NO744292L - - Google Patents

Description

The present invention relates to a method for the production of the new antibiotics S 7481/F-1 and S 7481/F-2.

The peculiarity of the method according to the invention for the production of the new antibiotics S 7481/F-1 and S 7481/F-2 is that a strain of the mushroom species Cylindrocarpon lucidum Booth respectively. the fungus Trichoderma polysporum (Link ex Pers.) Rifai is grown on

or in a nutrient medium, the new antibiotics are isolated from the fermentation broth in a manner known per se by extractive and/or adsorptive working methods and then purified chromatographically or by means of countercurrent distribution.

The new strain of the fungus Cylindrocarpon lucidum Booth used according to the invention was isolated from a soil sample found in Wisconsin, USA and a culture thereof was deposited in the United States Department of Agriculture (Northers Research and Development Division), Peoria, 111., USA under number NRRL 5760.

The new strain NRRL 5760 of Cylindrocarpon lucidum Booth agrees

largely consistent with the original description (C. Booth 1966;

The Genus Cylindrocarpon; Mycological Paper No. 104, 21). On potato dextrose agar, the strain NRRL 5760 forms neither sticky dysspores nor microconidia. Chlamydiaspore-active cells in the macroconidia are likewise lacking. The banana-shaped macrokenidia, usually divided by three transverse walls, are 28 - 45 x 4.8 - 5.7^u in size and are thus significantly smaller than indicated in the above description (45 - 65 x 6 - 6.5^u). A further important difference consists in the growth rate, as while this Typus should form 1 - 1.5 cm large colonies in the course of 7 days, the strain NRRL 5760 grows in the course of the same time to 7 - 8 cm large colonies, as on potato-dextrose agar almost exclusively colorless to brown substrate mycelium is formed. The conidiophores with the phialides form already after a few days in dense groups and bunches, branch penicillium- to verticillium-like and over time form macroconidia that collect as a slimy, beige mass.

The new strain of the fungus species Trichoderma polysporum used in accordance with the invention was isolated from a soil sample found in Norway and a culture thereof was deposited in the United States Department of Agriculture (Northern Research and Development Division), Peoria, 111., USA under no. NRRL 8044.

The new strain NRRL 8044 of Trichoderma polysporum (Link ex Pers). Rifai grows on 2% malt extract agar at 6 to 33°C and the growth optimum is at 24°C, as pure white, velvety on the surface and unfolded colonies with a transverse dimension of 40 - 50 mm are formed in the course of 12 days. At the highest temperature that still allows good growth (33°C), on the other hand, no aerial mycelium is formed above the head, but instead a very strongly folded colony. With increasing cultivation temperature, a growing yellow pigment forms on the underside of the colony, which diffuses into the agar at 27°C and more pronouncedly at 30 - 33°C.

The new strain of Trichoderma polysporum needs 15 - 20 days at 24°C on malt extract agar to form the conidiophores, which are both scattered but also in denser groups, above all on the edge of the culture vessel. The morphology of the bifruit form deviates in some features slightly from the description for this species (M.A. Rifai 1969: A REVISION OF THE GENUS TRICHODERMA in Mycological Papers, No. 116). Thus, the main branch of the conidia-bearers only reaches a transverse measurement of 2.0 - 3.8 ^a, while that for the species is indicated as 4 6.3 ^u. The phialides in the new strain of Trichoderma polysporum are, especially at the end of their development, elongated in length and this is typical for this species. Finally, the conidia are somewhat smaller than stated in the species description, namely 2.0 - 3.1 x 1.5 - 2.0 compared to 2.4 - 3.8 x 1.8 2.2^1. The present Trichoderma polysporum strain NRRL 8044 forms under all the mentioned cultivation conditions

no true chlamydospores.

The new mushroom strains NRRL 5760 and NRRL 8044 can be grown on many types of nutrient substrates, which contain the usual nutrients for mushrooms. Thus, these strains use the nutrients normally used for the carbon-heterotrophic organisms, e.g. sucrose, glucose, maltose, lactose, starch, malt extract as a carbon source, organic and inorganic nitrogen-containing compounds, such as corn starch, soy peptone, yeast or meat extracts, sodium nitrate, ammonium sulfate, ammonium nitrate, amino acids, etc., as a nitrogen source as well as the usual mineral salts and trace substances .

The new antibiotics S 7481/F-1 and S 7481/F-2 can be produced

in such a way that

1. A liquid medium is inoculated with a conidia and mycelium suspension

of the strain NRRL 5760 and the culture is incubated for 9-13 days, preferably 11 days, at 24-30°C, preferably 27°C, and at a pH value of 4.8-8.8, preferably at 8.1. Cultivation takes place in penicillin bottles under aerobic conditions in a surface culture.

2. A liquid medium is inoculated with a conidia and mycelium suspension

of the strain NRRL 8044 and the culture is incubated for 11 - 18 days, preferably 13 days, at 25 - 30°C, preferably at 27°C, and at a pH value of 4.3 - 6.2, preferably at 5.6 in a steel fermentation vessel under stirring (170 revolutions per minute) and aeration

(1 liter of air/minute/liter of nutrient solution).

As soon as a maximum quantity of antibiotics has been produced, these are extracted from the culture broth in a manner known per se by extractive and/or adsorptive methods.

A method that has proven to be appropriate consists in the extraction of the mycelium obtained from the cultivation of the mushroom strain NRRL 5760, optionally after previous mechanical division with 90% methanol, subsequent evaporation of methanol and extraction of the resulting aqueous mixture with ethylene chloride several times. To this extract can be added the ethylene chloride extract which is recovered from the cultivation filtrate. However, other water-immiscible solvents can also be used, e.g. chloroform, ethyl acetate, methylene chloride or butyl acetate.

The organic solution is evaporated to dryness and the residue is defatted either by chromatography, e.g. on silica gel with chloroform with a little methanol, e.g. in the ratio 98:2, or by distribution with petroleum ether and methanol, which has been mixed with an amount of water sufficient for phase separation. In the first case, the fractions that are antibiotically active against Aspergillus niger are combined, evaporated to dryness and the residue chromatographed in methanolic solution on "Sephadex LH 20". In the second case, the methanolic-aqueous phase is concentrated, optionally after the addition of further water, until the main amount of the methanol has been distilled off, after which the aqueous mixture is extracted with ethylene chloride, or another of the above-mentioned, water-immiscible solvents, the the resulting organic solution is evaporated to dryness and the residue is chromatographed in methanolic solution on "Sephadex LH 20". The further separation and purification of the two antibiotics takes place by renewed chromatography on aluminum oxide or silica gel, or by subsequent application of both adsorbents.

A further method, which has proven to be appropriate, consists in the extraction of the culture broth obtained during the cultivation of mushroom strain NRRL 8044 with the help of a water-immiscible solvent, possibly after previous mechanical division. Ethylene chloride can preferably be used as a solvent, but chloroform, ethyl acetate, methylene chloride or butyl acetate can also be used. The organic phase is separated, dried and evaporated. The residue is chromatographed, preferably with a methanol solution on "Sephadex LH 20", as the fractions active against Aspergillus niger are combined and chromatographed once more on aluminum oxide with toluene to which 10 - 50% ethyl acetate has been added. The detection of the active substances also takes place here with activity samples against Aspergillus niger. The purification of the two antibiotics takes place by

renewed chromatography.

For the production of the new antibiotics, strains can also be used, which can be obtained e.g. by selection or mutation under the influence of ultraviolet or X-rays or by the application of other precautions, e.g. when treating laboratory cultures with suitable chemicals.

The new antibiotics S 7481/F-1 and S 7481/F-2 are distinguished by interesting chemotherapeutic and pharmacological properties and can therefore be used as medicine. Thus, both antibiotics inhibit the growth of Aspergillus niger. In particular, the substance S 7481/F-1 is distinguished by an immunosuppressive effect. Both S 7481/F-1 and S 7481/F-2 also show anti-inflammatory properties. It should be emphasized that both antibiotics are not cytostatically active.

The immunosuppressive effect of S 7481/F-1 can be shown on

following way:

a) Local hemolysis in gel in vitro, the brain sample:

Inhibition of hemolysis in % compared to control samples constitutes wood

a dose of 2 x 60 mg/kg i.p. 99%, at a dose of 1 x 150 mg/kg p.o. 90% and at a dose of 3 x 200 mg/kg p.o. 99.5%. Both the formation of immunoglobulin M and immunoglobulin G antibodies is inhibited.

b) Hemagglutination test (HAT) in mice:

The anti-sheep erythrocyte antibodies are determined.

The inhibition is expressed in suppression index (SI), with the non-treating control sample being indicated by 1.00. SI = 0.3 after p.o. 5 x 200 mg/kg.

c) Skin transplantation sample in mice:

In H-2 histoin-tolerant mice, the survival time extension of the skin graft is prolonged compared to untreated control animals after a dose of 10 x 200 mg/kg'p.o. to some extent: approx. 16 days longer than the control samples. d) Experimental allergic encephalomyelitis (EAE) in rats: The experimentally induced nerve tissue damage, which paralyzed 8 of

10 animals in untreated control animals, gave the same number

experimental animals that had been treated with 13 x 25 mg/kg i.p., a 100% protection.

e) Oxazolone test in mice:

The decrease in ores swelling is expressed as suppressive index

(SAY); SI = 0.6 after 6 x 75 mg/kg p.o.

f) Cytopenia in bone marrow and blood:

The high oral intake of 6 x 500 mg/kg does not clearly show any

depression of the blood-forming cells in the bone marrow and no leukopenia in the blood on the 1st, 3rd and 7th day after the last substance supply.

Due to their immunosuppressive action, the compound can

S 7481/F-l is used for the prophylaxis and treatment of diseases that are used in connection with influencing the defense reaction in a negative way.

The new antibiotics S 7481/F-1 S 7481/F-2 also have an anti-arthritis effect. Thus they work, e.g. in Freund-Adjuvans-Arhtritis-La.tenztidsorsok in rats strongly anti-swelling in doses of approx. 50 mg/kg body weight.

A similar effect is observed in Freund-Adjuvant-Arthritis therapy trials in rats in doses of approx. 50 mg/kg/day.

Due to their anti-arthritis effect, these antibiotics can be used for the prophylaxis and treatment of arthritis and rheumatic diseases.

The doses used naturally vary according to the type of antibiotic, the method of administration and the condition to be treated. Usually, however, satisfactory results are achieved in experimental animals with a dose of 10 to 20 mg/kg body weight. If necessary, this dose can be given in 2 to 4 partial doses or also as a slow-release form. For larger mammals, the daily dose is approximately 50 to 900 mg. For oral administration, the partial doses can e.g. contain approximately 25 to 300 mg of the new antibiotics alongside solid and liquid carrier substances.

As medicine, the new antibiotics can be administered alone or in suitable preparation form with pharmacologically indifferent excipients. 1 the following examples which will illustrate the invention in more detail, all temperature indications are in °C.

Example 1.

o

10 liters of nutrient solution which per liter contains 30 g sucrose, 10 grams corn-stop, 3 grams NaNO^, 1 gram K^HPC^, 0.5 g MgS04 x 7H20, 0.5 gram KC1 and 0.01 gram FeS04 x 7H20 inoculated with .100 ml of a conidial and mycelial suspension of the strain NRRL 5760 and incubated in penicillin bottles of 700 ml volume each for 11 days at 27°C.

The mycelium separated from the culture broth is extracted by crushing and stirring with 3.5 liters of 90% methanol and the crushed mycelium separated by suction on a filter from the solvent is treated a further 2 times in the same way with 90% methanol. The combined filtrates are evaporated in a vacuum at 40° C. bath temperature to such an extent that the liquid mainly consists only of water. The resulting mixture is extracted 6 times with the same volume of ethylene chloride by shaking, after which the combined ethylene chloride solutions are purified by shaking with water and evaporated in vacuo at 40°C bath temperature. The residue thus obtained is chromatographed on 250 grams of silica gel (silica gel 60 "Merck", grain size 0.063 - 0.200 mm) using chloroform with an addition of 2% methanol as eluent, which is collected in 200 ml fractions. The fractions that are antibiotically active in the plate diffusion test against Aspergillus niger are combined, evaporated to dryness in the described manner and chromatographed after dissolving 10 grams of "Sephadex LH 20" in methanol with the same solvent, after which the 20 ml fractions as for the same sample are combined as above has been shown to be antibiotically effective against Aspergillus niger. In the examination in thin-layer chromatogram, e.g. with the help of silica gel on "Polygram" foils and hexane-acetone (1:1) as a fluid, it turns out that the rest of the methanol solution evaporated in the manner described above mainly consists of the two new substances S 7481/F-l and S 7481/ F-2. They are separated and purified at the same time, their mixture being chromatographed again using a thousandfold amount of silica gel of the above quality and chloroform to which 2% methanol has been added. The examination of the eluate fractions, whose volume in ml is half as large as the weight of silica gel in grams, by thin layer chromatogram shows that the substance S 7481/F-l first appears in the eluate followed by a mixture of the two substances and then by uniform S 7481/F -2. Further quantities of the two substances can be recovered from the mixture under the same conditions by repeated chromatography.

The new antibiotic S 7481/F-l has the following properties:

Amorphous, colorless powder with melting point 137 - 140°C (decomposition);

/ a7p° -236° (CHC13, C 0.5)

UV: see fig. 1.

IR: see fig. 2.

Proton NMR: see fig. 3.

13

C-NMR: see the table.

Mass spectrum (CEC mass spectrometer 21-1108, ion source E.B.: Introduction system: direct, electron energy: 70 eV, ion acceleration: 4 kV, temperature of the ion source, 290°C, pressure: 5.IO<-6>Torr,

Peak-Matching procedure, reference substance "PCR 8" /Tris-pentadecafluoroheptyl-1,3,5-triazine, M=1184, 9374/: hSyest mass peak m/e = 1183.831 (+ 0.004), corresponding to the mass of the ion C62 <H>109N11<<>^11' Pro<^u^tet from the elimination of water from the real molecule C62<H>iiiNn<0>i2<*>LSolubility: easily soluble in most common organic solvents, practically insoluble in petroleum ether and water. Appearance in thin-layer chromatogram (substance stains made

-visible with iodine vapor):

a) Silica gel on glass plates: chloroform + 4% methanol as emulsifier: Rf = 0.52. b) Silica gel on foils "polygram"; hexane/acetone 1 : 1 as eluent: Rf = 0.37; chloroform/acetone 2 : 1 as eluent: Rf = 0.34.

Elemental analysis (the sample dissolved in benzene, the solution evaporated to dryness, the residue dried in high vacuum for 5 hours at 100°C): found C 61.8 H 9.4 N 13.0 O 15.7%.

The values found agree with the gross formula,

<C>62<H>111<N>11°12-

In the hydrolyzate, which is obtained by boiling the compound S 7481/F-1 with 6 N hydrochloric acid (20 hours), the amino acid alanine, α-aminobutyric acid, N-methylleucine, N-methylglycine and valine can be detected. The

new antibiotic S 7481/F-2 has the following properties:

Amorphous, colorless powder with melting point 127 - 130°C ;(;cleavage) • /o/£° = -243° (chloroform, c = 0.5)

UV: see fig. 4.

IR: see fig. 5.

Proton NMR: see fig. 6.

Mass spectrum (recording conditions see antibiotic S 7481/F-l): HSyest mass peak m/e = 1169.815 (+ 0.006), corresponding to the mass of the ion C5i<H>io7Nll°ll'Proc^u^tet from the elimination of water from the molecule<Gg >1<H>l09<N>ii<o>i2<*>

Solubility: easily soluble in most common organic solvents-: practically insoluble in petroleum ether and water.

Conditions in thin-layer chromatogram (substance spots made visible with j odd vapour): a) Silica gel on glass plates; chloroform + 4% methanol as eluent: Rf =. 0.46.

b) .Silica gel on foils "polygram"; hexane/acetone 1 : 1 as eluent: Rf = 0.28; chloroform/acetone 2 : 1 as liquid

mean: Rf = 0.26.

Elemental analysis (sample dried for 5 hours in high vacuum at 100°C): Found C 61.7 H 9.1 N 13.1 O 16.5%.

The values found agree with the gross formula<C>61<H>109<N>11°12'

The conidia and mycelium suspension used as starting material for the inoculation is prepared from a culture of the originally isolated strain NRRL 5760, which was grown for 10 days on an agar medium, which per liter contained 20 grams of malt extract, 20 grams of agar, 4 grams of garlic extract and demineralized water. The conidia and mycelium were absorbed in physiological saline solution. This suspension serves for inoculation of the above nutrient solution.

Example 2:

2 50 liters of a nutrient solution that per liter contained 30 grams sucrose, 10 grams corn=stop, 3 grams NaNO^/1 gram K2HP04, 0.5

gram MgS04x 7H2O, 0.5 gram KC1 and 0.01 gram FeSC>4x 7H2O were inoculated with 2.5 liters, conidial and mycelial suspension of strain NRRL 5760 and incubated in penicillin bottles of each 700 ml room content for 11 days at 27 °C.

From the culture broth, the mycelium is separated by centrifugation and homogenized in an ultra-fast mixer with 80 liters of 90% methanol, then the methanolic extract solution is separated by centrifugation

-from the solid and the latter is extracted two more times in the same way with 90% methanol. The combined extract solutions are concentrated in vacuo at 30 - 40°C to 15 litres. The remaining aqueous phase is extracted 8 times with the same volume of ethylene chloride. Each of the ethylene chloride extracts is washed with 5 liters of water. The ethylene chloride solutions are evaporated after union in vacuum at 30 - 40°C. For degreasing, the extract residue is distributed between petroleum ether and 90% methanol in such a way that, after dissolving in 2 liters of 90% methanol, it is successively shaken 3 times with each time 2 liters of petroleum ether (boiling point 30 - 35°C), which is in 3 shaking funnels, after which the 3 petroleum ether phases, one after the other, are extracted 2 times with 2 liters of 90% methanol each time. 3 liters of water are added to the combined methanolic solutions and the mixture is concentrated in vacuo at 30 - 40°C to a volume of 2 litres. The aqueous concentrate is extracted by shaking out 5 times with 2 liters of ethylene chloride each time, the ethylene chloride solutions are washed with 0.5 liters of water each time, combined and evaporated to dryness in vacuum at 30 - 40°C. The residue is subjected to chromatography on a twenty-fold amount of "Sephadex LH 20", whereby methanol is used as eluent. The eluate is collected in 20 ml fractions and those of the fractions that prove antibiotically active against Aspergillus niger are combined and evaporated in a vacuum at 30 - 40°C

to dryness. The residue is then chromatographed on seventy-fold the amount of aluminum oxide (neutral) with activity level 1, whereby mixtures of toluene and ethyl acetate are used for elution. With toluene to which 15% ethyl acetate has been added, in the first 10 fractions (the volume of the fractions: half as many liters as the adsorbent in kg) first the main amount of the substance S 7481/F-l appears, followed by a mixture of S 7481/F-l and 7481 /F-2.. A further amount of the same mixture is isolated together with a little extraneous substance by subsequent elution with toluene-ethyl acetate (1:1). The detection of the substances in the eluate fractions takes place by thin-layer chromatography, e.g.? using silica gel on "polygram" foils and hexaneacetone 1:1) as a fluid. From the united

fractions, which contain the substance S 7481/F-1 in uniform form, the substance is recovered by evaporation in vacuum at 30 - 40°C and subsequent drying of the residue in high vacuum at 50°C. The combined eluate fractions, which contain the mixture, are likewise evaporated under the same conditions. For the isolation of additional S 7481/F-1 and of uniform S 7481/F-2, the residue is subjected to chromatography on a thousand-fold amount of silica gel, with chloroform, which contains 2% methanol, being used as eluent as this and the further measures were described in example 1-

Example 3.

500 liters of a nutrient solution, which per liter contained 40 g glucose, 5 grams casein peptone, 5 g MgS04xWH^ O, 2 grams KH2PC>4

3 grams of NaNO^-, 0.5 grams of KC1, 0.01 grams of FeSO4 and demineralized water, was inoculated with 50 liters of a pre-culture of the strain NRRL

8044 and incubated in a steel fermentation vessel under rotation (170 revolutions per minute) and aeration (01 liter of air/minute/liter of nutrient solution) for 13 days at 27°C.

500 liters of culture broth are crushed in an ultra-fast mixer and extracted several times with 500 liters of 1,2-dichloroethane each time. The organic phase is separated, concentrated over sodium sulphate and evaporated in vacuo. The evaporation residue is chromatographed on a hundredfold amount of "Sephadex LH 20" with methanol. The anti-Aspergillus niger active fractions are combined and the mixture is chromatographed on the one hundred to two hundred double quantity of nCytral aluminum oxide (activity level I). The elution takes place with toluene to which 10 - 50% ethyl acetate has been added. Predominantly by-products are eluted first, then S 7481/F-1 and then S 7481/F-2. The detection of the active substances in the fractions also takes place here by activity testing against Aspergillus niger. For purity control, thin-layer chromatographic analysis is carried out on silica gel plates with chloroform-methanol (96:4), the antibiotic compounds being made visible with iodine vapour. The pure fractions are combined and triturated with hexane, the antibiotic compound with S 7481/F-1 and S 7481/F-2 being obtained in the form of a white amorphous

o

powder if dried in high vacuum at 50°C.

The pre-culture used as starting material is obtained in the following way: The spore and mycelial suspension used for inoculation is prepared from a culture of the originally isolated strain NRRL 8044, which was grown for 21 days at 27°C on an agar medium which per liter contained 20 grams of malt extract, 20 grams of agar, 4 grams of yeast extract and demineralized water. The spores and mycelium in this culture were immersed in physiological saline solution.

With this suspension, 50 liters of a nutrient solution, which

had the same composition as in the 500 liter steel fermenter, inoculated and incubated in a steel fermenter under agitation (200 revolutions per minute) and aeration (1 liter of air/minute/liter of nutrient solution) for 3 days at 27°C . This fermentation solution serves as seed material for the 500 liter steel fermentation vessel.

Claims (3)

1. Procedure for the production of the new antibiotics S 7481/F-1 and S 7481/F-2, characterized in that a new strain of the mushroom species Cylindrocarpon lucidum Booth respectively. of the fungal strain Trichoderma polysporum (Link ex Pers.) or one of their mutants is grown on or in a nutrient medium and then the new antibiotics S 7481/F-1 and S 7481/F-2 are isolated from the nutrient medium and isolated in a known manner . 2. Method as stated in claim 1, characterized in that the strain NRRL 5760 is used as a new strain of the mushroom species Cylindrocarpon lucidum Booth. 3. Method as stated in claim 1, characterized in that the strain NRRL 8044 is used as a new strain of the mushroom species Trichoderma polysporum (Link ex Pers.).