CH617964A5 - Process for the preparation of new aminocyclitol antibiotics - Google Patents

Abstract

New analogues of gentamicin C1, C2 and C1a as well as compounds thereof which are acylated on the 1-amino, 3-amino and 2'-amino groups by a lower omega -amino- alpha -hydroxyalkanoyl group are prepared. For this, a nutrient medium is produced containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol added in the presence of a mutant microorganism which is incapable of synthesising the said aminocyclitol itself but which is capable of incorporating the said aminocyclitol into the said new aminocyclitol, and if desired, the product obtained is acylated with an ester of a lower omega -(N-benzyloxycarbonyl)amino- alpha -hydroxyalkanoic acid and then the benzyloxycarbonyl group is removed by catalytic hydrogenolysis. The new compounds possess an antibacterial activity, in particular towards gentamicin-resistant microorganisms; they are useful as antibacterial agents.

Description

This invention relates to a process for the preparation of anti-biotics of the amino-cyclitol type of formula I

_ch20-c-nhch2 (ch2) nch0hc0-0-n if in which Ri, R3 and Rs each represent a hydrogen atom, or else one of Ri, R3 and Rs represents an œ-amino- group a-hydroxy-lower alkanoyl of formula N2NCH2 (CH2) „CHOHCO— in which n is equal to 0 or to 1, the other groups Ri, R3 and Rs being hydrogen atoms; R2 represents a hydrogen atom or a hydroxy group; Rs represents a hydrogen atom, a hydroxyl group or a halogen atom (including fluorine, chlorine, bromine and iodine), except that when R2 is a hydrogen atom, Rs n ' is not a hydroxy group and is based on the amino groups in positions 1 and 3; and Rr> and R7 each represent a hydrogen atom or a methyl group.

Compounds of formula I are prepared in which Ri, R3 and Rs are hydrogen atoms, the process of the invention which is based on the method described in the US patent.

no. 3,669,838 to Shier et al. The process of the invention consists in cultivating a nutritive medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an added aminocy-clitol derivative of formula:

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4

in which Ri, R2, R3 and Rs have the meanings given above, and where Ri 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 aminocyclitol itself but capable of incorporating said aminocyclitol in the compound of formula I, particularly a mutant of Micromonospora purpurea designated by Micro-

where n has the meaning given above. Then the resulting mixture of the compounds of formula I is subjected, where one of the groups Ri, R3 and Rs is the group co- (N-benzyloxy-carbonyl) amino-a-hydroxy-lower alkanoyl:

ch20-c-nhch2 (ch2) nchohco-

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

As indicated above, when a compound of formula I is used as the starting substance in the acylation reaction, where each of the groups R 1, R 3 and R 5 is a hydrogen atom, a mixture of the three isomeric mono-amides is obtained in which one of the hydrogen atoms of the amine groups, Ri, R3 or Rs, is replaced by the group a) - (N-benzyloxycarbonyl) amino-a-hydroxy-lower alkanoyl. 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-hydroxysuc-cinimide ester, preferably at a temperature of from -10 ° C to 4-10 °. C, and in an aqueous solution of an inert organic solvent, for example tetrahydrofuran, dioxane, dimethyl ether of ethylene glycol, dimethylacetamide, 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. , propylene glycol dimethyl ether, etc.

The aminocyclitols of formula II in which Ri, R2 and R3

monospora purpurea ATCC 31 119 and to separate the product from the culture medium. The compounds of formula I are produced in which Ri and R3 are both a hydrogen atom when using the aminocyclitol of formula II where Ri and R3 represent a 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 in an antibiotic when aminocyclitol is added to the nutrient medium.

The compounds of formula I, where one of the groups Ri, R3 and Rs represents a group (o-amino-a-hydroxy-alka-lower nucleus), are prepared by a process in principle described by Konishi et al. In the patent USA

no. 3,780,018 which consists in reacting the compound of formula I where each of the groups Ri, R3 and Rs is a hydrogen atom, with an N-hydroxy-succinimide ester of formula:

iv are hydrogen atoms and Rs is fluorine or iodine atom, are new compounds and can be prepared as described below. Other aminocyclitols of formula II, which are also useful in the practice of the present invention, are known compounds. Those are:

Streptamine sulfate [Peck et al., J. Am. Chem. Soc. 68, 776 (1946)];

2-epistreptamine dihydrochloride [Suami et al.,

J. Org. Chem. 3.2831 (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. [These last two compounds are described by Testa et al., J. Antibiotics 27, 917-921 (1974)].

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

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 with the aid of a pharmaceutical support by suspension, either in the form of a free base or under forms non-toxic, pharmaceutically acceptable acid addition salts in an inert carrier such as polyethylene glycol, either by form of 60 tablets or encapsulation for oral administration, alone or with suitable adjuvants, or they can 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 into compounds

10

15

\ He

V-CH2 ° "C-,, n v

^ 1

-ch20-c-nhch2 (ch2) nch0hc0-0-n

35

40

50

5

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known; 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.

Preparation of new intermediaries Preparation 1

We put 2-deoxy-1,6: 3,4-dicarbonylstreptamine [Umezawa et al., Bull. Chem. Soc. (Jap.) 44.1411-1415 (1971)] (47 g, 0.21 mole) suspended in 500 ml of Pyridine and the stirred suspension is treated with 45 ml of methanesulfonyl chloride. After cooling, the mixture is diluted with approximately 3 liters of methanol and the product is filtered and dried, which gives 39 g of 2-deoxy-5-methanesulfonyl-1,6: 3,4-dicarbonylstreptamine, m.p. 264-266 ° C.

Heated at 125 ° C for 24 hours, a mixture of 2 g (0.0069 mole) of 2-deoxy-5-methanesulfonyl-1,6: 3,4-dicarbo-nylstreptamine, described above, and 4 , 8 g (0.032 mol) of sodium iodide in 70 ml of dimethylformamide, then it is brought to dryness. The 2,5-dideoxy-5-iodo-1,6: 3,4-dicarbonylstreptamine is mixed with 30 ml of 6N hydrochloric acid, the mixture is refluxed for 2.5 hours and cooled and cooled. evaporated to dryness under vacuum. The crude 2,5-dideoxy-5-iodostreptamine dihydrochloride is dissolved in 30 ml of acetic anhydride, the solution is treated with 5.25 g of sodium acetate and the mixture is heated to reflux for 2.5 hours. Then the mixture is cooled, poured into 200 ml of water and extracted with chloroform. The chloroform extracts, after washing once with a sodium thiosulfate solution, once with brine, once with water and by evaporation to dryness, provide an oil which is crystallized from ethanol, which gives two crops totaling 1.1 g of N, N / -diacetyl-2,5-dideoxy-5-iodostrepta-mine, mp 256-258 ° C. Hydrolysis of the latter by heating to reflux with aqueous hydrochloric acid and separation in a basic medium provides 2,5-dideoxy-5-iodo-streptamine.

Preparation 2

39 g (0.12 mole) of 2-deoxy-5-methanesulfonyl-1,6: 3,4-dicarbonylstreptamine, described in preparation 1 above, are placed in approximately 200 ml of 6N hydrochloric acid, the mixture is heated the mixture on a steam bath for 2 hours, evaporated to dryness in vacuo, mixed with 200 ml of isopropyl alcohol and evaporated to dryness once again. The residual oil is triturated with methanol, cooled, and the solid is collected which is recrystallized from methanol, which gives 2-deoxy-5-methanesulfonylstrep-tamine dihydrochloride, m.p. 208-210 ° C.

Analysis for C7H16N2O5S • 2HC1:

C H N

Calculated 26.84 5.79 8.94 Found 26.77 5.76 9.17

A solution of 28.7 g (0.09 mole) of 2-deoxy-5-methanesulfonylstreptamine in 45 ml of water and 90 ml of 2N sodium hydroxide is cooled in an ice bath and treated dropwise , with stirring, by a solution of 45 ml of benzyl chloroformate in 80 ml of toluene, added by a dropping funnel, and by 160 ml of 2N sodium hydroxide added by another dropping funnel. When the addition is complete, the mixture is stirred for an additional 15 minutes, diluted with about 50 ml of toluene, stirred for 3 hours and filtered. Recrystallization of the solid from ethanol provides 4.0 g of N, N'-dicarbobenzoxy-2-deoxy-5-methanesulfonylstreptamine, m.p. 198-201 ° C.

Analysis for C23H28N2O9S:

VS

H

NOT

Calculated

54.32

5.55

5.51

Found

54.75

5.61

5.60

The reaction of the latter with potassium fluoride in benzene or acetonitrile containing a crown ether, for example 1,4,7,10,13,16-hexa-oxacyclo-octadé-cane, using the mode procedure described by Liotta et al., J. Am. Chem. Soc. 96, 2250-2252 (1974) provides N, N'-dicarbobenzoxy-2,5-dideoxy-5-fluorostreptamine which, by hydrolysis by an aqueous mineral acid, provides 2,5-dideo-xy-5-fluorostreptamine.

Mutation process In the following operating modes, various media are used, constituted as follows:

Middle 1

N-Z Amine g / 1

Glucose 10

Soluble starch 20

Yeast extract 5

N-Z-Amine-Type A (Difco) 5

CaCOa 1

Agar 15

Middle 2

Germination medium (in distilled water)

Beef extract 0.3%

Tryptone 0.5%

Dextrose 0.1%

2.4% soluble starch

0.5% yeast extract

CaCOa 0.4%

Middle 3

Soy-glucose g / 1

Soy flour 30

Dextrose (Cerelose) 40

CaC03 1

Medium 4 TGE (trypticase-glucose extract)

g / 1

Trypticase-glucose extract 5.0

Trypticase-peptone 3.0

Glucose 1.0

Agar 15.0

Middle 5

Production environment

Beef extract 0.3%

0.5% yeast extract

Soy flour 0.5%

Maltose 0.1%

Starch 2,4r

0.1 'Casamino Acid

CaCO2 0.4% C0Cl2 • 6H2O 1 mg / liter s

10

15

20

25

30

35

40

45

50

55

60

65

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Medium 6 Streptomycin g / 1 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 no.

NRRL 2953 and maintained on N-Z amine inclined agar cultures (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 revolutions per minute 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 Ci, C2 and Cu, 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. until colonies are seen (generally 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 inhibition zone on the B. subtilis plate are transferred from the original plate (no B. subtilis) to cultures on inclined agar. of medium 1 and incubated until total growth is seen.

These potential non-producing mutants are then put in the presence of 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 deoxystreptamine solution (500 jig / 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 the 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 the VIB mutant and deposited on January 13, 1975 at the American Type Culture Collection, 12 301 Parklawn Drive, Rockville, Md. 20852, under the name of Micromonospora purpurea ATCC 31 119, is used in the production of antibiotics of the type gentamicin as described below.

Biosyntheses with M. purpurea ATCC 31119

Example 1

Cultures of the mutant organism are maintained on inclined cultures of NZ 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 revolutions per minute at 28-29 ° C and filtered air is blown 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 aseptically is transferred aseptically into 130 liter fermenters containing 70 liters of sterile germination medium 2 and 0.31 g per liter of streptamine sulfate suspended in 1 ml 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 exhibiting 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 V3). 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.

To identify the antibiotic components during the purification procedures, the chromatographic mobility values obtained by paper chromatography and thin layer chromatography for the genamicamines Ci, C2 and Cia and for each of the corresponding aminocyclitol homologs prepared are determined. as indicated above, where the chromatographic mobility (hereinafter referred to as MC) is expressed as:

distance of the component from the origin

MC =

distance of gentamicin Ci from the origin

5

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The chromatographic systems used are as follows:

System 1

Whatman paper no. 1 saturated with 0.95 M sulfate-bisulfate and top-down in 80% aqueous ethanol plus 1.5% NaCl, and subsequent bioautographer using B. subtilis as the test organism.

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 are located 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 Ci, 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-pyranosyl- (1-6) -0- [2,6-diamino-6-C-methyl-2,3,4, 6-tetradesoxy-ot-D-erythroglucopyranosyl- (1-4)] - D-streptamine; and

0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyrano-syl- (1-6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD- erythro-glucopyranosyl- (l-> 4)] - D-streptamine.

Table I

MC

System I

MC

System II

Gentamicin Ci

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 has antibacterial activity, is suspended in 200 ml of methanol and 10 ml of concentrated ammonium hydroxide (28%), and the mixture is stirred for 30 minutes and we filter it. This is repeated two more times, and the filtrates are 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 × 28 cm. The column is prepared in the form of a suspension using the lower phase of the mixture of isopropyl alcohol (1) xhloroform (2): 17% aqueous ammonium hydroxide (1). The column is developed with this solvent and 50 ml fractions are collected.

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, has a molecular ion and main fragments each greater by 16 mass units (that is to say by one oxygen atom) than those obtained for gentamicin Ci , as following:

Reference:

gentamicin Ci: M + 477,420,360,350, 347, 322,319,304 Component 1: M + 493,436, 376,366, 363,338, 335, 320.

This substance is transformed 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, giving 22 mg of component 1, 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyra-nosyl- (1-6) - 0- [2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l-> 4)] - D-strepta-mine, under form of the heptasulfated and decahydrated di-base, pf > 300 ° C.

Analysis for (C2iH43Ns08) 2 • 7H2SO4 • IOH2O:

îs Calculated Found

27.22 27.15

H

6.53 6.67

NOT

7.55 7.79

12.09 12.76

Fractions 10-13 provide a more polar positive component to ninhydrin, called 0-3-deoxy-4-C-methyl-3-20 methylamino-PL-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 peaks characteristic of the sugars corresponding to gentamicin Cia, at 129 (purpurosa-mine) and 160 (garosamine) and is called component 3, O-3-deoxy-4-C-methyl -3-methylamino-PL-arabinopyranosyl-30 (1-6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-gluco-pyranosyl- (1 -> 4) ] -D -streptamine.

Alternatively, the new component 1 is isolated as follows: a mixture of the crude antibiotic base obtained as above (0.9 g) is dissolved in 7 ml of water and the pH is adjusted to 4.5 with IN sulfuric acid. The solvent is passed through a column of strong anion exchange resin (IRA 401) in OH ~ form (dimensions of the bed = 0.7 × 10 cm). The column is eluted with water and the eluate is evaporated in vacuo 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 in vacuo, leaving an oily residue weighing 140 mg. The mass spectrum of this substance corresponds to that of fractions 8 and 9 45 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 ^ -50 L-arabinopyranosyl- (l- »6) -0- [2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1-4)] - D-streptamine and is summarized in Table II.

Table II

55 ô

Integration

Attribution

5,87,5,60

2

OCHO epimers

5.22

12

H exchangeable

3,09,3,15

6

NCH3X2

2.9-4.8

13

-CHO X 6, -CHN X 5, -CH2O

60 1.9-2.6

4

-CH2CH2-

1.72

6

CHsC-, CHBCH

Alternatively, a 4 g sample of the raw antibiotic salt 65 is dissolved in 50 ml of water and passed through an AG1X8 anion exchange resin (1x27 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

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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: concentrated ammonium metha-nokhydroxide (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.

By cultivating an appropriate aminocyclitol with the mutant Micromonospora purpurea ATCC 31119, in germination 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-pL-arabinopyranosyl- (1-6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4 , 6-tetradesoxy-aD-erythro-glucopyranosyl- (1-4)] epistreptamine (MC versus gentamicin Ci: System 1 = 0.59, System 2 = 0.63); 0-3-deoxy-4-C-methyl-3-methylamino- ß-L-arabinopyranosyl- (l-> 6) -0- [2,6-diamino-6-C-methyl-2,3,4 , 6-tetradeoxy-aD-erythro-glucopyranosyl- (1-> 4)] epistreptamine; and 0-3-deoxy-4-C-methyl-3-methylamino-pj-L-arabinopyranosyl- (l- ^ 6) -0- [2,6-diamino-2,3,4,6-tetradesoxy- aD-erythro-glucopyranosyl- (l ^> 4)] epistreptamine are obtained by using epistreptamine instead of streptamine in the fermentation procedure.

Example 3

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)] - 2,5-dideoxy-streptamine (MC versus gentamicin Ci: System 1 = 0.95, System 2 = 0.98); 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (l-> 6) -0- [2,6-diamino-6-C-methyl-2,3,4,6 -tetradesoxy-aD-erythro-glucopyranosyl- (1H> 4)] - 2,5-dideoxystreptamine (MC compared to gentamicin Ci):

System 1 = 0.66, System 2 = 0.73); and 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD- erythro-glucopyranosyl- (1-4)] - 2,5-dideoxystreptamine are obtained using dideoxystreptamine in place of streptamine in the fermentation procedure.

The same 0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (1-6) -0- [2-amino-6-methylamino-6-C-methyl-2,3, 4,6-tetradeoxy-cc-D-erythro-glucopyranosyl- (1 ^ 4)] - 2,5-dideoxystreptamine; and 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6>) -0- [2,6-diamino-6-C-methyl-2,3,4, 6-tetradeoxy-aD-erythro-glucopyranosyl- (l- »4)] - 2,5-dideoxystreptamine described above and having the same MC values as given above are obtained using 6,7-diazobicyclo [3.2 .1] octane-2,4-diol] (exo, exo) instead of streptamine in the fermentation procedure.

Example 4

0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1 ^ 6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4, 6-tetradeoxy-aD-erythro-glucopyranosyl- (1—> 4)] - 5-iodo-2,5-dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (l ~> 6) -0- [2,6-diamino-6-C-methyl-2,3,4,6 -tetradesoxy-ce-D-erythro-glucopyranosyl- (1-4)] - 5-iodo-2,5-dideoxy-streptamine; and 0-3-deoxy-4-C-methyl-3-methylamino-ß-L-arabinopyranosyl- (1-6) -0- [2,6-diamino-2,3,4,6-tetradesoxy- aD-erythro-glucopyranosyl- (l-> 4)] - 5-iodo-2,5-dideoxystreptamine are obtained by using 5-iodo-2,5-dideoxystreptamine in place of streptamine in the fermentation procedure.

Example 5

0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (W6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6- tetradesoxy-aD-erythro-glucopyranosyl- (1-> 4)] - 5-fluoro-2,5-dideoxystreptamine; 0-3-deoxy-4-C-methyl-

3-methylthino-ß-L-arabinopyranosyl- (l-> 6) -0- [2,5-diamino-6-C-methyl-2,3,4,6-tetradesoxy-αD-erythro-gIucopyranosyl- (l -> 4)] - 5-fluoro-2,5-dideoxystreptamine; and O-3-deoxy-

4-C-methyl-3-methylamino-pL-arabinopyranosyl- (l-> 6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l- » 4)] - 5-fluoro-2,5-dideoxystrep-tamine are obtained by using 5-fluoro-2,5-dideoxy-streptamine in place of streptamine in the fermentation procedure.

Example 6

Following a procedure similar to that described in Example 1 and using 0.10 g per liter of 2-deoxystrepta-mine in a 10 liter fermentation in production medium 5, in the presence of M. purpurea ATCC 31 119, and by separating the product as above, 0.51 g of crude substance is obtained which, by chromatography on silica gel, provides as main component a substance whose identity of chromatographic mobilities shows that it is identical to gentamicin Ci, i.e. 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-2-deoxystreptamine.

Synthesis of amino-hydroxy-lower alkanoyl substituted derivatives

Example 7

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- (1 -> 6) -0- [2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro ~ glucopy-ranosyl- (ls> 4)] - D -streptamine, described above in Example 1 (component 1), dissolved in 5 ml of 50% aqueous tetrahydrofuran, and treated with 208 mg (0.59 mmol) of the N-hydroxysuccinimide ester S - (-) -Y- (N-benzyl-oxycarbonyl) amino-a-hydroxybutyric acid (US Patent No. 3,780,018 to Konishi et al.), and the mixture was stirred at 5 ° C for 20 hours. Then the mixture is concentrated to 10 ml under

8

s

10

15

20

25

30

35

40

45

50

55

60

65

empty. 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, which leaves 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- (N-benzyloxycarbonyl) -amino-a-hydroxybutyric acid as before. The treatment of the reaction mixture provides an additional 435 mg of the crude product which is combined with the 513 mg previously obtained and is 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) mixture. Seven passages in this solvent system are necessary, and after having eluted the product band which is visible with ultraviolet, 229.5 mg of a crude product of the 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 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: hydroxide d concentrated ammonium (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%) mixture (1: 1: 1), which provides three components: A, 90.9 mg; B, 59.1 mg; and C, 48.5 mg, which are the amide derivatives of the acid S - (-) - Y- (N-benzyloxycarbonyl) -amino-a-hydroxybutyrique in positions 2 ', 1 and 3 respectively of Ô-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) -] - 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: concentrated ammonium methanokhydroxide (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. The addition of 10 ml of ethanol precipitates l- [S - (-) - Y-amino-a-hydroxybutyryl] -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- (l- »4)] - D- streptamine as 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 Ci = 0.73) .

617964

Analysis for C25H50O10N6 • 5H2SO4:

C H N

Calculated 27.67 5.57 7.75 Found 27.50 5.58 7.42

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-pL-arabinopyranosyl- (1-6) -0- [2-amino-6-C-methyl-2,3,4,6-tetradesoxy-aD-ery-thro-glucopyranosyl- (1-) 4)] - D-streptamine in the form of the tetrasulfated and heptahydrated di-base, pf 237-241 ° C with decomposition; TLC, Rf = 0.33 [silica gel, chloroform: methanol: concentrated ammonium hydroxide (28%): water, l: 4: 2: l; reference Rf for gentamicin Ci = 0.73],

Analysis for (C2sH5oN60io) 2 • 4H2SO4 • 7H2O:

VS

H

NOT

Calculated

35.16

7.20

9.84

Found

35.38

7.08

9.49

Component C provides 26 mg of 3- [S - (-) -y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-6) ) -0- [2-amino-6-methylamino-6-C-me-thyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l-> 4)] - D-streptamine in the form of pentasulfated and trihy-drated 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 Ci = 0.73].

Analysis for (C25HsoN60io) 2 • 5H2SO4 • 3H2O:

C H N

Calculated 34.64 6.74 9.67 Found 34.35 6.38 8.60

By proceeding in a similar manner to that described in Example 7, using respectively the antibiotics described in Examples 2,3,4 and 5 and the N-hydroxysuccinimide ester of S - (-) - Y acid. - (N-benzyloxycarbonyl) amino-a-hydro-xybutyric or the pentafluorophenyl ester of N- (benzyl-oxycarbonyl) - (S) -isoserine [acid (S) -ß-amino-a-hydroxypro-pionic, described by Haskell et al., Carbohydrate Research, 28, 273-280 (1973)], the following compounds of formula I are prepared in the same way:

Example 8

l - [(S) -P-amino-a-hydroxypropionyl] -0-3-deoxy-4-C-me-thyl-3-methylamino-PL-arabinopyranosyl- (l-> 6) -0-] 2, 6-dia-mino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyra-nosyl- (l-> 4)] epistreptamine and 2 '- [(S) -ß-amino -a-hydroxypro-pionyl] -0-3-deoxy-4-C-methyl-3-methylamino-PL-arabino-pyranosyl- (l- »6) -Ó-] 2,6-diamino-6-C- methyl-2,3,4,6-tetra-deoxy- <xD-erythro-glucopyranosyl- (l- »4] -epistreptamine.

Example 9

l- [S - (-) - Y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino- | 3-L-arabinopyranosyl- (l-> 6) -0- ] 2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l- »4) -2,5-dideoxystreptamine and 2 '- [S - (-) - Y-amino- a-hydro-xybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-PL-ara-binopyranosyl- (li> 6) -0- [2,6-diamino-2,3,4, 6-tetradeoxy-aD-erythro-glucopyranosyl- (l-> 4)] - 2,5-dideoxystreptamine.

9

s

10

15

20

25

30

35

40

45

50

55

60

65

617,964

10

Example 10

l - [(S) -P-amino-a-hydroxypropionyl] -0-3-deoxy-4-C-me-thyl-3-methylammo-pL-arabinopyranosyl- (l- »6) -0- [2, 6-dia-mino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l ^> 4)] -

5-iodo-2,5-dideoxystreptamine and 2 '- [(S) -ß-amino-a-hydroxy-s propionyl] -0-3-deoxy-4-C-methyl-3-methylamino ^ -L-ara -binopyranosyl- (l-> 6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l- »4] -5-iodo-2,5- dideoxystrepta-mine.

Example 11

l- [S - (-) - Y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-mes-thyl-3-methylthino-ß-L-arabinopyranosyl- (l-> 6) -0 - [2-amino-

6-methylamino-6-C-methyl-2,3,4,6-tetradesoxy-a-erythro-glu-copyranosyl- (1H.4)] - 5-fluoro-2,5-dideoxystreptamine and 2 '- [S - (-) - Y-amino-a-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methyl-amino-ß-L-arabinopyranosyl- (1 ^ 6) -0- [2-amino-6 -methylamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (l-> 4)] - 5-fluoro-2,5-dideoxystrepta-mine.

Antibacterial test results

We test 0-3-deoxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (l- »6) -0- [2-amino-6-methylamino-6-C-me-thyl-2 , 3,4,6-tetradesoxy-a ~ D-erythro-glucopyranosyl- (l- »4)] - D-streptamine described above in the example and called component 1 by comparing it to gentamicin vis-à-vis against 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 phosphate-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 tubes of 13x100 ml. 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 jig / ml.

Table III *

Minimum inhibitory concentration (ng / ml)

Organization

Component I

Gentamicin io 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

is Escherichia coli JR89

50

50

Escherichia coli K12 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

20 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

25 Pseudomonas aeruginosa A-20741

Inactive

Inactive

Pseudomonas aeruginosa A-20897

12.5

Inactive

* Grown in tiyptose-phosphate broth.

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), is carried out. ) -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 (Ci, C2 and Cia) and gentamicin Ci, and a comparison is made between 1-, 3- and 2 '- [S - (-) -Y-amino-cc-hy- droxybutyryljamides of component 1, described above in example 21, and called respectively compound 1 (1-HABA), component 2 (3-HABA) and component 1 (2'-HABA), 40 and 1-, 3 -, and 2 '- [S - (-) - Y-amino-a-hydroxybutyryl] amides of gentamicin Ci (all described by Konishi et al. in US Patent No. 3,780,018 issued December 18, 1973 ) and called respectively Ci (1-HABA), Ci (3-HABA) and Ci (2 / -HABA). The results are given 45 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 IV *

Test organisms

Compound

1

2

3

4

5

6

Gentamicin

Ci, C2, Cla

= £ 0.024

0.39

50

12.5

25

> 100

Gentamine Ci

0.049

0.78

50

12.5

25

> 100

Component I

0.098

1.56

6.25

0.78

3.13

25

Ci (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

Ci (3-HABA)

3.13

25

> 100

100

> 100

> 100

Component I

(3-HABA)

6.25

50

100

50

50

> 100

Ci (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.

B

Claims (7)

617964 CLAIMS 1. Process for the preparation of a new compound of formula I i ch-nh-r in which R1, R3 and R8 each represent a hydrogen atom, R2 represents a hydrogen atom or a hydroxy group; R5 represents a hydrogen atom, a hydroxy group or a halogen atom, except that when R2 is a hydrogen atom, Rs is not a hydroxy group in eis relative to the amino groups in positions 1 and 3; and R6 and R7 each represent a hydrogen atom or a methyl group, or one of its acid addition salts, characterized in that a nutritive medium containing carbohydrates is cultivated, a source of assimilable nitrogen , essential salts and a corresponding added aminocyclitol in the presence of a mutant microorganism and the product is isolated from the culture medium, said aminocyclitol having the formula II nh-r '. ii ho ^ nh-r-, in which Ri and R3 represent hydrogen atoms or together represent a single bond joining the two nitrogen atoms of the amino groups and R2 and Rs have the above meanings, said mutant microorganism being ch3nh 40 unable to synthesize said aminocyclitol itself but being able to incorporate said aminocyclitol into the compound of formula I, under the preceding conditions. 2. Method according to claim 1, characterized in that the microorganism strain is Micromonospora purpu- 45 rea ATCC 31 119. 3 617,964 amino-a-hydroxy-lower alkanoyl, characterized in that the process of claim 1 is carried out and the o is reacted in which n is equal to 0 or to 1 and the benzyloxycarbonyl group of the resulting product is subjected to hydrogenolysis by hydrogen on a catalyst. 3. Method according to claim 1 or 2, for preparing 0-3-deoxy-4-C-methyl-3- (methylamino) ^ - L-arabinopyranosyl- (l-> 6) -0- [2-amino -6- (methyIamino) -6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-gIucopyranosyl- so (l-> 4)] - D-streptamine and 0-3-deoxy-4-C-methyl-3- (methyl-amino) -ß-L-arabinopyranosyl- (l-> 6) -0- { 2,6-diamino-6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1 ^> 4)] - D-streptamine, characterized in that the added aminocyclitol is streptamine. ss 4. Method according to claim 1 or 2 for preparing 0-3-deoxy-4-C-methyl-3- (methylamino) ^ - L-arabmopyranosyl- (l-> 6) -0- [2-amino- 6- (methylamino) -6-C-methyl-2,3,4,6-tetradesoxy-aD-erythro-glucopyranosyl- (1-> 4)] - D-2,5-dideoxystreptamine; 0-3-deoxy-4-C-60 methyl-3- (methylamino) -pL-arabinopyranosyl- (l-> 6) -0- [2,6-diamino-6-C-methyl-2,3, 4,6-tetradeoxy-cc-D-erythro-gIuco-pyranosyl- (l-54)] - D-2,5-dideoxy-streptamine and 0-3-deoxy-4-C-methyl-3- (methylamino ) -pL-arabinopyranosyl- (l-> 6) -0- [2,6-diamino-2,3,4,6-tetradesoxy-aD-erythro-gluco-65 pyranosyl- (l- * 4)] - D -2,5-dideoxystreptamine, characterized in that the added aminocyclitol is 2,5-dideoxy-streptamine. 5. Process for producing a compound of formula i in which however one of Ri, R3 and Rs represents a group co- 6. Method according to one of claims 1 to 4, characterized in that the free base obtained is converted into one of its acid addition salts. 7. Method according to claim 5, characterized in that compound of formula I with an N-hydroxysuccinimide ester of formula IV iv transforms the free base obtained into one of its acid addition salts.