WO1982000587A1 - New metabolites,processes for their production and their use - Google Patents

New metabolites,processes for their production and their use Download PDF

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Publication number
WO1982000587A1
WO1982000587A1 PCT/EP1981/000121 EP8100121W WO8200587A1 WO 1982000587 A1 WO1982000587 A1 WO 1982000587A1 EP 8100121 W EP8100121 W EP 8100121W WO 8200587 A1 WO8200587 A1 WO 8200587A1
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WO
WIPO (PCT)
Prior art keywords
strain
spectrum
see
methanol
compound according
Prior art date
Application number
PCT/EP1981/000121
Other languages
English (en)
French (fr)
Inventor
Ag Sandoz
Original Assignee
Tscherter H
Dreyfuss M
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tscherter H, Dreyfuss M filed Critical Tscherter H
Priority to FI820652A priority Critical patent/FI820652A7/fi
Publication of WO1982000587A1 publication Critical patent/WO1982000587A1/en
Priority to DK168082A priority patent/DK168082A/da

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to the compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7.
  • S 41062/F-1, S 41062/F-6 and/or S 41052/F-7 are obtained by cultivating an S 41062/F-1 and/or S 41062/F-6 and/or S 41062/F-7 producing strain, e.g. of the fungal genus Crypto sporiopsis in the presence of a culture medium.
  • the compounds S 41062/F-1, S 41062/F-5 and S 41062/F-7 exhibit the following approximate characteristics.
  • S 41062/F-1 is a neutral cyclic hexapeptide, which bears a branched, amide linked, saturated (C-16) fatty acid radical.
  • S 41062/F-1 has the following structure:
  • Solubility easily soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DMSO) ; difficulty soluble in water, chloroform, ethyl acetate, ether, benzene, hexane; - the compound was stable in aqueous methanol at pH 4 to 7. Under alkaline or strongly acidic conditions decomposition with loss of anti-fungal activity occurred.
  • DMSO dimethylsulfoxide
  • Degradation products were obtained with retention times the same as or similar to e.g. aspartic acid, hydroxyproline/allo-hydroxy- proline, serine and ammonia as well as a further amino acid with retention time between those of leucine and tyrosine.
  • Iodine vapour or other suitable spray reagents can be used to detecr the substances.
  • a suitable spray reagent can for example be prepared by dissolving 100 mg of ferric chloride in 1 ml of ethyl acetate and adding 100 ml of concentrated nitrate-free sulphuric acid.
  • the completely solvent-free thin layer chromatogram was sprayed with the above-mentioned spray reagent and then heated using an incandescent heater until strong brown spots appeared.
  • the process according to the invention may be effected by known methods.
  • a sub-culture of the S 41062/F-1, S 41062/F-6 and/or S.41062/F-7 producing strain was deposited on 2.1.80 at the American Type Culture Collection (ATCC) , Rochville, Maryland, USA and is available to the public under the culture number ATCC 20594.
  • the culture is also available from Sandoz Ltd., Basle, Switzerland.
  • S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing strains may be used which may be obtained from the parent strain of ATCC 20594 or NRRL 12192 by mutation e.g. by radiation, treatment with conventional mutagenic substances, or by selection. Characteristics of the strain ATCC 20594 (and NRRL 12192)
  • the following physiological and morphological features are characteristic for the S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing Cryptosporiopsis strain ATCC 20594: Growth Occurs well on agar media preferably at lower temperatures. The minimal and maximal temperatures for growth are 0° and 31-33°C respectively with optimal growth between 15-24 °C.
  • Table 3 shows the growth characteristics dependent on incubation temperature and medium.
  • Colony diameters (average of 6 colonies in mm) were measured after a 14-day incubation period on petri dishes seeded punctate and serve as a measure of growth rate.
  • MA-medium MA-mediuia without agar
  • the pH of which can be adjusted with hydrochloric acid or sodium hydroxide ATCC 20594 will grow quite well at pH 2.0 with shaking (180 rpm) and at a temperature of 21-22°C whereas at a pH above 9.3 no growth takes place. Good growth occurs throughout the pH region of 2.3 to 8.0.
  • the hyaline single celled microconidia are very small and. cylindrical to slender clavate, whereby the broader end is rounded, the narrower truncate.
  • the colourless to weakly amber coloured single celled macroconidia on the other hand are large and normally allantoid. One end is rounded and the other has a protruding truncate base.
  • the culture conditions can have a marked influence on the formation and. size of the micro- and macro-conidia as summarised in the following Table 4.
  • the mature macro-conidia are filled with globular material which turns intensively red with Sudan III dye and deep black with Sudan-black B. Micro-conidia do not stain.
  • the germination of macro-conidia is optimal at 24°C on MA-medium in the narrow pH range of 3.1 to 3.8 (70-80% germination). Above pH 5.5 the germination rate is under 5%. The germination rate of the micro-conidia was less than 1% under all conditions tested.
  • the new strain ATCC 20594 can be cultivated at a suitable temperature on various conventional nutrient media as aerobic surface or immersion cultures. As soon as a sufficient amount of the new compound S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 has been produced in the culture which may e.g. be ascertained by the activity towards Candida sp., the mycelium may be separated from the culture broth and extracted in conventional manner.
  • a preferred isolation procedure comprises homogenising the mycelium with methanol and separating and concentrating the liquid phase. Subsequent extraction is effected with a water-immiscible organic solvent e.g. ethyl acetate followed by removal of the solvent in vacuum, dissolution of the residue in methanol and after scouring, e.g. with hexane, evaporation to dryness in vacuum.
  • a water-immiscible organic solvent e.g. ethyl acetate followed by removal of the solvent in vacuum,
  • S 41062/F-1, S 41062/F-6 and/or S 41052/F-7 can be isolated and purified in conventional manner, e.g. employing chrpmatographic techniques or countercurrent partitioning.
  • the invention also concerns fermentation broths which are obtained during the cultivation of the S 41062/F-1, S 41062/F-6 and/or S 41062/F-7 producing strains of Cryptosporiopsis.
  • the compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7 exhibit antibiotic activity. They exhibit a growth inhibiting effect towards micr-organisms such as Yeasts and fungi, but no significant activity towards gram positive and gram negative bacteria.
  • the wide activity against yeasts and fungi is particularly noticeable against-various human pathogenic Candida strains.
  • Table 5 indicates the minimum inhibition concentration (MIC) of S 41062/F-1, F-6 and F-7 against various micro-organisms determined in known manner in a series dilution test in a malt extract medium, incubation temperature 27°C over 43 to 72 hours.
  • mice and rats which have been pre-treated with estradiol-benzoate are infected intra-vaginally and then treated on 5 successive days either orally or parenterally (mouse) or intravaginally (rat) with test substance.
  • the success of treatment is determined by the presence or absence of fungus in the vagina or from the uterus.
  • Antimycotic activity is observed on local application at concentrations of 2% and upward.
  • Systemic activity is observed in vivo on sub-cutaneous application in a dosage range of from ca. 5 to 50 mg/kg animal body weight.
  • the compounds S 41062/F-1, S 41062/F-6 and S 41062/F-7 are therefore useful as antibiotics.
  • the dosage will, of course, vary depending on the compound employed, mode of administration and condition to be treated. However, in general, satisfactory results are obtained when administered at a daily dosage of from 5 mg to about
  • Unit dosage forms suitable for oral admini stration comprise from about 75 mg to about maximally 1500 mg, e.g. 250 mg, of the compounds admixed with a solid or liquid pharmaceutical carrier or diluent.
  • the invention therefore also concerns a method of treating diseases or infections caused by yeasts and fungi using the compound S-41062/F-1, S-41062/F-6 and/or S-41062/F-7 and also these
  • the compounds may be administered preferably orally or parenterally suitably in admixture with conventional pharmaceutically acceptable diluents and carriers, and, optionally, other excipients.
  • compositions also form part of the invention.
  • the following examples illustrate the invention. All temperatures are in degrees Centigrade.
  • EXAMPLE 1 Cultivation of Strain ATCC 20594 in a Glass Fermenter
  • the spore and mycelium suspension for inoculation is produced from a slant culture of strains ATCC 20594 which is obtained after 21 days incubation at 21° on an agar medium of the following composition.
  • Spores and mycelia of the culture obtained under a) are suspended in physiological common salt solution and used to inoculate 1 litre of the following preculture medium in a 2 litre Erlenmeyer flask.
  • Malt extract (syrup) 20 g Yeast extract 4 g
  • Demineralised water to 1 litre.
  • the preculture obtained under b) is used to inoculate 10 litres of the following production culture medium in a 14 litre glass fermenter.
  • Demineralised water to 1 litre Prior to sterilisation the pH is adjusted to 3.8 to 4.1 with IN HCl/NaOH.
  • Inciibation is performed for 5 days at 21° with stirring (150 rpm) and aeration (1 litre/min/litre medium).
  • EXAMPLE 2 Cultivation of Strain ATCC 20594 in a
  • 250 ml of an inoculum thus prepared are used for inoculation of 50 litres of preculture medium of the same composition as described in Example 1b) in a 75 litre steel fermenter.
  • Example 1c 3,000 litres of production culture medium composed as in Example 1c) in a 4,500 litre steel ferm enter are inoculated with the intermediate culture. Inoculation is carried out for 5 days at 21°, 0.5 bar and an air rate of 1.0 litre/min/litre medium.
  • 300 litres of fermentation broth (obtained according to Example 2) are adjusted to pH 6-7 with dilute sulphuric acid or sodium hydroxide and the mycelium precipitate separated from the culture filtrate with a Westfalia separator.
  • the wet mycelium is homogenised in a Dispax reactor with 10 times the quantity of methanol and the liquid phase separated off. This procedure is repeated twice with 90% aqueous methanol.
  • the filtrates are combined, freed from methanol in vacuum and then extracted three times with ethyl acetate and once with ethyl acetate/isopropanol (4:1).
  • the extracts are freed from solvent in vacuum and the residue dissolved in 10 times the quantity of 90% aqueous methanol. After scouring with hexane the polar phase is again evaporated to dryness in vacuum.
  • the fractions active against Candida albicans, nos. 8 and 9 which contain primarily the compounds S 41062/F-1 and S 41062/F-7 are combined and chromatographed on 1.5 kg of Sephadex LH 20 in methanol (at 25 ml per fraction) whereby the two metabolites were considerably enriched in the fraction 42 to 62.
  • the concentrated residues containing S 41062/F-1 are dissolved in ethanol, combined, treated with active charcoal and filtered to clarity through a layer of talc.
  • the almost colourless filtrate is concentrated under a mild vacuum and slowly mixed with acetone, whereupon S 41062/F-1 is obtained as a white and, damp with solvent, as a highly hygroscopic compound in microcrystalline form. M.p. 235-240° (gradual decomposition).
  • fractions 10 and 11 which were obtained from the first chromatogram and which contain predominantly S 41062/F-6, are combined and then separated on 500 g of kieselgel Merck (0.04 - 0.06 mm) in methylene chloride/methanol (4:1) with collection of 80 ml fractions.
  • the fractions active against Candida albicans are evaporated to dryness in vacuum and the residues tested for purity by thin-layer chromatography.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Botany (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/EP1981/000121 1980-08-15 1981-08-12 New metabolites,processes for their production and their use WO1982000587A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
FI820652A FI820652A7 (fi) 1980-08-15 1981-08-12 Uudet metaboliitit, mentelmä niiden valmistamiseksi ja niiden käyttö.
DK168082A DK168082A (da) 1980-08-15 1982-04-14 Fremgangsmaade til fremstilling af metaboliter

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CH619680 1980-08-15
CH6196/80 1980-08-15
CH7330/80801001 1980-10-01
CH733080 1980-10-01

Publications (1)

Publication Number Publication Date
WO1982000587A1 true WO1982000587A1 (en) 1982-03-04

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ID=25699156

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Application Number Title Priority Date Filing Date
PCT/EP1981/000121 WO1982000587A1 (en) 1980-08-15 1981-08-12 New metabolites,processes for their production and their use

Country Status (10)

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EP (1) EP0057724A1 (enEXAMPLES)
JP (1) JPS57501266A (enEXAMPLES)
DK (1) DK168082A (enEXAMPLES)
ES (1) ES8306506A1 (enEXAMPLES)
GR (1) GR75310B (enEXAMPLES)
IL (1) IL63571A0 (enEXAMPLES)
IT (1) IT1138149B (enEXAMPLES)
PT (1) PT73516B (enEXAMPLES)
WO (1) WO1982000587A1 (enEXAMPLES)
YU (1) YU198481A (enEXAMPLES)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4765656A (en) * 1985-10-15 1988-08-23 Gao Gesellschaft Fur Automation Und Organisation Mbh Data carrier having an optical authenticity feature and methods for producing and testing said data carrier
EP0405997A1 (en) * 1989-06-30 1991-01-02 Merck & Co. Inc. Antibiotic agent
US5310726A (en) * 1990-03-19 1994-05-10 Merck & Co., Inc. Lipopeptide compounds
US5386010A (en) * 1990-03-19 1995-01-31 Merck & Co., Inc. Lipopeptide compounds
US5428009A (en) * 1990-07-16 1995-06-27 Merck & Co., Inc. Lipopeptide derivatives
US6613738B1 (en) * 1998-08-10 2003-09-02 Hmv Corporation Cyclic lipopeptide from Cryptosporiopsis quercina possessing antifungal activity

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0021685A1 (en) * 1979-06-08 1981-01-07 Eli Lilly And Company A-30912 H-type antibiotics, their preparation and use
EP0031220A1 (en) * 1979-12-13 1981-07-01 Eli Lilly And Company Derivatives of cyclic peptide nuclei
EP0031221A1 (en) * 1979-12-13 1981-07-01 Eli Lilly And Company Cyclic peptide nuclei
EP0031662A1 (en) * 1979-12-13 1981-07-08 Eli Lilly And Company Derivatives of cyclic peptide nuclei
EP0032009A1 (en) * 1979-12-13 1981-07-15 Eli Lilly And Company Derivatives of cyclic peptide nuclei

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0021685A1 (en) * 1979-06-08 1981-01-07 Eli Lilly And Company A-30912 H-type antibiotics, their preparation and use
EP0031220A1 (en) * 1979-12-13 1981-07-01 Eli Lilly And Company Derivatives of cyclic peptide nuclei
EP0031221A1 (en) * 1979-12-13 1981-07-01 Eli Lilly And Company Cyclic peptide nuclei
EP0031662A1 (en) * 1979-12-13 1981-07-08 Eli Lilly And Company Derivatives of cyclic peptide nuclei
EP0032009A1 (en) * 1979-12-13 1981-07-15 Eli Lilly And Company Derivatives of cyclic peptide nuclei

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Experientia, Volume 32, Fasc. 3, 15th March 1976, (Basle, CH) R.J.J.Ch. LOUSBERG et al. "Isolation of (-)-Cryptosporiopsin, a Chlorinated Cyclopentenone Fungitoxic Metabolite from Phialophora Asteris f.sp. Helianthi", see pages 331-333 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4765656A (en) * 1985-10-15 1988-08-23 Gao Gesellschaft Fur Automation Und Organisation Mbh Data carrier having an optical authenticity feature and methods for producing and testing said data carrier
EP0405997A1 (en) * 1989-06-30 1991-01-02 Merck & Co. Inc. Antibiotic agent
AU639698B2 (en) * 1989-06-30 1993-08-05 Merck Sharp & Dohme Corp. Antibiotic agent
US5310726A (en) * 1990-03-19 1994-05-10 Merck & Co., Inc. Lipopeptide compounds
US5386010A (en) * 1990-03-19 1995-01-31 Merck & Co., Inc. Lipopeptide compounds
US5428009A (en) * 1990-07-16 1995-06-27 Merck & Co., Inc. Lipopeptide derivatives
US6613738B1 (en) * 1998-08-10 2003-09-02 Hmv Corporation Cyclic lipopeptide from Cryptosporiopsis quercina possessing antifungal activity

Also Published As

Publication number Publication date
PT73516B (en) 1983-02-08
YU198481A (en) 1983-10-31
IT1138149B (it) 1986-09-17
JPS57501266A (enEXAMPLES) 1982-07-22
IL63571A0 (en) 1981-11-30
DK168082A (da) 1982-04-14
IT8123488A0 (it) 1981-08-12
ES504747A0 (es) 1983-06-01
PT73516A (en) 1981-09-01
EP0057724A1 (en) 1982-08-18
GR75310B (enEXAMPLES) 1984-07-13
ES8306506A1 (es) 1983-06-01

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