DE2362707A1 - ANTHRACYCLINE ANTIBIOTIC AND METHOD FOR MANUFACTURING THE SAME - Google Patents

Description

DJpl.-Jng. Dr. itir. / ^

Frank Arnold Wed>

Patent attorney

Frankfart am Main 70

AND PROCEDURE FOR HERSO) BIiLUIiG DESSEIiBM

The present invention relates to a new anthracycline / anti-blotlcum andX process for the production of the same The ^ -> compound is a product of the breeding of the

Strains Actlnomadura carmlnata sp.noν and represents / according to the invention)

(daiv (hat that < ie ^Y ö

istall ines powder won red color; they ^Y molecular weight _c a / of compound

T. ^ 185-187 ° C (with decomposition), the vist In water,

lower alcohols, dimethylformamide and pyridines are readily soluble in Ethyl acetate, benzene, diethyl ether and petroleum ether are insoluble, the color of their aqueous solutions changes from yellow in an acid medium up to. Violet, in an alkaline medium, hers Solution in sulfuric acid is purple in color; her ultraviolet and visible absorption spectrum is given by the following Characterizes maxima;

409884/1434

236, 256, 293, 464, 478, 492, 512, 525 mn (in ethanol)

S'cL 221, 542, 471, 150, 214, 246, 280, 200 or 1 cm * * * * #

/ Uax ^ ⁵⁸ ' ⁵ °° » ⁵² °»

¹²⁰ > ¹¹ ° »^ ^{0 or} * ·
the infrared absorption spectrum of the antibiotic has the following absorption bands:

990, 1020, 1100, 1120, 1200, 1 ^ 00, 1420, 1450, 1540,

1630, 1710, 3000, 3400, 3600 cm "" ¹ , during the hydrolysis in ^v 0.2 n- ^ ä ^ rigex) hydrochloric acid solution, an aglyconiaite anthracycle and a carbon hydrate fragment is formed.

fpnai ^ akolosischeri z-.B. & lsj The antibiotic can serve as * Wifckstoff, ^ - r ~ ^ yföstat ikw ,

as well as in research in the field of biology at the Study of intracellular molecular processes can be used

at the same time
The invention relates ν process for the preparation of the new AnthrazykliaA.ntibiotlkums. In this process, according to the invention, the antibiotic-producing strains Actinomar dura carmlnata sp.nov. cultivated under the conditions of submerged fermentation on the fermentation media, which carbon-4,

lind

^V nitrogen sources, inorganic salts, with the antibiotic in the mycelium of the producer strain accumulates, 'it is isolated from the *.

the
Was ISatersucht ^v root Hr. 4281 of the Actlnomadura culture

carminata sp. nov., in the collection of the Institute for Research for new antibiotics from the Academy of Medical Knowledge ^ -

409884 / U34

sc & aften the USSE is deposited under the Hummer INA 4281.

Strain No. 4281 forms on most synthetic nutrient media a sparse, whitish aerial mycelium. The spurs

form density. Accumulations that can be destroyed with difficulty and consist of a chain of spores that lead to a Curls or tight knots. curled up · The Spore chains are arranged mono-oeally · The spores are smooth and about 1.5-2 times larger than vegetative hyphae. It was not possible to detect the formation of spores on the substrate pycelium. In hydrolysates of the cell envelopes of the Staiifes Ur · 4281 the Meso-Dlamlnopimelic Acid detected »The substrate mycelium on synthetic nutrient media has a colorless to red-purple color or brownish tone, which is due to the formation of a coloring Pigments of the anthracyl group conditional · No melanoid pigments are formed «

Next heiü. becomes a characteristic of stamp No. 4281 its growth on various diagnostic nutrients given:

Morphology: the spore chains are formed monopodularly on the aerial mycelium on short or fairly long spore carriers. These chains are usually curled up in a curl or compressed into tight balls, sometimes they stay straight. The amount of sporea in the chain is

and

6 to 14, the spores are smoothly ^v rounded "

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i »η» Nt? «, λ von flange (composition * soluble starch - 20 g, E ₂ HPO ₄ - 0.5 g, MgSO ₄ - 0.5 g, EHO ₃ -1g, NaCl - 0.5 g, FeSO ^. - 10 mg, agar-agar - 30 g, tap water 11) · The growth is scanty to moderate Ig; the lymph mycelium is scanty and sometimes absent; the substrate mycelium is colorless to pale purple? the pigment forms poorly, the medium is uncolored

Nutrient medium from Czapek (composition: slbharoee - 30 g, -2 g, K ₂ HPO ₄ - 1 g, MgSO _4. 7H ₃ O - 0.5 g, KCl - 0.5 g,

FeSO ₁ * - OfOI g, agar-agar - 350 g, distilled water - 1 1). That

■ and shade;

Aerial mycelium eats the substrate mycelium sparingly or moderately / of white ν is colorless to purple, ■ * in the latter Trap, the medium is slightly purple in color «

(Compositionί Glycerin -

ml, NaNO ₅ -2 g, K ₂ HPO ₄ " ¹ S» ^ ⁰ H. ' ° »5 S» KCl- 0.5 g, FeSO ₄ -10 mg, agar-agar - 30 g _f distilled water - 11 ) The growth is sparse, the aerial mycelium is absent or has the shape of a thin, whitish tinge, the substrate mycelium is brownish or reddish, the medium is not colored iy ^ faiflflaflimn von ^ ΐ ^ Ρ8ί1 Τ1Ι- ^ ί ^η ^ SE-I Composition of glucose

- 20 g, Z ₂ KP% - ° i5 g> KNO ^ - 1 gf NaCl- 0.3 g, MgCO, - 0.3 g, CaCO ₅ - 0.5 g, FeSO ₄ - 0.1 g, agar -Agar - 30 g, tap water

- 11) The growth is like on the nutrient medium of Lindenbein

jp (composition: glucose -

g, asparagine - 0.5 g, Κ ₂ ΕΦ ° 4 - ° »5 S _f agar agar - 30 g, distilled

A0988A / H3A

tes water - 1 I) * The growth is rather scanty, the aerial mycelium is absent or has the shape of a thin whitish tinge; the substrate myxellum is brownish or reddish «Hafaragar (composition: oat flakes - 20 g, agar-agar g, tap water - 11) · The growth is good, the air

and
mycelium is well developed ^v rosy to pink in color; the substrate mycelium is weak, purple to red-purple in color. The culture medium is uncolored or slightly colored

Organic nutrient medium No. 2 from Ga ₁ USQ (composition: Hottlnger-Bcmillon (700 mg% amino nitrogen) - 50 ml, peptone 5 g, NaCl -5 g, glucose - 10 g, agar-agar - 50 g, tap water -11). The air cells are unstained, the substrate my cells are brown-violet or violet-black, the nutrient medium is uncolored.

N ^ fa Vl <? <Llum of, fi m qyscm ,; (Meat extract - 50 ml, peptone 4 g, NaCl - 2.5 gi, yeast extract - 5 ml, glucose 10 g, agar agate 30 g, tap water - 1 1). The aerial mycelium is absent, the substrate mycelium is purple-black, the nutrient medium is unstained.

Iflffil T ¹ IiIflfl W ¹ H fl mL'fc yeast and time ^ qytrakt (Bacto-yeast extract ( "Dlfko" ■ from the USA.) - g M, bacto-time _z extract - 10 g, Bacto dextrose -4 g, distilled water - 1 1, Bakto-Agar 20 g) · The aerial mycelium is well developed, fluffy, rosy j the subfetrate mycelium is brown-llla, the nutrient medium is uncolored.

Melanolde pigments are missing »

Carbon consumption - fructose, xyIose, bhamnose, arablnose are moderate; Raffinose, mannitol, glycerine, inositol

409884 / U34

and sucrose weakly consumed

Properties ? inhibits the growth of St.

aurfus 209, its mutant U3? -2, Sarclna lutea, Bae.mycoldes, Bac.

ιis inhibited andj.

paracoli 509 ^ --v ~ a weak effect on E. coli. K-13 exercised.

Due to the characteristics of sporulation and the presence of meso-dlamino-pimellic acid in the 2ellenhtllen Rechneny

is the examined strain belonging to the genus Actlnomadura Zechevaller et Zechev · 1970 to, Among the representatives of this genus was the formation of pigments of the anthracycline group has not been detected,

will,
which was taken as an occasion for the strain 4281 as a new kind of

Actlnomadura carmlnata sp «nov to describe

The anti-biotic pollution can be realized on the nutrient media which are glucose, glycerine, Galactose, fructose, starch, ehamnose ', as organic Sources of nitrogen can include soy and pea flour, cottonseed, peanutuji, and sunflower cakes. As organic Salts are used sodium chloride, ammonium sulfate, ammonium chloride, Calcium carbonate, substituted potassium hydrogen phosphate The cultivation of the antibiotic pro -

one
duzlerenden tribal on ^v Permentatlonsnahrmedlum the following composition by göLUhrt (in weight · ^): soy flour 1-5, glucose O, 5-3f starch 1-3 _f ammonium Ulf at 0.05-0.5, sodium chloride from 0.2 to 0 , 5 calcium carbonate 0.2-0.6 »

The -.-- method according to the invention for

Isolation of the anthracycalnantilblot is expediently carried out in the following steps:

Ä09884 / U34

Separation of the mycelium of the producer strain from the culture fluid

slgkelt, extraction of the antibiotic from the% zellum by means of "

^{v of} a water-miscible organic solvent , concentration of the solution obtained, extraction of the

by means of

Antibiotic from the concentrated solution • a water-immiscible ₍ organic solvent, - concentration of the solution obtained, its purification on silica with subsequent elution and concentration of the eluate obtained and precipitation of the antibiotic with diethyl ether, petroleum ^^ ether or η-hexane »extraction of the Antibiotic with water with subsequent lyophilization of the aqueous solution.

/ ■ follow the steps,

The method ^. ^ Is preferably * * J.

Isolation of the Anthracykllnantlblotikums by allowing the culture liquid to stand at a pH of 3.0- £, 0 and a Tempeicatur of 40-55 ⁰ C, separation of the mycelium of the product ent t amme s from the culture liquid, extraction of the antibiotic from the obtained natural solution with a water-immiscible, organic solvent,. Concentrate the antibiotic solution obtained, purify it on silica with subsequent elution and concentrate the eluate obtained and precipitate the antibiotic with diethyl ether, petroleum _v ^ afhtr or n-hexane,. Extraction of the antibiotic with water with subsequent lyophilization of the aqueous solution,

(Willj

In animal experiments it could be shown that the anthracycin anthracite

409884/1434

blotlkum has a high cytostatic effectiveness » The Anthracycllnantlblotlkum was in the form of its water

Solution mice with (transplant ion tumors at a concentration

in

tion of the solution of 1.5 mg of the above-mentioned antibiotic per 1 ml

observed,

administered intravenously with distilled water. Bs became ν that the antibiotic inhibits the development of lymphosarcoma of the strain Lio-I from the forestomach tumor of the strain 01, -, from the bronchogenic lung tumor of the Stallies BL *

The anthracyclinantblotlkum inhibits the development of sarcoma 180 and lymph diadenosis BK / Ly m to a lesser extent, it is practically ineffective compared to the Ehr 1 Ich-Kar ζ Inom · The LDcq äes anthracyclinantlblotlkum amounts to intravenous administration of the aqueous solution of the mentioned concentration to white mouse 5 »7 (3.4-4.0) mg / kg, with subcutaneous application - 3f8 (5.2-4.6) mg / kg and with oral administration - 7 $ 3 (5» 8-9, 0) mg / kg.

The antibiotic becomes proportionate in the gastrointestinal tract well absorbed "

The antibiotic possesses cumulative properties · When administering _{the maxlma} i hears contractual dosla

its effect /; after 72 hours. The indicators of the funkji

the tlonal kidney and liver condition, the sugar and electrolyte blood levels, ^ Tetrocardlographic indicators change after the introduction of the anthracyclinic blotlcum, even in toxic

409884/1434

do not see cans ^ essential \ "/

In tests on dogs it was found that the Tobacco effect of the anthracyclinic biotlum is due to its inhibitory effect on blood formation. The animals go to administration - the antibiotic in the form of a solution in waßülgeÄ lethal doses of the aplasia of the bone marrow based · The intensity of Blutblldungshemmung 1st of -Eöhe the introduced dose and the duration of antibiotic medication dependent.

The process for making the new anthracyclnantiblotlkuüBwwill carried out as follows:

The culture of the producer tribe Aktlnomadura carmlnata sp «, nov · is in the active state on the agarized nutrient medium of the the following composition (in% by weight) stored and maintained:

Oatmeal 1.0-5.0

Agar-agar 1.0-3.0

pH after sterilization VfO

The culture Actlnomadura carmlnata sp »nov · is at one Temperature of 37 ° C grown for 7 days

· The culture of producer ens t amme s obtained is ^ grown by the submersion in spores flask for 3 days at a temperature of 37 ^ö 0 on shaker tables, the / min, a rotating motion at a speed of 200-220 U, perform the spore flasks each containing 200 ml fermentation medium of the following composition (in% by weight):

409884 / U3A

Soy flour 1.0-5.0

Glucose 1.0-5.0

Sodium chloride 0.2-0.5

Calcium carbonate 0.2-0.5

pH 7.0 after sterilization

Liter of the culture of the producer strain is brought from the spores · piston into the fermenter inoculator Pedal _e

80 liters of fermentation medium of the following composition (in% by weight) are added to the fermenter inoculator:

Soy flour 1.0-5.0

Glucose 1.0-5.0

sodium chloride 0.2-0.5

Calcium hydrogen phosphate 0.01-0.05

Calcium carbonate 0.2-0.5

pH after sterilization 7.0

Sterilization is during carried out for 45 minutes at a temperature of 120 ⁰ C.

The sperm whale oil is used as an antifoam agent.

The culture of the producer strain is grown in the fermenter-inoculator for 3 days at a temperature of 37 ° C. The pressure in the apparatus is 0.3-0.5 at, the ventilation rate - 1 1 air 5e 1 1 fermentation medium per minute. The ^{lead author} ^ / ird under

by-.. "

constant stirring ^v led. The speed of the agitator rotation is 240 U / mln _e

The inoculate «lrd in an amount of 80 l from the fermenter

409884 / U34

Inoculator In a steel ferment he transferred. The cultivation is in Eermentervbel a temperature of 27 ° C during the first 24 hours and from 57 ⁰ C during the after

The first days The pressure in the apparatus is 0.5 atmospheres

Aeration rate - 1 1 air per 1 1 fermentation medium per minute. The process is carried out with constant stirring. The speed of the clock mechanism rotation is 220 U / mln, Fermentation duration 120 hours »The submerged cult

a ..

tion of the antibiotic in the fermentation will follow ν nutrient medium Composition (in wt.%) Carried out.

Soy flour 1 »0-5.0

Glucose 0.5-5.0

Staike 1 »0-5.0

Ammonium sulfate 0.05-0.5

sodium chloride 0.2-0.5

Calcium carbonate 0.2-0.6

pH after sterilization 7.0

The sterilization is carried out wäheeiid 45 minutes at a temperature of 120 ⁰ C ·

When the culture Aotlnomadura carmlnata sp.nov.

in the liquid fermentation medium under the conditions of the sub-

C— ~ 7 calibrated '
Biersfermtntatlon ^v the Anthracycllnantlblotikum gammelt in the Ifyzellum of the producer strain), ^at ·

The culture fluid of the producer strain is Frame filter press filtered (FiltriermaterIaI 1st Beifing- und '

409884/1434

Nettle) · The further isolation of the anthracycalnant biotlum

he follows

ν from the mycelium, and the native solution, which

does not contain an antibiotic is discarded · From the obtained The mycelium of the producer strain becomes the antibiotic with organic solvents which are miscible with water, for example with

Acetone, extracted It is also possible to carry out the extraction with nlede-

how to carry out or ren alcohols, ^v methanol / ethanol. The received Solution is concentrated under vacuum, and the antibiotic is turned off by means of organic solvents which are immiscible with water, e.g. Chloroform, methylene chloride or ethyl acetate at a pH value extracted from 6.9-7.2. The antibiotic solution obtained is concentrated and further purification is carried out by chromatographers of the concentrated antibiotic solution on aqueous Silicic acid through · The eluate obtained, which is the antibiotic contains, is concentrated under vacuum j and the antibiotic becomes precipitated from petroleum ether, diethyl ether ad.gr η-hexane * Dtr

"φ

The precipitate is separated off and dried under vacuum at a temperature not exceeding 40 ^° C. The antibiotic is extracted from the dried precipitate with distilled water. The weighing antibiotic solution is lyophilized.

The isolation of the Anthrazyklinantlblotikums can also be realized aas the natlven solution · For this purpose, the culture is flüssigkeit''bei a pH of 3.0-5.0 and a temperature of 40-55 C. ^c. .- * held · Under these «conditions the antibiotic goes out of the mycelium into the watery one

409884/1434

Phase * in the native solution over · Then the cooling fluid is filtered off · The mycelium, which does not contain any antibiotic, is discarded · The antibiotic is extracted from the native solution with a water-immiscible organic solvent at a pH of 6 _f @ -7 , 2 extracted ¹

The solution obtained is described by the above procedure further processed.

For a better understanding of the present invention, the following specific examples are given
Bftlsplel 1

The culture Actlnomadurcu carminata sp.nov of the producer

one

Strain is in the active state on ν agarized nutrient medium of the following composition (in g and% by weight) ent

Oat flour 20 g (2%)

Agar-agar 18 g (1.8 ^)

Tap water 1 1

pH 7.0 after sterilization.

The spore material is kept at a temperature of 37 ° C during 7 Sagei bred

For submerged cultivation of the producer strain, a liquid Permentation medium of the following composition (in g and used:

Soy flour 10 g (1%)

Glucose 10 g (1%)

Sodium chloride 5 g (0.5%)

409884 / U34

Calcium carbonate 3 g (0
Tap water 1 1

pH after sterilization 7fO

Fit,

Flask of 750 ml ν which de 200 ml Fermentatlonsmedium said composition contain, be seeded according to the method mentioned above with the Xnokulat "The culture

the
is kept in flasks at a temperature of 37 ° C for 3 days

Shakes Ischen bred) with a rotating movement at a speed of 200-220 rpm. «2 g of inoculate from the above-mentioned flasks are put into the

regiments inoculator introduced · ^D ei "5ermenter inoculator of having
1 content becomes * 80 1 fermentation medium. loaded:

(in g and% by weight)

Soy flour 1O g (1%)

Glucose 10 g (1%)

Sodium chloride 3 g (0.3%)

Calcium hydrogen phosphate 0.25 g (0.025%) Tap water 1 1

pH 6.7 after sterilization

Sterilization rules: 120 ⁰ C - 45 min

The culture is kept in the fermenter-inoculator for 3 days at a temperature of 37 ° C. The pressure in the apparatus is 0 * 3-0.5 atj the aeration rate - 1 1 air per 1 1 fermentation medium per minute »The mixture is - with

stirred at a speed of 240 U / mm- \

409884 / U3A

The cultivation of the antibiotic is made in permenters stainless steel of 2,000.! content carried out which 800 1 fermentation medium contain the following composition (in g and% by weight):

Soy flour 10 g (1%)

Glucose 7 g (0.7%)

Starch 15 g Ci> 5 %)

Ammonium sulfate 1 g (0.1%)

sodium chloride 1 g (0.1%)

Calcium carbonate 1 g (0.1%)

Tap water 1 1

pH 6.5 after sterilization

conditions: sterilization ν ι 120 ⁰ C, 45 min.

In the fermenter he is 80 l inoculate from the fermenter inoculator given. . '

Cultivation is carried out at a temperature of 37 ° C. for the first 24 hours of cultivation, afterwards at 28 ° C. . The pressure in the apparatus is 0.5 at, the ventilation rate 1 air per 1 1 Mahrmedium per minute · The mixture is:, · -χ ..- _ά . stirred at a speed of 220 rpm.

During the cultivation, the sterile sperm whale oil is used in an amount of 0.5. Vol. % Added. '. - '■:., The fermentation time is 120 hours

409884 / U34

Example 2.

With the spore inoculate, -spore flasks ft ^ O ml

sown what 200 ml]? ermentatlonsni84lum the following Zuaam composition (mg and wt.%) contain: (Hottinger- «Boulllon, the Contains up to 28 mg% ammonia

Glucose 10 g (1%)

Sodium chloride 5 g (O | 5%)

Peptone 5 S (0 »5%)

Calcium carbonate 5 g (0.5%) tap water 1 1 pH after sterilization 7 _f O

The culture is in ^ spore flasks at a temperature of

37 ⁰ C for 2 days grown on shaking tables, which perform a rotating movement at a speed of 200-220 U / mln «

a

In 'fermenter inoculator made of stainless steel of capacity
1 ν are given 80 % ferment at lonsmedl to:. -? (in g

and weight *%)

Glycerine 20 g (2%) Males spring water 10 g (1%) Ammonium chlorine ld 2 g (0.2%) potassium chloride 3 g (0.3%) Calcium carbonate 5 g (0.5%) tap water 1 1 pH after sterilization 6.5

409884/1434

conditions: _n “ sterilization, • 120 ⁰ C, 4-5 min.

The fermenter inoculator is sown with the culture from the spore flasks in an amount of 2 liters. The culture is grown for days from 30-32 ° C. The pressure in the apparatus is 0.3-0.5 at, the aeration rate is 1 liter of air 1 1 nutrient medium per

one minute. The mixture is / moved by y Ruhr plant at a speed of 200-240 rpm.

The cultivation of the Anthsazykllnantlblkums is in one Fermenter <£ 000 l) carried out. Be in the fermenter 1 fermentation at ion medium of the following composition (in g and Weight,%) introduced:

Soy flour 10 g (1%)

Starch 20g (2%)

Ammonium sulfate 2 g (0.2%) sodium chloride 2 g (0.256) Calcium carbonate 2 g (0.2%) tap water 1 1 pH after sterilization 6.5

conditions: sterilization </ 120 ⁰ C, 45 min.

The per ment he is from the Ferment © r inoculator with 80 ^ oculate sown.

The cultivation wlÄd at a temperature of 28-30 ⁰ C performed · The pressure in the apparatus is at 0.5, the Belüftungageschwindigkelt - 1 1 1 1 air 3e nutrient medium per minute ·

409884/1434

The medium comes with a. Stirred speed of rpm. The fermentation time is 120 hours «

Example 3

For submerged cultivation of the producer strain in spore flasks, a fermentation medium will followW Composition (in g # & GeWo%) used:

Soda flour 10 g (1%) Glucose 10 g (1%)

Sodium chloride 3 g (0.3%) Calcium carbonate 3 g (0.3%) Tap water 1 1

pH 7 »O after sterilization

The culture wild at a temperature of 37 ° C during Days in spore flasks on Schüttelt Ischen, which perform a rotating movement »

The culture is taken from the spore flasks in an amount of 2

a capacity

introduced into ν fermenter inoculator of 15O 1 ν, in which is 80 1 nutrient medium of the following composition (in g and Weight *%) are:

Glycerine 20 g (2%)

Throat spring water 10 g sodium chloride 2 g (0.2%) sodium chloride 3 g (0.3%) calcium carbonate 5 S (0.5%) tap water 1 1

A0988A / U3

pH 6.5 after sterilization

Conditions: Sterilization - 'ν 120 ° C, 45 min

The inoculum is grown in the fermenter inoculator ^V age 3 at ^T C · The pressure in the apparatus amounts to 0.5 at the loading

air flow rate - 11 air 1 1 nutrient medium per minute each

one of

at ν speed of the agitator rotation 240 rpm The KuI-

one

activation of the antibiotic is carried out in ^ fermenters / ^; 2000 1- <~) with a batch of 1000 1 fermentation medium of the following

Composition (in g and% by weight) carried out Soy flour 10 gi (1%) Glycerine I5 g (1.5%)

Glucose 4 g (0.4%)

Ammonium sulfate 1 g (0.1%) Ammonlumchlorld 1 g (0.1%) Calcium carbonate 1 g (0.1%) Tap water 1 1

pH after sterilization 7 $ 2

The fermenter becomes from the fermenter inoculator with 80 Inoculate sown "

The cultivation is carried out at a temperature of 28-30 C ⁹ "The pressure in the apparatus amounts to 0.5 at which Belüftungßgeschwlndlgkelt - 1 1 1 1 The air nutrient medi to is per minute · the mixture by an agitator at a rate of

U / mln reached The duration of the procedure is 120 hours *

409884 / U34

Example 4.

700 1 culture liquid produced during the cultivation of the strain

Actinoma carminata sp. Nov · No. 4281 may be obtained

of

drops off. 17 kg "moist mycelium obtained are mixed with 85 1

the _M

Acetone extracted · 80 1 acetone solution containing the antibiotic are concentrated under vacuum. The aqueous antiblotic solution that remained after the acetone distillation is treated with 5 liters of carbon tetrachloride at a pH of 4.5, then the antibiotic is extracted with chloroform at pH 6.8 ¹ . The extraction is carried out three times. For an extraction you need 5.5 l of chloroform. The chloroform extracts are combined. The total volume ^of extract is 16 1. The extract

is concentrated under vacuum to a volume of 0.6 l.

Another cleaning of the concentrated antiblotic solution

is carried out on a column with aqueous silica "

500 g of silica are introduced into the column in chloroform. Hach the Antiblotikugftorptlon from 0.6 1 more concentrated Antibiotic solution in chloroform is the column first with 2 1

one es / carbon tetrachloride, then with 1.5 l ^ mixture of chloroform and

d h i

ιd anach y one

Acetone In the ratio of 11: 4, with 1.5 liters of mixed us chloroform,

Acetone and methyl alcohol in a ratio of 11t 1x0.5 washed. Dl · iüluierung of ^ \ '"

V antibiotic is made with an iron mixture ^of chloroform »Azetoa

and methyl alcohol in a ratio of 11: 1: 1. 2.5 1 fraction which contains ^ Twerden up to a volume of 50ml concentrated _f the antibiotic and the antibiotic is precipitated with hexane-η

40988A / U34

17 g of precipitate containing 30% antibiotic are obtained. The precipitate is dried at a temperature of at most 40 ^{° C.}

8.5 g of precipitate will dissolve in the mortar, of which 0.7 1 Add water in small portions. The solution obtained is separated from the precipitate and lyophilized. 2.6 g of powder of red color are obtained

The anthracycalnantlbiotlkum obtained contains 1200 units of action when titrated to Bac.eubtllls

Example · 5 ·

700 1 culture fluid produced during the cultivation of the Actlnomadur * earminata sp.nov. No. · Wurd obtain 4281 "are with 18% aqueous hydrochloric acid solution to a pH of 4 _t 5, and acidified, with stirring, while at 5O ⁰ C one hour

one

left standing · The culture fluid is filtered through a frame filter press filtered (filter material 1st Belting and nettle cloth). The mycelium is discarded. Natural solution obtained from 600 l the antibiotic is treated with 600 l of methylene chloride at pH 7.2 extMahlert

55 1 antidote solution in methylene chloride are up to

^el nem volume of 0.5 l concentrated and the concentrated anti-

ΘΪΏ.ΟΙ *

blotikumlÖsung is based on v. column chromatographed with aqueous silica

300 g of aqueous silica are in Methylenchlorltd in the Column introduced. After the anti-blot solution from 0.5 1

409884 / U3 4

concentrated solution in methylene chloride, the column is first

one with 1.5 l carbon tetrachloride, then with 1.2 l ^v mixture / Täus

finally one of it chloroform and acetone in a ratio of 1: 4, V with 1.2 1 mixture from chloroform, acetone and methyl alcohol in a ratio of 11: 1:

0.5 washed
lEluie runs des .

DleTAntLbLo tikums is carried out with a mixture of chloroform, acetone and methyl alcohol in the ratio of 11: 1: 1 »2.0 1 fraction containing the antibiotic is concentrated under vacuum up to a volume of 60 ml, and the antibiotic is precipitated with petroleum ether. The resulting precipitate is separated off and dried under vacuum at a temperature of at most 40 ^° C. The weight of the dried substance is

- *! der d erart;
carries 9.6 g of _# gTBTg ^ obtained substance are ground in the Moüse *, adding 0.8 l of distilled water in small portions · The aqueous solution is separated from the precipitate and lyophilized · Mftn receives 2.85 g of powder of red color · the Antarazyklinantibiotikum obtained contains 1100 ^w irkungselnheLten in mg in the titration of Bac · · εübtills

Example 6

800 1 in the cultivation of the Actinomadura strain

° arminata sp. Nov. No. 4281 obtained culture liquid are removed

(Man

kg of moist mycelium. ^ xtrahlert ^ with 60 liters

Acetone. 55 1 acetone solution, that is, the antibiotic

Vaporizing >

Concentrated under vacuum »The nacn ^ cLes Az · tones ...... lagging behind aqueous antibiotic solution is mixed with 4 1.

A0988A / U34

Carbon tetrachloride

V agile at pH 4.0-5.0 % then the antibiotic is extracted with methylene chloride at pH 7.2. The extraction is repeated three times. 4 liters of methylene chloride are used for each extraction. The solutions obtained are combined, washed with water and concentrated to a volume of 0.5 l. Further purification is carried out by chromatography

one .. .. .. ..

carried out on ν column with aqueous silica "

500 g of aqueous silica are added to the column in chloroform given. - After antibiotic absorption from 0.5 1 concentrated Antibiotic solution 1 Methylenchlorld gets the column first with

one esy

1.5 l chloroform, then with 1.2 l ^v mixture / from chloroform and acetone

_M finally one esj

in a ratio of 11: 4 / with 1.2 X ^v mixture / of chloroform, acetone and methyl alcohol in a ratio of 11: 1: 0.5

a «" · *

Dl "intlblotlkumelutl ^v is carried out with V. Mixture of chloroform, acetone and methyl alcohol in the ratio of 11: 1: 1. 1.5 1

en * ^Λ

Fraction, which contains the antibiotic / up to a volume of 50 concentrated ml, and the antibiotic wlrdmit Dläthyläther like "This gives 12 g precipitate, 30% antibiotic contains · The precipitate under vacuum at a temperature not exceeding 40 ⁰ C dried ·

_c des ... "«

öigverhaltenen precipitate · are ground in a mortar to give 0.5 1 of distilled water in small portions admits «D» solution obtained is separated from the precipitate and lyophlllslert "Mftn 2 g of powder of red ^ar ν ^one The AnthrazykllxiantlblotlkUB obtained contains 1200 effective units in mg during the titration on Bao "practiced"

409884 / U3A

Claims (5)

PATENT CLAIM: AnthracycllnJAntlblotikum, characterized that it is the product of the cultivation of the tribes an Actinomadura carmlnata sp.nov · 1st and crystalline powder of from the red color, molecular weight 545.5 and 'Schmp. 185-187 ° C ,. the (with decomposition) represents "in water, lower alcohols, Dimethylformamide and pyridine readily soluble in ethyl acetate, benzene, Chloroform, diethyl ether and petroleum ether is insoluble, the aqueous solutions of which change from yellow in an acidic medium to violet in an alkaline medium, the solution of which in sulfuric acid is colored violet, the ultraviolet and visible absorption spectrum of which is characterized by the following maxima.
I / max ff f 221, 542, 471, 150, 214, 246, 280, 200 or ? t 150, 120, 110, 30 or,
of the infrared absorption spectrum. has the following absorption bands: 990, 1020, 1100, i | 2O _t 1200, 1300, 1420, 1450, 154O, 1610, 1710, 30000, 3400, 3600 cm * " ¹ , and that during hydrolysis in 0.2 η aqueous hydrochloric acid solution. "in structure
Aglycon with anthracycline / / and a carbohydrate & iragment forms. 2. Process for the production of the Anthrecykllnntlbiotlkums 409884 / U34 according to claim 1, characterized in that the antibiotic producing strains Actinomadura carminata sp. nov. are cultivated under the conditions of submerged fermentation on fermentation media, which carbon, Containing nitrogen sources and inorganic salts, the antibiotic being in the mycelium of the producer strain can be accumulated and the said antibiotic isolated therefrom. 3. The method according to claim 2, characterized in that the cultivation of the antibiotic-producing strains is carried out on a fermentation medium which (in% by weight): soy flour - 1-5, Contains glucose 0, -3, starch 1-3, ammonium sulfate 0.05-0.5, sodium chloride 0.2-0.5, calcium carbonate 0.2-0.6. 4. The method according to claims 2 and 3, characterized in that the anthracycline antibiotic is isolated in the following steps: Separation of the mycelium of the producer strain from the culture liquid, Extraction of the antibiotic from the mycelium with a water-miscible organic solvent, Concentration of the obtained, solution, Extraction of the antibiotic from the concentrated solution with a water-immiscible organic solvent , Concentrating the obtained solution, Purification on silica with subsequent elution, concentration of the eluate obtained and precipitation of the antibiotic with ethyl ether, petroleum ether or n-hexane, and extraction of the antibiotic with water with subsequent lyophilization of the aqueous solution. 409884/1434 5. The method according to claims 2 and 3, characterized in that the isolation of the anthracycline Antibiotic in the following steps: Allowing the cooling liquid to stand at a pH of 3.0-5.0 and a temperature of 40-55 ° C, Separation of the mycelium of the producer strain from the culture fluid, Extraction of the antibiotic from the preserved native Solution with an organic solvent that is immiscible with water, Concentrating the antibiotic solution obtained, Purification of the concentrated solution on silica with subsequent elution and concentration of the obtained Eluate and precipitation of the antibiotic with diethyl ether, petroleum ether or n-hexane, and extraction of the antibiotic with water with subsequent lyophilization of the aqueous solution. 409884 / H34