DE1620375A1 - Process for the production of lysergic acid - Google Patents

Description

Process for the preparation of lysergic acid The present invention relates to a process for the production of lysergic acid by rearrangement of 6-methyl- # 8,9-ergolen-8-carboxylic acid.

This rearrangement according to the invention is caused by the action of bases accomplished on the 6-methyl- # 8,9-ergolen-8-carboxylic acid, preferably in water or an organic solvent which is inert under the conditions of the rearrangement or a mixture of these. The rate of rearrangement can be increased by heating increase. Inorganic bases such as ammonia or are suitable for the rearrangement Alkali metal hydroxides or organic bases such as pyridine.

The isolation of the 6-methyl- # 8,9-ergolen-8-carboxylic acid caused by the rearrangement according to the invention lysergic acid obtained from its solutions is carried out according to methods known per se, preferably using a strongly acidic ion exchanger, for example based on sulphonic acid resins such as Dowex 50 or Amberlite IR 120.

The 6-methyl-8, 9-ergolen-8-carboxylic acid used as the starting compound is used as the main product in the saprophytic cultivation of a new mushroom standard, reis, his mutants, who made it Sclerotia on the grass Paspalum dilatatum grown in the vicinity of Coimbra (Portugal) can be obtained could get.

Samples of this fungus strain were obtained from the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, Ill., deposited there and received the number NRRL 3080. This new strain of Claviceps Paspali Stevens et Hall is in contrast to all other tribes described so far this type of fungus (see e.g. Proc. Roy. Soc. Serials B, 155, 33) in vitro for conidia formation, able to. It is namely for the execution of microbiological processes in the technical A measure of great importance as to whether the fungus in question forms conidia or not. With the help of xonidia, it is possible to isolate single spores and thereby to arrive at genetically uniform material.

Conidia can also be obtained by means of X-ray and UV irradiation, as well as It is much easier to generate and isolate mutants with chemicals than from sterile ones uycel. Furthermore, glowing Paspalum plants can be infected with conidia and act on them Way on the natural host sclerotia are generated, which are sur conservation of Tribal. One can for example from this durable material at any time to gain fresh vaccine when the in vitro cultures degenerate after repeated passages. Conidia can also be lyophilized or can be stored indefinitely after freeze-drying, which is also the preservation of a tribe. Ultimately, however, one is suitable for inoculation Nutrient solution a conidia suspension is best because the amount of vaccination is the most suitable can be dosed and because it is the easiest way to work sterile. A sterile mycelium, on the other hand, has to be homogenized in a mixer at the beginning Isolation and propagation of the new strain takes place in the following way: From the Inside a sclerotium, a small piece of tissue is removed under sterile conditions and placed on wort agar transferred (Composition: 250 ml unhopped light beer wort (dry matter 17%), 18 g agar - agar, dist. Water ad 1 L (pH 5.2)]. A circular shape develops Colony that reaches 15 mm in diameter after 14 days at 24 ° C. she consists of an approx. 1 mm thick pseudosclerotial skin lying on top of the agar Structure and above it a cushion of white air marcel. A brown dye diffuses into the agar. Iceine conidia are formed.

This colony is cut into pieces with a spatula and into a Transfer a test tube with 12 cm3 of the following agar medium: 500 ml beer wort Cornsteep-Solids 60 g lactic acid 1 ml salmia solution up to pH 4.8.

Agar-agar 20 g dist. Water ad 1 L.

A small colony of initially ceissem, later red-brown mycelium. After 10 days, conidia begin at the hyphae tips to pinch off.

After 20 days there are enough conidia to create a watery one Prepare a suspension with which 20 slant tubes (same agar as above) can be inoculated. These cultures are incubated at 240 ° C.

The conidia germinate after 24-36 hours. After 6 days the agar surface is Evenly covered by a fine, white mycelium, after 10 days one is brown gray, finely furrowed mycelial cover formed, which lies closely on the agar and is only short Has aerial hyphae. Conidia are pinched off at these. After 12 days arise several places in the mycelium centers at which small droplets of a red-brown Liquid are excreted. The droplets reach a through knife from 1 - 3 mm and soon become milky-cloudy with numerous conidia. After i6-i8 The conidia formation is practically complete within days. A sloping agarcture in a test tube 2 cm in diameter with 12 ml agar medium contains approx. 109 Conidia.

For breeding in submerged culture, a preculture such as The following is prepared: A 4.5 percent aqueous malt extract solution is used as the medium with pH 5.4 used. One liter of this solution is poured into a 2 L Erlenmeyer flask Sterilized for 20 minutes at 1100 C, then with 6,108 conidia from a 15 day period inoculated into old agar culture and on a rotary shaker for 3 days incubated at 240 ° C. A dense culture of fine mycelium flakes is created. the Flakes are made up of a loose tangle of hyphae and have a diameter of 2 4 mm.

No alkali can be detected.

For the production of larger quantities of preculture, glass fermenters are used Contains 10 L each of the same medium, inoculated with 6,109 conidia each and with 3 days 230 e with aeration with 6 L air per minute and stirring at 300 r.p.m. incubated. A silicone emulsion is used to combat foam. The fermenters obtained in this way cultures are of the same nature as the shaking cultures. For the main culture The following nutrient solution turned out to be in 1 L L of distilled water Sorbitol 50 g succinic acid 36 g KH2PO4 2 g MgS04 0.3 g FeSO4. 7 H20 1 mg ZnSO4. 7 H2O 10 mg and has been adjusted to pH 5.4 with NH4OIH, is particularly useful.

This nutrient solution is inoculated with 10% of a 3-day-old preculture and in portions of 100 ml in 500 ml Erlenmeyer flasks on a reciprocating shaking Machine incubated at 23 ° C. Other cultures are in an analogous way in one 170 L stainless steel fermenter containing nutrient medium. It will aerated with 170 L air per minute and initially at 70, later 180 r.p.m. touched. A silicone emulsion is used to combat foam.

In this way, cultures emerge from numerous. similar Mycelium particles. These have a diameter of approx. 5 mm and have a spherical, compact core of approx. 1 mm diameter made of pseudoparenchymatic tissue. This The core has star-like, approx. 2 mm long appendages made up of parallel Hyphae. At the end of the approx. 10-day culture period, the mycelium is dark brown, and that The filtrate was intensely colored red-brown. The pH value changes only insignificantly.

The sulture filtrate obtained in this way has a colorimetric certain total alkaloid content of 620 mg / L, based on a molecular weight of 300. The composition of the alkaloid mixture determined by paper chromatography is roughly: 6-methyl- # 8,9-ergolen-8-carboxylic acid and small amounts of lysergic acid and 86.5% isolysergic acid lysergic acid amide 3.9% isolysergic acid amide 3.9 9% ergobasin 1.0% Ergobaslnln 0.5% Clavin alkaloids 4.2% By treating the conidia of the Fungal strain tERL 3080 with ethyleneimine and ultraviolet light on known per se Way, whereby the survival rate is 0.2 to 2%, one obtains a mutant with higher production capacity of 6-methyl- # 8,9-ergolen-8-carboxylic acid, samples of this Mutants have been obtained from the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, Ill., Deposited and received the Number NRRL 3167.

By growing the mutant in the shaking culture described above a yield of 3330 mg of oesamtalkaloids is obtained per liter Culture filtrate made up of 89% 6-methyl- # 8,9-ergolen-8-carboxylic acid, low narrow Lysergic and Isolysergic Acid and 11% Clavin Alkaloids.

In the fermenter culture described above, a total of 2090 mg total alkaloids per liter of culture filtrate is achieved with the same Composition as in the shaking culture filtrate.

The culture filtrate obtained in this way can be processed with a base Lysergic acid can be obtained directly or 6-methyl- # 8,9-ergolen-8-carboxylic acid are isolated and these are rearranged by treatment with bases in lysergic acid.

This is used for direct basic processing of the culture filtrate evaporated and the remaining residue, if necessary with heating with bases, preferably with dilute ammonia solutions or solutions of alkali metal hydroxides treated.

The rearrangement according to the invention can also be carried out in this way, for example that you can get the 6-methyl-8,9-ergolen-8-carboxylic acid from the evaporation residue of the culture filtrate extracted with dilute alcoholic ammonia and then carries out the rearrangement.

Another preferred method is that the culture filtrate with a start acidic cation exchanger like Dowex 50 or Amberlite IR 120, treats the ion exchanger, for example, with dilute aqueous ammonia eluted and the eluate containing 6-methyl- # 8,9-ergolen-8-carboxylic acid for rearrangement used. This rearrangement can be achieved by simply heating the basic solution or be carried out after adding further bases.

The isolation of the obtained by the rearrangement according to the invention Lysergic acid can be carried out by methods known per se, such as by setting the pH of the solution to the isoelectric point of lysergic acid, but is preferably the isolation was carried out using a strongly acidic ion exchanger.

In the following examples showing the implementation of the process explained, but in no way limit the scope of the invention all temperatures in degrees Celsius; the melting points are corrected.

For example: a. 6-methyl- # 8,9-ergolen-8-carboxylic acid 5 L culture filtrate of the new strain of Claviceps paspali Stevens et Hall with a colorimetric certain juice content of ergoline derivatives of approx. 500 mg / L (based on a mol. of 300). and a pH of 5, are passed through a column slurried with water of, 590 g Amberlite IR 120 (H + form; diameter of the column 2.8 cm; height 115 cm). filtered. The thermosetting rate is 500 ml / hour.

After washing the column with 1 L of water, the 6-methyl- # 8,9-ergolen-8-carboxylic acid becomes with 5 percent Ammonia elutes.

The flow through, collected in fractions of 500 ml each, is indicated by fluorescence in the W. and color reaction according to Keller (FeCl3 glacial acetic acid, 112504 conc.) on the content tested on ergoline derivatives. The first four fractions (total 2 L) steam to 500 ml at 13 mm Hg and a bath temperature of 300, the solution is made with glacial acetic acid to a pH value of 5.5, filtered from the precipitated resin (cellar color reaction negative), the filtrate is concentrated in vacuo to approx. 25 ml, 20 ml of methanol are added, the solution boils briefly and lets stand at 50 for a few hours. The crystallized After filtering off the acid, it is washed with water and methanol and at 800 2 Hrs. Dried in vacuo. The next seven fractions of the ammonia run-through after the same work-up give a further amount of crystallized 6-methyl- # 8,9-ergolen-8-carboxylic acid.

The crystallized products are used to purify the crude acid united, in 5 percent. dissolved alcoholic ammonia, the solution after filtration with 2N. Acetic acid adjusted to pH 5.5, warmed briefly on the water bath, which crystallized Acid filtered off after a few hours, washed with water and methanol and in vacuo dried at 80 °. M.p. 243-245 ° (dec.); [α] D = -180 ° (0.1N · NaOH).

Color reaction according to Keller, van Urk and Ehrlich as with lysergic acid ; Thin layer chromatogram on silica gel with Alcohol / 25 percent Ammonia (9: 1) as a flow agent: Rf value = 0.4 to 0.45. The stain gives when spraying turns blue with Ehrlich's reagent.

UV spectrum: # # 0.1N NaOH g £ = 217.5 / 4.56; 282 / 3.79; 292 / 3.74, minimum at 252 mµ. IR spectrum: characteristic bands at 3340, 2275 (broad), 1674 and 1580 cm 1 (in NuJol). b. LXsergic acid 500 mg from the culture filtrate of the new Claviceps paspalum strain isolated crude, crystallized 6-methyl- # 8,9-ergolen-8-carboxylic acid are heated in 10 ml of 2N sodium hydroxide solution for 2 hours on a water bath, the hot solution treated with activated charcoal and the filtrate with dilute hydrochloric acid and glacial acetic acid set a pH of 5.5. After a few hours the crystallized lysergic acid becomes filtered off, washed with water and methanol and dried at 800 in vacuo: M.p. 245-247 ° (dec.).

The lysergic acid thus obtained has all of the literature for this compound chemical and physical properties described.

Example 2: 10 1 of the culture filtrate obtained as described above are evaporated on a water bath and the residue with heating to about 500 extracted with 3 x 500 ml of 5% alcoholic ammonia solution. The extract is in a rotary evaporator to half the original volume geent, the one left behind concentrated solution adjusted to pH 5.5 with glacial acetic acid, treated with activated charcoal and the filtrate is concentrated to approx. 20 ml in vacuo, crystallization commencing. The mixture is left to stand for a few hours at 5 °, the precipitate is filtered off, with water and methanol washed, dried at 800, and by recrystallization or reprecipitation cleaned. The lysergic acid thus obtained melts at 245-247 ° and has all in the Chemical and physical properties described in the literature for this compound.

Example 3: 10 1 of the culture filtrate obtained as described above are stirred with 500 g Amberlite IR 120 (H + form) for approx. 12 hours treated, the ion exchanger is filtered off and the filtrate is treated again as described above using 250 g of the same cation exchanger. The color reaction according to Keller of a sample evaporated to dryness (5 ml) to the after twice treatment with the exchange resin resulting culture solution falls negative. The two portions of the ion exchanger are each twice during Stirred for 4 hours with the same amount of water by weight. Everyone is now dealt with Portion of the ion exchanger three times with the same weight of a 5% aqueous Ammonia solution with exclusion of carbon dioxide for 12 hours at 5-10 °. the individual Extracts are placed in a water bath at approx.

1000 in the rotary evaporator to half of the original volume concentrated, the concentrates adjusted to pH 5.5 with glacial acetic acid, possibly small amounts precipitated resin filtered off and concentrated in vacuo to 15-20 ml and continue as in Example 2, the lysergic acid is isolated and purified.

Instead of the ion exchanger used here, other strong ones can also be used acidic ion exchangers such as B. Dowex 50 can be used.

Claims (10)

Claims: 1) Process for the production of lysergic acid, characterized in that 6-methyl- 8,9-ergolen-8-carboxylic acid of the formula rearranged by the action of bases in lysergic acid and isolated from the solution. 2) Method according to claim 1 and 2, characterized in that one 6-Methyl- # 8,9-ergolen-8-carboxylic acid in water or under the conditions of Rearrangement of inert organic solvents or a mixture thereof in lysergic acid surrounded. 3) Method according to claim 1 and 2, characterized in that one the rate of rearrangement is increased by heating. 4) Method according to claim 1, 2 and 3, characterized in that that the isolation of the lysergic acid obtained by rearrangement from its solutions takes place using a strongly acidic ion exchanger. 5) Method according to claim 1, characterized in that as Starting material a solution of 6-methyl- # 8,9-ergolen-8-carboxylic acid in alcoholic Ammonia is used, obtained by evaporation of the culture filtrate of Clavioeps paspali Stevens et Hall NRRL 3080 or its mutants and extraction of the residue with alcoholic ammonia has been obtained. 6) Method according to claim 1, characterized in that as Starting material a solution of 6-methyl- # 8,9-ergolen-8-carboxylic acid in aqueous Ammonia is used by treating the culture filtrate of Claviceps paspali Stevens et Hall NRRL 3080 or its mutants with a cation exchanger and Eluting the exchanger with aqueous ammonia has been obtained. 7) Process for the production of lysergic acid and isolysorgic acid, thereby marked that the in vitro dor coniZ dia formation is lacking in the fungus strain NRRL 3080 the Spooios claviceps paspali Stevens et Hall or its mutants - in saprophytiacher Culture grows and lysorgic acid and isolysergic acid by alkaline work-up of the culture filtrate isolated. 8) Method according to claim 7, characterized in that the culture filtrate is evaporated and the remaining residue is treated with bases. 9) Method according to patent claim 8, characterized by the fact that one is predunnton as Bano Uses ammonia and carries out the treatment at elevated temperature. 10) Method according to claim 8, characterized in that inan used as a base alkali metal hydroxides.