KR790001980B1 - Process for preparation of 4,6-di-(aminoglycosil)-1,3-diamino cyclitol 1-n-substituted derivatives - Google Patents

Abstract

Bactericidal, title compd., gentamicin C1, C1a, and B, verdamicin, and antibiotic G-52 were prepd. Thus, sisomicin in 1N H2SO4 at pH 5 was treated with MeCHO and then reduced with Na(CN)BH3 to give 1-N-ethylsisomicin useful in bactericide prepns. contg. 1-15mg/kg body wt. Treatment of sisomicin sulfate with Ac2O gave 1-N-acetylsisomicin which showed min. inhibitory concns. of 0.3-3.0 mg/kg against strains of Escherichia coli.

Description

Method for preparing 1-N-substituted derivatives of 4, 6-di- (aminoglycosyl) -1,3-diaminocyclitol

The present invention relates to a process for the preparation of 1-N-substituted derivatives of 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol which are effective as antibacterial agents.

A wide range of antimicrobials that are chemically classified as 4, 6-di- (aminoclicosyl) -1, 3-diaminocyclitol are known in the literature. Valuable antimicrobials in this group are those wherein the amino cyclitol is 2-deoxystrepamine or derivatives thereof having amino functional groups at the 1 and 3 positions, and particularly valuable antimicrobial agents are 4, 6-di- (aminoglyco 6) The aminoglycosyl group at the 6-position in the 2-deoxystrepamines is a compound showing a transamiminyl group. Among the 4-aminoglycosyl-6-garoseamimin-2-deoxystrepamines, gentamicin B, B ₁ , C ₁ , C ₁ a, C ₂ , C ₂ aC ₂ b and X ₂ , sisomycin, Antibiotics such as verdamycin, antibiotics G-418, G-52, JI-20A and JI-20B.

The present invention relates to compounds wherein the amino group at position 1 of 2-deoxy streptamine or a derivative thereof is optionally N-substituted at 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol. It is about. 1-N-substituted derivatives of these 4, 6-di- (aminoglycosyl) -2-deoxy streptamine or derivatives thereof are valuable widespread antibacterial agents.

The invention also 4, 6-di- (amino glycosyl) 1,3-diamino-hour cycle erythritol gentamicin A, gentamicin B, gentamicin B _l, genda mitomycin C _1, gentamicin C ₁ a, Genta Mycin C ₂ , gentamicin C ₂ a, gentamicin C ₂ b, gentamicin X ₂ , sisomycin, verdamycin, tobramycin, antibiotic G-41 ₈ , antibiotic 66-40B, antibiotic 66-40D, antibiotic JI- 20A, antibiotic JI-20B, antibiotic G-52, mutamycin 1, mutamycin 2, mutamycin 4, mutamycin 5 and 1-N-substituted derivatives of mutamycin 6 and pharmaceutically nontoxic To acid addition salts thereof, wherein the substituent is -CH ₂ X, wherein X is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylhamkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl and the aliphatic group has up to 7 carbon atoms Case be substituted with unexposed and hydroxy is substituted on different carbon atoms stab.

CH ₂ X substituents in the compounds of the present invention include ethyl, n-propyl, n-butyl, β-methylpropyl, n-pentyl, β-methylbutyl, ν-methylbutyl and β, β-dimethylpropyl, n- Hexyl, δ-methylpentyl, β-ethylbutyl, ν-ethylbutyl, n-heptyl, ε-methylheptyl, β-ethylpentyl, ν-ethylpentyl, δ-ethylpentyl, ν-propylbutyl, n-octyl, Straight and branched chain alkyl groups such as iso-octyl, β-ethylhexyl, δ-ethylhexyl, ε-ethylhexyl, β-propylpentyl, ν-propylpentyl;

alkenyl groups such as β-propenyl, β-methylpropenyl, β-butenyl, β-methyl-β-butenyl, β-ethyl-β-hexenyl;

Cyclic groups such as cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cyclopentyl ethyl;

Aromatic groups such as 0-, m-, p-methylbenzyl;

ε-hydroxypentyl, β-hydroxy-νmethylbutyl, β-hydroxy-β-methylpropyl, δ-hydroxybutyl, β-hydroxypropyl, δ-hydroxypropyl, W-hydroxyoctyl Linear or branched alkyl groups substituted with hydroxy;

straight and branched chain alkyl groups substituted with amino such as ε-aminopentyl, β-aminopropyl, ν-aminopropyl, δ-aminobutyl, β-amino-νmethylbutyl and W-aminooctyl;

N-methyl, N-ethyl and N-propyl derivatives such as ε-methylaminopentyl, β-methylaminopropyl, β-ethylaminopropyl, δ-methylaminobutyl, β-methylamino-ν-methylbutyl and Derivatives of these alkyl groups mono-N-alkylated, such as W-methylaminobutyl;

β-hydroxy-ε-aminopentyl, ν-hydroxy-ν-methyl-δ-aminobutyl, β-hydroxy-δ-aminobutyl, β-hydroxy-ν-aminopropyl and β-hydroxy-β Linear and branched alkyl groups di-substituted with amino and hydroxy, such as -methyl-ν-aminopropyl;

β-hydroxy-ε-methylaminopentyl, ν-hydroxy-ν-methyl-δ-methylaminobutyl, β-hydroxy-δ-methylaminobutyl, β-hydroxy-ν-ethylaminopropyl and β- Derivatives of these alkyl groups mono-N-alkylated, such as hydroxy-β-methyl-ν-methylaminopropyl.

Among these compounds, 1-N-CH ₂ X-derivatives containing garasaminyl as a 6-aminoglycoside group and preferably 2-deoxystreptamine as 1,3-diaminocyclitol are suitable. Do.

2-deoxystreptamine is present in all compounds of the invention listed above except mutamicin. 1,3-diaminocyclitol hexes in each of 1-N-CH ₂ X-mutamycin 1, 2, 4, 5 and 6 are respectively streptamine, 2, 5-dideoxystrepamine, 2 -Epi-streptamine, 5-amino-2, 5-dideoxystreptamine and 5-epi-2-deoxystreptamine.

1-N-CH ₂ X-4-amino glycosyl-6-garosemanyl- ₂ -deoxystrepamine prepared according to the present invention is a gentamicin B, B ₁ , C ₁ , C ₁ a, C ₂ , C ₂ a, C ₂ b and X ₂ , sisomycin, verdamycin, derivatives of the antibiotics G-418, JI-20A, JI-20B and G-52, defined by the following structural formula (I).

In the above structural formula,

X is as described above,

Y represents an aminoglycosyl functional group selected from the following groups

.

Another useful 1-N-CH ₂ X-4, 6-di (aminoglycosyl) -2-deoxy-streptamine according to the present invention includes the following compounds of formula (II), (III) and (IV) have.

1-N-CH ₂ X-Tobramycin of Formula (II)

X in the above structural formula is as described above.

1-N-CH ₂ X-antibiotic 66-40D of formula III, belonging to the preferred compounds according to the invention.

X in the above structural formula is as described above.

1-N-CH ₂ X-gentamycin A and 1-N-CH ₂ X-antibiotic 66-40B of formula IV.

In the above structural formula

X is as described above,

Y 'is

1-N-CH ₂ X-mutamycin according to the present invention is 1-N-CH ₂ X-4-aminoglycosyl-6-garosaminyl-1,3-diaminocycle of formula (V) Includes Ritol.

In the above structural formula

X is as described above,

In 1-N-CH ₂ X-Dutamycin 1, V ₂ and W ₅ are hydrogen, W ₂ and V ₅ are hydroxy,

W ₁ , V ₂ , W ₅ and V ₅ in 1-N-CH ₂ X-mutamicin ₂ are hydrogen,

In 1-N-CH ₂ X-mutamycin 4 W ₂ and W ₅ are hydrogen, V ₂ and V ₅ are hydroxy,

In 1-N-CH ₂ X-mutamycin 5 W ₂ , V ₂ and W ₅ are hydrogen, V ₅ is amino,

In 1-N-CH ₂ X-mutamycin 6 W ₂ , V ₂ and V ₅ are hydrogen and W ₅ is hydroxy.

The terminal bond in which the group is not represented in the above structural formula represents a hydrogen atom.

1-N-CH ₂ X-4, 6-di- (aminoglycosyl) -1, 3-diamino, also defined by structural formulas (I), (II), (III), (IV) and (V) Pharmaceutically toxic acid addition salts of cyclitol are also included within the scope of the present invention, which salts are usually obtained by conventional methods of neutralizing the free base to pH 5 with an appropriate acid. Suitable acids include acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, acetic acid, propionic acid, maleic acid, ascorbic acid, citric acid and the like. The physical properties of the acid addition salts of 1-N-CH ₂ X-4, 6-di- (aminoglycosyl) -l, 3-diaminocyclitol according to the invention are white solids, soluble in water and most polar and It is insoluble in nonpolar organic solvents, and is sometimes soluble in most polar solvents and sometimes insoluble in nonpolar organic solvents.

1-N-CH ₂ X-4, 6-di- (aminoglycosyl) -1, 3 of structural formulas (I), (II), (III), (IV) and (V) prepared according to the invention Diaminocyclitol and its pharmaceutically nontoxic phosphate addition salts generally exhibit broad antimicrobial activity. In particular, 1-N-lower alkyl derivatives have improved antimicrobial activity over these compounds against microorganisms resistant to these parent compounds. Thus, for example, these compounds are more active against microorganisms that inactivate the parent antibiotic, for example by acetylation of the 3-amino group and / or adenylation of the 2 ''-hydroxyl group. Some of these also exhibit anti-protozoa, anti-amoeba and antiparasitic activity.

Preferred among the compounds according to the invention are 4-aminoglycosyl-6-garoseamimin-2-deoxystrepamine gentamicin B, gentamicin B ₁ , gentamicin C ₁ , gentamicin C ₁ a, genta There are 1-N-substituted derivatives of mycin C ₂ , gentamicin X ₂ , sisomycin, verdacin, antibiotic J1-20A, antibiotic JI-20B, antibiotic G-52 and antibiotic G-418, among which gentamicin C ₁ Most preferred are gentamicin C ₁ a, sisomycin, verdamycin and derivatives of the antibiotic G-52. Another particularly useful compound is the 1-N-substituted derivative of antibiotic 66-40D.

X in the 1-N-substituent is preferably selected from hydrogen, alkyl, hydroxyalkyl, aminoalkyl, aminohydroxyalkyl, phenyl or benzyl and the aliphatic group has up to 7 carbon atoms and is substituted with amino and hydroxy groups They are substituted on different carbon atoms. Preferred of these are hydrogen, alkyl, aminoalkyl and hydroxyalkyl having up to 7 carbon atoms and aminohydroxyalkyl with up to 3 carbon atoms, with substituents on different carbon atoms.

Particularly useful compounds according to the invention are those wherein X is hydrogen, methyl, ethyl and propyl, with methyl and ethyl being preferred. Particularly valuable compounds are the 1-N-CH ₂ X-4-aminoglycosyl-6-garosamimin- ₂ -deoxystrepamines of formula (I), wherein X is lower alkyl having 1 to 3 carbon atoms, in particular 1-N-lower alkyl derivatives and structural formulas of gentamicin C ₁ , gentamycin C ₁ a, gentamycin C ₂ , gentamycin C ₂ a, gentamycin C ₂ b, sisomycin, verdamycin and antibiotic G-52 (III) 1-N-lower alkyl-antibiotic 66-40D, and these derivatives are Gram-positive bacteria (e.g., staphylococcus aureus and Gram-negative bacteria (e.g., Escherichia coli and Pseudomonas aeruginosa, and a broad spectrum of antimicrobial agents that are also active against bacteria that are resistant to 1-N-unsubstituted precursors, are particularly effective 1-N-ethylveramycin and 1-N. Lower alkylcysomycins such as 1-N-methylcysomycin, 1-N- (n-pro )-Sisomycin, 1-N- (n-butyl)-sisomycin, and 1-N-ethyl sisomycin exhibiting activity against Gram-negative bacteria resistant to 1-N-unsubstituted precursors. Particularly useful compounds are 1-N-ethylgentamicin C ₁ a, 1-N-ethylgentamicin C ₁ , 1-N-ethyl antibiotic G-52, 1-N- (n-propyl) -Verdamycin, 1-N- (δ-aminobutyl) -sisomicin, 1-N-methylverdamycin, 1-N- (n-butyl) -veramycin, 1-N- (S-2-hydroxy 4-Aminobutyl) -gentamycin C ₁ , 1-N- (S-2-hydroxy-4-aminobutyl) -sisomicin and 1-N- (S-2-hydroxy-4-aminobutyl )-Verdamycin.

Most of the aforementioned 1-N-unsubstituted 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol antibiotics which are starting materials of the 1-N-substituted derivatives according to the present invention are known. It is. Of gentamicin, gentamicin X ₂ is known as gentamicin X in the literature so that the starting materials referred to herein as gentamicin C ₂ a are separated according to Preparation Example 1 below.

The starting materials referred to here as gentamicin C ₂ b are separated according to Preparation Example 2 and are designated in the literature as gentamicin C ₂ a having the same structural formula.

Isolation, properties and planar coordination of gentamicin C ₂ are described in US Pat. No. 3,651,042.

Antibiotics 66-40B and 66-40D, their preparation, isolation, properties and coordination are described in Belgian Patent No. 811,370. Antibiotics are produced together with sisomycin, the main product of fermentation of Micromonospora inyoensis (described in British Patent Application No. 1,274,518) and can be separated from fermentation media by special chromatographic separation. have.

Mutamycins 1, 2, 4, 5, and 6 can be prepared by culturing a mutant strain of micromonospora diuresis (herein referred to as micromonospora diuresis strain 1550F-1G) in an aqueous nutrient medium. have. This mutant strain cannot produce antibiotics when cultured in an aqueous nutrient medium under aerobic conditions without 1,3-diaminocyclitol. However, when a compound of this kind is added to the fermentation medium, mutamycin is produced. When 2-deoxystrepamine is added to the fermentation broth, known antibiotic sisomycin is produced.

The essential 1,3-diaminocyclitols that must be present in fermentation broth to obtain mutamycin are as follows.

Streptamine; To get mutamycin 1,

2, 5-dideoxystreptamine; To get mutamycin 2,

2-epistrepamine; To get mutamycin 4,

2, 5-dideoxy-5-aminostreptamine; In the year of getting mutamycin 5,

5-epi-2-deoxystreptamine; To get mutamycin 6.

Streptamine is a known compound, wherein 2,5-dideoxystrepamine can be obtained according to the invention of 6 in the preparation using hydrazine instead of monomethylhydrazine. 2-Epistreptdetsu. It may be prepared according to the method of Suami (Journal of Organic Chemistry 33, No. 7, 2831 2834 (1968)).

2,3-dideoxy-5-aminostreptamine is obtained by decomposing N, N-diacyl derivative of 2-deoxystreptamine with periodate and treating the resulting dialdehyde under alkaline conditions with nitromethane. And the nitro group is inserted at the 5 position, the nitro group is reduced to an amino group, and hydrolyzed N, N-diacyl functional groups to obtain a free amine.

5-Epi-2-deoxystreptamine is prepared according to the method of 1, 3, 4, 6-detracetyl-5-epi-2-deoxystreptamine [Hasagagawa and Sable, Tetrahedron, 25, 35-67 (1060), can be prepared by hydrolyzing esters and amide groups with 6N hydrochloric acid.

The preparation of mutamycin is described in Preparation Example 10.

Mother microorganisms (Micromonospora diuresis) have been deposited under No. NRRL 3292 with the Department of Agriculture's Northern R & D Department in Peoria, Illinois, United States, and described in the Journal of Antibiotics (Japan) Vol. XXIII, No. 11, 551-558 (1970), M. second. Published by Weinstein].

Micromonospora diureticis strain 1550 F-1G exhibits similar growth characteristics to the parental micromonospora diuresis. Cultures of M. diuresis strain 1550F-1G have also been deposited under the number NRRL 5742.

The following table shows the taxonomic, biochemical and morphological properties of microorganisms. The following systems and references are used to describe the formation of colors: The name of the birth is twofold. One is named after the "Descriptive Color Name Dictionary" (Taylor, Notch and Granville, issued by the United States Container Corporation, 1950), which has a color number corresponding to the color name. Name of color referring to synonyms developed in the "Color Harmony Manual" [4th Edition, 1958, United States Container Corporation], and the other developed by the National Standards Agency Circular 553, 1955. 11.1 (United States of America). And number.

M diureticosis strain 1550F- when incubated on agar medium containing 3% NZamine type A (peptone produced from the enzymatic digestion of casein and used as a nitrogen source), 1% dextrose and 1.5% agar. 1G NRRL 5742 is insufficiently grown to characterize.

TABLE I

Description of Colonies of Micromonospora Diureticis Strain 1550F-1GNRRL 5742 on Different Media or Culture Conditions

TABLE II

Utilization of Nitrogen Source by Micro Monospora Diuresis strain 1550F-1G NRRL 5742

TABLE III

Use of Carbohydrates by Micro Monospora Diureticis Strain 1550F-lG NRRL 5742

The preparation method of the present invention is a hydride donor-reducing agent of one of the above 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol which may have an amino protecting group at any position other than the 1 position. In the presence of an aldehyde of the structural formula X'-CHO, wherein X 'is a group defined above in which the amino or hydroxy groups present may be protected, and if necessary remove all protecting groups present in the molecule. In the final reaction step, characterized in that the derivative or pharmaceutically toxic acid addition salts are separated, 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol gentamicin A, gentamicin B, Gentamycin B ₁ , gentamycin C ₁ , gentamycin C ₁ a, gentamycin C ₂ , gentamycin C ₂ a, gentamycin C ₂ b, gentamycin X ₂ , sisomycin, veramycin, tobramycin, antibiotic G -418, antibiotic 66-40B, 1-N-Substitution of Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, Mutamycin 1, Mutamycin 2, Mutamycin 4, Mutamycin 5, and Mutamycin 6 Derivatives, wherein the substituents are —CH ₂ X, wherein X is as described above, and methods for preparing pharmaceutically toxic acid addition salts thereof.

1-N-unsubstituted-4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol selectively concentrates 1-amino functional groups with aldehyde and simultaneously reduces 1-N-alkyl-4 This method of obtaining 6-di- (aminoglycosyl) -1,3-diaminocyclitol antibacterial agents is always carried out at room temperature in the presence of air (although advantageously in the presence of an inert gas such as argon or nitrogen). . Advantageously this reaction ends in a short time, usually within 30 minutes, as measured by thin layer chromatography.

Hydride donor reducing agents useful in the process include dialkylaminoboranes (e.g. dimethylaminoborane, diethylaminoborane, preferably morpholinoborane), tetraalkylammonium cyano borohydrides (e.g. tetrabutylammonium cya Novolohydrides), alkali metal borohydrides (eg sodium borohydride) and preferably alkali metal cyanoborohydrides (eg lithium cyanoborohydride and sodium cyano borohydride).

This reaction proceeds conveniently in an inert solvent. As used herein, "inert solvent" means that 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol starting materials and reactants are dissolved and do not interfere with the reaction under reaction conditions to minimize side reactions. Refers to organic or inorganic solvents. Anhydrous aprotic solvents sometimes advance the reaction favorably (using anhydrous neutral solvents such as tetrahydrofuran when morpholinoborane is used as the hydride donor-reducing agent).

Although the present invention may also use co-solvent systems that are miscible with water, such as aqueous dimethylformamide, aqueous hexamethylphosphoramide, aqueous tetrahydrofuran and aqueous ethylene glycol dimethyl ether, usually lower alkanols, preferably water Or in a protic solvent such as aqueous lower alkanols (eg aqueous methanol, aqueous ethanol).

The reaction proceeds conveniently at pH 1-11, preferably 2-5, with a pH in the range 2.5-3.5. Preferred acidic media can also be obtained by adding organic or inorganic acids to 4, 6-di- (amino glycosyl) -1, 3-diaminocyclitol, whereby acid addition salts are formed. As the acid, an organic acid such as acetic acid, trifluoroacetic acid, or p-toluene-sulfonic acid, or an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or nitric acid may be used, and among them, sulfuric acid is most conveniently used. Also in the process according to the invention, a solution or suspension of 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol (eg sisomycin) in a protic solvent (eg water), It is also convenient to prepare the acid addition salt of the starting material by adding the desired acid (eg sulfuric acid) until the pH of the solution is adjusted to the desired pH.

Representatives useful for the present reaction in the aldehydes of formula X'CHO as described above include linear and branched alkyl aldehydes (e.g. formaldehyde, acetaldehyde, n-propanal, n-butanal, 2-methylpropanal). , n-pentinal, 2-methylbutanal, 3-methylbutanal, 2, 2-dimethylpropanal, n-hexenal, 2-ethylbutanal, n-heptanal, and n-octanal) Kenyl aldehydes (e.g., propenal, 2l methylpropenal, 2-butenal, 2-methyl-2-butenal, 2-ethyl-2-hexenal), cyclic aldehydes (e.g., cyclopropanecarbaldehyde, cyclopentane Carbaldehyde, cyclopentaneacetaldehyde, cyclohexanecarbaldehyde), benzaldehyde (e.g. 0-, m- and p-toluenecarbaldehyde and phenylacetaldehyde), hydroxy substituted straight-chain and branched alkylaldehyde (e.g. 5-hydroxypentanal, 2-hydroxy-3-methylbutanal, 2-hydroxy-2- Methylpropanal, 4-hydroxybutanal, 2-hydroxypropanal, ε-hydroxyoctanal), straight and branched chain alkylaldehydes substituted with amino (e.g. 5-aminopentanal, 2-aminopropanal, 3 Aminopropanal, 4-aminobutanal, 2-amino-3-methylbutanal, ε-aminooctanal and its mono-N-alkyl derivatives), and straight and branched chain alkylaldehydes substituted with amino and hydroxy; Examples: 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.

In this reaction, when the aldehyde has a chiral center, each colon can be used separately or together as a racemate, and each diastereomer or mixture thereof can be obtained.

Aldehydes that can be used in the present reaction are known compounds or can be readily prepared from known compounds according to methods known in the literature. Thus, for example, alkyl aldehydes (eg, 2-hydroxy-5-amino-pentanal) substituted with hydroxyl and amino functional groups may be protected by acetamido or phthalimido using known methods. It can be prepared from aminoaldehyde acetals (e.g. aminobutanal diethylacetal) by removing the acetal groups by decomposition to obtain N-protected aminoaldehydes (e.g. 4-amino-butanal diethyl acetal) Conversion to N-phthalimido derivatives and acid hydrolysis to give 4-phthalimido butanal). Treatment of N-protected aminoaldehyde with hydrocyanic acid yields the corresponding N-protected aminoalkyl hydroxynitrile (e.g. 2-hydroxy-5-phthalimido valeronitrile) and the catalytic conversion source (e.g. N-protected amino-hydroxyaldehyde, an aldehyde reagent used in the present invention (e.g., 2-, using hydrogen or reduced with a hydride in the presence of palladium) (e.g., di-isobutylaluminum hydride). Hydroxy-5-phthalimido pentanal) is obtained.

Hydride of 1-N-unsubstituted-4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol to minimize side reactions when using aminoaldehyde as a reagent in carrying out this reaction When treated with donor and aldehyde to give the corresponding 1-N-substituted derivatives of 4, 6-di- (amino glycosyl) -1, 3-diaminocyclitol, the amino functionality in the aldehyde before carrying out the reaction For example, it is preferable to protect with an acyl protecting group such as acetamido, phthalimido and the like, and then remove the N-protecting group generated in the compound. It is also advantageous, but generally not necessary, to protect the hydroxyl groups in the aldehydes having hydroxyl groups as the reaction proceeds.

It is also possible to use acetal or hemiacetal of the aldehyde in an acidic medium which produces the required aldehyde.

A convenient way to carry out this reaction is to protic solution of a 1-N-unsubstituted-4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol antibacterial agent such as sisomycin or verdamycin. The acid addition salt of the starting compound is prepared by adjusting the pH of the solution to about 2-5 by adding an acid such as dilute sulfuric acid. The acid addition salts produced when the pH of the solution is about 5 usually contain about 1 equivalent of acid for each amino group of 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol (e.g. For example, 2.5 moles of sulfuric acid per mole of sisomycin). After preparing the acid addition salt solution, at least 1 molar equivalent, preferably excess molar amount of aldehyde (e.g. acetaldehyde, propanal or butanal) is added and 1 molar equivalent (starting material 4, 6) in a short time of about 5 minutes. Hydride donor of di- (aminoglycosyl) -1, 3-diaminocyclitol) -reducing agent Preferably alkali metal cyanoborohydride, usually sodium cyanoborohydride, is added . This reaction is often terminated within 30 minutes as measured by thin layer chromatography and the corresponding 1-N-substituted derivatives of 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol (eg 1-N-ethylisomycin or 1-N-ethylveramycin). The separation and purification of the derivatives thus produced are carried out by known methods, ie precipitation, extraction, preferably by chromatography.

Thus, the present invention reacts aminoglycosides with aldehydes (preferably in excess) and hydride donor-reducing agents in situ to produce mono-N-substituted derivatives (e.g., 1-N-ethylcysomycin) as the main product. In the starting materials 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol, the 1-amino group bonded to the second carbon atom is bound to the other and other amino atoms. It provides a convenient and process process in a single vessel that is alkylated beyond a group.

It is also possible to partially use N-protected 4, 6-di- (amino glycosyl) -1, 3-diaminocyclitol as starting materials. In general, 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol having a -CH ₂ -NH ₂ group as the 6 'site is present at this position because the group is most reactive during the protection process. N-protected. Sisomycin may be protected at the 6 'position or at the 2', 6 'position or at the 2', 3 ', 6'-position. Other amino protecting groups are 4, 6-di- (aminoglycosyl) -1, 3 '' in 3-diaminocyclitol, having 3 ''-amino and 4 ''-hydroxy groups in cis position with one another. It may also be a bridging group such as carbonyl at the 4 '' position. Thus, for example, as a starting material, 1-N-unsubstituted-4, 6-di-, in which the amino functional group on the β'-carbon atom is N-protected (eg, 6'-Nt-butoxycarbonylsisomicin) (Aminoglycosyl) -1, 3-diaminocyclitol, or N-protected aminofunctional groups at C-2 'and C-3 positions (e.g., 2, 3-di-N-trifluoroacetylgenta Mycin C ₁ ) 1-N-unsubstituted-4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol can be used and the corresponding partially N-protected 1-N-substituted derivatives (E.g., 1-N-ethyl-6'-Nt-butoxycarbonylcysomycin and 1-N-ethyl-2 ', ε-di-N-trifluoroacetylgentamicin C _{1, respectively} ) , N-protecting groups are removed according to known methods to give 1-N-CH ₂ X compounds according to the invention (e.g. 1-N-ethylcysomycin and 1-N-ethylgentamicin C _{1 respectively} ). .

The required starting materials protected with amino groups are prepared according to methods analogous to or identical to Preparation Example 3. As used herein, a "protecting group" refers to a group which is inert to the following chemical reactions but which can be easily removed after the chemical reaction is completed. Examples of such aminoprotecting groups are benzyl, 4-nitrobenzyl, triphenylmethyl, 2, 4-dinitrophenyl: acyl groups (e.g. acetyl, propionyl and benzoyl), alkoxycarbonyl groups (e.g. methoxycarbo Neyl, ethoxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, t-butoxycarbonyl and 2-iodoethoxycarbonyl) and arylalkoxycarbonyl groups (e.g. carbenzyloxy and 4 Methoxybenzyloxycarbonyl group).

In the blocking process, the protecting group is usually in the form of acid imidazole derivatives and acid azides, or active such as ethylthiotrifluoroacetate, N-benzyloxycarbonyloxy, succinimide or P-nitrophenyl trichloromethylcarbonate Used in the form of esters. Thus, it can be said that the blocking group is derived from the compound BgLg, where Bg becomes a blocking group such as the acid portion of the active ester and Lg is a leaving group such as imidazole.

Alternatively, the above reaction may be progressed according to a method of reducing the generated Schiff base after forming the 1-N- Schiff base derivative. Method for preparing 1-N-substituted derivatives of 4, 6-di- (amino glycosyl) -1,3-diaminocyclitol (substituent is -CH ₂ X and X is as described above) Is a 1-N = CHX'-substituted 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol wherein all NH ₂ -groups and NHCH ₃ groups are protected by X 'described above Reduction of the NC-double bond in the derivative is followed by removal of all protecting groups in the molecule and separation into the desired derivative itself or pharmaceutically toxic acid addition salts.

4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol, in which a 6-aminoglycosyl group is a transamiminyl, usually forms an oxazolidine ring simultaneously with the 1-N-Shifs base group. Thus having the same 3 ''-N-4 ''-0-protecting group as the 1-N-substituent of the starting material. Thus, for example, 2 ', 3-di-N-trifluoro acetylgentamicin C ₁ is reacted with an aldehyde (e.g. benzaldehyde, phenylacetaldehyde or acetaldehyde) to correspond to the corresponding 3'',4'' Starting materials of oxazolidine-1-ylidene Schiff base (e.g. 1-N-3``-N-4 ''-0-di-benzylidene-2 ', 3'-di-N-trifluor Loacetylgentamicin C <sub>1,</sub> 1-N-3``-N-4 ''-0-di-phenethylidene-2 ', 3'-di-N-trifluoroacetylgentamycin C ₁ and 1 -N-3``-N-4 ''-0-diethylidene-2 ', 3-di-N-trifluorouroacetylgentamicin C <sub>1</sub> ), which is converted to sodium borohydride and methanolic sodium Reduction with methoxide to the corresponding 1-N-CH <sub>2</sub> X-3 &quot;, 4 &quot; -oxazolidine (e.g., 1-N-benzyl-3 &quot; -N-4 &quot; -0-benzylidene respectively) Gentamicin C <sub>1</sub> , 1-N-phenethyl-3``-N-4 ''-0-phenethylidenegentamycin C ₁ and 1-N-ethyl-3 ''-N-4 ''-0 -Ethylidene gentamycin C ₁ ), which is then reacted with an acid to 1-N-CH ₂ X compounds according to (eg 1-N-benzylgentamicin C ₁ , 1-N-phenethylgentamicin C ₁ and 1-N-ethylgentamicin C _{1, respectively} ) are obtained.

The following preparations illustrate the preparation of various necessary starting materials and the examples further illustrate the invention.

[Production Example 1]

Gendamicin C ₂ a

Isolation of Gentamicin C ₂ a from Co-produced Antibiotic

96 g of gentamicin base (prepared from sulfate obtained according to Example 4 of U.S. Patent No. 3, 091, 572), an upper layer formed by mixing methanol, chloroform and 17% ammonium hydroxide in a volume ratio of 1: 2: 1 Dissolve in 400 ml and add 1/10 of this solution to each of the first 10 tubes of a backflow extractor equipped with 500 ml of tube. Fill all the tubes, including the first 10 tubes, with the bottom layer of the solvent mixture. Solvent reservoirs are installed for each movement to deliver 40 ml of the top layer to the tube (1) and to be instrumented for 500 movements. At the end of the transfer, samples of all eighth tubes were transferred to Schreiher and Schile. Chromatography using the bottom layer of the solvent mixture on 589 (No. 2). After developing for 16 hours and drying the filtrate, a filtrate is coated on an agar plate inoculated with Staphylococcus oreus (ATCC 65389), and sprayed twice with a conventional ninhydrin solution, followed by heating. The agar plate is incubated overnight at 37 ° C. and then combined with a solution of a tube containing a substance that moves, such as gentamicin C ₁ (eg, tubes 290 to 360).

In tube 290 360 is replaced with a new tube containing 40 ml of the top layer and 40 ml of the bottom layer, then the instrument is repositioned to move further 2800 times and chromatographed as described above. 16 in tube 1 were combined and concentrated in vacuo to yield 1.3 g of gentamycin C ₂ a having the following properties.

(a) Molecular weight 463, Experimental Formula C ₂₀ H ₄ , N ₅ O ₇ (measured by mass spectrometry)

(b) [α] ²⁶ _D + 114 ° + 5 (concentration in water 0.3% using sodium ray)

); 1.17(3H₃s, C-

); 2.477 (3H, s, N-

); 2.47(3H, s, n-

); 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''5q); 5.04(1H, d, J=4Hz, H-1'') 5.13(1H, d, J=3.5Hz, H-1').(c) pmr spectrum; pmr (ppm) (D ₂ O): δ 0.99 (3H, d, J = 6, 5 Hz, CH-

); 1.17 (3H ₃ s, C-

); 2.477 (3H, s, N-

); 2.47 (3H, s, n-

); 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 ''5q); 5.04 (1H, d, J = 4 Hz, H-1'') 5.13 (1H, d, J = 3.5 Hz, H-1').

The emission of the secondary methyl group at δ 0.99 ppm shows that H-6 ′ appears as a doublet (J = 6.5 Hz) at δ 2.81 ppm.

[Production Example 2]

Gentamicin

Isolation of Gentamicin C ₂ b from Co-produced Antibiotic

The major gentamicin C components (C ₁ , C ₂ and C ₁ a) were separated according to Example 2 of U.S. Patent No. 3,651,042 and contained significantly overlapping gentamycin C ₁ and C ₂ free bases. Combine with the fraction (50 g of gentamicin C mixture overlaps 53.4 g). 1.5 g of this gentamicin C ₁ and C ₂ mixture is placed in a column containing 50 g of silica gel in a solvent system of chloroform: methanol: 15% ammonium hydroxide (1: 2: 1). The column is eluted with the same solvent system, and then chromatographed on a silica gel plate using a solvent system of chloroform: methanol: 22% ammonium hydroxide (1: 2: 1) as a developing agent. Combine the gentamicin C ₂ b (fraction 39 to 57 [410 mg]) with a fraction containing a mixture of gentamicin C ₁ and C ₂ . Fractions 39 to 57 were fractionated on silica gel again with a solvent system of chloroform: methanol: 7% ammonium hydroxide (1: 2: 1) and measured by thin layer chromatography, containing pure gentamicin C ₂ b with the following properties: (98 to 130) are combined (amount: 45 mg).

[α] ²⁶ _D + 165.5 ° (concentration 0.3%, H ₂ O):

); 2.40(3H, s, N-

); 2.55(3H, s, N-

); 5.12(1H, d, J=4Hz, H-1''); 5.22(1H, d, J.3Hz, H-1').Mass spectrum, m / e 463 (M + 1) &lt; + &gt; 446, 445, 433, 350, 332, 322, 304, 333, 305, 287, 191, 173, 163, 145, 160, 142, 118, 143; pmr (ppm) (D ₂ O) _: δ 1.25 (3H, s C-

); 2.40 (3H, s, N-

); 2.55 (3H, s, N-

); 5.12 (1H, doublet, J = 4 Hz, H-1 &quot;); 5.22 (1H, doublet, J. 3 Hz, H-1 ').

Pure gentamycin C ₂ b is a silica gel plate, developed zero chloroform: methanol: 22% ammonium hydroxide (1: 2: 1) by thin layer chromatography using the solvent system from gentamicin C ₁ and C ₂ by his mobility of Can be discerned. The approximate Rf value in the solvent system is as follows.

[Manufacture example 3]

Optionally protected di- (aminoglycosyl) -1,3-diaminocyglytol

A. 2 ', 3', 6'-tri-N-butoxycarbonyl-3``-N-4 ''-O-carbonyl sisomycin

Penta-N-carbenzoxyoxysomycin

25 g of sisomycin and 13 g of sodium carbonate are dissolved in 625 ml of distilled water, stirred at 25 ° C., then 100 ml of carbobenzoxy chloride is added and stirred for 16 hours. The solid is filtered, washed well with water, dried under vacuum and washed again with hexane to give penta-N-carbenzoxyoxysomycin (62 g) as a colorless amorphous solid.

Melting Point: 165-173 ° C

[α] ²⁶ _D + 96.2 ° (CH ₃ OH)

IR: V max (CHCl ₃ ) 3400, 1720, 1515, 1215, 1050, 695 cm ⁻¹ .

NMR: δ (CDCI ₃ ) 1.03 (3H, broad singlet, 4 ''-C-CH ₃ ); 3.02 (3H, broad singlet, 3 ''-NCH ₃ ); 5.02 (10H, broad singlet, CH ₂ C ₆ H ₅ ); 3.28, 3.30 ppm (25H, broad singlet-CH ₂ C ₆ H ₅ ).

2. Tetra-N-carbenzoxy-3 ''-N-4 ''-O-carbonyl-sisomicin

5 g of penta-N-carbenzoxy-sisomicin is dissolved in 50 ml of dimethylformamide and stirred, then 250 ml of sodium anhydride is added and stirred under argon at room temperature for 2 hours. Filter and add glacial acetic acid (2 ml) to the filtrate and then concentrate in vacuo. The residue is extracted with chloroform (20 ml, first passed through basic alumina), washed with water and then dehydrated with sodium sulfate. This solution is evaporated to give tetra-N-carbenzoxy-3 &quot; -N-4 &quot; -O-carbonyl-sisomicin as an amorphous powder (3.5 g).

Melting point: 210-213 ° C., [α] ²⁶ _D +68.8 (C = 0.22)

IR: ν _max (nusol) 3, 550, 1, 840, 1, 790, 1, 580 cm ^-1 .

NMR: δ (CDCl ₃ ) 1.34 (3H, singlet, 4 ''-CH ₃ ); 2.68 (3H, singlet, 3 ''-N-Me); 5.04 (8H, broad singlet, -CH ₂ C ₆ H ₅ ).

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

In a solution of 10.1 g of 1, 3, 2 ', 6'-tetra-N-benzyloxycarbonyl-3''-N-4''-0-carbonyl-sisomicin, dissolved in 200 ml of tetrahydrofuran, 1 L of liquid ammonia (redistilled further with sodium) is added. 6 g of sodium is added to the stirred solution in small pieces, stirred for 3 hours, and then ammonium chloride is added to destroy the excess sodium. The solvent is evaporated under a nitrogen stream, the residue is dissolved in water, passed through a medium of Amberlite IRC-50 resin (H ⁺ type), the resin is washed well with water and the product is eluted with 2N ammonium hydroxide solution. The ammonia eluate is evaporated in vacuo to give about 4 g of 3 ''-N-4 ''-0-carbonyl-sisomicin.

IR: ν _max . Nusol 1,745 cm ^-l

This product can be used for the next reaction without further purification. However, chromatography on silica gel using a lower layer of chloroform: methanol: ammonium hydroxide (1: 1: 1) solvent system as the eluent provides a very pure sample.

4. 2, 3, 6-tri-N-t-butoxycarbonyl-3``-N-4 ''-O-carbonyl-sisomicin

1.4 g (3 mmol) of 23 ''-N-4 ''-O-carbonyl-sisomicin was dissolved in 10 ml of 50% aqueous methanol containing triethylamine (3.5 mmol) and then stirred with t-part. Toxylcarbonyl azide (3.5 mmol) is added dropwise. The mixture is stirred for 2 days at room temperature. 5 ml of Amberlite 1RA-401S (OH ⁻ ) ion exchange resin is added together with 5 ml of methanol and stirred for half an hour. The resin was filtered off, washed with methanol, the filtrate was concentrated, and the residue was chromatographed on a silica gel (60-100 mesh, 20.0 g) column using a solvent system of chloroform: methanol: ammonium hydroxide (30: 10: 0.4). do. Homogeneous fractions containing the title compound are collected, the solvent is removed under vacuum and the residue is dissolved in methanol and precipitated with excess ether. Filtration and drying separate the solid product.

B. 2 ', 3-N-trifluoroacetyl gentamycin C ₁ .

1.2'N-trifluoroacetyl gentamycin C ₁

1.7 g of gentamicin C ₁ was dissolved in 20 ml of methanol, cooled to 4 ° C. and 0.46 ml (0.5% g) of ethyl thioltrifluoroacetate was added with stirring.

After reacting for 2 hours, the solution was concentrated in vacuo to obtain a residue, which was chromatographed on 80 g of silica gel using the lower layer of the mixture of chloroform: methanol: water: ammonium hydroxide (10: 5: 4: 1) as the eluent. Fractions containing the main ingredient are combined and concentrated to give 1.4 g of product.

Melting point 108-111 ° C, [α] ²⁶ _D = + 128 ° (concentration 0.3%, H ₂ O)

Elemental Analysis for C ₂₃ H ₄₂ N ₅ O ₈ F ₃ H ₂ O

Calculation: C46.69, H7.50, N11.84, F9.63

Found: C46.66, H7.65, N11.60, F9.24

2. 2 ', 3'-di-N-trifluoroacetyl gentamycin C ₁

0.66 g of the product in 1 was dissolved in 10 ml of methanol, cooled to 4 ° C., and then a solution of 0.148 ml (0.182 g) of ethyl thioltrifluoroacetate was dissolved in 3 ml of methanol. The reaction mixture is stirred for about 16 hours, concentrated in vacuo to give a residue, and then chromatographed according to the method 1 above on 30 g of silica gel.

The column is analyzed by thin layer chromatography, the appropriate fractions are combined and concentrated to yield 0.32 g of the title compound.

Melting point: 121 to 129 ° C, [α] ²⁶ _D = 121 ° (concentration 0.3%, H ₂ O) Elemental analysis for C ₂₅ H ₄₁ N ₅ O ₉ F ₆

Calculated Value: C44.84, H6.17, N10.46

Found: C44.94%, H6.35%, N10.17

C. 6'-N-trifluoroacetyl-sisomicin

After dissolving 20 g of sisomycin in 1.2 L of anhydrous methanol, 6 ml of ethyl thiol trifluoroacetate was added dropwise to 60 ml of methanol over 3 hours with stirring. The reaction was carried out at room temperature for 18 hours and the solvent was removed in vacuo to yield 23.8 g of product with about 95% purity.

Mass Spec.: Other clear peaks at m / e 543M ⁺ , m / e 413, 395, 385, 362, 223, 126:

NMR: (60MH ₀ , D ₂ O) δ 5.37 (doublet, J = 2Hz, H-1 ′); 5.12 (doublet, J = 4 Hz, H-1 ''); 4.96 (wide singlet, H-4); 2.57 (single line, N—CH ₃ ); 1.26 (single line, C-CH ₃ ).

D. 6'-Nt-butoxycarbonyl gentamycin C _1a

Dissolve 2.69 g of gentamicin C _1a in 60 ml of methanol: water (1: 1), cool to 5 ° C, add 1.815 ml of triethylamine, and add 1.91 g of t-butoxycarbonyl azide dropwise with stirring. do. The mixture is stirred at 5 ° C. for 18 hours, 20 ml Amberlite IRA-401S resin (type OH) is added, stirred for 30 minutes, filtered and the filtrate is dried under vacuum. The crude product is chromatographed on 350 g silica gel using a solvent-based lower layer of 2: 1: 1 chloroform: methanol: ammonium hydroxide as the eluent. Take 3 ml fractions and analyze their components by TLC. Fractions containing the main reaction product are combined and evaporated to afford the title compound (0.42 g, 13%).

[α] ²⁶ _D + 137 ° (CH ₃ OH),

PMR: δ 1.23 (3H, S, C—CH ₃ ); 1.45 (9H, S,

C- (CH3); 2.53 (3H, S, N-CH 3); δ 5.08 (2H,

Redundant doublet, J to 3.5 Hz, H-1 'and H-1' ') ppm.

Mass spectrometry: m / e 550 [(M + 1) ⁺ ] and m / e 549 (M ⁺ ).

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

1 g of gentamicin B is dissolved in 30 ml of 50% aqueous methanol and cooled to 5 ° C. 0.297 g-butoxycarbonyl azide was added dropwise with stirring, followed by dropwise addition of 0.186 ml of triethylamine, followed by stirring for 18 hours. The reaction mixture was evaporated in vacuo to give a residue, which was chromatographed on 100 g of silica gel using a lower layer of a 2: 1: 1 chloroformmethanol: ammonium hydroxide solvent system as the eluent. Collect 2 ml fractions and analyze the column by TLC. Fractions containing similar substances (fractions 180 to 230) are combined and evaporated to yield 0.830 g of 6-N-t-butoxy carbonyl gentamicin B having the following properties.

PMR: (60 MHz, D ₂ O) δ1.21 (3H, S, C-CH ₃ );

1.42 (9H, S, C (CH ₃ ) ₃ ); 2.53 (3H, S, N-CH ₃ );

5.2 (1H, doublet, J 4.5 Hz, H-1 &quot;); 5.23 (1H, doublet, J = 3.0 Hz, H-1 ') ppm.

[Production Example 4]

Preparation of Aldehyde Intermediates

A. 2-acet amido-3-hydroxyoctanal

The amino group in 2-amino-3-hydroxyoctanoic acid was protected by conversion to an acetamido group, and the resulting 2-amido-3-hydroxyoctanoic acid was esterified with methanol and the resulting 2-acetamido- The 3-hydroxyoctanoic acid methyl ester is reduced using di-isobutylaluminum hydride according to a known method to give 2-acetamido-3-hydroxy octanal.

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

Diethylacetal of 2-acetoxy-4-acetamidobutanal obtained by treating diethylacetal of 2-hydroxy-4-aminobutanal with acetic anhydride in pyridine was treated with sodium anhydride and methyliodide. Diethylacetal of acetoxy-4- (N-methylacetamido) butanal was obtained, followed by treatment with acid to remove the acetal protecting group, thereby obtaining 2-acetoxy-4- (N-methylacetamido) butanal. To obtain.

Production Example 5

Preparation of Mutamycin

A. Fermentation of MicromonoSporta Diureticosis Strain 1550F-1G NRRL 5742

Preparation of Inoculum

Germination stage 1

M. under sterile conditions. The lyophilic purification culture of diureticosis strain 1550F-1G (or cells obtained by gradient culture) is then added to a shake flask containing 100 ml of sterilization medium.

The flask and its contents are incubated for 2 to 5 days at 35 ° C. on a rotary shaker (280 r.p.m. 2 inch stroke).

Germination stage 2

25 ml of the fermentation broth of germination stage 1 are aseptically transferred to a 2 L shake flask containing 500 ml of the sterile germination medium. The flask and its contents are incubated at 28 ° C. for 3 days on a rotary shaker (280 r.p.m, 2 inch stroke).

Fermentation stage

500 ml of the inoculum obtained in the germination step 2 is transferred to a fermentation tank of 14 L containing 9.5 L of the following sterilization medium.

Before sterilizing the medium, the pH is adjusted to 8 and an aqueous solution containing 8.0 g of streptamine is added. The contents are fermented at 2.5 atmospheres with agitation of 4.5 liters of air per minute with agitation at 250 r.p.m for 48 to 240 hours under aerobic conditions. The pH of the fermentation broth varies slightly during the production of antibiotics, ranging from about 6.8 to 7.3.

Analyze antibiotic production according to the analytical method for sisomycin and collect the product when the production peaks.

(Fermentation is usually completed in about 7 days)

Peak production of mutamycin 1, 2, 4, 5 and 6 is about 5-50 mcg / ml.

B. Isolation of Mutamycin 1

7.0 g of oxalic acid is added to the broth from Preparation Example A with stirring and acidified to pH 2.0 with 6N sulfuric acid. The mixture is stirred for about 15 minutes and filtered using an appropriate filtrate. The filtrate is neutralized with 6N ammonium hydroxide (pH7) and the filtrate is passed through a 1.0 liter ammonium cation exchange resin column (e.g., Amberlite IRC-50 Rom and Haas, Pennsylvania, Philadelphia), discard the used juice after use. Is eluted with 2N ammonium hydroxide to collect approximately 100 ml of fraction. This column eluate is analyzed by disk testing each fraction against Staphylococcus Oreus ATCC6538P. The effective fractions are combined, evaporated under vacuum to about 100 ml, and lyophilized to give a solid product. The product is treated several times with warm methanol, powdered, filtered and the filtrate is evaporated to give a residue. The product is chromatographed on 25 g of silica gel using a solvent-based lower layer of chloroform: methanol: ammonium hydroxide (1: 1: 1) as eluent, and the fractions showing antibiotic activity are combined and evaporated to obtain mutamycin 1.

Mass spectrometry: (M + 1) ⁺ m / e 464, 118, 142, 160, 127, 161, 179, 189, 207, 315, 333, 320, 338, 348, 366, 378, 446.

C. In a similar manner, streptamine is prepared in the same amount of 2, 5-dideoxystrepamine, 2-epi-strepamine, 2-5-dideoxy-5-aminostreptamine or 5-epi-2 Substitution with deoxystreptamine and treatment with Preparation Examples A and B yields mutamycin 2, 4, 5 and 6, respectively.

Mass spectrometry data:

Mutamycin 2: (M + 1) ⁺ m / e 432, 127, 118, 142, 160, 129, 147, 157, 175, 255, 274, 283, 301, 288, 306, 316, 336, 346, 414, 431.

Mutamycin 4: (M + 1) ⁺ m / e 464, 127, 118, 142, 160, 161, 179, 189, 207, 315, 333, 320, 338, 348, 366, 378, 446.

Mutamycin 5: (M + 1) ⁺ m / e 447, 127, 118, 142, 160, 144, 162, 190, 270, 288, 316, 303, 321, 331, 349, 446.

Mutamycin 6: (M + 1) ⁺ m / e 448, 145, 163, 173, 191, 127, 118, 142, 160, 271, 289, 299, 317, 304, 322, 332, 350, 447.

Example 1

1-N-substituted sisomycin

A. 1-N-ethylcysomycin

To a solution of 5 g of sisomycin in 250 ml of water, 1 N sulfuric acid is added until the pH is about 5. 2 ml of acetaldehyde is added to this sisomycin sulfate addition salt solution, and after stirring for 10 minutes, 0.85 g of sodium cyanoborohydride is added. Containing by processing and freeze-dried 1-ethyl-N- seesaw ^{azithromycin-:} Continue stirring 15 minutes at room temperature and was concentrated to make the capacity of about 100ml and then basic ion exchange resin under vacuum () Amberlite IRA401S (OH example) Get the residue The residue is purified by chromatography on 200 g of silica gel using a lower layer of chloroform-methanol-7% ammonium hydroxide (2: 1: 1) solvent system as the eluent. Analogous eluates are combined and concentrated in vacuo to yield a residue containing 1-N-ethylcysomycin (amount: 1.25 g). Again on 100 g of silica gel, a solvent system of chloroform-methanol-3.5% ammonium hydroxide (1: 2: 1) was used as the eluent and purified further by chromatography. The eluates are combined, passed through a basic ion exchange resin column and lyophilized to yield 0.54 g of 1-N-ethylcisomycin.

[α] ²⁶ _D + 164 ° (0.3%, H ₂ O)

pmr (ppm) (D ₂ O): δ 1.05 (3H, t, J = 7 Hz, -CH ₂ CH ₃ );

1.19 (3H, S, -C-CH ₃ ); 2.5 (3H, S, N-CH ₃ );

4.85 (1H, m, = CH-); 4.95 (1H, doublet, J = 4 Hz, H ₁ ''); 5.33 (1H, doublet, J = 2.5 Hz, H ₁ ).

Mass spectrometry: (M + 1) ⁺ m / e476, 127, 154, 160, 173, 191, 201, 219, 256, 299, 317, 332, 345, 350, 360, 378, 390, 400.

B. Other solutions such as acetic acid, trifluoroacetic acid, P-toluenesulfonic acid, hydrochloric acid, phosphoric acid or nitric acid, instead of adding 1N sulfuric acid to a solution of sisomycin in water according to the method of Example 1A until the pH is about 5 Acids can also be used.

The acidified aqueous solution is treated with acetaldehyde and sodium cyano borohydride and the reaction product is purified as above to give 1-N-ethylcysomycin.

C. Alternatively, using the same amount of other hydride donor-reducing agent in place of sodium cyano borohydride in the method of Example 1A, for example, 1 mole of morpholinoborane, tetrabutylammonium cyanoborohydride or sodium Borohydrides are used to obtain 1-N-ethalsisomycin.

D. 1-N-methylsisomicin

To a solution of 4.64 g of sisomycin in 180 ml of water, add 1N sulfuric acid until the pH of the solution is about 5. 1.2 ml 37% formaldehyde solution is added, stirred for 10 minutes, and then 460 mg of sodium cyanoborohydride is added. The reaction solution is passed through a column of basic ion exchange resin (e.g., Amberlite IRA 401S (OH-type) and freeze-dried.) The residue is dried over silica gel with chloroform-methanol-7% ammonium hydroxide (2: 1: 1). The lower layer of the solvent system was chromatographed using the eluent and the eluates containing 1-N-methylcysomycin were combined and evaporated under vacuum to obtain a residue of 1-N-methylcysomycin.

[α] ²⁶ _D + 153 ° (0.3%, H ₂ O)

Mass spectrometry: (M + I) ⁺ m / e 462, 127, 140, 150, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336 (W), 346, 376, 386.

E. 1-N- (n-propyl) sisomicin

In a similar manner to Example 1A, a solution of dissolving sulfuric acid addition salts of sisomycin in water was treated with propanal and sodium cyanoborohydride and separated and purified similarly to 1-N- (n-propyl) seesaw. Obtain mycin.

[α] ²⁶ _D + 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-CH ₃ ); 2.45 (3H, S, -N-CH ₃ ); 4.82 (1H, m, = CH-); 4.90 (1H, doublet, J = 4HZ, H ₁ ''); 5.78 (1H, doublet, J = 2HZ, H ₁ ').

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

F. 1-N- (n-butyl) sisomycin

To a solution of 3 g of sisomycin in 200 ml of water, add 1 N sulfuric acid until the pH is about 3.5, add 1.5 ml of n-butanal, stir for 10 minutes, and then add 450 mg of sodium cyanoborohydride. Stirring was continued for 1 hour and concentrated under vacuum to be a dose of about 100ml of this basic ion exchange resin (for example, Amberlite IRA 401S (OH ^- is passed through a column of a) and freeze-drying of the thus obtained residue was purified by 140g Chromatography on silica gel with a lower layer of chloroform-methanol-7% ammonium hydroxide (2: 1: 1) solvent system Fractions containing 1-N- (n-butyl) sisomycin are combined and evaporated under vacuum to 1-N. A residue containing-(n-butyl) sisomicin is obtained.

[α] ²⁶ _D + 129 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.82 (3H, t, J = 7HZ, -CH ₂ CH ₃ ); 1.15 (3H, S, CH ₃ ); 2.46 (3H, S, -N- CH 3) 4.82 (1H, m, = CH -); 4.92 (1H, doublet, J = 4HZ, H ₁ ''); 5.29 (1H, doublet, J = 2HZ, H ').

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

G. Other 1-N-alkyl, 1-N-alkenyl, 1-N-aralkyl sisomycin

According to the method of Example 1A, an equivalent amount of the following alkylaldehyde is used instead of acetaldehyde.

2-methylpropanal

2. n-pentanal

3. 3-ethylbutanal

4. 2-methylbutanal

5. 2,2-dimethylpropanal

6. 2-ethylbutanal

7. n-octanal

8. Propane

9. 2-ethyl-2-hexenal

10. benzaldehyde and

11.Phenylacetaldehyde

In each case, the reaction was carried out in a similar manner to Example 1A, separated and purified to obtain the following compounds, respectively.

1.1-N- (β-methylpropyl) sisomicin

2. 1-N- (n-pentyl) sisomicin

3. 1-N- (γ-methylbutyl) sisomicin

4. 1-N- (β-methylbutyl) sisomicin

5. 1-N- (β, β-dimethylpropyl) sisomicin

6. 1-N- (β-ethylbutyl) sisomicin

[α] ²⁶ _D + 121 ° (0.4%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.15 (6H, t, 6.5HZ, CH ₂ -CH ₃ ); 2.4 (3H, S, N-CH ₃ ); 4.78 (1H, m, = CH-); 4.88 (1H, doublet, 4.0 HZ, H ₁ ''); 5.22 (1H, doublet, 2.0HZ, H ₁ ')

Mass spectrometry: (M + 1) m / e 532, 127, 160, 210, 229, 256, 275, 355, 373, 388, 401, 406, 416, 446, 456.

7. 1-N- (n-octyl) sisomicin

8. 1-N- (β-propenyl) sisomicin

9. 1-N- (β-ethyl-β-hexenyl) sisomicin

10. 1-N-benzyl soymycin

11. 1-N-phenylethylsisomicin

H. 1-N- (hydroxyalkyl) -sisomicin

In Example 1A an equivalent amount of the following aldehyde is used instead of acetaldehyde.

5-hydroxypentanal

2. 2-hydroxypropanal

3. 2-hydroxy-3-methylbutanal

4. 2-hydroxy-2-methylpropanal

5. 4-hydroxy butanal

6. ε-hydroxy octanal and

7. 2-hydroxy-4-pentanal

Reaction, separation and purification in a similar manner to Example 1A yield the following products, respectively.

1.1-N- (ε-hydroxypentyl) sisomicin

2. 1-N- (β-hydroxypropyl) sisomicin

3. 1-N- (β-hydroxy-γ-methylbutyl) sisomicin

4. 1-N (β-hydroxy-β-methylpropyl) sisomicin

5. 1-N- (δ-hydroxybutyl) -sisomicin

6. 1-N- (W-hydroxyoctyl) sisomicin

7. 1-N- (β-hydroxy-δ-pentenyl) sisomicin

I. 1-N- (δ-aminobutyl) sisomicin

To a solution of 3 g of sisomycin in 120 ml of water is added dropwise 1N sulfuric acid until the pH is about 5. 60 ml of dimethylformamide is added to a sisomycin sulfate addition salt solution, followed by a solution of 2 g of 4-phthal amidobutanal in 10 ml of dimethylformamide. After continuing stirring for 10 minutes, 420 mg of sodium cyanoborohydride was added, and after about 20 minutes, the reaction solution was added to 1 liter of anhydrous methanol with stirring, and sulfuric acid of 1-N- (δ-phthalimidobutyl) sisomicin was added. The precipitate containing the salt is collected by filtration.

The precipitate is dissolved in water and purified by passing the aqueous solution through a basic ion exchange resin. The residue was obtained by evaporation in vacuo and chromatographed on silica gel with the lower layer of chloroform-methanol-7% ammonium hydroxide (2: 1: 1) solvent mixture as eluent and then evaporated to evaporate 1-N- (δ-phthal). A residue containing imidobutyl) -sisomicin is obtained.

To 0.5 g of 1-N- (δ-phthalimidobutyl) -sisomicin is added 5 ml of 5% ethanol hydrazine hydrate and heated to reflux for 3 hours. The reaction solution is poured into a large amount of tetrahydrofuran and filtered to collect a precipitate containing 1-N- (δ-aminobutyl) sisomycin by filtration.

Compounds in this example can be obtained as follows.

(1) 4-acetamidobutyraldehyde

5 mg of 4-acetamidobutyraldehyde diethylacetal are dissolved in 75 ml distilled water and 5 ml of 1N sulfuric acid is added. This solution was checked by thin layer chromatography at room temperature and left at room temperature until hydrolysis was completed.

The solution is neutralized with sodium bicarbonate, saturated with sodium chloride and extracted with chloroform. The chloroform extract is distilled to obtain a residue containing 4-acetamidobutyraldehyde, which can be used without further purification in the next reaction.

(2) 1-N- (δ-acetamidobutyl) sisomicin

0.1 N sulfuric acid is added to a solution of 3 g of sisomycin in 120 ml of distilled water until the pH is about 5. 6 g of δ-acetamidobutyraldehyde is added and after 10 minutes 600 g of solid sodium cyanoborohydride is added. After 2 hours the solution is concentrated in small portions and poured into methanol. Then filtered to collect the precipitate was dissolved in water, Amberlite IRA 401-S (OH ^-) is passed through the ion exchange resin column.

The eluate is evaporated and the residue is chromatographed on silica gel using the bottom layer of chloroform: methanol: 7% ammonium hydroxide solvent mixture followed by evaporation to give a residue containing 1-N- (δ-acetamidobutyl) sisomycin. .

(3) 1-N- (δ-aminobutyl) sisomicin

1-N- (δ-acetamidobutyl) sisomycin obtained in the above example was treated with 10% potassium hydroxide solution at 100 ° C. for 3 hours and neutralized with Amberlite IRC-50 ion exchange resin, followed by neutralization with 2N hydroxide. Elution with ammonium solution. The eluate is concentrated and the resulting residue is taken up in water and lyophilized to give 1-N- (δ-aminobutyl) sisomicin.

[α] ²⁶ _D + 109 ° (0.3%, H ₂ O).

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

J. Other 1-N- (aminoalkyl) sisomicins and 1-N- (hydroxyaminoalkyl) sisomicins

The sulfuric acid addition salt of sisomycin is reacted with sodium cyano borohydride in dimethylformamide solution followed by an aldehyde substituted with amino according to a method analogous to Example 1I.

1.3-phthalimidopropanal

2. 5-talimidopentanal

3. 2-phthalimidopropanal

4. 2-Hydroxy-5-phthalimidopentanal

5. 3-Methyl-3-hydroxy-4-phthalimidobutanal

6. 2-hydroxy-4-phthalimidobutanal

7. 2-phthalimido-3-methylbutanal

8. 2-hydroxy-3-phthalimidopropanal

9. 2-hydroxy-2-methyl-3-phthalimidopropanal and

10. 8-phthalimidooctane.

Reaction, separation and purification were carried out according to a similar method as in Example 1A, to obtain the following products, respectively.

1. 1-N- (γ-phthalimidopropyl) sisomycin

2. 1-N- (ε-phthalimidopentyl) sisomycin

3. 1-N- (β-phthalimidopropyl) sisomycin

4. 1-N- (β-hydroxy-ε-phthalimidopentyl) sisomycin

5. 1-N- (γ-methyl-γ-hydroxy-δ-phthalimidobutyl) sisomycin

6. 1-N- (β-hydroxy-δ-phthalimidobutyl) sisomycin

7. 1-N- (β-phthalimido-γ-methylbutyl) sisomycin

8. 1-N- (β-hydroxy-γ-phthalimidopropyl) sisomycin

9. 1-N- (β-hydroxy-β-methyl-γ-phthalimidopropyl) sisomycin

10. 1-N- (W-phthalimidooctyl) sisomycin.

The above N-phthalimidoalkyl sisomycin derivatives are treated with ethanolic hydrazine hydrate as described in Example 1I to give the following compounds, respectively.

1. 1-N- (γ-aminopropyl) sisomycin

2. 1-N- (ε-aminopentyl) sisomycin

3. 1-N- (β-aminopropyl) sisomycin

4. 1-N- (β-hydroxy-ε-aminopentyl) sisomycin

5. 1-N- (γ-methyl-γ-hydroxy-δ-aminobutyl) sisomycin

6. 1-N- (β-hydroxy-δ-aminobutyl) sisomycin

7. 1-N- (β-amino-γ-methylbutyl) sisomycin

8. 1-N- (β-hydroxy-γ-aminopropyl) sisomycin

9. 1-N- (β-hydroxy-β-methyl-γ-aminopropyl) sisomycin and

10. 1-N- (W-aminooctyl) sisomycin

K. Alkylaminoalkylsisomicins and other hydroxy amino alkyl sisomycins

(1) 1-N- (β-methylaminoethyl) sisomycin

In a similar manner to Example 1A, a solution of sisomycin in water is treated with 1N sulfuric acid and 2- (N-methylacetamido) acetaldehyde followed by sodium cyanoborohydride. This is separated to give 1-N- (β- (N-methylacetamido) -ethyl) sisomycin.

The N-acetylated intermediate was reacted with 10% sodium hydroxide solution at 100 ° C. for 3 hours, then poured into Amberlite IRC 50 ion exchange resin, eluted with 2M ammonium hydroxide, and the eluate was concentrated in vacuo to 100 ml. Dosing and lyophilization yields a residue containing 1-N- (β-methylamino ethyl) sisomycin.

(2) (2) 2-acetamido-3-hydroxy octanal or 2-acet amido instead of 2- (N-methylacetamide) acetaldehyde as the aldehyde reagent according to the method of Example 1K (1) above. Corresponding 1-N- (aminoalkyl) sisomycin (eg: 1-N- (β-amino-γ-hydroxyoctyl) sisomycin and 1-N using other aldehydes such as -4-pentanal -(β-amino-δ-pentenyl) sisomycin are obtained respectively.

L. 1-N- (β-aminoethyl) sisomycin

1-N- (γ-aminopropyl) sisomycin

According to another method of Example 1I, 0.1N sulfuric acid was added to the sisomycin solution to pH about 5, and then the solution was treated with β-acetamido acetaldehyde followed by solid sodium cyanoborohydride. Isolation and purification give 1-N-acetamidoethyl) sisomycin.

This 1-N- (β-acetamiethylethyl) isocyancin was hydrolyzed with 10% potassium hydroxide solution and separated and purified according to a method similar to Example 1-I- (3) above to obtain 1-N- ( β-aminoethyl) sisomycin.

Mass spectrometry: (M + 1) ⁺ m / e 49l, 160, 169, 187, 206, 216, 234, 256, 283, 314, 325, 334, 347, 360, 370, 375, 405, 415.

Following the above method, 1-N- (γ-acetamidopropyl) sisomycin was obtained using γ-acetamidopropanal instead of β-acetamido acetaldehyde and hydrolyzed with 10% potassium hydroxide solution. Isolation and purification afford 1-N- (γ-aminopropyl) sisomycin.

Example 2

1-N-substituted-gentamycin C _1a

A. 1-N-ethylgentamycin C _1a

A solution of 5 g of gentamicin C _1a in 125 ml of water is added with 1N sulfuric acid to pH 5.2, followed by 2 ml of acetaldehyde, stirring for 5 minutes, and then 1 g of sodium cyano borohydride. Concentration of the solution under continued for 30 minutes and stirred at room temperature and then vacuum at a dose of approximately 75ml and the solution basic ion exchange resin (e.g. Amberlite IRA401S (OH ^- was passed through a column of a) freeze-dried 1-N -The residue containing ethyl gentamycin C _1a is obtained.

Purified by chromatography on 200 g of silica gel using a lower layer of chloroform-methanol-7% ammonium hydroxide (2: 1: 1) solvent system as eluent. The eluates are combined and concentrated in vacuo to give a residue containing 1-N-ethylgentamicin C _1a (amount: 0.95 g). This 1-N-ethylgentamicin C _1a was further purified by chromatography on 100 g of silica gel using chloroform-methanol-3.5% ammonium hydroxide (1: 2: 1) solvent system as eluent again. Similar eluates were combined and treated with basic ion exchange resin and lyophilized to yield 0.42 g of 1-N-ethylgentamicin C _1a .

[α] ²⁶ _D + 118 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 1.06 (3H, t, J = 7 Hz, -CH ₂ CH ₃ ); 1.19 (3H, s, C-CH ₃ ); 2.51 (3H, s, -N-CH ₃ ); 4.97 (1H, doublet, J = 4 Hz, H ₁ ''); 5.16 (1H, doublet, J = 3.5 Hz, H ₁ ').

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

B. Instead of adjusting the pH to about 5.2 with 1N sulfuric acid in Example 2A, add polyacid, such as acetic acid, P-toluenesulfonic acid, trifluoroacetic acid, hydrochloric acid, hydrochloric acid, phosphoric acid or nitric acid.

This acidified gentamicin C _1a solution was treated with acetaldehyde and sodium cyanoborohydride according to the method of Example 2A, separated and purified to give 1-N-ethylgentamicin C _1a .

C. Other aldehyde reagents in the process of Example 2A, instead of acetaldehyde, for example formaldehyde, n-propanal, n-butanal, n-octanal, hydroxy acetaldehyde, 4-hydroxybutanal and phenyl Using acetaldehyde the corresponding 1-N-alkylgentamicin C _1a , for example 1-N-methylgentamicin C _1a , 1-N- (n-propyl) gentamycin C _1a , 1-N- (n -Butyl) gentamycin C _1a , 1-N- (n-octyl) gentamycin C _1a , 1-N- (β-hydroxyethyl) gentamycin C _1a , 1-N- (δ-hydroxy butyl) genta Mycin C _la and 1-N- (phenylethyl) gentamycin C _la are obtained respectively.

Example 3

1-N-substituted-versamycin

A. 1-N-ethylveramycin

To a solution of 0.5 g of Verdamycin in 65 ml of water, add 1N sulfuric acid until the pH is about 4.9, and then add 0.2 ml of acetaldehyde. Stir for 5 minutes, add 65 mg of sodium cyanoborohydride, concentrate in vacuo to a volume of about 10 ml and pour in 50 ml of methanol with stirring. The precipitate was collected by filtration to collect 1-N-ethylversamycin, which was purified by chromatography on 100 g of silica gel using chloroform-methanol-3.5% ammonium hydroxide (1: 2: 1) solvent system as the eluent. Similar fractions are collected and the fractions containing the main component are evaporated in vacuo to give a residue containing 1-N-ethylversamycin.

This was further purified by chromatography on 7 g of silica gel using a lower layer of chloroform-methanol-7% ammonium hydroxide (2: 1: 1) solvent system as an eluent, and then fractions were collected and evaporated under vacuum to evaporate 1-N-ethyl. Obtain a residue of verdamycin.

Yield: 50 mg

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

B. Other aldehyde intermediates such as formaldehyde, n-propanal, n-butanal, n-octanal, hydroxyacetaldehyde, 4-hydroxybutanal and phenylacetaldehyde in the method of Example 3A instead of acetaldehyde Are separated and purified accordingly to obtain the following compounds, respectively.

1-N-methylvermycin and

1-N- (n-propyl) verdamycin,

[α] ²⁶ _D + 122 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.98 (3H, t, J = 7 Hz, CH ₂ CH ₃ ); 1.19 (3H, s, C-CH ₃ ); 1.16 (3H, doublet, J = 6 Hz, CH-CH ₃ ); 4.81 (1H, m, = CH-); 4.97 (1H, doublet, J = 4.0 Hz, H ₁ ''); 5.30 (1H, doublet, J = 2.0 Hz, = H ₁ ').

Mass spectrometry: (M + 1) ⁺ m / e 528, 141, 160, 168, 187, 205, 215, 233, 270, 346, 355, 373, 504.

1-N- (n-butyl) verdamycin.

[α] ²⁶ _D + 121 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.8 (3H, t, J = 6.5 Hz, CH ₂ -CH ₃ ); 2.45 (3H, s, NCH ₃ ); 4.8 (1H, m, C = CH-); 4.92 (1H, doublet, J = 4.0 Hz, H ₁ ''); 5.25 (1H, doublet, J = 2.0 Hz, H ₁ ',).

Mass spectrometry: (M + 1) ⁺ m / e 518, 141, 160, 182, 201, 219, 229, 247, 270, 341, 360, 378, 387, 388, 418, 442.

1-N- (β-hydroxyethyl) verdamycin

1-N- (δ-hydroxybutyl) verdamycin and

1-N-phenylethylverdamycin.

Example 4

1-N-substituted-gentamycin C ₁

A. 1-N-ethylgentamycin C ₁

According to a method similar to Example 1A, 1N sulfuric acid was added to a solution of 5 g of gentamicin C ₁ in 250 ml of water until the pH of the solution was about 5, and the acidic solution was added to acetaldehyde and sodium cyanoborohydride. Treatment, separation and purification to afford 1-N-ethyl gentamycin C ₁ .

[α] ²⁶ _D + 114 ° (0.35, H ₂ O).

pmr (ppm) (D ₂ O): δ 1.03 (3H, t, 7 Hz, -CH ₂ CH ₃ ); 1.03 (3H, doublet, J = 6.5 Hz, -CH-CH ₃ ); 1.17 (3H, s, C-CH ₃ ); 2.32 (3H, s, 6'N-CH ₃ ); 2.49 (3H, s, 3``-NHCH ₃ ); 4.94 (1H, doublet, J = 4 Hz, H ₁ ''); 5.13 (1H, doublet, 'J = 3.5 Hz, H ₁ ').

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

B. Other aldehyde reagents, such as formaldehyde, n-propanal, n-butanal, n-octanal, hydroxy acetaldehyde, 4-hydroxybutanal and phenylacetaldehyde in place of acetaldehyde in Example 4A 1-N-methylgentamicin C ₁ , 1-N- (n-propyl) gentamicin C _1, 1-N- (n-butyl) gentamicin C _{1, 1} -N- (n-octyl) gentamycin C ₁ , 1-N- (β-hydroxyethyl) gentamycin C _1.

[α] ²⁶ _D + 98.0 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.99 (3H, d, J = 6.5 Hz, 6-CH ₃ ); 1.13 (3H, s, 4 ''-CH ₃ ); 2.28 (3H, s, 6, -NCH ₃ ); 2.45 (3H, s, 3 ''-NCH ₃ ); 4.97 (1H, doublet, J = 4 Hz, H ₁ ''); 5.11 (1H, d, J = 3.5 Hz, H ₁ '')

Mass spectrometry: (M + 1) ⁺ m / e 522, 446, 404, 394, 391, 376, 373, 366, 363, 348, 345, 333, 286, 235, 217, 207, 189, 160, 157.

ν _max (KBR) 3, 300, 1, 060 cm ^-1 .

1-N- (δ-hydroxybutyl) gentamycin C ₁ and

1-N- (phenylethyl) gentamycin C ₁ .

[α] ²⁶ _D + 99.4 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 0.99 (3H, d, V = 6.5 Hz, 6′-CH ₃ ); 1.10 (3H, s, 4 ''-CH ₃₎ ; 2.28 (3H, s, 6'-NCH ₃ ); 2.43 (3H, s, 3``-NCH ₃ ); 4.88 (1H, doublet, J = 4 Hz, H ₁ '); 5.08 (1H, doublet, J = 3.5 Hz, H ₁ '); 7.33 (5H, s, -C ₆ H ₅ ).

Mass spectrometry: (m + 1) ⁺ m / e 582, 506, 464, 454, 451, 436, 433, 426, 423, 408, 405 (w), 393, 295, 286, 277, 267, 249, 160 , 157.

ν _max (KBR) 3, 300, 1, 050, 1, 030 cm ^-1

To obtain each.

Example 5

1-N-Substituted Antibiotics G-52

A. 1-N-ethyl antibiotic G-52

Dissolve 875 mg of antibiotic G-52 in 40 ml of distilled water and add 1N sulfuric acid until the solution has a pH of about 3.5.

0.7 ml of acetaldehyde are added, the reaction mixture is stirred for 10 minutes, and then 100 mg of sodium cyanoborohydride is added. After analyzing the reaction solution by thin layer chromatography, when the starting material antibiotic G-52 was completely reacted (about 10 minutes), the solution was concentrated in a vacuum of 35 to 40 ° C. to a volume of about 10 ml. This concentrated solution is passed through a basic ion exchange resin and lyophilized to obtain a residue containing 1-N-ethyl antibiotic G-52.

Purified by chromatography on a silica gel column (4 ′ × 1/2 ′) using a lower layer of chloro-methanol-7% ammonium hydroxide (2: 1: 1) solvent system as eluent. Eluents (measured by thin layer chromatography) are combined and the eluate containing the main component is concentrated in vacuo to give a residue containing 1-N-ethyl antibiotic G-52 (yield 60 mg), again chloroform-methanol Chromatography using a lower layer of a -7% ammonium hydroxide (1: 2: 1) solvent system as eluent yields further 35 mg of a residue containing 1-N-ethyl antibiotic G-52. The residue is combined and passed through a basic ion exchange resin column (e.g., Amberlite IRA401S) and lyophilized to yield 90 mg of 1-N-ethyl antibiotic G-52.

[α] ²⁶ _D + 122.1 ° (0.3%, H ₂ O)

pmr (ppm) (D ₂ O): δ 1.06 (3H, t, J = 6.5 Hz, 1N-CH ₂ CH ₃ ); 1.21 (3H, s, 4``-C-CH ₃ ); 2.30 (3H, s, 3 ''-N-CH ₃ ); 2.50 (3H, s, 6'-N-CH ₃ ); 4.94 (1H, m, H ₄ '); 4.97 (1H, doublet, J = 4.0 Hz, H ₁ ″); 5.34 (1H, doublet, J = 2.5 Hz, H ₁ ').

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

B. Use of other aldehyde reagents such as formaldehyde, n-propanal, n-butanal, n-octanehydroxy acetaldehyde, 4-hydroxybutanal and phenylacetaldehyde in place of acetaldehyde in Example 5A above; According to the embodiment, the purified product was purified by 1-N-methyl-antibiotic G-52, 1-N- (n-propyl) -antibiotic G-52, 1-N- (n-butyl) -antibiotic G-52, 1 -N- (β-hydroxyethyl) -antibiotic G-52, 1-N- (δ-hydroxybutyl) -antibiotic G-52 and 1-N-phenylethyl-antibiotic G-52 are obtained.

Example 6

A. Following a method analogous to Example 1A, each of the following solutions of 4, 6-diaminoglycosyl-1, 3-diamino cyclitol in water was treated with sulfuric acid followed by acetaldehyde and sodium cyanoborohydro. do.

Gentamicin A,

2. gentamicin B,

3. gentamicin B _1,

4. gentamicin C _1,

5. gentamicin C _1a,

6. gentamicin C _2,

7. gentamicin C _2a,

8. gentamicin C _2b,

9. gentamicin X _2,

10. antibiotic G-418,

11.antibiotic JI-2A,

12. Antibiotic JI-20B,

13. Tobramycin

14. antibiotic 66-40B,

15. antibiotic 66-40D,

16. Mutamycin 1,

17. mutamycin 2,

18. mutamycin 4,

19.mutamicin 5,

20.Mutamicin 6.

The reaction product was separated and purified in a similar manner to Example 1A to give the following corresponding 1-N-ethyl compounds, respectively.

1.1-N-ethylgentamicin A,

2. 1-N-ethylgentamicin B,

3. 1-N-ethylgentamycin B _l ,

4. 1-N-ethylgentamycin C ₁ ,

5. 1-N-ethylgentamycin C _1a ,

6. 1-N-ethylgentamycin C ₂ ,

7. 1-N-ethylgentamicin C _2a,

8. 1-N-ethylgentamycin C _2b ,

9. 1-N-ethylgentamicin X _2,

10. 1-N-ethyl antibiotic G-418,

11. 1-N-ethyl antibiotic JI-20A,

12. 1-N-ethyl antibiotic JI-20-B,

13. 1-N-ethyltobramycin,

14. 1-N-ethyl antibiotic 66-40 B,

15. l-N-ethyl antibiotic 66-40 D,

16. 1-N-ethylmutamycin 1,

17. 1-N-ethylmutamycin 2,

18. 1-N-ethylmutamycin 4,

19. 1-N-ethylmutamycin 5,

20. 1-N-ethylmutamycin 6.

B. Propanal instead of acetaldehyde in Example 6A is used to obtain the corresponding 1-N- (n-propyl) derivatives of each of 4, 6-diaminoglycosyl-1, 3-diaminocyclitol.

C. Each of the starting materials 4, 6-diaminoglycosyl-1, 3-diamino cyclitol described in Example 6A was treated according to the method of one of 1K in Example 1A to obtain a corresponding 1-N thereof. Obtain an alkyl derivative.

Example 7

Process for preparing 1-N-substituted-4, 6-di- (aminoglycosyl) -1, 3-diamino cyclitol via selectively blocked intermediates

A. Preparation of 1-N-ethylgentamicin C ₁ via 2 ′, 3-di-N-substituted intermediates

240 mg of 2 ', 3-di-N-trifluoro acetyl gentamicin C ₁ is dissolved in 10 ml of water-methanol (2: 1) and then 1N sulfuric acid is added to adjust the pH of the solution to 3.5. 0.19 ml of acetaldehyde is added and stirred for 10 minutes, followed by 27 mg of sodium cyanoborohydride, followed by further stirring for 10 minutes. It was evaporated under vacuum to obtain a residue containing 1-N-ethyl-2, 3-di-N-trifluoro acetylgentamicin C ₁ , which was then dissolved in 50 ml of concentrated ammonium hydroxide solution and left at room temperature for 24 hours. do. The mixture is evaporated in vacuo and chromatographed on silica gel (12 g) using the bottom layer of a mixture of chloroform-methanol-10% ammonium hydroxide solution (2: 1: 1) as eluent. Similar fractions analyzed by thin layer chromatography were combined and evaporated in vacuo to give a residue containing 1-N-ethylgentamicin C ₁ (winch 80 mg).

B. Preparation of 1-N-ethylcysomycin via 6'-N-substituted intermediate

6'-N-t-butoxycarbonyl sisomycin prepared by a method similar to Preparation 3D is treated with sulfuric acid in aqueous methanol according to a method similar to Example 7A and then reacted with acetaldehyde and sodium cyanoboro hydride.

The reaction mixture is left at room temperature for 30 minutes and evaporated in vacuo to give 1-N-ethyl-6'-N-t-butoxycarbonylsisomicin. The residue was dissolved in trifluoroacetic acid, left for 10 minutes, and added to excess anhydrous methanol, and then the precipitate of trifluoroacetic acid salt of 1-N-ethylcisomycin generated was filtered. This was chromatographed on silica gel similarly to Example 7A using a lower layer of chloroform-methanol-ammonium hydroxide solvent system to give 1-N-ethylcisomycin.

C. Preparation of 1-N-methylsisomicin from 3 ''-N-4 ''-O-carbonyl-sisomicin

5 g of 3 ''-N-4 ''-O-carbonyl-sisomicin is dissolved in 300 ml of distilled water and treated with 1N sulfuric acid until the pH is 2.5. 2 ml of 37% formaldehyde solution is added and after 10 minutes, a solution of 5 ml of 500 mg sodium cyanoborohydride is added dropwise. After 1 hour, the mixture was evaporated in vacuo to reduce the volume to 1/2 and the pH of the concentrate was adjusted to pH 11 by adding 1N sodium hydroxide, followed by evaporation to dryness. The residue was extracted with methanol (3 × 100 ml) and the methanol extracts were combined, diluted with an equal amount of chloroform, filtered and evaporated to dryness to afford crude product 1-N-methyl-3 ''-N-4'-O-carbonyl- Obtains sisomycin.

The crude product was treated with 10% potassium hydroxide solution at 100 ° C. for 5 hours, cooled, passed through Amberlite IRC-50 (H ⁺ ) ion exchange resin, eluted with 2N ammonium hydroxide solution, and the eluate was concentrated. Lyophilization yields crude product 1-N-methyl-sisomicin.

This crude product is chromatographed on silica gel (300 g) with chloroform-methanol-7% ammonium hydroxide (2: 1: 1) to give 1-N-methylsisomicin.

[α] ²⁶ _D + 153 ° (0.3%, H ₂ O).

Mass spectrometry: (M + 1) ⁺ m / e 462, 127, 140, 159, 160, 171, 187, 205, 256, 285, 303, 318, 331, 336 (w), 346, 376, 386.

D. Preparation of 1-N-methylversamycin via 3 '', 4 ''-N-O-oxazolidone intermediate

(1) To 1 g of verdamycin aqueous solution, sodium carbonate is added until the pH is about 8-9. A solution of 5 g of P-nitro phenylchloro carbonate dissolved in 25 ml dimethylformamide was added dropwise with stirring over 3 hours, at which time the sodium carbonate solution was further added so that the pH of the reaction mixture was maintained at about 8-9.

After the addition reaction is complete, stirring is continued for 16 hours at pH 8-9. The mixture is evaporated in vacuo and the resulting residue is extracted several times with hot chloroform, the extracts are combined and evaporated, and chromatographed on 100 g of silica gel with a lower layer of a 2: 1: 1 chloroform-methanol-ammonium hydroxide solvent system as the eluent. . These analogous fractions are combined and evaporated to give verdamycin 3 '', 4 ''-N, O-oxazolidone.

(2) The oxyzolidone derivatives were treated with sulfuric acid, formaldehyde and sodium cyanoborohydride in water according to a method similar to that of Example 1D, and purified by 1-N-methylveramycin-3 '', 4 '. '-N, O-oxazolidone is obtained.

(3) 0.2 g of 1-N-methylversamycin-3 '', 4 ''-N, O-oxazolidone was dissolved in 10 ml of 2N sodium hydroxide in a solution, heated to reflux for 5 hours, and then acetic acid was added to the solution. Neutralize and evaporate to give a residue.

This residue is chromatographed on 10 g silica gel with the bottom layer of a 2: 1: 1 chloroform-methanol-15% ammonium hydroxide mixture and the similar fractions analyzed by this are combined and evaporated to give 1-N-methyl vermycin.

Example 8

Preparation of 1-N-benzylgentamicin C ₁ via tri-N-protected-1-N-schiffbase intermediate

(1) 0.3 g of 2 ', 3-di-N-trifluoroacetylgentamycin C ₁ is dissolved in 12 ml of ethanol, 0.9 ml of benzaldehyde is added thereto, followed by stirring for 3 hours and evaporation under vacuum. The residue is dissolved in 0.8 ml chloroform and this solution is added dropwise to 25 ml hexane-ether (3: 1).

The precipitate was isolated by filtration and dried in vacuo to give 0.38 g of 1-N-3``-N-4 ''-O-bis-benzylidine-2 ', 3-di-N-trifluoroacetylgentamicin C ₁ is obtained. Melting Point: 128 to 134 ° C

[α] ²⁶ _D + 74.6 ° (0.26%, ethanol)

(2) 1.37 g of the product obtained in Example 8 (1) was dissolved in 100 ml of ethanol and then added to a solution of a 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 to pH about 3 with hydrochloric acid, and then stir for further 16 hours. The solution is extracted with ether and the ether layer is separated and decanted. Ammonium hydroxide is added to the aqueous phase until it is basic and then evaporated in vacuo to give a residue. The residue is extracted with 35 ml of hot ethanol and the extracts are combined and evaporated under vacuum. The residue thus obtained is chromatographed on 75 g of silica gel with a lower layer of a solvent system of chloroform-methanol-ammonium hydroxide-water of 2: 1: 0.2: 0.8. The similar fractions analyzed by thin layer chromatography are combined and evaporated to give a residue containing 1-N-benzylgentamicin C ₁ . Melting Point '83 to 88 ℃

[α] ²⁶ _D + 90 ° (0.3%, H ₂ O).

pmr (ppm) (D ₂ O): δ 1.03 (3H, d, J = 7HZ, HC-CH ₃ ); 1.16 (3H, S, C-CH ₃ ); 2.27 (3H, S, N-CH ₃ ); 2.50 (3H, S, N-CN ₃ ); 4.7 (D ₂ O + Ph CH ₂ N ⁻ ); 4.92 (1H, doublet, J = 4HZ, H-1 &quot;); 5.08 (1H, α, J = 3.5HZ, H-1 '); 7.43 (5H, S, aromatic H).

Mass spectrometry: (M + 1) m / e568, 440, 437, 412, 394, 379, 28I, 263, 253, 235, 160, 157.

(3) In the above method, 1-N-propylgentamicin C ₁ and 1-N-phenylethylgentamicin C ₁ are obtained by using other aldehydes such as propionaldehyde or phenylacetaldehyde instead of benzaldehyde.

Example 9

Acid addition salt

A. Sulfate (sulphate addition salt)

Dissolve 5. g of 1-N-ethylcysomycin in 25 ml of water and adjust the pH of this solution to 4.5 using 1N sulfuric acid. It is poured into about 300 ml of methanol with vigorous shaking, shaken continuously for about 10 to 20 minutes and filtered.

The precipitate is washed with methanol and dried in vacuo at about 60 ° C. to give 1-N-ethylcysomycin sulfate.

In this way, sulfates of the compounds of Examples 1 to 8 can be prepared.

B. Hydrochloride

It is dissolved in 25 g of 5.0 g of 1-N-ethyl verdamycin, acidified to pH 5 by addition of 2N hydrochloric acid, and lyophilized to obtain 1-N-ethyl verdamycin hydrochloride.

In this way, the hydrochloride salts of the compounds of Examples 2 to 8 can be prepared.

The present invention consists of at least one 1-N-substituted derivative of 4, 6-di- (aminoglycosyl) 1, 3-diamino cyclitol as defined below, together with a pharmaceutically toxic carrier or coating. Pharmaceutical compositions.

In addition, the present invention is a gene for gentamicin A, gentamycin B, gentamycin B ₁ , gentamycin C ₁ , gentamycin C _1a , gentamycin C ₂ , gentamycin C _2a , gentamicin C _2b , GentamicinX ₂ , sisomycin, verdamycin, tobramycin, antibiotic G-48, antibiotic 66-40B, antibiotic 66-40D, antibiotic JI-20A, antibiotic JI-20B, antibiotic G-52, mutamycin 1 1-N-substituted derivatives of 4, 6-di- (aminoglycosyl) -1, 3-diaminocyclitol selected from mutamycin 2, mutamycin 4, mutamycin 5 and mutamycin 6 Or a pharmaceutically innocuous acid addition salt thereof, which is administered in an amount that is non-toxic and antimicrobially effective. Wherein the substituent is —CH ₂ X wherein X is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylamino hydroxy alkyl, Phenyl, benzyl or tolyl and aliphatic groups have up to 7 carbon atoms and when substituted with amino and hydroxy have substituents on other carbon atoms.

로서 Z는 상술한 X로 나타내는 그룹이며, 단 Z는 카보닐그룹과 함께 부착되어 S-3-아미노-2-하이드록시-프로피오닐 또는 S-4-아미노-2-하이드록시 부티릴 이외의 기이고토브라 마이신의 경우에는 S-5-아미노-2-하이드록시발레릴 이외의 기이다.In addition, the substituent

Z is a group represented by X as described above, provided that Z is attached with a carbonyl group to form a group other than S-3-amino-2-hydroxy-propionyl or S-4-amino-2-hydroxy butyryl. In the case of gotobramycin, it is group other than S-5-amino-2-hydroxyvaleryl.

The compounds according to the invention show activity against a wide variety of microorganisms, especially Gram-negative bacteria, which are resistant to 1-N-unsubstituted starting materials. Thus, the compounds of the present invention can be used alone or in combination with other antibiotics to inhibit or reduce the growth of bacteria in various environments.

For example, the compound is used to disinfect dental and medical instruments infected with glassware, Staphylococcus oreus, or other bacteria that may be blocked by the compounds of the present invention. The activity of the compounds of the present invention against Gram-negative bacteria also makes them useful in eliminating infections caused by Gram-negative bacteria such as Proteus and Pseudomonas spp.

1-N-substituted derivatives of 4, 6-di- (aminoglycosyl) -1, 3-diamino cyclitol, such as 1-N-ethylcysomycin and 1-N-ethylveramycin, It is used to treat medicines, particularly bovine mastitis and Salmonella-induced diarrhea in livestock such as dogs and cats.

In general, the dosage depends on the age and weight of the animal to be treated, the method of administration and the degree of infection of the bacteria. In general, the dosages of the derivatives of 4, 6-di- (aminoglycosyl) -1, 3-diamino cyclitol, which are used for bacterial infection control, are corresponding to 1-N-unsubstituted-4, 6-di-. It will be similar to the required dosage of (aminoglycosyl) -1, 3-diaminocyclitol.

The compounds of the present invention may be administered orally and may also be administered topically in the form of a lotion, which is an aqueous, non-aqueous or emulsion in the form of hydrophilic and hydrophobic ointments, or in the form of a cream. Pharmaceutical carriers useful for preparing these formulations include materials such as water, oils, greases, polyesters, polyols, and the like.

Formulations for oral administration include tablets, capsules, elixirs, etc., and may be mixed with feed. Such dosage forms are most effective for treating bacterial infections of the gastrointestinal tract that cause diarrhea when infected.

In general, topical preparations contain an active compound according to the invention at about 0.1 to 3.0 g per 100 g of ointment, cream or lotion and are administered 2 to 5 times per day.

The antimicrobial agents of the present invention are used in liquid preparations such as solutions or suspensions for use in the ears and eyes and may also be administered parenterally via intramuscular injection. Injectable solutions or decoctions are usually administered 2 to 4 times per day, with an amount of 1 to 15 mg of antimicrobial agent per kg of body weight. The exact dosage depends on the condition and severity of the infection, the strength of the infecting microorganism against the antimicrobial agent and the individual characteristics of the animal species to be treated.

Claims (1)

4, 6-di- (aminoglycosyl) -1,3-diaminocyclitol, which may have an amino protecting group at any other position, is represented by the structures X'-CHO in the presence of a hydride donor-reducing agent. 4, 6-di (amino glycosyl) -1, 3-diaminocyclitol (gentamicin A, gentamicin B, gentamicin), characterized by hydro treatment (removing all protecting groups present in the molecule, if necessary). B _l , gentamycin C ₁ , gentamycin C _1a , gentamycin C ₂ , gentamycin C _2a , gentamycin C _2b , gentamycin X ₂ , sisomycin, versamycin, tobramycin, antibiotic G-418, antibiotic 66 -40 B, antibiotic 66-40 D, antibiotic JI-20 A, antibiotic JI-20 B, antibiotic G-52, mutamycin 1, mutamycin 2, mutamycin 4, mutamycin 5, and mutamycin A process for preparing the 1-N-substituted derivatives of 6) and pharmaceutically toxic acid addition salts thereof. here Substituents are —CH ₂ X where X is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylamino hydroxyalkyl, phenyl , Benzyl or tolyl, and the aliphatic group has up to 7 carbon atoms, and when substituted with amino and hydroxy groups, has a substituent on another carbon atom, X 'is a group defined above as X, where the amino or hydroxy groups present therein can be protected.