Preparation method of Fmoc-Gly-Ser {Psi (Me, Me) Pro}-OH pseudo dipeptide

Abstract

The invention discloses a preparation method of Fmoc-Gly-Ser {Psi (Me, Me) Pro}-OH pseudo dipeptide, and particularly relates to a preparation method of Fmoc-Gly-Ser {Psi (Me, Me) Pro}-OH pseudo dipeptide, which comprises the following steps: by taking Fmoc-Gly-OPFP as an initial raw material, carrying out azeotropic distillation condensation on the initial raw material and 2, 2-dimethoxypropane, and then salifying, purifying, curing and crystallizing to obtain a chiral Fmoc-Gly-Ser {Psi (Me, Me) Pro}-OH pseudo dipeptide refined product. According to the method, the reaction time is remarkably shortened, the raw material utilization rate is increased, a special purification method is matched, the product quality is stable and controllable, and the preparation method is short in overall route, economical, efficient and convenient to industrialize. Meanwhile, the preparation method can also be used for preparing other peptide compounds of the same type, and the method is wide in adaptability.

Description

Technical Field

[0001] The present invention relates to a method for preparing Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide, and particularly to an industrialized method for preparing Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide. Background Art

[0002] As an amino acid derivative, Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide is commonly used in the preparation of polypeptide drugs such as exenatide and liraglutide.

[0003]

[0004] The known synthesis methods of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide are as follows:

[0005]

[0006] The preparation method disclosed in CN107176970 uses Fmoc-Gly-OH and L-serine benzyl ester as starting materials. First, it is catalyzed by acidic resin for condensation, and then debenzylated by Pd-C catalysis to obtain the target product. This method uses a large amount of precious metal catalysts in the deprotection step, with poor comprehensive benefits and high equipment requirements. The preparation method disclosed in CN116621789 uses Fmoc-Gly-OH as the starting material and L-serine methyl ester as the starting material. First, it is catalyzed by pyridinium p-toluenesulfonate for condensation, and then hydrolyzed by sodium hydroxide to obtain the target product. The hydrolysis degree of this method is difficult to control, with many side reactions, greatly reducing the utilization rate of raw materials. Other preparation methods (DOI: 10.1016 / j.tetlet.2017.05.027) require column chromatography separation, with low conversion rate, large solvent consumption and cumbersome operation. Therefore, the above preparation methods are not suitable for industrialization. Summary of the Invention

[0007] Object of the Invention: The present invention aims to provide a method for preparing Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide that is convenient for industrialization.

[0008] Technical Solution: The method for preparing Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH pseudo-dipeptide according to the present invention comprises the following steps:

[0009]

[0010] (1) The reaction of compound Fmoc-Gly-OPFP with L-serine gives the intermediate Fmoc-Gly-Ser-OH;

[0011] (2) The intermediate Fmoc-Gly-Ser-OH reacts with 2,2-dimethoxypropane under the catalysis of pyridinium p-toluenesulfonate through azeotropic distillation reaction to obtain the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH, and then the purified product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is obtained through purification.

[0012] The present invention shortens the reaction time and improves the utilization rate of the raw material Fmoc-Gly-Ser-OH by methods such as azeotropic distillation.

[0013] Preferably, the reaction solvent for the azeotropic distillation in step (2) is selected from one or more of tetrahydrofuran, hexane, ethyl acetate, and cyclohexane.

[0014] More preferably, the azeotropic reaction solvent is selected from ethyl acetate.

[0015] Preferably, the mass ratio of the reaction solvent for the azeotropic distillation in step (2) to Fmoc-Gly-Ser-OH is 1:(5 - 15).

[0016] More preferably, the mass ratio of the reaction solvent for the azeotropic distillation to Fmoc-Gly-Ser-OH is 1:9.

[0017] More preferably, the mass ratio of ethyl acetate, the reaction solvent for the azeotropic distillation, to Fmoc-Gly-Ser-OH is 1:(5 - 15).

[0018] Even more preferably, the mass ratio of ethyl acetate, the reaction solvent for the azeotropic distillation, to Fmoc-Gly-Ser-OH is 1:9.

[0019] Preferably, the reaction temperature for the azeotropic distillation in step (2) is 75 - 90 °C.

[0020] More preferably, the reaction temperature for the azeotropic distillation is 80 °C.

[0021] More preferably, the azeotropic distillation reaction is carried out until the residue of the raw material Fmoc-Gly-Ser-OH does not exceed 5.0%.

[0022] Preferably, in step (2), the molar ratio of pyridinium p-toluenesulfonate to Fmoc-Gly-Ser-OH is (0.1 - 0.5):1.

[0023] More preferably, the molar ratio of pyridinium p-toluenesulfonate to Fmoc-Gly-Ser-OH is 0.15:1.

[0024] Specifically, in terms of the molar amount or mass of Fmoc-Gly-Ser-OH, Fmoc-Gly-Ser-OH and 2,2-dimethoxypropane are in 9 times the mass of ethyl acetate, catalyzed by 0.15 times the molar amount of pyridinium p-toluenesulfonate, heated to 80 °C for azeotropic distillation, and the reaction is carried out until the raw material residue is ≤5.0%. After completion, the reaction solution is subjected to post-treatments such as extraction and concentration to obtain an oily concentrate of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH with a purity of more than 85%.

[0025] Preferably, the purification method described in step (2) includes the following steps:

[0026] (I) Salifying the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH with a nitrogen-containing organic base;

[0027] (II) Freeing the salt obtained in step (I) with an acid and crystallizing to obtain the refined product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH.

[0028] The present invention realizes the purification of the target product by the method of salifying with a nitrogen-containing organic base, and combines the method of slurrying with a mixed solvent to solidify the refined oily target product.

[0029] More preferably, the nitrogen-containing organic base described in step (I) is selected from one or more of dicyclohexylamine, diisopropylamine, and di-tert-butylamine.

[0030] Even more preferably, the nitrogen-containing organic base is selected from dicyclohexylamine.

[0031] Further preferably, the molar ratio of the nitrogen-containing organic base to the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH in step (I) is (0.5 - 1.5):1.

[0032] Even more preferably, the molar ratio of the nitrogen-containing organic base to the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is 0.98:1.

[0033] Even further preferably, the molar ratio of the nitrogen-containing organic base dicyclohexylamine to the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is (0.5 - 1.5):1.

[0034] Still further preferably, the molar ratio of the nitrogen-containing organic base dicyclohexylamine to the crude product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is 0.98:1.

[0035] More preferably, the reaction solvent for salt formation in step (I) is selected from one or more of tetrahydrofuran, ethanol, and ethyl acetate.

[0036] Even more preferably, the reaction solvent for salt formation is a mixed solution of tetrahydrofuran and ethanol.

[0037] Even more preferably, the mass ratio of tetrahydrofuran to ethanol in the reaction solvent for salt formation is 10:1.

[0038] More preferably, the reaction temperature for salt formation in step (I) is 10 - 30 °C.

[0039] Specifically, based on the molar amount of the crude Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH, the crude Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is dissolved in a mixed solution of tetrahydrofuran - ethanol (10:1, m / m), 0.98 times the molar amount of dicyclohexylamine (DCHA) is added dropwise, and the mixture is stirred at 10 - 30 °C for precipitation, followed by filtration to obtain the Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH·DCHA salt.

[0040] More preferably, the crystallization method in step (II) is to slurry-crystallize the free product obtained by acid liberation in a mixed solvent selected from ethyl acetate - n-heptane, tetrahydrofuran - isopropyl ether, and tetrahydrofuran - n-heptane to obtain the refined product.

[0041] Even more preferably, the mixed solvent is selected from tetrahydrofuran - isopropyl ether.

[0042] Even more preferably, the mass ratio of tetrahydrofuran to isopropyl ether in the mixed solvent is 1:4.

[0043] Even more preferably, the crystallization method is to first solidify the free product obtained by acid liberation at 25 - 45 °C and then crystallize it at 0 - 10 °C to obtain the refined product.

[0044] Temperature is a key factor in crystallization. During the solidification process, if the temperature is too high, the oily product will separate from the solvent and it is difficult to solidify; if the temperature is too low, it is easy to precipitate on the wall in an amorphous form. During the crystallization process, media such as dry ice and liquid nitrogen can be appropriately added to make the internal temperature of the crystallization system lower than the temperature of the reaction kettle wall, promoting the precipitation of product particles in a granular form for easy filtration.

[0045] Even more preferably, the solidification temperature is 30 - 35 °C.

[0046] Even more preferably, the crystallization temperature is 0 - 5 °C.

[0047] More preferably, the crystallization method is to dissolve the free product obtained by acid dissociation in tetrahydrofuran at 25-45 °C, then add isopropyl ether for heat preservation and solidification, and then crystallize at 0-10 °C to obtain the refined product.

[0048] More preferably, the salt obtained in step (I) is dissociated by citric acid.

[0049] Specifically, after the Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH·DCHA salt is dissociated by 10% m / m citric acid and the solvent is removed under reduced pressure, an oily concentrate is obtained. The oily concentrate is dissolved in tetrahydrofuran and stirred at 30-35 °C for heat preservation. At the same time, isopropyl ether with a mass 4 times that of tetrahydrofuran is added. After the addition is complete, continue to stir at a constant temperature until oil droplets appear in the emulsion (the oil and the solvent seem to be layered but not completely layered), and then dry ice is slowly added, and solids will precipitate. At this time, the internal temperature of the solvent is 10-15 °C lower than the temperature of the reactor wall. The whole system is cooled to 0-5 °C, stirred at a constant temperature, filtered, and the filter cake is dried to obtain the refined product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH.

[0050] Preferably, the starting material Fmoc-Gly-OPFP is obtained by the alkali-catalyzed reaction of Fmoc-Gly-OH with a chlorinating reagent and pentafluorophenol.

[0051] More preferably, the chlorinating reagent is selected from thionyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, and oxalyl chloride.

[0052] Even more preferably, the chlorinating reagent is thionyl chloride.

[0053] More preferably, the base is selected from trimethylamine, triethylamine, morpholine, and pyridine.

[0054] Even more preferably, the base is pyridine.

[0055] More preferably, the starting material Fmoc-Gly-OPFP is prepared by the following stepwise reaction:

[0056] Step 1: React Fmoc-Gly-OH with a chlorinating reagent to obtain an acyl chloride;

[0057] Step 2: React the acyl chloride obtained in Step 1 with pentafluorophenol under the basic condition provided by pyridine to obtain Fmoc-Gly-OPFP.

[0058] More preferably, the molar ratio of Fmoc-Gly-OH to the chlorinating reagent, pentafluorophenol, and the base is 1:(1.1-1.3):(1.1-1.3):(1.3-1.6).

[0059] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0060] The preparation method designed by the present invention significantly shortens the reaction time through azeotropic distillation, improves the utilization rate of raw materials, and cooperates with a unique purification method. The product quality is stable and controllable, making the overall route of the preparation method short, economical and efficient, and facilitating industrialization. At the same time, this preparation method can also be used for the preparation of other similar peptide compounds, and the method has a wide adaptability. Brief description of the drawings

[0061] Figure 1 It is the HPLC chromatogram of the in-process control detection for the synthesis method in Example 3 after reacting for 8 hours (raw material residue: 4.62%);

[0062] Figure 2 It is the HPLC chromatogram of the purity detection for the refined product obtained by the purification method in Example 3 (product purity: 99.84%);

[0063] Figure 3 It is the HPLC chromatogram of the optical purity detection for the refined product obtained by the purification method in Example 3 (isomer content: 0.02%);

[0064] Figure 4 It is the HPLC chromatogram of the in-process control detection for the synthesis method in Comparative Example 1 after reacting for 8 hours (raw material residue: 24.62%);

[0065] Figure 5 It is the HPLC chromatogram of the purity detection for the refined product obtained by the purification method in Comparative Example 2 (product purity: 93.65%);

[0066] Figure 6 It is the HPLC chromatogram of the optical purity detection for the refined product obtained by the purification method in Comparative Example 2 (isomer content: 0.58%). Detailed implementation manners

[0067] The technical solutions of the present invention will be further described below in conjunction with the examples.

[0068] Example 1: Synthesis of Fmoc-Gly-OPFP

[0069] In a 100L reaction kettle equipped with mechanical stirring and a thermometer, 36 kg of toluene and 3.0 kg of Fmoc-Gly-OH were added, and 1.44 kg of thionyl chloride and 0.009 kg of DMF were added dropwise within 0.5 hour. After maintaining the temperature for reaction for 4 hours, the excess toluene was removed under reduced pressure. 2.23 kg of pentafluorophenol and 2.4 kg of pyridine were added successively. After adding, after maintaining the temperature for 3 hours, the reaction solution was filtered, and the filter cake was rinsed twice to obtain a white wet product. The wet product was dried overnight in a blast drying oven at 50 °C to obtain 5.97 kg of white crude Fmoc-Gly-OPFP.

[0070] Example 2: Synthesis of Fmoc-Gly-Ser-OH

[0071] In a 100 L reactor equipped with mechanical stirring and a thermometer, 5.97 kg of crude Fmoc-Gly-OPFP and 24.0 kg of acetone were added, and the mixture was stirred while maintaining the temperature. 1.59 kg of L-Ser-OH was added to 21.4 kg of 10% Na 2 CO 3 aqueous solution, stirred until clear, and then added dropwise to the acetone solution of Fmoc-Gly-OPFP. After reacting for 14 hours while maintaining the temperature, 45 kg of ethyl acetate was added, and the pH value of the system was adjusted to 2 using 3.09 kg of concentrated hydrochloric acid. The ethyl acetate phase was separated and washed successively with water and brine. After evaporating all the ethyl acetate under reduced pressure, 24 kg of tetrahydrofuran was added for recrystallization. The mixture was filtered, and the filter cake was rinsed twice to obtain a white wet product. The wet product was dried overnight in a forced-air drying oven at 50 °C to obtain 3.8 kg of pure Fmoc-Gly-Ser-OH.

[0072] Example 3: Synthesis, Purification and Crystallization of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH

[0073] In a 100 L reactor equipped with mechanical stirring and a thermometer, 3.8 kg of pure Fmoc-Gly-Ser-OH, 34.2 kg of ethyl acetate, 5.2 kg of 2,2-dimethoxypropane and 0.42 kg of pyridinium p-toluenesulfonate were added. The temperature was raised to 80 °C and azeotropic distillation was carried out for 8 hours. Samples were taken for in-process control. At this time, the residual raw material was 4.62%( Figure 1 ), and the temperature was lowered to 25 °C. 19.2 kg of methyl tert-butyl ether was added for extraction, and the phases were separated. The organic phase was dried with anhydrous sodium sulfate for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure to obtain 8.2 kg of an oily concentrate of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH.

[0074] In a 100 L reactor equipped with mechanical stirring and a thermometer, 8.0 kg of the oily concentrate of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH, 38 kg of tetrahydrofuran and 3.8 kg of ethanol were added, and the mixture was stirred until clear. 1.75 kg of dicyclohexylamine was added, and the mixture was stirred at room temperature for 5 hours, then filtered, and the filter cake was rinsed twice to obtain a white wet product. The wet product was dried overnight in a forced-air drying oven at 50 °C to obtain 3.4 kg of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH·DCHA salt.

[0075] In a 100L reactor equipped with mechanical stirring and a thermometer, 3.4 kg of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH·DCHA salt, 30.6 kg of methyl tert-butyl ether, and 25.3 kg of 10% m / m aqueous citric acid solution were added. After stirring, the methyl tert-butyl ether phase was separated and washed successively with water and brine. Anhydrous sodium sulfate was added for drying for 30 minutes, followed by filtration. The filtrate was concentrated under reduced pressure until dry. The concentrate was dissolved in 6.8 kg of tetrahydrofuran and heated to 35°C. While maintaining the temperature, 27.3 kg of isopropyl ether was added. After the addition, vigorous stirring was continued for 2 hours while maintaining the temperature, and then a small amount of solid viscous material appeared. Stirring was continued for another 3 hours, and the solid viscous material became powdery. After filtration, the filter cake was rinsed twice to obtain a white wet product. The wet product was dried overnight in a forced-air drying oven at 50°C to obtain 2.66 kg of white refined Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH, with a purity of 99.84%( Figure 2 ), and the isomer content: 0.02%( Figure 3 ).

[0076] Example 4: Application expansion of the preparation method (preparation of Fmoc-Gly-Cys{Psi(Me,Me)Pro}-OH)

[0077] In a 1000 mL four-necked flask equipped with mechanical stirring and a thermometer, 50 g of pure Fmoc-Gly-Cys-OH, 450 g of ethyl acetate, 65 g of 2,2-dimethoxypropane, and 5.5 g of pyridinium p-toluenesulfonate were added. The temperature was raised to 80°C, and azeotropic distillation was carried out until the raw materials no longer decreased. Then the temperature was lowered to 25°C. 200 g of methyl tert-butyl ether was added for extraction, and after phase separation, the organic phase was dried with anhydrous sodium sulfate for 30 minutes, followed by filtration. The filtrate was concentrated under reduced pressure to obtain 107 g of an oily concentrate of Fmoc-Gly-Cys{Psi(Me,Me)Pro}-OH.

[0078] All of the concentrate was dissolved in 400 g of tetrahydrofuran and 40 g of ethanol. 22.93 g of dicyclohexylamine was added, and after stirring while maintaining the temperature, filtration was carried out to obtain a white wet product, which was dried overnight in a forced-air drying oven at 50°C.

[0079] The dried solid was treated with 300 g of 10% m / m aqueous citric acid solution, concentrated, and then crystallized with 50 g of tetrahydrofuran and 200 g of isopropyl ether. After filtration, rinsing, and drying, 35 g of white refined product was obtained, with a purity of 99.78% and an isomer content of 0.05%.

[0080] Comparative Example 1: Synthesis of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH

[0081] In a 1000 mL four-necked flask equipped with mechanical stirring and a thermometer, 50 g of pure Fmoc-Gly-Ser-OH, 450 g of ethyl acetate, 58 g of 2,2-dimethoxypropane and 4.7 g of pyridinium p-toluenesulfonate were added. The temperature was raised to 80 °C and the mixture was kept under reflux with stirring for 8 hours. Samples were taken for in-process control, and at this time, the residual raw material was 24.62%( Figure 4 ).

[0082] Comparative Example 2: Purification of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH

[0083] In a 1000 mL four-necked flask equipped with mechanical stirring and a thermometer, 50 g of the oily concentrate of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH obtained in Example 3 was added, dissolved in 100 g of tetrahydrofuran, and the temperature was raised to 35 °C. 400 g of isopropyl ether was added while maintaining the temperature. After the addition was complete, the mixture was vigorously stirred for 5 hours while maintaining the temperature, filtered, and rinsed. The wet product was subjected to the above operation again for second crystallization purification, filtered, rinsed, and dried to obtain a light brown solid with a purity of 93.65%( Figure 5 ), and the isomer content was 0.58%( Figure 6 ).

Claims

1. A method for preparing a pseudo-dipeptide of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH, characterized in that: The following steps are involved: (1) The compound Fmoc-Gly-OPFP reacts with L-serine to obtain the intermediate Fmoc-Gly-Ser-OH; (2) The intermediate Fmoc-Gly-Ser-OH and 2,2-dimethoxypropane are reacted by azeotropic distillation under the catalysis of pyridinium p-toluenesulfonate to obtain a crude Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH product, which is then purified to obtain the refined Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH product.

2. The preparation method according to claim 1, characterized in that: The reaction solvent for the azeotropic distillation in step (2) is selected from one or more of tetrahydrofuran, hexane, ethyl acetate and cyclohexane.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the reaction solvent of the azeotropic distillation to Fmoc-Gly-Ser-OH in step (2) is 1:(5-15).

4. The preparation method according to claim 1, characterized in that: The reaction temperature of the azeotropic distillation in step (2) is 75-90°C.

5. The preparation method according to claim 1, characterized in that: The purification method described in step (2) comprises the following steps: (I) salting the crude Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH with a nitrogen-containing organic base; (II) The salt obtained in step (I) is freed by acid and crystallized to obtain the refined product of Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH.

6. The preparation method according to claim 5, characterized in that: The nitrogen-containing organic base described in step (I) is one or more selected from dicyclohexylamine, diisopropylamine and di-tert-butylamine.

7. The preparation method according to claim 5, characterized in that: The molar ratio of the nitrogen-containing organic base described in step (I) to the crude Fmoc-Gly-Ser{Psi(Me,Me)Pro}-OH is (0.5-1.5):

1.

8. The preparation method according to claim 5, characterized in that: The reaction solvent for forming the salt in step (I) is selected from one or more of tetrahydrofuran, ethanol and ethyl acetate.

9. The preparation method according to claim 5, characterized in that: The crystallization method described in step (II) is to crystallize the free product obtained by acid liberation in a mixed solvent selected from ethyl acetate-n-heptane, tetrahydrofuran-isopropyl ether, and tetrahydrofuran-n-heptane to obtain the refined product.

10. The preparation method according to claim 9, characterized in that: The crystallization method is to solidify the free product obtained by acid liberation at 25-45°C first, and then crystallize at 0-10°C to obtain the refined product.

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