DD201322A1 - PROCESS FOR THE PREPARATION OF EPSILON RHODOMYCINONE - Google Patents

Abstract

The invention relates to a process for the preparation of epsilon-rhodomycinone, a precursor of anthracycline antibiotics, which are of chemotherapeutic value for the chemotherapy of tumor, virus and bacterial diseases in humans and livestock. The microorganism used for the production of epsilon-Rhodomycinons is an antibiotic-blocked mutant of the species Streptomyces griseus and bears the name Zimet 43 665. The aim of the invention is the economic production of epsilon-Rhodomycinon as a precursor for anthracycline antibiotics with potentially cancerostatic, antiviral and antibacterial properties. The object is achieved in that formed by aerobic submerged fermentation of the strain ZIMET 43 665 of the species Streptomyces griseus in media with suitable C, N sources and mineral salts epsilon-rhodomycinone, isolated by suitable methods from the mycelium and subsequently purified.

Description

234895 2

Title of the invention

Process for the preparation of epsilon-rhodomycinone

Field of application of the invention

The invention relates to a process for the preparation of epsilon-Bhodomycinon by microbiological means, Epsilon-Rhodomycinon is a precursor of various antibiotically active substances, in the chemotherapy of tumor, virus and bacteria-induced diseases ¹ in humans and livestock of potential Benefits are «

Characteristic of the known technical solutions

It is known that epsilon-rhodomycinone is formed in addition to a variety of other anthracyclinones and anthraoyline in 6 or 7 different Streptomyceten species »In all cases, from the formed mixtures of anthracyclinones and anthracyclines, individual components for detailed structure-activity studies are not or only to be obtained with the lowest yields by means of separation processes which are difficult to realize industrially.

Epsilon rhodomycinones have hitherto been identified in fermentation solutions of the following microorganism species: Streptomyces purpurasoens (Brockmann and Pranck, 1955, Chem. Ber. 88, 1792).

, Str.eptomyces peucetius (DiMarco and Arcamone, 1975 · Arzneimittelforsch · 25, 368-375)

Streptomyces coeruleorubidus (Blumauerova et al · 1978 "Folia Microbiol., £ 3, 255-260) <

Streptomyces griseoruber 4620 (Podojil et al. Folia Microbiol .1980 _f., £ 5, 464)

Streptomyces violaceus (Fleck et al _os 1973 "GDR-WP 100 494) Actinomyces roseoviolaceus (loshimoto et al", 1980 "J _e Antibiotics, 33, 1150)

Streptomyces spec. J _e 14 (Mc Guire et al., 1980, Antimicrob. Agents Chemother., 1_8, 454 ~ 69)

It is dartiberhinaus been described that epsilon rhodomycinone as starting material for partial syntheses (Lin et al., 1980. J ₄ Med. Chem., 2J3j 1242-44) and the Mutasynthesis (Matsuzawa et al, "1980" J _{e Antibiotics?} ^ 3.5 1341-47) can be used. The prerequisite for this is the economical production of the epsilon-II-hymodomyeinone, which is not possible with the aid of the previously known methods,

Object of the invention

The invention aims at the economically favorable production of epsilon-rhodomyeinone

Explanation of the essence of the invention

The invention has the object zn reason, epsilon-Rhodoraycinor produced by a process ^ which avoids the disadvantages of the known modes of representation "has been surprisingly found ^ that a selected by Mutageneae from a population of Streptomyces griseus YES 3933 antibioticageblockter microorganism it gesta TFET _s epsilon- Rhodomyeinone from easily accessible and cheap starting materials in good "yields produce

Under aerobic and sterile culture conditions, the genetically modified microorganism or its variants are grown in a liquid nutrient medium with appropriate sources of carbon, nitrogen and mineral salts. The microorganism used in this process is available under Regiiernummer ZIlET 43 665 in the ZIWEET Depository.

23 48 9 5 2

Center for Microbiology, Central Institute for Microbiology and Experimental Therapy of the DDH Academy of Sciences, 69 Jena, Beutenbergstraße 11.

The cultivation of the strain ZIMET 43'665 and its variants is carried out under aerobic conditions. An earth lyophilic dried spore material or of glucose / gelatin (5 # ig) lyophilized mycelium fragments are at suitable agar media and then inoculated into liquid, previously sterilized culture media, and the resulting mycelium is in a known manner at a temperature of 25-37 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 is. The Hährmedium consists of carbon and nitrogen sources and inorganic salts. As carbon sources, starch, glucose, glycerin, dextrin, mannitol, sucrose, soybean oil and soybean meal can be used. Nitrogen sources other than the above-mentioned nitrogenous substrates include dry yeast, meat peptone and gasein. Good results can be achieved with the addition of mineral salts. The latter favor the course of the fermentation depending on the nutrient medium used. In complex media containing various flours, additions of calcium carbonate, sodium phosphate or potassium phosphate have proved to be beneficial.

The isolation of the anthracycline precursor epsilon-rhodomycinone is carried out by extracting the anthracyclinone from the myoel with organic solvents at pH 5.0, re-extracting it after concentrating the extracts from the aqueous phase with organic, water-immiscible solvents, and precipitated from the extract concentrates by trituration with petroleum ether with stirring, the solvent is removed by filtration, washed with petroleum ether, then dissolved in chloroform and säulenchromatografie ^ purified.

Λ 8 9 5 2

The thus isolated substance is a dark red crystalline substance, the check easily »ether in acetone, ethanol and glacial acetic acid, moderately in benzene and chloroform, and little in petroleum and water dissolves · The melting point of the substance is between 207-211 ⁰ C ₈ From the high-resolution mass spectrum, the empirical formula is 0 ₂ ρ ^Η 2θ ° 9 · The molecular weight is found 428, 1115; calculated 428, 1107 (mass) spectrum). Tlc on Kieselgelfolien characterization revealed in the course of the system chloroform: acetone = 10: 1 Rf value of 0,6O _4, the other hand is obtained an Rf value of 0.20 when as carrier material of silica gel 60 after buffering with 0.5 N! NaHC0 ~ and used as running system chloroform: acetone = 1: 1. The absorption maxima in the visible spectral range JL (E) in chloroform / cyclohexane = 1 * 10 are 471 (5933), 484 (6766), 496 (7494), 518 (5620), 534 (5100) nnu The IR spectrum (in KBr) shows characteristic maxima

-1 1598 (quinonecarbonyl) and 1735 em, (carbomethoxy-G-group).

As a result of this and because of the high-resolution mass spectrum with the characteristic fragmentation m / z 428; 41Oj (M ⁺ ) and 392; 376; 368; 36O; 357; 35O; 339; 334; 333 the structure of the epsilon-rhodomycinone was assumed (Brookmann and Boldt, 1961 "Chem _a Ber., 94, 2174; Brockmann and Wimmer, 1965." Ch. Em. Berger, 98, 2797; Brookman Jr. et al., 1965. Chem _e Ber _e , Reed and Reid, 1963 "Tetrahedron, 1, 9, 1817; Brockmann et al.," 1968. Tetrahedron Lett., 4719).

embodiments

1. Two 500 ml breast bottles each contain 80 ml of the Prezuoht medium

G-glucose 1, 50 *

Soy meal 1,50 ί> sodium chloride 0,50 % Calcium carbonate 0,10 % primary potassium phosphate 0 ^ 03 ' % in tap water

23489.5 '2

Sterilization: 35 min. At 110 ^° C. After sterilization, the acidity is at pH 6.3.

Each steep-breast bottle is inoculated with a spore and / or mycelium suspension which is prepared with sterile, isotonic saline solution from a 10 to 14 day old culture of the strain ZIMET 43 665 grown in the test tube on the following culture medium:

sucrose 0,300 % dextrin $ 1,500 urea $ 0.010 yeast extract 0.100 Ί » Peptone (bact.) 0.500% sodium chloride 0.050 # ferrous sulfate 0.001 # primary potassium 0.050 # phosphate Agar-agar (washed) 2,000% in aqua distillata

Sterilization: 40 min. At 116 ^° C. After sterilization, the acidity is between pH 6.5 and pH 7.0. The inoculated cultures are Vorzucht on a shaker at a frequency of 180 rev / min, incubated for 48 hours at 28 ⁰ C. 8 ml of a pre-culture incubated in this way are used to inoculate 500 ml steep-breasted bottles, each containing 80 ml of the following production medium:

Glucose $ 2.0

Soybean meal $ 2.0

Sodium chloride 0.5%

Caleium carbonate 0.3 #

in ι tap water

Sterilization: 35 min. At 110 ^° C. The acidity after sterilization is pH 6.3 »

The temperature during the fermentation is 28 C and the shaking frequency at 180 U / min. After 72 to 96 hours of fermentation, the highest yield of epsilon-ehodomycinone is achieved (10.5 mg of crude product per liter of culture fluid) *

2348-95 2

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

Glucose 3,000 %

Soymeal 3,000 #

Galcium carbonate 0,300 %

Sodium chloride 0.300%

Ferric chloride hydrate 0.025 %

in tap water

Sterilization: 35 min. At 110 ° G _e The acidity is after sterilization at pH 6 _S 5 »

3 · Proceed as in the 2 example with the difference that de:

Cultivation agar, the pre-breeding and the production medium have the following composition:

Cultivation agar sucrose 2.0 %

Dry yeast 0.1 <fo

Sodium nitrate '0.2 %

Magnesium sulfate 0.2%

secondary potassium phosphate 0.2 ί>

Agar-agar 1, 5 ⁰ Io

in tap water

Sterilization: 40 min. At 116 ^° C. Acidity is after sterilization at pH 7 _? 0®,

Vorzucht medium

Bacto Peptone 0.60 <$>

Dry yeast 0.30 # calcium nitrate

hydrate 0.05 % in tap water

Sterilisierungt 30 min at 120 * ⁰ Cj pH'7,2 after sterilizing,

Production medium

Glucose 4.00G> #

Dry yeast 1,500 <$>

Sodium chloride 0.200 #

Galcium carbonate 0.100 f>

sec.Calphium phosphate 0-100 #

234895

magnesium sulfate 0.010 la ferrous sulfate 0.001 # zinc sulfate 0.001 # copper sulphate 0.001 % in tap water

Sterilization for 30 min. At 120 ⁰ C, glucose is sterilized separately at 110 ⁰ C for 20 min. The acidity is thereafter at pH 7.0.

4. The procedure is as in Example 3 with the difference that the pre-culture and production medium has the following composition:

Prefab Medium Dextrin 3.00 #

Corn steep liquor 0.40 % Calcium carbonate 0.30% Ammonium sulfate 0.10 % Casein 0.50 %

sek.Kaliumphosphat 0.01% in tap water sterilization: 35 minutes at 120 ⁰ C. acidity after Sterili.

Sieren: pH 7.0. dextrin 3.00 Production medium calcium carbonate 0.60 Corn steep liquor 0.80 casein 0.50 ammonium sulfate 0.10 sek.Kaliumphosphat 0.01 in tap water

Sterilization: - 35 min. At 120 ^° C., acidity after sterilization: pH 7.0 »

5. The method differs from that of Example 2 in that the culture to be inoculated is developed on the following semi-solid medium:

23Ä895

Oatmeal 2.0 agar-agar 2 _s 0

in aqua distillata

The sterilization is carried out in the usual way and the acidity is at pH 7.0. Subsequently, the pre-growing medium described in Example 2 is inoculated and with the inoculum thus obtained, 500 ml steep breast bottles are inoculated containing 80 ml of the production nutrient medium composed as follows:

Glucose 1.0%

Potato Starch 1.0% Soy Meal 1.0 #

Dry yeast 0.5 ί sodium chloride 0.5 % calcium carbonate 0.3 # in tap water

Sterilization: 40 min. At 110 ^° C., awake of sterilization, the acidity is at pH 7.0,

6 × With a culture of strain ZIMET 43 665 from a semi-solid nutrient medium, as described in Example 5, 400 ml of the pre-seeded liquid shown in FIG. 1, which is contained in a 2 l glass flask are inoculated. It is incubated for 48 hours, at 28 ⁰ C on a Sehüttelgerät with a frequency of 180 U / min, The 400 ml of culture broth thus obtained are used for inoculation of 20 1 of the production medium described in Example 1, in a 32 l ~ glass fermenter During the fermentation, which can be carried out with a stirring speed of 400 rpm, and with an air supply of 15 liters per minute, foaming is controlled by the addition of small quantities of sunflower oil or other silicone-containing antifoaming agents »

234895 2

7 · As cultivation agar is the used as described in Example 5, the Vorzucht in the listed in the 1 _# sample medium, first for 24 hours in a 500 ml wide-necked bottle with 80 ml content and subsequently for a further 24 hours in the same medium in a 2 l glass flask with 400 ml contents is carried out before a 400 l V2A tank can be inoculated containing the production medium described in 1, Example * The stirring speed is set to 280 rpm, and the air supply 400 l / min, set.

After acidifying the culture solution to pH 5.0 by means of HCl and after separating the solid from the liquid phase (culture filtrate / mycelium), the culture filtrate is discarded and the mycelium is extracted 4 times with methanol, concentrated in vacuo to aqueous phase, with water diluted and subsequently reextracted exhaustively with chloroform. The re-extract is dried over Na ₂ SO ₄ . concentrated in vacuo, then taken up in a little chloroform and precipitated with a 10 to 20-fold excess of Eetroläther · The precipitated crude product is filtered off with suction and washed with Eetroläther «The yield is 10.5 mg / 1 culture fluid · The content of the crude product and the composition of anthracyelinone mixture present in the crude product was determined by UV spectroscopy · single column chromatography on buffered gel with chloroform or * chloroformate in a ratio of 10: 1 gives 4.3 mg of pure epsilon-rhodomycinone / 1 "

Claims (1)

234895 2 invention claim A process for the preparation of epsilon-fihodomycinon in a microbiological way, characterized durch, d a f ^ an antibiotic-blocked mutant of the species Streptomypes g risens, strain ZIlSET 43 665, under aerobic conditions in liquid nutrient media containing a carbon, a nitrogen source and Mineral salts contained, cultivated at a temperature of 25 to 37 ⁰ C, over a period of 2 to 6 days, and the formed epsilon-Shodomycinon from Myoel at an aoidity of pH 3 to pH 9, 5, extracted by conventional solvent, hereinafter Concentrated, precipitated and chromatographically purified by conventional methods »