DK146298B - METHOD OF ANALOGUE FOR PREPARING 1-N-SUBSTITUTED DERIVATIVES OF 4,6-DI- (AMINOGLYCOSYL) -1,3-DIAMINOCYCLITOLS OR ACID ADDITION SALTS THEREOF - Google Patents

Description

146298

The present invention relates to an analogous process for the preparation of novel 1-N-substituted derivatives of the 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitoles gentamicin B, gentamicin B, gentamicin C, gentamicin C , gentamicin C-, gentamicin C2 ^ in gentamicin sisomicin, verdamycin, Antibiotic G-418, Antibiotic 66-4OB,

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 -ch2x where X is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl or benzyl, which aliphatic groups have up to 7 C atoms and if substituted with both amino and hydroxy, the substituents base on different carbon atoms, or pharmaceutically acceptable acid addition salts thereof, and the process is characterized by the characterizing part of claim 1.

Widespread antibacterial agents are known which can be chemically classified as 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols. Valuable antibacterial agents of this group are those wherein the aroinocyclitol is 2-de-oxystreptamine or a derivative thereof with amino functions at positions 1 and 3. Particularly valuable antibacterial compounds among the 4,6-di- (aminoglycosyl) -2-deoxystreptamines are those in which the aminoglycosyl group at position 6 is a garosaminyl group. Within the class of 4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines there are antibiotics such as gentamicins B, B 2, C 2, Cla, C 2 'C 2a' C 2b and X 2, sisomicin, verdamicin, Antibiotic G -418, Antibiotic G-52, Antibiotic JI-20A, and Antibiotic JI-20B.

The novel 1-N substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols or acid addition salts thereof prepared by the present analogous process have been found to be broad-spectrum antibacterial agents exhibiting beneficial activity over for many organisms, in particular gram-negative organisms, and distinguishes in this respect compared to known antibacterial agents, as is more fully documented below, wherein the compounds of this invention are similar to known 1-N-substituted 4,6-di - (aminoglycosyl) -1,3-diaminocyclitols, wherein the 1-N substituent is one acyl group. The derivatives are distinguished in particular by being active against gram-negative organisms resistant to the corresponding known 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols.

Included among the substituents which may be the group CH 2 X in the novel compounds are l-20 chain and branched alkyl groups such as ethyl, n-propyl, n-butyl, ε-methylpropyl, n-pentyl, δ -methylbutyl, γ-methylbutyl and β, β-dimethylpropyl, n-hexyl, δ-methyl-pentyl, N-ethylbutyl, γ-ethylbutyl, n-heptyl, 6-methyl-heptyl, N-ethylpenty 1, γ-ethylpentyl, δ-ethylpenty 1, γ-propylbutyl, n-octyl, iso-octyl, δ-ethylhexyl, δ-ethylhexyl, £-ethylhexyl, δ-propylpentyl and γ-propylpentyl, alkenyl groups such as β-propenyl, β-methylpropenyl , O-butenyl, β-methyl-O-butenyl and O-ethyl-O-hexenyl, hydroxy-substituted straight-chain and branched-chain alkyl groups, such as 3 -hydroxypentyl, β-hydroxy-V-methylbutyl (i-hydroxy-β-methylpro pyl, S -hydroxybutyl, β -hydroxypropyl, Y -hydroxypropyl and β-hydroxy-octyl, amino-substituted straight-chain and branched-chain alkyl groups such as β-aminopentyl, (3-aminopropyl, V-aminopropyl, S-aminobutyl, pyamiro). -Y-methylbutyl and β-amino-octyl and mono-N-alkylated derivatives thereof, such as N-methyl, N-ethyl and N-propyl derivatives, e.g. 1'-methyl-aminopentyl, β-methyl-amonipropyl, p-ethylaminopropyl, δ-methylaminobutyl, β-methylamino-γ-methylbutyl and ω-methylaminobutyl, amino and hydroxydisubstituted 1-chain and branched alkyl groups such as β -hydroxy-6-aminopentyl, Y-hydroxy-Y ~ methyl-6-aminobutyl ,. β-hydroxy-5β-aminobutyl, β-hydroxy-γ-aminopropyl and β-hydroxy-β-methyl-γ-aminopropyl, and mono-N-alkylated derivatives thereof, such as β-hydroxy-C-methylaminopentyl, γ-hydroxy -Y-methyl-δ-methylaminobutyl, β-hydroxy-δ-methylaminobutyl, β-hydroxy-γ-ethylaminapropyl and β-hydroxy-β-methyl-γ-methyl-aminopropyl.

The compounds are preferably 1-N-Cl 2 X derivatives containing garosaminyl as 6-aminoglycoside group and especially containing 2-deoxystreptamine as 1,3-diamino-cyclitol.

2-Deoxystreptamine is present in all of the foregoing compounds prepared by the process of the invention except the mutamicins.

The 1,3-Diaminocyclitol core in each of the 1N-CH2X mutamines 1, 2, 4, 5 and 6 are streptamine, 2.5-20 dideoxy streptamine, 2-epi-streptamine, 5-amino-2,5-dideoxy, respectively. streptamine and 5-epi-2-deoxystreptamine.

The 1N-CH2X-4-Aminoglycosyl-6-garosaminyl-2-deoxystrep taminins produced by the present process are the derivatives of gentamicin B, gentamicin B 2, gentamicin C 2, gentamicin Cla, gentamicin C 2, gentamicin C 2a /? entamicin C2b 'sisomisin, verdaminicin, Antibiotic G-418, Antibiotic JI-20A, Antibiotic JI-20B and Antibiotic G-52, which compounds are defined by the following structural formula I:

? H2 j NHCH2X

T-x 35 ® L-o * \ 4 146298 wherein X has the former meaning and wherein Y is an aminoglycosyl group selected from the group consisting of: & a NH0 GH3

2 2 J

NH2-- “i-n: ch2x- i-n-gh2x.

ie N '\ F / gentamicin B) nJ γ / gentamicin B ^)

OH OH

10 CH3ffl- |! 3 [ζ (j 1-N-GH2X- Ιζ. J> | _ (i 1-N-CH2X- gentamicin C ^) N \ v gentamicin C ^) NH2 NH2

15 CH CH

m2 - Lr - nh2 /> L (i 1 -N "CH2X- KJ> L (i 1-Ν-0Η2χ-gentamicin C ^). l \ j gentamicin C ^) 20 NH2 NH2 ch2nhch3 / \ l (i 1- N-GH X-25 N | -gentamicin C ^) nh2 CH CH NH, -i-nh2 * 30 (i 1-N-CH2X- (i 1-N-CH X- \ ii, / verdamicin) n. Sisomicin ; nh2 nh2

CH NH CH

__i nh2 kf ^ OH (i (i i-N-C

Antibiotic „j / V 9H Antibiotic

Ho ^ -γ jτ_20Α) ho -y ji_2ob) nh2 nh2 146298 5 CH "NHCH, I 2 3 HO-- U l-S-OHJE- (1.55 \ -Y ££" · *** "" tlim HH2 and NH2

Other useful 1N-CH2X-4,6-di- (aminoglycosyl) -2-deoxystreptamines prepared by the process of the invention include 1N-CH2X Antibiotic 66-40D 10 of the following Formula III (which is among the preferred compounds). ): ch2nh2 nh2 NH2

III

wherein X has the meaning previously defined and 1-N-CH2X Antibiotic 66-4oB of the following Formula IV:

NH

fH2NH2 L_1 HHGH „X

(1V OH

wherein X has the meaning previously defined.

The 1-N-CH2X mutamicins produced by the process of the invention include 1-N-CH2X-4-amino-glycosyl-6-garosaminyl-1,3-diaminocyclitols of the following formula V: 5 ™ 2m2 F2 V2

J- ^ J-L NHCH 'X

NH2 w5

10 V

0

HO J-Q

Wherein X has the meaning previously defined and 2 5

in 1-N-CH 2 X-mutamicin 1 represents V and W hydro-2,5Z

gene and W and V hydroxy, 20 in 1N-CH2X-mutamicin 2 represents W2, V2, and V5 hydrogen, 25 in 1N-CH2 X-mutamicin 4 represents W and W hydro-2,52 gene and V and V hydroxy, in 1-N-CH-X-mutamicin 5 denotes W2, V2 and 5 ^ hydrogen and V amino, and in 1N-CH2X-mutamicin 6 denotes W2, V2 and V ~ * hydrogen, while WD denotes hydroxy.

In the structural formulas given herein, bond terminations without listed substituents denote hydrogen atoms.

The pharmaceutically acceptable acid addition salts of the 1N-CH2X-4,6-di- (aminoglycosyl) -1,3-diaminocyclitols of formulas I, II, III, IV and V are prepared in a manner known per se, such as by neutralizing the free 35 base with the acid in question, usually to pH ca. 5. Suitable acids for this purpose include acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, acetic acid, propionic acid, butyric acid, ascorbic acid and citric acid. The acid addition salts of the 1 ^ NC 2 Xi, 6-di- (aminoglycosyl) -1,3-diaminocyclitolols can be characterized as white solids which are water soluble, sparingly soluble in most other polar solvents and insoluble in non-soluble. polar organic solvents.

The 1N-CH2X-4,6-di- (aminoglycosyl) -1,3-diaminocyclicols as defined by formulas I, III, IV and V as well as their non-toxic pharmaceutically acceptable acid-1Q addition salts exhibit, as mentioned generally, a broad spectrum antibacterial action. In particular, the 1-N-alkyl derivatives have improved antibacterial effects compared to the corresponding 1-N unsubstituted antibiotics, which are especially manifested in an increased activity of the compounds against the 1-N resistant organisms. -substituted compound. Thus, the compounds are e.g. more active against organisms that inactivate the corresponding 1-N unsubstituted antibiotics by acetylation of the 3-amino group 20 and / or adenylylation of the 2 "hydroxy1 group. Some of these compounds also exhibit anti-protozoal, anti-amoebic and anthelmintic properties.

A preferred group of compounds are the 1-N-substituted derivatives of the 4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines gentamicin B, gentamicin B ^, gentamicin C ^, gentamicin C ^, gentamicin Q., sisomicin, verdamicin, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52 and Antibiotic G-418, of which the derivatives of gentamicin C ^, gentamicin C ^ a, sisomycin, verdamicin and Antibiotic G-52 are the most preferred . Other particularly useful compounds are the 1-N-substituted derivatives of Antibiotic 66-40B.

In the 1-N substituent, X is preferably selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, aminohydroxyalkyl, phenyl or benzyl, which aliphatic groups have up to 7 carbon atoms and, if substituted by both amino and hydroxy, carry the substituents of various carbon atoms. Of these, the preferred groups are hydrogen, alkyl, aminoalkyl and hydroxyalkyl having up to 7 carbon atoms and aminohydroxyalkyl having up to 3 carbon atoms and bearing the substituents on various carbon atoms.

Particularly useful compounds are those wherein X

represents hydrogen, methyl, ethyl and propyl, and preferably methyl and ethyl. A particularly valuable group is the 1-N-CH 2 X-4-aminoglycosyl-6-garosaminyl-2-deoxystreptamines of Formula I, wherein X represents an alkyl group 10 having 1-3 carbon atoms, especially 1-N- (C alkyl) - derivatives of gentamicin C1, gentamicin C2a, gentamicin C2, gentamicin C2a, gentamicin, sisomicin, verdamycin and Antibiotic G-52, as well as 1N-1S-C6alkyl) Antibiotic 66-40D with Formula III, which derivatives are 15 broad-spectrum antibacterial agents active against gram-positive bacteria (e.g., Staphylococcus aureus) and gram-negative bacteria (e.g., Escherichia coli and Pseudomonas aeruginosa), as detected by stand dilution experiments, including bacteria resistant to the 1-N unsubstituted precursors. Particularly useful are 1-N-ethylverdamicin (physical data for this and the following particularly listed compounds, see examples below) and 1-N-alkyl isomicines, e.g. 1-N-methyl-sisomicin, 1-N- (n-propyl) sisomicin, 1-N- (n-butyl) sisomicin, and preferably 1-N-ethyl-sisomicin exhibiting activity against gram negative organisms resistant to the 1-N unsubstituted precursors of the compounds. Other particularly useful compounds are 1-N-ethyl-gene-tamicin C- a, 1-N-ethyl-gentamicin C ^, 1-N-ethyl-Antibiotic G-52, 1-N- propyl) -verdamicin, 1-N- (δ-aminobutyl) -isomicin, 1-N- (n-butyl) -verdamicin, and 1-N- (S-2-hydroxy-4-aminobutyl) -gentamicin C-.

Most of the aforementioned 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol antibiotics from which the present 1-N-substituted derivatives can be prepared are known. The starting compounds herein referred to as gentamicin C2a and C1 are isolated and characterized as set forth below in Preparations 1 and 2.

U6298 9

The isolation, properties, and planar configuration of gentamicin C2 are disclosed in the disclosure to U.S. Pat. Patent No. 3,651,042.

Antibiotic 66-40B and Antibiotic 66-40D, their preparation, isolation, properties and configuration are disclosed in the description of Belgian Patent No. 811,370.

These antibiotics are prepared together with sisomycin, which is the main product of the fermentation of Micro-monospora inyoensis (disclosed in the specification of British Patent No. 1,274,518), and can be separated from the fermentation medium using special chromatographic separation technique.

Mutamicin 1, 2, 4, 5 and 6, the configuration of which is shown above, can be prepared by culturing a mutant strain of Micromonospora inyoensis herein designated Micromonospora inyoensis strain 1550F-1G in an aqueous nutrient medium. This mutant strain is unable to form an antibiotic when cultured under submerse aerobic conditions in an aqueous nutrient medium without the 1,3-diaminocyclitol building block. However, when certain such compounds are added to the fermentation medium, the mutamicins are formed. When 2-deoxystreptamine is added to the fermentation, the known antibiotic sisomycin is formed.

The necessary 1,3-diaminocyclitols which must be present during the fermentation to obtain the mutamicins are the following: streptamine to mutamicin 1 2,5-dideoxystreptamine to mutamicin 2 · 2-epistreptamine to mutamicin 4 2,5-dideoxy- 5-aminostreptamine for mutamicin 5 5-epi-2-deoxystreptamine for mutamicin 6.

The preparation of the mutamicins is described in generally available Swedish patent application no.

7409945-8.

Of the preparation methods a) to f) of the 1-N-substituted derivatives of the present invention, the process a) gives a corresponding 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, which may have amino protecting groups. is any member of any amino group other than that of the 1-position treated with an aldehyde of the formula

X'-CHO

wherein X1 is a group as defined for X above, wherein any amino or hydroxy groups present may be protected, in the presence of a hydride donor reducing agent, and, if required, all protecting groups present in the molecule are removed per se method, which last process step is followed by isolation of the derivative as such or as a pharmaceutically acceptable acid addition salt.

This process whereby the 1-amino group of a 1-N unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol is selectively condensed with an aldehyde and simultaneously reduced in situ to form an antibacterially active 1N alkyl-4,6-di- (aminoglycosyl) -1,3-diaminocytol, is usually carried out at room temperature in the presence of atmospheric air, but it may advantageously be carried out under an inert atmosphere, e.g. argon or nitrogen. The reaction is advantageously completed in a short time, usually less than 30 minutes, as determined by thin layer chromatography.

Hydride donor reducing agents which can be used in this process include dialkylaminoboranes (e.g., dimethylaminoborane, diethylaminoborane and preferably morpholinoborane), tetraalkylammonium cyanoborohydrides (e.g. tetrabutylammonium cyanoborohydride), alkali metal borohydrides, alkali metal cyanoborohydrides (e.g., lithium cyanoborohydride and sodium cyanoborohydride).

The process is conveniently carried out in an inert solvent. By "inert solvent" is meant any organic or inorganic solvent in which the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol starting materials and reagents are soluble and will not interfere with the reaction process. 11 146298 conditions for this, so that there is a minimum of competing side reactions. Although anhydrous aprotic solvents can sometimes be used advantageously in the process (such as tetrahydrofuran, when using morpholinoborane as a 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 (e.g., aqueous methanol, aqueous ethanol), but other water-miscible co-solvent systems such as aqueous dimethylformamide, aqueous hexa may also be used. -methylphosphoramide, aqueous tetrahydrofuran and aqueous ethylene glycol dimethyl ether.

The process is conveniently carried out at a pH 15 in the range 1-11, preferably 2-5, and proceeds best in the range 2.5 to 3.5. The preferred acidic medium can be obtained by adding an organic or inorganic acid to the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol.

Thereby, acid addition salts are formed from the compounds. 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 may be used. It is most appropriate to use sulfuric acid. According to a preferred embodiment 25 of the process, it is also convenient to prepare the acid addition salt 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 -diaminocyclitol (e.g., sisomicin) in a protic solvent (e.g., water) until the pH of the solution is adjusted to the desired pH.

Typical aldehydes of the formula X'CHO, wherein X 'has the aforementioned meaning which can be used in the process, include straight or branched alkyl aldehydes such as formaldehyde, acetaldehyde, n-propanal, n-butanal, 2-methylpropanal, n pentanal, 2-methyl-butanal, 3-methylbutanal, 2,2-dimethylpropanal, n-hexanal, 2-ethylbutanal, n-heptanal and n-octanal, alkenylaldehydes such as propenal, 2-methylpropenal, 2-butenal, 2-methyl-2-butenal, and 2-ethyl-2-hexenal, benzaldehyde and phenylacetaldehyde, hydroxy subsubstituted, straight-chain or branched alkyl aldehydes such as 5-hydroxypentanal, 2-hydroxy-3-methylbutanal, 2 -hydroxy-2-methylpropanal, 4-hydroxybutanal, 2-hydroxypropanal and 8-hydroxyoctanal, amino substituted straight or branched alkyl aldehydes such as 5-aminopentanal, 2-aminopropanal, 3-aminopropanal, 4-aminopalan amino-3-methylbutanal and 8-amino-10octanal, and mono-N-alkyl derivatives thereof, and amino- and hydroxydisubstituted straight-chain electricity 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 15 and 2-amino-3-hydroxyoctanal, and mono-N-alkyl derivatives thereof.

If the aldehyde has a chiral center, this method can use the individual enantiomers separately or together as a racemate, and 20 corresponding diastereoisomers or a mixture thereof will be obtained.

The aldehyde reagents which can be used in the process are either known compounds or they can be readily prepared from known compounds using methods well known in the art. Thus, e.g. alkyl aldehydes substituted with both hydroxy and amino groups (e.g. 2-hydroxy-5-aminopentanal) are prepared from an aminoaldehyde acetal (e.g. 4-aminobutanaldiethyl acetal) by protecting the amino group therein as an acetamido or phthalimido group under use. - Consideration of known methods followed by removal of the acetal function by acid hydrolysis to give an N-protected aminoaldehyde (e.g., by transfer of 4-aminobutanaldiethyl acetal in the corresponding N-phthalimido derivative, as by acid hydrolysis yields 4-phthalimidobuta-3 5 nal). Treatment of the N-protected aminoaldehyde with hydrogen cyanide gives the corresponding N-protected aminoalkylhydroxynitrile (e.g., 2-hydroxy-5-phthalimido-valeronitrile), as by catalytic reduction (e.g., with hydrogen in the presence of palladium) or by hydride reduction (e.g., with diisobutyl aluminum hydride) yields an N-protected aminohydroxyaldehyde (e.g., 2-hydroxy-5-5 phthalimidopentanal), which is an aldehyde reagent used in this process.

In carrying out the process in the manner in which a 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitol is treated with a hydride donor and an aldehyde to obtain the corresponding 1-N-substituted derivative of a 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, to minimize competing side reactions when an aminoaldehyde is used as a reagent, it is preferred to protect the amino group of the aldehyde, e.g. with an acyl blocking group, such as acetamido or phthali-mido, before carrying out the process, and then removing the N-protecting group in the compound thus formed. It may also be advantageous to protect the hydroxy group in hydroxy-containing aldehydes by carrying out the procedure. However, this is usually not necessary.

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

A convenient way of practicing the method comprises preparing a solution of an antibacterial 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol (e.g., sisomicin or verda-micin). in a protic solvent (preferably water) and adjusting the pH of the solution to from ca. pH 2 to approx. pH 5 with an acid (usually dilute sulfuric acid) to form the necessary acid addition salt of the starting compound. When the pH of the solution is approx. 5, the acid addition salt thus produced usually contains one equivalent acid for each amino function of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol (e.g., per mole of sisomicin is 2.5 moles of sulfuric acid to present). Following the preparation of the acid addition salt solution, at least one mole equivalent and preferably a large molar excess of the desired aldehyde (e.g. acetaldehyde, propanal or butanal) is added followed by a short time (usually about 5 minutes) of the addition of ca.

5 mole equivalent (based on starting 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol) of a hydride donor-reductant agent, preferably an alkali metal cyanoborohydride, usually sodium cyanoborohydride. The reaction is often completed in less than 30 minutes, as demonstrated by thin-length chromatography, and the corresponding 1-N-substituted derivative of a 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitol ( eg 1-N-ethylsisomicin or 1-N-ethylverdamicin). Isolation and purification of the derivative thus produced is carried out using known techniques such as precipitation, extraction and preferably chromatographic technique.

Said process thus provides a convenient one-container process whereby an aminoglycoside is reacted in situ with an aldehyde (preferably in excess amounts) and with a hydride donor reducing agent to produce, as the main product, a mono-N substituted derivative ( for example, 1-N-ethylsisomycin), wherein the process 1-amino group attached to a secondary carbon atom is usually alkylated preferably compared to other amino groups attached to primary and other secondary carbon atoms in 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 said process. In general, the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols having a group -CH 2 N 2 as the 6 'moiety may be N-protected at this position, since this group is the most reactive. Sisomicin may be protected at position 6 'or at positions 2' and 6 'or at positions 21, 3 and 6'. Other amino protecting groups may be bridging groups at positions 3 ", 4", such as carbonyl, in the 4,6-di- (aminoglycosyl) -1,3-diaminocyclic tolerances having 3 "amino and 4" -hydroxy group in mutual cis position. Thus, for example, use as a starting compound a 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol wherein the amino group at the 6'-carbon atom is N-protected (e.g., 6'-Nt-butoxycarbo Nylsisomicin) or a 1-N-unsubstituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol / wherein the amino groups at C-2 'and C-3 are N-protected (eg 2', 3 -di-N-trifluoro-10-acetylgentamicin C ^) and the corresponding partially N-protected 1-N-substituted derivative (e.g.

1N-ethyl-6'-Nt-butoxycarbonylsisomicin and 1-N-ethyl-2 ', 3-di-N-trifluoroacetylgentamicin (C), which after removal of the N-protecting groups, in a manner known per se, The NO 2 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 similar or identical to those set forth below in Preparation 3. As used herein, the terms "blocking group" or "protecting group" refer to groups which render the blocked or protected amino groups inert to subsequent chemical manipulation, but which can be readily removed after performing the chemical manipulation. 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 Butoxycarbonyl and 2-iodoethoxycarbonyl, and arylalkoxycarbonyl groups such as carbobenzyloxy and 4-methoxybenzyloxycarbonyl groups.

In the blocking process, the protecting group is usually used in the form of an acidic imidazole derivative and acid azide or as active esters such as ethylthiol trifluoroacetate, N-benzyloxycarbonyloxysuccinimide or p-nitrophenyltrichloroethyl arbonate. Blocking groups can thus be described as being derived from a compound BgLg where Bg becomes the blocking group such as the acid moiety of an active ester and Lg is a leaving group such as imidazole.

Alternatively, the process described above may be carried out in a manner which allows formation of a 1-N (Schiff base) derivative, after which the Schiff base thus obtained is reduced. N-substituted derivatives of the aforementioned 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols 10 assume that the N, C double bond in a Ι-Ν ^ ϊ ^ Χ'-substituted derivative of a corresponding 4 , 6-di- (aminoglycosyl) - 1,3-diaminocyclitol, wherein all groups of NH 2 are protected and groups of NHCH 2 may be protected, wherein X 'is a group as defined for X above, wherein any present amino or hydroxy group may be protected, whereupon all protecting groups present in the molecule are removed in a manner known per se, after which the desired derivative is isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

2Q The 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols wherein the 6-aminoglycosyl group is garosaminyl usually has a 3 "-N-4" -O protecting group identical to the 1-N substituent in the starting material because the oxazoli diene ring is formed simultaneously with the 1-N (Schiffsk base) group. Thus, e.g. 21,3-di-N-trifluoroacetyl-gentamicin by reaction with an aldehyde (e.g., benz-aldehyde, phenylacetaldehyde or acetaldehyde) to the corresponding 3 ", 4" oxazolidin-1-ylidene (Schiff base) starting material for this process (e.g., 1-N, 3 ", 4" -N, G-dibenzylidene-2 ', 3-di-N-trifluoroacetylgentamicin C ^, 1-N, 3 ", 4" -N, O-diphenethylidene-2 ', 3-di-N-trifluoroacetylgentamicin Cog 1-N, 3 ", 4" -N, O-diethylidene-21,3-di-N-trifluoroacetylgentamicin (C sodium borohydride and methanolic sodium methoxide give the corresponding 1N-CH2X-3 ", 4" oxazolidine (e.g., 1N-benzyl-3 ", 4" -N, O-benzylidene gentamicin, 1N-phenethyl-3 " , 4 "-N, O-phenethylidene-gentamicin and 1N-ethyl-3", 4 "-N, 0-ethyl-idengentamicin (C)), which, upon treatment with acid, gives a 17 146298 (e.g., 1-N-benzyl-gentamicin C C., 1-N-phenethylgentamicin, and 1-N-ethyl-gentamicin C ^).

The novel 1-N-substituted derivatives of the 5,6-di (aminoglycosyl) -1,3-diaminocyclitols as defined by formulas I, III, IV and V may also be prepared by a process (the present process c) , characterized in that a 1-N-substituted derivative of a corresponding 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitol, wherein one or more amino groups may be

II

protected, and the 1-N substituent is -CX ", where X" represents hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylamino-hydroxyalkyl, phenyl, benzyl or hydrocarbyloxy, which aliphatic groups have up to 7 carbon atoms and, if stubstituted with both amino and hydroxy, carry the substituents on various carbon atoms, and wherein any amino or hydroxy group present may be protected, treated with an amide-reducing hydride reagent, all of which are present in the molecule. protecting groups are removed in a manner known per se, after which the desired derivative is isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

The process is usually carried out in a non-reactive organic solvent, thinking of a solvent in which the starting compounds and the amide-reducing reagent are soluble and which do not react with said reagent, so that there is a minimum of competing side reactions. Non-reactive organic solvents which are very suitable for the reduction process are, for example, ethers such as dioxane, tetrahydrofuran and diethylene glycol dimethyl ether.

Preferred amide reducing hydride reagents are 35 aluminum hydrides and boron hydrides, including lithium aluminum hydride, lithium trimethoxy aluminum hydride, aluminum hydride, diborane, diisoamylborane and 9-borabicyclo [3,3,1> nonane.

C .: v 146298 18

It is generally preferred to use your borane as the amide reducing agent, except when the starting compound has a double bond such as e.g. in 1-N-acylsisomicin, 1-N-acylverdamiein, 1-N-acyl-Antibiotic 66-40B, 1-Nracyl-? Antibiotic, 66-40D and 1-N-acyl - Antibiotic G-52, which compounds are suitably reduced · by lithium aluminum hydride.

. When X "represents hydrocarbyloxy, such as t-butoxy,

'. and the amide is subjected to reduction, the corresponding 10-N-methyl compound is formed. Q

In this process, where a 1NCX "-4,6-di- (aminoglycosyl) ri, 3-diaminocyclitol is reduced to the corresponding 1NC3 X derivative, if the acyl side chain of the -1-N-acy intermediate has a chiral center, the individual stereoisomers are used separately or in a mixture thereof, and the corresponding diastereoisomers or a mixture thereof are widely obtained.

A process (the present process -: - d) for the preparation of the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclito-7; - wherein the eubstituent is straight-chain alkyl of up to 5 C atoms. consists of: a corresponding 4,6-di- (aminogly-eosyl) -1,3-diaminocyclytol having amino-protecting groups at any amino group other than that of the 1-position, and wherein the 1-amino group may be activated in a manner known per se, with an alkylating agent containing the straight-chain alkyl group of up to 5 C atoms and a leaving group upon which protecting groups present in the molecule and, if required, present * activating group or groups are removed in a manner known per se, and then the derivative is isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

. Examples of alkylating agents which, with advantage 35, can be used in this process are alkyl iodide, alkyl bromide, dialkyl sulfate, alkyl fluorosulfonate and alkyl p-foluenesulfonate, wherein the alkyl group is the requisite 1-chain alkyl group having up to 5 carbon atoms. Other alkylating agents in which the alkyl group preferably has 1 or 2 carbon atoms are trialkylanilinium hydroxide, trialkyloxonium fluorborate, trialkylsulfonium fluorborate or trialkylsulfoxonium fluorborate. All of these alkylating agents contain a group which is readily removed, such as Br, I, OSC, F, dialkylaniline or dialkyl ether.

The amino group at position 1 of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol may be free or activated in a manner known per se. An example of an activating group is trifluoromethylsulfonyl. These activating groups can be introduced into the molecule by reaction of a 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol having amino protecting groups at any position other than the 1-position, e.g. 3 ", 4" -N, O-carbonyl-2 ', 3,6'-tri-N-t-butoxy-carbonyl isomicin, with a compound giving the activating group such as trifluoromethylsulfonyl chloride.

The 1-amino group may also be alkylated via the corresponding di- (2-cyanoethyl) derivative derived by treating 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, which has amino protecting groups at any other amino group than that in the 1 position, with acrylonitrile. The 1-N-di- (2-cyanoethyl) derivative thus prepared is then alkylated with one of the alkylating agents listed above, followed by removal of the cyanoethyl groups.

Said process according to the invention is practiced under conditions similar to those used in the well known direct alkylation methods for amines.

Yet another method (the present process e) for preparing the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-di-aminocyclitols wherein the substituent is methyl wherein a corresponding 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol having amino protecting groups at any amino group other than that at the 1 position is reacted with formaldehyde and a cyclic imide, preferably succinimide, and the compound thus obtained is treated with a hydride-donor reducing agent, preferably sodium borohydride, and all protecting groups present in the molecule are removed in a manner known per se, after which the derivative is isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

Yet another method (present method f) for preparing the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclytols, wherein the substituent is methyl, consists in the fact that a corresponding 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol having amino protecting groups at any amino group other than that at the 1 position is reacted with formaldehyde in the presence of formic acid and all present in the molecule protecting groups are removed in a manner known per se, after which the derivative is isolated as such or in the form of a pharmaceutically acceptable acid addition salt.

The formation of the 1-N-methyl substituent with formaldehyde and formic acid is well known as the Eschweiler-Clarke reaction.

Of the above processes, the process (a) useful for the preparation of all the compounds of the present invention is particularly suitable as it uses readily available starting materials, proceeds readily, and yields good yields. Therefore, it is preferred according to the invention that method a) be used.

Furthermore, it is preferred according to the invention that the particularly active 1-N-ethylisomicin be prepared which is isolated as such or as a pharmaceutically acceptable acid addition salt.

In general, the dose administered depends on the compounds of the age and weight of the species of animal being treated, the mode of administration, and the type and severity of the bacterial infection to be avoided or reduced. In general, the dose of the subject derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols will correspond to the dose requirements of the corresponding 1-N unsubstituted 4,6-di- (aminoglycosyl) -1. 3-diaminocyclitoler.

The compounds of this invention may be administered orally. They may also be used locally in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions which may be aqueous / non-aqueous or of the emulsion type or in the form of creams. Pharmaceutical carriers useful in the preparation of such compositions will include, e.g. such substances as water, oils, fats, polyesters and polyols.

For oral administration, the subject compounds may be in the form of e.g. tablets, capsules or elixirs, or they can be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective in treating bacterial infections of the gastrointestinal tract, which infections cause diarrhea.

In general, the compositions for topical use contain from ca. 0.1 to approx. 3.0 g of the subject compounds per 100 g ointment, cream or lotion. The preparations for topical application are usually applied gently to lesions of approx. 2 to approx. 5 times a day.

The present antibacterial agents may be used in liquid form such as solutions or suspensions, for use in the ears and eyes and may also be administered parenterally by intramuscular injection. The injectable solution or suspension will usually be administered with from ca. 1 mg to approx. 15 mg antibacterial agent per kg. body weight per day divided into approx. 2 to approx. 4 doses.

The exact dose depends on the stage and strength of the infection, the sensitivity of the infected organism to the antibacterial substance and the individual characteristics of the species of animal being treated.

The following preparations exemplify the preparation of many of the required starting materials, 3Q and the following examples further detail the process of the invention.

Preparation 1 Gentamicin C2a 35 Separation of gentamicin C2a from co-produced antibiotics.

226,6298 96 g of gentamicin base (prepared from the sulfate salt obtained by the procedure of Example 4 of U.S. Patent 3,091,572) are dissolved in 400 ml of the upper phase formed when methanol, chloroform and 17% am -5 monium hydroxide is mixed in the volume ratio of 1: 2: 1. One tenth of the solution is added to each of the first ten tubes in a 500x80 ml tube countercurrent tractor. All the tubes, including the first ten, are filled to their capacity with the lower phase of the solvent mixture described above. The solvent reservoir is added to give 40 ml of the upper phase to tube # 1 for each transfer. The device is set to 500 transfers. Once the transfers have been completed, each eighth tube is chromatographically tested (double-determined) on Schleicher and Schuell 15 Paper No. 589 using the lower phase of the solvent mixture described above.

The chromatograms are allowed to develop in approx. 16 hours, after which the papers are dried. One piece of paper is placed on an agar plate seeded with Staphylococcus aureus (A.T.

20 C.C. 6538P), the second paper is sprayed with the usual ninhydrin solution and heated to develop. The agar plate is incubated overnight at 37 ° C and the solution is combined from tubes containing the material which migrates like gentamicin C pipe No. 290-360.

25 Pipes Nos 290-360 are replaced with fresh tubes containing 40 ml of the upper phase and 40 ml of the lower phase. The apparatus is set to a further 2800 transfers and the chromatographic procedure described above is repeated. The contents of tubes # 1-16 are combined and concentrated in vacuo to give 1.3 g of gentamicin C2a with the following properties: (a) A molecular weight of 463 as determined by mass spectrometry, which is in accordance with an empirical formula Ο_ηΗ. (B) a specific optical rotation measured at the D-line of sodium at 26 ° C at + 114 ° - 5 ° in water at a concentration of 0.3%, and (c) ) a proton magnetic resonance spectrum (pmr) as follows: pmr (ppm) (D20): 50.99 (3H, d, J = 6.5 Hz, CH-CH3), CH-CH3), 1.17 (3H , S, C-CH 3), 2.47 (3H, s, N-CH 3), 2.51 (1H, d, J = 10.5 Hz, H-3 "), 3.75 (1H, q, J = 10.5, 4 Hz, H-2 "), 4.00 (1H, d, J = 12 Hz, H-5" 5 equiv), 5.04 (1H, d, J = 4 Hz, H1), 5.13 (1H, d, J = 3.5 Hz, H-1 ').

Irradiation of the secondary methyl group at 60.99 ppm shows H-6 'as a doublet (J = 6.5 Hz) at 62.81 ppm.

10

Preparation 2 Gentamicin

Separation of gentamicin from co-produced antibiotics.

The major components of gentamicin C (C-, C₂, and C ^a) are separated as described in U.S. Pat. patent specification no.

3,651,042, Example 2, and the fractions containing mainly overlaps of free base gentamicins C C and C₂ (500g of gentamicin C mixture gives 53.4g of 20 overlaps) are pooled. 1.5 g of this gentamicin C1 and C2 mixture are added to a column containing 50 g of silica gel produced in a solvent system comprising chloroform: methanol: 15% 1s ammonium hydroxide (1: 2: 1).

The column is eluted with the same solvent system and the 25 eluted fractions are examined by thin-layer chromatography on silica gel plates using the solvent system chloroform: methanol: 22% ammonium hydroxide (1: 2: 1) as the developer. The fractions containing a mixture of gentamicins and C2 together with gentamicin 30 C2b (cargo; ions 39-57 [410 mg]) are combined. Fractions 39-57 are again chromatographed over silica gel using the solvent system chloroform: methanol: 7% ammonium hydroxide (1: 2: 1) and fractions (98-130) containing pure gentamicin C2tj as determined by thin layer chromatography are combined (yield 45 mg) and n have the following constants [dip + 165.5 (c = 0.3%, H 2 O), mass spectrum m / e 463 (M + 1), 446, 445, 433, 350, 332, 322, 304, 333, 305, 287, 191, 173, 163, 145, 160, 142, 24, 146 pm (ppm) (D 2 O): 51.25 (3H, s, C-CH 3H, s, N-CH 3), 2.55 (3H, s, N-CH 3), 5.12 (1H, d, J = 4 Hz, H1 "), 5.22 (1H, d, J = 3 Hz, H-1 ').

Pure gentamicin can be distinguished from gentamicin 5 and C3 by its motility by thin layer chromatography using silica gel plates and a solvent system of chloroform: methanol: 22% ammonium hydroxide (1: 2: 1) as the developer. The approximate R ^ values in this system are as follows: 10 gentamicin 0.47 gentamicin C2 0.47 gentamicin 0.35

Preparation 3 Selectively blocked 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitols.

A. 2 ', 3,61-Tri-N-t-butoxycarbonyl-3 ", 4" -N, O-carbonylsisomycin 1. Penta-N-carbobenzoxysomycin.

20 g of Sisomicin and 13 g of sodium carbonate are dissolved in 625 ml of distilled water. Carbobenz oxychloride (100 ml) is added to the stirred solution at 25 ° C and the mixture is stirred for 16 hours. The solid is filtered off, washed thoroughly with water, dried in vacuo and then washed with hexane to give penta-N-carbobenzoxysomycin (62 g) as a colorless amorphous solid. Mp: 165-173 ° C, [<x] J6: + 96.2 ° (CH-.0H). IR: (CHCl1) 3400,

U J 1 IuciX J

1720, 1515, 1215, 1050, 695 cm -1.

NMR: δ (CDCl3) 1.03 (3H, broad singlet, 4 "-C-CH3), 3.02 (3H, broad singlet, 3" -NCH3), 5.02 (10H, broad singlet, CH2CgHg) , 3.28, 3.30 ppm (25H, broad singlet, -CH2C6H5> · 2. Tetra-N-carbobenzoxy-3,1,4,4-N, O-carbonyl-sisomicin.

Dissolve 5 g of Penta-N-carbobenzoxy-isomicin in 50 ml of dimethylformamide, add 250 mg of sodium hydride to the stirred solution and stir the reaction under argon at room temperature for 2 hours. Filter, and to the filtrate is added glacial acetic acid (2 ml) and then concentrated in vacuo. The residue is extracted with chloroform (200 ml), which has already passed through basic alumina), the extract is washed with water and dried over 5 sodium sulfate. The solution is evaporated to give tetra-N-carbobenzoxy-3 ", 4" -N, O-carbonyl-sisomicin as an amorphous powder (3.5 g).

Mp: 210-213 ° C, [α] δ 6: + 68.8 (c = 0.22) IR ·· (nujol) 3550, 1840, 1760, 1580 cm ”1 max 10 NMR: 6 (CDCl13) 1.34 (3H, singlet, 4 "-CH 3), 2.68 (3H, singlet, 3" -N-Me), 5.04 (8H, broad singlet, -Cl 2 CgHg).

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

To a solution of 1,3,2 ', 6, -tetra-N-benzyloxy-carbonyl-3 ", 4"' N-, O-carbonyl-sisomicin (10.1 g) in tetrahydrofuran (200 ml ) 1 liter of liquid ammonia (redistilled from sodium) is added. Add 6 g of sodium to the stirred solution in small pieces. After 3 hours of stirring, the excess sodium is destroyed by the addition of ammonium chloride. The solvents are allowed to evaporate under a stream of nitrogen. The residue is dissolved in water and passed through a medium of "Amberlite" ® IRC-50 resin (H-form), and the resin is washed well with water and the product is eluted with 2N ammonium hydroxide solution. The ammonium eluate is evaporated in vacuo to give the title compound (3 ", 4" -N, O-carbonyl-sisomicin). Yield approx.

4 g. IR: v (nujol) 1745 cm ”1. The product was used in the subsequent steps without further purification. However, a very pure sample can be obtained by chromatographing the product over silica gel using the lower phase of a chloroform: methanol: concentrated ammonium hydroxide solvent system (1: 1: 1) as the eluent.

4. 2 ', 3,6'-Tri-N-t-butoxycarbonyl-3 ", 4" -N, O-carbonyl-isomicin.

Dissolve 3 ", 4" N, O-carbonyl-sisomicin (1.4 g, 3 mmol) in 10 ml of 50% aqueous methanol containing triethylamine (3.5 mmol). T-Butoxycarbonylazide ( 3.5 mmol (26 146298) is added dropwise with stirring. The mixture is stirred for 2 days at room temperature. Add 5 ml of Amberlite® IRA-401S (OH -) ion exchange resin with 5 ml of methanol and stir for ½ hour. The resin is removed by filtration and washed with methanol. The filtrate is concentrated and the residue is chromatographed 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. The homogeneous fractions containing the title compound are collected and the solvent removed by evaporation in vacuo. The residue is dissolved in methanol and the excess ether is precipitated. The solid product is isolated by filtration and dried.

B. 2 ', 3-Di-N-trifluoroacetyl-gentamicin C

1. 2'-N-trifluoroacetyl-gentamicin

Dissolve 1.7 g of Gentamicin C in 20 ml of methanol, cool the mixture to 4 ° C and add 0.46 ml (0.563 g) of ethylthiol trifluoroacetate with stirring. The reaction is allowed to continue for 2 hours and the solution 20 is concentrated to a residue in vacuum. The product is chromatographed on 80 g of silica gel using the lower phase of a mixture of chloroform: methanol: water: ammonium hydroxide in volume ratio of 10: 5: 4: 1 as eluent. The fractions containing the main component are combined and concentrated to give 1.4 g of the title compound, mp: 108-111 ° C, [α] 6: + 128 ° C

(c = 0.3%, H 2 O). Analysis for C23H42N5 ° 8F3, H2O:

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. 21,3-Di-N-trifluoroacetyl-gentamicin C

0.66 g of the product of step 1 is dissolved in 10 ml of methanol, the mixture is cooled to 4 ° C and 0.148 ml (0.182 g) of ethyl thiol trifluoroacetate dissolved in 3 ml of methanol is added. The reaction mixture is stirred for approx. 16 hours and concentrated to a residue in vacuo. The product is chromatographed on 30 g of silica gel as described in Step 1. The column is examined by thin layer chromatography, the appropriate fractions are combined and concentrated to give 0.32 g of the title compound, mp: 121-129 ° C, [a] Δ 6: 121 ° (c = 0.3%, H 2 O). Analysis for C 25 H 4 N 5 O 9 F 6: Calculated: C = 44.84%, H = 6.17%, N = 10.46%.

Found: C = 44.94%, H = 6.35%, N = 10.17%.

Example 1 1-N-substituted sisomicin.

10 A. 1-N-ethylsisomicin.

To a solution of 5 g of sisomicin in 250 ml of water is added IN sulfuric acid until the pH of the solution is adjusted to approx. 5. To the resulting solution of siso-micin sulfuric acid addition salt is added 2 ml of acetaldehyde, stirred for 10 minutes, then 0.85 g of sodium cyanoborohydride is added. Stirring is continued at room temperature for 15 minutes, after which the solution is concentrated in vacuo to a volume of approx. 100 ml, the solution is treated with a basic ion exchange resin (e.g., "Amber-2Q lite" ® IRA 401S (OH)) and then lyophilized to a residue comprising 1-N-ethyl-sisomicin.

Purify by chromatography on 200 g of silica gel, eluting with the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) -25 system. Uniform eluates, as determined by thin layer chromatography, are combined and the combined eluates of the main component concentrated in vacuo to a residue comprising 1-N-ethylsisomicin (yield 1.25 g). Further purify by again chromatographing on 100 g of silica gel eluting with a chloroform: methanol: 3.5% ammonium hydroxide (1: 2: 1) system. The combined uniform eluates (as determined by thin layer chromatography) are passed through a column of basic ion exchange resin and the eluate lyophilized to give 1-N-ethyl isomicin 35 (yield 0.54 g), [α] 3%, H 2 O), pmr (ppm) (D 2 O): 61.05 (3H, t, J = 7 Hz, -CH2 CH3), 1.19 (3H, s, -C-CH3), 2.5 (3H , s, N-CH 3), 4.85 (1H, m, = CH-), 4.95 28 148298 (1H, d, J = 4Hz, H1 "), 5.33 (1H, d, J = 2 Mass spectrum: (M + 1) + m / e 476 also m / e 127, 154, 160, 173, 191, 201, 219, 256, 299, 317, 332, 345, 350, 360, 378, 390, 5,400.

B. 1-N-methylsisomicin.

To a solution of 4.64 g of sisomicin in 180 ml of water is added IN sulfuric acid until the solution has a pH of approx. 5. Add 1.2 ml of 37% aqueous formaldehyde, stir for 10 minutes, then add 460 mg of sodium cyanoborohydride. The reaction solution is passed through a column of a basic ion exchange resin (e.g., "Amber-lite" ® IRA 401S (OH form)) and lyophilized. The resulting residue is chromatographed on silica gel in the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) solvent mixture. Uniform eluates containing essentially 1-N-methylsisomicin as determined by thin layer chromatography are combined.

Evaporate in vacuo to a residue of 1-N-methylsisomycin.

[α] p 6 + 153 ° (0.3%, H 2 O).

Mass spectrum: (M + 1) + m / e 462 also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 2 5 386.

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

In a similar manner to that described in Example 1A, the sulfuric acid addition salt of sisomicin in water 30 is treated with propanal and sodium cyanoborohydride. The resulting product is isolated and purified in a manner similar to that described to give 1-N- (n-propyl) sisomicin.

[α] δ 6 + 140 ° (0.3%, H 2 O), pmr (ppm) (D 2 O): 60.83 (3H, t, J = 7 Hz, 35 -CH 2 CH 3), 1.14 (3H, s , -C-CH 3), 2.45 (3H, s, -N-CH 2), 4.82 (1H, m, = CH-), 4.90 (1H, d, J = 4 Hz, H ), 5.78 (1H, d, J = 2 Hz, H1 ').

4 *

Mass spectrum: (M + 1) m / e 490 29 146298 also 127, 160, 168, 187, 205, 215, 233, 256, 313, 331, 346, 359, 364, 374, 404, 414.

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

To a solution of 3 g of sisomicin in 200 ml of water 5 is added IN sulfuric acid until the solution has a pH of approx.

3.5. Add 1.5 ml of n-butanal, stir for 10 minutes, then add 450 mg of sodium cyanoborohydride. Stirring is continued for 1 hour, then the solution is concentrated in vacuo to a volume of approx. 100 ml. This solution is passed through a column of a basic ion exchange resin (e.g., "Amberlite" ® IRA 401S (OH)) and lyophilized. The resulting residue is chromatographed on 140 g silica gel in the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) solvent mixture. Uniform fractions containing 1-N- (n-butyl) sisomicin as determined by thin layer chromatography are combined and the combined eluates are evaporated in vacuo to a residue comprising 1-N- (n-butyl) sisomicin.

[a] 26 + 129 ° (0.3%, H 2 O), pmr (ppm) (D 2 G) 60.82 (3H, t, 20 J = 7 Hz, -CH 2 CH 3), 1.15 (3H, s, C -CH3), 2.46 (3H, s, -N-CH3), 4.82 (1H, m, = CH-), 4.92 (1H, d, J = 4 Hz, H , 29 (1H, d, J = 2 Hz, H ').

^ 4-

Mass spectrum: (M + 1) m / e 504, also m / e 127, 160, 182, 201, 219, 25 229, 247, 256, 327, 345, 360, 373, 388, 418, 428.

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

In the procedure of Example 1A, acetaldehyde is replaced by equivalent amounts of each of the following 3Q aldehydes: 1. 2-ethylbutanal, 2. propenal.

In each case, the reaction is carried out in a manner similar to that described in Example 1A, and the resulting products are isolated and purified in a manner similar to that described to give: 1. 1-N- (β-ethylbutyl) sisomicin , [a] 26 + 121 ° (c = 0.4%, H 2 O), pmr (ppm) (D 2 O): 60.75 (6H, 146298 t, 6.5 Hz, CH 2 -CH 3), 2.4 (3H, s, N-CH 3), 4.78 (1H, m, = CH-), 4.88 (1H, d, 4.0 Hz, H 2 '), 5.22 (1H, d, 2 , 0 Hz, H 2).

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- (β-propenyl) sisomicin, [α] + 147 ° (0.4, CH 3 OH), nmr (D 2 O) 61.18 (s, 3H, C-CH 3), 2.48 (s, 3H) , N-CH 3), 3.75 (1H, dd, J = 4.11 Hz, H-2 "), 3.95 (1H, d, J = 13 Hz, H-5e), 4.84 (1H , m, H-4 '), 4.94 (1H, d, J = 4 Hz, H1 "), 5.30-5.0 (3H, m, H1 *, = CH2), 5.8 ( 1H, m, -CN = CH 2), MS m / e 488 (M).

15 F. 1-N- (6-Aminobuty1) sisomicin.

To a solution of 3 g of sisomicin in 120 ml of water is added dropwise IN sulfuric acid until the pH of the solution is adjusted to approx. 5. To the aqueous solution of the 2q sulfuric acid addition salt formed by sisomicin is added 60 ml of dimethylformamide followed by a solution of 2 g of 4-phthalimidobutanal in 10 ml of dimethylformamide. Stirring is continued for 10 minutes, then 420 mg of sodium cyanoborohydride is added. After approx. For 20 minutes, the reaction solution is added to 1 liter of anhydrous methanol with stirring and by filtration the resulting precipitate comprising the sulfuric acid addition salt of 1-N- (δ-phthalimidobutyl) sisomicin is collected.

It is purified by dissolving the precipitate in water and passing through the aqueous solution through a basic ion exchange resin. Evaporate in vacuo to a residue, chromatograph the residue over silica gel eluting with the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) solvent mixture and evaporate the 33 combined, uniform eluates to a residue comprising 1-N- (δ-phthalimidobutyl) sisomicin.

To 0.5 g of 1-N- (δ-phthalimidobutyl) sisomicin is added 5 ml of 5% ethanolic hydrazine hydrate and refluxed for 3 hours. The reaction solution is poured into a large volume of tetrahydrofuran and the resulting precipitate comprising 1-N- (δ-aminobutyl) sisomicin is collected by filtration.

The compound of this example may alternatively be prepared as follows: (1) 4-Acetamidobutyraldehyde.

Dissolve 5 g of 4-Acetamidobutyraldehyde diethyl acetal in 75 ml of distilled water and 5 ml of 1N sulfuric acid. The solution is allowed to stand at room temperature until the hydrolysis is completely determined by thin layer chromatography. The solution is neutralized with sodium bicarbonate, then the solution is saturated with sodium chloride and extracted with chloroform. The combined chloroform extracts are distilled to a residue comprising 4-acetamidobutyraldehyde which can be used without further purification in the subsequent procedure.

(2) 1-N- (δ-Acetamidobutyl) sisomicin.

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

(3) 1-N- (δ-Aminobutyl) sisomicin.

The 1-N- (δ-acetamidobutyl) sisomicin obtained in the previous example is treated with 10% aqueous sodium hydroxide at 100 ° C for 3 hours, then neutralized with "Amberlite" ® IRC-50 ion exchange resin and eluent. With 2N aqueous ammonium hydroxide. The eluted is concentrated and the resulting residue is dissolved in water and lyophilized to give 1-N- (δ-aminobutyl) sisomycin.

[Α] 20 D + 109 ° (c = 0.3%, H 2 O).

Mass spectrum: (M + 1) + m / e 519 also 127, 160, 197, 216, 234, 244, 256, 262, 342, 360, 375, 388, 393, 403, 443.

G. 1-N- (β-Aminoethyl) sisomicin and 1-N- (γ-aminopropyl) - 10 sisomicin.

In a similar manner to the alternative procedure described in Example 1F, an aqueous solution of sisomicin to which 0.1N sulfuric acid is added is added until the pH of the solution is approx. 5, with β-acetamidoacetal-X 5 dehyde followed by solid sodium cyanoborohydride. The resulting product is isolated and purified in a manner similar to that described to give 1-N- (β-acetamido-ethyl) sisomicin.

2Q 1-N- (β-acetamidoethyl) sisomicin is hydrolyzed with 10% aqueous potassium hydroxide, and the resulting product is isolated and purified in a similar manner to that described in section (3) of the alternative procedure of Example 1F to give of 1-N- (β-aminoethyl) sisomicin.

Mass spectrum: (M + 1) + m / e 491, 25 also 160, 169, 187, 206, 216, 234, 256, 283, 314, 325, 334, 347, 360, 370, 375, 405, 415.

In the above method, by substituting β-acetamidoaldehyde with γ-acetamidopropanal 1-N- (γ-acetamidopropyl) sisomicin, which, when hydrolyzed with 10% aqueous potassium hydroxide, is isolated and purified in the manner described, yields 1-N- (γ-aminopropyl) sisomicin.

[≤ x] p 6 + 127 ° (H 2 O), 35 nmr (D 2 O): S 1.2 (3H, s, -C-CH 3), 2.5 (3H, s, N-CH 3), 3.8 ( 1H, dd, J = 4, 10.5 Hz, H2 "), 4.0 (1H, d, J = 12.5Hz, H5" e), 4.85 (1H, m, -OCH-), 4 , 95 (1H, d, J = 4Hz, H1 "), 5.34 (1H, d, J = 2.5Hz, δ).

* · +

Mass spectrum: Μ + 1 m / e 504,> also 160, 202, 220, 230, 248, 256, 328, 346, 361, 374, 379, 389, 407, 419 and 429.

Example 2 1-N-ethylgentamicin Cla.

To 5 g of gentamicin C 2 a in 125 ml of water is added IN sulfuric acid until the pH of the solution is approx. 5.2. Then 2 ml of acetaldehyde is added. The solution is stirred for 5 minutes, then 1 g of sodium cyanoborohydride is added. Stirring is continued at room temperature for 30 minutes, the solution is concentrated in vacuo to a volume of approx.

75 ml, the solution is passed through a column of a basic ion exchange resin (e.g., "Amberlite" ® IRA. 401S (OH)), whereupon lyophilized to a residue comprising 1-N-ethylgentamicin c 2 a.

Purify by chromatography on 200 g of silica gel and elute with the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) system.

Uniform eluates as determined by thin layer chromatography are combined and the combined eluates of the major component are concentrated in vacuo to a residue comprising 1-N-ethylgentamicin C 2 a (yield 0.95 g). Further purification by repeated chromatography of 1-N-ethylgentamycin Cla on 100 g of silica gel and elution with chloroform: methanol: 3.5% ammonium hydroxide (1: 2: 1) system. The combined uniform eluates (as determined by thin layer chromatography) are treated with a basic ion exchange resin and the eluate is lyophilized to give 1-N-ethyl-gentamicin C 2 a (0.42 g), [α] p 6 + 118 ° (c = 0.3%, H 2 <D), pmr (ppm) (D 2 O): δ 1.06 (3H, t, J = 7Hz, -CH 2 CH 3), 1.19 (3 H, s, -C-CH 3), 2 , 51 (3H, s, -N-CH 3), 4.97 (1H, d, J = 4Hz, H +), 5.16 (1H, d, J = 3.5Hz, H +).

Mass spectrum: (M + 1) + m / e 478, 35 also m / e 129, 154, 160, 173, 191, 201, 219, 258, 301, 317, 319, 329, 332, 347, 350, 360, 378 and 402.

34

Example 3 1-N-substituted verdamicin.

A. 1-N-ethylverdamicin.

To 0.5 g of verdamicin in 65 ml of water is added IN sulfuric acid until the pH of the solution is adjusted to approx. 4.9, then 0.2 ml of acetaldehyde is added. The solution is stirred for 5 minutes, with 65 mg of sodium cyanoborohydride added, the solution concentrated in vacuo to a volume of approx. 10 ml and the solution is poured into 50 ml of methanol 1Q with stirring. By filtration, the resulting precipitate comprising 1-N-ethylverdamicin is collected. Purify by chromatography on 100 g of silica gel eluting with a chloroform: methanol: 3.5% ammonium hydroxide (1: 2: 1) system. Uniform fractions as determined by thin layer chromatography are collected and the combined fractions containing the major component are evaporated in vacuo to a residue comprising 1-N-ethylverdamicin. Further, purify by repeated chromatography on 7 g of silica gel eluting with the lower phase of a chloroform: methanol: 2Q 7% ammonium hydroxide (2: 1: 1) system. The uniform eluates are combined and evaporated in vacuo to a residue of 1-N-ethylverdamicin (yield 50 mg).

f.

Mass Spectrum: (M + 1) m / e 490, also m / e 141, 154, 160, 173, 191, 25 201, 219, 270, 313, 317 (w), 331, 332, 341, 350 (w) , 357, 359, 360, 378, 390, 414.

B. In the procedure of Example 3A, acetaldehyde is replaced by propanal and butanal ·. Each of the resultant products is isolated and purified in a similar manner to obtain 3 (1S of 1-N- (n-propyl) verdamicin, [α] 3δ + 122 ° (c = 0.3%, H 2 O), respectively. pmr (ppm) (D2 <D): δ 0.88 (3H, t, J = 7Hz, CH2 CH3), 1.19 (3H, s, C-CH3), 1.16 (3H, d, J = 6Hz , CH-CH 3), 4.81 (1H, m, = CH-), 4.97 (1H, d, J = 4.0Hz, H "), 5.30 (1H, d, J = 2.0Hz , = H.1).

35 x + x

Mass spectrum: (M + 1) m / e 528, also m / e 141, 160, 168, 187, 205, 146298 35 215, 233, 270, 346, 355, 373, 504, and Ι-N- (n- butyl) verdamicin, [α] δ + 121 ° (c = 0.3%, Η 2 <0), pmr (ppm) (D 2 O): δ 0.8 (3H, t, J = 6.5Hz, CH CH3), 2.45 (3H, s, NCH3), 4.8 (1H, m, C = CH-), 4.92 (1H, d, J = 4.0 Hz, H1 "), 5, 25 (1H, d, J = 2.0Hz, H

Mass spectrum: (M + 1) + m / e 518 also m / 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

In a similar manner to that described in Example 1A, 5 g of gentamicin are treated in 250 ml of water with 1N sulfuric acid until the pH of the solution is about 5. Then the acidified solution is treated with acetaldehyde and sodium cyanoborohydride in a similar manner to that described, and the resulting product is isolated and purified to give 1-N-26-ethylgentamicin C ^, [α] D + 114 (c = 0 (Ppm) (D 2 O): δ 1.03 (3H, t, 7Hz, -CH 2 CH 3), 1.03 (3 H, d, J = 6.5 Hz, -CH-CH 3), 1.17 (3H, s, C ~CH3), 2.32 (3H, s, 6'N-CH3), 2.49 (3H, s, 3 "-NHCHCH), 4.94 (1H, d, J = 4Hz, H +), 5.13 (1H, d, J = 3.5Hz, H +).

Mass Spectrum: (M + 1) + m / e 506, also m / e 154, 157, 160, 173, 191, 201, 219, 286, 317, 329 (w), 347, 350, 360, 375, 430 .

B. In the procedure of Example 4A, another corresponding aldehyde reagent is used instead of acetaldehyde. Each of the resulting products is isolated and purified in a similar manner to obtain 1-N-methylgentamicin C ^, δ (D₂O) 1.04 (3H, d, J = 6.5Hz, 6'-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,, 2 "= 4Hz 'E ±">' and 5.13 ppm (1H, d, 1 # 2 * = 3'5Hz 'Hi') 'M + m / e 491 , 35 also 416, 384, 364, 361, 346, 343, 336, 333, 318, 315, 303, 286, 205, 187, 177, 160, 159, 157, 1-N- (β-hydroxyethyl) gentamicin C + 98.0 ° (c = 0.3%, H 2 O), pmr (ppm) (D 2 O): δ 0.99 (3H, d, J = 6.5).

Hz, 6 '"CH3), 1.13 (3H, s, 4" -CH3), 2.28 (3H, s, β'-ΝΟ +), 2.45 (3H, s, 3 "-NCH3) , 4.97 (1H, d, J = 4Hz, H +) and 5.11 (1H, d, J = 3.5Hz, H ").

Mass spectrum: (M + 1) m / e 522, also m / e 446, 404, 394, 391, 376, 373, 366, 363, 348, 345, 333, 286, 235, 217, 207, 189, 160 , 157, v (KBr) 3300, 1060 cm -1, and 1-N- (phenyl max max ethyl) gentamicin C C, [α] D + 99.4 (c = 0.3%, ^ 0) , pmr (ppm) (030): δ 0.99 (3H, d, J = 6.5Hz, 6'-CH 3), 1.10 (3H, s, 4n-CH 3), 2.28 (3H, s, 6'-NCH 3), 2.43 (3H, s, 3 "- NCH 3), 4.88 (1H, d, J = 4Hz, H 2), 5.08 (1H, d, J = 3, ) And 7.33 (5H, s, -CgH5).

Mass spectrum: (M + 1) + m / e 582, also m / e 506, 464, 454, 451, 436, 15 433, 426, 423, 408, 405 (w), 393, 295, 286, 277, 267 , 249, 160, 156, (KBr) 3300, 1050, 1030 cm "1.

Example 5 1-N-Ethyl Antibiotic G-52.

Dissolve 875 mg of Antibiotic G-52 in 40 ml of distilled water and add IN sulfuric acid until the pH of the solution is adjusted to approx. 3.5. 0.7 ml of acetaldehyde is added, the reaction mixture is stirred for 10 minutes, then 100 mg of sodium cyanoborohydride is added. The reaction solution is assayed by thin layer chromatography, and when the antibiotic G-52 used as starting material is found to be completely reacted (i.e., about 10 minutes), the solution is concentrated in vacuo at ca. 35-40 ° C to a volume of approx. 10 ml. The concentrated solution is passed through a basic ion exchange resin, whereupon lyophilized to a residue comprising 1-N-ethyl-Antibiotic G-52. Purify by chromatography on a silica gel column eluting with the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (2: 1: 1) system. Uniform eluates as determined by thin layer chromatography are combined and the combined eluates of the major component are concentrated in vacuo to a residue comprising 1-N-Ethyl Antibiotic G-52 (yield 60 mg). The overlapping eluates of the preceding chromatography are further purified by chromatography on silica gel eluting with the lower phase of a chloroform: methanol: 7% aqueous ammonium hydroxide (1: 2: 1) system to give a further residue of 35 mg comprising 1-N-ethyl Antibiotic G-52. The combined residues of 1-N-ethyl Antibiotic G-52 are passed through a column of basic ion exchange resin (eg, "Amberlite" ® IRA 40 IS) and the eluate lyophilized to give 1-N-ethyl-Anti-10 biotic G-52 (yield 90 mg), [α] + + 122.1 ° (c = 0.3%, H 2 O), pmr (ppm) (D 2 O): δ 1.06 (3H, t, J = 6 , 5Hz, 1N-CH2 ~ CH3), 1.21 (3H, s, 4 "-C-CH3), 2.30 (3H, s, 3" -N-CH3), 2.50 (3H, s, 6'-N-CH 3), 4.94 (1H, m, H4 '), 4.97 (1H, d, J = 4.0Hz, H "), 5.34 (1H, d, J = 2, 5Hz, H,).

· * · -F. X

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 20 In a similar manner to that described in Example 1A, each of the following 4,6-diaminoglycosyl-1,3-diaminocyclitols is treated in water with sulfuric acid followed by acetaldehyde and sodium cyanoborohydride: 1. gentamicin B, 25 2 antibiotic JI-20B, 3. mutamicin 2, 4. mutamicin 6.

Each of the resulting products is isolated and purified in a similar manner to that described in Example 1A to give the corresponding 1-N-ethyl compound, i.e.: 1. 1-N-ethylgentamicin B, [a ] + 6 + 119.7 ° (c, in water)

Mass spectrum; m / e [MH] + 380, 352, 334, 378, 350, 35 332, 219, 191, nmr (60 MHz D 2 O): δ 5.55 (H-1 ',

J1 = 3'0Hz) 5'05 Ji "2" = 4Hz) in 2.9 (N-CH3), 1.05-1.5 (2C-CH3).

2. 1-N-Ethyl Antibiotic JI-20B, [α] ρ6 + 112.5 ° (H 2 O) nmr (D 2 O): δ 1.1 (3H, t, J = 7Hz, CH2 ~ CH3), 1.22 (3H, s, C-CH 3), 1.3 (3H, d, -CH-CH 3), 5.95 (1H, d, J = 4Hz, H 2 '), 5.35 (1H, J = 3.5Hz,

H ').

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,

10 m.p. 80-84 ° C

pmr (D 2 O): δ 1.06 (t, J = 7 Hz, 3.1 N-CH 2 CH 3), 1.19 (s, 3,4 "-CH 3), 2.50 (s, 3, 3" - N-CH3), 2.53 (d, J2 ", 3" = 10.5Hz, 1, H3 "), 3.75 (dd, J1n, 2n = 4Hz, J211.3" = 10.5Hz, 1, H2 "), 3.83 (d, H5" eq 5 "2x 12Hz, 1, Hg"), 4.86 (m, 1, H4 *), 4.98 (d, H2 ", 1" = 4Hz , 1, H 2 ') and 5.10 (d, J 1' 2 '= 2 / 5Hz, 1, H 2 /).

Mass spectrum: m / e 459, 384, 329, 316, 311, 301, 282, 203, 185, 175, 160, 142, 127.

4. 1-N-ethylmutamicin 6, m.p. 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 (δ) D20

5.14 d, J = 2.5Hz 1 * -H

5.00 d, J = 4.1Hz 1 "-H

4.9 wide singlet 4'-H

4.38 wide singlet 5-H

3.93 d, J = 12.5Hz 5 "e-H

3.78 q. 2 "-H

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

3.21 (1H) wide singlet 6'-H

2.65 d, J = 11.0Hz 3 "-H

2.52 singlet 3 "-N-CH 3 1.22 singlet 4'-0-0Η3 1.07 triplet 1-N-CH 2 -CH 3 cmr (D 2 O): ppm: δ 149.8, 102.9, 97, 4, 97.0, 83.9, 80.5, 73.2, 70.1, 69.6, 68, 63.9, 54.5, 47.1, 47.0, 43.1, 40, 8, 37.5, 33.0, 25.6, 22.4, 14.6.

Example 7

Preparation of 1-N-substituted 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols via selectively blocked intermediates.

1Q A. 1-N-ethylgentamicin via one 21,3-di-N-substituted intermediate.

Dissolve 240 mg of 21,3-di-N-trifluoroacetylgentamicin in 10 ml of water / methanol (2: 1) and adjust the pH of the solution to ca. 3.5 by addition of 1N sulfuric acid.

0.19 ml of acetaldehyde is added, stirred for 10 minutes, then 27 mg of sodium cyanoborohydride is added and the reaction is stirred for a further 10 minutes. The reaction mixture is evaporated in vacuo to a residue comprising 1-N-ethyl-2 ', 3-di-N-trifluoroacetylgentamicin C

2Q The residue is dissolved in 50 ml of concentrated ammonium hydroxide and the solution is allowed to stand at room temperature for 24 hours. The mixture is evaporated in vacuo and the resulting residue is chromatographed over silica gel (12 g) by eluting with the lower phase of a mixture of chloroform: methanol: 10% aqueous ammonium hydroxide (2: 1: 1). Uniform fractions (as determined by thin layer chromatography) are combined and the combined eluates are evaporated in vacuo to a residue comprising 1-N-ethylgentamicin (yield 80 mg) with the same characteristics as in Example 3Q 4a.

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

In a similar manner to that described in Example 7A, 6'-N-t-butoxycarbonyl isomicin in aqueous methanol is treated with sulfuric acid and then with acetaldehyde and sodium cyanoborohydride. The reaction mixture is allowed to stand at room temperature for 30 minutes and then evaporated in vacuo to give 1-N-ethyl-61-N-t-butoxycarbonylsilicin. The residue is dissolved in trifluoroacetic acid and the solution is allowed to stand for 10 minutes. Then anhydrous methanol is added to an excess, the resulting precipitate of the trifluoroacetic acid salt of 1-N-ethyl isomycin is filtered and chromatographed over silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system in a similar manner as the one described in Example 7A to give 1-N-ethylsisomicin with the same characteristics as given in Example 1A.

C. 1-N-Methylsisomicin from 3 ", 4" -N> O-Carbony1-siso-micin.

3 ", 4" -N, O-carbony1-sisomicin (5 g) in distilled water (300 ml) is treated with 1N sulfuric acid until the pH of the solution is 2.5. Aqueous formaldehyde (37% 1 s) (2 ml) is added and after 10 minutes a solution of sodium cyanoborohydride (500 mg) in water 20 (5 ml) is added dropwise. After 1 hour, the volume of the solution is reduced to half by evaporation in vacuo, the pH of the concentrated solution is adjusted to 11 by addition of 1N sodium hydroxide solution and the solution is evaporated to dryness. The residue is extracted with methanol (3 x 100 ml) and the combined methanol extracts are diluted with an equal volume of chloroform, filtered and evaporated to dryness to give crude 1-N-methyl-3 ", 4" -N, O-carbonylsisomicin.

The crude product is treated with 10% aqueous potassium hydroxide at 100 ° C for 5 hours. The cooled solution is passed down through an "Amberlite" ® IRC-50 (H +) ion exchange resin and eluted with 2N aqueous ammonium hydroxide, and the eluted is concentrated and lyophilized to give crude 1-N-methylsisomicin.

Chromatography of the crude material on silica gel (300 g) in chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) gives 1-N-methylsisomicin. [a] + 153 ° (0.3%, EUO).

Δ +

Mass spectrum: (M + 1) m / e 462, also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 5 386th

Example 8 1-N-Benzylgentamicin via a tri-N protected 1-N-Schiff's base intermediate.

10 (1) 0.3 g of 21,3-di-N-trifluoroacetylgentamicin are dissolved in 12 ml of ethanol and 0.9 ml of benzaldehyde is added. The reaction mixture is stirred for 3 hours and then evaporated in vacuo. The residue is dissolved in 0.8 ml of chloroform and the solution is added dropwise to 25 ml of hexane / ether (3: 1).

The resulting precipitate is separated by filtration and dried in vacuo to give 1-N, 3 ", 4" -Nr0-bis-benzy-lidin-2 ', 3-di-N-trifluoroacetylgentamicin C C · (yield 0, 38 g), m.p. 128-134 ° C, [α]: + 74.6 ° (c = 0.26%, ethanol).

20 (2) 1.37 g of the product obtained in Example 8 (1) are dissolved in 100 ml of ethanol and added 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, the mixture is acidified to a pH of ca. 3 with hydrochloric acid, then stir for an additional 16 hours. The solution is extracted with ether, the ether layer is separated and discarded. To the aqueous phase is added ammonium hydroxide until it is basic and then evaporated in vacuo to a residue. The residue is extracted with 35 ml of warm ethanol. The extracts are combined and evaporated in vacuo. The resulting residue is chromatographed over 75 g of silica gel eluting with the lower phase of a chloroform: methanol: ammonium hydroxide: water (2: 1: 0.2: 0.8) solvent system. Uniform fractions as determined by thin layer chromatography are combined and evaporated to a residue comprising 1-N-benzylgentamicin Cir. Mp: 83-88 ° C, [α] 6 + 90 ° (c = 0.3%, H ppm) (D20): δ 1.03 (3H, d, J = 7Hz, HC-CH-j), 1.16 (3H, 146298 42 s, C-CH 3) δ 2.27 (3H, s, N -CH3), 2.50 (3H, s, N-CH3), 4.7 (D2O + PhCH2N-), 4.92 (1H, d, J = 4Hz, H1 "), 5.08 (1H, d , J = 3.5Hz, H-1 '), 7.43 (5H, s, aromatic H).

Mass spectrum: (M + 1) + m / e 568, 5 also m / e 440, 437, 412, 394, 379, 281, 263, 253, 235, 160, 157.

Example 9 1-N-substituted 4,6-diaminoglycosyl-1,3-diaminocyclic tolerances prepared by hydride reduction of the corresponding 10-N-acyl ester ivat.

1-N- (S-S-amino-p-hydroxybutyl) gentamicin C1.

98 mg of 1-N- (S-δ-amino-β-hydroxybutyryl) gentamicin is suspended in 8 ml of tetrahydrofuran. 14 ml of 1N diborane in tetrahydrofuran are added and refluxed for 6 hours under an atmosphere of nitrogen. Carefully add 2 ml of water to decompose any excess diborane and evaporate. The resulting residue is dissolved in hydrazine hydrate and refluxed under an atmosphere of nitrogen for 16 hours.

The solution is evaporated and the residue is extracted with hot aqueous ethanol. The combined ethanol extracts are evaporated and the resulting residue is chromatographed over 10 ml of silica gel eluting with the lower phase of a chloroform: methanol: concentrated ammonium hydroxide 25 (2: 1: 1) solvent system. Uniform fractions as determined by thin layer chromatography are combined and evaporated to give 1-N- (SS-amino-p-hydroxybutyl) gentamicin (yield 14.5 mg), mp: 93-98 ° C, [α] + 72.4 ° (c = 0.35%, H 2 O), pmr (ppm) (D 2 O) δ 1.18 (3H, d, J = 7Hz, CH-CEL 2), 1.24 (3H, s, C-CH 3), 2.49 (3H, s, N-CH 3), 2.54 (3H, s, N-CH 3), 5.07 (1H, d, J = 3.5 Hz, H , 24 (1H, d, J = 3.5Hz, H-1 ').

Mass spectrum: (M + 1) + m / e 565, also m / e 528, 516, 490, 437, 434, 410, 35 397, 278, 250, 232, 160, 157.

In an analogous manner, 1-N- (SY-methylamino-β-hydroxypropyl) -gentamicin B, pmr: D20: δ 1.21 (3H, s, 146298 43 C-CH ) - 2.62 (3H, s, 3 "-N-CH3), 5.02 (1H, d, J = 4.5Hz, H1"), 5.12 (1H, d, J = 3.0Hz, H1 *).

Example 10 1-N-Methylsisomicin from 3 ", 4" -N, O-carbonyl-2 ', 3,6'-tri-Khfc-butoxycarbonyl-isomicin.

3 ", 4" -N, O-carbonyl-2 ', 3,6'-tri-N-t-butoxycarbonylsisomicin (0.77g) is dissolved in tetrahydrofuran (20ml) and the solution is cooled in an ice bath. Methyl β-fluorosulfonate (0.12 g) is added and the solution is allowed to warm to room temperature. The solvent is removed and the residue is dissolved in trifluoroacetic acid. After 5 minutes at room temperature, the trifluoroacetic acid is removed in vacuo and the residue is treated with 10% potassium hydroxide solution at 100 ° C for 5 hours.

The cooled solution is passed down through a column of "Amberlite" ® IRC-50 (H +) ion exchange resin and eluted with 2N aqueous ammonium hydroxide. The eluted is concentrated and lyophilized to give the crude title product.

The crude material is chromatographed on silica gel with the lower phase of a chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) solvent mixture to give 1-N-methylsisomicin. [α] D + 153 ° (0.3%, 25 H + 0).

Mass spectrum (M + 1) m / e 462, also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 386.

Example 11 1-N-Methylsisomicin.

2 ', 3,6' -Tri-Nt-butoxycarbonyl-3 ", 4" -N, O-carbonyl-sisomicin (5g) in ethanol (100ml) is treated with 37% aqueous formaldehyde (1ml) and ammonium formate (5 g) and the solution is refluxed for 24 hours. The solution is diluted with water (200 ml) and extracted with chloroform (3 x 100 ml). The combined extracts are evaporated to dryness and the residue containing 2 ', 3,6'-tri-Nt-butoxycarbonyl-3 ", 4" -N, O-carbonyl-1-N-methyl-s in soikin 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 crude product is eluted with 2N aqueous 10 ammonium hydroxide. The combined eluted fractions are evaporated to dryness in vacuo and the residue is chromatographed on silica gel (200 g) in the lower phase of a chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) solvent system to give of 1-N-methylsisomicin. [a] D: 15 + 153 ° (0.3%, H + 0).

^ +

Mass spectrum: (M + 1) m / e 462, also m / e 122, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 386 .

Example 12 1-N-Methylsisomicin.

2 1,3,6 '-Tri-Nt-butoxycarbonyl-3 "crude" -N, O-carbonylsisomicin (0.77 g) is treated in tetrahydrofuran (25 ml) with methylamine (101 mg) and trifluoromethylsulfonic acid -25 anhydride (290 mg) at 0 ° C for 18 hours. The solution is evaporated to dryness and the residue is dissolved in dimethylformamide (10 ml) and stirred with methyl iodide (300 mg) and potassium carbonate (130 mg) for an additional 18 hours. The solvent is removed by evaporation and the residue is treated with 30% aqueous potassium hydroxide at 100 ° C for 12 hours. The cooled solution is passed through a column of "Amberli tea" ® IRC 50 (H +) ion exchange resin. The crude product is eluted with 2N aqueous ammonium hydroxide. The combined eluate is evaporated to dryness in vacuo and the residue chromatographed on silica gel (200 g) in the lower phase of a chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) solvent system to give 1 N-methylsisomi- cin. [α] δ 6: + 153 ° (0.3%, H 2 O).

Mass spectrum: (M + 1) + m / e 462, also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 5 386.

Example 13 1-N-Methylsisomicin.

2 ', 3,6' -Tri-Nt-butoxycarbonyl-3 ", 4" τΝ, 0-carbonylsisomicin (0.77 g) in tetrahydrofuran (20 ml) is treated with 37% aqueous formaldehyde (3 ml) and succinimide (170 mg) at room temperature for 18 hours. The solution is dropped into a diethyl ether / hexane (1: 1) mixture and the precipitate is collected by filtration. This material is treated with sodium borohydride (200 mg) in ethanol (20 ml) 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 through + IRC 50 (H) ion exchange resin and the crude product is eluted with 2N aqueous ammonium hydroxide. The combined eluate is evaporated to dryness in vacuo and the residue is chromatographed on silica gel (200g) in the lower phase of a chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) solvent system to give 1-N-methylsisomicin. [α] D 20: + 153 ° (0.3%, H 2 O).

and 3 Mass Spectrum: (M + 1) m / e 462, also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376 , 386.

Example 14 -a n 1-N-Methylsisomicin.

21.3, 6'-Tri-Nt-butoxycarbonyl-3 "; 4" -N, O-carbonylsisomicin (0.77 g) is dissolved in dichloromethane (100 ml) with acrylonitrile (0.25 g) and is left at room temperature for 24 hours. The solvent is removed in vacuo leaving a residue which is dissolved in dimethylformamide and treated with methyl iodide (180 mg) at 50 ° C for 12 hours.

46

The solvent is removed and the residue is treated with 10% aqueous potassium hydroxide at 100 ° C for 8 hours. The cooled solution is passed through a column of "Amberlite" ® IRC 50 (H +) ion exchange resin and the crude product is eluted with 5 2N aqueous ammonium hydroxide. The combined eluate is evaporated to dryness in vacuo and the residue is chromatographed on silica gel (200 g) in the lower phase of a chloroform: methanol: 7% ammonium hydroxide (2: 1: 1) solvent system to obtain methylsisomicin. [α] D: 10 + 153 ° (0.3%, ELO).

^ +

Mass spectrum: (M + 1) m / e 462, also m / e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 386 .

The following table shows the minimum male concentration (MIC) for some of the compounds. The experiments were performed in Mueller-Hinton Broth (pH 7.2) using stand methods.

The In Vitro Minimum Inhibitory Concentration (MIC) Designation of Compounds in the Table: 20 a) Novel Compounds of the Invention:

Compound 1: 1-N-ethylgentamicin Cla Compound 2: 1-N-ethylgentamicin C

Compound 3: 1-N-Ethyl Antibiotic G-52 Compound 4: 1-N- (4-amino-2 (S) -hydroxybutane) - gentamicin C

Compound 5: 1-N-hydroxyethylgentamicin C

Compound 6: 1-N-phenethylgentamicin C

Compound 7: 1-N-ethylverdamicin Compound 8: 1-N-ethylsisomicin

Compound 9: 1-N- (2-ethylbutyl) -isomicin Compound 10: 1-N- (4-aminobutyl) -isomicin Compound 11: 1-N-ethylgentamicin B Compound 12: 1-N-ethyl antibiotic JI-20B Compound 13: 1N-ethylmutamicin 2

Compound 14: 1- N -ethylmutamicin 6 b) Compounds of the prior art:

Compound 15: 1-N- (S-4-amino-2-hydroxybutyryl) - gentamicin C1a (U.S. Pat.

Compound 16: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin (U.S. Pat.

3,780,018)

Experimental Procedure

Prepare and sterilize 2000 ml of Mueller-Hinton-10 nutrient solution adjusted to pH 7.2. Transfer 5 ml of the sterile medium to each of 120 sterile 16 x 150 mm cotton swab test tubes. Arrange the tubes in 10 groups each on 12 tubes. Add a control tube to each group. Add to 4 each tube 10 cells of one of the bacterial strains to be tested.

15 Add to each group an aqueous solution of the antibiotic to be tested, forming the following final concentrations per ml. tubes: 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 20 (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.

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

Table 30 Escherichia coli Forb. 1 Forb. 2 Forb. 3 Forb. 4 W677 / R55 - 3.0 0.3 0.03 JR 66 3.0 3.0 0.3 3.0 JR 88 3.0 .7.5 3.0 3.0 JR 90 0.75 17, 5 7.5 3.0 35 LA290 R55 0.3 0.75 0.3 3.0 R5 / W677 - 0.3 17.5 0.75 HL97 / W677 - 3.0 0.75 3.0

Swidinsky 4195 0.75 3.0 3.0 7.5 146298 48

Table (continued)

Escherichia coli Forb. 1 Forb. 2 Forb. 3 Forb. 4

St. Michael 589 0.3 0.75 0.75 3.0

Baker 2 0.3 0.3 0.3 3.0 F14-BK 0.075 0.3 0.3 3.0 1574-1 0.075 0.3 3.0 3.0 ATCC 10536 0.075 0.3 0.3 0.75

Pseudomonas

Travers 1 3.0 7.5 3.0 17.5 10 Stone 130 3.0 - 7.5 17.5

Stone 138 7.5 17.5 7.5 7.5

Capetown 18 3.0 17.5 7.5 3.0

Shreveport 3796> 25 17.5> 25> 25

Stone 20 0.05 0.3 0.03 0.3 15 Stone 39 0.3 3.0 3.0 3.0

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

Georgetown 3694 3.0 0.75 3.0 7.5 3020 3.0 0.075 0.3 0.3

Oklahoma 6 - 3.0 0.75 3.0 AD 17 0.075 0.75 3.0 3.0 25 Ad 18 0.075 0.3 3.0 3.0

Providencia 164 17.5> 25> 25> 25

Proteus mirabillis

Harding 0.3 3.0 7.5 3.0 30 Straightening Membel 0.75 3.0 17.5 17.5 Straightening Anderson -> 25> 25> 25

Serratia

Dalton 0.75 0.3 0.3 3.0

Salmonella 35 Group B typhim 0.3 3.0 3.0 3.0 146298 49

Table (continued)

Escherichia coli Forb. 5 Forb. 6 Forb. 7 Forb. 8 W677 / R55 3.0 0.75 0.3 0.3 JR 66 3.0 0.75 0.3 0.3 5 JR 88 3.0 0.75 3.0 0.8 JR 90 3.0 0.75 3.0 0.3 LA290 R55 3.0 0.75 0.3 0.3 R5 / W677 3.0 7.5 3.0 17.5 HL97 / W677 0.01 -> 25> 25 10 Swidinsky 4195 3.0 17.5 3.0 3.0

St. Michael 589 3.0 7.5 0.8 0.3

Baker 2 3.0 17.5 0.3 0.3 F14-BK 3.0 0.75 0.3 0.3 1574-1 3.0 7.5 0.3 0.3 15 ATCC 10536 0.75 0.75 0.1 0.08

Pseudomonas

Travers 1 7.5 - 3.0 0.8

Stone 130 17.5 - 3.0 0.8

Stone 138 17.5 - 3.0 0.8 20 Capetown 18 7.5 - 3.0 0.3

Shreveport 3796 17.5 - 3.0> 25

Stone 20 0.3 - 0.03 0.03

Stone 39 3.0 - 0.8 0.3

St. Michael 762 3.0 - 3.0 0.3 25 1395 17.5 - 3.0 0.3 NRRL 3223 3.0 - 0.8 0.3 GN 315 3.0

Klebsiella

Georgetown 3694 3.0 - 0.3 0.3 30 3020 0.75 - 0.3 0.3

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

Providencia 35 164> 25 - 17.5 17.5 146298 50

Table (continued)

Proteus Forb. 5 Forb. 6 Forb. 7 Forb. 8 mirabillis

Harding 3.0 - 0.8 0.3 5 Straightening Membel 17.5 - 0.8 0.3 Straightening Anderson> 25 -> 25> 25

Serratia

Dalton 3.0 - 0.3 0.9

Salmonella Group B typhim 3.0 - 0.8 3.0

Escherichia coli Forb. 9 Forb. 10 Forb. 11 Forb. 12 W677 / R55 - 0.3 3.0 3.0 JR 66 0.075 0.3 3.0 7.5 JR 88 - 0.3 3.0 17.5 15 JR 90 0.75 0.3 3.0 LA290 R55 0.3 0.3 3.0 7.5 R5 / W677 17.5 3.0> 25 17.5 HL97 / W677 17.5

Swidinsky 4195 0.75 3.0> 25> 25 20 p.m. Michael 589 0.3 0.75 7.5 17.5

Baker 2 0.3 0.3 3.0 17.5 F14-BK 0.3 0.3 3.0 0.75 1574-1 0.3 0.3 3.0 0.75 ATCC 10536 0.3 0 , 03 0.3 0.3 25 Pseudomonas

Travers 1 17.5 0.3 3.0 3.0

Stone 130 17.5 0.3 7.5

Stone 138 17.5 0.3 7.5

Capetown 18 7.5 0.3 3.0 30 Shreveport 3796> 25> 25> 25 17.5

Stone 20 0.03 0.03 0.3 0.075

Stone 39 3.0 0.3 3.0 3.0

St. Michael 762 7.5 0.3 3.0 3.0 1395 17.5 0.3 3.0 7.5 35 NRRL 3223 3.0 0.03 3.0 3.0 GN 315> 25> 25> 25 > 25 51 148 2 9 8

Table (continued)

Klebsiella Forb. 9 Forb. 10 Forb. 11 Forb. 12

Georgetown 3694 7.5 0.03 3.0 7.5 5 3020 0.3 0.03 3.0 0.75

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

Providencia 10 164 -> 25 7.5> 25

Proteus mirabillis

Harding 3.0 0.75 17.5 17.5 Straightening Membel - 3.0 3.0 17.5 Straightening Anderson -> 25> 25> 25 15 Serratia

Dalton 17.5 3.0 7.5 17.5

Salmonella

Group B typhim 3.0 0.75 3.0 3.0

Escherichia coli Forb. 13 Forb. 14 Forb. 15 Forb. 16 20 W677 / R55 0.075 0.125 17.5 7.5 JR 66 0.075 1 7.5 7.5 JR 88 0.075 0.25 17.5 17.5 JR 90 0.3 0.5 17.5 7.5 LA290 R55 0.075 0.25 7.5 7.5 R5 / W677 17.5> 16 HL97 / W677 8

Swidinsky 4195 0.3 1 - 7.5

St. Michael 589 0.3 2 7.5 7.5

Baker 2 0.3 1 7.5 17.5 30 F14-BK 0.075 0.125 3.0 7.5 1574-1 0.075 0.25 - 7.5 ATCC 10536 0.03 0.125 3.0 17.5

Pseudomonas

Travers 1 0.3 0.125 17.5 7.5 35 Stone 130 0.3 0.5> 25 17.5

Stone 138 0.3 0.5> 25 17.5

Capetown 18 0.3 0.25> 25 17.5

Shreveport 3796 7.5 4 -> 25

Claims (4)

5 p.m. Michael 762 0.3 0.25> 25 7.5 1395 0.3 0.25> 25 7.5 NRRL 3223 0.075 0.25> 25 17.5 GN 315> 25> 16 Klebsiella 10 Georgetown 3694 0.3 0.125 3.0 7.5 3020 0.3 0.06 3.0 7.5 Oklahoma 6 0.3 0.25 - - AD 17 0.075 0.25 3.0 7.5 AD 18 0.075 0.06 3.0 7.5 15 Providencia 164 3.0 8 Proteus mirabillis Harding 3.0 0.5 - 17.5 trial Membel 0.3 0.5> 25> 25 20 trial Anderson> 25 16 - - Serratia Dalton 0.75 0.5 3, 0 7.5 Salmonella Group B typhim 0.3 0.25 3.0 7.5 25 1. Analogous Process for Preparation of 1-N-Substituted Derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitolines gentamicin B, gentamicin B 2, gentamicin C 2, gentamicin Cla, gentamicin C 2, gentamicin C 2a , gentamicin sisomicin, verdamicin, Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 35 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is 1.6298 wherein X is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl or benzyl, which aliphatic groups have up to 7 C atoms and, if substituted with both amino and hydroxy, the substituents bear on various carbon atoms, or pharmaceutically acceptable acid addition salts thereof, characterized in that a) a corresponding 4,6-di- (aminoglycosyl) -1,3-diamine-10 nocyclitol, which may have amino protecting groups at any other amino group other than that in the 1-position is treated with an aldehyde of the formula: X * -CHO wherein X 'is a group as defined for X above wherein any amino or hydroxy group present may be protected, in the presence of a hydride donor reducing agent, where necessary, all protecting groups present in the molecule are removed in a manner known per se, b) the N, C double bond in a 1-N = CHX 'substituted derivative of a corresponding 4.6 di- (aminoglycosyl) -1,3-di-aminocyclitol in which all groups NH2 are protected and groups NHCH3 may be protected, wherein X 'is a group as defined for X above, wherein any amino or hydroxy group present may be protected, reduced, and all protecting groups present in the molecule are removed in a manner known per se, c) a 1-N-substituted derivative of a corresponding 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, wherein one or more amino groups may be protected, and the 1-N-substituent r -CX "where X" is hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or hydrocarbyloxy having aliphatic groups having up to 7 C atoms and, if substituted with both amino and hydroxy, the substituents carry on different carbon atoms, and wherein any amino or hydroxy group present may be protected, treated with an amide-reducing hydride reagent, whereupon all protecting groups present in the molecule are removed on and off. 5 d) for the preparation of the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclytols wherein the substituent is straight-chain alkyl of up to 5 C atoms , a corresponding 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol having amino protecting groups at any amino group other than that at the 1-position and wherein the 1-amino group may be activated per se method, reacted with an alkylating agent agent containing the straight-chain alkyl group having up to 5 C atoms and a leaving group, whereupon protecting groups present in the molecule and, if required, activating groups or groups present in a manner known per se, e) prepare the the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, wherein the substituent is methyl, a corresponding 4.6-di- (aminoglycosyl) -1,3-diaminocyclitol, having amino protecting groups at any amino group other than that at the 1-position, reacting with formaldehyde and a cyclic imide, and the compound thus obtained is treated with a hydride donor reducing agent and all protecting groups present in the molecule are removed in and for or f) for the preparation of the aforementioned 1-N-substituted derivatives of 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols wherein the substituent is methyl, a corresponding 4.6-di- (aminoglycosyl) -1,3-diaminocyclite ol having amino protecting groups at any amino group other than that at the 1-position are reacted with formaldehyde in the presence of formic acid, and all protecting groups present in the molecule are removed in a manner known per se, and that according to said methods a) to f) the derivative is isolated as such or as a pharmaceutically acceptable acid addition salt.