((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method and application

Abstract

This invention discloses ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method, and its applications, relating to the field of chemical synthesis technology. By providing a dipeptide hydrochloride with tyrosine and leucine as key structural units, the synthesis method of this dipeptide hydrochloride is simple, the reaction conditions are mild, and the product has high purity, high yield, and low impurity content. When the above-mentioned dipeptide hydrochloride provided by this invention is used as a partial active fragment of polypeptide products, it can also significantly improve the purity and yield of polypeptide products.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, and more specifically, to a ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method, and its application. Background Technology

[0002] Tyrosine is a non-essential amino acid that plays multiple roles in the human body. It is converted into various biological substances through different metabolic pathways, such as dopamine, adrenaline, and thyroxine. These substances are closely related to neurotransmission control and metabolism, therefore, research on tyrosine metabolism helps in understanding the pathogenesis of certain diseases.

[0003] Amino acid drugs containing leucine are mainly used for amino acid infusion and comprehensive amino acid preparations. They are used to diagnose and treat children with idiopathic hyperglycemia and glucose metabolism disorders, biliary liver disease with decreased secretion, anemia, poisoning, muscular dystrophy, poliomyelitis, neuritis, and mental illness. Amino acids and amino acid derivatives have been widely used in drug development, attracting active research and development from researchers both domestically and internationally.

[0004] However, due to the complex structure and abundant reactive sites of amino acids, when multiple amino acid complexes are involved in drug development, it is easy to encounter situations such as low yield, low purity, and high impurity content of the reaction products.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method, and its application.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, the chemical structural formula of which is as follows:

[0009]

[0010] Secondly, the present invention provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as described in the foregoing embodiments, comprising condensing BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to obtain a reaction intermediate, and then subjecting the reaction intermediate to a deprotection reaction.

[0011] Thirdly, the present invention provides the use of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as described in the foregoing embodiments, or ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the preparation method of any of the foregoing embodiments, in the preparation of polypeptide products.

[0012] The present invention has the following beneficial effects:

[0013] This invention provides ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method, and its application. By providing a dipeptide hydrochloride with tyrosine and leucine as key structural units, the synthesis method of this dipeptide hydrochloride is simple, the reaction conditions are mild, and the product has high purity, high yield, and low impurity content. When the above-mentioned dipeptide hydrochloride provided by this invention is used as a partial active fragment of polypeptide products, it can also significantly improve the purity and yield of polypeptide products. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The high-performance liquid chromatogram of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of the present invention;

[0016] Figure 2 The 1H NMR spectrum of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of this invention;

[0017] Figure 3 The mass spectrum of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of the present invention;

[0018] Figure 4 The mass spectrum of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of the present invention;

[0019] Figure 5The mass spectrum of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0022] In a first aspect, the present invention provides ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, the chemical structural formula of which is as follows:

[0023]

[0024] Secondly, the present invention provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as described in the foregoing embodiments, comprising condensing BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to obtain a reaction intermediate, and then subjecting the reaction intermediate to a deprotection reaction.

[0025] In an optional implementation, the chemical structural formula of the reaction intermediate is:

[0026]

[0027] In an optional embodiment, the preparation method of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride includes the following steps:

[0028] S01, condensation reaction

[0029] In an optional embodiment, the condensation reaction process includes: dissolving BOC-O-benzyl-L-tyrosine in a solvent for the condensation reaction and cooling it to 0-5°C, then adding a condensing agent, a racemic inhibitor, and a condensation accelerator, reacting at 10-15°C for 20-40 min, and then adding L-leucine benzyl ester hydrochloride and a condensation accelerator to carry out the condensation reaction.

[0030] The purpose of cooling down during the dissolution of BOC-O-benzyl-L-tyrosine is to better control the reaction temperature when adding the condensing agent.

[0031] After BOC-O-benzyl-L-tyrosine is dissolved, a condensing agent, a racemic inhibitor, and a condensing accelerator are added to the system, and the system is reacted at a certain temperature for a period of time to adjust the system to a suitable intermediate state for the condensation reaction. Then, L-leucine benzyl ester hydrochloride is added to carry out the condensation reaction with BOC-O-benzyl-L-tyrosine, and a condensing accelerator is added at the same time to assist the smooth progress of the condensation reaction.

[0032] Preferably, in order to ensure the structure and purity of the final obtained dipeptide hydrochloride, the mass ratio of BOC-O-benzyl-L-tyrosine to L-leucine benzyl ester hydrochloride is 1:0.70 to 0.75. For example, the mass ratio of BOC-O-benzyl-L-tyrosine to L-leucine benzyl ester hydrochloride can be any value among 1:0.70, 1:0.71, 1:0.72, 1:0.73, 1:0.74 or 1:0.75, or a range between any two values, or any other point value within the above range.

[0033] Preferably, the solvent for the condensation reaction includes any one of N,N-dimethylformamide and tetrahydrofuran. In order to ensure the purity of the final obtained dipeptide hydrochloride, and at the same time ensure the yield and purity of the obtained reaction intermediate, the solvent for the condensation reaction is more preferably N,N-dimethylformamide (DMF).

[0034] Preferably, the mass ratio of the solvent in the condensation reaction to BOC-O-benzyl-L-tyrosine is 5.5 to 6.0:1, for example, it can be any value among 5.5:1, 5.6:1, 5.7:1, 5.8:1, 5.9:1 or 6.0:1, or a range between any two values, or any other point value within the above range.

[0035] Preferably, the condensing agent includes either TBTU or HBTU. To ensure the purity of the final obtained dipeptide hydrochloride, and at the same time to ensure the yield and purity of the obtained reaction intermediate, the condensing agent is more preferably TBTU.

[0036] TBTU stands for O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid. HBTU stands for O-benzotriazole-tetramethylurea hexafluorophosphate.

[0037] Preferably, the racemic inhibitor includes any one of 1-hydroxybenzotriazole and N-hydroxy-7-azobenzotriazole. In order to ensure the purity of the final obtained dipeptide hydrochloride, and at the same time ensure the yield and purity of the obtained reaction intermediate, the racemic inhibitor is more preferably 1-hydroxybenzotriazole (HOBT).

[0038] Preferably, the condensation accelerator includes any one of N,N-diisopropylethylamine and pyridine. In order to ensure the purity of the final obtained dipeptide hydrochloride, and at the same time ensure the yield and purity of the obtained reaction intermediate, the condensation accelerator is more preferably N,N-diisopropylethylamine (DIPEA).

[0039] Preferably, before adding L-leucine benzyl ester hydrochloride, in order to adjust the system to reach the intermediate state of the reaction, the mass ratio of condensing agent, racemic inhibitor, and condensing accelerator to BOC-O-benzyl-L-tyrosine is 1-1.05:0.35-0.4:0.5-0.55:1.

[0040] Preferably, the mass ratio of the condensation accelerator added together with L-leucine benzyl ester hydrochloride to BOC-O-benzyl-L-tyrosine is 0.3–0.4:1. When the condensation accelerator added later is N,N-diisopropylethylamine (DIPEA), N,N-diisopropylethylamine can neutralize the hydrochloric acid in L-leucine benzyl ester hydrochloride, thereby promoting the smooth progress of the condensation reaction.

[0041] Preferably, during the condensation reaction with the addition of L-leucine benzyl ester hydrochloride, there is no need to heat or maintain the temperature of the reaction system. The reaction system is allowed to naturally heat to room temperature and continue to react. This method has mild reaction conditions and is simple to operate.

[0042] Preferably, the reaction can be terminated by thin-layer chromatography (TLC) spotting. The condensation reaction is considered to be complete when the reaction of the raw material BOC-O-benzyl-L-tyrosine is observed to be complete.

[0043] SO2, extraction

[0044] In an optional embodiment, the material following the condensation reaction is further extracted before obtaining the reaction intermediate.

[0045] The choice of solvent in the extraction process has a significant impact on the purity and yield of the reaction intermediate obtained by the condensation reaction. Preferably, in order to ensure the purity and yield of the reaction intermediate, the good solvent used for extraction is methyl tert-butyl ether, and the poor solvent is petroleum ether.

[0046] Preferably, the extraction process includes: adding a good solvent and an alkaline material to the material after the condensation reaction for extraction; washing the separated organic phase with an acidic material; washing the separated organic phase with an alkaline material; and stirring the separated organic phase with a poor solvent to induce crystallization.

[0047] The material after the condensation reaction is the overall reaction system without BOC-O-benzyl-L-tyrosine after TLC spotting.

[0048] During the extraction process, a good solvent is added to dissolve the reaction intermediates in the material after the condensation reaction. An alkaline material is used to dissolve the condensing agent, racemic inhibitor, and other acidic byproducts in the material after the condensation reaction. The separated organic phase is washed with an acidic material to remove the condensing agent and other alkaline byproducts. Then, it is washed with an alkaline material again to remove the residual acidic byproducts in the organic phase. Finally, a poor solvent is added to the organic phase to stir and crystallize, and the reaction intermediates precipitate from the organic phase.

[0049] Preferably, in order to ensure the yield and purity of the reaction intermediate during the extraction process, the mass ratio of the good solvent to BOC-O-benzyl-L-tyrosine is 7-8:1, and the mass ratio of the poor solvent to BOC-O-benzyl-L-tyrosine used during stirring and crystallization is 12.5-13.5:1.

[0050] For example, the mass ratio of the good solvent to BOC-O-benzyl-L-tyrosine can be any value among 7:1, 7.2:1, 7.4:1, 7.6:1, 7.8:1, or 8:1, or a range between any two values, or any other point value within the aforementioned range.

[0051] For example, the mass ratio of the poor solvent used during stirring crystallization to BOC-O-benzyl-L-tyrosine can be any value among 12.5:1, 12.7:1, 12.9:1, 13.1:1, 13.2:1, or 13.5:1, or a range between any two values, or any other point value within the above range.

[0052] Preferably, the acidic material is a hydrochloric acid solution, and the concentration of hydrochloric acid in the hydrochloric acid solution is 0.2 to 0.8N. For example, it can be any value among 0.2N, 0.3N, 0.4N, 0.5N, 0.6N, 0.7N, and 0.8N, or a range between any two values, or any other point value within the above range.

[0053] Preferably, the mass ratio of the acidic material to BOC-O-benzyl-L-tyrosine is 4.5 to 5.5:1, for example, it can be any value among 4.5:1, 4.7:1, 4.9:1, 5.1:1, 5.3:1 or 5.5:1, or a range between any two values, or any other point value within the above range.

[0054] Preferably, the alkaline material includes any one of sodium bicarbonate, sodium carbonate, or sodium hydroxide, with sodium bicarbonate being the most preferred.

[0055] Preferably, the alkaline material added together with the good solvent is a 2-3% sodium bicarbonate solution, and the mass ratio of the 2-3% sodium bicarbonate solution to BOC-O-benzyl-L-tyrosine is 18-19:1.

[0056] Preferably, the alkaline material added during the washing process is a 0.5-1.5% sodium bicarbonate solution, and the mass ratio of the 0.5-1.5% sodium bicarbonate solution to BOC-O-benzyl-L-tyrosine is 4.5-5.5:1.

[0057] HOBT in the system can be completely removed by adding alkaline material twice. Since most of the HOBT has been removed after the first wash, the concentration of alkaline material can be reduced in the second wash to reduce raw material consumption.

[0058] Preferably, in order to ensure that the reaction intermediate can be completely precipitated, the stirring and crystallization time is 2 to 4 hours.

[0059] In an optional embodiment, in order to improve the yield of the reaction intermediate, the filter cake obtained after crystallization and filtration is further subjected to pulping and filtration with a poor solvent, and the filter cake obtained by pulping and filtration is dried to obtain the reaction intermediate.

[0060] Preferably, the mass ratio of the undesirable solvent to BOC-O-benzyl-L-tyrosine in the pulping and filtration process is 7.5 to 8.0:1, for example, it can be any value among 7.5:1, 7.6:1, 7.7:1, 7.8:1, 7.9:1 or 8.0:1, or a range between any two values, or any other point value within the above range.

[0061] Preferably, the temperature for drying to obtain the reaction intermediate is 45–55°C, more preferably 50°C.

[0062] SO3, deprotection reaction

[0063] In an optional embodiment, the deprotection reaction includes mixing and reacting a reaction intermediate with a deprotection solvent system; the deprotection solvent system includes a deprotection reagent and a deprotection solvent.

[0064] Preferably, the concentration of the reaction intermediate in the deprotection solvent system is 0.11–0.18 mol / L, the volume ratio of the deprotection reagent to the mass ratio of BOC-O-benzyl-L-tyrosine is 2.5–2.6 L:1 kg, and the remainder in the deprotection solvent system is the deprotection solvent.

[0065] To ensure the efficiency of the deprotection reaction, the concentration of the reaction intermediate in the deprotection solvent system is controlled within the aforementioned range, allowing the reaction to proceed smoothly and rapidly. To maintain the concentration of the reaction intermediate within this range during the deprotection reaction, this can be achieved by adjusting the amount and frequency of addition of the deprotection solvent.

[0066] For example, after mixing the reaction intermediate and the deprotection reagent, the mass of the deprotection solvent that needs to be added is calculated based on the current concentration. After the reaction has been going on for a period of time, a solid will appear in the system, affecting the reaction rate of the deprotection reaction. Therefore, the deprotection solvent can be added to the system again to improve the conversion rate of the deprotection reaction of the reaction intermediate.

[0067] Preferably, the deprotecting agent includes a tetrahydrofuran hydrochloride solution or an ethyl acetate hydrochloride solution; to improve the purity and yield of the final obtained dipeptide hydrochloride, the deprotecting agent is more preferably a tetrahydrofuran hydrochloride solution.

[0068] Preferably, the concentration of hydrochloric acid in the tetrahydrofuran hydrochloride solution is 2 to 4N, for example, it can be any value of 2N, 3N or 4N, or a range between any two values, or any other point value within the above range.

[0069] Preferably, the deprotection solvent includes dichloromethane or ethyl acetate; to improve the purity and yield of the final obtained dipeptide hydrochloride, dichloromethane is more preferably used as the deprotection solvent.

[0070] Preferably, the deprotection reaction temperature is 22–28°C, and the deprotection reaction can be carried out at room temperature, such as 25°C.

[0071] The deprotection reaction conditions in the synthesis process of this invention are mild and the operation is simple. At the same time, the obtained dipeptide hydrochloride has high purity, high yield and low impurity content, making it suitable for large-scale production of dipeptide hydrochloride.

[0072] In an optional embodiment, after the deprotection reaction is completed, the deprotected material is obtained, the deprotected material is separated, and the separated solid material is pulped, filtered and dried.

[0073] The process of separating the material after the deprotection reaction can be carried out by vacuum filtration. The solid material obtained is a filter cake. The filter cake is pulped, filtered and dried to obtain ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride.

[0074] Preferably, the solvent for pulping includes either ethyl acetate or tetrahydrofuran.

[0075] Preferably, the mass ratio of solvent to solid material in the pulping process is 1:0.15 to 0.25, and the pulping time is 40 to 80 minutes.

[0076] Preferably, the drying temperature during the process of obtaining ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is 45-55°C.

[0077] In an optional embodiment, the yield of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is >75%, the purity is ≥99%, and the maximum content of single impurities is <0.4%.

[0078] Preferably, the yield of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is >85%, the purity is ≥99%, and the content of the maximum single impurity is <0.2%.

[0079] In an optional embodiment, the reaction process of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is as follows:

[0080]

[0081] Thirdly, the present invention provides the use of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as described in the foregoing embodiments, or ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the preparation method of any of the foregoing embodiments, in the preparation of polypeptide products.

[0082] Preferably, the polypeptide product can be, for example, any of the amino acid inhibitors or carriers.

[0083] Example 1

[0084] This embodiment provides ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, the chemical structural formula of which is as follows:

[0085]

[0086] This embodiment also provides a method for preparing the above-mentioned ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, the specific process of which is as follows:

[0087]

[0088] The specific steps are as follows:

[0089] S01, condensation reaction

[0090] Weigh 90 kg of BOC-O-benzyl-L-tyrosine and add it to 513 kg of N,N-dimethylformamide, then cool it to 0-5 °C. Then add 32.5 kg of HOBT, 93.3 kg of TBTU and 47.0 kg of DIPEA in sequence, and maintain the reaction at 10-15 °C for 30 min. Then add 63.7 kg of L-leucine benzyl ester hydrochloride and 31.3 kg of DIPEA, and allow the temperature to rise naturally to room temperature.

[0091] Using TLC, the condensation reaction was considered complete when the reaction of the raw material BOC-O-benzyl-L-tyrosine was observed to be finished.

[0092] SO2, extraction

[0093] Add 1660 kg of 2.5% sodium bicarbonate solution and 666 kg of methyl tert-butyl ether to the material obtained after the condensation reaction in step S01 for extraction. After extraction, separate to obtain an organic phase. Add 450 kg of 0.5N hydrochloric acid solution to the organic phase for washing. The resulting aqueous phase has a pH of 1. Add 450 kg of 1% sodium bicarbonate solution to the organic phase for washing. The resulting aqueous phase has a pH of 8. Add 1170 kg of petroleum ether to the organic phase and stir to crystallize for 3 hours.

[0094] After crystallization, the system was filtered, and the obtained filter cake was then slurried with 702 kg of petroleum ether. The solid obtained after slurrying and filtration was placed in a forced-air drying oven and dried at 50℃. After drying, 131.4 kg of white to pale yellow solid was obtained, which is the reaction intermediate. The reaction intermediate is benzyl((S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)-L-leucine ester.

[0095] SO3, deprotection reaction

[0096] The reaction intermediate obtained in step S02 was added to 1741.1 kg of dichloromethane, followed by 228.8 L of 3N tetrahydrofuran hydrochloride solution. The reaction was carried out at 25 °C. After 1 hour when a solid appeared in the system, 889.1 kg of dichloromethane was added until the reaction was completed. The reaction was considered complete when the reaction intermediate was observed to have reacted completely by TLC.

[0097] After the deprotection reaction was completed, the obtained deprotected material was filtered to separate the solid material into a filter cake. The filter cake was slurried with 658 kg of ethyl acetate for 1 hour and then filtered. The filter cake obtained by the second filtration was placed in a vacuum oven at 50°C and dried to obtain 103.5 kg of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride.

[0098] The purity of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this embodiment was determined by high performance liquid chromatography, and the results were as follows: Figure 1 The results are shown. (From...) Figure 1 It can be seen that the purity of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this embodiment is 99.5%, and the yield is 88.5%.

[0099] The intermediate obtained in step S02 was subjected to high-performance liquid chromatography (HPLC) under the same conditions as described above. The yield of the reaction intermediate benzyl((S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)-L-leucine ester was 94%, and the HPLC purity was 99.4%.

[0100] The ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this embodiment was analyzed using nuclear magnetic resonance spectroscopy to obtain the following results: Figure 2 The 1H NMR spectrum is shown. The ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this embodiment was analyzed using a Tandem Quadrupole mass spectrometer to obtain the following results. Figures 3-5 The mass spectrum shown is from... Figures 2-5 As can be seen, the structure of the product prepared by the method provided in this embodiment is as follows:

[0101] Its chemical name is ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride.

[0102] Example 2

[0103] This embodiment provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride. The steps are similar to those in Example 1, except that the deprotecting solvent in step S03 is ethyl acetate.

[0104] Comparative Example 1

[0105] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the good solvent in step S02 is ethyl acetate.

[0106] Comparative Example 2

[0107] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that in step S02, the good solvent is ethyl acetate and isopropanol in a volume ratio of 10:1.

[0108] Comparative Example 3

[0109] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that in step SO2, the good solvent is ethyl acetate and the poor solvent is n-heptane.

[0110] Comparative Example 4

[0111] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that in step S02, the good solvent is methyl tert-butyl ether and isopropanol in a volume ratio of 10:1.

[0112] Comparative Example 5

[0113] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting agent in step S03 is thionyl chloride and the deprotecting solvent is methanol.

[0114] Comparative Example 6

[0115] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting agent in step S03 is oxaloyl chloride and the deprotecting solvent is methanol.

[0116] Comparative Example 7

[0117] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting solvent in step S03 is methanol.

[0118] Comparative Example 8

[0119] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting agent in step S03 is thionyl chloride and the deprotecting solvent is ethanol.

[0120] Comparative Example 9

[0121] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting agent in step S03 is oxaloyl chloride and the deprotecting solvent is ethanol.

[0122] Comparative Example 10

[0123] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting solvent in step S03 is ethanol.

[0124] Comparative Example 11

[0125] This comparative example provides a method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, with steps similar to those in Example 1, except that the deprotecting solvent in step S03 is tetrahydrofuran.

[0126] Experimental Example 1

[0127] ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride was prepared according to the methods provided in Examples 1 and Comparative Examples 1-4. The yield and purity of the reaction intermediate benzyl((S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)-L-leucine ester obtained in step S02 were investigated, and the effect of changes in the yield and purity of the reaction intermediate on the final product ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride was verified.

[0128] The purity of the reaction intermediates was determined by high performance liquid chromatography (HPLC), using BOC-O-benzyl-L-tyrosine, L-leucine benzyl ester hydrochloride, DIPEA, and N,N-dimethylformamide as standard substrates. The results are shown in Table 1.

[0129] Table 1 Purity and yield of reaction intermediates

[0130] Yield / % purity / % Impact on the final product Example 1 94 99 No impact Comparative Example 1 67 96 influential Comparative Example 2 61 98 No impact Comparative Example 3 69 96 influential Comparative Example 4 78 99 No impact

[0131] Table 1 shows that the choice of good and bad solvents in the extraction process has a significant impact on the purity and yield of the reaction intermediates. Comparative Examples 1 and 3 have low purity, which affects the purity of the final product when the obtained reaction intermediates are further subjected to deprotection reactions, making it difficult to control the single impurity content of the final product below 0.5%. Comparative Examples 2 and 4 also have low yields of the reaction intermediates, which affects the yield of the final product.

[0132] Experimental Example 2

[0133] ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride was prepared according to the methods provided in Examples 1 and 2 and Comparative Examples 5-11, and its impurity content and purity were determined by high performance liquid chromatography (HPLC). ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, L-leucine benzyl ester hydrochloride, and the deprotected solvent were used as standard substrates for analysis, and the results are shown in Table 2.

[0134] Table 2. Purity, Yield, and Impurity Content of the Final Product

[0135] Yield / % purity / % Maximum single impurity content / % Example 1 88 99 0.19 Example 2 78 99 0.37 Comparative Example 5 69 97 2.2 Comparative Example 6 68 97 2.2 Comparative Example 7 66 98 1.0 Comparative Example 8 66 96 1.9 Comparative Example 9 69 96 2.1 Comparative Example 10 68 97 1.1 Comparative Example 11 62 99 0.67

[0136] As shown in Table 2, the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the method provided in the embodiments of the present invention has high yield, high purity, and low maximum single impurity content. In Comparative Examples 5-11, due to adjustments in the deprotection reagent and / or deprotection solvent, the yield and purity of the final active ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride decreased, and the impurity content increased.

[0137] This invention provides a ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, its preparation method, and its application. The preparation method is simple, the reaction conditions are mild, and the product has high purity, high yield, and low impurity content. When the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided by this invention is used as a partial active fragment of polypeptide products, it can also significantly improve the purity and yield of polypeptide products.

[0138] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, characterized in that, The reaction involves a condensation reaction of BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to obtain a reaction intermediate, followed by a deprotection reaction of the reaction intermediate. The deprotection reaction includes mixing the reaction intermediate with a deprotection solvent system; the deprotection solvent system includes a deprotection reagent and a deprotection solvent. The deprotecting agent includes a tetrahydrofuran hydrochloride solution or an ethyl acetate hydrochloride solution; the deprotecting solvent includes dichloromethane or ethyl acetate. Before obtaining the reaction intermediate, the material after the condensation reaction is further extracted; the extraction process includes: adding a good solvent and an alkaline material to the material after the condensation reaction for extraction, washing the separated organic phase with an acidic material, washing the separated organic phase with an alkaline material, and stirring the separated organic phase with a poor solvent to precipitate crystals. The good solvent used for extraction is methyl tert-butyl ether, and the poor solvent is petroleum ether; It also includes slurrying and filtering the filter cake obtained after crystallization with a poor solvent, and drying the filter cake obtained by slurry filtration to obtain the reaction intermediate; The chemical structural formula of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is as follows: 。 2. The preparation method according to claim 1, characterized in that, The concentration of the reaction intermediate in the deprotection solvent system is 0.11~0.18 mol / L, the volume ratio of the deprotection reagent to the mass ratio of BOC-O-benzyl-L-tyrosine is 2.5~2.6L:1kg, and the remainder in the deprotection solvent system is the deprotection solvent.

3. The preparation method according to claim 1, characterized in that, The deprotection reagent is a tetrahydrofuran hydrochloride solution.

4. The preparation method according to claim 3, characterized in that, The concentration of hydrochloric acid in the tetrahydrofuran hydrochloride solution is 2~4N.

5. The preparation method according to claim 1, characterized in that, The deprotection solvent is dichloromethane.

6. The preparation method according to claim 1, characterized in that, The deprotection reaction is carried out at a temperature of 22~28℃.

7. The preparation method according to claim 1, characterized in that, After the deprotection reaction is completed, the deprotected material is obtained. The deprotected material is then separated, and the separated solid material is pulped, filtered, and dried.

8. The preparation method according to claim 7, characterized in that, In the process of pulping, filtering and drying the separated solid material, the pulping solvent includes either ethyl acetate or tetrahydrofuran.

9. The preparation method according to claim 8, characterized in that, The mass ratio of the solvent to the solid material in the pulping process is 1:0.15~0.25, and the pulping time is 40~80 min.

10. The preparation method according to claim 7, characterized in that, During the process of pulping, filtering and drying the separated solid material, the drying temperature is 45~55℃.

11. The preparation method according to claim 1, characterized in that, The mass ratio of good solvent to BOC-O-benzyl-L-tyrosine is 7~8:1, and the mass ratio of poor solvent to BOC-O-benzyl-L-tyrosine used during stirring and crystallization is 12.5~13.5:

1.

12. The preparation method according to claim 1, characterized in that, The acidic material is a hydrochloric acid solution, and the concentration of hydrochloric acid in the hydrochloric acid solution is 0.2~0.8N.

13. The preparation method according to claim 1, characterized in that, The mass ratio of the acidic material to BOC-O-benzyl-L-tyrosine is 4.5~5.5:

1.

14. The preparation method according to claim 1, characterized in that, The alkaline material includes any one of sodium bicarbonate, sodium carbonate, or sodium hydroxide.

15. The preparation method according to claim 1, characterized in that, The alkaline material added together with the good solvent is a 2-3% sodium bicarbonate solution, and the mass ratio of the 2-3% sodium bicarbonate solution to BOC-O-benzyl-L-tyrosine is 18-19:

1.

16. The preparation method according to claim 1, characterized in that, The alkaline material added during the washing process is a 0.5-1.5% sodium bicarbonate solution, and the mass ratio of the 0.5-1.5% sodium bicarbonate solution to BOC-O-benzyl-L-tyrosine is 4.5-5.5:

1.

17. The preparation method according to claim 1, characterized in that, The stirring and crystallization time is 2-4 hours.

18. The preparation method according to claim 1, characterized in that, The mass ratio of the unsuitable solvent to BOC-O-benzyl-L-tyrosine in the pulping and filtration process is 7.5~8.0:

1.

19. The preparation method according to claim 1, characterized in that, The drying temperature for drying the filter cake obtained by pulping and filtration is 45~55℃.

20. The preparation method according to claim 1, characterized in that, The condensation reaction process includes: dissolving the BOC-O-benzyl-L-tyrosine in a solvent for the condensation reaction and cooling it to 0~5℃, then adding a condensing agent, a racemic inhibitor and a condensation accelerator, reacting at 10~15℃ for 20~40 min, and then adding the L-leucine benzyl ester hydrochloride and the condensation accelerator to carry out the condensation reaction.

21. The preparation method according to claim 1 or 20, characterized in that, The mass ratio of BOC-O-benzyl-L-tyrosine to L-leucine benzyl ester hydrochloride is 1:0.70~0.

75.

22. The preparation method according to claim 20, characterized in that, The solvent for the condensation reaction includes any one of N,N-dimethylformamide, tetrahydrofuran, and dichloromethane.

23. The preparation method according to claim 1, characterized in that, The solvent for the condensation reaction is N,N-dimethylformamide.

24. The preparation method according to claim 20, characterized in that, The mass ratio of the solvent in the condensation reaction to the BOC-O-benzyl-L-tyrosine is 5.5~6.0:

1.

25. The preparation method according to claim 20, characterized in that, The condensing agent includes either TBTU or HBTU.

26. The preparation method according to claim 20, characterized in that, The condensing agent is TBTU.

27. The preparation method according to claim 20, characterized in that, The racemic inhibitor includes either 1-hydroxybenzotriazole or N-hydroxy-7-azobenzotriazole.

28. The preparation method according to claim 20, characterized in that, The racemic inhibitor is 1-hydroxybenzotriazole.

29. The preparation method according to claim 20, characterized in that, The condensation accelerator includes any one of N,N-diisopropylethylamine and pyridine.

30. The preparation method according to claim 20, characterized in that, The condensation accelerator is N,N-diisopropylethylamine.

31. The preparation method according to claim 20, characterized in that, Before the addition of the L-leucine benzyl ester hydrochloride, the mass ratio of the condensing agent, racemic inhibitor, and condensing accelerant to the BOC-O-benzyl-L-tyrosine is 1~1.05:0.35~0.4:0.5~0.55:

1.

32. The preparation method according to claim 20, characterized in that, The mass ratio of the condensing agent added together with the L-leucine benzyl ester hydrochloride to the BOC-O-benzyl-L-tyrosine is 0.3~0.4:

1.

33. The preparation method according to claim 1, characterized in that, The yield of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is >75%, the purity is ≥99%, and the content of the maximum single impurity is <0.4%.

34. The preparation method according to claim 1, characterized in that, The yield of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is >85%, the purity is ≥99%, and the content of the maximum single impurity is <0.2%.

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