CH615459A5 - Process for the preparation of a metabolite - Google Patents

Description

615459

2nd

PATENT CLAIM Process for the production of the new metabolite S 14750 / A in free form or in the form of a salt, characterized in that the strain NRRL 8077 of the Actino-mycetspecies Streptomyces hygroscopicus (Jansen 1931) Waksman and Henrici, 1948 is grown on or in a nutrient medium and then isolate the metabolite S 14750 / A in free form or in the form of a salt from the fermentation broth in which the cell material is digested.

Medium growth properties

Mycelium shape

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30th

35

The present invention relates to a process for the preparation of the new metabolite S 14750 / A in free form or in the form of a salt.

According to the invention, the new metabolite S 14750 / A is obtained in the form of the acid or in the form of the salts by cultivating strain NRRL 8077 from the Actinomycetes species Streptomyces hygroscopicus (Jansen 1931) Waksman and Henrici, 1948 on or in a nutrient medium and the new metabolites in the form of the acid or in the form of a salt from the fermentation broth in which the cell material is digested.

The metabolite S 14750 / A in the form of the acid can optionally be converted into the salt form in a manner known per se. Examples of suitable salts are alkali metal and alkaline earth metal salts.

The above-mentioned strain of Streptomyces hygroscopicus (Jansen 1931) Waksman and Henrici, 1948 was isolated from a soil sample found in Vergei, Alicante in Spain and a sample thereof on January 30, 1975 at the United States Department of Agriculture (Northern Research and Development Division) , Peoria, St., USA, is deposited and is available to the public under the cultural number NRRL 8077.

The NRRL 8077 strain grows well on organic and synthetic media. The spore carriers are monopodial (sometimes sympodial) with narrow, compact spirals with 2 to 5 turns. They often form dense clumps that have a pseudoverticillates appearance. The spores are oval to elliptical, 1.0 to 1.3 microns long and 0.8 to 0.9 microns wide,

their surface is wart-like. A pronounced hygroscopic behavior was observed on some, especially organic media, in that black, shiny spots are formed by exudates on agar cultures when the spores become moist.

Using the Waksman classification system (Waksman, The Actinomycetes, Vol. 2,328-334, 1961), strain NRRL 8077 could be identified as Streptomyces hygroscopicus (Jansen 1931) Waksman and Henrici, 1948. The growth properties of the NRRL 8077 strain on normal biological media and its carbon assimilation are shown in the following tables. 55

Growth characteristics

45

50

Medium growth properties

Mycelium shape

Malt yeast extract

Agar oats

W: good, yellow-brown spira a

LM: gray-beige to mouse gray 2-5

(G 3 ih) * turns

LP: none

W: good, leather yellow to spira a yellow-brown

Agar

Strength

Agar

10th

Glycerin 15 asparagine agar

LM: gray-beige to mouse gray (G 3 ih) *

LP: none

W: medium, colorless to slightly brown

LM: white to ash gray (G 5 fe) *

LP: none

W: medium, leather yellow to slightly yellow-brown

LM: white to ash gray (G 5 fe) *

LP: light yellow pigment

2-5

Coils

Spira a 2-5

Coils

Spira a 2-5

Coils

W: growth LM: aerial mycelium LP: soluble pigment

*: Reference with reference to the «Color-wheels system»

Assimilation of carbon compounds growth carbon source

Glucose, arabinose, sucrose, xylose, starch, inositol, mannitol, fructose rhamnose, raffinose, galactose, glycerin, cellobiose, dextrin, mannose cellulose, salicin, inulin, culcitol,

Sorbitol

*** = good growth and recovery ** = medium growth and recovery - = no growth or recovery

The new strain NRRL 8077 has the following physiological properties:

Nitrate reduction Starch hydrolysis Cellulose decomposition Tyrosine reaction Milk coagulation Milk peptone formation Gelatin liquefaction

Melanin formation positive positive negative negative negative strong positive positive, slightly yellow

Pigment negative eo

65

Strain NRRL 8077 is cultivated using methods known per se and essentially consists of cultivating said strain on a suitable medium and under suitable conditions and then the metabolite S 14750 / A formed in the course of breeding in free form or in To separate the form of its salts. The strain NRRL 8077 can be grown after any aerobic surface or immersion cultivation.

At the start of cultivation, the pH of the fermentation medium should be between 6.8 and 7.3. The optimum temperature for breeding can be between 25 and 35 °. The ventilation of the breeding can fluctuate between wide limit values. The maximum yield of the metabolite S 14750 / A is obtained after cultivation for 4 to 7 days, this time

3rd

615459

span mainly depends on the medium used.

In order to avoid a delay in the production of the metabolite S 14750 / A, the vegetative form is preferably used for the inoculation of the production medium instead of the spore form of the microorganism. Accordingly, a vegetative inoculum of the microorganism is first generated by inoculating a small amount of a culture medium with a spore suspension of strain NRRL 8077.

The fermentation media are said to mainly contain an assimilable carbon source, an assimilable nitrogen source and optionally mineral salts and growth factors, all of which can be supplied in the form of well-defined products or complex mixtures found in biological products of various origins. Media based on glucose are particularly suitable. Organic and inorganic nitrogen-containing compounds such as peptone, yeast or meat extracts, ammonium nitrate, ammonium sulfate, amino acids etc. are also suitable as nitrogen sources.

Trace elements that are required for optimal growth of the organism used to produce the metabolite S 14750 / A should also be contained in the culture medium. Such trace elements usually occur as impurities in the other constituents of the medium in amounts which are sufficient for the growth requirement of the strain NRRL 8077 used according to the invention.

The isolation of the new metabolite can be carried out according to methods known per se, e.g. B. be carried out in the following way:

As soon as a maximum amount of the metabolite S 14750 / A has been produced during cultivation, the mycelium fraction in the culture broth is optionally digested, e.g. B. with an Ultraturrax, and the metabolite obtained by extractive and / or adsorptive methods in a known manner. However, the mycelium can also be centrifuged out of the culture broth first, the culture filtrate and the mycelium extracted separately after digestion and the extracts processed individually or combined.

One method that has proven to be preferred is the separation of the culture broth in culture filtrate and mycelium by centrifugation. The culture filtrate is then adjusted to pH 9 with 1 N sodium hydroxide solution, filtered and with a water-immiscible solvent, such as. B. ethyl acetate, 1,2-dichloroethane, chloroform, dichloromethane, toluene or benzene extracted at 20-30 °. The extract obtained in this way is then freed from the solvent in vacuo at a low temperature, preferably 20-60 °. The mycelium is digested and extracted at 20-30 ° with methanol and with a 9: 1 mixture of methanol and water using an Ultra-Turrax. After centrifuging off the cell material and clarifying, the combined methanol extracts are evaporated in vacuo at 20-40 ° to water, water possibly being added. After adjusting the aqueous phase to pH 9, the mixture is extracted again with a water-immiscible organic solvent as described above and the organic phase is evaporated. The two crude extracts thus obtained, which contain the new metabolite S 14750 / A preferably as the sodium salt, are now combined. For further purification, chromatography can be carried out on Sephadex LH-20 with methanol and / or on fine-grained silica gel. Chromatography on silica gel Merck with a grain size of 0.2-0.5 mm has preferably been used. Chloroform, chloroform-acetone or chloroform-methanol mixtures can be used for elution. A mixture of toluene with 1% triethylamine with the addition of increasing amounts of acetone (2-50%) has proven to be particularly suitable. The

The ratio of adsorbent to crude extract can be varied within the range of 30-100: 1. The metabolite can be recognized in the fractions either by thin layer chromatography or by its activity against Staphylococcus aureus. The already quite pure material obtained after the chromatography is a mixture of acid and sodium salt. For conversion to the pure sodium salt, the pure fractions are in a water-immiscible organic solvent which is resistant to bases, such as. B. chlorinated hydrocarbons, hydrocarbons, ether, preferably chloroform, dissolved and shaken with aqueous sodium hydroxide solution. The sodium salt of the metabolite S 14750 / A dissolved in the organic phase is isolated by evaporation in vacuo at 20-40 ° and purified by crystallization. When crystallizing from acetone, the sodium salt of S 14750 / A is obtained, which contains the crystal acetone, from which the crystal solvent can be removed by repeated pulling up with benzene in vacuo at 20-50 °. The pure Na salt thus obtained is amorphous. To prepare the free acid, the Na salt is dissolved in a water-immiscible organic solvent, preferably chloroform, dichloromethane or 1,2-dichloroethane, shaken with a dilute aqueous solution of a strong acid, e.g. B. HCl, H2SO4, H3PO4 at 0-20 °, then washes out the strong acid and evaporates the organic phase in vacuo at 20-40 °. The acid thus obtained is amorphous. The amorphous acid, the amorphous Na salt and the crystal acetone-containing Na salt of the new metabolite S 14750 / A show the following properties:

Acid Na salt Na salt amorphous crystallized with acetone

Mp 91-100 ° 122-128 ° 160-165 °

an0 in methanol + 4.3 ° + 7.2 °

in chloroform -5.5 ° + 15.2 °

UV (methanol) end absorption end absorption

The elemental composition of the acid gives the following values:

C 64.4% H 9.7% 0 26.2%

The IR spectrum of the acid in CH2CI2 is shown in FIG. 1 and the IR spectrum of the amorphous Na salt in CH2CI2 is shown in FIG. 2. The NMR spectrum of the acid (100 MHz) in deuterochloroform with tetramethylsilane as the internal standard is shown in FIG. 3, the NMR spectrum of the amorphous Na salt in FIG. 4.

Sodium salt and acid are readily soluble in organic solvents such as toluene, benzene, dichloromethane, chloroform, ethyl acetate, butyl acetate, ethanol, methanol. Both compounds are very difficult to dissolve in water. In the thin-layer chromatogram, the acid and sodium salt on silica gel plates have the same Rf values. The table shows the Rf values on silica gel plates, Merck, Kieselgel 60, F 254 0.25 mm layer thickness, in comparison to some other ionophores.

Rf values in Flm

1

2nd

3rd

S 14750 / A

0.32

0.74

0.55

Nigericin

0.17

0.22

0.35

X-206

0.27

0.71

0.56

A-204

0.36

0.43

0.59

Grisorixin

0.20

0.34

0.33

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15

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40

45

50

55

60

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615459

4th

Superplasticizer (varnish) l: toluene-acetone (8: 2) + 1% triethylamine superplasticizer (Flm) 2: chloroform-ethyl acetate (l: 1)

Superplasticizer (Flm) 3: chloroform-methanol (95: 5)

Iodine or a solution of 0.2% Ce (SC> 4) 2 in 50% sulfuric acid (warm to 120-130 °) is used to make the spots visible.

The new Metabolit S 14750 / A in its free form or in the form of its salts is suitable for human and veterinary purposes.

The new metabolite S 14750 / A shows in the form of the acid or its salts, e.g. B. the Na salt, antibacterial effect against gram-positive organisms and fungi. The following table shows the minimum inhibitory concentrations (MIC values) against some microorganisms.

acid

Na salt

Staphylococcus aureus

1

0.03

Micrococcus lysodeikticus

0.01

0.01

Helminthosporium sp.

1000

30th

The metabolite S 14750 / A or its salts can be used for human medical purposes as a medicament alone, both in pure form and as a crude concentrate or in the corresponding pharmaceutical forms for topical, oral, enteral or parenteral administration.

The new metabolite S 14750 / A is also effective against various coccidia species and species. This effectiveness is demonstrated under experimental conditions using the example of Eimeria tenella, an important coccidiosis pathogen in poultry.

The tests were carried out on day-old chicks in a 10-day test. The chick groups received a feed medicated with Metabolit S 14750 / A in various concentrations and were orally infected with 200,000 sporulated E. tenella oocysts 48 hours after the start of feeding on the 3rd day of life. On the 7th day after infection, the test was stopped and the efficacy was determined, taking into account the criteria of mortality, degree of lesion in the caecum and weight gain in comparison to infected-untreated and healthy-untreated control animals.

It shows that Metabolit S 14750 / A in feed concentrations of more than 30 ppm. the course of infection of E.tenella in the appendix is interrupted or inhibited, which is expressed in comparison to the control animal groups both in the mortality and degree of lesion of the appendix and in a favorable weight gain, which is in the range of healthy chicks.

The new Metabolit S 14750 / A is effective against coccidiosis infections when added to chickens' feed. Infections are effectively combated with quantities of 5 to 50 g of Metabolit S 14750 / A per ton of feed.

Furthermore, the Metabolit S 14750 / A and its salts as a feed additive show that even in low concentrations they improve feed conversion in ruminants. This effect was demonstrated in vitro using an artificial rumen. With this method, substances can be tested for an inhibition of methanogenesis and / or a shift in fatty acid formation in favor of propionate. Rumen juice is removed from the rumen of a ruminant and passed through at 39 ° with constant passage of CO2

Gauze filtered. This rumen juice is then incubated together with the feed used by the animal and containing the substance to be examined. After the incubation period has ended, 2 samples are taken from the gas space and 5 are analyzed by gas chromatography for methane and carbon dioxide. The liquid phase is then examined for its fatty acid content. The comparison to a sample without added substance gives a parameter for the effectiveness. For example, a small addition of a metabolite 10 S 14750 / A results in a reduction of up to approx. 95% of the methane production. With a constant fermentation rate, the production of propionic acid increases, especially with a higher addition of metabolite S 14750 / A.

The Metabolit S 14750 / A and its salts can therefore be used as a feed additive. The feed administered expediently has a content of 1 to 300 mg of active ingredient per kg of feed.

The easiest way to administer the new metabolite S 14750 / A and its salts for veterinary purposes is to add them to the animal feed. However, they can also be administered in other ways. For example, they can be placed in tablets, impregnating agents, boluses or capsules and given to the animals in doses. The preparation of the new metabolite 25 S 14750 / A and its salts in such dosage forms can be carried out according to methods which are generally known in the field of veterinary pharmacy.

In the following examples, which explain the execution of the method but are not intended to limit the scope of the invention in any way, all the temperatures are given in degrees Celsius.

example 1

A well sporulating agar culture of strain 35 NRRL 8077 is washed with 5 ml of sterile 0.90% saline, whereby a cloudy spore suspension is obtained. One milliliter of the spore suspension is used to inoculate a preculture medium with the following composition:

40

Pharmamedia 25 g / 1 1

Glucose 25 g / 1 j> Medium I

and least Water ad 11 y

Adjust to pH 7 with NaOH or H2SO4. 45 The preculture medium is sterilized in an autoclave at 120 ° for 20 minutes. The preculture is incubated with aeration at 27 ° for 3 days (shaking machine 200 rpm).

In 500 ml Erlenmeyer containing a nutrient solution-50 containing the composition,

55

60

Starch (soluble

Glycerin

Soybean flour

Com steep liquor

Table salt

Calcium carbonate and dist. water

Ï 0.0 g / I 5.0 g / 1 10.0 g / 1 5.0 g / 1 3.0 g / 1 3.5 g / 1 adi 1

Medium II

is inoculated with a pre-culture. The nutrient solution was previously adjusted to a pH of 7.0 to 7.2 and sterilized at 120 ° for 20 minutes in an autoclave.

The fermentation period is 5 days under the same conditions as for the pre-culture.

The agar culture of strain NRRL 8077 used as the starting material is obtained by using a culture medium of the following composition:

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615 459

Glucose malt extract yeast extract agar (Bacto) and dist. water

4.0 g / 1 10.0 g / 1 4.0 g / 1 20.0 g / 1 adi 1

Medium III

inoculated with a spore suspension of strain NRRL 8077.

Before sterilization, the medium is adjusted to pH 8.3-8.6 (with NaOH), then sterilized at 120 ° (20 minutes). Then the pH is adjusted to 6.9-7.3 if necessary.

10th

Example 2

The stock culture is grown in two 300 ml Erlenmeyer, each containing 50 ml of medium III. Incubate as a stand culture at 27 ° for 20 days. 100 ml of sterile saline solution are added to each of the two is Erlenmeyer and the agar cultures are ground to a fine mixture using an Ultra-Turrax. The two spore suspensions are then combined and used to inoculate 501 of medium I in a steel fermenter. This preculture is incubated for 3 days at 270 with aeration (0.5 l / min. / Per 1 medium), the medium being stirred (200 rpm). 501 of the preculture are used to inoculate 5001 of medium II in a steel fermenter tank. Incubate for 4 days at 270 with aeration (0.5 l / min. / Per 1 25 medium) at 0.5 atmospheric pressure, the medium being stirred (130 rpm).

480 liters of culture broth are centrifuged, resulting in approx. 4001 culture filtrate and approx. 80 kg mycelium. The culture filtrate is adjusted to pH 9 with 1 N sodium hydroxide solution and clear filtered through filter aid Celite 545. The filtrate is extracted three times at 20 ° with 500 liters of ethyl acetate each time and the extracts are washed with 100 liters of water. After evaporating the organic phase in vacuo at 20-40 °, approx. 320 g of crude extract are obtained. The mycelium is first extracted with 100 liters of 35 methanol, then twice with 70 liters of methanol-water (9: 1) for 1 hour at 20-25 °, the cells being disrupted at the same time using an Ultra-Turrax device. The cell material is then centrifuged off and the solution is clearly filtered through Celite 545. After adding 40-100 liters of water to the filtrate, the mixture is evaporated in vacuo at 20-40 ° to a volume of 50 liters, adjusted to pH 9 with 1N sodium hydroxide solution and then extracted six times with 40 liters of ethyl acetate each. After washing the organic phase with 20 liters of water, the mixture is evaporated to dryness in vacuo at 20-40 ° and about 160 g of mycelium extract are obtained. The two extracts are combined and dissolved in a little of a mixture of toluene-acetone (98: 2) + 1% triethylamine, on a column of 20 kg of silica gel 60 (Merck), particle size 0.2-0.5 mm, 15 cm column diameter, applied. Then with toluene-acetone (98: 2), (95: 5), (9: 1) and (4: 1), each with 1% triethylamine added, in fractions of 50 liters at a flow rate of 60 liters per Hour eluted. The fractions are evaporated in vacuo at 20-40 ° and the residue is checked for the content of S 14750 / A by thin layer chromatography and / or biologically (against Staphylococcus aureus). The active fractions, approx. 68 g, are combined and chromatographed on 40 times the amount of silica gel 60, Merck, particle size 0.06-0.2 mm. Toluene-acetone (98: 2) + 1% triethylamine to toluene-acetone (9: 1) + 1% triethylamine first eluted predominantly inactive accompanying substances. The new metabolite S 14750 / A is eluted in a highly enriched form as a mixture of acid and sodium salt with toluene-acetone (4: 1) + 1% triethylamine to toluene-acetone (1: 1) + 1% triethylamine. To convert to the pure Na salt, 40 g of the above fraction are dissolved in 500 ml of chloroform, shaken three times with 250 ml of 2N aqueous NaOH solution, the organic phase is washed twice with 100 ml of water, dried over sodium sulfate and evaporated in a vacuum at 20-40 °. The crude Na salt obtained in the form of a light yellow residue is crystallized from 1.5 times the amount of acetone, the metabolite S 14750 / A being obtained in the form of almost colorless acetone-containing crystals. To remove the crystal acetone, pull up several times with benzene in vacuo. The Na salt thus obtained is an almost colorless amorphous powder after drying in a high vacuum at 60 °.

To produce the acid, 2 g of Na salt are dissolved in 500 ml of chloroform and extracted twice with 200 ml of 2N aqueous hydrochloric acid. After washing the organic phase twice with 100 ml of water each time, the organic phase is dried over sodium sulfate and evaporated in vacuo at 20-40 °. After drying in a high vacuum at 60 °, the acid S 14750 / A is obtained in the form of an almost colorless amorphous powder.

2 sheets of drawings