DD156374A1 - PROCESS FOR PREPARING AN ANTIBIOTICUM - Google Patents

Abstract

The invention relates to a method for producing an antibiotic which is of potential use for the chemotherapy of viral and bacterial diseases in humans and livestock. In particular, the invention relates to a method for producing the antibiotic granaticin methyl ester using the microorganism Streptomyces lateritius, strain ZIMET 43 646. The object of the invention is to obtain a potentially virostatic and antimicrobial antibiotic with low toxicity and lack of cross-resistance to other clinically used antibiotics, to expand the range of such Pharmaca. The object is achieved in that by aerobic submerged fermentation of the strain ZIMET 43 646 in media with suitable feedstocks granaticin methyl ester is formed, isolated with suitable methods of mycelium and culture filtrate, purified and recovered.

Description

Title of the invention

Process for the preparation of an antibiotic ^

Field of application of the invention

The invention relates to a process for the preparation of an antibiotically active substance _? which is of potential use in the chemotherapy of viral and bacterial diseases in humans and livestock. In particular, the invention relates to the nature of a pigment antibiotic Sj which, because of its physico-chemical properties, is termed granaticin methyl ester and is first produced by means of a microbiological production process.

Characteristic of ^; known technical solution

To! Type of naphthoquinone antibiotics. Natural products belonging to (dip naphthocyclinones or isochromanquinones) are Ze Be Granaticin, Granaticin B _}, Litmocidin, Litmomycin, Antibioticum Yir-141} Antibioticum 1321 and its components A and B ^ Antibioticum Zg and Zgg, as well as the Antibioticum XK-fy6 _s furthermore Dihydro- Granaticin and the methyl ester of, Dihydro-Grana.ticin, the granatic acid _$ o < _? ß _f y ~ Naphthociclinonj purpuromycin _3, the Rubromycine "the griseorhodin and Grar" natomycine A ₃ C ₅ D and E ₀ the Griseusine A and B are still known Recently, * Microbiological ^production: procedures for Naphthoehinon antibiotics are with previously The following microorganism species have become known: Proactinomyces cyaneusj Streptomyces olivaceus ViAJCSMAN ₁ Streptomyces litmogenes _s Actinomyces litmocidini, Actinomyces anthooyaneusj Streptomyoes thermoviolaceus var * Wr-141, Streptomyces granaticolor ^ Streptomyces violaceo'ruber and Streptomyces arenae _e

Aim of the invention

The invention is used to produce eines.neuen Antibioticmns from DER structural class of _{naphthoquinones%} as well as its derivatives and SAIKE ₅ around the pallet potentially virostatic and antimicrobial compounds by a new Antibiotic to expand · "

Explanation of the essence of the invention

The invention has for its object _{y to provide} a technologically simple method j. which allows _s to produce the new antibiotic granaticin methyl ester with the aid of a microorganism of easily accessible and cheap starting materials in good yields.

In accordance with the invention, the object is achieved in that a microorganism or "its mutants or variants are bred under aerobic and sterile culture conditions in a liquid nutrient medium with appropriate carbon, nitrogen and mineral salts" The microorganism, which finds application in the inventive method was rn.it help. well-known mutagenesis and selection methods from populations of the wild strain selected by the granatomycin producer ο This wild strain is isolated from a soil sample from the Baltic Sea island Hiddensee (Fährinsel) and at the depository for patent strains, the GDR at the Central Institute for Microbiology and experimental therapy _$ . Jena, the Academy of Sciences of the GDR under the number ZIEET 43 62? deposited. _: "

The tribe ZIIyIET 43 62? is a representative of the species Strep_tomj £ es. iäiSi ^ ÜiiS (Sveshnikova) Pridham, Hesseltine and Benedict 1958 and is extensively characterized in the patent application YIF G 12d / 216.415 «Those from the tribe ΖΙΜΕΪ 43 62? by mutagenesis and selection obtained mutant with biosynthetic capacity for the new antibiotic to Granaticin-methyl. ester is in the depository for patents of the GDR in the Central Institute of Microbiology and experimental

-i-ΐ'ΙΊΊ

Therapy, Jena, the Academy of Sciences of the GDR under the number ZIMET 43 646 deposited,. , The cultivation of the strain ZIMET 43 646 and its mutants and variants is carried out under aerobic conditions. An'Erde lyophilic dried spore material or lyophilized in glucose broth mycelial fragments are vaccinated for the cultivation in suitable agar media and liquid nachfolgend.in, previously sterilized culture media, and the resulting mycelium is (in known manner at a temperature of 25-37 ⁰ C. Preferably 28 ⁰ C) cultivated over a period of 2 to 6 days (preferably 4 days) at an acidity "at the beginning of the fermentation process at pH 6.2 to pH 7 $ 0 and at the end of the process between pH 7" 5 and pH 8 The nutrient medium consists of carbon and nitrogen sources as well as inorganic salts. As carbon sources, glucose, starch, mannose, glycerine, dextrin, soya flour and other flours can be used. Nitrogen sources include soybean meal, soybean oil, dry yeast, meat peptone and casein. Good results can be achieved with the addition of mineral salts. The latter favor the course of fermentation depending on the nutrient medium used. In complex media containing various flours, additions of calcium carbonate, sodium phosphate, and potassium phosphate, respectively, have been found to be beneficial.

Fermentation of the Granaticin ~ MethyIester, ZIIvIET 43 646 can be carried out in narrow neck bottles of various contents, in round bottom flasks and glass fermenters, and in V2A steel tanks.

The isolation of the granatic-methyl ester is carried out by isolating the mycelium from the culture solution and washing the mycelium with aqueous alkaline solution, after combining the washing solution with the mercury titrate after adjusting to a slightly acidic pH Value with the help of organic or inorganic acids in the combined antibiotic-containing; Solution, after extraction

of ^ antibiotic with HxiLfe a Rait water-immiscible or sparingly miscible Extraktionsm.itteIs ₅ after concentration thereof to a Mininialvolumen and after replacing the Extrak ™ tionsmlttels with the aid of azeotropic distillation with water after Eeextr action of the active ingredient mixture ^ by means of a V / ater not or less miscible extract of higher density than water, after separation of the ree extract, after concentration of the extract of eee to a minimum volume, after chromatographic purification and separation on dextran gel the fraction containing the granaticin methyl ester is collected separately and from this the antibiotic is evaporated of the solvent .. won. With unfavorable fermentation with respect to the granaticin methyl ester to be recovered, and the associated enhanced. Separation between these and the granatic-methylester is achieved by adding a low-boiling hydrocarbon fraction to the extractate, filtering off the precipitate which has precipitated, and concentrating the precipitate and subjecting it to azeotropic distillation is "the aqueous suspension is' then as shown. o <.a _{e s} reextracted the antibiotic thereby transferred into the organic phase, purified by gel chromatography and freed from the remaining accessory ingredients.

Granaticin methyl ester is an intense red substance, with slightly acidic properties and has indicator nature, dissolves well in practically all organic solvents, but poorly in water. "The summation formula is ι ^ p3 ^ 24 ^ 11 ^? the molecular weight is 476 »The amorphous substance has a W absorption in methanol of 285 _s 525 and 560 nm, with shoulder at 490 nm. The maxima of the IR spectrum (in KBr) are: "74Oy 775, 910, .960, 990, 10.75, x 1140, 1170, 1190, 1260, 140O ₅ 1440, 1560, 1605, 1740, 2925, 3440 cdT ¹ (Fig.1).

The granaticin methyl ester differs from the known microbial metabolites granate ic.in and garnetic acid in the acid by the methoxy signal at 3 * 70-3f80 ppm in the proton

resonance spectrum and of 4-deoxy-garnetic acid methyl ester (^ dihydrogranaticin methyl ester) by the signal at 4.75 ppm "or * the lack of signals at 3 * 00 and 2.34 ppm (protons 4a and 4e im Dihydrogranaticin-methyl ester)!. (Fig.2).

The process for producing the granaticin methyl ester is supplemented by, but not limited to, the following embodiments.

Embodiment 1

The culture solution of Streptomyces lateritius mutant, strain ZIMBT 43 646, is separated in the original state and the separated mycelium is incubated with the same volume of alkaliated water from pH 7 to 10, preferably pH 7.5 to pH 8.0, as the broth washed out. Culture filtrate and washing solution are combined and adjusted with acid to a pH of 4.2 ί 1.0. It is then extracted with a third and in the second operation with a fifth of the reference volume of acetic acid ethyl ester. The extract is concentrated under mild conditions in vacuo and the organic phase is replaced by water by azeotropic distillation. "The aqueous suspension is diluted to one-tenth to one-fifth of the reference volume with water and re-adjusted to pH 3 0 »5 by means of mineral acid, preferably by means of dilute sulfuric acid, with an extractant of higher density than water, preferably chlorohydrocarbons, especially chloroform.The chloroform extract is dried and concentrated to give a final volume of 0.5-2% of the reference volume of culture solution reached.

AusführungsbeispieLv2

The ethyl acetate extract is not in contrast to the embodiment 1 in the aqueous suspension of the anti-

^e 7 ^f 7 η η

I i υ /

biotics transferred _? but after concentrating on i $ of the reference volume, with five times the amount of a low-boiling hydrocarbon Sj, preferably with petroleum ether _? mixed and the failed Miederschlag is after a. Off. abfiltierte some hours The filtrate is concentrated to the original volume of ethyl acetate ~ concentrate, and by tracking of water, an aqueous suspension of the antibiotic is obtained © This is _s set forth in Example 1 _$ © further processed

Embodiment 3

The obtained according to Examples 1 or © 2 chloroform extract concentrate is added in portions to 100 ml of gel columns applied which 2 kg of gel contain and then eluted with chloroform "The second exiting the column _s red colored fraction! Contains the granaticin Methylestero DIESSE Fraction is concentrated to a minimum volume "'For cleaning, in particular a faster-running accompanying component." Is the cleaning and separation operation on dextran gel, preferably Sephadex IH 20, repeated with chloroform in' chloroform '?

The antibiotically uniform chloroform solution of Granaticin ~ ^; Metbyl ester is gently brought to dryness or the granaticin methyl ester is displaced from the eluate concentrate by the addition of five to ten times the amount of petroleum ether _$ . filtered off and dried *

Due to the physico-chemical characterization of the pigment Antibibticums from the strain ZIMET 43 646 of the species Streptomyoes, l§jfce_ritiu £ identity with the structure of the granaticin methyl ester can be found * The produced according to the proposed method granaticin methyl ester can be thus, for the first time as Natural product from fermentation solutions of the Mut ante ZIMET. 43 & h & of the species Stregtom ^ ce ^ late ^ ci. "* Tius and thus produce on an industrial scale.

Fresh fermentation solutions of grana "ticin-methylesterj ZIMET 43 646 _s as well as their iso ™

Granatiein methyl esters possess antibiotic activity. - Preference is given to representatives of gram-positive and certain Gram-negative bacteria inhibiting their growth. Against yeasts and fungi, however, the Granaticin methyl ester displays a distinctly weaker growth-inhibiting effect. "

In Table 1, the "Minimum Inhibitory Concentration" (MIC) of the granaticin methyl ester is compiled against representatives of gram-positive, gram-negative and mycobacteria. It is clear that the gram-positive spore-forming agent, Bacillus subtills. ATCG 6633, which has the highest sensitivity to the microbially produced granaticin methyl ester, depending on the medium * shows a surprisingly strong activity against a representative of Gram-negative bacteria, Proteus morganii (strain SG 464), while for the strain OX 19 of the species Proteus vulgaris the MIC value is more than 10 times higher under comparable conditions «

Table 1

Minimal Henun concentration (MK) of the granaticin methyl ester against Gram-positive Gram-negative bacteria and mycobacteria in a standardized agar diffusion assay

Test organism MJ-IK (mcg / diffusion zone)

Bacillus subtilis ATGC 6633 ¹ ^ 0.2

Bacillus subtilis ATGC 6633 ² ^, 0.1

Micrococcus flavus ATGC 10240. 0.9

Mycobacterium around phlei SG 346 2 _s 5

Escherichia coli C 600 ' y5 ₉ O

Proteus morgariii SG- 464 '0 _s 4

Proteus vulga'ris OX 19. , 5 * 0

Pseudomonas aeruginosa NGTC 10701> 5 ₅ O

Salmonella gallinarum ATCC 9184 ·> 5 _} 0

1) Complex Medium (Peptone Soybean Extract Agar)

2) Synthetic medium (ammonium tartrate agar)

Claims (1)

· * Ι'ίΊΊ invention claim Process for the preparation of an antibiotic, characterized in that the microorganism Streptomyces lateritius (Sveshnikova) Pridham et al. 1958, strain ZIMET 43 646 under aerobic conditions in liquid nutrient media containing a carbon and nitrogen source and mineral salts, "at a temperature between 25 and 37 ⁰ C, preferably 28 ⁰ C during a period of 2 to 6 days, preferably 4 days, is cultivated and the antibiotic granaticin methyl ester formed in this case is extracted both from the mycelium and from the culture filtrate, subsequently enriched with customary methods, purified from unwanted accompanying substances and recovered. lenu ... ^ .. Seitßn tables