DD252616B1 - PROCESS FOR THE PRODUCTION OF CERULENINE ON MICROBIOLOGICAL PATHS - Google Patents

Description

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Field of application of the invention

The invention relates to a process for the preparation of a microbial active ingredient that exhibits antibiotic activity against fungi, yeasts and gram-positive bacteria, and moreover, as an inhibitor of fatty acid synthetase and polyketide synthetase as a research agent in hybrid biosynthesis, can lead to the production of novel hybrid antibiotics used in chemotherapy of tumor, virus and microorganism-induced diseases in humans and livestock are of potential use. The antibiotic and enzyme inhibitor is an optically active keto-epoxydodecadienamide called cerulenin, which is becoming increasingly important in the microbiological and pharmaceutical industries because of its ready-to-use for the targeted selection of high-performance antibiotic strains. The field of application of the invention is thus in the pharmaceutical research and industry.

Characteristic of the known technical solutions

Cerulenin was first isolated and described in 1960 as an antifungal antibiotic from fungal cultures of the species Cephalosporium caerulens (Hata, T. et al., Jpn. Bacteriol., 15, 1057). For reviews on the production and properties of cerulenin (Omura, S., 1976. Bacteriol, Rev., 40, 681; Omura, S., 1981. In Lowenstein, JM, Methods in Enzymology, Vol. 72, 520-532, Masuma, R. et al., 1982. J. Antib. 35, 184). It is known that so far only a few types of fungi are capable of biosynthesis of cerulenin.

Helicoceras oryzae, H. certidis, H. plantaginis, H. nymphaerum are known generators of helicocerin identical with Cerulenin (Engl. Pat. 1,154,133 1969). Cephalosporium caerulens (JP 65/9588 1965), Acrocylindrium oryzae (JP 70/21 638 1970) and Sartoriafumigata (JP 72241561972) are further formers of cerulenin, which together have the disadvantage that during fermentation except the desired Cerulenin several severely separable steroidal Antibiotics, in particular helolic acid, be synthesized, which complicate the isolation of Cerulenin.

It is also known that the addition of zeolites during fermentation promotes cerulenin biosynthesis in cephalosporium cornulens (JP 57206380 1982).

Due to the numerous applications and the importance of cerulenin as a specific enzyme inhibitor, several methods for the total synthesis of (± (cerulenin (Boeckmann, R. Kl and Thomas, EW, 1979. J. Am. Chem. Soc., 101, 897; Corey .

E.J. and Williams, D.R., 1977. Tetrahedron Lett., 1977, 3847; Jakubowski, A.A. et al., 1982. J. Org. Chem., 47, 1992) and a process for producing optically active (+) - cerulenin have been developed (Sveta, N. et al., 1979. Tetrahedron Lett., 1979, 2039 ). However, the syntheses lead to a series of chemical reactions with sometimes only moderate yields, so that a synthesis does not currently appear economical.

While the basic structure of the antibiotic has been known for some time (Omura, S. et al., 1969. Chem. Pharm. Bull., 1969, 2361; Arison, B. et al., 1974. J. Antib., 27, 28 ), the absolute configuration of cerulenin as (2R, 3S) -2,3-epoxy-4-keto-7.10.trans, trans-dodecadienamide (Figure 1) has been elucidated only in recent years (Sveta, N. et al., 1979. Tetrahedron Lett., 1979, 2039; Poughny, JR and Sinary, P., 1978. Tetrahedron Lett, 1978, 3031; Vigneron, JP and Blanchard, JM, 1980. Tetrahedron Lett. 1980, 1739).

Object of the invention

The invention aims to produce the antibiotic and enzyme inhibitory cerulenin. Thus, the range of known manufacturing processes for the increasingly important in pharmaceutical research and industry cerulenin is to be extended by an original process that avoids the disadvantages of known production methods.

Explanation of the essence of the invention

The invention has for its object to provide a technologically simple method that allows cerulenin using a previously unknown microorganism strain exclusively and without difficult to separate, steroidal companion antibiotics from easily accessible and inexpensive starting materials in good yields. According to the invention the object is achieved in that under mutagenesis from a belonging to the fungi imperfecti population IP 45 is grown under aerobic and sterile culture conditions in a liquid nutrient medium with carbon, nitrogen sources and mineral salts. The microorganism used in this process has been deposited in the ZIMET Depository for Microorganisms, in the Central Institute for Microbiology and Experimental Therapy of the GDR Academy of Sciences, 6900 JENA, Beutenbergstrasse 11, under the registration number ZIMET 43807 and differs from known cerulenin-formers by morphological-physiological characteristics and in that during the fermentation no Helvolsäure and other steroidal antibiotics are synthesized as difficult to separate by-products. Cultures of strain IP 45 grown on suitable agar media are inoculated into liquid, previously sterilized nutrient media and the resulting mycelium is added in a manner known per se at a temperature between 25 and 37 ° C for a period of 2 to 4 days, preferably 2 days cultured at the beginning of the fermentation process between pH6.2 and pH 6.6 and at the end of the process between pH 5.0 and pH 6.6.

Glucose, glycerin and sucrose can be used as carbon sources in the suture media. Nitrogen sources include dry yeast, meat peptone and sodium nitrate. Good results can be achieved with the addition of mineral salts and Zeosorb molecular sieves. The latter promote the course of fermentation as selective adsorbents and exchangers. In complex media, additions of calcium carbonate, sodium phosphate or potassium phosphate have proved to be advantageous. The fermentation of the cerulenin ZIMET 43807 can be carried out in breast bottles and round-bottomed flasks of various contents, in the glass fermentor and in V2A steel tanks. Isolation of the cerulenin is carried out by extracting the antibiotic from the culture filtrate with organic, water-immiscible or poorly-miscible solvents, reextracting the extracts with a water-immiscible solvent, drying over anhydrous sodium sulfate, drying to dryness is then taken up in an organic nonpolar solvent and purified by column chromatography. From the crystalline crude product, which results after distilling off the solvent of the combined antibiotic fractions of the column chromatography, the cerulenin is obtained by recrystallization as a pure crystalline compound. Properties of cerulenin

Colorless, microcrystalline substance of carbon tetrachloride. M.p .: 90-93 ⁰ C, molecular formula: C ₁₂ H ₁₇ NO _3, MW: 223, IaI I ^s - -12 ° C (chloroform, с = 1.0). Solubility: Readily soluble in chloroform, carbon tetrachloride, benzene and ethyl acetate, moderately soluble in water.

Mass spectrum m / z: M ⁺ gef .: 223.1185; Re: 223.1208; Fragmentation m / z: 206 (M ⁺ -NH ₃ ); 179 (M ⁺ -CONH ₂ , base peak); 142 (M ⁺ -C ₆ H ₉ ). IR spectrum (КВг) сітГ ¹ : 3380,3210 (-NH ₂ , amide); 1720 (C = O); 1663 (C = O, amide); 1610 (C = C); 967 ( ^H C = C _n ). ¹³ C-NMR spectrum: The data of the ¹³ C-NMR analysis are summarized in Tab. They are characteristic of cerulenin (Funabashi, H. et al., 1983. Tetrahedron Letters, 24, 2673) having the structure (2R, 3S) -2,3-epoxy-4-keto-7,10-trans, transdodecadienamide (formula, Fig. 1).

The advantages of the production method described here for the enzyme inhibitor cerulenin are summarized

- In the provision of a microorganism, in addition to cerulenin no other, difficult to separate from this drug substances in the fermentation medium or enriches in the mycelium;

- the ability to make available, in good yields and using cheap feedstock, a biofematic chemical of significant importance to the pharmaceutical research and industry, for the construction of hybrid antibiotics with a novel structure and mode of action as well as for the selection of industry High performance strains of potential use;

In a stereospecific biosynthesis of the optically active Ceruienin, which is obtained during the fermentation with the relevant for the biochemical action of the enzyme inhibitor configuration in high yield;

In the simple isolation of the cerulenin from the culture filtrate of the builder without the need to separate the helolic acid or other steroidal antibiotics.

embodiments

The invention is explained in more detail by the following embodiments, but not limited. 1. Two 500ml steep breast bottles each contain 80 ml of the following pre-breeding medium:

Glucose 2.0%

Peptone 0.5%

Meat extract 0,5%

Dry yeast 0.3%

Sodium chloride 0.5%

Calcium carbonate 0.3%

in tap water

Sterilization: 35 minutes at 110 ^° C. The acidity after sterilization is at pH 6.3. Each steep breast bottle is inoculated with mycelium fragments of Cerulenin-Former IP 45, which was grown on the following proximal bottom:

Malt extract 3.0%

Agar-agar 1.2%

in tap water

Sterilization: 35min. at 115 ° C. The acidity after sterilization is pH 6.3. The inoculated pre-breeding cultures are 48h. incubated at 28 ° C on a shaker with a frequency of 180 U / min. 8 ml of a pre-breeding culture incubated in this way are used to inoculate 500 ml steep-breasted bottles, each containing 80 ml of the following production medium:

Glycerine 3.0%

Glucose 1.0%

Peptone 0.5%

Sodium chloride 0.2%

Zeosorb molecular sieve

4 A powdered 1.0%

in tap water

Sterilization: 35min. at 115 ° C. The acidity after sterilization is pH 6.3. During the fermentation, the temperature is at 28 ⁰ C and the shaking frequency is 180U / min. With a culture of cerulenin builder IP 45 from a solid medium, inoculate 80 ml of the mentioned liquid pre-breeding medium, which is contained in a 500 ml Steilbrustflasche. It breeds 48h. at 28 ⁰ C on a shaking table with a shaking frequency of 180U / min. 12 ml of the culture broth thus obtained are used to inoculate 400 ml of the same pre-growing medium, which is contained in a 2I glass flask. It breeds another 48hrs. at 28 ⁰ C at a shaking frequency of 180U / min before the thus obtained 400ml culture broth for inoculation of 201 of the described production medium serve. The latter is contained in a 321 glass fermenter. During the fermentation, with a stirring speed of 400 rpm, and with an air supply of 151 / min. carried out, the foaming can be controlled by adding small amounts of Antafron. Culture broth obtained from a fermentation batch in the 321 glass fermenter is centrifuged and the supernatant culture filtrate is exhaustively extracted with chloroform. The combined chloroform extracts are dried over sodium sulfate, the solvent is distilled off in a vacuum rotary evaporator, the oily residue is taken up in a little chloroform and washed on silica gel (0.063-0.2 mm) with chloroform, chloroform / ethyl acetate = 95/5 and chloroform / ethyl acetate = 90 /. 10 chromatographed. Fractions containing thin layer chromatography cerulenin are concentrated to dryness in a vacuum rotary evaporator. This gives 2.7 g of crystalline Cerulenin crude product, which can be recrystallized for ultrafine purification from benzene / petroleum ether and then from carbon tetrachloride

 The procedure is as in Example 1 with the difference that the production medium has the following composition:

Glucose 4.0%

Peptone 1.0%

in tap water

Sterilization: 35 minutes at 115 ° C. The acidity after sterilization is pH 6.3. The procedure is as in Example 1 with the difference that the culture medium has the following composition:

potato extract 2.0% glucose 1.0% peptone 0.1% Agar Agar 1.2% in tap water 35min. at 12O ⁰ C, natural. Table 1

¹³ C-NMR data of cerulenin (CDCI3) compared to references by Funabashi et al. / I /. The chemical shifts are related to tetramethylsilane as internal standard.

C atom Chemical shift 1) Own measurement 167.3 C-1 167.27 55.4 C-2 55.33 58.4 C-3 58.36 202.0 C-4 201.97 40.9 C-5 40.84 26.0 C-6 25.95 125.8 C-7 125.79 127.8 C-8 127.78 129.2 C-10 129.20 130.7 C-11 130.66 35.5 C-9 35.41 17.9 C-12 17.82

/ 1 / H. Funabashi, S. Lwasaki and S. Okuda Tetrahedron Letters 24 (1983) 2673-2676

Claims (2)

A process for the preparation of cerulenin in a microbiological way using a fungi imperfecti attributable strain, characterized in that used for the fermentation of the fungal microorganism IP45 strain ZIMET 43807, - The cultivation carried out under aerobic and sterile Submersbedingungen with the addition of Zeosorb molecular sieve and - The subsequent extraction is carried out using trichloromethane. 2. The method according to claim 1, characterized in that the cultivation is carried out with the addition of powdered Zeosorb molecular sieve 4A.