DE2710694C2 - Aminocyclitol glycosides, processes for their production and preparations containing these compounds - Google Patents

Description

The diaminocyclitols of FR-PS 22 03 808 could not be incorporated into an antibiotic if one the same D "mutants of S. fradiae and S. rimosus, form paromomycine, used as in the above Publication.

In "Biochemistry", Volume 13, No. 25, Pages 5073-5078 (1974), it is stated that all attempts were unsuccessful S were the pseudodisaccharides known as neamine, paromamine, and 6-kanosaminido-2-deoxystreptamine using the D "mutants of S. fradiae, S. rimosus, form paromomycine and S. kanamyceticus.

It was also found that 5-0-jSD-ribopyranosyl-2,4-dideoxystreptamine do not produce any antibiotics can, if the D ~ mutants of S. fradiae and S. rimosus, form paromomycinus, according to the above the procedure described.

The previous publications also show that diaminocyclitols or pseudodisaccharides, which in known antibiotics such as streptomycin, bluensomycin and hygromycin are present by the above Mutants cannot be introduced into antibiotics.

The already cited publication in "The Journal of Antibiotics", Volume XXVI, No. 10, mentions e.g. B. that Bluensamin, Streptidin, Bluensidin, Hyosamine and Actinamine when using the D mutants of S. fradiae, S. rimosus, form paromomycinus and S. kanamyceticus cannot be incorporated into antibiotics.

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

Furthermore, it has been found that a diaminocyclitol which can be introduced into an antibiotic with a mutant strain in accordance with the method of the cited US patent specification cannot necessarily also be introduced into another mutant strain. Examples are also the VeröffentU voltage in "The Journal of Antibiotics", Vol XXVI, no. Point 10., where a D ~ mutant of S. rimosus, shape paromomycinus, supplied with 2-Epistreptamin no antibiotic during this subunit by a D "mutant of S. fradiae and S. kanamyceticus was introduced into antibiotics.

But the same D ~ of the same D "mutant of S. kanamyceticus generated with no antibiotic Streptamin - Mutant fradiae S. or S. rimosus, shape paromomycinus, brir _a t this diaminocyclitol in antibiotics Finally show the previous publications.. that the structure of a hypothetical antibiotic cannot be predicted in any case when a diaminocyclitol is used in conjunction with a mutant strain. This fact is also confirmed by the above publication in The Journal of Antibiotics, Vol. XXVI, No. 12 where it is stated that the antibiotics obtained when using a D ~ mutant of S. kanamyceticus with 1-N-methyl-deoxystreptamine or Myo-inosa-1,3-diamine are "not the expected products" but different ones Connections are.

From the above it appears that no valid prediction about the possible introduction of a

certain diaminocyclitol into an antibiotic when using a certain mutant strain can be made according to the method of US Pat. No. 3,669,838. In no case can it be predicted whether that Procedure will ever be successful or what the exact structure of the antibiotic that one is given will be hopes to manufacture.

Hence the claim in "J. Org. Chem. ", Vol. 40, No. 4, pp. 456-461 (1975) that if appropriate 2,4-Dideoxystreptamine with the help of a bio-conversion process into neomycins, paromomycins and rii> ostamycin could be introduced, to be viewed as extraordinarily vague and indefinite and is lacking in any way reasonable and valid basis.

Surprisingly, it has now been found that, according to the invention, 2,4-dideoxystreptamine is replaced by D ~ mutants by S. fradiae and S. rimosus, form paromomycinus, can be introduced into new antibiotics. This is all the more surprising when one takes into account that experiments carried out with 2,4-dideoxystreptcinin in The presence of a D ~ mutant of S. kanamyceticus did not yield any antibiotic.

According to the prior art, the results achieved according to the invention with 2,4-dideoxystreptamine are thus obtained not to be expected.

The invention also provides a process for the preparation of the compounds according to the invention, the is characterized in that in a nutrient medium, which in addition to a soluble carbohydrate, one Source of assimilable nitrogen and essential mineral salts 2,4-dideoxystreptamine of the formula

HO

HO

or an acid addition salt thereof, either

a) D "Streptomyces fradiae ATCC 21401 breeds and 6-deoxy-neomycin B and C from the culture medium wins or

b) D "Streptomyces rimosus, form paromomycinus, ATCC 21484 grows and from the culture medium 6-deoxy-paromomycin I and II wins,

optionally in the form of their acid addition salts.

When using the method according to the invention, the antibiotic is obtained in the form of a free base, regardless of whether the 2,4-dideoxystreptamine was present as a free base or as a salt. Is the antibiotic called salt if desired, all that is needed is a suitable organic or inorganic acid, such as. B. sulfuric acid, to be converted to a pharmaceutically useful acid addition.

The microorganisms used for the process according to the invention are D Streptomuos fradiae ATCC 21401 and D ^ Streptomyccs rimosus, form paromomycinus, ATCC 21484, both of which are described in US Pat. No. 3,669,838 are described. With D Streptomyces fradiae ATCC 21401, the 6-Dcsoxyncomycin according to the invention and D Streptomyces rimosus, form paromomycinus, ATCC 21484 is used to produce the 6-deoxyparomomycins used.

The microorganism is grown in a known manner in a nutrient medium with a suitable pH value, which z. B. glucose, casein hydrolyzate, (NH ₄ ), 11PO ₄ , MgSU ₄ 7 11.0, FeSO ₄ · 7 H, O, CuSO ₄ · 5 H _: 0 and CaCO, and then added to another nutrient medium that contains the diaminocyclitol - as a free base or as an acid addition salt - and z. B. contains soybean meal, helcextrakt, NaCl, CaCO, and glucose and has also been adjusted to the appropriate pH. The culture is then inkubicrt at least for 5 days at a temperature of about 28 ° to 30 ⁰ C with shaking and good ventilation. to achieve optimal formation of the desired 6-deoxyneomycin or 6-deoxyparomomyein.

6-Dcsoxyneomycin B and 6-deoxyncomycin C and 6-deoxyparomomyein I and 6-deoxypuromomycin II are obtained from 6-deoxyneomycin or 6-deoxyparomomycin by known methods, e.g. B. by separating with the aid of paper chromatography.

The diaminocyclitol used is a known compound which is described in GB-PS 1,445,675. According to the process of this patent specification, it is obtained by ID- (1.3,5 / 2) -l, 5-diazido-2,3-cyclohexanediol, made from lL-l.S-Di-O-tosyl-I ^ .SAVcyclohexantetrol and sodium azide, in the presence of a Catalyst, such as. B. Raney-Nickei, hydrogenated.

The aminocyclitol glycosides according to the invention have valuable antibiotic properties.

Their antibacterial effectiveness against various test organisms is determined by the following Experiments explained.

a) Antibacterial activity of 6-deoxyneomycin and 6-deoxyparomomycin

The antibiotic activity of the compounds according to the invention was examined by measuring Quantities of the respective test compounds in sterile water were placed on an agar medium.

The increasing concentrations of test compounds contained in agar culture media were then in Cast petri dishes; various organisms previously grown in a suitable medium and at -20 ° C in a 1: 1 mixture of nutrient medium and glycerine were mechanically with placed in a multi-inoculator in each dish. The dishes were then incubated at 37 ° C. for 14 hours and then examined for the growth of the organisms.

The concentration of the antibiotic that was just enough to prevent the bacteria from growing was recorded and designated 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 paromomycin, each in sulfate form. The numbers in Tables Ϊ and ΪΪ correspond to the amount of free base.

Table I.

organism

Amount in ag / ccm that prevented growth

Neomycin 6-deoxy-paromomycin 6-deoxy

neomycin paromo

mycin

Escherichia coli W 3110, ATCC 27325 2.5 1.25 5.0 40 Proteus mirabilis 5.0 5.0 1.25 20th Staphylococcus aureus, ATCC 9144 2.5 3.75 1.25 20th Sarcina Iutea 2.5 2.5 5.0 40 Klebsieila edwardsii 7.5 10 2.5 40 Shigella sonnei C 631978 2.5 3.75 2.5 40 Salmonella typhimurium LT 2 ATCC 19585 5.0 5.0 2.5 40

The results show that by removing the hydroxyl group at the 6-position of the aminocyclitol moiety of neomycin (this gives 6-deoxyneomycin) the antibacterial effectiveness of neomycin overall is not changed significantly. It is particularly pointed out that 6-deoxyneomycin against Escherichia coli W 3110 shows a better antibacterial effect than neomycin.

b) Effectiveness of 6-deoxyneomycin B and 6-deoxyneomycin C.

To determine the antibacterial effectiveness of 6-deoxyneomycin B and 6-deoxyneomycin C. the procedure described above was repeated using D.S.T. Agar was used as the nutrient medium; the M.LC.-words were compared to neomycin and 6-deoxyneomycin.

Table II

organism S. Hs.; Iierichia coli Bristol Amount in; jg / ecm, through which prevented growth 6-dcsoxy- 6-Dcsox left 6-dcsoxy- Proteus mirabilis Neomycin Neomy Neomy ncomycin ncomycin U neomycin C ¹⁰ Staphylococcus aureus cin B cin C 2.5 2.5 5 Salmonella lyphimurium LT 2 0.62 0.62 80 5 5 5 1.25 1.25 10 5 2.5 20th 0.62 0.62 80 10 5 20th 2.5 2.5 40

These results show that 6-deoxyneomycin B is somewhat less effective than neomycin B, however 6-deoxyneomycin C has better potency than neomycin C.

For therapeutic purposes, the compounds of the invention are normally used as pharmaceutical or veterinary preparation applied in the form of a dosage unit that of the respective Mode of administration is adapted; Such a preparation comprises at least one compound according to the invention as Active ingredient in association with a carrier or additive for this compound. For oral administration 2ü the preparation can z. B. as a coated or uncoated tablet, as a hard or soft gelatin capsule, as Suspension or as a syrup. Other possible forms of the preparation are suppositories rectal or vaginal use, solutions or suspensions for parenteral use or creams and ointments for external use.

The following examples illustrate the preparation of the compounds according to the invention.
25th

example 1
Preparation of 6-deoxyneomycin base and its sulfate

a) 6-deoxyneomycin in the form of a free base

D 'Streptomyces fradiae ATCC 21401 was left in a flask on the following medium as previously used a pH of 7.2 to 7.3 had been adjusted, grow for 48 hours:

Glucose i g

Caesar Hydroivsat! S.

(NH ₄ I ₂ HPO ₄ 1 g

Mg SO ₄ · 7 H, 0.05 g

Fe SO ₄ · 7 Η, Ο 0.005 g

Cu SO ₄ · 5 H ₂ O 0.005 g

CaCO ₃ 1 g
Water to 100 ecm

This culture was shaken and kept at 28 ° C for the specified time. Then became a 10% inoculum from this culture is added to the following nutrient medium, which was also applied beforehand a pH of 7.2 to 7.3 had been set:

glucose Ig Soybean meal 2.5 g Yeast extract 0.5 g NaCl 0.5 g CaCO ₃ 0.2 g 2,4-dideoxystreptamine dihydrochloride 0.025 g Water to 100 ecm

The cultures were placed in Erlenmeyer flasks at 28 ° to 30 ° C in a rotating shaker incubated with good ventilation.

The formation of 6-deoxyneomycin started after 2 days and was optimal after 5 days.
Control cultures which did not contain 2,4-dideoxystreptamine dihydrochloride did not produce 6-deoxyneomycin.

The culture was then centrifuged and the supernatant liquid poured through a column in which a weakly acidic, carboxyl group (polymethacrylic type) containing cation exchange resin medium Porosity ("Amberlite® IRC-50") was in ammonium form. This measure was carried out twice, to extract the 6-deoxyneomycin and unchanged 2,4-dideoxystreptamine dihydrochloride. the The column was washed out with water and the 6-deoxyneomycin with a 2N ammonium hydroxide solution eluted in water. The eluate was concentrated in vacuo on a rotary evaporator and then placed in a Sephadex® G-10 column. By eluting with a 0.1m ammonium hydroxide solution

Z. / LKJ

became 6-deoxyneomycin in the first fraction and 2,4-dideoxystreptamine dihydrochloride in the subsequent fractions obtain.

In this way, 6-deoxyneomycin was obtained as a free base in an amount equivalent to 83 units of activity (1 unit = 1; ig / cc neomycin). That means that about 22% of the originally used 2,4-Dideoxystrcptamine dihydrochloride were incorporated into the 6-deoxyneomycin thus obtained.

This was the maximum achievable efficiency, and a longer incubation time was the amount of 6-Desoxyneomycin increase nich ^first

In an alternative procedure, the crude 6-deoxyneomycin was chromatographed on Whatman paper by paper No. y MM purified that developed with a 4: 1 solution of methanol and ammonium hyaroxide became. The location of the 6-deoxyneomycin was visualized by first adding 0.25% sodium hypochlorite in water, then absolute ethanol and then 1% soluble starch and 1% potassium iodide in water and air-dried between each application.

The R _r value of 6-deoxyneomycin in this experiment was found to be 0.30.

For comparison purposes, the following R, values were determined using the same solvent systems

R _r value

Neomycin 0.26

2-deoxystreptamine 0.58

2,4-dideoxystreptamine 0.62, _{| (}

b) 6-deoxyneomycin sulfate

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

Mü ^of 6-Desoxyneomycin sulfate = + 43 ° (c = 1.0, water).

Example 2
Separation of 6-deoxyneomycin into 6-deoxyneomycin B and 6-deoxyneomycin C

The 6-deoxyneomycin prepared according to Example 1 was separated into 6-deoxyneomycin B and 6-deoxyneomycin C by paper chromatography, a mixture of tert-butanol, butanone, 6.5N ammonium hydroxide solution and methanol in a ratio of 3 : 16: 6: 1 was used .

6-B and 6-Desoxyneomycin Desoxyneomycin C were determined using the method described above on the paper.

The Rr values of 6-deoxyneomycin B and C were then determined on Whatman chromatography paper 3 MM using a solvent system composed of methanol and ammonium hydroxide in a ratio of 4: 1 and compared with those of the corresponding epimers of neomycin; the following results were obtained:

R _r value

6-deoxyneomycin B 0.29

6-deoxyneomycin C 0.21

Neomycin B 0.25

Neomycin C 0.165

Example 3 Production of 6-deoxyparomomycin and its epimers I and II

D "Streptomyces rimosus, form paromomyoinus, ATCC 21484 was left in a flask for two to three days grow on the following medium previously adjusted to pH 7.5:

Soybean meal 1 g Casein hydrolyzate 0.25 g

CaCO ₃ 0.5 g

Glucose 1 g

NaCl 0.5 g

NH ₄ Cl 0.167 g

Water to 100 ecm

This culture was shaken and kept at 28 ° C. for the specified period of time. Then became a 10% inoculum from this culture was 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 Erlcnmeyer-Kolbcn at 28 ° to 30 ⁰ C on a rotary shaker with good aeration. Then the 6-deoxyparomomycin was recovered and purified according to the method of Example la).

Purification by paper chromatography under the conditions of Example 1 showed that the 6-Des-5 oxyparomomycin had an Rp value of 0.30.

For comparison purposes, it should be mentioned that the R _r value of paromomycin was determined to be 0.27 in an identical chromatography procedure.

On Whatman chromatography paper 3 MM using a solvent system of methanol and ammonium hydroxide in a ratio of 4: 1 were now the R, values of the 6-deoxyparomomycins I and ίο Il determined and compared with those of the corresponding epimers of paromomycin, with the following results received:

R ₁ value

6-deoxyparomomycin I 0.34

6-Dejoxyparomomycin II 0.275

Paromomycin I 0.3

Paromomycin II 0.22

Claims (3)

20 30 40 patent claims: 1. The aminocyclitol glycosides a) 6-deoxy-neomycin B, b) 6-deoxy-neomycin C, c) 6-deoxy-paromomycin I and d) 6-deoxy-paromomycin II the general formula OH OH H with the following assignment and meaning of the substituents R ¹ , R ² and K ³ : • 45 50 R ¹ R ² R ³ a) H CH ₂ NH ₂ NH ₂ b) CH ₂ NH ₂ H NH ₂ O H CH ₂ NH ₂ OH d) CH ₂ NH ₂ H OH 55 60 and their pharmaceutically acceptable acid addition salts. 2. Process for the preparation of the compounds according to claim 1, characterized in that in a nutrient medium which, in addition to a soluble carbohydrate, is a source of assimilable nitrogen and essential mineral salts 2,4-dideoxystreptamine of the formula HO HO NH ₂ NH ₂ or an acid addition salt thereof, either a) D "Streptomyces fradiae ATCC 21401 breeds and 6-deoxy-neomycin B from the culture medium and C wins or b) D ~ Streptomyces rimosus, form paromomycins, ATCC 21484 breeds and extracts 6-deoxy-paromomycin I and II from the culture medium,
optionally in the form of their acid addition salts. 3. Pharmaceutical or veterinary preparations containing at least one compound according to Claim 1 and usual carriers and additives. The invention relates to new aminocyclitol glycosides with pharmacological activity and a method for the preparation of these compounds and pharmaceutical and veterinary medicinal products containing these compounds Preparations. The compounds according to the invention are defined by claim 1 These compounds can also be present as epimers or as mixtures of epimers. Epimers and their mixtures are also encompassed by the present invention. The chemical structures of the compounds of the invention are similar to those of neomycin or paromomycin. For this reason they are used in this description as neomycin or paromomycin derivatives designated. "Neomycin" in this description is a mixture of neomycin B and neomycin C, obtained by culturing Streptomyces fradiae ATCC 21401_. Similarly, "Paromomycin" denotes a mixture of F aromomycin I and Paromomycin II obtained by cultivating Streptomyces rimosus, Form paromomycinus, ATCC 21484. The invention also relates to pharmaceutical or veterinary preparations that contain at least contain one of the compounds according to the invention in conjunction with conventional carriers or additives. It has been found that the aminocyclitol glycosides according to the invention have a valuable antibiotic activity show and are therefore suitable for the treatment of diseases that make the growth pathogenic Bacteria are caused, such as. B. Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Sarcina lutea, Klebsiella edwardsii, Shigella sonnei, Salmonella typhimurium. The compounds according to the invention can prevent the growth of pathogenic bacteria in one of these Bacteria-infected patients can be inhibited if the patient is given at least one of these compounds administered. A few years ago it was observed that certain bacterial strains compared to most commercially available Antibiotics resist; gev; rden are increasing the therapeutic value of these antibiotics when half was lowered; some of the bacteria have become so strong that they do not affect any of the respond to common antibiotics. It is therefore imperative and in the general interest to develop new antibiotics that can destroy such organisms which have become resistant to the known antibiotics. It is known that the inactivation of antibiotics is due to the destruction or alteration of the 1 ~ s. tibiotic molecule is due to enzymes that attack the molecule at certain vulnerable points. In particular, the aminocyclotitol glycoside antibiotics, which are used to an ever increasing extent, are used often inactivated by resistant bacteria. Those of some organisms which normally act on this Antibiotics that respond to resistance may develop through phosphorylating, acylating, or adenylylating Enzymes that 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 It is therefore desirable to manufacture antibiotics that are less non-essential hydroxyl and Have amino groups. From US-PS 36 69 838 is known that of microorganisms that normally antibiotics with a Generate aminocyclitol subunit, e.g. B. neomycin or paromomycin, mutants are produced who lack the ability of aminocyclitol subunit biosynthesis because they use the aminocyclitol pathway is disturbed and therefore no antibiotic can be formed. However, if a suitable aminocyclitol molecule is added to the nutrient medium of these aminocyclitol-negative mutants to, they can make an antibiotic using this molecule. The results published so far, however, allow any prediction as to whether this bio-conversion process will be used a particular diaminocyclitol is incorporated into an antibiotic, seem impossible. In the literature numerous cases are even described in which this process for the production of new Antibiotics was ineffective. The process described above cannot therefore be applied generally to the production of new semi-synthetic or use completely synthetic antibiotics. This fact is z. B. also in "The Journal of Antibiotics", Volume XXVI, No. 10, pages 551-561 (1973), confirmed where experiments with 29 different aminocyclitols are described by the process of the aforementioned US patent should be incorporated into an antibiotic. Of the 29 aminocyclitols tested in this way, only streptamine and 2-EpisUeptamine could be obtained with the help of the Purpose used 2-deoxystreptamine negative (D ") mutants of Streptomyces fradiac, Streptomyces rimosus. Form paromomycinus and Streptomyces kanamyceticus introduced into an antibiotic