DE2946523A1 - Antibacterial and antiviral antibiotic viriplanin - obtained by culturing Ampullariella regularis strain - Google Patents

Abstract

The new antibiotic viriplanin (I) has the following properties: (a) consists of C, H, O and N; (b) soluble in 2N NaOH, 2N H2SO4, DMF and DMSO, and sparingly soluble in water, ether and hydrocarbons; (c) is ppted. from neutral solvents as a red powder with a m.pt. of 210-215 deg.C (decomp.); (d) has a Rf value of 0.23 on neutral silica gel plates in CHCl3/MeOH (80:20); (e) molecular wt. ca. 2000; (f) gives 2-desoxy-L-fucose on acid hydrolysis; (g) gives mesaconic acid when hydrolysed in a strong acid and then a basic medium; and (h) gives 7-O-methylnogalarol and nogalarene when treated with 2.5N HCl in MeOH. (I) is obtained by culturing Ampullariella regularis SE47 (ATCC 31417) under aerobic conditions, pref. at 20-40 deg.C and pH 6-8, and isolating (I) from the culture broth or the mycelium. (I) has antibacterial activity against Gram-positive bacteria and antiviral activity against herpes viruses (e.g. herpes simplex).

Description

Antiviral antibiotic, process for its manufacture

and its therapeutic use. The invention relates to a new one Anthracycline group antibiotic, process for its preparation and its Use as an antibacterial and antiviral agent.

The antibiotic according to the invention, hereinafter referred to as viriplanin, inhibits the growth of gram-positive bacteria such as Bacillus subtilis ATCC 6633 iuid is effective against herpes viruses in vitro and in vivo.

Viriplanin and its salts can therefore be used alone or in combination with other antibiotic or antiviral compounds for prevention or combating bacterial or viral infections.

Viriplanin has the following chemical and physical properties: 1. The substance turns red when it is precipitated from neutral solutions amorphous or microcrystalline powder, which is in the range of about 210-215 ° C below Decomposition melts.

2. The elemental analysis has the empirical formula (C20-22 29-31 N ° 9-11) n in unison.

It must be pointed out that in the case of higher molecular weight natural substances the error range of the elemental analysis (approx. 0.8% for the individual determination) none precise calculation of the empirical formula allows (R.B. Woodward, Angew. Chem. 69, pp. 50-51 (1957).

3. Viriplanin dissolves in 2N aqueous NaOH with violet, in 2N aqueous Sulfuric acid with an orange-yellow color. In contrast, it is sparingly soluble in water.

It is readily soluble in dimethylformamide and dimethyl sulfoxide, but less sparingly soluble in lower alcohols such as methanol and ethanol and in chloroform it is in ethers and hydrocarbons.

4. The IR absorption spectrum of the viriplanin shows, when it is processed into KBr pellets, absorption bands at the following wavelengths (expressed in reciprocal centimeters): Band frequency Int. (Wave numbers in cm 3420 5 2980 s 2970 s 1715 s 1655 m 1625 s 1570 5 1545 (shoulder) s 1445 s 1410 s 1385 s 1305 s 1240. s 1205 5 1175 s 1115 s 1090 s 1050 s 1005 s 975 5 875 m 855 m 835 m 805 m The IR band intensities are denoted by s, m and w. An s-band has at least 2/3 the intensity of the strongest band in the spectrum, an m-band an intensity in the range between 1/3 and 2/3 of the strongest band and a w-band less than 1/3 the intensity of the strongest Gang up. These estimates are made on the basis of percent permeability.

5. The UV absorption spectrum in methanol shows the following absorption maximaa (abscissa: wavelength in nrl; ordinate: extinction) (c = 0.00195 in methanol) When sulfuric acid was added to the solution in methanol, the following absorption maxima were obtained When aqueous sodium hydroxide solution is added to the methanol solution, the following absorption maxima are obtained: 6. W values: Table 1 shows the values of the compound on neutral silica gel 60 plates (Merck, Darmstadt, Federal Republic of Germany) in comparison with fluorescein and nogalamycin.

Table 1 RF value of | compare | Fluorescent Nogalaycin cein Viriplanin mobile solvent Chloroform + methanol 95 + 5 0 0.06 0.09 90 + 10 0 0.09 | 0.11 80 + 20 0.23 0.31 0.47 50 + 50 0.68 0.36 0.68 Chloroform + methanol Ammonia *) 40 + 6 + 1 0.09 0.66 0 80 + 20 + 9 (Glacial acetic acid) 0.49 0.29 0.68 *) Aqueous solution containing 25% by weight of ammonia RF value of | compare | Fluorescent Eluent Viriplanin Nogalamycin cein 90 + 10 + 1 (Formic acid) 0.06 0.36 0.09 Methanol 0.85 0.17 0.74 Butanol + glacial acetic acid + water 60 + 20 + 20 0.55 0.22 0.88 7 The determination of the molecular weight of the viriplanin yields a molecular weight of approx. 2,000 using the Sephadex superfine method (mobile phase phosphate buffer pH 7 or 6 m urea solution).

In aqueous solutions, micelle formation results in much higher Faked molecular weights.

This is calculated from the nitrogen content of the elemental analysis Equivalent weight to approx. 450-550.

Viriplanin is an orange-red amorphous or microcrystalline powder, which has the same absorption maxima in the UV-VIS spectrum as nogalamycin. It shows but a clearly low extinction and has in all of the systems examined by us deviating RF values in the thin-layer chromatogram.

The acid hydrolysis of viriplanin provides i.a.

2-deoxy-L-fucose. Viriplanin becomes a strong acidic and first then subjected to a basic hydrolysis, can be in the hydrolysis mixture detect mesaconic acid and several red components.

After viriplanin had been broken down with 2.5N hydrochloric acid in methanol, the compounds 7-0-methylnogalarol and nogalarene (see formula below) known from the literature were isolated from the methanolysate. These compounds were also released from the antibiotic nogalamycin when they were broken down in the same way.

7-0-methylnogalorol: R = nogalarene: R = In addition to a strong antibacterial effect, viriplanin shows a pronounced effect against herpes simplex viruses.

Those previously described as effective against herpes simplex viruses Substances are either nucleosides (e.g. 5-iodo-2'-deoxyuridine) that act as antimetabolites frequently cause unwanted side effects, or they are much weaker effective compounds such as N-1-adamantyl-N-t2- (dimethylamino) ethoxy7-acetamide hydrochloride.

The following are experiments to investigate and evaluate the virustatic Effect of viriplanin described.

1. Cell culture tests The virustatic effect was determined in cell cultures of the substances in comparative infection titer determinations in plaque reduction tests (Schwöbel, W .: Archiv ges. Virusforsch. 14, 99-112, 1963) and titer reduction test (Tonew, M., Tonew, E .: Archiv ges. Virusforsoh. 33, 319-329, 1971).

The tests were evaluated in comparison to virus-free and substance-free cell controls and substance-free virus controls.

a) Plaque Reduction Test Mouse fibroblast cells grown in plastic dishes are inoculated with a suitable virus dilution and after an incubation period of 45 min. at 37 ° C. in a CO2 incubator with a layer of cell culture medium and methyl cellulose, which contains the test substance in the appropriate concentrations contains. After a further incubation of 48 hours at 37 ° C., the lines become Stained with neutral red and the virus plaques counted.

b) Titer Reduction Test Monkey kidney cells grown in plastic dishes are inoculated with an appropriate virus concentration and after an incubation period of 45 min. at 37 0C in the CO 2 incubator with cell culture medium that the Contains test substance in appropriate concentrations. After another incubation from 30 hours

at 370C the cultures are frozen at-250C and thawed again. The infectivity titer is then determined in the treated and untreated cultures in monkey kidney cells grown in microtiter plates by the method determined by Takatsy (Acta med. Hung. 1, 35, 1954).

The results with viriplanin are compiled in the tables below.

Plaque reduction test ug / ml reduction in plaque I pay 10 100 1 100 0.1 50 0.0 0 Titer reduction test Dose treated untreated titer reduction / µg / ml 10 3.0a) 6.0 3.0 1 4.0 6.0 2.0 a) Cell culture infectious units (ZXID50) log (10) per 0.2 ml 2. Animal experiments a) Mouse 4-6 week old mice were infected intracerebrally with herpes simplex virus and 3 hours later also treated intracerebrally with suitable concentrations of the test substance. In further experiments, mice whose immune defense was suppressed by cytostatics were infected intraperitoneally with herpes simplex virus and then also treated intraperitoneally with test substance. Such treatment increased the mean survival time and reduced the mortality of the infected mice.

b) Rabbit cornea tests were carried out in rabbits based on the Method of B.R. Jones and K. Al-Hussaini (Trans.

Ophthal. Soc. 83, 613-631, 1963). The intensity of the Infection was related to number of herpes lesions conjunctivitis, keratitis and anterior chamber exudation assessed. the with treated with a 1% suspension of the test substance (in DSO and buffer solution) Rabbit eyes showed either no lesions or significantly less pronounced lesions than the control eyes treated only with buffer solution and DSO.

The antibiotic represents one after the experiments described above represents and shows significant progress in the treatment of viral infections it also has a strong antibacterial effect.

Viriplanin is formed by the strain Ampullariella regularis SE-47, which was isolated from a soil sample in Kenya by baiting with Pinus pollen (Jacaranda-Coffee Research Station). The strain was made at the American Type Culture Collection, 12301 Parklawn Drive, Rockville Maryland 20852, USA, under number ATCC 31417 deposited on July 24, 1978.

It has the following characteristics: Mycelium: approx. 0.3 - 1.4 / u wide, branched, partly meandering; occasionally with nodular, thickened sections; no real one Aerial mycelium present; on some culture media, especially with abundant sporangia formation, Colonies with a short, hoop-like coating.

Sporangia: mostly bottle-shaped-cylindrical with truncated to slightly arched end, usually longer than pulpy, often narrow and lana, some also more compact and bell-shaped; about 4-11 x 5 -10 µ; on suitable culture media (e.g. oatmeal agar, Potato and carrot agar) in a dense layer on the colony surface.

Spores: rod-shaped, about 0.6 - 1 x 2 - 3.5 µ, flagellated; in the sporangium arranged in longitudinal, straight parallel rows; swarm in water about 45 minutes from the sporangia.

Melanin formation on tyrosine agar + milk agar + gelatin + PEH agar + Nitra treduct ion + gelatin liquefaction + milk peptonization + pyrosine dissolution Growth at 150 + 200 ° +++ 27 ° +++ 32 ° +++ 37 ° ++ 420 salt tolerance 2 % ++ 3% + 4% starch hydrolysis + L-arabinose ++ D-fructose ++ D-glucose ++ i-inositol Lactose ++ D-mannitol raffinose L-rhamnose ++ sucrose ++ D-xylose ++ growth on different culture media: oat flakes W good yeast agar SM brown orange (HaH) "LM" ++, ripe-like, thin coating SP brown-orange Spg ++ yeast-starch- W good agar SM brown-red (E) "LM" ++, mature-like, thin coating SP red-brown Czapek agar W moderate (Cz) SM pale brown-red "LM" ++, ripe-like, thin coating SP brownish pink casamino peptone- W good Czapek agar SM red-brown to dark orange-brown (CPC) "LM" ++, mature, thinner Coating SP strong red-brown to orange-brown Spg + N03- agar W moderate SM brown LM " ++, mature, thin coating SP dark brown nitrite from nitrate ++ tyrosine agar W moderate (Ty) SM brown LM -SP dark brown crystal dissolution -Skim milk agar W good (Ca) SM orange brown LM -SP brown milk peptonized Potato glucose W good agar SM strong red-brown (KG) LM -SP orange potato-carrot- W good mineral salt agar SM brown orange (KMS) "LM" +++, ripe-like coating SP brown orange Spg +++ potato oblique W well area SM brown-orange, bulging colony LM -SP brown-orange glucose-asparagine- W good Agar SM rust-red (DA) "LM" ++, mature, thin coating SP brownish orange Malt extract yeast- W moderate to good agar (MHG) SM strong brown-red medium no. 2 "LM" ++, mature, thin coating SP strong dark brown-red starch salt agar W good (StS) SM strong brownish orange medium No. 4 "LM" ++, like a ripening, thin coating SP orange glycerine-asparagine- W good agar (C; A) SM orange medium No. 5 LM SP yellow-orange Peptone iron yeast W moderate extract agar SM dark brown (PEH) SP brown black medium No. 6 W moderate glycerine tyrosine agar brown Medium No. / SM brown Of the Strain SE 47 is due to its mostly bottle-shaped sporangia and rod-shaped Planospores characterized as representatives of the species Ampullariella regularis. He shows good agreement with this species in the other characteristics as well, but falls due to the production of an orange to brownish-red dye that is found in of the co-examined reference culture (C.B.S. 193.64, Couch) is not available and is also not mentioned in the species description (COUCH, 1963).

Abbreviations: W = growth SP = substrate-discoloring SM = substrate mycelium Dye LM = aerial mycelium Spg = sporangia When testing for usability of various Carbon sources mean ++ growth as well as on mineral salt agar with glucose or better - growth like on pure mineral salt agar or less.

Culture media: Potato Carrot Mineral Salt Agar (KMS): grated Potatoes and carrots each 40 g, boiled in 500 ml H20 for 30 minutes, strained, made up to 500 ml; Mineral salt solution 500 ml Agar 15 g.

Mineral salt solution and media No. 2, 4, 5, 6 and 7 according to SHIRLING and GCTTLIEB, Int. J. Syst. Bact. 16: 313-340 (1966).

Glycose asparagine agar (DA) according to WAKSMAN, The Actinomycetes II, Baltimore 1961, p. 328.

For the composition of the other culture media, see SCHÄFER, D .; Contributions on the classification and taxonomy of the Actinoplanaceae ", Dissertation, Marburg 1973 (especially p. 42 f.).

The cultures were kept at room temperature and diffuse daylight and logged after four to six weeks of growth.

The fermentation process for the production of viriplanin can be done with Using solid, semi-solid or liquid culture media. Preferred aqueous liquid nutrient media are used.

The inoculation of the culture media is carried out according to generally accepted methods, e.g. via inclined tubes or flask cultures.

The culture takes place under aerobic conditions and can according to the general common methods such as using shake cultures e.g. in shake flasks, from air-moving cultures or from submerged cultures. Preferred the cultivation takes place in aerobic submerged process in aerated fermenters, e.g. in common submerged fermentation tanks.

It is possible to carry out the culture continuously or batchwise. The process is preferably carried out batchwise.

The culture can be carried out in all nutrient media known to be be used for the cultivation of microorganisms of the order of the Actinomycetales.

The nutrient medium must have one or more assimilable carbon sources and sources of nitrogen as well as mineral salts, these products being in the form of defined individual components, but also in the form of complex mixtures, which in particular represent biological products of various origins are available can.

All customary carbon sources can be used as the carbon source. For example, starch, molasses, whey powder, dextrin, sugar such as sucrose, Called maltose, glucose and glycerin. All the usual sources of nitrogen are used organic and inorganic nitrogen sources in question. For example, soybean meal, Cottonseed flour, lentil flour, pea flour, soluble and insoluble vegetable proteins, Corn steeple, yeast extract, peptones and meat extract as well as ammonium salts and Nitrates, e.g. NH4Cl, (NH4) 2S04, NaN03 and KN03 are listed. The mineral salts, which should be contained in the nutrient medium, provide e.g.

the following ions: Mg ++, Na +, K +, Ca ++, NH4 +, Cl-, SO42-, PO43- and NO3- as well Ions of the usual trace elements such as Cu, Fe, Mn, Mo, Zn, Co, Ni. If the carbon or nitrogen sources or the water used does not have enough these salts, or contains trace elements, it is advisable to prepare the nutrient medium accordingly complete. The composition of the nutrient media can be varied within wide limits. Art and composition of the nutrient media will generally depend on which Components are each available particularly cheaply.

The pH of the growing cultures should preferably be between about 6 and about 8, especially 6.5 and 7.5, are held. Too much pH drop in the acidic range by adding an organic or inorganic base, preferably avoided by CaCO3. As usual in fermentation technology, an automatic pH regulation can also be carried out with the sterile organic or inorganic acid, e.g. H2S04, or sterile lye, e.g. NaOH in Injected into the culture solution at intervals.

It is useful to ensure that the microorganisms are sufficient be brought into contact with oxygen as well as the nutrients. This can be done after the generally customary methods such as shaking and stirring.

The growth temperature can be between about 20 and about 40 ° C., preferably between 25 and 35 ° C, particularly preferably it is around 280 ° C. The duration cultivation can be varied widely, e.g. the composition of the nutrient medium and the curing temperature play a role. The respective optimal conditions can easily be determined by anyone skilled in the microbiological field.

It has been found that the amount of is in the culture broth enriching antibiotic generally their maximum about 2 to 8, preferably Reached 3 to 6 days after the start of cultivation.

As in general with microbiological processes, foreign infections of the culture media are avoided. The usual precautions are taken for this purpose, such as sterilization of the culture media, the culture vessels and those necessary for ventilation Air. Both steam and dry sterilization can be used to sterilize the device be used.

If undesirable amounts of foam are produced during cultivation, the usual chemical foam suppressants, e.g. liquid fats and oils, Oil-water emulsions; Paraffins, higher alcohols such as octodecanol, silicone oils, polyoxyethylene or polyoxypropylene compounds are added. Foam can also be done with the help of the conventional mechanical devices (which e.g. use centrifugal forces) are damped or be eliminated.

Viriplanin can generally be obtained from the mycelium or from the culture medium common physico-chemical methods can be isolated. The insulation can e.g. according to the usual methods of extraction, precipitation, chromatography or partitioning take place.

The isolated antibiotic can also be used with the help of the methods mentioned be finely cleaned. Care must be taken with all isolation and cleaning operations care must be taken to ensure that pH values of about 7.0 or more, preferably between 6.0 and 8.0, are maintained will. Organic and preferably inorganic can be used to increase the pH Bases are used, e.g. ammonia, alkali and alkaline earth hydroxides, alkali and Alkaline earth carbonates and hydrogen carbonates, e.g. KOH, NaOH, Na2CO3, NaHCO3 and CaC03.

In order to prevent the isolation and cleaning methods mentioned above Find out fractions in which viriplanin is in the highest concentration or in which If purity is present, the usual physico-chemical methods, e.g. B. Measure the UV band at 480 nm, the RF values or preferably the study of the antibacterial Activity. The investigation is particularly favorable in the present case the activity against Bacillus Subtilis ATCC 6633 using the usual plate test (see e.g. P. Klein, Bacteriological Basics of Chemotherapeutic Laboratory Practice, Springer-Verlag, Göttingen (1957), pages 86 ff.).

Isolation and purification of viriplanin can e.g.

in the case that a liquid aqueous nutrient medium is used, such as be carried out as follows: The culture filtrate obtained by centrifuging the culture broth Can be used with a water-immiscible organic solvent such as n-butanol or chloroform, with the resulting aqueous phase being discarded will. The organic phase can expediently be strongly concentrated in vacuo or mixed with a solvent in which viriplanin is not soluble.

The resulting raw antibiotic can be centrifuged or Filtraticn separated from the mother liquor and with an organic solvent, in which viriplanin is insoluble, such as n-hexane or carbon tetrachloride and dried in vacuo.

The manufacture of Viriplanin and its purification is carried out by the following Examples explained.

Unless otherwise stated, percentages are percentages by weight.

Examples 1a (fermentation) The sporangia of a 10 day at 200C grown agar slant culture of the strain Ampuliariella regularis SE-47 on the culture medium Bacto-Pepton (Difco) 0.25% casein hydrolyzate, acidic (Oxoid) 0.25% K2HP04 x 3 H20 0.1% KCl 0.05% MgSO4 x 7 H20 0.05% pH 7.0 FeS04 0.01% cane sugar 3.0% agar (Difco) 2.0% are suspended in 15 ml of sterile 0.9% NaCl solution. Ten 1 l Erlenmeyer flasks, each containing 140 ml of sterile "nutrient solution C" of the composition Corn starch (Maizena) 2.0% glucose 1.0% N-Z-Amine (Sheffield Chemical) 0.5% pH 7.2 yeast extract (Ohly) containing 1.0% CaC03 0.2% are added with 1.0 ml of the sporangia suspension inoculated and kept on the rotary shaker at 240C for 5 days.

Example 1b (work-up) The viscous ones obtained according to Example la Culture broths were combined and with 3 times the volume of methanol carried out. The mycelium and other precipitated products were on the centrifuge separated and discarded. The clear, supernatant solution was in vacuo to Syrup consistency concentrated, with distilled water to a volume of 500 ml diluted and extracted twice with 250 ml of n-butanol at pH 7.0. The severed ones organic phases were combined and concentrated to 1/20 of the volume in vacuo. The resulting red precipitate of crude viriplanin was separated from the mother liquor, washed with carbon tetrachloride and dried in vacuo at room temperature. 0.25 g was obtained.

Example 2a (fermentation) 1 l Erlenmeyer flask, each containing 140 ml sterile "nutrient solution A" of the composition soy flour, defatted 3.0% glycerine, pure 3.0% CaC03 contain 0.3% tap water, with 1.0 ml each of the in example 1 described sporangia suspension of Ampullariella regularis SE-47 inoculated and Maintained on the shaker at 28 ° C. for 4 days.

Three glass fermenters each with 8 liters of nutrient solution of the composition K2HPO4 x 3 H20 0.1% NaCl 0.1% MgS04 x 7 H20 0.05% FeS04 0.001% Before adding the CaCO3 0.2% CaCO§ adjusted to pH 7.0 yeast extract (Ohly) 1.5% cane sugar 2.0% glucose 0.5% tap water is sterilized at 1200C and after cooling with 140 each ml of the prepared as described above, grown for 4 days in "nutrient solution A" Shake cultures of the strain Ampullariella regularis SE-47 inoculated. While stirring (300 revolutions / min. From 0 to 24 hours; 400 revolutions j min. From 24 hours) and aeration (4 ltr. air / min.) The fermenter was 65 hours at one temperature kept at 28 ° C. The pH of the culture broth was 7.8 to 8.0 after this time. The mycelium volume was 15.5 t.

Example 2b (work-up) The culture broths prepared according to Example 2a the three glass fermenters were combined and placed in the overhead centrifuge at 3000 revolutions Centrifuged / min.

The clear filtrate (18 liters) was adjusted to pH 7.0 with 1 N H2S04 and extracted 2 times with 9 l of n-butanol each time. The separated from the aqueous phase, The combined organic phases were concentrated to 3 liters in vacuo at 20 ° to 30 ° C. The resulting fine-grained red precipitate of raw antibiotic was filtered off, washed with carbon tetrachloride and in a vacuum dried at room temperature. The dry product yield (1st fraction) was 11.75 g.

The mother liquor obtained after separation of the 1st fraction was concentrated down to 300 ml. This resulted in the excretion of further red precipitate of crude viriplanin, which has been worked up as described above and as the 2nd fraction provided a dry product yield of 3.24 g.

The mycelium obtained after centrifuging the culture broth was resuspended in 180 l of methanol, stirred gently at room temperature for one hour and again separated in the overflow centrifuge. The red colored clear methanolic The extract was concentrated to the syrup consistency in a vacuum at 20 to 350C, with Diluted 400 ml of distilled water and exhaustively (15 x) extracted with n-butanol. The separated and combined organic phases were reduced to 100 in vacuo ml concentrated. The resulting fine-grained red precipitate of raw antibiotic was filtered off, washed with carbon tetrachloride and in vacuo at room temperature dried. The dry product yield was 6.65 g.

Example 2c (work-up): 3 liters of culture filtrate were passed through a Lewapol column (MEA 1864), 52 cm x 7.5 cm given. It is made with aqua bidist. rewashed until the Pass is colorless.

Then inactive accompanying substances with 10, 30 and 50% aqueous. MeOH eluted. The active ingredient is eluted from the column with pure MeOH. After evaporation 1.33 g of crude product are obtained. The active ingredient content is as high as that of the 1st fraction in example 2b.

Example 2d (work-up) 1.8 l of butanolic solution according to example 2b are mixed with 1.8 l of petroleum ether and twice with 90 ml of phosphate buffer each time pH 7.0 extracted. The organic phase is discarded, the aqueous one again extracted with pure petroleum ether. The aqueous phase is mixed with 3.8 g of sodium chloride 180 ml of MeOH and 180 ml of chloroform were added. After the segregation of the system the lower organic phase contains the antibiotic. Receives after evaporation one 620 mg of enriched material.

Example 2e (work-up) 1.00 g of the according to Example 2b, fraction 1, obtained antibiotic is in a mixture of 100 ml MeOH and 100 ml chloroform solved. 50 ml of phosphate buffer containing 2% NaCl are added to the solution.

Two phases are formed. Enriches in the lower organic phase the antibiotic. The lower phase is 2 x with freshly prepared upper phase washed and evaporated. 465 mg of enriched material are obtained.

Example 3 Pure preparation of 25 g of the crude product obtained according to Example 2b are extracted twice with 150 ml of chloroform-methanol-concentrated ammonia mixture (120: 30: 1) and filtered. The residue is washed out again with 200 ml of the solvent mixture, the combined filtrates are evaporated to dryness in vacuo (13.6 g).

10 g of the above extract were on a 100 x 6 cm column of neutral Silica gel 60 (0.063-0.2 mm grain size, Merck, Darmstadt, Federal Republic of Germany) chromatographed, first with 2 1 chloroform-methanol-concentrated ammonia mixture (95: 5: 1) and then eluted with a chloroform-methanol-concentrated ammonia mixture (120: 30: 1) became.

The red main zone yields 1.4 g of pure in thin-layer chromatography Viriplanin and another 4.9 g of highly enriched material. Viriplanin was used in Thin layer chromatogram and identified by bioautography.

The pure product has the properties given on pages 2-7.

Claims (6)

Claims Antibiotic Viriplanin, which a) consists of carbon, There is hydrogen, oxygen and nitrogen, b) in 2N aqueous NaOF, in 2N aqueous Sulfuric acid, in dimethylformamide and dimethyl sulfoxide well, in water, ether and Difficult hydrocarbons: is soluble, c) precipitated from neutral solvents, is a red powder with a melting point of about 210 to 2150C (decomposition), d) on neutral silica gel plates in the solvent mixture chloroform / methanol (80/20) has an Rf value of 0.23, e) has a molecular weight of about 2000, f) at the acid hydrolysis including 2-deoxy-L-fucose, g) with hydrolysis in strongly acidic and then basic medium including mesaconic acid and hl during degradation with 2.5 N hydrochloric acid in methanol 7-0-methylnogalarol and nogalarene supplies. 2. Process for the preparation of the antibiotic viriplanin, thereby characterized in that the strain Ampullariella regularis SE 47 (ATCC 31417) under aerobic conditions in a common culture medium and grows the antibiotic isolated from the culture broth or the mycelium by generally customary methods and cleans if necessary. 3. The method of claim 2, wherein the growth temperature at 20 to 400C and the pH of the growing culture is 6 to 8. 4. Medicines characterized by an antibiotic content Viriplanin. 5. Use of the antibiotic viriplanin in combating bacterial and viral infections. 6. Use of the antibiotic viriplanin in combating infections caused by herpes viruses.