DE1442217A1 - Process for the production of new active ingredients - Google Patents

Description

New registration documents specific to Äön Druck der Offenlei ^ gescarrrt "- us. Sign: 20 130-BR / ro -

CIBA AKTIENGESELLSCHAFT, BASEL (SWITZERLAND)

Case 5089 / 1-4 / E
Germany

Process for the production of new active ingredients.

There are already a large number of materials of biological origin, especially microorganisms

° ferrous growth substances have been isolated, for example

C ^ the Perrichromes, the Coprogen, the Perrioxamines.

σ> It has now been found that one originates from PiIz

New Unteriaoen {^ 7iiA ^ ^ 2Hr ssu3ae ₉ j ^ _{finite> A90M. v} .4.a. \ mn I

of the Aspergillaceae family a new iron-containing growth substance, ferrirubin, and the corresponding iron-free compound, desferrubin. Ferrirubin-producing strains are in particular Penicillium variabile M 2733, Spicaria sp. M 4622 and Paecilomyces variotl M 4646 _# The present invention therefore relates to the use of these strains for the production of ferrirubin and desferrirubin by means of customary breeding conditions.

The named strains are in the Eidg. Techn. University, Institute for Special Botany, Zurich, as well as in our laboratories under the specified designation kept. The strains were determined according to K.B. Raper and .Ch.Thorn, A Manual of the Penicillia, Baltimore, The Williams & Wilkins Co., 1949.

Penicillium variabile M 2733 was isolated from a soil sample from the Botanical Garden in Zurich. Of the Trunk is to be classified in the section Biverticillata-Symmetrica of the penicillia. There perithecia or sclerotia are absent, the colonies have a typically velvety surface and the back of the colonies often red-orange to is brown in color, the 'strain belongs to the Penicillium series purpurogenum. It is characteristic of the ETH strain M2733 furthermore the formation of sterile, yellow aerial mycelia, which is why the isolated strain of the species Penicillium variabile Sppp 1912 is to be assigned. The description of this kind at ~

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Raper and Thorn (i.e. pp. 642-644} is detailed and stem M 2733 fits well into the range of variation of the taxon line up.

Spicaria sp. M 4622 was isolated from a sample of an infected SUssmostes from the Central Office for Fruit and Potato Utilization, Wadenswil, Canton of Zurich. The organism shows a yellowish-sand-colored sporulation; the long, pointed sterigms arise in Wirtein and show strongly diverging tendencies; the conidia arise in chains at the ends of the sterigms. Such forms are to be assigned to the genus Spicaria Harz I87I (Raper and Thorn, pp. 24-25). A species identification within the genus Spicaria is not possible with certainty, since the principles of species differentiation are not fixed. The isolation is therefore to be referred to as Spicaria species strain ETH M 4622.

Paecilomyces varioti M 4646 was isolated at the Federal Research Institute in Wädenswil, Canton of Zurich. The i The organism forms abundantly, brownish-yellow, elongated ova- · le conidia at the ends of long, pointed cnidia, which are vertically arranged and strong diverge. The organism is to be assigned to the genus' Paecilomyces Bainier I907 and therein to the species Paecilomyces varioti Bainer 1907, perhaps the only very variable species of the genus (cf. Raper and Thorn, pp. 688-692).

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Perrirubin is a reddish-brown substance that has so far only been obtained in an amorphous state. It is very hygroscopic. Analysis values: C = $ 49.01; H = $ 6.57; N = $ 12.70; Pe = $ 5.25; 0 = $ 26.47 (calc.); Acetyl = $ 0. . The UV spectrum in ethanol shows maxima at 216 ηιμ (log ^E lcm τ ² ^ 58); 252 πιμ (log E ¹ ^ _n = 2.34) and 450 mμ (log E ₁ = 1.52). The IR spectrum in potassium bromide shows bands at 842, 945, 990 (shoulder), 1040, IO66, 1125 (shoulder), 1195 (shoulder), 1239, 1282 (shoulder), 1341 (shoulder), 1366, l46o, 1533, 1647, 2938, 3085 (shoulder), 3420 ^{cm -1} ZVgI. Pig. I.

The following Rf values are shown in the paper chromatogram obtained: 0.42 in system I (n-butanol-glacial acetic acid-water, 4: lsi) and 0.73 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)

In the acidic hydrolysis of perrirubin, in contrast to ferrichrome, ferrichrysin and perricrocin, none is produced Acetic acid, but 3 moles of trans-5-hydroxy-3-methylpentene (2) acid.

When treating Perrirubin with bases or acids or with substances that form iron complexes removes the iron from the molecule, forming a colorless powder called desferrirubin. If you have this "

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with 6-n. Hydrochloric acid hydrolyzes and the hydrolysis products
Catalytically hydrogenated (for the purpose of reducing any hydroxylamino groups that may be present), three ninhydrin-positive products are obtained, which on the basis of paper chromatography analysis (mobile phase phenol-water 8: 2 and butanol-glacial acetic acid-water 4: 1: 1 and staining ipit ninhydrin)
as well as ornithine in the analysis according to Moore and Stein,
Glycine and Serine can be identified. Glycine and serine
are present in a molecular ratio of 1: 2. Elsen-free
Perrichrom, with 6-n. Hydrochloric acid hydrolyzed and catalytic I

hydrogenated, gives not three, but only two ninhydrin- i

positive products, namely glycine and ornithine. Coprogen ^:

is the only ninhydrin-positive hydrolysis product ·.

Ornithine. '

In perrirubin the amino acids are serine · (2 · mol), j

Glycine (1 mol) and §-hydroxyornithine (3 mol) to a cyoli- j

see Hexapeptld connected. The hydroxyornithine is N (C) -acy- ^: lated; the hydroxamic acid residues formed in this way are iron

complex bound.

I perrirubin is assigned the following formula: j

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CH ₂ OH

R = HOCH ₂ -CH ₂ -C = CH- (trans)

The acid connected to the 3 ornithine residues has therefore j follow the structure of trans-5-hydroxy-3-methyl-pentene- (2) - i

acid ί

HOCh ₀ -CH ₀ -C = CH-COOH. j

. CH ί

The gross formula of pearl ruby is therefore Cj ,, HghO ₁₇ NqPe. During the catalytic hydrogenation of perrirubin, the trans-5-hydroxy-3-methyl-pentene (2) acid is hydrogenated without the remaining part of the molecules being attacked. The result is hexahydroferrubine, the acid component of which is 5-hydroxy-3-methyl-valeric acid. Hexahydroferri- »ruby is identical to the Hexahydroferrirhodin obtained by hydrogenation of Perrirhodin (cf. note no.

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P l4 42 220.9 - Case 5157 / 1-3 / E) · The IR absorption spectrum of the Compound in Nujol is shown in FIG. In the UV absorption spectrum Hexahydroferrirubin is the strongest one Absorption band of ferrirubin at 252 Γημ disappeared and the broad maximum at 450 πιμ shifted to 425-430 Γημ. 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. P 14 42 073-6 - Case 4972 / 1-4 / 5038). The Rf values des hex & hydroferrirubins in systems I and ΐί are 0.57 and 0.80 compared to 0.52 and 0.81 of ferrirubin; (yellow-orange spot in contrast to the reddish-brown 'spot of ferrirubin). If you remove from the hexahydro: ferrirubin the iron, for example by means of 8-hydroxyquinoline, cleaves in the obtained Desferri-hexahydro-

ferrirubin produces 5-hydroxy-3 ~ by mild acid hydrolysis methyl-valeric acid residues, acetylated (on nitrogen and oxygen) and selectively cleaves the O-acetyl groups from, e.g. by means of ammonia, one obtains desferrichrysin, which can be converted into ferrichrysin with iron (III) salt. The ferrichrysin thus obtained from ferrirubin shows in the paper chromatogram in systems I and II the same RF values like authentic ferrichrysin.

The elemental analysis of the iron-free ferrirubin found the following values: C = 51.12; *

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H = 7.29; N = 13.00; Acetyl = 0 #.

A major difference compared to the iron-free sideramines Ferrichrysin, Ferricrocin and Perrichrome consists in the rate of hydrolysis of the hydroxamic acid bonds. The latter three compounds lose their ability to bind complexes to form iron (III) ions completely after approx. 18 minutes when warming with hydrochloric acid in a boiling water bath. With iron-free ferrirubin the intensity of the iron chloride reaction only decreases noticeably after about 2 hours. A similarly high level of resistance Against acid was previously in the sideramine series only with biologically inactive ferrichrome A was observed. The other properties of perrirubin agree with those of Perrichrome A does not match. Perrichrome A is an acid with three free carboxyl groups (ß-methylglutaconic acid .reste), while ferrirubin has no titratable groups.

. In the NMR spectrum, recorded with a Varian spectrograph in DpO, desferrirubin exhibits the following signals on (see. Fig. 2):. -

1.86 ^ ppm (14 H); 2.38 ppm (4 H); 2.92 ppm (1H); 3.79 PPm (10H); 4.39 ppm (4 H); 6.03 PPm (2H).

Information on the chemical shifts in

δ values compared to tetramethylsilane = 0. The through integration obtained proton numbers mean pure ratio- *

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numbers, where the smallest signal was assumed to be 1. All signals form broad signal clusters without clear resolution * Auto pile at 3.79 and 6.03 ppm loading \ sit distinct double peaks, but are apparently not pure doublet.

With the addition of ferric chloride at neutral pH, the ferrirubin is recovered from desferrirubin. . ;

Derivatives of perrirubin and desferrirubin 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 W-hydroxyalkenoyl groups than the 5- Hydroxy-3-methyl-pentenoyl group, for example propionyl, butyryl, pentanoyl, pentenoyl, 5-hydroxypentenoyl. The first-mentioned derivatives are obtained when ferrirubin or hexahydroferrirubin is treated with acylating agents, for example with acid anhydrides or acid halides. like acetic anhydride or acetyl chloride, and! the ester is isolated and, if desired, the iron elimi- \ defined. The deacyl compound is obtained by deacylation \,

i;

the Desferrirubins or Hexahydrodesferrirubins in mild \ acidic medium, eg in the presence of dilute mineral acids. The cyclic hexapeptide obtained in this way can be aoyllerted in a known mannerj leave O-ayl groups formed in the process "

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- ίο -

split off with ammonia, for example in methanol. The acyl analogue of desferrirubin can be mixed with iron salts be converted into the corresponding iron complex.

Ferrirubin, desferrirubin and analogues are growth substances for various microorganisms] they can therefore used in the culture of such organisms.

These compounds are anti-anemic Properties and, accordingly, can be used as remedies. Also desferrirubin and its derivatives have valuable pharmacological properties. So prevent they cause 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 hemosiderosis. You can accordingly used in medicine.

The pharmaceutical preparations contain the compounds mentioned in a mixture with an organic one or inorganic pharmaceutical carrier suitable for enteral or parenteral use. the Preparations are made from the starting material according to the usual methods. Suitable carriers are substances which do not react with desferrirubin, such as GeIa-: tine, lactose, glucose, sodium chloride, starch, magnesium stearate, talc, vegetable oils, benzyl alcohol, gum, Polyalkylene glycols, cholesterol or other known medi-

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zjin porters.

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

Ferrirubin, desferrirubin and derivatives of these compounds are obtained if ferrirubin-forming compounds are used Strains of the Aspergillaceae family in an aqueous nutrient solution that is a source of carbon and nitrogen as well as containing inorganic salts, cultured under aerobic conditions, ferrirubin and / or from the culture filtrate Desferrirubin is isolated and, if desired, ferrirubin is converted into the iron-free compound or hydrogenation products or derivatives of these compounds. ; Since the formation of ferrirubin and / or des-

' ι

■ ι

ferrirubins favored by a nutrient medium poor in iron is, it is advisable to breed under iron deficiency, preferably with an iron content of at most 10 "'mol / Liters.

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 such as ornithine, peptides, Proteins and their breakdown products such as peptone or tryptone5

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Meat extracts, water-soluble fractions of cereal grains, such as maize or wheat, distillation residues from alcohol production, yeast, seeds, in particular the rapeseed and soya plants, the cotton plant, ammonium salts, nitrates. In terms of 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.

The cultivation takes place aerobically, for example, in resting surface culture, or preferably submerged while shaking or stirring with air or oxygen in shake bottles or the well-known fermenters. As a temperature a temperature between 18 and 40 ° C, preferably 27 ° C, is suitable the nutrient solution generally after 12-16 days. . The ferrirubin activity can be microbiological using the modified Bonifas test (see zahner et al.,

Areh.Microbiol. ^, 325 ff. [1960]). As ί
A ferrimycin solution of 0.1 mg / ml is used for the test solution

Salary, as a test strain Staphylococcus aureus Rosenbach.

The ferrirubin concentration of the cooling solution can also be determined optically. Be for this purpose 5 ml culture liquid with 1.5 g sodium chloride, 1 ml 0.1 # ferric sulfate solution and 5 ml benzyl alcohol for 5 minutes shaken, centrifuged, the organic phase filtered '

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and the absorbance of the organic phase is determined at 440 ιτιμ. An extract prepared in the same way from uninoculated nutrient solution is used as a comparison sample.

After 12-16 days, the cultures reach an activity equivalent to 50-200 mg of pure ferrirubin.

■ At the end of the cultivation one can per liter Add 1 g of ferric chloride to the nutrient solution, taking the existing Desferrirubin is converted into ferrirubin. One separates the ttjfcei "from the culture filtrate, preferably in the presence a filter aid, e.g. Hyflo-Supercel, after which the Most of the ferrirubin 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 ferrirubin from the .XuItuFIltrat proceed and, for example, use one of the working methods specified below or a combination 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,

8 0.9 80 6/07

- i4 -

dilute acetic acid-methanol or water-methanol-ice- vinegar-butanol mixtures. This has proven to be particularly advantageous for the elution of a frankonite or norite adsorbate Proven mixture of water (4 parts by volume) and pyridine (1 part by volume) »

2) Perrirubin can also be removed from an aqueous solution by means of organic solvents. For these extraction processes, higher organic alcohols, such as benzyl alcohol or isopropyl alcohol, have proven particularly useful. In two cases, an inorganic salt, for example ammonium sulfate or sodium chloride, is added to the aqueous phase. From the organic \. Ferrirubin can be obtained in enriched form in extracts either by evaporating off the solvent or by precipitation using a suitable organic solvent, for example ether, petroleum ether or ethyl acetate.

5) Another method of enriching Perrirubin is that it is placed 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 enriching ferrirubin is chromatography, such as adsorption chromatography on various materials, e.g. on Norit, aluminum oxide, magnesium silicates, silica gel, CaI - **

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• Calcium sulfate, as well as partition chromatography with cellulose, '■ starch, silica gel, celite and the like. As carrier substances.

5) Furthermore, the ferrirubin can by countercurrent ■

distribution according to Craig between two immiscible solvent phases be enriched. The following solvent system has proven to be particularly effective: n-butanol

ί Benzyl alcohol-Ο, ΟΟΙ-η. Hydrochloric acid-aqueous, saturated at 19 ^:

te sodium chloride solution (9: 9: 15: 5). !

In addition to ferrirubin, desferrirubin is formed by the named strains and can therefore also be derived directly from the KuIturfiltrat be isolated. Advantageously removed: iron present in the culture filtrate during isolation, for example by adding iron complex-forming substances, e.g., 8-oxyquinoline. ;

The iron complex-forming substances can be added to the culture broth immediately after the fermentation has ended. Advantageously, the addition is carried out at a later stage of the work-up to any entrained by agents who used iron (III) also. Ions to "" remove.. I

The isolation of desferrirubin from the *

Culture solution is carried out according to the methods mentioned for the isolation of ferrirubin.

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

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Example 1 ;

The strain Penicillium variabile M 2733 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 ^, 7 H ₃ O) and 0 * 5 g calcium chloride. The nutrient solution is sterilized at 120 ° for 20 minutes. It is inoculated with a spore suspension of rice cultures in dist. Water + $ 5 tween

The content of ferrirubin in the culture medium is determined as follows:

10 ml culture filtrate + 3 g sodium chloride + 10 μl benzyl alcohol are shaken well and centrifuged. The organic phase is filtered, ^{shaken with 3-fold} volume of ether and pH 8 buffer and centrifuged again. In the aqueous extract, the extinction at 440 ηιμ is determined and the mg of crude perrirubin read off using a calibration curve.

After 12-16 days, 1 g of ferric chloride per liter is added to the cultures and filtered with the addition of 2% 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 with

i. ■.

Sodium sulfate dried, filtered and with 3 volumes "' ■

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Aether displaced. The benzyl alcohol-ether mixture extracted until discoloration with distilled water. The aqueous extracts are combined, the remains of benzyl alcohol removed with ether and lyophilized to give a red powder.

10.4 g of crude product are a 15 ^ -stufigen ■ subjected to craig distribution. A mixture of benzyl alcohol, n-butyl alcohol, sat. watery Sodium chloride solution and 0.001-n. aqueous hydrochloric acid in a volume ratio of 18: 18: 10: 30.

The red-brown dye forms a single distribution maximum in step 128. This corresponds to an approximate distribution coefficient of K = 5.0.

The fractions II6-14o are combined. By adding 2 volumes of ether, ferrirubin is forced into the aqueous phase. This is separated and the or- GaniSQhe SchiGht shaken out three more times with a little water. The combined aqueous solutions are made with sodium chloride about half saturated and shaken out several times with phenol-chloroform (1 kg of phenol to 1 liter of chloroform), whereby the dye passes into the organic phase. The extracts are clarified on a Celite column with the double volume of ether and shaken out four times with a little Walser, the brownish-red dye in the aqueous phase goes. The aqueous solutions are washed "

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several times with ether to completely remove the phenol and evaporate it in vacuo. The red-brown amorphous residue (1.245 g) is further purified by reprecipitation from methanol-ether. The pure connection is very hygroscopic. ■

The elemental analysis found: C = $ 49.01; H = $ 6.57; N = $ 12.70; Pe = $ 5.25; 0 = $ 26.47 (calc.); Acetyl = $ 0.

Absorption spectrum in ethanol: A _Q "

Max

4cm = ² '58bÄ _max = 252 "μ. (log ^

: A _Q "= 216 πιμ; Max

450 up (log E ^ _1n = 1.52). ..-; - \

The IR spectrum in potassium bromide shows i.a.

Bands at 842, 945, 990 (shoulder), 1040, 1066, 1125 '

(Shoulder), 1195 (shoulder), 1239, 1282 (shoulder), 134l \

(Shoulder), 1366, 1460, 1533, 1647, 2938, 3085 (shoulder),!

-1 ·

3420 cm, see Pig. I.

Perrirubin was found in the paper chromatogram with ^;

! compared to other sideramines, see table. 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 methanol ical solution of the comparison subsections (starting points 1, 3, 5 and 7) and the ferrirubin (starting points 2, 4, "6 and 8)

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applied. 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.

Table 1

Sideramine Rf (System I) Rf (System II). Ferrichrome 0.28 0.30 Ferricrocin 0.29 0.28 Ferrichrysine 0.26 0.34 Coprogen 0.43 0.55 Ferrirubin 0.42 0.73

Example 2:

The strain Penicillium variabile M 2733 is 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 3 times with 1/20 volume of chloroform-phenol (1 part by volume: 1 part by weight), · separates the organic phase and filtered through Celite. Then 3 volumes are added to the organic solution

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Ether and extract it several times with distilled water until it becomes discolored. The aqueous extracts are combined, washed with ether to remove phenol residues, freed from ether in vacuo and then lyophilized. The crude product obtained is as in Example 1 cleaned. "'

In the same way as described in Example 1, perrirubin is obtained if the strain Spicaria sp. M 4622 or the strain Paecilomyees varioti M 4646 (at 33) and the culture medium is processed as described.

Example 5;

435 mg ferric Rubin are dissolved in 30 ^m l of water and 1.0 g 8-hydroxyquinoline dissolved in ca, I5 ml of methanol was added '. After 24 hours Stirring at room temperature, the black precipitate (hydroxyquinoline iron complex) is filtered off and the pale yellowish piltrate is extracted three times with jühloroform in order to remove the excess reagent. The aqueous solution is then evaporated, and the iron-free perrirubin is obtained as a pale yellow amorphous residue. Yield 39 ° ^m S

A dissolved in DPO · sample of the compound shows the NMR spectrum (taken on a Varian-spectrograph ', model \ 60) the following signals:

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1.86 ppm (14 H); 2.38 ppm (4 H); 2.92 ppm (1H);

3 * 79 Ρ? «(10 H); 4.39 ppm (4H); 6.03 PPm (2H). Information on the chemical shifts in 4-values compared to tetraraethylsilane = 0. The proton numbers obtained by integration represent pure ratios, the smallest signal being assumed to be 1 *. All signals ■■ form broad signal clusters with no clear resolution. The signal clusters at 3> 79 and 6.03 ppm have clear double maxima, but are evidently not pure doublets.

The following fourths are found in the elemental analysis: C 51.12; H 7.29; N 13.00; Acetyl 0 #.

Example 4: j

150 mg of iron-free ferrirubin are mixed with 4 ml of j. 6-n. Hydrochloric acid melted in a glass tube and heated to 110 for 24 hours. The evaporation residue of the hydrolyzate is dissolved in a little water and diluted with Peinsprit; approx. 30 ό1 and hydrogenated in the presence of 50 mg of prehydrogenated platinum oxide overnight. A sample of the evaporation return! The status is checked by two-dimensional paper chromatography (phenol-water 8: 2 and butanol-glacial acetic acid-water; 4: 1: Ij ninhydrin) and by analysis according to Moore and Stein for amino acids. Both methods show the presence '

called ornithine, serine and glycine. Serine and glycine are:

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present in a molecular ratio of 2: 1.

Example 5 *

28 rag iron-free perrirubin, dissolved in 1 ml · methanol, are mixed with 25 mg iron chloride in 2 ml methanol mixed. Immediately a deep purple-black appears Color, which after buffering the solution with a bit of crystalline. Sodium acetate turns reddish brown. After 1 hour Standing is evaporated to dryness in vacuo, the residue in 5 | ml of water dissolved, about half saturated with sodium chloride and extracted the perrirubin with benzyl alcohol. The extracts are sat. Washed sodium chloride solution and dried with sodium sulfate. The solution is with Aether diluted and the sideramine extracted again with water. Evaporation of the ether-washed extracts gives perrirubin, approx. 25 mg of orange-red powder. By Paper chromatography in solvent systems I and II (see Tab. 1) it is shown that the won in this way Iron complex is identical to the "directly isolated.

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Example 6 ::

To a slurry of 100 mg 10 percent. j

I palladium carbon in 5 ml of methanol is given a solution of;

149 mg of ferrirubin in 5 ml of methanol and stir the mixture

at room temperature in a hydrogen atmosphere. The ; Hydrogen uptake is very rapid at first. After approx. 1 hour approx. 3 mol of hydrogen have been absorbed and the hydrogenation comes to a standstill. The catalyst is now filtered off. A solution of 100 mg of iron (III) chloride in methanol is added to the orange-brown piltrate to remove any reduced 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.Wasser and the solution is repeated several times with phenol-chloroform (Ig: 1 ml) shaken out. The phenol-chloroform extract, filtered through Celite, is mixed with 2 vol. Ether diluted, three times with dist. Shaken out water and the aqueous solutions washed with ether in a vacuum steams. Hexahydroferrirubin is obtained as an orange-yellow amorphous powder; Yield: 132 mg. UV absorption maxima , in torment fuel: high final absorption at 220 ιτιμ (log £ over 4); Maximum at 425-430 ηιμ (log £ 3.5). IR spectrum in Nujöl, see Pig. 3. In paper chromatography they are Rf values in systems I and II:

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I II

Perrirubin 0.52 0.81 Hexahydroferrirubin 0.57. 0.80

Perrirubin gives a reddish-brown, the hydrogenation «·.

product a yellow-orange stain.

Example 7 '· '

Ϊ g of raw hexahydroferrirubin is in 40 ml Dissolved water and mixed with 1.5 g of 8-hydroxyquinoline dissolved in 15 ml of methanol. After 24 hours Stir at 20 ° the precipitated iron (III) oxinate is filtered off. The Piltrat is shaken out 5 times with chloroform and then evaporated to dryness in vacuo. 900 mg are obtained Desferri-hexahydroferrirubin "of the formula II

₃
CH-CH ₂ -CH ₂ OH _CH ^

^ ² CO-NH-CH ₂ - CO CH-CH ₂ -CH ₂ OH

.ι,, / ■ ^a Λ te

HO-N- (CH ₀ ), - CH NH

NH 'CH- (CHg) ₃ -N-OH

CO · CO

HOCH ₂ -CH NH II

NH CH-CH ₂ OH

CO-CH-NH-L-CO

(CH ₂ J ₃ -N-Co-CH ₂ -CH-CH ₂ -CH ₂ OH OH CH ₃

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as an almost amorphous colorless powder which ^{shows color reaction with perrichloride ^ '' vlf} ^ S§fMaÄi «^ ötioi) όΐή © viölest ©» Im

LMWH spectrum, which in Dgl the following signals:

(ppm) breakdown 0.93 d (J - 5 cps)

1.2-2.2 b

2.4
3.4 - 4,:

is recorded, one finds

Number of H assignment

■ 9 methyl groups of the 5-hydroxy-3-methyl-pentanoic acid residues.

β- and 7-CH _{2 of} the N-hydroxy-ornithine residues; 7-CH _{2 of} the 5-hydroxy-3-methyl-pentanoic acid residues; ß-CH of the 5-hydroxy-3-methylpentanoic acid residues.

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

IB £ -CH the N-Hydroxy-

orni ^ remnants; CH _{2 of} the glycine residue; CH _{2 of} the serine residues; £ -CH _{2 of} the 5-hydroxy-3-methylpentanoic acid residues.

a-CH the N-hydroxy-ornithine and serine residues.

Example 8;

t i 900 mg Besferri-hexahydroferrirubin are with 30 »1 day Hydrochloric acid heated to approx. 95 ° in a water bath. In At intervals of approx. 5 minutes, a drop of the solution is added to the spot plate with 2 drops of 2 percent. methanolic

chloride solution added. Initially one occurs

ORIGINAL INSPECTED, 809806/0764 ~~~ "~

strong violet color reaction. After 50 minutes, the color reaction is only very pale. The hydrolysis is stopped and the solution, which contains the cyclo- (L ~ & -hydroxyornithyl-glycyl-L- £ -hydroxyornithyl-L-seryl-L - & - hydroxyornithyl-L-seryl), evaporated in vacuo. The residue is acetylated with 10 ml each of acetic anhydride and pyridine for 5 hours at room temperature. The reaction mixture is again evaporated in vacuo. For partial ammonolysis of the Q-acetyl bonds formed, the reaction product is left to stand in 60 ml of cold-saturated methanolic ammonia for K hours. The evaporation residue of this solution, which contains the desferrichrysin and by-products, is then reacted in methanolic solution with 1 g of iron (III) chloride, the deep violet solution with krlst. Sodium acetate buffered until the color changes to orange-brown, and the solution evaporated after standing for 4 hours. The residue is dissolved in water, about 10 $ sodium chloride is added and shaken several times. Phenol-chloroform (Ig: 1 ml) and filtered the initially cloudy phenol-chloroform solution through a column of 10 g of Celite. The clear filtrate is diluted with ether and extracted three times with dist. Water. The aqueous extracts, washed well with ether, are evaporated in vacuo and the crude product is purified by a Craig distribution over 50 stages. The solvent system used is a '

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• Mixture of 9 parts of n-butanol, 9 parts of benzyl alcohol, 15 ! Divide 0.001-n. Hydrochloric acid and 5 parts sat. water Sodium chloride solution. The main component of the mixture is most enriched in stage 19, which is a distribution coefficient of 0.62 (authentic Perrichrysin: k «0.63).

Fractions 12 to 25 are worked up in the usual way with phenol-chloroform. 202 mg are obtained Perrichrysin, which is further purified by crystallization from ethyl alcohol. The red-orange crystals decompose like "authentic perrichrysin" slowly at approx. 250-260 ° without melting.

¹ The product behaves in paper chromatography in systems I and II exactly like authentic perrichrysin (see Table 2), and the IR absorption spectrum agrees with that of authentic perrichrysin

completely agree. Paper chromatography:

Table 2

System I.
Silica gel
plate Filter paper System II
filter
paper Perrichrysin, auth.
Conversion product Rf 0.129
Rf 0.129 0.39
0.39 0.32
0.32

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In the antagonism test against ferrimycin A, the product behaves like natural perrichrysin.

809806/0764

Claims (3)

- 29 claims: 1. The use of Penicillium variabile M 2733 »Spicaria sp. M 4622 or Paecilomyces varioti M 4646 for her production of ferrirubin and desferrirubin by means of the usual cultivation conditions. 2. Ferrirubin ^ the Fe (III) complex of cyclo-feeryl) _p glycyl- [N ^p -hydroxy-N ^ - (trans-5-hydroxy-3-methyl-pentenyl) -ornithylJ ^ J. 3. Desferrirubin of the formula cyclo- £ (seryl) p-glycyl- [N ^ -hydroxy-N ^ - (trans-5-hydroxy-3-methyl-pentenyl) -ornithyl] ₃ ). New documents (Art. 7 ξ Τ Abe. 2 No. I sentence 3 of the ÄmJerungsg · «· of 4.9. J9« 7) 809806/0 7 64