DE4134168A1 - New anionophoric antibiotics obtd. from Streptomyces auriantiacus IMET 43917 - effective against viruses, Gram positive bacteria and fungi - Google Patents

Abstract

Antibiotic IMET 4570 of formula (I) and its derivs. are new, where the derivs. include cpds. where (a) the COOH gp. is esterified or converted to an acid halide, amide, hydroxamate or salt, (b) the OH gp. is esterified, halogenated or etherified, or (C) the NMe2 gp. is converted to an acid-addn. or quat. ammonium gp. USE - (I) is active against viruses (no details given), Gram-positive bacteria (e.g. Bacillus subtilis ATCC 6633) and certain fungi (esp. phytopathogenic fungi; no details given). It is an anionophore and may be used to prepare other pharmaceuticals.

Description

The invention relates to a novel anionophore antibiotic of the type one Phase transfer mediator for lipophilic anions, which belongs to the group of oligoether Antibiotics is assigned, as well as on a process for producing the same Hydrolysis of the biotechnologically using a new one Microorganism, Streptomyces aurantiakus, producible Pamamycin complex. The new antibiotic is itself or in the form of its partially synthetically produced Derivatives of interest as a pharmaceutically active substance. The Field of application of the invention lies in the microbiological-pharmaceutical Research and in the pharmaceutical industry.

Macrodiolids form a relatively small group e.g. T. ionophore effective antibiotics which are similar in their biosynthesis, structure and action to the representatives of polyether and macrotetrolidide antibiotics (Kondo, S. et a .., J. Antibiot. 41; 1196-1204 (1988); Kondo, S. et al. Tetrahedron Lett. 28, 5861-5864 (1987); JW Westley, Polyether Antibiotics Vol. 1 and 2, Marcel Dekker 1982 and 1983). The antibiotic Pamamycin 607, first described in 1987 (Kondo, S. et al., Tetrahedron Letters 28, 5861-5864 (1987), is the first natural product to phase change lipophilic anions such as MnO ₄ - from a weakly acidic aqueous solution into an organic phase (eg toluene / butanol; 5: 3: v / v). Antibacterial and antifungal properties have been described for the substance [Agric. Biol. Chem. 51 (1987)] how the new molecular principle of action of facilitating the transport of certain anions in lipophilic phases (e.g. biomembranes) can be used for the development of pharmaceuticals. To clarify this question, it is necessary not only the effect of the already known To investigate natural product pamamycin on living cells and organisms, but also to find further anionophores or to obtain them from pamamycin by chemical modification.

The invention has for its object to provide a new anionophore antibiotic To make it available and to manufacture it effectively. The connection should due to their new structure for extraction antibiotic or otherwise effective preparations as well as starting material for semi-synthesis novel agents with anionophores and other properties of interest be.

It has now surprisingly been found. that that of Streptomyces aurantiakus IMET43917 synthesized antibiotic mixture from Pamamycin 607 and 621 by acid hydrolysis into an antibacterial, antifungal and antiviral effective, new Antibiotic can be split.

The invention thus relates to:

The compound of formula I.

in which R _{1 is} hydrogen, a carboxyl, hydroxyl, ester, amide or hydroxamate group, a halide or a salt with inorganic or organic cations. and R _{2 represents} hydrogen, a hydroxyl, ester or ether group or a halide or the compound of the formula is converted into a salt of the dimethylamino group.

•   - Streptomyces aurantiakus IMET43917.
• - A process for the preparation of the compound of formula 1, thereby characterized in that Streptomyces aurantiakus IMET43 917 or its Mutants and variants are cultivated until the antibiotic mixture is mixed Pamamycin 607 and 621 accumulate in the culture broth.
• The use of the compound of general formula 1 as a medicament, especially as an antibacterial, antiviral and antifungal agent.

The invention is described in detail below, particularly in its preferred embodiments. Furthermore, it is determined by the content of the Claims determined.

The microorganism Streptomyces aurantiakus IMET43 917 was developed in the Core collection of the Central Institute for Microbiology and Experimental Therapy the Academy of Sciences, Jena, according to the number given the terms of the Budapest Treaty on September 11, 1989.

According to the invention the object is achieved in that under aerobic and sterile Culture conditions in a liquid nutrient medium with appropriate Carbon, nitrogen sources and mineral salts the new microorganism Streptomyces aurantiakus lMET43 917 (Rossi-Doria 1891; Gasparini 1892; Krassilnikov; isol. in China) or its variants and mutants.  

All are considered mutants and variants of Streptomyces aurantiakus IMET43 917 those microorganisms of the species that are able to mix the antibiotics To synthesize pamamycin 607 and 621.

Cultivation of the Streptomyces aurantiakus IMET43 917 strain and its Mutants and variants occur under aerobic conditions. In 2.5 to 7.5%, preferably 5% glucose-gelatin solution lyophilically dried mycelium fragments are inoculated into suitable nutrient media and the mycelium formed is in known manner at temperatures between 23 ° C and 37 ° C, in particular between 26 and 30 ° C, over a period of 48 hours to 144 hours an acidity between pH 5.0 and 9.5, especially pH 6.5-7.5, cultivated until the antibiotic mixture Pamamycin 607 and 621 has accumulated in the culture broth. The formation of the antibiotic mixture can be according to the expert known methods, such as. B. by measuring the biological activity or by thin-layer chromatographic separation, etc.

The nutrient medium consists of carbon, nitrogen sources and inorganic salts. For the fermentation of the Streptomyces aurantiakus IMET43 917 strain, it is possible to work in steep-breasted bottles and round-bottom flasks of different volumes, in glass fermenters and V ₂ A steel tanks.

B: Obtaining the antibiotic IMET4570.

The new antibiotic to be manufactured was called IMET4570.

The production of pamamycin as a raw material for the extraction of the Antibiotic IMET4570 is done by separating the biomass at the end of the Fermentation, by extraction with organic solvents such as B. methanol or acetone, concentration of the extract, re-extraction with water-insoluble organic solvents such as B. chloroform and chromatographic Separation of by-products of the fermentation from the residue of the concentrated chloroform extract using conventional chromatographic Adsorbents or carrier materials such. B. silica gels or organophilic  Dextran gels. By sequential application of column chromatography on silica gel and gel chromatography on organophilic dextran gel is one of Impurities free mixture of Pamamycine 607 and 621 (molecular weight 607 and 621; Ratio 1: 1 according to HPLC; Account, S. et al. J. Antibiot. 41; 1196-1204, 1988) received. The chemical identity of Streptomyces aurantiakus IMET43 917 formed pamamycin complex of component 607 and 621 with the already published pamamycin (Kondo, S. et al., J. Antiobiot. 41; 1196-1204, 1988) was beyond any doubt by mass spectrometric and NMR investigations approved.

Various hydrolysis products of the pamamycin complex are obtained as a mixture by heating the pamamycin complex with methanolic potassium hydroxide solution under reflux, neutralizing the reaction product and extracting with chloroform or ethyl acetate. The antibiotic IMET4570 obtained by hydrolysis of pamamycin is separated from hydrolysis by-products by preparative column chromatography on silica gel using CHCl ₃ / MeOH as the eluent. If necessary, a further fine cleaning can be carried out by column chromatography on silica gel RP-8 (methanol) or column chromatography on organophilic dextran gel, e.g. B. LH-20 (methanol) can be made.

After purification, the compound of formula 1 can be modified synthetically will.

On the one hand, the carboxyl group of the substituent R _{1 can be} derivatized by esterifying, halogenating, converting the carboxyl group into an amide or hydroxamate group or synthesizing a salt with inorganic or organic cations.

On the other hand, the hydroxyl function of the substituent R _{2 can be} derivatized by esterifying, halogenating or etherifying the hydroxyl function.

In addition, the compound of formula 1 using acids and quaternary Converted ammonium compounds into a salt of the dimethylamino group will.

The structure of the new antibiotic IMET4570 shown in Formula 1 was determined by mass spectrometric investigations (Tab. 1) and high-field NMR investigations (see Fig. 2: 200 MHz ¹ H NMR spectrum; see Fig. 3: 50 MHz ¹³ C- NMR spectrum).

The structure of the antibiotic is shown by the data shown in Table 1 Sufficiently characterized IMET4570.

The antibiotic IMET4570 is effective against viruses, gram-positive bacteria, e.g. B. Bacillus subtilis ATCC 6633 (minimum inhibitory concentration 3 µg / punch hole in the agar plate Diffusion test) as well as against some fungi, preferably phytopathogenic fungi effective in the smallest amounts. As a characteristic of its anionophores Properties IMET4570 transports methyl orange (as a chromogenic anion) an aqueous solution (pH 5.0) in an organic phase from toluene / butanol (5: 3; v / v).

Embodiments

The invention is explained in more detail by the following exemplary embodiments.

example 1

Inclined agar test tubes from the Streptomyces aurantiakus IMET43 917 strain are used to inoculate a pre-culture medium of the following composition (g / l) in 500 ml steep-breasted bottles (80 ml content, 28 ° C. Rotary shaker, 48 hours cultivation time): Glucose 15; Soy flour 15; CaCO ₃ 1; NACl 5; KH ₂ PO ₄ ; pH 6.5. The second pre-breeding is carried out on the same pre-breeding medium in 51 bottles with 400 ml content (48 hours; 28 ° C, rotary shaker 100 rpm). A medium of the following composition is used to enlarge the scale to 20 l and the main culture in 150 l fermenters:
Soy flour 20 g / l;
Glucose 20 g / l;
HaCl 5 g / l;
CaCO ₃ 3 g / l.

The main culture is carried out in 150 l tank fermenters for 2 to 8 days, preferably 5 days. The mycelium is then separated off and extracted with three times the wet weight of the mycelium with methanol for 5 to 24 hours, preferably 10 hours, with stirring at room temperature. Then the extracted mycelium is separated and the methanolic extract is concentrated in a vacuum rotary evaporator. The aqueous residue is re-extracted with 10 l of chloroform and the dried extract is evaporated to dryness. The residue is purified in portions by chromatography on silica gel 60 (0.063-0.2 mm; 10 g of crude substance / 300 g of silica gel) using ethyl acetate as the first eluent, followed by elution with a mixture of acetone-MeOH (9: 1). Bioactive fractions can be recognized by their inhibitory effect on Bacillus subtilis ATCC 6633 and a chromatographic R ₁ of 0.0-0.4 - on silica gel foils (Merck, acetone as eluent) are concentrated to dryness in a rotary evaporator. 3-4 g of pamamycin as a mixture of the components with molecular weight 607 and 621 in a ratio of approx. 1: 1 are obtained as a colorless waxy mass from 150 l fermentation solution.

Example 2

2 g of pamamycin complex are refluxed with 100 ml of 2N KOH in methanol / water (9: 1; v / v) for 5 hours. The mixture is then acidified to pH 5 and the solution is extracted four times with 150 ml of ethyl acetate each time. The combined extracts are dried over Ha ₂ SO ₄ and evaporated to dryness. The residue (1.8 g) is dissolved in a little chloroform and applied to a silica gel column (silica gel 60, 1 m × 2 cm; CHCl ₃ / MeOH; 9: 1; v / v) and mixed with CHCl ₃ / MeOH (8: 2; v / v) eluted. First, nitrogen-free cleavage products of pamanycin as well as products of partial hydrolysis (opening one of the two ester bonds) elute. The antibiotic lMET4570 appears on further elution with a mixture of CHCl ₃ / MeOH (1: 1; v / v) and can be used in thin-layer chromatography of eluates due to its blue coloration with vanillin / H ₂ SO ₄ conc. can be detected (R ₁ 0.2 - 0.4; silica gel film Merck, CHCl ₃ / MeOH; 8: 2; v / v). Yield: 0.9 g.

An additional purification can be done by chromatography on reverse phase silica gel (Merck RP-8 or RP-18) using methanol or methanol-water Mixtures are made as eluents.

Table 1

Claims (10)

1. Compound of formula 1 in which
R _{1 is} hydrogen, a carboxyl, hydroxyl, ester, amide or hydroxamate group, a halide or a salt with inorganic or organic cations, and R _{2 is} hydrogen, a hydroxyl, ester or ether group or a halide or the Compound of formula 1 is converted into a salt of the dimethylamino group. 2. Streptomyces aurantiakus IMET43 917. 3. A process for the preparation of the compound of formula 1, characterized characterized in that Streptomyces aurantiakus IMET43 917 or its Mutants and variants are cultivated until the antibiotic mixture is mixed Pamamycin 607 and 621 accumulate in the culture broth. 4. The method according to claim 3 or 4, characterized in that Streptomyces aurantiakus IMET43 917 at a temperature of 20 ° C to 37 ° C is cultivated.   5. The method according to claim 3 or 4, characterized in that Streptomyces aurantiakus lMET43 917 for a period of three to six Days cultivated. 6. The method according to claim 3, 4 or 5, characterized in that Streptomyces aurantiakus IMET43 917 through aerobic and sterile fermentation is obtained in liquid nutrient media that contain a carbon, Contain nitrogen source and mineral salts. 7. The method according to one of claims 3 to 6, characterized in that by alkaline hydrolysis of the compound of Pamamycine 607 and 621 and chromatography on silica gel the compound of formula 1 as a uniform Structure is gained. 8. Use of the compound according to general formula 1 for Use as a medicine. 9. Use of the compound of formula 1 for the preparation of a antibacterial, antiviral or antifungal drug. 10. Use of the compound of general formula 1 according to claim 1 as Synthesis building block suitable for the partial synthetic production of Pharmaceuticals.