BE569746A - - Google Patents

Description

       

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   The present invention relates to a novel water-soluble antibiotic which, in what follows, will be referred to as A 9578, its compasants, salts and derivatives, as well as pharmaceutical preparations containing these compounds. The invention also relates to a process for the preparation of these substances and mixtures of substances.



   The new antibiotic A 9578 is formed during the cultivation of a new strain of actinomycetes which was isolated from a test sample made in the soil near Boston, Massachusetts, United States of America, and which has been stored in the laboratories of the Applicant and at the Federal Polytechnic School (Zurich Switzerland), Special Botanical Institute, under the designation A 9578.



   Streptomyces A 9578 belongs to the Streptomyces griseoflavus (Krainsky) family of Waksman and Henrici, but differs from other representatives of this family by the formation of the new water-soluble antibiotic. So far only one strain of Streptomyces griseoflavus is known to produce an antibiotic. In this case, this is called gray-oflavin # Y. Waga "J. Antibiotios Japan", A 6, 66 (1953), which however already differs from the new antibiotic A 9578 by its solubility in organic solvents. The aerial mycelium of Streptomyces A 9578 is ash gray.

   The spore carriers are branched and form numerous spirals usually comprising 2 to 5 turns. The spores themselves are 1.0-1.4 ux 0.7-0.9 # in size and have about 0.2 [gamma] long quills on their surface which go to a point and are only little widened at the base. Growth depends relatively little on temperature, and the fungus thrives at 18 as well as at 40; however the optimum growth is between 25 and 32.



   To give other characteristics, the growth of Streptomyces A 9578 on various nutrified media will be described in what follows. Nutrient media 1 to 7 as well as 10 were prepared according to W. Lindenhein, "Arch. Pikrobiol." 17, 361 (1952).



  1) Synthetic celose Veiled, colorless growth, no aerial mycelium.



  2) Synthetic medium to liquidate: Sediment, reddish flakes. Well formed pelicle with gray-white veil-like mycelium. Substratum yellow-brown to dark yellow.



  3) Celose-glucose: Growth puncture at the beginning, rough after 12 days pale yellow.



  4) Asparagine Glucose Agar Growth punctiform at first, later veiled, yellowish. Velvety aerial mycelium, at first brownish-gray, later ash-gray. Substratum yellow.



  5) Calcium Malate Agar -. Growth hazy yellow-white, later dark yellow. Aerial mycelium scanty, farinaceous, white as flour. Substratum light brown.



  6) Agar medium (gelatin) at 18 C: Very little growth, very slow liquefaction (0.5 cm after 30 days).



  7) Starch agar: Veiled growth of milky white, no aerated mycelium. Starch hydrolysis 1 cm after 5 days.



  8) Potatoes Growth punctate at first, pale yellow, later pustular. Velvety aerial mycelium, yellow-white to ash-gray. Substratum colored in brownish-gray.



  9) Carrots Very scanty growth. Scanty aerial mycelium. Substratum not discolored.



  10) Sunflower milk: Ring growth and colorless surface film.



   Gray-white aerial mycelium. Slow hydrolysis without coagulation; turned-

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 blue ground.



  Tyrosinase reaction: Negative.



   Streptomyces A 9578 shows good growth, according to the method of TG Pridham and D. Gottlieb, "J. Bacteriology" 56, 107 (1948), when the following carbon sources are used: L-xylose, L-arabinos, L-rhamnose, saccha- rose, raffinose, inu line, D-mannite, D-sorbite, mesoinosite, salicin, D-fructose.



   With regard to the preparation of the antibiotic A 9578, the present invention is not limited to the use of Streptomyces A 9578 or other strains corresponding to the description, but it also relates to the u - Use of varieties of these organisms, as obtained, for example, by selection or mutation, in particular under the influence of ultraviolet radiation or X-rays, or the action of nitrogen mustard.



   To obtain the antibiotic A 9578, a strain of streptomycetes exhibiting the properties of Streptomyces A 9578 is aerobically cultivated, for example in an aqueous nutrient solution containing carbohydrates, nitrogen compounds as well as inorganic salts, until that this solution exhibits a notable antibacterial action, then the antibiotic A 9578 is then isolated.



   As assimilable carbohydrates, or envisages for example glucose, sucrose, lactose, mannite, starch as well as glycerin.



  Nitrogenous nutrients and, where appropriate, growth promoting substances include: amino acids, peptides and proteins, as well as their degradation products such as peptones or tryptones, as well as meat extracts, soluble fractions in the water of grains of cereals such as corn and wheat, distillation residues resulting from the preparation of alcohols, yeasts, beans, in particular soybeans, seeds, for example cotton, etc. but also ammonium salts and nitrates. Like other inorganic salts, the nutrient solution may contain, for example, chlorides, carbonates, alkali and alkaline earth metal sulfates, magnesium, iron, zinc and manganese.



   The cultivation takes place aerobically, that is to say, for example in surface culture at rest or, preferably, in an immersed manner with shaking or agitation with air or oxygen in flasks. stirred or in known fermenters. As a temperature, a temperature between 18 and 40 is suitable. By operating in this way, the nutrient solution exhibits a noticeable antibacterial effect, generally after one and a half to five days.



   To isolate the antibiotic A 9578, the following methods can, for example, be used: the mycelium is separated from the culture filtrate, after which the major part of the antibiotic is found in said filtrate. However, significant amounts of antibiotic remain which are adsorbed to the mycelium.



  It is therefore advantageous to properly extract the latter by washing. Suitable for this purpose are in particular water and aqueous organic solvents, such as alcohols, for example aqueous methanol.



   To extract the antibiotic from the culture filtrate and to purify it, various methods are suitable which can be used alone or in combination. It has proved to be advantageous, during these operations, to maintain the culture solution at a pH of between 3 and 5.



   1) Adsorption agents, for example activated carbons, such as norite, activated earths such as fuller's earth or fluridine, or resin-based adsorbents, such as asmite, can be used. Elution of the adsorbates advantageously takes place with mixtures of organic solvents miscible with water and water or aqueous acids, for example with mixtures of water and methanol, water and pyridine, dilute acetic acid and methanol, or water, methanol, glacial acetic acid and butanol. It is particularly proven

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 advantageous for the elution of an adsorbate of norite, a mixture of water (2 parts by volume), methanol (1 part by volume), glacial acetic acid (1 part by volume) and butanol (2 parts by volume).



   2) A second method for separating the antibiotic from the culture filtrate consists in adsorbing the antibiotic on cation exchangers, in which case in particular resins containing acid groups, such as Amgerilite IRC-50, are suitable. The latter can be used both in the acid form and in the sodium form, although a mixture of these two forms in a volumetric proportion of 1: 2 has in particular proved its worth. Elution advantageously takes place with dilute acids, for example with a methanolic solution of hydrochloric acid.



   3) In addition, the basic antibiotic can also be precipitated directly from the culture filtrate, for example by reaction with an organic acid of the pioric acid type. By treating these precipitates with salts of organic bases, for example with triethylammonium sulfate, or with dilute acids, the antibiotic is obtained: in the form of the corresponding salt. In this case, it is possible to work both in an aqueous medium and in a solvent miscible with water, such as methanol or acetone. This conversion of the poorly soluble salts into easily soluble salts of the antibiotic is carried out either with the aid of mineral acids or on ion exchange resins, for example on Amberlite IRA-400.



   4) An enrichment of the antibiotic can also be obtained by adding to aqueous solutions or to aqueous alcoholic solutions of salt, an excess of organic solvents miscible with water, such as acetone, dioxane, etc., them. salts then being precipitated in solid form.



   5) Another method of enriching the antibiotic consists of extracting aqueous solutions of said antibiotic with solutions of phenol in chloroform, in which case both the pH of the aqueous solution and the phenol content of the chloroform solution. are subject to variations.



  When, for example, a distribution is carried out between a solution containing 100 g of phenol per 100 cm3 of chloroform, and an aqueous phase with a pH of 1 to 6, the antibiotic is then found almost exclusively in the organic phase, while when a solution containing only 33 g of phenol per 100 cm 3 of chloroform is used, said antibiotic is withdrawn almost completely from the aqueous phase, simply at a pH of between 4 and 6. If the expression coefficient of distribution of antibiotic the ratio of the concentration in the organic phase to the concentration in the aqueous phase, it then emerges from the above indications that the distribution coefficient increases as the phenol content of the organic phase increases and decreases as the pH of the aqueous phase decreases.

   Since it is thus possible to establish in this system any coefficient of distribution of the entibiotic, it is possible by combining a small number of distribution operations, to separate a large quantity of inactive impurities.



   6) Another enrichment method for the antibiotic is offered by chromatography, for example adsorption chromatography on different materials, for example on norite, aluminum oxide, magnesium silicates , solica gel, calcium sulfate, as well as distribution chromatography with cellulose, starch, silica gel, celite and the like as carrier substances, or also chromatography on resins ion exchangers, for example on "Dowex 50", Amberlite IRC-50 and the like. Partition chromatography on cellulose, using the solvent system butanol (4 parts by volume), glacial aetic acid (1 part by volume) and water (5 parts by volume), for example, has proved particularly advantageous.



   7) In addition, the antibiotic can be enriched by counter-current chromatography following Craig between two immiscible solvent phases. In

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 In this case, the following solvent systems have proved to be particularly advantageous: a) Secondary butanol - normal 1/10 buffer solution of ammonium acetate, with a pH of 4.68. b) Normal 1/10 buffer solution of ammonium acetate, pH 4.6 - 10% solution of phenol in chloroform. The distribution coefficient of the antibiotic in system b) and consequently the situation of the maximum activity in the distribution can be modified on demand, on the one hand, by varying the pH of the buffer solution. and, on the other hand, by varying the phenol content of the organic phase.



   In order to obtain an even purer preparation it has been found appropriate to combine the indicated enrichment methods as follows: From the culture filtrate, the antibiotic is adsorbed on an Amberlite buffered ion exchanger. IRC-50 and eluted with a methanolic solution of hydrochloric acid. The eluates are concentrated in vacuo at a pH of 5, whereby an aqueous concentrate of the antibiotic is obtained which, in terms of its volume, corresponds to approximately 1/100 of the culture solution. .

   This concentrate is distributed according to method 5) indicated above, several times between mixtures of phenol and chloroform, on the one hand, and aqueous solutions of a variable pH, on the other hand, which causes that after drying by refrigeration a preparation is obtained which, in relation to the cultured filtrate, has an increased specific activity of 500 to 1000.



  By treating such a preparation according to method 6) indicated above (distribution chromatography on cellulose), and according to method 7), one can obtain the basic antibiotic A 9578, largely unitary, preferably under the form of a salt.



   As shown by tests carried out by chromatography on paper, the antibiotic A 9578 consists of at least two components, namely the main component A and the secondary component B. These components are even more separated. from each other during the enrichment process described, especially during partition chromatography on cellulose and during countercurrent partitioning.



   The two components of antibiotic A 9578 are defined in paper chromatography by a direct comparison of their R values with the R values of a series of known antibiotics (2 to 11) in systems A to G. In system H, pass-through chromatography is carried out. The numbers indicated are in the table which follows the spreads, expressed in cm, of the antibiotics after 16 hours of chromatography. Antibiotics are detected autobiographically using Staphylecoccus aureus or Bacillus subilis.

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<tb> tème <SEP> 1a <SEP> 1b <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10 <SEP > 11
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<tb> A <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0.92 <SEP> 0 , 07 <SEP> 0
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<tb> B <SEP> 0.49 <SEP> 0.63 <SEP> 0.05 <SEP> 0.05 <SEP> 0.17 <SEP> 0.02 <SEP> 0.02 <SEP> 0 , 66 <SEP> 0.55 <SEP> 0.92 <SEP> 0.32 <SEP> 0.22
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<tb> C <SEP> 0.34 <SEP> 0.58 <SEP> 0.22 <SEP> 0 <SEP> 0.02 <SEP> 0.22 <SEP> 0.03 <SEP> 0.72 <SEP> 0.62 <SEP> 0.93 <SEP> 0.39 <SEP> 0.11
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<tb> D <SEP> 0.05 <SEP> 0.15 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> x <SEP> x <SEP> 0.92 <SEP> 0
<tb>
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<tb> E <SEP> 0.32 <SEP> 0.32 <SEP> 0 <SEP> 0.10 <SEP> 0.22 <SEP> 0 <SEP> 0 <SEP> x <SEP> x <SEP > 0.86 <SEP> 0,

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<tb> F <SEP> 0.47 <SEP> 0.47 <SEP> 0.22 <SEP> 0.14 <SEP> 0.12 <SEP> 0.04 <SEP> 0.05 <SEP> 0 , 49 <SEP> 0.42 <SEP>, <SEP> 0.91 <SEP> 0.43 <SEP> 0.36
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<tb> G <SEP> 0.74 <SEP> 0.74 <SEP> 0.07 <SEP> 0.02 <SEP> x <SEP> x <SEP> 0.94 <SEP> 0.61 <SEP > 0.69
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<tb> H <SEP> 2.7 <SEP> 7.6 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> (14, <SEP> (8.8) <SEP> 27 <SEP> 1
<tb>
 A Butanol saturated with water B Mixture of butanol, glacial acetic acid and water (4: 1: 5) (upper phase) @ @ C Butanol saturated with water + 2% p-toluene acid -sulfonic D Butanol saturated with water + 2% piperidine E Mixture of butanol, pyridine and water (6: 4: 3).
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  80% ethanol + 1.5% sodium chloride, Whatman N 4 paper impregnated with 0.95-molar sodium sulfate solution + 0.05-molar sodium hydrosulfate solution.



   G Mixture of butanol, ethanol and water (1: 1: 2).



   H Mixture of butanol, butyl acetate, glacial acetic acid and water (10: 3: 1.3: 14.3) (upper phase)
1a A 9578 base A
1b A 9578 base B
2 Streptomycin
3 Ristocetin A
4 Ristocetin B
5 Neomycin B
6 Viomycin
7 Chlorotetracycline
8 Hydroxytetracycline
9 Actinomycin J
10 Cycloserine
11 Grisein x Antibiotic distributed over the entire spreading path () Unclear position
Upon electrophoresis on paper in a 0.1-molar solution of an acetate-buffer, pH 4.6, the antibiotic A 9578 migrates to the cathode.

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  The rate of migration is about half that of streptomycin.



   Antibiotic A 9578 component A is a yellow-orange powder, which dissolves readily in water, dissolves well in methanol, and dissolves poorly in most organic solvents.



   Base A provides the following color reactions: FeCl3: red-brown FeCl3-K3Fe (CN) 6: Ninhydrin blue: pale purple The Sakaguchi, Maltol and Ehrlich-Morgan tests are negative. An aqueous solution of component A exhibits absorption maxima in the ultraviolet spectrum at 215 min / (E 1% 1 cm = 312) and at 315 mu (E 1 cm = 42).



   The free base of antibiotic A 9578 is easily accessible from its salts, from sulphate for example, by reaction-in medium
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 aqueous on barium hydroxide, by neutralization of barite in exoesa # didicsde of car1x # am3io, 7th by staysaraticndu Izédpa: ê of ca.eb # ace of k.:r;llUl and. barium sulfate and L3D] eimnt the free base using refrigeration drying. More simply, the preparation from the salts takes place using a strong basic anion exchanger, for example the hydroxylated form of the product which is commercially available under the name "Dowex 2".



   One of the properties of the antibiotic A9578 which reproduces most often is a marked minimum of stability between pH 6 and pH 9. At 2000-and at a pH value of 7 and 8 the solutions of the antibiotic lose, at during 48 hours most of their activity while at the same temperature and pH 1 to 5 and 10 to 13 they remain fully effective.



   The salts of antibiotic A 9578 and its components are derived from known inorganic and organic salts, for example from those of hydrochloric acid, sulfuric acids and phosphoric acids, acetic, propionic, valerian, palmitic or oleic, succinic, citric, mandelic, glutamic or pantothenic acids. They are neutral or acidic salts. Their preparation takes place by causing the corresponding acids to act on the free base, or by double decomposition of salts, for example of sulphate! A 9578 with calcium pantothenate.



   Antibiotic A 9578 exhibits very high antibiotic activity against the various test organisms. In the so-called agar transverse trait test, it is active against the following test organisms: Micrococcus pyogenes, var. aureus, Streptococcus viridans, Streptococcus faccalis, Corynebacterium diphteriae, Escherichia coli, Bacillus megatherium and Bacillus subtilis.



   The antibiotic A 9578 is also active in vivo. When administered subcutaneously 5 times, at a rate of 10 mg per kg, to mice infected * with Sttphylococcus pyogenes, var. aureus, 100% survivors are observed and for a dose of 5 times 5 mg per kg, 50% survivors are observed. When the mice are infected with Escherichia coli, 50% of survivors are observed by administering to them 5 times, by the subcutaneous route, 50 mg of antibiotic per kg.



   The toxicity of the antibiotic is minimal. Thus, for example, a subcutaneous administration of 1000 mg per kg is tolerated without damage by mice. Higher doses have not yet been proven.



     The antibiotic A 9578, its components, salts or derivatives can be

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 for use as medicaments, for example in the form of pharmaceutical preparations comprising the indicated compounds in admixture with a pharmaceutical carrier material, organic or inorganic, suitable for enteral, parenteral or local application. For this carrier material, substances which do not react with the new compounds are considered, such as, for example, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, benzyl alcohols, gums. , polyalkylene glycols, petroleum jelly, cholesterin or other known excipients.

   Pharmaceutical preparations can be presented, for example, in the form of tablets, dragees, powders, ointments, creams, suppositories, or in liquid form in the form of solutions, suspensions or emulsions. . Where appropriate, they are sterilized and / or contain auxiliary substances such as preservatives, stabilizers, wetting agents or emulsifiers.



  They may also contain still other therapeutically valuable substances.



   The invention also relates, as new industrial products, to the compounds obtained by carrying out the process defined above.



   The invention is described in more detail in the following non-limiting examples, in which the temperatures are indicated in degrees centigrade.



  Example 1
The culture of Streptomyces A 9578 is carried out according to the submerged culture method. A nutrient solution is used containing per liter of tap water 20 g of soybean flour and 20 g of mannite. The nutrient solution is sterilized in the seed flask or in the fermenters for 20 to 30 minutes under pressure of an effective atmosphere. The sterilized nutrient solution has a pH of 7.5 to 8.0. Seeding takes place with an amount of up to 10% of a partially sporulating vegetative culture of the organism. While shaking well or with agitation, one incubates, at 27, in the fermenters, while passing per minute, in the cultures, about 2 volumes of sterile air per volume of solution.

   After 48 to 120 hours of incubation, the culture solution reaches the maximum inhibitory value vis-à-vis the test organisms (Bacterium subtilis, Bacterium megatherium, Micrococcus pyogenes, var. Aureus). The culture is interrupted, the pH is brought to 4.5 by the addition of dilute sulfuric acid, then by filtration or centrifugation, the mycelium and other solid components are separated from the solution containing the major part of the. antibiotic, by adding, if necessary, to the culture solution, before filtration, approximately 1% of a filtration aid, for example Hyflo supercel. The filter residues are washed with water and with aqueous methanol, then the washings are combined with the culture filtrates.



   If, instead of the nutrient solution indicated above, a solution containing the following nutrients per liter of water is used, then after similar cultivation and treatment, culture filtrates having such high antibiotic activity.

   
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<tb> a) <SEP> Glucose <SEP> 10 <SEP> g
<tb> Soy <SEP> <SEP> flour <SEP> 10 <SEP> g
<tb> Sodium <SEP> <SEP> <SEP> 5 <SEP> g
<tb> Nitrate <SEP> of <SEP> sodium <SEP> 1 <SEP> g
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<tb> b) <SEP> Glycerin <SEP> 20 <SEP> g
<tb> Soy <SEP> <SEP> flour <SEP> 10 <SEP> g
<tb> Sodium <SEP> <SEP> <SEP> 5 <SEP> g
<tb> Nitrate <SEP> of <SEP> sodium <SEP> 1 <SEP> g
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 10 <SEP> g
<tb>
 

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<tb> c) <SEP> Glucose <SEP> 10 <SEP> g
<tb> Soy <SEP> <SEP> flour <SEP> 10 <SEP> g
<tb> Corn <SEP> steep <SEP> liquor <SEP> (water <SEP> of <SEP> swelling <SEP> of <SEP> corn) <SEP> 20 <SEP> g <SEP>
<tb> Sodium <SEP> <SEP> <SEP> 5 <SEP> g
<tb> Nitrate <SEP> of <SEP> sodium <SEP> 1 <SEP> g
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 10 <SEP> g
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   <SEP> Lactose <SEP> 20 <SEP> g
<tb> Soluble <SEP> distillers <SEP> 20 <SEP> g
<tb> Sodium <SEP> <SEP> <SEP> 5 <SEP> g
<tb> Nitrate <SEP> of <SEP> sodium <SEP> 1 <SEP> g
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 Example 2.



   By adding a dilute solution of sodium hydroxide, three liters of a culture filtrate obtained according to Example 1 are adjusted to a pH of 7.5, then 50 g of activated carbon (Norite A) are added. It is stirred mechanically for one hour, whereby all of the substance showing antibiotic activity is adsorbed by the carbon. This active substance is separated from the completely inactive solution, by filtering, advantageously by adding a little of a filter aid, such as Hyflo Supercel for example.



  The charcoal is then poured into 500 cm3 of a mixture of 4 parts by volume of water and one part by volume of pyridine, the mixture is stirred mechanically for half an hour and then filtered, to further extract at a repeat, in the same way, of the coal residue. The eluates contain all of the antibiotic activity.



  Example 3.



   By adding a dilute solution of sodium hydroxide, three liters of a culture filtrate obtained according to Example 1 are adjusted to a pH of 7.5 and then the whole is taken up immediately, with a flow rate of 0.5 liter per hour, on a column of Amberlite IRC-50 (hydrogenated form), the length and diameter of which are respectively 30 and 5 cm. The antibiotic is then completely adsorbed. The column is washed with 3 liters of water. For the elution, one liter of 0.4-normal hydrochloric acid is used. The first 500 em3 of the eluate are antibiotically inactive, while the next 500 cm3 contain all of the activity. In order to remove excess hydrochloric acid, this active eluate is percolated through an Amberlite IR-4B column.

   By subjecting the percolate to refrigeration drying, the enriched antibiotic A 9578 is obtained in the form of a highly active amorphous powder.



  Example 4.



   By adding a dilute solution of sodium hydroxide, six liters of a culture filtrate obtained according to Example 1 are adjusted to a pH of 7.5 and then immediately percolated through an Asmite 173 column with a length of of 14 cm and a diameter of 4.5 cm. The antibiotic is then completely adsorbed.



  The adsorbent resin is then washed with 3 liters of water and the antibiotic eluted with one liter of a mixture of methanol and normal hydrochloric acid (1: 1).



  The eluate which contains all of the activity is concentrated in vacuo to a volume of 15 cm 3 in vacuo. To this solution is added 75 cm3 of a 0.25-normal methanolic solution of hydrochloric acid and the mixture is poured into 10 = liters of acetone, whereby the hydrochloride of the antibiotic precipitates.



  It is filtered and washed with acetone. In order to further purify it, it is dissolved in 150 cc of methanol and the slightly cloudy solution filtered by adding celite. After evaporating the filtrate in vacuo at 25, the hydrochloride of antibiotic A 9578 is obtained in the form of an amorphous powder.



  Example 5.

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   Thirty liters of a culture filtrate obtained according to Example 1 are adjusted to a pH of 4.5 and then concentrated to 2 liters in a thin layer evaporator. The concentrate is adjusted to pH 8 by the addition of a dilute sodium hydroxide solution, then filtered by adding celite.



  The clear filtrate is adjusted to a pH of 5 and, while stirring, 1.5 liters of a hot 5% aqueous picric acid solution are added thereto. After allowing the precipitate formed to stand for several hours at 0, it is separated by filtration while adding 50 g of Celite. The filtrate has very little antibiotic activity. The filtration residue is then stirred three times with 800 cm 3 of cold acetone each time, then filtered. The filtrate is concentrated in vacuo to 80 cc, whereby the antibiotic picrate and excess picric acid precipient. After separation, 8.5 g of dry substance are obtained.



   To isolate the antibiotic as the hydrochloride, 2.5 g of the dry substance mentioned are dissolved in 30 cm3 of methanol. Next, a mixture of 1 cc of concentrated hydrochloric acid and 10 em3 of acetone is added first, followed by 500 cc of ether, whereby the hydrochloride precipitates quantitatively. By dissolving the precipitate several times in a hydrochloric solution of methanol and by precipitating with ether, the last picric residues can be removed from said precipitate. Finally, the hydrochloride is dissolved in as little methanol as possible, filtered and evaporated in vacuo. 714 mg of the hydrochloride of antibiotic A 9578, which is very antibiotically active, is obtained.



  Example 6.



   With 5.5 kg of Hyflo supercel, 600 liters of a culture filtrate obtained according to Example 1 are stirred, adjusted to a pH of a value of 4.0 using 2.5 liters of binormal hydrochloric acid, and then filter. The filtration residue is washed with 100 liters of water. The clear filtrate is stirred for 45 minutes with 7 kg of previously treated nor.ite. The pretreatment of norite takes place by stirring it several times with normal hydrochloric acid and then washing neutral with water. The antibiotic-laden norite is separated by filtration. The filtrate does not contain antibiotic activity. The norite adsorbate is washed twice with 200 liters of water, by stirring and filtration. The washing liquid does not contain activity.

   Elution takes place by stirring the activated carbon twice for one hour with a mixture of normal butanol, methanol, glacial acetic acid and water in a volumetric ratio of 2: 1: 1: 2, the activated carbon being separated by filtration. After combining the eluates (140 liters + 46 liters), they are mixed well with 96 liters of n-butyl acetate. The aqueous phase which forms is separated and the organic phase is then washed with 1.2 liters of water. After having combined the aqueous phases (65.5 liters) they are washed, by stirring, successively with 72 liters of a mixture of normal butanol and n-butyl acetate in a volumetric ratio of 1: 2. , with 48 liters of ethyl acetate and finally with 24 liters of ether. The organic phases are discarded.



  The remaining aqueous phase (44 liters), which contains all of the antibiotic activity, is concentrated to no more than 30, in a Quickfitt evaporator, to a volume of 5.45 liters. From this highly active, black-brown concentrate, the antibiotic is obtained by refrigeration drying as 509 g of a brown powder. This material exhibits, relative to the culture filter, a specific antibiotic activity 30 to 50 times stronger.



  Example 7.



   To isolate the antibiotic A 9578 from 300 liters of a culture filtrate obtained according to Example 1, one part by volume of Amberlite IRC-50, in the hydrogenated form, is mixed with two parts by volume. Amberlite IRC-50, in sodium form. 6.3 liters of this mixture are poured into a glass column. The height to diameter ratio of the resin bed is 8: 1.

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  The culture filtrate is adjusted to a pH of 4.0 with binormal hydrochloric acid and is percolated, at a rate of passage of 0.2 liters per minute per liter of resin, through said resin, which causes an orange-brown area to form in the upper two-thirds of the column. The resin is then washed with 30 liters of water and with 60 liters of 80% methanol. The product that passes and the washing liquids contain little antibiotic activity. Elution takes place in two portions with a total of 37 liters of a mixture of 8 parts by volume of methanol and 2 parts by volume of normal hydrochloric acid.

   The two portions are adjusted to a pH of 5.0 with the aid of a penta-normal solution of sodium hydroxide, combined and then concentrated, at a temperature at most equal to 30, to a volume of 2 liters in a circulating evaporator. The aqueous concentrate is adjusted to a pH of 5.6 and, in order to remove little insoluble matter, it is filtered through Hyflo-superoel.



  This filtrate (2.3 liters) contains substantially all of the antibiotic activity of the culture solution. It is extracted in 4 portions with a total of 500 cm3 of a mixture of phenol and chloroform containing 100 g of phenol in 100 cm3 of chloroform. The aqueous phase is discarded. The extract (500 am3) obtained with the mixture of phenol and chloroform is diluted with one liter of chloroform, then extracted three times with each time 500 om3 of a decinormal solution of an ammonium acetate buffer. with a pH of 4.60, which makes it possible to withdraw from the organic phase the colored impurities which are inactive from the antibiotic point of view. The chloroform phase is then extracted with 300 + 2 times 100 cm3 of a decinormal solution of hydrochloric acid.

   The red-colored acidic extract formed which contains the antibiotic is adjusted to a pH of 3.5 with potassium bicarbonate and back extracted with 4 portions of 50 cm3 of a mixture of phenol and chloroform (100 g: 100cm3). The extract obtained with the mixture of phenol and chloroform is filtered through celite. While shaking vigorously, 50 cm3 of water, 500 cm3 of ether and 300 cm3 of petroleum ether are added to the clear filtrate (200 om3) of red color. After separating the aqueous phase, the organic phase is then washed twice with 50 cm 3 of water. After combining the aqueous extracts, they are extracted twice with 500 cm3 of ether and once with 500 cm3 of benzene, and then lyophilized.



  2.60 g of an orange-brown powder are obtained, which has a high antibiotic activity. Compared to the starting material, this material exhibits a specific antibiotic activity 500 to 1000 times greater (activity per unit weight of dry substance) o On Whatman N 1 paper, this material is present in the normal butanol system: n-butyl: glacial acetic acid water = 10: 3: 1.3: 14.3, during autobiographical development with Staphylococcus aureus, 2 spots of substance. The slower migrating antibiotic substance is referred to as base A, and the substance that migrates two and a half times faster is referred to as base B. Base A provides a blue colored reaction on the paper when touching with a mixture of Fecl3 and K3feCl6.



  Example 8.



   In 1.5 liters of water, 338 g of an antibiotic preparation obtained according to Example 6 are dissolved. 150 g of crystalline ammonium sulphate are added to the whole and the solution is extracted with one liter + twice. 500 cm3 of a mixture of phenol and chloroform containing 100 g of phenol in 100 cm3 of chloroform.



  After combining the extracts obtained with the mixture of phenol and chloroform, they are extracted with 750 cm3 of normal hydrochloric acid in order to then filter them through a layer of celite. While stirring, to the clear red-brown filtrate are added 600 cm3 of water, 4 liters of ether and finally 4 liters of petroleum ether. The aqueous phase is separated and the organic phase is then extracted with twice 200 cm3 of water. After combining the aqueous phases (one liter), they are washed twice with one liter of ether, and then freeze-dried. 136 g of a brown powder are obtained which, relative to the starting material, shows a specific antibiotic activity increased by a factor of 2.

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  Example 9.



   In 55 cm3 of a decinormal solution of an ammonium acetate buffer with a pH of 4.60, 550 mg of a highly active antibiotic preparation (base B) obtained is dissolved. according to Example 1 and extracted four times with 20 cm3 of a mixture of phenol and chloroform containing 100 g of phenol in 400 cm3 of chloroform. The organic extracts are washed in return with twice 15 cm 3 of the buffer solution. The organic extract (80cm3) containing the antibiotic is diluted with 40cm3 of chloroform, washed again with 60cm3 of the buffer solution, and then filtered through a double pleated filter.



  The dark red filtrate is extracted with 30 + 20 + 10 cm3 of 0.2-normal hydrochloric acid. The acidic solution containing the antibiotic is diluted with 50 cm3 of water, then extracted to exhaustion with 2 times 20 ± 10 cm3 of a mixture of phenol and chloroform containing 100 g of phenol in 100 cm3 of chloroform. After combining the extracts obtained with the mixture of phenol and chloroform, they are filtered through a double pleated filter and extracted with 20 cm3 of water, 200 cm3 of ether and 100 cm3 of petroleum ether. After having separated the aqueous phase, the organic phase is then extracted with twice 15 cm 3 of water. After combining the aqueous extracts; they are washed twice with 50 cm3 of ether and once with 50 cm3 of benzene, and then lyophilized.

   117 mg of a reddish-brown powder are obtained which, based on the starting material, shows an approximately five-fold enrichment of the antibiotic activity.



  Example 10.



   On 127 g of Whatman N 1 cellulose powder, 700 mg of an antibiotic preparation obtained according to Example 7 are chromatographed. As eluting agent, the upper phase of a mixture of 4 parts of butanol, d. 'one part of glacial acetic acid and 5 parts of water. After separating the upper phase, 10% by volume of pure butanol is added thereto. The substance is kneaded with a tenfold amount of cellulose powder and poured as a powder onto the column. The flow rate is 15 to 20 cm3 per hour.



  Fractions up to 40 cm3 are collected. The various fractions are extracted with 50 cm3 of petroleum ether. The separated aqueous phase is washed with benzene and then lyophilized. Most of the antibiotic activity is found in fractions 7 and 8 (121 mg) and in fractions 10 to 13 (142 mg).



  According to the examinations carried out by chromatography on paper, fractions 7 and 8 contain mainly base B and fractions 10 to 13 mainly base A.



  Example 11.



   In a Craig distribution apparatus, 3 g of an antibiotic preparation are distributed in 100 stages in the secondary butanol-solution-decinormal buffer solution of ammonium acetate with a pH of 4.68. obtained according to Example 8. Each unit contains 100 cm3 of an upper phase and 100 cm3 of a lower phase. Is the substance placed in the unit? 3. The treatment is carried out by adding to the mixture of the two phases a double volume of petroleum ether while lyophilizing the aqueous phase. Dark fractions 3 to 11 contain little antibiotic activity. Fractions 12 to 20 (631 mg), which are yellow-orange in color, contain most of the antibiotic activity (especially base A).

   Fractions 21 to 40 which are yellow in color are weakly active and contain a mixture of base A and base B in which the second predominates.



  Example 12.



   In a Craig distribution apparatus, one distributes in 29 stages, in the system 1/20-normal buffer solution of ammonium acetate, with a pH of 4.58, - 10% of phenol in the chloroform, 200 mg of an antibiotic preparation (base A) obtained according to Example 10. Each stage contains 10 cm3 of an upper phase and 10 cm3 of a lower phase. Most of

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 the antibiotic activity is found in fractions 6 to 15. These latter (about 200 cm3) are combined, 400 cm3 of ether and 300 cm3 of petroleum ether are added thereto, then shaken. After having separated the aqueous phase of red-orange color, it is washed with chloroform and extracted with 20 + 3 times 10 cm3 of a mixture of 100 g of phenol in 100 cm3 of chloroform.

   While stirring, to the extract obtained with the mixture of phenol and chloroform, 20 cm3 of water, 300 cm3 of ether and 200 cm3 of petroleum ether are added. The red-colored aqueous phase is separated, washed with plenty of ether and with benzene, and then freeze-dried. 86.4 mg of base A are obtained as a yellow powder which is soluble in water. Ultraviolet spectrum in water: # max 215 mu
 EMI12.1
 (E m = 312), 9,315 1 (E 1 cm = Q-2).



  Colored reactions: FeCl3: red-brown;
Fec13 + K3Fe (CN) 6: blue; Ninhydrin: weakly positive
Negative: Sakaguchi, Maltol, Elson-Morgan.



   CLAIMS.



   1. A process for the preparation of a novel antibiotic, its components, salts and derivatives, characterized in that a strain of actynomycetes exhibiting the properties of Streptomyces A 9578, or a mutation, is cultivated in a nutrient solution. of this strain, until said solution exhibits a noticeable antibiotic effect and the antibiotic A 9578 is then isolated from the nutrient solution and, if desired, divided into its components and / or that salts or derivatives of these compounds are prepared.



   The present process can be further characterized by the following points:
1) Actinomycetes strain A 9578, or a mutation of this strain, is used.



   2) The culture is carried out under aerobic conditions, preferably in culture immersed in an aqueous medium containing assimilable carbohydrates, nitrogen compounds, inorganic salts, as well as, where appropriate, substances promoting the growth. growth.



   3) Cultivation takes place for 36 to 120 hours, at a temperature between 18 and 40, preferably at 27.



   4) The culture filtrate is separated from the mycelium at a pH between 3 and 5.



   5) The antibiotic is isolated from the culture solution by adsorption using activated carbon, activated earths or resin adsorbents.



   6) The adsorbed antibiotic is extracted with an acid eluting agent.



   7) An organic solvent miscible with water or an aqueous mixture of such a solvent is used as the elution liquid.



   8) A mixture of methanol and a dilute aqueous solution of acetic acid is used as elution liquid.



   9) The eluate is concentrated in vacuo at low temperature and the antibiotic is precipitated from the concentrate with acetone.



   10) The antibiotic is isolated from the culture solution by adsorption using a cation exchanger.

** ATTENTION ** end of DESC field can contain start of CLMS **.


    

Claims (1)

11) The antibiotic is adsorbed by means of a weakly acidic ion exchanger containing carboxylic groups and is eluted from it with <Desc / Clms Page number 13> an acid eluting agent. 12) The antibiotic is adsorbed using a buffered ion exchanger containing carboxylic groups and eluted therefrom with an acid eluting agent. 13) The antibiotic is enriched by partitioning between solutions of phenol in chloroform and aqueous solutions. 14) The phenol content of the chloroform solution is varied. 15) The pH value of the aqueous solution is varied. 16) The antibiotic is enriched using chromatography. 17) The antibiotic is enriched using chromatography on cellulose. 18) The antibiotic is enriched using a distribution between two immiscible solvent phases. 19) The antibiotic is precipitated in an aqueous medium using an organic acid of the picric acid type and the precipitate is isolated. 20) The sparingly soluble salt of the antibiotic is reacted with an inorganic acid, in an aqueous medium or in organic solvents miscible with water, preferably in methanol or acetone, with the organic acid of the antibiotic. type of picric acid, then isolates the inorganic salt of the antibiotic that has formed. 21) The inorganic salts of the antibiotic, preferably the sulphate, are reacted on an inorganic base, the cation of which forms with the anion of the antibiotic salt a precipitate which is hardly soluble in aqueous medium, for example on water. barium hydroxide, then isolates the base of the antibiotic which has formed in the free state. 22) Using an anion exchange resin, the salts of the antibiotic are converted into the free base and the latter is isolated. 23) The isolation and treatment methods described under 4) to 22) are suitably combined. II. As new industrial products: 24) The new antibiotic A 9578 as well as its components and salts and derivatives. 25) Component A of antibiotic A 9578. 26) Component B of antibiotic A 9578. 27) The hydrochloride of antibiotic A 9578, as well as components A and B. 28) Sulfates of antibiotic A 9578, as well as components A and B. 29) Phosphates of antibiotic A 9578, as well as components A and B. 30) Pantothenate from antibiotic A 9578, as well as components A and B. 31) Pharmaceutical preparations containing the antibiotic A 9578, its components, salts or derivatives, or mixtures thereof.