DE1923885A1 - Antibiotic substances and processes for their manufacture - Google Patents

Description

1923835

P 19 25 885.8

Soc. Farmaceutici Italia

Milan / Italy

Antibiotic substances and processes for their manufacture.

The present invention relates to antibiotic substances and their derivatives.

In particular, the invention relates to a process for the production of antibiotic substances which have the properties of pines indicator and which are called dihydrodaunomycin and paunosaminyldaunomycin, as well as their salts and their aglycone dihydrodaunomycinone, which are produced on cultures of Streptomyces peucetius var. Carneus. The new antibiotic Substances dihydrodaunomycin, daunosaminyldaunomyein and dihydrodaunomycinone, which are also called B-III, B-107 and B-112, have interesting pharmacological activities, in particular

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antitumoral, antibacterial, antiviral and antiprotozoal Activity. The new strain Streptomyces peucetius var. Carneus, which produces the new antibiotics, is also used Streptomyces P.I.65 called (index number of the strain collection of Societd Farmaceutici Italia). He became the plant pathologist Institute of the University of Milan with index number I.P.I. 1950, at the American Type Culture Collection, Rockville, Maryland 20852, U.S.A. under number A.T.C.C. 21354 and at the Commonwealth Mycological Institute, Ferry Lane, Kew, Surrey, England with the number I.H.I. deposited; it has the following properties:

Microscopic properties .

The vegetative mycelium that forms on the usual culture media consists of thin (0.5-0.9 m / j thickness), more or less long and branched hyphae. These branches carry thicker hyphae (1.1 -1.6 mm in thickness), namely the conidiophores that are often combined in bundle form and end hook-shaped. The conidia are round with a diameter of 1.8-3.3 m / U and are arranged first in chains and then freely.

Under the electron microscope the conidia show an almost spherical shape with irregular outlines and warty surface.

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Macroscopic properties

Table I shows the properties observed on the respective culture soils, the microorganism being allowed to grow at 28 ° C and observations J>, 8, 15 and 30 days after inoculation *

Biochemical properties

Gelatin: total hydrolysis

Starch: total hydrolysis

Nitrates: no reduction to nitrites

HpS: poor development

Melanoid pigments: no development

Milk: no peptonization, no coagulation.

The microorganism utilizes: tyrosine, d-xylose, maltose, Mannose, mannitol, glycerin, glucose, sucrose, trehalose, Raffinose and fructose; are not used: lactose, adonit, rhamnose, sorbitol, 1-arabinose, meso-inositol and Esculin.

The strain produces in liquid aerated submerged culture antibiotic substances.

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Table I.

Cultural characteristics of Streptomyces peucetius var. Carneus

Med ium growth Aerial mycelium vegetative soluble Mycelium Pigments Malt yeast small together very sparse plentiful, intensive, extract- flowing Kolo lich, smooth, yellowish, first Agar never with folds gray-green, then red Red Yellow, (after ger harder he no spi yellow then red Hesseltine have a patina, ralen and Brown and colleagues * Γ plentiful Whisk Bennet sparse, absent moderate, absent A at all in the form of wine pink isolated yellow little one Colonies Emerson- moderate, absent moderate, pale red Agar in the form of colorless lich, small to- Brown merge send Kolo nien, patina lichen-like, not hard Potato- plentiful, plentiful, plentiful, intensive, Aga? (after even blue green- meat-. first Hesseltine, smoothness gray, hyphae coloured, reddish- and colleagues patina the hook smooth hard yellow, then shaped and patina bright then ball • organge end in a shape Peptone plentiful, absent plentiful, abivesend Agar in together colorless + KNO, flowing small Colonies

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BATH ORIGINAL

Portion of table I

Czapeck-
Agar plentiful,
in together
flowing
small ones
Colonies sparse
white-gray,
slightly wadded
Hyphae that
hook or
ball-shaped
end up plentiful,
lively
pink flesh
coloured absent Asparagine
Glucose-
Agar sparse,
in small
isolated
Colonies sparse,
whitish-pink
very decaying
a short one
Mycelium without
Hook at the end sparse,
colorless absent Glycerine
Glycine
A at all plentiful,
smooth hard
patina absent plentiful,
of yellow
until easy
orange absent Strength-
A at all sparse,
in small
isolated
Colonies moderate, of
blue green
to gray
green sparse,
colorless,
then yellow
lich-pink absent gelatin moderate, on
the waiter
area absent moderate, of
colorless
to yellow
lich plentiful,
brown to
deep
black milk sparse absent moderate, ring
shaped on
the waiter
area,
salmon pink sparse,
pink

⁺ Kessel tine and coworkers. 195 ² I-, Ann.NYAcad.Sci. 60, pages 156-151.

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Identification of the tribe

The description of the examined microorganism made it possible to assign it to the species Retinaculum apertum, the classification system of Pridham and colleagues. (Appl. Mierobiol. 6, 1958, page 52) to be assigned. In Baldaccl's classification (Gior, di Mierobiol. 6, 1958, page 10) the microorganism belongs to the Albosporeus series; In the Waksman ¹ see system (The Actionomycetes, Vol. 2, 1961, page 129) the microorganism belongs to the Ruber series.

A comparison between the properties of the microorganism and those of the species that make the quoted systematic Belonging to groups (taxa) shows that none of them has such properties as those of the microorganism examined correspond.

The comparative figures in comparison with the species producing similar substances are given in Table II. The table shows the data from Streptomyces cinereoruber, Streptomyces cinereoruber var. Fructo fermentans, Streptomyces caespitosus and Streptomyces antibioticus are indicated, although they are not included in the groups indicated above belong. In the following, the differences to the species that do not contain any substances of the investigated are listed Type. The new microorganism is different

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differs from the species Streptomyces albosporeus (Waksman, The Actinomycetes, Vol. 2, 1961, page 171) in that the latter reduces the nitrates, does not form any soluble pigments and no hydrogen sulfide; it differs from the species Streptomyces eirmaraornensis (Waksman, The Äctinomycetes, Vol. 2, 1961, pages 195-196) and by Streptomyces fradiae (Waksman * The Äctinomycetes, Vol. 2, 196l _p pages 211-212) because of the color of the vegetative mycelium and the air mycelium of the species Streptomyces ruber (Waksman, The Äctinomycetes, Vol. 2, 1961, page 271), because the latter causes the milk to curdle, does not form soluble pigments and does not form hydrogen sulfide.

It differs from Streptomyces rubescens (Waksman, The Actinomycetes, Vol. 2, I96I, page 271) because of the color des Luftmyoels and because Streptorayces rubescens is not soluble Forms pigment and does not form hydrogen sulfide; it differs from Streptomyces oidiosporus (Waksman, The Actinomycetes, Vol. 2, 1961, p. 251)> because it doesn't reduce nitrates and doesn't peptonize milk * Plus Streptomyces oidiosporus does not form any soluble pigments.

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1923385

Tabel

II

Comparison between Streptomyces peucetius var. Carneus and species which produce substances similar to the new antibiotic obtainable according to the invention.

Streptomyces
peucetius
var. carneus Streptomyces
purpurascens Streptomyces
bibiliae Streptomyces
cinereoruber Sporophores straight or
hook-shaped spiral
shaped spiral
shaped straight or.
hook-shaped Spurs almost round,
warty, 1.8- oval, sta
chely, 0.8-
1 / U times oval, smooth,
0.7-1 / U times vegetati
ves mycelium colorless to
yellow Red,
sometimes with
lichens
possible end
see Red corals
Red yellow Red- '
Brown Aerial mycelium blue-green to
gray-green white-reddish White ash gray reduction
the nitrates
Milk (pep.
Coag.)
L-xylose
L-arabinose
L-rhamnose
Fructose
Sucrose
Lactose
Raffinose
D-mannitol
D-sorbitol mm
+ j • 5- ί Educated
Antibio
tika Dihydrodauno-
mycin, dauno-
saminy! dauno
mycin and di-
hydrodauno-
mycinon Rodomycin Cynerubin Rodomyc in

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Table II continued

Strept. c i-nereo -
ruber var.fructo
fermentans Strept.
caespitosus Strept.
niveoruber Strept.
galilaeus Sporo-
phores straight or
hook-shaped whorled spiral
shaped Spirals
shapely Spurs ova, smooth,
0.7-1 / U times oval, smooth
0.5-1.3 / rev
times 0 ₃ y-
0.5 / rev smooth smooth vegetati
ves mycelium yellow-red-brown cream colored
to brown
to yellow
reddish carmine carmine Aerial mycelium ash gray yellowish
white-gray whitish know up
ash gray reduction
the nitrates
Milk (pep.
Coag.)
L-xyIose
L-arabinose
L-rhamnose
Fructose
Sucrose
Lactose
Raffinose
D-mannitol
D-sorbitol ι \ j \ Educated
Antibiotics Cynerubin Mytomycin Cynerubin Cynerubin

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Posting of table II

Streptomyces Streptomyces Streptomyces Streptomyces antibioticus A II65 (Ashe- A 220 (Ashe- DOA 1205 shov et al., shov et al., (Brockmann, Antib. other Antib. other Chem.Ber.92, Chemoth. 4, Chemoth. 4, -I88O, 1959 58O, 1965). 58O, 1954) Sporo- just not be not be Spirals phores wrote wrote shaped Spurs smooth, round not be not be not be lich wrote wrote wrote vegetati creamy yellow not loading not be brick red, ves mycelium rubbed wrote wine red Luftmyoel know up not be not be red-gray mouse gray wrote wrote reduction the nitrates /. / / / Milk (pep. Coag.) + / / + L-xylose / / / / L-arabinose / / / / L-rhamnose / / / / Fructose / / / / Sucrose / / / / Lactose / / / / Raffinose / '/ / / D-mannitol / / / / D-sorbitol / / / / Educated Cynerubin Aklavin Rutilantine Pyrromycin Antibiotics
ka

+ = positive reaction - = negative reaction / = information is missing

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From this it can be concluded that the microorganism studied differs from all previously known species that form similar Substanaen., differs and to a large extent Sense it also distinguishes see from all the kinds that too belong to the taxonomic subgroups of the genus and to which this Staraiü is to be regarded as belonging. The Streptomyces peucetius var. Carneus differs from the Streptomyces peucetius (Giornale di Microbiologia, Vol. 11, 1965, page 109) because

a) it forms an abundant, gray-green aerial mycelium on the yeast extract tyrosine medium _s be it the vegetative mycelium, be it the colorless backside of the culture,

b) on the Bennet and Czapeck ¹ media, the vegetative mycelium showed strong colors with wine-colored tints on the first medium and flesh-colored tints on the second

α) on the Emerson medium the vegetative mycelium has a tightness and takes on a typical fleece-like appearance and the cultural back is colorless,

d) on the starch and salt mediuro a plentiful, forms blue-green aerial mycelium with pure gray-green tints,

©) No aerial mycelia formation on the potato glucose medium is detected,

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f) the antibiotics dihydrodaunomycin (B-IIl), daunosaminyldaunomycin (B-I07) and dihydrodaunoraycinone (B-I12) in liquid submerged culture.

From this it can be concluded that the microorganism examined is a variant of Streptomyces peucetius, for which the name Streptomyces peucetius var. Carneus is proposed. The microorganism Streptomyces peucetius var. Carneus can be stored on the same medium by successive transfers ψ on medium B (composition as described in Example 1) or by freeze-drying a suspension of the mycelium of a culture.

By fermenting the new microorganism Streptomyces peucetius var. carneus are grown in a culture medium containing carbon, nitrogen and mineral salt sources, some new antibiotic substances with anti-cancer, antibacterial, antiviral and antiprotozoal activity ^ produced.

The preparation of the Dihydrodaunomycins, Daunosaminyldaunomycins and Dihydrodaunomycinons in particular by cultivation of Streptomyces peucetius var. Carneus in a liquid culture medium which was sterilized in advance, under aerobic conditions at a temperature of 25 to 55 ⁰ C for a period 4-7 days at a pH 6.5 to 8 carried out. The culture medium is

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consists of a carbon and nitrogen source and mineral salts.

Starch, dextrin, glucose, Glycerin, mannitol, maltose, soft grain, grains, soybean oil and soy flour can be used. Come as a source of nitrogen In addition to some of the nitrogenous substances mentioned above, dry yeast, meat peptone and casein can also be used.

Good results can also be achieved when using ammonium salts such as nitrate, sulfate and sec. Phosphate. The mineral salts useful in the production of the antibiotics can vary depending on the medium used.

Potassium carbonate is almost always present and can as well Chlorides, sulfates, phosphates etc. of sodium, potassium, magnesium, manganese, else, copper, zinc and cobalt added will.

The fermentation can take place in Erlenmeyer flasks or in laboratory or industrial fermenters of various capacities be performed. When the fermentation is over, the antibiotic substances dihydrodaünomycin, Daunosaminyldaunomycin and Dihydrodaunomycinone with appropriate Solvents extracted from the culture broth under suitable conditions, separated by chromatography and then cleaned. The amount of antibiotics that are in

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the culture broth is present becomes spectrophotometric and then compared with antibiotic samples of known titers.

Physico-chemical properties

The antibiotic substances dihydrodaunomycin _s daunosaminyldaunomycin and dihydrodaunomycinone have the following chemical structural formula

OCH ₃ 0

OH 0-R "

OH

where in the antibiotic D ihydrod aunomyc in R = ^ and

NH "OH H is the remainder of daunosaminyl - CH - CH ₂ - c - C - C - CH ₅

H H

0 ~

means; for the antibiotic daunosaminyldaunomycin R = O and R. means the remainder daunosaminyl and a second Daunosaminylrest is linked to one of the free hydroxyl groups of the molecule like a glucoside; and the antibiotic

OH
Dihydrodaunomycinone R = * and R, a hydrogen atom

means.

Dihydrodaunomycin hydrochloride gives on elemental analysis

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the following values:

empirical formula: C ₀₁₇ H, .. O _n Ji.HCl

d (p \ IU

found: C 57.16, H 6.01, N 2.68, Cl 5.86 % calcd: C 57.29, H 5.71, N 2.47, Cl 6.26 %

The absorption spectrum in the UV and in the visible range, measured in methanol, shows absorption maxima in the
the following wavelengths:

at 233 nyu EJ £ _m = 590 at 479 nyu EJ £ _m = 220

at 252 m / Ue \% «430 at 496 m / U e} ^ = 223

/ I cm ' / 1 ran ■ *.

at 286 m / UE} ^ = l40 at 528 m ₇ ue} ^ = 130
/ 1 cm / 1 cm

The IR spectrum in KBr shows pronounced bands at the following wavelengths (in ^ u): 2.94; 3.44; 6 _P 19; 6.32; 6.95; 7.11; 7.25; 7.8o; 8.28; 8.95; 9.40; 9.95; 1O, 18;
11.5; 12.30; 12.65; 13.18.

The daunosaminyl daunomycin hydrochloride shows on elemental analysis the following values:

empirical formula: ^C 33 ^H 4o ° l2 ^N 2 * ^2HC1

found: C 54.42, H 6.17, N 3.64, Cl 9.43 % calcd: C 54.32, H 5.80, N 3.84, Cl 9.72 %

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The absorption spectrum in the U.V. and in the visible area shows absorption maxima at the following wavelengths:

at 255.5 m / U e} Jf _m = 515 at 477 m / U e} J _n = * 168

/ L a / 1 cni

at 251 nyu E * J _n - 564 at 497 nyu E * J _n = 171

at 290 nyu eJ -J _n = 121 at 551 nyu E * J _n = 95

In the IR absorption spectrum one notices bands at the the following wavelengths (in nyu): 2.95 (broad); 5.44 (broad); 5.80; 6.18; 6.5Oj 7.10; 7.78; 8.24; 8.95; 10.05; 12.20; 12.50; 15.10.

Dihydrodaunomycinone can not only be obtained from the kitchen broths des Streptomyces peucetius var. carneus, but also by acid hydrolysis of the antibiotic dihydrodaunomycin. Its elemental analysis gives the following values:

empirical formula: ^c pi ^H 20 ⁰ 8

Found: C 62.85; H 5.16; OGH ₃ 7.75 % calculated: C 62.90, H 5.02, OCH, 7.75 %

The absorption spectrum in the U.V. and in the visible range, measured in methanol, shows absorption maxima in the the following wavelengths:

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at 253 nyu E * ^ = 78O at 252 m / U eJ J _n = 6IO at 285 m / U EJ J _n = 185

at 479 nyu E * J _n = 295

at 496 m a

at 527 m / rev

cm

310

= 180

In the IR absorption spectrum one notices bands at the the following wavelengths (in / u): 2.94; 3.44; 6.19; 6.35; 6.92; 7.08; 7.30; 7.84; 8.09i 8.31; 9.38; 9.80; 10.20; 11.38; 12.50; 13.18.

The following examples are presented to the present invention explain in more detail, but without this being restricted to this.

example 1

. A strain Streptomyces peucetius var carneus the prepared suspension is required from a freeze-dried stock to inoculate ^5-1 J slant nutrient media with the following composition:

Sucrose 2. % sec. Potassium phosphate 0.2 pounds Sodium nitrate 0.2 % Magnesium sulfate
heptahydrate 0.2 % Dry yeast 0.5 % Tap water on 100

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The uncorrected pH value of the medium is approximately 7. Agar-Difco is added to the nutrient medium in an amount of 2% (w / v), based on the volume of the nutrient medium. The nutrient medium is then sterilized in an autoclave at 110 ° C. for 20 minutes. The inoculated oblique tubes are incubated for 7 days at 28 ° C and then used to inoculate the flasks with the vegetative phase, which each contain 60 ml of medium A, which has the following composition:

Bacto-Peptone 0.5 %

Dry yeast 0.5 %

Calcium nitrate 0.05 %

Tap water to 100

The uncorrected pH of the medium is about 7. The flasks are autoclaved at 110 ° C for 20 minutes sterilized. Each piston comes with part of the surface layer of the mycelium corresponding to 1/4 of the surface of the inclined tube, which is filled with 2 ml of distilled water had been homogenized, inoculated.

The flasks are placed on a rotary shaker (240 rpm, eccentricity 35 mm) and incubated at 28 ° C.

After 48 hours of incubation, the flasks are used for inoculation the production phase flask is used, each of which contains "-40 ml of medium B with the following composition:

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Glucose 6 %

Dry yeast 2.5 %

Sodium chloride 0.2 %

Potassium carbonate 0.2 %

sec. Kailum phosphate 0.1 ji

anhydrous magnesium. _m ν

sulfate o, oi ^

Iron sulfate heptahydrate 0.001 %

Zinc sulfate heptahydrate 0.001 %

Copper chloride 0.001 %

Ethylene diamino tetra

acetic acid sodium salt 0.012 %

Tap water to 100

The uncorrected pH of the medium is 7. Man

sterilized in an autoclave for 20 minutes at HO ⁰ Cj the glucose is sterilized separately for 20 minutes at HO ^{0 C.}

2 ml of the cultures are used to inoculate the production flasks used during the vegetative phase. Me fermentation conditions are the same as with the vegetative cultures »After 120 hours of incubation the cultures contain 114 / Ug / ml Total pigments (calculated as dihydrodaunomycin based on the absorption at 496 myu of the ethanol extract of an aliquot Part of the culture), which is based on 21 / Ug / ml dihydrodaunomycin, 4 / Ug / ml daunosaminyldaunomycin and 10 / Ug / ml dihydrodaunomycinone exist.

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Example 2

The procedure is as in Example 1 with the difference that for In the vegetative and productive phase, the nutrient media L or P are used, which have the following composition:

Vegetative medium L :

Glucose 1 %

Plant extract broth 1.5 % enzymatic casein hydrolyzate 0.3 %

Tap water to 100

pH not corrected. 20 minutes sterilization at 120 ° C.

Productive medium D ;

Glucose 6 %

Marc 2.5 %

Sodium chloride 0.2 %

Potassium carbonate 0.2 %

sec. Potassium phosphate 0.1 %

anhydrous magnesium sulfate 0.01 %

, Iron sulfate heptahydrate 0.001 % zinc sulfate heptahydrate 0.001 % tap water to 100

The pH is brought to 7 with dilute NaOH. 20 minutes sterilization at 110 ^° C.

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The maximum concentration of dihydrodaunomycin, des Daunosaminyldaunomycins and Dihydrodaunomycinons reached on the 6th day of fermentation and is 66 / Ug / ml Total pigments containing 21 / Ug / ml Dihydrodaunomycin, 3 / Ug / ml DaunosaminyldaunomyG in and 8 / Ug / ml Dihydrodaunomycin

Example 3

Proceed as described in Example 1, with the difference that a culture medium with the following composition is used for the productive phase:

glucose 7 % Chickpea flour 6.6 % Potassium carbonate 0.2 % Sodium chloride 0.2 % sec. Potassium phosphate 0.1 % Magnesium sulfate
heptahydrate 0.02 Iron sulfate heptahydrate 0.001 Zinc sulfate heptahydrate 0.001 Leituneswasser on 100

20 minutes sterilization at 120 ^° C.

After 5 days a concentration of 72 / Ug / ml total pigments, containing 22 / Ug / ml dihydrodaunomyein, 4 / Ug / ml Daunosaminyldaunomycin and 9 / Ug / ml dihydrodaunomycinone, achieved.

9.0 8 a 8 S / 1 7 5 ö

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Example 4

A culture of Streptomyces peucetlus var. Carneus on the usual nutrient medium, as described in Example 1, was for inoculating 500 ml of the liquid nutrient medium for the vegetative phase, also described in Example 1, which contained in 2000 ml flasks made of embossed refractory glass is used.

The mycelium of the slant was removed prior to inoculation homogenized. The flask is rotated for 48 hours at 28 ° C on a rotary shaker (120 rpm, eccentricity 25 mm) incubated.

500 ml of the vegetative culture obtained are used for inoculating 50 1 of a nutrient medium which is in an 80 1 stainless steel permenter and has the following composition:

Peptone 0.5 %

Dry beer yeast 0.3 %
Calcium nitrate tetrahydrate 0.05 %
Tap water to 100

This fermenter is powered by a turbine stirrer at 250 rpm. stirred at an air supply of 0.7 1 per 1 culture medium per minute and at a temperature of 27 ⁰ C. After 48 hours of incubation using · 25 1 of mycelium suspension obtained

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for inoculation of 500 l of the permentation culture medium, which is used in a 800 liter container is included. The nutrient medium has the following composition:

glucose 6th % Dry yeast 2.5 % Sodium chloride 0.2 * Potassium carbonate 0.2 % sec. Potassium phosphate 0.1 % Magnesium sulfate 0.0] L % Iron sulfate heptahydrate 0.001 % Copper chloride 0.001 % Tap water on 100

The culture medium is sterilized beforehand at 120 ° C and after the inoculation is carried out with two turbine stirrers at 220 rpm. stirred, with an air supply of 0.7 1 per 1 nutrient medium and per minute at a temperature of 27 ° C. After 10-140 hours After treatment, a content of 69 / Ug / ml total pigments is achieved. 50 1 filtered culture broth, the 69 / Ug / ml Total pigments, contained, corresponding to a total amount of 3.45 g, with 6U hydrochloric acid to a pH of 2 set and 5 1 chloroform are added. The mass is shaken for 20 minutes and after separation of the phases the chloroform extract is taken up and evaporated to dryness. The residue washed with petroleum ether, which consists of 874 mg of a red-brown product after two recrystallizations from glacial acetic acid, 240 mg

9 0 988S / T75 0

■ - 24 -

■ ■ - ο Γ 1 ²⁰ ° ^C dihydrodaunomycinone, which melts at 250-232 C j {jW-J _D

+ 75 ° (e - 0 * 05 in dioxane). The aqueous acidic phase, which 52 / Ug / ml contains total pigments, 32 tiger ammonia adjusted to pH 7.5 and mixed with a & r from 20 ml of sailcylaldehyde and 200 ml of methyl alcohol added. After shaking for 30 minutes, it will turn on one Adjusted pH to 8.6 and extracted with 10 1 chloroform with shaking for 1 hour. After the organic Phase was separated, the extraction is repeated twice with a total of δ 1 solvent.

The combined extracts are concentrated to approximately 1.5 liters under reduced pressure; they contain 2.58 g total pigments (determined spectrophotometrically). This solution is then evaporated, the residue is taken up in a little methylene chloride and placed on a column of 75 S silica. The column is eluted with the solvent system methylene chloride: petroleum ether: methanol (100: 50: 10). Two colored zones are formed. From the more slowly descending colored zone, 1.65 g of the amorphous salicylidene derivative of dihydrodaunomycin are obtained. This product is recrystallized from methanol and gives 1.23 g of crystals which melt at 165-168 ° C *. Calculated for C- ^ H ₅₅ O _n N: C 64.44, H 5.58, N 2.21,

OCH, 4.90 % found: C 64.24, H 5.6l, N 2.13,

OCH, 4.86 %. 909 885/1750 -25-

To obtain the hihydrodaunomyoin hydrochloride, 1 g of salicylidene derivative is dissolved in 50 ml of chloroform and extracted three times with 15 ml of 0.1N HCl each time. The colored substance migrates completely into the aqueous phase, which is made alkaline with 0.1N NaOH to a pH value of 8.6 and then extracted with chloroform until the extracts are only slightly colored. The combined chloroform extracts are dried over anhydrous sodium sulfate and concentrated in vacuo to about 10 ml; To this end, 2.9 ml of 0.6N HCl in methanol and 10 parts by volume of anhydrous ethyl ether are added. The resulting ¹ precipitate is recrystallized from methanol «dioxane. The yield of dihydrodaunomycin hydrochloride is 0.52 g, melting point 204-206 ° C. (dec.);

- »ο

fotl ²⁰ ° = ^{+ 188} ° ( ^e = °> ¹ ^ ⁱⁿ methanol). By eluting LJ _D

the first colored zone of the above-mentioned silica column becomes 800 mg of a pigment mixture amorphous product obtained.

The product is dissolved in 40 ml of chloroform and mixed three times with extracted 10 ml of 0.1N HCl each time. This way everyone will Pigments transferred into the aqueous phase, which brought to a pH of 8.6 with 0, IN NaOH and with chloroform is extracted. It is extracted three times with a total of 100 ml of solvent. The combined extracts are dried over anhydrous sodium sulfate, concentrated under vacuum to a volume of approximately 5 ml, and made up with 1.5 ml 0.6N HCl in methanol and 10 parts by volume of anhydrous ethyl

9 0 9 8 8 5/1750 - 26 -

ether shifted. 400 mg of a precipitate are obtained, which consists of crude hydrochloride which is purified by chromatography on a column of 100 g cellulose powder (Whatman CF 11), buffered with M / 15 phosphate buffer to a pH of 5.4. The eluent used is an M / 15 phosphate buffer solution saturated to pH 5.4 Butyl alcohol. "Fractions of 25 ml each are taken up .

From fractions 21-50, 85 mg of a product are obtained, some of which consists of daunosaminy! Daunomycin »

Fractions 5I - 78 contain pure daunosaminyldaunomycin. These fractions are combined and 350 ml of ethyl ether and 300 ml of petroleum ether are exposed. It is then extracted three times with water, a total of 450 ml. The aqueous phase, in which the total particles are contained, is _{extracted with chloroform at a pH of 5 3} 4 and this extract is discarded. The aqueous phase is adjusted to pH 8.6 with 0.1N NaOH and then extracted with chloroform. "Three extractions are carried out with a total of 600 ml. The combined chloroform extracts are dried over anhydrous sodium sulfate and concentrated in vacuo to approximately 20 ml. 1.5 ml of Ο, βΝ HCl in methanol and 10 parts by volume of anhydrous ethyl ether are added to the concentrate, with 200 mg of amorphous daunosarainyldaunomycin hydrochloride being kept. 909885/1750 -27- /

The wnkristallisierte from methanol-isopropanol precipitation yields 150 mg Daunosamlnyldaunomycinhydrochloridkristalle, mp 200 ⁰ C. (Dec.); & \ ° methanol).

909895/1750

Claims (1)

Claims 1. Microbiological process for the production of the new antibiotic substances DlhydrodaunomycIn, Daunosaminyldaunomycln and Dlhydrodaunomyclnon and their salts with pharmaceutically acceptable acids, characterized in that the new microorganism Streptomyces peucetlus νar. carneus under aerobic conditions In a liquid culture medium containing a carbon source, a nitrogen source and mineral salts, at a temperature of 25 to 35 ° C for a Period of 4 to 7 days at a pH between 6.5 and 8 is cultivated, and the formed antibiotics of the general formula OCH 0 - R, where in the case of the antibiotic Dihydrodaunomycln RR ₁ the remainder Daunosamlnyl NH ₀ OH H -CH-CH ₀ -CCC-CH, H H OH and means; with the antibiotic Daunosaminyldaunomycln R = O 9 0 9 8 8 5/1750 - 29 - and R ₁ don radical Daunosaminyl and a second Daunosaminylreet is glucosidally linked to one of the free hydroxyl groups of the molecule, and at Antibioti cum dihydrodaunomycinone R ■ and R _n a hydrogen atom means, isolated from the distant entation broth, purified and, if necessary, converted into salts. 2. New antibiotic substance of the general formula OH i, w OH 0-R ₁ wherein R and R. have the meaning given in claim 1 have, as well as their salts with pharmaceutically acceptable acids. J). Dihydrodaunomycin. 4. Dihydrodaunomyoin hydrochloride. 5. Daunosaminyldaunomycln. 6. Daunosaminyl daunomycin hydrochloride. 7. Dihydrodaunomycinone. 8. Pharmaceutical composition with antitumoral, antiνiral, antibacterial and antiprotozoal activity, characterized in that it contains one or more antibiotic substances according to claims 2 to 7 in the form of a mixture with a suitable binder. 90988 5/17 50