DE1089512B - Production of actinomycin Z - Google Patents

Description

The present invention relates to the production of a new antibiotic, hereinafter referred to as "Actinomycin Z «, its components, derivatives and fission products.

The antibiotic actinomycin Z is produced in the culture of a new strain of Streptomyces fradiae, hereinafter described as strain NRRL 2765. It was collected from one in Horsham, England Soil sample is isolated and is processed in our laboratories and in the Swiss Federal Institute of Technology, Institute for Special Botany, Zurich, kept under the designation A 20 675.

Streptomyces fradiae NRRL 2765 forms a dark red cinnamon brown "brownish-gray aerial mycelium. The individual Spores are smooth. The spore chains form open, regular spirals of usually more than five turns. They are monopodial and have a long, straight main axis. When breeding on peptone-containing The strain NRRL 2765 does not cause melanoid discoloration in culture media. The growth is relatively little depending on the temperature; The fungus develops well at both 18 and 40 ° C, but that is not the case Optimal between 25 and 32 ° C.

For further characterization, the growth of the strain NRRL 2765 on various Culture media described. The nutrient media 1 to 7 and 10 were according to W. Lindenbein, Arch. MikrobioL, 17, 361 [1952] produced.

1. Synthetic agar

Growth point-like to pimple, deep yellow or chestnut brown. Air mycelium velvety, white-gray to brownish gray. Substrate deep yellow to dark brown.

2. Synthetic solution

Sediment, flakes milk white to light brown. Pellicle punctiform, white-yellow. Substrate light brown.

3. Glucose agar

Growth thin, veil-like, white-yellow. Aerial mycelium is not formed. Discolored substrate.

4. Glucose asparagine agar

Growth thin, veil-like, initially yolk yellow, later golden yellow to brownish yellow. Velvet mycelium, cinnamon brown to brownish gray.

5. Calcium malate agar

Growth punctiform to pimple, yolk yellow. Air mycelium velvety, snow-white to cinnamon-brown. Substrate brownish yellow.

6. Gelatin prick (18 ° C)

Surface growth point-like, brownish-yellow. Aerial mycelium is not formed. The substrate is chestnut brown. Liquefaction after 20 days 0.6 cm. after 30 days 1.1 cm.

Production of actinomycin Z

Applicant:
CIBA Aktiengesellschaft, Basel (Switzerland)

Representative: Dipl.-Ing. E. Splanemann, patent attorney, Hamburg 36, Neuer Wall 10

Claimed priority:
Switzerland from July 25, 1958 and June 5, 1959

Dr. Ernst Gäumann, Vladimir Prelog, Zurich,

and Dr. Ernst Vischer, Basel (Switzerland),

have been named as inventors

7. Thickness plate

Growth pustular, golden yellow. Aerial mycelium sparse, dusty, forming a fine coating, chalk-white. Hydrolysis after 8 days 0.7 cm.

8. Potatoes

Growth wrinkled, light brown. Aerial mycelium sparse, light gray to brownish gray. Substrate not discolored.

9. Carrots

Growth wrinkled, light brown. Aerial mycelium sparse, chalk-white. Substrate dark brown.

10. Litmus milk

Growth sparse, pellicles thin, light yellow. No peptonization or coagulation. Litmus red.

The strain NRRL 2765 is the same with regard to the appearance of the spores, the color of the air mycelium and the formation of spirals Spore chains and melanoid discoloration of peptone-containing culture media are identical to S. fradiae and are therefore counted provisionally of this type. It differs from the well-known producers of neomycin and fradicin Strains of S. fradiae (cf. German Patent 831754 and S.A. Waksman, "Neomycin", Rutgers University Press [1953]), as the following table shows:

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3 S. fradiae S. fradiae 4th S. fradiae S. fradiae (Original description) No. 3535 No. 3554 NRRL 2765 tribe branched hyphae, branched hyphae, short, closed branched hyphae morphology without spirals without spirals Spirals on the with spirals on the of the Luftmycels Ends of Ends of Sporophores Sporophores rapid expiry, none rapid evaporation, none rapid condensation .., none rapid evaporation, Gelatin stick Pigments; Point Pigments; flaky Pigments; flaky Pigments easily shaped, cream growth growth maroon; light brown punctiform, brownish yellow Peptonization; strength strength no Litmus milk slightly alkaline Peptonization; Peptonization; Peptonization; p _H equal p _H equal slightly sour Growth orange; Growth braid Growth braid Growth wrinkled, potato no soluble good; orange; no good; orange; no light brown; no Pigment; little soluble pigment; soluble pigment; soluble pigment; Aerial mycelium little aerial mycelium little aerial mycelium little aerial mycelium thin, veil-like, sparse wax quite ample lush growth; Starch agar colorless, with pink tum; Aerial mycelium Growth with pink Aerial mycelium chalk Aerial mycelium; sparse, pink with Aerial mycelium with White, good hydrolysis white border; white edges; good hydrolysis good hydrolysis good hydrolysis Aerial mycelium Aerial mycelium Aerial mycelium Aerial mycelium cinnamon Glucose asparagine sea shell pink light brown purple sea shell pink brown to brown Agar gray-violet

The present invention is not limited to the manufacture of the antibiotic ActinomycinZ Use of strain NRRL 2765 or other strains corresponding to the description is restricted, but also relates to the use of variants of these organisms, as they are e.g. B. by selection or Mutation, especially when exposed to ultraviolet or X-rays or nitrogen mustard oils, be won.

Actinomycin Z is markedly different from the known antibiotics of the neomycin group and fradicin, which are produced by the above-mentioned strains of S. fradiae. The neomycins are glycoside-like compounds that are active against bacteria, while fradicin, a compound of the empirical formula C3oH ₃₄ N ₄ 0 ₄ , is not active against bacteria, but has a fungicidal effect (cf. Waksman and Lechevalier, "Actinomycetes and their Antibiotics", 1953 ).

Actinomycins are a group of closely related red, crystalline, anti-bacterial agents highly effective antibiotics, the molecule of which is made up of a dye part and a peptide part. Until now The actinomycins A, B, C, F, I, J and X and their components have become known.

Is actinomycin A (see U.S. Patent 2,378,876) a compound formed by Streptomyces antibioticus that crystallizes in vermilion platelets. In the Hydrolysis with hydrochloric acid provides the 5-amino acids, L-threonine, sarcosine, L-proline, D-valine and N-methyl-L-valine (cf. Ber., 87, 1045 [1954]).

Actinomycin B, obtained from an unspecified Streptomyces strain (cf. Todd et al., J. Chem. Soc. [1950], 2946), is also a red, crystallized substance which is divided into two main components B ₁ and B ₂ can be divided and proved to be identical to actinomycin X or its components X ₁ and X ₂ (cf. Brockmann, Ber., 87, 1045 [1954]). X ₁ contains the same amino acids as actinomycin A, but in a different molar ratio, while X ₂ also contains oxyproline (cf. Brockmann, Ber., 92, 1296 [1959]).
Actinomycin C (cf. German patents 912 010, 925 064, 930 579 and 934715) is a compound formed by Streptomyces chrysomallus, which crystallizes in alizarin red bipyramids and consists of components C ₁ , C ₂ , C ₃ and two secondary components. In addition to the amino acids already mentioned, the main component C _{2 also contains isoleucine, while the main component C 3} does not contain D-valine, but also contains isoleucine; C ₁ is identical to actinomycin A.

Actinomycin F (main components F ₁ and F ₃ ; cf. German patent specification 1 021131 and Naturw., 43, 131 [1956]) is an antibiotic that is obtained by adding sarcosine to the nutrient solutions of actinomycin-forming streptomycetes; the components F ₁ and F ₃ differ from the previously mentioned actinomycins in that they contain no L-proline; for component F _{3 there} is also no VaHn; both components contain isoleucine.

Actinomycin I (cf. German Patent 934 715 and Naturw., 41.65 [1954]) contains actinomycin I ₁ , which is identical to actinomycin A _{, as the main component, and I 0} , which has the same amino acids but in a different molar ratio , and some other minor components.

Actinomycin J ₁ (cf. J. Antibiot. Jap., 2, 181 [1949]) is identical to actinomycin B (cf. Bossi et al., HeIv. Chim. Acta41, 1645 [1958]), while J _{2 is} a mixture of J ₁ with an antibiotic inactive compound.

All of the mentioned actinomycins differ in that

their amino acid components of actinomycin Z.

The antibiotic actinomycin Z consists of at least six components Z ₀ , Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ , which can be separated by means of paper chromatography and which are different from the components of the actinomycins C, X and I, on the all known natural actinomycins can be traced back. The corresponding data are in the following table

I 089 512

_{compiled, with the RC 2} values (= ratio of the distance on paper of an unknown actinomycin to the distance of actinomycin C ₂ ) being specified for the individual components according to a proposal by Brockmann and Gröhne (Chemische Belichte, 87, 1036 [1954]) :

Actinomycin Z
'RC ₂ value Z
approx.
0.35 No. ₁
0.39 Z ₂
0.78 Z ₃
1.63 Z ₄
2.36 Z ₅
2.55 Actinomycin C
RC ₂ value Ci
0.63 C ₂
1.00 C ₃
1.52 Actinomycin I.
RC ₂ value Ii
0.65 Actinomycin X Xo
0.20 Xi
0.65 X ₂
1.05

Organic solvents which are at least partially soluble in water, such as acetone, butanol or methyl ethyl ketone.

You can also see the cultures directly, without previous 5 Separate the mycelium, extract in the manner indicated.

A further enrichment can be achieved by repeatedly extracting the antibiotic-containing organic extracts first with an acidic aqueous solution with a ο p _H below 5 and then with an alkaline aqueous solution with a p _H above 8, the main amount of the antibiotic activity being in remains of the organic phase from which actinomycin Z is isolated. As acidic aqueous solutions, dilute acids such as acetic acid, hydrochloric acid or sulfuric acid, or buffer solutions such as citrate or phosphate buffers, and as alkaline aqueous solutions, dilute alkalis, such as sodium hydroxide or potassium hydroxide, or buffers are suitable and the like.. proven a solvent system consisting of ^ao D ^as raw actinomycin Z may be selected from the concentrates sodium metakresotinat and n is a mixture of 1 Volum- such extracts by addition of petroleum ether, pentane, part of di-n-butyl ether and 3 parts by volume of -Butyl acetate. Hexane and the like precipitated directly as an amorphous red powder

The nutrient solution contains, for example, inorganic salts.

wise chlorides, nitrates, carbonates, sulphates of alkalis, a good cleaning method for the new anti-

Alkaline earths, magnesium, iron, zinc, manganese. ^{Carbohydrates 2} 5 biotic represents the chromatography, whereby the material sources are mainly carbohydrates, such as. B. aluminum oxide has proven particularly effective as an adsorbent. Glucose, sucrose, lactose and starch. As nitrogen- The recovery of the pure antibiotic in crystalline-containing compounds and optionally to be added form is taken, for. B. from organic solvents, growth-promoting substances are z. B. called: Amino as from acetone, methanol, ethanol, chloroform, acetonic acids and their mixtures, peptides and proteins as well as 3o methanol mixtures, acetone-ether mixtures or

Acetone-petroleum ether mixtures. The same solvents or aqueous organic solutions, such as dilute alcohols, dilute solutions are used for recrystallization Acetone, etc.

Actinomycin Z crystallizes in red fine rods. 260-264 ° C; [a] D = -314 ° C (c = 0.246 in chloroform). The elemental analysis gives the following values: C = 54.88 o / ,,, 54.65%; H = 6.42 / ", 6.57 ° / ₀ ; N = 12.25 ° / _0, 12.03 "/"; O (calculated) = 26.60 ⁰ I ₀ . Its UV absorption shake flasks or the well-known fermenters. Than 4 <> spectrum shows bands at 242 πιμ (log e ₁ ^1,. * = 2.34), temperature is one between 18 and 4O ⁰ C. at 429 my. (log £ ^ = 2.25) and at 443 πιμ (log A significant antibacterial effect shows the nutritional E ₁ ¹ ^ ₁ = 2.27). In the infrared spectrum, recorded in solution generally after 24 to 96 hours. Potassium bromide (see Fig. 1) are bands among others

To isolate the antibiotic z. B. the following visible at the following wavelengths: 2.89, 3.36, 3.40, 5.70, Method used: The mycelium is separated from the culture «6,05,6,30,6,60,6,67,7,07,7,31,7,58,7,65,8,36,9,00 , 9.12, filtrate, after which the main amount of the antibiotic in the 9.39, 13.30 and 14.40 μ.

Culture filtrate is found. However, to isolate the six components of the Actino-

significant amounts of the antibiotic are adsorbed on the mycelium. mycins Z is suitable for chromatography. As adsorbent-es it is therefore advantageous to wash the latter thoroughly. Aluminum oxide or cellulose are particularly suitable for this purpose organic, at least partially proven, are particularly suitable. Also the distribution between two not or water soluble solvents such as alcohols, e.g. B. Meth- only partially miscible phases is used for enrichment anol, ethanol and butanols, or ketones, e.g. B. acetone of the individual components is very useful. Advantage- and methyl ethyl ketone. These mycelium extracts are carefully prepared using the countercurrent method worked either directly or after prior concentration. As a particularly favorable solvent system Apply vacuum to the culture filtrate. One extracts the 55 has become a mixture of an aqueous solution of sodium mixture with a water-immiscible organic / 5-naphthalene sulfonate with isopropyl ether. These Niche solvents, such as esters of lower fatty acids, the methods mentioned can be used individually or in combination for example ethyl acetate or amyl acetate, carbon can be used together. For the pure display Hydrogen, e.g. B. Benzene, chlorinated hydrocarbons- the six actinomycin Z components have the following substances, e.g. B. ethylene chloride, methylene chloride or a combination of 6 has proven to be particularly beneficial:

their hydiolysates, such as peptone or tryptone, meat extracts, water-soluble fractions of cereal grains, such as maize and wheat, of distillation residues in the Alcohol production, from yeast, beans, in particular the soy plant, from seeds, for example cotton 35 plant.

The cultivation takes place aerobically, for example in dormant surface culture or preferably submerged while shaking or stirring with air or oxygen in

Chloroform, ketones, e.g. B. methyl propyl ketone, methyl amyl ketone or diisobutyl ketone, alcohols such as butyl alcohols or amyl alcohols, ethers, e.g. B. ethyl ether, diisopropyl ether, dibutyl ethers or glycol ethers and the like. Instead of solvent extraction of cultures 65 or in combination with such as a further cleaning operation you can also use the antibiotic Gain adsorption, for example on activated carbon or on activated earths, such as fuller earth or floridin, and

1. Chromatography on aluminum oxide, where components Z ₀ and Z ₁ can be separated from one another and from components Z ₂ to Z ₅ ;

2. Countercurrent distribution, component Z _{5 being} separated off;

3. Chromatography on cellulose, the components Z ₂ , Z ₃ and Z _{4 being} separated from one another.

The components of actinomycin Z can be found in

subsequent extraction of the adsorbate z. B. be obtained with a 7 ° crystalline form, preferably

the solvents and solvent mixtures specified for the crystallization of actinomycin Z are used will. These components are characterized in more detail below:

Actinomycin Z ₀

Orange-brown microcrystalline powder; decomposes at 250 ° C; UV absorption in fine spirits: bands at 236 ηιμ (log E ₁ ¹ , ^ = 2.44) and at 437 πιμ (log E ₁ ** = 2.17).

Actinomycin Z ₁

Orange-red crystals; M.p. 256 to 260 ° C (dec.); [a] _D = -362 ° (c = 0.185 in chloroform); Elemental analysis: C = 53.97%, H = 6.79%, N = 12.30%, (CH ₃ ) N = 7.48%, CH ₃ O = 0%; UV absorption spectrum in fine spirits: bands at 240 ΐημ (log E ₁ ¹ ^ = 2.25), 427 πιμ (log E ₁ ¹ ^ = 2.00) and at 442 ηιμ (log E ₁ ¹ ^ = 2.01) .

Actinomycin Z ₅

Red crystals; M.p. 261 to 267 ° C (dec.); [d \ _D = - 284 ° (c = 0.244 in chloroform); Elemental analysis: C = 55.71%, H = 6.44%, N = 12.25%; UV absorption spectrum in fine spirits: bands at 240 πιμ (log E ₁ ¹ ^ = 2.40), at 428 ΐημ (log E / £ = 2.21) and at 443 πιμ (log E ₁ ¹ ^ = 2, 24).

During the acid hydrolysis of actinomycin Z and its components Z ₀ , Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₈ , among others, the amino acids N-methylvaline, valine, N-methylalanine, sarcosine and threonine are formed.

The antibiotic actinomycin Z has a very high antibiotic effectiveness against various Test organisms. If one uses as a test method in vitro dilution series (powers of ten) in glucose broth, which are incubated for 24 hours at 37 ° C, the following inhibitory concentrations result:

Test organisms

Micrococcus pyogenes, var. Aureus

Micrococcus pyogenes, var.aureus, peni-

cillin resistant

Streptococcus pyogenes

Streptococcus viridans

Streptococcus faecalis

Corynebacterium diphtheriae

Bacillus megatherium

Mycobacterium tuberculosis *)

Entamoeba histolytica **)

Trichomonas fetus ***)

Inhibitory

concentration
/ ig / cm ³

10

10

10

10

10

10

10

100

1000

<40

*) Cultivated in Kirchner's synthetic medium with 5% bovine albumin; Read the growth after 2 weeks.
**) culture in Bacto-Entamoeba medium; Reading of the amoebicidal effect after 24 hours.

***) Cultivated in broth with 10% horse serum at 37 ° C; Reading after 4 days.

The development of influenza virus on isolated membranes of the chicken chorioallantois from 14-day hatching eggs is still inhibited ^{at a concentration of 10 μg / cm s.}

In addition to the manufacturing processes for the antibiotic actinomycin Z and for its components Z ₀ , Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ , the present invention also relates to the compounds mentioned themselves, the conversion products obtained by means of hydrogenation or oxidation and the Fission products, as they are, for. B. can be obtained in the hydrolysis of the antibiotic actinomycin Z.

The antibiotic actinomycin Z, its components Z ₀ , Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ , the above-mentioned conversion and cleavage products or mixtures thereof can be used as remedies, e.g. B. in the form of pharmaceutical preparations, use. These contain the

ίο said compounds mixed with one for that enteral, parenteral or local application suitable pharmaceutical organic or inorganic carrier material. Substances that do not react with the new compounds, such as

z. B. gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, benzyl alcohol, gum, polyalkylene glycols, Petroleum jelly, cholesterol, or other known excipients. Pharmaceutical preparations can e.g. B. as tablets, dragees, powders, ointments, creams, suppositories or in liquid foim as solutions, Suspensions or emulsions are present. If necessary, they are sterilized and / or contain auxiliary materials, such as preservatives, stabilizers, wetting agents or emulsifiers. You can also do others therapeutically contain valuable substances.

The invention is described in the following examples, without thereby limiting the Subject of the invention is intended. The temperatures are given in degrees Celsius.

example 1

One prepares a nutrient solution of composition: 20 g of soya flour, 20 g of mannitol and 11 tap water, and sets it to p _H 7.8. This or a multiple thereof is ^{filled into 500 cm 3} Erlenmeyers (with 100 cm ³ nutrient solution each) or in 500 l fermenters (with 300 l nutrient solution each) and sterilized for 20 to 30 minutes at 1 atm. Then up to 10% of a partially sporulating, vegetative culture of the strain NRRL 2765 is inoculated and incubated with good shaking or stirring and in the fermenters with aeration (with about 1 volume of sterile air per volume of nutrient solution per minute) at 27 °. After 70 to 120 hours of growth, the cultures are filtered with the addition of a filter aid, depending on the volume, through a suction filter or through a filter press or a rotating filter, thus removing the mycelium and other solid components from the antibiotic aqueous solution.

Example 2

If the nutrient solutions described below are used instead of the medium given in Example 1 a), b), c) or d), after an analogous sterilization, inoculation with the strain NRRL 2765, incubation is obtained at 27 ° and filtration, aqueous antibiotic solutions:

a) 10 g galactose, 5 g peptone, 3 g meat extract (Oxo Lab Lemco) and 11 g tap water; p _H before sterilization: 7.5.

b) 20 g of malt extract, 20 cm ^{3 of} corn steep liquor, 5 g of sodium chloride, 0.2 g of sec. Potassium hydrophosphate, 2 g calcium carbonate and 11 tap water; p _H before sterilization: 7.5.

c) 20 g glycerine, 2.5 g glycocolla, 1.0 g sodium chloride, 0.1 g sec. Potassium hydrophosphate, 0.1 g crystall. Ferrous sulfate, 0.1 g crystall. Magnesium sulfate, 0.5 g calcium carbonate and 11 tap water; p _H before sterilization: 7.5.

d) 20 g glycerine, 1 g casein hydrolyzate, 10 g potassium nitrate, 5 g sec. Potassium hydrophosphate, 5 g sodium chloride, 5 g

Magnesium sulfate, 10 mgcryst. Ferrous sulfate and 1 tap water; p _H before sterilization: 7.5.

Example 3

The filter residue of a 120-1 batch obtained according to Example 1 or 2 is stirred with 151 acetone and nitrated again. This is repeated twice, whereupon the antibiotic-containing acetone solution egg combined, concentrated in vacuo to 5 l and combined with the culture filtrate. This solution is with 701 ethyl acetate ίο pulled out, with the entire antibacterial activity going into the organic phase. The extract comes with Washed water, evaporated to 5 l in vacuo and then several times with 0.5N acetic acid and with 2N sodium hydroxide solution shaken out. Finally, the ethyl acetate solution is dried over sodium sulfate and concentrated they in vacuo to 21. By adding 11 petroleum ether the crude actinomycin Z is precipitated in the form of an orange-red powder. (Yield: 11.5 g.)

ao example 4

9.5 g of the crude actinomycite Z obtained according to Example 3 are chromatographed on a column of 260 g of neutral aluminum oxide of activity III. The column is eluted with 500 cm ³ abs each. Benzene and chloroform and with 700 cm ^{3 of} a mixture of chloroform-methanol (98: 2). With the first two solvents only colorless, inactive by-products are removed, while the chloroform-methanol eluate is colored deep red and, after evaporation in vacuo, gives 4.5 g of a red, amorphous, biologically active residue. By crystallization from a mixture of acetone and ether, the pure actinomycin Z is obtained in the form of red-orange crystals which melt at 260 to 264 ° with decomposition. The elemental analysis gives the following values: C = 54.88 ° / ₀ , H = 6.42% and N = 12.25%, O (calculated) = 26.45%. The UV spectrum recorded in fine spirits shows the following absorption maxima:

log E ₁ ¹

1% cm

242
2.34

429
2.25

443 πιμ.
2.27

component RC ₂ value Z ₀ 0.35 No. ₁ 0.39 Z ₂ 0.78 Z ₃ 1.63 Z ₄ 2.36 Z ₅ 2.55

In the infrared absorption spectrum (see Fig. 1) there are, among other things, bands at 2.89, 3.36, 3.40, 5.70, 6.05, 6.30, 6.60, 6.67, 7.07, 7.31, 7.58, 7.65, 8.36, 9.00, 9.12, 9.39, 13.30 and 14, 40 µ.

The paper chromatographic analysis of the crystalline actinomycin Z (round filter method with 10% aqueous solution of sodium metacresotinate as stationary and with a mixture of 3 parts by volume of butyl acetate and 1 part by volume of dibutyl ether as the mobile phase) shows that it consists of at least six components (Z ₀ , Z ₁ , Z ₂ , Z ₃ , Z ₄ and Z ₅ ), which _{show the following RC 2} values (RC ₂ = ratio of the distance traveled by the unknown substance to the distance traveled by component C ₂ of actinomycin C):

65 Example 5

A solution of 30 mg actinomycin Z in a mixture of 1 cm ^{3 of} conc. Hydrochloric acid and 1 cm ^{3 of} water are melted in a glass tube and 20 hours

heated to 110 to 120 °. After cooling, the hydrolysis mixture is filtered and dried in vacuo evaporated. The residue is examined using two-dimensional paper chromatography, with the following Solvent systems are used:

a) water-saturated phenol and

b) a mixture of n-butanol, glacial acetic acid and water im Volume ratio 4: 1: 1.

The paper chromatograms are performed on the one hand with ninhydrin and on the other hand with pyridine and p-nitrobenzoyl chloride stained. In this way, the amino acids N-methylvaline, VaHn, N-methylalanine, sarcosine and threonine can be detected.

Example 6

A solution of 750 mg of crystalline actinomycin Z obtained according to Example 4 in 20 cm ^{3 of} benzene is placed on a column of 100 g of aluminum oxide (activity III according to Brockmann, neutral). 100 cm ³ each of benzene and chloroform are allowed to run through the column, no elution taking place. The chromatogram is then developed with a mixture of chloroform and methanol (99: 1), three red zones being formed. The lowest one can be ^{eluted with 120 cm 3 of} this mixture and consists of the actinomycins Z ₂ , Z ₃ , Z ₄ and Z ₅ . Another 500 cm ³ of the mixture mentioned elute the second zone, which consists of uniform actinomycin Z ₁ . This is crystallized from acetone-ether and forms orange-red crystals with a melting point of 256 to 260 ° (decomp.); \ a \ _D = -362 ° (c = 0.185 in chloroform); Elemental analysis: C = 53.97%, H = 6.79%, N = 12.30%, (CH ₃ ) N = 7.48%, CH ₃ O = 0%; U V absorption spectrum in fine spirits: A _max = 240 ηιμ (log E ₁ ¹ ^ = 2.25), 427 ηιμ (log E / _c * = 2.00), 442 mti (log E ₁ ^1. * = 2, 01). The IR spectrum agrees completely with that of the actinomycin-Z mixture.

The third zone of the chromatogram is ^{eluted with 150 cm 3 of} a chloroform-methanol mixture (97: 3). It consists of a uniform actinomycin Z ₀ . This is obtained from acetone ether as an orange-brown, microcrystalline powder which decomposes at about 250 °. In the paper chromatogram it behaves like actinomycin Z ₁ , but the zone is less sharp. Its UV absorption spectrum in fine spirits shows bands at 236 πιμ (log E ₁ ¹ ^ 2.44) and at 437 πιμ (log E ₁ ¹ ^ = 2.17).

Example 7

390 mg of the mixture of components Z ₂ , Z ₃ , Z ₄ and Z ₅ of actinomycin Z obtained according to Example 6 are subjected to a hundred-stage countercurrent distribution, the following solvent mixture being used: 2% strength aqueous solution of sodium naphthalene / 3-sulfonate as the stationary phase and a mixture of 4 parts by volume of isopropyl ether and 1 part by volume of ethyl acetate as the mobile phase. After a hundred transfers there are three substance and color maxima in fractions 6, 55 and 88. The contents of distribution vessels 0 to 15, 41 to 65 and 81 to 92 are combined. Fractions 0 to 15 contain actinomycin Z ₁ and impurities, while fractions 41 to 65 _{contain a mixture of actinomycins Z 2} , Z ₃ and Z ₄ , which is obtained from acetone-ether in fine red crystals, which are at approx Slowly decompose at 260 °. [a] _D ² ° = -296 ° (c = 0.257 in chloroform); UV absorption spectrum in fine spirits: / l _ma x = 240 ΐημ (log E ₁ ¹ ,. * = 2.36), 428 ταμ (log E ₁ ^ = 2.17), 441 ταμ log E ₁ ^ * = 2, 18). Fractions 81 to 92 are treated with acetone and ether to give pure crystalline actinomycin Z ₅

009 608/311

obtain. M.p. 261-267 ° (dec.); [d \ _D = -284 ° (c = 0.244 in chloroform); UV absorption spectrum in fine spirits: bands at 240 ταμ (log E / _c * = 2.40), at 428 ταμ (log E ₁ ¹ ^ = 2.21) and at 443 ηιμ (log E ₁ ^1. * = 2.24 ); Elemental analysis: C = 55.71%, H = 6.44%, N = 12.25%, O (calculated) = 25.60%.

Example 8

30 g of cellulose ^{powder are stirred with 120 cm 3 of} a 10% strength aqueous sodium metacresotinate solution and then poured into a chromatography column 2.6 cm in diameter. The excess aqueous solution is removed, whereupon the column is washed through with a mixture of 3 parts by volume of butyl acetate and 1 part by volume of dibutyl ether. 280 gm actinomycin Z is dissolved in 15 cm ^{3 of} this solvent mixture and this solution is applied to the column and eluted further with the same mixture, the following fractions being worked up individually and examined by paper chromatography:
Parliamentary group 1

40 cm ³ actinomycin Z ₅ , uniform;
Group 2

40 cm ³ mixture of actinomycin Z ₄ , Z ₃ and Z ₂ ;
Parliamentary group 3

180 cm ³ mixture of actinomycin Z ₃ , Z ₂ and Z ₁ ;

Group 4

130 cm ³ almost exclusively colorless by-products;
Parliamentary group 5

300 cm ³ actinomycin Z ₁ , uniform.

Example 9

According to the method described in Example 8, 250 mg of a mixture of actinomycin Z ₂ , Z ₃ and Z ₄ obtained according to Example 7 are chromatographed on 30 g of cellulose powder, fractions of 20 cm ^{3 of} solvent each being collected. Fractions 4, 6 and 10 contain actinomycin Z ₄ , Z ₃ and Z _{2, which are} uniform according to paper chromatography. These fractions are processed individually. Treatment with acetone-ether gives the actinomycins Z ₄ , Z ₃ and Z ₂ in crystalline form.

Example 10

25 mg of the mixture of actinomycins Z ₂ , Z ₃ and Z ₄ (cf. Example 7) are separated on four sheets of Whatman paper no. 1 according to the method described in Example 8. The zones of the individual components are cut out and eluted three times with alcohol. The evaporation residues of the eluates, which contain abundant sodium metacresotinate, are dissolved in ethyl acetate and washed three times with dilute sodium carbonate solution and water. The dried solutions are evaporated and the residues on columns of about 1 g of aluminum oxide are cleaned again. About 5 mg each of the pure actinomycins Z ₃ and Z ₄ are thus obtained. Actinomycin Z ₂ is obtained in an amount of less than 1 mg.

Determination of the amino acids. About 3 to 5 mg each of the pure actinomycins Z ₀ , Z ₁ , Z ₃ , Z ₄ and Z ₅ are concentrated with 0.5 ml of water and 0.5 ml. Hydrochloric acid melted in a small tube and hydrolyzed overnight at about 110 °. The hydrolysates are evaporated in vacuo and each dissolved in about 100 μl of water. The amino acids are determined by two-dimensional paper chromatography. Solvents: 1. Phenol saturated with water, 2. Butanol — glacial acetic acid — water (4: 1: 1). By means of the ninhydrin reaction, the same five amino acids can be detected in the hydrolyzates of all actinomycin Z components, namely threonine, sarcosine, N-methylalanine, valine and N-methylvaline. Proline is never found.

Claims (1)

Claim: The use of Streptomyces fradiae NRRL 2765 or one of its properties Microorganism for the production of the antibiotic actinomycin Z or its components by conventional biological cultivation, extraction of the antibiotic from the culture filtrate, if necessary Division into components and pure representation. Considered publications:
German patent specifications No. 831 754,912 010,925 064, 930 579.934 715.1021131; U.S. Patents Nos. 2,378,876, 2,667,441, 2,686,749, 2,698,821, 2,799,622; Chem. Zentralblatt, 1952, p. 4315; 1953, p. 5198; 1954, P. 10506; 1956, p. 13161; 1951, II, p. 1304; 1951.1, Pp. 1019 to 1020; 1956, p. 9488; 1950, II, pp. 56 and 308; 1952, p. 2690. Legacy Patents Considered:
German Patent No. 1060 091, 1 sheet of drawings © 009 608/311 9.60