DE1442073A1 - Process for the production of new active ingredients - Google Patents

Description

New for the Druolc of the Offenlegungsschrift intended •. Registration documents - us. Character * 20 086-BR / pf

UA2073

ÖIBA AKTIENGKESELISOHAi ¹ O ?, BASEIi (SWITZERLAND)

Note P U 42 073.6

Oasis 4972 / 1-4 / 5038
Germany

Process for the production of new active ingredients

Made of materials "of biological origin, in particular Microorganisms, a large number of ferrous growth substances have already been isolated, for example the Jerrichrome, the Ooprogen, the Ferrioxamine.

It has now been found that new strains of As ^ pergillus, namely Aspergillus melleus M 2853 and

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Aspergillus terreu3 M 4785, a new ferrous growth substance, the ierrichrysin, and the corresponding egg-free Compound, desferriohrysin, can win. object of the present application are therefore fferriohrysinj Des-.ferriohrysin and derivatives and salts of these "compounds, processes for their preparation and the new Aspergillus strains melleus M 2853 and Aspergillus terreus M 4785,

Ferrichrysin is a red-brown, crystalline substance which is readily soluble in water and methanol, even in the cold, and is more difficult to dissolve in absolute ethanol. The compound has the elemental composition ö »43137 #f H β 6.08 io \ H * 16.72 i>% fe« 7.09 ^ f 0 = »26.74 $> (calc.) The UV spectrum in * Water shows a maximum at 420 m ^ (log

Egg /? ™ = »1 * 48). The IR spectrum in potassium bromide shows i.a. ι a

Bands open at 725, 783, 975, 1060V 1140, 1200 (shoulder), 1218 (shoulder), 1237, 1275 (shoulder), 1340 (shoulder), 1368, 1430 (shoulder), 1445 (shoulder) _v 1465, 1495 ( Shoulder), 1517, 1585, 1652, 2930, 3070 (shoulder), 3420 cm " ¹ , see Pig. 1.

The crystals decompose at about 270 ° with a black coloration without melting. In the paper chromatogram the following Rf values are obtained! 0.26 in system I (n-butanol-glacial acetic acid-water, 4i1ii) and 0.34 in system II (tert-butanol-0.004-n. Hydrochloric acid-saturated aqueous sodium

90 9 ß 1 7 / ης öc_ .

ORIGINAL INSPECTED

, I -T τ * r M 'Vf

. {jhlor solution, 2i1 »1f paper impregnated with acetone-water-fairy saturated aqueous sodium chloride solution, 6; 3ii).

During the acid hydrolysis of the B 3? Errichrysins arise as volatile products 3 moles of acetic acid. (Bernsteineäu3Pe and i-amino-5-hydroxylamino-pentane, the essential Constituent parts of the ferrloxamines are in the hydrolysis chemistry des ferriohrysin not detectable).

In the treatment of Ferrichrysins with bases or acids or with ¹ Eieenkomplex-forming materials, such as 8-hydroxy-ohinolin, the iron from the molecule is removed, leaving the Desferriohrysin is formed. This is a colorless powder that has not yet been crystallized. It is readily soluble in water, methanol, ethanol, more heavily in butanol, and almost insoluble in acetone, chloroform and hypophilic solvents. The elemental analysis gives the following values »ö 46.36 $> \ H 6.98 ^ i H 15.65 i> \ 0 31.01 (calc.), Which corresponds to a sum formula O ₂ QH ^ qNqO ₁ . is equivalent to. The UV spectrum is not particularly characteristic and only shows final absorption at 214 mu. In the IR spectrum (recorded in potassium bromide) there are bands at 2.94 / *, 3.43, «, 6.10 ^, 6.53, «, 7, Oy ,, 7.32 ^, 8.10 / ·, 8.30 / ,, 8.62 ^ ₄ ,, 9.60 /» and 10.14 / * recognizable (see Fig. 3) .

The NMR spectrum of desferrichrysine in urifluoroacetic acid shows the following signals »

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OFUGiNAL INSPECTED

2.07 ppmj wide maximum corresponds to 4 η protons; ₂

Groups.
2 »54 ppm; Singlet corresponds to 3 η protons; Methyl in

Ac e thy dr oxams aur e - Gkrupp ierungen. 4.00 ppm; broad maximum corresponds to 2 η protons. 4.2-5.1 ppm; broad accumulation of bands with recognizable maxima at 4.33, 4.83 and 5.00 ppm; total approx. 3 η protons.
8.02 ppm; corresponds to approx. 3 η protons; peptidic HH.

The number η in the above information probably means 3, corresponding to the probably identical 3 basic units from which the molecule is built. If iron chloride is added to a solution of desferrichrysin, an intense violet-red color is produced. The ferrichrysin can be isolated from the reaction solution. If one desferrichrysin with 6-n. Hydrochloric acid hydrolysed and the hydrolysis products (for the purpose of reducing any hydroxylamino groups present) are catalytically hydrogenated, in addition to 3 moles of acetic acid, three ninhydrin-positive products are obtained which, with phenol-water (4: 1) as a mobile phase, have the following Rf values in the paper chromatogram: 0.21; 0.30 and 0.38. The investigation according to the method of Moore and Stein [d. H. Spackmann, WH Stein, S. Moore, Analytical Chemistry ^ O, 1190 (1958) J shows that

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ORSGlMAL INSPECTED

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it is ornithine, serine and glycine in a molecular ratio 3 * 2i.V acts. If the acidic hydrolysis product becomes without subsequent catalytic hydrogenation investigated directly, no ornithine is detectable.

In ferrichrysine, the amino acids glycine (1 mol), serine (2 mol) and <f-hydroxyornithine (3 mol) are linked to form a cyclic peptide. The hydroxyornithine is U (ο) -acetylatedj the iron is complexed to the 3 hydroxamic acid residues formed in this way. The overall formula of the iron-free IPerrichrysin is therefore! Ri - ($ T-acetyl) -cyclofglycyl, (seryl) 2> (i -hydroxyornithyl),}. lerrichrysin is assigned the following structural formula:

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OBSGlNAL IMSPECTEO

HE

CH ₂ OH

Ferrichrysin is a growth substance for various microorganisms and can therefore be used in the
Culture of such organisms can be used, the following table shows the relative growth of Microbaeterium
lacticum strain ATCC 8181, measured by the extinction of the culture, when various amounts of perrichrysin are added to the culture medium »

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Tabel

time in
Hours. Ferriehrysin in ^. / Liter O 1 10 31.6 100 22nd
25th
28
31
46 0.177
0.222
0.244
0.249
0.257 0.230
0.280
0.330
0.342
0.359 0.362
0.512
0.598
0.645
0.788 0.459
0.802.
0.941
1.040
1.346 0.319
0.644
0.974
1.115
1.503

Ferrichrysin has anti-anaemic properties and can accordingly be used as a remedy in human and Veterinary medicine can be used. Desferrichrysin also has valuable pharmacological properties. So it prevents the deposition of iron-containing pigments in the tissue or, in cases of pathological deposition of iron in the organism an excretion of iron, e.g. B. in hemochromatosis and hemosiderosis. It can be used accordingly in human and veterinary medicine be used.

Like other sideramines, ferrichrysin is able to reduce the antibacterial effects of antibiotics Group of the sideromycins belong to, competitively lift against gram-positive pathogens (antisideromycin effect).

With regard to the biological effects mentioned, ferrichrysin shows certain similarities with others Sideramines, such as B. the -Ferrioxaminen, the lerrichromes, and the coprogen. On the other hand, it differs from these substances in its physicochemical properties in a characteristic way, which is illustrated below.

1) Perrichrysin differs from the ferrioxamines, which are produced by actinomycete strains, not only in that no succinic acid and no i-amino-5-hydroxylamino-pentane can be detected among the hydrolysis products, but also in its spectroscopic properties. Its IR spectrum differs from that of the ferrioxamines in essential points and the maximum of the UV spectrum for ferrichrysin is shorter-wave (approx. 420 u ) than for lerrioxamines (approx. 45Oy *).

2) fferrichrom obtained by breeding Ustilago sphaerogena differs only slightly from ferrichrysin in terms of spectroscopy. In the IH spectrum Ferrichrome shows a medium strong maximum at 1275 cm ", which appears only as a weak shoulder in the case of perrichrysin.

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Instead of a broad band at 1060 cm for ferrichrysin there are two well separated maxima at 1025 in JPerrichrom and 1068 cm ~ available. In the MR spectrum, the Desferr.ichrysin a broad maximum at 4.00 ppm and a broad one

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Accumulation of bands in the 4.2-5.1 ppm range with maxima at 4.33? 4> -83 and 5.00 ppm while the iron-free Ferrichrome in this area maxima at 4.33; Shows 4.52 (shoulder) and 4.74 ppm. They differ in solubility Ferrichrysin and ferrichrome, on the other hand, are clear: In contrast to ferrichrysin, ferric chromium is almost in cold methanol insoluble, it only dissolves in approx. 1000 times the amount of methanol after prolonged boiling. Also the hydrolysis products are different. Bisen-free ferrichrome, with 6-n. Hydrochloric acid hydrolyzed and catalytically hydrogenated, does not provide three, but only two ninhydrin-positive products, namely glycine and ornithine. There are also differences in paper chromatography present: Ferrichrome has the Bf values in systems I and II 0.19 and 0.25, respectively.

3) Ferrichrome A, a companion of ferrichrome, has, in contrast to ferrichrysin, no significant antagonistic effect on ferrimycin. During hydrolysis, it does not produce 3 moles of acetic acid, but 3 moles of ß-methylglutaconic acid. In the paper chromatogram in systems I and II, Ferrichrom A shows Rf values of 0.12 and 0.18, and thus migrates about half as far as ferrichrysine. Finally, ferrichrome A behaves differently than ferrichrysin in the Craig distribution: in the benzyl alcohol-n-butanol-saturated aqueous sodium chloride solution-0.001-n system. aqueous hydrochloric acid 9-9: 5-15 over 95 levels, it accumulates in levels 3-15, while perrichrysin appears in fractions 36-60. _in

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HA2073

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4) Neither the IR spectrum nor hydrolysis products of Coprogen are known. The elemental composition, however, is clearly different from that of fferrichrysin; for coprogen is indicated i C = 50.9 ^ JH s 6.9 * /; N = • 10.2 $>% Fe = 6.7 "p. Coprogen decomposes" already at 205 ° C. In the paper chromatogram, Coprogen shows the Rf values 0.45 (system I) and 0.55 (system II).

lerrichrysin, desferrichrysin and derivatives and salts of these compounds are obtained when using Aspergillus melleus M 2853 or Aspergillus terreus M 4785 or ierrichrysin-producing mutants of these strains in an aqueous nutrient solution, which is a source of carbon and nitrogen as well as containing inorganic salts, cultured under aerobic conditions, from the culture filtrate ferrichrysin and / or desferrichrysin isolated and, if desired, converted 3? errichrysin into the iron-free compound or derivatives or salts this makes connections. Like other sideramines, perrichrysin or desferrichrysin is also used in larger quantities formed only when the jfes producing strains under Cultivated iron deficiency, d. H. in nutrient solutions that contain less iron than normally used in breeding used by microorganisms. Um, a good yield at To achieve errichrysin or desferrichrysin, the iron content of the nutrient medium must therefore be kept low.

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The Aspergillus melleus M 2853 strain was isolated from the air and the Aspergillus terreus M 4785 strain from soil. The two strains are produced in our laboratories and in the Bidg. ! Technical University, Institute for Special Botany, Zurich, kept under the name given. When cultivated on malt extract agar, both strains are characterized by a vegetative mycelium. from septate and branched hyphae, from whose foot cells conidiophores grow out in the form of rounded, swollen heads. The little heads carry finished cells, sterigms, in a dense arrangement, from which the conidia are cut off in unbranched chains by the formation of transverse walls. As a result, both strains are clearly identified as belonging to the genus Aspergillus Michell 1729 (cf. Thorn et Church, "The Aspergilli", Baltimore 1926). The further classification took place according to Thorn and Raper ¹¹ A manual of the Aspergilli ", Baltimore, 1945. The determination of the group _T membership can be made either on the basis of color characteristics or on the basis of morphological criteria. Both ways result in the membership of strain M 2853 to Aspergillus ochraceus group: Within the Aspergillus ochraceus group, the classification is based primarily on the color of the sporulating mycelium, the formation of the sclerotia and the shape of the conidia

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- 12 -. ■

Conidial heads, the sparse occurrence of sclerotia and the smooth, rounded conidia, strain M 2853 is characterized as Aspergillus melleus.

The coloring and morphological features of · ■ Strain M 4785 result in belonging to the Aspergillus terreus group and within the group to the species Aspergillus terreus thorn »'

Purely the cultivation of Aspergillus melleus M-2853 or Aspergillus terreus M 4785 come as carbon and nitrogen sources . B. glucose, sucrose, lactose, starch, alcohols such as mannitol and glycerin, amino acids, e.g. B. ornithine, peptides, proteins and their breakdown products such as peptone or tryptone, meat extracts, water-soluble fractions of cereal grains such as corn or wheat, distillation residues from alcohol production, yeast, seeds, in particular the rapeseed and soy plants, the cotton plant, ammonium salts, Nitrates and urea. In inorganic salts, the nutrient solution can contain, for example, chlorides, carbonates, sulfates, nitrates of alkalis, alkaline earths, magnesium, zinc and manganese as well as traces of iron (<10 " ⁷ mol / liter).

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

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♦ 'H42073

Shake flasks or the well-known]? Ermentern. As .temperature a value between 18 and 40 0, preferably 27 G. A substantial errichrysin effect is suitable shows the nutrient solution generally after 4-12 days. The Eerrichrysin activity can be microbiological using the modified Bonifas test (see zahner et al., Arch. Mikrobiol. j56, 325 ff. £ 196O]) can be determined. A perrimycin solution with a content of 0.01 mg / ml is used as the test solution, and Staphylococcus aureus as the test strain.

The perrichrysin concentration of the culture solution can also be determined optically. For this purpose, 5 ml of culture liquid with 1.5 g of sodium chloride, 1 ml of 0.1% perrisulfate solution and 5 ml of benzyl alcohol are shaken for 5 minutes, centrifuged, the organic phase is filtered and the absorbance of the organic phase is determined at 420 f . An extract prepared in the same way from uninoculated nutrient solution is used as a comparison sample.

After 4-12 days the cultures reach an activity which corresponds to 300-400 mg of pure perrichrysin.

1 g of i'errichloride per liter is added to the culture. Iron can also be added towards the end of the fermentation. The mycelium is separated from the KuIturfiltrat, after which most of the sideramine is found in the culture filtrate. Nevertheless, significant amounts of it remain on

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-H-

Mycelium adhere. It is also advantageous to wash it off well. For this purpose, for example, water or aqueous alcohols, wis aqueous methanol *

Pur the isolation of lerrichrysin from the culture filtrate can proceed according to methods known to sioh and z. B. use one of the following working methods or combinations thereof.

1) One can use adsorbents, e.g. B. activated carbons such as forite, activated earths such as prankonite, Puller earth or ploridine, or resin adsorbers such as Asmitr. The elution of the adsorbates is expediently carried out with mixtures of water-miscible organic solvents Water, e.g. Bo with water-methanol, water-pyridine, diluted Acetic acid-methanol or water-methanol-glacial acetic acid-butanol mixtures. This has proven to be particularly advantageous for the elution of a prankonite or E “oritadsorba, tes Mixture of water (4 parts by volume) and pyridine (1 tolum part) proven.

2) Perrichrysin can also be removed from an aqueous solution using organic solvents. Purely these extraction processes have higher organic Alcohols e.g. B. Benzyl alcohol or isopropyl alcohol * especially proven. Appropriately, the aqueous

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UA2073.

Phase an inorganic salt, ζ. B. ammonium sulfate or sodium chloride added. From the obtained organic extracts Ferrichrysin either by evaporation of the solvent or by precipitation by means of a SITUATE "Neten organic solvent, e.g., B. ether, petroleum ether or ethyl acetate, are obtained in an enriched form.

3) Another method of enrichment of Ferriehrysin is that it is between a aqueous solution and a solution of phenol in chloroform, the phenol content of the chloroform solution can be varied.

4) Another method for the enrichment of ferrichrysin is chromatography, such as adsorption chromatography on different materials, e.g. B. Norit, aluminum oxide, magnesium silicates, silica gel, Calcium sulfate, as well as partition chromatography with cellulose, starch, silica gel, gelite and the like as carrier substances, or chromatography on ion exchange resins, e.g. B. on Dowex-50, Amerlite IRC-50 and the like.

- 16 909817 / ηκρς

5) Furthermore, the ferrichrysin can be mixed between two immiscible solvent phases by countercurrent distribution according to Oraig be enriched. The following solvent-- ' system has proven itself particularly with n-butanol-benzyl alcohol-0.001-n. Hydrochloric acid aqueous solution of jetrium chloride saturated at 19 ° C9 $ 9 * 15 * 5)

Desferrichfysin is produced by Aspergillus alongside ferrichrysin melleus M 2853 and Aspergillus terreus M 4785 and can therefore also be isolated directly from the culture filtrate will. It is advantageous to remove any iron present in the culture filtrate during the isolation, for example by adding it Iron complex-forming substances, e.g. B. 8-oxy-quinoline or Ethylenediamine tetraacetic acid.

The iron complex-forming substances can be added to the culture broth immediately after the fermentation has ended. The addition advantageously takes place in a later stage of the Work-up to remove any iron (III) ions introduced by the agents used.

The desferrichrysin is isolated from the culture solution in accordance with the method for isolating ferrichrysin mentioned methods.

The invention is illustrated in the following examples described. The temperatures are given in degrees Celsius.

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example 1 i

The older Aspergillus melleua M 2853 strain is grown in a well-ventilated submerged culture at 27 °. A nutrient solution is used which contains 20 g of glucose, 5 g of 1-asparagine, 1 g of secondary potassium phosphate, 1 g of crystalline per liter of tap water. Magnesium sulphate (MgSO ^. 7 H ₂ O), 0.5 g calcium chloride and 2 mg. Contains zinc sulphate. The iron content of tap water is 20-30 ^ / liter. The nutrient solution is sterilized at 120 ° for 20 minutes. Inoculation is done with a spore suspension containing 150-180 million spores per liter.

4-8! Days will be added to the cultures 1 g JFerrichlorid "added per liter and with addition of 2 $> Celite filtered to the culture are now per liter of 300 g of sodium chloride and the clear solution was extracted three times with 1/10 volume of benzyl alcohol. The organic Phase is dried with sodium sulfate and mixed with 3 volumes of ether. The benzyl alcohol-ether mixture is extracted with a small amount of distilled water until the aqueous phase is only pale red in color. The aqueous extract is lyophilized, a red powder being obtained In a yield of 300-400 mg / liter, containing 30% of the activity of the starting material.

18.9 g of crude perrichrysin mixture are subjected to a Craig distribution over 95 stages, the sub-, punch into the first 7 glasses of an automatic distribution

9Q9fli7 / nRRS 1 1

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apparatus (glass content 25 ml per phase) is filled. as Distribution system is a mixture of benzyl alcohol, n-butyl alcohol, sat. aq. Sodium chloride solution and 0.001-n. aqueous hydrochloric acid in a volume ratio of 9 * 9 * 5 * 15.

The red-brown dye is essentially separated into a main and a secondary component, which have their distribution maxima in fractions 46 and 75, respectively. This corresponds to an approximate distribution coefficient of K .. = 0.9 or Kp ⁼ 5 »8.

Fractions 36-60 are combined, and twice the volume of ether is added, and the aqueous phase is added severed. The organic layer is used three more times shaken out some water, the combined aqueous extracts about half saturated with sodium chloride and several times with Phenol-chloroform (1 kg of phenol to 1 liter of chloroform) extracted. The extracts are mixed with semi-saturated liatrium chloride solution washed. The initially cloudy solution is clarified on a short Celite column, then with a double column Volume of ether diluted and shaken out several times with a little water, Y / obei the brown-red dye again in the aqueous Phase goes.

The aqueous extracts, washed several times with ether, are evaporated in vacuo and the residue is dried. 8.2 g of purified ferrichrysin are obtained. For the Crystallization, 16g of such an evaporation residue

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dissolved in approx. 200 ml of hot absolute ethyl alcohol (distilled over sodium). Crystallization sets in quickly and 11.6 g of crystalline ferric chrysin precipitate in the form of red-brown rods. The mother liquors become dry evaporated and redissolved in absolute alcohol. 0.9 g of crystals are obtained no more than.

For analysis, a sample is recrystallized again from absolute alcohol. The red-brown rods turn black at 270 ° without melting. Analysis! öef. 0 43.37 H 6.08 Fe 7.09 N 16.72 #. Absorption spectrum in water: * ___ «415 m / * f log E Jz « 1 »48

If the alcohol is not completely dry for the crystallization, partly amorphous, partly irregular Lumps of crystallizing hydrates.

Ferrichrysin is readily soluble in both water and cold methanol. Also leaves in absolute ethyl alcohol amorphous ferrichrysine can still be absorbed quite well (up to approx. 5 ^). Ferrichrysin separates from such a solution but quickly in red-brown short sticks that hardly become matted on the nutsch. These crystals only dissolve difficult and after prolonged heating in absolute alcohol.

The IR spectrum shows a. a. Bands at 725, 783, 975, 1060, 1140, 1200 (shoulder), 1218 (shoulder), 1237, 1275 (shoulder), 1340 (shoulder), 1368, 1430 (shoulder), 1445 (Shoulder), 1465, 1495 (shoulder), 1517, 1585, 1652, 2930,

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3070 (shoulder), 3420 cm " ¹ cf. Ji ¹ Ig. 1.

The paper chromatogram in system I (n-butanol-glacial acetic acid-water, 4s 1 s 1) gives the Rf value 0.20, im System II (tert-butanol-0.04-n. Hydrochloric acid-saturated aqueous sodium chloride solution, 2: 1 si, paper impregnated aqueous sodium chloride solution saturated with acetone-water 6: 3ϊ1) the Rf value 0.34-

iTerrichrysin is also formed when the strain Aspergillus terreus M 4785 is bred and from it. Insulated growth substance.

Example 2i

4 g crist. lerrichrysin are dissolved in 100 ml of water dissolved, 3 g of 8-hydroxyquinoline in 36 ml of methanol were added and stirred for 24 hours "at room temperature black precipitate filtered off and the pale yellow mitrat extracted three times with chloroform to remove excess Remove reagent. The aqueous phase is evaporated in a vacuum. 3.3 g of desferrichrysin are obtained M 2853 in the form of an almost colorless residue. Ss is readily soluble in water, methanol, ethanol, more difficult in butanol, almost insoluble in acetone, chloroform and lipophilic solvents. The elemental analysis gives the following values:

■ U42Ö73

, - 21 -

C 46.36 i>% H 6.98 # | N 15.65 ⁰ H O 31.01 # (^ er.), Which corresponds to a Suiumenformei OpqH.qNqO ^ -. The UY spectrum is not particularly characteristic and only shows final absorption at 214 m / ·. In the IR spectrum (recorded in potassium bromide) there are bands at 2.94y *, 3.43, *, 6.10 *, 6.53 ^, 7.04 *, 7.32 ^, 8.10 ^, 8 , 30 *, »8.62 ^, 9.60 ^ and 10.14 A recognizable (see Fig. 3).

The NMR spectrum of this compound in rifluoroacetic acid shows the following signals:

2.07 ppm} broad maximum corresponds to 4 η protons; GHp-

Groups.
2.54 ppmj singlet corresponds to 3 η protons; Methyl in

Acethydroxamic acid groups. 4.00 ppmj wide maximum corresponds to 2 η protons. 4.2-5.1 ppmj wide accumulation of bands with recognizable maxima at 4.33; 4.83 and 5.00 ppm; total approx. 3 η protons.

8.02 ppm? corresponds to approx. 3 η protons; peptidic NH. The number η in the above information probably means 3, corresponding to the likely identical ones three fine units of which the molecule is built up.

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9098 ιΪ / n approx c

Example 3t

Aspergillus melleus M 2853 is grown using the submerged method in a nutrient solution that contains 20 g of glucose per liter of tap water ? 5 g L-asparagine, 1 g sec. Salium phosphate, 1 g crystall. Contains magnesium sulphate (MgSO ^. 7 HgO), 0.5 g calcium chloride and 2 mg zinc sulphate. The nutrient solution is sterilized in the inoculation flasks or fermenters for 20-30 minutes at 1 atm. Then it is inoculated with a spore suspension as in Example 1. Incubation is carried out with good shaking or stirring at 27 °, the cultures in the fermenters at about 2%. Air per volume of solution per minute. After about 96 hours of incubation, the culture solution has reached the highest content of desferrichrysin. This is determined by means of the Antisideromyein-iOestes. Since the nutrient solution contains small amounts of iron, the desferrichrysin formed is partly in the form of its Bisen complex.

340 liter active culture broth of Aspergillus melleus M 2853 are 85 g of 8-Qxychinolin dissolved in 1.6 liters of methanol, mixed, "oh an hour with addition of 2 fo Hyflo-Supercel as a filter aid removed by filtration, the mycelium. The FiItrat is to remove excess 8-oxyquinoline through a 5 liter column

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Amberlite IR-45, OH ^ form, percolated. The pH of the percolate is adjusted to 7.5 by adding hydrochloric acid. The active culture solution thus obtained is then with Saturated sodium chloride and extracted with benzyl alcohol. The desferrichrysin is obtained after evaporation of the extracts in the form of a pale yellow powder *

20 mg desferrichrysin are 3 hours with 1 ml 6-n. Hydrochloric acid heated and the evaporation residue dissolved in 96% ethanol, in the presence of a platinum oxide catalyst (Adams) hydrogenated. In the reaction product one can detect 3 ninhydrin-positive products, which in the paper chromatogram with phenol-water (4: 1) as the superplasticizer, the following Rf values show «0.21} 0.30 and 0.38.

Example 4:

70 mg of desferrichrysin are dissolved in 2 ml of methyl alcohol and treated with 40 mg of crystalline. Ferric chloride in 2 ml of methyl alcohol mixed. Immediately a deep black-violet color develops, which upon the addition of something crystalline. Matrium acetate turns orange-red. This solution is evaporated to dryness in vacuo, the residue with hot extracted absolute alcohol and filtered off from the sodium chloride. The Filtr ^ t is evaporated, the residue in approx.

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Oj, 5 ml abs. Dissolved alcohol, filtered and allowed to crystallize ^{at O 0.} 53 mg of crystalline are obtained. ferricrichrysin, which in the paper chromatogram is uniform in two solvent systems and is identical to the original ferricrysin.

20 mg perrichrysin are in 3 ml 1,2 "-n. Dissolved hydrochloric acid and heated it in a boiling water bath for 5 minutes. the initially red-brown solution becomes almost colorless. It is evaporated to dryness in vacuo. Leaves in the residue neither succinic acid nor 1-amino-5-hydroxylaminopentane prove.

20 mg ferriohrysin are mixed with 20 # sulfuric acid heated to reflux. The hydrolysis solution is distilled, diluting it a few times with water collected in good cooling, the volatile Contains acids, is neutralized on phenolphthalain using sodium hydroxide solution and evaporated to dryness in a vacuum. By reacting the residue with p-phenylphenacyl bromide one obtains the p-phenylphenacyl acetate amounts, the three moles Acetic acid.

20 mg desferrichrysin are 3 hours. With 1 ml 6-n. Hydrochloric acid is heated and the evaporation residue, dissolved in 96% ethanol, is hydrogenated in the presence of a platinum oxide catalyst (Adams). 3 ninhydripositive products can be detected in the reaction product, which show the following Rf values in the paper chromatogram with phenol-water (4t1) as the mobile phase: 0.21; 0.30 and 0.38.

909 M1 7 / nt ο c ²⁵ ~

Example 5 s

25 mg of iron-free Ierrichrysin are heated to 110 ° for 20 hours with 1 ml of concentrated hydrochloric acid and water. The evaporation residue of the hydrolysis solution is dissolved in a little water and diluted with 20 ml of fine spirits. This solution is hydrogenated in the presence of 20 mg of pre-hydrogenated PtOp in order to reduce hydroxylamine to amino groups. The hydrogenation product is investigated according to Moore and Stein [DH Spackmann, WH Stein, S. Moore, Analytical Chemistry 3 , 0 »1190 (1958) 3. It results in the presence of the amino acids ornithine, serine and green in a molecular ratio of 3 * 2j1.

Example 6t

The strain Aspergillus melleus M 2853 is based on the following nutrient solution

Gflucose 20th G MgSO ₄ . 7th H ₂ 0 1 , 0 G Asparagine 5 G CaOl ₂ 0 , 5 G K ₂ HPO ₄ 1 , 0 G ZnSO ₄ . 7th H ₂ 0 2 mg

Tap water 1000 ml

. - 26 909ft 1 7 / η κ Q c

cultured with the addition of variable amounts of IPerrisulphate. In the antagonism test and by extinction measurement, "at 410 m / l (cultures saturated with SaGl, 1 mg / ml S'eCl" are added and the extract is extracted with the same amount of benzyl alcohol) used for measurement) of 3 errichrysingehalt _s determined in parallel therewith, a determination of the Mycelgewichtes the results are shown in Figure 2 summarized on the abscissa is the amount of the nutrient solution ferric sulfate in |?... £ / liter, on the ordinate the activity in The unbroken line refers to the values on the 5th day, the dashed line to those on the 2nd day.

The addition of 80 liters / liter ferric sulfate decreases the formation of IPerrichrysin from 550 mg / mother to below 5 mg / liter.

90 SfM 7 / η

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Claims (10)

- 27 - j patent claims: Process for obtaining the new growth substances perrichrysin and desferrichrysin, characterized in that daae man Aspergillus melleus M 2853 or Aspergillus terreus M 4785 in the usual way in an aqueous nutrient solution, which contains a carbon and nitrogen source and inorganic salts, grows under aerobic conditions Perrichrysin or Desferrichrysin is isolated from the culture filtrate and, if desired, metal salts obtained are incorporated into the Free hydroxamic acid or the free hydroxamic acid converted into metal salts. 2. The method according to claim 1, characterized in that after completion of the cultivation an iron salt for Culture solution there. 3. The method according to claims 1 and 2, characterized in that the active substances by means of Benzyl alcohol extracted from the culture filtrate. 4 · Method according to claims 1 and 2, characterized characterized in that the active substances are extracted using phenol-chloroform. 909817/05 ^ 5 New documents {Art 711 At *. 2 No. 1 set * 3 <m kntmsnotom. ν. U42073 5. The method according to claims 1-4, characterized in that that the raw active substances are saturated by means of countercurrent distribution in the benzyl alcohol-n-butanoI system. wKssr. Natriurachlorldlosung-O / OOl-n. "Hydrochloric acid (9 * 9 * 5» 15) cleans. 6, method according to claims 1-5 »characterized in that one ferrlehrysin from absolute ethanol recrystallized. 7. The method according to claim 1, characterized in that for the purpose of obtaining Besferrichrysin the culture filtrate Adds iron complex-forming substances 8. "Method according to claims 1 and 7 * thereby characterized in that the culture filtrate 8-Qxy-quinoline *. adds « 9 · Method according to claim 1, characterized in that- > ι net that one can make Ferrichrysln by means of acids or bases or iron complex-forming substances which remove iron. 10. Perrichrysin, the Fe (III! Complex of cyclo-glycoyl- (seryl> ₂ - (N ^ -hydroxy-R * -acetyl-ornithyi), 3. 11 · Desferrichrysin of the formula Cyül © - [glyoyl- {seryl) g-. j , 1 _β 909 617/0585 ί