DE1157735B - Manufacture of ferrimycin - Google Patents

Description

GERMAN

PATENT OFFICE

C 23640 IVa / 30h

REGISTRATION DATE: MARCH 14, 1961

NOTICE THE REGISTRATION AND ISSUE OF EDITORIAL: NOVEMBER 21, 1963

In the patent 1058 216 a new water-soluble antibiotic A 9578 is described, which by breeding of the actinomycete strain S. griseoflavus A 9578 or its mutants.

It has now been found that the same antibiotic, which is now called ferrimycin, and its various components, such as B. the ferrimycins A, A1, A2 and B, also in breeding of a strain S. lavendulae, A 21510.

The strain A 21510 was isolated from a soil sample which was collected in the canton of Obwalden, Switzerland. was collected, and is in the Institute for Special Botany of the Federal Institute of Technology Zurich deposited under the designation A 21510. The organism has the following characteristics: The spores have a smooth surface: the aerial mycelium is pale carmine to cinnamon brown to brown-gray; the spore chains form open, irregular loops and spirals at the ends of longer straight or longer ones only slightly wavy sporophores; when growing on peptone-containing culture media, a black-brown, melanoid discoloration. The growth is relatively little temperature dependent, both at 18 as well as at 40 ° C, the fungus develops well, but the optimum is between 25 and 32 ° C.

For further characterization, the growth of Streptomyces lavendulae, strain A 21510, described on various nutrient media. The nutrient media 1 to 7 and 10 were after W. Lindenbein, Arch. Mikrobiol., 17, p. 361 (1952). Media 11 and 12 did Recipe from Pridham et al. (Antibiotics Annual, 1956/1957, p. 947). The manufacture of Medium 13 was carried out as described by Tresner and Danga (J. Bacteriology, 76, p. 239 [1958]) and that of medium 14 according to the Manual of Methods for Pure Culture Study of Bacteria, Committee on Bacteriological Technics of Soz. Amer. Bacteriol., Biotechn. Publications, Geneva, N.Y. (1946).

1. Synthetic agar:

Growth thin, veil-like, white-yellow; Aerial mycelium very sparse, just a dusty coating forming.

2. Synthetic solution:

Sediment, flakes white-yellow to light yellow.

3. Glucose agar:

Growth thin, veil-like or pustular, light brown to dark brown; Partial substrate discolored dark brown; Aerial mycelium initially producing ferrimycin

Applicant:
CIBA Aktiengesellschaft, Basel (Switzerland)

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

Claimed priority:
Switzerland of March 18, 1960 (No. 3065)

Dr. Ernst Gäumann, Zurich,

Dr. Ernst Vischer, Basel,

and Dr. Hans Bickel, Binningen (Switzerland),

have been named as inventors

Snow-white with pale carmine spots, later gradually changing to cinnamon brown to brown-gray darkening.

4. Glucose asparagine agar:

Growth thin, veil-like to pustular, light brown to dark brown; Substrate discolored light brown; Aerial mycelium velvety pale carmine to cinnamon brown.

5. Calcium malate agar:

Growth thin, veil-like, light brown with sparse pale carmine aerial mycelium.

6. Gelatin prick (27 ° C):

Growth in the prick dark brown or brown-yellow; Substrate discolored dark brown; Aerial mycelium is absent; Liquefaction absent or only very sparse.

7. Thickness plate:

Growth sparse, punctiform or pustular, white-yellow; Hydrolysis after 10 days 11 mm.

8. Potato:

Growth initially thin, veil-like, later wrinkled, light brown to dark brown to brown-black; Substrate discolored dark brown; Aerial mycelium sparse, pale carmine punctiform.

309 749/371

3 4

9. Carrot: Breeding takes place aerobically, for example in

Very sparse growth. resting surface culture, or preferably sub-

mers with shaking or stirring with air or

10. Litmus milk: oxygen in shaker bottles or the familiar ones

Growth sparse, ring-shaped, pustular, light brown ₅ fermenters. A suitable temperature is a brownish yellow; sparse coagulation and economical between 18 and 40 ⁰ C. An essential antibaklichePeptonisierung; Litmus mostly red, but the nutrient solution shows some material effects in blue. generally after 1 to 5 days.

11 Yeast extract agar · ■ To isolate the ferrimycins, z. B. fol-

'Growth thin,' veil-like to wrinkled, light- ¹⁰ & *** ^ f ^ T ^dienei V The mycelium is separated from brown to dark brown; Substrate little dark culture filtrate, after which the majority of the distant brown discolored; Aerial mycelium very well developed, ¹ ^ ¹¹ * ™ culture filtrate is found. It remains velvety, pale carmine to cinnamon brown. ^ ^he spit considerable amounts of ferrimycins on

Mycelium adsorbed. It is therefore advantageous to do the latter

12. Carvajal oatmeal agar: _{15 gu} t wash. Water and growth are suitable for this, thin, veil-like, light yellow; Air-aqueous organic solvents, such as alcohols, mycelium velvet, initially chalky white, later z. B. aqueous methanol.

cinnamon brown. To remove the ferrimycins from the culture filtrate,

13. Peptone-Iron-Agar: VT ^ V ^ Y ^{separate from each other} ^ u are separated

. . so different methods are suitable, which individually or m

Positive response. Combination can be used with each other.

14. Nitrate Broth: It has been shown to be beneficial to the culture solution Nitrates are reduced to nitrites. during these operations on a pH between

3 and 5 to hold.

In the features described, specifically morpho 25 1. One can use adsorbents, e.g. B. Logy of the spores and the spore chains, color of activated carbon, such as Norit, activated earth, such as Fuller-Luftmycels and ability to form melanin with earth or floridin, or resin adsorbers such as asmit. Growth on peptone-containing nutrient media (cf. Ett- The adsorbates are expediently eluted with linger et al., Arch. Mikrobiol., 31, p. 326 [1958]) mixtures of water-miscible organic if the strain A 21510 agrees with Streptomyces lavens 3 ° solvents with water or aqueous acids, dulae match. He is therefore provisionally this z. B. with water-methanol, water-pyridine, verGus counted. diluted acetic acid-methanol or water-methanol

A number of other representatives are known to have glacial acetic acid-butanol mixtures. As particularly beneficial of the genus S. lavendulae produce antibiotics. for the elution of a noritol adsorbate the As will be shown below, there are 35 different mixtures of water (2 parts by volume), methanol (1 Vodiese known substances in characteristic lum part), glacial acetic acid (1 volume part) and butanol (2 volume way from the ferrimycins. parts) proved.

It is also known that Streptomycetenstämme 2. A second method for separating the ferric

occasionally degenerate and many originally mycine from the culture filtrate consists in that one described strains no longer available today 40 the antibiotic activity on cation exchangers are (see. "Applied Microbiology", 6, p. 52 ff. [1958]). adsorbed, particularly containing acid groups Finding another species that ferrimycin resins, such as "Amberlite IRC-50", are suitable. The latter produce, thus means an enrichment that can both in the acid form and in the sodium technology, form can be used, but has become a mixture

According to the invention, be-ferrimycins are grown in a volume ratio of 1: 2 to produce the 45 of these two forms one of the above descriptions is particularly effective. The elution is expedient speaking Streptomycetenstamm in aqueous, one with dilute acids, z. B. with methanolic Carbon and nitrogen source and optionally hydrochloric acid.

growth-promoting substances and inorganic salts 3. The basic ferrimycins can also be used

containing nutrient solution aerobically until this is a substantial 50 precipitated directly from the culture filtrate, z. B. Liehe shows antibacterial effect, and then isolated from it by means of reaction with an organic acid the ferrimycins. Picric acid type. By treating this

As a carbon source z. B. assimilable precipitations with salts of organic bases, e.g. B. with Carbohydrates such as glucose, sucrose, lactose, triethylammonium sulfate, or with dilute acids Mannitol, starch, and glycerin are in question. As a stick- 55 the antibiotic is in the form of the appropriate Substantial nutrients and possibly growth salt obtained. You can do this both in aqueous Promoting substances may be mentioned: amino acids, peptides medium as well as in a water-miscible medium and proteins, as well as their degradation products, such as solvents such as methanol or acetone, work. Peptone or tryptone, also meat extracts, water- This transfer of the sparingly soluble salts in soluble fractions of cereal grains, such as maize and 60 readily soluble salts of ferrimycins, is made with either Wheat, of distillation residues of alcohol mineral acids or of ion exchange resins, z. B. production, of yeast, beans, in particular that of »Amberlite IRA-400, carried out. Soy plant, from seeds, for example the cotton 4. An enrichment of the Ferrimycine is also

plant, etc., but also ammonium salts and nitrates. achieved in that one aqueous or alcoholic From Other inorganic salts can use the nutrient aqueous solutions of the salt with an excess solution, for example, chlorides, carbonates, sulfates of organic, water-miscible solutions of Alkalis, alkaline earths, magnesium, iron, zinc agents such as acetone, dioxane, etc., added, the and contain manganese. Salts are precipitated in solid form.

5. Another method of enriching and / or separating the ferrimycins is that aqueous solutions of the same are extracted with solutions of phenol in chloroform, both the pn of the aqueous solution and the phenol content of the chloroform solution being varied. So is the antibiotic activity z. B. with a distribution between a solution containing ^{100 g of phenol per 100 cm 3 of} chloroform, and an aqueous phase of pH 1 to 6 almost exclusively in the organic phase, while when using a solution containing 100 cm ^{3 of} chloroform contains only 33 g of phenol, is almost completely withdrawn from the aqueous phase only at a pn between 4 and 6. If one understands the ratio of concentration in the organic phase to concentration in the aqueous phase by the distribution coefficient of the ferrimycins, it follows from the above data that the distribution coefficient increases with increasing phenol content of the organic phase and decreases with decreasing Ph of the aqueous phase. Since it is thus possible to set any desired distribution coefficient of the ferrimycins in this system, a large number of inactive impurities can be separated off by combining a few distribution operations.

6. Another enrichment and / or separation method for the ferrimycins is chromatography, such as adsorption chromatography on various materials, e.g. B. Norit, aluminum oxide, magnesium silicates, silica gel, calcium sulfate, and partition chromatography with cellulose, starch, silica gel, Celite and the like. B. on "Dowex 50", "Amberlite IRC-50" and the like. For example, partition chromatography on cellulose using the solvent system butanol (4 parts by volume), glacial acetic acid (1 part by volume) and water (5 parts by volume) and chromatography are suitable Strongly acidic ion exchangers such as "Dowex 50 WX ₂ " in ammonium acetate buffer (pH 4.5) of increasing concentration have proven particularly effective.

7. Furthermore, the ferrimycins cannot by countercurrent distribution according to Craig between two miscible solvent phases are enriched and / or separated from one another. The following solvent systems have particularly proven themselves:

a) sec. Butanol - ^ ₁₀ n-ammonium acetate buffer of Ph 4.68.

b) ^1-η Ao ammonium acetate from the pn 4.6 to 10 ° / ₀ by weight solution of phenol in chloroform. The distribution coefficient of the antibiotic in system b) and thus the position of the maximum activity in the distribution can be changed as desired on the one hand by changing the pn of the buffer solution and on the other hand by varying the phenol content of the organic phase.

c) Benzyl alcohol (200 parts by volume) - n-butanol (100 parts by volume) - 1 n-hydrochloric acid (6 parts by volume) - Water (300 parts by volume) - aqueous sodium chloride solution (60 parts by volume) saturated at 19 ° C.

8. A good method for enriching and / or separating the ferrimycins is electrophoresis. It is carried out as high-voltage electrophoresis at 500 to 4000VoIt. Has particularly proven itself the countercurrent process. The ferrimycins present as a cation are retained locally, by moving the motion forced by the electric field by means of an oppositely directed Electrolyte flow precisely compensated. Substances with a different electrical mobility leave the separation apparatus at both ends of the electrodes.

For the production of largely pure preparations, the following combination of the listed has been found Proven enrichment process. The ferrimycins are made from the culture filtrate on the buffered ion exchanger "Amberlite IRC-50" adsorbed and eluted with methanolic hydrochloric acid. The eluates are concentrated at pn 5 in vacuo to give an aqueous concentrate of ferrimycins which by volume approximately one hundredth of the culture solution is equivalent to. This is done several times between phenol-chloroform mixtures according to the above-mentioned method 5 on the one hand and aqueous solutions of varying pH on the other hand distributed, wherein after freeze-drying, a preparation is obtained which, with respect to the lyophilized culture filtrate, has a Has specific effectiveness increased by a factor of 500 to 1000. By treating one Preparations according to the above-mentioned methods 6, 7 and 8 can be largely uniform preparations the ferrimycins are preferably obtained in the form of a salt.

As can be seen from paper chromatographic studies, the antibiotic is ferrimycin of two main components, namely the base A and the base B. The former can in turn be converted into the Separate components A1 and A2. These various components are described in the course of the Enrichment process largely separated from each other, especially in partition chromatography on cellulose, in chromatography on ion exchange resins and in countercurrent distribution.

The two main components Ferrimycin A and B are defined by a paper chromatography direct comparison of their Rf values with the Rf values of a number of known antibiotics (2 to 11) in systems A to G. System H is continuously chromatographed. The numbers given there are the distances of the antibiotics measured in centimeters after a chromatogram lasting 16 hours. The antibiotics are detected bioautographically with Staphylococcus aureus or Bacillus subtilis.

system la Ib 2 3 4th 5 6th 7th 8th 9 10 11 A. 0
0.49
0.34
0.05 0
0.63
0.58
0.15 0
0.05
0.22
0 0
0.05
0
0 0
0.17
0.02
0 0
0.02
0.22
0 0
0.02
0.03
0 0
0.66
0.72
X 0
0.55
0.62
X 0.92
0.92
0.93
0.92 0.07
0.32
0.39 0
0.22
0.11
0 B. C. D.

la 7th 2 3 4th 5 6th 7th 8th 9 10 11 system 0.32
0.47
0.74
2.7 Ib 0
0.22
0.07
0 0.10
0.14
0 0.22
0.12
0 0
0.04
0.02
0 0
0.05
0 X
0.49
X
(14.5) 8th 0.86
0.91
0.94
27 0.43
0.61 0.12
0.36
0.69
1 E. 0.32
0.47
0.74
7.6 X
0.42
X
(8.8) F. G H

A water-saturated butanol,

B butanol — glacial acetic acid — water (4: 1: 5)
(upper phase),

C water-saturated butanol + 2% p-toluenesulphonic acid,

D water-saturated butanol + 2% piperidine,
E butanol — pyridine — water (6: 4: 3),

F 80% ethanol + 1.5% sodium chloride, Whatman No. 4 impregnated with 0.95 molar sodium sulfate + 0.05 molar sodium hydrosulfate,

G butanol — ethanol — water (1: 1: 2),

H butanol — butyl acetate — glacial acetic acid — water
(10: 3: 1.3: 14.3) (upper phase),

Analysis:

C.

is H

Fe

Equivalent weight.

P & MCS

Ultraviolet spectrum

(Express)

la Ferrimycin A, Ib Ferrimycin B, 2 Streptomycin, 3 Ristocetin A, 4th Ristocetin B, 5 Neomycin B, 6th Viomycin, 7th Chlortetracycline, 8th Oxytetracycline, 9 Actinomycin J, 10 Cycloserine, 11 Grisiness, χ Antibiotics distributed over the entire course, O Position out of focus.

Ferrimycin A1

47.63%

6.72%
4.88%

1078
7.87

229 ηιμ (336)
f 319πιμ (37)
\ 425 πιμ (27.6)

Ferrimycin A 2

48.77%
6.65%
4.82%

984
7.73

227 πιμ (332) 319 πιμ (37)
425 ηιμ (25)

In paper electrophoresis in 0.1 molar acetate buffer of pH 4.6, ferrimycin A and B migrate against the cathode. The migration speed is about half that of streptomycin.

Ferrimycin A, as it is e.g. B. is obtained by means of electrophoresis or chromatography on ion exchangers or cellulose, is a red-brown powder that is readily soluble in water, methanol, dimethylformamide, benzyl alcohol, glacial acetic acid or phenol-chloroform mixtures. The infrared spectrum is shown in FIG. The ultraviolet spectrum (in water) shows a maximum at 318 ηιμ (EW = 47.2) and inflections at 229 and 400 πιμ. The elemental analysis of the hydrochloride gives the following values: C = 50.68%, H = 6.99%, N = 13.45%, Fe (grav.) = 3.55%, Fe (color.) = 3.66% , Cl = 2.75%.

The Ferrimycins A1 and A 2 differ physically and chemically only insignificantly from each other. The corresponding values of highly enriched Preparations obtained by cellulose chromatography in acetic acid butanol-water systems are summarized in the following table:

In FIG. 5, the ultraviolet spectra (in water: c = 5 x 10- ³⁾ of the purified Ferrimycins A (curve 1, Absorbance E = A) and the component ferrimycin A ₂ (curve 2, E = A-0.1) and ferrimycin A ₁ (curve 3, E = A-0.2).

The paper chromatographic behavior of ferrimycin A, A1, A2 and B is shown in FIG. Of the Proof was made bioautographically with Staphylococcus aureus. It means

I = system butanol - glacial acetic acid - water (4: 1: 5)

10 hours,
II = system butanol — butyl acetate — glacial acetic acid

Water (100: 30: 13: 143) 24 hours,
TII = like II, but 60 hours,

1 = crude product containing ferrimycin A and B,

2 = ferrimycin B,

3 = ferrimycin A,

4 = ferrimycin Al,

5 = ferrimycin A2.

In the chromatography of highly purified Ferrimycin Α preparations on cellulose columns with a saline Tert.-butanol-hydrochloric acid mixture produces the pure Ferrimycins Al and A2 in the form of the dihydrochlorides obtain. Their properties are summarized in the following table:

Ferrimycin A ^ dihydrochloride

Microanalysis

C 46.70%, 46.44%

H 6.79%, 6.77%

N 12.73%, 12.80%

Fe 5.27%, 5.34%

Cl 6.43%, 6.31%

(C) CH ₃ 1.95%, 2.15%

Titration (HeIv., 37, p. 1872 [1954])

a)
b)
c)

4.11
7.92
11.4

Equivalent weight

1129

9 10

UV absorption (H ₂ O) acid precipitated in the form of amorphous salts. With ninhydrin

in is formed in a pyridine-containing one upon prolonged heating

■ "Ulf ·! Ti ,, Jl /",. ,,., ".. - τλ · in ι. Ι. ■. ·

319 mu, E ^1% 28 3 ßutanol a blue-violet color. The color reactions

/ im _m n τ? 1 ™ ολ '/ ι according to Sakaguchi and Molisch as well as the anthrone

4JU DILI, £ 1 ™ Z0.4 «it · -KT χ ι τ ·· J τ- ·

5 tests are negative. Neutral solutions of the Ferrimy IR absorption (KBr): one does not react with potassium ferrocyanide, against it

Compare Figure 7 weakly acidic instantaneous to give

Berlin blue. Berlin blue stains also arise

Craig's distribution with ferricyanide ferric chloride reagent, while addi-

Verteilungskoeffizientbei 25 ⁰ C in the system n-butanone ¹⁰ set V _0n potassium ferricyanide alone no appreciable nol-benzyl alcohol 0.001 N HCl-saturated color reaction and the addition of ferric chloride mainly a

Aqueous NaCl solution (10: 5: 15: 3) 0.372 causes brown coloring. Red colored Ferrimycm solutions

change their color when added dropwise from stronger

Paper chromatography mineral acid reversible from violet-red to colorless.

Rf value in the system tert.-butanol O, 004 ^n-1 ^ HCl ^When ISF ^atz _T ^vo _fi ⁿ 2N sodium hydroxide solution is ferric hydroxide -gesättigtewäßrigeNaCl solution (2: 1: 1); Whatman precipitated. Leaves concentrated methanohsche one No. 1 paper impregnated with acetone Η, Ο- ^solution S ^e n ^v ° ⁿ 8-hydroxyquinol on aqueous Lösaturated aqueous NaCl solution (6: 3: 1) .. 0.59 solutions of the ferrimycins act so divide

rapidly dark colored, crystalline precipitates of

". .. ^. ,,,,., so iron hydroxyquinoline from. Deliver the mother liquors

Fernmycm Α, dihydrochloride _{after washing with} chloroform during evaporation the

Microanalysis of iron-free ferrimycins in the form of almost colorless ones

C 45.78% powder based on the addition of ferric chloride in the red

H 6.77% colored ferrimycins can be reconverted. the

N 12.75% 25 Ferrimycins underreact with sodium dithionite

Fe 5.29% discoloration. When the solution is left on the

Cl 6.23% air returns the original color.

(C) CH ₃ 2.16% In the acid hydrolysis of ferrimycin A can

including succinic acid, l-amino-5-hydroxyl-30 amino-pentane, <5-aminovaleric acid, cadaverine and Ammonia can be detected.

The free bases of the ferrimycins are easily accessible from their salts, from the sulfate z. B. by Reaction in an aqueous medium with barium hydroxide, 35 neutralization of the excess barite with carbon dioxide and separation of the barium carbonate and sulfate precipitate and isolation of the free base ϊτν AVi f "^ T4 η" »by means of freeze drying. It is easier to manufacture

u v adsorption <n ₂ uj position from the salts using a strong

λτηαχ 231 ΐημ, E} * _n 328 40 basic anion exchanger, z. B. the OH form

■ Ämeu: 319 ηιμ, E} 1 30 of the under the name Dowex-2 in the trade

λτηαχ 435 πιμ, Ε}? “26.2 located product.

tr Al · «r (KU Λ Fine of the most striking properties of the Ferrimy-

iK adsorption ικ, ογ ;. _{dne is} ^ _pronounced stability minimum between

Compare Fig. 8 45 p _H 6 and 9. Solutions lose at 20 ⁰ C and at

Craig's distribution ^a ™ ^Ph ™ \ ⁷ ™ ίΙ · Tf ^ ^ / tunden? ⁿ

^{6 &} most of their activity while they are at the

Distribution coefficient at 25 ⁰ C in the system n-Bu- same temperature at a p _H of 1 to 5 and

tanol — benzyl alcohol — 0.001 n-HCl — saturated 10 to 13 remain largely effective.

aqueous NaCl solution (10: 5: 15: 3) 0.175 50 The antibiotic effectiveness of Ferrimycins is

". ,. ,. through the iron-containing growth substances, the ferrioxamines,

Paper chromatography antagonized. In this respect differ

Rf value in the system tert-butanol-0.004 n-HCl the ferrimycins of all known, also of

- saturated aqueous NaCl solution (2: 1: 1); What- strains of the genus Streptomyces lavendulae produman No. 1 paper impregnated with acetone-H ₂ O- 55 adorned antibiotics.

saturated aqueous NaCl solution (6: 3: 1) .. 0.47 The salts of the ferrimycins are derived from the

known inorganic and organic salts,

The dihydrochlorides of the pure ferrimycins A _1, for example hydrochloric acid, sulfuric acids and A _2, have about 20,000 times the antibiotic activity with respect to the lyophilized culture and phosphoric acids, the acetic, propionic, valerian and filtrates. 60 Palmitic or oleic acid, succinic acid, lemon-The ferrimycin dihydrochloride are very soluble acid, mandelic acid, glutamic acid or pantothenin water, methanol, 90% phenol, phenolic acid. They represent neutral or acidic salts. Your Her-chloroform mixtures, dimethylformamide and Mestellung takes place by the action of the corresponding thylcellosolve, soluble to moderately soluble in glacial acetic acid, acids on the free base or by double Umalcohol, benzyl alcohol and pyridine and insoluble in 65 formation of salts, for example of ferrimycin-butanol, chloroform, ethyl acetate and less polar sulfate with pantothenic acid calcium. Solvents. They are made from aqueous solutions. The Ferrimycins have a very high anti-

with picric acid, pure ammonium and picrolon - biotic effectiveness against various test

Titration (HeIv., 37, P. 1872 [1954]) pi a)
b)
c) ~ Ci ₁ SC equivalent to
weight 4.04
7.91
11.5 1 1086

Organisms. The following table summarizes the activities of ferrimycin A, Al and A 2. The measured zones of inhibition in millimeters are indicated by measure obtained in the agar plate with paper washers of 6 mm diameter at impregnation with 0, l% ig ^e ri solutions.

Test organisms

Staph. aureus

Streptococcus faecalis.,

Escherichia coli

Shigella sonnei

Pasteurelle pestis

Bac. megatherium

Klebsial type A

Base A Base Al 26th 27 8th 0 10.5 10.5 13.5 14th 12th 12th 20th 20th 23 22.5

Base A2

26.5 9 11 14 12 20 22.5

Incubation, the culture solution has the greatest inhibitory value against the test organisms (B. subtilis, B. megatherium, Micrococcus pyogenes, var. aureus). The culture is interrupted, the pH is brought to 4.5 and by adding dilute sulfuric acid separates the mycelium and other solid components from those containing the majority of the ferrimycins Solution by means of filtration or centrifugation, where appropriate the culture solution before

ίο Filtration about 1% of a filter aid, e.g. "Hyflo Supercel" is added. The filter residue it is washed with water and with aqueous methanol and the washing liquids are combined with the culture nitrate.

If, instead of the nutrient solution given above, one uses those that per liter of tap water contain the following nutrients, culture filtrates are obtained after analogous cultivation and processing of similar high antibiotic effectiveness.

b)

The ferrimycins are also effective in vivo. So z. B. with ferrimycin A, Al and A2 at five ao single subcutaneous administration of 0.1 mg / kg to those infected with Staphylococcus pyogenes, var. aureus Mice 100% and at a dose of 0.05 mg / kg five times 60% survivors were observed.

The toxicity of the ferrimycins is low. So z. B. the subcutaneous application of 1000 mg / kg of Mice endured without harm. Higher doses have not yet been tested.

The antibiotic ferrimycin, its components A, Al, A2 and B, their salts or derivatives can be used as 3 ° Remedies, e.g. B. in the form of pharmaceutical preparations, use. These include the above Compounds in a mixture with a pharmaceutical suitable for enteral, parenteral or local administration organic or inorganic carrier material. For the same, such substances come into question, who do not respond to the new connections, such as B. gelatin, lactose, starch, magnesium stearate, Talc, vegetable oils, benzyl alcohol, gum, polyalkylene glycols, petroleum jelly, cholesterol or others 40 known excipients. The pharmaceutical preparations can e.g. B. as tablets, coated tablets, Powders, ointments, creams, suppositories or in liquid form as solutions, suspensions or Emulsions are present. If necessary, they are sterilized and / or contain auxiliaries such as preservatives, Stabilizing, wetting or emulsifying agents. You can also use other therapeutically useful substances to isolate the ferrimycins from 3001 of the above

Contain substances. Culture filtrates are 1 part by volume of »Amberlite IRC-50«

The invention is illustrated in the following example 50 in the Η-form with 2 parts by volume »Amberlite IRC-50«

described in more detail. The temperatures are mechanically mixed in Celsius in the Na form. 6.31 this

degrees indicated. Mixtures are poured into a glass column. That

_B · ■ j height-diameter ratio of the resin bed

^p carries 8: 1. The culture filtrate is treated with 2N hydrochloric acid

The cultivation of the S. lavendulae, A 21510, is stopped after 55 pH 4.0 and with a flow rate Submerged procedure carried out. One uses speed of 0.21 per minute per liter of resin by the a nutrient solution that percolates 20 g of resin per liter of tap water, with two thirds in the top Rapeseed meal, 10g glucose, Ig calcium carbonate and the column forms an orange-brown zone. That 0.2 g sec. Contains potassium phosphate. The resin nutrient solution is then mixed with 301 water and 601 is washed in the inoculation flasks or in the fermenters 60-80% methanol. The pass and sterilized for 20 to 30 minutes at 1 atm. The washing liquids contain little antibiotic

Glucose 10 g

Soy flour 10 g

Sodium chloride 5 g

Sodium nitrate Ig

Glycerin 20g

Soy flour 10 g

Sodium chloride 5 g

Sodium nitrate Ig

Calcium carbonate 10 g

Glucose 10g

Soy flour 10 g

Corn steep liquor (corn steep liquor) 20 g

Sodium chloride 5 g

Sodium nitrate Ig

Calcium carbonate 10 g

d) lactose 20g

Distillers solubles 20 g

Sodium chloride 5 g

Sodium nitrate Ig

e) Corn steep liquor (corn steep liquor) 20 g

Malt extract 20 g

Sodium chloride 5 g

sec. Potassium phosphate 0.2 g

Calcium carbonate 2 g

sterilized nutrient solution shows a pH of 7.5 to 8.0. Inoculation takes place with up to 10% of a partial sporulating vegetative culture of the organism. Incubate with good shaking or stirring at 27 °, with cultures in fermenters with about 2 volumes of sterile air per volume of solution in the Minute. After 48 to 120 hours

table activity. Elution takes place in two portions with a total of 371 of a mixture of 8 parts by volume Methanol and 2 parts by volume of 1N hydrochloric acid.

of the aqueous eluate concentrate obtained, 1 kg of "Hyflo" is added while stirring and within 2 hours with 1.81 of a mixture of 11 parts phenol and 1 part water. Then one leaves

Stir for another hour, after which it is sucked off. The filtrate contains about 2 to 5 7o of the activity and is discarded. The well squeezed filter cake is stirred twice with ether and once with 41 of a mixture of 8 1 of acetone and ether and 21 respectively JS suction, is then washed and the residue on the filter successively with 51 per acetone and 4] chloroform. All washing solutions are practically inactive. The filter cake, moist with chloroform, is then introduced into a mixture of phenol-chloroform 1: 1 (w / v) with stirring and stirred for 1 hour. 151 chloroform are then added in a steady stream over the course of 1 hour. The "Hyflo" is sucked off and washed out twice with 21% phenol-chloroform 1:11 (w / v) and once with 21% chloroform. The residue still contains 5 to 10 7 _{0 of} the activity.

The combined active filtrates (221) are at 25 ° concentrated to 41 and the concentrate within 30 minutes in a mixture of 41 ether, 8 1 petrol ao dripped in ether and 400 g of "Hyflo". After a further 30 minutes, suction is applied. The filter cake will Washed out with 21 ether and twice with 11 acetone. All filtrates contain only traces of activity. «5

The filter cake is stirred three times for 10 minutes with 1.5 l of methanol each time, suction filtered and for Finally washed with 0.51 methanol on the suction filter. The washed-out "Hyflo" does not contain any Activity more.

The dark brown filtrates (4 to 4.51) are gently turned at 20 to 30 ° in a water jet vacuum Evaporated dry. The still sticky residue is dried in a high vacuum for 20 hours. This results in 75 to 88 g of a red-brown, highly active ferrimycin preparation. The activity yield, based on the starting material, is 90 7o

6 g of the ferrimycin preparation obtained, which, based on the lyophilized culture filtrate, has an approximately 1000-fold antibiotic activity and contains both ferrimycin A and ferrimycin B, is distributed countercurrently over 115 levels according to Craig. The apparatus consists of 120 units. It is charged per unit with 100 cm ³ upper and 100 cm ³ lower phase of a mixture equilibrated at 19 ° of benzyl alcohol (200 parts by volume), n-butanol (100 parts by volume), 1N hydrochloric acid (6 parts by volume), water (200 parts by volume ) and at 19 ° saturated, aqueous sodium chloride solution (60 parts by volume). The first two units contain only solvent. In each of the following three units, 2 g of substance are entered and then distributed 115 times at 19 °. The number of shaking movements per distribution is thirty, the duration of the pauses is 15 minutes.

When the distribution is complete, the 118 fractions obtained are stored at -10 °. For the purpose of testing, 10 cm ^{3 of the} upper and 10 cm ^{3 of the} lower phase are removed from every third fraction and these are shaken with 50 cm ^{3 of} petroleum ether. The separated aqueous phase (10 cm ³ ), which now contains all of the hydrophilic material, is freed from any organic solvents still adhering to it by brief evacuation. The test solutions obtained in this way are used, on the one hand, for colorimetric evaluation (extinction at 425 μm in 1 cm cuvettes [see FIG. 6, curve I]) and for the ninhydrin color reaction. To carry out the latter, 0.5 cm ^{3 of} test solution and 0.5 cm ^{8 of} ninhydrin reagent, prepared according to the instructions of S. Moore and WH Stein, J. Biol. Chem., 211, p. 907 (1954), mixed, 15 Heated to 100 ° for minutes, ^{diluted with 5 cm 3 of} alcohol-water (1: 1) and then measured in a spectrophotometer at 570 μm (curve 2). For biological testing, the test solutions are diluted 1:50. Curve 3 shows the in vitro activities against Staphylococcus aureus obtained in the plate diffusion test with respect to an arbitrary standard. The active fractions 40 to 70 contain, as can be shown by paper chromatography, ferrimycin A, while the ferrimycin B is in fractions 75 to 100. A substantial enrichment can be achieved. The antibiotically active fractions 25 to 39, 40 to 48, 49 to 55, 56 to 70 and 71 to 95 are combined and shaken with the same volume of petroleum ether. The red colored ingredients are driven into the aqueous phases. They are isolated from these in the manner described in Example 12 using phenol-chloroform. The amounts obtained and the concentrations and yields of antibiotic activity achieved in relation to the starting material are tabulated below.

fraction lot
in mg At
enriching
factor Activity
yield Ferri
mycin , ₀ 25 to 39
40 to 48
49 to 55
56 to 70
71 to 95 492
254
211
257
938 0.55
4.77
9.22
3.49
1.72 4.6%
20.4%
32.8%
15.1%
27.2% A.
B.

2 g of the ferrimycin A preparation obtained (fractions 49 to 55), which, based on the lyophilized culture filtrate, has an antibiotic activity that is about 9000 times higher, are placed on a 70 cm x 7.14 cm ³ column of a strongly acidic ion exchanger » Dowex 50 WX ₂ "(100/200 mesh) chromatographed. The ion exchanger is cleaned beforehand according to the instructions of Hirs et al., J. Biol. Chem., 219, p. 623 (1956), and after conversion into the ammonium form for 3 days with 0.2 molar ammonium acetate buffer of pH 4.50 at 17 ° equilibrated at a flow rate of 100 cm ³ per hour. The substance is then dissolved in 20 cm ^{3 of} buffer solution, carefully applied to the column and then eluted with the same buffer solution and flow rate. After an elution period of 20 hours, the system is switched to gradient elution. The 0.2 molar ammonium acetate buffer entering the column is continuously concentrated in a 1-1 mixing chamber by an equally large inflow of 2 molar ammonium acetate buffer at pH 4.50 and its elution effect is continuously increased. The eluate is collected in fractions of 40 cm ³ on an automatic fraction collector. The chromatogram is evaluated by measuring the extinction at 425 ΐημ, paper chromatography and biological testing. The fractions combined according to this evaluation are worked up in the manner described with phenol-chloroform. From fractions 195 to 216, 44 mg of ferrimycin A are obtained, which has an antibiotic activity that is about 16,000 times greater than that of the lyophilized culture filtrate, as a brown-red powder.

Microanalysis

C 48.65%, H 7.09%, N 12.95%, Fe 4.5 (Γ%, Cl 6.10%, (C) CH ₃ 1.99% (Roth-Kulm), amino-N 2.33% (Van Slyke).

Titration

pK & cs (HeIv., 37, p. 1872 [1954]): 4.18; 7.88 Equivalent weight: 1106

Absorption spectrum

λη, α, χ 228ηιμ, Eil 282
Amas 219 ΐημ, E il 28.2
Xmax 425 ΐημ, E il 22.6

"Reduction value" according to C. S. Hanes, Biochem. J., 23, p. 99 (1929)

1.7 cm ^{3 of} Vioo n sodium thiosulfate

Bound hydroxylamine according to T. Emery and _ao JB Neilands, Nature, 184, p. 1632 (1959)

0.83 moles of NH ₂ OH per atom of Fe.

400 mg of ferrimycin A obtained in this way are chromatographed on a column (87 cm × 23.8 cm ² ) packed dry with 830 g of Whatman cellulose powder. To prepare the chromatogram, first 5.3 liters of methanol-water-saturated aqueous sodium chloride solution (5: 2: 3) mixture are added over the course of 70 hours and then 3 liters of the organic phase of a mixture of tert-butanol over the course of 70 hours. Pass 0.02 N hydrochloric acid saturated sodium chloride solution (2: 1: 1) through the column. The column is then equilibrated for 6 days at a flow rate of 30 ml / hour. aqueous sodium chloride solution (2: 1: 1) saturated with the organic phase of a mixture of tert-butanol — 0.001 η-hydrochloric acid — serving as a mobile phase. The substance is rubbed in a mortar with 5 g of washed cellulose powder and 30 ml of superplasticizer to form a homogeneous paste, which is applied to the column. Three colored zones develop over the course of the chromatogram (75 hours). Shortly before the exit of the first zone, the eluate begins to be collected in fractions of 10 ml in the automatic fraction collector. Prezone: Fraction No. 20 to 37; Main zone: parliamentary group no. 38 to 65; Post zone: Fraction No. 75 to 95. The combined eluate fractions are shaken with 10 percent by volume of water and the same volume of ether. The deep red-colored ingredients are extracted from the aqueous phases with phenol-chloroform (1: 1), the extracts are washed extensively with 0.01 η-hydrochloric acid containing 10% sodium chloride, the extracts are dried by passage over Celite and the ingredients are precipitated from the dry extracts by adding ether-petroleum ether with thorough stirring and at low temperature to "Hyflo Supercel". The substances precipitated on the "Hyflo Supercel" are washed extensively with ether by stirring and suction filtration and then eluted with a little methanol. The methanolic solutions are gently concentrated to dryness in a vacuum. _{37 mg of a mixture of ferrimycin A 1} and an inactive by-product are obtained from the pre-zone (20 to 37), 291 mg of pure ferrimycin Aj dihydrochloride from the main zone (38 to 65) and 5 mg of pure ferrimycin A _a - from the post-zone Dihydrochloride in the form of a brown-red powder. For analysis, it is dried over phosphorus pentoxide-potassium hydroxide at 25 ° / 0.001 torr for 50 hours. The data determined are summarized in the following table.

Ferrimycin Aj. dihydrochloride Microanalysis

C 46.70%, 46.44%

H 6.79%, 6.77%

N 12.73%, 12.80%

Fe 5.27%, 5.34%

Cl 6.43%, 6.31%

(C) CH ₃ 1.95%, 2.15%

Titration (HeJv., 37, p. 1872 [1954]) equivalent to
weight PKi ₁ C 1129 a)
b)
c) ~ 4.11
7.92
-11.4

UV absorption (H ₂ O)

l _m ax 229m [i, Eil 317
λτηαχ 319 πιμ, E ü 28.3
λνιαχ 430 ΐημ, Ε if »26.4

IR absorption (KBr)
Compare FIG. 7

Craig distribution

Partition coefficient at 25 ° in the n-butanol — benzyl alcohol system — 0.001 n-HCl — saturated aqueous NaCl solution (10: 5: 15: 3) 0.372

Paper chromatography

Rf value in the system tert-butanol — 0.004 n-HCl — saturated aqueous NaCl solution (2: 1: 1); Whatman No. 1 paper impregnated with acetone _{— H a} O — saturated aqueous NaCl alloy (6: 3: 1) 0.59

Ferrimycin A ₂ dihydrochloride

C 45.78%

H

N

Fe

α

(QCH ₃

12.75% 5.29% 6.237ο 2.16 V ₀

Titration (HeIv., 37, p. > 1872 [1954]) equivalent to
weight PK ^ _£ ί 1086 a)
b)
c) ~ 4.04
7.91
-11.5

UV absorption (H ₂ O)

Imax 231 ηιμ, Ε i? "328

Xuax 319 ηιμ, E} 1 30

Xmax 435 ηιμ, E ü 26.2

IR absorption

Compare FIG. 8

Craig distribution

Partition coefficient at 25 ° in the n-butanol — benzyl alcohol system — 0.001 n-HCl — saturated aqueous NaCl solution (10: 5:15: 3) 0.175

Paper chromatography

Rf value in the system tert-butanol — 0.004 n-HCl — saturated aqueous NaCl solution (2: 1: 1); Whatman No. 1 paper impregnated with acetone-H ₂ O-saturated aqueous NaCl solution (6: 3: 1) 0.47

IO The dihydrochlorides of the pure ferrimycins A ₁ and A ₂ have an antibiotic activity about 20,000 times that of the lyophilized culture filtrate.

Claims (1)

PATENT CLAIM: The use of Streptomyces lavendulae A 21510 for the production of the antibiotic ferrimycin or its components by conventional biological breeding, extraction of the antibiotic from the culture filtrate, pure preparation and production of its salts. Considered publications:
German interpretation document No. 1 058 216. In addition 3 sheets of drawings © 309 749/371 11.63