DD201321A1 - PROCESS FOR PREPARING EPSILONIC ISORHODOMYCINONE - Google Patents

Abstract

The invention relates to a process for the production of epsilon-isorhodomycinone, a precursor of anthracycline antibiotics, which are of potential use for the chemotherapy of tumorous, viral and bacterial diseases in humans and livestock. The microorganism used for the production of the epsilon-isorhodomycinone is a mutant of the species Streptomyces violaceus and bears the name ZIMET 43649. The aim of the invention is to obtain a precursor of an antibiotic with potentially cancerostatic, antiviral and antibacterial properties as a starting substrate for semisynthesis and mutasynthesis to the Range of such Pharmaca expand. The object is achieved in that formed by aerobic submerged fermentation of the strain ZIMET 43649 of the species Streptomyces violaceus in media with suitable C, N sources and mineral salts epsilon-isorhodomycinone, isolated by suitable methods from the mycelium and subsequently purified.

Description

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Title of the invention

Process for the preparation of epsilon-isorhodomycinone

Field of application of the invention

The invention relates to a process for the preparation of epsilon-Isorhodomycinon microbiologically _e epsilon Isorhodomycinon is a precursor of various antibiotic substances that induced in the chemotherapy of tumor, viral and bacterial diseases in human and livestock of potential benefits are"

Characteristic of the known technical solutions

It is known that epsilon-isorhodomycinone is formed in trace amounts from the following microorganism species in addition to a variety of other anthracyclinones and anthracyclines. In all cases, it is not possible to obtain individual components from the mixtures of anthracyclines and anthracyclinones formed for detailed structure-weifungsuntersuchungen or only in the lowest yields by means of commercially difficult separation process. "Epsilon-isorhodomyoinone have been identified in fermentation solutions following anthracycline-formers: Streptomyces purpurascens (Brockmann and Franck, 1955. Ohem Beri, 88, 1792. # 1818; Brockman and Boldt, 1961, Chem. Ber., Ht. ₉ 2174, 2187) Actinomyces roseoviolaceus A 529 (IFO 13O8I) (Yoshimoto et al al *, 1980 _# J ₀ Antibiotics,

Object of the invention

The invention serves the economically favorable production of epsilon-isorhodomycinone «

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Explanation of the essence of the invention

It is surprisingly found that a microorganism selected by mutagenesis from a population of Streptomyces violaceus allows epsilon-isorhodomycinone to be readily available and cheap To produce starting materials in good yields ·

Under aerobic and sterile culture conditions, the genetically modified microorganism or its variants are cultured in a liquid nutrient medium with appropriate carbon, nitrogen sources and mineral salts. The microorganism used in this process is under the registration number ZIMBT 43,649 and the ZIMET microorgans deposit components 1, smen, Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the GDR, 6900 Jena, Beutenbergstrasse 11, has been deposited «

The cultivation of the strain ZIMST 43 649 and its mutants and variants is carried out under aerobic conditions · lyophilized spore material or glucose gelatin (5%) lyophilized mycelial fragments are inoculated into suitable agar media and subsequently into liquid, previously sterilized nutrient media and the resulting mycelium is cultured in a conventional manner at a temperature between 23 and 37 0, preferably 28 ⁰ O, over a period of 2 to 6 days, preferably 4 days, at an acidity at the beginning of the Permentationsprozesses 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 carbon and nitrogen sources, as well as of inorganic salts» The carbon sources can be starch, glucose, glycerine, dextrin, mannitol, sucrose , Soybean oil and soybean meal are used. "In addition to the above-mentioned nitrogen-containing substrates," nitrogen oxides "also come from" dry yeasts " fe, meat peptone and casein in irage «

Good results can be achieved with the addition of mineral salts · The latter promote the course of fermentation in

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Dependence on the nutrient medium used · In complex media containing various flours, additions of calcium carbonate, Na-phosphafc or »potassium phosphate have proved to be advantageous * The fermentation of the strain ZIMBT 43 649 ^ nn in steep-breasted bottles and round-bottomed flasks of various contents, in the glass fermenter and in V2A tanks ·

Isolation of the anthracycline vector epsilon-isorhodo-mycinone is carried out by extracting the anthracyclinone from the mycelium with organic solvents at pH 5, reextracting it with water-immiscible solvents after concentration of the extracts, and concentrating it from the extract concentrates by adding petroleum ether Stirring is precipitated, the solvent is removed by filtration, washed with petroleum ether, subsequently dissolved in chloroform and purified by column chromatography.

Epsilon isorhodomycinone is a dark red substance which crystallizes out of benzene / acetone and dissolves well in acetone, lower alcohols, glacial acetic acid, benzene and chloroform, but not in petroleum ether or water. »The anthracyclinone has indicator properties and is red in acidic solutions, al The melting point of the substance is between 220 and 226 ⁰ O. From the high-resolution mass spectrum results in the empirical formula ^ö 22 ^H 20 ° 16 · ® ^{& 3} molecular weight is: found 444.1148; Calculated 444.1056 (mass spectrum) · The thin-layer chromatographic characterization on silica gelpolymers yielded an Rf value of 0> 60 in the chloroform: acetone = 10: 1 system. On the other hand, an Rf value of 0.15 »is obtained when using silica gel as support material 60 after buffering with 0.5 η NaHCOo and as running system chloroform: acetone a 1: 1 are used * The absorption maxima in the ultraviolet spectral range Jl ^ _8x in chloroform / cyclohexane a 1:10 are 492 (934-5), 512 (14721), 523 (17665), 550 (17409), 565 (20354) nnu The IR spectrum (in KBr) shows; characteristic maxima at 1595 (quinoneearbonyl) and 1734 (COOCH ₃ ) om "" ¹

Because of this and because of the high-resolution mass spectrum

with the characteristic fragmentation m / z 444 (M ⁺ ) as well as 428, 426, 408, 384, 376 _? 355 * 349 * 339 * 33S ₉ 337 * 310 was adopted from the blockial dialysis © structure of epsilon · »isorhodomycinone confirmed (Brocbmana and Boldt 1961, Ghenu ^Ber · J2ä» 2174; Brockmami and Wimmer 1965 $ Ghem «Ber», $ 5 ₉ 2797; Brook man, Jr, "et al", ₈ 1965 "; Caem · Ber © $ Q _S 1260; Reed and Reid 1963; Tetrahedron ^ ^ ₈ 1817; Brockmann et al · '1968, Tetrahedron Lett _ea 4719) 5

embodiments

1 "Two 500 ml bottles of sucrose contain 80 ml of each of the pre-breeding medium

Glucose '1.5 %

Soybean meal 1.5 %

Ha-Ochloride O _S 5% Oa-carbonate 0.1 %

Primary K-phosphate 0 _? 03 %

Xn tap water

Sterilization $ 35 min _ö at 110 ⁰ Ce, After sterilization the acidity is at pH 6 _g 3. ©

Each Reagent bottle is presented by a spore «« mycelium suspension inoculated with sterile _$ which ^ isotonic saline from a test tube on the following culture-medium breeds ge et en j, 10 to 14-day-old culture of the tribe Zimeo? 43 649 is produced?

Oatmeal 1.0%

oatmeal ^

ground 1 ₈ .0 %

Agar Agar . 2.0%

is tap water sterilization 35 minutes at 120 ° © 0 _s nature except * The inoculated Vorzuchfc-Kultuxen 48 hrs at 28 ⁰ _e G on a shaker with a Frsquena of 180 / min "bebrütetο 8 ml of a so hatched serves VorzuchtkuTbur

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for inoculation of 500 ml steep-breasted bottles each containing 80 ml of the following production medium;

Glucose 2 ^ 0 %

Soybean meal 2.0 %

Na ~ Chloride 0.5%

Oa-carbonate 0.3%

in tap water

Sterilization: 35 min * at 115 ⁰ O, pH 6.3 * The temperature during the fermentation is preferably at 28 ⁰ O and the shaking frequency at 180 / min ·

After 96 hours of fermentation, the highest anthracycline content is reached (80 mg / 1) ·

2 »The procedure is the same as in Example 1, except that the production medium is composed as follows:

glucose 1.0 % Strength 1.0% soy flour 1.0 % baker's yeast 0.5% Na Ohlorid 0.5 % Oa-carbonate 0.3%

in tap water Sterilization: 40 min · at 116 ⁰ O, pH 6.2 «

With a culture of the strain ZIMET 43 649 from a solid medium as described in the 1 # example, inoculated 80 ml of the liquid pre-breeding medium mentioned in Example 1, which is contained in a 500 ml Steilbrustflasche »incubated 24 hrs at 28 ⁰ O on a shaker with a frequency of 180 U / 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 2 1 glass flask. Be incubated for a further 24 Stdi at 28 ⁰ O and at a shaking frequency of 180 U / min, "before the 400 ml of culture broth thus obtained for inoculation of 20 1 of Example 1

serve Produktionsmediuais described "The latter is a 32 l '~ jar fermenter containing" During the fermentatio which was carried out with an air supply of 15 I / min _e with a Rührgesehwindigkeit of 400 U / min® and the formation of foam by Zusafcs small quantities of sunflower oil or other ^ silikonaltlger foam control controlled © 20 1 culture solution with hydrochloric acid to a pH of 5 _? 0 is set and, after separation of the mycelium, the Kulturfilfcrat obtained is discarded The mycelium is extracted _s times with methanol in vacuo bis.zur aqueous phase concentrated _$ 4 diluted with water and then with chloroform exhaustively re-extracted © The R.eexferakt is, after drying over Na ₂ SO ,, concentrated in vacuo ₉ subsequently taken up in a little chloroform and with a 10 to 20 *. The excess crude product is filtered off with suction and washed with petroleum ether. The crude product obtained is 87% purity with a yield of 92 ₈ 8 mg / liter fermentation solution. It contains a mixture of epsilon-1-rhodomycinone and epsilon-isorhodomycinone Behavior 1s1 ₀ Content of the crude product and the percentage composition of AgIykongemisches were determined by UV spectroscopy, "Single column chromatography on buffered (acidic or alkaline) silica gel with chloroform 'bsw» chloroform / methanol in the ratio 20g1 to 1s1 results in 60 mg epsilon-ehodomycinone / epsilon Isolation of pure epsilon-isorhodomycinone from the mixture is achieved by separation of epsilon-rhodomycinone by repeated column chromatography on NaHCO3-buffered silica gel with chloroform / Methanol in the ratio 1sO to 1s1 _O

In contrast, the isolation of pure epsilon-rhodomycinone from the mixture is extremely difficult and is achieved by appropriate Säulenchromateogrs, fie only with considerable yield lossesHö

«7

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The method differs from that of the example in that the stock ZIMBT 43 649 after inbred culture is inoculated into a 400 l V2A tank in a 2 l glass broth instead of a 32 l glass fermenter. Example production medium contains · The stirring speed is 280 rpm · and the air supply is set to 400 1 / min. From 350 l of culture solution, 80 mg of pure epsilon-rhodomycinone / epsilon-isorhodomycinone mixture are obtained per liter, according to the method described in Example 3, from which the epsilon-isorhodomycinone can be separated in the manner described in Example 3 »

The advantages of the production process for epsilon-isorhodo «mycinone persist in

- The simple recovery of a precursor of potentially cancerostatic anthracycline Antibiotioa, which can lead to new anthracycline antibiotics using partial synthesis and mutasynthesis, which were previously not a therapeutic impact test or the structure-activity study was not accessible

- The extension of the range of microorganism mutants, which prefer to synthesize a precursor (anthracyclinone) of anthracycline antibiotics as a single component and accumulate in the mycelium

- the use of mutagenesis mutants of Streptomyoes violaoeus . Strain ZIMET 43 649, which is capable of forming a mixture of epsilon-Bhodomycinon and epsilon-isorhodomyoinone, from which the individual component epsilon-isorhodomycinone can be separated easily and in surprisingly high yield, so that otherwise in the isolation of anthracyclines and Anthracyclinoneη usual effort in separation and cleaning operations greatly reduced

in the preparation of a precursor which has not hitherto been sufficiently producible by microbiological means, different epsilon-isorhodomycinone glycosides have been tested in cancer chemotherapy

due to feialender manufacturing process was not possible

making available a Msher for Mutasynthesis, the bioconversion and / or semi-synthesis nonexistent at "· thracyclinonSf with the aid of a further ₈ biomodifizierte Strukturderivafce of Anthracite © y®llo." antibiotics prepared and evaluated, their therapeutically ^ toxikologisohen effects can be studied

the surprisingly high yield of anthracyclinone in comparison with other known anthracyolinone production processes, with simultaneous use of cheap fermentation feedstocks.

Claims (1)

9 ·. 234896 O invention claim Process for the preparation of epsilon ^ isorhodomycinone in a microbiological manner, characterized in that a mutant of Streptomyces violaceuA strain ZIMBG? 43 649, cultivated under aerobic conditions in liquid nutrient media containing a source of carbon and nitrogen and mineral salts, at a temperature of between 25 and 37 ⁰ O, for a period of 2 to 6 days, and the formed epsilon-isorhodo * Mycinone is extracted from the Myoel at an acidity between pH 3 and pH 9 »by means of conventional solvents, then concentrated by conventional methods, precipitated and purified by chromatography ·