DD230019A1 - PROCESS FOR THE PREPARATION OF CHANOCLAVIN-I - Google Patents

Abstract

It is the preparation of the alkaloid Chanoclavin-I with the help of the strain Claviceps purpurea (Fr.) Tul. IBP 180, ZIMET PA 138, its mutants or variants. The strain forms a total alkaloid mixture of 500-600 μg per ml of culture solution of which at least 90% of the alkaloids can be shaken out with organic solvents. In addition to the chanoclavin-I, which makes up about 80% of the alkaloid mixture, chanoclavin-1-aldehyde is formed (about 20% of the total alkaloid mixture). Other alkaloids do not occur. The strain is grown in aerobic fermentation in a liquid nutrient medium containing an assimilable carbon source, an assimilable nitrogen source and mineral salts in an emersed and / or submerged culture at a temperature of 20-30 degrees C and a pH of 4.0-6 , 2 for a period of 7-35 days. More than 95% of the alkaloids formed are released into the nutrient medium from which the alkaloids are isolated.

Description

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Process for the preparation of chanoclavin-I

The invention relates to a process for the preparation of chanoclavin-I.

Field of application of the invention

The Clavinalkaloide are precursors in the biosynthesis of the ergot alkaloids with peptide side chain. They are formed by different claviceps strains. Some members of the clavinal alkaloids have prolactin inhibitor activity. A use as anti-Parkinson agents is also suitable. Clavinalkaloids are valuable starting materials for partial syntheses. Starting from chanoclavin-I, modified secoergolines and derivatives of tetracyclic ergoline can be obtained. This opens up new possibilities for the pharmacological use of ergot alkaloids.

Characteristic of the known technical solutions

Chanoclavin-I has hitherto been accessible only in small quantities. In the literature three methods of recovery using microorganisms have been described.

The strain Claviceps paspali Li 342 / SE 60 forms in saprophytic culture 400 , ag total alkaloid per ml of culture solution. 40 % of this amount is chanoclavin-I (Pharmacie 20, 523-524 (1965)). In this method, predominantly lysergic acid derivatives are formed in addition to chanoclavin-I, so that the work-up is difficult.

ZC.GEZ.1973 * S3J_52-5

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Italian authors report a strain with the designation PI 82. It makes 150 - 200 / ug chanoclavin / ml. They describe the formation of "giant colonies" whose different sectors have great variability in the ability to form alkaloids. One type of sector may even lose its ability to form alkaloids completely (Fermentation Advances 1969 , 611-628). In this method, it is not clear whether chanoclavin-I or a mixture of chanoclavin isomers is formed.

In a third work, the formation of 158 mg chanoclavin-1/1 heme is described. The authors cultivated the fungus. Balänsia strangulans 38 days in an emersed culture (J. Gen. Microbiol., VY3, 119-126 (1979)). This and the aforementioned method have the disadvantage of a long fermentation duration, since alkaloid is formed only in emerser cultivation.

Object of the invention

It is the object of the invention to prepare the alkaloid chanoclavin-I according to a favorable process in order to obtain its pharmaceutic. table use, optionally via partial synthetic transformations.

Explanation of the essence of the invention

The invention has for its object to develop a process for the preparation of chanoclavin-I, according to which the production of the alkaloid can also be done in submerged! Cultivation and in which no interfering with the workup secondary alkaloids are formed.

According to the invention for the production of chanoclavin-I the strain Claviceps purpurea (Fr.) TuI. IBP 180, its mutants or variants used. The cultivation for the characterization of Claviceps purpurea (Pr ..) TuI. IBP 180 took place on or in

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the media compiled in Table 1. The new strain has the following macroscopic and microscopic properties:

Macroscopic appearance

Age of the cultures: 4 weeks

Agar IL 126: Single colonies: '0 2-3 cm, sharply attached, crater-shaped, height 0.5-1.0 cm.

Color: gray with a light, light coating. No fluorescence in UV light

Agar NL 829: single colonies from 0 1.5 - 2.0 cm, spicy

attached, crater-shaped rugged, raised.

Height: about 0.3 - 1.2 cm Color: light reddish-purple No fluorescence in UV light

Microscopic appearance

Agar NL 126: Age: 4 weeks

Thin hyphae, septate, central or wall-shaped plasma. The large round to oval cells contain z. T. vacuoles.

Agar UL 829: Age: 4 weeks

There is a mixture of filamentous hyphae and nodular thickened hyphae with central plasma. Bubble-distended, almost isodiametric cells, which are mostly vacuolated, are also observed.

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Preculture NL 720: age 7 days

Short, 4 - cell hyphae, nodular, septate, central plasma. The hyphae are partially swollen. Occasionally, hyphae appear with wall-shaped plasma.

Hyphae 0: 7.5 - 12.5 / um; 0 of the bubonic swelling: 12.5 - 15 / um.

Main culture EL 720: Age 3 days.

Short hyphae from 4 - TO segments, nodose-thickened, plasma-rich. 0 7.5 - 13 / um; 0 of swollen swelling: 15 - 22.5 / um

 Age: 7 days

Short hyphae from 3-10 segments, plesary, nodular, many vesicular swollen cells. 0 of hyphae 10-13 / um; 0 of the blistering swelling 15-13 / Um. Age: 14 days

Short hyphae from 3-8 segments. The cells are knot-like thickened, pläsmareich and isolated with vacuole, very many large, round to oval cells. Cell size: 15-20 / um. The cell walls are heavily thickened.

The cultivation takes place at 24 - 1 ⁰ C. The shake cultures were incubated on Rundschütteltischen at 240 rpm.

The aerobic fermentation of the fungus for production is expediently carried out in liquid nutrient media in an emersed and / or submerged culture. The nutrient media contain an assimilable C source, such as sucrose, glucose,

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Mannitol, sorbitol, glycerin, citric or succinic acid, an assimilable IT source such as ammonia, asparagine, peptone, casein hydrolyzate or an ammonium salt, as well as mineral salts. The fermentation can take place, for example, in Fernbach, Erlenmeyer or shake flasks or in the permentor.

The cultivation is generally one to three stages. It is advantageous if the preculture is 4-14 days old and the inoculum ratio preculture: main culture is 1: 5 to 1 :. The main culture is 7-20 days. The pH can be between 4.0 and 6.2, the temperature between 20 and 30 ⁰ G. It is particularly expedient to carry out a fermentation at 22-25 ^° C. and pH 5.2 in the usual way.

22-25 ^° C and pH 5.2-5.9. The stammering attitude can be up

The new strain Clavic-eps purpurea (Fr.) TuI. IBP 180 is deposited with the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR in Jena under the name ZIl ₁ IET PA 138. It forms a total alkaloid mixture of 500-600 , ag / ton of culture solution of which at least 90 % of the alkaloids can be shaken out in organic solvents and consist of about 80 % of chanoclavin-I and about 20 % of chanoclavin-1-aldehyde. Chanoclavin-I-aldehyde was previously only accessible synthetically, it is here for the first time as Uaturstoff isolated. The alkaloids are obtained from the nutrient media in a known manner.

Optionally, to increase the yield of chanoclavin-I, the chanoclavin-1-aldehyde is reduced in a simple manner known per se to chanoclavin-I. For this z ' are suitable . For example, complex hydrides such as NaBH, (see Example 4).

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The new strain is distinguished by the fact that only a short fermentation period is required, that it releases more than 95 % of the alkaloids formed in the surrounding nutrient medium and that no costly separation of Nebenalkaloiden · is required.

Execution ^ examples

The following examples are intended to illustrate the invention. The nutrient media used are shown in Table 1.

Example 1 · .

Starting from canned food of the tribe Claviceps purpurea (Pr.) TuI. IB? 180 seed cultures were seeded into 100 ml UL 720 medium in 500 ml shake flasks. The flasks were 7 days at 24 - i ⁰ C incubated at 240 rpm. 10 % of the cultures obtained were used to inoculate in each case a 500 ml shake flask with 100 ml medium HL 720. After incubation for 14 days under the conditions mentioned, the main cultures cultivated contained between 450 and 550 μg total alkaloid per ml of culture filtrate. 75 - 80 % of the alkaloid was chanoclavin-I. The remainder was identified as chanoclavin-1-aldehyde.

Example 2

Starting from the precultures mentioned in Example 1, main cultures were inoculated with medium NL 833. Pure 100 ml of medium used 10 ml preculture suspension. After incubation at 24 Htägiger - 1 ⁰ C and 240 rpm were temperature 500 - 600, ug formed alkaloid -per ml. The spectrum corresponded to that mentioned in Example 1.

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Example 3

To identify or isolate the alkaloids, appropriate amounts of the culture solution were made alkaline and extracted three times with chloroform. When working up the total suspension or the mycelium was extracted with? / Acid (2 %) (Ultra-Turrax), degreased and the alkaloid after alkalization also taken up in chloroform. After concentration in vacuo, the residue was taken up in chloroform and analyzed by preparative layer chromatography. As eluent systems were used:

a) CHCl ₃ / methanol = 8: 2

b) Methanol (only for the separation and isolation of the aldehyde)

The separating material used was silica gel P ^ ocja "Merck" in a layer thickness of 1 mm.

The isolated chanoclavin-I possessed after recrystallization from acetone a melting point of 215 ⁰ C (Boetius hot stage), The specific rotation (measured in pyridine at c = 1, 20 ⁰ C, D-line of sodium) was -238 °. Further identification was carried out by means of IR, NMR and mass spectra. The comparative chromatographic analysis (TLC, silica gel FF ^, ^, "Merck", 0.7 mm layer thickness) showed agreement with authentic comparison material. The following Rp values were determined:. ,

1. CHCl-,: tert. Butanol = 3: 1 (in 15 % ammonia atmosphere): 0.39

2. Methanol: 0.12

3. CHCl ₃ : methanol = 8 :. 2: 0.08

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in the mass spectrum of chanoclavin-I were u _o a. observe the following peaks:

m / e 256, 1586 (calc. 256, 1576) C ₁₆ H ₂₀ N ₂ O, M ⁺ ; 237; 183.0935 (calc. 183.0922) C ₁₂ H ₁₁ N ₂ ; 168; 167; 155; 154th

Among other things, the nuclear resonance spectrum contained the signals (/ "values in ppm, solvent CD, OD, HIDs as standard):

1.85. Vinyl CH ₃ singlet 2.42 N-methyl singlet 3.85-3.95 H to C ₁₀ multiplet 4.13 -CHgO Group singlet 5.3-5.4 Vinyl hydrogen doublet

(For numbering of the individual C atoms, see Figure 1).

The amorphous chanoclavin-1-aldehyde was purified twice by preparative TLC under the conditions already mentioned. The identification was made in comparison to synthetic material (prepared according to Naidoo et al., Chemical Communications 1970 , 471-2) by means of IR, NMR and mass spectroscopy. As R ^ values were determined

1. CHCl ₃ : methanol = 8: 2: 0.44

2. methanol ·: 0.27

With van Urk's reagent, the aldehyde gives a turquoise color after a few minutes.

- 1 In the IR spectrum, the aldehyde band appears at 1685 cm (solvent: CHCl ·,).

The NMR spectrum of the chanoclavine-1-aldehyde showed inter alia the following signals (cT values in ppm, solvent

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CD, ΟΏ, HMDS as standard):

1.95 Vinyl CH ₃ singlet 2.52 N-Methyl Singlet. 4.33 H to C ₁₀ multiplet 8.0 NH of the indole ring singlet 9.46 H of the aldehyde , singlet

For numbering of the individual C atoms, see Figure 1).

The mass spectrum of chanoclavin-1-aldehyde showed u. a. the following peaks:

m / e 254.1414 (calc. 254.1419) M ⁺ , C ₁₆ H ₁₈ N ₂ O; 237.1407 (calc. 237.1392), C ₁₆ H ₁₇ U ₃ ; 223.1010 (223.0907), C ₁₅ H ₁₃ NO; 194,1017 (calculates 194,070) ^ C ₁₄ H ₁₂ N; 168; .155; 154th

After deuteration with DpO, 2 hydrogen atoms were exchanged. The molecular peak disappeared, but at m / e 256 the new M peak appeared. It can be seen that 2 hydrogen atoms are interchangeable.

Example 4

4OO -500 mg of crude halide were dissolved in 50 ml of aqueous methanol. About 200 mg of sodium borohydride were added to this mixture and the mixture was heated to 60 ^° C. for 30 minutes. At the end of this reaction, no more chanoclavine-1-aldehyde was detectable using the chromatographic methods mentioned in Example 3; it was only the stain of chanoclavin-I to see.

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Table 1 :

Compilation; the nutrient solutions used (data in g / l)

NL 720 15.0 - 1.0 - ML 833 20.0 - • 1.0 - 0.3 - NL 126 - 5.0 - 5.0 - WL 829 10.0 - 0.25 - sucrose 200.0 - 0.25 • 0.3 ' 300.0 - 0.25 0,010 1000.0 - - 1 00,0 0.5 0,020 1000.0 wort - 0,010 - 0.0308 - 150 ml - - 1.0 0.02 30.0 ammonium citrate 0, Ό308 1.0 - - 0.25 asparagine 0.1 5.4 1000.0 5.4 yeast extract 1000.0 NaOH 30.0 NaOH CaOTO ₃ ) ₂ - uniform at KH ₂ PO ₄ 6.0 (NH ₄ ) ₂ HPO ₄ 5.8-6.0 NaOH MgSO _4. 7H ₂ O NaOH 30 min. FeSO ₄ . 7H ₂ O each ZnSO .. 7 HpO 110 ⁰ C. KGL Distilled water ad Agar Agar pH before the Sterilize prf-sizing with autoclaving

Claims (4)

217 invention claim 1. A process for the preparation of chanoclavin-I, characterized in that the strain Claviceps purpurea (Pr.) TuI. IBP 180, ZIMET PA 138, its mutants or variants are used, wherein the yield of Chanoclavin-I is optionally increased by subsequent reduction of the resulting Chanoclavin-I aldehyde. 2. Method according to item 1, characterized in that the strain Claviceps purpurea (Pr.) TuI. 180, ΖΙΜΞΤ PA 138, in aerobic fermentation in a liquid culture medium containing an assimilable carbon source, an assimilable nitrogen source and mineral salts in an emersed and / or submerged culture at a temperature of 20 - 30 ⁰ C and a pH of 4, 0 - 5.2 is cultivated for a period of 7-35 days and worked up from the nutrient medium in a known per se to chanoclavin-1-aldehyde and / or chanoclavin-I. 3. Method according to points 1 and 2, characterized in that the perforation
pH 5.2 - 5.9 is performed. records that the perforation at 22 - 25 ⁰ C and 4. The method according to points 1-3, characterized in that the reduction of the chanoclavin-I aldehyde is carried out in the isolated crude caloid with complex hydrides. Hienu1 eii formulas L 'J. ui -'-