Method for preparing hydrazine compound

Abstract

A method for preparing a hydrazine compound. The method comprises: using an imine reductase to catalyze a reduction reaction between a substrate ketone compound as shown in formula I or formula III and a substrate hydrazine compound as shown in formula II, to obtain a product hydrazine compound. The imine reductase is used to perform the synthesis of the hydrazine compound, the synthesis process is simple and economical, various hydrazine compounds can be directly synthesized, and fewer substituent groups are present on the N atoms of the resulting hydrazine compounds. In addition, the method can be used for industrial amplification, is low in cost and high in purity, and achieves true green chemistry.

Description

Method for preparing hydrazine compounds

[0001] This application is based on the Chinese application with CN application number 202311335104.3 and application date October 16, 2023, and the Chinese application with CN application number 202410177669.1 and application date February 8, 2024, and claims priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the technical field of enzyme catalysis reaction, in particular to a method for preparing hydrazine compounds. Background Art

[0003] Chiral hydrazines, acylhydrazides, aromatic hydrazines, and other hydrazine compounds are very important compounds with a wide range of applications in biology, agriculture, and dyes. They are also important intermediates in the synthesis of many heterocyclic compounds. Some compounds containing hydrazide structures have potential pharmaceutical activity, some of which are used clinically as active pharmaceutical ingredients (APIs).

[0004] Patent applications (WO2013083606A1, US2009286812A1) report a method for reacting ketones with hydrazine hydrate and then adding sodium borohydride to produce hydrazine compounds. Patent application (WO2012069948A1) reports the reaction of ketones with hydrazine hydrate in the presence of hydrogen and platinum-carbon. Reference (Angew. Chem., Int. Ed. 2019, 58, 15767) reports a method for reducing hydrazones to produce hydrazine compounds under cobalt and zinc catalysis. Reference (Angew. Chem., Int. Ed. 2015, 54, 5112) reports a method for reducing hydrazones to produce hydrazine compounds under nickel catalysis. Reference (ACS Catal. 2015, 5, 6086-6089) reports a method for reacting ketones and hydrazine under palladium trifluoroacetate catalysis to produce hydrazine compounds.

[0005] There are currently no reported methods for enzymatically reacting ketone compounds with hydrazine compounds to produce hydrazine compounds. Furthermore, existing metal-catalyzed methods often result in metal residues, are ligand-dependent, and produce hydrazine compounds with protecting groups such as aromatic groups and benzoyl groups, which complicates further reactions. Enzyme-catalyzed reactions require simple environments and contain fewer impurities. Therefore, it is crucial to provide a method for preparing hydrazine compounds using enzyme-catalyzed reactions.

[0006] Summary of the Invention

[0007] The main purpose of the present invention is to provide a method for preparing hydrazine compounds to solve the problem that the hydrazine compounds obtained by the existing methods have too many substituents on the N atoms, which interferes with their subsequent application effects.

[0008] To achieve the above object, according to one aspect of the present invention, a method for preparing a hydrazine compound is provided, comprising: using imine reductase to catalyze the following reduction reaction of a substrate ketone compound as shown in Formula I or Formula III and a substrate hydrazine compound as shown in Formula II to obtain a product hydrazine compound;

[0009] Wherein, R1 and R2 are each independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C 10 Cycloalkyl or C5-C 10 or R1 and R2 are combined to form a C3-C8 substituted or unsubstituted cycloalkyl, a C3-C8 substituted or unsubstituted heterocycloalkyl or a C9-C 15 R4, R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C 10 Cycloalkyl, C5-C 10 a substituted or unsubstituted heterocycloalkyl group, When cycloalkyl, phenyl or benzocycloalkyl is substituted, one or more H atoms on the cycloalkyl or benzocycloalkyl are arbitrarily substituted by halogen atoms or alkyl; one or more C atoms on the heterocycloalkyl are arbitrarily substituted by N, O, P or S atoms; when heterocycloalkyl is substituted, one or more H atoms on the heterocycloalkyl are arbitrarily substituted by halogen atoms, benzyl, tert-butyloxycarbonyl or alkyl; R3 is selected from hydrogen, C1-C4 alkyl, C6-C 10 aromatic groups, C1-C4 hydroxyalkyl groups, substituted or unsubstituted C1-C4 acyl groups, substituted or unsubstituted C6-C 10 Arylmethyl, tert-butyloxycarbonyl, benzyloxycarbonyl or When the acyl group or the arylmethyl group is substituted, one or more H atoms on the acyl group or the arylmethyl group are optionally replaced by a halogen atom, a phenyl group or an alkyl group.

[0010] Furthermore, the imine reductase is selected from any one of the imine reductases shown in Table 1.

[0011] Furthermore, R1 and R2 in the substrate ketone compound are independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C6 cycloalkyl or C5-C6 substituted or unsubstituted heterocycloalkyl; or R1 and R2 are combined to form C5-C6 substituted or unsubstituted cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl or C9-C 13 R4, R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C6 cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl, When the cycloalkyl group, phenyl group or benzocycloalkyl group is substituted, one or more H atoms on the cycloalkyl group or benzocycloalkyl group are arbitrarily replaced by F atoms, Cl atoms or methyl groups; one or more C atoms on the heterocycloalkyl group are arbitrarily replaced by N or O atoms; when the heterocycloalkyl group is substituted, one or more H atoms on the heterocycloalkyl group are arbitrarily replaced by Cl atoms, F atoms, benzyl, tert-butyloxycarbonyl or methyl groups.

[0012] Furthermore, R3 in the substrate hydrazine compound is selected from hydrogen, C1-C2 alkyl, C6 aromatic, C1-C2 hydroxyalkyl, benzyl, tert-butyloxycarbonyl, benzyloxycarbonyl,

[0013] Furthermore, the substrate ketone compound includes any one of the following:

[0014] Furthermore, the substrate hydrazine compound includes any one of the following: NH2NH2·H2O, NH2NHBoc, NH2NHCbz, NH2NH2Bn,

[0015] Furthermore, in the reaction system of the reduction reaction, the molar ratio of the substrate ketone compound to the substrate hydrazine compound is 10-100:50-200.

[0016] Furthermore, the mass ratio of the substrate ketone compound to the imine reductase is 1:1-10.

[0017] Furthermore, the reaction system of the reduction reaction also includes: glucose dehydrogenase, D-glucose, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and buffer.

[0018] The present invention utilizes the technical solution of imine reductase to synthesize hydrazine compounds using substrates of specific structures. This simple and economical synthesis process allows for the direct synthesis of a variety of hydrazine compounds, with the resulting hydrazine compounds containing fewer substituents on their nitrogen atoms. Furthermore, the process is well suited for industrial scale-up, with low cost and high purity, achieving true green chemistry. DETAILED DESCRIPTION

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0020] As mentioned in the background technology, ketone compounds are reacted with hydrazine compounds in the prior art, and the synthesis of hydrazine compounds is carried out by a multi-metal catalytic method. This synthesis method has metal residues, depends on ligands, and all that is generated are hydrazine compounds with protecting groups such as aromatic groups and benzoyl groups, which brings difficulties to further reactions. The conditions of the enzyme-catalyzed reaction are milder and do not introduce more impurities. In addition, there is no or only one substituent on the N atom in the generated hydrazine compounds, which provides convenience for the subsequent utilization of the compound. Thus, the application proposes a method for synthesizing hydrazine compounds using enzyme-catalyzed reactions, so as to achieve the purpose of efficient and industrialized production of hydrazine compounds.

[0021] In a first typical embodiment of the present invention, a method for preparing a hydrazine compound is provided, comprising: using imine reductase to catalyze the following reduction reaction of a substrate ketone compound as shown in Formula I or Formula III and a substrate hydrazine compound as shown in Formula II to obtain a product hydrazine compound;

[0022] Wherein, R1 and R2 are each independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C 10 Cycloalkyl or C5-C 10 or R1 and R2 are combined to form a C3-C8 substituted or unsubstituted cycloalkyl, a C3-C8 substituted or unsubstituted heterocycloalkyl or a C9-C 15 R4, R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C 10 Cycloalkyl, C5-C 10 a substituted or unsubstituted heterocycloalkyl group, When cycloalkyl, phenyl or benzocycloalkyl is substituted, one or more H atoms on the cycloalkyl or benzocycloalkyl are arbitrarily substituted by halogen atoms or alkyl; one or more C atoms on the heterocycloalkyl are arbitrarily substituted by N, O, P or S atoms; when heterocycloalkyl is substituted, one or more H atoms on the heterocycloalkyl are arbitrarily substituted by halogen atoms, benzyl, tert-butyloxycarbonyl or alkyl; R3 is selected from hydrogen, C1-C4 alkyl, C6-C 10aromatic groups, C1-C4 hydroxyalkyl groups, substituted or unsubstituted C1-C4 acyl groups, substituted or unsubstituted C6-C 10 Arylmethyl, tert-butyloxycarbonyl, benzyloxycarbonyl or When the acyl group or the arylmethyl group is substituted, one or more H atoms on the acyl group or the arylmethyl group are optionally replaced by a halogen atom, a phenyl group or an alkyl group.

[0023] Ketone compounds and hydrazine compounds generate hydrazones. Under the action of imine reductase, the active hydrogen of NAD(P)H undergoes nucleophilic addition to the C=N bond of the hydrazone, generating a nitrogen anion. The surrounding electropositive amino acids or water molecules then provide protons to generate hydrazine compounds, thereby obtaining product hydrazine compounds with no or only one substituent on the N atom. In a preferred embodiment, the imine reductase is an NAD(P)H-dependent enzyme. During the catalytic process of this type of enzyme, NAD(P)H is consumed as hydrogen and converted into NAD(P) + In addition, in order to save costs, glucose is oxidized to the corresponding gluconolactone and the electrons are transferred to NAD(P) + , so that the coenzyme can be recycled (see the reaction formula below).

[0024] A series of imine reductases are screened by participating in the catalytic reaction of ketone compounds and hydrazine compounds to generate hydrazine compounds, with the ability to catalyze the generation of hydrazine compounds as the screening condition.

[0025] By utilizing the above enzyme to carry out a reaction, the hydrazone generated by the substrate ketone compound and the hydrazine compound is reduced to obtain the desired product hydrazine compound with no or only one substituent on the N atom. This allows for a simpler and more convenient synthesis of the hydrazine compound, better subsequent application of the compound, and is more suitable for industrial production.

[0026] Any imine reductase capable of performing the above reaction is applicable to the present application. In a preferred embodiment, the imine reductase is selected from any one of the imine reductases shown in Table 1.

[0027] Table 1:

[0028] Any ketone compound substrate that can be used to perform a reduction reaction to generate a hydrazine compound using the above enzyme is applicable to the present application. In a preferred embodiment, R1 and R2 in the substrate ketone compound are independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C6 cycloalkyl or C5-C6 substituted or unsubstituted heterocycloalkyl; or R1 and R2 are combined to form C5-C6 substituted or unsubstituted cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl or C9-C 13 R4, R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C6 cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl, When the cycloalkyl group, phenyl group or benzocycloalkyl group is substituted, one or more H atoms on the cycloalkyl group or benzocycloalkyl group are arbitrarily replaced by F atoms, Cl atoms or methyl groups; one or more C atoms on the heterocycloalkyl group are arbitrarily replaced by N or O atoms; when the heterocycloalkyl group is substituted, one or more H atoms on the heterocycloalkyl group are arbitrarily replaced by Cl atoms, F atoms, benzyl, tert-butyloxycarbonyl or methyl groups.

[0029] Any hydrazine compound substrate that can be used to perform a reduction reaction to generate a hydrazine compound using the above enzyme is suitable for the present application. In a preferred embodiment, R3 in the substrate hydrazine compound is selected from hydrogen, C1-C2 alkyl, C6 aromatic group, C1-C2 hydroxyalkyl, C1-C4 acyl, substituted or unsubstituted arylmethyl, tert-butyloxycarbonyl, benzyloxycarbonyl, or

[0030] In a preferred embodiment, the substrate ketone compound includes any one of the following:

[0031] In a preferred embodiment, the substrate hydrazine compound includes any one of the following: NH2NH2·H2O, NNH2NHBoc, NH2NHCbz, NH2NH2Bn,

[0032] Any substrate and enzyme addition amount for the hydrazine compound synthesis reaction using imine reductase are applicable to the present application. From the perspective of improving reaction efficiency, in a preferred embodiment, in the reaction system of the reduction reaction, the molar ratio of the substrate ketone compound to the substrate hydrazine compound is 10-100:50-200; the mass ratio of the substrate ketone compound to the imine reductase is 1:1-10; specifically, the mass ratio of the substrate ketone compound to the imine reductase is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0033] In the process of using imine reductase to catalyze the reaction of substrate ketone compounds and substrate hydrazine compounds to produce product hydrazine compounds with no or only one substituent on the N atom, a chemical substance that provides electrons is required. Any chemical substance that can provide electrons is suitable for this application. In a preferred embodiment, the reaction system of the above-mentioned reduction reaction also includes: glucose dehydrogenase, D-glucose, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and a buffer. In a preferred embodiment, the buffer is Tris-HCl, the concentration of Tris-HCl is 100 mM, and the pH value of Tris-HCl is 7.5-10.0.

[0034] The above-mentioned nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate can provide reducing hydrogen for the synthesis reaction of the hydrazine compound of the present application, and can also complete its coenzyme cycle by participating in the reaction of glucose dehydrogenase catalyzing D-glucose to produce gluconolactone, thereby reducing the cost of the reaction, as shown in the following reaction formula.

[0035] Any reaction conditions capable of completing the synthesis reaction of hydrazine compounds are applicable to this application. Considering the efficiency of the reduction reaction and the energy consumption, in a preferred embodiment, the reduction reaction time is 16-20h; the reduction reaction temperature is 20-40°C. Under the reaction temperature and time conditions, the reaction efficiency is high and the energy consumption is relatively low. Any acid-base environment capable of completing the synthesis reaction of hydrazine compounds is applicable to this application. Considering the conditions suitable for the reduction reaction of the reactants, in a preferred embodiment, the pH value of the reduction reaction is 7.5-10.

[0036] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0037] Note: The following methods for detecting the conversion rate of the hydrazine compound include GC detection and HPLC detection. The data obtained can represent the conversion rate of the enzyme-catalyzed reaction used in the preparation method of this application. The selection of the detection method depends on the state of the product. If the product has a low boiling point (≤200°C), GC detection is used; if the product has a high boiling point (greater than 200°C), HPLC detection is used.

[0038] The following examples in which the chiral configurations of the products are marked are examples in which a single chiral product can be generated. Hydrazine compounds with a single chirality are a very important class of structural units, existing in drugs, agrochemicals, natural products, and chiral catalysts, and have a wide range of applications and important significance.

[0039] Example 1:

[0040] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of acetone to imine reductase in the enzyme solution was 1:8), and then 11.5 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 11.5 mg (100 mmol) acetone 12 mg (120 mmol) of hydrazine hydrate was added to 2 mL with 100 mM Tris-HCl (pH 8.5) and shaken at 30°C for 20 h. 500 μL of the system was terminated with 100 μL of 0.1 M sodium hydroxide solution, centrifuged at 12,000 rpm for 10 min, and extracted with ethyl acetate. The conversion rate was determined by GC or LC-MS. Table 2 shows the conversion rate.

[0041] Table 2:

[0042] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0043] Example 2:

[0044] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 5.48 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 5.48 mg (20 mmol) 3-cyclopentyl-3-oxopropionitrile (dissolved in 10 μL DMSO), 20 mg (200 mmol) of hydrazine hydrate, and 100 mM Tris-HCl pH 9.0 were added to 2 mL and shaken at 25°C for 16 h. 500 μL of the system was taken and terminated with 500 μL of acetonitrile. After thorough shaking and mixing, the reaction was centrifuged at 12,000 rpm for 10 min. The conversion rate was determined by HPLC or LC-MS and is shown in Table 3 below. Selected enzymes were used for chiral detection. For example, enzymes 4, 37, and 72 primarily produced R-configured products, while enzymes 5, 18, 24, 17, and 25 primarily produced S-configured products.

[0045] Table 3:

[0046] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0047] Example 3:

[0048] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:6), and then 15.68 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 15.68 mg (80 mmol) cyclohexanone (dissolved in 10 μL DMSO), 9.6 mg (96 mmol) of hydrazine hydrate, and 100 mM Tris-HCl, pH 8.0, were added to 2 mL. The mixture was shaken at 20°C for 16 h. 500 μL of the system was terminated with 100 μL of 0.1 M sodium hydroxide solution, centrifuged at 12,000 rpm for 10 min, and extracted with ethyl acetate. The conversion was determined by GC or LC-MS, as shown in Table 4 below.

[0049] Table 4:

[0050] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0051] Example 4:

[0052] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 14.6 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 14.6 mg (50 mmol) beta-tetralone (dissolved in 10 μL DMSO), 5 mg (50 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 7.5, to 2 mL. Shake and react at 35°C for 18 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 5 below.

[0053] Table 5:

[0054] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0055] Example 5:

[0056] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 3.64 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 3.64 mg (10 mmol) 6,8-difluoro-3,4-dihydro-1H-2-naphthalenone (dissolved in 10 μL DMSO), 10 mg (100 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 7.5, to 2 mL. Shake at 30°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 6 below.

[0057] Table 6:

[0058] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0059] Example 6:

[0060] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:8), and then 8.12 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 8.12 mg (20 mmol) 1-benzyl-3-methyl-4-piperidone (dissolved in 10 μL DMSO), 10 mg (100 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 10.0, to 2 mL. Shake at 40°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 7 below.

[0061] Table 7:

[0062] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0063] Example 7:

[0064] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:5), and then 5.12 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 5.12 mg (20 mmol) 1-(tetrahydro-2H-pyran-4-yl)ethanone (dissolved in 10 μL DMSO), 10 mg (100 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 9.0, to 2 mL. Shake at 30°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 8 below.

[0065] Table 8:

[0066] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0067] Example 8:

[0068] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:8), and then 6.72 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 6.72 mg (20 mmol) 1-(tetrahydro-2H-pyran-4-yl)ethanone (dissolved in 10 μL DMSO), 10 mg (100 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 8.0, to 2 mL. Shake and react at 30°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 9 below.

[0069] Table 9:

[0070] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0071] Example 9:

[0072] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:8), and then 4 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 4 mg (20 mmol) 1-(tetrahydro-2H-pyran-4-yl)ethanone (dissolved in 10 μL DMSO), 10 mg (100 mmol) hydrazine hydrate, and 100 mM Tris-HCl, pH 9.0, to 2 mL. Shake at 30°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. Conversion rates determined by HPLC or LC-MS are shown in Table 10 below.

[0073] Table 10:

[0074] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0075] Example 10:

[0076] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:5), and then 13.44 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 13.44 mg (60 mmol) 1-(tetrahydro-2H-pyran-4-yl)ethanone (dissolved in 10 μL DMSO), 10 mg (100 mmol) of hydrazine hydrate, and 100 mM Tris-HCl, pH 9.0, were added to 2 mL. The mixture was shaken at 30°C for 16 h. 500 μL of the system was terminated with 100 μL of 0.1 M sodium hydroxide solution. After thorough shaking and mixing, the mixture was extracted with ethyl acetate and the conversion rate was determined by GC or LC-MS. Table 11 shows the following.

[0077] Table 11:

[0078] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0079] Example 11:

[0080] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:5), and then 19.6 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 19.6 mg (100 mmol) cyclohexanone (dissolved in 10 μL DMSO), 52.8 mg tert-butyloxycarbonylhydrazine (200 mmol), and 100 mM Tris-HCl, pH 9.0, to 2 mL. Shake and react at 30°C for 18 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. Conversion rates determined by HPLC or LC-MS are shown in Table 12 below.

[0081] Table 12:

[0082] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0083] Example 12:

[0084] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:5), and then 19.6 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 19.6 mg (100 mmol) cyclohexanone (dissolved in 10 μL DMSO), 66.4 mg of benzyloxycarbonylhydrazine (200 mmol), and 100 mM Tris-HCl, pH 9.0, to make up to 2 mL. Shake at 30°C for 18 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. Conversion rates determined by HPLC or LC-MS are shown in Table 13 below.

[0085] Table 13:

[0086] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0087] Example 13:

[0088] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 7.2 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 7.2 mg (50 mmol) 2-butanone 12 mg (120 mmol) of hydrazine hydrate was added to 2 mL with 100 mM Tris-HCl, pH 8.5, and the mixture was shaken at 37°C for 16 h. 500 μL of the system was terminated with 100 μL of 0.1 M sodium hydroxide solution, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. The conversion rate was determined by GC or LC-MS, as shown in Table 14 below.

[0089] Table 14:

[0090] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0091] Example 14:

[0092] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 4.8 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 4.8 mg (20 mmol) acetophenone (dissolved in 10 μL DMSO), 20 mg (200 mmol) of hydrazine hydrate, and 100 mM Tris-HCl, pH 8.5, to 2 mL. Shake and react at 37°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. The conversion rate is shown in Table 15 below, as determined by HPLC or LC-MS.

[0093] Table 15:

[0094] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0095] Example 15:

[0096] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of acetone to imine reductase in the enzyme solution was 1:8), and then 4.48 mg GDH, 21.6 mg Glu, 1.33 mg NAD +, 1.53mg NADP + , 4.48 mg (20 mmol) trifluoroacetone 12 mg (120 mmol) of hydrazine hydrate was added to 2 mL with 100 mM Tris-HCl, pH 9.0, and the mixture was shaken at 30°C for 20 h. 500 μL of the system was terminated with 100 μL of 0.1 M sodium hydroxide solution, centrifuged at 12,000 rpm for 10 min, and extracted with ethyl acetate. The conversion rate was determined by GC or LC-MS. Table 16 shows the following.

[0097] Table 16:

[0098] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0099] Example 16:

[0100] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:6), and then 3.92 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 3.92 mg (20 mmol) cyclohexanone (dissolved in 10 uL DMSO), 35.2 mg (200 mmol) tert-butyl hydrazine Make up to 2 mL with 100 mM Tris-HCl, pH 9.0, and shake at 37°C for 16 h. Terminate the reaction with 500 μL of the system and 100 μL of 0.1 M sodium hydroxide solution. Mix thoroughly by shaking, then centrifuge at 12,000 rpm for 10 min. GC or LC-MS analysis of the conversion yield is shown in Table 17 below.

[0101] Table 17:

[0102] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0103] Example 17:

[0104] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 7.84 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 7.84 mg (40 mmol) cyclohexanone (dissolved in 10 μL DMSO), 43.2 mg (200 mmol) phenylhydrazine Make up to 2 mL with 100 mM Tris-HCl, pH 8.5, and shake at 37°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, shake thoroughly, and centrifuge at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 18 below.

[0105] Table 18:

[0106] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0107] Example 18:

[0108] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:5), and then 15.68 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 15.68 mg (80 mmol) cyclohexanone (dissolved in 10 μL DMSO), 30.4 mg (200 mmol) 2-hydroxyethylhydrazine Make up to 2 mL with 100 mM Tris-HCl, pH 9, and shake at 37°C for 16 h. Terminate the reaction with 100 μL of 0.1 M sodium hydroxide solution in 500 μL of the system. Mix thoroughly by shaking, then centrifuge at 12,000 rpm for 10 min. GC or LC-MS analysis of the conversion rate is shown in Table 19 below.

[0109] Table 19:

[0110] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0111] Example 19:

[0112] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 3.92 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 3.92 mg (20 mmol) cyclohexanone (dissolved in 10 μL DMSO), 12.3 mg (200 mmol) benzylhydrazine (NH2NH2Bn), and 100 mM Tris-HCl, pH 8.5, to make up to 2 mL. Shake and react at 37°C for 16 h. 500 μL of the system was taken and terminated with 500 μL of acetonitrile. After thorough shaking and mixing, the reaction was centrifuged at 12,000 rpm for 10 min. The conversion rate was determined by HPLC or LC-MS, as shown in Table 20 below.

[0113] Table 20:

[0114] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0115] Example 20:

[0116] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 3.92 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 3.92 mg (20 mmol) cyclohexanone (dissolved in 10 μL DMSO), 54.5 mg (200 mmol) benzoyl hydrazide Make up to 2 mL with 100 mM Tris-HCl, pH 9.0, and shake at 37°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, shake thoroughly, and centrifuge at 12,000 rpm for 10 min. HPLC or LC-MS analysis of the conversion yield is shown in Table 21 below.

[0117] Table 21:

[0118] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0119] Example 21:

[0120] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 3.92 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 3.92 mg (20 mmol) cyclohexanone (dissolved in 10 μL DMSO), 29.6 mg (200 mmol) acetic acid hydrazide Make up to 2 mL with 100 mM Tris-HCl, pH 9.0, and shake at 37°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shake, and centrifuge at 12,000 rpm for 10 min. Analyze by LC-MS. Alternatively, terminate the reaction with 100 μL of 0.1 M sodium hydroxide solution, centrifuge at 12,000 rpm for 10 min, extract with ethyl acetate, and analyze by GC. The conversion rate is shown in Table 22 below.

[0121] Table 22:

[0122] In the above table, + represents a conversion rate of 0.01 to 1%, ++ represents a conversion rate of 1 to 10%, +++ represents a conversion rate of 10 to 30%, ++++ represents a conversion rate of 30 to 50%, and +++++ represents a conversion rate of more than 50%.

[0123] Example 22:

[0124] In a 10 mL reaction bottle, 306 imine reductase enzyme solutions as shown in Table 1 were added (the mass ratio of substrate ketone to imine reductase in the enzyme solution was 1:10), and then 3.92 mg GDH, 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 1.96 mg (10 mmol) cyclohexanone (dissolved in 20uL DMSO), 50.5mg (100mmol) 9-fluorenylmethoxycarbonylhydrazine Make up to 2 mL with 100 mM Tris-HCl, pH 9.0, and shake at 37°C for 16 h. Terminate the reaction with 500 μL of acetonitrile, thoroughly shaken, and centrifuged at 12,000 rpm for 10 min. The reaction was analyzed by LC-MS. The conversion rates are shown in Table 23 below.

[0125] Table 23:

[0126] Example 23:

[0127] We selected 16 different ketones and reacted them with hydrazine hydrate under the action of enzymes 1-20: in a 10 mL reaction bottle, the imine reductase enzyme solution with enzyme numbers 1-20 as shown in Table 1 was added (the mass ratio of acetone to imine reductase in the enzyme solution was 1:10), and then 1 wt GDH (the mass ratio of 1 ketone compound), 21.6 mg Glu, 1.33 mg NAD + , 1.53mg NADP + , 20mmol ketone (dissolved in 10uL DMSO), 20mg (200mmol) hydrazine hydrate, 100mM Tris-HCl pH9.0 to 2mL, shake at 30°C for 16h. Take 500uL of the system and terminate the reaction with 500uL acetonitrile, shake thoroughly and mix, centrifuge at 12000rpm for 10min and send for detection (HPLC or LC-MS), or take 500uL of the system and terminate the reaction with 100uL 0.1M sodium hydroxide solution, centrifuge at 12000rpm for 10min, extract with ethyl acetate and send for detection (GC). The detection conversion rate is shown in the following Tables 24-25.

[0128] Table 24:

[0129] Table 25:

[0130] As can be seen from the above description, the above-described embodiments of the present invention achieve the following technical effects: providing a method for directly reacting a ketone compound with a hydrazine compound under the catalysis of imine reductase to produce a hydrazine compound having no or only one substituent on the nitrogen atom. This synthesis process is simple and economical, and can directly synthesize a variety of hydrazine compounds. Furthermore, this technical solution has mild conditions, readily available and low-cost raw materials and catalysts, simple operation, environmental friendliness, and suitability for industrial production.

[0131] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrazine compound, characterized in that: The method comprises: Using imine reductase to catalyze the following reduction reaction of a substrate ketone compound as shown in formula I or formula III and a substrate hydrazine compound as shown in formula II to obtain a product hydrazine compound; Wherein, R1 and R2 are each independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C 10 Cycloalkyl or C5-C 10 or R1 and R2 are combined to form a C3-C8 substituted or unsubstituted cycloalkyl, a C3-C8 substituted or unsubstituted heterocycloalkyl or a C9-C 15 A substituted or unsubstituted benzocycloalkyl group; R4 and R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C 10 Cycloalkyl, C5-C 10 A substituted or unsubstituted heterocycloalkyl group, When the cycloalkyl group, the phenyl group or the benzocycloalkyl group is substituted, one or more H atoms on the cycloalkyl group or the benzocycloalkyl group are arbitrarily substituted with a halogen atom or an alkyl group; One or more C atoms on the heterocycloalkyl group are arbitrarily substituted by N, O, P, or S atoms. When the heterocycloalkyl group is substituted, one or more H atoms on the heterocycloalkyl group are arbitrarily substituted by halogen atoms, benzyl, tert-butyloxycarbonyl, or alkyl groups; R3 is selected from hydrogen, C1-C4 alkyl, C6-C 10 aromatic group, C1-C4 hydroxyalkyl group, substituted or unsubstituted C1-C4 acyl group, substituted or unsubstituted C6-C 10 Arylmethyl, tert-butyloxycarbonyl, benzyloxycarbonyl or When the acyl group or the arylmethyl group is substituted, one or more H atoms on the acyl group or the arylmethyl group are arbitrarily substituted with a halogen atom, a phenyl group or an alkyl group.

2. The method according to claim 1, characterized in that: The imine reductase is selected from any one of the imine reductases shown in Table 1.

3. The method according to claim 1, characterized in that R1 and R2 in the substrate ketone compound are independently selected from hydrogen, cyano, Pyridine, furan, C1-C4 alkyl, C5-C6 cycloalkyl or C5-C6 substituted or unsubstituted heterocycloalkyl; or R1 and R2 are combined to form C5-C6 substituted or unsubstituted cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl or C9-C 13 A substituted or unsubstituted benzocycloalkyl group; R4 and R5 are each independently selected from trifluoromethyl, cyano, arylmethyl, methoxy, substituted or unsubstituted phenyl, C1-C4 alkyl, C5-C6 cycloalkyl, C5-C6 substituted or unsubstituted heterocycloalkyl, When the cycloalkyl group, the phenyl group or the benzocycloalkyl group is substituted, one or more H atoms on the cycloalkyl group or the benzocycloalkyl group are arbitrarily substituted by F atoms, Cl atoms or methyl groups; One or more C atoms on the heterocycloalkyl group are arbitrarily replaced by N or O atoms. When the heterocycloalkyl group is substituted, one or more H atoms on the heterocycloalkyl group are arbitrarily replaced by Cl atoms, F atoms, benzyl, tert-butyloxycarbonyl or methyl.

4. The method according to claim 1, characterized in that: R3 in the substrate hydrazine compound is selected from hydrogen, C1-C2 alkyl, C6 aromatic, C1-C2 hydroxyalkyl, benzyl, tert-butyloxycarbonyl, benzyloxycarbonyl, 5. The method according to claim 3, characterized in that: The substrate ketone compound includes any one of the following:

6. The method according to claim 4, characterized in that The substrate hydrazine compound includes any one of the following: NH2NH2·H2O, NH2NHBoc, NH2NHCbz, NH2NH2Bn, 7. The method according to claim 1, characterized in that In the reaction system of the reduction reaction, the molar ratio of the substrate ketone compound to the substrate hydrazine compound is 10-100:50-200.

8. The method according to claim 1, characterized in that: The mass ratio of the substrate ketone compound to the imine reductase is 1:1-10.

9. The method according to claim 1, characterized in that: The reduction reaction system also includes: glucose dehydrogenase, D-glucose, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and buffer.

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