CH630118A5 - Process for the preparation of the antibiotic N-acetylthienamycin - Google Patents

Description

The invention relates to a method for producing a new antibiotic in the form of a new antibiotic substance, which is referred to here as N-acetylthienamycin. The new antibiotic available by the method can be in diluted form, as a raw concentrate or in pure form.

The invention has for its object to provide a method for producing a new and valuable antibiotic, which inhibits the growth of various gram-negative and gram-positive microorganisms in a highly effective manner, by fermentation of nutrient media with a microorganism.

The new antibiotic N-acetylthienamycin is produced according to the invention as defined in claim 1. The cultivation of Streptomyces cattleya in a nutrient medium also leads to the formation of thienamycin. Acetylation of thienamycin is another method for the production of N-acetylthienamycin. The production of thienamycin by fermentation of Streptomyces cattleya is described in U.S. Patent Application Serial No. 526 992 and in the U.S. patent application Serial No. 613 822, which in turn is a partial continuation application of U.S. Patent Application Serial No. 534 382.

Based on extensive classifying studies, the microorganism Streptomyces cattleya isolated from a soil sample was identified as Actinomycete and designated MA-4297 in the culture collection by Merck & Co., Inc., Rahway, New Jersey. A culture of this microorganism is permanently on November 18, 1974 in the culture register of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, has been assigned with the identification number NRRL 8057.

The characteristic morphological and cultural properties of Streptomyces cettleya are summarized in Table I.

Table I

Morphology-The sporophores are compact spirals that appear as side and end branches on the aerial mycelium. The spores are ellipsoidal to cylindrical in shape, have a size of 0.9 pi X 1.2 [x and occur in chains of more than 10.

Cultural characteristics Tomato porridge oatmeal agar

Vegetative growth - back - brownish, flat, spreading;

Aerial mycelium - orchid colors (10 gc), mixed with white; Soluble pigment - none.

Czapek dox agar (sucrose nitrate agar)

Vegetative growth - colorless, flat, spreading; Aerial mycelium - sparse, white with pink tinge;

Soluble pigment - none.

Egg albumin agar

Vegetative growth - brownish with gray-orchid color-

approach, flat, spreading;

Aerial mycelium - orchid colors (10 gc) mixed with lighter

Tones of orchid color and some white;

Soluble pigment - none.

Glycerin-asparagine agar

Vegetative growth - back - brownish with gray

pink approach, flat, spreading;

Aerial mycelium orchid colors (10 gc) mixed with something

White;

Soluble pigment - none.

Yeast extract glucose + salt agar

Vegetative growth — brownish with a gray-pink tinge; Aerial mycelium orchid colors (10 gc) mixed with pink

White;

Soluble pigment — none.

Yeast extract-malt extract agar

Vegetative growth — brownish;

Aerial mycelium - orchid colors (10 gc), mixed with pink

White;

Soluble pigment - none.

Peptone Iron Yeast Extract Agar

Vegetative growth - brownish;

Aerial mycelium - none;

Soluble pigment - weak browning of the medium; Melanin - negative;

H2S development - negative.

Nutrient agar

Vegetative growth — light brown;

Aerial mycelium - none;

Soluble pigment - none.

Nutrient starch agar

Vegetative growth - cream-colored to brownish; Aerial mycelium - none;

Soluble pigment - none;

Hydrolysis of starch - moderate.

Nutritional gelatin agar

Vegetative growth - cream-colored;

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Aerial mycelium - none;

Soluble pigment - none;

Liquefaction of gelatin - massive.

Vertical gelatin tube

Vegetative growth - brownish;

Aerial mycelium - none;

Soluble pigment - none;

Liquefaction of gelatin - moderate.

Potato wad

Vegetative growth - moderate, brownish;

Aerial mycelium - scanty, grayish-pink-white;

Soluble pigment - none.

Loeffler's blood serum

Vegetative growth - cream-colored;

Aerial mycelium - none;

Soluble pigment - none;

Liquefaction - none.

Skimmed milk agar

Vegetative growth - brownish;

Aerial mycelium - scanty, whitish;

Soluble pigment - weak browning of the medium; Hydrolysis of casein - positive.

Litmus milk

Vegetative growth - brownish to brown;

Aerial mycelium - none;

Color - no soluble pigment, litmus indicator turns bluish;

Coagulation and / or peptonization - partial peptonization, becomes alkaline.

Skimmed milk

Vegetative growth - brownish;

Aerial mycelium - none;

Soluble pigment - none;

Coagulation and / or peptonization - partial peptonization, becomes alkaline.

Tyrosine agar

Vegetative growth - brownish;

Aerial mycelium mixture of orchid colors (10 gc) and

White;

Soluble pigment - none;

Decomposition of tyrosine - positive.

Unless stated otherwise, all of the above characteristics were determined after three weeks of incubation at 18 ° C. The media used in these studies were approximately neutral (pH 6.8 to 7.2). The color names used in the description correspond to the definitions in “Color Harmony Manual”, 4th edition (1958), published by the Container Corporation of America, Chicago, Illinois.

Streptomyces cattleya was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown for three weeks at 28 ° C on basal synthetic medium (Pridham and Gottlieb) containing the carbohydrate in question in a concentration of 1%. The media used in these studies were approximately neutral (pH 6.8 to 7.2). Table II shows the utilization of these carbohydrates by Streptomyces cattleya, where + means good growth, ± poor growth and -no growth on the carbohydrate in question.

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Tables

glucose

+

Maltose

±

Arabinose

-

Mannitol

+

Cellulose

-

Mannose

±

Fructose

±

Raffinose

Inositol

-

Rhamnose

Lactose

-

Sucrose

■

Xylose

±

The following applies to the extent of growth with temperature changes, the oxygen requirement and the effect on nitrate of the microorganism:

Temperature range (yeast extract glucose + salt agar);

28 ° C - good

37 ° C — moderate

50 ° C - no growth

Oxygen demand (needle vaccination culture in yeast extract glucose salt agar);

aerobic.

Nitrate reduction - positive

The new antibiotic N-acetylthienamycin, produced according to the invention, is produced in aerobic fermentation of suitable aqueous nutrient media under controlled conditions by inoculation with the organism Streptomyces cattleya. The same aqueous media that are also suitable for the production of other antibiotics are suitable for the production of N-acetylthienamycin. Such media contain C and N sources which can be assimilated by the microorganism, and also inorganic salts.

In general, carbohydrates such as sugar, e.g. Glucose, fructose, maltose, sucrose, xylose, mannitol and the like, and starches such as cereals, e.g. Oats, rye, corn starch, corn flour and the like can be used alone or together with other sources of assimilable carbon in the nutrient medium. The exact amount of carbohydrates in the medium depends in part on the other components of the medium; in general, however, the amount of carbohydrates varies between 1 and 6% by weight of the medium. These C sources can be used individually, or several such C sources can be combined in the medium. In general, many protein substances can be used as N sources in the fermentation process. Suitable N sources are e.g. Yeast hydrolysates, primary yeast, soybean meal, cottonseed meal, casein hydrolyzates, corn steep liquor, soluble slurry ingredients or tomato paste and the like. The N sources can be used alone or in combination with one another in amounts of 0.2 to 6% by weight of the aqueous medium.

The inorganic nutrient salts that can be added to the culture media include the usual salts that provide sodium, potassium, ammonium, calcium, phosphate, sulfate, chlorine, carbonate ions and other ions. This subheading also includes trace metals such as cobalt, manganese, iron and magnesium.

The culture media described in the examples are for illustration only; you can use a variety of media.

The fermentation is conveniently carried out at a temperature of 20 to 37 ° C; However, for best results, the fermentation is preferably carried out at a temperature of 22 to 30 ° C. The pH of the culture medium suitable for growing the culture of Streptomyces cattleya and for producing N-acetylthienamycin can vary in the range from 6.0 to 8.0.

The new antibiotic N-acetylthienamycin arises both in surface culture and in submerged culture. According to the invention, however, the fermentation is carried out in submerged culture.

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The fermentation for producing the antibiotic is expediently carried out on a small scale by inoculating a nutrient medium with the culture producing the antibiotic and continuing the fermentation after transfer to a production medium for several days in a shaking machine at a constant temperature of about 24 ° C.

The fermentation is expediently started in a sterilized flask filled with nutrient medium over one or more stages of the development of the inoculum. Any combination of C and io N sources can be used as the nutrient medium for the inoculum level. The inoculation flask is e.g. Shaken in a constant temperature chamber at about 28 ° C for a day or two until the culture is satisfactory, and part of the resulting culture is used to inoculate either a two-step inoculum or to inoculate the production medium. Intermediate inoculation flasks, when used, can be developed in essentially the same way, i.e. part of the flask content of the last inoculation stage is used to inoculate the production medium. The inoculated flasks are conveniently shaken at constant temperature for several days, and at the end of the incubation period the contents of the flask can be centrifuged or filtered.

For work carried out on a large scale, the fermentation is preferably carried out in suitable fermenters which are provided with a stirrer and a system for venting the fermentation medium. According to this method, the nutrient medium can be mixed in the fermenter and sterilized by heating to a temperature up to approximately 120 ° C. After cooling, the sterilized medium can be inoculated with a previously grown inoculum of the culture and the fermentation can be e.g. Continue for 3 to 5 days, with stirring and / or aeration of the nutrient medium and at a temperature of approximately 24 ° C. This method for the production of N-acetylthienamycin is particularly suitable for the 3s production of large amounts of the antibiotic.

Physical and chemical properties of N-acetyl-thienamycin

An NMR spectrum of N-acetylthienamycin at 100 MHz shows the following maxima: 40

CH3-CH (OH)

01.27, d, 3H, J «6.5; 01.98, S, 3H; 02.94, m, 2H; 03.17, m, 2H; 03.38, t, 2H, Jss 6.5; 03.38, m, 1H;

04.20, m, 2 H.

The NMR spectrum of a combined solution of N-acetylthienamycin obtained by fermentation and thienamycin obtained by acetylation cannot be distinguished from the spectra of the individual solutions. A pKa of 3.3 ± 0.1 for the COOH group in N-acetylthienamycin was determined from the dependence of the value for Xmax on the pH.

In paper electrophoresis in 0.1 molar potassium phosphate buffer solution (pH 7) using paper No. 2043-B from Schleicher and Schuell at a voltage gradient of 50 V / cm, both the N-acetylthienamycin obtained by fermentation and the one migrate N-acetylthienamycin obtained by acetylation of thienamycin in the course of 20 minutes at 10 ° C. over a distance of 2.7 cm to the anode. The antibiotic is localized by bioautography to Vibrio percolans ATCC 8461 (without drying the paper in between) and the migration is measured from the point of application to the middle of the inhibition zone.

Thin layer chromatography using sheets coated with cellulose and a solvent mixture of 70% ethanol and 30% water shows an Rf for the N-acetylthienamycin obtained by fermentation and for the acetylation of thienamycin of 0.7, detected by bioautography on Vibrio percolans ATCC 8461. The Rr value refers to the distance from the point of origin to the center of bioactivity, divided by the distance from the point of origin to the solvent front.

The IR spectrum of N-acetylthienamycin is shown in the drawing.

N-Acetylthienamycin is believed to have the following molecular structure:

O

S-CH2-CH2-NH-C-CH3

(I)

COOH

N-Acetylthienamycin is also characterized by the antibiotic spectrum profile that follows Escherichia coli. This test is carried out using the Bauer-Kirby disc diffusion method,

which is only changed in relation to the depth of the agar layer,

which in this case is 2 mm. The results given as the diameter of the zone of inhibition in millimeters can be found in Table III. Table III shows the antibiotic spectrum profile for N-acetylthienamycin and also for the compound Enterobacter obtained by acetylating thienamycin. cloacae

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Klebsiella pneumoniae

Table III Test organism

Staphylococcus aureus

Bacillus subtilis

Merck No.ATCC No.

MB2985 -MB2314 -

MB 964 6633

Antibiotic resistance *

60 Proteus mirabilis Proteus morganii Serratia

65

Pseudomonas aeruginosa

MB2884 MB2964 MB2482

MB2921 MB2922

MB2646 MB2647

MB2830

MB2833

MB2840

MB2824 MB2835 MB3286

P, C P

P P

P, C

p, c p, c

P, C

P, C P, c P, C

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Test organism

Merck No.

N-acetyl-thienamy-cin (a) 9.85 y per disc **

N-acetyl-thienamy-cin (b) 10.35 y per disc **

Staphylococcus aureus

MB2985 MB2314

37 37

37 37

Bacillus subtilis

MB 964

43

44.5

Escherichia coli

MB2884 MB2964 MB2482

31.5 28.5 28

31.5 29.5 28

Klebsiella pneumoniae

MB2921 MB2922

28 27

28.5 30

Enterobacter cloacae

MB2646 MB2647

26.5 29

27 29

Proteus mirabilis

MB2830

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26

Proteus morganii

MB2833

25.5

25th

Serratia

MB2840

27th

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Pseudomonas aeruginosa

MB2824 MB2835

MB3286

23 11

(unset) 0

24 11

(unset) 0

* P = penicillins represented by ampicillin. C = cephalosporins represented by cephalothin.

** The weight calculations are based on an assumed E7 ™, 30i nm - 290 and an extinguishability by hydroxylamine of 96% for the pure material.

(a) Produced by fermentation of Streptomyces cattleya.

(b) Made by acetylating thienamycin.

N-acetylthienamycin shows in vivo activity against gram-negative and gram-positive organisms and is therefore suitable for combating bacterial infections in humans and animals. To determine the in vivo activity, N-acetylthienamycin is dissolved in 0.01 molar sodium phosphate buffer solution with a pH of 7.0 and diluted with this solution to five four-fold concentrations of the antibiotic for the investigation. Female white Swiss mice with an average weight of approximately 21 g are infected intraperitoneally with the test organism suspended in broth. The number of organisms injected is determined according to standardized plate counting methods. At the time of infection and 6 hours later, some of the mice are treated with the antibiotic intraperitoneally. Five mice are used for each concentration of the antibiotic examined. Each test includes controls of five mice for each of the different dilutions of the infecting culture to calculate the number of organisms lethal to 50% of the infected, untreated mice (LD50). This calculation is made based on survival data on the seventh day after infection, and at this point the amount of antibiotic that should protect 50% of the infected mice is also calculated (ED50).

All mice so infected and not treated with the antibiotic die within 48 hours of infection. The effectiveness of N-acetylthienamycin is shown in Table IV.

Table IV

Efficacy studies in mice (a)

Organism Staphylococcus aureus 2949

No.LD50 13

Form of administration, cans i.p. X 20)

ED50, mg / kg / dose (c) 0.050

(a) CD-1, female mice, body weight 21 g.

(b) Indicates treatment at the time of infectious dose administration and 6 hours thereafter.

^ Weight of N-acetylthienamycin based on the estimated value E1ft> cmj 301 = 290 and an extinction by hydroxylamine of 96% for the pure material.

N-acetylthienamycin is a valuable antibiotic that is active against a wide variety of gram-positive and gram-negative bacteria and is therefore used in both human and veterinary medicine. The compound made according to the invention can be used as an antibacterial drug for the treatment of infections caused by gram-positive or gram-negative bacteria, e.g. against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae. The antibacterial agent described can also be used as an additive to animal feed, for food preservation and as a disinfectant. For example, it can be used in aqueous compositions in concentrations of 0.1 to 100 parts of antibiotic per million parts of solution, or preferably in concentrations of 1 to 10 parts of antibiotic per million parts of solution, to inhibit the growth of harmful bacteria on medical and dental equipment to destroy and inhibit, and as a bactericid for technical applications, for example in water-based paints, in the white water of paper mills and to inhibit the growth of harmful bacteria.

The antibiotic described can be used in a wide variety of pharmaceutical preparations as the sole active ingredient or in combination with one or more other antibiotics or with one or more pharmacologically active substances. As an example of the former case, an aminocyclitol antibiotic, such as gentamicin, can be given at the same time in order to expand the antibacterial spectrum and to minimize the possibility of the occurrence of resistant organisms. As an example of the latter case, diphenoxylate and atropine can be combined in dosage forms for the treatment of gastrointestinal diseases. The antibiotic can be used in the form of capsules, tablets, powders, liquid solutions or as a suspension or elixir. It can be given orally, locally, intravenously or intramuscularly.

Oral delivery tablets and capsules may be in unit dosage form and conventional excipients such as binders e.g. Syrup, acacia resin, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone, fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine, lubricants, e.g. Magnesium stearate, talc, polyethylene glycol, silica, disintegrants, e.g. Potato starch or safe wetting agents such as sodium lauryl sulfate. Tablets can be coated by methods known per se. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, etc., or as a dry product for mixing with water or other carriers before use. Such liquid preparations can be conventional additives such as suspending agents e.g. Sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose

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cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, e.g. Lecithin, sorbitan monooleate or acacia resin, non-aqueous vehicles such as edible oils, e.g. Almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol, preservatives, e.g. p-Hydroxybenzoic acid methyl or propyl ester or sorbic acid. Suppositories can contain conventional suppository bases such as cocoa butter or other glycerides.

Means for injection can be provided in unit dose form in ampoules or in multiple dose form in containers with the addition of preservatives. The agents can take the form of suspensions, solutions, emulsions in oily or aqueous vehicles and contain formulation agents such as suspending, stabilizing and / or dispersing agents. The active ingredient may also be in powder form and, before use, with a carrier, e.g. sterile, pyrogen-free water.

The agents may also be in a form suitable for absorption by the mucous membranes of the nose and throat or the bronchial tissues, e.g. as powder or liquid spray or inhalant, lozenges, pharynx paints, etc. For the treatment of the eyes or ears, the preparations can be provided as individual capsules, in liquid or semi-liquid form or used as drops, etc. For local application, they can be present in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints, powders, etc.

In addition to a carrier, the agents can contain other constituents, such as stabilizers, binders, oxidation retardants, preservatives, lubricants, suspending agents, viscosity-influencing agents or flavorings and the like.

In veterinary medicine, such as in the treatment of chickens, cows, sheep, pigs and the like, the agents can e.g. are formulated as preparations for injecting into the breast that are long-term effects or release the active ingredient quickly.

The dosing schedule and mode of administration largely depend on the condition of the animal to be treated, the weight of the animal, the sensitivity of the infecting microorganism and the stage of infection, with parenteral administration being preferred for general infections and oral administration for intestinal infections.

For the treatment of bacterial infections in humans, the described compound can be administered orally or parenterally by conventional methods for the treatment with antibiotics in amounts of 2 to 600, preferably 5 to 100 mg / kg / day and preferably divided into individual doses, e.g. be given three to four times a day. Unit dosage forms can be used, e.g. Contain 25, 250, 400, 800 or 1000 mg of active ingredient together with physiologically acceptable carriers or extenders. The dose units can be in the form of liquid preparations, such as solutions or suspensions, or as solid substances in tablets or capsules. The optimal dose for each individual case depends on the type and severity of the infection, smaller doses being used for the treatment of children; all such design rules are known to the person skilled in the art.

The invention also encompasses the non-toxic, pharmaceutically acceptable salts of N-acetylthienamycin, e.g. the pharmacologically acceptable salts that are formed with inorganic or organic bases. These include e.g. Metal salts derived from alkali or alkaline earth hydroxides, carbonates or bicarbonates, e.g. Sodium, potassium, ammonium and calcium salts, and also salts of primary, secondary or tertiary amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkali

anolamines, lower dialkanolamines, lower alkylenediamines, N, N-diaralkyl-lower alkylenediamines, aralkylamines, amino-substituted lower alkanols, lower alkanols substituted by lower N, N-dialkylamino groups, amino-, polyamino- and guanidino-substituted amine-containing lower alkane acids . Typical examples are salts derived from sodium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine , Ethylenediamine, N, N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminethanol, 2-amino-2-methylpropanol- (l), theophylline, N-methylglucamine and the like are derived.

The salts of the compound described can be prepared by known methods. So you get e.g. the mono salts, such as the monosodium salt, by reacting 1 equivalent of sodium hydroxide with 1 equivalent of the product of the process according to the invention in a suitable solvent. Mixed salts with divalent cations can also be prepared by combining 1 mol of a divalent base with 1 mol of the product of the process according to the invention and 1 equivalent of another acid. Salts can also be prepared by reacting 1 equivalent of a base with a divalent cation, such as calcium hydroxide, with 1 equivalent of the product of the process according to the invention. These salts are pharmacologically acceptable, non-toxic derivatives that can be used as active ingredients in suitable pharmaceutical unit dosage forms. They can also be combined with other drugs to get agents with a wide range of activities.

The fermentation liquids containing the antibiotic obtained by the methods described here have activities in the range of about 0.1 to 4 y / ml. The cleaning of the antibiotic preparations and the extraction of the antibiotic can be carried out according to various methods. One such method is to filter filtered fermentation liquid containing N-acetylthienamycin through a column of a strong cation exchange resin. Examples of such resins are those of the sulfonate type with a styrene-divinylbenzene skeleton, e.g. the core polystyrene resin "Dowex" -50 X 2 (manufactured by Dow Chemical Co., Midland, Michigan) containing sulfonic acid groups in the sodium form. Other representative representatives of the class of strong cation exchange resins are the following: "Dowex-50 X 4," Dowex "-50 X 8 (manufactured by Dow Chemical Co., Midland, Michigan)," Amberlite "IR120 (manufactured by Rohm & Haas Co., Philadelphia, Pennsylvania), Duo-lite C25D (manufactured by Chemical Process Co., Redwood City, California), "Permutit" Q (manufactured by Permutit Co., Birmingham, New Jersey), "lonac" C- 249 (manufactured by lonac Chemical Co., Birmingham, New Jersey) and «Am-berlite» 200.

The outlet of the cation exchange resin containing the antibiotic N-acetylthienamycin can optionally be further cleaned by other cleaning methods. Thienamycin remains adsorbed on the strong cation exchange resin. Accordingly, the process for separating these two compounds is clearly distinguishable.

One such method is to adsorb N-acetylthienamycin on a strongly basic anion exchange resin. Examples of such strongly basic anion exchange resins are those with a styrene-divinylbenzene skeleton, e.g. the quaternized ammonium resin "Dowex" -l X 2 (manufactured by Dow Chemical Co., Midland, Michigan) in the polystyrene core in the chloride form. Other representative members of this class of strongly basic ion exchange resins

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are: "Duolite" A-40, A-42, A-101, A-102 and A-l 14 (can be further processed after a number of process steps

manufactured by Chemical Process Co., Redwood City, California); in which the following chromatographic measurements

"Amberlite" IRA-400, IRA-401 and IRA-410 (manufactured by Dien are used: anion exchange resins from polystyrene

from Rohm and Haas, Washington Square, Philadelphia 5, rol-trimethylammonium type (e.g. «Dowex» -l X 2 in the Chlo-

Pennsylvania). The antibiotic 5 ridform contained in the eluate, polymeric adsorbent (e.g. XAD-2, a poly-

N-acetylthienamycin can be further purified by using styrene resin), «Dowex» -l X 4 in the chloride form and gel filter

it passes through a column which is filled with an acrylic ester position resin (e.g. "Bio-Gel" P-2, a polyacrylic acid amide resin).

of medium polarity, such as XAD-7 or The bioactivity of the eluates is measured by using the

8, or by a column with a non-polar, hydro-eluate using Staphylococcus aureus ATCC

phobic, cross-linked polystyrene-divinylbenzene polymer 10 6538P as test organism or, if the purity allows it,

is like XAD-1,2 and 4, preferably XAD-2. (XAD- due to the extinction extinguished by hydroxylamine

1,2,4,7 and 8 are analyzed by Rohm and Haas, Wash. A preferred method of obtaining Ington Square, Philadelphia 5, Pennsylvania.) N-acetylthienamycin from fermentation liquids is available in Ta-

Further purified N-acetylthienamycin is obtained e.g. belle V stated.

by gel filtration through polyacrylic acid amide gel with a 15 In the examples below, embodiments are

Ren size that explains molecules with molecular weights of more than men of the inventive method. The example

1800 excludes, like "Bio-Gel" P-2 (manufactured by Bio Rad, le, however, are used only for illustration; the invention encompasses

Richmond, California). also functionally equivalent embodiments of the method

A preferred method of obtaining purified rens. Therefore, any modification of the

N-Acetylthienamycin is to consider a solution of the antibiotic forms that cums to form an identical product, like the filtered fermentation liquid, the pH of which leads as an analogous method. The described here

has been set to 4 to 5 to pass through a column, embodiments can be significantly modified,

which is a strong cation exchange resin of the sulfonate type and minor deviations fall within the scope of the inventions.

Contains sodium form («Dowex» -50 X 4). The catch-up.

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Table V

Flow chart of the cleaning procedure for the antibiotic N-acetylthienamycin

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The percentages given below are by weight unless otherwise stated.

Analytical method for antibiotic N-acetylthienamycin

I. Bioanalysis

Antibacterial activity analyzes are carried out by the following disc diffusion method using Vibrio percolans ATCC 8461 or Staphylococcus aureus ATCC 6538P as the test organism.

Panels with Vibrio percolans ATCC 8461 are manufactured as follows:

A freeze-dried culture of Vibrio percolans ATCC 8461 is suspended in 15 ml of a sterile medium containing 8 g / 1 Difco broth and 2 g / 1 yeast extract in distilled water (hereinafter abbreviated as NBYE). The culture is incubated overnight in a rotating shaker at 28 ° C. This culture is used to inoculate the surface of slant tubes containing 1.5% agar in NBYE and the inoculated slant tubes are incubated overnight at 28 ° C and then stored in the refrigerator.

The cooled oblique tubes made from a single freeze-dried culture are used for a period of up to 4 weeks after their preparation as follows: An eyelet with seed from the oblique tube is dispersed in a 250 ml Erlenmeyer flask in 50 ml NBYE. The culture is incubated overnight in a rotating shaker at 28 ° C and then diluted to a density that results in 50 percent light transmission at 660 nm. 33.2 ml of this diluted culture are added to a volume of 11 NBYE containing 15 g agar and kept at 46 ° C. The inoculated, agar-containing medium is poured into petri dishes made of plastic of 100 × 15 mm in quantities of 5 ml per dish, cooled and kept at 2 to 4 ° C. up to 5 days before use.

Plates with Staphylococcus aureus ATCC 6538P are made as follows:

A culture of the analytical organism Staphylococcus aureus ATCC 6538P developed overnight in nutrient broth containing 0.2% yeast extract is diluted with nutrient broth containing 0.2% yeast extract to a suspension with an optical transmission of 55% at a wavelength of 660 nm. This suspension is added to «Difco» nutrient agar, which has been supplemented with 2.0 g / 1 Difco yeast extract and is at a temperature of 47 to 48 ° C, giving a mixture which contains 33.2 ml of the Contains suspension per liter of agar. 5 ml of this suspension are poured into 85 mm diameter petri dishes and these plates are cooled and kept at 4 ° C until use (5 days at most).

Samples of the antibiotic to be analyzed are diluted to a suitable concentration with pH 7 phosphate buffer solution. Filter paper discs with a diameter of 6.35 or 12.7 mm are immersed in the test solution and placed on the surface of the analysis plates. The plates are incubated overnight at 37 ° C, after which the diameter of the zone of inhibition is determined in millimeters. The diameter of the inhibition zone in millimeters determines the relative strength.

II. Absorbance extinguished by hydroxylamine

The proportion of the absorbance measured at 301 nm, which can be attributed to the content of impure samples in the antibiotic, is determined by the selective extinction of this absorbance (with simultaneous inactivation of the antibiotic activity) by reaction with dilute hydroxylamine.

Samples containing the antibiotic to be tested are prepared in 0.01 molar potassium phosphate buffer solution (pH 7) so that they have an initial A30i between 0.1 and 1.0. Freshly prepared, neutralized hydroxylamine (NH2OH • HCl + NaOH to a final pH of 7) is added to a final concentration of 10 mmolar, after which the reaction is allowed to proceed at room temperature for at least 30 minutes. If the A301 value obtained in this way is subtracted from the original reading (after correction for the dilution by the added reagent), the absorbance extinguished by hydroxylamine is obtained. Solutions of pure N-acetylthienamycin show an absorbance of 96.0% which can be extinguished by hydroxylamine.

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example 1

A tube containing a freeze-dried culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents are suspended in a tube containing 0.7 ml of sterile Davis-15 salts of the following composition:

20th

Davis salts sodium citrate k2hpo4 kh2po4

(NHd) 2S04 MgS04 • 7H20 distilled water

0.5 g 7.0 g 3.0 g 1.0 g 0.1 g 1000 ml.

25 0.2 ml of this suspension are used to inoculate a culture slant tube with medium A (with agar) of the following composition:

Medium A

30 yeast autolysate («Ardamine» *) 10.0 g glucose 10.0 g

Phosphate buffer ** 2.0 ml

MgS04 • 7H20 0.05 g distilled water 1000 ml

35 pH adjusted to 6.5 with NaOH.

* "Ardamine": Yeast Products Corporation ** Phosphate buffer solution KH2P04 91.0 g

Na2HP04 95.0 g

40 distilled water 1000 ml

25.0 g of agar per liter are added for inclined tubes.

The inoculated slant tube is incubated at 28 ° C for 8 days and then stored at 4 ° C.

45 A portion of the spores and aerial mycelium of this slant tube are used to inoculate a 250 ml Erlenmeyer inoculation flask with a deflector, which contains 50 ml Medium A (without agar). This inoculation flask is shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for 2 days, after which a satisfactory culture has developed.

Fifteen 250 ml Erlenmeyer flasks, each containing 40 ml of medium B, are inoculated with 1 ml of the culture from the inoculation flask. Medium B has the following composition:

20.0 g

55 Medium B corn flour soluble

Slurry ingredients soybean meal 60 sodium citrate CaCl2 • 2H20 MgS04 • 7H20 CoC12 ■ 6H20 FeS04-7H20 65 polyglycol 2000 **

distilled water pH adjusted to 6.5 with NaOH. ** Polyglycol 2000: Dow Chemical Co.

10.0 15.0 4.0 0.5 0.1 0.01g 0.01g 0.25% by volume 1000 ml g

g g

9

630118

These 15 production flasks are shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for 53 hours. At the time of harvesting (after 53 hours) the fermentation liquids of the 15 flasks are combined and a sample is centrifuged for analysis. Before the analysis, the pH of the centrifuged fermentation liquid is adjusted from 6.5 to 5.9 with sodium hydroxide solution.

Analyzes with Staphylococcus aureus ATCC 6538P and Vibrio percolans ATCC 8461 are carried out on analysis plates using 12.7 mm disks which are immersed in the supernatant liquid of the centrifuged fermentation liquid.

The analysis results are the following:

Activity against ATCC 6538P, activity against ATCC 8461, zone of inhibition, mm zone of inhibition, mm

39/44 SH 35/44 SH

SH = a little out of focus.

200 ml of the filtered fermentation liquid are adjusted to a pH of 8.0 and adsorbed on 10 ml of «Dowex» -1X 2 resin in the chloride form at a rate of 2 ml / min, the sequence being in the form of 10 fractions of 20 ml each.

The adsorbate is eluted with a mixture of 90% by volume of methanol and 10% by volume of water, which contains 3% by weight of ammonium chloride, and the eluate is collected in 10 fractions of 5 ml each. The eluate fractions Nos. 1 to 6 are combined and concentrated in vacuo to drive off the methanol. The concentrate is analyzed according to the disk diffusion method using 12.7 mm diameter disks containing 100 ml x antibiotic solution against Staphylococcus aureus MB-2985, whereby a zone of inhibition of 28 mm is obtained.

The MB-2985 analysis plates are manufactured as follows:

A culture of MB-2985 in brain-infusion medium, developed overnight with shaking at 37 ° C., is diluted to 20,000 times and applied to the surface of 10 ml of brain-heart infusion agar in a Petri dish with a diameter of 85 mm. 1.27 mm diameter disks containing 100 | xl antibiotic solution are placed on the plates, and the plates are incubated at 37 ° C. for 18 hours. The zones of inhibition are read in millimeters.

Example 2

A tube containing a freeze-dried culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents used to inoculate a 250 ml Erlenmeyer inoculation flask provided with a deflector and containing 50 ml of medium A of the following composition:

20.0 10.0 15.0 4.0 0.5 0.1 0.01g 0.01g 0.25% by volume 1000 ml g g g g g g

Medium B Maize flour soluble slurry constituents Soybean meal s Sodium citrate CaCl2 • 2H20 MgS04 • 7H20 CoC12 • 6H20 FeS04-7H20 io Polyglycol 2000 **

distilled water pH adjusted to 6.5 with NaOH.

** Polyglycol 2000: Dow Chemical Co.

These flasks are shaken at 28 ° C at 220 rpm 15 (stroke 5.1 cm) for 3 days and analyzes are carried out during the fermentation. The analyzes are carried out on standard analytical plates with Staphylococcus aureus ATCC 6538P and Vibrio percolans ATCC 8461 using 1.27 cm discs which are immersed in the excess liquid of the centrifugate of the fermentation liquid. Before the analysis, the pH of this fermentation liquid is adjusted as shown in the table below. The results are as follows:

25 age, h

Activity against ATCC 6538P Activity against 30 ATCC 8461

Initial pH value set to sh = somewhat unset 35 h = unset

Medium A

Yeast autolysate («Ardamine» *) 10.0 g

Glucose 10.0 g

Phosphate buffer ** 2.0 ml

MgS04-7H20 0.05 g distilled water 1000 ml pH adjusted to 6.5 with NaOH.

* "Ardamine": Yeast Products Corporation ** Phosphate buffer solution KH2P04 91.0 g

Na2HP04 95.0 g distilled water 1000 ml

This inoculation flask is shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for two days, after which a satisfactory culture has developed.

Fifteen 250 ml Erlenmeyer flasks, each containing 40 ml of medium B, are inoculated with 1 ml of the culture from the inoculation flask. Medium B has the following composition:

48

53

72

33 / 39h 31 / 38h 21 / 27h

35sh / 46h 36sh / 44h 33sh / 38h 5.2 5.1 4.8 6.1 6.2 6.9

After 53 hours, the fermentation liquids are combined and filtered. 590 ml of filtrate (pH 5.9) are obtained. The pH of the filtrate is adjusted to 7.0, after which 40 5.9 mg of ethylenedinitrile tetraacetic acid (EDTA) are added.

580 ml of this filtrate (pH 7.0) are adsorbed on 130 ml of «Dowex» -l X 2 resin in the chloride form, the process being collected in the form of 2 fractions of 290 ml each. Then the adsorbate is washed with 130 ml of demineralized water. The washed adsorbate is kept overnight in the refrigerator and then eluted with 5% saline, collecting 6 fractions of 50 ml each.

The percentage recovery of the initial bioactivity, determined according to the disk plate method, is given below 50:

Fraction eluate fractions 55 No. 1 to 5

Vibrio percolans ATCC 8461

26%

Staph. aureus ATCC 6538P

1%

The antibiotic N-acetylthienamycin is found in fractions No. 1 to 5 of the NaCI eluate. Fraction No. 4 is analyzed by the disk diffusion method using 1.27 cm diameter disks containing 100 (xl antibiotic solution) against Staphylococcus aureus MB-2985, whereby an inhibition zone diameter of 29 mm is obtained. 2985 are produced by the method described in Example 1.

65

Example 3

A tube with a freeze-dried culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the

630 118

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Content suspended in 0.8 ml sterile Davis salts of the following composition:

Davis salts

Sodium citrate 0.5 g

K2HP04 7.0 g

KH2P04 3.0 g

(NH4) 2so4 1.0 g

MgS04-7H20 0.1 g distilled water 1000 ml.

This suspension is used to inoculate four slant tubes with medium A (with agar) of the following composition:

Medium A

Yeast autolysate («Ardamine» *) 10.0 g glucose 10.0 g

Phosphate buffer ** 2.0 mg

MgS04-7H20 0.05 g distilled water 1000 ml pH adjusted to 6.5 with NaOH.

* "Ardamine": Yeast Products Corporation ** Phosphate buffer solution KHoP04 91.0 g

Na2HP04 95.0 g distilled water 1000 ml

25.0 g of agar per liter are added for inclined tubes.

The inoculated slant tubes are incubated at 28 ° C for one week and then stored at 4 ° C.

10 ml of medium A (without agar) are aseptically transferred to one of these inclined tubes, the spores and the aerial mycelium are scraped in suspension, and 1.2 ml of this suspension are used to inoculate three 2 i-Erlenmeyer flasks equipped with deflection elements, each of which Contain 500 ml of medium A (without agar). These inoculation flasks are shaken at 28 ° C at 160 rpm for 24 hours, after which a satisfactory culture has developed.

The cultures from these inoculation flasks are pooled and used to inoculate 4671 Medium A (without agar) in a 7561 stainless steel fermenter. This fermenter works for 24 hours at 28 ° C, stirring at 130 rpm and passing 283 l of air per minute. Anti-foaming agent (Polyglycol 2000 from Dow Chemical Corp.) is added as needed, but not in amounts greater than 0.1%. The pH determinations have the following results:

Age, h 0 24

pH 6.3 6.4

4541 of the culture in this seeding fermenter are used to inoculate 40821 Medium E in a 56701 stainless steel fermenter. Medium E has the following composition:

Medium E

Cerelose 25.0 g

Corn steep liquor (on a wet basis) 15.0 g soluble slurry constituents 10.0 g

Cottonseed medium (Pharmamedia) 5.0 g

CoCl2 • 6H2 0.01g

CaC03 (after pH adjustment) 3.0 g

Polyglycol 2000 0.25%

Tap water 1000 ml pH adjusted to 7.3 with NaOH.

This fermenter is operated for 138 hours at 24 ° C with stirring at 70 rpm and passing air at a rate of 1.54 m3 / min. If necessary, additional anti-foaming agent (Polyglycol 2000) is added, but not in amounts of more than 0.1%. Antibacterial analyzes are carried out with the following results:

Age, h pH ATCC No. 6633

(9.5 mm discs), inhibition zone, mm

0

6.9

0

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6.3

0

36

6.0

0

48

5.9

0

60

6.0

23

72

5.9

-

84

6.0

21st

96

6.2

-

108

6.5

35

120

6.6

36

132

6.7

41

138

6.7

39

The 40821 fermentation liquid is filtered through a 76 cm filter press with the addition of a filter aid in an amount of 4 parts by weight per 100 room parts. 46 g of the sodium salt of ethylenedinitrile tetraacetic acid (EDTA) are added to the filtrate. The filtrate is cooled to 6 ° C, adjusted to a pH of 4.5 ± 0.2 and kept at 6 ° C. The cold filtrate is introduced at a speed of 481 / min into a 4801 column with «Dowex» -50 X 4 Na + (300-840 n). After a preliminary run of 14001, a run-off of 18.91 is opened, the pH of which is adjusted to 7.08 with sodium hydroxide solution, and which is stored at 5 ° C.

A column 3.8 cm in diameter filled with 300 ml "Dowex" -l X 2 (150-300 (x) in the chloride form

is washed with 600 ml of demineralized water at 5 ° C. 41 of the cold effluent from the «Dowex» -50 X 4 column are passed through this column at a rate of 30 ml / min. Then the column is washed with 300 ml of 25-jimolar EDTA solution. The antibiotic N-acetylthienamycin is eluted at a rate of 15 ml / min at 5 ° C with 900 ml of 5% saline solution, which is 0.01 molar in potassium phosphate buffer (pH 7.0) and 25 fimolar in EDTA . 14 fractions of 75 ml each are collected and analyzed for their biological activity using the disk diffusion method. The eluate fraction Nos. 3 to 9, the amount of which is 525 ml, are combined and concentrated to 115 ml in vacuo. The concentrate contains 65% of the total bioactive material introduced into the "Bow-ex" -L X 2 Cl ~ column.

The concentrate of the «Dowex» -L X 2 Cl ~ eluate is introduced at 5 ° C into a column with a diameter of 3.8 cm, which is loaded with 450 ml of pre-washed XAD-2. The XAD-2 is column by column with 4 column volumes each 1) 0.001 molar EDTA solution, 2) in NaOH, 3) demineralized water, 4) in HCl, 5) demineralized water, 6) methanol, 7) acetone, 8) demineralized water and pre-washed with 2250 ml of 5% NaCl solution, which is 25-molar of EDTA, before use.

After the sample has been introduced into the column, two portions of 25 ml of water are added. The column is developed with demineralized water at a flow rate of 10 ml / min at 5 ° C. A first 400 ml fraction and then 11 further 75 ml fractions are collected. The pH of each fraction is adjusted between 6.9 and 7.13 with in sodium hydroxide solution or in hydrochloric acid. Fractions Nos. 3 to 9, which contain 46% of the total bioactive material fed into the XAD-2 column,

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are combined to a total volume of 490 ml. One (NH4) 2S04 1.0 g

45 ml sample is used for bioanalysis according to the standard Schei MgSÖ4 • 7H20 0.1 g bend diffusion method against Staphylococcus aureus ATCC distilled water 1000 ml.

Taken from 6538P. The remaining 445 ml are under

Vacuum concentrated to 50 ml. 5 This suspension is used to make four slant tubes

Inoculate the 50 ml concentrate of the XAD-2 eluate at 5 ° C. with medium A (with agar) of the following composition at a rate of 1 ml / min into a column of:

1.5 cm in diameter, the medium A is loaded with 40 ml of pre-washed "Dowex" -l X 4 Cl ~ (particle sizes <37 n)

is. The «Dowex» -l X4 Cl ~ resin (<37 | x, determined by de-10 yeast autolysate («Ardamine» *) 10.0 g of water)) has a speed of 10.0 g before use with a glucose of 1 ml / min in columns with 240 ml of 0.2 mol phosphate buffer * 2.0 ml of saline solution, which is 0.005 molar of NH4C1 and 0.1 mm MgS04-7H20 0.05 g lar of NH4OH, and then washed distilled water at the same rate 1000 ml with 120 ml demineralized water. 15 pH adjusted to 6.5 with NaOH.

Following this sample, two 5 ml portions of “Ardamine”: Yeast Products Corporation demineralized water are added to the column. The Ko ** phosphate buffer solution lonne is developed at 5 ° C. at a rate of 0.92 ml / min KH2P04 91.0 g with 0.07 molar saline solution, the 0.005 molar Na2HP04 95.0 g of NH4C1 and 0, Is 1 mmolar at NH4OH. Frak 20 distilled water 1000 ml ions of 8.6 to 9.3 ml are collected. The fractions, which begin with an outlet volume of 600 ml and with a Vo- For inclined tubes, 25.0 g agar per liter are added.

lumens of 710 ml ends are combined; they contain 98%. The inoculated slant tubes are incubated for a week at 28 ° C in the entire "Dowex" -L X 4 column and then stored at 4 ° C.

bioactive material. This liquid is vacuumed on 25 10 ml of medium A are aseptically concentrated on one of these sloping 2 ml. tubes transferred, the spores and the aerial mycelium are in

The «Dowex» -l X 4 concentrate is scraped into a column with suspension, and 1.2 ml of this suspension will combine! Entered a diameter of 2.2 cm, which uses 225 ml to inoculate three 21-Erlenmey “Bio-Gel” P-2 (37-74 | x) with a deflection element with an exclusion limit of 1800 flasks, each of 500 ml of Medium A (without agar) dalton (previously determined by decanting distilled 30. These inoculation flasks are charged for 24 hours at 28 ° C water). shaken at 160 rpm, whereupon a satisfactory

Before use, the "Bio-Gel" P-2 column with culture has been developed.

225 ml of 1 molar saline and then washed with 100 ml of demineralized water. The cultures from these inoculation flasks are combined and a rate of 1 ml / min at 5 ° C. with demineralized 35 is used to develop 4671 Medium A (without agar) in a 7561 water, with 2 ml fractions being collected inoculating fermenter made of stainless steel. This will be. The eluate fraction fermenter collected from 104 to 128 ml works for 24 hours at 28 ° C., with 83% of the 130 rpm introduced into the “Bio-Gel” P-2 column being stirred and 283 liters of air per minute passes through. After ten bioactivity and are combined and concentrated to 1.58 ml. Foam contraceptives (Polyglycol 2000 from Dow

A column with 50 ml of XAD-2 (1.6 cm X 27 cm) is manufactured by 40 Chemical Corp.), but not in amounts of more than 0.1%, and added in columns with 200 ml of 1 mmolar. The pH determinations have the following

EDTA solution, in sodium hydroxide solution, demineralized water, in results:

HCl, demineralized water, methanol, acetone and demineralized water. The concentrate of the "Bio-Alter, h _0_ 24 Gel" P-2 eluate, which contains 44.4 hydroxylamine-erasable 45 pH 6.3 6.4 optical density units, is added to the XAD-2 at 5 ° C -

Column entered, whereupon two portions of 2 ml each of the culture in this inoculation fermenter are used.

poured mineralized water. The column is used to inoculate 40821 Medium E in a 56701 fermenter at a rate of 1 ml / min with demineralized stainless steel water. Medium E has washed the following until the UV extinction of the washing liquids at 50 composition:

300 nm has decreased to 0.060. The column is washed at a rate of 1 ml / min with 50% methanol in demineralized E mineralized water, fractions of 1 ml each of cerelose being collected. The fractions with an absorbance in corn steep liquor (on a wet basis)

300 nm of more than 0.1 are combined and 55 soluble slurry constituents are concentrated in vacuo. The product obtained is N-acetylthienamycin with cottonseed medium (Pharmamedia)

II, 6 optical density units that can be extinguished by hydroxylamine - CoCl2 • 6H20. CaC03 (after pH adjustment)

Example 4 Polyglycol 2000

A tube with a freeze-dried culture of strepto- «o tap water myces cattleya NRRL 8057 is opened aseptically and the pH is adjusted to 7.3 with NaOH.

Content suspended in 0.8 ml sterile Davis salts of the following composition: This fermenter is kept at 24 ° C. for 138 hours

Stirring at 70 rpm and passing air with a Davis salts 65 speed of 1.54 m3 / min. As needed

Sodium citrate 0.5 g of another anti-foaming agent (Polyglycol 2000), but not

K2HP04 7.0 g added in amounts of more than 0.1%. Antibacterial

KH2P04 3.0 general analysis performed with the following results:

25.0

G

15.0

G

10.0

G

5.0

G

0.01

G

3.0

G

0.25

%

1000

ml

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Age, h pH ATCC No. 6633

(9.5 mm discs), inhibition zone, mm

0

6.9

0

24th

6.3

0

36

6.0

0

48

5.9

0

60

6.0

23

72

5.9

-

84

6.0

21st

96

6.2

-

108

6.5

35

120

6.6

36

132

6.7

41

138

6.7

39

The 40821 fermentation liquid is filtered through a 76 cm filter press with the addition of a filter aid in an amount of 4 parts by weight per 100 room parts. 46 g of the sodium salt of ethylenedinitrile tetraacetic acid (EDTA) are added to the filtrate. The filtrate is cooled to 6 ° C, adjusted to a pH of 4.5 ± 0.2 and kept at 6 ° C. The cold filtrate is introduced at a speed of 481 / min into a 4801 column with «Dowex» -50 X 4 Na + (300-840 n). After a preliminary run of 14001, a runoff of 18.91 is collected, the pH of which is adjusted to 7.08 with sodium hydroxide solution, and which is stored at 5 ° C.

A column with a diameter of 3.8 cm, which is filled with 300 ml «Dowex» -l X 2 (150-300 | i) in the chloride form,

is washed with 300 ml of demineralized water of 5 CC, 41 of the cold effluent from the «Dowex» -50 X 4 column are passed through this column at a rate of 30 ml / min. The column is then washed with 350 ml of 25 molar EDTA solution and eluted at 5 ° C. at a rate of 15 ml / min with 900 ml of 5% NaCl solution which contains 0.01 molar Tris. HCl (pH 7) and 25- {imolar to EDTA. Fractions of 75 ml each are collected and analyzed using the disk diffusion method against Staphylococcus aureus ATCC 6538P. Fractions Nos. 4 to 10, which contain 47% of the introduced bioactivity, are added to 42.5 ml of the sample which had been taken from the combined fractions from the first XAD-2 adsorption for analysis in Example 3. These combined fractions are concentrated in vacuo to 100 ml and adjusted to a pH of 6.32 with hydrochloric acid.

A column with a diameter of 3.8 cm, which is loaded with 450 ml XAD-2 resin, is column by column with 4 column volumes in succession 1) 0.001 molar EDTA solution, 2) in NaOH, 3) demineralized water, 4 ) Pre-washed in HCl, 5) demineralized water, 6) methanol, 7) acetone, 8) demineralized water and washed before use with 2250 ml 5% NaCl solution which is 25- (xmolar of EDTA). The above concentrate becomes into the XAD-2 column, whereupon two portions of 5 ml of demineralized water are poured in. The column is developed at 5 ° C. with a rate of 10 ml / min with demineralized water ml and further fractions of 75 ml each were collected and analyzed according to the disk diffusion method Fractions Nos. 9 to 15, which contain 22% of the bioactivity introduced into the XAD-2 column, are combined and concentrated in vacuo to 56 ml.

A column measuring 21 cm X 1.7 cm, which is charged with 40 ml "Dowex" -l X 4C1- (<37 | i, determined by decanting water), is used at a rate of 1 ml / min before use with 240 ml of 0.2 molar NaCl solution, which is 0.005 molar of NH4C1 and 0.1 mmolar of NH4OH, and then washed at the same speed with 120 ml of demineralized water.

The XAD-2 concentrate is introduced into the column, whereupon two portions of 2 ml of demineralized water and then two portions of 2 ml of elution buffer are added. The column is eluted at a rate of 1 ml / min at 5 ° C. with a 0.07 molar NaCl solution which is 0.005 molar in NH4C1 and 0.1 mmolar in NH4OH. Fractions of 10 ml each are collected and analyzed using the disk diffusion method. The eluate fractions collected from 544 to 647 ml contain apparent 100% of the bioactivity entered and are combined with one another and concentrated to 2.3 ml in vacuo. The concentrate contains 36.4 optical density units that can be erased by hydroxylamine.

A 2.2 cm X 62 cm column loaded with 225 ml Bio-Gel P-2 resin (37-74 | i) with an exclusion limit of 1800 daltons is loaded with 225 ml 1 molar saline before use and then washed with 100 ml of demineralized water. The “Dowex” -l X 4 concentrate is added to the column, after which two portions of 2 ml of demineralized water are poured in. The column is developed at 5 ° C. at a rate of 1 ml / min with demineralized water, fractions of 2 ml each being collected, which are then analyzed using the disk diffusion method. The fractions from 124 to 129 ml, which contain 7.04 optical density units which can be erased by hydroxylamine, are combined and concentrated in vacuo to 2 ml of an aqueous solution of the product N-acetylthienamycin. The fractions collected from 117 to 123 ml and from 130 to 139 ml of eluate are combined to a solution of the product N-acetylthienamycin which contains 13.7 optical density units which can be erased by hydroxylamine.

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1 sheet of drawings

Claims (3)

630 118 1. Process for the preparation of the new antibiotic N-acetylthienamycin, characterized in that, under submerged aerobic conditions, Streptomyces cattleya is cultivated in an aqueous, assimilable C and N sources and nutrient medium containing inorganic salts, and the N-acetylthienamycin formed is isolated. 2. The method according to claim 1, characterized in that one uses the microorganism Streptomyces cattleya NRRL 8057. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2 for the production of non-toxic, pharmaceutically acceptable salts of N-acetylthienamycin by reaction with inorganic or organic bases. The discovery of the remarkable antibiotic properties of penicillin has sparked great interest in this area, which has led to the discovery of many other valuable antibiotic substances; such antibiotics are e.g. other penicillins, cephalosporins, streptomycin, bacitracin, teracyclins, chloramphenicol, erythromycins and the like. In general, the antibacterial activity of each of these antibiotics does not extend to certain clinically important pathogenic bacteria. Some of them only work against gram-positive types of bacteria. Furthermore, in the course of the widespread use of previously known antibiotics for the treatment of bacterial infections, the acquired resistance has led to the emergence of difficult resistance problems. The shortcomings of the known antibiotics have therefore prompted further research to find other antibiotics which are active against a broad range of pathogens and also against resistant strains of particular microorganisms.