CH620944A5 - Process for the preparation of new aminocyclitol aminoglycoside derivatives - Google Patents

Description

The invention also relates to the process for the preparation of non-toxic addition salts of the compounds of formula I such as, for example, sulfate.

The compounds of formula I can exist in the form of epimers and mixtures of these epimers. The preparation of the epimers and mixtures of epimers in question therefore also forms part of the present invention.

The compounds discussed here have a chemical structure similar to that of neomycin or paromomy-cine. For this reason, the compounds of formula I will hereinafter be designated as derivatives of neomycin or of paromomycin, namely:

Deoxy-6 neomycin B when R [represents hydrogen, R2 represents CH2NH2 and R represents the group A in which R3 represents NH2.

Deoxy-6 neomycin C when R [represents CH2NH2, R2 represents hydrogen and R represents the group A in which R3 represents NH2.

The mixture of these two epimers as obtained by the process described below will be named, subsequently, 6-deoxy-neomycin.

Deoxy-6 paromomycin I when R] represents hydrogen, R2 represents CH2NH2 and R represents the group A in which R3 represents OH.

Deoxy-6 paromomycin II when R] represents CH2NH2, R2 represents hydrogen and R represents the group A in which R3 represents OH.

The mixture of these two epimers as obtained by the process described below will be named, subsequently, deoxy-6 paromomycin.

35

40

50

55

60

The mixture of dideoxy-3 ', 4' neomycin and its derivatives will denote, thereafter, the non-separated mixture of compounds in which Rt and R2, which are different, represent hydrogen or CH2NH2 and R represents group B, this mixture being obtained according to the method described below.

“Neomycin” will be used subsequently to designate the mixture of neomycin B and neomycin C obtained by culture of Streptomyces fradiae.

Likewise, “paromomycin” will be used, thereafter, to designate the mixture of paromomycin I and paromomycin II obtained by culture of Streptomyces rimosus forma paromomycinus.

As will be demonstrated below, it has been found that the present aminoglycoside derivatives of aminocyclitol exhibit a remarkable antibiotic activity capable of making them useful in the treatment of conditions caused by the development of pathogenic bacteria such as, for example:

Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Sorcina lutea, Klebsiella edwardisii, Shigella sonneì, Salmonella typhimurium.

It has been observed, for a few years, that certain bacterial strains have developed resistance to most of the antibiotics marketed, having the effect of reducing their therapeutic value by more than half. On the other hand, some bacteria are even today, powerful enough to resist all known antibiotics.

It is therefore of primary necessity and of general interest to create new antibiotics capable of destroying organisms which have become resistant to commonly used antibiotics.

We know that the inactivation of antibiotics is due to the destruction or modification of the antibiotic molecule by enzymes that attack it in certain vulnerable places.

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In particular, the aminoglycoside antibiotics of aminocyclitol, which are used on an increasingly large scale, are subject to inactivation by resistant bacteria. The resistance developed by certain organisms normally sensitive to these aminocyclitol aminoglycoside antibiotics is caused by phosphorylating, acylating or adenylating enzymes which attack the free hydroxyl and amino groups of the antibiotic (Ann. Rev. Biochem., 42,47 , 1973).

Removal of hydroxyl groups will make the antibiotic insensitive to inactivation at these locations. It is therefore desirable to produce antibiotics with fewer non-essential hydroxyl or amino groups.

During tests carried out with the compounds obtained according to the invention, it has been found that the mixture of dideoxy-3 ', 4' neomycin and its derivatives exhibits a powerful antibiotic activity with regard to organisms resistant to other aminocyclitol aminoglycoside antibiotics, especially with regard to neomycin and gentamicin.

Thus, it has been demonstrated that the mixture of dideoxy-3 ′, 4 ′ neomycin and its derivatives is active with regard to strains of Escheri-chia coli known for their resistance to gentamicin and to neomycin due respectively to the 'adenylylant enzyme in position 2 "and the phosphorylating enzyme in position 3'.

For this reason, the mixture of dideoxy-3 ', 4' neomycin and its derivatives, defined above, constitutes among the antibiotics covered by formula I, the preferred antibiotic.

It is known from United States Patent No. 3,669,838 that it is possible to form mutants of microorganisms reputed to produce antibiotics containing an aminocyclitol sub-motif such as, for example, neomycin or paromomycin, these mutants being incapable of biosynthesizing the aminocyclitol sub-motif following a deficiency in the preparation of aminocyclitol so that no antibiotic can be produced.

However, such negative aminocyclitol mutants have the ability to use an appropriate aminocyclitol molecule when added to a nutrient medium to form an antibiotic.

We also know that it is possible to use this technique by substituting a pseudodisaccharide for the aminocyclitol sub-motif.

However, from the results cited in existing publications, it is not possible to predict whether any diaminocyclitol or a pseudodisaccharide will be incorporated into an antibiotic by the bioconversion technique cited above.

In fact, numerous examples have been described in the literature where this method of obtaining new antibiotics has proved ineffective.

Consequently, the process described above cannot be generalized for the production of new semi-synthetic or fully synthetic antibiotics.

This fact is confirmed, for example, in "The Journal of Antibiotics", Vol. XXVI, N ° 10, p. 551-561 (1973) which describes a description of trials undertaken with 29 different aminocyclitols with a view to incorporating them into an antibiotic by the method of the United States patent cited above.

Among the 29 aminocyclitols thus studied, it was found that only streptamine and epi-2 streptamine were incorporated by means of negative deoxy-2 streptamine (D-) mutants of Streptomyces fradiae, Streptomyces rimosus forma paromomycinus and Streptomyces kanamyceticus used for this purpose.

Likewise, the diaminocyclitols cited in French Patent No. 2,203,808 could not be incorporated into an antibiotic using the same D- mutants of S. fradiae and S. rimosus forma paramomycinus as those used in the above reference.

In "Biochemistry", Vol. 13, No. 25, p. 5073-5078 (1974) it is reported that no success has been recorded in attempts to incorporate the pseudodisaccharides known as neamine, paromamine or 6-kanosaminido-5 5-deoxy-2 streptamine using D ~ S mutants fradiae, S. rimosus forma paromomycinus and S. kanamyceticus.

At the same time, "The Journal of Antibiotics", Vol. XXVIII, N ° 8, p. 573-579 (1975) reports that gentamines, which are pseudodisaccharides, were not incorporated into antibiotics using a D ~ mutant of Micromonospora inyoensis.

It has also been found that O-βD-ribopyranosyl-5 dideoxy-2,4 streptamine is incapable of producing antibiotics using D "mutants of S. fradiae and S. rimosus forma paromomycinus according to the method cited above.

It has also been demonstrated in previous publications that diaminocyclitols or pseudodisaccharides present in already known antibiotics, such as streptomycin, bluensomycin and hygromycin, cannot be incorporated into antibiotics by means of the mutants mentioned above.

For example, we published in "The Journal of Antibiotics", Vol. XXVI, No. 10, cited above, that bluensamine, streptidine, bluensidine, hyosamine and actinamine are not incorporated into antibiotics by the D- mutants of S. fradiae, S. rimosus forma paromomycinus and S. kanamyceticus when these mutants are used for this purpose.

Likewise, neamine and paromamine, which are found respectively in neomycin and paromomycin, were not incorporated into antibiotics when a 30 D- mutant of S. fradiae was used ("Biochemistry": reference cited above). ).

Furthermore, it has also been observed that a diaminocyclitol or a pseudodisaccharide, which is successfully incorporated into an antibiotic using a mutant strain according to the method of the aforementioned United States patent, will not necessarily be incorporated into an antibiotic by a different mutant strain. Examples have been cited in the previous reference from "The Journal of Antibiotics", Vol. XXVI, No. 10, which shows that a D- mutant of S. 40 rimosus forma paromomycinus does not produce an antibiotic with epi-2-streptamine whereas a mutant D "of S. fradiae and S. kanamyceticus are able to incorporate this sub-motif into antibiotics.

In addition, the same D- mutant of S. kanamyceticus with streptamine does not provide any antibiotics, while the same D- mutant of S. fradiae and S. rimosus forma paromomycinus incorporates this diaminocyclitol in antibiotics.

With regard to neamine, this pseudodisaccharide makes it possible to obtain ribostamycin when a D ~ mutant of S. ribosidificus is used as cited in "The Journal of Antibiotics", Vol. XXVI, N ° 12, p. 784-785 (1973) while neamine is incapable of being incorporated into an antibiotic when a D- mutant of S. fradiae is used, for example.

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55 Finally, previous publications have also shown that the structure of a hypothetical antibiotic cannot in any way be predicted when using a diaminocyclitol or pseudodisaccharide with a mutant strain. This fact is clearly demonstrated in "The Journal of Antibiotics", 6 () Vol. XXVI, No. 12, cited above, where it is reported that antibiotics obtained, when a D "" mutant of S. kanamyceticus is used with N-methyl-1 deoxystreptamine or myo-inosadiamine-1,3, "Are not the products hoped for" but other compounds. Likewise, neamine and a D ~ mutant of 65 M. inyoensis provides sisomicin; however, this mutant and paromamine, a different pseudodisaccharide, also produces sisomicin ("The Journal of Antibiotics" Vol. XXVIII, No. 8, cited above).

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It has also been shown that a D- mutant of S. ribosidifi-cus, in the presence of neamine, produces only ribostamycin but not a neomycin analog as might be expected ("The Journal of Antibiotics", Vol. XXVI , N ° 12 cited above).

It is clear from the above that it is not possible to make any valid prediction about the possible incorporation of a diaminocyclitol or a particular pseudodisaccharide using a certain mutant strain according to the method of aforementioned United States of America patent. In any case, it is not possible to predict whether this method will be valid or what will be the exact structure of the antibiotic that we hope to obtain.

Consequently, the assertion advanced in J. Org. Chem. Flight. 40, No. 4, p. 456-461 (1975) that "2,4-dideoxy streptamine could be incorporated into neomycins, paromomycins and ribostamycin using a bioconversion technique" can be considered excessively vague, undefined and devoid of any valid and reasonable basis.

It has now been found, quite surprisingly, that 2,4-dideoxy streptamine can be incorporated into new antibiotics by means of D- mutants of S. fradiae and S. rimosus forma paromomycinus and that a mixture of pseudodisaccharides, a mixture of gentamines, can also be incorporated into new antibiotics using a D- mutant of S. rimosus forma paromomycinus.

This discovery is all the more surprising since tests carried out with 2,4-dideoxy streptamine in the presence of a D- mutant of S. kanamyceticus produced no antibiotic and that a mutant of S. fradiae was unable to '' incorporate the gentamine mixture in question into an antibiotic.

All the results obtained with 2,4-dideoxy streptamine and the mixture of gentamines make the invention completely unpredictable from the state of the art.

The compounds of formula I are prepared by cultivating a negative deoxystreptamine mutant of Streptomyces in an appropriate medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and a compound of formula

(HA)

or an acid addition salt of this compound, formula in which X = Y = H, or X = -OH and Y = a group of formula

R,

(IIB)

H.

or an acid addition salt of these compounds.

Using this method, the antibiotic is obtained in the form of a free base, whether the diaminocyclitol or the mixture of gentamines is in the form of a free base or in the form of a salt. If it is desired to use the antibiotic obtained in the form of a salt, it is sufficient to treat it with an appropriate organic or inorganic acid, such as for example sulfuric acid, to obtain a non-toxic addition salt.

The microorganisms used in the present invention are D- Streptomyces fradiae ATCC 21401 and D ~ Streptomyces 5 rimosus forma paromomycinus ATCC 21484 both described in US Patent No. 3,669,838: D ~ Streptomyces fradiae ATCC 21401 is used to produce deoxy-6 neomycin and D "Streptomyces rimosus forma paromomycinus ATCC 21484 is used for the preparation of deoxy-6 paromomycin and the mixture of dideoxy-3 ', 4' neomycin and its derivatives.

According to known techniques, a culture of the microorganism is carried out in a nutritive medium having the appropriate pH value and containing, for example glucose, casein hydrolyzate-sat, (NH4) 2HP04, MgS04 • 7H20, FeS04 • 7H20, CuSO4 • 5H20, CaCO3 then this culture is added to another culture medium containing the diaminocyclitol of formula IIA or the mixture of gentamines of formula IIB both in the form of the free base or of addition salt and, for example, soybean meal, yeast extract, NaCl, CaCO3 and glucose, this medium again being at the appropriate pH value. The culture medium is incubated at a temperature of about 28-30 ° C while shaking and keeping under good aeration for at least 5 days to achieve optimal production of 6-deoxy-neomycin, 6-deoxy-paromomycin or mixture of dideoxy-3 ', 4' neomycin and its derivatives, which it is desired to obtain.

Deoxy-6 neomycin B and deoxy-6 neomycin C as well as deoxy-6 paromomycin I and deoxy-6 paromomycin II will be obtained from deoxy-6 neomycin and deoxy-6 paromomycin, respectively. conventional, for example by separation using paper chromatography.

The isomers constituting the mixture of 3 ', 4', neomycin and its derivatives can also be separated according to known methods.

The diaminocyclitol of formula IIA is a known compound having been described in French patent n ° 2,270,235. It can be obtained according to the method described in the above-mentioned French patent 40, that is to say by hydrogenating in presence of a catalyst, for example Raney nickel, lD- (1,3,5 / 2) -1,5-diazido-2,3-cyclohexanediol obtained from lL-1,5-di-0 -tosyl-1,2,5 / 3-cyclohexanetétrol and sodium azide.

As regards the mixture of gentamines of formula 45 IIB, it can be obtained from commercial gentamicin sulfate according to the method of COOPER et al. described in J. Chem. Soc. (c) 1971,960.

As mentioned previously, it has been found that the 50 aminoglycoside derivatives of aminocyclitol obtained according to the invention exhibit an advantageous antibiotic activity.

The results described below illustrate this antibacterial activity, with regard to various organisms tested:

55 a) Antibacterial activity of deoxy-6 neomycin and deoxy-6 paromomycin

The antibiotic activity of these aminoglycoside derivatives of aminocyclitol was tested by adding, to nutritive agar, measured quantities of the compound to be studied in sterile water. 60 The agar containing increasing concentrations of the test compound is poured into petri dishes. Mechanically, by means of a multinoculator, each Petri dish is inoculated with several different organisms cultivated beforehand in an appropriate medium and maintained at -20 ° C. in 65 in a 1: 1 mixture of nutritive medium and glycerol. The dishes are then incubated at 37 ° C for 14 hours and the development is observed.

Record the minimum concentration of antibiotic that

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inhibits the development of bacteria and is called "minimum inhibitory concentration" or C.I.M.

The results obtained in this test were recorded in Table I below in comparison with neomycin and paromomycin, both in the form of sulfate.

The figures listed in Tables I and II correspond to the quantity of free base.

Table I

[ig / ml required to prevent development

Organization (a)

neo deoxy-6

paro-

paro-

mycine neo mony-

momy-

Escherichia coli W3110

mycine cine cyne i.e. ATCC 27325

2.5

1.25

5.0

40

Proteus mirabilis

i.e. NCIB 11355

5.0

5.0

1.25

20

Staphylococcus aureus

i.e. ATCC 9144

2.5

3.75

1.25

20

Sarcina lutea i.e.

NCIB 11356

2.5

2.5

5.0

40

Klebsiella edwardsii

7.5

10

2.5

40

Shigella sonnei C6319

78 i.e. NCIB 11357

2.5

3.75

2.5

40

Salmonella typhimurium

LT2 i.e. ATCC 19585

5.0

5.0

2.5

40

(a) ATCC, American Type Culture Collection; NCIB, National Collection of Industriai Bacteria (Great Britain)

neomycin with respect to Escherichia coli W3110 ie ATCC 27325 is superior to that of neomycin.

These results show that the suppression of the group b) Activity of deoxy-6 neomycin B and of deoxy-6 neo-hydroxyl at position 6 of the aminocyclitol entity of neomycin C.

cine, which corresponds to deoxy-6 neomycin, does not modify The antibacterial activity of deoxy-6 neo- has been studied

significantly the overall antibacterial activity of mycin B and deoxy-6 neomycin C by the same neomycin method. than that described above using D.S.T. as

It will be noted that the antibacterial activity of the deoxy-6 nutrient medium.

35 The C.I.M. was recorded in comparison with neomycin and deoxy-6 neomycin:

Table II Organization

Escherichia coli Bristol i.e. NCIB 11351 Proteus mirabilis i.e. NCIB 11355

Staphylococcus aureus i.e. ATCC 9144 Salmonella typhy-murium i.e. ATCC 19585

(xg / ml required to prevent development neomycin neomycin neomycin deoxy-6

0.62

1.25

0.62

2.5

B

0.62

1.25

0.62

2.5

deoxy-6 deoxy-6

neomycin neomycme neomycme B C

80

10

80

40

2.5

2.5

2.5

10

20

20

We can conclude from these results that deoxy-6 neomycin B is somewhat less active than neomycin B but that deoxy-6 neomycin C is more potent than neomycin C.

c) Activity of the mixture of dideoxy-3 ', 4'neomycin and its derivatives

The antibacterial activity of the mixture of dideoxy-3 ', 4' neomycin and its derivatives was determined according to the method described below.

D-Streptomyces rimosus forma paromomycinus ATCC 21484 is incubated at 30 ° C. on a nutritive agar plate containing 50 (μg / ml of a mixture of gentamines of formula IIB. After three days, the plate is covered with agar seeded with an organism to be tested and the production of antibiotics is observed by an inhibition zone around the Streptomyces. This inhibition zone is measured and compared with that of a control consisting solely of nutritive agar.

As a comparison, a similar test was also carried out with 50 μg / ml of neamine in addition to the nutritive agar instead of the mixture of gentamines of formula IIB and using 65 also D - Streptomyces rimosus forma paromomycinus ATCC 21484. We have found that the antibiotic thus produced corresponds to neomycin.

The results obtained are given in the following table:

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Table IH

Organization

Escherichia coliPT2

Escherichia coli ML 1629

i.e. NCIB 11354

Escherichia coli ML 1410

i.e. NCIB 11353

Escherichia coli JR 66

i.e. NCIB 11352

Zone of inhibition (in mm) when the nutrient agar is added with:

Nothing Neamine Mixture of gentamines of formula III

11

0

0

25

20

25

These results show that the mixture of dideoxy-3 ', 4' neomycin and its derivatives is active with regard to organisms which are resistant to neomycin. It should also be mentioned that the E. coli PT2 organism is also resistant to gentamicin.

The therapeutic compositions can be presented in any form suitable for administration in human or veterinary therapy. As regards the administration units, these may take the form, for example, of a coated or uncoated tablet, a capsule, a capsule, a suspension or a syrup for oral administration.

The therapeutic compositions can also be presented in the form of a suppository for rectal administration, an ovum for vaginal administration, a solution or a suspension for parenteral administration or a cream or 'an ointment for topical administration.

Whatever the route of administration chosen, the therapeutic compositions will be prepared by combining at least one of the compounds of formula I with a pharmaceutical vehicle or an appropriate excipient.

The following nonlimiting examples illustrate the preparation of the compounds of formula I:

Example 1

Preparation of deoxy-6 neomycin base and its sulfate, a) Deoxy-6 neomycin in the form of free base

In a flask, a culture of the strain D "Streptomyces fradiae ATCC 21401 is carried out for 48 hours on the following medium previously adjusted to pH 7.2-7.3:

g

Glucose 1

Casein hydrolyzate 1

(NH4) 2HP04 1

MgS04 • 7H20 0.05

FeS04-7H20 0.005

CuS04 • 5H20 0.005

CaC03 1

Water up to 100 ml

This culture is shaken and maintained at 28 ° C for the time period indicated above. A 10% concentration of the culture inoculum is then added to the following nutrient medium adjusted to pH 7.2 to 7.3:

Cultures are incubated in Erlenmeyer flasks at 28 ° C to 30 ° C and shaken, with good aeration, on a rotary apparatus.

The production of deoxy-6 neomycin begins after 2 days 5 and is maximal after 5 days.

Control cultures not containing 2,4-dideoxy-streptamine hydrochloride do not produce 6-deoxy-neomycin.

The culture medium is then centrifuged and the supernatant liquid is poured into a column containing a weakly acidic cation exchange resin, of carboxylic type (polymeth-acrylic) and of medium porosity (Amberlite IRC-50, Amber-lite is a brand deposited), this resin being in ammonium form.

i5 This operation is carried out twice to extract the deoxy-6 neomycin and the 2,4-dideoxy streptamine dihydrochloride intact. The column is washed with water and the deoxy-6 neomycin is eluted with an aqueous solution of 2N ammonium hydroxide. The eluate is concentrated in vacuo using a rotary evaporator and then poured into a Sephadex G-10 column. Eluting with a 0.1 molar solution of ammonium hydroxide, 6-deoxy neomycin is obtained in the first fractions and 2,4-dideoxy-2,4 streptamine hydrochloride in the following fractions.

In this way, an amount of deoxy-6 neomycin is produced in the form of a free base representing 83 units of activity (1 unit = 1 (μg / ml of neomycin). This result corresponds to incorporation into deoxy-6 neomycin thus obtained from approximately 22% of the initial 2,4-dideoxy-2,4-streptamine hydrochloride.

This figure represents the maximum activity obtained and a subsequent incubation no longer provides deoxy-6 neomycin.

According to an alternative, the crude deoxy-6 neomycin is purified by chromatography on Whatman No. 3 MM paper developed with a 4/1 solution of methanol / ammonium hydroxide. Deoxy-6 neomycin is localized by means of an aqueous 0.25% sodium hypochlorite solution, then with absolute ethanol and finally with an aqueous solution of 1% soluble starch and 1% d potassium iodide, each application being separated by air drying.

In this test, the Rf of deoxy-6 neomycin is equal to 0.30.

For comparison purposes, it will be mentioned that the Rf values of neomycin, 2-deoxy-2 streptamine and 2,4-dideoxy-streptamine with the same solvent system are:

30

35

45

Neomycin Deoxy-2 streptamine Dideoxy-2,4 streptamine

55

Glucose

Soybean flour yeast extract NaCl CaC03

2,4-dideoxy streptamine dihydrochloride Water up to 100 ml g 1

2.5 0.5 0.5 0.2 0.025

Rf 0.26 0.58 0.62

60

b) Deoxy-6 neomycin sulfate

The deoxy-6 neomycin obtained as described above is converted into its sulfate by reaction with the equivalent amount of dilute sulfuric acid.

[a] ^ 6-deoxy neomycin sulfate: + 43 ° (c = 1.0; water)

c) Structure of deoxy-6 neomycin

The structure of the antibiotic compound of formula I was determined in the form of the free base obtained previously by comparison with the natural antibiotic, neomycin, formed when the 2-deoxystreptamine is incubated in the appropriate medium. The presumed product deoxy-6 neomycin will be called Compound X below.

The methanolysis of neomycin as well as that of Compound X using a 10% methanolic solution of hydrogen chloride for two hours at reflux, provides two pro-

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main products, one of them corresponding to the methylneobiosaminide produced from the two compounds.

The other product, hereinafter called Z, is different in the two antibiotics, Compound Z obtained from neomycin migrating less than product Z obtained from Compound X during a chromatography test using a solution 4/1 methanol / ammonium hydroxide as solvent.

The structure of each product Z was confirmed by mass spectrometry of the per-trimethyl-silyl derivatives.

Although it is not possible to obtain molecular ions, the spectrum of product Z obtained from neomycin and that of product Z obtained from Compound X are very similar except that two peaks in the spectrum of first (m / e 343 and 460) are moved back 88 units of mass in the second (m / e 255 and 372).

This corresponds to a loss of OSi (CH3) 3 and a replacement with hydrogen.

The structure of product Z, obtained from neomycin therefore corresponds to neamine and the structure of product Z, obtained from Compound X, corresponds to deoxy-6 neamine.

The acetylation of the two products Z in methanol followed by acid hydrolysis with a 3N hydrochloric acid solution at reflux for 10 hours, provides, in each case, two main products. During a paper chromatography, it appears that one of these products is the same in both cases, whether it is obtained from product Z of neomycin or from product Z of Compound X. This common product must be considered to correspond to diamino-2,6 dideoxy-2,6 D-glucose. As for the two other compounds, these are identical, in chromatography, one to 2-deoxy-streptamine obtained from neamine, the other to 2,4-dideoxy-streptamine from deoxyneamine.

These results lead to the conclusion that Compound X corresponds to deoxy-6 neomycin.

Example 2

Separation of deoxy-6 neomycin into deoxy-6 neomycin B and deoxy-6 neomycin C.

The deoxy-6 neomycin, obtained according to the method described in Example 1, is separated into deoxy-6 neomycin B and deoxy-6 neomycin C by paper chromatography using a 3/16/6/1 mixture of tertbutanol / butanone / 6.5 N ammonium hydroxide solution / methanol as solvent.

Deoxy-6 neomycin B and deoxy-6 neomycin C are detected on paper using the same method as that described above.

Using the solvent system methanol and ammonium hydroxide in the proportion 4 to 1 on 3 MM Whatman chromatographic paper, the Rf values of the deoxy-6 neomycins B and C were determined in comparison with the corresponding epimers of neomycin and the following results were obtained:

Soybean flour casein hydrolyzate CaC03; Glucose NaCl NH4C1

Water up to 100 ml g 1

0.25

0.5

1

0.5 0.167

10

15

30

35

This culture is shaken and maintained at 28 ° C for the time period indicated above. A 10% concentration of the inoculum of this culture is then added to a medium identical to that mentioned above but containing in addition 0.025 g of 2,4-dideoxy-2,4-streptamine hydrochloride.

Cultures are incubated in Erlenmeyer flasks at 28 ° to 30 ° C and shaken, with good aeration, on a rotary apparatus.

The subsequent operations for preparing and purifying 6-deoxy-paromomycin are exactly the same as those described in Example 1a above.

Purification by paper chromatography under the same conditions as those described in Example 1 above shows that the Rf of deoxy-6 paromomycin is 0.30.

For comparison purposes, it should be mentioned that the Rf of paromomycin in an identical chromatographic test is 0.27.

Using as a solvent system methanol and ammonium hydroxide in the proportion 4 to 1 on 3 MM Whatman chromatographic paper, the Rf values of deoxy-6 paromomycins I and II were determined in comparison with the corresponding epimers of paromomycin and the following results were obtained:

Deoxy-6 paromomycin I Deoxy-6 paromomycin II Paromomycin I Paromomycin II

0.34 0.275 0.3 0.22

45

50

55

Deoxy-6 neomycin B Deoxy-6 neomycin C Neomycin B Neomycin C

0.29 0.21 0.25 0.165

Example 3

Preparation of deoxy-6 paromomycin and its epimers I and II.

In a flask, a culture of the strain D ~ Streptomyces rimosus forma paromomycinus ATCC 21484 is carried out for two to three days on the following medium previously adjusted to pH 7.5:

65

Example4

Preparation of the mixture of dideoxy-3 ', 4' neomycin and its derivatives a) Mixture of gentamines of formula IIB

First a mixture of gentamines of formula IIB is prepared from the gentamicin sulphate sold. First, convert this salt to its free base and then evaporate to obtain an oil. To this oil is added a solution of hydrochloric acid in methanol, prepared by adding hydrochloric acid to dry methanol, and the solution thus obtained is refluxed for two hours. The methanolic hydrochloric acid is removed in vacuo and the resulting oil is taken up in a little water. This solution is then passed through a column of Amberlite I.R.A. 400 to obtain the free base of the desired gentamines. The various fractions thus collected are evaporated to dryness and separated on the one hand into methyl garosaminide and on the other hand into a mixture of gentamines by chromatography on silica gel using a 1/1/1 mixture of methanol / chloroform / hydroxide ammonium solvent. In this chromatographic separation, the Rf of methyl garosaminide is 0.8 and that of the mixture of gentamines is 0.2.

b) Mixture of dideoxy-3 ', 4' neomycin and its derivatives

In a flask, a culture of the strain D "Streptomyces rimosus forma paromomycinus ATCC 21484 is carried out for two to three days on the following medium previously adjusted to pH 7.5:

9

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g

Soybean meal 1

0.25 casein hydrolyzate

CaCo3 0.5

Glucose 1

NaCl 0.5

NH4C1 0.167

This culture is shaken and maintained at 28 ° C during the time period mentioned above. A concentration of 10% of the inoculum of this culture is then added to a medium identical to that mentioned above and adjusted to pH 7.2 but containing in addition 50 [xg of the mixture of gentamines obtained previously.

Cultures are incubated in Erlenmeyer flasks at 30 ° C

and shake them, under good aeration, on a rotary device for 5 days. The mixture of 3 ', 4' neomycin and its derivatives, obtained in crude form, is purified by chromatography on paper using Whatman paper No. 3 MM 5 developed with a 4/1 solution of methanol / ammonium hydroxide. The location of the mixture of 3 ', 4' neomycin and its derivatives is determined by means of sodium hypochlorite at 0.25% in water, then with absolute ethanol followed by an aqueous solution at 1 % soluble starch and 1% 10 potassium iodide by air drying between each application.

In this test, the Rf of the mixture of dideoxy-3 ', 4' neomycin and its derivatives is 0.25.

For purposes of comparison, it will be mentioned that the Rf of the mixture of gentamines of formula IIB in an identical chromatographic test is between 0.5 and 0.7.

VS

Claims (6)

620 944 2 CLAIMS 1. Process for the preparation of new aminoglyco-sides derivatives of aminocyclitol corresponding to the general formula: CH OH GOLD 10 (I) OH in which Rt and R2, which are different, represent hydrogen or a group ch2 nh2, and R is chosen from the groups: 'H in which R3 represents nh2 or oh and R4 represents R5 ch3 I I -ch — nh2 or —ch — nhch3, R5 representing hydrogen or a methyl group, as well as their non-toxic addition salts, characterized in that a negative deoxy-streptamine mutant of Streptomyces is cultivated in a medium nutrient containing a soluble carbohydrate, an assimilable source of nitrogen, essential mineral salts and a compound of formula NH, YO NH, HO (IIA) X or an acid addition salt of this compound, formula in which X = Y = H, or X = -OH and Y = a group of formula (IIB) these fradiae ATCC 21401 or D ~ Streptomyces rimosus forma paromomycinus ATCC 21484 or its salts when x = h, and 35 D "Streptomyces rimosus forma paromomycinus ATCC 21484 when x = -oh, the free base thus obtained being subsequently treated, if desired, with an organic or inorganic acid to obtain a non-acid addition salt toxic. 2. Method according to claim 1 characterized in that D " 40 Streptomyces fradiae ATCC 21401 is cultured in the presence of 2,4-dideoxy streptamine or one of its addition salts in order to obtain 6-deoxy-neomycin in the form of the free base. 3. Method according to claim 1 characterized in that 45 D "Streptomyces rimosus forma paromomycinus ATCC 21484 2,4-dideoxy streptamine hydrochloride to obtain 6-deoxy neomycin in free base form. 4. Method according to claim 1 characterized in that D- Streptomyces rimosus forma paromomycinus ATCC 21484 so is cultivated in the presence of the mixture of gentamines to obtain the mixture of dideoxy-3 ', 4' neomycin and its derivatives, in the form of free base. 5. Method according to claim 1 characterized in that the organic acid is sulfuric acid. 55 6. Deoxy-6 neomycin as prepared according to the method of claim 2. 7. Mixture of dideoxy-3 ', 4' neomycin and its derivatives as prepared according to the method of claim 4. 60 The present invention relates to a process for the preparation or an acid addition salt of these compounds, in order to obtain, under the new aminoglycoside derivatives of aminocyclitol having free base form, the compounds of formula I, said mutating a pharmacological activity which can be represented negative deoxystreptamine of Streptomyces being D ~ Streptomy- by the general formula: 620 944 (I) in which Rj and R2, which are different, represent hydrogen or CH2 NH2 and R is chosen from the groups: (AT) NEL (B) in which R3 represents NH2 or OH and R4 represents R, CH3 the I CH-NH2 or CH-NHCH3 in which R5 represents hydrogen or methyl.