A method for synthesizing Nα-Fmoc-isoleucine-α-aminoisobutyric acid
Through a new synthesis method, the synthesis steps of Nα-fluorene methoxycarbonyl-isoleucine-α-aminoisobutyric acid are simplified, the purification and yield of the product are improved, the problems of complex synthesis, low yield and difficulty in purification in the prior art are solved, and efficient and low-cost production is achieved.
Patent Information
- Application Number
- CN202411949372.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art has multiple disadvantages in the synthesis of Nα-fluorene methoxycarbonyl-isoleucine-α-aminoisobutyric acid, including low yield, difficulty in purification and complex synthesis steps, resulting in low product quality and high production costs.
A new synthetic method is adopted, which includes the following steps: first react 2-aminoisobutyric acid with a silicon protective reagent to produce N,O-bistrialkylsilyl-protected 2-aminoisobutyric acid; secondly, Nα-fluorenylmethoxycarbonyl-isoleucine and 2-cyano-2-(hydroxyimino)acetate under the action of a condensation agent to form activated ester; then, two intermediates are reacted, and the silicon protecting group is removed under the action of the quenching reagent, and finally purified to obtain the target product.
This method simplifies the synthesis steps, improves the controllability of the reaction and the ease of purification of the product, reduces production costs, is suitable for large-scale production, and effectively avoids the formation of dipeptide impurities of Fmoc-isoleucyl-isoleucine and Nα-fluorenyl-isoleucine symmetric anhydrides.
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Figure CN119751547A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of synthesis of pharmaceutical intermediates, and more specifically to a method for synthesizing Nα-fluorenylmethoxycarbonyl-isoleucine-α-aminoisobutyric acid. Background Art
[0002] Nα-Fmoc-Ile-Aib-OH) belongs to the difficult sequence dipeptide "Difficult Sequences". Dipeptides or even short peptides containing α-aminoisobutyric acid are an important molecular building block for the modification and optimization of the amino acid sequence of peptide drugs. In recent years, with the rapid development of glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists), especially the successive launch of blockbuster drugs Semaglutide and Tirzepatide, the market capacity of this type of molecular building block has grown rapidly and has a bright future. Therefore, the research and development of the synthesis technology of this type of molecular building block has important social and economic significance.
[0003] Generally, the synthesis of dipeptides can be represented by the following general formula:
[0004]
[0005] In the above general formula, R1 includes but is not limited to tert-butyl, benzyl, fluorenylmethyl, R2 and R4 are amino acid side chains, R3 includes but is not limited to hydrogen atom, methyl, ethyl, benzyl, tert-butyl, and R5 is a hydrogen atom or an active ester group.
[0006] In the above general formula, when R1 is tert-butyl, benzyl, or fluorenylmethyl, the amino protecting groups in general formula (1) are Boc-, Z-, or Fmoc-, respectively; when R3 is methyl, ethyl, benzyl, or tert-butyl, the carboxyl protecting groups in general formula (2) are methyl ester, ethyl ester, benzyl ester, or tert-butyl ester, respectively.
[0007] In the above general formula, the active ester group represented by R5 is commonly an asymmetric anhydride, HOBt (1-hydroxybenzotriazole) and its analogs, HOAt (N-hydroxy-7-azabenzotriazole) and its analogs, HOSu (N-hydroxysuccinimide) and its analogs. Since HOBt and its analogs, HOAt and its analogs have poor water solubility or limited solubility, they must be washed with alkaline water for multiple times after the synthesis to control their residual amount.
[0008] The reaction described in the above general formula is more suitable for the synthesis of low steric hindrance dipeptides, but the reaction effect is not very good for highly steric hindrance Cα,α-dialkyl substituted amino acids, the yield is low or even no reaction. At the same time, the α-amino group in the above general formula (1) and the α-carboxyl group in the general formula (2) must be protected at the same time, and after the amide bond is formed, the α-carboxyl protecting group in the general formula (3) is removed by alkaline hydrolysis or hydrogenolysis. This increases the synthesis steps by at least two steps, which puts great pressure on the total yield and cost.
[0009] Since Fmoc- is an alkali-sensitive group, the synthesized fully protected dipeptide is not suitable for alkaline hydrolysis, and hydrogenolysis also has certain side reactions. Therefore, the combination of Fmoc- with methyl ester and ethyl ester has serious defects, and the combination of Fmoc- with benzyl ester is also limited. The combination of Fmoc- with tert-butyl ester is acceptable for the target product involved in this patent, but there is also the disadvantage of increasing the synthesis steps by at least two steps.
[0010] If R5 is an active ester and R3 is a hydrogen atom, the peptide bond formation of the general formula (1) and the general formula (2) usually needs to be carried out in water or a water-containing solvent. However, the stability of the active ester in the general formula (1) in water has certain problems, and the reaction yield is not ideal.
[0011] In summary, the existing literature or processes have various disadvantages for the synthesis of Nα-Fmoc-isoleucine-α-aminoisobutyric acid, which leads to limited synthesis yield and difficulty in product purification. Therefore, it is necessary to develop more effective synthesis technology in order to obtain higher quality Nα-Fmoc-isoleucine-α-aminoisobutyric acid to meet market needs. Summary of the invention
[0012] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for synthesizing Nα-Fmoc-isoleucine-α-aminoisobutyric acid.
[0013] In order to solve the above problems, the technical solution provided by the present invention is as follows:
[0014] A method for synthesizing Nα-Fmoc-isoleucine-α-aminoisobutyric acid, comprising the following steps:
[0015] Step (1), reacting 2-aminoisobutyric acid with a silicon protecting agent in a solvent to obtain a N,O-bistrialkylsilyl protected 2-aminoisobutyric acid solution, i.e., intermediate B;
[0016] Step (2), Nα-Fmoc-isoleucine and ethyl 2-cyano-2-(hydroxyimino)acetate react in a solvent under the action of a condensing agent to synthesize Nα-Fmoc-isoleucine-Oxyma activated ester solution, i.e., intermediate A;
[0017] Step (3), reacting intermediate A with intermediate B, adding a quenching agent to remove the silicon protecting group after the reaction, and refining to obtain the target product Nα-fluorenylmethoxycarbonyl-isoleucine-α-aminoisobutyric acid;
[0018] The specific reaction formula is as follows:
[0019]
[0020] Wherein R is a C1-C3 alkyl group.
[0021] As a preferred embodiment of the technical solution of the present invention, the silicon protection agent in step (1) is selected from any one of trimethylsilyl chloride, triethylsilyl chloride, hexamethyldisilazane, hexamethyldisilazane, and N,O-bis(trimethylsilyl)acetamide.
[0022]
[0023] As a preferred embodiment of the technical solution of the present invention, when the silicon protecting reagent trimethylchlorosilane, triethylchlorosilane, hexamethyldisilazane, hexamethyldisilazane urea, or N,O-bis(trimethylsilyl)acetamide is used in step (1), an organic base is used in the reaction, and the organic base used is selected from any one of triethylamine, N,N-diisopropylethylamine, tributylamine, pyridine, and collidine; more preferably, when the silicon protecting reagent trimethylchlorosilane, triethylchlorosilane is used in step (1), an organic base is used in the reaction, and the organic base used is selected from any one of triethylamine, N,N-diisopropylethylamine, tributylamine, pyridine, and collidine; more preferably, when the silicon protecting reagent hexamethyldisilazane, hexamethyldisilazane urea, or N,O-bis(trimethylsilyl)acetamide is used in step (1), the organic base is omitted, and the silicon protecting reagent is mixed with α-aminoisobutyric acid and the reaction temperature is kept at 50-100°C for reaction, or even refluxed.
[0024] As a more preferred embodiment of the technical solution of the present invention, the molar ratio of 2-aminoisobutyric acid to the silicon protective agent in step (1) is 1:1.5-2.5, and the amount of the silicon protective agent is preferably such that the α-amino group and α-carboxyl group of the 2-aminoisobutyric acid react completely and form a clear solution.
[0025] As a more preferred embodiment of the technical solution of the present invention, the molar ratio of 2-aminoisobutyric acid to the organic base in step (1) is 1:1.5-2.5.
[0026] As a more preferred embodiment of the technical solution of the present invention, when adding the organic base in step (1), the internal temperature needs to be controlled at 15-20°C, and the temperature of the reaction after the mixing of the reaction materials is completed is 50-100°C.
[0027] As a more preferred embodiment of the technical solution of the present invention, the solvent in step (1) is selected from tetrahydrofuran, dichloromethane or ethylene glycol dimethyl ether, and the type of solvent is preferably determined according to the boiling point of the silicon protection reagent.
[0028] As a more preferred embodiment of the technical solution of the present invention, the amount of the solvent used in step (1) is 3 to 10 times the mass of 2-aminoisobutyric acid, and the type and amount of the solvent selected are based on obtaining a clear solution after the reaction.
[0029] As a preferred embodiment of the technical solution of the present invention, the molar ratio of Nα-fluorenylmethoxycarbonyl-isoleucine / ethyl 2-cyano-2-(hydroxyimino)acetate / condensation agent in step (2) is 1:1.0-1.2:1.0-1.5.
[0030] As a more preferred embodiment of the technical solution of the present invention, the coupling reagent in step (2) is selected from any one of DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), among which DCC is the cheapest and has the lowest cost of use among the three, while EDCI is the most convenient to use and the by-products can be removed by post-treatment water washing, and DIC is between the two.
[0031]
[0032] As a more preferred embodiment of the technical solution of the present invention, the solvent in step (2) is selected from tetrahydrofuran, dichloromethane or ethylene glycol dimethyl ether.
[0033] As a more preferred embodiment of the technical solution of the present invention, the amount of solvent used in step (2) is 3 to 10 times the mass of Nα-Fmoc-isoleucine.
[0034] As a preferred embodiment of the technical solution of the present invention, the molar ratio of the bistrialkylsilyl-protected 2-aminoisobutyric acid in step (3) to the Nα-fluorenylmethoxycarbonyl-isoleucine in step (2) is 0.8 to 3.0:1.
[0035] As a more preferred embodiment of the technical solution of the present invention, when adding intermediate B in step (3), the internal temperature is controlled to be 5-10°C, and the reaction solution of intermediate B in step (1) is preferably cooled to 5-10°C before use.
[0036] As a more preferred embodiment of the technical solution of the present invention, the molar ratio of the organic base in step (3) to the Nα-fluorenylmethoxycarbonyl-isoleucine in step (2) is 0.8-2.0:1.
[0037] As a more preferred embodiment of the technical solution of the present invention, the quenching reagent in step (3) is one or more of water, methanol, ethanol, and isopropanol. The use of a mixed solvent has a beneficial effect of removing impurities.
[0038] As a more preferred embodiment of the technical solution of the present invention, the solvent used for the crystallization or recrystallization of Nα-fluorenylmethoxycarbonyl-isoleucine-α-aminoisobutyric acid is one or more of ethanol, methanol, isopropanol, tetrahydrofuran, ethyl acetate, ether, isopropyl ether, methyl tert-butyl ether or petroleum ether.
[0039] Finally, the isoleucine in the technical solution of the present invention is selected from any one of L-isoleucine, D-isoleucine and DL-isoleucine.
[0040] If there is a conflict between the Chinese name and the structural formula of the compound in the present invention, the structural formula shall prevail, unless there is an obvious error in the structural formula.
[0041] The beneficial effects of the technical solution of the present invention include:
[0042] (1) Due to the use of a temporary silicon protecting group and starting from isoleucine, the present invention reduces and simplifies the synthesis steps compared to conventional methods;
[0043] (2) Each step of the synthesis is simple and reproducible. Intermediates A and B are directly used in step 3 without purification. After the reaction, the temporary silicon protecting group can be removed by simple and mild quenching, which simplifies the subsequent crystallization process.
[0044] (3) Most importantly, since the α-amino group and α-carboxyl group of 2-aminoisobutyric acid are not exposed at the same time during the synthesis process, the intermediates A and B in step (3) are also homogeneous reactions, which not only eliminates the generation of dipeptide impurities of Fmoc-isoleucyl-isoleucine, but also prevents the generation of symmetrical anhydride of Nα-fluorenylmethoxycarbonyl-isoleucine.
[0045] (4) The product is easy to purify, has low production cost, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Figure 1 This is the HNMR spectrum of Fmoc-Ile-Aib-OH obtained in the specific implementation mode 1 of the present invention. DETAILED DESCRIPTION
[0047] The present invention is described below with reference to examples, but the present invention is not limited thereto. In the art, simple replacement or improvement made by technicians in the field to the present invention belongs to the technical solution protected by the present invention.
[0048] Embodiment 1:
[0049] 1. Synthesis of 2-aminoisobutyric acid protected by bis-silyl group (Intermediate B)
[0050] Add 500g of dichloromethane to a dry and clean 1000mL reaction bottle, then add 50g of 2-aminoisobutyric acid (molecular weight 103.12g / mol, molar number 485mmol), 123g of triethylamine (molecular weight 101.19g / mol, molar number 1212.2mmol), cool to 15-20℃ in an ice-water bath, and drop 131.7g of trimethylchlorosilane (molecular weight 108.64g / mol, molar number 1212.2mmol) for 0.5 hours. After the dropwise addition is completed, the oil bath is heated to 50℃ and kept warm for 5.0 hours until all the solids are dissolved, and the reaction is terminated. The final reaction solution is a red clear solution, and the reaction solution is cooled to 25℃ and nitrogen protection is used for standby.
[0051] 2. Synthesis of Fmoc-Ile-OH activated ester (Intermediate A)
[0052] To a dry and clean 500 mL reaction bottle, add 85.7 g (molecular weight 353.42 g / mol, molar number 242.5 mmol) of Fmoc-Ile-OH (Nα-fluorenylmethoxycarbonyl-isoleucine), 37.9 g (molecular weight 142.11 g / mol, molar number 266.8 mmol) of ethyl 2-cyano-2-(hydroxyimino)acetate (abbreviated as Oxyma), and 600 g of dichloromethane, stir at 25°C to dissolve the solid, and cool to 15°C. While maintaining the internal temperature at 15-20°C, 60.0 g of dicyclohexylcarbodiimide (DCC) (molecular weight 206.33 g / mol, molar number 291 mmol) was added in batches. After the addition, the mixture was kept at 15-20°C and the reaction was continued for 0.5 hour. The mixture was naturally heated to room temperature (28-32°C) and reacted for 4 hours. The reaction was completed when the HPLC control showed that Fmoc-Ile-OH was <0.5%. The mixture was protected by nitrogen and used for standby.
[0053] 3. Synthesis of Fmoc-Ile-Aib-OH
[0054] Cool all the reaction liquid of Fmoc-Ile-OH activated ester to 5-10°C, under nitrogen protection and keeping the internal temperature at 5-10°C, slowly add it dropwise to the 2-aminoisobutyric acid solution protected by the double silicon group, and then add 49.1g of triethylamine (molecular weight 101.19g / mol, molar number 485mmol), continue to keep warm for 0.5 hours after addition, naturally warm to room temperature (30°C ± 2) and keep warm for 6 hours, and the HPLC control shows that Fmoc-Ile-OH activated ester <1.0% reaction is over. Slowly add 200g of methanol to the reaction liquid and stir for 2 hours. The reaction liquid is concentrated under reduced pressure in a 40-45°C water bath until no fraction is distilled out. Add 800g of ethyl acetate to the residual oil and dissolve it, then add 400g of 10% saline solution and wash twice. The organic phase is concentrated under reduced pressure to dryness to obtain 131.4g of residual oil. The residual oil was recrystallized with 500 g ethyl acetate / petroleum ether (1:1), and the obtained wet product was dried under vacuum at 50° C. to obtain 94.6 g of dry product, with a total yield of 89.0%, HPLC 99.7%, and a maximum single impurity ≤0.1%.
[0055] Embodiment 2:
[0056] 1. Synthesis of 2-aminoisobutyric acid protected by bis-silyl group (Intermediate B)
[0057] To a dry and clean 1000mL reaction bottle, add 400g of tetrahydrofuran, then add 50g of 2-aminoisobutyric acid (molecular weight 103.12g / mol, molar number 485mmol), cool to 15-20°C in an ice-water bath, add 156.6g of hexamethyldisilazane (molecular weight 161.40g / mol, molar number 970mmol), heat the oil bath to 80-100°C and keep the reaction for 5.0 hours until all the solids are dissolved, and the reaction is terminated. The final reaction solution is a red clear solution. The reaction solution is cooled to 25°C and protected with nitrogen for standby use.
[0058] 2. Synthesis of Fmoc-Ile-OH activated ester (Intermediate A)
[0059] Add 85.7g of Fmoc-Ile-OH (molecular weight 353.42g / mol, molar number 242.5mmol), 41.4g of ethyl 2-cyano-2-(hydroxyimino)acetate (abbreviated as Oxyma) (molecular weight 142.11g / mol, molar number 291mmol), and 600g of tetrahydrofuran to a dry and clean 500mL reaction bottle, stir and dissolve the solid at 25°C, and cool to 15°C. Keep the internal temperature at 15-20°C, add 45.9g of diisopropylcarbodiimide (DIC) (molecular weight 126.20g / mol, molar number 363.8mmol) in batches, keep the temperature at 15-20°C after addition, continue to react for 0.5 hours, naturally raise the temperature to room temperature (28-32°C) and react for 4 hours. The HPLC control shows that Fmoc-Ile-OH <0.5% and the reaction is over, and nitrogen protection is used for standby.
[0060] 3. Synthesis of Fmoc-Ile-Aib-OH
[0061] Cool all the reaction liquid of Fmoc-Ile-OH activated ester to 5-10°C, under nitrogen protection and keeping the internal temperature at 5-10°C, slowly add it dropwise to the 2-aminoisobutyric acid solution protected by the double silicon group, and then add 58.8g of trimethylpyridine (molecular weight 121.18g / mol, molar number 485mmol), continue to keep warm for 0.5 hours after addition, naturally warm to room temperature (30°C ± 2) and keep warm for 6 hours, and the HPLC control shows that Fmoc-Ile-OH activated ester <1.0% reaction is over. Slowly add 200g of water to the reaction liquid and stir for 2 hours. The reaction liquid is concentrated under reduced pressure in a 40-45°C water bath until no fraction is distilled out. Add 800g of ethyl acetate to the residual oil and dissolve it, then add 400g of 10% saline solution and wash twice. The organic phase is concentrated under reduced pressure to dryness to obtain 135.7g of residual oil. The residual oil was recrystallized with 500g ethyl acetate / petroleum ether (1:1) to obtain a wet product, which was dried in vacuo at 50°C to obtain 96.77g of a dry product with a total yield of 91.0%, HPLC 99.6%, and a maximum single impurity ≤ 0.1%.
[0062] Embodiment 3:
[0063] 1. Synthesis of 2-aminoisobutyric acid protected by bis-silyl group (Intermediate B)
[0064] Add 150g of ethylene glycol dimethyl ether to a dry and clean 1000mL reaction bottle, then add 50g of 2-aminoisobutyric acid (molecular weight 103.12g / mol, molar number 485mmol), cool to 15-20℃ in an ice-water bath, add 246.7g of N,O-bis(trimethylsilyl)acetamide (molecular weight 203.43g / mol, molar number 1212.5mmol), heat to 80-100℃ in an oil bath and keep warm for 5.0 hours until all the solids are dissolved, and the reaction is terminated. The final reaction solution is a red clear solution. Cool the reaction solution to 25℃ and protect it with nitrogen for standby use.
[0065] 2. Synthesis of Fmoc-Ile-OH activated ester (Intermediate A)
[0066] To a dry and clean 500 mL reaction bottle, add 85.7 g of Fmoc-Ile-OH (molecular weight 353.42 g / mol, molar number 242.5 mmol), 41.4 g of ethyl 2-cyano-2-(hydroxyimino)acetate (abbreviated as Oxyma) (molecular weight 142.11 g / mol, molar number 291 mmol), and 600 g of ethylene glycol dimethyl ether, stir at 25° C. to dissolve the solid, and cool to 15° C. While maintaining the internal temperature at 15-20°C, 69.7 g (molecular weight 191.71 g / mol, molar number 363.8 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added in batches. After the addition, the mixture was kept at 15-20°C for 0.5 hour. The mixture was naturally heated to room temperature (28-32°C) and reacted for 4 hours. The reaction was completed when the HPLC control showed that Fmoc-Ile-OH was <0.5%. The mixture was protected by nitrogen and used for standby.
[0067] 3. Synthesis of Fmoc-Ile-Aib-OH
[0068] Cool all the reaction liquid of Fmoc-Ile-OH activated ester to 5-10°C, under nitrogen protection and keeping the internal temperature at 5-10°C, slowly add it dropwise to the 2-aminoisobutyric acid solution protected by the double silicon group, and then add 62.68g of diisopropylethylamine (molecular weight 129.25g / mol, molar number 485mmol), continue to keep warm for 0.5 hours after addition, naturally warm to room temperature (30°C ± 2) and keep warm for 6 hours, and the HPLC control shows that Fmoc-Ile-OH activated ester <1.0% reaction is over. Slowly add 200g of water to the reaction liquid and stir for 2 hours. The reaction liquid is concentrated under reduced pressure in a 40-45°C water bath until no fraction is distilled out. 132.6g of residual oil is obtained, 800g of ethyl acetate is added to dissolve, and then 400g of 10% saline solution is added to wash twice. The organic phase is concentrated under reduced pressure to dryness to obtain a residual oil. The residual oil was recrystallized with 500g ethyl acetate / petroleum ether (1:1) to obtain a wet product, which was dried under vacuum at 50°C to obtain 99.96g of a dry product with a total yield of 94.0%, HPLC 99.5%, and a maximum single impurity ≤ 0.1%.
[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. A method for synthesizing Nα-Fmoc-isoleucine-α-aminoisobutyric acid, characterized in that: The synthesis method comprises the following steps: Step (1), reacting 2-aminoisobutyric acid with a silicon protecting agent in a solvent to obtain a N,O-bistrialkylsilyl protected 2-aminoisobutyric acid solution, i.e., intermediate B; Step (2), Nα-Fmoc-isoleucine and ethyl 2-cyano-2-(hydroxyimino)acetate react in a solvent under the action of a condensing agent to synthesize Nα-Fmoc-isoleucine-Oxyma activated ester solution, i.e., intermediate A; Step (3), reacting intermediate A with intermediate B, adding a quenching agent to remove the silicon protecting group after the reaction, and refining to obtain the target product Nα-fluorenylmethoxycarbonyl-isoleucine-α-aminoisobutyric acid; The specific reaction formula is as follows: Wherein R is a C1-C3 alkyl group.
2. The method according to claim 1, characterized in that The silicon protecting agent in step (1) is selected from any one of trimethylsilyl chloride, triethylsilyl chloride, hexamethyldisilazane, hexamethyldisilazane, and N,O-bis(trimethylsilyl)acetamide.
3. The method according to claim 1, characterized in that The silicon protection agent in step (1) is selected from any one of hexamethyldisilazane, hexamethyldisilazane urea, and N,O-bis(trimethylsilyl)acetamide, and the reaction temperature in step (1) is 50-100°C.
4. The method according to claim 2, characterized in that An organic base is used in the reaction in step (1), and the organic base used in step (1) is selected from any one of triethylamine, N,N-diisopropylethylamine, tributylamine, pyridine, and collidine. When the organic base is added in step (1), the internal temperature is controlled to be 15-20° C., and the temperature of the heating reaction is 50-100° C.
5. The method according to claim 4, characterized in that In the step (1), the molar ratio of 2-aminoisobutyric acid, silicon protection reagent and organic base is 1:1.5-2.5:1.5-2.
5.
6. The method according to claim 1, characterized in that The solvent in step (1) is selected from tetrahydrofuran, dichloromethane or ethylene glycol dimethyl ether; the amount of the solvent used is 3 to 10 times the mass of 2-aminoisobutyric acid.
7. The method according to claim 1, characterized in that The molar ratio of Nα-fluorenylmethoxycarbonyl-isoleucine, ethyl 2-cyano-2-(hydroxyimino)acetate and condensing agent in step (2) is 1:1.0-1.2:1.0-1.
5.
8. The method according to claim 7, characterized in that The condensation agent in step (2) is selected from any one of DCC, DIC and EDCI.
9. The method according to claim 1, characterized in that The solvent in step (2) is selected from tetrahydrofuran, dichloromethane or ethylene glycol dimethyl ether; the amount of the solvent used is 3 to 10 times the mass of Nα-fluorenylmethoxycarbonyl-isoleucine.
10. The method according to claim 1, characterized in that An organic base is used in the reaction in step (3), and the organic base used in step (3) is selected from any one of triethylamine, N,N-diisopropylethylamine, n-tributylamine, pyridine, and collidine; the molar ratio of the organic base in step (3) to Nα-fluorenylmethoxycarbonyl-isoleucine in step (2) is 0.8 to 2.0:1; the molar ratio of 2-aminoisobutyric acid in step (1) to Nα-fluorenylmethoxycarbonyl-isoleucine in step (2) is 0.8 to 3.0:
1.
11. The method according to claim 1, characterized in that The quenching reagent in step (3) is one or more of water, methanol, ethanol, and isopropanol; the refining is crystallization or recrystallization, and the solvent used is one or more of ethanol, methanol, isopropanol, tetrahydrofuran, ethyl acetate, ether, isopropyl ether, methyl tert-butyl ether or petroleum ether.