> + 119.7° (c, 1 in water)

Mass spectrum m/e [MH]<+> 380, 352, 334, 378, 350, 332, 219, 191, nmr (60 MHz D2O): δ 5.55 (H-1', JlV <=> 3'° HZ)' 5.05 (H_1"' Jl"'2" <=> 4 <Hz>)' 2.9 (N-CH 3 ), 1.05-1.5 (2C-CH 3 ).

2. 1-N-ethyl-Antibiotic JI-20B,

[a]<*6> + 112.5 (H 2 O)

nmr (D 2 O): δ 1.1 (3H, t, J=?Hz, CH 2 -CH 3 );

1.22 (3H, s, C-CH3); 1.3 (3H, d, -CH-CH3);

4.95 (1H, d, J=4 Hz, Hy); 5.35 (1H, J = 3.5Hz, Hy). Mass spectrum M<+> + 1 m/e 524, also 154, 160, 173, 175, 191, 201, 219, 304, 317, 332, 333, 350, 360.

3. 1-N-ethylmutamicin 2,

Temp. 80-84°C

pmr (D20): £ 1.06 (t , J=7 Hz, 3, 1-N-CH2CH3), 1.19 (s, 3, 4"-CH3), 2.50 (s, 3, 3" -N-CH3), 2.53 (d, J2",3<n> = 10.5 Hz, 1, Hy), 3.75

(dd, J-l" ,2" = 4 Hz; J2» ,3" = 10.5 Hz, 1, H2<»>), 3.83 (d, Hy e<q> y 2x 12 Hz, 1, Hy ), 4.86

(m, 1, Hy), 4.98 (d, Hyy = 4 Hz, i, Hy) and 5.10 (d, J^y = 2.5 Hz, 1, H1'). Mass spectrum: m/e 459, 384, 329, 3l6, 311, 301, 283, 203, 185, 175, 160, 142, 127.

4- l -N-ethylmutamicin 6,

Temp. 118-122°C (dec.)

Mass spectrum (M)+ m/e 475, (M + 1)<+> m/e 476, Monosaccharides: m/e 160, 127

2-deoxystreptamines: m/e 219, 201, 191, 171 Disaccharides: m/e 355, 317, 299,

m/e 378, 350, 322

pmr (£) D20

Example 7 Preparation of HJ-?substituted-4,6-di-(aminoglycosyl)-1,3-di-aminocyclitols via selectively blocked intermediates A. 1-N-ethylgentamic in via a 2', 3-di-N- substituted intermediate product

Dissolve 240 mg of 2*,3-di-N-trifluoroacetylgentamicin C 1 in

10 ml water-methanol (2:1) and adjust the pH of the solution to 3.5 by adding 1 N sulfuric acid. Add 0.19 ml of acetaldehyde, stir for 10 minutes and then add 27 mg of sodium cyanoborohydride and stir the reaction mixture for another 10 minutes. Evaporate the reaction mixture in vacuo to a residue comprising 1-N-ethyl-2',3-di-N-trifluoroacetylgentamicin C 2 . Dissolve the preceding residue in 50 ml of concentrated ammonium hydroxide and let the solution stand at room temperature for 24 hours. Evaporate the mixture in vacuo and chromatograph the resulting residue above. silica gel (12 g) and elute with the lower phase of a mixture of chloroform-methanol-10% aqueous ammonium hydroxide (2:1:1). Combine the equal fractions (as determined by thin layer chromatography) and evaporate the combined eluates in vacuo to a residue comprising 1-N-ethylgentamicin Ci (yield 80 mg) with the same characteristics as stated in Example 4A.

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

In the same manner as described in Example 7A, treat 6'-N-t-butoxycarbonylsisomicin (prepared in the same manner as Preparation 3D) in aqueous methanol with sulfuric acid, then acetaldehyde and sodium cyanoborohydride. Allow the reaction mixture to stand at room temperature for 30 minutes and then evaporate in vacuo to obtain 1-N-ethy1-6<1->N-t-butoxycarbonylsisomicin. Dissolve the residue in trifluoroacetic acid and leave the solution for 10 minutes. Then add an excess of anhydrous methanol, filter the resulting precipitate of the trifluoroacetic acid salt of 1-N-ethylsisomicin and chromatograph over silica gel and employ the lower phase of a chloroform-methanol-ammonium hydroxide solvent system in the same manner-as described in Example 7A, whereby 1-N-ethylsisomicin is obtained with the same characteristics as stated in example IA.

C. 1- N- methylsisomiciri from 3"- N- 4"- O- carbonyl- sisomicin

- 5 g of 3"-N-4,,-0-carbonyl-sisomicin in 300 ml of distilled water is treated with 1 N sulfuric acid until the pH of the solution is 2.5. 37% aqueous formaldehyde (2 ml) is added, and after 10 minutes, a solution of 500 mg of sodium cyanoborohydride in 5 ml of water dropwise. After 1 hour, the volume of the solution is reduced to half by evaporation in vacuum, the pH of the concentrated solution is adjusted to 11 by the addition of 1 N sodium hydroxide solution, and the solution is evaporated to dryness. The residue extracted with 3 x 100 ml methanol and the combined methanol extracts diluted with an equal volume of chloroform, filtered and evaporated to dryness to give impure 1-N-methyl-3"-N-4"-O-carbonyl-sisomicin.

The impure product is treated with 10% aqueous potassium hydroxide at 100° C. for 5 hours. The cooled solution is passed through an Amberlite IRC-50 (H<+>) ion exchange resin and eluted with 2 N aqueous ammonium hydroxide and the eluent is concentrated and lyophilized to form impure 1-N-methyl-sisomicin.

Chromatography of the impure material on silica gel (300 g) in chloroform-methanol-7% ammonium hydroxide (2:1:1) gives 1-N-methyl-sisomicin. (ct)J<6> + 153° (0.3% H 2 O) ;

Mass spectrum: (M + 1)<+> m/e 462

also w/e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336j[w) , 346, 376, 386.

D. 1- N- methylverdamicin via a 3", 4"- N- O- oxazolidone intermediate

(1) To an aqueous solution of 1 g of verdamicin, add sodium carbonate until the pH is within the range of 8 to 9. Add a solution of 5 g of p-nitrophenyl chlorocarbonate in 25 ml of dimethylformamide dropwise with stirring over .3 hours while the pH of the reaction mixture is maintained in the range 8 to 9 by adding more sodium carbonate solution. After the addition is complete, continue stirring at pH 8 to 9 for 16 hours. Evaporate the mixture in vacuo, extract the resulting residue several times with hot chloroform, combine and evaporate the extracts and chromatograph the resulting residue over 100 g of silica gel and elute with the lower phase of a (2:1:1) chloroform-methanol-concentrated ammonium hydroxide solvent system. Combine and evaporate the equal fractions as determined by thin layer chromatography to give verdamicin-3",4"-N,0-oxazolidone. (2) In the same manner as described in Example ID, treat the preceding oxazolidone derivative in water with sulfuric acid, formaldehyde and sodium cyanoborohydride. Isolate and purify the resulting product in the same manner as previously described, whereby 1-N-methylverdamic in-3", 4"-N,O-oxazolidone is obtained. (3) Dissolve 0.2 g of 1-N-methylverdamicin-3",4"-N,0-oxyzolidone in 10 ml of 2 N sodium hydroxide solution. Heat under reflux for 5 hours, neutralize the reaction mixture with acetic acid and evaporate to a residue. Chromatograph the residue over 10 g of silica gel and elute with the lower phase of a (2:1:1) system of chloroform-methanol-15% ammonium hydroxide. Combine and evaporate the equal fractions as determined by thin layer chromatography to a residue, whereby 1-N-methylverdamicin is obtained.

Example 8

1- N- benzylqentamicin C ^ - via a tri- N- protected 1- N- Schiff base intermediate

(1) Dissolve 0.3 g of 2*,3-di-N-trifluoroacetylgentamicin C^ in 12 ml

ethanol and add 0.9 ml of benzaldehyde. Stir the reaction mixture in

3 hours and evaporate in vacuo. Dissolve the resulting residue in

0.8 ml chloroform and add the solution dropwise to 25 ml hexane-ether (3:1). Separate the resulting precipitate by filtration and dry in vacuo to give 1-N-3"-N-4"-O-bis-benzylidine-2",3-di-N-trifluoroacetylgentamicin (yield = 0.38 g), sm .p. 128 - 134° C, (a)£<6> + 74.6° (c=0.26%, ethanol).

(2) Dissolve 1.37 g of the product obtained in example 8(1)

in 100 ml of ethanol and add to a stirred mixture of 1.37 g of sodium methoxide and 1.94 g of sodium borohydride in 100 ml of ethanol. Stir for 3 hours, acidify the mixture to a pH of approx. 3 with hydrochloric acid pg tube

then for 16 hours. Extract the solution with ether, separate and discard the ether layer. Add ammonium hydroxide to the aqueous phase until basic, then evaporate in vacuo to a residue. Extract the residue with 35 g of hot ethanol. Combine the extracts

and evaporate in vacuo. Chromatograph the resulting residue over 75 g of silica gel and elute with the lower phase of a chloroform-methanol-hydroxide-water (2:1:0.2:0.8) solvent system. Combine equal fractions as determined by thin-layer chromatography! and evaporate to a residue comprising 1-N-benzylgentamicin Cpjm. p. 83 .- 88° C, (a}£<6> + 90° (c=0.3%, H20) ; pmr (ppm)

(D 2 O): & 1.03 (3H, d, J=7Hz, HC-CH 3 ); 1.16 (3H, s, C-CH3);

~ T, 21 (3H, s, N-CH3); 2.50 (3H, s, N-CH3); 4.7 (D2<0><+> PhCH2N ); 4.92 (1H, d, J=4Hz, H-1"); 5.08 (1H, d, J=3.5 Hz, H-1'); 7.43 (5H, s, aromatic H);

Mass spectrum: (M + 1)<+> m/e 568

also w/e 440, 437, 412, 394, 379, 281, 263, 253, 235, 160, 157.

Example 9

1- N- substituted- 4, 6- diaminoglycosy1- 1, 3- diaminocyclitols prepared by hydril reduction of the corresponding 1- N- acyl derivatives _A«- 1- N- ( S-( S- amino- g- hydroxybutyl) - gentamicin

(1) Suspend 98 mg of 1-N-(S-5-amino-8-hydroxybutyryl)-gentamicin in 8 ml of tetrahydrofuran. Add 14 mL of 1N diborane in tetrahydrofuran and heat at reflux for 6 hours under a nitrogen atmosphere. Carefully add 2 ml of water to decompose any excess diborane and evaporate. Dissolve the resulting residue in hydrazine hydrate and heat to reflux temperature under a nitrogen atmosphere for 16 hours. Evaporate the solution and extract the residue with hot aqueous ethanol. Evaporate the combined ethanol extracts and chromatograph the resulting residue over 10 ml of silica gel, eluting with the lower phase of a chloroform-methanol-concentrated ammonium hydroxide (2:1:1) solvent system. Combine and evaporate the equal fractions as determined by thin-layer chromatography to give 1-N-(S-6-amino-g-hydroxybutyl)-gentamicin C ± (yield 14.5 mg), m.p. 93 - 98° C, (ct)^r +72.4°

(c=0.35%, H 2 O); pmr (ppm) (D 2 O) δ 1.18 (3H, d, J=7Hz, CH-CH 3 );

1.24 (3H, s, C-CH3); 2.49 (3H, s, N-CH 3 ); 2.54 (3H, s, N-CH3);

5.07 (1H, d, J=3.5Hz, H-1<n>); 5.24 (1H, d, J=3.5Hz, H-1').

Mass spectrum: (M + 1)<+> m/e 565

also ra/e 528, 516, 490, 437, 434, 410, 397, 278, 250, 232, 160, 157.

Prepare in the same way 1-N-(S-Y-methylamino-|3-hydroxypropyl)-gentamicin B,

pmr: D 2 O: 1.21 (3H, s, C-CH.^) ; 2.33 (3H, s, 3,,,-N-CH 3 );

2.62 (3H, s, 3"-N-CH3); 5.02 (1H, d, J=k, 5 Hz, H-1<»>); 5.12

(1H, d, J=3.0 Hz, H-1').

Example 10

1- N- methylsisomicin from 3"- N- 4"- 0- carbonyl- 2', 3, 6'- tri- t-butoxycarbonyl- sisomicin

Dissolve 0.77 g of 3"-N-4"-O-carbonyl-2',3,6'-tri-N-t-butoxycarbonyl-sisomicin in 20 ml of tetrahydrofuran and cool in an ice bath. Add 0.12 g of methyl fluorosulfonate and allow the mixture to warm to room temperature. Remove the solvent and dissolve the residue in trifluoroacetic acid. After 5 minutes at room temperature, remove the trifluoroacetic acid in vacuo and treat the residue with 10% potassium hydroxide solution at 100° C for 5 hours. /"'

Pass the cooled solution down a column of Amberlite IRC-50 (H ) ion exchange resin and elute with 2 N aqueous ammonium hydroxide. Concentrate the eluent and lyophilize, whereby the impure titrated product is obtained. ..Chromatograph the impure material on silica gel and elute the lower phase with a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system, whereby 1-N-methyl-sisomicin is obtained.

(a}£6 + 153° (0.3%, H2O); Mass spectrum: (M + 1)+ m/e 462

also w/e 127, 140, 159, 160, 177, 187, 205,

256, 285, 303, 318, 331, 336(w), 346, 376, 386.

Example 11

1- N- methylsisomycin

5 g of 2',3,6'-tri-N-t-butoxycarbonyl-3"-N-4"-O-carbonylsisomicin in 100 ml of ethanol are treated with 1 ml of 37% aqueous formaldehyde and 5 g of ammonium formate and the solution is heated under reflux -run for 24 hours. 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<1->tri-N-t-butoxycarbonyl-3"-N-4"-0-carbonyl-1-N-methyl-sisomicin is dissolved in trifluoroacetic acid and after 5 minutes at room temperature, the solvent is removed in vacuo. The residue is treated with 10% potassium hydroxide (25 ml) at 100° C. for 5 hours. The cooled solution is passed down through a column of Amberlite IRC 50 (H<+>) ion exchange resin and the impure product is eluted with 2 N aqueous ammonium hydroxide. The combined eluent is reduced to dryness in vacuo and the residue is chromatographed on 200 g of silica gel and eluted with the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system, whereby 1-N-methylsisomicin is obtained. (a) D + 153° (0.3%, H 2 O); Mass spectrum: (M + 1)<+> m/e 462

also w/e 122, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336(w), 346, 376, 386.

Example 12

1- N- methylsisomycin

Treat 0.77 g of 2',3,6'-tri-N-t-butoxycarbonyl-3"-N-4"-carbonyl-sisomicin in 25 ml of tetrahydrofuran with 101 mg of methylamine and 290 mg of trifluoromethylsulfonic anhydride at 0° C. for 18 hours. Reduce the solution to dryness and dissolve the residue in 10 ml of dimethylformamide and stir with 300 mg of methyl iodide and 130 mg of potassium carbonate for a further 18 hours. Remove the solvent by evaporation and treat the residue with 10% aqueous potassium hydroxide at 100° C. for 12 hours. Pass the cooled solution through a column of Amberlite IRC, 50 (H ) ion exchange resin. The impure product is then eluted with 2 N aqueous ammonium hydroxide. The combined eluent is reduced to dryness in vacuo and the residue is chromatographed on 200 g of silica gel and eluted in the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system, whereby 1-N-methylsisomicin is obtained. (a)^<6> + 153° (0.3%, H 2 O);

Mass spectrum: (M + 1)<+> m/e 462

also w/e 127, 140, 159, 160, 177, 187, 205,

256, 285, 303, 318, 331, 336(w), 346, 376, 386.

Example 13

1- N- methylsisomycin

0.77 g of 2',3,6,-tri-N-t-butoxycarbonyl-3"-N-4"-0-carbonylsisomicin in 20 ml of tetrahydrofuran is treated with 3 ml of 37% aqueous formaldehyde and 170 mg of succinimide at room temperature in 18 hours. The solution is poured into a mixture of diethyl ether-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 5 hours. The ethanol is removed in vacuo and the residue is treated with 10% aqueous potassium hydroxide for 12 hours at 100° C. The cooled solution is passed down a column of Amberlite IRC 50 (H<+>) ion exchange resin and the impure product is eluted with 2 N aqueous ammonium hydroxide. The combined eluent is reduced to dryness in vacuo and the residue is chromatographed on silica gel (200 g) and eluted with the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system, whereby 1-N- methylsisomycin. (°0D

153° (0.3%, H 2 O);

Mass spectrum: (M + 1) m/e 462

also w/e 127, 140, 159, 160, 177, 187, 205,

256, 285, 303, 318, 331, 336(w), 346, 376, 386.

Example 14

1- N- methylsisomycin

Dissolve 0.77 g of 2',3, 6'-tri-N-t-butoxycarbonyl-3n-N-4"-0-carbonylsisomicin in 100 ml of dichloromethane with 0.25 g of acrylonitrile and allow the mixture to stand at room temperature for 24 hours. Remove the solvent in vacuo and dissolve the residue in dimethylformamide and treat with 180 mg of methyl iodide at 50° C. for 12 hours. Remove the solvent and treat the residue with 10% aqueous potassium hydroxide at 100° C. for 18 hours. The cooled solution is passed down through a column of Amberlite IRC 50 (H ) ion exchange resin and the crude product is eluted with 2 N aqueous ammonium hydroxide. The combined eluent is reduced to dryness in vacuo and the residue is chromatographed on 200 g of silica gel and eluted with the lower phase of a chloroform-methanol-7 % ammonium hydroxide (2:1:1) solvent system,, whereby 1-N-methylsisomicin is obtained. (a)^<6> + 153° (°'3%' H20^J

Mass spectrum: (M + 1)+ m/e 462

also w/e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336(w), 346, 376, 386.

The compounds according to the invention are broad-spectrum antibacterial agents which advantageously exhibit activity against many organisms, in particular gram-negative organisms, which are resistant to their 1-N-unsubstituted precursor. Thus, the compounds according to the invention can be used alone or in combination with other antibiotic agents to prevent growth or reduce the number of bacteria in different environments. They can:, e.g. is used to disinfect laboratory glassware, dental and medical equipment contaminated with Staphylococcus aureus or other bacteria that can be inhibited by the compounds according to the invention. The activity of the compounds of the invention against gram-negative bacteria makes them useful in combating infections caused by gram-negative organisms, e.g. species of Proteus and Pseudomonas. The 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, e.g. 1-N-ethylsisomicin and 1-N-ethylverdamicin are also used in veterinary medicine, in particular in the treatment of mastitis in cattle and Salmonella-induced diarrhea in domestic animals such as dogs and cats.

The following table indicates the minimum inhibitory concentration (MIC) of certain compounds according to the invention. The experiments were carried out in Mueller-Hinton cast (pH 7.2) using standard procedures.

In vitro minimum inhibitory concentration (MIC)

Label the connections in the table:

a) New compounds produced according to the present invention:

Compound 1: 1-N-ethylgentamicin C,

X3

Compound 2: 1-N-ethylgentamicin

Compound 3: 1-N-ethyl Antibiotic G-52

Compound 4"- 1-N-(4-amino-2(S)-hydroxybutanj-gentamicin Compound 5= 1-N-hydroxyethylgentamicin C-^

Compound 6: 1-N-phenethylgentamicin C1

Compound 7- 1-N-ethylverdamicin

Compound 8 = 1-N-ethylsisomicin

Compound 9: 1-N-(2-ethylbutyl)-sisomicin

Compound 10: 1-N-(4-aroinobutyl)-sisomycin

Compound 11: 1-N-ethylgentamicin B

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

Compound 13: 1-N-ethylmutamicin 2

Compound 14: l-N-ethylmutamicin 6

b) Compounds known from the prior art:

Compound 15: 1-N-(S-4-amino-2-hydroxybutyryl)-genta micin Cla (US patent 3,796,699)

Compound 16: 1-N-(S-4-amino-2-hydroxybutyryi)-gentamicin C1 (US Patent 3,780,018)

Test method

Prepare and sterilize 200 ml of Mueller-Hinton nutrient solution adjusted to pH 7>2. Transfer 5 ml of the sterile medium to each of 120 sterile 16 x 150 mm test tubes with cotton plugs. Arrange the tubes in IO groups of 12 tubes each. Add to each tube lO^ cells of one of the bacterial strains to be tested. Add to each group an aqueous solution of the antibiotic to be tested, forming the following final concentrations per tube: .50 mcg/ml, 25 mcg/ml, 10 mcg/ml, 5 mcg/ml, 2 mcg/ml,

1 mcg/ml, 0.5 mcg/ml, 0.2 mcg/ml, 0.1 mcg/ml, 0.05 mcg/ml,

0.01 mcg/ml, 0.005 mcg/ml and 0.0 mcg/ml (control). Incubate the tubes for 24 hours at 37°C. Visually determine for each group of tubes the lowest concentration of antibiotic that inhibits bacterial growth and the highest concentration of antibiotic that allows bacterial growth.

Determine the minimum inhibitory concentration of the test antibiotics against each of the test bacteria by calculating the mean value between the two values found above.

Generally, the dose administered of the compounds of the invention will depend on the age and weight of the animal species

being treated, method of administration and the type and condition of the bacterial infection to be prevented or reduced. In general, the dose of the derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols to combat a given bacterial infection will be similar to the dose required of the corresponding 1-N-unsubstituted-4,6-di-(aminoglycosyl )-1,3-diaminocyclitols.

The compounds according to the invention can be administered orally.

They can also be applied topically in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions that can be aqueous, non-aqueous

mostly of the emulsion type or in the form of creams. Pharmaceutical

carriers useful in the preparation of such formulations will include e.g. such components as water, oils, fats, polyesters, polyols and the like.

For oral administration, the compounds according to the invention can be composed in the form of tablets, capsules, elixirs or the like or they can even be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective_^__ for the treatment of bacterial infections in the gastrointestinal tract, which infections cause diarrhoea.

In general, topical formulations will contain from 0.1 to

3.0 g of the compounds according to the invention per 100 g ointment, creams or lotion. The topical formulations are usually applied gently to lesions 2 to 5 times per day. day.

The antibacterial agents according to the invention can be used

in liquid form such as solutions, suspensions and the like as ear and eye preparations and can also be administered parenterally via intramuscular injection. The injectable solution or suspension will usually be administered with from 1 mg to about 15 mg antibacterial agent per

kg body weight per day divided into 2 to 4 doses. The exact dose depends on the degree and condition of the infection, the susceptibility of the infecting organism to the antibacterial agent and the individual characteristics of the animal species being treated.

Claims (2)

1. Analogous process for the preparation of 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitolene gentamicin B, gentamicin B^, gentamicin C^, gentamicin C^a, genta micin cy gentamicin C2a, gentamicin C^b' s^-som:'-c:i:n' verdamicin, Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is wherein X is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl or benzyl, which aliphatic radicals have up to 7 carbon atoms and, if X is substituted with amino and hydroxy, the substituents bear different carbon atoms, and pharmaceutically acceptable acid addition salts thereof, characterized in that one of the following procedures A to G, Å is used) one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diamino cyclitols which may have amino-protecting groups in any position other than the 1-position are treated with an aldehyde of the formula where X' is a group as defined for X and where any amino or hydroxy group present may be protected, in the presence of a hydride donor reducing agent, and, if necessary, that all protecting groups present in the molecule are removed, B) reduction of N, The C double bond in a 1-N=CHX'-substituted derivative of one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, in which all NH2~ groups are protected and NHCH^ groups may be protected, where X' is a group as defined for X, in which any amino or hydroxy group present may be protected, and that all present protecting groups in the molecule are removed, C) a 1-N-substituted derivative of one of the above-mentioned 4,6-di-r(aminoglycosyl)-1,3- diaminocyclites, in which one or more amino groups may be protected and the 1-N substituent is 0 where X" is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-r . alkylamiiroalkyl, aminohydroxyalkyl, N-alkylaminobnydroxyalkyl, phenyl, benzyl or hydrocarbyloxy, which aliphatic radicals have up to 7 carbon atoms, and if X is substituted with amino and hydroxy, the substituent carries different carbon atoms, and in which any amino or hydroxy group present may be protected, treated with an amide-reducing hydride reagent, and if desired/ that all protective groups present in the molecule are removed, . D) that for the preparation of the above-mentioned 1-N-substituted derivatives of .4,6-di-(aminoglycosyl)-1,3-diaminocyclitols in which the substituent is straight-chain alkyl with up to 5 carbon atoms, that one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols , which exhibits a mLno protecting groups in any position other than the 1-position, and in which the 1-amino group may be activated, is reacted with an alkylating agent containing a straight-chain alkyl group of up to 5 carbon atoms and one leaving group, and that they protecting groups, and if the desired activating group or groups present in the molecule are removed, E) that for the production of the above-mentioned 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols in which the substituent is methyl, that one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which exhibit amino-protecting groups in any position other than the 1-position, is reacted with formaldehyde and a cyclic imide, after which the compound thus obtained is treated with a hydride-donor-reducing agent, and that all protective groups present in the molecule are removed, F) that for the preparation of the same compounds as indicated in point E, that one of the above-mentioned 4,6-di«{aminoglycosyl)-1,3-diaminocyclitols exhibiting amino-protecting groups in any position other than the 1-position is reacted with formaldehyde in the presence of formic acid and that all protective groups present in the molecule are removed. which process alternatives A to F are followed by isolation of the derivative as such or as a pharmaceutically acceptable acid addition salt. 2. Method according to claim 1, c a racter e r in particular that 1-N-ethylsisomicin. is prepared and isolated as such or as a pharmaceutically acceptable acid addition salt.