CS235340B2 - Method of ergopeptine derivatives production - Google Patents

Description

The present invention relates to a process for the preparation of novel ergopeptin derivatives having valuable pharmacological properties and which can be used as active ingredients of medicaments.

It is an object of the present invention to provide a process for the preparation of ergopeptin derivatives of formula (I)

R 1 represents a methyl group and

Kg represents an isobutyl or benzyl group, or

R1 is ethyl and

R 8 is benzyl or

R 1 represents an isopropyl group and

Rg is an isopropyl group, a sec-butyl group, an isobutyl group or a benzyl group, and their acid addition salts by culturing a strain of Claviceps purpurea producing the corresponding 9 * methylenderivative or a strain derived from Claviceps purpurea which consists in strain add 1 to 5 g / L of L-thiazolidine-4-car235340

235340 2 of a boxylic acid or a salt thereof, cultivation is carried out at 22-26 ° C and the resulting compounds are optionally converted into their acid addition salts.

Suitable salts for forming salts are, for example, hydrochloric acid, sulfuric acid, malelic acid, fumaric acid and tartaric acid.

Both Claviceps purpurea strains and those strains which can be obtained from the parent Claviceps purpurea strain by mutation under the action of radiation or by mutagenic agents or selections can be used. Such strains are known and available to the public.

For example, Claviceps purpurea NRRL 12043 is used as the preferred ergotamine / ergotaminin mutant strain to produce 9'-thiaergotamine and 9'-thieergotamine. The culture of this strain was deposited on September 20, 79 in the United States Department of Agriculture (Northern Regional Research Center). , Peoria, 111., Sp. st. a. NRRL culture number 12043. '

For the production of 9'-thiaergocristin and 9'-thiaergocristinin, a preferred strain producing ergocristin / ergocristin of the mutant Claviceps purpurea NRRL · 12044 is used. Its culture was deposited on 29 September 79 at the above deposition site under the designation NRRL 12044.

Strains useful for making other compounds of Formula 1 are known and are also available to the public.

Characterization and fermentation of strains NRRL · 12043 and 12044 _from

(a) Characteristics of strain NRRL 12043

The starting fungus strain is isolated from sclerotium containing ergotamine, which was found on a brome (Bromus) in the Vallis region (Switzerland). The multi-stage mutagenesis process using ultraviolet radiation and chemicals yields the strain NRRL 12043 used according to the invention and can be characterized as follows:

A small piece of mycelium strain NRRL 12043 is inoculated into the center of an agar plate with the following composition:

g sucrose, 10 g asparagine, 0.5 g casamino acid (Difco), 0.25 g KHgPO4,

0.25 g MgSO4.7HgO, 0.125 g KCl, 16 mg FeSO4.7HgO, 10 mg ZnSO4.7HgO, 15 g agar and distilled water to 1 liter. The pH was adjusted to 6.0 with 25% ammonia solution. The medium is sterilized at 120 ° C for 20 minutes. At 24 ° C, colonies with a diameter of 3 cm were formed after 7 days, colonies with a diameter of about 5 cm were formed after 14 days, and colonies with a diameter of about 8 cm were formed after 20 days. This colony is arched and has slight folds. The margin is diffueni. The color of the colony is beige purple in the middle and beige gray at the edge. A 20 day old colony contains about 1 x 10 6 conidia per tin. These conidia are oval and have dimensions ranging from 6 to 12 χ 4 to 7 µn.

b) Characterization of strain 12044

The starting fungus strain was isolated from sclerotia on a Shiite in North America and this strain contains ergocristin. The multi-step mutagenesis process using ultraviolet rays and chemicals yields the NRRL 12044 strain which is used according to the invention and can be characterized as follows:

A small piece of mycelium strain NRRL 12044 is inoculated into the center of an agar plate with the following composition:

g of malt extract (Wander), 30 g of potatoes (Stocki, Knorr), 5 g of agar and distilled water to 1 liter. The pH is 5.3 to 5.7. The culture medium is sterilized at 120 ° C for 20 minutes. After 7 days at 24 ° C a colony of about 2 cm is formed, after 14 days a colony of about 4 cm and after 20 days a colony of about 5 cm,

The mycelium is domed flat and is covered with white, cotton-like mesh of hyphae protruding into space. A cratery bump about 1 cm in diameter is formed in the center. Radial folds emanate from this bump. The mycelium covering the agar is violet colored. Concentric zones with stronger pigmentation and concentric zones with weaker pigmentation are formed. The edge is diffuse. A 20-day-old mycelium contains per 1 cn / about 1.10 ® conidia. These conidia vary considerably in size (5 to 12 x 3 to 7 µm; however, mostly 5 to 6 x, 3 µm). The mycelium does not contain any alkaloids.

Reproduction and vaccine production

(a) Strain NRRL 12043

Reproduction and production of the vaccine strain NRRL · 12043 may be carried out by suspending conidia from one of the colonies described above in sterile water and inoculating sloping agar tubes with agar broth containing: 70g malt extract (Wander) , 30g potatoes (Stocki, Knorr), 15g agar and distilled water up to 1 liter. The pH value is 5.3 to 5.7. the broth is sterilized at 120 ° C for 20 minutes.

After 14 days, such a slant agar culture (12 ml of nutrient broth in a tube of 18 mm diameter and 20 cm in length, was placed obliquely (to create a wedge) of about 1 x 10 @ -4 Conidia. From such a slant agar culture, conidia were suspended in sterile water. at a concentration of 10 to 10 ' spores / ml, 0.5 ml of this suspension is inoculated evenly with sloping agar tubes with the same culture medium and cultured at 24 DEG C. for 14 days. Deň: 32 ° C.

(b) NRRL · 12044

Reproduction and production of vaccine strain NRRL 12044 may be carried out by:. According to one of the colonies described above, they are suspended in sterile water and inoculated with slurry agar in tubes containing the above-described broth. After 18 days, such a slant agar culture (12 ml of nutrient broth in a tube of 18 mm diameter and 20 cm length, placed obliquely (to wedge) contains about 1 to 2 × 10 conidia. Conidia from such a slant agar culture are suspended in sterile water and in this case a concentration of 10 to 10 .mu.m / ml is achieved, in each case 0.5 ml of this suspension is inoculated evenly with sloping agar tubes with the same medium and cultured at 24 DEG C. for 14 days. stored at -40 ° C.

Preliminary cultures

In carrying out the process according to the invention, it has proven advantageous to grow the spores first through the previous stage or through the previous stage before the production broth is inoculated. Initially, nutrient medium is to be selected, in which the spores are well germinated and the mycelium grows rapidly. Suitable such media are: a rich source carbon monosaccharide or disaccharide, optionally in combination with a polyalcohol, an amino acid or ammonium salt nitrogen source, mineral salts, trace elements, and complex plant seed ingredients. The pH is preferably adjusted to between 5 and 7. In the first step, the broth is richly inoculated with conidia and then incubated on a shaker or fermenter of various sizes at 22-26 ° C for 4-7 days. This results in a dense culture of fluffy, cotton-like 2 to 3 mm measuring, unpigmented mycelial flakes consisting of long hyphae having a diameter of 3 to 6 µm.

Main cultures

A part of the culture which has been obtained in one or, if appropriate, several previous steps is seeded with a production nutrient medium which preferably has a composition such that a high mycella weight is achieved in a short time. Sucrose has proven to be the best and cheapest source of carbon, ammonium salts such as oxalate, citrate, succinate and formate as the nitrogen source. In addition, mineral salts and iron and zinc are to be added as trace elements. Cultivation is carried out either in Erlenmeyer flasks or in fermenters of different sizes. Good aeration is important. The optimum temperature is 24 ° C.

At the beginning of the formation of alkaloids, usually between day 3 and day 6 of the culture of the main culture, 1 to 5 g / liter of L-thiazolidine-4-carboxylic acid or some of its salts such as potassium, sodium or ammonium salts are added to the main culture. . The cultures are then fermented for a further 6 to 12 days. Over a period of 9 to 18 days, a dense culture is formed, consisting mainly of 0.5 to 3 mm long and 0.1 to 1 mm thick cylindrical compact mycelium particles in addition to fine flakes. The mycelium particles exhibit an image of plectenchymatic tissue from spherical or polyhedral to low cylindrical, thick and heavily vacuolized cells having a size of 6 to 12 x 6 to 14 µm, in particular 8x9 µm, which is permeated by long hyphae at a distance of up to 100 µm . These particles are pigmented with a pigmented color. The culture filtrate is also colored suddenly.

When the weight gain and alkaloid content of the mycelium is reduced, the alkaloids can be extracted from all the slurry cultures using organic solvents by known methods, or the mycelium can be separated by filtration or centrifugation from the filtrate and both parts extracted separately. The major proportion of peptide alkaloids is enriched in the mycelium.

Isolation of compounds of formula I

These sulfur-containing ergotpeptins can be isolated from culture suspension, culture filtrate or mycelium by known extraction methods. Purification is accomplished using conventional purification operations, such as precipitation from active solvents with inactive solvents or conversion to salts that are difficult to dissolve. Especially suitable are known purification procedures, such as chromatography on aluminum oxides, silica gel Sephedex Ui-20, etc., in each case in suitable solvent systems. As well as the known ergotpeptins, these novel 9'-thiaergopeptins are sensitive to daylight.

In the free form and in polar solvents, the compounds are isomerized to the other form until a certain equilibrium is reached.

In the form of a salt which can be produced, for example, by the action of methanesulfonic acid, 9'-thiaergotamine and 9'-thiaergocristine are stable for a long time.

The compounds obtained according to the invention can, if desired, be converted into other compounds of formula I by known methods.

Thus, the compounds of formula I can be alkylated at the 1-position and / or the 2-position or the 6-position.

Further, the compounds of formula I may be reduced at the 9,10 position.

In addition, the compounds of formula I may be halogenated in the 2-position.

The compounds produced according to the invention have not been described in the literature. These compounds have interesting pharmacological properties in animal experiments and can therefore be used as medicaments.

In particular, these compounds show a stimulating effect on dopamine receptors. Dopaminergic properties were detectable in rats induced by injecting 6-hydroxydopamine into the substantia migra unilateral damage to the substantia nigra in the nigro-neosterial dopamine pathway, at doses between about 0.5 to 100 mg / kg (U. Ungerstedt method,

Acta phyeiol. acand. Suppl. 367. 69-93 (1971). After administration of the drug, distinct activation was detectable by the rats performing rotational movements in the direction of the non-denervated side.

The novel substances can be used for the treatment of parkinsonism, by virtue of their dopaminergic properties.

Furthermore, these novel compounds have the effect of inhibiting prolactin secretion.

Thus, these compounds inhibit implantation (entrapment of the fertilized egg in the uterine mucosa) in rats after eubcutaneous administration of doses between 0.01 and 1 mg / kg and lactation using doses of 1 to 10 mg / kg p.o. [cf. Experiments 1330 (1978) 1.

These novel substances can be used for inhibiting lactation, galactorrhosis, for the treatment of hyperprolactinic hypogonadism and acromegaly, or for the treatment of prolactinomas, for example, due to the intrinsic properties of inhibiting prolactin secretion.

Furthermore, the novel compounds in the sleep-wake cycle in a chronically implanted rat from about 3 mg / kg i.p. or 10 mg / kg p.o. shorten the paradoxical sleep time and prolong the wakeful state [cf. J. M. Vigouret et al., Pharmacology 16, 1, 156-173 (1978)]. In addition, these compounds cause an increase in glucose metabolism in the brain nuclei of the sensory information at 0.3 mg / kg i.p. (Method according to I. Solokoff, Journal of Cerebral Blood Flow and Metabolism 1981, 1_, 7-36, by E. Savaki et al., Brain Research 1982, 233. 347 and J. McCulloch et al., Journal of Cerebral Blood Flow and Metabolism 1981, 1, 33-136).

Based on these results, the compounds are now suitable for the treatment of senile dementia, especially in the advanced stage and for increasing vigilance.

In addition, the compounds of the invention possess vasoconstrictor activity that can be demonstrated in vitro on spiral bands of the Arteria carotis axterna of a dog (E. Muller-Schweinitzer, Naunyn-Schmiedeberg ' Arch. Pharmacol., 292, 113-118 (1976)) using doses. from 10 nM / liter.

Because of their vasoconstrictive effect, the new agents are used to treat migraine.

Furthermore, they induce these substances in a rat with a damaged brain and spinal cord [Brit. J. Pharmac. Chemother.30 (1967), 78-87, using a dose of from about 5 to 50 µm / kg i. V. A long lasting presoric effect associated with an increase in blood pressure. In a cat (Angelologica 3 '(1966), 77-99), these agents cause severe, dose-dependent, long-lasting vascular constriction at doses of about 0.5 to about 50 µg / kg i.

Due to this activity, these substances can be used as agents for toning veins.

Finally, the compounds of the invention have a serotoninergic effect based on the results in the sleep cycle. In view of this effect and the results in the Ungerstedt model, these compounds can be used as antidepressants, especially in the elderly.

In the following examples which illustrate the invention in more detail, all temperatures are in degrees Celsius and are not corrected.

Example I

9'-thiaergotamine and 9'-thiaergotamine

1. Cultivation of strain NRRL 12043

(a) Pre-culture:

Q

The conidia of Clavlceps purpurea strain NRRL 12043 (about 10) on sloping agar is suspended in 10 ml of sterile water. Inoculate 200 ml of the pre-culture medium using 1 ml of this suspension of constituents as follows:

sucrose

Ammonium hypochlorite Ca (NO ₃ ) ₂ i4 H ₂ 0 kh ₂ po ₄ MgSO ₄ .7 h ₂ o KC1

FeSO ₄ .7 H ₂ 0 ZnSO ₄ .7 H ₂ 0 distilled water g / ltr

100 ALIGN!

0.25

0.25

0.125

0.0166

0.0068 to 1 liter in a 500 ml Erlenmeyer. The pH was adjusted to 6.5 with NH ₄ OH. The soil is sterilized at 120 ° C for 20 minutes. This pre-culture is shaken at 24 ° C with rotation (180 rpm, 5 cm circumference). At the end of growth, the culture has a pH of 5.3 and the dry mycelium weight is 22 g / liter.

(b) Main culture:

Inoculate 50 ml of the main culture composition with 10 ml of pre-culture ee:

Nutrient media Nucleic acid Ammonium oleate Ca (NO ₃ ) ₂ .4 H ₂ 0 MgSO ₄ . 7 H ₂ O kh ₂ after ₄ KCl

FeSO ₄ .7 H ₂ O ZnSO ₄ .7 H ₂ 0 distilled water g / liter

240

9.6

2.5

0.625

0.625

0.312

0.0252

0.0102 to 1 liter contained in a 500-ml Erlenmeyer; the pK value was adjusted to 6.2 with 25% ammonium hydroxide. the broth is sterilized at 110 ° C for 20 minutes.

This culture is incubated at 24 ° C on a rotating shaker (180 rpm, 5 cm perimeter). After 4 days of culture, 90 mg of L-thiazolidine-4-carboxylic acid is added to the culture. The incubation of the culture is continued for 10 days on a rotary shaker. After completion of the fermentation, the cultures are filtered off. The hycelium is gently dried. The dry mycelium weight of the main culture is 85 g / liter. The alkaloid content is 12 mg / g. The proportion of 9'-thiaergotamine and 9'-thiaergotamine is 10% of the total alkaloid content.

2. Isolation of the title compounds

500 g of dry mycelium are homogenized twice with 1600 ml of methanol and 2% concentrated ammonia, as well as twice with 1200 ml of methanol for 5 minutes each using an Ultra-Turrax, and the combined filtrates are evaporated in vacuo at a maximum bath temperature of 40 ° C (about 50 g of wine red residue). This residue is dissolved in 500 ml of methanol and the solution is filtered through a 10 cm diameter chromatography tube through 500 g of alumina activity II. The reaction mixture was washed with 2000 ml of methanol and all the filtrate was evaporated to dryness in vacuo (25 g of a yellow foam was obtained). This residue was now separated on a 200-fold amount of silica gel 60 (Merck) in methylene chloride and 2% methanol by chromatography. The residue fractions were examined by thin-layer chromatography and those fractions that contained only 9'-thiaergotamine corresponding spots (cf. Rj values in Table 1) were pooled.

9´-thiaergotamine:

Those fractions containing 9-thiergotamine according to thin layer chromatography and in accordance with the corresponding R odpovídající value, are dissolved in ethyl acetate, the solution is filtered through a pad of talc and, after concentration of the filtrate in vacuo, a small amount of hexane is added. Recrystallization from the same solvent gave pure white to yellowish crystals. Melting point: from 199 ° C slow decomposition; [α] D = -165 ° (c = 0.5 in chloroform).

9'-thiaergotamine methanesulfonate:

180 mg of 9'-thiaergotamine is dissolved in a small amount of ethanol and an alcoholic solution containing 29 mg of methanesulfonic acid is added to the solution, which crystallizes the alkaloid as methanesulfonate. Recrystallization from methanol gave a pure white salt, m.p. = + 76 ° (c = 0.51 in methanol).

'-thiaergotaminin:

The thin-layer chromatographed fractions according to the thin-layer chromatogram having a uniform Rf value (see Table 1) are dissolved in ethyl acetate, combined and filtered through a pad of talc to give a clear filtrate. A small amount of hexane was added to the concentrated solution to precipitate the alkaloid as white crystals. Recrystallization from the same solvent gave the pure compound. Melting point: from 199 DEG decomposition; = + 343 ° (c = 0.55 in chloroform).

Table 1

Thin layer chromatography R _f values compared to ergotamine (Et) and ergotaminine (Et-in)

Layer and solvent system

Thia-Et

Thia-Et-in Et-in mixture of ethyl acetate and sec. Butanol (9: 1) mixture of toluene and isopropanol (9: 1) mixture of toluene and isopropanol (19: 1).

silica gel ^{* 2} ) mixture of teluene and isopropanol (85:15) mixture of methylene chloride and methanol (93: 7)

0.44 0.34 0.68 0.59 0.58 0.50 0.69 0.63 0.30 0.24 0.58 0.46

0.15 0.11 0.43 0.31 0.25 0.22 0.65 0.52

Merck F 254 (Type E) alumina plates ² ) Merck F 254 silica gel plates.

On thin-layer chromatography plates, the spots appeared as blue-violet spots at ultraviolet light at 254 nm and as pale blue spots at 366 nm. They can also be detected by iodine vapors as brown spots.

Uro-ergoteptide-typing reagent is excellently suited for spotting fabrics.

After spraying, these compounds show a blue-violet color, which is more intense due to the action of daylight or water-jet (Hauch-Konig).

Example 2

9'-thiaorgocristin and 9'-thiaergocristinin

Cultivation of HRRL 12044 strain

The strain 12044 ee is cultivated in an analogous manner to that described in Example 3. The dry mycelium of the main culture is 80 g / liter. The alkaloid content is 10 mg / g. The proportion of the title compounds is 8% of the total alkaloid content.

2. Isolation of the title compounds:

500 g of dry mycelium are homogenized twice with 1500 ml of methanol and 2% concentrated ammonia in each case and homogenized twice with 1500 ml of methanol for 5 minutes each using an ultraturrax. The combined filtrates are evaporated in vacuo at a maximum bath temperature of 40 ° C (190 g of wave red residue). After dissolution in 900 ml of methanol, the solution obtained is filtered with a 10 µm layer of 600 g of alumina (basic, 11th grade activity). layer was washed with 2000 mL of methanol and the filtrate was evaporated to dryness in vacuo (108 g).

This residue was now partitioned in 3,000 g of Sephadex LH-20 in methanol, collecting 200 ml fractions and evaporated. The residue fractions containing the new thia-ergoteptides according to thin layer chromatography were intoxicated (526 mg) and re-chromatographed on (20 g of Sephadex LH-20 in methanol to give 156 mg of a pure mixture of 9'-thiaergocristin and 9 g). The two compounds were separated by known chromatographic methods on silica gel 60 (Merck) using methylene chloride and 2% methanol.

9'-thiaargskristin:

Fractions which, according to thin-layer chromatographic analysis and in accordance with its value (cf. Table 2), contain only 9 ' -thiergocristine are combined and crystallized from benzene (28 mg). Melting point 146 ° C (slow decomposition) = -176 ° (c = 0.5 in chloroform).

9'-thiaergocristinin:

The chromatographic fractions, which by TLC appears to be uniform and to stain a substance in accordance with the respective values of R _f (see Table 2) were combined and crystallized from a mixture of ethanol and ethyl acetate. Mp 209 DEG-210 DEG C. with decomposition; [.alpha.] D = + 344 DEG (c = 0.51 in CHCl3).

Table 2

R ^ values determined by thin-layer chromatography compared to ergocristin (Ec) and ergocristinin (Ec-in) w values

Layer and solvent - Thia-Ec-Ec Thia-Ec-in-Ec-system ethyl acetate mixture

and sec. butanol (9: 1) 0.68 0.63 0.78 0.70 toluene / isopropanol (9: 1) 0.68 0.62 0.69 0.66 toluene / isopropanol (19: 1) 0.50 0.42 0.58 0.47 silica gel ²⁾ toluene / isopropanol mixture (85:15) 0.37 0.28 0.56 0.48 methylene chloride / methanol (93: 7) 0.47 0.41 0.70 0.60

Explanation to Table 2:

Merck F 254 (type e) Merck F 254 finished alumina plates

The same applies to the detection of fabric spots as in Example 1.

Example 3

In an analogous manner to that described in Example 1 or 2, 9'-thia-6-ergecryptine can also be prepared [m.p. 203-205 ° C (decomposition); [= + 46 ° (c = 0.7 in dimethylformamide)] and 9'-thia-α-ergocryptinine [m.p. 228-230 ° C (decomposition); [α] D = + 281 ° (c = 0.8 in dimethylformamide)] by using a strain producing a mixture of ergocryptin and ergocryptinin .

Claims (1)

SUBJECT OF THE INVENTION A process for the preparation of ergopeptin derivatives of the general formula (I) in which either R 1 represents a methyl group and R 8 is isobutyl or benzyl, or R1 is ethyl and R 8 is benzyl or R 1 represents an isopropyl group and Rg is an isopropyl group, a sec-butyl group, an isobutyl group or a benzyl group, in free base or acid addition salt form, by culturing a strain of Claviceps purpurea producing the corresponding 9'-methylenderivative or a strain derived from Claviceps purpurea, 1 to 5 g / l of L-thiazolidine-4-carboxylic acid or a salt thereof is added to a liquid culture of this strain, cultivation is carried out at a temperature of 22 to 26 ° C and the resulting compounds are optionally converted into their editoral salts and acids.