NO146811B - PROCEDURE FOR THE PREPARATION OF AMINOCYCLLITOL ANTIBIOTICS - Google Patents

Description

The present invention relates to the production of aminocyclitol antibiotics with the formula:

where R 2 and R 1 - are both hydrogen or both are hydroxy, R 8 and R 1 are each hydrogen or methyl; as well as acid addition salts thereof.

Preparation of compounds of this type is described in US patent no. 3,669,838. This method includes growing a suitable mutant microorganism in a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential mineral salts, and an added aminocyclitol derivative, but where this microorganism is not able to synthesize said aminocyclitol as such, but is in capable of incorporating said aminocyclitol into compounds of formula I. More specifically, a mutant of Micromonospora purpurea with the designation Micromonospora purpurea ATCC 31.119 is cultivated, after which the product is isolated from the culture medium. According to the method in US patent no. 3,669,838, the nature of the mutant is such that it cannot synthesize the aminocyclitol subunit from a nutrient medium to thereby form the antibiotic itself, but can incorporate said subunit into an antibiotic when the aminocyclitol is added to the nutrient medium.

It has been found that a further mutant of M. purpurea 31.119, namely M. purpurea ATCC 31.164, is also capable of incorporating an aminocyclitol into antibiotics of the formula I type.

The present method is thus characterized by the fact that in a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an amino-cyclitol compound with the formula:

where R 2 and have the same meaning as stated above and R 2

is amino or hydroxy, grows Micromonospora purpurea ATCC 31.164, and, if desired, converts an obtained free base into an acid addition salt thereof.

The compounds of formula I have been tested in antibacterial samples of the standard serial dilution type and have been found to have antibacterial activity, especially against gentamicin-resistant organisms. The compounds can thus be used as antibacterial agents.

Compounds of formula I can primarily be used for oral,

topical or parenteral administration, and can be prepared for use with a pharmaceutical carrier in a suspension,

and the compounds can either be used in their free form

bases or as pharmaceutically acceptable, non-toxic acid addition salts, in an inert carrier such as polyethylene glycol,

or by preparing tablets or capsules for oral administration, either alone or with suitable diluents,

or alternatively they can be processed into regular creams or ointments for topical application.

The structure of compounds of formula I was established by means of studies of their chromatographic properties, which were determined by thin-layer chromatography analysis, their nuclear magnetic resonance, as well as mass spectra, by decomposition into known compounds; and by agreement between calculated and found values by an analysis of the elements present.

Results of antibacterial tests

The compound 0-3-deoxy-4-C-methyl-3-methylamino-(3-L-arabino-pyranosyl-(1->-6)-0-[2-amino-6-methylamino-6-C-methyl -2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1-*-4)]-D-streptamine described in example 1 and designated component 1,

was tested in comparison with gentamicin against a variety of microorganisms using the following procedure.

Stock solutions of each compound containing 200 meg/ml base were prepared in distilled water and filter sterilized. Cultures of the test organisms were grown for 24 hours at

37°C in 10 ml tubes containing tryptose phosphate or Mueller-Hinton medium. Each culture was adjusted with medium to an optical density of 0.1 in a "Spectronic 20" apparatus (approximately 10 g cells/ml). The adjusted cultures were diluted to 1:500 in medium and were then used as inoculum (final cell concentration approx. 2 x 10 5 cells/ml). The compounds were tested for antibacterial activity by a single tube dilution method. Two parallel serial dilutions were carried out in the medium from the stock solutions themselves, and 0.2 ml of each concentration was placed in seventeen 13 x 100 mm tubes. All tubes were inoculated with 0.2 ml of an appropriately diluted culture (final cell concentration per tube is approx. 10 cells/ml).• Minimum inhibitory concentrations (lowest concentration that did not produce visible growth) were read after 16 hours of incubation at 37° C.

The results are set out in Table I below. The compounds were considered to be inactive at inhibitory concentrations of more than 100 mcg/ml.

The same component 1 described in Example 1, namely 0-3-deoxy-4-C-methyl-3-methylamino-|3-L-arabinopyranosyl-(1-*-6)-0- [ 2-amino-6- methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1-+4)]-D-streptamine was tested in comparison with the gentamicin complex (C,, C„ and C, ) and gentamicin C^, and the 1-, 3- and 2'-[S-(-)-γ-amino-α-hydroxy-butyryl]-amides of component 1, described below in Example 4, and respectively denoted component 1 (1-HABA), component 2

(3-HABA) and component 1 (2'-HABA). These were tested in comparison with the corresponding 1-, 3- and 2'-[S-(-)-γ-amino-cx-hydroxybutyryl]-amides of gentamicin (all described in US Patent No. 3,780,018), and denoted respectively

(1-HABA) , C1 (3-HABA) and C-^ (21-HABA) . These results are set out in Table II below where the test organisms 1, 2, 3,

4, 5 and 6 indicate B. subtilis ATCC 6633, S. aureus Smith,

E. coli JR 76.2, Ent. cloacae A-20960, K. pneumoniae A-

20636 and Ps. aeruginosa A-20897, respectively.

Mutation processes

In the following procedures, the following media were used:

The organism used, namely Micromonospora purpurea, was obtained from the US. Dept. of Agriculture as NRRL 2953, and this was maintained on N-Z-amine agar slants (medium 1). Fermentation in the medium itself was carried out in flasks containing germination medium 2 for 4 days at 37°C in a rotating shaker. From this first stage starting medium, a 10% inoculum was transferred to the fermentation medium (medium 2), and the fermentation was continued as mentioned above at 28°C for 7 days.

To establish the ability of the organism to biosynthesize gentamicin in the absence of added deoxystreptamine, a third-stage fermentation was performed using a 5% inoculum in a

10 liter fermenter tank in a soybean-glucose medium (3)

at 28°C, stirring at 200 rpm and bubbling through at 2 liters/minute using filtered air. After 6

24 hours, the contents of the tank were acidified at pH 2.0 with 6N sulfuric acid, filtered, and a 500 ml portion neutralized with ammonium hydroxide and passed through an "IRC-50" ion exchange resin (Na<+->form). The column was then washed with water and eluted with

2N sulfuric acid. Using the method from US patent 3,091,572, a 300 mg sample of impure gentamicin was isolated, which was found to be biologically active and which contained three components corresponding to C,, C„ and C, by thin-layer chromatography

x £ xa

graph examination.

In order to mutate the organism, different cultures were cultivated

in a medium 2 (37°C for 3 days), and the resulting cells harvested by centrifugation, washed and resuspended in buffered saline. This suspension was treated with a mutagenic agent, namely N-methyl-N'-nitro-N-nitroso-guanidine. Samples of the mutant culture were plated in medium 4 at 37°C until colonies were obtained (this usually took a week). Colonies were picked and transferred to parallel plates (medium 4), and one set was overlaid with a spore suspension of B. subtilis. After incubation at 37°C for 18 to 20 hours, the colonies showing no zone of inhibition on the B. subtilis plate were transferred from the main plate (no B. subtilis) to medium 1 agar slants and incubated until full growth was obtained).

These potential non-producing mutants were then exposed to deoxystreptamine in an attempt to stimulate antibiotic biosynthesis in the following manner. Cultures of possible mutants were streaked as bands on the surface of plates with medium 4,

and they were then incubated at 37°C until a good weight was obtained (this took from approx. 3 to 4 days). Filter paper discs were then dipped in a solution of deoxystreptamine (500 mcg/ml) and placed on top of the streak culture. After incubation for 24 hours, the surface of the plates was inoculated with B. subtilis using the overlay technique, and the incubation was

continued for another 18 to 20 hours. The isolates that showed zones of inhibition surrounding the disk were designated as deoxystreptamine mutants. One such mutant designated VIB/is "located in""the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 as Micromono-

spora purpurea ATCC 31.119, and this was used for the production of gentamicin-type antibiotics as described below.

The latter organism itself was subjected to the same mutation procedure as described above, and samples of mutagenic cultures were plated in medium 4 at 37°C

until good colonies were obtained (this took about a week).

The colonies were then plated out on parallel plates containing streptomycin test agar (medium 6).

One set served as a master plate for later recovery, while the other set contained 25 µg/ml streptamine sulfate and a spore suspension of B. subtilis was used as the test organism. The plates were incubated at 37°C for 24 hours and examined for zones of inhibition. Those showing the largest zones were transferred from the master plate to agar slants of N-Z-amine medium (medium 1) and incubated for a week at 37°C.

The mutants that incorporated small amounts of streptamin sul-

vessels were then screened with streptamine and scyllo-inosose at 500 pg/ml as a starting point. Maintenance cultures were transferred to flasks containing medium 2, incubated at 37°C in a rotary shaker for 7 days. The flasks were periodically assessed for antibiotic activity via the so-called disk diffusion test, where B. subtilis was used as the test organism.

The isolates that showed zones of inhibition around the disk were designated as streptamine or scylloinosis mutants. One such mutant, designated VIB-3P, has been submitted to the American Type Culture Collection, 12301 Parklawn Drice, Rockville,

Md. 20851 as Micromonospora purpurea ATCC 31.164, and

this is used for the production of aminocyclitol antibiotics

of the streptamine, deoxystreptamine and dideoxystreptamine type as described below.

The following examples illustrate the invention.

Biosynthesis with M. purpurea ATCC 31. 164

Example: 1

The mutant organism was maintained on N-Z-amine agar slants (medium 1) from which the culture was transferred to flasks containing 500 ml of germination medium 2. The flasks were incubated at 28°C for 4 days in a rotary shaker with a rotation speed of 225 rpm ./minute.

A 10% (v/v) inoculum from the germination stage was aseptically transferred to a 14 liter fermentation tank containing 9 liters of sterile germination medium 2. The medium was stirred at 450 rpm at 28-29°C and bubbled through with filtered air in a amount of 5 litres/minute. During the inoculation itself, 200 mg/litre of streptamine sulphate was added as a suspension in sterile distilled water. Fermentation continued for 8 days.

A 24-hour, 10-liter inoculum was prepared as described above, was aseptically transferred to a 130-liter fermentation tank containing 70 liters of sterile germination medium 2, and 0.31 g/liter of streptamine sulfate suspended in sterile distilled water was added. Aerobic fermentation was carried out at 29°C for 7 days.

The fermentation was terminated by adding 10N sulfuric acid to pH 2.0, and a filtration was carried out using filter aid to remove mycelium. The filtered medium was adjusted to pH 7.0 and 1.56 g of oxalic acid was added per grams of calcium carbonate in the medium to remove calcium. The medium became

■set aside overnight, and the clear medium was decanted and passed over a "Bio-Rex 70" (weak cation exchanger) resin in the Na<+> form using 14 g of resin per liter medium. The column was then washed with distilled water and eluted with

2N sulfuric acid. All fractions that had antibiotic activity were combined, neutralized and concentrated under vacuum below 50°C to the point where a salt out crystallization was obtained (about 1/3 volume). The pH was then adjusted to 10.5, and four volumes of acetone were added to the precipitated inorganic substances which were removed by filtration. The filtrate was adjusted to pH 5.0 with 6N sulfuric acid, concentrated under vacuum to approx. 1/20 of the original volume and then cooled. A white crystalline substance melting above 300°C was collected by filtration and from its characteristics on thin layer chromatography, its infrared spectrum it was found to be identical to streptamine sulfate. From two 10-liter fermentations that were processed as described above, a total of 0.7 g of streptamine sulfate was obtained, and from two 80-liter fermentations, a total of 21 g of streptamine sulfate was obtained at this stage.

The filtrate was further concentrated, and 10 volumes of methanol were added and a first impure antibiotic was obtained. From two 10 liter fermentations you got 7 g, and from two 80 liter fermentations you got 2 4 g.

For the purpose of identifying antibiotic components during the purification methods, chromatographic mobility values were determined on paper and thin-layer chromatography for the gentamicins C, C, and C, and for each of the corresponding aminocyclitol analogs prepared as described above, where the chromatographic mobility (hereinafter referred to as CM.) is expressed as: c M _ distance of the component from the starting point distance for gentamicin C, from the starting point The chromatographic systems used were the following: System 1 - Whatman No. 1 paper saturated with 0.95 molar sulfate-bisulfate and developed with a falling liquid front in 80% aqueous ethanol + 1.5% NaCl and subsequent bioautography by

use B. subtilis as the test organism.

System 2 - Silica gel "F 254" plate developed in a lower phase of chloroform (1): methanol (1): concentrated (28%) ammonium hydroxide (1). The components were determined using a ninhydrin injection and heating. The CM values of the most important antibiotic compounds of formula I compared to a reference gentamicin complex, all relative to gentamicin C , are shown in Table III. The compounds designated as Component 1, Component 2 and Component 3 are the following, respectively: 0-3-deoxy-4-C-methyl-3-methylamino-p-L-arabinopyranosyl-(1+6)-0-[2- amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1-*4)]-D-streptamine. 0-3-deoxy-4-C-methyl-3-methylamino-(3-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl-2,3,4, 6-tetradeoxy-α-D-erythro-glucopyranosyl-(1-»-4)]-D-streptamine, and 0-3-deoxy-4-C-methyl-3-methylamino-p-L-arabinopyranosyl-(1+6)- O-[2,6-diamino-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1-^-4)]-D-streptamine.

From the aforementioned 10 liter fermentation tanks, the solid impurity (7 g) showing antibacterial activity was suspended in 200 ml of methanol and 10 ml of concentrated (28%) ammonium hydroxide, after which the mixture was stirred for 30 minutes and then filtered. This was repeated twice and the filtrates were combined and concentrated under vacuum to give a pale yellow oil weighing 0.9 g. The "used salts" were in total

essentially free of antibiotic activity.

The oily compound was mixed with 4 g of silica gel ("Davison grade 923", 100-200 mesh)" and added to a silica gel column measuring 1.8 x 28 cm. The column was prepared from a slurry using the lower phase of isopropyl alcohol (1) rchloroform (2): 17% aqueous ammonium hydroxide (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 on removal of the solvent. The mass spectrum of this compound, which was designated Component 1, showed a molecular ion and major fragments that were all 16 mass units (ie one oxygen atom) larger than that obtained for gentamicin C, as follows: Reference gentamicin C^: M<+ > 477, 420, 360, 350, 347, 322,

319, 304

Component 1: M<+> 493, 436, 376, 366, 363, 338,

335, 320.

This material was converted to its sulphate salt by dissolving it in ethanol and then 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-methyl-3-methylamino-|3-L-arabinopyranosyl-(l->-6) -O- [ 2-amino-6-methyl-amino-6-C-methyl-2 ,3,4, 6-tetradeoxy-α-D-erythroglucopyranosyl-(1->-4)] -D-streptamine as dibase :hepta -sulphate: decahydrate, melting point >300°C.

Analysis:

Meant for

Fractions 10-13 gave a more polar ninhydrin component which was designated 0-3-deoxy-4-C-methyl-3-methylamino-3-arabinopyranosyl-(1->-6)-O-[2,6-diamino-6-C -methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine. This was component 2 which showed antibiotic activity.

Fractions 15-26 gave a third more polar component - which showed antibiotic activity. Mass spectrum of this component showed characteristic sugar peaks corresponding to gentamicin Cj, _a at 129- (purpurosamine) and 160 (garosamine) and it was designated 0-3-deoxy-4-C-methyl-3-methylamino-3-L-arabinopyrano- syl-(1-s-6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine, component 3.

Alternatively, the new Component 1 can be isolated as follows: a mixture of the impure antibiotic obtained as described above (0.9 g) was dissolved in 7 ml of water, and pH

was adjusted to 4.5 with IN sulfuric acid. The solution was passed over a strong anionic 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 treated with 50 ml of the lower phase of a solvent consisting of 17% aqueous ammonium hydroxide:isopropyl alcohol:chloroform (1:1:2). The solvent was poured off and concentrated under vacuum to give an oil weighing 140 mg. Mass spectrum of this material corresponded to that found for fractions 8 and 9 above, i.e. M<+> 493, 436, 376, 366, 363, 338, 335, 320.

The nuclear magnetic resonance spectrum for Component 1 was also consistent with the proposed structure, and corresponded to 0-3-deoxy-4-C-methyl-3-methylamino-|3-L-arabinopyrano-syl-(1+6)-0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine and is summarized in Table IV.

Alternatively, a 4 gram sample of the impure antibiotic salt can be dissolved in 50 ml of water and passed over an anion exchange resin "AG 1 x 8" (resin layer 1 x 27 cm). The antibiotic was eluted with water, and all active fractions were pooled and evaporated under vacuum. A further desalting was carried out on the residue by extraction with methanolic sodium hydroxide. The liberated base was mixed with 7 g of silica gel and added to a 50 g silica gel column. This column was developed with chloroform:methanol:concentrated (28%) ammonium hydroxide (3:4:2), and 25 mL fractions were collected. The early fractions yielded a new less polar Component 1,

but it was mixed with several less polar impurities, which was evident by thin-layer chromatography. These fractions were pooled and the concentrate applied to a 50 g silica gel column as described 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 component free of impurities. On removal of the solvent, the resulting oil was converted to the sulfate salt in ethanolic sulfuric acid as described earlier, and 0.124 g of Component 1 was obtained as the dibase:heptasulfate decahydrate, melting

point >300°C as described above.

Example 2

The same procedure as described in example 1 was used in that 0.10 g/litre of 2,5-dideoxystreptamine was used in two 80 liter fermentation tanks and six 10 liter fermentation tanks where production medium 5 was used where 0.5% tryptone was used instead of soybean meal and 2% cerelose was used instead of starch. The fermentation was carried out with M. purpurea ATCC 31.164, and isolation of the product was carried out as before. Two residues of 0.68 g and 0.13 g respectively were obtained, which were combined and chromatographed on silica gel plates. Three bands were developed whose mass spectra showed that they were respectively the following compounds: Component C^: 0-3-deoxy-4-C-methyl-3-(methylamino)-Ø-L-ara-binopuranosyl-(1+6) -0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-2,5-dideoxystreptamine , Component C^ : 0-3-deoxy-4-C-methyl-3-(methylamino)-(3-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl -2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-2,5-dedeoxystreptamine, and Component C±a: 0-3-deoxy-4-C-methyl- 3-(methylamino)-p-L-arabinopyranosyl-(1+6)-O-[2,6-diamino-2,3,4,6-tetradeoxy-α-D-erythroglucopyranosyl-(1+4)]-D-2, 5-dideoxystreptamine Mass spectra of the above three components C^, C^ and C^a showed the following mass peaks: Component C^: M+ 461, 404, 344, 334, eel, 306, 303, 288, 160 and 157. Component C2: M<+> 447, 404, 334, 330, 317, 306, 288, 160 and 143. Component C, la: M+ 433, M +1 434, 404, 334, 316, 306, 303, 288, 275 , 160 and 129.

The Rf values by thin-layer chromatography analysis on silica gel plates using a lower phase of chloroform (1) :methanol (1) :concentrated ammonium hydroxide (1) as developing solvent were 0.43, 0.37 and 0.29 for the components C,, C„ and C, , respectively.

-L 2. -L a

Example 3

The same procedure as described in example 1 was used, and 0.50 g/liter of 2-amino-1,3,4,6-cyclohexanepentol was used

(1,3,5-cis) in three 10 liter fermentation tanks in production medium 5 plus 0.1% added phyton in the presence of M. purpurea ATCC 31.164. Isolation of the product was carried out as be-

written earlier and yielded 214 mg of a material which, by chromatographic analysis and from its mass spectrum, proved to be identical to "Component 1" described in Example 1 above, namely 0-3-deoxy-4-C-methyl- 3-(methylamino)-p-L-arabinopyransoyl-(1+6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl -(1+4)]-D-streptamine. The mass spectrum showed the following mass peaks: M<+ >493, 457, 436, 383, 376, 366, 363, 346, 344, 338, 335, 321,

318, 261, 160 and 157.

Example 4

A solution of 270 mg (0.54 mmol) of 0-3-deoxy-4-C-methyl-3-methylamino-3_L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6 -C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine, described in example 1 (Component 1), was dissolved in 5 ml of 50% aqueous tetrahydrofuran and the solution was cooled to 5°C in an ice bath. It was then treated with 208 mg (0.59 mmol) of the N-hydroxysuccinimide ester of S-(-)-γ-(N-benzyloxycarbonyl)-amino-ct-hydroxybutyric acid (Konishi et al., US Patent 3,780. 018), and the mixture became

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 evaporated, and a residue of 513 mg of crude product was obtained, 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 S-(-)-γ-(N-benzyloxycarbonyl)-amino-α-hydroxybutyric acid as previously described. Work-up of the reaction mixture gave a further 435 mg of crude product which was combined with the previously mentioned 513 mg. The pooled product was chromatographed on seven 40 x 20 cm silica gel plates that were 1 mm thick. The system was developed with chloroform: methanol concentrated (28%) ammonium hydroxide (3:1:1) (lower phase). Seven

passes in this solvent were necessary, and after elution of the product band visible under ultraviolet light, 229.5 mg of an impure mixture of the three mono-acylated products was obtained.

The mixture of acylated products was placed on three 40 x 20 cm silica gel plates 1 mm thick, and the plates were developed five times with chloroform:isopropanol:concentrated (28%) ammonium hydroxide (4:1:1) (lower phase), twice with chloroform:iso<p>ropanol:concentrated (28%)ammonium hydroxide (3:1:1) (lower phase) and nine times with chloroform:methanol:concentrated (28%)ammonium hydroxide (4:1:1) (lower phase ). Under ultraviolet irradiation three distinct bands could be distinguished and these were separately excised and eluted from silica gel with chloroform:methanol:concentrated (28%) ammonium hydroxide (1:1:1) (lower phase). Three components were obtained: A 90.9 mg, B 59.1 mg and C 48.5 mg, which were the S-(-)-y-(N-benzyloxycarbonyl)-amino-α-hydroxybutyric acid amide derivatives in the 2'-

The 1- and 3-positions, respectively, of 0-3-deoxy-4-C-methyl-3-methylamino-p-L-arabinopyranosyl-(1+6)-O-[2-amino-6-methylamino-6-C- methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl- -

(1+4)]-D-streptamine. The R^ values for components A, B and C when these were developed five times on silica gel with chloroform: methanol: concentrated (28%) ammonium hydroxide (4:1:1)

(lower phase) was the following:

A - 0.48

B - 0.62

C - 0.70

Component B (the 1-amide, 56.1 mg) was dissolved in 25 ml of 50% aqueous ethanol and 20 mg of 10% palladium on charcoal, and the mixture was shaken in a Parr hydrogenator at a pressure of approx. 4 kg/cm 2 for 5 1/2 hours, after which the catalyst was removed by filtration. Evaporation of the solvent gave 34.3 mg of a white solid 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 gave a precipitate of l-[S-(-)-γ-amino-α-hydroxybutyryl]-0-3-deoxy-4-C-methyl-3-methylainino-|3-L-arabinopyranosyl- (1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-ct-D-erythro-glufcopyranosyl-(1+4)]-D- streptamine as the pentasulfate salt (32 mg), melting point 230-235°C (decomposition), thin-layer chromatography R.^ = 0.18 (silica gel, chloroformmethanol:concentrated (28%)-ammonium hydroxide:water, 1:4:2:1, R ^ gentamicin standard = 0.73).

Analysis: Calculated for <C>25<H>50°10N6"5H2S04:

Components A and C were treated in a similar manner. To walk

47 mg of 2'-[S-(-)-γ-amino-α-hydroxybutyryl]-O-3-deoxy-4-C-methyl-3-methylamino-ø-L-arabinopyranosyl-(1+6)- O-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine as the bisbase•tetrasulfate• heptahydrate, melting point 237-241°C (decomposition): thin-layer chromatography R^ = 0.33 (silica gel, chloroform: methanol : concentrated (28%) ammonium hydroxide: water, 1:4:2:1, R^ gentamicin C, standard = 0, 73).

Analysis: Calculated for (<C>25<H>50N6°10^2"4H2S04*7H2°:

Component C gave 26 mg of 3-[S-(-)-γ-amino-α-hydroxybutyryl]-0-3-deoxy-4-C-methyl-3-methylamino-|3-L-arabinopyranosyl- (1+ 6) - 0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1+4)]-D-streptamine as bisbase•penta -sulfate • trihydrate, melting point 220-230°C (decomposition), thin-layer chromatography R^ = 0.30 (silica gel, chloroform: methanol: concentrated (28%) ammonium hydroxide: water, 1:4:2:1, R^ gentamicin standard = 0.73).

Analysis: Calculated for (<C>25<H>50<N>6°10^2"<5>H2S04'3H20:

Claims (1)

Process for the preparation of aminocyclitol antibiotics with the formula: ~ • where R 2 and R 1 - are both hydrogen or both are hydroxy, R 8 and R 1 are each hydrogen or methyl; as well as acid addition salts thereof, characterized in that in a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol compound with the formula: wherein R 2 and R 1 have the same meaning as indicated above and R 1 is amino or hydroxy, grows Micromonospora purpurea ATCC 31.164, and, if desired, converts an obtained free base into an acid addition salt thereof.