DE1123436B - Process for the production of new growth substances - Google Patents

Description

Made from materials of biological origin, in particular plant material, animal organs and microorganisms, a large number of biologically and physiologically active substances have already been isolated. Such substances are used, among other things, to accelerate the growth of microorganisms, in particular Yeasts. For example, extracts are used to accelerate growth and fermentation processes in technical fermentations Soybean meal (French patent 786 564), uncooked whey (German patent 717 997), Yeast or malt extracts (French patent 870 386) added. Furthermore, according to the German Patent specification 864 140 a growth substance can be obtained from bacteria, which the growth of yeast in promotes in the same way as vitamin H, but is not identical to it.

It is also already known that iron-containing growth substances can be obtained from microorganisms, namely Ferrichrom Coprogen and Ferregens Factor (Bact. Rev., 21, p. 101 [1957]).

It has now been found that from plant organisms, in particular actinomycetes, and their Extracts growth substances can be obtained in pure or enriched form, the following ferrioxamine to be named.

The ferrioxamines are nitrogen and iron containing organic compounds. They have a brownish red Color and are easily soluble in acids and strongly polar solvents such as water, dimethylformamide, Glycol, ethylene glycol monomethyl ether, and in lower aliphatic alcohols such as methanol. They are also in higher aliphatic alcohols as well as in aromatic alcohols and phenols limited solubility, e.g. B. in butanol, benzyl alcohol, phenol. The hydrolysates contain the ferrioxamines ninhydrin-positive substances. From a chemical point of view, the ferrioxamines are a group of antibiotics which are called "sideramycins" are related. The sideramycins include those that contain iron Antibiotics Grisein, Albomycin, the Ferrimycine, the Antibiotic 1787 (H. Thrum, Naturwiss. 44, p. 561 [1957]) and the substances L.A. 5937 (P. Sen si and M. T. Timbal, Antibiotics & Chemotherapy, 9, p. 160 [1959]).

The crude ferrioxamine obtained, for example, from the fermentation of streptomycetes is usually a mixture of different components. Ferrioxamine B is the main product of the Fermentation of S. pilosus Ettlinger et al. NRRL 2857 (ETH 21748) formed ferrioxamines. Besides are further substances called Ferrioxamine A, C, process for the production of new growth substances

Applicant: CIBA Aktiengesellschaft, Basel (Switzerland)

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

Claimed priority:

Switzerland of September 25 , 1959 (No. 78 652, 78 653) and March 18, 1960 (No. 3063, 3064)

Dr. Ernst Gäumann, Dr. Vladimir Prelog, Zurich,

Dr. Hans Bickel, Binningen,

and Dr. Ernst Vischer, Basel (Switzerland),

have been named as inventors

D ₁ , D ₂ , E and F are designated. When Craig's distribution from the culture filtrate (2 to lOtägige fermentation at 27 ⁰ C) by extraction with phenol-chloroform (Ig: ImI) crude product obtained five fractions can be extracted with an absorbance at 425 μιη in Figure is shown first. In the chromatography on the ion exchange resin Dowex 50 WX _2, pure ferrioxamine B hydrochloride is obtained from main fraction III as a brown-red powder (FIG. 2, fractions 93 to 125).

Ferrioxamine B hydrochloride is soluble in water and strongly polar organic solvents. It behaves according to paper chromatography and in the multiple distribution as a uniform substance of similar polarity as the ferrimycins A ₁ and A ₂ , but differs from them by its

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significantly greater stability. In a weakly acetic acid solution, it migrates in electrophoresis with only a slightly slower migration speed than that. For ferrioxamine B hydrochloride in highly enriched form the following properties were found: microanalysis after 48 hours at 20 ⁰ C / 0.001 mm: C48,04 »A» H 7.41%, N11,21%, Cl 5.25%, Fe 7.67%, P0%. Titration: pK * _M cs (HeIv., 37, p. 1872 [1954]) 9.74; Equivalent weight 704. Absorption spectrum in H ₂ O: X _ma x 430 πιμ with E \ t> - 39.0. The infrared spectrum in Nuyol shows, among other things, bands at 3230, 2900, 1640, 1573, 1461, 1377, 1260, 1225, 1185, 1132, 1028 cm- ¹ , double band: 989, 975, 935, 810, 750 cm " ¹ (s Fig. 3) Multiple distribution, paper chromatography and paper electrophoresis: see Table 1, Figs.

During hydrolysis with dilute hydrochloric acid, succinic acid, l-amino-5-hydroxylamino-pentane, Cadaverine and hydroxylamine can be detected. Will hydriodic acid for the If hydrolysis is used, no l-amino-5-hydroxylamino-pentane and no hydroxylamine are produced. With 2,4-Dinitrofluorobenzene forms ferrioxamine B, a 2,4-dinitrophenyl derivative.

Further iron-containing, reducing compounds with antisideramycin activity can be isolated by chromatography on ion exchangers from the secondary fractions II, IV and V, which are obtained in the Craig distribution of the crude ferrioxamine mixture (see FIG. 1). According to their behavior in the paper bromatogram (Fig. 4) and on the ion exchanger (Fig. 5), they are referred to as Ferrioxamines A, C, D ₁ , D ₂ , E and F.

The ferrioxamines A and C isolated from fractions II and IV (cf. FIG. 1) behave physically and chemically very similar to ferrioxamine B. A proves in the paper chromatogram and in the multiple distribution is slightly more polar than B. For C it is the other way round. This finding also corresponds to the slightly increased basicity of A compared to B as well as its electrophoretic one which is slightly greater Migration speed in weakly acetic acid solution or the smaller one compared to B. Basicity and electrical mobility of C (Fig. 5). A and C continue to show similar IR spectra and Solubility properties such as Ferrioxamine B and, up to now, could only have been able to do so as hydrochloride can be obtained in amorphous form.

Ferrioxamine A hydrochloride is a brown-red powder that dissolves well in water, methanol, ethanol, glacial acetic acid and dimethylformamide. It is insoluble in ether, acetone, ethyl acetate and chloroform. Rf in the solution system i: 0.35, in the solution system V: 0.21 (see Tab. 1). Distribution coefficient in System VI: 0.111 (see Tab. 1). .. Paper electrophoresis see Figure 5. Microanalysis: C44,21 ° / _0, H 7.52% N ,, 12.63%, Fe 7.95%, Cl 5.93%. Titration: pK * _M cs 9.89; Equivalent weight 634. UV spectrum in water: max. 430 πιμ (E [* _m = 37). IR spectrum in potassium bromide shows the following bands (s = strong band, m = middle band, w = weak band): 2.92 μ (s), 3.42 μ (m), 6.10 μ (s) , 6.32 μ (s), 6.88 μ (m), 7.30 μ (w), 7.92 μ (w), 8.10 μ (w), 8.49 μ (w), 8 , 98 µ (w), 9.55 µ (w), 10.15 µ (w), 10.67 µ (w) (see. Fig. 11).

Ferrioxamine A gives a positive reaction with ninhydrin. The iron bond in Ferrioxamine A becomes removed from the complex when treated with mineral acid or strong alkalis. Iron-free Ferrioxamine A is colorless. It can be returned to ferrioxamine A with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex. For further characteristic properties see table 1.

Ferrioxamine C hydrochloride shows almost the same solubility properties as A. Rf in the solvent system I: 0.54, in the solvent system V: 0.37

ίο (see Tab. 1). Paper electrophoresis see Fig. 5, partition coefficient in system VI: 0.489 (see Table 1). Microanalysis: C 48.33%, H 7.92%, N 10.20%, Cl 5.15%, Fe 6.82%. Titration: pK * _M cs 8.88; Equivalent weight 762. UV spectrum in water:

max. 430 ΐημ (E} * „= 39). The IR spectrum in Potassium bromide shows bands at 2.92 μ (s), 3.43 μ (s), 5.85 μ (m), 6.10 μ (s), 6.33 μ (s), 6.87μ (s), 7.30μ (m), 7.95μ (m), 8.23μ (w), 8.52μ (m), 9.65 µ (w), 13.23 µ (m) (see. Fig. 12).

Ferrioxamine C gives a positive color reaction with ninhydrin. The iron bond in Ferrioxamine C becomes removed from the complex when treated with mineral acid or strong alkalis. Iron-free Ferrioxamine C is colorless. It can be returned to ferrioxamine C with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex.

_{The more lipophilic ferrioxamines D 1} , D ₂ and E isolated from fraction V (see electrophoresis and titration as neutral compounds (cf. Table 1, FIGS. 4 and 5). Ferrioxamine D, which is isolated from a uniformly appearing band in ion exchange chromatography as the fastest migrating substance (see FIG. 8), can be converted into the more lipophilic ferrioxamine, which crystallizes from methanol ether in elongated prisms, by simple distribution between chloroform and water Separate D ₁ and the only very small amount of ferrioxamine D ₂ . Ferrioxamine D ₁ is readily soluble in water, methanol, ethanol, glacial acetic acid, methyl cellosolve and choroform, and is sparingly soluble in ether, acetone, ethyl acetate, pyridine and dimethylformamide. It crystallizes from methanol ether in red needles. F. after three crystallizations 194 to 200 ⁰ C. Rf in the solvent system I:

0.73, R ₁ in the solvent system V: 0.72 (vg). Table 1). Distribution coefficient in system VI: 1.80 (see Table 1). Electrophoresis see Figure 5. Microanalysis: C 49.31%, H 7.47%, N 12.37%, Cl 0%, Fe 7.66%. Titration: no acidic or basic functions can be determined. UV spectrum in water: Xmax 430 ηιμ (E} * _m = 44). The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μ (s), 3.06 μ (s), 3.25 μ (w), 3.43 μ (s), 6.08 μ (s) , 6.35 μ (s), 6.86 μ (s), 7.30 μ (m), 7.94 μ (m), 8.20 μ (w), 8.49 μ (w), 8 , 83μ (w), 9.00μ (w), 9.65μ (w), 10.00μ (w), 10.31μ (w), 10.67μ (w), 12.20 μ (w), 13.30 μ (m) (see Fig. 13). Ferrioxamine D ₁ does not react color with ninhydrin. The iron bond in Ferrioxamine Dj is removed from the complex when it is treated with mineral acid or strong alkalis. Eisenfreifis Ferrioxamine D ₁ is colorless. It can be returned to ferrioxamine D ₁ with ferric chloride . It also reacts with other metal ions to form the

corresponding metal complexes, for example the greenish copper complex.

Ferrioxamine D ₂ : The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μ (s), 3.43 μ (πι), 6.08 μ (s), 6.36 μ (s), 6.90 μ (s), 8.49 μ (w), 8.87 μ (w), 9.65 μ (w), 10.05 μ (w), 10.70 μ (w), 13.22 μ ( w) (see. Fig. 16). R _t in solvent system I: 0.64; Rf in the solvent system V: 0.68 (see Table 1). Paper electrophoresis see Fig. 5.

Ferrioxamine E, which has a more differentiated IR spectrum than the other ferrioxamines (see. Fig. 14), differs from these also in its poor solubility in water and methanol.

Microanalysis: C 49.80 ° / ₀ , H 7.37 ° / ₀ , N 12.48 ° / ₀ , Cl 0 ° / ₀ , Fe 8.14 ° / ₀ . Titration: no acidic or basic functions can be determined. UV spectrum in water: Xmax 430 Γημ (EH = 42). The IR spectrum in potassium bromide shows, among other things, bands at 2.92 μ (s), 3.02 μ (s), 3.45 μ (s), 5.96 μ (s), 6.15 μ (s) , 6.36 μ (s), 6.90 μ (s), 7.10 μ (m), 7.15 μ (m), 7.39 μ (m), 7.82 μ (w), 7 , 98 μ (m), 8.45 μ (w), 8.54 μ (w), 8.85 μ (w), 901 μ (w), 9.20 μ (w), 9.98 μ ( m), 10.07 μ (w), 10.23 μ (w), 10.43 μ (w), 10.71 μ (w), 11.87 μ (w), 13.20 μ (m) , 13.66 µ (w) (see Fig. 14).

Ferrioxamine E does not react color with ninhydrin. The iron bond in Ferrioxamine E is turned off removed from the complex when treated with mineral acid or strong alkalis. Iron-free Ferrioxamine E is colorless. It can be returned to ferrioxamine E with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex.

Ferrioxamine F, which, according to its behavior in paper chromatography and in countercurrent distribution, also belongs to the lipophilic group (D ₁ , D ₂ , E), however, in contrast to D ₁ , D ₂ and E, shows basic properties and is isolated as the hydrochloride ( see Figs. 4 and 5).

Ferrioxamine F hydrochloride is readily soluble in water, methanol, pyridine, glacial acetic acid, ethanol, dimethylformamide; Slightly soluble in chloroform, insoluble in ethyl acetate, acetone and ether. Microanalysis: C 50.44%, H 7.29%, N 10.53%, Cl 4.10%, Fe 5.57%. Rf in the solvent system V: 0.80 (see Table 1). Distribution coefficient in System VI: 3.12 (see Table I). Electrophoresis see Fig. 5. Titration: pKxcs 9.75, equivalent weight 695. The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μ (s), 3.45 μ (m), 6.10 μ (s ), 6.37 μ (s), 6.92 μ (m) 7.40 μ (w), 7.97 μ (w), 8.50 μ (w), 8.88 μ (w), 9 , 72 µ (w), 10.10 µ (w), 10.70 µ (w), 13.75 µ (w) (see Fig. 15). UV spectrum in water: Xmax 430 ηιμ, P 1% _ "Μ

Ferrioxamine F gives a positive reaction with ninhydrin. The iron bond in Ferrioxamine F is turned off removed from the complex when treated with mineral acid or strong alkalis. Iron-free ferrioxamine F is colorless. It can be returned to ferrioxamine F with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex.

In the following table 1 are characteristic physical data of the previously isolated Ferrioxamine compiled for comparison.

Tabel

Ferrioxamine Paper electro
phoresis Paper chro
RfI matography
Rf V Multiple
distribution
KVI Absorbance
at 430 Γημ
F i% Titr
pKmcs ation
equivalent to a) b) c) d) ⁿ To e) weight A. 13.6 0.35 0.21 0.11 37 9.89 634 B. 13.0 0.44 0.29 0.23 39 9.74 704 C. 12.3 0.54 0.37 0.49 39 8.88 762 D ₁ 3.9 0.73 0.72 1.80 44 (neutral) D ₂ 3.9 0.64 0.48 (neutral) E. 3.9 0.68 0.59 1.59 42 (neutral) F. 12.5 0.80 3.12 34 9.75 695

a) Centimeter running distance in 0.33 η-acetic acid, after 4 ^1/2 hours at 220 V. In comparison, fructose migrates 3.9 cm.

b) Rf I: Rf value in system I: n-butanol — glacial acetic acid — water (4: 1: 5).

c) Rf V: R _f value in system V: tert-butanol — water — saturated aqueous sodium chloride solution — 0.1 N hydrochloric acid (50: 25: 25: 1), paper impregnated with acetone — water — saturated aqueous sodium chloride solution (6: 3: 1).

d) KVI: partition coefficient in system VI: n-butanol - benzyl alcohol - water - saturated aqueous sodium chloride solution - 0.1 n-hydrochloric acid (200: 100: 300: 60: 3). Distribution of 10 mg over 34 stages of 3 per cm ^{3 of} the organic and aqueous phase at 23 to 25 ⁰ C. Evaluation by absorbance (2 cm ³ of diluted with alcohol in 10 cm ³ fractions) at 425 Γημ.

e) W. Simon et al., HeIv., 37, p. 1872 (1954).

Table 2 gives comparative chemical and physical data for ferrioxamine B and related Growth substances and some sideramycin antibiotics (grisein A, albomycin and ferrimycin A) are compiled.

The salts of the basic ferrioxamines A, B, C and F are derived from the well-known inorganic and organic salts, for example from hydrochloric acid, sulfuric acids and phosphoric acids, the Acetic, propionic, valeric, palmitic or oleic acid, succinic acid, citric acid, tartaric acid, mandelic acid, Glutamic acid or pantothenic acid. They represent neutral or acidic salts. Their production he follows

Tabel

substance C. Anal;
H «Enwen
N e ( ⁰ Io)
Cl Fe mole
kular
weight d)
PK * MCS Abso
Ä-max rption
EJ * _m Proven
Hydrolysis products ^e ) Ferrimycin A ¹ 48.65 7.09 12.95 6.10 4.56 1106 ^a ) 4.18; 228 282 NH ₃ , succinic acid, 7.88 319 28.2 1 -amino-5-hydroxyl- 425 22.6 amino-pentane, o-aminovalerian- acid, cadaverine, (0.83) Krist. Substance with ληιαχ 227 and 323 ηιμ, Proline and non-identical detectable ninhydrin positive substances Grisein A ² ) 43.95 5.65 12.97 5.14 1034 ^a ) 265 108 Methyluracil, 420 28.9 Glutamic acid Albomycin ³ ) 4.16 1270- Methyluracil, serine, 1346 ") Ornithine Ferrioxamine 48.04 7.41 11.21 5.25 7.67 704 ^a ) 9.74 430 39.0 NH ₃ , succinic acid, B-hydro 1 - amino-5-hydroxyl- chloride aminopentane, Cadaverine, o-amino (1.2) valeric acid, Hydroxylamine, no Wisteria, ornithine or Serine Ferrichrome *) 44.02 5.90 16.55 7.35 725 ^c ) 425 39.4 NH ₃ , glycine, ornithine 2.89 FerrichromA ⁴ ) 44.75 5.80 11.18 5.3 1100 ^c ) 440 33.8 Serine, glycine, ornithine 3.01 Coprogen ⁶ ) 50.96 6.83 10.26 6.61 440 36.6

a) By titration.

b) According to Fe, SO ₁ and NH, content.

c) Determined by two independent methods.

d) W. Simon, E. Kovats, L. H. Chopard-dit-Jean & E. Heilbronner, HeIv., 37, p. 1872 (1954).

e) Colorimetrically according to Csaky ") determined" hydroxylamine values " per atom of Fe. The values given in brackets are uncorrected.

') Patent application C 21 506 (Case 4287/1 + 2 / E).

² ) FA Kuehl et al., J. Amer. Chem. Soc, 73, p. 1770 (1951).

³ ) GF Gause, Brit. Med. J., 1955, p. 1177.

«) JB Neilands, Bact. Rev., 21, p. 101 (1957).
⁵ ) CW Hesseltine et al., J. Amer. Chem. Soc, 74, p. 1362 (1952).
o) TZ Csaky, Acta Chem. Scand., 2, p. 450 (1948).

by double conversion of salts, for example of ferrioxamine sulfate with pantothenic acid calcium or by anion exchange on an ion exchanger, for example from ferrioxamine chloride a strongly basic ion exchanger like IRA-400 in the sulphate form.

The ferrioxamines have growth-promoting properties for a large number of organisms.

They have such an effect on Bacillus subtilis, Micrococcus pyogenes var. Aureus, and Saccharomyces cerevisiae, Ustilago sphaerogena and Chlamydomonas eugametos. As an example, in table 3 Results of experiments with Ustilago sphaerogena (smut fungus) and with Chlamydomonas eugametos (Green algae), using an enriched preparation of ferrioxamine B:

Tabel

Relative growth
compared to an untreated control
Ferrioxamine B additive ^ g / cm ³ ) 10 1 0.1 247% 0.01 O 100 540% 405% 98% 158% 100% 271% 136% 98% 100% 280%

0.001

Ustilago sphaerogena (24-hour culture) ..
Chlamydomonas eugametos (4-day culture)

Other organisms, e.g. B. Representatives of the genus Arthrobacter, such as Arthrobacter terregens and Arthro-Π1

bacter flavescens, only develop in the presence of ferric dioxamines. In these cases

The ferrioxamines therefore have a similar vitamin character as it does for the active substances Ferrichrom, Terregens Factor and Coprogen has been shown (Bact. Rev., 21, p. 101 [1957]).

Another biological property of ferrioxamines is that they are able to reduce the antibacterial effects of antibiotics that the Group belonging to the Sideramycins, lifts competitively against gram-positive pathogens.

The antisideramycin effect of ferrioxamines occurs compared to the antibiotics albomycin, grisein, A1787, ferrimycin, and A 22765, all of which also crossed Have resistance to being gris. It is surprising that the ferrioxamines have the effect of the grisein-like Abolish sideramycins against gram-positive bacteria, but not against gram-negative bacteria.

The antagonism between the ferrioxamines on the one hand and the sideramycins, the antisideramycin activity can be detected both “in vitro” and “in vivo”. The antagonizing one Effect of ferrioxamine B against various antibiotics is from the following Table. The test organisms used were: Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The antibiotics to be tested were tested in the modified Bonifas test, the one below has been tested against ferrioxamine B (1 mg / ml).

Tabel 4th Staphylococcus Ash
richia 30th aureus coli Antibiotics Disinhibition of the test organisms competitive 0 ¹ ) Bacillus 35 Ferrimycin subtilis competitive no Grisiness competitive competitive no (40,000 IU / mg) competitive no Albomycin competitive competitive η 40 A 1787 competitive no no A22 765 ² ) competitive no no Neomycin competitive no no Streptomycin no no no Streptothricin no no 0 45 Viomycin no penicillin no no

') 0 means that the antibiotic in question is against this Test organism shows almost no effect.

² ) S. aureofaciens Duggar A 22765 strain.

Other antibiotics not listed in the table that are not influenced by ferrioxamine are: Acetomycin, Actinomycin C, X, I and Z, Angolamycin, Carbomycin, Chartreusin, Chlortetracycline, Cycloserine, Cincrubine, Desertomycin, Erythromycin, Exfoliatin, Granaticin, Holomycin, Leucomycin, Megacidin, Methymycin, narboinycin, novobiocin, olcandomycin, oxytetracycline, picromycin, rhodomycin, Spiramycins, Streptogramin, Tetriomycins and Thiolutin.

The in vitro antisideramycin action of ferrioxamines is well suited for qualitative detection and quantitative determination of ferrioxamines, adding the in and of itself to the determination of synergistic active substances developed test by Bonifas (V. Bonifas, Experientia, 8, p. 234 [1952]) can be applied accordingly. For this purpose test plates, e.g. B. Petri dishes that have a layer of a suitable agar medium, with Bacillus subtilis Cohn emend. Prazmowski or Staphylococcus aureus Rosenbach inoculated. Strips of filter paper (5 mm wide), e.g. B. from Whatman no. 1 paper coated with a solution of a sideramycin antibiotic, e.g. B. one Solution of 10j / ml ferrimycin in methanol, soaked are hung up. At right angles to it, strips of the same width are placed over it, the one with the one on ferrioxamine B-content solution to be tested are soaked. After 9 to 15 hours of incubation at 36 ° C an influence on the antibiotic effect of ferrimycin is easy to see: in the inhibition zone, which forms from the strips with the antibiotic, one is created at the intersection of the two strips wedge-shaped constriction, its shape and dimensions under standard conditions for quantitative Determination of the ferrioxamines used (see. Fig. 10). If a solution has both ferrioxamine as well as ferrimycin, it must be heated to 60 ° C for 30 minutes at neutral pH. This will destroys the antibiotic effect of ferrimycin, while the ferrioxamine effect is retained and consequently can be quantified.

In the antisideromycin test, B of all ferrioxamines was found to be the most effective. With Staphylococcus aureus as the test organism, the other ferrioxamines show respect to B following activities: A 51%, C 16%, D ₁ 8%, D ₂ 3%, 4% E and F 30%.

The already mentioned substances ferrichrome, terregens factor and coprogen are similar in their Vitamin character for Arthrobacter terregens and Arthrobacter flavescens the ferrioxamines. On the other hand is the growth-promoting effect on Bacillus that is typical of the ferrioxamines described above subtilis, Micrococcus pyogenes, Saccharomyces cerevisiae, Ustilago sphaerogena and Chlamydomonas eugametos not known for the three substances ferrichrome, terregens factor and coprogen. It is also nothing about an antagonistic effect of these substances on the antibacterial activity of the Sideramycin antibiotics known.

In paper chromatography, ferrichrome clearly differs from the ferrioxamines B to F in a direct comparison in system V (cf. Tab. 1), but shows a similar R _f value to ferrioxamine A in this system Paper electrophoresis in 0.33 n-acetic acid can easily be distinguished from the strongly basic ferrioxamine A. The terreg factor contains only traces of iron and is therefore different from all of the listed ferrioxamines. On the basis of its analytical data, coprogen can be easily distinguished from the ferrioxamines A, B, D and E. The ratio of% C to% N is 4.97 in the case of coprogen, and 3.5 to 4.3 in the case of the ferrioxamines mentioned. With the ferrioxamines C and F this difference is not so pronounced (% C to% N = 4.74 to 4.78). However, the ferrioxamines C and F are strong bases which are isolated as hydrochlorides, while coprogen, according to the data available, is a neutral substance (Journ. Amer. Chem. Soc, 74, p. 1362 [1952]).

The ferrioxamines, their derivatives and cleavage products as well as salts of these compounds are obtained, if you get the new growth substances from plant organisms or their extracts

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known methods, taking into account the chemical and physical data given above and using the antisideramycin test isolated and, if desired, salts, derivatives or Produces cleavage products of the new compounds.

The starting materials for the production of ferrioxamines are z. B. higher plants such as Dicotyledoneae, e.g. B. the genus Solanaceae, for example Solanum lycopersicum or the genus UmbeUiferae, for example Daucus carota L. and Monocotyledoneae, e.g. B. Commelinaceae, such as Rheoe discolor, and algae cultures, e.g. B. from Chlamydomonas eugametos, or especially cultures of microorganisms, e.g. B. of representatives of the genus Streptomyces, of bacteria, z. B. from B. subtilis or from yeasts, e.g. B. from Saccharomyces cerevisiae. The antisideramycin effect can be seen in the crude extract or culture filtrate by means of the above-mentioned test.

A particularly preferred source are cultures of Streptomycetes, which on the basis of the von Ettlinger et al. (Arch. Mikrobiol., 3, p. 326 [1958]) assigned to the following species are: Streptomyces griseoflavus (Krainsky) Waksman et Henrici, Streptomyces lavendulae (Waksman et Curtis) Waksman et Henrici, Streptomyces galilaeus Ettlinger et al., Streptomyces pilosus Ettlinger et al., Streptomyces polychromogenes

ίο Hagemann, Penasse et Teilion, Streptomyces viridochromogenes (Krainsky) Waksman et Henrici, Streptomyces aureofaciens Duggar, Streptomyces olivaceus (Waksman) Waksman et Henrici, Streptomyces griseus (Krainsky) Waksman et Henrici, Streptomyces glaucescens Gause et al.

The following table shows the species-determining characteristics of the ferrioxamine-forming Streptomycete strains evident.

morphology
the spurs Tabel 5 Morphology of the air mycelium Melanoides
pigment characteristics
Art Spurs with short
Spines Color of
Luftmycels Spore chains with open regulari
gen spirals, often more than six
Turns is missing S. griseoflavus
(Krainsky)
Waksman et Henrici Spores with fine
fragile
Hair Ash gray Spore chains with open regulari
gen spirals, usually more than six
Turns before
act S. pilosus Ettlinger
et al. Spurs with short
Spines Ash gray Spore chains with open regulari
gen spirals, often more than six
Turns before
act S. viridochromogenes
(Krainsky)
Waksman et Henrici smooth Light Blue Spore chains branched monopodially,
straight or wavy is missing S. olivaceus (Waksman)
Waksman et Henrici smooth Ash gray Spore chains branched monopodially,
with irregular open
Spirals is missing S. aureofaciens Duggar smooth Ash gray Spore chains branched monopodially,
long straight main axis, open
regular spirals, usually more than
six turns before
act S. galilaeus Ettlinger
et al. smooth Ash gray Spore chains with open, irregular
moderate spirals at the ends
long straight pieces before
act S. lavendulae-
(Waksman et Curtis)
Waksman et Henrici smooth Pale carmine
cinnamon brown Spore chains straight or wavy is missing S. polychromogenes
Hagemann et al. smooth Pale carmine
cinnamon brown Spore chains wavy, sympodial ver
branched tufts without spirals is missing S. griseus Waksman
et Henrici Yellowish-
greenish
Gray

Cultures of the microorganisms mentioned are advantageously used to obtain larger amounts of ferrioxamines. The above-mentioned Streptomyces strains, which can easily be grown on a larger scale, have proven particularly useful in this regard. The present invention is not limited to the use of the representatives of the species mentioned, but also relates to the use of ferrioxamine-forming strains of other species and in particular of variants of all these organisms, as they are, for. B. be obtained by selection or mutation, in particular from ultraviolet or X-rays or from nitrogen mustard oils.

To obtain the Ferrioxamine on a larger scale z. B. a strain exhibiting the properties of the streptomycetes listed above,

ζ. Β. in aqueous, containing carbohydrates, nitrogenous compounds and inorganic salts Nutrient solution grown aerobically until it shows a substantial ferrioxamine effect, and the ferrioxamines isolated on this. But you can also grow plants such as green algae or bacteria such as B. subtilis and isolate ferrioxamines from it in pure or enriched form. For the breeding of the said Microorganisms come as assimilable carbohydrates z. B. glucose, sucrose, lactose, mannitol, Starch as well as glycerin in question. As nitrogenous nutrients and possibly growth-promoting Substances may be mentioned: amino acids, peptides and proteins as well as their breakdown products, such as peptone or Tryptone, also meat extracts, water-soluble parts of cereal grains, such as maize and wheat, from Distillation residues from alcohol production, from yeast, seeds, in particular from the rapeseed and soy plants, of the cotton plant, etc., but also ammonium salts and nitrates. From other inorganic salts the nutrient solution can contain, for example, chlorides, carbonates, sulphates of alkalis, alkaline earths, magnesium, Contains iron, zinc and manganese.

The cultivation takes place aerobically, for example in resting surface culture or preferably submerged with shaking or stirring with air or oxygen in shaker bottles or the known fermenters. As temperature example, one between 18 and 4O ⁰ C. A suitable essential nutrient solution Ferrioxaminwirkung shows the case generally after 2 to 10 days at streptomycetes. 0.1% ferric chloride is added to the culture and the mycelium is separated from the culture filtrate, after which the majority of the ferrioxamines are found in the culture filtrate. Nevertheless, significant amounts of it remain adsorbed on the mycelium. It is therefore advantageous to wash out the latter well. For example, water and / or aqueous organic solvents, such as alcohols, e.g. B. aqueous methanol.

In a similar way, bacteria, e.g. B. B. subtilis, and the culture filtrate as Use source for isolation of ferrioxamines.

For the isolation of the ferrioxamines from the materials mentioned, in particular the culture filtrates from Fungal or bacterial cultures, you can proceed according to methods known per se and z. Legs use the following working methods or combinations thereof.

1. One can use adsorbents, e.g. B. Activated carbons such as Norit, activated earths such as Frankonite, Fuller's earth or floridine, or resin adsorbers such as Asmit. The adsorbates are expediently eluted with mixtures of water-miscible organic solvents with water, e.g. B. with water-methanol, Water-pyridine, dilute acetic acid-methanol or water-methanol-glacial acetic acid-butanol mixtures. This has proven to be particularly advantageous for the elution of a frankonite or norite adsorbate Mixture of water (4 parts by volume) and pyridine (1 part by volume) proved.

2. A second method for separating the ferrioxamines is that they are adsorbed on cation exchangers, especially acid groups containing resins such as Dowex 50 are suitable. The latter can be in the acid form as well can be used in the sodium form, but a mixture of these two forms has also proven useful. the Elution is conveniently done with acidic agents,

z. ß. with hydrochloric acid or acidic buffer solution: n.

3. Furthermore, the ferrioxamines can be removed from an aqueous solution by means of organic solvents will. Higher organic alcohols, e.g. B. benzyl alcohol or isopropyl alcohol, particularly proven. The aqueous phase expediently becomes an inorganic one Salt, e.g. ammonium sulfate or sodium chloride, added. From the obtained organic The ferrioxamines can be extracted either by evaporation of the solvent or by precipitation by means of a suitable organic solvent, e.g. B. ether, petroleum ether or ethyl acetate, in enriched form can be obtained.

4. An enrichment of the ferrioxamines is also achieved in that one concentrated, aqueous or alcoholic-aqueous solutions of the salt with an excess of organic, water-miscible Solvents such as acetone, dioxane, etc., are added, the salts being precipitated in solid form.

5. Another method of enriching the Ferrioxamines is that they are between an aqueous solution and a solution of phenol in chloroform, both the pH of the aqueous solution and the phenol content of the chloroform solution can be varied. Is one understood by that Partition coefficient of ferrioxamines is the ratio of concentration in the organic phase to the concentration in the aqueous, it follows that the distribution coefficient with increasing phenol content the organic phase increases and decreases with decreasing Ph of the aqueous phase. Because it thus it is possible to have any distribution coefficient of ferrioxamines in this system you can make a large part of it inactive by combining a few distribution operations Separate impurities.

6. Another method for the enrichment and / or separation of the ferrioxamines is chromatography represents how adsorption chromatography on various materials, e.g. B. Norit, aluminum oxide, Magnesium silicates, silica gel, calcium sulfate, as well as partition chromatography with cellulose, Starch, silica gel, celite and the like as carrier substances, or chromatography on ion exchange resins, z. On Dowex 50, Amberlite IRC-50 and the like.

7. Furthermore, the ferrioxamines cannot, due to countercurrent distribution according to Craig between two miscible solvent phases are enriched. The following solvent systems have been used particularly proven:

a) Benzyl alcohol - 20% aqueous solution of ammonium sulfate;

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

8. Finally, preparative electrophoresis on a column with support material is well suited for Purification, enrichment and separation of ferrioxamine preparation. This is called high voltage electrophoresis performed at 500 to 4000 volts.

Another improvement is that this process is based on the so-called countercurrent principle performs. The ferrioxamines A, B, C and F present as cations are on the

Support column held in place by observing its motion caused by the electric field precisely compensated by means of a flow of the electrolyte running in the opposite direction. This ensures that substances with a different electrical mobility attach the support column to the two Leave the electrode ends.

The individual Ferrioxamines are considered pure and uniform substances in the form of an amorphous Obtained in powder or as crystals. The following methods have proven to be special for their extraction proven suitable:

Lyophilization of an aqueous or alcoholic solution.

Cases from an aqueous, alcoholic or phenolic solution with lipophilic organic Solvents miscible with the solvent containing the ferrioxamine; particularly suitable for this precipitation are lower alkyl ketones such as acetone, methyl ethyl ketone, Ethers such as diethyl ether, diisobutyl ether, and hydrocarbons such as pentane, hexane, petroleum ether.

Crystallization from suitable solvent mixtures such as alcohol - ether, e.g. B. methanol

- Diethyl ether, or mixtures of water and organic solvents, at least are partially miscible with water, e.g. B. water

- acetone, water - glacial acetic acid, etc.

The ferrioxamines, their derivatives and the salts of these compounds can be used as growth substances for various Organisms are used, either alone or in the form of special preparations. They also have pronounced anti-anemic properties, for example in the case of bleeding anemia and iron deficiency anemia, and accordingly can be used as remedies in the form of preparations will.

The preparations contain the ferrioxamines, their salts or derivatives together with a suitable one Carrier material. For the same purpose, such substances come into question that those with the new compounds are not react, such as B. gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, benzyl alcohols, Gum, polyalkylene glycols, petroleum jelly or cholesterol. Such preparations can be in solid form, z. B. as a powder, or in liquid form as solutions, suspensions or emulsions. Possibly are they sterilized and / or contain auxiliary substances such as preservatives, stabilizers, wetting agents or Emulsifier. They can also contain other therapeutically valuable substances.

1 shows a countercurrent distribution of crude ferrioxamine over 80 stages with 100 cm ^{3 of} organic and 100 cm ^{3 of} aqueous phase in the system n-butanol-benzyl alcohol-saturated aqueous saline solution-n-hydrochloric acid (100: 200: 300: 60: 6) . - • - • - Extinction at 425 ηιμ. HJ antisideramycin activity in millimeters (modified Bonifas test);

FIG. 2 shows in the chromatogram of fraction III of FIG. 1 on Dowex 50-WX ₂ with ammonium acetate buffer as eluent;

Fig. 3 shows the IR spectrum of ferrioxamine B in Nujol;

4 shows the ferrioxamines in the paper chromatogram in the system tert-butanol — water — saturated aqueous sodium chloride solution - 0.1 N hydrochloric acid (50:

25: 25: 1), paper impregnated with acetone — water— saturated aqueous sodium chloride solution (6: 3: 1);

5 shows paper electrophoresis of the ferrioxamines, centimeter running distance in 0.33 η-acetic acid after 4V for ₂ hours at 220V;

FIG. 6 shows in the chromatogram of fraction II from FIG. 1 on Dowex 50-WX ₂ with ammonium acetate as buffer solution. - · - · extinction at 425 πιμ;

FIG. 7 shows a chromatogram of fraction IV ο from FIG. 1 on Dowex 50-WX ₂ with ammonium acetate buffer as the eluent. - · - · - absorbance at 425 ΐημ;

FIG. 8 shows a chromatogram of fractions 2 to 5 which is obtained in both cases when fraction V of FIG. 1 is chromatographed on Dowex 50-WX ₂ with ammonium acetate buffer as the eluent. - ■ - · extinction at 425 ΐημ;

FIG. 9 shows a chromatogram of fractions 48 to 55 which is obtained in both cases when fraction V of FIG. 1 is chromatographed on Dowex 50-WX ₂ with ammonium acetate buffer as the eluent. - · - · extinction at 425 ηιμ;

Figure 10 shows the antagonism between ferrioxamines and ferrimycin in the modified Bonifas test. The hatched areas show the inhibition the growth of the test organism;

11 shows the IR spectrum of ferrioxamine A. in potassium bromide;

Fig. 12 shows the IR spectrum of ferrioxamine C in potassium bromide;

13 shows the IR spectrum of ferrioxamine D ₁ in potassium bromide;

Fig. 14 shows the IR spectrum of ferrioxamine E in potassium bromide;

15 shows the IR spectrum of ferrioxamine F ³ ^ in potassium bromide;

16 shows the IR spectrum of ferrioxamine D ₂ in potassium bromide.

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

The cultivation of Streptomyces pilosus, strain NRRL 2857, is carried out by the submerged method. A nutrient solution is used which contains 20 g of soy flour and 20 g of mannitol per liter of tap water. The nutrient solution is sterilized in the inoculation flask or in the fermenters for 20 to 30 minutes at 1 atm. The sterilized nutrient solution has a pH of 7.2 to 7.6. Inoculation is carried out with up to 20 ° / ₀ of the said organism a partially sporulated vegetative culture. It is incubated with good shaking or stirring at 24 to 30 °, the cultures in fermenters being aerated with about 2 volumes of air per volume of solution per minute. After 48 to 240 hours of incubation, the culture solution has reached the highest content of ferrioxamine B. Interrupting the culture is 0.1% ferric chloride, separating the mycelium and other solid components from the bulk of the ferrioxamine B containing solution by filtration or centrifugation from, optionally wherein the culture solution before filtration about 1 ° / ₀ of a filter aid, z. B. Hyflo Supercel is added. The filter residues are washed with water or aqueous methanol and the washing liquids are combined with the culture

filtrate. The culture filtrate obtained is under constant stirring at 2 ° / ₀ alumina, for example frankonite offset. After thorough mixing, the mixture is filtered off and the absorption process is repeated once or twice with the resulting filtrate. The filter residues are combined and washed several times with water and aqueous methanol. The filter residues are then eluted two to three times with a pyridine-water mixture (1: 4). The eluate, which has been clarified by filtration, is concentrated in vacuo. The concentrates obtained can either be further processed directly (see Example 3), or a mixture of the ferrioxamines can be isolated therefrom in crude form by means of freeze-drying.

If one uses in place of the above nutrient solution those that per liter of tap water the contain the following nutrients, culture filtrates are obtained after analogous cultivation and processing with a similarly high content of ferrioxamine B.

a) sucrose 20 g

Sodium citrate 0.9 g

Ammonium acetate 3 g

sec. Potassium phosphate 3 g

Magnesium sulfate 0.8 g

Copper sulfate 0.01 mg

Manganese chloride 0.07 mg

Ferricitrate 20 mg

b) raw glucose 10 g

Soy flour 10 g

Cornsteep liquor 20 g

Table salt 5 g

Sodium nitrate Ig

Lime 10 g

c) Rapeseed extraction meal 20 g

Raw glucose 10 g

sec. Potassium phosphate 0.2 g

Lime 10 g

d) flax flour 40 g

Raw glucose 10 g

sec. Potassium phosphate 0.2 g

Lime 10 g

Instead of the specified strain of the species Streptomyces pilosus, the following strains can be used (the strains in the institute for special Botany, Swiss Federal Institute of Technology, Zurich, preserved). After analog breeding and Working up, culture filtrates with a similarly high ferrioxamine content are obtained.

tribe
No. Streptomyces species S. olivaceus 7 346 S. olivaceus 7 437 S. aureofaciens Duggar 22 083 S. aureofaciens 22 765 S. galilaeus Ettlinger et al. 18 822 S. lavendulae (Waksman et Curtis) 14 677 Waksman et Henrici S. lavendulae 21510 S. polychromogenes Hagemann et al. 21837 S. polychromogenes 23 217 S. polychromogenes 23 310 S. griseus Waksman et Henrici 10 112 S. griseus 13 495 S. griseus 7 419

Example 2

tribe
No. Streptomyces species 9 578 S.griseoflavus (Krainsky) Waksman et Henrici 15311 S.griseoflavus 11 686 S. pilosus Ettlinger et al. 23 258 S. pilosus 23 305 S. pilosus 17 635 S. viridochromogenes (Krainsky) Waksman et Henrici 18 055 S. viridochromogenes 6 445 S. olivaceus (Waksman) Waksmann et Henrici

The strain A 23 978 of the species Streptomyces aureofaciens (Institute for Special Botany, Eidgenössische Technische Hochschule, Zurich) is grown submerged on a nutrient solution that is per liter of tap water Contains 20 g of malt extract, 20 g of distillers solubles. The breeding and processing takes place in the same way as in Example 1, culture filtrates with a similarly high ferric dioxamine content being obtained.

3 °

Example 3

60 l of culture are produced and worked up in the manner shown in Example 1. The won Pyridine-water eluate (approx. 6 l) is concentrated to 31 in vacuo. In this concentrate are 870 g Dissolved ammonium sulfate. The solution is by filtration or centrifugation, optionally with the addition of 1 ° / o Hyflo Supercel, clarified. By shaking three to four times with benzyl alcohol or isopropyl alcohol the ferrioxamines are converted into the organic solvent. The organic phases are combined and dried with the aid of sodium sulfate. There is an excess of ether or ethyl acetate and the precipitated ferrioxamines are filtered off. The addition of filter aids, z. B. Hyflo Supercel, before precipitation facilitates the recovery of the precipitate. For this the ferrioxamines can be washed out with methanol or water. These eluates are evaporated or lyophilized, and so the ferric oxamines are obtained in enriched form.

Example 4

A culture filtrate obtained according to Example 1 or 2 is added per liter of 20 g of sodium chloride. The clear solution is _{extracted three times with 10} volumes of chloroform-phenol mixture (1 part by volume of chloroform, 1 part by weight of phenol). The organic phases are combined, filtered with the addition of Hyflo Supercel and mixed with an excess of ether. The ferrioxamines are converted into the aqueous components by shaking them out several times with a little water. The aqueous phases obtained are combined and extracted twice with ether to completely remove the phenol. An orange to brown-red preparation of ferrioxamines which can be broken down by paper chromatography is obtained by freeze-drying.

209 508/299

Example 5

400 g of Hyflo Supercel and 200 ml of a 10% strength aqueous ferric sulfate solution are added to the culture solution while stirring and the mixture is filtered. After adding 3.6 kg of sodium chloride, the filtrate is extracted in a countercurrent extractor with 21 phenol-chloroform (1 g: 1 ml), the extract is dried over sodium sulfate and left in a well-stirred suspension of 20 g of Hyflo Supercel in the course of 1 hour 2 1 ether and 101 petroleum ether pour in. After filtering the powdery mixture of filter aid and precipitate and washing it out with about 21 ether, it is eluted ^{five times with 600 cm 3 of methanol each time.} The combined eluates yield 10 g of crude ferrioxamine in the form of a brown-red powder on gentle evaporation.

Example 6

4 g of crude ferrioxamine are distributed in the system n-butanol — benzyl alcohol — water — saturated aqueous sodium chloride solution — 1 N hydrochloric acid (100: 200: 300: 60: 6) over 80 stages of 100 cm ^{3 of} organic and 100 cm ^{3 of} aqueous phase . The distribution is evaluated by biological testing and by measuring the extinction at 425 m: 2 cm ^{3 of the} upper and lower phases are ^{removed from every fourth unit and mixed with 32 cm 3 of} methanol, a homogeneous solution being obtained, the concentration of which is suitable for both tests (see Fig. 1). The distribution fractions, which are grouped into four groups according to this evaluation, contain, as can be shown by paper chromatography, in addition to the main product ferrioxamine B (in distribution fraction III), further antisideramycin-active, red-colored compounds designated as ferrioxamine A, C, D ₁ , D ₂ , E and F. will.

40

45

The distribution fraction III is shaken with 31 petroleum ether. The deep red colored aqueous phase is washed with chloroform, mixed with sodium chloride up to a concentration of 10% and extracted exhaustively with phenol-chloroform (1 g: 1 ml). The phenol-chloroform extract is washed several times with 10 ° / ₀ sodium chloride containing 0.01 N hydrochloric acid, filtered through a small column of 20 g of Celite and the contents precipitated by addition of 25 g of Hyflo Supercel, 500 cm ³ of ether and 11 Petroleum ether with stirring at 0 °. The powdery mixture of filter aid and precipitate is washed well with ether and then eluted with a little methanol. With gentle evaporation, 982 mg of crude ferrioxamine B are obtained in the form of a brown-red powder from the methanol eluate.

The distribution fractions II, IV and V are worked up in the same way.

Parliamentary group no. Distribution
fraction Most important
Ferrioxamine 6 to 18
19 to 32
33 to 62
63 to 80 II
III
IV
V A.
B.
C.
D ₁ , D ₂ , E and F

see and filled with cellulose powder, vertical glass column 1.5 m high and 2.6 cm diameter of the zone electrophoresis according to J. Porath (Biochim. et Biophys. Acta, 22, p 151 [1956]). _{7 3} n-acetic acid is used as the electrolyte solution. The substance is dissolved in 20 cm ^{3 of} water and the deep red-colored solution is applied to the column at the upper end of the anode. The orange-red colored, about 10 cm high ferrioxamine zone migrates at a voltage of 1600 volts and a current of 30 mA at a rate of 3.9 cm / hour towards the cathode at the lower end of the column. In order to increase the separating effect of the column, this electrical migration is compensated for by an electrolyte flow in the opposite direction, whereby the active ingredient, which is easily recognizable due to its inherent color, is held locally in the column. Accompanying substances, which have a greater or lesser electrical migration speed than the active ingredient, are in the cathode or. Anode compartment drifted off and removed from the column. After 5 to 6 days, under these conditions, the active ingredient has passed through a column of liquid 4 to 5 m in length. Now it aborts the electrophoresis and the column eluted with 7 ₃ n-acetic acid, fractions are collected of 15 cm ^3. These are examined individually. The strongly red-colored, biologically active fractions are combined. 10 are employed to ° / ₀ sodium chloride and exhaustively extracted with a mixture of phenol-chloroform (1 part by weight, 1 part by volume). The combined extracts are washed several times with V100 "hydrochloric acid, which contains 10% sodium chloride, and then nitrated through Celite. Filter aid (Hyflo Supercel) is added to the deep red-colored anhydrous filtrate and, while stirring, five times the volume of ether-petroleum ether (1: 1), the active ingredient precipitating on the filter aid, which is filtered off and washed with plenty of ether. The active substance is then eluted with a little methanol and the red solution is evaporated to dryness in vacuo at 20 ° Preparation of ferrioxamine B twice enriched with the starting material. It is a red amorphous powder which is soluble in water, methanol, butanol, benzyl alcohol, phenol, dimethylformamide and acetic acid and contains carbon, hydrogen, oxygen, nitrogen and iron the following properties: pK (66 ° / ₀ aqueous methyl cellosolve) = 9.54; equivalent weight = 645; ultraviolet absorption sm maximum at 430 µm (log EU = 1.51); See Fig. 3 for the infrared spectrum.

The hydrolysis of such a preparation in 6N hydrochloric acid (6 hours at 110 °) gives a Mixture containing five ninhydrin-positive substances. These show on paper chromatography the following Rf values:

Example 7

1 g of a preparation of ferrioxamine B obtained according to Example 6 is supplied in a cooling jacket

65

Mixed solvent n-butanol — ice Substance no. vinegar - water
(4: 1: 1) Phenol — H ₂ O (8: 2) 0.37 1 0.68 0.34 2 0.40 0.10 3 0.35 0.05 4th 0.30 0.04 5 0.20

Example 8

865 mg of crude ferrioxamine B from partition fraction III (cf. Example 6) are chromatographed on a column (66 cm · 7.14 cm ² ) of Dowex 50-WX ₂ (100/200 mesh) at 15 to 17 ^l . D ^ r ion exchanger is previously according to the instructions of Hi rs et al. (J. Biol. Chem., 219, p. 623 [1956]), brought to the column in the ammonium form by sedimentation and treated with 0.2 M ammonium acetate buffer of Ph 4.60 for 48 hours at a flow rate of 200 ml / Equilibrated hour. The in 9 cm ³ of said buffer solution to the column applied substance was developed with 0.2 M p _H 4.6 buffer for 72 hours (100 ml / hour). The eluent is then continuously concentrated using a 1-1 mixing vessel with 2 M ammonium acetate buffer of Ph 4.7 (gradient elution) and the eluate is collected ^{in fractions of 40 cm 3 shortly before the exit of the first colored zone.} The fractions combined according to the extinction measurement at 425 μm (see FIG. 2) are extracted in the manner already described using phenol-chloroform, ether-petroleum ether-Hyflo Supercel and methanol.

fraction lot
mg substance 13 to 18
93 to 125
185 to 197 62
544
36 not identified
Ferrioxamine B hydrochloride
not identified

Ferrioxamine B is soluble in water, methanol, alcohol, phenol, dimethylformamide and glacial acetic acid, poorly soluble in butanol and practically insoluble in chloroform, acetone, ether and ethyl acetate. Microanalysis after 48 hours at 20 ° / 0.001 mm:

C 48.04 ° / ₀ , H 7.41%, N II, 21 ° / ₀ , Cl 5.25 ° / ₀ , P 0%, S 0%, Fe 7.67% • titration: pK _M cs = 9.74, equivalent weight 704. Absorption (H ₂ O): X _ma .x 430 ηιμ, E \ * _m = 39.0. TR spectra see Fig. 3. R _f value in system I: 0.44, in system V: 0.29 (see table 1), distribution coefficient in system VI: 0.228 (see table 1).

Ferrioxamine B gives a positive reaction with ninhydrin. It can be easily done with ferric chloride-potassium ferricyanide reagent (J. M. Barton et al., Nature, 170, p. 249 [1952]). On the other hand, it can be discolored with sodium dithionite be reduced. The colorless solutions obtained thereby gain rapidly on standing in air their original red color back. The iron bound in Ferrioxamine B is part of the complex deprived of the action of mineral acids, strong alkalis or 8-oxyquinoline.

Iron-free Ferrioxamine B (Desferrioxamine B) is

ao colorless. It can be converted back into ferrioxamine with ferric chloride. Also with other metal ions Desferrioxamine B reacts to form the corresponding metal complexes, for example des greenish colored copper desferrioxamine.

Example 9

20 mg of a preparation of ferrioxamine B obtained according to Example 8 are dissolved in 3 cm ^{3 of} 1.2 N hydrochloric acid and heated for 5 minutes in a boiling water bath. The initially red-brown solution becomes almost colorless. It is evaporated to dryness in vacuo. The residue is then dissolved in 0.2 cm ^{3 of water and 0.01 cm 3} each of this solution is used for the subsequent paper chromatographic analysis. A mixture of 7 parts by volume of n-butanol and 3 parts by volume of 6η-hydrochloric acid is used as the flow agent. The following substances could be identified:

substance Rf a b C. d Cadaverine
Fe ⁺ + 0.12
0.17
0.25
0.34
0.68
0.84
0.93 Pale yellow
yellow Gray purple
Gray purple
Bluish Deep red
Deep red
Pale red
Red - 1 -amino-5-hydroxyl-
aminopentane
Hydroxylamine
Unknown substance ...
Fe +++
Unknown substance ... Purple red

In the table, a means the inherent color of the substances recognizable on the paper, b means color reaction with Ninhydrin, c color reaction with triphenyltetrazolium chloride + sodium hydroxide solution, d color reaction with the Czaki reagent. (All reagents according to the "Handbook of Paper Chromatography" by I. M. Hais and K. Macek, Verlag Gustav Fischer, Jena, 1958.)

If 57% hydroiodic acid (4 hours, 110 °) is used instead of hydrochloric acid during hydrolysis, for example, hydroxylamine, l-amino-5-hydroxylaminopentane and Fe + ++ are missing in the paper chromatogram. But there is more cadaverine available.

A second portion of ferrioxamine B is hydrolyzed with 1,2η-hydrochloric acid, as described above. The hydrolysis mixture is then extracted with ether. The extract is dried with sodium sulfate and steam it up. Succinic acid can be obtained in crystalline form from the residue.

Example 10

^{A solution of 450 mg of 2,4-dinitrofluorobenzene in 26 cm 3 is added} to a solution of 450 mg of a ferrioxamine B preparation obtained according to Example 8 and 450 mg of sodium hydrogen carbonate in water

Ethanol. After standing for 4 hours at room temperature, the alcohol is driven off in vacuo. The excess reagent is removed by shaking with ether. The deep red-brown dinitrophenylferrioxamine B can now easily be shaken out with n-butanol (ferrioxamine B itself remains in the aqueous phase under the same conditions). The butanol extract, washed with water and then dried, is evaporated in vacuo. The residue is extracted with acetone and the acetone solution is filtered off from an insoluble brown residue (114 mg). The dinitrophenyl-ferrioxamine B can be precipitated as an amorphous orange-red powder from the acetone solution, which has been concentrated to about 5 cm ^3. Yield 338 mg.

In paper electrophoresis in dilute acetic acid, the dinitrophenyl derivative migrates at 1000 volts 5.5 cm in 2 hours, while ferrioxamine B migrates 18.5 cm under the same conditions.

Example 11

To isolate the ferrioxamines A, C, D ₁ , D ₂ , E and F, the distribution fractions II, IV and V (cf. Example 6) obtained in the countercurrent distribution of crude ferrioxamine are isolated on Dowex 50-WX ₂ (200/400 mesh ) chromatographed with ammonium acetate buffer as eluent. The resin is first cleaned according to the instructions of Hirs et al. (CHW Hirs, S. Moore, WH Stein, J. Biol. Chem., 219, p. 623 [1956]). It is brought onto the column in the ammonium form by sedimentation and prior to the substance application for about 70 hours with the buffer solution used for elution at a flow rate of 20 ml / cm ² / hour. equilibrated. The substances are applied to the column in a 1 to 10% solution, depending on their solubility, and the chromatograms are developed at 23 to 25 ° and at a flow rate of 5 to 15 ml / cm ² / hour. with ammonium acetate buffer of pH 4.5 in increasing concentration (gradient elution). The chromatograms are evaluated by measuring the extinction of the eluate fractions (30 to 40 cm ³ ) at 425 μm. The correspondingly combined eluate fractions are extracted exhaustively with phenol-chloroform (1 g: 1 cm ^{3) after the addition of 10% common salt.} The deep red-colored extracts are washed extensively with 0.01 η-hydrochloric acid containing 10% sodium chloride, filtered through a layer of Celite and the ingredients are precipitated from the clear filters by adding ether and petroleum ether onto Hyflo Supercel. The mixture of substance and carrier is washed with ether to remove phenol and then eluted with a little methanol. The ferrioxamines are obtained from the methanol eluates during gentle evaporation in the form of brown-red hygroscopic powders, which are dried over phosphorus pentoxide for 50 hours at 25 ° / 0.03 mm for analysis.

5 g of fraction II (cf. Example 6) are chromatographed on a Dowex-WX ₂ column of 90 cm × 22 cm ² using the method described above (FIG. 3). Buffer solution at the beginning: 0.1m ammonium acetate pn 4.5. Throughput speed: HOml / hour. Volume of the eluate fractions: 35 to 40 cm ³ . Change to gradient elution with mixing chamber of 41 and 1.75m ammonium acetate buffer of pH 4.70 for fraction No. 90.

Parliamentary group no.

283 to 300
301 to 325

326 to 352

353 to 385

xo 416 to 445

weight
mg

302
488

393
452
415

ingredients

Ferrioxamine A hydrochloride Ferrioxamine B + A hydrochloride

I not closer

I characterized substances

Ferrioxamine A hydrochloride is a brown-red powder, which dissolves well in water, methanol, ethanol, glacial acetic acid and dimethylformamide. It is insoluble in

Ether, acetone, ethyl acetate and chloroform. Ri im Solution system i: 0.35, in solution system V: 0.24 (see Table 1). Distribution coefficient in system VI:

0.111 (see Table 1). Paper electrophoresis see Fig. 5.

Microanalysis: C 44.21%, H 7.52%, N 12.63%,

ao Fe 7.95%, Cl 5.93%. Titration: pK _MC s 9.89, equivalent weight 634. UV spectrum in water: ληαχ 430 πιμ, (E il = 37). The IR spectrum in potassium bromide shows the following bands (s = strong band, m = middle band, w = weak band): 2.92μ (s \ 3.42μ (m), 6.10μ (s), 6, 32μ (s), 6.88 μ (m), 7.30 μ (w), 7.92 μ (w), 8.10 μ (w), 8.49 μ (w), 8.98 μ ( w), 9.55 µ (w), 10.15 µ (w), 10.67 µ (w) (see. Fig. 11).

Ferrioxamine A gives a positive reaction with ninhydrin. The iron bond in Ferrioxamine A becomes removed from the complex when treated with mineral acid or strong alkalis. Iron-free Ferrioxamine A is colorless. It can be returned to ferrioxamine A with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex.

Example 12

5 g of fraction IV (cf. Example 6) are chromatographed on a Dowex 50-WX ₂ column according to the method described in Example 11 (cf. FIG. 7). Buffer solution at the beginning: 0.1m ammonium acetate Ph 4.5. Throughput speed: 110 ml / hour.

Volume of the eluate fractions: 35 to 40 cm ³ . Change to gradient elution with Im ammonium acetate of Ph 4.6 in fraction 720, with 2m ammonium acetate of pn 4.7 in fraction 963:

50 fraction No. weight ingredients mg 34 to 46 135 I not further characterized 55 54 to 68 226 j fabrics 218 to 284 - yellow green fluorescent sub do not punch isolated 780 to 830 430 Ferrioxamine B hydrochloride 860 to 900 194 Ferrioxamine C hydrochloride 60 1034 to 1080 360 unspecified substance

Ferrioxamine C shows approximately the same solubility properties as A. Rf in the solvent system I: 0.54, in the solvent system V: 0.37 (see Table 1). Paper electrophoresis vg). Fig. 5, partition coefficient in system VI: 0.489 (see Table 1).

Microanalysis: C 48.33%, H 7.92%, N 10.20%, Cl 5.15%, Fe 6.82%. Titration: pK _M cs 8.88; Equivalent weight 762. UV spectrum in water: Imax 430 ηιμ (E} * „= 39). The IR spectrum in potassium bromide shows, among other things, bands at 2.92 μ (s), 3.43 μ (s), 5.85 μ (m), 6.10 μ (s), 6.33 μ (s) , 6.87 μ (s), 7.30 μ (m), 7.95 μ (m), 8.23 μ (w), 8.52 μ (m), 9.65 μ (w), 13 , 23 μ (m) (see. Fig. 12).

Ferrioxamine C gives a positive color reaction with ninhydrin. The iron bond in Ferrioxamine C becomes removed from the complex when treated with mineral acid or strong alkalis. Iron-free Ferrioxamine C is colorless. It can be returned to ferrioxamine C with ferric chloride. It reacts also with other metal ions to form the corresponding metal complexes, for example des greenish copper complex.

Example 13

148 g of fraction V (cf. Example 6) are initially pre-fractionated ^{on a 150 cm-79 cm 2 column.} The chromatogram is developed at a flow rate of 2 to 3 l / hour for 24 hours with 0.1m, 0.2m, 0.6m and for 100 hours with 1.8m ammonium acetate buffer of pH 4.7. Fractions of 5 l are collected. The combined fractions 2 to 5 contain mainly ferrioxamine D ₁ , D ₂ and E (44 g), 48 to 55 mainly ferrioxamine F (8.4 g).

Rechromatography of fractions 2 to 5

5 g of fractions 2 to 5 are chromatographed under the same conditions as fraction II, however without gradient elution (see. Fig. 8).

Parliamentary group no.

48 to 56
66 to 80

weight
mg

980
989

ingredients

Ferrioxamine D ₁ and D ₂
Ferrioxamine E.

_{The mixture of D 1} and D ₂ (980 mg) isolated from fractions 48 to 56 with phenol-chloroform is dissolved in 100 cm ^{3 of} water, saturated with sodium chloride and extracted twice with the same volume of chloroform. After drying over sodium sulfate, the chloroform extract leaves 697 mg of ferrioxamine D ₁ on evaporation. The aqueous phase is washed extensively with chloroform and then extracted with phenol-chloroform. When the extract is worked up in the usual way, 95 mg of ferrioxamine D _{2 are obtained} . Ferrioxamine D ₁ is readily soluble in water, methanol, ethanol, glacial acetic acid, methyl cellosolve and chloroform, and is sparingly soluble in ether, acetone, ethyl acetate, pyridine and dimethylformamide. It crystallizes from methanol-ether in red needles. F. after three crystallizations 194 to 200 °. Rf in solvent system I: 0.73, Rf in solvent system V: 0.88 (see Table 1). Distribution coefficient in system VI: 1.80 (see Table 1). Electrophoresis see Fig. 5.

Microanalysis: C 49.31%, H 7.47%, N 12.37%, Cl0%, Fe 7.66% · Titration: no acidic or basic Functions can be determined. UV spectrum in water:

Imax 430 Πΐμ (E [I = 44).

The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μ (s), 3.06 μ (s), 3.25 μ (w), 3.43 μ (s), 6.08 μ (s) , 6.86 μ (s), 7.30 μ (m), 7.94 μ (m), 8.20 μ (w), 8.49 μ (w), 8.83 μ (w), 9 , 00 μ (w), 9.65 μ (ν / \ 10.00 μ (w), 10.31 μ (w), 10.67 μ (w), 12.20 μ (w), 13.30 μ (m) (cf. Fig. 13). Ferrioxamine D ₁ does not react color with ninhydrin. The iron bond in ferrioxamine D ₁ is removed from the complex when it is treated with mineral acid or strong alkalis. Iron-free ferrioxamine D ₁ is colorless. It can be recycled with ferric chloride in ferric oxamine D _1. It also reacts with other metal ions to form the corresponding metal complexes,

ίο for example the greenish copper complex.

Ferrioxamine D ₂ : The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μ (s), 3.43 μ (m), 6.08 μ (s), 6.36 μ (s), 6.90 μ (s), 8.49 μ (w), 8.87μ (νν), 9.65μ (ιν), 10.05 μ (w), 10.70 μ (w), 13.22 μ (ν) (see Fig. 16).

Rf in solvent system I: 0.64; Rf in the solvent system V: 0.68 (see Table 1). Paper electrophoresis see Fig. 5.
Ferrioxamine E: that obtained from fraction 66-80

ao Ferrioxamine E (898 mg) is soluble in glacial acetic acid, in the Most solvents, especially poorly or insoluble in water and methanol. By twice Dissolving in a lot of water and acetone results in a microcrystalline powder.

R ₁ values in system I: 0.68, in system V: 0.59. Paper electrophoresis see Fig. 5. Distribution coefficient in system VI: 1.59 (see Table 1).

Microanalysis: C 49.80%, H 7.37%, N 12.48%, Cl0%, Fe 8.14%. Titration: no acidic or basic functions can be determined. UV spectrum in water: X _max 430 ΐημ (E}? „= 42).

The IR spectrum in potassium bromide shows, among other things, bands at 2.92μ (8), 3.02μ (5), 3.45μ (s), 5.96μφ, 6.15μ (5), 6.36μ (8) , 6.90μ ( ₅ ), 7.10μ (ΐη), 7.15μ (πι), 7.39μ (ηΐ), 7.82μ ^), 7.98μ (ΐη), 8.45μ (ιν), 8.54 μ (ιν), 8.85μΜ, 9.01 μ (w), 9.20 μ (ιν), 9.98μ (ΐη), 10.07 μ (W), 10.23 μ (w) , 10.43μ (w), 10.71μ (w), 11.87μ (νν), 13.20μ (πι), 13.66μ (νν) (see Fig. 14). Ferrioxamine E does not react color with ninhydrin.

The iron bond in Ferrioxamine E is removed from the complex when it is with mineral acid or strong alkalis is treated. Iron-free Ferrioxamine E is colorless. It can with ferric chloride in Ferrioxamine E can be recycled. It also reacts with other metal ions to form the corresponding one Metal complexes, for example the greenish copper complex.

Rechromatography of fractions 48 to 55

5 g of fractions 48 to 55 are processed under the same conditions as fractions 2 to 5 (see above). rechromatographed. Buffer solution at the beginning: 0.5 M ammonium acetate pH 4.7. Change to gradient elution with mixing chamber of 41 and inflow of 2 m ammonium acetate buffer of pH 4.7 for fraction 663 (see Fig. 9). Fractions 821 to 880 contain 1.12 g of material, which consists predominantly of ferrioxamine F. consists. For further purification, this material is distributed in a Craig countercurrent distribution in the n-butanol-water system Subjected to over 20 levels (10/10 mJ). The content of the elements is then extracted with 80 ml petroleum ether each, discards the petroleum ether phases and lyophilizes the aqueous raffinates. With the material obtained from elements 1 to 4 (570 mg) the distribution is repeated (30 levels). From element 5, 64 mg are obtained by paper chromatography uniform ferrioxamine F hydrochloride in the form of a brown-red powder. Ferrioxamine F hydrochloride

209 508/299

is easily soluble in water, methanol, pyridine, glacial acetic acid, Ethanol, dimethylformamide, slightly soluble in chloroform, insoluble in ethyl acetate, acetone and ether.

Microanalysis: C 50.44%, H 7.29%, N 10.53%, Cl 4.10%, Fe 5.57%. R ₁ in the solvent system V: 0.80 (see Table 1). Distribution coefficient in System VI: 3.12 (see Table 1). Electrophoresis see Fig. 5. Titration: pKjncs 9.75, equivalent weight 695.

The IR spectrum in potassium bromide shows, among other things, bands at 2.95 μφ, 3.45 μ (m), 6.19 μ (5), 6.37μ (5), 6.92μ (πι), 7.40μ (», 7.97μ ^), 8.50μΜ, 8.88μ (W), 9.72μ (W), ΙΟ, ΙΟμ (νν), 10.70μ ^), 13.75μ ^) (see Fig. 15).

UV spectrum in water: X _max 430 ΐημ (E \ * _m = 34). Ferrioxamine F gives a positive reaction with ninhydrin. The iron bond in Ferrioxamine F is removed from the complex when it is treated with mineral acid or strong alkalis. Iron-free Ferrioxamine F is colorless. It can be returned to ferrioxamine F with ferric chloride. It also reacts with other metal ions to form the corresponding metal complexes, for example the greenish copper complex.

Example 14

100 g of fresh baker's yeast is suspended in 1 l of methanol and stirred vigorously for 30 minutes. The suspension is filtered and the methanolic solution is concentrated in vacuo to 200 cm ³ . ^{200 cm 3 of} water are added to this concentrate and the solution is clarified by filtration or centrifugation. The clarified solution is extracted three times with a chloroform-phenol mixture (1 part by volume of chloroform, 1 part by weight of phenol). A little filter aid (Hyflo Supercel) is added to the combined organic phases and an excess of ether is added to this suspension with constant stirring. The precipitated crude ferrioxamine is separated off together with the filter aid, washed with ether and dried. The ferrioxamines are eluted from the filter aid with methanol. The methanolic solution is gently evaporated in vacuo, the ferrioxamines being obtained in the form of an active red-brown resin which can be purified by means of paper chromatography.

Preparations with a similarly high content of ferrioxamines can be obtained by replacing the Baker's yeast a commercially available yeast extract, pressed brewer's yeast or fresh yeast cultures as described worked up.

Example) 15

The green alga Chlamydomonas eugametos is submerged on a nutrient solution with the following composition grown at 24 °:

Magnesium sulfate 2.5 g

K ₂ HPO ₄ 1.25 g

Urea 1.05 g

Tap water 1000 ml

Trace element solution 10 ml

Trace element solution content per 1000 ml:

CaCl ₂ 8.35 g

H ₃ BO ₃ 11.42 g

FeSO ₄ -7 H ₂ O 4.98 g

ZnSO ₄ · 7 H ₂ O 8.82 g

MnCl ₂ -4 H ₂ O 1.44 g

H ₂ MoO ₄ 0.71g

CuSO ₄ · 5 H ₂ O 1.57 g

Co (NO ₃ ) ₂ 6H ₂ O 0.49 g

Complexone III 3g

The nutrient solution is sterilized in the culture vessels for 20 to 30 minutes at 1 atm. The inoculation occurs with up to 20% of a submerged culture. the

ίο Cultures are continuously aerated in the 4-1 shaking flasks with V ₄ volumes of air per volume of solution per minute. After a 4- to 12-day culture under constant light, the bright green cultures are worked up. The cultures are unfiltered with 2% clay, e.g. B. Frankonit, offset and vigorously mixed. The suspension is filtered with the addition of filter aid and the solution is again mixed with 2% clay, stirred and filtered off. The combined filter residues are washed out with water and methanol and then eluted with a pyridine-water mixture (1: 4). The azeotropic mixture is concentrated in vacuo and the ferrioxamines are isolated from the concentrates by freeze-drying. The red-brown preparations can be further enriched in the manner indicated above.

Example 16

Young, approximately 50 cm high tomato plants are processed into pressed juice in a juicer. 11 pressed juice is freed from chlorophyll by extracting three times with ethyl acetate. To the brown aqueous phase 290 g of ammonium sulfate are added, the resulting solution is filtered and then extracted three times Benzyl alcohol. The combined organic phases are dried by adding sodium sulfate and treated with an excess of ethyl acetate. The resulting precipitate is washed with ether and then taken up in a little water. Freeze drying is used to obtain Finally, an effective ferrioxamine preparation, which, as shown in the previous examples, can work up.

Preparations of similar activity are obtained if, instead of tomato plants, leaves of Rheoe discolor can be used.

Example 17

The cultivation of Bacillus subtilis is carried out according to the submerged method. A nutrient solution is used which contains 10 g of raw glucose, 5 g of peptone, 3 g of meat extract, 5 g of table salt and 10 g of lime per liter of tap water. The cultures are incubated at 33 to 37 ° with aeration and with constant shaking or stirring. After 48 to 96 hours of incubation, the unfiltered cultures are mixed with 2% clay (Frankonit), stirred vigorously and then clarified by filtration or centrifugation. The sediment is washed with methanol and then eluted with a pyridine-water mixture (1: 4). The eluate contains the majority of the ferrioxamines that, as described above, isolated.

Claims (7)

PATENT CLAIMS: 1. Process for the production of ferrous growth substances from plant material, thereby draws that the ferrioxamines, their derivatives and cleavage products and salts of these compounds are isolated from microorganisms or their extracts by methods known per se, taking into account the chemical and physical properties of these compounds and using the antisideramycin test and, if desired, salts, derivatives or breakdown products of the ferrioxamines. 2. The method according to claim 1, characterized in that from cultures of algae, yeast, bacilli or streptomycetes, the ferrioxamines A, B, C, D ₁ , D ₂ , E, F or a mixture of individual components by means of adsorption, extraction, precipitation , Crystallization, lyophilization, partitioning between solvents, chromatography and / or electrophoresis by checking the presence of ferrioxamines in the starting material and in the individual work-up operations using the antisideramycin test. 3. Process according to Claims 1 and 2, characterized in that the starting material is used Cultures of Streptomyces griseoflavus (Krainsky) Waksman et Henrici A 9578 or Streptomyces pilosus NRRL 2857 were used. 4. The method according to claims 1 to 3, characterized in that the ferrioxamines are _{adsorbed on the ion exchanger Dowex 50-WX 2} and eluted with ammonium acetate buffer of pn 4.5 to 4.6 in a molarity of 0.2 to 2.0 . 5. Process according to claims 1 to 4, characterized in that the ferrioxamines with Solutions of phenol in chloroform are extracted. 6. Process according to Claims 1 to 5, characterized in that the ferrioxamines are used distributed between two immiscible solvent phases. 7. Process according to Claims 1 to 6, characterized in that the ferrioxamines are used enriched by means of high voltage electrophoresis according to the countercurrent principle. Considered publications:
German Patent Nos. 717 997, 864 140;
French patents nos. 786 564, 870 386. In addition 3 sheets of drawings © 209 508/299 1.