DE2710694A1 - NEW AMINOGLYCOSIDE AMINOCYCLITOL DERIVATIVES, THEIR PRODUCTION AND USE - Google Patents

Description

PATENTANWÄLTE 97 1PlPQt

Dlpl.-Ing. P. WIRTH Dr. V. SCH MIE D-KOWARZIK DipT-Ing. G. D AN N E N B E R G · Dr. P. WE IN H O LD · Dr. D. G U D E L

TELEPHONE: C089) ^Μ " ^Μ S ₁ EGFR ₁ EDSTnAGSE

335025 8000 MÖNCHEN

Wd / Eh

Br.

LAHAZ

Avenue Pierre 1er de Serbie, 39

F - 75 <"> Οβ Paris / France

New Aninoftlykosid-aminocyclitol-Deriyate, their production and Verv; endunn ;.

The present invention relates to aminocyclitol derivatives, and in particular it relates to novel amino-glycoside-aminocyelitol derivatives with pharmacological activity such as a process for the preparation of these compounds.

The inventive aminoglycoside aminocyclitol derivatives can be represented by the following general formula:

709837/0996

CH_OH

² OR

OH

-In this formula, K ^ and ΙΪ2 have different meanings and stand for hydrogen or CHpIIHp, and H is one of the following groups:

CH ₂ R ₃ .

NH.

HH.

709837/0996

t Rc CH,

I ⁵ I ³

in which K _{5 is} NH ₂ or OH and H _{4 is} CHNH ₂ or CHNHCH ₅ , where K _{5 is} hydrogen or a Kethy! group.

The pharmaceutically acceptable acid addition salts of these derivatives also fall within the scope of the present invention.

The compounds of the formula I can also be present as epimers or as mixtures of epimers.

Epimers and their mixtures are also supported by the present Invention includes.

The chemical structures of the compounds according to the invention resemble those of neomycin or paromomycin. For this reason, compounds of the formula I are referred to in this description as Neomycin or paromomycin derivatives referred to in following '»»' iron:

6-DeoxyneoLiycin B, when R _{1 is} hydrogen, Rp is CHpNH ₂ and R is the group A, in which R 1 is the group NHp; 6-Deoxyneomycin C, when R _{1 is} CH ₂ NH ₂ , R _{2 is} hydrogen and R is the group A, in which R ₁ is the group NH 2; the mixture of these two epimers obtained according to the invention is referred to as 6-deoxyneomycin.

6-Deoxyparomomycin I, when R _{1 is} hydrogen, R _{2 is} CH ₂₂ and K is the group A, in which is the group OH; 6-Deoxyparomomycin II, when H ₁ for CH ₂ NH ₂ , Ji ₂ for oxygen

709837/0996

and H represents the group A in which R 1 is the group OH; the mixture of these two epimers obtained according to the invention is called 6-deoxyparomomycin.

The mixture of 3 ', 4'-l) ideoxyneomycin and its derivatives produced according to the invention denotes an undivided combination of compounds in which R ₁ and Rg have different meanings and are hydrogen or CHpNKo and R is group B.

In this description, “neomycin” is to be understood as meaning a mixture of neomycin 3 and neomycin C which is obtained by culturing (= growing) Streutorayces fradiae .

Similarly, “Paromoinycin” denotes a mixture of Paromomycin I and Paromomycin II, which is obtained by cultivating Streptomyces rimo3us , form paromomycin .

The present invention is also intended to provide a pharmaceutical or veterinary medicinal preparation which as the main active ingredient at least one of the epimers according to formula I, ^ a pharmaceutically usable acid addition salt thereof or a mixture of these üpimeren respectively. their pharmaceutical useful acid addition salts in association with pharmaceutical carriers or additives for these compounds contains.

709837/0996

271069;

Another object of the present invention is to provide a process for the manufacture of pharmaceutical or veterinary Preparations in which at least one of the üpimoren according to formula I, a pharmaceutically acceptable acid addition salt the same or a mixture of these üpimeren respectively. of their pharmaceutically acceptable acid addition salts with a suitable one Carrier or additive is combined.

It has been found that the aminoglycoside aminocyclitol derivatives according to the invention show valuable antibiotic activity and are therefore suitable for the treatment of diseases caused by the growth of pathogenic bacteria, such as, for example, Escherichia coli , Proteus mirabilis , Staphylococcus aureus . Sarcina lutea , Klebsiella edv / ardsii , Shi ^ ella sonnei , Salmonella typhinurium .

The derivatives according to the invention can make Yfechstun more pathogenic Bacteria in a patient infected by these bacteria are inhibited if the patient is given at least one of the epimers according to formula Γ, a pharmaceutically usable acid addition salt thereof or a mixture of these epimers or their pharmaceutically acceptable acid addition salts administered.

A few years ago it was observed that certain strains of bacteria have become resistant to most commercially available antibiotics, making the therapeutic .. 'ert this anti-

709837/0996

biotics has been cut by more than half; some of the bacteria are even so heavily designed that they do not apply to any of the common Respond to antibiotics.

It is therefore imperative and in the general interest to develop new antibiotics that can target such organisms can destroy which have become resistant to the known antibiotics.

It is known that the inactivation of antibiotics is due to the destruction or modification of the antibiotic molecule by enzymes that attack the molecule at certain vulnerable points.

Especially the aninoglycoside aminocyclitol antibiotics that are used in are increasingly used, are often inactivated by resistant bacteria. Those of some organisms which normally respond to these aminoglycoside aminocyclitol antibiotics responding, developed resistance may be caused by phosphorylating, acylating, or adenylylating enzymes attack the free hydroxyl and amino groups of the antibiotic (Ann.Rev.Biochem., 42, p. 47, 1973).

By removing the hydroxyl groups, the antibiotic becomes immune to inactivation at these sites. It is therefore, it is desirable to produce antibiotics that have fewer non-essential hydroxyl and amino groups.

709837/0996

Al

Tests with the compounds according to the invention have shown that the mixture of 3 ¹ »4'-dideoxyneomycin and its derivatives has a clear antibiotic effect on organisms which have developed resistance to other arainoglycoaid-aminocyclitol antibiotics, and in particular to neomycin and gentamicin.

It has been shown that the mixture of 3 ¹ , 4 ^! -l> ideoxyneomycin or its derivatives also acts on Escherichia coli-Stäiume, which - because of their 2 ^w -adenylylierenden respectively. 3'-phosphorylating Knzyms - are resistant to gentamicin and neomycin.

For this reason, the above-described mixture of 3 ¹ _f 4'-I) ideoxyneomycin or its derivatives represented by Porinel I is the preferred antibiotic according to the invention.

From US Pat. No. 3,669,838 it is known that microorganisms, which normally produce antibiotics with an aminocyclitol subunit, such as neomycin or paromomycin, Mutants can be made which lack the ability to biosynthesize aminocyclitol subunit because of them the aminoc ^ clitol pathway is disturbed and therefore no antibiotic can be formed.

However, if you add this aminocyclitol-negative to the nutrient medium Mutants have a suitable aminocyclitol molecule, so they can Form an antibiotic using this molecule.

709837/0996

Us is also known that in this process, a pseudodisaccharide instead of the aninocyclitol subunit (= 3ubunit) can be used / grounded. .

However, the results published so far allow any prediction whether a certain diaminocyclitol is obtained with this bio-conversion process or pseudodioaccharide is introduced into an antibiotic may seem impossible.

In the literature numerous cases are even described in which this process for the production of new antibiotics is ineffective was.

The process described above cannot therefore be applied generally to the production of new semi-synthetic or completely synthetic ones Use antibiotics.

This fact is also confirmed, for example, in "The Journal of Antibiotics ¹¹ , Volume XXVI, No. 10, pages 551-561 (1973), where experiments with 29 different aminocyclitols are described, which were prepared by the method of the above-mentioned US patent Antibiotic should be introduced.

Of the 29 aminocyclitols tested in this way, only streptamine could and 2-] ipistreptanin with the help of those used for this purpose

709837/0996

2-Deoxystreptamine-negative (D ") mutants of Strcptonyces fradiae , Streptomyces rimosua . Form paronomycinus, and Streptomyce3 kanarayceticus can be introduced into an antibiotic.

The diaminocyclitols of French patent 2 203 808 could not be incorporated into an antibiotic if the same D ~ mutants of S, fradiae and S. rimosus , form paromonycinus , were used as in the above-mentioned publication.

In "Biochemistry", Volume 13, No. 25, pages 5073-5078 (1974), it is stated that all attempts were unsuccessful as neamine, paromamine and 6-kanosaminido ~ 2-deoxystreptamine known pseudodisaccharides with the help of the D ~ -Ijutanten of

S. fradiae . S. rimosus , form paromomycinus , and S. kanamyceticus .

"The Journal of Antibiotics", Volume XXVIII, No. 8, pages 573-579 (1975) also states that gentamines, which are pseudodiaaccharides, were not introduced into antibiotics by a D ~ mutant of Micromonospora inyoensis.

It was also found that 5-0-βD-ribopyranosyl-2,4-dideoxystreptamine cannot produce antibiotics if the D ~ mutants of. S. fradiae and S. rimosus . Porm paromonycinus , using the procedure described above.

709837/0996

The previous publications also show that diaminocyclitols or pseudodisaccharides, which are found in known antibiotics, such as streptomycin, bluensomycin and hy ^ romycin, are present, cannot be introduced into antibiotics by the mutants mentioned above.

The already cited publication in "The Journal of Antibiotics", Volume XXVI, No. 10, mentions, for example, that Bluensamin, Streptidin, Blu ^ nsidin, Hyosarnin and Actinamin when using the D "" - I.utants of S. fradiae . S. riraosus . Form paromomycinus , and S. kanamyceticus cannot be incorporated into antibiotics.

Also neamine and paromamine, which respectively in neomycin. Paroiaoraycin are found, were not introduced into antibiotics by a D ~ mutant of S. fradiae (see "Biochemistry" above).

It has also been found that a diaminocyclitol or pseudodisaccharide that can be introduced into an antibiotic with a mutant strain according to the method of the US patent mentioned cannot necessarily also be introduced with another mutant strain. Examples can also be found in the publication in "The Journal of Antibiotics", Volume XXVI, No. 10, where a D ~ - !: utant from S. rimosus , form paromomycinus , with 2-epistreptanin did not provide antibiotic during this subunit was introduced into antibiotics by a D ^- mutant of S. fradiae and 0. kanamyceticus.

709837/0996

The same ΐΓ mutant from S. kanamyceticus does not produce an antibiotic with otreptamine, but the same D ~ mutant from S. fradiae or S, riiiosuB , form paromomycinus , introduces this diarainocyclitol into antibiotics.

As described in "The Journal of Antibiotics", Vol. XXVI, No. 12, pp. 784-785 (1973), the pseudodisaccharide neamine can provide ribostamycin when using a D "" lutant of S. ribosidificus ; with the help of a D "* mutant of, for example, S. fradiae , however, neamine cannot be introduced into an antibiotic.

Finally, the previous publications show that the structure of a hypothetical antibiotic cannot be predicted in any case if a diaminocyclitol or a pseudodisaccharide is used together with a mutant stann. This fact is also confirmed by the above-mentioned publication in "The Journal of Antibiotics", Volume XXVI, No. 12, where it is stated that the antibiotics which are produced when using a D ~ mutant of S. kanamyceticus with 1-N- Methyldeoxystreptamine or Myo-inosa-1,3-diamine are "not the expected products" but other compounds. Similarly, Neamin and a D ~ -Hutant from M. inyoen-SJB produce sisomicin as a product, while the same mutant together with paromamine, another pseudodisaccharide, also forms sisomicin ("The Journal of Antibiotics", Volume XXVIII, No. 8 ; see above).'

709837/0996

It was also shown that a D ~ -i.utant from S. ribor.idificua in the presence of neamine only supplies ribostamycin and no analogue of neomycin, as was actually to be expected (see above literature reference in "The Journal of Antibiotics", volume XXVI, No. 12).

From the above it appears that there is no valid prediction about the possible incorporation of a particular dianinocyclitol or pseudodisaccharide into an antibiotic when using a certain mutant strain according to the method U.S. Patent 3,669,838 cited above. In no case can it be predicted whether the procedure will take place at all will be successful, or what will be the exact structure of the antibiotic that nan hopes to manufacture.

Hence the assertion in "J. Org. Chem.", Volume AO, No. 4, pages 456-461 (1975) _t that 2,4-dideoxystreptamine may be incorporated into neomycins, paromomycins and ribostamycin by means of a bio-conversion process could be viewed as extraordinarily vague and indefinite and has no reasonable or valid basis.

Surprisingly, it has now been found that, according to the invention, 2,4-dideoxystreptamine can be replaced by D ~ mutants of S. fradiae and S. rimosus . Form paromomycinus , as well as a mixture of pseudodisaccharides, ie of gentamines, can be introduced into new antibiotics by a D ~ mutant of S. rimosus , form paromomycinu3 _t.

709837/0996

This is all the more surprising when one takes into account that experiments carried out with 2,4-dideoxystreptamine in the presence of an L "~ mutant of S. kanamyceticus yielded no antibiotic, and that a D ~" mutant of S . fradiae was unable to incorporate the mixture of gentamines in question into an antibiotic.

According to the prior art, the results achieved according to the invention with 2,4-dideoxystreptaain and the mixture of gentamines were not to be expected.

The compounds of the invention are prepared by cultivating a deoxystreptamine negative Hutante from Streptomyces in a suitable medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and:

a) either 1D- (1,3,5 / 2) -1,5-diamino-2,3-cyclohexanediol or 2,4-Dideoxystreptamine of the following formula:

II

HO

BO

or an acid addition salt thereof, e.g., the dihydrochloride; or

709837/0996

b) a mixture of gentamines of the following general terms Formula:

III

in which H. has the same meaning as in group B. of the formula I, or an acid addition salt thereof.

When using the inventive method, the antibiotic obtained in the form of a free base, regardless of whether the diaminocyclitol of the formula II or the mixture of gentamines according to formula III was present as a free base or as a salt. If the antibiotic is desired as a salt, all that is needed is with a suitable organic or inorganic acid such as sulfuric acid to a pharmaceutically useful one Acid addition salt to be implemented.

709837/0996

Microorganisms for the process according to the invention are D "" Stroplomycerü frndiae ATCC 21401 and D ~ .Stroptoinyocn rino ::; '» , form paromomycine , ATCC 21484, both of which are described in US Pat. No. 3,669,858. The 6-deoxyneomycin according to the invention is produced with D ~ ütreptoin.yceo fradiae ATCC 21401, and D ~ Streptomycea rinosus . Form paromor.ycinus , ATCC 21484 is used for the production of 6-deoxyparoiaomycin and the mixture of 3 ', 4'-dideoxynec-iycin and its derivatives.

The microorganism is cultivated in a known manner in a nutrient medium with a suitable pH-value, such as glucose, casein hydrolyzate, (NH ^) ₂ IIPO ₄ , MgSO ₄ .7H ₂ O, PeSO ₄ .7H ₂ O, CuSO-. 5HgO urci CaCO ^ contains, and then added to another nutrient medium that contains the maininocyclitol of formula II or the mixture of gentamines wet formula III - both as a free base or as an acid addition salt - as well as, for example, soybean meal, ilefe extract, HaCl, CaCO ₅ and glucose and has also been adjusted to the appropriate pH. The culture is then incubated for at least 5 days at a temperature of about 3O ⁰ C with shaking and good ventilation in order to optimally form the desired 6-deoxyneomycin, 6-deoxyparonomycin or the mixture of 3 ', 4'-dideoxyneoraycin and its To achieve derivatives.

6-Deoxyneomycin B and 6-Deoxyneomycin C and 6-Deoxyparoiüomycin I and 6-Deoxyparomomycin II become from G-Deoxyneomycin

709837/0996

respectively 6-Deoxyparononiycin obtained using known processes, e.g. by separation using paper chromatography.

The isomers, including the mixture of 3 ', 4'-dideoxyneomycin and its derivatives can also be separated in known terms.

The diaininocyclitol of Formula II is a known compound which is described in British Patent 1,445,675. According to the process of this patent specification, it is obtained by adding 1D- (1,3 _f 5/2) -1, S-diazido-Z ^ -cyclohexanediol, prepared from 1L-1,5-M-0-tosyl-1, 2,5 / 3-cyclohexanetetrol and sodium azide, hydrogenated in the presence of a catalyst such as Raney nickel.

The wiping of gentamines according to formula III can help the method of Cooper et al, "J. Chem. Soc.% (c) 1971, p 960, made from commercially available gentamicin sulfate.

Already mentioned, have the amino glycoside-aminocyclitol derivatives according to the invention valuable antibiotic properties.

Their antibacterial effectiveness against various tea organisms is illustrated by the experiments below.

709837/0996

a) Antibacterial effectiveness_of 6-I) eoxyjieomy_cin and 6-deoxy_- paromom ^ cin

The antibiotic effectiveness of the amino-glycoside-arainocyclitol derivatives according to the invention was examined by placing measured flips of each test compound in sterile. 'acser on an agar medium.

The agar media containing increasing concentrations of solid compound were then poured into Petri dishes; Various organisms, which had previously been grown in a suitable medium and stored at -20 ° C. in a 1: 1 mixture of culture medium and glycerol, were mechanically added to each dish using a multi-inoculator. Then the plates were incubated for 14 hours at 37 ⁰ C and then, ^r examined for da3 V ach3tuin of organisms.

The concentration of the antibiotic that was just enough to Prevent the growth of bacteria was recorded and referred to as the "minimum inhibitory concentration" (M.I.C.).

The results obtained in this experiment are summarized in Table I and compared with the results obtained with Neomycin and Paromoraycin each in sulfate form were obtained. The numbers in Tables I and II correspond to the amount of free base.

709837/0996

-vo
ll
Table I. 2.5 L. Paromo-
m.ycin / I U03 * Amount in 5.0 jiLß / ccm, through which the
was prevented 5.0 Growth organism Neomycin 6-deoxy
neomycin 1.25 6-i) eoxy-
paromo-
mycin Escherichia coli
W 3110 2.5 1.25 1.25 40 Froteus mirabilis 5.0 5.0 5.0 20th Staphylococcus
aureus 2.5 3.75 2.5 20th Sarcina lutea 2.5 2.5 2.5 40 Klebsiella edwnrdsii 7.5 10 2.5 40 Shigella sonnei
C 631978 3.75 40 Salmonella typhi-
murium LT 2 5.0 40

The results show that by removing the hydroxyl group in the 6-position of the alainocyclitol moiety, dea neomycins (6-Deoxyneomycin is obtained as a result) the antibacterial effectiveness of the neomycin is not significantly changed will. It is particularly pointed out that 6-deoxyneomycin is better antibacterial than Escherichia coli W 3110 ». Effect shows as neomycin.

b) Effective mkeityon_6 ~ pgoxyneomycin_B_und_6-Deoxyneom ^ cin_C

For reading the antibacterial effectiveness of 6-deoxyneomycin B and * 6-J) eoxyneoiaycin C followed the procedure described above repeated, with E.S.T. Agar used as a culture medium became; the M. 1.C. fourth got neomycin and 6-deoxyneomycin compared. 709837/0996

Table II

Quantity in ii ^ / ccm, durci. which prevents the './achstun

became

Keo- Heo- 6-Deoxy- 6-j) co; -: y-Heomycin mycin b-Deoxy- neomycin neo :: ../ an organism nycin BC neomycin BC

Hacherichia

coli Bristol 0.6? 0.62 80 2.5 2.5 5 Protexis

mirabilia 1.25 1.25 10 5 5 5 staphylo-

COCC W S

aureus 0.62 0.62 80 5 2.5. 20 Salmonella

typhimurium 2.5 2.5 40 10 5 20

These results make 6-Deoxyneor.iycin B slightly less is effective as neomycin 3, but that G-docxyneomyein C oii: c has better effectiveness than neomycin C.

c) iiilksankeit der__i ^ churiij aus_3_ | __4'-Dideoxvneom ^ cin_und

The antibacterial effectiveness of I.üschun, ·; from 3 ', 4' -Dioeoxyneomycin and its derivatives was determined in the following way

D ~ Streptomyceo riniosus , form paroi; -iomycinus , ATCC 21484 was incubated at 30 ° C. on an agar medium containing 50 μg / ccva of a mixture of Gentaainen ^ em according to ormel III. l: After three days an agar plate inoculated with a test organism was placed on the culture medium, and the formation of the antibiotics was due to an inhibition. ^ nsone around the i) treptoi; iycf.? .- *. lutan th recognizable. This Inhibierunjjo ^ cne was, ^ eHfisncn Lind uiii;

709837 / 099Ö

compared to a control that beatand au & dv.ui Agiir-Kühr boden alone.

For comparison, a similar Versiich was carried out in which the agar Näbrboden - instead of the mixture of iTentaminen gemasy formula III - 50 g./ccrn neamine added v / ur ^<:; .- η; I) " Streptonyces rirnosus , I'Oru paror, o:.:; / cinu 3, ATCC 21484 was also used. The antibiotic thus obtained turns out to be neomycin.

The results are summarized in Table III.

coli PT ₂ 1629 Tabel III Neamin Mix of
Gentamines
/ tenäsr. .'orncl III coli ML 1410 0 21 organism coli HL 66 Inliibierun _r .; Rcone in i. ;;: i, if ^:.:
A * η ν - '.''■''Λν ^ ο d η η zwt ' · "■■" ■ η ν / ';"'■■""" !: 0 25th Escherichia coli JR nothing 0 20th Escherichia 0 0 25th Escherichia 0 Escherichia 0 0

These results show that the mixture of 3 ', 4'-3ideoxyneomycin and its derivatives have a good effect on organisms that are resistant to neomycin. Ka will noted that Ilscherichia coli also opposed PTp Gentamicin is resistant.

709837/0996

"*" 27106 ^f U

ouch

For therapeutic purposes, the compounds according to the invention are normally used as a pharmaceutical3 or veterinary ο preparation in the form of a dosage unit which is adapted to the particular mode of administration; such a rr ' ^: mrat ui.ifaCt at least one compound according to the invention air; ", / ir ·: - substance in association: with a carrier or additive for the compound. For oral administration, the preparation can be, for example, as a coated or non-coated tablet, as a hard or calibrated gelatin capsule, as a suspension or Other possible forms of the preparation are guppositories for rectal or vaginal use, solutions or suspensions for parenteral use or creams and ointments for external use.

The following examples explain the preparation of the inventive Links.

Example 1; Preparation of 6-deoxyneoinycin base and its Sulfate

^a) 6-Deox ^ ^ neon clninPorineinerf reien3ase In a flask was allowed to Ώ "Streptomyces fradiae ATCC 21401 grown on the following medium, which had been previously adjusted to a pH-./ert of 7» 2 to 7.5 48 hours :

Glucose 1 g

Casein hydrolyzate 1 g

(MH ₄ 'J ₂ IIPO ₄ 1 g

Mg SO ₄ .7H ₂ O 0.05 g

Pe SO ₄ .7H ₂ O 0.005 g

709837/0996

f ^AD

27106G4

Cu SO ₄ .5H ₂ O 0.005 β

CaCO ₅ 1 g

Water to 100 ecm

This culture was shaken for the specified time period and kept at 2ü ° C. Then a 10% inoculum was made this culture added to the following Tlührmediura, daa before has also been adjusted to a pH value of 7.2 to 7 »5 was:

Glucose 1 g

Soybean meal 2.5 g

Yeast extract 0.5 g

NaCl 0.5 g

CaCO, 0.2g

2,4-dideoxy3treptamine

dihydrochloride 0.025 g

V / ater to 100 ecm

The cultures were incubated in Erlenmeyer flasks at 28 ° C to 3O ⁰ C in a rotary shaker with good ventilation.

The formation of 6-deoxyneomycin started after 2 days and was optimal after 5 days.

Comparison cultures which do not contain 2, / i-dideoxystrcptamine dihydrochloride. did not form 6-7) eoxyneomycin.

709837 / 099b

The culture was then centrifuged and the supernatant liquid poured through a column in which there is a weakly acidic, carboxyl group (polymethacrylic type) containing cation exchange resin medium porosity ("Amberlite IRC-50") in Ammonium form. This iilassnahue was carried out twice, around the 6-deoxyneonycine and unchanged 2,4-dideoxystreptanine dihydrochloride to extract. The column was washed out with ./a3ser and 6-Deoxyneomycir with a 2N ammonium hydroxide solution eluted in './asser. The eluate was under a vacuum concentrated in a rotary evaporator and then added to a Sephadex G-10 column. By eluting with a 0.1m Ammonium hydroxide solution became 6-deoxyneomyein in the first fraction and in the subsequent fractions 2,4-dideoxystreptaniine dihydrochloride obtain.

On top of this, 6-deoxyneomycin was used as a free base in one Obtained amount corresponding to 83 units of effectiveness (1 unit = 1 µg / cc neomycin). That means that about 22 / S des 2,4-dideoxystreptamine dihydrochloride originally used were incorporated into the 6-deoxyneomycin thus obtained.

This was the maximum achievable effectiveness and a longer incubation time failed to increase the amount of 6-deoxyneomycin.

In an alternative method, the crude 6-deoxyneomycin was chromatographed on Whatman paper no. 3 MM

709837/0996

nigt, that with a 4: 1 solution; of methanol and Ammoniuiahydroxyd was developed. The location of the 6-Deoxyneomycins was visualized by first 0.25 / $ llatriumhypochlorit in v / ater, then absolute ethanol and then 1 i "soluble Jtärke and 1> * potassium iodide in V / Aoser applying an end and between the individual plots each dried in the air.

It was found that the Rf-Y / ert of 6-deoxyneomycin in these; Attempt was 0.30.

For comparison purposes, the same solvents were used the following HF values are determined:

Kf- .. ^r ert

Neomycin 0.26

2-deoxystreptamine 0.58

2,4-dideoxystreptamine 0.62

The 6-deoxyneomycin prepared in the manner described above was converted into the corresponding sulfate salt it was reacted with an equivalent amount of dilute sulfuric acid.

of 6-deoxyneomycin sulfate = + 43 ° (c = 1.0, water) D

709837/0996

After the antibiotic compound of formula I on the above ., also been prepared in the form of a free base its structure was determined by comparing it with the natural one Compared antibiotic neomycin obtained by incubating 2-deoxystreptamine in a suitable indium.

The presumed 6-deoxyneomycin is referred to as Compound X hereinafter.

Both neomycin and compound X were each 2 hours under reflux with a 10 # strength methanolic hydrogen chloride solution treated and two main products were obtained; one of these products, that of both compounds was formed was Hethylneobiosaminid.

The other compound, hereinafter referred to as Product Z, was different for the two antibiotics; the one with neomycin The product Z obtained migrated during the chromatography, which was carried out with a 4: 1 methanol / ammonium hydroxide solution as the eluent was carried out, less strongly than the product Z of the compound X.

The structures of the products Z were each determined by mass spectrometry of the pertrimethylsilyl derivatives.

Although the molecular ions were not obtained, they were Spectra of the product Z from neomycin and of the product Z from

709837/0996

Compound X very similar; there were only 2 maxima from the spectrum of the first-mentioned product (n / e 343 and 460) in the the latter product moved down by 88 masoene units (m / e 255 and 372). This leaves a loss of OSi (CH ^), and recognize exchange by hydrogen.

The structure of the product Z from neomycin thus corresponds to that of neamine, and the product Z of the compound X has the same Structure like 6-deoxyneamine.

By acetylating the products Z in methanol and then-

de 10-hour hydrolysis with a 3N hydrochloric acid solution

under reflux conditions, two main products are produced again obtain.

The paper chromatography revealed that apparently one of these products was the same in both cases, i.e. so / ohl of Z from neomycin as was also formed by Z from compound X; this product was apparently 2,6-diarnino-2,6-dideoxy-D-glucose. The other two products were respectively by chromatography as 2-deoxyatreptamine from Neamine. as 2,4-dideoxystreptamine confirmed by deoxyneamine.

These results suggest that the Compound X is 6-deoxyneomycin.

709837/0996

At a pi el 2: Separation of 6-Deox.yneorrycin into 6-Peoxyneom.ycin B. and 6-dcoxynooinycin C

The 6-Deoxyneomycin prepared according to Example 1 was through
Paper chromatography in 6-deoxyneomycin B and 6-deoxyneomycin C separated, a mixture of tert-butanol, butanone, 6,5n-Al: ammonium hydroxide solution and methanol in a ratio of 3: 16: 6: 1 being used as the solvent .

6-Deoxyneomycin B and 6 -]) eoxyneo: nycin C were obtained using the
procedure described above noted on paper.

The Rf values of the 6-deoxyneomycins B and C were then determined on V / hatman chromatography paper 3MM using a solvent system composed of methanol and aluminum oxide hydroxide in a ratio of 4: 1, and with those of the corresponding piners of neomycin
compared; the following results were obtained:

Rf value 6-deoxyneomycin B. 0.29 6-Deoxyneomycin C 0.21 Neomycin B 0.25 Neomycin C 0.165 Example 3: Manufacture of 6-Deoxyparomomycin and its Epimers I and II In a flask one left D " Streptomyces riraosus, form paromo-

mycinus, ATCC 21484, can be grown for two to three days on the following medium that has previously been adjusted to pH 7.5 was:

709837/0996

Λ 2710694 Soybean meal IJ
1 ß Casein hydrolyzate 0.25 β CaCO, 0.5 β glucose 1 ß NaCl 0.5 β NH ₄ Cl 0.167 β

Water to 100 ecm

This culture was shaken uring v / the specified period and held at 28 ⁰ C. A 10% inoculum from this culture was then placed in a medium which corresponded to the medium described above, but additionally contained 0.025 g of 2,4-dideoxystreptamine dihydrochloride.

The cultures were incubated in Erlenmeyer flasks at 28 ° to 30 ⁰ C on a rotary shaker with good aeration.

Then the 6-deoxyparomomycin according to the method of Example 1 a) washed and cleaned.

Purification by paper chromatography under the conditions of Example 1 showed that the 6-deoxyparomomycin a Rf value of 0.30.

For comparison purposes, it should be mentioned that the Hf value of Paromomycin found to be 0.27 using an identical chromatography procedure.

709837/0996

On V / hatman chromatography paper 3 MIu using a Solvent system of methanol and ammonium hydroxide in proportion 4: 1, the Rf- '. 7ths of the 6-deoxyparomomycins I and II and compared with those of the corresponding epimers of paromomycin, the following results being obtained:

I. Rf value 6-deoxyparoniomycin II 0.34 6-deoxyparomomycin 0.275 Paromomycin I. 0.3 Paromomycin II 0.22

Example 4: Manufacture /; the mixture of 3 ¹ .4'-Bideoxyneomycin and its derivatives

^a ) Mi ££] 2H2 £ LY.22 Gentamines 2 £ ^π ^ § £

A mixture of gentanines according to Formula III was first produced made from commercially available gentamicin sulfate, this salt was first converted into the free base and then evaporated, whereby an oil was obtained. A solution of hydrochloric acid in methanol (prepared by adding hydrogen chloride acid to dry methanol) and the resulting solution heated under reflux for two hours. The methanolic Hydrochloric acid was removed in vacuo and the oil thus obtained was taken up in a little water and filtered through an "Amberlite I.R.A. 400 "containing column passed to the free base of the mixed To win gentamines. The individual fractions thus collected were evaporated to dryness and chromatographed on silica gel using a 1: 1: 1 mixture of methanol,

709837/0996

Chloroform and Amraoniurahydroxyd as solvents in Methylgarosaiainid and mixed gentamines separately. This chromatographic separation gave a lif-V / ert of 0.8 for Methylgarosamiiiid and of 0.2 for the mixture of gentamines.

D ~ Streptorr.yces rimosus was found in a flask. Form paroraomycinus , ATCC 21484, grow for two to three days on the following medium, which had previously been adjusted to a pH - '. Vert of 7.5:

Soybean meal 1 g

Casein hydrolyzate 0.25 g

CaCO ₃ 0.5 g

Glucose 1 g

NaCl 0.5 g

HH ₄ Cl 0.167 g

Water to 100 ml.

The culture was shaken and kept at 28 ^° C. for the specified period of time. A 10% inoculum from this culture was then placed in a medium which corresponded to the medium described above, but had been adjusted to a pH of 7.2 and in addition 50 / g of the mixture of Contained gentamines.

Cultures were incubated in ^ rlenmeyer flask at 30 ⁰ C on a rotary evaporator with good aeration five days.

709837/0996

/ so obtained ^w

The crude mixture of 3 ¹ , 4'-dideoxyneomycin and its derivatives was determined by paper chromatography on Whatman paper no. 3MM that was developed with a 4: 1 solution of methanol and ammonium hydroxide. The place of the mixture of 3 ''4'-Dideoxyneomycin and its derivatives was visualized by 0.25 i> sodium hypochlorite in the Y / ater, then absolute ethanol and finally 1 ^c /> soluble starch 1 and i "potassium iodide in V / ater applied i <<d air-dried between each application.

This was for the mixture of 3 ', 4'-dideoxyneomycin and its derivatives have an Rf -7ert of 0.25.

For comparison, it should be mentioned that with an identical chromatography process for the mixture of gentamines according to Formula III gave an Rf-Y.'ert of 0.5 to 0.7.

709837/0996

Claims (12)

Patent claims: 1. 'Aminoglycoside arainocyclitol derivatives of the general - ^ ^P0rmel: "CH ₂ OU OR in which R ₁ and R _{2 have} different meanings and are hydrogen or CH ₂ NH ₂ and Ii is one of the following groups: A = or I ⁵ CH- CH I ³ CH-N I I in which R _{5 is} NH ₂ or OH and R _{4 is} CH-NH ₂ or CH-NHCH ₅ , where Rc is hydrogen or a methyl group; and the pharmaceutically acceptable acid addition salts of these derivatives. 709837/0996 ORIGINAL INSPECTED a, 2. derivative or /. Acid addition salt according to Claim 1, characterized in that R is the group A in which FU stands for NH ₂ , and R "and R _? have different meanings and represent hydrogen or CH ₂ IJH ₂ . 3. derivative or. Acid addition salt according to Claim 1, characterized in that R is the group A in which _{R 1 stands for TIH 2} and R _{1 stands} for hydrogen and R _{2 stands} for CH ₂ IIH ₂ . 4. derivative or acid addition salt according to claim 1, characterized in that R is the group A in which R is _{0 for NII} and R _{1 is} CH ₂ NH ₂ and R _{2 is} hydrogen. 5. acid addition salt according to claim 2, characterized in that it is the sulfate. 6. derivative or acid addition salt according to claim 1, characterized in that R is the group B, R ₁ and R _{2 have} different meanings and are hydrogen or CH ₂ NH ₂ . 7. Process for the preparation of aminoglycoside aminocyclitol derivatives or their pharmaceutically usable acid addition salts according to claims 1-6, characterized in that a -deoxystreptamine negative mutant of Streptomyces cultured in a suitable medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential inner salts and: 709837/09 9 8 a) either 2,4-mdeoxystreptarain of the formula -MH. or an acid addition salt thereof contains, and on this wise the compounds in the form of the free base of formula I, in which R stands for group A, wins, b) or a mixture of gentamines of the following general ones ? orrael or an acid addition salt thereof contains: CH, in which R _{4 stands} for CH-NH ₂ or CH-NHCH, where Rc is hydrogen or a methyl group, and in this way, in the form of the free base, the compounds of the formula I in which R stands for group B are obtained, 709837/0996 "^" 271069; where as deoxystreptainin-negative mutants of otreptoinyce: when using 2,4-Diueoxystrcptarnin or its: ¹ Ualze D ~ ^ treptomycos fradiae ATCC 214 01 or D ~ Streptonyces rimosus , form paroir.oniycinu3 , ATCC 21484 and when using the mixed origin of U -entaininen D ~ Streptomyces rii.iosus . Form paromonr / cinus , ATCC 21484 are used and the free base obtained is optionally treated with a suitable organic or inorganic acid and converted into the corresponding pharmaceutically acceptable acid addition salt. 8. The method according to claim 7, characterized in that nan D " Streptonyces fradiae Ai ¹ CC 21401 is cultivated in the presence of 2,4-dideoxystreptamine or one of its acid addition salts and 6-deoxyneomycin wins in the form of the free base. 9. The method according to claim 7, characterized in that D ~ Streptomyce3 fradiae ATCC 21401 is cultivated in the presence of 2,4-dideoxystreptamine dihydrochloride and 6-deoxyneomycin is obtained in the form of the free base. 10. The method according to claim 7, characterized in that D ~ Streptomyces rimosua . Form paromomycinus , ATCC 21 484 cultivated in the presence of the mixture of gentamines and the mixture of 3 ¹ , 4'-dideoxyneomycin and its derivatives in the form of the free rabbit wins. 709837/0996 ~ * ~ 271069; 11. Ancestors according to claim 7, characterized in that one as inorganic? Acid iJchv / efelüMure used. 12. Pharmaceutical or veterinary preparation, characterized in that there is at least one derivative according to claims 1-6 or a pharmaceutical as active ingredient Breakable acid addition of these in association with a pharmaceutical carrier or additive for it contains. 709837/0996