Preparation method of Fmoc-Trp (Boc)-OH

Abstract

The invention provides a preparation method of Fmoc-Trp (Boc)-OH, which comprises the following steps: a) carrying out reaction on tryptophan and fluorenylmethoxycarbonyl succinimide under the action of alkali to obtain an intermediate compound as shown in a formula (3); reacting with benzyl halide under the action of alkali to obtain an intermediate compound as shown in a formula (4); then reacting with di-tert-butyl dicarbonate ester under the action of a catalyst to obtain an intermediate compound as shown in a formula (6); and finally, performing deprotection to obtain the Fmoc-Trp (Boc)-OH. According to the method provided by the invention, Fmoc-Trp (Boc)-OH is synthesized through four-step reaction, the process of adding Fmoc to H-Trp (Boc)-OH is avoided, the process is simplified, the operation is simple, the reaction condition is mild, the industrial production difficulty is reduced, expensive raw materials are not involved, the production cost is favorably controlled, and the obtained product is high in purity and high in yield.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a method for preparing Fmoc-Trp(Boc)-OH. Background Art

[0002] Fmoc-Trp(Boc)-OH is a commonly used intermediate in the synthesis of peptides such as semaglutide and telpotide. The Chinese name of Fmoc-Trp(Boc)-OH is N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butyloxycarbonyl-L-tryptophan, and its molecular formula is C 31 H 30 N2O6, CAS No. 143824-78-6, has the following structural formula:

[0003]

[0004] In the Fmoc-Trp(Boc)-OH synthesis route disclosed in the prior art, the intermediate H-Trp(Boc)-OH is first obtained, and then Fmoc is added to obtain the final product Fmoc-Trp(Boc)-OH; however, H-Trp(Boc)-OH is unstable during the Fmoc addition process and will produce impurity Fmoc-Trp-OH, while Fmoc-Trp-OH can hardly be removed from Fmoc-Trp(Boc)-OH, thereby affecting the yield and purity of Fmoc-Trp(Boc)-OH.

[0005] For example, CN202110672717 discloses two routes for synthesizing Fmoc-Trp(Boc)-OH, the first of which is:

[0006]

[0007] The second type is:

[0008]

[0009] The synthesis route disclosed in CN202211006819 is:

[0010]

[0011] The above synthetic route not only has low yield and purity, but also has complicated steps. Summary of the invention

[0012] In view of this, the present invention provides a method for preparing Fmoc-Trp(Boc)-OH. The method provided in the present application has simple steps, mild reaction conditions, and the obtained Fmoc-Trp(Boc)-OH has high purity and high yield.

[0013] The present application provides a method for preparing Fmoc-Trp(Boc)-OH, comprising the following steps:

[0014] a) tryptophan reacts with fluorenylmethoxycarbonyl succinimide under the action of a base to obtain an intermediate compound represented by formula (3);

[0015]

[0016] b) The intermediate compound represented by formula (3) reacts with a benzyl halide under the action of a base to obtain an intermediate compound represented by formula (4):

[0017]

[0018] c) the intermediate compound represented by formula (4) reacts with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound represented by formula (6):

[0019]

[0020] d) Deprotecting the intermediate compound represented by formula (6) to obtain Fmoc-Trp(Boc)-OH.

[0021] In some specific implementations, in step a), the base is an inorganic base;

[0022] The molar ratio of tryptophan, fluorenylmethoxycarbonyl succinimide and the base is 1:(0.8-1.3):(1-2.5).

[0023] In some specific implementations, in step a), the reaction temperature is 25°C to 35°C, and the reaction time is 2h to 6h;

[0024] The reaction medium is water and an organic solvent, and the organic solvent is selected from ethyl acetate, acetone or tetrahydrofuran.

[0025] In some specific implementations, in step b), the base is an inorganic base;

[0026] The molar ratio of the intermediate compound represented by formula (3), benzyl halide and base is 1:(1 to 1.5):(1.1 to 2.0).

[0027] In some specific implementations, in step b), the base is selected from sodium carbonate or potassium carbonate.

[0028] In some specific implementations, in step b), the reaction temperature is 60°C to 80°C, and the reaction time is 2h to 4h;

[0029] The medium of the reaction is an organic solvent, and the organic solvent is selected from acetonitrile or ethyl acetate.

[0030] In some specific implementations, in step c), the catalyst is 4-dimethylaminopyridine;

[0031] The molar ratio of the intermediate compound represented by formula (4), the catalyst and di-tert-butyl dicarbonate is 1:(0.040-0.045):(1-2).

[0032] In some specific implementations, in step c), the reaction temperature is 10°C to 35°C, and the reaction time is 0.5h to 2h;

[0033] The reaction medium is an organic solvent, and the organic solvent is selected from dichloromethane, acetonitrile, ethyl acetate or tetrahydrofuran.

[0034] In some specific implementations, the step d) specifically includes:

[0035] Under the action of a catalyst, the intermediate compound represented by formula (6) reacts with hydrogen to obtain Fmoc-Trp(Boc)-OH.

[0036] In some specific implementations, in step d), the catalyst is selected from Pd / C, Raney nickel or Pd / BaSO4;

[0037] The weight ratio of the intermediate compound represented by formula (6) to the catalyst is 1:(0.01-0.1);

[0038] The reaction temperature is 20°C to 30°C, and the reaction time is 16h to 20h.

[0039] The preparation method of Fmoc-Trp(Boc)-OH provided in the present application comprises the following steps: a) tryptophan reacts with fluorenylmethoxycarbonyl succinimide under the action of a base to obtain an intermediate compound shown in formula (3); b) the intermediate compound shown in formula (3) reacts with a benzyl halide under the action of a base to obtain an intermediate compound shown in formula (4); c) the intermediate compound shown in formula (4) reacts with di-tert-butyl dicarbonate under the action of a catalyst to obtain an intermediate compound shown in formula (6); d) the intermediate compound shown in formula (6) is deprotected to obtain Fmoc-Trp(Boc)-OH. The method provided by the present application uses tryptophan (H-Trp-OH) as the starting material, first adds Fmoc, then carries out esterification reaction with benzyl chloride, then carries out Boc reaction, and finally carries out hydrogenation reaction, and synthesizes Fmoc-Trp(Boc)-OH in 4 steps, avoiding the process of adding Fmoc to H-Trp(Boc)-OH, simplifying the process, and making the operation simple, and the reaction conditions mild, reducing the difficulty of industrial production, and not involving precious raw materials, which is conducive to controlling production costs, and the obtained Fmoc-Trp(Boc)-OH has high purity and high yield. Experimental results show that the purity of Fmoc-Trp(Boc)-OH prepared by the method provided by the present application is greater than 98.0%, and the yield is greater than 75%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 HPLC spectrum of the intermediate Fmoc-Trp-OH prepared in Example 1;

[0041] Figure 2 HPLC spectrum of the intermediate Fmoc-Trp-OBZ1 prepared in Example 1;

[0042] Figure 3 HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZ1 prepared in Example 1;

[0043] Figure 4 HPLC spectrum of Fmoc-Trp(Boc)-OH prepared in Example 1;

[0044] Figure 5 This is the infrared spectrum of Fmoc-Trp(Boc)-OH prepared in Example 1;

[0045] Figure 6 HPLC spectrum of the intermediate Fmoc-Trp-OH prepared in Example 2;

[0046] Figure 7 HPLC spectrum of the intermediate Fmoc-Trp-OBZ1 prepared in Example 2;

[0047] Figure 8HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZ1 prepared in Example 2;

[0048] Fig. 9 HPLC spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2;

[0049] Fig.10 This is the infrared spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2;

[0050] Fig.11 This is the NMR spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2. DETAILED DESCRIPTION

[0051] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0052] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0053] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0054] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0055] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0056] The present application provides a method for preparing Fmoc-Trp(Boc)-OH, comprising the following steps:

[0057] a) tryptophan reacts with fluorenylmethoxycarbonyl succinimide under the action of a base to obtain an intermediate compound represented by formula (3);

[0058]

[0059] b) The intermediate compound represented by formula (3) reacts with a benzyl halide under the action of a base to obtain an intermediate compound represented by formula (4):

[0060]

[0061] c) the intermediate compound represented by formula (4) reacts with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound represented by formula (6):

[0062]

[0063] d) Deprotecting the intermediate compound represented by formula (6) to obtain Fmoc-Trp(Boc)-OH.

[0064] The present invention uses tryptophan (H-Trp-OH) as a starting material, first adds Fmoc, then performs an esterification reaction with benzyl chloride, then performs a Boc reaction, and finally performs a hydrogenation reaction. The four-step reaction synthesizes Fmoc-Trp(Boc)-OH, avoids the process of adding Fmoc to H-Trp(Boc)-OH, simplifies the process, and has simple operation and mild reaction conditions, reduces the difficulty of industrial production, does not involve expensive raw materials, is conducive to controlling production costs, and the obtained Fmoc-Trp(Boc)-OH has high purity and high yield.

[0065] The present invention uses tryptophan (H-Trp-OH) and fluorenylmethoxycarbonyl succinimide (Fmoc-Osu) as raw materials, reacts under the action of a base to obtain an intermediate Fmoc-Trp-OH shown in formula (3), and the reaction process is as follows:

[0066]

[0067] H-Trp-OH has the structure of formula (1), and the present application has no special restrictions on its source; Fmoc-Osu has the structure of formula (2), and the present application has no special restrictions on its source.

[0068] In some specific implementations, in step a), the base is an inorganic base, including but not limited to sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, etc., and can be one or more thereof. When the base is a combination of multiple substances, the present application has no special restrictions on its type. In some specific implementations, the base is selected from sodium carbonate, sodium bicarbonate or potassium carbonate.

[0069] In some specific implementations, in step a), the medium of the reaction is water and an organic solvent, wherein water is used to dissolve tryptophan and alkali, and the organic solvent is used to dissolve fluorenylmethoxycarbonyl succinimide. The present application has no particular restrictions on the ratio of water and organic solvent, and the ratio of tryptophan and fluorenylmethoxycarbonyl succinimide is selected to provide medium conditions for the reaction. In some specific implementations, the organic solvent is selected from ethyl acetate, acetone or tetrahydrofuran.

[0070] In some specific implementations, the molar ratio of tryptophan, fluorenylmethoxycarbonyl succinimide and the base is 1:(0.8-1.3):(1-2.5), preferably 1:(0.9-1.2):(1.5-2), and more preferably 1:0.96:2.

[0071] Specifically, the present application first dissolves water, alkali and H-Trp-OH, then adds an organic solvent and Fmoc-Osu to react to obtain the intermediate Fmoc-Trp-OH shown in formula (3). In some specific implementations, Fmoc-Osu is added at 25°C to 35°C for reaction. Adding Fmoc-Osu at a temperature can make Fmoc-Osu react thoroughly and improve the reaction yield. In some specific implementations, the reaction temperature is 25°C to 35°C, preferably 28°C to 32°C, and the reaction time is 2h to 6h, preferably 3h to 5h.

[0072] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0073] Acid was added to the reaction system to adjust the pH to 2-3, and the organic layer was collected after separation;

[0074] The organic layer is washed, dried, concentrated, crystallized, centrifuged and dried to obtain the intermediate Fmoc-Trp-OH represented by formula (3).

[0075] The present application has no special restrictions on the acid, which can be hydrochloric acid. The present application has no special restrictions on the washing method, for example, saturated sodium chloride can be used for washing 3 times. The present application has no special restrictions on the drying method, for example, sodium sulfate can be used for drying. The present application has no special restrictions on the crystallization method, for example, the temperature can be lowered to 20°C to 25°C for crystallization. The present application has no special restrictions on the drying after centrifugation, for example, it can be dried at 50°C to 55°C.

[0076] After obtaining the intermediate compound represented by formula (3), it is reacted with benzyl halide under the action of a base to obtain the intermediate compound represented by formula (4). Taking benzyl bromide as an example, the reaction process is as follows:

[0077]

[0078] The benzyl halide includes but is not limited to benzyl bromide or benzyl chloride, preferably benzyl bromide. The present application has no particular limitation on its source.

[0079] In some specific implementations, in step b), the base is an inorganic base, including but not limited to sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, etc., and can be one or more thereof. When the base is a combination of multiple substances, the application has no special restrictions on its type. In some specific implementations, the base is selected from sodium carbonate or potassium carbonate.

[0080] In some specific implementations, in step b), the reaction medium is an organic solvent. In some specific implementations, the organic solvent is selected from acetonitrile or ethyl acetate.

[0081] In some specific implementations, the molar ratio of the intermediate compound represented by formula (3), the benzyl halide and the base is 1:(1-1.5):(1.1-2.0), preferably 1:1.1:1.5.

[0082] Specifically, the present application first mixes the intermediate compound represented by formula (3) with an organic solvent under stirring conditions, then adds a base and a benzyl halide, and reacts by heating under stirring conditions to obtain the intermediate Fmoc-Trp-OBZl represented by formula (4).

[0083] In some specific implementations, the temperature when adding the benzyl halide is 25°C to 30°C, the temperature of the temperature-raising reaction is 60°C to 80°C, and the time is 2h to 4h. The reaction temperature after adding the benzyl halide is controlled to be 60°C to 80°C, preferably 70°C to 80°C, which can ensure a rapid reaction without adding too many impurities. When the reaction temperature is lower than this temperature, the reaction time is long, and the raw materials cannot react completely, and the impurities increase.

[0084] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0085] After the reaction is completed, water is added to the reaction system, and the organic layer is collected after separation;

[0086] The organic layer is washed, dried, concentrated, crystallized, centrifuged and dried to obtain the intermediate compound Fmoc-Trp-OBZl represented by formula (4).

[0087] The present application has no special restrictions on the washing method, for example, saturated sodium chloride can be used for washing 3 times. The present application has no special restrictions on the drying method, for example, sodium sulfate can be used for drying. The present application has no special restrictions on the crystallization method, for example, the temperature can be lowered to 20°C to 25°C and a solvent such as petroleum ether can be added for crystallization. The present application has no special restrictions on the drying after centrifugation, for example, it can be dried at 50°C to 55°C.

[0088] After obtaining the intermediate compound Fmoc-Trp-OBZ1 shown in formula (4), it is reacted with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound shown in formula (6). The reaction process is as follows:

[0089]

[0090] Di-tert-butyl dicarbonate, with a chemical formula of (Boc)2O and a structural formula as shown in formula (5), reacts with the intermediate compound Fmoc-Trp-OBZl shown in formula (4) under the action of a catalyst to generate Fmoc-Trp(Boc)-OBZl shown in formula (6).

[0091] In some specific implementations, in step c), the catalyst is 4-dimethylaminopyridine.

[0092] In some specific implementations, in step c), the reaction medium is an organic solvent. In some specific implementations, the organic solvent includes but is not limited to ethyl acetate, dichloromethane, acetonitrile or tetrahydrofuran, and is preferably acetonitrile, ethyl acetate, dichloromethane or tetrahydrofuran.

[0093] In some specific implementations, the molar ratio of the intermediate compound represented by formula (4), the catalyst and di-tert-butyl dicarbonate is 1:(0.040-0.045):(1-2), preferably 1:0.042:1.2.

[0094] Specifically, the present application first mixes the intermediate compound represented by formula (4) with an organic solvent under stirring, then adds a catalyst, and reacts under stirring to obtain the intermediate Fmoc-Trp(Boc)-OBZl represented by formula (6).

[0095] In some specific implementations, the temperature when adding the catalyst is controlled to be 20°C to 25°C. Adding the catalyst at too high a temperature will result in an increase in impurities. In some specific implementations, the temperature after adding the catalyst is 10°C to 35°C, preferably 20°C to 25°C, and the reaction time is 0.5h to 2h.

[0096] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0097] After the reaction is completed, water is added to the reaction system, and the organic layer is collected after separation;

[0098] The organic layer was washed, dried and concentrated to obtain the intermediate compound Fmoc-Trp(Boc)-OBZ1 represented by formula (6).

[0099] The present application has no particular limitation on the washing method, for example, saturated sodium chloride can be used for washing three times. The present application has no particular limitation on the drying method, for example, sodium sulfate can be used for drying.

[0100] After obtaining the intermediate compound Fmoc-Trp(Boc)-OBZ1 shown in formula (6), it is deprotected to obtain Fmoc-Trp(Boc)-OH shown in formula (7). The reaction process is as follows:

[0101]

[0102] Specifically, the present application can use a hydrogenation method for deprotection, which specifically includes the following steps:

[0103] Under the action of a catalyst, the intermediate compound represented by formula (6) reacts with hydrogen to obtain Fmoc-Trp(Boc)-OH.

[0104] In some specific implementations, the catalyst includes but is not limited to Pd / C, Raney nickel or Pd / BaSO4, etc., preferably Pd / C. In some specific implementations, the weight ratio of the intermediate compound represented by formula (6) to the catalyst is 1:(0.01-0.1), preferably 1:0.05.

[0105] In some specific implementations, in step d), the reaction medium is an organic solvent. In some specific implementations, the organic solvent includes but is not limited to methanol, ethanol, ethyl acetate, dichloromethane, etc., preferably dichloromethane.

[0106] Specifically, the present application first mixes the intermediate compound shown in formula (6) with an organic solvent under stirring, then adds a catalyst, replaces the nitrogen, and then introduces hydrogen, and reacts under stirring to obtain Fmoc-Trp(Boc)-OH shown in formula (7). In some specific implementations, the reaction temperature is 20°C to 30°C, and the reaction time is 16h to 20h. If the reaction temperature is too low, the reaction time will be long; if the reaction temperature is too high, impurities will increase and it will be difficult to remove.

[0107] After the reaction is completed, the obtained reaction product is subjected to post-treatment, specifically, comprising the following steps:

[0108] After the reaction is completed, the reaction system is filtered, the filtrate is concentrated, and an organic solvent is added for crystallization. The solid obtained by centrifugation is washed by adding an organic solvent under stirring, centrifuged, and the solid is dried to obtain Fmoc-Trp(Boc)-OH.

[0109] In some specific implementations, the present application has no particular restrictions on the solvent used for the crystallization, which can be a mixed solution of ethyl acetate and n-hexane, cooled to 0°C to 5°C for crystallization. The present application has no particular restrictions on the solvent used for the washing, which can be n-hexane. The present application has no particular restrictions on the drying after the centrifugation, for example, it can be dried at 15°C to 20°C.

[0110] After the reaction was completed, the obtained solid was subjected to HPLC analysis, infrared analysis and nuclear magnetic resonance analysis. The results showed that it was Fmoc-Trp(Boc)-OH, the purity of which could reach more than 98%, and the yield of which could reach more than 75%.

[0111] The preparation method of Fmoc-Trp(Boc)-OH provided in the present application is further described below in conjunction with examples.

[0112] Example 1

[0113] (1) Preparation of Fmoc-Trp-OH:

[0114] 200 kg of pure water was added to a 1000 L reactor, 17 kg of sodium carbonate was added under stirring, 20 kg of tryptophan (H-Trp-OH) represented by formula (1) was added under stirring and completely dissolved, 270 kg of ethyl acetate was added, and the temperature was controlled at 25°C to 30°C. 35 kg of fluorenylmethoxycarbonyl succinimide (Fmoc-Osu) represented by formula (2) was added at once, and the temperature was controlled at 25°C to 30°C for 2 hours. The pH value was controlled at 8 to 9. The TLC raw material H-Trp-OH was completely reacted. 48 kg of hydrochloric acid was added to the system, acidified to pH = 2 to 3, separated, and the ethyl acetate layer was collected. The ethyl acetate layer was washed with saturated sodium chloride for 3 times. After washing, sodium sulfate was added, dried, filtered, and concentrated until a large amount of solid precipitated. The temperature was lowered to 20°C to 25°C for crystallization for 2 hours, centrifuged, and dried at 50°C to 55°C to obtain 40.2 kg of the intermediate Fmoc-Trp-OH solid represented by formula (3), see Figure 1 , Figure 1 This is the HPLC spectrum of the intermediate Fmoc-Trp-OH prepared in Example 1. Yield: 96.2%, HPLC purity: 99.89%.

[0115]

[0116] (2) Preparation of Fmoc-Trp-OBZ1:

[0117] 402 kg of ethyl acetate was added to a clean, anhydrous 2000 L reactor, and 40.2 kg of Fmoc-Trp-OH prepared in step (1) was added under stirring. 15 kg of sodium carbonate and 17.8 kg of benzyl bromide were added, and stirring was started. The temperature was raised to 70°C to 75°C and the reaction was continued for 4 hours. The TLC raw material Fmoc-Trp-OH was completely reacted. The reaction system was cooled to 20°C to 30°C, 500 kg of water was added to the reaction solution and stirred to dissolve, and then the mixture was allowed to stand for separation. The ethyl acetate layer was collected, and the ethyl acetate layer was washed with saturated sodium chloride for 3 times. After washing, sodium sulfate was added, dried, filtered, and concentrated until a large amount of solid precipitated. The temperature was lowered to 20°C to 25°C, 100 kg of petroleum ether was added, and crystallized for 2 hours. The mixture was centrifuged and dried at 50°C to 55°C to obtain 44.8 kg of the intermediate Fmoc-Trp-OBZl solid represented by formula (4). Figure 2 , Figure 2 This is the HPLC spectrum of the intermediate Fmoc-Trp-OBZ1 prepared in Example 1. Yield: 92%, HPLC purity: 99.74%;

[0118]

[0119] (3) Preparation of Fmoc-Trp(Boc)-OBZ1:

[0120] 448 kg of ethyl acetate was added to a clean, anhydrous 2000 L reactor, and 44.8 kg of Fmoc-Trp-OBZl prepared in step (2) was added under stirring, and the solution was stirred until the solution was clear. 22.7 kg of di-tert-butyl dicarbonate ((Boc)2O) represented by formula (5) was added, and the reaction temperature was controlled to 20°C to 25°C. 0.45 kg of 4-dimethylaminopyridine catalyst was added, and the temperature was controlled to 20°C to 25°C for 2 hours. According to TLC, the raw material Fmoc-Trp-OBZl was completely reacted. 500 kg of water was added to the system, and the liquid was separated. The ethyl acetate layer was collected, and the ethyl acetate layer was washed with saturated sodium chloride three times. After washing, sodium sulfate was added, dried, filtered, and concentrated to dryness to obtain 50 kg of the intermediate Fmoc-Trp(Boc)-OBZl represented by formula (6). Figure 3 , Figure 3 HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZ1 prepared in Example 1. Yield: 93.5%, HPLC purity 99.78%;

[0121]

[0122] (4) Preparation of Fmoc-Trp(Boc)-OH:

[0123] Add 500 kg of dichloromethane and 50 kg of Fmoc-Trp(Boc)-OBZl to a 1000 L reactor, stir to dissolve, add 2 kg of Pd / C, replace with nitrogen, introduce hydrogen, control the temperature at 25°C to 30°C for 15 h, and the raw material Fmoc-Trp(Boc)-OBZl is completely reacted by TLC. After filtering the Pd / C, concentrate the filtrate to dryness, add 80 kg of ethyl acetate and 200 kg of n-hexane, cool to 0°C to 5°C for crystallization for 8 h, centrifuge, add 70 kg of n-hexane to the centrifuged solid, stir and wash for 2 h, centrifuge the centrifuged solid and dry it at 15°C to 20°C to obtain 36 kg of Fmoc-Trp(Boc)-OH solid represented by formula (7), see Figure 4 and Figure 5 , Figure 4 HPLC spectrum of Fmoc-Trp(Boc)-OH prepared in Example 1, Figure 5 This is the infrared spectrum of Fmoc-Trp(Boc)-OH prepared in Example 1. Yield: 84.3%, HPLC purity: 99.74%.

[0124]

[0125] Example 2

[0126] (1) Preparation of Fmoc-Trp-OH:

[0127] Add 600 kg of pure water to a 2000 L reactor, add 50 kg of sodium carbonate under stirring, add 60 kg of H-Trp-OH under stirring, stir to completely dissolve, add 800 kg of ethyl acetate, control the temperature at 25°C to 30°C, add 104 kg of Fmoc-Osu at one time, control the temperature at 25 to 30 degrees to react for 3 hours, control the pH at 8 to 9, and the TLC raw material H-Trp-OH is completely reacted. Add 150 kg of hydrochloric acid to the system, acidify to pH = 2 to 3, separate the liquids, collect the ethyl acetate layer, wash the ethyl acetate layer with saturated sodium chloride three times, add sodium sulfate after washing, dry, filter and concentrate until a large amount of solid precipitates, cool to 20°C to 25°C for crystallization for 2 hours, centrifuge, and dry at 50-55°C to obtain 120 kg of intermediate Fmoc-Trp-OH solid, see Figure 6 , Figure 6 HPLC spectrum of the intermediate Fmoc-Trp-OH prepared in Example 2, yield: 95.7%, HPLC purity: 99.82%

[0128] (2) Preparation of Fmoc-Trp-OBZ1:

[0129] 1200 kg of acetonitrile was added to a clean, anhydrous 3000 L reactor, and 120 kg of Fmoc-Trp-OH prepared in step (1) was added under stirring, 45 kg of sodium carbonate was added, and 39 kg of benzyl chloride was added. The stirring was started, and the temperature was raised to 70°C to 75°C for 4 hours. The TLC raw material Fmoc-Trp-OH was completely reacted; the reaction system was cooled to 20°C to 30°C, 500 kg of water was added to the reaction solution, and 1000 kg of ethyl acetate was added and stirred to dissolve, and then the mixture was allowed to stand for separation, and the ethyl acetate layer was collected. The ethyl acetate layer was washed with saturated sodium chloride for 3 times. After washing, sodium sulfate was added, dried, filtered, and concentrated until a large amount of solid precipitated. The temperature was lowered to 20-25°C, 300 kg of petroleum ether was added, and crystallization was performed for 2 hours. The reaction was centrifuged and dried at 50°C to 55°C to obtain 133 kg of the intermediate Fmoc-Trp-OBZl solid, see Figure 7 , Figure 7 HPLC spectrum of the intermediate Fmoc-Trp-OBZ1 prepared in Example 2, yield: 91.5%, HPLC purity: 99.75%

[0130] (3) Preparation of Fmoc-Trp(Boc)-OBZ1:

[0131] 1330 kg of tetrahydrofuran was added to a clean, anhydrous 3000 L reactor, and 133 kg of Fmoc-Trp-OBZl prepared in step (2) was added under stirring. After stirring, 53 kg of (Boc)2O was added, and the temperature was controlled to 20°C to 25°C. 1.33 kg of 4-dimethylaminopyridine was added, and the temperature was controlled to 20°C to 25°C for reaction for 3 h. The raw material Fmoc-Trp-OBZl was completely reacted by TLC. 500 kg of water was added to the system, and 500 kg of ethyl acetate was added to extract and separate the liquids. The ethyl acetate layer was collected, and the ethyl acetate layer was washed with saturated sodium chloride three times. After washing, sodium sulfate was added to dry the layer, and then filtered and concentrated to dryness to obtain 155.6 kg of the intermediate Fmoc-Trp(Boc)-OBZl oily product. Figure 8 , Figure 8 HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZ1 prepared in Example 2, yield: 98%, HPLC purity 99.71%.

[0132] (4) Preparation of Fmoc-Trp(Boc)-OH:

[0133] Add 1556 kg of ethyl acetate and 155.6 kg of Fmoc-Trp(Boc)-OBZl to a 3000 L reactor, stir to dissolve, add 7.78 kg of Pd / C, replace with nitrogen, introduce hydrogen, control the temperature at 30°C to 35°C for 18 hours, TLC shows that the raw material Fmoc-Trp(Boc)-OBZl is completely reacted, filter the Pd / C, concentrate the filtrate to about 300 L, add 600 kg of n-hexane, cool to 0°C to 5°C, crystallize for 8 hours, centrifuge, add 200 kg of n-hexane to the centrifuged solid, stir and wash for 2 hours, centrifuge the centrifuged solid and dry it at 15°C to 20°C to obtain 118 kg of Fmoc-Trp(Boc)-OH solid, see Fig. 9 , Fig.10 and Fig.11 , Fig. 9 HPLC spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2, Fig.10 This is the infrared spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2, Fig.11 This is the NMR spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2. Yield: 88.8%, HPLC purity: 99.66%.

[0134] Embodiment 3-4

[0135] This example investigates the effect of the inorganic base in step (1) on the intermediate Fmoc-Trp-OH. Specifically, referring to the preparation method provided in step (1) of Example 1, the type of inorganic base is changed, and the other preparation steps and process parameters are the same as those in Example 1. The results of step (1) are shown in Table 1.

[0136] Table 1

[0137]

[0138]

[0139] Example 5, Comparative Examples 1 to 4

[0140] This comparative example investigates the effect of the inorganic base in step (2) on the intermediate Fmoc-Trp-OBZl. Specifically, the preparation method provided in step (2) of reference example 1 is used, only the type of inorganic base is changed, and the other preparation steps and process parameters are the same as those in example 1. After testing, the purity and yield of Fmoc-Trp-OBZl are shown in Table 2 below.

[0141] Table 2

[0142] Inorganic base Reaction time Yield purity Example 1 Sodium carbonate 4h 92％ 99.74％ Example 5 Potassium carbonate 4h 93.7％ 98.68％ Comparative Example 1 Sodium bicarbonate 28h 88.4％ 96.27％ Comparative Example 2 Potassium hydroxide 3h 93.2％ 93.14％ Comparative Example 3 Potassium bicarbonate 20h 89.5％ 95.53％ Comparative Example 4 Sodium hydroxide 3h 92.8％ 94.11％

[0143] Examples 6-7, Comparative Examples 5-6

[0144] This example and comparative example investigated the effect of the reaction temperature in step (2) on the intermediate Fmoc-Trp-OBZ1. Specifically, the preparation method provided in step (2) of reference example 1 was used, and only the type of reaction temperature was changed. The other preparation steps and process parameters were the same as those in example 1. After testing, the yield and reaction time of Fmoc-Trp-OBZ1 are shown in Table 3 below.

[0145] Table 3

[0146] Reaction temperature Reaction time Yield purity Example 6 76℃～80℃ 4h 90％ 99.35％ Example 1 70℃～75℃ 4h 92％ 99.74％ Example 7 65℃～69℃ 8h 85％ 98.45％ Comparative Example 5 50℃～55℃ 14h 63％ 96.32％ Comparative Example 6 30℃～35℃ No complete response within 48 hours 36％ 88.5％

[0147] Example 8, Comparative Example 7

[0148] This example and comparative example investigated the effect of the organic solvent in step (2) on the intermediate Fmoc-Trp-OBZ1. Specifically, the preparation method provided in step (2) of reference example 1 was used, only the type of organic solvent was changed, and the other preparation steps and process parameters were the same as those in example 1. After testing, the yield and purity of Fmoc-Trp-OBZ1 are shown in Table 4 below.

[0149] Table 4

[0150] Organic solvents Yield purity Example 1 Ethyl acetate 92％ 99.74％ Example 8 Acetonitrile 91％ 99.68％ Comparative Example 7 Tetrahydrofuran 63％ 96.43％

[0151] Embodiments 9-10

[0152] This example and comparative example investigated the effect of the organic solvent in step (3) on the intermediate Fmoc-Trp(Boc)-OBZl. Specifically, the preparation method provided in step (3) of reference example 1 was used, only the type of organic solvent was changed, and the other preparation steps and process parameters were the same as those in example 1. After testing, the yield and purity of Fmoc-Trp(Boc)-OBZl are shown in Table 5 below.

[0153] Table 5

[0154] Organic solvents Yield purity Example 1 Ethyl acetate 93.5％ 99.78％ Example 9 Acetonitrile 91％ 99.36％ Example 2 Tetrahydrofuran 98％ 99.71％ Example 10 Dichloromethane 94.5％ 99.45％

[0155] Examples 11 to 14, Comparative Example 8

[0156] This example investigates the effect of the reaction temperature in step (4) on the intermediate Fmoc-Trp(Boc)-OH. Specifically, the preparation method provided in step (4) of reference example 1 was used, and only the reaction temperature was changed. The other preparation steps and process parameters were the same as those in example 1. After testing, the purity and reaction time of Fmoc-Trp(Boc)-OH are shown in Table 6 below.

[0157] Table 6

[0158]

[0159]

[0160] In summary, the present invention provides a method for preparing Fmoc-Trp(Boc)-OH, which is suitable for scale-up, requires simple equipment, has good reaction efficiency, low production cost, and the purity of the final product is greater than 98.0%, and the yield is greater than 75%.

[0161] The above is only 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 principle 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 Fmoc-Trp(Boc)-OH, comprising the following steps: a) tryptophan reacts with fluorenylmethoxycarbonyl succinimide under the action of a base to obtain an intermediate compound represented by formula (3); b) The intermediate compound represented by formula (3) reacts with a benzyl halide under the action of a base to obtain an intermediate compound represented by formula (4): c) the intermediate compound represented by formula (4) reacts with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound represented by formula (6): d) Deprotecting the intermediate compound represented by formula (6) to obtain Fmoc-Trp(Boc)-OH.

2. The preparation method according to claim 1, characterized in that: In the step a), the base is an inorganic base; The molar ratio of tryptophan, fluorenylmethoxycarbonyl succinimide and the base is 1:(0.8-1.3):(1-2.5).

3. The preparation method according to claim 2, characterized in that: In the step a), the reaction temperature is 25°C to 35°C and the reaction time is 2h to 6h; The reaction medium is water and an organic solvent, and the organic solvent is selected from ethyl acetate, acetone or tetrahydrofuran.

4. The preparation method according to claim 1, characterized in that: In the step b), the base is an inorganic base; The molar ratio of the intermediate compound represented by formula (3), benzyl halide and base is 1:(1 to 1.5):(1.1 to 2.0).

5. The preparation method according to claim 4, characterized in that: In the step b), the base is selected from sodium carbonate or potassium carbonate.

6. The preparation method according to claim 4, characterized in that: In the step b), the reaction temperature is 60°C to 80°C and the reaction time is 2h to 4h; The medium of the reaction is an organic solvent, and the organic solvent is selected from acetonitrile or ethyl acetate.

7. The preparation method according to claim 1, characterized in that: In the step c), the catalyst is 4-dimethylaminopyridine; The molar ratio of the intermediate compound represented by formula (4), the catalyst and di-tert-butyl dicarbonate is 1:(0.040-0.045):(1-2).

8. The preparation method according to claim 7, characterized in that: In the step c), the reaction temperature is 10°C to 35°C and the reaction time is 0.5h to 2h; The reaction medium is an organic solvent, and the organic solvent is selected from dichloromethane, acetonitrile, ethyl acetate or tetrahydrofuran.

9. The preparation method according to claim 1, characterized in that: The step d) specifically comprises: Under the action of a catalyst, the intermediate compound represented by formula (6) reacts with hydrogen to obtain Fmoc-Trp(Boc)-OH.

10. The preparation method according to claim 9, characterized in that: In step d), the catalyst is selected from Pd / C, Raney nickel or Pd / BaSO4; The weight ratio of the intermediate compound represented by formula (6) to the catalyst is 1:(0.01-0.1); The reaction temperature is 20°C to 30°C, and the reaction time is 16h to 20h.

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