JPWO2018207888A1 - Manufacturing method of ramelteon - Google Patents

Abstract

The present invention provides a simple and efficient method for producing the sleep-inducing agent ramelteon. According to the present invention, asymmetric reduction of easily available enal (E) -2- [1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde To produce optically active aldehyde (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde by using Can be manufactured.

Description

  The present invention relates to a method for producing ramelteon.

  As a method for producing ramelteon used as a sleep-inducing agent, the following methods are known.

  1) Using 1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-one with a phosphate derivative, (E) -2- [1,2,6,7- After conversion into tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetonitrile, reducing the nitrile functional group to an amine compound, asymmetric hydrogenation of the olefin, and the obtained optically active substance (Patent Literature 1) by subjecting a compound of formula (1) to a reduction reaction and then acylating the compound to obtain ramelteon.

  2) 1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-one was converted to (E) -2- [1,2,6,7- using phosphoric ester derivative. After conversion to tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetonitrile, hydration of the nitrile function to amide function, olefin isomerization and asymmetric hydrogenation To induce ramelteon (Non-Patent Document 1).

  3) Racemic 2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine is salt-formed using an optically active carboxylic acid derivative, A method of optically resolving a carboxylic acid salt (Patent Document 2)

  4) Salt formation of racemic 2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetic acid using an optically active amine derivative, A method of optically resolving an amine derivative salt (Patent Document 3).

Japanese Patent No. 4358917 Chinese Patent Application Publication No. 104529959 Chinese Patent Application No. 10154445

Tetrahedron: Asymmetry, 2006, 17, 184-190.

  However, the prior arts (1) and (2) are not suitable for industrial-scale implementation because expensive transition metal catalysts are used for high-pressure hydrogenation that requires special equipment. In addition, since the prior arts (3) and (4) are optical resolution methods, unnecessary enantiomers occupying half of the weight of the substrate must be discarded, and these are also not efficient methods.

  It is an object of the present invention to provide a simple and efficient method for producing ramelteon with respect to the above prior art.

  As a result of intensive studies, the present inventors have found a simple and efficient method for producing ramelteon from readily available starting materials and which does not require special equipment such as high-pressure hydrogenation, and have completed the present invention.

で表されるエナールを不斉還元することにより、下記式（２）；

で表される光学活性アルデヒドを製造し、続いてこれを下記式（４）；

で表されるラメルテオンに変換することを特徴とするラメルテオンの製造法。[1] The following formula (1);

By asymmetric reduction of the enal represented by the following formula (2);

To produce an optically active aldehyde represented by the following formula (4):

A method for producing ramelteon, which comprises converting into ramelteon represented by the formula:

（式中、Ｒは置換基を有しても良い炭素数１〜２０のアルキル基、置換基を有しても良い炭素数２〜２０のアルケニル基、置換基を有しても良い炭素数７〜２０のアラルキル基、置換基を有しても良い炭素数６〜２０のアリール基、カルボキシル基、又は１Ｈ−テトラゾール基を表す。＊は不斉炭素原子を表す。Ａ^-はカウンター陰イオンを表す。）で表される光学活性ピロリジン塩誘導体を不斉有機触媒に用いて、下記式（６）；

で表わされるＨａｎｔｚｓｈエステルを還元剤に用いて行うことを特徴とする、［１］に記載のラメルテオンの製造法。[2] The asymmetric reduction is performed 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. . the aralkyl group having 7 to 20, an optionally substituted aryl group having 6 to 20 carbon atoms, a carboxyl group, or 1H- tetrazole a group * is an asymmetric carbon atom .A ^- the counter anion Using an optically active pyrrolidine salt derivative represented by the following formula (6):

The method for producing ramelteon according to [1], wherein the method is performed using a Hantzsh ester represented by the formula (1) as a reducing agent.

[3] In the optically active pyrrolidine salt derivative (5), R is a diphenylmethyl group, a (trimethylsilyloxy) diphenylmethyl group, or a (hydroxy) diphenylmethyl group, the absolute configuration is R, and A ^- is The method for producing ramelteon according to [2], which is trichloroacetate or trifluoroacetate.

[4] The method for producing ramelteon according to [1], wherein the asymmetric reduction is performed using enone reductase.

[5] The method for producing ramelteon according to [4], wherein the enone reductase is OYE2, NemA, YersER, or NCR-R.

で表される光学活性アミンに変換した後、これをプロピオニル化することを特徴とする、［１］〜［５］のいずれかに記載のラメルテオンの製造法。[6] The optically active aldehyde (2) is represented by the following formula (3):

The method for producing ramelteon according to any one of [1] to [5], wherein after converting into an optically active amine represented by formula (1), this is propionylated.

[7] The method for producing ramelteon according to [6], wherein the method for converting into the optically active amine (3) is performed using a transaminase in the presence of an amino group donor.

[8] The method for producing ramelteon according to [7], wherein the amino group donor is isopropylamine, and the transaminase is ω-transaminase.

[9] The method for producing ramelteon according to [7], wherein the amino group donor is α-phenethylamine, and the transaminase is ω-transaminase.

[10] The method for producing ramelteon according to [7], wherein the amino group donor is an α-amino acid, and the transaminase is an ω-transaminase.

[11] The method for producing ramelteon according to any one of [7] to [10], wherein the transaminase is reacted in an organic solvent.

[12] The method for producing ramelteon according to any one of [7] to [11], wherein the transaminase immobilized on a resin is reacted in an organic solvent.

で表されるα，β−不飽和ニトリルとヒドリド還元剤を反応させて製造したものである、［１］〜［１２］のいずれかに記載のラメルテオンの製造法。[13] The enal (1) is represented by the following formula (7):

The method for producing ramelteon according to any one of [1] to [12], which is produced by reacting an α, β-unsaturated nitrile represented by the formula (1) with a hydride reducing agent.

で表されるエナール（（Ｅ）−２−［１，２，６，７−ｔｅｔｒａｈｙｄｒｏ−８Ｈ−ｉｎｄｅｎｏ［５，４−ｂ］ｆｕｒａｎ−８−ｙｌｉｄｅｎｅ］ａｃｅｔａｌｄｅｈｙｄｅ）。[14] The following formula (1);

((E) -2- [1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde).

  According to the present invention, ramelteon can be easily and efficiently produced.

The temporal stability of the conversion rate in the production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine using an enzyme flow reactor was investigated. It is a graph shown.

  Hereinafter, the method for producing ramelteon according to the present invention will be described in detail.

  First, raw materials, intermediate products, and target products used in the production method of the present invention will be described.

  As the starting material of the present invention, for example, an α, β-unsaturated nitrile represented by the following formula (7), that is, (E)-(1,6,7,8-tetrahydro-2H-indeno [5,4- b] furan-8-ylidene) acetonitrile can be used. Here, for example, the compound (7) is a carbanion prepared from diethyl cyanomethylphosphonate and sodium hydride according to the method described in Japanese Patent No. 4081161, and 1,2,6,7-tetrahydro-8H-indeno [ It can be easily produced by reacting 5,4-b] furan-8-one.

  Further, enal, 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 described below. It is represented by equation (1). Here, the compound (1) is a novel compound not described in the literature.

  The optically active aldehyde which is an intermediate product of the present invention, that is, (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde is described below. It is represented by equation (2).

  The optically active amine, ie, (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine, which is an intermediate product of the present invention, is , And is represented by the following equation (3).

  The product of the present invention, ramelteon, 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 equation (4).

  Preferred embodiments of the present invention are shown in the drawings as follows. Also, in the present application, “the production of the optically active aldehyde represented by the formula (2) and the subsequent conversion to the ramelteon represented by the formula (4)” includes the step 3 from the optically active aldehyde (2). And a method of producing ramelteon (4) from optically active aldehyde (2) through steps 4 and 5 from the optically active aldehyde (2).

  The steps will be described below in order.

(Step 1)
In this step, the enal represented by the above formula (1) is produced by reacting the α, β-unsaturated nitrile represented by the above formula (7) with a hydride reducing agent.

  Examples of the hydride reducing agent include lithium aluminum hydride, diisobutyl aluminum hydride, sodium bis (2-methoxyethoxy) aluminum hydride, lithium tri (tert-butoxy) aluminum hydride, borane, lithium borohydride, and hydrogen hydride. Examples include sodium borohydride, potassium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, lithium triethylborohydride, catecholborane, and the like, and preferred is diisobutylaluminum hydride.

  The upper limit of the amount of the hydride reducing agent used is preferably 10 times the molar amount of the compound (7), and more preferably 5 times the molar amount. The lower limit is 0.5-fold molar amount, more preferably 1-fold molar amount, relative to compound (7).

  The reaction solvent in this step is not particularly limited as long as it does not affect the reaction, and specifically, for example, tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol dimethyl ether Solvents such as pentane, hexane, heptane and methylcyclohexane; aromatic hydrocarbon solvents such as benzene, toluene, xylene, ethylbenzene and mesitylene; N, N Amides such as -dimethylformamide, N, N-dimethylacetamide, N, N-diethylacetamide, N-methyl-2-pyrrolidone, N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, and hexamethylphosphoramide Solvent; urea and dimethyl propylene urea solvent; phosphonic acid triamide system such as hexamethylphosphoric triamide solvent or the like can be used. These may be used alone or in combination of two or more. When two or more kinds are used in combination, the mixing ratio is not particularly limited. Preferred is an aromatic hydrocarbon solvent, and more preferred is tetrahydrofuran, 4-methyltetrahydropyran, or toluene.

  The upper limit of the amount of the solvent used is preferably 100 times, more preferably 50 times, and particularly preferably 20 times, the weight of the compound (7). The lower limit is preferably 0.1-fold weight, more preferably 0.5-fold weight, and particularly preferably 1-fold weight, relative to compound (7). In such a range, the cost is not too high, and the post-processing is simple.

  The reaction temperature in this reaction is not particularly limited and may be set as appropriate. However, in order to reduce the generation of by-products, 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, but 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 in this step is not particularly limited, but it is preferable to add a hydride reducing agent to a solution containing the compound (7) and a solvent.

  As the treatment after the reaction, a general treatment for obtaining a product from the reaction solution may be performed. For example, when the reaction solution after 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 common extraction solvent, for example, ethyl acetate, diethyl ether, methylene chloride, toluene, hexane, or the like. Good. The desired product is obtained by distilling off the reaction solvent and the extraction solvent from the obtained extract by operations such as heating under reduced pressure and vacuum drying, or by using a drying agent such as anhydrous magnesium sulfate. The target product thus obtained has a sufficient purity that can be used in the subsequent steps, but for the purpose of further increasing the purity, crystallization, fractional distillation, column chromatography, and other general purification techniques. The purity may be further increased.

(Step 2)
In this step, the optically active aldehyde represented by the formula (2) is produced by asymmetric reduction of the enal represented by the formula (1). Here, the enal (1) may be produced according to the method described in Step 1, or even if produced by any other method 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.

  First, a method of using a reducing agent in the presence of an asymmetric organic catalyst will be described.

Examples of the asymmetric organic catalyst include (2S, 5S) -2-tert-Butyl-3-methyl-5-benzyl-4-oxoimidazolidinium trichloroacetate, (R) -2- (tert-Butyl) -3-methyl- 4-oxoimidazolidinium trichloroacetate, (2S, 5S) -5-Benzyl-3-methyl-2- (5-methyl-2-furyl) -2-imidazolidinium trichloroacetate, (5S) -2,3-trimethylate phenylmethyl-4-oxoimidazolidinium trichloroacetate, (2S, 5S) -2-tert-Butyl- -Methyl-5-benzyl-4-oxoimidazolidinium trifluoroacetate, (R) -2- (tert-Butyl) -3-methyl-4-oxoimidazolidinium trifluoroacetate, (-2-S-yl-, 5-S-yl-, 5-S-yl-, 5-S-yl-S-yl-S- (2-S-yl)-(2-S-yl) 5-methyl-2-furyl) -2-imidazolidinium trifluoroacetate, (5S) -2,2,3-Trimethyl-5-phenylmethyl-4-oxoimidazolidinium trifluoroacetate, and the like.
And the following formula (5):

And the like. Preferred are optically active pyrrolidine salt derivatives represented by the formula (5).

  Here, in the formula (5), R represents 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, or 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, and may have a substituent. An alkyl group having 1 to 20 (more preferably 1 to 10 carbon atoms), a carboxyl group, or a 1H-tetrazole group is preferred. Examples of the substituent 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 and a piperidinyl group; and these substituents may be used alone or in combination of two or more.

  R is preferably methyl, ethyl, isopropyl, methoxymethyl, (1-pyrrolidinyl) methyl, vinyl, benzyl, phenyl, diphenylmethyl, (trimethylsilyloxy) diphenylmethyl, (hydroxy) It is 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.

  In the formula (5), * represents an asymmetric carbon atom, and the absolute configuration may be either an S configuration or an R configuration, and preferably the absolute configuration is an R configuration.

In the formula (5), 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. Further, a counter anion supported by a polymer may be used, and specific examples include a cation exchange resin in which the counter anion is a sulfonate.

  In addition, the optically active pyrrolidine salt derivative, the optically active pyrrolidine in the reaction system, hydrogen chloride, hydrogen bromide, sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, trichloroacetic acid, It may be prepared by mixing acids such as trifluoroacetic acid, acetic acid and 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, particularly preferably 3 times the weight. The lower limit is preferably 0.001 times by weight, more preferably 0.01 times by weight, and particularly preferably 0.1 times by weight, relative to the compound (1).

  The upper limit of the amount of the asymmetric organic catalyst to be used is preferably 10-fold weight, more preferably 5-fold weight, and particularly preferably 1-fold weight, relative to the compound (1). The lower limit is preferably 0.001 times by weight, more preferably 0.01 times by weight, and particularly preferably 0.1 times by weight, relative to the compound (1). In such a range, the cost is not too high, and the post-processing is simple.

  Examples of the reducing agent include hydrogen gas, formic acid, sodium formate, triethylammonium formate, and a 1,4-dihydropyridine derivative, and preferably a 1,4-dihydropyridine derivative. More preferably, the 1,4-dihydropyridine derivative has the following formula (6):

Is a Hantzsh ester represented by the formula: Diethyl 1,4-Dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate.

  The upper limit of the amount of the reducing agent used is preferably 100 times, more preferably 50 times, and particularly preferably 10 times the weight of the compound (1). The lower limit is preferably 0.1-fold weight, more preferably 0.5-fold weight, and particularly preferably 1-fold weight, relative to the compound (1). In such a range, the cost is not too high, and the post-processing is simple.

  The solvent in this step is not particularly limited as long as it does not affect the reaction, and specifically, for example, water; methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, ethylene glycol and the like Alcohol solvents such as tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, 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 pentane, hexane, heptane and methylcyclohexane; benzene, toluene, xylene and ethylbenzene Aromatic solvents such as methylene chloride and mesitylene; halogen solvents such as methylene chloride and 1,2-dichloroethane; sulfoxide solvents such as dimethyl sulfoxide; N, N-dimethylformamide, N, N-dimethylacetamide; Amide solvents such as N-diethylacetamide, N-methyl-2-pyrrolidone, N-ethyl-2-pyrrolidone, N-methyl-ε-caprolactam, hexamethylphosphoramide; urea solvents such as dimethylpropyleneurea; A phosphonic acid triamide-based solvent such as methylphosphonic acid triamide can be used. These may be used alone or in combination of two or more. When two or more kinds are used in combination, the mixing ratio is not particularly limited. Preferred are ether solvents; ester solvents; aromatic hydrocarbon solvents; and nitrile solvents, more preferably tetrahydrofuran, 4-methyltetrahydropyran, ethyl acetate, toluene, or acetonitrile, and particularly preferably tetrahydrofuran and toluene. Or acetonitrile.

  The upper limit of the amount of the solvent used is preferably 100 times, more preferably 50 times, and particularly preferably 20 times, the weight of the compound (1). The lower limit is preferably 0.1-fold weight, more preferably 0.5-fold weight, and particularly preferably 1-fold weight, relative to the compound (1). In such a range, the cost is not too high, and the post-processing is simple.

  The reaction temperature in this reaction is not particularly limited and may be appropriately set, but in order to reduce the generation of by-products, 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, but 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 (1), the asymmetric organic catalyst, the reducing agent, and the solvent in this step is not particularly limited.

  As the treatment after the reaction, a general treatment for obtaining a product from the reaction solution may be performed. For example, when the reaction solution after 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 common extraction solvent, for example, ethyl acetate, diethyl ether, methylene chloride, toluene, hexane, or the like. Good. When the reaction solvent and the extraction solvent are distilled off from the obtained 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, but for the purpose of further increasing the purity, crystallization, fractional distillation, column chromatography, and other general purification techniques. The purity may be further increased.

  Subsequently, a method using enone reductase will be described.

  Production of an optically active aldehyde represented by the above formula (2) by allowing enone reductase having the ability to stereoselectively reduce the carbon-carbon double bond of the enal represented by the above formula (1) to act. I 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 an optically active aldehyde (2). .

  The enone reductase may be not only the enzyme itself, but also a cell of a microorganism that produces the enzyme, a culture solution of the microorganism, or a treated product of the microorganism, as long as it has the desired reducing activity. It also includes a transformant into which a DNA encoding an enzyme having a reducing activity derived from the microorganism is introduced.

  The treated cells of the microorganisms are not particularly limited, and include, for example, dried cells, treated with a surfactant, treated with a lytic enzyme, immobilized cells or a cell-free extract obtained by crushing the cells. it can. Furthermore, an enzyme that catalyzes an asymmetric reduction reaction may be purified from a 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.

  As the enone reductase having the ability to stereoselectively reduce the carbon-carbon double bond at the enone site of the enal (1) to produce an optically active aldehyde (2), an old yellow color which is generally recognized as an enone reductase The enzyme includes an enzyme (Old Yellow Enzyme), and examples of such an enzyme include an oxidoreductase classified into EC 1.6.99 according to the enzymatic classification method of the International Union of Biochemistry and Molecular Biology. As oxidoreductases classified into EC1.6.99, EC1.6.9.11: NADPH dehydrogenase, EC1.6.9.2: NAD (P) H dehydrogenase (quinone), EC1.6.99. 3: an oxidoreductase classified as NADH dehydrogenase, EC 1.6.99.5: NADH dehydrogenase (quinone), or EC 1.6.99.6: NADPH dehydrogenase (quinone), preferably EC 1.6. 0.99.1: NADPH dehydrogenase.

  As the above-mentioned EC 1.6.99.1: NADPH dehydrogenase, specifically, genus Candida, genus Kluyveromyces, genus Saccharomyces, or genus Schizosaccharomyces, etc. And those derived from bacteria such as genus Bacillus, genus Escherichia, genus Pseudomonas, genus Yersinia, genus Zymomonas, and preferably Saccharomyces macesa saccharomyces maca. ), Kluyveromyces, Bacillus, Eche Examples include those derived from microorganisms such as the genus Escherichia, the genus Pseudomonas, the genus Yersinia, the genus Zymomonas, and the most preferred examples are Saccharomyces cerevisiae and Saccharomyces cerevisiae. Seth lactis (Kluyveromyces lactis), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Yersinia Berukobieri (Yersinia bercovieri), Zymomonas mobilis (Zymomonas mobilis) Is included.

  As the enone reductase, for example, OYE2, OYE3 (from Saccharomyces cerevisiae; described in WO 2006/129628), and KYE (from Kluyveromyces lactisCath. , 349, 1521 (2007)), YqjM (derived from Bacillus subtilis; described in J. Biol. Chem., 278, 19891 (2003)), NemA (Escherichia coli). Biol. Pharm. Bull. 20, 110 (1997)), XeaA, XenE, etc. (Pseudomonas putida) Pseudomonas putida), described in Appl.Environ.Microbiol., 74, 6703 (2008), and YersER (derived from Yersinia bercovieri; described in Adv. Synth. Catal., 349, Adv. Synth. NCR-R (derived from Zymomonas mobilis; described in Biotechnol. Bioeng., 98, 22 (2007)) and the like, preferably OYE2, NemA, YersER, or NCR-R, and more preferably. Is NemA, YersER, or NCR-R, and more preferably NemA.

The microorganism that produces the enzyme can usually be obtained from a stock that is easily available or purchased. For example, it is available from the following culture collection:
-Incorporated Administrative Agency National Institute of Technology and Evaluation Biotechnology Division, Biological Genetic Resources Division (NBRC) (2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba 292-0818)
-RIKEN BioResource Center, Microbial Material Development Section (JCM) (2-1-1, Hirosawa, Wako-shi, Saitama 351-0198)
・ German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Marschorder Weg 1b, D-38124 Brunswick, Germany)

  The enone reductase (for example, OYE2) used in the present invention has a known amino acid sequence (for example, J. Biol. Chem. 268, 6097-6106 (as long as it has the desired reductase activity). 1993), one or more (for example, 40, preferably 20, more preferably 15, more preferably 10, more preferably 5, 4, 3, Or two or less) amino acids may be substituted, inserted, deleted and / or added polypeptides. Alternatively, when the amino acid sequence is compared with a known sequence, the homology is 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, further preferably 98% or more, or It may have an amino acid sequence of 99% or more. Further, an additional amino acid sequence may be bound to the amino acid sequence of the reductase used in the present invention, as long as it has the desired reductase activity. For example, a tag sequence such as a histidine tag or an HA tag can be added. Alternatively, it can be a fusion protein with another protein. Peptide fragments may be used as long as they have the desired reductase activity.

  In the present invention, when a transformant containing DNA encoding enone reductase is used, the optically active aldehyde (2) can be produced more efficiently. Unless otherwise specified, genetic manipulations such as DNA isolation, vector preparation, and transformation described in the present specification are described in Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular biology (Green Publishing Associates and Wiley-Interscience) can be carried out by a method described in a written document such as Molecular Biology.

  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 a vector include a plasmid vector, a phage vector, a cosmid vector, and the like, and a shuttle vector capable of gene exchange with another host strain can also be used.

Such a vector usually contains control factors such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter and the like in the case of Escherichia coli, and is operable with the DNA of the present invention. It can be suitably used as an expression vector containing an expression unit linked to the expression vector. For example, pSTV28 (manufactured by Takara Bio Inc.), pUCNT (WO 94/03613) and the like can be mentioned.
The “regulator” refers to a base sequence having a functional promoter and any associated transcription elements (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  “Operably linked” means that various regulatory elements such as a promoter and an enhancer that regulate the expression of a gene and the gene are operably linked in a host cell. It is well known to those skilled in the art that the type and kind of the control factor can be changed depending on the host.

  The host organism used to express each enzyme is not particularly limited as long as it can be transformed with an enzyme expression vector containing DNA encoding each enzyme and can express the enzyme into which the DNA has been introduced. . Among these, bacteria are preferable from the viewpoint of introduction and expression efficiency, and Escherichia coli (Escherichia coli) is particularly preferable.

  The vector containing the DNA encoding the reductase used in the present invention can be introduced into a host microorganism by a known method. For example, when Escherichia coli is used as a host microorganism, the vector is introduced into a host cell by using a commercially available Escherichia coli HB101 (hereinafter, E. coli HB101) competent cell (manufactured by Takara Bio Inc.). can do.

  As an example of a transformant containing a DNA encoding an enzyme that reduces the carbon-carbon double bond at the enone site of enal (1), E. coli is transformed with vector pTSYE2. E. coli HB101 obtained by transformation. E. coli HB101 (pTSYE2), obtained in a similar manner. coli HB101 (pTSYE3), E. coli. coli HB101 (pNKYE), E. coli. E. coli HB101 (pNYqjM); E. coli HB101 (pNNemA); E. coli HB101 (pNXenA); coli HB101 (pNXenE); E. coli HB101 (pNYersER); coli HB101 (pNNCR-R) and the like.

  Further, in the present invention, by using a transformant containing both a DNA encoding an enzyme having a desired reducing activity and a DNA encoding a polypeptide having a coenzyme regeneration ability, more efficient use is possible. The optically active compound of the present invention can be produced. For example, a transformant containing both a DNA encoding an enzyme that reduces a carbon-carbon double bond at the enone site of an enal (1) and a DNA encoding a polypeptide capable of regenerating a coenzyme, 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 ability to regenerate a coenzyme are incorporated into the same vector, and these are inserted into a host cell. In addition, these two types of DNAs can be obtained by incorporating these two types of DNAs into two types of vectors having different incompatibility groups, respectively, and introducing the two types of vectors into the same host cell. When a host cell that releases the enzyme outside the cell is used, or when a crushed solution of the host cell is used for the reaction, 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 coenzyme regeneration ability may be introduced into different host cells, respectively, and these host cells may be cultured in the same culture medium or using different culture mediums.

As the polypeptide having a coenzyme regeneration ability, NAD ⁺ (oxidized nicotinamide / adenine dinucleotide) or NADP ⁺ (oxidized nicotinamide / adenine dinucleotide phosphate) is converted to NADH (reduced nicotinamide / adenine dinucleotide). Alternatively, an oxidoreductase capable of converting to NADPH (reduced nicotinamide / adenine dinucleotide phosphate) is preferable.
Examples of such enzymes include hydrogenase, formate dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase. Preferably, formate dehydrogenase and glucose dehydrogenase are used.

  Examples of the formate dehydrogenase include, for example, Candida, Kloeckera, Pichia, Lipomyces, Pseudomonas, Moraxella, and Hoaxella hum. (Hyphomicrobium), Paracoccus, Thiobacillus, Ancylobacter, and the like, and particularly enzymes derived from microorganisms, such as Thiobacillus sp., Which is obtained from the enzyme Thiobacillus sp. No.

  Examples of the glucose dehydrogenase include enzymes derived from microorganisms such as Bacillus, Lactobacillus, and Pediococcus, and in particular, Bacillus megaterium and Lactobacillus plan. Examples include enzymes obtained from taram (Lactobacillus plantarum), Lactobacillus pentosus (Lactobacillus pentosus), and Pediococcus parvulus.

  Examples of the enzyme include Cryptococcus sp. (JP-A-2006-262767), Gluconobacter sp. (J. Bacteriol., 184, 672-678, (2002)), and Saccharomyces. Glucose dehydrogenase and Cryptococcus derived from the genus (Methods Enzymol., 89, 159-163, (1982)), the genus Lactobacillus, the genus Pediococcus (WO 2009/041415). Glucose-6-phosphorus derived from the genera Aspergillus, Pseudomonas Dehydrogenase are known (Arch. Biochem. Biophys., 228,113-119 (1984)).

  Next, the reduction reaction of enal (1) using enone reductase will be described.

  In the case of the reduction reaction using enone reductase, an appropriate solvent and an enzyme source such as the substrate enal (1), the microorganism, its culture, or its processed product are mixed, and the mixture is stirred and shaken while adjusting the pH. Or leave it still.

In order to promote the reduction reaction of enal (1) using enone reductase, an enzyme for reducing NAD ⁺ and / or NADP ⁺ to each reduced form and a substrate for the reduction are co-present in the reaction solution. It is preferred to carry out the reaction. For example, glucose dehydrogenase is used as an enzyme to reduce to a reduced form, and glucose is used as a substrate for reduction, or formate dehydrogenase is used as an enzyme to reduce to a reduced form, and formic acid is used as a substrate for reduction. It is good to coexist.

  In general, the reaction can be promoted by adding a coenzyme such as NADH or NADPH required for a reduction reaction by a biological method. In this case, these are usually added directly to the reaction solution.

Furthermore, adding a surfactant such as Triton (manufactured by Nakarai Tesque Co., Ltd.), Span (manufactured by Kanto Kagaku Co., Ltd.), or Tween (manufactured by Nacalai Tesque Co., Ltd.) to the reaction solution also promotes the reduction reaction. It is effective.
Further, an organic solvent may coexist in the reaction system. If a water-insoluble organic solvent such as ethyl acetate, butyl acetate, isopropyl ether, toluene, hexane, or the like is added to the reaction solution, the inhibition of the reaction by the substrate and / or the alcohol product that is a 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.

  As a reaction solvent, an aqueous medium such as water or a buffer is usually used. Examples of the buffer include a potassium phosphate buffer and a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer.

  As the enzyme source, a culture solution containing cells of the above microorganisms is usually used as it is for the reaction. The culture solution may be used after being concentrated. When components in the culture solution have an adverse effect on the reaction, it is preferable to use cells obtained by processing the culture solution by centrifugation or the like, or treated cells.

  The substrate, enal (1), may be added all at once in the early stage of the reaction, or may be added in divided portions as the reaction progresses.

  The upper limit of the reaction temperature is preferably 60 ° C, 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, and more preferably 9 as an upper limit. 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 is preferably 50% (W / V) as an upper limit, and more preferably 10% (W / V). is there. The lower limit is preferably 0.01% (W / V), and more preferably 0.1% (W / V).

  The upper limit of the substrate concentration in the reaction solution is preferably 50% (W / V), and more preferably 30% (W / V). The lower limit is preferably 0.01% (W / V), and more preferably 0.1% (W / V).

  The reaction is usually carried out while shaking or stirring with aeration.

  The reaction time is appropriately determined depending on the substrate concentration, the amount of the microorganism, and other reaction conditions. The upper limit of the reaction time is preferably 336 hours, more preferably 168 hours. The lower limit is preferably 0.1 hour, and more preferably 1 hour.

  In order to promote the reduction reaction, it is preferable to add an energy source such as glucose, ethanol, or isopropanol to the reaction solution. The upper limit of the amount added is preferably 50% (W / V), and more preferably 30% (W / V). The lower limit is preferably 0.1 (W / V), and more preferably 0.5% (W / V).

  The method for removing the optically active aldehyde (2) generated by the reduction reaction is not particularly limited. Ethyl acetate, toluene, t-butyl methyl ether, hexane, n-butanol, directly from the reaction solution or after separating cells and the like, Extraction with a solvent such as dichloromethane, dehydration, and purification by distillation, silica gel column chromatography, or the like can easily provide a highly pure optically active aldehyde (2).

  The optically active aldehyde (2) thus obtained is included in the scope of the present invention even if it is converted into ramelteon (4) using any conversion method. Preferably, a method of condensing the optically active aldehyde (2) with propionic amide and reducing it (Step 3), or converting the optically active aldehyde (2) to an optically active amine (3) (Step 4), followed by propyronylation (Step 5). Hereinafter, each step will be described in detail.

(Step 3)
In this step, the optically active aldehyde represented by the formula (2) is condensed with propionic amide and reduced to produce the ramelteon represented by the formula (4). Specifically, for example, Adv. Synth. Catal 2013,355,717-733. In a diglyme solution in the presence of propionamide, [Rh (COD) Cl] ₂ as a metal catalyst, and Xantphos (4,5-Bis (diphenylphosphino) -9,9-dimethylxanthene) as a ligand, The desired product is obtained by adding hydrogen gas.

  The target product obtained in this way has sufficient purity, but for the purpose of further increasing the purity, crystallization, fractional distillation, column purification and other general purification techniques, to further increase the purity. Is also good.

(Step 4)
In this step, an optically active amine represented by the above formula (3) is produced from the optically active aldehyde represented by the above formula (2). Here, the optically active aldehyde (2) may be produced according to the method described in Step 2, or even if produced by any other method 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 method using hydrogenation 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). Method.

  The method using ammonia and a hydride reducing agent is described, for example, in J. Am. Org. Chem. , 2010, 75, 5470-5577. May be followed. That is, the target product can be obtained by adding aqueous ammonia and ammonium acetate to an ethanol solvent and reducing it with sodium cyanoborohydride.

  The method for hydrogenation in the presence of ammonia and a metal catalyst may be, for example, the method described in Japanese Patent No. 4059978. That is, the target product can be obtained by adding ammonia gas and hydrogen gas in the presence of a homogeneous transition metal catalyst such as nickel, rhodium, palladium, and iridium. Or, Catalysts 2015, 5, 2258-2270. In other words, the desired product can be obtained by adding ammonia gas and hydrogen gas in the presence of a ruthenium catalyst supported on alumina.

  Further, the method of using formic acid in the presence of the metal catalyst may be, for example, according to the method described in WO 2016/096905. That is, the target product can be obtained by using ammonium formate in the presence of a palladium catalyst.

  Further, a method for transamination in the presence of an enzyme (transaminase) will be described. That is, a transaminase having an ability to generate an 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, whereby An optically active amine represented by the formula (3) is produced.

  Any transaminase may be used as long as it has an ability to generate an optically active amine (3) from an optically active aldehyde (2) in the presence of an amino group donor. Further, the transaminase may be not only the enzyme itself, but also a cell itself of a microorganism producing the enzyme, a culture solution of the microorganism, or a processed product of the cell, as long as it has a desired transamination activity. . It also includes a transformant into which a DNA encoding an enzyme having a reducing activity derived from the microorganism is introduced.

  The treated cells of the microorganisms are not particularly limited, and include, for example, dried cells, treated with a surfactant, treated with a lytic enzyme, immobilized cells or a cell-free extract obtained by crushing the cells. it can. Furthermore, an enzyme that catalyzes an asymmetric reduction reaction may be purified from a 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 includes a carrier binding method, a cross-linking method, and an entrapping method, and any of them may be used.

  Further, the transaminase may be reacted in water, or may be reacted in an organic solvent, that is, the reaction may be carried out using an organic solvent in the reaction system or using only the organic solvent. When only an organic solvent is used, it is expected that the reactivity will be improved by adding a small amount of water. When a solvent is used, examples of the solvent include alcohol solvents such as glycerol, ethylene glycol, methanol, ethanol, 1-propanol and 2-propanol; sulfoxide solvents such as dimethyl sulfoxide; and amide solvents 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; Ketone solvents such as butanone and methyl isobutyl ketone; diethyl ether, dinormal propyl ether, diisopropyl ether, dinormal butyl ether, methyl isopropyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, cyclo Ether solvents such as ethyl methyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane; nitrile solvents such as acetonitrile and propionitrile; halogen solvents such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane And the like. 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; and more preferred solvent is methyl-tert-butyl ether.

  Examples of the transaminase having the ability to generate an optically active amine (3) from an optically active aldehyde (2) in the presence of an amino group donor include, for example, EC 2.6 according to the enzymatic classification method of the International Union of Biochemistry and Molecular Biology. 1.1. An enzyme group classified as X is included.

  In a particularly preferred embodiment of the present invention, the transaminase is preferably what is commonly referred to as ω-transaminase. ω-transaminase refers to one having an activity of generating an amine other than an α-amino acid.

  As the ω-transaminase, specifically, the genus Arthrobacter, the genus Bacillus, the genus Pseudomonas, the genus Vibrio, the genus Alcaligenes, the genus Mesorhizobium Examples include bacteria derived from bacteria such as the genus Chromobacterium, the genus Caulobacter, and the genus Rhodobacter.

  Preferably, Arthrobacter sp., Bacillus megaterium, Pseudomonas sp., Vibrio fluvialis, Vibrio itroes genus resectus, Alstrobac resactus sect. -Denetrificans (Alcaligenes denitrificans), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas corrugata (Pseudomonas corrugata), Mesorhizobium sp. Seumu (Chromobacterium violaceum), Caulobacter-Kuresentasu (Caulobacter crescentus), and the like Rhodobacter Sufaeroidesu (Rhodobacter sphaeroides) is.

  The most preferred examples are Arthrobacter sp. KNK168 (Arthrobacter sp. KNK168), Bacillus megaterium, Bacillus megaterium, Pseudomonas sp. KNK425, Pseudomonas sp. -Vibrio fluvialis JS17, Arthrobacter citreus, Alcaligenes denitrificans Y2k-2 (Alcaligenes denitrificans Y2k-2), Pseudomonas fluorescens K8P8-K8-N8K-N8K-K8N-K8K-N8K-N8K-K8N-K8-K8-K8-K8N-K8-K8K-N8K-K8N-K8-K8N-K8-K8K-K8N8K-K8N8K-K8PK8K-K8PK8K-K8PK8K-K8N8K-K8PK8N-K8P-K8PK8K-K8K-K8N8K-K8N8K-K8PK8N8K-K8PK8N8K8K-K18P-K8N-K8_K8_K8_K8_K8_8 luorescens KNK08-18), Pseudomonas Korugata 10F6 (Pseudomonas corrugata 10F6), Mesorhizobium sp LUK (Mesorhizobium sp.LUK), Chromobacterium violaceum DSM 30191 (Chromobacterium violaceum DSM 30191), Bacillus megaterium SC6394 (Bacillus megaterium SC6394) And Caulobacter crescentus CB15, and Rhodobacter sphaeroides DSM 158 (Rhodobacter sphaeroides DSM 158).

  These ω-transaminases are described, for example, in Iwasaki et al. , Biotechnol. Lett. (2003) 25, 1843-1846, Shin et al. , Biotechnol. Bioeng. (1997) 55, 348-358, Shin and Kim, Book of Abstracts, 217th ACS National Meeting, Anaheim, Calif. , March 21-25, (1999) 180, Shin and Kim, Biosc. Biotechnol. Biochem. (2001) 65, 1782-1788 and Shin and Kim, Biotechnol. Bioeng. (1998) 60, 534-540, M.P. Shaheer et al. , Appl. Microbiol. Biotechnol. (2012) 94, 1163-1171, Sam et al. , ACS catal. (2012) 2,993-1001, Rudat et al. , AMB Express (2012) 2:11, Par et al. , Biotechnol. Bioeng. , (2011) 108, 1479-1493, International Publication No. 2012/1224639, International Publication No. 2012/043653, International Publication No. 2012/043622, International Publication No. 2012/007548, International Publication No. 2011/026556, and the like. It is described in.

These microorganisms can usually be obtained from a stock that is easily available or purchased. For example, it is available from the following culture collection:
-Incorporated Administrative Agency National Institute of Technology and Evaluation Biotechnology Division, Biological Genetic Resources Division (NBRC) (2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba 292-0818)
-RIKEN BioResource Center, Microbial Material Development Section (JCM) (2-1-1, Hirosawa, Wako-shi, Saitama 351-0198)
・ German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) (Marschorder Weg 1b, D-38124 Brunswick, Germany)

  In addition, as long as the transaminase used in the present invention has the desired transamination activity, one or more transaminases (for example, 40, preferably 20, more preferably It may be a polypeptide in which 15, more preferably 10, more preferably 5, 4, 3, or 2 or less amino acids have been substituted, inserted, deleted and / or added. Alternatively, the amino acid sequence has a homology of 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, further preferably 98% or more, or 99% or more with a known amino acid sequence. It may have the above amino acid sequence. In addition, an additional amino acid sequence may be bound to the amino acid sequence of the reductase used in the present invention as long as it has transamination activity. For example, a tag sequence such as a histidine tag or an HA tag can be added. Alternatively, it may be a fusion protein with another protein. Further, as long as it has the desired reductase activity, it may be a peptide fragment.

  In the present invention, when a transformant containing a DNA encoding transaminase is used, the optically active amine (3) can be produced more efficiently. Unless otherwise specified, genetic manipulations such as DNA isolation, vector preparation, and transformation described in the present specification are described in Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular biology (Green Publishing Associates and Wiley-Interscience) can be carried out by a method described in a written document such as Molecular Biology.

  The vector used for the above 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 a vector include a plasmid vector, a phage vector, a cosmid vector, and the like, and a shuttle vector capable of gene exchange with another host strain can also be used.

For example, in the case of Escherichia coli, such a vector usually contains regulatory factors such as a lacUV5 promoter, a trp promoter, a trc promoter, a tac promoter, an lpp promoter, a tufB promoter, a recA promoter, and a pL promoter. It can be suitably used as an expression vector containing an expression unit linked as possible. For example, pSTV28 (manufactured by Takara Bio Inc.), pUCNT (WO 94/03613) and the like can be mentioned.
The “regulator” refers to a base sequence having a functional promoter and any associated transcription elements (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  “Operably linked” means that various regulatory elements such as a promoter and an enhancer that regulate the expression of a gene and the gene are operably linked in a host cell. It is well known to those skilled in the art that the type and kind of the control factor can be changed depending on the host.

  The host organism used to express each enzyme is not particularly limited as long as it can be transformed with an enzyme expression vector containing DNA encoding each enzyme and can express the enzyme into which the DNA has been introduced. . Among these, bacteria are preferable from the viewpoint of introduction and expression efficiency, and Escherichia coli (Escherichia coli) is particularly preferable.

  The vector containing the DNA encoding the reductase used in the present invention can be introduced into a host microorganism by a known method. For example, when Escherichia coli is used as a host microorganism, the vector is introduced into a host cell by using a commercially available Escherichia coli HB101 (hereinafter, E. coli HB101) competent cell (manufactured by Takara Bio Inc.). can do.

  Since an amination reaction using a transaminase is generally a reversible reaction, the reaction generally stops apparently at an equilibrium point. By combining these known methods for eliminating the reaction equilibrium, the reaction using the polypeptide of the present invention can be improved. For example, as described in WO 2007/1399055, by using alanine as an amino group donor, by-product pyruvate 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. Similarly, by using alanine as an amino group donor, pyruvic acid by-produced is removed with pyruvate decarboxylase (WO 2007/093372), and a method using alanine dehydrogenase (US2009 / 0117627A1) (US2008 / 0213845A1), a method using acetobutyric acid synthase (Biosci. Biotechnol. Biochem. 72 (11), 3030-3033 (2008)), and acetone produced using isopropylamine. The removal method is also effective.

  When a plurality of enzymes are conjugated to eliminate the above reaction equilibrium, by using a transformant that produces the plurality of enzymes in the same host cell, the optically active compound of the present invention can be more efficiently used. Can be manufactured. The transformant can be obtained by incorporating DNAs encoding a plurality of necessary enzyme genes into the same vector and introducing the same into the same host cell. In addition, these two types of DNAs can be used in a plurality of vectors having different incompatibility groups. And introduced into the same host cell.

  Any amino group donor can be used as long as it produces the optically active amine represented by the formula (3) in the presence of an enzyme source. Specific examples include α-phenethylamine, 2-butylamine, 2-pentylamine, 2-heptylamine, 3-heptylamine, n-ethylamine, n-propylamine, n-butylamine, n-amylamine, isopropylamine, isobutylamine , Α-amino acids (eg, glycine, alanine, etc.), 3-amino-1-phenylbutane, benzylamine, β-phenethylamine, cyclohexylamine, and optically active forms thereof. Among them, α-phenethylamine, isopropylamine, or α-amino acid (for example, alanine) is preferable, and α-phenethylamine or α-amino acid is more preferable.

Preferred combinations of ω-transaminase and amino group donor include the following.
1) When the amino group donor is isopropylamine, preferably, it is Arthrobacter sp., Pseudomonas sp., Vibrio fluvialis, or Pseudomonas fluorescens (Pseudomonas fluorescens). fluorescens), more preferably Arthrobacter sp.
2) When the amino group donor is α-phenethylamine, it is preferably Arthrobacter sp., Pseudomonas sp., Vibrio fluvialis, or Pseudomonas fluorescens (Pseudomonas sp.). Pseudomonas fluorescens, more preferably Arthrobacter sp., Pseudomonas sp., Or Pseudomonas fluorescens, and Pseudomonas fluorescens, and Pseudomonas s. .).
3) When the amino group donor is an α-amino acid (particularly alanine), preferably, it is Arthrobacter sp., Pseudomonas sp., Vibrio fluvialis, or Pseudomonas. -Fluorescence (Pseudomonas fluorescens), more preferably Arthrobacter sp. Or Pseudomonas sp. MV37 (Pseudomonas sp. MV37).

  Next, a transamination reaction of an optically active aldehyde (2) using a transaminase will be described.

  In the case of a transamination reaction using an enzyme source, an appropriate solvent and a substrate such as the optically active aldehyde (2), the microorganism, a culture thereof, or a processed product thereof are mixed, and the mixture is stirred and shaken under pH adjustment. Finally or leave it still.

  As a reaction solvent, an aqueous medium such as water or a buffer is usually used. Examples of the buffer include a potassium phosphate buffer and a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer.

  As the transaminase, a culture solution containing cells of the above microorganisms may be used for the reaction as it is, or the culture solution may be concentrated and used. When components in the culture solution have an adverse effect on the reaction, it is preferable to use cells obtained by processing the culture solution by centrifugation or the like, or treated cells.

  The optically active aldehyde (2) as a substrate may be added all at once in the early stage of the reaction, or may be added in portions as the reaction progresses.

  The upper limit of the reaction temperature is preferably 60 ° C, 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, and more preferably 9 as an upper limit. 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 is preferably 50% (W / V) as an upper limit, and more preferably 10% (W / V). is there. The lower limit is preferably 0.01% (W / V), and more preferably 0.1% (W / V).

  The upper limit of the substrate concentration in the reaction solution is preferably 50% (W / V), and more preferably 30% (W / V). The lower limit is preferably 0.01% (W / V), and more preferably 0.1% (W / V).

  The reaction is usually carried out while shaking or stirring with aeration.

  The reaction time is appropriately determined depending on the substrate concentration, the amount of the microorganism, and other reaction conditions. The upper limit of the reaction time is preferably 336 hours, more preferably 168 hours. The lower limit is preferably 0.1 hour, and more preferably 1 hour.

  In order to promote the reduction reaction, it is preferable to add an energy source such as glucose, ethanol, or isopropanol to the reaction solution. The upper limit of the amount added is preferably 50% (W / V), and more preferably 30% (W / V). The lower limit is preferably 0.1 (W / V), and more preferably 0.5% (W / V).

  Further, it is also effective to add a surfactant such as Triton (manufactured by Nakarai Tesque Co., Ltd.), Span (manufactured by Kanto Chemical Co., Ltd.), or Tween (manufactured by Nakarai Tesque Co., Ltd.) to the reaction solution.

  Further, in order to avoid the inhibition of the reaction by the substrate and / or the alcohol product which is a product of the reduction reaction, water-insoluble organic solvents such as ethyl acetate, butyl acetate, isopropyl ether, toluene and hexane (water-insoluble organic solvents) Solvent) may be added to the reaction solution.

  Further, for the purpose of increasing the solubility of the substrate, a water-soluble organic solvent (water-soluble organic solvent) such as methanol, ethanol, acetone, tetrahydrofuran, and dimethyl sulfoxide may be added.

  The above reaction produces an optically active amine. The generated optically active amine can be isolated from the reaction mixture by a known method such as extraction, distillation, recrystallization, or column separation.

  For example, after adjusting the pH to acidic, 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 and the like. With a suitable solvent, the unreacted substrate and the ketone compound corresponding to the amino group donor generated by the transamination reaction can be selectively removed while leaving the generated optically active amino compound in the aqueous phase.

  The produced optically active amino compound and unreacted amino group donor can be extracted with a common organic solvent, for example, by adjusting the pH to basic.

  The generated optically active amino compound and the unreacted amino group donor can be separated, for example, by distillation.

  The target product thus obtained has a sufficient purity that can be used in the subsequent steps, but for the purpose of further increasing the purity, general crystallization, fractional distillation, phase transfer washing, column chromatography, etc. The purity may be further increased by a suitable purification technique. Preferably, hydrogen chloride, hydrogen bromide, sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, oxalic acid, formic acid, acetic acid, benzoic acid, p-nitrobenzoic acid, trifluoroacetic acid, tartaric acid, dibenzoyltartaric acid, This is a method in which a salt is formed with an acid such as mandelic acid or camphorsulfonic acid to carry out crystallization. Specifically, for example, a method described in International Publication WO 2006/030739, that is, a method of crystallizing the hydrochloride of the optically active amine (3) from n-butanol.

(Step 5)
In this step, the optically active amine represented by the formula (3) produced by the method described in the step 4 is further propionylated to produce a ramelteon represented by the formula (4).

  As the method for propionylation, 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 inorganic bases such as lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium hydrogen carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate; 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 and calcium hydride; trimethylamine, triethylamine, tributylamine, diisopropylethyl Min, N- methylpyrrolidine, N- methylmorpholine, 1,8-diazabicyclo [5,4,0] undec-7-ene, pyridine, quinoline, include amines such as imidazole. These may be used alone or in combination of two or more. When two or more kinds are used in combination, the mixing ratio is not particularly limited. Preferably, it is sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, or tributylamine, and more preferably, sodium hydroxide, potassium carbonate, or triethylamine.

  The upper limit of the amount of the base used is preferably 100 times, more preferably 50 times, and particularly preferably 10 times the weight of the compound (3). The lower limit is preferably 0.1-fold weight, more preferably 0.5-fold weight, and particularly preferably 1-fold weight, relative to compound (3). In such a range, the cost is not too high, and the post-processing is simple.

  The upper limit of the amount of the propionylating agent to be used is preferably 50 times, more preferably 10 times, and particularly preferably 5 times the weight of the compound (3). The lower limit is preferably 0.5-fold weight, more preferably 0.8-fold weight, and particularly preferably 1-fold weight, relative to the compound (3). In such a range, the cost is not too high, and the post-processing is simple.

  The solvent in this step is not particularly limited as long as it does not affect the reaction, and specifically, for example, water; tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether (MTBE) , Ethylene glycol dimethyl ether, ether solvents such as 4-methyltetrahydropyran; nitrile solvents such as acetonitrile and propionitrile; ester solvents such as ethyl acetate, n-propyl acetate and isopropyl acetate; pentane, hexane, heptane and methyl 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 methyl isobutyl ketone; methylene chloride; Solvents such as dimethyl sulfoxide; N, N-dimethylformamide, N, N-dimethylacetamide, N, N-diethylacetamide, N-methyl-2-pyrrolidone, N-ethyl Amide solvents such as -2-pyrrolidone, N-methyl-ε-caprolactam and hexamethylphosphoramide; urea solvents such as dimethylpropylene urea; phosphonic acid triamide solvents such as hexamethylphosphonic acid triamide; . These may be used alone or in combination of two or more. When two or more kinds are used in combination, the mixing ratio is not particularly limited. Preferably water; ether solvents such as tetrahydrofuran, methyltetrahydrofuran, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol dimethyl ether, and 4-methyltetrahydropyran; ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone. A nitrile solvent such as acetonitrile and propionitrile, more preferably water, tetrahydrofuran, acetone or acetonitrile, particularly preferably tetrahydrofuran or acetonitrile.

  The upper limit of the amount of the solvent used is preferably 100 times, more preferably 50 times, and particularly preferably 20 times, the weight of the compound (3). The lower limit is preferably 0.1-fold weight, more preferably 0.5-fold weight, and particularly preferably 1-fold weight, relative to compound (3). In such a range, the cost is not too high, and the post-processing is simple.

  The reaction temperature in this reaction is not particularly limited and may be appropriately set, but in order to reduce the generation of by-products, 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, but 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 order of mixing the compound (3), the propionylating agent, the base, and the solvent in this step is not particularly limited.

  As the treatment after the reaction, a general treatment for obtaining a product from the reaction solution may be performed. For example, the reaction solution after completion of the reaction is added to water or an aqueous solution of an acid such as hydrochloric acid or sulfuric acid, and an extraction operation is performed using a common extraction solvent such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane and the like. When the reaction solvent and the extraction solvent are distilled off from the obtained extract by an operation such as heating under reduced pressure, the desired product (4) is obtained. Preferably, the target substance precipitated by adding water to the reaction solution is separated by filtration and washed with water, tetrahydrofuran, acetone, acetonitrile, ethyl acetate, methylene chloride, toluene, heptane and the like. The thus obtained target product, ramelteon (4), has a sufficient purity. However, in order to further increase the purity, general purification methods such as crystallization, fractional distillation, and column chromatography are used. The purity may be further increased.

  This application claims the benefit of priority based on Japanese Patent Application No. 2017-094201 filed on May 10, 2017. The entire contents of the specification of Japanese Patent Application No. 2017-094201 filed on May 10, 2017 are incorporated herein by reference.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the present invention in any way.

(Example 1) Production of (E) -2- [1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde (E)-(1,6) A solution comprising 7,8-tetrahydro-2H-indeno [5,4-b] furan-8-ylidene) acetonitrile (purity: 97.6% by weight, 252.5 mg, 1.25 mmol) and toluene (10 mL) The mixture was cooled to 70 ° C., and a diisobutylaluminum hydride / toluene solution (1.0 mol / L, 2.5 mL, 2.5 mmol) was added dropwise over 15 minutes. After stirring for 16 hours while the temperature was naturally raised to 25 ° C., ethyl acetate (5 mL) was added (reaction yield: 75%). Water (30 mL), toluene (20 mL) and concentrated hydrochloric acid (5 mL) were added for extraction. The aqueous layer was further extracted twice with toluene (20 mL), and the combined organic layers were dried over anhydrous magnesium sulfate. After the desiccant was filtered off, the solvent was distilled off from the filtrate under reduced pressure, and hexane (10 mL) was added to the residue for crystallization. The crystals were collected by filtration under reduced pressure, washed with hexane (10 mL), and dried in vacuo to give the title compound as a red solid (68.7 mg, purity: 93.8 wt%, isolated yield: 26%).
¹ H NMR (CDCl ₃ ): δ 10.05 (1 H, d), 7.13 (1 H, d), 6.90 (1 H, d), 6.33 (1 H, d), 4.67 (2 H, d) dd), 3.41 (2H, dd), 3.33 (2H, m), 3.10 (2H, m)

Example 2 Production of (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde Production of (E) -2- [ 1,2,2 6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde (400 mg, 2 mmol), Diethyl 1,4-Dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate (608 mg, 2.4 mmol), a solution composed of (R) -2- (diphenylmethyl) pyrrolidine (47.5 mg, 0.2 mmol), tetrahydrofuran (3 mL), trifluoroacetic acid (22.8 mg, 0.2 mmol) at 20 ° C. The mixture was stirred for 16 hours (reaction yield: 8 %). The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography to give the title compound as a pale-yellow oil (260.0 mg, purity: 93.3%, isolation yield: 60%, optical purity: 85.2% ee).
¹ H NMR (CDCl ₃ ): δ 9.85 (1 H, s), 6.98 (1 H, d), 6.64 (1 H, d), 4.60-4.52 (2 H, m), 3. 70 (1H, m), 3.15-3.09 (2H, m), 2.90-2.81 (3H, m), 2.81-2.61 (1H, m), 2.50- 2.40 (1H, m), 1.76-1.70 (1H, m)

(Optical purity analysis conditions)
Column: Daicel Chiral Cell OD-H 4.6 × 250 mm
Eluent: ethanol / hexane = 2/98 (v / v)
Column temperature: 30 ° C
Flow rate: 1.0 mL / min.
Detector: UV 210nm
Retention time: S-form = 14.1 minutes, R-form = 17.6 minutes

(Examples 3 to 20) Production of (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (E) -2- [1, 2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde (200 mg, 1 mmol), Diethyl 1,4-Dihydro-2,6-dimethyl-3,5-pyridinedicalboxylate ( (304 mg, 1.2 mmol), a solvent, an asymmetric organic catalyst, and an acid were added thereto, and the results obtained by changing various conditions are shown below.

ＴＦＡ：トリフルオロ酢酸
ＴＣＡ：トリクロロ酢酸

TFA: trifluoroacetic acid TCA: trichloroacetic acid

ＴＨＦ：テトラヒドロフラン
４−ＭＴＨＰ：４−メチルテトラヒドロピラン
ＤＭＡ：ジメチルアセトアミド

THF: tetrahydrofuran 4-MTHP: 4-methyltetrahydropyran DMA: dimethylacetamide

(Examples 21 to 24) Production of (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (E) -2- [1, 2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde (0.05 mmol), D-glucose (0.15 mmol), NAD ⁺ (0.05 μmol), NADP ⁺ (0.05 μmol) was stirred at 30 ° C. overnight in 1 mL of potassium phosphate buffer (pH 7.5) in the presence of enone reductase and glucose dehydrogenase shown below. The results are shown below. The conversion rate to (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde was determined by extracting 200 μL of the reaction solution with 1 mL of ethyl acetate. Thereafter, it was confirmed by gas chromatography under the following conditions. Optical purity was analyzed under the following conditions.

(Conversion rate analysis conditions)
Column: RESTEK, Rtx®-35 Amine 30 mm x 0.25 mm ID
Eluent: ethanol / hexane = 2/98 (v / v)
Column temperature: 220 ° C
Injector temperature: 200 ° C
Detector temperature: 200 ° C
Carrier gas: He (150 kPa)
Detection: FID

(Optical purity analysis conditions)
Column: Daicel Chiral Cell OD-H 4.6 × 250 mm
Eluent: ethanol / hexane = 2/98 (v / v)
Column temperature: 30 ° C
Flow rate: 1.0 mL / min.
Detector: UV 210nm

Example 25 Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine (S)-(1,6) 7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehydride (purity: 94.2% by weight, 360 mg, 1.68 mmol), ammonium acetate (2.59 g, 33.6 mmol) 317 mg (5.0 mmol) of sodium cyanoborohydride was added at 25 ° C. to a solution consisting of water, ethanol (30 mL) and 25% by weight aqueous ammonia (10 mL), and the mixture was heated to 70 ° C. and stirred for 6 hours. After cooling to room temperature, a 30% by weight aqueous sodium hydroxide solution (1.344 g, 10.1 mmol) was added, and ethanol was distilled off under reduced pressure. 4-Methyltetrahydropyran (15 mL x 2) was added to the obtained residue for extraction, and the organic layers were combined, washed twice with 10 mL of saturated saline, and concentrated under reduced pressure to give a yellow oil (810.0 mg). (Purity: 12.3% by weight, yield: 29%).
Subsequently, purification was performed by a phase transfer washing operation. Ethyl acetate (10 mL) and water (5 mL) were added to the yellow oil (810.0 mg), and the pH was adjusted to 0.1 with concentrated hydrochloric acid. After separating the organic layer, the aqueous layer was adjusted to pH = 12 by adding a 30% by weight aqueous sodium hydroxide solution, and extracted twice with ethyl acetate (10 mL). The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give the title compound as a colorless oil (purity: 93.3% by weight, transfer washing recovery: 97%).
¹ H NMR (CDCl ₃ ): δ 6.87 (1 H, d), 6.53 (1 H, d), 4.51-4.42 (2 H, m), 3.16-3.03 (3 H, m) ), 2.80-2.67 (4H, m), 2.15 (1H, m), 1.93-1.87 (3H, m), 1, 70 (1H, m), 1.50 ( 1H, m)

(Example 26) Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine (S)-(1,6) 7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (0.05 mmol), α-phenethylamine (0.075 mmol), pyridoxal phosphate (0.5 μmol), and ω-transaminase Was stirred overnight at 30 ° C. in 1 mL of potassium phosphate buffer (pH 7.5). After completion of the reaction, 200 μL of the reaction solution was adjusted to pH = 12 by adding a 30% by weight aqueous solution of sodium hydroxide, extracted with 1 mL of ethyl acetate, and analyzed by gas chromatography under the following conditions to find (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine was produced.

(Conversion rate analysis conditions)
Column: RESTEK, Rtx®-35 Amine 30 mm x 0.25 mm ID
Column temperature: 220 ° C
Injector temperature: 200 ° C
Detector temperature: 200 ° C
Carrier gas: He (150 kPa)
Detection: FID

(Example 27) Preparation of freeze-dried bacterial cells of ω-transaminase-expressing Escherichia coli The following ω-transaminase gene, enzyme A: Arthrobacter sp. KNK168 (Japanese Patent No. 5410089, SEQ ID NO: 25), Enzyme B: Pseudomonas sp. KNK425 (Japanese Patent No. 5410089, SEQ ID NO: 2), Enzyme C: Pseudomonas fluorescens KNK08-18 (Pseudomonas fluorescens) (Japanese Patent No. 5410089) , Enzyme D: Pseudomonas sp. MV37 (Japanese Patent No. 5878871, SEQ ID NO: 2), Enzyme E: Vibrio fur Alice JS17 (Vibrio fluvialis JS17) (sequence described in Appl. Microbiol. Biotechnol. 61.463-471 (2003)) was converted to a NdeI recognition site downstream of the lac promoter of pUCNT (WO 94/03613). It was inserted between the cloning sites to obtain a recombinant vector. Using the recombinant vector, E. coli E. coli was used. E. coli HB101 (manufactured by Takara Bio Inc.) was transformed to obtain a recombinant Escherichia coli. The obtained recombinant Escherichia coli is cultured in a 2 × YT medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH 7.0) containing 200 μg / ml ampicillin, and the cells are collected. did. The obtained cells were frozen in methanol in which dry ice had been dissolved, and then dried overnight with a freeze dryer (Tokyo Rikakikai, FDS-1000) to obtain freeze-dried cells.

(Example 28) Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine using ω-transaminase (water reaction) , 1 enzyme method)
(S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (0.05 mmol), amino group donor ((S) -α-phenetylamine) (0.075 mmol) (hereinafter, S-PEA) or (R) -α-phenethylamine (0.075 mmol) (hereinafter, R-PEA) or isopropylamine (0.375 mmol) (hereinafter, ISPA) 0.5 mL of potassium phosphate buffer (pH 7.5) to which 5 mg of lyophilized cells of pyridoxal phosphate (0.5 μmol) and ω-transaminase (any of enzymes A, B, C, D and E) were added. And stirred at 30 ° C. overnight. After completion of the reaction, 200 μL of the reaction solution was adjusted to pH = 12 by adding a 30% by weight aqueous solution of sodium hydroxide, extracted with 1 mL of ethyl acetate, and analyzed by gas chromatography under the same conditions as in Example 26. Shown in As a result, (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine was produced by all enzymes.

(Example 29) Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine using ω-transaminase (water reaction) 3 enzyme method)
L-lactic acid dehydrogenase PALDH derived from Pediococcus acidilactici JCM8797 strain and glucose dehydrogenation derived from Bacillus megaterium IAM1030 described in Example 13 of WO 2007/1399055. Recombinant E. coli co-expressing the enzyme GDH was cultured in 2 × YT medium (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl, pH 7.0) containing 200 μg / ml ampicillin, The cells were collected by centrifugation. After the obtained cells were frozen in methanol in which dry ice was dissolved, the cells were dried overnight with a freeze dryer (Tokyo Rika Kikai, FDS-1000) to obtain freeze-dried PAL / GDH cells.

(S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (50 μmol), an amino group donor (L-alanine (561 μmol), or D-alanine (561 μmol)), pyridoxal phosphate (0.5 μmol), D-glucose (333 μmol), NAD ⁺ (1.3 μmol), and freeze-dried ω-transaminase cells (enzymes A, B, C, D, E) Any of the above) was stirred at 30 ° C. for 4 hours in 1 mL of potassium phosphate buffer (pH 7.5) to which 5 mg of PAL.GDH freeze-dried cells 2.5 mg were added.

  After the completion of the reaction, a 30% by weight aqueous solution of sodium hydroxide was added to 200 μL of the reaction solution to adjust the pH to 12, and the mixture was extracted with 1 mL of ethyl acetate, and analyzed by gas chromatography under the same conditions as in Example 26. Shown in As a result, (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine was produced by all enzymes.

Example 30 Production of (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine using ω-transaminase (in a solvent) reaction)
(S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde (0.05 mmol), amino group donor ((S) -α-phenetylamine) (0.075 mmol) (S-PEA)) in 0.5 mL of water-saturated MTBE to which 0.5 μL of a 0.5 M aqueous pyridoxal phosphate solution adjusted to pH 7.5 and 5 mg of lyophilized ω-transaminase cells (enzyme D) were added. The mixture was stirred at 30 ° C. for 3 hours to react. After the reaction was completed, the reaction solution was analyzed by gas chromatography under the same conditions as in Example 26, and the results are shown in Table 6. As a result, (S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine was generated.

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 7.5 g (manufactured by Mitsubishi Chemical Corporation) twice with 7.5 mL of 0.1 M KPB (potassium phosphate buffer) (pH 7.5), 250 mg of lyophilized TPM cells of TPM and 20 mg of PLP (pyridoxal phosphate) Was dissolved in 10 mL of 0.1 M KPB (pH 7.5), and the mixture was stirred at room temperature for 2 hours. After removing water with a pipette, nitrogen was blown and dried 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 immobilized resin was packed in a glass tube of Φ5 mm × 50 mm to prepare an immobilized resin column.

Solution A was MTBE solution of 100 mM (S)-(1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) acetaldehyde, and solution B was water-saturated. A 100 mM (S) -α-phenetylamine solution was prepared using the MTBE solution. Using a syringe pump (YSP-101, manufactured by YMC), solution A and solution B are each fed at 0.025 mL / min, mixed in a T-shaped mixing section, and fixed at 30 ° C. The solution was passed through a modified 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 used. The conversion rate was calculated from the ratio of -yl) ethanamine 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 to the target compound was maintained stably from 40 minutes to 180 minutes after the start of the liquid sending.
When commercializing ramelteon production using an enzyme flow reactor, it is possible to increase the conversion rate by using a method such as extending the column length, increasing the number of passages, or reducing the flow rate. is there.

Example 32 Production of Ramelteon ((S) -N- [2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethyl] propionamide) S) -2- (1,6,7,8-tetrahydro-2H-indeno [5,4-b] furan-8-yl) ethanamine (purity: 93.3% by weight, 60.0 mg, 0.28 mmol) , Acetonitrile (5 mL), triethylamine (56 mg, 0.56 mmol), and propionyl chloride (31 mg, 0.34 mmol) were stirred at 25 ° C. for 1 hour. After adding water (5 mL) to make a homogeneous solution, acetonitrile was distilled off under reduced pressure. Ethyl acetate (15 mL) was added to the residue for extraction, and the organic layer was further washed twice with water (5 mL) and concentrated under reduced pressure to obtain a white solid (75.9 mg, purity: 89.3% by weight, Reaction yield: 95%, optical purity: 83.5% ee).
Ethyl acetate (1 mL) was added to and dissolved in the white solid (60 mg, 0.23 mmol), and hexane (5 mL) was added for crystallization. After cooling to 5 ° C. and stirring for 30 minutes, the crystals were filtered off under reduced pressure, washed with hexane (5 mL) and dried in vacuo to give the title compound as white crystals (38.1 mg, purity: 97.3% by weight). , Crystallization recovery: 97%, optical purity: 97.9% ee).
¹ H NMR (CDCl ₃ ): δ 6.95 (1 H, d), 6.61 (1 H, d), 5.4 (1 H, brs), 4.59 (1 H, dd), 4.55 (1 H, d) dd), 3.36 (2H, m), 3.24-3.10 (3H, m), 2.89 (1H, dd), 2.77 (1H, dd), 2.20 (1H, m) ), 2.17 (2H, q), 2.03 (1H, m), 1.85 (1H, m), 1.82 (1H, m), 1.14 (3H, t)

(Optical purity analysis conditions)
Column: Daicel Chiral Cell OD-H 4.6 × 250 mm
Eluent: ethanol / hexane = 1/9 (v / v)
Column temperature: 30 ° C
Flow rate: 1.0 mL / min.
Detector: UV 210nm
Retention time: S-form = 12.3 minutes, R-form = 16.0 minutes

Claims (14)

Formula (1) below;

By asymmetric reduction of the enal represented by the following formula (2);

To produce an optically active aldehyde represented by the following formula (4):

A method for producing ramelteon, which comprises converting into ramelteon represented by the formula: The asymmetric reduction is performed 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. . the aralkyl group having 7 to 20, an optionally substituted aryl group having 6 to 20 carbon atoms, a carboxyl group, or 1H- tetrazole a group * is an asymmetric carbon atom .A ^- the counter anion Using an optically active pyrrolidine salt derivative represented by the following formula (6):

The method for producing ramelteon according to claim 1, wherein the reaction is performed using a Hantzsh ester represented by the following formula: In the optically active pyrrolidine salt derivative (5), R is a diphenylmethyl group, a (trimethylsilyloxy) diphenylmethyl group, or a (hydroxy) diphenylmethyl group, the absolute configuration is R, A ⁻ is trichloroacetate, 3. The method for producing ramelteon according to claim 2, which is trifluoroacetate.   The method for producing ramelteon according to claim 1, wherein the asymmetric reduction is performed using enone reductase.   The method for producing ramelteon according to claim 4, wherein the enone reductase is OYE2, NemA, YersER, or NCR-R. The optically active aldehyde (2) is represented by the following formula (3);

The method for producing ramelteon according to any one of claims 1 to 5, wherein the compound is converted to an optically active amine represented by the formula (1) and then propionylated.   The method for producing ramelteon according to claim 6, wherein the method for converting into the optically active amine (3) is performed using transaminase in the presence of an amino group donor.   The method for producing ramelteon according to claim 7, wherein the amino group donor is isopropylamine, and the transaminase is ω-transaminase.   The method for producing ramelteon according to claim 7, wherein the amino group donor is α-phenethylamine, and the transaminase is ω-transaminase.   The method for producing ramelteon according to claim 7, wherein the amino group donor is an α-amino acid, and the transaminase is an ω-transaminase.   The method for producing ramelteon according to any one of claims 7 to 10, wherein the transaminase is reacted in an organic solvent.   The method for producing ramelteon according to any one of claims 7 to 11, wherein the transaminase immobilized on a resin is reacted in an organic solvent. The enal (1) is represented by the following formula (7);

The method for producing ramelteon according to any one of claims 1 to 12, which is produced by reacting an α, β-unsaturated nitrile represented by the formula (1) with a hydride reducing agent. Formula (1) below;

((E) -2- [1,2,6,7-tetrahydro-8H-indeno [5,4-b] furan-8-ylidene] acetaldehyde).

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