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

Abstract

The invention discloses ((S)-2-amino-3-(4-(benzyloxy) phenyl) propionyl)-L-leucine benzyl ester hydrochloride as well as a preparation method and application thereof, and relates to the technical field of chemical synthesis. The dipeptide hydrochloride with tyrosine and leucine as key structural units is provided, the synthesis method of the dipeptide hydrochloride is simple, the reaction condition is mild, the product is high in purity, high in yield and low in impurity content, and when the dipeptide hydrochloride is used as part of active fragments of polypeptide products, the purity of the polypeptide products is greatly improved. The purity and the yield of polypeptide products can also be obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and in particular to ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride and a preparation method and application thereof. Background Art

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

[0003] Amino acid drugs containing leucine are mainly used as amino acid infusions and comprehensive amino acid preparations. They are used to diagnose and treat children with idiopathic hyperglycemia and glucose metabolism disorders, biliary liver disease with reduced secretion, anemia, poisoning, muscular dystrophy, poliomyelitis, neuritis and mental illness. Amino acids and amino acid derivatives have been widely used in the field of drug development and have been actively researched and developed by researchers at home and abroad.

[0004] However, due to the complex structure of amino acids and the rich reactive sites, in the process of drug development, if multiple amino acids are involved, it is very easy for the reaction products to have low yield, low purity and high impurity content.

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

[0006] The object of the present invention is to provide a ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride and a preparation method and application thereof.

[0007] The present invention is achieved in that:

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

[0009]

[0010] In a second aspect, 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 aforementioned embodiment, comprising subjecting BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to a condensation reaction to obtain a reaction intermediate, and then subjecting the reaction intermediate to a deprotection reaction.

[0011] In a third aspect, the present invention provides a method of preparing a polypeptide product using ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride according to the aforementioned embodiment, or ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the preparation method according to any of the aforementioned embodiments.

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

[0013] The present invention provides ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride and a preparation method and application thereof. By providing a dipeptide hydrochloride with tyrosine and leucine as key structural units, the synthesis method of the dipeptide hydrochloride is simple, the reaction conditions are mild, and the product has high purity, high yield and low impurity content. When the dipeptide hydrochloride provided by the present invention is used as a partial active fragment of a polypeptide product, the purity and yield of the polypeptide product can also be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 The HPLC 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 hydrogen nuclear magnetic resonance spectrum of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in Example 1 of the present 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 5This is 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. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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

[0023]

[0024] In a second aspect, 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 aforementioned embodiment, comprising subjecting BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to a condensation reaction to obtain a reaction intermediate, and then subjecting the reaction intermediate to a deprotection reaction.

[0025] In an optional embodiment, the chemical 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 comprises 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 condensation reaction solvent and cooling to 0-5°C, then adding a condensation agent, a racemization inhibitor and a condensation promoter, reacting at 10-15°C for 20-40 minutes, and then adding L-leucine benzyl hydrochloride and a condensation promoter to carry out a condensation reaction.

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

[0031] After BOC-O-benzyl-L-tyrosine is dissolved, a condensation agent, a racemization inhibitor and a condensation promoter are added to the system and reacted at a certain temperature for a period of time. The system can be adjusted to a reaction intermediate state suitable for the condensation reaction, and then L-leucine benzyl ester hydrochloride is added to carry out a condensation reaction with BOC-O-benzyl-L-tyrosine, and a condensation promoter 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 dipeptide hydrochloride finally obtained, the mass ratio of BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride is 1:0.70-0.75. For example, the mass ratio of BOC-O-benzyl-L-tyrosine and 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 value between any two values, or any other point value within the above range value.

[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 dipeptide hydrochloride finally obtained and the yield and purity of the reaction intermediate obtained, the solvent for the condensation reaction is more preferably N,N-dimethylformamide (DMF).

[0034] Preferably, the mass ratio of the solvent for 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 value between any two values, or any other point value within the above range value.

[0035] Preferably, the condensing agent includes any one of TBTU and HBTU. In order to ensure the purity of the dipeptide hydrochloride finally obtained and the yield and purity of the reaction intermediate obtained, the condensing agent is more preferably TBTU.

[0036] TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate. HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate.

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

[0038] Preferably, the condensation promoter includes any one of N,N-diisopropylethylamine and pyridine. In order to ensure the purity of the dipeptide hydrochloride finally obtained and the yield and purity of the reaction intermediate obtained, the condensation promoter 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 the condensation agent, the racemization inhibitor and the condensation promoter 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 promoter added together with L-leucine benzyl ester hydrochloride to BOC-O-benzyl-L-tyrosine is 0.3-0.4: 1. When the condensation promoter added later is N,N-diisopropylethylamine (DIPEA), N,N-diisopropylethylamine can neutralize the hydrochloric acid in L-leucine benzyl ester hydrochloride to promote the smooth progress of the condensation reaction.

[0041] Preferably, during the condensation reaction by adding L-leucine benzyl ester hydrochloride, there is no need to heat or insulate the reaction system. The reaction system is naturally heated to room temperature and the reaction continues. This method has mild reaction conditions and is simple to operate.

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

[0043] S02, Extraction

[0044] In an optional embodiment, the process further includes extracting the material after the condensation reaction before obtaining the reaction intermediate.

[0045] The choice of solvent in the extraction process has a great influence on the purity and yield of the reaction intermediates obtained by the condensation reaction. Preferably, in order to ensure the purity and yield of the reaction intermediates, 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, adding an acidic material to the separated organic phase for washing, adding an alkaline material to the separated organic phase for washing, and adding a poor solvent to the separated organic phase for stirring and 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 in the good solvent, and the alkaline material is used to dissolve the condensation agent, racemization inhibitor and other acidic by-products in the material after the condensation reaction. The separated organic phase is washed with an acidic material to remove the condensation promoter and other alkaline by-products, and then washed with an alkaline material to remove the acidic by-products remaining in the organic phase again. Finally, a poor solvent is added to the organic phase to stir and crystallize, and the reaction intermediates are precipitated from the organic phase.

[0049] Preferably, in order to ensure the yield and purity of the reaction intermediates in the extraction process, the mass ratio of the good solvent to BOC-O-benzyl-L-tyrosine is 7 to 8:1, and the mass ratio of the poor solvent used in the stirred crystallization to BOC-O-benzyl-L-tyrosine is 12.5 to 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 value between any two values, or any other point value within the above range values.

[0051] For example, the mass ratio of the poor solvent to BOC-O-benzyl-L-tyrosine used in the stirred crystallization 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 value between any two values, or any other point value within the above range value.

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

[0053] Preferably, the mass ratio of the acidic material to BOC-O-benzyl-L-tyrosine is 4.5-5.5:1, 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 value between any two values, or any other point value within the above range value.

[0054] Preferably, the alkaline material comprises any one of sodium bicarbonate, sodium carbonate or sodium hydroxide, preferably sodium bicarbonate.

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

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

[0059] In an optional embodiment, in order to improve the yield of the reaction intermediate, the method further comprises pulping and filtering the filter cake obtained by filtration after crystallization using a poor solvent, and drying the filter cake obtained by pulping and filtration to obtain the reaction intermediate.

[0060] Preferably, the mass ratio of the poor 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 value between any two values, or any other point value within the above range value.

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

[0062] S03, deprotection reaction

[0063] In an optional embodiment, the deprotection reaction comprises mixing the reaction intermediate with a deprotection solvent system; the deprotection solvent system comprises 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 mass ratio of the volume of the deprotection reagent to BOC-O-benzyl-L-tyrosine is 2.5-2.6 L:1 kg, and the balance in the deprotection solvent system is the deprotection solvent.

[0065] In order to ensure the reaction efficiency of the deprotection reaction, the concentration of the reaction intermediate in the deprotection solvent system is controlled within the above range so that the reaction process can proceed smoothly and quickly. In order to ensure that the concentration of the reaction intermediate in the deprotection solvent system is stable within the above 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 the reaction intermediate and the deprotection reagent are mixed, the mass of the deprotection solvent that needs to be added is calculated based on the current concentration. After a period of reaction, solids 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 increase the conversion rate of the deprotection reaction of the reaction intermediate.

[0067] Preferably, the deprotection agent includes a tetrahydrofuran hydrochloride solution or a hydrochloric acid ethyl acetate solution; in order to improve the purity and yield of the dipeptide hydrochloride finally obtained, the deprotection agent is more preferably a tetrahydrofuran hydrochloride solution.

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

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

[0070] Preferably, the reaction temperature of the deprotection reaction is 22-28°C, and the deprotection reaction can be carried out at room temperature, for example, 25°C.

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

[0072] In an optional embodiment, after the deprotection reaction is completed, a material after the deprotection reaction is obtained, the material after the deprotection reaction is separated, and the solid material obtained by separation is slurried, filtered and dried.

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

[0074] Preferably, the slurry solvent includes any one of ethyl acetate and tetrahydrofuran.

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

[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 single impurity content 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 maximum single impurity content 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] In a third aspect, the present invention provides a method of preparing a polypeptide product using ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride according to the aforementioned embodiment, or ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the preparation method according to any of the aforementioned embodiments.

[0082] Preferably, the polypeptide product may be, for example, any one of an amino acid inhibitor and a carrier.

[0083] Example 1

[0084] This embodiment provides a ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, the chemical structure 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, and the specific process 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, and 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, keep the reaction at 10-15°C for 30 minutes, then add 63.7 kg of L-leucine benzyl ester hydrochloride and 31.3 kg of DIPEA, and naturally raise the temperature to room temperature for reaction.

[0091] Using TLC spot plate, it was observed that the raw material BOC-O-benzyl-L-tyrosine had reacted completely, and it was considered that the condensation reaction was completed.

[0092] S02, Extraction

[0093] To the material after the condensation reaction obtained in step S01, 1660 kg of 2.5% sodium bicarbonate solution and 666 kg of methyl tert-butyl ether were added for extraction. After extraction, an organic phase was separated and washed with 450 kg of 0.5 N hydrochloric acid solution. The pH of the aqueous phase obtained after washing was 1. 450 kg of 1% sodium bicarbonate solution was added to the organic phase for washing. The pH of the aqueous phase obtained after washing was 8. 1170 kg of petroleum ether was added to the organic phase and stirred for crystallization for 3 hours.

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

[0095] S03, deprotection reaction

[0096] The reaction intermediate obtained in step S02 is added to 1741.1 kg of dichloromethane, and then 228.8 L of 3N hydrochloric acid tetrahydrofuran solution is added, and the reaction is carried out at 25°C. After solid appears in the system for 1 hour, 889.1 kg of dichloromethane is added until the reaction is completed. The reaction is completed when the TLC spot plate shows that the reaction intermediate has reacted completely.

[0097] After the deprotection reaction is completed, the material obtained after the deprotection reaction is filtered and separated to obtain a solid material as a filter cake. The filter cake is slurried with 658 kg of ethyl acetate for 1 hour and then filtered. The filter cake obtained by filtering again is 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 example was tested by high performance liquid chromatography to obtain Figure 1 The results are shown. Figure 1 It can be seen that the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this example has a purity of 99.5% and a yield of 88.5%.

[0099] The intermediate obtained in step S02 was subjected to HPLC detection under the same conditions as above, and the yield of the reaction intermediate benzyl ((S)-3-(4-(benzyloxy)phenyl)-2-((tert-butyloxycarbonyl)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 example was detected by nuclear magnetic resonance spectrometer to obtain Figure 2 The ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride provided in this example was detected by a Tandem Quadrupole mass spectrometer to obtain Figures 3 to 5 The mass spectrum shown. Figures 2 to 5 It can be seen that the structure of the product prepared by the method provided in this embodiment is:

[0101] The 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, and the steps are similar to those in Example 1, except that: in step S03, the deprotection solvent 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, and the steps are 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, and the steps are similar to those in Example 1, except that the good solvent in step S02 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, and the steps are similar to those in Example 1, except that in step S02, 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, and the steps are 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, and the steps are similar to those in Example 1, except that in step S03, the deprotection reagent is thionyl chloride and the deprotection 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, and the steps are similar to those in Example 1, except that in step S03, the deprotection reagent is oxalyl chloride and the deprotection 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, and the steps are similar to those in Example 1, except that the deprotection 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, and the steps are similar to those in Example 1, except that in step S03, the deprotection reagent is thionyl chloride and the deprotection 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, and the steps are similar to those in Example 1, except that in step S03, the deprotection reagent is oxalyl chloride and the deprotection 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, and the steps are similar to those in Example 1, except that the deprotection 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, and the steps are similar to those in Example 1, except that the deprotection solvent in step S03 is tetrahydrofuran.

[0126] Test Example 1

[0127] According to the method provided in Example 1 and Comparative Examples 1 to 4, ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride was prepared, and 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 studied, 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 intermediate was detected by high performance liquid chromatography using BOC-O-benzyl-L-tyrosine, L-leucine benzyl ester hydrochloride, DIPEA and N,N-dimethylformamide as standard substrates to obtain the results 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] As can be seen from Table 1, the selection of good solvent and poor solvent in the extraction process has a great influence on the purity and yield of the reaction intermediate. The purity of Comparative Examples 1 and 3 is relatively low. When the obtained reaction intermediate is further subjected to subsequent deprotection reaction, the purity of the final product is affected, so that the single impurity of the final product cannot be well controlled below 0.5%. The yields of the reaction intermediates obtained in Comparative Examples 2 and 4 are both relatively low, which affects the yield of the final product.

[0132] Test Example 2

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

[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 can be seen from Table 2, the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the method provided in the embodiment of the present invention has high yield, high purity and low maximum single impurity content. In Comparative Examples 5 to 11, due to the adjustment of 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 were reduced, and the impurity content was increased.

[0137] The present invention provides a ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride and a preparation method and application thereof. The preparation method is simple, the reaction conditions are mild, 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 the present invention is used as a partial active fragment of a polypeptide product, the purity and yield of the polypeptide product can also be significantly improved.

[0138] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride, characterized in that Its chemical structure is as follows:

2. A method for preparing ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as claimed in claim 1, characterized in that: The method comprises the steps of subjecting BOC-O-benzyl-L-tyrosine and L-leucine benzyl ester hydrochloride to a condensation reaction to obtain a reaction intermediate, and then subjecting the reaction intermediate to a deprotection reaction.

3. The preparation method according to claim 2, characterized in that: The chemical structural formula of the reaction intermediate is:

4. The preparation method according to claim 2 or 3, characterized in that: The deprotection reaction comprises mixing the reaction intermediate with a deprotection solvent system; the deprotection solvent system comprises a deprotection reagent and a deprotection solvent; Preferably, the concentration of the reaction intermediate in the deprotection solvent system is 0.11-0.18 mol / L, the mass ratio of the volume of the deprotection reagent to the BOC-O-benzyl-L-tyrosine is 2.5-2.6 L:1 kg, and the balance in the deprotection solvent system is the deprotection solvent; Preferably, the deprotection reagent comprises a tetrahydrofuran hydrochloride solution or a hydrochloric acid ethyl acetate solution; more preferably a tetrahydrofuran hydrochloride solution; Preferably, the concentration of hydrochloric acid in the tetrahydrofuran hydrochloric acid solution is 2 to 4N; Preferably, the deprotection solvent comprises dichloromethane or ethyl acetate; more preferably dichloromethane; Preferably, the reaction temperature of the deprotection reaction is 22-28°C.

5. The preparation method according to claim 4, characterized in that: After the deprotection reaction is completed, the material after the deprotection reaction is obtained, the material after the deprotection reaction is separated, and the separated solid material is pulped, filtered and dried; Preferably, the slurry solvent includes any one of ethyl acetate and tetrahydrofuran; Preferably, the mass ratio of the pulping solvent to the solid material is 1:0.15-0.25, and the pulping time is 40-80 min; Preferably, the drying temperature is 45-55°C.

6. The preparation method according to claim 2, characterized in that: Before obtaining the reaction intermediate, the method further includes extracting the material after the condensation reaction; Preferably, the good solvent used for extraction is methyl tert-butyl ether, and the poor solvent is petroleum ether; Preferably, the extraction process comprises: adding a good solvent and an alkaline material to the material after the condensation reaction for extraction, adding an acidic material to the separated organic phase for washing, adding an alkaline material to the separated organic phase for washing, and adding a poor solvent to the separated organic phase for stirring and crystallization; Preferably, the mass ratio of the good solvent to BOC-O-benzyl-L-tyrosine is 7 to 8:1, and the mass ratio of the poor solvent used in the stirring crystallization to BOC-O-benzyl-L-tyrosine is 12.5 to 13.5:1; Preferably, the acidic material is a hydrochloric acid solution, and the concentration of hydrochloric acid in the hydrochloric acid solution is 0.2-0.8N; preferably, the mass ratio of the acidic material to BOC-O-benzyl-L-tyrosine is 4.5-5.5:1; Preferably, the alkaline material comprises any one of sodium bicarbonate, sodium carbonate or sodium hydroxide; 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; 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; Preferably, the stirring crystallization time is 2 to 4 hours.

7. The preparation method according to claim 6, characterized in that: The method further comprises: using a poor solvent to pulp and filter the filter cake obtained by filtration after crystallization, and drying the filter cake obtained by pulping and filtering to obtain the reaction intermediate; Preferably, the mass ratio of the poor solvent to BOC-O-benzyl-L-tyrosine in the beating and filtering process is 7.5 to 8.0:1; Preferably, the drying temperature is 45-55°C.

8. The preparation method according to claim 2 or 6, characterized in that: The condensation reaction process comprises: dissolving the BOC-O-benzyl-L-tyrosine in a condensation reaction solvent and cooling the mixture to 0-5° C., adding a condensation agent, a racemization inhibitor and a condensation promoter, reacting the mixture at 10-15° C. for 20-40 minutes, and then adding the L-leucine benzyl ester hydrochloride and the condensation promoter to carry out a condensation reaction; Preferably, the mass ratio of the BOC-O-benzyl-L-tyrosine to the L-leucine benzyl ester hydrochloride is 1:0.70-0.75; Preferably, the solvent for the condensation reaction includes any one of N,N-dimethylformamide, tetrahydrofuran, and dichloromethane, more preferably N,N-dimethylformamide; Preferably, the mass ratio of the solvent of the condensation reaction to the BOC-O-benzyl-L-tyrosine is 5.5-6.0:1; Preferably, the condensing agent includes any one of TBTU and HBTU, more preferably TBTU; Preferably, the racemization inhibitor includes any one of 1-hydroxybenzotriazole and N-hydroxy-7-azobenzotriazole, more preferably 1-hydroxybenzotriazole; Preferably, the condensation-accelerating agent includes any one of N,N-diisopropylethylamine and pyridine, more preferably N,N-diisopropylethylamine; Preferably, before adding the L-leucine benzyl ester hydrochloride, the mass ratio of the condensation agent, the racemization inhibitor and the condensation promoter to the BOC-O-benzyl-L-tyrosine is 1-1.05:0.35-0.4:0.5-0.55:1; Preferably, the mass ratio of the condensation-accelerating agent added together with the L-leucine benzyl ester hydrochloride to the BOC-O-benzyl-L-tyrosine is 0.3-0.4:

1.

9. The preparation method according to claim 2, characterized in that: The yield of the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is greater than 75%, the purity is greater than 99%, and the maximum single impurity content is less than 0.4%; Preferably, the yield of ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride is greater than 85%, the purity is greater than or equal to 99%, and the maximum single impurity content is less than 0.2%.

10. Use of the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride as claimed in claim 1, or the ((S)-2-amino-3-(4-(benzyloxy)phenyl)propionyl)-L-leucine benzyl ester hydrochloride prepared by the preparation method according to any one of claims 2 to 9 in the preparation of polypeptide products.

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