WO1999063102A1 - ANTIDIABETIC ACTIVE COMPOUND(S) WF19353 AND PROCESS FOR THEIR FERMENTATIVE PREPARATION USING A FUNGUS OF THE GENUS $i(HELICOMYCES) - Google Patents
ANTIDIABETIC ACTIVE COMPOUND(S) WF19353 AND PROCESS FOR THEIR FERMENTATIVE PREPARATION USING A FUNGUS OF THE GENUS $i(HELICOMYCES) Download PDFInfo
- Publication number
- WO1999063102A1 WO1999063102A1 PCT/JP1999/002708 JP9902708W WO9963102A1 WO 1999063102 A1 WO1999063102 A1 WO 1999063102A1 JP 9902708 W JP9902708 W JP 9902708W WO 9963102 A1 WO9963102 A1 WO 9963102A1
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- Prior art keywords
- substance
- methanol
- reaction
- exchangeable
- max
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- 241000872577 Helicomyces Species 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 11
- 150000001875 compounds Chemical class 0.000 title abstract description 8
- 238000002360 preparation method Methods 0.000 title description 4
- 241000233866 Fungi Species 0.000 title description 3
- 230000003178 anti-diabetic effect Effects 0.000 title description 3
- 239000003472 antidiabetic agent Substances 0.000 title description 3
- 239000000126 substance Substances 0.000 claims abstract description 112
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- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 5
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- 230000001225 therapeutic effect Effects 0.000 claims abstract 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 109
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 75
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 60
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 48
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 claims description 40
- 238000006243 chemical reaction Methods 0.000 claims description 40
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 30
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 claims description 21
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 17
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 16
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- 238000000862 absorption spectrum Methods 0.000 claims description 8
- OZECDDHOAMNMQI-UHFFFAOYSA-H cerium(3+);trisulfate Chemical compound [Ce+3].[Ce+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O OZECDDHOAMNMQI-UHFFFAOYSA-H 0.000 claims description 8
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 claims description 8
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- 238000002329 infrared spectrum Methods 0.000 claims description 8
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- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- 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
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Definitions
- the present invention relates to new bioactive compounds, hereinafter entitled WF19353 substances which are useful as a medicament.
- the present invention relates to new bioactive compounds, WF19353 substances.
- WF19353 substances which have the inhibitory activity against gluconeogenesis, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which are useful as antidiabetic or antiobesic agents, and to a use thereof as a medicament.
- one object of this invention is to provide the novel compounds, WF19353 substances which are of use for treating and preventing diabetes, obesity and the like.
- Another object of this invention is to provide a process for production of the WF19353 substances by fermentation of a WF19353 substances- producing strain belonging to the genus Helicomyces in a nutrient medium.
- a further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF19353 substances.
- Still further object of this invention is to provide a use of the WF19353 substances for treating and preventing diabetes, obesity and the like.
- the WF19353 substances comprise 4 components, which are referred to as WF19353F substance, WF19353B substance, WF19353A substance and WF19353D substance, respectively, and, in this specification, the expression "WF19353 substance” refers to one of the WF19353 substances.
- the WF19353 substances can be produced by fermentation of the WF19353 substances-producing strain belonging to the genus Helicomyces such as Helicomyces sp. No. 19353 in a nutrient medium.
- the production of the WF19353 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 WF19353 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 fungal strain No.19353 was originally isolated from a decayed leaf sample, collected at Kawauchi-mura, Futaba-gun, Fukushima-ken, Japan. This organism grew restrictedly on various culture media, and formed dark brown colonies. On the Miura's LCA medium (Miura, K. and M. Kudo: Trans. My col. Soc. Japan, 11:116-118, 1970), the strain produced helical conidia from vegetative hyphae. The conidia were hyaline, multicellular, coiled and planate. Teleomorph was not observed. Its mycological characteristics were as follows.
- Conidia were borne on sessile denticles of vegetative and aerial hyphae, or on short lateral branches. Conidiogenous cells were acrogenous or intercalary, monoblastic or polyblastic, pale brown, smooth, cylindrical, and denticulate. The denticles were 2-5 x 1-2 ⁇ m in size. Conidia were holoblastic, solitary, hyaline, smooth, helical, coiled 2-3 times in two dimensions, planate, and 13-18(-25) ⁇ m in diameter. They were sometimes uncoiled and formed filamentous shapes. Conidial filaments were 5-12 septate and 1-2 ⁇ m thick.
- Chlamydospores were formed intercalary, solitary but aggregated at old cultures, pale brown, smooth, subglobose to pyriform, and 5-9 ⁇ m in diameter. Vegetative hyphae were smooth, septate, pale brown and branched. The hyphal cells were cylindrical and 2-4 ⁇ m in width.
- Strain No.19353 was able to grow at the temperature range from 5 to 31 °C, with the growth optimum at 24 to 27°C. These temperature data were determined on potato dextrose agar (made by NISSUI).
- Potato dextrose G Restrictedly, 1.5-2.5 cm agar (Difco 0013)
- S Circular, convex, felty, radiately sulcate or wrinkly, partly hygroscopic, formed no anamorph, brownish gray (6F2) to grayish brown (6F3), and white to yellowish white
- Czapek's solution G Very restrictedly, 1.0-1.5 cm agar S: Circular, submerged, thin, plane, formed no anamorph, yellowish gray (4B2) to grayish yellow (4C3)
- Corn meal agar G Restrictedly, 1.5-2.5 cm (Difco 0386)
- S Circular, thin, plane, submerged, hygroscopic, formed no anamorph, dark gray (1F1) at the center, and brownish gray (4F2) to olive brown (4F3) at the margin
- MY20 agar* G Very restrictedly, 0.5-1.0 cm
- Oatmeal agar G Very restrictedly, 1.0-1.5 cm
- G growth, measuring colony size in diameter S : colony surface
- the WF19353 substances are produced when the WF19353 substances- producing strain belonging to the genus Helicomyces 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 peanut powder, yeast extract, peptone, gluten meal, cotton seed flour, soybean powder, 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 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 salts, copper salts, zinc salts, iron salts, or 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 35 °C, for a period of about 24 hours to 120 hours, which may be varied according to fermentation conditions and scales.
- the culture broth is then subjected for recovery of the WF19353 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.
- the WF19353F substance as obtained has the following physico-chemical properties:
- Soluble methanol, acetic acid
- the WF19353B substance as obtained has the following physico-chemical properties:
- Soluble methanol, acetic acid
- HPLC High Performance Liquid Chromatography
- the WF19353A substance as obtained has the following physico-chemical properties:
- Soluble methanol, dimethyl sulfoxide, acetic acid Slightly soluble: water Insoluble: ethyl acetate, n-hexane Color reaction:
- the WF19353D substance as obtained has the following physico- chemical properties:
- Soluble methanol, dimethylsulf oxide, acetic acid Slightly soluble: water Insoluble: ethyl acetate, n-hexane Color reaction:
- the WF19353 substances possesses pharmacological activities such as the inhibitory activity against gluconeogenesis, and the like, and therefore are useful for the treatment and prevention of diabetes, obesity and the like.
- the WF19353 substances may be useful for various diseases because of its useful pharmaceutical activity such as the inhibitory activity against gluconeogenesis, and so on.
- Hepatocytes were prepared from 24 hours starved male Wistar rat (150- 200g) by the collagenase perfusion technique. Cells were cultured in
- glucose produced into the medium was determined enzymatically.
- Gluconeogenesis rate was performed as glucose value derived from pyruvate.
- WF19353 A substance 0.21 ⁇ g/ml
- 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 the WF19353 substance, 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, corn 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.
- the dosage of therapeutically effective amount of the WF19353 substance varies 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 the WF19353 substance per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of the WF19353 substance per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of the WF19353 substance per kg weight of human being is generally given for treating.
- the following examples are given for the purpose of illustrating the present invention.
- Example 1 Fermentation of strain WF19353 for the production of the WF19353F substance and the WF19353B substance:
- a loopful of the slant culture (strain WF19353) was inoculated into 30ml of sterilized seed medium consisting of sucrose 4%, glucose 1%, soluble starch
- Etsu Chemical Co., Ltd. 0.05% in each of six 100ml Erlenmeyer flasks.
- the flasks were incubated at 25 °C for 5 days on a rotary shaker (220 rpm, 5.1 cm-throw) and then inoculated (5%) into 160ml of the same sterilized seed medium in each of sixteen 500ml Erlenmeyer flasks. And the flasks were incubated at 25 °C for 2 days on a rotary shaker (220 rpm, 5.1 cm-throw).
- the resultant seed culture was inoculated (3%) into 20 liters of sterilized production medium in each of four 30-liter jar fermenters .
- the production medium was composed of glucose 1%, modified starch 6%, cotton seed flour 1%, soybean flour 1%, KH 2 P0 4 1.6%, Na 2 HP0 4 12H 2 0 1.2%, Adekanol
- the fermentation was carried out at 25 ⁇ C for 5 days under aeration of 20 liters/minute and agitation of 250 rpm.
- the culture broth was filtrated with an aid of diatomaceous earth. And then, 50 liters of methanol was added to the mycelium obtained thus and the mixture was allowed to stand for about 2 hours with stirring at room temperature. The resultant mixture was filtrated with an aid of diatomaceous earth.
- the filtrate was diluted with an equal volume of water and passed through a column (5 liters) of DIAION HP20 (Mitsubishi Chemical Co., Ltd.) packed with water. The column was washed with 50% aqueous methanol (15 liters) and then eluted with methanol (20 liters). The eluate was concentrated in vacuo to an aqueous solution (2 liters) and 3 liters of water was added to the solution.
- This solution was passed through a column (2 litters) of YMC-GEL (ODS-AM120-S50, YMC Co., Ltd.) packed with water.
- the column was eluted with 20% aqueous acetonitrile (13 liters) and then 25% aqueous acetonitrile (7.5 liters). The elution was monitored by analytical HPLC indicated below2). Fractions containing the WF19353F substance and the WF19353B substance were combined respectively.
- the portion corresponding to the WF19353F substance was diluted with an equal volume of water and passed through a column (2 liters) of YMC-
- the column was washed with water and eluted with 95% aqueous methanol.
- the eluate was concentrated in vacuo to dryness and dissolved in small amount of methanol.
- About twice amount of ethyl acetate was added to the solution and allowed to stand at 4°C for about 12 hours to yield 478 mg of the WF19353F substance as a powder.
- the portion corresponding to the WF19353B substance was diluted with an equal volume of water and passed through a column (2 liters) of YMC- GEL (ODS-AM 120-S50) packed with water. The column was eluted with 35% aqueous methanol containing 0.1% trifluoroacetic acid (11 liters). The portion corresponding to the WF19353B substance was diluted with an equal volume of water and passed through a column (1 liter) of YMC-GEL (ODS-
- strain WF19353 Fermentation of strain WF19353 for the production of the WF19353A substance and the WF19353D substance: A loopful of the slant culture (strain WF19353) was inoculated into 30 ml of sterilized seed medium consisting of sucrose 4%, glucose 1%, soluble starch 2%, cotton seed flour 3%, soybean flour 1.5%, KH 2 P0 4 1%, CaC0 3 0.2%, Adekanol LG-109 (Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70 (Shin-Etsu Chemical Co., Ltd.) 0.05% in each of six 100 ml Erlenmeyer flasks.
- the flasks were incubated at 25 °C for 5 days on a rotary shaker (220 rpm, 5.1 cm-throw) and then inoculated (5%) into 160 ml of the same sterilized seed medium in each of sixteen 500 ml Erlenmeyer flasks. And the flasks were incubated at 25 °C for 2 days on a rotary shaker (220 rpm, 5.1 cm-throw). The resultant seed culture was inoculated (3%) into 20 liters of sterilized production medium in each of four 30-liter jar fermenters.
- the production medium was composed of glucose 1%, modified starch 6%, cotton seed flour 1%, soybean flour 1%, KH 2 P0 4 1.6%, Na 2 HP0 4 -12H 2 0 1.2%, Adekanol LG- 109 0.05% and Silicone KM-70 0.05%.
- the fermentation was carried out at 25°C for 5 days under aeration of 20 liters/minute and agitation of 250 rpm.
- Retention time WF19353D 7.0 min
- the portion corresponding to the WF19353A substance was diluted with an equal volume of water and passed through a column (350 ml) of YMC- GEL (ODS-AM 120-S50) packed with water.
- the column was washed with 25% aqueous acetonitrile (1 liters) and eluted with 30% aqueous acetonitrile
- the portion corresponding to the WF19353A substance was diluted with an equal volume of water and passed through a column (30 ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness and given the crude powder.
- the portion corresponding to the WF19353D substance was diluted with an equal volume of water and passed through a column (180 ml) of YMC- GEL (ODS-AM 120-S50) packed with water.
- the column was washed with water (540 ml) and 25% aqueous acetonitrile (540 ml) and then eluted with 30% aqueous acetonitrile.
- trifluoroacetic acid was added to the portion corresponding to the WF19353D substance (Final concentration was 0.2%.).
- the solution was diluted with an equal volume of water and passed through a column (38 ml) of YMC-GEL (ODS-AM 120-S50) packed with water.
- the column was washed with water and 25% aqueous methanol containing 0.1% trifluoroacetic acid. Then it was eluted with 40% methanol containing 0.1% trifluoroacetic acid.
- the portion corresponding to the WF19353D substance was diluted with an equal volume of water and passed through a column (5 ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness, and given the 4.8 mg of WF19353D substance as a white powder.
- Fig.l and Fig.2 show ⁇ -NMR spectrum and 13 C-NMR spectrum, respectively, of WF19353F substance in DMSO-d6.
- Fig.3 and Fig.4 show ⁇ -NMR spectrum and 13 C-NMR spectrum, respectively, of WF19353B substance in DMSO-d6.
- Fig.5 and Fig.6 show ⁇ -NMR spectrum and 13 C-
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Abstract
The present invention provides new bioactive compounds WF19353 substances which have the inhibitory activity against gluconeogenesis, and a process for producing the same by fermentation of a WF19353 substances-producing strain belonging to the genus Helicomyces in a nutrient medium and recovering the WF19353 substances. Also provided are a pharmaceutical composition containing the WF19353 substance(s) and carrier(s), a use of the WF19353 substance(s) as a medicament and use of the WF19353 substance(s) for the manufacture of a medicament for therapeutic treatment or prevention of diabetes or obesity in human or animal.
Description
ANTIDIABETIC ACTIVE COMPOUND(S) WF 19353 AND PROCESS FOR THEIR FERMENTATIVE PREPARATION USING A FUNGUS OF THE GENUS
HELICOMYCES
DESCRIPTION
NOVEL COMPOUND
TECHNICAL FIELD
The present invention relates to new bioactive compounds, hereinafter entitled WF19353 substances which are useful as a medicament.
DISCLOSURE OF INVENTION
The present invention relates to new bioactive compounds, WF19353 substances.
More particularly, it relates to novel compounds, WF19353 substances which have the inhibitory activity against gluconeogenesis, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which are useful as antidiabetic or antiobesic agents, and to a use thereof as a medicament.
Accordingly, one object of this invention is to provide the novel compounds, WF19353 substances which are of use for treating and preventing diabetes, obesity and the like.
Another object of this invention is to provide a process for production of the WF19353 substances by fermentation of a WF19353 substances- producing strain belonging to the genus Helicomyces in a nutrient medium.
A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF19353 substances.
Still further object of this invention is to provide a use of the WF19353 substances for treating and preventing diabetes, obesity and the like.
The WF19353 substances comprise 4 components, which are referred to as WF19353F substance, WF19353B substance, WF19353A substance and WF19353D substance, respectively, and, in this specification, the expression "WF19353 substance" refers to one of the WF19353 substances.
The WF19353 substances can be produced by fermentation of the WF19353 substances-producing strain belonging to the genus Helicomyces such as Helicomyces sp. No. 19353 in a nutrient medium.
It is to be understood that the production of the WF19353 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 WF19353 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.
Characteristics of producing strain No.19353
The fungal strain No.19353 was originally isolated from a decayed leaf sample, collected at Kawauchi-mura, Futaba-gun, Fukushima-ken, Japan. This organism grew restrictedly on various culture media, and formed dark brown colonies. On the Miura's LCA medium (Miura, K. and M. Kudo: Trans. My col. Soc. Japan, 11:116-118, 1970), the strain produced helical conidia from vegetative hyphae. The conidia were hyaline, multicellular, coiled and planate. Teleomorph was not observed. Its mycological characteristics were as follows.
Cultural characteristics on various agar media are summarized in Table
1. Culture on potato dextrose agar grew restrictedly, attaining 1.5-2.5 cm in diameter two weeks later at 25 °C. This colony surface was convex, felty, radiately sulcate or wrinkly, partly hygroscopic, brownish gray to grayish brown, and white to yellowish white at the margin. The reverse color was brownish gray to grayish brown, and pale gray to yellowish gray at the margin.
Conidial structures were not observed. Colonies on corn meal agar spread with similar rate as on potato dextrose agar under the same conditions. The surface was plane, thin, submerged, hygroscopic, dark gray at the center, and brownish gray to olive brown at the margin. The reverse was olive gray to olive. Conidial structures were not produced.
The morphological characteristics were determined from the cultures on the Miura's LCA plate. Conidia were borne on sessile denticles of vegetative and aerial hyphae, or on short lateral branches. Conidiogenous cells were acrogenous or intercalary, monoblastic or polyblastic, pale brown, smooth, cylindrical, and denticulate. The denticles were 2-5 x 1-2 μm in size. Conidia were holoblastic, solitary, hyaline, smooth, helical, coiled 2-3 times in two dimensions, planate, and 13-18(-25) μm in diameter. They were sometimes uncoiled and formed filamentous shapes. Conidial filaments were 5-12 septate and 1-2 μm thick. Chlamydospores were formed intercalary, solitary but aggregated at old cultures, pale brown, smooth, subglobose to pyriform, and 5-9 μm in diameter. Vegetative hyphae were smooth, septate, pale brown and branched. The hyphal cells were cylindrical and 2-4 μm in width.
Strain No.19353 was able to grow at the temperature range from 5 to 31 °C, with the growth optimum at 24 to 27°C. These temperature data were determined on potato dextrose agar (made by NISSUI).
We compared the above morphological characteristics with fungal taxonomic criteria by von Arx (J. A. von Arx: The Genera of Fungi - Sporulating in Pure Culture. 3rd ed., pp. 315, J. Cramer, Vaduz, 1974) and by Goos (R. D. Goos: Mycologia 79: 1-22, 1987). As the consequence, the strain No.19353 was considered to belong to the hyphomycete genus Helicomyces Link 1809. Thus, we identified this isolate as one strain of the genus Helicomyces , and named it Helicomyces sp. No.19353. The strain has been deposited to the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, as FERM BP-6358 (deposited date: May 15, 1998).
Table 1. Cultural characteristics of strain No.19353.
Media Cultural characteristics
Malt extract agar* G: Restrictedly, 1.5-2.5 cm
S: Circular, plane, thin, hygroscopic, formed no anamorph, brown (6E5) to dark brown
(6F5) at the center, and yellowish gray (4B2) at the margin
R: Olive brown (4E4-4F4) at the center, and yellowish gray (4B2) to grayish yellow
(4B3) at the margin
Potato dextrose G: Restrictedly, 1.5-2.5 cm agar (Difco 0013) S: Circular, convex, felty, radiately sulcate or wrinkly, partly hygroscopic, formed no anamorph, brownish gray (6F2) to grayish brown (6F3), and white to yellowish white
(4A2) at the margin
R Brownish gray (5F2) to grayish brown
(5F3), and pale gray (1B1) to yellowish gray
(4B2) at the margin
Czapek's solution G: Very restrictedly, 1.0-1.5 cm agar S: Circular, submerged, thin, plane, formed no anamorph, yellowish gray (4B2) to grayish yellow (4C3)
R: Yellowish gray (4B2) to grayish yellow
(4C3)
Sabouraud dextrose G: Restrictedly, 1.5-2.5 cm agar (Difco 0190) S: Circular, raised, felty, radiately sulcate, formed no anamorph, dark brown
(6F3-6F4), and white to yellowish white
(4A2) at the margin R: Light brown (6D3) to dark brown (6F3), and yellowish gray (4B2) at the margin
Emerson Yp Ss agar G: Restrictedly, 1.5-2.5 cm (Difco 0739) S : Circular, plane , felty, wrinkly, partly hygroscopic, formed no anamorph, grayish brown (5F3) to brown (5E4), yellowish gray
(4B2) or reddish brown (9E4) at the margin
R: Yellowish gray (4B2) to brownish gray
(4D2)
Corn meal agar G: Restrictedly, 1.5-2.5 cm (Difco 0386) S: Circular, thin, plane, submerged, hygroscopic, formed no anamorph, dark gray (1F1) at the center, and brownish gray (4F2) to olive brown (4F3) at the margin
R: Olive gray (2F2) to olive (2E3)
MY20 agar* G: Very restrictedly, 0.5-1.0 cm
S: Circular, raised, felty to cottony, formed no anamorph, brownish gray (6E2-7E2)
R: Dark gray (1F1)
Oatmeal agar G: Very restrictedly, 1.0-1.5 cm
(Difco 0552) S: Circular, plane, felty, formed no anamorph, blackish blue (19F5-20F5), and dark brown
(6F8) at the margin
Abbreviation : G: growth, measuring colony size in diameter S : colony surface
R: reverse. * : The compositions of malt extract agar, Czapek's solution agar and MY20 agar were based on JCM Catalogue of Strains (Nakase, T., 6th ed., pp.617, Japan Collection of Microorganisms, the Institute of Physical and Chemical Research, Saitama, 1995).
These characteristics were observed after 14 days of incubation at 25 °C. The color descriptions were based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., pp. 252, Methuen, London, 1978).
Production of the WF19353 substances
The WF19353 substances are produced when the WF19353 substances- producing strain belonging to the genus Helicomyces 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 peanut powder, yeast extract, peptone, gluten meal, cotton seed flour, soybean powder, 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 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 salts, copper salts, zinc salts, iron salts, or 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 35 °C, for a period of about 24 hours to 120 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 WF19353 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.
The WF19353F substance as obtained has the following physico-chemical properties:
Appearance: white powder Molecular formula : C^CINA Elementary Analysis:
Calcd for C36H42C1N708-3H20
C 54.72, H 6.12, N 12.41 Found:
C 54.97, H 6.10, N 12.04 Molecular weight:
ESI-MS(negative):m/z 734(M-H)~, 736(M+H)+ Molecular weight: 736.22 Melting point:
225 - 230 °C (dec) Specific rotation:
[α]D(23°C) +40 ° (c=0.5, in dimethyl sulf oxide) Ultraviolet absorption spectrum: λmax (methanol): 233 nm (ε=53700) λmax (0.01N HCl-methanol): 319 nm (ε=7700) and 240nm (ε=62000) λmax (0.01N NaOH-methanol) : 304nm (ε=28000) and 230nm (ε=55000)
Solubility:
Soluble: methanol, acetic acid
Slightly soluble: water
Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: cerium sulfate reaction, iodine vapor reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid.water 0.5
F254** (4:1:2, v/v)
** made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL) Mobile phase: 18% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate: 1 ml/min Detection: UV at 210 nm Retention time: 6.0 minutes ***: made by DAISO CO., LTD.
Infrared Spectrum: vmax (KBr): 3390, 2980, 2940, 2910, 1630, 1540, 1500, 1440, 1380, 1280, 1200, 1100, 1040, 1010 cm"1 H Nuclear magnetic resonance spectrum: (500 MHz, DMSO-d6) δH
9.05 (IH, d, J=7Hz, exchangeable), 8.85 (IH, s), 8.15 (IH, br s, exchangeable), 8.06 (IH, d, J=8Hz), 7.75 - 7.70 (2H, m), 7.50 (IH, dd, J=8Hz and J=8Hz), 7.45 (IH, d, J=2Hz), 7.34 (IH, d, J=8Hz), 7.08 (IH, d, J=8Hz), 6.32 (IH, br d, J=5Hz, exchangeable), 5.16 (IH, br s), 5.07 (IH, dd, J=7Hz and J=4Hz), 4.96 (IH, br s), 4.77 (IH, m), 4.59 (IH, m), 4.11 (IH, m),
3.98 (IH, m), 3.96 (IH, s), 3.30 (IH, m), 3.00 (IH, m), 2.06 (3H, s), 2.04 (IH, m), 1.83 (3H, s), 1.81 (IH, m), 1.64 (3H, s), 1.29 (3H, s) in Fig. 1 13C Nuclear magnetic resonance spectrum:
(125 MHz, DMSO-d6) δC 169.9 (s), 168.3 (s), 166.1 (s), 165.4 (s), 158.3 (s), 157.4 (s), 150.2 (d), 146.6
(s), 142.0 (s), 140.9 (s), 135.4 (s), 132.6 (s), 129.8 (d), 129.0 (d), 128.9 (d), 128.8 (d), 127.0 (d), 126.6 (s), 126.5 (s), 126.0 (d), 123.9 (s), 123.5 (s), 117.7 (t), 115.4 (d), 75.8 (s), 69.1 (d), 67.6 (d), 59.9 (d), 56.1 (d), 45.4 (t), 45.4 (t), 38.3 (t), 26.9 (q), 23.4 (q), 22.9 (q), 20.5 (q) as shown in Fig. 2 Nature: amphoteric substance,
the WF19353B substance as obtained has the following physico-chemical properties:
Appearance: white powder Molecular formula : C^CIN Elementary Analysis:
Calcd for C37H44ClN708-5/2H20
C 55.88, H 6.21, N 12.33 Found:
C 55.96, H 6.34, N 12.00 Molecular weight:
ESI-MS (negative) : m/z 748(M- H)~ ,750 (M + H)+ Molecular weight: 750.25 Melting point: 230 °C (dec) Specific rotation:
[α]D(23°C) +37 ° (c=0.5, in dimethyl sulf oxide) Ultraviolet absorption spectrum: λmax (methanol) : 232 nm (ε=62900) λmax (O.OlNHCl-methanol) : 320 nm (ε=8300) and 240 nm (ε=70200) λmax (0.01N NaOH-methanol) : 304nm (ε=33000) and 230nm (ε=67000)
Solubility:
Soluble: methanol, acetic acid
Slightly soluble: water
Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: cerium sulfate reaction, iodine vapor reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6
F254** (4:1:2, v/v)
* * made by E. Merck
High Performance Liquid Chromatography (HPLC): Condition: Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL)
Mobile phase: 18% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate: 1 ml/min Detection: UV at 210 nm Retention time: 9.4 minutes
*** : made by DAISO CO., LTD. Infrared Spectrum: vmax (KBr): 3400, 2940, 1650, 1645, 1635, 1540, 1500, 1440, 1380, 1280, 1200, 1160, 1140, 1045, 910 cm 1 H Nuclear magnetic resonance spectrum:
(500 MHz, DMSO-d6) δH 9.05 (IH, d, J=7Hz, exchangeable), 8.86 (IH, s), 8.17 (IH, br s, exchangeable), 8.06 (IH, d, J=8Hz), 7.75 - 7.70 (2H, m), 7.51 (IH, dd, J=8Hz and J=8Hz), 7.44 (IH, d, J=2Hz), 7.35 (IH, d, J=8Hz), 7.08 (IH, d, J=8Hz), 6.33 (IH, br d, J=5Hz, exchangeable), 5.16 (IH, br s), 5.07 (IH, dd,
J=7Hz and J=4Hz), 4.97 (IH, br s), 4.76 (IH, m), 4.61 (IH, m), 4.11 (IH, m), 3.98 (IH, m), 3.97 (IH, s), 3.30 (IH, m), 3.00 (IH, m), 2.67 (IH, m), 2.47 (IH, m), 2.04 (IH, m), 1.83 (3H, s), 1.81 (IH, m), 1.63 (3H, s), 1.28 (3H, s), 0.95 (3H, t, J=7Hz) in Fig. 3 13C Nuclear magnetic resonance spectrum:
(125 MHz, DMSO-d6) δC 169.8 (s), 168.2 (s), 165.9 (s), 165.4 (s), 158.3 (s), 157.4 (s), 150.2 (d), 146.6 (s), 142.0 (s), 140.9 (s), 137.6 (s), 135.4 (s), 129.8 (d), 129.0 (d), 128.9 (d), 128.8 (d), 126.9 (d), 126.6 (s), 126.4 (s), 126.0 (d), 123.9 (s), 123.5 (s), 117.7 (t), 115.4 (d), 75.7 (s), 69.1 (d), 67.6 (d), 59.8 (d), 56.2 (d), 45.4 (t), 45.4 (t),
38.2 (t), 27.0 (q), 26.2 (t), 23.4 (d), 19.9 (q), 13.2 (q) as shown in Fig. 4 Nature: amphoteric substance,
the WF19353A substance as obtained has the following physico-chemical properties:
Appearance: white powder Molecular formula :
C38H46C1N708 Molecular weight:
ESI-MS: m/z 762(M-HT, 764 (M + H)+ Molecular weight: 764.27 Melting point:
210-215 °C (dec) Specific rotation:
[α]D(23°Q +31 ° (c=0.3, in dimethyl sulfoxide) Ultraviolet absorption spectrum: λmax (methanol): 232 nm (ε=49000) λmax (0.01N HCl-methanol): 320 nm (ε=6200) and 240 nm (ε=62000) λmax (0.01N NaOH-methanol): 230 nm (ε=48000) Solubility:
Soluble: methanol, dimethyl sulfoxide, acetic acid Slightly soluble: water
Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: iodine vapor reaction, cerium sulfate reaction, Dragendorff reaction Negative: Molish reaction
Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6
F254** (4:1:2, v/v)
* * made by E. Merck
High Performance Liquid Chromatography (HPLC):
Condition:
Column: YMC-Pack Pro C18 AS-303, S-5 120A (4.6 mmφ x 250 mmL****)
Mobile phase: 30% aqueous acetonitrile containing 50 mM sodium- phosphate buffer (pH 5.8) and 5 mM SDS)
Flow rate: 1 ml/minute Detection: UV at 210 nm Retention time: 14.0 minutes ****: YMC Co., Ltd. Infrared Spectrum: vmax (KBr): 3370, 2970, 1640, 1540, 1490, 1440, 1270, 1020, 770 cm'1 H Nuclear magnetic resonance spectrum:
(500 MHz, DMSO-d6) δH 9.17 (IH, d, J=7Hz, exchangeable), 8.87 (IH, s), 8.16 (IH, br s, exchangeable), 8.07 (IH, d, J=9Hz), 7.90 (IH, dd, J=9Hz and J=2Hz), 7.73
(IH, m), 7.53 - 7.48 (2H, m), 7.33 (IH, d, J=9Hz), 7.28 (IH, d, J=9Hz), 6.39
(IH, br d, J=4Hz, exchangeable), 5.14 (IH, br s), 5.09 (IH, dd, J=7Hz and
J=4Hz), 4.89 (IH, br s), 4.79 (IH, m), 4.65 (IH, m), 4.11 (IH, m), 4.01 (IH, m), 3.97 (IH, s), 3.73 (3H, s), 3.32 (IH, m), 2.99 (IH, m), 2.67 (IH, m), 2.46
(IH, m), 2.05 (IH, m), 1.82 (IH, m), 1.80 (3H, s), 1.63 (3H, s), 1.29 (3H, s),
0.95 (3H, t, J=7Hz) in Fig. 5
13C Nuclear magnetic resonance spectrum:
(125 MHz, DMSO-d6) δC 169.8 (s), 168.1 (s), 165.8 (s), 165.2 (s), 159.5 (s), 157.4 (s), 150.1 (d), 146.6
(s), 141.8 (s), 140.3 (s), 137.7 (s), 135.3 (s), 129.3 (d), 129.1 (d), 129.1 (d), 129.1 (d), 127.2 (d), 126.5 (s), 126.3 (s), 125.7 (d), 125.4 (s), 125.1 (s), 117.8 (t), 111.4 (d), 75.7 (s), 69.2 (d), 67.7 (d), 59.7 (d), 56.4 (d), 55.8 (q), 45.43 (t), 45.37 (t), 38.3 (t), 27.0 (q), 26.2 (t), 23.4 (q), 19.9 (q), 13.2 (q) as shown in Fig. 6
Nature: amphoteric substance,
and, the WF19353D substance as obtained has the following physico- chemical properties:
Appearance: white powder Molecular formula : C37H44C1 708
Molecular weight:
ESI-MS (negative): m/z 748(M-H)~, 750 (M + H)+ Molecular weight: 750.24 Melting point: 230 - 235 °C (dec)
Specific rotation:
[α]D(23 °C) +35 ° (c=0.1 , in dimethyl sulfoxide) Ultraviolet absorption spectrum: λmax (methanol) : 228 nm (ε =43000) λmax (0.01N HCl-methanol): 240 nm (ε=41000) and 320 nm(ε=3900) λmax (0.01N NaOH-methanol): 230 nm (ε=41000) Solubility:
Soluble: methanol, dimethylsulf oxide, acetic acid Slightly soluble: water Insoluble: ethyl acetate, n-hexane
Color reaction:
Positive: iodine vapor reaction, cerium sulfate reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6 F254** (4:1:2, v/v)
** made by E. Merck High Performance Liquid Chromatography (HPLC): Condition: Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL)
Mobile phase: 22% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate: 1 ml/min Detection: UV at 210 nm Retention time: 7.0 minutes
*** : made by DAISO CO., LTD. Infrared Spectrum: vmax (KBr): 3370, 2970, 1640, 1490, 1440, 1270, 1200, 1140, 1020, 770 cm"1 H Nuclear magnetic resonance spectrum:
(500 MHz, DMSO-d6) δH 9.08 (IH, br s, exchangeable), 8.87 (IH, s), 8.26 (IH, br s, exchangeable), 8.07 (IH, d, J=8Hz), 7.95 (IH, br d, J=9Hz), 7.74 (IH, m), 7.54 - 7.48 (2H, m), 7.33 (IH, d, J=9Hz), 7.28 (IH, d, J=8Hz), 6.22 (IH, br s, exchangeable), 5.14 (IH, br s), 5.08 (IH, dd, J=7Hz and J=6Hz), 4.89 (IH, br s), 4.70 (IH, m),
4.51 (IH, m), 4.10 (IH, m), 3.99 (IH, s), 3.96 (IH, m), 3.73 (3H, s), 3.30 (IH, m), 3.05 (IH, m), 2.05 (IH, m), 2.05(3H,s), 1.82 (IH, m), 1.80 (3H, s), 1.67 (3H, s), 1.29 (3H, s) in Fig. 7 13C Nuclear magnetic resonance spectrum: (125 MHz, DMSO-d6) δC
168.3 (s), 166.3 (s), 165.2 (s), 159.5 (s), 157.3 (s), 150.1 (d), 146.6 (s), 141.8 (s), 140.3 (s), 135.3 (s), 129.5 (d), 129.2 (d), 129.1 (d), 129.0 (d), 127.2 (d), 126.5 (s), 125.7 (d), 125.3 (s), 125.0 (s), 117.7 (t), 111.3 (d), 75.8 (s), 69.0 (d), 67.5 (d), 60.3 (d), 55.8 (q), 55.4 (d), 45.4 (t), 45.3 (t), 38.2 (t), 26.8 (q), 23.3 (q), 22.7 (q), 20.6 (q) as shown in Fig. 8 Nature: amphoteric substance.
Biological properties of the WF19353 substances
The WF19353 substances possesses pharmacological activities such as the inhibitory activity against gluconeogenesis, and the like, and therefore are useful for the treatment and prevention of diabetes, obesity and the like.
And further, the WF19353 substances may be useful for various diseases because of its useful pharmaceutical activity such as the inhibitory activity against gluconeogenesis, and so on.
As examples for showing biological activities of the WF19353 substances, some biological data are explained in the following.
Effect of WF19353 substance on rat hepatocytes gluconeogenesis
Hepatocytes were prepared from 24 hours starved male Wistar rat (150- 200g) by the collagenase perfusion technique. Cells were cultured in
William's E medium containing 5% (v/v) fetal bovine serum, O.lmg/ml kanamycin for 6 hours at 96-well tissue culture plates. Cells were washed with phosphate-buffered saline and incubated with Dulbecco's Modified Eagle's Medium without glucose, supplemented with 20 mM pyruvate, lxlO"7 M glucagon, 0.1 mg/ml kanamycin and 1% (v/v) fetal bovine serum. After
15 hours, glucose produced into the medium was determined enzymatically. Gluconeogenesis rate was performed as glucose value derived from pyruvate.
The half-maximal inhibitory concentration of WF19353 substances on rat hepatocytes gluconeogenesis was as follows.
WF19353 A substance 0.21 μg/ml
WF19353 B substance 0.24 μg/ml
WF19353 D substance 0.25 μg/ml
WF19353 F substance 0.33 μg/ml
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 the WF19353 substance, 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, corn 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 the WF19353 substance varies 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 the WF19353 substance per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of the WF19353 substance per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of the WF19353 substance per kg weight of human being is generally given for treating.
The following examples are given for the purpose of illustrating the present invention.
Example 1 : Fermentation of strain WF19353 for the production of the WF19353F substance and the WF19353B substance:
A loopful of the slant culture (strain WF19353) was inoculated into 30ml of sterilized seed medium consisting of sucrose 4%, glucose 1%, soluble starch
2%, cotton seed flour 3%, soybean flour 1.5%, KH2P04 1%, CaC03 0.2%, Adekanol LG-109 (Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70 (Shin-
Etsu Chemical Co., Ltd.) 0.05% in each of six 100ml Erlenmeyer flasks. The flasks were incubated at 25 °C for 5 days on a rotary shaker (220 rpm, 5.1 cm-throw) and then inoculated (5%) into 160ml of the same sterilized seed medium in each of sixteen 500ml Erlenmeyer flasks. And the flasks were incubated at 25 °C for 2 days on a rotary shaker (220 rpm, 5.1 cm-throw).
The resultant seed culture was inoculated (3%) into 20 liters of sterilized production medium in each of four 30-liter jar fermenters . The production medium was composed of glucose 1%, modified starch 6%, cotton seed flour 1%, soybean flour 1%, KH2P04 1.6%, Na2HP04 12H20 1.2%, Adekanol
LG-109 0.05% and Silicone KM-70 0.05%. The fermentation was carried out at 25 <C for 5 days under aeration of 20 liters/minute and agitation of 250 rpm.
The production of the WF19353F substance and the WF19353B substance in the fermentation broth was monitored by HPLC analysis indicated below 1).
1): Analytical HPLC condition
Column : YMC Pack Pro C18 AS-303, S-5 120A (4.6 mmφ x 250 mmL** **)
Mobile phase : 30% aqueous acetonitrile containing 50mM sodium- phosphate buffer (pH 5.8) and 5mM SDS Temperature : 70^ Flow rate : lml/min Detection : UV at 210 nm
Retention time : WF19353F 5.0 min WF19353B 5.6 min * ** *: YMC Co., Ltd.
Isolation of the WF19353F substance and the WF19353B substance:
After the culture was completed, the culture broth was filtrated with an aid of diatomaceous earth. And then, 50 liters of methanol was added to the mycelium obtained thus and the mixture was allowed to stand for about 2 hours with stirring at room temperature. The resultant mixture was filtrated with an aid of diatomaceous earth. The filtrate was diluted with an equal volume of water and passed through a column (5 liters) of DIAION HP20 (Mitsubishi Chemical Co., Ltd.) packed with water. The column was washed with 50% aqueous methanol (15 liters) and then eluted with methanol (20 liters). The eluate was concentrated in vacuo to an aqueous solution (2 liters) and 3 liters of water was added to the solution. This solution was passed through a column (2 litters) of YMC-GEL (ODS-AM120-S50, YMC Co., Ltd.) packed with water. The column was eluted with 20% aqueous acetonitrile (13 liters) and then 25% aqueous acetonitrile (7.5 liters). The elution was monitored by analytical HPLC indicated below2). Fractions containing the WF19353F substance and the WF19353B substance were combined respectively.
The portion corresponding to the WF19353F substance was diluted with an equal volume of water and passed through a column (2 liters) of YMC-
GEL (ODS-AM 120-S50) packed with water. The column was eluted with 25% aqueous acetonitrile (8 liters). The portion corresponding to the WF19353F substance was diluted with an equal volume of water and passed through a column (1 liter) of YMC-GEL (ODS-AM 120-S50) packed with water. The column was washed with water and eluted with 30% aqueous methanol containing 0.1% trifluoroacetic acid. The portion corresponding to the WF19353F substance was diluted with an equal volume of water and passed through a column (180ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness and
dissolved in small amount of methanol. About twice amount of ethyl acetate was added to the solution and allowed to stand at 4°C for about 12 hours to yield 478 mg of the WF19353F substance as a powder.
The portion corresponding to the WF19353B substance was diluted with an equal volume of water and passed through a column (2 liters) of YMC- GEL (ODS-AM 120-S50) packed with water. The column was eluted with 35% aqueous methanol containing 0.1% trifluoroacetic acid (11 liters). The portion corresponding to the WF19353B substance was diluted with an equal volume of water and passed through a column (1 liter) of YMC-GEL (ODS-
AM 120-S50) packed with water. The column was washed with water and eluted with 25% aqueous acetonitrile. The portion corresponding to the WF19353B substance was diluted with an equal volume of water and passed through a column (350ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness and dissolved in small amount of methanol. About twice amount of ethyl acetate was added to the solution and allowed to stand at 4<C for about 12 hours to yield 512 mg of the WF19353B substance as a powder.
2): Analytical HPLC condition
Column : YMC Pack Pro C18 AS-303, S-5 120A (4.6 mmφ x 150 mmL****)
Mobile phase : 18% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate : lml/min Detection : UV at 210 nm Retention time : WF19353F 6.0 min WF19353B 9.4 min
****: YMC Co., Ltd.
Example 2:
Fermentation of strain WF19353 for the production of the WF19353A substance and the WF19353D substance:
A loopful of the slant culture (strain WF19353) was inoculated into 30 ml of sterilized seed medium consisting of sucrose 4%, glucose 1%, soluble starch 2%, cotton seed flour 3%, soybean flour 1.5%, KH2P04 1%, CaC03 0.2%, Adekanol LG-109 (Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70 (Shin-Etsu Chemical Co., Ltd.) 0.05% in each of six 100 ml Erlenmeyer flasks.
The flasks were incubated at 25 °C for 5 days on a rotary shaker (220 rpm, 5.1 cm-throw) and then inoculated (5%) into 160 ml of the same sterilized seed medium in each of sixteen 500 ml Erlenmeyer flasks. And the flasks were incubated at 25 °C for 2 days on a rotary shaker (220 rpm, 5.1 cm-throw). The resultant seed culture was inoculated (3%) into 20 liters of sterilized production medium in each of four 30-liter jar fermenters. The production medium was composed of glucose 1%, modified starch 6%, cotton seed flour 1%, soybean flour 1%, KH2P04 1.6%, Na2HP04-12H20 1.2%, Adekanol LG- 109 0.05% and Silicone KM-70 0.05%. The fermentation was carried out at 25°C for 5 days under aeration of 20 liters/minute and agitation of 250 rpm.
The production of the WF19353A substance and the WF19353D substance in the fermentation broth was monitored by HPLC analysis indicated below 3).
3): Analytical HPLC condition
Column : YMC Pack Pro C18 AS-303, S-5 120A (250 x 4.6 mmφ x 250 mmL****)
Mobile phase : 30% aqueous acetonitrile containing 50 mM sodium- phosphate buffer (pH 5.8) and 5 mM SDS
Temperature : 70 °C
Flow rate : 1 ml/min
Detection : UV at 210 nm
Retention time : WF19353A 14.0 min
WF19353D 12.1 min
****: YMC Co., Ltd.
Isolation of the WF19353A substance and the WF19353D substance: After the culture was completed, the culture broth was filtrated with an
aid of diatomaceous earth. And then, 50 liters of methanol was added to the mycelium obtained thus and the mixture was allowed to stand for about 2 hours with stirring at room temperature. The resultant mixture was filtrated with an aid of diatomaceous earth. The filtrate was diluted with an equal volume of water and passed through a column (5 liters) of DIAION HP20
(Mitsubishi Chemical Co., Ltd.) packed with water. The column was washed with 50% aqueous methanol (15 liters) and then eluted with methanol (20 liters). The eluate was concentrated in vacuo to an aqueous solution (2 liters) and 3 liters of water was added to the solution. This solution was passed through a column (2 litters) of YMC-GEL (ODS-AM120-S50, YMC Co.,
Ltd.) packed with water. The column was eluted with 20% aqueous acetonitrile (13 liters), 25% aqueous acetonitrile(7.5 liters) and then 30% aqueous acetonitrile(5.5 liters). The elution was monitored by analytical HPLC indicated below (WF19353A was used Method No. 4) and WF19353D was used Method No. 5)). Fractions containing the WF19353A substance and the WF19353D substance were combined respectively.
4): Analytical HPLC condition
Column : YMC Pack Pro C18 AS-303, S-5 120A (4.6 mmφ x 250 mmL* ***) Mobile phase : 30% aqueous acetonitrile containing 50 mM sodium- phosphate buffer (pH 5.8) and 5 mM SDS. Temperature : 70 °C Flow rate : 1 ml/min Detection : UV at 210 nm Retention time :
WF19353A 14.0 min WF19353D 12.1 min
5): Analytical HPLC condition Column : DAISOPAK SP-120-5-ODS-AP (150 x 4.6 mmφ x 250mmL***)
Mobile phase : 22% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate : lml/min
Detection : UV at 210 nm
Retention time : WF19353D 7.0 min
The portion corresponding to the WF19353A substance was diluted with an equal volume of water and passed through a column (350 ml) of YMC- GEL (ODS-AM 120-S50) packed with water. The column was washed with 25% aqueous acetonitrile (1 liters) and eluted with 30% aqueous acetonitrile
(1 liters). The portion corresponding to the WF19353A substance was combined and trifluoroacetic acid (trifluoroacetic acid) was added (Final concentration was 0.2%.) .Then this solution was diluted with an equal volume of water and passed through a column (60 ml) of YMC-GEL (ODS- AM 120-S50) packed with water. The column was washed with 30% aqueous methanol containing 0.1% trifluoroacetic acid and eluted with 40% aqueous methanol containing 0.1% trifluoroacetic acid. The portion corresponding to the WF19353A substance was diluted with an equal volume of water and passed through a column (30 ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness and given the crude powder. It was dissolved in small amount of methanol and further purified by preparative HPLC, using YMC-pack ODS-AM SH-343 (250 x 20 mm I.D., YMC Co., Ltd.) with 30% aqueous acetonitrile containing 0.5% NaH2P04-2H20 as a mobile phase and a flow rate 15 ml / minute. Fractions containing the WF19353A substance were collected and diluted with an equal volume of water and passed through a column (5 ml) of YMC-GEL (ODS- AM 120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness and given the 11.0 mg of the WF19353A substance as a white powder.
The portion corresponding to the WF19353D substance was diluted with an equal volume of water and passed through a column (180 ml) of YMC- GEL (ODS-AM 120-S50) packed with water. The column was washed with water (540 ml) and 25% aqueous acetonitrile (540 ml) and then eluted with 30% aqueous acetonitrile. trifluoroacetic acid was added to the portion corresponding to the WF19353D substance (Final concentration was 0.2%.). The solution was diluted with an equal volume of water and passed through a column (38 ml) of YMC-GEL (ODS-AM 120-S50) packed with water. The
column was washed with water and 25% aqueous methanol containing 0.1% trifluoroacetic acid. Then it was eluted with 40% methanol containing 0.1% trifluoroacetic acid. The portion corresponding to the WF19353D substance was diluted with an equal volume of water and passed through a column (5 ml) of YMC GEL (ODS-AM120-S50) packed with water. The column was washed with water and eluted with 95% aqueous methanol. The eluate was concentrated in vacuo to dryness, and given the 4.8 mg of WF19353D substance as a white powder.
BRIEF DESCRIPTION OF DRAWINGS
Fig.l and Fig.2 show Η-NMR spectrum and 13C-NMR spectrum, respectively, of WF19353F substance in DMSO-d6. Fig.3 and Fig.4 show Η-NMR spectrum and 13C-NMR spectrum, respectively, of WF19353B substance in DMSO-d6. Fig.5 and Fig.6 show Η-NMR spectrum and 13C-
NMR spectrum, respectively, of WF19353A substance in DMSO-d6. Fig.7 and Fig.8 show Η-NMR spectrum and 13C-NMR spectrum, respectively, of WF19353D substance in DMSO-d6.
Claims
1. WF19353 substances of the following physico-chemical properties, which comprise WF19353F substance, WF19353B substance, WF19353A substance and WF19353D substance :
(1) WF19353F substance: Appearance: white powder Molecular formula : C36H42C1N708
Elementary Analysis:
Calcd for C36H42C1N708-3H20
C 54.72, H 6.12, N 12.41 Found: C 54.97, H 6.10, N 12.04
Molecular weight:
ESI-MS (negative): m/z 734(M-H)", 736(M+H)+ Molecular weight: 736.22 Melting point: 225 - 230 ┬░C (dec)
Specific rotation:
[╬▒]D(23 ┬░C) +40 ┬░ (c=0.5 , in dimethyl sulfoxide) Ultraviolet absorption spectrum: ╬╗max (methanol): 233 nm (╬╡=53700) ╬╗max (0.01N HCl-methanol): 319 nm (╬╡=7700) and 240 (╬╡=62000) ╬╗max (0.01N NaOH-methanol): 304 nm (╬╡=28000) and 230 (╬╡=55000) Solubility:
Soluble: methanol, acetic acid Slightly soluble: water Insoluble: ethyl acetate, n-hexane
Color reaction:
Positive: cerium sulfate reaction, iodine vapor reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid.water 0.5
F254** (4:1:2, v/v)
** made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL) Mobile phase: 18% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate: 1 ml/min Detection: UV at 210 nm
Retention time: 6.0 minutes * ** : made by DAISO CO., LTD. Infrared Spectrum: vmax (KBr): 3390, 2980, 2940, 2910, 1630, 1540, 1500, 1440, 1380, 1280, 1200, 1100, 1040, 1010 cm 1 H Nuclear magnetic resonance spectrum (Fig. 1) :
(500 MHz, DMSO-d6) ╬┤H 9.05 (IH, d, J=7Hz, exchangeable), 8.85 (IH, s), 8.15 (IH, br s, exchangeable), 8.06 (IH, d, J=8Hz), 7.75 - 7.70 (2H, m), 7.50 (IH, dd, J=8Hz and J=8Hz), 7.45 (IH, d, J=2Hz), 7.34 (IH, d, J=8Hz), 7.08 (IH, d,
J=8Hz), 6.32 (IH, br d, J=5Hz, exchangeable), 5.16 (IH, br s), 5.07 (IH, dd, J=7Hz and J=4Hz), 4.96 (IH, br s), 4.77 (IH, m), 4.59 (IH, m), 4.11 (IH, m), 3.98 (IH, m), 3.96 (IH, s), 3.30 (IH, m), 3.00 (IH, m), 2.06 (3H, s), 2.04 (IH, m), 1.83 (3H, s), 1.81 (IH, m), 1.64 (3H, s), 1.29 (3H, s) 13C Nuclear magnetic resonance spectrum ( Fig. 2) :
(125 MHz, DMSO-d6) ╬┤C 169.9 (s), 168.3 (s), 166.1 (s), 165.4 (s), 158.3 (s), 157.4 (s), 150.2 (d), 146.6 (s), 142.0 (s), 140.9 (s), 135.4 (s), 132.6 (s), 129.8 (d), 129.0 (d), 128.9 (d), 128.8 (d), 127.0 (d), 126.6 (s), 126.5 (s), 126.0 (d), 123.9 (s), 123.5 (s), 117.7 (t), 115.4 (d), 75.8 (s), 69.1 (d), 67.6 (d), 59.9 (d), 56.1 (d), 45.4 (t), 45.4 (t),
38.3 (t), 26.9 (q), 23.4 (q), 22.9 (q), 20.5 (q) Nature: amphoteric substance,
(2) WF19353B substance:
Appearance: white powder Molecular formula : ^CINA Elementary Analysis:
Calcd for C37H44ClN708-5/2H20
C 55.88, H 6.21, N 12.33 Found:
C 55.96, H 6.34, N 12.00 Molecular weight:
ESI-MS (negative): m/z 748(M-H)-,750(M+H)+ Molecular weight: 750.25 Melting point: 230 ┬░C (dec) Specific rotation:
[╬▒]D(23 ┬░C) +37 ┬░ (c=0.5 , in dimethyl sulfoxide) Ultraviolet absorption spectrum: ╬╗max (methanol) : 232 nm (╬╡=62900) ╬╗max(0.01N HCl-methanol): 320nm(╬╡=8300) and 240nm (╬╡=70200) ╬╗max (0.01N NaOH-methanol): 304nm(╬╡=33000) and 230 nm (╬╡=67000)
Solubility:
Soluble: methanol, acetic acid Slightly soluble: water Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: cerium sulfate reaction, iodine vapor reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6
F254** (4:1:2, v/v)
* * made by E. Merck
High Performance Liquid Chromatography (HPLC):
Condition:
Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL) Mobile phase: 18% aqueous acetonitrile containing 0.1% trifluoroacetic acid Flow rate: 1 ml/min
Detection: UV at 210 nm Retention time: 9.4 minutes *** : made by DAISO CO., LTD. Infrared Spectrum: vmax (KBr): 3400, 2940, 1650, 1645, 1635, 1540, 1500, 1440, 1380,
1280, 1200, 1160, 1140, 1045, 910 cm 1 H Nuclear magnetic resonance spectrum (Fig. 3) :
(500 MHz, DMSO-d6) ╬┤H 9.05 (IH, d, J=7Hz, exchangeable), 8.86 (IH, s), 8.17 (IH, br s, exchangeable), 8.06 (IH, d, J=8Hz), 7.75 - 7.70 (2H, m), 7.51 (IH, dd,
J=8Hz and J=8Hz), 7.44 (IH, d, J=2Hz), 7.35 (IH, d, J=8Hz), 7.08 (IH, d, J=8Hz), 6.33 (IH, br d, J=5Hz, exchangeable), 5.16 (IH, br s), 5.07 (IH, dd, J=7Hz and J=4Hz), 4.97 (IH, br s), 4.76 (IH, m), 4.61 (IH, m), 4.11 (IH, m), 3.98 (IH, m), 3.97 (IH, s), 3.30 (IH, m), 3.00 (IH, m), 2.67 (IH, m), 2.47 (IH, m), 2.04 (IH, m), 1.83 (3H, s), 1.81 (IH, m), 1.63 (3H, s), 1.28 (3H, s),
0.95 (3H, t, J=7Hz) 13C Nuclear magnetic resonance spectrum (Fig. 4) :
(125 MHz, DMSO-d6) ╬┤C 169.8 (s), 168.2 (s), 165.9 (s), 165.4 (s), 158.3 (s), 157.4 (s), 150.2 (d), 146.6 (s), 142.0 (s), 140.9 (s), 137.6 (s), 135.4 (s), 129.8 (d), 129.0 (d), 128.9 (d),
128.8 (d), 126.9 (d), 126.6 (s), 126.4 (s), 126.0 (d), 123.9 (s), 123.5 (s), 117.7 (t), 115.4 (d), 75.7 (s), 69.1 (d), 67.6 (d), 59.8 (d), 56.2 (d), 45.4 (t), 45.4 (t), 38.2 (t), 27.0 (q), 26.2 (t), 23.4 (d), 19.9 (q), 13.2 (q) Nature: amphoteric substance,
(3) WF19353A substance: Appearance: white powder Molecular formula :
C38H46C1N708 Molecular weight:
ESI-MS: m/z 762(M-H)~,764 (M + H)+ Molecular weight: 764.27 Melting point:
210-215 ┬░C (dec) Specific rotation:
[╬▒]D(23┬░Q +31 ┬░ (c=0.3, in dimethyl sulfoxide) Ultraviolet absorption spectrum: ╬╗max (methanol): 232 nm (╬╡=49000) ╬╗max (0.0 IN HCl-methanol): 320 nm (╬╡=6200) and 240 nm (╬╡=62000) ╬╗max (0.01 N NaOH-methanol) : 230 nm (╬╡ =48000) Solubility:
Soluble: methanol, dimethyl sulfoxide, acetic acid Slightly soluble: water
Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: iodine vapor reaction, cerium sulfate reaction, Dragendorff reaction Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6
F254** (4:1:2, v/v)
* * made by E. Merck
High Performance Liquid Chromatography (HPLC):
Condition:
Column: YMC-Pack Pro C18 AS-303, S-5 120A (4.6 mmφ x 250 mmL****)
Mobile phase: 30% aqueous acetonitrile containing 50 mM sodium- phosphate buffer (pH 5.8) and 5 mM SDS)
Flow rate: 1 ml/minute Detection: UV at 210 nm Retention time: 14.0 minutes ****: YMC Co., Ltd. Infrared Spectrum: vmax (KBr): 3370, 2970, 1640, 1540, 1490, 1440, 1270, 1020, 770 cm"1
*H Nuclear magnetic resonance spectrum (Fig. 5) : (500 MHz, DMSO-d6) ╬┤H 9.17 (IH, d, J=7Hz, exchangeable), 8.87 (IH, s), 8.16 (IH, br s, exchangeable), 8.07 (IH, d, J=9Hz), 7.90 (IH, dd, J=9Hz and J=2Hz), 7.73 (IH, m), 7.53 - 7.48 (2H, m), 7.33 (IH, d, J=9Hz), 7.28 (IH, d, J=9Hz), 6.39 (IH, br d, J=4Hz, exchangeable), 5.14 (IH, br s), 5.09 (IH, dd, J=7Hz and J=4Hz), 4.89 (IH, br s), 4.79 (IH, m), 4.65 (IH, m), 4.11 (IH, m), 4.01 (IH, m), 3.97 (IH, s), 3.73 (3H, s), 3.32 (IH, m), 2.99 (IH, m), 2.67 (IH, m), 2.46
(IH, m), 2.05 (IH, m), 1.82 (IH, m), 1.80 (3H, s), 1.63 (3H, s), 1.29 (3H, s), 0.95 (3H, t, J=7Hz)
13C Nuclear magnetic resonance spectrum (Fig. 6) : (125 MHz, DMSO-d6) ╬┤C 169.8 (s), 168.1 (s), 165.8 (s), 165.2 (s), 159.5 (s), 157.4 (s), 150.1 (d), 146.6 (s), 141.8 (s), 140.3 (s), 137.7 (s), 135.3 (s), 129.3 (d), 129.1 (d), 129.1 (d), 129.1 (d), 127.2 (d), 126.5 (s), 126.3 (s), 125.7 (d), 125.4 (s), 125.1 (s), 117.8 (t), 111.4 (d), 75.7 (s), 69.2 (d), 67.7 (d), 59.7 (d), 56.4 (d), 55.8 (q), 45.43 (t), 45.37 (t), 38.3 (t), 27.0 (q), 26.2 (t), 23.4 (q), 19.9 (q), 13.2 (q) Nature: amphoteric substance, and,
(4) WF19353D substance: Appearance: white powder
Molecular formula :
C37H44C1N708 Molecular weight:
ESI-MS (negative) : m/z 748(M- H)~ , 750 (M + H)+ Molecular weight: 750.24
Melting point:
230 - 235 ┬░C (dec) Specific rotation:
[╬▒]D(23┬░C) +35 ┬░ (c=0.1, in dimethyl sulfoxide) Ultraviolet absorption spectrum: ╬╗max (methanol) : 228 nm (╬╡ =43000) ╬╗max (0.01N HCl-methanol): 240 nm (╬╡=41000) and 320 nm(╬╡=3900) ╬╗max (0.01N NaOH-methanol): 230 nm (╬╡=41000) Solubility: Soluble: methanol, dimethylsulf oxide, acetic acid
Slightly soluble: water
Insoluble: ethyl acetate, n-hexane Color reaction:
Positive: iodine vapor reaction, cerium sulfate reaction, Dragendorff reaction
Negative: Molish reaction Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica Gel 60 n-butanol:acetic acid:water 0.6
F254** (4:1:2, v/v)
* * made by E. Merck High Performance Liquid Chromatography (HPLC): Condition:
Column: DAISOPAK SP-120-5-ODS-AP*** (4.6 mmφ x 150mmL) Mobile phase: 22% aqueous acetonitrile containing 0.1% trifluoroacetic acid
Flow rate: 1 ml/min Detection: UV at 210 nm
Retention time: 7.0 minutes ***: made by DAISO CO., LTD. Infrared Spectrum: vmax (KBr): 3370, 2970, 1640, 1490, 1440, 1270, 1200, 1140, 1020, 770 cm"1
XH Nuclear magnetic resonance spectrum (Fig. 7) :
(500 MHz, DMSO-d6) ╬┤H 9.08 (IH, br s, exchangeable), 8.87 (IH, s), 8.26 (IH, br s, exchangeable), 8.07 (IH, d, J=8Hz), 7.95 (IH, br d, J=9Hz), 7.74 (IH, m), 7.54 - 7.48 (2H, m), 7.33 (IH, d, J=9Hz), 7.28 (IH, d, J=8Hz), 6.22 (IH, br s, exchangeable),
5.14 (IH, br s), 5.08 (IH, dd, J=7Hz and J=6Hz), 4.89 (IH, br s), 4.70 (IH, m), 4.51 (IH, m), 4.10 (IH, m), 3.99 (IH, s), 3.96 (IH, m), 3.73 (3H, s), 3.30 (IH, m), 3.05 (IH, m), 2.05 (IH, m), 2.05(3H, s), 1.82 (IH, m), 1.80 (3H, s), 1.67 (3H, s), 1.29 (3H, s) 13C Nuclear magnetic resonance spectrum (Fig. 8) :
(125 MHz, DMSO-d6) ╬┤C 168.3 (s), 166.3 (s), 165.2 (s), 159.5 (s), 157.3 (s), 150.1 (d), 146.6 (s), 141.8 (s), 140.3 (s), 135.3 (s), 129.5 (d), 129.2 (d), 129.1 (d), 129.0 (d), 127.2 (d), 126.5 (s), 125.7 (d), 125.3 (s), 125.0 (s), 117.7 (t), 111.3 (d), 75.8 (s), 69.0 (d), 67.5 (d), 60.3 (d), 55.8 (q), 55.4 (d), 45.4 (t), 45.3 (t), 38.2 (t), 26.8 (q), 23.3 (q), 22.7 (q), 20.6 (q) Nature: amphoteric substance.
2. A process for production of the WF19353 substances, which comprises culturing Helicomyces sp. No. 19353 (FERM BP-6358) in a nutrient medium and recovering the WF19353 substances.
3. Biological pure culture of Helicomyces sp. No. 19353 (FERM BP-6358).
4. A pharmaceutical composition containing the WF19353 substance(s).
5. A use of the WF19353 substance(s) as a medicament.
6. A method for treating or preventing diabetes or obesity, which comprises administrating the WF19353 substance(s) to human or animal.
7. Use of the WF19353 substance(s) for the manufacture of a medicament for therapeutic treatment or prevention of diabetes or obesity in human or animal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP54687799A JP2002509445A (en) | 1998-05-29 | 1999-05-21 | New compound |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPP3803A AUPP380398A0 (en) | 1998-05-29 | 1998-05-29 | Novel compounds |
| AUPP3803 | 1998-05-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999063102A1 true WO1999063102A1 (en) | 1999-12-09 |
Family
ID=3808050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1999/002708 WO1999063102A1 (en) | 1998-05-29 | 1999-05-21 | ANTIDIABETIC ACTIVE COMPOUND(S) WF19353 AND PROCESS FOR THEIR FERMENTATIVE PREPARATION USING A FUNGUS OF THE GENUS $i(HELICOMYCES) |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP2002509445A (en) |
| AU (1) | AUPP380398A0 (en) |
| WO (1) | WO1999063102A1 (en) |
-
1998
- 1998-05-29 AU AUPP3803A patent/AUPP380398A0/en not_active Abandoned
-
1999
- 1999-05-21 JP JP54687799A patent/JP2002509445A/en active Pending
- 1999-05-21 WO PCT/JP1999/002708 patent/WO1999063102A1/en active Application Filing
Non-Patent Citations (1)
| Title |
|---|
| No relevant documents disclosed * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002509445A (en) | 2002-03-26 |
| AUPP380398A0 (en) | 1998-06-25 |
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