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WO1995018119A1 - NOUVEAU COMPOSE F-10463a - Google Patents

NOUVEAU COMPOSE F-10463a Download PDF

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Publication number
WO1995018119A1
WO1995018119A1 PCT/JP1994/002202 JP9402202W WO9518119A1 WO 1995018119 A1 WO1995018119 A1 WO 1995018119A1 JP 9402202 W JP9402202 W JP 9402202W WO 9518119 A1 WO9518119 A1 WO 9518119A1
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WO
WIPO (PCT)
Prior art keywords
compound
excipient
pharmaceutically acceptable
effective amount
therapeutically effective
Prior art date
Application number
PCT/JP1994/002202
Other languages
English (en)
Japanese (ja)
Inventor
Takeshi Ogita
Futoshi Nara
Kazuhiko Tanzawa
Tsuyoshi Hosoya
Kouhei Furuya
Original Assignee
Sankyo Company, Limited
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 Sankyo Company, Limited filed Critical Sankyo Company, Limited
Priority to AU12805/95A priority Critical patent/AU1280595A/en
Publication of WO1995018119A1 publication Critical patent/WO1995018119A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/36—Compounds containing oxirane rings with hydrocarbon radicals, substituted by nitrogen atoms

Definitions

  • the present invention relates to a novel compound g′-lU463a which inhibits sphingomyelinase and is fiff) as various medicines, and a method for producing the same.
  • the product ffl of Inyuichi Leukin 10 (hereinafter referred to as “1L-1 / 3”) is diverse and is generally considered to be an essential biological material for maintaining homeostasis.
  • abnormalities in the regulation of IL-1 / 3 production and the overproduction of IL-11 (3) cause various diseases.
  • tumor necrosis factor (hereinafter, referred to as It has the effect of killing certain types of tumor cells or virus-infected cells and enhancing the bacterial action of granulocytes, but also has an excessive amount of TNF- ⁇ . When produced, it is a major cause of several diseases.
  • both cytokines are a major cause of septic shock caused by endotoxin (LPS) entering the body [ ⁇ J y KJ eL al. Science, 234, 470 (1986); Tracey KJ et al. Ature. (Lundon), 330, GC2 (1987)], other granulomas [Kobayashi K. eL al. J. Immunol., 134, ⁇ 8 (198 5)], meningococcal meningitis and malaria infection [Curfs JHA] eL al. J. Exp.
  • LPS endotoxin
  • IL-11 and TNF- are involved in the development and progression of chronic inflammatory diseases such as rheumatoid arthritis (RA), which activate lymphocyte infiltration in synovial tissues and promote synovial cell proliferation. , And destruction of chondrocytes and promotion of bone resorption by activating osteoclasts ffl (Mizel SB et al. Proc.NaLl. Acad. Sci. USA 78, 2474 (198U), Miyasaka N. eL al. Arthritis Rheum., 31, 480 (1988), Arend W.l '. And Uayer J. -M.
  • RA chronic inflammatory diseases
  • RA rheumatoid arthritis
  • Glucocorticoid used as a therapeutic agent Is known to have a part of its effect on the suppression of the production of these site forces. 1 [Lew We. EL al. J. lrnmu nol., 140, 1895 (1988)], Glucocorti Coides have the disadvantage of inducing various serious side effects due to their various physiological actions.
  • IL-10 and TNF- ⁇ adhere to monocytes to endothelial cells and migrate subendothelium [Pober J.S.eL al. J. Immunol., 137, 1893 (1986), Nlken N.A.eL al.
  • IL-1 / 3 and TNF- ⁇ promote platelet activating factor (PAF) production induce tissue factor on the endothelial cell membrane surface, decrease trombombomodulin protein C, and increase plasminogen activity. Suppression of the production of ⁇ ⁇ ⁇ ⁇ 1 ⁇ 1 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ B B ⁇ B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B , 83, 4533
  • IL_1 and TNF-tt are deeply involved in mesangial cell proliferation and substrate regeneration, which are the main causes of glomerulonephritis [Werber 11. I. eL al. J. Immunol., 138, 32 ⁇ 7 (1987), Baud L. et al. Kidney Int., 41, 60 ⁇ (1992)].
  • TNF- ⁇ causes extreme weight loss and loss (cachexia) in chronic infectious disease and cancer patients by suppressing adipocyte lipoprotein lipase activity and causing anorexia (cachexia). It is called cacheclin (cachecLin) [Beutler B. et al. Nature (London), 316, 552 (1985)].
  • TNF-ci transduces transcription from the viral genome terminus of LT1V inserted into chromosome LT through the transcription factor N1'- ⁇ B.
  • N1'- ⁇ B transcription factor
  • fulminant hepatitis [Muto Y. et al. Lancet II, 72 (1988)], asthma, and idiopathic pulmonary fibrosis [Kelley J. Am. Rev. Respir. Dis., 141 (3), 765 (1990)], AKDS (adulL respiratory di stress syndrome) [Millar A. eL al. Lancet II, 712 (1989)], etc., Autoimmune thyroid disease Katsumi Eguchi et al. Approach from the latest medicine 1 From the site force, Mejiriki Rubiyu Inc., 38 (1991)], Lyme disease [Habicht GS tal. J.
  • Sphingomyelinase is an enzyme that decomposes sphingomyelin into ceramide and phosphorylcholine using sphingomyelin, one of the choline-containing lipids contained in the cell membrane system and nucleus in the body, as a substrate.
  • This enzyme was initially found as one of the lysosomal hydrolases having an optimal pH in the acidic region, but recently, the activity of the enzyme having an optimal PH in the neutral region has been demonstrated in microsomal fractions, , 693, 53 (1982), T-Koizumi K. and Kojima KJ Biochem., 93, 1803 (1986). It is thought that various enzymes are actually involved in the metabolism of sphingomyelin in the living body.
  • ceramide is further hydrolyzed by ceramidase to produce fatty acids and sphingosine.
  • sphingomyelins are metabolized in mammals to form ceramides and sphingosins [Schneider PB and Kennedy li. PJ Lipid Kes., (J, 58 ( ⁇ 8)]
  • This ceramide persphingosin, a degradation product of sphingomyelin has been shown to be involved in the control of cell proliferation and differentiation and the signaling mechanisms closely related to them. Kiyohide and Keiko Koizumi Protein Nucleic Acid Enzyme, 36, ⁇ 29 (1991)], and this reaction pathway is called the sphingomyelin pathway.
  • inhibitors of sphingomyelinase activity can block these TNFa and IL-1 signal transductions, and pathological conditions involving these site power-ins. Can be improved.
  • the sphingomyelinase reaction itself promotes the uptake of LDL and denatured LDL involved in the development of atherosclerosis into peripheral cells, increasing cholesterol / ester synthesis and its intracellular accumulation (Stein O. et al. liiochim. Biochim. Acta., 1126, 291 (1992), Chatter jee SJ Biol. Chem., 268, 3401 (1993)], and are expected to be involved in the development of this disease state.
  • sphingomyelinase reduces sphingomyelin content in the apical membrane on the dinusoid side in the proximal tubule of the kidney, and reduces the uptake of phosphate and sugars that function in an Na-dependent manner. [Vrtovsnik F. et al. Kidney International., 41, 983 (1992)].
  • specific inhibitors of sphingomyelinase are anti-HIV agents, anti-diabetic agents, anti-atherosclerotic agents, anti-osteoporosis agents, anti-thrombotic agents, pile inflammatory agents, immunosuppressants, diuretics, and Can be used as a prophylactic or therapeutic agent for respiratory disease, thyroid disease, Alzheimer's disease, hepatitis, nephritis, leukemia, and caxonia.However, no specific and potent inhibitor of sphingomyelinase has been found to date. .
  • soluble IL-11 receptors and IL-1 receptor antagonists have been found as substances that specifically inhibit the IL-1 / 3 action, and patients with septic shock and Symptom improvement has been seen in RA patients.
  • the present inventors searched for substances having sphingomyelinase inhibitory activity from microbial metabolites.
  • the present inventors have found that a novel compound G-10463a having an inhibitory activity on isase is produced, and completed the present invention.
  • the F-104 (53a) of the present invention has the following structural formula and properties.
  • Examples of the strain belonging to the genus Dasyscyphus used in the present invention include Dasyscyphus moll issimus (Lasch) Dennis SANK13892 strain (FEKM bP-4491), and the microbiological properties of this strain are as follows. .
  • SANK13892 was isolated from a dead herb stem collected in Aomori Prefecture in May 15J91.
  • the ascidian disc is a flat, disc-shaped disk with a maximum diameter of 0.2 countries and is grayish orange to yellow. Hair on the periphery of the ascus disc.
  • the outer skin layer is composed of cells of a pale brown, thin-film appearance, which looks like a polygonal tissue.
  • the medulla layer is tightly entangled and consists of hyphae slightly parallel to the outer wall, presenting entangled bacterial tissue.
  • the hair grows straight from the outermost layer of the outer skin layer, has a cylindrical shape, has a thin film, a large number of partition walls, a maximum length of 2 ⁇ . And a thickness of 3.5 ⁇ . Its tip is almost blunt. The surface is almost smooth, and resinous amorphous substances are scattered and adhere.
  • the ascus is formed by a clamp and is cylindrical, 8-spore, 58-66 X 5-B. 5 ⁇ m in size. Its tip turns blue with Melzer reagent.
  • the lateral thread is a thin spear, measuring 74.5-10 ⁇ X 3.5-4 ⁇ and extending about 20 ⁇ above the asci.
  • Ascospores are rod-shaped to spindle-shaped, single-celled, colorless and are 18-25 x 2.5-5 ⁇ m in size.
  • the separation medium used for isolating the novel strain of the present invention is a medium containing a material selected from a carbon source, a nitrogen source, an inorganic ion, an organic nutrient source, and the like as appropriate, and is either a synthetic or natural medium. But it can be used.
  • F-10463a is obtained by culturing the strain in an appropriate medium and collecting from it.
  • the nutrient source known nutrients conventionally used for culturing fungi can be used.
  • a carbon source glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean ash, etc. can be used.
  • soybean flour, corn steep liquor, dust extract, malt extract, potato, ammonium sulfate, sodium nitrate and the like can be used as a nitrogen source.
  • inorganic salts such as calcium carbonate and phosphate, and appropriately add organic and inorganic substances that promote the growth of the strain and promote the production of F-10463a.
  • the culture method the liquid culture method, particularly the submerged culture method, is the most suitable, as is the method for producing general antibiotics. The cultivation is performed under aerobic conditions, and the appropriate temperature for cultivation is 15-30 ° C, but in most cases, it is cultivated around 23.
  • F-10463a usually reaches a high level at 5- ⁇ mouth in shaking culture.After completion of the culture, the amount of F-10463a present in the cells of the culture or in the oral fluid is reduced to the volume of acetone Extraction is performed by adding and mixing an organic solvent such as cetonitrile. The solid portion present in the extract is separated by filtration or centrifugation using diatomaceous earth as a filtration operation aid, and F-104Ma present in the filtrate or supernatant is determined by using the sphingomyelinase inhibitory activity as an index. Extraction and purification are performed using the physicochemical properties for 1H.
  • F-10463a present in this extract can be obtained by first removing the contaminating organic solvent by a concentration operation, and then removing the immiscible organic solvent, for example, n-butanol. , Methylethyl ketone, ethyl acetate, black form, ethylene chloride, methylene chloride, etc., alone or in combination.
  • an adsorbent for example, activated carbon or an adsorbent resin such as Amberlite XA II-2, XAD-4 (manufactured by Rohm and Haas), Diaion H-10, HP-20, CH P-20P, HP-50 (Mitsubishi Kasei Co., Ltd.) etc. can be used.
  • the F-10463a thus obtained was further subjected to adsorption column chromatography using a carrier such as silica gel or florisil, distribution column chromatography using Sephadex LH-20 (manufactured by Pharmacia), Sephadex G It can be purified by gel filtration chromatography using -25 (manufactured by Pharmacia) and high-performance liquid chromatography using normal-phase and reverse-phase columns.
  • Sphingomyelinase inhibitory activity can be measured by the following method ( ⁇ Lipid Kes. 2 ⁇ , 456 (1979)).
  • the sphingomyelinase reaction is performed by mixing 50 ⁇ l of the substrate solution prepared in this manner with 10 ⁇ l of the sample solution and mixing the microsomal fraction (25,000 000 g 100,000 xg, protein concentration 3 to 4 mgZm 1) 40 ⁇ l was added as an enzyme solution, and incubation was carried out at 37 ° C for 40 minutes. After the reaction is completed, add 500 ⁇ l of chloroform: methanol (2: 1, V / V) for extraction and perform extraction.
  • sphingomyelinase activity is calculated as a value obtained by subtracting the measured value obtained by subtracting the MgC required for the sphingomyelinase reaction from the measured value.
  • the main culture was performed as follows. Sterile each 5ml who is the seed culture The culture medium 1 0 () nil of the above composition into an Erlenmeyer flask fifteen including 500 ml, 2 3 "(: between 7 days, shaking Rotary 2 00R P m The culture was carried out on a machine.
  • the preparation of the sphingomyelinase enzyme preparation was basically carried out according to the method of (; att S. (Bio hem. Biophys. Res. Commun. 68, 235 (1976)). Using rat brain as a source, its microsomal fraction was first prepared as follows: 10 Wistar Imamichi male rats ( ( J weeks old) were exsanguinated from the carotid artery, Immediately after removing the cerebellum, buffer A (0.25 sucrose, ImM ⁇ ⁇ ), 1 mM, pre-cooled to 4
  • the sphingomyelinase activity was measured as follows using a mixed micellar system with reference to the method of Hostetler KY and Yazaki PJ (Lipid Res. 2 ⁇ , 456 (1979)).
  • sphingomyelin cattle, 20 mM, Sigma
  • was dried to dryness with nitrogen gas 200 ⁇ l of 1 M tris-monohydrochloric acid ( ⁇ 7.5) and 40 ⁇ l of 10% ( ⁇ / V) Triton X—100, 20 ⁇ l of 0.5 ⁇ MgCl 2 , and 1.24 ml of H 20 were added, and incubated at 48 ° C. for 30 minutes.
  • sonication is performed twice for 15 seconds under the output power of 20W,
  • a mixed micelle system 101 [ ⁇ -methyl
  • sphingomyelinase reaction mix 10 ⁇ l of the sample solution with 0 ⁇ l of the substrate solution prepared in this way, add 10 ⁇ l of the enzyme solution described above, and incubate for 37 and 40 min. Went by. After the completion of the reaction, 500 1 of Kuguchi-holm: methanol (2: 1, ⁇ / ⁇ ) is added and extraction operation is performed. The obtained aqueous layer 150 ⁇ 1 is added to a 3 m1 Bikofuro''40 (Packard Japan). And [ 14C ] phosphorylcholine as a reaction product was measured with a liquid scintillation counter. The sphingomyelinase activity is calculated as the value obtained by subtracting the measured value excluding MgC required for the sphingomyelinase reaction from this measured value.
  • the concentration of F-10463a required to inhibit the sphingomyelinase reaction by 50% as determined by this method was 20 g / roi.
  • the culture solution of 15 Erlenmeyer flasks was adjusted to pH 3 with GN hydrochloric acid, and then centrifuged at 3,000 rotations for 10 minutes.
  • 5 UU ml of 80% acetone was added, and the mixture was stirred for 1 hour.
  • the mixture was centrifuged at 30 ° C. for 10 minutes, and the supernatant was concentrated under reduced pressure to remove acetone. It was then extracted twice with an equal volume of ethyl acetate.
  • the ethyl acetate layer was washed with a saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 430 rag of a brown syrup.
  • the novel compound F-10463a of the present invention is an anti-HIV agent, an anti-diabetic agent, an anti-atherosclerotic agent, an anti-osteoporosis agent, an anti-thrombotic agent, an anti-inflammatory agent, an immunosuppressant, a diuretic, and a respiratory disease. It can be used as a prophylactic or therapeutic agent for thyroid disease, Alzheimer's disease, hepatitis, nephritis, leukemia, and caxonia.
  • compositions of the present invention used to treat the above conditions comprise a mixture of a medically indicated carrier and a therapeutically effective amount of F-10463a.
  • the composition can be administered in various forms, such as tablets, capsules, granules, powders, syrups, or injections, drops, suppositories, etc. Oral administration can be mentioned.
  • compositions of the present invention When administered as an injection or infusion, the therapeutic compositions of the present invention are pyrogen-free and in the form of a parenterally acceptable aqueous solution.
  • parenterally acceptable preparations prepared in view of PH, isotonicity and stability are within the skill of the art.
  • Dosage and administration regimens for treatment of the above conditions will depend on factors that may affect the action of the drug, such as the patient's symptoms, weight, sex, age, diet, severity of any infection, time of administration, and other factors. It can be determined by the attending physician in view of factors that may have a clinical impact.
  • the dose is about 0.01 to 1 rag to ⁇ mg per day for an adult, and these can be administered once or in several divided doses.
  • about 0.01 mg to 10 mg can be administered by subcutaneous injection, intramuscular injection, or intravenous injection.

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

Abstract

L'invention concerne un nouveau composé F-10463a représenté par la formule (I) et utile en tant que principe actif anti-HIV, antidiabétique, antiartérioscléreux, antiostéoporeux, antithrombotique, anti-inflammatoire, immunodépresseur, diurétique, et pour prévenir ou traiter les maladies respiratoires, thyroïdiennes, la maladie d'Alzheimer, l'hépatite, la néphrite, la leucémie et la cachexie.
PCT/JP1994/002202 1993-12-27 1994-12-26 NOUVEAU COMPOSE F-10463a WO1995018119A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12805/95A AU1280595A (en) 1993-12-27 1994-12-26 Novel compound f-10463a

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP33061393 1993-12-27
JP5/330613 1993-12-27

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WO1995018119A1 true WO1995018119A1 (fr) 1995-07-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999030739A1 (fr) * 1997-12-16 1999-06-24 Sankyo Company, Limited Remede contre la leucemie
WO2001060809A1 (fr) * 2000-02-18 2001-08-23 Sankyo Company, Limited Derives 4,5-epoxy-6-hydroxy-2-cyclohexene-1-one 6-substitues et intermediaires utilises dans leur production

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03157386A (ja) * 1989-07-31 1991-07-05 Merck & Co Inc 新規なＨＭＧ―ＣｏＡ合成酵素阻害剤

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03157386A (ja) * 1989-07-31 1991-07-05 Merck & Co Inc 新規なＨＭＧ―ＣｏＡ合成酵素阻害剤

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999030739A1 (fr) * 1997-12-16 1999-06-24 Sankyo Company, Limited Remede contre la leucemie
WO2001060809A1 (fr) * 2000-02-18 2001-08-23 Sankyo Company, Limited Derives 4,5-epoxy-6-hydroxy-2-cyclohexene-1-one 6-substitues et intermediaires utilises dans leur production

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Publication number Publication date
AU1280595A (en) 1995-07-17

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