Method for producing Fmoc-Trp(Boc)-OH

Abstract

To provide a method for producing Fmoc-Trp(Boc)-OH that simplifies the steps, uses mild reaction conditions, and yields high purity and high yield. [Solution] The method comprises the steps of: reacting tryptophan with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of a base to obtain an intermediate of formula (3); reacting with benzyl halide under the action of a base to obtain an intermediate of formula (4); reacting with di-tert-butyl dicarbonate under the action of a catalyst to obtain an intermediate; and deprotecting to obtain Fmoc-Trp(Boc)-OH. JPEG2026079752000029.jpg64168

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and specifically relates to a method for producing Fmoc-Trp(Boc)-OH.

Background Art

[0002] Fmoc-Trp(Boc)-OH is an intermediate commonly used in the synthesis of polypeptides such as Semaglutide and Tirzepatide. Fmoc-Trp(Boc)-OH has the chemical name: N-α-fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan, molecular formula: C ,

[0005] H 30 N2O6, CAS number: 143824-78-6, and its structural formula is as follows.

Chemical

[0003] In all the synthetic routes of Fmoc-Trp(Boc)-OH disclosed in the prior art, after obtaining the intermediate H-Trp(Boc)-OH, Fmoc is linked to obtain the final product Fmoc-Trp(Boc)-OH. However, H-Trp(Boc)-OH is unstable during the Fmoc linking process, and the impurity Fmoc-Trp-OH is generated. Since it is almost impossible to remove Fmoc-Trp-OH from Fmoc-Trp(Boc)-OH, there is a problem that it affects the yield and purity of Fmoc-Trp(Boc)-OH.

[0004] For example, CN202110672717 discloses two routes for synthesizing Fmoc-Trp(Boc)-OH. The first route is as follows.

Chemical

[0005] The second route is as follows: [ka]

[0006] CN202211006819 discloses the following synthesis routes. [ka]

[0007] The above synthesis route has problems not only with reduced yield and purity, but also with the complexity of the procedure. [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, the present invention provides a method for producing Fmoc-Trp(Boc)-OH that simplifies the steps, uses mild reaction conditions, and yields high purity and high yield.

[0009] According to this application, a method for producing Fmoc-Trp(Boc)-OH is provided, comprising the following steps a) to d): [ka] Step a) Reacting tryptophan with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of a base to obtain the intermediate compound shown in formula (3) above, [ka] Step b) involves reacting the intermediate compound shown in formula (3) above with benzyl halide under the action of a base to obtain the intermediate compound shown in formula (4) above. [ka] Step c) of reacting the intermediate compound shown in the above formula (4) with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound shown in the above formula (6); Step d) of deprotecting the intermediate compound shown in the above formula (6) to obtain Fmoc-Trp(Boc)-OH.

[0010] In some specific embodiments, in step a), the base is an inorganic base, and the molar ratio of tryptophan, N-(9-fluorenylmethoxycarbonyloxy)succinimide, and the base is 1:(0.8 - 1.3):(1 - 2.5).

[0011] In some specific embodiments, in step a), the reaction is carried out at a temperature of 25°C to 35°C for 2 to 6 hours, and the reaction medium is an organic solvent selected from the group consisting of ethyl acetate, acetone, and tetrahydrofuran and water.

[0012] In some specific embodiments, in step b), the base is an inorganic base, and the molar ratio of the intermediate compound shown in formula (3), benzyl halide, and the base is 1:(1 - 1.5):(1.1 - 2.0).

[0013] In some specific embodiments, in step b), the base is sodium carbonate or potassium carbonate.

[0014] In some specific embodiments, in step b), the reaction is carried out at a temperature of 60°C to 80°C for 2 to 4 hours, and the reaction medium is an organic solvent such as acetonitrile or ethyl acetate.

[0015] In some specific embodiments, in step c), the catalyst is 4-dimethylaminopyridine, and the molar ratio of the intermediate compound shown in formula (4), the catalyst, and di-tert-butyl dicarbonate is 1:(0.040 - 0.045):(1 - 2).

[0016] In some specific embodiments, in step c), the reaction is carried out at a temperature of 10°C to 35°C for 0.5 to 2 hours, and the reaction medium is an organic solvent selected from the group consisting of dichloromethane, acetonitrile, ethyl acetate, and tetrahydrofuran.

[0017] In some specific embodiments, step d) includes reacting the intermediate compound shown in formula (6) with hydrogen gas under the action of a catalyst to obtain Fmoc-Trp(Boc)-OH.

[0018] In some specific embodiments, in step d), the catalyst is selected from the group consisting of Pd / C, Raney nickel, and Pd / BaSO4, the weight ratio of the intermediate compound shown in formula (6) to the catalyst is 1:(0.01 - 0.1), and the reaction is carried out at a temperature of 20°C to 30°C for 16 to 20 hours.

[0019] The present invention provides a method for producing Fmoc-Trp(Boc)-OH, comprising the steps of: a) reacting tryptophan with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of a base to obtain the intermediate compound shown in formula (3); b) reacting the intermediate compound shown in formula (3) with benzyl halide under the action of a base to obtain the intermediate compound shown in formula (4); c) reacting the intermediate compound shown in formula (4) with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound shown in formula (6); and d) deprotecting the intermediate compound shown in formula (6) to obtain Fmoc-Trp(Boc)-OH. In the present invention, Fmoc-Trp(Boc)-OH is synthesized by a four-step reaction starting with tryptophan (H-Trp-OH), first linking it with Fmoc, then esterifying it with benzyl chloride, then linking it with Boc, and finally undergoing a hydrogenation reaction. According to the method of this application, the process of linking Fmoc to H-Trp(Boc)-OH is avoided, the process is simplified, the operation is easy, the reaction conditions are mild, the difficulty of industrial production is reduced, valuable raw materials are not required, production costs are reduced, and Fmoc-Trp(Boc)-OH can be obtained in high purity and high yield. Experimental results have shown that Fmoc-Trp(Boc)-OH produced by the method of this application has a purity of 98.0% or higher and a yield of 75% or higher. [Brief explanation of the drawing]

[0020] [Figure 1] This is the HPLC chromatogram of the intermediate Fmoc-Trp-OH prepared in Example 1. [Figure 2] This is the HPLC chromatogram of the intermediate Fmoc-Trp-OBZl prepared in Example 1. [Figure 3] This is the HPLC chromatogram of the intermediate Fmoc-Trp(Boc)-OBZl prepared in Example 1. [Figure 4] This is the HPLC chromatogram of Fmoc-Trp(Boc)-OH prepared in Example 1. [Figure 5]This is the infrared spectrum of Fmoc-Trp(Boc)-OH produced in Example 1. [Figure 6] This is the HPLC chromatogram of the intermediate Fmoc-Trp-OH prepared in Example 2. [Figure 7] This is the HPLC chromatogram of the intermediate Fmoc-Trp-OBZl prepared in Example 2. [Figure 8] This is the HPLC chromatogram of the intermediate Fmoc-Trp(Boc)-OBZl prepared in Example 2. [Figure 9] This is the HPLC chromatogram of Fmoc-Trp(Boc)-OH prepared in Example 2. [Figure 10] This is the infrared spectrum of Fmoc-Trp(Boc)-OH produced in Example 2. [Figure 11] This is the nuclear magnetic resonance spectrum of Fmoc-Trp(Boc)-OH prepared in Example 2. [Modes for carrying out the invention]

[0021] Here, the expression "one or more of the following" should be understood to include, unless otherwise specified in the context and usage, the objects listed individually after the expression alone, as well as various combinations of two or more of the listed objects. The expression "and / or" should be understood to have the same meaning when combined with three or more of the listed objects, unless otherwise specified in the context.

[0022] The use of the terms “include,” “possess,” and “contain” (including their grammatical synonyms) should generally be interpreted as open and non-restrictive, i.e., not excluding other elements or steps not listed, unless otherwise specified in the context.

[0023] It should be understood here that, as long as the invention is implementable, the order of the steps or the order in which specific actions are performed is not important. Also, two or more steps or actions may be performed simultaneously.

[0024] Any use of examples or exemplary expressions such as “for example,” “including,” or “including” in this specification is intended solely to better illustrate the invention and, unless otherwise specified, does not limit the scope of the invention. No expression in this specification should be construed as indicating that any element not for which protection is claimed is essential for the practice of the invention.

[0025] Furthermore, all numerical ranges and parameters used to define the present invention are approximate values, and although relevant values ​​in specific examples are shown as accurately as possible, it is unavoidable that any numerical values ​​inherently contain variability due to individual test methods. Therefore, unless otherwise specified, all ranges, quantities, numerical values ​​and percentages used in this disclosure should be understood to be "approximate." As used herein, "approximate" usually means that the actual numerical value is within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range.

[0026] According to this application, a method for producing Fmoc-Trp(Boc)-OH is provided, comprising the following steps a) to d): [ka] Step a) Reacting tryptophan with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of a base to obtain the intermediate compound shown in formula (3) above, [ka] Step b) involves reacting the intermediate compound shown in formula (3) above with benzyl halide under the action of a base to obtain the intermediate compound shown in formula (4) above. [ka] Step c), reacting the intermediate compound shown in formula (4) above with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound shown in formula (6) above, Step d) Deprotect the intermediate compound shown in formula (6) above to obtain Fmoc-Trp(Boc)-OH.

[0027] In this application, tryptophan (H-Trp-OH) is used as a starting material, first linked with Fmoc, then esterified with benzyl chloride, then a Boc reaction, and finally a hydrogenation reaction. Fmoc-Trp(Boc)-OH is synthesized through four reaction steps, and the step of linking Fmoc to H-Trp(Boc)-OH is avoided, resulting in a simplified process, easy operation, mild reaction conditions, reduced difficulty in industrial production, elimination of valuable raw materials, reduced production costs, and the advantage of obtaining Fmoc-Trp(Boc)-OH in high purity and high yield.

[0028] In this application, as shown in the reaction process below, tryptophan (H-Trp-OH) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-Osu) are used as starting materials, and the reaction is carried out under the action of a base to obtain the intermediate Fmoc-Trp-OH shown in formula (3). [ka]

[0029] H-Trp-OH has the structure represented by formula (1), and the source of its supply is not particularly limited in this application. Fmoc-Osu has the structure represented by formula (2), and the source of its supply is not particularly limited in this application.

[0030] In some specific embodiments, the base in step a) is an inorganic base, and is not limited to, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, and may be one or more of these. If the base is a combination of multiple substances, this application does not particularly limit the types thereof. In some specific embodiments, the base is selected from the group consisting of sodium carbonate, sodium bicarbonate, and potassium carbonate.

[0031] In some specific embodiments, the reaction medium in step a) is water and an organic solvent, where water is used to dissolve tryptophan and the base, and the organic solvent is used to dissolve N-(9-fluorenylmethoxycarbonyloxy)succinimide. In this application, the ratio of water to organic solvent is not particularly limited and may be selected according to the ratio of tryptophan to N-(9-fluorenylmethoxycarbonyloxy)succinimide, as long as it provides the necessary media conditions for the reaction. In some specific embodiments, the organic solvent is selected from the group consisting of ethyl acetate, acetone, and tetrahydrofuran.

[0032] In some specific embodiments, the molar ratio of tryptophan to N-(9-fluorenylmethoxycarbonyloxy)succinimide to 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.

[0033] Specifically, in this application, water, a base, and H-Trp-OH are first dissolved, and then an organic solvent and Fmoc-Osu are added and reacted to obtain the intermediate Fmoc-Trp-OH shown in formula (3). In some specific embodiments, Fmoc-Osu is added and reacted at a temperature of 25°C to 35°C. Adding Fmoc-Osu at this temperature ensures the complete reaction of Fmoc-Osu and improves the reaction yield. In some specific embodiments, the reaction temperature is 25°C to 35°C, preferably 28°C to 32°C, and the reaction time is 2 to 6 hours, preferably 3 to 5 hours.

[0034] After the reaction is complete, the reaction product is post-processed. Specifically, this includes the steps of adding acid to the reaction system to adjust the pH to 2-3, recovering the organic layer after liquid-liquid separation, and washing, drying, concentrating, crystallizing, centrifuging, and drying the organic layer to obtain the intermediate Fmoc-Trp-OH shown in formula (3).

[0035] In this application, the acid is not particularly limited and may be hydrochloric acid. In this application, the washing method is not particularly limited and may be washed three times using saturated sodium chloride, for example. In this application, the drying method is not particularly limited and may be dried using sodium sulfate, for example. In this application, the crystallization method is not particularly limited and may be performed by cooling to 20°C to 25°C, for example. In this application, the drying after centrifugation is not particularly limited and may be performed at 50°C to 55°C, for example.

[0036] After obtaining the intermediate compound shown in formula (3), it is reacted with benzyl halide under the action of a base to obtain the intermediate compound shown in formula (4). Taking benzyl bromide as an example, the reaction process is as follows. [ka]

[0037] Here, examples of benzyl halides include benzyl bromide and benzyl chloride, but are not limited to these, with benzyl bromide being preferred. This application does not particularly limit the source of the benzyl halide.

[0038] In some specific embodiments, the base in step b) is an inorganic base, and may include, but is not limited to, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, and may be one or more of these. If the base is a combination of multiple substances, the type is not particularly limited in this application. In some specific embodiments, the base is sodium carbonate or potassium carbonate.

[0039] In some specific embodiments, the reaction medium in step b) is an organic solvent. In some specific embodiments, the organic solvent is acetonitrile or ethyl acetate.

[0040] In some specific embodiments, the molar ratio of the intermediate compound shown in formula (3), benzyl halide, and base is 1:(1~1.5):(1.1~2.0), preferably 1:1.1:1.5.

[0041] Specifically, in this application, the intermediate compound shown in formula (3) and an organic solvent are first mixed under stirring, then a base and benzyl halide are added, and the reaction is carried out by raising the temperature under stirring to obtain the intermediate Fmoc-Trp-OBZl shown in formula (4).

[0042] In some specific embodiments, the temperature at which benzyl halide is added is 25°C to 30°C, and the heating reaction is carried out at a temperature of 60°C to 80°C for 2 to 4 hours. By controlling the reaction temperature after the addition of benzyl halide to 60 to 80°C, preferably 70 to 80°C, a rapid reaction can be ensured without increasing the amount of impurities. If the reaction temperature is lower than this, the reaction time will be longer, the raw materials may not react completely, and the amount of impurities may increase.

[0043] After the reaction is complete, the reaction product is post-processed. Specifically, this process includes adding water to the reaction system after the reaction is complete, recovering the organic layer after liquid-liquid separation, and washing, drying, concentrating, crystallizing, centrifuging, and drying the organic layer to obtain the intermediate compound Fmoc-Trp-OBZl shown in formula (4).

[0044] In this application, the washing method is not particularly limited, and for example, it can be washed three times using saturated sodium chloride. In this application, the drying method is not particularly limited, and for example, it can be dried using sodium sulfate. In this application, the crystallization method is not particularly limited, and for example, it can be carried out by cooling to 20°C to 25°C and adding a solvent such as petroleum ether to crystallize. In this application, the drying method after centrifugation is not particularly limited, and for example, drying can be carried out at 50°C to 55°C.

[0045] After obtaining the intermediate compound Fmoc-Trp-OBZl shown in formula (4), it is reacted with di-tert-butyl dicarbonate under the action of a catalyst, as shown in the following reaction process, to obtain the intermediate compound shown in formula (6). [ka]

[0046] Di-tert-butyl dicarbonate, having the chemical formula (Boc)2O and the structural formula shown in formula (5), reacts with the intermediate compound Fmoc-Trp-OBZl shown in formula (4) under the action of a catalyst to produce Fmoc-Trp(Boc)-OBZl shown in formula (6).

[0047] In some specific embodiments, the catalyst in step c) is 4-dimethylaminopyridine.

[0048] In some specific embodiments, the reaction medium in step c) is an organic solvent. In some specific embodiments, the organic solvent is, but is not limited to, ethyl acetate, dichloromethane, acetonitrile, tetrahydrofuran, and is preferably acetonitrile, ethyl acetate, dichloromethane, or tetrahydrofuran.

[0049] In some specific embodiments, the molar ratio of the intermediate compound shown in formula (4), the catalyst, and di-tert-butyl dicarbonate is 1:(0.040~0.045):(1~2), preferably 1:0.042:1.2.

[0050] Specifically, in this application, the intermediate compound shown in formula (4) and an organic solvent are first mixed under stirring, then a catalyst is added, and the reaction is carried out under stirring to obtain the intermediate Fmoc-Trp(Boc)-OBZl shown in formula (6).

[0051] In some specific embodiments, the catalyst is added at a temperature of 20°C to 25°C. If the temperature at which the catalyst is added is too high, the amount of impurities may increase. In some specific embodiments, the reaction temperature after catalyst addition is set to 10°C to 35°C, preferably 20°C to 25°C, and the reaction time is set to 0.5 to 2 hours.

[0052] After the reaction is complete, the reaction product is post-processed. Specifically, this process includes adding water to the reaction system after the reaction is complete, recovering the organic layer after liquid-liquid separation, and washing, drying, and concentrating the organic layer to obtain the intermediate compound Fmoc-Trp(Boc)-OBZl shown in formula (6).

[0053] In this application, the washing method is not particularly limited, and for example, it may be washed three times using saturated sodium chloride. In this application, the drying method is not particularly limited, and for example, it may be dried using sodium sulfate.

[0054] After obtaining the intermediate compound Fmoc-Trp(Boc)-OBZl shown in formula (6), deprotection is performed as shown in the following reaction process to obtain Fmoc-Trp(Boc)-OH shown in formula (7). [ka]

[0055] Specifically, the deprotection in this application is carried out by a hydrogenation method, which specifically includes the step of reacting the intermediate compound shown in formula (6) with hydrogen gas under the action of a catalyst to obtain Fmoc-Trp(Boc)-OH.

[0056] In some specific embodiments, the catalyst is not limited to the following, but examples include Pd / C, Raney nickel, and Pd / BaSO4, with Pd / C being preferred. In some specific embodiments, the weight ratio of the intermediate compound shown in formula (6) to the catalyst is 1:(0.01~0.1), preferably 1:0.05.

[0057] In some specific embodiments, the reaction medium in step d) is an organic solvent. In some specific embodiments, the organic solvent is, but is not limited to, methanol, ethanol, ethyl acetate, dichloromethane, and is preferably dichloromethane.

[0058] Specifically, in this application, first, the intermediate compound shown in formula (6) and an organic solvent are mixed under stirring, then a catalyst is added, nitrogen gas is purged, and hydrogen gas is passed through, and the reaction is carried out under stirring to obtain Fmoc-Trp(Boc)-OH shown in formula (7). In a specific embodiment, the reaction is carried out at a temperature of 20°C to 30°C for 16 to 20 hours. If the reaction temperature is too low, the reaction time may be prolonged. If the reaction temperature is too high, impurities will increase and become difficult to remove.

[0059] After the reaction is complete, the reaction product is post-processed. Specifically, this process includes filtering the reaction system after the reaction is complete, concentrating the resulting filtrate, adding an organic solvent to crystallize it, and adding an organic solvent to the solid obtained by centrifugation, washing it under stirring, centrifuging it, and drying the resulting solid to obtain Fmoc-Trp(Boc)-OH.

[0060] In some specific embodiments, the solvent used for crystallization in this application is not particularly limited, and a mixed solution of ethyl acetate and n-hexane can be used, and crystallization can be carried out by cooling to 0°C to 5°C. In this application, the solvent used for washing is not particularly limited, and may be n-hexane. In this application, the drying after centrifugation is not particularly limited, and can be dried at a temperature of, for example, 15°C to 20°C.

[0061] After the reaction was complete, the resulting solid was subjected to HPLC analysis, infrared analysis, and nuclear magnetic resonance analysis. The results revealed that the obtained solid was Fmoc-Trp(Boc)-OH, with a purity of over 98% and a yield of over 75%.

[0062] The method for producing Fmoc-Trp(Boc)-OH according to this application will be further explained below with reference to examples.

[0063] [Example 1] (1) Preparation of Fmoc-Trp-OH: 200 kg of pure water was placed in a 1000 L reaction vessel, 17 kg of sodium carbonate was added under stirring, and 20 kg of tryptophan (H-Trp-OH) shown in formula (1) was added under stirring. After stirring until completely dissolved, 270 kg of ethyl acetate was added, and while controlling the temperature to 25-30°C, 35 kg of N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-Osu) shown in formula (2) was added all at once. The reaction was carried out for 2 hours while controlling the temperature to 25-30°C, and the pH value was controlled to 8-9. Complete reaction of the starting material H-Trp-OH was confirmed by TLC. 48 kg of hydrochloric acid was added to the system to acidify the pH to 2-3, and the mixture was separated. The ethyl acetate layer was recovered, washed three times with saturated sodium chloride, then sodium sulfate was added and dried, filtered, and the filtrate was concentrated until a large amount of solid matter precipitated. The mixture was cooled to 20-25°C, crystallized for 2 hours, then centrifuged and dried at 50-55°C to obtain 40.2 kg of the intermediate Fmoc-Trp-OH shown in formula (3) as a solid. Referring to Figure 1, Figure 1 shows the HPLC spectrum of the intermediate Fmoc-Trp-OH produced in Example 1, with a yield of 96.2% and an HPLC purity of 99.89%. [ka]

[0064] (2) Manufacturing of Fmoc-Trp-OBZl: 402 kg of ethyl acetate was placed in a clean, anhydrous 2000 L reaction vessel, and 40.2 kg of Fmoc-Trp-OH produced in step (1) was added under stirring. Then, 15 kg of sodium carbonate and 17.8 kg of benzyl bromide were added, and stirring was started. The internal temperature was raised to 70°C to 75°C, and the reaction was allowed to proceed for 4 hours. After confirming that the Fmoc-Trp-OH starting material had completely reacted by TLC, the reaction system was cooled to 20°C to 30°C, 500 kg of water was added to the reaction solution and stirred until clarified, then allowed to stand and separated. Next, the ethyl acetate layer was recovered, washed three times with saturated sodium chloride, then sodium sulfate was added and dried. The mixture was then concentrated until a large amount of solid precipitated, cooled to 20°C-25°C, 100 kg of petroleum ether was added and crystallized for 2 hours, followed by centrifugation and drying at 50°C-55°C to obtain 44.8 kg of the intermediate Fmoc-Trp-OBZl shown in formula (4) as a solid. Referring to Figure 2, Figure 2 shows the HPLC spectrum of the intermediate Fmoc-Trp-OBZl produced in Example 1, with a yield of 92% and an HPLC purity of 99.74%. [ka]

[0065] (3) Manufacturing of Fmoc-Trp(Boc)-OBZl: 448 kg of ethyl acetate was placed in a clean, anhydrous 2000 L reaction vessel, and 44.8 kg of Fmoc-Trp-OBZl prepared in step (2) was added under stirring. The mixture was stirred until the solution became clear, and 22.7 kg of di-tert-butyl((Boc)2O) dicarbonate shown in formula (5) was added. The reaction temperature was controlled to 20°C to 25°C, 0.45 kg of 4-dimethylaminopyridine catalyst was added, and the reaction was carried out for 2 hours while controlling the temperature at 20°C to 25°C. TLC confirmed that the starting material Fmoc-Trp-OBZl had completely reacted. 500 kg of water was added to the system, liquid-liquidate was separated, the ethyl acetate layer was recovered, washed three times with saturated sodium chloride, then dried with sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain 50 kg of the intermediate Fmoc-Trp(Boc)-OBZl shown in formula (6) as an oily substance. Referring to Figure 3, Figure 3 shows the HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZl produced in Example 1, with a yield of 93.5% and an HPLC purity of 99.78%. [ka]

[0066] (4) Preparation of Fmoc-Trp(Boc)-OH: 500 kg of dichloromethane and 50 kg of Fmoc-Trp(Boc)-OBZl were placed in a 1000 L reaction vessel and stirred until clarified, then 2 kg of Pd / C was added. After purging with nitrogen gas, hydrogen gas was passed through. The reaction was carried out for 15 hours while controlling the temperature to 25°C to 30°C, and the complete reaction of the starting material Fmoc-Trp(Boc)-OBZl was confirmed by TLC. After removing Pd / C by filtration, the filtrate was concentrated to dryness, 80 kg of ethyl acetate and 200 kg of n-hexane were added, and crystallization was carried out for 8 hours while cooling to 0°C to 5°C. Centrifugation was performed, and 70 kg of n-hexane was added to the solid obtained from centrifugation, and the mixture was stirred and washed for 2 hours. The solid obtained from centrifugation was dried at 15°C to 20°C to obtain 36 kg of Fmoc-Trp(Boc)-OH shown in formula (7) as a solid. Referring to Figures 4 and 5, Figure 4 shows the HPLC spectrum of Fmoc-Trp(Boc)-OH produced in Example 1, and Figure 5 shows the infrared spectrum of Fmoc-Trp(Boc)-OH produced in Example 1, with a yield of 84.3% and an HPLC purity of 99.74%. [ka]

[0067] [Example 2] (1) Preparation of Fmoc-Trp-OH: 600 kg of pure water was placed in a 2000 L reaction vessel, and 50 kg of sodium carbonate was added under stirring. Then, 60 kg of H-Trp-OH was added under stirring and stirred until completely dissolved. After that, 800 kg of ethyl acetate was added and the temperature was controlled to 25-30°C. Next, 104 kg of Fmoc-Osu was added all at once and the reaction was carried out for 3 hours while controlling the temperature to 25-30°C, controlling the pH to 8-9, and confirming that H-Trp-OH had completely reacted by TLC. 150 kg of hydrochloric acid was added to the system to acidify the pH to 2-3, liquid-liquid was separated, the ethyl acetate layer was recovered, washed three times with saturated sodium chloride, then sodium sulfate was added and dried, filtered, and the filtrate was concentrated until a large amount of solid material precipitated. After cooling to 20-25°C and crystallization for 2 hours, it was centrifuged and dried at 50-55°C to obtain 120 kg of the intermediate Fmoc-Trp-OH as a solid. Referring to Figure 6, which shows the HPLC spectrum of the intermediate Fmoc-Trp-OH produced in Example 2, the yield was 95.7% and the HPLC purity was 99.82%.

[0068] (2) Manufacturing of Fmoc-Trp-OBZl: 1200 kg of acetonitrile was placed in a clean, anhydrous 3000 L reaction vessel. Under stirring, 120 kg of Fmoc-Trp-OH produced in step (1) was added, followed by the addition of 45 kg of sodium carbonate and 39 kg of benzyl chloride, and stirring was started. The internal temperature was raised to 70°C to 75°C and the reaction was carried out for 4 hours. Afterward, TLC was used to confirm that the starting material Fmoc-Trp-OH had completely reacted. The reaction system was cooled to 20°C to 30°C, 500 kg of water was added to the reaction solution, 1000 kg of ethyl acetate was added and stirred until clarified, then allowed to stand and separated. Next, the ethyl acetate layer was recovered, washed three times with saturated sodium chloride, dried with sodium sulfate, concentrated until a large amount of solid precipitated, cooled to 20°C to 25°C, 300 kg of petroleum ether was added and crystallized for 2 hours, then centrifuged and dried at 50°C to 55°C to obtain 133 kg of the intermediate Fmoc-Trp-OBZl as a solid. Referring to Figure 7, Figure 7 shows the HPLC spectrum of the intermediate Fmoc-Trp-OBZl produced in Example 2, with a yield of 91.5% and an HPLC purity of 99.75%.

[0069] (3) Manufacturing of Fmoc-Trp(Boc)-OBZl: 1330 kg of tetrahydrofuran was placed in a clean, anhydrous 3000 L reaction vessel, and 133 kg of Fmoc-Trp-OBZl prepared in step (2) was added under stirring. The mixture was stirred until the solution became clear, and 53 kg of (Boc)2O was added. The temperature was controlled to 20°C to 25°C, and 1.33 kg of 4-dimethylaminopyridine was added. The mixture was reacted for 3 hours while maintaining the temperature at 20°C to 25°C, and TLC confirmed that the starting material Fmoc-Trp-OBZl had reacted completely. 500 kg of water was added to the system, and 500 kg of ethyl acetate was added for extraction. The mixture was separated, the ethyl acetate layer was recovered, washed three times with saturated sodium chloride, dried with sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain 155.6 kg of the intermediate Fmoc-Trp(Boc)-OBZl as an oily substance. Referring to Figure 8, which shows the HPLC spectrum of the intermediate Fmoc-Trp(Boc)-OBZl produced in Example 2, the yield was 98% and the HPLC purity was 99.71%.

[0070] (4) Preparation of Fmoc-Trp(Boc)-OH: 1556 kg of ethyl acetate and 155.6 kg of Fmoc-Trp(Boc)-OBZl were placed in a 3000 L reaction vessel and stirred until clarified, then 7.78 kg of Pd / C was added. After purging with nitrogen gas, hydrogen gas was passed through. The reaction was carried out for 18 hours while controlling the temperature at 30°C to 35°C, and the complete reaction of Fmoc-Trp(Boc)-OBZl was confirmed by TLC. After removing Pd / C by filtration, the filtrate was concentrated to approximately 300 L and allowed to dry, 600 kg of n-hexane was added, and crystallization was carried out for 8 hours while cooling to 0°C to 5°C. Centrifugation was performed, and 200 kg of n-hexane was added to the solid obtained from centrifugation and stirred and washed for 2 hours. The solid obtained from centrifugation was dried at 15°C to 20°C to obtain 118 kg of Fmoc-Trp(Boc)-OH as a solid. Referring to Figures 9, 10, and 11, Figure 9 shows the HPLC spectrum of Fmoc-Trp(Boc)-OH produced in Example 2, Figure 10 shows the infrared spectrum of Fmoc-Trp(Boc)-OH produced in Example 2, and Figure 11 shows the nuclear magnetic resonance spectrum of Fmoc-Trp(Boc)-OH produced in Example 2. The yield was 88.8% and the HPLC purity was 99.66%.

[0071] [Examples 3-4] In these examples, the effect of the inorganic base in step (1) on the intermediate Fmoc-Trp-OH was investigated. Specifically, the manufacturing steps and process parameters were the same as in Example 1, except that the type of inorganic base was changed, based on the manufacturing method in step (1) of Example 1. The results in step (1) are shown in Table 1 below.

[0072] [Table 1]

[0073] [Example 5, Comparative Examples 1-4] In these comparative examples, the effect of the inorganic base in step (2) on the intermediate Fmoc-Trp-OBZl was investigated. Specifically, the manufacturing steps and process parameters were the same as in Example 1, except that the type of inorganic base was changed based on the manufacturing method in step (2) of Example 1. The results of the experiment, including the purity and yield of Fmoc-Trp-OBZl, are shown in Table 2 below.

[0074] [Table 2]

[0075] [Examples 6-7, Comparative Examples 5-6] In these examples and comparative examples, the effect of the reaction temperature in step (2) on the intermediate Fmoc-Trp-OBZl was investigated. Specifically, the production method in step (2) of Example 1 was used, with only the reaction temperature being changed; all other production steps and process parameters were the same as in Example 1. The yield and reaction time of Fmoc-Trp-OBZl obtained from the experiment are shown in Table 3 below.

[0076] [Table 3]

[0077] [Example 8, Comparative Example 7] In these examples and comparative examples, the effect of the organic solvent in step (2) on the intermediate Fmoc-Trp-OBZl was investigated. Specifically, the manufacturing steps and process parameters were the same as in Example 1, except that the type of organic solvent was changed, based on the manufacturing method in step (2) of Example 1. The yield and purity of Fmoc-Trp-OBZl obtained from the experiment are shown in Table 4 below.

[0078] [Table 4]

[0079] [Examples 9-10] In this example and comparative example, the effect of the organic solvent in step (3) on the intermediate Fmoc-Trp(Boc)-OBZl was investigated. Specifically, the manufacturing steps and process parameters were the same as in Example 1, except that the type of organic solvent was changed based on the manufacturing method in step (3) of Example 1. The yield and purity of Fmoc-Trp(Boc)-OBZl obtained from the experiment are shown in Table 5 below.

[0080] [Table 5]

[0081] [Examples 11-14, Comparative Example 8] In these examples, the effect of the reaction temperature in step (4) on the intermediate Fmoc-Trp(Boc)-OH was investigated. Specifically, the production method in step (4) of Example 1 was used, with only the reaction temperature being changed; all other production steps and process parameters were the same as in Example 1. The results of the experiment, including the purity of Fmoc-Trp(Boc)-OH and the reaction time, are shown in Table 6 below.

[0082] [Table 6]

[0083] Based on the above, the present invention provides a method for producing Fmoc-Trp(Boc)-OH that is suitable for scale-up. This synthesis method requires simple equipment, has high reaction efficiency, relatively low production costs, and the purity of the final product reaches 98.0% or higher, with a yield of 75% or higher.

[0084] The above description represents only preferred embodiments of the present invention. Those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

【Request Item 1】 【Chemistry 1】 Step a) involves reacting tryptophan with N-(9-fluorenylmethoxycarbonyloxy)succinimide under the action of a base to obtain the intermediate compound shown in formula (3) above, 【Chemistry 2】 Step b) involves reacting the intermediate compound shown in formula (3) above with benzyl halide under the action of a base to obtain the intermediate compound shown in formula (4) above. 【Transformation 3】 Step c) involves reacting the intermediate compound shown in formula (4) above with di-tert-butyl dicarbonate under the action of a catalyst to obtain the intermediate compound shown in formula (6) above. A method for producing Fmoc-Trp(Boc)-OH, comprising step d) deprotecting the intermediate compound shown in formula (6) above to obtain Fmoc-Trp(Boc)-OH.

2. In step a), the base is an inorganic base, The manufacturing method according to claim 1, characterized in that the molar ratio of tryptophan, N-(9-fluorenylmethoxycarbonyloxy)succinimide, and base is 1:(0.8-1.3):(1-2.5).

3. In step a), the reaction is carried out at a temperature of 25°C to 35°C for 2 to 6 hours. The production method according to claim 2, characterized in that the reaction medium is an organic solvent selected from the group consisting of ethyl acetate, acetone, and tetrahydrofuran, and water.

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

5. The manufacturing method according to claim 4, characterized in that in step b), the base is sodium carbonate or potassium carbonate.

6. In step b), the reaction is carried out at a temperature of 60°C to 80°C for 2 to 4 hours. The method for producing the product according to claim 4, characterized in that the reaction medium is an organic solvent which is acetonitrile or ethyl acetate.

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

8. In step c), the reaction is carried out at a temperature of 10°C to 35°C for 0.5 to 2 hours. The production method according to claim 7, characterized in that the reaction medium is an organic solvent selected from the group consisting of dichloromethane, acetonitrile, ethyl acetate, and tetrahydrofuran.

9. The manufacturing method according to claim 1, characterized in that step d) includes reacting the intermediate compound shown in formula (6) with hydrogen gas under the action of a catalyst to obtain Fmoc-Trp(Boc)-OH.

10. In step d), the catalyst is Pd / C, Raney nickel, and Pd / BaSO4. ４ Selected from the group consisting of, The weight ratio of the intermediate compound to the catalyst shown in formula (6) is 1:(0.01 to 0.1), The manufacturing method according to claim 9, characterized in that the reaction is carried out at a temperature of 20°C to 30°C for 16 to 20 hours.

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