DD213691A1 - METHOD FOR PRODUCING A STIMULATOR OF CYODIUM DIFFERENTIATION - Google Patents

Abstract

The invention relates to a process for the preparation of a cytodifferentiation stimulator which stimulates industrially important microorganisms both in their morphological differentiation and in their biosynthetic properties for anthracycline antibiotics which are of interest in cancer chemotherapy and is therefore of potential use in the production of active substances in the pharmaceutical industry. In particular, the invention relates to a process for the preparation of a heretofore unknown, low-molecular compound which, based on the chemical constitution analysis, can be referred to as stereochemically uniform 2- (6'-methylheptanolyl) -3-hydroxymethyl-4-butanolide. Preparation of stimulator ST43683 becomes strain ZIMET43683 Streptomyces viridochromogenes. By aerobic submerged fermentation of strain ZIMET43683 in media containing suitable C, N sources and mineral salts, the stimulator is formed, isolated from the culture filtrate by extractive techniques, and subsequently purified by chromatographic techniques. The microbiological activity is determined using the indicator mutant Streptomyces griseus ZIMET 43682.

Description

Process for the preparation of a stimulator of cytodifferentiation

Field of application of the invention

The invention relates to a process for the fermentative production of a stimulator of cytodifferentiation in industrially important microorganisms, which is able to stimulate both the expression of morphological features and the formation of secondary metabolites and therefore in the microbiological and pharmaceutical industries in microbial drug production of potential use »

In particular, the invention relates to the production of an effector which induces and stimulates the biosynthesis of the anthracycline antibiotics used in the chemotherapy of cancers «

Characteristic of the known technical solutions

It is known that microorganisms produce specific signal substances which bring about the activation of cytodifferentiation, including the secondary metabolism, or regulate the coordinated course of these processes. So far, the following effectors have been found in streptoraycetes, d * h, in the most important group of microorganisms for industrial active ingredient production in o, g. Senses known?

- the inductor of Streptomycinbiosynthese and aerial mycelium * "education griseue at Streptomyoes (AI EIIiEH et al," 1976, Bioorg Khim (Moscow), 2, 1142-1147; KHOKHLOY et al _e> 1973 Uov® Acta Leopoldina Suppl *. 7, 289-298J TOVAROVA et al *, 1970, Izvest, Akad * Nauk SSSR (Ser "Biol") 2s

427-34; KHOlQiLOY et al., 1973 _S Z. Alig ₀ Microbiol. 647-655) described as so-called Α-factor (2- (6-methylheptanoyi) -3-hydroxymethyl * 4-butanolide) in the literature;

the inducer of staphylomyoin production by Strepto myces virglniae, a C 1-5 -butyrolactone derivative (YANAGIMOTO et al., 1971, J. Ferment, Technol, 49j 611-614, IMAGIMOTO et al., 1979, Hakkogogaku, 57 * 6-14 ) j

the C-factor in Streptomyces griseus "is a protein effector of spore formation (BIRO et al., 1980, Eur · J · Biochem., 103 , 359-363);

- the antibiotic pamamycin, an inducer of air methylation in Streptopype 3 alboniger of previously unknown structure (McCMS and POGELL, 1979, J · Äntibiot * 32, 673-678);

the sporulation pigment of Streptomyces venezuelae (SCRIBNEIi et al., 1973, Appl. Microbiol., 2, 873-879),

the fethylenomycin, an inhibitor of air myoegen formation in SCPI plasmid-deficient mutants (KIHBY et al., 1973, Nature, 254, 265-267) J

r * ·: the oosynthesis factor I, a flavin derivative of unknown structure, effective as a coenzyme of a dehydrogenase step of tetracycline biosynthesis (EuLLER et al., 1960, J. Amer, Chem. Soc. 82, 5002) and

the 4,5-dihydroxydecanoic acid 4α-laotons (L-factors) as inducers of leukemomycin and sporulation in blocked mutants of Streptomyoes griseus (ERITT et al., 1981, Z _e Alig. Mikrofciol., 22, 91-95 , GRAEE et al., 1982, J. An'tibiot. 21 » 57-62, (JRAi ¹ E et al., 1981. DDR-WP C 12 P / 232 491 2)

Called the active ingredients mentioned, also stimulators, effectors or bioregulators have been studied to aufsufinden possibilities for their use in the control of i ^l ermentationspro.zessen and for their use as drugs in terms of their structure and mode of operation *

From the totality of streptomycetes. hitherto known bioregulators are the L- and A-factors as well as the

considered to be the closest prior art

It should be noted on the one hand, that the previously known L-factors (^, S-Dihidroxydeoansäure- ^ lactoae) have a comparatively low stimulation activity®

On the other hand, the original process for the production of the Α-factor, a fermentation process, yields this compound in spite of elaborate work-up steps only in very low yields. "In the meantime, the chemical synthesis of the A-factor has been successful (KLEINER et al, 1977, Bioorg However, this stimulator falls as raoemat.

From the chemically synthesized, racemic A - factor is further chemical reduction by a mixture of several stereoisoraerer derivatives with reduced C6-keto ~ group, d, h. a mixture of several 2 (6 ^l -methylheptanolyl) - »3-hydroxymethyl-4-butanolides. This derivative mixture has no biological activity against streptomycin-producing strains of Streptomyces gyiseus (KLEINER et al., 1977, Bioorg. Khira. (Moscow), 2, 424-426; ONOPRIENKO et al., 1979, Abstracts Int , by Antibiotics, Weimar / GDR, May 14-18, p.C-14).

Object of the invention

The invention serves to produce a novel stimulator of cytodifferentiation, in particular leukaemomycin production, in Streptomyces strains in order to expand the range of such bioregulators to a microbiologically producible representative of potent benefit for biotechnological adaptations in the microbiological industry. The stimulator to be produced, when added to industrially important Streptomycetes cultures that are unable to differentiate either aerial mycelia or spores and can not synthesize anthracycline antibiotics, is intended to activate and promote both differentiation and anthracycline biosynthesis.

Explanation of the essence of the invention

The invention has the object augrunde to describe a technologically simple process by means of which a new stimulator of cytodifferentiation, the o # g "meets requirements, prepared from a microorganism by cultivating it on an easily accessible medium using cheaper starting materials, with the help a specific method (Eritt et al. 1982, Z * Allg. Microbiol. 2 £, 91-95) and can be isolated and purified by means of oxy-chromatographic methods. "

According to the invention, the object is achieved by culturing a microorganism or its mutants and variants under aerobic and sterile culture conditions in a liquid currency medium with appropriate carbon and nitrogen sources and mineral salts. The microorganism which is used in this process is from a population the species Streptomyoes viridoohromogenea isolated and under the registration number ZIMST 43683 in the depository center of the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 6900 Jena, Beutenbergstr »11, has been deposited

The cultivation of the strain ZIMET 43683 and its mutants and variants takes place under aerobic conditions. Lyophilizates to earth "phil dried spore material or"(#ig 5) lyophilized mycelium fragments are inoculated onto appropriate nutrient agar and subsequently in liquid, previously sterilized culture media, and the resulting mycelium is of glucose / gelatin in a known manner at a temperature between 25 ⁰ C and 37 ⁰ O, preferably 28 ⁰ C, over a period of 2 to 6 days, preferably 4 days, cultured at an acidity at the beginning of the fermentation process between pH 6.2 and pH 7.0 and at the end of the process between pH 7.5 and pH 8.3 * The nutrient medium consists of sources of carbon and phosphorus as well as inorganic salts. As sources of carbon and nitrogen it is possible to use the starting materials customary in the antibiotics industry. The fermentation of the artist; ZIMET 43683 can be used in steep-breasted bottles and round-bottomed flasks of various contents, in glass fermenters and in V2A steel tanks.

be guided.

The culture solution obtained in the fermentation is separated by means of a separator into mycelium and culture filtrate. Preferably, the latter is mixed at an acidity of pH 5 to pH 7 for the extraction with organic solvents, in particular butyl acetate. Concentration of the Butylaoetatextrakte a crude product is obtained, which adsorbs on dry cellulose powder, subsequently brought back into solution by elution with water and further enriched by reextraction with ether. The resulting product is purified by preparative thin-layer chromatography until gas-chromatographic homogeneity. The high-purity material so obtained presents at room temperature, a colorless oil is · in the mass spectrum (high resolution mass spectrometer JEOI JMS-D-100, direct inlet system, 120 ⁰ C, 75 ev electron current) occur as listed in Fig. 1 characteristic fragments "The characteristic maxima in the IR spectrum (Hicolet Fourier transform instrument) are: (GDCl ₃ ) (οπΓ ¹ ): 1010, 1036, 10, 10, 12, 12, 12, 12, 12, 12, 12, 13, 13, 14, 17, ^max · 1520, 1767, (saturated 5-ring lactone ), 3625 (together with 1036s primary 0H ~ group) and 3440 (together with 1080: intramolecular Η bond, secondary 0H group) »A C0 band characteristic of the Α factor at 1720 cnT ¹ (Mort, 1981, Tetrahedron Lett., 22, 3431-32) is absent in the co-presence of a Co-OH group. The TJV-Spelstfcrum shows a weak absorption at 215 mn (£ - 120) «

The gross composition of the substance obtained according to the invention, which was designated ST 43683, was determined to be C ₁₃ H ₂₄ O 3 and its molecular weight is 244. These parameters, as well as the relative configuration, are indicated by the 200 MHz ¹ H NMR data (Bruker ViIP 200 / SY) confirms (^ f ⁰⁰⁰¹ S ppm: H2, 8.65, J ₂₃ * 9.4; J ₂₆ = 4 · 6; H3, 2.76; J ₃₄₃ »8.1; ⁸ · ⁶ ? ^J 35a ^{= 5} "^{8; J} 3,5b = ⁶ × ^{2 "H4a> 4e42i J} 4a4b *" ⁸ ^ 3.97; H5a, 3.75; J ₅ ^ ₅₁₃ '-10.6; H6, 4:03; -J y ₆ * 6 * 7; HT, H8, H9, H10, 1.1-1.75, H11, 1.49, J _{11 12} = 6 * 8, H12, H13, 0 * 87j coupling s constant in Hz) .The relatively high fourth of the coupling «·

constant J ₂₃ = 9 x 4 Hz is consistent with the trans arrangement of the substituents, corresponding to a 2S, 3S or _f 2R, 3R configuration of the chiral centers on the butyrolactone ring (Sevastionoff u. Pfau, 1967, Bull. Soc., Chinu Prance ig62, 4162-71).

The assignment of the structure is additionally supported by the ¹ ^ values (25.16 MHz Varian XL-100/15)! C ^ ^CDCl 3 ^{Ppm: Cl} , 177.79 (0) 02, 49.46 (d); C3, 40.34 (d); C4, 68.82 (t); C5, 62.88 (t); C6, 71.14 (d); 07, 34.25 (t) j C8, 26.72 (t); C9, 27.62 (t); 010., 39.24 (t); 011, 28.22 (d); C12, 22.73 CO; C13, 22.73 (q).

On silica gel films, St 43683 shows the following R _f values: 0.26 (CHCWMeOH, 95: 5, v / v) and 0.18 (benzene / ether, 1: 1,

According to its physicochemical characterization, the new substance ST 43683 represents a 2- (6 ^l -methylheptanolyl) -3-hydroxymethyl-4-butanolide. The novelty of this substance is seen in the fact that as a result of the described process, going beyond the prior art , a 2- (6'-methylheptanolyl) -3-hydroxymethyl-4-butanolide of very high stereochemical purity is obtained with respect to the 02 and 03 configurations.

The substance ST 43683 is, as surprisingly found, biologically active. It is capable of activating and stimulating both biochemical and morphological differentiation in strains of the leucaemomycin producer Streptomyoes griseus . According to the in Eritt et al., 1982, Z. AlIg. Microbiol., 22 ^ (2), 91 ^ -95 (see below), the stimulator ST 43683 has a 5-fold stronger stimulation activity in comparison with the known L-factors (4,5-dihydroxydecanoic acid-4-lactones) in terms of biosynthesis as well as morphological differentiation. Thus, with the present method for the first time an A-factor-like molecule can be produced by microbiological means, which shows biological activity in strains of the Leukaemomyoin-forming agent Streptomyoes griseus . ,

Evidence of the effect of the stimulator of cytodifferentiation

ST 43683 is carried out with the aid of a special method which allows to detect both the stimulation of aerial mycelium and sporulation as well as that of leukemomycin biosynthesis. "Details of the microbiological analysis are described by Eritt et al., 1982, Z. Al. Microbiol, 22j 91-95. The indicator strain used is a mutant of Streptomyces griseus that is genetically blocked both in the differentiation of the aerial mycelium and the spores, as well as in anthraeycline biosynthesis. The indicator strain necessary for carrying out the microbiological analysis of the stimulator ST 43683 has been deposited under the registration number ZIMET 43682 in the depository of the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 6900 Jena, Beutenberg 11, Aitraße 11.

embodiments

In the following embodiments, the invention will be explained in more detail «

1 × mycelium fragments or spores of the stimulator-forming agent ZIMBT 43683, which had been cultivated for 10 days on Streptomyces normal oatmeal agar, are used as inoculum for pre-culture cultures in the following submerser medium:

0.6 % Bacto-peptone; 0.3 # yeast extract; 0.05 # CaCO-; Tap water ad 100 #; pH 7.2; Sterilization; 120 ⁰ C, 30 Mn, The vorzuohtkulturen are in 100 ml Steiltrustflaschen ä * 20 ml filling and are incubated for 48 hours, at 28 ⁰ C on a shaker (180 U / min). The submerged mycelium obtained in this way is used by incremental scale enlargement to inoculate 400 l-type perforators with submersible medium of the following composition: 3 % glucose; 1 ^c h soy flour; 0.3 # WaCl; 0.3% CaCO ₃ ; pH 7.0 after sterilization (125 ⁰ C, 60 min ·) · During the fermentation at 27 ⁰ C to 2 $ ⁰ C, which is carried out with a stirring speed of 260 rev / min and an air supply of 400 l / min, can the foaming by addition of small amounts of silicone-containing foam protection agent

Each fermentation period of 100 hours determines the highest content of ST 43683 »

300 l of a 100% solution of strain ZIMET 43683, obtained according to example 1, are adjusted to pH 5.0 or pH 7.0, separated by a separator into myeel and potassium turfiltrate and corresponding to the one in FIG processed schema,

After extraction of the culture solution with 0.2 vol. Butylacetat and concentration of the solution 80 g of residue are obtained », which is dissolved in 300 ml of diethyl ether and treated with 200 g of cellulose powder for column chromatography. After removal of the ether © in vacuo, the Gelluloseadsorbat is filled into a column and eluted with 4 1 of water. The aqueous solution is then extracted 3 times with 2 1 of ether, After drying the solution and evaporation of the solvent remain about »5 g of residue, from which by preparative thin-layer chromatography on Silufol-silica gel using Benzen / Bther (1: 1 , v / v, two-fold run of the chromatogram), an enriched fraction of ST 43683 is obtained. The yield is 280 mg. The second fine purification is carried out by preparative thin-layer chromatography on Silufol-Kieselge. Films using CHCl.sub.3 / methanol (95: 5, v / v). Yields 60 mg. In a third phase of thin-phase radiography (silufol, benzene / ether, 1: 1, v / v), 17 mg of the pure stimulator ST 43683 are obtained. The purity of the product was demonstrated by gas-chromatographic analysis (OV 101, uaschrom Q, 140 ° column temperature). The product obtained was used for the investigation of the biolgic effect.

pH 7.5 and pH 8.3, is cultured and

- the new, stereoohemically uniform stimulator ST 43683 formed by the sum formula C- 3 ^H 2ZI 4 as well as the structure of a 2- (6'-methylheptanolyl) -3-hydroxymethyl-4-butanolide; in accordance with a 2S, 3S or 2RjSS order, has a trans configuration of the substituents on C2 and G3, extracted from the potassium turfiltrate at an acidity of pH 5 to pH 7 using conventional organic solvents, hereinafter referred to as purified per se known chromatographic methods *

2. The method according to item 1, characterized daduroh that the cultivation of the strain ZIMET 43683 in 400 1-fermentors at 28 ⁰ G over a period of 4 days and under an air supply of 400 l / min.

For this 2 pages illustrations.

2261 ^ 0

m / z .19 & 1377

m / z 145.0494

_{m / z}

m / z 1160471 (or the enantiomeric (2R, 3R) form) ICsH & Og *

Fig. 2 Processing scheme

100 hours, - »Cultures Streptpmvces yiridpohrpmo ^ enes

I yO2

Culture fluidity 300 1 mycelium

Extraction with 0 «2 vol» butyl acetate

I residue (80 g)

Dissolve in E-fc & er (300 ml) Misohen with 200 g of cellulose 'powder for the column & filtrate

Evaporation of the solvent

Elution of Zellaloseadsorbates with

4: 1 water

Aqueous Lb'aun p

Extraction with

3x2 1 ether

I Hüokstand (5 g)

Preparative TLC, Silufol φenzien / Ether _> 1: 1, v / v}

Active fraction C280 mg; 2 times run)

Preparative TLC, Silufol CHCWMeOH, 95s5, v / v

Active fraction C60

Preparative DC, Silufol Benzene / Aoeton, 1: 1, v / v, 3-fold run

% pure product (17

Claims (1)

invention claim A method of producing a cytodifferentiation stimulator in microbiological ways, characterized in that - The microorganism Streptomyoes viridoohromoffenes ZIMET 43683 under aerobic and sterile conditions in liquid nutrient media containing a carbon and a nitrogen source and mineral salts, at a temperature of 25 ⁰ C to: 37 ⁰ C for a period of 2 days to 6 days in a Acidity, the beginning of fermentation between pH 6.2 and pH 7.0 and at the end of this process between