DE4303684A1 - Musacins, novel metabolites from Streptomyces sp., and chemical derivatives having pharmacological action, a process for their preparation and their use - Google Patents

Abstract

The invention relates to the natural substances musacins, which are synthesised by Streptomyces sp. during fermentation and released into the mycelium or the culture supernatant, and the chemical derivatives prepared based on musacins, a preparation process, the use of the musacins and derivatives of the musacins as pharmacological active compounds and the microorganism Streptomyces griseoviridis DSM 7429.

Description

The invention relates to the natural substances Musacine, which Streptomyces sp. synthesized during the fermentation and into the mycelium or the Culture supernatant are given, as well as those based on the Musacine prepared, chemical derivatives, a manufacturing process, the use the muscacins and derivatives of muscacins as pharmacologically active substances and the microorganism Streptomyces griseoviridis DSM 7429.

Secondary metabolites of microorganisms become successful for treatment used by infectious diseases. Secondary metabolites are to low-molecular compounds, their formation in "biosynthetic One-way streets, which branch off from primary metabolism, take place and their Function for the respective producer is unclear. To date, about 8,000 from cultures of various microorganisms (especially fungi and bacteria of the Genus Streptomyces) isolated secondary metabolites. Secondary metabolites from streptomycetes have also been around for a long time known.

The indication area of microbial active substances has been on in the meantime Diseases that are not infectious diseases (eg tumor therapy, Immunomodulation or for the regulation of the solid change) and on the Plant protection (herbicides and insecticides) expanded. Secondary metabolites For example, streptomycetes are widely distributed in different ways Application areas, such. As a cholesterol-lowering or anti-tumor agent used. However, the active ingredients are often still with defects afflicted, characterized by unsatisfactory levels of action, too high Toxicity and / or unwanted side effects.

The object of the invention is to microbial agents with improved To seek properties and new mechanisms of action.

This object is achieved by fermentation of Streptomyces sp. in a nutrient solution with carbon and nitrogen source and the usual inorganic salts until the muscacins in the mycelium and accumulate in the culture, subsequent isolation of the muscellins from the mycelium and the culture and optionally a chemical derivatization of the Musacine. The Musacine and their chemical derivatives possess pharmacological and thus therapeutic efficacy and can be beneficial as compounds with anthelminthic or antiviral properties be used.

The invention relates to:

• 1. A compound of the general formula in the independently of each other
  R ¹ = hydrogen, R ¹ and R ⁴ together form a bond,
  R ² and R ^{3 are} hydrogen or R ² and R ³ together represent a chemical bond,
  R ^{4 is} hydrogen or R ⁴ and R ¹ together form a bond or R ⁴ and R ⁵ together represent a chemical bond,
  R ⁵ , R ⁶ and R ^{7 are} hydrogen or R ⁵ and R ⁴ , R ⁵ and R ⁶ or R ⁶ and R ⁷ together represent a chemical bond, or
  R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , which may be the same or different, a saturated or unsaturated, branched or unbranched or cyclic C ₁ -C ₂₀ alkyl ester, a substituted or unsubstituted aromatic Ester, a heteroalkyl ester or an alkyl or arylsulfonic acid ester.
• 2. A process for the preparation of a compound of formula I which is characterized characterized in that Streptomyces sp. cultured in a nutrient medium until a compound of formula I accumulates in the culture and these from the Nutrient medium is isolated.
• 3. A use of a compound of formula I as a pharmacological effective substance.
• 4. Streptomyces sp. DSM 7429.

In the following the invention is described in detail, in particular in their preferred embodiments. Furthermore, the invention is characterized by the content of Claims determined.

The compounds of the formula I are called muscacins.  

The compounds according to the invention are preferred by Streptomyces sp of Streptomyces griseoviridis, and more preferably of Streptomyces griseoviridis DSM 7429 synthesized.

Streptomyces sp. is isolated from a soil sample.

For the purification of the fungus from the soil, it is suspended in a dilution series with a physiological NaCl solution (0.9%). The various dilutions (10 ° -10 ⁶ ) are then applied to a fungal agar z. B. plated on a malt extract glucose base. After incubation of the cultures at 25 ° C for 3-15 days, fungal colonies are formed, which can be isolated and isolated by several successive purification steps.

The determination of the fungal genus Streptomyces sp. is determined by morphological and taxonomic criteria known to those skilled in the art Methods made.

Characteristic of Streptomyces griseoviridis DSM 7429 are the following criteria to be analyzed macroscopically or by microscope [culture of the strain ISP (International Streptomyces Project) medium]:
Color of aerial mycelium: pale carmine to blue red, substrate mycelium: light brown; SEM image: round-ellipsoidal spores (1.0 × 1.4 μ) with a smooth surface; Spore chains are slightly spirally wound, in irregular loops of rarely more than 3 turns, which grow out directly from the substrate mycelium or from the main axis of the spore chains.

Due to successive isolation and purification steps can be detected by Streptomyces sp. isolate a fungal colony, which makes the Musacine very efficiently into the culture supernatant and is referred to as the main producer.  

The main producer is a fungal isolate which is a compound of the Formula I in a 10 to 100 times increased amount compared to isolates of same fungus produced or in the culture supernatant.

The strongly producing fungal colony is increased. An isolate of Streptomyces sp. was at the German Collection of Microorganisms and Cell Cultures according to the rules of the Budapest Treaty on 26.01.1993 under the number DSM 7429 deposited.

The strain Streptomyces sp. DSM 7429 has brown red spore chain, which in Spirals are arranged. The surface of the roundish-ellipsoidal spores appears smooth. Based on morphological data, strain DSM 7429 as Streptomyces griseoviridis.

In a nutrient solution containing a carbon source and a nitrogen source as well as the usual inorganic salts, Streptomyces sp., Preferably Streptomyces griseoviridis and more preferably Streptomyces griseoviridis DSM 7429, produces a compound of general formula I in which independently
R ¹ = hydrogen,

R ¹ and R ⁴ together form a bond,
R ² = hydrogen or R ² and R ^{3 form} a common double bond,
R ³ = hydrogen or R ³ and R ^{2 form} a common double bond,
R ⁴ = hydrogen or R ⁴ and R ¹ together form a bond or R ⁴ and R ^{5 form} a common double bond,
R ⁵ = hydrogen or R ⁵ and R ^{4 form} a common double bond or R ⁵ and R ^{6 form} a common double bond,
R ⁶ = hydrogen or R ⁶ and R ^{5 form} a common double bond or R ⁶ and R ^{7 form} a common double bond,
R ⁷ = hydrogen or R ⁷ and R ^{6 form} a common double bond.

"Together form a bond" means that the respective substituents close to the ring.

These naturally produced compounds are preferred Links.

Instead of the strain DSM 7429 can of course also its mutants and Variants are used, as far as they synthesize this compound. Such Mutants can be prepared in a manner known per se by physical means, For example, irradiation, such as ultraviolet or X-rays or chemical mutagens, such as ethyl methanesulfonate (EMS), 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl-N'-nitro-N- nitrosoguanidine (MNNG).

Screening for mutants and variants that are a compound of the formula synthesize, according to the following scheme:

• Separation of the mycelium after fermentation;
• Adsorption of the culture filtrate on a polystyrene adsorber resin;
• Elution with 50% methanol;
• - Concentration and analysis by thin layer chromatography on silica gel; as eluent is a chloroform-methanol mixture (Ratio 9: 1) used;
• Detection by means of spraying with a mixture of anisaldehyde and Sulfuric acid.

The fermentation conditions described below apply to Streptomyces sp., Streptomyces griseoviridis and the deposited isolate.

Preferred carbon sources for aerobic fermentation are assimilable carbohydrates and sugar alcohols, such as glucose, lactose or D-mannitol and carbohydrate-containing natural products such. B. malt extract. When Nitrogenous nutrients are considered: amino acids, peptides and Proteins and their degradation products, such as peptones or tryptones, further Meat extracts, ground seeds, for example of maize, wheat, beans, Soybean or cotton plant, distillation residues from alcohol production, Meat flours or yeast extracts, but also ammonium salts and nitrates. On Inorganic salts, the nutrient solution, for example, chlorides, carbonates, Sulfates or phosphates of alkali or alkaline earth metals, iron, tin, cobalt and Manganese included.

The formation of a compound of the formula I proceeds particularly well in a nutrient solution which contains about 0.2 to 5%, preferably 1 to 4%, soya flour and 0.2 to 5%, preferably 1 to 4% of mannitol, in each case based on the Weight of the entire nutrient solution. Similarly good results are obtained with a nutrient solution, the glycerol in concentrations of 0.5 to 6%, preferably 2 to 4%, soybean flour in concentrations of 0.1 to 4%, preferably 0.5 to 2%, K ₂ HPO ₄ in concentrations of 0.01 to 6%, preferably 0.5 to 2%, NaCl in concentrations of 0.01 to 6%, preferably 0.5 to 2% and MgSO ₄ in concentrations of 0.01 to 3%, preferably contains 0.1 to 1%, in each case based on the weight of the entire nutrient solution. The cultivation is carried out aerobically, that is, for example, submerged with shaking or stirring in shake flasks or fermenters, optionally with the introduction of air or oxygen. It can be carried out in a temperature range of about 18 to 35 ° C, preferably at about 25 to 35 ° C, in particular at 28 to 32 ° C. The pH range should be between 6 and 8, advantageously between 6.5 and 7.5. The microorganism is cultivated under these conditions generally over a period of 24 to 300 hours, preferably 36 to 140 hours.

Advantageously cultivated in several stages, d. H. you first set one or several precultures ago in a liquid nutrient medium, which then in the actual production medium, the main crop, for example in the Volume ratio 1: 10, to be inoculated. The preculture is obtained z. B., by inoculating a spiked mycelium into a nutrient solution and about 36 to 120 hours, preferably 48 to 72 hours, grow. The spurred mycelium can be obtained, for example, by adding the strain about 3 to 40 Days, preferably 4 to 10 days, on a solid or liquid medium, For example, yeast-malt agar or soybean-mannitol agar grows.

The course of fermentation can be determined by the pH of the culture or of the Mycelium volume and by chromatographic methods, such. B. Thin Layer Chromatography or High Pressure Liquid Chromatography or Monitoring the biological activity to be monitored. A connection of general formula I is contained both in the mycelium and in the culture filtrate.  

The isolation of one of the compounds of the invention from the Culture medium or mycelium is carried out by known methods Consideration of chemical, physical and biological Properties of the products. It can z. B. the adsorption, ion exchange or Gel filtration methods are applied.

To test the metabolite concentration in the culture medium or in the individual Isolation levels may be thin layer chromatography, for example Silica gel with butanol / glacial acetic acid / water, ethyl acetate / methanol / water or Chloroform / methanol can be used as the eluent. The detection at the thin-layer chromatographic separation can, for example, by Dyeing agents such as anisaldehyde or by biological testing, z. B. with viruses or nematodes, wherein the amount of the formed substance appropriately compared with a calibration solution.

To isolate a compound of formula I are culture broth and mycelium of individual fermentations first z. B. by filtration or centrifugation separated. The culture filtrate is passed over an adsorber resin, such. B. XAD® Adsorber the Fa. Amberlite given to which bind the compounds of formula I and with a solvent / water mixture, eg. For example, methanol / water 4: 1, eluted can be.

The pure isolation of a compound of formula I is carried out by chromatography on suitable materials, preferably on silica gel, alumina, Ion exchangers or adsorber resins, by subsequent elution with organic solvents or solvent mixtures, such as. B. Acetic acid alkyl esters, mixtures of alkyl acetates with a lower one Alkanol, chloroform or methylene chloride or mixtures of these solvents with lower alkanols, optionally with water, or a for Ion exchange resins suitable pH or salt gradients, such as for example, common salt or tris- (hydroxymethyl) -aminomethane-HCl (Tris buffer) by contamination of the biologically active fractions. But a cleaning is  also by extractive methods such as liquid / liquid extraction or solid / liquid Extraction possible.

The compounds according to the invention have pharmacological activity, especially against viruses and nematodes.

The compounds of the formula I which are synthesized by Streptomyces sp., Preferably by Streptomyces griseoviridis and particularly preferably by Streptomyces griseoviridis DSM 7429, then partially isolated or isolated, can be used for the chemical modification on the substituents R ¹ to R ⁷ .

As Arylsulfonsäureester phenyl radicals are defined, optionally substituted with up to 3 radicals from the group C ₁ -C ₃ alkyl, halogen, C ₁ -C ₃ alkoxy.

The esterification reaction is carried out on the R ² and / or R ³ substituents. The esters are prepared by methods known to those skilled in the art.
Chemically synthesized:

Saturated or unsaturated, branched or unbranched, cyclic or open-chain C ₁ -C ₂₀ -alkyl esters, substituted or unsubstituted aromatic esters or heteroaryl esters.

Preferably synthesized are saturated or unsaturated, branched or unbranched, cyclic or open-chain C ₁ -C ₁₀ -alkyl esters, hydroxy, amino, C ₁ -C ₁₀ -alkylamino, oxialkyl, sulforyl, C ₁ -C ₁₀ -alkylsulfonyl , halo or thio-substituted aromatic esters, wherein the substituents on the aromatic ring may be the same or different and the aromatic is preferably a benzoyl or aminobenzoyl derivative.

The conversion of a compound of the formula I into one of the chemically prepared derivatives synthesized by Streptomyces sp., Preferably by Streptomyces griseoviridis and particularly preferably by Streptomyces griseoviridis DSM 7429, takes place in a manner known per se. Thus, the esters of the compound can be prepared by reacting the muscazines with acids catalysed by dicyclohexylcarbodimide or with acid chlorides in pyridine / CH ₂ Cl ₂ . Also with acid anhydrides, the respective esters can be produced.

Compounds of formula I are in the solid state and in solutions in the pH range between pH 3 and 8, in particular pH 5 and 7, stable and let thus familiarize themselves with common galenic preparations.

A compound of the formula I is suitable because of its valuable pharmacological properties very good for use as a drug. In particular, they are effective against viruses and nematodes.

A compound of formula I has biological activity by having a anthelmintic action against z. B. Plathelminthes (flatworms) and Nemathelminthes (roundworms) shows. Within these tribes is one Compound of formula I in particular effective against Haemonchus, Trichostrongylus, Ostertagia, Cooperia, Chabertia, Strongyloides, Oesophagostomum, Hyostrongylus, Ancylostoma, Ascaris and Heterakis. In addition, a compound of formula I has biological activity by having a antiviral effect z. Against adenoviruses, poxviruses, herpesviruses, mysoviruses, Renamyxoviruses and piconaviruses shows.

A compound of the general formula I can in principle be used as such in Substance can be administered. Preferably, the use in mixture with suitable excipients or carrier material. As a carrier material can at Veterinary medicines are the usual feed mixtures or, in humans, all used pharmacologically acceptable carrier materials and / or excipients become.

The application also relates to pharmaceutical preparations of a compound the formula I.  

The pharmaceutical compositions according to the invention are generally oral or parenteral administered, but also a rectal application is possible in principle. suitable solid or liquid galenic preparations are, for example Granules, powders, tablets, dragees, (micro-) capsules, suppositories, syrups, Emulsions, suspensions, aerosols, drops or injectable solutions in Ampoule form and preparations with Protahierter drug release, in whose Production usually carriers and additives and / or auxiliaries such as Explosive, binding, coating, swelling, lubricating or lubricating agents, Flavorings, sweeteners or solubilizers find use. As commonly used carriers or excipients z. For example, magnesium carbonate, Titanium oxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, Starch, Vitamins, Cellulose and their Derivatives, Animal Vegetable Oils, Polyethylene glycols and solvents such as sterile water, alcohols, Called glycerine and polyhydric alcohols.

Optionally, the dosage units for oral administration be encapsulated to delay the task or for a longer time Period, such as by coating or embedding the Particle active ingredient in suitable polymers, waxes or the like.

Preferably, the pharmaceutical preparations are in dosage units prepared and administered, each unit as an active ingredient contains certain dose of a compound of formula I. At fixed Dosage units such as tablets, capsules and suppositories may take this dose up to about 200 mg, but preferably about 0.1 to 100 mg, and at Injection solutions in ampoule form up to about 200 mg, but preferably about 0.5 to 100 mg, per day.

The daily dose to be administered depends on the body weight, age, Sex and condition of the mammal. In some circumstances, however, too be higher or lower daily doses. The administration of the Daily dose can be given by single dose in the form of a single  Dosage unit or in several smaller dosage units as well by multiple subdivided doses at specific intervals.

The medicaments according to the invention are prepared by adding a or several compounds of the formula I with conventional carrier and optionally additives and / or auxiliaries in or a suitable Dosage form brings.

In the following examples, the invention will be further explained. Percentages are by weight, mixing ratios Liquids are by volume unless otherwise stated were made.

Examples 1. a) Preparation of a spore suspension of the producer strain Streptomyces sp. DSM 7429

100 ml of nutrient solution [10 g starch, 2 g (NH ₄₎ SO ₄₁ 1 g K ₂ HPO _4, 1 g MgSO ₄ × 7 H ₂ O, 1 g NaCl, 2 g CaCO ₃₁ 2.5 ml of a trace element solution (3 g Calcium chloride, 1 g of ferric citrate, 0.2 g of manganese sulfate, 0.1 g of zinc chloride, 0.025 g of copper sulfate, 0.02 g of sodium tetraborate, 0.004 g of cobalt chloride, 0.01 g of sodium molybdate in 1 L of distilled water), with water to 1 l, pH before sterilization 7.3] in a 300 ml sterile Erlenmeyer flask are inoculated with the strain DSM 7429 and incubated for 72 hours at 28 ° C and 150 rpm on a rotary shaker. Subsequently, 20 ml of culture liquid in a sterile 500 ml Erlenmeyer flask with the nutrient medium of the above composition, to which an additional 20 g of agar / l is added for solidification, evenly distributed and decanted. The cultures are incubated at 30 ° C for 10 to 14 days. The spores of a flask produced after this time are washed off with 500 ml of deionized water containing one drop of a commercially available nonionic surfactant (eg Triton X 100, from Serva), reused immediately or at 50 ° C. at -22 ° C. Glycerol stored.

b) Production of a culture or a preculture of the producer strain DSM 7429 in Erlenmeyer flask

A sterile 300 ml Erlenmeyer flask with 100 ml nutrient solution (2 g soy flour, 2 g Mannitol, make up to 1 liter with water, pH = 7.2 before sterilization) with a culture grown on a slant tube or with 0.2 ml Spore suspension inoculated and on a shaker at 150 rpm and Incubated at 30 ° C for about 72 hours.

For inoculating 10 and 100 liter fermenters, a 48 hour old one is enough Submerged culture (inoculation amount approx. 5%) from the same nutrient solution.

2. Preparation of the compound of the general formula

A 10 l fermenter is operated under the following conditions: Nutrient medium: 30 g / l glycerol, 2 g / l casein peptone, 1 g / l K ₂ HPO ₄ , 1 g / l NaCl, 0.5 g / l MgSO ₄ × 7 H ₂ O, 5 ml of a trace solution [3 g CaCl ₂ × 2 H ₂ O, 1 g Fe-III citrate, 0.2 g MnSO ₄ , 0.1 g ZnCl ₂ , 0.025 g CuSO ₄ × 5 H ₂ O, 0.02 g Na ₂ B ₄ O ₇ × 10 H ₂ O, 0.004 CoCl ₂ , 0.01 g NaMoO ₄ × 2 H ₂ O, to 1 l with redist. Fill with water; pH = 7.2 (before sterilization)].

Incubation period: 72 hours incubation temperature: 30 ° C stirrer: 250 Up Ventilation: 4 l air / min

By repeated addition of few drops of ethanolic polyol solution, the Foaming can be suppressed. The production maximum is after approx. Reached 72 hours.  

3. Isolation of the compound of general formula I.

After completion of the fermentation of DSM, the culture broth is added of about 2% filter aid (eg Celite). It can be following Schemes are worked up:

4. Characterization of compounds of the formula I. Musacin A (4,5-Dihydroxy-octa-2,6-dienoic acid-2,3-dihydroxy-propyl ester)

Musacin A is obtained from fraction V (161 mg) by separation twice Sephadex LH 20 (column 3 x 100 cm, MeOH) in an amount of 37 mg (4.0 mg / L) isolated as a yellowish oil.

C ₁₁ H ₁₈ O ₆ (246.3)

DCI-MS (200 eV): m / e = 264 (100%, M + + NH ₃ + H +), 246 (22%, M +).
El-MS (70 eV): m / e = 176 (7%, M + HC ₄ H ₇ O⁺), 155 (4% MC ₃ H ₇ O ₃ +), 84 (93%, C ₄ H ₄ O ₂ ), 71 (56%, C ₄ H ₇ O), 57 (27%, C ₃ H ₅ O).

UV: (MeOH) λ _max (ε): 209 (9500)
(MeOH + Hcl) λ _max (ε): 208 (9600)
(MeOH + NaOH) λ (ε): 217 (12800)
CD: (MeOH) λ _extr. ([R] ²² ): 228 (+10400) Rotation: [α] = +23.2 (c = 0.48, MeOH)

¹ H-NMR (500 MHz, CD ₃ OD): δ = 1.71 (ddd, J = 6.5, 1.5 and 1, 8-CH ₃ ), 3.56 (dd, J = 5 and 2.5, 3'-H), 3.85 (tt, J = 5.5 and 5.5, 2'-H), 3.98 (dd, J = 7 and 6, 5-H), 4.13 (m, 4-H), 4.13 (ddd, J = 11.6 and 1.5 , 1'-H _a ), 4.22 (ddd, J = 11.4 and 1.5, 1'-H _b ), 5.53 (ddq, J = 15.5, 7 and 1.5, 6-H), 5.73 (dqd, 15.5, 6.5 and 1, 7-H), 6.10 (dd, J = 15.5 and 1.8, 2-H), 7.07 (dd, J = 15.5 and 4.5, 3-H) ppm.

¹³ C-NMR (125.7 MHz, CD ₃ OD): δ = 16.6 (q, C-8), 62.6 (t, C-3 '), 65.2 (t, C-1'), 69.8 (d, C) 2 '), 73.8 (d, C-4), 75.0 (d, C-5), 120.6 (d, C-2), 128.1 (d, C-7), 130.0 (d, C-6), 148.3 (d, C-3), 166.6 (s, C-1) ppm.

b) Musacin B 4,5-dihydroxy-octa-2,6-dienoic acid-4-hydroxy-5-oxo-tetrahydrofuran-3-y l methylester

From fraction II (1.12 g) is first chromatographed on silica gel (Column 4.5 x 30 cm, EA / MeOH gradient = 95: 5-9: 1) an intermediate fraction (52 mg), from which after purification on Sephadex LH 20 (column 3 × 100 cm, MeOH) 42 mg (4.5 mg / l) of Musacin B as a yellowish oil become.

C ₁₃ H ₁₈ O ₇ (286.3)

DCI-MS (200 eV): m / e = 304 (100%, M + + NH ₃ + H +), 286 (95%, M +)
El-MS (70 eV): m / e = 216 (17%, M⁺HC ₄ H ₇ O), 188 (3%, M + HC ₄ H ₇ O-CO), 116 (12%, C ₅ H ₈ O ₃ ), 84 (100%, C ₄ H ₄ O ₂ ), 71 (81%, C ₄ H ₇ O), 69 (62%, C ₄ H ₅ O), 57 (49%, C ₃ H ₅ O).

IR (cm⁻ ¹ ): = 3420 br, 2920 w, 1780 s, 1722 s, 1660 m, 1380 w, 1270 m, 1180, 1135 m, 1005 m, 720 w.

UV: (MeOH) λ _max (ε): 206 (9000)
(MeOH + HCl) λ _max (ε): 207 (8800)
(MeOH + NaOH) λ _max (ε): 214 (8200)
CD: (MeOH) λ _extr. ([R] ²² ): 220 (+15 700) Rotation: [α] = -10.1 (c = 0.38, MeOH)

¹ H-NMR (500 MHz, CD ₃ OD): δ = 1.71 (ddd, J = 6.5, 1.5 and 0.5, 8-CH ₃ ), 2.77 (m, 3'-H), 3.98 (dd, J = 7 and 6, 5-H), 4.06 (dd, J = 9.5 and 9.5, 4'-H _a ), 4.14 (ddd, J = 5.5, 5 and 1.5, 4-H), 4.30 (dd, J = 11.5 and 6.5, 5'-H _a ), 4.32 (d, J = 10, 2'-H), 4.41 (dd, J = 11.5 and 4.5, 5'-H _b ), 4.44 (dd, J = 9.5 and 8, 4'-H _b), 5:53 (ddq, J = 15.7 and 1.5, 6-H), 5.73 (dqd, J = 15, 6.5, and 1, 7-H), 6:09 (dd, J = 15.5 and 2, 2 -H), 7.08 (dd, J = 15.5 and 4.5, 3-H) ppm.

¹³ C-NMR (50.3 MHz, CD ₃ OD): δ = 18.1 (q, C-8), 45.0 (d, C-3 '), 63.3 (t, C-5'), 68.2 (t, C) 4 '), 69.9 (d, C-2'), 75.2 (d, C-4), 76.4 (d, C-5), 121.6 (d, C-2), 129.6 (d, C-7), 131.5 (d, C-3), 150.3 (d, C-3), 167.7 (s, C-1), 178.6 (s, C-1 ') ppm.

c) Musacin C

From fraction III (1.1 g) is obtained by chromatography on silica gel 473 mg (50.7 mg / l) of a mixture of Musacin B and Musacin C by means of a yellowish oil, wherein the ratio of the components is approximately 1: 1. For characterization is derivatized.  

d) acetylation

66 mg of the mixture are dissolved in 20 ml of acetic anhydride and admixed with 10 mg of N, N-dimethylaminopyridine (DMAP). It is stirred for 2 hours at room temperature and then hydrolyzed with 20 ml of ice water. The reaction mixture is extracted 3 times with 20 ml of CHCl ₃ , and the combined organic phases are washed 3 times with 20 ml of aqueous NaHCO ₃ solution and once with H ₂ O. The combined aqueous phases are extracted again with 20 ml CHCl ₃ and the entire organic phase dried over Na ₂ SO ₄ . After filtration of the drying agent is concentrated in vacuo to give 96 mg of crude product. This is separated by chromatography on silica gel (column 2 × 45 cm, CHCl ₃ / MeOH = 98: 2). 12.7 mg (27%) of Musacin B triaceate and 25.2 mg (50%) of Musacin C-tetraacetate can be isolated as each colorless oil.

Musacin B triacetate 4.5-diacetoxy-octa-2,6-dienoic acid 4-acetoxy-5-oxo-tetrahydrofuran-3-y l methylester

C ₁₉ H ₂₄ O ₁₀ (412.4)

DCI-MS (200 eV): m / e = 430 (100%, M + NH ₃ + H +)
El-MS (70 eV): m / e = 342 (10%, M + HC ₄ H ₇ O), 300 (84%, M + HC ₄ H ₇ OC ₂ H ₂ O), 258 (15%, M + HC ₄ H ₇ O-2 × C ₂ H ₂ O), 216 (8%, M + HC ₄ H ₇ O-3 × C ₂ H ₂ O), 157 (16%, C ₇ H ₉ O ₄ ) , 126 (90%, C ₆ H ₆ O ₃ ), 113 (22%, C ₆ H ₉ O ₂ ), 84 (44%, C ₄ H ₄ O ₂ ), 71 (56%, C ₄ H ₇ O ), 57 (38%, C ₃ H ₅ O), 43 (100%, C ₂ H ₃ O).

IR (cm⁻ ¹ ): = 3440w, 2920m, 2860w, 1800m, 1750s, 1665w, 1380m, 1230s, 1180m, 1100m, 1025, 800w.

UV: (MeOH) λ _max (ε): 204 (14100)
(MeOH + HCl) λ _max (ε): 205 (15300)
(MeOH + NaOH) λ _max (ε): 214 (11800), 288 (2000)
CD: (MeOH): no measurable effectRotary value: [α] = 45.5 (c = 0.56, CHCl ₃ )

¹ H-MR (500 MHz, CDCl _3): δ = 1.71 (dd, J = 6.5 and 1, 8-CH ₃₎ (, 2.03 2.05 and 2:09 (3 × s, 3 × Acetyl-CH _3), 3.12 m, 3'-H), 4.17 (dd, J = 11.5 and 5.5, 5'-H _a ), 4.30 (dd, J = 11.5 and 4.5'-H _b ), 4.37 (dd, J = 10 and 1.5, 4'-H _a ), 4.44 (dd, J = 10 and 6, 4'-H _b ), 5.40 (verd., 5-H and 6-H), 5.54 (ddd, J = 5, 3.5 and 1.5, 4-H), 5.60 (d, J = 8, 2'-H), 5.83 (m, 7-H), 5.95 (dd, J = 15.5 and 1.5, 2-H), 6.83 (dd, J = 15.5 and 5, 3-H) ppm.

¹³ C-NMR (125.7 MHz, CDCl _3): δ = 17.8 (C-8), 20.4, 20.8 and 21.0 (3 x Acetyl-CH _3), 37.3 (C-3 '), 61.1 (C-5') , 67.5 (C-2 '), 67.5 (C-4'), 72.8 (C-4), 74.2 (C-5), 122.4 (C-2), 123.9 (C-6), 133.0 (C -7), 142.4 (C-3), 165.0 (C-1), 169.6, 169.7 and 169.9 (3x acetyl-C = O), 171.4 (C-1 ') ppm.

e) esterification with p-bromobenzoyl chloride

37 mg of Musacin B and C are dissolved in 10 ml of dry pyridine and with ice cooling with 142 mg of p-bromobenzoyl chloride. To Warming to room temperature is stirred for another 24 hours and the Reaction mixture with the addition of 10 ml of toluene finally in vacuo concentrated. 280 mg of crude product are obtained from the after chromatography Silica gel (column 2.5 × 43 cm, EA / Hex = 1: 2) 29 mg (50%) of the Musacin B-esters and 7 mg (11%) of the Musacin C ester are isolated.

4.5-di-O-p-brombenzoyloxy-octa-2,6-dienoic acid-4-O-p-brombenzoyloxy-S - oxo tetrahydrofuran-3-yl-methylester

C ₃₄ H ₂₇ O ₁₀ Br ₃ (834.7)

El-MS (70 eV): m / e = 834 (2%, M +), 764 (3%, MC ₄ H ₆ O), 183 (100%, C ₇ H ₃ OBr), 76 (8%, C ₆ H ₄ ).
IR (cm⁻ ¹ ): = 3420w, 2950w, 2910m, 2840w, 1795m, 1720s, 1660w, 1590m, 1480w, 1395in, 1260s, 1170m, 1100s, 1010s, 965w, 845w, 750m.

UV: (MeOH) λ _max (ε): 204 (74 500), 246 (64 300)
(MeOH + Hcl) λ _max (ε): 204 (73,000), 246 (62,400)
(MeOH + NaOH) λ _max (ε): 207 (48 600), 243 (60 300)
CD: (MeOH) λ _extr. ([R] ²² ): 213 (+10 200), 226 (+1700), 252 (+8100)

¹ H-NMR (200 MHz, CDCl ₃ ): δ = 1.75 (dd, J = 6.5 and 1.5, 8-CH ₃ ), 3.26 (m, 3'-H), 4.35 (dd, J = 5 and 2, 5'-H), 4.43 (dd, J = 10 and 1.5, 4'-H _a ), 4.55 (dd, J = 10 and 6, 4'-H _b ), 5.58 (ddq, J = 15, 7.5 and 1.5, 6-H), 5.77 (dd, J = 7.5 and 3.5, 5-H), 5.86 (reported 4-H), 5.92 (dqd, J = 15, 6.5 and 1, 7-H), 6.04 ( dd, J = 15.5 and 1.5, 2-H), 7.01 (dd, J = 15.5 and 5, 3-H), 7.58 (m, benzoyl-H), 7.88 (m, benzoyl-H) ppm.

4.5-di-O-p-brombenzoyIoxy-octa-2,6-dienoic acid-3-O-p-brombenzoyloxy-2 - O-p- bromobenzoyloxymethyl-3-methoxycarbonyl-propyl ester

C ₄₂ H ₃₄ O ₁₂ Br ₄ (1050.3)

El-MS (70 eV): m / e = 1050 (0.04%, M⁺), 980 (0.04%, MC ₄ H ₆ O), 850 (0.03%, M⁺-C ₇ H ₄ O ₂ Br ), 513 (4%, C ₂₀ H ₁₇ O ₆ Br ₂ ), 200 (29%, C ₇ H ₄ O ₂ Br), 183 (100%, C ₇ H ₃ OBr), 155 (28%, C ₆ H ₃ Br).

UV: (MeOH) λ _max (ε): 205 (109 000), 245 (110,000)
(MeOH + HCl) λ _max (ε): 205 (95,800), 246 (99,700)
(MeOH + NaOH) λ _max (ε): 208 (74 500) 242 (108 000)
CD: (MeOH) λ _extr. ([R] ²² ): 215 (+10 200), 240 (+20 000)

¹ H-NMR (300 MHz, CDCl ₃ ): δ = 1.76 (dd, J = 6.5 and 1.5, 8-CH ₃ ), 3.10 (m, 3'-H) 3.66 (s, 6'-CH ₃ ), 4.30 (dd, J = 11.5 and 7.5, 4'-H _a ), 4.45 (dd, J = 11.5 and 5.5, 4'-H _b ), 4.52 (dd, J = 11.5 and 6, 5'-H _a ) , 4.60 (dd, J = 11.5 and 7, 5'-H _b), 5:55 (d, J = 3.5, 2'-H), 5:59 (ddq, J = 15.5, 7.5 and 1.5, 6-H), 5.79 (dd, J = 7.5 and 4, 5-H), 5.90 (ddd, J = 5.5, 4 and 1.5, 4-H), 5.98 (dqd, J = 15.5, 6.5 and 1, 7-H), 6.10 ( dd, J = 16 and 2, 2-H), 7.07 (d, J = 16 and 5.5, 3-H), 7.59 (m, benzoyl-H), 7.87 (m, benzoyl-H) ppm.

f) Musacin C tetraacetate 4.5-diacetoxy-octa-2,6-dienoic acid-3-acetoxy-2-acetoxymethyl-3- methoxycarbonyl-propyl

C ₂₂ H ₃₀ O ₁₂ (486.5)

DCI-MS (200 eV): m / e: 504 (100%, M + NH ₃ + H +).
El-MS (70 eV): m / e: 416 (2%, M + HC ₄ H ₇ O), 374 (39%, M + HC ₄ H ₇ OC ₂ H ₂ O), 314 (12%, M + HC ₄ H ₇ OC ₂ H ₂ OC ₂ H ₄ O ₂ ), 272 (4%, M + HC ₄ H ₇ O-2 × C ₂ H ₂ OC ₂ H ₄ O ₂ ), 231 (88%, C ₁₀ H ₁₅ O ₆ ), 189 (10%, C ₈ H ₁₃ O ₅ ), 147 (2%, C ₆ H ₁₁ O ₄ ), 126 (66%, C ₆ H ₆ O ₃ ), 84 (36%). , C ₄ H ₄ O ₂ ), 71 (18%, C ₄ H ₇ O), 43 (100%, C ₂ H ₃ O)
IR (cm⁻ ¹ ): = 3440w, 2990m, 2920w, 2850w, 17455, 1660w, 1440w, 1370m, 1225s, 1170m, 1030m, 970w, 800w.

UV: (MeOH) λ _max (ε): 205 (14 500)
(MeOH + HCl) λ _max (ε): 204 (15 300)
(MeOH + NaOH) λ _max (ε): 214 (15 800)
CD: (MeOH) λ _extr. ([R] ²² ): 209 (-2400)
Rotation: [a] = +15.4 (c = 0.5, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ): δ = 1.70 (dd, J = 6.5 and 1, 8-CH ₃ ), 2.01, 2.03, 2.05 and 2.07 (4 x s, 4 x acetyl-CH ₃ ), 2.76 (m ,, 3'-H), 3.74 (s, 6'-CH _3), 4.16 (dil., 4'-H _a, 4'-H _b), 4.25 (dd, J = 11.5 and 5.5, 5'-H _b), 5.20 (d, J = 3.5, 2'-H), 5:39 (dil., 5-H and 6-H), 5:53 (ddd, J = 5.0, 3.5 and 1.5, 4 -H), 5.82 (m, 7-H), 5.98 (dd, J = 15.5 and 1.5, 2-H), 6.85 (dd, J = 15.5 and 5, 3-H) ppm.

¹³ C-NMR (125.7 MHz, CDCl _3): δ = 17.8 (C-8), 20.5, 20.7, 20.8 and 21.0 (4 x Acetyl-CH _3), 39.4 (C-3 '), 52.5 (C-6 '), 60.6 (C-4'), 61.5 (C-5 '), 69.5 (C-2'), 72.8 (C-4), 74.2 (C-5), 122.9 (C-2), 123.8 ( C-6), 133.0 (C-7), 141.9 (C-3), 165.1 (C-1), 169.0, 169.7 and 2 × 169.9 (4 × acetyl-C = O), 170.4 (C-1 ') ppm.

g) Musacin D (5 - ((S) -1-hydroxy-but-2-enyl) -2,5-dihydrofuran-2-one)

Chromatography of fraction I (148 mg) on silica gel (column 3 × 40 cm, EA / hexane = 4: 1) initially gives 56 mg of a crude product of muscacin D (1). 28 mg (3.0 mg / l) of the muscazin D are isolated after two separate separations on Sephadex LH 20 (column 3 × 100 cm, MeOH and column 3 × 100 cm, CHCl ₃ ) as a white solid.

C ₈ H ₁₀ O ₃ (154.1)

MP: 68 ° C.
DCI-MS (200 eV): m / e = 173 (100%, M + NH ₃ + H +).
El-MS (70 eV): m / e = 136 (0.4%, MH ₂ O), 84 (16%, M + HC ₄ H ₇ O), 71 (100%, C ₄ H ₇ O), 53 ( 18%, C ₄ H ₅ ).
IR: δ = 3420s, 3100w, 2950m, 2910m, 2890m, 1760m, 1740s, 1670w, 1600m, 1450w, 1335m, 1260w, 1170m, 1090m, 840m cm⁻ ¹ .

UV: (MeOH) λ _max (ε): 206 (8300)
(MeOH + HCl) λ _max (ε): 206 (8700)
(MeOH + NaOH) λ _max (ε): 210 (5200), 323 (9500)
CD: (MeOH) λ _extr. ([R] ²² ): 227 (27 800) Rotation: [α] = +182.0 (c = 0.91, CHCl ₃ )

¹ H-NMR (200 MHz, CDCl ₃ ): ε = 1.75 (ddd, J = 6.5, 1.5 and 1, 8-CH ₃ ), 2.67 (s, br, 5-OH), 4.40 (dd, J = 6.5 and 4.5, 5-H), 5.01 (ddd, J = 4.5, 2 and 1.5, 4H), 5.52 (ddq, J = 15, 6.5 and 1.5, 6H), 5.88 (dqd, J = 15, 6.5 and 1, 7H), 6.19 (dd, J = 6 and 2, 2-H), 7.52 (dd, J = 6 and 1.5, 3-H) ppm.

¹³ C-NMR (50.3 MHz, CDCl _3): δ = 17.8 (7, C-8), 72.1 (d, C-5), 85.7 (d, C-4), 122.9 (d, C-2), 127.5 (d, C-6), 130.8 (d, C-7), 153.2 (d, C-3), 172.9 (s, p-1) ppm.

h) Musacin F (4-hydroxy-5- (1-hydroxy-but-2-enyl) -tetrahydrnfuran-2-one)

By chromatography on silica gel (column 2 × 30 cm, EA) is obtained Fraction IV (142 mg) 9 mg Musacin F (1.0 mg / l) in the form of a colorless oil.

C ₈ H ₁₂ O ₄ (172.2)
El-MS (70 eV): m / e = 172 (3%, M), 101 (17%, MC ₄ H ₇ O), 84 (44%, M + HC ₄ H ₇ OH ₂ O), 71 ( 100%, C ₄ H ₇ O).
IR (cm⁻ ¹ ): = 3420 br, 2930 w, 17805, 1585 in, 1200 in, 1100 w, 1060 w, 970 w.

UV: final absorption

Rotation: [α] = +10.28 (c = 0.32, CHCl ₃ )
¹ H-NMR (200 MHz, CD ₃ OD): δ = 1.74 (ddd, J = 6.5, 1.5 and 1.5, 8-CH ₃ ), 2.30 (dd, J = 18 and 1.5, 2-H _a ), 2.89 (dd, J = 18 and 7, 2H _b ), 4.24 (m, 5-H), 4.29 (dd, J = 3 and 1.5, 4-H), 4.45 (ddd, J = 7, 1.5 and 1.5, 3 -H), 5.53 (ddq, J = 15.5, 6 and 1.5, 6-H), 5.86 (dqd, J = 15.5, 6.5 and 1.5, 7-H) ppm.
¹³ C-NMR (50.3 MHz, CD ₃ OD): δ = 18.1 (q, C-8), 39.5 (t, C-2), 68.0 (d, C-3), 72.7 (d, C-5) , 92.4 (d, C-4), 130.0 (d, C-6), 130.0 (d, C-7), 179.1 (5, C-1) ppm.

i) Musacine G, H, I, J and K

From fraction IV (383 mg) are purified by chromatography on silica gel (Column 4.5 × 30 cm, EA / MeOH = 9: 1) 150 mg (16.1 mg / l) in the form of a yellowish oil, wherein a mixture of five components is present. to Separation is derivatized.

acetylation

40 mg of the muscacines G to K are dissolved in 10 ml of acetic anhydride and admixed with 5 mg of DMAP. After 2.5 hours of stirring at room temperature, it is hydrolyzed with 10 ml of ice-water and extracted 3 × 10 in CHCl ₃ . The combined organic phases are washed 3 times with 10 ml of aqueous NaHCO ₃ solution and 1 × with 10 in H ₂ O and the entire aqueous phase is extracted again with 10 ml of CHCl ₃ . The organic phase is dried over Na ₂ SO ₄ , filtered from the drying agent and concentrated in vacuo. This gives 75 mg of crude product which is chromatographed on silica gel (column 2 × 45 cm, EA / Hex 2: 1). Thus 19.3 mg of a triacetate mixture of the muscacines G and H and 14.4 mg of a tetraacetate mixture of the muscacines I, J and K can be separated off.

k) Musacin G and H triacetates

C ₁₉ H ₂₄ O ₁₀ (412.4)

DCI-MS (200 eV): m / e = 430 (100%, M + NH ₃ + H +).
El-MS (70 eV): m / e = 412 (2%), 370 (2%), 326 (15%), 268 (8%), 242 (4%), 157 (20%), 152 ( 41%), 110 (20%), 43 (100%, C ₂ H ₃ O).

SM 450 A triacetate

¹ H-NMR (200 MHz, CDCl ₃ ): δ = 1.23 (d, J = 6.5, 8-CH ₃ ), 2.05 to 2.20 (3 x s, 3 x acetyl-CH ₃ ), 3.15 (m, 3 -H), 4.15 to 4.55 (v, 4'- and 5'-H), 5.10 (m ,, J = 6.5 and 4, 7-H), 5.45 (ddd, J = 6.5, 4 and 1, 6- H), 5.63 (d, J = 8.5,
2-H), 5.90 (v, 5-H), 5.96 (v, 3-H), 6.07 (m, J = 15.5, 6.5 and 0.5, 5-H), 6.84 (ddd, J = 15.5, 5 and 1.5, 2-H) ppm.

¹³ C-NMR (50.3 MHz, CDCl _3): δ = 20.4 (q, C-8), 20.00 to 21.3 (3 × q, 3 x Acetyl-CH _3), 37.3 (d, C-3 '), 61.1 (t, C-5 '), 67.5 (d, C-2'), 67.7 (t, C-4 '), 69.5 (d, C-7), 74.4 (d, C-4), 121.0 (i.e. , C-2), 131.3 (d, C-6), 136.0 (d, C-5), 145.1 (d, C-3), 165.1 (5, C-1), 169.5 to 170.2 (3 x s, 3x acetyl-C = O), 171.4 (s, C-1 ') ppm.

Musacin H triaceate

¹ H-NMR (200 MHz, (200 MHz, CDCl ₃ ): δ = 1.32 (d, J = 6.5, 8-CH ₃ ), 2.05 to 2.20 (3 x s, 3 x acetyl-CH ₃ ), 3.15 ( m, 3'-H), 4.15 to 4.55 (v, 4'- and 5'-H), (td, J = 6.5 and 1.5, 7-H), 5.37 (+ d, J = 6.5 and 1.5, 7 -H), 5.63 (d ₁ J = 8.5, 2'-H), 5.78 (dd, J = 5.5 and 0.5, 6-H), 5.94 (m ,, J = 16, 2-H), 6.0 (v , 5-H), 6.40 (ddd, J = 15.5, 11 and 1.4-H), 7.26 (dd, J = 12 and 4.5, 3-H) ppm.

¹³ C-NMR (50.3 MHz, CDCl _3): δ = 15.2 (q, C-8), 20.0 to 21.3 (3 × q, 3 x Acetyl-CH _3), 37.3 (d, C-3 '), 61.0 (t, C-5 '), 67.6 (d, C-2'), 67.8 (t, C-4 '), 70.3 (d, C-7) 71.8 (d, C-6), 121.6 (d, C-2), 126.1 and 126.2 (d, C-4), 134.5 and 134.6 (d, C-5), 144.2 (d, C-3), 165.9 (s, C-1), 169.5 to 170.2 (3 × s, 3 × acetyl-C = O), 171.6 (s, C-1 ') ppm.

l) Musacin I, J and K tetraacetates

C ₂₂ H ₃₀ O ₁₂ (486.5)

DCI-MS (200 eV): m / e = 504 (10%, M + NH ₃ + H +).
El-MS (70 eV): m / e = 486 (0.6%), 455 (0.8%), 427 (100%), 400 (8%), 353 (10%), 311 (5%), 231 ( 76%), 189 (5%), 152 (4%), 110 (6%), 43 (32%, C ₂ H ₃ O).
¹ H-NMR (200 MHz, CDCl _3): δ = 1.22 (d, J = 6.5), 1:32 (d J = 6.5.), 2.04 2.05 2.06 2.10 2.11 2.13 2.16 2.19 (8 × s, 8 × acetyl-CH _3), 2.79 m, 2.97 m, 3.78 s, 4.19 m, 4:52 m, 5.10 m, 5.23 (dd, J = 4 and 1), 5:42 m, 5.83 m, 6.40 m, 6.87 m , 7.25 in ppm.

¹³ C-NMR (50.3 MHz, CDCl _3): δ = 15.2 (q), 20.0 (q), 20.5 (q), 20.6 (q), 20.7 (q), 21.0 (q), 21.0 (q), 21.1 (q), 21.3 (q), 39.4 (d), 39.5 (d), 4.10 (d), 52.5 (d), 60.7 (t), 61.3 (t), 61.4 (t), 61.5 (t), 66.8 (t), 68.8 (d), 69.5 (d), 69.6 (d), 70.3 (d), 71.8 (d), 74.4 (d), 121.3 (d), 122.0 (d), 126.2 (d), 131.4 (d), 134.6 (d), 135.7 (d), 143.8 (d), 144.7 (d), 165.3 (s), 166.0 (s), 169.1 (s), 169.1 (s), 169.5 (s), 169.9 (s), 170.1 (s), 170.1 (s), 170.3 (s), 170.5 (s), 171.6 (s).

m) esterification of Musacin D with (R / S) -α-phenylbutyric acid

Musacin D is reacted with one enantiomer of α-phenylbutyric acid. For this purpose, 6 mg of Musacin D are dissolved in 10 ml of CH ₂ Cl ₂ and treated with 8 mg of DCC, 5 mg (DMAP) and 64 mg of α-phenylbutyric acid. It is stirred for 2.5 hours at room temperature and the reaction is then stopped by adding 10 ml of MeOH. The reaction mixture is concentrated in vacuo and purified on silica gel (column 2 × 45 cm, CHCl ₃ / MeOH = 98: 2). This gives 3.4 mg (29%) of the ester A from the reaction with (R) -α-phenylbutyric acid and 1.7 mg (15%) of the ester B from the corresponding reaction with (S) -α-phenylbutyric acid.

5 - ((S) -1- (R) -2-phenylbutyrnxy) -but-2-enyl) -2,5-dihydrofuran-2-one (A)

C ₁₈ H ₂₀ O ₄ (300.4)

El-MS (70 eV): m / e = 300 (6%, high resolution calculated for C ₁₈ H ₂₀ O ₄ and found 300.1361, M +), 217 (4%, MC ₄ H ₃ O ₂ ), 147 (10% , C ₁₀ H ₁₁ O), 119 (100%, C ₉ H ₁₁ ), 91 (94%, C ₇ H ₇ ).

¹ H-NMR (200 MHz, CDCl ₃ ): δ = 0.89 (t, J = 7.5, 4'-H), 1.62 (ddd, J = 6.5, 1.5 and 1, 8-CH ₃ ), 1.81 (m, 3'-H _a ), 2.09 (m, 3'-H _b ), 3.44 (t, J = 7.5, 2'-H), 5.06 (ddd, J = 4, 2 and 1.5, 4-H), 5.30 (ddq, J = 15, 7 and 1.5, 6-H) ₁ 5.48 (dd, J = 7 and 4, 5-H), 5.63 (dqd, J = 15, 6.5 and 1, 7-H), 6.18 (6.18) dd, J = 5.5 and 2, 2-H), 7.28 (dil., 3-H and phenyl-H) ppm.

5 - ((S) -1 - ((S) -2-phenylbutyroxy) -but-2-enyl) -2,5-dihydrofuran-2-one (B)

C ₁₈ H ₂₀ O ₄ (300.4)

El-MS (70 eV): m / e = 300 (4%, high resolution calcd for C ₁₈ H ₂₀ O ₄ and found 300.1361, M⁺), 217 (3%, MC ₄ H ₃ O _2), 147 (8 %, C ₁₀ H ₁₁ O), 119 (100%, C ₉ H ₁₁ ), 91 (97%, C ₇ H ₇ ).

¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.89 (t, J = 7.5, 4'-H), 1.71 (dd, J = 6.5 and 1, 8-CH ₃ ), 1.81 (m, 3 ') -H _a ), 2.09 (m, 3'-H _b ), 3.45 (t, J = 7.5, 2'-H), 4.95 (ddd, J = 4.5, 2 and 1.5, 4-H), 5.39 (dil , 5-H), 5.41 (ddq, J = 14.5, 7.5 and 1, 6-H), 5.82 (dqd, J = 14.5, 6.5 and 1.5, 7-H), 6.03 (dd, J = 6 and 2 , 2-H), 7.08 (dd, J = 6 and 1.5, 3-H), 7.30 (m, phenyl-H) ppm

n) Acetylation of Musacin A

15 mg of Musacin A are dissolved in 5 ml of acetic anhydride, combined with 5 mg of N, N-dimethylaminopyridine and stirred for 1 hour at room temperature. The reaction mixture is hydrolyzed with 5 ml of ice-water and extracted 3 × with 5 ml of CHCl ₃ . The combined organic phases are washed 3 times with 5 in aqueous NaHCO ₃ solution and 1 × with 5 ml H ₂ O. The combined aqueous phases are shaken again with 5 ml of CHCl ₃ and the entire organic phase dried over Na ₂ SO ₄ . After filtration of the drying agent is concentrated in vacuo and receives 34 mg of crude product. Chromatography on silica gel (column 2 × 30 cm, EA / hexane = 4: 1) gives 12.8 mg (51%) of Musacin A tetraacetate as a colorless oil.

4.5-diacetoxy-octa-2,6-dien acid 2,3-diacetoxy-propyl

C ₁₉ H ₂₆ O ₁₀ (414.5)

El-MS (70 eV): m / e = 344 (2%, MC ₄ H ₆ O), 302 (30%, MC ₄ H ₆ OC ₂ H ₂ O), 242 (10%, MC ₄ H ₆ OC ₂ H ₂ OC ₂ H ₄ O ₂ ), 239 (2%, MC ₇ H ₁₁ O ₅ ), 200 (8%, MC ₄ H ₆ O- 2 × C ₂ H ₂ OC ₂ H ₄ O ₂ ), 159 (86%, C ₇ H ₁₁ O ₄ ), 126 (80%, C ₆ H ₆ O ₃ ), 117 (4%, C ₅ H ₉ O ₃ ), 113 (14%, C ₆ H ₉ O ₂ ) , 84 (36%, C ₄ H ₄ O ₂ ), 57 (4%, C ₃ H ₅ O), 43 (100%, C ₂ H ₃ O).
IR: δ = 3450 w, 2930 br, 1745 s, 1665 w, 1370 w, 1370 in, 12305, 1170 m, 1050 m, 970 m cm⁻ ¹

¹ H-NMR (200 MHz, CHCl ₃ ): δ = 1.73 (dd, J = 6.5 and 1, 8-CH ₃ ), 2.06, 2.08, 2.10 and 2.12 (4 × s, 4 × acetyl-CH ₃ ), 4.17 (dd, J = 12 and 5.5, 1'-H _a ), 4.31 (verd., 3'-H _a and 3'-H _b ), 4.37 (dd, J = 12 and 4.5, 1'-H _b ), 5.29 (tt, J = 5 and 4.5, 2'-H), 5.42 (dil., 5-H and 6-H), 5.57 (ddd, J = 5, 3.5 and 1.5, 4-H), 5.85 (dqd, J = 14.5, 6.5 and 2.5, 7-H), 6.02 (dd, J = 15.5 and 1.5, 2-H) ₁ 6.87 (dd, J = 15.5 and 5, 3-H) ppm.

¹³ C-NMR (50.3 MHz, CDCl ₃ ): δ = 17.9 (q, C-8), 20.7, 20.9, 20.9 and 21.0 (4 x q, 4 x acetyl-CH ₃ ), 62.3 (t ₁ C-3 ;), 62.5 (t, C-1 '), 69.0 (q, C-2') ₁ 72.9 (d, C-4), 74.3 (q, C-5), 122.9 (q, C-2), 123.9 (q, C-7), 141.9 (q, C-3), 165.0 (S, C-1), 169.6, 169.8, 170.0 and 170.4 (4 x S, 4 x acetyl-C = O) ppm.

o) Acetylation of Musacin B

15 mg of Musacin B are dissolved in 5 ml of acetic anhydride, admixed with 5 mg of DMAP and stirred at room temperature for 2 hours. It is then hydrolyzed with 5 ml of ice-water and extracted 3 × with 5 ml of CHCl ₃ . The combined organic phases are washed 3 × with 5 ml aqueous NaHCO ₃ solution and 1 × with H ₂ O, and the combined aqueous phases are extracted again with 5 ml CHCl ₃ . The entire organic phase is dried over Na ₂ SO ₄ , filtered from the drying agent and concentrated in vacuo. This gives 22 mg of crude product, from which after chromatography on silica gel (column 1 × 30 cm, CHCl ₃ / MeOH = 31: 1) and preparative thin-layer chromatography (eluent CHCl ₃ / MeOH = 31: 1) 2.8 mg (13%) of the Musacin B triacetate be obtained as a colorless oil.

4,5-diacetoxy-octa-2,6-dienoic acid 4-acetoxy-5-oxo-tetrahydrofuran-3-y l methylester

C ₁₉ H ₂₄ O ₁₀ (412.4)

El-MS (70 eV): m / e = 300 (0.3%, M + HC ₄ H ₇ -C ₂ H ₂ O) ₁ 215 (0.4%, MC ₄ H ₇ O- 3 × C ₂ H ₂ O) , 84 (4%, C ₄ H ₄ O ₂ ), 71 (4%, C ₄ H ₇ O), 43 (100%, C ₂ H ₃ O).

¹ H-NMR (500 MHz, CDCl ₃ ): δ = 1.71 (dd, J = 6.5 and 1, 8-CH ₃ ), 2.02, 2.05 and 2.08 (3 x s, 3 x acetyl-CH ₃ ), 2.99 ( m ,, 3'-H), 4.11 (dd, J = 9.5 and 9.5, 4'-H _a ), 4.28 (dd, J = 11.5 and 6, 5'-H _a ), 4.36 (dd, J = 11.5 and, 5'-H 4.5 _b), 4:51 (dd, J = 9.5 and 8.5, 4'-H _b), 5:39 (dil., 5-H and 6-H), 5:41 (d, J = 9.5, 2 '-H), 5.54 (ddd, J = 5.5, 3.5 and 1.5, 4-H), 5.82 (dqd, J = 14.5, 6.5 and 2.5, 7-H), 5.99 (dd, J = 15.5 and 1.5, 2 -H), 6.86 (dd, J = 15.5 and 5.5, 3-H) ppm.

p) Biological Activity of Musacins Antiviral activity

The antiviral activity of the compounds according to the invention is determined in vitro Try tested. For this purpose, the compounds of the invention in various dilutions to cell cultures of HeLa and Vero cells in Given microtiter plates. After 3 hours, the cultures with various human pathogenic viruses (eg adenovirus herpes simplex I, Myxovirus, Picorna rhinovirus 2). 48 to 72 hours after infection the therapeutic success is microscopic and cytopathogenic effect after neutral red images (color test according to Finter) determined photometrically (Finter, N.B., In "Interferones", N.B. Finter et al., North Holland Publishing Co., Amsterdam, 1966). The minimum concentration at which about half of the infected cells show no cytopathogenic effect is considered minimal Hemine concentration (MIC) is considered.  

The results are summarized in the following table:

Claims (13)

1. Compound of general formula I. in the independently of each other
R ¹ = hydrogen, R ¹ and R ⁴ together form a bond,
R ² and R ^{3 are} hydrogen or R ² and R ³ together represent a chemical bond,
R ^{4 is} hydrogen or R ⁴ and R ¹ together form a bond or
R ⁴ and R ⁵ together represent a chemical bond,
R ⁵ , R ⁶ and R ^{7 are} hydrogen or R ⁵ and R ⁴ , R ⁵ and R ⁶ or R ⁶ and R ⁷ together represent a chemical bond or
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , which may be the same or different, a saturated or unsaturated, branched or unbranched or cyclic C ₁ -C ₂₀ alkyl ester, a substituted or unsubstituted aromatic Ester, a heteroalkyl ester or an alkyl or arylsulfonic acid ester. 2. Compound of formula I for use as a medicament. 3. A pharmaceutical composition containing the compound of the formula I and one or more pharmaceutical carriers. 4. A process for the preparation of a compound of general formula I according to Claim 1, characterized in that Streptomyces sp. in a nutrient medium is cultured until a compound of formula I in the Culture accumulates and this is isolated from the nutrient medium. 5. The method according to claim 4, characterized in that Streptomyces griseoviridis is cultured in a nutrient medium. 6. The method according to claim 4, characterized in that Streptomyces griseoviridis DSM 7429 is cultured in a nutrient medium. 7. The method according to one or more of claims 4 to 6, characterized characterized in that the nutrient medium 0.2 to 5% soybean meal and 0.2 to Contains 50% mannitol, in each case based on the weight of the total Nutrient solution. 8. The method according to one or more of claims 4 to 7, characterized in that the nutrient medium 0.5 to 6% glycerol, 0.1 to 4% soybean flour, 0.01 to 6% K ₂ HPO ₄ , 0.01 to 6% NaCl, 0.01 to 3% MgSO ₄ , in each case based on the weight of the entire nutrient solution. 9. The method according to one or more of claims 4 to 8, characterized characterized in that the cultivation at 18 to 50 ° C and in your pH range is between 6 and 8.   10. Use of a compound of formula I according to claim 1 as pharmacologically active substance. 11. Use according to claim 10, characterized in that a Compound of formula I is used as an antiviral agent. 12. Use according to claim 10, characterized in that a Compound of formula I is used as an anthelminthic active ingredient. 13. Streptomyces griseoviridis DSM 7429 and its variants and Mutants, as far as these variants and mutants are a compound of Produce formula I.