DE1442220A1 - Process for the production of new active ingredients - Google Patents

Description

Today for the printing of the publication enclosed with "An- aeldungeunterlagen - us «Zeiobem 20 157-BR / ro -

, 1442220 CIBA AKTIENGESELLSCHAFT, BASEL (SWITZERLAND)

Case 5157 / I-3 / E

Germany

Process for the production of new active ingredients.

There are already a large number of materials of biological origin, in particular microorganisms ferrous growth substances have been isolated, for example the ferrichrome, the coprogen, the ferrioxamine,

It has now been found that Aspergillus strains a new ferrous growth substance, the Ferrirho-

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din, and the corresponding iron-free compound, the desferrirhodin, can win. Ferrirhodin-producing strains are ZwBv Aspergillus versicolor ETH M 3636 and Aspergillus nidulans ETH M 2734. The present invention is hence the use of these strains to produce ferrirhodine and desferrirhodin, by means of usual cultivation conditions, perrirhodin and desferrirhodin are in the Breeding of the named strains in addition to the growth substance Perri- " crocin and desferricrocin are formed. ·

The named strains are in the Federal Institute of Technology, Institute for Special Botany, Zurich, and in our laboratories under the specified Designation retained. They have also been registered in the United States Department of Agriculture, Agricultural Research Service, Peoria, Illinois, under numbers NRRL 3052 (= M 3636) and NRRL 3055 (= M 2734).

The Aspergillus species was determined according to CH.Tho and K.B. Raper, A Manual of Aspergilli, The Williams. and Wilkins Co., Baltimore, 1945.

Both strains were isolated from a Rieselboden from Münster, Westphalia.

Aspergillus versicolor M 3636 is characterized by 'green, partly also flesh-colored conidia cups, not colored, ' smooth conidial support walls and a double series of sterigms, furthermore by spherical vescicles and the absence of

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Perithecia marked as representatives of the Aspergillus versicolor group. It is because of the prickly conidia assigned to the species Aspergillus versicolor (Vuill.) Tiraboschi 19O8 (I.e. p.192).

Aspergillus nldulans M 2734 forms in pure culture Perithecia with red-brown ascospores, making its affiliation to the Aspergillus nidulans group is assured. The two, approx. 1μ wide longitudinal strips on the smooth Ascospores are typical of the species Aspergillus nidulans (Eidam) Wint. in Rabh. 1884. Strain M 2734 only differs insignificant from the description in Thorn and Raper, namely in the mostly somewhat paler pigmentation and in the little wider longitudinal ridges.

Perrirhod.in is a red-brown, crystalline substance. It has a similar structure to the sideramines ferrichrom, ferrichrom A, perricrocin, and ferrichrysin and ferrirubin, which are all iron complexes attached to i-N-acyl-i-N-L-hydroxy-ornithine residues contain bound and in which the ^ -hydroxyornithine with one or two other amino acids is linked to a cyclic hexapeptide. The building blocks of the sideramines mentioned are shown in the table below :

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Tabel

Sideramine £ -N-hydroxy-
ornithine Serine Glycine Acid residue Perrichrome 3 0 3 3 acetyl Perricrocin 3 1 2 3 acetyl Perrichrysin 3 2 1 3 acetyl Perrichrome A 3 2 1 3 ß-methyl glutaconyl Ferrirhodin 3 2 1 3 I ice Perrirubin 3 2 1 3 I trans

The acid component of ferrirhodine could be elucidated in the sense of formula I (ice) by succeeding after hydroxamic acid cleavage by means of periodic acid there is lactone of 5-hydroxy-3-methyl-pentenoic acid (II) and to deduce its constitution from the UV, IR and especially the NMR spectrum.

HOCH ₂ -CH ₂ -C = CH-CO-

I II

In the IR absorption spectrum (in carbon tetrachloride) the compound shows a strong band at 170 cm ", which can be ascribed to a carbonyl group (lactone, ester). A high UV absorption maximum at 216-217 πιμ (loci: = 3.92

BATH ORIGINAL

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in Peinsprit) shows that there is a double bond to the carbony group is conjugated. The spectrum also shows a high absorption maximum at 254 πιμ and in the visible area broad maximum at 445-450 ιημ. The NMR signals (in carbon tetrachloride) are very well resolved and can be clearly assigned to the individual hydrogen atoms. the chemical shifts are given in δ values, spin-spin interactions in cps. The following mean: d = doublet, t = triplet, q = quartet. The following signals can be found:

2.00 ppm (d, J = 1.2 cpsj 3 H),

2.3β ppm (t, J = 6.3 cps; 2 H),

4.31 ppm (t, J = 6.3 cpsj 2 H),

5.68 ppm "(q, J = 1.2 cpsj 1 H).

Internal reference substance: tetramethylsilane

The signal of methyl group 3 next to the double bond is due to the proton at C-2 as a result of 1,3 interaction split via the double bond into a doublet with a small coupling constant. The same interaction on the other hand, splits the signal of the single proton at C-2 - with the same coupling constant - into a quartet. Besides that the signals for the methylene groups are found at C-4 and C-5 at 2.36 and 4.31 ppm, respectively, which are due to spin-spin interaction as a triplet with the normal coupling constant of approx. 6 cps. appear.

Based on these spectroscopic data, the constitution of the degradation lactone is very likely

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given in the sense of formula II.

Ferrirhodine is assigned the following formula III:

CH CO Mi CH

HH (CH) R (CH) \ ₀

/ Vx ₀ A \

/ VI ₁ / CW \

CH (CH) UT 0PΌ C

, _χ _ Xi.3 / \

CH- (CH ₉ ), -UT ^ - 0 TPe Ό CH-CH ₀ OH

BH "/ CO.

CO KH

^ NH CO CH

CH ₂ OH

R = OH-CH ₀ -CH ₀ -C = CH- '. ■

d. d j

(ice) ^CH 3 III

i Ferrirhodin has the elemental composition C = x 49.28 %; H = 6.38 %} N = 12.30 %; Fe = 6.25 %, these data correspond to the gross formula Ο ^, Η, - ^ Ο, “N _Q Fe. The UV spec-

1 Ψ

trum in Feinprit shows the following maxima: 215 mp- (lög _L E _T ■ ' =

i j χ cm

2.55); 252 ΐημ (log * \ ^ «2.33); ^ 5 Γημ (log E ^ J _m = 1.58).

The maximum at 252 ιημ absent in Acethydroxa- maten Ferrichrome, Ferrichrysin and ferricrocin, as with

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the pearl oxamines. It is very likely characteristic of iron trihydroxamates with α, ß-unsaturated acid components, as it is found not only in ferrirhodine but also in perrichrome A (ß-methylglutaconic acid as acid component). The same maximum is also found with ferrirubin and with coprogen with a previously unknown acid component. The IR spectrum in potassium bromide has, inter alia, bands at 852, "933, 988, IO38, 1142, II90, 1244, 1355, 1460, 1539, 1660, 2945, 3440 cm" ¹ , see FIG. 1.

The crystals decompose at about 270 ^° C., turning black, without melting. The following Rf values are obtained in the paper chromatogram: 0.43 in system I (n-butanol-glacial acetic acid-water, 4: 1: 1) and 0.88 in system II (tert.-butanol-0.004-n. Hydrochloric acid saturated aqueous sodium chloride solution, 2: 1: 1; paper impregnated with acetone-water-saturated aqueous sodium chloride solution, 6: 3: 1). Under the same test conditions, the Rf values for ferrichrome are 0.28 (system I) and 0.30 (system II), for coprogen 0.43 (system I) and 0.55 (system II), for perrichrysin 0.26 (System I) and 0.34 (System II), for perrirubin 0.42 (System I) and 0.73 (System II) and for perricrocin 0.29 (System I) and 0.28 (System II) .

When treating ferrirhodine with 3ases or acids or with substances that form iron complexes, the iron is removed from the molecule, whereby the colorless

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Desferrirhodin is formed.

In the catalytic hydrogenation of ferriridine the cis-5-hydroxy-3-methyl-pentene (2) acid is hydrogenated, without attacking the rest of the molecule. This gives hexahydroferrirhodine, its acid component is 5-hydroxy-3-methyl-valeric acid. The hexahydroferrirhodin is identical to hexahydroferrirubin, obtained by hydrogenation of perrirubin, its Acid component is trans-5-hydroxy-3-methyl-pentene (2) acid (see note number C 301Ö0 IVa / 6b _ Case 5089 / 1-4 / E). The IR spectrum of the hexahydro compound in Nuj oil is in .Fig. 2 shown. In the UV spectrum of hexahydroferrirhodine the absorption band at 254 πιμ has disappeared and the absorption band in the visible shifted to 425-430 πιμ. The absorption curve is practically congruent with that of ferrichrysin (this has instead of the three 5-hydroxy-3-methyl-valeric acid residues three acetyl residues, see note no. C 28460 IVa / 6a - Case 4972 / 1-4 / 5038). the Rf values of the hexahydroferrirhodine in systems I and II are 0.57 and 0.80 compared to 0.65 and 0.85 of the ferrirhodine (yellow-orange spot in contrast to the reddish-brown spot in contrast to the reddish-brown spot of Ferrirhodin).

If the iron is removed from the hexahydroferrirhodine, for example by means of 8-hydroxyquinoline, it splits

BATH ORIGINAL

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in the obtained des-ferri-hexahydroferrirhodin by mild acid hydrolysis of the 5-hydroxy-3-methyl-valeric acid residues ab, acetylated (on nitrogen and oxygen) and selectively splits off the O-acetyl groups, e.g. using ammonia, see above Desferriehrysin is obtained, which can be converted into Perrichrysin with iron (III) salt. That from Ferrirhodin The perrichrysin obtained shows the same Rf values as authentic in the paper chromatogram in systems I and II Perrichrysin.

Ferrirhodin is recovered from desferrirhodin when iron ions are added.

Derivatives of Perrirhodins and Desferrirhodins and their hydrogenation products are, for example, compounds in which the hydroxyl group of the serine residues is esterified, or in which the acyl group of the hydroxamic acid residues is substituted by other acyl groups, for example by lower alkanoyl or lower alkenoyl or other ω-hydroxy-alkenoyl groups than the 5-hydroxy-3-methylpentenoyl group, e.g. propionyl, butyryl, pentanoyl, pentenoyl, 5-hydroxy-pentenoyl. The former derivatives are obtained when ferrirhodine or hexahydroferrirhodine are treated with acylating agents, e.g. acid anhydrides or acid halides, such as acetic anhydride or acetyl chloride, and the esters isolated and, if desired, eliminates the iron. The deacyl compound is obtained through

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- ίο -

Deacylation of desferrirhodin or hexahydrodesferrirhodin in a tired acidic medium, e.g. in the presence of diluted Mineral acids. The cyclic hexapeptide obtained in this way can be acylated in a known manner; thereby formed O-acyl groups can be with ammonia, for example in Split off methanol. The acyl analog of desferrirhodine can be converted into the corresponding iron complex with iron salts be transferred.

Ferrlrhodin, Desferrirhodin and analogues are • growth substances for various microorganisms; you can therefore used in the culture of such organisms.

The iron-containing compounds have anti-anemic properties and can accordingly be used as remedies be used. Desferrirhodin and its derivatives also have valuable pharmacological properties. In this way they prevent the deposition of iron-containing pigments in the tissue or, in cases of pathological iron deposition an excretion of iron in the organism, e.g. in hemochromatosis and hemoslderosis. You can accordingly used in medicine. ■

Like other sideramines, Ferrlrhodin is also Im * states the antibacterial effects of antibiotics that belong to the group of sideromycins, competitively lift off against gram-positive pathogens (antisideromycin effect).

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Ferrirhodin, Desferrirhodin as well as derivatives of these compounds are obtained by using ferrirhodin-forming compounds Aspergillus strains in an aqueous nutrient solution, which is a carbon and nitrogen source and inorganic salts contains, cultured under aerobic conditions, isolated from the culture filtrate ferrirhodin and / or desferrirhodin and, if desired, ferrirhodin is converted into the iron-free compound or hydrogenation products or derivatives thereof Establishes connections. Because the formation of Ferrirhodins and / or Desferrirhodins by a nutrient medium poor in iron is favored, it is advisable to breed under iron deficiency, preferably with an iron content of at most 10 moles / liter.

The following carbon and nitrogen sources can be considered for breeding the strains: carbohydrates, e.g. glucose, sucrose, lactose, starch, alcohols such as mannitol and glycerine, amino acids, e.g. ornithine, peptides, Proteins and their breakdown products, such as peptone or tryptone, meat extracts, water-soluble parts of cereal grains, such as corn or wheat, distillation residues from alcohol production, yeast, seeds, especially from the rapeseed and soy plants, of the cotton plant, ammonium salts, nitrates. The nutrient solution can, for example, contain inorganic salts Chlorides, carbonates, sulfates, nitrates of alkalis, alkaline earths, magnesium, zinc and manganese as well as traces of

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Contain iron.

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 well-known fermenters. As a temperature a suitable temperature between 18 and 40 ° C, preferably 27 C. A significant sideramine effect is shown by the Nutrient solution generally after 8-12 days.

The ferrirhodin activity can be microbiologically determined by means of the modified Bonifas test (cf.Zahner et al., Arch. Microbiol. JjjS, 325 ff. [1960]). as A ferrimycin solution of 0.1 mg / ml is used as the test solution Salary, as a test strain Staphylococcus aureus Rosenbach.

The ferrirhodin concentration of the culture solution can also be determined optically. Be for this purpose 5 ml culture liquid with 1.5 g sodium chloride, 1 ml 0.1 $ - iger ferric sulfate solution and 5 ml benzyl alcohol shaken for 5 minutes, centrifuged off, the organic phase filtered and the absorbance of the organic phase determined at 410 πιμ. An extract prepared in the same way from uninoculated nutrient solution is used as a comparison sample.

After 8-12 days the cultures reach an activity which corresponds to 100-200 mg / liter of pure ferrirhodine.

At the end of the cultivation, 1 g of ferric chloride per liter can be added to the nutrient solution, with the existing

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Desferrirhodin in Ferrirhodin tiberge: ". '/. Rt is. The iron addition can also take place at any other point in time of the fermentation or also staggered. You separate that Mycelium from the culture filtrate, preferably in the presence of one Filter aid, e.g. Hyflo-Supercel, after which the main amount des ferrirhodine is found in the culture filtrate. Nevertheless, significant amounts of it stick to the mycelium. It is therefore advantageous to wash it off well. For this purpose, for example, water or aqueous alcohols are suitable, like aqueous methanol.

Methods known per se can be used to isolate ferrirhodine from the culture filtrate and e.g. one of the following working methods or use combinations thereof.

1) Adsorbents can be used, e.g. activated carbons such as Norit, activated earths such as Frankonit, 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. with water-methanol-, water-pyridine- Acetic acid-methanol or water-methanol-glacial acetic acid-butanol mixtures. As particularly advantageous for elution of a frankonite or norite adsorbate, the mixture of water (4 parts by volume) and pyridine (1 part by volume) proven.

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- l4 -

2) Furthermore, the ferrirhodin can be removed from an aqueous solution by means of organic solvents. Higher organic alcohols, e.g. benzyl alcohol or isopropyl alcohol, are particularly suitable for these extraction processes proven. Appropriately, an inorganic salt, e.g. ammonium sulfate or sodium chloride, is added to the aqueous phase. added. The ferrirhodin can be obtained from the organic extracts obtained either by evaporating the Solvent or by precipitation using a suitable organic solvent, e.g. ether, petroleum ether or ethyl acetate, can be obtained in enriched form.

3) Another method of enriching the '

Ferrirhodins consists in putting it between an aqueous solution and a solution of phenol in chloroform distributed, the phenol content of the chloroform solution can be varied. -.-..-

4) Another method for enriching ferrirhodine is chromatography, such as adsorption chromatography on various materials, e.g. on 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.

5) Ferrirhodin can be weltered by countercurrent distribution according to Craig between two immiscible solvent phases. The following solution

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medium system has proven particularly useful: n-butanol-benzyl alcohol-OjOOl-n. Hydrochloric acid aqueous sodium chloride solution saturated at 19 (9: 9: 15: 5).

Desferrirhodin is used next to Ferrirhodin by the called Aspergillus strains and can therefore also be isolated directly from the culture filtrate. Advantageous iron present in the culture filtrate is removed during isolation, for example by adding iron complexes Substances, e.g. 8-oxy-quinoline.

The iron complex-forming substances can be added to the cooling broth immediately after the fermentation has ended. The addition advantageously takes place at a later stage of the work-up to include any agents used also remove entrained iron (III) ions.

The isolation of desferrirhodin from the culture solution is carried out according to the methods mentioned for the isolation of ferrirhodine.

The pharmaceutical preparations contain ferrirhodin, desferrirhodin, their hydrogenation products and / or Derivatives of these compounds in admixture with an organic or inorganic pharmaceutical carrier suitable for enteral or parenteral use is suitable. The preparations are made from the starting material according to the usual Methods made. Suitable carriers are substances that do not react with active substances, such as gelatin,

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Lactose, glucose, sodium chloride, starch, magnesium stearate, talc, vegetable oils, benzyl alcohols, rubber, polyalkylene glycols, Cholesterol or other known medicinal carriers.

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

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- 17 Example- 1:

The strain Aspergillus versicolor M J>6j> 6 is grown in a well-ventilated submerged culture at 27 °. A nutrient solution is used which contains 20 g of glucose (iron-free), 5 g of L-asparagine, 1 g of secondary potassium phosphate, 1 g of crystalline per liter of distilled water. Contains magnesium sulfate (MgSO ₁ ^, 7.H ₂ O) and 0.5 g calcium chloride. The nutrient solution is sterilized at 120 ° for 20 minutes. It is vaccinated with a spore suspension that contains 100-200 million spores per liter.

After 8-12 days, 1 g of ferric chloride per liter is added to the cultures and filtered with the addition of 2ff celite. The culture filtrate is now saturated with sodium chloride and the clear solution is extracted twice with 1/10 volume of benzyl alcohol. The organic phase is dried with sodium sulfate, filtered and mixed with 3 volumes of ether. The benzyl alcohol-ether mixture is extracted with distilled water until it is discolored. The aqueous extracts are combined, the residues of benzyl alcohol are removed with ether and lyophilized, a red powder being obtained.

10.6 g of crude product are a 137-stage

Subjected to craig distribution. A mixture of 1.8 liters of benzyl alcohol, 1.8, is used as the two-phase solvent system Liter of n-butyl alcohol, 1.0 liter of saturated aqueous

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Sodium chloride solution and 3 liters of 0.001-n. aqueous hydrochloric acid .

In the course of the distribution, the red-brown dye is essentially divided into a main component and a secondary component separated. The main component has a maximum in level 52 (K approx. 0.5), the secondary component in Level 123 (K approx. 11). The processing of the fractions 4O-6O yields 1.29 S ferricrocin (cf. Application No. C 3016I IVa / 6b Case 5O88 / I-3 / E).

The fractions IO6-I3O are combined. By adding ether, the ferrirhodin is forced into the aqueous phase. This is separated off and the organic layer is extracted twice more with water. The combined aqueous solutions are approximately half saturated with sodium chloride and extracted several times with phenol-chloroform (1 kg of phenol to 1 liter of chloroform). The cloudy red-brown phenol-chloroform extracts are clarified on a column of Celite, double the volume of ether is added and the extract is extracted several times with distilled water. The aqueous solutions are washed several times with ether in order to completely remove the phenol, and they are evaporated in vacuo. 3 ² H ™ S of a red-brown crystalline residue are obtained. By recrystallization from methanol-ether, the ferrirhodine is obtained in the form of shiny red-brown crystals; Decomposition point approx. 270 °.

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Analysis: Found C $ 49.28; H $ 6.38; Pe $ 6.25; Ν $ 12.30. Ber. for C ₄₁ H ₆₄ O ₁₇ N ₉ Pe

C $ 48.71; H $ 6.38; N $ 12.47 ;. Pe $ 5.53 Absorption spectrum in fine spirits: Λ „_. = 215 ηιμ (log

Max

(log B ^ _m - 1.56).

The IR spectrum in potassium bromide shows, inter alia, bands at 852, 933, 988, 1038, 1142, 1190, 1244, 1355, 1460, 1539, 1660, 2945, 3440 cm " ¹ , cf. Pig. I.

Perrirhodin was compared with other sideramines in the paper chromatogram, see Table 2. The method is as follows: A sheet of Whatman No. 1 filter paper is washed with a mixture of acetone, water and saturated aqueous Sodium chloride solution (volume ratio 6: 3: 1) impregnated and air-dried for 10-15 minutes. On the The starting line is alternately a drop of a methanolic solution of the reference substance (starting points 1, 3, 5 and 7) and of ferrirhodine (starting points 2, 4, 6 and 8). The chromatogram lasts for about 20 hours developed with the superplasticizer tert-butanol-0.004-n. Hydrochloric acid sat. Sodium chloride solution (2: 1: 1). the Sideramine spots are recognized by their own color. Despite the relatively small difference in the Rf values Some sideramines result in a clear distinction using the method described.

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Table 2

Sideramine Rf (I) Rf (II) Ferrichrome 0.28 0.30 Ferrichrysine 0.26 0.3 ^ Ferricrocin .0.29 0.28 Ferrirhodin 0.43 0.88 Coprogen 0.43 0.55 Ferrirubin 0.42 0.73

Example 2:

The strain Aspergillus versicolor M 3636 becomes like grown in Example 1, the culture medium mixed with iron chloride and filtered with the addition of Celite. One extracts the culture filtrate obtained is separated twice with 1/10 volume of chloroform-phenol (1 part by volume: 1 part by weight) the organic phase and filtered through Celite. Then the organic solution is mixed with 4 volumes of ether and extract it several times with distilled water until it becomes discolored. The aqueous extracts are combined for removal washed of phenol residues with ether, in vacuo freed from ether and then lyophilized. The crude product obtained is purified as in Example 1. In the same way as in example 1

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perrirhodin is obtained when Aspergillus nidulans M 273 ^ · is cultivated and the culture medium is as described worked up.

Example 5:

150 mg crystall. Perrirhodin are dissolved in 10 ml of water and 200 mg of 8-hydroxy-quinoline, dissolved in 3 ^m l of methanol was added. After stirring for 20 hours, the black precipitate is filtered off and the excess reagent is removed from the piltrate by shaking it out several times with chloroform. The pale reddish aqueous solution contains the desferrirhodin. The solution is mixed with 150 mg of periodic acid in 5 ml of water and left to stand for two hours. Then with conc. Hydrochloric acid made strongly acidic and extracted continuously with ether for 2 hours. The ethereal solution is dried and evaporated and the residue is distilled at 12 mm pressure and a block temperature of 120-140. About 40 mg of a yellow oil are obtained, which is uniform in the thin-layer chromatogram (Rf 0.50 in benzene-ethyl acetate 1: 1 on silica gel). Coloring by spraying with sulfuric acid and then with 1% potassium permanganate solution white spot visible on brownish background. _max πιμ in Peinsprit 217, log £ = 3.92.

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■ \) (CO) in carbon tetrachloride: 173O cm ".

Nuclear Magnetic Resonance Spectrum: 2.00 ppm (d, J = 1.2 cps; 3 H),

2.36 ppm (t, J = 6.3 cps; 2 H),

4.31 ppm (t, J = 6.3 cps; 2 H),

5.68 ppm (q, J = 1.2 cps j 1 H).

According to this, it is the lactone of 5-hydroxy-3-methyl-pentenoic (2) acid.

Example 4:

10 mg perrirhodin are in 1 ml 6-n. Dissolved hydrochloric acid and extracted continuously with ether for 3 hours, after which the aqueous solution is iron-free. 0.3 ml of conc. Hydrochloric acid and melt the colorless solution in a glass tube. After 18 hours of heating at 110 °, it is evaporated in vacuo and a sample of the residue was analyzed according to Moore and Stein. Serine and glycine can be detected. At basic A trace of ornithine is found in amino acids. Another unidentifiable peak is apparently £ -N-hydroxyornithine attributable to.

The bulk of the hydrolyzate is dissolved in 2 drops of water and 5 ml of fine spirits and with 20 mg of platinum dioxide Hydrogenated for 24 hours. The Moore and Stein analysis of the hydrogenation product shows the presence of serine and glycine in an approximate ratio of 1.3: 1, as well

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Ornithine and ammonia (approx. 2: 1), the sum of which corresponds to about three times the molecular amount of the detected glycine. The appearance of ammonia in the hydrogenated hydrolyzate is due to the decomposition of part of the $ -N-'hydroxyornithine to explain during hydrolysis.

Example 5 '·

12 mg perrirhodin are mixed with 1 ml 57 percent. Hydroiodic acid melted in a glass tube and heated for 6 hours. heated to 110 °. The reaction mixture is evaporated in vacuo, a deep brown residue being obtained. This is dissolved in a little water, dried, redissolved and dried until the hydrolyzate is practically colorless is.

The analysis according to Moore and Stein shows the presence of the following amino acids:

0.20 serine, 1.00 glycine, 1.55 alanine, 2.95 ornithine, 0.03 NH ₅ .

The numbers indicate the molecular ratio based on glycine = 1.00.

The glycine yield is quantitative. Serine is changed to a large extent, the converted Serine occurs almost entirely as alanine in the hydrolyzate and can be quantified as such. the

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Sum of serine alanine + corresponds with good reproducibility 90 $ ^de s theoretical serine value.

Ornithine is produced by reductive hydrolysis of the hydroxylamine derivative educated and recorded almost quantitatively. Ammonia occurs only in traces (1-2 $ of ornithine).

Example 6:

To a slurry of 100 mg of 10 $ palladium carbon a solution of 149 mg is added to 5 ml of methanol Ferrirhodin in 5 ml of methanol and stir the mixture • Room temperature in a hydrogen atmosphere. The hydrogen uptake is very quick at first. After approx. 1 hour approx. 3 mol of hydrogen have been taken up and the hydrogenation takes place to a standstill. The catalyst is now filtered off. A solution of 100 mg is added to the orange-brown filtrate Iron (III) chloride in methanol is added to reduce the amount at best Replace iron and buffer the solution by adding some crystalline. Sodium acetate. After 4 hours Standing, the solution is evaporated in vacuo, the residue is taken up in water and the solution is repeated several times with phenol-chloroform (1: 1) shaken out. The phenol-chloroform extract filtered through Celite is diluted with 2 vol. Of ether, three times with dist. Shaken out water and evaporated the aqueous solutions washed with ether in vacuo.

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The hexahydroferrirhodine is obtained as an orange-yellow amorphous powder. Yield: 132 mg.

UV absorption maxima in Peinsprit: high final absorption at 220 ΐημ (log £ over 4); Maximum at 425-430 ηιμ. (log £ 3.5). IR spectrum in Nujöl, see 'Fig. 2.

In paper chromatography, the Rf values in systems I and II are: ■

I II

Ferrirhodine 0.65 0.85

Hexahydroferrirhodine 0.57 0.80

Ferrirhodin gives a reddish-brown spot, the hydrogenation product a yellow-orange spot.

Example 7:

1 g of crude hexahydroferrirhodine is dissolved in 40 ml of water and 1.5 g of 8-hydroxyquinoline, dissolved in 15 ml of methanol, mixed. After stirring for 24 hours at the precipitated iron (III) oxinate is filtered off. That The filtrate is extracted 5 times with chloroform and then evaporated to dryness in vacuo. 900 mg of desferrihexahydroferrirhodine are obtained of formula IV

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CH,

CH-CH ₂ -CH ₂ -OH

co

I /

HO-N- (CH ₂ ) -OH

CO-NH-CH-CO CH.

CH-CH ₂ -CH ₂ OH

ι ²

CO.

NH

CO

NH CH- (CH ₂ ) -N-OH

CO

CH-CH ₀ OH ■ IV 2

CO-CH-NH-CO

JN-CO-CH ₂ -CH-CH ₂ -CH ₂ Oh OH CH,

as an almost amorphous colorless powder that shows a violet color reaction with ferric chloride (~ L% in methanol). The following signals are found in the NMR spectrum, which is recorded in heavy water (D _{p 0):}

(ppm) split 0.93 d (J = 5 cps)

1.2-2.2 b

2.4

b b

Number of H

21

6 18 Assignment .

Methyl groups of the 5-hydroxy-3-methyl-pentane acid residues.

ß- and 7-CH- the N-hydroxy-ornitnine residues; γ-CH- der - 5-hydroxy-3rmethylpentanoic acid residues; ß-CH of the 5-hydroxy-3-methylpentanoic acid residues.

ct-CHp of the 5-hydroxy-3-methylpentanoic acid residues.

<£ -CH _{2 of} the N-hydroxy-ornithm radicals;

the glycine residue;

8 0 9 8 0 5/0960

(ppm) Splitting number of H allocation

the serine residues; p of the 5-hydroxy-3-methylpentanoic acid residues.

4,3 b 5 'a-CH of the N-hydroxy-or

nithine and serine residues.

Example 8:

900 mg desferri-hexahydroferrirhodin are used with 30 ml of N-hydrochloric acid heated to approx. 95 ° in a water bath. At intervals of about 5 minutes, one drop of the solution is placed on the spot plate with 2 drops of 2 # strength methanolic iron (III) chloride solution offset. Initially there is a strong purple color reaction. After 50 minutes the 'color reaction just very pale. The hydrolysis is stopped and the solution which contains the cyclo-tL - ^ - hydroxy-ornithyl-glycyl-L - ^ - hydroxy-ornithyl-L-seryl-L - ^ - hydroxy-ornithyl-L-seryl) contains, evaporated in vacuo. The residue is acetylated with 10 ml each of acetic anhydride and pyridine during 5 hours at room temperature. The reaction mixture is again evaporated in vacuo. For partial ammonolysis of the O-acetyl bonds formed becomes the reaction product left to stand in 60 ml of cold-saturated methanolic ammonia for 4 hours. The evaporation residue of this solution, the which contains desferrichrysin and byproducts is on it in methanolic solution with Ig iron (III) chloride reacted

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sets, the deep purple solution with krist. Sodium acetate buffered until the color changes to orange-brown and the solution evaporated after standing for 4 hours. The • Dissolve the residue in water, add about 10 $ sodium chloride and shake several times with phenol-chloroform (lg: 1 ml) and the initially cloudy phenol-chloroform solution is filtered through a column of 10 g Celite. The clear piltrate is diluted with ether and extracted three times with distilled water. The aqueous extracts, washed well with ether, are evaporated in vacuo and the crude product is purified through a Craig distribution over 50 levels. as In the solvent system, a mixture of 9 parts of n-butanol, 9 parts of benzyl alcohol, 15 parts of 0.001 N-hydrochloric acid is used and 5 parts of saturated aqueous sodium chloride solution. The main component of the mixture is in the stage 19 most enriched, which corresponds to a distribution coefficient of 0.62 (authentic perrichrysin: k = 0.63).

Fractions 12 to 25 are more common

Way worked up with phenol-chloroform. 202 mg are obtained Ferrlchrysih, which is further purified by crystallization from ethyl alcohol. The red-orange crystals decompose like authentic ferrichrysin, slowly at approx. 25O-26O without melting.

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In paper chromatography in systems I and II, the product behaves exactly like authentic perrichrysin (cf. table below), and the IR absorption spectrum corresponds completely to that of authentic perrichrysin.
Paper chromatography:

Tabel

System I. Filter paper System II Silica gel pl. 0.39 Filter paper Perrichrysin
auth. Rf 0.129 0.39 0.32 Transformation
product Rf 0.129 0.32

In the antagonism test against perrimycin A, the product behaves like natural ferrichrysin.

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Claims (3)

Patent claims: 1. Use of Aspergillus versicolor ETH M or Aspergillus nidulans ETH M 273 ^ for the production of Ferrirhodin and Desferrirhodin by means of the usual cultivation conditions » 2. Ferrirhodin, the Fe (III) complex of cyclo- £ (seryl) p glycyl - [] Sr -hydroxy-N - (eis-5-hydroxy-3-methyl-pentenyl) -orni- 3. Desferrirhodin, the compound of the formula Cyclop seryl) p-glycyl- [N ^ -hydroxy-N ^ - (cis-5-hydroxy-3-methyl-pentenyl) -ornithyl] } ». . . 8 09805/0960
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