FI75188C - FOERFARANDE FOER FRAMSTAELLNING AV TERAPEUTISKT ANVAENDBARA 9'-TIAERGOPEPTINDERIVAT. - Google Patents

Description

75188

Process for the preparation of therapeutically useful 9'-thia-ergopeptin derivatives

Divided separated from patent application 822670 5

The invention relates to a process for the preparation of therapeutically useful 9'-thia-ergopeptin derivatives of formula (I)

io f1 ° H ^ I

CO-NH - 4 ^ S ^ TJ ^ (I) wherein R 1 is methyl or isopropyl and their acid addition salts, which process is characterized by culturing a Claviceps purpurea strain producing a compound of formula (I) on carbon - and in a culture medium containing a nitrogen source, mineral protection and trace elements; at the beginning of alkaloid formation, after 3-6 days, L-thiazolidine-4-carboxylic acid or a salt thereof is added to the culture; the culture is continued for 6 to 12 days, after which the compound of formula (I) is extracted from the culture broth, the culture from the filtrate or mycelium by methods known per se, and the obtained compound of formula (I) is converted, if desired, into its acid addition salt.

30 This method can be performed as follows:

For example, for the production of 91-thia-ergotamine and 9'-thiaergotamine, the strain Claviceps purpurea Mutante NRRL 12043 is used as the primary ergotamine / ergotamine-producing strain. Its culture was recorded on 20.9.

35 1979 to the United States Department of Agriculture (Northern Regional Research Center), Peoria, 111. USA under farm number NRRL 12043.

2 75188 Claviceps purpurea Mutante NRRL 12044 is used as the primary ergocristine / ergo-christinin-producing strain for the preparation of 9'-thia-ergocristine and 9'-thia-ergocristinine.

3 1979 to the above deposit under crop number NRRL 12044.

Characteristics and preparation of strains NRRL 12043 and 12044 using a) Characteristics of strain NRRL 12043 10 The starting fungal strain was isolated from mycelium gum containing ergotamine found in Bromus hay, a municipality called WAllis (Switzerland). Using several mutagenic treatment steps using ultraviolet radiation and chemicals, the strain NRRL 12043 used according to the invention was obtained, which can be characterized as follows:

A small piece of mycelium strain NRRL 12043 is planted in the center of an arag plate with a medium having the following composition: 20 60 g sucrose, 10 g asparagine, 0.5 g Casamino acids (Difco), 0.25 g KH 2 PO 4, 0.25 g MgSO 4 4.7H 2 O , 0.125 g KCl, 16 mg FeSO 4, 4.71 ^ 0, 10 mg ZnSO 4 .7H 2 O. 15 g of agar and distilled water to 1 liter. The pH is adjusted to 6.0 with 25% ammonia solution. The medium is sterilized for 25 minutes at 120 ° C. After seven days at 24 ° C, a column with a diameter of about 3 cm is formed, after 14 days with a diameter of about 5 cm and after 20 days with a diameter of about 8 cm. The column is convex and slightly wrinkled. The border is diffuse. The center of the column is beige-purple and the edge is beige-gray. The 20-day-old column contains about 1x10 condoms per cm. These are oval and have dimensions of 6-12 x 4-7 ^ u.

b) Characteristics of strain 12044 The parent fungal strain was isolated from ryegrass found in rich North Ame-35 containing ergocristine. Using several mutagenic treatment steps using ultraviolet irradiation and chemicals, strain NRRL 12044 for use according to the invention was obtained, which can be characterized as follows:

II

3 75188

A small piece of mycelium strain NRRL 12044 is planted in the center of an agar plate with a medium having the following composition: 70 g malt extract (Wander), 30 g potatoes (Stocki, 5 Knorr), 15 g agar and distilled water to 1 liter, pH 5.3 - 5.7. The medium is sterilized for 20 minutes at 120 ° C. After seven days at 24 ° C, a column with a diameter of about 2 cm is formed, after 14 days with a diameter of about 4 cm and after 20 days with a diameter of about 5 cm.

10 The mycelium is uniformly convex and covered with a white web of mushroom webs rising into the air. A crater-like bump with a diameter of about 1 cm is formed in the center. It emits radial folds. The mycelium on the agar is stained purple. Concentric areas with the strongest coloration and areas with the weakest coloration are formed. The border is diffuse. The 20-day-old mycelium contains 1 x g of 10 conidia per cm. The dimensions of these differ considerably (5-12 x 3-7 ^ u; in most cases, however, 5-6 x 3 ^ u). Felt-20 to does not contain any alkaloids.

Reproduction and inoculation_ (a) NRRL 12043

Addition of strain NRRL 12043 and inoculation can be accomplished by slurrying conidia from one of the 25 columns described above in sterilized water and inoculating them into slant agar tubes containing agar medium containing: 70 g malt extract (Wander), 30 g potatoes (Stocki, Knorr) , 15 g of agar and distilled water to 1 liter. The pH is 5.3 to 5.7. The medium is sterilized for 30 to 20 minutes at 120 ° C. After 14 days, such a culture of oblique agar (12 ml of medium in a reagent glass 18 mm in diameter and 20 cm long, wedge-shaped) 9 contains about 1x10 conidia. From such an oblique agar culture, the conidia are slurried in sterilized water and thus quenched with a concentrate of 10 to 10 spores / ml. In the sloping agar tubes containing the medium, 0.5 ml of this suspension is evenly inoculated. These cultures are incubated at 24 ° C for 14 days. They are then stored at -40 ° C. (b) NRRL 12044 The addition of strain NRRL 12044 and the preparation of the inoculum may be carried out by slurrying the conidia from one of the columns described above in sterilized water and inoculating them into slant agar tubes containing the agar medium described above. After 18 days, such a culture of 10 oblique agar (12 ml of medium in a reagent glass 18 mm in diameter and 20 cm long, g-wedge-shaped) contains about 1-2 x 10 conidia. The conidia are slurried from such an oblique agar in sterilized water to produce a concentrate with 10 to 7 15 10 spores / ml. Sloped agar tubes containing the same medium are evenly inoculated with 0.5 ml of this suspension. These cultures are incubated at 24 ° C for 14 days. They are then stored at -40 ° C.

Precultures In carrying out the method according to the invention, it has proved advantageous to cultivate the spores first in the precursor (s) before implantation into the production medium. The first step is to choose a medium in which the spores germinate well and the mycelium grows rapidly. Suitable for such a medium are: a high-concentration carbon source in the form of a mono- or disaccharide, optionally in combination with a polyalcohol, a nitrogen source in the form of an amino acid or an ammonium salt, further inorganic salts, trace elements and additive complexes obtained from plant seeds. Adjusting the pH to between 30 and 7 is preferred. In the first stage, a large amount of conidia are inoculated into the medium and then germinated in a shaker or in special fermentation vessels at 22-26 ° C for 4-7 days. A fluffy, cotton-like, 2-3 mm long pigment-free mycelium clumps result in a 35 micron dense culture with long fungal filaments 3-6 microns in diameter.

Tue 75188 Main crops

A part of one or possibly more of the 5 precursor cultures is inoculated into a production medium having a preferred composition such that the weight of the mycelium is increased in a short time. Sucrose has proven to be the best and cheapest carbon source, with ammonium salts such as oxalate, citrate, succinate and formate as the nitrogen source. The medium must still contain mineral salts and iron and zinc as trace elements. Cultures are carried out either in Erlenmeyer flasks or in fermentation vessels of different sizes. Good ventilation is important. The optimum temperature is 24 ° C.

At the beginning of alkaloid formation, usually between the third and the sixth day of the main culture, 1-5 g / l of L-thiazolidine-4-carboxylic acid or a salt of this acid such as potassium, sodium or ammonium salt is added to the main culture 15. The cultures are then further incubated for 6-12 days. Within 9-18 days, a dense culture is formed, consisting mainly of cylindrical compact mycelium particles 0.5-3 mm long and 0.1-1-1 mm thick, together with fine fluff. The mycelium particles resemble a plectenchymatic tissue with spherical or poly-shaped - short-cylindrical, thick-walled and highly cellular cells, measuring 6-12 x 6-14 μu, mainly 8 x 9 ^ -u, through which up to 100 μl passes. long mushroom strings.

They are browned. The culture filtrate is also brownish in color.

As the weight of the mycelium increases and the alkaloid content decreases, the alkaloids can be extracted from the whole culture broth by known methods with organic solvents or the manure is separated from the filtrate by filtration or centrifugation, and each part is extracted separately. Most of the peptide alkaloids are enriched in mycelium.

Isolation of Compounds of Formula (I) These sulfur-containing ergopeptins can be isolated from culture broth, culture filtrate or mycelium by known extraction methods. Purification is accomplished using conventional purification steps, such as, for example, precipitating the active moiety with inactive solvents or converting to sparingly soluble salts. Particularly suitable are known purification methods, such as, for example, chromatography on alumina, silica gel, Sephadex LH-20, etc., in each case with suitable solvent mixtures. Like the known ergopeptins, these new 91-tia ergopeptins are sensitive to daylight. In the free form and in polar solvents, they are isomerized up to a certain equilibrium state to the second form in question.

In the salt form, which can be prepared e.g. with methanesulfonic acid, 9'-thia-ergotamine and 9'-thia-ergocristine retain their stability for a relatively long time.

The compounds of the invention have not been described in the literature so far. They show interesting pharmacological properties in animal experiments and can therefore be used as medicaments.

In particular, these compounds have been shown to stimulate dopamine receptors. Dopaminergic properties could be demonstrated in rats in which 6-hydroxido-pamine injection into the black nucleus caused unilateral damage to the dopamine pathway of the nigro-neosteriatum at doses ranging from about 0.5 to 100 mg / kg / U. Understedt method, Acta Physiol, Scand. Suppl. 367 (1971) 69 -93 /. The effect of the substance after administration, a distinct activation was detected, so that the rats went around the side in the direction of the nerve was damaged.

30 Based on their dopaminergic properties, new agents may find use in the treatment of Parkinson's disease.

Furthermore, the new compounds have an inhibitory effect on prolactin secretion. Thus, in rats, they prevent implantation at subcutaneous doses of 0.01 to 1 mg / kg 35 and milk secretion at doses of 1 to 10 mg / kg orally / liter. Reference: Experientia 34 (1978) 1330 /.

7 75188

Based on their inhibitory properties for prolactin secretion, the new agents may find use in, for example, the prevention of milk secretion, the prevention of milk leakage, the treatment of glandular underdevelopment and giant hemorrhage caused by prolactin-bleeding, or prolactinomy.

Furthermore, the new agents cause sleep / wake cycle in a chronically implanted rat at a dose of about 3 mg / kg i.p. or at a dose of 10 mg / kg p.o. starting from a reduction in the apparent duration of 10 sleeps and an increase in wakefulness / see

J.M. Vigouret et al., Pharmacology 16 (1) (1978) 156-1737. In addition, at a dose of 0.3 mg / kg i.p. an increase in glucose metabolism in the neural nuclei of sensory information processing. (See L. Solokoff, Journal of Cerebral Blood Flow and Metabolism 1981, 1, 7-36, HE Sa-vaki et al., Brain research 1982, 233, 347 and J. McCulloch et al., Journal of Cerebral Blood Flow and metabolism 1981, 1, 1 33-136).

Based on these results, the new agents are suitable for the treatment of aging boredom, especially in the early stages and to increase vitality.

In addition, the compounds of the invention have a vasoconstrictive effect, which can be demonstrated in vitro with helical strips of canine Arteria carotis externa / E. Miiller-25 Schweinitzer, Naunyn-Schmiedeberg's Arch. Pharmacol. 292 (1976) 113-1187, from doses of 10 nM / l.

Due to their vasoconstrictive effect, new substances are used to treat migraines.

Furthermore, they cause in rats whose brain and spinal cord are damaged / Brit. J. Pharmac. Chemother. 30 (1,967), 78-87 /, at a dose of about 5 to about 50 μg / kg i.v., for a long-lasting antihypertensive effect with an increase in blood pressure. In Mellander's cat / Angiologi-ca 3 (1966), 77-997, they cause a strong, dose-dependent and long-lasting narrowing of the vasculature at doses of about 0.5 to about 50 ug / kg i.a.

8 75188 Based on this effect, substances can be used as intravenous agents.

Finally, the compounds of the invention have a serotonergic effect based on the results of the sleep / wake-up cycle. In view of this effect and the results of the Ungerstedt experimental model, they can be used as analgesics for depressive conditions, especially in the elderly population.

In the following examples / which further illustrate the invention, all reported temperatures are in degrees Celsius and are uncorrected.

Example 1 9'-thia-ergotamine and 9'-thia-ergotamine 1. Culture of strain NRRL 12043 a) Preculture 15 Conidia (about 10) of a slant agar culture of strain NRRL 12043 of Glaviceps purpurea are suspended in 10 ml of sterilized water. 1 ml of this conidia suspension is inoculated into 200 ml of preculture medium with the following composition: 20 g / liter

Sucrose 100

Profile 10

Ammonium oxalate 3

Ca (NO 3) 2 * 4 H 2 O 1 25 KH 2 PO 4 0.25

MgSO 4 - 7 H 2 O 0.25 KCl 0.125

FeSO 4 * 7 H 2 O 0.0166

ZnSO 4 * 7 H 2 O 0.0068 30 distilled water ad 1 liter in a 500 ml Erlenmeyer flask; The pH is adjusted to 6.5 with NH 4 OH. The medium is sterilized for 20 minutes at 120 °. This preculture is shaken at 24 ° for five days (180 rpm 5 cm). At the end of the growth, the pH of the culture 35 is 5.3 and the dry weight of the mycelium is 22 g / l.

Il 9 751 88 (b) Main culture 10 ml of the preculture is inoculated into 50 ml of the main culture medium with the following composition: g / liter 5 Sucrose 240

Ammonium oxalate 9.6

Ca (NO 3) 2 '4 H 2 O 2.5

MgSO 4 - 7 H 2 O 0.625 KH 2 PO 4 0.625 10 KCl 0.312

FeSC> 4 · 7 H2® 0.0252

ZnSO. · 7 H „0 0.0102 4 2 distilled water ad 1 liter .on in a 500 ml Erlenmeyer flask; The pH is adjusted to 6.2 with 25% 10-15 NH 4 OH. The medium is sterilized for 20 minutes at 110 °.

This culture is incubated at 24 ° on a rotary shaker (180 rpm, circumference 5 cm). After an incubation period of four days, 90 mg of L-thiazole-20-didine-4-carboxylic acid are added per culture. The cultures are incubated in a rotary shaker for another 10 days. At the end of the fermentation, the cultures are filtered separately. The mycelium is dried carefully. The dry weight of the main culture is 85 g / l. The alkaloid content is 12 mg / g. 9'-thia-ergotamine + 9'-thia-ergotamine-25 n accounts for 10% of the total alkaloid content.

2. Isolation of the title compounds 500 g of dry mycelium are homogenized twice with 1 600 ml portions of a mixture of methanol and 2% concentrated ammonia and twice with 1 200 ml portions of methanol for five minutes each time, Ultra- In a Tur-Rax apparatus and the combined filtrates are evaporated to dryness in vacuo at a bath temperature of not more than 40 ° (about 50 g of a burgundy residue). This residue is dissolved in 500 ml of methanol and filtered through a chromatographic tube of 35 cm through a 500 g portion of alumina, Activation-Ttsstufe II (Woelm), then washed with 20 ° C ml of methanol. and the whole of the filtrate is evaporated to dryness in vacuo (25 g of a yellow foam). This residue is then separated from the chromatography by a 200-fold amount of Kieselgel 60 (Merck) with a mixture of methylene chloride + 2% methanol. Fractional residues are examined by thin layer chromatography and fractions containing only spots corresponding to 9'-thia-ergotamine (cf. Rf values in Table 1) are pooled.

9'-thia-ergotamine: 10 Fractions containing only 9'-thia-ergotamine by thin-layer chromatographic analysis and corresponding to their Rf value are dissolved in ethyl acetate, filtered through a pad of talc until clear and, after concentration in vacuo, a little hexane is added to crystallize the alkaloid. 15 separate. Recrystallization from the same solvent gives pure white to yellowish crystals. Sp. From 199 °, slowly decomposing; mp = -165 ° (c = 0.5 in chloroform).

91-thia-ergotamine-methanesulfonate; 180 mg of 9'-thia-ergotamine are dissolved in a small amount of ethanol and an alcoholic solution containing 28 mg of methanesulfonic acid is added, whereby the alkaloid crystallizes out as the methanesulfonate. Recrystallization of the methanol list gives a pure white salt. Sp. From 211 ° to 25 ° C, dec. = + 76 ° (c = 0.51 in methanol).

9'-thia-ergotaminiini:

The chromatogram fractions with a uniform spot and Rf value obtained by flash chromatography (see Table 1) are dissolved in ethyl acetate, combined and filtered through a Kirk-30 pad. A little hexane is added to the concentrated solution, whereupon the alkali precipitates as white crystals. Recrystallization from the same solvent gives the pure compound. Sp. From 199 °, decomp.; = + 343 ° (c = 0.55 in chloroform).

35 Table 1

Thin chromatographic Rf values compared to ergotamine (Et) and 11 75188 ergotamine (Et-in)

Layer and Tia- Et Tia- Et-in solvent_ Et__Et-in__ 5 Alox ^

Ethyl ester / sec. Butanol 0.44 0.34 0.68 0.59 9: 1

Toluene / isopropanol 0.58 0.50 0.69 0.63 9: 1 10 Toluene / isopropanol 0.30 0.24 0.58 0.46 19: 1 2)

Silica gel

Toluene / isopropanol 0.15 0.11 0.43 0.31 85:15 15 CH Cl / methanol 0.25 0.22 0.65 0.52 93: 7 1)

Aluminum oxide fertilizer plates Merck F 254 (Typ E) 2)

Kieselgel-Fertigplatten Merck F 254 20 On thin plates, the spots appear in UV light at 254 nm as blue-violet and at 366 nm as light blue spots. Using iodine vapors, they are also identifiable as brown spots.

The van Urk reagent so-25, which is typical of ergopeptins, is also excellent for detecting spots. After spraying, these compounds appear as blue-violet in color, which is enhanced by daylight or by evaporation with queen water vapor.

Example 2 9'-thia-ergocristine and 91-thia-ergocristinine 1 · Culture of strain NRRL 12044

Strain NRRL 12044 is cultured according to Example 1. The dry mycelia weight of the main culture is 80 g / l. The alkaloid content is 10 mg / kg. The title compounds account for 8 'of the total alkaloid content.

2. Isolation of the title compounds 12 751 88 500 g of dry mycelium are homogenized twice with 1,500 ml portions of a mixture of methanol and 2% concentrated ammonia and twice with 1,500 ml portions of methanol, five minutes each time, Ultra-Turrax -lait-5 in tea. The combined filtrates are evaporated to dryness in vacuo at a bath temperature of not more than 40 ° (190 g of a burgundy residue). After dissolving in 900 ml of methanol, it is filtered through a 10 cm thick layer of 600 galumium oxide (basic, activity grade 2), then washed with 2,000 ml of methanol and the filtrate is evaporated to dryness in vacuo (108 g). This residue is now separated into methanol with 3,000 g of Sephadex LH-20 and the 200 ml fractions are collected and evaporated to dryness. The fraction residues containing 15 new thia-ergopeptides as determined by thin layer chromatography are combined (526 mg) and chromatographed once more with 120 g of Sephadex LH-20 in methanol to give 156 mg of pure 9'-thia-ergocristine / - Kristin mixture. Separation of these two compounds was carried out by known chromatographic methods on silica gel Kieselgel 60 (Merck) eluting with a mixture of methylene chloride and 2% methanol.

91-thia-ergocristine:

Fractional residues which, based on thin layer chromatographic analysis and their corresponding Rf value (cf. Table 2), contain only 9'-thia-ergocristine are combined and crystallized from benzene (28 mg). Sp. 146 °, sintering slowly with decomposition; [.alpha.] D @ 20 = -176 DEG (c = 0.5 in chloroform).

9 * -Tia-ergocristinine:

The chromatogram fractions, which are uniform by thin-layer chromatography and have a spot corresponding to the corresponding Rf values (see Table 2), are combined and crystallized from an ethanol-ethyl acetate mixture. Sp. 209-210 ° with decomposition; Z®6 / ^ 0 = + 344 ° (c = 0.51 in CHCl3).

Table 2 35 Thin chromatographic Rf values compared to ergocristin (Ec) and ergocristinine (Ec-in).

13,751 88

Layer and Tia- Et Tia- Ec-in solvent Ec Ec-in

Alox1)

Acetate ester / sec.butanol 0.68 0.63 0.78 0.70 5 9: 1

Toluene / isopropanol 0.68 0.62 0.69 0.66 9: 1

Toluene / isopropanol 0.50 0.42 0.58 0.47 19: 1 2) 10 Silica gel

Toluene / isopropanol 0.37 0.28 0.56 0.48 85:15 CH2Cl2 / methanol 93: 7 0.47 0.41 0.70 0.60 1) 15 Aluminum oxide fertilizer plates Merck F 254 (Typ e) 2 )

Kieselgel-Fertigplatten Merck F 254

Detection of spots of matter as in Example 1.

Claims (1)

751 88 14 A process for the preparation of therapeutically useful 9'-thiaergopeptin derivatives of the formula 5 (I) p OH ΓΓΪΓ1 fl 0. ί J CO-NH-4 ^ A oJ-Ia0 (i> 10 (il Jl N-CH , H CH2-C6H5 3 HN_11 wherein R1 is methyl or isopropyl, and for the preparation of these acid addition salts, characterized in that a Claviceps purpurea strain producing a compound of formula (I) is cultured with a source of carbon and nitrogen, mineral salts and at the beginning of alkaloid formation, after 3-6 days, L-thiazolidine-4-carboxylic acid or a salt thereof is added to the culture, the culture is continued for 6-12 days, after which the compound of formula (I) is extracted from the culture broth, the culture filtrate or mycelium by methods known per se and the compound of formula (I) obtained is, if desired, converted into its acid addition salt.