DD282711A5 - METHOD FOR PRODUCING AN ANTIBIOTICS - Google Patents

Abstract

The invention relates to a process for the preparation of a new antibiotic which is of potential use as an antiinfective, coccidiostat and antiviral for the therapy of infectious diseases. The object of the invention is to produce the novel polyene antibiotic, which has been designated diffusomycin and has a broad spectral antibacterial effect, in an effective process as a pure product. The object associated with this object is achieved by growing a microorganism of the species Streptomyces albus, preferably strain Streptomyces albus JA 3453-D, on a complex medium with carbon and nitrogen sources and inorganic salts under submerged cultivation conditions and removing the antibiotic diffusomycin from the mycelium and the culture filtrate is extracted by means of organic solvents and then purified by chromatographic methods to homogeneity. {Polyene antibiotic; Diffusomycin; Production by fermentation; Streptomyces albus strain JA 3453, broad spectrum antibacterial activity; anti-infective; coccidiostat; Virostatikum; in vitro activity; Drug of potential use}

Description

For this 2 pages drawings

Field of application of the invention

The invention relates to a process for the preparation of a novel antibiotic which is of potential use as an anti-infective agent, coccidostat and antiviral for the treatment of infectious diseases in humans, livestock and plants. The field of application of the invention is thus in the pharmaceutical research and industry.

Characteristic of the known state of the art

Despite the large number of antibiotics currently isolated from microorganisms, animals and plants, the search for new antibiotic agents continues unabated, with new emphasis on antiparasitic (eg coccidiostats) and antiviral agents. I have the following disadvantages or the following options for the ongoing search for new antibiotics and for improved methods for their production:

- Insufficient efficacy of existing drugs against old and new problem germs, where the development of resistance under antibiotic selection pressure can lead to the complete inactivity of common preparations;

- high toxicity and unwanted side effects of bek.wnter preparations;

- Find new preparations with improved use values including development of methods for their economical production and cheaper

- Possibility of a partial and total synthetic modification of new natural active substances or provision of such substances as "lead structures" and associated possibility for each appropriate adaptation of structure-activity relationships.

Finding new antibiotics and characterizing their mechanism of action is of great importance for the detection of the molecular causes of diseases (eg viral diseases) and the cellular sites of action of pathogenic cells (microbes, parasites, viruses).

Polyenes form a group of unsaturated and partially nitrogen-containing macrocyclic lactones and lactams, which are known above all for their antifungal activity (BERDY, J. et al., CRC hundbock of antibiotic compounds, Vol. II, pp. 165-313). Individual representatives of this class of drugs, such as Amphotericin Bund Nystatin, have gained access to the treatment of dermatomycoses as well as phytofungicides in agriculture. No information is yet available on the use of polyenes as cocaudostats and antivirals (BERDY, J. et al., Supra, p.169-175 (198O), FINLAY and RADICS, cn, J. Chem. 58, 579-590 [1980]).

Object of the invention

The invention serves to prepare a new antibiotic to broaden the range of known antibacterial, coccidiostatic and virostatic antibiotics by a representative of a novel, original structure which is of potential use as a therapeutic agent for the control of diseases.

-2- 282 Explanation of the nature of the invention

The invention has for its object to describe a technologically simple, microbiological process by means of which a new antibiotic can be obtained using a bacterial microorganism using a cheap culture medium and purified to homogeneity.

According to the invention the object is achieved in that a microorganism of the species Streptomyces albus, preferably the strain Streptomyces albus JA 3453-D, under aerobic and sterile culture conditions in a liquid medium with carbon and nitrogen sources such as glucose, starch or soybean meal and with in the fermentation industry usual mineral additives to the medium is cultivated (submersed fermentation).

The microorganism JA 3453-D is in the GDR depository for microorganisms, Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences, DDR - 6900 Jena, Beutenbergerstr. 14, under registration number ZIMET 43859.

Cultivation and maintenance of the microorganism of the species Str. Albus used, preferably of the strain ZIMET 43859, is carried out as follows:

In glucose / gelatin (5%) lyophilized mycelial fragments are inoculated on agar and then in liquid, previously sterilized nutrient media of the above-mentioned nature, and the resulting mycelium at a temperature between 20 ° C and 38 ⁰ C, preferably 26 ⁰ C, on a period of 2 days to 6 days, preferably 4 days, cultured at a pH which is between pH 5.5 and pH 8.3 at the beginning of the fermentation. The fermentation of the Str. Albus strain used in each case can be carried out in steep-breasted bottles and round-bottomed flasks of various contents, in glass fermentors or V2A-Taη <s.

The detection of the new antibiotic in the culture filtrate is carried out after extraction of defined volumes with CHCl ₃ , concentration of the extract to dryness and resolvation in a defined amount of methanol by means of the agar plate diffusion test using Bacillus subtilis ATCC 6633 as test germ. In the process, sharply defined zones are obtained at the edges, within which the antibiotic partially inhibits bacterial growth. The inhibitory effect of the sharply demarcated antibiotic leads to their formation of light scattering in relation to the environment of significantly altered "diffuse" zones of inhibition, in which a greatly reduced growth of test positive or gram negative test microorganisms is likewise recognized.For the name of the new antibiotic from Streptomyces albus, preferably from the strain Str. albus JA 3453, therefore, the name diffusomycin was chosen.

The preparation of pure antibiotic diffusomycin is carried out by extracting the culture filtrate with chloroform and the mycelium with acetone. From the acetone solution diluted with water antibiotic diffusomycin can be obtained by extraction with CHCl ₃ . The extraction from the combined CHCl ₃ extraction is carried out in a manner known per se for lipophilic substances by chromatography on silica gel (column chromatography, thin-layer chromatography) using organic solvents as eluent. Further purification is carried out by column chromatography on organophilic dextrins using acetone as the eluent. In this way, high-purity antibiotic diffusomycin is obtained, which is characterized by the following physicochemical characteristics: Properties: pale yellowish, waxy mass, melting point 54 ⁰ C to 56 ⁰ C (uncorrected) Molecular weight: (FAB-MS) m / z 655: C ₃₆ H ₄₉ N ₃ Os Characteristic fragment peaks were obtained by EI-MS: gef. m / z 593.3504 (, 3465 calcd for C ₃₄ H ₄₇ N ₃ O ₆ ; M ⁺ -COz-H ₂ O). m / z 575.3349 (, 3359 calcd for C ₃₄ H ₄₆ N ₃ O ₆ ; M ⁺ -CO ₂ H ₂ O). ₍₂ -H ₂ O-CH ₃ OH M ⁺ -CH, 3302 for C ₃₃ H ₄₃ N ₃ O _6). Found m / z 561.3207. m / z 543.3183; Found (3097 calcd for C ₃₃ H ₄₁ N ₃ O ₄ M ⁺ -CO ^ H ₂ O-CH ₃ OH.). m / z 453.2762 (, 2627 calcd. for C ₂₆ H ₃₆ N ₃ O ₄ ) Stability: Stable in acetone or dimethyl sulfoside

 Chemical analysis: nitrogen content gef. 6.99%; 6.75% Rf values (Silufol silica gel films): 0.4 (benzene / acetone; 3: 5; v / v)

0.35 (CHCl ₃ / MeOH; 9: 1, v / v).

 IR spectrum (cm - ') IKBr): 500; 645; 700; 750; 810; 830; 890; 900; 950; 960; 992; 1040; 1080; 1100; 1120; 1180; 1290; 1330;

1380; 1385; 1395; 1430; 1450; 1510; 1535; 1645; 1695; 1826; 2930; 2960; 3350; UV spectrum (EtOH) (nm): 335,268,277,290 (triene chromophore) Optical rotation: [a] _D = 36.0 ° (0.5 dm, 10 mg, 1 ml) Figure 1 shows the 400 Hz 'H proton spectrum

Fig.2 shows the 100 MHz C ^l3-NMR spectrum of the antibiotic Diffusomycin Antimicrobial activity upon application of 50 \ ig Diffusomycin in the agar diffusion test:

Inhibition zone diameters of 16 nm to 45 mm diameter (sharply delimited partial inhibition) were determined with the following test germs: Bacillus aubtilis ATCC 6633; Staphylococcus aureus SG 511; Escherichia soli mutabile SG 458; Serratia marcescens SG 621; Proteus vulgaris SG 2 (OX 19); Escherichia coli C 600; Klebsiella aerogenes SG 117; Pseudomonas aquinosa SG 137; Pseudomonas aeruginosa K 799.

The in vitro antiviral activities of the antibiotic diffusomycin in single-stage replication cycle experiments with influenza A / strain WSN; Herpes simplex hominis virus type 1 strain Kupka and Vaciniaviru3 strain Lister in chicken embryonic and RH cells is shown in Table 1.

Table 2 shows the results of plaque reduction experiments with antibiotic diffusomycin in influenza Α virus, herpes simplex hominis virus type 1 and vaccinia virus in chicken embryonic cells (TONEW, E. and TONEW, M., ZbI. Bakt. I, Orig.

211, 437-444 (1969); TONEW, M. u. GLÜCK, B., J. Basic Microbiol. 26, 173-189 (1986); REED, J. u. Münch, H., Am. J. Hyg. 27, 493-497 (1938).

Table 3 shows the results of the coccidiostatic efficacy test on Brutei according to WIESNER, J. (Diss. FSU Jena, 1973).

The advantages of the invention are seen as follows:

- By the inventive method an innovative in its chemical structure antibiotic of the type of polyene lactams is produced by fermentation.

- As shown by the results of the test for antibacterial efficacy, it is an antibiotic of a new type of effect, as a broad spectral partial growth inhibition occurs, which is an essential characteristic of diffusomycin compared to a variety of known antibiotics.

- According to the results of the in vitro antiviral and coccidiostatic studies, the antibiotic has a good efficacy against test organisms and at the same time a low toxicity to living cells.

- Under defined conditions, eg. B. in acetonic solution, or dissolved in dimethyl sulfoxide, the antibiotic diffusomycin is long-term chemically stable with the exclusion of atmospheric oxygen.

- The antibiotic diffusomycin is formed as a single component. A presence of bioactive by-products of similar structure (congeners) has not been found. The separation of a coproduced representative of another type of structure (pentalenolactone) succeeds easily when using chromatographic methods.

Aüsführungsbelspiel

Mycelium fragments of the strain Str., Bus ZIMET 43859, which have been lyophilically dried, are used to inoculate an agar medium of the following composition (g / l) for streptomycetes:

Baker's yeast 15; Corn starch 10; NaCl 5; Meat peptone 1; Agar 20;

Inoculum cultures are obtained by using mycelium pieces of agar culture or freeze-dried mycelia as stock culture in a medium of the following composition (g / l):

Soy flour 40; Glucoso 40; NaCl 2.5; NaCO ₃ 5; KH ₂ PO ₄ 0.25; pH6,3.

The second preculture takes place on the same medium. After 48 hours incubation of the preculture in 500 ml Stoilbrustflaschen

(80ml content, 25 ° C, rotary table, 180 rpm), the stepwise scale magnification by transfer of culture samples in 5-L-Steil breast bottles and a further 24 hours incubation at 27 ⁰ C is performed.

For cultivation in 500 ml steep breast bottles or in fermentors, a medium of the following composition is used

Soy flour 40; Glucose 20; Potato starch 30; NH ₄ NO ₃ 2; CaCO ₃ 2; pH 6.2 (before sterilization).

The ventilation is done with sterile air (1: 1). The fermentation time is 72 hours to 120 hours and the stirring speed in fermentors 280 rpm. A foaming can be controlled in a conventional manner by adding small amounts of Polypropyienglykol.

After completion of the fermentation, the My: el is separated by separators. From 2Ol fermentation solution about 0.7 kg of wet mycelial residue are obtained, which is suspended in 51 acetone overnight. After extracting extracted mycelium, the acetone extract is diluted with 51% water and extracted twice with 51CHCl ₃ each together with the culture filtrate. After drying the CHCl ₃ extract with Na ₂ SO ₄ , the solvent is removed in vacuo and the residue (oil, 50 g to 100 g) in 20 g portions on a silica gel column (300 g silica gel) using benzene / acetone (3 : 5, v / v) as the eluent chromatographed. Bioactive zones, recognizable either by the marked partial inhibition of bacterial growth using Bacillus subtllis ATCC 6633 in the usual agar plate diffusion test or by characteristic staining with 3% vanillin in conc. H ₂ SO ₄ when spotted on Chromatogrammfolien (silufole, silica gel) are combined. Yield: 4g to 5g (oil).

For fine cleaning, the crude product is applied to chromatogram plates silica gel H (1 mm layer thickness, air-dry) (20 × 20 cm, O, 5 g / plate), which are developed with benzene / acetone (3: 5, v / v). The substance-containing zones determined by the abovementioned color reaction with 3% vanillin / H ₂ SO ₄ are eluted with acetone and concentrated.

Subsequently, the resulting product (about 2 g) is applied to silufol films (silica gel) developed with CHCl3 / M00H (9: 1, v / v). The substance zone containing the antibiotic diffusomycin is eluted with acetone. Yield: 0.5 g, purity about 85%.

Ultrapure antibiotic diffusomycin is finally obtained by applying 0.1 g of diffusomycin to silica gel plates and developing twice in the benzene / acetone (5: 3, v / v) system. The substance is eluted from the corresponding zones with acetone. The residue of the product obtained after concentration is dissolved in a little acetone and subjected to column chromatography (column 15 cm × 0.6 cm) of organophilic dextran gel using acetone as the eluent. Diffusomycin appears before the impurities in the eluate and is obtained after concentration of the corresponding bioactive fractions in vacuo as a pale yellowish, waxy solid. The solution of the antibiotic Diffusomycin in acetone at 4 ⁰ C in the absence of atmospheric oxygen for more than 6 months at room temperature for at least 3 weeks, lasting.

Table 1

Results of the antiviral effect of diffusomycin in single stage replicate cycle trials with influenza A / strain WSN, herpes simplex hominis virus type 1 strain Kupka and vaccinia virus strain Lister in chicken embryonic and RH cells.

Virus strain

cell

influenza

HSVTypi Kupka

Vaccinia Lister

HEZ

HEZ

HEZ

Influenza A / WSN

HSVTypi Kupka

Vaccinia Lister

RH

RH

RH

Conc. Titanium reduction in 1OgI ₀ TCID ₆ Q % in in 0.2 ml for virus control Hem mcg / ml mung 125 6.33 > 99.9 62.5 6.0 > 99.9 31.25 4.17 > 99.9 15.62 3.0 > 99.9 125 6.17 > 99.9 62.5 6.0 > 99.9 31.25 3.67 > 99.9 15.62 2.5 99.7 125 4.5 > 99.9 62.5 2.5 99.7 31.25 2.0 99.0 15.62 1.65 97.2 250 6.5 > 99.9 125 6.33 > 99.9 62.5 3.23 > 99.9 31.25 3.0 99.9 250 6.17 > 99.9 125 5.67 > 99.9 62.5 3.75 > 99.9 31.25 2.5 99.7 250 4.0 > 99.9 125 1.6 97.5 62.5 1.0 90 31.25 0.33 n.s.

Mean values from 3 experiments; n. s. = not significant HEZ = chicken embryonic cells; RH = permanent human kidney cells

Table 2

Results of the PIaquer reduction experiments with diffusomycin and influenza Α virus, herpes simplex hominis Virtis type 1 and vaccinia virus in chicken embryonic cells

Virus strain Concentration Plaqueria calculated to

in mcg / ml untreated virus control in%

influenza 125 97 A / WSN 62.5 83 31.25 67 15.62 51 Herpes Kupka 125 91 simplex 62.5 84 hominis 31.25 62 Type 1 15.62 49 vaccinia 125 87 Lister 62.5 71 31.25 62 15.62 43

Titerbestimmungen from 3 attempts

Table 3

Results of testing diffusomycin for coccidiostatic activity against Eimaria tenalla strain WC AM-P 20 in intra-lantanto infected chick embryos

concentration groups Latalität lethality Testergebnio in the test Strength in the experi the Kon (Pg / embryo) ment control group 1. Acute toxicity 100 10 100% - 33 10 70% - LD ₆₀ = 28 [ig 11 10 10% - 2. Coccidiostasis 10 17 6% 100% +++ 3 18 0% 100% +++ 1 22 0% 100% +++

+++ = ρ <0,001 (four-field test after FISHER)

Legends to the figures Fig.1: 400MHz '' H NMR spectrum of the antibiotic diffusomycin (Varian XLAA-400) Fig.2: 100MHz NMR spectrum of the antibiotic diffusomycin (Varian XLAA-400)

Claims (7)

1. A process for the preparation of an antibiotic, referred to as Dif .'usomycin, on mikrob ellem way under aerobic conditions, characterized in that a microorganism of the species Streptomyces albus in liquid nutrient media containing a carbon and a nitrogen source and mineral salts in a Temperature of 2O ⁰ C to 38 ⁰ C over a period of 2 days to 6 days cultivated and the antibiotic from the mycelium and the culture filtrate is isolated. 2. The method according to claim 1, characterized in that the microorganism Streptomyces albus JA 3453-D is used. 3. The method according to claim 1 and 2, characterized in that is used as the carbon and / or nitrogen source glucose, starch or soybean meal. 4. The method according to claim 1 to 3, characterized in that the fermentation is carried out at 26 ⁰ C over a period of 4 days. 5. The method according to claim 1 to 4, characterized in that the microorganism used bsi a pH value, which is at the beginning of the fermentation process between pH 5.5 and pH 8.3, is cultured. 6. The method according to claim 1 to 5, characterized in that the antibiotic is recovered from the mycelium and the culture filtrate by means of organic solvents and purified by chromatographic methods to homogeneity. 7. The method according to claim 1 to 6, characterized in that the antibiotic dikffusomycin has the gross formula C ₃₅ H ₄₉ N ₃ O ₉ and is characterized by ¹³ C and ¹ H-NMR spectral data as Polyenlactam structure.