Preparation method of (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile

Abstract

The invention provides a preparation method of (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile, which comprises the following steps: carrying out reduction reaction on 1-((2-ethoxyl) imino)-2, 3-dihydro-1H-indene-4-nitrile or a mixture containing 1-oxo-2, 3-dihydro-1H-indene-4-nitrile and ethanolamine under the catalysis of imine reductase to obtain (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile, namely (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile or (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile. The compound shown in the formula I is obtained, namely (S)-1-((2-ethoxyl) amino)-2, 3-dihydro-1H-indene-4-nitrile. By adopting the method disclosed by the invention, a target compound can be efficiently and accurately synthesized, and when imine reductase is used as a catalyst in the reaction, the synthesis of chiral reductive amine can be realized under a very mild condition. The catalytic reaction provided by the invention has high stereoselectivity, and a target compound with a single stereostructure can be accurately synthesized.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a method for preparing (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile. Background Art

[0002] Ozamod is a potential sphingosine phosphate 1 receptor agonist jointly developed by the Scripps Research Institute and Celgene Corporation in the United States for the treatment of multiple sclerosis (MS) and ulcerative colitis. It can induce sequestration of peripheral blood lymphocytes and reduce the number of activated lymphocytes circulating in the gastrointestinal tract. (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile (the compound shown in Formula I) is a key intermediate in the synthesis of Ozamod. Currently, the methods for preparing and synthesizing (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile (the compound shown in Formula I) in the existing technology have some problems, including low yield, complex steps, or the use of expensive metal catalysts, resulting in high synthesis costs and environmental pollution.

[0003] Therefore, there is an urgent need to develop a green, environmentally friendly and efficient method to prepare (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile, which can help reduce costs, increase yields and provide a feasible solution for the simple synthesis of this compound. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. In order to solve the above problems, a green, environmentally friendly and efficient method has been developed to prepare (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile. The present invention uses enzyme engineering technology to synthesize the compound for the first time. Specifically, the inventors constructed Escherichia coli expressing imine reductase, ketoreductase and glucose dehydrogenase. By expressing imine reductase, ketoreductase / glucose dehydrogenase in Escherichia coli, and under the synergistic action of coenzyme cycle reaction substrates and coenzymes, the target compound (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile (compound of formula I) was successfully synthesized. Compared with chemical synthesis methods, this method has the following advantages:

[0005] (1) No expensive and environmentally polluting metal catalysts are required; compared to the catalysts used in chemical synthesis methods, this method utilizes imine reductase to achieve the synthesis of chiral reduced amines under very mild conditions, avoiding the use of expensive catalysts;

[0006] (2) The E. coli expression system constructed through genetic recombination technology provides an efficient enzyme expression platform, making the synthesis process of the target compound more efficient and controllable;

[0007] (3) The synthesis of chiral reductive amination is chemically very difficult, but through the catalytic action of imine reductase, the chemical synthesis process can be achieved under mild conditions, thereby greatly simplifying the synthesis steps and improving the product yield, purity and optical purity.

[0008] Therefore, in the first aspect of the present invention, the present invention provides a method for preparing a compound shown in Formula I, characterized in that it comprises: 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile, or a mixture comprising 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and ethanolamine, undergoing a reduction reaction under the catalysis of imine reductase to obtain the compound shown in Formula I.

[0009]

[0010] The method described in the present invention can efficiently and accurately synthesize the target compound (compound shown in Formula I). ​​The inventors have found through a large number of experimental studies that when the reaction adopts imine reductase as a catalyst, the synthesis of chiral reduced amine can be achieved under very mild conditions. This catalytic reaction has a high degree of stereoselectivity and can accurately synthesize a single chiral form of the compound, ensuring the optical purity and quality of the synthesized product. Therefore, this method not only has high synthesis efficiency, but also can ensure the optical purity and quality of the product, providing a reliable and sustainable solution for the preparation of the compound.

[0011] According to an embodiment of the present invention, the imine reductase is produced by expression in a first wet bacterial cell. The inventors have discovered through extensive experimental research that only by using the recombinant bacteria constructed with a gene encoding a specific imine reductase provided by the present invention can the efficient synthesis of chiral reduced amines and the accurate synthesis of a target compound having a single stereostructure with high purity, enantiomeric excess (ee), and yield be achieved.

[0012] According to an embodiment of the present invention, the first wet cell is selected from but not limited to recombinant Escherichia coli wet cells, and may also be selected from other bacteria to construct recombinant wet cells.

[0013] According to an embodiment of the present invention, the imine reductase has the amino acid sequence shown in SEQ ID NO: 2.

[0014] MKPTLTVIGAGRMGSALIKAFLQSGYTTTVWNRTKAKSEPLAKLGAHLADTVRDAVKRSDIIVVNVLDYDTSDQLLRQDEVTRELRGKLLVQLTSGSPALAREQETWARQHGIDYLDGAIMATPDFIGQAECALLYSGSAALFEKHRA VLNVLGGATSHVGEDVGHASALDSALLFQMWGTLFGTLQALAISRAEGIPLEKTTAFIKLTEPVTQGAVADVLTRVQQNRLTADAQTLASLEAHNVAFQHLLALCEERNIHRGVADAMYSVIREAVKAGGHGKDDFAILTRFLK(SEQID NO:2)

[0015] It should be noted that the "amino acid sequence of imine reductase" described in the present invention is derived from Myxococcus stipitatus.

[0016] According to an embodiment of the present invention, the nucleotide encoding the imine reductase has the sequence shown in SEQ ID NO: 1.

[0017] ATGAAACCGACCCTGACCGTTATTGGCGCTGGCCGTATGGGCTCCGCACTGATTAAAGCATTCCTGCAATCTGGCTACACGACCACGGTGTGGAACCGTACCAAAGCCAAAAGCGAACCGCTGGCAAAACTGGGCGCACATCTGGCTGATACGGTGCGTGACGCCGTTAAACGCAGCGATATTATCGTGGTTAATGTGCTGGATTATGACACCTCTGATCAGCTGCTGCGCCAAGACGAAGTGACGCGTGAACTGCGCGGCAAACTGCTGGTTCAGCTGACCAGCGGTTCTCCGGCACTGGCTCGTGAACAGGAAACGTGGGCGCGCCAACATGGCATTGATTATCTGGACGGTGCGATCATGGCCACCCCGGATTTTATTGGCCAGGCAGAATGCGCTCTGCTGTACAGTGGTTCCGCGGCCCTGTTCGAAAAACACCGTGCTGTCCTGAATGTGCTGGGCGGTGCCACCAGCCATGTCGGCGAAGATGTTGGTCATGCCTCAGCACTGGACAGCGCCCTGCTGTTTCAGATGTGGGGCACCCTGTTCGGTACGCTGCAAGCACTGGCTATTTCTCGCGCAGAAGGCATCCCGCTGGAAAAAACCACGGCGTTTATCAAACTGACCGAACCGGTCACCCAGGGTGCCGTTGCAGATGTCCTGACCCGTGTTCAGCAAAATCGCCTGACCGCAGACGCTCAGACGCTGGCAAGTCTGGAAGCTCATAACGTGGCGTTCCAACACCTGCTGGCCCTGTGTGAAGAACGTAATATCCATCGCGGTGTTGCGGATGCCATGTACTCCGTTATTCGTGAAGCGGTCAAAGCCGGCCACGGTAAAGATGACTTTGCAATTCTGACCCGCTTCCTGAAATAA(SEQ ID NO:1)

[0018] According to an embodiment of the present invention, in the mixture comprising 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and ethanolamine, the molar ratio of the 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile to the ethanolamine is 1:(0.5-1.5). Therefore, the target compound can be produced with high purity and yield in the presence of imine reductase.

[0019] According to an embodiment of the present invention, the reduction reaction is carried out in the presence of a hydrogen supply system. Therefore, NADH / NADPH can be continuously generated through the coenzyme cycle, thereby promoting the formation of the target product and improving the yield of the target product.

[0020] According to an embodiment of the present invention, the hydrogen supply system includes at least one of an additional enzyme, a coenzyme cycle reaction substrate, and a coenzyme. The coenzyme cycle reaction substrate serves to provide the hydrogen atoms and electrons required for the reaction, thereby carrying out the redox reaction. The additional enzyme serves to circulate the coenzyme, promoting the conversion of the coenzyme nicotinamide adenine dinucleotide NAD+ or nicotinamide adenine dinucleotide phosphate NADP+ to reduced nicotinamide adenine dinucleotide NADH or reduced nicotinamide adenine dinucleotide phosphate NADPH. Therefore, in the presence of the additional enzyme, the coenzyme cycle reaction substrate, and the coenzyme, the hydrogen supply system can circulate the coenzyme, thereby promoting the continuous reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0021] According to an embodiment of the present invention, the additional enzyme is selected from ketoreductase or glucose dehydrogenase. Ketoreductase has the ability to reduce ketone functional groups, transferring hydrogen atoms and electrons to NAD+ or NADP+; glucose dehydrogenase functions in the hydrogen supply system by converting glucose into gluconic acid and producing the reduced coenzyme NADH or NADPH.

[0022] According to an embodiment of the present invention, the substrate of the coenzyme cycle reaction is selected from isopropanol or glucose. Therefore, it can provide chemical groups for hydrogen atom and electron transfer, be oxidized or reduced by enzymes, and thus participate in the redox process in the reaction.

[0023] According to an embodiment of the present invention, the coenzyme is selected from nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+). In the hydrogen supply system, NAD+ and NADP+ act as oxidants, accepting hydrogen atoms (H + ) and electrons (e - ), thus acting as a catalyst in the reaction. Through this process, NAD+ is reduced to NADH and NADP+ is reduced to NADPH.

[0024] According to an embodiment of the present invention, the additional enzyme is selected from ketoreductase, the substrate for the coenzyme cycle reaction is selected from isopropanol, and the coenzyme is selected from nicotinamide adenine dinucleotide. Thus, a cyclic reaction of the hydrogen supply system can be achieved, thereby promoting the continuous progress of the reduction reaction described in the present invention, generating the target product, and improving the yield and purity.

[0025] According to an embodiment of the present invention, the additional enzyme is selected from glucose dehydrogenase, the substrate for the coenzyme cycle reaction is selected from glucose, and the coenzyme is selected from nicotinamide adenine dinucleotide phosphate. Thus, a cyclic reaction of the hydrogen supply system can be achieved, thereby promoting the continuous progress of the reduction reaction described in the present invention, generating the target product, and improving the yield and purity.

[0026] According to an embodiment of the present invention, the ketoreductase is produced by expression in a second wet cell.

[0027] According to an embodiment of the present invention, the glucose dehydrogenase is produced by expression of the third wet bacteria.

[0028] According to an embodiment of the present invention, the second wet cell and the third wet cell are also selected from but not limited to recombinant Escherichia coli wet cells, and can also be selected from other bacteria to construct recombinant wet cells.

[0029] According to an embodiment of the present invention, the reduction reaction is carried out in the presence of a buffer. Therefore, it can play an important role in maintaining pH stability, maintaining ionic strength, and providing a stable solution environment in the reduction reaction system, thereby promoting the reaction and improving the quality and yield of the product.

[0030] According to an embodiment of the present invention, the pH value of the buffer solution is 5-9.

[0031] According to an embodiment of the present invention, the buffer is selected from at least one of a citric acid buffer, a TricHCl buffer solution, a phosphate buffer and a TEOA buffer.

[0032] According to an embodiment of the present invention, the concentration of the imine reductase in the reduction reaction system is 0.1 g / L to 5 g / L. Therefore, the reaction of 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile or a mixture comprising 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and ethanolamine can be promoted to completion, thereby increasing the yield of the target product.

[0033] According to an embodiment of the present invention, the concentration of the imine reductase in the reduction reaction system is 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.3 g / L, 1.5 g / L, 1.8 g / L, 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, 3 g / L, 3.3 g / L, 3.5 g / L, 3.8 g / L, 4 g / L, 4.3 g / L, 4.5 g / L, 4.8 g / L or 5 g / L.

[0034] According to an embodiment of the present invention, the concentration of the 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile or 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile in the reduction reaction system is 1 g / L to 100 g / L, for example, 1 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L.

[0035] According to an embodiment of the present invention, the concentration of the ketoreductase in the reduction reaction system is 0.04 g / L to 2 g / L. Therefore, a cyclic reaction of the hydrogen supply system can be ensured, thereby promoting the continuous progress of the reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0036] According to an embodiment of the present invention, the concentration of the ketoreductase in the reduction reaction system is 0.04 g / L, 0.08 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L or 2 g / L.

[0037] According to an embodiment of the present invention, the concentration of glucose dehydrogenase in the reduction reaction system is 0.1 g / L to 5 g / L. Therefore, the cyclic reaction of the hydrogen supply system can be ensured, thereby promoting the continuous progress of the reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0038] According to an embodiment of the present invention, the concentration of glucose dehydrogenase in the reduction reaction system is 0.1g / L, 0.3g / L, 0.5g / L, 0.8g / L, 1g / L, 1.3g / L, 1.5g / L, 1.8g / L, 2g / L, 2.3g / L, 2.5g / L, 2.8g / L, 3g / L, 3.3g / L, 3.5g / L, 3.8g / L, 4g / L, 4.3g / L, 4.5g / L, 4.8g / L or 5g / L.

[0039] According to an embodiment of the present invention, the concentration of the coenzyme in the reduction reaction system is 0.2 g / L to 1 g / L. Therefore, the cyclic reaction of the hydrogen supply system can be ensured, thereby promoting the continuous progress of the reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0040] According to an embodiment of the present invention, the concentration of the coenzyme in the reduction reaction system is 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L.

[0041] According to an embodiment of the present invention, the volume concentration of the isopropyl alcohol in the reduction reaction system is 5% to 20%. Therefore, the cyclic reaction of the hydrogen supply system can be ensured, thereby promoting the continuous progress of the reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0042] According to an embodiment of the present invention, the volume concentration of the isopropanol in the reduction reaction system is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0043] According to an embodiment of the present invention, the concentration of glucose in the reduction reaction system is 7.5 g / L to 37.5 g / L. Therefore, the cyclic reaction of the hydrogen supply system can be ensured, thereby promoting the continuous progress of the reduction reaction of the present invention, generating the target product, and improving the yield and purity.

[0044] According to an embodiment of the present invention, the concentration of glucose in the reduction reaction system is 7.5g / L, 10g / L, 12g / L, 14g / L, 16g / L, 18g / L, 20g / L, 22g / L, 24g / L, 26g / L, 28g / L, 30g / L, 32g / L, 34g / L, 36g / L or 37.5g / L.

[0045] According to an embodiment of the present invention, the reduction reaction is carried out at 25-40°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0046] According to an embodiment of the present invention, the reduction reaction is carried out under magnetic stirring.

[0047] According to an embodiment of the present invention, the reduction reaction time is 15 to 30 hours, for example, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours or 30 hours.

[0048] According to an embodiment of the present invention, the method for preparing the compound of formula I further comprises: extracting the product of the reduction reaction, and drying the extracted product to obtain the compound of formula I.

[0049] According to an embodiment of the present invention, the extractant in the extraction treatment is selected from ethyl acetate.

[0050] According to an embodiment of the present invention, the desiccant in the drying process is selected from anhydrous sodium sulfate.

[0051] According to an embodiment of the present invention, the first wet cell is prepared by fermenting a recombinant Escherichia coli carrying a nucleotide sequence expressing the imine reductase to obtain the first wet cell. The method can produce recombinant Escherichia coli expressing the imine reductase, i.e., the first wet cell.

[0052] According to an embodiment of the present invention, the OD of Escherichia coli 600 When the value is 3 to 4, the recombinant E. coli after fermentation is collected to obtain the first wet bacteria. At this time, the bacteria concentration is high and the enzyme activity is good.

[0053] According to an embodiment of the present invention, the recombinant Escherichia coli carrying the nucleotide sequence expressing the imine reductase is obtained by introducing a first vector carrying the nucleotide sequence expressing the imine reductase into a recipient Escherichia coli.

[0054] According to an embodiment of the present invention, the recipient Escherichia coli may further include a second vector carrying a nucleotide sequence for expressing ketoreductase or a nucleotide sequence for expressing glucose dehydrogenase.

[0055] According to an embodiment of the present invention, the first vector and the second vector are respectively selected from pET-28a.

[0056] According to an embodiment of the present invention, the nucleotide sequence expressing the ketoreductase is shown as SEQ ID NO: 3.

[0057] ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGCAATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCGCGCTGATGTAGGTGAACGCGCTGC CAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGATGCTTCTGATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGTTGTCAACAATGCCGGCATCGGTGTTGT CAAGAGTGTTGAAGATACCACAACTGAAGAATGGCACAAGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCCAACGTATGAAGAATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCTATGGTATGGTTGGTGATCCAAGTGTGGGTGCATACAACGCTTCAAAAGGTGCTGTCCGTATTATGTCTAAATCAGCTGCCTTGGATTGCGCT TTGAAGGACTACGATGTTTCGGGTTAACACTGTTCATCCAGGTCCGATCAAGACACCAATGCTTGACGATGTTGAAGGGGCAGAAGAAATGTGGTCACAGCGGACCAAGACACCAATGGG TCATATCGGTGAACCTAACGATATCGCTTGGGTCTGTGTTTACCTGGCATCTGGCGAATCTAAATTTGCCACTGGTGCAGAATTCGTTATCGATGGTGGATGGACTGCTCAATAA(SEQ ID NO:3)

[0058] According to an embodiment of the present invention, the nucleotide sequence expressing the glucose dehydrogenase is shown in SEQ ID NO:4.

[0059] (SEQ ID NO:4)

[0060] According to an embodiment of the present invention, the second wet cell is obtained by fermenting a recombinant Escherichia coli carrying a nucleotide sequence expressing the ketoreductase. The method can be used to prepare a recombinant Escherichia coli expressing the ketoreductase, i.e., the second wet cell.

[0061] According to an embodiment of the present invention, the OD value of the recombinant Escherichia coli carrying the nucleotide sequence expressing the ketoreductase after fermentation is 600When the value is 3 to 4, the recombinant E. coli after fermentation is collected to obtain the second wet bacterial cells. At this time, the bacterial cell concentration is high and the enzyme activity is good.

[0062] According to an embodiment of the present invention, the third wet cell is obtained by fermenting a recombinant Escherichia coli carrying a nucleotide sequence expressing the glucose dehydrogenase. Using the method described above, a recombinant Escherichia coli expressing glucose dehydrogenase, i.e., the third wet cell, can be prepared.

[0063] According to an embodiment of the present invention, the OD value of the recombinant Escherichia coli carrying the nucleotide sequence expressing the glucose dehydrogenase after fermentation is 600 When the value is 3 to 4, the recombinant E. coli after fermentation is collected to obtain the third wet bacterial cell. At this time, the bacterial cell concentration is higher and the enzyme activity is better.

[0064] According to an embodiment of the present invention, the nucleotide sequence expressing the ketoreductase is shown as SEQ ID NO: 3.

[0065] According to an embodiment of the present invention, the nucleotide sequence expressing the glucose dehydrogenase is shown in SEQ ID NO:4.

[0066] In the second aspect of the present invention, the present invention provides the use of the compound of formula I prepared by the method described in the first aspect in the preparation of Ozamod.

[0067] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0068] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0069] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0070] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0071] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0072] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0073] Example 1: Preparation of recombinant Escherichia coli wet cells

[0074] The imine reductase gene sequence (shown in SEQ ID NO: 1), ketoreductase gene sequence (shown in SEQ ID NO: 3), and glucose dehydrogenase gene sequence (shown in SEQ ID NO: 4) treated with Nde I and Xho I restriction endonucleases were ligated with a pET-28a empty vector treated with the same restriction endonucleases and transformed into Escherichia coli (calcium chloride chemical transformation). Single clones were identified and sequenced to obtain recombinant strains 1, 2, and 3, respectively, containing the aforementioned gene sequences. These strains were then stored separately. Glycerol tubes containing recombinant strains 1, 2, and 3 were removed from the -80°C laboratory freezer and thawed. The bacterial suspension was streaked onto a resistant LB solid plate (containing 50 ng / μl kanamycin) using an inoculating loop in a clean bench. The plate was then incubated inverted at 37°C overnight. Colonies were selected and inoculated into 5 ml of LB liquid medium (containing 50 ng / μl kanamycin) and incubated at 37°C, shaking at 220 rpm for approximately 16 hours. 5 ml of bacterial solution was inoculated into 100 ml of LB medium (containing 50 ng / μl kanamycin) and cultured at 37°C, 220 r / min, and shaken until OD 600 The value was 0.6-0.8, the temperature was lowered to 25 ° C, and isopropyl-β-D-thiogalactoside (IPTG) solution was added to a final concentration of 0.2 mmol / L, and then the induction culture was continued for 16-20 h until the OD 600 The value was set to 3-4. The precipitated cells were collected by centrifugation at 4000 r / min for 30 min to obtain the recombinant Escherichia coli wet cells expressing imine reductase, the recombinant Escherichia coli wet cells expressing ketoreductase and the recombinant Escherichia coli wet cells expressing glucose dehydrogenase respectively.

[0075] Example 2: Preparation of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile

[0076] (1) Option 1

[0077] Synthesis route:

[0078]

[0079] Synthesis process:

[0080] In a 25 mL reaction flask, 100 mg of 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile, 500 mg of the recombinant E. coli wet cells expressing imine reductase prepared in Example 1 (the concentration of the recombinant E. coli wet cells is 5 g / L, and each 1 mg of the recombinant E. coli wet cells contains 0.1 mg of imine reductase), and 10 mg of the recombinant E. coli wet cells expressing ketoreductase prepared in Example 1 (the reaction mixture can also be prepared according to the literature Hummel W, Grger H. Strategies for regeneration of nicotinamide coenzymes emphasizing self-sufficient closed-loop recycling systems. J Biotechnol., 2014, 191:22-31). The final concentration of recombinant E. coli expressing ketoreductase in the reaction system was 1 g / L (0.2 mg of ketoreductase per 1 mg of recombinant E. coli wet cells), 0.5 mL of isopropanol, and 10 mg of nicotinamide adenine dinucleotide (NAD+). TricHCl buffer (0.1 M, pH 8.5) was added to a volume of 10 mL. After magnetic stirring at 35°C for 24 hours, the reaction was inactivated with 5 mL of ethyl acetate and extracted with 10 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate solid, and concentrated to yield 91 mg of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile as a white solid. The product had a purity of 99%, an ee greater than 99%, and a yield of 90%.

[0081] The hydrogen spectrum of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile is characterized as follows:

[0082] 1H NMR (400MHz, DMSO-d6): δ (ppm) 7.50-7.31 (m, 3H), 4.04 (m, 1H), 3.62 (q, 2H, J = 5.5Hz), 3.57-3.43 (m, 1H), 3.27 (ddd, 1H, J1 = 17.6Hz, J2 = 9.1Hz, J3 = 5 .0Hz), 3.15-2.85 (m, 1H), 2.74 (m, 2H), 2.58 (dtd, 1H, J1=9.0Hz, J2=5.5Hz, J3=5.3Hz, J4=3.6Hz), 2.20 (ddd, 1H, J1=13.4Hz, J2=8.9Hz, J3=4.6Hz).

[0083] (2) Option 2

[0084] Synthesis route:

[0085]

[0086] Synthesis process:

[0087] In a 25 mL reaction flask, 80 mg of 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and 30 mg of ethanolamine, 200 mg of the recombinant Escherichia coli wet cells expressing the imine reductase prepared in Example 1 (the concentration of the recombinant Escherichia coli wet cells is 20 g / L, and each 1 mg of the recombinant Escherichia coli wet cells contains 0.1 mg of imine reductase), and 25 mg of the recombinant Escherichia coli wet cells expressing glucose dehydrogenase prepared in Example 1 (the reference Chaparro-Riggers JF, Rogers TA, Va'zquez Figueroa E & Bommarius AS (2007) Comparison of three enoate reductases and their potential use for biotransformations. Adv Synth Catal 349, 1521-1531. Recombinant Escherichia coli expressing glucose dehydrogenase was prepared at a concentration of 2.5 g / L. Each mg of recombinant E. coli wet cells contained 0.2 mg of glucose dehydrogenase, 300 mg of glucose, and 5 mg of nicotinamide adenine dinucleotide (NADP+). Phosphate buffer (0.1 M, pH 7.5) was added to a volume of 10 mL. The mixture was stirred at 30°C with magnetic stirring and a saturated aqueous sodium bicarbonate solution was added dropwise to adjust the pH to 7-9. The stirring time was 15-24 hours. The reaction was then inactivated and extracted with 10 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate solid, and concentrated to yield 93 mg of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile as a white solid. The product had a purity of 99%, an ee greater than 99%, and a yield of 90%.

[0088] The hydrogen spectrum of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile is characterized as follows:

[0089] 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.50-7.31 (m, 3H), 4.04 (m, 1H), 3.62 (q, 2H, J = 5.5Hz), 3.57-3.43 (m, 1H), 3.27 (ddd, 1H, J1 = 17.6Hz, J2 = 9.1Hz, J3 = 5 .0Hz), 3.15-2.85 (m, 1H), 2.74 (m, 2H), 2.58 (dtd, 1H, J1=9.0Hz, J2=5.5Hz, J3=5.3Hz, J4=3.6Hz), 2.20 (ddd, 1H, J1=13.4Hz, J2=8.9Hz, J3=4.6Hz).

[0090] Comparative Example: Synthesis of (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile using different imine reductases

[0091] (S)-1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-indene-4-carbonitrile was prepared using the method described in Example 2, except that the recombinant Escherichia coli wet cells expressing the imine reductase were replaced with commercially available imine reductase powder. The specific process is as follows:

[0092] 1) In a 25 mL reaction flask, add 100 mg of 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile, 200 mg of imine reductase powder ES-IRED-101 (purchased from Shangke Biopharmaceuticals (Shanghai) Co., Ltd.), 10 mg of the recombinant E. coli cells expressing ketoreductase prepared in Example 1 (the final concentration of the recombinant E. coli cells expressing ketoreductase in the reaction system is 1 g / L (0.2 mg of ketoreductase per 1 mg of recombinant E. coli cells), 0.5 mL of isopropanol, and 10 mg of nicotinamide adenine dinucleotide (NAD+). Add TricHCl buffer solution (0.1 M, pH 8.5) to a volume of 10 mL. Incubate at 30°C with magnetic stirring for 24 hours. No product is produced as detected by HPLC.

[0093] 2) In a 25 mL reaction flask, add 80 mg of 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and 30 mg of ethanolamine, 200 mg of imine reductase powder ES-IRED-101 (purchased from Shangke Biopharmaceuticals (Shanghai) Co., Ltd.), 25 mg of wet cells of recombinant E. coli expressing glucose dehydrogenase prepared in Example 1 (the concentration of the wet cells of recombinant E. coli expressing glucose dehydrogenase is 2.5 g / L, and each mg of recombinant E. coli wet cells contains 0.2 mg of glucose dehydrogenase), 300 mg of glucose, and 5 mg of nicotinamide adenine dinucleotide (NADP+). Add phosphate buffer (0.1 M, pH 7.5) to a volume of 10 mL. Incubate at 30°C with magnetic stirring for 24 hours. No product is produced according to HPLC detection.

[0094] Referring to the experimental methods of the above two schemes, the imine reductase powder ES-IRED-101 was replaced with imine reductase powders ES-IRED-102 to ES-IRED-117 (a total of 16 enzyme powders, purchased from Shangke Biopharmaceutical (Shanghai) Co., Ltd.) for reduction reaction, but no target product was obtained.

[0095] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a compound of formula I, characterized in that: include: 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile, or a mixture comprising 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile and ethanolamine, undergoes a reduction reaction under the catalysis of imine reductase to obtain the compound represented by formula I.

2. The method according to claim 1, characterized in that The imine reductase is produced by expressing in a first wet cell; Optionally, the first wet cells are selected from recombinant Escherichia coli wet cells; Optionally, the imine reductase has the amino acid sequence shown in SEQ ID NO: 2; Optionally, the nucleotide encoding the imine reductase has the sequence shown in SEQ ID NO: 1; Optionally, the molar ratio of the 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile to the ethanolamine is 1:(0.5-1.5).

3. The method according to claim 1, characterized in that The reduction reaction is carried out in the presence of a hydrogen supply system; Optionally, the hydrogen supply system includes at least one of an additional enzyme, a coenzyme cycle reaction substrate, and a coenzyme; Optionally, the additional enzyme is selected from ketoreductase or glucose dehydrogenase; Optionally, the coenzyme cycle reaction substrate is selected from isopropanol or glucose; Optionally, the coenzyme is selected from nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate; Optionally, the additional enzyme is selected from ketoreductase, the coenzyme cycle reaction substrate is selected from isopropanol, and the coenzyme is selected from nicotinamide adenine dinucleotide; Optionally, the additional enzyme is selected from glucose dehydrogenase, the coenzyme cycle reaction substrate is selected from glucose, and the coenzyme is selected from nicotinamide adenine dinucleotide phosphate; Optionally, the ketoreductase is produced by expression in a second wet cell; Optionally, the glucose dehydrogenase is produced by expression in a third wet cell.

4. The method according to claim 1, wherein The reduction reaction is carried out in the presence of a buffer; Optionally, the pH value of the buffer is 5 to 9; Optionally, the buffer is selected from at least one of a citric acid buffer, a TricHCl buffer solution, a phosphate buffer and a TEOA buffer.

5. The method according to claim 1 or 4, characterized in that The concentration of the imine reductase in the reduction reaction system is 0.1 g / L to 5 g / L; Optionally, the concentration of the 1-((2-hydroxyethyl)imino)-2,3-dihydro-1H-indene-4-carbonitrile or 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile in the reduction reaction system is 1 g / L to 100 g / L.

6. The method according to claim 3 or 4, characterized in that The concentration of the ketoreductase in the reduction reaction system is 0.04 g / L to 2 g / L; Optionally, the concentration of the glucose dehydrogenase in the reduction reaction system is 0.1 g / L to 5 g / L; Optionally, the concentration of the coenzyme in the reduction reaction system is 0.2 g / L to 1 g / L; Optionally, the volume concentration of the isopropanol in the reduction reaction system is 5% to 20%; Optionally, the concentration of glucose in the reduction reaction system is 7.5 g / L to 37.5 g / L.

7. The method according to claim 1, characterized in that The reduction reaction is carried out at 25-40°C; Optionally, the reduction reaction is carried out under magnetic stirring; Optionally, the reduction reaction time is 15 to 30 hours.

8. The method according to claim 2, characterized in that The first wet cell is prepared by the following method: Fermenting the recombinant Escherichia coli carrying the nucleotide sequence expressing the imine reductase to obtain the first wet cell; Preferably, the OD of E. coli 600 The value is 3 to 4, and the recombinant Escherichia coli after fermentation is collected to obtain the first wet bacteria; Optionally, the recombinant Escherichia coli carrying the nucleotide sequence for expressing the imine reductase is obtained by introducing a first vector carrying the nucleotide sequence for expressing the imine reductase into a recipient Escherichia coli.

9. The method according to claim 8, characterized in that The recipient Escherichia coli may further include a second vector carrying a nucleotide sequence expressing a ketoreductase or a nucleotide sequence expressing a glucose dehydrogenase; Optionally, the first vector and the second vector are respectively selected from pET-28a; Optionally, the nucleotide sequence expressing the ketoreductase is shown in SEQ ID NO: 3; Optionally, the nucleotide sequence expressing the glucose dehydrogenase is shown as SEQ ID NO:

4.

10. The method according to claim 3, characterized in that The second wet cell is obtained by fermenting a recombinant Escherichia coli carrying a nucleotide sequence expressing the ketoreductase; Optionally, the third wet cell is obtained by fermenting a recombinant Escherichia coli carrying a nucleotide sequence expressing the glucose dehydrogenase; Optionally, the nucleotide sequence expressing the ketoreductase is shown in SEQ ID NO: 3; Optionally, the nucleotide sequence expressing the glucose dehydrogenase is shown as SEQ ID NO:4.

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