WO1995006646A1 - Substances br-050 et br-051 inhibant la resorption osseuse - Google Patents

Substances br-050 et br-051 inhibant la resorption osseuse Download PDF

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Publication number
WO1995006646A1
WO1995006646A1 PCT/JP1994/001445 JP9401445W WO9506646A1 WO 1995006646 A1 WO1995006646 A1 WO 1995006646A1 JP 9401445 W JP9401445 W JP 9401445W WO 9506646 A1 WO9506646 A1 WO 9506646A1
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WO
WIPO (PCT)
Prior art keywords
bone resorption
methanol
strain
culture
shows
Prior art date
Application number
PCT/JP1994/001445
Other languages
English (en)
Japanese (ja)
Inventor
Terumi Kagamizono
Akiko Maejima
Akira Kawashima
Tomoko Akiyama
Tsutomu Ando
Kazuhide Isogai
Shigeo Morimoto
Original Assignee
Taisho Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Taisho Pharmaceutical Co., Ltd. filed Critical Taisho Pharmaceutical Co., Ltd.
Priority to AU75462/94A priority Critical patent/AU7546294A/en
Publication of WO1995006646A1 publication Critical patent/WO1995006646A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention relates to a compound having a bone resorption inhibitory action c
  • calcitonin is a peptide having a molecular weight of about 3,400 in which 32 amino acids are bound, its absorption from the gastrointestinal tract was low, and it had to be used as an injection.
  • an orally administrable drug, rather than an injection is ideal for patients, and its appearance has been awaited. Have been. Disclosure of the invention
  • the present inventors have isolated a large number of strains from nature and studied various cultures of the strains.As a result, they have found that a compound produced by a certain strain has a strong bone resorption inhibiting action, and It was completed.
  • Is a compound represented by H ⁇ _H FC (hereinafter, BR- 050, A when A is c u is referred to when BR- 051 of c o).
  • strains that produce BR-050 and BR-051 are newly isolated strains from plants collected by the present inventors in Awano-cho, Kamitsuga-gun, Tochigi Prefecture, and have the name of microorganism, fPenicil lifer superimpositus TF-0402J and accession number. Deposited as "FE RM BP-4755" with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. The bacteriological properties of this strain are shown below.
  • This strain grows well on potato-glucose, oatmeal, malt extract, Yp Ss ⁇ Sablo agar medium, etc., and forms spores with potato-glucose, oatmeal, YpSs, cornmeal, LCA (Miura) Good or moderate on agar medium. Observation by an optical microscope of colonies formed by this strain on LC A (Miura) agar medium at 26 ° C for 17 days shows that the hyphae are highly branched with septa, and the conidiophores are aerial mycelia. It is unbranched and rarely branched in rare cases, and a single or 3 to 6 conidium-forming cells (fiaraids) grow from its tip.
  • Conidiophores are colorless, surface is smooth, width is 3.5-5.5 m at base, length is 55-125 im for unbranched, 100-225 m2 for branched and rarely more Obviously Obviously, Fiaraid is colorless, smooth surface, tapered cylindrical shape, length 20 ⁇ 47 / zm, width 2.5 ⁇ 4.5 111 at base, 1.0 ⁇ 1.5 at tip
  • Conidia are colorless, one septum, rarely lacks a septum, surface is smooth, spindle-shaped, 8-24 / m in length, 1.5-4.2 m, diagonally displaced slightly from the tip of the phialid It sticks and chains, but rarely becomes a sticky mass.
  • the culture was extended to 3 weeks, but no form of teleomorph was observed.
  • Table 1 shows the results of macroscopic observations when the cells were cultured on various media for 26 and 14 days.
  • the colors used are based on the system color names in the Japan Standards Association and JIS Color Name Book (1985).
  • This strain grows in Sabouraud's liquid medium at pH 6.0 in the range of 9-31 ° C, and the optimal temperature is 25-29 ° C.
  • This strain grows in ⁇ 2 ⁇ 10 at 26 ° C in Yp S s liquid medium,
  • the optimal pH is 6-7.
  • this strain Based on the above morphological characteristics and culture characteristics, this strain
  • the strain was closest to superimpositus and was found to exhibit properties.
  • the strain was named fPenicil lifer suTDerimpositus TF-0402.
  • BR-050 and BR-501 are similar to the production of general fermentation products, and the Penicilliier super impositus TF-0402 strain is cultured under aerobic conditions in a medium containing various nutrients. It is done by doing.
  • a liquid medium is mainly used as a medium, and glucose, sucrose, molasses, starch, etc. are used alone or in combination as a carbon source.
  • a nitrogen source meat extract, oatmeal, yeast extract, soybean powder, polypeptone, etc. are used alone or in combination.
  • organic substances and inorganic salts that promote the growth of this strain and promote the production of BR-0500 and BR-051 can be added as necessary.
  • Adekanol, silicon, and the like can be used as the defoaming agent.
  • the cultivation method is aerobic cultivation such as shaking culture and aeration-agitation culture (suitable for pH 2 to 10, 9 to 31 ° C for 3 to 6 days, preferably pH 6 to 7, 25 Incubate at ⁇ 29 ° C for 5 days.
  • aerobic cultivation such as shaking culture and aeration-agitation culture (suitable for pH 2 to 10, 9 to 31 ° C for 3 to 6 days, preferably pH 6 to 7, 25 Incubate at ⁇ 29 ° C for 5 days.
  • BR-0500 and BR-051 produced by this culture may be isolated according to a general method for collecting fermentation products. For example, the following method is effective.
  • the culture solution is extracted with an organic solvent such as acetone.
  • the solution is transferred to a non-water-soluble organic solvent such as ethyl acetate, benzene, or chloroform, and concentrated to a syrup.
  • a non-water-soluble organic solvent such as ethyl acetate, benzene, or chloroform
  • the syrup is dissolved again in an organic solvent such as benzene, ethyl acetate, acetone, methanol, chloroform, and the like, and subjected to silica gel column chromatography, gel filtration, column chromatography, and high-performance liquid chromatography to obtain the compound of the present invention.
  • silica gel column chromatography gel filtration, column chromatography, and high-performance liquid chromatography
  • Fig. 1 shows the results measured by the KBr method.
  • Fig. 4 shows the results measured by the KBr method.
  • FIG. 1 shows an infrared absorption spectrum of BR-050 measured by the KBr method.
  • Figure 13 shows the 13 C-NMR spectrum of 050.
  • FIG. 4 shows the infrared absorption spectrum of BR-051 measured by the KBr method.
  • the culture solution of 200L Jar Amen for 1 unit of 120L and 50L Jar Amentor for 3 units of 90L was filtered and separated into supernatant and cells, and the supernatant was adsorbed.
  • resin Diaion HP-20 (trade name; manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) 10L]
  • wash with 20L of purified water and elute the fraction eluted with a mixed solution of acetone / water (50:50).
  • the active substance was eluted with 20 L of acetone.
  • the cells were extracted twice with 15 L of acetone.
  • the supernatant-derived acetone eluate and the cell-derived acetone extract were combined, concentrated under reduced pressure, and the obtained residue was extracted twice with 15 L ethyl acetate.
  • the ethyl acetate fraction was dehydrated with anhydrous sodium sulfate and concentrated to dryness to obtain 85.8 g of a brown oily substance.
  • the mixture was eluted with the mixed solvent of 5) and concentrated to dryness to obtain 32.8 g of a brown oily substance.
  • the obtained sample was subjected to gel filtration with SEPHADEX LH-20 (trade name, manufactured by Pharmacia) prepared with n-hexane-dichloromethane-methanol (5: 5: 1), and the active fraction was collected and concentrated to dryness 9 g of a solid, brown oil was obtained.
  • the obtained sample was dissolved in 15 ml of chloroform, and adsorbed on a column (0.8 L, solvent: chloroform) filled with silica gel. After washing with 1.6 L of black-mouthed form, the fraction eluted with a mixed solvent of black-mouthed form-methanol (99: 1) was removed. Then, elution was carried out with a mixed solvent of black form-methanol (98: 2), and the mixture was concentrated to dryness to obtain 4 g of a brown oily substance.
  • SEPHADEX LH-20 trade name, manufactured by Pharmacia
  • Carrier silica gel (Sensyu Chemical Co., Ltd.) Solvent composition 1% methanol, 99% black form
  • Carrier ODS-silica gel (Sensyu Chemical Co., Ltd.) Solvent composition 73% acetonitrile, 27% water
  • the compound of the present invention Since the compound of the present invention has an excellent inhibitory action on bone resorption, it is useful as a therapeutic drug for diseases based on abnormal bone metabolism such as osteoporosis and hypercalcemia.
  • the compounds of the invention can be administered orally in dosage forms such as tablets, pills, capsules, granules and the like, which are manufactured according to conventional pharmaceutical techniques.
  • dosage forms such as tablets, pills, capsules, granules and the like, which are manufactured according to conventional pharmaceutical techniques.
  • additives such as ordinary bulking agents and binders.
  • the dosage of the compound of the present invention for a treated patient may vary depending on the age of the patient, the type and condition of the disease, and the like. Normally, 0.5 to 700 mg, preferably 1 to 50 mg per day or several times per day. Ability to administer in divided doses ⁇ Can.
  • Test example (Bone resorption inhibitory action)
  • a 5-day-old heron femur and tibia are removed, cut into small pieces with scissors, and 5% fetal blood
  • the mixture was stirred for 30 seconds in ⁇ -minimum essential medium (one MEM) containing Kiyoshi (FBS).
  • ⁇ -minimum essential medium one MEM
  • FBS Kiyoshi
  • the supernatant of the cell suspension containing the separated osteoclasts was placed in a 96-well plate containing ivory slices sliced to a diameter of 6 mm and a thickness of 150 / zm. We sowed to be we1 1.
  • the number of P and pits stained under a microscope was counted, and the number of pits in the compound-added group was calculated assuming that the number of pits formed in the control group without the compound was 100%. It was determined the bone resorption inhibitory concentration (IC 5. value).
  • the compound of the present invention has an IC 5 .
  • the values were 0.01 ⁇ gZml for BR-050 and 0.05 g / m1 for BR-051.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Des composés représentés par la formule générale (I), offrent une excellente inhibition de la résorption osseuse et se révèlent donc utiles comme remèdes pour des maladies dues à un métabolisme osseux anormal, telles que l'ostéoporose et l'hypercalcémie. Dans cette formule, A représente CH(OH) ou C=O.
PCT/JP1994/001445 1993-09-03 1994-09-01 Substances br-050 et br-051 inhibant la resorption osseuse WO1995006646A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75462/94A AU7546294A (en) 1993-09-03 1994-09-01 Bone resorption inhibiting substances br-050 and br-051

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP5/219833 1993-09-03
JP21983393 1993-09-03
JP6/68591 1994-04-06
JP6859194 1994-04-06

Publications (1)

Publication Number Publication Date
WO1995006646A1 true WO1995006646A1 (fr) 1995-03-09

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1994/001445 WO1995006646A1 (fr) 1993-09-03 1994-09-01 Substances br-050 et br-051 inhibant la resorption osseuse

Country Status (2)

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AU (1) AU7546294A (fr)
WO (1) WO1995006646A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090326A1 (fr) * 2004-03-24 2005-09-29 The Kitasato Institute Antibiotique fki-1778 et procédé servant à produire celui-ci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4913392A (fr) * 1972-04-05 1974-02-05

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4913392A (fr) * 1972-04-05 1974-02-05

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090326A1 (fr) * 2004-03-24 2005-09-29 The Kitasato Institute Antibiotique fki-1778 et procédé servant à produire celui-ci

Also Published As

Publication number Publication date
AU7546294A (en) 1995-03-22

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