DD271528A1 - PROCESS FOR THE MANUFACTURE OF AKLAVIKETONE - Google Patents

Abstract

The invention relates to a process for the preparation of Aklaviketon on a microbiological route. The aim of the invention is the oeconomically favorable production of Aklaviketon as a precursor for mutasynthesis and partial syntheses of anthracycline antibiotics with potential cancerostatic, antiviral, antibacterial, immunosuppressive and ergotropic properties. The object associated with this aim is achieved by forming aklaviketone by aerobic submerged fermentation of the strain Streptomyces galilaeus S 383 in media containing a carbon source, a nitrogen source and mineral salts, isolating it from the mycelium and subsequently purifying it by chromatography.

Description

Spectroscopic properties: UV / VIS: λ ™ _χ (27β), (290), 435 nm (CHCl ₃ )

IR: Vn ₁ MiBSO, 1655, 1675, 17 38

(c = o region, CHCl ₃ )

MS: m / z Μΐ found: 410.09b8; ber. 410, 1009 (CJeH ₁₈ O ₈ ); 392.0895 (MH ₂ O) T, 361.0681 (MH ₂ O-OCH ₃ ) *

¹ H NMR: see Table 1 Structural formula: see Figure 1 The data presented characterize aklaviketone as a pure substance.

embodiments By the following embodiments, the invention will be explained, but not limited. Example 1: Two 500-rpl slippers each contain 80 ml of the following pre-breeding medium:

Glucose 1.5%

Soybean meal 1.6%

Sodium chloride 0.5% Calcium carbonate 0.1% Potassium hydrogen phosphate 0.3%

in tap water (sterilization: 35 minutes at 11O ⁰ C) After sterilization, the acidity is at pH 6.3.

Each steep-vial is inoculated with a spore suspension prepared with sterile, isotonic saline from a 10 days-to-14 days culture of strain S383 grown in a test tube on the following culture medium:

Oatmeal 2%

Agar-agar 2%

in tap water (sterilization: 40 minutes at 120 ° C)

After sterilization, the acidity is adjusted to pH 7.0. The inoculated Vorzucht-cultures are incubated on a shaker at a frequency of 180 rev / min for 48 hours at 28 ⁰ C. 8 ml of 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%

Sujame flour i ', 0%

Sodium chloride 0.5% Calcium carbonate 0.3%

in tap water (sterilization: 3b minutes at 11O ⁰ C) The acidity after sterilization is pH 6.3.

The temperature during the fermentation is 28 ⁰ C and the shaking frequency at 180U / min. After 72- to 96-hour fermentation, the highest yield of Aklaviketon is achieved.

Example 2:

With a culture of strain S 383 from a semi-solid medium, as described in Example 1, 400 ml of the liquid pre-emergence medium mentioned in Example 1, which is contained in a 2 l glass flask, are inoculated. It is incubated for 48 hours at 28 ° C on a shaker with a frequency of 180 U / min. The 4CO ml culture broth thus obtained are used to inoculate 20 liters of the production medium described in Example 1, which is contained in a 32 l glass fermentor. During the fermentation, which can be carried out with a stirring speed of 400 rpm and an air supply of 15 l / min, foaming is controlled by the addition of small quantities of sunflower oil or silicone-containing antifoaming agent.

Example 3:

With the media described in Example 1, the Vorzucht is first carried out for 24 hours in a 500 ml Steilbrustflasche with 80ml content and then for another 24 hours in the same medium in a 2-liter glass flask with 400ml content before a 400- Can be inoculated I-V2 A steel tank containing the production medium described in Example 1. The stirring speed is 280 rpm and the air supply is 400 l / min.

Example 4: From 5I of the culture solution obtained according to Examples 1, 2 and 3, the mycelium is separated by separation and the culture filtrate

discarded. The mycelium is extracted several times with acetone, the acetone extracts combined and concentrated to 300cm ³ .

After adding 500 ml of chloroform and the same amount of water, the chloroform phase separates, which is separated,

dried and concentrated to a small volume. By adding petroleum ether is a brown precipitate as

Crude product precipitated (404 mg). The subsequent purification is carried out by column chromatography on silica gel. For this purpose, the crude product> n one

small amount of the mixture chloroform-methanol (90:10, v / v) and placed on a small column of silica gel 60, given. The

Elution is carried out with the same solvent system chloroform-methanol (90:10, v / v). It is separated into several yellow, red and blue ions. Aklaviketon wanders as a red zone. After unification of Fractions containing aklaviketone after thin-layer chromatography, the solution is concentrated to dryness,

dissolved with a little chloroform, the residue and the pure Aklaviketon by adding petroleum ether like.

Table 1: ¹ H NMR data of Aklaviketone in CDCl ₃ (100 MHz)

Assignment of protons * Chemical shift, ppm (J, Hz)

ΗΛ 7.78 dd (A)

H-2 7.64 t (B)

H-3 7.33 dd (C)

(Jab = 8, Jac = 1.6, Jbc ¹ = 8)

H-11 7.70 s

4-OH 12,65 s

'6-OH 13.94 s, br

8-CH ₂ 2.81 and 3.40 (AB)

10-H 4,21 d (X)

(Jab = 10, Jax = 1.8, Jb _x = O)

CH ₂ (C ₂ H ₆ ) 1.67 m

CH ₃ (C ₂ H ₆ ) 1.09 t

(JcH, / CHi = 7.5)

COOCH ₃ 3.69 s

Abbreviations: β slngulet, dd doublet of doublets, t triplet, q quartet, m multiple, br wide signal * numbering 8. Formula

Claims (2)

1. A process for the preparation of Aklaviketon on a microbial route, characterized in that the strain Stroptomyces galilaeus S383 under aerobic conditions in liquid Nährmedier containing a carbon, a nitrogen source and mineral salts, at a temperature of 25 ⁰ C to 37 ⁰ C is cultivated for a period of 2 days to 6 days and the resulting Aklavikoton extracted from the mycelium using conventional solvents, then concentrated by conventional methods, precipitated and purified by chromatography. '• 2nd A method according to claim 1, characterized in that the fermentation is carried out at a temperature of 28 ⁰ C and over a period of 3 days. For this 1 page formula Field of application of the invention The invention relates to a process for the preparation of Aklaviketon on microbiological Weje. Aklaviketone is a suitable precursor for mutagenic or semisynthetic production of various novel antibiotic active substances which are of potential use in the chemotherapy of tumor, human and farm animal related diseases, tumor and human, and as immunopuppressants and ergotropics.
The field of application of the invention is thus in the chemical-pharmaceutical research and industry. Characteristic of the known state of the art Aklaviketone is a new product. A process for its preparation is not known. As an anthracycline precursor with a keto group in the C ₇ position, only maggiemycin has heretofore been known (McGUIRE, J. et al., 1980. Adv. Biotechnol., [Proc. Int., Ferm., Symp.) 6th, 3,117-122]. Maggiemycin differs from aklaviketone by a hydroxyl group in C _(1- position.) Anthracyclinones with hydroxyl groups in C, position (YOSHIMOTO, A. et al., 1980) have been used as starting materials for mutasyntheses or partial syntheses of aniracyclin arbiiotics. J. Antibiot., 33, 1150-1157; OKI, T. et al., 1380. J. Antibiot. 33, 1331-134 OJYOSHIMOTO ₁ A. et al., 1980. J. Antibiot., 33, 1558-1166; YOSHIMOTO. A. et al., 1981, J. Antibiov, 34, 1492-1494, U.S. Pat., Appl., EP 62, 321, Oct. 13, 1982) and anthracyclines (ARCAMONE, F .: doxorubicin, Academic Press, New York 190 i). Object of the invention The invention aims at the economically favorable production of AMaviketon as a precursor for mutasynthesis and partial syntheses of anthracycline antibiotics with potential cancerostatic, antiviral, antibacterial, immunosuppressive and orgotropic properties. Explanation des.Wesens of the invention The object of the invention is to describe a microbiological process which can be used to produce aklaviketone as a starting material for mutase syntheses or partial syntheses of anthracycline antibiotics. According to the invention this object is achieved in that the microorganism Streptomyces galilaeus S383 is cultivated under aerobic and sterile culture conditions in a liquid nutrient medium with appropriate carbon, nitrogen sources and mineral salts. The microorganism which is used in this process is deposited under the registration number ZIMET 43855 in the GDR depository for microorganisms, Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences, GDR-6900 Jena, Beutenbergstraße 11. Surprisingly, this microorganism could be isolated after mutagen treatment from a Streptomycetes strain, which was known as a generator of pyrromycinones and ε-Pyrromycinonglykosiden. The cultivation of the strain ZIMET 43 855 and its variants takes place under aerobic conditions. Of glucose / gelatin (5%) lyophilized mycelium fragments are inoculated onto appropriate Agarnährmedien and subsequently in liquid, previously sterilized culture media, and the resulting mycelium is in a known manner at a temperature between 25 ⁰ C and 37 ° C, preferably 28 ⁰ C, over a period of 2 days to 6 days, preferably 4 days, cultured at an acidity which is between pH 6.2 and pH 7.0 at the beginning of the fermentation process and between pH 7.5 and pH 8.3 at the end of the process , The nutrient medium consists of carbon and nitrogen sources as well as organic salts. The carbon source used is starch, glucose, glycerol, dextrin, mannitol, sucrose, soybean oil and / or soybean meal. As nitrogen source, in addition to the above-mentioned nitrogen-containing substrates, dry yeast, meat peptone and / or casein are also suitable. Good results are obtained with addition of mineral salts. The latter favor the course of the fermentation depending on the nutrient medium used. In complex media, additions of calcium carbonate, sodium phosphate or potassium phosphate have proved to be advantageous. The isolation of the obtained new, aklaviketone called substance is carried out by known methods by extraction of the mycelium with organic solvents, precipitation with petroleum ether and subsequent chromatofjraphische cleaning. The substance obtained in this way is a yellow-brown powder. It is characterized by the following properties: empirical formula: C ₂ 2H | ₈ Oe. The compound is decomposable and does not have a clear melting point. It is readily soluble in organic solvents such as chloroform, acetone, methanol but insoluble in water and petroleum ether.