CH618215A5 - Process for the preparation of aminocyclitol antibiotics - Google Patents

Abstract

Aminocyclitol analogues of gentamicin C1, C2 and C1a and the corresponding compounds which are acylated on the 1-amino, 3-amino and 2'-amino groups with a lower omega -amino- alpha -hydroxyalkanoyl group are prepared. For this, a nutrient medium is prepared containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol added with the mutant Micromonospora purpurea ATCC 31 164. If desired, the product is acylated with a lower omega -(N-benzyloxycarbonyl)amino- alpha -hydroxyalkanoic acid ester and then the benzyloxycarbonyl group is subjected to catalytic hydrogenolysis.

Description

The present invention relates to a process for the preparation of antibiotics of the aminocyclitol type corresponding to formula I

R-

r

CH-NH-R

NH-R,

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4

in which R ,, R3 and R8 each represent a hydrogen atom; or one of Rj, R3 and R8 represents a co-amino-a-hydroxy-lower alkanoyl group of formula

H2NCH2 (CH2) nCHOHCO

in which n is equal to 0 or to 1, the other groups R15 R3 and Rg being hydrogen atoms; R2 and R5 represent a hydrogen atom or a hydroxy group; except that when R2 is a hydrogen atom, R5 is not a hydroxy group and is with respect to the amino groups in positions 1 and 3 and R6 and R7 each represent a hydrogen atom or a methyl group.

Compounds of formula I are prepared in which Rb R3 and Rg are hydrogen atoms, by the method described in the US patent. No. 3,669,838 to Shier et al. This method consists in cultivating a nutritive medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an added aminocycli-tol derivative of formula:

single bond. According to the procedure described by Shier et al., The nature of the mutant is such that it is unable to synthesize the aminocyclitol subunit from a nutrient medium to thereby produce the antibiotic, but that it can incorporate this last s in an antibiotic when aminocyclitol is added to the nutrient medium.

It has been found that another mutant than M. purpurea ATCC 31 119, that is to say M. purpurea ATCC 31 164, is also capable of incorporating into the antibiotics of formula I above, an aminocyclitol of formula:

Ha

NH-R.

II

HH-Rn in which R1; R2, R3 and R5 have the meanings given above, and where R! and R3 can additionally represent a single bond joining the two nitrogen atoms of the amine groups, in the presence of an appropriate mutant microorganism, said mutant microorganism being unable to synthesize said aminicyclitol itself but being able to incorporate said aminocyclitol in the compound of formula I, particularly a mutant of Micromonospora purpurea designated by Micromonospora purpurea ATCC 31 119 and in separating the product from the culture medium. The compounds of formula I are produced in which R] and R3 are both a hydrogen atom when using the aminocyclitol of formula II where Ri and R3 represent a

Wherein R2 'and R5' are each a hydrogen atom or a hydroxy group and R / is an amine or hydroxy group.

The process which makes it possible to prepare the compounds of formula I where R6 and R7 each represent a hydrogen atom m or a methyl group; R2 and R5 each represent a hydrogen atom or a hydroxy group; Rj, R3 and R8 represent a hydrogen atom, is used in the presence of M. purpurea ATCC 31 164 using the above operating mode, and consists in cultivating a nutritive medium containing carbohydrates, a source of assimilable nitrogen, salts essential and an aminocyclitol of formula IIa in the presence of M. purpurea ATCC 31 164.

The compounds of formula I, where one of the groups R 1, R 3 and R 8 represents a group co-amino-α-hydroxy-lower alkanoyl, can be prepared by the method described by Konishi et al. in the U.S. patent N 3,780,018, which consists in reacting the compound of formula I in which each of the groups R 1, R 3 and R 8 is a hydrogen atom, with an N-hydroxysuccinimide ester of formula:

0

^ 11

M CH20-C-NHCH2 (CH2) nCH0HC0-0-N

IV

where n has the meaning given above, then the mixture resulting from the compounds of formula I is subjected where one of the groups Rl R3 and Rg is the group co- (N-benzyloxycarbo-nyl) amino-a-hydroxy-lower alkanoyl:

/ \

Ì

_.CH20-C-NHCH2 (CH2) nCHOHCO-

5

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.10

the other two being hydrogen atoms, upon hydrogenolysis of the benzyloxycarbonyl group by hydrogen on a catalyst.

The N-hydroxysuccinimide esters of formula IV form a category of compounds which is generally known).

As indicated above, when a compound of formula I is used as the starting material in the acylation reaction where each of the groups Rb R3 and R8 is a hydrogen atom, a mixture of the three possible isomeric mono-amides is obtained in which one of the hydrogen atoms of the amine groups, R [, R3 or R8, is replaced by the co- (N-benzyloxycarbonyl) amino-a-hydroxy-alka-lower nucleus group. When one wants a separate characterization and study of these products, they must obviously be separated from each other.

The acylation reaction can be carried out by reacting equivalent amounts, in moles, of the compound of formula I and of the N-hydroxysuccinimide ester, preferably at a temperature of from -10 ° C to + 10 ° C, and in an aqueous solution of an inert organic solvent, for example tetrahydrofuran, dioxane, dimethyl ether of ethylene glycol, dimethyl acetamide, dimethylformamide, dimethyl ether of propylene glycol, etc.

The hydrogenolysis of the benzyloxycarbonyl group can be carried out on a palladium on carbon catalyst in an organic solvent miscible with inert water, for example methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether. , dimethyl ether of propylene glycol, etc.

The compounds of formula I have been tested in a conventional antibacterial test with serial dilution, and have been found to have antibacterial activity, particularly against organisms resistant to gentamicin. The compounds are thus useful as antibacterial agents.

The compounds of formula I are essentially intended for oral, local or parenteral administration and can be prepared, for their use, using a pharmaceutical support by suspension, either in the form of a free base or in the form pharmacologically acceptable, non-toxic acid addition salts in an inert carrier such as polyethylene glycol, either by tablet form or encapsulation for oral administration, alone or with suitable adjuvants, or they may be introduced into classic creams or jellies for local application.

The molecular structures of the new compounds have been assigned on the basis of the study of their chromatographic characteristics determined by thin layer chromatography (TLC), their nuclear magnetic resonance spectrum (NMR) and their mass spectrum; by degradation to known compounds; and by the correspondence between the calculated and found values for their elementary analyzes.

The following specific examples are representative of how to prepare the compounds and how to carry out the process of the invention, without the invention being limited to these examples.

Mutation process

In the following procedures, various media are used, constituted as follows: 60

Middle 1:

35

45

5 (1

N — Z Amine g / l

Glucose

10

Soluble starch

20

Yeast extract

5

N-Z-Amine-Type A (Difco)

5

CaC03

1

Agar

15

Medium 2: Germination medium (in distilled water)

Beef extract 0.3%

Tryptone 0.5%

Dextrose 0.1%

2.4% soluble starch

0.5% yeast extract

CaC03 0.4%

Medium 3: Soy-glucose g / l

Soy flour 30

Dextrose (Cerelose) 40

CaC03 1

Medium 4: TGE (trypticase-glucose extract) g / l

Trypticase-glucose extract 5.0

Trypticase-peptone 3.0

Glucose 1.0

Agar 15.0

Medium 5: Production medium

Beef extract 0.3%

0.5% yeast extract

Soy flour 0.5%

Maltose 0.1%

Starch 2.4%

0.1% Casamino Acid

CaC02 0.4% CoCl2 • 6H20 1 mg / liter

Medium 6: Streptomycin g / l test agar

Beef extract 1.5

Yeast extract 3.0

Peptone 6.0

Agar 15.0

The organism Micromonospora purpurea is obtained from the United States Department of Agriculture under the number NRRL 2953 and it is maintained on agar cultures inclined N-Z amine (Medium 1). Fermentation is carried out by immersion in flasks containing the germination medium 2 for 4 days at 37 ° C. on a rotary agitator. From the seeds of this first step, a 10% inoculum is transferred to the germination medium (medium 2) and the fermentation is continued as above at 28 ° C for 7 days.

To establish the organism's ability to biosynthesize gentamicin in the absence of added deoxystreptamine, a third stage fermentation is carried out using a 5% inoculum in a 10 liter fermenter in a soy and glucose medium ( medium 3) at 28 ° C, stirring at 200 rpm and blowing 2 liters of filtered air per minute. After 6 days, the contents of the tank are acidified to pH 2.2 with 6N sulfuric acid, filtered and neutralized a portion of 500 ml with ammonium hydroxide and passed over an exchange resin of IRC 50 ions (Na + form). Then the column is rinsed with water and eluted with 2N sulfuric acid. By following the procedure described in the US patent. No. 3,091,772, a 300 mg sample of crude gentamicin, which is found to be biologically active and which contains three components similar to gentamicin Cl9 C2 and Cla, is isolated by TLC examination.

In order to obtain a mutant of the organism, the broth cultures are cultured in medium 2 (37 ° C. for 3 days) and the resulting cells are harvested by centrifugation, washed and put back in suspension in buffered saline. This suspension is treated with the mutagenic agent, N-methyl-N'-nitro-N-nitroso-guanidine. Samples of the mutagenized culture are cultured on a medium plate at 37 ° C.

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6

until colonies are flown (usually about a week). The colonies are transplanted onto two series of plates (medium 4), one series of which is covered with a suspension of B. subtilis spores. After incubation at 37 ° C for 18 to 20 hours, the “transplants” which show no zone of inhibition on the B. subtilis plate are transferred from the original plate (no B. subtilis) to agar cultures. inclined with medium 1 and incubated until total growth is seen.

These potential non-producing mutants are then put in the presence of m deoxystreptamine in an antibiotic biosynthesis stimulation test, as follows. Stock cultures of the potential mutants are streaked onto the surface of medium plates 4 and incubated at 37 ° C until growth is seen (approximately 3 to 4 days). Then filter paper discs are immersed in a solution of deoxystreptamine (500 ng / ml) and they are placed above the culture streaks. After incubation for 24 hours, the plate surface is inoculated with B. subtilis using the overlay technique, and incubation is continued for an additional 18-20 hours. Isolates with zones of inhibition around the disc are referred to as deoxystreptamine mutants. One of these mutants, called mutant VIB and deposited at American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, under the name of Micromonospora purpurea ATCC 31 119, is used in the production of gentamicin-type antibiotics as described above below.

This latter organism is then itself subjected to the same mutation operating procedure as that described above, and samples of the mutagenized culture are cultured on a plate, on medium 4, at 37 ° C. until what we see colonies (about a week). The colonies are transplanted into two sets of plates containing streptomycin test agar (medium 6). 35

One of the series serves as an original plate for subsequent recovery while the second series contains 25 µg / ml streptamine sulfate and a suspension of B. subtilis spores,

forming an upper layer as a test organism. These plates are incubated at 37 ° C for 24 hours and examined for areas of inhibition. Those with the largest areas are transferred from the original plate onto cultures on an inclined agar of N-Z amine (medium 1) and they are incubated for one week at 37 ° C.

Mutants incorporating lower levels of 45 streptamine sulfate are then tested with streptamine and scyllo-inosose at 500 µg / ml as a base. The mother cultures are transferred to flasks containing medium 2 plus the above intermediates and incubated at 37 ° C on a rotary shaker for 7 days. Antibiotic activity in the vials is periodically determined by the disc diffusion test method using B. subtilis as the test organism. Isolates with zones of inhibition around the disc are designated as mutants of streptamine or scyllo-inososis. A te1 mutant, called a VIB-3P mutant and deposited on 55 July 11, 1975 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20851, under the name of Micro-monospora purpurea ATCC 31 164, is used for the production of the antibiotic aminocyclitols of the streptamine, deoxystreptamine and disexoxystreptamine type as described below under Ml.

Biosyntheses ivec M. purpurea ATCC 31164

<i5

Example 1

Cultures of the mutant organism are maintained on inclined cultures of N-Z amine agar (medium 1) from which transfers are carried out in flasks containing 500 ml of germination medium 2. The flasks are incubated at 28 °. C for 4 days on a rotary shaker (5 cm stroke) at 225 rpm.

A 10% (volume / volume) inoculum from the germination stage is aseptically transferred into 14-liter fermenters containing 9 liters of sterile germination medium 2. They are stirred at 450 rpm at 28-29 ° C and filtered air is injected at 5 liters per minute. At the time of inoculation, 200 mg per liter of streptamine sulfate are added as a suspension in sterile distilled water. The fermentation continues for 8 days.

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

The fermentation is stopped by adding 10N sulfuric acid to pH 2.0 and by filtration using a filter aid to collect the mycelia. The pH of the filtered broth is adjusted to 7.0 and 1.56 g of oxalic acid are added per gram of calcium carbonate present in the medium, to remove the calcium. Then allowed to stand overnight, and the clarified broth is decanted and passed over a Bio-Rex 70 resin (weak cation exchanger) in Na + form, using about 14 g of resin per liter of broth. Then the column is washed with distilled water and eluted with 2N sulfuric acid. All the fractions with antibiotic activity are combined, neutralized and concentrated in vacuo below 50 ° C., until the crystallization of the salt becomes evident (at a volume of approximately 1/3). Then the pH is adjusted to 10.5, and four volumes of acetone are added to precipitate the mineral matter which is removed by filtration. The pH of the filtrate is adjusted to 5.0 with 6N sulfuric acid, concentrated in vacuo to about 1/20 of the initial volume and cooled. A white crystalline crop melting above 300 ° C. is collected by filtration, which, by its properties in thin layer chromatography and its infrared spectrum, appears to be identical to streptamine sulfate. From two 10-liter fermentations treated as above, a total of 0.7 streptamine sulfate is obtained, and from two 80-liter fermentations, a total of 21 g of streptamine sulfate is collected at this stage.

The filtrate is further concentrated and 10 volumes of methanol are added which provides the first crude solid antibiotic. From two 10 liter fermentations, 7 g are obtained, and from two 80 liter fermentations, 24 g are obtained.

In order to identify the antibiotic components during the purification procedures, the chromatographic mobility values obtained by paper chromatography and thin layer chromatography for the genticamines Cu Q and Cla and for each of the corresponding aminocyclitol homologs prepared as indicated above, where the chromatographic mobility (hereinafter referred to as MC) is expressed as:

jyjç _ distance of the component from the origin distance of gentamicin Q from the origin.

The chromatographic systems used are as follows:

System 1 - Whatman paper n ° 1 saturated, with 0.95M sulfate bi-sulfate and developed top-down in 80% aqueous ethanol plus 1.5% NaCl, and subsequent bioautograph using B. subtilis as organism test.

System 2 - Plate of silica gel F 254 developed in the lower phase of the chloroform mixture (1): methanol (1): concentrated ammonium hydroxide (28%) (1). The components

7

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are localized with a ninhydrin spray and heating.

The MC values of the main antibiotic components of the present invention with respect to a reference gentamicin complex, values which are all given with respect to gentamicin C, are given in Table I, where the compounds designated by component 1, component 2 and component 3 are respectively:

0-3-deoxy-4-C-methyl-3-methylamino-PL-arabino-pyranosyl- (1 - ^ 6) -0- [2-amino-6-methylamino-6-C-methyl-2,3, 4,6-tetradeoxy-aD-erythro-glucopyranosyl- (1-4)] -D-streptamine;

0-3-deoxy-4-C-methyl-3-methylamino-pL-arabino-pyranosyI- (l— »ó ^ O-pjó-diamino-ó-C-methyl ^ .S ^ ó-tetra-deoxy-aD -erythro-glucopyranosyHl- ^ jD-streptamine; and

0-3-deoxy-4-C-methyl-3-methylamino-PL-arabino-pyranosyl- (l - ^ - O - ^. Ó-diamino ^ SAó-tetradesoxy-aD-erythro-glucopyranosyl ^ l—> 4) ] -D-streptamine.

Table I ■

MC System I

MC System 2

Gentamicin C!

1

1

Gentamicin C2

0.89

0.83

Gentamicin Cia

0.50

0.67

Component 1 (Main)

0.96

0.92

Component 2 (Low)

0.76

0.75

Component 3 (Low)

0.50

0.61

The crude solid (7 g) from the 10 liter fermenters, which 1 () exhibits antibacterial activity, is suspended in 200 ml of methanol and 10 ml of concentrated ammonium hydroxide (28%), and stir the mixture for 30 minutes and filter it. 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" are essentially devoid of antibiotic activity.

The oily base is mixed with 4 g of silica gel (Davison, quality 923.74-149 microns) and it is introduced into a column of silica gel measuring 1.8 X 28 cm. The column 4 is prepared in the form of a suspension using the lower phase of the isopropyl alcohol mixture (1): chloroform (2): 17% aqueous ammonium hydroxide (1). The column is developed with this solvent and 50 ml fractions are collected. 45

Fractions 8 and 9 contain a single ninhydrin positive component which provides 20 mg as a pale yellow oil upon removal of the solvent. The mass spectrum of this substance, called component 1, presents a molecular ion and main fragments each greater by 16 units of 50 mass (that is to say of an oxygen atom) than those obtained for gentamicin. C ,, as follows:

Reference: gentamicin Q: M + 477,420,360, 350, 347, 322,319,304

Component 1: M + 493.436, 376.366, 363.338, 335.320. „We transform this substance into its sulfate by dissolving it in ethanol and adding a few drops of ethanol containing sulfuric acid. The resulting white solid is collected and dried to give 22 mg of component 1, 0-3-deoxy-4-C-methyl-3-methylamino ^ -L-arabinopyranosyl- () ll (l- ^ 6 ) -0 ~ [2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetra-deoxy-aD-erythro-glucopyranosyl- (1 ^ 4)] - D-streptamine,

in the form of the heptasulfated and decahydrated di-base, m.p. > 300 ° C.

65

Analysis, calculated for (C2iH43N508) 2 • 7H2 S04 • 10H20: C 27.22; H 6.53; N7.55; S 12.09 Found: C27.15; H6.67; N7.79; S 12.76.

Fractions 10-13 provide a positive component to the more polar ninhydrin called 0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopyranosyl- (l—> 6) -0- [ 2,6-diamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1 ^ -4)] - D-streptamine, component 2 which exhibits antibiotic activity.

Fractions 15-26 have a third, more polar component with antibiotic activity. The mass spectrum of this component has characteristic peaks of sugars corresponding to gentamicin Cla, at 129 (purpurosamine) and 160 (garosamine) and is called component 3, O-3-deoxy-4- C-methyl-3 -mithylamino-ß- L-arabinopyranosyl- (/ -> 6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l- * 4)] - D -streptamine.

Alternatively, the new component 1 is isolated as follows: a mixture of the raw antibiotic base obtained as above (0.9 g) is dissolved in 7 ml of water and the pH is adjusted to 4.5 with acid sulfuric IN. The solvent is passed through a column of strong anion exchange resin (IRA 401) in OH "form (bed dimensions = 0.7 × 10 cm). The column is eluted with water and the eluate is evaporated. under vacuum at 35 ° C. The resulting residue is triturated with 50 ml of the lower phase of a solvent composed of 17% aqueous ammonium hydroxide, isopropyl alcohol and chloroform (1: 1: 2). The solvent is decanted and concentrated under vacuum, which leaves an oily residue weighing 140 mg. The mass spectrum of this substance corresponds to that of fractions 8 and 9 above, that is to say M + 493,436,376, 366,363,338 , 335.320.

The nuclear magnetic resonance spectrum of component 1 is also compatible with the proposed structure corresponding to 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (l—> 6) -0- [2 -amino-6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1—> 4)] - D-streptamine and is summarized in Table II.

Table II ô

5.87, 5.60 5.22

3,09,3,15

2.9-4.8

1.9-2.6

Integration Attribution

2 OCHO epimers

12 H exchangeable 6 NCH3 X 2

13 -CHO X 6, -CHN X 5, -CH20 4 -CH2CH2-

Alternatively, a 4 g sample of the raw antibiotic salt is dissolved in 50 ml of water and passed through an AG1X8 anion exchange resin (1 X 27 cm resin bed). The antibiotic is eluted with water and all the active fractions are combined and evaporated in vacuo. The salt is removed from the residue by extraction with methanolic sodium hydroxide. The released base is mixed with 7 g of silica gel and it is introduced on a column of 50 g of silica gel. This column is developed with the chloroform: methanol: concentrated ammonium hydroxide (28%) (3: 4: 2) mixture and fractions of 25 ml are collected. The first fractions provide the new least polar component 1, but mixed with several less polar impurities as shown by thin layer chromatography. These fractions are combined and the concentrated product is introduced onto a column of 50 g of silica gel as above. This column is developed with the chloroform: methanol: concentrated ammonium hydroxide (28%) (5: 3: 1) mixture and fractions of 25 ml are collected. Fractions 6-12 contain the desired component free of obvious impurities. The oil resulting from the removal of the solvent is converted into sulphate in ethanolic sulfuric acid as described above, which gives 0.124 g of component 1 in the form of the heptasulphated and decahydrated di-base, m.p. > 300 ° C, described above.

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By cultivating an appropriate aminocyclitol with the mutant Micromonospora purpurea ATCC 31 164, in germinator medium 2 and by separating the products described above in Example 1, the following compounds of formula I are prepared in the same way:

Example 2

0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopy-ranosyl- (l- * 6) -0- [2-amino-6-methylamino-6-C-methyl-2 , 3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- {1 - ^> 4)] epi-streptamine (MC versus gentamicin Q: System 1 = 0.59, System 2 = 0.63); 0-3-deoxy-4-C-methyl-3-methyl-amino-ß-L-arabinopyranosyl- (1 - ^> 6) - 0- [2,6-diamino-6-C-methyl-2, 3,4,6-tetradeoxy-αD-erythro-glucopyranosyl- (1— * 4)] epistreptamine \ and 0-3-deoxy-4-C-methyl-3-methyl-amino-ß-L-arabinopyranosyl- ( / - »6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l - ^> 4) epistreptamine are obtained using epistreptamine instead of streptamine in the fermentation procedure.

Example 3

Following a procedure similar to that described in Example 1 and using 0.10 g per liter of 2-deoxy-strept-amine in a 10 liter fermentation in production medium 5, in the presence of M. purpurea ATCC 31 164, and by separating the product as above, we obtain 0.51 g of crude substance which, by chromatography on silica gel, provides as main component a substance whose identity of chromatographic mobilities shows that it is identical to gentamicin C [, i.e. 0-3-deoxy-4-C-methyl-3- (methylamino) -fi-L-arabinopyranosyl- (l—> 6) -0- [2-amino -6- (methylamino) -6- C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1 ^ 4)] - D-2-deoxystreptamine

Example 4

Following a procedure similar to that described in Example 1 and using 0.10 g per liter of 2,5-dideoxy-streptamine in two ferments of 80 liters and six fermentations of 10 liters in the production medium 5 in which replaced the soy flour with 0.5% tryptone and the starch with 2% Cerelose, in the presence of M. purpurea ATCC 31 164, and by separating the product as before, two residues are obtained, 0, 68 g and 0.13 g, which are combined and which are chromatographed on silica gel plates, which gives three bands, the mass spectra of which show that they correspond respectively to component Q: 0-3- deoxy-4-C-methyl-3- (methyl lamino) -ß-L-arabinopyranosyl- (1 ^> 6) -0- [2-amino-6- (methylamino) -6-C-methyl-2,3 , 4,6-tetradesoxy-aD-erythro-glucopyranosyl- (/ -> 4)] - D-2,5-dideoxystreptamine; component C2: 0-3-deoxy-4-C-methyl-3- (methylamino) -fi-L-arabinopyranosyl- (1— »6) -0-] 2,6-diamino-6- C-methyl- 2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl (1-4)] - D-2,5-dideo-oxystreptamine; and the component Cla: 0-3-deoxy-4-C-methyl-3- (mJthylamino) -ß-L-arabinopyranosyl- (l—> 6) -0-] 2,6-di-amino-2,3 , 4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1 ^ * 4)] - D-2,5-dideoxystreptamine. The mass spectra for the three components Cx, C2 and Cla indicated above include the following peaks: Component Ct: M + 461,404,344, 334, 331, 306,303,288,160 and 157. Component C2: M + 447,404,334, 330,317,306,288,160 and 143. Component Cla: M + 433, M + +1 434,404,334,316,306,303,288,275,160 and 129.

The Rf values, by TLC on silica gel plates using the lower phase of the chloroform mixture (l): metha-nol (l): concentrated ammonium hydroxide (1) as development solvent, are 0.43.0 , 37, and 0.29 for components C !, C2 and Cla respectively.

Example 5

Following a procedure similar to that described in Example 1 and using 0.50 g per liter of 2-amino-1,3,4,5,6-cyclohexanepentol (1,3,5cis) in three fermentations of 10 5 liters in production medium 5 additionally containing 0.1% phytone, in the presence of M. purpurea ATCC 31 164, and by separating the product as before, 214 mg of substance is obtained which is revealed by its mobility chromatographic and by its mass spectrum, be identical to "component 1" 10 described in Example 1 above, that is to say 0-3-deoxy-4-C-methyl-3- (methylamino ) -fi-L-arabinopyranosyl- (7—6) -0- [2-amino-6- (methylamino) -6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l—> 4)] - D-streptamine. The mass spectrum shows the following peaks: M + 493,457,436,383,376, 15,366,363, 346,344,338,335,321,318, 261,160 and 157.

Synthesis of amino-hydroxy-lower alkanoyl substituted derivatives

Example 6

Cooled to 5 ° C in an ice bath, a solution of 270 mg (0.54 mmol) of 0-3-deoxy-4-C-methyl-3-methyl-amino-ß-L-arabinopyranosyl- ( l—> 6) -0- [2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopy-ranosyl- (l—> 4)] -D-streptamine, described above in Example 25 (component 1), dissolved in 5 ml of tetrahydrofuran auquex at 50%, and it is treated with 208 mg (0.59 mmol) of the ester of S- (-) -y- (N-benzy-Ioxycarbonyl) amino-a-hydroxybutyric acid N-hydroxysuccinimide (US Patent No. 3,780,018 to Konishi et al.), and the mixture was stirred 5 ° C m for 20 hours. Then the mixture is concentrated to 10 ml under vacuum. 25 ml of n-butanol and 10 ml of water are added, and the layers are separated. The aqueous layer is again washed with 10 ml of n-butanol. The combined organic layers are evaporated, leaving a residue of 513 mg of crude product which is left aside.

The aqueous layer is evaporated to dryness, which gives 304 mg of a residue which is dissolved in 25 ml of 50% aqueous tetrahydrofuran and which is treated with an additional 208 mg of the N-hydroxysuccinimide ester. S - (-) - y-4 (1 (N-benzyloxycarbonyl) -amino-a- hydroxybutyric acid as before. Treatment of the reaction mixture provides an additional 435 mg of the crude product which is combined with the 513 mg previously obtained and they are chromatographed on 7 plates of silica gel 40 × 20 cm with a thickness of 1 mm. The system is developed with the lower phase of the chloroform: methanol: 28% concentrated ammonium hydroxide (3: 1 : 1) Seven passages in this solvent system are necessary, and after having eluted the band of the product which is visible with ultraviolet, 229.5 mg of a crude product of the 50 three monoacylated products are obtained.

The mixture of acylated products is deposited on three plates of silica gel 40 × 20 cm and 1 mm thick and the plates are developed 5 times with the lower phase of the mixture 55 chloroform: isopropanol: concentrated ammonium hydroxide (28 %) (4: 1: 1), twice with the lower phase of the chloroform mixture: isopropanol: concentrated ammonium hydroxide (28%) (3: 1: 1) and 9 times with the lower phase of the chloroform mixture: methanol : concentrated ammonium hydroxide 60 (28%) (4: 1: 1). 3 distinct bands are obtained visible under ultraviolet radiation which are cut separately and which are eluted from silica gel with the lower phase of the chloroform: methanol: concentrated ammonium hydroxide (28%) (1: 1: 1), which provides three components: A, 90.9 mg; B, 65 59.1 mg; and C, 48.5 mg, which are the amide derivatives of S - (-) - Y- (N-benzyloxycarbonyl) - amino-a-hydroxybutyric acid in positions 2 ', 1 and 3 respectively from 0-3 -deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6) -0- [2-

9

618215

amino-6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-a-D-erythro-glucopyranosyl- (1-4)] - D-streptamine. The Rf values for components A, B and C, when developing 5 times on silica gel with the lower phase of the chloroform system: methanol: concentrated ammonium hydroxide (28%) (4: 1: 1),

are:

A-0.48 B-0.62 C-0.70

Component B is stirred (1-amide, 56.1 mg) dissolved in 25 ml of 50% aqueous ethanol and 20 mg of 10% palladium on carbon in a Parr stirrer at 3.75 atmospheres for 5.5 hours, after which the catalyst is removed by filtration on a filter aid. Evaporation of the solvent provides 34.3 mg of a white glass which is dissolved in 2.5 ml of water and which is treated with 11.4 mg of sulfuric acid in 0.1 ml of water. Addition of 10 ml of ethanol precipitates 1-lS - (-) - y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopyranosyl - (7—> 6) 0 [2-amino-6-methylamino-6- C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glu-copyranosyl- (1- ^ 4)] - D -streptamine in the form of pentasulfate (32 mg), mp 230-235 ° C with decomposition, TLC, Rf = 0.18 (silica gel, chloroform: methanol: concentrated ammonium hydroxide (28%): water, 1: 4: 2: 1; Reference Rf for gentamicin C] = 0.73).

Analysis, calculated for C25H50O10N6 • 5H2S04:

C 27.67; H 5.57; N7.75 Found C 27.50; H 5.58; N7.42.

Components A and C are treated in the same way. A provides 47 mg of 2 '- [S - (-) - y-amino-a-hydroxy-butyryl] -0-3-deoxy-4-C-methyl- 3-methylamino-fi-L-arabinopyranosyl- (l- ^ 6) -0- [2-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-ery-} 5 thro-glucopyranosyl- (1 ^> 4)] - D-streptamine in the form of the tetrasulfated and heptahydrated di-base, mp 237-241 ° C with decomposition; TLC, Rt = 0.33 [silica gel, chloroform: me-thanol: concentrated ammonium hydroxide (28%): water, 1: 4: 2: 1; Gentamicin reference Rf Q = 0.73]. 4 "

Analysis, calculated for (C25H50N6O10) 2 • 4H2S04 ■ 7H20:

C 35.16; H 7.20; N9.84 Found C 35.38; H 7.08; N9.49

Component C provides 26 mg of 3- [S - (-) - y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopyranosyl- (l ^ > 6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (7-h> 4)] - D-streptamine under form of the pentasulfated and trihydrated di-base, pf 220-230 ° C with decomposition; TLC, Rf = 0.30 [silica gel, chloroform: methanol: concentrated ammonium hydroxide (28%): water, 1: 4: 2: 1; Gentamicin reference Rf Q = 0.73]. 5

Analysis, calculated for (C25H50N6O10) 2 • 5H2S04 • 3H20:

C 34.64; H 6.74; N9.67 Found C 34.35; H 6.38; N 8.60.

6

By proceeding in a similar manner to that described in Example 6, using respectively the antibiotics described in Examples 2 and the N-hydroxysuccinimide ester of S - (-) - v- (N-benzyloxycarbonyl acid). ) amino-a-hydroxybutyric or the pentafluorophenyl ester of N- (benzyloxycarbonyl) - (S) -isoserine [(S) -ß-amino-a-hydroxypropionic acid, described by Haskell et al., Carbohydrate Research, 28,273- 280 (1973)], the following compounds of formula I are prepared in the same way:

5

Example 7

1 -f (S) -fi-amino-a-hydroxypropionyl] -0-3-deoxy-4-C-methyl-3-methylaminô-fi-L-arabinopyranosyl- (l- ^ 6) -0-] 2, 6-diamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopy-10 ranosyl- (1 ^> 4)] epistreptamine and 2 '- [(S) -ß-amino- a-hydroxy-propionyl] -0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabi-nopyranosyl- (1— * 6) - O-] 2,6-diamino-6- C -methyl-2,3,4,6-tetra-deoxy-aD-erythro-glucopyranosyl- (1 ^> 4)] -epistreptamine

15 Example 8

l- [S - (-) - y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopyranosyl- (1— »6) -0-] 2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1- ^ 4) -2,5-dideoxystreptamine and 2 '- [S - (-) - y-amino-a -2o hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-fi-L-arabinopyranosyl- (/ -> 6) -0- [2,6-diamino-2,3,4,6 -tetradesoxy-a- D-erythro-glucopyranosyl- (1- ^ 4)] -2,5-dideoxystreptamine

Example 9

25 1 - [(S) -fi-amino-a-hydroxypropionyl] -0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1 ^> 6) -0- [2-amino -6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1 ^> 4)] -2-deoxystreptamine and 2 '- [(S) -ß-amino -a-hydroxypropionyl] -0-3-deoxy-4-C-methyl-3-30 mithylamino-ß-L-arabinopyranosyl- (1 ^> 6) -0-2-amino-6-me-thylamino-6- C-methyl-2,3,4,6-tetradesoxy-aD-erythro-gluco-py ranosyl- (1 ^> 4)] - 2-deoxystreptamine.

Antibacterial test results

We test 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (l—> 6) -0- [2-amino-6-methylamino-6-C-methyl-2,3 , 4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1—> 4)] - D-streptamine described above in example 1 and called component 1 by comparing it to gentamicin vis-à-vis a number of microorganisms according to the following procedure.

Stock solutions of each compound containing 200 g / ml of base are prepared in distilled water and are sterilized by filtration. Cultures of the test microorganisms are cultured for 24 hours at 37 ° C. in 10 ml tubes of phos-phate-tryptose or Mueller-Hinton broth. Each culture is adjusted with broth to an optical density of 0.1 on a spectronic 20 (about 108 cells / ml). The adjusted cultures are diluted 1: 500 in the broth for use as an inoculum (final cell concentration of approximately 2 X 105 cells / ml). The antibacterial activity of the test compounds is tested by a single series tube dilution method. Original dilutions are prepared in double serial dilution in broth from stock solutions of drugs, and 0.2 ml of each drug concentration is placed in 17 13 X 100 ml tubes. All tubes are inoculated with 0.2 ml of the appropriate diluted culture (final cell concentration per tube = 105 cells / ml). Minimum inhibitory concentrations (lower drug concentrations with no visible growth) are determined after a 16 hour incubation at 37 ° C.

The results are given in Table III below. Inactive compounds are considered at inhibitory concentrations above 100 µg / ml.

618,215

Table IIP

Organization

10

Minimum inhibitory concentration fUg / ml)

Component I

Gentamicin

Staphylococcus aureus Smith

0.78

0.195

Escherichia coli Vogel

3.13

3.13

Escherichia coli W677 / HJR66

50

inactive

Escherichia coli JR 35

3.13

6.25

Escherichia coli JR 76.2

6.25

100

Escherichia coli JR89

50

50

Escherichia coli Kl 2 ML 1629

3.13

1.56

Enterobacter cloacae A-20960

1.56

25

Klebsiella pneumoniae 39645

1.56

0.78

Klebsiella pneumoniae A-20636

3.13

50

Proteus mirabilis MGH-1

6.25

1.56

Providencia 164

100

inactive

Providencia stuartii A-20894

100

100

Pseudomonas aeruginosa MGH-2

3.13

0.39

Pseudomonas aeruginosa A-20717

12.5

<12.5

Pseudomonas aeruginosa A-20741

Inactive

Inactive

Pseudomonas aeruginosa A-20897

12.5

Inactive

"Grown in tryptose-phosphate broth.

We make a comparison test of this same component 1 described above in Example 1, 0-3-deoxy-4-C-methyl-3-methylamino- ß-L-arabinopyranosyl- (1-6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l—> 4)] - D-streptamine, with the complex of gentamicin (Cb Q and Cla) and gentamicin Q, and a comparison trial is carried out between the 1-, 3- and 2 '- [S - (-) - Y-amino- <x-hydroxybutyryl] amides component 1, described above in Example 6, and respectively called compound 1 (1-HABA),

: 5 component 2 (3-HABA) and component 1 (2'-HABA), and the 1-, 3-, and 2 '- [S - (-) - Y-amino-Ct-hydroxybutyryl] corresponding amides of the gentamicin C! (all described by Konishi et al. in U.S. Patent No. 3,780,018 issued December 18, 1973) and called respectively C! (1-HABA) C, (3-HABA) "and C, (2'-HABA). The results are given in Table IV below, where the test organisms 1,2,3,4,4 and 5 respectively designate 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.

Table IVe

Test organisms

Compound 1

2

3

4

5

6

Gentamicin

C „C2, Cla ^ 0.024

0.39

50

12.5

25

> 100

Gentamine Q 0.049

0.78

50

12.5

25

> 100

Component I 0.098

1.56

6.25

0.78

3.13

25

C, (1-HABA) 0.39

3.13

12.5

3.13

6.25> 100

Component I

(1-HABA) 0.78

6.25

12.5

6.25

12.5

> 100

Cj (3-HABA) 3.13

25

> 100

100

> 100

> 100

Component I

(3-HABA) 6.25

50

100

50

50

> 100

C] (2'-HABA) 6.25

50

100

25

50

> 100

Component I

(2'-HABA) 1.56

25

50

25

50

> 100

"Grown in Mueller-Hinton broth.

VS

Claims (5)

618,215 CLAIMS 1. Process for the preparation of a compound of formula I in which R15 R3 and R8 each represent an amino atom) -ß-L-arabinopyranosyl (1-6) -0- [2-amino-6- (methyl- of hydrogen, and R2 and R5 each represent a amino atom) - 6-C-methyl-2,3,4,6-tetradesoxy-aD-erythroglucopy-drogen or a hydroxy group, R6 and R7 represent 45 ranosyl- (1— »4)] - D-2-deoxystreptamine, characterized in that each one a hydrogen atom or a methyl group and its that the added aminocyclitol is 2-deoxystreptamine. acid addition salts, characterized in that a medium is cultivated 3. Method according to claim 1 making it possible to prepare nutritious containing carbohydrates, a source of assimilable nitrogen, 0-3-deoxy-4-C- methyl-3 (methylamino) -PL-arabinopyra- nosyl- (l- »6) -0- [2-amino-6 (methylamino) -6-C-methyl-5» 2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l— »4 )] - D-streptamine or Ó-3-deoxy-4-C-methyl-3- (methylamino) -PL-arabinopyranosyl- (1-6) -0- [2,6-diamino-6-C- methyl-2,3,4,6-tetradesoxy-cx-D-erythro-glucopyranosyl- (l—> 4)] - D-streptamine, characterized in that the added aminocyclitol is 55 streptamine. 4. The method of claim 1 for preparing 0-3-deoxy-4-C-methyl-3- (methylamino) -PL-arabinopyra-nosyl- (1-6) -0- [2-amino-6 - (methylamino) -6-C-methyl - (, 0 2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyI- (l—> 4)] - D-2,5-dideoxystreptamine; 0-3 -deoxy-4-C-methyl-3-in which R2 'and R5' are each a hydrogen atom or (m0thylamino) -ß ~ L-arabinopyranosyl- (1 ^ 6) -0- [2,6-diamino a hydroxy group and R, ′ is an amine or 6-C-methyl-2,3,4,6-tetradesoxy-αD-erythro-glucopyranosyl group hydroxy in the presence of Micromonospora purpurea ATCC (1—> 4)] - D-2,5-dideoxystreptamine or 0-3-deoxy-4-C- 31 164, and if desired, the free base obtained is converted into () s methyl-3- (methylamino) -p-L-arabinopyranosyl- (1-6) -0-one of its acid addition salts. [2,6-diamino-2,3,4,6-tetradesoxy-a-D-erythro-glucopyranosyl- 2. Method according to claim 1 for preparing (1— »4)] - D-2,5-dideoxystreptamine, characterized in that gentamicin C1 (0-3-deoxy-4-C-methyl-3- ( methyl- the aminocyclitol added is 2,5-dideoxystreptamine. essential salts and an aminocyclitol of formula HO NH. 618,215 5. Method according to claim 1 for preparing 0-3-deoxy-4-C-methyl-3- (methylamino) -PL-arabino-pyranosyl- (l-> 6) -0- [2-amino-6 - (methylamino) -6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l—> 4) -D-streptamine characterized in that the added aminocyclitol is 2-amino -1,3,4,5,6-cyclohexanepentol (1,3,5-cis). 6. Process for the preparation of a compound of formula I in which one of the symbols Rb R3 and R "represents a lower co-amino-a-hydroxyalkanoyl radical, characterized in that the process according to claim 1 is carried out and that we do; react the compound obtained with an N-hydroxysuccin-imide ester of formula / V -CHo0-C-NHCH_ (CH_) CHOHCO-O-N 2 2 2 n \ IV in which n is equal to zero or 1, and the benzyloxycarbonyl group of the resulting product is subjected to hydrogen-genolysis by hydrogen on a catalyst.