DD200571A1 - METHOD FOR THE PRODUCTION OF ERGOPEPTINES WITH MODIFIED PROLINTEIL - Google Patents

Abstract

The invention is important for medicine and for the pharmaceutical industry. The aim is to achieve an improvement of known therapeutic options or an expansion to new indications by providing new stable ergopeptins. The invention is therefore based on the object to produce ergopeptins by directed fermentation, in which the proline portion of the peptide fragment is replaced by an analog of these amino acids. According to the invention, an amino acid analogous to proline is added to a submerged ergopeptin-forming strain in the growth phase of the mycelium and the strain is cultured in the presence of this amino acid; the modified ergopeptins are isolated from the nutrient medium. For example, ergosin-forming strains of Claviceps purpurea are used. For the preparation of 1'sz-methyl-5'Alpha-isobutyl-9'thiaergopeptin (Thiaergosin) is used as the proline analog amino acid 1,3-thiazolidine-4-carboxylic acid.

Description

Process for the preparation of ergopeptins with modified proline part

The invention relates to a process for the preparation of ergopeptins with modified proline moiety.

Area of application of the invention

Of the more than 50 known natural ergot alkaloids, the representatives of the peptide alkaloids are of great importance in internal medicine and gynecology, such as ergotamine and the representatives of the brgotoxin group (ergocornine, ergocristine, ergokryptin). Ergosine is very close to the ergotamine and differs from the latter by replacing the amino acid phenylalanine with the amino acid leucine in the peptide side chain. Ergosin also has an uterotonic and vasoconstrictive effect. The ergosine and its 9,10-dihydro derivatives and other partial synthetic derivatives can be used as agents for migraine, arterial hypertension and peripheral vascular diseases. Thiazolidine-4-ocarboxylic acid is also a potent compound used successfully in the fight against cancer.

The invention is of interest to the medical and pharmaceutical industries. A combination of the lysergic acid derivatives with the thiazolidine-4-ocarbonsäure new therapeutic and indication options are to be developed.

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Characteristic of the known technical solutions

In the peptide part of the ergopeptins modified alkaloids are known and accessible in principle in two ways. Once, the peptide side chain can be synthesized completely synthetically (J. Rutsohmann and PA Stadler, Handbook of Experimental Pharmacology, Vol. 49, p. 29 (1978)). On the other hand, it is possible to influence the composition of the alkaloid mixture qualitatively by controlled fermentation. The chemical synthesis of homologues of ergotamine (replacement of the proline with pipecolic acid) is tedious and leads to unstable preparations. A controlled fermentation by addition of cyclolspezifischen amino acids and amino acid analogues, parasitic and submersed, has been repeatedly described. Claviceps purpurea strains which form ergotamine or ergotoxins or amino acid auxotrophic mutants obtained therefrom (Federal Republic of Germany Patent Laid-Open 28 16 773, Int Cl ² : C 07 D 257/08, H. Kobel and JJ · Sanglier in US Pat "Antibiotic and other secondary metabolites", 5th FEMS Symposium, Academic Press p 233 (1978), E. Beacco et al., Experientia 3 £, 1291 (1978); N, Crespi-Perellino, Ebcperientia 32, 217 (1978); 1981)). So far, during the fermentation, such amino acids or analogues have been added to the culture medium which can replace the amino acid adjacent to the lysergic acid and the second amino acid in the peptide portion of the peptide alkaloids consisting of three amino acids.

Ergopeptins with modified proline moiety produced by controlled fermentation are not known to date.

Aim of the invention;

It is the object of the invention to achieve an improvement of known therapeutic options or the development of new indications by providing new stable ergopeptides with modified peptide part.

3 depiction of the essence of the invention

231978

The invention has for its object to produce ergopeptins by directed fermentation, in which the proline part of the peptide fragment is replaced by an analog of this amino acid.

According to the invention, a submerged ergopeptin-forming strain is used for the production of ergopeptins with a modified proline moiety, to which an amino acid analogous to proline is added in the growth phase of the mycelium, and the strain is cultured in the presence of this amino acid. For example, ergosin-forming strains are used, in particular the strain Claviceps purpurea (Fr.) TuI. IBP 179 »its mutants or variants · This strain is deposited with the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR in Jena under the name IMET PA 136 as well as with the German Collection of Microorganisms in Göttingen under the number DSM 1871. Its use for the production of ergosine / ergosinin is known (see, for example, European Patent Application 0 022 973, IntCe ³ : C 12 P 17/18). The strain is able to saprophytically produce ergosine, which is almost free of minor alkaloids, and is characterized by stability, good fermentation properties and high alkaloid production, whereby 95 % of the alkaloids formed are excreted in the surrounding nutrient solution. Another example of a strain which can be used in the method according to the invention is Claviceps purpurea (Fr.) TuI. MUT 168. He is deposited with the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR in Jena under the name IMET PA 134. He also produces ergosin / ergosinin in the same culture as his mutants or variants (see GDR Patent 129,801, Int Cl ² : C 12 D 13/02).

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For the production of the ergosin analog alkaloid with modified proline part, the aerobic fermentation of the felt is expediently carried out in liquid nutrient medium in submerged culture. The nutrient media contain an assimilable C source, such. Sucrose, glucose, mannitol, sorbitol, glycerol, citric or succinic acid, an assimilable N source such as ammonia, asparagine, peptone, casein hydrolyzate or an ammonium salt, as well as mineral salts.

The cultivation takes place, for example, in a shake flask or in a fermenter, it is carried out in one to three stages. It is advantageous if the preculture is 4 - 14 days old and the inoculum ratio is pre-: main culture 1: 5 to 1: 20. The duration of the main culture is 7-18 days. The pH can be between "0 and 6.2, the temperature 20-30 ⁰ C. 4th The fermentation is advantageously carried out at 22-25 ^'0 C and pH 5.2 to 5.9.

The amino acid analogous to the proline is added to the culture medium in a concentration of 1-10 g / l, preferably 3-6 g / l of nutrient solution in the growth phase of the mycelium, which is generally on the 1st to 5th day of the main culture. Suitable amino acids are in particular 1,3-thiazolidine-4-carboxylic acid, 3,4-dehydroproline and 4-hydroxyproline. For example, the process according to the invention for the preparation of the novel compound 2 ^l β-methyl-5'cC-isobutyl-9'-thiaergopeptin ("Thiaergosin") is used.

To determine the appropriate time to complete the reaction, on the one hand the alkaloid concentration is determined spectrophotometrically after staining with van Urk's reagent at 560 nm and on the other hand determined the ratio between unmodified and modified alkaloids by thin-layer chromatography separation, elution and staining. In general, the reaction is stopped after a total culture time of 14-25 days.

231978

The mycelium is discarded after removal of the nutrient solution. This is made alkaline with ammonia or sodium hydroxide until pH 9-10 and extracted with suitable organic solvents, preferably chlorinated hydrocarbons. The organic phase is concentrated and the alkaloid mixture is separated into the individual components by chromatography. The identification of the individual alkaloids is carried out by the usual analytical methods (UV, IR, MS, NMR).

The process according to the invention makes it possible for the first time to prepare stable bregopeptins with a modified proline portion by controlled fermentation and thus to improve known therapeutic options or to develop new indications. The new compound thiaergosin has a blocking effect on the receptors and affects the chronic arterial peripheral circulatory disorders.

Ausführun ^ sbeispiele

The following examples are intended to illustrate the invention. The nutrient media used are summarized in Tab.

Example 1;

Starting from canned food of the clan Claviceps purpurea

(Fr.) TuI. IBP 179 are inoculated into precursors of 100 ml NL 849 medium in 500 ml shake flasks. The pistons become the 7 days at 24 ⁰ C ± 1 ⁰ C incubated at 240 rpm. 10 500 ml flasks, each containing 100 ml of NL 720 medium, are inoculated with 10 ml each of the preculture. After 1 day of fermentation, 5 g / L L-1 _{t of} 3-thiazolidine-4-carboxylic acid are added to the flask. After another 10 days of incubation, the cultures are harvested and the nutrient solution worked up. From 1 l culture medium 250 mg Peptidalka-

231978 8

75 % of Ergosin / Ergosinin and 25 % Thiaergosin / Thiaergosinin.

Example 2:

10 500 ml flasks, each containing 100 ml of nutrient solution NL 833, are inoculated with 10 ml of the preculture mentioned in Example 1. On the 2nd day of fermentation will be the. Flask 5 g / l L-1 ^ -Thiazolidin ^ -carboxylic added. On the 14th day of the fermentation, the flasks are worked up under the conditions mentioned in Example 1. The nutrient solution contains 400 mg / l of a peptide-alkaloid mixture.

Example 3:

For the qualitative determination of the alkaloids, 10 ml of the nutrient solution are alkalized and extracted with chloroform. The extract is concentrated to 5 ml and aliquots are separated by layer chromatography.

Solvent systems:

I chloroform: methanol (9: 1)

II Chloroform: methanol: glacial acetic acid (80: 15: 5) III ethyl acetate: methanol (95: 5)

Rp values in system I, II, III corresponding to ergosine: 0.45; 0.52, 0.22. Thiaergosin: 0.51 ;, 0.54; 0.44. Ergosinine: 0.76, 0.66; 0.59. Thiaergosinine: 0.85; 0.77; 0.69. Thiaergosin and Thiaergosinin can be converted into each other. They show strong fluorescence in UV light and give a deep blue tint with van ürk * s reagent.

The separated after layer chromatography on silica gel zones are scraped, extracted and then mixed with van Urk's reagent. After centrifugation, a

_ ₇ _ 231978 8

colored solution, which is determined colorimetrically in the usual way. In 1 ml of the culture filtrate obtained according to Example 2, 300 / μg ergosin / ergosinin and 100 / ug thiaergosin / thlaergosinin were determined.

For identification, the crude halide mixture of 1 liter of culture filtrate is separated by repeated layer chromatography on silica gel using solvent systems I, II, III. From the 2 ^{l of} β-methyl-5 ^F oT-isobutyl-9'-thiaergopeptin (Thiaergosin) 50 mg could be obtained. The Keller's and Van Urk's reactions were positive, and the compound showed the typical lysergic acid. UV absorption: UV ?? ^H 240, 312 nm and a minimum at 270 nm. Alkaline hydrolysis yielded lysergic acid and pyruvate. In the mass spectrum, the following characteristic fragments were obtained: m / e no M ⁺ peak, 298 (cyclol group); 267 (lysergic acid amide) j 228 (lactam); 221; 207; 196; 185; 180; 172; 164; 154; 88th

The sulfur-containing key fragments of the peptide part differ from the corresponding ergosine peaks by 18 mass units: m / e 298 (C ^ H ^ gNgO ^ S, Found: 298.0981, calc .: 298.0973); 228 (C ₁₀ H ₁₆ N ₂ O ₂ S, Found: 228.0923, calc .: 222.0918); 185 (C ₇ H ₉ N ₃ O ₂ S, Found: 185.0386, calc .: 185.0388); 172 (CgHgN ^ S, Found: 172.0307, calc .: 172.0301); also m / e 88 compared to m / e 70 in ergosine.

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Table 1: Composition of the nutrient solutions used Data in g / l

NL 849 NL 720 15.0 NL 833 20.0 sucrose 300.0 200.0 1.0 300.0 1.0 skimmed milk 100 ml - 0.25 - 0.25 ammonium citrate 10.0 0.3 0.3 Ca (NOo) ₂ 0,010 0,010 KH ₂ PO ₄ 0.25 0.0308 0.0308 MgSO _4. 7H ₂ O 0.3 0.1 r -. PeSO ₄ . 7H ₂ O 1000.0 1000.0 ZnSO ₄ . 7H ₂ O KCl - - 5.8-6.0 5.4 Aqua Dist ad · 1000.0 NaOH NaOH pH before the uniform 30 min 110 ⁰ C Sterilize 5.4 pH adjustment with NaOH autoclaving Each

978

CH ₃ OH

H CO-NH c

CH ₃

CH.

2 ¹ p-methyl-5'oC-isobutyl 9'-thiaergopeptine

99.1111

Claims (10)

- & - 231978 Invention ungsana pruch .1. Process for the preparation of ergopeptins with modified proline moiety, characterized in that a submerged ergopeptin-forming strain is used, to which in the growth phase of the mycelium an amino acid analogous to the proline is added, the strain is cultured in the presence of this amino acid and the modified ergopeptins are isolated from the nutrient medium , 2. The method according to item 1, characterized in that the proline analog amino acid is added in a concentration of 1-10 g / l of nutrient solution. 3. The method according to item 2, characterized in that the proline analog amino acid in a concentration of 3-6 g / l of nutrient solution is added. 4. The method according to item 1-3, characterized in that as the proline analog amino acid 1,3-thiazolidine-4-carboxylic acid is used. 5. The method according to item 1 to 4, characterized in that a submersed ergosin forming strain is used. 6. The method according to item 5, characterized in that the strain Claviceps purpurea (Pr.) TuI. IBP 179, its mutants or variants used. 7. The method according to item 5, dadurok characterized in that the strain Claviceps purpurea (Pr.) TuI. MUT 168, its mutants or variants, used. 8. The method according to item 1-7, characterized in that the strain in aerobic fermentation in a liquid nutrient medium containing an assimilable carbon source, an assimilable nitrogen source and mineral salts in submerged cultivation in a 231978 8 Temperature of 20 - 30 ⁰ C and a pH of 4 »0-6,2 cultured, on 1 .. - 5 · · day of the main culture an amino acid analogous to the proline added, cultivated in the presence of this amino acid for another 7-18 days and isolated the modified ergopeptins from the nutrient medium · 9. The method according to item 8, characterized in that the fermentation at 22 - 25 ⁰ C and pH 5.2 - 5.9 is performed. 10. The method according to item 1 to 9, characterized in that thiaergosin is produced. For this ISeite formulas