WO1995018142A1 - Wf15604 substances - Google Patents

Wf15604 substances Download PDF

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Publication number
WO1995018142A1
WO1995018142A1 PCT/JP1994/002192 JP9402192W WO9518142A1 WO 1995018142 A1 WO1995018142 A1 WO 1995018142A1 JP 9402192 W JP9402192 W JP 9402192W WO 9518142 A1 WO9518142 A1 WO 9518142A1
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Prior art keywords
substance
salts
wf15604a
substances
liters
Prior art date
Application number
PCT/JP1994/002192
Other languages
French (fr)
Inventor
Toshihiro Shibata
Motoaki Nishikawa
Tomoko Sato
Shigehiro Takase
Michio Yamashita
Seiji Hashimoto
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.)
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Priority claimed from GB9326520A external-priority patent/GB9326520D0/en
Priority claimed from GB9405532A external-priority patent/GB9405532D0/en
Application filed by Fujisawa Pharmaceutical Co., Ltd. filed Critical Fujisawa Pharmaceutical Co., Ltd.
Priority to AU13709/95A priority Critical patent/AU1370995A/en
Priority to JP7517896A priority patent/JPH09511902A/en
Publication of WO1995018142A1 publication Critical patent/WO1995018142A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
    • C07J53/002—Carbocyclic rings fused
    • C07J53/004—3 membered carbocyclic rings
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal 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
    • C12P33/00—Preparation of steroids
    • 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

  • This invention relates to new bioactive compounds, WF15604A substance and WF15604B substance, hereinafter, referred to briefly as WF15604 substances, or salts thereof which are useful as a medicament.
  • the present invention relates to new compounds, WF15604A substance and WF15604B substance, and pharmaceutically acceptable salts thereof which have inhibitory activity on cholesterol biosynthesis as well as antifungal activity, to a process for preparation thereof, to a pharmaceutical composition containing the same, to a use of the same as a medicament, and to a method for treating hypercholesterolaemic states, hyperlipoproteinaemic states and their associated conditions as well as fungal infections.
  • one object of this invention is to provide WF15604 substances (WF15604A substance and/or WF15604B substance) or pharmaceutically acceptable salts thereof which inhibit cholesterol biosynthesis as well as fungal growth, and therefore are capable of lowering blood serum cholesterol levels and blood lipid levels as well as treating fungal infections in human beings or animals.
  • Another object of this invention is to provide a process for production of the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof by fermentation of a WF15604 substances-producing strain such as Sporormiella minima No. 15604 in a nutrient medium.
  • a further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof.
  • Still further object of this invention is to provide a use of the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof as a medicament and a method for treating hypercholesterolaemic states, hyperlipoproteinaemic states and their associated conditions, and fungal infections in human beings or animals.
  • LDL-C low density lipoprotein cholesterol
  • the WF15604 substances or salts thereof inhibit cholesterol biosynthesis and fungal growth. They lower concentrations of cholesterol in blood. Thus it is useful for treating hypercholesterolaemic states and hyperlipoproteinaemic states (e. g. atherosclerosis), and their associated conditions (e. g. angina, myocardial infarction, cerebral vascular occlusion, arterial aneurism, peripheral vascular disease, recurrent pancreatitis and xanthomas) as well as fungal infections.
  • hypercholesterolaemic states and hyperlipoproteinaemic states e. g. atherosclerosis
  • their associated conditions e. g. angina, myocardial infarction, cerebral vascular occlusion, arterial aneurism, peripheral vascular disease, recurrent pancreatitis and xanthomas
  • the WF15604 substances can be produced by fermentation of the WF15604 substances-producing strain such as Sporormiella minima No. 15604 in a nutrient medium.
  • the production of the WF15604 substances 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 the WF15604 substances 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 with N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.
  • the fungus strain No. 15604 was originally isolated from a soil sample, collected at Mt. Kiyosumi, Chiba-ken, Japan. This organism grew very rapidly on various culture media, and formed dark green to brown colonies. Strain No.15604 formed teleomorph, consisting of ascomata, on some agar media. The asci were bitunicate and cylindrical, the ascospores had gelatinous sheath, and each cell of ascospore had an elongated germ slit. On the basis of its morphological characteristics, the strain appears to belong to the ascomycetes genus Sporormiella Ellis et Everhart 1892 - Its mycological characteristics were as follows.
  • the morphological characteristics were determined on the basis of the cultures on LCA agar - ' after four weeks. Perithecia were scattered or loosely aggregated, immersed or superficial, subglobose to nearly pyriform, 130 - 200 x 110 - 180 ⁇ m, smooth, and dark brown to black. Asci were eight-spored, cylindrical, and 60 - 90 x 20 - 30 ⁇ m. Ascospores were obliquely bi- or tri-seriate, four-celled, cylindrical, 23 - 30 x 4 - 5 ⁇ m, broadly rounded at end, straight or curved, ranging from hyaline when young through brown, and transversely septate. Constrictions at septa were broad and deep.
  • the vegetative hyphae were smooth, septate, hyaline to brown, and branched.
  • the hyphal cells were cylindrical and 1 - 4 ⁇ m in diameter.
  • Strain No.15604 was able to grow at the temperature range from 6 to 36 °C with the growth optimum at 23 to 31 °C. These temperature data were determined on potato dextrose agar (made by NISSUI, Japan).
  • strain No.15604 resembled Sporormiella minima Ahmed et Cain 1972. And above characteristics corresponded with this species discription by
  • strain No.15604 was identified as a strain of Sporormiella minima and has been deposited to the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, as FERM BP-4507 (deposited date: December 21, 1993).
  • Potato dextrose G Spreading broadly, 6.5 - 7.5 cm agar (Difco 0013) S: Circular, sulcate, thin, formed immature ascomata, yellowish brown (5F4), yellowish white (3A2) at the edge R: Grayish brown (5F3), yellowish white (1 A2) at the edge
  • Czapek's solution G Spreading broadly, 5.0 - 6.0 cm agar (Raper and S:. Irregular, plane, thin, formed immature Thorn 1949) ascomata, yellowish white (1A2) R: Yellowish white (1 A2)
  • Oatmeal agar G Spreading broadly, >7.5 cm (Difco 0552)
  • S Sulcate, felty to cottony, did not form .
  • R Brownish gray (4D2), pale yellow (4A3) at the edge
  • Emerson Yp Ss agar G Spreading broadly, >7.5 cm (Difco 0739)
  • S Plane, thin, felty, formed ascomata, greenish gray (30C2), orange gray (5B2) at the center, greenish white (30A2) at the edge
  • R Dark green (30F3), orange white (5A2) at the center, greenish white (30A2) at the edge
  • Corn meal agar G Spreading broadly, 6.5 - 7.0 cm (Difco 0386) S: Circular, plane, thin, felty, did not form teleomorph, dark green (30F4) R: Dark green (30F4)
  • MY20 agar* G Spreading broadly, 5.0 - 7.0 cm S: Irregular, raised, felty, did not form teleomorph, dull green (30E3) R: Dull green (30F4)
  • G growth, measuring colony size in diameter
  • S colony surface
  • R reverse * MY20 agar: 5 g peptone, 3 g yeast extract, 3 g malt extract, 200 g glucose and 20 g agar per liter of water
  • the WF15604 substances are produced when the WF15604 substances-producing strain 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, glycerin, or the like.
  • the preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, corn 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, amino acid, or the like.
  • ammonium salts e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.
  • urea 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.
  • sodium or calcium carbonate sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, cobalt salts, or the like.
  • a defoaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil, 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 fermenter, 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 the WF15604 substances 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.
  • WF15604 substances may be converted to their salts since they are acidic substances.
  • the salts of the WF15604 substances can be prepared by a conventional manner, during or after the recovery and purification of the WF15604 substances.
  • Suitable salts , of the WF15604 substance 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.
  • WF15604A substance has the following physico-chemical properties:
  • Soluble pyridine, dimethyl sulfoxide
  • the WF15604A substance is inferred to have the following plane structural formula.
  • WF15604B substance has the following physico-chemical properties:
  • WF15604 substances or salts thereof are potent inhibitors of cholesterol biosynthesis. Therefore, they can lower blood serum cholesterol levels and blood lipid levels and are useful for the treatment of hyperlipemia and atherosclerosis.
  • HepG2 cells (HB 8065, a human hepatoma cell line) were obtained from the American Type Culture Collection. Cholesterol synthesis in HepG2 cells was determined according to the method of Brown et al. (J. Biol. Chem. 253, 1121-1128, 1978) with some modifications. HepG2 cells were grown in flasks containing Eagle's modified minimum essential medium with non-essential amino acids supplemented with pyruvate (1 mM), penicillin G (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum (FBS) in a humidified incubator (5% C0 2 ) at 37 °C.
  • FBS fetal bovine serum
  • the cells On day 0, the cells (3 x 10 5 cells/well) were seeded into 35 mm 6-well plastic culture dishes (2 ml well). On day 4, the medium was replaced with 1 ml of fresh medium containing 10% human lipoprotein-deficient serum instead of FBS and the cells were preincubated for 2 hours at 37 °C with WF15604 substances (WF15604A substance or WF15604B substance) dissolved in 2 ⁇ l of dimethylsulfoxide (DMSO). DMSO (2 ⁇ l) was added to the control culture. Then, 1 mM [1- C]acetic acid, sodium salt (37 MBq/mmol, DuPont/NEN Research Products) was added to the medium and incubated at 37 °C for 2 hours.
  • WF15604 substances WF15604A substance or WF15604B substance
  • DMSO dimethylsulfoxide
  • the cells were washed with phosphate-buffered saline (PBS), pH 7.4 and then dissolved in 1 ml of aqueous 15% KOH.
  • PBS phosphate-buffered saline
  • the non-saponifiable lipids were extracted twice with 2 ml of petroleum ether, evaporated to dryness, resuspended in 1ml of 50% acetone in 95% ethanol containing 0.1% cold cholesterol as a carrier.
  • Sterols were precipitated by the addition of 1 ml of 0.5% digitonin in 50% ethanol, filtered on a glass filter.
  • WF15604A substance or WF15604B substance Antifungal activities of WF15604 substances (WF15604A substance or WF15604B substance) were measured by micro-broth dilution method in 96 well multi-trays employing yeast nitrogen base dextrose medium.
  • a 50 ⁇ l sample solution with serial 2-fold dilutions was added a 50 ⁇ l of microorganism suspension in saline to yield a final concentration of 1 x 10 colony forming units/ml.
  • the Candida and Aspergillus cultures were incubated at 37°C for 22 hours. After incubation, the growth of microorganism in each well was determined by measuring the turbidity. The results were shown as IC ⁇ Q value in which concentration the turbidity was half of that in the well without sample.
  • WF15604 substances or salts thereof are used in the form of conventional pharmaceutical preparation which contains said substance, as an active ingredient, in admixture with pharmaceutically acceptable carriers such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral and external(topical) administration.
  • pharmaceutically acceptable carriers such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral and external(topical) administration.
  • the pharmaceutical preparations may be in solid form such as tablet, granule, powder, capsule, suppository, solution, suspension, syrup, emulsion, lemonade, lotion, ointment, gel, and the like.
  • auxiliary substances stabilizing agents, wetting agents and other commonly used additives such as lactose, stearic acid, magnesium stearate, terra alba, sucrose, corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cacao butter, ethylene glycol, tartaric acid, citric acid, fumaric acid, and the like.
  • the dosage of the WF15604 substances may vary from and also depend upon the age, conditions of the patient, a kind of diseases, etc.. In general, amount between about 0.1 mg and about 1,000 mg or 42 . l fi . PCT/JP94/02192
  • an average single dose of about 0.1 mg, 1 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200 mg, 250 mg of the WF15604 substances may be used in treating hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions.
  • aqueous seed medium 160 ml containing sucrose 4%, cotton seed flour 2%, dried yeast 1%, peptone 1%, KH 2 P0 4 0.2%, CaC0 3
  • the cultured broth (60 liters) was extracted with 60 liters of acetone by intermittent mixing.
  • the acetone extract was filtered with the aid of diatomaceous earth and 120 liters of water was added.
  • the resultant cake was reextracted with 20 liters of acetone.
  • the acetone extract which was filtered with the aid of diatomaceous earth, was concentrated in vacuo to 9 liters and 18 liters of water was added.
  • These extracts were combined and passed through a column (5 liters) of Diaion HP-20 (Mitsubishi Chemical Ind. Co., Ltd.). The column was washed with water and 50% aqueous methanol, and then eluted with methanol.
  • the column was washed with 50% aqueous methanol and 70% aqueous methanol, and then eluted with 80% aqueous methanol.
  • An active fraction(l liter) was concentrated in vacuo to an aqueous solution, adjusted to pH 2.0 with 1N-HC1 and extracted with an equal volume of ethyl acetate, twice.
  • the extract was washed with water and concentrated in vacuo to dryness.
  • the resultant pale yellowish product was dissolved in a small volume of methanol. After standing at 4°C overnight, WF15604A substance was obtained as colorless prisms (122 mg).
  • the cultured broth (360 liters) obtained by the similar method described above was extracted with 360 liters of acetone by intermittent mixing.
  • the acetone extract was filtered with an aid of diatomaceous earth and 600 liters of water was added.
  • the resultant cake was reextracted with 60 liters of acetone.
  • the acetone extract was filtered with an aid of diatomaceous earth and 180 liters of water was added.
  • WF15604A substance (869 mg, crystallized from methanol) was purified by the same method as described in Example 1.
  • the eluate containing WF15604B substance (45 liters) was adjusted to pH 6.0 with IN NaOH and then concentrated in vacuo to 13 liters.
  • This solution was subjected to a column (1 liter) of YMC gel (ODS- AM 120-S50, YMC Co., Ltd.). The column was washed with acetonitrile-methanol-0.015M (NH 4 ) 2 HP0 4 -H 3 P0 4 buffer (pH 6.0)
  • This active fraction(0.7 liters) was concentrated in vacuo to an aqueous solution.
  • the aqueous solution was adjusted to pH 2.0 with IN HC1 and extracted with an equal volume of ethyl acetate, twice.
  • the extract was washed with water and concentrated in vacuo to dryness.
  • the resultant pale yellowish product was dissolved in a small volume of methanol. After standing at 4°C overnight, WF15604B substance was obtained as colorless needles (46 mg).

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Abstract

The invention relates to new compounds, WF15604 substances or salts thereof, having antifungal activity and inhibitory activity on cholesterol biosynthesis, to a process for the preparation thereof and to a pharmaceutical composition comprising the same, said compounds having formula (I).

Description

DESCRIPTION
WF15604 SUBSTANCES
TECHNICAL FIELD
This invention relates to new bioactive compounds, WF15604A substance and WF15604B substance, hereinafter, referred to briefly as WF15604 substances, or salts thereof which are useful as a medicament.
DISCLOSURES OF INVENTION
The present invention relates to new compounds, WF15604A substance and WF15604B substance, and pharmaceutically acceptable salts thereof which have inhibitory activity on cholesterol biosynthesis as well as antifungal activity, to a process for preparation thereof, to a pharmaceutical composition containing the same, to a use of the same as a medicament, and to a method for treating hypercholesterolaemic states, hyperlipoproteinaemic states and their associated conditions as well as fungal infections.
Accordingly, one object of this invention is to provide WF15604 substances (WF15604A substance and/or WF15604B substance) or pharmaceutically acceptable salts thereof which inhibit cholesterol biosynthesis as well as fungal growth, and therefore are capable of lowering blood serum cholesterol levels and blood lipid levels as well as treating fungal infections in human beings or animals.
Another object of this invention is to provide a process for production of the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof by fermentation of a WF15604 substances-producing strain such as Sporormiella minima No. 15604 in a nutrient medium.
A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof.
Still further object of this invention is to provide a use of the WF15604 substances (WF15604A substance and/or WF15604B substance) or salts thereof as a medicament and a method for treating hypercholesterolaemic states, hyperlipoproteinaemic states and their associated conditions, and fungal infections in human beings or animals.
High levels of blood cholesterol and blood lipids are conditions which are involved in the onset of arteriosclerosis. It is well known that inhibitors of cholesterol biosynthesis are effective in lowering the level of blood plasma cholesterol, especially low density lipoprotein cholesterol (LDL-C). It has now been established that lowering LDL- C levels affords protection from coronary heart disease.
The WF15604 substances or salts thereof inhibit cholesterol biosynthesis and fungal growth. They lower concentrations of cholesterol in blood. Thus it is useful for treating hypercholesterolaemic states and hyperlipoproteinaemic states (e. g. atherosclerosis), and their associated conditions (e. g. angina, myocardial infarction, cerebral vascular occlusion, arterial aneurism, peripheral vascular disease, recurrent pancreatitis and xanthomas) as well as fungal infections.
The WF15604 substances can be produced by fermentation of the WF15604 substances-producing strain such as Sporormiella minima No. 15604 in a nutrient medium.
It is to be understood that the production of the WF15604 substances 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 the WF15604 substances 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 with N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.
Particulars of Sporormiella minima No. 15604 is as follows:
Characteristics of producing strain No. 15604
The fungus strain No. 15604 was originally isolated from a soil sample, collected at Mt. Kiyosumi, Chiba-ken, Japan. This organism grew very rapidly on various culture media, and formed dark green to brown colonies. Strain No.15604 formed teleomorph, consisting of ascomata, on some agar media. The asci were bitunicate and cylindrical, the ascospores had gelatinous sheath, and each cell of ascospore had an elongated germ slit. On the basis of its morphological characteristics, the strain appears to belong to the ascomycetes genus Sporormiella Ellis et Everhart 1892 - Its mycological characteristics were as follows.
Cultural characteristics on various agar media are summarized in Table 1. Culture on Emerson Yp Ss agar grew spreading broadly, attaining more than 7.5 cm in diameter after two weeks at 25 °C . This colony surface was plane, thin, felty, and greenish gray to orange gray. The reverse was dark green to orange white. Ascomata were observed. Colonies on malt extract agar grew spreading broadly, attaining 5.5 - 6.5 cm in diameter under the same conditions. The surface was plane, thin, and dark green. The reverse was dark green. Ascomata were not observed.
The morphological characteristics were determined on the basis of the cultures on LCA agar - ' after four weeks. Perithecia were scattered or loosely aggregated, immersed or superficial, subglobose to nearly pyriform, 130 - 200 x 110 - 180 μm, smooth, and dark brown to black. Asci were eight-spored, cylindrical, and 60 - 90 x 20 - 30 μm. Ascospores were obliquely bi- or tri-seriate, four-celled, cylindrical, 23 - 30 x 4 - 5 μm, broadly rounded at end, straight or curved, ranging from hyaline when young through brown, and transversely septate. Constrictions at septa were broad and deep. Segments were readily separable at the central septum and easily separable at the other septa. Cells of ascospore were nearly equal in size. Terminal cells were very slightly narrow toward the ends. Germ slits were nearly parallel with a kink near the middle. Gelatinous sheath were hyaline.
The vegetative hyphae were smooth, septate, hyaline to brown, and branched. The hyphal cells were cylindrical and 1 - 4 μm in diameter.
Strain No.15604 was able to grow at the temperature range from 6 to 36 °C with the growth optimum at 23 to 31 °C. These temperature data were determined on potato dextrose agar (made by NISSUI, Japan).
According to the taxonomic criteria of the genus Sporormiella, strain No.15604 resembled Sporormiella minima Ahmed et Cain 1972. And above characteristics corresponded with this species discription by
Ahmed et Cain ' *■' without ascus size. Then strain No.15604 was identified as a strain of Sporormiella minima and has been deposited to the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, as FERM BP-4507 (deposited date: December 21, 1993).
(to be continued)
Table 1. Cultural characteristics of strain No. 15604
Medium Cultural characteristics
Malt extract agar G: Spreading broadly, 5.5 - 6.5 cm (Blaskeslee 1915) S: Irregular, plane, thin, did not form teleomorph, dark green (30F4), yellowish white (2A2) at the edge R: Dark green (30F5), yellowish white (1A2) at the edge
Potato dextrose G: Spreading broadly, 6.5 - 7.5 cm agar (Difco 0013) S: Circular, sulcate, thin, formed immature ascomata, yellowish brown (5F4), yellowish white (3A2) at the edge R: Grayish brown (5F3), yellowish white (1 A2) at the edge
Czapek's solution G: Spreading broadly, 5.0 - 6.0 cm agar (Raper and S:. Irregular, plane, thin, formed immature Thorn 1949) ascomata, yellowish white (1A2) R: Yellowish white (1 A2)
Sabouraud dextrose G: Spreading broadly, 6.5 - 7.5 cm agar (Difco 0190) S: Irregular, raised, sulcate, wrinkly at the center, did not form teleomorph, light yellow (4A4) R: Grayish yellow (4B5)
(to be continued) Medium Cultural characteristics
Oatmeal agar G: Spreading broadly, >7.5 cm (Difco 0552) S: Sulcate, felty to cottony, did not form . teleomorph, gray (1E1), pale orange (5 A3) at the edge R: Brownish gray (4D2), pale yellow (4A3) at the edge
Emerson Yp Ss agar G: Spreading broadly, >7.5 cm (Difco 0739) S: Plane, thin, felty, formed ascomata, greenish gray (30C2), orange gray (5B2) at the center, greenish white (30A2) at the edge R: Dark green (30F3), orange white (5A2) at the center, greenish white (30A2) at the edge
Corn meal agar G: Spreading broadly, 6.5 - 7.0 cm (Difco 0386) S: Circular, plane, thin, felty, did not form teleomorph, dark green (30F4) R: Dark green (30F4)
MY20 agar* G: Spreading broadly, 5.0 - 7.0 cm S: Irregular, raised, felty, did not form teleomorph, dull green (30E3) R: Dull green (30F4)
Abbreviation: G: growth, measuring colony size in diameter S: colony surface R: reverse * MY20 agar: 5 g peptone, 3 g yeast extract, 3 g malt extract, 200 g glucose and 20 g agar per liter of water
These characteristics were observed after 14 days of incubation at 25 °C. The color descriptions were based on the Methuen Handbook of Colour^3 \
References:
(1) Ahmed, S. I. and R. F. Cain: Revision of the genera Sporormia and Sporormiella, Canadian Journal of Botany, 50, pp. 419 - 477, 1972.
(2) Miura, K. and M. Y. Kudo: An agar-medium for aquatic Hyphomycetes, Transactions of the Mycological Society of Japan, 11, pp. 116 - 118, 1970.
(3) Koraerup, A. and J. H. Wanscher: Methuen Handbook of Colour (3rd ed.), 525p., Methuen, London, 1978
Production of the WF15604 substances
The WF15604 substances are produced when the WF15604 substances-producing strain 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, glycerin, or the like.
The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, corn 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, 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 2 . g .
as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, cobalt salts, or the like.
If necessary, especially when the culture medium foams seriously a defoaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil, 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 fermenter, 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 the WF15604 substances 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.
WF15604 substances may be converted to their salts since they are acidic substances. The salts of the WF15604 substances can be prepared by a conventional manner, during or after the recovery and purification of the WF15604 substances.
Suitable salts, of the WF15604 substance 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. WF15604A substance has the following physico-chemical properties:
Appearance:
Colorless prisms Molecular formula :
Figure imgf000011_0001
Elementary analysis: •
Calcd for C^H^O-j • CH3OH:
C 67.86, H 8.82 (%) Found:
C 67.81, H 9.15 (%) Molecular weight:
FAB-MS m/z 539 (M + Na)
HRFAB-MS 539.2951 C30H44O7 • Na=539.2984
Melting point:
181 - 190 °C (dec.) Specific rotation:
[α] D (23 °C) +115 ° (c=1.0 in pyridine)
Ultraviolet absorption spectrum: end absorption Solubility:
Soluble: pyridine, dimethyl sulfoxide
Slightly soluble: water
Insoluble: n-hexane Color reaction:
Positive: eerie sulfate reaction and iodine vapor reaction
Negative: ninhydrin reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60W* dichloromethane:methanol (10: 1 , v/v) 0.38
* made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: acetonitrile - methanol - water - phosphoric acid = 70:15:15:0.1
Column: YMC ODS-AM-303** (S-5, 12θA ODS, 4.6mmID x 250mm)
Row rate: 1.0 ml/minute
Detection: UV (210 πm)
Retention time: 7.3 minutes
** trade name: YMC Co. Infrared absorption spectrum: vmax (KBr): 3470' 2960' 2880' 2610' 1720' 1640' 1460' 1380'
1290, 1270, 1240, 1190, 1140, 1080, 1020, 990, 890 cm" 1
H Nuclear magnetic resonance spectrum: (400 MHz, pyridine-d5) δR
5.26 (IH, br d, J=6.5Hz), 5.23 (IH, s), 4.98 (IH, dd, J=7 and 9Hz),
4.87 (IH, br s), 4.83 (IH, br s), 3.20 (IH, dd, J=7 and 17Hz), 3.00
(IH, dd, J=9 and 17Hz), 2.98 (IH, m), 2.60 (IH, d, J=20Hz), 2.54
(IH, d, J=20Hz), 2.30 - 2.11 (4H, m), 1.95 (IH, m), 1.88 (3H, s),
1.90 - 1.77 (2H, m), 1.71 (IH, d, J=6Hz), 1.61 (IH, m), 1.49 (IH, d, J=6Hz), 1.43-1.13 (5H, m), 1.08 (3H, d, J=7Hz), 1.07 (3H, d,
J=7Hz), 0.95 (3H, d, J=6Hz), 0.82 (3H, s) as shown in Fig. 1
13 C Nuclear magnetic resonance spectrum:
(100 MHz, pyridine-d5) δc
208.5 (s), 207.2 (s), 178.7 (s), 156.6 (s), 106.7 (t), 74.0 (s), 72.0 (d), 68.5 (d), 58.5 (d), 57.1 (d), 54.5 (d), 50.1 (s), 47.9 (t), 47.6 (t), 47.5 (t), 41.3 (s), 40.7 (s), 35.8 (d), 34.5 (t), 34.1 (d), 33.9 (s),
31.2 (t), 30.7 (t), 28.3 (t), 22.1 (q), 22.0 (q), 20.7 (t), 18.5 (q),
16.3 (q), 10.5 (q) as shown in Fig. 2 Nature:
Acidic substance.
From the above physical and chemical properties, the WF15604A substance is inferred to have the following plane structural formula.
Figure imgf000013_0001
WF15604B substance has the following physico-chemical properties:
Appearance:
Colorless needles Molecular formula : 30H44°5 Elementary analysis:
Calcd for C3QH4405 • 1 2H20:
C 72.99, H 9.19 (%) Found:
C 73.35, H 9.54 (%) Molecular weight:
FAB-MS m/z 507 (M + Na) (484.68: C3()H4405)
Melting point:
218.1 - 221.5 °C Specific rotation:
[α] D (23 °C) -74 ° (c=0.54 in pyridine)
Ultraviolet absorption spectrum: end absorption
240 nm (sh.) in methanol 240 nm (sh.) in acidic methanol 250 nm (sh.) in alkaline methanol Solubility:
Soluble: pyridine, dimethyl sulfoxide, methanol Slightly soluble: chloroform
Insoluble: water Color reaction:
Positive: eerie sulfate reaction and iodine vapor reaction
Negative: Ehrlich reaction, Molish reaction and ninhidrin reaction Thin layer chromatography (TLC):
- - —
Stationary phase Developing solvent Rf value
Silica Gel 60W* dichloromethane:methanol (10:1, v/v) 0.45
* made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Mobile phase: acetonitrile - methanol - water - phosphoric acid = 70:15:15:0.1
Column: YMC ODS-AM-303** (S-5, 12θA ODS, 4.6mmID x 250mm)
Flow rate: 1.0 ml/minute
Detection: UV (210 nm)
Retention time: 12.3 minutes
** trade name: YMC Co. Infrared absorption spectrum: vmax (KBr): 3420' 3080' 2960' 2870' 1700' 1640' 1470' 1380' 1360, 1340, 1300, 1280, 1260, 1130, 1090, 1060, 1010, 990,
890 cm"
H Nuclear magnetic resonance spectrum: (400 MHz, pyridine-d5) δjj
5.31 (IH, m), 5.05 (IH, m), 4.86 (IH, br s), 4.84 (IH, br s), 4.74 (IH, m), 3.59 (IH, m); 3.18 (IH, dd, J=16 and 7Hz), 3.07 (IH, dd, J=16 and 10Hz), 2.87 (IH, m), 2.31-2.03 (6H, m), 2.00-1.90 (2H, m), 1.76 (IH, m), 1.75 (3H, s), 1.68 (IH, d, J=5Hz), 1.61 (IH, d, J=5Hz), 1.64-1.41 (4H, m), 1.36-1.18 (2H, m), 1.07 (3H, d, J=7Hz), 1.06 (3H, d, J=7Hz), 0.99 (3H, d, J=6Hz), 0.77 (3H, s) as shown in Fig. 3 1 C Nuclear magnetic resonance spectrum:
(100 MHz, pyridine-d5) δc
209.5 (s), 178.8 (s), 156.7 (s), 142.6 (s), 116.3 (d), 106.6 (t), 72.8 (d), 69.3 (d), 57.7 (d), 53.3 (s), 48.9 (d), 48.4 (t), 46.8 (t), 42.4 (s), 38.7 (d), 38.4 (s), 36.3 (d), 36.0 (s), 34.7 (t), 34.1 (d), 31.3 (t),
29.0 (t), 26.8 (t), 23.8 (t), 23.6 (t), 22.1 (q), 22.0 (q), 18.6 (q),
17.1 (q), 9.9 (q) as shown in Fig. 4
Nature:
Acidic substance.
WF15604 substances or salts thereof are potent inhibitors of cholesterol biosynthesis. Therefore, they can lower blood serum cholesterol levels and blood lipid levels and are useful for the treatment of hyperlipemia and atherosclerosis.
Now, in order to show the utility of the WF15604A substance, the test data on cholesterol biosynthesis inhibiting activity and antifungal activity of the WF15604A substance are shown in the following.
Test 1 Inhibition of cholesterol synthesis in HepG2 cells
HepG2 cells (HB 8065, a human hepatoma cell line) were obtained from the American Type Culture Collection. Cholesterol synthesis in HepG2 cells was determined according to the method of Brown et al. (J. Biol. Chem. 253, 1121-1128, 1978) with some modifications. HepG2 cells were grown in flasks containing Eagle's modified minimum essential medium with non-essential amino acids supplemented with pyruvate (1 mM), penicillin G (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum (FBS) in a humidified incubator (5% C02) at 37 °C. On day 0, the cells (3 x 105 cells/well) were seeded into 35 mm 6-well plastic culture dishes (2 ml well). On day 4, the medium was replaced with 1 ml of fresh medium containing 10% human lipoprotein-deficient serum instead of FBS and the cells were preincubated for 2 hours at 37 °C with WF15604 substances (WF15604A substance or WF15604B substance) dissolved in 2 μl of dimethylsulfoxide (DMSO). DMSO (2 μl) was added to the control culture. Then, 1 mM [1- C]acetic acid, sodium salt (37 MBq/mmol, DuPont/NEN Research Products) was added to the medium and incubated at 37 °C for 2 hours. After incubation, the cells were washed with phosphate-buffered saline (PBS), pH 7.4 and then dissolved in 1 ml of aqueous 15% KOH. To each dissolved cell lysate was added 1 ml of 15% KOH in 95% ethanol and then samples were saponified for 1 hour at 75 °C. The non-saponifiable lipids were extracted twice with 2 ml of petroleum ether, evaporated to dryness, resuspended in 1ml of 50% acetone in 95% ethanol containing 0.1% cold cholesterol as a carrier. Sterols were precipitated by the addition of 1 ml of 0.5% digitonin in 50% ethanol, filtered on a glass filter.
The radioactivity of digitonin-precipitable [ C] -labeled sterols formed was counted with a liquid scintillation counter (TRI-CARB 1500, Packard Instrument Co.).
Results:
Substance Inhibition of cholesterol synthesis
WF15604A substance 0.004 μg/ml
WF15604B substance 0.005 μg/ml
Test 2 Antifungal activity:
Antifungal activities of WF15604 substances (WF15604A substance or WF15604B substance) were measured by micro-broth dilution method in 96 well multi-trays employing yeast nitrogen base dextrose medium. To a 50 μl sample solution with serial 2-fold dilutions was added a 50 μl of microorganism suspension in saline to yield a final concentration of 1 x 10 colony forming units/ml. The Candida and Aspergillus cultures were incubated at 37°C for 22 hours. After incubation, the growth of microorganism in each well was determined by measuring the turbidity. The results were shown as IC^Q value in which concentration the turbidity was half of that in the well without sample.
Results:
Organisms IC^Q (μg/ml)
WF15604A substance WF15604B substance
Candida albicans FP578 50.0 1.6
Aspergillus fumigatus 1305 3.0 12.5
For therapeutic administration, WF15604 substances or salts thereof are used in the form of conventional pharmaceutical preparation which contains said substance, as an active ingredient, in admixture with pharmaceutically acceptable carriers such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral and external(topical) administration.
The pharmaceutical preparations may be in solid form such as tablet, granule, powder, capsule, suppository, solution, suspension, syrup, emulsion, lemonade, lotion, ointment, gel, and the like.
If needed, there may be included in the above preparations auxiliary substances, stabilizing agents, wetting agents and other commonly used additives such as lactose, stearic acid, magnesium stearate, terra alba, sucrose, corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cacao butter, ethylene glycol, tartaric acid, citric acid, fumaric acid, and the like.
While the dosage of the WF15604 substances may vary from and also depend upon the age, conditions of the patient, a kind of diseases, etc.. In general, amount between about 0.1 mg and about 1,000 mg or 42 . l fi . PCT/JP94/02192
even more, preferably between about 1 mg and about 200 mg per day may be administered to a patient. An average single dose of about 0.1 mg, 1 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200 mg, 250 mg of the WF15604 substances may be used in treating hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions.
The following Examples are given for the purpose of illustrating this invention in more detail.
Example 1
(1) Fermentation :
An aqueous seed medium (160 ml) containing sucrose 4%, cotton seed flour 2%, dried yeast 1%, peptone 1%, KH2P04 0.2%, CaC03
0.2% and Tween 80 0.1% was poured into a 500-ml Erlenmeyer flask and sterilized at 120 °C for 30 minutes. A loopful of Sporormiella minima No. 15604 (FERM BP-4507) was inoculated from a slant culture into the flask. The flask was cultured on a rotary shaker (220 rpm, 5.1 cm-throw) at 25 °C for 4 days. The resultant seed culture was inoculated to 20 liters of sterile production medium consisting of starch acid hydrolysates 6%, cotton seed flour 1%, peanut powder 1%, dried yeast 2%, Na2HP04 • 12H20 0.5%, Adecanol LG-109
(deforming agent , Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70 (deforming agent, Shin-Etsu Chemical Co., Ltd.) 0.05% in a 30-liter jar fermenter. Fermentation was carried out at 25°C for 7 days under aeration of 20 liters/minute and agitation of 250 rpm.
(2) Isolation :
The cultured broth (60 liters) was extracted with 60 liters of acetone by intermittent mixing. The acetone extract was filtered with the aid of diatomaceous earth and 120 liters of water was added. The resultant cake was reextracted with 20 liters of acetone. The acetone extract, which was filtered with the aid of diatomaceous earth, was concentrated in vacuo to 9 liters and 18 liters of water was added. These extracts were combined and passed through a column (5 liters) of Diaion HP-20 (Mitsubishi Chemical Ind. Co., Ltd.). The column was washed with water and 50% aqueous methanol, and then eluted with methanol. To the eluate (19.5 liters) was added 20 liters of water, and then pH was adjusted to 3.0 with 1N-HC1. The mixture was passed through a column (2 liters) of Diaion SP-207 (Mitsubishi Chemical Ind. Co., Ltd.).
After washing with 50% aqueous methanol, water, 40% aqueous acetonitrile containing 0.05% phosphoric acid and 50% aqueous acetonitrile containing 0.05% phosphoric acid, the active fractions were eluted with 60% aqueous acetonitrile containing 0.05% phosphoric acid and 70% aqueous acetonitrile containing 0.05% phosphoric acid. This eluate (10 liters) was adjusted to pH 6.0 with lN-NaOH and an equal volume of water was added. The mixture was subjected to a column (1 liter) of YMC gel (ODS-AM 120-S50, YMC Co., Ltd.). The column was washed with 50% aqueous methanol and 70% aqueous methanol, and then eluted with 80% aqueous methanol. An active fraction(l liter) was concentrated in vacuo to an aqueous solution, adjusted to pH 2.0 with 1N-HC1 and extracted with an equal volume of ethyl acetate, twice. The extract was washed with water and concentrated in vacuo to dryness. The resultant pale yellowish product was dissolved in a small volume of methanol. After standing at 4°C overnight, WF15604A substance was obtained as colorless prisms (122 mg).
Example 2
(1) Fermentation :
Fermentation process was carreid out as a similar manner as described in Example 1.
(2) Isolation :
The cultured broth (360 liters) obtained by the similar method described above was extracted with 360 liters of acetone by intermittent mixing. The acetone extract was filtered with an aid of diatomaceous earth and 600 liters of water was added. The resultant cake was reextracted with 60 liters of acetone. The acetone extract was filtered with an aid of diatomaceous earth and 180 liters of water was added.
These extracts were combined and passed through a column (30 liters) of Diaion HP-20 (Mitsubishi Chemical Ind. Co., Ltd.). The column was washed with water, 50% aqueous methanol and 70% aqueous methanol, and then eluted with methanol.
To the eluate (260 liters) was added 260 liters of water and passed through a column (12 liters) of Diaion SP-207 (Mitsubishi Chemical Ind. Co., Ltd.). After washing with 50% aqueous methanol, water and stepwisely by solvent systems of 40%, 50% and 60% aqueous acetonitrile containing 0.05% phosphoric acid, WF15604A substance and WF15604B substance were eluted with 70% and 80% aqueous acetonitrile containing 0.05% phosphoric acid, respectively.
WF15604A substance (869 mg, crystallized from methanol) was purified by the same method as described in Example 1.
The eluate containing WF15604B substance (45 liters) was adjusted to pH 6.0 with IN NaOH and then concentrated in vacuo to 13 liters. This solution was subjected to a column (1 liter) of YMC gel (ODS- AM 120-S50, YMC Co., Ltd.). The column was washed with acetonitrile-methanol-0.015M (NH4)2HP04-H3P04 buffer (pH 6.0)
(40:10:50) and eluted with acetonitrile-methanol-0.015M (NH4)2HP04-H3P04 buffer (pH 6.0) (50:10:40).
To this eluate (3.3 liters) was added 0.7 liters of water and subjected to a column (1 liter) of YMC gel (ODS-AM 120-S50, YMC Co., Ltd.). After washing with acetonitrile-methanol-water (40:15:45) containing 0.05% phosphoric acid, acetonitrile-methanol-water (50:15:35) containing 0.05% phosphoric acid and acetonitrile-methanol-water (60:15:25) containing 0.05% phosphoric acid, the active fraction was eluted with acetonitrile-methanol-water (70:15:15) containing 0.05% phosphoric acid.
This active fraction(0.7 liters) was concentrated in vacuo to an aqueous solution. The aqueous solution was adjusted to pH 2.0 with IN HC1 and extracted with an equal volume of ethyl acetate, twice. The extract was washed with water and concentrated in vacuo to dryness. The resultant pale yellowish product was dissolved in a small volume of methanol. After standing at 4°C overnight, WF15604B substance was obtained as colorless needles (46 mg).

Claims

1. WF15604A substance having the following chemical formula:
Figure imgf000022_0001
or salts thereof.
2. A process for production of WF15604A substance or salts thereof, which comprises culturing a WF 15604 A substance-producing strain in a nutrient medium and recovering the same.
3. Biological pure culture of Sporormiella minima No. 15604 (FERM BP-4507).
4. A pharmaceutical composition containing WF15604A substance or salts thereof.
5. A use of WF15604A substance or salts thereof as a medicament.
6. A method for treating or preventing hypercholesterolaemic or hyperlipoproteinaemic states and associated conditions which comprises administrating WF 15604 A substance or salts thereof to human beings or animals.
7. Use of WF15604A substance or salts thereof for the manufacture of a medicament for therapeutic treatment or prevention of hypercholesterolaemic or hyperlipoproteinaemic states and associated conditions in human beings or animals.
8. A compound of Claim 1 for use as a medicament.
PCT/JP1994/002192 1993-12-29 1994-12-22 Wf15604 substances WO1995018142A1 (en)

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