Preparation method of N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid

Abstract

The present invention belongs to the technical field of compound preparation, and specifically provides a method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid, which comprises the following steps: step S1, causing L-isoleucine to undergo a substitution reaction with a fluorenylmethoxycarbonyl-type amino protecting agent to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine; step S2, causing the 2-aminoisobutyric acid to undergo a condensation reaction with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine under the action of a condensing agent to obtain N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid. According to the preparation method of the embodiment of the present invention, chiral L-isoleucine is reacted with an Fmoc-type amino protecting agent, and after the amino protection is completed, it is condensed with 2-aminoisobutyric acid to obtain the target product N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid (Fmoc-Ile-Aib-OH), which has a short route and low synthesis cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound preparation, and more specifically, relates to a method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid. Background Art

[0002] Medium-molecular compounds (molecular weight 500 to 2000) are the focus of drug development because they can inhibit protein-protein interactions. However, peptides themselves are difficult to develop into pharmaceuticals because they generally have low drug-like properties (metabolic stability or membrane permeability).

[0003] In recent years, it has been found that by cyclization of peptides or the use of non-natural amino acids with methyl groups in peptides, metabolic stability or membrane permeability is greatly improved, and cyclic peptide compounds containing non-natural amino acids are effective in inhibiting protein-protein interactions. It is known that among cyclic peptides containing non-natural amino acids, cyclic peptides containing substituted amino acids in particular have drug-like properties. In addition, cyclic peptides containing non-natural amino acids are a class of drug-like molecules with membrane permeability and metabolic stability, and their druggable characteristics have further increased their attention.

[0004] Therefore, the synthesis and development of cyclic peptides containing unnatural amino acids is of great value. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing a non-natural dipeptide, namely N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid (hereinafter sometimes referred to as Fmoc-Ile-Aib-OH), which is simple to operate, has a short route and is suitable for industrial production.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid according to an embodiment of the present invention comprises the following steps:

[0008] Step S1, L-isoleucine is subjected to a substitution reaction with a fluorenylmethoxycarbonyl amino protecting agent (hereinafter, sometimes referred to as an Fmoc amino protecting agent) to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine;

[0009] Step S2, allowing the 2-aminoisobutyric acid to undergo a condensation reaction with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine in the presence of a condensing agent to obtain N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0010] In some embodiments of the present invention, the substitution reaction is carried out in a first solvent under the action of a first base, the first solvent is one or more of dichloromethane, N,N'-dimethylformamide, N,N'-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone, and the first base is one or more of triethylamine, diisopropylethylamine, pyridine, p-dimethylaminopyridine, and diazabicyclic.

[0011] Furthermore, the Fmoc-based amino protecting agent is 9-fluorenylmethyl chloroformate (ie, Fmoc-Cl), 9-fluorenylmethyl-N-succinimidyl carbonate (ie, Fmoc-OSu) or a mixture thereof.

[0012] Furthermore, the molar ratio of L-isoleucine to the Fmoc amino protecting agent and the first base is 1:(1-1.5):(1-5.0), the reaction temperature of the substitution reaction is 0-80° C., and the reaction time is 1-40 hours.

[0013] In some embodiments of the present invention, step S2 includes:

[0014] Step S21, allowing the 2-aminoisobutyric acid to react with a silicon protective agent in a second solvent under the action of a second base;

[0015] Step S22, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and a condensing agent are continuously added to the reaction system of step S21, so that the protected 2-aminoisobutyric acid and N-(9-fluorenylmethoxycarbonyl)-L-isoleucine undergo a condensation reaction under the action of the condensing agent to generate the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0016] Furthermore, the second solvent is one or more of N,N'-dimethylformamide, N,N'-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, dichloromethane, ethyl acetate, acetonitrile, n-heptane, tetrahydrofuran, petroleum ether, and ether; the silicon protective agent is one or more of trimethylsilyl chloride, tert-butyldimethylsilyl chloride, triethylsilyl chloride, benzyltriethylsilyl chloride, triisopropylsilyl chloride, and tert-butyldiphenylsilyl chloride; the second base is one or more of triethylamine, diisopropylethylamine, pyridine, p-dimethylaminopyridine, and diazabicyclic.

[0017] Furthermore, the molar ratio of the 2-aminoisobutyric acid to the silicon protective agent and the second base is 1:(1-5):(1-10), the reaction temperature of step S21 is 0-80° C., and the reaction time is 1-40 hours.

[0018] Furthermore, the condensing agent is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, N,N-carbonyldiimidazole, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0019] Furthermore, in step S2, the molar ratio of 2-aminoisobutyric acid, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and condensing agent is 1:(1.0-1.5):(1.0-3.0), the reaction temperature is 0-60° C., and the reaction time is 4-20 hours.

[0020] Furthermore, the step S2 further includes:

[0021] Step S23, after the condensation reaction is completed, water is added to the reaction solution for washing, filtering, and drying to obtain a crude product, and the crude product is slurried and purified with a mixed solvent of ethyl acetate / petroleum ether to obtain the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid.

[0022] The above technical solution of the present invention has at least one of the following beneficial effects:

[0023] According to the preparation method of the embodiment of the present invention, chiral L-isoleucine is reacted with an Fmoc-type amino protecting agent, and after the amino protection is completed, it is condensed with 2-aminoisobutyric acid to obtain the target product N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucine aminocarboxyl)-2-aminoisobutyric acid (Fmoc-Ile-Aib-OH), which has a short route and low synthesis cost;

[0024] In addition, the reaction conditions of each step are controllable, the operation is simple, the total yield is high, the route is feasible, and it is suitable for industrial production;

[0025] Furthermore, the carboxyl group of 2-aminoisobutyric acid is protected by silane using a silicon protective agent, and then condensed with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine, which can avoid the generation of unnecessary by-products, improve the yield, and reduce the difficulty of post-processing;

[0026] Furthermore, carrying out the condensation reaction under the action of a condensation agent can, on the one hand, promote the improvement of the reaction efficiency, and on the other hand, reduce the occurrence of chiral racemization. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0028] The following first describes in detail the preparation method of N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid according to an embodiment of the present invention.

[0029] The method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid according to an embodiment of the present invention comprises the following steps:

[0030] Step S1, allowing L-isoleucine to undergo a substitution reaction with an Fmoc-type amino protecting agent to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine;

[0031] Step S2, allowing the 2-aminoisobutyric acid to undergo a condensation reaction with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine in the presence of a condensing agent to obtain N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0032] Specifically, the synthesis route is shown in the following formula (1):

[0033] (1)

[0034] According to the preparation method of the embodiment of the present invention, chiral L-isoleucine is reacted with an Fmoc-type amino protecting agent, and after the amino protection is completed, it is condensed with 2-aminoisobutyric acid to obtain the target product N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucine aminocarboxyl)-2-aminoisobutyric acid (Fmoc-Ile-Aib-OH), the route is short, and the synthesis cost is low; in addition, the reaction conditions of each step are controllable, the operation is simple, the total yield is high, the route is feasible, and it is suitable for industrial production.

[0035] Below, each step is explained in detail.

[0036] (i) Step S1, allowing L-isoleucine to undergo a substitution reaction with an Fmoc-type amino protecting agent to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine.

[0037] That is, the preparation method of the present invention uses L-isoleucine as a starting material, and firstly causes it to undergo a substitution reaction with an Fmoc-type amino protecting agent to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine.

[0038] In some embodiments of the present invention, the substitution reaction is carried out in a first solvent under the action of a first base. The solvent is conducive to improving the reaction efficiency, and under alkaline conditions, the removal of hydrogen from the carboxylic acid group at the end of L-isoleucine can be promoted, thereby promoting the substitution reaction.

[0039] Among them, the first solvent can be one or more of dichloromethane, N,N'-dimethylformamide, N,N'-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone, preferably any one of dichloromethane and dimethyl sulfoxide, which is more convenient for concentration and removal in the post-processing stage, and can be recycled and reused after recovery.

[0040] In addition, the first base can be, for example, one or more of triethylamine, diisopropylethylamine, pyridine, p-dimethylaminopyridine, and diazabicyclo. Among them, diisopropylethylamine is preferred because it has little effect on the chirality of the raw materials and the product.

[0041] The Fmoc-type amino protecting agent may be Fmoc-Cl, Fmoc-OSu or a mixture thereof, preferably Fmoc-Osu, which has a higher reaction conversion rate and a higher yield.

[0042] Furthermore, the molar ratio of L-isoleucine to the Fmoc amino protecting agent and the first base is 1:(1-1.5):(1-5.0).

[0043] That is to say, relative to L-isoleucine, using a slightly higher amount of the Fmoc-type amino-protecting agent and a slightly higher amount of the first base than the chemical equivalent is not only more conducive to promoting the reaction, improving the yield and reaction rate, but also does not cause a large amount of excess and waste of raw materials. Preferably, the molar ratio of L-isoleucine to the Fmoc-type amino-protecting agent (such as Fmoc-OSu) and the first base (such as diisopropylethylamine) is 1:1.05:1.5.

[0044] Wherein, the reaction temperature of the substitution reaction is 0 to 80° C., and the reaction time is 1 to 40 hours. Preferably, the reaction temperature is room temperature, and the reaction time is 5 hours. Too high a temperature and prolonged reaction time will increase the generation of by-products. Moreover, the reaction conditions of the reaction are mild and controllable.

[0045] After the reaction is completed, the product can be washed with dilute hydrochloric acid, washed with brine, dried, and the first solvent evaporated to obtain a crude intermediate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine. The crude product is slurried with acetonitrile / water (preferably 1 / 10) to obtain N-(9-fluorenylmethoxycarbonyl)-L-isoleucine.

[0046] (ii) Step S2, allowing the 2-aminoisobutyric acid to undergo a condensation reaction with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine in the presence of a condensing agent to obtain N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0047] That is, after obtaining N-(9-fluorenylmethoxycarbonyl)-L-isoleucine, it is further subjected to a condensation reaction with 2-aminoisobutyric acid to generate the target product N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0048] In some embodiments of the present invention, step S2 includes:

[0049] Step S21, allowing the 2-aminoisobutyric acid to react with a silicon protective agent under the action of a second base in a second solvent;

[0050] Step S22, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and a condensing agent are continuously added to the reaction system of step S21, so that the protected 2-aminoisobutyric acid and N-(9-fluorenylmethoxycarbonyl)-L-isoleucine undergo a condensation reaction under the action of the condensing agent to generate the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid.

[0051] That is, the carboxyl group of 2-aminoisobutyric acid is first protected by a silicon protective agent, and then a condensation reaction is carried out with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine, thereby avoiding the reaction between the carboxyl group and the amino group in N-(9-fluorenylmethoxycarbonyl)-L-isoleucine to produce unnecessary by-products, reducing the difficulty of post-processing, and at the same time, the silicon protective group can also be naturally deprotected under the action of the second base after the condensation reaction to form the target product.

[0052] In addition, carrying out the condensation reaction under the action of a condensation agent can, on the one hand, promote the improvement of the reaction efficiency, and on the other hand, reduce the occurrence of chiral racemization.

[0053] The second solvent may be one or more of N,N'-dimethylformamide, N,N'-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, dichloromethane, ethyl acetate, acetonitrile, n-heptane, tetrahydrofuran, petroleum ether, and ether, preferably acetonitrile. The acetonitrile has a better yield and can be removed by distillation, which reduces wastewater and is an environmentally friendly solvent choice.

[0054] In addition, the silicon protective agent can be one or more of trimethylchlorosilane, tert-butyldimethylchlorosilane, triethylchlorosilane, benzyltriethylchlorosilane, triisopropylchlorosilane, and tert-butyldiphenylchlorosilane. Among them, tert-butyldiphenylchlorosilane is preferred, which can be used in a smaller amount, reduce the feed ratio, and obtain a higher yield. For example, compared with trimethylchlorosilane, it can use two less equivalents, and the yield is higher (relative to chlorosilane, the yield can be increased by 10-15%).

[0055] In addition, as the second base, for example, it can be one or more of triethylamine, diisopropylethylamine, pyridine, p-dimethylaminopyridine, and diazabicyclic. Pyridine is preferred. Pyridine is more friendly to the amino protecting group Fmoc-, reduces the alkalinity of the reaction system, reduces the risk of deprotection, and has a higher yield.

[0056] Furthermore, the molar ratio of the 2-aminoisobutyric acid to the silicon protective agent and the second base is 1:(1-5):(1-10), preferably 1:3:5.

[0057] The reaction temperature of step S21 is 0-80° C., for example, it can be carried out at room temperature, and the reaction time is 1-40 hours, for example, it can be 16 hours.

[0058] Further, the condensing agent is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, N,N-carbonyldiimidazole, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate. Among them, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is preferred, which not only has a higher yield, but also can effectively avoid the problem of chiral racemization.

[0059] Furthermore, in step S2, the molar ratio of 2-aminoisobutyric acid, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine, and condensing agent is 1:(1.0-1.5):(1.0-3.0). Preferably, it is 1:1.05:1.5. At this ratio, high yield, low input ratio, and low cost can be taken into account.

[0060] In addition, the reaction temperature of step S2 is 0-60°C, for example, it can be carried out at 30°C, and the reaction time is 4-20 hours, for example, it can be 16 hours. The reaction efficiency is high, the operation is simple, and the equipment requirements are low.

[0061] Furthermore, the step S2 further includes:

[0062] Step S23, after the condensation reaction is completed, water is added to the reaction solution for washing, filtering, and drying to obtain a crude product, and the crude product is slurried and purified with a mixed solvent of ethyl acetate / petroleum ether to obtain the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid.

[0063] The following is a detailed description of the method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid of the present invention through specific examples.

[0064] Example 1

[0065] (1) Preparation of reaction intermediate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine

[0066] In a three-necked flask, dissolve 145 g of L-isoleucine in 800 mL of dimethyl sulfoxide, add 258 g of 9-fluorenylmethyl chloroformate, add 120 g of pyridine, maintain the internal temperature at 25-30°C and react for 5 h.

[0067] TLC monitoring showed that the raw material reaction was complete. The reaction solution was washed with 3.0 L of water, filtered, and then washed twice with 500 mL of aqueous solution. The product was dried in an oven and slurried with a mixed solvent of acetonitrile and water to obtain 286 g of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine with a yield of 78%.

[0068] The NMR data of the intermediate product are as follows:

[0069] 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.2Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 5.27 (d, J = 8.7Hz, 1H), 4.43 (dd, J = 15.4, 5.9 Hz, 3H), 4.26 (t, J = 7.0 Hz, 1H), 2.00 (s,1H), 1.51 (d, J = 7.4 Hz, 1H), 1.24 (dd, J = 13.8, 8.1 Hz, 1H), 1.08 – 0.81(m, 5H).

[0070] The detection results were consistent with the structure of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine.

[0071] (2) Preparation of N-(N-(9-Fluorenylmethoxycarbonyl)-L-isoleucylcarboxyl)-2-aminoisobutyric acid

[0072] In a three-necked flask, 103 g of 2-aminoisobutyric acid was dissolved in 800 mL of N,N'-dimethylformamide, and then 324 g of trimethylsilyl chloride and 645 g of diisopropylethylamine were added, and the mixture was reacted at room temperature for 6 h.

[0073] Then, 367 g of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and 229 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added thereto, and the mixture was reacted at 25-30° C. for 16 h.

[0074] After TLC showed that the reaction was complete, the reaction solution was washed with 3.0 L of water, filtered, and then washed twice with 500 mL of water, and dried in an oven to obtain a crude product of N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid (Fmoc-Ile-Aib-OH). The crude product was slurried with a mixed solvent of ethyl acetate and petroleum ether to obtain 294 g of Fmoc-Ile-Aib-OH, with a yield of 65% and an optical purity ee value of 97%.

[0075] The NMR data of the product are as follows:

[0076] 1 H NMR (400 MHz, DMSO) δ 12.18 (s, 1H), 8.11 (s, 1H), 7.90 (d, J =7.5 Hz, 2H), 7.75 (t, J = 7.1 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (dd, J= 9.6, 5.9 Hz, 3H), 4.41 – 4.16 (m, 3H), 3.89 (t, J = 8.6 Hz, 1H), 1.70 (d, J= 6.5 Hz, 1H), 1.44 (d, J = 7.2 Hz, 1H), 1.34 (d, J = 4.4 Hz, 6H), 1.16 –1.02 (m, 1H), 0.83 (dd, J = 17.3, 7.1 Hz, 6H).

[0077] The detection results were consistent with the structure of Fmoc-Ile-Aib-OH. Example 2

[0078] (1) Preparation of reaction intermediate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine

[0079] In a three-necked flask, dissolve 145 g of L-isoleucine in 800 mL of dichloromethane, add 337 g of 9-fluorenylmethyl-N-succinimidyl carbonate, add 150 g of N,N'-diisopropylethylamine, maintain the internal temperature at 25-30°C and react for 5 h.

[0080] After the reaction of the raw materials was completed as monitored by TLC, 3.0 L of water was added to the reaction solution for washing. After separation, the reaction solution was washed twice with 500 mL of saturated brine. After concentration, the solution was slurried with a mixed solvent of acetonitrile and water to obtain 330 g of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine with a yield of 90%.

[0081] The NMR data of the intermediate product are as follows:

[0082] 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.2Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 5.27 (d, J = 8.7Hz, 1H), 4.43 (dd, J = 15.4, 5.9 Hz, 3H), 4.26 (t, J = 7.0 Hz, 1H), 2.00 (s,1H), 1.51 (d, J = 7.4 Hz, 1H), 1.24 (dd, J = 13.8, 8.1 Hz, 1H), 1.08 – 0.81(m, 5H).

[0083] The detection results were consistent with the structure of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine.

[0084] (2) Preparation of Fmoc-Ile-Aib-OH

[0085] In a three-necked flask, 103 g of 2-aminoisobutyric acid was dissolved in 800 mL of acetonitrile, and then 450 g of tert-butyldimethylsilyl chloride and 395 g of pyridine were added and reacted at room temperature for 6 h.

[0086] Then, 367 g of N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and 570 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added thereto, and the mixture was reacted at 35-40° C. for 16 h.

[0087] TLC showed that the reaction was complete. After the reaction solution was concentrated, 3.0 L of water was added for washing, filtered, and then washed twice with 500 mL of water, and dried in an oven to obtain Fmoc-Ile-Aib-OH. The crude product was slurried with a mixed solvent of ethyl acetate and petroleum ether to obtain 362 g of Fmoc-Ile-Aib-OH, with a yield of 80% and an optical purity ee value of 99%.

[0088] The NMR data of the product are as follows:

[0089] 1 H NMR (400 MHz, DMSO) δ 12.18 (s, 1H), 8.11 (s, 1H), 7.90 (d, J =7.5 Hz, 2H), 7.75 (t, J = 7.1 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (dd, J= 9.6, 5.9 Hz, 3H), 4.41 – 4.16 (m, 3H), 3.89 (t, J = 8.6 Hz, 1H), 1.70 (d, J= 6.5 Hz, 1H), 1.44 (d, J = 7.2 Hz, 1H), 1.34 (d, J = 4.4 Hz, 6H), 1.16 –1.02 (m, 1H), 0.83 (dd, J = 17.3, 7.1 Hz, 6H).

[0090] The detection results were consistent with the structure of Fmoc-Ile-Aib-OH.

[0091] It should be noted that each step in the above embodiments is independent of each other, that is, step (1) of embodiment 1 and step (2) of embodiment 2 can be combined to generate a new embodiment. Similarly, step (1) of embodiment 2 can be combined with step (1) of embodiment 2 to generate a new embodiment. And so on. The steps of the above embodiments can be arbitrarily combined to generate new embodiments. These combinations should be understood to be within the scope of the present application.

[0092] It can be seen from the above examples that the method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid (Fmoc-Ile-Aib-OH) of the present invention has the advantages of simple operation, short route, low cost and high yield.

[0093] In summary, the preparation method of N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid of the present invention has the advantages of short synthesis route, simple operation, low cost and high yield.

[0094] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid, characterized in that: The steps include: Step S1, allowing L-isoleucine to undergo a substitution reaction with a fluorenylmethoxycarbonyl-type amino protecting agent to generate N-(9-fluorenylmethoxycarbonyl)-L-isoleucine; Step S2, condensing 2-aminoisobutyric acid with N-(9-fluorenylmethoxycarbonyl)-L-isoleucine in the presence of a condensing agent to obtain N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid. The step S2 comprises: Step S21, allowing the 2-aminoisobutyric acid to react with a silicon protective agent under the action of a second base in a second solvent, wherein the second solvent is acetonitrile, the second base is pyridine, and the silicon protective agent is tert-butyldimethylchlorosilane; Step S22, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and a condensing agent are continuously added to the reaction system of step S21, so that the protected 2-aminoisobutyric acid and N-(9-fluorenylmethoxycarbonyl)-L-isoleucine undergo a condensation reaction under the action of the condensing agent to generate the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucinecarboxyl)-2-aminoisobutyric acid, wherein the condensing agent is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

2. The preparation method according to claim 1, characterized in that: The substitution reaction is carried out in a first solvent under the action of a first base, wherein the first solvent is one or more of dichloromethane, N,N'-dimethylformamide, N,N'-dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone, and the first base is one or more of triethylamine, diisopropylethylamine, pyridine, p-dimethylaminopyridine, and diazabicyclic.

3. The preparation method according to claim 2, characterized in that: The fluorenylmethoxycarbonyl amino protecting agent is 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl-N-succinimidyl carbonate or a mixture thereof.

4. The preparation method according to claim 2, characterized in that: The molar ratio of L-isoleucine to the fluorenylmethoxycarbonyl amino protective agent and the first base is 1:(1-1.5):(1-5.0), the reaction temperature of the substitution reaction is 0-80°C, and the reaction time is 1-40 hours.

5. The preparation method according to claim 1, characterized in that: The molar ratio of the 2-aminoisobutyric acid to the silicon protective agent and the second base is 1:(1-5):(1-10), the reaction temperature of step S21 is 0-80° C., and the reaction time is 1-40 hours.

6. The preparation method according to claim 1, characterized in that: In the step S2, the molar ratio of the 2-aminoisobutyric acid, N-(9-fluorenylmethoxycarbonyl)-L-isoleucine and the condensing agent is 1:(1.0-1.5):(1.0-3.0), the reaction temperature is 0-60° C., and the reaction time is 4-20 hours.

7. The preparation method according to claim 1, characterized in that: The step S2 further comprises: Step S23, after the condensation reaction is completed, water is added to the reaction solution for washing, filtering, and drying to obtain a crude product, and the crude product is slurried and purified with a mixed solvent of ethyl acetate / petroleum ether to obtain the N-(N-(9-fluorenylmethoxycarbonyl)-L-isoleucylaminocarboxyl)-2-aminoisobutyric acid.