CA2057651A1 - Agistatins, novel metabolites from fusarium sp., a process for their preparation, and their use - Google Patents
Agistatins, novel metabolites from fusarium sp., a process for their preparation, and their useInfo
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- CA2057651A1 CA2057651A1 CA002057651A CA2057651A CA2057651A1 CA 2057651 A1 CA2057651 A1 CA 2057651A1 CA 002057651 A CA002057651 A CA 002057651A CA 2057651 A CA2057651 A CA 2057651A CA 2057651 A1 CA2057651 A1 CA 2057651A1
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/08—Bridged systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P3/06—Antihyperlipidemics
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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Abstract
Abstract of the disclosure Agistatins, novel metabolites from Fusarium sp., a process for their preparation, and their use With the aid of a Fusaxium strain, it is possible to produce novel metabolites of the formula I below in which R1 is hydrogen or hydroxyl, R2 is hydroxyl or an oxo group, R3 is methoxy or hydroxyl or, together with R1, forms an ether bridge, and R4 is hydrogen or hydroxyl, R5 is hydrogen or, together with R4, is an additional bond, and R6 is hydrogen or, together with R3, is an additional bond.
In particular, the metabolites are to be employed for inhibiting cholesterol biosynthesis.
In particular, the metabolites are to be employed for inhibiting cholesterol biosynthesis.
Description
5 ~ æ ~
HOECHST ARTIENGESELLSCHAFT HOE 90/F 37~ Dr.Sl/pe Description Agistatins, novel meta~olites from Fusarium 8p., a process for their pr~paration, and their use It has been found that Fusaxium strains can produce novel metabolites, the agi~ta~ins, which have pharmacological and theref ore therapeutic activity and can be employed particularly advantageou~ly ~8 cholesterol biosynthesi~
inhibitors having corresponding pha~m~cological utility.
The invention therefore relates to:
1. A process for the preparation of the com~ounds of the formula I
~5 ~ I
in which is hydrogen or hydroxyl r R2 is hydroxyl or an oxo group, R3 is methoxy or hydroxyl or, together with Rl, forms an eth~r bridge, and R4 is hydrogen or hydxoxyl, R5 is hydrogen or, together with R4, is an additional bond, and R5 is hydrogen or, together with R3, i an additional bond , which comprises culturing Fusarium DSM 5983 in a nutrient medium until the compounds of the formula I
are accumulated in the culture.
~7~ ~
HOECHST ARTIENGESELLSCHAFT HOE 90/F 37~ Dr.Sl/pe Description Agistatins, novel meta~olites from Fusarium 8p., a process for their pr~paration, and their use It has been found that Fusaxium strains can produce novel metabolites, the agi~ta~ins, which have pharmacological and theref ore therapeutic activity and can be employed particularly advantageou~ly ~8 cholesterol biosynthesi~
inhibitors having corresponding pha~m~cological utility.
The invention therefore relates to:
1. A process for the preparation of the com~ounds of the formula I
~5 ~ I
in which is hydrogen or hydroxyl r R2 is hydroxyl or an oxo group, R3 is methoxy or hydroxyl or, together with Rl, forms an eth~r bridge, and R4 is hydrogen or hydxoxyl, R5 is hydrogen or, together with R4, is an additional bond, and R5 is hydrogen or, together with R3, i an additional bond , which comprises culturing Fusarium DSM 5983 in a nutrient medium until the compounds of the formula I
are accumulated in the culture.
~7~ ~
2. A use of the compound of the formula I a~ choles-terol bio~ynthesis inhibitors, and pharmacological utilization thereof.
The following text will describe the invention in detail, in particular in its preferred embodLmen~s. The ~nvention is furthermore determined by the con~ents of the patent claLms.
The compounds according to the invention ar~ preferably produced with ~usarium sp. DSM 5983. The strain was isolated from a soil sample from Hawaii and deposi~ed on June 6, 1990, under the abovementioned number, at the Deutsche Sammlung von Nikroorganismen ~German Collection of Microorganisms] in compliance with the provi~ions of the Budapest Convention.
The strain Fusarium sp. DSM 5983 has a white aerial mycelium and white conidia. It forms ~he macro- and microconidia and chlamydospores which are characteristic of Fusaria.
In a nutrient solution which contains a carbon source and a nitrogen sour~e as well as the con~entional inorganic salts, Fusarium sp., preferably DSM 5983, produce~ the compounds of the formula I. Instead of strain DSM 5983, it is, of course, also possible to 4mploy the mutant~ and variants thereof, a~ long a~ they synthesize this ~om-pound. Such mutants can be produced in a manner known per se using physical means, for e~ample irr~diation, such as with ultraviolet rays or ~-ray~, or chemi-~al mutagens, for example ethyl methanesulfonate (E~S), 2-hydroxy methoxybenzophenone ~OB~ or N-methyl-N~-nitro-N-nitroso-guanidine (MNNG).
Preferred car~on ~ources which are 5uitable for the aerobic fermentation are carbohydrate and ~ugar alcohol~
2~7~ ~
~ 3 --which can be assimilated, such as glucose, lactose or D-mannitol, and carbohydrate-containing natural product~, such as malt extract. The following are suitable as nitrogen-containing nutrients: amino acids, peptides and proteins, and their degradation produc~s such as peptones or tryptones, furthermore meat extracts, ground seeds, for example of maize, wheat, beans, soybeans or of the cotton plant, distillation residue~ of alcohol produc-tion, meat meals or yeast extracts, but also ammonium salts and nitrates. Inorganic ~alt6 which the nutrient solution may contain are, for example, chlorides, carbo-nates, sulfates or phosphates of the alkali metals or of the alkaline earth metals, iron, zinc, cobalt and manganese.
~he compounds of the formula I are particularly readily formed in a nutrient solution which contains approxi-mately 0.2 to 5 ~, preferably 1 to 4 %, of malt extract and 0.02 to 0.5 %, preferably 0.1 to 0.4 ~, of yeast extract, as well as 0.2 to 5.0 %, preferably 0.5 ~o 2 %, of glucose and 0.01 to 0.1 ~, preferably 0.04 to 0.04 to 0.06 %, of (NH4)2HPO4, in each case based on the weight of the entire nutrient solution. Culturing is carried out under aerobic conditions, that i~ to say, for example, submerse conditions with shaking or stirring in shaker ~5 flasks or fermenters, if necessary while passing in air or oxygen. Culturing can be carried out in a temperature range from approximately 15 to 30C, preferably at approximately 20 to 30C, in particular at 23 to 28C.
The pH range should be between 1 and 7, advantageously between 2.5 and 4.5. In general, the microorganism is cultured under these conditions over a period of 24 to 300 hours, preferably 3Ç to 140 hours.
It is advantageous to carry out the culture in several steps, i.e. one or more precultures are first e~tablished in a liquid nutrient medium, and the actual production medium, the main culture, is then inoculated with these, for example in a ratio by volume of 1:10. The preculture r,~
is obtained, for example, by inoculating a nutrient solution with a sporulating mycelium and allowing it to grow for approximately 36 to 120 hours, preferably from 48 to 72 hours. The sporulating mycelium can be obtained for e~ample by allowing the strain to grow for apprvxi-mately 3 to 40 days, preferably 4 to 10 days, on a solid or liquid medium, for example yeast/malt agar or potato/
dextrose agar.
The course of the fermentation can be monitored with the aid of the pH of the culture or of ~he volume of myce-lium, and also by chromatographic methods, for example thin-layer chromatography or high pressure liquid chroma-tography, or by assays of the biological activity. The compounds of the formula I are contained both in the mycelium and in the culture filtrate.
~he compounds mentioned are isolated from the culture medium by known methods, taking into account the chemi-cal, physical and biological properties of the products.
For an assay of the metabolite concentration in the culture medium or in the individual isolation steps, it is possible to use thin-layer chromatography, for example on silica ~el using butanol/glacial acetic acid/water or ethyl acetate/methanol/water mixtures as the eluent. In the case of separation by means of thin-layer chromato-graphy, detection can be effected, for example, bystaining reagents such as anis~ldehyde, or by bioassays, for example as a cholesterol biosynthesis inhibitor, the amount of substance formed advantageously being compared with a calibration solution.
To isolate the compounds I, culture liquor and mycelium are first extracted with non-polar organic solvents, for example n-hexane, petroleum ether or halogenated hydro-carbons such as, for example, chloroform etc., so as to remove the non-polar impurities. This is followed by extraction with a more polar organic solvent, for example lower alcohols, acetone and/or ethyl acetate, and also - 5 _ mixtures of these solvents.
The isolation of the pure compounds of the formula I is carried out on sui~able materials, preferably, for example, on silica gel, aluminum oxide, ion exchangers or adsorber resins, followed by elution with organic, polar solvents or solvent mixtures, fo.r example alkyl acetates, mixtures of alkyl acetates with a lower alkanol, chloro-form or methylene chloride, or mixtures of these 801vent8 with lower alkanols, if appropriate also with water, or a pH or salt gradient which is ~uitable for ion-exchanger resins, for example sodium chloride or tris(hydroxy-methyl)aminomethane HCl (tris buffer), and combination of the biologically active fractions.
In the solid state and in solutions in the pH range between 1 and 9, and particularly 3 and 7, the compounds of the formula I are stable and can therefore be incorporated in pharmaceutical preparations.
The compounds according to the invention can be used as lipid regulators, in particular for inhibiting the biosynthesis of cholesterol.
The compounds of the formula I can be used for the prophylaxis and treatment of diseases which are based on an increased cholesterol level, in particular of coronary heart diseases, arteriosclerosis and sLmilar diseases.
The use also relates to pharmaceutical preparations of the compounds of the formula I.
In the preparation of pharmaceuticals, it is also pos-sible to use pharrnaceutically acceptable additives, such as diluents and/or excipients, in addition to the active substance. These are underskood as meaning physiologic-ally acceptable substances which, after having been mixed with the active substance, bring the latter into a form which is suitable for administration.
Examples of suitable solid or li~uid pharmaceutical preparation forms are tablets, coated tablets, powders, capsules, suppositories, syrups, emulsions, suspensions, drops or solutions for injection, as well as preparations with a protracted release of active substance. Examples of excipients or diluents which are frequently used are various sugars or types of starch, cellulose derivatives, magnesium carbonate, gelatin, animal and vegetable oilR, polyethylene glycols, water or other suitable solvents, and also buffers which contain water and which can be made isotonic by adding glucose or salts.
In addition, other additives which can be used in the pharmaceutical preparations according to the invention are, if appropriate, surface-active agents, colorants and flavorings, stabilizers and al~o preservatives. It is also possible to use pharmacologically acceptable poly-meric excipients, for example polyphenylpyrrolidone, or other pharmaceutically acceptable additives, for example cyclodextrin or polysaccharides. In particular, the compounds can also be co~bined with additives which bind bile acid, in particular non-toxic/ basic anion exchan-gers which cannot be absorbed in the gastrointestinal tract.
The preparations can be administered orally, rectally or parenterally. The preparations can preferably be prepared in dosage units; examples of suitable dosage units are, in particular, tablets, capsules or suppositories. Each dosage unit, in particular for oral administration, can contain up to 1000 mg, but preferably 10 to 100 mg, of ~0 the active ingredient. However, it is also possihle to use dosage units which are above or below this amount and which, if required, are to be divided or multiplied prior to administration.
If appropriate, the dosage units for oral administration can be microencapsulated so as to slow down release, or extend it over a longer period, for example by coating or embedding the particula~e active substance in suitable polymers, waxes or the like.
Parenteral administration can be effected using liquid dosage forms, such as sterile solutions and suspensions which are intended for in~ramuscular or subcutan~ous injection. Dosage forms of this type are prepared by dissolving, or suspending, a measured amount of active substance in a suitable physiologically acceptable diluent, for example an aqueous or oily medium, and sterilizing the solution, or ~he suspension, if appropriate with the concomitan~ use of suitable stabilizers, emulsifiers and/or preservatives and/or antioxidants.
The preferred administration form, in particular with regard to long-term therapy, i8 the oral adminis~ration form, which facilitates the preven~ion and therapy of the abovementioned diseases substantially.
The pharmaceutical preparations are prepared by generally customary processes. The dosage schedule may depend on the type, age, weight, sex and medical condition of the patient or person at risk.
The examples which follow will describe the invention in greater detail. Percentages are by weight, unless other-wise defined.
Examples:
1. a) Preparation of a spore suspension of the producer strain:
100 ml of nutrient solution (60 g of malt extract, 2 g of yeast extract, 10 g of glucose, 0.5 g of (NH4)2HPO4 in 1 1 of tap water, pH before sterilization 6.0) in a ~terile 500 ml Erlenmeyer flask are inoculated with strain DSM
5983 and the culture is incubated on a rotary shaker for 72 hours at 25C and 140 rpm. In a sterile 500 ml Erlenmeyer flask, 20 ml of culture liquid are subsequently uniformly distributed in the nutrient mQdium (potato infusion 4.0 g/l [infusion of 200 g of potatoes], 20.0 g/l D-glucose, pH before sterilization 5.6~, to which 15 g of agar/l had additionally been add2d for solidification, and the mixture is decanted. The cultures are incubated for 10 to 14 days at 25C. The spores of one flask which have been formed after this time are rinsed with 500 ml of demineralizad water containing one drop o~ a commercially available non ionic surfactant (for example Triton0 X 100, manufactured by Serva), and either reused immediately or stored in 50 % glycerol at 5 b) Production of a culture, or precul~ure, of theproducer strain in an Erlenmeyer flask A sterile 500 ml Erlenmeyer flask containing 100 ml of the nutrient solution described under a) is inoculated with a culture which has been yrown on agar slants or with 0.2 ml of spore suspension, and the mixture is incubated on a rotary shaker at 140 rpm and 25C. Maximum production of the compounds of the formula I is attained after approximately 120 hours. To inoculate 10 and 100 l fermenters, it is sufficient to use a 72 hour old submerse culture (inoculum approximately 5 %) from the same nutrient ~olution.
2. Preparation of the compounds of the formula I:
A 10 l fermenter is operated under the following condi-tions:
0 Nutrient medium: 20 g/l of malt extract 10 g/l of glucose 2 g/l of yeast extract 0,5 g/l of (NH")2HPO4 pH 6.0 (before sterilization) 5 Incubation time: 120 houra 2~
Incubation temperature: 25C
5tirring speed: 200 rpm Aeration: 2 1 of air/min.
Foam formation can be suppressed by repeatedly adding a few drops of ethanolic polyol solution. Maximum produc-tion takes place after approximately 96 hours. The pH
during harvesting is approximately 3.5. The yields ar~
approximately 80 mg/l of the compounds of the formula I.
The following text will describe the invention in detail, in particular in its preferred embodLmen~s. The ~nvention is furthermore determined by the con~ents of the patent claLms.
The compounds according to the invention ar~ preferably produced with ~usarium sp. DSM 5983. The strain was isolated from a soil sample from Hawaii and deposi~ed on June 6, 1990, under the abovementioned number, at the Deutsche Sammlung von Nikroorganismen ~German Collection of Microorganisms] in compliance with the provi~ions of the Budapest Convention.
The strain Fusarium sp. DSM 5983 has a white aerial mycelium and white conidia. It forms ~he macro- and microconidia and chlamydospores which are characteristic of Fusaria.
In a nutrient solution which contains a carbon source and a nitrogen sour~e as well as the con~entional inorganic salts, Fusarium sp., preferably DSM 5983, produce~ the compounds of the formula I. Instead of strain DSM 5983, it is, of course, also possible to 4mploy the mutant~ and variants thereof, a~ long a~ they synthesize this ~om-pound. Such mutants can be produced in a manner known per se using physical means, for e~ample irr~diation, such as with ultraviolet rays or ~-ray~, or chemi-~al mutagens, for example ethyl methanesulfonate (E~S), 2-hydroxy methoxybenzophenone ~OB~ or N-methyl-N~-nitro-N-nitroso-guanidine (MNNG).
Preferred car~on ~ources which are 5uitable for the aerobic fermentation are carbohydrate and ~ugar alcohol~
2~7~ ~
~ 3 --which can be assimilated, such as glucose, lactose or D-mannitol, and carbohydrate-containing natural product~, such as malt extract. The following are suitable as nitrogen-containing nutrients: amino acids, peptides and proteins, and their degradation produc~s such as peptones or tryptones, furthermore meat extracts, ground seeds, for example of maize, wheat, beans, soybeans or of the cotton plant, distillation residue~ of alcohol produc-tion, meat meals or yeast extracts, but also ammonium salts and nitrates. Inorganic ~alt6 which the nutrient solution may contain are, for example, chlorides, carbo-nates, sulfates or phosphates of the alkali metals or of the alkaline earth metals, iron, zinc, cobalt and manganese.
~he compounds of the formula I are particularly readily formed in a nutrient solution which contains approxi-mately 0.2 to 5 ~, preferably 1 to 4 %, of malt extract and 0.02 to 0.5 %, preferably 0.1 to 0.4 ~, of yeast extract, as well as 0.2 to 5.0 %, preferably 0.5 ~o 2 %, of glucose and 0.01 to 0.1 ~, preferably 0.04 to 0.04 to 0.06 %, of (NH4)2HPO4, in each case based on the weight of the entire nutrient solution. Culturing is carried out under aerobic conditions, that i~ to say, for example, submerse conditions with shaking or stirring in shaker ~5 flasks or fermenters, if necessary while passing in air or oxygen. Culturing can be carried out in a temperature range from approximately 15 to 30C, preferably at approximately 20 to 30C, in particular at 23 to 28C.
The pH range should be between 1 and 7, advantageously between 2.5 and 4.5. In general, the microorganism is cultured under these conditions over a period of 24 to 300 hours, preferably 3Ç to 140 hours.
It is advantageous to carry out the culture in several steps, i.e. one or more precultures are first e~tablished in a liquid nutrient medium, and the actual production medium, the main culture, is then inoculated with these, for example in a ratio by volume of 1:10. The preculture r,~
is obtained, for example, by inoculating a nutrient solution with a sporulating mycelium and allowing it to grow for approximately 36 to 120 hours, preferably from 48 to 72 hours. The sporulating mycelium can be obtained for e~ample by allowing the strain to grow for apprvxi-mately 3 to 40 days, preferably 4 to 10 days, on a solid or liquid medium, for example yeast/malt agar or potato/
dextrose agar.
The course of the fermentation can be monitored with the aid of the pH of the culture or of ~he volume of myce-lium, and also by chromatographic methods, for example thin-layer chromatography or high pressure liquid chroma-tography, or by assays of the biological activity. The compounds of the formula I are contained both in the mycelium and in the culture filtrate.
~he compounds mentioned are isolated from the culture medium by known methods, taking into account the chemi-cal, physical and biological properties of the products.
For an assay of the metabolite concentration in the culture medium or in the individual isolation steps, it is possible to use thin-layer chromatography, for example on silica ~el using butanol/glacial acetic acid/water or ethyl acetate/methanol/water mixtures as the eluent. In the case of separation by means of thin-layer chromato-graphy, detection can be effected, for example, bystaining reagents such as anis~ldehyde, or by bioassays, for example as a cholesterol biosynthesis inhibitor, the amount of substance formed advantageously being compared with a calibration solution.
To isolate the compounds I, culture liquor and mycelium are first extracted with non-polar organic solvents, for example n-hexane, petroleum ether or halogenated hydro-carbons such as, for example, chloroform etc., so as to remove the non-polar impurities. This is followed by extraction with a more polar organic solvent, for example lower alcohols, acetone and/or ethyl acetate, and also - 5 _ mixtures of these solvents.
The isolation of the pure compounds of the formula I is carried out on sui~able materials, preferably, for example, on silica gel, aluminum oxide, ion exchangers or adsorber resins, followed by elution with organic, polar solvents or solvent mixtures, fo.r example alkyl acetates, mixtures of alkyl acetates with a lower alkanol, chloro-form or methylene chloride, or mixtures of these 801vent8 with lower alkanols, if appropriate also with water, or a pH or salt gradient which is ~uitable for ion-exchanger resins, for example sodium chloride or tris(hydroxy-methyl)aminomethane HCl (tris buffer), and combination of the biologically active fractions.
In the solid state and in solutions in the pH range between 1 and 9, and particularly 3 and 7, the compounds of the formula I are stable and can therefore be incorporated in pharmaceutical preparations.
The compounds according to the invention can be used as lipid regulators, in particular for inhibiting the biosynthesis of cholesterol.
The compounds of the formula I can be used for the prophylaxis and treatment of diseases which are based on an increased cholesterol level, in particular of coronary heart diseases, arteriosclerosis and sLmilar diseases.
The use also relates to pharmaceutical preparations of the compounds of the formula I.
In the preparation of pharmaceuticals, it is also pos-sible to use pharrnaceutically acceptable additives, such as diluents and/or excipients, in addition to the active substance. These are underskood as meaning physiologic-ally acceptable substances which, after having been mixed with the active substance, bring the latter into a form which is suitable for administration.
Examples of suitable solid or li~uid pharmaceutical preparation forms are tablets, coated tablets, powders, capsules, suppositories, syrups, emulsions, suspensions, drops or solutions for injection, as well as preparations with a protracted release of active substance. Examples of excipients or diluents which are frequently used are various sugars or types of starch, cellulose derivatives, magnesium carbonate, gelatin, animal and vegetable oilR, polyethylene glycols, water or other suitable solvents, and also buffers which contain water and which can be made isotonic by adding glucose or salts.
In addition, other additives which can be used in the pharmaceutical preparations according to the invention are, if appropriate, surface-active agents, colorants and flavorings, stabilizers and al~o preservatives. It is also possible to use pharmacologically acceptable poly-meric excipients, for example polyphenylpyrrolidone, or other pharmaceutically acceptable additives, for example cyclodextrin or polysaccharides. In particular, the compounds can also be co~bined with additives which bind bile acid, in particular non-toxic/ basic anion exchan-gers which cannot be absorbed in the gastrointestinal tract.
The preparations can be administered orally, rectally or parenterally. The preparations can preferably be prepared in dosage units; examples of suitable dosage units are, in particular, tablets, capsules or suppositories. Each dosage unit, in particular for oral administration, can contain up to 1000 mg, but preferably 10 to 100 mg, of ~0 the active ingredient. However, it is also possihle to use dosage units which are above or below this amount and which, if required, are to be divided or multiplied prior to administration.
If appropriate, the dosage units for oral administration can be microencapsulated so as to slow down release, or extend it over a longer period, for example by coating or embedding the particula~e active substance in suitable polymers, waxes or the like.
Parenteral administration can be effected using liquid dosage forms, such as sterile solutions and suspensions which are intended for in~ramuscular or subcutan~ous injection. Dosage forms of this type are prepared by dissolving, or suspending, a measured amount of active substance in a suitable physiologically acceptable diluent, for example an aqueous or oily medium, and sterilizing the solution, or ~he suspension, if appropriate with the concomitan~ use of suitable stabilizers, emulsifiers and/or preservatives and/or antioxidants.
The preferred administration form, in particular with regard to long-term therapy, i8 the oral adminis~ration form, which facilitates the preven~ion and therapy of the abovementioned diseases substantially.
The pharmaceutical preparations are prepared by generally customary processes. The dosage schedule may depend on the type, age, weight, sex and medical condition of the patient or person at risk.
The examples which follow will describe the invention in greater detail. Percentages are by weight, unless other-wise defined.
Examples:
1. a) Preparation of a spore suspension of the producer strain:
100 ml of nutrient solution (60 g of malt extract, 2 g of yeast extract, 10 g of glucose, 0.5 g of (NH4)2HPO4 in 1 1 of tap water, pH before sterilization 6.0) in a ~terile 500 ml Erlenmeyer flask are inoculated with strain DSM
5983 and the culture is incubated on a rotary shaker for 72 hours at 25C and 140 rpm. In a sterile 500 ml Erlenmeyer flask, 20 ml of culture liquid are subsequently uniformly distributed in the nutrient mQdium (potato infusion 4.0 g/l [infusion of 200 g of potatoes], 20.0 g/l D-glucose, pH before sterilization 5.6~, to which 15 g of agar/l had additionally been add2d for solidification, and the mixture is decanted. The cultures are incubated for 10 to 14 days at 25C. The spores of one flask which have been formed after this time are rinsed with 500 ml of demineralizad water containing one drop o~ a commercially available non ionic surfactant (for example Triton0 X 100, manufactured by Serva), and either reused immediately or stored in 50 % glycerol at 5 b) Production of a culture, or precul~ure, of theproducer strain in an Erlenmeyer flask A sterile 500 ml Erlenmeyer flask containing 100 ml of the nutrient solution described under a) is inoculated with a culture which has been yrown on agar slants or with 0.2 ml of spore suspension, and the mixture is incubated on a rotary shaker at 140 rpm and 25C. Maximum production of the compounds of the formula I is attained after approximately 120 hours. To inoculate 10 and 100 l fermenters, it is sufficient to use a 72 hour old submerse culture (inoculum approximately 5 %) from the same nutrient ~olution.
2. Preparation of the compounds of the formula I:
A 10 l fermenter is operated under the following condi-tions:
0 Nutrient medium: 20 g/l of malt extract 10 g/l of glucose 2 g/l of yeast extract 0,5 g/l of (NH")2HPO4 pH 6.0 (before sterilization) 5 Incubation time: 120 houra 2~
Incubation temperature: 25C
5tirring speed: 200 rpm Aeration: 2 1 of air/min.
Foam formation can be suppressed by repeatedly adding a few drops of ethanolic polyol solution. Maximum produc-tion takes place after approximately 96 hours. The pH
during harvesting is approximately 3.5. The yields ar~
approximately 80 mg/l of the compounds of the formula I.
3. Isolation of the compounds of the formula I:
When the fermentation of DSM 5983 has ended, the culture liquor is filtered with the addition of approximately 2 %
of filter aid (for example Celite~. Working-up can be effected following the diagrams below:
lo ~ ~ ~ 7 ~ ;3~
Working-up/i~olation Diagram 1: Culture supexnatant _ Culture _ Filtration ~-~ Culture supernatant Adsorption on Amberlite XAD-16 (20-25% by volume of the amount applied) _ ~
Washing with water (50 % by volume) Elution with 80 % MeOH
/
Crude product . I , ND
silica gel CHCl3/MeOli ( 50 : 1 ) Sephadex LH-20 in MeOH
Silica gel CHCl3/NeOH/n-hexane (20 : 1 : 5) Compounds of the formula I
Working-up/isolation Diagram 1: Mycelium -r3 ~L
NycelLum I
Ext lction with acetone ~3 I ~
Chromatography on silica gel CHCl3/MeOH (50 : 1) /
Sephadex LH-20 in MeOH
Silica gel CHCl3/MeOH/n-hexane (20 : 1 : 5) Compounds of the formula I
7 ~
When the fermentation of DSM 5983 has ended, the culture liquor is filtered with the addition of approximately 2 %
of filter aid (for example Celite~. Working-up can be effected following the diagrams below:
lo ~ ~ ~ 7 ~ ;3~
Working-up/i~olation Diagram 1: Culture supexnatant _ Culture _ Filtration ~-~ Culture supernatant Adsorption on Amberlite XAD-16 (20-25% by volume of the amount applied) _ ~
Washing with water (50 % by volume) Elution with 80 % MeOH
/
Crude product . I , ND
silica gel CHCl3/MeOli ( 50 : 1 ) Sephadex LH-20 in MeOH
Silica gel CHCl3/NeOH/n-hexane (20 : 1 : 5) Compounds of the formula I
Working-up/isolation Diagram 1: Mycelium -r3 ~L
NycelLum I
Ext lction with acetone ~3 I ~
Chromatography on silica gel CHCl3/MeOH (50 : 1) /
Sephadex LH-20 in MeOH
Silica gel CHCl3/MeOH/n-hexane (20 : 1 : 5) Compounds of the formula I
7 ~
4. Bioassay as cholesterol biosynthesis inhibitors:
a) In-vitro determination:
HEP-G2 cell monolayers in lipoprotein-free nutrient medium are preincubated for one hour with appropriate concentrations of the test substances of the formula I.
The l4C-tagged biosynthesis precursor [14CJsodium acetate is added, and incubation is then continued for 3 hours.
After this r some of the cells are subjected to alkaline hydrolysis after previously adding an internal ~tandard of 3H cholesterol. The lipids of the hydrolyzed cells are extracted with a chloroform/me~hanol mixture. Support cholesterol is added, and this lipid mixture is separat~d by preparative thin-layer chromatography, the cholesterol band is stained and then isolated, and the amount of 14C-cholesterol formed from the 14C-precursor is determined by scintigraphy. In an aliquot of the cells, cell protein was determined, so that the amount of 14C-cholesterol formed from l4C-precursor per mg of cell protein can be calculated per cell unit. To compare the inhibitory action of an added test preparation, the control is used, so that the inhibition of cholesterol biosynthesis at a particular molar concentration of the test preparation in ~he medium can be indicated directly. In aliquots of the cell culture, the integrity of the cell culture and the absence of damage to cells caused by action of prepara-tions is assessed morphologically (light microscopy) and measured biochemically by determining the secretion of lactate dehydrogenase into the incubation medium.
Lovostatin was used as standard preparation. The extent of cholesterol biosynthesis inhibition by the compounds of the formula I where R = H is, at a concentration of 10-8 mol/l, 41 %.
~he inhibition of chole~terol biosynthesis at 10-7 ~ol/l lovostatin is 74 %, and at 10-8 mol/l 48 %.
- 13 - 2~$~
b) In-vivo determination:
Inhibition of hepatic cholesterol biosynthesis effect the reduction of serum lipids, as can be demonstrated in chronic tests on male rats. The tes~ preparations in polyethylene glycol 400 were administered daily in the morning to groups of male rat~ of the strain HOE: WISKf (SPF 71) of starting weight of approximat01y 240 g, by gavage, the control group in question only receiving the vehicle. 24 hours after the last administration and after 24 hours~ fasting, blood samples were taken, and the lipoproteins in the serum obtained from a pooled group of rats were separated by means of the preparative ultra-centrifuge technique. In ~his process, the following density limi~s were used for the separation of VLDL, LDL
and HDL:
VLDL 1. 006 LDL 1.04 HDL 1.21 To determine the cholesterol and the triglycerides, fully-enzymatic Boehringer/Mannheim methods were used, and to determine the protein, Lowry and coworkers' method was 1l sed.
The values measured for the compounds of the formula I in comparison with clofibrate are listed below:
o h Cl~ U~
~ ~ o tq ~ ,1 O ~t` o h ~~ _ a) ;>l l ~ ~Ct~ o~
_I
~:1 ~D CO
~ l o~ o~
~:
S~ ~ r~ ~
a ~ ~ l ~n ~1 ~
~ o a D ~
o ~ ~ ~ U~
l l a) ~
~n ~o o o ~~ O
$ a ~ ,, 0 h O ~ ~ .
~ r~
a) In-vitro determination:
HEP-G2 cell monolayers in lipoprotein-free nutrient medium are preincubated for one hour with appropriate concentrations of the test substances of the formula I.
The l4C-tagged biosynthesis precursor [14CJsodium acetate is added, and incubation is then continued for 3 hours.
After this r some of the cells are subjected to alkaline hydrolysis after previously adding an internal ~tandard of 3H cholesterol. The lipids of the hydrolyzed cells are extracted with a chloroform/me~hanol mixture. Support cholesterol is added, and this lipid mixture is separat~d by preparative thin-layer chromatography, the cholesterol band is stained and then isolated, and the amount of 14C-cholesterol formed from the 14C-precursor is determined by scintigraphy. In an aliquot of the cells, cell protein was determined, so that the amount of 14C-cholesterol formed from l4C-precursor per mg of cell protein can be calculated per cell unit. To compare the inhibitory action of an added test preparation, the control is used, so that the inhibition of cholesterol biosynthesis at a particular molar concentration of the test preparation in ~he medium can be indicated directly. In aliquots of the cell culture, the integrity of the cell culture and the absence of damage to cells caused by action of prepara-tions is assessed morphologically (light microscopy) and measured biochemically by determining the secretion of lactate dehydrogenase into the incubation medium.
Lovostatin was used as standard preparation. The extent of cholesterol biosynthesis inhibition by the compounds of the formula I where R = H is, at a concentration of 10-8 mol/l, 41 %.
~he inhibition of chole~terol biosynthesis at 10-7 ~ol/l lovostatin is 74 %, and at 10-8 mol/l 48 %.
- 13 - 2~$~
b) In-vivo determination:
Inhibition of hepatic cholesterol biosynthesis effect the reduction of serum lipids, as can be demonstrated in chronic tests on male rats. The tes~ preparations in polyethylene glycol 400 were administered daily in the morning to groups of male rat~ of the strain HOE: WISKf (SPF 71) of starting weight of approximat01y 240 g, by gavage, the control group in question only receiving the vehicle. 24 hours after the last administration and after 24 hours~ fasting, blood samples were taken, and the lipoproteins in the serum obtained from a pooled group of rats were separated by means of the preparative ultra-centrifuge technique. In ~his process, the following density limi~s were used for the separation of VLDL, LDL
and HDL:
VLDL 1. 006 LDL 1.04 HDL 1.21 To determine the cholesterol and the triglycerides, fully-enzymatic Boehringer/Mannheim methods were used, and to determine the protein, Lowry and coworkers' method was 1l sed.
The values measured for the compounds of the formula I in comparison with clofibrate are listed below:
o h Cl~ U~
~ ~ o tq ~ ,1 O ~t` o h ~~ _ a) ;>l l ~ ~Ct~ o~
_I
~:1 ~D CO
~ l o~ o~
~:
S~ ~ r~ ~
a ~ ~ l ~n ~1 ~
~ o a D ~
o ~ ~ ~ U~
l l a) ~
~n ~o o o ~~ O
$ a ~ ,, 0 h O ~ ~ .
~ r~
5.a) Characterization of the compound of the formula I
o~ ~
Agistatin A ~figure) was isolated as an amorphou6 solid and is solu~le in CH2Clz, CHC13, ether, ethyl acetate, acetone, ethanol and isopropanol. Agistatin ~ i8 insoluble in n pentane, n-hexane and cyclohexane.
Melting point: 73C
Optical rotation: [~ 2U = _ 36.2 (c = 0.9; methanol) Thin-layer chromatography:
Silica gel 60, F254: chloroform/methanol (9:1 vsv) Rf 0.70 n-Butanol/acetic acid/water (top phase) (4:1:5, v:v:v):Rf 0.75 Staining behavior: orcinol: brown; anisaldehyde/sulfuric acid: brown.
EI MS: m/e = 242 (8 %, M~, high re~olution: 242.1154, C12H1~Os), 224 (10 %, M-~2O), 210 (10~), 182 (32%) 168 t46%)~ 140 (24%3, 13g (~4~)~ 111 (64%~, ~5 (9 Molecular mass: 242-27 (C12Hl8O5) IR (KBr): 34~0, 2960, 2920, 1730, 1880 cml UV (MeOH): A = 203 nm ~ - 3250;224l1~00),261(1000)nm + HCL:~ = 203(~ = 4450);224~1750),271(900~nm ~NaOH:A = 211~ = 4700,271(1600,303(1450)nm H NMR (200 MHz, CDCl3):
= 0~98(t,3H,J = 7v0 Hz,1 1'H3~ 32-1,84~m,2~,1~H2),1.85~m,1H, 6-H);
1,94-2.26(m,2H,7-H2);2944(d,lH,J6~H,6-3~5 Hz,~-OH); ~B2~dd,1H, J3~3b-14,5 Hz, J3~,2= 2,5 Hz,3-Ha);2,76(s,1H,4a-OH);3,32(dd,1H,J3~ 14.5 Hz, J3b2= 4 Hz,3-Hb);3.42(s,3H,g-H3);4,09(dd,1H,J~,~.~ s 3,5 andJ = 3,5 Hz,5-H);5.13(dd,1H, J2,3b= 4 HZ~J2~a~= 2~5 Hz,2-H);5~61(dd,lH,J = 2~-andS.O Hz, 8-H).
.
.
~ ~ rj ~ d, 13c NMR ( 50 MHz, CDCl3):
~ = 11,6 (q, G11); 24.3 (t, C-10~; 2~,1 (t, e-7); 34~(d,C-8);43~8(t,C-3);5~o~(q~
G9);71.5(d, G5),73~6(s,C-4a);100.U(~,C-2~ .3~,C-~);146~1(s,C-8a);
205.5(s,C-4).
Elemental analysis: calculatedO C 59.53 H 7.43 found: C 59.56 H 7.38 b) Characterization of the compound of ~he formula I:
1 ~o HO ~ C~3 OH
Agistatin B (figure):
Empirical formula: C11H18O4 Molecular mass: 214.26 Consistency: colorless, cry~talline Soluble in: MeOH, EtOH
Insoluble in: n-pentane, n-hexane Melting point: l6lC
IR (KBr): 3440, 3400, 2940, ll~0, 1085, 930 cm Elemental analy~is: found calculated C 61.46 61.66 H 8.39 8.47 Optical rotations [~]DO = +38.2; (c = l.0) Mass spectrum: m/z = 214 (22 %, Mt, high resolution:
2l4.l205, C1lH1ôO4) 196(4 %, M-18),186(4 %, M-28),170(6 %, M-44),167 (14 %, M-47),142(66 %, M-72),113(66 ~, M-101), 95(99 %, M-119) 3C NMR (50.3 MHz, CD30D):
,9 (q, C-11); 22,2 5t, C-10); 26.1 (t, C-7); 2~.~ (t, C:-8); 37,~; ~d, 1::-6);
40,8(t, G2);67,4(S, G4); 6908 (d, G3); 71.5 (d, G~)i 74.5 (d, C-9); 91,2 (d, C-l) H NMR (500 MHz, CDCl3):
= 0"94 (t, 3H, Jt1,10= 7.~ Hz, 1 l-H~); 1.3~1,80 (m, 7H, 4tl, 7~Ha~ 8-H2, 10-H2);
1~97 (ddd, 1H, J2-.2b = 14 Hz, J2~,3 = 3.5 Hz, J2nl = 2 Hz, 2-Ha); 2.09 ~hr s, 1H, OH~;
2~14 ~br s, 1 H, OH); 2.55 (ddd, 1H, `12h,2. = 14 Hz, J2b,3 Z 10 Hz, J2b,1 = 2 Hz, 2-Hb);
3,81 (m, 1H, 9-H); 3.99 ~dd, lH, J3,2b - 10 Hz, J3,2~, = 3 ~z, 3-~1); 4,19 (br s, 1~1, 5 H); 4.96 (dd, 1H, J=2and 2 Hz, 1-H).
c) Characterization of the compound of +~he formula I
3 ~ CH3 C)H
OH
Agistatin D (figure):
Empirical formula: C11H14O4 Molecular mass: 2l0.23 Consistency: white powder Soluble in: CH2Clz, CHCl3, ether, EtO~c, acetone, EtOH, isopropanol Insoluble in: n-pentane, n-hexane, cyclohe~ane Melting point: 93C
UV~MeOH)nm l~): 201(31743,227(1132),289~6942)nm (MeOH/HCI) (~): 202 (5234), 225 (2339), 288 (7128) nrn (MeOH/NaOH) (~): 215 (8037), 274 (4770), 305 (4937) nm IR (I<Br): 33~0 br, 2950, 29~0, 2860, 1~80, 1B5:), 1620, 1600, 1585, 1~10, 1280, 100~ cm Optical rotation (MeOH): [~]DO = ~252-4; (c = 0.55 Mass spectrum: m/z - 210 ~84 %, M+) High resolution: calculated for C1lH14O4 2l0.0892 found 210.08937 - 18 ~ 7~
132(44 %,M-18),163~8~ %, M-47),13~100 %, M-71), 137(80 %, M-73),126~75 %, M-84~,71~87 %, M-139), 55(76 %, M-155) 3C NMR (50 MHz, d6-acetone):
9(q~C-11);25~1(t,C-9);26~7(t,C-7);35~8(d,C-~);70~4(d,C-5~;
72~1~s~-4a);105.5(d,C-3);117~4(~C-8);1~0,2~s~c-8a);15904(d~c-2);
192.1(s,C-4) H NMR (200 MHz, CDC13) ~ = 0~98(~,3H,J= 7~0 Hz,10-H3);1~36-1~72~m,2~,9-H2);1~5~m,1H, 6-H);
2~04 2.38(m,2H,7-H2); ~50(s,1H,5-OH);2.82(s,1H,4a-OH);425(s,1H,5-H);
5,54(d,1H,J2= 6,0 Hz,3H);6.00(dd,1H,J= 2.5 and5,0 Hz,8-H);7~45(d,1H, J3= 6~0 Hz,2H) d) Characteri~ation of ~he compound of the formula I
OH
, ~ ~ CH3 3 OH .5 l H
Agistatin E (figure):
Empirical formula: CllH15O5 Molecular mas~: 228.24 Consistency: colorless, c~ystalline Soluble in: MeOH, EtOH, acetone, CHCl3 Insoluble in: n-pentane, n-hexane, n-heptane Melting point: 124C
UV (MeOH/NaOH) 214 (2929), 266 (14798), 304 (3794) nm nm (~):
IR (KBr): 3430, 2970, 2940, 1745, 1460, 1435, 1350, 1270 cm Optical rotation (MeOH): [~D = ~107-7; (c = 0.44) Mass spectrum: m/z = 228 (4 %, M~) - 19 2~7~
~igh resolution: calculated ~or CllHl605 228 09972 found 228 . 09972 210 (4 %, M-18), 181 (17 %, M-47), 164 (14 ~6, M-~4), 140 (54 %, M-88), 139 (52 %, M-~9), 111 (62 %, M-l 17), 83 (79 %, M-145), 55 171 %, M-173), 43 (100 96, M-185) 3C NMR (50.3 MHz, CDCl3) s o' = 11.6 (q, C-11); 22,2 (t, C-7); 24.2 (t, C-10); 32~,4 (t, C-8); 35,4 (d, C-6);
44,6 (t, C-2~; 74~8 (d, C-5); 75,5 Is, C-4); 92,3 (d, C-1); 9802 (s, C-9~; 206,4 (s, C-3) 1H NMR ( 2 O O MHZ , CDC13 ):
~5 = 0,92 (t, 3H, J = 7~,0 HZ, 1 1 -H3); 1,40 (m, 2H, 1 0-H2); 1,~0 (m, 1 H, 6-HJ; 1.65 (m, 2H, 7-H2); 1.75/1098 (m, 2H, 8-H2), 2~70 Is, 11i, 9-OH); 2~8312,.90 (dd, ~H, JA,~ 19.0 HZ~ JA1= ~IB1 = 2.0 HZ, 2-HAand 2-H~ ,74 (s, 1H, 4-OH); 3.94 (s, 1H, 5-H); 5.48(dd, 1 H, J1 2-HA = J1 2.HB3 = 2"0 Hz, 1-H)
o~ ~
Agistatin A ~figure) was isolated as an amorphou6 solid and is solu~le in CH2Clz, CHC13, ether, ethyl acetate, acetone, ethanol and isopropanol. Agistatin ~ i8 insoluble in n pentane, n-hexane and cyclohexane.
Melting point: 73C
Optical rotation: [~ 2U = _ 36.2 (c = 0.9; methanol) Thin-layer chromatography:
Silica gel 60, F254: chloroform/methanol (9:1 vsv) Rf 0.70 n-Butanol/acetic acid/water (top phase) (4:1:5, v:v:v):Rf 0.75 Staining behavior: orcinol: brown; anisaldehyde/sulfuric acid: brown.
EI MS: m/e = 242 (8 %, M~, high re~olution: 242.1154, C12H1~Os), 224 (10 %, M-~2O), 210 (10~), 182 (32%) 168 t46%)~ 140 (24%3, 13g (~4~)~ 111 (64%~, ~5 (9 Molecular mass: 242-27 (C12Hl8O5) IR (KBr): 34~0, 2960, 2920, 1730, 1880 cml UV (MeOH): A = 203 nm ~ - 3250;224l1~00),261(1000)nm + HCL:~ = 203(~ = 4450);224~1750),271(900~nm ~NaOH:A = 211~ = 4700,271(1600,303(1450)nm H NMR (200 MHz, CDCl3):
= 0~98(t,3H,J = 7v0 Hz,1 1'H3~ 32-1,84~m,2~,1~H2),1.85~m,1H, 6-H);
1,94-2.26(m,2H,7-H2);2944(d,lH,J6~H,6-3~5 Hz,~-OH); ~B2~dd,1H, J3~3b-14,5 Hz, J3~,2= 2,5 Hz,3-Ha);2,76(s,1H,4a-OH);3,32(dd,1H,J3~ 14.5 Hz, J3b2= 4 Hz,3-Hb);3.42(s,3H,g-H3);4,09(dd,1H,J~,~.~ s 3,5 andJ = 3,5 Hz,5-H);5.13(dd,1H, J2,3b= 4 HZ~J2~a~= 2~5 Hz,2-H);5~61(dd,lH,J = 2~-andS.O Hz, 8-H).
.
.
~ ~ rj ~ d, 13c NMR ( 50 MHz, CDCl3):
~ = 11,6 (q, G11); 24.3 (t, C-10~; 2~,1 (t, e-7); 34~(d,C-8);43~8(t,C-3);5~o~(q~
G9);71.5(d, G5),73~6(s,C-4a);100.U(~,C-2~ .3~,C-~);146~1(s,C-8a);
205.5(s,C-4).
Elemental analysis: calculatedO C 59.53 H 7.43 found: C 59.56 H 7.38 b) Characterization of the compound of ~he formula I:
1 ~o HO ~ C~3 OH
Agistatin B (figure):
Empirical formula: C11H18O4 Molecular mass: 214.26 Consistency: colorless, cry~talline Soluble in: MeOH, EtOH
Insoluble in: n-pentane, n-hexane Melting point: l6lC
IR (KBr): 3440, 3400, 2940, ll~0, 1085, 930 cm Elemental analy~is: found calculated C 61.46 61.66 H 8.39 8.47 Optical rotations [~]DO = +38.2; (c = l.0) Mass spectrum: m/z = 214 (22 %, Mt, high resolution:
2l4.l205, C1lH1ôO4) 196(4 %, M-18),186(4 %, M-28),170(6 %, M-44),167 (14 %, M-47),142(66 %, M-72),113(66 ~, M-101), 95(99 %, M-119) 3C NMR (50.3 MHz, CD30D):
,9 (q, C-11); 22,2 5t, C-10); 26.1 (t, C-7); 2~.~ (t, C:-8); 37,~; ~d, 1::-6);
40,8(t, G2);67,4(S, G4); 6908 (d, G3); 71.5 (d, G~)i 74.5 (d, C-9); 91,2 (d, C-l) H NMR (500 MHz, CDCl3):
= 0"94 (t, 3H, Jt1,10= 7.~ Hz, 1 l-H~); 1.3~1,80 (m, 7H, 4tl, 7~Ha~ 8-H2, 10-H2);
1~97 (ddd, 1H, J2-.2b = 14 Hz, J2~,3 = 3.5 Hz, J2nl = 2 Hz, 2-Ha); 2.09 ~hr s, 1H, OH~;
2~14 ~br s, 1 H, OH); 2.55 (ddd, 1H, `12h,2. = 14 Hz, J2b,3 Z 10 Hz, J2b,1 = 2 Hz, 2-Hb);
3,81 (m, 1H, 9-H); 3.99 ~dd, lH, J3,2b - 10 Hz, J3,2~, = 3 ~z, 3-~1); 4,19 (br s, 1~1, 5 H); 4.96 (dd, 1H, J=2and 2 Hz, 1-H).
c) Characterization of the compound of +~he formula I
3 ~ CH3 C)H
OH
Agistatin D (figure):
Empirical formula: C11H14O4 Molecular mass: 2l0.23 Consistency: white powder Soluble in: CH2Clz, CHCl3, ether, EtO~c, acetone, EtOH, isopropanol Insoluble in: n-pentane, n-hexane, cyclohe~ane Melting point: 93C
UV~MeOH)nm l~): 201(31743,227(1132),289~6942)nm (MeOH/HCI) (~): 202 (5234), 225 (2339), 288 (7128) nrn (MeOH/NaOH) (~): 215 (8037), 274 (4770), 305 (4937) nm IR (I<Br): 33~0 br, 2950, 29~0, 2860, 1~80, 1B5:), 1620, 1600, 1585, 1~10, 1280, 100~ cm Optical rotation (MeOH): [~]DO = ~252-4; (c = 0.55 Mass spectrum: m/z - 210 ~84 %, M+) High resolution: calculated for C1lH14O4 2l0.0892 found 210.08937 - 18 ~ 7~
132(44 %,M-18),163~8~ %, M-47),13~100 %, M-71), 137(80 %, M-73),126~75 %, M-84~,71~87 %, M-139), 55(76 %, M-155) 3C NMR (50 MHz, d6-acetone):
9(q~C-11);25~1(t,C-9);26~7(t,C-7);35~8(d,C-~);70~4(d,C-5~;
72~1~s~-4a);105.5(d,C-3);117~4(~C-8);1~0,2~s~c-8a);15904(d~c-2);
192.1(s,C-4) H NMR (200 MHz, CDC13) ~ = 0~98(~,3H,J= 7~0 Hz,10-H3);1~36-1~72~m,2~,9-H2);1~5~m,1H, 6-H);
2~04 2.38(m,2H,7-H2); ~50(s,1H,5-OH);2.82(s,1H,4a-OH);425(s,1H,5-H);
5,54(d,1H,J2= 6,0 Hz,3H);6.00(dd,1H,J= 2.5 and5,0 Hz,8-H);7~45(d,1H, J3= 6~0 Hz,2H) d) Characteri~ation of ~he compound of the formula I
OH
, ~ ~ CH3 3 OH .5 l H
Agistatin E (figure):
Empirical formula: CllH15O5 Molecular mas~: 228.24 Consistency: colorless, c~ystalline Soluble in: MeOH, EtOH, acetone, CHCl3 Insoluble in: n-pentane, n-hexane, n-heptane Melting point: 124C
UV (MeOH/NaOH) 214 (2929), 266 (14798), 304 (3794) nm nm (~):
IR (KBr): 3430, 2970, 2940, 1745, 1460, 1435, 1350, 1270 cm Optical rotation (MeOH): [~D = ~107-7; (c = 0.44) Mass spectrum: m/z = 228 (4 %, M~) - 19 2~7~
~igh resolution: calculated ~or CllHl605 228 09972 found 228 . 09972 210 (4 %, M-18), 181 (17 %, M-47), 164 (14 ~6, M-~4), 140 (54 %, M-88), 139 (52 %, M-~9), 111 (62 %, M-l 17), 83 (79 %, M-145), 55 171 %, M-173), 43 (100 96, M-185) 3C NMR (50.3 MHz, CDCl3) s o' = 11.6 (q, C-11); 22,2 (t, C-7); 24.2 (t, C-10); 32~,4 (t, C-8); 35,4 (d, C-6);
44,6 (t, C-2~; 74~8 (d, C-5); 75,5 Is, C-4); 92,3 (d, C-1); 9802 (s, C-9~; 206,4 (s, C-3) 1H NMR ( 2 O O MHZ , CDC13 ):
~5 = 0,92 (t, 3H, J = 7~,0 HZ, 1 1 -H3); 1,40 (m, 2H, 1 0-H2); 1,~0 (m, 1 H, 6-HJ; 1.65 (m, 2H, 7-H2); 1.75/1098 (m, 2H, 8-H2), 2~70 Is, 11i, 9-OH); 2~8312,.90 (dd, ~H, JA,~ 19.0 HZ~ JA1= ~IB1 = 2.0 HZ, 2-HAand 2-H~ ,74 (s, 1H, 4-OH); 3.94 (s, 1H, 5-H); 5.48(dd, 1 H, J1 2-HA = J1 2.HB3 = 2"0 Hz, 1-H)
Claims (6)
1. A process for the preparation of the compounds of the formula I
I
in which R1 is hydrogen or hydroxyl, R2 is hydroxyl or an oxo group, R3 is methoxy or hydroxyl or, together with R1, forms an ether bridge and R4 is hydrogen or hydroxyl, R5 is hydrogen or, together with R4, is an additional bond, and R6 is hydrogen or, together with R3, is an additional bond, which comprises culturing Fusarium sp. DSM 5983 in a nutrient medium until the compounds of the formula I
are accumulated in the culture.
I
in which R1 is hydrogen or hydroxyl, R2 is hydroxyl or an oxo group, R3 is methoxy or hydroxyl or, together with R1, forms an ether bridge and R4 is hydrogen or hydroxyl, R5 is hydrogen or, together with R4, is an additional bond, and R6 is hydrogen or, together with R3, is an additional bond, which comprises culturing Fusarium sp. DSM 5983 in a nutrient medium until the compounds of the formula I
are accumulated in the culture.
2. The process as claimed in claim 1 , in which the nutrient medium contains 0.2 to 5 % of malt extract, 0.1 to 0.5 % of yeast extact, 0.5 to 2 % of glucose and 0.01 to 0.1 % of (NH4)2HPO4, in each case based on the weight of the entire nutrient solution.
3. The process as claimed in claim 1 or 2, wherein culturing takes place at 18 to 30°C.
4. The process as claimed in one or more of claims 4 to 3, wherein culturing takes place in the pH range between 1 and 7.
5. The use of the compounds of the formula I as claimed in claim 1 as cholesterol biosynthesis inhibitors.
6. Fusarium sp. DSM 5983 as well as its variants and mutants, as long as they produce compounds of the formula I as claimed in claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4040147 | 1990-12-15 | ||
| DEP4040147.2 | 1990-12-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2057651A1 true CA2057651A1 (en) | 1992-06-16 |
Family
ID=6420444
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002057651A Abandoned CA2057651A1 (en) | 1990-12-15 | 1991-12-13 | Agistatins, novel metabolites from fusarium sp., a process for their preparation, and their use |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0492318A2 (en) |
| JP (1) | JPH04368378A (en) |
| AU (1) | AU8976791A (en) |
| CA (1) | CA2057651A1 (en) |
| HU (1) | HUT61336A (en) |
| IL (1) | IL100368A0 (en) |
-
1991
- 1991-12-13 IL IL100368A patent/IL100368A0/en unknown
- 1991-12-13 CA CA002057651A patent/CA2057651A1/en not_active Abandoned
- 1991-12-13 AU AU89767/91A patent/AU8976791A/en not_active Abandoned
- 1991-12-13 JP JP3329372A patent/JPH04368378A/en active Pending
- 1991-12-13 HU HU913946A patent/HUT61336A/en unknown
- 1991-12-13 EP EP91121422A patent/EP0492318A2/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| AU8976791A (en) | 1992-06-18 |
| EP0492318A2 (en) | 1992-07-01 |
| JPH04368378A (en) | 1992-12-21 |
| HUT61336A (en) | 1992-12-28 |
| HU913946D0 (en) | 1992-02-28 |
| IL100368A0 (en) | 1992-09-06 |
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