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WO1984001574A1 - Process for preparing physiologically active substance fa-5859 and its derivatives - Google Patents

Process for preparing physiologically active substance fa-5859 and its derivatives Download PDF

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Publication number
WO1984001574A1
WO1984001574A1 PCT/JP1982/000409 JP8200409W WO8401574A1 WO 1984001574 A1 WO1984001574 A1 WO 1984001574A1 JP 8200409 W JP8200409 W JP 8200409W WO 8401574 A1 WO8401574 A1 WO 8401574A1
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WIPO (PCT)
Prior art keywords
reaction
acid
compound
salt
water
Prior art date
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PCT/JP1982/000409
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French (fr)
Japanese (ja)
Inventor
Susumu Shinagawa
Tsuneo Kanamaru
Mitsuko Asai
Original Assignee
Takeda Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Priority to PCT/JP1982/000409 priority Critical patent/WO1984001574A1/en
Priority to US06/442,368 priority patent/US4521432A/en
Priority to CA000416173A priority patent/CA1190166A/en
Priority to DE8282110866T priority patent/DE3263167D1/en
Priority to EP82110866A priority patent/EP0080695B1/en
Priority to KR1019830001950A priority patent/KR900008248B1/en
Publication of WO1984001574A1 publication Critical patent/WO1984001574A1/en
Priority to US06/939,152 priority patent/US4767781A/en
Priority to US07/423,555 priority patent/US4948534A/en

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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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • 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
    • C12R2001/66—Aspergillus

Definitions

  • the present invention relates to a method for producing a novel physiologically active substance F A-58559 and a derivative thereof having an excellent fatty acid decomposition inhibitory action.
  • Decanoyl 'Carnitine decanoyl- (10) -l-ca-rniti-ne
  • 2-bromopalmitoyl-Coenzyme A I- ⁇ t, ri tz et al., Proc. Proceedings of the National Academy of Sciences U.S.A. S. A. 54, p. 122, pp. 1965] is known; however, it has not been clinically evaluated in terms of toxicity and action.
  • Methyl-2-tetradecylglycide and 2-tetradecylglycide (me thy 1-2-tetradecylg-lycidate, 2-tetradecylglycidate) [GF Tutwiler et al., Diabetes (Vol. 28, p. 24, pp. 1979 and Method-in. Methods in Enzymology (see p. 72, p. 533, pp. 1989))
  • the present invention relates to a method for producing a novel physiologically active substance FA-585-9 and a derivative thereof.
  • the present invention relates to (1)
  • R2 represents a protecting group other than an acetyl group.
  • a compound represented by the general formula CH 3, wherein the compound C 2] or a salt thereof is subjected to a protecting group elimination reaction and, if necessary, to an acetylation reaction.
  • insulin deficiency promotes the release of fatty acid in adipose tissue due to insulin deficiency, thereby increasing the supply of fatty acids to the liver and, at the same time, increasing the production of ketone bodies due to the accelerated decomposition of fatty acids.
  • they exhibit a so-called keton deficiency.
  • glucose is poorly used in extrahepatic tissues, and the generated ketones are used as an energy source. Therefore, if the breakdown of fatty acids is prevented, glucose utilization is expected to increase with a decrease in ketone bodies, which is expected to result in a decrease in blood sugar levels.
  • Specific inhibitors of fatty acid degradation are based on a new mechanism.
  • the present inventors have conducted various studies for the purpose of obtaining a derivative of the physiologically active substance FA-589, and found that a deacetyl derivative obtained by hydrolyzing the physiologically active substance FA-589 is novel. It has been found that it is a substance and has a remarkable inhibitory effect on fatty acid degradation, and is referred to as a physiologically active substance deacetyl (FA-5589).
  • the present inventors have further studied various methods for chemically synthesizing the bioactive substance deacetyl-FA-589 and the bioactive substance FA-589, and have found that the general formula
  • R 2 has the same meaning as described above.
  • examples of the protective group other than the acetyl group represented by R2 include t-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-dibenzyloxycarbonyl Bonyl and the like.
  • any method for decomposing an amide bond can be applied.
  • the acid include inorganic acids such as sulfuric acid and hydrochloric acid
  • examples of the base include rhodium hydroxide, sodium hydroxide and barium hydroxide.
  • ion exchange resin examples include Dowex-50 (manufactured by Dow Chemical Company, USA), Amberlight IR-120 (manufactured by ROHM 'And'Haas' Comparator, USA), Diamond-SKIA, SKIB (Mitsubishi Chemical Corporation) Industrial Co., Ltd., Japan).
  • the reaction is preferably carried out in an aqueous solution, and more preferably in a mixed solvent of water and methanol, ethanol, butanol or the like as a solvent.
  • the reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 10 ° C, for about 30 minutes to 30 hours, and more preferably for about 3 to 16 hours.
  • the above base is preferably carried out in an aqueous solution, and more preferably in a water-containing solution, for example, a mixed solvent of water, methanol, ethanol, butanol and the like.
  • the reaction is usually carried out at about 60 to 200 ° C., more preferably at about 90 to: I 20 ° C., for about 30 minutes to 30 hours, more preferably at about 3 to 1 hour. It takes 6 hours.
  • the above-mentioned ion-exchange resin is used, it is carried out by heating the ion-exchange resin in a state of being suspended in an aqueous solution of the raw material compound.
  • the reaction is usually carried out at about 60 to 200 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.
  • the catalytic reduction reaction is carried out in water or alcohol such as methanol or ethanol.
  • Hydrogen gas is introduced in the presence of a catalyst such as hydrogen, and pressurization is performed as necessary.
  • the temperature of the catalytic reduction reaction is about 0-5 °. c, preferably about 2 0-3 0
  • the reaction time is about 0.5 to 5 hours, more preferably about 1 to 3 hours.
  • the acid used in the acid treatment include bromoiodic monoacetic acid, monoacetic acid hydrochloride, dioxane hydrochloride, trifluoroacetic acid, and methanesulfone.
  • Trifluor ⁇ -methanesulfonic acid and the like.
  • the temperature of the acid treatment is about 10
  • reaction time is about 30 minutes to 24 hours, and is suitably changed depending on the acid used.
  • acetylate for example, the above compound, acetic anhydride, acetyl chloride
  • Lide or acetic acid is converted to methyl carbonate, ethyl chlorocarbonate,
  • Alkoxycals such as chill, chlorocarbonate, ⁇ -pill, and isobutyl chlorocarbonate
  • the anhydride of sulphonic acid obtained by reaction with bonyl chloride is converted into water or water and water.
  • Organic bases such as mines or hydroxides, oxides, bicarbonates, etc. of alkali metal, alkaline earth metal such as sodium, potassium, potassium hydroxide, etc.
  • the reaction temperature is about -10 ° C to 50 ° C, which is preferable.
  • the compound [3] of the present invention or a salt thereof is subjected to a trimethylation reaction.
  • the reaction is carried out in an organic solvent such as acetone, dioxane, tetrahydrofuran, dimethylformamide and the like.
  • an alkali metal such as sodium, potassium, or calcium, or a hydroxide or oxide of an alkali earth metal may be coexisted, if necessary.
  • the reaction temperature is about -10 ° C to 50 ° C, preferably about 0 ° C to 20 ° C.
  • Each target product produced by each of the above reactions can be collected and purified by commonly used separation and purification means, for example, methods such as chromatography and recrystallization.
  • the compound [3] or a salt thereof is subjected to a trimethylation reaction, and then, if necessary, any hydrolysis method for decomposing an amide bond can be applied to the hydrolysis reaction.
  • any hydrolysis method for decomposing an amide bond can be applied to the hydrolysis reaction.
  • it can be carried out by using an acid, a base, an ion exchange resin or the like.
  • the acid include inorganic acids such as sulfuric acid and hydrochloric acid.
  • Examples of the base include potassium hydroxide, sodium hydroxide and paricum hydroxide.
  • the ion exchange resin include: Dowex-50 (manufactured by Dow Chemical Company, USA), Amberlight IR-120 (manufactured by ROHM 'And-Haas' Company, USA), Diamond-SKIA, SKI B (Mitsubishi Chemical Industries, Japan).
  • the reaction is preferably carried out in an aqueous solution, and more preferably in a mixed solvent of water and methanol, ethanol, butanol or the like as a water-containing solvent.
  • the reaction is' usually about 6 0 to 2 0 0 ° C, further preferably about 9 0 ⁇ 1 2 0 ° C, about 3 0 minutes to 3 0 hours, preferably Furthermore at about 3 to 1 6 hours It is.
  • the reaction is usually carried out at about 60 to 20 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably about 3 to: I 6 Done in time.
  • the reaction is carried out by heating the ion exchange resin suspended in an aqueous solution of the raw material compound.
  • the reaction is usually performed at about 60 to 200 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably for about 3 to 16 hours.
  • deacetyl FA-5589 or a salt thereof from the reaction mixture, commonly used means such as ion exchange resin, adsorption, concentration, and crystallization are used.
  • ion exchange resin ion exchange resin
  • concentration concentration
  • crystallization ion exchange resin
  • the target substance can be isolated in either a difficult form or a salt, but it is easier to separate and collect it in the form of a salt.
  • a strongly acidic ion exchange resin or the like is used to adsorb the target substance, and elution is performed using, for example, hydrochloric acid or the like.
  • the target substance can be obtained by collecting the indicated categories.
  • the reaction solution is more easily concentrated under reduced pressure to remove the excess salt, and then methanol, ethanol, etc.
  • Jechirue - solvent salted £ salts of the present substance can be obtained.
  • a crystal such as ether.
  • an acid or a base forming the salt may be adsorbed on the ion-exchange resin, for example, by suspending an ion-exchange resin or the like.
  • the free form of deacetyl FA-585-99 or FA-585-99 free form can form a salt, which can be converted into a pharmaceutically acceptable salt by conventional means. Wear.
  • the acid for forming the; ⁇ include hydrochloric acid, sulfuric acid, nitric acid, oxalic acid, acetic acid, succinic acid, citric acid, and fumaric acid.
  • Compound [ 2 ] used as a starting compound in the method of the present invention can be produced, for example, by the following reaction steps.
  • Compound [ 3 ] can be produced, for example, by the following reaction steps.
  • R3 represents a protecting group which differs from the protecting group shown by R2 in the nature at the time of elimination.
  • the protecting group represented by R3 includes, for example, benzyloxycarbonyl, t-butoxycarbonyl, p-nitrobenzyloxycarbonyl, F> —methoxybenzyloxycarbonyl, trityl, tolu, t— Amyloxy carbonyl, isoborniroquine carbonyl, diphenylphosphinyl, 0-di-to- ⁇ -phenylsulfenyl, phthaloyl and the like.
  • the reaction temperature at which organic bases such as rare amines and pyridines may be used is from ⁇ 20 to 3 (TC, preferably from about ⁇ 10 to 0 °).
  • the compound [5j may be substituted with ethyl acetate, methyl acetate, tetrahydrofuran, dioxane, acetate nitrolinole, getyl ether.
  • Diazomethane may be blown directly into the reaction solution, or a solution saturated with an organic solvent such as getyl ether or ethyl acetate may be added to the reaction solution.
  • a silver salt such as benzoic acid or acetic acid is added to a methanol solution of the compound [6] with trimethylamine, triethylamine, N-methylmorpholine. , N-ethylethyl morpholine, pyridine, etc. in a solution of an organic base at about -5 to 50 ° C, preferably about 0 ° C to 27 ° C, and about 30 minutes to 10 ° C in the dark. This is done by acting on time.
  • Compound [8] can be produced by saponifying compound [7].
  • the saponification is carried out, for example, in water or an organic solvent such as methanol, ethanol, dioxane, tetrahydrofuran, or acetonitrile, or in a mixed solvent of water and these organic solvents.
  • compound [7] and hydroxides such as sodium, potassium, and balium at about ⁇ 10 ° C. to 50 ° C., preferably at about 0 ° C. to 27 ° C. for about 1 hour.
  • the reaction is carried out for 5 hours.
  • the compound [2] can be produced by subjecting the compound [9] to a trimethylation reaction.
  • the trimethylation reaction can be carried out in the same manner as in the reaction for subjecting the compound [3 to the above-mentioned trimethylation reaction.
  • the conversion of the compound [8] into the compound [10] can be carried out in the same manner as in the above-mentioned reaction of subjecting the compound [2] to the protecting group elimination reaction.
  • Compound [11] can be produced by subjecting compound [10] to an acetylation reaction.
  • the acetylation reaction can be carried out in the same manner as the acetylation reaction in which the compound [ 2 ] is converted to a protective group elimination reaction and, if necessary, subjected to the acetylation reaction.
  • Compound [3] can be produced by subjecting compound [11] to a protecting group elimination reaction.
  • the protecting group elimination reaction can be carried out in the same manner as in the reaction for producing compound [9] from compound [8] described above.
  • Each target product produced by each of the above reactions can be collected and purified by commonly used separation and purification means, for example, methods such as mouth chromatography and recrystallization.
  • the compound [8] can be used as a synthetic intermediate when producing a valuable compound.
  • salts of those compounds may be used.
  • the salt include sodium, potassium, calcium, barium, triethylamine, pyridine, hydrogen chloride, hydrogen bromide, and hydrogen iodide.
  • the inhibitory activity of Detatyl FA-5859 a concentration at which the degradation activity was reduced to 50 in comparison with no addition of the inhibitor, was 4-8.
  • Acute toxicity of deacetil; FA-585-9 The LL> 50 value was more than 40 o / T ⁇ by intravenous injection in mice.
  • Decetyl FA-585-9 or a salt thereof is useful, for example, as a fatty acid degradation inhibitor.
  • Deacetyl: FA-5589 or a salt thereof may be used as a fatty acid degradation inhibitor, for example, for the treatment of diabetes in mammals (eg, mice, rats, humans, etc.). About 0.2 to 200 ⁇ as a daily dose.
  • Ma when administering decetyl FA-5589 or a salt thereof, it is orally prepared in the form of tablets, granules, capsules, liquids and the like by conventional means, or parenterally as an injection, for example. Can be administered.
  • deacetyl FA-5589 can be used as a biochemical reagent for elucidating the metabolism of fatty acids.
  • Dacetel F A—5859 to the reaction solution! Carnitine deficiency can be easily created by this method, so that the role of carnitine in fatty acid oxidation can be clarified.
  • the uptake of fatty acids into mitochondria was inhibited by the addition of deacetel FA-5589. By doing so, it is possible to clarify the role of peroxisomes and the relationship between peroxisomes and the oxidation process in mitochondria.
  • the reaction system usually used in the fatty acid oxidation reaction is used, and it varies depending on the concentration of the intracellular granule component, but generally, it is about 0.1 / ⁇ to 100. A 0 / ⁇ concentration is advantageously used.
  • Decetyl FA-5589 is also a useful substance as a synthetic intermediate for a compound that exhibits a further advantageous fatty acid decomposition inhibitory action.
  • the reaction was stopped by the addition of 70% perchloric acid 0.5 was the measurement of enzymatic activity by measuring the generated 14 C0 2.
  • the inhibitory activity of the compound FA-5859 was 1% at 250 ⁇ / ⁇ ⁇ , 25% at 500 ig / m £, and 10% as compared to the case without the inhibitor. 0 0 At 36% inhibition.
  • Acute toxicity L 50 values of f 1 A- 5 8 5 9 was on 4 0 0 / mouse on intravenous injection.
  • FA-58 ⁇ 9 or a salt thereof is useful, for example, as a fatty acid degradation inhibitor.
  • FIII-589 or a salt thereof for the treatment of diabetes in mammals (eg, mice, rats, humans, etc.) About 0.2 to 200 / is administered as a daily dose.
  • tablets, granules, capsules, etc. may be used in a conventional manner. It can be administered orally in the form of preparations, liquids, etc., and parenterally, for example, as an injection.
  • — 5859 can be used as a biochemical reagent to elucidate the metabolism of fatty acids.
  • FA- since 5 8 5 9 may make it easier for the carnitine deficiency by adding to the reaction solution, it is possible to further clarify the role of carnitine in fatty acid oxidation.
  • the physiological roles of Mi Tokondoria and Peruokishizomu in fatty acid oxidation are largely unknown, by inhibiting the uptake of fat acids to Mi Tokondoria FA- 5 8 5 9 was added and the pel Okishizomu It is possible to elucidate the role and implications of the oxidation process in perokinsome and mitochondria.
  • FA-589 is also a useful substance as a synthetic intermediate for a compound that exhibits a further advantageous fatty acid decomposition inhibitory action.
  • Ndiloxy carbonylamino—r t monobutoxycarbonylaminobutyric acid obtained in Reference Example 1 was dissolved in 10 trifluoroacetic acid and left at room temperature for 10 minutes. The reaction solution was dried under reduced pressure, and the residue was further dried under reduced pressure. This was dissolved in 10 sodium hydroxide 7 , cooled to 0 ° C., added with dimethyl sulfate 0.05 S, and stirred at 0 ° C. for 1 hour and then at room temperature for 1 hour.
  • the reaction mixture was subjected to column chromatography (60) with the power of Amberlite IR-120 (H ⁇ type), washed with water and eluted with ⁇ ⁇ ⁇ ⁇ ⁇ -ammonia water, from 150 »to 220a eluted fraction was concentrated under reduced pressure to collect the residual Tomebutsu dried under vacuum 0.4 4 other (L) -? - benzyl O gold Lupo sulfonyl Amino - r - DOO Rimechirua Mi no - ⁇ the a aminobutyric acid Obtained as a paste. Thin layer chroma Togurafi one (carrier: Nri dim 6 OF 2 5 4, Mezoreku Co. West Germany]
  • Acetonitrile was distilled off, and the residue was passed through a column (80 ⁇ ) of Dowex 50X2 (11+ type). The column was washed with zK300a and eluted with 0.5 N ammonia. The eluted fraction from 220 W to 240 A was collected, concentrated under reduced pressure, and the residue was dissolved in 50 W water and freeze-dried. The obtained soft material was dissolved in 2.8 W of IN-hydrochloric acid and dried under reduced pressure. The residue is crystallized using methano-lugetyl ether to obtain crystals of (L) -r-trimethylamino- ⁇ -acetylaminobutyric acid / hydrochloride (FA-585-9. Hydrochloride). Was. Yield 5 10 «9. Melting point 2 2 2 to 2 2 3 (decomposition),
  • the FA-585-9 educt 1.60 was dissolved in constant boiling hydrochloric acid 4 ⁇ ⁇ and left at 95 ° C for 16 hours.
  • the reaction solution was concentrated under reduced pressure, a small amount of water was added to the residue, and the mixture was concentrated again under reduced pressure.
  • a mixed solvent of methanol and diethyl ether was added to the residue, and the precipitated crystals were collected by filtration.
  • 1.20 ⁇ of diacetel_FA—5859 dihydrochloride was obtained.
  • Absorption spectrum No specific absorption in the ultraviolet and visible regions from 210 nm to 700 nm.
  • the physiologically active substances deacetyl FA-585-99 and the physiologically active substance FA-585-99 have an excellent inhibitory effect on fatty acid degradation. It is used as a therapeutic agent for diabetes in mammals such as humans and humans, and the substance is used as a biochemical reagent for elucidating the metabolism of fatty acids.

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Abstract

Novel physiologically active substances, deacetyl FA-5859 and FA-5859, represented by the formula (I), wherein R1 represents hydrogen or an acetyl group, and the salts thereof can be chemically synthesized. These compounds show an excellent fatty acid decomposition-inhibiting effect, and are utilized as diuretics for mammals and as a biochemical reagent for clarifying the mechanism of fatty acid metabolism.

Description

明 細 書  Specification
生理活性物質 FA— 5 8 5 9およびその誘導体の製造法  Production method of bioactive substance FA-5 8 5 9 and its derivatives
技術分野  Technical field
本発明は、 優れた脂肪酸分解阻害作用を有する新規生理活性物質 F A - 5 8 5 9およびその誘導体の製造法に関するものである。  TECHNICAL FIELD The present invention relates to a method for producing a novel physiologically active substance F A-58559 and a derivative thereof having an excellent fatty acid decomposition inhibitory action.
景技術  Landscape technology
従来、 脂肪酸分解阻害作用を有する化合物としては、 4 -ペンテノ ン 酸 ( 4— pen teno ic acid ) P. C. Hoi 1 andら ， ノくィ ォケ ミ カル' ジャーナル（ Biochemical Journal ) 1 3 6卷 1 5 7頁及び 1 7 3頁， 1 9 7 3年， H, S. A. Sherrattら ， バイオケ ミカル ' ファーマコロジ - ( Biochemi cal Pharmacology ) 2 5巻 7 4 3頁， 1 9 7 6年参照 ) 〕 ， ノヽィ ポグリ ン （ Hypogl yc in ) 〔 H. S. A.' S erra 11 ら ，バイ オケ iカル ' ファーマコロジ一 ( Bi ochemi ca 1 Pharmaco 1 ogy ) 2 5 巻 7 4 3頁， 1 9 7 6年参照〕 ， デカノ ィ ール ' カルニチン （ decanoyl 一 (十)一 c a rn i t i ne )及び 2—ブロモノ ノレ ミ ト ィ 一ノレ · コェ ンザィ ム A ( 2 - bromopalmi toyl CoA ) 〔 I. Β· t，r i tzら， プロシ一ディ ングス ' ォブ ' ザ ' ナショナル ' アカデ ミ ー ' ォブ ' サイエンス （ Proceedings of the National Academy of Sciences)U. S. A. 5 4巻 1 2 2 6頁， 1 9 6 5年参照〕などが知られている力;、 毒性及び作用の 点から臨床的には^ されていない。 メチル— 2 —テ ト ラデシルグリ シ デ一 ト及び 2—テ トラデ i ノレグリ シデ一 ト（me thy 1 - 2 - tetradecylg- lycidate,2-tetradecylgl ycidate )〔 G. F. Tutwi ler ら， ディ ァ ベ-テス （ Diabetes ) 2 8卷 2 4 2頁， 1 9 7 9年及びメ ソー ド - ィ ン .ェンザィ モロジ一 ( Me t hods i n Enzymology ) 7 2巻 5 3 3頁， 1 9 8 1年参照〕は脂肪酸分解阻害作用を有し、 且つ経口で血瑭低ト作  Conventionally, compounds having an action of inhibiting fatty acid degradation include 4-pentenoic acid PC Hoi 1 and et al., Biochemical Journal, Vol. Pp. 7 and 173, 1973, H, SA Sherratt et al., Biochemical 'Pharmacology-(Biochemical Pharmacology), Vol. 25, pp. 73, 1976)]], Nodi Pogli (See HSA'S erra 11 et al., Bio Orchestral Pharmaco 1 ogy, Vol. 25, pp. 743, 1976), Decanoyl 'Carnitine (decanoyl- (10) -l-ca-rniti-ne) and 2-bromopalmitoyl-Coenzyme A (I-Βt, ri tz et al., Proc. Proceedings of the National Academy of Sciences U.S.A. S. A. 54, p. 122, pp. 1965] is known; however, it has not been clinically evaluated in terms of toxicity and action. Methyl-2-tetradecylglycide and 2-tetradecylglycide (me thy 1-2-tetradecylg-lycidate, 2-tetradecylglycidate) [GF Tutwiler et al., Diabetes (Vol. 28, p. 24, pp. 1979 and Method-in. Methods in Enzymology (see p. 72, p. 533, pp. 1989)) Has an inhibitory effect and is orally low in blood
WIPO 用を示すことが知られている。 WIPO It is known to indicate
発明の開示 Disclosure of the invention
太発明は、 新規生理活性物質 F A— 5 8 5 9およびその誘導体の製造 法に関する。  The present invention relates to a method for producing a novel physiologically active substance FA-585-9 and a derivative thereof.
さらに詳しくは、 本発明は (1)、 一般式  More specifically, the present invention relates to (1)
CH3 CH 3
CH3 -Ne-CH2 -CH-CH2 -COO0 〔 2 〕 CH 3 -Ne-CH 2 -CH-CH 2 -COO 0 [2]
CH3 H-R2 CH 3 HR 2
〔式中、 R2はァセチル基以外の保護基を示す。 〕で表わされる化合物 C 2 〕またはその塩を保護基脱離反応に付し、 必要によりァセチル化反 応に付すことを特徵とする一般式 CH3 [In the formula, R2 represents a protecting group other than an acetyl group. A compound represented by the general formula CH 3, wherein the compound C 2] or a salt thereof is subjected to a protecting group elimination reaction and, if necessary, to an acetylation reaction.
CH3 -Ne -CH2 -CH-CH2 -COO® l 〕 CH 3 -Ne -CH 2 -CH-CH 2 -COO® l)
CH3 NR -Ri 〔式中、 Riは水素またはァセチル基を示す。 〕で表わされる化合物CH 3 NR -Ri wherein Ri represents hydrogen or an acetyl group. A compound represented by the formula
〔 1 〕またはその塩の製造法および (2)、 式 (1) or a method for producing the salt thereof and (2)
NHs -CH2 -CH-CH2— COOH NHs -CH 2 -CH-CH 2 — COOH
I ': 3 〕 H -COCH3 で表わされる化合物〔 3 ：!またはその塩を卜 リメチル化反応に付し、 必 要により加水分解反応に付すことを特徴とする一般式〔 1 〕  I ′: 3] A compound represented by the general formula [1], wherein a compound [3 :! or a salt thereof represented by H—COCH3 is subjected to a trimethylation reaction and, if necessary, to a hydrolysis reaction.
CH3 CH 3
CH3 -N@-CH2 -CH-CH2-COOG 〔 1 J CH 3 -N @ -CH 2 -CH-CH 2 -COO G (1 J
CH3 NR -Ri CH 3 NR -Ri
〔式中、 Riは水素またはァセチル基を示す。 〕で表わされる化合物 〔 1 〕またはその塩の製造法に関する。 [Wherein Ri represents hydrogen or an acetyl group. ] Or a salt thereof.
OMFI OMFI
、 λ r n 近年、 次第に増加を続ける糖尿病及びその合併症に対して、 新しい作 用機作に基き、 且つより有効な糖尿病治療薬の開発が'期待されている。 , Λ rn In recent years, the development of more effective antidiabetic drugs based on new mechanisms has been expected for diabetes and its complications, which are increasing gradually.
すなわち、 糖尿病においては、 ィンス リン欠乏のため脂肪組織での脂肪 酸の遊離が促進されるので、 肝臓への脂肪酸供給が増加し、 同時に脂肪 酸の分解も亢進するためケトン体の生成も上昇し、 いわゆるケ 卜ン欠症 を呈する。 また肝臓外組織ではグルコースの利用が悪く 、 生成したケ 卜 ン体をエネルギー源としている。 したがって脂肪酸の分解を阻止すれば、 ケ トン体の減少にともないグルコースの利用が亢進され、 結果的に血糖 値の低下をきたすことが期待され、 脂肪酸分解の特異的阻害剤は新しい 機作に基く新規糖尿病治療薬として利用しうるものと考えられる。 この ような事情に鑑み、 本発明者らは新規糖尿病治療薬の開発を目的として、 種々検索したところ、 ェメ リセラ属またはァスペルギルス属に属する微 生物の培養物中に脂肪酸分解を阻害する物質を見い出し、 これを単離し rこところ、該物質は新規物質であること、 また優れた脂肪酸分解阻！^用を有 することを見い出し、 これを生理活性物質 F A— 5 8 5 9と称すること  That is, in diabetes, insulin deficiency promotes the release of fatty acid in adipose tissue due to insulin deficiency, thereby increasing the supply of fatty acids to the liver and, at the same time, increasing the production of ketone bodies due to the accelerated decomposition of fatty acids. However, they exhibit a so-called keton deficiency. In addition, glucose is poorly used in extrahepatic tissues, and the generated ketones are used as an energy source. Therefore, if the breakdown of fatty acids is prevented, glucose utilization is expected to increase with a decrease in ketone bodies, which is expected to result in a decrease in blood sugar levels.Specific inhibitors of fatty acid degradation are based on a new mechanism. It is thought that it can be used as a new diabetes therapeutic agent. In view of such circumstances, the present inventors have conducted various searches with the aim of developing a novel therapeutic agent for diabetes, and found that a substance that inhibits fatty acid degradation in a culture of a microorganism belonging to the genus Emelysera or Aspergillus. Found and isolated this, the substance is a novel substance, and excellent fatty acid decomposition inhibition! ^ It is found that it has a use, and it is called bioactive substance F A-585 9
し/  I /
本発明者らは、 該生理活性物質 F A - 5 8 5 9の誘導体を得る目的で 種々研究したところ、 該生理活性物質; F A - 5 8 5 9を加水分解して得 られたデァセチル体は新規物質であり、 しかも顕著な脂肪酸分解阻害作. 用を有することを見い出し、 これを生理活性物質デァセチル; F A - 5 8 5 9と称することとした。  The present inventors have conducted various studies for the purpose of obtaining a derivative of the physiologically active substance FA-589, and found that a deacetyl derivative obtained by hydrolyzing the physiologically active substance FA-589 is novel. It has been found that it is a substance and has a remarkable inhibitory effect on fatty acid degradation, and is referred to as a physiologically active substance deacetyl (FA-5589).
本発明者らは、 さらに、 生理活性物質デァセチル F A - 5 8 5 9およ び生理活性物質 F A - 5 8 5 9の化学合成法について種々研究したとこ ろ、 一般式  The present inventors have further studied various methods for chemically synthesizing the bioactive substance deacetyl-FA-589 and the bioactive substance FA-589, and have found that the general formula
ΟΜΡί .. .. /IPO CH3 ΟΜΡί .. .. / IPO CH 3
CH3 - N@ - CH2 -CH -CH2 -COOe 2 〕 CH 3 - N @ - CH 2 -CH -CH 2 -COO e 2 ]
CH3 H -R2 CH 3 H -R 2
〔式中、 R2は前記と同意義を有する。 〕で表わされる化合物〔 2 〕ま たはその塩を保護基脱離反応に付し、 必要によりァセチル化反応に付す ことにより、 また式 [Wherein, R 2 has the same meaning as described above. By subjecting the compound [2] or a salt thereof represented by the formula (1) or a salt thereof to an acetylation reaction, if necessary, to obtain a compound represented by the formula
NH2 -CH2 -CH-CH2 - COOH NH 2 -CH 2 -CH-CH 2 -COOH
L 3 ·  L 3 ·
NH - COCHg で表わされる化合物;： 3 〕またはその塩をト リメチル化反応に付し、 必 要により加水分解反応に付すことにより、 有利に一般式  The compound represented by NH—COCHg ;: 3] or a salt thereof is subjected to a trimethylation reaction, and if necessary, to a hydrolysis reaction, thereby advantageously obtaining a compound represented by the general formula:
CH3 CH 3
CH3 - Q -CH2 -CH -CH2 -COO0 〔 1 〕 CH 3 -Q -CH2 -CH -CH 2 -COO 0 [1]
CH3 NH -R i CH 3 NH -R i
〔式中、 Riは前記と同意義を有する。 〕で表わされる化合物〔 1 〕ま たはその塩を製造することができることを見い出し、 さらに研究した結 果、 本発明を完成した。 Wherein Ri has the same meaning as described above. [1] or a salt thereof can be produced, and as a result of further research, the present invention has been completed.
本明細書においては、 化合物、' 1 ：!において Riが水素である化合物 を 「生理活性物質デァセチル F A— 5 8 5 9」 とあるいは単に 「デァセ チル F A— 5 8 5 9」 と、 化合物 '： 1 .において R]_がァセチル基であ る化合物を Γ生理活性物質 F A - 5 8 5 9」 とあるいは単に 「F A - 5 8 5 9」 とそれぞれ称することもある 3 In the present specification, a compound, in which Ri is hydrogen in the compound '1 :!', is referred to as “bioactive substance deacetyl FA—5859” or simply “deacetyl FA—5859” and the compound “: . 1 in R] _ is Asechiru groups der Ru compounds Γ physiologically active substance FA - 5 8 5 9, "one had simply" FA - 5 8 5 9 "and 3, sometimes referred to respectively
上記一般式中、 R2で表わされるァセチル基以外の深護基としては、 たとえば t一ブチルォキシカルボニル ， ベ ンジルォキンカルボニル ， p —メ トキシベンジルォキ Ϊ カルボニル ， p —二 ト ロべンジルォキシカル ボニルなどが挙げられる。  In the above general formula, examples of the protective group other than the acetyl group represented by R2 include t-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-dibenzyloxycarbonyl Bonyl and the like.
_ OMPI 本発明方法である化合物〔 2 〕またはその塩を保護基脱離反応に付す には、 たとえば加水分解 ，又は接触還元，酸処理等の'従来ぺプチド合成 反応に適用されている手段を適用しうる。 該加水分解反応は、 アミ ド結 合を分解するすべての方法を適用することができる。 たとえば、 酸，塩 基 ，ィオン交換樹脂などを用いることにより行なうことができる。 該酸 としては、 たとえば硫酸，塩酸などの無機酸が挙げられ、 該塩基として は、 たとえば水酸化力リ ゥム ，水酸化ナト リ ウム ，水酸化バリゥムなど が挙げられ、 該ィオン交換樹脂としては、 たとえばダウエックス - 5 0 (ダウケミカル社製 ，米国） ， アンバ—ライ ト I R— 1 2 0 (ローム ' アン ド 'ハース ' コンパ —社製 ，米国） ，ダイヤイ オン— S K I A ， S K I B (三菱化成工業株式会社製 ， 日本）などが挙げられる。 _ OMPI In order to subject the compound [2] or a salt thereof to the protecting group elimination reaction according to the method of the present invention, means conventionally used in peptide synthesis reactions such as hydrolysis, catalytic reduction, and acid treatment are applied. sell. As the hydrolysis reaction, any method for decomposing an amide bond can be applied. For example, it can be carried out by using an acid, a base, an ion exchange resin or the like. Examples of the acid include inorganic acids such as sulfuric acid and hydrochloric acid, and examples of the base include rhodium hydroxide, sodium hydroxide and barium hydroxide. Examples of the ion exchange resin include For example, Dowex-50 (manufactured by Dow Chemical Company, USA), Amberlight IR-120 (manufactured by ROHM 'And'Haas' Comparator, USA), Diamond-SKIA, SKIB (Mitsubishi Chemical Corporation) Industrial Co., Ltd., Japan).
上記の酸を用いる場合には、 水溶液中で該反応を行なうのが好ましく、 また含 溶媒としてたとえば水とメタノー ル ，エタノール ， ブタノール 等との混合溶媒中で行なうのが好ましい。 反応は、 通常約 6 0 〜 2 0 0 °C、 さらに好ましくは約 9 0 〜 1 0 °Cで、 約 3 0分〜 3 0時間、 さら に好ましくは約 3 〜 1 6時間で行なわれる。  When the above acid is used, the reaction is preferably carried out in an aqueous solution, and more preferably in a mixed solvent of water and methanol, ethanol, butanol or the like as a solvent. The reaction is usually carried out at about 60 to 200 ° C, more preferably at about 90 to 10 ° C, for about 30 minutes to 30 hours, and more preferably for about 3 to 16 hours.
上記の塩基を用いる場合には、 水溶液中で行なうのが好ましく、 さら に含水 、 例えば水とメタノール ， エタノー ル ， ブタノ ー ル等との混 合溶媒中で行なうのが好ましい。 該反応は、 通常約 6 0 〜 2 0 0 °C、 さ らに好ましくは約 9 0〜： I 2 0 °し'で、 約 3 0分〜 3 0時間、 さらに好ま しくは約 3 〜 1 6時間で行なわれる。  When the above base is used, it is preferably carried out in an aqueous solution, and more preferably in a water-containing solution, for example, a mixed solvent of water, methanol, ethanol, butanol and the like. The reaction is usually carried out at about 60 to 200 ° C., more preferably at about 90 to: I 20 ° C., for about 30 minutes to 30 hours, more preferably at about 3 to 1 hour. It takes 6 hours.
上記のィオン交換樹脂を用いる場合には、 原料化合物の水溶液に該ィ オン交換樹脂を懸濁させた状態で加温することにより行なわれる。 該反 応は通常約 6 0 〜 2 0 0 °C、 さらに好ましくは約 9 0 〜 1 2 0 °Cで約 3 0分〜 3 0時間、 さらに好ましくは約 3 〜 1 6時間で行なわれる。  When the above-mentioned ion-exchange resin is used, it is carried out by heating the ion-exchange resin in a state of being suspended in an aqueous solution of the raw material compound. The reaction is usually carried out at about 60 to 200 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably at about 3 to 16 hours.
OMPI 、 409 OMPI, 409
- 6 - 該接触還元反応は、水又はメタノ ール ，ェタノ ール等のアルコール中  -6-The catalytic reduction reaction is carried out in water or alcohol such as methanol or ethanol.
で、 又は水とアルコールとの混合溶媒中 ， パラジウム黑，パラジウム炭 , Or in a mixed solvent of water and alcohol, palladium 黑, palladium charcoal
素等の触媒の存在下水素ガスを導入し、 必要に応じて加圧して行なわれ る。 該接触還元反応の温度は、 約 0 〜 5 o 。c、 好ましくは約2 0 〜 3 0 Hydrogen gas is introduced in the presence of a catalyst such as hydrogen, and pressurization is performed as necessary. The temperature of the catalytic reduction reaction is about 0-5 °. c, preferably about 2 0-3 0
で、 反応時間は約 0. 5 〜 5時間、 さらに好ましくは約 1 〜 3時間で行 なわれる。 該酸処理に用いられる酸としては、 たとえば臭 i K素一酢酸， 塩酸一酢酸，塩酸一ジォキサン ， ト リ フルォロ酢酸， メタンスルホン  The reaction time is about 0.5 to 5 hours, more preferably about 1 to 3 hours. Examples of the acid used in the acid treatment include bromoiodic monoacetic acid, monoacetic acid hydrochloride, dioxane hydrochloride, trifluoroacetic acid, and methanesulfone.
ト リ フルォ σメタンスルホン酸等があげられる。 酸処理の温度は約一 1 0  Trifluor σ-methanesulfonic acid and the like. The temperature of the acid treatment is about 10
てから 5 0 。C，好ましくは約 0 から 2 5てで、 反応時間は約 3 0分か ら 2 4時間の間で、 用いた酸により適当に変えるのが望ましい。 50 since then. C, preferably about 0 to 25 hours, and the reaction time is about 30 minutes to 24 hours, and is suitably changed depending on the acid used.
化合物〔 2 〕またはその塩を保護基脱離反応に付し得られた化合物を  A compound obtained by subjecting compound [2] or a salt thereof to a protecting group elimination reaction,
ァセチル化するには、 たとえば上記化合物と、 無水酢酸，ァセチルクロ To acetylate, for example, the above compound, acetic anhydride, acetyl chloride
ラィ ド又は酢酸をク口ル炭酸メチル ， ク ロル炭酸ェチル ， ク σル炭酸ブ Lide or acetic acid is converted to methyl carbonate, ethyl chlorocarbonate,
チル ， クロル炭酸プ σピル ， クロル炭酸ィ ソブチル等のアルコキシカル Alkoxycals such as chill, chlorocarbonate, σ-pill, and isobutyl chlorocarbonate
ボニルクロライ ドと反応して得られる薛酸の無水物とを、 水又は水とァ The anhydride of sulphonic acid obtained by reaction with bonyl chloride is converted into water or water and water.
セ トン ， ジォキサン ， ァセ トニト リ ル ， ジメチルホルムア ミ ド ， テ トラ Setone, dioxane, acetonitrile, dimethylformamide, tetra
ハイ ドロフラン等の有機溶媒との混合溶媒中反応させることによって達 Reaction in a mixed solvent with an organic solvent such as hydrofuran
成される。 反応に際してはピリ ジン ， ト リ ェチルァ ミ ン ， ト リ メチルァ Is done. In the reaction, pyridine, triethylamine, trimethylamine
ミン等の有機塩基又はナト リ ウム ， カ リ ウム ，力ルシゥム等のアル力リ 金属，アルカ リ土類金属の水酸化物，酸化物，重炭酸塩等を脱酸剤とし  Organic bases such as mines or hydroxides, oxides, bicarbonates, etc. of alkali metal, alkaline earth metal such as sodium, potassium, potassium hydroxide, etc.
て共存させてもよい。 反応温度としては約- 1 0 °Cから 5 0 ¾ ,好まし May coexist. The reaction temperature is about -10 ° C to 50 ° C, which is preferable.
くは約 0でから 2 5 °Cである。 Or about 0 to 25 ° C.
本発明方法である化合物〔 3 〕またはその塩をト リメチル化反応に付  The compound [3] of the present invention or a salt thereof is subjected to a trimethylation reaction.
すには、 たとえば化合物〔 3 〕またはその塩とジメチル硫酸，メチルブ For example, compound [3] or a salt thereof and dimethyl sulfate or methyl
ロマイ ド ，メチルク σライ ドまたはメチルョ一ダイ ドとを水中又は水と Romide, methyl chloride or methyl chloride in water or water
R£A R £ A
C.、 '?1C., '? 1
Ά、 '- 。 Ά, '-.
- ァセ 卜二 ト リ ノレ ， ジォキサン ， テ ト ラハイ ドロフラン ， ジメチルホルム アミ ド等の有機溶媒中で反応させることによって行なわれる。 反応に際 しては必要に応じてナ ト リ ウム ， カ リ ウム ， カル ウム等のアル力 リ金 属，アル力リ土類金属の水酸化物，酸化物等を共存させてもよい。 反応 温度は約- 1 0°Cから 5 0°C ，好ましくは約 0°Cから 2 0°Cである。 - The reaction is carried out in an organic solvent such as acetone, dioxane, tetrahydrofuran, dimethylformamide and the like. During the reaction, an alkali metal such as sodium, potassium, or calcium, or a hydroxide or oxide of an alkali earth metal may be coexisted, if necessary. The reaction temperature is about -10 ° C to 50 ° C, preferably about 0 ° C to 20 ° C.
上記の各反応によって生成した各目的物は、 通常用いられる分離 ，精 製手段，たとえばクロマ トグラフィ ー ，再結晶などの方法によって採取, 精製することができる。  Each target product produced by each of the above reactions can be collected and purified by commonly used separation and purification means, for example, methods such as chromatography and recrystallization.
化合物〔 3 〕またはその塩をト リメ チル化反応に付し、 ついで必要に より行なわれる加水分解反応は、アミド結合を分解するすべての方法を適 用することができる。たとえば、酸，塩基，イオン交換樹脂などを用いること により行なうことができる。 該酸としては、 たとえば硫酸，塩酸などの 無機酸が挙げられ、 該塩基としては、 たとえば水酸化カリクム ，水酸化 ナト リ クム ，水酸化パリ クムなどが挙げられ、 該ィオン交換樹脂として は、 たとえばダウエックス- 5 0 (ダウケミカル社製，米国） ， アンバ 一ライ ト I R— 1 2 0 ( ローム ' アン ド - ハ—ス ' コンパ二—社製，米 国） ，ダイヤイ オン— SKIA , SKI B (三菱化成工業株式会社製， 日本）などが挙げられる。  The compound [3] or a salt thereof is subjected to a trimethylation reaction, and then, if necessary, any hydrolysis method for decomposing an amide bond can be applied to the hydrolysis reaction. For example, it can be carried out by using an acid, a base, an ion exchange resin or the like. Examples of the acid include inorganic acids such as sulfuric acid and hydrochloric acid. Examples of the base include potassium hydroxide, sodium hydroxide and paricum hydroxide. Examples of the ion exchange resin include: Dowex-50 (manufactured by Dow Chemical Company, USA), Amberlight IR-120 (manufactured by ROHM 'And-Haas' Company, USA), Diamond-SKIA, SKI B (Mitsubishi Chemical Industries, Japan).
上記の酸を用いる場合には、 水溶液中で该反応を行なうのが好ましく、 また含水溶媒としてたとえば水とメタノ ール ， エタノール ， ブタノ 一ル 等との混合溶媒中で行なうのが好ましい。 反応は、'通常約 6 0〜2 0 0 °C、 さらに好ましくは約 9 0〜 1 2 0°Cで、 約 3 0分〜 3 0時間、 さら に好ましくは約 3〜1 6時間で行なわれる。 When the above-mentioned acid is used, the reaction is preferably carried out in an aqueous solution, and more preferably in a mixed solvent of water and methanol, ethanol, butanol or the like as a water-containing solvent. The reaction is' usually about 6 0 to 2 0 0 ° C, further preferably about 9 0~ 1 2 0 ° C, about 3 0 minutes to 3 0 hours, preferably Furthermore at about 3 to 1 6 hours It is.
上記の塩基を用いる場合には、 水溶液中で行なうのが好ましく、 さら に含水溶媒、 例えば水とメタノール ， エタノール ， ブタノ ール等との混  When the above-mentioned base is used, it is preferable to carry out the reaction in an aqueous solution, and further, to mix a water-containing solvent such as water with methanol, ethanol, or butanol.
C ?I ヽ ν,.ίΓΟ 合溶媒中で行なうのが好ましい。 該反応は、 通常約 6 0〜 2 0 o°C\ さ らに好ましくは約 9 0〜 1 2 0°Cで、 約 3 0分〜 3 0時間、 さらに好ま しくは約 3〜： I 6時間で行なわれる。 C? I ν ν, .ίΓΟ It is preferable to carry out in a combined solvent. The reaction is usually carried out at about 60 to 20 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably about 3 to: I 6 Done in time.
上記のイオン交換樹脂を用いる場合には、 原料化合物の水溶液に該ィ オン交換樹脂を懸濁させた伏態で加温することにより行なわれる。 該反 応は通常約 6 0〜 2 0 0°C、 さらに好ましくは約 9 0〜 1 2 0 °Cで約 3 0分〜 3 0時間、 さらに好ましくは約 3〜 1 6時間で行なわれる。  When the above-mentioned ion exchange resin is used, the reaction is carried out by heating the ion exchange resin suspended in an aqueous solution of the raw material compound. The reaction is usually performed at about 60 to 200 ° C., more preferably at about 90 to 120 ° C., for about 30 minutes to 30 hours, more preferably for about 3 to 16 hours.
反応液からデァセチル F A— 5 8 5 9またはその塩を得るには、 通常 用いられる手段、 たとえばィオン交換樹脂，吸着，濃縮，結晶化などが 用いられる。 反応液から目的物を採取する場合には、 遊難形あるいは塩 のいずれでも単離できるが、 塩の形で分離採取する方が容易である。 反応終了液から目的物質を得る具体例としては、 たとえば強酸性ィォ ン交換樹脂等を利用し、 目的物質を吸着させ、 例えば塩酸等を利用して 溶出を行いニンヒ ドリン反応で ( 1生を示す区分を集めることによって目 的物質を得ることが出来るが、 例えば塩 による加水分解反応液からは 更に簡単に反応液を減圧濃縮し過剰の塩 ¾を留去した後、 メタノ —ル ， ェタノ—ル ， ジェチルェ—テル等の溶媒を加え結晶として本物質の塩 £ 塩を得ることが出来る。 In order to obtain deacetyl FA-5589 or a salt thereof from the reaction mixture, commonly used means such as ion exchange resin, adsorption, concentration, and crystallization are used. When the target substance is collected from the reaction solution, it can be isolated in either a difficult form or a salt, but it is easier to separate and collect it in the form of a salt. As a specific example of obtaining the target substance from the reaction completed solution, for example, a strongly acidic ion exchange resin or the like is used to adsorb the target substance, and elution is performed using, for example, hydrochloric acid or the like. The target substance can be obtained by collecting the indicated categories. For example, from the hydrolysis reaction solution with salt, the reaction solution is more easily concentrated under reduced pressure to remove the excess salt, and then methanol, ethanol, etc. Le, Jechirue - solvent salted £ salts of the present substance can be obtained. However as a crystal, such as ether.
このようにして、 遊離または塩の形のデァセチル F A— 5 8 5 9また は F A— 5 8 5 9が得られる。  In this way, deacetyl FA-585-59 or FA-585-59 in free or salt form is obtained.
デァセチル F A - 5 8 5 9または F A - 5 8 5 9の塩を遊離形に変換 することもできる。 この方法は、 たとえばイオン交換樹脂等を利.吊して、 該塩を形成している酸または塩基をィオン交換樹脂に吸着させればよい。 デァセチル F A - 5 8 5 9遊離形または F A— 5 8 5 9遊離形は、塩を 形成し得るので、 常套手段により薬理的に許容し得る塩とすることがで きる。 該;^を形成させる酸としては、 たとえば塩酸，硫酸，硝酸 '蓚酸， 酢酸，コハク酸 ，クェン酸 ， フマ一ル酸などが挙げられる。 It is also possible to convert the salt of deacetyl FA-585-9 or FA-585-9 to the free form. In this method, an acid or a base forming the salt may be adsorbed on the ion-exchange resin, for example, by suspending an ion-exchange resin or the like. The free form of deacetyl FA-585-99 or FA-585-99 free form can form a salt, which can be converted into a pharmaceutically acceptable salt by conventional means. Wear. Examples of the acid for forming the; ^ include hydrochloric acid, sulfuric acid, nitric acid, oxalic acid, acetic acid, succinic acid, citric acid, and fumaric acid.
本発明方法において原料化合物として用いられる化合物〔 2 〕は、 た とえば以下の反応工程により製造することができる。 Compound [ 2 ] used as a starting compound in the method of the present invention can be produced, for example, by the following reaction steps.
R3 - NH - CH2 -CH-COOH 〔 4 〕 R 3 -NH-CH 2 -CH-COOH [4]
NH-R2 NH-R 2
1  1
R3 -NH-CH2 -CH-C0^0^CO-0-C2H5 〔 5 〕 R 3 -NH-CH 2 -CH-C0 ^ 0 ^ CO-0-C 2 H 5 [5]
NH-R2 NH-R 2
1  1
R3 - NH - C - CH - CO CHN2 〔 6 〕 R 3 -NH-C-CH-CO CHN 2 [6]
NH-R2 NH-R 2
1  1
R3 -NH-CH2 - CH- CH2 -COOCH3 〔 7 〕R 3 -NH-CH 2 -CH- CH 2 -COOCH3 [7]
H-R2 HR 2
R3 -NH-CH2 - CH- CH2 - COOH C 8 〕 R 3 -NH-CH 2 -CH- CH 2 -COOH C 8]
NH-R2 NH-R 2
1  1
NH2 -CH2 -CH-CH2 -COOH C 9 〕 NH 2 -CH 2 -CH-CH 2 -COOH C 9]
NH-R2 NH-R 2
CH3 上 On CH 3
CH3 -N@-CH2 -CH-CH -COO0 〔 2 〕 CH 3 -N @ -CH 2 -CH-CH -COO 0 [2]
CH3 H-R2 上記化合物〔 4 〕は、 たとえば i/ンセシス （ Synthesis：)第 2 6 6 貝 ， 1 9 8 1年に記載された方法によりあるいはそれと同様の方法によ り製造することができる。 CH 3 HR 2 The above compound [4] can be produced, for example, by the method described in i / Synthesis (Synthesis :) No. 2666, 1998, or by a method similar thereto.
化合物〔 3 〕は、 たとえば以下に示す反応工程により製造することが できる。 Compound [ 3 ] can be produced, for example, by the following reaction steps.
R3 -NH-CH2 -CH-CH2 -COOH C 8 〕 R 3 -NH-CH 2 -CH-CH 2 -COOH C 8]
NH-R2 NH-R 2
Figure imgf000011_0001
J 2 0409
Figure imgf000011_0001
J 2 0409
一 10 —  One 10 —
R3 -NH-CH2 -CH-CH2 - COOH 〔10〕R 3 -NH-CH 2 -CH-CH 2 -COOH (10)
H2 H 2
R3 - H-CH2 -CH-CH2 -COOH R 3 -H-CH 2 -CH-CH 2 -COOH
NH-COCH3 H2 -CH2 -CH-CH2 -COOHNH-COCH3 H 2 -CH 2 -CH-CH 2 -COOH
H-COCH3 上記式中、 R2は前記と同意義を有する。 R3は、 R2で示される保 護基と脱離の際の性質を異にする保護基を示す。 該 R3で示される保護 基としては、 たとえばベンジルォキ カルボニル ， t一ブ トキシカルボ ニル , p —ニ ト ロべンジルォキシカルボニル , F> —メ トキ'ンベンジルォ キシカルボニル ， ト リ チル , ト ル ， t—ア ミルォキ カルボニル ， ィ ソボルニルォキンカルボニル , ジフエニルホスフィニル ， 0 —二 ト σフ ェニルスルフエ二ル ， フタロイルなどが挙げられる。  H-COCH3 In the above formula, R2 has the same meaning as described above. R3 represents a protecting group which differs from the protecting group shown by R2 in the nature at the time of elimination. The protecting group represented by R3 includes, for example, benzyloxycarbonyl, t-butoxycarbonyl, p-nitrobenzyloxycarbonyl, F> —methoxybenzyloxycarbonyl, trityl, tolu, t— Amyloxy carbonyl, isoborniroquine carbonyl, diphenylphosphinyl, 0-di-to-σ-phenylsulfenyl, phthaloyl and the like.
化合物〔 4 〕から化合物〔 5 〕を製造するには、 たとえば化合物：4〕  To produce compound [5] from compound [4], for example, compound: 4]
(ニク口ル: ^酸ェチル , ク σル炭酸メチル ， クロノレ炭酸プ口ビル 3， サコ ' 炭酸ブチル ， ク口ル炭酸ィ ソブチル等 ¾サ Γ7 -7 f ドを^ ： , 酢メチル ， ジォキサン ，テ ト ラハイ ド マ -'ン ， ァ トニ卜 リ .. ' 璣 媒中で作用させることによって行た れ · U¾ ¾、 \ -メ チ—'レモルホ リ ン ， N—ェチルモルホ リ ン ， ト .ノ'ェチ 'レア ン ， ト リ .乂 チ (Nichol: ^ ethyl, chloromethyl carbonate, chronole carbonate carbonate 3, coco's butyl carbonate, isobutyl butyl carbonate, etc. ¾ 7 -7 f ^:, methyl acetate, dioxane, Tetrahydrin, atonitrile .. 'This is done by acting in a solvent. · U¾, \ -methyl-'remorpholine, N-ethylmorpholine, tono. 'Echi' Lean, Tri.
'·レア ミ ン ， ピリジ ン等の有機塩基を共 させてもよい 応温度は^- 2 0でから 3 (TC ,好ましくは約- 1 0でから 0¾である。 The reaction temperature at which organic bases such as rare amines and pyridines may be used is from −20 to 3 (TC, preferably from about −10 to 0 °).
化合物 ':： 6 jは、 化合物-、 5 〕にジァゾメタ ンを反応させることに  Compound ':: 6j reacts compound-, 5] with diazomethane
り製造される。 たとえば、 化合物〔 5 jを酢酸ェチル ，酢酸メチ ， テ トラハイ ドロフラン， ジォキサン，ァセ卜ニ卜リノレ，ジェチルエーテル ?¾ Manufactured. For example, the compound [5j may be substituted with ethyl acetate, methyl acetate, tetrahydrofuran, dioxane, acetate nitrolinole, getyl ether.
媒中約一 2 0 から 3 0 Cで好ましくは钓一 1 ϋ Cから 2 5 じ-? -' 1 一 CMPI _About 1 to 20 ° C in the medium, preferably about 1 to 1 ° C to 25 じ-?-1 CMPI _
'',、 W IrO 時間から 2 4時間ジァゾメタンと反応させる。 ジァゾメタンは反応液に 直接吹きこむか、 又はジェチルエーテル，酢酸ェチル等'の有機溶媒に飽和 させた溶液を反応液に加えても良い。 '' ,, W IrO Time from reacting with 2 4 hours Jiazometan. Diazomethane may be blown directly into the reaction solution, or a solution saturated with an organic solvent such as getyl ether or ethyl acetate may be added to the reaction solution.
化合物〔 6 〕から化合物〔 7 〕を製造するには、 たとえば化合物〔6〕 のメタノ —ル溶液に安息香酸 ，酢酸等の銀塩をト リメチルァ ミ ン ， ト リ ェチルア ミ ン ， N —メチルモルホ リ ン ， N —ェチルモルホリ ン , ピリ ジン 等の有機塩基に溶かした溶液を約 - 5 から 5 0 °Cで好ましくは約 0 °C .から 2 7 °Cで、 暗所で約 3 0分から 1 0時間作用させることによって行 なわれる。  To produce the compound [7] from the compound [6], for example, a silver salt such as benzoic acid or acetic acid is added to a methanol solution of the compound [6] with trimethylamine, triethylamine, N-methylmorpholine. , N-ethylethyl morpholine, pyridine, etc. in a solution of an organic base at about -5 to 50 ° C, preferably about 0 ° C to 27 ° C, and about 30 minutes to 10 ° C in the dark. This is done by acting on time.
化合物〔 7 〕をケン化することにより化合物〔 8 〕を製造することが できる。 該ケン化は、 たとえば水，又はメタノ 一ル ，エタノ ール ， ジォ キサン ，テ トラハイ ドロフラン ， ァセ トニ ト リル等の有機溶媒中で、 又 は水とこれら有機溶媒との混合溶媒中で、 化合物〔 7 〕とナ ト リ ウム ， カリウム ，バリ ゥム等の水酸化物とを約- 1 0 °Cから 5 0 °C ，好ましく は約 0 °Cから 2 7 °Cで約 1時間から 5時間反応させることによって行な われる。  Compound [8] can be produced by saponifying compound [7]. The saponification is carried out, for example, in water or an organic solvent such as methanol, ethanol, dioxane, tetrahydrofuran, or acetonitrile, or in a mixed solvent of water and these organic solvents. And compound [7] and hydroxides such as sodium, potassium, and balium at about −10 ° C. to 50 ° C., preferably at about 0 ° C. to 27 ° C. for about 1 hour. The reaction is carried out for 5 hours.
化合物〔 8 〕から化合物 C 9 〕を製造するには、 R3 を選択的に脱離 する方法が適用される。 その方法はぺプチド合成に通常用いられる方法 で、 前記した化合物〔 2 〕を保護基脱離反応に付す反応と同様に行なう ことができる。  In order to produce compound C 9] from compound [8], a method of selectively eliminating R 3 is applied. This method is a method usually used for peptide synthesis, and can be carried out in the same manner as in the above-mentioned reaction of subjecting compound [2] to a protecting group elimination reaction.
化合物〔 9 〕をト リメチル化反応に付すことによりヽ 化合物〔 2 〕を 製造することができる。 該ト リメチル化反応は、 前記した化合物〔 3 をト リメチル化反応に付す際の反応と同様に行なうことができる。  The compound [2] can be produced by subjecting the compound [9] to a trimethylation reaction. The trimethylation reaction can be carried out in the same manner as in the reaction for subjecting the compound [3 to the above-mentioned trimethylation reaction.
化合物〔 8 〕を化合物〔 1 0 〕に変換するには、 前記した化合物〔2〕 を保護基脱離反応に付す反応と同様に行なうことができる。 化合物〔 1 0 〕をァセチル化反応に付すことにより、 化合物〔 1 1 〕 を製造することができる。 該ァセチル化反応は、 前記した化合物〔 2 〕 を保護基脱離反応に ftし、 必要によりァセチル化反応に付す反応におけ る該ァセチル化反応と同様に行なうことができる。 The conversion of the compound [8] into the compound [10] can be carried out in the same manner as in the above-mentioned reaction of subjecting the compound [2] to the protecting group elimination reaction. Compound [11] can be produced by subjecting compound [10] to an acetylation reaction. The acetylation reaction can be carried out in the same manner as the acetylation reaction in which the compound [ 2 ] is converted to a protective group elimination reaction and, if necessary, subjected to the acetylation reaction.
化合物〔 1 1 〕を保護基脱離反応に付すことにより、 化合物〔 3 〕を 製造することができる。 該屎護基脱離反応としては、 前記した化合物 〔 8〕から化合物〔 9〕を製造する際の反応と同様に行なうことができ る。  Compound [3] can be produced by subjecting compound [11] to a protecting group elimination reaction. The protecting group elimination reaction can be carried out in the same manner as in the reaction for producing compound [9] from compound [8] described above.
上記各反応によって生成した各目的物は、 通常用いられる分離，精製 手段、 たとえばク口マトグラフィー ，再結晶などの方法によって採取 ， 精製することができる。  Each target product produced by each of the above reactions can be collected and purified by commonly used separation and purification means, for example, methods such as mouth chromatography and recrystallization.
上記から明らかなごとく、 化合物〔 8 〕は、 有 ¾な化合物を製造する 際の合成中間体として用いることができる。  As is clear from the above, the compound [8] can be used as a synthetic intermediate when producing a valuable compound.
上記反応工程において、 それらの化合物の塩を用いてもよい。 該塩と してはたとえば、 ナ ト リ ウム ， カ リ ウム ， カル ゥム ， バリ ウム ， ト リ ェチルァ ン ， ピリ ジン ，塩化水素，臭化水素，ヨウ化水素などが挙げ られる。  In the above reaction step, salts of those compounds may be used. Examples of the salt include sodium, potassium, calcium, barium, triethylamine, pyridine, hydrogen chloride, hydrogen bromide, and hydrogen iodide.
次に生理活性物質デァセチル FA- 5 8 5 9の脂肪酸分解阻害能を、 ラッ ト肝臓ホモジネ— 卜のミ トコン ドリァ画分を用い、 日本生化学会編 「生化学実験講座」 第 9卷、 脂質の代謝、 7 5頁（ 1 9 7 5年東京 学 同人）記載の方法、 及び I. B. Fritzら、 プロシ—ディングス . ォブ - ザ - ナショナル ' アカデミー 'ォブ 'サイエンス（ Proceedings of the National Academy of Sciences, U.S.A. ) 5 4^1 2 2 6 頁， 1 9 6 5年に記載の方法に準じて測定した。 すなわち、 S D系ラッ ト （ 7週令，雄）を 2 4時間絶食後、 放血によって屠殺し、 直ちに肝臓 を取り出し 1 0倍量（w/v)の 5mMト リスー塩酸緩衝液（ pH 7.5 )を含 む 0.2 5 M蔗糖溶液を加え、 テフロン棒付ホモゲナイザーでホモジナイ ズした。 6 0 0 x ^ , 2 0分間の遠心上澄みを 3 0， 0 0 0 x ^， 3 0分 間遠心し、 生じたペレツ 卜を上記の蔗糖溶液に、 肝臓湿重量 0.2 /0.5 溶液となる濃度に懸濁し、 その
Figure imgf000015_0001
に用いた。 リ ン酸力 リ クム緩衝液 ( pH 7.5 ) 3 0 ΐποϊβ, KC£ 3 0 0 imole, Mg 。 3 <mo 1 e , 簾糖 1 2 0 imol e, L一リ ンゴ酸 0.0 3 iinole, Next, using the mitochondrial fraction of rat liver homogenate, the bioactive substance deacetyl FA-589-59 was tested for its ability to inhibit fatty acid degradation using the mitochondrial fraction of rat liver, edited by the Biochemical Society of Japan, Vol. Metabolism, pp. 75 (1975, Tokyo Gakudo), and IB Fritz et al., Procedings of the National Academy of Sciences, Procedings of the National Academy of Sciences, USA) 5 4 ^ 1 2 2 6 pages, was measured according to the method described in 1 9 6 5 years. That is, an SD rat (7-week-old, male) was fasted for 24 hours, sacrificed by exsanguination, and immediately treated with liver. Then, a 0.25 M sucrose solution containing 10 volumes ( w / v ) of 5 mM tris-hydrochloride buffer (pH 7.5) was added, and homogenized with a homogenizer equipped with a Teflon rod. Centrifuge the supernatant at 600 x ^, 20 min for 30 min, then centrifuge at 0,000 x ^, 30 min.Pet the resulting pellets in the above sucrose solution at a concentration to give a liver wet weight 0.2 / 0.5 solution. Suspended in the
Figure imgf000015_0001
It was used for. Phosphoric acid liquid buffer (pH 7.5) 30 ΐποϊβ, KC £ 300 imole, Mg. 3 <mo 1 e, Fructose 1 2 0 imol e, L-Lingic acid 0.0 3 iinole,
ATP 3 imole, L一カルニチン 3 mol e， コェンザィ ム A ( Co enzyme A ) 0.6 imo 1 e， NAD 7.5 imole , 1— 14C -ノ レ ミチン酸（ 0. 2 iCi , 牛血清アルブミ ンをモル比 1 ： 5になるように添加した溶液 pH 7. 5 ) 0.6 zmole, 水又は阻害剤を含有する水溶液 0. lr^及び酵素 溶液 0.5m^からなる反応混液 2.5m をハイアミ ンハイ ドロォキサイ ド 10 — X〔パッカード社製 (：オランダ） 〕を¾した^紙をつるした密閉試験 管中に入れ、 3 7 'C、 2 0分間好気的に振 it反応した。 7 0 %過塩素酸 0.4¾を添加して反応を止めた後生成した 14 CO 2を測定することによ つて活性の測定をした。 该化台物デァセチル FA— 5859の阻害活性す なわち阻害剤無添加の場合に比絞して分解活性を 5 0 咀害する濃度は 4〜 8 であった。 ATP 3 imole, L one-carnitine 3 mol e, Koenzai arm A (Co enzyme A) 0.6 imo 1 e, NAD 7.5 imole, 1- 14 C - Bruno Le Michin acid (0. 2 ICI, the molar ratio of bovine serum albumin 1: 7.5 Solution pH 7.5) 0.6 zmole, water or aqueous solution containing inhibitor 0. lr ^ and enzyme solution 0.5m ^ 2.5m reaction mixture was added to high-amin hydroxide 10 — X [Packard (made in the Netherlands)] was placed in a hanged closed test tube with ^ paper and shaken aerobically at 37'C for 20 minutes. After the reaction was stopped by adding 0.4% of 70% perchloric acid, the activity was measured by measuring the generated 14 CO 2 . The inhibitory activity of Detatyl FA-5859, a concentration at which the degradation activity was reduced to 50 in comparison with no addition of the inhibitor, was 4-8.
デァセチル； F A— 5 8 5 9の急性毒性 LL>50値は、 マウス静脈内注射 で 4 0 o /T^以上であった。 Acute toxicity of deacetil; FA-585-9 The LL> 50 value was more than 40 o / T ^ by intravenous injection in mice.
デァセチル F A - 5 8 5 9 またはその塩は、 たとえば脂肪酸分 解阻害剤として有用である。 デァセチル； FA— 5 8 5 9またはその塩 を脂肪酸分解阻害剤として用いるには、 たとえば哺乳動物（例、 マウス， ラッ 卜 ，人など）の糖尿病の治療を目的として、 デァセチル: F A 5 8 5 9 として約 0.2ないし 2 0 0 ^を 1 日投与量として投与する。 ま た、 デァセチル F A— 5 8 5 9 またはその塩を投与するにあたっては、 常套手段によってたとえば錠剤，顆粒剤， カプセル剤'，液剤などの剤型 にして経口的に、 またたとえば注射剤として非経口的に投与することが できる。 Decetyl FA-585-9 or a salt thereof is useful, for example, as a fatty acid degradation inhibitor. Deacetyl: FA-5589 or a salt thereof may be used as a fatty acid degradation inhibitor, for example, for the treatment of diabetes in mammals (eg, mice, rats, humans, etc.). About 0.2 to 200 ^ as a daily dose. Ma In addition, when administering decetyl FA-5589 or a salt thereof, it is orally prepared in the form of tablets, granules, capsules, liquids and the like by conventional means, or parenterally as an injection, for example. Can be administered.
また、 デァセチル F A — 5 8 5 9は、 脂肪酸の代謝を解明する上 での生化学的試薬として使用することができる。 たとえば、 デァセ テル F A— 5 8 5 9を反応液中に添！]することによりカルニチン欠乏状 態を容易に作りうるので、 脂肪酸酸化におけるカルニチンの役割をよリ —層明確にすることができる。 また、 脂肪酸酸化における ミ トコン ドリ ァとペルォキ j /ゾームとの玍理的役割はほとんど明らかにされていない ので、 ミ トコン ドリアへの脂肪酸の取り込みをデァセテル F A— 5 8 5 9 を添加して阻害することによって、 ペルォキシゾームの役割およびペル ォキシゾームとミ トコン ドリァでの酸化過程とのかかわりを明らかにす ることが可能である。 これらの場合、 脂肪酸酸化反応で通常用いられる 反応系を用いて、 細胞内穎粒成分の濃度によっても異なるが、 一般的に はデァセチル F A— 5 8 5 9約 0. 1 /诚〜 1 0 0 0 /诚濃度が有利 に用いられる。  In addition, deacetyl FA-5589 can be used as a biochemical reagent for elucidating the metabolism of fatty acids. For example, add Dacetel F A—5859 to the reaction solution! Carnitine deficiency can be easily created by this method, so that the role of carnitine in fatty acid oxidation can be clarified. In addition, since the physical role of mitochondria and Peroki j / somes in fatty acid oxidation has not been elucidated, the uptake of fatty acids into mitochondria was inhibited by the addition of deacetel FA-5589. By doing so, it is possible to clarify the role of peroxisomes and the relationship between peroxisomes and the oxidation process in mitochondria. In these cases, the reaction system usually used in the fatty acid oxidation reaction is used, and it varies depending on the concentration of the intracellular granule component, but generally, it is about 0.1 / 诚 to 100. A 0 / 诚 concentration is advantageously used.
デァセチル F A - 5 8 5 9は、 さらに有利な脂肪酸分解阻害作用を示 す化合物の合成中間体としても有用な物質である。  Decetyl FA-5589 is also a useful substance as a synthetic intermediate for a compound that exhibits a further advantageous fatty acid decomposition inhibitory action.
また、生理活性物質 F A - 5 8 5 9の脂肪酸分解且害能の測定を、 ラッ ト肝臓ホモジネートを用い、 日本生化学会編「生化学実験講座」 第 9巻 脂質の代謝， 7 5頁（ 1 9 7 5年， 東京化学同人）記載の方法に準じて 以下に記載の方法で行った。 すなわち、 S ϋ系ラッ ト （ 7週令，雄）を 2日間絶食後、 放血によって屠殺し、 直ちに肝臓を取り出し 1 0倍量 ( W/V ) の 5 mMト リス一塩酸緩衝液（ pH 7. 5 )を含む 0. 2 5 M蔗 糖溶液を加え、 テフロン棒付ホモジナイザーでホモジナイ ズする。 In addition, measurement of fatty acid degradation and harmful ability of the physiologically active substance FA-585-99 was performed using rat liver homogenate, and edited by the Biochemical Society of Japan, “Biochemical Experiment Course”, Vol. 9, Lipid Metabolism, page 75 (1 The method was described below according to the method described in 975, Tokyo Chemical Dojin. That is, Sϋ rats (7-week-old, male) were fasted for 2 days, sacrificed by exsanguination, and the liver was immediately removed and 10-fold (W / V) 5 mM tris-hydrochloride buffer (pH 7). 0.25 M Add the sugar solution and homogenize with a homogenizer with a Teflon rod.
6 0 0 X g， 2 0分間の遠心上澄みを 3 0, O O O x g' 3 0分間遠心し、 玍じたペレットを上記の蔗糖溶液に、 肝臓湿重量 0.2 g / 0.5m^溶液と なる濃度に懸濁し、 その 0.5m ^を酵素液として反応に用いた。  Centrifuge the supernatant at 600 X g for 20 minutes and centrifuge it for 30 minutes at 30 x OOO xg 'for 30 minutes, and add the pellet to the above sucrose solution to a concentration that will give a liver wet weight of 0.2 g / 0.5 m ^. The suspension was used, and 0.5 ml of the suspension was used as an enzyme solution in the reaction.
リ ン酸カ リ ゥム緩衝液 ( pH 7.5 ) 3 0 imole, Cl 30 0 ί mole, Phosphate buffer (pH 7.5) 30 imole, Cl 300 mole
A P 3 μ mole , MgCl 2 3 μ mole ， 蔬糖 l 2 0 mole， l -1 C パルミ チン酸（ 0.1 Ci ,牛血清アルブ ミ ンをモル比 1 ： 5になるよ うに添加した溶液 PH 7.5 ) 0.6 mole , L カル-チン （ L— carnitine ) 0.6 mole , コェンザィ ム A(Co enzyme A ) 0.6 mole ， ォキサル酢酸 0.2 mole ， 水又は阻害剤を含有する水溶液 0. 及び酵素溶液 0. から成る <¾5混液 3. Om^をハイァミ ンハイ ドロ ォキサイ ド 1 0—X〔パッカード社（ オランダ）製〕を浸した JiSをつ るした密閉試験管中に入れ、 3 7°C 2 0分間好気的に振盪反応した。 AP 3 μmole, MgCl 2 3 μmole, Vegetable l 20 mol, l- 1 C Palmitic acid (0.1 Ci, bovine serum albumin added at a molar ratio of 1: 5 PH 7.5) <¾5 consisting of 0.6 mole, L-carnitine, 0.6 mole, Coenzyme A, 0.6 mole, oxalacetic acid, 0.2 mole, aqueous solution containing water or inhibitor 0, and enzyme solution 0. Mixture 3. Put Om ^ into a sealed test tube with JiS soaked with Hyamine Hydroxide 10-X [Packard (Netherlands)] and aerobically shake at 37 ° C for 20 minutes. Reacted.
7 0 %過塩素酸 0. を添加して反応を止めた後、 生成した14 C02 を 測定することによって酵素活性の測定をした。 該化合物 F A— 5 8 5 9 の阻害活性は、 阻害剤無添加の場合に比較して、 2 5 0 μ /^ε で 1 Ί % , 5 0 0 ig/m£で 2 5 % , 1 0 0 0
Figure imgf000017_0001
で 3 6 %阻害であった。 f1 A— 5 8 5 9の急性毒性 L 50値は、 マウス静脈内注射で 4 0 0 / 上上であった。 The reaction was stopped by the addition of 70% perchloric acid 0.5 was the measurement of enzymatic activity by measuring the generated 14 C0 2. The inhibitory activity of the compound FA-5859 was 1% at 250 μ / ^ ε, 25% at 500 ig / m £, and 10% as compared to the case without the inhibitor. 0 0
Figure imgf000017_0001
At 36% inhibition. Acute toxicity L 50 values of f 1 A- 5 8 5 9 was on 4 0 0 / mouse on intravenous injection.
FA- 5 8 δ 9またはその塩は、 たとえば脂肪酸分解阻害剤として有 用である。 F Α— 5 8 5 9またはその塩を脂肪酸分解阻害剤として用い るには、 たとえば哺乳動物（例、 マウス ， ラッ ト ，人など）の糖尿病の 治療を目的として、 P A— 5 8 5 9 として約 0.2ないし 2 0 0 / を 1日投与量として投与する。 また、 FA— 5 8 5 9またはその塩を投与 するにあたっては、 常套手段によってたとえば錠剤， 顆粒剤， カプセル 剤，液剤などの剤型にして経口的に、 またたとえば注射剤として非経口 的に投与することができる。 FA-58δ9 or a salt thereof is useful, for example, as a fatty acid degradation inhibitor. To use FIII-589 or a salt thereof as a fatty acid degradation inhibitor, for example, as PA-589, for the treatment of diabetes in mammals (eg, mice, rats, humans, etc.) About 0.2 to 200 / is administered as a daily dose. In addition, when administering FA-5589 or a salt thereof, tablets, granules, capsules, etc. may be used in a conventional manner. It can be administered orally in the form of preparations, liquids, etc., and parenterally, for example, as an injection.
また、 — 5 8 5 9は、 脂肪酸の代謝を解明する上での生化学的試 薬として することができる。 たとえば、 F A— 5 8 5 9を反応液中 に添加することによってカルニチン欠乏状態を容易に作りうるので、 脂 肪酸酸化におけるカルニチンの役割をより一層明確にすることができる。 また、 脂肪酸酸化における ミ トコンドリァとペルォキシゾームとの生理 的役割はほとんど明らかにされていないので、 ミ トコンドリァへの脂肪 酸の取り込みを F A— 5 8 5 9を添加して阻害することによって、 ペル ォキシゾームの役割およびペルォキンゾームと ミ ト コン ド リ ァでの酸化 過程とのかかわりを明らかにすることが可能である。 これらの場合、 脂 肪酸酸化反応で通常用いられる反応系を用いて、 細胞内顆粒成分の濃度 によっても異なる力;、 一般的には F A— 5 8 5 9約 0. 5 /mi〜 5 0 ^ /mi濃度が有利に用いられる。 In addition, — 5859 can be used as a biochemical reagent to elucidate the metabolism of fatty acids. For example, FA- since 5 8 5 9 may make it easier for the carnitine deficiency by adding to the reaction solution, it is possible to further clarify the role of carnitine in fatty acid oxidation. Moreover, since the physiological roles of Mi Tokondoria and Peruokishizomu in fatty acid oxidation are largely unknown, by inhibiting the uptake of fat acids to Mi Tokondoria FA- 5 8 5 9 was added and the pel Okishizomu It is possible to elucidate the role and implications of the oxidation process in perokinsome and mitochondria. In these cases, using a reaction system usually used in the fatty acid oxidation reaction, a force that varies depending on the concentration of the intracellular granule component; generally, FA—585 / about 0.5 / mi to 50 ^ / mi concentration is advantageously used.
F A- 5 8 5 9は、 さらに有利な脂肪酸分解阻害作用を示す化合物の 合成中間体としても有用な物質である。  FA-589 is also a useful substance as a synthetic intermediate for a compound that exhibits a further advantageous fatty acid decomposition inhibitory action.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
以下に参考例および実施例を挙げて、 本発明をさらに具体的に説明す る  Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples.
参考例 1.  Reference example 1.
(1) (L )一な 一ベンジルォキ カルボニルァ ミ ノ 一/?— t —ブ ト キ 'ンカ ルボニルァ ミノプロピオン酸 6. 1 を酢酸ェチル 1 0 0 Wに溶解し， ― 1 0。Cに冷却してこれに N—メチルモルホリ ン 1. 8 ^とクロル炭酸ェチ ル 1. 9 とを加えて - 1 0 ¾で 1時間かきまぜた。 不溶部をろ' iし、 ろ 液に大過剰のジァゾメタン -ジェチルエーテル溶液を加え 0 °Cで 1時間  (1) (L) One benzyloxy carbonylamino 1 /? — T—butoxycarbonylaminopropionic acid 6.1 was dissolved in 100 mL of ethyl acetate, and −10. After cooling to C, N-methylmorpholine 1.8 ^ and chlorocarbonate 1.9 were added, and the mixture was stirred at-10 ° for 1 hour. Filter the insoluble part, add a large excess of diazomethane-getyl ether solution to the filtrate, and add 0 ° C for 1 hour
\―…、 \ ―…,
\ - ついで室温で 1 2時間かきまぜた。 溶媒を減圧留去し、残留物をメタノ ール 5 0 Wに溶解し、 これに安息香酸の銀塩 2 0 0 «9のト リェチルアミ ン溶液 2 を加え室温ついで暗所で 4時間かきまぜた。 不溶物をろ別し てろ液を減圧濃縮し、 残留物を酢酸ェチル 1 0 0 ^に溶解した。 この酢 酸ェチル溶液を 1 0 %クェン酸水， 5 炭酸水素ナト リ ウム ，水の順で 洗い無水硫酸ナ卜 リゥ厶で乾燥した。 酢酸ェチルを留去すると、（L)一 ベンジルォキシカルボニノレアミノ 一 r一 t —ブ卜キシ力ルボニルァ ミノ 酸メチルエステルの結晶が析出した。 酢酸ェチル -石油エーテルより再 結晶した。 収量 4.8 （ 7 3 % )。 \- Then, the mixture was stirred at room temperature for 12 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in methanol 5 0 W, the mixture was stirred for 4 hours at room temperature in the dark followed added thereto the door Ryechiruami down solution 2 of a silver salt 2 0 0 «9 benzoic acid. The insoluble material was filtered off, the filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate 100 ^. This ethyl acetate solution was washed with 10% aqueous solution of citric acid, sodium hydrogencarbonate and water in that order, and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off, and crystals of (L) methyl 1-benzyloxycarboninoleamino-r-t-butoxyl-carbonylamino acid were precipitated. Recrystallized from ethyl acetate-petroleum ether. Yield 4.8 (73%).
融点 1 0 0 — 1 0 1°C ， 〔o 4 + 6.0。（ c = 1 ， ジメチルホルムア ミ ド中） Mp 1 0 0 - 1 0 1 ° C, [o 4 + 6.0. (C = 1, in dimethylformamide)
元素分析 ： C18H2606N2 として fc Elemental analysis: C 18 H 26 0 6 N 2 as fc
計算値 ： C ， 5 9.0 0 ； H , 7.1 5 ； N ， 7.6 5 (¾)  Calculated values: C, 59.00; H, 7.15; N, 7.65 (¾)
実側値 ： C , 5 9.3 0 ； H ， 7.0 7 ； N , 7.7 4 (¾)  Actual value: C, 59.30; H, 7.07; N, 7.74 (¾)
(2) 上記で得た（L) 一 一べンジルォキ 'ンカルボニルァ ミ ノ - r - t - ブトキ カルボニルァ ミノ酪酸メチルエステル 3.6 0 をメタノール 20 i ^に溶解し、 0°Cで 1 N -水酸化ナト リ ウム 1 2； ^を加えた。 室温で 3 時間かきまぜた後、 クェン酸で中和し、 酢酸ェチル 1 0 を加えた。 酢酸ェチル溶液を水洗し、 無水硫酸ナ ト リ ゥムで乾燥後減圧濃縮した。 析出した結晶をろ取し、 酢酸ェチルより再結晶し、 （L)— ベンジル ォキシカルボニルア ミ ノ ー r 一 t —ブ ト キ カルボニルァ ミ ノ酷酸の結 晶を得た。 収量 2.9 6 （ 8 5 % )。 (2) Dissolve (L) 1-benzyloxyaminocarbonyl-r-t-butoxycarbonylaminobutyric acid methyl ester 3.60 obtained above in methanol 20 i ^, and add 1N-NaOH at 0 ° C. Lithium 12; ^ was added. After stirring at room temperature for 3 hours, the mixture was neutralized with citric acid, and ethyl acetate (10) was added. The ethyl acetate solution was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The precipitated crystals were collected by filtration and recrystallized from ethyl acetate to obtain crystals of (L) -benzyloxycarbonylamino-r-t-butoxycarbonylamino-acid. Yield 2.96 (85%).
融点 1 3 6 - 1 3 7°C ， 〔 〕 4 + 1 2.2° ( c = 1 ， ジメチルホルムァ ミ ド中）。 Melting point 1336-1337 ° C, [] 4 + 12.2 ° (c = 1, in dimethylformamide).
元素分析： Cl7 406N2 として 計箕値： C , 5 7.9 4 ； H ， 6.8 7 ； N ， 7.9 5 %) Elemental analysis: as Cl 7 4 0 6 N 2 (Calculated value: C, 57.94; H, 6.87; N, 7.95%)
実測値 ： C , 5 7· 9 7 ； H， 6.7 6 ； N , 8.1 3 (¾)  Observed value: C, 57.97; H, 6.76; N, 8.13 (¾)
参考例 2. Reference example 2.
参考例 1で得られた（L)— ：ンジルォキ カルボニルァ ミノ — r — t 一ブトキシカルボニルァ ミノ酪酸 0· 7 5 をト リフルォロ酢酸 1 0 に溶解し、 室温で 1 0分間放置した。 反応液を減圧乾固し残留物をさ らに減圧下乾燥した。 これを 1 0 水酸化ナト リウム 7 に溶解し、 0 °Cに冷却してジメチル硫酸 0. S 5； ^を加え 0 °Cで 1時間ついで室温で 1 時間かきまぜた。 反応液をアンバ—ライ ト I R— 1 2 0 (H÷型） の力 ラムクロマ トグラフィ ー （ 6 0 ） に付し、 水洗後 Ν—アンモニア水で 溶出し、 1 5 0 »から 2 2 0 a までの溶出画分を集めて減圧濃縮し、 残 留物を減圧下乾燥して 0.4 4 タの （L)- ?—ベンジルォキンカルポニル ァミノ — r —ト リメチルァ ミ ノー —ア ノ酪酸をァメ状物質として得 た。 薄層クロマ トグラフィ 一（担体 ： ンリ カゲル 6 O F 2 5 4 ， メゾレク 社西ドイツ〕 0.75 (L) —: Ndiloxy carbonylamino—r—t monobutoxycarbonylaminobutyric acid obtained in Reference Example 1 was dissolved in 10 trifluoroacetic acid and left at room temperature for 10 minutes. The reaction solution was dried under reduced pressure, and the residue was further dried under reduced pressure. This was dissolved in 10 sodium hydroxide 7 , cooled to 0 ° C., added with dimethyl sulfate 0.05 S, and stirred at 0 ° C. for 1 hour and then at room temperature for 1 hour. The reaction mixture was subjected to column chromatography (60) with the power of Amberlite IR-120 (H ÷ type), washed with water and eluted with ア ン モ ニ ア -ammonia water, from 150 »to 220a eluted fraction was concentrated under reduced pressure to collect the residual Tomebutsu dried under vacuum 0.4 4 other (L) -? - benzyl O gold Lupo sulfonyl Amino - r - DOO Rimechirua Mi no - § the a aminobutyric acid Obtained as a paste. Thin layer chroma Togurafi one (carrier: Nri dim 6 OF 2 5 4, Mezoreku Co. West Germany]
1 ) Rf = 0.1 3 ( π —プロパノ ール ：水 = 4 ： 1 )  1) Rf = 0.1 3 (π-propanol: water = 4: 1)
2 ) Rf = 0.2 6 ( n—プロパノ ール ：水： 1 5 N—アンモニア =  2) Rf = 0.26 (n—propanol: water: 15 N—ammonia =
7 0 : 2 8 ： 2 ) o  7 0: 2 8: 2) o
参考例 3. Reference example 3.
(1) 参考例 1で得られた（L)— 一べンジルォキシカルボニルァ ミ ノ - r - t ーブ トキシカルボニルァミノ酪酸 1.4 を 8 0 %メタノ - 'レ水 3 に溶かし、 パラジウム黒の存在下接触還元を行った。 触媒をろ别 してメタノ 一ルを留去し、残留物に 8 4 0 ^の炭酸水素ナト リ ウムと水 1 0 とを加えて溶かした。 この溶液に 0ででァセトニト リル 1 0 « と 無水酢酸 0, 5 とを加え、 0 °Cで 1時間室温で 1 2時間かきまゼた ァ セトニト リルを留去して、殘留物にジェチルェ一テル 5 0 を加えて洗 い、 水層を 0. 1 N塩酸で中和した。 酷酸ェチル 5 で3回抽出し、 酢 酸ェチル層を無水硫酸ナ卜 リ ゥムで乾燥した。 酢酸エヂルを留去して石 油べンジソで結晶ィ匕し、酢酸ェチルから再結晶し、 （L)— ―ァセチル ァ ミ ノ — ？" — t —ブ ト キンカルボニルァ ミ ノ酪酸の結晶を得た。 (1) Dissolve the (L) -benzyloxycarbonylamino-r-t-butoxycarbonylaminobutyric acid 1.4 obtained in Reference Example 1 in 80% methano-water 3 and add palladium black. Was carried out in the presence of. The catalyst was filtered to remove methanol, and the residue was dissolved by adding 840 ^ sodium hydrogen carbonate and water 10. Acetonitrile 10 «and acetic anhydride 0.5 were added to the solution at 0, and the mixture was stirred at 0 ° C for 1 hour and room temperature for 12 hours. Cetonitrile was distilled off, and the residue was washed with 50 parts of ethyl acetate and the aqueous layer was neutralized with 0.1 N hydrochloric acid. Extraction was carried out three times with severe ethyl acetate, and the ethyl acetate layer was dried with anhydrous sodium sulfate. Ethyl acetate was distilled off, crystallized with petroleum benzene and recrystallized from ethyl acetate, and (L) —— acetylamino—? "-T-butoxycarbonylaminobutyric acid crystals were obtained.
収量 0.8 8 ^ ( 8 6 % )。 融点 1 4 0 — 1 4 1 °C。  Yield 0.88 ^ (86%). Melting point 140-141 ° C.
〔 〕 4+ 2 6.0。（ c = 0.9 ， ジメチルホルムア ミ ド中）。 [] 4 + 2 6.0. (C = 0.9, in dimethylformamide).
元素分析 '· C11H20O5N2 として Elemental analysis' · As C11H20O5N2
計算値 ： C ， 5 0.7 5 ； H , 7. 7 5 ； N , 1 0.7 6 (¾) 実測値 ： C ， 5 0.5 3 ； H , 7.3 5 ； N , 1 0.6 8 (%)  Calculated value: C, 50.75; H, 7.75; N, 10.76 (¾) Actual value: C, 50.53; H, 7.35; N, 10.68 (%)
(2) 上記で得られた（L)-^—ァセチルァ ミ ノ — r— t 一ブトキンカル ボニルァ ミ ノ酪酸 7 0 0 をト リ フルォ口酢酸 1 0 に溶解し室温で 3 0分間放置した。 ト リフルォロ酢酸を留去して残留物を減圧下乾燥し、 1 0 0 の水に溶解しダウエックス 5 0 X 2 (H +型）のカラム （ 3 0 0 ) に流した。 水洗した後 0.5 Nアンモニアで溶出し、 溶出液を減圧濃縮し た。 析出した結晶をろ取してメタノ ールより再結晶し、 （L)— -ァセ チルァ ミ ノ — r -ア ミ ノ酪酸の結晶を得た。 収量 3 4 8 W。  (2) The above-obtained (L)-^-acetylamino-rt-butoxyquinoneaminobutyric acid 700 was dissolved in trifluoroacetic acid 10 and left at room temperature for 30 minutes. Trifluoroacetic acid was distilled off, and the residue was dried under reduced pressure. The residue was dissolved in water (100) and passed through a column (300) of Dowex 50X2 (H + type). After washing with water, the mixture was eluted with 0.5 N ammonia, and the eluate was concentrated under reduced pressure. The precipitated crystals were collected by filtration and recrystallized from methanol to obtain crystals of (L) -acetylamino-r-aminobutyric acid. Yield 3 4 8 W.
融点 1 7 7 - 1 7 8で （分解） ， 〔《 — 1 5.4° ( c = 0.7 0.1 - 塩酸） Melting point 1777-1778 (decomposition), [<< — 15.4 ° (c = 0.7 0.1-hydrochloric acid)
元素分析： C6H1203N2 として As C 6 H 12 0 3 N 2 : Elemental analysis
計箕値 ： C ， 4 4.9 9 ； H , 7. 5 5 ； N 1 7. 4 9 {%)  Minor value: C, 44.99; H, 7.55; N17.49 {%)
実測値 ： C , 4 4.8 3 ； H ， 7. 6 2 ； N 1 7. 2 2 (¾) 実施例 1.  Measured value: C, 44.83; H, 7.62; N17.22 (¾) Example 1
(1) 参考例 2で得られた（L)一^—ベンジルォキ カルボニルア ミ ノ 一 r ー ト リ メチルァ ミノ /? アミ /酪酸 0.3 9 を 5.7 N塩酸 5 に溶 一 Ci.iFI(1) Reference Example obtained in 2 (L) one ^ -? Benjiruoki carbonyl A Mi Roh one r over door Li Mechirua Mino / soluble one Ami / butyric acid 0.3 9 to 5.7 N hydrochloric acid 5 Ci.iFI
VvLrO 解し、溶解液を 9 0°Cで 3 0分間加温した。 反応液を減圧濃縮し、残留 物に少量の水を加えて再び減圧濃縮した。 残留物にメタノ —ルとジェチ ルェ—テルとの混合溶媒を加え、析出する結晶をろ取して 2 2 8 の (L)— rート リメチルァ ミ ノ—^一ァミ ノ酪酸（デァセチル F A— 5 8VvLrO After dissolving, the lysate was heated at 90 ° C. for 30 minutes. The reaction solution was concentrated under reduced pressure, a small amount of water was added to the residue, and the mixture was concentrated again under reduced pressure. A mixed solvent of methanol and ethyl ether was added to the residue, and the precipitated crystals were collected by filtration. The (L) -trimethylamino-^-aminoaminobutyric acid (decetyl FA) in 228 — 5 8
5 9 )を得た。 融点 2 1 9 — 2 2 0 °C (分解） ， 59) was obtained. Melting point 2 19 — 220 ° C (decomposition),
〔a〕2 D 4十 6.7。 （ c - 1 , N—醉酸中）。 [A] 2 D 4 Ten 6.7. (C-1, N-in drunk acid).
元素分析 ： C7H1802N2C12 として Elemental analysis: as C7H 18 0 2 N 2 C1 2
計算値 ： C , 3 6.0 5 ； H , 7.7 7 ； N ， 1 2.0 1 ； C 1 530.40(¾) , 実側値 ： C ， 3 5· 9 6 ； H， 7.5 8 ； N , 1 1.8 8 ； Ci， 30.13(¾)0 (2) 実施例 4で得られたデァセチル F A - 5 8 5 9の 2塩酸塩 6 9 9 ^9 を水 2 0 Wに溶かし 0ででかきまぜながら重炭酸ナト リ ウム 1.1 ，無 7_酢酸 0.4 5 Wをァセトニト リル 1 0 » に溶かした溶液を加えた。 0。C で 1時間 ，室温で終夜かきまぜた。 ァセ トニト リルを留去し、 ダウエツ クス 5 0 X 2 (11+型） のカラム （ 8 0 ^ )に流した。 zK 3 0 0 aでカ ラムを洗い、 0.5 Nアンモニアで溶出した。 2 2 0 Wから 2 4 0 a の溶 出画分をあつめて減圧濃縮し、 残渣を 5 0 Wの水に溶かして凍結乾燥し た。 得られたァメ伏物質を I N -塩酸 2.8 Wに溶かし、 減圧乾固した。 残渣をメタノ —ルージェチルェ一テルを用いて結晶化し、 （L)一 r一卜 リ メチルァ ミ ノ — ^―ァセチルァ ミ ノ酪酸 ·塩酢塩 ( FA - 5 8 5 9 . 塩酸塩 )の結晶を得た。 収量 5 1 0 «9。 融点 2 2 2 ~ 2 2 3で（分解），Calculated: C, 3 6.0 5; H , 7.7 7; N, 1 2.0 1; C 1 5 30.40 (¾), the value of experiment: C, 3 5 · 9 6 ; H, 7.5 8; N, 1 1.8 8 Ci, 30.13 (¾) 0 (2) Dissolve the dihydrochloride 699-1 ^ 9 of deacetyl FA-585-99 obtained in Example 4 in 20 W of water and stir with sodium bicarbonate to remove sodium bicarbonate. The solution was prepared by dissolving 0.15 um and 0.45 W of 7-acetic acid in acetonitrile 10 ». 0. The mixture was stirred for 1 hour at room temperature and overnight at room temperature. Acetonitrile was distilled off, and the residue was passed through a column (80 ^) of Dowex 50X2 (11+ type). The column was washed with zK300a and eluted with 0.5 N ammonia. The eluted fraction from 220 W to 240 A was collected, concentrated under reduced pressure, and the residue was dissolved in 50 W water and freeze-dried. The obtained soft material was dissolved in 2.8 W of IN-hydrochloric acid and dried under reduced pressure. The residue is crystallized using methano-lugetyl ether to obtain crystals of (L) -r-trimethylamino-^-acetylaminobutyric acid / hydrochloride (FA-585-9. Hydrochloride). Was. Yield 5 10 «9. Melting point 2 2 2 to 2 2 3 (decomposition),
〔α〕 — 20.0° ( c = 0.75 ，水） [Α] — 20.0 ° (c = 0.75, water)
元素分析： C9Hi903N2Clとして As C 9 Hi 9 0 3 N2Cl: elemental analysis
計算値 ： C， 4 5.2 8 ； H， 8.0 2 ； N 1.7 3 ； C1 4.8 5 (¾) 実測値： C， 4 5.1 3 ； H， 8.2 4 ； N 1.7 9 ； C 1 4.7 3 (¾) 実施例 2. (1) 参考例 3で得られた（L)一 ―ァセチルア ミノ ーア ーァ ミ ノ酪酸Calculated value: C, 45.28; H, 8.02; N1.73; C14.85 (¾) Actual value: C, 45.13; H, 8.24; N1.79; C14.73 (¾) Implemented Example 2. (1) (L) 1-acetylamino-aminobutyric acid obtained in Reference Example 3
2 8 8 を 1 0 %7_K酸化ナト リ ウム 8 に溶解した。 この溶液を 0°Cに 冷却してジメチル硫酸 0.8 を加え、 0°Cで3 0分間ついで室温で3 0 分間かきまぜた後ダウエックス 5 0 X 2 (H+型） のカラムクロマトグ ラフィ 一（ 8 に付した。 カラムを水洗後 0.5 N -アンモニア水で 溶出し、 2 0 5 Wから 2 4 0 までの溶出画分をあつめて減圧濃縮した。 残留物を N -塩酸 1.5 に溶解し再び減圧乾固した。 乾固物をメタノ ― ルージェチルェ一テルから結晶化し、 （L)— Γ—ト リ メチルァ ミノ —^ -ァセチルァ ミ ノ酪酸 '塩酸塩（ FA— 5 8 5 9 '塩酸塩）の結晶を得 た。 収量 2 2 0 ^。 融点 2 1 7 — 2 1 8°C (分解） ， 〔な〕 4— 20.2° ( c = 0.9 6 ，水中） 288 was dissolved in 10% 7_K sodium oxide 8. This solution was cooled to 0 ° C, 0.8 ml of dimethyl sulfate was added, and the mixture was stirred at 0 ° C for 30 minutes and then at room temperature for 30 minutes, and then subjected to Dowex 50 X 2 (H + type) column chromatography (8+). After washing the column with water, the column was eluted with 0.5 N aqueous ammonia, and the eluted fractions from 205 W to 240 W were collected and concentrated under reduced pressure.The residue was dissolved in 1.5 N-hydrochloric acid and dried again under reduced pressure. The dried product was crystallized from methano-lugetyl ether to form crystals of (L) -trimethylamino-^-acetylaminobutyric acid 'hydrochloride (FA-5589' hydrochloride). Yield 2 20 ^ Melting point 2 17 — 2 18 ° C (decomposition), [N] 4 — 20.2 ° (c = 0.96, in water)
元素分析： C9H1903N2C1として As C 9 H 19 0 3 N 2 C1: Elemental analysis
計算値 ： C， 4 5.2 8 ； H , 8.0 2 ； N , 1 1.7 3 ； C 1， 1 4.8 5 実測値 ： C， 4 4.9 7 ； H , 8.0 2 ； N , 1 1.4 9 ； C 1 , 1 4.8 4 (%)0 (2) 上記で得られた（L)— r一ト リメチルアミノ ー ?—ァセチルァミノ 酪酸 '塩酸塩（ F A - 5 8 5 9塩酸塩） 1 0 0 を 5.7 N塩酸 5 に溶 解し、 1 0 0°Cで 6時間処理した。 反応液を减圧濃縮し、残留物に少量 の水を加えて再び減圧濃縮した。 残留物にメタノ—ルとジェチルェ —テ ルとの混合溶媒を加え、析出する結晶をろ取して 9 5 の（L) - r - ト リ メチルァ ミ ノ —^ —ァ ミ ノ酪酸 · 2塩酸塩（デァセチル FA— 5 8 5 9 · 2塩酸塩）の結晶を得た。 融点 2 1 8 - 2 1 9で （分解） ， 〔α + 6.3° ( c = 1 , 1 N—酢酸中） Calculated value: C, 45.28; H, 8.02; N, 11.73; C1, 14.85 Actual value: C, 44.97; H, 8.02; N, 11.49; C1, 1 4.8 4 (%) 0 (2) Dissolve the (L) -r-trimethylamino-?-Acetylaminobutyric acid'hydrochloride (FA-585-99 hydrochloride) 100 obtained above in 5.7 N hydrochloric acid 5 Disassembled and treated at 1000C for 6 hours. The reaction solution was concentrated under reduced pressure, a small amount of water was added to the residue, and concentrated under reduced pressure again. A mixed solvent of methanol and getyl ether was added to the residue, and the precipitated crystals were collected by filtration. 95 (L) -r-trimethylamino-^-aminobutyric acid · 2HCl Crystals of a salt (deacetyl FA-5585-9 dihydrochloride) were obtained. Melting point 2 18-2 19 (decomposition), [α + 6.3 ° (c = 1, 1N in acetic acid)
元素分析： C7H1802N2C12 として As C 7 H 18 0 2 N 2 C1 2: Elemental analysis
計箕値 ： C， 3 6.0 5 ； H } 7.7 7 ； , 1 2.0 1 ； C 1 , 3 0.4 0 (%) 実測値 ： C , 3 6. 1 2 ； H , 7.8 8 ； N， 1 1.8 5 ； C1， 3 0.1 1 {%) u 実施例 4. Calculated value: C, 36.05; H } 7.77 ;, 12.01; C1, 30.40 (%) Actual value: C, 36.12; H, 7.88; N, 11.85 ; C1, 3 0.1 1 {%) u Example 4.
FA- 5 8 5 9遊離体 1.6 0 を定沸点塩酸 4 Οπ^に溶解し、 9 5°C で 1 6時間放置した。 反応液を減圧濃縮し、 残留物に少量の水を加えて 再び減圧濃縮した。 残留物にメタノールとジェチルェ一テルとの混合溶 媒を加え、 析出する結晶をろ取した。 メタノールより再結晶すると、 1. 2 0 ^のデァセテル _F A— 5 8 5 9の 2塩酸塩が得られた。  The FA-585-9 educt 1.60 was dissolved in constant boiling hydrochloric acid 4Οπ ^ and left at 95 ° C for 16 hours. The reaction solution was concentrated under reduced pressure, a small amount of water was added to the residue, and the mixture was concentrated again under reduced pressure. A mixed solvent of methanol and diethyl ether was added to the residue, and the precipitated crystals were collected by filtration. When recrystallized from methanol, 1.20 ^ of diacetel_FA—5859 dihydrochloride was obtained.
融点 2 1 9〜2 2 0°C ,〔α〕 2 + 6.3。 ( c = 1.0 , lN -酢酸） 元素分析： C7H1802N2C12 Melting point 219-220 ° C, [α] 2 +6.3. (C = 1.0, lN - acetic acid) Elemental analysis: C 7 H 18 0 2 N 2 C1 2
理論値： C 3 6.0 5 ； H 7.77 ； N 1 2.0 1 ； C1 30.4 0 (¾)  Theoretical value: C 3 6.05; H 7.77; N 1 2.01; C1 30.40 (¾)
実測値 ： C 3 6.0 9 ； H 7.7 2 ,· N 1 1.8 1 ； C 12 9.8 0 (5¾  Observed value: C36.09; H7.72, N11.81; C129.80 (5¾
吸収スぺク トル ： 2 1 0 nmから 7 0 0 nm までの紫外および可視領域 には特徵的な吸収は示さない。 Absorption spectrum: No specific absorption in the ultraviolet and visible regions from 210 nm to 700 nm.
産業上の利用可能性 Industrial applicability
生理活性物質デァセチル FA— 5 8 5 9および生理活性物質 F A- 5 8 5 9は、 優れた脂肪酸分解阻害作用を有しており、 該物質またはそれ らの塩は、 たとえばマウス ， ラ： / ト ，人等の哺乳動钧の糖尿病治療剤と して利用され、 また、 該物質は、 脂肪酸の代謝を解明するための生化学 的試薬として使用される。  The physiologically active substances deacetyl FA-585-99 and the physiologically active substance FA-585-99 have an excellent inhibitory effect on fatty acid degradation. It is used as a therapeutic agent for diabetes in mammals such as humans and humans, and the substance is used as a biochemical reagent for elucidating the metabolism of fatty acids.
一 ο:'.·'?ιOne ο: '. ·'? Ι
、 Vv I O , Vv I O

Claims

請 求 の 範 囲 The scope of the claims
1. —般式  1. —general formula
CH3 CH 3
CH3 -N@-CH2 - CH- CH2 -COO® CH 3 -N @ -CH 2 -CH- CH 2 -COO®
CH3 腿 - R2 CH 3 thigh-R 2
〔式中、 R2はァセチル基以外の保護基を示す。 〕で表わされる化合物 またはその塩を保護基脱離反応に付し、 必要によりァセチル化反応に付 すことを特徵とする一般式 [Wherein, R 2 represents a protecting group other than an acetyl group. A compound represented by the general formula, which is subjected to a protecting group elimination reaction and, if necessary, to an acetylation reaction.
CH3 CH 3
CH3 -N@-CH2 -CH-CH2 -COO® CH 3 -N @ -CH 2 -CH-CH 2 -COO®
CH3 NH-Ri CH 3 NH-Ri
〔式中、 ； Riは水素またはァセチル基を示す。 〕で表わされる化合物ま たはその塩の製造法。 Wherein Ri represents hydrogen or an acetyl group. ] Or a salt thereof.
2,  2,
丽2 -CH2 - CH - C -COOH 丽2 -CH 2 -CH-C -COOH
NH-COCH3 で表わされる化合物またはその塩を卜 リ メチル化反応に付し、 必要によ り加水分解反応に付すことを特徵とする一般式  A general formula characterized in that a compound represented by NH-COCH3 or a salt thereof is subjected to a trimethylation reaction and, if necessary, to a hydrolysis reaction.
CH3 CH 3
CH3 -N@-CH2 -CH-CH2 -COO0 CH 3 -N @ -CH 2 -CH-CH 2 -COO 0
CH3 NH-Ri CH 3 NH-Ri
〔式中、 Riは水素またはァセチル基を示す。 〕で表わされる化合物ま たはその塩の製造法。 [Wherein Ri represents hydrogen or an acetyl group. ] Or a salt thereof.
、 ,
PCT/JP1982/000409 1981-11-26 1982-10-15 Process for preparing physiologically active substance fa-5859 and its derivatives WO1984001574A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
PCT/JP1982/000409 WO1984001574A1 (en) 1982-10-15 1982-10-15 Process for preparing physiologically active substance fa-5859 and its derivatives
US06/442,368 US4521432A (en) 1981-11-26 1982-11-17 Physiologically active substance FA-5859, its derivative, their production and use
CA000416173A CA1190166A (en) 1981-11-26 1982-11-23 Physiologically active substance fa-5859, its derivative, their production and use
DE8282110866T DE3263167D1 (en) 1981-11-26 1982-11-24 Physiologically active substance fa-5859, its derivative; their production and antidiabetic agents containing said compounds
EP82110866A EP0080695B1 (en) 1981-11-26 1982-11-24 Physiologically active substance fa-5859, its derivative; their production and antidiabetic agents containing said compounds
KR1019830001950A KR900008248B1 (en) 1982-07-28 1983-05-07 Process for preparing physiologically active substance fa-5859
US06/939,152 US4767781A (en) 1981-11-26 1986-12-08 Derivatives of beta-amino-gamma-trimethylammonio-butyrate and their production and use
US07/423,555 US4948534A (en) 1981-11-26 1989-10-16 Derivatives of β-amino-γ-trimethylammonio-butyrate and their production and use

Applications Claiming Priority (1)

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Country Link
WO (1) WO1984001574A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Izumiya Nobuo, Ono Motonori, Kato Tetsuo, Aoyagi Motohiko (Authors), "Gosei Kagaku Series, Peptide Gosei" 30. October. 1975 (30.10.1975) Maruzen Kabushiki Kaisha P.14-15 *

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