WO2018207888A1 - Procédé de production de rameltéon - Google Patents

Procédé de production de rameltéon Download PDF

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WO2018207888A1
WO2018207888A1 PCT/JP2018/018199 JP2018018199W WO2018207888A1 WO 2018207888 A1 WO2018207888 A1 WO 2018207888A1 JP 2018018199 W JP2018018199 W JP 2018018199W WO 2018207888 A1 WO2018207888 A1 WO 2018207888A1
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ramelteon
transaminase
represented
producing
optically active
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PCT/JP2018/018199
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Japanese (ja)
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西山　章
紀幸 伊藤
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/80—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/81—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B53/00—Asymmetric syntheses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B61/00—Other general methods

Definitions

  • the present invention relates to a method for producing ramelteon.
  • the following methods are known as a production method of ramelteon used as a sleep inducing agent.
  • Tetrahedron Asymmetry, 2006, 17, 184-190.
  • the prior arts (1) and (2) are not suitable for implementation on an industrial scale because they use an expensive transition metal catalyst and perform high-pressure hydrogenation that requires special equipment.
  • the prior arts (3) and (4) are optical resolution methods, unnecessary enantiomers accounting for half of the substrate weight must be discarded, and these are also not efficient methods.
  • the problem to be solved by the present invention with respect to the above prior art is to provide a simple and efficient production method of ramelteon.
  • the asymmetric reduction is represented by the following formula (5): Wherein R is an alkyl group having 1 to 20 carbon atoms which may have a substituent, an alkenyl group having 2 to 20 carbon atoms which may have a substituent, and a carbon number which may have a substituent. Represents an aralkyl group having 7 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms which may have a substituent, a carboxyl group, or a 1H-tetrazole group, * represents an asymmetric carbon atom, and A ⁇ represents a counter anion.
  • the optically active pyrrolidine salt derivative represented by the following formula (6) The method for producing ramelteon according to [1], wherein the Hantzsh ester represented by the formula is used as a reducing agent.
  • R is a diphenylmethyl group, (trimethylsilyloxy) diphenylmethyl group, or (hydroxy) diphenylmethyl group, the absolute configuration is R-form, and A ⁇ is The method for producing ramelteon according to [2], which is trichloroacetate or trifluoroacetate.
  • optically active aldehyde (2) is represented by the following formula (3): The method for producing ramelteon according to any one of [1] to [5], which is converted to an optically active amine represented by the following formula and then propionylated.
  • the enal (1) is represented by the following formula (7);
  • ramelteon can be easily and efficiently produced.
  • an ⁇ , ⁇ -unsaturated nitrile represented by the following formula (7) that is, (E)-(1,6,7,8-tetrahydro-2H-indeno [5,4- b] furan-8-ylidene) acetonitile can be used.
  • the compound (7) can be synthesized with a carbanion prepared from diethyl cyanomethylphosphonate and sodium hydride, 1,2,6,7-tetrahydro-8H-indeno [ It can be easily produced by reacting 5,4-b] furan-8-one.
  • enal which is an intermediate product of the present invention, that is, (E) -2- [1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde is It is represented by Formula (1).
  • the compound (1) is a novel compound not described in any literature.
  • optically active aldehyde which is an intermediate product of the present invention, ie, (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde is It is represented by Formula (2).
  • an optically active amine which is an intermediate product of the present invention, that is, (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanolamine is Is represented by the following formula (3).
  • ramelteon which is the product of the present invention, that is, (S) -N- [2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethyl] Propionamide is represented by the following formula (4).
  • the preferred embodiment of the present invention is represented as follows in the figure. Further, in the present application, “manufacturing the optically active aldehyde represented by the formula (2) and subsequently converting it into the ramelteon represented by the formula (4)” includes the step 3 from the optically active aldehyde (2). And a method for producing ramelteon (4) from optically active aldehyde (2) via steps 4 to 5 are included.
  • the enal represented by the formula (1) is produced by reacting the ⁇ , ⁇ -unsaturated nitrile represented by the formula (7) with a hydride reducing agent.
  • Examples of the hydride reducing agent include lithium aluminum hydride, diisobutylaluminum hydride, sodium bis (2-methoxyethoxy) aluminum hydride, lithium tri (tert-butoxy) aluminum hydride, borane, lithium borohydride, hydrogenation.
  • Examples thereof include sodium borohydride, potassium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, lithium triethylborohydride, catecholborane and the like, preferably diisobutylaluminum hydride.
  • the upper limit of the amount of the hydride reducing agent used is preferably 10 times the molar amount relative to the compound (7), more preferably 5 times the molar amount. As a minimum, it is 0.5 time mole amount with respect to the said compound (7), More preferably, it is 1 time mole amount.
  • the reaction solvent in this step is not particularly limited as long as it does not affect the reaction.
  • tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol dimethyl ether Ether solvents such as 4-methyltetrahydropyran; aliphatic hydrocarbon solvents such as pentane, hexane, heptane and methylcyclohexane; aromatic hydrocarbon solvents such as benzene, toluene, xylene, ethylbenzene and mesitylene; N, N Amino acids such as dimethylformamide, N, N-dimethylacetamide, N, N-diethylacetamide, N-methyl-2-pyrrolidone, N-ethyl-2-pyrrolidone, N-methyl- ⁇ -caprolactam, hexamethylphosphoramide, etc.
  • phosphonic acid triamide system such as hexamethylphosphoric acid triamide solvents such as; system solvent; urea and dimethyl propylene urea-based solvent. These may be used alone or in combination of two or more.
  • the mixing ratio is not particularly limited.
  • it is an aromatic hydrocarbon solvent, more preferably tetrahydrofuran, 4-methyltetrahydropyran, or toluene.
  • the upper limit of the amount of the solvent used is preferably 100 times the weight of the compound (7), more preferably 50 times the weight, and particularly preferably 20 times the weight.
  • the lower limit is preferably 0.1 times the weight of the compound (7), more preferably 0.5 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the reaction temperature in this reaction is not particularly limited and may be set as appropriate.
  • the upper limit is preferably 120 ° C, more preferably 80 ° C, and particularly preferably 40 ° C. ° C.
  • the lower limit is preferably ⁇ 100 ° C., more preferably ⁇ 80 ° C., and particularly preferably ⁇ 70 ° C.
  • the reaction time in this reaction is not particularly limited and may be appropriately set.
  • the upper limit is preferably 100 hours, more preferably 50 hours, and particularly preferably 25 hours.
  • the lower limit is preferably 0.1 hour, more preferably 0.5 hour, and particularly preferably 1 hour.
  • a general process for obtaining a product from the reaction solution may be performed.
  • a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane, or the like.
  • the reaction solvent and the extraction solvent are distilled off from the obtained extract by an operation such as heating under reduced pressure, vacuum drying, or the use of a desiccant such as anhydrous magnesium sulfate, the desired product is obtained.
  • the target product thus obtained has a sufficient purity that can be used in the subsequent steps.
  • the target product is obtained by a general purification method such as crystallization, fractional distillation, or column chromatography. Further, the purity may be further increased.
  • the optically active aldehyde represented by the formula (2) is produced by asymmetric reduction of the enal represented by the formula (1).
  • the enal (1) may be produced according to the method described in the step 1, or even produced by other methods is included in the scope of the present invention.
  • Examples of the method for asymmetric reduction include a method using a reducing agent in the presence of an asymmetric organic catalyst, and a method using enone reductase.
  • Examples of the asymmetric organic catalyst include (2S, 5S) -2-tert-Butyl-3-methyl-5-benzoyl-4-oxomidazolidinium trichloroacetate, (R) -2- (tert-Butyl) -3-methyl- 4-oxoimidolidium trichloroacetate, (2S, 5S) -5-Benzyl-3-methyl-2- (5-methyl-2-furyl) -2-imidazolidinium trichloroacetate, (5S) -2,2,3-Trimethyl phenylmethyl-4-oxoimidazolidinium trichloroacetate, (2S, 5S) -2-tert-Butyl- -Methyl-5-benzyl-4-oxomidazolidinium trifluoroacetate, (R) -2- (tert-Butyl) -3-methyl-4-oxomimidazolidinium trifluoroacetate, (2S, 5S) -3 Mac
  • An optically active pyrrolidine salt derivative represented by formula (5) is preferable, and an optically active pyrrolidine salt derivative represented by formula (5) is preferable.
  • R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and a substituent.
  • An alkyl group, a carboxyl group, or a 1H-tetrazole group having 1 to 20 (more preferably 1 to 10 carbon atoms) is preferable.
  • substituents examples include an aromatic hydrocarbon group having 6 to 10 carbon atoms such as a phenyl group; an alkoxy group having 1 to 6 carbon atoms such as a methoxy group and an ethoxy group; a hydroxy group; a trimethylsilyloxy group and a triethylsilyloxy group A 5- to 7-membered heterocyclic group such as a pyrrolidinyl group or a piperidinyl group; and the like, and these substituents may be combined with one or more.
  • aromatic hydrocarbon group having 6 to 10 carbon atoms such as a phenyl group
  • alkoxy group having 1 to 6 carbon atoms such as a methoxy group and an ethoxy group
  • a hydroxy group such as a trimethylsilyloxy group and a triethylsilyloxy group
  • a 5- to 7-membered heterocyclic group such as a pyrrolidinyl group or a piperidinyl group
  • R is preferably methyl, ethyl, isopropyl, methoxymethyl, (1-pyrrolidinyl) methyl, vinyl, benzyl, phenyl, diphenylmethyl, (trimethylsilyloxy) diphenylmethyl, (hydroxy) A diphenylmethyl group, a carboxyl group, or a 1H-tetrazole group, more preferably a diphenylmethyl group, a (trimethylsilyloxy) diphenylmethyl group, or a (hydroxy) diphenylmethyl group, and still more preferably a diphenylmethyl group.
  • * represents an asymmetric carbon atom
  • the absolute configuration may be either S-form or R-form, and preferably the absolute configuration is R-form.
  • a ⁇ represents a counter anion.
  • the counter anion is not particularly limited, but is preferably chloride ion, bromide ion, sulfate ion, phosphate ion, mesylate, tosylate, triflate, trichloroacetate, trifluoroacetate, acetate, or benzoate, and more preferably Is trichloroacetate or trifluoroacetate.
  • supported by the polymer may be sufficient, for example, the cation exchange resin etc. in which a counter anion is a sulfonate are mentioned.
  • optically active pyrrolidine salt derivative includes optically active pyrrolidine in the reaction system, hydrogen chloride, hydrogen bromide, sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, trichloroacetic acid, You may prepare by mixing acids, such as a trifluoroacetic acid, an acetic acid, and a benzoic acid.
  • the acid is preferably trifluoroacetic acid or trichloroacetic acid, and more preferably trifluoroacetic acid.
  • the upper limit of the amount of the acid used is 10 times the weight of the optically active pyrrolidine, more preferably 5 times the weight, and particularly preferably 3 times the weight.
  • the lower limit is preferably 0.001 times weight, more preferably 0.01 times weight, and particularly preferably 0.1 times weight with respect to the compound (1).
  • the upper limit of the amount of the asymmetric organic catalyst used is preferably 10 times the weight of the compound (1), more preferably 5 times the weight, and particularly preferably 1 times the weight.
  • the lower limit is preferably 0.001 times weight, more preferably 0.01 times weight, and particularly preferably 0.1 times weight with respect to the compound (1). In such a range, the cost is not excessive and post-processing is simple.
  • Examples of the reducing agent include hydrogen gas, formic acid, sodium formate, triethylammonium formate, 1,4-dihydropyridine derivatives and the like, with 1,4-dihydropyridine derivatives being preferred.
  • the 1,4-dihydropyridine derivative is more preferably the following formula (6);
  • Hantzsh ester represented by: Diethyl 1,4-Dihydro-2,6-dimethyl-3,5-pyridine dicarboxylicboxylate.
  • the upper limit of the amount of the reducing agent used is preferably 100 times the weight of the compound (1), more preferably 50 times the weight, and particularly preferably 10 times the weight.
  • the lower limit is preferably 0.1 times the weight of the compound (1), more preferably 0.5 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the solvent in this step is not particularly limited as long as it does not affect the reaction. Specifically, for example, water; methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, ethylene glycol, etc.
  • Alcohol solvents such as tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol dimethyl ether, 4-methyltetrahydropyran and the like; nitrile solvents such as acetonitrile and propionitrile; Ester solvents such as ethyl acetate, n-propyl acetate, isopropyl acetate; aliphatic hydrocarbon solvents such as pentane, hexane, heptane, methylcyclohexane; benzene, toluene, xylene, ethylbenzene Aromatic hydrocarbon solvents such as Zen and mesitylene; halogen solvents such as methylene chloride and 1,2-dichloroethane; sulfoxide solvents such as dimethyl sulfoxide; N, N-dimethylformamide,
  • the mixing ratio is not particularly limited.
  • the upper limit of the amount of the solvent used is preferably 100 times the weight of the compound (1), more preferably 50 times the weight, and particularly preferably 20 times the weight.
  • the lower limit is preferably 0.1 times the weight of the compound (1), more preferably 0.5 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the reaction temperature in this reaction is not particularly limited and may be set as appropriate.
  • the upper limit is preferably 150 ° C., more preferably 100 ° C., and particularly preferably 50 ° C.
  • the lower limit is preferably ⁇ 80 ° C., more preferably ⁇ 30 ° C., and particularly preferably 0 ° C.
  • the reaction time in this reaction is not particularly limited and may be appropriately set.
  • the upper limit is preferably 100 hours, more preferably 50 hours, and particularly preferably 25 hours.
  • the lower limit is preferably 0.1 hour, more preferably 0.5 hour, and particularly preferably 1 hour.
  • the mixing order of compound (1), asymmetric organic catalyst, reducing agent and solvent in this step is not particularly limited.
  • a general process for obtaining a product from the reaction solution may be performed.
  • a general process for obtaining a product from the reaction solution may be performed.
  • a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane, or the like. Good.
  • the reaction solvent and the extraction solvent are distilled off from the resulting extract by an operation such as heating under reduced pressure, the desired product is obtained.
  • the target product thus obtained has a sufficient purity that can be used in the subsequent steps.
  • the target product is obtained by a general purification method such as crystallization, fractional distillation, or column chromatography. Further, the purity may be further increased.
  • An optically active aldehyde represented by the above formula (2) is produced by acting an enone reductase having the ability to stereoselectively reduce the carbon-carbon double bond of the enal represented by the above formula (1). To do.
  • Any enone reductase may be used as long as it has the ability to stereoselectively reduce the carbon-carbon double bond at the enone site of enal (1) to produce optically active aldehyde (2). .
  • Enon reductase may be not only the enzyme itself, but also the microbial cell itself of the microorganism that produces the enzyme, the culture solution of the microbial cell, or the processed microbial cell product as long as it has the desired reducing activity. Moreover, the transformant into which DNA which codes the enzyme which has the reduction activity derived from the said microorganisms was introduce
  • transduced is also included.
  • the microorganism-treated product of the microorganism is not particularly limited, and examples thereof include dry cells, surfactant-treated products, lysed enzyme-treated products, immobilized cells, or cell-free extracts obtained by disrupting cells. it can. Furthermore, an enzyme that catalyzes the asymmetric reduction reaction may be purified from the culture and used.
  • microorganisms may be used alone or in combination of two or more.
  • these microorganisms may be immobilized and used by a known method.
  • the old yellow which is usually recognized as an enone reductase Examples include an enzyme (Old Yellow Enzyme), such as an oxidoreductase classified as EC 1.6.99 according to the enzyme classification method of the International Union of Biochemistry and Molecular Biology. As oxidoreductases classified into EC1.6.99, EC1.65.99.1: NADPH dehydrogenase, EC1.65.99.2: NAD (P) H dehydrogenase (quinone), EC1.69.99.
  • NADH dehydrogenase oxidoreductase classified into NADH dehydrogenase, EC1.65.99.5: NADH dehydrogenase (quinone), or EC1.65.99.6: NADPH dehydrogenase (quinone), preferably EC1.6 .99.1: NADPH dehydrogenase.
  • the EC 1.6.99.1 NADPH dehydrogenase include the genus Candida, the genus Kluyveromyces, the genus Saccharomyces, the genus Schizosaccharomyces, and the like. And those derived from bacteria such as Bacillus genus, Escherichia genus, Pseudomonas genus, Yersinia genus, Zymomonas genus, and preferably Saccharomyces saccharomyces (Saccharomyces saccharomyces).
  • Genus, Kluyveromyces genus, Bacillus genus, Esh examples include those derived from microorganisms such as Escherichia genus, Pseudomonas genus, Yersinia genus, Zymomonas genus, and the most preferred examples are Saccharomyces cerevisiae cerevisiae and Saccharomyces cerevisiae cerevisiae. Seth lactis (Kluyveromyces lactis), Bacillus subtilis (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Yersinia bermobies i. is) is included.
  • Examples of the enone reductase include OYE2, OYE3 (derived from Saccharomyces cerevisae, described in International Publication No. 2006/129628), and KYE (derived from Kluyveromyces lactis C. certis). , 349, 1521 (2007)), YqjM (derived from Bacillus subtilis, described in J. Biol. Chem., 278, 19891 (2003)), NemA (Escherichi coli) Origin: Biol.Pharm.Bul.20, 110 (1997)), XeaA, XenE, etc.
  • YersER derived from Yersinia bercoviaeri, described in Adv. Synth. Catal., 349, 1521.
  • NCR-R derived from Zymomonas mobilis, described in Biotechnol. Bioeng., 98, 22 (2007)
  • OYE2 derived from Zymomonas mobilis, described in Biotechnol. Bioeng., 98, 22 (2007)
  • OYE2 derived from Zymomonas mobilis, described in Biotechnol. Bioeng., 98, 22 (2007)
  • NemA derived from Zymomonas mobilis, described in Biotechnol. Bioeng., 98, 22 (2007)
  • NemA derived from Zymomonas mobilis
  • YERSER derived from Zymomonas mobilis
  • NCR-R derived from Zymomonas mobilis, described in Biotechnol. Bioeng., 98, 22 (2007)
  • Microorganisms that produce enzymes can usually be obtained from stocks that are readily available or purchased. For example, it can be obtained from the following culture collections. ⁇ National Biotechnology Headquarters, Biotechnology Headquarters, National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture 292-0818) ⁇ RIKEN BioResource Center Microbial Materials Development Office (JCM) (2-1, Hirosawa, Wako, Saitama 351-0198) German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Marschuder Weg 1b, D-38124 Brunswick, Germany)
  • the enone reductase (for example, OYE2) used in the present invention has its known amino acid sequence (for example, J. Biol. Chem. 268, 6097-6106 (for example) as long as it has the desired reductase activity. 1993) (one amino acid sequence of OYE2) (for example, 40, preferably 20, more preferably 15, more preferably 10, more preferably 5, 4, 3, Or a polypeptide having 2 or less amino acids) substituted, inserted, deleted and / or added.
  • the homology is 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, more preferably 98% or more, or It may have 99% or more amino acid sequence.
  • a tag sequence such as a histidine tag or an HA tag can be added.
  • it can be a fusion protein with another protein.
  • a peptide fragment may be sufficient.
  • the optically active aldehyde (2) can be more efficiently produced by using a transformant containing DNA encoding enone reductase.
  • genetic manipulations such as DNA isolation, vector preparation, and transformation described in this specification are described in Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in. It can be carried out by a method described in a document such as Molecular Biology (Green Publishing Associates and Wiley-Interscience).
  • the vector used for the above transformant is not particularly limited as long as it can express the gene encoding the reductase used in the present invention in a suitable host organism.
  • examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host strains can also be used.
  • Such vectors for example in the case of E. coli, usually contain regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc., and are operable with the DNA of the present invention. It can be suitably used as an expression vector comprising an expression unit linked to the.
  • pSTV28 manufactured by Takara Bio Inc.
  • pUCNT International Publication No. 94/03613
  • the “regulatory factor” refers to a base sequence having a functional promoter and any related transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).
  • operably linked means that various regulatory elements such as promoters and enhancers that regulate gene expression and the gene are linked in a state where they can operate in the host cell.
  • regulatory elements such as promoters and enhancers that regulate gene expression and the gene are linked in a state where they can operate in the host cell.
  • type and kind of the control factor can vary depending on the host.
  • the host organism used for expressing each enzyme is not particularly limited as long as it is transformed by an enzyme expression vector containing DNA encoding each enzyme and can express the enzyme into which the DNA has been introduced. .
  • bacteria are preferable from the introduction and expression efficiency, and Escherichia coli (Escherichia coli) is particularly preferable.
  • a vector containing a DNA encoding a reductase used in the present invention can be introduced into a host microorganism by a known method.
  • Escherichia coli as the host microorganism, the vector is introduced into the host cell by using a commercially available Escherichia coli HB101 (hereinafter, E. coli HB101) competent cell (manufactured by Takara Bio Inc.). can do.
  • Examples of transformants containing DNA encoding an enzyme that reduces the carbon-carbon double bond at the enone site of enal (1) include the vector pTSSYE2 and E. coli.
  • E. coli obtained by transforming HB101.
  • E. coli HB101 pTSYE2
  • E. coli obtained in the same manner. coli HB101 pTSYE3
  • E. coli. coli HB101 pNKYE
  • E. coli. coli HB101 pNYqjM
  • E. coli. coli HB101 pNYersER
  • E. coli. and E. coli HB101 pNNCR-R.
  • a transformant containing both a DNA encoding an enzyme having a target reducing activity and a DNA encoding a polypeptide having a coenzyme regeneration ability can be produced.
  • a transformant containing both a DNA encoding an enzyme that reduces the carbon-carbon double bond at the enone site of enal (1) and a DNA encoding a polypeptide having a coenzyme regeneration ability is Both the DNA encoding the enzyme that reduces the carbon-carbon double bond at the enone site of (1) and the DNA encoding the polypeptide having the coenzyme regeneration ability are incorporated into the same vector, and this is incorporated into the host cell.
  • these two types of DNAs can be incorporated into two different vectors of different incompatibility groups, and the two types of vectors can be introduced into the same host cell.
  • an enzyme that reduces the carbon-carbon double bond at the enone site of enal (1) is used.
  • the encoding DNA and the DNA encoding the polypeptide having the ability to regenerate coenzyme may be introduced into different host cells, and these host cells may be cultured in the same culture solution or using different culture solutions.
  • NAD + oxidized nicotinamide / adenine dinucleotide
  • NADP + oxidized nicotinamide / adenine dinucleotide phosphate
  • NADH reduced nicotinamide / adenine dinucleotide
  • an oxidoreductase having the ability to convert to NADPH is preferable.
  • enzymes include hydrogenase, formate dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase.
  • formate dehydrogenase and glucose dehydrogenase are used.
  • Examples of the formate dehydrogenase include Candida, Kloeckera, Pichia, Lipomyces, Pseudomonas, Moraxella, and Hyphomicrobium.
  • Examples include enzymes derived from microorganisms such as the genus Hyphomicrobium, the genus Paracoccus, the genus Thiobacillus, and the genus Ansylobacter, and the enzyme obtained from Thiobacillus sp. Can be mentioned.
  • glucose dehydrogenase examples include enzymes derived from microorganisms such as the genus Bacillus, the genus Lactobacillus, and the genus Pediococcus, and in particular, Bacillus megaterium, Lactobacillus plan Examples thereof include enzymes obtained from Talam (Lactobacillus plantarum), Lactobacillus pentosus (Lactobacillus pentosus), and Pediococcus parvulus.
  • microorganisms such as the genus Bacillus, the genus Lactobacillus, and the genus Pediococcus, and in particular, Bacillus megaterium, Lactobacillus plan
  • examples thereof include enzymes obtained from Talam (Lactobacillus plantarum), Lactobacillus pentosus (Lactobacillus pentosus), and Pediococcus parvulus.
  • Examples of the enzyme include the genus Cryptococcus (Japanese Patent Laid-Open No. 2006-262767), the genus Gluconobacter (J. Bacteriol., 184, 672-678, (2002)), and Saccharomyces (Saccharomyces).
  • Glucose dehydrogenase and cryptococcus from the genus Methodhods Enzymol., 89, 159-163, (1982)
  • Lactobacillus genus Lactobacillus genus
  • Pediococcus genus International Publication No. 2009/041415
  • Glucose-6-li from the genera, Aspergillus, Pseudomonas Dehydrogenase are known (Arch. Biochem. Biophys., 228,113-119 (1984)).
  • an appropriate solvent and substrate enal (1) the above microorganism, a culture thereof, or a processed product thereof, etc. are mixed and stirred and shaken under pH adjustment. Or leave it alone.
  • the reaction solution contains an enzyme that reduces NAD + and / or NADP + to each reduced form and a substrate for the reduction. It is preferable to carry out the reaction.
  • glucose dehydrogenase as the enzyme that reduces to the reduced form
  • glucose coexists as the substrate for reduction
  • formate dehydrogenase as the enzyme that reduces to the reduced form
  • formic acid as the substrate for reduction
  • the reaction can be promoted by adding a coenzyme such as NADH or NADPH that is required for a reduction reaction by a biological method.
  • a coenzyme such as NADH or NADPH that is required for a reduction reaction by a biological method.
  • these are usually added directly to the reaction solution.
  • a surfactant such as Triton (manufactured by Nacalai Tesque Co., Ltd.), Span (manufactured by Kanto Chemical Co., Ltd.), Tween (manufactured by Nacalai Tesque Co., Ltd.)
  • an organic solvent may coexist in the reaction system. If an organic solvent insoluble in water such as ethyl acetate, butyl acetate, isopropyl ether, toluene, hexane or the like is added to the reaction solution, inhibition of the reaction by the substrate and / or the alcohol that is the product of the reduction reaction can be avoided.
  • the solubility of the substrate can be increased by adding a water-soluble organic solvent such as methanol, ethanol, acetone, tetrahydrofuran, or dimethyl sulfoxide.
  • reaction solvent an aqueous medium such as water or a buffer solution is usually used.
  • buffer include potassium phosphate buffer and tris (hydroxymethyl) aminomethane-hydrochloric acid buffer.
  • a culture solution containing the microorganisms is usually used for the reaction as it is. You may concentrate and use a culture solution. In addition, when the components in the culture solution adversely affect the reaction, it is preferable to use microbial cells or processed microbial cells obtained by treating the culture solution by centrifugation or the like.
  • Enal (1) as a substrate may be added all at once at the beginning of the reaction, or may be added in divided portions as the reaction proceeds.
  • the temperature during the reaction is preferably 60 ° C as an upper limit, more preferably 40 ° C.
  • the lower limit is preferably 10 ° C, more preferably 20 ° C.
  • the pH during the reaction is preferably 10 as an upper limit, more preferably 9.
  • the lower limit is preferably 2.5, and more preferably 5.
  • the amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates, and the upper limit is preferably 50% (W / V), more preferably 10% (W / V). is there.
  • the lower limit is preferably 0.01% (W / V), more preferably 0.1% (W / V).
  • the substrate concentration in the reaction solution is preferably 50% (W / V) as the upper limit, more preferably 30% (W / V).
  • the lower limit is preferably 0.01% (W / V), more preferably 0.1% (W / V).
  • the reaction is usually performed with shaking or stirring with aeration.
  • the reaction time is appropriately determined depending on the substrate concentration, the amount of microorganisms, and other reaction conditions.
  • the upper limit of the reaction time is preferably 336 hours, and more preferably 168 hours.
  • the lower limit is preferably 0.1 hour, more preferably 1 hour.
  • an energy source such as glucose, ethanol or isopropanol
  • the upper limit of the addition amount is preferably 50% (W / V), more preferably 30% (W / V).
  • the lower limit is preferably 0.1 (W / V), more preferably 0.5% (W / V).
  • the method for taking out the optically active aldehyde (2) produced by the reduction reaction is not particularly limited, but ethyl acetate, toluene, t-butyl methyl ether, hexane, n-butanol,
  • a high-purity optically active aldehyde (2) can be easily obtained by extraction with a solvent such as dichloromethane, followed by dehydration and purification by distillation or silica gel column chromatography.
  • optically active aldehyde (2) thus obtained is included in the scope of the present invention even if it is derived into ramelteon (4) by any conversion method.
  • each process is demonstrated in detail.
  • ramelteon represented by the formula (4) is produced by condensing and reducing the optically active aldehyde represented by the formula (2) with propionic acid amide.
  • ramelteon represented by the formula (4) is produced by condensing and reducing the optically active aldehyde represented by the formula (2) with propionic acid amide.
  • a metal catalyst for example, Adv. Synth. Catal 2013, 355, 717-733.
  • propionamide, [Rh (COD) Cl] 2 as a metal catalyst
  • Xantphos 4,5-Bis (diphenylphosphino) -9,9-dimethylxanthene
  • the target product thus obtained has a sufficient purity, but for the purpose of further increasing the purity, the purity can be further increased by a general purification method such as crystallization, fractional distillation, column chromatography and the like. Also good.
  • the optically active amine represented by the formula (3) is produced from the optically active aldehyde represented by the formula (2).
  • the optically active aldehyde (2) may be produced according to the method described in Step 2, or it may be produced by other methods, and is included in the scope of the present invention.
  • Specific conversion methods include, for example, a method using ammonia and a hydride reducing agent, a hydrogenation method in the presence of ammonia and a metal catalyst, a method using formic acid in the presence of a metal catalyst, and a transamination in the presence of an enzyme (transaminase). The method of doing is mentioned.
  • the desired product is obtained by adding aqueous ammonia and ammonium acetate in an ethanol solvent and reducing with sodium cyanoborohydride.
  • the method for hydrogenation in the presence of ammonia and a metal catalyst may be performed according to the method described in Japanese Patent No. 4059978, for example. That is, the desired product can be obtained by adding ammonia gas and hydrogen gas in the presence of a transition metal catalyst such as homogeneous nickel, rhodium, palladium, iridium or the like. Or, Catalysts 2015, 5, 2258-2270. In other words, the target product can also be obtained by adding ammonia gas and hydrogen gas in the presence of a ruthenium catalyst supported on alumina.
  • a transition metal catalyst such as homogeneous nickel, rhodium, palladium, iridium or the like.
  • Catalysts 2015, 5, 2258-2270 Catalysts 2015, 5, 2258-2270.
  • the target product can also be obtained by adding ammonia gas and hydrogen gas in the presence of a ruthenium catalyst supported on alumina.
  • the method using formic acid in the presence of the metal catalyst may be according to the method described in International Publication No. 2016/096905. That is, the desired product can be obtained by using ammonium formate in the presence of a palladium catalyst.
  • transaminase a method for transamination in the presence of an enzyme (transaminase) will be described. That is, the transaminase having the ability to generate the optically active amine represented by the formula (3) is allowed to act on the optically active aldehyde represented by the formula (2) in the presence of an amino group donor. An optically active amine represented by the formula (3) is produced.
  • transaminase Any transaminase may be used as long as it has an ability to produce an optically active amine (3) in the presence of an amino group donor from the optically active aldehyde (2).
  • the transaminase may be not only the enzyme itself, but also the microbial cell itself, the culture solution of the microbial cell, or the processed microbial cell product as long as it has the desired transamination activity.
  • transduced is also included.
  • the microorganism-treated product of the microorganism is not particularly limited, and examples thereof include dry cells, surfactant-treated products, lysed enzyme-treated products, immobilized cells, or cell-free extracts obtained by disrupting cells. it can. Furthermore, an enzyme that catalyzes the asymmetric reduction reaction may be purified from the culture and used. These may be used alone or in combination of two or more.
  • these microorganisms may be immobilized and used by a known method.
  • the immobilization method generally include a carrier binding method, a crosslinking method, and a comprehensive method, and any of them may be used.
  • the transaminase may be reacted in water, and may be reacted in an organic solvent, that is, the organic solvent may be present in the reaction system or may be reacted using only the organic solvent.
  • the organic solvent may be present in the reaction system or may be reacted using only the organic solvent.
  • solvents such as glycerol, ethylene glycol, methanol, ethanol, 1-propanol, and 2-propanol
  • sulfoxide solvents such as dimethyl sulfoxide
  • amide systems such as N, N-dimethylformamide Solvents
  • aliphatic hydrocarbon solvents such as n-hexane and n-heptane
  • ester solvents such as ethyl acetate, isopropyl acetate and butyl acetate
  • aromatic hydrocarbon solvents such as benzene, toluene and xylene
  • acetone, 2- Ketone solvents such as butanone and methyl isobutyl ketone
  • Preferred solvents include dimethyl sulfoxide, N, N-dimethylformamide, ethyl acetate, isopropyl acetate, butyl acetate, toluene, xylene, diethyl ether, dinormal propyl ether, diisopropyl ether, dinormal butyl ether, methyl isopropyl ether, methyl tert -Butyl ether and ethyl-tert-butyl ether are mentioned, and more preferred solvent is methyl-tert-butyl ether.
  • the transaminase having the ability to produce an optically active amine (3) from the optically active aldehyde (2) in the presence of an amino group donor is, for example, EC 2.6 according to the enzyme classification method of the International Union of Biochemistry and Molecular Biology. .1. An enzyme group classified as X is mentioned.
  • the transaminase is generally called ⁇ -transaminase.
  • the ⁇ -transaminase refers to one having an activity to generate amines other than ⁇ -amino acids.
  • ⁇ -transaminase examples include the genera Arthrobacter, Bacillus, Pseudomonas, Vibrio, Alcaligenes, and Mesorhizobium. Examples include those derived from bacteria such as the genus Chromobacterium, the genus Caurobacter, and the genus Rhodobacter.
  • Arthrobacter sp. Bacillus megaterium, Pseudomonas sp., Vibrio fluvialis, Arthrobacter re-bacter ⁇ Dentricificans (Alcaligenes denitrificans), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas corrugata (Pseudomonas corrugata), Mesozobium sp.
  • Violaceum Chromobacterium violaceum
  • Caulobacter-Kuresentasu Caulobacter crescentus
  • Rhodobacter Sufaeroidesu Rhodobacter sphaeroides
  • Arthrobacter sp. KNK168 Arthrobacter sp. KNK168
  • Bacillus megaterium Pseudomonas sp. KNK425 (Pseudomonas sp. KNK425)
  • Pseudomonas sp. ⁇ Furubiarisu JS17 (Vibrio fluvialis JS17)
  • Arthrobacter citreus Arthrobacter citreus
  • Alkaligenes denitrificans Y2k-2 Alkaligenes denitrificans Y2k-2 (Alcaligenes denitrificans Y2k-2)
  • Pseudomonas P eu 8 KNu fluor 8 as fluorescens KNK08-18 Pseudomonas Korugata 10F6 (Pseudomonas corrugata 10F6)
  • Mesorhizobium sp LUK Mesorhizobium sp LUK
  • megaterium SC6394 Bacillus megaterium SC6394
  • Caulobacter crescentus CB15 Calobacter crecentus CB15
  • Rhodobacter sphaeroides DSM 158 Rhobacter sphaeroides DSM 158
  • microorganisms can usually be obtained from stocks that are easy to obtain or purchase. For example, it can be obtained from the following culture collections. ⁇ National Biotechnology Headquarters, Biotechnology Headquarters, National Institute of Technology and Evaluation (NBRC) (2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture 292-0818) ⁇ RIKEN BioResource Center Microbial Materials Development Office (JCM) (2-1, Hirosawa, Wako, Saitama 351-0198) German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Marschuder Weg 1b, D-38124 Brunswick, Germany)
  • the transaminase used in the present invention has one or more (for example, 40, preferably 20, more preferably) in the known amino acid sequence as long as it has the desired transamination activity.
  • a polypeptide in which 15 amino acids, more preferably 10, more preferably 5, 4, 3, or 2 amino acids) are substituted, inserted, deleted and / or added may be used.
  • the amino acid sequence is 85% or more homologous to a known amino acid sequence, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, more preferably 98% or more, or 99%. You may have the above amino acid sequence.
  • the optically active amine (3) when a transformant containing DNA encoding transaminase is used, the optically active amine (3) can be produced more efficiently.
  • genetic manipulations such as DNA isolation, vector preparation, and transformation described in this specification are described in Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in. It can be carried out by a method described in a document such as Molecular Biology (Green Publishing Associates and Wiley-Interscience).
  • the vector used for the transformant is not particularly limited as long as it can express the gene encoding the transaminase used in the present invention in an appropriate host organism.
  • examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host strains can also be used.
  • such a vector usually contains regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc., and operates with the DNA of the present invention. It can be suitably used as an expression vector comprising expression units that are ligated together.
  • pSTV28 manufactured by Takara Bio Inc.
  • pUCNT International Publication No. 94/03613
  • the “regulatory factor” refers to a base sequence having a functional promoter and any related transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).
  • operably linked means that various regulatory elements such as promoters and enhancers that regulate gene expression and the gene are linked in a state where they can operate in the host cell.
  • regulatory elements such as promoters and enhancers that regulate gene expression and the gene are linked in a state where they can operate in the host cell.
  • type and kind of the control factor can vary depending on the host.
  • the host organism used for expressing each enzyme is not particularly limited as long as it is transformed by an enzyme expression vector containing DNA encoding each enzyme and can express the enzyme into which the DNA has been introduced. .
  • bacteria are preferable from the introduction and expression efficiency, and Escherichia coli (Escherichia coli) is particularly preferable.
  • a vector containing a DNA encoding a reductase used in the present invention can be introduced into a host microorganism by a known method.
  • Escherichia coli as the host microorganism, the vector is introduced into the host cell by using a commercially available Escherichia coli HB101 (hereinafter, E. coli HB101) competent cell (manufactured by Takara Bio Inc.). can do.
  • an amination reaction using transaminase is generally a reversible reaction, the reaction generally stops at an equilibrium point.
  • the reaction using the polypeptide of the present invention can be improved.
  • alanine as an amino group donor, pyruvate by-product is conjugated with lactate dehydrogenase and glucose dehydrogenase for coenzyme regeneration.
  • a method of converting to lactic acid and eliminating the reaction equilibrium is effective.
  • a method using alanine as an amino group donor and removing by-product pyruvate with pyruvate decarboxylase International Publication No.
  • the optically active compound of the present invention can be more efficiently obtained by using a transformant that produces the plurality of enzymes in the same host cell.
  • the transformant can be obtained by incorporating DNAs encoding a plurality of necessary enzyme genes into the same vector and introducing them into the same host cell, and by combining these two types of DNA with a plurality of vectors having different incompatibility groups. And can be obtained by introduction into the same host cell.
  • Any amino group donor can be used as long as it generates the optically active amine represented by the formula (3) in the presence of an enzyme source.
  • Specific examples include ⁇ -phenethylamine, 2-butylamine, 2-pentylamine, 2-heptylamine, 3-heptylamine, n-ethylamine, n-propylamine, n-butylamine, n-amylamine, isopropylamine, isobutylamine.
  • ⁇ -amino acids eg, glycine, alanine, etc.
  • 3-amino-1-phenylbutane benzylamine, ⁇ -phenethylamine, cyclohexylamine, and optically active forms thereof.
  • ⁇ -phenethylamine, isopropylamine, or ⁇ -amino acid eg, alanine
  • ⁇ -phenethylamine or ⁇ -amino acid is more preferable.
  • Examples of preferred combinations of ⁇ -transaminase and amino group donors include the following. 1) When the amino group donor is isopropylamine, preferably Arthrobacter sp., Pseudomonas sp., Vibrio fluviaris, or Pseudomonas fluorescens (Pseudomona) fluorescens), and more preferably, Arthrobacter sp.
  • amino group donor is ⁇ -phenethylamine
  • Arthrobacter sp. Pseudomonas sp., Vibrio flubialis, or Pseudomonas fluorescens (Pseudomonas fluorescens) Pseudomonas fluorescens, more preferably Arthrobacter sp., Pseudomonas sp., Pseudomonas fluorescens, and more preferably p. Pseudomonas fluorescens. .).
  • the amino group donor is an ⁇ -amino acid (especially alanine)
  • it is preferably Arthrobacter sp., Pseudomonas sp., Vibrio fluviaris, or Pseudomonas. -Fluorescence (Pseudomonas fluorescens), More preferably, Arthrobacter sp. (Arthrobacter sp.) Or Pseudomonas sp. MV37 (Pseudomonas sp. MV37).
  • reaction solvent an aqueous medium such as water or a buffer solution is usually used.
  • buffer include potassium phosphate buffer and tris (hydroxymethyl) aminomethane-hydrochloric acid buffer.
  • a culture solution containing the cells of the above microorganisms may be used for the reaction as it is, or the culture solution may be concentrated.
  • the components in the culture solution adversely affect the reaction, it is preferable to use microbial cells or processed microbial cells obtained by treating the culture solution by centrifugation or the like.
  • optically active aldehyde (2) as a substrate may be added all at once at the beginning of the reaction, or may be added in divided portions as the reaction proceeds.
  • the temperature during the reaction is preferably 60 ° C as an upper limit, more preferably 40 ° C.
  • the lower limit is preferably 10 ° C, more preferably 20 ° C.
  • the pH during the reaction is preferably 10 as an upper limit, more preferably 9.
  • the lower limit is preferably 2.5, and more preferably 5.
  • the amount of the enzyme source in the reaction solution may be appropriately determined according to the ability to reduce these substrates, and the upper limit is preferably 50% (W / V), more preferably 10% (W / V). is there.
  • the lower limit is preferably 0.01% (W / V), more preferably 0.1% (W / V).
  • the substrate concentration in the reaction solution is preferably 50% (W / V) as the upper limit, more preferably 30% (W / V).
  • the lower limit is preferably 0.01% (W / V), more preferably 0.1% (W / V).
  • the reaction is usually performed with shaking or stirring with aeration.
  • the reaction time is appropriately determined depending on the substrate concentration, the amount of microorganisms, and other reaction conditions.
  • the upper limit of the reaction time is preferably 336 hours, and more preferably 168 hours.
  • the lower limit is preferably 0.1 hour, more preferably 1 hour.
  • an energy source such as glucose, ethanol or isopropanol
  • the upper limit of the addition amount is preferably 50% (W / V), more preferably 30% (W / V).
  • the lower limit is preferably 0.1 (W / V), more preferably 0.5% (W / V).
  • a surfactant such as Triton (manufactured by Nacalai Tesque), Span (manufactured by Kanto Chemical Co., Ltd.), Tween (manufactured by Nacalai Tesque) to the reaction solution.
  • an organic solvent insoluble in water insoluble organic solvent such as ethyl acetate, butyl acetate, isopropyl ether, toluene, hexane). Solvent may be added to the reaction solution.
  • an organic solvent soluble in water such as methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide can be added.
  • the optically active amine is generated by the above reaction.
  • the produced optically active amine can be isolated from the reaction mixture by a known method such as extraction, distillation, recrystallization, or column separation.
  • ethers such as diethyl ether and diisopropyl ether
  • esters such as ethyl acetate and butyl acetate
  • hydrocarbons such as hexane, octane and benzene
  • halogenated hydrocarbons such as methylene chloride, etc.
  • the produced optically active amino compound and unreacted amino group donor can be extracted with a common organic solvent in the same manner, for example, by adjusting the pH to basic.
  • the produced optically active amino compound and the unreacted amino group donor can be separated by, for example, distillation.
  • the target product thus obtained has a sufficient purity that can be used in the subsequent steps.
  • crystallization, fractional distillation, solution washing, column chromatography, etc. are generally used.
  • the purity may be further increased by a simple purification method.
  • crystallization is performed by forming a salt with an acid such as mandelic acid or camphorsulfonic acid.
  • an acid such as mandelic acid or camphorsulfonic acid.
  • a ramelteon represented by the formula (4) is produced by further propionylating the optically active amine represented by the formula (3) produced by the method described in the step 4.
  • a propionylating agent such as propionyl chloride, propionyl bromide, or propionic anhydride may be used in the presence of a base.
  • Examples of the base include lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium hydrogen carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate and the like; lithium Methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxide, sodium methoxide, sodium ethoxide, sodium isopropoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide, potassium isopropoxide, potassium tert -Alkoxides such as butoxide; metal hydrides such as sodium hydride, potassium hydride, calcium hydride; trimethylamine, triethylamine, tributylamine, diisopropylethyl Amine, N- methylpyrrolidine, N- methylmorpholine, 1,8-diazabicyclo [5,4,0] undec-7-ene, pyridine, quinoline, include
  • the mixing ratio is not particularly limited.
  • Sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, or tributylamine is preferable, and sodium hydroxide, potassium carbonate, or triethylamine is more preferable.
  • the upper limit of the amount of the base used is preferably 100 times the weight of the compound (3), more preferably 50 times the weight, and particularly preferably 10 times the weight.
  • the lower limit is preferably 0.1 times the weight of the compound (3), more preferably 0.5 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the upper limit of the amount of the propionylating agent used is preferably 50 times the weight of the compound (3), more preferably 10 times the weight, and particularly preferably 5 times the weight.
  • the lower limit is preferably 0.5 times the weight of the compound (3), more preferably 0.8 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the solvent in this step is not particularly limited as long as it does not affect the reaction.
  • Ether solvents such as ethylene glycol dimethyl ether and 4-methyltetrahydropyran
  • nitrile solvents such as acetonitrile and propionitrile
  • ester solvents such as ethyl acetate, n-propyl acetate and isopropyl acetate
  • Aliphatic hydrocarbon solvents such as cyclohexane
  • Aromatic hydrocarbon solvents such as benzene, toluene, xylene, ethylbenzene, and mesitylene
  • Ketone solvents such as acetone, methyl ethyl ketone, and
  • the mixing ratio is not particularly limited.
  • water ether solvents such as tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol dimethyl ether, 4-methyltetrahydropyran; ketone solvents such as acetone, methyl ethyl ketone, methyl isobutyl ketone
  • a nitrile solvent such as acetonitrile or propionitrile, more preferably water, tetrahydrofuran, acetone, or acetonitrile, and particularly preferably tetrahydrofuran or acetonitrile.
  • the upper limit of the amount of the solvent used is preferably 100 times the weight of the compound (3), more preferably 50 times the weight, and particularly preferably 20 times the weight.
  • the lower limit is preferably 0.1 times the weight of the compound (3), more preferably 0.5 times the weight, and particularly preferably 1 times the weight. In such a range, the cost is not excessive and post-processing is simple.
  • the reaction temperature in this reaction is not particularly limited and may be set as appropriate.
  • the upper limit is preferably 150 ° C., more preferably 100 ° C., and particularly preferably 50 ° C.
  • the lower limit is preferably ⁇ 80 ° C., more preferably ⁇ 30 ° C., and particularly preferably 0 ° C.
  • the reaction time in this reaction is not particularly limited and may be appropriately set.
  • the upper limit is preferably 100 hours, more preferably 50 hours, and particularly preferably 25 hours.
  • the lower limit is preferably 0.1 hour, more preferably 0.5 hour, and particularly preferably 1 hour.
  • the mixing order of the compound (3), propionylating agent, base and solvent in this step is not particularly limited.
  • a general process for obtaining a product from the reaction solution may be performed.
  • the reaction solution after completion of the reaction is added to water or an aqueous acid solution such as hydrochloric acid or sulfuric acid, and an extraction operation is performed using a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane or the like.
  • a general extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane or the like.
  • the target product precipitated by adding water to the reaction solution is filtered off and washed with water, tetrahydrofuran, acetone, acetonitrile, ethyl acetate, methylene chloride, toluene, heptane, or the like.
  • the target product, ramelteon (4), obtained in this way has sufficient purity, but for the purpose of further increasing the purity, a general purification method such as crystallization, fractional distillation, column chromatography or the like is used. Further, the purity may be further increased.
  • TFA trifluoroacetic acid
  • TCA trichloroacetic acid
  • THF Tetrahydrofuran 4-MTHP: 4-methyltetrahydropyran DMA: dimethylacetamide
  • Example 27 Preparation of lyophilized cells of E. coli expressing ⁇ -transaminase
  • enzyme A Arthrobacter sp. KNK168 (Patent No. 5410089, SEQ ID NO: 25)
  • Enzyme B Pseudomonas sp. KNK425 (Pseudomonas sp. KNK425)
  • Enzyme C Pseudomonas fluorescens KNK08-18 (Pseudomonas fluorescens) (Patent No.
  • Enzyme D Pseudomonas sp. MV37 (Pseudomonas sp. MV37) (Patent No. 5878871, SEQ ID NO: 2)
  • Enzyme E Vibrio flu Alice JS17 (Vibrio flavialis JS17) (sequence described in Appl. Microbiol. Biotechnol. 61.463-471 (2003)), NdeI recognition site and multi-site of pUCNT (International Publication No. 94/03613) downstream of lac promoter
  • the recombinant vector was obtained by inserting between the cloning sites.
  • E. coli E. coli using the recombinant vector.
  • coli HB101 (Takara Bio Inc.) was transformed to obtain recombinant E. coli.
  • the obtained recombinant Escherichia coli was cultured in 2 ⁇ YT medium (tripton 1.6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0) containing 200 ⁇ g / ml ampicillin, and the cells were collected. did.
  • the obtained cells were frozen in methanol in which dry ice was dissolved, and then dried overnight with a freeze dryer (Tokyo Rika Kikai Co., Ltd., FDS-1000) to obtain freeze-dried cells.
  • Example 29 Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanolamine using ⁇ -transaminase (reaction in water 3 enzyme method) L-lactic acid dehydrogenase PALDH derived from Pediococcus acidilactici JCM8797 and glucose dehydrogenase derived from Bacillus megaterium IAM1030 described in Example 13 of International Publication No. 2007/139055 Recombinant E.
  • ⁇ -transaminase reaction in water 3 enzyme method
  • L-lactic acid dehydrogenase PALDH derived from Pediococcus acidilactici JCM8797
  • glucose dehydrogenase derived from Bacillus megaterium IAM1030 described in Example 13 of International Publication No. 2007/139055 Recombinant E.
  • GDH coli co-expressing the enzyme GDH was cultured in 2 ⁇ YT medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0) containing 200 ⁇ g / ml ampicillin, Bacteria were collected by centrifugation. The obtained cells were frozen in methanol in which dry ice was dissolved, and then dried overnight with a freeze dryer (Tokyo Rika Kikai, FDS-1000) to obtain PAL / GDH freeze-dried cells.
  • 2 ⁇ YT medium tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0
  • Bacteria were collected by centrifugation.
  • the obtained cells were frozen in methanol in which dry ice was dissolved, and then dried overnight with a freeze dryer (Tokyo Rika Kikai, FDS-1000) to obtain PAL / GDH freeze-dried cells.
  • Example 31 Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine using an enzyme flow reactor Diaion HP2MG After washing twice 7.5 g (manufactured by Mitsubishi Chemical) with 7.5 mL of 0.1 M KPB (potassium phosphate buffer) (pH 7.5), 250 mg of TPM freeze-dried cells and 20 mg of PLP (pyridoxal phosphate) 10 mL of 0.1 M KPB (pH 7.5) in which was dissolved was added and stirred at room temperature for 2 hours. After removing water with a pipette, nitrogen was blown to dry until moisture disappeared to prepare an enzyme-immobilized resin. When the protein content in the removed water was measured, 38% of the added protein was adsorbed on the resin. The immobilization resin was packed in a glass tube of ⁇ 5 mm ⁇ 50 mm to produce an immobilization resin column.
  • KPB potassium phosphate
  • solution A 100 mM (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde, MTBE solution
  • solution B was saturated with water
  • YSP-101 syringe pump
  • liquid A and liquid B are fed at 0.025 mL / min, mixed in a T-shaped mixing section, and fixed at 30 ° C.
  • the solution was passed through a fluorinated resin column.
  • the eluted reaction solution was analyzed by gas chromatography under the same conditions as in Example 26, and (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8 was obtained.
  • the conversion rate was calculated from the ratio of -yl) ethanolamine and (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde.
  • the results are shown in Table 7 and FIG.
  • the conversion activity into the target compound was stably maintained from 40 minutes to 180 minutes after the start of liquid feeding.

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Abstract

La présente invention concerne un procédé de production simple et efficace de rameltéon, qui est un agent induisant le sommeil. Selon la présente invention, le rameltéon peut être produit en un procédé à plusieurs étapes comprenant une étape dans laquelle le (E)-2-[1,2,6,7-tétrahydro-8H-indéno[5,4-b]furan-8-ylidène]acétaldéhyde, qui est un énal facilement disponible, est réduit de manière asymétrique pour produire du (S)-(1,6,7,8-tétrahydro-2H-indéno[5,4-b]furan-8-yl)acétaldéhyde, qui est un aldéhyde optiquement actif.
PCT/JP2018/018199 2017-05-10 2018-05-10 Procédé de production de rameltéon WO2018207888A1 (fr)

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JP2007185133A (ja) * 2006-01-12 2007-07-26 Daicel Chem Ind Ltd アミントランスアミナーゼ、および該アミントランスアミナーゼを利用した光学活性アミンの製造方法
JP2009526531A (ja) * 2006-02-13 2009-07-23 ロンザ・アーゲー 光学活性キラルアミンの調製方法
WO2011108672A2 (fr) * 2010-03-04 2011-09-09 高砂香料工業株式会社 Catalyseur d'hydrogénation asymétrique homogène
US20110294171A1 (en) * 2007-07-11 2011-12-01 Dsm Ip Assets B.V. Preparation of a saturated aldehyde
CN104109143A (zh) * 2013-04-22 2014-10-22 上海阳帆医药科技有限公司 一类褪黑激素(mt1-mt2)受体激动剂及其制备方法与用途

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JPH1180106A (ja) * 1997-09-05 1999-03-26 Takeda Chem Ind Ltd 光学活性化合物の製造法
JP2007185133A (ja) * 2006-01-12 2007-07-26 Daicel Chem Ind Ltd アミントランスアミナーゼ、および該アミントランスアミナーゼを利用した光学活性アミンの製造方法
JP2009526531A (ja) * 2006-02-13 2009-07-23 ロンザ・アーゲー 光学活性キラルアミンの調製方法
US20110294171A1 (en) * 2007-07-11 2011-12-01 Dsm Ip Assets B.V. Preparation of a saturated aldehyde
WO2011108672A2 (fr) * 2010-03-04 2011-09-09 高砂香料工業株式会社 Catalyseur d'hydrogénation asymétrique homogène
CN104109143A (zh) * 2013-04-22 2014-10-22 上海阳帆医药科技有限公司 一类褪黑激素(mt1-mt2)受体激动剂及其制备方法与用途

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