CH636642A5 - Process for the preparation of the antibiotic BL580 delta - Google Patents

Description

636 642

PATENT CLAIM Process for the production of the antibiotic BL580 A with the formula:

O*

and the pharmaceutically acceptable cationic salts thereof, characterized in that Streptomyces hygroscopicus NRRL 8180 is submerged in an aqueous nutrient medium which contains assimilable sources of carbon, nitrogen and inorganic salts under aerobic conditions until substantial antibiotic activity has been formed in this medium, then the antibiotic BL580 A is obtained from this medium and, if necessary, converted into a 65 pharmaceutically acceptable cationic salt.

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The invention relates to a process for the production of the new antibiotic BL580 A by fermentation; the invention also includes the preparation of the pharmaceutically acceptable cationic salts of this antibiotic. The antibiotic BL580 A can be produced in a diluted form, as a raw concentrate and in pure crystalline form. The effects of this new antibiotic as an anticoccidial agent, as well as its chemical and physical properties, distinguish it from previously described antibiotics.

According to the invention, the antibiotic is produced by the method according to the patent claim.

The antibiotic BL580 A can be described by the structural formula given below. An alpha-substituent lies behind the paper plane and is denoted by a dashed line (bond), while a

ß-substituent protrudes from the paper plane and is denoted by a wedge-like bond (- «- bond).

rc rc

T

<\ # o

O— &

Sz

636 642 4

The new antibiotic is an organic carboxylic acid growth on asparagine dextrose agar, Hickey and and can therefore form salts with non-toxic pharmaceutically acceptable Tresner agar, inorganic salt starch agar and Bennett's cations. The preparation of such salts agar, weak growth on Czapek solution agar.

expediently takes place by mixing the free acid of the antibiotic with stoichiometric amounts of corresponding 5 aerial mycelium

Cations, expediently in a neutral solvent. The aerial mycelium is yellowish and is in the Sporulàtionszo-tel. Suitable cations for this are the greyish sodium, potassium, calalenes, their color ranging from deer-colored (4 ig) to beaver-cium, magnesium or ammonium ions and dyeing cations (4 Ii) to ash-colored (5 fe). The sporulation zones of organic amines, such as tri (lower alkyl) amine cations (become black and hygroscopic in older cultures, for example triethylamine, triethanolamine) or procain. The io cationic salts of the antibiotic BL580 A are in the general- soluble pigments mean crystalline solids, which dissolve in water relatively- In most media no soluble pigments, on sig poorly, in most common organic yeast extract Bennett and potato dextrose -Agar yellowish

Solvents, such as methanol, ethyl acetate, acetone, chlorine-soluble pigments, which are only present in small quantities, form, heptane, ether or benzene, but are soluble. 15

The new antibiotic, to which the designation BL580 A base color has been assigned, arises during the cultivation of a generally yellowish shade on most new strains of Streptomyces hygroscopicus under controlled media.

conditions, and this strain also produces the well-known antibiotics BL580alpha and BL580ß (U.S. Patent 20 Various Physiological Responses

3 812 249). This new strain is a complete gelatin by treatment of nitrates are reduced to nitrites

S. hygroscopicus NRRL 5647 with N-methyl-N '-nitro-N "-nitro- liquefaction, no formation of melanoid pigments on peptone-sucuanidine-derived mutant. A living culture of the new iron agar, complete peptonization on purple milk within microorganism was found in the Culture Collection Labora- of 7 days, sodium chloride tolerance in the growth medium tory, Northern Utilization Research and Development Divi- 25 Ïï7%, however <10%, carbon utilization according to Pridham and sion, United States Department of Agriculture, Peoria, Illinois, Gottlieb, J. Bacteriol. 56,107-114 (1948): Good utilization of deposited and added to the permanent culture collection there - adonite, d-galactose, d-fructose, d-raffinose, salicin, d-xylose, from there under the accession number and Dextrose, poor or no utilization of d-melezi-

NRRL 8180 freely available. tose, d-melibiosis, 1-arabinose, i-inositol, lactose, d-mannitol,

A determination of the physiological, morphological 30 1-rhamnose, sucrose and d-trehalose.

and cultural characteristics of NRRL 8180 according to Dr. H.D. Tresner,

Lederle Laboratories Division, American Cyanamid Company, Micromorphology

Pearl River, New York, reveals that these features are essentially the same as those of NRRL 5647. Spore-bearing elements grown from aerial mycelium are the same as those observed in tightly-wound spirals with multiple windun-general descriptions of the microorganism end up. The spores are mostly isodiometric, cylin-eighth diagnostic properties are the same as for drish and phalangiform and have dimensions of the microorganism NRRL 5647 in US Pat. No. 3,812,249 - 0.6-0.7 x 0.7 -0.8 pm The spores are due to an electro-light, this description, however, is smooth in the following of the microscopic examination. The spore sheaths are repeated for simplicity. are fine wrinkled.

The determination of the cultural characteristics, physiological 40 On the basis of the observed general characteristics Han characteristics and morphological characteristics of the microorganism, the microorganism NRRL 8180 is a mus NRRL 8180 according to the methods described by representatives from a large group of Streptomycetes, its Shirling and Gottlieb in boarding school. Journ. of system. Bacteriol. 16, characteristic features of gray spores, spiral-shaped 313-340 (1966) are described in more detail. The underlined spore chains, smooth-walled spores and the lack of Mela-descriptive color information and the color plate designations are 45 pigments. The hygroscopic nature of the cultures was according to Jacobsen et al. from Color Harmony together with all their morphological and physiological

Manual, 3rd edition (1948), Container Corp. of America, seeing properties makes them a typical tribe of

Chicago, Illinois. The description details fin- Streptomyces hygroscopicus, as well as corresponding determinations, which can be found in the following Tables I to IV according to H.D. Tresner and E.J. Backus, "A Broadened Concept of so the Characteristics of Streptomyces hygroscopicus", Appi.

Strength of growth microbiol. 4,243-250 (1956) and H.D. Tresner, E.J. Backus and

Good growth on yeast extract, Kuster oatmeal, J.A. Hayes, "Morphological Spore Types in the Streptomyces tomato paste, oatmeal and potato dextrose agar, maize hygroscopic-like complex", Appi. Microbiol. 15,637-639

(1967) show.

Table I

Culture characteristics of Streptomyces hygroscopicus NRRL 8180 Incubation time: 14 days Temperature: 28 ° C

Medium extent of aerial mycelium soluble underside remarks

Color growth and / or spores pigment

Czapek solution low agar

Trace of a whitish aerial mycelium, no sporulation, no colorless to whitish

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medium

Due to the growth of mycelium and / or spores, the pigment is soluble

Color undersides

Remarks

Yeast extract agar good

Kuster-oat curl - good agar

Asparagine dextrose - medium agar

Hickey and mediocre

Tresner agar inorganic medium

Salts-starch agar

Tomato paste oat well flour agar

Bennet agar mediocre

Potato-dextrose-good agar whitish aerial mycelium that turns fawn-colored (4 ig) to beaver-colored (4 Ii) in sporulation zones; strong sporulation of whitish aerial mycelium, which becomes fawn-colored (4 ig) to beaver-colored (4 Ii) in sporulation zones; strong sporulation of whitish aerial mycelium that turns ash-colored (5 fe) to fawn-colored (4 ig) in sporulation zones; moderate sporulation of whitish to yellowish aerial mycelium, which becomes fawn-colored (4 ig) to beaver-colored (4 Ii) in sporulation zones; strong sporulation of whitish to yellowish aerial mycelium, which becomes fawn-colored (4 ig) to beaver-colored (4 Ii) in sporulation zones; strong sporulation of whitish to yellowish aerial mycelium, which becomes fawn-colored (4 ig) to beaver-colored (4 Ii) in sporulation zones; very strong sporulation of whitish aerial mycelium, which becomes beaver-colored (4 II) in sporulation zones; strong sporulation of whitish to yellowish aerial mycelium, which becomes ashen (5 fe) to fawn (4 ig) in sporulation zones; moderate sporulation yellowish, slight none none none none none yellowish, slightly yellowish, slightly bamboo-colored (2 fb)

light mustard bamboo (2fb)

bamboo colors (2 fb)

pastel yellow (1 db)

yellow-

maple (3ng)

bamboo colors (2 fb)

yellow-

maple (3ng)

blackish hygroscopic areas in central colony areas blackish hygroscopic areas in central colony areas; yellowish exudate in marginal zones blackish hygroscopic areas in central colony zones extensive hygroscopic areas in central colony zones blackish hygroscopic areas in central colony zones extensive hygroscopic areas in central colony zones, yellowish exudate in peripheral zones blackish hygroscopic areas in central colony zones blackish hygroscopic areas in central colony zones

Table II

Micromorphology of Streptomyces hygroscopicus NRRL 8180

Medium aerial mycelium and / or spore structures Spore appearance Spore size Spore surface

Kuster-Oats- Spores grow from the aerial mycelium. The spores are mostly 0.6 to 0.7 (in x due to a flaked agar that has branches, isodiametric, 0.7 to 0.8 p.m electron microscope-

which, in tightly wound spirals, cylindrical, phalanx-like, see determinations smoothly;

The spore sheaths end with several passages and are finely wrinkled

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Table III

Different physiological reactions of Streptomyces hygroscopicus NRRL 8180

medium

Incubation (28 ° C) growth rate physiological reactions organic nitrate broth organic nitrate broth gelatin

Peptone Iron Agar Purple Milk

Yeast extract agar plus (4,7,10 and 13%) NaCl

7 days good

14 days good

7 days good

24 to 48 hours good

7 days good

10 days good

Nitrates are reduced to nitrites

Nitrates are reduced to nitrites complete liquefaction no melanoid pigments are formed complete peptonization

NaCl tolerance equal to or above 7%, but below

10%

Table IV

Carbon utilization behavior of Streptomyces hygroscopicus NRRL 8180 Incubation: 10 days Temperature: 28 '

Carbon source

Recovery *

Adonit

1-arabinose

Dextran d-fructose i-inositol

Lactose d-mannitol d-melezitose d-melibiosis d-raffinose

1-rhamnose

Salicin

Sucrose d-trehalose d-xylose

Dextrose

Negative control

3 0 3 3 0 0

0

1 1 3 0 3 0 0 3 3 0

* 3 = good recovery 2 = sufficient recovery 1 = bad recovery 0 = no recovery

The production of the antibiotic BL580 A is of course not limited to this particular microorganism or the organisms that fully meet the growth behavior described above and the microscopic properties mentioned for NRRL 8180. The invention therefore also includes the use of mutants which are formed from the microorganism NRRL 8180 by various means, such as, for example, by X-ray radiation, ultraviolet radiation, nitrogen mustard, actinophages or the like.

The antibiotic BL580 A is an extremely effective active ingredient for combating coccidial infections in warm-blooded animals. The antibiotic BL580 A is also significantly less toxic than the antibiotic BL580al-pha (the structure of which is given in NL-PS 74 02 938. The effectiveness of the antibiotic BL580 A as an anti-cocidiosis agent is shown in the following in-vivo Studies shown for which the following poultry feed is used:

Dicalcium phosphate

1.2%

ground limestone

0.5%

stabilized fat

4.0%

dehydrated alfalfa (17% protein)

2.0%

20 corn gluten flour (41% protein)

5.0%

Menhaden fish meal (60% protein)

5.0%

Soybean meal (44% protein)

30.0%

ground yellow corn, fine substances to 100%

25 The vitamin given in the above poultry feed mix

Amino acid premix is made from the following formulation. The quantities refer to units per

Kilograms of poultry feed.

30 Butylated hydroxytoluene

125 mg dl-methionine

500 mg

Vitamin A

3300 I.E.

Vitamin D3

11001.C.E.

Riboflavin

4.4 mg

35 vitamin E.

2.2 IU

niacin

27.5 mg

Pantothenic acid

8.8 mg

Choline chloride

500 mg

Folic acid

1.43 mg

40 menadione sodium bisulfate

1.1 mg

Vitamin B12

11 mcg

50

55

Vitamin-amino acid premix

Trace minerals

Sodium chloride

0.5% 0.1% 0.3%

ground yellow corn, fine substances on 5 gm

Mixed coccidial infections of Eimeria tenella and Eimeria acervulina

A mixed inoculum of 5000 oocytes sprouted from Eimeria acervulina and a sufficient number of oocytes from Eimeria tenella, so that mortality of 85 to 100% results in untreated controls, groups of seven-day-old chicks are added to the goiter by direct inoculation. During the entire duration of the experiment, the chicks can eat poultry feed and drink water at will. Two days after the inoculation, the various chick groups in the experiment were offered a feed containing active ingredients, which was composed of the poultry feed and several doses of the antibiotic BL580 A. The tests are terminated 10 days after the inoculation. The animals are weighed and sacrificed, after which their intestinal tract is examined for damage. The results obtained in this experiment are shown in Table V below. The results show that the administration of 125 ppm or 250 ppm of antitotic BL580 A together with the feed to infected chicks results in 100% survival of these infected chicks. The experimental data also show that administering 30 ppm or 60 ppm of antibiotic BL580 A to infected chicks together with the feed gives a very strong suppression of the damage caused by Eimeria tenella and Eimeria acervulina.

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Table V

Concentration on the number of% surviving% animals with reduced damage

BL580 A in feed, ppm animals Animals Eimeria tenella Eimeria acervulina

0 60 17 0 0

250 5 100 100 100

125 5 100 100 100

60 24 91.6 46 100

30 25 60 0 64

Table VI

BL580 A concentration in feed, ppm

Number of experimental animals

% surviving animals

% Animals with reduced damage tenella acervulina necatrix brunetti maxima

0

15

0

0

0

60

0

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120

15

100

100

100

100

100

100

60

15

100

40

93

100

48

80

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15

100

7

7

86

0

21st

Mixed coccidia infection of Eimeria tenella, Eimeria acervulina, Eimeria necatrix, Eimeria brunetti and Eimeria maxima

A commercial vaccine (Coccivac D, Sterwin Laboratories, Opalika, Alabama), which contains a mixture of at least five types of Eimeria coccidia, is administered to chicks in an amount 70 times the normal immunization dose. The vaccine is given to groups of seven-day-old chicks by direct inoculation into the goiter of all animals. The animals had free access to water and the above-mentioned poultry feed for the entire duration of the experiment. Two days after the inoculation, the various chick groups in the trial were offered feed containing active ingredients consisting of the poultry feed and several doses of BL580 A antibiotic. The investigations are terminated 10 days after inoculation, the animals being weighed, killed and their intestinal tracts examined for damage or injuries. The results obtained are summarized in Table VI. The results show that administration of 120 ppm of antibiotic BL580 A to infected chicks together with the feed leads to a 100% survival of the infected chicks. Furthermore, at this active ingredient concentration there is also a very strong suppression of damage or injuries caused by Eimeria tenella, Eimeria acervulina, Eimeria necatrix, Eimeria brunetti and Eimeria maxima.

Fermentation process

The microorganism Streptomyces hygroscopicus NRRL 8180 can be grown in a large number of different liquid culture media. Media suitable for the formation of this antibiotic BL580 A contain an assimilable carbon source, e.g. Starch, sugar, molasses or glycerin, an assimilable nitrogen source, e.g. Protein, protein hydrolyzate, polypeptides, amino acids or corn steep liquor, as well as inorganic anions and cations, such as potassium, magnesium, calcium, ammonium sulfate, carbonate, phosphate and chloride. Trace elements, such as boron, molybdenum and copper, are present as impurities in other components of the medium. Aeration in tanks and flasks is carried out by passing sterile air through the fermenting medium or by blowing sterile air onto the surface of the medium. In addition, mechanical means provide movement in tanks. If required, a defoamer can be added, for example 1% octadecanol in lard oil.

Inoculum production

A Streptomyces hygroscopi- shaking flask inoculum

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eus NRRL 8180 is produced by inoculating 100 ml of sterile liquid medium in 500 ml flasks with scraped or washed-off spores from a slant agar culture. A suitable medium is, for example:

Soybean flour glucose

Corn steep liquor

CaCOa

water

1.0% 2.0% 0.5% 0.3% to 100%

45

50

The flasks are incubated at a temperature of 25 to 29 ° C, preferably 28 ° C, with vigorous agitation on a rotary shaker for 48 to 96 hours.

Two 100 ml portions of this inoculum are then used to inoculate 12 liters of the same sterile medium in a 20 liter flask. This inoculum is incubated with movement and ventilation with sterile air for 36 to 64 hours at 25 to 29 ° C., preferably 28 ° C.

The inoculum obtained in this way is then used to inoculate 300 liters of the same sterile medium in a fermentation tank. This inoculum is incubated with movement and aeration with sterile air for 36 to 64 hours at 25 to 29 ° C., preferably 28 ° C.

The inoculum obtained is then used to inoculate a 4,000 liter fermentation tank containing 3,000 liters of a sterile medium of the following composition:

Corn steep liquor

Soybean flour

Cornstarch

CaCÛ3

water

0.5% 1.0% 4.0% 0.1% to 100%

55 The above medium is fermented for 100 to 200 hours at a temperature of 27 to 32 ° C while moving with a stirrer and aeration at a rate of 0.4 to 0.8 liters of air per liter of medium per minute. A defoamer, for example Hodag 60 FD82, is usually also added, in an amount of about 1.3 liters per 1000 liters of medium.

Cleaning process

After the fermentation has ended, the fermentation mash containing the anti-65 biotic BL580 A is combined with about half of its volume of ethyl acetate and the whole is then stirred for 2 to 3 hours. Then add about 8% diatomaceous earth and filter the mixture through a plate and frame

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Filter press. The filter cake is washed on the press with ethyl acetate. The ethyl acetate extracts are collected and concentrated to a syrup in a distiller.

The syrup obtained in this way is washed with a quantity of heptane twice its volume and then stored at 4 ° C. overnight. The supernatant liquid is decanted and concentrated to a gummy residue.

The gummy concentrate is treated with 10 liters of methanol, after which the whole is cooled for several hours using dry ice. The mixture is then filtered through a sintered glass filter on which there is a layer of diatomaceous earth, washing with cold methanol. The methanol solution is evaporated to dryness under vacuum.

A chromatography column is then prepared which contains activated carbon in an amount of approximately 1 liter of coal per 50 g of feed. The dried residue is dissolved in methylene chloride in an amount of 40 g per liter and added to the column. The methylene chloride eluate is collected as a single fraction and evaporated to dryness. The residue obtained is then mixed with methanol and the whole is then stored in a cold room with dry ice in order to reduce the temperature to -10 ° C for 15 minutes. After 15 minutes, the solidified oil is filtered off and the methanol-soluble material is evaporated to dryness in vacuo, giving an oil.

This oil is then dissolved in a minimal amount of methylene chloride, after which silica gel is added, the whole is concentrated to dryness and finally applied to a dry silica gel column. The column is developed first with 10% ethyl acetate in benzene and then with 20% ethyl acetate in benzene. Then let the column run off. The column is divided into 10 equal parts (including the feed). Core samples are removed along the length of the entire column at Rf values of 0.05, 0.15, 0.25,0.35 etc. and with an appropriate volume of a mixture of ethyl acetate, methylene chloride and methanol (2: 2: 1 ) eluted. At locations where the antibiotic overlaps, a core sample is collected from each VS of an RF unit. The point at which the antibiotic comes is determined by thin-layer chromatographic examination of the kernel eluates using commercially available thin-layer chromatogram plates (Silplate-22 from Brinkmann Instrument Co., Westbury, New York 11590). The respective zones are detected by charring in the presence of sulfuric acid.

The section of the column which contains a material with Rf values from 0.11 to 0.35 is cut out of the column and slurried in a mixture of ethyl acetate, methylene chloride and methanol (2: 2: 1 by volume). The resulting mixture is filtered, washed with a further solvent mixture and then concentrated to dryness in vacuo. The resulting residue is dissolved in tert-butanol, whereupon the whole is filtered and freeze-dried. In this way, a fluffy solid is obtained.

A two-phase system is produced by mixing n-heptane, methanol, ethyl acetate and water (3000: 1500: 75: 37, by volume). A certain type of diatomaceous earth (Celatom from Eagle-Picher Industries, Cincinnati, Ohio) is then mixed with the lower phase of this system in an amount of 800 g to 600 ml of the lower phase and a column is then packed with this mixture in certain proportions (diameter 7 , 5 cm). A mixture of diatomaceous earth, lower phase and lyophilized product (40 g: 30 ml: 13.8 g) is used as the column feed. The charged column is developed using the lower phase, collecting 25 ml fractions. To detect the active substance-containing fractions, selected fractions are subjected to thin-layer chromatography using gel plates, working with a mixture of chloroform and ethyl acetate (1: 1) as the developing agent and the actual detection is carried out by carbonization. Fractions 90 to 150 are pooled and evaporated to give the antibiotic BL580 A.

The invention is further illustrated by the following examples.

example 1

Production of the inoculum

An exemplary medium used to grow the first stage inoculum is made from the following ingredients:

Soybean flour 1.0 g

Glucose 2.0 g

Corn steep liquor 0.5 g

CaCOs 0.3 g

Water to 100 ml

Using washed or scraped off spores from an agar slope from Streptomyces hygroscopicus NRRL 8180, two 500 ml flasks are then inoculated, each containing 100 ml of the above medium in sterilized form. The flasks are then placed on a rotary shaker and mixed vigorously for 72 hours at a temperature of 28 ° C.

The flask inoculum formed above is then transferred to a 19 liter glass flask containing 12 liters of the same sterile medium. This second inoculum is aerated with sterile air while allowed to grow for 48 hours at a temperature of 28 ° C.

The second inoculum produced in the above manner is then transferred to a 380 liter fermentation tank containing 300 liters of the same sterile medium. This third inoculum is aerated with sterile air in an amount of 1 liter of air per liter of medium per minute, the whole being stirred with a stirrer at a speed of 173 revolutions per minute. The batch is allowed to grow at 28 ° C for 48 hours. The pH at this time is 6.9 to 7.0.

Example 2 Fermentation

The following components are used to produce a fermentation medium:

Corn steep liquor 0.5 g

Soybean meal 1.0g

Corn starch 4.0 g

CaCOî 0.1g

Water to 100 ml

3000 liters of a fermentation medium of the above formulation are sterilized in a 4000 liter tank for 60 minutes at 120 ° C. After sterilization, the medium has a pH of 6.4 to 6.5. This medium is then inoculated with 300 liters of the third inoculum prepared according to Example 1. The fermentation is carried out at 28 to 30 ° C, using 4.0 liters of Hodag FD82 as defoamer. The batch is aerated with 0.6 liters of sterile air per liter of mash and per minute. The mash is stirred with a stirrer running at a speed of 150 revolutions per minute. After a total fermentation of 138.5 hours, the mash is harvested.

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Example 3

Isolation and cleaning

2550 liters of the fermented mash produced according to Example 2, which has a pH of 7.4, are combined with 1275 liters of ethyl acetate and the whole is then stirred for 2.5 hours. Then add 8 percent by weight diatomaceous earth. The mixture thus obtained is then filtered in several portions with stirring through a few frame presses. The water-ethyl acetate filtrates are combined, giving a total of 3250 liters of material, which is then left to separate the layers. This results in 1000 liters of ethyl acetate extract. After filtering each portion of the mixture of mash, ethyl acetate and diatomaceous earth through the corresponding press, the filter cake present on the press is washed with ethyl acetate. The ethyl acetate washes are combined and separated to give 535 liters of ethyl acetate wash. The above 1000 liters of ethyl acetate extract and 535 liters of ethyl acetate wash are combined and then concentrated to 225 liters in a 1515 liter still. This 225 liter material is then concentrated in a 190 liter distillation device to a volume of 20 liters. Subsequently, these 20 liters of material are further concentrated in a glass still, which leads to a syrup.

The syrup obtained in the above manner is kept at 4 ° C. for 48 hours and then stirred with twice the volume of heptane. The mixture is then left to stand at 4 ° C. overnight. The supernatant liquid is then decanted and then concentrated to a gummy residue.

The gummy residue is mixed with 10 liters of methanol and the mixture is cooled for several hours using dry ice. The mixture is then filtered through a sintered glass filter which contains a layer of diatomaceous earth, washing with cold methanol. The combined filtrates and washing liquids are then evaporated to dryness in vacuo, giving 1353.5 g of residue.

The residue obtained in the above manner (1353.5 g) is dissolved in methylene chloride in such a way that a concentration of 40 g per liter results. Subsequently, a chromatography column is packed with 27.07 liters of carbon granulate with a grain size of 0.83 x 0.44. The methylene chloride solution above is then passed through this column at a flow rate of 375 to 400 ml per minute. The methylene chloride eluate is collected as a single fraction and evaporated to dryness to give 153 g of residue. The residue is mixed thoroughly with 8 to 9 liters of methanol. The resulting mixture is then cooled in a cold room using dry ice to -10 "Cab and kept for 15 minutes at -10 ° C. The resulting solidified oil is filtered off and the methanol filter is concentrated to dryness in vacuo, which gives 781.4 g of an oil.

To produce a dry packed chromatography column, 4 kg of silica gel are placed in a plastic column with a circumference of 30.5 cm. 200 g of the above oil are then dissolved in a minimal amount of methylene chloride. Then 300 g of silica gel are added, the whole is mixed thoroughly and the mixture thus obtained is then concentrated to dryness in vacuo. The dried mixture thus obtained is applied to the column, the upper end of the column being provided with a certain amount of sea sand so as to prevent the bed from mixing during the elution. The plastic column is placed in a glass bowl as a support and base. The column is eluted with 12 liters of 10% ethyl acetate in benzene. The column is left to dry

run, whereupon they are eluted with 7.6 liters of 20 percent ethyl acetate in benzene. After collecting the appropriate fractions, the column is allowed to run dry. The column is then purged with nitrogen. The antibiotic activity is determined by examining appropriate fractions using Streptococcus pyogenes NY5. The column is divided into 10 equal parts (including the column charging). Core samples are taken along the length of the entire column at Rf values of 0.05.0.15.0.25.05.35 ... etc. At each point where the antibiotic bands overlap, samples are collected at every Vs of an Rf unit. Each core sample is diluted with 10 ml of a mixture of ethyl acetate, methylene chloride and methanol (2: 2: 1) and the samples are examined by thin-layer chromatography using a mixture of ethyl acetate and chloroform (1: 1), each with 30 ml used and the detection is carried out with carbonization with sulfuric acid. The section of the column with Rf values from 0.11 to 0.35 is removed and slurried in a mixture of ethyl acetate, methylene chloride and methanol (2: 2: 1). The mixture is filtered and washed with the same solvent mixture, after which the material obtained is evaporated to dryness in vacuo. The residue is dissolved in tert-butanol, and subsequent filtration and lyophilization of the material obtained gives 26.7 g of product. A two-phase system is produced by mixing n-heptane, methanol, ethyl acetate and water (3000: 1500: 75: 37, by volume). 800 g of acid-washed diatomaceous earth are then mixed with 600 ml of the lower phase of this solvent system and a 125 cm3 glass column is then packed with this mixture in portions. The feed containing 40 g of diatomaceous earth, 30 ml of lower phase and 13.8 g of the above lyophilized material is placed in the form of a mixture. The loaded column is developed with the upper phase of the solvent system and 25 ml fractions are collected. The position of the desired compound is determined by examining appropriate fraction samples in the manner described above by thin-layer chromatography. Fractions 90 to 150 are combined and lyophilized, whereby the product obtained is 2.75 g of antibiotic BL580 A, which is predominantly in the form of the sodium salt.

Example 4

Production and isolation of the antibiotic BL580 A in the form of the free acid

The sodium partial salt of the antibiotic BL580 A is prepared and isolated by the processes described in Examples 1 to 3. A two-phase system is then produced by mixing heptane, methanol, ethyl acetate and water (3000: 1500: 80: 40 parts by volume). A glass column is then packed with a mixture of 800 g of diatomaceous earth and 600 ml of the lower phase of the above system. The feeding is done in the form of a mixture of 28 g diatomaceous earth, 21 ml lower phase and 10.9 g sodium partial salt of the antibiotic BL580 A. The column is developed using the upper phase. Fractions with a volume of 90 ml each are collected. Selected fractions are chromatographed on Silplate® F-22,

whereby a mixture of ethyl acetate and chloroform is used as the developing agent and the actual detection of the antibiotic BL580 A is carried out by charring. Fractions 29 to 39, which contain the antibiotic BL580 A, are combined and evaporated to remove the solvent. By dissolving the residue obtained in tert-butanol and then lyophilizing the solution obtained in this way, 4.79 g of material are obtained.

800 mg of this lyophilized material is stirred in 300 ml

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a two-phase system consisting of water, ether and petroleum ether (2: 1: 1). The pH of the solution is adjusted to 2.5 with stirring using 1 normal hydrochloric acid. The organic phase is separated off and washed three times with an equal volume of water. The solvent extract is then concentrated in vacuo to a residue. Subsequent dissolution of the residue in tert-butanol and subsequent lyophilization of the solution obtained gives 657 mg of antibiotic BL580 A in the form of the free acid.

The free acid of the antibiotic BL580 A results in the following analytical values in a microanalytical analysis: C = 61.10, H = 8.9, Ash = 0. The free acid has a specific rotation value [alpha] o5 = + 21 ° ± 1 ° ( c = 0.9 in methanol).

The free acid of the antibiotic BL580 A shows characteristic absorptions in the infrared region of the spectrum at the following wavelengths: 2.95; 5.88; 8.35; 8.60; 9.00; 9.12; 9.43; 10.05 and 10.47 p ..

An infrared absorption spectrum of the free acid of the antibiotic BL580 A in the form of a KBr pellet is shown in Fig. 4 of the drawing.

A PMR spectrum of the free acid of the antibiotic B1580 A is shown in Fig. 5 of the drawing.

A 13C NMR spectrum of the free acid of the antibiotic BL580 A is given in Fig. 6 of the drawing.

Example 5

Production of the sodium salt of the antibiotic BL580 A

The free acid of BL580 A (1 g) is dissolved in 300 ml of a mixture of ether and petroleum ether (1: 1). This solution is then added to 200 ml of water to form a two-phase system. The pH of the solution is then adjusted to 10.0 with stirring by adding 0.1 normal sodium hydroxide solution, whereupon the organic phase is separated off and evaporated to a residue under vacuum. The residue is dissolved in 10 ml of ether and 20 ml of petroleum ether (boiling range 30 to 70 ° C.) are added to the solution. The solution obtained is then inoculated with a crystal of the sodium salt of BL580 A and then allowed to evaporate slowly at a temperature of 4 ° C. until a crystalline solid forms. The crystals are collected on a filter, washed with cold petroleum ether and air-dried to give 323 mg of BL580 A sodium salt.

This sodium salt of the antibiotic BL580 A melts at 157 to 161 ° C and has the following elementary analysis: C = 60.99; H = 8.47; Na = 1.95: [alphass5 = + 6 ° ± 1 ° (c = 1.153 in methanol). The sodium salt of BL580 A shows characteristic absorptions in the infrared region at the following wavelengths: 6.27.7.28.9.0.9.13.9.23.9.48 and 10.60 p ..

An infrared absorption spectrum of the sodium salt of BL580 A in the form of a KBr pellet is shown in Fig. 1 of the drawing.

A 13C NMR spectrum of the sodium salt of BL580 A is given in Fig. 2 of the drawing.

A PMR spectrum of the sodium salt of BL580 A is shown in Fig. 3 of the drawing.

Example 6

Preparation and isolation of the p-bromophenacyl ester of BL580A

The sodium partial salt of the antibiotic BL580 A is prepared and isolated according to the procedure of Examples 1 to 3. 1 g of this sodium salt of BL580 A, 834 mg of p-bromophenacyl bromide, 600 mg of lithium carbonate and 20 ml of dry dimethylformamide are placed in a flask and the whole is allowed to react therein at a temperature of 37 ° C. for 16 hours. Then 4 volumes of chloroform are added and the resulting suspension is filtered. The filtrate is evaporated to remove the solvent in vacuo to give a syrupy residue. Using a solvent system consisting of heptane, ethyl acetate, methanol and water (2000: 25: 1000: 17), a distribution column with diatomaceous earth is then produced. It works with 120 g diatomaceous earth and 90 ml lower phase of the above solvent system for the column. A mixture of the above syrupy residue, 12 g of diatomaceous earth and 9 ml of the lower phase is then added to the column. The column is developed with the upper phase. Fractions with a volume of 10 ml each are collected. The location of the activity in the fractions is determined by thin layer chromatography. Fractions 22 to 41 are combined and concentrated to a residue under vacuum. The residue is dissolved in 50 ml of methanol and the solution is filtered. The filtrate is heated on a steam bath and mixed with 10 ml of water, after which the mixture is allowed to slowly cool to 4 ° C. The resulting crystals are collected, resulting in 566 mg of p-bromophenacyl ester of BL580 A.

A microanalytical analysis of the p-bromophenacyl ester of BL580 A reveals the following analysis values: C = 58.80; H = 7.80; Br = 8.64. The ester has a specific rotation value [alpha] d = + 63 ° ± 2 ° (CHCL at 0.51%).

The p-bromophenacyl ester of BL580 A shows characteristic absorptions in the infrared region at the following wavelengths: 2.95; 5.88; 6.30; 8.20; 8.45; 8.60; 9.00; 9.15 (broad); 9.37 (broad); 9.62; 10.06 and 10.37.

The p-bromophenacyl ester of BL580 A monohydrate has a molecular weight of 1114 + 0.3% as determined by X-ray diffraction.

An infrared absorption spectrum of the p-bromophenacyl ester of BL805 A in the form of a KBr pellet is shown in FIG. 7 of the drawing.

An ultraviolet absorption spectrum of the p-bromophenacyl ester of BL580 A in the form of a solution in methanol with •

a concentration of 33.84 micrograms per milliliter of solvent is shown in Figure 8 of the drawing.

A PMR spectrum of the p-bromophenacyl ester of BL580 A is given in Fig. 9 of the drawing.

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9 sheets of drawings