KR20040095889A - Methods for the Preparation of Tetrasubstitued Pyrazins by Utilizing Baker's Yeast - Google Patents

Abstract

PURPOSE: Methods for preparing tetrasubstitued pyrazines using baker's yeast are provided, thereby easily and environment-friendly preparing tetrasubstitued pyrazines in water under mild condition. CONSTITUTION: The method for preparing tetrasubstitued pyrazines of formula (2) using baker's yeast comprises the steps of: (1) preparing a solution of baker's yeast; and (2) dissolving beta-keto alpha-oxime carbonyl derivatives of formula (1) in hydrophilic organic solvent and adding the solution into the baker's yeast solution to carry out chemoselective microbial reduction, wherein R1 is methyl, ethyl or phenyl; R2 is hydrogen, methyl or benzyl; R3 is methoxy, ethoxy, benzyloxy or phenylamine; the organic solvent is selected from benzene, toluene and hexane.

Description

Methods for the preparation of pyrazine compounds using Baker's Yeast {Methods for the Preparation of Tetrasubstitued Pyrazins by Utilizing Baker's Yeast}

The present invention relates to a method for preparing a pyrazine compound represented by the following Chemical Formula 2 by a new and environmentally friendly process by a chemical selective reduction reaction using a baker's yeast, which is a microorganism.

In the above formula, R ¹ is methyl (CH ₃ ), ethyl (CH ₃ CH ₂ ) or phenyl (C ₆ H ₅ ), and R ³ is methoxy (CH ₃ O), ethoxy (CH ₃ CH ₂ O ), Benzyloxy (C ₆ H ₅ CH ₂ O) or phenylamine (C ₆ H ₅ NH).

Compounds useful as medicines, pesticides, fragrances, etc., in most cases have a pure optical activity, and new drugs are being developed in recent years, many of them have a complex structure having an optical active center.

There are many limitations and difficulties in synthesizing such compounds into optically pure isomers. For example, use of expensive metal catalysts or additional reaction steps are required to obtain purely the desired conformational compounds purely, complex purification processes for the separation of racemates, etc. In many cases, no product of the desired structure can be obtained.

Synthesis using stereocatalytic biocatalyst is one of the very effective methods to solve this problem. Research in this field has been actively researched since the 1980s, and has been developed with repeated attempts to use biocatalysts such as enzymes and microorganisms in pure organic chemical reactions.

As a synthesis method using such a biocatalyst, many studies have been published in which an optically active secondary alcohol is produced by asymmetrically reducing a carbonyl compound using an enzyme or a microorganism containing the enzyme. Among several possible biotransformations, microbial reduction reactions are being investigated as a tool for the synthesis of optically active alcohols from carbonyl compounds. In particular, the baker's yeast, Baker Yeast (BY) (BY) (Saccharomyces cerevisiae) is one of the most frequently used microorganisms due to the mild conditions, ease of handling and wide bioavailability in carrying out the asymmetric reduction reaction.

Recently, reduction methods for C = N and N-O bonds in the oxime molecular structure have been studied. As a result, several reports have been published. Among them, reduction reaction to amine (Tetrahedron Letters, 31, 5555 (1990)) was reported using oxime compound as a substrate, and reduction reaction to amino alcohol (Microbiol. & Biotech., 14, 247 (1998)) was reported. Hydrolysis to ketones (J. Chem. Soc. Perkin Trans. 1, 2056 (1991)).

In addition, a ketone group obtained by selectively reducing only a ketone group by using ( E ) -1-phenyl-1,2-alkandione 2- ( O -methyloxime) as a substrate having a ketone group and an oxime group in one molecule Examples of reducing compounds in one molecule of oxime groups (Tetrahedron Asymmetry, 11, 2107, (2000)), but this method is limited to compounds in which the side chain at carbon position 2 is an alkyl group having 1 to 4 carbon atoms, In particular, no examples of oxime groups causing chemiselective reduction reactions have yet been reported.

Pyrazine heterocyclic compounds prepared by the process of the present invention are widely used as intermediates in the field of medicinal chemistry. In addition, their derivatives are of interest because of their chemotherapeutical activity, in particular 2,5-double substituted pyrazine biosynthesized from amino acids are known as common units of various marine natural products with cell proliferation inhibitory and anticancer properties. ( Org. Lett. 2000, 2 , 33).

Recently, pyrazinamide ( J. Clin. Microbiol. 2002, 40 , 501) and pyrazinester ( Antimicrob. Agents Chemother. 1998, 42 , 462) have been shown to have antituberculosis activity in vitro and in vivo . It is known that there is an antituberculosis activity, and interest in heterocyclic compounds having such pyrazine structures has been greatly increased.

Methods for preparing such pyrazine compounds are disclosed in Korean Patent Publication Nos. 1997-10750 and Heterocycles, 27, 1123 (1988), Syntheric Communications, 3, 225 (1973) and J. Chem. Soc. 1, 1474 (1959). The process described in the above-mentioned patents and documents uses a catalyst containing hydrogenated silver, or a metal such as SnCl ₂ , TiCl ₃ , Pd-C / H _2, or the like. The method of synthesis is difficult to process or the metal or organic metals remaining after the reaction must be treated, there is a disadvantage that is not environmentally friendly.

The present invention has been completed to solve the above-mentioned problems, an object of the present invention to prepare a pyrazine compound of formula 2 useful in the field of medicinal chemistry, etc., the reaction conditions are gentle, easy to handle and environmentally friendly method To provide.

The present invention relates to a method for preparing a pyrazine compound by chemoselective microbial reduction using baker yeast, as shown in Scheme 1 below, the compound of formula 1 is contacted with baker's yeast The present invention relates to a novel preparation method for preparing a pyrazine compound represented by the following Chemical Formula 2 wherein four functional groups are substituted.

In which R ¹ is methyl (CH ₃ ), ethyl (CH ₃ CH ₂ ) or phenyl (C ₆ H ₅ );

R ² is hydrogen (H), methyl (CH ₃ ) or benzyl (C ₆ H ₅ CH ₂ );

R ³ is methoxy (CH ₃ O), ethoxy (CH ₃ CH ₂ O), benzyloxy (C ₆ H ₅ CH ₂ O) or phenylamine (C ₆ H ₅ NH).

Since the chemoselective reduction reaction of the present invention is carried out under mild reaction conditions, the desired pyrazine compound can be produced more easily and more environmentally friendly than general chemical synthesis by using the chemoselective reduction method according to the present invention.

Hereinafter, the configuration and operation of the present invention in more detail, but the present invention is not limited thereto.

As used herein, the term "chemically selective reduction reaction" refers to the selective reduction of only one functional group, that is, the oxime group, even if two functional groups, a ketone group and an oxime group, capable of reacting with a microorganism are simultaneously present in one molecule. it means.

The manufacturing method of the present invention

1) preparing an aqueous solution of Baker's Yeast, and

2) dissolving the beta-keto alpha-oximecarbonyl derivative of Formula 1 in a hydrophilic organic solvent and adding it to the Baker yeast aqueous solution to perform a chemoselective reduction reaction.

<Formula 2>

Wherein R ¹ , R ² and R ³ are as mentioned in Scheme 1 above.

When the reaction is complete, the product is obtained by conventional purification methods such as gas chromatography / mass spectrometry (GC / MSD) confirming the completion of the reaction and then extraction with chloroform or methylene chloride.

The baker's yeast used in the manufacturing method according to the present invention can be used as well as the general baking baker's yeast available on the market, as well as the baker's yeast (IMBY) which is commercially available for the chemical reagent and the support for the baker's yeast.

In addition, in the production method of the present invention, sugar may be added in the step of preparing an aqueous solution of the baker's yeast in order to more efficiently perform the filtration process of separating the product from the reaction mixture after the reaction. At this time, examples of sugars that may be added include saccharose, D-glucose and D-galactose, and saccharose is preferred.

When sugar is added, the ratio of the baker's yeast: sugar: beta-keto alpha-oximecarbonyl compound is most preferably about 2 g: 3 g: 0.4 mmol, and the amount of the solvent used to prepare an aqueous solution of the baker yeast is preferable. Silver is about 30 ml to 100 ml, preferably about 60 ml.

The solvent used to prepare the aqueous solution of Baker Yeast is preferably water, and also using a mixture of water in a volume ratio of 10: 1 to 1:10 with a nonpolar organic solvent such as benzene, toluene or n-hexane There is no significant difference in the yield of the product.

Hydrophilic organic solvents used to dissolve beta-keto alpha-oximecarbonyl derivatives of Formula 1 include, but are not limited to, alcohols such as methanol, ethanol, propanol and butanol, but all solvents that do not affect the reaction include Can be used.

The temperature of the chemoselective reduction reaction is suitably 20 ° C. to 50 ° C., preferably 30 ° C. to 35 ° C., and the chemoselective reduction reaction is suitably performed when the pH is maintained at about 5 to 7 during the chemoselective reduction reaction. , Product yields are highest when maintained at slightly acidic, especially about pH 5. The maintenance of weak acidity is controlled by the frequent addition of 1N HCl and 1N NaOH solutions during the chemoselective reduction reaction. In addition, phosphate buffer solution (pH = 5.0, 6.0, 7.0) may be used as the reaction solvent for maintaining the weak acidity of the reaction solution, but the yield is relatively low.

Examples of compounds of formula (2) which may be prepared by the preparation process according to the invention include 3,6-dimethylpyrazine-2,5-dicarboxylic acid diethyl ester, 3,6-dimethylpyrazine-2,5-dica Carboxylic acid dimethyl ester, 3,6-diethylpyrazine-2,5-dicarboxylic acid dimethyl ester, 3,6-dimethylpyrazine-2,5-dicarboxylic acid bisphenylamide and 3,6-dimethylpyrazine -2,5-dicarboxylic acid dibenzyl esters, but is not limited thereto.

Methods for the preparation of compounds of formula 1 which are starting materials of the invention are described in Tetrahedron Asymmetry, 11, 2107, (2000), J. Amer. Chem. Soc. 60 , 1328, (1938) and Synthesis, 850, (1985), and the general scheme is as follows.

Where

R ¹ is methyl (CH ₃ ), ethyl (CH ₃ CH ₂ ) or phenyl (C ₆ H ₅ );

R ³ is methoxy (CH ₃ O), ethoxy (CH ₃ CH ₂ O), benzyloxy (C ₆ H ₅ CH ₂ O) or phenylamine (C ₆ H ₅ NH).

As such, the method of the present invention obtains a pyrazine compound from a beta-keto alpha-oxime carbonyl derivative which can be easily synthesized by a known general method in a simple one step reaction, thus requiring a special apparatus or using a metal catalyst. It is a new method that is significantly more advanced than the new method, and is especially environmentally friendly. The product can also be conveniently purified by conventional techniques such as filtration, extraction, evaporation, chromatographic separation and combinations thereof.

The starting materials used in the present invention and the process for preparing substituted pyrazine derivatives of the formula (2) according to the present invention are described in more detail by the following examples, but the present invention is not limited by the following examples.

<Example 1>

Preparation of 2-Hydroxyyi-3-oxobutyric acid ethyl ester (R ¹ = CH ₃ , R ² = H, R ³ = CH ₃ CH ₂ O in Formula 1) .

7.3 ml (7.50 g, 58 mmol) of 3-oxobutyric acid ethyl ester was dissolved in 8.4 ml (2.5 eq) of glacial acetic acid (glac.AcOH) and sodium nitrite (NaNO) in an ice-filled reaction vessel. ₂ ) 1.2 eq (40 wt% solution in 10 ml of water) was slowly added dropwise. After the addition, the reaction was performed at room temperature for 2 hours, and when the reaction was terminated, ice water and sodium carbonate (Na ₂ CO ₃ ) (2.5 eq) were added, and extracted three times with an ethyl acetate solvent.

After filtration under reduced pressure, the filtrate was dried over anhydrous sodium sulfate (Na ₂ SO ₄ ), concentrated and confirmed by gas chromatography / mass spectrometry (GC / MSD), followed by silica gel column chromatography (hexane: ethyl acetate = 3: 1). Separation and purification afforded the title compound as a colorless liquid (yield 44%).

MS: m / z 159 (M ⁺ ), 131, 113, 86, 70, 54; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 9.78 (s, 1H), 4.39 (q, J = 7.14 Hz, 3H), 2.42 (s, 1H), 1.35 (t, J = 7.14 Hz, 2H ); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 194.47, 162.21, 151.49, 62.95, 25.76, 14.36.

<Example 2>

Of 2-Methoxyimino-3-oxobutyric acid ethyl ester (R ¹ = CH ₃ , R ² = CH ₃ , R ³ = CH ₃ CH ₂ O in formula 1) Produce.

2 g (12.57 mmol) of the oxime compound obtained in Example 1 were stirred with 1.43 ml (1.2 eq) of dimethylsulfate (Me ₂ SO ₄ ) under acetone solvent (20 ml). Potassium carbonate (K ₂ CO ₃ ) (0.6 eq) was slowly added dropwise in an ice vessel, followed by reaction at room temperature for 1 hour. After completion of the reaction, the mixture was filtered under reduced pressure, dried over anhydrous magnesium sulfate (MgSO ₄ ), concentrated and confirmed by gas chromatography / mass detector, and then purified by silica gel column chromatography (hexane: ethyl acetate = 20: 1) to obtain the title compound. Was obtained as a white solid (yield 98.7%).

MS: m / z 173 (M ⁺ ), 128, 100, 70, 58; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 4.33 (q, J = 7.14 Hz, 3H), 4.10 (s, 3H), 2.40 (s, 3H), 1.33 (t, J = 7.14 Hz, 2H ); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 193.26, 161.57, 150.45, 64.77, 62.51, 25.55, 14.41.

<Example 3>

2-benzyloxyimino-3-oxobutyric acid ethyl ester (R ¹ = CH ₃ , R ² = C ₆ H ₅ CH ₂ , R ³ = CH ₃ CH in formula 1) Preparation of ₂ O).

2 g (12.57 mmol) of the oxime compound obtained in Example 1 were stirred with 1.79 ml (1.2 eq) of benzyl bromide (BnBr) in acetone solvent (20 ml). Potassium carbonate (0.55 eq) was slowly added dropwise in an ice vessel, followed by reaction for 1 hour at room temperature. After the reaction was completed, the mixture was filtered under reduced pressure, dried over anhydrous magnesium sulfate, concentrated to confirm by GC / MSD, and purified by silica gel column chromatography (hexane: ethyl acetate = 20: 1) to obtain the title compound as a white transparent oil. Obtained (yield 87.4%).

MS: m / z 249 (M ⁺ ), 204, 105, 91 (100), 77; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 7.40-7.25 (m, 5H), 5.31 (s, 2H), 4.34 (q, J = 7.14 Hz, 3H), 2.36 (s, 1H), 1.30 (t, J = 7.15 Hz, 2H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 193.29, 161.56, 150.89, 136.27, 128.97, 128.88, 128.57, 78.98, 62.49, 25.63, 14.44.

<Example 4>

Preparation of 2-Hydroxyyi-3-oxobutyric acid methyl ester (R ¹ = CH ₃ , R ² = H, R ³ = CH ₃ O in Formula 1).

2.8 ml (3 g, 0.026 mol) 3-oxo-butyric acid methyl ester, 3.7 ml (2.5 eq) glacial acetic acid and NaNO ₂ / H ₂ O according to the synthesis method described in Example 1 (1.2 eq) was reacted to give the title compound as a colorless liquid (yield 87.6%).

MS: m / z 145 (M ⁺ ), 128, 113, 86, 70, 54; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 3.89 (s, 3H), 2.45 (s, 3H).

Example 5

Preparation of 2-Methoxyimino-3-oxobutyric acid methyl ester (R ¹ = CH ₃ , R ² = CH ₃ , R ³ = CH ₃ O) in formula (I).

The oxime compound obtained in Example 4 was reacted with 0.78 ml (1.2 eq) of Me ₂ SO ₄ and K ₂ CO ₃ (0.6 eq) in acetone (10 ml) solvent to obtain the title compound as a white solid (yield 92.6%). ).

¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 4.09 (s, 3H), 3.85 (s, 3H), 2.38 (s, 3H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 192.98, 161.78, 150.13, 64.63, 52.82, 25.35.

<Example 6>

Preparation of 2-Hydroxyyi-3-oxopentanoic acid methyl ester (R ¹ = CH ₃ CH ₂ , R ² = H, R ³ = CH ₃ O in Formula 1) .

3 g (0.023 mol) of 3-oxopentanoic acid methyl ester was dissolved in 10.0 ml of glacial acetic acid and reacted with NaNO ₂ / H ₂ O (1.2 eq) according to the synthesis method described in Example 1. The compound was obtained as a yellow transparent liquid (yield 63.0%).

¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 10.61 (s, 1H), 3.89 (s, J = 7.38 Hz, 3H), 2.81 (q, 3H), 1.28 (t, J = 7.38 Hz, 2H ).

<Example 7>

Of 2-Methoxyimino-3-oxopentanoic acid methyl ester (R ¹ = CH ₃ CH ₂ , R ² = CH ₃ , R ³ = CH ₃ O in Formula 1) Produce.

1.1 g (0.007 mol) of the oxime compound obtained in Example 6 was reacted with 0.81 ml (1.2 eq) of dimethyl sulfate and potassium carbonate (0.6 eq) in 11 ml of acetone to obtain the title compound as a colorless liquid (yield 94.1% ).

¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 4.08 (s, 3H), 3.86 (s, 3H). 2.81 (q, J = 7.2 Hz, 3H), 1.12 (t, J = 7.2 Hz, 2H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 195.93, 161.92, 149.55, 64.54, 52.80, 31.16, 7.73.

<Example 8>

2-Hydroxyyi-3-oxo-N-phenylbutanamide, R ¹ = CH ₃ , R ² = H, R ³ = C ₆ H ₅ NH Manufacture).

TDO (2,2,6-Trimethyl-4H-1,3-dioxin-4-one, 14.4 ml) in a mixed solution of dry xylene (150 ml) and 9.3 g (1 eq) of aniline , 1.1 eq) was added thereto, and the mixture was stirred for 2 hours while heating to reflux. After cooling to room temperature, the mixture was filtered under reduced pressure and recrystallized from ethyl acetate / n-hexane to obtain 3-oxo-N-phenylbutanamide (5-oxo-N-phenylbutanamide) in a yield of 52.0%.

9.16 g (51.7 mmol) of 3-oxo-N-phenylbutanamide thus obtained was dissolved in glacial acetic acid (50 ml) and slowly added dropwise NaNO ₂ / H ₂ O (1.2 eq) in an ice bath. After reaction at 10 ° C. for about 30 minutes, cold water was added, filtered under reduced pressure and recrystallized from benzene / cyclohexane to give the title compound as a yellow solid (yield 50%).

MS: m / z 206 (M ⁺ ), 189, 146, 120, 93, 77, 65, 51; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 10.97 (s, 1H), 7.60-7.20 (m, 5H, aromatic), 2.57 (s, 3H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 200.53, 187.50, 161.79, 143.90, 135.76, 129.67, 126.68, 121.79, 26.81.

Example 9

2-Methoxyimino-3-oxo-N-phenylbutanamide, R ¹ = CH ₃ , R ² = CH ₃ , R ³ = C ₆ H ₅ NH).

1.5 g of the compound obtained in Example 8 was reacted with 1.03 ml (1.5 eq) of dimethylsulfate and 1.51 g (1.5 eq) of potassium carbonate in an acetone solvent to give the title compound as a yellow solid, according to the synthesis method described in Example 2. Yield 97.4%).

MS: m / z 220 (M ⁺ ), 177, 146, 119, 92, 77, 65, 58; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) major; 7.81 (s, 1 H), 7.56-7.00 (m, 5 H), 4.07 (s, 3 H), 2.38 (s, 3 H) minor; 8.17 (s, 1 H), 7.56-7.00 (m, 5 H), 3.99 (s, 3 H), 2.55 (s, 3 H).

<Example 10>

2-Hydroxymino-3-phenyl-propionic acid ethyl ester (R ¹ = C ₆ H ₅ , R ² = H, R ³ = CH ₃ CH ₂ O in Formula 1) Manufacture).

According to the synthesis method described in Example 1, 19.2 g (0.1 mol) of 3-oxo-3-phenylpropionic acid ethyl ester was dissolved in 20.0 ml of acetic acid to give NaNO ₂ / H ₂ O ( 1.2 eq) to give the title compound as a white solid (yield 83.4%).

¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 7.88-7.29 (m, 5H), 4.27 (q, J = 6.0 Hz, 3 H), 1.22 (t, J = 6.0 Hz, 2H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 191.23, 161.58, 149.46, 135.04, 134.65, 133.85, 130.77, 129.54, 129.41, 128.61, 62.87, 14.22.

<Example 11>

2-Methoxyimino-3-phenyl-propionic acid ethyl ester in Formula 1, R ¹ = C ₆ H ₅ , R ² = CH ₃ , R ³ = CH ₃ CH ₂ O) manufacture.

According to the synthesis method described in Example 2, 2 g of the compound obtained in Example 10 was reacted with 1.28 ml (1.5 eq) of dimethylsulfate and 1.87 g (1.5 eq) of acetone solvent (25 ml) to give the title compound a colorless liquid. Obtained as a state (yield 56.4%).

MS: m / z 188 (M ⁺ ), 188, 129, 105 (100), 77, 51.

<Example 12>

2-Hydroxyyi-3-oxobutyric acid benzyl ester (R ¹ = CH ₃ , R ² = H, R ³ = C ₆ H ₅ CH ₂ O in Formula 1) Manufacturing.

19.2 g of 3-oxobutyric acid benzyl ester was dissolved in 14.3 ml (2.5 eq) of glacial acetic acid and reacted with NaNO ₂ / H ₂ O (1.2 eq) according to the synthesis method described in Example 1. The compound was obtained as a clear yellow liquid (yield 71.0%).

MS: m / z 221 (M ⁺ ), 204, 115, 107, 91 (100), 77, 65; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 9.74 (s, 1 H), 7.38-7.24 (m, 5H), 5.33 (s, 2H), 2.37 (s, 3H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 194.57, 162.07, 151.30, 134.86, 129.05, 128.73, 68.36, 25.78.

Example 13

2-Methoxyimino-3-oxobutyric acid benzyl ester, R ¹ = CH ₃ , R ² = CH ₃ , R ³ = C ₆ H ₅ CH ₂ O Manufacture).

According to the synthesis method described in Example 2, 2 g of the oxime compound obtained in Example 12 was reacted with 1.28 ml (1.5 eq) of dimethylsulfate and 1.87 g (1.5 eq) of potassium carbonate in acetone solvent (25 ml) to give the title compound a colorless property. Obtained as a liquid state (yield 94.8%).

MS: m / z 236 (M ⁺ ), 204, 129, 91, 65; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 7.40-7.37 (m, 5H), 5.33 (s, 2H), 4.10 (s, 3H), 2.41 (s, 3H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 192.97, 161.33, 150.06, 134.99, 128.82, 128.76, 128.48, 67.79, 64.65, 25.40.

<Example 14>

3,6-dimethylpyrazine-2,5-dicarboxylic acid diethyl ester, R ¹ = CH ₃ , R ³ = CH ₃ CH ₂ O) manufacture.

2 g of baker's yeast and 3 g of saccharose were added to 60 ml of water and stirred at 30 ° C. for 30 minutes, followed by 63.7 mg (0.4 mmol of 2-hydroxyimino-3-oxobutyl acid ethyl ester obtained in Example 1). 0.5 ml ethanol solution) was added. The reaction solution was stirred for 1.5 days while adjusting the pH of the reaction solution to 5.0 with an appropriate amount of 1N HCl solution and 1N NaOH solution.

The reaction mixture was filtered under reduced pressure, and the filtrate was saturated with NaCl and extracted three times with chloroform. The extract was dried over sodium sulfate, concentrated to confirm by gas chromatography, separated by silica gel column chromatography (hexane: ethyl acetate = 5: 1), and recrystallized with ethyl acetate / n-hexane to give 23.5 mg (yield 46.6%) of the title compound. Was obtained as a white solid.

Melting point 83.6-88.0 ° C .; IR (KBr): (C = O) 1711.0 cm "¹; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 4.49 (q, J = 7.11 Hz, 4H), 2.8 (s, 6H), 1.45 (t, J = 7.11 Hz, 3H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ 165.47, 151.15, 144.47, 62.78, 22.68, 14.57; MS m / z 252 (M ⁺ ), 223, 208, 180 (100), 151, 134, 106, 81, 67.

C ₁₂ H ₁₆ N ₂ O ₄ [M ⁺ ] by HRMS, Theorem 252.1110; Found 252.1151

Anal for C ₁₂ H ₁₆ N ₂ O ₄ : Theoretical C, 57.13; H, 6.39; N, 11.10; 0, 25.37; Found C, 57.95; H, 6. 46; N, 10.58; 0, 25.01.

<Example 15>

3,6-dimethylpyrazine-2,5-dicarboxylic acid diethyl ester, R ¹ = CH ₃ , R ³ = CH ₃ CH ₂ O) manufacture.

3 g of montmorillonite K10 and 2 g of baker's yeast were added to 100 ml of water, stirred in a reactor at 30 ° C. for 1.5 hours, and then filtered to obtain baker's yeast (IMBY) immobilized on the support. In the same manner as in Example 14, 30.1 g of saccharose and 63.7 mg (0.4 mmol) of 2-hydroxyimino-3-oxobutyl acid ethyl ester obtained in Example 1 were reacted with a 0.5 ml ethanol solution for 3.5 days for 10.1 mg ( Yield 20.0%) of the title compound.

<Example 16>

3,6-dimethylpyrazine-2,5-dicarboxylic acid diethyl ester, R ¹ = CH ₃ , R ³ = CH ₃ CH ₂ O) manufacture.

As in Example 14, 2 g of baker's yeast, 3 g of saccharose, and 69.3 mg (0.4 mmol) of 2-methoxyimino-3-oxobutyl acid ethyl ester obtained in Example 2 were reduced at pH 5 for 1.5 days to obtain 26.7 mg. (Yield 52.8%) of the title compound was obtained as a white solid.

<Example 17>

3,6-dimethylpyrazine-2,5-dicarboxylic acid diethyl ester, R ¹ = CH ₃ , R ³ = CH ₃ CH ₂ O) manufacture.

As in Example 14, 2 g of Baker Yeast, 3 g of Saccharose and 99.7 mg (0.4 mmol) of 2-benzyloxyimino-3-oxobutyl acid ethyl ester obtained in Example 3 were reduced for 2 days at pH 5 for 20.2 mg. (Yield 40.0%) of the title compound was obtained as a white solid.

Example 18

Of 3,6-dimethylpyrazine-2,5-dicarboxylic acid dimethyl ester (R ¹ = CH ₃ , R ³ = CH ₃ O in Formula 2) Produce.

2 g of baker's yeast, 3 g of saccharose and 58 mg (0.4 mmol) of 2-hydroxyimino-3-oxobutyl acid methyl ester obtained in Example 4 were reduced to 2 days at pH 5 for 27 days as in Example 14. (Yield 31.0%) of the title compound was obtained as a white solid.

Melting point 133.2-138.0 ° C .; ¹ H NMR (300 MHz, CDCl ₃ ): δ 4.03 (s, 6H), 2.84 (s, 6H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 165.66, 151.65, 143.98, 53.60, 22.77; MS m / z 224 (M ⁺ ), 194, 166 (100), 135, 107, 67.

C ₁₀ H ₁₂ N ₂ O ₄ [M ⁺ ] by HRMS, Theoretical 224.08; Found: 225.22,

Anal for C ₁₀ H ₁₂ N ₂ O ₄ : Theoretical C, 53.57; H, 5.39; N, 12.49; 0, 28.54; Found C, 53.94; H, 5.54; N, 11.95; O, 27.82.

Example 19

Of 3,6-dimethylpyrazine-2,5-dicarboxylic acid dimethyl ester (R ¹ = CH ₃ , R ³ = CH ₃ O in Formula 2) Produce.

As in Example 14, 2 g of baker's yeast, 3 g of saccharose, and 63.7 mg (0.4 mmol) of 2-methoxyimino-3-oxobutyl acid methyl ester obtained in Example 5 were reduced to 29 days at pH 5 for 29 mg. (Yield 65.1%) of the title compound was obtained as a white solid.

Example 20

3,6-Diethylpyrazine-2,5-dicarboxylic acid dimethyl ester, R ¹ = CH ₃ CH ₂ , R ³ = CH ₃ O) manufacture.

2 g of baker's yeast, 3 g of saccharose and 63.7 mg (0.4 mmol) of 2-hydroxyimino-3-oxopentanoic acid methyl ester obtained in Example 6 were reduced in a pH of 5 for 12 days as in Example 14, and then 12 mg (Yield 23.2%) of the title compound was obtained as a white solid.

¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 4.01 (s, 6H), 3.11 (q, J = 7.52 Hz, 4H), 1.26 (t, J = 7.50 Hz, 3H); MS m / z 252 (M ⁺ ), 220, 192 (100), 177, 160, 132, 57.

C ₁₂ H ₁₆ N ₂ O ₄ [M ⁺ ] by HRMS, 252.11. Found: 253.25.

Example 21

3,6-Diethylpyrazine-2,5-dicarboxylic acid dimethyl ester, R ¹ = CH ₃ CH ₂ , R ³ = CH ₃ O) manufacture.

As in Example 14, 2 g of baker's yeast, 3 g of saccharose and 69.3 mg (0.4 mmol) of 2-methoxyimino-3-oxopentanoic acid methyl ester obtained in Example 7 were reduced for 12 days at pH 5 for 12.2 mg (Yield 24.2%) of the title compound was obtained as a white solid.

<Example 22>

Preparation of 3,6-dimethylpyrazine-2,5-dicarboxylic acid bisphenylamide (R ¹ = CH ₃ , R ³ = C ₆ H ₅ NH in Formula 2).

2 g of baker's yeast, 3 g of saccharose and 82.5 mg (0.4 mmol) of 2-hydroxyimino-3-oxo-N-phenylbutanamide obtained in Example 8 were reduced for 5 days at pH 5 as in Example 14. 31.3 mg (Yield 45.2%) of the title compound were obtained as a yellow solid.

Melting point: 224.0-226.5 ° C .; ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 9.96 (s, 1H), 7.77-7.16 (m, 10H), 3.10 (s, 6H); ¹³ C NMR (75 MHz, CDCl ₃ ): δ (ppm) 161.79, 151.39, 143.21, 137.80, 129.55, 125.19, 120.32, 32.33, 30.11, 29.77, 23.79, 23.10, 14.53; MS m / z 346 (M ⁺ ), 329, 226, 198, 157, 93.

C ₂₀ H ₁₈ N ₄ O ₂ [M ⁺ ] by HRMS, 346.14. Found 347.24.

<Example 23>

Preparation of 3,6-dimethylpyrazine-2,5-dicarboxylic acid bisphenylamide (R ¹ = CH ₃ , R ³ = C ₆ H ₅ NH in Formula 2).

2 g of baker's yeast, 3 g of saccharose and 88.1 mg (0.4 mmol) of 2-methoxyimino-3-oxo-N-phenylbutanamide obtained in Example 9 were reduced in pH 5 for 4.5 days as in Example 14. 17.6 mg (25.4% yield) of the title compound were obtained as a yellow solid.

<Example 24>

Preparation of 3,6-dimethylpyrazine-2,5-dicarboxylic acid dibenzyl ester (R ¹ = CH ₃ , R ³ = C ₆ H ₅ CH ₂ in Formula ₂ ).

2 g of baker's yeast and 3 g of saccharose and 88.5 mg (0.4 mmol) of 2-hydroxyimino-3-oxobutyl acid benzyl ester obtained in Example 12 were reduced to 8 days at pH 5 for 8 days as in Example 14. (Yield 10.7%) of the title compound was obtained as a white solid.

Melting point: 109.3-110.6 ° C .; ¹ H NMR (CDCl ₃ ): δ (ppm) 7.48-7.26 (m, 10H), 5.45 (s, 4H), 2.75 (s, 6H); ¹³ C NMR (300 MHz, CDCl ₃ ): δ (ppm) 165.29, 151.36, 144.32, 135.49, 129.10, 129.03, 128.95, 68.31, 30.12, 22.77; MS m / z 377 (M ⁺ ), 270, 242 (100), 199, 136, 108, 91, 65.

C ₂₂ H ₂₀ N ₂ O ₄ [M ⁺ ], by HRMS, 376.41. Found 377.16.

<Example 25>

Preparation of 3,6-dimethylpyrazine-2,5-dicarboxylic acid dibenzyl ester (R ¹ = CH ₃ , R ³ = C ₆ H ₅ CH ₂ in Formula ₂ ).

2 g of baker's yeast and 3 g of saccharose and 94.1 mg (0.4 mmol) of 2-methoxyimino-3-oxobutyl acid benzyl ester obtained in Example 13 were reduced to 6 days at pH 5 for 28 days as in Example 14. (Yield 37.5%) of the title compound was obtained as a white solid.

According to the preparation method of the present invention, a pyrazine derivative of the formula (2), which is a heterocyclic compound useful in the manufacture of a pharmaceutical, can be environmentally friendly and easily prepared.

Claims (12)

1) preparing an aqueous solution of Baker's Yeast, and 2) dissolving the beta-keto alpha-oximecarbonyl derivative of Formula 1 in a hydrophilic organic solvent and adding the same to the baker's yeast aqueous solution to perform chemoselective microbial reduction. , A method for producing a pyrazine compound substituted with four functional groups represented by the formula (2). <Formula 1> <Formula 2> In which R ¹ is methyl (CH ₃ ), ethyl (CH ₃ CH ₂ ) or phenyl (C ₆ H ₅ ); R ² is hydrogen (H), methyl (CH ₃ ) or benzyl (C ₆ H ₅ CH ₂ ); R ³ is methoxy (CH ₃ O), ethoxy (CH ₃ CH ₂ O), benzyloxy (C ₆ H ₅ CH ₂ O) or phenylamine (C ₆ H ₅ NH). The process according to claim 1, wherein the chemoselective reduction reaction is carried out at a pH of 5.0 to 7.0. The method of claim 1 or 2, wherein the solvent used in step 1 comprises water, a buffer solution having a pH of 5.0 to 7.0 and a mixed solution of water and an organic solvent (volume ratio = 10: 1 to 1:10). The manufacturing method selected from the group. 4. A process according to claim 3 wherein the organic solvent is a nonpolar organic solvent selected from the group consisting of benzene, toluene and hexane. The method of claim 1, wherein the baker's yeast is selected from the group consisting of baker's yeast, which is readily available on the market, baker's yeast marketed for chemical reagents, and baker's yeast immobilized on a support. The method according to claim 5, wherein the baker's yeast fixed on the support is baker's yeast (IMBY) fixed on montmorillonite K10. The method of claim 1, wherein further sugar is added in step 1. 8. A process according to claim 7, wherein the sugar is selected from the group consisting of saccharose, D-glucose and D-galactose. The process according to claim 1, wherein the hydrophilic organic solvent for dissolving the beta-keto alpha-oximecarbonyl derivative of formula 1 is an alcohol selected from the group consisting of methanol, ethanol, propanol and butanol. 8. The process according to claim 7, wherein the ratio of baker yeast: sugar: beta-keto alpha-oximecarbonyl compound participating in the reaction is 2g: 3g: 0.4 mmol. The method according to claim 2, wherein the pH is maintained at 5.0 by using 1N HCl and 1N NaOH. The production method according to claim 1, wherein the reaction temperature is 20 ° C to 50 ° C and the reaction time is 1 day to 6 days.