NO148558B - ANALOGY PROCEDURE FOR THE PREPARATION OF AMINOGLYCOSIDE AMINOCYCLITOL DERIVATIVES - Google Patents

Description

This invention relates to an analogue method for the production of new aminoglycoside-aminocyclitol derivatives which have pharmacological activity.

The aminoglycoside-aminocyclitol derivatives which are prepared can be represented by the general formula: where R x and R ^ , which are different, are hydrogen or CH 2 NH 2 and R is selected from the group consisting of:

where R3 means NH2 or OH and R4 means

Rc CH,

r in CHNH2 or CHNHCH^,

R,, means hydrogen or methyl.

The pharmaceutically acceptable acid addition salts of these derivatives are also included within the scope of the invention.

The compounds of formula I can exist in the form of epimers and mixtures of said epimers.

Such epimers and mixtures thereof are included within the scope of the present invention.

The compounds produced according to the present invention have chemical structures that are of a similar nature to the structures of neomycin or paromomycin. For this reason, the compounds of formula I will hereafter be designated as neomycin or paromomycin derivatives as indicated in the following: 6-deoxyneomycin B when R^ means hydrogen, R2 means CH2NH2 and R means the group A where R^ means NH2-

6-deoxyneomycin C when R^ means CH2NH2, R2 means hydrogen and R means the group A where R^ means NH2.

The mixture of these two epimers, as obtained by the method described herein, will hereafter be referred to as 6-deoxyneomycin.

6-deoxyparomomycin I when R^ means hydrogen, R2 means CH2NH2 and R means the group A where R3 means OH.

6-deoxyparomomycin II when R^ means CH2NH2, R2 means hydrogen and R means the group A where R^ means OH.

The mixture of these two epimers, as obtained by the method described herein, will hereafter be referred to as 6-deoxyparomomycin.

The mixture of 3<1>,4<1->dideoxyneomycin and its derivatives denotes the unseparated composition of compounds where R^ and R2, which are different, means hydrogen or CH2NH2 and R means the group B, and said mixture is obtained by the method described below .

"Neomycin" will hereafter be used to denote the mixture of neomycin B and neomycin C obtained by cultivation of Streptomyces fradiae.

similarly, "paromomycin" will hereafter be used for

to denote the mixture of paromomycin I and paromomycin II obtained by cultivation of streptomyces rimosus forma paromomycinus.

A pharmaceutical or veterinary medicinal preparation may contain as a significant active ingredient at least one of the epimers of formula I or a pharmaceutically acceptable acid addition salt thereof or a mixture of said epimers or their pharmaceutically acceptable acid addition salts, in combination with a pharmaceutical carrier or an inert formulation agent.

Pharmaceutical or veterinary medicinal preparations can be prepared by mixing at least one of the epimers of formula I or a pharmaceutically acceptable acid addition salt thereof or a mixture of said epimers or of their pharmaceutically acceptable acid addition salts with a suitable carrier or an inert formulation agent.

The aminoglycoside-aminocyclitol derivatives that are prepared

according to the invention, which will later be demonstrated, have been found to provide valuable antibiotic activity suitable to make them useful in the treatment of diseases caused by the growth of pathogenic bacteria such as Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Sarcina lutea, Klebsiella edwardsii, Shigella sonnei. Salmonella typhimurium.

The new compounds can be used to inhibit growth

of pathogenic bacteria in a host in need of such inhibition, and for this purpose at least one of the epimers of formula I or a pharmaceutically acceptable acid addition salt thereof or a mixture of said epimers or their pharmaceutically acceptable acid addition salts is administered to said host.

It has been observed for some years that certain strains of bacteria have developed resistance to most commercialized antibiotics with the result that the therapeutic value of these antibiotics has been reduced by more than half, and certain bacteria are now even strong enough to resist all the known antibiotics.

It is thus very necessary and of general interest

to create new antibiotics capable of destroying organisms that have become resistant to the antibiotics currently used.

It is known that this inactivation of antibiotics

is due to destruction or alteration of the antibiotic molecule with

enzymes that attack this molecule at certain vulnerable points.

In particular, the aminoglycoside-aminocyclitol antibiotics, which are increasingly used, are prone to being inactivated by resistant bacteria. The resistance developed by certain organisms normally susceptible to these aminoglycoside-aminocyclitol antibiotics is due to phosphorylating, acylating or adenyrating enzymes which attack the free hydroxyl and amino groups of the antibiotic (Ann. Rev. Biochem., 42,

47, 1973).

Removal of the hydroxyl groups will render the antibiotic immune to inactivation at these sites. It is therefore desirable to produce antibiotics with less unimportant hydroxyl and amino groups.

During the course of experiments carried out with the compounds prepared according to the invention, the mixture of 3<1>,4<1->dideoxyneomycin and its derivatives was found to give clear antibiotic activity against organisms resistant to other aminoglycoside-aminocyclitol antibiotics. and more especially neomycin and gentamicin..

It was thus demonstrated that the mixture of 3',4'-dideoxyneomycin and its derivatives are active against Escherichia coli strains known to be resistant to gentamicin and neomycin by the action of the 2<11->adenylylating enzyme and the 3< 1->phosphorylating enzyme.

For this reason, the mixture of 3<1>,4<1->dideoxyneomycin and its derivatives, referred to above, is the antibiotic covered by formula I which is the preferred antibiotic according to the invention.

It is known from U.S. Patent No. 3,669,838 that mutants of microorganisms known to produce antibiotics containing an aminocyclitol subunit, such as e.g. neomycin or paromomycin, which lack the ability to biosynthesize the aminocyclitol subunit due to a deficiency in the aminocyclitol pathway so that no antibiotic can be formed.

However, such aminocyclitol-negative mutants have the ability to utilize a suitable aminocyclitol molecule when this is added to the nutrient medium so that an antibiotic can be formed.

It is also known that this technique can be utilized by inserting a pseudodisaccharide into the aminocyclitol subunit.

From the results discussed in the existing publications, however, it is not possible to predict whether a given diaminocyclitol or a pseudodisaccharide will be incorporated into an antibiotic by the aforementioned bio-conversion technique.

In reality, numerous cases have been described in the literature where this method of producing new antibiotics has proven to be ineffective.

The method described above cannot therefore be generalized for the production of new semi-synthetic or fully synthetic antibiotics.

This is e.g. confirmed in "The Journal of Antibiotics", Vol. XXVI, No. 10, pp. 551-561 (1973), where a description is given of experiments carried out with 29 different aminocyclitols for incorporation into an antibiotic by the method of the above-cited U.S. patent.

Among the 29 aminocyclitols thus tested, it was found that only streptamine and 2-epistreptamine were incorporated by the 2-deoxystreptamine-negative (D~) mutants of Streptomyces fradiae, Streptomyces rimosus forma paromomycinus

and Streptomyces kanamyceticus used for this purpose.

It likewise failed to incorporate the diaminocyclitols cited in French patent document No. 2,203,808 into an antibiotic using the same D~ mutants of S.fradiae and of S.rimosus forma paromomycinus that were used in the aforementioned reference.

In "Biochemistry", Vol. 13, No. 25, pp. 5073-5078 (1974), reported that no success was achieved when attempts were made to incorporate the pseudodisaccharides known as neamine, paromamine or 6-canosaminido-2-deoxystreptamine, using of D mutants of S. fradiae, S. rimosus forma paromomycin and S. kanamyceticus.

similarly, it is mentioned in "The Journal of Antibiotics", Vol. XXVIII, No.8, pp. 573-579 (1975), that gentamines, which are pseudodisaccharides, were not incorporated into antibiotics using a D mutant of Micromonospora inyoensis .

It has also been found that 5-O-£D-ribopyranosyl-2,4-dideoxystreptamine is unable to form antibiotics using D~ mutants of S. fradiae and of S. rimosus forma paromomycinus in accordance with the aforementioned approach.

Previous publications have also shown that diaminocyclitols or pseudodisaccharides present in already known

antibiotics, such as streptomycin, bluensomycin and hygromycin,

cannot be incorporated into antibiotics using the above-cited mutants.

It is e.g. featured 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.

similarly, neamine and paromamine, found in neomycin and paromomycin respectively, were not incorporated into antibiotics using a D mutant of S. fradiae ("Biochemistry": reference cited above).

Furthermore, it has also been observed that a diaminocyclitol or a pseudodisaccharide, which can be incorporated into an antibiotic using a mutant strain in accordance with the method of the above-cited U.S. patent, will not necessarily be incorporated into an antibiotic with a different mutant strain. tribe. Examples are given in the aforementioned reference from "The Journal of Antibiotics", Vol. XXVI, No. 10, which shows that a D mutant of S. rimosus forma paromomycinus with 2-epistreptamine does not form any antibiotic while a D~ mutant of S. fradiae and of S. kanamyceticus is able to incorporate this subunit into antibiotics.

Furthermore, the same D mutant of S. kanamyceticus with streptamine does not provide any antibiotic while the same D~ mutant of S. fradiae and of S. rimosus forma paromomycinus incorporates this diaminocyclitol into antibiotics.

With respect to neamine, this pseudodisaccharide can provide ribostamycin when a D~ mutant of S. ribosidificus is used, as stated in "The Journal of Antibiotics", Vol.XXVI, No. 12, pp. 784-785 (1973), while neamine is not able to be incorporated into an antibiotic when, e.g. a D mutant of S. fradiae is used.

Finally, previous publications have also shown that the structure of a hypothetical antibiotic cannot in any case be predicted when a diaminocyclitol or a pseudodisaccharide is used together with a mutant strain. This is clearly shown in "The Journal of Antibiotics", Vol. XXVI, No. 12, cited above, where it is discussed that the antibiotics obtained when a D~ mutant of S. kanamyceticus is used together with 1-N-methyl-deoxystreptamine or myo-inosa-1,3-diamine are not the "expected products" but other connections. Likewise, neamine and a D~ mutant of M. inyoensis provide sisomicin, but this mutant together with paromamine, a different pseudodisaccharide, also forms sisomicin ("The journal of Antibiotics", vol. XXVIII, No. 8, cited above).

It has also been shown that a D mutant of S. ribosidificus in the presence of neamine provides only ribostamycin, but not an analogue of neomycin as would be expected ("The Journal of Antibiotics", Vol. XXVI, No. 12, cited above ).

It is clear from the foregoing that no firm prediction can be made as to the possible incorporation of a particular diaminocyclitol or pseudodisaccharide into an antibiotic using a given mutant strain in accordance with the method of the above-cited U.S. patent. It is in no case possible to predict whether this method will be tenable or what will be the exact structure of the antibiotic that one hopes to produce.

Therefore, the claim in J. Org. Chem., Vol.40, No.4,

pp. 456-461 (1975), that 2,4-dideoxystreptamine can be incorporated into neomycins, paromomycins and ribostamycin by a bio-conversion technique is regarded as extremely unclear, indefinite and devoid of any reasonable and tenable basis.

It has now been rather unexpectedly found, in accordance with the present invention, that 2,4-dideoxystreptamine can be incorporated into new antibiotics with b~ mutants of S. fradiae and of S. rimosus forma paromomycinus and that a mixture of pseudodisaccharides, i.e. a mixture of gentamines, can also be incorporated into new antibiotics with a D~" mutant of S. rimosus forma paromomycinus.

This discovery becomes even more surprising when one takes

whereas experiments carried out with 2,4-dideoxystreptamine in the presence of a D~ mutant of S. kanamyceticus did not produce any antibiotic and that a D mutant of S. fradiae was unable to incorporate the mixture of gentamines in question in an antibiotic.

All the results obtained with 2,4-dideoxystreptamine and the mixture of gentamines make the invention completely unexpected in relation to the state of the art.

The new compounds of formula I according to the invention are prepared by culturing the deoxystreptamine-negative

the mutant

a) Streptomyces rimosus forma paromomycinus ATCC 21484, in the presence of 1D-(1, 3, 5/2)-1,5-diamino-2,3-cyclohexanediol

or 2,4-dideoxystreptamine of the formula;

or an acid addition salt thereof, e.g. a hydrochloride, or in the presence of a mixture of gentamines of the general formula i

or an acid addition salt thereof, where R. has the same meanings as in group B in formula I, or

b) D-Streptomyces fradiae ATCC 21401 in the presence of 2,4-dideoxystreptamine of formula II or an acid addition

salt thereof, in a medium containing a soluble carbohydrate, a source of assimilable nitrogen and essential mineral salts, after which the obtained free base is optionally reacted with an organic or inorganic acid to a pharmaceutically acceptable acid addition salt, and/or an obtained mixture of compounds of formula You are separated.

When the method described here is used, the antibiotic obtained is in the form of a free base, regardless of whether the diaminocyclitol of formula II or the mixture of gentamines of formula III is in the form of a free base or in the form of a salt. If it is desired to use the resulting antibiotic as a salt, it is sufficient to react it with a suitable organic or inorganic acid, e.g. sulfuric acid, to obtain a pharmaceutically acceptable acid addition salt.

The microorganisms used in the present invention are D Streptomyces fradiae ATCC21401 and D~ Streptomyces rimosus forma paromomycinus ATCC21484, both described in U.S. Patent No. 3,669,838: D~ Streptomyces fradiae ATCC21401 is used to produce 6-deoxyneomycin according to the invention and D Streptomyces rimosus forma paromomycinus ATCC21484 are used in the production of the 6-deoxyparomomycin and the mixture of 3<1>,4<1->dideoxyneomycin and its derivatives according to the invention.

In accordance with known technology, the microorganisms are grown in a nutrient medium with a suitable pH value and containing e.g. glucose, casein hydrolyzate, (NH4)2HP04, MgS04-7H20, FeS04-7H20, CuS04*5h20 and CaCO^, and it is then added to another growth medium containing the diaminocyclitol of formula II or the mixture of gentamines of formula III, both in the form of a free base or an acid addition salt, and e.g. soybean meal, yeast extract, NaCl, CaCO-j and glucose, and again at a suitable pH value. The culture medium is incubated at a temperature of approx. 28-30°C with shaking and good aeration for at least 5 days to achieve optimal production of the desired 6-deoxyneomycin, 6-deoxyparomomycin or of mixtures of 3<1>,4<1->dideoxyneomycin and its derivatives.

6-deoxyneomycin B and 6-deoxyneomycin C and 6-deoxyparomomycin I and 6-deoxyparomomycin II are obtained from 6-deoxyneomycin and 6-deoxyparomomycin respectively by conventional methods, e.g. by separating them using paper chromatography.

The isomers that make up the mixture of 3<1>,4'-dideoxyneomycin and its derivatives can also be separated by conventional methods.

The diaminocyclitol of formula II is a known compound described in British Patent No. 1,445,675. It can be achieved by proceeding according to the method described in the aforementioned British patent, namely by in the presence of a catalyst, e.g. Raney's nickel, to hydrogenate 1D-(1,3,5/2)-1,5-diazido-2,3-cyclohexanediol obtained from 1L-1,5-di-0-tosyl-1,2,5/3- cyclohexanethetrol and sodium azide.

With regard to the mixture of gentamines of formula III, this can be obtained from commercial gentamicin sulfate by the method of COOPER et al., described in j.Chem. Soc. (c) 1971, 960.

As mentioned above, it has been found that the amine glycoside-aminocyclitol derivatives produced according to the invention provide valuable antibiotic activity.

This antibacterial activity against various test organisms is illustrated by the following data: a) Antibacterial activity of 6-deoxysyneomycin and 6-deoxy-garomomYcIn

These aminoglycoside-aminocyclitol derivatives according to the invention were tested with regard to antibiotic activity by adding measured quantities of the investigated compound in sterile water to nutrient agar.

The agar, containing increasing concentrations of the compound under investigation, was then poured into petri dishes, and to each of these were mechanically added with a multi-inoculator different organisms, which had previously been cultured in a suitable medium and stored at -20 °C in a 1 to 1 mixture of growth medium and glycerol. The dishes were incubated at 37°C for 14 hours and examined for growth.

The concentration of antibiotic that just inhibited the growth of the bacteria was written down and referred to as the "minimum inhibitory concentration" or M.I.C.

The results recorded in this test are listed in Table I in comparison with the results for neomycin and paromomycin, both in sulphate form.

The numbers given in Tables I and II correspond to the amount of free base.

These results show that the removal of the hydroxyl group in the 6-position of the aminocyclitol part of neomycin, which corresponds to 6-deoxyneomycin, does not produce any significant change in the overall antibacterial activity of neomycin. It can be noted that the antibacterial activity of 6-deoxyneomycin against Escherichia coli W 3110 is better than that of neomycin.

b) Activity_of_6-deoxyvneomvicin B_and_ 6-deoxyvneomvicin c

The antibacterial activity of 6-deoxyneomycin B and

6-Deoxyneomycin C was tested by the same method as that described above, using D.S.T. agar as nutrient medium, and values for M.I.C. was recorded by comparison with neomycin and 6-deoxyneomycin:

From these results, it can be concluded that 6-deoxyneomycin B is somewhat less potent than neomycin B, but that 6-deoxyneomycin c is more potent than neomycin c.

c) Activity_of_the_mixture_of_3^i4 derivatives<->

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

D~ Streptomyces rimosus forma paromomycinus ATCC 21484 was incubated at 30°C on a nutrient agar plate containing 50 µl. g/ml of a mixture of gentamines of formula III. After 3 days, this plate was covered with agar seeded with a test organism, and formation of antibiotics was shown by a zone of inhibition around the Streptomyces. This zone of inhibition was measured and compared to the zone of a comparison consisting of nutrient agar alone.

As a comparison, a similar test was also carried out with 50 µg/ml of neamine as a supplement to nutrient agar, instead of the mixture of gentamines of formula III, and also using D~ Streptomyces rimosus forma paromomycinus ATCC 21484. Thus formed antibiotic was found to be neomycin.

The results obtained are indicated in the following table:

These figures show that the mixture of 3<1>,4'-dideoxyneomycin and its derivatives according to the invention are active against organisms resistant to neomycin. It can be added that the organism E.coli PT2 is also resistant to gentamicin.

It will be understood that for therapeutic use the compounds produced according to the invention will usually be administered in the form of a pharmaceutical or veterinary medicinal preparation in a dosage unit form that is suitable for the administration method used, the preparation as active ingredient comprising at least one of the compounds prepared according to the invention together with a pharmaceutical carrier or an inert formulation agent. For oral administration, the preparation can take the form of, e.g. a coated or uncoated tablet, a hard- or soft-gelatin capsule, a suspension or a syrup. The preparation may alternatively take the form of a suppository for rectal or vaginal administration, a solution or suspension for parenteral administration or, a cream or ointment for topical administration.

The following examples illustrate the method according to the invention:

Example 1

Preparation of 6-deoxyneomycin base and its sulfate

a) 6-deox_Yneomycin in free base form

In a flask, D~ Streptomyces fradiae ATCC 21401 was grown

for 48 hours on the following medium which had previously been adjusted to a pH value of 7.2-7.3:

This culture was shaken and kept at 28°C for the indicated time period. A 10% inoculation of the culture was then added to the following growth medium which had previously been adjusted to a pH value of 7.2-7.3:

Cultures were incubated in Erlenmeyer flasks at 28 to 30°C on a rotating shaker and with good aeration.

The formation of 6-deoxyneomycin began after 2 days and

was optimal after 5 days.

Control cultures containing no 2,4-dideoxystreptamine dihydrochloride did not produce any 6-deoxyneomycin.

The culture medium was then centrifuged and the supernatant was poured through a column containing a weakly acidic, carboxylic (polymethacrylic) type of cation exchange resin of medium porosity ("Amberlite" IRC-50, "Amberlité1 is a registered trademark) in ammonium form. This operation was performed twice to extract the 6-deoxyneomycin and unchanged 2,4-dideoxystreptamine dihydrochloride. The column was washed with water and the 6-deoxyneomycin was eluted with a 2 N ammonium hydroxide solution in water. The eluate was concentrated in vacuo on a rotary evaporator and was then applied to a Sephadex G-10 column.Elution with 0.1 M ammonium hydroxide solution gave 6-deoxyneomycin in the first fractions and 2,4-dideoxystreptamine dihydrochloride in the subsequent fractions.

By this method, a quantity of 6-deoxyneomycin was formed in free base form which represented 83 units (1 unit = 1 µg/ml neomycin) of activity. This corresponds to an incorporation in the thus obtained 6-deoxyneomycin of approximately 22% of the originally used 2,4-dideoxystreptamine dihydrochloride.

This represents the maximum activity obtained and further incubation did not result in any increase in 6-deoxyneomycin.

As an alternative procedure, the crude 6-deoxyneomycin was purified by paper chromatography on Whatman No 3 MM paper with a 4/1 methanol/ammonium hydroxide solution. The location of the 6-deoxyneomycin was visualized using 0.25% sodium hypochlorite in water, then absolute ethanol followed by 1% soluble starch and 1% potassium iodide in water with air drying between each application.

In this experiment, the Rf value was - for 6-deoxyneomycin

found to be 0.30.

It can be mentioned, for comparison, that the Rf values for neomycin, 2-deoxystreptamine and 2,4-dideoxystreptamine with the same system of solvents are:

b) 6-deoxy_YneomYcin sulfate

The 6-deoxyneomycin obtained as described above was

converted into its sulphate salt by reaction with an equivalent quantity of dilute sulfuric acid.

24 o

[o?]D for 6-deoxyneomycin sulfate = +43 (c = 1.0, water)

c) Structure_of 6-deoxYneomvc:in

The structure of the antibiotic compound of formula I

in free base form obtained above was determined by comparison with the natural antibiotic, neomycin, formed when 2-deoxystreptamine is incubated in an appropriate medium.

The predicted 6-deoxyneomycin is hereafter referred to as compound X.

Methanolysis of both neomycin and compound X with 10% methanolic hydrogen chloride solution for 2 hours under reflux gave two major products, one of which corresponds to methylneobiosaminide and was formed from both compounds. The second product, hereafter referred to as Z, was different in the two antibiotics, in that the Z-compound from neomycin migrated less than the Z-product from compound X in the chromatographic sample using a 4/1 methanol/ammonium hydroxide solution as the elution solvent .

Confirmation of the structure for each Z was achieved by mass spectrometry of the per-trimethylsilyl derivatives.

Although molecular ions were not obtained, the spectra of the Z compound from neomycin and of the Z compound from compound X were very similar, except that two peaks from the spectrum of the former (m/e 343 and 460) were shifted 88 mass units lower in the latter (m/e 255 and 372). This corresponds to a loss of OSi (CHg^ and replacement with hydrogen.

The structure of Z from neomycin thus corresponds to neamine

and the structure of Z from compound X corresponds to 6-deoxyneamine.

Acetylation of both Z compounds in methanol followed by acid hydrolysis with a 3 N hydrochloric acid solution under reflux for 10 h gave in each case two major products. By paper chromatography it turned out that one of these products was the same in both cases, whether it was obtained from X starting from neomycin or Z starting from compound X, and it must be assumed to be 2,6-di-amino-2 ,6-dideoxy-D-glucose. The other two compounds were found to be chromatographically identical to 2-deoxystreptamine from neamine and 2,4-dideoxystreptamine from deoxyneamine.

These results lead to the conclusion that compound X corresponds to 6-deoxyneomycin.

Example 2

Separation of 6-deoxyneomycin into 6-deoxyneomycin B and 6-deoxyneomycin C

The 6-deoxyneomycin obtained by following the procedure described in Example 1 was separated into 6-deoxyneomycin B and 6-deoxyneomycin C by paper chromatography using a 3/16/6/1 mixture of tert-butanol/butanone/ 6.5 N ammonium hydroxide solution/methanol as solvent.

6-deoxyneomycin B and 6-deoxyneomycin c were detected on

the paper using the same method as described above.

Using as a solvent system methanol and ammonium hydroxide in the ratio 4 to 1 on 3MM Whatman chromatography paper, the Rf values for the 6-deoxymycins B and C were determined in comparison with the corresponding epimers of neomycin, and the following results were obtained:

Example 3

Preparation of 6-deoxyparomomycin and its I and II epimers

In a flask, D~Streptomyces rimosus forma paromomycinus ATCC 21484 was grown for 2 to 3 days on the following medium previously adjusted to a pH value of 7.5:

This culture was shaken and kept at 28°C for the indicated time period. A 10% inoculum of this culture was then added to a medium identical to that stated above, except that it additionally contained 0.025 g of 2,4-dideoxystreptamine dihydrochloride.

Cultures were incubated in Erlenmeyer flasks at 28° to 30°C on a rotating shaker with good aeration.

The subsequent operations in the preparation and purification of the 6-deoxyparomomycin were exactly the same as those described in Example 1a above.

Purification by paper chromatography under the same conditions as those described in the preceding Example 1 showed that the Rf value of 6-deoxyparomomycin was 0.30.

For comparison, it can be mentioned that the Rf value of paromomycin in an identical chromatographic sample was 0.27.

By using as a solvent system methanol and ammonium hydroxide in the ratio 4 to 1 on 3MM Whatman chromatography paper, the Rf values of the 6-deoxyparomomycins I and II were determined in comparison with the corresponding epimers of paromomycin,

and the following results were obtained:

Example 4

Preparation of the mixture of 3', 4'-dideoxyneomycin and its derivatives

a) Mixture of_2e2tamines_m^d_for^nel_iii A mixture of gentamines of formula III was first prepared from commercially available gentamicin sulfate. This salt was first converted to its free base and evaporated to obtain

an oil. A solution of hydrochloric acid in methanol, prepared by adding hydrochloric acid to dry methanol, was added. to this oil and the solution thus obtained was refluxed for 2 hours. The methanolic hydrochloric acid was removed under vacuum and the resulting oil was taken up in a little water and passed through a column of "Amberlite" I.R.A. 400 to provide the free base of the desired gentamines. The various fractions thus collected were evaporated to dryness and separated into methyl garosaminide and mixed gentamines by chromatography on silica gel using a 1/1/1 methanol/chloroform/ammonium hydroxide mixture as eluent. In this chromatographic separation, methylgarosaminide exhibited an Rf value of 0.8 and the mixture of gentamines an Rf value of 0)2.

b) Mixture of 3', 4'-dideoxyneomycin and its derivatives In a flask, D~ Streptomyces rimosus forma paromomycinus ATCC 21484 was grown for 2 to 3 days on the following medium previously adjusted to a pH value of 7.5 :

This culture was shaken and kept at 28 C for the indicated time period. A 10% inoculation of this culture was then added to a medium identical to that stated above, adjusted to pH 7.2, except that it additionally contained 50 µg of the previously obtained mixture of gentamines.

Cultures were incubated in Erlenmeyer flasks at 30°C on a rotating shaker and with good aeration for 5 days. The thus obtained crude mixture of 3<1>,4<1->dideoxyneomycin and its derivatives was purified by paper chromatography on Whatman No 3MM paper developed with a 4/1 methanol/ammonium hydroxide solution. The location of the mixture of 3<1>,4<1->dideoxyneomycin and its derivatives was made visible using 0.25% sodium hypochlorite in water, then absolute alcohol followed by 1% soluble starch and 1% potassium iodide in water with air drying between each “addition.

In this test, the Rf value of the mixture of 3',4'-dideoxyneomycin and its derivatives was found to be 0.25.

For comparison, it can be mentioned that the Rf value for the mixture of gentamines of formula III in an identical chromatographic sample was from 0.5 to 0.7.

Claims (4)

1. Analogy method for the production of new antibiotically active aminoglycoside-aminocyclitol derivatives with the general formula where and , which are different, means hydrogen or CI^NI^ and R is selected from the group consisting of: where R^ means NH2 or OH and R^ means where R,, means hydrogen or methyl, and pharmaceutically acceptable acid addition salts thereof, characterized by growing the deoxystreptamine-negative mutant a) D-Streptomyces rimosus forma paromomycinus ATCC 21484 in the presence of 2,4-dideoxystreptamine of formula: or an acid addition salt thereof, or in the presence of a mixture of gentamines of the general formula where R. means where R5 means hydrogen or methyl, or an acid addition salt thereof, or b) D-Streptomyces fradiae ATCC 21401 in the presence of 2,4-dideoxystreptamine of formula II or an acid addition salt thereof, in a medium containing a soluble carbohydrate, a source of assimilable nitrogen and essential mineral salts, after which the free base obtained is optionally reacted with an organic or inorganic acid to a pharmaceutically acceptable acid addition salt, and/or a mixture obtained of compounds of formula I is separated . 2. Method according to claim 1, characterized in that D~Streptomyces fradiae ATCC21401 is cultivated in the presence of 2,4-dideoxystreptamine or an acid , addition salt thereof to obtain 6-deoxyneomycin in free base form. 3. Method according to claim 1, characterized in that D~ Streptomyces fradiae ATCC21401 is grown in the presence of 2,4-dideoxystreptamine dihydrochloride to obtain 6-deoxyneomycin in free base form. 4. Method according to claim 1, characterized in that d" Streptomyces rimosus forma paromomycinus ATCC21484 is grown in the presence of the mixture of gentamines to obtain the mixture of 3',4'-dideoxyneomycin and its derivatives in free base form.