Preparation method and application of chiral amine and salt thereof

Abstract

The invention relates to a preparation method and application of chiral amine and salt thereof. Reacting the compound 1 in the presence of imine reductase, NADP (Nicotinamide Adenine Dinucleotide Phosphate), glucose dehydrogenase, glucose, a solvent and a cosolvent to obtain a compound 2 and a compound 2-A; and converting the compound 2 and the compound 2-A into the chiral amine TL-010-2. The method combines biological enzyme catalysis and chemical methods, has the advantages of high yield and high purity, and is more suitable for large-scale industrial production. Compared with an existing traditional chemical method, the method has the advantages that the enzymolysis process is simple, post-treatment is easy, the reaction condition temperature is low, operation is easy and convenient, control is easy, existing processing equipment can be utilized, equipment investment is reduced, the use amount of energy and organic solvents can be reduced, and green production is achieved.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a chiral amine and a salt thereof. Background Art

[0002] Previously, the applicant developed a series of process routes for preparing (R)-praziquantel from the key chiral intermediate CP-5, and also developed a process route for producing CP-5 using lipase. Subsequently, the key intermediate TL-010-02 or a protected compound containing N-Boc protection was prepared through several routes, and then further reacted through several steps to prepare high-quality (R)-praziquantel.

[0003] The production process for obtaining high-purity CP-5 is shown in Scheme 1, which is involved in U.S. Patent US9139859B2 published on September 22, 2015 and WO2021 / 047566A1 published on March 18, 2021.

[0004]

[0005] Solution 1

[0006] There are several methods for converting CP-5 to the key intermediate TL-010-2 and subsequently to (R)-praziquantel.

[0007] Among the various methods for producing (R)-praziquantel, a promising method is to asymmetric hydrogenate the appropriate material to introduce the desired (R)-chirality. Czarnocki and his collaborators (Roszkowshki, P.; Maurin, JK; Czarnocki, Z. Tetrahedron, Asymmetry, 2006, 17, 1415) have reported such a substrate generated from phenylethylamine (see Scheme 2). However, the asymmetric transfer hydrogenation of imine compound 4 to compound 5 is low in yield and general in enantioselectivity. The current recrystallization to improve the ee value further reduces the overall yield to 24%.

[0008] Therefore, there is still room for further improvement.

[0009]

[0010] Solution 2 Summary of the invention

[0011] An object of the present invention is to provide a method for preparing chiral amines and their salts with high yield and purity.

[0012] Another object of the present invention is to provide a method for preparing (R)-praziquantel, wherein the method for preparing a key chiral intermediate has the advantages of high yield and purity.

[0013] In order to achieve the above object, the present invention adopts the following technical solution:

[0014] One aspect of the present invention provides a method for preparing a chiral amine, comprising the following steps:

[0015] (1) reacting compound 1 in the presence of imine reductase, NADP, glucose dehydrogenase, glucose, a solvent and a cosolvent to obtain compound 2 and / or compound 2-A;

[0016] (2) converting the compound 2 and / or the compound 2-A into chiral amine TL-010-2 ((1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline);

[0017] Wherein, the structural formula of the compound 1 is:

[0018] The structural formula of the compound 2 is:

[0019] The structural formula of the compound 2-A is:

[0020] The structural formula of the chiral amine TL-010-2 is:

[0021] According to some embodiments, the initial concentration of compound 1 is 30-100 g / L, for example, the initial concentration of compound 1 is 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. According to some further embodiments, the initial concentration of compound 1 is 50-100 g / L.

[0022] According to some embodiments, in step (1), the pH value is controlled to be maintained at 5 to 7 during the reaction, for example, the pH value is 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. According to some other embodiments, the pH value is controlled to be maintained at 6 to 6.5 during the reaction. The pH value during the reaction can be maintained at the desired pH value by adding alkali solution, and the alkali solution includes but is not limited to sodium carbonate, sodium bicarbonate, potassium carbonate, calcium carbonate, sodium sulfite, sodium acetate, sodium sulfide, ferrous sulfide, sodium silicate, sodium phosphate, etc.

[0023] According to some embodiments, in step (1), the reaction temperature is controlled to be 25-35° C., for example, the reaction temperature is 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C. or 35° C. According to some further embodiments, the reaction temperature is controlled to be 25-30° C.

[0024] According to some embodiments, the solvent is PBS buffer or water.

[0025] According to some embodiments, the cosolvent is one or more of ethyl acetate, methanol, isopropanol, dimethyl sulfoxide, toluene, isopropyl ether, and methyl tert-butyl ether. According to some further embodiments, the cosolvent is toluene.

[0026] According to some embodiments, the amount of the cosolvent is to facilitate the reaction, and can be added according to the conventional amount in the art. According to some specific embodiments, the feed volume of the cosolvent is more than 10% of the total volume of the reaction raw materials. Further, the feed volume of the cosolvent is more than 20% of the total volume of the reaction raw materials, such as 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50% and the like. Further, the feed volume of the cosolvent is 20% to 40% of the total volume of the reaction raw materials. Further, the feed volume of the cosolvent is 25% to 40% of the total volume of the reaction raw materials. Further, the feed volume of the cosolvent is 30% of the total volume of the reaction raw materials.

[0027] According to some embodiments, the feed mass of the NADP is 0.07% to 1.5% of the feed mass of the compound 1, for example, 0.07%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45% or 1.5%. According to some further embodiments, the feed mass of the NADP is 0.1% to 1.5% of the feed mass of the compound 1.

[0028] According to some embodiments, the feeding molar amount of glucose is 1 to 3 times the feeding molar amount of compound 1, for example, the feeding molar amount of glucose is 1, 1.5, 2, 2.5 or 3 times the feeding molar amount of compound 1. According to some further embodiments, the feeding molar amount of glucose is 2 to 3 times the feeding molar amount of compound 1.

[0029] According to some embodiments, the feed mass of the glucose dehydrogenase is 25% to 75% of the feed mass of the compound 1, for example, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. According to some further embodiments, the feed mass of the glucose dehydrogenase is 40% to 60% of the feed mass of the compound 1, more preferably 50%.

[0030] According to some embodiments, the imine reductase is fed in the form of enzyme powder, whole cells, cell lysis solution or supernatant obtained by separation of cell lysis solution. The whole cells, cell lysis solution or cells in the supernatant obtained by separation of cell lysis solution can be made of commonly used Escherichia coli and Pichia pastoris expressing imine reductase, and the enzyme powder is the powder of imine reductase obtained by separation and purification of Escherichia coli and Pichia pastoris expressing imine reductase.

[0031] According to one embodiment, when the imine reductase is fed in the form of enzyme powder, the feeding mass of the imine reductase enzyme powder is 0.5 to 5 times the feeding mass of the compound 1, for example, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5. .2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times or 5.0 times. According to some further embodiments, the feed mass of the imine reductase enzyme powder is 1 to 5 times the feed mass of the compound 1. Further, the feed mass of the imine reductase enzyme powder is 1.5 to 5 times the feed mass of the compound 1. Further, the feed mass of the imine reductase enzyme powder is 1.5 to 3 times the feed mass of the compound 1.

[0032] According to another embodiment, when the imine reductase is fed in the form of whole cells or cell lysate, the feed mass of the whole cells or the feed mass of the cells in the cell lysate is 1 to 10 times the feed mass of the compound 1, for example, 1 time, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times. According to some further embodiments, the feed mass of the whole cell is 3 to 10 times the feed mass of the compound 1. Further, the feed mass of the whole cell is 3 to 5 times the feed mass of the compound 1.

[0033] According to another embodiment, when the imine reductase is fed in the form of a supernatant obtained by separating a cell lysate, the amount of the supernatant is 4 to 10 mL of the supernatant per 1 g of the compound 1, for example, 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL, 10 mL of the supernatant is added per 1 g of the compound 1. According to some further embodiments, the amount of the supernatant is 4 to 8 mL of the supernatant per 1 g of the compound 1. Further, the amount of the supernatant is 4 to 6 mL of the supernatant per 1 g of the compound 1.

[0034] According to some specific embodiments, the enzyme activity of the supernatant obtained by separating the cell lysis solution is 2 to 5 u / g.

[0035] According to some specific embodiments, the supernatant separated from the cell lysis solution is obtained by mixing whole cells expressing imine reductase with a solvent in a ratio of 1:3 to 6, rupturing, and centrifuging, wherein the solvent is PBS buffer or water.

[0036] The imine reductase and the cells expressing it, glucose dehydrogenase, compound 1 and other raw materials in this application can be prepared by conventional methods in the art, or can also be purchased commercially. For example, the imine reductase can be obtained from Tongli Biotechnology, brand TL-IRED; the glucose dehydrogenase can be obtained from Tongli Biotechnology, brand TL-GDH. Among them, the glucose dehydrogenase is fed in the form of glucose dehydrogenase crude enzyme solution. The density of the glucose dehydrogenase crude enzyme solution is 1kg / m 3 , the concentration in the reaction system of the present invention is generally 50 g / L.

[0037] According to some embodiments, after the reaction of step (1) is completed, centrifugation or reduced pressure distillation is performed, dichloromethane and / or methanol is added, stirred, filtered, and the organic phase is collected; dichloromethane and / or methanol is added to the collected filter cake, and the organic phase is slurried, filtered, and collected, and this step is performed once or multiple times; the collected organic phases are combined and directly used in the reaction of step (2).

[0038] According to some embodiments, the compound 2 and the compound 2-A react in the presence of hydrazine hydrate and methanol to obtain the chiral amine TL-010-2. Studies have found that both the compound 2 and the compound 2-A can be converted into TL-010-2 under the action of hydrazine hydrate, so the organic phase after post-treatment in step (1) is preferably directly used for hydrazine hydrate deprotection without further purification.

[0039] According to some further embodiments, the molar amount of the hydrazine hydrate is 2 to 4 times, and further 2.5 to 3.5 times, the molar amount of the compound 1.

[0040] The asymmetric reduction of compound 1 has high yield and high ee value, and is therefore significantly better than that reported by Czarnocki.

[0041] According to still other embodiments, the salt of the chiral amine is hydrochloride, hydrobromide, oxalate, acetate, lactate, phosphate, fumarate, tartrate, citrate, gluconate, sulfate, methanesulfonate and other sulfonic acids.

[0042] According to some embodiments, the preparation method further comprises the step of converting the chiral amine TL-010-2 into TL-010-2 dihydrochloride in the presence of hydrogen chloride ethanol solution and methanol, wherein the structural formula of the TL-010-2 dihydrochloride is:

[0043] According to some further embodiments, the specific steps of preparing the TL-010-2 hydrochloride from the chiral amine TL-010-2 are as follows:

[0044] (1) adding the chiral amine TL-010-2 and methanol into a reactor and stirring at room temperature;

[0045] (2) in a dissolved or undissolved state, adding dropwise an EtOH solution containing 20 to 25 wt.% HCl into the reactor at a temperature of 15 to 30° C.;

[0046] (3) After the addition is complete, reflux and stir at 75-85°C for 15-25 minutes;

[0047] (4) After the reaction is completed, the TL-010-2 hydrochloride is obtained by post-treatment.

[0048] According to some embodiments, the preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline dihydrobromide (TL-010-2 dihydrobromide) in the presence of hydrobromic acid and methanol, wherein the structural formula of the TL-010-2 dihydrobromide is:

[0049] According to some embodiments, the preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline oxalate (TL-010-2 oxalate) in the presence of oxalic acid and methanol, wherein the structural formula of the TL-010-2 oxalate is:

[0050] According to some embodiments, the preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline maleate (TL-010-2 maleate) in the presence of maleic acid and methanol, wherein the structural formula of the TL-010-2 maleate is:

[0051] The second aspect of the present invention provides a chiral amine salt, the structural formula of which is

[0052] The third aspect of the present invention provides a method for preparing (R)-praziquantel, comprising the following steps:

[0053] 1) preparing the chiral amine TL-010-2 or its salt according to the above preparation method;

[0054] 2) Using the chiral amine TL-010-2 or its salt as a raw material, the (R)-praziquantel is prepared.

[0055] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0056] The present invention combines bio-enzyme catalysis and chemical methods, has the advantages of high yield and high purity, and is more suitable for large-scale industrial production. Compared with the existing traditional chemical methods, the present invention has the advantages of simple enzymatic hydrolysis process, easy post-processing, reaction conditions and temperature, simple and convenient operation, easy control, and can use existing processing equipment to reduce equipment investment, and can reduce the use of energy and organic solvents to achieve green production. In particular, the method of the present invention helps to solve the unresolved industrial problem of separation and purification of high-purity left-handed praziquantel, and paves the way for the preclinical and clinical evaluation of the drug properties of left-handed praziquantel and the entry of left-handed praziquantel produced in large-scale industrialization into the international market. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 HPLC spectra of the enzyme reaction products, compound 2 and compound 2-A;

[0058] Figure 2 is the NMR-H spectrum of compound 2;

[0059] Figure 3 is the NMR-H spectrum of the chiral amine free base TL-010-2;

[0060] Figure 4 This is the chiral purity HPLC spectrum of TL-010-2 dihydrochloride;

[0061] Figure 5is the NMR-H spectrum of TL-010-2 dihydrochloride;

[0062] Figure 6 This is the NMR-C13 spectrum of TL-010-2 dihydrochloride. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to the following embodiments. The implementation conditions adopted in the embodiments can be further adjusted according to specific requirements, and the unspecified implementation conditions are usually the conditions in routine experiments. The slight differences in the experimental data of the following embodiments are due to rounding during calculation. In the table below, "ND" means that the corresponding substance was not detected in the test.

[0064] The overall reaction route of this application includes the following steps:

[0065]

[0066]

[0067] Example 1 Substrate concentration screening

[0068] Table 1

[0069] Material Name Feeding amount Compound 1 100.0mg DMSO (Dimethyl Sulfoxide) 0.4mL IRED (Imine Reductase) Enzyme Powder 20mg NADP 0.7mg GDH (glucose dehydrogenase) 0.05mL glucose 80.7mg PBS buffer, pH 5.6 2.7 / 1.4 / 0.4mL

[0070] operate:

[0071] 1) Take three 10 mL single-mouth reaction bottles, add the materials in Table 1 (different substrate concentrations are approximately: 30 g / L, 50 g / L, 100 g / L), stir magnetically, and react at 30 °C;

[0072] 2) Samples were taken after 22 hours of reaction and HPLC was used for central control;

[0073] 3) Samples were taken after 44 hours of reaction and then controlled by HPLC;

[0074] 4) Sampling after 70h reaction and central control HPLC;

[0075] 5) Add 10 mL of acetonitrile to the reaction solution, stir for 10 min, filter, and evaporate the mother liquor to dryness under reduced pressure;

[0076] 6) Add DCM (dichloromethane): 10 mL, water: 20 mL, stir for 10 min, let stand to separate the liquids, add anhydrous sodium sulfate to dry the organic phase, evaporate the organic phase under reduced pressure, and pass it through a column.

[0077] The data of the central control HPLC and the product compound 2 after column are shown in Table 2.

[0078] Table 2

[0079]

[0080] The results showed that the reaction was better at a substrate concentration of 50 g / L. The products after column LC-MS and 1 H-NMR showed the target structure.

[0081] Example 2 - pH coarse screening

[0082] Table 3

[0083] Material Name Feeding amount Compound 1 100.0mg DMSO 0.4mL IRED Enzyme Powder 30mg NADP 0.7mg GDH 50 μl glucose 80.7mg PBS buffer 2.7mL

[0084] operate:

[0085] 1) Take 5 10 mL single-mouth reaction bottles, add the materials in Table 3 (substrate concentration is about 30 g / L), add buffers of different pH values, stir magnetically, and react at 30 °C;

[0086] 2) Samples were taken after 22 hours of reaction and HPLC was used for central control;

[0087] 3) Samples were taken after 46 hours of reaction and then controlled by HPLC;

[0088] 4) Samples were taken after 70 hours of reaction and the samples were controlled by HPLC.

[0089] The in-process HPLC data are shown in Table 4.

[0090] Table 4

[0091]

[0092] The results of this experiment show that as time goes on, the product increases and the pH of the system tends to be alkaline, which increases the amount of substrate 2-A. This is a dynamic change, suggesting that it is necessary to constantly monitor and adjust the reaction process. It is important to control the pH value between 5.0 and 7.0. The pH value needs to be finely screened later.

[0093] Example 3 - pH Fine Screen

[0094] Table 5

[0095] Material Name Feeding amount Compound 1 400.0mg DMSO 1.6mL IRED Enzyme Powder 120mg NADP 2.8mg GDH 0.2mL glucose 322.7mg PBS buffer 5.8mL

[0096] operate:

[0097] 1) Take 4 10 mL single-mouth reaction bottles, add the materials in Table 5 (substrate concentration is about 50 g / L), add buffers of different pH values, stir magnetically, and react at 30 °C;

[0098] 2) Samples were taken after 24 hours of reaction and then controlled by HPLC;

[0099] 3) Combine the four reactions, add DCM (20 mL) and water (20 mL), stir for 10 min, filter (pad with a large amount of diatomaceous earth), let the mother liquor stand for separation, discard the organic phase, and evaporate the aqueous phase to dryness under reduced pressure at 50°C;

[0100] 4) DCM (30 mL) was added, slurried at room temperature for 30 min, and filtered to obtain white solid compounds 2 and 2-A. The structures were confirmed by LC-MS test MS (+): 311.

[0101] The in-process HPLC data and the detection data of compounds 2 and 2-A are shown in Table 6.

[0102] Table 6

[0103]

[0104] The results show that the reaction is preferably carried out at pH 6.0-6.5.

[0105] Example 4 Reaction Temperature Screening

[0106] The feeding table of this embodiment is the same as that of embodiment 3. The pH of the buffer solution of this embodiment is 6.0. The experiment is completed according to the method of embodiment 3 at different reaction temperatures. Samples are taken at 24 and 48 hours of reaction, respectively, and HPLC is controlled. The experimental data are shown in the following table 7:

[0107] Table 7

[0108]

[0109]

[0110] The results show that the optimal reaction temperature is 25-30°C.

[0111] Example 5 Screening of Imine Reductase Dosage

[0112] In this embodiment, except that the dosage of imine reductase powder is different (120 mg / 200 mg / 320 mg / 400 mg / 600 mg (0.3 w / w, 0.5 w / w, 0.8 w / w, 1.0 w / w, 1.5 w / w)) and the dosage of glucose is 646.4 mg, the other ingredients in the feeding table are the same as those in Example 4.

[0113] operate:

[0114] 1) Take 5 10 mL single-mouth reaction bottles, add various materials and enzyme powder in different proportions, stir magnetically, and react at 30 °C;

[0115] 2) Samples were taken after 24 hours of reaction and then controlled by HPLC;

[0116] 3) Samples were taken after 48 hours of reaction and the samples were controlled by HPLC.

[0117] The in-process HPLC data are shown in Table 8.

[0118] Table 8

[0119]

[0120] The results showed that the in-process control results of enzyme powder dosages of 0.3w / w, 0.5w / w, 0.8w / w, and 1.0w / w were similar. As the enzyme dosage increased, the substrate tended to react completely.

[0121] Example 6 Feeding Escherichia coli expressing imine reductase and screening for appropriate cell dosage

[0122] Table 9

[0123] Material Name Feeding amount Compound 1 400.0mg DMSO 1.6mL IRED Cells 400, 800, 1200, 1600 mg NADP 2.8mg GDH 0.2mL glucose 646.4mg PBS buffer, pH 6.5 5.2, 4.8, 4.4, 4.0 mL

[0124] operate:

[0125] 1) Take 4 10 mL single-mouth reaction bottles, add the materials in the feeding table respectively (the substrate concentration in this example is about 50 g / L), stir magnetically, and react at 30° C.;

[0126] 2) Samples were taken after 24 hours of reaction and then controlled by HPLC;

[0127] 3) Samples were taken after 48 hours of reaction and the samples were controlled by HPLC.

[0128] The in-process HPLC data are shown in Table 10.

[0129] Table 10

[0130]

[0131]

[0132] The results show that when the cell dosage is 4w / w, the raw materials can basically react completely.

[0133] Example 7: Feeding with Escherichia coli cell lysate expressing imine reductase and screening the amount of cell lysate

[0134] Table 11

[0135]

[0136] Wherein, the cell disruption solution is prepared by: 5 g cells / 20 mL PBS buffer (pH 6.5), and the cells are disrupted by ultrasound or other methods to obtain the cell disruption solution.

[0137] operate:

[0138] 1) Take a 10 mL single-mouth reaction bottle, add the materials in the feeding table 11 respectively, stir magnetically, and react at 30°C;

[0139] 2) Samples were taken after 24 hours of reaction and then controlled by HPLC;

[0140] 3) Samples were taken after 48 hours of reaction and then controlled by HPLC;

[0141] The in-process HPLC data are shown in Table 12.

[0142] Table 12

[0143]

[0144] The results show that: with a cell disruption solution of 20w / w (corresponding to a cell dosage of 4w / w), the raw materials can basically react completely. Comparing the reactions of cells and cell disruption solution, the in-process control results are similar.

[0145] Example 8 Cosolvent Screening

[0146] Table 13

[0147] Material Name Feeding amount Compound 1 4.0g Solvent 8mL IRED Cells 4.0g NADP 28mg GDH 2mL glucose 6.5g 0.1M PBS buffer (pH 6.5) 55.5mL

[0148] operate:

[0149] 1) Take 11 100 mL three-necked reaction bottles, add the materials in the feeding table 13 (substrate concentration is about 50 g / L), wherein the co-solvents are the co-solvents shown in the following table 14, stir magnetically, and react at 27°C;

[0150] 2) The initial pH is 6.3-6.5, and the pH is adjusted to 6.3 with 1N dilute hydrochloric acid. The pH value will decrease during the reaction. The pH value of the reaction solution is stabilized at 6.3 by adding 20wt.% sodium carbonate solution;

[0151] 3) Samples were taken after 24 hours of reaction and HPLC was used for central control;

[0152] 4) Samples were taken after 48 hours of reaction and the samples were controlled by HPLC.

[0153] 5) The reaction was carried out with toluene or isopropyl ether as a co-solvent, evaporated to dryness under reduced pressure at 50°C, slurried with MeOH, filtered, and chirality measured with MeOH.

[0154] The experimental data are shown in Table 14.

[0155] Table 14

[0156]

[0157] In Table 14 above, the initial screening results indicate that DMSO, MTBE, isopropyl ether, and toluene are good co-solvents. Further enlarged comparative experiments revealed that the addition of water-soluble co-solvents would make the reaction worse, and the effects of some co-solvents were low, so toluene was finally selected as the co-solvent.

[0158] Example 9: Further fine screening pH

[0159] Table 15

[0160] Material Name Feeding amount Compound 1 4.0g Toluene 4mL IRED Cells 4.0g NADP 28mg GDH 2mL glucose 6.5g 0.1M PBS buffer 19.5mL

[0161] operate:

[0162] 1) Take three 100 mL three-necked jacketed reaction bottles, add the materials in the feeding table respectively (the substrate concentration in this example is about 100 g / L), stir magnetically, and react at 27°C;

[0163] 2) The pH value will decrease during the reaction. By adding 20% ​​sodium carbonate solution, the pH value of the reaction solution is stabilized at 6.2, 6.3, and 6.4;

[0164] 3) Samples were taken after 14 h of reaction and HPLC / chirality control was performed;

[0165] 4) Samples were taken after 37h of reaction and subjected to HPLC / chirality control.

[0166] The central control data is shown in Table 16.

[0167] Table 16

[0168]

[0169]

[0170] The results showed that the reaction system was sensitive to pH, and the optimal pH was around 6.3.

[0171] Example 10 NADP dosage screening

[0172] Table 17

[0173] Material Name Feeding amount Compound 1 20.0g Toluene 60.0mL IRED Cells 20.0g NADP 280, 210, 140, 70, 14 mg GDH 10.0mL glucose 32.3g 0.1M PBS buffer 57.7mL

[0174] operate:

[0175] 1) Take 5 250mL three-necked jacketed reaction bottles, add the materials in the feeding table respectively (the substrate concentration in this example is about 100g / L), stir mechanically, and react at 27°C;

[0176] 2) The pH value will decrease during the reaction. By adding 20% ​​sodium carbonate solution, the pH value of the reaction solution is stabilized to 6.3;

[0177] 3) Samples were taken after 24 hours of reaction and the test results were shown in Table 18.

[0178] Table 18

[0179]

[0180] The results showed that the reaction was faster and more complete with the increase of NADP dosage.

[0181] Example 11 Glucose dosage screening

[0182] Table 19

[0183] Material Name Feeding amount Compound 1 20.0g Toluene 20.0mL IRED Cells 20.0g NADP 140.0mg GDH 10.0mL glucose 32.3、24.8、17.4g

[0184]

[0185] operate:

[0186] 1) Take three 250mL three-necked jacketed reaction bottles, add the materials in the feeding table (substrate concentration is about 100g / L), stir mechanically, react at 27°C, and stabilize the pH value of the reaction solution at 6.3 by dropping 20% ​​sodium carbonate solution;

[0187] 2) Samples were taken after 24 hours of reaction and the results were analyzed by HPLC. See Table 20.

[0188] Table 20

[0189]

[0190] The results show that the data results with a glucose equivalent of 2.6eq are relatively good, and the 2.6eq reaction is preferred.

[0191] Example 12: Feeding with the clear liquid of Escherichia coli cell lysate expressing imine reductase

[0192] Table 21

[0193] Material Name Feeding amount Compound 1 100.0g Toluene 300.0mL IRED enzyme serum 400.0mL NADP 350.0mg GDH 50.0mL glucose 161.3g

[0194] The IRED enzyme supernatant is a supernatant obtained after centrifugal sedimentation of the cell disrupted liquid, wherein the enzyme activity of the IRED enzyme supernatant is 3.7u / g.

[0195] operate:

[0196] 1) In a 3.0L four-necked jacketed reaction flask, add the materials in the feeding table (substrate concentration is about 100g / L), stir with a paddle machine, react at 27°C, and stabilize the pH value of the reaction solution at 6.3 by dropping 20% ​​sodium carbonate solution;

[0197] 2) Samples were taken after 15 h, 23 h, 39 h, 47 h and 63 h of reaction and the test results were shown in Table 22.

[0198] Table 22

[0199]

[0200] The results showed that the reaction process was stable when the enzyme supernatant was used instead of the cell disruption solution, and the reaction was basically complete after 63 hours. In addition, since the enzyme supernatant contained PBS buffer, not adding the buffer here had no negative effect on the reaction.

[0201] Example 13. Preliminary test of enzyme reaction amplification

[0202] Table 23

[0203]

[0204]

[0205] operate:

[0206] 1) Add the materials in the feeding table to a 500 mL three-necked jacketed reaction bottle (the substrate concentration in this example is 100 g / L), stir mechanically, and react at 27° C.

[0207] 2) Stabilizing the pH value of the reaction solution to 6.3 by adding 20% ​​sodium carbonate solution;

[0208] 3) Samples were taken after 22h, 40h and 45h of reaction and then controlled by HPLC;

[0209] 4) After the reaction is completed, put it in the refrigerator for post-processing screening. The central control data is shown in Table 24.

[0210] Table 24

[0211]

[0212] This experiment shows that when the cell and substrate feeds are both 100 g / L, only 0.9% of the substrate raw materials remain within 45 hours, and the reaction is basically complete.

[0213] Example 14 Enzymatic product post-treatment steps

[0214] Solution 1:

[0215] 20 g of compound 1 was used as a substrate and reacted for 45 hours according to the method of Example 13 to obtain an enzymatic reaction solution. The enzymatic reaction solution was directly centrifuged, and the mother liquor was evaporated under reduced pressure at 50°C until about 40 mL (2 v / w) of solvent remained. Methanol (200 mL, 10 v / w) was added and evaporated twice, and about 40 mL (2 v / w) of solvent remained each time. Methanol (200 mL, 10 v / w) was added and stirred for 15 h, and filtered. The centrifugal filter cake was added with methanol (200 mL, 10 v / w), slurried with methanol, and filtered. Combine the methanol phases, add 85% hydrazine hydrate (0.21 mol, 3.0 eq) to the methanol phase, stir at an external temperature of 80°C for 1 h, cool the reaction solution in an ice-water bath, a small amount of solid precipitated, filter out the solid, and evaporate most of the solvent from the mother liquor at 40°C under reduced pressure (remaining about 60 mL, 3 v / w). Add DCM (200 mL, 10 v / w) and evaporate under reduced pressure until the system remains about (60 mL, 3 v / w), then add DCM (400 mL, 20 v / w), slurry at room temperature overnight, and quantitative HPLC detection shows that the yield of TL-010-2 is 89.3%.

[0216] Option 2:

[0217] 40 g of compound 1 was used as a substrate and reacted for 45 hours according to the method of Example 13 to obtain an enzymatic reaction solution. Most of the solvent and water (about 80 mL, 2 v / w) were evaporated from the enzymatic reaction solution under reduced pressure at 50 ° C., and the solution was dried with DCM 3 times and methanol once (each time the system was dried, the remaining solvent was about 80 mL, 2 v / w), methanol (400 mL, 10 v / w) was added and slurried for 15 h, filtered, and the methanol phase was collected. 85% hydrazine hydrate (0.42 mol, 3.0 eq) was added to the methanol phase and stirred at an external temperature of 80°C for 1 h. The reaction solution was cooled in an ice-water bath. A small amount of solid precipitated and was filtered off. Most of the solvent was evaporated from the mother liquor at 40°C under reduced pressure (about 120 mL, 3 v / w remained). DCM (400 mL, 10 v / w) was added and evaporated under reduced pressure until about (120 mL, 3 v / w) remained in the system. DCM (800 mL, 20 v / w) was then added and slurried at room temperature overnight. The yield of TL-010-2 was 85.2% as determined by quantitative HPLC.

[0218] Option 3:

[0219] 20g of compound 1 was used as a substrate, and the reaction was carried out according to the method of Example 13 for 45 hours to obtain an enzymatic reaction liquid. The enzymatic reaction liquid was directly centrifuged, and a small amount of solid floated between the toluene and water layers, and centrifuged again to collect the filter cake and the mother liquor. The mother liquor was evaporated at 50°C under reduced pressure to remove toluene and water, methanol was added for pulping, filtered, and the methanol phase was collected. 85% hydrazine hydrate (0.21mol, 3.0eq) was added to the methanol phase, and the external temperature was 80°C and stirred for 1h. The reaction liquid was cooled in an ice water bath, and a small amount of solid precipitated. The solid was filtered out, and the mother liquor was evaporated at 40°C under reduced pressure to remove most of the solvent (about 60mL, 3v / w remaining), DCM (200mL, 10v / w) was added and evaporated under reduced pressure until the system remained about (60mL, 3v / w), and then DCM (400mL, 20v / w) was added, and pulped overnight at room temperature. The yield of TL-010-2 was 44.64% as determined by quantitative HPLC.

[0220] The filter cake was slurried with methanol (200 mL, 10 v / w), filtered, and the methanol phase was collected. 85% hydrazine hydrate (0.21 mol, 3.0 eq) was added to the methanol phase, and the external temperature was 80 ° C. Stirring for 1 h, the reaction solution was cooled in an ice water bath, and a small amount of solid precipitated. The solid was filtered out, and the mother liquor was evaporated at 40 ° C. Most of the solvent (about 60 mL, 3 v / w) was removed by decompression, and DCM (200 mL, 10 v / w) was added and evaporated under reduced pressure until the system remained about (60 mL, 3 v / w), and then DCM (400 mL, 20 v / w) was added, and slurry was kept at room temperature overnight. The yield of TL-010-2 was 44.64% after testing.

[0221] The total yield of the mother liquor and filter cake in this scheme is 89.28%.

[0222] Example 15 Preparation of 5 kg chiral amine intermediate TL-010-2

[0223] 1. Enzymatic synthesis of compounds 2 and 2A and post-treatment of products

[0224] 1) Compound 1 (2.32 Kg, 7.99 mol, 1 eq), glucose (3.744 Kg, 20.78 mol, 2.6 eq), NADP (8.12 g, 0.0103 mol, 0.00074 eq), toluene (3 v / w), GDH (solution, 0.5 v / w), IRED enzyme solution (4 v / w) were added to a 50 L multi-port reactor equipped with mechanical stirring. The total volume of the reaction raw materials was 23.5 L, and the mixture was reacted at 29 ° C with mechanical stirring;

[0225] 2) using a potentiometric automatic titrator to stabilize the reaction pH at 6.30 by adding 10% sodium carbonate solution;

[0226] 3) After 42 hours of reaction, samples were taken and HPLC was used for central control; the sampling method after sampling was as follows: 0.3 mL of the reaction solution was added to a 3 mL centrifuge tube, and 2.0 mL of DMSO was added. There was a slight release of heat, and the supernatant was taken after centrifugation. 0.3 μL was injected, and the concentration was 210 nm; the central control index was as follows: if compound 1 ≤ 2.0% according to the area normalization method, the reaction endpoint was considered.

[0227] 4) After the reaction is completed, the enzyme reaction solution is post-treated according to the method of Scheme 1 of Example 14 and the methanol phase is combined and directly used in the next reaction.

[0228] 2. Synthesis of TL-010-2

[0229] 1) Add the combined methanol phase from step 1 to the reactor, add 85% hydrazine hydrate (1.411 Kg, 23.97 mol, 3 eq), heat to 80°C, and stir to react for 1 hour.

[0230] 2) Take a sample and send it to IPC. If the compound 2 / 2-A is less than 0.1% according to the area normalization method, it is the end point of the reaction.

[0231] 3) Stop heating, let it cool naturally, and stir overnight.

[0232] 4) Vacuum distillation (temperature 40°C) to remove most of the solvent until the residual volume is about 3v / w. Vacuum distill with DCM twice, adding 5v / w of DCM each time, evaporating until the residual volume is 3v / w, adding 20v / w of DCM, and slurrying at room temperature (15°C) overnight.

[0233] 5) The system was cooled to 6°C, filtered, and the filter cake was rinsed with cold DCM (5°C, 2 v / w).

[0234] 6) The mother liquor of step 5 is concentrated to a residue and product 1 is isolated.

[0235] 7) Add DCM 3 v / w to the filter cake of step 5 and stir at room temperature (15° C.) overnight.

[0236] 8) Cool to 6°C, filter, and rinse the filter cake with cold DCM (5°C, 2 v / w).

[0237] 9) The mother liquor is concentrated to a residue and product 2 is isolated.

[0238] 10) The mother liquor product 1 and product 2 were combined to obtain the first batch of oily products (oil) of 0.962 Kg (5.929 mol, 74.2%). The filter cake was washed with DCM (2 v / w) and dried to obtain the second batch of solid products of 0.1212 Kg (0.747 mol, 9.3%). The total comprehensive yield was 1.0832 Kg (6.677 mol, 83.6%).

[0239] According to the purity of TL-010-2 free radical, the purity of the first batch of products was 90.6%, 0.8716Kg (5.373mol, 67.2%), and the purity of the second batch of solid products was 99.1%, 0.1201Kg (0.740mol, 9.3%), and the combined total weight was 0.9917Kg (6.113mol, 76.4%).

[0240] 3. Synthesis of TL-010-2 hydrochloride

[0241] 1) Add TL-010-2 (1.0 eq) and EtOH (10.0 v / w) to a 20 L four-necked reaction bottle with a magnetic rod, and stir magnetically at room temperature to dissolve the system;

[0242] 2) Cool the system to 10±5°C, slowly dropwise add 10% HCl in EtOH solution (4.401 kg, 27.74 mol, 5 eq), a large amount of solid precipitates during the dropwise addition, and control the temperature to <30°C;

[0243] 3) After the addition is complete, reflux at 80°C with stirring for 0.5 h, then slowly cool to room temperature and stir for 12 h;

[0244] 4) After 2 h, the system was cooled to 5±3°C and stirred for 1.0 h, and then quickly filtered under nitrogen protection. The filter cake was rinsed with cold EtOH (5±3°C, 2.0 v / w), and then dried at 50°C under reduced pressure until the product had a constant weight and a moisture content of ≤1.0%, to obtain a light yellow or white solid. The mother liquor was weighed, and the content was detected to calculate the mother liquor loss;

[0245] 5) After integrating the two batches of materials, including the mother liquor and purity correction, the total product output was 1.133 Kg (purity 98.3%, chiral purity>98%, yield 78%).

[0246] Example 16 Preliminary stability test of TL-010-2 free base and hydrochloride

[0247] The TL-010-2 free base and TL-010-2DiHCl prepared in Example 16 were tested for storage stability, and the results are shown in Table 25. Among them, the purity of TL-010-2 free base dropped to about 90% after being stored at 5±3°C for 60 days, and the purity of TL-010-2 free base dropped to below about 66% after being stored at 25±2°C and 60±5%RH for 60 days. The purity of TL-010-2DiHCl can be maintained at more than 98.9% when stored at 5±3°C for 60 days and at 25±2°C and 60±5%RH for 60 days. Therefore, TL-010-2DiHCl can be used for long-term storage.

[0248] Table 25

[0249]

[0250]

Claims

1. A method for preparing a chiral amine and a salt thereof, characterized in that: The steps include: (1) reacting compound 1 in the presence of imine reductase, NADP, glucose dehydrogenase, glucose, a solvent and a cosolvent to obtain compound 2 and / or compound 2-A; (2) converting the compound 2 and / or the compound 2-A into chiral amine TL-010-2 ((1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline); Wherein, the structural formula of the compound 1 is: The structural formula of the compound 2 is: The structural formula of the compound 2-A is: The structural formula of the chiral amine TL-010-2 is:

2. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: The initial concentration of the compound 1 is 30-100 g / L; or, In step (1), the pH value is controlled to be maintained at 5 to 7 during the reaction; or, In step (1), the reaction temperature is controlled to be 25-35°C; or, The solvent is PBS buffer or water; or, The co-solvent is one or more of ethyl acetate, isopropanol, dimethyl sulfoxide, toluene, isopropyl ether, methyl tert-butyl ether, and methanol; or, The mass of the NADP feed is 0.07% to 1.5% of the mass of the compound 1 feed; or, The feed mass of the glucose dehydrogenase is 25% to 75% of the feed mass of the compound 1; or The molar amount of glucose fed is 1 to 3 times the molar amount of compound 1 fed.

3. The method for preparing a chiral amine and a salt thereof according to claim 2, characterized in that: The initial concentration of the compound 1 is 50-100 g / L; or, In step (1), the pH value is controlled to be maintained at 6 to 6.5 during the reaction; or, In step (1), the reaction temperature is controlled to be 25-30°C; or, The co-solvent is toluene; or, The mass of the NADP is 0.1% to 1.5% of the mass of the compound 1; or The feed mass of the glucose dehydrogenase is 40% to 60% of the feed mass of the compound 1; or The molar amount of glucose fed is 2 to 3 times the molar amount of compound 1 fed.

4. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: The imine reductase is derived from Escherichia coli cells or Pichia pastoris and is fed in the form of enzyme powder, whole cells, cell disrupted liquid, or supernatant separated from the cell disrupted liquid; When the imine reductase is fed in the form of enzyme powder, the feeding mass of the imine reductase enzyme powder is 0.5 to 5 times the feeding mass of the compound 1; When the imine reductase is fed in the form of whole cells or cell lysate, the feeding mass of the whole cells or the feeding mass of the cells in the cell lysate is 1 to 10 times the feeding mass of the compound 1; When the imine reductase is fed in the form of a supernatant obtained by separation of cell lysate, the feeding amount of the supernatant is 4 to 10 mL of the supernatant per 1 g of the compound 1.

5. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: After the reaction in step (1) is completed, centrifuge or distill under reduced pressure, add dichloromethane and / or methanol, stir, filter, and collect the organic phase; add dichloromethane and / or methanol to the collected filter cake, slurry, filter, and collect the organic phase, this step is repeated once or multiple times; the collected organic phases are combined and directly used in the reaction of step (2).

6. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: The compound 2 and the compound 2-A react in the presence of hydrazine hydrate and methanol to obtain the chiral amine TL-010-2.

7. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: The salt of the chiral amine is hydrochloride, hydrobromide, oxalate, acetate, lactate, phosphate, fumarate, tartrate, citrate, gluconate, sulfate, methanesulfonate or other sulfonates.

8. The method for preparing a chiral amine and a salt thereof according to claim 1, characterized in that: The preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline dihydrochloride (TL-010-2 dihydrochloride) in the presence of a hydrogen chloride ethanol solution and methanol, wherein the structural formula of the TL-010-2 dihydrochloride is: or, The preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline dihydrobromide (TL-010-2 dihydrobromide) in the presence of hydrobromic acid and methanol, wherein the structural formula of the TL-010-2 dihydrobromide is: or, The preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline oxalate (TL-010-2 oxalate) in the presence of oxalic acid and methanol, wherein the The preparation method further comprises the step of converting the chiral amine TL-010-2 into (1R)-(-)-1-aminomethyl-1,2,3,4-tetrahydroisoquinoline maleate (TL-010-2 maleate) in the presence of maleic acid and methanol, wherein the structural formula of the TL-010-2 maleate is:

9. A salt of a chiral amine, characterized in that: Its structural formula is 10. A method for preparing (L-praziquantel), characterized in that: It includes the following steps: 1) preparing the chiral amine TL-010-2 or its salt according to the preparation method according to any one of claims 1 to 8; 2) Using the chiral amine TL-010-2 or its salt as a raw material to prepare the (R)-praziquantel.

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