Method for preparing nitrogen-containing heterocyclic compound and derivative thereof by enzymatic-chemical cascade method

Abstract

A method for preparing a nitrogen-containing heterocyclic compound and a derivative thereof by an enzymatic-chemical cascade method, comprising: reacting an alcohol, an amine, an alcohol dehydrogenase, a flavin molecule and a coenzyme in a solvent to obtain the nitrogen-containing heterocyclic compound and the derivative thereof; compared with the prior art, the method is a green and economical enzymatic-chemical cascade method, and is used for synthesizing nitrogen-containing heterocyclic compounds and derivatives thereof; compared with a common toxic chemical catalyst, the alcohol dehydrogenase is selected as a catalyst in the method, which has the characteristics of high substrate specificity, no pollution, high catalytic efficiency, no toxic solvents and simple post-treatment.

Claims

1. A method for preparing a nitrogen-containing heterocyclic compound and a derivative thereof by an enzymatic-chemical cascade method, comprising: reacting an alcohol, an amine, an alcohol dehydrogenase, a flavin molecule and a coenzyme in a solvent to obtain the nitrogen-containing heterocyclic compound and the derivative thereof; wherein, the nitrogen-containing heterocyclic compound is selected from the group consisting of ##STR00022## ##STR00023## the alcohol is selected from the group consisting of benzyl alcohol, p-methoxybenzyl alcohol, 2-furanmethanol, 2-thiophene methanol, 2-pyridine methanol, n-octanol, benzyl alcohol, p-nitrobenzyl alcohol, cyclohexanol, phenylpropanol, 2-amino-1-propanol, cyclohexyl methanol, cinnamyl alcohol and phenylethanol; the amine is selected from the group consisting of o-phenylenediamine, o-aminophenol, 2-amino-1-butanol, 2-amino-1-propanol, 3-aminopropanol, 3-amino-2-methylpropane-1-ol and 6-(3,4 diaminophenyl)-4,5 dihydro-5-methyl-3(2H)-phthalazinone; the coenzyme is any one or a combination of NADP+ and NAD+; the flavin molecule is any one of the synthetic flavin analog shown in formula I, ##STR00024## wherein, R.sub.1 and R.sub.2 are each indendently selected from hydrogen, methyl, trifluoromethyl, methoxy, halogen atom, nitro or amino; R.sub.3 is selected from hydrogen, C.sub.1-C.sub.5 alkyl, phenyl or benzyl; and X- is selected from halide ion, nitrate or trifluoromethanesulfonate.

2. The method according to claim 1, wherein the alcohol dehydrogenase is any one or a combination of ethanol dehydrogenase, horse liver alcohol dehydrogenase, yeast alcohol dehydrogenase and mannitol dehydrogenase.

3. The method according to claim 1, wherein the synthetic flavin analog is any one of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 8-chloro-1,10-ethylidene isoalloxazine chloride and 1,10-ethylidene isoalloxazine chloride.

4. The method according to claim 1, wherein the solvent is an aqueous buffer solution.

5. The method according to claim 1, wherein the reaction is performed at a pH of 4 to 10 and 30? C. to 70? C.for 2 hours to 60 hours.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The advantages of the above and/or other aspects of the present invention will become more apparent by further explaining the present invention with reference to the following drawings and detailed description.

(2) FIG. 1 is a reaction schematic diagram of Embodiment 1, wherein HLADH is a horse liver alcohol dehydrogenase and F4 is a synthetic flavin analog.

(3) FIG. 2 is a hydrogen spectrum of a product 2-phenylbenzimidazole in Embodiment 1.

(4) FIG. 3 is a carbon spectrum of the product 2-phenylbenzimidazole in Embodiment 1.

(5) FIG. 4 is a hydrogen spectrum of a product 2-(4-methoxyphenyl)benzimidazole in Embodiment 2.

(6) FIG. 5 is a carbon spectrum of the product 2-(4-methoxyphenyl)benzimidazole in Embodiment 2.

(7) FIG. 6 is a hydrogen spectrum of a product 2-furol-benzimidazole in Embodiment 3.

(8) FIG. 7 is a carbon spectrum of the product 2-furol-benzimidazole in Embodiment 3.

(9) FIG. 8 is a hydrogen spectrum of a product 2-thienyl-benzimidazole in Embodiment 4.

(10) FIG. 9 is a carbon spectrum of the product 2-thienyl-benzimidazole in Embodiment 4.

(11) FIG. 10 is a hydrogen spectrum of a product 2-pyridyl-benzimidazole in Embodiment 5.

(12) FIG. 11 is a carbon spectrum of the product 2-pyridyl-benzimidazole in Embodiment 5.

(13) FIG. 12 is a hydrogen spectrum of a product 2-heptyl-benzimidazole in Embodiment 7.

(14) FIG. 13 is a carbon spectrum of the product 2-heptyl-benzimidazole in Embodiment 7.

(15) FIG. 14 is a hydrogen spectrum of a product 2-cyclohexyl-benzimidazole in Embodiment 8.

(16) FIG. 15 is a carbon spectrum of the product 2-cyclohexyl-benzimidazole in Embodiment 8.

(17) FIG. 16 is a hydrogen spectrum of a product 2-phenyl-benzothiazole in Embodiment 9.

(18) FIG. 17 is a carbon spectrum of the product 2-thienyl-benzimidazole in Embodiment 9.

DETAILED DESCRIPTION

(19) The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The reagents and materials used are commercially available unless otherwise specified.

(20) The present invention will be further described in detail below with reference to the specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In the following embodiments, concentrations of alcohol, amine, flavin molecule and coenzyme all refer to final concentrations in the system; and a dosage of the alcohol dehydrogenase is relative to the whole reaction system.

(21) A method for producing nitrogen-containing heterocyclic compounds and derivatives thereof of the present invention uses an alcohol as a substrate, uses an NAD.sup.+-dependent horse liver alcohol dehydrogenase to catalyze the production of aldehyde with a catalytic amount of synthetic flavin analog and coenzyme in an oxygen or air atmosphere, and the generated aldehyde reacts with the amine to generate the nitrogen-containing heterocyclic compound and the derivative thereof under the chemical oxidation of the synthetic flavin analog.

(22) In the following embodiments, the enzyme activity of the horse liver alcohol dehydrogenase is 5 U/mL.

Embodiment 1

(23) Benzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze benzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated benzaldehyde reacted with 1,2-phenylenediamine to generate 2-phenylbenzimidazole, and the reaction schematic diagram was shown in FIG. 1. Benzyl alcohol was generated into benzaldehyde in a regeneration reaction system composed of synthetic flavin analog and coenzyme by using the horse liver alcohol dehydrogenase as a catalyst. The generated benzaldehyde continued to react with 1,2-phenylenediamine, and a final product 2-phenylbenzimidazole was generated under the oxidation catalysis of the synthetic flavin analog.

(24) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of benzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 48 hours. The yield was 99% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 2 and FIG. 3.

(25) ##STR00006##

Comparative Example 1

(26) As in Embodiment 1, the other amounts of the test were kept constant, but the amount of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride was changed to 0.1 mM, and the reaction lasted for 48 hours. The yield was 68% through quantitative analysis by HPLC.

Comparative Example 2

(27) As in Embodiment 1, the other amounts of the control test were kept constant, but the amount of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride was changed to 0.2 mM. The reaction lasted for 48 hours. The yield was 76% through quantitative analysis by HPLC.

Embodiment 2

(28) 4-methoxybenzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze 4-methoxybenzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated p-methoxybenzaldehyde reacted with 1,2-phenylenediamine to generate 2-(4-methoxyphenyl)benzimidazole.

(29) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of p-methoxybenzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 4 hours. The yield was 99% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 4 and FIG. 5.

(30) ##STR00007##

Embodiment 3

(31) 2-furaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze 2-furanmethanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated 2-furaldehyde reacted with 1,2-phenylenediamine to generate fuberidazole.

(32) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of 2-furanmethanol, 1 mM of NAD.sup.+, 1 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 12 hours. The yield was 88% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 6 and FIG. 7.

(33) ##STR00008##

Comparative Example 3

(34) As in Embodiment 3, the other amounts of the test were kept constant, but the amount of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride was changed to 0.5 mM, and the reaction lasted for 24 hours. The yield was 67% through quantitative analysis by HPLC.

Embodiment 4

(35) 2-thiophene methanol was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze 2-thiophene methanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated 2-thiophene formaldehyde reacted with 1,2-phenylenediamine to generate 2-(2-thienyl)-1H-benzimidazole.

(36) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of 2-thiophene methanol, 1 mM of NAD+, 1 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield was 57% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 8 and FIG. 9.

(37) ##STR00009##

Embodiment 5

(38) 2-pyridinecarboxaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze 2-pyridinemethanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated 2-pyridinecarboxaldehyde reacted with 1,2-phenylenediamine to generate 2-(2-pyridyl)-1H-benzimidazole.

(39) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of 2-pyridinemethanol, 1 mM of NAD+, 1 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield was 87% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 10 and FIG. 11.

(40) ##STR00010##

Comparative Example 4

(41) As in Embodiment 5, the other amounts of the test were kept constant, but the amount of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride was changed to 2 mM, and the reaction lasted for 24 hours. The yield was 63% through quantitative analysis by HPLC.

Embodiment 6

(42) Cinnamaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze cinnamyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated cinnamaldehyde reacted with 1,2-phenylenediamine to generate 2-(2-phenylvinyl)-1H-benzimidazole.

(43) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of cinnamyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 12 hours. The yield was 82% through quantitative analysis by HPLC.

(44) ##STR00011##

Embodiment 7

(45) Octanal was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze n-octanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated octanal reacted with 1,2-phenylenediamine to generate 2-(2-heptyl)-benzimidazole.

(46) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of n-octanol, 1 mM of NAD.sup.+, 1 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 12 hours. The yield was 72 through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 12 and FIG. 13

(47) ##STR00012##

Embodiment 8

(48) Cyclohexyl formaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze cyclohexyl methanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated cyclohexyl formaldehyde reacted with 1,2-phenylenediamine to generate 2-(cyclohexyl)-1H-benzimidazole.

(49) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of cyclohexyl methanol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 48 hours. The yield was 91% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 14 and FIG. 15.

(50) ##STR00013##

Embodiment 9

(51) Benzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze benzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated benzaldehyde reacted with 2-aminobenzenethiol to generate 2-phenylbenzothiazole.

(52) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of benzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of o-aminophenol were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield was 18% through quantitative analysis by HPLC. A NMR of the product was shown in FIG. 16 and FIG. 17.

(53) ##STR00014##

Embodiment 10

(54) Benzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze benzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated benzaldehyde reacted with 2-aminophenol.

(55) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of benzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of ortho-aminophenol were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. No 2-phenylbenzoxazole was detected.

(56) ##STR00015##

Embodiment 11

(57) 4-nitrobenzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze p-nitrobenzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated 4-nitrobenzaldehyde reacted with 1,2-phenylenediamine.

(58) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of p-nitrobenzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 1,2-phenylenediamine were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield was 15% through quantitative analysis by HPLC.

(59) ##STR00016##

Embodiment 12

(60) Phenyl acetaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze phenylethanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated phenyl acetaldehyde reacted with 2-amino-1-butanol.

(61) In a shaker a 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of phenylethanol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 2-amino-1-butanol were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield of generated 2-ethyl-5-phenyl-1H-pyrrole was 92% through quantitative analysis by HPLC.

(62) ##STR00017##

Embodiment 13

(63) Cyclohexanecarboxaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze cyclohexanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated cyclohexanecarboxaldehyde reacted with 3-aminopropanol.

(64) In a shaker a 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of cyclohexanol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 3-aminopropanol were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield of generated 5,6,7,8-tetrahydroquinoline was 40% through quantitative analysis by HPLC.

(65) ##STR00018##

Embodiment 14

(66) Benzenepropanal was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze phenylpropanol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated benzenepropanal reacted with 3-amino-methylpropane-1-ol.

(67) In a shaker a 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of phenylpropanol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 3-amino-methylpropane-1-ol were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield of generated 3-benzyl-5-benzhydrylpyridine was 50% through quantitative analysis by HPLC.

(68) ##STR00019##

Embodiment 15

(69) 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride used as a catalyst for regenerating NAD.sup.+ was coupled with a horse liver alcohol dehydrogenase to catalyze 2-amino-1-propanol, and the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride was used as an oxidizing agent for further oxidation reaction.

(70) In a shaker at 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of 2-amino-1-propanol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride and 5 U/mL of horse liver alcohol dehydrogenase were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield of generated 2,5-dimethyl pyrazine was 45% through quantitative analysis by HPLC.

(71) ##STR00020##

Embodiment 16

(72) 4-methoxybenzaldehyde was prepared by coupling 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as a catalyst for regenerating NAD.sup.+ with a horse liver alcohol dehydrogenase to catalyze p-methoxybenzyl alcohol. By using the 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride as an oxidizing agent, the generated 4-methoxybenzaldehyde reacted with 6-(3,4-diaminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one.

(73) In a shaker a 30? C. and 200 rpm, in 2 mL of 100 mM potassium phosphate buffer with a pH of 7, 5 mM of 4-methoxybenzyl alcohol, 1 mM of NAD.sup.+, 0.5 mM of 7-trifluoromethyl-N1,N10-vinyl isoalloxazine chloride, 5 U/mL of horse liver alcohol dehydrogenase and 6 mM of 6-(3,4-diaminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one were added, and the reaction solution was communicated with outside air. The reaction lasted for 24 hours. The yield of generated drug intermediate pimobendan was 50% through quantitative analysis by HPLC.

(74) ##STR00021##

(75) The present invention provides the idea and the method for preparing the nitrogen-containing heterocyclic compound and the derivative thereof by the enzymatic-chemical cascade method. There are many methods and ways to realize the technical solutions. The above are only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skills in the art can make some improvements and embellishments without departing from the principle of the present invention, and these improvements and embellishments should also be regarded as falling with the scope of protection of the present invention. All the unspecified components in the embodiments can be realized by the prior art.