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WO1994010324A1 - Ws79089 substances from streptosporangium - Google Patents

Ws79089 substances from streptosporangium Download PDF

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Publication number
WO1994010324A1
WO1994010324A1 PCT/JP1993/001504 JP9301504W WO9410324A1 WO 1994010324 A1 WO1994010324 A1 WO 1994010324A1 JP 9301504 W JP9301504 W JP 9301504W WO 9410324 A1 WO9410324 A1 WO 9410324A1
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WO
WIPO (PCT)
Prior art keywords
methanol
substance
λmax
reaction
magnetic resonance
Prior art date
Application number
PCT/JP1993/001504
Other languages
French (fr)
Inventor
Toshiro Iwamoto
Motoaki Nishikawa
Nobuharu Shigematsu
Sumio Kiyoto
Masakuni Okuhara
Yasuhisa Tsurumi
Original Assignee
Fujisawa Pharmaceutical Co., Ltd.
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 Fujisawa Pharmaceutical Co., Ltd. filed Critical Fujisawa Pharmaceutical Co., Ltd.
Priority to JP51088094A priority Critical patent/JP3304358B2/en
Publication of WO1994010324A1 publication Critical patent/WO1994010324A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/62—Streptosporangium

Definitions

  • This invention relates to new bioactive compounds, hereinafter referred to WS79089 substances or salts thereof which are useful as a medicament.
  • the present invention relates to novel compounds, WS79089 substances or salts thereof which have endothelin converting enzyme inhibiting activity, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which is useful as endothelin converting enzyme inhibiting agents, and to a use thereof as a medicament.
  • one object of this invention is to provide novel compounds, WS79089 substances or salts thereof which are of use for treating and preventing acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis, and the like.
  • WS79089 substances include WS79089-1 substance, WS79089-2 substance, WS79089-3 substance, WS79089-4 substance and WS79089- 5 substance, and they are collectively referred to as WS79089 substances.
  • WS79089-1 substance, WS79089-2 substance and WS79089-4 substance may be produced by fermentation process of a certain microorganism.
  • another object of this invention is to provide a process for production of WS79089-1 and/or WS79089- 2 substance and/or WS79089-4 substance by fermentation of a WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging to the genus Streptosporangium in a nutrient medium.
  • a further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, WS79089 substances or salts thereof.
  • Still further object of this invention is to provide a use of WS79089 substances or salts thereof for treating and preventing acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis, and the like.
  • Suitable salts of WS79089 substances are conventional pharmaceutically acceptable salts and include a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, etc.), and the like.
  • a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenedi
  • WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance can be produced by fermentation of a WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging to the genus Streptosporangium such as Streptosporangium sp. No. 79089 in a nutrient medium.
  • WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only.
  • This invention also includes the use of any mutants which are capable of producing WS79089-1 and/or WS79089- 2 substance and/or WS79089-4 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-ray, ultra ⁇ violet radiation, treatment which N-methyl-N'-nitro-N- nitrosoguanidine, 2-aminopurine, and the like.
  • Streptosporangium sp. No. 79089 is as follows: (1) Taxonomic characteristics of Streptosporangium sp. No. 79089
  • the strain No. 79089 was isolated from a soil sample obtained from Tateyama-shi, Chiba-ken, Japan.
  • the methods and media described by Shirling and Gottlieb ⁇ 1 ) and Waksman 2) were employed for the taxonomic study.
  • the observations were made after 14 to 21 day's cultivation at 30°C.
  • the morphological observations were made on the cultures grown on yeast extract-malt extract agar and oatmeal agar using an optical microscope and a scanning electron microscope.
  • the color names used in this study were taken from the Methuen Handbook of Colour ⁇ .
  • the temperature range for growth and NaCl tolerance were determined on yeast extract-malt extract agar.
  • the preparation of cells and detection of the isomer of diaminopimelic acid was performed by procedure of Becker et ar- 4 '. Detection of whole-cell sugar was performed by the method of Lechevalier and Lechevalier ⁇ 5) . Phospholipid composition was determined by the method of Lechevalier et al ⁇
  • the vegetative mycelium developed well without fragmentation.
  • the aerial mycelium branched and produced globose sporangia.
  • the sporangia were globose and 5.5- 11 ⁇ m (usually 7- 9 ⁇ m) in diameter with a short sporangiophore.
  • Sporangiospores were formed by septation of a coiled hyphae within the sporangium. They were spherical to oval, 0.6-1.0 x 0.8-1.4 ⁇ m, 20 to 50 spores per sporangium and nonmotile.
  • DL-Diaminopimelic acid was detected in the whole-cell hydrolysates of strain No. 79089.
  • Rhamnose, ribose, madurose, mannose, glucose and galactose were detected as whole-cell sugar, placing this strain in the cell wall type 3B.
  • the diagnostic phospholipids detected were phospholipids containing glucosamine, phosphatidylethanolamine, phosphatidylinositol and diphosphatidyl glycerol, placing it in the type P4 phospholipid group.
  • the strain No. 79089 is considered to belong to the genus Streptosporangium Couch ⁇ 7 ' 8) . Therefore, this strain was designated as Streptosporangium sp. No. 79089.
  • Yeast extract-malt G good extract agar A: abundant, pale red (9A3) R: reddish brown (9D6) S: reddish
  • Inorganic salts-starch G poor agar A: none
  • Glycerol-asparagine G poor agar A: none
  • Peptone-yeast extract- G moderate iron agar A: none
  • Starch-yeast extract G good agar* A: moderate, white to reddish white (8A2)
  • Glucose-asparagine G poor agar A: thin, white
  • G growth
  • A aerial mycelium
  • R reverse side color
  • S soluble pigment
  • Starch-yeast extract agar comprises soluble starch 1%, yeast extract 0.2% and agar 1.5% (pH 7.3).
  • Lechevalier, M. P. and H. Lechevalier Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol. 20, 435-443, 1970
  • WS79089-1 substance and/or WS79089-2 substance and/or WS79089-4 substance are produced when WS79089-1 substance and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging the genus Streptosporangium is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.).
  • the preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose or glycerin, or the like.
  • the preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, com steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like.
  • ammonium salts e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.
  • urea or amino acid or the like.
  • the carbon and nitrogen sources though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.
  • medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium slats, copper salts, zinc salts, or cobalt salts, or the like.
  • a defaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added.
  • Agitation and aeration of the culture mixture may be accomplished in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, and the like.
  • the fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 30°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.
  • the culture broth is then subjected for recovery of WS79089-1 substance, WS79089-2 substance and WS79089-4 substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof.
  • WS79089-3 substance may be produced by subjecting WS79089-1 substance or WS79089-2 substance to hydrolysis with alkali.
  • WS79089-5 substance also may be produced by subjecting WS79089-4 substance to hydrolysis with alkali.
  • corresponding salt may be obtained according to the alkali used in the process.
  • salt may be converted to free type, WS79089-3 substance, per se or WS79089-5 substance, per se by a conventional manner.
  • the salt of WS79089 substances can be prepared by a conventional manner, during or after the recovery and purification of WS79089 substances.
  • WS79089-1 substance as obtained has the following physico- chemical properties:
  • Soluble chloroform, dichloromethane, ethyl acetate
  • HPLC High Performance Liquid Chromatography
  • Condition Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
  • Flow rate 1.0 ml/minute
  • Detection UV 235 nm
  • Retention time 9.4 minutes ** trade name: made by E. Merck
  • Ultraviolet absorption spectrum ⁇ max (methanol) ( ⁇ ): 220 (36800), 245 (38600), 266 (sh) (34100),
  • WS79089-2 substance as obtained has the following physico- chemical properties:
  • Soluble water, methanol
  • WS79089-3 substance (free type, WS79089-3 substance, per se) as obtained has the following physico-chemical properties:
  • WS79089-4 substance as obtained has the following physico- chemical properties:
  • Soluble chloroform, dichloromethane, ethyl acetate
  • WS79089-5 substance (free type, WS79089-5 substance, per se) as obtained has the following physico-chemical properties:
  • WS79089 substances possess pharmacological activities such as endothelin converting enzyme inhibiting activity, and the like, and therefore are useful for the treatment and prevention of acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure , rheumatoid arthritis, osteoarthritis, and the like.
  • the enzyme was obtained from bovine carotid endothelial cells.
  • Cultured cells (about 1 x 10 cells) were scraped off, washed with calcium, magnesium PBS (phosphate buffered saline) and homogenized with a polytron (made by Brinkman Instrument) at setting maximal speed for 3 x 10 sec on ice in 10 ml of 0.25 M sucrose containing 25 mM HEPES buffer (pH 7.4).
  • ET-1 Human big endothelin- 1 (hereinafter, endothelin- 1 is referred to as ET-1) (50 ng) was incubated with the cell homogenate preparation (20- 30 ⁇ g protein) for 16 hours at 37°C in 250 ⁇ l of 50 mM Tris-HCl buffer (pH 7.0) containing 0.5 mM p-chloromercuriphenylsulfonic acid and 0.05 mM diisopropylfluorophosphate.
  • ET-1 Human big endothelin- 1
  • reaction mixture was diluted with 80 ⁇ l of RIA buffer [50 mM Tris- HC1 buffer (pH 7.0) containing 0.1 % BSA, 0.1 % Triton X-100 and 1 mM EDTA] and mixed with 50 ⁇ l of anti-ET-1 serum (antisera against the C-terminal peptide of ET-1 (16-21), final dilution, 1:20,000) and 50 ⁇ l of 125 I-ET-1 (37 KBq/lOml) and then incubated for 2 hours at 4°C.
  • RIA buffer 50 mM Tris- HC1 buffer (pH 7.0) containing 0.1 % BSA, 0.1 % Triton X-100 and 1 mM EDTA
  • DMEM Dulbecco's modified minimum essential medium
  • Fibroblasts 1.5 x plastic flask. After 3 days, medium was removed, cells were washed with 20 ml DMEM, and then 11.25 ml DMEM supplemented with bovine serum albumin (3 mg/ml) and Interleukin l ⁇ (Genzyme Co.,
  • FITC Fluorescein isothiocyanate
  • Collagen Gijutsu Kenshukai Collagen Gijutsu Kenshukai, Tokyo, Japan, 1 mg/ml
  • Tris-HCl buffer 25 ⁇ l of 80 mM o-phenanthrolin was added, 70 ⁇ l of ethanol and 30 ⁇ l of Tris-HCl buffer were added, mixed and centrifuged at 3000 rpm for 10 min.
  • Fluorescene excitation 485 nm, emission 538 nm
  • kidneys (600 g) were dissected to be free of their capsules and their combined volume was measured by displacement in 150 ml of ice-cold 10 mM phosphate buffer (pH 7.0). Unless specified, all the following procedures were carried out at 0-4 °C.
  • the kidneys were minced with scissors and then homogenized with a polytron (made by Brinkman PT-20, maximal speed for 6 x 20 sec) in 100 ml of 10 mM phosphate buffer (pH 7.0). The homogenate was centrifuged at 20,000 x g for 60 minutes at 4°C.
  • the pellet was resuspended in 250 ml buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.0) and then 250 ml of Triton X-100 was added and the suspension stirred for 3 hours at room temperature before centrifugation at 100,000 x g for 60 minutes.
  • the supernatant, containing the NEP was dialyzed twice at 4°C against 50 liters of 5 mM Tris-HCl pH 7.5, 0.05% Triton X-100.
  • the dialyzed enzyme was chromatographed at 4°C on a column (26 x 2.6 cm) of DEAE Toyopeari 650 M equilibrated in 5 mM Tris-HCl pH 7.5, 0.05% Triton X-100.
  • the column was washed with same buffer (5 mM Tris-HCl pH 7.5, 0.05% Triton X-100).
  • the column was then developed with 900 ml of linear gradient of 0-0.3 M NaCl in 10 mM Tris-HCl, 0.05% Triton X-100 buffer (pH7.5) at a flow rate of 1 ml/min.
  • the fractions containing the NEP activity were pooled and stored at 4°C.
  • Reaction solution contains 50 ⁇ l of 50 mM HEPES pH 6.5, 50 ⁇ l of leucine aminopeptidase (Sigma Type III-CP, porcine kidney cytosol) diluted 1/100 in water, 50 ⁇ l of NEP prepared as described above, 50 ⁇ l of 20 ⁇ M GH-Ala-Ala-Phe-MCA (made by Peptide Institute, Osaka), 50 ⁇ l of water and 10 ⁇ l of sample solution in a total volume of 210 ⁇ l. Blank values were obtained by using water (100 ⁇ l) instead of NEP and leucine aminopeptidase under identical experimental conditions. The reaction was stopped by the addition of 10 ⁇ l of 20 % acetic acid. MCA fluorescence was measured with Titertek Fluoroskan II (excitation 355 nm, emission 460 nm).
  • mice Male Sprague-Dawley rats (250-300 g) were anesthetized with pentobarbital sodium (Nembutal- Abbott, 50 mg/kg of body weight).
  • a femoral artery catheter was implant for monitoring arterial pressure and heart rate, and a femoral vein catheter was used for injection of saline or drugs.
  • Blood pressure and heart rate was recorded from the femoral artery through a cannula (PE-50) connected to a pressure transducer (Nihon Koden, MPU-0.5A) coupled to a Biophysiograph RM 6000 system.
  • Sodium salt of WS79089-3 substance was markedly attenuated the hypertensive effect of human big ET-l (lnmol/kg, iv) by the inhibiting the conversion of human big ET-1 to human ET-1.
  • the pharmaceutical composition of this invention can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains WS79089 substance(s) or its(their) pharmaceutically acceptable salt(s), as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral administrations.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotion, gel, cream, and any other form suitable for use.
  • the carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening, solubilizing and coloring agents and perfumes may be used.
  • the composition for applying the composition to human, it is preferable to apply it by intravenous, intramuscular, topical or oral administration.
  • dosage of therapeutically effective amount of WS79089 substance(s) vary(ies) from and also depends upon the age and condition of each individual patient to be treated, in the case of individual patient to be treated, in the case of intravenous administration, a daily dose of 0.01 - 10 mg of WS79089 substance(s) per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of WS79089 substance(s) per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of WS79089 substance(s) of human being is generally given for treating.
  • a loopful of Streptosporangium sp. No. 79089 on mature slant culture was transferred into a 225-ml Erlenmeyer flask containing 60 ml of sterile seed medium composed of corn starch 1%, glycerin 1 %, glucose 0.5%, cotton seed flour 1%, dried yeast 0.5%, corn steep liquor 0.5% and CaCO ⁇ 0.2%.
  • the medium was adjusted to pH 6.5 prior to addition of CaCO-i.
  • These flasks were shaken on a rotary shaker (220 rpm, 5.1 cm-throw) for 3 days at 30°C.
  • the resultant seed culture (3.2 ml) was transferred into each 500-ml Erlenmeyer flask containing 160 ml of production medium which contained starch 5%, potato protein 1%, wheat germ 1%, soy bean powder 0.5 %, ZnCl 2
  • the whole cultured broth (7.5 liters) was extracted with an equal volume of acetone at neutral pH, and filtered with the aid of diatomaceous earth. After the filtrate was concentrated in vacuo to give an aqueous solution (1 liter), an aqueous solution was adjust to pH 2.0 with 6N HCl and then extracted twice with 1 liter of ethyl acetate. Two liters of ethyl acetate extract was concentrated under reduced pressure after dehydration over anhydrous Na S0 . The resultant oily materials were applied to a Silicar CC-4 (Mallinckrodt) column chromatography (2 liters).
  • the column was washed with 1 liter of chloroform and the first active fractions were eluted with 2 liters of chloroform and the second active fractions were eluted with 2 liters of chloroform-methanol (100:1).
  • the first active fractions containing WS79089-2 were pooled and concentrated in vacuo to give deep red powder. After washing the deep red powder with 5 ml of methanol, and methanol insoluble material was evaporated to dryness to give deep red pure powder of WS79089-2 substance (36 mg).
  • the second active fractions containing WS79089-1 substance were pooled and concentrated under reduced pressure to give red oily residue.
  • WS79089-1 substance 50 mg was solubilized with 5 ml of aqueous IN NaOH solution at room temperature for 30 minutes for subjecting hydrolysis reaction.
  • the hydrolysed product was diluted with 50 ml of water and desalted on a Diaion HP-20 column (100 ml, Mitsubishi Chemical Ind.). The column was washed with 200 ml of water, then a sodium salt of WS79089-3 substance was eluted with 100 ml of methanol.
  • the active fraction was concentrated in vacuo to give a sodium salt of WS79089-3 substance (32 mg) as deep red powder.
  • WS79089-2 substance (20 mg) was solubilized with 2 ml of aqueous IN NaOH solution at room temperature for 30 minutes for subjecting hydrolysis reaction.
  • the conversion product was diluted with 20 ml of water and desalted on a Diaion HP-20 column (40 ml, made by Mitsubishi Chemical Lid.). The column was washed with 80 ml of water, then a sodium salt of WS79089-3 substance was eluted with 40 ml of methanol.
  • the active fraction was concentrated in vacuo to give a sodium salt of WS79089-3 substance (14 mg) as deep red powder.
  • a sodium salt of WS79089-3 substance (70 mg) was dissolved in 50 ml of water. The solution was adjusted to pH 3.5 with IN HCl , from which WS79089-3, per se, free type was extracted with 50 ml of ethyl acetate. This procedure was repeated. The ethyl acetate layers were accumulated, dried over anhydrous Na 2 S0 4 , and concentrated in vacuo to give WS79089-3 substance, per se, free type (50 mg).
  • the whole cultured broth obtained by a method described in Example 1 was extracted with an equal volume of acetone at neutral pH, and filtered with the aid of diatomaceous earth, and then added 150 liters of water.
  • the filtrate was passed through a column (20 liters) of Diaion HP-20.
  • the column was washed with 100% methanol and then eluted with 100% acetone.
  • the eluate 60 liters was evaporated to dryness under reduced pressure.
  • the residue was dissolved in chloroform (100 ml) and applied to a Silica gel 60 column (2 liters). The column was developed with 3 liters of chloroform.
  • the active fractions (from 2 liters to 3 liters) containing WS79089-4 substance were combined and then concentrated to a small volume of solution under reduced pressure.
  • the materials obtained were mixed with Silica gel 60 (25 g) and evaporated under reduced pressure to remove organic solvent.
  • the resultant dry powder was applied to a top of same silica gel column (500 ml) which was pre-packed with n- hexane.
  • the column was washed with 1 liter of n-hexane and with 2.5 liters of a mixture of n-hexane-ethyl acetate (2:1), and eluted with 2 liters of a mixture of n-hexane-ethyl acetate (1:1).
  • the active fractions were collected and concentrated under reduced pressure to give deep red powder of WS79089-4 substance (106 mg)
  • WS79089-4 substance (25 mg) was suspended in 2 ml of aqueous IN NaOH solution and shook gently at room temperature for 60 minutes.
  • the conversion product was desalted on a Diaion HP-20 column (40 ml, Mitsubishi Chemical Ind.). The column was washed with 200 ml of water, then a sodium salt of WS79089-5 substance was eluted with 40 ml of methanol. The active fraction was concentrated in vacuo to give sodium salt of WS79089-5 substance (25 mg) as deep red powder.
  • Sodium salt of WS79089-5 substance (24 mg) was dissolved in 3 ml of water. The solution was adjusted to pH 3.5 with IN HCl, from which a free type of WS79089-5 substance was extracted with 2 ml of ethyl acetate. This procedure was repeated. The ethyl acetate layers were accumulated and concentrated under reduced pressure to give a free type of WS79089-5 substance (19 mg).

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Abstract

This invention relates to novel compounds, WS79089 substances or salts thereof, having endothelin converting enzyme inhibiting activity, to processes for the preparation thereof and to a pharmaceutical composition comprising the same.

Description

DESCRIPTION
WS 79089 SUBSTANCES FROM STREPTOSPORANGIUM.
TECHNICAL FIELD
This invention relates to new bioactive compounds, hereinafter referred to WS79089 substances or salts thereof which are useful as a medicament.
DISCLOSURE OF INVENTION
The present invention relates to novel compounds, WS79089 substances or salts thereof which have endothelin converting enzyme inhibiting activity, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which is useful as endothelin converting enzyme inhibiting agents, and to a use thereof as a medicament.
Accordingly, one object of this invention is to provide novel compounds, WS79089 substances or salts thereof which are of use for treating and preventing acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis, and the like.
WS79089 substances include WS79089-1 substance, WS79089-2 substance, WS79089-3 substance, WS79089-4 substance and WS79089- 5 substance, and they are collectively referred to as WS79089 substances. WS79089-1 substance, WS79089-2 substance and WS79089-4 substance may be produced by fermentation process of a certain microorganism. Accordingly, another object of this invention is to provide a process for production of WS79089-1 and/or WS79089- 2 substance and/or WS79089-4 substance by fermentation of a WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging to the genus Streptosporangium in a nutrient medium. A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, WS79089 substances or salts thereof.
Still further object of this invention is to provide a use of WS79089 substances or salts thereof for treating and preventing acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis, and the like.
Suitable salts of WS79089 substances are conventional pharmaceutically acceptable salts and include a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, etc.), and the like.
WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance can be produced by fermentation of a WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging to the genus Streptosporangium such as Streptosporangium sp. No. 79089 in a nutrient medium.
It is to be understood that the production of WS79089-1 and/or WS79089-2 substance and/or WS79089-4 substance is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only. This invention also includes the use of any mutants which are capable of producing WS79089-1 and/or WS79089- 2 substance and/or WS79089-4 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-ray, ultra¬ violet radiation, treatment which N-methyl-N'-nitro-N- nitrosoguanidine, 2-aminopurine, and the like.
Particulars of Streptosporangium sp. No. 79089 is as follows: (1) Taxonomic characteristics of Streptosporangium sp. No. 79089
(i) Materials and methods
The strain No. 79089 was isolated from a soil sample obtained from Tateyama-shi, Chiba-ken, Japan. The methods and media described by Shirling and Gottlieb^1 ) and Waksman 2) were employed for the taxonomic study. The observations were made after 14 to 21 day's cultivation at 30°C. The morphological observations were made on the cultures grown on yeast extract-malt extract agar and oatmeal agar using an optical microscope and a scanning electron microscope. The color names used in this study were taken from the Methuen Handbook of Colour^. The temperature range for growth and NaCl tolerance were determined on yeast extract-malt extract agar. The preparation of cells and detection of the isomer of diaminopimelic acid was performed by procedure of Becker et ar-4'. Detection of whole-cell sugar was performed by the method of Lechevalier and Lechevalier^5). Phospholipid composition was determined by the method of Lechevalier et al^\
(i) Results and discussion
The vegetative mycelium developed well without fragmentation. The aerial mycelium branched and produced globose sporangia. The sporangia were globose and 5.5- 11 μm (usually 7- 9 μm) in diameter with a short sporangiophore. Sporangiospores were formed by septation of a coiled hyphae within the sporangium. They were spherical to oval, 0.6-1.0 x 0.8-1.4 μm, 20 to 50 spores per sporangium and nonmotile.
The results of cultural characteristics are shown in Table 1. Strain No. 79089 required B vitamins for growth, so that it grew poorly on some synthetic agar media. On yeast extract-malt extract agar and oatmeal agar, the strain formed white to pale red aerial mycelium. Reverse side color was yellowish white, light orange, red and reddish brown. Reddish soluble pigments were produced in yeast extract-malt extract agar and oatmeal agar. Both reverse mycelial pigments and soluble pigments were pH sensitive, changing from red to yellowish orange with addition of 0.05N HC1. Melanoid pigments were not produced in tryptone-yeast extract broth, peptone-yeast extract-iron agar and tyrosine agar.
DL-Diaminopimelic acid was detected in the whole-cell hydrolysates of strain No. 79089. Rhamnose, ribose, madurose, mannose, glucose and galactose were detected as whole-cell sugar, placing this strain in the cell wall type 3B. The diagnostic phospholipids detected were phospholipids containing glucosamine, phosphatidylethanolamine, phosphatidylinositol and diphosphatidyl glycerol, placing it in the type P4 phospholipid group.
Physiological properties are shown in Table 2.
From the morphological and chemical characteristics described above, the strain No. 79089 is considered to belong to the genus Streptosporangium Couch^7'8). Therefore, this strain was designated as Streptosporangium sp. No. 79089.
A lyophilized sample of the Streptosporangium sp. No. 79089 has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan as PERM BP-4009 (deposited date: 11 September 1992).
(to be continued)
Table 1 Cultural characteristics of the strain No. 79089
Medium Cultural characteristics
Yeast extract-malt G: good extract agar A: abundant, pale red (9A3) R: reddish brown (9D6) S: reddish
Oatmeal agar G: moderate
A: abundant, white to pale red (7 A3)
R: pastel red (7A4)
S: pale orange
Inorganic salts-starch G: poor agar A: none
R: white to yellowish white (4A2)
S: none
Glycerol-asparagine G: poor agar A: none
R: white to yellowish white (4A2)
S: none
Peptone-yeast extract- G: moderate iron agar A: none
R: light orange (5A4)
S: none
Tyrosine agar G: poor
A: none
R: white to yellowish white (4A2)
S: none
( to be continued) Medium Cultural characteristics
Nutrient agar G: moderate
A: none
R: pale yellow (3A3)
S: none
Starch-yeast extract G: good agar* A: moderate, white to reddish white (8A2)
R: brownish red (8C6)
S: pale orange
Sucrose-nitrate agar G: poor
A: thin, white
R: white to yellowish white (4A2)
S: none
Glucose-asparagine G: poor agar A: thin, white
R: white to yellowish white (4A2)
S: none
Abbreviation : G: growth, A: aerial mycelium,
R: reverse side color, S: soluble pigment
* Starch-yeast extract agar comprises soluble starch 1%, yeast extract 0.2% and agar 1.5% (pH 7.3).
(to be continued) Table 2 Physiological characteristics of the strain No.79089
Figure imgf000009_0001
+ : positive, ± : weekly positive, - : negative
* Basal medium was Pridham-Gottlieb's inorganic medium (ISP medium No. 9) ^ , containing Hayakawa's B-vitamins^. Literature Cited:
(1) Shirling, E. B. and D. Gottlieb: Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol.16,313-340, 1966
(2) Waksman, S. A.: The actinomycetes Vol. 2: Classification, identification and description of genera and species: The Williams and Wilkins Co., Baltimore, 1961
(3) Kornerup, A. and J. H. Wanscher: Methuen Handbook of Colour, Methuen, London, 1978
(4) Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates: Appl. Microbiol. 12, 421-423, 1964
(5) Lechevalier, M. P. and H. Lechevalier: Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol. 20, 435-443, 1970
(6) Lechevalier, M. P., C. De Bievre and H. A. Lechevalier: Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem. Syst. Ecol. 5, 249-260, 1977
(7) Buchanan, R. E. and N. E. Gibbons: Bergey 's Manual of Determinative Bacteriology, 8th edition: The Williams and W lkins Co., Baltimore, 1974
(8) Williams, S. T: Bergey's Manual of Systematic Bacteriology, Vol. 4. Williams and Wilkins, Baltimore, 1989
(9) Hayakawa, M and H. Nonomura: Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J. Ferment. Technol., 65, 501-509, 1987.
Production of WS79089 substances
WS79089-1 substance and/or WS79089-2 substance and/or WS79089-4 substance are produced when WS79089-1 substance and/or WS79089-2 substance and/or WS79089-4 substance-producing strain belonging the genus Streptosporangium is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.).
The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose or glycerin, or the like.
The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, com steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like.
The carbon and nitrogen sources, though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.
When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium slats, copper salts, zinc salts, or cobalt salts, or the like.
If necessary, especially when the culture medium foams seriously a defaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, and the like.
The fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 30°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.
When the fermentation is completed, the culture broth is then subjected for recovery of WS79089-1 substance, WS79089-2 substance and WS79089-4 substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof.
WS79089-3 substance may be produced by subjecting WS79089-1 substance or WS79089-2 substance to hydrolysis with alkali. WS79089-5 substance also may be produced by subjecting WS79089-4 substance to hydrolysis with alkali. By such hydrolysis with alkali, corresponding salt may be obtained according to the alkali used in the process. Thus obtained salt may be converted to free type, WS79089-3 substance, per se or WS79089-5 substance, per se by a conventional manner.
Generally, the salt of WS79089 substances can be prepared by a conventional manner, during or after the recovery and purification of WS79089 substances.
WS79089-1 substance as obtained has the following physico- chemical properties:
Appearance:
Deep red powder Molecular formula:
C27H20°9 Melting point:
186 - 193°C Molecular weight:
HR-FABMS: m/z 489.1204 (M + H)+ (calculated: 489.1185)
Solubility:
Soluble: chloroform, dichloromethane, ethyl acetate
Slightly soluble: n-hexane
Insoluble: water Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction
(to be continued) Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.26 trifluoroacetic acid (0.1%) Silica gel 6OF25 * chloroform : methanol=100:l 0.13
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%) Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm) Flow rate: 1.0 ml/minute Detection: UV 235 nm Retention time: 9.4 minutes ** trade name: made by E. Merck Ultraviolet absorption spectrum: λmax (methanol) (ε): 220 (36800), 245 (38600), 266 (sh) (34100),
300 (sh) (13200), 345 (sh) (5400), 479(16600) nm λmax (methanol-O.lN HCl) (ε): 220 (36500), 245 (39200), 266 (sh)
(34800), 300 (sh) (13700), 345 (sh) (5400), 477(17500) nm λmax (methanol-O.lN NaOH) (ε): 222 (sh) (39700), 245 (43900), 300 (sh) (11800), 345 (sh) (7800), 500(15700) nm Infrared spectrum: vmax (KBr): 3457' 2931' 2849' 1656' 1616' 1540' 1474' 1456' 1438, 1420, 1369, 1304, 1243, 1217, 1175, 1136, 1067, 1038,
983, 956, 923, 860, 843, 804, 766, 753, 720, 701, 656, 586, cm'
H Nuclear magnetic resonance spectrum: (400 MHz, CD2C12) δ
14.11 (IH, s), 13.01 (IH, s), 11.99 (IH, s), 7.86 (IH, d, J=8Hz), 7.70 (IH, t, J=8Hz), 7.34 (IH, d, J=8Hz), 6.85 (IH, s), 5.38 (IH, broad s), 4.86 (IH, m), 4.03 (3H, s), 3.19 (IH, broad d, J=16Hz), 3.12 - 2.94 (3H, m), 1.59 (3H, d, J=6Hz) as shown in Fig. 1 1 3 C Nuclear magnetic resonance spectrum:
(100 MHz, CD2C12) δ
188.7 (s), 187.7 (s), 170.5 (s), 163.0 (s), 159.9 (s), 158.6 (s), 151.9 (s), 144.9 (s), 143.8 (s), 141.4 (s), 136.6 (d), 133.2 (s), 130.3 (s), 125.3 (d), 123.1 (s), 119.5 (d), 119.1 (d), 117.9 (s), 117.3 (s), 115.9 (s), 108.3 (s), 76.7 (d), 63.3 (q), 61.0 (d), 38.6 (t), 34.9 (t), 20.9 (q) as shown in Fig. 2.
WS79089-2 substance as obtained has the following physico- chemical properties:
Appearance:
Deep red powder Melting point:
Above 260°C Molecular formula :
C29H22°10 Molecular weight:
FABMS: m/z 531 (M + H)+ Solubility:
Soluble: chloroform, dichloromethane
Slightly soluble: n-hexane, methanol
Insoluble: water Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.21 trifluoroacetic acid (0.1%) Silica gel 60F2r4* chloroform : methanol= 100:1 0.55
* made by E. Merck High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%) Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm) Flow rate: 1.0 ml/minute Detection: UV 235 nm Retention time: 14.0 minutes ** trade name: made by E. Merck Ultraviolet absorption spectrum: λmax (methanol) (ε): 222 (sh) (29300), 245 (31300), 266 (sh) (25000), 300 (sh) (10100), 345 (sh) (4300), 479(12100) nm λmax (methanol-O.lN HCl) (ε): 222 (sh) (29500), 245 (32900), 266 (sh) (26900), 300 (sh) (11200), 345 (sh) (4500), 477(13800) nm λmax (methanol-O.lN NaOH) (ε): 222 (sh) (34600), 245 (38300), 300 (sh) (10600), 345 (sh) (6900), 500 (13800) nm Infrared spectrum: vmax (KBr): 2949' 1744' 1663' 1617' 1509' 1452' 1419' 1364' 1312, 1295, 1268, 1246, 1217, 1177, 1164, 1127, 1085, 1066,
1029, 1018, 982, 947, 920, 899, 861, 807, 793, 755, 584 cm
H Nuclear magnetic resonance spectrum: (400 MHz, CD2C12) δ
14.11 (IH, s), 12.96 (IH, s), 12.00 (IH, s), 7.85 (IH, dd, J=8,
1.5Hz), 7.68 (IH, t, J=8Hz), 7.32 (IH, dd, J=8, 1.5Hz), 6.74 (IH, s), 6.46 (IH, t, J=3Hz), 4.84 (IH, m), 3.90 (3H, s), 3.31 (IH, dd,
J=17, 2Hz), 3.04 (lH,dd, J=17, 4Hz), 2.95 (2H, m), 1.91(3H, s),
1.55 (3H, d, J=6Hz) as shown in Fig. 3
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD2C12) δ
188.9 (s), 187.7 (s), 170.5 (s), 170.4 (s), 163.1 (s), 160.0 (s), 158.5 (s), 152.2 (s), 144.3 (s), 141.3 (s), 139.7 (s), 136.7 (d), 133.2 (s), 131.9,(s), 125.4 (d), 123.1 (s), 119.2 (d), 119.1 (d), 118.2 (s), 117.3 (s), 116.5 (s), 108.5 (s), 76.7 (d), 63.0 (q), 62.8 (d), 35.6 (t), 34.9 (t), 21.2 (q), 20.9 (q) as shown in Fig. 4. Sodium salt of WS79089-3 substance as obtained has the following physico-chemical properties:
Appearance:
Deep red powder Molecular formula :
C27H21°10Na Solubility:
Soluble: water, methanol
Slightly soluble: chloroform
Insoluble: n-hexane Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.48 trifluoroacetic acid (0.1%) Silica gel 60F254* chloroform : methanol= 100:1 0.00
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 1.0 ml/minute
Detection: UV 235 nm
Retention time: 4.5 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction
Infrared spectrum: vm maQVx (KBr): 3373, 2922, 2854, 1615, 1592, 1456, 1370, 1301,
1244, 1193, 1164, 1128, 1070, 1036, 974, 928, 857, 802, 765, 695, 604 cm'1 H Nuclear magnetic resonance spectrum: (400 MHz, CD3OD) δ
7.73 (IH, d, J=8Hz), 7.62 (IH, t, J=8Hz), 7.20 (IH, d, J=8Hz), 6.68
(IH, s), 5.27 (IH, broad s), 4.15 (IH, m), 3.98 (3H, s), 3.38 (IH, dd, J=5, 13Hz), 3.11 (lH,dd, J=7, 13Hz), 2.99 (IH, dd, J=2, 16Hz),
2.76 (IH, dd, J=2, 16Hz), 1.25 (3H, d, J=6Hz) as shown in Fig. 5
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD3OD) δ
188.9 (s), 188.7 (s), 176.4 (s), 163.4 (s), 161.4 (s), 159.1 (s), 153.1 (s), 144.1 (s), 143.9 (s), 139.5 (s), 137.2 (d), 134.6 (s), 134.4 (d), 125.3 (d), 124.5 (d), 123.2 (s), 120.7 (s), 119.6 (d), 118.2 (s), 118.1 (s), 117.1 (s), 70.0 (d), 63.5 (q), 61.3 (d), 44.4 (t), 38.8 (t), 23.6 (q) as shown in Fig. 6.
WS79089-3 substance (free type, WS79089-3 substance, per se) as obtained has the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
190 - 195°C Molecular formula :
C27H22°10 Molecular weight:
FABMS: m/z 507 (M + H)+ Solubility:
Soluble: methanol
Slightly soluble: chloroform
Insoluble: n-hexane Ultraviolet absorption spectrum: λmax (methanol) (ε): 224 (39300), 245 (31200), 266 (sh) (26600), 313 (14200), 486(14500) nm λmax (methanol-O.lN HCl) (ε): 222 (35500), 245 (33100), 266 (29800), 300 (sh) (15500), 477(16100) nm λmax (methanol-O.lN NaOH) (ε): 222 (41200), 245 (sh) (27900), 270 (31200), 313 (sh) (9600), 405 (sh) (6100), 530(15700) nm Infrared spectrum: vm lllα!JλY (KBr): 3212, 2970, 1716, 1615, 1568, 1474, 1455, 1414,
1366, 1298, 1244, 1187, 1167, 1130, 1069, 1037, 1014, 993, 928,
766 cm"
H Nuclear magnetic resonance spectrum: (400 MHz, DMSO-d6) δ
12.90 (IH, s), 7.80 - 7.72 (2H, m), 7.36 (IH, dd, J=8, 1.5Hz), 6.78
(IH, s), 5.21 (IH, broad s), 3.93 (3H, s), 3.91 (IH, m), 3.05 - 2.95
(2H, m), 2.85 - 2.72 (2H, m), 1.13 (3H, d, J=6Hz) as shown in Fig.
7
13 C Nuclear magnetic resonance spectrum:
(100 MHz, DMSO-d6) δ
187.1 (s), 186.9 (s), 171.3 (s), 161.4 (s), 156.7 (s), 156.3 (s), 151.4 (s), 143.8 (s), 141.5 (s), 139.5 (s), 136.4 (d), 132.7 (s), 131.3 (s), 124.4 (d), 123.3 (d), 122.0 (s), 118.5 (s), 118.4 (d), 116.9 (s), 116.6 (s), 115.7 (s), 67.1 (d), 62.5 (q), 58.6 (d), 44.0 (t), 37.7 (t), 23.8 (q) as shown in Fig. 8.
WS79089-4 substance as obtained has the following physico- chemical properties:
Appearance:
Deep red powder Melting point:
230 - 233°C Molecular formula :
C27H20°8 Elemental analysis: Calcd: for
Figure imgf000018_0001
C, 68.64; H, 4.27 (%) Found:
C, 67.98; H, 4.33 (%) Molecular weight: FABMS: m/z 473 (M + H)+ Solubility:
Soluble: chloroform, dichloromethane, ethyl acetate
Slightly soluble: n-hexane, methanol
Insoluble: water Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.02 trifluoroacetic acid (0.1%) Silica gel 60F2 4* chloroform : methanol= 100:1 0.76
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 1.0 ml/minute
Detection: UV 235 nm
Retention time: 31.0 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Ultraviolet absorption spectrum: λmax (methanol) (ε): 220 (30900), 245 (29400), 266 (sh) (27500), 300 (sh) (11900), 345 (sh) (4100), 479(14700) nm λmax (methanol-O.lN HCl) (ε): 220 (30900), 245 (30000), 266 (28200), 300 (sh) (11900), 345 (sh) (4100), 477(14700) nm λmax (methanol-O.lN NaOH) (ε): 220 (31700), 245 (31400), 300 (sh) (9670), 345 (sh) (5800), 500 (14100) nm Infrared spectrum: vmax (KBr): 2936' 1733' 1662' 1616' 1568' 1475' 1457' 1439' 1417, 1368, 1321, 1306, 1266, 1242, 1227, 1204, 1169, 1127, 1082, 1064, 1038, 1012, 986, 951, 925, 899, 863, 854, 842, 792,
753, 731, 705, 661, 640, 600, 590, 580 cm -1
H Nuclear magnetic resonance spectrum: (400 MHz, CD2Cl2) δ
14.11 (IH, s), 13.03 (IH, s), 11.96 (IH, broad s), 7.81 (IH, dd, J=7.5, 1.5Hz), 7.65 (IH, t, J=8Hz), 7.28 (IH, dd, J=8.5, 1.5Hz), 6.74 (IH, s), 4.76 (IH, m), 3.87 (3H, s), 3.41 (IH, m), 2.97 (2H, m), 2.83 (IH, m), 2.65 (IH, m), 2.38 (IH, m), 1.55 (3H, d, J=6Hz) as shown in Fig. 9.
C Nuclear magnetic resonance spectrum: (100 MHz, CD2Cl2) δ
188.5 (s), 188.2 (s), 170.7 (s), 163.0 (s), 160.4 (s), 158.5 (s), 151.7 (s), 149.9 (s), 146.6 (s), 141.1 (s), 136.5 (d), 133.4 (s), 131.0 (s), 125.0 (d), 122.6 (s), 118.9 x 2 (d and s), 117.4 (s), 117.2 (d), 114.5 (s), 107.8 (s), 76.3 (d), 61.7 (q), 34.9 (t), 30.3 (t), 23.1 (t), 20.9 (q) as shown in Fig. 10.
WS79089-5 substance (free type, WS79089-5 substance, per se) as obtained has the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
225 - 228°C Molecular formula :
C27H22°9 Molecular weight:
FABMS: m/z 491 (M + H)+ Solubility:
Soluble: methanol, dichloromethane
Slightly soluble: water
Insoluble: n-hexane Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value RP-18 WF254s* 85% methanol containing 0.31 trifluoroacetic acid (0.1%) Silica gel 60F2^4* chloroform : methanol=100:l 0.00
* made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 9.6 ml/minute
Detection: UV 235 nm
Retention time: 9.6 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Ultraviolet absorption spectrum: λmax (methanol) (ε): 224 (38600), 245 (28100), 266 (23600), 313 (14300), 486 (13200) λmax (methanol-O.lN HCl) (ε): 222 (32900), 245 (27800), 266 (25100), 300 (sh) (14200), 477(13000) nm λmax (methanol-O.lN NaOH) (ε): 222 (39000), 245 (sh) (27100), 270 (26500), 313 (sh) (8300), 405 (sh) (4400), 515(12300) nm Infrared spectrum: v^QY (KBr): 2932, 1704, 1642, 1614, 1474, 1457, 1437, 1415,
1366, 1304, 1245, 1167, 1134, 1068, 1037, 1009, 987, 934, 863, 794,
756, 732, 706, 655, 602, 590, 569, 560 cm"1
H Nuclear magnetic resonance spectrum: (400 MHz, CD30D:CD2C12=1:1) δ
7.85 (lH,dd, J=8, 1Hz), 7.70 (IH, t, J=8Hz), 7.33 (IH, dd, J=8, 1Hz), 6.86 (IH, s), 4.11 (IH, m), 3.89 (3H, s), 3.09 (lH,dd, J=13, 5Hz), 3.01 (lH,dd, J=8, 13Hz), 2.92 (IH, broad s), 2.75 (2H, broad t, J=6.5Hz), 1.26 (3H, d, J=6Hz) as shown in Fig. 11. 1 3 C Nuclear magnetic resonance spectrum:
(100 MHz, CD3OD: CD2C12=1:1) δ
189.2 (s), 188.4 (s), 173.3 (s), 163.0 (s), 158.0 (s), 157.3 (s), 152.4 (s), 147.5 (s), 145.7 (s), 142.6 (s), 136.9 (d), 133.5 (s), 132.1 (s), 125.5 (d), 122.9 (s), 122.8 (d), 119.5 (d), 118.8 (s), 117.6 (s), 117.4 (s), 115.0 (s), 69.3 (d), 61.8 (q), 45.1 (t), 30.0 (t), 23.6 (t), 23.4 (q) as shown in Fig. 12.
Biological properties of WS79089 substances
WS79089 substances possess pharmacological activities such as endothelin converting enzyme inhibiting activity, and the like, and therefore are useful for the treatment and prevention of acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure , rheumatoid arthritis, osteoarthritis, and the like.
As examples for showing biological activities of WS79089 substances, some biological data are explained in the following.
Test 1 Endothelin converting enzyme (ECE) inhibition assay
Preparation of Cell Homogenate
The enzyme was obtained from bovine carotid endothelial cells.
Q
Cultured cells (about 1 x 10 cells) were scraped off, washed with calcium, magnesium PBS (phosphate buffered saline) and homogenized with a polytron (made by Brinkman Instrument) at setting maximal speed for 3 x 10 sec on ice in 10 ml of 0.25 M sucrose containing 25 mM HEPES buffer (pH 7.4).
Measurement of ECE activity
Human big endothelin- 1 (hereinafter, endothelin- 1 is referred to as ET-1) (50 ng) was incubated with the cell homogenate preparation (20- 30 μg protein) for 16 hours at 37°C in 250 μl of 50 mM Tris-HCl buffer (pH 7.0) containing 0.5 mM p-chloromercuriphenylsulfonic acid and 0.05 mM diisopropylfluorophosphate. And then, obtained 20 μl of reaction mixture was diluted with 80 μl of RIA buffer [50 mM Tris- HC1 buffer (pH 7.0) containing 0.1 % BSA, 0.1 % Triton X-100 and 1 mM EDTA] and mixed with 50 μl of anti-ET-1 serum (antisera against the C-terminal peptide of ET-1 (16-21), final dilution, 1:20,000) and 50 μl of 125I-ET-1 (37 KBq/lOml) and then incubated for 2 hours at 4°C. After 2 hours incubation added 0.5 ml of Amerlex-M donkey anti- rabbit (made by Amersham Japan, 1/4 cone), vortex mixed and incubated for 10 minutes at room temperature. After magnetic separation (Amerlex-M system) the supernatant was removed by decant and immunocomplex was counted in a gamma counter (Packard Auto Gamma Model 5650).
Results
IC50 (μg/ml) values of WS79089 substances against ECE
WS79089 substances IC50 (μg/ml)
Figure imgf000023_0001
Human skin fibroblast
Cells were obtained from explants of skin of a normal healthy volunteer, grown in Dulbecco's modified minimum essential medium (DMEM) supplemented with 10 % fetal calf serum, penicillin (50 μ/ml) and streptomycin (50 μ ml) at 37°C in a humid atomosphere (5% C02-95% air). Cells were subcultured by trypsinization and used between passage 5 to 15.
Collagenase induction
Fibroblasts (1.5 x
Figure imgf000024_0001
plastic flask. After 3 days, medium was removed, cells were washed with 20 ml DMEM, and then 11.25 ml DMEM supplemented with bovine serum albumin (3 mg/ml) and Interleukin lβ (Genzyme Co.,
U.S.A., 100 u/ml) was added and cultured for 3 days. The medium was collected, centrifuged at 1500 rpm for 5 minutes to eliminate cell fragments and stored at -20°C until use. On use, one milliliter of cultured supernatant was activated with 200 μl of trypsin (Sigma, 100 μg/ml), followed by addition of 200 μl of soybean trypsin inhibitor
(Sigma, 400 μg/ml).
Assay
All reactions were performed in U-shaped microtiter plates. Twenty-five microliters of inhibitor were serially diluted in 0.1 M Tris-HCl buffer (pH 7.5) containing 0.4 M NaCl and 10 mM CaCl2.
Fifty microliters of FITC (Fluorescein isothiocyanate)-collagen (Collagen Gijutsu Kenshukai, Tokyo, Japan, 1 mg/ml) solution and 25 μl of 80 mM o-phenanthrolin was added, 70 μl of ethanol and 30 μl of Tris-HCl buffer were added, mixed and centrifuged at 3000 rpm for 10 min. Fluorescene (excitation 485 nm, emission 538 nm), derived from the eliminated FITC by collagenase, in the supernatant was measured and inhibitory percent by test sample was calculated.
(to be continued) IC^Q (μg/ml) values of WS79089 substances against Collagenase
WS79089 substances IC5Q (μg/ml)
Figure imgf000025_0001
Test 3 NEP inhibition assay
Preparation of Neutral Endopeptidase (NEP)
Forty Sprague-Dawley male rats (6 week old) were sacrificed by C02 gas generated from dry ice. Eighty kidneys (600 g) were dissected to be free of their capsules and their combined volume was measured by displacement in 150 ml of ice-cold 10 mM phosphate buffer (pH 7.0). Unless specified, all the following procedures were carried out at 0-4 °C. The kidneys were minced with scissors and then homogenized with a polytron (made by Brinkman PT-20, maximal speed for 6 x 20 sec) in 100 ml of 10 mM phosphate buffer (pH 7.0). The homogenate was centrifuged at 20,000 x g for 60 minutes at 4°C. The pellet was resuspended in 250 ml buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.0) and then 250 ml of Triton X-100 was added and the suspension stirred for 3 hours at room temperature before centrifugation at 100,000 x g for 60 minutes. The supernatant, containing the NEP was dialyzed twice at 4°C against 50 liters of 5 mM Tris-HCl pH 7.5, 0.05% Triton X-100. The dialyzed enzyme was chromatographed at 4°C on a column (26 x 2.6 cm) of DEAE Toyopeari 650 M equilibrated in 5 mM Tris-HCl pH 7.5, 0.05% Triton X-100. The column was washed with same buffer (5 mM Tris-HCl pH 7.5, 0.05% Triton X-100). The column was then developed with 900 ml of linear gradient of 0-0.3 M NaCl in 10 mM Tris-HCl, 0.05% Triton X-100 buffer (pH7.5) at a flow rate of 1 ml/min. The fractions containing the NEP activity were pooled and stored at 4°C.
Measurement of NEP activity using microplates for enzyme inhibitor detection.
Reaction solution contains 50 μl of 50 mM HEPES pH 6.5, 50 μl of leucine aminopeptidase (Sigma Type III-CP, porcine kidney cytosol) diluted 1/100 in water, 50 μl of NEP prepared as described above, 50 μl of 20 μM GH-Ala-Ala-Phe-MCA (made by Peptide Institute, Osaka), 50 μl of water and 10 μl of sample solution in a total volume of 210 μl. Blank values were obtained by using water (100 μl) instead of NEP and leucine aminopeptidase under identical experimental conditions. The reaction was stopped by the addition of 10 μl of 20 % acetic acid. MCA fluorescence was measured with Titertek Fluoroskan II (excitation 355 nm, emission 460 nm).
IC50 (μg/ml) values of WS79089 substances against NEP
WS79089 substances IC5Q (μg/ml)
Figure imgf000026_0001
Test 4
The effect of sodium salt of WS79089-3 substance on the pressor activity of human big ET-1 in vivo
Method
Male Sprague-Dawley rats (250-300 g) were anesthetized with pentobarbital sodium (Nembutal- Abbott, 50 mg/kg of body weight). A femoral artery catheter was implant for monitoring arterial pressure and heart rate, and a femoral vein catheter was used for injection of saline or drugs. Blood pressure and heart rate was recorded from the femoral artery through a cannula (PE-50) connected to a pressure transducer (Nihon Koden, MPU-0.5A) coupled to a Biophysiograph RM 6000 system. Three groups of rats were injected with the following: human big ET-1 (1 nmol/kg) alone (n=4), human big ET-1 (1 nmol/kg) with sodium salt of WS79089-3 substance (3.2 mg/kg) iv bolus injection (n=4).
Results
Sodium salt of WS79089-3 substance was markedly attenuated the hypertensive effect of human big ET-l (lnmol/kg, iv) by the inhibiting the conversion of human big ET-1 to human ET-1.
(to be continued)
Data are means ± S.E.M.
Significant differences between the groups are indicated; * p<0.05, **p<0.01.
Figure imgf000028_0001
The pharmaceutical composition of this invention can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains WS79089 substance(s) or its(their) pharmaceutically acceptable salt(s), as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral administrations. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotion, gel, cream, and any other form suitable for use.
The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening, solubilizing and coloring agents and perfumes may be used.
For applying the composition to human, it is preferable to apply it by intravenous, intramuscular, topical or oral administration. While the dosage of therapeutically effective amount of WS79089 substance(s) vary(ies) from and also depends upon the age and condition of each individual patient to be treated, in the case of individual patient to be treated, in the case of intravenous administration, a daily dose of 0.01 - 10 mg of WS79089 substance(s) per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of WS79089 substance(s) per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of WS79089 substance(s) of human being is generally given for treating.
Following examples are given for the purpose of illustrating the present invention in more detail.
Example 1
Production of WS79089-1 substance and WS79089-2 substance
(1) Fermentation
A loopful of Streptosporangium sp. No. 79089 on mature slant culture was transferred into a 225-ml Erlenmeyer flask containing 60 ml of sterile seed medium composed of corn starch 1%, glycerin 1 %, glucose 0.5%, cotton seed flour 1%, dried yeast 0.5%, corn steep liquor 0.5% and CaCO^ 0.2%. The medium was adjusted to pH 6.5 prior to addition of CaCO-i. These flasks were shaken on a rotary shaker (220 rpm, 5.1 cm-throw) for 3 days at 30°C. The resultant seed culture (3.2 ml) was transferred into each 500-ml Erlenmeyer flask containing 160 ml of production medium which contained starch 5%, potato protein 1%, wheat germ 1%, soy bean powder 0.5 %, ZnCl2
0.01 % and CaCO^ 0.2%. The medium was adjusted to pH 6.5 prior to addition of CaCO^. These fifty flasks were incubated at 30°C for 96 hours on a rotary shaker set at 220 rpm (5.1 cm-throw).
(2) Isolation
The whole cultured broth (7.5 liters) was extracted with an equal volume of acetone at neutral pH, and filtered with the aid of diatomaceous earth. After the filtrate was concentrated in vacuo to give an aqueous solution (1 liter), an aqueous solution was adjust to pH 2.0 with 6N HCl and then extracted twice with 1 liter of ethyl acetate. Two liters of ethyl acetate extract was concentrated under reduced pressure after dehydration over anhydrous Na S0 . The resultant oily materials were applied to a Silicar CC-4 (Mallinckrodt) column chromatography (2 liters). The column was washed with 1 liter of chloroform and the first active fractions were eluted with 2 liters of chloroform and the second active fractions were eluted with 2 liters of chloroform-methanol (100:1). The first active fractions containing WS79089-2 were pooled and concentrated in vacuo to give deep red powder. After washing the deep red powder with 5 ml of methanol, and methanol insoluble material was evaporated to dryness to give deep red pure powder of WS79089-2 substance (36 mg). The second active fractions containing WS79089-1 substance were pooled and concentrated under reduced pressure to give red oily residue. The oily residue was applied to a Lobar pre-packed LiChroprep Si 60 (40-63 μm) column ( made by E. Merck, size C; 440-37). The column was washed with 400 ml of chloroform-methanol (100:1) and WS79089-1 substance was eluted with 700 ml of chloroform-methanol (100:1). The active fractions containing WS79089-1 substance were pooled and concentrated in vacuo to give deep red powder of WS79089-1 substance (125 mg).
Example 2
Conversion of WS79089-1 substance to WS79089-3 substance
WS79089-1 substance (50 mg) was solubilized with 5 ml of aqueous IN NaOH solution at room temperature for 30 minutes for subjecting hydrolysis reaction. The hydrolysed product was diluted with 50 ml of water and desalted on a Diaion HP-20 column (100 ml, Mitsubishi Chemical Ind.). The column was washed with 200 ml of water, then a sodium salt of WS79089-3 substance was eluted with 100 ml of methanol. The active fraction was concentrated in vacuo to give a sodium salt of WS79089-3 substance (32 mg) as deep red powder.
Example 3
Conversion of WS79089-2 substance to WS79089-3 substance
WS79089-2 substance (20 mg) was solubilized with 2 ml of aqueous IN NaOH solution at room temperature for 30 minutes for subjecting hydrolysis reaction. The conversion product was diluted with 20 ml of water and desalted on a Diaion HP-20 column (40 ml, made by Mitsubishi Chemical Lid.). The column was washed with 80 ml of water, then a sodium salt of WS79089-3 substance was eluted with 40 ml of methanol. The active fraction was concentrated in vacuo to give a sodium salt of WS79089-3 substance (14 mg) as deep red powder.
Example 4
Conversion of a sodium salt of WS79089-3 substance to a free type of WS79089-3 substance
A sodium salt of WS79089-3 substance (70 mg) was dissolved in 50 ml of water. The solution was adjusted to pH 3.5 with IN HCl , from which WS79089-3, per se, free type was extracted with 50 ml of ethyl acetate. This procedure was repeated. The ethyl acetate layers were accumulated, dried over anhydrous Na2S04, and concentrated in vacuo to give WS79089-3 substance, per se, free type (50 mg).
Example 5
The whole cultured broth obtained by a method described in Example 1 (75 liters) was extracted with an equal volume of acetone at neutral pH, and filtered with the aid of diatomaceous earth, and then added 150 liters of water. The filtrate was passed through a column (20 liters) of Diaion HP-20. The column was washed with 100% methanol and then eluted with 100% acetone. The eluate (60 liters) was evaporated to dryness under reduced pressure. The residue was dissolved in chloroform (100 ml) and applied to a Silica gel 60 column (2 liters). The column was developed with 3 liters of chloroform. The active fractions (from 2 liters to 3 liters) containing WS79089-4 substance were combined and then concentrated to a small volume of solution under reduced pressure. The materials obtained were mixed with Silica gel 60 (25 g) and evaporated under reduced pressure to remove organic solvent. The resultant dry powder was applied to a top of same silica gel column (500 ml) which was pre-packed with n- hexane. The column was washed with 1 liter of n-hexane and with 2.5 liters of a mixture of n-hexane-ethyl acetate (2:1), and eluted with 2 liters of a mixture of n-hexane-ethyl acetate (1:1). The active fractions were collected and concentrated under reduced pressure to give deep red powder of WS79089-4 substance (106 mg)
Example 6
Conversion of WS79089-4 substance to WS79089-5 substance
WS79089-4 substance (25 mg) was suspended in 2 ml of aqueous IN NaOH solution and shook gently at room temperature for 60 minutes. The conversion product was desalted on a Diaion HP-20 column (40 ml, Mitsubishi Chemical Ind.). The column was washed with 200 ml of water, then a sodium salt of WS79089-5 substance was eluted with 40 ml of methanol. The active fraction was concentrated in vacuo to give sodium salt of WS79089-5 substance (25 mg) as deep red powder.
Sodium salt of WS79089-5 substance (24 mg) was dissolved in 3 ml of water. The solution was adjusted to pH 3.5 with IN HCl, from which a free type of WS79089-5 substance was extracted with 2 ml of ethyl acetate. This procedure was repeated. The ethyl acetate layers were accumulated and concentrated under reduced pressure to give a free type of WS79089-5 substance (19 mg).

Claims

1. WS79089 substances or salts thereof which include
WS79089-1 substance having the following physico-chemical properties:
Appearance:
Deep red powder Molecular formula:
C27H20°9 Melting point:
186 - 193°C Molecular weight:
HR-FABMS: m/z 489.1204 (M + H)+ (calculated: 489.1185)
Solubility:
Soluble: chloroform, dichloromethane, ethyl acetate
Slightly soluble: n-hexane
Insoluble: water Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.26 trifluoroacetic acid (0.1%) Silica gel 60F ^4* chloroform : methanol=100:l 0.13
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%) Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm) Flow rate: 1.0 ml/minute Detection: UV 235 nm Retention time: 9.4 minutes ** trade name: made by E. Merck Ultraviolet absorption spectrum: λmax (methanol) (ε): 220 (36800), 245 (38600), 266 (sh) (34100),
300 (sh) (13200), 345 (sh) (5400), 479(16600) nm λmax (methanol-O.lN HCl) (ε): 220 (36500), 245 (39200), 266 (sh)
(34800), 300 (sh) (13700), 345 (sh) (5400), 477(17500) nm λmax (methanol-O.lN NaOH) (ε): 222 (sh) (39700), 245 (43900), 300 (sh) (11800), 345 (sh) (7800), 500(15700) nm Infrared spectrum: vmaγ (KBr): 3457, 2931, 2849, 1656, 1616, 1540, 1474, 1456,
1438, 1420, 1369, 1304, 1243, 1217, 1175, 1136, 1067, 1038,
983, 956, 923, 860, 843, 804, 766, 753, 720, 701, 656, 586, cm"1
H Nuclear magnetic resonance spectrum: (400 MHz, CD2Cl2) δ
14.11 (IH, s), 13.01 (IH, s), 11.99 (IH, s), 7.86 (IH, d, J=8Hz),
7.70 (IH, t, J=8Hz), 7.34 (IH, d, J=8Hz), 6.85 (IH, s), 5.38 (IH, broad s), 4.86 (IH, m), 4.03 (3H, s), 3.19 (IH, broad d, J=16Hz),
3.12 - 2.94 (3H, m), 1.59 (3H, d, J=6Hz) as shown in Fig. 1
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD2C12) δ
188.7 (s), 187.7 (s), 170.5 (s), 163.0 (s), 159.9 (s), 158.6 (s), 151.9 (s), 144.9 (s), 143.8 (s), 141.4 (s), 136.6 (d), 133.2 (s), 130.3 (s), 125.3 (d), 123.1 (s), 119.5 (d), 119.1 (d), 117.9 (s), 117.3 (s), 115.9 (s), 108.3 (s), 76.7 (d), 63.3 (q), 61.0 (d), 38.6 (t), 34.9 (t), 20.9 (q) as shown in Fig. 2,
WS79089-2 substance having the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
Above 260°C Molecular formula :
C29H22°10 Molecular weight:
FABMS: m/z 531 (M + H)+ Solubility:
Soluble: chloroform, dichloromethane
Slightly soluble: n-hexane, methanol
Insoluble: water Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.21 trifluoroacetic acid (0.1%) Silica gel 60F2^4* chloroform : methanol= 100:1 0.55
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%) Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm) Flow rate: 1.0 ml/minute Detection: UV 235 nm Retention time: 14.0 minutes ** trade name: made by E. Merck Ultraviolet absorption spectrum: λmax (methanol) (ε): 222 (sh) (29300), 245 (31300), 266 (sh) (25000), 300 (sh) (10100), 345 (sh) (4300), 479(12100) nm λmax (methanol-O.lN HCl) (ε): 222 (sh) (29500), 245 (32900), 266 (sh) (26900), 300 (sh) (11200), 345 (sh) (4500), 477(13800) nm λmax (methanol-O.lNNaOH) (ε): 222 (sh) (34600), 245 (38300), 300 (sh) (10600), 345 (sh) (6900), 500 (13800) nm Infrared spectrum: vmax (KBr): 2949' 1744' 1663' 1617' 1509' 1452' 1419' 1364' 1312, 1295, 1268, 1246, 1217, 1177, 1164, 1127, 1085, 1066,
1029, 1018, 982, 947, 920, 899, 861, 807, 793, 755, 584 cm"1
H Nuclear magnetic resonance spectrum: (400 MHz, CD2C12) δ
14.11 (IH, s), 12.96 (IH, s), 12.00 (IH, s), 7.85 (IH, dd, J=8,
1.5Hz), 7.68 (IH, t, J=8Hz), 7.32 (IH, dd, J=8, 1.5Hz), 6.74 (IH, s), 6.46 (IH, t, J=3Hz), 4.84 (IH, m), 3.90 (3H, s), 3.31 (IH, dd,
J=17, 2Hz), 3.04 (lH,dd, J=17, 4Hz), 2.95 (2H, m), 1.91(3H, s),
1.55 (3H, d, J=6Hz) as shown in Fig. 3
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD2C12) δ
188.9 (s), 187.7 (s), 170.5 (s), 170.4 (s), 163.1 (s), 160.0 (s), 158.5 (s), 152.2 (s), 144.3 (s), 141.3 (s), 139.7 (s), 136.7 (d), 133.2 (s), 131.9,(s), 125.4 (d), 123.1 (s), 119.2 (d), 119.1 (d), 118.2 (s), 117.3 (s), 116.5 (s), 108.5 (s), 76.7 (d), 63.0 (q), 62.8 (d), 35.6 (t), 34.9 (t), 21.2 (q), 20.9 (q) as shown in Fig. 4.
sodium salt of WS79089-3 substance having the following physico- chemical properties:
Appearance:
Deep red powder Molecular formula :
C27H21°10Na Solubility:
Soluble: water, methanol Slightly soluble: chloroform Insoluble: n-hexane Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.48 trifluoroacetic acid (0.1%) Silica gel 60F254* chloroform : methanol= 100:1 0.00
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 1.0 ml/minute
Detection: UV 235 nm
Retention time: 4.5 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Infrared spectrum: vmav (KBr): 3373, 2922, 2854, 1615, 1592, 1456, 1370, 1301,
1244, 1193, 1164, 1128, 1070, 1036, 974, 928, 857, 802, 765, 695,
604 cm'1
H Nuclear magnetic resonance spectrum: (400 MHz, CD3OD) δ
7.73 (IH, d, J=8Hz), 7.62 (IH, t, J=8Hz), 7.20 (IH, d, J=8Hz), 6.68 (IH, s), 5.27 (IH, broad s), 4.15 (IH, m), 3.98 (3H, s), 3.38 (IH, dd, J=5, 13Hz), 3.11 (lH,dd, J=7, 13Hz), 2.99 (IH, dd, J=2, 16Hz), 2.76 (IH, dd, J=2, 16Hz), 1.25 (3H, d, J=6Hz) as shown in Fig. 5
13 C Nuclear magnetic resonance spectrum:
Figure imgf000037_0001
188.9 (s), 188.7 (s), 176.4 (s), 163.4 (s), 161.4 (s), 159.1 (s), 153.1 (s), 144.1 (s), 143.9 (s), 139.5 (s), 137.2 (d), 134.6 (s), 134.4 (d), 125.3 (d), 124.5 (d), 123.2 (s), 120.7 (s), 119.6 (d), 118.2 (s), 118.1 (s), 117.1 (s), 70.0 (d), 63.5 (q), 61.3 (d), 44.4 (t), 38.8 (t), 23.6 (q) as shown in Fig. 6,
WS79089-3 substance (free type, WS79089-3 substance, per se) having the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
190 - 195°C Molecular formula :
C27H22°10 Molecular weight:
FABMS: m/z 507 (M + H)+ Solubility:
Soluble: methanol Slightly soluble: chloroform Insoluble: n-hexane Ultraviolet absorption spectrum: λmax (methanol) (ε): 224 (39300), 245 (31200), 266 (sh) (26600),
313 (14200), 486(14500) nm λmax (methanol-O.lN HCl) (ε): 222 (35500), 245 (33100), 266
(29800), 300 (sh) (15500), 477(16100) nm λmax (methanol-O.lN NaOH) (ε): 222 (41200), 245 (sh) (27900), 270 (31200), 313 (sh) (9600), 405 (sh) (6100), 530(15700) nm Infrared spectrum: vm Illααγλ (KBr): 3212, 2970, 1716, 1615, 1568, 1474, 1455, 1414,
1366, 1298, 1244, 1187, 1167, 1130, 1069, 1037, 1014, 993, 928,
766 cm"1
H Nuclear magnetic resonance spectrum: (400 MHz, DMSO-d6) δ
12.90 (IH, s), 7.80 - 7.72 (2H, m), 7.36 (IH, dd, J=8, 1.5Hz), 6.78 (IH, s), 5.21 (IH, broad s), 3.93 (3H, s), 3.91 (IH, m), 3.05 - 2.95 (2H, m), 2.85 - 2.72 (2H, m), 1.13 (3H, d, J=6Hz) as shown in Fig. 7
13 C Nuclear magnetic resonance spectrum:
(100 MHz, DMSO-d6) δ
187.1 (s), 186.9 (s), 171.3 (s), 161.4 (s), 156.7 (s), 156.3 (s), 151.4 (s), 143.8 (s), 141.5 (s), 139.5 (s), 136.4 (d), 132.7 (s), 131.3 (s), 124.4 (d), 123.3 (d), 122.0 (s), 118.5 (s), 118.4 (d), 116.9 (s), 116.6 (s), 115.7 (s), 67.1 (d), 62.5 (q), 58.6 (d), 44.0 (t), 37.7 (t), 23.8 (q) as shown in Fig. 8,
WS79089-4 substance having the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
230 - 233°C Molecular formula :
C27H20°8 Elemental analysis: Calcd: for C^yH^Og
C, 68.64; H, 4.27 (%) Found:
C, 67.98; H, 4.33 (%) Molecular weight:
FABMS: m/z 473 (M + H)+ Solubility:
Soluble: chloroform, dichloromethane, ethyl acetate Slightly soluble: n-hexane, methanol Insoluble: water
(to be continued) Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.02 trifluoroacetic acid (0.1%) Silica gel 60F2^4* chloroform : methanol= 100:1 0.76
* made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 1.0 ml/minute
Detection: UV 235 nm
Retention time: 31.0 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Ultraviolet absorption spectrum: λmax (methanol) (ε): 220 (30900), 245 (29400), 266 (sh) (27500), 300 (sh) (11900), 345 (sh) (4100), 479(14700) nm λmax (methanol-O.lN HCl) (ε): 220 (30900), 245 (30000), 266 (28200), 300 (sh) (11900), 345 (sh) (4100), 477(14700) nm λmax (methanol-O.lN NaOH) (ε): 220 (31700), 245 (31400), 300 (sh) (9670), 345 (sh) (5800), 500 (14100) nm Infrared spectrum: vmQY (KBr): 2936, 1733, 1662, 1616, 1568, 1475, 1457, 1439,
1417, 1368, 1321, 1306, 1266, 1242, 1227, 1204, 1169, 1127, 1082, 1064, 1038, 1012, 986, 951, 925, 899, 863, 854, 842, 792,
753, 731, 705, 661, 640, 600, 590, 580 cm'1
H Nuclear magnetic resonance spectrum: (400 MHz, CD2C12) δ 14.11 (IH, s), 13.03 (IH, s), 11.96 (IH, broad s), 7.81 (IH, dd, J=7.5, 1.5Hz), 7.65 (IH, t, J=8Hz), 7.28 (IH, dd, J=8.5, 1.5Hz), 6.74 (IH, s), 4.76 (IH, m), 3.87 (3H, s), 3.41 (IH, m), 2.97 (2H, m), 2.83 (IH, m), 2.65 (IH, m), 2.38 (IH, m), 1.55 (3H, d, J=6Hz) as shown in Fig. 9.
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD2C12) δ
188.5 (s), 188.2 (s), 170.7 (s), 163.0 (s), 160.4 (s), 158.5 (s), 151.7 (s), 149.9 (s), 146.6 (s), 141.1 (s), 136.5 (d), 133.4 (s), 131.0 (s), 125.0 (d), 122.6 (s), 118.9 x 2 (d and s), 117.4 (s), 117.2 (d), 114.5 (s), 107.8 (s), 76.3 (d), 61.7 (q), 34.9 (t), 30.3 (t), 23.1 (t), 20.9 (q) as shown in Fig. 10,
and
WS79089-5 substance (free type, WS79089-5 substance, per se) has the following physico-chemical properties:
Appearance:
Deep red powder Melting point:
225 - 228°C Molecular formula :
C27H22°9 Molecular weight:
FABMS: m/z 491 (M + H)+ Solubility:
Soluble: methanol, dichloromethane Slightly soluble: water Insoluble: n-hexane
(to be continued) Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
RP-18 WF254s* 85% methanol containing 0.31 trifluoroacetic acid (0.1%) Silica gel 60F2c4* chloroform : methanol= 100:1 0.00
* made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: 80% methanol containing trifluoroacetic acid (0.1%)
Column: LiChrospher 100 RP-18** (5μm) (250 x 4 mm)
Flow rate: 9.6 ml/minute
Detection: UV 235 nm
Retention time: 9.6 minutes
** trade name: made by E. Merck Color reaction:
Positive: eerie sulfate reaction, iodine vapor reaction
Negative: ninhydrin reaction Ultraviolet absorption spectrum: λmax (methanol) (ε): 224 (38600), 245 (28100), 266 (23600), 313 (14300), 486 (13200) λmax (methanol-O.lN HCl) (ε): 222 (32900), 245 (27800), 266 (25100), 300 (sh) (14200), 477(13000) nm λmax (methanol-O.lN NaOH) (ε): 222 (39000), 245 (sh) (27100), 270 (26500), 313 (sh) (8300), 405 (sh) (4400), 515(12300) nm Infrared spectrum: vmax (KBr): 2932' 1704' 1642' 1614' 1474' 1457' 1437' 1415' 1366, 1304, 1245, 1167, 1134, 1068, 1037, 1009, 987, 934, 863, 794,
7 T.56, 732, 706, 655, 602, 590, 569, 560 cm
Ii H Nuclear magnetic resonance spectrum: (400 MHz, CD30D:CD2C12=1 :1) δ
7.85 (lH,dd, J=8, 1Hz), 7.70 (IH, t, J=8Hz), 7.33 (IH, dd, J=8, 1Hz), 6.86 (IH, s), 4.11 (IH, m), 3.89 (3H, s), 3.09 (lH,dd, J=13, 5Hz), 3.01 (lH,dd, J=8, 13Hz), 2.92 (IH, broad s), 2.75 (2H, broad t, J=6.5Hz), 1.26 (3H, d, J=6Hz) as shown in Fig. 11.
13 C Nuclear magnetic resonance spectrum:
(100 MHz, CD3OD: CD2C12=1:1) δ
189.2 (s), 188.4 (s), 173.3 (s), 163.0 (s), 158.0 (s), 157.3 (s), 152.4 (s), 147.5 (s), 145.7 (s), 142.6 (s), 136.9 (d), 133.5 (s), 132.1 (s), 125.5 (d), 122.9 (s), 122.8 (d), 119.5 (d), 118.8 (s), 117.6 (s), 117.4 (s), 115.0 (s), 69.3 (d), 61.8 (q), 45.1 (t), 30.0 (t), 23.6 (t), 23.4 (q) as shown in Fig. 12.
2. A process for production of WS79089-1 substance as defined above and/or WS79089-2 substance as defined above, and/or WS79089-4 substance as defined above, or a salt thereof, which comprises culturing a WS79089-1 substance and/or WS79089-2 substance and/or WS79089- 4 substance-producing strain belonging to the genus Streptosporangium in a nutrient medium and recovering the same.
3. A process of Claim 2, in which said strain is Streptosporangium sp. No. 79089 (PERM BP-4009) .
4. Biological pure culture of Streptosporangium sp. No. 79089.
5. A method of preparing WS79089-3 substance as defined above by subjecting WS79089-1 substance as defined above or WS79089-2 substance as defined above or a salt thereof to hydrolysis reaction.
6. A pharmaceutical composition containing WS79089 substance(s) as defined above or a salt thereof.
7. Use of WS79089 substance(s) as defined above or a salt thereof for the manufacture of a medicament for therapeutic treatment or prevention of acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis in human or animal.
8. A method for treating or preventing acute renal failure, myocardial infarction, subarachnoid hemorrhage, asthma, hypertension and some other peripheral circulatory failure, rheumatoid arthritis, osteoarthritis which comprises administrating WS79089 substance(s) as defined above or a salt thereof to human or animal.
9. WS79089 substance or salts thereof for use as a medicament.
PCT/JP1993/001504 1992-10-26 1993-10-19 Ws79089 substances from streptosporangium WO1994010324A1 (en)

Priority Applications (1)

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JP51088094A JP3304358B2 (en) 1992-10-26 1993-10-19 WS79089 substance from Streptosporangium

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GB929222485A GB9222485D0 (en) 1992-10-26 1992-10-26 Ws79089substances

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4293546A (en) * 1980-07-24 1981-10-06 Smithkline Corporation Anthracycline antibiotics produced by Streptosporangium fragilis Shearer sp. nov. ATCC 31519

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4293546A (en) * 1980-07-24 1981-10-06 Smithkline Corporation Anthracycline antibiotics produced by Streptosporangium fragilis Shearer sp. nov. ATCC 31519

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 110, no. 25, 19 June 1989, Columbus, Ohio, US; abstract no. 228218c, OHUCHI, SHOKICHI ET AL.: "New angiotensin converting enzyme inhibitors,SF2513A,B and C, produced by Streptosporangium nondiastaticum." page 326; *
CHEMICAL ABSTRACTS, vol. 116, no. 5, 3 February 1992, Columbus, Ohio, US; abstract no. 37872a, HAYAKAWA,MASAYUKI ET AL.: "New methods for the highly selective isolation of Streptosporangium and Dactylosporangium from soil." page 421; *
J.FERMENT.BIOENG., vol. 72, no. 5, 1991, pages 327 - 333 *
MEIJI SEIKA KENKYU NENPO, vol. 27, 1988, pages 46 - 54 *

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JP3304358B2 (en) 2002-07-22
GB9222485D0 (en) 1992-12-09

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