Preparation method of vortioxetine intermediate

Abstract

The invention relates to a preparation method of a vortioxetine intermediate, and particularly provides a method for reducing a compound shown as a formula (I) into a compound shown as a formula (II) by using nitroreductase. Wherein the nucleotide sequence of the nitroreductase is as shown in SEQ ID NO. 1. The preparation method provided by the invention has the advantages of mild reaction conditions, high safety, no use of a heavy metal catalyst, environmental protection and low cost, and is suitable for industrial production. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical intermediates, and particularly relates to a method for preparing a vortioxetine intermediate. Background Art

[0002] Vortioxetine was jointly developed by Lundbeck of Denmark and Takeda Pharmaceutical of Japan. It was approved by the U.S. Food and Drug Administration (FDA) on September 30, 2013 for the treatment of major depressive disorder (MDD). Vortioxetine mainly exerts its antidepressant effect by increasing the concentration of serotonin (5-HT) in the central nervous system (CNS). Compared with other selective 5-HT reuptake inhibitors (SSRIS) or serotonin-norepinephrine reuptake inhibitors (SNRIS), vortioxetine has almost no effect on norepinephrine and dopaminergic neurons. Several clinical trials have shown that vortioxetine has good efficacy, safety and tolerability in the treatment of MDD. The chemical name of vortioxetine is: 1-(2-((2,4-dimethylphenyl)sulfanyl)phenyl)piperazine, and the chemical structure is as follows:

[0003]

[0004] The main synthetic routes of vortioxetine disclosed at present are:

[0005]

[0006] 2-((2,4-dimethylphenyl)thio)aniline is a key intermediate for preparing vortioxetine. The commonly used method for preparing the intermediate currently disclosed is to prepare it by chemical reduction reaction of 2-(2,4-dimethylphenylthio)nitrobenzene. Patent application CN104628676A discloses a method for preparing the vortioxetine intermediate compound, that is, using 10% palladium carbon as a catalyst and reducing the nitro group with hydrogen. This method has few by-products, simple post-processing and purification, and high yield. However, hydrogen is flammable and explosive, and there are great safety hazards, and the metal catalyst palladium used causes high production costs. In addition, patent application CN105283442A also discloses another method for preparing the vortioxetine intermediate compound, which uses an iron powder / acetic acid system for reduction. This method uses iron powder, which will produce a large amount of iron mud and metal-containing wastewater, which is not conducive to the reaction and environmental protection.

[0007] In summary, the synthetic methods of vortioxetine key intermediate 2-((2,4-dimethylphenyl)thio)aniline reported so far all have various problems, such as the use of metal catalysts such as palladium carbon, which is costly; the use of hydrogen reduction has great safety risks and is limited in scale-up production. The use of iron powder reduction produces a large amount of iron powder waste residue, and has high requirements for equipment stirring, making it difficult to scale up, and the difficulty of post-processing separation and purification is large, which limits scale-up production.

[0008] Therefore, it is necessary to develop a safe, efficient, environmentally friendly, economical and high-yield method for reducing a nitro group to an amino group, especially a method for reducing a nitro group of 2-((2,4-dimethylphenyl)thio)aniline to an amino group. Summary of the invention

[0009] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a bioreduction method for preparing vortioxetine intermediate 2-((2,4-dimethylphenyl)thio)aniline. The method uses a nitroreductase of a specific sequence to reduce the nitro group in the raw material and can effectively and efficiently convert the nitro group into an amino group in the presence of a coenzyme. The method has short steps, simple operation, mild reaction conditions, high safety, no heavy metal catalysts, green and environmentally friendly, good product quality, high yield, and low cost.

[0010] In one aspect, the present invention provides a method for preparing a compound as shown in formula (II):

[0011]

[0012] The method comprises: a compound represented by formula (I) is subjected to a reduction reaction in a buffer solution under the action of nitroreductase or recombinant nitroreductase wet bacteria to obtain a compound represented by formula (II); wherein the nucleotide sequence encoding the nitroreductase is shown in SEQ ID NO.1, and the amino acid sequence of the nitroreductase is shown in SEQ ID NO.2.

[0013] In some embodiments, the method for preparing the compound of formula (II) of the present invention comprises the following specific steps:

[0014] Add the compound shown in formula (I), nitroreductase or recombinant nitroreductase wet bacteria, additional enzyme wet bacteria, coenzyme cycle reaction substrate, coenzyme, and buffer into the reaction container, stir until the reaction is complete, and obtain the compound shown in formula (II) after post-treatment.

[0015] In some embodiments, the additional enzyme wet cells of the present invention are ketoreductase wet cells or glucose dehydrogenase wet cells; the coenzyme cycle reaction substrate is isopropanol or glucose; when ketoreductase wet cells are used, isopropanol is used as the substrate of the coenzyme cycle reaction; when glucose dehydrogenase wet cells are used, glucose is used as the substrate of the coenzyme cycle reaction.

[0016] In some embodiments, the coenzyme described in the present invention is nicotinamide adenine dinucleotide NAD+ or nicotinamide adenine dinucleotide phosphate NADP+.

[0017] In some embodiments, the buffer of the present invention is selected from a citrate buffer, a TricHCl buffer solution, a phosphate buffer, a TEOA buffer or any combination of the foregoing buffer solutions.

[0018] In some embodiments, the pH value of the buffer solution of the present invention is 5-9, preferably 6.5-7.5.

[0019] In some embodiments, the preparation step of the recombinant nitroreductase wet bacteria of the present invention includes: adding restriction site base sequences to both ends of the nitroreductase gene (nucleotide sequence and corresponding amino acid sequence are SEQ: 1 and SEQ: 2, respectively), amplifying to obtain the target gene fragment; then using restriction endonucleases to perform double enzyme digestion on the target gene fragment and the expression vector pet28a, respectively, and cutting the gel to recover the target gene fragment and the vector pet28a fragment; then using T4 DNA ligase to connect the target gene fragment and the pet28a vector fragment, adding the connection product to an electroporation cup containing Escherichia coli BL21 (DE3) electrocompetent cells, immediately electroporating on an electroporator, and then immediately transferring to ice, adding LB agar medium preheated to 37°C, and mixing; transferring the mixed culture medium to a culture tube, and culturing at 200 rpm and 37°C; spreading the incubated transformation liquid on a kanamycin LB agar plate, and culturing in a 37°C incubator; picking transformants on the coated plate for PCR verification, The positive strains were verified; the recombinant engineering strains with correct sequencing results were activated on a plate, and then a single colony was picked and inoculated into a conical flask containing a culture medium containing kanamycin and cultured at 37°C; then the seed liquid was converted into a fresh LB culture medium at a volume ratio of 2%, cultured at 37°C, and cultured with shaking at 220 rpm until the absorbance OD600 value of the bacterial liquid at a wavelength of 600 nanometers reached 0.6 to 0.8, and then isopropyl-β-D-thiogalactoside was added, and the culture was continued at 25°C and centrifuged, the supernatant was discarded, and the recombinant Escherichia coli wet cells were collected.

[0020] In some embodiments, the concentration range of the compound represented by formula (I) of the present invention is 2g / L to 50g / L, the concentration range of the wet cells of recombinant Escherichia coli expressing the nitroreductase is 1g / L to 50g / L, and the concentration range of the coenzyme is 0.2 to 1g / L.

[0021] In some embodiments, the concentration range of the ketoreductase wet cells of the present invention is 0.2 g / L to 10 g / L, and the volume concentration range of isopropanol as a substrate for the coenzyme cycle reaction is 5% to 20%; the concentration range of the glucose dehydrogenase wet cells is 0.5 g / L to 25 g / L, and the concentration range of glucose as a substrate for the coenzyme cycle reaction is 7.5 g / L to 375 g / L.

[0022] In some embodiments, the reaction temperature of the reduction reaction of the present invention is 20°C to 45°C, preferably 30°C to 35°C.

[0023] In another aspect, the present invention provides a nitroreductase, wherein the nucleotide sequence of the nitroreductase is shown as SEQ ID NO.1, and the amino acid sequence of the nitroreductase is shown as SEQ ID NO.2.

[0024] Compared with the prior art, the present invention has the following beneficial technical effects: by using a nitroreductase of a specific sequence to reduce the nitro group in the raw material and in the presence of a coenzyme, the nitro group can be effectively and efficiently converted into an amino group, and during the reaction process, the raw material will not undergo a side reaction to produce other by-products, the impurity content is low, the product conversion rate is high, the overall process is green and environmentally friendly, and the cost is low.

[0025] The foregoing description only summarizes certain aspects of the present invention, but is not limited to these aspects. These aspects and other aspects will be described in more detail and complete below.

[0026] Detailed description of the invention

[0027] Definitions and general terms

[0028] Certain embodiments of the present invention are now described in detail, examples of which are illustrated by the accompanying structural formulae and chemical formulae. The present invention is intended to cover all alternatives, modifications and equivalent technical solutions, which are all included within the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present invention (including but not limited to defined terms, term applications, described techniques, etc.), the present invention shall prevail.

[0029] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0030] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0031] Unless otherwise indicated, the following definitions used herein shall apply. For purposes of the present invention, chemical elements are consistent with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry can be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0032] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0033] Unless otherwise specified or there is a clear conflict in context, the articles "a", "an", and "the" as used herein are intended to include "at least one" or "one or more". Therefore, these articles as used herein refer to articles that refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated for use or use in the implementation of the described embodiment.

[0034] In the present invention, NADH represents reduced coenzyme I; NADPH represents reduced coenzyme II; mmol / L or mM represents millimole per liter; μM represents micromole per liter; μL represents microliter; ℃ represents degrees Celsius; pH represents acidity or alkalinity; s represents seconds, min represents minutes; h represents hours; rpm represents revolutions per minute; U represents enzyme activity unit; SDS-PAGE represents sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pET26b(+) is an expression vector; PCR represents polymerase chain reaction; OD600 represents the absorbance at a wavelength of 600nm; LB liquid culture medium represents Luria-Bertani liquid culture medium.

[0035] In the present invention, the substrate conversion rate refers to the percentage of the mass difference of the substrate before and after the reaction to the mass of the substrate before the reaction.

[0036] In the present invention, the "coenzyme circulation system" refers to a circulation system in which the coenzyme consumes hydrogen atoms when participating in the catalytic reaction of nitroreductase, and is converted from NADH or NADPH to NAD+ or NADP+; lactate dehydrogenase catalyzes the dehydrogenation of the substrate lactate or glucose dehydrogenase catalyzes the dehydrogenation of the substrate glucose to produce free hydrogen atoms, which are donated to NAD+ or NADP+; NAD+ or NADP+ is converted back to NADH or NADPH, and can participate in the catalytic reaction of nitroreductase again.

[0037] The ketoreductase wet cell of the present invention is known, and can also be prepared according to document ① (Hummeeiw, Grger H. Strategies for regeneration of nicotinamide coenzymes emphasizing self-sufficient closed-loop recycling systems. J Biotechnol, 2014, 191: 22-31). The glucose dehydrogenase wet cell is known, and can be prepared separately according to document ② (Chaparro-Riggers JF, Rogers TA, Va′zquez-Figueroa E & Bommarius AS (2007) Comparison of three enoatereductases and their potential use for biotransformations. Adv Synth Catal 349, 1521–1531).

[0038] 1 H NMR spectra were recorded using a Bruker 400 MHz or 600 MHz nuclear magnetic resonance spectrometer. 1H NMR spectra were obtained in CDC13, D2O, DMSO-d6, CD3OD or acetone-d6 as solvents (in ppm) and TMS (0 ppm) or chloroform (7.26 ppm) as reference standards. When multiple peaks are present, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), ddd (doublet of doublet of doublets), dddd (doublet of doublet of doublet of doublets), dt (doublet of triplets), tt (triplet of triplets). Coupling constants, J, are expressed in Hertz (Hz).

[0039] The measurement conditions of low-resolution mass spectrometry (MS) data are: Agilent 6120 quadrupole HPLC-M (column model: Zorbax SB-C18, 2.1×30 mm, 3.5 microns, 6 min, flow rate of 0.6 mL / min. Mobile phase: 5%-95% (CH3CN containing 0.1% formic acid) in (H2O containing 0.1% formic acid)), electrospray ionization (ESI), at 210 nm / 254 nm, with UV detection.

[0040] The purity of the compounds was tested using Agilent 1260 pre-HPLC or Calesep pump 250 pre-HPLC (column model: NOVASE P50 / 80 mm DAC) with UV detection at 210 nm / 254 nm.

[0041] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0042] Specific implementation methods

[0043] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail, but the present invention is not limited to these embodiments.

[0044] Example 1: Preparation of recombinant Escherichia coli wet cells

[0045] (1) Preparation of recombinant nitroreductase wet cells

[0046] The restriction site base sequences of Nco I and Xho I were added to both ends of the nitroreductase gene (nucleotide sequence and corresponding amino acid sequence were SEQ: 1 and SEQ: 2, respectively), and the target gene fragment was amplified. Then, the target gene fragment and the expression vector pet28a were double-digested with two restriction endonucleases, Nco I and Xho I, respectively, and the target gene fragment and the vector pet28a fragment were recovered by gel cutting. Then, the target gene fragment and the pet28a vector fragment were connected overnight using T4 DNA ligase. 5 microliters of the connection product were transferred to an electroporation cup containing 50 microliters of Escherichia coli electrocompetent cells BL21 (DE3). Perform electroporation on an electroporator, then immediately transfer to ice, add 1 mL of LB agar medium preheated to 37°C, and mix well; then transfer the mixed medium to a 2 mL culture tube and culture at 200 rpm and 37°C for one hour; take 150 microliters of the incubated transformation liquid and spread it on an LB agar plate containing 100 microliters per mL of kanamycin, and culture it in a 37°C incubator overnight for 16 hours. The next day, pick the transformants on the coated plate for PCR verification, and send the positive strains for sequencing verification. The strains whose sequencing results are consistent with the SEQ:1 sequence are selected as genetic engineering bacteria for recombinant nitroreductase and as subsequent research objects.

[0047] The recombinant nitroreductase engineering strain constructed above was activated on a plate. On the second day, a single colony was picked and transferred to a conical flask containing 15mL culture medium containing kanamycin, and cultured at 37°C for 16h. Subsequently, the seed solution was transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220rpm. When the absorbance OD600 value of the bacterial solution at a wavelength of 600 nanometers reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce protein expression, and cultured at 25°C and 220rpm for 18h. Centrifuge (9000rpm, 10min), discard the supernatant, collect the recombinant nitroreductase wet bacteria, and store at -20°C.

[0048] (2) Preparation of wet bacteria of recombinant glucose dehydrogenase

[0049] The restriction site base sequences of Nde I and Xho I were added to both ends of the gene of glucose dehydrogenase (nucleotide sequence and corresponding amino acid sequence were SEQ: 3 and SEQ: 4, respectively), and the target gene fragment was amplified. Then, the target gene fragment and the expression vector pet28a were double-digested with two restriction endonucleases, Nde I and Xho I, respectively, and the target gene fragment and the vector pet28a fragment were recovered by gel cutting. Then, the target gene fragment and the pet28a vector fragment were connected overnight using T4 DNA ligase. 5 microliters of the connection product were transferred to an electroporation cup containing 50 microliters of Escherichia coli electrocompetent cells BL21 (DE3). Perform electroporation on an electroporator, then immediately transfer to ice, add 1 mL of LB agar medium preheated to 37°C, and mix well; then transfer the mixed medium to a 2 mL culture tube and culture at 200 rpm at 37°C for one hour; take 150 microliters of the incubated transformation liquid and spread it on an LB agar plate containing 100 microliters per mL of kanamycin, and culture it in a 37°C incubator overnight for 16 hours. The next day, pick the transformants on the coated plate for PCR verification, and send the positive strains for sequencing verification. The strains whose sequencing results are consistent with the SEQ:3 sequence are selected as genetic engineering bacteria for recombinant glucose dehydrogenase and as subsequent research objects.

[0050] The recombinant glucose dehydrogenase engineering strain constructed above was activated on a plate. On the second day, a single colony was picked and transferred to a conical flask containing 15 mL of culture medium containing kanamycin and cultured at 37°C for 16 hours. The seed solution was then transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220 rpm. When the absorbance OD600 value of the bacterial solution at a wavelength of 600 nanometers reached 0.6 to 0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2 mM to induce protein expression, and cultured at 25°C and 220 rpm for 18 hours. Centrifuge (9000 rpm, 10 min), discard the supernatant, collect the wet bacteria of the recombinant glucose dehydrogenase, and store at -20°C.

[0051] (3) Preparation of recombinant ketoreductase wet cells

[0052] The ketoreductase (nucleotide sequence and corresponding amino acid sequence are SEQ: 5 and SEQ: 6, respectively) gene was added with Nde I and Hind III restriction site base sequences at both ends to amplify the target gene fragment. The target gene fragment and expression vector pet28a were then double-digested with Nde I and Hind III restriction endonucleases, and the target gene fragment and vector pet28a fragment were recovered by gel cutting. T4 DNA ligase was then used to connect the target gene fragment and pet28a vector fragment overnight. 5 microliters of the ligation product was transferred to an electroporation cup containing 50 microliters of E. coli electrocompetent cells BL21 (DE3). Perform electroporation on an electroporator, then immediately transfer to ice, add 1 mL of LB agar medium preheated to 37°C, and mix well; then transfer the mixed medium to a 2 mL culture tube and culture at 200 rpm at 37°C for one hour; take 150 microliters of the incubated transformation liquid and spread it on an LB agar plate containing 100 microliters per mL of kanamycin, and culture it in a 37°C incubator overnight for 16 hours. The next day, pick the transformants on the coated plate for PCR verification, and send the positive strains for sequencing verification. The strains whose sequencing results are consistent with the SEQ:5 sequence are selected as genetic engineering bacteria for recombinant ketoreductase and as subsequent research objects.

[0053] The recombinant ketoreductase engineering strain constructed above was activated on a plate. On the second day, a single colony was picked and transferred to a conical flask containing 15mL culture medium containing kanamycin, and cultured at 37°C for 16h. Subsequently, the seed solution was transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220rpm. When the absorbance OD600 value of the bacterial solution at a wavelength of 600 nanometers reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce protein expression, and cultured at 25°C and 220rpm for 18h. Centrifuge (9000rpm, 10min), discard the supernatant, collect the recombinant ketoreductase wet bacteria, and store at -20°C.

[0054] Example 2: Preparation of 2-((2,4-dimethylphenyl)thio)aniline

[0055] In a 25mL reaction bottle, add 100mg 2-(2,4-dimethylphenylthio)nitrobenzene, 50mg recombinant nitroreductase wet bacteria (wet bacteria concentration is 5g / L, each mg wet bacteria contains 0.1mg enzyme), 10mg recombinant ketoreductase wet bacteria (wet bacteria concentration is 1g / L, each mg wet bacteria contains 0.2mg enzyme), 0.5mL isopropanol, 10mg nicotinamide adenine dinucleotide NADP+; add sodium phosphate buffer (0.1M, pH=8.5) to a volume of 10mL. After magnetic stirring at 35℃ for 24 hours, use ethyl acetate to inactivate the reaction and extract, dry the organic phase with excess anhydrous sodium sulfate, filter to remove the sodium sulfate solid, and concentrate to obtain 2-((2,4-dimethylphenyl)thio)aniline with a yield of 90% and a purity of 98%.

[0056] 1 H NMR (600MHz, CDCl3) δ7.40 (dd, J=7.7, 1.5Hz, 1H), 7.28-7.23 (m, 1H), 7.04 (s, 1H), 6.89 (d, J=8.0Hz, 1H), 6.82 (d d,J=8.0,1.1Hz,1H),6.79(td,J=7.5,1.3Hz,1H),6.75(d,J=8.0Hz,1H),4.25(s,2H),2.44(s,3H),2.31(s,3H).

[0057] Example 3: Preparation of 2-((2,4-dimethylphenyl)thio)aniline

[0058] In a 25mL reaction bottle, add 100mg 2-(2,4-dimethylphenylthio)nitrobenzene, 200mg recombinant nitroreductase wet bacteria (wet bacteria concentration is 20g / L, each mg wet bacteria contains 0.1mg nitroreductase), 25mg recombinant glucose dehydrogenase wet bacteria (wet bacteria concentration is 2.5g / L, each mg wet bacteria contains 0.2mg enzyme), 300mg glucose, 10mg nicotinamide adenine dinucleotide NADP+; add sodium phosphate buffer (0.1M, pH=7.0-7.5) to a volume of 10mL. After magnetic stirring at 30°C for 24 hours, use ethyl acetate to inactivate the reaction and extract, dry the organic phase with excess anhydrous sodium sulfate, filter to remove the sodium sulfate solid, and concentrate to obtain 2-((2,4-dimethylphenyl)thio)aniline with a yield of 92% and a purity of 99%.

[0059] Comparative Example

[0060] In a 25mL reaction bottle, add 100mg 2-(2,4-dimethylphenylthio)nitrobenzene, 20mg nitroreductase (ES-NTR-101, Shangke Biopharmaceutical Shanghai Co., Ltd.), 25mg recombinant glucose dehydrogenase wet bacteria (the concentration of wet bacteria is 2.5g / L, and each mg of wet bacteria contains 0.2mg enzyme), 300mg glucose, 10mg nicotinamide adenine dinucleotide NADP+; add sodium phosphate buffer (0.1M, pH=7.5) to a volume of 10mL. After 24 hours of magnetic stirring at 30℃, HPLC detection showed no reaction and no product in the system.

[0061] In the comparative example, nitroreductase ES-NTR-101 was replaced with nitroreductase ES-NTR-102 to ES-NTR-112 (purchased from Shangke Biopharmaceutical (Shanghai) Co., Ltd.) in turn for reduction reaction, but no target product was obtained in the system.

[0062] Conclusion: Not all types of nitroreductases can reduce 2-(2,4-dimethylphenylthio)nitrobenzene to 2-((2,4-dimethylphenyl)thio)aniline, but the nitroreductase of the present invention having the amino acid sequence shown in SEQ ID NO.2 can better reduce 2-(2,4-dimethylphenylthio)nitrobenzene to 2-((2,4-dimethylphenyl)thio)aniline (vortioxetine intermediate) with higher product yield and purity.

[0063] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for preparing a compound as represented by formula (II), It includes: The compound represented by formula (I) is subjected to reduction reaction in a buffer solution under the action of nitroreductase or recombinant nitroreductase wet bacteria to obtain a compound represented by formula (II); The nucleotide sequence encoding the nitroreductase is shown as SEQ ID NO.1, and the amino acid sequence of the nitroreductase is shown as SEQ ID NO.

2.

2. The method for preparing the compound of formula (II) according to claim 1, comprising the steps of: Add the compound shown in formula (I), nitroreductase or recombinant nitroreductase wet bacteria, additional enzyme wet bacteria, coenzyme cycle reaction substrate, coenzyme, and buffer into the reaction container, stir until the reaction is complete, and obtain the compound shown in formula (II) after post-treatment.

3. The method for preparing the compound of formula (II) according to claim 2, characterized in that: The additional enzyme wet cells are ketone reductase wet cells or glucose dehydrogenase wet cells; the coenzyme cycle reaction substrate is isopropanol or glucose; when ketone reductase wet cells are used, isopropanol is used as the substrate of the coenzyme cycle reaction; when glucose dehydrogenase wet cells are used, glucose is used as the substrate of the coenzyme cycle reaction.

4. The method for preparing the compound of formula (II) according to claim 2 or 3, characterized in that: The coenzyme is nicotinamide adenine dinucleotide NAD+ or nicotinamide adenine dinucleotide phosphate NADP+.

5. The method for preparing the compound of formula (II) according to claim 1 or 2, characterized in that: The buffer is selected from a citric acid buffer, a Tric-HCl buffer solution, a phosphate buffer, a TEOA buffer or any combination of the aforementioned buffer solutions.

6. The method for preparing the compound of formula (II) according to claim 1 or 2, characterized in that: The pH value of the buffer solution is 5 to 9, preferably 6.5 to 7.

5.

7. The method for preparing the compound of formula (II) according to claim 2 or 3, characterized in that: The preparation steps of the recombinant nitroreductase wet bacteria include: adding restriction site base sequences to both ends of the nitroreductase gene (nucleotide sequence and corresponding amino acid sequence are SEQ: 1 and SEQ: 2, respectively), amplifying to obtain the target gene fragment; then using restriction endonucleases to perform double enzyme digestion on the target gene fragment and the expression vector pet28a, respectively, and cutting the gel to recover the target gene fragment and the vector pet28a fragment; then using T4 DNA ligase to connect the target gene fragment and the pet28a vector fragment, adding the connection product to an electroporation cup containing Escherichia coli BL21 (DE3) electrocompetent cells, immediately electroporating on an electroporator, and then immediately transferring to ice, adding LB agar medium preheated to 37°C, and mixing; transferring the mixed culture medium to a culture tube, and culturing at 200 rpm and 37°C; spreading the incubated transformation liquid on a kanamycin LB agar plate, and culturing in a 37°C incubator; picking transformants on the coated plate for PCR verification, The positive strains were verified; the recombinant engineering strains with correct sequencing results were activated on a plate, and then a single colony was picked and inoculated into a conical flask containing a culture medium containing kanamycin and cultured at 37°C; then the seed liquid was converted into a fresh LB culture medium at a volume ratio of 2%, cultured at 37°C, and cultured with shaking at 220 rpm until the absorbance OD600 value of the bacterial liquid at a wavelength of 600 nanometers reached 0.6 to 0.8, and then isopropyl-β-D-thiogalactoside was added, and the culture was continued at 25°C and centrifuged, the supernatant was discarded, and the recombinant Escherichia coli wet cells were collected.

8. The method for preparing the compound of formula (II) according to claim 2 or 3, characterized in that: The concentration range of the compound represented by formula (I) is 2g / L to 50g / L, the concentration range of the wet cells of recombinant Escherichia coli expressing the nitroreductase is 1g / L to 50g / L, and the concentration range of the coenzyme is 0.2 to 1g / L.

9. The method for preparing the compound of formula (II) according to claim 2 or 3, characterized in that: The concentration range of the ketoreductase wet bacteria is 0.2g / L to 10g / L, and the volume concentration range of isopropanol as a substrate for the coenzyme cycle reaction is 5% to 20%; the concentration range of the glucose dehydrogenase wet bacteria is 0.5g / L to 25g / L, and the concentration range of glucose as a substrate for the coenzyme cycle reaction is 7.5g / L to 375g / L.

10. The method for preparing the compound of formula (II) according to claim 2 or 3, characterized in that: The reaction temperature of the reduction reaction is 20°C to 45°C, preferably 30°C to 35°C.

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