CH357833A - Process for the preparation of dihydrostreptomycin - Google Patents

Description

  

      Method for producing dihydrostreptomycin Dihydrostreptomycin is a substance useful for medical treatment because it has a strong and broad antibacterial spectrum like streptomycin and is less toxic and more stable than the latter.

   It is known that dihydrostreptomycin, which is similar to streptomycin in its antibacterial properties, has the advantage over the latter that it is less toxic to the nervous system. Up to now, dihydrostreptomycin has been obtained by hydrogenating the formyl radical of streptomycin and has been marketed as a sulfate or hydrochloride. These salts are colorless or white crystalline or powdery substances.

   They are odorless or almost odorless, taste slightly bitter, and their aqueous solutions are levorotatory; an aqueous solution of the sulfate crystals shows a rotation of [a] 25 = -88 (c = 1). It is known that dihydrostreptomycin has a broad antibacterial spectrum against gram-positive, gramne: gative and acid-fast bacteria.



  Up to now, dihydrostreptomycin has been produced by reducing the S. treptomycin isolated from the culture medium of Streptomyces griseus (see US Pat. No. 2498574). This method requires very pure streptomycin, otherwise a considerable loss in the reduction is inevitable.



  No report has yet been published on the direct production of dihydrostreptomycin by culturing dihyd @ rostreptomycin-producing microorganisms.



  It has now been found that dihydrostreptomycin is obtained in a simple manner while improving the yield and lowering the production costs if a strain of a dihydrostreptomycin-producing microorganism of the genus Streptomyces is cultivated aerobically in an aqueous nutrient medium.



  The dihydrostreptomycin produced in this way can be isolated from the culture medium.



  Any strain of any type belonging to the streptomycetes which provides dihydrostreptomycin can be used. So z. B. a new species called Streptomyces. humidus nov. sp. No. <B> 23572 </B> can be used for this purpose. This species has the characteristic properties listed in the table below.

   (The color names marked with Rdg refer to Ridgways Color Standards and Nomenclature).
EMI0002.0001
  
    <I> Streptomyces <SEP> humidus <SEP> strain <SEP> 23 <SEP> 572 </I>
<tb> culture properties
<tb> medium <SEP> soluble <SEP> remarks
<tb> growth <SEP> air mycelium <SEP> and <SEP> spores <SEP> pigment
<tb> Czapek agar <SEP> colorless <SEP> white <SEP> none <SEP> Plenty of <SEP> with <SEP> small <SEP> moist
<tb> glucose-asparagine- <SEP> colorless <SEP> white <SEP> to <SEP> smoke gray <SEP> none <SEP> black <SEP> spots <SEP> mixed up,
<tb> Agar <SEP> (Rdg <SEP> XLVI)

   <SEP> 21 "-d <SEP> which <SEP> <SEP> gradually <SEP> over
<tb> or <SEP> wine red- <SEP> the <SEP> whole <SEP> surface <SEP> spread reddish yellow <SEP> th. <SEP> back <SEP> creamy reddish (Rd'g <SEP> XL, <SEP> 17 "'- d) <SEP> yellow <SEP> (Rdg <SEP> XXX <SEP> 19" -d) <SEP> or
<tb> yellowish-red-yellow <SEP> (Rd, - <SEP> XXX,
<tb> 19 "-b) <SEP> later <SEP> chamois-leather yellow <SEP> becoming <SEP> (Rd- <SEP> XXX
<tb> 19 "-b).
<tb> Starch agar <SEP> colorless <SEP> white <SEP> to <SEP> pale <SEP> none <SEP> reverse side <SEP> creamy reddish yellow
<tb> Smoke gray <SEP> (Rdg <SEP> XXX <SEP> 19 "-b).
<tb> (Rdg <SEP> XLVI, <SEP> 21 "" - f) <SEP> Weak <SEP> hydrolysis.
<tb> Calcium Maiat Agar <SEP> colorless, <SEP> poor, <SEP> white <SEP> none
<tb> later <SEP> red-yellow <SEP> yellow <SEP> becoming
<tb> (Rdg <SEP> IV <SEP> 19-d)

  
<tb> glycerine nitrate <SEP> agar <SEP> colorless <SEP> poor, <SEP> white <SEP> none
<tb> Dextrose Nitrate Agar <SEP> colorless <SEP> poor, <SEP> white <SEP> none
<tb> Bouillon Agar <SEP> colorless <SEP> none <SEP> none
<tb> Gelatine <SEP> colorless <SEP> none <SEP> none <SEP> moderate <SEP> liquefaction.
<tb> Potatoes <SEP> colorless <SEP> white <SEP> to <SEP> smoke gray <SEP> none <SEP> damp <SEP> black <SEP> spots
<tb> (Rdg <SEP> XLVI, <SEP> 21 <B> 1 "</B> -d) <SEP> observed.
<tb> Carrots <SEP> colorless <SEP> white <SEP> to <SEP> smoke gray <SEP> none
<tb> (Rd, - <SEP> XLVI, <SEP> 21 <B> 1 "</B> -d)
<tb> Yeast extract agar <SEP> colorless <SEP> white <SEP> to <SEP> light <SEP> none <SEP> partially <SEP> moist <SEP> becoming.
<tb> Yellowish gray
<tb> (Rd <B> g </B> <SEP> XLVI, <SEP> 17 "" - b)

  
<tb> Whole <SEP> egg <SEP> colorless <SEP> white <SEP> none <SEP> weak <SEP> peptonization.
<tb> milk <SEP> colorless <SEP> white <SEP> none
<tb> Glycerin-Asparaginat- <SEP> colorless <SEP> white <SEP> to <SEP> smoke gray <SEP> none
<tb> Agar <SEP> (Rdg <SEP> XLVI, <SEP> 21 "" - d)
<tb> Peptone Nitrate Broth <SEP> colorless <SEP> Nitrate reduction.
<tb> The <SEP> aerial mycelium <SEP> of this <SEP> strain <SEP> shows <SEP> spirals <SEP> of <SEP> 0.8-12 <SEP>, u, <SEP> and <SEP> oval <SEP> Conidia <SEP> from <SEP> 1-1,5 <SEP> 1u <SEP> X <SEP> 1,5-2p ..

         The carbon consumption, measured by the Pridham method, is as follows:
EMI0002.0004
  
    d (+) - Xylose <SEP> ++ <SEP> d (+) - Raffinose <SEP> - <SEP> Salicin <SEP> ++
<tb> 1 (+) - Arabinose <SEP> + <SEP> + <SEP> + <SEP> Inulin <SEP> - <SEP> Na-acetate <SEP> 1 (+)

  -Rhamnose <SEP> + <SEP> + <SEP> + <SEP> d-mannitol <SEP> + <SEP> + <SEP> + <SEP> sodium citrate <SEP>
<tb> d-fructose <SEP> +++ <SEP> d-sorbitol <SEP> - <SEP> Na-succinate <SEP> +
<tb> d-galactose <SEP> +++ <SEP> dulcit <SEP> sucrose <SEP> - <SEP> dl-inositol <SEP> - <SEP> control <SEP> maltose <SEP> + <SEP> + <SEP> +
<tb> lactose <SEP> ++
<tb> - <SEP> = <SEP> no <SEP> growth <SEP> +++ <SEP> = <SEP> good <SEP> growth <SEP> ++ <SEP> = <SEP> acceptable <SEP> growth
<tb> + <SEP> = <SEP> little <SEP> growth <SEP> <SEP> = <SEP> questionable <SEP> growth The new species characterized with the above-mentioned properties was discovered by Nakazawa and Shibata Streptomyces humidus nov. <RTI

   ID = "0003.0006"> sp. called. In the following table, a comparison of this species with Streptomyces hygroscopicus, which is similar in characteristics to the aforementioned species, is made:

    
EMI0003.0010
  
    Medium <SEP> Str. <SEP> humidus <SEP> Str. <SEP> hygroscopicus
<tb> nutrient agar <SEP> colorless <SEP> growth <SEP> cream <SEP> colored, <SEP> later <SEP> yellowish
<tb> with <SEP> yellowish <SEP> brown <SEP> back
<tb> glucose-asparagine <SEP> back side <SEP> cream-red-yellow <SEP> or <SEP> yellowish- <SEP> growth <SEP> cream <SEP> colored <SEP> to <SEP> straw-yellow,
<tb> red-yellow, <SEP> later <SEP> chamois-leather-colored <SEP> becomes <SEP> later <SEP> weak <SEP> chrome-yellow <SEP> to, <SEP> brownish dend, <SEP> aerial mycelium <SEP> white <SEP> to <SEP> smoke gray <SEP> or <SEP> orange, <SEP> aerial mycelium <SEP> white <SEP> to <SEP> light yellowish wine yellow- <SEP> reddish-yellow <SEP> gray
<tb> potato <SEP> growth <SEP> colorless <SEP> aerial mycelium <SEP> white <SEP> growth <SEP> cream <SEP> colored,

   <SEP> later <SEP> yellowish
<tb> to <SEP> smoke gray <SEP> gray <SEP> to <SEP>; slightly <SEP> brownish. <SEP> No <SEP> air mycelium <SEP> or <SEP> trace <SEP> of <SEP> white Streptomyces hygroscopicus produces hygroscopin [see J. Agr. Chem. Soc. (Japan) 28, 296 (1954) and Antibiotics und Chemotherapie 3, 1268 1953)], carbomycin [see Antibiotics and Chemotherapie 3, 899 (1953)], fear mycin [see J.

       Antibiotics, Japan 7, 113 (1954)] and hygromycin [see Antibiotics and Chemotherapy 3, <B> 1 </B> 268 (<B> 1 </B> 953)].



       Streptomyces humidus, on the other hand, produces dihydro-streptomycin.



  The strain belonging to Streptomyces humidus mentioned above is hygroscopic, but some strains belonging to the same species do not have this property. Likewise, some do not form aerial mycelium and others produce pigment.



       Dihydrostreptomycin-producing strains of other species can also be used for the same purpose if their properties are Streptomyces humidus nov. sp. are not very similar.



  As is observed in microorganisms, especially in Streptorayceten, their behavior changes slightly spontaneously in the culture medium or they are artificially changed. This is why the identification of a species is so difficult that any mere description of the properties of a species has no definite meaning for the identification of that species. Mutants of dihydrostreptomycin-producing species, such as those described e.g. B. by treatment with X-rays, ultraviolet rays and Che mikalien are used.



  Characteristic properties of some mutants produced from Streptomyces humidus by the usual mutation agents, such as ultraviolet radiation, with the separation of individual spores, are shown in the following table:

    
EMI0003.0050
  
    <I> Mutants <SEP> of <SEP> Streptomyces <SEP> humidus <SEP> nov. <SEP> sp. <SEP> No. <SEP> 23 <SEP> 572 </I>
<tb> Medium <SEP> 23 <SEP> 572 <SEP> G <SEP> I <SEP> 23 <SEP> 572 <SEP> Y <SEP> 23 <SEP> 572 <SEP> W
<tb> glucose asparagine agar <SEP> tea green <SEP> light orange yellow <SEP> white
<tb> Starch agar <SEP> white <SEP> light orange yellow <SEP> white
<tb> Dextrose Nitrate <SEP> Agar <SEP> white <SEP> cream-colored <SEP> white
<tb> Potato <SEP> tea green <SEP> capuchin-reddish yellow
<tb> later <SEP> orange-yellow
<tb> Glycerine asparaginate agar <SEP> white <SEP> light <SEP> orange yellow <SEP> white Most of the strains producing dihydrostreptomycin do not resemble Streptomyces griseus in their properties and are both against

      Dihydro-streptomycin and streptomycin resistant. In contrast to the antibiotics obtained by reducing streptomycin, the antibiotics obtained by cultivating dihydrostreptomycin generating microorganisms are generally negative for the maltol reaction. The maltol reaction is a color reaction of streptomycin with phenol reagent,

      which is generated by hydrolysis of streptomycin by means of alkali [Schenk et al., J. Amer. Chem. Soc. 67, 2277 (1945), Boxer, J. Biol. Chem. 169, 153 (1947), Eiseman et al., Anal. Chem. 21, 1507 (1949)]. These properties can advantageously be used to separate different strains. It can e.g.

   B. the desired strains can be selectively separated by the dilution method or by cultivation in a medium containing dihydrostreptomycin.



  As a carbon source, for. B. starch, lactose, sucrose, dextrin, glycerine and maltose are needed. As a nitrogen source, organic or inorganic nitrogen-containing substances can be used, such as. B. Soybean flour, meat extract, peptones, peanut powder; Casein, amino acids, yeast, bran, corn mash, cottonseed powder, nitrates, urea or ammonium compounds.

   Small amounts of inorganic salts and growth-promoting substances can also be added to the nutrient solution. Other suitable sources of growth are mycelia of a strain belonging to Penicillium or e.g. B. its culture medium. Any nutrient medium suitable for the cultivation of Streptomyces griseus serves the same purpose. A suitable parent substance can be added if necessary.



  The culture medium can be solid or liquid; submerged and aerobic cultivation is most advantageous for industrial purposes.



  When Streptomyces humidus is used as the dihydro-streptomycin-producing microorganism and the cultivation is carried out under submerged aerobic conditions, it can preferably be carried out at a temperature of about 24 to 30 ° C. for a period of 3 to 8 days. But the temperature and the time period must be adapted to the other culture conditions. When using other dihydrostreptomycin-producing microorganisms, the most suitable conditions for their cultivation are selected in each case.



  The dihydrostreptomycin produced in this way can be isolated in optimum purity from a nutrient medium containing dihydrostreptomycin with the aid of various physical or chemical methods. A high degree of purity for the intended use is achieved here.



  To isolate the dihydrostreptomycin from the nutrient medium and separate from the impurities, you can z. For example, use can be made of the differences between dihydrostreptomycin or its salts and the impurities in terms of adsorbability, solubility, the distribution coefficient between two solvents and the strength with which ions are bound.

    In the case of cultivation in liquid medium, the antibiotic can be concentrated in the culture medium up to a concentration of several thousand micrograms per em3; however, the dihydrostreptomycin can be isolated from a solution containing only 10 μ / cm 3.



  In addition to dihydrostreptomycin, the substance obtained raw from the culture medium naturally contains various impurities such as carbohydrates, protein substances, salts, animal and vegetable substances, mycelia from microorganisms and their conversion products. Each of these impurities contains z. B. the following substances: Carbohydrates: starch, sugar, water-soluble carbohydrate.



  Protein substances: amino acids, casein, meat extract, protein, peptones.



  Animal and vegetable substances: protein substances, fats, fatty acids, lipoids, cellulose. Salts: ammonium salts and amines, salts of alkali metals, of calcium, magnesium, iron, manganese, copper, zinc. Transformation products of microorganisms: enzymes, protein substances, sugars, polyhydric alcohols, fatty acids.



  In addition to these substances, compounds such as urea can be obtained by synthesis.



  The composition of the impurities is very complex, but this varies in quality and quantity depending on the extent and type of raw materials used. The nutrient medium can also contain other impurities such as pH regulators, precipitants, adsorbents.



  The dihydrostreptomycin can be separated from the mixture from the impurities mentioned in various ways.



  One of these agents makes use of the difference between dihydrostreptomycin and the impurities in terms of solubility. In the case of liquid culture, a dihydrostreptomycin solution is obtained by filtering the fermentation mixture because it is preferably in the liquid part. But a smaller proportion of the active compound is found in the solid components and can be collected from it by extraction with hot water.

   In this case, impurities in the protein series can be eliminated by adjusting the pH to the isoelectric point of the protein or by adding substances which precipitate the impurities before, after or with the simultaneous removal of the mycelium. If the pH of the fermentation mixture is high, the addition of an acidic substance is appropriate to make the protein easier to precipitate. If the concentration of dihydrostreptomycin in the solution is sufficiently high, the antibiotic can be precipitated by simply adding a suitable precipitant.



       If the concentration of the dihydrostreptomycin is not sufficient, part of the solvent will be evaporated. As a precipitant z. B. phosphotungstic acid, alkyl or aralkylsulfonic acids, acidic azo compounds and polyhalophenol compounds are used. All of these form salts with dihydrostreptomycin.

   Some other compounds can be used and most of them are acidic compounds with carboxyl groups. In some cases the salts of dihydrostreptomycin so formed can be double or complex salts. Also, as will be described later, ion exchangers can be used as a kind of precipitant. In order to accelerate the precipitation of the active substance or the impurities, one can also salt out, if necessary.



  If solid cultures are used, the solid material is extracted with water, an aqueous solvent or another suitable solvent to obtain a solution of dihydro-streptomycin. If the fermentation mixture has previously been converted into a solid substance, this can be worked up as described above; in this case, however, the extraction is carried out at a suitable pH.



  Another means for separating the dihydrostreptomycin consists in making use of the difference between the antibiotic active compound and the impurities in terms of adsorbability. Dihydro- streptomycin is z in the culture filtrate. B. adsorbed by adding a suitable adsorbent. If necessary, use can be made of adsorption chromatography. As an adsorbent z. B.

   Activated charcoal, alum earth and silica gel are used. When the adsorbent is removed, most of the impurities remain in the liquid part. The adsorbent is then eluted with a suitable solvent, the non-elutable impurities remaining in the adsorbent. The elution is generally carried out with an acidic solvent. Chromatography results in a more precise separation, but this method requires more complicated procedures.



  Differences between the antibiotic active compound and the impurities in terms of the partition coefficient between two solvents can also be used for the separation. As the solvent, water or an aqueous solvent and an organic solvent immiscible with the latter can be used. In this case the active compound tends to disperse in the aqueous solvent when the solvents are strongly acidic. If, on the other hand, the solvents are neutral or basic and a higher fatty acid, picric acid and the like are also present, the active compound is transferred to the organic solvent layer.

   Accordingly, it is possible to take up the active compound in the material in an organic solvent and then transfer it to water or an aqueous solvent. As organic solvents, for. B. butanols, pentanols, phenols, lower fatty acid esters, methyl isobutyl ketone, diethyl ether and halogenated hydrocarbons, such as chloroform, carbon tetrachloride, are used.

   This method can be used in the form of a partition chromatography or a countercurrent partition method. The separation can also be carried out in such a way that use is made of the differences between the antibiotically active compound and the impurities with regard to the strength of the ion-binding capacity. This is z. B. achieved by bringing a solution of the active compound with an ion exchanger in contact.

   In this case, an anion exchanger adsorbs anionic impurities and leaves the active compound in the solution, while a cation exchanger adsorbs the antibiotic active compound. In the latter case, cationic impurities are also adsorbed, but the latter adsorption can, if a suitable ion exchanger is used,

       kept as small as possible. The sulfonic acid type resins adsorb e.g. B. easily the impurities, but the carboxylic acid type resins have little tendency here for. The ion exchangers can be added using the mixing method; the use of a column for the resin is a suitable method in some cases. This method can be used for a dilute solution of the antibiotic active compound as for the culture filtrate.



  Dialysis can also be used to remove the high molecular weight contaminants, but the process is complicated.



  The above-mentioned agents can be used separately or in combination, depending on the quality of the material and the intended use of the product. One can also proceed in such a way that one z. B. the culture medium is initially filtered to remove the test components or the substances precipitated after cultivation ent.

   The antibiotic active compound in the filtrate is adsorbed on a cation exchanger and then eluted with an acidic solvent. In this way, a concentrated solution of the active compound is achieved which contains only a small amount of impurities. If necessary, the solution is further concentrated and subjected to adsorption chromatography in order to remove almost all impurities. The antibiotic can be precipitated from the concentrated solution with a suitable agent.

   The precipitate is then dissolved in a suitable organic solvent and an acid added, whereupon the corresponding acidic salt of dihydrostreptomycin is obtained. When the product is recrystallized, the acid salt is obtained in an almost pure state.



       All work processes such as filtering, washing, heating, cooling, mixing, distilling, evaporating and drying can be carried out one after the other or at the same time.



  In order to prevent the decomposition of the antibiotic active compound. It is advisable to work under conditions that are not extremely acidic or extremely alkaline. You can, of course, heat it if necessary; but it is preferable not to heat too long. In comparison with other antibiotics, dihydro-streptomycin is relatively stable in the culture medium, so that all manipulations can be carried out without difficulty.



  The dihydrostreptomycin thus obtained was identified on the basis of the following data: <I> A. Physical and chemical properties 1. The infrared absorption spectrum was determined from the following derivatives of both dihydrostreptomycin available in accordance with the invention (hereinafter referred to as antibiotic 21 572) and commercial dihydrostreptomycin. I.

   Sulfate, II. Streptidine picrate, <B> 111. </B> α-methyl-pentaacetyldihydrostreptobio @ aminide. The IR spectra of the derivatives of dihydrostreptomycin obtained according to the invention are identical to the IR spectra of the corresponding derivatives of dihydrostreptomycin commercially available.



  All spectra are measured in nujol paraffin oil with a sodium chloride prism.



  2. The ultraviolet spectra of the sulfate of antibiotic 23 572 and those of a commercial sample of dihydrostreptomycin sulfate agree well, no specific adsorption was observed.



  3. The analytical values of the sulfate of the antibiotic 23 572 agree well with the theoretical values of the dihydrostreptomycin sulfate. The values for dihydrostreptomycin sulfate (C21H41012N7) 2-3 H2S04) are: Calculated: C 34.42%; H 6.0511 / G; N 13.3804; S 6,561 / 0 Found:

         C 34.45 "/ o; H 6.4211 / o; N 12.981 / o; S 6.43% 34.110 / 0; 6.54 0/0 4. The sulfate of the antibiotic 23 572 shows a rotation of (a ) 25 - 87.4 (C = 1, H, 0), while the dihydrostreptomycin sulfate shows a rotation of (a) 17 - 86.0.



  The specific rotation of dihydrostreptomycin was determined by I. A. Solomons et al. in Science 109, 515 (1949) with (a) D-88 and by F. J. Wolf et al. in J. Am. Chem. Soc. 68, 2163 (1946) given (a) D - 88.5.



  5. The sulfate of antibiotic 23 572 melts with decomposition and blackening at 250 to 255 C, the dihydrostreptomycin sulfate also melts with decomposition and black coloring at 250 to 255 C.



  6. Both the antibiotic 23 572 and the dihydrostreptomycin behave negatively towards the maltol reaction, streptomycin reacts against it.



  7. The antibiotic <B> 23572 </B> the dihydro-, streptomycin and streptomycin behave positively towards the Sakabguschi reaction.



  B. Solutions of the sulfate from the antibiotic <B> 23572 </B> of the dihydrate, rostreptomycin sulfate and streptomycin sulfate in 0.1N NaOH in a concentration of 5 mg / cm3 were left to stand at room temperature for 24 and 48 hours and the antibacterial activity of each antibiotic determined by the dilution method.

   The following values resulted:
EMI0006.0073
  
    immediately <SEP> after <SEP> after <SEP> 24 <SEP> hours <SEP> after <SEP> 48 <SEP> hours
<tb> resolution
<tb> Sulphate <SEP> of the <SEP> antibiotic <SEP> 23 <SEP> 572 <SEP> <B> 10000 </B> <SEP> E / cm3 <SEP>> <SEP> <B> 10000 </ B> <SEP> E / cm3 <SEP> 15 <SEP> 000 <SEP> E / cm3
<tb> Dihydrostreptomycin sulfate <SEP> 15 <SEP> 000 <SEP> E / cm3 <SEP>> <SEP> 10000 <SEP> E / cm3 <SEP> 15 <SEP> 000 <SEP> E / em3
<tb> Streptomycin sulfate <SEP> 10000 <SEP> E / em3 <SEP> <<SEP> 100 <SEP> E / cm3 <SEP> 75 <SEP> E / cm3 Under alkaline conditions, the sulfate of the antibiotic is <B> 23572 as stable as that. Dihydrostreptomycin sulfate.



  9. 30 mg / cm3 of semicarbazide was added to each of the aqueous solutions of the sulfate of the antibiotic <B> 23572, </B> of the dihydrostreptomycin sulfate and the streptomycin sulfate and, after standing for 4 hours at room temperature, the antibacterial activity of each antibiotic against B. subtilis was determined by Dilution method determined.

   The following results were achieved:
EMI0006.0082
  
    No <SEP> addition <SEP> from <SEP> 4 <SEP> hours <SEP> after <SEP> addition
<tb> Semicarbazide <SEP> of the <SEP> semicarbazide
<tb> Sulphate <SEP> of the <SEP> antibiotic <SEP> 23 <SEP> 572 <SEP> <B> 35000 </B> <SEP> E / cm3 <SEP> 35 <SEP> 000 <SEP> E / em3
<tb> (10 <SEP> mg / cm3)
<tb> Dihydrostreptomycin sulfate <SEP> 35 <SEP> 000 <SEP> U / cm3 <SEP> <B> 50000 </B> <SEP> U / cm3
<tb> (10 <SEP> mg / cm3)
<tb> Streptomycin sulfate <SEP> (10 <SEP> mg / cm3) <SEP> 35 <SEP> 000 <SEP> E / cm3 <SEP> <<SEP> 5 <SEP> 000 <SEP> E / cm3 A solution of 30 mg / cm3 semicarbazide shows an effectiveness of 75 U / cm3.



  10.1 mg / cm3 cysteine was added to each of the aqueous solutions of the sulfate of the antibiotic 23 572, the dihydrostreptomycin sulfate and the streptomycin sulfate and, after standing for 4 hours at room temperature, the antibacterial effectiveness of each antibiotic against B. subtilis was determined by the dilution method. The following results were achieved:

    
EMI0007.0015
  
    No <SEP> additional <SEP> from <SEP> 4 <SEP> hours <SEP> after <SEP> addition
<tb> Cysteine <SEP> of the <SEP> cysteine
<tb> Sulphate <SEP> of the <SEP> antibiotic <SEP> 23 <SEP> 572 <SEP> 15 <SEP> 000 <SEP> E / cm3 <SEP> 15 <SEP> 000 <SEP> E / cm3
<tb> (5 <SEP> mg / cm3)
<tb> Dihydrostreptomycin sulfate <SEP> 15 <SEP> 000 <SEP> U / cm3 <SEP> 15 <SEP> 000 <SEP> U / cm3
<tb> (5 <SEP> mg / cm3)
<tb> Streptomyein sulfate <SEP> (5 <SEP> mg / cm3) <SEP> 15 <SEP> 000 <SEP> U / cm3 <SEP> 7 <SEP> 500 <SEP> U / cm3
<tb> A <SEP> solution <SEP> of <SEP> 1 <SEP> mg / cm3 <SEP> cysteine <SEP> showed <SEP> an <SEP> effectiveness <SEP> of <SEP> 100 <SEP> E / cms.

         The sulfate of the antibiotic 23 572 does not lose any of its effectiveness through the addition of semicarbazide or cysteine; it behaves in the same way as dihydrostreptomycin sulfate. 11.

   The qualitative reaction to the formyl residue is negative, as can be seen from the following table:
EMI0007.0021
  
    Fehling's <SEP> phenol conc. <SEP> Silver mirror reagent <SEP> <B> H @ so ° </B> <SEP> reaction
<tb> Sulphate <SEP> of the <SEP> antibiotic <SEP> 23 <SEP> 572 <SEP> negative <SEP> no <SEP> discoloration <SEP> brown, <SEP> clear
<tb> Dihydrostreptomycin sulfate <SEP> negative <SEP> no <SEP> discoloration <SEP> brown, <SEP> clear
<tb> streptomycin sulfate <SEP> brown <SEP> deep <SEP> brown <SEP> color <SEP> black-brown
<tb> Precipitation <SEP> Precipitation 12. Streptidine picrate was obtained from the antibiotic 23 572 according to the method of F. H. Stodola et a1. [J.

   At the. Chem. Soc. 73, 2290 (1951)] was obtained in the following way: 2 g of the hydrochloride from the anti-biotic <B> 23572 </B> were dissolved in <B> 100 </B> cm3 of anhydrous methanol which contained 10/9 hydrogen chloride. After standing for 48 hours at room temperature, 200 cm3 of ether were added to the solution with stirring and the white crystals (streptidine hydrochloride) formed were separated off by centrifugation. The crystals were dissolved in 20 cm3 of water and a saturated picric acid solution was added.

   After standing for several hours, the precipitated crystals were separated off and recrystallized from water. 400 mg of streptidine picrate were obtained as yellowish needles with a melting point of 271-273 C.

   Analysis for C8H1 $ O4N6-2 C6HH3N307 showed: Calculated: C, 33,340 / 9; H 3.360 / 0; N 23.33% Found: C 33.46%; H 3.440; 9;

          N 23.49% The infrared absorption spectrum of Picrates agreed with that of Picrates made from dihydrostreptomycin as described under 1.

    The liquid above the above streptidine hydrochloride was neutralized with a solution of sodium hydroxide in methyl alcohol, the sodium chloride formed was separated off by centrifugation and the solvent was distilled off under reduced pressure. A white powder remained. This residue was dissolved in 20 cm 3 of pyridine and 7 cm 3 of acid anhydride was added.

   After standing overnight, water was added to the reaction mixture and the solvent was distilled off. The residue was recrystallized from methanol. It was obtained in the form of white needles with a melting point of 194.5 C α-methyl-pentaacetyldihydrostreptobiosaminide. The yield was 700 mg.

   The analysis for C13H19NO8 (CH.C0) 5 (OCH3) resulted in: Calculated: C 51.15% -; H 6.62%; N 2.490 / 0 Found: C 51.09% -; H 6, 13-0 / 9;

          N 2.40%. The rotation (a) = -120.0 (c = 1%, D in chloroform).



  The infrared absorption spectrum of the compound obtained was in good agreement with the α-methyl-pentaacetyldihydrostreptobiosaminide obtained from commercial dihydrostreptomycin, as already described under 1.



  The ss-methyl-pentaacetyldihydrostreptobiosaminide was obtained from antibiotic 23 572 by the method of N. G. Brink [J. At the. Chem. Soc. <B> 68.2557 </B> (1946)] in the following way:

   10 g of the hydrochloride of the antibiotic 23 572 were dissolved in 500 cm3 of methanol containing 1% hydrogen chloride. After the solution had stood at room temperature overnight, the solvent was distilled off under reduced pressure. The residue was dissolved in 750 cm3 of methanol and 450 cm3 of ether was added.

   The solution was passed through a column which was charged with 210 g of acid-washed alum earth which had been impregnated with a methanol-ether mixture in a ratio of 2: 1. The column was washed with 1000 cm3 of a methanol-ether mixture in the ratio <B> 3: 2 </B>. The flowed out solution was evaporated to dryness under reduced pressure.

   The residue was acetylated by allowing it to stand overnight with 10 cm3 of acetic anhydride and 10 cm3 of pyridine at room temperature. Water was then added and the solution was evaporated to dryness in vacuo. The residue was dissolved in chloroform and the chloroform solution was washed with water, dilute sulfuric acid and finally with water.

   After the chloroform had distilled off, the brownish solid residue was boiled with 100 cm3 ether for 2 minutes and the ether solution was separated off by decanting. The same procedure was repeated again. The ether-insoluble fraction was crystallized from methanol and yields a-methyl-pentaacetyldihydrostreptobiosaminide with a melting point of 194.5 C.

   The ether solution was concentrated to approximately 50 cm3 and petroleum ether was added, giving white crystals. Crystallization from methanol yielded approximately 100 mg of ss-methylpentaacetyldihydrostreptobiosaminide with a melting point of 155-156 C. No melting point depression was observed when it was mixed with the ss-isomer prepared from dihydrostreptomycin by the method just given was.

   The analysis gave for C "H19NOs (CH.CO) 5 (OCH3): C 50.710 / 0; H 6.620 / a; N 2.6511 / o. The calculated values are: C 51.15%; H 6.620 / 0; N 2.4911 / o. (A) = -36. (C = 1 / chloroform).

    D The infrared spectrum of the product obtained was in good agreement with that of the β-methyl-pentaacetyldihydrobiosaminide obtained from dihydro-streptomycin.



  A solution of 260 mg of a-methyl-pentaacetyl-dihydrostreptobiosaminide, which was obtained from the antibiotic <B> 23572 </B>, in 20 cm3 of 10 N / od hydrochloric acid was refluxed for 3 hours. After cooling, the brownish solution was decolorized with charcoal and evaporated to dryness in vacuo.

   The residue was acetylated at room temperature with 1 cm 3 of acetic anhydride and 3 cm 3 of pyridine. After adding water, the solution was evaporated to dryness. The residue was dissolved in 50 cm3 of a benzene-petroleum ether mixture in a ratio of 7: 3.

   The solution was passed through a column charged with 4 g of an acid washed alum earth impregnated with petroleum ether, the acetylation product being adsorbed on the acid washed alum earth.

    The column was treated with 100 cm3 of a 7: 3 benzene / chloroform mixture to elute the substance in question. The effluan was concentrated to approximately 10 cm3 and 30 cm3 of ether were added, which resulted in crystals - Recrystallization from chloroform-ether gave 25 mg of needles of pentaacetyl-N-methyl-L-glucosamine with a melting point of 158-159 C.

      Analysis showed: C 50.71%; H 6.14 11 / a; N 3.25 0/0. The calculated values for C17H "N010 are:

         C 50.62%; H 6.250 / 0; N 3.4711 / o. (a) D = -102 (c = 0.7% in chloroform). The infrared absorption spectrum of the product obtained was in complete agreement with that of pentaacetyl-N-methyl-L-glucosamine,

      which was obtained from dihydrostreptomycin by the method described. No melting point depression was observed when mixed with pentaacetyl-N-methyl-L-glucosamine obtained from dihydro-streptomycin.



  13. Antibiotic 23 572 was hydrolyzed with alkali under conditions similar to those used for the hydrolysis of hydroxystreptomycin and described by F. H. Stodola et al. [J. At the. Chem. Soc. 73, 2290 (1951)]. Accordingly, a solution of 2 g of the hydrochloride of the antibiotic 23 572 in 40 cm 3 of 11 NaOH was heated to 100 ° C. for 3 hours. The reaction mixture was acidified with hydrochloric acid and worked up.

   However, neither a substance with a positive reaction to maltot nor a substance which can be extracted under acidic conditions could be found, i.e. the antibiotic 23 572 behaves differently than streptomycin and hydroxystreptomycin in this test. The same test gave the same result with dihydrostreptomycin.



  14. A warm solution of methyl orange was added to an aqueous solution of 50,000 y / em3 of the antibiotic 23 572 until no more precipitate was formed. The precipitate was filtered off and recrystallized from methanol-water (1: 3). The helianthat was obtained in the form of flakes with a melting point of 222-225 C (potency: 340 streptomycin units per mg).

   The analysis for dihydrostreptomycin heliantate (C03H $ 00., 1N10s3) gave: Calculated: C 50.46%; H 5.7911 / o; N 14.95%; S 6.4011 / o Found:

         C 50.19%; H 5.93%; N 14.93%; S 6.801 / 0 50.84114; 5.95% 15. The paper distribution chromatography of streptomycin and dihydrostreptomycin was published by F. H. Stodola et al. [J. At the. Soc. 73, 2290 (1951)].

   Butanol, saturated with water, with 2 / o p-toluenesulfonic acid and 2 / o piperidine was used as solvent. Both compounds were differentiated with the help of the bioautogram.



  Paper chromatography was applied to the above two compounds and the antibiotic 23 572 by the same method. The bio-autographs were tested with B. subtilis as test microorganisms. It was found that the antibiotic showed a growth-preventing zone in the same place as the dihydro-streptomycin.



  16. The crystallographic constants of the sulphate of the antibiotic 23 572 were compared with those of F. J. Wolf et al. [Science 109, 515 (1949)] reported dihydrostreptomycin sulfates.

    
EMI0009.0025
  
    Sulfate <SEP> from <SEP> dihydrostreptomycin sulfate
<tb> antibiotic <SEP> 23 <SEP> 572
<tb> 1.545 <SEP> <SEP> 0.002 <SEP> 1.552 <SEP> <SEP> 0.002
<tb> 1.556 <SEP> <SEP> 0.005 <SEP> 1.558 <SEP> <SEP> 0.004
<tb> y <SEP> 1.564 <SEP> <SEP> 0.002 <SEP> 1.566 <SEP> <SEP> 0.002
<tb> <I> B.

   <SEP> Chemotherapeutic <SEP> effectiveness </I> The growth-inhibiting effect of the antibiotic on streptomycin-sensitive strains of human tubercle bacilli (H 37 Rv) was almost of the same order of magnitude as that of dihydrostreptomycin; it is inhibited in vitro of growth in a concentration of 1-2 y per ml.

   However, there is no inhibition of growth on the streptomycin-resistant strains of the human tubercle bacillus. The antibiotic effect of the antibiotic 23 572 on tubercle bacilli in vitro was considerably high.



  <I> Toxicity </I> The LD.ö values in mice (CFi) of antibiotic sulfate and dihydrostreptomycin sulfate were calculated using the Litchfield and Wilcoxon method.

    
EMI0009.0047
  
    Application <SEP> sulfate <SEP> of <SEP> antibiotic <SEP> 23 <SEP> 572 <SEP> dihydrostreptomycin sulfate
<tb> LDso <SEP> LD6o
<tb> Intravenous <SEP> injection <SEP> 250 <SEP> (219-285) <SEP> mg / kg <SEP> 240 <SEP> (215-268) <SEP> mg / kg
<tb> Subcutaneous <SEP> injection <SEP> 2000 <SEP> (1710-2340) <SEP> mg / kg <SEP> 1800 <SEP> (1450 = 2250) <SEP> mg / kg
<tb> Intraperitoneal <SEP> injection <SEP> <B> 1900 </B> <SEP> (1500-2390) <SEP> mg / kg <SEP> 1700 <SEP> (1430-2020) <SEP> mg / kg
<tb> <I> C. <SEP> Antibacterial <SEP> Spectrum </I> The sulfate of the antibiotic 23 572 was compared with the dihydrostreptomycin sulfate on the market to find out which amount by weight resulted in complete inhibition of the microorganisms used for the test.



  The following results were achieved:
EMI0009.0051
  
    <I> Antibacterial <SEP> spectrum </I>
<tb> Sulfate <SEP> from <SEP> dihydrostreptomycin sulfate
<tb> Microorganisms <SEP> Antibiotic <SEP> 23 <SEP> 572 <SEP> from <SEP> trade
<tb> mcg / cm3 <SEP> mcg / cm3
<tb> Staphylococcus <SEP> aureus <SEP> 1 <SEP> 1
<tb> Terajima
<tb> Bacillus <SEP> subtilis <SEP> (PCI-219) <SEP> 0.5 <SEP> 0.5
<tb> Salmonella <SEP> typhosa <SEP> 64 <SEP> 64
<tb> Shigella <SEP> dysenteriae <SEP> 1 <SEP> 1
<tb> Vibrio <SEP> cholerae <SEP> 8 <SEP> 8
<tb> Protews <SEP> vulgaris <SEP> 8 <SEP> 8
<tb> Escherichia <SEP> coli <SEP> 4 <SEP> 4
EMI0010.0001
  
    <I> Antibacterial <SEP> spectrum </I> <SEP> (continued)

  
<tb> Sulfate <SEP> from <SEP> dihydrostreptomycin sulfate
<tb> Microorganisms <SEP> Antibiotic <SEP> 23 <SEP> 572 <SEP> from <SEP> trade
<tb> mcg / cm3 <SEP> mcg / cm3
<tb> Pseudomonas <SEP> aeruginosa <SEP> 32 <SEP> 32
<tb> Aspergillus <SEP> niger <SEP>> <SEP> 100 <SEP>> <SEP> 100
<tb> Penicillium <SEP> notatum <SEP>> <SEP> 100 <SEP>> <SEP> 100
<tb> Saccharomyces <SEP> cerevisiae <SEP>> <SEP> 100 <SEP>> <SEP> 100
<tb> Candida <SEP> albicans <SEP>> <SEP> 100 <SEP>> <SEP> 100
<tb> Mycobacterium <SEP> 607 <SEP> 2.0 <SEP> 2.0
<tb> Mycobacterium <SEP> avium <SEP> 1.0 <SEP> 1.0 In the following examples, the effect of antibiotics was determined using the cylinder-plate method,

   where Bacillus subtilis (PCI 219) was used as the testing organism, with the exception of cases where the testing method was specifically noted. <I> Example 1 </I>
EMI0010.0008
  
    Glucose <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 2.0%
<tb> meat extract <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.6%
<tb> peptone <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> <B> 1.0 () / o </B>
<tb> Table salt <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.6%
<tb> calcium carbonate <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.6'0 / 0 500 liters of a nutrient solution with the above components were sterilized by heating in a fermentation vessel.

   A strain belonging to Streptomyces humidus was inoculated and aerobically cultivated for 96 hours at 27-28 ° C. with stirring. The culture medium showed the following antibacterial effect:

    500 mcg / cm3 against B. subtilis. <I> Example 2 </I>
EMI0010.0016
  
    Corn mash <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.01 / o
<tb> strength <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.011 / o
<tb> peptone <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.51 / o
<tb> Potassium dihydrogen phosphate <SEP>. <SEP> 0.1%
<tb> Magnesium Sulphate <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.05% <SEP>.
<tb> calcium carbonate <SEP>. <SEP>. <SEP>. <SEP>.

   <SEP> 0.3% 50 cm3 of this nutrient solution were sterilized in the usual way in a shaking vessel. A strain belonging to Streptomyces humidus was inoculated and cultured aerobically at 27 ° C. for 4 days with shaking.

   The antibacterial activity of the culture medium reached 1200 mcg / cm3 against B. subtilis. <I> Example 3 </I> Strain H-120, which belongs to Streptomyces humidus, was cultivated aerobically in various nutrient solutions which consisted of sieve waste from cereals. The results are shown in the following table.

   Each of the cultures described in Examples 3 to 8 was carried out with 50 cm3 of the culture medium in a vessel with 300 cm3 capacity with shaking at 26-28 ° C.
EMI0010.0036
  
    '<SEP> Maximum <SEP> time <SEP> to <SEP> to
<tb> Medium <SEP> Effect <SEP> of <SEP> Reaching <SEP> the <SEP> maximum
<tb> Dihydrostreptomycin <SEP> effect
<tb> mcg / cm3 <SEP> hours
<tb> Starch bouillon <SEP> 78 <SEP> 120
<tb> Glucose Broth <SEP> 225 <SEP> 120
EMI0011.0001
  
    (Continuation)

  
<tb> Maximum <SEP> time <SEP> to <SEP> to
<tb> Medium <SEP> Effectiveness <SEP> from <SEP> reaching <SEP> the <SEP> maximum
<tb> Dihydrostreptomycin <SEP> effect
<tb> mcg / cm3 <SEP> hours
<tb> Starch bouillon <SEP> 211 <SEP> 144
<tb> + <SEP> 3.0% <SEP> CaC03
<tb> Glucose Broth <SEP> 286 <SEP> 120
<tb> + <SEP> 3 <SEP>% <SEP> <B> CaCO3 </B>
<tb> 1. <SEP> 227 <SEP> 120e
<tb> 2. <SEP> 246 <SEP> 96
<tb> 3. <SEP> 329 <SEP> 120
<tb> 4. <SEP> 242 <SEP> 120
<tb> 2. <SEP> + <SEP> yeast extract <SEP> 315 <SEP> 120
<tb> <B> 950/0 </B>
<tb> 1. <SEP> Glucose <SEP> 1.0% <SEP> 2. <SEP> Corn mash <SEP> 3.0% <SEP> 3. <SEP> Glucose <SEP> 2.0% <SEP > 4. <SEP> Strength <SEP> 0.5%
<tb> soybean <SEP> starch <SEP> 3;

  0% <SEP> meat <SEP> meat extract <SEP> 0.3 <SEP>%
<tb> flour <SEP> 1.00 / 0 <SEP> peptone <SEP> 0.50 / a <SEP> extract <SEP> 0.60 / a <SEP> yeast extract <SEP> 0.5%
<tb> peptone <SEP> 0.5% <SEP> K2HP04 <SEP> 0.1% <SEP> peptone <SEP> 1.0% <SEP> peptone <SEP> 0.50 / 0
<tb> NaCl <SEP> <B><I>0.5110</I> </B> <SEP> mgS04 <SEP> * <SEP> 7 <SEP> H @ O <SEP> 0.051 / o <SEP > NaCl <SEP> <B> 0.61V0 </B> <SEP> Glucose <SEP> <B> 1.011 / 0 </B>
<tb> CaC0 .;

   <SEP> 0.3% <SEP> CaCO3 <SEP> <B> 0.31) 4 </B> <SEP> CaCO <B> 3 </B> <SEP> 0.6% <SEP> NaCI < SEP> <B> 0.3% </B> <I> Example 4 </I>
EMI0011.0002
  
    <I> Source medium </I>
<tb> strength <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0%
<tb> K., HP04 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.10 / 0
<tb> M-S04 <SEP> * <SEP> <B> 711 </B> 2 <B> 0 </B> <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.05 <SEP>%
<tb> CaC03 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.3 <SEP>%
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>.

   <SEP> 7.0 grain mash and soybean meal were added as a nitrogen source at various rates to the aqueous starting medium, strain H-120 of Streptomyces humidus was added and cultivated aerobically.



  The results can be seen in the following table:
EMI0011.0007
  
    Nitrogen source <SEP> Maximum <SEP> time <SEP> to <SEP> for
<tb> Effect <SEP> of <SEP> reaching <SEP> the <SEP> maximum
<tb> Corn mash <SEP> soybean meal <SEP> dihydrostreptomycin <SEP> effect
<tb>% <SEP> o <SEP> mcg / cm3 <SEP> hours
<tb> 3.0 <SEP> 1.0 <SEP> 468 <SEP> 120
<tb> - <SEP> 3.0 <SEP> 288 <SEP> 144
<tb> 3.0 <SEP> 2.0 <SEP> 499 <SEP> 144
<tb> 1.5 <SEP> 1.0 <SEP> 417 <SEP> 120
<tb> 3.0 <SEP> 0.5 <SEP> 372 <SEP> 144
<tb> 3.0 <SEP> 1.0 * <SEP> 968 <SEP> 144
<tb> Soybean meal <SEP> became <SEP> by <SEP> heating <SEP> to <SEP> 100o <SEP> 1 <SEP> hour <SEP> for <SEP> with <SEP> the <SEP> tenfold <SEP > Volume <SEP>
<tb> 5, öiger <SEP> hydrochloric acid <SEP> hydrolyzed.

         <I> Example 5 </I>
EMI0011.0008
  
    <I> Source medium </I>
<tb> strength <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0%
<tb> K @ HP04 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.10 / 0
<tb> MgS04 <SEP> '<SEP> 7 <SEP> H20 <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.0511 / o
<tb> CaCO3 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.3%
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 7.0 Streptomyces humidus strain H-120 has been identified on various.

       Aqueous media which, in addition to the starting medium, consist of grain mash, soybean meal and other organic and inorganic nitrogen sources as essential nitrogen sources, cultivated aerobically. In some cases, soy bean meal was omitted.

   The results are shown in the following table:
EMI0012.0001
  
    Nitrogen source <SEP> Maximum <SEP> time <SEP> to
<tb> Effect <SEP> from <SEP> to <SEP> reaching
<tb> Grain mash <SEP> Soybean meal <SEP> Other <SEP> Dihydrostreptomycin <SEP> the <SEP> maximum
<tb> (HCl hydrolyzate) <SEP> effect
<tb> Mycelium <SEP> from <SEP> 23 <SEP> 572 <SEP> (moist) <SEP> mcg / cm3 <SEP> hours
<tb> 3.0 <SEP> 1.0 <SEP> 2.0%, <SEP> 660 <SEP> 144
<tb> 3.0 <SEP> 1.0 <SEP> NH4N03 <SEP> 0.10 / 0 <SEP> 326 <SEP> 120
<tb> 3.0 <SEP> 1.0 <SEP> CO (NH2) 2 <SEP> 0.10 / 0 <SEP> 55 <SEP> 96
<tb> 3.0 <SEP> 1.0 <SEP> CO (NH2) 2 <SEP> 0.1% <SEP> 60 <SEP> 96
<tb> Bacon oil <SEP> <B> 1.01 / 0 </B>
<tb> 3.0 <SEP> 1.0 <SEP> (NHIHP04 <SEP> 0.10 / 0 <SEP> 411 <SEP> 120
<tb> 3.0 <SEP> 1.0 <SEP> KN03 <SEP> 0.10 / 0 <SEP> 600 <SEP> 96
<tb> 3.0 <SEP> 1.0 <SEP> NH4C <B> 1 </B> <SEP> 0,

  10/0 <SEP> 327 <SEP> 120
<tb> 3.0 <SEP> 1.0 <SEP> <B> (NH4) 2S04 </B> <SEP> 0.10 / 0 <SEP> 396 <SEP> 144
<tb> 3.0 <SEP> - <SEP> Casein <SEP> <B> 1.00 / 0 </B> <SEP> 500 <SEP> 120
<tb> 3.0 <SEP> - <SEP> Casein: -'-- <SEP> 1.0 <SEP> 0/0 <SEP> 1000 <SEP> 144
<tb> 1.0 <SEP> 1.0 <SEP> rice bran <SEP> 2.00 / a <SEP> 30 <SEP> 168
<tb> - <SEP> casein <SEP> became <SEP> 1 <SEP> hour <SEP> for <SEP> at <SEP> 100a <SEP> C <SEP> with <SEP> the <SEP> approximately <SEP > ten times the <SEP> volume <SEP> of <SEP> <B> 5111 "</B> <SEP> HCl <SEP> hydrolyzed.

         <I> Example 6 </I>
EMI0012.0002
  
    <I> Source medium </I>
<tb> Corn mash <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0%
<tb> Soybean meal <SEP> (HCl hydrolyzate) <SEP>. <SEP> 1.0%
<tb> K2HP04 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.10 / 0
<tb> MgS04 <SEP> * <SEP> 7H20 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0; 05 <SEP>%
<tb> <B> CaCO3 </B> <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.3%
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 7.0 strain Q-97 of:

          Streptomyces humidus was aerobically cultivated on the starting aqueous medium in which the carbon source was variously changed.

   The results are shown in the following table:
EMI0012.0009
  
    Maximum <SEP> time <SEP> to <SEP> for
<tb> carbon source <SEP> effectiveness <SEP> of <SEP> reaching <SEP> the <SEP> maximum
<tb> Dihydrostreptomycin <SEP> effectiveness
<tb> mcg / cm3 <SEP> hours
<tb> Dextrin <SEP> 3.0% <SEP> 1127 <SEP> 120
<tb> Thickness <SEP> 1.00 / <B> 0 </B> <SEP> 600 <SEP> 120
<tb> Strength <SEP> 3.0% <SEP> 844 <SEP> 120
<tb> Glucose <SEP> 1.00 / 0, <SEP> 592 <SEP> 120
<tb> Glucose <SEP> <B> 3.0%, </B> <SEP> 440 <SEP> 120
<tb> Glycerin <SEP> <B> 3.0%. </B> <SEP> 1050 <SEP> 144
<tb> Lactose <SEP> 3.0% <SEP> 90 <SEP> 96
<tb> Mannose <SEP> 3.00 / 0 <SEP> 1368 <SEP> 144
<tb> Maltose <SEP> 3.0%.

   <SEP> 90 <SEP> 120
<tb> Galactose <SEP> 3.0% <SEP> 1870 <SEP> 144
<tb> Fructose <SEP> <B> 3.0 (1 / @ </B> <SEP> 1390 <SEP> 144
<tb> Sucrose <SEP> 3.0 <SEP> / 0 <SEP> 63 <SEP> 144 <I> Example 7 </I> Strain Q-97 of Streptomyces humidus was grown on various aqueous media, in which corn mash and soybean meals the sources of nitrogen were and dextrin was the source of carbon, cultured aerobically. However, the composition of the inorganic salts has been changed several times.

   The results are shown in the following table:
EMI0013.0010
  
    <I> Source medium </I>
<tb> Dextrin <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0%
<tb> Corn mash <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.011 / o
<tb> Soybean meal <SEP> (HCl hydrolyzate) <SEP>. <SEP> 1;

  00/0
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 7.0
EMI0013.0011
  
    Inorganic <SEP> salts <SEP> Maximum <SEP> time <SEP> to
<tb> Effectiveness <SEP> from <SEP> to <SEP> achievement
<tb> K <SEP> IHPO, 4 <SEP> <U> M9S04.7H20 <SEP> CaC03 </U> <SEP> Dihydrostreptomycin <SEP> the <SEP> maximum
<tb> effectiveness
<tb> Other <SEP> mcg / cm3 <SEP> hours
<tb> - <SEP> 0.05 <SEP> 0.3 <SEP> - <SEP> 717 <SEP> 120
<tb> 0.1 <SEP> - <SEP> 0.3 <SEP> - <SEP> 675 <SEP> 120
<tb> 0.1 <SEP> 0.05 <SEP> 0.6 <SEP> - <SEP> 1230 <SEP> 144
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> - '@ <SEP> 700 <SEP> 144
<tb> 0.1 <SEP> 0.05 <SEP> 1.0 <SEP> - <SEP> 1260 <SEP> 120
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> ZnS04 <SEP> '<SEP> 7H20 <SEP> 0.0010 / 0 <SEP> 639 <SEP> 144
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> MnS04 <SEP> * <SEP> 7H20 <SEP> 0,

  002% <SEP> 555 <SEP> 144
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> FeS04 <SEP> * <SEP> 7 <SEP> H20 <SEP> 0.0021 / o <SEP> 1165 <SEP> 120
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> Na.s04 <SEP> 0.10 / <B> 0 </B> <SEP> 1099 <SEP> 144
<tb> (anhydrous)
<tb> 0.1 <SEP> 0.05 <SEP> 0.3 <SEP> CuS04 <SEP> '<SEP> 5 <SEP> H20 <SEP> 0.0020 / 0 <SEP> <B> 1038 < / B> <SEP> 120
<tb> Neutralization <SEP> of the <SEP> hydrolyzate <SEP> of <SEP> soybean meal <SEP> and <SEP> setting <SEP> of <SEP> pH <SEP> was <SEP> with <SEP> potassium hydroxide <SEP > carried out.

         <I> Example 8 </I>
EMI0013.0012
  
    <I> Medium </I>
<tb> Dextrin <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0 <SEP>%
<tb> Corn mash <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 3.0%
<tb> HCl hydrolysates <SEP> of the <SEP> soybean flour <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 1.0%
<tb> K2HP04 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.10I0
<tb> MgS04 <SEP> * <SEP> 7 <SEP> H20 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 0.0511 / o
<tb> CaCO3 <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> <B> 0.3711 / 9 </B>
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP>.

   <SEP> 7.0 The mutants obtained by treating Streptomyces humidus in various ways were cultivated nerobically in the above medium. The results are shown in the following table:

    
EMI0013.0018
  
    Maximum <SEP> time <SEP> to <SEP> for
<tb> strains <SEP> effectiveness <SEP> of <SEP> reaching <SEP> the <SEP> maximum
<tb> Dihydrostreptomycin <SEP> effectiveness
<tb> meg / cm3 <SEP> hours
<tb> 23 <SEP> 572 <SEP> (original) <SEP> 660 <SEP> 144
<tb> B <SEP> - <SEP> 9 <SEP> 460 <SEP> 144
<tb> A <SEP> - <SEP> 113 <SEP> 846 <SEP> 120
EMI0014.0001
  
    (Continuation)

  
<tb> Maximum <SEP> time <SEP> to <SEP> to
<tb> strains <SEP> effect <SEP> of <SEP> reaching <SEP> the <SEP> maximum
<tb> Dihydrostreptomycin <SEP> effectiveness
<tb> mcg / cm3 <SEP> hours
<tb> C <SEP> - <SEP> 46 <SEP> 792 <SEP> 120
<tb> E <SEP> - <SEP> 96 <SEP> 433 <SEP> 144
<tb> H <SEP> - <SEP> 106 <SEP> 1220 <SEP> 144
<tb> K <SEP> - <SEP> 34 <SEP> 1248 <SEP> 144
<tb> UD- <SEP> 1 <SEP> 1308 <SEP> 144
<tb> Q <SEP> - <SEP> 97 <SEP> 1296 <SEP> 144
<tb> VD <SEP> 886 <SEP> 144
<tb> N <SEP> 931 <SEP> 144
<tb> Tu <SEP> 574 <SEP> 120
<tb> <B> VP </B> <SEP> 976 <SEP> 120
<tb> Vu <SEP> 1235 <SEP> 144
<tb> L <SEP> 840 <SEP> 120
<tb> Y <SEP> 966 <SEP> 120
<tb> G <SEP> 374 <SEP> 120
<tb> Q <SEP> 1085 <SEP> 120 <I> Example 9 </I>
EMI0014.0002
  
    <I> Medium </I>
<tb> glucose <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 2.0 () / o
<tb> meat extract <SEP>. <SEP>.

   <SEP> 0.6%
<tb> peptone <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 1.0%
<tb> NaCl <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> <B> 0.6- /, </B>
<tb> CaC0, <SEP>. <SEP>. <SEP>. <SEP>. <SEP> <B> 0.611 / 0 </B>
<tb> pH <SEP>. <SEP>. <SEP>. <SEP>. <SEP>. <SEP> 7.0 A strain belonging to Streptomyces humidus was submerged for 96 hours. and cultured aerobically in a tank.

   7 kg (10/0) of active charcoal were added to 700 liters of the filtrate of the culture medium [pH: 8.5, effectiveness: 35 U / cm3 (dilution unit) against E. coli] and the mixture was stirred for 30 minutes to adsorb the active compound. The active charcoal was eluted with 10 times its volume of methanolic hydrogen chloride for 30 minutes at a pH of 2.0. 140 liters of eluate with an effectiveness of 100 U / cm3 were obtained in this way.

   The elution was repeated once more, with 70 liters of eluate with an efficiency of 35 U / cm3 being obtained. The combined eluates were made almost neutral with n NaOH up to a pH of 6.5 and concentrated in vacuo at low temperature to about 300 cm3. During this procedure, the precipitated sodium chloride was removed and then 3 liters of anhydrous acetone was added to the concentrated solution.

   The precipitated active compound was dried in vacuo to give a white powder. The yield was 147 g or 6011 / o with a potency of 100 U / mg. The product obtained is negative for the maltol reaction, but positive for the Sakaguchi reaction.



  <I> Example 10 </I> Purification by ion exchanger a) 500 liters of the culture filtrate [pH 7.7, effectiveness: 350 U / cm3 (dilution unit) against E. Coli], which was obtained as in Example 9, were carried out by a tower filled with 30 liters of Amberlite IRC-50 (H-type, registered trademark) was run at a rate of 1.8 liters per minute.

   The effectiveness of the effluent against E. coli was less than 10 U / cm3. The resin was washed with water, eluted with 0.5N HCl and 36 liters of the colored eluate with an activity of 3500 U / cms against E. coli were collected. The eluate was adjusted to a pH of 6.5 with n NaOH and concentrated to 1 liter at low temperatures with occasional removal of the deposited sodium chloride.

   The effectiveness was 160,000 U / cm3, the yield was 91.5%.



  b) 4.1 kg of oxalic acid were added to 748 liters of the cultivation filtrate obtained according to Example 9 (content of dihydrostreptomycin approximately 2.200 meg / cm3) and the precipitate obtained was separated off by a De Laval separator. The pH was adjusted to approximately 7.0 with a sodium hydroxide solution and the resulting precipitate was also separated.

   The solution was passed through a tower filled with 6.3 liters of Amberlite IRC-50 (Na type) at a rate of 40 liters / hour, whereupon the effluent only showed an effectiveness of 12-6 meg / cm3. The heart was washed with water and eluted in active compound by passing 4% hydrochloric acid through it at a rate of 3 liters / hour.



  20 liters of the eluate thus obtained were neutralized by adding Amberlite IR-45 (OH type) or Amberlite IRA-400 (OH type) and concentrated to 2.5 liters under reduced pressure at low temperature. The effectiveness was <B> 368000 </B> meg / cm3.



  <I> Example 11 </I> Extraction with an organic solvent to 1 liter of the filtrate of the cultivation obtained according to Example 10 b) with an effectiveness of 1001 meg / em3 against B. subtilis was 400 cm3 n-Butyl alcohol or isoamyl alcohol containing 3 1 / o lauric acid was added, the aqueous layer was adjusted to a pH of 7.5 and the mixture was shaken for 20 minutes.

   After the butylalcohol or isoamylalcohol layer had been separated off, the same extraction was carried out again. The combined butyl alcohol or isoamyl alcohol solutions were shaken with dilute sulfuric acid in water in such a way that the resulting aqueous layer was 120 cm 3 and its pH was 2.0. After repeating the same extraction, the combined extracts were shaken with ether in order to remove the lauric acid contained therein.

   The effectiveness of the 25 cm3 solution thus obtained was 2891 mcg / cm3 against B. subtilis, the yield was 72.5%. <I> Example 12 </I> Chromatography on Active Charcoal. 1 liter of a solution obtained according to example 10 cz) with an effectiveness of 160,000 U / cm3 (dilution units) against E.

       coli was increased with n HCl. The pH was adjusted to 3.5 and sent through a tower 8 cm wide and 50 cm high, which was charged with 50 g of active carbon impregnated with water at a pH of 3.5. The effluent was divided into parts of 300 cm3. The tower was treated with distilled water adjusted to pH 3.5 with hydrochloric acid. Fractions 1-11 contained sodium chloride and impurities, while the others contained the active compound as shown below.



  The fractions 13-21 were combined, adjusted to a pH of 6.5 by adding Amberlite IRA-400 (OH type) and concentrated under reduced pressure at low temperature. Ten times its volume of anhydrous acetone was added to the concentrated solution, and the resulting precipitate was separated from the liquid by decanting and dried. 101 g of the hydrochloride were obtained as a colorless amorphous substance.

   This corresponds to 783.3 mcg / mg of dihydrostreptomycin. A solution of 6 g of M9S04 was added to a solution of 10 g of this product in 50 cm3 of water. 7H20 in 7 cm3 water. The mixture was adjusted to a pH of 6.0 with dilute sulfuric acid, filtered, and methanol was added dropwise with stirring until the solution became cloudy and the temperature of the mixture was kept at 50-55.degree.

   A few crystals of dihydrostreptomycin sulfate were added to the cloudy solution and the solution was allowed to stand at 50-55 ° C., whereupon the solution separated crystals in a short time (about 5 minutes) and became clear at the same time. Further methanol was added until the solution became slightly cloudy again and the mixture was allowed to stand as above. The procedure was continued in this manner until the amount of methanol reached 100 cm 3 and the solution became clear.

   The temperature of the solution was then reduced to room temperature, the crystals were filtered off and these were stirred for 5 minutes in 50% strength methanol. The crystals were filtered off, washed with methanol and dried at 50 ° C. under reduced pressure.

    The yield was 8.9 g (738 mcg / mg as dihydro-streptomycin). Several recrystallizations increased the potency to approximately 780 mcg / mg as dihydrostreptomycin. It was found in the above process that M9S04: has the effect of destroying substances similar to histamine contained in the material.



  <I> Example 13 </I> 5 cm3 of the concentrate of the active compound obtained in Example 10 b) (potency 368000 mcg / cm3) were diluted with water up to 50 cm3 and a warm, saturated solution of methyl orange was added to no more precipitate was formed. After cooling: the precipitate was filtered off and repeatedly recrystallized from aqueous methanol, whereupon 2.1 g of the helianthate of the active compound were obtained.

   A suspension of the helianthate in a small amount of water was adjusted to a pH of 2.0 with dilute sulfuric acid, whereupon the active compound dissolved to form the sulfate. The solution was filtered to remove the precipitated methyl orange and treated with a small amount of activated carbon. Ten times its volume of anhydrous acetone was added to the colorless solution obtained, whereupon 0.6 g of the sulfate of the active compound separated out.

 

Claims (1)

PATENT CLAIM Process for the production of dihydrostreptomycin, characterized in that a strain of a dihydrostreptomycin-producing microorganism of the genus Streptomyces is cultivated aerobically in an aqueous nutrient medium. SUBClaims 1. The method according to claim, characterized in that cultivation is carried out until a concentration of at least 10 y dihydrostreptomycin / cm3 is present in the culture medium. 2. Process according to claim, characterized in that cultivation is carried out submerged and aerobically and the dihydrostreptomycin formed is isolated from the culture medium. 3. The method according to claim and sub-claims 1 and 2. 4. The method according to claim and sub-claim 2, characterized in that Streptomyces humidus or a mutation or variation of this type is cultivated. 5. Method according to claim and sub-claim 4, characterized in that cultivation is carried out at a temperature of 24-30 C for 3-8 days and the dihydrostreptomycin formed is isolated from the culture medium. 6. The method according to the dependent claims 2, 3, 4 and 5, characterized in that the isolation of the dihydrostreptomycin or its salts takes place by means of an adsorbent. 7. The method according to the dependent claims 2, 3, 4 and 5, characterized in that the isolation of the dihydrostreptomycin or its salts is carried out by extracting the antibiotic with a water-immiscible organic solvent. B. Process according to dependent claims 2, 3, 4 and 5, characterized in that the isolation of the dihydrostreptomycin takes place by the formation of a water-insoluble compound of the antibiotic. 9. The method according to the dependent claims 2, 3, 4 and 5, characterized in that the isolation of the dihydrostreptomycin takes place by means of chromatographic adsorption. 10. The method according to the dependent claims 2, 3, 4 and 5, characterized in that the isolation of the dihydrostreptomycin takes place by means of ion exchangers. 11. Method according to dependent claim 6, characterized in that activated carbon is used as the adsorbent. 12. The method according to dependent claim 7, characterized in that the extraction is carried out under weakly alkaline conditions. 13. The method according to dependent claim 7, characterized in that the water-insoluble organic solvent is n-butanol or isoamyl alcohol. 14. The method according to dependent claim 8, characterized in that the agent used to form a water-insoluble salt of the antibiotic is methyl orange.