DK143111B - PROCEDURE FOR PREPARING AMINOCYCLOTOL COMPOUNDS OR ACID ADDITION SALTS - Google Patents

Description

(11) PRESENTATION 1431 1 1 DENMARK ln, -c '·' ® J * Hp t (21) Application No. 65 1/76 (22) Filed on 17 · Feb. 1976 (24) Unwind 1 Feb 7 1976 (44) The application presented and the petition published on JO. Π13.Γ · 1 9 @ 1

DIRECTORATE OF

PATENT AND TRADE MARKET <30> Wont be requested from it

Feb 18 1975 * 550275 * US

Sep 22 1975 * 615595 * US

<71> STERLING DRUG INC., 9O Park Avenue, New York, N.Y., US.

(72) Inventor: Sol Jacob Dautn, 468 West Lawrence Street, Albany, New York, US: Robert La Grone TTlarke, 5 Sunset Drive, Delmar, New York, US.

(74) Plenipotentiary in the proceedings:

The Hofman-Bang & Bout engineering firm (54) Process for the preparation of aminocyclytol compounds or acid addition salts thereof.

The invention relates to a process for the preparation of novel aminocyclitol compounds having the general formula I or acid addition salts thereof according to the preamble of claim 1, which is characterized by the characterizing part of claim 1.

The culture method used is similar to that described in U.S. Patent No. 5,669,858. As a microorganism, a mutant of Micromonospora purpurea, preferably the organism designated Micromonospora purpurea ATTCC 51 119, is used.

8

The compounds of formula I wherein one of R, R and R is a 2-amino-cc-hydroxy-lower alkanoyl group are prepared by the method described in U.S. Patent No. 3,780,018, and consisting of reacting a compound of formula I, wherein R, Ir and R are all hydrogen, with an N-hydroxysuccinimide ester of the formula

// CH20-C-NHCH2 (CH2) nCHOHCO0-N

\ = / V-1 -iv

ISLAND

wherein n is 0 or 1. The resulting mixture of

„4« 2 Q

a scheme of formula I wherein one of R, R and R is q, - (N-Benzyloxy-carbonyl) amino-α-hydroxy-lower-alkanoyl group: - (CH 2) -CH 2 -C-NHCH 2 (CH 2) nCHOHCO- and the other two are hydrogen, are then subjected to hydrogenolysis of the benzyloxycarbonyl group with hydrogen over a catalyst.

As indicated above, a mixture of the three possible isomeric 1 g monoamides is obtained in which the R, R, and R amine hydrogen atoms respectively are replaced by the W- (N-benzyloxycarbonyl) amino-α-hydroxy-lower alkanoyl group. Of course, when individual characterization and examination of these products is desired, they must be separated from one another. The acylation reaction is carried out by reacting molar equivalent amounts of the compound of formula I and the N-hydroxysuccin imide ester, preferably at a temperature of from about -10 to about 10 ° C and in an aqueous solution of an inert organic solvent, e.g. tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dimethylacetamide, dimethylformamide, propylene glycol dimethyl ether and the like.

3 163111

The hydrogenolysis of the benzyloxyearbonyl group is carried out over a palladium-on-carbon catalyst in an inert, water-miscible organic solvent, for example, methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether and the like.

The aminocyclitols used in the practice of the invention are known compounds. These are, for example: streptamine sulfate [Peck et al., J. Am. Chem. Soc. 68, 776 (1946)]; 2-Epistreptamine dihydrochloride [Suami et al., J, Org. Chem. 33, 2831 (1968)]; 2,5-dideoxystreptamine dihydrochloride, m.p. > 300 ° C; and 6,7-diazabicyclo [3,2,1] octane-2,4-diol (exo, exo), m.p. 185 - 193 ° C [The latter two compounds are reported by Testa et al., J. Antibiotics 27, 917-921 (1974)].

The N-hydroxysuccinimide esters of formula IV are a generally known class of compounds.

The compounds of formula I have been tested by a standard successive dilution assay and have been shown to have antibacterial activity, especially against gentamicin-resistant organisms. Thus, the compounds are useful as antibacterial agents.

The compounds of formula I are mainly intended for oral, topical or parenteral administration and can be prepared for use with a pharmaceutical carrier by suspension, either in the form of their free bases or as pharmaceutically acceptable, non-toxic acid addition salts, in an inert carrier such as polyethylene glycol. , or by tableting or encapsulation for oral administration either alone or with suitable additives, or alternatively they may be formulated with conventional creams or vaselines for topical application.

4 143111

The molecular structures of the compounds of formula I were established on the basis of their chromatographic properties determined by thin-layer chromatographic analysis, their magnetic nucleation (NMR) and mass spectrum, decomposition to known compounds, and agreement between calculated and found values for elemental analyzes of the compounds.

The following exemplary embodiments serve to elucidate in detail the process of the invention and the preparation of the ndgangafn-pbl except for it.

MUT A TTOTSrSl'MEcEmEs

The following procedures used different media composed as follows:

Medium 1: N-Z Arnin g / 3_

Glucose 10

Soluble Starch 20 Yeast Extract 5 N-Z-Amine Type A (Difco) 5

CaCO1

Agar 15

Medium 2: Sprouting medium (in distilled water) Meat extract 0.3 <fo

Tryptone 0.5 1 °

Dextrose 0.1 $

Soluble starch 2.4 in »Yeast extract 0.5 ia

CaCO 3 0.4 1 °

Medium 3: Soybean Glucose Eli

Soybean meal 30

Dextrose (Cerelose) 40

Ca003 1 5 143111

Medium 4: TGE g / l

Trypticase glucose extract 5.0

Trypticase peptone 3.0

Glucose 1.0

Agar 15.0

The organism Micromonospora purpurea "was obtained from the U.S.

Dept, of Agriculture as NERL 2953 and held on N-Z-amine oblique substrates (Medium 1). Submerged ferments were carried out in flasks containing germ medium (Medium 2) for 4 days at 37 ° C on a rotary shaker. In this first stage, a 10 per cent transfer was made. graft material to the germinating medium (Medium 2) and fermentation was continued as above at 28 ° C for 7 days.

In order to establish the organism's ability to biosynthesize gentamicin in the absence of added deoxystreptamine, a third-stage fermentation was performed using a 5 per cent. graft in a 10 liter fermentor in a soybean glucose medium (Medium 3) at 28 ° C, stirring at 200 rpm. and aerated at 2 liters / min. with filtered air. After 6 days, the tank contents were acidified to pH 2.0 with 6N sulfuric acid, filtered and a 500 ml aliquot neutralized with ammonium hydromide and passed through an "IRC-50" ion exchange resin (Na + form). The column was then rinsed with water and eluted with 2N sulfuric acid. According to the procedure described in U.S. Patent No. 3,091,572, a 300 mg sample of crude gentamicin was found which was found to be biologically active and contained three components corresponding to gentamicin-C1, -Cg and - C ^ a by thin layer chromatographic examination.

For mutation of the organism, liquid cultures were grown in Medium 2 (37 ° C for 3 days) and the resultant cells harvested by centrifugation, washed and resuspended in buffered saline. This suspension was treated with the mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine. Samples of the mutagenic culture were placed on plates in Medium 4 at 37 ° C until colonies were evident (usually about a week). Colonies were seeded on similar plates (Medium 4), one set of which was poured with a spore suspension of B. subtilis. After incubation at 37 ° C for 18-20 hours, the 6 143111 "seeds" showing no inhibition zone on the B. subtilis plate were transferred from the main plate (no B. subtilis) to Medium 1 oblique substrates and incubated until full. growth was evident.

Pieces of Potentially Non-Producing Mutant Strains Then were exposed to deoxystreptamine in an attempt to stimulate antiBiotic Biosynthesis as follows. Stock cultures of the potential mutant strains were plated as Bands on the surface of Medium 4 plates and incubated at 37 ° C until growth was evident (about 3-4 days). Eilter paper slices were then dipped in a solution of deoxystreptamine (500 µg / ml) and placed on top of the culture line. After incubation for 24 hours, the plate surface was seeded with B. suBtilis by the overlay technique and the incubation was continued for an additional 18-20 hours. Isolates showing inbibition zones around the disc were designated as deoxystreptamine mutants. One such mutant, designated YIB Mutant and deposited in the American Type Culture Collection, 12301 Parklawn Brive, Rockville, Md. 20852 as Micromonospora purpurea ATCC 31 119 was used to prepare gentamicin-type antibiotics as described below.

BIOSYNTHESES WITH M. PURPUREA ATCC 31 119 Example 1

The mutant organism was kept on N-Z-amine agar slant substrates (Medium 1) from which transfers were made to flasks containing 500 ml of germinating medium (Medium 2). The flasks were incubated at 28 ° C for 4 days on a rotary shaker (5 cm stroke) at 225 rpm.

10% by volume seed graft from the germination stage Was aseptically transferred to 14 liter fermentors containing 9 liters of sterile germ medium (Medium 2). Bisse Stirred at 450 rpm. at 28-29 ° C and aerated with filtered air at 5 liters / minute. At the time of inoculation, 200 mg / liter of streptamine sulfate was added as a suspension in sterile distilled water. The fermentation was continued for 8 days.

10 liter 24 hour old seed material prepared as above was aseptically transferred to 130 liter fermenters containing 70 liters of sterile germ medium (Medium 2) and 0.31 g / liter streptamine sulfate suspended in sterile distilled water was added. Aero fermentation was performed at 29 ° C for 7 days.

The ferments were terminated by adding 10N sulfuric acid to pH

2.0 and filtration using a filter aid for removing mycelia. The filtered liquid "was adjusted to pH 7.0 and 1.56 g of oxalic acid per gram of calcium carbonate present in the medium was removed to remove calcium. The mixture was allowed to stand overnight and the clarified liquid was decanted off and passed through" Bio-Rex 70 "(weak cation exchange resin) in the Na + form using about 14 g of resin per liter of liquid. The column was then washed with distilled water and eluted with 2N sulfuric acid. All fractions showing antibiotic activity were combined, neutralized and concentrated in vacuo at below 50 ° C to the point where salt crystallization became apparent (about one-third volume), the pH was then adjusted to 10.5 and 4 volumes of acetone added to precipitate inorganic material which was removed by filtration The filtrate was adjusted to pH 5.0 with 6N sulfuric acid, concentrated in vacuo to about 1/20 of the original volume and cooled.

A white crystalline material precipitated, which was collected by filtration and melted at over 300 ° C. Based on its thin-layer chromatographic properties and its infrared spectrum, it was found to be identical to streptamine sulfate. From two 10-liter fermentations, processed as above, a total of 0.7 g of streptamine sulfate was obtained, and from two 80 liter of fermentations a total of 21 g of streptamine sulfate was obtained in this step.

The filtrate was further concentrated and 10 volumes of methanol were added to give the first crude solid antibiotic. From two 10 liter fermentations 7 g was obtained and from two 80 liter fermentations 24 g was obtained.

In order to identify antibiotic components during the purification procedures, chromatographic mobility values obtained by paper chromatography and thin layer chromatography for gentamicin-C ^, -Cg and -C-] α and for each of the corresponding amino-cyclitol analogs prepared as above were determined. Inn 8 143111 matographic mobility (hereinafter referred to as CM) is expressed as: q jj - the distance of the component from the origin of the gentamicin-O

The chromatographic systems used were as follows: S ^ s t em_ 1_:

Whatman No. 1 paper saturated with 0.95M sulfate hydrogen sulfate and developed downwards in 80 per cent. aqueous ethanol + 1.5 in ° NaCl and subsequent bioautography using B. subtilis as a sample or genome sme.

System 2:

Silica gel I 254 plate developed in the lower phase of 1: 1: 1 chloroform / methanol / concentrated ammonium hydroxide ($ 28). The components were localized with a nihhydrin spray after heating.

The CM values of the predominant antibiotic components of the example in comparison with a reference e-gentamic in-complex, all relative to gentamicin-C-1, are shown in Table I, where the compounds are designated component 1, component 2 and component 3 is meant to be respectively: 0-3-deoxy-4-C-methyl-5-methylamino-pI-arabinopyranosyl- (1 * 6) - O- [2-amino-6-methylamino-6-C-thyme- 2,5,4,6-tetradeoxy-α-D-γ-thro-glucopyranosyl- (1 * 4)] -D-streptamine; 0-5-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1 * 6) -0- [2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy -aD-erythroglucopyrano-syl- (1 * 4) j -D-streptamine; and 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1 * 6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-α-D glucopyranosyl- (1 * 4)] - D-streptamine.

9 143111

TABLE I

C.M. System 1 C.M. System 2 G-entamyclin-C1-1 G-entamycin-02 0.89 0.83

Gentamicin C, 0.50 0.67

Component 1 (predominantly) 0.96 0.92

Component 2 (minor) 0.76 0.75

Component 3 (minor) 0.50 0.61

In the 10 liter fermentor ", the crude solid (7 g) showing antibacterial activity was suspended in 200 ml of methanol and 10 ml of concentrated ammonium hydroxide (28 $), and" the mixture was stirred for 30 minutes and filtered. This was repeated two more times, and the filtrates were combined and concentrated in vacuo to give a pale yellow oil weighing 0.9 g. The "spent salts" were essentially free of antibiotic activity.

The oily base was mixed with 4 g of silica gel (Davison grade 923, particle size 0.074 - 0.149 mm) and placed on a silica gel column measuring 1.8 x 28 cm. The column was prepared as a slurry using the lower phase of isopropyl alcohol / chloroform / 17 per cent. aqueous ammonium hydroxide (1: 2: 1). The column was developed with this solvent and 50 ml fractions were collected.

Fractions 8 and 9 contained a single ninhydrin positive component which gave 20 mg as a pale yellow oil after removal of the solvent. The mass spectrum of this material, designated Component 1, showed a molecular ion and major fragments, each of which were 16 mass units (i.e., an oxygen atom) larger than that obtained from gentamicin-C1 as follows:

Reference gentamicin C ^: M + 4-77, 420, 60, 350, 347, 322, 319, 304

Component 1: M 493, 436, 376, 366, 363, 338, 335, 320.

This material was converted to its sulfate salt by dissolving in ethanol by adding a few drops of ethanol containing sulfuric acid. The resulting white solid was collected and dried to give 22 mg of component 1, 0-3-deoxy-4-C-thyme-3-methylamino-β-1-arabinopyranosyl- (1- * 6) - O- [2-amino-6-methylamino-6-O-methyl-2,3, 4, 6-tetradeoxy-u-D-erythro-glueopyranysole - (1 "* 4)] - D-streptamine as di -base * heptasulfate * decahydrate, m.p.

> 300 ° C.

Analysis calculated for 0: 0 27.22 - H 6.53 - N 7.55 - S 12.09

Found: C 27.15 - H 6.67 - N 7.79 - S 12.76.

The actions 10-13 yielded a more polar ninhydrin positive component, designated component 2, 0-3-deoxy-4-O-methyl-3-methylamino-PL-arabino-pyranosyl- (1-6> -0) [2,6-Diamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl (1M)] D-streptamine, which showed antibiotic activity.

Fractions 15-26 provided a third more polar component which exhibited antibiotic activity. The mass spectrum of this component showed characteristic sugar maxima similar to gentamicin-CήO at 129 (purpurosamine) and 160β (garosamine), and it is designated component 3, 0-3-deoxy-4-C-methyl 1-3-methyl thyro-6- L-arabinopyranosyl- (1- * 6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-oc-D-erythro-glucopyranosyl- (1- * 4) 1- D-streptamine.

Alternatively, the new component 1 was isolated as follows: A mixture of crude antibiotic base, prepared as above (0.9 g), was dissolved in 7 ml of water and the pH was adjusted to 4.5 with 1N sulfuric acid. The solution was passed through a strong anion exchange column ('IRA 401') in the OH form (layer size = 0.7 x 10 cm). The column was eluted with water and the eluate evaporated in vacuo at 35 ° C.

The resulting residue was triturated with 50 ml of the lower phase 11 of a solvent composed of 17 per cent. aqueous ammonium hydroxide / isopropyl alcohol / chloroform (1: 1: 2). The solution was decanted off and concentrated in vacuo to give an oil residue weighing 140 mg. The mass spectrum of this material corresponds to the spectrum of fractions 8 and 9 above, i.e.

M + 493, 436, 376, 366, 363, 338, 335, 320.

The magnetic chemistry resonance spectrum of component 1 also matched the assumed structure corresponding to 0-3-deoxy-4-C-methyl-3-methyl-lamino - β-L-ar abino pyr ano sy 1- (1- * 6) O- [2-Amino-6-methylamino-6-C-methyl-2,3,4,5-tetradeoxy-αD-erythro-glueopyranosyl- (1- * 4)] - D-streptamine and is summarized in Table II.

TABLE II

_S_ Integration _ Attribution_ 5.87; 5.60 2 anomeric OCHO 'groups

5.22 12 interchangeable H

3.09; 3.15 6 NCH3 x 2

2.9 - 4.8 13 -OHO x 6, -CHN x 5, -OHgO

1.9 - 2.6 4 -CH2CH2-

1.72 6 -CH₂O-, CH₂CH

Alternatively, a 4 g sample of the crude antibiotic salt was dissolved in 50 ml of water and passed through an anion exchange resin "AG1X8" (resin layer 1 x 27 cm). The antibiotic was eluted with water and all active fractions combined and evaporated under vacuum. Further desalting was performed on the residue by extraction with methanolic sodium hydroxide. The released base was mixed with 7 g of silica gel and placed on a 50 g of silica gel column. This column was developed with chloroform / methanol / concentrated (28 #) ammonium hydroxide (3: 2: 1) and 25 ml fractions were collected. Early fractions gave the new minor polar component 1, but mixed with several minor polar impurities as evidenced by thin layer chromatography. These fractions were combined and the concentrate was put on a 50 g silica gel column as above. This column was developed with chloroform / methanol / concentrated (28 #>) ammonium hydroxide (5: 3: 1) and 25 ml fractions were collected. Fractions 6-12 contained the desired compo 12 free of obvious impurities. After removal of the solvent, the resulting oil was converted to the sulfate salt in ethanolic sulfuric acid as described above to give 0.124 g of component 1 as the di-base * heptasulfate * decahydrate, m.p. > 300 ° C, as described above.

Ted culture of a suitable aminocyclitol with the mutant strain Micromonospora purpurea AICC 31 119 in germination medium (Medium 2) and isolation of the products as described above in Example 1 are similarly prepared by the following compounds of formula I:

Example 20 0-3-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1- * 6) -0- [2-amino-6-methylamino-6-C-methyl 2,3,4 »6-tetrahydroxy-αE-erythro-glucopyranosyl (1-4)] epistreptamine (CM relative to gentamicin-C 2 s System 1 = 0.59, System 2 = 0.63) ; 0-3-Deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1- * 6) -0- [2,6-diamino-6-C-methyl-2,3,4 »6- tetradeoxy-.alpha.-D-erythro-glueopyranosyl- (M-1 -)] epistreptamin; and 0-3-deoxy-4-C-methyl-3-methylamino-β-1-arabinopyranosyl- (1-6) -O- [2,6-diamino-2,3,4,6-tetradeoxy-αD -erythro-glucopyranosyl- (1- * 4)] - epistreptamine obtained by using epistreptamine instead of streptamine in the fermentation procedure.

Example 30 0-3-Deoxy-4-O-methyl-5-methylamino-p-1-arabinopyranosyl- (1-6) -O- [2-amino-6-methylamino-6-C-methyl-2, 3,4,6-tetradeoxy-αD-erythro-glucopyranosyl- (1H)] -2,5-dideoxystreptamine (CM relative to gene tamicin-C ^: System 1 = 0.95 »System 2 = 0.98) ; 0-3-Deoxy-4-C-methyl-3-methylamino-p-arabinopyranosyl- (1- * 6) -0- [2,6-diamino-6-C-methyl-2,3 »4» 6- tetradeoxy-α-E-erythro-glucopyranosyl (1- * 4)] - 2,5-cLideoxy-streptamine (CM relative to gentamicin-C C: System 1 = 0.66,

System 2 = 0.73); and 0-3-deoxy-4 "C-methyl-3-methylamino-pL-arabino-pyranosyl- (1-6) -O- / 2,6-diamino-2,3,4,6-tetradeoxy-αD -erythro-gluco-pyranosyl- (1-4) 1-2,5-dideoxystreptamine obtained by using di-deoxystrepamine instead of streptamine in the fermentation procedure.

13 143111

The same 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6) -O- / S-amino-6-methylamino-6-C-methyl-2,3,4 , 6-tetradeoxy-αD-erythroglucopyranosyl- (144) 7-2,5-dideoxystreptamine and 0-3-deoxy-4- C-methyl-3-methylamino-PL-arabinopyranosyl- (1 ^ 6) -0- / 5 , 6-Diamino-6-C-methyl-2,3 »4,6-tetradeoxy-oc-D-erythro-glucopyranosyl- (144)] - 2,5-dideoxystreptamine as described above and with the same CM values as indicated above were obtained using 6,7 * diazabicyclo [-3.2, 1] octane-2,4-diol (exo, exo) in place of streptamine in the fermentation procedure.

SYNTHESY AE AMINO-HYDROXY-MVERE-ALTCANPYL DERIYATES

Example 4

A solution of 270 mg (0.54 ml) of 0-3-deoxy-4-C-methyl-3-methyl-amino-β-L-arablnopyranoyl- (1- + 6) -O- [2-amino] 6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy- [xD-erythro-glucopyranosyl- (1-H-)] -D-strep -amine, prepared as described above in Example 1 (component 1), dissolved in 5 ml of 50% aqueous tetrahydrofuran, was cooled to 5 ° C in an ice bath and treated with 208 mg (0.59 millimole) of the N-hydroxysuccinimide ester of S - (-) - Y- (N-benzyloxycarbonyl ) amino-α-hydroxybutyric acid (Koni-shi et al. U.S. Patent No. 3,780,018) and the mixture was stirred at 5 ° C for 20 hours. The mixture was then concentrated to 10 ml in vacuo. 25 ml of n-butanol and 10 ml of water were added and the layers were separated.

The aqueous layer was washed again with 10 ml of n-butanol. The combined organic layers were evaporated leaving a residue of 513 mg of crude product which was set aside.

The aqueous layer was evaporated to dryness to give 304 mg of residue which was dissolved in 25 ml of 50 # aqueous tetrahydrofuran and treated with an additional 208 mg of the N-hydroxysuccinimide ester of 5- (_) - Y- (N-benzyloxycarbonyl) -amino-cc-hydroxybutyric acid as before. Workup of the reaction mixture yielded an additional 435 mg of crude product which was combined with the previously obtained 513 mg and chromatographed on seven 40 mm x 20 cm silica gel plates. The system was developed with a solution system consisting of the lower phase of a mixture of chloroform / methanol / concentrated (28 $) ammonium hydroxide (3: 1 * 1). Seven passages in this solvent system were necessary and, after elution of the product band which was ultraviolet visible, 229.5 mg of a crude mixture of the three monoacylated products was obtained.

The mixture of acylated products was placed on three 1 mm thick 40 x 20 cm silica gel plates and developed five times with the lower phase of chloroform / isopropanol / concentrated (28 f) ammonium hydroxide (4: 1: 1). twice with the lower phase of chloroform / iso-propanol / concentrated (28 fo) ammonium hydroxide (3: 1: 1) and nine times with the lower phase of chloroform-methanol / concentrated (28 fo) ammonium hydroxide (4: 1: 1 ). Three distinct bands visible under ultraviolet irradiation were obtained which were excised separately and eluted from the silica gel with the lower phase of chloroform / methanol / concentrated (28 fo) ammonium hydroxide (1: 1: 1) to give three components: A 90.9 mg; B, 59.1 mg; and C 48.5 mg which is 5- (-) - Y- (N-benzyloxycarbonyl) amino-α-hydroxybutyric acid amide derivatives in 2'-, 1- and 3-position of 0-3-deoxy, respectively -4-C-methyl-3-methylamino-β-1-arabinopyranosyl- (1-> 6) -0- [2-amino-6-methylamino-6- C-methyl-2,3,4,6-tetradeoxy -αD-erythro-glucopyranosyl- (1Ή)] -D-streptamine. The R ^ values of components A, B and C after development five times on silica gel with the lower phase of a system consisting of chloroform / methanol / concentrated (28 f) ammonium hydroxide (4: 1: 1) were: A - 0.48 B - 0.62 C - 0.70

Component B (1-amide, 56.1 mg) dissolved in 25 ml of 50 µl aqueous ethanol and 20 mg of 10 µl palladium-on-charcoal was shaken on a Parr shaker, maintained at a hydrogen pressure of 3.85 kp / cm 2 for 5 1/2 hours, after which the catalyst was removed by filtration through filter aid. Evaporation of the solvent gave 34.3 mg of a white glassy substance which was dissolved in 2.5 ml of water and treated with 11.4 mg of sulfuric acid in 0.1 ml of water. Addition of 10 ml of ethanol precipitated 1- [S - (-) - γ-amino-o: -hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methyl-o-β-1-arabino pyrano silk 1- (1-> 6) -O- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl- (1Ή)] - 143111 D-streptamine as the pentasulfate salt (32 mg), m.p. 230 DEG-235 DEG C. (decomp.); thin layer chromatography, R f = 0.18 (silica gel; chloroform / methanol / concentrated (28 ¢) ammonium hydroxide / water (1: 4: 2: 1); gentamicin C ^ standard = 0.73).

Analysis calculated for C 25 H 50 ° 10 N 6 * 5H 2 SO 4: C 27.67 - H 5.57 - N 7.75

Found: C 27.50 - H 5.58 - N 7.42.

Components A and C were treated in the same way. A gave 47 mg of 2 '- [S - (_) _ Y_amino-α-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1-6) -0- [ 2-Amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl (1-4)] -D-streptamine as the bis-base * tetrasulfate · heptahydrate , m.p. 237 - 241 ° C (decomp); thin layer chromatography, R f = 0.33 (silica gel; chloroform / methanol / concentrated (28 ¢) ammonium hydroxide / Tooth (1: 4: 2: 1); R

Analysis calculated for (C₂ ^H ^ 0NgO.j0) 2 * 4H₂SO ^ · H₂O: B 35.16 - H 7.20 - N 9.84

Round: C 35.38 - Ξ 7.08 - N 9.49.

Component C gave 26 mg of 3- [S - (-) -γ-amino-oc-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (1-> 6) - O- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl 1- (1-> 4)] -D-streptamine as bis-base. The pentasulfate * trihydrate, m.p.

220 DEG-230 DEG C. (decomp.); thin layer chromatography, R f = 0.30 [silica gel; chloroform / methanol / concentrated ($ 28) ammonium hydroxide / water (1: 4: 2: 1); R gentamicin C1 standard = 0.73].

Analysis calculated for (Cr 2 H 2 ON 2 O 3) δ · 5H 2 SO 4 · 350.0: C 34.64 - H 6.74 - N 9.67 Round: C 34.35 - H 6.38 - N 8 , 60th

16 163111

ANTIBACTERIAL TEST RESULTS

The compounds of Example 1 prepared 2-hydroxygentamicin-C ^, 2-hydroxygentamicin-Cg, and 2-hydroxygentamicin-C-yCCg complex (hereinafter referred to as Oxy-C-, Oxy-Cg and Oxy-C ^, respectively). Cg) and the compounds of Example 3 prepared from Example 3 and 5-deoxygentamicin-C1 / C2 / C2_a complex (hereinafter referred to as Deoxy-C1 and Deoxy-C1 / Cg / C with gentamicin-C2, gentamicin-Cg and gentamicin-C - ^ / C2 / Cla complex (hereinafter referred to as GC · ^, G-Cg and GC - ^ / Cg / C following the following procedure.

Stock solutions of each compound containing 200 μg / ml base were prepared in distilled water and filter sterilized. Cultures of the sample organisms were grown for 24 hours at 37 ° C in 10 ml glass with tryptose phosphate or Miller-Hinton liquid. Each culture was adjusted with optical density fluid 0.1 on a Spectronic

ISLAND

20 (approximately 10 cells / ml). The set cultures were diluted 1: 500 in liquid for use as graft material (final cell culture).

C

concentration approximately 2 x 10 3 cells / ml). The test compounds were tested for antibacterial activity by a single series of dilution methods. Double dilution rows were prepared in liquid from the stock solutions and 0.2 ml of each antibiotic concentration was placed in 17 13 x 100 mm glass. All glasses were seeded with 0.2 ml of the appropriate diluted culture (final cell concentration per glass = 105 cells / ml). Minimum inhibitory concentrations (lowest antibiotic concentration showing no visible growth) were read after 16 hours of incubation at 37 ° C.

The results are listed in Table III below. The compounds were considered inactive at inhibitory concentrations above 100 µg / ml.

17 143111 i to>, σι vo to to oo co to _ vo w h m - · »c— c— cm o to o» oc \ j cm 10 - - o in - o

ΦΗ OH I ItOHlHCMOOtOCMVOHHI I

P o cd

H

o to „l \ <Tt VO to CO VO lhio od

>, CM H LO OJ O- to * * O

w o «<D - in to» * to C \ l C \ l o · o '-' v oh i i in to icmcmohcmhhho i i

Φ H PO

CM CO o to VO COM3 O O tO tfl CO

rj CM ΙΟ Ο ΙΟ ΙΟ 0 - IOO O O LO LO ΟΙ ·> * s H ·> Ο O *> tOO ““ OOHH · * · * · *

O OH \ HlOlOHCMtoOHHlO ^ HHO

—I (TifOOfO vo vovooooototovo

o tOHOH OlO lOlOOOOOHHiO

I .. ·> H “OO *> tOO · * · * Η Η Η H

Ο ΟίΟΛίΟΙΟΗΗΟΙΙΟΗΗ ^ Λ, Λ tOfOH

CD

-I H

a! \ CTttOOVO VO VO to O CTt

<! dj foHOto to lOlOOOOOfO

ha q ·> “H“ OO ”to to“ “HOOO *

3j \ OfOA.HlOlOHC \ IC \ IHH / \ HHHOl I

Cd - (H

w i O

Η · Ι I

ΟΙ ΟΙ O

H Cl

Ol • J, Μ I CM

Μ I I o coto to to tOfOlOIOtrt VO

rq CD I r'l '' '. 0-H LO CM tOHOJHCMO to <i * π-j i bd --a * µ O · * LfN · * · »· * · * ·» 000 · “

H Cl OO OlOlOHVOOJHfOVOlOVOHtOiO I Η I I

Oil Cl ΦΙ »O I

£ j! i c ^ ro voto to to vo vo oororovo ω

Ή | >, hr \ Η Ο ΙΟ ·> to Ctl CM ΙΟ to OOHHtO

• HI li Ctl · »·> O» (t | O ““ ““ “OtOHH ·>“ “CQ

I OO OIOHHHlOHVOVOHHlOOl / v, fOfOH § HI>

a! I rn tO ΙΛ to fOfOtOIOtOOO O to two to P

5! >, O-H HCMOHHCM.HCMOO OCMCMCM ji rji bd H * * Ο * · »O * ·» · * λ * ΗΗΟΗ * A- * • HI OO Λ.ΙΓ \ \ 0 \ Q ft

Sl g

-C

ft i -C t> - Η Φ o vo o \ ot -d- ra vo to ω go ζτ- c \ i Λ | o (Ti VO to Η O o o i ^ p O O <T I CM CM CM K. ft VO (Ti CM CM tD C I I I O r · ** rj vo (Μ I I (ΤΟ <'ί <1 << 2ϋ> c

Φ p h> vooJCroS vo P

s · Η Cd cm o HHcdcdcdcdcd

o3 H. Ha) (UQ5tt1 · ^ CQ CQ CQ CQ CQ H

• h cocDtsiovocnscdcdcdHPcdogogo

r * Jrf) [s. | r \ ^ 0I) W ΟΉτΙΗ IhH ϋ (η ΰ Ih β * P

cd cq o vo tf οί D <Η δ C Ch cd · · η · ητ) · η · η φ

§) ^ |> ^ HHHWOOOPCCbDbpbpbpbp X

U φ Hi | cdP © CCCCC C

O ίπ * rl * H · Η · Η * H Ή O Η Η W ^ μ CHHr_ | HHH ΦΦ · Η .ΗΦΦΦΦΦ P cd o o o o o o · cCS >> cdcdcdcdcd

ooooooPftft · οο ..... 'Oh

....... C · »CCCCQCQtQCQCQ Cd 18 143111

In the field of antibiotics where microorganisms develop resi? resistance to antibiotic therapy by mutation, progress is measured by the ability of new antibiotics to control microorganisms that are for some reason resistant to known antibiotics. Comparisons of the test results in the above Table III between 2-hydroxygentamicin-C- and gentamicin-C-, 2-hydroxygentamic in-C ^ and gentamicin-C ^, 2-hydr oxygentamic in-C - - / C₂ complex, respectively and gentamiGin-C - ^ / C ^ / C ^ complex, 5-deoxygentamicin-C ^ and gentamicin-C ^ as well as 5-deoxygentamicin-Cj / Op / C · ^ complex and gentamicin-C ^ / C2 / Clax complex shows that the compounds of the invention are active against from 4 to 8 microorganisms that are resistant to the corresponding gentamicin. These comparisons are summarized in Table III-A below, where the organisms listed in Table III are numbered in sequence and identified by the numbers thus assigned, and where better activity for a particular stated 2-hydroxy or 5-de oxygentamic in or complex thereof in comparison with the corresponding gentamicin or complex thereof is indicated by +.

19 143111

Organism OxyC1 Oxy-C2 Oxy-c ^ / C., Deoxy-c ^ Deoxy-c ^ / CVC, 2 3 ++ 4- 4 5 + + 4- 6 + 7 8 + + 4- 4- 9 ++ + 10 11 12 + + 4- 4- 13 + + 4. + 14 +++ + 4.

15 + 16 • 17 18

The same component 1 as described above in Example 1, 0-3-de-ozy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1- * 6) -0- [2-amino-6-methylamino -6-C-methyl-2,3,4,6-te tradeoxy-cc-D-ery thro-glucopyrano-sil- (1- * 4)] - D-streptamine, was tested in comparison with gentamicin complex ( C1-C2 and G3a) and gentamicin-C1; and the 1-, 3- and 2 '- [S - (-) - γ-amino-α-hydroxy-butyryl] amides of component 1, described above in Example 4 and designated component 1 (1-HABA), component 1, respectively (3-HABA) and component 1 (2'-HABA), were tested in comparison with the corresponding 1-, 3- and 2, - [S - (-) - γ-amino-α-hydroxybutyrryl] amides of gentamicin-C1 (all described by Konishi et al. in U.S. Patent No. 3,780,018, issued December 18, 1973), designated (1-HABA), (3-HABA) and (2'-HABA, respectively). .

These results are set forth in Table IY below, where test organisms 1, 2, 3, 4, 5 and 6 identify B. subtilis ATCC 6633, S. aureus Smith, E. coli JR 76.2, Ent. cloacae A-20960, K. pneumoniae A-20636 and Ps. aeruginosa A-20987.

TABLE · 17 *

Test organisms

Compound 1 23456

Gentamicin-C ^, -Cg, -C ^ & 0.024 0.39 50 12.5 25> 100

Gentami cin-C, 0.049 0.78 50 12.5 25> 100

Component 1 0.098 1.56 6.25 0.78 3.15 C1 (1-HABA) 0.39 3.13 12.5 3.13 6.25> 100

Component 1 (1-HABA) 0.78 6.25 12.5 6.25 12.5> 100 01 (3-ΞΑΒΑ) 3.13 25> 100 100> 100> 100

Component 1 (3-HABA) 6.25 50 100 50 50> 100 01 (21-HABA) 6.25 50 100 25 50> 100

Component 1 (2'-HABA) 1.56 25 50 25 50> 100 * Cultured in Mueller-Hinton liquid.

Claims (2)

21. A3111 1. K CH-NH-Rb tS RO -NH> 4 n. NH-R5 _ ~~ r2 R-5> A-- (y H-NH-R1 -n \ CH3NH -ch3 OH .... In Ί ~ TQ Ί wherein R and R are either all hydrogen or one of R, ks R 1 and R are a ω-amino-α-hydroxy-alkanoyl group of the formula H 2 N-CH 2 (CH 2) n-OH-O-OH 0 ... III Ί Ύ o where n is 0 or 1 and the other two of R, R 1 and R represent the hy-OC drug, R and R 3 are hydrogen or hydroxy, except that when 2 R R is hydrogen, R 2 is not hydroxy in the cis position to amino groups - 7 in 1 - and 3-position, and R and R 'each mean hydrogen or methyl, or acid addition salts thereof, characterized in that in a nutrient medium containing carbohydrates, a source of assimilated nitrogen, necessary salts and a corresponding added aminocyclitol with the formula HO-, NH-R5 *, YV -7 \\ HO ^ _NH-r1- ... II wherein R and R are hydrogen or together form a single 2 <5 bond which connects the two nitrogen atoms , and R and R 2 have the above meaning, cultivate a mutant microorganism of the species Mi-cromonospora purpurea, which is unable to synthesize said aminocyclitol itself, but is able to incorporate it into the compound of formula I under the said conditions, except Micromonospora purpurea ATCC 31164, to form a compound of formula I wherein R 1, R 2 and R 2 are all hydrogen and R 2, R 2, R 2 and R have the above meaning and the compound formed, if desired, is reacted with an N-hydroxysuccinimide ester of formula: / Λ <RTI ID = 0.0> (CH2-C-NHCH2 </RTI> (CH2) nCHOHCO0-N2 ... IV wherein n is 0 or 1 and the benzyloxycarbonyl group of the resulting compound is subjected to hydrogenolysis with hydrogen over a catalyst to form a compound of Formula I wherein one of R, R-1 and R is a ω-amino-α-hydroxy-alkanoyl group of formula III and, if desired, a formed free base is transferred into an acid addition salt thereof. A process for preparing aminocyclitol compounds of the general formula: r7 Process according to claim 1, characterized in that the microorganism strain is Micromonospora purpurea ATCC 31119. A process according to claim 1 or 2 for the preparation of 0-3-deoxy-4-C-methyl-3- (methylamino) -β-L-arabinopyranosyl. - (1-> 6) -0- [2-amino-6- (methylamino) -6-C-methyl-2,3,4,6-tetradeoxy-αDe-rythro-glucopyranosyl- (1Vi-)] -D streptamine, 0-3-deoxy-4-C-methyl-